The bioisosteric modification of pyrazinamide derivatives led to potent antitubercular agents: Synthesis via click approach and molecular docking of pyrazine-1,2,3-triazoles

Article abstract of DOI:10.1016/j.bmcl.2019.126846

Tuberculosis remains as a major public health risk which causes the highest mortality rate globally and an improved regimen is required to treat the drug-resistant strains. Pyrazinamide is a first-line antitubercular drug used in combination therapy with other anti-TB drugs. Herein, we describe the modification of pyrazinamide structure using bioisosterism and rational approaches by incorporating the 1,2,3-triazole moiety. Three sets of pyrazine-1,2,3-triazoles (3a-o, 5a-o and 9a-l) are designed, synthesized and evaluated for their in vitro inhibitory potency against mycobacterium tuberculosis H₃₇Rv. The pyrazine-1,2,3-triazoles synthesized through the bioisosteric modification displayed improved activity as compared to rationally modified pyrazine-1,2,3-triazoles. Among 42 title compounds, seven derivatives demonstrated significant anti-tubercular activity with the MIC of 1.56 μg/mL, which are two-fold more potent than the parent compound pyrazinamide. Further, the synthesized pyrazinamide analogs demonstrated moderate inhibition activity against several bacterial strains and possessed an acceptable in vitro cytotoxicity profile as well. Additionally, the activity profile of pyrazine-1,2,3-triazoles was validated by performing the molecular docking studies against the Inh A enzyme. Furthermore, in silico ADME prediction revealed good oral bioavailability for the potent molecules.

Full text of DOI:10.1016/j.bmcl.2019.126846

Journal Pre-proofs
The bioisosteric modification of pyrazinamide derivatives led to potent antitu-
bercular agents: Synthesis via click approach and molecular docking of pyra-
zine-1,2,3-triazoles
Rajkumar Reddyrajula, Udayakumar Dalimba
PII:
S0960-894X(19)30820-0
DOI:
https://doi.org/10.1016/j.bmcl.2019.126846
BMCL 126846
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
1 September 2019
14 November 2019
18 November 2019
Please cite this article as: Reddyrajula, R., Dalimba, U., The bioisosteric modification of pyrazinamide derivatives
led to potent antitubercular agents: Synthesis via click approach and molecular docking of pyrazine-1,2,3-triazoles,
Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126846
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The bioisosteric modification of pyrazinamide derivatives led to
potent antitubercular agents: Synthesis via click approach and
molecular docking of pyrazine-1,2,3-triazoles
Rajkumar Reddyrajula a, Udayakumar Dalimba a*
aOrganic Chemistry Laboratory, Department of Chemistry, National Institute of Technology
Karnataka, Surathkal, Srinivasanagar, Mangalore-575025, India.
*Corresponding author email: udayaravi80@gmail.com, udayakumar@nitk.ac.in
Abstract
Tuberculosis remains as a major public health risk which causes the highest mortality
rate globally and an improved regimen is required to treat the drug-resistant strains.
Pyrazinamide is a first-line antitubercular drug used in combination therapy with other anti-TB
drugs. Herein, we describe the modification of pyrazinamide structure using bioisosterism and
rational approaches by incorporating the 1,2,3-triazole moiety. Three sets of pyrazine-1,2,3-
triazoles (3a-o, 5a-o and 9a-l) are designed, synthesized and evaluated for their in vitro
inhibitory potency against mycobacterium tuberculosis H37Rv. The pyrazine-1,2,3-triazoles
synthesized through the bioisosteric modification displayed improved activity as compared to
rationally modified pyrazine-1,2,3-triazoles. Among 42 title compounds, seven derivatives
demonstrated significant anti-tubercular activity with the MIC of 1.56 μg/mL, which are two-
fold more potent than the parent compound pyrazinamide. Further, the synthesized
pyrazinamide analogs demonstrated moderate inhibition activity against several bacterial
strains and possessed an acceptable in vitro cytotoxicity profile as well. Additionally, the
activity profile of pyrazine-1,2,3-triazoles was validated by performing the molecular docking
studies against the Inh A enzyme. Furthermore, in silico ADME prediction revealed good oral
bioavailability for the potent molecules.
Key words: Pyrazinamide, Bioisosterism, 1,2,3-triazole, Mycobacterium tuberculosis,
molecular docking.

