Antileishmanial potential of fused 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiols: Synthesis, biological evaluations and computational studies

Article abstract of DOI:10.1016/j.bmcl.2017.06.053

A series of newer 1,2,4-triazole-3-thiol derivatives 5(a–m) and 6(a–i) containing a triazole fused with pyrazine moiety of pharmacological significance have been synthesized. All the synthesized compounds were screened for their in vitro antileishmanial and antioxidant activities. Compounds 5f (IC₅₀?=?79.0?μM) and 6f (IC₅₀?=?79.0?μM) were shown significant antileishmanial activity when compared with standard sodium stibogluconate (IC₅₀?=?490.0?μM). Compounds 5b (IC₅₀?=?13.96?μM) and 6b (IC₅₀?=?13.96?μM) showed significant antioxidant activity. After performing molecular docking study and analyzing overall binding modes it was found that the synthesized compounds had potential to inhibit L. donovani pteridine reductase 1 enzyme. In silico ADME and metabolic site prediction studies were also held out to set an effective lead candidate for the future antileishmanial and antibacterial drug discovery initiatives.

Full text of DOI:10.1016/j.bmcl.2017.06.053

Accepted Manuscript
Antileishmanial potential of fused 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiols:
Synthesis, biological evaluations and computational studies
Sanjeev R. Patil, Ashish Asrondkar, Vrushali Patil, Jaiprakash N. Sangshetti,
Firoz A. Kalam Khan, Manoj G. Damale, Rajendra H. Patil, Anil S. Bobade,
Devanand B. Shinde
PII:
S0960-894X(17)30655-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.053
BMCL 25087
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 March 2017
6 April 2017
21 June 2017
Please cite this article as: Patil, S.R., Asrondkar, A., Patil, V., Sangshetti, J.N., Kalam Khan, F.A., Damale, M.G.,
Patil, R.H., Bobade, A.S., Shinde, D.B., Antileishmanial potential of fused 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-
thiols: Synthesis, biological evaluations and computational studies, Bioorganic & Medicinal Chemistry Letters
(2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.053
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Antileishmanial potential of fused 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiols:
Synthesis, biological evaluations and computational studies
Sanjeev R. Patila,b, Ashish Asrondkarb, Vrushali Patilb, Jaiprakash N. Sangshettic, Firoz A.
Kalam Khanc, Manoj G. Damaled, Rajendra H. Patile, Anil S.Bobadeb, Devanand B. Shindef*
aDepartment of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad 431 004, MS, India
bHaffkine Institute for Training, Research and Testing, Parel, Mumbai-400 012, MS, India
cY.B. Chavan College of Pharmacy, Aurangabad 431 001, MS, India.
d Shri Bhagwan College of Pharmacy, Aurangabad 431 003, MS, India.
eDepartment of Biotechnology, Savitribai Phule Pune University, Pune MS India
f Shivaji University, Vidyanagar, Kolhapur-416004, MS, India
*Corresponding author E-mail: dbssanjeev2015@rediffmail.com
Tel No.: 91-231-2609060 , Fax No.: 91-231-2692333
A series of newer 1,2,4-triazole-3-thiol derivatives 5(a-m) and 6(a-i) containing a triazole fused
with pyrazine moiety of pharmacological significance have been synthesized. All the synthesized
compounds were screened for their in vitro antileishmanial and antioxidant activities.
Compounds 5f (IC50= 79.0 µM) and 6f (IC50= 79.0 µM) were shown significant antileishmanial
activity when compared with standard sodium stilbogluconate (IC50= 490.0 µM). Compounds 5b
(IC50= 13.96 µM) and 6b (IC50= 13.96 µM) showed significant antioxidant activity. After
performing molecular docking study and analyzing overall binding modes it was found that the
synthesized compounds had potential to inhibit L. donovani pteridine reductase 1 enzyme. In
silico ADME and metabolic site prediction studies were also held out to set an effective lead
candidate for the future antileishmanial and antibacterial drug discovery initiatives.
Keywords: Pyrazine; 1,2,4-Triazole; Antioxidant activity; Antileishmanial activity; Cytotoxicity
activity; Molecular docking, ADMET analysis.
1

