Novel thiomorpholine tethered isatin hydrazones as potential inhibitors of resistant Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bioorg.2021.105133

Novel chemotherapeutic agents against multidrug resistant-tuberculosis (MDR-TB) are urgently needed at this juncture to save the life of TB-infected patients. In this work, we have synthesized and characterized novel isatin hydrazones 4(a-o) and their thi

Full text of DOI:10.1016/j.bioorg.2021.105133

Bioorganic Chemistry 115 (2021) 105133
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel thiomorpholine tethered isatin hydrazones as potential inhibitors of
resistant Mycobacterium tuberculosis
,
*
Sivanandhan Karunanidhia, Balakumar Chandrasekarana, Rajshekhar Karpoormatha
,
,
Harun M. Patela b, Francis Kayambaa, Srinivas Reddy Merugua, Vishal Kumara,
Sanjeev Dhawana, Babita Kushwahaa, Mavela Cleopus Mahlalelaa
a Department of Pharmaceutical Chemistry, Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal (Westville), Durban 4000,
South Africa
b R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur (Dhule) 425405, Maharashtra, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Novel chemotherapeutic agents against multidrug resistant-tuberculosis (MDR-TB) are urgently needed at this
juncture to save the life of TB-infected patients. In this work, we have synthesized and characterized novel isatin
hydrazones 4(a-o) and their thiomorpholine tethered analogues 5(a-o). All the synthesized compounds were
initially screened for their anti-mycobacterial activity against the H37Rv strain of Mycobacterium tuberculosis
(MTB) under level-I testing. Remarkably, five compounds 4f, 4h, 4n, 5f and 5m (IC50 = 1.9 µM to 9.8 µM) were
found to be most active, with 4f (IC50 = 1.9 µM) indicating highest inhibition of H37Rv. These compounds were
further evaluated at level-II testing against the five drug-resistant strains such as isoniazid-resistant strains (INH-
R1 and INH-R2), rifampicin-resistant strains (RIF-R1 and RIF-R2) and fluoroquinolone-resistant strain (FQ-R1) of
MTB. Interestingly, 4f and 5f emerged as the most potent compounds with IC50 of 3.6 µM and 1.9 µM against RIF-
R1 MTB strain, followed by INH-R1 MTB strain with IC50 of 3.5 µM and 3.4 µM, respectively. Against FQ-R1 MTB
strain, the lead compounds 4f and 5f displayed excellent inhibition at IC50 5.9 µM and 4.9 µM, respectively
indicating broad-spectrum of activity. Further, molecular docking, ADME pharmacokinetic and molecular dy-
namics simulations of the compounds were performed against the DNA gyrase B and obtained encouraging
results.
Synthesis
Multidrug-resistant tuberculosis
Isatin
Docking
MD simulations
1. Introduction
and rifampicin [2]. However, poor patient compliance owing to long-
term treatment regimen and lack of new broad spectrum, potent anti-
Tuberculosis (TB), a communicable disease mainly caused by a single
infectious microorganism Mycobacterium tuberculosis (MTB), and is
known for one of the top 10 causes of death worldwide [1]. A recent
global report (2019) on tuberculosis by world health organization
(WHO) documented about 1.5 million deaths among 10 million cases of
TB that clearly alarming the severity of this deadly disease [1]. More-
over, a syndemic interaction between acquired immuno deficiency
syndrome (AIDS) and TB has become an epidemic, since 20% of the
deaths are due to co-infection of TB in HIV-positive patients. Current
treatment of TB composed of initial two months of intensive stage
therapy with four important first-line oral anti-TB drugs (isoniazid,
rifampicin, pyrazinamide and ethambutol) followed by four months of
continuation stage management with the administration of isoniazid
mycobacterial drugs has greatly contributed to the spontaneous emer-
gence of drug-resistant TB such as multidrug-resistant TB (MTR-TB;
refers to the resistant against isoniazid and rifampicin) and extremely
drug resistant TB (XDR-TB; a MDR-TB with resistance towards fluo-
roquinolones and/or any second-line anti-TB drugs) [3]. Steady rise in
MDR-TB and XDR-TB, especially in developing countries has further
complicated the treatment and management of TB, causing serious
health and socio-economic concerns. In addition to the existing chal-
lenges, recent emergence of totally drug resistant TB (TDR-TB; resistant
against all currently available anti-TB drugs) [4,5] has become a greatest
threat to human kind. It is also very important to note that there are no
new drugs introduced into the market for the treatment of MDR/XDR-TB
for the past four decades except Bedaquiline, which got provisional
* Corresponding author.
E-mail addresses: karpoormath@ukzn.ac.za, rvk2006@gmail.com (R. Karpoormath).
https://doi.org/10.1016/j.bioorg.2021.105133
Received 23 May 2021; Received in revised form 22 June 2021; Accepted 26 June 2021
Available online 3 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
approval in the year 2012 by US-FDA [6]. This is the latest development
in the current TB management and this scenario indicates that there is an
urgent need to discover novel drug scaffolds which can resolve the drug
resistance problems with broad-spectrum of activity, and devoid of
serious side effects.
alone [24]. In the subsequent year, the same research group [25] re-
ported a higher inhibitory activity for the ciprofloxacin tethered isatin
hybrid. Sriram et. al., [26] demonstrated Gatifloxacin hybridized isatin
(II) as promising anti-mycobacterial compound with MTB DNA gyrase
inhibition (Fig. 1). Furthermore, several isatin hybrids (III-VI) have
displayed the encouraging anti-mycobacterial activity profiles [27-30]
(Fig. 1).
Structure-based drug design (SBDD) is one of the successful ap-
proaches in the new drug discovery involving the use of 3D structures of
the target proteins [7-9]. DNA gyrase including Mycobacterium species is
a tetrameric holoenzyme with two A (GyrA) and two B (GyrB) subunits
that are responsible for maintaining the topology of the DNA duplex
during the replication process. GyrA subunit involved in the breakage
and reunion of the DNA, whereas the GyrB subunit exhibits an ATP-ase
activity. In the absence of ATP, the DNA gyrase enzyme catalyzes only to
the relaxation of the supercoiled DNA. DNA gyrase inhibition using
Hydrazone, a bioactive linker is found mostly in the heterocyclic
medicinal compounds. Sriram et al., [31] investigated the anti-
mycobacterial activity of isonicotinyl hydrazones and reported potent
inhibition against MTB H37Rv strain. Küçükgüzel et al., [32] also iden-
tified some hydrazone derivatives as effective anti-mycobacterial com-
pounds. Similarly,
a momentous candidate, thiomorpholine can
improve the anti-mycobacterial activity of heterocyclic compounds
through a synergistic effect. Sutezolid (VI), a thiomorpholine containing
anti-mycobacterial compound (Fig. 1) is presently under the clinical
development as a drug candidate for the treatment of XDR-TB [33].
These inspiring investigations and in continuation of our research to-
wards the discovery of novel anti-mycobacterial agents [34-36], we
envisaged to design, synthesize and evaluate potential anti-
mycobacterial activities of novel isatin-based analogues.
fluoroquinolone antibiotics is
a clinically validated therapeutic
approach to treat drug-resistant bacterial infections [10]. MTB DNA
gyrase has recently attracted greater attention as potential drug target
for the new generation of anti-TB drugs to combat MDR-TB, and
fluoroquinolone-resistant MTB. There are at least two types of gyrase
inhibitors reported in the literature with potent activity against MTB
[11-13] such as fluoroquinolones and aminopiperidines as GyrA in-
hibitors, while aminopyrazinamides, thiazolopyridine ureas,
aminobenz-imidazoles and pyrrolamides reported as GyrB inhibitors
[14]. GyrB is the primary target for clinically important drug Novobi-
ocin, the only approved GyrB inhibitor [15]. In 2013, the co-crystallized
DNA GyrB ATPase protein (PDB ID: 4B6C) with bound inhibitor (ami-
nopyrazinamide) was reported, which paved the way to understand the
structural interactions at the DNA GyrB active-site for the discovery of
novel anti-TB agents [16].
2. Results and discussion
2.1. Chemistry
The synthesis of the final thiomorpholine tethered isatin hydrazones
was achieved in higher yield through a facile and straightforward three
step reactions employing a microwave reactor (CEM Discover, Explorer-
12 Hybrid, Microwave conditions: 80 ◦C at 150 psi) except for the final
step. Indoline-2,3-dione [isatin; 1a] or 5-chloro-indoline-2,3-dione [5-
chloro-isatin; 1b] was treated individually with hydrazine hydrate
(50–60%) in methanol to yield respective 3-hydrazonoindoline-2-ones
(2a and 2b) via a simple condensation reaction [37,38]. Under micro-
wave irradiation, the free primary amino group present in 2a or 2b
reacted with various aldehydes (3) in acidic medium to form respective
Schiff-bases 4(a-o) in good yields [39,40]. The tethering of
It is well documented in literature that isatin (1H-indole-2,3-dione) is
one of the versatile building block or scaffolds in medicinal chemistry
due to its wide range of pharmacological activities including anti-viral
[17], anti-angiogenic [18], anti-cancer [19], anti-malarial [20], anti-
microbials [21,22]. In particular, tryptanthrin (I) (Fig. 1), an isatin hy-
bridized natural product reported as a potent anti-mycobacterial agent
[23]. Feng et al., documented that isatin tethered balofloxacin exhibited
improved anti-mycobacterial activity against H37Rv than balofloxacin
Fig. 1. Literature reported anti-mycobacterial agents to design of novel isatin hybrids.
2

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
thiomorpholine to 4(a-o) was achieved using 34% formaldehyde as a
source of methylene (–CH2) linker in ethanol, yielding the final target
compounds 5(a-o), quantitatively (Scheme 1).
weights. All the spectral images (FT-IR, 1H NMR, 13C NMR and repre-
sentative HRMS) of the compounds are provided in the supporting
information.
The expected structures of the isatin hydrazones 4(a-o) and the
thiomorpholine tethered final compounds 5(a-o) were confirmed based
2.2. Biological studies
on their spectral data. In FT-IR spectra, the appearance of the vibrational
ꢀ 1
bands around 1510 cmꢀ 1 for N H bending, 1597 cm (C O stretch),
–
–
–
2.2.1. Anti-mycobacterial studies under Level-I (MTB H37Rv) evaluation
In vitro anti-mycobacterial evaluation of all the newly synthesized
intermediates 4(a-o) and the final compounds 5(a-o) was carried out at
Infectious Disease Research Institute (IDRI) within the National Institute
of Allergy and Infectious Diseases (NIAID) screening program (Bethesda,
MD, USA). The minimum inhibitory concentration (MIC) was deter-
mined against M. tuberculosis strain H37Rv grown under aerobic condi-
tions by using a dual read-out (OD590 and fluorescence) assay protocol
[41,42]. All the synthesized compounds exhibited significant anti-
mycobacterial activity profile against the tested MTB H37Rv strain
under level-I screening program (Table 1). Remarkably, 5-nitro-thio-
phene substituted hybrid 4f presented highest potency against MTB
1714 cmꢀ 1 (C N stretch), and 3133 cm for N H stretching sup-
ꢀ 1
–
–
–
ported the formation of compounds 4(a-o). This was further substanti-
ated from the 1H NMR spectra, wherein a distinctive singlet peak
resonating around δ 8.37 ppm to δ 8.94 ppm was attributed to the Schiff
base proton (-N = CH–) and isatin-NH appeared as singlet between δ
10.09 ppm and δ 11.02 ppm, respectively confirming the formation of 4
(a-o). Apart from aromatic or heterocyclic carbon signals, the 13C NMR
spectra displayed a characteristic carbon signal of Schiff base carbon (-N
= CH–) between δ 152 ppm to δ 162 ppm, validating the formation of
compounds 4(a-o).
–
The absence of the singlet signal corresponding to N H proton of
isatin moiety and appearance of new methylene (–CH2–) signal at δ 4.49
ppm in 1H NMR spectra of 5(a-o) confirmed the anticipated structures of
final compounds. Further, new triplet signal in the aliphatic region
around δ 2.58 ppm, thiomorpholine protons at δ 2.84 ppm and the most
characteristic Schiff base protons (-N = CH–) resonating between δ 8.51
ppm and δ 9.01 ppm authenticated the formation of 5(a-o). This has
been further characterized by 13C NMR of 5(a-o) which demonstrated
the significant methylene carbon (–CH2–) signal appeared in the range
of δ 62.06 ppm to δ 62.79 ppm and the presence of carbon signals (δ
26.93 ppm and δ 55.73 ppm) corresponding to the thiomorpholine
moiety. In addition, the respective accurate mass signals displayed by
the HRMS data confirmed the formation of final compounds 5(a-o),
which revealed the positive correlation with the expected molecular
H
37Rv strain with a promising IC50 value of 1.9 µM and MIC at 2.3 µM. A
couple of other compounds, 4h bearing 5-nitro-furyl substitution and
the bioisosteric pair 4n containing 5-nitro-thiophene substituent,
exhibited relatively higher MTB H37Rv inhibition with IC50 values 7.6
µM and 9.8 µM, respectively. Further, 4h and 4n demonstrated
impressive MIC values at 12 µM and 17 µM, respectively indicating their
encouraging anti-mycobacterial potency. The unsubstituted thiophene
hybridized compound 4d revealed a moderate inhibition with IC50 value
of 56 µM. Remaining compounds in this series bearing other sub-
stitutions such as aromatic or six-membered heterocyclic rings were
found to be poor or moderately active. It was observed that compounds
having 5-nitro-thiophene/furyl substitutions indicated best activity
against the MTB H37Rv strain.
Scheme 1. Synthesis of thiomorpholine tethered isatin hydrazones. Reagents and conditions: (i) Hydrazine hydrate, methanol, microwave irradiation, 80 ◦C, 2 mins,
150 psi; (ii) Substituted aromatic/heteroaromatic aldehydes (3), ethanol, microwave irradiation, 80 ◦C, 10 mins, 150 psi; (iii) Thiomorpholine, formaldehyde,
ethanol, stir, RT, 2 h.
3

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
Table 1
Anti-mycobacterial activity data of the synthesized compounds 4(a-o) and 5(a-o).
Compound
M. tuberculosis a (H37Rv)
Compound
M. tuberculosis a (H37Rv)
Compound
M. tuberculosis a (H37Rv)
MIC
IC50
IC90
MIC
IC50
101
IC90
MIC
IC50
50
IC90
50
>200
>100
>100
>200
>200
>200
4i
4e
4f
4a
>100
29
60
2.3
1.9
3.4
>100
>100
>100
4b
4c
4j
>200
113
56
>200
>200
92
>200
180
65.4
135
4g
4k
88
56
12
7.6
10
>200
>50
>50
4h
4d
4l
Compound
M. tuberculosis a (H37Rv)
Compound
M. tuberculosis a (H37Rv)
Compound
M. tuberculosis a (H37Rv)
MIC
IC50
132
IC90
MIC
IC50
190
IC90
MIC
7.0
IC50
3.9
IC90
>200
>200
>200
>200
6.9
5f
4m
4n
5b
17
9.8
17
>200
140
>200
>200
66
150
5g
5c
5d
>200
>200
190
95
>200
>200
81
32
75
9.0
6.4
8.4
4o
5h
5i
>200
120
>200
>200
190
>200
5a
5e
Compound
M. tuberculosis a (H37Rv)
Compound
M. tuberculosis a (H37Rv)
Compound
M. tuberculosis a (H37Rv)
MIC
IC50
190
IC90
MIC
5.6
IC50
3.9
IC90
4.7
MIC
IC50
IC90
>200
>200
Stdb
0.0067
–
–
(continued on next page)
4

