Synthesis, antimicrobial and antioxidant evaluation, and molecular docking study of 4,5-disubstituted 1,2,4-triazole-3-thiones

Article abstract of DOI:10.1016/j.molstruc.2020.128273

There is a growing need for new antibiotics and antifungal drugs to combat pathogenic bacteria and fungi. In order to develop potential antimicrobial agents, some 1,2,4-triazole-3-thiones were synthesized from the reaction of hydrazides with isothiocyanates under optimized conditions in deep eutectic solvent of potassium carbonate-glycerol (1:5 M ratio). Blocking properties of all products were assessed on a variety of Gram-positive and Gram-negative bacterial as well as fungal pathogens. Good to excellent inhibitory effects especially against fungi were observed with all synthesized compounds. 5-(4-Hydroxyphenyl)-4-(4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3b) showed significant antioxidant activity according to the results obtained from 2,2-diphenyl-1-picrylhydrazylf (DPPH) free radical scavenging experiments. The possible interaction mechanism of synthetic triazoles with 1IYL enzyme on Aspergillus fumigatus was investigated by molecular docking method. A complete agreement was found between experimental data and theoretical calculations. Hydrogen bond acceptor strength of N-1 in 1,2,4-triazole rings was the main cause of the observed differences.

Full text of DOI:10.1016/j.molstruc.2020.128273

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Synthesis, antimicrobial and antioxidant evaluation, and molecular docking study of
4,5-disubstituted 1,2,4-triazole-3-thiones
Hamid Beyzaei, Maryam Ghanbari Kudeyani, Hojat Samareh Delaram, Reza Aryan
PII:
S0022-2860(20)30598-6
DOI:
https://doi.org/10.1016/j.molstruc.2020.128273
MOLSTR 128273
Reference:
To appear in: Journal of Molecular Structure
Received Date: 21 October 2019
Revised Date: 13 April 2020
Accepted Date: 14 April 2020
Please cite this article as: H. Beyzaei, M.G. Kudeyani, H.S. Delaram, R. Aryan, Synthesis, antimicrobial
and antioxidant evaluation, and molecular docking study of 4,5-disubstituted 1,2,4-triazole-3-thiones,
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Synthesis, antimicrobial and antioxidant evaluation, and molecular docking study of 4,5-
disubstituted 1,2,4-triazole-3-thiones
Hamid Beyzaei*, Maryam Ghanbari Kudeyani, Hojat Samareh Delarami, Reza Aryan
CRediT Author Statement
Hamid Beyzaei: Supervision, Writing- Original Draft, Writing- Reviewing and Editing. Maryam Ghanbari
Kudeyani: Methodology, Investigation. Hojat Samareh Delarami: Software, Data Curation. Reza Aryan
Conceptualization, Formal Analysis.

Synthesis, antimicrobial and antioxidant evaluation, and molecular docking study of 4,5-
disubstituted 1,2,4-triazole-3-thiones
Hamid Beyzaei*, Maryam Ghanbari Kudeyani, Hojat Samareh Delarami, Reza Aryan
Department of Chemistry, Faculty of Science, University of Zabol, Zabol, Iran

Synthesis, antimicrobial and antioxidant evaluation, and molecular docking study of 4,5-
disubstituted 1,2,4-triazole-3-thiones
Hamid Beyzaei*, Maryam Ghanbari Kudeyani, Hojat Samareh Delarami, Reza Aryan
Department of Chemistry, Faculty of Science, University of Zabol, Zabol, Iran
∗Corresponding author: Hamid Beyzaei; E-mail: hbeyzaei@yahoo.com and hbeyzaei@uoz.ac.ir;
Tel.: +98 543 123 2186; Fax: +98 543 123 2180.
E-mail addresses: hbeyzaei@yahoo.com (H. Beyzaei), ghanbari.m.chemist@gmail.com (M.
Ghanbari
Kudeyani),
hsd102012@gmail.com
(H.
Samareh
Delarami),
rezaaryanchemist@yahoo.com (R. Aryan).
1