Leishmaniasis is a group of diseases caused by infection with intracellular species of the parasitic
protozoan of the genus Leishmania with different clinical forms ranging from cutaneous
leishmaniasis (CL) with skin lesions to visceral leishmaniasis (VL) with enlargement of liver,
spleen, and bone marrow dysfunctions. According to the World Health Organization,
leishmaniasis is an uncontrolled tropical disease with high morbidity and mortality rates in
africa, asia, and the america.1 The drugs currently in use are expensive, require long term
treatment,2 display high liver and heart toxicities, develop clinical resistance after few weeks of
treatment and currently contribute to increase leishmaniasis-AIDS co-infections in some
countries3 and hence there is a need for new antileishmanials with improved efficacy and less
side effects. Oxidative and free-radical-mediated processes reactions seem to play an important
role in the progression of various diseases, including cancer and coronary heart disease. Radical
damage has also been implicated in neurodegeneration occurring both through normal aging
processes and in diseases such as Alzheimer's disease.4 Thus, there is also a need for
development of safe and effective antioxidants to prevent radical-mediated damage.
The therapeutic effects of 1,2,4-triazole containing compounds have been well studied for
a number of pathological conditions including inflammation, cancer, pain, tuberculosis or
hypertension. Moreover, synthesis of triazoles fused to another heterocyclic ring has attracted
wide spread attention due to their diverse applications as antimicrobial,5 molluscicidal agents,6
antidepressant,7 pesticides, herbicides,8 antiviral,9 antitumorial,10 analgesic,11 anti-inflammatory
agents.12 Among these, the commonly known systems are generally triazoles fused to
thiadiazines, pyridines, pyridazines, pyrimidines, pyrazines and triazines. Although there are not
many triazoles fused to thiadiazines, a number of them are incorporated into a wide variety of
therapeutically important compounds possessing a broad spectrum of biological activities such as
anticancer,13 antibacterial,14 anti-fungal,15 anti-inflammatory,16 diuretic,17 analgesic,18
hypoglycemic19 agents. Schiff bases are important organic compounds and well known
intermediate for the preparation of azetidinone, thiazolidinone, formazone, aryl acetamide and
many other derivatives. These are the compounds contain characteristic CH=N group. They are
used as pigments and dyes, catalysts, intermediates in organic synthesis and as polymer
stabilisers.20 Schiff bases have also been shown to exhibit a broad range of biological activities.
In view of these marked activities of fused [1,2,4] triazole [3,4-b] [1,3,4] thiadiazine
derivatives21 and in continuation of our previously reported studies on heterocyclic derivatives
2

of pyrazinamide22, it was contemplated to synthesize the title compounds and evaluate their
biological potency.
As part of our efforts on the development of a new route for the preparation of
biologically active molecules 23 and, considering the importance of biological properties of schiff
base herein, we described an efficient and simple method for the synthesis of 1,2,4-triazole-3-
thiol derivatives 5(a-m) and 6(a-i) containing triazole fused with pyrazine moiety (Scheme 1).
Scheme 1. Synthesis of titled compounds 5(a-m) and 6(a-i)
In the present study, a series of schiff bases of 1,2,4-triazole 3-thiol possessing pyrazine
nucleus 5(a-m) have been synthesized by the reaction of 4-amino-5-(pyrazin-2-yl)-4H-1,2,4-
triazole-3-thiol and substituted benzaldehydes.24 The condensation between pyrazinamide (1)
and hydrazine hydrate gave pyrazine-2-carbohydrazide (2) which on further cyclization with
carbon disulfide in presence of base potasium hydroxide in ethanol as a solvent gave
intermediate 5-(pyrazin-2-yl)-1,3,4-oxadiazole-2-thiol (3). The intermediate (3) was refluxed
with hydrazine hydrate in ethanol produced key intermediate 4-amino-5-(pyrazin-2-yl)-4H-1,2,4-
triazole-3-thiol (4) which on further reaction with substituted aromatic aldehydes and
3

