Synthesis and docking studies of pyrazine-thiazolidinone hybrid scaffold targeting dormant tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2016.03.055

The persistence of Mycobacterium tuberculosis (MTB) in dormant stage assists the pathogen to develop resistance against current antimycobactrial drugs. To address this issue, we report herein the synthesis of N-(4-oxo-2 substituted thiazolidin-3yl) pyrazi

Full text of DOI:10.1016/j.bmcl.2016.03.055

Accepted Manuscript
Synthesis and docking studies of pyrazine-thiazolidinone hybrid scaffold tar-
geting dormant tuberculosis
T.S. Chitre, K.D. Asgaonkar, P.B. Miniyar, A.B. Dharme, M.A. Arkile, A.
Yeware, D. Sarkar, V.M. Khedkar, P.C. Jha
PII:
S0960-894X(16)30275-X
http://dx.doi.org/10.1016/j.bmcl.2016.03.055
BMCL 23698
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 December 2015
10 March 2016
15 March 2016
Please cite this article as: Chitre, T.S., Asgaonkar, K.D., Miniyar, P.B., Dharme, A.B., Arkile, M.A., Yeware, A.,
Sarkar, D., Khedkar, V.M., Jha, P.C., Synthesis and docking studies of pyrazine-thiazolidinone hybrid scaffold
targeting dormant tuberculosis, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/
j.bmcl.2016.03.055
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Synthesis and docking studies of pyrazine-thiazolidinone hybrid scaffold targeting dormant
tuberculosis.
T.S. Chitre*a, K. D. Asgaonkara, P. B. Miniyarb, A. B. Dharmea, M. A. Arkilec, A. Yewarec,
D.Sarkarc , V. M. Khedkarc,d and P. C. Jhae
a. AISSMS College of Pharmacy, Kennedy Road, Pune, Maharashtra, India.
b. Sinhgad Institute of Pharmacy, Narhe, Pune, Maharashtra, India.
c. Combichem-Bioresource Center, OCD, National Chemical Laboratory, Pune, Maharashtra,
India.
d. School of Health Sciences, University of KwaZulu Natal, Westville Campus, Durban 4000,
South Africa.
e. School of Chemical Sciences, Central University of Gujarat, Sector-30, Gandhinagar-38200,
Gujarat, India.
*Corresponding Author:
Dr. Trupti S. Chitre
Associate Professor
All India Shri Shivaji Memorial Society’s College of Pharmacy,
Kennedy Road, Pune
India
Email:chitretrupti@rediffmail.com
Tel. No: 091-020-26058204
091-09860993381

Abstract: The persistence of Mycobacterium tuberculosis (MTB) in dormant stage assists the
pathogen to develop resistance against current antimycobactrial drugs. To address this issue, we
report herein the synthesis of N-(4-oxo-2 substituted thiazolidin-3yl) pyrazine-2-carbohydrazide
derivatives designed by following the molecular hybridization approach using pyrazine and
thiazolidenone scaffolds.
The compounds were evaluated against MTB H37Ra and
Mycobacterium bovis BCG in dormancy model. Most of the compounds had IC50 values in 0.3-1
µg/ml range. The active compounds were further tested for anti-proliferative activity against
THP-1, Panc –1, A549, and MCF – 7 cell lines using MTT assay and exhibited no significant
cytotoxicity. We also report molecular docking studies using active analogs and MTB -
Decaprenylphosphoryl-β-D-ribose-2’-epimerase (DprE1) to rationalize the biological activity
and to provide an insight into the probable mechanism of action and binding mode of hybridized
structures. The results obtained validate the use of molecular hybridization approach and also
suggest that reported compounds can provide a novel pharmacophore to synthesize lead
compounds against dormat MTB.
Key words: Tuberculosis, dormant, pyrazine, thiazolidenone, hybrid design, docking