Abstract
There is a growing need for new antibiotics and antifungal drugs to combat pathogenic bacteria and fungi. In order
to develop potential antimicrobial agents, some 1,2,4-triazole-3-thiones were synthesized from the reaction of
hydrazides with isothiocyanates under optimized conditions in deep eutectic solvent of potassium carbonate-glycerol
(1:5 molar ratio). Blocking properties of all products were assessed on a variety of Gram-positive and Gram-
negative bacterial as well as fungal pathogens. Good to excellent inhibitory effects especially against fungi were
observed with all synthesized compounds. 5-(4-Hydroxyphenyl)-4-(4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-
thione (3b) showed significant antioxidant activity according to the results obtained from 2,2-diphenyl-1-
picrylhydrazylf (DPPH) free radical scavenging experiments. The possible interaction mechanism of synthetic
triazoles with 1IYL enzyme on Aspergillus fumigatus was investigated by molecular docking method. A complete
agreement was found between experimental data and theoretical calculations. Hydrogen bond acceptor strength of
N-1 in 1,2,4-triazole rings was the main cause of the observed differences.
Keywords 1,2,4-Triazole-3-thione, Deep eutectic solvent, Glycerol, 1IYL, Molecular docking, Antimicrobial and
antioxidant evaluation.
Introduction
Triazoles are an important class of aromatic five-membered heterocyclic compounds with a wide variety of
biological activities. They consist of two structural isomers: 12,3- and 1,2,4-triazole. Compounds based on 1,2,4-
triazole scaffold show diverse biological and pharmacological activities [1-3]. 1,2,4-Triazole-3-thiones occupy a
unique place among compounds containing 1,2,4-triazole ring. Lesinurad is a selective inhibitor of uric acid
transporter 1 (URAT1) and organic anion transporter 4 (OAT4) indicated for the treatment of hyperuricemia [4].
Prothioconazole is a systemic fungicide that can be used alone or combined with other seed and foliar treatments [5].
The synthesized compounds having 1,2,4-triazole-3-thione skeleton have been reported to possess biological and
pharmacological activities such as anticonvulsant, urease inhibition, antioxidant, analgesic. anti-parasitic, antiulcer,
anticancer, anti-HIV, anti-tuberculosis, antiamoebic, antigiardial, antiepileptic, anti-inflammatory, antidepressant
and anxiolytic [6-16]. This wide range of activities has encouraged chemists to design new synthetic methods.
Cyclocondensation of hydrazinecarbothioamides or thiosemicarbazide with aldehydes, ethyl benzimidate or aroyl
halides, reaction of methyl 3-acyldxithiocarbazates with amines, thermolysis of thiosemicarbazones, condensation of
aroylthiosemicarbazides, reaction of 2-hydrazinoethanol with aroyl isothiocyanate, and reaction of hydrazides and
isothiocyanates are a few examples [17-25].
Green chemistry is a branch of chemistry, which directs chemical processes to improve the quality of life of all
living organisms. One of the main goals of green chemistry is the development of the use of environmentally
friendly solvents rather than harmful solvents. Solvents such as water, glycerol, supercritical carbon dioxide and
polyethylene glycol have been suggested for this purpose. Recently, the use of deep eutectic solvents (DESs) has
been expanding due to their diverse applications [26]. Glycerol (glycerin)/potassium carbonate is a green catalytic
media that its efficiency has been proven in the synthesis of different heterocyclic compounds [27, 28].
2

Molecular docking studies are efficient computational procedures which can predict how a ligand interacts with the
protein's binding site. Some (2R,3R)-1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-(N-substitutied)-2-butanole
derivatives were designed and synthesized as antifungal agents by Wu et al., their inhibitory mechanism was
determined with molecular docking [29]. A synthesized hybrid of ciprofloxacin and 1,2,4-triazole-3-thione
derivative showed better inhibitory activities against Candida albicans versus itraconazole, it strongly binds to
active site of target enzyme according to reasonable docking scores [30]. A novel synthetic series of 1H-1,2,4-
triazole was evaluated for their potential antifungal activities, the interactions of the most effective compounds in
binding site of protein were studied by molecular docking methods [31].
In this context, one-pot synthesis of 4,5-disubstituted 1,2,4-triazole-3-thione derivatives 3a-i was reported. They
were prepared via cyclocondensation of various aryl hydrazides 1a-e with isothiocyanates 2a-e in the presence of
glycerol/K2CO3 as catalyst and reaction media. In vitro inhibitory potential of synthesized compounds was evaluated
on pathogens including five Gram-negative and four Gram-positive bacteria as well as three fungi. In addition, their
free radical scavenging capacity was assessed to determine antioxidant activity. The interaction of all derivatives in
active site of 1IYL was explored to determine their possible inhibitory mechanism against Aspergillus fumigatus
strain.
Material and Methods
Chemicals
All yields refer to isolated products. Melting points were recorded on a Kruss type KSP1N melting point apparatus
and are uncorrected. The reaction progress was monitored by aluminum TLC plates pre-coated with silica gel 60
with fluorescent indicator F254 using n-hexane/ethyl acetate (8:2, v/v) as the desired mobile phase. The resulted
TLC plates were visualized under UV radiation (254 nm). IR spectra of compounds were recorded on a Bruker
Tensor-27 FT-IR spectrometer using KBr disks. 1H and 13C NMR spectra of products in DMSO-d6 were recorded on
a Bruker FT-NMR Ultra Shield-400 spectrometer (400 and 100 MHz, respectively). Elemental analyses for C, H, N
and S atoms were performed on a Termo Finnigan Flash EA microanalyzer. UV/Vis spectroscopy was performed
using a Jenway 6405 UV/Vis spectrophotometer.
General procedure for the synthesis of 1,2,4-triazole-3-thiones 3a-i
A mixture of both 1 mmol of hydrazides 1a-i and isothiocyanates 2a-i were added to 1 g of prepared in situ
glycerol/K2CO3 (5:1 molar ratio) DES. The contents were vigorously stirred at 100 °C until the end of the reaction
was determined by TLC analysis. The reaction mixture was cooled downed to room temperature; 10 ml of ethanol
was added with stirring to it. The precipitates were filtered off, washed respectively with ethanol (5 ml) and water (5
ml), dried in an oven. The products 3a-i were obtained without need to further purification.
4-(4-Nitrophenyl)-5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (3a)
Yield: 0.23 g, 78%; m.p.: 278-280 °C (Lit. [19]: 285 °C); reaction time: 10 h; IR (KBr) ν: 3428 (NH), 1646 (C=N),
1
1591, 1511, 1328, 1242 (C=S), 1175, 1112, 753, 687 cm-1; H NMR (400 MHz, DMSO-d6) δ: 11.83 (s, 1H, NH),
3