Tuberculosis (TB), a chronic bacterial disease, caused by Mycobacterium tuberculosis
(Mtb). TB affects the respiratory site (pulmonary TB), but it can also affect variant sites of the
body (extrapulmonary TB) including lymphatic system, central nervous system, and
circulatory system. In 2017, around two million people died due to TB, which includes half
million cases co-infected with HIV.1 In the last few decades there is a gradual increment in
tuberculosis cases due to development of MDR-TB (multidrug-resistant) and XDR-TB
(extensively drug-resistant).2,3 Treatment for this MDR and XDR-TB is more expensive, less
effective, much longer, and causes several side effects. Recently, Bedaquiline, a diaryl
quinoline-based molecule4,5 (approved by the US-FDA) and Delamanid,6 an imidazole analoge
(approved by the European Medicines Agency) are employed to treat pulmonary MDR-TB in
adults but the treatment resulted adverse side effects. The molecules which are under clinical
trials at present are Pretomanid,7 a nitroimidazole derivative, Diamine analogue,8 and Salicylic
acid derivative (I-A09). However, toxic side-effects of bedaquiline led to significant death rates
in clinical testing.9 Thus, there is an emergent need to develop more effective drug regimens
with minimum side effects and shorter duration of treatment. The most promising molecular
design strategies to develop effective drugs are (i) Structural or bioisosteric modification of the
established drug molecule;10 (ii) Molecular hybridization of two or more potent
pharmacophores;11,12 iii) In silico drug design based on protein-ligand interactions;13 and (iv)
the rational screening of various molecular units.14,15 Pyrazinamide is a first-line antitubercular
drug, which is often used in combination with either isoniazid, rifampicin and ethambutol or
streptomycin. It is a prodrug, which will be converted into the pyrazinoic acid in presence of
pyrazinamidase enzyme under acidic conditions (pH: 5-6). Pyrazinoic acid inhibits the FAS-I
(fatty acid synthase enzyme), which plays an important role in fatty acid synthesis.16 Recent
literature reports demonstrate the promising antitubercular profile of several modified
pyrazinamide derivatives (Figure 1). For example, N-benzylpyrazine-2-carboxamide
alkylamino derivatives (I) exhibit superior inhibition activity against Mtb when compared with
pyrazinamide (MIC range 4.6-10 μM).17 A series of pyrazinyl ferrocenyl-derived complexes
(II) are reported as moderate antitubercular agents in two different growth media GAST-Fe and
7H9-ADC.18 Further, Zitko et al. modified the pyrazinamide structure by incorporating the 4-
arylthiazol-2-amine scaffold and among these 6-chloro-N-(4-(4-fluorophenyl)thiazol-2-
yl)pyrazine-2-carboxamide (III) showed significant anti-TB activity with MIC of 0.78
μg/mL.19 Similarly, a new set of N-substituted 5-chloropyrazine-2-carboxamides (IV)
demonstrated excellent inhibition activity against Mtb H37Rv, Mycobacterium kansasii and
two strains of Mycobacterium avium.20 These reports on promising antitubercular activity of

PZA derivatives motivated us to modify the PZA structure by incorporating the active
pharmacophoric 1,2,3-triazole unit with the aim of enhancing the antitubercular activity.
R
O
O
O
S
N
N
N
N
N
N
N
Cl
N
H
R
N
H
N
H
N
N
H
Fe
III
II
F
I
O
N
O
N
HN
N
N
N
H
n
N
R
N
O
N
Cl
N
N
N
IV
N
N
O
O
N
VI
OH
OH
O
N
N
N
Cl
V
OAc
OAc
AcO
O
O
Ar
O
O
R
N
O
N
S
N
N
VIII
N
OAc
N
VII
R3
H
S
N
N
N
N
N
O
O
R1
N
O
O
HO
OH
HO
N
N
R
N
R1
O
IX
X
O
R2
N
N
N
H
N
N
Cl
R
XI
Figure 1. Reported pyrazinamide and 1,2,3-triazole derivatives with antitubercular activity.
The 1,2,3-triazole moiety is a key scaffold and it finds enormous significance in the
drug discovery due to various pharmacological applications of triazole derivatives. It can
actively participate in hydrogen bonding and dipole-dipole interactions through two ring
nitrogens, N2 and N3 and also possesses a large dipole moment (5D).21 In fact, 1,2,3-triazole
is an integral core structural moiety in several marketed drugs like cefatrizine, tazobactam,
carboxyamidotriazole (CAI) and TSAO etc. A few recent letters demonstrated the promising
antitubercular activity of 1,2,3-triazole derivatives.22,23 For instance, dibenzo[b,d]thiophene
based 1,2,3-triazole derivatives exhibited promising inhibitory activity against against Mtb
H37Rv.24 A series of N-phenyl-1,2,3-triazole derivatives exhibit excellent antitubercular
activity with the MIC values ranging from 2.5 to 0.62 μg/mL.25 Similarly, biphenyl

incorporated 1H-1,2,3-triazoles displayed good inhibitory potency against Mtb H37Rv strain.26
Recently, benzofuran salicylic acid-1,2,3-triazole derivative I-A09 (V) has been found to
possess good antitubercular activity, which is presently under clinical trials.27 Compounds VI
– IX are the other 1,2,3-triazole derivatives recently reported for their antitubercular activity27.
In our previous work, we demonstrated the excellent inhibitory potency of two set of 1,2,3-
triazole derivatives against Mtb H37Rv strain: phenothiazine incorporated 1,2,3-triazoles (X)28
and quinoline-1,2,3-triazole derivatives (XI)29, most of the compounds showed the MIC of 1.56
μg/mL. Thus, the promising antitubercular activity of 1,2,3-triazoles motivated us to modify
the pyrazinamide by incorporating the 1,2,3-triazole ring to enhance the antimycobacterial
activity. Accordingly, we have designed and synthesized a series of novel pyrazine-1,2,3-
triazole derivatives bearing an ester linker. (Figure 2).
O
N
NH2
N
Pyrazinamide (1)
Firstline Anti-TB Drug
Incorporation of
1,2,3-Triazole
O
X
O
R
N
N
N
Y
3a-o
Rational
Modification
Bioisosteric
Modification
R
N
N
N
O
S
N
N
X
X
N
H
O
R
N
N
N
N
Y
9a-l
5a-o
Figure 2. Design strategy for new pyrazine-1,2,3-triazoles (3a-o, 5a-o and 9a-l).
Next, we attempted the bioisosteric and rational modification of the ester linker with an amide
and 1,3,4-oxadiazole-2-thiol linkers respectively and explored the effects of these structural
modifications on the inhibitory activity against Mtb H37Rv strain. Bioisosteric modification or
bioisosterism is one of the important methodologies in drug design and it involves the
replacement of a functional group by a new functionality with similar chemical or physical