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is prevalent in all parts of the
world. It is one of the world’s deadliest communicable diseases due to high virulence and ability
of MTB to enter into a dormant state which can subsequently undergo reactivation. The long-
term persistence of MTB in dormant stage assists the pathogen to develop resistance against
current antimycobactrial drugs. Thus, there is need for development of drugs which can target
1-3
the dormant MTB and help in eradication of the disease.
Mycobacterium bovis (M.bovis) is
also considered to be a disease causing strain in humans exposed to infected material or
immunodeficiency. Hence identification of Bacillus Calmette-Guerin (BCG), an attenuated
derivative of virulent strain of M.bovis is also considered clinically significant. 4
Molecular hybridization approach has been an area of interest towards development of agents
against MTB. Most important hybridized structures include clinically used drugs such as
rifamycin, ethambutol and isoniazid clubbed with other hydrophobic structure such as cinnamyl
acid derivatives.5-11 In this letter, we report novel chemical structures as antitubercular agents
based on the hybridization of pyrazine and thiazolidine derivatives.
Previous studies have indicated promising antitubercular activity for pyrazine and thiazolidine
12-19
derivatives against active strain.
Pyrazinamide mannich bases, pyrazine 2-carbohydrazides
and pyrazine-2-carboxamide derivatives have been reported to have MIC values in lower
micromolar range. Similarly thiazolidinone derivative have been reported to have MIC values in
the micromolar range with some compounds having activity in submicromolar range.
Thus, it was of interest to evaluate the activity of hybrids of pyrazine and thiazolidine derivatives
against dormant mycobacterium. Since some of our compounds demonstrated promising activity
we also investigated their probable mechanism by performing molecular docking study using
Decaprenylphosphoryl-β-D-ribose-2’-epimerase (DprE1) as the target receptor.20
The starting materials, pyrazine-2-carboxylic acid/ 3-aminopyrazine – 2-carboxylic acid/ 5-
methylpyrazine-2-carboxylic acid were purchased from Spectrochem (Mumbai, India) and their
identity was confirmed by IR, 1H NMR and Mass spectra. N-(4-oxo-2 substituted thiazolidin-
3yl) pyrazine-2-carbohydrazide derivatives (4a-4w) were synthesized in three steps as shown in

scheme 1. Briefly, substituted pyrazine-2-carboxylic acid derivative were converted to
carbohydrazides using hydrazine hydrate. Substituted pyrazine-2-carbohydrazides were then
converted to their respective hydrazine using substituted benzaldehydes. Lastly, carbohydrazones
were reacted with with thioglycolic acid in the presence of catalytic amount of ZnCl2 to obtain
final hybrid compounds.14,19,21 In total 23 compounds were synthesized (4a- 4w; Fig. 1). Final
compounds were purified using column chromatography and structures of all the compounds
were confirmed by IR, 1H NMR and mass spectroscopy (supplementary data). Purity of the
compounds was determined by TLC/HPLC. The melting points were found in the range of 145-
167 0C.
N
N
CONHNH2
N
N
COOH
R2
Hydrazine Hydrate 99.98%
2
R1
R2
R1
1
Ethanol
X
CHO
(Substituted benzaldehydes)
O
N
N
CONHN C
H
X
CH2COOH
HS
DMF
N
CONH
R2
N
S
3
R2
R1
X
R1
N
4
Scheme 1. Synthetic route for N-(4-oxo-2 substituted thiazolidin-3yl) pyrazine-2-
carbohydrazide derivatives (4a-4w)
R2
N
N
R1
O
CONH N
S
X

