Spectroscopic, quantum chemical studies, Fukui functions, in vitro antiviral activity and molecular docking of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide

Article abstract of DOI:10.1016/j.molstruc.2016.04.088

The molecular structural parameters and vibrational frequencies of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide have been obtained using density functional theory technique in the B3LYP approximation and CC-pVDZ (5D, 7F) basis set. Detailed vibrationa

Full text of DOI:10.1016/j.molstruc.2016.04.088

Journal of Molecular Structure 1119 (2016) 188e199
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Spectroscopic, quantum chemical studies, Fukui functions, in vitro
antiviral activity and molecular docking of 5-chloro-N-(3-nitrophenyl)
pyrazine-2-carboxamide
a
,
b
c
d
S.H. Rosline Sebastian , Monirah A. Al-Alshaikh , Ali A. El-Emam ,
e
,
*
f
f
g
C. Yohannan Panicker , Jan Zitko , Martin Dolezal , C. VanAlsenoy
a
Department of Physics, Karpagam University, Eachanari, Coimbatore, Tamilnadu, India
Christhu Jyothi Public School, Rajakkad, Idukki, Kerala, India
Department of Chemistry, College of Sciences, King Saud University, Riyadh, 11451, Saudi Arabia
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi Arabia
Department of Physics, Fatima Mata National College, Kollam, Kerala, India
Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic
Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium
b
c
d
e
f
g
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 February 2016
Received in revised form
The molecular structural parameters and vibrational frequencies of 5-chloro-N-(3-nitrophenyl)pyrazine-
2-carboxamide have been obtained using density functional theory technique in the B3LYP approxi-
mation and CC-pVDZ (5D, 7F) basis set. Detailed vibrational assignments of observed FT-IR and FT-Raman
bands have been proposed on the basis of potential energy distribution and most of the modes have
wavenumbers in the expected range. In the present case, the NH stretching mode is a doublet in the IR
1
April 2016
Accepted 25 April 2016
Available online 28 April 2016
ꢀ
1
ꢀ1
spectrum with a difference of 138 cm and is red shifted by 76 cm from the computed value, which
indicates the weakening of NH bond resulting in proton transfer to the neighboring oxygen atom. The
molecular electrostatic potential has been mapped for predicting sites and relative reactivities towards
electrophilic and nucleophilic attack. The hyperpolarizability values are calculated in order to find its role
in nonlinear optics. From the molecular docking study, amino acids Asn161, His162 forms H-bond with
pyrazine ring and Trp184, Gln19 shows H-bond with C]O group and the docked ligand, title compound
forms a stable complex with cathepsin K and the results suggest that the compound might exhibit
Keywords:
DFT
Antiviral activity
Cytotoxicity
Pyrazine
Spectroscopic
Molecular docking
inhibitory activity against cathepsin K. Moderate in vitro antiviral activity with EC50 at tens of
detected against feline herpes virus, coxsackie virus B4, and influenza A/H1N1 and A/H3N2.
mM was
©
2016 Elsevier B.V. All rights reserved.
1
. Introduction
prodrug it is metabolized via mycobacterial enzyme pyr-
azinamidase to form pyrazinoic acid [7]. Pyrazinoic acid accumu-
lates intracellularly and lowers pH in mycobacterial cell, which
leads to inhibition of membrane transport and depletion of energy
[8]. Derivatives of heterocyclic compounds such as pyridazine, py-
rimidine, and pyrazine are valuable chemotherapeutic agents [4,6].
The pyrazine ring is present in a number of naturally occurring
compounds in the forms of pteridines and antibiotics. Pyrazine-2-
caboxamides was recognized as the first line antimycobacterial
agent exhibiting a promising antibacterial and antifungal activity.
Pyrazine-2-carboxamide may be used in the treatment of multi-
drug resistant tuberculosis, a major growing problem among HIV-
infected patients [9]. The title compound of 5-chloro-N-(3-
nitrophenyl)pyrazine-2-carboxamide was shown to possess
The pyrazine moiety is an important part of many clinically used
drugs, including anticancer, diuretic [1], antidiabetic, antith-
rombotic, antidepressants or anti-infective (antituberculotics, bac-
tericides and fungicides) agents and offers many possibilities in
drug development [2e4]. Some N-phenylpyrazine-2-carboxamides
with various substituents both on the pyrazine and phenyl core
were described to possess significant antimycobacterial activity [5].
Pyrazinamide, a nicotinamide analogue, plays an important role in
TB-therapy [6] and has multiple mechanisms of action and as a
*
Corresponding author.
E-mail address: cyphyp@rediffmail.com (C.Y. Panicker).
http://dx.doi.org/10.1016/j.molstruc.2016.04.088
022-2860/© 2016 Elsevier B.V. All rights reserved.
0

S.H.R. Sebastian et al. / Journal of Molecular Structure 1119 (2016) 188e199
189
in vitro whole cell antimycobacterial activity against M. tuerculosis
H37Rv with minimum inhibitory concentration of 3.13 g/mL
11 M). Several pyrazine derivatives proved to be clinically sig-
chemical shifts as d values in parts per million (ppm) are indirectly
referenced to tetramethylsilane (TMS) via the solvent signal (see
Scheme 1).
m
(
m
nificant antiviral drugs. For examples, flutimide [10] and its ana-
logues [11] are potent and species selective inhibitors of influenza
virus endonuclease. Active metabolite (ribofuranosyltriphosphate)
of favipiravir (T-705) is an inhibitor of RNA-dependent RNA poly-
merase, effective against RNA viruses [12]. Telaprevir is a serine
protease inhibitor used in the treatment of hepatitis C [13]. The
presence of pyrazine-2-carboxamide motif in favipiravir and
telaprevir led us to examine the antiviral activity of the title com-
pound, which itself is an N-substituted pyrazine-2-carboxamide.
2.3. Antiviral evaluation
Antiviral activity in cell culture was assessed by cytopathic effect
(CPE) reduction assays using a broad panel of viruses [15,16]. To
perform the tests, the virus was added to semiconfluent cell cul-
tures in 96-well plates and, simultaneously, serial dilutions of the
test compound were added. The plates were incubated until clear
CPE was reached (typically 3e6 days). The antiviral activity was
expressed as EC50, which is the effective concentration causing 50%
reduction of CPE compared non-treated infected cells. The CPE was
evaluated by visual microscopy and/or by standard colorimetric
formazan-based MTS cell viability assay. Viruses examined on
Crandell-Rees Feline Kidney (CRFK) cells are: feline coronal virus
and feline herpes virus. Viruses examined on human embryonic
lung fibroblast (HEL) cells are: herpes simplex virus type 1 (HSV-1),
a thymidine kinase-deficient (TK) HSV-1 KOS strain resistant to
acyclovir, herpes simplex virus type 2 (HSV-2), vaccinia virus, hu-
man adenoviurs type 2 and vesicular stomatitis virus (VSV). Viruses
examined on human cervix carcinoma (HeLa) cells are: VSV, Cox-
sackie B4 virus and respiratory syncytial virus (RSV). Viruses
examined on African Green Monkey (Vero) cells are: para-
influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie B4 virus
and Punta Toro virus. Viruses examined on Madin-Darby canne
kidney (MDCK) cells: human influenza A/H1N1, A/H3N2 and B vi-
ruses. Finally, activity against human immunodeficiency virus (HIV)
type 1 and type 2 was studied in human MT-4 lymphoblast cells.
Unless stated otherwise, the tested strains and cell for cultiva-
tion were obtained from the American Type Culture Collection
2
. Experimental
2.1. Synthesis of 5-chloro-N-(3-nitrophenyl)pyrazine-2-
carboxamide
The title compound was prepared as described previously [14]
by convenient two-step synthesis using 5-hydroxypyrazine-2-
carboxylic acid (5-hydroxy-POA; Sigma-Aldrich, Darmstadt, Ger-
many) as a starting compound. The 5-chloro-N-(3-nitrophenyl)
pyrazine-2-carboxamide was obtained as white solid in 67% yield
(
after all purification steps). The identity and purity of the com-
ꢁ
pound was checked by melting point (223.5e225.1 C;
2
ꢁ
1
13
24.5e225.6 C) in literature [14] and H and C NMR spectra.