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
Table 1 (continued)
Compound
M. tuberculosis a (H37Rv)
Compound
M. tuberculosis a (H37Rv)
Compound
M. tuberculosis a (H37Rv)
MIC
IC50
IC90
MIC
IC50
IC90
MIC
IC50
IC90
5j
5m
>200
>200
100
190
>200
>200
37
87
23
47
34
77
5k
5l
5n
5o
a
Concentration in µM.
b
Std is Rifampicin.
Recent literature reports indicated that thiomorpholine moiety
enhanced the antimicrobial and mycobacterial potency through syner-
gistic effect [29,43]. To further improve the anti-mycobacterial potency
of 4(a-o) series, we envisaged to incorporate thiomorpholine moiety to
initially evaluated against five drug-resistant isolates namely INH-R1,
INH-R2, RIF-R1, RIF-R2, and FQ-R1 of MTB under aerobic conditions.
The results of level-II testing data indicated once again that compounds
4f and 5f presented considerable activity against all the tested drug-
resistant strains (Table 2), which was followed by 4n and 5m. Unam-
biguously, the thiomorpholine-tethered analogue 5f exhibited an
interesting and potent inhibition of rifampicin resistant R1 (RIF-R1)
strain with an IC50 of 1.9 µM that is nearly equal to the standard drug
rifampicin (IC50 = 1.2 µM). Against RIF-R2 strain, 5f demonstrated
– –
N H via a lipophilic methylene (–CH2) linker, resulting in a
isatin
series of novel compounds 5(a-o). The anti-mycobacterial screening
results of 5(a-o) revealed compound 5f with the best inhibition against
MTB H37Rv strain with IC50 value of 3.9 µM and MIC of 7.0 µM. How-
ever, it was observed that the activity of compound 5f was quite lower
than 4f. Interestingly, both 4f and 5f had one common feature that is 5-
nitro-thiophenyl substitution in their structures which contributed
positively and substantiating the role of electron-withdrawing nitro
higher potency (IC50 = 8.4 µM) than the rifampicin (IC50 > 50 μM)
indicating the potential anti-TB activity against rifampicin-resistant
MTB strains. Importantly, 5f showed higher inhibition with IC50 3.4
µM (against INH-R1 strain) and 5.3 µM (against INH-R2 strain) than the
group on
π-excessive 5-membered heterocyclic system towards the
accomplishment of higher anti-mycobacterial potency. An equipotent
compound 5 m containing unsubstituted furyl ring displayed IC50 value
of 3.9 µM and MIC of 5.6 µM, respectively. Bioisosteric replacement of
the unsubstituted furyl in compound 5 m with 5-nitro substituted furyl
ring resulted the compound 5 h with one-fold lower anti-mycobacterial
activity (IC50 = 6.4 µM). Though the compound 5n had 5-nitro-thio-
phenyl substitution on the chloroisatin scaffold, it could only to show
a moderate inhibition at IC50 23 µM. Similarly, compound 5o (IC50 = 47
µM) with pyridine substituent displayed no significant activity, which
standard drug isoniazid (IC50 > 200 μM) which authenticated for their
prospective use in INH-resistant TB therapy. Furthermore, 5f displayed
higher inhibition of FQ-R1 strain with the promising IC50 of 4.9 µM
which is considerably potent than the standard drug levofloxacin (IC50
= 12 µM) indicating 5f could be a lead compound effective against XDR-
TB. Investigation of SAR of the corresponding intermediate 4f desig-
nated a moderate inhibition against rifampicin resistant R1 (RIF-R1)
strain with an IC50 of 3.6 µM, a two-fold lower activity compared to 5f.
Remarkably, 4f demonstrated 10-fold higher potency (IC50 = 5.3 µM)
could be attributed to the
π-deficient pyridine system for the reduced
than the standard reference rifampicin (IC50 > 50 μM) against RIF-R2
potency. All the remaining compounds in this series displayed little or no
anti-mycobacterial activity. Level-1 anti-mycobacterial screening of the
thirty compounds led to the identification of five most active com-
pounds, which were considered for further assessment for level-II anti-
mycobacterial screening against drug-resistant MTB strains.
strain. The same compound 4f demonstrated equipotent anti-TB activ-
ity (IC50 = 3.5 µM) as compared to 5f against INH-R1 strain. Against
INH-R2 strain, 4f presented an outstanding inhibition (IC50 = 4.6 µM)
than the standard drug isoniazid (IC50 > 200 μM) which validated its
anti-TB potential. Moreover, 4f exhibited two-fold higher inhibition of
FQ-R1 strain (IC50 = 5.9 µM) than the standard drug levofloxacin (IC50
= 12 µM) thereby signifying the intermediate 4f could be effective
against XDR-TB strain. Thus, this work resulted in the discovery of two
promising leads 4f and 5f as highly potent anti-mycobacterial com-
pounds, which can be further optimized as potential inhibitors of MDR-
and XDR-resistant MTB strains (Fig. 2).
2.2.2. Anti-mycobacterial studies under Level-II (Drug-resistant MTB
strains) evaluation
With an encouraging anti-mycobacterial result from level-I
screening, five most active compounds 4f, 4h, 4n, 5f and 5m were
further considered for level-II screening to evaluate their efficacy under
different circumstances such as varied oxygen conditions, activity
against multidrug-resistant mycobacterial isolates and other strains of
mycobacterial species. In level-II testing, these five compounds were
5

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
Table 2
Anti-mycobacterial activity data of selected compounds against five drug-resistant isolates of M. tuberculosis.
Compound
INH-R1a
INH-R2b
RIF-R1c
RIF-R2d
FQ-R1e
MIC
IC50
IC90
MIC
IC50
IC90
MIC
IC50
IC90
MIC
IC50
IC90
MIC
IC50
IC90
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
4f
5.0
21
3.5
4.4
21
8.6
21
4.6
11
7.5
9.2
20
3.6
4.4
7.7
1.9
11
7.7
11
19
5.3
25
24
20
5.9
22
21
4h
16
14
17
31
27
35
41
4n
22
14
16
43
24
43
24
56
24
59
130
16
28
>100
23
5f
5.5
42
3.4
5.9
29
14
5.3
16
9.6
24
14
17
8.4
25
18
4.9
27
5m
26
28
20
28
25
38
39
44
32
Rifampicin
Isoniazid
Levofloxacin
0.018
>200
1.2
0.0084
>200
0.64
0.022
>200
1.4
0.0065
>200
1.4
0.0047
>200
0.84
0.012
>200
1.4
2
1.2
0.15
0.59
2.3
0.21
0.91
>50
0.62
1.1
>50
0.54
0.6
>50
0.6
1.2
0.027
0.35
20
0.013
0.36
12
0.039
0.47
22
0.17
0.76
a
b
c
INH-R1 was derived from H37Rv and is a katG mutant (Y155* = truncation).
INH-R2 is strain ATCC35822.
RIF-R1 was derived from H37Rv and is a nrpoB mutant (S522L).
RIF-R2 is strain ATCC35828.
d
e
FQ-R1 is a fluoroquinolone-resistant strain derived from H37Rv and is a gyrB mutant (D94N). INH, isoniazid; RIF, rifampicin; FQ, Fluoroquinolone.
Fig. 2. Anti-mycobacterial activity of the most active compounds 4f and 5f.
2.2.3. MIC assay under hypoxic (low) oxygen condition and MBC
determination
condition (IC50 = 5.2 µM). Moreover, 5f displayed highest inhibition
(MBC = 7 µM) than 4f, which is also evident for the promising anti-
mycobacterial activity (Table 3).
The promising five compounds (4f, 4h, 4n, 5f and 5m) were evalu-
ated against MTB H37Rv grown under hypoxic and normal oxygen
conditions. The low oxygen recovery assay (LORA) protocol was fol-
lowed for the evaluation under hypoxic conditions [44], whereas the
minimum bactericidal concentration (MBC) and MIC were determined
against MTB H37Rv grown under aerobic conditions in 7H9-Tw-OADC
medium. The outcome of these experiments revealed compound 4f
most potent under low oxygen level at an exciting IC50 of 0.65 µM and at
normal oxygen level with IC50 1.6 µM. Further, the MBC value obtained
for 4f was also notable (IC50 = 11.5 µM). Similarly, the thiomorpholine
tethered compound 5f also exhibited comparatively high inhibition at
normal oxygen level with an impressive IC50 of 2.2 µM than the hypoxic
2.2.4. Cytotoxicity and intracellular anti-mycobacterial activity assays
Cytotoxicity and intracellular anti-mycobacterial activity of the five
most active compounds were assessed by employing THP-1 human
monocytic cell line, and THP1 cells infected with MTB, respectively
[45]. As it was obvious for the isatin-based medicinal compounds were
cytotoxic [46], the tested compounds showed moderate cytotoxicity. Of
the tested compounds, the intracellular activity of 4f and 5f was higher
at IC50 of 6.0 µM and 6.5 µM, respectively which specified higher po-
tency (Table 3).
6

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
Table 3
Anti-mycobacterial activity (under hypoxic and under aerobic), MBC, cytotoxicity and intracellular activity of the selected compounds (4f, 4n, 4h, 5f and 5m) against
M. tuberculosis H37Rv grown under various conditions.
Compound
Anti-mycobacterial activity
Under Low Oxygena
Minimum Bactericidal
Cytotoxicity c IC50
Intracellular
Concentrationb (MBC,
μM)
(μM)
Activity (against
Under aerobic
M. tuberculosis) d
MIC
M)
IC50
(
μ
M)
IC90
M)
MIC
M)
IC50
(
μ
M)
IC90
M)
IC50
M)
IC90
M)
(μ
(μ
(μ
(μ
(μ
(
μ
4f
> 200
130
80
0.65
8
13
4.5
1.6
20
4.4
11.5
17
0.39^
2.9
6
16
4n
30
41
> 50
23
18
35
4h
24
42
23
12
12
3.8
14
22
5f
50
5.2
16
33
4.4
2.2
17
4.8
7
0.84^
5.2
6.5
15
10
5m
39
24
41
44
28
19
Rifampicin
Metronidazole
Staurosporine
Isoniazid
0.13#
200$
ND
0.00041#
29$
0.0065#
110$
ND
0.0096#
> 200$
ND
0.00072#
> 200$
ND
0.0025#
> 200$
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.23
ND
ND
ND
0.29
ND
ND
0.018
ND
ND
ND
ND
ND
ND
ND
a
Organisms grown under hypoxic conditions were assessed using LORA assay.
Organisms were grown under aerobic conditions in 7H9-Tw-OADC medium.
Cytotoxicity was determined using the human monoocytic (THP-1) cell line.
Intracellular activity was determined using THP1 infected with M. tuberculosis.
Calculated averages for rifampicin for each run (number of replicates 6).
Metronidazole was run as a control once in each run.
b
c
d
#
$
^
compounds found to be cytotoxic; ND: Not determined.
2.2.5. Anti-mycobacterial evaluation against other disease relevant
mycobacterial species
studies into the Mycobacterial ATP synthase enzyme binding pocket.
Docking methodology was validated by measuring RMSD of the co-
crystalized (internal) ligand and extracted internal ligand of the
docked target protein–ligand complex structure, which served as a
control docking model as shown in Fig. 3. The docking result showed
that Glide SP docking evaluated the optimal orientation of the co-
crystallized ligand. RMSD value of 1.123 suggested that the methodol-
ogy was perfect for predicting the binding affinity for unknown ligands.
Docking result demonstrated that potent compounds 4f, 4n, 4h, 5f, 5m
also shown significant docking score with GyrB ATPase domain as
shown in Table 5. The indolin-2-one derivative 4f showed the hydrogen
bond interaction with the Asp79 and Arg141 via the NH and NO2
functional group. Similarly, N-substituted indolin-2-one derivative 5f
showed hydrogen bond interaction with the Arg141 and Glu48 via the
NO2 functional group and nitrogen of thiomorpholine ring as given in
Fig. 4. .
The compounds 4f, 4h, 4n, 5f and 5m were also screened for their in
vitro anti-TB activity against other disease-relevant Mycobacterial species
such as Mycobacterium abscessus and Mycobacterium avium using MABA
method [47]. .
The activity data demonstrated a moderate anti-mycobacterial ac-
tivity for the compound 5 m towards M. avium (IC50 = 50 µM) than
M. abscessus (MIC = 84 µM). However, remaining compounds did not
show notable inhibition against the screened Mycobacterial species
(Table 4).
2.2.6. Structure-activity relationship (SAR) studies
Fig. 2 presents anti-TB spectrum of activity profiles of the most active
compounds 4f and 5f. The SAR analysis indicated the plausible reason
for the highest inhibition and could be attributed to the presence of more
reactive 5-membered heterocyclic thiophene moiety substituted with
highly electron-withdrawing nitro (NO2) group. In the absence of nitro
substitution on five-membered thiophene/furyl ring, a decrease in the
activity was observed. A systematic SAR study through the analysis of
level-I and level-II results paved a way towards the discovery of the lead
compounds 4f and 5f, wherein the latter being the most active against
the multidrug-resistant strains of MTB, exclusively.
2.3.2. MM-GBSA binding free energy analysis
MM-GBSA binding free energy analysis was carried out of the nine
potent protein–ligand complexes along to assess the affinity of ligands to
the target proteins. The binding free energies (ΔG Bind) evaluated by this
method are more efficient than the Glide score values for the assortment
of protein–ligand complexes. The primary energy components, such as
Coulomb or Electrostatics Interaction energy (ΔG Bind Coulomb), Lipo-
philic Interaction energy (ΔG Bind Lipo), Generalized Born electrostatic
solvation energy (ΔG Solv-GB) and van der Waals interaction energy (ΔG
Bind vdW) altogether contribute to the analysis of MM-GBSA-based rela-
tive binding affinity. The binding energies and the contributing factors
calculated for the protein dock complexes are mentioned in Table 6.
Among all the studied complexes, 4i complex showed high binding free
energies (ΔG Bind = -58.49 Kcal/mol). Among the in vitro tested potent
compounds; 5f (ΔG Bind = -53.48 Kcal/mol), 4n (ΔG Bind = -51.93 Kcal/
mol), 4f (ΔG Bind = -51.93 Kcal/mol), and 4h (ΔG Bind = -50.63 Kcal/
mol) have shown significant binding free energy (Table 6). Correlation
between docking score and binding free energy is given in Fig. 5.
2.3. In silico study
2.3.1. Molecular docking studies
The molecular docking tool, GLIDE, was used for ligand docking
Table 4
Anti-mycobacterial activity of the selected compounds (4f, 4n, 4h, 5f and 5m)
against other disease-relevant Mycobacterial species.
Compound
M. abscessusa
M. aviumb
MIC (
μ
M)
IC50
(μ
M)
IC90
(μ
M)
MIC (μM)
4f
> 200
> 200
190
> 200
> 200
> 200
180
> 200
> 200
> 200
> 100
90
> 200
100
4n
2.3.3. ADME prediction and drug likeliness
4h
> 200
> 100
50
In order to describe the drug-likeliness of the hybrids 4(a-o) and 5(a-
o), we screened them for Lipinski’s rule of five, wherein the molecular
properties will be correlated with the oral bioavailability of the
respective molecules. QikProp of Schrodinger Maestro-12.1 [48] was
employed for the computation of various parameters and the results are
5f
> 100
98
5m
84
Rifampicin
3.3
2.1
3.1
0.1
a
b
M. abscessus subsp. bollettii 103.
M. avium subsp. avium 2285 (S).
7

