A novel method for the synthesis of 1,2,4-triazole-derived heterocyclic compounds: enzyme inhibition and molecular docking studies

Journal of the Iranian Chemical Society

https://doi.org/10.1007/s13738-019-01848-3

ORIGINAL PAPER

A novel method for the synthesis of 1,2,4‑triazole‑derived heterocyclic

compounds: enzyme inhibition and molecular docking studies

Naheed Riaz¹· Muhammad Iftikhar¹· Muhammad Saleem¹· Aziz‑ur‑Rehman²· Ishtiaq Ahmed³·

Muhammad Ashraf¹· Shahnawaz¹· Jameel Rehman¹· Mariya al‑Rashida⁴

Received: 5 October 2019 / Accepted: 27 December 2019

Abstract

Two series of new N-aryl/aralkyl derivatives (9a–q) of 2-(4-ethyl-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazol-3-ylthio)aceta-

mide and N-aryl/aralkyl derivatives (10a–q) of 2-(4-phenyl-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazol-3-ylthio)acetamide

were synthesized. The methods included successive conversions of thiophen-2-acetic acid (a) into its respective ester,

hydrazide and N-aryl/aralkyl 1,3,4-triazole. The target compounds (9a–q; 10a–q) were obtained by the reaction of N-aryl/

aralkyl 1,3,4-triazole (5, 6) with various electrophiles, (8a–q), in N,N-dimethyl formamide (DMF) and sodium hydroxide

at room temperature. The characterization of these compounds was done by FTIR, ¹H-, ¹³C-NMR, EI-MS and HR-EI-MS

spectral data. All compounds were evaluated for their enzyme inhibitory potentials against electric eel acetylcholinesterase,

AChE (10f, 10d; IC₅₀values 32.26 0.12, 45.72 0.11 µM, respectively), equine butyrylcholinesterase, BChE (9d, 9l, 9b,

10d, 10h; IC₅₀values 12.52 0.19, 12.52 0.19, 21.72 0.18, 23.62 0.22, 24.52 0.21 µM, respectively), jack bean urease

(10i, 10n, 9e; IC₅₀values 7.27 0.05, 7.35 0.04, 8.79 0.05 µM, respectively) and yeast α-glucosidase enzymes (9o, 10i;

IC₅₀values 62.94 0.19, and 69.46 0.15 µM, respectively). The molecular docking studies supported these fndings. This

study provides cheaper bioactive triazole amides as promising future lead molecules.

Keywords Triazole amides · Synthesis · Characterization · NMR spectroscopy · Mass spectrometry · Enzyme inhibition ·

Molecular docking studies

Introduction

1,2,4-triazole-derived synthetic drugs, like etizolam, alpra-

zolam, furacylin, are used for the treatment of infamma-

tion, as muscle relaxants and anxiety disorders, respectively.

1,2,4-Triazoles mostly are synthetic aromatic compounds

possessing an extensive chemistry [1, 2]. These compounds

with mercapto substitution on ring sy stems exhibit a diver-

sity of biological activities like antitumor [3 ], fungicidal [4],

antibacterial, antidepressant [5], anticonvulsant [6 ], antican-

cer [7], and antioxidant [8 ], while some are hypotensive [9].

In addition, they are reported to exhibit enzyme inhibition

activities against thymidine phosphorylase [10], carbonic

anhydrase and cholinesterase [11], glycogen phosphory-

lase [12 ], histone deacetylase [13 ], angiotensin converting

enzyme [14], cyclooxygenase [15 ], lipase and α-glucosidase

[16], nitric oxide synthase [17 ], and CYP19 aromatase [18].

The previous reports revealed that 1,2,4-triazole-derived

motifs are strong candidates for future drug discovery and

development.

During the last few decades, pharmaceutical and agro-

chemical industries have been focusing on the synthesis

of nitrogen-rich heterocyclic compounds. Among them,

* Naheed Riaz

nrch322@yaoo.com; nrch322@iub.edu.pk

1

Department of Chemistry, Baghdad-ul-Jadeed Campus,

The Islamia University of Bahawalpur, Bahawalpur 63100,

Pakistan

2

Department of Chemistry, Government College University,

Lahore 54000, Pakistan

3

Karlsruhe Institute of Technology (KIT), Institute for Biological

Interfaces (IBG-1), Hermann-von-Helmholtz-Platz,

76344 Eggenstein-Leopoldshafen, Karlsruhe, Germany

4

Department of Chemistry, Forman Christian College (A

There is a demand to address the new complex biologi-

cal malfunctions, such as neurodegeneration, dementia, and

Chartered University), Ferozepur Roa, Lahore 54600,

Pakistan

Vol.:(0123456789)

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Journal of the Iranian Chemical Society

cancer for the betterment of health across the world. There-

fore, medicinal chemists are designing chemicals that are

able to interact with two or more targets to produce synergis-

tic clinical efects, decreasing the risks and lack of compli-

ance associated with combinations of several drugs. In this

regard, enzymes remain prime targets for the drug design

because the altering enzyme activity has the immediate and

defned efects. Recent multitarget-directed ligand (MTDL)

approaches in medicinal chemistry have combined inhibitors

of cholinesterases (ChE) to combat the loss of neurotrans-

mitters in Alzheimer’s disease (AD) [19].

was added to the reaction mixture. A reddish brown oily

product was generated at basic pH (8–9 maintained by

Na₂CO₃) and was extracted with CHCl₃to get compound

1. The physical and spectroscopic data of 1 are as follows:

Reddish brown oil; yield: 71%; IR (KBr) v_max: 3022,

1

2928, 1755, 1597 cm⁻¹; H-NMR (500 MHz, CDCl₃): δ

1.15 (t, J = 6.5 Hz, 3H, CH₃–CH₂–O), 3.45 (s, 2H, H-6),

4.43 (q, J = 6.5 Hz, 2H, CH₃–CH₂–O), 6.53 (d, J = 3.0 Hz,

1H, H-3), 6.83 (dd, J = 3.5, 3.0 Hz, 1H, H-4), 7.10 (d,

J=3.5 Hz, 1H, H-5); ¹³C-NMR (125 MHz, CDCl₃): δ 20.1

(CH₃), 35.3 (C-6), 63.3 (CH₂), 127.3 (C-4), 128.0 (C-3),

131.8 (C-5), 141.4 (C-2), 166.9 (C-7); HR-EI-MS (m/z):

170.0410 [M]⁺calculated for C₈H₁₀SO₂; 170.0402.

Structure-based drug design for a single enzyme target

has been facilitated with crystal structures enabling the com-

putational searches to identify lead compounds for refne-

ment. With such large-scale computational approaches

including analysis of of-target activity, combining suitable

pharmacophores for enzyme combinations such as ChE for

AD is entirely feasible. However, such compounds still have

to be synthesized and tested experimentally, to confrm the

predicted inhibitory efects against each of the targets [11].

The present work was designed to explore a new series

of biologically active mercapto N-aryl/aralkyl acetamide

triazoles, which may assist in designing new synthetic

drug leads; therefore, N-aryl/aralkyl derivatives (9a–q) of

2-(4-ethyl-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazol-3-

ylthio)acetamide and N-aryl/aralkyl derivatives (10a–q) of

2-(4-phenyl-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazol-3-

ylthio)acetamide were synthesized in a series of steps and

were subsequently screened against electric eel acetylcho-

linesterase (AChE), equine butyrylcholinesterase (BChE),

jack bean urease and yeast α-glucosidase enzymes.

Synthesis of 2‑(thiophen‑2‑yl)acetohydrazide (2)

Ethyl thiophene-2-acetate (2; 0.05 mol, 7.5 ml) and

0.05 mol (2.4 ml) of hydrazine hydrate (80%) were stirred

for 4–5 h in 100-ml round-bottom fask till the completion

of the reaction. The light yellow crystalline product was

fltered, washed with n-hexane and dried. The physical and

spectroscopic data of 2 are as follows:

Yellow crystals; yield 76%; M.P.: 108–110 °C; IR

(KBr) v_max: 3417, 3320, 3024, 2935, 1692, 1533 cm⁻¹;

¹H-NMR (500 MHz, CDCl₃): δ 3.45 (s, 2H, H-6), 4.02 (s,

1H, –NH), 6.53 (d, J = 3.0 Hz, 1H, H-3), 6.83 (dd, J = 3.5,

3.0 Hz, 1H, H-4), 7.10 (d, J = 3.5 Hz, 1H, H-5), 8.90 (s,

2H, –NH₂); ¹³C-NMR (125 MHz, CDCl₃): δ 35.5 (C-6),

127.3 (C-4), 128.0 (C-3), 129.8 (C-5), 135.4 (C-2), 168.9

(C-7); HR-E-IMS (m/z): 156.0357 [M]⁺calculated for

C₆H₈SN₂O; 156.0350.

Materials and methods

Synthesis of 4‑ethyl‑1‑(2‑(thiophen‑2‑yl)acetyl)

thiosemicarbazide (3)

¹H- and ¹³C-NMR spectra were recorded on Bruker instru-

ment operating at 500 and 125 MHz, respectively, using

TMS as an internal standard. IR spectra were recorded on

Shimadzu 460 FTIR spectrometer using KBr pellets. Mass

spectra were measured on JMSA 500 mass spectrometer

and JMS H × 110 spectrometer with data system. Melting

points were found on Gallen Kemp electrothermal appara-

tus. All the chemicals and solvents of analytical grade were

purchased from local supplier of Sigma, Aldrich and Alfa

Aesar. During synthesis, reaction progress was monitored

using TLC.

Carbohydrazide (2, 2.5 g, 0.02 mol) was dissolved in 25 ml

of ethanol in a 100-ml round-bottom fask. Ethyl isothio-

cyanate (0.02 mol) was added in the reaction mixture, and

contents were refuxed for 3–4 h. Precipitated fnal product

was cooled, fltered, washed and dried. The physical and

spectroscopic data of 3 are as follows:

Amorphous solid; yield 79%; M.P.: 121–123 °C; IR

1

(KBr) v_max: 3325, 2938, 1697, 1601 cm⁻¹; H-NMR

(500 MHz, CDCl₃): δ 1.15 (t, J=6.4 Hz, 3H, CH₃–CH₂–N),

3.41 (s, 2H, H-6), 3.53 (q, J= 6.4 Hz, 2H, CH₃–CH₂–N),

6.52 (d, J=3.0 Hz, 1H, H-3), 6.85 (dd, J=3.5, 3.0 Hz, 1H,

H-4), 7.11 (d, J = 3.5 Hz, 1H, H-5); ¹³C-NMR (125 MHz

CDCl₃): δ 20.1 (CH₃), 36.3 (C-6), 39.5 (CH₂), 126.3 (C-4),

129.0 (C-3), 131.8 (C-5), 138.4 (C-2), 171.0 (C-7), 180.6

(C=S); HR-EI-MS (m/z): 243.0510 [M]⁺calculated for

C₉H₁₃S₂N₃O; 243.0499.

Synthesis of ethyl 2‑(thiophen‑2‑yl)acetate (1)

Thiophen-2-acetic acid (a; 10 g) was dissolved in an

excess amount of ethanol in a round-bottom fask. Six ml

of H₂SO₄was added to the fask and the reaction mixture

was refuxed for 3–4 h. Excess amount of distilled water

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Journal of the Iranian Chemical Society

Amorphous powder; yield 78%; M.P.: 123–125 °C;

Synthesis of 4‑phenyl‑1‑(2‑(thiophen‑2‑yl)acetyl)

thiosemicarbazide (4)

1

IR (KBr) v_max: 3033, 2929, 1601, 1524 cm⁻¹; H-NMR

(500 MHz, CDCl₃): δ 3.25 (s, 2H, H-6′), 6.57 (d, J=2.8 Hz,

1H, H-3′), 6.98 (dd, J = 3.2, 2.8 Hz, 1H, H-4′), 7.18 (d,

J = 3.2 Hz, 1H, H-5′), Ar–H, [7.01–7.09 (m, 5H)], 13.6

(s, SH, 1H); ¹³C-NMR (125 MHz, CDCl₃): δ 35.3 (C-6′),

126.9 (C-4′), 128.0 (C-3′), 131.0 (C-5′), 136.0 (C-2′), Ar–C

[125.8 (2 × CH), 128.1 (2 × CH), 129.1 (CH), 136.4 (C)],

141.2 (C-5), 151.0 (C-3); HR-EI-MS (m/z): 273.0381 [M]⁺

calculated for C₁₃H₁₁S₂N₃; 273.0394.

