Synthesis, biological evaluation and molecular modeling study of 3,4-disubstituted 5-mercapto-1,2,4-triazoles

Article abstract of DOI:10.14233/ajchem.2016.19379

Based on the outcome of computational docking to the active site of cytochrome P450 14α-demethylase (CYP51), diverse 3,4-disubstituted 5-mercapto-1,2,4-triazoles were prepared and screened for antioxidant and antifungal activities. The docking study of synthesized compounds showed promising binding affinity towards docked enzyme, sterol 14α-demethylase(CYP51) from trypanosome cruzi obtained from a RCSB protein data bank (PDB ID: 3KHM). The synthesized compounds were characterized by IR, ¹H NMR and Mass spectral data. Among the novel synthesized compounds IV-6, IV-1 and IV-2 showed maximum antifungal activity against A. Niger and C. albicans organism when compared the standard fluconazole. For antioxidant activity, all the compounds showed moderate activity but compound IV-6 and IV-7 showed significant activity when compared to standard ascorbic acid.

Full text of DOI:10.14233/ajchem.2016.19379

Asian Journal of Chemistry; Vol. 28, No. 3 (2016), 508-512
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2016.19379
Synthesis, Biological Evaluation and Molecular Modeling
Study of 3,4-Disubstituted 5-Mercapto-1,2,4-triazoles
1,*
2
3
SUBHAS SAHOO , C.B. MAHENDRA KUMAR and C. MALLIKARJUNA SETTY
1
2
3
Department of Pharmaceutical Chemistry, Pulla Reddy Institute of Pharmacy, Annaram (V), Jinnaram (M), Dundigal-502 313, India
Department of Pharmaceutical Chemistry, St. Mary's College of Pharmacy, Secunderabad-500 025, India
Department of Pharmaceutics, The Oxford College of Pharmacy, Bangalore-560 068, India
*Corresponding author: E-mail: subash_myid@yahoo.com
Received: 26 May 2015; Accepted: 20 July 2015;
Published online: 5 December 2015;
AJC-17640
Based on the outcome of computational docking to the active site of cytochrome P450 14α-demethylase (CYP51), diverse 3,4-disubstituted
-mercapto-1,2,4-triazoles were prepared and screened for antioxidant and antifungal activities. The docking study of synthesized compounds
5
showed promising binding affinity towards docked enzyme, sterol 14α-demethylase(CYP51) from trypanosome cruzi obtained from a
1
RCSB protein data bank (PDB ID: 3KHM). The synthesized compounds were characterized by IR, H NMR and Mass spectral data.
Among the novel synthesized compounds IV-6, IV-1 and IV-2 showed maximum antifungal activity against A. niger and C. albicans
organism when compared the standard fluconazole. For antioxidant activity, all the compounds showed moderate activity but compound
IV-6 and IV-7 showed significant activity when compared to standard ascorbic acid.
Keywords: 1,2,4-Triazoles, Antioxidant activity, Antifungal activity, Molecular docking study.
used broadly and efficiently. For example, voriconazole,
fluconazole and itraconazole, currently play an important role
in the treatment of invasive fungal infection. These antifungal
drugs act by inhibiting CYP51, a necessary enzyme in the
biosynthesis of ergosterol, through a mechanism in which the
heterocyclic nitrogen atom (N-4 of triazole) binds to the heme
iron atom [13]. These efficient analogues are ideally suited
for further alteration to obtain more efficacious antibacterial
compounds.
Various biologically active compounds having therapeutic
activities and diverse drawback lead to the scientist to prepare
most biologically effective compounds with minimal side
effects. In this paper, we have tried to report some novel synthe-
sized 3,4-disubstituted 5-mercapto-1,2,4-triazoles and screened
for their antioxidant activity as well as docking for antifungal
activity.
INTRODUCTION
At the present scenario, antioxidants awaken researcher’s
attention in both medicinal plants and synthetic compounds.
Antioxidant properties of diverse compounds were investigated
by employing various in vitro systems, interaction of 2,2-
diphenyl-1-picrylhydrazyl (DPPH) and scavenging of
superoxide radical, microsomal NADPH-dependent inhibition
of lipid peroxidation (LP), microsomal ethoxyresorufin
O-deethylase (EROD) activity [1].Antioxidants inhibit or delay
oxidation, which appears to have a role in the prevention of
many diseases [2]. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free
radical scavenging measurements were used to determine
antioxidant capacity with ascorbic acid as standard.