8.26 (d, J = 8.1 Hz, 2H, H-3′,5′), 7.90 (s, 2H, H-2″,6″), 7.78 (d, J = 8.1 Hz, 2H, H-2′,6′), 7.57 (s, 3H, H-3″,4″,5″)
ppm; 13C NMR (100 MHz, DMSO-d6) δ: 117.3 (C-3′,5′), 125.9 (C-2″,6″), 126.2 (C-3″,5″), 129.2 (C-1″), 129.8 (C-
2′,6′), 131.7 (C-4″), 141.5 (C-1′), 143.0 (C-4′), 145.3 (C-5), 159.6 (C-3); Anal. Calcd. for C14H10N4O2S: C 56.37, H
3.38, N 18.78, S 10.75. Found: C 56.41, H 3.39, N 18.75, S 10.73.
5-(4-Hydroxyphenyl)-4-(4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3b)
Yield: 0.24 g, 76%; m.p.: 180-182 °C; reaction time: 8 h; IR (KBr) ν: 3454 (NH), 3338 (OH), 1657 (C=N), 1586,
1
1501, 1331, 1245 (C=S), 1178, 1112, 851, 746, 691 cm-1; H NMR (400 MHz, DMSO-d6, ppm) δ: 10.36 (brs, 1H,
NH), 10.00 (s, 1H, OH), 8.17 (d, J = 7.7 Hz, 2H, H-3′,5′), 7.88 (d, J = 7.7 Hz, 2H, H-2′,6′), 7.80 (d, J = 7.2 Hz, 2H,
H-2″,6″), 6.84 (d, J = 7.2 Hz, 2H, H-3″,5″) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 115.3 (C-3″,5″), 123.3 (C-1″),
124.0 (C-3′,5′), 125.2 (C-2′,6′), 130.4 (C-2″,6″), 141.5 (C-1′), 143.8 (C-4′), 146.2 (C-5), 161.2 (C-4″), 166.2 (C-3)
ppm; Anal. Calcd. for C14H10N4O3S: C 53.50, H 3.21, N 17.83, S 10.20. Found: C 53.54, H 3.19, N 17.88, S 10.17.
4-(4-Nitrophenyl)-5-(4-(pyridin-4-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3c)
Yield: 0.24 g, 80%; m.p. > 300 °C (Lit. [32]: 320-322 °C); reaction time: 9 h; IR (KBr) ν: 3415 (NH), 3213, 2362,
1
1642 (C=N), 1605, 1556, 1500, 1462, 1327, 1244 (C=S), 1173, 1111, 846, 728, 696, 646, 502 cm-1; H NMR (400
MHz, DMSO-d6) δ: 10.97 (brs, 1H, NH), 8.77 (d, J = 3.9 Hz, 2H, H-3″,5″), 8.19 (d, J = 8.7 Hz, 2H, H-3′,5′), 7.81-7-
89 (m, 4H, H-2′,6′,2″,6″) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 122,0 (C-2″,6″), 124.1 (C-3′,5′), 125.7 (C-2′,6′),
127.6 (C-1″), 139.8 (C-1′), 143.8 (C-4′), 146.0 (C-5), 150.7 (C-3″,5″), 164.9 (C-3) ppm; Anal. Calcd. for
C13H9N5O2S: C 52.17, H 3.03, N 23.40, S 10.71. Found: C 52.21, H 3.05, N 23.41, S 10.66.
5-(4-(Furan-2-yl)phenyl)-4-(4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3d)
Yield: 0.22 g, 76%; m.p. 185-186 °C (Lit. [33]: 187-188 °C); reaction time: 8 h; IR (KBr) ν: 3418 (NH), 2361, 1629
1
(C=N), 1551, 1510, 1408, 1334, 1267 (C=S), 1188, 1110, 844, 704 cm-1; H NMR (400 MHz, DMSO-d6) δ: 11.55
(brs, 1H, NH), 8.26 (d, J = 8.7 Hz, 2H, H-3′,5′), 7.99 (s, 1H, H-5″), 7.77 (d, J = 8.7 Hz, 2H, H-2′,6′), 7.17 (s, 1H, H-
3″), 6.75 (s, 1H, H-4″) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 112.8 (C-4″), 113.4 (C-3″), 117.3 (C-3′,5′), 125.9
(C-2′,6′), 139.0 (C-5″), 140.7 (C-2″), 141.5 (C-1′), 144.3 (C-4′), 146.7 (C-5), 159.0 (C-3) ppm; Anal. Calcd. for
C12H8N4O3S: C 50.00, H 2.80, N 19.44, S 11.12. Found: C 49.95, H 2.80, N 19.46, S 11.09.
4,5-Bis(4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3e)
Yield: 0.32 g, 93%; m.p. 249-250 °C; reaction time: 9 h; IR (KBr) ν: 3479 (NH), 1627 (C=N), 1509, 1301, 1260
(C=S), 1176, 1107, 838, 624 cm-1; 1H NMR (400 MHz, DMSO-d6) δ: 14.35 (s, 1H, NH), 8.34 (d, J = 7.4 Hz, 2H, H-
3″,5″), 8.31 (d, J = 7.0 Hz, 2H, H-3′,5′), 8.10 (d, J = 7.4 Hz, 2H, H-2″,6″), 7.75 (d, J = 7.0 Hz, 2H, H-2′,6′) ppm; 13
C
NMR (100 MHz, DMSO-d6) δ: 117.1 (C-3′,5′), 123.0 (C-3″,5″), 127.7 (C-2″,6″), 130.4 (C-2′,6′), 131.2 (C-1′), 134.6
(C-1″), 145.3 (C-4′), 146.8 (C-5), 150.5 (C-4″), 159.1 (C-3) ppm; Anal. Calcd. for C14H9N5O4S: C 48.98, H 2.64, N
20.40, S 9.34. Found: C 48.95, H 2.63, N 20.44, S 9.32.
4