properties. It is generally employed to enhance the bioactivity, reduce the toxicity and improve
the bioavailability of the parent compound30. In this direction, we synthesized three sets of
pyrazine-1,2,3-triazole derivatives (3a-o, 5a-o and 9a-l) and screened these molecules for their
antitubercular activity.
O
O
Y
X
Y
X
a
b
O
O
R
N
N
N
O
2a
2b
2c
: X = N, Y = N;
: X = N, Y = CH;
: X = CH, Y = CH;
3a-o
Y
X
OH
O
O
1a
1b
1c
: X = N, Y = N;
: X = N, Y = CH;
: X = CH, Y = CH;
Y
X
Y
N
H
c
b
N
H
R
N
N
N
X
4a
4b
4c
: X = N, Y = N;
: X = N, Y = CH;
: X = CH, Y = CH;
5a-o
3a
3b
3c
3d
3e
3f
5a
5b
5c
5d
5e
5f
: X = N, Y = N, R = 4-cyano benzyl;
: X = N, Y = N, R = 4-nitro benzyl;
: X = N, Y = N, R = 4-fluoro benzyl;
: X = N, Y = N, R = 4-trifluoro methyl benzyl;
: X = N, Y = N, R = 2-fluoro benzyl;
: X = N, Y = CH, R = 4-cyano benzyl;
: X = N, Y = CH, R = 4-nitro benzyl;
: X = N, Y = CH, R = 4-fluoro benzyl;
: X = N, Y = CH, R = 4-trifluoro methyl benzyl;
: X = N, Y = CH, R = 2-fluoro benzyl;
: X, Y = CH, R = 4-cyano benzyl;
: X = N, Y = N, R = 4-cyano benzyl;
: X = N, Y = N, R = 4-nitro benzyl;
: X = N, Y = N, R = 4-fluoro benzyl;
: X = N, Y = N, R = 4-trifluoro methyl benzyl;
: X = N, Y = N, R = 2-fluoro benzyl;
: X = N, Y = CH, R = 4-cyano benzyl;
: X = N, Y = CH, R = 4-nitro benzyl;
: X = N, Y = CH, R = 4-fluoro benzyl;
: X = N, Y = CH, R = 4-trifluoro methyl benzyl;
: X = N, Y = CH, R = 2-fluoro benzyl;
: X, Y = CH, R = 4-cyano benzyl;
3g
3h
5g
5h
3i
3j
3k
3l
5i
5j
5k
5l
: X, Y = CH, R = 4-nitro benzyl;
: X, Y = CH, R = 4-nitro benzyl;
3m
3n
3o
5m
5n
5o
: X, Y = CH, 4-fluoro benzyl;
: X, Y = CH, R = 4-trifluoro methyl benzyl;
: X,Y = CH, R = 2-fluoro benzyl;
: X, Y = CH, 4-fluoro benzyl;
: X, Y = CH, R = 4-trifluoro methyl benzyl;
: X,Y = CH, R = 2-fluoro benzyl;
Scheme 1 : Synthetic protocol for the synthesis of title compounds 3a-o and 5a-o. Reagents
and conditions: a) EDC.HCl, HOBt, DiPEA, DMF, rt, 16h, yield: 75%; b) R-Br, NaN3,
sodium ascorbate, copper (II)sulfate, t-BuOH/H2O, rt, 4h; c) i) SOCl2, DCM, Cat.
DMF, 0 oC to rt, 10 min; ii) TEA, propargyl amine, DCM, 0 oC to rt.
The pyrazine-1,2,3-triazole analogs were synthesized using three different synthetic
routes. Our initial strategy focused on the modification of pyrazinamide (1) structure, a first-
line anti-TB drssug, by incorporating the 1,2,3-triazole moiety through an ester linker. The
synthesis started with esterification of pyrazine-2-carboxylic acid (1a) using EDC.HCl,
propargyl alcohol, and ester activating agent HOBt which afforded prop-2-yn-1-yl pyrazine-2-
carboxylate 2a. In the final step, we employed the Huisgen 1,3-dipolar cycloaddition reaction
(click reaction)31 in which the alkyne (2a) was reacted with the substituted benzyl bromides in

presence of copper (II)sulphate pentahydrate to get (1-benzyl-1H-1,2,3-triazol-4-yl)methyl
pyrazine-2-carboxylate derivatives (3a-e).
In order to investigate the importance of pyrazine ring on antitubercular activity of
compounds 3a-e, we have planned to synthesize the compounds 3f-j and 3k-o by replacing the
pyrazine ring with pyridine and benzene rings respectively. Accordingly, a similar protocol
(Scheme 1) was employed by reacting pyridine/benzene carboxylic acids 1b/1c with propargyl
alcohol to get intermediate 2a/2b which are then subjected to click reaction to afford
corresponding ester analogs 3f-j and 3k-o. Next, we designed and synthesized the amide
analogs (5a-o) by bioisosteric modification where in, the ester group in compounds 3a-o was
replaced with its bioisoster amide group. The key intermediate N-(prop-2-yn-1-yl)pyrazine-2-
carboxamide (4a) was synthesized by classical amide formation reaction of pyrazine-2-
carboxylic acid (1a) with thionyl chloride and propargyl amine. The click chemistry protocol
was followed with compound 4a to synthesize the target amide analogs (5a-o). In the final set
of compounds, the ester linker was replaced with 1,3,4-oxadiazole-2-thiol linker, a rational
structural modification, to get 2-(((1-(benzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5-(pyrazin-2-
yl)-1,3,4-oxadiazole derivatives (9a-l). The synthetic strategy for these analogs is depicted in
Scheme 2.
N
N
N
O
N
Y
N
NH2
Y
N
Y
N
SH
S
a
b
N
O
O
H
6a
6b
7a
7b
: Y = N
: Y = CH
: Y = N
: Y = CH
8a
8b
: Y = N
: Y = CH
c
9a
: Y = N, R = 4-cyano benzyl
9b
: Y = N, R = 4-nitro benzyl
: Y = N, R = 4-fluoro benzyl
9c
9d
N
S
N
: Y = N, R = 4-(trifluoromethyl) benzyl
: Y = N, R = 2-fluoro benzyl
R
Y
N
N
N
9e
9f
O
N
: Y = N, R = 4-methyl benzyl
9g
9h
: Y = CH, R = 4-cyano benzyl
: Y = CH, R = 4-nitro benzyl
9a-l
9i
9j
9k
9l
: Y = CH, R = 2-fluoro benzyl
: Y = CH, R = 4-(trifluoromethyl) benzyl
: Y = CH, R = 2-fluoro benzyl
: Y = CH, R = 4-methyl benzyl
Scheme 2: Synthetic scheme for the synthesis of title compounds 9a-l. Reagents and
conditions: a) CS2, KOH, Ethanol, reflux, 16h; b) propargyl bromide, NaH, DMF, 0
oC to rt, 6h, yield: 85.9 %; c) NaN3, sodium ascorbate, copper(II)sulfate, t-
BuOH/H2O, rt, 4h.