2.2. Spectra measurement
The FT-IR spectrum (Fig. 1) was recorded using KBr pellets on a
DR/Jasco FT-IR 6300 spectrometer in KBr pellets with a spectral
ꢀ1
resolution of 4 cm . The FT-Raman spectrum (Fig. 2) was obtained
on a Bruker RFS 100/s, Germany. For excitation of the spectrum the
emission of a Nd:YAG laser was used, excitation wavelength
(
(
ATCC). The strain A/HK/7/87 (A/H3N2) was obtained from J. Neyts
Katholieke Universiteit Leuven, Leuven, Belgium).
1
064 nm, maximal power 150 mW, measurement of solid sample.
2
.4. Evaluation of in vitro cytotoxicity
One thousand scans were accumulated with a total registration
time of about 30 min. The spectral resolution after apodization was
2
Palo Alto, CA, USA) at 500 MHz for H and 125 MHz for C. The
spectra were recorded in DMSO-d at ambient temperature. The
ꢀ
1
Compound's cytotoxic activity was measure as a control test of
cm . NMR spectra were recorded on Varian VNMR S500 (Varian,
1
13
the cell viability during the antiviral screening assays. Cytotoxicity
was determined in uninfected cells, which were incubated with
serial compound dilutions for 72 h. CC50, that is compound con-
centration producing 50% cytotoxic effect, was determined by
colorimetric formazan-based MTS assay. Alternatively, the toxicity
was expressed as minimal cytotoxic concentration (MCC), which
represented the tested compound concentration causing minimal
changes in cell morphology as detected microscopically.
6
3. Computational details
Calculations of the title compound were carried out with the
Gaussian09 program [17] using B3LYP/CC-pVDZ (5D, 7F) basis set to
predict the molecular structure and frequencies and a scaling factor
of 0.9613 had to be used for obtaining a considerably better
agreement with experimental data [18]. The hybrid B3LYP func-
tional is a combination of the Becke's three parameter exchange
functional [19] and Lee-Yang-Parr correlation functional [20,21].
The cc-pVDZ basis sets have had redundant functions removed and
have been rotated in order to increase computational efficiency
[22]. Structural parameters corresponding to the optimized ge-
ometry of the title compound (Fig. 3) are given in Table 1. The as-
signments of the calculated frequencies are done using Gaussview
[23] and GAR2PED [24] software. With respect to the carboxamide
moiety, C10eO25eN11eH24 two different conformations are
possible AI and BII (Fig. 4). The energies of the conformation AI
are ꢀ1321.94142319 a.u. and that of BII is ꢀ1321.95789451 a.u. The
Fig. 1. FT-IR spectrum of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide.

190
S.H.R. Sebastian et al. / Journal of Molecular Structure 1119 (2016) 188e199
ꢁ
Scheme 1. Synthesis of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamides. Conditions: (a) anhydrous toluene, 90 C, 1 h; (b) anhydrous acetone, TEA, RT.
bond lengths C
.3706, 1.4027 Å and the corresponding reported values are 1.5248,
.2486, 1.3521, 1.4109 Å [27]. For the title compound, the pyrazine
bond lengths, C eC , C eN , C eN , C eC , C eN and C eN are
.3991, 1.3401, 1.3222, 1.4058, 1.3303 Å and the corresponding re-
4
eC10, C10eO25, C10eN11, C13eN11 are 1.5081, 1.2235,
1
1
4
5
5
6
1
6
1
2
2
3
4
3
1
ported values are 1.3955, 1.3482, 1.3521, 1.4109, 1.3532, 1.3477 [27].
The CeN bond lengths in the pyrazine ring of the title compound
are much shorter than the normal CeN sing bond that is referred to
1.49 Å [27] and the same results are shown for the two CeC bonds
lengths in the pyrazine ring and are also smaller than that of the
normal CeC single bond of 1.54 Å [28]. The bond lengths
C
10eN11 ¼ 1.3706 and C13eN11 ¼ 1.4027 Å are also shorter than the
normal CeN single bond of 1.49 Å, which confirms this bond to
have some character of a double or conjugated bond [29].
For the title compound C]O bond length is 1.2235 Å and the
corresponding reported values are 1.2253 Å [30], 1.2486 Å [27] and
1.2253 Å [31] and according to literature [32,33] the changes in
bond lengths in C]O and CeN are consistent with the following
interpretation: that is, hydrogen bond decreases the double bond
character of C]O bond and increases the double bond character of
CeN bond. For the title compound the NeO bond lengths are 1.2236
and 1.2257 Å which was in agreement with the reported values
1.2245 and 1.2237 Å [34]. The CeNeO angles are reported as 117.7
Fig. 2. FT-Raman spectrum of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide.
ꢁ
and 117.5 [34] whereas for the title compound, the angles are 117.3
ꢁ
and 117.7 .
At N11 position, the angles C13eN11eH24 is 117.5, C10eN11eH24 is
ꢁ
1
13.8 and C13eN11eC10 is 128.7 . This asymmetry of angles at N11
position indicates the weakening of N11eH24 bond resulting in
proton transfer to the oxygen atom O25 [35]. At C
4
position the
ꢁ
angles C
5
eC
4
eN
3
is increased by 1.4 and N
3
eC
4
eC10 is reduced by
ꢁ
ꢁ
1
.1 from 120 and this asymmetry reveals the interaction between
the amide moiety and the pyrazine ring. At C10 position, the bond
ꢁ
ꢁ
angles are C
N
4
eC10eN11 ¼ 112.8 ,
C
4
eC10eO25 ¼ 121.1 and
ꢁ
11eC10eO25 ¼ 126.1 and this asymmetry gives the interaction
between carbonyl group and the neighbouring pyrazine ring.
4.2. IR and Raman spectra
The observed IR, Raman bands, calculated (scaled) frequencies
and assignments are given in Table 2.
Fig. 3. Optimized geometry of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide.
4
.2.1. NO
Nitro benzene shows NO
2
modes
least energy is for BII and this conformation is taken for the further
theoretical calculations in the article.
2
stretching modes in the region
ꢀ
1
1535 ± 30 and 1345 ± 30 cm [36] in the present case bands
ꢀ1
ꢀ1
observed at 1566, 1345 cm in the IR spectrum, 1565, 1345 cm in
the Raman spectrum and 1568, 1342 cm theoretically as assigned
4
. Results and discussion
ꢀ1
as NO
2
stretching modes [36]. Both the NO
2
stretching modes,1568,
In the following discussion the phenyl and pyrazine rings are
designated as Ph and Pz.
ꢀ1
1
2
8
342 cm
are IR and Raman active with IRintensities, 361.12,
70.96 and Raman activities, 178.80, 120.14 and have PEDs 54 and
3%. In aromatic compounds the deformation modes of NO are
2
ꢀ
1
4
.1. Geometrical parameters
assigned at 850 ± 60, 740 ± 50 and 540 ± 70 cm [36]. For the title
compound, bands observed at 811, 724, 526 cm (IR), 810, 730,
530 cm (Raman) and 808, 726, 528 cm are assigned as NO
deformation modes. According to the calculations, the deformation
modes of NO are IR active with IR intensities, 26.99, 29.49 and
ꢀ1
ꢀ1
ꢀ1
The CeC bond lengths in the phenyl ring lie in the range
.3930e1.4083 Å and for benzene the CeC bond length is 1.3993 Å
25] and for benzaldehyde 1.3973 Å [26]. In the present case, the
2
1
[
2

S.H.R. Sebastian et al. / Journal of Molecular Structure 1119 (2016) 188e199
191
Table 1
Optimized geometrical parameters of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide.