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
Fig. 3. The impeccably overlapped conformation of the docked ligand B5U (purple-coloured carbon backbone) with respect to its crystallized conformation (green-
coloured carbon backbone) obtained from the bioactive complex structure 4B6C. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
be>80% and QPPCaco values were > 500 with the exception of 4f, 4h,
Table 5
4n, 5f, 5 h, and 5n. The partition coefficient (QPlogPo/w) and water
Glide SP docking score of the synthesized compounds.
solubility (QPlogS) are also important parameters for the absorption and
Compound
Code
Docking
Score
Glide
Glide
Glide Energy
distribution of the drugs [52]. QPlogPo/w and QPlogS values were
computed, which ranged from 1.033 to 3.401 and ꢀ 4.365 to ꢀ 0.303,
respectively. Thus, QikProp predicted the physico-chemically crucial
descriptors and pharmaceutically relevant properties, all of which
established that the hybrids confer good drug-like properties (Table 7
and footnote) and can be considered for the further drug development
events.
Rewards
Emodel
4k
4f
ꢀ 7.785753
ꢀ 7.723262
ꢀ 7.723262
ꢀ 7.613359
ꢀ 7.604208
ꢀ 7.532954
ꢀ 7.506493
ꢀ 7.428364
ꢀ 7.424273
ꢀ 7.397044
ꢀ 7.354074
ꢀ 7.340918
ꢀ 7.333291
ꢀ 7.288588
ꢀ 7.262156
ꢀ 7.189469
ꢀ 7.176897
ꢀ 7.10449
ꢀ 2.357329
ꢀ 2.482856
ꢀ 2.482856
ꢀ 2.435352
ꢀ 2.016184
ꢀ 2.553628
ꢀ 2.586952
ꢀ 2.154589
ꢀ 2.668566
ꢀ 1.70923
ꢀ 58.680619
ꢀ 59.216648
ꢀ 59.216648
ꢀ 58.15461
ꢀ 69.982567
ꢀ 58.3836
ꢀ 42.229731
ꢀ 43.842531
ꢀ 43.842531
ꢀ 41.869961
ꢀ 51.69332
ꢀ 42.20331
ꢀ 38.379974
ꢀ 44.141524
ꢀ 40.288377
ꢀ 51.766265
ꢀ 42.504819
ꢀ 45.544755
ꢀ 48.858482
ꢀ 46.504151
ꢀ 43.975905
ꢀ 41.692272
ꢀ 36.874597
ꢀ 45.64237
ꢀ 37.296104
ꢀ 53.305054
ꢀ 44.348117
ꢀ 45.570659
ꢀ 47.53827
ꢀ 48.558695
ꢀ 53.610616
ꢀ 39.778039
ꢀ 47.863721
ꢀ 47.713697
ꢀ 47.696196
ꢀ 49.724288
4n
4l
5f
4h
4g
4i
ꢀ 54.185238
ꢀ 61.148719
ꢀ 55.929295
ꢀ 67.573204
ꢀ 57.837476
ꢀ 63.44352
ꢀ 67.868708
ꢀ 64.171054
ꢀ 62.303222
ꢀ 58.709521
ꢀ 54.333397
ꢀ 61.625476
ꢀ 51.197577
ꢀ 68.163663
ꢀ 60.682799
ꢀ 61.909195
ꢀ 64.268893
ꢀ 65.6246
2.3.4. Molecular dynamic simulation study
4d
5b
4o
4j
A molecular docking study was performed using the rigid crystal
structure of GyrB ATPase domain. Hence, we have evaluated target and
lead compounds interactions in the dynamic behavior using molecular
dynamic simulation to obtain the stable binding conformation. Com-
pound 5f in complex with GyrB ATPase domain was considered for the
molecular dynamic simulation for 10 ns, using simple point charge
(SPC) water mode. The Root Mean Square Deviation (RMSD), Root
Mean Square Fluctuation (RMSF), and Protein-Ligand Contacts were
analyzed from the MD simulation trajectories to study thermodynamic
conformational stability during 10 ns period. MD simulation trajec-
tories’ RMSD analysis denotes the protein backbone’s stability, when
bound with the specific ligand within the dynamic condition. It also
provides brief insights into its structural conformation during the MD
simulation. Lower RMSD value throughout the MD simulation suggests
the higher stability of the protein–ligand complex, whereas higher
RMSD value shows comparatively low stability of the protein–ligand
complex [34,39].
ꢀ 2.335253
ꢀ 1.73906
5a
5m
4b
4a
4m
5d
4e
5h
5e
5g
5i
ꢀ 1.973769
ꢀ 2.146985
ꢀ 1.982486
ꢀ 2.392884
ꢀ 2.665586
ꢀ 2.118613
ꢀ 2.801185
ꢀ 2.073834
ꢀ 2.264101
ꢀ 2.117143
ꢀ 1.673494
ꢀ 1.467624
ꢀ 1.663096
ꢀ 2.604747
ꢀ 2.03999
ꢀ 7.035323
ꢀ 7.003703
ꢀ 6.965062
ꢀ 6.927207
ꢀ 6.884912
ꢀ 6.705468
ꢀ 6.696261
ꢀ 6.691652
ꢀ 6.596076
ꢀ 6.536609
ꢀ 6.494694
ꢀ 6.169308
5j
5n
4c
5o
5c
5 k
5 l
ꢀ 68.78346
ꢀ 53.698382
ꢀ 63.272763
ꢀ 64.697257
ꢀ 61.291287
ꢀ 65.784868
ꢀ 2.038783
ꢀ 1.863428
ꢀ 1.460659
The RMSD graph result of compound 5f is shown in Fig. 6. Initially,
ligand was unstable at around 3 ns with RMSD of the graph line showed
an increasing trend from 0 to 10 ns with RMSD value of 4.4 Å. After 4 ns,
the promising result was observed, and the graph line was stable till 10
ns. The overall RMSD analysis revealed that fluctuations in a graph
during 10 ns simulation are within the standard range of RMSD. Ligand
RMSD of 1–3.6 Å indicates that the compound 5f bound tightly within
the cavity of GyrB ATPase domain. The RMSF value represents the
mobility and flexibility of each protein residue during the entire simu-
lation. Greater the RMSF values indicate more flexibility during the MD
simulation, while the lower value of RMSF reflects the stability of the
system [34]. Compound 5f-GyrB ATPase domain complex yielded little
fluctuations up to 2 Å, which is perfectly acceptable Fig. 7.
summarized in Table 7. Remarkably, all the synthesized hybrid com-
pounds were found to be in agreement with Lipinski’s rule of five [49]
and Jorgensen’s rule of three as well. The pharmacokinetic ADME
properties play significant roles in the determination of the safety and
efficacy of drug-like compounds. Human intestinal absorption (HIA) and
Caco-2 permeability (QPPCaco) parameters are the best markers of the
absorption of the drug in the intestine and Caco-2 monolayer penetra-
tion, respectively. HIA data are the sum of bioavailability and absorption
evaluated from the ratio of excretion or cumulative excretion in urine,
bile, and feces [50]. Moreover, QPPCaco permeability parameter acts as
a crucial feature regulating the metabolism of drugs [51]. The predicted
percentages of human oral absorption for the hybrids were found to
Protein-ligand interactions can be monitored throughout the
8

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
Fig. 4. Binding interaction of the compounds 4f (A: 3D view; B: 2D view) and 5f (C: 3D view; D: 2D view) with GyrB ATPase domain.
simulation (Fig. 8). Nitrogen of thiomorpholine ring of compound 5f is
forming direct hydrogen bond with Asn52 and water mediated hydrogen
bond with Val99 and Val125. Residue Glu65 played a crucial role in
forming a hydrogen bond with nitro functional group and water medi-
ated hydrogen bond with hydrazone nitrogen of compound 5f. Arg82
results indicated that both 4f and 5f were mostly equipotent against the
drug-resistant MTB strains, however closer analysis of the data revealed
that 4f was specifically more active against INH-R1 and R2 (IC50 = 3.5
and 4.6 µM), while compound 5f was explicitly found best active against
rifampicin-resistant (RIF-R1) strain with a potent IC50 value 1.9 µM,
followed by isoniazid-resistant INH-R1 (IC50 = 3.4 µM) and INH-R2
(IC50 = 5.3 µM) strains, respectively. Moreover, 4f and 5f established
interesting IC50 at 5.9 µM and 4.9 µM, respectively against
fluoroquinolone-resistant (FQ-R1) MTB strain that indicated the possi-
bility of further exploiting these compounds for developing potential
XDR anti-TB agents. These exciting activity profiles of the lead com-
pounds 4f and 5f suggested that they can be further optimized to
develop highly potent anti-mycobacterial drugs for the treatment of
both MDR-TB and XDR-TB infections. Molecular docking results
revealed crucial ligand-protein interactions, while MD simulation sug-
gested that the complex was stable for 10 ns in the GyrB ATPase domain.
In silico computation of pharmacokinetic properties of all the synthe-
sized compounds were found to be in agreement within the acceptable
ranges. Based on the identified lead structures 4f and 5f, further syn-
thesis and anti-mycobacterial screening against multidrug-resistant TB
strains are currently in progress to obtain novel drug candidates
exhibiting potency at nanomolar ranges, while retaining the safety
profiles.
formed π-π stacking with thiophene ring of the compound 5f.
Additionally, the Fig. 9 shows the total number of specific contacts
protein makes with ligand throughout the trajectory. The contribution
of amino acids in each trajectory frame of 10 ns MD simulation as shown
in the bottom panel of Fig. 9, which represent the number of contacts
and their density (the darker shade of orange shows more than one
contact in that frame). Key interaction seen during each frame was with
hydrophobic residue Ile84 and Pro85, which was consistent during the
complete simulation process. Other interactions were also found with
Val99, Val125, Ile171 and His89, which were not consistent during the
simulation.
3. Conclusion
In summary, isatin hydrazones 4(a-o) and their thiomorpholine an-
alogs 5(a-o) were synthesized, characterized and screened at level-I
against MTB H37Rv for their anti-mycobacterial activity. From the two
series, five most active compounds 4f, 4h, 4n, 5f and 5m were selected
for level-II screening. Compounds 4f and 5f emerged as the most potent
compounds exhibiting highest inhibition against both the normal
(H37Rv) and drug-resistant MTB strains, which could be attributed to the
presence of 5-nitrothiophene hydrazone moiety in their structures. The
4. Experimental
All the chemicals used in this research work were purchased from
9

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
Sigma-Aldrich and Merck Millipore, South Africa. All the solvents,
except those of reagent grade, were dried and purified when necessary,
according to previously published methods. The progress of the re-
actions and the purity of the compounds were monitored by thin-layer
chromatography (TLC) on pre-coated silica gel plates procured from E.
Merck and Co. (Darmstadt, Germany). The melting points of the syn-
thesized compounds were determined using a Thermo Fisher Scientific
(IA9000, UK) digital melting point apparatus and are uncorrected. The
IR spectra were recorded on a Bruker Alpha FT-IR spectrometer (Bill-
erica, MA, USA) using the ATR technique. The 1H NMR and 13C NMR
spectra were recorded on a Bruker AVANCE 400 and 100 MHz (Bruker,
Rheinstetten/Karlsruhe, Germany) spectrometer, respectively using the
solvent DMSO‑d6. The chemical shifts (δ) reported are given in parts per
million (ppm) and the coupling constants (J) are in Hertz (Hz) values
with respect to TMS as an internal standard. The spin multiplicities are
reported as s = singlet, d = doublet, t = triplet, dd = doublet of doublet
and m = multiplet. HRMS spectra were recorded on an Autospec mass
spectrometer with electron impact at 70 eV.
Table 6
Binding free energy components for the protein ligand complexes calculated by
MM-GBSA analysis.
Compound
Code
MMGBSA (Kcal/mol)
Prime
Energy
ΔG Bind
ΔG
Coulomb
ΔG Lipo
ΔG
Solv_GB
ΔG vdW
Code
4k
4f
ꢀ 53.81
ꢀ 51.93
ꢀ 51.93
ꢀ 44.73
ꢀ 53.48
ꢀ 50.63
ꢀ 48.14
ꢀ 58.49
ꢀ 39.62
ꢀ 50.31
ꢀ 54.47
ꢀ 52.76
ꢀ 53.12
ꢀ 48.16
ꢀ 51.82
ꢀ 51.44
ꢀ 46.7
ꢀ 11.33
ꢀ 1.41
ꢀ 1.41
ꢀ 3.36
ꢀ 7.42
ꢀ 5.37
ꢀ 10.64
ꢀ 13.49
ꢀ 1.23
ꢀ 1.88
ꢀ 11.58
ꢀ 1.8
ꢀ 17.58
ꢀ 17.25
ꢀ 17.25
ꢀ 20.72
–22.26
ꢀ 16.07
ꢀ 16.98
ꢀ 20.31
ꢀ 19.35
–23.49
ꢀ 18.6
22.26
15.37
15.37
21.25
15.47
16.45
23.79
26.76
19.76
29.32
23.04
20.89
27.31
24.99
21.43
28.12
16.01
24.37
21.45
22.87
23.29
24.61
28.09
29.41
23.56
20.51
27.62
30.32
27.42
31.63
ꢀ 44.51
ꢀ 46.02
ꢀ 46.02
ꢀ 46.89
ꢀ 57.62
ꢀ 43.1
ꢀ 14335.05
ꢀ 14333.05
ꢀ 14333.05
ꢀ 14335.9
ꢀ 14309.4
ꢀ 14326.58
ꢀ 14320.36
ꢀ 14348.02
ꢀ 14324.85
ꢀ 14286.36
ꢀ 14332.3
ꢀ 14322
4n
4l
5f
4h
4g
4i
ꢀ 41.75
ꢀ 48.9
4d
5b
4o
4j
ꢀ 44.01
ꢀ 59.51
ꢀ 44.61
ꢀ 53.24
ꢀ 57.14
ꢀ 54.26
ꢀ 51.2
ꢀ 20.15
ꢀ 21.18
ꢀ 20.21
ꢀ 19.29
ꢀ 18.73
ꢀ 19.68
ꢀ 18.67
ꢀ 17.47
ꢀ 20.01
ꢀ 17.89
ꢀ 19.89
–23.01
–23.79
ꢀ 20.72
ꢀ 15.5
5a
5m
4b
4a
4m
5d
4e
5 h
5e
5g
5i
ꢀ 1.07
ꢀ 0.37
ꢀ 0.86
ꢀ 12.35
ꢀ 1.09
ꢀ 1.67
ꢀ 3.11
ꢀ 9.44
ꢀ 0.04
ꢀ 0.1
ꢀ 14309.15
ꢀ 14298.68
ꢀ 14315.2
ꢀ 14335.05
ꢀ 14331.81
ꢀ 14302.68
ꢀ 14314.99
ꢀ 14306.45
ꢀ 14296.43
ꢀ 14294.48
ꢀ 14315.31
ꢀ 14295.47
ꢀ 14314.87
ꢀ 14320.96
ꢀ 14304.51
ꢀ 14302.88
ꢀ 14309.22
ꢀ 14313.41
ꢀ 45.87
ꢀ 42.29
ꢀ 52.05
ꢀ 39.38
ꢀ 54.07
ꢀ 52.29
ꢀ 54.42
ꢀ 60.21
ꢀ 62.72
ꢀ 58.36
ꢀ 39.02
ꢀ 54.67
ꢀ 55.34
ꢀ 54.35
ꢀ 44.28
4.1. Synthesis of compounds 2a and 2b
ꢀ 49.58
ꢀ 37.73
ꢀ 57.77
ꢀ 44.5
Compounds 2a and 2b were synthesized in good yields according to
the reported procedure [37,38].
ꢀ 47.39
ꢀ 50.53
ꢀ 51.04
ꢀ 52.38
ꢀ 45.1
ꢀ 0.92
ꢀ 1.99
ꢀ 2.8
4.2. General procedure for the synthesis of (Z)-3-(((E)-arylidene)
hydrazono)indolin-2-one 4(a-o)
5j
5n
4c
5o
5c
5 k
5 l
ꢀ 6.17
ꢀ 8.2
ꢀ 51.05
ꢀ 46.6
ꢀ 21.25
ꢀ 19.54
ꢀ 20.43
–22.19
The stirred solution of hydrazonoindolones 2a or 2b (2 mmol) in
ethanol (5 ml) was placed in a microwave reactor vial (10 ml capacity),
corresponding aldehydes 3 (2.4 mmol), and 3 drops of acetic acid were
added subsequently irradiated at 80 ◦C for 10 min in the CEM micro-
wave reactor. Upon completion of the reaction, the mixture was filtered,
washed with ethanol and dried to obtain the desired 3-arylidenehydra-
zono indolin-2-ones 4(a-o) in good yields.
ꢀ 7.75
ꢀ 9.91
ꢀ 16.43
ꢀ 52.66
ꢀ 53.57
ΔG Bind: Binding free Energy; ΔG Coulomb: Coulomb or Electrostatics Interaction
energy; ΔG Lipo: Lipophilic Interaction energy; ΔG Solv_GB: Generalized Born
electrostatic solvation energy, ΔG vdW : Van der Waals Interaction energy.
Fig. 5. The correlation plot between MM-GBSA (ΔG Bind) values (primary y-axis) and docking score (secondary y-axis) of the synthesized compounds.
10