The synthesis involves the cyclization of commercial pyrazine/pyridine-2-carbohydrazide
(6a/6b) with carbon disulfide in presence of potassium hydroxide to get 5-(pyrazin/pyidin-2-
yl)-1,3,4-oxadiazole-2-thiol (7a/7b). Alkylation of 7a/7b with propargyl bromide under basic
conditions gave the intermediate 2-(prop-2-yn-1-ylthio)-5-(pyrazin/pyridin-2-yl)-1,3,4-
oxadiazole 8a/8b. The pre-final intermediate (8a/8b) was subjected to 1,3-dipolar
cycloaddition reaction with aryl bromides in the presence of copper (II) sulfate pentahydrate,
sodium salt of L- ascorbic acid and sodium azide in t-butanol/water (1:1, v/v) to get the target
compounds(9a-l). The esterification/amide formation of acid yielding free-final intermediates
(2a, 2b and 2c/4a, 4b and 4c) was evidenced by the disappearance of acid peak and appearance
of new peaks for alkynyl–CH, doublet for –CH2 and triplet for alkynyl–CH due to meta
coupling (4J coupling) as well additional amide peak for amide derivatives in their respective
1H NMR spectra. In addition, the mass spectrum of the intermediates showed [M+H]+ peaks,
further supporting the structure of these molecules. Lastly, the cyclization to form pyrazine-
1,2,3-triazoles from respective intermediates was interpreted by various spectroscopic means.
1
For example, the H NMR spectrum of compound 3a exhibited a singlet peak at δ 8.39 ppm
which corresponds to the 1,2,3-triazole proton. Further, the methylene protons of O-CH2 and
phenyl-linked -CH2 resonated as singlet peaks at δ 5.734 and 5.462 ppm respectively. The 13C
NMR spectrum of compound 3a displayed two individual signals at 128.5 and 163.9 ppm due
to 1,2,3-triazole (-CH-) and carbonyl (-C=O-) carbons respectively, which confirmed the
formation of the 1,2,3-triazole ring with ether linker. The structure of amide analog (5a) was
confirmed by its 1H NMR spectrum wherein a broad singlet and a doublet appeared at δ 8.37
and 4.76 ppm corresponding to amide (-CONH-) and methylene (–CH2-N-) protons
respectively. ESI mass spectra of compounds 3a and 5a showed [M+H]+ peaks at m/z 321.10
and 320.10, which are in agreement with their molecular formula C16H12N6O2 and C16H13N7O
respectively. The detailed analytical and spectral data of all the intermediates and title
compounds are summarized in the experimental section and the substitution patterns of target
compounds are given in Table 3. The representative spectra (Figures S1 – S28) of the target
compounds are presented in the supporting information.
The in vitro antitubercular activity of pyrazine-1,2,3-triazole analogs (3a-o, 5a-o and
9a-l) was assessed against the Mtb H37Rv (ATCC27294) strain using MABA method.32
Pyrazinamide, a first line anti-TB drug was taken as the standard for comparison of the
inhibition activity. The MIC values of target compounds are in the range 1.56 – 50.0 μg/mL
and seven target compounds are more potent than the pyrazinamide 1.