5-(4-Nitrophenyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (3f)
Yield: 0.22 g, 74%; m.p.: 271-273 °C (Lit. [34]: 252-254 °C); reaction time: 6 h; IR (KBr) ν: 3481 (NH), 1630
(C=N), 1591, 1480, 1301, 1252 (C=S), 1175, 1106, 842, 750, 624 cm-1; 1H NMR (400 MHz, DMSO-d6) δ: 14.27 (s,
1H, NH), 8.34 (d, J = 6.7 Hz, 2H, H-3″,5″), 8.11 (d, J = 6.7 Hz, 2H, H-2″,6″), 7.48 (m, 3H, H-3′,4′,5′), 7.38 (m, 2H,
H-2′,6′) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 122.9 (C-3″,5″), 125.32 (C-4′), 129.1 (C-2′,6′), 129.6 (C-3′,5′),
129.8 (C-2″,6″), 134.6 (C-1′), 135.4 (C-1″), 147.7 (C-5), 150.7 (C-4″), 166.5 (C-3) ppm; Anal. Calcd. for
C14H10N4O2S: C 56.37, H 3.38, N 18.78, S 10.75. Found: C 56.41, H 3.39, N 18.74, S 10.77.
4-(4-Fluorophenyl)-5-(4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3g)
Yield: 0.22 g, 64%; m.p.: 229-231 °C; reaction time: 5 h; IR (KBr) ν: 3419 (NH), 1691 (C=N), 1600, 1500, 1459,
1417, 1281, 1237 (C=S), 1011, 966, 853, 773, 695, 607, 543 cm-1; 1H NMR (400 MHz, DMSO-d6) δ: 14.40 (s, 1H,
NH), 8.37 (d, J = 7.2 Hz, 2H, H-3″,5″), 8.16 (d, J = 7.2 Hz, 2H, H-2″,6″), 7.45 (d, J = 7.8 Hz, 2H, H-2′,6′), 7.34 (d, J
= 7.8 Hz, 2H, H-3′,5′) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 116.6 (C-3′,5′), 122.9 (C-3″,5″), 126.3 (C-2″,6″),
130,5 (C-1′), 131.4 (C-2′,6′), 134.4 (C-1″), 146.2 (C-5), 149.7 (C-4″), 160.6 (C-3), 164.5 (C-4′) ppm; Anal. Calcd.
for C14H9FN4O2S: C 53.16, H 2.87, N 17.71, S 10.14. Found: C 53.21, H 2.88, N 17.74, S 10.10.
5-(4-Nitrophenyl)-4-(p-tolyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3h)
Yield: 0.27 g, 88%; m.p.: 169-170 °C; reaction time: 9 h; IR (KBr) ν: 3414 (NH), 1695 (C=N), 1607, 1511, 1458,
1
1398, 1327, 1225 (C=S), 1097, 1011, 967, 842, 775, 723, 672, 624, 594 cm-1; H NMR (400 MHz, DMSO-d6) δ:
13.80 (s, 1H, NH), 8.33 (d, J = 6.9 Hz, 2H, H-3″,5″), 8.12 (d, J = 6.9 Hz, 2H, H-2″,6″), 7.25 (d, J = 5.9 Hz, 2H, H-
2′,6′), 7.13 (d, J = 5.9 Hz, 2H, H-3′,5′), 2.32 (s, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 21.2 (CH3),
113.8 (C-3″,5″), 128.8 (C-3′,5′), 129.5 (C-2″,6″), 130.1 (C-2′,6′), 131.6 (C-1′), 132.8 (C-4′), 135.5 (C-1″), 150.4 (C-
5), 151.7 (C-4″), 168.4 (C-3) ppm; Anal. Calcd. for C15H12N4O2S: C 57.68, H 3.87, N 17.94, S 10.26. Found: C
57.62, H 3.85, N 17.97, S 10.30.
4-Ethyl-5-(4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3i)
Yield: 0.20 g, 80%; m.p.: 174-176 °C; reaction time: 5 h; IR (KBr) ν: 3427 (NH), 1697 (C=N), 1607, 1514, 1396,
1327, 1241 (C=S), 1020, 967, 819 cm-1; 1H NMR (400 MHz, DMSO-d6) δ: 13.69 (s, 1H, NH), 8.32 (d, J = 6.7 Hz,
2H, H-3″,5″), 8.13 (d, J = 6.7 Hz, 2H, H-2″,6″), 4.40 (q, J = 7.5 Hz, 2H, CH2), 1.12 (t, J = 7.5 Hz, 3H, CH3) ppm;
13C NMR (100 MHz, DMSO-d6) δ: 13.8 (CH3), 63.5 (CH2), 113.9 (C-3″,5″), 129.7 (C-2″,6″), 130.1 (C-2′,6′), 136.4
(C-1″), 151.4 (C-5), 151.8 (C-4″), 166.7 (C-3) ppm; Anal. Calcd. for C10H10N4O2S: C 47.99, H 4.03, N 22.39, S
12.81. Found: C 48.02, H 4.05, N 22.38, S 12.77.
Biological assay
Gram-negative bacterial strains including Salmonella enterica subsp. enterica (PTCC 1709), Pseudomonas
aeruginosa (PTCC 1310), Shigella dysenteriae (PTCC 1188), Acinetobacter baumannii (PTCC 1855), Escherichia
5