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
Table 7
The drug likeliness and in silico ADME properties of 4(a-o) and 5(a-o) calculated using QikProp.
Entry
Drug likeliness (Lipinski’s rule of five)
In silico ADME by QikProp
Molecular
weight
QPlogP
O/Wa
H-
H-bond
Violation of
Lipinski’s
rule
QPlogSb
QPlogHERGc
QPPCacod
QPPMDCKe
QPlogKhsaf
% human
oral
Violation
of rule of
three
bond
donor
acceptor
absorptiong
4a
4b
4c
4d
4e
4f
279.298
309.324
250.259
255.294
239.233
300.291
250.259
284.231
313.743
343.769
284.704
289.739
273.678
334.736
284.704
394.490
424.517
365.452
370.486
354.426
415.484
365.452
399.423
428.935
458.962
399.897
404.931
388.871
449.929
399.897
2.231
2.317
1.227
2.128
1.489
1.415
1.238
1.033
2.705
2.813
1.816
2.588
1.919
1.840
1.777
2.525
2.451
1.301
2.461
1.892
1.652
1.386
1.276
2.998
3.401
2.033
2.977
2.378
2.135
1.758
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5.75
6.50
6.50
5.00
5.50
6.00
6.50
6.50
5.75
6.5
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
ꢀ 3.194
ꢀ 3.499
ꢀ 2.492
ꢀ 3.126
ꢀ 2.288
ꢀ 3.099
ꢀ 2.491
ꢀ 2.598
ꢀ 3.828
ꢀ 3.797
ꢀ 3.336
ꢀ 3.800
ꢀ 2.897
ꢀ 3.920
ꢀ 3.144
ꢀ 1.858
ꢀ 1.608
ꢀ 0.303
ꢀ 3.122
ꢀ 1.313
ꢀ 1.534
ꢀ 0.761
ꢀ 1.254
ꢀ 2.833
ꢀ 4.365
ꢀ 1.839
ꢀ 2.706
ꢀ 2.065
ꢀ 2.321
ꢀ 1.506
ꢀ 5.366
ꢀ 5.339
ꢀ 5.350
ꢀ 5.306
ꢀ 5.039
ꢀ 5.263
ꢀ 5.345
ꢀ 5.263
ꢀ 5.308
ꢀ 4.902
ꢀ 5.481
ꢀ 5.201
ꢀ 4.868
ꢀ 5.411
ꢀ 5.236
ꢀ 6.160
ꢀ 6.020
ꢀ 5.651
ꢀ 6.092
ꢀ 6.201
ꢀ 5.981
ꢀ 5.817
ꢀ 6.213
ꢀ 6.290
ꢀ 6.515
ꢀ 6.217
ꢀ 5.964
ꢀ 6.099
ꢀ 5.995
ꢀ 5.821
1287.917
1098.810
599.360
1225.380
1164.595
173.470
613.566
199.110
1262.277
1695.044
675.204
1217.922
1138.978
143.574
719.589
948.479
837.312
630.381
790.597
784.503
142.208
441.444
143.231
790.810
761.076
602.086
934.259
769.902
154.992
399.574
650.318
547.750
284.483
1133.156
583.280
102.239
291.779
86.448
ꢀ 0.206
ꢀ 0.196
ꢀ 0.497
ꢀ 0.263
ꢀ 0.448
ꢀ 0.309
ꢀ 0.495
ꢀ 0.461
ꢀ 0.102
ꢀ 0.143
ꢀ 0.375
ꢀ 0.163
ꢀ 0.358
ꢀ 0.200
ꢀ 0.390
ꢀ 0.508
ꢀ 0.592
ꢀ 0.997
ꢀ 0.509
ꢀ 0.722
ꢀ 0.649
ꢀ 0.906
ꢀ 0.833
ꢀ 0.370
ꢀ 0.260
ꢀ 0.769
ꢀ 0.399
ꢀ 0.597
ꢀ 0.547
ꢀ 0.819
95.66
94.93
83.84
94.67
90.54
75.30
84.09
74.14
100
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
4g
4h
4i
1567.580
2164.534
798.639
2775.490
1403.968
189.250
854.116
942.709
803.023
567.796
1456.302
770.261
139.415
415.365
123.566
1933.578
1851.636
1428.073
4277.038
1878.658
389.882
920.604
4j
100
4k
4l
6.50
5.00
5.50
6.00
6.50
8.75
9.50
9.50
8.00
8.50
9.00
9.50
9.50
8.75
9.50
9.50
8.00
8.50
9.00
9.50
88.22
100
4m
4n
4o
5a
5b
5c
5d
5e
5f
92.89
76.32
88.48
95.01
93.60
84.67
93.22
89.93
75.13
82.40
73.00
96.37
100
5g
5h
5i
5j
5k
5l
88.60
100
5m
5n
5o
92.53
78.64
83.80
a
Predicted octanol/water partition co-efficient log p (acceptable range from ꢀ 2.0 to 6.5).
Predicted aqueous solubility in mol/L (acceptable range: ꢀ 6.5 to 0.5).
b
c
d
e
f
Predicted IC50 value for blockage of HERG K + channels (concern below ꢀ 5.0).
Predicted Caco-2 cell permeability in nm/s (acceptable range: <25 is poor and >500 is good).
Predicted apparent MDCK cell permeability in nm/s (acceptable range: <25 is poor and > 500 is good).
Prediction of binding to human serum albumin (acceptable range: ꢀ 1.5 to 1.5).
g
Percentage of human oral absorption (<25% is poor and > 80% is high).
Fig. 6. Time-dependent Protein-ligand RMSD plot (Angstrom) of the compound 5f with GyrB ATPase domain.
4.2.1. (Z)-3-(((E)-4-methoxybenzylidene)hydrazono)indolin-2-one (4a)
J = 7.71 Hz, 1H, ArH), 7.94–7.97 (d, J = 8.76 Hz, 2H, ArH), 8.03–8.05
Crimson red solid; Yield: 82%; mp: 192–194 ◦C; FTIR (ATR, νmax
,
(d, J = 7.52 Hz, 1H, ArH), 8.63 (s, 1H, N C-H), 10.82 (s, 1H, –NH) ppm;
–
–
cmꢀ 1): 1457 (Aromatic C C stretch), 1510 (N H bend), 1597 (C O),
13C NMR (100 MHz, DMSO‑d6): δ = 55.51, 110.71, 114.74, 116.61,
122.27, 126.03, 128.84, 130.99, 133.33, 144.81, 150.63, 161.93,
162.60, 164.71 ppm.
–
–
–
–
–
1
–
–
–
1714 (C N), 3133 (N H stretch); H NMR (400 MHz, DMSO‑d6): δ =
3.85 (s, 3H, –OCH3), 6.89–8.90 (t, J = 7.76 Hz, 1H, ArH), 7.02–7.06 (t, J
= 7.54 Hz, 1H, ArH), 7.12–7.14 (d, J = 8.80 Hz, 2H, ArH), 7.37–7.41 (dt,
11

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
Fig. 7. Time-dependent Protein RMSF plot (Angstrom) of the compound 5f with GyrB ATPase domain.
Fig. 8. Simulation Interactions Diagram, 2D binding interaction of the compound 5f with GyrB ATPase domain along with bar diagram.
4.2.2. (Z)-3-(((E)-3,4-dimethoxybenzylidene)hydrazono)indolin-2-one
(4b)
4.2.4. (Z)-3-(((E)-thiophen-2-ylmethylene)hydrazono)indolin-2-one (4d)
Crimson red solid; Yield: 72%; mp: 202–204 ◦C; FTIR (ATR, νmax
,
Crimson red solid; Yield: 79%; mp: 221–223 ◦C; FTIR (ATR, νmax
,
cmꢀ 1): 1454 (Aromatic C C stretch), 1539 (N H bend), 1605 (C O),
–
–
–
–
–
cmꢀ 1): 1449 (Aromatic C C stretch), 1500 (N H bend), 1606 (C O),
1735 (C N), 3088 (N H stretch); H NMR (400 MHz, DMSO‑d6): δ =
1
–
–
–
–
–
–
–
–
1
–
–
–
1709 (C N), 3133 (N H stretch); H NMR (400 MHz, DMSO‑d6): δ =
6.88–6.90 (d, J = 7.80 Hz, 1H, ArH), 7.01–7.05 (t, J = 7.52 Hz, 1H,
ArH), 7.26–7.28 (m, 1H, ArH), 7.36–7.40 (m, 1H, ArH), 7.77–7.78 (d, J
= 3.24 Hz, 1H, ArH), 7.93–7.95 (d, J = 5.00 Hz, 1H, ArH), 8.00–8.02 (d,
3.85 (s, 3H, –OCH3), 3.86 (s, 3H, –OCH3), 6.88–6.90 (d, J = 7.84 Hz, 1H,
ArH), 7.01–7.05 (t, J = 7.72 Hz, 1H, ArH), 7.11–7.13 (d, J = 8.20 Hz,
J = 7.60 Hz, 1H, ArH), 8.91 (s, 1H, N C-H), 10.81 (s, 1H, NH) ppm; 13C
–
–
ArH), 7.35–7.39 (dt, J = 7.62, 0.90 Hz, 1H, ArH), 7.52–7.55 (m, 2H,
–
–
ArH), 7.99–8.01 (d, J = 7.52 Hz, 1H, ArH), 8.56 (s, 1H, N C-H), 10.82
NMR (100 MHz, DMSO‑d6): δ = 110.81, 116.63, 122.24, 128.81,
(s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 55.47, 55.69,
110.30, 110.71, 11.72, 116.58, 122.27, 123.941, 126.12, 128.76,
133.36, 144.78, 149.09, 150.48, 152.46, 161.41, 164.71 ppm.
128.92, 133.63, 135.28, 138.42, 144.99, 151.34, 157.07, 164.66 ppm.
4.2.5. (Z)-3-(((E)-furan-2-ylmethylene)hydrazono)indolin-2-one (4e)
Yellow solid; Yield: 75%; mp: 209–211 ◦C; FTIR (ATR, νmax, cmꢀ 1):
–
–
–
–
–
4.2.3. (Z)-3-(((E)-pyridin-3-ylmethylene)hydrazono)indolin-2-one (4c)
1462 (Aromatic C C stretch), 1543 (N H bend), 1619 (C O), 1731
–
Brick red solid; Yield: 78%; mp: 201–203 ◦C; FTIR (ATR, νmax, cmꢀ 1):
(C N), 3136 (N H stretch); 1H NMR (400 MHz, DMSO‑d6): δ =
–
–
–
–
–
–
–
1459 (Aromatic C C stretch), 1552 (N H bend), 1613 (C O), 1726
6.78–6.80 (dd, J = 3.44 Hz, 1.76 Hz, 1H, ArH), 6.88–6.90 (d, J = 7.88
–
(C N), 3156 (N H stretch); 1H NMR (400 MHz, DMSO‑d6): δ =
Hz, 1H, ArH), 7.02–7.05 (t, J = 7.54 Hz, 1H, ArH), 7.33–7.34 (d, J =
–
–
6.88–6.90 (d, J = 7.80 Hz, 1H, ArH), 6.99–7.03 (t, J = 7.56 Hz),
7.37–7.41 (t, J = 7.64 Hz, 1H, ArH), 7.57–7.60 (m, 1H, ArH), 7.84–7.86
(d, J = 7.52 Hz, 1H, ArH), 8.35–8.37 (d, J = 7.84 Hz, 1H, ArH), 8.65 (s,
3.48 Hz, 1H, ArH), 8.07–8.09 (d, J = 9.96 Hz, 1H, ArH), 8.55 (s, 1H,
N
C-H), 10.83 (s,1H, NH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ =
–
–
110.73, 113.14, 116.67, 119.73, 122.25, 129.01, 133.65, 144.87,
148.04, 149.03, 150.82, 151.32, 164.62 ppm.
1H, N C-H), 9.08 (s, 1H, ArH), 10.82 (s, 1H, NH) ppm; 13C NMR (100
–
–
MHz, DMSO‑d6): δ = 110.85, 116.21, 122.41, 124.28, 128.90, 129.33,
133.88, 135.17, 145.13, 150.18, 152.33, 157.47, 164.32 ppm.
12

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
Fig. 9. Protein-Ligand contacts showing good contacts (darker shades) with the amino acid residues over 10 ns time period of simulation of the compound 5f with
GyrB ATPase domain.
–
4.2.6. (Z)-3-(((E)-(5-nitrothiophen-2-yl)methylene)hydrazono)indolin-2-
one (4f)
Hz, 1H, ArH), 7.83–7.86 (m, 2H, ArH), 8.56 (s, 1H, N C-H), 10.91 (s,
–
1H, NH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 110.99, 114.15,
116.11, 119.07, 122.45, 129.06, 134.3.65, 145.37, 147.25, 150.24,
150.48, 152.85, 164.11 ppm.
Brown solid; Yield: 71%; mp: 241–243 ◦C; FTIR (ATR, νmax, cmꢀ 1):
–
–
–
–
–
1434 (Aromatic C C stretch), 1526 (N H bend), 1613 (C O), 1725
–
(C N), 3144 (N H stretch); 1H NMR (400 MHz, DMSO‑d6): δ =
–
–
6.89–6.381 (d, J = 7.84 Hz, 1H, ArH), 7.05–7.09 (t, J = 7.44 Hz, 1H,
4.2.9. (Z)-5-chloro-3-(((E)-4-methoxybenzylidene)hydrazono)indolin-2-
one (4i)
ArH), 7.40–7.44 (m, 1H, ArH), 7.7–7.79 (m, 1H, ArH), 8.20–8.21 (d, J =
4.36 Hz, 1H, ArH), 8.87 (s, 1H, N C-H), 10.90 (s,1H, NH) ppm; 13C
Crimson red solid; Yield: 80%; mp: 265–267 ◦C; FTIR (ATR, νmax
,
–
–
cmꢀ 1): 1456 (Aromatic C C stretch), 1511 (N H bend), 1606 (C O),
–
–
–
–
–
NMR (100 MHz, DMSO‑d6): δ = 110.98, 115.98, 121.98, 122.45,
1
–
–
–
128.64, 134.09, 140.11, 145.25, 149.29, 150.67, 155.72, 164.04 ppm.
1736 (C N), 3143 (N H stretch); H NMR (400 MHz, DMSO‑d6): δ =
3.86 (s, 3H, –OCH3), 6.91–6.93 (d, J = 8.40 Hz, 1H, ArH), 7.15–7.17 (d,
J = 8.76 Hz, 2H, ArH), 7.44–7.46 (dd, J = 8.36 Hz, 2.20 Hz, 1H, ArH),
7.92–7.94 (d, J = 8.76 Hz, 2H, ArH), 8.00–8.01 (d, J = 2.02 Hz, 1H,
4.2.7. (Z)-3-(((E)-pyridin-4-ylmethylene)hydrazono)indolin-2-one (4g)
Yellow solid; Yield: 76%; mp: 245–247 ◦C; FTIR (ATR, νmax, cmꢀ 1):
ArH), 8.68, (s, 1H, N C-H), 10.96 (s, 1H, NH) ppm; 13C NMR (100 MHz,
–
–
–
–
–
–
1427 (Aromatic C C stretch), 1556 (N H bend), 1614 (C O), 1725
–
–
(C N), 3091 (N H stretch); 1H NMR (400 MHz, DMSO‑d6): δ =
DMSO‑d6): δ = 55.57, 112.32, 114.91, 117.73, 125.75, 125.80, 128.01,
–
–
6.80–6.90 (d, J = 7.76 Hz, 1H, ArH), 6.97–7.01 (t, J = 7.76 Hz, 1H,
131.10, 143.55, 150.00, 162.90, 163.41, 164.41 ppm.
ArH), 7.37–7.41 (dt, J = 7.4 Hz, 0.8 Hz, 1H, ArH), 7.67–7.69 (d, J =
7.52 Hz, 1H, ArH), 7.84–7.86 (dd, J = 5.96 Hz, 2H, ArH), 8.51 (s, 1H,
4.2.10. (Z)-5-chloro-3-(((E)-3,4-dimethoxybenzylidene)hydrazono)
indolin-2-one (4j)
–
N
C-H), 8.76–8.78 (dd, J = 5.88 Hz, 2H, ArH), 10.91 (s,1H, NH) ppm;
–
13C NMR (100 MHz, DMSO‑d6): δ = 111.07, 116.12, 122.58, 128.96,
Crimson red solid; Yield: 78%; mp: 285–287 ◦C; FTIR (ATR, νmax
,
cmꢀ 1): 1456 (Aromatic C C stretch), 1507 (N H bend), 1616 (C O),
–
–
–
–
–
130.52, 133.24, 134.41, 144.29, 145.52, 151.38, 154.43, 164.18 ppm.
1
–
–
–
1739 (C N), 3153 (N H stretch); H NMR (400 MHz, DMSO‑d6): δ =
4.2.8. (Z)-3-(((E)-(5-nitrofuran-2-yl)methylene)hydrazono)indolin-2-one
(4 h)
3.87 (s, 3H, –OCH3), 3.88 (s, 3H, –OCH3), 6.90–6.92 (d, J = 8.36 Hz, 1H,
ArH), 7.16–7.18 (d, J = 8.28 Hz, 1H, ArH), 7.43–7.46 (dd, J = 8.36 Hz,
Red solid; Yield: 73%; mp: 236–238 ◦C; FTIR (ATR, νmax, cmꢀ 1):
1.88 Hz, 1H, ArH), 7.52–7.54 (d, J = 8.44 Hz, 1H, ArH), 7.58 (s, 1H, 1H,
–
–
–
–
–
–
1460 (Aromatic C C stretch), 1550 (N H bend), 1614 (C O), 1740
ArH), 8.08 (d, J = 1.84 Hz, 1H, ArH), 8.66 (s, 1H, N C-H), 10.96 (s, 1H,
–
–
(C N), 3149 (N H stretch); 1H NMR (400 MHz, DMSO‑d6): δ =
NH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 55.25, 55.78, 109.60,
111.81, 112.31, 117.81, 124.84, 125.75, 125.96, 125.33, 132.73,
143.51, 149.14, 150.39, 152.87, 163.43, 164.45 ppm.
–
–
6.89–6.91 (d, J = 7.84 Hz, 1H, ArH), 7.00–7.04 (t, J = 7.52 Hz, 1H,
ArH), 7.40–7.44 (dt, J = 7.75, 0.9 Hz, 1H, ArH), 7.54–7.55 (d, J = 3.96
13