Contagious microbial diseases remain vital problems for
humanity, because confrontation to an amount of antimicrobial
agents among the variety of clinically important species of
microorganisms [like methicillin-resistant Staphylococcus
aureus (MRSA)] has turned into an important universal health
problem [3-5]. Hence, there will constantly be a crucial need
to find out new antimicrobial agents.
EXPERIMENTAL
All the chemicals were of synthetic grade and commer-
cially procured from SD Fine chemicals, Hyderabad. Melting
points were determined in open capillary method and were
uncorrected. Purity of the compound was checked on silica
Gel TLC plates. IR spectra were recorded on FT-IR8400S,
Fourier transform (SHIMADZU) infrared spectrophotometer
1
,2,4-Triazole derivatives were found to posses a variety
of pharmacological activities like anti-HIV [6,7], antimicrobial
8-10], antiviral [11], anti-inflammatory [12], antioxidant, etc.
Among the azoles, especially triazole antifungal agents were
[

Vol. 28, No. 3 (2016)
Synthesis and Molecular Modeling Study of 3,4-Disubstituted 5-Mercapto-1,2,4-triazoles 509
using KBr disc method. The proton magnetic resonance spectra
benzo[e][thiadiazine-1,1-dioxide (IV-3): Yield 61 %, m.p.
220-221 °C; TLC solvent chloroform: ethanol (8:2), R value:
1
(
3
H NMR) were recorded on Perkin Elmer spectrophotometer-
00 MHz in DMSO-d , chemical shifts are reported as parts
f
-
1
6
0.61. IR (KBr, νmax, cm ): 3314.66 (NH), 2604.84 (SH),
1
per million (ppm) using TMS as an internal standard.
3098.67 (aromatic-H) 1600.11 (C=N) 1122.27 (C-O-C). H
Preparation of methyl 2-(naphthalen-6-yloxy)acetate
and acetohydrazide (I and II) [14]: Appropriate quantities
of acid (0.1 mol) and methanol (50 mL) was introduced into a
clean and dry round bottom flask and stirred well for 10 min
to the above mixture, few drops of concentrated sulfuric acid
were added and the reaction mixture was refluxed for 6 h. The
reaction mixture was concentrated by distilling the excess
ethanol under reduced pressure and treated with saturated
solution of sodium bicarbonate. The ester formed in the
reaction was used for the preparation of hydrazides directly.
The appropriate ester (0.1 mol) was dissolved in 50 mL of
ethanol in a clean dry round-bottomed flask and to this
hydrazine hydrate (0.1 mol) was added. The reaction mixture
was then refluxed for a period of 15 to 18 h. The excess ethanol
was distilled off under reduced pressure. The resultant mixture
was then poured into ice cold water and the obtained solid
was filtered, recrystallized from ethanol.
NMR δ (ppm): 4.289 (1H, -NH, 2° amine), 4.73 (1H, -NH, 2°
amine) 3.325 (1H, -SH), 7.266-7.994 (9H, -Ar-H), 5.33 (2H-
CH
2
aliphatic ring
N-(5-Mercapto-3-[(naphthalen-6-yloxy) methyl]-4H-
1,2,4-triazol-4-yl)isonicotinamide (IV-4): Yield 60 %, m.p.
190-191 °C; TLC solvent chloroform: ethanol (8:2), R value:
)
. 6.97 (2H-CH
2
), Mass m/z 538.8.
f
-1
0.54. IR (KBr, νmax, cm ): 3305.94 (NH), 1667.50 (C=O),
2619.30 (SH), 3118.29 (aromatic-H) 1624.37 (C=N) 1118.54
(C-O-C). H NMR δ (ppm): 10.08 (1H, -NH, 2° amine), 4.748
(2H-CH ), 3.321 (1H, -SH), 7.721-8.713 (11H, -Ar-H),
1
2
4-(5-Mercapto-3-[(naphthalen-6-yloxy)methyl]-4H-
1,2,4-triazol-4-yl)benzoic acid (IV-5): Yield 70 %, m.p. 171-
172 °C; TLC solvent chloroform: ethanol (8:2), R value: 0.53.
f
-1
IR (KBr, νmax, cm ): 3458.66 (OH), 1661.47 (C=O), 2659.99.
