Discovery of 1,2,3-triazole based quinoxaline-1,4-di-N-oxide derivatives as potential anti-tubercular agents

Article abstract of DOI:10.1016/j.bioorg.2020.103955

A series of thirty one novel 2-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline-1,4-dioxide (7a-l), 3-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6-chloro-2-methylquinoxaline-1,4-dioxide (8a-l) and 2-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6,7-dichloro-3-methylquinoxaline-1,4-dioxide (9a-g) analogues were synthesized, characterized using various analytical techniques and single crystal was developed for the compounds 8 g and 9f. Synthesized compounds were evaluated for in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv strain and two clinical isolates Spec. 210 and Spec. 192. The titled compounds exhibited minimum inhibitory concentration (MIC) ranging from 30.35 to 252.00 μM. Among the tested compounds, 8e, 8 l, 9c and 9d exhibited moderate activity (MIC = 47.6 – 52.0 μM) and 8a exhibited significant anti-tubercular activity (MIC = 30.35 μM). Furthermore, 8e, 8 l, and 9d were found to be less toxic against human embryonic kidney, HEK 293 cell lines. Finally, a docking study was also performed using MTB DNA Gyrase (PDB ID: 5BS8) for the significantly active compound 8a to know the exact binding pattern within the active site of the target enzyme.

Full text of DOI:10.1016/j.bioorg.2020.103955

Journal Pre-proofs
Discovery of 1,2,3-triazole based quinoxaline-1,4-di-N-oxide derivatives as
potential anti-tubercular agents
Singireddi Srinivasarao, Adinarayana Nandikolla, Amaroju Suresh,
Augustynowicz-Kopec Ewa, Agnieszka Głogowska, Balaram Ghosh, Banoth
Karan Kumar, Sankaranarayanan Murugesan, Sravani Pulya, Himanshu
Aggarwal, Kondapalli Venkata Gowri Chandra Sekhar
PII:
S0045-2068(19)32256-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.103955
YBIOO 103955
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
30 December 2019
12 February 2020
17 May 2020
Please cite this article as: S. Srinivasarao, A. Nandikolla, A. Suresh, A-K. Ewa, A. Głogowska, B. Ghosh, B.
Karan Kumar, S. Murugesan, S. Pulya, H. Aggarwal, K. Venkata Gowri Chandra Sekhar, Discovery of 1,2,3-
triazole based quinoxaline-1,4-di-N-oxide derivatives as potential anti-tubercular agents, Bioorganic Chemistry
(
2020), doi: https://doi.org/10.1016/j.bioorg.2020.103955
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Discovery of 1,2,3-triazole based quinoxaline-1,4-di-N-oxide derivatives as
potential anti-tubercular agents
Singireddi Srinivasarao,[a] Adinarayana Nandikolla,[a] Amaroju Suresh,[a] Augustynowicz-Kopec
Ewa,[b] Agnieszka Głogowska,[b] Balaram Ghosh,[c] Banoth Karan Kumar,[d] Sankaranarayanan
Murugesan,[d] Sravani Pulya,[c] Himanshu Aggarwal[a] and Kondapalli Venkata Gowri Chandra
Sekhar[a]
aDepartment of Chemistry, Birla Institute of Technology and Science, Pilani, Hyderabad Campus, Jawahar Nagar, Kapra
Mandal, Hyderabad – 500 078, Telangana, India.
bMicrobiology Department, National Tuberculosis and Lung Diseases Research Institute, 01-138 Warsaw, Poland.
cDepartment of Pharmacy, Birla Institute of Technology and Science, Pilani, Hyderabad Campus, Jawahar Nagar, Kapra
Mandal, Hyderabad-500 078, Telangana, India.
dMedicinal Chemistry Research Laboratory, Department of Pharmacy, Birla Institute of Technology and Science, Pilani,
3
33 031, Rajasthan, India.
Abstract:
yl)methoxy)carbonyl)-3-methylquinoxaline-1,4-dioxide (7a-l), 3-(((1-(substituted phenyl)-1H-1,2,3-
triazol-4-yl)methoxy)carbonyl)-6-chloro-2-methylquinoxaline-1,4-dioxide (8a-l) and 2-(((1-
substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6,7-dichloro-3-methylquinoxaline-1,4-
A
series of thirty one novel 2-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-
(
dioxide (9a-g) analogues were synthesized, characterized using various analytical techniques and
single crystal was developed for the compounds 8g and 9f. Synthesized compounds were evaluated
for in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv strain and two clinical
isolates Spec. 210 and Spec. 192. The titled compounds exhibited minimum inhibitory concentration
(
MIC) ranging from 30.35 to 252.00 µM. Among the tested compounds, 8e, 8l, 9c and 9d exhibited
moderate activity (MIC = 47.6 – 52.0 µM) and 8a exhibited significant anti-tubercular activity (MIC
30.35 µM). Furthermore, 8e, 8l, and 9d were found to be less toxic against human embryonic
kidney, HEK 293 cell lines. Finally, a docking study was also performed using MTB DNA Gyrase
PDB ID: 5BS8) for the significantly active compound 8a to know the exact binding pattern within
the active site of the target enzyme.
=
(