Bond lengths (Å)
C1eC2
C2eN3
C4eC5
C5eH8
N11eC13
C12eC14
C14eC16
C15eH19
C18eN21
1.4058
1.3303
1.3991
1.0915
1.4027
1.3939
1.3961
1.0858
1.4844
C1eN6
C2eH7
C4eC10
C10eN11
N11eH24
C12eH26
C14eH17
C16eC18
N21eO22
1.3222
1.0921
1.5081
1.3706
1.0181
1.0932
1.0914
1.3936
1.2236
C1eCl9
N3eC4
C5eN6
C10eO25
C12eC13
C13eC15
C15eC18
C16eH20
N21eO23
1.7508
1.3443
1.3401
1.2235
1.4083
1.4045
1.393
1.088
1.2257
ꢁ
Bond angles ( )
C2eC1eN6
123.3
C2eC1eCl9
118.9
N6eC1eCl9
117.9
C1eC2eN3
C2eN3eC4
C5eC4eC10
N6eC5eH8
C4eC10eO25
C10eN11eH24
C13eC12eH26
N11eC13eC15
C12eC14eH17
C13eC15eH19
C14eC16eH20
C15eC18eN21
C18eN21eO23
120.3
117.4
119.7
118.0
121.1
113.8
119.4
122.9
119.6
121.3
122.6
118.0
117.3
C1eC2eH7
N3eC4eC5
C4eC5eN6
C1eN6eC5
N11eC10eO25
C13eN11eH24
C14eC12eH26
C12eC13eC15
C16eC14eH17
C18eC15eH19
C18eC16eH20
C16eC18eN21
O22eN21eO23
121.3
121.4
121.6
116.2
126.1
117.5
119.8
119.5
119.9
120.6
119.7
118.5
125.1
N3eC2eH7
N3eC4eC10
C4eC5eH8
118.4
118.9
120.3
112.8
128.7
120.8
117.7
120.5
118.0
117.7
123.5
117.7
C4eC10eN11
C10eN11eC13
C13eC12eC14
N11eC13eC12
C12eC14eC16
C13eC15eC18
C14eC16eC18
C15eC18eC16
C18eN21eO22
mode has an IR intensity of 194.89 and Raman activity of 181.54
with a PED of 80%. Raju et al. [41] reported the C]O modes at 1654,
ꢀ1
7
95, 580 (IR), 1659, 798, 577 cm (DFT), for a similar derivative.
4.2.3. NH and CN modes
The NeH stretching mode is expected in the region
ꢀ
1
ꢀ1
3
500e3300 cm and the NeH deformations at around 1500 cm
ꢀ
1
and 1250 cm [39,42] and for the title compound, these modes are
assigned at 3462, 3324 (IR), 3450 cm (Raman), 3400 cm (DFT)
stretching), 1508, 1219 cm (DFT). For the title compound, the
ꢀ1
ꢀ1
ꢀ
1
(
theoretical calculation gives a PED of 99% for the NH stretching
mode with IR intensity of 97.13 and Raman activity of 226.17. The
NH stretching mode is a doublet in the IR spectrum with a differ-
ꢀ
1
ꢀ1
ence of 138 cm and is red shifted by 76 cm from the computed
value, which indicates the weakening of NH bond resulting in
proton transfer to the neighboring oxygen atom. The reported
values of NeH modes are at 1587, 1250, 650 (IR), 1580, 1227,
ꢀ
1
6
52 cm (DFT) [43]. In the present case, the NeH out-of-plane
mode is assigned 655 cm theoretically [36] and the reported
values for a similar derivative are 762 (IR), 757 cm (DFT) [41]. The
CeN stretching modes are expected in the region 1100e1300 cm
44] and in the present case the bands at 1110, 924 cm in IR, 1110,
18 cm in Raman, 1219, 1111, 921 cm theoretically are assigned
ꢀ1
ꢀ1
ꢀ1
ꢀ1
[
9
ꢀ1
ꢀ1
as these modes which are in agreement with literature [41].
Fig. 4. Possible conformations of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide
with respect to the dihedral angle C10eO25eN11eH24.
Aromatic nitro compounds show a CeN stretching vibration
ꢀ1
ꢀ1
near [44] 870 cm and the band at 897 cm theoretically is
assigned as the CeN stretching mode of nitro group attached with
the phenyl ring for the title compound. Out of the four CeN
stretching modes, corresponding to the mode 1219 (DFT), the IR
intensity is 39.14 and Raman activity is 342.14 with a PED of 47%
and experimentally no bands are observed and for the mode at 897
(DFT), the IR intensity and Raman activity are low and the corre-
sponding bands are absent experimentally as expected. But for the
modes at 1111, 921 (DFT), the IR intensities and Raman activities are
moderate and bands are observed experimentally in both the
spectra with PEDs 48 and 40%. The CeN stretching modes are re-
4
1
[
8.25 with PEDs, 51, 62, 40%. The reported values of NO
593, 1431 (stretching), 744 (IR), 772, 736 (IR), 779, 737 cm (DFT)
34] and at 1621, 1582, 1526, 1328 (IR), 1601, 1585, 1561, 1532, 1347,
2
modes are
ꢀ
1
ꢀ1
ꢀ1
1343, 1332 cm (DFT) [37] and at 744 (IR), 821, 716 cm (DFT)
[38].
4.2.2. C ¼ O modes
For the title compound, the C]O stretching and deformation
ꢀ
1
modes are assigned at 1694 (IR), 1696 (Raman), 1699 cm (DFT)
and 764 (IR), 666 (Raman), 664, 760 cm
agreement with reported literature [34,39,40]. The C]O stretching
ꢀ1
ꢀ1
ꢀ1
(DFT) which are in
ported at 1258, 1245, 1103, 897 cm (DFT) [34], 1215 cm (DFT)
[45].

192
S.H.R. Sebastian et al. / Journal of Molecular Structure 1119 (2016) 188e199
Table 2
Calculated (scaled) wavenumbers, observed IR, Raman bands and assignments of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide.
B3LYP/CC-pVDZ (5D, 7F)
IR
(cm
Raman
Assignmentsa
y
(cm 1
ꢀ
)
IR
I
R
A
y
ꢀ1
)
y
(cm
ꢀ1
)
e
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
9
9
9
9
9
9
8
8
8
8
7
7
7
7
6
6
6
6
6
5
5
5
5
4
4
4
4
3
3
3
2
2
2
2
1
1
9
8
5
5
3
2
400
147
121
088
078
071
052
699
615
578
568
545
517
508
452
421
409
342
327
305
255
240
225
219
200
135
111
092
064
050
96
73
60
46
30
21
97
89
69
08
88
79
60
26
98
73
64
55
18
76
39
28
05
87
28
19
08
81
47
08
95
47
34
21
67
23
5
8
4
2
7
97.13
22.91
4.69
0.79
8.82
226.17
25.17
120.94
80.38
165.25
99.36
59.30
181.54
144.39
84.72
178.80
593.16
51.12
745.17
16.82
118.81
66.86
120.14
152.37
62.07
60.40
4.37
40.58
342.14
137.32
6.75
18.17
51.34
9.61
0.50
4.43
80.12
0.69
1.87
1.54
5.31
0.47
1.99
3.58
5.04
1.21
2.81
19.30
2.52
0.80
1.23
7.22
1.16
12.59
8.49
0.31
5.32
3462, 3324
e
3450
e
y
y
y
y
y
y
y
y
y
y
y
y
y
d
d
d
y
y
y
y
d
d
y
y
y
d
y
y
y
d
d
d
NH(99)
CHPh(99)
CHPh(97)
CHPz(99)
CHPh(97)
CHPz(99)
CHPh(99)
C ¼ O(80)
3120
3090
e
3118
e
e
6.09
7.83
3071
3052
1694
1618
e
e
3055
1696
1620
1585
1565
1540
e
194.89
2.72
37.97
361.12
32.79
7.53
604.75
19.98
41.23
113.37
270.96
90.39
68.15
29.34
9.91
12.88
39.14
38.71
2.70
163.70
83.15
3.47
54.94
54.86
0.31
0.25
1.89
10.82
8.39
5.34
Ph(52),
Ph(49),
y
y
NO
NO
2
(29)
(18)
2
1566
1540
e
2
NO (54),
y
Ph(27)
Pz(64),
Pz(77)
NH(46),
CHPh(20),
CHPz(21),
Ph(44),
dCHPz(15)
e
e
y
CN(20)
Ph(57)
Pz(53)
1452
1424
e
1457
1425