Initially, we incorporated diversely substituted 1,2,3-triazole chain on to the pyrazine
core through an ester linker to probe the SAR. Later, the pyrazine core was replaced with either
a pyridine or a phenyl ring. Accordingly, three sets of ester derivatives viz 3a-e, 3f-j and 3h-o
carrying pyrazine, pyridine and phenyl rings respectively were investigated to understand the
activity profile of the molecules. Incorporation of the 1,2,3-triazole ring, as seen in 3a-e
resulted no improvement in the inhibition activity (in comparison with pyrazinamide) against
Mtb H37Rv. However, three compounds 3a, 3c and 3d with 4-cyano benzyl, 4-fluoro benzyl
and 4-trifluoromethyl benzyl substituents on the triazole ring respectively displayed similar
activity (3.12 μg/mL) as that of the pyrazinamide. Compound 3e with a 2-fluoro benzyl
substitution is less active than its positional isomer 3c, indicating that the position of flouro is
important for the antitubercular activity of ester analogs. Also, 4-nitro benzyl substituted
derivative 3b displayed reduced activity. In order to investigate the vital role of the pyrazine
ring on antitubercular activity, we have screened the pyridine (3f-j) as well as the phenyl (3k-
o) derivatives against the H37Rv strain. In general, this structural modification substantially
reduced the inhibition activity particularly in the case of phenyl derivatives. All the pyridine
and phenyl derivatives (3f-o) are less active than the pyrazinamide. Three compounds 3g, 3i
and 3k carrying 4-nitro benzyl, 4-trifluoromethyl benzyl and 4-cyano benzyl substituents
respectively displayed moderate activity with the MIC of 12.5 μg/mL.
In order to explore the effect of an amide functionality towards the antitubercular
activity, we have synthesized and evaluated the amide derivatives 5a-o which are the
bioisosteric analogs of the ester derivatives 3a-o. The strategy followed for the structural
modification among the amide derivatives is similar to that we followed in the case of ester
derivatives, where in the pyrazine core is replaced with either pyridine or phenyl rings which
resulted three sets of amide derivatives (5a-e, 5f-j and 5k-o). The bioisosteric modification
demonstrated a significant enhancement of the inhibition activity. Compounds 5a-d with MIC
of 1.56 μg/mL are 2-fold and 2 to 4-fold more potent than the pyrazinamide and their ester
analogs respectively. The 4-fluorobenzyl derivative (5c) is more active than its positional
isomer 5e, the activity profile is similar to that observed in the case of respective ester analogs,
3c and 3e. The replacement of the pyrazine ring with a pyridine ring did not alter the activity
of the compounds, except compound 5h which showed a much lower activity (MIC = 25
μg/mL). However, the antitubercular activity decreased significantly when the pyrazine ring is
replaced with a phenyl ring; the MIC values of the phenyl derivatives (5k-o) are in the range
12.5 – 50 μg/mL. It is interesting to note that all the pyrazine and pyridine amide derivatives
(5a-j) are more potent than respective ester derivatives (3a-j).

In these molecules, the ester functionality of the parent compounds is replaced with a
1,3,4-oxadiazole-2-thiol linker. We have synthesized the pyrazine and the pyridine analogs in
this series but not the phenyl analogs based on the lower activity shown by the corresponding
ester (3k-o) and amide (5k-o) analogs. This rational structural modification turned out to be
unsuccessful in terms of the inhibition activity of the compounds against the Mtb strain. All the
1,3,4-oxadiazole-2-thiol analogs except 9f are less potent than the pyrazinamide. Overall, the
SAR for the pyrazine-1,2,3-triazole core with different linkers suggested that there is scope in
this region for improving in vitro inhibition activity against Mtb H37Rv.
The in vitro antibacterial activity of newly synthesized pyrazine-1,2,3-triazole analogs
(3a-o, 5a-o and 9a-l) were examined against E. coli, S. aureus, and P. aeruginosa using the
dilution method33 and MIC values are represented in Table 1. Ciprofloxacin was also tested as
a reference drug. The compounds demonstrated better inhibition activity against gram-positive
bacteria S. aureus than other tested bacterial strains. Particularly, the amide derivatives (5a-o)
designed through bioisosteric approach exhibited potent antibacterial activity which is
comparable with that of the standard drug, against S. aureus. Ester analogs (3a-o) and
oxadiazole analogs (9a-l) showed moderate antibacterial activity. The replacement of pyrazine
ring with pyridine or benzene resulted loss in antibacterial activity against three strains; the
SAR is similar to that observed in antitubercular studies. These results signify the biological
importance of the amide functionality in the pyrazine-1,2,3-triazole derivatives. Among the
tested compounds, 5b carrying a 4-NO2 group is the most potent antibacterial agent.
Compounds 5a and 5f with a cyano (CN) functional group on the phenyl ring are moderately
active against all the tested microbial strains.
Table 1. In vitro growth inhibition activity of the title compounds (3a-o, 5a-o, and 9a-l)
against Mtb, escherichia coli, staphylococcus aureus and pseudomonas aeruginosa.
S.No Comp
Mtb (MIC)
E. Coli
S.aureus
P.aeruginosa
MIC (μg/mL) MIC (μg/mL) MIC (μg/mL)
μg/mL
μM
9.74
18.36
9.95
8.59
19.9
78.29
1
2
3
4
5
6
3a
3b
3c
3d
3e
3f
3.12
6.25
3.12
3.12
6.25
25
50
25
12.5
25
25
50
25
25
50
50
12.5
25
12.5
25
25
25
50
25

7
3g
3h
3i
12.5
50
36.80
>100
34.5
25
-
25
50
25
50
50
50
50
-
8
9
12.5
25
25
25
25
-
25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
3j
80.05
39.26
>100
80.31
>100
80.31
4.88
50
3k
3l
12.5
50
25
50
3m
3n
3o
5a
5b
5c
5d
5e
5f
25
50
-
25
25
-
50
50
25
25
6.25
6.25
12.5
12.5
12.5
6.25
12.5
12.5
6.25
25
25
50
-
25
50
6.25
6.25
12.5
25
12.5
6.25
6.25
25
12.5
25
25
50
-
1.56
1.56
1.56
1.56
3.12
1.56
1.56
25
6.25
3.12
6.25
12.5
6.25
6.25
6.25
12.5
12.5
6.25
25
4.59
4.99
4.31
9.99
4.90
5g
5h
5i
4.61
80.30
4.31
1.56
3.12
12.5
50
5j
10.02
39.39
>100
>100
>100
>100
16.60
15.76
33.84
59.60
>100
8.53
5k
5l
50
5m
5n
5o
9a
9b
9c
9d
9e
9f
50
50
50
-
50
25
50
50
25
50
25
-
50
-
50
6.25
6.25
12.5
25
50
-
25
12.5
25
25
25
-
25
50
50
3.12
25
12.5
50
50
-
12.5
50
25
50
25
-
9g
9h
9i
66.59
63.22
>100
25
25
50
50