Corresponding author
Tel.: +91 40 66303527 E-mail: kvgc@hyderabad.bits-pilani.ac.in; kvgcs.bits@gmail.com
1

Keywords: quinoxaline-1,4-di-N-oxide, 1,2,3-triazole, anti-mycobacterial activity, cytotoxicity.
1
. Introduction
Mycobacterium tuberculosis is a pathogenic bacteria and causative agent of Tuberculosis (TB). TB is
a contagious and infectious disease. Mostly, it affects the lungs (pulmonary TB) but can also affect
other sites (extra pulmonary TB) in the body. TB remains the leading cause of death among
infectious diseases worldwide. According to the 2019 WHO report it is estimated that 1.4 million
people die throughout the world from TB [1].
Quinoxaline derivatives have a broad scope in medicinal chemistry as the compounds have
diverse biological activities, such as antimicrobial, antiviral, anticancer, antifungal, and antitubercular
[2-3]. In the last three decades, several quinoxaline derivatives like 2-oxo derivatives, mono, and di-
N-oxide derivatives and their biological activities have been reported [4]. The pyrazine ring nitrogen,
when oxidized, gives the quinoxaline-1,4-di-N-oxide. These N-oxides are endowed with antiviral,
anticancer, antiprotozoal, and antibacterial activities [5-9]. Quinoxaline-1,4-di-N-oxide (A) is a
common sub-structure of many biologically and pharmacologically active compounds. Several
reports reveal that the compounds with various substituents at 2,3,6 and 7th position exhibit
promising anti-TB activity (B to G) [5-16]. Pan et al. synthesized thirty-one compounds of
quinoxaline-1,4-di-N-oxides variously substituted at the C-2 position, and their antimycobacterial
MIC ranged from 0.39 to 50 μg/mL. From this reported work, B and C emerged to be the most active
anti-TB agents with MTB MIC 0.39 and 1.56 μg/mL, respectively [10]. Sainza et al. reported
quinoxaline-l,4-di-N-oxide derivatives with different substituents at 2,3,6 and 7th position as
selective hypoxia agents. Some of these products gave good results as anti-tuberculosis agents [9-11].
9
9%
MTB
inhibition
was
observed
in
different
7-chloro-3-(para-substituted)
phenylaminoquinoxaline-2-carbonitrile-1,4-di-N-oxides [12] while 100 % growth inhibition was
observed in 6,7-dichloro-2-(ethoxycarbonyl)-3-methylquinoxaline-1,4-dioxide and 3-acetamide-6,7-
dichloroquinoxaline-2-carbonitrile-1,4-di-N-oxide derivatives [11,12]. Jaso et al. reported 2-acetyl
and 2-benzoyl-6(7)-substituted quinoxaline-1,4-di-N-oxide derivatives with MTB MIC ranging from
3
.3 to 62.5 μM against MTB H37Rv strains. Same group reported twenty-nine new 6 (7)-substituted
quinoxaline-2-carboxylate-1,4-dioxides with MTB MIC ranging from 0.10 to >6.25 μg/mL [13-15].
Seven 2-benzoylquinoxaline-1,4-di-N-oxide derivatives and twenty-seven 2-acetylquinoxaline-1,4-
di-N-oxides showed MTB MIC from 0.20 to 99.91 μg/mL against MTB H37Rv [15-16]. Torres et
al., reported 1,4-di-N-oxide-quinoxaline-2-ylmethylene isonicotinic acid hydrazides with MTB MIC
2

ranging from 0.58 to 90.84 μM against MTB H37Rv and H is the most potent agent with MTB MIC
.58 μM [17]. Quinoxaline-N-oxide based anti-TB agents are depicted in Figure 1.
0
Recently, few selected derivatives were found to be active against the single drug-resistant strains of
MTB and in macrophage model [17]. The two different molecules F and G were successful in vivo in
a murine model. In non-replicating (NRP) bacteria, compounds F and G showed significant activity.
If the action on NRP bacteria translates to in vivo conditions, it leads to shorter anti-TB treatment.
Under this assumption, quinoxaline-1,4-di-N-oxides may represent a new class for very good orally
active anti-TB drugs [18] with a potency to shorten the treatment period.
O-
4
O-
O-
O-
H
5
N+
N+
N+
N+
F
F Cl
Cl
N
O
6
7
3
2
H
N
+
N+
O-
N+
O-
N+ CN
O-
N
S
S
1
8
A
C
D
O-
B
O
O
O
quinoxaline 1,4-dioxide
MTB MIC = 0.39 µg/mL
MTB MIC = 12.5 µg/mL
MTB MIC = 1.56 µg/mL
O-
N+
O-
N+
O-
N+
O
O-
N+
O
O
N
H
Cl
Cl
Cl
Cl
F
N
O
O
N
N+
O-
O
N+
O-
N+
O-
N+
O-
F
G
E
H
MTB MIC = 0.39 µM
MTB MIC = 0.20 µg/mL
MTB MIC = 0.10 µg/mL
MTB IC50 = 0.58 µM
Figure 1: A is the numbering system in quinoxaline-1,4-dioxide and B-H are examples of N-oxide
based anti-tubercular agents
1
,2,3-triazole and their derivatives exhibited different biological activities viz., antibacterial,
antifungal, antiviral, antiallergic, anti-HIV, anti-inflammatory, anticonvulsant, and β-Lactamase
inhibition [19-22]. 1,2,3-triazole ring based compounds exhibit the dipole character moderately; they
are rigid, capable of hydrogen bonding, and are also stable in in vivo conditions [23-24]. The in vivo
and in vitro inhibitory activities of triazole based derivatives are very strong against MTB. To date,
many triazole based anti-TB agents are published, and some of them (I-N) are showcased in Figure 2
[25-29].
3