Sr. Comp
No code
X
R1
R2
Sr.
No
Comp
code
4m
4n
X
R1
R2
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
-C6H5
-H
-H
-H
-H
-H
-H
-NH2 13
-NH2 14
-NH2 15
-NH2 16
-NH2 17
-NH2 18
-NH2 19
-NH2 20
-NH2 21
-NH2 22
3- NO2C6H5
4- NO2C6H5
4- OHC6H5
-CH3 -H
-CH3 -H
-CH3 -H
4- ClC6H5
2,4- ClC6H5
3- NO2C6H5
4- NO2C6H5
4- OHC6H5
4o
4p
3-OC2H5,4 -OHC6H5 -CH3 -H
4q
4-OCH3 C6H5
-CH3 -H
-CH3 -H
4r
3,4-OCH3 C6H5
4g
4h
4i
3- OC2H5,4 -OHC6H5 -H
4s
3- OCH3,4 -OHC6H5 -CH3 -H
3,4-OCH3 C6H5
4- FC6H5
-H
-H
-H
4t
4- N(CH3)2 C6H5
4- OHC6H5
-CH3 -H
4u
-H
-H
-H
-H
-H
-H
10 4j
11 4k
12 4l
4- N(CH3)2 C6H5
-C6H5
4v
4- ClC6H5
-CH3 -H
-CH3 -H
23
4w
4- NO2C6H5
4- ClC6H5
N-(4-oxo-2 substituted thiazolidin-3yl) pyrazine-2-carbohydrazide derivatives were screened for
in vitro antitubercular activity against MTB H37Ra (ATCC 25177) and M.bovis BCG (ATCC
35743). Preliminary antitubercular screening was performed at concentrations, 30, 10 and
3µg/mL (Supplementary material). The XTT Reduction Menadione assay (XRMA) which is a
well established anti-tubercular screening protocol (for dormancy model) was used for screening
the compounds, against MTB H37Ra. 22 For M.bovis BCG, Nitrate reductase assay protocol was
used .3 The antitubercular drugs, rifampicin, pyrazinamide and isoniazid were used as reference
standards. The MIC and IC50 are presented only for compounds which showed more than 90%
inhibition at 30µg/ml concentration (Table 1) in dormant assay for MTB H37Ra and M. bovis
BCG.3 To evaluate the selectivity towards mycobacterium, the derivatives were tested
(Supplementary data Table 2) against Mycobacterium smegmatitis as well as antibacterial
screening using two Gram Positive (S. aureus and B. subtillus) and two Gram Negative bacteria
(P. flurescence and E.coli) was carried out. 23 The compounds, 4a, 4g, 4h, 4m, 4n, 4p,4s, 4t, 4v
and 4w showed more than 90 % inhibition of MTB H37Ra and M.bovis BCG. Compound 4p,
displayed MIC = 26.56 µg/mL and IC50 value of 0.337 µg/mL against MTB H37Ra, whereas 4v

showed, MIC = 28.82 µg/mL and IC50 value of 3.26 µg/mL against M.bovis BCG respectively.
None of the derivatives showed inhibition of Mycobacterium smegmatitis. According to the data,
substitution in phenyl ring with hydroxyl and ethoxy groups has favored activity against MTB
H37Ra and presence of 4-Chloro has shown more activity against M.bovis BCG. In general
presence of 4-Chloro, 4- Nitro and substitution with hydroxyl and ethoxy/methoxy groups in
ortho positions on phenyl ring have seem to give better activity. Remaining 13 compounds have
shown % inhibition in a range of around 50 to 80% and hence were not studied further. The
mentioned 10 compounds were then further examined for toxicity in a non adherent, human
monocytic (THP-1) cell line, in adherent human pancreas epithelioid (Panc –1), adherent human
lung epithelial (A549) cell line and in human breast epithelial (MCF – 7) cell line by MTT assay
(Table 1). 24
Table 1
Selected derivatives with their antimycobacterial activity and cytotoxicity.
Comp. M.tuberculosis
code
M.bovis BCG
THP 1 cell line
Panc 1 cell line A 549 cell line MCF 7
aMIC aIC50
bMIC bIC50
cMIC cIC50
dMIC dIC50 eMIC eIC50 fMIC fIC50
(µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml (µg/ml) (µg/ml) (µg/ml)
4a
4g
4h
4m
4n
4p
4s
28.03
28.64
25.82
29
0.909
3.46
24.4
12.9
23.42
23.45
9.89
23.45
9.46
5.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
89.65
68.88
85.41
>100
>100
>100
86.88
>100
>100
97.66
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
77.04 >100
84.17 >100
80.02 >100
>100 >100
>100 >100
>100 >100
62.26 >100
>100 >100
>100 >100
>100 >100
81.33
80.77
86.17
>100
>100
>100
70.22
>100
>100
65.6
29.42
29.23
18.52
28.8
0.718
4.29
27.53
26.56
27.87
0.528
0.337
0.997
15.92
12.25
29.4
4t
28.402 2.31
21.8
3.26
4.93
4v
4w
28.82
29.02
1.092
1.466
8.48
9.38