40
41
42
43
44
45
9j
9k
50
50
>100
>100
34.30
25.34
11.74
9.41
50
50
252
*
25
50
12.5
*
50
25
225
*
9l
12.5
3.12
6.25
3.12
PZA
*
*
*
STM
INN
2
2
4
PZA: Pyrazinamide, STM: Streptomycin; INN: Ciprofloxacin (standard antibiotic); -: Not
detected; *: Not screened.
The compounds which exhibited potent antitubercular activity (MIC ≤ 1.56 μg/mL)
were examined for the in vitro cytotoxicity profile against Vero cell line using MTT assay.34,35
Diminution in cell viability was noticed with different concentrations (15.625, 31.25, 62.5, 125,
250, and 500 μl/mL) of each active compound when compared with untreated control cells
(Figure 3). The tested pyrazine-1,2,3-triazole analogs exhibited higher IC50 values which
indicated that these compounds are non-toxic to the normal cell lines. The IC50, selectivity
index (SI) and % growth inhibition of the tested compounds are presented in Table 2. Only two
compounds 5b and 5f exhibited a measurable cytotoxicity with 20.52 and 17.69 % growth
inhibition respectively at a concentration of 62.5 μl/mL. Further, high selectivity index (>200)
of the compounds confirm their nontoxic nature: selectivity index (>10) is generally considered
as nontoxic. These experiments revealed that the compounds with potent antitubercular activity
and low cytotoxicity could be suitable for further development of new anti-TB leads.
Figure 3. Cell Vaibility vs Concentration (of Mtb active compounds with MIC 1.56 μg/mL).

Table 2. IC50, selective index (SI), and % growth inhibition of the active compounds.
S.No Compound
IC50a (μl/mL)
533±1.5
SIb
% growth Inhibition
1
2
3
4
5
6
7
5a
5b
5c
5d
5f
341.6
258.1
317.7
219.2
255.9
298.3
215.7
12.33
20.52
5.9
402.7±3.3
495.7±0.8
342±4.1
8.4
17.69
7.08
7.42
399.2±5.4
465.4±0.7
336.5±1.2
5g
5i
[a] IC50: Concentration of the compound corresponding to 50% inhibition of the VERO cell
lines. [b] SI = IC50 against Vero cells/MIC against Mtb H37Rv.
To gain an insight into the key binding interactions of pyrazine-1,2,3-triazole
derivatives within the active site of the protein, we performed the molecular docking studies
against enzyme InhA of Mtb using Auto-dock Vina 1.1.2.36 The crystal structure of Inh A (PDB
ID: 4TZK) in complex with 641 was retrieved from the PDB, which was resolved at 1.62 Å by
X-ray diffraction and used as the template. The docking results clearly indicated significant
binding interactions between the amide analogs (Figure 4) and the InhA of Mtb protein which
further supports the higher in vitro activity profile shown by these derivatives as compared to
respective ester or 1,3,4-oxadiazole-2-thiol analogs. The binding site interaction of compounds
3d, 5d and 9d in the active pocket of Inh A enzyme is shown in Figure 5. One of the most
active compounds, 5d bound deep in the active pocket and exhibited two H-bond interactions:
i) carbonyl oxygen of the pyrazine core interacts with Thr 196 (Bond length: 2.77 Å) residue
and ii) nitrogen of the pyrazine ring binds with Gly 96 (2.56 Å). Further, a pi-pi stacking
interaction is seen between the phenyl ring and Tyr 158 residue, which is a crucial amino acid
residue that interacts with the long chain fatty acyl substrates required for mycolic acid
synthesis in mycobacteria.37 Additionally, it formed an aromatic stacking interaction with Phe
149 residue as well. These strong binding interactions of 5d could result in the significant

inhibition of the target enzyme. Hence, the in vitro potency of the molecule (MIC = 1.56
μg/mL) is further validated by its favorable binding interactions with the receptor site.
Figure 4. Binding mode of active amide derivatives in the Mtb InhA active site; 5a: Green;
5b: Grey; 5c: Yellow; 5d: Red; 5f: Brown red; 5g: White; 5i: Pink; (5j: Dark blue and
5m: Blue with lower Mtb activity differ in their orientation as compared to the active
compounds).
The best docked pose of compound 3d, which is the ester analog of 5d, exhibited two
hydrogen bonds and a pi-pi stacking interaction with Lys 165 (2.39 Å), Ile 21 (2.35 Å) and Phe
149 residues respectively, which revealed that the molecule does not follow the binding pattern
of 5d. Further, the molecule did not interact with the most important residue Tyr 158 which
could be the reason for its lower in vitro inhibition activity (MIC = 3.12 μg/mL) than that of
the amide analog 5d. Similarly, compound 9d (the 1,3,4-oxadiazole-2-thiol analog of 3d)
which did not bind with Tyr 158 residue displayed lower in vitro inhibition activity (MIC: 6.25
μg/mL). The binding interactions of another amide derivative 5b is shown in Figure 6. The
compound exhibited two H-bonds: the nitrogen of pyrazine ring and the oxygen of the nitro
group respectively interacting with Lys 165 (2.25 Å) and Gly 104 (2.50 Å) residues. Further,
the 1,2,3-triazole ring formed two pi-pi stacking interactions with Tyr 158 and Phe 149
residues.