S
O N
2
O N
2
HN
HN
O
N
N
N
F
N
O
O
O
N
N
O
N
N
Cl
N
J
K
I
N
MTB IC50 = 2.20 µg/mL
MTB MIC = 0.25 µg/mL
MTB MIC = 0.78 µg/mL
N
N
N
N
O
N
N
N
Cl
N
N
N
N N
N
H
N
N
1
1
O
N
L
M
O
MTB MIC = 0.25 µg/mL
MTB MIC = 3.125 µg/mL
MTB MIC = 0.62 µg/mL
Figure 2: Some of the triazole based derivatives as anti-tubercular agents
Given the facts mentioned above about antitubercular activity observed in 1,2,3-triazoles and
quinoxaline-1,4-di-N-oxides as well as in continuation to our ongoing efforts towards developing
efficacious anti-TB agents, we designed the molecules of the current research work by hybridizing
these two active heterocyclic systems together into a single entity as outlined in Figure 3 [24]. We
used ester linkage, as the compounds with ester group exhibited most promising anti-Tb activity (F
and G of Figure 1) compared to other linkers. Various functionalities such as amide, alkyl and
heterocycles can be varied. [30]
O-
N
O-
N
N
O
N
Cl
N
+
+
X
Y
O
N Ar
HN
O
F
O
N
N
+
-
+
-
N
O
Cl
N
O
N
O
O N
2
Target
H
G
J
X, Y = H, H
Cl, H
Cl, Cl
Ar = Substituted phenyl
MTB MIC = 0.10 µg/mL
MTB MIC = 0.25 µg/mL
=
=
Figure 3: Synthetic strategy of the title compounds
2
. Results and Discussion
Chemistry
The designed target molecules were synthesized in five steps (Scheme-1). Firstly, N-oxide
intermediate (3a-c) was prepared via azides (2a-c). Ethyl acetoacetate was treated with propargyl
4

alcohol in toluene at 110 °C to get trans esterified compound 5. Compound 5 on treatment with
various substituted phenyl azides yielded 1H-1,2,3-triazoles (6a-l) [31]. Compound 6 upon reacting
with various N-Oxide intermediates (3a-c) in the presence of triethylamine formed quinoxaline-1,4-
dioxides (7a-l, 8a-l, and 9a-h). The synthesized compounds were purified, and structural elucidation
1
13
was done with mass spectrometry, H NMR and C NMR. From NMR data, the respective signals of
protons and carbons were verified with their coupling constants, multiplicities, and chemical shift
values. The final reports nearly matched with the theoretical values within the range of ± 0.05.
Preparation of Intermediate 3a-c
X
N
X
Y
NH2
X
Y
N3
(i)
(ii)
+O
N
Y
NO2
NO2
O-
1
2
3a-c
3a; X,Y=H,H
3b; X,Y=Cl,H
3c; X,Y=Cl,Cl
X
Y
N
O
N+
O-
O
O
O
O
O
O
(iii)
(iv)
3a-c
(v)
O
O
N
Ar
N
O
N
4
5
6a-l
Ar= Substituted phenyl
O-
O
X
Y
N+
O
N Ar
N
N
N+
O-
7
a-l; X,Y=H,H
a-l; X,Y=Cl,H
a-g; X,Y=Cl,Cl
8
9
Scheme 1: Synthetic scheme of titled quinoxaline-1,4-diN-oxide derivatives
Reagents and conditions: (i) NaNO (1.50 eq), NaN (1.50eq), 6N HCl (8 wt/v), 0 °C, 2 h. (ii)
2
3
toluene (30 wt/v), 110 °C, 24 h. (iii) propargyl alcohol (10.0eq), toluene, 24 h. (iv) substituted
different aromatic azides, sodium ascorbate (10 mol %), CuI (10 mol%), CuSO .5H O (10 mol %),
4
2
t
BuOH: H O (2:1), rt, 16 h. (v) N-oxide intermediate (3a-c) (1.2 eq), triethylamine, rt, 16 h.
2
In-vitro Mycobacterium tuberculosis screening
5