Two grams (0.012 mol) of carbohydrazide (3) was dissolved

in 25 ml of ethanol in a 100-ml round-bottom fask. Two

ml of phenyl isothiocyanate (0.012 mol) was added in the

reaction mixture, and the contents were refuxed and stirred

for 3–4 h. Precipitated crude product was cooled, fltered,

washed and dried. The physical and spectroscopic data for

4 are as follows:

Amorphous powder; yield 68%; M.P.: 119–120 °C; IR

(KBr) v_max: 3317, 3031, 2929, 1685, 1601, 1524 cm⁻¹;

¹H-NMR (500 MHz, CDCl₃): δ 3.13 (s, 2H, H-6), 6.48 (d,

J = 3.0 Hz, 1H, H-3), 6.69 (dd, J = 3.2, 3.0 Hz, 1H, H-4),

7.10–7.15 (m, 5H, Ar–H), 7.19 (d, J=3.2 Hz, 1H, H-5); ¹³C-

NMR (125 MHz, CDCl₃): δ 34.3 (C-6), 126.9 (C-3), 128.0

(C-4), 129.8 (C-5), (C-2) 136.4, Ar–C [125.0 (2 × CH),

127.3 (2×CH), 128.1 (CH), 128.3 (C)], 168.5 (C-7), 180.6

(C=S); HR-EI-MS (m/z): 291.0505 [M]⁺calculated for

C₁₃H₁₃S₂N₃O; 291.0500.

Synthesis of compounds (8a–q)

Calculated amount of each of the aryl/aralkyl amines (7a–q,

11.0 mmol) separately was added to 10 ml distilled water

in an iodine fask. Basic pH (9–10) was adjusted by add-

ing 5% Na₂CO₃solution. The reaction mixture was stirred

for 10 min at 0–5 °C, and at the same temperature, it was

supplemented drop-wise with bromoacetyl bromide (1.0 ml,

11.0 mmol). The reaction fask was then shaken vigorously

till the formation of precipitates. The precipitates were

stirred for further 45 min, and the solid product was fltered,

washed with distilled water and dried to yield the respective

electrophiles, N-aralkyl/aryl-substituted-2-bromoacetamides

(8a–q).

Synthesis of 4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑1,2,

4‑triazole‑3‑thiol (5)

4-Ethyl-1-(2-(thiophen-2-yl)acetyl)thiosemicarbazide (3)

was dissolved in 10% aqueous NaOH in a 100-ml round-

bottom fask and was refuxed for 4 h. The reaction mixture

was poured into ice cold water, acidifed with conc HCl to

pH 4–6 and the precipitated product was cooled, fltered,

washed and dried. The physical and spectroscopic data for

5 are as follows:

General method for the synthesis of compounds

(9a–q)

Compound 5 (0.1 mmol, 0.002 g) was dissolved in 10 ml of

DMF in a 50-ml round-bottom fask. The solution was then

supplemented with sodium hydride (0.002 g, 0.1 mmol),

and the mixture was stirred for 30 min at room tempera-

ture, followed by the slow addition of electrophiles (8a–q,

each separately) while stirring for 2–3 h. Distilled water was

then added to the reaction mixture and the products were

recovered by fltration or solvent extraction according to the

product nature.

Amorphous powder; yield 79%; M.P.: 121–123 °C;

1

IR (KBr) v_max: 3032, 2925, 1593, 1543 cm⁻¹; H-NMR

(500 MHz, CDCl₃): δ 1.10 (t, J=6.5 Hz, 3H, CH₃–CH₂–N),

3.25 (s, 2H, H-6′), 3.98 (q, J=6.5 Hz, 2H, CH₃–CH₂–N),

6.50 (d, J=3.0 Hz, 1H, H-3′), 6.85 (dd, J=3.4, 3.0 Hz, 1H,

H-4′), 7.10 (d, J=3.4 Hz, 1H, H-5′), 13.6 (s, SH, 1H); ¹³C-

NMR (125 MHz, CDCl₃): δ 20.7 (CH₃), 35.3 (C-6′), 39.5

(CH₂), 126.3 (C-4′), 129.0 (C-3′), 131.8 (C-5′), 138.4 (C-2′),

140.4 (C-5), 149.7 (C-3); HR-EI-MS (m/z): 225.0380 [M]⁺

calculated for C₉H₁₁S₂N₃; 225.0394.

N‑(2,3‑dimethylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)

‑4H‑1,2,4‑triazol‑3‑ylthio)acetamide (9a)

Amorphous powder; yield: 71%; M.P.: 135–137 °C; IR

(KBr) v_max: 3020, 2920, 1696, 1597, 1535 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.15 (t, J=6.5 Hz, 3H, CH₃–CH₂–N),

2.13 (s, CH₃, 3H), 2.28 (s, CH₃, 3H), 3.83 (q, J = 6.5 Hz,

2H, CH₃–CH₂–N), 4.01 (s, 2H, H-6′), 4.17 (s, 2H, H-2″),

thiophene-H [6.57 (d, J = 2.8 Hz, 1H, H-3′), 6.98 (dd,

J = 3.0, 2.8 Hz, 1H, H-4′), 7.18 (d, J = 3.0 Hz, 1H, H-5′],

Ar–H [7.10–7.20 (m, CH, 3H)], 7.97 (s, 1H, NH); ¹³C-NMR

(125 MHz, CDCl₃): δ 13.4 (CH₃), 14.1 (CH₃), 19.7 (CH₃),

31.9 (C-6′), 32.1 (C-2″), 45.8 (CH₂), 127.3 (C-4′), 128.0

(C-3′), 131.8 (C-5′), 141.4 (C-2′), Ar–C [122.8 (C-4‴),

Synthesis of 4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑1,

2,4‑triazole‑3‑thiol (6)

4-Phenyl-1-(2-(thiophen-2-yl)acetyl)thiosemicarbazide (4)

was dissolved in 10% aqueous NaOH in 100-ml round-bot-

tom fask and refuxed for 4 h. The progress of the reaction

was monitored using TLC. The reaction mixture was poured

into ice cold water, acidifed to pH 4–6 using concentrated

HCl, and the precipitated product was cooled, filtered,

washed and dried. The physical and the spectroscopic data

of compound 6 are as follows:

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Journal of the Iranian Chemical Society

(s, 2H, H-2″), thiophene-H [6.86 (d, J= 2.8 Hz, 1H, H-3′),

6.90 (dd, J = 3.0, 2.8 Hz, 1H, H-4″), 7.18 (d, J = 3.0 Hz,

1H, H-5′)], Ar–H [7.23–7.26 (m, 3H, H-3′-5′), 7.80 (s,

1H, NH); ¹³C-NMR (125 MHz, CDCl₃): δ 13.6 (CH₃),

16.2 (CH₃), 20.1 (CH₃), 30.1 (C-6′), 36.3 (C-2″), 42.5

(CH₂), thiophene-C [126.6 (C-4′), 130.0 (C-3′), 134.9

(C-5′), 136.1 (C-2′)], Ar–C [125.0 (C-5‴), 125.2 (C-3‴),

126.2 (C-4‴), 126.8 (C-2‴), 129.0 (C-6‴, 137.3 (C-1‴)],

150.5 (C-5), 154.6 (C-3), 167.3 (C-1″); HR-EI-MS (m/z):

386.1224 [M]⁺calculated for C₁₉H₂₂S₂N₄O; 386.1235.

125.4 (C-5‴), 125.8 (C-6‴), 130.4 (C-2‴), 135.7 (C-3‴),

137.9 (C-1‴)], 151.0 (C-5), 155.0 (C-3), 166.9 (C-1″); HR-

EI-MS (m/z): 386.1226 [M]⁺calculated for C₁₉H₂₂S₂N₄O;

386.1235.

N‑(2,4‑dimethylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (9b)

Amorphous powder; yield: 69%; M.P.: 129–130 °C; IR

(KBr) v_max: 3030, 2915, 1696, 1596, 1535 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.12 (t, J=6.5 Hz, 3H, CH₃–CH₂–N),

2.11 (s, CH₃, 3H), 2.24 (s, CH₃, 3H), 3.81 (q, J=6.5 Hz, 2H,

CH₃–CH₂–N), 4.00 (s, 2H, H-6′), 4.13 (s, 2H, H-2″), thio-

phene-H [6.57 (d, J=2.8 Hz, 1H, H-3′), 6.98 (dd, J=3.0,

2.8 Hz, 1H, H-4′), 7.18 (d, J = 3.0 Hz, 1H, H-5′)], Ar–H,

[7.23 (br s, 1H, H-3‴), 7.33 (d, J=8.0 Hz, 1H, H-5‴), 7.38

(d, J = 8.0 Hz, 1H, H-6‴)], 7.98 (s, 1H, NH); ¹³C-NMR

(125 MHz, CDCl₃): δ 13.6 (CH₃), 16.2 (CH₃), 20.1 (CH₃),

31.1 (C-6′), 36.5 (C-2″), 44.4 (CH₂), 126.4 (C-4′), 130.1

(C-3′), 132.5 (C-5′), 138.2 (C-2′), Ar–C [126.1 (C-5‴),

127.2 (C-6‴), 127.2 (C-3‴), 128.4 (C-2‴), 129.6 (C-4‴),

136.7 (C-1‴)], 151.6 (C-5), 155.0 (C-3), 166.5 (C-1″); HR-

EI-MS (m/z): 386.1224 [M]⁺calculated for C₁₉H₂₂S₂N₄O;

386.1235.

N‑(3,4‑dimethylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (9e)

Amorphous powder; yield: 69%; M.P.: 127–129 °C; IR

1

(KBr) v_max: 3026, 2928, 1693, 1591, 1535 cm⁻¹; H-

NMR (500 MHz, CDCl₃): δ 1.04 (t, J = 6.5 Hz, 3H,

CH₃–CH₂–N), 1.94 (s, 3H, CH₃), 2.03 (s, 3H, CH₃), 3.80

(q, J = 6.5 Hz, 2H, CH₃–CH₂–N), 4.02 (s, 2H, H-6′),

4.12 (s, 2H, H-2″), thiophene-H, [6.54 (d, J = 2.8 Hz,

1H, H-3′), 6.91 (dd, J = 2.9, 2.8 Hz, 1H, H-4′), 7.09 (d,

J = 2.9 Hz, 1H, H-5′)], Ar–H [7.23 (br s, 1H, H-2‴), 7.33

(d, J=8.0 Hz, 1H, H-5‴), 7.38 (d, J=8.0 Hz, 1H, H-6‴)],

7.90 (s, 1H, NH); ¹³C-NMR (125 MHz, CDCl₃): δ 14.6

(CH₃), 19.3 (CH₃), 21.3 (CH₃), 31.4 (C-6′), 35.4 (C-2″),

46.1 (CH₂), thiophene-C [126.4 (C-4′), 130.0 (C-3′),

134.9 (C-5′), 138.7 (C-1′)], Ar–C [125.2 (C-5‴), 126.2

(C-6‴), 127.2 (C-2‴), 128.3 (C-3‴), 128.7 (C-4‴), 137.1

(C-1‴)], 151.2 (C-5), 153.7 (C-3), 168.5 (C-1″); HR-EI-

MS (m/z): 386.1227 [M]⁺calculated for C₁₉H₂₂S₂N₄O;

386.1235.

N‑(2,5‑dimethylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (9c)

Amorphous powder; yield: 69%; M.P.: 126–127 °C; IR

(KBr) v_max: 3025, 2910, 1685, 1580, 1540 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.08 (t, J=6.5 Hz, 3H, CH₃–CH₂–N),

2.15 (s, 3H, CH₃), 2.29 (s, 3H, CH₃), 3.85 (q, J=6.5 Hz, 2H,

CH₃–CH₂–N), 4.01 (s, 2H, H-6′), 4.12 (s, 2H, H-2″), thio-

phene-H [6.52 (d, J=3.0 Hz, 1H, H-3′), 6.80 (dd, J=3.5,

3.0 Hz, 1H, H-4′), 7.15 (d, J = 3.5 Hz, 1H, H-5′)], Ar–H,

[7.01 (br s, 1H, H-6‴), 6.97 (d, J = 7.8 Hz, 1H, H-3‴),

7.37 (d, J=7.8 Hz, 1H, H-4‴), 7.79 (s, 1H, NH); ¹³C-NMR

(125 MHz, CDCl₃): δ 13.6 (CH₃), 16.2 (CH₃), 20.1 (CH₃),

30.1 (C-6′), 35.3 (C-2″), 44.5 (CH₂), 126.6 (C-4′), 130.0

(C-3′), 134.9 (C-5′), 136.1 (C-2′), Ar–C [125.0 (C-3‴),

125.2 (C-4‴), 126.2 (C-6‴), 126.8 (C-2‴), 129.0 (C-5‴),

137.3 (C-1‴)], 150.5 (C-5), 154.6 (C-3), 167.3 (C-1″); HR-

EI-MS (m/z): 386.1227 [M]⁺calculated for C₁₉H₂₂S₂N₄O;

386.1235.