coli (PTCC 1399), Gram-positive bacterial strains including Streptococcus pyogenes (PTCC 1447), Staphylococcus
epidermidis (PTCC 1435), Listeria monocytogenes (PTCC 1297), Bacillus cereus (PTCC 1665) and fungi including
Aspergillus fumigatus (PTCC 5009), Candida albicans (PTCC 5027) and Fusarium oxysporum (PTCC 5115) were
prepared from the Persian Type Culture Collection (PTCC), Karaj, Iran. Broth microdilution and streak plate
methods were applied for antimicrobial testing according to the previously described procedures [27]. DPPH free
radical scavenging method was also used to determine the antioxidant activity of synthetic heterocycles [27]. All
biological tests were repeated at least three times. The results were reported as the mean of three independent
experiments.
Computational details
Geometrical optimization of 1,2,4-triazole compounds was performed using the Gaussian 09 program package [35]
at the B3LYP level of theory [36]. Vibrational frequency calculations were also performed at the same level to
ensure that the structures are local minima.
For molecular docking of 1,2,4-triazole compounds, we selected the X-ray structure of N-myristoyltransferase from
Candida albicans (CaNMT, PDB ID: 1IYL) from the Protein Data Bank as fungal target. The molecular docking
was performed using MGL tools 1.5.6 including the AutoDock4.2 scoring function to predict the interactions
between ligand and receptor [37]. Then, the ligand and receptor was considered flexible and rigid, respectively. The
best-docked conformation of the ligand-receptor complex is according to the lowest binding energy.
A blind docking was performed to recognize the binding sites of 1IYL receptor. For 1IYL grid size was set at
126×126×126 Å in the x, y, and z-axes, respectively, with 0.375 Å grids spacing to cover the whole receptor. The
interaction energies between each amino acid with the best pose of docked 1,2,4-triazoles into 1IYL binding site
were calculated by Molegro Molecular Viewer 2.5 (MMV) (http://www.molegro.com/mmv-product.php).
Results and Discussion
Chemistry
1,2,4-Triazole-3-thiones 3a-i were synthesized through reaction of aryl hydrazides 1a-e with isothiocyanates 2a-e,
as shown in Scheme 1 and Fig. 1. Reactions were progressed in a fixed and optimized 5:1 molar ratio of
glycerol/K2CO3 at 100 °C for 5-10 h.
Scheme 1 Synthesis of 1,2,4-triazole-3-thione derivatives 3a-i
6