All the synthesized compounds were tested for their capacity to inhibit the growth of three
different M. tuberculosis strains. Among the three, one of them was reference strain M. tuberculosis
H37Rv ATTC 25618. The remaining were clinical strains isolated from TB patients [32-33]. MTB
strain spec. 210 was resistant to p-aminosalicylic acid, rifampicin, ethambutol, isoniazid (INH), while
the other strain (Spec. 192) was fully sensitive to the administrated TB drugs [32]. The minimum
inhibitory concentration (MIC) for the tested compounds was performed by a classical test-tube
method of successive dilution in Youmans’ modification of the Proskauer and Beck liquid medium
containing 10% of bovine serum [33]. INH was taken as reference drug. The stock solutions of
compounds and antibiotic were prepared in dimethyl sulfoxide (DMSO, Sigma Aldrich) and then
diluted in liquid medium. The final concentrations of tested compounds and INH were in the range of
3
.1–100 μg/ml. Mycobacterium strains were grown in Lowenstein-Jensen medium slants. Colonies
were suspended with sterile distilled water containing 5 mm glass beads and vortexed during 45 s.
The supernatant was harvested and adjusted to 0.5 McFarland (corresponding to approx.
8
1
,5ꢀ×ꢀ10 ꢀCFU/ml) with a nephelometer (Becton, Dickinson & Company, Franklin Lakes, MD, USA).
The, prepared inoculum was then added to each tube containing the tested compounds and antibiotics
5
in various concentration, so the final inoculum size was 5x10 CFU/ml. The test tubes were incubated
at 37 °C for 21 days. The growth of mycobacteria was assessed using a visual method. The MIC
value was determined as the lowest concentration of the drug at which no growth of microorganisms
was observed (no turbidity). The negative and positive control consisted of medium without
inoculum and medium inoculated with the same amount of bacteria respectively.
The result of in vitro studies on MTB is outlined in Table 1. The activity ranged from < 12.5 to >
1
00 μg/mL (30.35 to >252 µM).
6

O
N
O
X
Y
O
N
N
N
R
N
O
7(a-l); X,Y = H, H
8(a-l); X,Y = Cl, H
9(a-g); X,Y = Cl, Cl
Table 1: Results of antimycobacterial screening of titled compounds
MIC µM (µg/mL)
against MTB
H37Rv
MIC µM
(µg/mL) against
MTB Spec. 192
MIC µM (µg/mL)
against MTB
Spec. 210
Entry
X
Y
R
Series-I
7
a
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Ethyl
132.50 (50)
123.33 (50)
>252.94 (>100)
>242.84 (>100)
109.59 (50)
>236.77 (>100)
126.47 (50)
60.71 (25)
132.50 (50)
123.33 (50)
>252.94 (>100)
>242.84 (>100)
109.59 (50)
>236.77 (>100)
126.47 (50)
60.71 (25)
132.50 (50)
123.33 (50)
>252.94 (>100)
>242.84 (>100)
109.59 (50)
>236.77 (>100)
126.47 (50)
60.71 (25)
7
b
7
c
4-Fluoro
4-Chloro
4-Bromo
4-nitro
7
d
7
e
7
f
7
g
2- fluoro
2-chloro
2-nitro
7
h
7
i
59.19 (25)
59.19 (25)
59.19 (25)
7
j
3-nitro
59.19 (25)
59.19 (25)
59.19 (25)
7
k
3-trifluoromethyl
3,5-dichloro
224.54 (100)
97.06 (50)
224.54 (100)
97.06 (50)
112.27 (50)
194.12 (100)
7
l
Series-II
8
a
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
30.35 (12.5)
56.83 (25)
30.35 (12.5)
56.83 (25)
58.16 (25)
56.02 (25)
50.94 (25)
54.72 (25)
30.35 (12.5)
56.83 (25)
8
b
4-ethyl
4-fluoro
4-chloro
4-bromo
4-nitro
8
c
58.16 (25)
58.16 (25)
8
d
56.02 (25)
56.02 (25)
8
e
50.94 (25)
>203.76 (>100)
54.72 (25)
8
f
54.72 (25)
7