N‑(3,5‑Dimethylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (9f)

Amorphous powder; yield: 67%; M.P.: 127–129 °C; IR

1

(KBr) v_max: 3020, 2925, 1697, 1591, 1530 cm⁻¹; H-

NMR (500 MHz, CDCl₃): δ 1.04 (t, J = 6.5 Hz, 3H,

CH₃–CH₂–N), 1.94 (s, 3H, CH₃), 2.05 (s, 3H, CH₃), 3.83

(q, J = 6.5 Hz, 2H, CH₃–CH₂–N), 4.01 (s, 2H, H-6′), 4.12

(s, 2H, H-2″), thiophene-H, 6.62 [(d, J=2.8 Hz, 1H, H-3′),

6.85 (dd, J = 3.0, 2.8 Hz, 1H, H-4′), 7.10 (d, J = 3.0 Hz,

1H, H-5′)], Ar–H [7.23 (br s, 1H, H-2‴), 7.33 (br s, 1H,

H-4‴), 7.35 (br s, 1H, H-6‴)], 7.98 (s, 1H, NH); ¹³C-

NMR (125 MHz, CDCl₃): δ 14.6 (CH₃), 16.3 (CH₃), 20.3

(CH₃), 31.4 (C-6′), 33.2 (C-2″), 45.1 (CH₂), thiophene-C

[126.4 (C-4′), 130.0 (C-3′), 134.9 (C-5′), 138.7 (C-2′)],

Ar–C [125.2 (C-2‴), 126.2 (C-4‴), 127.2 (C-6‴), 128.3

(C-3‴), 130.7 (C-5‴), 137.1 (C-1‴)], 151.2 (C-5), 153.7

(C-3), 168.5 (C-1″); HR-EI-MS (m/z): 386.1227 [M]⁺cal-

culated for C₁₉H₂₂S₂N₄O; 386.1235.

N‑(2,6‑dimethylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (9d)

Amorphous powder; Yield: 62%; M.P.: 151–153 °C;

IR (KBr) v_max: 3025, 2915, 1689, 1570, 1540 cm⁻¹;

¹H-NMR (500 MHz, CDCl₃): δ 1.05 (t, J = 6.5 Hz, 3H,

CH₃–CH₂–N), 1.82 (s, 3H, CH₃), 1.93 (s, 3H, CH₃), 3.84

(q, J = 6.5 Hz, 2H, CH₃–CH₂–N), 4.00 (s, 2H, H-6′), 4.10

1 3

Journal of the Iranian Chemical Society

N‑phenyl‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑1,2,4

‑triazo‑3‑ylthio)acetamide (9g)

138.4 (C-1‴)], 150.3 (C-5), 154.6 (C-3), 166.5 (C-1″); HR-

EI-MS (m/z): 372.1060 [M]⁺calculated for C₁₈H₂₀S₂N₄O;

372.1078.

Amorphous powder; yield: 66%; M.P.: 147–150 °C; IR

(KBr) v_max: 3020, 2928, 1692, 1597, 1542 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.08 (t, J=6.5 Hz, 3H, CH₃–CH₂–N),

3.78 (q, J=6.5 Hz, 2H, CH₃–CH₂–N), 3.99 (s, 2H, H-6′),

4.09 (s, 2H, H-2″), thiophene-H, [6.54 (d, J = 3.0 Hz,

1H, H-3′), 6.83 (dd, J = 3.0, 2.8 Hz, 1H, H-4′), 7.10 (d,

J=2.8 Hz, 1H, H-5′)], Ar–H 7.24–7.27 (m, 5H, H-2‴–6‴),

8.01 (s, 1H, NH); ¹³C-NMR (125 MHz, CDCl₃): δ 16.2

(CH₃), 30.0 (C-6′), 35.0 (C-2″), 45.1 (CH₂), thiophene-C

[126.4 (C-4′), 130.2 (C-3′), 132.4 (C-5′), 138.9 (C-2′)],

Ar–C [125.2 (C-3‴,5‴), 127.1 (C-2‴,6‴), 128.4 (C-4‴),

129.6 (C-1‴)], 151.6 (C-5), 154.9 (C-3), 168.3 (C-1″); HR-

EI-MS (m/z): 358.0912 [M]⁺calculated for C₁₇H₁₈S₂N₄O;

358.0922.

N‑(4‑methylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑

1,2,4‑triazo‑3‑ylthio)acetamide (9j)

Amorphous powder; yield: 62%; M.P.: 122–124 °C; IR (KBr

disk,) v_max: 3020, 2920, 1696, 1597, 1535 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.04 (t, J=6.5 Hz, 3H, CH₃–CH₂–N),

2.76 (s, 3H, CH₃), 3.81 (q, J= 6.5 Hz, 2H, CH₃–CH₂–N),

4.01 (s, 2H, H-6′), 4.10 (s, 2H, H-2″), thiophene-H, [6.55 (d,

J=2.6 Hz, 1H, H-3′), 6.92 (dd, J=3.0, 2.6 Hz, 1H, H-4′),

7.18 (d, J = 3.0 Hz, 1H, H-5′), Ar–H [7.23 (d, J = 8.4 Hz,

2H, H-2‴,3‴), 7.33 (d, J = 8.4 Hz, 2H, H-5‴,6‴), 7.90

(s, 1H, NH); ¹³C-NMR (125 MHz, CDCl₃): δ 13.6 (CH₃),

22.1 (CH₃), 31.9 (C-6′), 35.7 (C-2″), 46.1 (CH₂), thiophene-

C [126.3 (C-4′), 128.1 (C-3′), 128.4 (C-5), 137.3 (C-2′)],

Ar–C [116.8 (C-3‴,5‴), 125.2 (C-2‴,6‴), 126.3 (C-4‴)

138.8 (C-1‴)], 150.3 (C-5), 154.5 (C-3), 168.4 (C-1″); HR-

EI-MS (m/z): 372.1060 [M]⁺calculated for C₁₈H₂₀S₂N₄O;

372.1078.

N‑(2‑methylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑

1,2,4‑triazo‑3‑ylthio)acetamide (9h)

Amorphous powder; yield: 73%; M.P.: 123–125 °C; IR

(KBr) v_max: 3016, 2924, 1696, 1590, 1532 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.08 (t, J=6.5 Hz, 3H, CH₃–CH₂–N),

2.24 (s, 3H, CH₃), 3.79 (q, J= 6.5 Hz, 2H, CH₃–CH₂–N),

4.01 (s, 2H, H-6′), 4.12 (s, 2H, H-2″), thiophene-H, [6.59

(d, J = 2.8 Hz, 1H, H-3′), 6.82 (dd, J = 3.0, 2.8 Hz, 1H,

H-4′), 7.15 (d, J=3.0 Hz, 1H, H-5′)], Ar–H 7.23–7.29 (m,

4H, H-3‴–6‴), 7.81 (s, 1H, NH); ¹³C-NMR (125 MHz,

CDCl₃): δ 17.0 (CH₃), 22.2 (CH₃), 30.4 (C-6′), 35.7 (C-2″),

46.1 (CH₂), thiophene-C [126.5 (C-4′), 131.8 (C-3′), 132.3

(C-5′), 136.6 (C-2′)], Ar–C [125.9 (C-4‴), 126.2 (C-5‴),

126.0 (C-3‴), 127.1 (C-6‴), 130.3 (C-2‴), 135.2 (C-1‴)],

151.9 (C-5), 155.0 (C-3), 167.3 (C-1″); HR-EI-MS (m/z):

372.1060 [M]⁺calculated for C₁₈H₂₀S₂N₄O; 372.1078.

Methyl‑(2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑1,2,4‑tria‑

zol‑3‑ylthio)acetamido)‑1‑benzoate (9k)

Amorphous powder; yield: 71%; M.P.: 173–175 °C; IR

(KBr) v_max: 3020, 2920, 1696, 1597, 1535 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.05 (t, J=6.5 Hz, 3H, CH₃–CH₂–N),

3.80 (q, J=6.5 Hz, 2H, CH₃–CH₂–N), 3.98 (s, 3H, –OCH₃),

4.02 (s, 2H, H-6′), 4.11 (s, 2H, H-2″), thiophene-H, [6.49

(d, J = 2.6 Hz, 1H, H-3′), 6.85 (dd, J = 3.4, 2.6 Hz, 1H,

H-4′), 7.11 (d, J=3.4 Hz, 1H, H-5′)], Ar–H [7.23 (m, 4H,

H-3‴–6‴), 7.90 (s, 1H, NH); ¹³C-NMR (125 MHz, CDCl₃):

δ 21.3 (CH₃), 31.2 (C-6′), 34.5 (C-2″), 45.1 (CH₂), 55.3

(OCH₃), thiophene-C [124.5 (C-3′), 128.7 (C-4′), 128.4

(C-5′), 136.2 (C-2′)], Ar–C [115.3 (C-4‴), 125.0 (C-3‴),

126.0 (C-5‴), 127.9 (C-6‴), 128.3 (C-2‴) 139.4 (C-1‴)],

148.3 (C-5), 152.0 (C-3), 169.4 (C-1″); HR-EI-MS (m/z):

416.0965 [M]⁺calculated for C₁₉H₂₀S₂N₄O₃; 416.0976.

N‑(3‑methylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑

1,2,4‑triazo‑3‑ylthio)acetamide (9i)

Amorphous powder; yield: 77%; M.P.: 120–122 °C; IR

(KBr) v_max: 3019, 2930, 1699, 1594, 1535 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.12 (t, J=6.5 Hz, 3H, CH₃–CH₂–N,),

2.12 (s, 3H, CH₃), 3.80 (q, J= 6.5 Hz, 2H, CH₃–CH₂–N),

4.00 (s, 2H, H-6′), 4.10 (s, 2H, H-2″), thiophene-H, [6.62 (d,

J=3.2 Hz, 1H, H-3′), 6.88 (dd, J=3.2, 2.8 Hz, 1H, H-4′),

7.19 (d, J=2.8 Hz, 1H, H-5′)], Ar–H [7.23 (s, 1H, H-2‴),

7.33 (m, 3H, H-4‴–6‴), 7.80 (s, 1H, NH); ¹³C-NMR

(125 MHz, CDCl₃): δ 17.0 (CH₃), 21.2 (CH₃), 30.4 (C-6′),

35.7 (C-2″), 46.1 (CH₂), thiophene-C [126.7 (C-4′), 137.3

(C-3′), 128.3 (C-5′), 138.0 (C-2′)], Ar–C [116.8 (C-4‴),

120.2 (C-5‴), 124.8 (C-2‴), 125.0 (C-6‴), 126.2 (C-3‴),

N‑(2‑ethylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑1,

2,4‑triazo‑3‑ylthio)acetamide (9l)

Amorphous powder; yield: 62%; M.P.: 133–135 °C; IR

(KBr) v_max: 3025, 2929, 1689, 1593, 1539 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.00 (t, J= 6.6 Hz, 3H, CH₃–CH₂),

1.05 (t, J=6.5 Hz, 3H, CH₃–CH₂–N), 2.60 (q, J=6.6 Hz,

2H, CH₃–CH₂), 3.83 (q, J = 6.5 Hz, 2H, CH₃–CH₂–N),

3.99 (s, 2H, H-6′), 4.12 (s, 2H, H-2″), thiophene-H,

[6.53 (d, J = 2.6 Hz, 1H, H-3′), 6.87 (dd, J = 3.5, 2.6 Hz,

1H, H-4′), 7.11 (d, J= 3.5 Hz, 1H, H-5′)], Ar–H 7.23 (m,

4H, H-3‴–6‴), 7.91 (s, 1H, NH); ¹³C-NMR (125 MHz,

1 3

Journal of the Iranian Chemical Society

CDCl₃): δ 13.6 (CH₃), 18.1 (CH₃), 22.0 (CH₂), 31.4 (C-6′),

34.7 (C-2″), 44.1 (CH₂), thiophene-C [126.5 (C-4′), 130.2

(C-3′), 132.2 (C-5′), 136.7 (C-2′)], Ar–C [124.7 (C-4‴),

125.9 (C-3‴), 126.2 (C-5‴), 127.1 (C-6‴), 134.5 (C-2‴),

138.4 (C-1‴)], 151.9 (C-5), 155.1 (C-3), 167.7 (C-1″); HR-

EI-MS (m/z): 386.1226 [M]⁺calculated for C₁₉H₂₂S₂N₄O;

386.1235.

(500 MHz, CDCl₃): δ 1.01 (t, J = 6.6 Hz, 3H, CH₃–CH₂),

1.05 (t, J = 6.5 Hz, 3H, CH₃–CH₂–N), 2.16 (q, J = 6.6 Hz,

2H, CH₃–CH₂), 2.23 (s, 3H, CH₃), 3.81 (q, J=6.5 Hz, 2H,

CH₃–CH₂–N), 4.00 (s, 2H, H-6′), 4.12 (s, 2H, H-2″), thio-

phene-H [6.58 (d, J = 3.2 Hz, 1H, H-3′), 6.80 (dd, J = 3.4,

3.2, Hz, 1H, H-4′), 7.15 (d, J = 3.4 Hz, 1H, H-5′)], Ar–H

7.30 (m, 3H, H-3‴–5‴), 7.89 (s, 1H, NH); ¹³C-NMR

(125 MHz, CDCl₃): δ 13.6 (CH₃), 13.9 (CH₃), 17.1 (CH₃),

22.4 (CH₂), 31.8 (C-6′), 37.4 (C-2″), 45.0 (CH₂), thio-

phene-C [126.5 (C-3′), 133.0 (C-4′), 135.8 (C-5′), 141.4

(C-2′)], Ar–C [125.2 (C-4‴), 126.1 (C-3‴), 126.9 (C-5‴),

127.5 (C-2‴), 127.6 (C-6‴) 136.9 (C-1‴)], 150.8 (C-5),

154.5 (C-3), 167.5 (C-1″); HR-EI-MS (m/z): 400.1380

[M]⁺calculated for C₂₀H₂₄S₂N₄O; 400.1391.