1410
1345
e
y
y
d
CHPh(21)
CN(15)
1345
1330
e
NO (83), y
2
Ph(80)
Pz(59), yPh(22)
CHPz(49),
CHPz(45),
Pz(55),
CN(47),
Pz(57),
1302
e
e
yPz(26)
1242
e
1242
e
dCHPh(39)
d
CHPh(10)
NH(40)
dCHPz(11)
e
e
d
e
1198
1140
1110
e
1136
1110
1088
e
CHPh(78)
CN(48),
Pz(46),
Ph(15),
CHPh(44),
Pz(52),
Ph(22),
CHPh(86), t
CHPz(80),
CHPh(72)
CN(40),
CHPz(50),
CHPh(80)
d
CHPz(15)
CN(11)
CHPh(58)
Ph(28)
Pz(24)
y
d
1068
1048
998
974
e
e
y
997
e
y
y
Ph(49)
Ph(10)
Pz(10)
e
g
g
g
y
g
g
d
d
g
t
d
944
e
945
e
t
924
e
918
e
y
Ph(12)
y
CN(39)
0.88
889
e
886
864
810
e
44.87
26.99
11.87
3.87
11.35
29.49
3.86
48.00
13.95
18.19
1.65
4.98
2.49
14.25
8.71
4.13
C ¼ O(21),
NO (51), Ph(10)
CHPh(55), Ph(18),
CC(18),
C ¼ O(13)
CCl(44),
C ¼ O(32)
CHPh(16)
C ¼ O(36),
CN(10)
NO (22),
NH(43),
dCN(15), dPz(17)
811
789
e
2
d
t
2
gNO (17)
776
e
Pz(52),
Pz(16),
g
y
g
764
724
e
d
730
700
e
2
gNO (62), g
t
t
d
t
d
d
g
d
d
Pz(37),
Ph(57),
Ph(25),
Ph(15),
Pz(67),
Ph(56),
g
g
d
g
gCCl(11)
675
e
666
e
2
d
C ¼ O(35)
CN(11)
CN(11)
NO (10)
NO (19)
C ¼ O(19)
CCl(17),
e
g
g
e
615
577
e
g
t
NH(10),
Ph(14),
580
541
526
e
g
2
CN(29),
t
Ph(39),
g
2
530
e
NO
NO
2
(40),
(39),
d
y
2.60
0.08
0.30
5.68
0.14
2.41
3.31
1.44
0.49
1.36
4.23
0.53
2.41
0.59
2.07
0.93
2
d
C ¼ O(10)
485
430
e
485
e
g
CCl(33),
t
Pz(31),
NO (17)
dCC(14),
g
CC(19)
0.19
t
d
t
d
d
d
Ph(66),
CCl(31),
Pz(85)
Ph(24),
CN(42),
CCl(37),
g
2
21.88
11.38
4.91
1.95
4.66
0.30
7.97
0.74
0.44
0.82
1.56
0.84
2.37
0.85
0.05
0.16
0.06
421
406
378
e
d
Ph(10)
410
e
dCN(34)
d
d
t
e
C ¼ O(16),
d
NO
CC(10)
CCl(12),
NO (36)
2
(28)
e
e
Pz(23),
d
e
294
250
e
g
CC(23),
Ph(23),
CN(19),
g
t
C ¼ O(15),
gNH(10)
e
d
t
d
t
d
t
t
d
t
t
t
CCl(22),
Ph(46),
CCl(13),
Ph(27),
CN(42),
CC(47),
d
d
2
e
gCC(13)
d
t
d
tNH(38)
t
d
e
221
e
Pz(16),
d
CC(22),
dPh(22)
e
NO (60)
2
e
130
95
CC(17),
d
C ¼ O(14)
e
e
e
Pz(50),
CC(25),
C ¼ O(26),
CC(15)
NO (19),
C ¼ O(31), NH(33)
t
C ¼ O(13)
0.43
0.80
1.22
0.30
e
e
CN(39),
dCC(18)
e
e
NO (58), t
2
e
e
NH(50),
t
2
tCC(11)
3
e
e
t
a
y-stretching;
d
-in-plane deformation;
g
I A
-out-of-plane deformation; t-torsion; Ph-phenyl ring; Pz-pyrazine ring; IR -IR intensity; R -Raman activity; potential energy
distribution (%) is given in brackets in the assignment column.

S.H.R. Sebastian et al. / Journal of Molecular Structure 1119 (2016) 188e199
193
4.2.4. CeCl modes
4.3. NMR spectra
The CeCl stretching vibrations give bands in the region
ꢀ
1
7
10e505 cm and for simple organic chlorine compounds the
The absolute isotropic chemical shielding was calculated by
B3LYP/GIAO model [53] and relative chemical shifts were then
ꢀ
1
CeCl absorptions are in the region 750e700 cm [36]. Renjith
et al. [46,47] reported the CeCl stretching modes in the region
estimated by using the corresponding TMS shielding:
calculated previously at the same theoretical level. Numerical
values of chemical shift calc(TMS) - calc together with
calculated values calc(TMS), are given in Tables 3 and 4. The
experimental values are: H NMR (500 MHz, DMSO)
NH), 9.14 (d, J ¼ 1.5 Hz,1H), 8.96 (d, J ¼ 1.3 Hz,1H), 8.92 (t, J ¼ 2.2 Hz,
scalc(TMS)
ꢀ
1
6
09e947 cm
and Resmi et al. [48] reported CeCl stretching
ꢀ
1
modes at 876, 644, 581 cm in Raman spectrum and at 877, 646,
88 cm theoretically. In the present case the CeCl stretching
mode is assigned at 764 cm in the IR spectrum and at 760 cm
theoretically with IR intensity 11.35, Raman activity 19.30 and a
PED of 44%.
d
calc
¼
s
s
ꢀ1
5
s
ꢀ1
ꢀ1
1
d: 11.27 (bs,1H,
13
1H), 8.31e8.26 (m,1H), 8.01e7.97 (m,1H), 7.67 (t, J ¼ 8.2 Hz,1H).
NMR (125 MHz, DMSO): : 161.78, 151.41, 148.1, 144.4, 143.5, 143.2,
39.5, 130.3, 126.9, 119.0 and 115.1.
Looking at the predicted and measured d
values for 13C NMR
C
d
1
4
.2.5. Pyrazine ring modes
The pyrazine CeH stretching modes are observed at 3090,
spectrum, the biggest difference can be observed for the carbox-
amide carbon (C10), followed by the chloro substituted pyrazine
ring carbon (C1). According to predicted values, C1 should have
ꢀ
1
ꢀ
1
3
071 cm in the IR spectrum and at 3088, 3071 cm theoretically
ꢀ
1
with PED 99% which are expected in the range 3000e3100 cm
[
(
1
49]. The pyrazine ring stretching modes are assigned at 1540, 1424
IR), 1540, 1425, 1302, 1198 (Raman) and in the range
545e1200 cm theoretically. Almost for all the modes the IR
higher d than C10 of carboxamide. In real world, it is the opposite
way. From the measured spectrum, we have assigned the carbox-
amide C10 to the peak of 161.78 ppm and chloro substituted C1 to
151.41 ppm. This assignment is based on the shifts of similar rele-
ꢀ1
intensity and Raman activities are high and the PED values are from
5
3% to 64%. Lukose et al. [27] reported pyrazine ring stretching
vant derivatives. For example the reported
boxamide carbon (measured in DMSO-d at ambient temperature)
are: N-phenylpyrazine-2-carboxamide ꢀ160.5 ppm (CDCl ) [54],
pyrazine-2-carboxamide e 165.0 ppm(DMSO-d ) [55].The typical
for chloro substituted carbon of pyrazine nucleus can be taken
d values for the car-
ꢀ
1
modes at 1545, 1153, 1059, 985 cm experimentally and at 1550,
6
ꢀ1
1518, 1193, 1152, 1045, 982 cm theoretically. The ring breathing
3
mode of the 1,4- substituted pyrazine ring is reported at 1120 (IR),
6
ꢀ1
ꢀ1
1124 (Raman), 1126 cm theoretically [50] and 1131 cm [51] and
d
1
13
for the title compound the ring breathing mode of the pyrazine ring
is assigned at 1092 cm
breathing mode has a IR intensity of 83.15 and PED value of 46%.
The CH deformation modes of the pyrazine ring are assigned at
from 2-chloropyrazine ꢀ148.6 ppm (DMSO-d
6
) [56]. The H and
C
ꢀ1
ꢀ1
(DFT) and 1088 cm
(IR). The ring
NMR signals were assigned to atoms based on expected shielding
and HeH coupling, together with comparison with published data
of relevant similar compounds.