(SH), 3030.10 (aromatic-H), 1604.10 (C=N) 1122.34 (C-O-
1
C). H NMR δ (ppm): 3.323 (1H, -SH), 4.765 (2H-CH
2
), 7.242-
7.877-(11H, -Ar-H), 10.298 (1H-OH).Mass m/z 391.
2
-(Naphthalen-6-yloxy)dithiocarbazinates (III) [14]:
4-(Naphthalen-1-yl)-3-[(naphthalen-6-yloxy) methy])-
An appropriate quantity of acid hydrazide (0.01 mol) was taken
in a clean and dry round bottomed flask. Carbon disulphide
and alcoholic potassium hydroxide (1.5 mol) was introduced
into the round bottomed flask containing acid hydrazide. The
reaction mixture was refluxed for a period of 3-4 h.After cooling,
the separated product was collected by filtration, washed with
water and dried.
4H-1,2,4-triazole-5-thiol (IV-6): Yield 68 %, m.p. 123-
125 °C; TLC solvent chloroform: ethanol (8:2), R value: 0.56.
f
-1
IR (KBr, νmax, cm ): 2933.82 (aliphatic-H), 3057.44 (aromatic-
1
H) 2637.26 (SH), 1598.40 (C=N), 1112.24 (C-O-C). H NMR
δ (ppm): 4.781 (2H-CH
2
), 3.312(1H, -SH), 6.778-8.069 (14H,
-Ar-H), Mass m/z 383.1.
N-(N,N-dimethylcarbamimidoyl)-5-mercapto-3-
[(naphthalene-2-yloxy)methyl]-4H-1,2,4-triazole-carboxa-
imidamide. (IV-7):Yield 61 %, m.p. 129-130 °C; TLC solvent
General procedure for the synthesis of 3,4-disubstituted
1
,2,4-triazole (IV 1-7) [15]: The appropriate dithiocarba-
zinates salt (0.01mol), substituted amines (0.01 mol) and water
5 mL) was transferred in a clean and dry round bottomed
flask and the reaction mixture was refluxed for 6 h till profuse
S gas evolves. The reaction mixture was further boiled for
.5 h at the same temperature.After reflux the reaction mixture
-1
chloroform:ethanol (8:2), R value: 0.60. IR (KBr, νmax, cm ):
f
(
3320.84 (NH), 2904.84 (aliphatic-H), 3098.67 (aromatic-H).
1
H NMR δ (ppm): 5.513 (1H, -NH, 2° amine), 4.765 (2H,
H
0
2
-NH, 2° amine), 3.347 (1H, -SH), 6.756 (2H-CH ), 7.210-7.872
2
(7H, -Ar-H), 2.929 (6H-CH ). Mass m/z 367.1.
3
was cooled and the solid material was separated by filtration.
The solid was washed with dil HCl (50 %, 25 mL) and cold
water (3 × 25 mL) and dried. The resultant compounds (IV
Free radical scavenging activity: The percentage of
antioxidant activity of each substance was assessed by DPPH
free radical assay. The measurement of the DPPH radical scaven-
ging activity was performed according to the methodology
described by Braca et al. [16]. One milliliter of the sample (0.75,
1.5 and 3 µg/mL) was added to 3 mL of 0.1 mmol methanol
solution of DPPH. When DPPH reacts with an antioxidant
compound, which can donate hydrogen, it is reduced. The
changes in colour (from deep violet to light yellow) were read
at 517 nm was determined after 0.5 h of reaction using a UV-
visible spectrophotometer and the percent inhibition activity
was calculated.
Molecular docking studies [17]: In the present study,
the X-ray crystal structure of the antifungal agent fluconazole
bound to sterol 14α-demethylase (CYP51) from trypanosome
cruzi obtained from a RCSB protein data bank (PDB ID:
3KHM). Resolution of protein structure with 464 amino acid
residues was 2.85 Å. The protein was further processed by
removing water and fluconazole. Docking simulation studies
were carried out in Autodock vina. Polar and aromatic
hydrogen’s and gasteiger charges were added in the protein
using MGLtools1.5.4 and the pdb file was subsequently
1
-7) were recrystallized by using ethanol as solvent.