Figure 5. Binding modes of lead compounds a) 3d, b) 5d and c) 9d within Inh A active
pocket.
Among ester derivatives, the replacement of pyrazine ring by either pyridine or phenyl
ring resulted in loss of activity against Mtb H37Rv. Compound 3a, an ester analog binds
strongly to the Inh A binding site through two hydrogen bonds; the nitrogen (N3) of the triazole
ring binds with Lys 165 (2.14 Å) and the nitrogen of the pyrazine ring interacts with Ile 21
(2.14 Å) as illustrated in Figure 7. Whereas compound 3f, the pyridine analog of 3a, showed
two H-bond interactions: carbonyl oxygen of the pyridine ring and nitrogen (N2) of triazole
ring interacting with Ala 22 (2.39 Å) and Lys 165 (2.24 Å) residues respectively. A similar
binding pattern is seen for the phenyl analog 3k which formed hydrogen bonding interaction
with Ser 94 (2.42 Å) and Lys 165 (2.21 Å) residues. In addition, all three compounds displayed
a prominent π-π stacking interaction between the benzene ring and Phe 149 residue. It can be
seen that these compounds form H-bonds with Lys 165 and a п-п stacking with Phe 149, but
the pyrazine ring in 3a is involved in the formation of a stronger H-bond interaction with a
bond length of 2.14 Å which provides a rational justification for the higher potency of pyrazine
derivatives over the pyridine and the phenyl derivatives. Hence, the docking results further
validate the activity profile observed in the in vitro studies of the potent molecules.

Figure 6. Illustration of structural interactions of compound 5b within Inh A active pocket.
In drug discovery, optimization of the ADME (Absorption, Distribution, Metabolism,
and Excretion) parameters of a new drug entity is the most challenging process and have a great
impact on its potentiality and bioavailability. The pharmacokinetic properties such as partition
coefficient of n-octanol and water, molecular weight, polar surface area, number of hydrogen
bond acceptors/donors/rotatable bonds and molecular volume of the final compounds (3a-o,
5a-o and 9a-l) were calculated using the Molinspiration online toolkit38 whereas the drug
likeness score was calculated using Molsoft online software.39 The computed ADME results
are outlined in Table 3. Octanol-water partition coefficient (lipophilicity) was estimated by
milogP, which affects the bioavailability, drug absorption, toxicity, hydrophobic drug-receptor
interactions and metabolism of molecules. Higher milogP values generally results with poor
absorption. It is evident from table 3 that all the Mtb active compounds (5a, 5b, 5c, 5d, 5f, 5g
and 5i) follow the Lipinski’s rule of five. Polar surface area (PSA) is another useful parameter
(should be <140 Å) correlated with drug transport properties includes Caco-2 monolayers
permeability, blood-brain barrier penetration and human intestinal absorption40 and the
calculated values are in the range of 57.12 – 131.42 A° with one exception (compound 9b

showed 141.25 A°). Thus, these ADME prediction of target compounds could be helpful for
further development of new antitubercular leads with good oral activity.
Figure 7. Binding modes of lead compounds a) 3a, b) 3f and c) 3k within Inh A active
pocket.
Table 3. Structural features and pharmacokinetic properties of target compounds (3a-o, 5a-o
and 9a-l).
S.No Comp.
X
Y
R
mi
PSAb HBAc HBD Rot Be
Volf
(A3)
Drug-
Vial-
logPa (<140 (<10)
(<5) Å)
4-CN 1.40 106.60
(0-15)
likeness ationsg
d
(<5)
1
3a
N
N
8
0
6
274.3
-0.15
0

2
3b
3c
3d
3e
3f
N
N
N
N
N
N
N
N
N
N
N
N
N
4-NO2 1.60 128.63
4-F 1.81 82.81
4-CF3 2.54 82.81
2-F 1.76 82.81
10
7
7
7
7
9
6
6
6
6
8
5
5
5
8
10
7
7
7
7
9
6
6
6
6
8
5
5
5
9
11
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
7
6
7
6
6
7
6
7
6
6
7
6
7
6
5
6
5
6
5
5
6
5
6
5
5
6
5
6
5
6
7
6
280.51
262.11
288.47
262.11
278.19
284.67
266.26
292.63
266.26
282.35
288.82
270.42
296.78
270.42
277.45
283.93
265.52
291.89
265.52
281.61
288.08
269.68
296.05
269.68
285.76
292.24
273.84
300.20
273.84
308.86
315.33
296.93
-0.19
0.33
-0.07
-0.09
0.66
0.49
1.09
0.63
-0.67
-0.17
-0.12
0.31
-0.13
0.00
0.26
0.28
0.78
0.15
0.30
0.88
0.76
1.36
0.70
1.02
-0.04
0.11
0.50
-0.16
0.31
-0.92
-0.56
-0.32
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
3
4
5
6
CH 4-CN 1.63 93.71
CH 4-NO2 1.84 115.74
7
3g
3h
3i
8
CH
CH 4-CF3 2.77 69.92
CH 2-F 1.99 69.92
4-F
2.04 69.92
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
3j
3k
3l
CH CH 4-CN 3.21 80.81
CH CH 4-NO2 3.41 102.85
3m
3n
3o
5a
5b
5c
5d
5e
5f
CH CH
CH CH 4-CF3 4.35 57.02
CH CH 2-F 3.57 57.02
4-F
3.62 57.02
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
4-CN 0.57 109.39
4-NO2 0.77 131.42
4-F
4-CF3 1.71 85.60
2-F 0.93 85.60
0.97 85.60
CH 4-CN 0.80 96.50
CH 4-NO2 1.00 118.53
5g
5h
5i
CH
CH 4-CF3 1.94 72.71
CH 2-F 1.16 72.71
4-F
1.21 72.71
5j
5k
5l
CH CH 4-CN 2.03 83.61
CH CH 4-NO2 2.24 105.64
5m
5n
5o
9a
9b
9c
CH CH
CH CH 4-CF3 3.17 59.82
CH CH 2-F 2.39 59.82
4-F
2.44 59.82
-
-
-
N
N
N
4-CN 1.80 119.22
4-NO2 2.00 141.25
4-F
2.21 95.43