ꢀ1
1242 (IR), 1242 (Raman), 1255, 1240 cm (DFT) with PEDs 49 and
4
ꢀ
1
5% (in-plane bending) and 944 (IR), 945 (Raman), 946, 897 cm
4.4. Nonlinear optical properties
(
[
DFT) (out-of-plane bending) with PEDs 80 and 50% as expected
36,40].
Nonlinear optics deals with the interaction of applied electro-
magnetic fields in various materials to generate new electromag-
netic fields, altered in wavenumber, phase, or other physical
properties [57]. Quantum chemical calculations have been shown
to be useful in the description of the relationship between the
electronic structure of systems and its NLO response [58]. The
computational approach allows the determination of molecular
NLO properties as an inexpensive way to design molecules by
analyzing their potential before synthesis and to determine high
order hyperpolarizability tensors of the molecules. The calculated
values of the dipole moment and polarizability are 6.8 Debye and
4
.2.6. Phenyl ring modes
For the title compound, the phenyl CeH stretching modes are
assigned at 3120, 3052 (IR), 3118, 3055 (Raman) and 3147, 3121,
3
are almost 100%. The phenyl ring stretching modes are expected in
the range 1615e1260 cm [36] and for the title compound, the
ꢀ1
078, 3052 cm (DFT) as expected [36]. The PEDs of these modes
ꢀ1
bands observed 1618, 1452, 1330 (IR), 1620, 1585, 1457, 1410
ꢀ
1
(
Raman) and in the range 1615e1327 cm theoretically. All the
phenyl ring stretching modes are IR and Raman active according to
the theoretical calculations and having PEDs in the range 44e80%.
The sixth phenyl ring stretching mode or ring breathing mode
appears as a weak band near 1000 cm
benzene and in the present case this mode is assigned at 973 cm
ꢀ
23
2
.61 ꢂ 10 e.s.u. The first order hyperpolarizability of the title
ꢀ
30
compound is calculated and is found to be 5.76 ꢂ 10 e.s.u which
is comparable with the reported values of similar derivatives
ꢀ1
in 1,3-di substituted
[27,50]. The calculated hyperpolarizability of the title compound is
ꢀ
1
4
4.31 times that of the standard NLO material urea
theoretically as expected [36,52]. For the title compound, the ring
breathing mode has low IR intensity (0.31) and Raman activity
(
8.12) according to the calculations and no band is observed
Table 3
ꢀ
1
Comparison of calculated and predicted shifts of 13C NMR spectrum of 5-chloro-N-
experimentally. The ring breathing mode is reported at 983 cm
theoretically by Raju et al. [41]. For the title compound, the in-plane
(3-nitrophenyl)pyrazine-2-carboxamide (sorted by decreasing d measured values).
and out-of-plane CeH modes of the phenyl ring are assigned at
Atom
Predicted
d
(ppm)
Measured
d
(ppm)
Difference d (ppm)
1
242, 1136 (IR), 1242, 1140, 1068, 1048 (Raman), 1240, 1135, 1064,
C10
C1
C18
C5
C2
C4
C13
C14
C12
C16
C15
157.60
163.33
151.35
147.80
143.60
143.45
140.26
129.88
123.19
121.28
116.87
161.78
151.41
148.04
144.40
143.46
143.18
139.48
130.24
126.87
118.98
115.05
ꢀ4.18
11.92
3.31
3.40
0.14
0.38
0.78
ꢀ0.36
ꢀ3.68
2.30
ꢀ
1
1050 cm (DFT) (in-plane deformation) and 889, 789 (IR), 886
ꢀ
1
(
Raman), 960, 930, 889, 788 cm (DFT) (out-of-plane deformation)
as expected [36]. The in-plane CH bending modes have PED values
of 39, 78, 58 and 44% and out-of-plane bending modes have these
values of 86, 72, 80 and 55%.
The RMS error of the observed IR and Raman modes are 3.55 and
.28 respectively for B3LYP/CC-pVDZ (5D, 7F) methods. The small
3
difference is due to the fact that experimental results belong to the
solid phase and theoretical calculations belong to gaseous phase.
1.83

194
S.H.R. Sebastian et al. / Journal of Molecular Structure 1119 (2016) 188e199
Table 4
Comparison of calculated and predicted shifts of 1H NMR spectrum of 5-chloro-N-
(
3-nitrophenyl)pyrazine-2-carboxamide (sorted by decreasing d measured values).
Atom
Predicted
d
(ppm)
Measured d (ppm)
H24
H8
H7
9.3363
9.8788
8.867
11.27
9.14
8.96
a
H19
H26
H20
H17
10.3734
7.3884
8.5686
7.9448
8.92
a
8.29
7.99
7.67
a
Middle of a multiplet.
(
0.13 ꢂ 10ꢀ30e.s.u) [59] The theoretical second order hyper-
polarizability was calculated using the Gaussian09 software and is
ꢀ37
equal to ꢀ20.23 ꢂ 10
e.s.u. We conclude that the title compound
and its derivatives are an attractive object for future studies of
nonlinear optical properties. For the title compound, the calculated
CeN distances in the molecular structure are intermediate between
those of CeN single and C]N double bond and therefore, the
calculated data suggest an extended
p-electron delocalization of
the pyrazine ring and carboxamide moiety [60] which is respon-
sible for the nonlinearity of the molecule.
Fig. 5. HOMO-LUMO plots of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide.
4.5. Frontier molecular orbitals
The Highest Occupied Molecular Orbital (HOMO) and the
group and nitrogen atoms (Fig. 6).
Lowest Unoccupied Molecular Orbital (LUMO) are the main orbital
take part in chemical stability. The HOMO represents the ability to
donate an electron, LUMO as an electron acceptor represent the
ability to obtain an electron. The HOMO-LUMO energy gap shows
that the energy gap reflects the chemical reactivity and the level of
conductivity of the molecule [61]. Smaller the value of energy gap,
the easier electron transfer occurs from HOMO to LUMO. Relatively
large gap means the molecule would not be kinetically stable [62].
In the present case the energy values of HOMO and LUMO
are ꢀ8.059 and ꢀ4.974 eV, respectively. The ionization energy and
The Fukui function is a local reactivity descriptor which gives
the preferred regions where a chemical species will change its
density when the number of electrons is modified. Hence, Fukui
function indicates the propensity of the electronic density to
deform at a given position upon accepting or donating electrons
[68e70] and the corresponding condensed or atomic Fukui func-
tions on the jth atom site are given as,
ꢀ
fj ¼ q ðNÞ ꢀ q ðN ꢀ 1Þ
j
j
þ
electron affinity can be expressed as: I ¼ -EHOMO and A ¼ -ELUMO
I ¼ 8.059 eV and A ¼ 4.974 eV. The different global descriptors are
given by, hardness ¼ ꢀ(I þ A)/2
¼ (I-A)/2, chemical potential
and electrophilicity index [63,64]. In the present case the
values of these descriptors are
:
fj ¼ q ðN þ 1Þ ꢀ q ðNÞ
j
j
h
m
h
i
2
fj0
1
2
u
¼
m
/2
h
¼
q ðN þ 1Þ ꢀ q ðN ꢀ 1Þ
j
j
h
¼ 1.543,
m
¼ ꢀ6.516 and
u
¼ 13.00
and the energy gap between HOMO and LUMO orbitals is 3.085 eV
Fig. 5 shows the distributions of the HOMO and LUMO orbitals and
In the above equations, q
j
is the atomic charge (evaluated from
Mulliken population analysis, electrostatic derived charge, etc.) at
jth atomic site is the neutral (N), anionic (N þ 1) or cationic (N ꢀ1)
chemical species. Chattarajet al. [71] have introduced the concept of
generalized philicity and it contains almost all information about
the known different global and local reactivity and selectivity
2
the HOMO is localized over the entire molecule except NO and the
nitrogen atom of the pyrazine ring (away from the chlorine atom)
and the LUMO is over the entire molecule, except the NH group.