isothiocyanates and resulted in 4-(benzylideneamino)-5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiols
5(a-m) and 1-(3-mercapto-5-(pyrazin-2-yl)-4H-1,2,4-triazol-4-yl)-3-phenylthiourea 6(a-i),
respectively. The structures of all newely synthesized compounds were established by 1H NMR,
13C NMR, FT-IR and mass spectroscopy (Supporting information). The mass spectrum of 5f
displayed the molecular ion peak (M+) peak at m/z 299, which was consistent with the product
structure. The 1H NMR spectrum of 5f exhibited singlet of proton at δ 14.30 which indicates the
presence of -SH group. Another singlet of one proton at δ 10.34 confirmed the formation of
Schiff base (-N=C-H) proton. The peak observed at δ= 9.43 ppm confirmed the presence of the
13
hydroxyl group. In the C NMR spectrum of compound 5f, characteristic peaks for C-SH and
N=C-H were observed at δ 162.35 and 163.60 ppm, respectively. C-OH peak at δ 166.48 ppm
indicated the presence of the hydroxyl group. The IR spectrum of 5f exhibited characteristic
absorption band at 1575.91 cm-1 for (C=N linkage) and all other general frequency bands were in
good agreement with the structure. The physicochemical properties are as shown in Table 1. The
isolated yields of synthesized compounds were in the range of 85.0–92.0%. Melting points were
determined in open capillary tubes and are uncorrected.
Table 1. The physicochemical properties of compounds 5(a-m) and 6(a-i)
Entry
R1
R2
Mol. Formula
Mol.
Wt.
Melting
range
Rf
value
Yield
(%)
(°C)
p-OC2H5
p-(N(CH)3)2
C4H4
m-OCH3 C16H16N6O2S
356.4
207-210
0.62
0.74
0.59
0.64
0.44
0.49
0.68
0.56
0.65
0.73
0.59
86.5
87.0
91.0
90.5
86.0
85.0
89.5
87.5
89.0
88.0
90.0
5a
5b
5c
5d
5e
5f
H
o-OH
H
C15H15N7S
C17H12N6S
325.39 192-194
332.38 220-222
298.32 189-192
314.32 266-269
298.32 205-208
300.32 178-180
307.33 189-192
327.32 180-183
351.21 266-269
310.38 179-181
o-OH
C13H10N6OS
C13H10N6O2S
C13H10N6OS
C13H9FN6S
C14H9N7S
o-OH
p-OH
H
p-OH
p-F
H
5g
5h
5i
p-CN
H
m-NO2
o-Cl
H
C13H9N7O2S
C13H8Cl2N6S
C14H15N6S
p-Cl
H
5j
p-C2H5
5k
4

CHO
p-Cl
H
H
H
C13H10N6S
C13H9ClN6S
C13H11N7S
282.32 280-282
316.77 224-227
0.64
0.59
0.42
0.52
0.82
0.74
0.62
0.54
0.64
0.68
0.52
92.0
90.5
86.5
87.0
91.0
90.5
86.0
85.0
89.5
87.5
89.0
5l
5m
6a
6b
6c
6d
6e
6f
H
329.4
186-188
m-F
p-F
H
C13H10FN7S2
C13H10FN7OS
C17H13N7S2
347.39 173-176
331.33 228-232
379.46 181-183
363.85 186-189
298.32 238-242
365.38 221-223
397.40 198-201
H
C4H4
p-Cl
o-Cl
o-F
H
H
C13H10ClN7S2
C13H9Cl2N7S2
C13H9F2N7S2
C14H10F3N7S2
p-Cl
p-F
H
6g
6h
6i
p-CF3
m-Cl
p-Cl
C13H9Cl2N7OS 382.23 222-226
Solvent of re-crystallization was ethanol; Eluents used in TLC were Ethyl acetate: n-hexane (8:2)
for all compounds.
All the synthesized compounds 5(a-m) and 6(a-i) were tested for their in vitro
antileishmanial activity against
a
culture of Leishmania donovani promastigotes
(NHOM/IN/80/DD8) using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay and sodium stibogluconate was used as standard drug.25 Compounds 5(a-
m) and 6(a-i) showed varying degrees of antileishmanial activities with IC50 ranging between
79.0 to 382.4 µM (Table 2). Compounds 5f, 6f and 5l were found to be most promising
compounds exhibiting IC50 value of 79.0, 79.0 and 95.2 µM, respectively when compared with
sodium stilbogluconate. All the synthesized compounds showed better activity than standard
sodium stibogluconate (IC50 = 490.0 µM) against L. donovani promastigotes. Structure activity
relationship revealed that the activity mainly depends upon the presence of substituent on phenyl
ring. In the first series 5(a-m), in the compound 5f (IC50= 79.0 µM), introduction of the hydroxyl
group at the para position of phenyl gives most active compound, however, fluoro group at para
position in compound 5g gives least active compound with IC50 value 382.4 µM. From the
series 6(a-i), the compound 6f having the chloro substitution at ortho and para position exhibited
excellent activity with IC50 value 79.0 µM, however, in compound 6e chloro group at para
position of phenyl ring displayed the least activity among all the synthesized compounds. All
5