8
g
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
2-fluoro
2-chloro
58.16 (25)
56.02 (25)
54.72 (25)
109.45 (50)
104.21 (50)
52.00 (25)
Series-III
58.16 (25)
56.02 (25)
54.72 (25)
109.45 (50)
104.21 (50)
52.00 (25)
58.16 (25)
56.02 (25)
109.44 (50)
109.45 (50)
104.21 (50)
52.00 (25)
8
h
8
i
2-nitro
8
j
3-nitro
8
k
3-trifluoromethyl
3,5-dichloro
8
l
9
a
Cl Cl
Cl Cl
Cl Cl
Cl Cl
Cl Cl
Cl Cl
Cl Cl
H
112.04 (50)
53.85 (25)
52.00 (25)
47.60 (25)
107.70 (50)
104.01 (50)
97.06 (50)
<22.59 (<3.1)
112.04 (50)
53.85 (25)
52.00 (25)
47.60 (25)
107.70 (50)
104.01 (50)
97.06 (50)
<22.59 (<3.1)
112.04 (50)
53.85 (25)
52.00 (25)
47.60 (25)
107.70 (50)
104.01 (50)
97.06 (50)
91.15 (12.5)
9
b
4-fluoro
4-chloro
4-bromo
2-fluoro
2-chloro
3,5-dichloro
-
9
c
9
d
9
e
9
f
9
g
Isoniazid
-
-
Among the thirty-one compounds screened, eight compounds (8a, 8e, 8f, 8i, 8l, 9b, 9c and 9d)
showed activity against MTB with MIC <55.00 µM. Two compounds (8a and 9d) inhibited MTB
with MIC <50.00 µM. Among these eight compounds, 8a (6-chloro-2-methyl-3-(((1-phenyl-1H-
1
,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline-1,4-dioxide) is the most active one with MIC 30.35
µM.
SAR of compounds 7a-l
SAR is explained based on the activity of compound 7a. Structural changes at the ortho, meta, and
para positions alter the anti-TB activity. Compound 7a was inhibiting the growth of MTB H37Rv
strain at 132.50 µM. In this series, the introduction of an electron-donating ethyl group at the 4th
position, activity remains unaltered. Presence of electron-withdrawing F and Cl on the phenyl ring at
para position resulted in decrease in the activity by two folds (7c, MIC >252.94 µM; 7d, MIC
>
242.84 µM) but the presence of bromo (7e) at para position increased the activity by one fold with
MIC 109.59 µM. The presence of electron-withdrawing nitro group at para position decreased the
activity by two folds (7f, >236.77 µM), but the nitro group at ortho and meta position increased the
8

activity by two folds (7i, MIC 59.19 µM; 7j, MIC 59.19 µM). With the introduction of the fluoro
group at the ortho position, activity remains unchanged compared to compound 7a, but the presence
of chloro (compound 7h) at the ortho position enhanced the activity by two folds with MIC 60.71
rd
th
µM. The presence of two Cl (compound 7l) at 3 and 5 position increased the activity by one fold
with MIC 97.06 µM. The introduction of the electron-withdrawing CF group (compound 7k) at meta
3
position decreased the activity by two folds with MIC 224.54 µM.
SAR of compounds 8a-l
SAR is explained based on the activity of compound 8a. Compound 8a was inhibiting the growth of
MTB H37Rv strain at 30.35 µM. With the presence of electron-donating ethyl group at para position
activity fell by two folds with MIC 56.83 µM. Introduction of electron-withdrawing groups viz., F,
Cl, and Br at the ortho, meta, and para position, either mono or di-substituted resulted in a decrease
in the activity by two folds. The presence of an electron-withdrawing nitro group at para position
(compound 8f) decreased the activity by two folds with MIC 54.72 µM while nitro group at meta
position (compound 8j) decreased the activity by four folds with MIC 109.45 µM. The introduction
of the electron-withdrawing CF group (compound 8k) at meta position reduced the activity by four
3
folds with MIC 104.21 µM. All these results showed that the insertion of electron-withdrawing
moiety decreases the anti-tubercular activity.
SAR of compounds 9a-g
In this series, two Cl groups were introduced on the quinoxaline-1,4-N-dioxide frame. SAR is
explained based on the activity of compound 9a (MIC 112.04 µM). With introduction of electron-
withdrawing halogens like viz., F, Cl, and Br at para position on the phenyl moiety, activity
increased by two folds (9b, MIC 53.85 µM; 9c, MIC 52.00 µM, and 9d, MIC 47.60 µM) but
presence of F and Cl at the ortho position, activity remains unchanged (9e, MIC 107.70 µM; 9f, MIC
rd
th
1
04.01 µM). The introduction of Cl at 3 and 5 positions resulted in the retention of the activity.
The presence of two Cls on quinoxaline-1,4-N-dioxide did not improve the anti-TB activity. Overall,
it was noticed that 6-chloro-3-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2-
methylquinoxaline-1,4-dioxide derivatives (8a-l) exhibited better anti-TB activity followed by 2-(((1-
(
substituted
derivative (7a-l) and 6,7-dichloro-2-(((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-
-methylquinoxaline-1,4-dioxide derivatives (9a-g). The schematic representation of SAR studies is
showed in Figure 4.
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline-1,4-dioxide
3
9