Rifam 0.0021 0.0014 0.04
0.004
0.0023
ND
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-picin
Isoni
-azid
0.075
0.0025 0.045
Pyrazi >10
namid
-e
ND*
>100
a Compounds were tested against MTB H37Ra strains using XTT-Menadione assay; b.
Compounds were tested against M.bovis BCG using Nitrate reductase assay; c. Cytotoxicity
studies on THP-, Panc 1, A549, and MCF 7 cell line using MTT assay.
ND*- not determined
Further, with the aim of rationalizing the antitubercular activity, in the absence of available
resources to carry out the enzyme-based experimental studies, a molecular docking study was
carried out for the most active analogues against Decaprenylphosphoryl-β-D-ribose-2’-epimerase
25,26,27
(DprE1) as the target receptor.
DprE1 is an essential component for cell growth and
survival, making it a potential target. Following the discovery of the benzothiazinones
(nitrobenzothiazinone-BTZ043) that binds covalently to DprE1 there has been a growing interest
in this target.28
Molecular docking study revealed that N-(4-oxo-2 substituted thiazolidin-3yl) pyrazine-2-
carbohydrazide derivatives investigated herein could snugly fit into the active site of DprE1 with
very similar orientations occupying positions very close to that of the native ligand in the crystal
structure and their resulting complexes were stabilized by a network of steric and electrostatic
interactions (Fig.2). Their binding energies were also found to be negative ranging from -
51.558kcal/mol to -36.395kcal/mol while the docking score ranged from -7.83 to -6.00 (native
ligand: -7.953). The docking score for currently known DprE1 inhibitor – nitrobenzothiazinone
(BTZ043) was found to be -8.621 with a binding energy of -55.288kcal/mol. A statistically
significant correlation was observed as well between the experimental antitubercular activity and
the molecular docking scores wherein the active analogues exhibited higher docking scores while
those with relatively low inhibition were also predicted to have a lower score. Furthermore a
detailed per-residue interaction analysis between the enzyme and the docked pyrazine-2-

carbohydrazides was carried out to identify the most significantly interacting residues through
which we can speculate regarding the detailed binding patterns in the cavity. For the sake of
brevity we have illustrated these results only for the most active analogue 4p while the results for
4a, 4g, 4h, 4m, 4n, 4s, 4t, 4v and 4w and their binding modes as, Figures 3S-11S respectively are
provided in the supplementary material.
Figure 2. Binding mode of 4p into the active site of DprE1 enzyme (the dotted lines signify the
hydrogen bonding interactions and the pi-pi stacking interactions are represented by green lines
while).
The lowest energy docked conformation of 4p (Fig. 2) revealed that the compound binds with an
overall binding energy of -51.558 kcal/mol making intimate contacts with the residues lining the
active site of the target enzyme DprE1 through significant bonded and non-bonded interactions.
The per residue-ligand interaction energy distribution showed an extensive network of favorable
van der Waals interactions with Lys418(-2.65 kcal/mol), Thr118(-1. 85 kcal/mol), Gly117(-
2.936 kcal/mol), Pro116(-2.164 kcal/mol) and Tyr60(-2.12 kcal/mol) residues through the central
thiazolidinone ring; with Lys367(-1.82 kcal/mol), Val365(-4.01 kcal/mol), Gln336(-2.27
kcal/mol), Leu317(-3.92 kcal/mol), His132(-3.33 kcal/mol) through the pyrazine heterocycle
while 3-ethoxy-4-hydroxyphenyl component of 4p was engaged in significant van der Waals
interactions with Asp389(-1.93 kcal/mol), Cys387(-3.04 kcal/mol), Leu363(-1.95 kcal/mol),
Asn324(-1.90 kcal/mol), Gly321(-1.82 kcal/mol), Phe320(-2.79 kcal/mol), Trp230(-1.96
kcal/mol) residues in the active site. Furthermore significant electrostatic interactions were
observed with Lys418 (-2.30 kcal/mol), Gly117 (-2.10 kcal/mol) and Pro116 (-1.73 kcal/mol) via