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
4.2.11. (Z)-5-chloro-3-(((E)-pyridin-3-ylmethylene)hydrazono)indolin-2-
one (4k)
was added to the reaction mixture and continued the stirring for addi-
tional 3 h at room temperature. Upon completion of the reaction (as
monitored by TLC), the formed precipitate was filtered, washed with
ethanol followed by diethyl ether and dried to obtain the 3-(((E)-aryli-
dene)hydrazono)-1-(thiomorpholino-methy)indolin-2-ones 5(a-o) in
excellent yields.
Crimson red solid; Yield: 73%; mp: 287–289 ◦C; FTIR (ATR, νmax
,
cmꢀ 1): 1455 (Aromatic C C stretch), 1552 (N H bend), 1612 (C O),
–
–
–
–
–
1
–
–
–
1736 (C N), 3155 (N H stretch); H NMR (400 MHz, DMSO‑d6): δ =
6.90–6.92 (d, J = 8.40 Hz, 1H, ArH), 7.43–7.46 (dd, J = 8.38 Hz, 2.18
Hz, 1H, ArH), 7.61–7.64 (m, 1H, ArH), 8.32–8.35 (m, 1H, ArH), 8.70 s,
–
–
1H, N C-H), 8.75–8.76 (d, J = 4.82, 1.52 Hz, 1H, ArH), 11.02 (s, 1H,
4.3.1. (Z)-3-(((E)-4-methoxybenzylidene)hydrazono)-1-(thiomorpholino-
methyl)indolin-2-one (5a)
NH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 112.47, 117.33, 124.38,
125.87, 128.04, 129.05, 133.30, 135.21, 143.91, 149.85, 150.22,
152.60, 158.89, 164.00.
Yellow solid; Yield: 83%; mp: 137–139 ◦C; FTIR (ATR, νmax, cmꢀ 1):
1
–
–
–
–
1457 (Aromatic C C stretch), 1600 (C O), 1717 (C N); H NMR (400
–
–
MHz, DMSO‑d6): δ = 2.57–2.59 (t, J = 4.88 Hz, 4H), 2.82–2.84 (t, J =
4.88 Hz, 4H), 3.86 (s, 3H, –OCH3), 4.49 (s, 2H, –CH2-), 7.12–7.14 (d, J =
8.64 Hz, 3H, ArH), 7.22–7.24 (d, J = 7.92 Hz, 1H, ArH), 7.45–7.49 (m,
1H, ArH), 7.95–7.97 (d, J = 8.72 Hz, 2H, ArH), 8.10–8.12 (d, J = 7.36
Hz, 1H, ArH), 8.65 (s, 1H, -N = CH) ppm; 13C NMR (100 MHz,
DMSO‑d6): δ = 26.93, 52.18, 55.52, 62.06, 110.96, 114.78, 116.01,
122.92, 125.97, 128.51, 131.11, 133.24, 145.76, 149.76, 162.28,
4.2.12. (Z)-5-chloro-3-(((E)-thiophen-2-ylmethylene)hydrazono)indolin-
2-one (4 l)
Crimson red solid; Yield: 69%; mp: 253–255 ◦C; FTIR (ATR, νmax
,
cmꢀ 1): 1440 (Aromatic C C stretch), 1504 (N H bend), 1603 (C O),
–
–
–
–
–
1
–
–
–
1722 (C N), 3088 (N H stretch); H NMR (400 MHz, DMSO‑d6): δ =
6.82–6.84 (d, J = 8.32 Hz, 1H, ArH), 7.21–7.24 (dd, J = 4.94 Hz, 3.70
Hz, 1H, ArH), 7.36–7.39 (dd, J = 8.36 Hz, 2.20 Hz, 1H, ArH), 7.74–7.75
162.70, 164.28 ppm; HRMS (ESI) m/z calcd. for
C22H22N4O2S,
(dd, J = 3.84 Hz, 0.76 Hz, 1H, ArH), 7.92–7.94 (d, J = 5.03 Hz, 1H,
394.1463: found 417.1361 [M + Na] +
–
–
ArH), 7.97–7.98 (d, J = 2.20 Hz, 1H, ArH), 8.90 (s, 1H, N C-H), 10.89
(s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 112.34, 117.79,
125.81, 128.41, 129.00, 132.94, 133.86, 136.13, 138.26, 143.68,
150.87, 158.6, 164.37 ppm.
4.3.2. (Z)-3-(((E)-3,4-dimethoxybenzylidene)hydrazono)-1-
(thiomorpholino-methyl)indolin-2-one (5b)
Crimson red solid; Yield: 72%; mp: 121–123 ◦C; FTIR (ATR, νmax
,
cmꢀ 1): 1466 (Aromatic C C stretch), 1604 (C O), 1718 (C N); 1H
–
–
–
–
–
–
4.2.13. (Z)-5-chloro-3-(((E)-furan-2-ylmethylene)hydrazono)indolin-2-
one (4 m)
NMR (400 MHz, DMSO‑d6): δ = 2.56–2.59 (t, J = 4.96 Hz, 4H),
2.82–2.84 (t, J = 5.02 Hz, 4H), 3.86 (s, 3H, –OCH3), 3.87 (s, 3H, –OCH3),
4.49 (s, 2H, –CH2-), 7.10–7.16 (m, 2H, ArH), 7.12–7.24 (d, J = 7.92 Hz,
1H, ArH), 7.46–7.48 (m, 1H, ArH), 7.54–7.57 (m, 2H, ArH), 8.06–8.08
(d, J = 7.36 Hz, 1H, ArH), 8.59 (s, 1H, -N = CH) ppm; 13C NMR (100
MHz, DMSO‑d6): δ = 26.93, 52.18, 55.49, 55.73, 62.06, 110.37, 110.90,
111.77, 115.96, 122.95, 124.10, 126.05, 128.43, 133.20, 145.74,
149.11, 149.54, 152.58, 161.77, 164.25 ppm; HRMS (ESI) m/z calcd. for
Brown solid; Yield: 70%; mp: 204–206 ◦C; FTIR (ATR, νmax, cmꢀ 1):
–
–
–
–
–
1458 (Aromatic C C stretch), 1534 (N H bend), 1616 (C O), 1741
–
(C N), 3152 (N H stretch); 1H NMR (400 MHz, DMSO‑d6): δ =
–
–
6.81–6.82 (m, 2H, ArH), 6.90–6.92 (d, J = 8.40 Hz, 1H, ArH), 7.36–7.37
(d, J = 3.48 Hz, 1H, ArH), 7.44–7.47 (dd, J = 8.36 Hz, 2.24 Hz, 1H,
–
–
ArH), 8.09–8.15 (m, 2H, ArH), 8.61 (s, 1H, N C-H), 10.97 (s, NH), ppm;
13C NMR (100 MHz, DMSO‑d6): δ = 112.31, 113.34, 117.83, 121.00,
125.79, 128.27, 133.00, 143.61, 148.61, 148.83, 150.83, 152.30,
164.35 ppm.
C
22H24N4O3S, 424.1569: found 447.1470 [M + Na] +
4.3.3. (Z)-3-(((E)-pyridin-3-ylmethylene)hydrazono)-1-(thiomorpholino-
methyl)-indolin-2-one (5c)
4.2.14. (Z)-3-(((E)-(5-nitrothiophen-2-yl)methylene)hydrazono)indolin-
2-one (4n)
Yellow solid; Yield: 72%; mp: 175–177 ◦C; FTIR (ATR, νmax, cmꢀ 1):
1
–
–
–
–
1468 (Aromatic C C stretch), 1599 (C O), 1724 (C N); H NMR (400
–
–
Reddish brown solid; Yield: 68%; mp: 222–224 ◦C; FTIR (ATR, νmax
,
MHz, DMSO‑d6): δ = 2.57–2.59 (t, J = 5.00 Hz, 4H), 2.82–2.85 (t, J =
5.02 Hz, 4H), 4.50 (s, 2H, –CH2-), 7.10–7.14 (t, J = 7.56 Hz, 1H, ArH),
7.24–7.26 (d, J = 7.92 Hz, 1H, ArH), 7.47–7.51 (m, 1H, ArH), 7.60–7.63
(dd, J = 7.90 Hz, 4.82 Hz, 1H, ArH), 7.91–7.93 (d, J = 7.48 Hz, 1H,
ArH), 8.37–8.40 (m, 1H, ArH), 8.68 (s, 1H, -N = CH), 8.68–8.76 (m, 1H,
ArH), 9.09–9.10 (d, J = 1.84 Hz, 1H, ArH) ppm; 13C NMR (100 MHz,
DMSO‑d6): δ = 26.93, 52.16, 62.13, 11.08, 115.62, 123.10, 124.35,
128.58, 129.15, 133.73, 135.26, 146.10, 149.40, 150.25, 152.45,
157.61, 163.91 ppm; HRMS (ESI) m/z calcd. for C19H19N5OS, 365.1310:
found 388.1210 [M + Na] +
cmꢀ 1): 1450 (Aromatic C C stretch), 1529 (N H bend), 1607 (C O),
–
–
–
–
–
1
–
–
–
1738 (C N), 3102 (N H stretch); H NMR (400 MHz, DMSO‑d6): δ =
6.92–6.94 (d, J = 4.28 Hz, 1H, ArH), 7.48–7.49 (d, J = 2.20 Hz, 1H,
–
–
ArH), 7.79–7.82 (m, 2H, ArH), 8.94 (s, 1H, N C-H), 10.09 (s, 1H, NH);
13C NMR (100 MHz, DMSO‑d6): δ = 112.60, 117.06, 117.32, 126.09,
128.35, 130.53, 133.68, 133.91, 144.25, 145.90, 149.03, 151.29,
156.32, 163.23 ppm.
4.2.15. (Z)-5-chloro-3-(((E)-pyridin-4-ylmethylene)hydrazono)indolin-2-
one (4o)
Brown solid; Yield: 71%; mp: 272–274 ◦C; FTIR (ATR, νmax, cmꢀ 1):
4.3.4. (Z)-1-(thiomorpholinomethyl)-3-(((E)-thiophen-2-ylmethylene)-
hydrazono)-indolin-2-one (5d)
–
–
–
–
–
1419 (Aromatic C C stretch), 1554 (N H bend), 1614 (C O), 1734
–
(C N), 3157 (N H stretch); 1H NMR (400 MHz, DMSO‑d6): δ =
Pale red solid; Yield: 74%; mp: 186–188 ◦C; FTIR (ATR, νmax, cmꢀ 1):
–
–
1
–
–
–
–
6.85–6.87 (d, J = 8.36 Hz, 1H, ArH), 7.39–7.42 (dd, J = 8.36 Hz, 2.24
1460 (Aromatic C C stretch), 1605 (C O), 1714 (C N); H NMR (400
–
–
Hz, 1H, ArH), 7.55–7.56 (d, J = 2.28 Hz, 1H, ArH), 7.77–7.78 (m, 2H,
MHz, DMSO‑d6): δ = 2.56–2.59 (t, J = 5.08 Hz, 4H), 2.81–2.84 (t, J =
4.95 Hz, 4H), 4.49 (s, 2H, –CH2-), 7.11–7.15 (t, J = 7.58 Hz, 1H, ArH),
7.22–7.24 (d, J = 7.88 Hz, 1H, ArH), 7.28–7.30 (m, 1H, ArH), 7.45–7.49
(m, 1H, ArH), 7.80–7.81 (d, J = 3.20 Hz, 1H, ArH), 7.97–7.98 (d, J =
5.00 Hz, 1H, ArH), 8.07–8.09 (d, J = 7.36 Hz, 1H, ArH), 8.94 (s, 1H -N =
CH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 26.94, 52.16, 62.07,
111.01, 116.03, 122.89, 128.56, 128.90, 133.39, 133.46, 135.54,
138.36, 145.94, 150.39, 157.40, 164.21 ppm; HRMS (ESI) m/z calcd. for
–
–
ArH), 8.48 (s, 1H, , N C-H), 8.73–8.74 (m, 2H, ArH), 10.98 (s, 1H, NH)
ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 112.62, 117.14, 121.93,
125.90, 127.80, 133.52, 139.92, 144.04, 148.74, 150.80, 156.80,
163.73 ppm.
4.3. General procedure for the synthesis of 3-(((E)-arylidene)
hydrazono)-1-(thiomorpholinomethyl)indolin-2-one 5(a-o)
C
18H18N4O3S2, 370.0922: found 393.0820 [M + Na] +
In a round bottom flask, 3-arylidinehydrazonoindoline-2one 4(a-o)
(1 mmol), formaldehyde (1.2 mmol) and ethanol (10 ml) were added
and stirred at room temperature for 10 min. Thiomorpholine (1.2 mmol)
14