Figure 4: Structural activity relationship of the three series of compounds.
In-silico predicted physicochemical properties
Currently, before approval of the drug into the market, ADMET properties of the drugs should be in
the optimum range. These parameters play a crucial role in the physicochemical properties of the
drugs after administration. To predict these parameters, several online servers / commercial softwares
were available [34]. QikProp [35-36] (commercial software with Schrodinger) was used for the
prediction of physico-chemical parameters of the titled new chemical entities. The significant
parameters were considered related to the Lipinski rule (Rule of five) and Jorgensen’s rule of three
[37] (descriptions of these rules is described in Table 2).
All the titled compounds possessed the predicted physicochemical properties within the acceptable
range as that of most of the marketed drugs. These compounds satisfy all the parameters related to
the Lipinski rule of five (except compound 9g in terms of molecular weight and log Po/w).
Jorgensen’s rule of three was also satisfied with the same set of compounds. Very few compounds
violated the solubility parameters (7j, 7i, 7g, 7f, and 7a). Overall, all the predicted parameters were
well within the range, and these compounds are not likely to encounter any problem in further
development, if any.
Table-2. In-silico predicted parameters of the titled compounds
1
0

Lipinski rule
Jorgensen’s rule of three
Log Violation LogS Metabolites PCaco Violations
Po/w
Code
MW
Donor
Accept
HB
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
0
HB
6
6
6
6
6
7
6
6
7
7
6
6
6
6
6
6
6
7
6
6
7
7
6
6
6
6
6
6
6
6
6
7
a
377.36
405.41
395.35
411.8
456.26
422.36
395.35
411.8
422.36
422.36
445.36
446.25
411.8
439.86
429.79
446.25
490.7
3.28
3.95
3.53
3.79
3.86
2.58
3.51
3.84
2.7
0
0
0
0
0
1
0
0
1
1
0
0
0
0
0
0
0
1
0
0
1
1
0
0
0
0
0
1
0
0
2
-5.294
-6.295
-5.694
-6.066
-6.181
-5.256
-5.431
-5.884
-5.175
-5.209
-7.611
-6.849
-6.055
-7.059
-6.459
-6.823
-6.943
-6.003
-6.226
-6.655
-5.933
-6.086
-7.557
-7.555
-6.701
-7.135
-7.474
-7.577
-6.846
-7.28
4
5
4
4
4
5
4
4
5
5
5
4
3
4
3
3
3
4
3
3
4
4
4
3
2
2
2
2
2
2
2
268.328
258.554
266.316
268.041
267.175
41.28
353.9
385.357
58.119
0
1
0
1
1
0
0
1
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
7
b
7
c
7
d
7
7
7
e
f
g
7
h
7
7
i
j
2.48
4.6
4.3
30.889
265.59
271.01
7
k
7
8
l
a
3.79
4.45
4.03
4.29
4.37
3.08
4.02
4.35
3.2
3.01
4.79
4.82
4.23
4.48
4.73
4.81
4.46
4.78
5.25
271.704
258.858
266.798
271.002
270.977
41.78
353.202
383.477
58.344
8
b
8
c
8
d
8
8
8
e
f
g
456.8
429.79
446.25
456.8
456.8
479.8
480.69
446.25
464.24
480.69
525.15
464.24
480.69
515.14
8
h
8
8
i
j
30.881
8
k
267.721
293.281
270.93
271.547
269.907
273.194
354.703
385.653
270.129
8
9
l
a
9
b
9
c
9
d
9
9
9
e
f
g
-8.261
Description: Lipinski rule- Number of violations of Lipinski’s rule of five. The rules are: mol MW < 500, logPo/w < 5,
donor HB ≤ 5, accept HB ≤ 10 and maximum 4 violations
Jorgensen’s rule of three - Number of violations of Jorgensen’s rule of three. The three rules are: logS> -5.7, PCaco>
2
2 nm/s, # Primary Metabolites < 7. Compounds with fewer (maximum 3) violations
In-silico molecular docking studies
Docking studies of the significantly active molecule (8a) was performed using the Glide module [38]
in Schrodinger. All docking calculations were performed using Extra Precision (XP) mode. A
scaling factor of 0.8 and a partial atomic charge of less than 0.15 was applied to the atoms of the
1
1

protein. Glide docking score (Table 3) was used to determine the best-docked structure from the
output. The interactions of these docked complexes were investigated further by using XP visualizer.
Validation of the docking protocol
The accuracy of the docking study was determined by finding the lowest energy conformation of the
co-crystallized ligand and resembles an experimental binding mode as determined by X-ray
crystallography. The docking procedure was verified by removing the co-crystallized ligand from the
binding site of the protein and re-docked the same co-crystallized ligand in the active binding site.
The hydrogen-bonding interactions and RMSD between the anticipated confirmation and the
observed X-ray crystallographic conformation was used for validation of the docking protocol. The
o
RMSD for the targeted protein was found to be 0.46A (Figure 5) indicated that the docking protocol
could be reliable for further study and ready for the docking of titled compounds.
Table-3. Scrutiny of amino-acid residues and water molecules contributed to the interactions with the
targeted protein- 5BS8
Code
H-bond
Halogen bond Glide score Glide energy
(PDB – 5BS8)
Interactions
(kcal/mol)
(kcal/mol)
8
a
-
ARG-128
ARG-128
-7.4
-48.5
Co-crystallized
ligand
ARG-128
ARG-128
ARG-128
-11.5
-67.7
(Salt bridge)
(Moxifloxacin)
*HOH-205,206,209,210
1
2