thiazolidinone ring; with Asp389 (-2.08 kcal/mol) and Leu363 (-2.18 kcal/mol), via 3-ethoxy-4-
hydroxyphenyl ring contributed to the enhanced binding affinity of 4p. This enhanced binding
affinity can also be attributed to the three prominent hydrogen bonding interactions: first
between oxygen (O) of amide linker and Gly117 residue at a distance of 1.76Å; the second
interaction was between oxygen (O) of thiazolidinone scaffold and Lys418 residue with a
bonding distance of 2.12Å while the third hydrogen bond was formed between hydroxy (OH)
group of the 3-ethoxy-4-hydroxyphenyl ring and Leu363 with a bonding distance of 2.13Å. The
complex of 4p with DprE1 was additionally stabilized by a crucial pi-pi stacking interactions
observed between the pyrazine heterocycle of 4p and His132 residue in the active site. These
type of hydrogen-bonding and the pi-pi (π- π) stacking interactions function as an "anchor",
guiding the 3D orientation of the ligand in its active site and aid the steric and electrostatic
interactions within. Thus a strong network of thermodynamic interactions observed between 4p
and DprE1 account for its good insilico binding and provides a clue for its significant invitro
antitubercular activity. These type of interactions were consistently observed for 4a, 4g, 4h, 4m,
4n, 4s, 4t, 4v and 4w as well but decreasing gradually with their observed anti-tubercular
activity. Further, Figure 12S presents an overlay of the binding mode of 4p over BTZ043 to
provide a comparison of their binding mode into the active site of DprE1 (please refer
supplementary material).
We have thus synthesized N-(4-oxo-2 substituted thiazolidin-3yl) pyrazine-2-carbohydrazide
derivatives using hybrid approach and evaluated them for antitubercular activity. The
synthesized compounds displayed promising antitubercular activity in dormancy model, wherein
all the compounds have shown more than 70% inhibition of MTB H37Ra and M.bovis BCG at
30µg/ml concentration. Few derivatives have even shown around 70 % inhibition of MTB
H37Ra and M.bovis BCG at 3µg/ml concentration. The derivative 4p, displayed MIC = 26.56
µg/mL and IC50 value of 0.337 µg/mL against MTB H37Ra, whereas 4v showed MIC = 28.82
µg/mL and IC50 value of 3.26 µg/mL against M.bovis BCG. A result of screening against
Mycobacterium smegmatitis and Gram positive as well as Gram negative bacterial strains,
indicated the selectivity of these derivatives towards MTB. Further the most active compounds
were evaluated for their cytotoxicity effect against, four human cell lines like, THP-1, Panc –1,

A549 and MCF –7 using MTT assay. The theoretical predictions from molecular docking studies
were found to be in harmony with the in vitro antitubercular data, suggesting that DprE1 could
be a potential target for these pyrazine-thiazolidinone derivatives. It could provide an insights
into specific bonded and non-bonded interactions guiding the activity which could be fruitfully
utilized for structure based optimization of this scaffold for the discovery of potent DprE1
inhibitors. The compounds could thus be further optimized for lead development.
Acknowledgments:
The authors are thankful to Dr. Ashwini R. Madgulkar, Principal, AISSMS College of Pharmacy,
for continuous motivation, support and providing necessary infrastructure to carry out this work.
We also thank Schrödinger Inc. for providing the Demo license of Schrödinger Suite that has
tremendously helped in this study and especially Craig Coel for the valuable support that has
tremendously helped in this study.
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Figure(s)
O
S
N
O
HN
N
N
C
R'
R"
X
Compound code 4a-4j: R'= -H, R"= -NH2
Compoundcode 4k-4t: R'= -CH3, R"= -H
Compound code 4u-4w: R'= H, R"= -H
X = -Cl, -NO2, -OH, -OCH3, -NH2
Figure 1. Structures of synthesized derivatives.
Figure 2. Binding mode of 4p into the active site of DprE1 enzyme (the dotted lines signify the
hydrogen bonding interactions and the pi-pi stacking interactions are represented by green lines
while).

*Graphical Abstract (for review)