N‑(4‑ethylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑1,

2,4‑triazo‑3‑ylthio)acetamide (9m)

Amorphous powder; yield: 62%; M.P.: 129–131 °C; IR

(KBr) v_max: 3035, 2934, 1682, 1581, 1550 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.01 (t, J= 6.6 Hz, 3H, CH₃–CH₂),

1.16 (t, J=6.5 Hz, 3H, CH₃–CH₂–N), 2.25 (q, J=6.6 Hz,

2H, CH₃–CH₂), 3.86 (q, J=6.5 Hz, 2H, CH₃–CH₂–N), 4.00

(s, 2H, H-6′), 4.10 (s, 2H, H-2″), thiophene-H [6.53 (d,

J=2.6 Hz, 1H, H-3′), 6.89 (dd, J=3.5, 2.6 Hz, 1H, H-4′),

7.11 (d, J=3.5 Hz, 1H, H-5′)], Ar–H [7.30 (d, J=8.2 Hz,

2H, H-2‴,3‴), 7.45 (d, J=8.2 Hz, 2H, H-5‴,6‴)], 7.88 (s,

1H, NH); ¹³C-NMR (125 MHz, CDCl₃): δ 13.7 (CH₃), 14.8

(CH₃), 21.2 (CH₂), 31.5 (C-6′), 39.3 (C-2″), 45.9 (CH₂),

thiophene-C [126.2 (C-3′), 126.7 (C-4′), 135.7 (C-5′), 137.3

(C-2′)], Ar–C [119.8 (C-3‴,5‴), 126.7 (C-2‴,6‴), 135.7

(C-4‴), 140.4 (C-1‴)], 150.3 (C-5), 154.5 (C-3), 166.4

(C-1‴); HR-EI-MS (m/z): 386.1226 [M]⁺calculated for

C₁₉H₂₂S₂N₄O; 386.1235.

N‑(2‑methoxyphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4

H‑1,2,4‑triazol‑3‑ylthio)acetamide (9p)

Amorphous powder; yield: 65%; M.P.: 141–143 °C; IR

1

(KBr) v_max: 3016, 2929, 1691, 1590, 1533 cm⁻¹; H-

NMR (500 MHz, CDCl₃): δ 1.09 (t, J = 6.5 Hz, 3H,

CH₃–CH₂–N), 3.81 (q, J =6.5 Hz, 2H, CH₃–CH₂–N), 4.00

(s, 3H, OCH₃), 4.10 (s, 2H, H-6′), 4.15 (s, 2H, H-2″), thio-

phene-H, [6.60 (d, J = 3.5 Hz, 1H, H-3′), 6.84 (dd, J = 3.5,

3.2 Hz, 1H, H-4′), 7.12 (d, J = 3.2 Hz, 1H, H-5′), Ar–H

[7.30 (m, 4H, H-3‴–6‴), 7.84 (s, 1H, NH); ¹³C-NMR

(125 MHz, CDCl₃): δ 14.4 (CH₃), 30.6 (C-6′), 36.4 (C-2″),

45.6 (CH₂), 57.6 (OCH₃), thiophene-C [126.4 (C-4″),

129.9 (C-3″), 132.1 (C-5″), 136.4 (C-2″)], Ar–C [114.5

(C-3‴), 121.5 (C-5‴), 126.7 (C-4‴), 127.9 (C-6‴), 136.4

(C-2‴), 152.2 (C-1‴)], 154.8 (C-5), 155.8 (C-3), 166.1

(C-1″); HR-EI-MS (m/z): 388.1019 [M]⁺calculated for

C₁₈H₂₀S₂N₄O₂; 388.1027.

N‑(4‑ethoxyphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑

1,2,4‑triazol‑3‑ylthio)acetamide (9n)

Amorphous powder; yield: 65%; M.P.: 135–137 °C; IR

(KBr) v_max: 3035, 2934, 1682, 1581, 1550 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.03 (t, J=6.5 Hz, 3H, CH₃–CH₂–N),

1.10 (t, J=6.5 Hz, 3H, CH₃–CH₂–O), 3.83 (q, J=6.5 Hz,

2H, CH₃–CH₂–N), 3.98 (q, J=6.5 Hz, 2H, CH₃–CH₂–O),

404 (s, 2H, H-6′), 4.12 (s, 2H, H-2″), thiophene-H, [6.51 (d,

J=2.6 Hz, 1H, H-3′), 6.78 (dd, J=3.2, 2.6 Hz, 1H, H-4′),

7.10 (d, J=3.2 Hz, 1H, H-5′)], Ar–H [7.35 (d, J=8.2 Hz,

2H, H-5‴,6‴), 7.38 (d, J = 8.2 Hz, 2H, H-2‴,3‴)], 7.91

(s, 1H, NH); ¹³C-NMR (125 MHz, CDCl₃, ppm): δ 14.1

(CH₃), 14.2 (CH₃), 31.5 (C-6′), 36.3 (C-2″), 44.5 (CH₂), 65.4

(CH₂), thiophene-C [126.2 (C-4′), 126.3 (C-3′), 126.9 (C-5′),

130.9 (C-2′)], Ar–C [114.4 (C-3‴,5‴), 125.1 (C-2‴,6‴),

136.9 (C-4‴), 150.5 (C-1‴)], 154.4 (C-5), 155.9 (C-3),

166.2 (C-1″); HR-EI-MS (m/z): 402.1170 [M]⁺calculated

for C₁₉H₂₂S₂N₄O₂; 402.1184.

N‑benzyl‑2‑(4‑ethyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑1,2,4

‑triazol‑3‑ylthio)acetamide (9q)

Amorphous powder; yield: 76%; M.P.: 118–120 °C; IR

(KBr) v_max: 3035, 2910, 1670, 1584, 1527 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.05 (t, J=6.5 Hz, 3H, CH₃–CH₂–N),

3.84 (q, J=6.5 Hz, 2H, CH₃–CH₂–N), 3.98 (s, 2H, H-6′),

4.10 (s, 2H, H-2″), thiophene-H, [6.58 (d, J = 3.0 Hz,

1H, H-3′), 6.85 (dd, J = 3.2, 3.0 Hz, 1H, H-4′), 7.15 (d,

J=3.2 Hz, 1H, H-5′)], Ar–H 7.23–7.34 (m, 5H, H-2‴–6‴),

7.99 (s, 1H, NH); ¹³C-NMR (125 MHz, CDCl₃): δ 13.6

(CH₃), 32.2 (C-6′), 35.1 (C-2″), 45.2 (CH₂), 46.3 (CH₂),

thiophene-C [126.3 (C-4′), 128.3 (C-3′), 128.2 (C-5′), 138.0

(C-2′)], Ar–C [114.5 (C-2‴, 6‴), 119.0 (C-4‴), 128.1

(C-3‴, 5‴), 137.3 (C-1‴)], 150.2 (C-5), 154.5 (C-3),

168.3 (C-1″); HR-EI-MS (m/z): 372.1061 [M]⁺calculated

for C₁₈H₂₀S₂N₄O; 372.1078.

N‑(2‑ethyl‑6‑methylphenyl)‑2‑(4‑ethyl‑5‑(thiophen‑2‑yl‑

methyl)‑4H‑1,2,4‑triazol‑3‑ylthio)acetamide (9o)

Amorphous powder; yield: 65%; M.P.: 121–122 °C; IR

(KBr) v_max: 3020, 2922, 1687, 1587, 1545 cm⁻¹; ¹H-NMR

1 3

Journal of the Iranian Chemical Society

N‑(2,5‑dimethylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10c)

Synthesis of compounds (10a–q)

In a 50-ml round-bottom fask, 4-phenyl-5-(thiophen-2-

ylmethyl)-4H-1,2,4-triazole-3-thiol (6; 0.002 g, 0.1 mmol)

was dissolved in 10 ml of DMF followed by the addition

of sodium hydride (0.002 g, 0.1 mmol). The mixture was

stirred for 30 min at 30 °C, and then the electrophiles

(8a–q, each separately) were added slowly to the mixture

with further stirring for 2–3 h. Distilled water was added

to the fask, and the products were recovered by fltra-

tion or solvent extraction according to the nature of the

product.

Amorphous powder; yield: 76%; M.P.: 119–121 °C; IR

(KBr) v_max: 3020, 2920, 1696, 1587, 1530 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 2.10 (s, 3H, CH₃), 2.18 (s, 3H, CH₃),

4.02 (s, 2H, H-6′), 4.12 (s, 2H, H-2″), thiophene-H [6.62 (d,

J=3.5 Hz, 1H, H-3′), 6.92 (dd, J=3.5, 2.8 Hz, 1H, H-4′),

7.18 (d, J=2.8 Hz, 1H, H-5′)], Ar–H 7.23 (br s, 1H, H-6‴),

7.33 (d, J = 8.6 Hz, 1H, H-4‴), 7.38 (d, J = 8.6 Hz, 1H,

H-3‴), Ph–N 7.49–7.52 (m, CH, 5H), 7.93 (s, 1H, NH);

¹³C-NMR (125 MHz, CDCl₃): δ 17.4 (CH₃), 20.3 (CH₃),

30.6 (C-6′), 35.7 (C-2″), thiophene-C [126.4 (C-2′), 130.5

(C-3′), 131.0 (C-5′), 136.5 (C-2′)], 124.2 (C-3‴), 125.0

(C-6‴), 126.1 (C-2‴), 127.1 (C-4‴), 132.1 (C-5‴), 135.6

(C-1‴), Ph–N [126.4 (2×CH), 127.1 (CH), 129.9 (2×CH),

152.4 (C)], 153.7 (C-5), 155.0 (C-3), 167.2 (C-1″); HR-EI-

MS (m/z): 434.1224 [M]⁺calculated for C₂₃H₂₂S₂N₄O;

434.1235.

N‑(2,3‑dimethylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10a)

Amorphous powder; yield: 74%; M.P.: 131–135 °C; IR

(KBr) v_max: 3020, 2914, 1696, 1596, 1542 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.90 (s, 3H, CH₃), 1.97 (s, 3H, CH₃),

3.87 (s, 2H, H-6′), 4.15 (s, 2H, H-2″), thiophene-H [6.54 (d,

J=2.8 Hz, 1H, H-3′), 6.88 (dd, J=3.0, 2.8, Hz, 1H, H-4′),

7.18 (d, J = 3.0 Hz, 1H, H-5′)], Ar–H 7.10–7.20 (m, 3H,

H-4‴–6‴), Ph–N 7.43–7.47 (m, 5H), 7.89 (s, 1H, NH);

¹³C-NMR (125 MHz, CDCl₃): δ 16.7 (CH₃), 22.3 (CH₃),

30.4 (C-6′), 35.0 (C-2′), thiophene-C [126.2 (C-4′), 130.2

(C-3′), 132.3 (C-5′), 136.6 (C-2′)], 120.2 (C-4‴), 120.7

(C-5‴), 124.6 (C-6‴), 126.2 (C-2‴), 126.8 (C-3‴), 132.3

(C-1‴), Ph–N [124.8 (CH), 126.6 (2×CH), 129.6 (2×CH),

149.4 (C)], 151.2 (C-5), 155.0 (C-3), 169.9 (C-1″); HR-EI-

MS (m/z): 434.1224 [M]⁺calculated for C₂₃H₂₂S₂N₄O;

434.1235.

N‑(2,6‑dimethylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10d)

Amorphous powder; yield: 70%; M.P.: 120–121 °C; IR

(KBr) v_max: 3031, 2934, 1697, 1581, 1522 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.94 (s, 3H, CH₃), 2.12 (s, 3H, CH₃),

4.01 (s, 2H, H-6′), 4.16 (s, 2H, H-2″), thiophene-H [6.52 (d,

J=2.8 Hz, 1H, H-3′), 6.88 (dd, J=3.0, 2.8 Hz, 1H, H-4′),

7.18 (d, J = 3.0 Hz, 1H, H-5′), Ar–H 7.10–7.20 (m, 3H,

H-3‴–5‴), Ph–N 7.44–7.47 (m, CH, 5H), 7.88 (s, 1H, NH);

¹³C-NMR (125 MHz, CDCl₃): δ 16.9 (CH₃), 22.6 (CH₃),

31.4 (C-6′), 35.6 (C-2″), thiophene-C [126.4 (C-4′), 130.4

(C-3′), 132.7 (C-5′), 136.3 (C-2′)], 123.6 (C-3‴), 125.4

(C-4‴), 129.8 (C-5‴), 126.7 (C-2‴), 132.6 (C-6‴), 134.9

(C-1‴), Ph–N [124.5 (2×CH), 127.1 (CH), 129.5 (2×CH),

138.6 (C)], 150.5 (C-5), 155.1 (C-3), 170.2 (C-1″); HR-EI-

MS (m/z): 434.1224 [M]⁺calculated for C₂₃H₂₂S₂N₄O;

434.1235.