Both the HOMO and LUMO are mainly localized on the pyrazine
ring, indicating that the HOMO-LUMO are mostly the
bonding type orbital.
p-anti-
4.6. Molecular electrostatic potential and Fukui functions
Molecular electrostatic potential (MEP) is related to the elec-
tronic density which is a very useful descriptor in understanding
sites for nucleophilic reactions or electrophilic attack [65e67]. To
visually consider the most probable sites of the title compound for
an interaction with nucleophilic and electrophilic species, MEP was
calculated at B3LYP/CC-pVDZ (5D, 7F) level of optimized geometry.
The electrophilic reactivities are visualized by red colour which
indicates the negative regions of the molecule and the nucleophilic
reactivity regions are coloured by blue, indicating positive regions
of the molecule (Fig. 6). In the present case, the electrophilic re-
gions are oxygen atoms and nucleophilic regions are mainly NH
Fig. 6. MEP plot of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide.

S.H.R. Sebastian et al. / Journal of Molecular Structure 1119 (2016) 188e199
195
descriptor, in addition to the information regarding electrophilic/
nucleophilic power of a given atomic site in the molecule. Toro-
a higher energy orbitals ꢀ0.33898, ꢀ0.28828, ꢀ0.27097, ꢀ0.26859
a.u and low occupation numbers 1.92043,1.90368,1.44780,1.87210.
Labbe et al. [72] proposed a dual descriptor (
defined as the difference between the nucleophilic and electro-
D
f(r)), which is
The bonding orbitals with low p characters are: n
(O23), with lower
1
(Cl
9
), n
1
(O22),
n
1
1
n (O25)
energy
philic Fukui function and is given by,
orbitals ꢀ0.93826, ꢀ0.80903, ꢀ0.81052, ꢀ0.71211 a.u and high
occupation numbers 1.99474, 1.80903, 1.98270, 1.97656.
h
i
þ
ꢀ
D
f ðrÞ ¼ f ðrÞ ꢀ f ðrÞ
Thus, a very close to pure p-type lone pair orbital participates in
the electron donation to the
s
*(C
4
eC
5 1 3
) orbital for n (N )/
D
f ðrÞ > 0, then the site is favoured for a nucleophilic attack,
s
*(C eC ), *(C eC ) orbital for n (N )/
s
s
*(C eC ), *(C eN )
p
4
5
1
2
1
6
1
2
1
6
whereas if
trophilic attack.
D
f ðrÞ < 0, then the site may be favoured for an elec-
3 9 1 6 1
orbital for n (Cl )/p*(C eN ), p*(C10eO25) orbital for n (N11)/
p
*(C10eO25),
s
*(N21eO23
)
orbital for n2(O22)/
*(N21eO22), and
2
orbital for n (O25)/s*(C10e N11) interaction in the compound. The
s
*(N21eO23),
Under this situation, the reactivity descriptors,
D
f(r) provides
s*(N21eO22) orbital for n (O23)/
s
s
*(C10e N11)
3
useful information on both stabilising and destabilising in-
teractions between a nucleophile and an electrophile and helps in
identifying the electrophilic/nucleophilic behaviour of a specific
site within a molecule. It provides positive value for nucleophilic
attack and a negative value for electrophilic attack and the results
are tabulated in Table 5. The behaviour of molecules as electro-
philes/nucleophiles during reaction depends on the local behaviour
of molecules.
results are tabulated in Table 7.
4.8. Molecular docking
Pyrazine-2-carboxamide derivatives showed antimicrobial ac-
tivity [76] and Cathepsin K is a lysosomal cysteine protease that has
pleiotropic roles in bone resorption, arthritis, atherosclerosis, blood
pressure regulation obesity and cancer. The extracellular cathepsin
K is an intestinal antibacterial factor with anti-inflammatory po-
tential and suggests that topical administration of cathepsin K
might provide a therapeutic option for patients with inflammatory
bowel disease [77]. Thus we selected the target macromolecule for
docking simulation. High resolution 3D crystal structure of
cathepsin K was downloaded from the Protein Data Bank website
(PDB ID: 1SNK). All molecular docking calculations were performed
on Auto Dock-Vina software [78]. The protein was prepared for
docking by removing the co-crystallized ligands, waters and co-
factors. The Auto Dock Tools (ADT) graphical user interface was
used to calculate Kollman charges and polar hydrogens. The ligand
was prepared for docking by minimizing its energy at B3LYP/CC-
pVDZ (5D, 7F) level of theory. Partial charges were calculated by
Geistenger method. The active site of the enzyme was defined to
include residues of the active site within the grid size of
40 Å ꢂ 40 Å ꢂ 40 Å. The docking protocol was tested by extracting
co-crystallized inhibitor from the protein and then redocking the
same. The docking protocol predicted the same conformation as
was present in the crystal structure with RMSD value well within
the reliable range of 2 Å [79]. Amongst the docked conformations of
the title ligand, one which binds well at the active site was analyzed
for detailed interactions in Discover Studio Visualizer 4.0 software.
The ligand binds at the active site of the substrate (Figs. 7 and 8) by
weak non-covalent interactions. Amino acids Asn161, His162 forms
H-bond with pyrazine ring and Trp184, Gln19 shows H-bond with
C]O group. The docked ligand title compound forms a stable
4.7. Natural bond orbital analysis
The natural bond orbital (NBO) calculations were performed
using NBO 3.1 program [73] as implemented in the Gaussian09
package at the DFT/B3LYP level and the possible intensive in-
teractions [74,75] are given in Table 6.
The strong intra-molecular hyper-conjugative interactions are:
C
s
N
p
4
eC
*(C
11 of n
*(N21eO23), N21eO22 from O23 of n
from O25 of n (O25)/ *(C10eN11) and the corresponding electron
densities and stabilization energies are: 0.03614, 0.04400,
.39466e, 0.32335, 0.05769, 0.62811, 0.06933e and 8.90, 10.08,
3.46, 67.12, 17.85, 150.88, 23.44 kJ/mol.
The bonding in terms of the natural hybrid orbitals with
5
from N
eC ), C eN
(N11)/
3
of n
from Cl
*(C10eO25), N21eO23 from O22 of n
(O23)/ *(N21eO22), C10eN11
1
(N
3
)/
s
of n
*(C
4
eC
5
), C
1
)/
eC
2
from N
eN ), C10eO25 from
(O22)/
6 1 6
of n (N )/
1
2
1
6
9
3
(Cl
9
p
*(C
1
6
1
p
2
3
p
2
s
0
1
3 9 2 3 2
considerable p characters are: n (Cl ), n (O22), n (O23), n (O25) with
Table 5
Values of the Fukui function considering Mulliken charges.
-
þ
j
Atom
f
j
f
Df
k
C1
C2
N3
C4
C5
N6
H7
H8
ꢀ0.074540
0.145306
0.150167
0.051460
0.127521
0.216175
ꢀ0.024170
ꢀ0.022890
0.212819
ꢀ0.055380
0.252383
0.106391
ꢀ0.058940
0.120622
0.138753
0.135301
ꢀ0.073060
0.024782
ꢀ0.100480
ꢀ0.077150
0.007088
ꢀ0.027570
ꢀ0.017230
ꢀ0.142830
0.068535
ꢀ0.083060
0.130076
0.204618
ꢀ0.100550
ꢀ0.294150
0.227258
ꢀ0.102580
ꢀ0.229950
0.209248
0.200820
0.172673
0.433824
ꢀ0.564460
ꢀ0.166890
0.314788
ꢀ0.197780
ꢀ0.192910
ꢀ0.139100
0.244906
0.168245
0.257879
0.260078
0.233355
ꢀ0.117540
ꢀ0.104300
0.320085
ꢀ0.202010
0.238988
ꢀ0.245860
ꢀ0.444320
0.175798
ꢀ0.230100
ꢀ0.446130
0.233419
0.223714
ꢀ0.040150
0.489206
ꢀ0.816850
ꢀ0.273290
0.373729
ꢀ0.318400
ꢀ0.331660
ꢀ0.274400
0.317961
0.143463
0.358357
0.337230
0.226267
ꢀ0.089970
ꢀ0.087070
0.462915
ꢀ0.270550
0.322046
complex with cathepsin K and gives a binding affinity (DG in kcal/
mol) value of ꢀ6.7 (Table 8). These preliminary results suggest that
the compound might exhibit inhibitory activity against cathepsin K.