other compounds from the series displays good to moderate antileishmanial activity compared to
the standard.
Cytotoxic study was performed on HeLa cell line and evaluated by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.26 The cells were seen
under the microscope (Ziess, Germany) at 10x magnification. The cytotoxic study against HeLa
cell line of compound 5f was carried out at concentration of 500 µg/mL. The compound 5f had
shown no cytotoxicity effect against HeLa cell lines to its tested concentration (Figure 1).
Table 2. Antileishmanial and antioxidant activity of compounds 5(a-m) and 6(a-i)
Entry
Entry
Antileishmanial Antioxidant
Antileishmanial Antioxidant
IC50 µM
IC50 µM
IC50 µM
IC50 µM
190.0
113.0
178.0
102.0
106.4
79.0
58.80
13.96
69.18
75.86
75.86
58.90
58.80
47.80
72.40
81.20
47.70
-
95.2
198.2
*
52.40
39.80
58.80
13.96
69.18
75.86
75.86
58.90
58.90
58.80
81.20
-
5a
5b
5c
5d
5e
5f
5l
5m
6a
6b
6c
6d
6e
6f
6g
6h
6i
287.3
322.1
336.3
*
382.4
99.0
5g
5h
5i
79.0
176.3
288.9
229.1
490.0
-
347.1
241.3
201.1
490.0
-
5j
5k
SS
AA
-
14.0
-
14.0
* No activity upto 500 µM; IC50 represents the mean values of three replicates; standard
errors were all within 10% of the mean; SS: Sodium stibogluconate; AA: Ascorbic acid.
6

Compound 5f
Control
Figure 1. Cytotoxic study on HeLa cell line of compound 5f.
All the compounds 5(a-m) and 6(a-i) were evaluated for antioxidant activity using free
radical scavenging activity DPPH (1,1-diphenyl-2-picryl-hydrazil) method27 and ascorbic acid
was used as standard (Table 2). All synthesized compounds has shown high to moderate
percentage of inhibition as compared to ascorbic acid. From the series 5(a-m), compound 5b
N(CH3)2 group at para position with inhibition concentration (IC50) 13.96 µM found to
equipotent with respect to standard ascorbic acid (IC50= 14.0 µM). Compounds 5m chloro
group at para position, 5k ethyl group at para position and 5h cyano group at para position with
IC50 39.8, 47.7 and 47.8 µM, respectively, showed comparatively significant antioxidant activity
with respect to standard ascorbic acid. The remaining compounds from the synthesized series
5(a-m) showed less activity compared to the standard. Similarly, from the series 6(a-i),
compound 6b having fluoro group at meta position of phenyl ring with EC50 13.96 µM was
found to most active compound compared with standard drug ascorbic acid. Remaining all the
compounds from this series was found to be less active.
Molecular docking study of synthesized compounds 5(a-m) and 6(a-i) were performed to
understand binding interactions with using Pteridine reductase 1 (PTR1) enzyme of L. donovani
(PDB ID: 2XOX)30 was performed Surflex-Dock module of Sybyl 2.1.1 package following
standard procedure. Pyrazine derivatives have been reported to act as antileishmanial by
inhibiting Pteridine reductase 1 enzyme.29 PTR1 is an essential enzyme for pterin salvage in the
Leishmania parasite, and can potentially be used as a target in the development of improved
therapies. PTR1 is a homotetramer with a subunit molecular mass of approximately 30 kDa.
PTR1 is able to catalyze the two-step reduction of folate to H2 folate and subsequently to H4
7