-(4-Fluorophenyl)-3-[(naphthalen-6-yloxy) methyl]-
H-1,2,4-triazole-5-thiol (IV 1): Yield 58 %, m.p. 116-
17 °C;TLC solvent chloroform: ethanol (8:2), R value: 0.62.
4
4
1
f
-1
IR (KBr, νmax, cm ): 3030.10 (aromatic-H), 2659.50 (SH),
1
1
635.10 (C=N) 1145.36 (C-O-C). H NMR δ (ppm): 3.328
(1H, -SH), 4.766 (2H-CH ), 7.188-7.896 (11H, -Ar-H), Mass
2
m/z 351.1.
4
-(5-Mercapto-3-[(naphthalene-2-yloxymethyl)-4H-
,2,4-triazol-4-yl]-N-(5-methyl-1,3,4-oxadiazol-2-yl)
benzenes sulfonamide (IV-2): Yield 62 %, m.p. 169-170 °C;
TLC solvent chloroform: ethanol (8:2), R value: 0.53. IR (KBr,
max, cm ): 3333.42 (NH), 2837.26 (aliphatic-H), 3095.30
aromatic-H), 2633.23 (SH), 1623.33 (C=N) 1110.09 (C-O-
1
f
-
1
ν
(
1
C). H NMR δ (ppm): 4.765 (1H, -NH, 2° amine), 4.252 (1H,
SH), 6.087-7.877 (11H, -Ar-H), 4.765 (2H-CH ), 2.289 (3H-
-
2
CH
3
).
6
-Choloro-7-[(5-mercapto-3-((naphthalene-2-yloxy)-
methyl)-4H-1,2,4-triazol-4-yl)sulphonyl]-3-4-dihydro-2H-

510 Sahoo et al.
Asian J. Chem.
converted to pdbqt format. Pre-optimized compounds were
also pre-processed similarly and converted to pdbqt format.
All the torsion angles in the small-molecules were set free so
as to perform flexible docking. Grid box of size 22 × 22 × 18
with 1 Å spacing was defined along x, y and z axis. The defined
grid box was large enough to cover an active site of the protein.
The analysis of binding free energy and interactions of ligands
with residues at an active site was carried out by using Pymol
and Discovery studio 3.5.
ester, it was further reacted with hydrazine hydrate to get the
aryloxy acid hydrazides (II).And then aryloxy acid hydrazides
were treated with carbon disulphide and alcoholic KOH to get
the potassium salt dithiocarbazinates (III). The diverse 3,4-
disubstituted 1,2,4-triazole (IV 1-7) were obtained by the ring
closure reaction, between the potassium salt dithiocarbazinates
and different drug moieties containing free amino group in
their lead structure.
A series of 3,4-disubstituted 5-mercapto-1,2,4-triazoles
were synthesized and their antifungal activities were screened
for human pathogenic fungi. The compounds IV-6, IV-1 and
IV-2 showed maximum antifungal activity against A. niger and
C. albicans organism when compared the standard fluconazole
Antifungal activity
Standard nutrient agar medium: Meat extract was taken
and made up the volume to 100 mL with water and to this
were added weighed quantities of peptone, salt and agar. The
contents were dissolved by heating and the mixture was filtered
and the pH was adjusted to 7.5. The medium was sterilized
by autoclaving at 121 °C for 15 min, cooled to 45 °C and then
poured in 20 mL quantities to petri dishes. A loopful of an
overnight broth culture was spread evenly over the whole part
with a sterile cotton-wool swab [18].
The culture plates were dried in the incubator with the lid
until its surface was free from visible moisture without further
delay; the known concentration of the drug applied with
adequate spacing to the surface of the culture plates with sterile
fine pointed forceps and pressed gently to ensure full contact
with the medium. It was then transferred to the incubator for
(
Table-1). The obtained results indicated that for antifungal
activity of these novel triazole derivatives it is very helpful to
introduce the aryl group to interact with a hydrophobic pocket
and also generates π-π stacking interaction with TYR A103
by traizole ring (Fig. 1, Table-2). For antioxidant activity, all
the compounds shows moderate activity but compound IV-6
and IV-7 showed significant activity when compared to
standard ascorbic acid (Table-3).