N‑(2,4‑dimethylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10b)

Amorphous powder; yield: 81%; M.P.: 116–117 °C; IR

(KBr) v_max: 3023, 2929, 1688, 1592, 1534 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.90 (s, 3H, CH₃), 1.97 (s, 3H, CH₃),

4.00 (s, 2H, H-6′), 4.15 (s, 2H, H-2″), thiophene-H [6.58

(d, J = 2.6 Hz, 1H, H-3′), 6.98 (dd, J = 2.8, 2.6 Hz, 1H,

H-4′), 7.18 (d, J=2.8 Hz, 1H, H-5′)], Ar–H [7.23 (br s, 1H,

H-3‴), 7.33 (d, J=8.2 Hz, 1H, H-6‴), 7.38 (d, J=8.2 Hz,

1H, H-5‴)], Ph–N 7.43–7.47 (m, 5H), 7.99 (s, 1H, NH);

¹³C-NMR (125 MHz, CDCl₃): δ 16.7 (CH₃), 22.4 (CH₃),

31.2 (C-6′), 36.5 (C-2″), thiophene-C [127.2 (C-4′), 130.1

(C-3′), 132.3 (C-5′), 132.5 (C-2′)], 119.6 (C-3‴), 124.7

(C-5‴), 126.1 (C-2‴), 126.4 (C-4‴), 128.4 (C-6‴), 136.7

(C-1‴), Ph–N [124.0 (2×CH), 128.4 (CH), 129.6 (2×CH),

138.6 (C)], 151.6 (C-5), 155.0 (C-3), 166.5 (C-1″); HR-EI-

MS (m/z): 434.1224 [M]⁺calculated for C₂₃H₂₂S₂N₄O;

434.1235.

N‑(3,4‑dimethylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10e)

Amorphous powder; yield: 71%; M.P.: 118–120 °C; IR

(KBr) v_max: 3032, 2931, 1698, 1580, 1522 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.91 (s, 3H, CH₃), 2.15 (s, 3H, CH₃),

4.05 (s, 2H, H-6′), 4.15 (s, 2H, H-2″), thiophene-H [6.59 (d,

J=3.2 Hz, 1H, H-3′), 6.82 (dd, J=3.2, 2.8 Hz, 1H, H-4′), 7.18

(d, J=2.8 Hz, 1H, H-5′)], Ar–H 7.25 (br s, 1H, H-2‴), 7.37 (d,

J=8.6 Hz, 1H, H-6‴), 7.42 (d, J=8.6 Hz, 1H, H-5‴), Ph–N

7.52–7.55 (m, 5H), 7.90 (s, 1H, NH); ¹³C-NMR (125 MHz,

CDCl₃): δ 16.7 (CH₃), 22.6 (CH₃), 31.8 (C-6′), 35.6 (C-2″),

thiophene-C [126.4 (C-2′), 131.4 (C-3′), 132.7 (C-5′), 136.3

(C-2′)], 123.6 (C-2‴), 124.9 (C-6‴), 125.4 (C-6‴), 126.7

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Journal of the Iranian Chemical Society

(C-2′)], 124.3 (C-4‴), 124.3 (C-3‴), 124.9 (C-5‴), 125.8

(C-6‴), 130.4 (C-2‴), 132.3 (C-1‴), Ph–N [126.6 (2×CH),

129.8 (CH), 130.3 (2×CH), 136.5 (C)], 152.1 (C-5), 155.0

(C-3), 167.2 (C-1″); HR-EI-MS (m/z): 420.1060 [M]⁺cal-

culated for C₂₂H₂₀S₂N₄O; 420.1078.

(C-4‴), 133.6 (C-3‴), 134.9 (C-1‴), Ph–N [125.5 (2×CH),

127.1 (CH), 129.5 (2×CH), 138.6 (C)], 150.5 (C-5), 155.1

(C-3), 170.2 (C-1″); HR-EI-MS (m/z): 434.1224 [M]⁺calcu-

lated for C₂₃H₂₂S₂N₄O; 434.1235.

N‑(3,5‑dimethylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10f)

N‑(3‑Methylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10i)

Amorphous powder; yield: 78%; M.P.: 117–119 °C; IR

(KBr) v_max: 3025, 2905, 1702, 1575, 1510 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.94 (s, 3H, CH₃), 2.12 (s, 3H, CH₃,),

4.02 (s, 2H, H-6′), 4.16 (s, 2H, H-2″), thiophene-H [6.55 (d,

J=2.6 Hz, 1H, H-3′), 6.80 (dd, J=3.2, 2.6 Hz, 1H, H-4′),

7.15 (d, J=3.2 Hz, 1H, H-5′)], Ar–H 7.23 (br s, 1H, H-2‴),

7.33 (s, 1H, H-4‴), 7.35 (s, 1H, H-6‴), Ph–N 7.11–7.15 (m,

CH, 5H), 7.86 (s, 1H, NH); ¹³C-NMR (125 MHz, CDCl₃): δ

16.9 (CH₃), 22.6 (CH₃), 31.4 (C-6′), 35.6 (C-2″), thiophene-

C [126.4 (C-4′), 130.4 (C-3′), 132.7 (C-5′), 136.3 (C-2′)],

123.6 (C-2‴), 124.5 (C-4‴), 124.9 (C-6‴), 126.7 (C-5‴),

132.6 (C-3‴), 134.9 (C-1‴), Ph–N [125.4 (2×CH), 127.9

(CH), 129.8 (2×CH), 138.6 (C)], 150.5 (C-5), 155.1 (C-3),

170.2 (C-1″); HR-EI-MS (m/z): 434.1224 [M]⁺calculated for

C₂₃H₂₂S₂N₄O; 434.1235.

Amorphous powder; yield: 81%; M.P.: 143–145 °C;

1

IR (KBr) v_max: 3019, 2921, 1696, 1590, 1532 cm⁻¹; H-

NMR (500 MHz, CDCl₃): δ 2.33 (s, 3H, CH₃), 4.01 (s, 2H,

H-6′), 4.17 (s, 2H, H-2″), thiophene-H [6.52 (d, J=2.8 Hz,

1H, H-3′), 6.89 (dd, J = 3.2, 2.8 Hz, 1H, H-4′), 7.18 (d,

J = 3.2 Hz, 1H, H-5′)], Ph–N 7.20–7.29 (m, 5H), 7.45 (s,

1H, H-2‴), 7.49 (d, J=8.0 Hz, 1H, H-4‴), 7.52 (dd, J=8.5,

8.0 Hz, 1H, H-5‴), 7.54 (d, J = 8.5 Hz, 1H, H-6‴), 7.69

(s, NH, 1H); ¹³C-NMR (125 MHz, CDCl₃): δ 20.1 (CH₃),

31.2 (C-6′), 35.5 (C-2″), thiophene-C [126.4 (C-4′), 130.1

(C-3′), 132.5 (C-5′), 136.7 (C-2′)], 120.2 (C-4‴), 124.7

(C-5‴), 126.7 (C-3‴), 127.2 (C-2‴), 128.3 (C-6‴), 138.0

(C-1‴), Ph–N [124.8 (2×CH), 126.1 (CH), 129.3 (2×CH),

138.4 (C)], 151.6 (C-5), 155.0 (C-3), 166.5 (C-1″); HR-EI-

MS (m/z): 420.1060 [M]⁺calculated for C₂₂H₂₀S₂N₄O;

420.1078.

N‑phenyl‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑1,2,4‑

triazol‑3‑ylthio)acetamide (10g)

N‑(4‑methylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10j)

Amorphous powder; yield: 81%; M.P.: 151–153 °C; IR

(KBr) v_max: 3021, 2910, 1701, 1577, 1514 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 4.01 (s, 2H, H-6′), 4.15 (s, 2H, H-2″),

thiophene-H [6.55 (d, J=2.6 Hz, 1H, H-3′), 6.80 (dd, J=3.2,

2.6 Hz, 1H, H-4′), 7.15 (d, J=3.2 Hz, 1H, H-5′)], Ar–H

7.23–7.25 (m, 5H, H-2‴–6‴), Ph–N 7.45–7.47 (m, 5H),

9.72 (s, NH, 1H); ¹³C-NMR (125 MHz, CDCl₃): δ 26.0

(C-6′), 35.7 (C-2″), thiophene-C [126.4 (C-4′), 131.1 (C-3′),

133.6 (C-5′), 136.9 (C-2′)], 123.6 (C-4‴), 125.0 (C-2‴,6‴),

130.9 (C-3‴,5‴), 134.4 (C-1‴), Ph–N [126.9 (2×CH), 127.9

(CH), 130.0 (2×CH), 136.9 (C)], 153.0 (C-5), 154.8 (C-3),

166.9 (C-1″); HR-EI-MS (m/z): 406.0909 [M]⁺calculated for

C₂₁H₁₈S₂N₄O; 406.0922.

Amorphous powder; yield: 81%; M.P.: 124–126 °C; IR

(KBr) v_max: 3024, 2916, 1685, 1590, 1530 cm⁻¹; ¹H NMR

(500 MHz, CDCl₃): δ 2.07 (s, 3H, CH₃), 3.83 (s, 2H,

H-6′), 4.17 (s, 2H, H-2″), thiophene-H [6.58 (d, J=2.8 Hz,

1H, H-3′), 6.82 (dd, J = 3.0, 2.8 Hz, 1H, H-4′), 7.11 (d,

J = 3.0 Hz, 1H, H-5′), Ph–N 7.22–7.29 (m, 5H), 7.53 (d,

J=8.2 Hz, 2H, H-2‴, 6‴), 7.57 (d, 2H, J=8.2 Hz, H-3‴,

5‴), 7.99 (s, 1H, NH); ¹³C-NMR (125 MHz, CDCl₃): δ 20.2

(CH₃), 30.8 (C-6′), 35.0 (C-2″), thiophene-C [126.5 (C-4′),

130.2 (C-3′), 129.6 (C-5′), 132.4 (C-2′)], 124.7 (C-3‴, 5‴),

126.5 (C-2‴, -6‴), 130.2 (C-4‴), 136.7 (C-1‴), Ph–N

[125.9 (2 × CH), 128.4 (CH), 129.3 (2 × CH), 138.9 (C)],

151.6 (C-5), 154.9 (C-3), 168.3 (C-1′); HR-EI-MS (m/z):

420.1060 [M]⁺calculated for C₂₂H₂₀S₂N₄O; 420.1078.

N‑(2‑methylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10h)

Methyl‑(2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑1,2,4‑

triazol‑3‑ylthio)acetamido) benzoate (10k)

Amorphous powder; yield: 81%; M.P.: 157–160 °C;

1

IR (KBr) v_max: 3025, 2929, 1695, 1597, 1535 cm⁻¹; H-

NMR (500 MHz, CDCl₃): δ 1.93 (s, 3H, CH₃), 4.02 (s, 2H,

H-6′), 4.14 (s, 2H, H-2″), thiophene-H [6.58 (d, J=2.8 Hz,

1H, H-3′), 6.80 (dd, J = 3.0, 2.8 Hz, 1H, H-4′), 7.10 (d,

J = 3.0 Hz, 1H, H-5′)], 7.23–7.25 (m, 4H, H-3‴–6‴),

Ph–N 7.42–7.48 (m, CH, 5H), 7.69 (s, NH, 1H); ¹³C-NMR

(125 MHz, CDCl₃): δ 19.9 (CH₃), 31.0 (C-6′), 34.7 (C-2″),

thiophene-C [126.3 (C-4′), 130.4 (C-3′), 131.5 (C-5′), 135.2

Amorphous powder; yield: 81%; M.P.: 170–173 °C; IR

(KBr) v_max: 3025, 2928, 1696, 1597, 1531 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 3.57 (s, 3H, OCH₃), 3.83 (s, 2H,

H-6′), 4.14 (s, 2H, H-2″), thiophene-H [6.52 (d, J=2.8 Hz,

1H, H-3′), 6.83 (dd, J = 3.2, 2.8 Hz, 1H, H-4′), 7.11 (d,

J=3.2 Hz, 1H, H-5′), Ar–H 7.23 (m, 4H, H-3‴-6‴), Ph–N

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Journal of the Iranian Chemical Society

7.43–7.49 (m, 5H), 7.88 (s, 1H, NH); ¹³C-NMR (125 MHz,

CDCl₃): δ 31.8 (C-6′), 35.0 (C-2″), 55.2 (OCH₃), thiophene-

C [126.5 (C-4′), 130.2 (C-3′), 129.6 (C-5′), 132.4 (C-2′)],

124.7 (C-4‴), 124.7 (C-3‴), 125.9 (CH), 126.5 (C-5‴),

127.1 (C-6‴), 130.2 (C-2‴), 136.7 (C-1‴), Ph–N [128.1

(2 × CH), 129.3 (CH), 129.6 (2 × CH), 138.9 (C)], 151.6

(C-5), 154.9 (C-3), 168.3 (C-1′); HR-EI-MS (m/z): 464.0964

[M]⁺calculated for C₂₃H₂₀S₂N₄O₃; 464.0976.