folate. This ability to reduce folate is the same reaction catalyzed by the dihydrofolate reductase
(DHFR) component of the DHFR-thymidylate synthase bifunctional polypeptide (DHFR-TS)
which is the presumptive primary cell target for antiprotozoal activity. 30-33
The synthesized compounds had shown good binding affinity as predicated by non
covalent interactions such as hydrogen bond interaction, van der Waal’s interaction, Pi-anion
interaction, Pi-Pi shaped interaction, and alkyl interaction. To represent the details of docking
score, terms like total score (total docking score), crash score (degree of inappropriate
penetration by the ligand into the protein and of interpenetration between ligand atoms that are
separated by rotatable bonds of compounds) and polar score gives (the contribution of the polar
non-hydrogen bonding interactions to the total score) are used and presented in Table 3. The
compounds 5(a-m) had shown shown overall very efficient binding mode and penetrating active
site cavity by forming the various interactions with active site residues such as GLU29, ASP259,
VAL260, ILE262, PHE263, CYS265, SER266, SER267, LYS268. In case of most active
compound 5f, active site residue ASP259 had formed the hydrogen bond interactions with OH
and SH The most active compound 5f had shown good docking score that is 4.31. groups of aryl
and triazole ring with a distance of 1.60 to 1.75Å respectively, whereas SER266 had formed the
hydrogen bond interactions with heterocyclic pyrazine moiety with a distance of 1.65Å (Figure
2). The synthesized series 6(a-i) had also shown good docking intections with Pteridine
reductase 1 enzyme. The most active compound 6f had shown good docking score that is 4.29.
The compounds 6(a-i) were penetrated into active site cavity by forming the various interactions
with active site residues such as GLU29, ASP259, ILE262, PHE263, SER266, SER267, and
LYS268. In case of most active compound 6f, amino acid residue ASP259 had formed the
hydrogen bond interactions with SH groups of triazole ring with a distance of 1.60 Å. The aryl
and pyrazine ring system had shown strong non covalent interactions such as Pi-Sigma, Pi-Pi-T-
Shaped and amide Pi-stacked interactions with active site amino acids such as ILE262 and
PHE263 (Figure 2). On the basis of activity data and molecular docking analysis, it was found
that synthesized compounds had potential to inhibit pteridine reductase 1 enzyme.
8

Table 3. Molecular docking stastics of synthesized compounds 5(a-m) and 6(a-i)
Entry
Total Score Crash
(Logki)
Polar
Entry
Total Score
(Logki)
4.11
Crash
Polar
3.12
3.90
3.95
3.83
3.19
4.31
3.85
3.83
3.62
4.05
3.84
-0.7790
-1.1928
-0.7190
-0.7051
-0.9636
-1.3329
-0.7980
-0.5621
-0.5467
-0.6038
-0.6622
3.19
2.60
2.65
2.70
3.35
2.97
1.94
2.27
3.41
3.27
1.84
-4.2131 2.25
-4.0137 2.94
-2.6578 2.31
-2.8695 2.10
-4.5349 4.21
-2.7988 4.23
-2.8185 3.85
-2.1991 2.19
-3.4021 2.30
-2.5470 1.74
-3.4660 2.99
5a
5b
5c
5d
5e
5f
5l
5m
6a
6b
6c
6d
6e
6f
3.01
2.65
2.86
3.53
2.79
2.81
5g
5h
5i
4.29
3.40
6g
6h
6i
2.54
5j
3.46
5k
Total score: Total docking score; Crash: Degree of inappropriate penetration by the ligand into
the protein and of interpenetration between ligand atoms that are separated by rotatable bonds;
Polar: Contribution of the polar non-hydrogen bonding interactions to the total score.
5f
6f
Figure 2: Binding modes of most active compounds 5f and 6f in the putative active site of PRT1
enzyme.
9