Cl9
C10
N11
C12
C13
C14
C15
C16
H17
C18
H19
H20
N21
O22
O23
H24
O25
H26
4.9. In vitro antiviral activity
As a complementary test, the title compound was tested for
potential activity against diverse DNA and RNA viruses of medical
importance (Section 2.3). Table 9 summarized detected antiviral
activities. Moderate activity with EC50 at tens of mM was detected
for feline herpes virus and coxsackie virus B4. Activity against
coxsackie B4 virus was detected in CRFK cells, but not in HeLa cells.
Similar activity was detected against two strains of influenza A,
both H1N1 (A/Virginia/ATCC3/2009) and H3N2 (A/HK/7/87). Ac-
cording to EC50 values determined by MTS assay, the antiviral ac-
tivity of the title compound was comparable to standard zanamivir
or ribavirin. Influenza A/H1N1 (A/PR/8) was resistant. Influenza B
virus (B/HK/5/72) was resistant. Other tested strains not explicitly

196
S.H.R. Sebastian et al. / Journal of Molecular Structure 1119 (2016) 188e199
Table 6
Second order perturbation theory analysis of Fock matrix in NBO basis corresponding to the intra-molecular bonds of the title compound.
Donor(i)
Type
ED/e
Acceptor(j)
Type
ED/e
E(2)a
E(j)-E(i)b
F(i,j)c
C1eC2
C1eN6
s
s
p
1.99175
1.98900
1.71624
C1eN6
C1eC2
C2eN3
s
s
p
*
*
*
0.02802
0.04400
0.33801
1.58
1.87
17.55
1.28
1.40
0.31
0.040
0.046
0.067
e
e
e
C4eC5
p*
0.29095
21.28
0.34
0.076
e
C1eCl9
s
e
1.98678
e
C2eN3
C5eN6
s
s
*
*
0.01617
0.01365
2.84
3.29
1.18
1.17
0.052
0.055
e
C4eC5
s
e
1.98687
e
N3eC4
C4eC10
s
s
*
*
0.02350
0.07087
1.49
2.09
1.23
1.15
0.038
0.045
e
e
e
C10eN11
s*
0.06933
1.77
1.21
0.042
e
C4eC10
s
e
1.97533
e
C2eN3
N3eC4
s
s
*
*
0.01617
0.02350
3.07
1.07
1.18
1.16
0.054
0.032
e
e
e
e
e
e
e
e
e
C4eC5
s*
s*
s*
0.03614
0.01365
0.02810
1.45
2.50
4.14
1.22
1.17
1.10
0.038
0.048
0.060
C5eN6
N11eC13
e
C10eN11
s
e
1.98925
e
C4eC5
N11eC13
s
s
*
*
0.03614
0.02810
1.54
1.96
1.37
1.26
0.041
0.045
e
e
e
C12eC13
s*
0.02002
1.60
1.38
0.042
e
C10eO25
C13eC15
p
p
e
1.97278
1.61698
e
C4eC5
C12eC14
C16eC18
p*
p*
p*
0.29095
0.32116
0.38571
4.13
19.50
21.08
0.36
0.28
0.28
0.037
0.067
0.069
e
C18eN21
s
e
1.99013
e
C13eC15
C14eC16
s
s
*
*
0.35624
0.01506
1.66
1.46
1.36
1.38
0.042
0.040
e
N21eO22
p
e
1.98470
e
C16eC18
N21eO22
p
p
*
*
0.38571
0.62811
4.61
7.11
0.44
0.30
0.045
0.049
e
LPN3
s
e
1.91230
e
C1eC2
C4eC5
s
s
*
*
0.04400
0.03614
8.81
8.90
0.89
0.92
0.080
0.082
e
e
e
C4eC10
s*
0.07087
2.31
0.79
0.038
e
LPN6
s
e
1.90124
e
C1eC2
C1eCl9
s
s
*
*
0.04400
0.07263
10.08
5.20
0.88
0.46
0.085
0.044
e
e
e
C4eC5
s*
0.03614
8.76
0.90
0.081
e
LPCl9
s
p
1.99474
1.97023
C1eC2
C1eC2
s
s
*
*
0.04400
0.04400
1.13
3.10
1.43
0.83
0.036
0.045
e
e
e
e
C1eN6
C1eN6
s*
0.02802
0.39466
5.09
0.83
0.29
0.058
0.060
n
1.92043
p
*
13.46
e
LPN11
s
e
1.63626
e
C10eO25
C13eC15
p
p
*
*
0.32335
0.35624
67.12
34.88
0.26
0.29
0.120
0.090
e
LPO22
s
e
1.98254
e
C18eN21
N21eO23
s
s
*
*
0.09412
0.05769
4.21
1.98
1.09
1.16
0.062
0.043
e
e
p
1.90368
C18eN21
N21eO23
s
*
*
0.09412
0.05769
10.40
17.85
0.57
0.64
0.069
0.096
e
e
s
e
LPO23
s
e
1.98270
e
C18eN21
N21eO22
s
s
*
*
0.09412
0.05657
4.14
1.98
1.09
1.17
0.061
0.043
e
e
e
p
e
n
1.90569
e
C18eN21
N21eO22
N21eO22
s
*
*
0.09412
0.05657
0.62811
10.16
17.48
0.57
0.65
0.13
0.068
0.096
0.128
s
1.44780
p
*
150.88
e
LPO25
s
e
1.97656
e
C4eC10
C10eN11
s
s
*
*
0.07087
0.06933
2.12
2.27
1.10
1.16
0.044
0.046
e
e
e
a
p
1.87210
C4eC10
s
*
*
0.07087
0.06933
18.61
23.44
0.66
0.71
0.100
0.117
e
e
C10eN11
s
E(2) means energy of hyper-conjugative interactions (stabilization energy in kJ/mol).
Energy difference (a.u.) between donor and acceptor i and j NBO orbitals.
F(i,j) is the Fock matrix elements (a.u.) between i and j NBO orbitals.
b
c
mentioned in Table 9 were completely resistant. The basis for the
antiviral effect of 5-chloro-N-(3-nitrophenyl)pyrazine-2-
carboxamide remains to be identified.
either CC50 (concentration reducing the cell viability by 50% as
measured by formazan-based colorimetric assay) or as MCC (min-
imum compounds concentration to cause microscopically detect-
able alteration of normal cell morphology). With the exception of
human MT-4 lymphoblasts (CC50 ¼ 13
m
ꢃ
M) and Madin-Darby
4
.10. In vitro cytotoxicity
canine kidney (MDCK) cells (MCC
20 M; although
m
CC50 > 100
mM), no cytotoxicity was detected up to the concen-
In vitro cytotoxicity of 5-chloro-N-(3-nitrophenyl)pyrazine-2-
tration of 100
m
M. This rather low or none cytotoxicity of 5-chloro-
carboxamide on different animal and human cell lines was evalu-
ated during the testing of antiviral activity as a control (see Section
2
N-(3-nitrophenyl)pyrazine-2-carboxamide is consistent with the
previously published study, which evaluated in vitro cytotoxicity of
.3 for the list of used cell lines). The toxicity was measured as

S.H.R. Sebastian et al. / Journal of Molecular Structure 1119 (2016) 188e199
197
Table 7
NBO results showing the formation of Lewis and non-Lewis orbital.