Figure 5. Superimposed view of the co-crystallized ligand in the active site of the protein -5BS8
Green color- Binding pose after docking, White color- X-ray native pose of ligand)
(
Figure 6. Co-crystallized ligand (Moxifloxacin) docked pose and interactions in the active site of the
protein-5BS8. [Color interpretation White- Hydrogen bond, Magenta – Salt bridge.
1
3

Figure 7. Significantly active compound 8a docked pose and interactions in the active site of the
protein-5BS8. [Color interpretation: Purple- Halogen bond]
The docking score of the significantly active compound 8a is low (Glide score-7.4 kcal/mol) as
compared with the co-crystallized ligand (Moxifloxacin) (Glide score -11.5 kcal/mol) (Table 3).
Analyzing the interactions of the molecule in the target protein revealed that the co-crystallized
ligand (Figure 6) showed one hydrogen bond interaction with the surrounded four water molecules
as well as another hydrogen bond interaction with amino acid residue ARG-128 of the target protein.
However, the significantly active molecule 8a exhibited two halogen bond interactions (Figure 7)
with the amino acid residue ARG-128 of the target protein and no hydrogen bond interactions as that
of the co-crystallized ligand. This might be the possible reason for the reduced docking score of the
significantly active moiety. Docking study and in-vitro anti-tubercular activity studies are correlating
the results. The significantly active compound exhibited the minimum inhibitory concentration of
3
0.35 µM among the titled compounds tested against all the mycobacterial cell lines i.e. MTB
H37Rv, MTB Spec 192, MTB Spec 210.
Single crystal X-ray crystallographic structure of compounds 8g and 9f
The suitable crystals of compounds 8g and 9f for single-crystal X-ray diffraction (SCXRD) study
were grown from the mixture of methanol and dichloromethane (1:3). The SCXRD measurements
were performed on the Rigaku XtaLAB P200 diffractometer using graphite monochromated Cu-Kα
1
4

radiation (λ = 1.54184 Å). The data was collected and reduced using CrysAlisPro (Rigaku Oxford
Diffraction) software. The data collection was carried out at 100 K, and the structures were solved
using Olex2 with the ShelX structure solution program using Direct Methods and refined with the
ShelXL refinement package using Least Squares minimization. The primary crystallographic data are
shown in Table 4.
The molecular structure of the compound 8g contains one linker per asymmetric unit (Figure 8) with
the chemical formula (C H ClFN O ), where one of the chlorine atoms (Cl3) has 80% occupancy
1
9
12
5
4
whereas the second chlorine atom (Cl4) has 20% occupancy. The structure crystallizes in the
monoclinic crystal system. On the other hand, the compound 9f crystallizes in the triclinic crystal
system, where two linkers are observed per asymmetric unit (Figure 8). Crystallographic data for the
compounds 8g and 9f has been deposited to the Cambridge Crystallographic Data Center, and the
corresponding deposition number is CCDC 1913106 and 1937576, respectively.
Table 4. Crystal data and structure refinement for compounds 8g and 9f
Identification code
8g
9f
Empirical formula
C H N O ClF
C H N O Cl
3
1
9
12
5
4
19 12
5
4
Formula weight
Temperature/K
428.790
293
480.70
100.0
Crystal system
Space group
Monoclinic
I 2/a
triclinic
P-1
a/Å
b/Å
c/Å
α/°
β/°
γ/°
Volume/Å
Z
29.8337(7)
4.7387(1)
27.6372(7)
90.000
110.662(3)
90.000
3655.8(2)
8
1.558
4.73450(10)
17.2736(2)
23.9809(3)
92.3020(10)
90.8230(10)
96.7280(10)
1945.77(5)
4
3
3
ρcalcg/cm
μ/mm-1
1.6408
4.629
2.305
F(000)
1752
983.2
Crystal size/mm3
Radiation
0.02 × 0.02 × 0.3
Cu Kα (λ = 1.54184)
10.6 to 159.8
0.3 × 0.05 × 0.02
Cu Kα (λ = 1.54184)
8.82 to 160
2
Θ range for data collection/°
-
37 ≤ h ≤ 35, -4 ≤ k ≤ 5, -34 ≤ l -5 ≤ h ≤ 3, -21 ≤ k ≤ 21, -30 ≤ l
Index ranges
≤
34
≤ 30
Reflections collected
9797
23507
Independent reflections
3823 [R = 0.0284, R
=
8200 [R = 0.0409, R
=
sigma
int
sigma
int
1
5