Early prediction of drug like properties of lead compounds is an important task as it
decides the time and cost of drug discovery and development. The many of the active agents who
has shown significant biological activity fails in clinical trials because of inadequate drug like
properties. 34 The drug like properties has been predicted by analyzing absorption, distribution,
metabolism, and elimination (ADME) parameters based on Lipinski’s rule of five and its
variants. 35,36 We had calculated and analyzed various physical descriptors and pharmaceutical
relevant properties for ADMET prediction by using FAFDrugs2 and data is summarized in
Table 4. All the compounds showed significant values for the various parameters analyzed and
showed good drug-like characteristics based on Lipinski’s rule of five and its variants. None of
the synthesized compounds 5(a-m) and 6(a-i) had violated the Lipinski’s rule of five its variants.
The value of polar surface area (PSA), log P and H/C ratio of synthesized compounds had
indicated for good oral bioavailability. The parameters, like number of rotatable bonds and
number of rigid bonds are linked with the intestinal absorption result and showed that all
synthesized compounds 5(a–m) and 6(a-i) had good absorption. Also, all the synthesized
compounds was found to be nontoxic.
Table 4: Pharmacokinetic parameters important for agents to have good oral bioavailability of
synthesized compounds 5(a-m) and 6(a-i).
Entry
MW
Log P
PSA
n-Rot
Bond
6
n-Rig
Bond
18
HBD HBA Rings
Ratio
H/C
0.56
Toxicity
356.40 2.31
325.39 1.97
100.10
84.89
1
1
8
6
3
3
NT
NT
5a
5b
4
3
3
3
3
3
3
4
3
4
18
23
18
18
18
18
19
19
18
18
0.53
0.47
0.61
0.69
0.61
0.61
0.57
0.76
0.69
0.46
348.38 2.76
298.32 1.61
314.32 1.31
298.32 1.61
300.31 2.04
307.33 1.77
327.32 2.33
351.21 3.21
310.37 2.46
101.90
101.90
122.10
101.90
81.65
2
2
3
2
1
1
1
1
1
7
7
8
7
6
6
8
6
6
3
3
3
3
3
3
3
3
3
NT
NT
NT
NT
NT
NT
NT
NT
NT
5c
5d
5e
5f
5g
5h
5i
105.40
121.60
81.65
5j
81.65
5k
10

310.33 1.71
316.76 2.55
329.40 2.11
98.72
81.65
4
3
5
19
18
18
1
1
3
7
6
6
3
3
3
0.57
0.61
0.69
NT
NT
NT
5l
5m
6a
125.44
347.39 2.24
331.32 2.08
379.46 3.26
363.84 2.76
398.29 3.41
365.38 2.38
397.40 3.12
382.22 3.25
125.44
110.42
125.44
125.44
125.44
125.44
125.44
110.42
5
3
5
5
5
5
6
3
18
20
23
18
18
18
18
20
3
3
3
3
3
3
3
3
6
6
6
6
6
6
6
6
3
3
3
3
3
3
3
3
0.76
0.76
0.52
0.76
0.84
0.84
0.85
0.84
NT
NT
NT
NT
NT
NT
NT
NT
6b
6c
6d
6e
6f
6g
6h
6i
MW: molecular weight; Log P: logarithm of partition coefficient of compound between n-
octanol and water; PSA: Polar surface area; n-Rot Bond: number of rotatable bonds; n-Rig Bond:
number of rigid bonds; HBA: hydrogen bond acceptors; HBD: hydrogen bond donor; and NT:
Non toxic.
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generated through processing of the transformations found in the Symyx Metabolite database
previously known as MDL, for each transformation, the differences between the structures of the
reactant. In order to make predictions of on the drug molecule the each atom environment is
calculated and Symyx Metabolite database mining is done to search similar environment. As
based on the interest of the study, only Phase I additions (defined as the addition of a single
oxygen atom; covering hydroxylation, oxidation and epoxidation), and eliminations (e.g.,
39
dealkylation, ester and amide hydrolysis) are engaged . For an addition, the atom neighboring
the added oxygen highlighted as a reaction center. One of the most active compound 5f (Figure
11