Bond(A-B) EDA% EDB% NBO s%
ED/ea
p%
58.07
1
.38
.76
s
C1eC2
1.99175 50.71 49.29 0.7121(sp )Cþ 41.93
1
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0.78802
e
e
0.7021(sp )C
36.22
63.78
e
1
.90
s
C1eN6
1.98900 40.87 59.13 0.6393(sp )Cþ 34.51
65.49
63.24
1
.71
0.90981
e
e
0.7690(sp )N
36.76
e
1
.00
p
C1eN6
1.71624 44.81 55.19 0.6994(sp )Cþ
0.00 100.0
0.00 100.0
1
.00
0.35976
e
e
0.7429(sp )N
e
3
.25
s
C1eCl9
1.98678 45.72 54.28 0.6762(sp )Cþ 23.49
76.51
84.86
5
1
.58)
.68
0.70066
e
e
0.7368(sp
Cl
15.14
e
s
C4eC5
1.98687 51.38 48.62 0.7168(sp )Cþ 37.35
62.65
63.46
1
.74
0.76390
e
e
0.6973(sp )C
36.54
e
2
.05
s
C4eC10
C10eN11
C10eO25
C13eC15
C18eN21
N21eO22
1.97533 52.16 47.84 0.7222(sp )Cþ 32.75
67.25
64.84
1
.84
0.70070
e
e
0.6917(sp )C
35.16
e
2
.10
s
1.98925 37.16 62.84 0.6096(sp )Cþ 32.17
67.83
64.25
1
.80
0.85690
e
e
0.7927(sp )N
35.75
e
Fig. 8. The docked protocol reproduced the co-crystallized conformation with H-bond
1
.00
p
1.97278 30.74 69.26 0.5544(sp )Cþ
0.00 100.0
0.00 100.0
(green), p-p (magenta) and H-bond receptor surface shown.
1
.00
0.37909
e
e
0.8322(sp )O
e
1
.00
p
1.61698 51.67 48.33 0.7188(sp )Cþ
0.00 100.0
0.00 100.0
1
.00
0.27731
e
e
0.6952(sp )C
e
3
.13
s
1.99013 37.62 62.38 0.6134(sp )Cþ 24.19
75.81
62.55
1
.67
0.82072
e
e
0.7898(sp )N
37.45
Table 8
e
1
.00
p
1.98470 40.36 59.64 0.6353(sp )Nþ
0.00 100.0
0.00 100.0
The binding affinity values of different poses of the title compound predicted by
Autodock Vina.
1
.00
0.43610
e
e
0.7723(sp )O
e
n1N3
1.91230
0.40443
e
e
e
e
sp2.49
e
sp2.50
e
28.63
e
71.37
e
Mode
Affinity (kcal/mol)
Distance from best mode (Å)
e
RMSD l.b.
RMSD u.b.
n1N6
1.90124
0.3868
e
e
e
e
28.48
e
71.52
e
e
1
2
3
4
5
6
7
8
9
ꢀ6.7
ꢀ6.7
ꢀ6.6
ꢀ6.4
ꢀ6.3
ꢀ6.3
ꢀ6.2
ꢀ6.2
ꢀ6.0
0.000
3.808
3.764
1.614
24.331
3.906
2.844
24.481
24.166
0.000
7.537
4.474
2.125
26.336
7.225
4.008
26.925
25.700
n1Cl9
1.99474
0.93826
e
e
e
e
sp0.18
e
sp99.99
e
sp1.00
85.00
e
15.00
e
e
n2Cl9
1.97023
0.33916
e
e
e
e
0.09
e
99.91
e
e
n3Cl9
1.92043
0.33898
e
e
e
e
0.00 100.0
e
e
sp1.00
e
e
n1N11
1.63626
0.28525
e
e
e
e
0.00 100.0
e
e
e
e
n1O22
1.98254
0.80903
e
e
e
e
sp0.29
e
sp99.99
77.33
e
22.63
e
e
5-chloro-N-pyrazine-2-carboxamides with various substituents on
the phenyl ring on human hepatocellular liver carcinoma cell line
HepG2, and the 3-nitrophenyl derivative was one of the least toxic
n2O22
1.90368
0.28828
e
e
e
e
0.24
e
99.76
e
e
e
sp0.29
e
sp99.99
e
sp1.00
e
sp0.62
e
n1O23
1.98270
0.81052
e
e
e
e
77.50
e
22.50
e
compounds with IC50 ¼ 32.7
mM [80].
e
n2O23
1.90569
0.28894
e
e
e
e
0.23
e
99.77
e
e
5. Conclusion
n3O23
1.44780
0.27093
e
e
e
e
0.00 100.0
e
e
e
The optimized geometries and vibrational wavenumbers of the
title compound have been determined using DFT/B3LYP/CC-pVDZ
n1O25
1.97656
0.71211
e
e
e
e
61.80
e
38.20
e
e
(5D, 7F) basis set. The vibrational spectra were then assigned and
n2O25
1.87210
0.26859
e
e
e
e
sp1.00
e
0.00 100.0
e
the modes were visualized through the GaussView program. The
NH stretching mode is a doublet in the IR spectrum and is red
shifted by 76 cm from the theoretical wavenumber which in-
e
e
a
ED/e in a.u.
ꢀ1
dicates the weakening of NH bond. The ring breathing mode of the
ꢀ
1
ꢀ1
pyrazine ring is assigned at 1092 cm (DFT) and 1088 cm (IR)
with IR intensity of 83.15 and PED value of 46%. The phenyl ring
ꢀ1
stretching mode or ring breathing mode is assigned at 973 cm
theoretically as expected and has low IR intensity (0.31) and Raman
activity (8.12) according to the calculations and no band is observed
experimentally. The HOMO and LUMO orbitals are mainly localized
on the pyrazine ring, indicating that these orbitals mostly the
p-
anti-bonding type orbital. From the MEP analysis it is evident that
the electrophilic regions are oxygen atoms and nucleophilic regions
are mainly NH group and nitrogen atoms. Stability of the molecule
arising from hyperconjugative interaction and charge delocaliza-
tion has been analyzed using natural bond orbital analysis. Mod-
erate in vitro antiviral activity with EC50 at tens of mM was detected
against feline herpes virus, coxsackie virus B4, and one strain of
influenza A/H1N1 and one strain of influenza A/H3N2. The docked
Fig. 7. Schematic for the ligand interaction with the active site of cathepsin K.

198
S.H.R. Sebastian et al. / Journal of Molecular Structure 1119 (2016) 188e199
Table 9
In vitro antiviral activity EC50 (m (mM) of 5-chloro-N-(3-nitrophenyl)pyrazine-2-carboxamide in comparison with standards.
M)a and cytotoxicityb
Entry
Feline herpes virus infected
CRFKc cells
Coxsackie virus B4 infected Veroc
cells
Influenza infected MDCKc cells
Feline herpes
CRFK toxicity
Coxsackie virus B4
Vero toxicity
Influenza A/H1N1/
Influenza A/
MDCK toxicity
A/Virginia/ATCC3/
H3N2 (A/HK/7/8/
7)
2009
MTS
CC50
MTS
MCC
CPE
MTS
CPE
MTS
CC50
MTC
1
2
3
st run
nd run
rd run
15
20
e
>100
>100
e
34
45
>100
>100
e
>100
>100
12
>100
24
21
45
35
9.4
6.2
e
>100
>100
>100
e
100
100
ꢃ20
100
>100
e
e
Ganciclovir
Ribavirin
Zanamivir
0.9
e
>100
>250
e
e
e
e
e
e
>250
e
8.9
8.5
19
8.5
6.4
20
6.9
6.6
6.8
>100
>100
ꢃ20
e
e
>100
a
Antiviral activity was expressed as EC50, defined as the compound concentration which reduces virus-induced cytopathic effect to 50% as determined microscopically
(
CPE), or by measuring cell viability in the formazan-based MTS assay (MTS).
Cytotoxicity measures: CC50 - concentration reducing the cell viability by 50% as measured by formazan-based colorimetric assay; MCC - minimum compounds con-
b
centration to cause microscopically detectable alteration of normal cell morphology.
c
CRFK e Crandell-Rees Feline Kidney cells; MDCK - Madin-Darby canine kidney cells; Vero - African Green Monkey cells.
ligand title compound forms a stable complex with cathepsin K and
gives a binding affinity value of ꢀ6.7 kcal/mol and this suggests that
the compound might exhibit inhibitory activity against cathepsin K.
O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J W. Ochterski, R.L. Martin, K.
Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dap-
prich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox,
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[
Acknowledgements
[
[
[
[
[
The authors would like to extend their sincere appreciation to
the Deanship of Scientific Research at King Saudi University for
funding this work through the Research Group Project No. PRG-
[
1436-23. Authors wish to thank Associate Professor Lieve Naesens
(
Rega Institute for Medical Research, Laboratory of Virology and
Chemotherapy, Leuven, Belgium) for coordination of antiviral
testing.
[25] K. Tamagawa, T. Iijima, M. Kimura, J. Mol. Struct. 30 (1976) 243.
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