0
.0513]
0.0422]
8200/0/561
Data/restraints/parameters
Goodness-of-fit on F2
3823/0/281
1.092
1.014
Final R indexes [I>=2σ (I)]
R = 0.0633, wR = 0.1429
R = 0.0399, wR = 0.1058
1
2
1
2
8g
9f
Figure 8. ORTEP diagram of crystals 8g and 9f
In vitro cytotoxicity studies
Compounds with MTB MIC < 60 µM (7h, 7i, 7j, 8a, 8e, 8f, 8i, 8l, 9b, 9c, and 9d) were subjected to
cytotoxicity studies against normal human cell lines, human embryonic kidney (HEK) cells. Cell
viability was measured by in vitro MTT assay [39]. Cells were exposed to compound treatment at a
range of concentrations 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.62, and 7.81 µM to determine
their IC values from the dose-response curve. Data represent mean values of measurements ± s.d.
5
0
(Figure 9). The obtained result clearly indicates selectivity of the compounds for mycobacterium
HEK-293T IC50 12hrs
HEK-293T IC50 12hrs
HEK-293T IC50 12hrs
1
20
120
100
80
60
40
20
0
120
100
80
60
40
20
0
8
8
8
8
8
a: IC50=62.71M
e: IC50=299.8M
f: IC50=229.4M
i: IC50=197.4M
l: IC50=277.1M
7
h: IC50=45.15M
i: IC50=71.75M
9b: IC50=73.45M
9c: IC50=115.6M
9d: IC50=285.7M
100
7
8
0
7j: IC50=107.7M
6
0
4
0
2
0
0
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
Log concentration (M)
Log concentration (M)
Log concentration (M)
(A)
(B)
(C)
over normal cell lines. The most potent compound 8a (100% bacterial inhibition concentration, MIC
30.35 µM) showed significant selectivity towards MTB over HEK cells (HEK IC = 62.71 µM,
=
5
0
IC100 = 125.42 µM, bacterial MIC = 30.35 µM; selective = 125.42/30.35 = ~ 4 fold) and that the
remaining compounds were even more selective for bacterial cells over normal human cell lines.
1
6

Figure 9: IC results of the 11 compounds by MTT assay. Graph (A) represents the IC values for
5
0
50
7
h, 7i, 7j , Graph (B) represents the IC values for 8a,8e,8f,8i,8l and graph (C) represents the IC
50 50
values for 9b,9c,9d on HEK-293T cells when treated with the compounds at concentration range of
-2000 µM (n=2) for 12 hrs. Data represents mean ±SD.
7
3
. Conclusion
In this work, quinoxaline-1,4-di-N-oxide analogues with three different series were synthesized; 2-
methyl-3-(((substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline-1,4-di-N-oxide
derivatives,
6-chloro-3-(((1-(substituted
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2-
methylquinoxaline-1,4-di-N-oxide derivatives and 6,7-dichloro-2-(((1-(4-fluorophenyl)-1H-1,2,3-
triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline-1,4-di-N-oxide derivatives by molecular
hybridization approach. Amongst the synthesized compounds, 6-chloro-3-(((1-(substituted phenyl)-
1
H-1,2,3-triazol-4-yl)methoxy)carbonyl)-2-methylquinoxaline-1,4-di-N-oxide derivatives showed
significant anti-TB results. One of the compounds, 8a, showed excellent MTB activity with MIC
0.35 µM. Further, the few most active compounds in the series did not exhibit high cytotoxicity
3
against HEK cell lines. Finally, the significantly active compound, standard drug, and co-crystallized
ligands were subjected to molecular docking studies to explore their putative binding interaction
pattern at the active site of MTB DNA Gyrase.
Supporting information summary
Experimental procedures for intermediates (chemistry) and spectroscopic / analytical data along with
1
13
representative HNMR and CNMR spectra are available in supporting information.
Acknowledgements
KVGCS and SM thank DBT, New Delhi [BT/IN/Spain/39/SMl2017-18] for providing financial
support. The financial assistance provided by DIST FIST grant (SR/FST/CSI-240/2012), New Delhi
is gratefully acknowledged. SS thanks CSIR for providing SRF fellowship. Central analytical lab
facilities of BITS Pilani Hyderabad campus are gratefully acknowledged.
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Declaration of interest:
Authors declare no conflict of interest
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Thirty-one 1,2,3-triazole analogues of Quinoxaline-1,4-di-N-oxide were synthesized.
Screened the compounds against MTB H37Rv, spec. 192 and spec. 210.
8a exhibited good anti-TB activity with MIC 12.5 μg/mL against the tested strains.
Compounds were docked to MTB DNA Gyrase to know the binding interactions.
Most active compounds were less toxic against the normal HEK 293 cell line.
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