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
4.3.5. (Z)-3-(((E)-furan-2-ylmethylene)hydrazono)-1-
(thiomorpholinomethyl)-indolin-2-one (5e)
144.45, 149.05, 163.00, 163.68 ppm; HRMS (ESI) m/z calcd. for
C21H21ClN4O2S, 428.1074: found 451.0972 [M + Na] +
Yellow solid; Yield: 72%; mp: 174–176 ◦C; FTIR (ATR, νmax, cmꢀ 1):
1
–
–
–
–
1462 (Aromatic C C stretch), 1628 (C O), 1720 (C N); H NMR (400
4.3.10. (Z)-5-chloro-3-(((E)-3,4-dimethoxybenzylidene)hydrazono)-1-
(thiomorpholino- methyl)indolin-2-one (5j)
–
–
MHz, DMSO‑d6): δ = 2.56–2.59 (t, J = 5.16 Hz, 4H), 2.81–2.84 (t, J =
4.95 Hz, 4H), 4.49 (s, 2H, –CH2-), 6.80–6.81 (m, 1H, ArH), 7.10–7.14 (t,
J = 7.60 Hz, 1H, ArH), 7.22–7.24 (d, J = 7.92 Hz, 1H, ArH), 7.35–7.36
(d, J = 3.48 Hz, 1H, ArH), 7.45–7.49 (m, 1H, ArH), 8.10–8.15 (m, 2H,
ArH), 8.57 (s, 1H, -N = CH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ =
26.93, 52.17, 62.08, 110.93, 113.22, 116.07, 120.06, 122.89, 128.66,
133.45, 145.84, 148.21, 148.99, 150.39, 151.07, 164.19 ppm; HRMS
yellow solid; Yield: 80%; mp: 210–212 ◦C; FTIR (ATR, νmax, cmꢀ 1):
1
–
–
–
–
1458 (Aromatic C C stretch), 1599 (C O), 1731 (C N); H NMR (400
–
–
MHz, DMSO‑d6): δ = 2.57 (s, 4H), 2.81 (s, 4H), 3.87 (s, 6H, –OCH3), 4.47
(s, 2H, –CH2-), 6.85 (s, 1H, ArH), 7.16 (s, 1H, ArH), 7.50–7.56 (d, 2H,
ArH), 8.23 (s, 2H, ArH), 8.65 (s, 1H, -N = CH) ppm; 13C NMR (100 MHz,
DMSO‑d6): δ = 26.91, 52.07, 55.59, 62.18, 112.62, 114.94, 117.20,
125.75, 126.56, 127.67, 127.89, 131.19, 132.52, 144.45, 149.05,
163.00, 163.68 ppm; HRMS (ESI) m/z calcd. for C22H23ClN4O3S,
458.1179: found 481.1080[M + Na] +
(ESI) m/z calcd. for C18H18N4O2S, 354.1150: found 377.1051 [M + Na]
+
4.3.6. (Z)-3-(((E)-(5-nitrothiophen-2-yl)methylene)hydrazono)-1-
(thiomorpholino-methyl)indolin-2-one (5f)
4.3.11. (Z)-5-chloro-3-(((E)-pyridin-3-ylmethylene)hydrazono)-1-
(thiomorpholinomethyl)-indolin-2-one (5 k)
Brick red solid; Yield: 75%; mp: 188–189 ◦C; FTIR (ATR, νmax, cmꢀ 1):
1456 (Aromatic C C stretch), 1609 (C O), 1724 (C N); H NMR (400
Pale red solid; Yield: 75%; mp: 192–194 ◦C; FTIR (ATR, νmax, cmꢀ 1):
1
–
–
–
–
–
–
1
–
–
–
–
MHz, DMSO‑d6): δ = 2.57–2.59 (t, J = 5.00 Hz, 4H), 2.81–2.84 (t, J =
4.64 Hz, 4H), 4.49 (s, 2H, –CH2-), 7.14–7.18 (t, J = 7.64 Hz, 1H, ArH),
7.24–7.26 (d, J = 7.96 Hz, 1H, ArH), 7.49–7.53 (t, J = 7.40 Hz, 1H,
ArH), 7.79–7.85 (m, 2H, ArH), 8.21 (s, 1H, ArH), 8.90 (s, 1H, -N = CH)
ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 26.94, 52.13, 62.17, 111.27,
115.72, 123.19, 128.62, 129.12, 130.51, 133.35, 134.18, 134.40,
136.42, 146.45, 153.47, 154.53 ppm; HRMS (ESI) m/z calcd. for
1458 (Aromatic C C stretch), 1602 (C O), 1723 (C N); H NMR (400
–
–
MHz, DMSO‑d6): δ = 2.57 (s, 4H), 2.82 (s, 4H), 4.48 (s, 2H, –CH2-),
7.08–7.12 (t, J = 7.56 Hz, 1H, ArH), 7.23–7.25 (d, J = 7.87 Hz, 1H,
ArH), 7.48–7.56 (m, 2H, ArH), 7.25–7.90 (m, 2H, ArH), 8.57 (s, 1H, -N
= CH) ppm: 13C NMR (100 MHz, DMSO‑d6): δ = 26.91, 52.06, 62.20,
112.88, 117.31, 120.98, 121.31, 125.80, 126.60, 128.27, 132.99,
144.51, 149.16, 152.53, 164.51, 178.37 ppm; HRMS (ESI) m/z calcd. for
C
18H17N5O3S2, 415.0773: found 438.0672 [M + Na] +
C
19H18ClN5OS, 399.0921: found 422.0819 [M + Na] +
4.3.7. (Z)-3-(((E)-pyridin-4-ylmethylene)hydrazono)-1-
(thiomorpholinomethyl) indolin-2-one (5 g)
4.3.12. (Z)-5-chloro-1-(thiomorpholinomethyl)-3-(((E)-thiophen-2-yl-
methylene)-hydrazono)-indolin-2-one (5 l)
Crimson red solid; Yield: 79%; mp: 222–224 ◦C; FTIR (ATR, νmax
,
Yellow solid; Yield: 72%; mp: 202–204 ◦C; FTIR (ATR, νmax, cmꢀ 1):
cmꢀ 1): 1463 (Aromatic C C stretch), 1599 (C O), 1724 (C N); 1H
1440 (Aromatic C C stretch), 1627 (C O), 1722 (C N); H NMR (400
1
–
–
–
–
–
–
–
–
–
–
–
–
NMR (400 MHz, DMSO‑d6): δ = 2.57–2.59 (t, J = 5.00 Hz, 4H),
2.82–2.85 (t, J = 5.04 Hz, 4H), 4.50 (s, 2H, –CH2-), 7.08–7.12 (t, J =
7.60 Hz, 1H, ArH), 7.24–7.26 (d, J = 7.92 Hz, 1H, ArH), 7.47–7.51 (m,
1H, ArH), 7.74–7.76 (d, J = 7.32 Hz, 1H, ArH), 7.87–7.88 (m, 2H, ArH),
8.51 (s, 1H, -N = CH), 8.79–8.80 (m, 2H, ArH) ppm; 13C NMR (100 MHz,
DMSO‑d6): δ = 26.94, 52.15, 62.16, 111.18, 115.41, 122.04, 123.09,
128.29, 133.89, 140.05, 146.22, 148.32, 150.71, 155.77, 163.64 ppm;
HRMS (ESI) m/z calcd. for C19H19N5OS, 365.1310: found 388.1209[M
+ Na] +
MHz, DMSO‑d6): δ = 2.56–2.58 (t, J = 4.76 Hz, 4H), 2.80–2.82 (t, J =
4.76 Hz, 4H), 4.48 (s, 2H, –CH2-), 7.25–7.27 (d, J = 8.48 Hz, 2H, ArH),
7.30–7.32 (m, 1H, ArH), 7.53–7.55 (dd, J = 8.48 Hz, 2.20 Hz, 1H, ArH),
7.84–7.85 (d, J = 3.28 Hz, 1H, ArH), 8.02–8.03 (d, J = 4.96 Hz, 1H,
ArH), 8.11–8.12 (d, J = 2.12 Hz, 1H, ArH), 9.01 (s, 1H, -N = CH) ppm;
13C NMR (100 MHz, DMSO‑d6): δ = 27.43, 52.64, 62.79, 113.07,
117.87, 127.27, 128.62, 129.41, 133.12, 134.24, 136.28, 138.80,
145.07, 150.21, 158.57, 164.32 ppm; HRMS (ESI) m/z calcd. for
C
18H17ClN4OS2, 404.0532: found 427.0432 [M + Na] +
4.3.8. (Z)-3-(((E)-(5-nitrofuran-2-yl)methylene)hydrazono)-1-
(thiomorpholino-methyl)indolin-2-one (5 h)
4.3.13. (Z)-5-chloro-3-(((E)-furan-2-ylmethylene)hydrazono)-1-
(thiomorpholino-methyl)-indolin-2-one (5 m)
Crimson red solid; Yield: 68%; mp: 195–197 ◦C; FTIR (ATR, νmax
,
Brick red solid; Yield: 68%; mp: 189–191 ◦C; FTIR (ATR, νmax, cmꢀ 1):
cmꢀ 1): 1463 (Aromatic C C stretch), 1628 (C O), 1721 (C N); 1H
1434 (Aromatic C C stretch), 1634 (C O), 1731 (C N); H NMR (400
1
–
–
–
–
–
–
–
–
–
–
–
–
NMR (400 MHz, DMSO‑d6): δ = 2.57–2.59 (t, J = 4.96 Hz, 4H),
2.82–2.85 (t, J = 5.02 Hz, 4H), 4.50 (s, 2H, –CH2-), 7.09–7.13 (t, J =
7.78 Hz, 1H, ArH), 7.24–7.26 (d, J = 7.92 Hz, 1H, ArH), 7.49–7.53 (m,
1H, ArH), 7.56–7.57 (d, J = 3.96 Hz, 1H, ArH), 7.86–7.87 (d, J = 3.96
Hz, 1H, ArH), 7.89–7.91 (dd, J = 7.52 Hz, 0.76 Hz, 1H, ArH), 8.58 (s,
1H, -N = CH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 26.93, 52.15,
62.20, 111.18, 114.15, 115.56, 1119.25, 123.07, 128.70, 134.14,
146.34, 147.31, 149.52, 150.17, 152.90, 163.72 ppm; HRMS (ESI) m/z
calcd. for C18H17N5O4S, 399.1001: found 400.1080 [M + H] +
MHz, DMSO‑d6): δ = 2.57–2.58 (d, J = 4.88 Hz, 4H), 2.80–2.82 (d, J =
5.28 Hz, 4H), 4.48 (s, 2H, –CH2-), 6.82–6.83 (m, 1H, ArH), 7.25–7.27 (d,
J = 8.52 Hz, 1H, ArH), 7.38–7.39 (d, J = 3.40 Hz, 1H, ArH), 7.53–7.55
(dd, J = 8.46 Hz, 2.14 Hz, 1H, ArH), 8.16 (d, J = 1.28 Hz, 2H, ArH), 8.63
(s, 1H, -N = CH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 26.90, 52.06,
62.20,112.62, 113.41, 117.31, 121.32, 126.60, 127.92, 132.73, 144.52,
148.76, 148.80, 149.88, 152.51, 163.88 ppm; HRMS (ESI) m/z calcd. for
C
18H17ClN4O2S, 388.0761: found 411.0659 [M + Na] +
4.3.14. (Z)-5-chloro-3-(((E)-(5-nitrothiophen-2-yl)methylene)
hydrazono)-1-(thiomorpholino-methyl)indolin-2-one (5n)
4.3.9. (Z)-5-chloro-3-(((E)-4-methoxybenzylidene)hydrazono)-1-
(thiomorpholinomethyl)indolin-2-one (5i)
Brown solid; Yield: 79%; mp: 206–208 ◦C; FTIR (ATR, νmax, cmꢀ 1):
Yellow solid; Yield: 82%; mp: 205–207 ◦C; FTIR (ATR, νmax, cmꢀ 1):
1438 (Aromatic C C stretch), 1604 (C O), 1728 (C N); H NMR (400
1
–
–
–
–
–
–
1
–
–
–
–
1458 (Aromatic C C stretch), 1625 (C O), 1733 (C N); H NMR (400
MHz, DMSO‑d6): δ = 2.56–2.58 (d, J = 5.0 Hz, 4H), 2.80–2.83 (d, J =
4.95 Hz, 4H), 4.48 (s, 2H, –CH2-), 6.82–6.83 (m, 1H, ArH), 7.25–7.27 (d,
J = 8.52 Hz, 1H, ArH), 7.38–7.39 (d, J = 3.48 Hz, 1H, ArH), 7.53–7.56
(dd, J = 8.48 Hz, 2.32 Hz, 1H, ArH), 8.16–8.17 (d, J = 1.88 Hz, 2H,
ArH), 8.64 (s, 1H, -N = CH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ =
26.91, 52.05, 62.20, 112.62, 113.41, 117.31, 121.34, 126.59, 127.92,
127.92, 132.72, 144.52, 148.72, 148.80, 149.89, 152.54, 163.88 ppm;
HRMS (ESI) m/z calcd. for C18H16ClN5O3S2, 449.0383: found 472.0283
–
–
MHz, DMSO‑d6): δ = 2.56–2.57 (d, J = 4.00 Hz, 4H), 2.81–2.82 (d, J =
4.72 Hz, 4H), 3.87 (s, 3H, –OCH3), 4.49 (s, 2H, –CH2-), 7.16–7.18 (d, J =
8.52 Hz, 1H, ArH), 7.25–7.28 (d, J = 8.64 Hz, 1H, ArH), 7.52–7.55 (dd,
J = 8.42 Hz, 1.54 Hz, 1H, ArH), 7.93–7.95 (d, J = 8.52 Hz, 1H, ArH),
8.05–8.06 (d, J = 1.40 Hz, 1H, ArH), 8.70 (s, 1H, -N = CH) ppm; 13
C
NMR (100 MHz, DMSO‑d6): δ = 26.91, 52.07, 55.59, 62.18, 112.62,
114.94, 117.20, 125.75, 126.56, 127.67, 127.89, 131.19, 132.52,
15