2
4 h at 37 °C. At the end of 24 h, the diameters of the zone of
inhibition produced were measured.
RESULTS AND DISCUSSION
For the synthesis of the target compounds, the reaction
sequences are outlined in Scheme-I. Herein, the aryloxy acetic
acid was prepared by reacting β-naphthol with sodium
hydroxide followed by addition of chloro acetic acid. At first
the powdered β-naphthol treated with sodium hydroxide. In
this exothermic reaction the solution mixture immediately
converted to the sodium salt of β-naphthol, which followed
by addition of freshly prepared chloro acetic acid produces
aryloxy acetic acid. Further the prepared aryloxy acetic acid
was treated with methanol and a few drops of H
corresponding ester (I), without separating the corresponding
2
SO
4
to get the
Fig. 1. Hydrophobic interaction (dotted lines) of Compound IV-6 at the
binding pocket of 14α-demethylase (CYP51) domain
O
O
O
O
MeOH
O
OH
O
NH2
OCH3
N
H
H SO4
2
NH NH2 , HCl
2
II
I
ETHANOL
KOH
CS2
N
N
SH
O
N
R
R-NH2
O
O
NH
S
HN
C
-
+
S K
IV 1-7
III
Scheme-I: Synthesis of 3,4-disubstituted 1,2,4-triazole (IV 1-7)

Vol. 28, No. 3 (2016)
Compound
Synthesis and Molecular Modeling Study of 3,4-Disubstituted 5-Mercapto-1,2,4-triazoles 511
TABLE-1
in vitro ANTIFUNGAL ACTIVITY OF SYNTHESIZED COMPOUNDS
*Inhibition zone diameter (mm)
Structure
A. nigier
C. albicans
5
0 µg
100 µg
50 µg
100 µg
N
N
SH
F
N
O
IV-1
7 ± 0.03
11 ± 0.01
7 ± 0.05
12 ± 0.06
N
N
SH
N
O
IV-2
7 ± 0.04
12 ± 0.03
8 ± 0.02
12 ± 0.04
S O
NH
O
O
N
N
CH3
N
N
SH
N
O
O
S
Cl
O
IV-3
6 ± 0.01
10 ± 0.06
7 ± 0.02
10 ± 0.05
O
S
NH
O
N
HN
N
SH
O
N
HN
O
IV-4
7 ± 0.04
11 ± 0.03
8 ± 0.02
9 ± 0.04
C
N
N
N
SH
N
O
IV-5
IV-6
4 ± 0.01
7 ± 0.06
–
05 ± 0.05
18 ± 0.03
COOH
SH
N
N
N
O
10 ± 0.02
17 ± 0.04
10 ± 0.05
N
N
SH
N
O
C NH
HN
C NH
IV-7
–
4 ± 0.03
6 ± 0.02
9 ± 0.04
N
H C
3
CH3
Fluconazole
DMF
9 ± 0.04
–
14 ± 0.03
–
8 ± 0.01
–
13 ± 0.01
–
–
= denotes no activity

512 Sahoo et al.
Asian J. Chem.
TABLE-2
ACKNOWLEDGEMENTS
PROTEIN LIGAND INTERACTIONS OF
3
,4-DISUBSTITUTED 5-MERCAPTO-1,2,4-TRIAZOLES
The authors express their thanks to Sri Ch Sathi Reddy,
Founder, Sri Gayathri Educational Society, Annaram and Dr.
V. Rama Mohan Gupta, Principal, Pulla Reddy Institute of
Pharmacy, Hyderabad, India for their help and encouragement.
Compound
Dock score
-10.1
-10.3
-10.6
-10.1
-10.9
-11.3
-8.8
IV-1
IV-2
IV-3
IV-4
IV-5
REFERENCES
1
2
3
.
.
.
C. Kus, G. Ayhan-Kilcigil, S. Özbey, F.B. Kaynak, M. Kaya, T. Çoban
and B. Can-Eke, Bioorg. Med. Chem., 16, 4294 (2008).