Fig. 1 Chemical structure of the synthesized 1,2,4-triazole-3-thiones
The reaction conditions were optimized in terms of solvent and temperature (Table 1). Reaction of 1 mmol of both
benzhydrazide and 4-nitrophenyl isothiocyanate in 2 g of solvent was considered as a model. Condensation of
intermediate carbothioamides to 1,2,4-triazole-3-thione derivatives occurred at a minimum temperature of 100 °C as
well as by-products were formed at higher temperatures; therefore, it was kept constant in all processes. No
condensation was observed in glycerol media (Entry 1). Traces of 1,2,4-triazoles were afforded in DESs containing
choline chloride (ChCl) as hydrogen bond acceptor and urea, glucose, citric acid and tartaric acid as hydrogen bond
receptors (Entries 2-5). Reaction yield was improved in choline chloride/glycerol (Entry 6). Reaction progress was
studied in different molar ratios of K2CO3/glycerol (Entries 7-12). The highest yield and shortest time of reaction
was achieved in K2CO3/glycerol (1:5 Molar, Entry 10).
Table 1 Optimization of the model reaction conditions for the preparation of 1,2,4-triazole-3-thione 3a
Entry
1
Solvent
Glycerol
Time (h)
2
Yield (%)
0
2
3
4
5
6
7
8
9
ChCl/urea 1:2
4
9
10
7
10
7
8
4
5
9
Trace
Trace
Trace
Trace
66
32
59
43
78
ChCl/Glucose 2:1
ChCl/Citric acid 1:1
ChCl/Tartaric acid 1:1
ChCl/glycerol 1:2
K2CO3/glycerol 1:2
K2CO3/glycerol 1:3
K2CO3/glycerol 1:4
K2CO3/glycerol 1:5
K2CO3/glycerol 1:6
K2CO3/glycerol 1:7
10
11
12
54
Trace
8
1
The chemical structure of all synthesized triazoles was characterized by FT-IR, H- and 13C-NMR spectroscopies
and elemental analysis. The presence of thiocarbonyl groups in triazole rings as predominant tautomer was proved
7

by their absorption bands in the region of 1225-1267 cm-1 and carbon-13 chemical shifts appeared at 159.0-168.4
ppm [38].
Among the various methods proposed for the preparation of 1,2,4-triazole-3-thiones, reaction of hydrazides and
isothiocyanates have been well developed. The reaction occurs in solvents such as benzene, diethyl ether, DMSO,
1,4-dioxane, dichloromethane and N,N-dimethyl acetamide [18, 25, 39-42]. Intermediate 1-acylthiosemicarbazides
produced during this reaction are usually separated, purified and used in the next step [32, 33, 43]. They are
converted to 1,2,4-triazole and 1,3,4-thiadiazole in the presence of basic or acidic catalysts, respectively [18, 25].
Caustic soda and potash are mainly applied under basic conditions. 1,2,4-Tiazole-3-thione derivatives have been
synthesized via a one-step reaction in short times under microwave irradiation [44].
It is predicted that the reaction proceeds in three steps. The first step is the formation of 1-acylthiosemicarbazides
2'a-i via the nucleophilic addition of hydrazides 1a-e to isothiocyanates 2a-e. In the next step, the intramolecular
attachment of NH group belonging to the isothiocyanate component to carbonyl group can produce 4,5-disubstituted
5-hydroxy-1,2,4-triazolidine-3-thiones 2''a-i. Finally, 1,2,4-triazole-3-thiones 3a-i are generated by the elimination
of water. The reaction times of isothiocyanates 2a-d are approximately 1.5-2 times longer than those of
isothiocyanates 2f-i. If the first step was the rate-determining step, the total reaction times should be reduced in the
presence of isothiocyanates containing strong electron withdrawing NO2 groups. As a result, the second stage is
predicted as the rate-determining step. In this step, the nucleophilic attack of NH bond to carbonyl group becomes
faster as the electron-attracting ability of substituents on isothiocyanates decreases; in fact, the nucleophilicity of NH
groups diminishes in the presence of adjacent electron withdrawing substituents.
Biological study
In vitro inhibitory potentials of 1,2,4-triazoles 3a-i were studied against a variety of pathogenic Gram-positive and
Gram-negative bacteria as well as fungi. The results were compared with those from gentamicin and terbinafine, and
presented as MIC (the Minimum Inhibitory Concentration), MBC (the Minimum Bactericidal Concentration) and
MFC (the Minimum Fungicidal Concentration) values in Tables 2 and 3.
Table 2 Antibacterial effects of 1,2,4-triazoles 3a-i
Products
3a
Antibiotic
Gentamicin
Bacterial
species
3b
3c
3d
3e
3f
3g
3h
3i
1310 MIC 1024
MBC 2048
1709 MIC 1024
MBC 1024
1399 MIC 512
MBC 512
1188 MIC 512
MBC 1024
128
256
256
256
2048
4096
2048
2048
32
256
512
128
256
1024
2048
512
512
256
1024
2048
2048
2048
1024
1024
-
-
-
-
2048
4096
2048
2048
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.063
0.063
8
8
8
8
-
-
-
-
-
-
-
2048
2048
-
-
2048 0.031
4096 0.063
2048 16
-
512
512
1024
2048
4096
-
1855 MIC
-
2048
2048
8