3) was analyzed for metabolic product prediction through an online web server of MetaPrint2D-
React for prediction of possible metabolic pathways and the predicted site of metabolism
prediction done for Human only. The plot of MetaPrint2D shown possible Site of metabolism of
individual atom neither indicated by NOR values (Normalized Occurrence Ratio). The higher the
NOR value indicates site frequently undergoes through particular metabolism phase I metabolic
reactions. 40, 41 The NOR ratio for 5f was observed as Red 0.66 ≤ NOR ≤ 1.00, Orange 0.33 ≤
NOR < 0.66, Green 0.15≤ NOR < 0.33, White (No color) 0.00 ≤ NOR < 0.15, and Grey Little/no
data. Figure 3 indicates that most of the atoms in skeleton of compound 5f are less prone to
metabolic deactivation marked by grey color.
Figure 3: Metabolic/Biotransformation pathways for 5f predicted by MetaPrint-2D React server.
In Summary, we have carried out the efficient synthesis of 5-(pyrazin-2-yl)-4H-1,2,4-
triazole-3-thiols 5(a-m) and 6(a-i). All the synthesized compounds have been investigated for
their in vitro antileishmanial and antioxidant activities. Among them compound 5f and 6f with
IC50 = 79.0 µM exhibited excellent antileishmanial activity, whereas compound 5b and 6b with
IC50 = 13.96 µM displayed excellent antioxidant activity. Compound 5f showed no cytotoxicity
to HeLa cell lines upto its highest tested concentrations 500 µg/mL. A molecular docking study
suggested good binding interactions of these compounds with Pteridine reductase 1 enzyme of
L. donovani. In silico ADME and metabolic sites prediction studies of synthesized compounds
indicated that compounds had potential to develop as good oral drug like candidate. These data
12

suggest that 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiols chemical scaffold can be used as a
template for further structure optimization for generating compounds with higher antileishmanial
and antioxidant activity.
Acknowledgements
The authors are grateful to the Dept. Of Chemical technology, Aurangabad And Haffkine
Institute, Mumbai, India for providing laboratory facilities. The authors are also thankful to the
Department of Biotechnology, Savitribai Phule Pune University, Pune and Dr. Rafiq Zakaria
Campus, Y.B. Chavan College of Pharmacy, Aurangabad, India for providing biological
activities.
13

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Captions
Scheme 1. Synthesis of titled compounds 5(a-m) and 6(a-i)
Table 1. The physicochemical properties of compounds 5(a-m) and 6(a-i)
Table 2. Antileishmanial and antioxidant activity of compounds 5(a-m) and 6(a-i)
Table 3. Molecular docking stastics of synthesized compounds 5(a-m) and 6(a-i)
Table 4. Pharmacokinetic parameters important for agents to have good oral bioavailability of
synthesized compounds 5(a-m) and 6(a-i)
Figure 1. Cytotoxic study on HeLa cell line of compound 5f
Figure 2. Binding modes of most active compounds 5f and 6f in the putative active site of PRT1
enzyme.
Figure 3. Metabolic/Biotransformation pathways for 5f predicted by MetaPrint-2D React server
17

Graphical Abstract
Antileishmanial potential of fused 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiols:
Synthesis, biological evaluations and computational studies
Sanjeev R. Patila,b, Ashish Asrondkarb, Vrushali Patilb, Jaiprakash N. Sangshettic, Firoz A.
Kalam Khanc, Manoj G. Damaled, Rajendra H. Patile, Anil S.Bobadeb, Devanand B. Shindef*
aDepartment of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad 431 004, MS, India
bHaffkine Institute for Training, Research and Testing, Parel, Mumbai-400 012, MS, India
cY.B. Chavan College of Pharmacy, Aurangabad 431 001, MS, India.
d Shri Bhagwan College of Pharmacy, Aurangabad 431 003, MS, India.
eDepartment of Biotechnology, Savitribai Phule Pune University, Pune MS India
f Shivaji University, Vidyanagar, Kolhapur-416004, MS, India
*Corresponding author E-mail: dbssanjeev2015@rediffmail.com
Tel No.: 91-231-2609060, Fax No.: 91-231-2692333
18