(500 MHz, CDCl₃): δ 1.38 (t, J=6.5 Hz, 3H, CH₃–CH₂–O),

3.92 (q, J=6.5 Hz, 2H, CH₃–CH₂–O), 3.97 (s, 2H, H-6′),

4.11 (s, 2H, H-2″), thiophene-H [6.58 (d, J = 2.8 Hz,

1H, H-3′), 6.82 (dd, J = 3.0, 2.8 Hz, 1H, H-4′), 7.11 (d,

J = 3.0 Hz, 1H, H-5′), Ph–N 7.22–7.29 (m, 5H), Ar–H

7.53 (d, J = 8.0 Hz, 2H, H-2‴, 6‴), 7.57 (d, J = 8.0 Hz,

2H, H-3‴, 5‴), 7.99 (s, 1H, NH); ¹³C-NMR (125 MHz,

CDCl₃): δ 14.4 (CH₃), 30.7 (C-6′), 34.3 (C-2″), 63.6 (CH₂),

thiophene-C [126.4 (C-4′), 130.9 (C-3′), 132.0 (C-5′),

136.4 (C-2′)], 114.6 (C-3‴, 5‴), 126.8 (C-2‴, 6‴), 128.5

(C-4‴), 130.5 (C-1‴), Ph–N [125.0 (2×CH), 129.4 (CH),

130.4 (2×CH), 152.3 (C)], 154.8 (C-5), 155.7 (C-3), 166.1

(C-1″); HR-EI-MS (m/z): 450.1171 [M]⁺calculated for

C₂₃H₂₂S₂N₄O₂; 450.1184.

N‑(2‑ethylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10l)

Amorphous powder; yield: 71%; M.P.: 129–130 °C; IR

(KBr) v_max: 3033, 2935, 1697, 1582, 1522 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.15 (t, J= 6.5 Hz, 3H, CH₃–CH₂),

2.63 (q, J=6.5 Hz, 2H, CH₃–CH₂), 4.01 (s, 2H, H-6′), 4.12

(s, 2H, H-2″), thiophene-H [6.50 (d, J=3.0 Hz, 1H, H-3′),

6.87 (dd, J=3.2, 3.0 Hz, 1H, H-4′), 7.10 (d, J=3.2 Hz, 1H,

H-5′)], Ar–H 7.26 (m, 4H, H-3‴–6‴), Ph–N 7.52–7.59 (m,

5H), 7.79 (s, 1H, NH); ¹³C-NMR (125 MHz, CDCl₃): δ 13.5

(CH₃), 24.0 (CH₂), 31.2 (C-6′), 35.4 (C-2″), thiophene-C

[126.3 (C-4′), 130.2 (C-3′), 132.4 (C-5′), 134.5 (C-2′)],

124.7 (C-4‴), 125.6 (C-3‴), 126.2 (C-5‴), 126.5 (C-6‴),

130.5 (C-2‴), 136.7 (C-1‴), Ph–N [125.9 (2×CH), 127.1

(CH), 129.7 (2×CH), 138.4 (C)], 151.1 (C-5), 151.9 (C-3),

167.7 (C-1″); HR-EI-MS (m/z): 434.1224 [M]⁺calculated

for C₂₃H₂₂S₂N₄O; 434.1235.

N‑(2‑ethyl‑6‑methylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑yl‑

methyl)‑4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10o)

Amorphous powder; yield: 68%; M.P.: 121–123 °C; IR

(KBr) v_max: 3020, 2925, 1694, 1591, 1535 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.16 (t, J= 6.4 Hz, 3H, CH₃–CH₂),

2.17 (s, 3H, CH₃), 2.63 (q, J=6.4 Hz, 2H, CH₃–CH₂), 4.01

(s, 2H, H-6′), 4.12 (s, 2H, H-2″), thiophene-H [6.52 (d,

J=2.8 Hz, 1H, H-3′), 6.88 (dd, J=3.0, 2.8 Hz, 1H, H-4′),

7.18 (d, J = 3.0 Hz, 1H, H-5′)], Ar–H 7.10–7.20 (m, 3H,

H-3‴–5‴), Ph–N 7.44–7.49 (m, 5H), 7.88 (s, 1H, NH);

¹³C-NMR (125 MHz, CDCl₃): δ 13.5 (CH₃), 17.6 (CH₃),

24.0 (CH₂), 31.2 (C-6′), 36.3 (C-2″), thiophene-C [126.4

(C-4′), 129.7 (C-3′), 130.2 (C-5′), 136.7 (C-2′)], 124.7

(C-3‴), 125.6 (C-4‴), 125.9 (C-5‴), 130.7 (C-6‴), 131.5

(C-2‴), 134.5 (C-1‴), Ph–N [126.2 (2×CH), 127.1 (CH),

130.5 (2×CH), 138.4 (C)], 151.9 (C-5), 155.1 (C-3), 167.7

(C-1″); HR-EI-MS (m/z): 436.1018 [M]⁺calculated for

C₂₂H₂₀S₂N₄O₂; 436.1027.

N‑(4‑ethylphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10m)

Amorphous powder; yield: 68%; M.P.: 121–125 °C; IR

(KBr) v_max: 3041, 2915, 1701, 1601, 1530 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 1.15 (t, J= 6.5 Hz, 3H, CH₃–CH₂),

2.43 (q, J=6.5 Hz, 2H, CH₃–CH₂), 4.01 (s, 2H, H-6′), 4.10

(s, 2H, H-2″), thiophene-H [6.53 (d, J=2.6 Hz, 1H, H-3′),

6.82 (dd, J=3.2, 2.6 Hz, 1H, H-4′), 7.17 (d, J=3.2 Hz, 1H,

H-5′)], Ph–N 7.23–7.29 (m, 5H), Ar–H 7.57 (d, J=8.2 Hz,

2H, H-2‴,6‴), 7.59 (d, J=8.2 Hz, 2H, H-3‴, 5‴), 7.90 (s,

1H, NH); ¹³C-NMR (125 MHz, CDCl₃): δ 13.5 (CH₃), 24.0

(CH₂), 31.2 (C-6′), 35.4 (C-2″), thiophene-C [126.3 (C-4′),

130.2 (C-3′), 132.4 (C-5′), 134.5 (C-2′)], 124.7 (C-3‴, 5‴),

126.2 (C-2‴, 6‴), 128.5 (C-4‴), 136.7 (C-1‴), Ph–N

[125.6 (2 × CH), 127.1 (CH), 129.7 (2 × CH), 138.4 (C)],

151.1 (C-5), 151.9 (C-3), 167.7 (C-1″); HR-EI-MS (m/z):

434.1224 [M]⁺calculated for C₂₃H₂₂S₂N₄O; 434.1235.

N‑(2‑methoxyphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10p)

Amorphous powder; yield: 63%; M.P.: 140–142 °C; IR

(KBr) v_max: 3016, 2929, 1691, 1590, 1533 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 3.65 (s, 3H, OCH₃), 4.00 (s, 2H,

H-6′), 4.12 (s, 2H, H-2″), thiophene-H [6.52 (d, J=2.8 Hz,

1H, H-3′), 6.88 (dd, J = 3.0, 2.8 Hz, 1H, H-4′), 7.18 (d,

J=3.0 Hz, 1H, H-5′)], Ar–H 7.12–7.18 (m, 4H, H-3‴–6‴),

Ph–N 7.44–7.47 (m, 5H), 7.49 (s, 1H, NH); ¹³C-NMR

(125 MHz, CDCl₃): δ 31.6 (C-6′), 36.4 (C-2″), 58.6 (OCH₃),

thiophene-C [126.6 (C-4′), 129.8 (C-3′), 132.3 (C-5′), 136.6

(C-2′)], 116.5 (C-3‴), 122.5 (C-4‴), 126.7 (C-5‴), 127.8

(C-6‴), 128.9 (C-2‴), 136.4 (C-1‴), Ph–N [125.2 (2×CH),

127.2 (CH), 129.1 (2×CH), 152.4 (C)], 154.7 (C-5), 155.8

(C-3), 166.6 (C-1″); HR-EI-MS (m/z): 436.1018 [M]⁺cal-

culated for C₂₂H₂₀S₂N₄O₂; 436.1027.

N‑(4‑ethoxyphenyl)‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑

4H‑1,2,4‑triazol‑3‑ylthio)acetamide (10n)

Amorphous powder; yield: 68%; M.P.: 129–131 °C; IR

(KBr) v_max: 3019, 2923, 1696, 1597, 1535 cm⁻¹; ¹H-NMR

1 3

Journal of the Iranian Chemical Society

N‑benzyl‑2‑(4‑phenyl‑5‑(thiophen‑2‑ylmethyl)‑4H‑1,2,4‑

triazol‑3‑ylthio)acetamide (10q)

10 min. It was followed by the addition of 100 µl freshly

prepared alcohol–phenol reagent. The contents were incu-

bated for further 15 min, and absorbance was recorded at

630 nm. The percentage inhibition and IC₅₀values of active

compounds were determined as mentioned above for AChE,

BChE.

Amorphous powder; yield: 61%; M.P.: 133–134 °C; IR

(KBr) v_max: 3035, 2910, 1690, 1584, 1527 cm⁻¹; ¹H-NMR

(500 MHz, CDCl₃): δ 4.02 (s, 2H, H-6′), 4.12 (s, 2H,

H-2″), 4.37 (s, 2H, CH₂), thiophene-H [6.54 (d, J=3.0 Hz,

1H, H-3′), 6.80 (dd, J = 3.2, 3.0 Hz, 1H, H-4′), 7.10 (d,

J=3.2 Hz, 1H, H-5′), Ar–H 7.34–7.39 (m, 5H, H-2‴–6‴),

Ph–N 7.45–7.49 (m, 5H), 7.89 9 s, 1H, NH); ¹³C-NMR

(125 MHz, CDCl₃): δ 28.2 (CH₂), 31.2 (C-6′), 33.1 (C-2″),

thiophene-C [124.3 (C-4′), 125.3 (C-3′), 127.2 (C-5′), 136.0

(C-2′)], 116.5 (C-2‴, 6‴), 126.9 (C-4‴), 128.2 (C-3‴, 5‴),

137.3 (C-1‴), Ph–N [125.0 (2 × CH), 127.8 (CH), 129.0

(2×CH), 129.0 (CH), 139.0 (C)], 150.2 (C-5), 154.5 (C-3),

168.3 (C-1″); HR-EI-MS (m/z): 420.1068 [M]⁺calculated

for C₂₂H₂₀S₂N₄O; 420.1078.

α‑Glucosidase assay

The yeast α-glucosidase inhibition assay was performed

according to the reported method [22] with some modi-

fcations. Total volume of the reaction mixture of 100 µl

contained 70 µl 50 mM phosphate bufer, pH 6.8, 10 µl of

0.5 mM test compound and 10 µl (0.057 units) enzyme per

well. The contents were mixed, pre-incubated for 10 min at

37 °C and pre-read at 405 nm. The reaction was initiated by

the addition of 10 µl of 0.5 mM substrate p-nitrophenylglu-

copyranoside. After 30 min of incubation, absorbance was

measured. All experiments were carried out in triplicate.

Acarbose was used as a positive control. The percentage

inhibition and IC₅₀values of active compounds were deter-

mined as mentioned above for AChE, BChE.

Enzyme inhibition assays

Cholinesterase assay

Acetylcholinesterase (AChE) and butyrylcholinesterase

(BChE) inhibition assays were performed using Ellman

method [20] with slight modifcations. Reaction mixture of

100 µl containing 60 µl 50 mM Na₂HPO₄bufer, pH 7.7 and

10 µl (0.5 mM) test compound and 10 µl enzyme (0.005 unit

AChE and 0.0005 units BChE) was added per well. The con-

tent was mixed and preread at 405 nm. After preincubation

at 37 °C for 10 min, the reaction was initiated by the addition

of 10 µl of 0.5 mM substrate (acetylthiocholine iodide or

butyrylcholine bromide) per well, followed by the addition

of 10 µl DTNB (0.5 mM per well). Incubation was continued

for further 30 min. Absorbance was measured at 405 nm on

Synergy HTX (BioTek, USA) 96-well plate reader. Eserine

was used as a positive control. The percent inhibition was

calculated as below. IC₅₀values of active compounds were

determined by assaying the enzyme with suitable dilutions

of the active compounds and computing the data with EZ-Fit

software (Perrella Int. USA).