MBC
1665 MIC 256
MBC 512
1435 MIC
MBC
1297 MIC 1024
MBC 1024
-
64
-
-
32
64
-
256
-
4096
2048
4096
512
2048
1024
1024
512
512
-
4096
1024
2048
-
-
-
-
-
-
-
-
-
-
-
-
-
4096 32
2048 0.25
2048
2048
512
1024
512
512
1024
1024
512
1024
1024
1024
-
2048
-
-
-
4
1
2
2
2
2
2
-
-
512
2048
4096
512
-
-
-
-
1447 MIC 4096
MBC 2048
256
256
512
512
512
1024
2048
4096
512
1024
512
– No noticeable antibacterial effect at concentration of 2048 μg ml-1, MIC (μg ml-1), MBC (μg ml-1).
Table 3 Antifungal effects of 1,2,4-triazoles 3a-i
Products
3a
Antifungal
Terbinafine
Fungal
species
3b
3c
3d
3e
3f
3g
3h
3i
5009 MIC 2048
MFC 4096
64
128
4
8
64
128
64
64
4
8
64
64
512
512
2
2
4
8
8
1
2
128
256
8
8
2
128
256
4
8
128
128
2048
4096
2
4
32
256 32
512 32
5115 MIC
MFC
5027 MIC
MFC
4
4
-
4
8
-
32
64
32
64
4
1024
2048
-
4
64
-
– No noticeable antifungal effect at concentration of 2048 μg ml-1, MIC (μg ml-1), MFC (μg ml-1).
At least a 4-nitrophenyl substituent is present at 4- or 5-positions of the 1,2,4-triazole ring due to antimicrobial
potential of aromatic nitro compounds [45]. They were incorporated via 4-nitrophenyl isothiocyanate and/or 4-nitro
benzohydrazide. Efficacy of 1,2,4-triazole derivative 3c was proven on all tested bacteria, even though the best
results was belonged to heterocycle 3b. Generally, antibacterial activity of triazoles 3a-d was more significant than
those of derivatives 3f-i. Derivatives 3a-c were the only blockers of Escherichia coli. No remarkable antibacterial
properties were observed with 1,2,4-triazole 3e having two 4-nitrophenyl groups. As expected, the synthesized
derivatives could effectively inhibit the growth of pathogenic fungi [46]. Aspergillus fumigatus and Fusarium
oxysporum were inhibited with all of them. The best antifungal effects were observed with triazole 3e. There was no
notable difference between derivatives 3a-d and 3f-i. Antioxidant property of all products was assessed against
DPPH radical. Acceptable effect was recorded only for triazole 3b containing 4-hydroxyphenyl substituent with IC50
of 9.19 μg ml-1. It is comparable to antioxidant activity of ascorbic acid (IC50 = 3.94 μg ml-1).
Molecular docking analyses
Molecular docking is a technique which helps to prove binding and interaction poses of ligands within the binding
pocket of macromolecules. Analogue of voriconazole containing 1,2,4-triazole ring showed excellent blocking
properties against ten fungal pathogens especially Aspergillus fumigatus strain, it can be strongly attached to the
enzyme lanosterol 14α-demethylase (CYP51) via its morpholine side chain [29]. A hybrid of fluoroquinolone and
1,2,4-triazole-3-thione with comparable antifungal activity to itraconazole can attach to Gly465, Cys470, Val311
and Leu312 amino acid residues via hydrophobic and hydrogen bonding interactions [30]. A synthetic 1,2,4-triazole
9