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
[M + Na] +
plates and incubated under aerobic conditions for 5 days. Growth was
measured by luminescence. Rifampicin was included in each plate and
metronidazole was included in each run as positive controls for aerobic
and anaerobic killing of MTB, respectively.
4.3.15. (Z)-5-chloro-3-(((E)-pyridin-4-ylmethylene)hydrazono)-1-
(thiomorpholino-methyl)-indolin-2-one (5o)
Yellow solid; Yield: 79%; mp: 194–196 ◦C; FTIR (ATR, νmax, cmꢀ 1):
1
–
–
–
–
–
–
1466 (Aromatic C C stretch), 1604 (C O), 1718 (C N); H NMR (400
4.4.3. Minimum bactericidal concentration (MBC) determination
MTB was grown aerobically to logarithmic phase and inoculated into
liquid medium containing four different compound concentrations with
a final maximum concentration of 2% DMSO. For test compounds (4f, 4
h, 4n, 5f, and 5 m) with MIC < 20 µM, the concentration selected were
10X MIC, 5X MIC, 1X MIC and 0.25X MIC. Cultures were exposed to
compounds for 21 days and cell viability measured by enumerating
colony forming units on agar plates on day 0, 7, 14 and 21. MBC was
defined as the minimum concentration required to achieve a 2-log kill in
21 days. For compounds with > 1-log kill, an assessment of time and/or
concentration-dependence was determined from the kill kinetics. DMSO
was used as a positive control for growth.
MHz, DMSO‑d6): δ = 2.58 (s, 4H), 2.84 (s, 4H), 4.49 (s, 2H, –CH2-),
7.09–7.13 (m, 1H, ArH), 7.24–7.26 (d, J = 7.92 Hz, 1H, ArH), 7.49–7.52
(m, 1H, ArH), 7.56–7.57 (d, J = 3.80 Hz, 1H, ArH), 7.86–7.87 (d, J =
3.68 Hz, 1H, ArH), 7.89–7.91 (d, J = 7.60 Hz, 1H, ArH), 8.58 (s, 1H, -N
= CH) ppm; 13C NMR (100 MHz, DMSO‑d6): δ = 26.93, 52.15, 62.20,
111.17, 114.14, 115.56, 119.24, 123.06, 128.69, 134.13, 146.33,
147.31, 149.53, 15017, 152.89, 163.71 ppm; HRMS (ESI) m/z calcd. for
C
19H18ClN5OS, 399.0921: found 422.0819 [M + Na] +
4.4. Biological studies
4.4.1. Anti-mycobacterial evaluation (MIC) under aerobic conditions by in
vitro
4.4.4. Intracellular activity evaluation [47]
The activity of compounds against intracellular bacteria was deter-
mined by measuring viability in infected THP-1 cell after 3 days in the
presence of test compounds. Test compounds (4f, 4 h, 4n, 5f, and 5 m)
were prepared as 10-point three-fold serial dilutions in DMSO. The
highest concentration of compound tested was 50 µM where compounds
were soluble in DMSO at 10 mM. For compounds with limited solubility,
the highest concentration was 200X less than the stock concentration e.
g. 25 µM for 5 mM DMSO stock, 5 µM for 1 mM DMSO stock. THP-1 cells
were cultured incomplete RPMI and differentiated into macrophage-like
cells using 80 nM PMA overnight at 37 ◦C, 5% CO2. THP-1 cells were
infected with a luminescent strain of H37Rv (which constitutively ex-
presses luxABCDE) at a multiplicity of infection of THP-1 and incubated
over night at 37 ◦C, 5% CO2. Infected cells were recovered using
Accutase/EDTA solution, washed twice with PBS to remove extracel-
lular bacteria and seeded into assay plates. Compound dilutions were
added to a final DMSO concentration of 0.5%. Assay plates were incu-
bated for 72 h at 37 ◦C, 5% CO2. Each run included isoniazid as a control.
Relative luminescent units (RLU) were measured using a Biotek Synergy
2 plate reader. The dose response curve was fitted using the Lev-
enberg–Marquardt algorithm. The IC50 and IC90 were defined as the
compound concentrations that produced 50% and 90% inhibition of
growth, respectively.
All the newly synthesized compounds 4(a-o) and 5(a-o) were
screened for their in vitro anti-mycobacterial activity against
M. tuberculosis H37Rv grown under aerobic conditions using a dual read-
out (OD590 and fluorescence) assay procedure [41,42,53]. The experi-
ment was carried out at Infectious Disease Research Institute (IDRI)
within the National Institute of Allergy and Infectious Diseases (NIAID)
screening program, Bethesda, MD, USA. Test compounds were prepared
as 10-point two-fold serial dilutions in DMSO and diluted into 7H9-Tw-
OADC medium in 96-well plates with a final DMSO concentration of 2%.
The highest concentration of compound was 200 µM and compounds
were soluble in DMSO at 10 mM. For compounds with limited solubility,
the highest concentration was 50X less than the stock concentration e.g.
100 µM for 5 mM DMSO stock, 20 µM for 1 mM DMSO stock. For potent
compounds, assays were repeated at lower starting concentrations. Each
plate included assay controls for background (medium/DMSO only, no
bacterial cells), zero growth (100 µM Rifampicin) and maximum growth
(DMSO only), as well as a rifampicin dose response curve (DRC). Plates
were inoculated with M. tuberculosis and incubated for 5 days. Growth
was measured by OD590 and fluorescence (Ex560/Em590) using a Bio-
Tek™ Synergy4 plate reader and calculated separately for OD590 and
RFU. MIC was determined on the basis of 10-point dose response curve
which was plotted as % growth. The MIC was defined as the minimum
concentration at which growth was completely inhibited and was
calculated from the inflection point of the fitted curve to the lower
asymptote (zero growth). In addition, DRC were generated using the
Levenberg-Marquardt algorithm and the concentrations that resulted in
4.4.5. Cytotoxicity assay [47]
The cytotoxicity of compounds was determined by measuring THP-1
cell viability after 3 days in the presence of test compounds. Test com-
pounds (4f, 4 h, 4n, 5f, and 5 m) were prepared as 10-point three-fold
serial dilutions in DMSO. The highest concentration of compound tested
was 50 µM where compounds were soluble in DMSO at 10 mM. For
compounds with limited solubility, the highest concentration was 200X
less than the stock concentration e.g. 25 µM for 5 mM DMSO stock, 5 µM
for 1 mM DMSO stock. Each plate included staurosporine as a control.
THP-1 cells were cultured incomplete RPMI and differentiated into
macrophage-like cells using 80 nM PMA overnight at 37 ◦C, 5% CO2.
Cells were inoculated into assay plates and cultured for 24 h before
compound dilutions were added to a final DMSO concentration of 0.5%.
Each run included staurosporine as a control. Assay plates were incu-
bated for 3 days at 37 ◦C, 5% CO2; growth was measured using the
CellTiter-Glo® Luminescent Cell Viability Assay (Promega) which uses
ATP as an indicator of cell viability. Relative luminescent units (RLU)
were measured using a Biotek Synergy 4 plate reader. The DRC was
fitted using the Levenberg–Marquardt algorithm. The IC50 was defined
as the compound concentration that produced 50% inhibition of growth.
50% and 90% inhibition of growth were determined (IC50 and IC90
,
respectively).
4.4.2. MIC determination under hypoxic (low) oxygen condition [46]
Test compounds (4f, 4 h, 4n, 5f, and 5 m) were prepared as 20-point
two-fold serial dilutions in DMSO and diluted into DTA medium in 96-
well plates to a final DMSO concentration of 2%. The highest concen-
tration of compound was 200 µM where compounds were soluble in
DMSO at 10 mM. For compounds with limited solubility, the highest
concentration was 50X less than the stock concentration e.g. 100 µM for
5 mM DMSO stock, 20 µM for 1 mM DMSO stock. Control compounds
were prepared as two-fold serial dilutions in DMSO and diluted into DTA
medium in 96-well plates with a final DMSO concentration of 2%. MTB
constitutively expressing the luxABCDE operon was inoculated into DTA
medium in gas-impermeable glass tubes and incubated for 18 days to
generate hypoxic conditions (Wayne model of hypoxia). At this point,
bacteria are in a non-replicating state (NRP stage 2) induced by oxygen
depletion. Oxygen-deprived bacteria were inoculated into compound
assay plates and incubated under anaerobic conditions for 10 days fol-
lowed by incubation under aerobic conditions (outgrowth) for 28 h.
Oxygen-deprived bacteria were also inoculated into compound assay
4.4.6. MIC determination against drug-resistant isolates of M.
Tuberculosis [42]
The MIC of compound was determined by measuring bacterial
16

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
growth after 5 days in the presence of test compounds. Test compounds
(4f, 4 h, 4n, 5f, and 5 m) were prepared as 10-point two-fold serial
dilutions in DMSO and diluted into 7H9-Tw-OADC medium in 96-well
plates with a final DMSO concentration of 2%. The highest concentra-
tion of compound was 200 µM where compounds were soluble in DMSO
at 10 mM. For compounds with limited solubility, the highest concen-
tration was 50X less than the stock concentration e.g. 100 µM for 5 mM
DMSO stock, 20 µM for 1 mM DMSO stock. Each plate included assay
controls for background (medium/DMSO only, no bacterial cells), zero
growth (100 µM rifampicin) and maximum growth (DMSO only), as well
as a DRC of rifampicin. Plates were inoculated with drug-resistant iso-
lates of MTB and incubated for 5 days and growth was measured by
OD590. To calculate the MIC, the 10-point DRC was plotted as % growth
and fitted to the Gompertz model using GraphPad Prism 5. The MIC was
defined as the minimum concentration at which growth was completely
inhibited and was calculated from the inflection point of the fitted curve
to the lower asymptote (zero growth). In addition dose response curves
were generated using the Levenberg-Marquardt algorithm and the
concentrations that resulted in 50% (IC50) and 90% (IC90) inhibition of
growth were determined, respectively.
(PPrep) module in Maestro software. Finally, the protein structure was
¨
minimized using the OPLS-2005 force field (Schrodinger, LLC, NY, USA,
2009) Glide’s Receptor Grid Generation module was used to generate the
receptor grid at the active site of co-crystalline ligand with the centered
dimension cubic grid box of 10 Ǻ × 10 Ǻ×10 Ǻ [55]. Finally, the low-
energy conformation of the ligands was selected and docked into the
grid generated from protein structures using standard precision (SP)
docking mode. The evaluation was carried out with a Glide SP docking
score and a single absolute best pose is produced as the output for a
specific ligand [54-56].
4.5.2. Binding free energy calculation
Molecular mechanics with generalized born surface area (MM/
GBSA) is the most popular method to estimate the ligand binding en-
ergies, which includes the OPLS3 power field and VSGB solvent model
[57]. The Prime MM-GBSA simulation was carried out by using the Glide
pose viewer file to calculate the total binding free energy. These poses
were taken as inputs for the energy minimization of the protein–ligand
complexes (Ecomplex), the free protein (Eprotein), and the free ligands
(Eligand). The binding free energy ΔGbind was determined according to
the following equation:
4.4.7. MIC determination against other disease-relevant mycobacterial
species [42,47]
▴Gbind = E. Complex (minimized) ꢀ E. ligand (minimized) ꢀ E.
receptor (minimized)
The MIC values was determined by measuring bacterial growth in
the presence of test compounds. Test compounds (4f, 4 h, 4n, 5f, and 5
m) were prepared as 20-point two-fold serial dilutions in DMSO and
diluted into 7H9-Tw-OADC medium in 96-well plates with a final DMSO
concentration of 2%. The highest concentration of compound was 200
µM where compounds were soluble in DMSO at 10 mM. For compounds
with limited solubility, the highest concentration was 50X less than the
stock concentration e.g. 100 µM for 5 mM DMSO stock, 20 µM for 1 mM
DMSO stock. Each plate included assay controls for background (me-
dium/DMSO only, no bacterial cells), zero growth (100 µM rifampicin)
and maximum growth (DMSO only), as well as DRC of rifampicin.
The MM/GBSA calculations are used to estimate relative binding
affinity of ligands to the receptor (reported in kcal/mol). As the MM/
GBSA binding energies are approximate free energies of binding, a more
negative value indicates stronger binding [58,59].
4.5.3. Molecular dynamic simulations
MD simulations for the best dock protein ligand complex was carried
out using the Desmond program, an explicit solvent MD package
¨
(version 3.1, Desmond Molecular Dynamics System, Schrodinger) along
with fixed optimized potentials for liquid simulation (OPLS 2005) force
field [60]. The system was built up for simulation using a predefined
water model (simple point charge, SPC) as solvent in an orthorhombic
box with periodic boundary conditions specifying the shape and size of
box as 10 Å × 10 Å × 10 Å distance. The desirable electrically neutral
system for simulation was built with 0.15 M NaCl (physiological con-
centration of monovalent ions) in 10 Å buffer regions between the
protein atoms and box sides using the system-built option. Steepest
Descent and limited-memory Broyden-Fletcher Goldfarb-Shanno algo-
rithms were applied in a hybrid manner to achieve the relaxation of the
system [61]. A constant 300 K temperature and 1 atm pressure was
maintained during the simulation using the Nose-Hoover thermostat
algorithm and Martyna-Tobias-Klein Barostat algorithm, respectively
[62]. Long-range and short-range coulombic interaction was controlled
using smooth particle mesh ewald method with 9.0 Å endpoint values
[62]. The simulation was achieved under NPT ensemble for 10 ns and
trajectory information was obtained with the rest of 10 ps applying the
Berendsen thermostat and barostat methods [62].
4.4.7.1. Mycobacterium abscessus. Plates were inoculated with M.
abscessus and incubated for 3 days at 37˚C and growth was measured by
OD590. To dose response curve was plotted as % growth and fitted to the
Gompertz model. The MIC was defined as the minimum concentration at
which growth was completely inhibited and was calculated from the
inflection point of the fitted curve to the lower asymptote (zero growth).
In addition, dose response curves were generated using the Levenberg-
Marquardt algorithm and the concentrations that resulted in 50% and
90% inhibition of growth was determined (IC50 and IC90, respectively).
Rifampicin was included once in each run.
4.4.7.2. Mycobacterium avium. Plates were inoculated with M. avium,
incubated for 5 days at 37˚C and Alamar blue was added to each well (10
µL of Alamar blue to 100 µL culture) and incubated for 24 h at 37 ˚C.
Plates were visually inspected and the color recorded for each well. MIC
was defined as the lowest concentration at which no metabolic activity
was seen (blue well).
Supporting Information
Spectral images of all of the synthesized compounds are provided in
the supporting information.
Declaration of Competing Interest
4.5. Computational study
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.5.1. Molecular docking
We have performed docking study of all the synthesized compounds
towards the GyrB ATPase domain. The ligands were prepared using
¨
LigPrep module (Schrodinger, LLC, NY, USA, 2009) by adding hydrogen
atoms, removing salt, generating stereoisomers, ionizing at pH (7 ± 2)
and determining valid 3D conformation [54]. Additionally, the geome-
try of the ligands was minimized using the standard molecular me-
chanic’s energy function OPLS_2005 force field. The crystal structure of
GyrB ATPase (PDB ID: 4B6C) was obtained from Protein Data Bank. The
protein structure was prepared using the Protein Preparation Wizard
Acknowledgments
Authors thank Discipline of Pharmaceutical Sciences, College of
Health Sciences, University of KwaZulu-Natal (UKZN), South Africa (SA)
for the necessary facilities. Authors sincerely thank National Institutes of
Health and the National Institute of Allergy and Infectious Diseases-
17

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
[20] Nisha, J. Gut, P.J. Rosenthal, V. Kumar, Kumar, β-amino-alcohol tethered 4-
aminoquinoline-isatin conjugates: Synthesis and antimalarial evaluation, Eur. J.
Med. Chem. 84 (2014) 566–573.
Bethesda, MD, (USA) for in vitro anti-mycobacterial activity character-
ization. Authors are thankful to Inkosi Albert Luthuli central hospital,
Durban, SA for antimicrobial screening and Centre for High Perfor-
mance Computing-Cape Town, SA for computational resources,
respectively. Author BC gratefully acknowledges National Research
Foundation (NRF), SA for the research funding in the form of NRF-
Innovation Post-Doctoral Research Fellowship (Grant No. 99546). RK
is also thankful to NRF-SA for funding the project (Grant No. 103728 and
112079). Authors express heartfelt thanks to Mr. Dilip Jagjivan and Dr.
Caryl Janse Van Rensburg (UKZN-SA) for their assistance in NMR and
HR-MS experiments, respectively.
[21] Z.H. Chohan, H. Pervez, A. Rauf, K.M. Khan, C.T. Supuran, Isatin-derived
antibacterial and antifungal compounds and their transition metal complexes,
J. Enzyme Inhib. Med. Chem. 19 (5) (2004) 417–423.
[22] S.K. Sridhar, M. Saravanan, A. Ramesh, Synthesis and antibacterial screening of
hydrazones, Schiff and Mannich bases of isatin derivatives, Eur. J. Med. Chem. 36
(7-8) (2001) 615–625.
[23] J.M. Hwang, T. Oh, T. Kaneko, A.M. Upton, S.G. Franzblau, Z. Ma, S.-N. Cho,
P. Kim, Design, synthesis, and structure–activity relationship studies of
tryptanthrins as anti-tubercular agents, J. Nat. Prod. 76 (2013) 354–367.
[24] L.-S. Feng, M.-L. Liu, B.o. Wang, Y. Chai, X.-Q. Hao, S. Meng, H.-Y. Guo, Synthesis
and in vitro anti-mycobacterial activity of balofloxacin ethylene isatin derivatives,
Eur. J. Med. Chem. 45 (8) (2010) 3407–3412.
Note:
[25] L.-S. Feng, M.-L. Liu, S. Zhang, Y. Chai, B.o. Wang, Y.-B. Zhang, K. Lv, Y. Guan, H.-
Y. Guo, C.-L. Xiao, Synthesis and in vitro anti-mycobacterial activity of 8-OCH3
ciprofloxacin methylene and ethylene isatin derivatives, Eur. J. Med. Chem. 46 (1)
(2011) 341–348.
International Patent Filed: PCT Int. Appl. (2018), WO 2018100484
A1 20180607.
[26] D. Sriram, A. Aubry, P. Yogeeswari, L.M. Fisher, Gatifloxacin derivatives:
Synthesis, anti-mycobacterial activities, and inhibition of Mycobacterium
tuberculosis DNA gyrase, Bioorg. Med. Chem. Lett. 16 (2006) 2982–2985.
[27] S.S. Karki, R. Hazare, S. Kumar, A. Saxena, A. Katiyar, Synthesis and antimicrobial
activity of some 3-substituted-2-oxindole derivatives, Turk J. Pharm. Sci. 8 (2011)
169–178.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105133.
[28] T. Aboul-Fadl, M. Abdel-Hamid, A. Youssef, Schiff bases of indoline-2,3-dione
(isatin) derivatives as efficient agents against resistant strains of Mycobacterium
tuberculosis, Der Pharma Chem. 7 (2015) 217–225.
References
[29] M. Biava, G. Cesare Porretta, D. Deidda, R. Pompei, A. Tafi, F. Manetti, Importance
of the thiomorpholine introduction in new pyrrole derivatives as anti-
mycobacterial agents analogues of BM 212, Bioorg. Med. Chem. 11 (4) (2003)
515–520.
[1] World health Organisation, Global Tuberculosis Report 2019, http://www.who.
int/tb/publications/global_report/en/ (accessed July 22, 2020).
[2] Treatment for TB Disease, Cent. Dis. Control Prev. http://www.cdc.gov/tb/topic/
treatment/tbdisease.htm (accessed July 22, 2020).
[30] M.R. Barbachyn, D.K. Hutchinson, S.J. Brickner, M.H. Cynamon, J.O. Kilburn, S.
P. Klemens, S.E. Glickman, K.C. Grega, S.K. Hendges, D.S. Toops, C.W. Ford, G.
E. Zurenko, Identification of a novel oxazolidinone (U-100480) with potent anti-
mycobacterial activity, J. Med. Chem. 39 (3) (1996) 680–685.
[3] E. Torfs, T. Piller, P. Cos, D. Cappoen, Opportunities for overcoming
Mycobacterium tuberculosis drug resistance : Emerging mycobacterial targets and
host-directed therapy, Int. J. Mol. Sci. 20 (2019) 2868.
[4] A. Zumla, P. Nahid, S.T. Cole, Advances in the development of new tuberculosis
drugs and treatment regimens, Nat. Rev. Drug Discov. 12 (5) (2013) 388–404.
[5] R.C. Goldman, K.V. Plumley, B.E. Laughon, The evolution of extensively drug
resistant tuberculosis (XDR-TB): History, status and issues for global control, Infect.
Disord. Drug Targets 7 (2007) 73–91.
[31] D. Sriram, P. Yogeeswari, K. Madhu, Synthesis and in vitro and in vivo anti-
mycobacterial activity of isonicotinoyl hydrazones, Bioorg. Med. Chem. Lett. 15
(20) (2005) 4502–4505.
[32] S¸.G. Küçükgüzel, S. Rollas, I. Küçükgüzel, M. Kiraz, Synthesis and anti-
mycobacterial activity of some coupling products from 4-aminobenzoic acid
hydrazones, Eur. J. Med. Chem. 34 (12) (1999) 1093–1100.
[6] R. Mahajan, Bedaquiline: First FDA-approved tuberculosis drug in 40 years, Int. J.
Appl. Basic Med. Res. 3 (1) (2013) 1, https://doi.org/10.4103/2229-516X.112228.
[7] S. Cherukupalli, C. Balakumar, R.R. Aleti, N. Sayyad, G.A. Hampannavar, S.
R. Merugu, H.R. Reddy, R. Banerjee, R. Karpoormath, Synthesis of 4,6-disubsti-
tuted pyrazolo[3,4-d]pyrimidine analogues: Cyclin-dependent kinase 2 (CDK2)
inhibition, molecular docking and anticancer evaluation, J. Mol. Struc. 1176
(2019) 538–551.
[33] Global Alliance for TB drug development, Study to evaluate the safety, tolerability,
pharmacokinetics of Sutezolid, https://clinicaltrials.gov/ct2/show/NCT03199313
(accessed July 22, 2020).
[34] H.M. Patel, M. Palkar, R. Karpoormath, Exploring MDR-TB inhibitory potential of
4-amino quinazolines as Mycobacterium tuberculosis N-acetylglucosamine-1-
phosphate uridyltransferase (GlmU MTB) inhibitors, Chem. Biodiv. 17 (8) (2020),
https://doi.org/10.1002/cbdv.v17.810.1002/cbdv.202000237.
[8] B. Chandrasekaran, S. Cherukupalli, S. Karunanidhi, A. Kajee, R.R. Aleti,
N. Sayyad, B. Kushwaha, S.R. Merugu, K.P. Mlisana, R. Karpoormath, Design and
synthesis of novel heterofused pyrimidine analogues as effective antimicrobial
agents, J. Mol. Struct. 1183 (2019) 246–255.
[35] M.S. Shaikh, A.M. Kanhed, B. Chandrasekaran, M.B. Palkar, N. Agrawal,
C. Lherbet, G.A. Hampannavar, R. Karpoormath, Discovery of novel N-methyl
carbazole tethered rhodanine derivatives as direct inhibitors of Mycobacterium
tuberculosis InhA, Bioorg. Med. Chem. Lett. 29 (16) (2019) 2338–2344.
[36] G.A. Hampannavar, R. Karpoormath, M.B. Palkar, M.S. Shaikh, B. Chandrasekaran,
Dehydrozingerone inspired styryl hydrazine thiazole hybrids as promising class of
anti-mycobacterial agents, ACS Med. Chem. Lett. 7 (7) (2016) 686–691.
[37] S.G. Shingade, S.B. Bari, U.B. Waghmare, Synthesis and antimicrobial activity of 5-
chloroindoline-2, 3-dione derivatives, Med. Chem. Res. 21 (7) (2012) 1302–1312.
[38] K.M. Khan, M. Khan, M. Ali, M. Taha, S. Rasheed, S. Perveen, M.I. Choudhary,
Synthesis of bis-Schiff bases of isatins and their antiglycation activity, Bioorg. Med.
Chem. 17 (22) (2009) 7795–7801.
[9] M. Batool, B. Ahmad, S. Choi, A structure-based drug discovery paradigm, Int. J.
Mol. Sci. 20 (2019) 2783.
[10] K. Mdluli, Z. Ma, Mycobacterium tuberculosis DNA gyrase as a target for drug
Discovery, Infect. Disord. Drug Targets 7 (2007) 159–168.
[11] T. Khan, K. Sankhe, V. Suvarna, A. Sherje, K. Patel, B. Dravyakar, DNA gyrase
inhibitors: Progress and synthesis of potent compounds as antibacterial agents,
Biomed. Pharmacother. 103 (2018) 923–938.
[12] V. Nagaraja, A.A. Godbole, S.R. Henderson, A. Maxwell, DNA topoisomerase I and
DNA gyrase as targets for TB therapy, Drug Discov. Today 22 (3) (2017) 510–518.
´
[13] S. Petrella, E. Capton, B. Raynal, C. Giffard, A. Thureau, F. Bonnete, P.M. Alzari,
[39] W.M. Eldehna, A. Altoukhy, H. Mahrous, H.A. Abdel-Aziz, Design, synthesis and
QSAR study of certain isatin-pyridine hybrids as potential anti-proliferative agents,
Eur. J. Med. Chem. 90 (2015) 684–694.
A. Aubry, C. Mayer, Overall structures of Mycobacterium tuberculosis DNA gyrase
reveal the role of a Corynebacteriales GyrB-specific insert in ATPase activity,
Structure 27 (4) (2019) 579–589.e5.
[40] H.E. Dweedar, H. Mahrous, H.S. Ibrahim, H.A. Abdel-aziz, Analogue-based design,
synthesis and biological evaluation of anticancer agents, Eur. J. Med. Chem. 78
(2014) 275–280.
[14] K. Chaudhari, S. Surana, P. Jain, H.M. Patel, Mycobacterium Tuberculosis (MTB)
GyrB inhibitors: An attractive approach for developing novel drugs against TB, Eur.
J. Med. Chem. 124 (2016) 160–185.
[41] A. Zelmer, P. Carroll, N. Andreu, K. Hagens, J. Mahlo, N. Redinger, B.D. Robertson,
S. Wiles, T.H. Ward, T. Parish, J. Ripoll, G.J. Bancroft, U.E. Schaible, A new in vivo
model to test anti-tuberculosis drugs using fluorescence imaging, J. Antimicrob.
Chemother. 67 (8) (2012) 1948–1960.
[15] S. Chopra, K. Matsuyama, T. Tran, J.P. Malerich, B. Wan, S.G. Franzblau, S. Lun,
H. Guo, M.C. Maiga, W.R. Bishai, P.B. Madrid, Evaluation of gyrase B as a drug
target in Mycobacterium tuberculosis, J. Antimicrob. Chemother. 67 (2) (2012)
415–421.
[42] J. Ollinger, M.A. Bailey, G.C. Moraski, A. Casey, S. Florio, T. Alling, M.J. Miller,
T. Parish, P.C. Karakousis, A dual read-out assay to evaluate the potency of
compounds active against Mycobacterium tuberculosis, PLoS ONE 8 (4) (2013)
e60531, https://doi.org/10.1371/journal.pone.006053110.1371/journal.
pone.0060531.g00110.1371/journal.pone.0060531.g00210.1371/journal.
pone.0060531.g00310.1371/journal.pone.0060531.g00410.1371/journal.
pone.0060531.g00510.1371/journal.pone.0060531.g00610.1371/journal.
pone.0060531.t00110.1371/journal.pone.0060531.t002.
[16] P.S. Hameed, S. Suresh, M. Kakoli, et al., Optimization of pyrrolamides as
mycobacterial GyrB ATPase inhibitors: structure-activity relationship and in vivo
efficacy in a mouse model of tuberculosis, Antimicrob. Agents Chemother. 58
(2014) 61–70.
[17] M.C. Pirrung, S.V. Pansare, K.D. Sarma, K.A. Keith, E.R. Kern, Combinatorial
optimization of isatin-β-thiosemicarbazones as anti-poxvirus agents, J. Med. Chem.
48 (8) (2005) 3045–3050.
[18] A.H. Abadi, S.M. Abou-Seri, D.E. Abdel-Rahman, C. Klein, O. Lozach, L. Meijer,
Synthesis of 3-substituted-2-oxoindole analogues and their evaluation as kinase
inhibitors, anticancer and antiangiogenic agents, Eur. J. Med. Chem. 41 (2006)
296–305.
[43] Y.U. Cebici, H. Bayrak, Y. Sirin, Synthesis of novel Schiff bases and azol-β- lactam
derivatives starting from morpholine and thiomorpholine and investigation of their
anti-tubercular, antiurease activity, acethylcolinesterase inhibition effect and
antioxidant capacity, Bioorg. Chem. 88 (2019), 102928.
[19] K. Kumar, S. Sagar, L. Esau, M. Kaur, V. Kumar, Synthesis of novel 1H–1,2,3-
triazole tethered C-5 substituted uracil–isatin conjugates and their cytotoxic
evaluation, Eur. J. Med. Chem. 58 (2012) 153–159.
[44] S.H. Cho, S. Warit, B. Wan, C.H. Hwang, G.F. Pauli, S.G. Franzblau, Low- oxygen-
recovery assay for high-throughput screening of compounds against nonreplicating
18

S. Karunanidhi et al.
Bioorganic Chemistry 115 (2021) 105133
¨
Mycobacterium tuberculosis, Antimicrob. Agents Chemother. 51 (4) (2007)
[54] S. Release, Protein Preparation Wizard, Epik, Schrodinger, LLC, New York, NY,
1380–1385.
2008.
[45] N. Andreu, A. Zelmer, T. Fletcher, P.T. Elkington, T.H. Ward, J. Ripoll, T. Parish, G.
J. Bancroft, U. Schaible, B.D. Robertson, S. Wiles, T.M. Doherty, Optimisation of
bioluminescent reporters for use with mycobacteria, PLoS ONE 5 (5) (2010)
e10777, https://doi.org/10.1371/journal.pone.0010777.
[55] T.A. Halgren, R.B. Murphy, R.A. Friesner, H.S. Beard, L.L. Frye, W.T. Pollard, J.
L. Banks, Glide: a new approach for rapid, accurate docking and scoring. 2.
Enrichment factors in database screening, J. Med. Chem. 47 (7) (2004) 1750–1759,
https://doi.org/10.1021/jm030644s.
[46] M.F. Abo-Ashour, W.M. Eldehna, A. Nocentini, A. Bonardi, S. Bua, H.S. Ibrahim, M.
[56] R.A. Friesner, J.L. Banks, R.B. Murphy, T.A. Halgren, J.J. Klicic, D.T. Mainz, M.
P. Repasky, E.H. Knoll, M. Shelley, J.K. Perry, D.E. Shaw, P. Francis, P.S. Shenkin,
Glide: a new approach for rapid, accurate docking and scoring. 1. Method and
assessment of docking accuracy, J. Med. Chem. 47 (7) (2004) 1739–1749, https://
doi.org/10.1021/jm0306430.
ˇ
M. Elaasser, V. Krystof, R. Jorda, P. Gratteri, S.M. Abou-Seri, C.T. Supuran, 3-
Hydrazinoisatin-based benzenesulfonamides as novel carbonic anhydrase
inhibitors endowed with anticancer activity: Synthesis, in vitro biological
evaluation and in silico insights, Eur. J. Med. Chem. 184 (2019), 111768.
[47] S.G. Franzblau, R.S. Witzig, J.C. McLaughlin, P. Torres, G. Madico, A. Hernandez,
M.T. Degnan, M.B. Cook, V.K. Quenzer, R.M. Ferguson, R.H. Gilman, Rapid, low-
technology MIC determination with clinical Mycobacterium tuberculosis isolates
by using the microplate alamar blue assay, J. Clin. Microbiol. 36 (2) (1998)
362–366.
[57] B. Vijayakumar, S. Parasuraman, R. Raveendran, D. Velmurugan, Identification of
natural inhibitors against angiotensin I converting enzyme for cardiac safety using
induced fit docking and MM-GBSA studies, Pharmacogn Mag 10 (Suppl 3) (2014)
S639–S644, https://doi.org/10.4103/0973-1296.139809.
[58] H.M. Patel, M. Shaikh, I. Ahmad, D. Lokwani, S.J. Surana, BREED based de novo
hybridization approach: generating novel T790M/C797S-EGFR tyrosine kinase
inhibitors to overcome the problem of mutation and resistance in non-small cell
lung cancer (NSCLC) [published online ahead of print, J. Biomol. Struct. Dyn. 39
(8) (2021) 2838–2856, https://doi.org/10.1080/07391102.2020.1754918.
[59] K.A. Casalvieri, C.J. Matheson, D.S. Backos, P. Reigan, Molecular docking of
substituted pteridinones and pyrimidines to the ATP-binding site of the N-terminal
domain of RSK2 and associated MM/GBSA and molecular field datasets, Data Brief
29 (2020) 105347, https://doi.org/10.1016/j.dib.2020.105347.
¨
¨
[48] Schrodinger-Maestro, Schrodinger Release, 2019-3.
[49] C. Balakumar, M. Ramesh, C.L. Tham, S.P. Khathi, F. Kozielski, C. Srinivasulu, G.
A. Hampannavar, N. Sayyad, M.E. Soliman, R. Karpoormath, Ligand- and structure-
based in silico studies to identify kinesin spindle protein (KSP) inhibitors as
potential anticancer agents, J. Biomol. Struct. Dyn. 36 (14) (2018) 3687–3704.
[50] Y.H. Zhao, J. Le, M.H. Abraham, A. Hersey, P.J. Eddershaw, C.N. Luscombe,
D. Boutina, G. Beck, B. Sherborne, I. Cooper, J.A. Platts, Evaluation of human
intestinal absorption data and subsequent derivation of a quantitative structure –
activity relationship (QSAR) with the Abraham descriptors, J. Pharm. Sci. 90 (6)
(2001) 749–784.
[60] Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2018-
¨
4. Maestro-Desmond Interoperability Tools, Schrodinger, New York, NY, 2018-4.
[51] S.P. Khathi, B. Chandrasekaran, S. Karunanidhi, C.L. Tham, F. Kozielski, N. Sayyad,
R. Karpoormath, Design and synthesis of novel thiadiazole-thiazolone hybrids as
potential inhibitors of the human mitotic kinesin Eg5, Bioorg. Med. Chem. Lett. 28
(17) (2018) 2930–2938.
[61] H.M. Patel, I. Ahmad, R. Pawara, M. Shaikh, S.J. Surana, In silico search of triple
mutant T790M/C797S allosteric inhibitors to conquer acquired resistance problem
in non-small cell lung cancer (NSCLC): a combined approach of structure-based
virtual screening and molecular dynamics simulation, J. Biomol. Struct. Dyn.
(2020) 1–15, https://doi.org/10.1080/07391102.2020.1734092.
[52] S.K. Talapatra, C.L. Tham, P. Guglielmi, R. Cirilli, B. Chandrasekaran,
R. Karpoormath, S. Carradori, F. Kozielski, Crystal structure of the Eg5-K858
complex and implications for structure-based design of thiadiazole-containing
inhibitors, Eur. J. Med. Chem. 156 (2018) 641–651.
[62] Z. Jin, Y. Wang, X.-F. Yu, Q.-Q. Tan, S.-S. Liang, T. Li, H. Zhang, P.-C. Shaw,
J. Wang, C. Hu, Structure-based virtual screening of influenza virus RNA
polymerase inhibitors from natural compounds: Molecular dynamics simulation
and MM-GBSA calculation, Comput. Biol. Chem. 85 (2020) 107241, https://doi.
org/10.1016/j.compbiolchem.2020.107241.
[53] R.J. Lambert, J. Pearson, Susceptibility testing: accurate and reproducible
minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC)
values, J. Appl. Microbiol. 88 (2000) 784–790.
19