Results and discussion

Chemistry

Ethyl 2-thiopheneacetate (1) was initially synthesized by

refuxing thiophen-2-acetic acid with ethanol for 3–4 h. The

oily product was then extracted with CHCl₃from the reac-

tion mixture at basic pH (8–9 with Na₂CO₃). The next step

was the synthesis of 2-(thiophen-2-yl)acetohydrazide (2) by

stirring 1 with hydrazine hydrate for 4–5 h at room tem-

perature. The resulting light yellow crystalline product was

fltered and washed with n-hexane. The ethanolic solution of

hydrazide (2) was refuxed (3–4 h) with ethyl isothiocyanate

and phenylisothiocyanate separately to get 4-ethyl-1-{2-

(thiophen-2-yl)acetyl}thiosemicarbazide (3) and 4-phenyl-

1-{2-(thiophen-2-yl)acetyl}thiosemicarbazide (4), respec-

tively, on cooling. Intramolecular cyclization was achieved

by refuxing 3 and 4 under alkaline (10% NaOH) conditions

yielded 4-ethyl-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazole-

3-thiol (5) and 4-phenyl-5-(thiophen-2-ylmethyl)-4H-1,2,4-

triazole-3-thiol (6), respectively, which were precipitated by

pouring the reaction mixtures into ice cold water and acidi-

fed to pH 4–6 with concentrated HCl [23, 24]. The N-aryl/

aralkyl-2-bromoacetamides (8a–q), the ot her precursors of

target compounds 9a–q and 10a–q, were synthesized by stir-

ring aryl/alkyl amines (7a–q) with 2-bromoacetyl bromide

in a basic medium (pH 9–10). Final step was the coupling

of compounds 5 and 6 separately with the precursors 8a–q

to get target compounds 9a–q and 10a–q. This task was

Control − Test

Inhibition (%) =

× 100

Control

Urease assay

Phenyl hypochlorite method was used to determine the

antiurease activities of the compounds (9a–q and 10a–q)

with slight modifcations [21]. The reaction mixture con-

tained 60 µl 200 mM phosphate bufer pH 8.0, 20 µl jack

bean urease enzyme and 5 µl test compound. Contents were

pre-incubated at 25 °C for 10 min, and then 15 µl of 20 mM

urea was added and incubation continued at 25 °C for further

1 3

Journal of the Iranian Chemical Society

accomplished by stirring the solutions of precursors 5 and

6 in N,N-dimethyl formamide (DMF, 10 mL) and adding

sodium hydride (0.002 g, 0.1 mmol). After 30 min stir-

ring, the reaction mixture was followed by the slow addi-

tion of the electrophiles, 8a–q, respectively, and was further

stirred at room temperature for 2–3 h. The reaction mixtures

were then supplemented with distilled water, and the end

products 9a–q and 10a–q were separated by fltration or

solvent extraction according to the nature of the products.

The structures of all the synthesized compounds (1–6) and

N-substituted derivatives (9a–q; 10a–q) were confrmed by

the analysis of spectral data as presented in the experimental

section.

The starting compound ethyl 2-thiopheneacetate (1) was

synthesized according to the literature [25], and its structure

was confrmed by ¹H- and ¹³C-NMR spectra which exhib-

ited additional peaks for –COOCH₂CH₃compared to the

NMR data of the starting material. The treatment of 1 with

hydrazine hydrate (NH₂–NH₂·H₂O) resulted in the forma-

tion of hydrazide derivative 2 in good yield (81%), which

was identifed as a key intermediate for the synthesis of tar-

get compounds. The ¹H-NMR spectrum of 2 did not show

any signals corresponding to ethoxy (–OCH₂CH₃) group;

instead, new singlets appeared at δ 4.02 (1H, –NH) and 8.90

1

(2H, –NH₂) attributed to –NHNH₂moiety. The H NMR

spectrum of compound 3 displayed signals for N-ethyl group

at δ 1.15 (t, 3H, N–CH₂–CH₃, J=6.4 Hz) and 3.53 (q, 2H,

N–CH₂–CH₃, J=6.4 Hz), while the spectrum of 4 substan-

tiated the presence of phenyl moiety due to a multiplet at δ

7.10–7.15 (m, 5H). The C=S carbon nucleus resonated at

δ 180.6 in the ¹³C-NMR spectrum of both the compounds

3 and 4. The resonance of thiol proton at δ 13.6 in the ¹H-

NMR spectra of the compounds 5 and 6, and the absence

of –NH and carbonyl carbon signals in their NMR spectra

The goal of the present study was: primarily, to synthe-

size new thiophene-1,2,4-triazoles (9a–q; 10a–q) containing

a variety of substituents and secondarily, to investigate the

synthesized compounds for their enzyme inhibitory poten-

tial against some enzymes of therapeutical importance like

AChE, BChE, urease, and α-glucosidase. Syntheses of

the intermediates and target compounds were carried out

according to the protocol as shown in Fig. 1.

EtOH, 3-4 Hrs

Reflux

O

3

NH -NH , 4-5 Hrs

2

O

3

O

2

5

1

6

7

6

7

Stirring, RT

S

OH

S

OEt

S

NH-NH₂

2

a

1

Et-N=C=S,

Ph-N=C=S

EtOH, Reflux, 3-4 Hrs

10% NaOH,

Reflux, 04 Hrs

1

3

O

3'

N

H

N

H

N

R₁

NH₂

5

1

5'

6

7

1'

6'

5

3

S

R

S

N

R

SH

7a-q

H

S

3 R = C₂H₅

4 R = C₆H₅

2-Bromoacetyl

bromide, Basic

medium, pH 9-10

5 R = C₂H₅

6 R = C₆H₅

DMF, NaOH 30 min at RT

H

N

R₁

Br

O

8a-q

Stirring 2-3 Hrs

1

3'

N

H

2''

5'

1'

^1''N R₁

6'

5

3

S

N

R

S

O

9a-q R = C₂H₅

10a-q R = C₆H₅

Fig. 1 Protocol for the synthesis of triazole amides from thiophene-2-acetic acid

1 3

Journal of the Iranian Chemical Society

substantiated the conversion of 3 and 4 into 1,2,4-triazol-

3-thiols in compounds 5 and 6. The syntheses of difer-

ent aryl/aralkyl substituted bromoacetamides (8a–q) were

carried out by the reaction of diferent aryl/aralkyl amines

(7a–q; Table 1) with bromoacetyl bromide. Owing to aro-

1535 cm⁻¹, attested for C-H (aromatic), C=O, C=N, and

C=C functionalities. In the ¹H NMR spectrum, signals at

δ 1.15 (t, 3H, CH₃–CH₂–N, J = 6.5 Hz) and 3.83 (q, 2H,

CH₃–CH₂–N, J=6.5 Hz) were assigned to N-ethyl group in

9a. Two singlet methylene protons resonating at δ 4.01 and

4.17 were attributed to thiophene-CH₂, and -S-CH₂, respec-

tively. In addition to the aromatic proton resonances at δ

7.10–7.20 (m, 3H, CH), the spectrum displayed two singlet

methyl signals at δ 2.13 (s, 3H, CH₃) and 2.28 (s, 3H, CH₃)

that confrmed the presence of 2,3-dimethyl aniline residue

in 9a. The resonance of nineteen carbons in the ¹³C-NMR

spectrum of 9a was in agreement with the molecular for-

mula. The multiplicity of these signals was established as

three methyl, three methylene, six methine and seven qua-

ternary carbons through DEPT experiment. Similarly, other

compounds (9b–q; 10a–q) were also characterized by IR,

1

matic ring in aryl/aralkyl amines, the H-, and ¹³C-NMR

spectra displayed additional signals for these groups. Syn-

thones 5 and 6 were coupled with aryl/aralkyl amines (8a–q)

to get target molecules 9a–q and 10a–q which were char-

acterized and confrmed by spectroscopic analyses, and the

data are presented in the experimental section. Compound

9a was synthesized as a dull white amorphous solid (yield

of 71%), which melted at 135–137 °C. The molecular for-

mula C₁₉H₂₂S₂N₄O was determined through HR-EI-MS

due to molecular ion [M]⁺peak at m/z 386.1226. The IR

spectrum displayed absorption bands at 3020, 1696, 1597,

Table 1 Aryl/aralkyl substituted amines

Compd

R

Compd

R

Compd

R

a

g

m

5'''

1'''

5'''

1'''

CH₃

Et

3'''

CH₃

b

c

h

i

n

o

5'''

1'''

5'''

1'''

H₃C

CH₃

3'''

EtO

CH₃

3'''

Et

5'''

1'''

5'''

1'''

5'''

1'''

CH₃

3'''

CH₃

3'''

CH₃

d

e

j

p

q

5'''

1'''

5'''

1'''

H₃C

OCH₃

3'''

CH₃

3'''

7'''

k

1'''

5'''

1'''

3'''

H₃C

3'''

COOCH₃

3'''

CH₃

–

f

l

–

5'''

1'''

5'''

1'''

3'''

Et

3'''

CH₃

1 3

Journal of the Iranian Chemical Society

¹H-NMR, ¹³C-NMR, and mass spectral data as described in

experimental section.

0.19, 23.62 0.22, 27.43 0.16, 24.52 0.21, 28.35

0.18 and 27.24 0.19 µM, respectively, with respect to

the reference standard eserine (Table 3). These compounds

exhibited activity due to the presence of dimethylphenyl

group attached to the nitrogen atom at ortho and para posi-

tion of acetamide moiety in these molecules. The ethyl

group attached to nitrogen atom at position 4 of 1,2,4-tria-

zole nucleus also enhanced the ortho and para alkyl group

attached to the nitrogen of acetamide moiety. Overall activ-

ity of the evaluated compounds was found signifcant.

Biology (biological studies)

Acetylcholinesterase activity

Compounds 9a–q; 10a–q were evaluated for their inhibitory

activities against AChE (Table 2). Results showed that the

compounds 9i, 9n, 10a, 10d, 10f, 10j and 10m were found

to be moderate inhibitors of AChE activity with IC₅₀values

of 73.54 0.16, 69.26 0.12, 165.26 0.17, 45.72 0.11,

32.26 0.12, 117.43 0.12, 72.53 0.13 µM, respectively.

The positive control, serine exhibited IC₅₀0.19 0.05 µM

in this assay. The activity of these compounds could be

attributed to the presence of dimethylphenyl group attached

to the nitrogen atom at meta position of acetamide moiety.

The phenyl group attached to nitrogen atom at position 4

of 1,2,4-triazole nucleus also enhanced the ortho and para

alkyl group attached to the nitrogen of acetamide moiety.

Overall activity of the synthesized compounds was moder-

ately good.

Urease activity

Compounds 9a–q; 10a–q were also evaluated for their antiu-

rease potential (Table 4). Compounds 9e, 10i and 10n were

found to be potent inhibitors with IC₅₀values 8.79 0.057,

7.27 0.05, 7.35 0.04 µM, respectively; thiourea as stand-

ard drug displayed IC₅₀value of 21.25 0.15. The SAR

study revealed that the activity of these compounds could

be attributed to ortho, meta methyl substitution at the phenyl

group attached to the nitrogen atom of acetamide moiety.

α‑Glucosidase activity

Butyrylcholinesterase activity

The compounds 9a–q; 10a–q were also evaluated for their

inhibitory studies against α-glucosidase, and the results are

shown in Table 5. Compounds 9o and 10i were found to

be good inhibitors with IC₅₀values of 62.94 0.19, and

The BChE activity of compounds 9a–q; 10a–q showed that

the compounds 9b, 9d, 9h, 9l, 10a, 10d, 10e, 10h, 10j and

10o were signifcantly promising inhibitors with IC₅₀values

21.72 0.18, 12.52 0.19, 19.75 0.22, 12.52 0.19, 27.24

Table 2 AChE inhibitory

Comp

AChE

Comp

AChE

profles of compounds 9a–q;

10a–q

Inhibition (%) at

0.5 mM

IC₅₀(µM)

Inhibition (%) at

0.5 mM

IC₅₀(µM)

9a

9b

9c

9d

9e

9f

9g

9h

9i

9j

9k

9l

9m

9n

9o

9p

9q

Eserine

45.32 0.15

21.49 0.12

42.15 0.16

45.56 0.17

76.92 0.19

32.59 0.12

37.54 0.12

37.36 0.14

81.53 0.19

74.12 0.17

72.35 0.23

71.24 0.24

74.53 0.19

82.24 0.17

27.62 0.14

74.58 0.21

48.75 0.15

91.46 1.25

–

10a

10b

10c

10d

10e

10f

10g

10h

10i

10j

10k

10l

10m

10n

10o

10p

10q

Eserine

85.29 0.21

31.24 0.13

18.46 0.12

89.52 0.18

24.51 0.14

87.64 0.19

25.75 0.15

32.38 0.17

43.51 0.14

86.64 0.19

53.21 0.16

43.58 0.12

87.31 0.18

32.48 0.12

38.36 0.14

44.15 0.14

25.3 0.15

165.26 0.17

–

45.72 0.11

–

32.26 0.12

–

113.62 0.15

–

73.54 0.16

157.32 0.13

216.72 0.18

283.53 0.19

145.84 0.15

69.26 0.12

–

165.42 0.18

–

0.19 0.05

117.43 0.12

452.42 0.15

–

72.53 0.13

–

91.46 1.25

0.19 0.05

1 3

Journal of the Iranian Chemical Society

69.46 0.15 µM, respectively. Other compounds showed

moderate to poor activities against the said enzyme.

N-phenyl group were making π–π stacked interaction with

Trp286. One of the triazole ring nitrogen atoms was mak-

ing a hydrogen bond with Ser293.

Molecular docking studies

BChE docking studies

AChE docking studies

For docking against BChE, crystal structure of human BChE

enzyme (PDB id: 4BDS, 2.1 Å) was downloaded from the

PDB. To validate the docking protocol, the ligand that had

co-crystallized with the enzyme was extracted and re-docked

into the same enzyme. The docking protocol was able to

reproduce the experimentally observed bound conformation

with rmsd of less than 2. The three most active BChE inhibi-

tors 9d, 9h and 9l were docked against BChE. For compound

9d, the NH of amide group was found to act as a hydrogen

bond donor toward Asp70. One of the ring nitrogen atoms

was acting as a hydrogen bond acceptor toward Gln71 and

Ser72. Several other non-covalent interactions were also

observed. Both methyl groups on the phenyl ring were found

to make π-alky interactions with Phe329 and Trp82. One of

the methyl group was additionally making a π-alkyl interac-

tion with Tyr332. The triazole ring was making a π-alkyl

interaction with Asp70. The phenyl ring was making a π–π

stacked interaction with Tyr332 (Fig. 4).