34
35
36
37
38
39
40
41
42
9d
9e
9f
-
-
-
-
-
-
-
-
-
N
N
N
4-CF3 2.94 95.43
2-F 2.16 95.43
4-CH3 2.49 95.43
8
8
0
0
0
0
0
0
0
0
0
7
6
6
6
7
6
7
6
6
323.30
296.93
308.56
313.01
319.49
301.09
327.45
301.09
312.71
-0.79
-0.57
-0.60
-0.31
-0.05
0.27
0
0
0
0
0
0
0
0
0
8
9g
9h
9i
CH 4-CN 2.56 106.33
CH 4-NO2 2.76 128.36
8
10
7
CH
CH 4-CF3 3.70 82.53
CH 2-F 2.92 82.53
CH 4-CH3 3.25 82.53
4-F
2.96 82.53
9j
7
-0.23
0.11
9k
9l
7
7
-0.03
a]
b]
Logarithm of partition coefficient between n-octanol and water (milog P). Polar surface
c]
d]
e]
area. Number of hydrogen bond acceptors. No. of hydrogen bond donors. Number of
rotational bonds. f] Volume. g] No. of violations from the Lipinski rule of five.
In the present work, we report on the structural modification of the first-line anti-
tubercular drug pyrazinamide using bioisosterism and rational approaches by incorporating the
1,2,3-triazole moiety in to the core structure. The core pyrazine and the 1,2,3-triazole rings are
hybridized through three different linkers in order to understand the SAR in these molecules.
Accordingly, three set of pyrazine-1,2,3-triazole hybrids 3a-o, 5a-o and 9a-l carrying an ester,
an amide and a 1,3,4-oxadiazole-2-thiol linkers respectively were synthesized through a click
chemistry approach. Further structural modification was carried out by replacing the pyrazine
core with a pyridine or a phenyl ring in order to explore the effect of pyrazine ring on the
inhibition activity of these hybrid molecules. The pyrazine-1,2,3-triazoles (5a-e) with an amide
linker are two-fold more potent than the pyrazinamide and are more potent than respective ester
and 1,3,4-oxadiazole-2-thiol analogs. Interestingly, these amide derivatives exhibited
significant antibacterial activity as well against all tested bacterial strains. The replacement of
the pyrazine core with a pyridine or a phenyl ring in general reduced the inhibition activity of
the molecules. The in vitro cytotoxicity assessment revealed that all the potent compounds are
non-toxic against VERO cell lines. The in vitro activity profile observed was further validated
by molecular docking studies of the active molecules against enzyme InhA. The active
molecules bind strongly with the receptor enzyme and most of the potent compounds interacted
with the crucial Tyr 158 residue. Further, in silico ADME calculations predicted good oral
bioavailability of the Mtb active compounds. Taken altogether, the in vitro anti-tubercular

activity, cytotoxicity and molecular docking results suggest that pyrazine-1,2,3-triazoles
carrying an amide linker are promising lead compounds for further development of new anti-
tubercular drugs.
Acknowledgements
Authors are thankful to NITK, Surathkal for providing the research facilities and to Dr.
Reddy’s institute of life sciences, HCU campus for providing analytical facilities.
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Table of contents
Table 1. In vitro growth inhibition activity of the title compounds (3a-o, 5a-o, and 9a-l)
against Mtb, escherichia coli, staphylococcus aureus and pseudomonas aeruginosa.
Table 2. IC50, selective index (SI), and % growth inhibition of the active compounds.
Table 3. Structural features and pharmacokinetic properties of target compounds (3a-o, 5a-o
and 9a-l).
Scheme 1 : Synthetic protocol for the synthesis of title compounds 3a-o and 5a-o. Reagents
and conditions: a) EDC.HCl, HOBt, DiPEA, DMF, rt, 16h, yield: 75%; b) R-Br, NaN3, sodium
ascorbate, copper (II)sulfate, t-BuOH/H2O, rt, 4h; c) i) SOCl2, DCM, Cat. DMF, 0 oC to rt, 10
min; ii) TEA, propargyl amine, DCM, 0 oC to rt.
Scheme 2: Synthetic scheme for the synthesis of title compounds 9a-l. Reagents and
conditions: a) CS2, KOH, Ethanol, reflux, 16h; b) propargyl bromide, NaH, DMF, 0 oC to rt,
6h, yield: 85.9 %; c) NaN3, sodium ascorbate, copper(II)sulfate, t-BuOH/H2O, rt, 4h.
Figure 1. Reported pyrazinamide and 1,2,3-triazole derivatives with antitubercular activity.
Figure 2. Design strategy for new pyrazine-1,2,3-triazoles (3a-o, 5a-o and 9a-l).
Figure 3. Cell Vaibility vs Concentration (of Mtb active compounds with MIC 1.56 μg/mL).

Figure 4. Binding mode of active amide derivatives in the Mtb InhA active site; 5a: Green;
5b: Grey; 5c: Yellow; 5d: Red; 5f: Brown red; 5g: White; 5i: Pink; (5j: Dark blue and 5m:
Blue with lower Mtb activity differ in their orientation as compared to the active compounds).
Figure 5. Binding modes of lead compounds a) 3d, b) 5d and c) 9d within Inh A active
pocket.
Figure 6. Illustration of structural interactions of compound 5b within Inh A active pocket.
Figure 7. Binding modes of lead compounds a) 3a, b) 3f and c) 3k within Inh A active
pocket.
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:

Highlights
 Bioisosteric modification of pyrazinamide structure was carried out.
 3 sets of pyrazine-triazole hybrids were synthesized via a click chemistry approach.
 7 compounds demonstrated better anti-TB activity than pyrazinamide.
 The compounds showed moderate inhibition activity against several bacterial strains.
 All the active compounds are non-toxic to the normal cell lines.