B. Halliwell, J.M. Gutteridge and C.E. Cross, J. Lab. Clin. Med., 119,
IV-6
IV-7
Fluconazole
-8.0
598 (1992).
L. Yu, S. Haley, J. Perret, M. Harris, J. Wilson and M. Qian, J. Agric.
Food Chem., 50, 1619 (2002).
TABLE-3
DPPH RADICAL SCAVENGING EFFECT AND
INHIBITION OF SYNTHESIZED COMPOUNDS
4. K.A. Metwally, L.M. Abdel-Aziz, E.-S.M. Lashine, M.I. Husseiny and
%
R.H. Badawy, Bioorg. Med. Chem., 14, 8675 (2006).
5
6
7
.
.
.
M.W. Khan, M.J. Alam, M.A. Rashid and R. Chowdhury, Bioorg. Med.
Chem., 13, 4796 (2005).
M. Hogberg, P. Engelhardt, L. Vrang and H. Zhang, Bioorg. Med. Chem.
Lett., 10, 265 (2000).
Y. Dong, T.K. Venkatachalam, R.K. Narla, V.N. Trieu, E.A. Sudbeck
and F.M. Uckun, Bioorg. Med. Chem. Lett., 10, 87 (2000).
Conc.
µg/mL)
Sample
Control
Scavenging
Compounds
(
Absorbance Absorbance effect (%)
3.00
0.2925
0.3663
0.4784
0.2802
0.3963
0.4403
0.2336
0.2746
0.3792
0.2529
0.2620
0.2979
0.2131
0.2204
0.3104
0.1640
0.2221
0.3422
0.0746
0.1979
0.3104
0.0520
0.1692
0.3728
1.1332
1.1332
1.1332
0.5247
0.5247
0.5247
0.5247
0.5247
0.5247
0.5247
0.5247
0.5247
0.6197
0.6197
0.6197
0.6197
0.6197
0.6197
0.6197
0.6197
0.6197
1.1332
1.1332
1.1332
74.18
67.67
50.99
46.59
24.47
16.08
55.47
47.66
27.73
51.80
50.06
43.22
65.61
64.43
49.91
73.53
64.16
44.77
87.96
74.88
49.91
95.41
85.06
67.10
IV-1
1.50
0
3
.75
.00
8. G. Mazzone, F. Bonina, R. Arrigo and G. Blandino, Farmaco Sci., 36,
IV-2
IV-3
IV-4
IV-5
IV-6
IV-7
1.50
181 (1981).
9
1
.
S. Narayanaswami and K. Richardson, European Patent, 96,569 (1983);
Chem. Abstr., 100, 139122t (1984).
0
3
.75
.00
0. M.B. Gravestock, European Patent, 94, 146 (1983); Chem. Abstr., 100,
39118w (1984).
1. E. De Clercq, J. Clin. Virol., 30, 115 (2004).
2. L. Navidpour, H. Shadnia, H. Shafaroodi, M. Amini, A.R. Dehpour
and A. Shafiee, Bioorg. Med. Chem., 15, 1976 (2007).
1.50
1
0
3
.75
.00
1
1
1.50
0
3
.75
.00
13. N.H. Georgopapadakou and T.J. Walsh , Antimicrob. Agents Chemother.,
40, 279 (1996).
14. S. Sahoo, P.K. Patwari, C.B. Mahender Kumar and C.M. Setty, Iranian
J. Pharm. Sci., 9, 51 (2013).
1
1
1.50
0
3
.75
.00
5. M. Koparir, A. Cansiz and A. Demirdag, Molecules, 9, 204 (2004).
6. A. Braca, N. De Tommasi, L. Di Bari, C. Pizza, M. Politi and I. Morelli,
J. Nat. Prod., 64, 892 (2001).
7. O. Trott and A.J. Olson, J. Computational Chem., 31, 455 (2010).
8. National Committee for Clinical Laboratory Standards (NCCLS), Perfor-
mance Standards forAntimicrobial Disk Susceptibility Tests:Approved
Standard M2-A6, National Committee for Clinical Laboratory Standards,
Wayne, PA, edn 6 (1999).
1.50
0
3
.75
.00
1
1
1.50
0.75
3.00
1.50
0.75
Ascorbic
acid