The crystal structure of AChE (PDB id: 4M0E) was down-

loaded from the PDB. Docking studies were carried out

using BioSolveIT’s LeadIT software. Discovery Studio

Visualizer [26] was used to visualize protein–ligand inter-

actions. The thiophenyl ring of compound 10f was making a

π-anion interaction with Glu292 (Fig. 2). One of the methyl

groups was making π-alkyl interaction with Phe297 and

Phe338, while the other methyl group was making π-alkyl

interaction withTyr341 and Tyr337. The phenyl ring was

making a π–π T-shaped interaction with Tyr124. The N-sub-

stituted phenyl ring was also making a π–π T-shaped inter-

action with Trp286. One of the triazole ring nitrogen atoms

was making a hydrogen bond with Ser293. The carbonyl

oxygen was making a hydrogen bond with Phe295.

Similar interactions were observed for compound 10d

(Fig. 3) with AChE, π-anion interaction was observed

between the thiophenyl ring and Glu292, while π-alkyl

interactions were observed between one of the methyl

groups and amino acids Phe338 and Tyr341, the other

methyl group was also making π-alkyl interactions with

Tyr341, Tyr72 and Trp286. The phenyl ring was making

π–π stacked interaction with Tyr341. Both triazole and

For compound 9l similar inter actions as in 9d were found

as can be seen from Fig. 5. One of the nitrogen atoms of

the triazole ring was making a hydrogen bond with Ser72,

whereas the triazole ring itself was making a π-anion interac-

tion with Asp70. The NH group of amide group was making

Table 3 BChE inhibitory

Comp

BChE

Comp

BChE

activities of compounds 9a–q;

10a–q

Inhibition (%) at

0.5 mM

IC₅₀(µM)

Inhibition (%) at

0.5 mM

IC₅₀(µM)

9a

9b

9c

9d

9e

9f

9g

9h

9i

9j

9k

9l

9m

9n

9o

9p

9q

Eserine

65.26 0.29

83.42 0.25

65.87 0.28

85.23 0.29

67.84 0.27

16.52 0.24

79.23 0.26

87.74 0.28

71.25 0.29

43.76 0.24

62.58 0.26

87.63 0.28

42.52 0.35

35.34 0.27

56.23 0.28

61.76 0.29

82.27 0.26

83.75 1.16

165.43 0.25

21.72 0.18

166.43 0.23

12.52 0.19

154.82 0.23

–

32.24 0.21

19.75 0.22

112.82 0.23

–

10a

10b

10c

10d

10e

10f

10g

10h

10i

10j

10k

10l

10m

10n

10o

10p

10q

Eserine

86.47 0.25

29.58 0.32

71.35 0.27

73.43 0.29

87.28 0.23

84.25 0.28

79.42 0.36

73.64 0.29

78.32 0.27

75.36 0.26

79.45 0.37

26.32 0.23

76.27 0.26

78.79 0.28

86.47 0.25

29.58 0.32

71.35 0.27

83.75 1.16

27.24 0.19

–

48.53 0.23

23.62 0.22

27.43 0.16

84.01 0.23

58.52 0.28

24.52 0.21

54.36 0.22

28.35 0.18

61.45 0.24

–

55.63 0.19

68.96 0.21

27.24 0.19

–

212.96 0.21

12.52 0.19

–

326.92 0.24

275.24 0.25

37.56 0.21

0.62 0.08

48.53 0.23

0.62 0.08

1 3

Journal of the Iranian Chemical Society

Table 4 Urease inhibitory

activity of compounds 9a–q;

10a–q

Comp

Urease

Comp

Urease

Inhibition (%) at

0.25 mM

IC₅₀(µM)

Inhibition (%) at

0.25 mM

IC₅₀(µM)

9a

9b

9c

9d

9e

9f

9g

9h

9i

9j

9k

9l

9m

9n

9o

9p

9q

Thiourea

15.3 0.14

11.6 0.13

15.2 0.15

8.4 0.15

98.45 0.12

14.2 0.13

7.4 0.12

–

10a

10b

10c

10d

10e

10f

10g

10h

10i

10j

10k

10l

10m

10n

10o

10p

10q

Thiourea

26.48 0.13

9.75 0.12

28.56 0.15

12.27 0.11

19.36 0.14

21.25 0.12

6.43 0.13

21.25 0.12

98.56 0.11

16.57 0.13

18.36 0.14

21.41 0.12

13.62 0.15

98.75 0.12

27.41 0.12

31.41 0.12

12.5 0.19

98.21 0.18

–

8.79 0.05

–

9.3 0.14

12.6 0.15

38.5 0.17

24.7 0.16

6.3 0.12

7.27 0.05

–

7.5 0.11

–

31.2 0.14

34.8 0.15

15.7 0.13

11.5 0.16

98.21 0.18

7.35 0.04

–

21.25 0.15

Table 5 α-Glucosidase

inhibitory activities of

compounds 9a–q; 10a–q

Comp

α-Glucosidase

Comp

α-Glucosidase

Inhibition (%) at IC₅₀(μM)

0.5 mM

9a

9b

9c

9d

9e

9f

9g

9h

9i

9j

9k

9l

9m

9n

9o

9p

9q

Acarbose

71.46 0.28

65.75 0.29

37.14 0.19

64.39 0.32

27.82 0.25

47.65 0.27

43.59 0.21

65.78 0.28

43.65 0.16

42.37 0.19

76.52 0.34

72.34 0.29

63.25 0.36

48.76 0.24

89.43 0.28

65.27 0.36

47.5 0.31

274.52 0.19

392.75 0.21

–

328.26 0.22

–

374.25 0.21

–

172.18 0.23

289.52 0.24

365.46 0.27

–

62.94 0.19

325.83 0.23

–

10a

10b

10c

10d

10e

10f

10g

10h

10i

10j

10k

10l

10m

10n

10o

10p

10q

Acarbose

46.53 0.27

35.24 0.28

68.46 0.23

78.34 0.26

18.32 0.25

46.45 0.28

43.36 0.23

32.54 0.27

97.68 0.27

28.26 0.23

12.39 0.21

22.54 0.25

63.35 0.26

78.52 0.27

17.5 0.21

31.5 0.27

17.5 0.11

65.73 1.93

–

319.28 0.16

174.57 0.19

–

69.46 0.15

–

385.14 0.18

195.16 0.19

–

65.73 1.93

375.82 1.76

a hydrogen bond with Asp70. The ethyl group substituted on

the phenyl ring was making a π-alky interaction with TRp82,

whereas the alkyl part of Ala328 was also making a π-alkyl

interaction with the phenyl ring. The phenyl ring was also

making π–π stacked interactions with Trp82.

For compound 9h, the thiophenyl ring was found to be

making π-anion and π-sigma interaction with Asp70; π–π

stacked interactions were observed between the triazole

ring and Trp82. A π-alkyl interaction was observed between

N-ethyl side chain and Trp82. The carbonyl oxygen was

1 3

Journal of the Iranian Chemical Society

Fig. 2 Docked conformation of 10f showing hydrogen bonds (green),

π-alkyl (light purple), π-anion (orange), and π–π stacked (purple)

interactions with AChE

Fig. 5 Docked conformation of 9 l with BChE showing hydrogen

bonds (green), π-alkyl (light purple), and π–π stacked (purple) inter-

actions

within hydrogen bonding distance of amino acids Gly116,

Gly117 and Ala199. The phenyl ring was found to be making

π–π T-shaped interactions with Phe329 and Trp231 (Fig. 6).

Urease docking studies

The crystal structure of urease (PDB id: 4GY7) was down-

loaded from the PDB. For compound 10i, hydrogen bond

was observed between the NH of amide group and the

amino acid Ala436. The amino acid Arg439 was found to

be involved in making hydrogen bonds with the ring nitrogen

atoms of the triazole ring and the ring sulfur of thiophene

group. Several non-bonded interactions were also observed.

The phenyl ring was found to be making a π-anion interac-

tion with Asp494 and π–π T-shaped interaction with aro-

matic rings of Trp495 (Fig. 7).

Fig. 3 Docked conformation of 10d with AChE showing hydro-

gen bonds (green), π-alkyl (light purple), π-anion (orange), and π–π

stacked (purple) interactions

For compound 10n the ethoxide side chain was found

to be making a π-alkyl interaction with Ile411. The phenyl

and triazole rings were making π-alkyl interactions with

Ala440. The triazole ring was additionally making a π-anion

interaction with Asp494, and a π–π T-shaped interaction

with His593. The NH group of amide group was making a

hydrogen bond with carboxymethylated amino acid Cme592

(Fig. 8).

α‑Glucosidase docking studies

Since crystal structure of α-glucosidase from Saccharomyces

cerevisiae is not yet available from the PDB, its homology

model was constructed and validated using crystal structure

of oligo-1,6-glucosidase as a tem plate, according to our pre-

viously reported procedure [27–29]. The most active inhibi-

tors 9o and 10i were docked using LeadIT software. Similar

binding orientations were found for both compounds. For

Fig. 4 Docked conformation of 9d with BChE showing hydro-

gen bonds (green), π-alkyl (light purple), π-anion (orange), and π–π

stacked (purple) interactions

1 3

Journal of the Iranian Chemical Society

Fig. 6 Docked conformation of 9 h with BChE showing hydro-

gen bonds (green), π-alkyl (light purple), π-anion (orange), and π–π

T-shaped and π–π stacked (purple) interactions

Fig. 8 Docked conformation of 10n with urease enzyme showing

hydrogen bonds (green), π-alkyl (light purple), π-anion (orange), and

π–π T-shaped (purple) interactions

Fig. 7 Docked conformation of 10i with urease showing hydrogen

bonds (green), π-anion (orange), and π–π T-shaped (purple) interac-

tions

compound 9o, π-alkyl interactions were observed between

ethyl side chain and amino acids Phe177 and Phe157. The

methyl group was making π-alkyl interaction with Tyr71

and His348. The phenyl ring was making π-anion interac-

tions with Asp214 and Glu276. The triazole ring was mak-

ing π-alkyl and π–π T-shaped interactions with Arg312 and

Phe300, respectively. One of the triazole nitrogen atoms was

making a hydrogen bond with Gln350. Another hydrogen

bond was observed between the NH of amide group and

amino acid Glu276 (Fig. 9).

Fig. 9 Docked conformation of 9o with yeast α-glucosidase showing

hydrogen bonds (green), π-alkyl (light purple), π-anion (orange), and

π–π T-shaped (purple) interactions

interactions with Ala278 and Arg312, respectively; both

rings were making π–π T-shaped interactions with Phe300.

Additionally, the thiophenyl ring was making a π-anion

interaction with His279. The thiophenyl ring and the

N-substituted phenyl ring were found to be in contact via

π–π stacked interaction. Hydrogen bonds were observed

between one of the triazole ring nitrogen atoms and Gln350,

between NH of amide group and Glu276, and between car-

bonyl group of amide and Arg439 (Fig. 10).

For compound 10i, the methyl group was found to be

making a π-alkyl interaction with Tyr71 and His111. The

phenyl ring was making π-anion and π–π T-shaped inter-

actions with amino acids Asp214 and Tyr71, respectively.

The thiophenyl ring and triazole rings were making π-alkyl

1 3

Journal of the Iranian Chemical Society

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Conclusions

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The targeted N-aryl/aralkyl derivatives (9a–q) of 2-(4-ethyl-

5-(thiophen-2-ylmethyl)-4H-1,2,4-triazol-3-ylthio)aceta-

mide and N-aryl/aralkyl derivatives (10a–q) of 2-(4-phe-

nyl-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazol-3-ylthio)

acetamide were synthesized in good yields, and these mole-

cules possessed various enzyme inhibitory activities moder-

ate to good and potent against AChE (9i, 9n, 10d, 10f, 10m

with IC₅₀values 73.54 0.16, 69.26 0.12, 45.72 0.11,

32.26 0.12, 72.53 0.13 µM, respectively), BChE (9b, 9d,

9l, 10a, 10d, 10e, 10h, 10j, 10o, IC₅₀values 21.72 0.18,

12.52 0.19, 12.52 0.19, 27.24 0.19, 23.62 0.22,

27.43 0.16, 24.52 0.21, 24.52 0.21, 28.35 0.18, and

27.24 0.19 µM, respectively), urease (9e, 10i, 10n IC₅₀

values 8.79 0.05, 7.27 0.05, 7.35 0.04 µM, respectively)

and α-glucosidase enzymes (9o, 10i with IC₅₀values of

62.94 0.19, and 69.46 0.15 µM, respectively). Therefore,

these studies led to the conclusion that the newly synthesized

molecules might serve as promising drug molecules for fur-

ther structural optimizations and drug designing studies.

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1 3

Article Doi

A novel method for the synthesis of 1,2,4-triazole-derived heterocyclic compounds: enzyme inhibition and molecular docking studies

DOI: 10.1007/s13738-019-01848-3

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