containing diflurophenyl and ethyl phenyl carbonate side chains could effectively interact with the active site of
CACYP51 using hydrophobic and Van der Waals forces according to the docking results [31].
In this study, molecular docking procedure was applied to determine interactions of 1,2,4-triazoles 3a-i in active site
of Aspergillus fumigatus N-myristoyltransferase (NMT). The NMT is an essential enzyme in antifungal agents [47].
It facilities the attachment of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine residue in living
organisms such as Aspergillus fumigatus and Candida albicans. Aspergillus fumigatus was selected as target fungus
because it was the only pathogen with relatively different MIC and MFC values. It should also be noted that
cytochrome P450 14 alpha-sterol demethylase (CYP51) (PDB IDs: 5V5Z and 1EA1) is also selected for the
molecular docking study, but the obtained results are not in agreement with the experimental data.
The results of the docking for the prepared 1,2,4-triazoles with 1IYL have been presented in Tables 4 and 5, as well
as Fig. 2 (A and B). As seen, the order of binding energies for the interaction of triazoles to 1IYL is 3e > 3c > 3b >
3f > 3g > 3i > 3d > 3a > 3h, which is in agreement with the order obtained from experimental MIC and MFC
values. These results show that the lowest binding energy of compounds to 1IYL corresponds to 3e by -8.68 kcal
mol-1. As shown in Fig. 2A, there are three conventional H-bonds between N-1, and nitro groups of 4-nitrophenyl
substituents at positions 4 and 5 of the 1,2,4-triazole ring 3e and Leu419, Tyr422 and Tyr210 amino acid residues,
while such interactions are not observed between the compounds 3h and 3i and the 1IYL receptor. Furthermore,
Tyr418, Pro219, Thr218, Arg423, Phe420 and Leu220 residues are placed close to the complex, allowing for
possible Van der Waals interactions.
Table 4 Molecular docking results for interaction between the triazole 3a-i and 1IYL enzyme
1,2,4-Triazoles
BE (kcal.mol-1)
-7.35
ki (μM) vdW+Hbond+ desolv
3a
3b
3c
3d
3e
3f
4.10
1.20
1.07
1.95
0.43
1.86
2.00
2.73
-
-8.46
-9.1
-8.08
-8.15
-9.20
-8.64
-9.55
-9.39
-8.56
-8.37
-
-7.79
-8.68
-7.82
3g
3h
3i
-7.77
-7.59
-6.83
Table 5 Hydrogen bonding interaction energies between the triazole 3a-i and responsive amino acid residues in
molecular docking
1,2,4-Triazoles
Amino acid
Tyr335
Energy (kcal mol-1)
Bond length (Å)
3a
3b
3c
-0.87
-2.50
-1.56
3.43
2.76
3.16
Leu419
Leu177
10

Thr211
Tyr107
Leu419
Tyr210
Tyr422
Leu419
Thr211
-
-1.00
-2.50
-2.5
-0.87
-0.15
-2.50
-2.5
-
3.40
2.70
2.78
3.43
3.34
2.71
2.95
-
3d
3e
3f
3g
3h
3i
-
-
-
Fig. 2 (A) 3D depiction of the 1,2,4-triazole 3e bound to active site on 1IYL. (B) The interaction between
the 1,2,4-triazole 3e and amino acid residues of 1IYL.
Conclusions
A series of 1,2,4-triazole-3-thiones were generated in glycerol/potassium carbonate as deep eutectic solvent. All
products were screened for their antifungal and antibacterial potentials. Significant inhibitory effects were observed
with them especially against fungal pathogens. Molecular docking calculations explored to identify ligand-protein
interactions. When 1IYL was used as target enzyme, computational results were strongly correlated with the data
obtained from the biological screening on Aspergillus fumigatus, which revealed compound 3e containing 4-
nitrophenyl substituents has the highest binding affinity towards the receptor. Hydrogen bonds between N-1 of
triazole rings and some amino acid residues on target enzyme are predominant interactions. These studies may be
helpful in the development of antifungal drug discovery.
11

Conflict of Interest
The authors declare that they have no conflict of interest.
CRediT Author Statement
Hamid Beyzaei: Supervision, Writing- Original Draft, Writing- Reviewing and Editing. Maryam Ghanbari
Kudeyani: Methodology, Investigation. Hojat Samareh Delarami: Software, Data Curation. Reza Aryan
Conceptualization, Formal Analysis.
Acknowledgment
This work was financially supported by the University of Zabol under grant number UOZ-GR-9618-10.
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Highlights
‹ Nine 4,5-disubstituted 1,2,4-triazole-3-thione derivatives were synthesized via a new and
efficient process.
‹ The in vitro inhibitory properties of all synthesized products were evaluated against four
Gram-positive and six Gram-negative pathogenic bacteria as well as three fungal
pathogens.
‹ The inhibitory activities of derivatives as MIC, MBC and MFC values were determined
by broth microdilution and streak plate methods.
‹ DPPH free radical scavenging experiments were applied to determine antioxidant activity
of synthetic heterocycles.
‹ Molecular docking method was applied to find interaction between synthetic compounds
and target enzyme (1IYL) on Aspergillus fumigatus.
‹ Hydrogen bond acceptor strength of N-1 in 1,2,4-triazole rings was the main factor that
caused the observed differences.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: