Design, synthesis and biological evaluation of novel Pseudomonas aeruginosa DNA gyrase B inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.103905

In the present study, we attempted to develop a novel class of compounds active against Pseudomonas aeruginosa (Pa) by exploring the pharmaceutically well exploited enzyme targets. Since, lack of Pa gyrase B crystal structures, Thermus thermophilus gyrase B in complex with novobiocin (1KIJ) was used as template to generate model structure by performing homology modeling. Further the best model was validated and used for high-throughput virtual screening, docking and dynamics simulations using the in-house database for identification of Pa DNA gyrase B inhibitors. This study led to an identification of three lead molecules with IC₅₀ values in range of 6.25–15.6 μM against Pa gyrase supercoiling assay. Lead-1 optimization and expansion resulted in 15 compounds. Among the synthesized compounds six compounds were shown good enzyme inhibition than Lead-1 (IC₅₀ 6.25 μM). Compound 13 emerged as the most potential compound exhibiting inhibition of Pa gyrase supercoiling with an IC₅₀ of 2.2 μM; and in-vitro Pa activity with MIC of 8 μg/mL in presence of efflux pump inhibitor; hence could be further developed as novel inhibitor for Pa gyrase B.

Full text of DOI:10.1016/j.bioorg.2020.103905

BioorganicChemistry100(2020)103905
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of novel Pseudomonas aeruginosa
DNA gyrase B inhibitors
⁎
Sridhar Jogulaa, Vagolu Siva Krishnaa, Nikhila Medaa, Vadla Balrajub, Dharmarajan Srirama,
a Department of Pharmacy, Birla Institute of Technology and Science-Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad 500078, India
b Albany Molecular Research Hyderabad Research Centre Private Limited, MN Park, Genome valley, Hyderabad 500078, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the present study, we attempted to develop a novel class of compounds active against Pseudomonas aeruginosa
(Pa) by exploring the pharmaceutically well exploited enzyme targets. Since, lack of Pa gyrase B crystal struc-
tures, Thermus thermophilus gyrase B in complex with novobiocin (1KIJ) was used as template to generate model
structure by performing homology modeling. Further the best model was validated and used for high-throughput
virtual screening, docking and dynamics simulations using the in-house database for identification of Pa DNA
gyrase B inhibitors. This study led to an identification of three lead molecules with IC50 values in range of
6.25–15.6 µM against Pa gyrase supercoiling assay. Lead-1 optimization and expansion resulted in 15 com-
pounds. Among the synthesized compounds six compounds were shown good enzyme inhibition than Lead-1
(IC50 6.25 µM). Compound 13 emerged as the most potential compound exhibiting inhibition of Pa gyrase
supercoiling with an IC50 of 2.2 µM; and in-vitro Pa activity with MIC of 8 µg/mL in presence of efflux pump
inhibitor; hence could be further developed as novel inhibitor for Pa gyrase B.
Pseudomonas aeruginosa
DNA gyrase B
Molecular docking
Molecular dynamics
Isothiazole
Oxazole
1. Introduction
target for antimicrobial agents. It belongs to a class of type II topoi-
somerase enzymes, a hetero-tetramer having two each subunits of A
Pseudomonas aeruginosa (Pa) is a rod-shaped, gram-negative bac-
terium belonging to the family Pseudomonadacea and emerging
worldwide as one of serious threats to human health [1]. A ubiquitous
environmental and most common nosocomial isolates constituting of
20% in case of pediatric intensive care unit (PICU) [2] and 10% [3] of
all hospital acquired infections. It is also termed as opportunistic human
pathogen as it colonizes in immune compromised patients, like those
with cystic fibrosis, cancer, or AIDS [4] rather than healthy individuals.
Treatment against this pathogen remains challenging because of de-
velopment of antibiotic resistance, an increasing threat to human
health. It exhibits intrinsic resistance i.e. constitutive expression of
AmpC b-lactamase and efflux pumps, combined with a low perme-
ability of the outer membrane and its remarkable ability to acquire
further resistance mechanisms to multiple groups of antimicrobial
agents, including β-lactams, aminoglycosides and fluoroquinolones [5].
Hence, there is an immediate need in the identification and synthesis of
novel drugs for potential targets that circumvents Pseudomonas infec-
tions.
and B. An ATP dependent enzyme, controls topological transitions [6]
by introducing negative super-coiling in double stranded closed circular
DNA. Negative supercoiling maintains DNA in under winding state,
helps in strand separation during replication.
The action of subunits A (GyrA) is inhibited by quinolones such as
nalidixic acid and ciprofloxacin, while those of the B subunits (GyrB)
are inhibited by coumarins such as coumermycin Al and novobiocin.
Targeting gyrase may have an edge over others as it arrests DNA re-
plication predominantly along with recombination, transcription and
repair. While GyrA domain was exploited extensively, the GyrB cata-
lytic domain has high scope for research. Pa has developed resistant
towards fluoroquinolones and other available drugs. Most of the present
drugs were specific to GyrA, but very few compounds were explored
towards GyrB. Novobiocin is the only aminocoumarin till date that was
approved against GyrB. The remarkable ability of Pa to acquire further
resistance mechanism to multiple groups of available antimicrobial
agents including β-lactams, aminoglycosides and fluoroquinolones
provides a huge scope for finding structurally different compounds.
Hence, the aim of present study is to identify new compounds acting
specifically on GyrB subunit of P. aeruginosa [7].
A strategic approach to antimicrobial drug discovery is to target
enzymes that are vital for the existence of the bacterium. In supports to
this, DNA gyrase is an essential enzyme and has proven to be a worthy
⁎ Corresponding author.
E-mail address: dsriram@hyderabad.bits-pilani.ac.in (D. Sriram).
https://doi.org/10.1016/j.bioorg.2020.103905
Received 23 October 2019; Received in revised form 29 April 2020; Accepted 30 April 2020
Availableonline04May2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

S. Jogula, et al.
BioorganicChemistry100(2020)103905
2. Results and discussion
Table 1
Molecular interactions of the Lead compounds and novobiocin.
2.1. Homology modeling
S. No
Name
XP G score (kcal/
mol)
Interactions
Pa GyrB structure was generated by using the homology modeling
by considering the Thermus thermophilus GyrB as a template [8]. Total of
20 structures were generated and 16th model with lowest discrete op-
timized potential energy (DOPE) score (−39058.39 kcal/mol−1), was
selected as the reliable one for further validation. It possess 98.5% re-
sidues in most favored regions of Ramachandran plot analysis which
suggesting the model was good with decent stereo-chemical quality [9].
ProSA analysis resulted in z-score of −7.78 revealed that the model is
well within the range of native conformations of X-ray crystal structures
and overall residue energies remain largely negative [10]. ProQ tool
assessment showed LG score of 4.945 represents the model is of ex-
tremely good quality [11]. The model structure was visualized by using
PyMOL [12].
1
Lead-1
−4.843
Asn48, Asp75, Gly79, Lys112,
Thr167
2
3
4
Lead-2
−3.141
−4.682
−3.119
Asp51, Lys112
Lead-3
Asn48, Lys112
Novobiocin
Asn48, Asp75,Asn109, Arg138
docking, 18 compounds were observed to show significant glide score
in SP docking hence re-docked using extra precision mode (XP) [14].
All compounds were evaluated for their activity using Pa DNA gyrase
supercoiling assay and found that Lead-1, Lead-2 and Lead-3 were ac-
tive with IC50 of 6.2 µM, 8.7 µM and 15.6 µM respectively. Molecular
interactions of the Lead and novobiocin were shown in Table 1 and
Fig. 2.
Based on the Lead-1 molecular interactions and its biological po-
tency, we have designed and synthesized 15 derivatives and also per-
formed the molecular docking and dynamic simulations. Out of all
synthesized derivatives, compound 13 [4-amino-N5-(2-((3-chloro-5-
(trifluoromethyl) benzyl)amino)-2-oxoethyl)-N5-(2-methylbenzo[d]oxazol-
6-yl)isothiazole-3,5-dicarboxamide] in complex with GyrB (Fig. 3)
showed hydrogen bonds with binding site residues such as Asn48,
Asp75, Gly79, Lys112, and Thr167. It also formed π-π interaction with
Phe106. Hydrogen bonds with Asn48 and Asp75 were retained by
compound 13 when compared with both novobiocin and Lead-1 in-
teractions. Residues, Gly79, Lys112, and Thr167 shown van der Waals
interactions in case of novobiocin, the same residues were enhancing
the binding affinity by forming the hydrogen bonds with compound 13.
The XP G score of compound 13 (−5.317) shown to be better com-
pared to novobiocin and Lead-1. Lead molecule and its derivatives have
obeyed to Lipinski rule of 5 (Table 1 in supporting information) [15].
Thus, Lead-1 and compound 13 were proposed as potential Pa DNA
GyrB inhibitors.
2.2. Binding site analysis
The generated tertiary structure of drug target is the initial re-
quirement for structure-based drug design. Incorporating ligands into
the homology model from the structural template enhances overall
accuracy of the predicted models which also helps in determining li-
gand binding pocket. The superposition of the overall atoms of the
target-template reflects the close resemblance with experimental one
and the interpreted lower RMSD of 0.81 Å show higher structural
conservation. The modeled structure in complex with novobiocin was
explored to find residues such as Asn48, Asp51, Glu52, Asp75, Asp78,
Gly79, Ile80, Pro81, Asp83, Ile96, Asn107, Lys112, Val120, Arg138,
Thr167 present within the 4 Å binding site region of novobiocin. Five
hydrogen bonds were observed with residues Asn48, Asp75, Asp83,
Lys105 and Arg138. The target-template sequence alignment showed
that these residues were conserved and were used to generate grid for
molecular docking studies (Fig. 1).
2.3. Virtual screening
2.4. Chemistry
A receptor grid of 20 Å X 20 Å X 20 Å was generated around the
novobiocin binding residues and in-house library molecules were al-
lowed to dock within the grid by virtual screening workflow protocol
[13]. Glide high throughput virtual screening identified 184 com-
pounds and these were re-docked using standard precision (SP)
The designed molecules were synthesized by following synthetic
approach that has been shown in Scheme 1, intermediate compounds
like 2-methylbenzo[d]oxazol-6-amine (III), bezylamines V(a – p) [17]
and 4-amino-3-carbamoylisothiazole-5-carboxylic acid (X) [16] are
Fig. 1. Model structure of Pseudomonas aerugi-
nosa GyrB, superimposed with template 1KIJ in
the presence of novobiocin co-crystal ligand
(RMSD = 0.81 Å). The color code representa-
tions for the picture as palegreen: template;
violet: target. Conserved active site residues of
target and template in the presence of novo-
biocin were labeled. The amino acids re-
presented in lines and novobiocin was in sticks.
2

S. Jogula, et al.
BioorganicChemistry100(2020)103905
Fig. 2. Molecular docking of lead molecules and novobiocin with the Pa gyrase B homology model. a) Lead-1 (yellow) at active site of enzyme, b) Lead-2 (grey), c)
Lead-3 (green), d) novobiocin (salmon red) docking confirmation at same active site and interactions with residues are in magenta color.
known and so followed literatures reported to synthesize them starting
with commercially available and less expensive materials. 2-methyl-
benzo[d]oxazol-6-amine (III) prepared in two steps, in first step 2-
Amino-5-nitrophenol (I) was treated with triethyl orthoacetate at reflux
temperature as a neat reaction without any use of solvent gave 2-me-
thyl-6-nitrobenzoxazole (II) in good yield. In step two, we used Zn/
NH4Cl condition for reducing nitro to amine which gave very good
conversion and also avoided handling Pd/H2 chemistry, obtained 2-
methylbenzo[d]oxazol-6-amine (III) with very good quality.
in four steps [16], in first step; 2-cyanoacetamide (VI) as treated with
sodium nitrite in presence of acetic acid and water to give compound
VII, which upon reacting with 4-methylbenzenesulphonyl chloride gave
compound VIII, which was cyclised using ethyl 2-mercaptoacetate to
produce compound IX. Hydrolysis of compound IX gave 4-amino-3-
carbamoylisothiazole-5-carboxylic acid (X).
Compounds V (a- p) were prepared by treating with 2-bromoace-
tylbromide in presence of triethylamine with corresponding benzyl
amines IV (a – p) [17]. Compound XI (a – p) were synthesized by N-
alkylation of 2-methylbenzo[d]oxazol-6-amine (III) with corresponding
4-amino-3-carbamoylisothiazole-5-carboxylic acid (X) was prepared
Fig. 3. Molecular interactions of compound l3 (blue color) shown in (a) 3D and (b) 2D representation.
3

S. Jogula, et al.
BioorganicChemistry100(2020)103905
Scheme 1. Synthetic protocol of the compounds, Reagents and conditions: (a) Triethyl orthoacetate, 110 °C, 12 h; (b) Zn, NH4Cl, THF:MeOH:H2O (1:1:1), 0–25 °C;
(c) 2-bromoacetylbromide, TEA, CH2Cl2, 0–25 °C, 2 h; (d) NaNO2, CH3COOH, H2O, 0–15 °C, 8 h; (e) 4-methylbenzenesulphonyl chloride, pyridine, 0–5 °C, 12 h; (f)
ethyl 2-mercaptoacetate, piperidine, ethanol, 0–5 °C, 12 h; (g) NaOH, H2O, ethanol, rt, 12 h; (h) Cs2CO3, DMF, 80 °C, 3 h; (i) TBTU, triethylamine, ethylacetate, 3 h.
Fig. 4. Chemical structures of Lead-1 and compounds 1 to 15.
4

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BioorganicChemistry100(2020)103905
Table 2
Synthesized compounds represented with substitutions along with their biolo-
gical activities.
Compound
R
IC50 μM
MICa
MICb
MICc
μg/mL μg/mL
μg/mL
Lead-1
4-Fluorobenzyl
2,6-Dimethoxybenzyl
1-Phenylethyl
2-Methoxybenzyl
3,4-Dichlorobenzyl
4-Methoxybenzyl
4-Chlorobenzyl
4-Bromobenzyl
3-(Trifluoromethyl)
benzyl
6.2
0.9
> 64
> 64
64
–
–
–
–
8
–
–
8
–
–
1
2
3
4
5
6
7
8
> 10
> 10
10.0
4.5
–
–
0.05 32
–
0.92
0.81
0.69
0.02
0.99
32
64
32
32
64
8
–
9.6
6.6
–
Fig. 5a. . Picture depicting the supercoiling assay of lead1. B - relaxed DNA; S -
relaxed DNA + enzyme + Novobiocin (10 μM); C - relaxed DNA + enzyme; I -
relaxed DNA + enzyme + lead1 (10 μM).
4.2
16
–
2.4
9
2,5-Dichlorobenzyl
3-Methylbenzyl
3,5-Dimethylbenzyl
3-Methyl-5-
3.2
0.53
0.56
0.08
> 64
> 64
> 64
> 64
16
–
16
–
10
11
12
> 10
> 10
7.4
–
–
–
–
(trifluoromethoxy)
benzyl
13
3-Chloro-5-
2.2
64
8
16
(trifluoromethyl)
benzyl
14
2-Methylbenzyl
3-Chlorobenzyl
> 10
5.2
> 64
> 64
–
–
15
0.87
16
16
a
b
c
Without efflux pump inhibitor.
With phenylalanyl arginyl β-naphthylamide.
With conessine.
Fig. 5b. Picture depicting the supercoiling assay of active compound 13. C -
relaxed DNA + enzyme; compound 13 at different concentrations 5, 2.5, 1.25
(μM) + relaxed DNA + enzyme.
compounds V (a – p) using cesium carbonate in DMF solvent. Final
coupling reaction was also tried by using Boc protected com-
pound X but failed to obtain desired product in good yield. Then the
coupling reaction was performed using 4-amino-3-carbamoylisothia-
zole-5-carboxylic acid (X) and corresponding secondary amines XI (a –
p) with 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetra-
fluoroborate (TBTU) and using ethylacetate as solvent to afford the
Lead-1 and 1 to 15 compounds (Fig. 4).
presence of efflux pump. Various chromosomally encoded efflux sys-
tems and outer membrane porins have been identified as important
contributors to resistance in Pa. A number of MDR RND efflux pumps
have been characterized in clinical isolates of P. aeruginosa, namely
MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM.
A
number of potent efflux pump inhibitors including phenylalanyl arginyl
β-naphthylamide (PAβN), carbonyl cyanide m-chloro- phenylhy-
drazone (CCCP), quinoline derivatives, conessine, and 1-(1-
Naphthylmethyl)-piperazine (NMP) have been reported to enhance
antibiotic activity against antibiotic- resistant Gram-negative bacteria.
In view of this we further tested selected compounds in presence of
PaβN and conessine and found that 2 to 8 times reduction in MIC
(Table 2).
2.5. Biological evaluation
All the synthesized compounds were screened for their in-vitro ac-
tivity using Pa DNA gyrase supercoiling assay kit (Inspiralis, Norwich,
UK) and results showed in (Table 2) [18,19]. Lead-1 possessing 4-
fluorobenzyl group exhibited good Pa DNA GyrB inhibitory activity
with an IC50 value of 6.2
0.9 μM. When, benzyl substitution of Lead-
1 possessed electron withdrawing groups such as 4-chloro (IC50
:
6.6
5.2
0.69 μM), 4-bromo (IC50: 4.2
0.02 μM) and 3-chloro (IC50:
2.6. Molecular dynamics
0.87 μM), the activity was not much deviated in comparison
with Lead-1. When Lead-1 benzyl group possessed electron releasing
groups such as 2,6-dimethoxy,1-phenylethyl, 2-methoxy, 4-methoxy, 3-
methyl, 3,5-dimethyl, 2-methyl, the activity was significantly reduced
with IC50 value of ≥ 10 μM. 3,4-dichlorobenzyl 3-(trifluoromethyl)
benzyl, 2,5-dichlorobenzyl and 3-chloro-5-(trifluoromethyl)benzyl
modifications on Lead-1 significantly enhanced the activity. Com-
pound 13 was found to be the most active with an IC50 of 2.2 µM
(Figs. 5a and 5b). Novobiocin was used as standard (positive control)
and relaxed DNA without enzyme as blank (negative control). In-vitro
activity of the synthesized molecules was evaluated against P. aerugi-
nosa using broth dilution method [20]. Most of the compounds showed
moderate activity as shown in Table 2. These molecules were not active
(IC50 value of > 10 µM) against DNA gyrase of Staphylococcus aureus,
Mycobacterium tuberculosis and Escherichia coli indicates that it specifi-
cally active against P. aeruginosa.
Molecular dynamics simulations were carried out closer to the
physiological environmental condition embedded with a system having
water molecules, temperature and pressure [21,22]. The binding or-
ientations of Lead-1 and compound 13 obtained after simulations
showed better correlation with the biologically active states. Moreover,
MD simulations quantify stability of the docked conformations. The
dynamical properties of docking complex were analyzed from trajec-
tories data obtained from 50 ns MD simulations through potential en-
ergy calculations, root mean square deviation (RMSD) and the stability
of P. aeruginosa DNA GyrB with Lead-1 and compound 13 complexes
was evaluated.
The root mean square deviation range for protein Cα and Lead-1 to
their initial conformation was an average of 5.5 Å and 3.7 Å respec-
tively, which was showed that after a small rearrangement from the
initial conformation of complex up to first 10 ns time, was stable during
entire MD simulations period (50 ns). Further the compound 13 has
also shown the stability as Lead-1 with in the binding site. Compound
13 was more stable during the 50 ns with the average RMSD of 2.6 Å
when compared with Lead-1. The RMSD plot analysis revealed that the
The molecules were further tested in-vitro against Pa by agar dilu-
tion method. In the initial screening none of the compounds showed
any appreciable activity (MIC of ≥ 32 μg/mL) even though showed
good DNA gyrase inhibition. The inactivity might be due to the
5

S. Jogula, et al.
BioorganicChemistry100(2020)103905
Fig. 6. Compound 13 RMSD plot with respect to time (50 ns).
Pa GyrB - compound 13 docking complex was stable in all 10,416
preparation workflow in the Maestro v9.6 (Schrodinger LLC, 2015). All
hydrogens were added which were subsequently minimized with opti-
mized potentials for liquid simulations (OPLS) 2005 force field and the
impact molecular mechanics engine. Minimization was performed re-
straining the heavy atoms with the hydrogen torsion parameters turned
off, to allow free rotation of the hydrogens setting the root mean square
deviation (RMSD) of 0.3 Å. Active site residues were obtained from the
template structure ligand interactions and a grid was generated around
centroid of these residues.
trajectories generated during 50 ns MD simulations time (Fig. 6).
3. Conclusion
In present work, molecular modeling, docking and dynamics simu-
lations were employed to identify inhibitors of Pseudomonas aeruginosa
DNA GyrB. Three molecules were found to be active with an IC50 below
15 µM. Of these three, the best molecule was taken as Lead-1 and
further optimized to synthesize fifteen novel compounds. These com-
pounds were further evaluated by DNA gyrase supercoiling assay and
observed six compounds were showing better IC50 values (2.2 –
6.25 µM) compared to lead, compound 13 (4-amino-N5-(2-((3-chloro-5-
(trifluoromethyl)benzyl)amino)-2-oxoethyl)-N5-(2-methylbenzo[d]oxazol-
6-yl)isothiazole-3,5-dicarboxamide) being best with IC50 2.2 µM. This
was further supported by molecular docking and dynamics simulations
showing that the compound 13 has better binding affinity and stability
throughout 50 ns simulations time. Hence we believe that compound
13 would be an interesting lead for further optimization to synthesize
novel Pa DNA GyrB inhibitor.
4.3. Virtual screening
Virtual screening is of key significance for the in-silico drug dis-
covery process to accelerate drug development, as it helps in the se-
lection of the best drug candidates. The prepared in-house database
molecules were docked into the binding sites of the protein by using
HTVS protocol for the assessment of protein–ligand binding affinities.
The filtered molecules from HTVS were exposed to Glide SP (standard
precision) docking which can dock tens to hundreds of ligands with
high accuracy. Post-docking minimization was implemented to opti-
mize the ligand geometries. Compounds with best docking and Glide
scores were then subjected to Glide XP (extra precision) docking for
further removal of false positives is achieved by extensive sampling and
progressive scoring, resulting in advanced enrichment. High oral
availability is often an important consideration for the development of
bioactive molecules as therapeutic agents. Significant descriptors and
pharmaceutically relevant properties (ADME properties) for the com-
pounds were predicted for the development of a successful drug.
The Pa DNA GyrB modeled structure was optimized by adding hy-
drogen atoms and energy minimization to improve favorable steric
contacts. A receptor grid of 20 Å x 20 Å x 20 Å was generated around
novobiocin binding site and directed towards molecular docking for
virtual screening from prepared ligand dataset of around 3000 com-
pounds (in-house database). The binding affinity, interaction with sur-
rounding residues, binding orientation of few compounds was observed
to be better when compared to novobiocin. The Lead-1 (IC50 of
6.25 µM) from the biological data has been taken for further optimi-
zation. The newly synthesized 15 analogues of Lead-1 were also per-
formed the molecular docking and dynamics simulation studies to
evaluate their mode of binding and stability in the binding pocket.
4. Experimental
4.1. Homology modeling
Homology models are useful in structure-based drug designing ap-
plications, especially when a crystallographic structure is unavailable.
Comparative structure modeling technique was implemented to predict
the tertiary structure of Pa DNA gyrase using Modeller 9v13. Pa DNA
gyrase B sequence (Q9I7C2) was retrieved from UniProt (http://
www.uniprot.org/) and performed the BLAST search against PDB to
identify the similar structural availability. Crystal structure of 43 K
ATPase domain of Thermus thermophilus GyrB in complex with novo-
biocin was chosen as structural template (1KIJ) with structural identity
of 46% through BLASTP against PDB analysis. CLUSTALX was used for
initial target–template alignment. The alignment file was converted to
Modeller input format (⁄. ali) and modeler python script was defined to
include heteroatoms during homology modeling. Twenty homology
models were constructed based on satisfaction of target–template spa-
tial restraints using Modeller9v13. The most reliable model was se-
lected through DOPE score evaluations. The selected model was eval-
uated through RAMPAGE for assessment of Ramachandran plot, ProSA
and ProQ. The validated Pa DNA gyrase model’s novobiocin binding
site was considered as target residues for the rational drug design and
they were visualized using PyMOL.
4.4. Chemistry
Reagents obtained from commercial sources were used directly
without further purification. Reactions were carried out under inert
atmosphere of nitrogen or argon. All the reactions were monitored by
thin layer chromatography (TLC) on silica gel 40 F254 (Merck,
Darmstadt, Germany) coated on aluminium plates. 1H and 13C NMR
spectra were recorded on a Bruker AM-400 NMR spectrometer, Bruker
4.2. Protein preparation
The Pa DNA GyrB model was preprocessed with the protein
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BioorganicChemistry100(2020)103905
BioSpin Corp., Germany. Chemical shifts are in parts per million (ppm)
using tetramethylsilane as internal standard.
CDCl3): δ 8.59 (s, 1H), 7.27–7.23 (t, J = 8.0 Hz, 1H), 7.18 (d,
J = 7.2 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.93–6.89 (t, J = 7.2 Hz,
1H), 4.25 (d, J = 5.6 Hz, 2H), 3.92 (s, 2H), 3.80 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 173.4, 156.3, 128.3, 128.1, 127.4, 122.3, 113.6,
55.8, 35.7, 28.6. MS (ESI) Calc. for [C10H12BrNO2 + H]+ = 259.00,
found 249.00.
4.4.1. Procedure for synthesis of 2-methyl-6-nitrobenzoxazole (II)
A mixture of Triethyl orthoacetate (80 mL) and 2-Amino-5-ni-
trophenol (I) (10.0 g) were refluxed for overnight under nitrogen, the
reaction mixture was cooled 0 °C. Precipitated solids were filtered and
obtained solids were charged with hexanes (100 mL), the slurry was
stirred for 30 min. Filtered the solids and dried to afford 8.20 g of 2-
methyl-6-nitrobenzoxazole (II) as pale yellow solid with the yield 76%.
Analytical data for II: mp = 170–173 °C; 1H NMR (400 MHz, CDCl3): δ
8.37 (d, J = 2.3 Hz, 1H), 8.26 (d, J = 2.3 Hz, 1H), 7.72 (d, J = 8.9 Hz,
1H), 2.71 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 168.7, 150.0, 146.8,
120.3, 119.3, 106.8, 100.5, 14.8. MS (ESI) Calc. for [C8H6N2O3 + H]+
= 179.14, found 179.09.
4.4.8. 2-Bromo-N-(3,4-dichlorobenzyl)acetamide (Ve)
Yield: 82%; yellow solid; mp = 206–208 °C; 1H NMR (400 MHz,
CDCl3): δ 8.86 (s, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.52 (s, 1H), 7.27
(d, J = 8.40 Hz, 1H), 4.30 (d, J = 6.0 Hz, 2H), 3.92 (s, 2H). 13C NMR
(100 MHz, CDCl3): δ 173.2, 144.7, 131.5, 130.2, 129.8, 128.3, 127.6,
42.4, 28.9. MS (ESI) Calc. for [C9H8BrCl2NO + H]+ = 298.91, found
298.90.
4.4.9. 2-Bromo-N-(4-methoxybenzyl)acetamide (Vf)
4.4.2. Procedure for synthesis of 2-methylbenzo[d]oxazol-6-amine (III)
Yield: 89%; brown solid; mp = 187–189 °C; 1H NMR (400 MHz,
CDCl3): δ 8.70 (s, J = 8.8 Hz, 1H), 7.19 (d, J = 8.40 Hz, 2H), 6.89 (d,
J = 8.40 Hz, 2H), 4.22 (d, J = 5.6 Hz, 2H), 3.88 (s, 2H), 3.72 (s, 3H).
13C NMR (100 MHz, CDCl3): δ 173.2, 156.6, 131.3, 130.8, 115.1, 55.2,
43.6, 28.9. MS (ESI) Calc. for [C10H12BrNO2 + H]+ = 259.00, found
259.10.
To
a
solution of 2-methyl-6-nitrobenzoxazole II (2.20 g,
12.36 mmol) in THF:MeOH:H2O (1:1:1, 30 mL) was charged NH4Cl
(123.6 mmol). Reaction mixture cooled to 0–5 °C, charged zinc powder
(123.6 mmol) portion wise, after the addition reaction mixture stirred
at 10–15 °C for 2 h (monitored by TLC). Reaction mixture filtered
through celite bed, filtrate charged with water (30 mL) and extracted
with EtOAc (2 × 30 mL). The combined organic layer was washed with
brine (20 mL), dried over anhydrous Na2SO4, filtered and evaporated
under reduced pressure to afford 2-methylbenzo[d]oxazol-6-amine III
as light brown solid with yield 78%. Analytical data for III: mp = 146 –
147 °C; 1H NMR (400 MHz, DMSO-d6): δ 7.27 (d, J = 8.40 Hz, 1H),
4.4.10. 2-Bromo-N-(4-chlorobenzyl)acetamide (Vg)
Yield: 74%; pale yellow solid; mp
=
203–205 °C; 1H NMR
(400 MHz, CDCl3): δ 8.82 (s, 1H), 7.40 (d, J = 8.40 Hz, 2H), 7.29
(d, J = 8.40 Hz, 2H), 4.28 (d, J = 5.6 Hz, 2H), 3.91 (s, 2H). 13C NMR
(100 MHz, CDCl3): δ 172.9, 136.8, 133.7, 131.8, 127.8, 43.1, 28.4. MS
(ESI) Calc. for [C9H9BrClNO + H]+ = 262.95, found 263.09.
6.74 (s 1H), 6.56 (d, J = 6.4 Hz, 1H), 5.25 (s, 2H), 2.48 (s, 3H); 13
C
NMR (100 MHz, DMSO-d6): δ 160.01, 151.83, 146.91, 131.38, 118.73,
111.53, 94.43, 13.85. MS (ESI) Calc. for [C8H8N2O + H]+ = 148.16,
found 148.08.
4.4.11. 2-Bromo-N-(4-bromobenzyl)acetamide (Vh)
Yield: 81%; brown solid; mp = 235–237 °C; 1H NMR (400 MHz,
CDCl3): δ 8.73 (s, 1H), 7.51 (d, J = 8.83 Hz, 2H), 7.22 (d, J = 8.83 Hz,
2H), 4.28 (d, J = 5.7 Hz, 2H), 3.86 (s, 2H). 13C NMR (100 MHz, CDCl3):
δ 172.9, 136.8, 131.9, 130.1, 122.8, 42.8, 28.8. MS (ESI) Calc. for
[C9H9Br2NO + H]+ = 306.90, found 307.00.
4.4.3. General procedure for preparation of compounds Va – Vp
Substituted Benzyl amine IV (a–p) (10 mmol) in dichloromethane
(50 mL) charged with triethylamine (12 mmol), and cooled to 0 °C. 2-
bromoacetylbromide (11 mmol) was added drop wise, continued stir-
ring for 2 h at room temperature (monitored by TLC). The resulting
mixture was concentrated under reduced pressure and the residue was
purified by column chromatography using EtOAc/Hexanes as eluent to
afford the desired product Va – Vp.
4.4.12. 2-Bromo-N-(3-(trifluoromethyl)benzyl)acetamide (Vi)
Yield: 72%; yellow solid; mp = 251–253 °C; 1H NMR (400 MHz,
CDCl3): δ 8.87 (s, 1H), 7.51 (s, J = 8.84 Hz, 1H), 7.46 (t, J = 6.2 Hz,
1H), 7.41 (d, J = 8.82 Hz, 1H), 7.34 (d, J = 8.82 Hz, 1H), 4.29 (d,
J = 5.7 Hz, 2H), 3.92 (s, 2H). 13C NMR (100 MHz, CDCl3): δ 173.7,
138.4, 130.9, 130.6, 127.8, 124.6, 123.5, 123.1, 42.7, 28.9. MS (ESI)
Calc. for [C10H9BrF3NO + H]+ = 296.98, found 297.04.
Analytical data of compounds Va – Vp:
4.4.4. 2-Bromo-N-(4-fluorobenzyl)acetamide (Va)
Yield: 82%; brown solid; mp = 163–165 °C; MS(ESI): 1H NMR
(400 MHz, CDCl3): δ 8.83 (s, 1H), 7.25 (d, J = 7.82 Hz. 2H), 7.04
(d, J = 8.82 Hz, 2H), 4.45 (d, J = 5.7 Hz, 2H), 3.96 (s, 2H). 13C NMR
(100 MHz, CDCl3): δ 170.84, 162.4, 133.2, 129.5, 115.73, 43.1, 28.4.
MS (ESI) Calc. for [C9H9BrNO + H]+ = 246.99, found 246.98.
4.4.13. 2-Bromo-N-(2,5-dichlorobenzyl)acetamide (Vj)
Yield: 70%; brown solid; mp = 179–181 °C; 1H NMR (400 MHz,
CDCl3): δ 8.83 (s, 1H), 7.52 (d, J = 8.21 Hz, 1H), 7.46 (s, 1H), 7.39 (d,
J = 8.19 Hz, 1H), 4.32 (d, J = 5.7 Hz, 2H), 3.90 (s, 2H). 13C NMR
(100 MHz, CDCl3): δ 174.7, 145.2, 132.2, 130.6, 130.2, 128.9, 128.5,
39.4, 28.8. MS (ESI) Calc. for [C9H8BrCl2NO + H]+ = 296.91, found
297.02.
4.4.5. 2-Bromo-N-(2,6-dimethoxybenzyl)acetamide (Vb)
Yield: 79%; white solid; mp = 181–183 °C; 1H NMR (400 MHz,
CDCl3): δ 7.14 (d, J = 8.83 Hz, 1H), 7.08 (d, J = 8.83 Hz, 2H), 6.27
(bs, 1H), 4.51 (d, J = 5.7 Hz, 2H), 3.89 (s, 2H), 2.37 (s, 6H). 13C NMR
(100 MHz, CDCl3): δ 170.8, 156.7, 128.2, 115.1, 104.7, 57.3, 30.8,
28.4. MS (ESI) Calc. for [C11H14BrNO3 + H]+ = 289.01, found 248.02.
4.4.14. 2-Bromo-N-(3-methylbenzyl)acetamide (Vk)
Yield: 70%; pale yellow solid; mp
=
162–164 °C; 1H NMR
(400 MHz, CDCl3): δ 8.79 (s, 1H), 7.32 (t, J = 6.1 Hz, 1H), 7.11 (s, 1H),
7.06 (d, J = 7.78 Hz, 1H), 7.03 (d, J = 7.8 Hz, 1H), 4.26 (d,
J = 5.8 Hz, 2H), 3.86 (s, 2H), 2.29 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 172.9, 138.2, 136.8, 129.1, 128.9, 127.4, 125.8, 45.1, 28.8, 22.1. MS
(ESI) Calc. for [C10H12BrNO + H]+ = 243.01, found 243.00.
4.4.6. 2-Bromo-N-(1-phenylethyl)acetamide (Vc)
Yield: 76%; brown solid; mp = 221–223 °C; 1H NMR (400 MHz,
CDCl3): δ 7.41–7.21 (m, 5H), 6.70 (s, 1H), 5.19–5.05 (m, 1H), 3.87 (d,
J = 6.6 Hz, 2H), 1.55 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 171,
134.3, 128.9, 127.1, 126.8, 53.2, 30.3, 28.4. MS (ESI) Calc. for
[C11H12BrNO + H]+ = 243.01, found 243.06.
4.4.15. 2-Bromo-N-(3,5-dimethylbenzyl)acetamide (Vl)
Yield: 76%; white solid; mp = 175– 177 °C; 1H NMR (400 MHz,
CDCl3): δ 8.77 (s, 1H), 7.21 (s, 1H), 7.16 (s, 1H), 7.12 (s, 1H), 4.25 (d,
J = 5.6 Hz, 2H), 3.84 (s, 2H), 2.27(s, 6H). 13C NMR (100 MHz, CDCl3):
δ 172.1, 142.1, 137.9, 128.6, 125.9, 44.2, 28.7, 21.6. MS (ESI) Calc. for
4.4.7. 2-Bromo-N-(2-methoxybenzyl)acetamide (Vd)
Yield: 65%; white solid; mp = 197–199 °C; 1H NMR (400 MHz,
7

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BioorganicChemistry100(2020)103905
4.4.23. Procedure for synthesis of 4-amino-3-carbamoylisothiazole-5-
[C11H14BrNO + H]+ = 257.02, found 257.08.
carboxylic acid (X)
4.4.16. 2-Bromo-N-(3-methyl-5-(trifluoromethoxy)benzyl)acetamide (Vm)
Yield: 78%; brown solid; mp = 241–243 °C; 1H NMR (400 MHz,
CDCl3): δ 8.79 (s, 1H), 7.13 (s, 1H), 7.09 (s, 1H), 7.07 (s, 1H), 4.24 (d,
J = 5.8 Hz, 2H), 3.82 (s, 2H), 2.31 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 175.2, 161.4, 143.2, 142.5, 130.1, 123.9, 113.5, 109.1, 43.3, 28.8,
21.8. MS (ESI) Calc. for [C11H11BrF3NO2 + H]+ = 326.99, found
327.01.
To a solution of ethyl 4-amino-3-carbamoylisothiazol-5-carboxylate
(0.1 mol) in ethanol (100 mL) was added an aqueous solution (60 mL)
of NaOH (0.2 mol) at room temperature and allowed to stir for over-
night. After completion of the reaction by TLC analysis, the solvent was
evaporated under reduced pressure to afford crude residue. The crude
material was dissolved in water and the aqueous layer was washed with
ethyl acetate (100 mL). Then, the aqueous layer acidified to pH 4 using
concentrated HCl and resulting precipitated solid was filtered and dried
under reduced pressure to afford 4-amino-3-carbamoylisothiazole-5-
carboxylic acid (X) as white solid with 80% yield.
4.4.17. 2-Bromo-N-(3-chloro-5-(trifluoromethyl)benzyl)acetamide (Vn)
Yield: 78%; white solid; mp = 206–208 °C; 1H NMR (400 MHz,
CDCl3): δ 8.88 (s, 1H), 7.69 (s, 1H), 7.47 (s, 1H), 7.42 (s, 1H), 4.29 (d,
J = 5.8 Hz, 2H), 3.92 (s, 2H). 13C NMR (100 MHz, CDCl3): δ 175.7,
145.1, 133.7, 132.4, 131.8, 123.5, 122.8, 120.9, 43.3, 28.8. MS (ESI)
Calc. for [C10H8BrClF3NO + H]+ = 330.94, found 331.02.
Analytical data of compound X: mp = 251–253 °C, 1H NMR
(400 MHz, DMSO-d6): δ 8.11 (s, 2H), 7.76 (s, 2H); 13C NMR (100 MHz,
DMSO-d6): δ 163.69, 162.71, 149.17, 148.87, 122.63. MS (ESI) Calc. for
[C5H5N3O3S + H]+ = 188.01, found 188.00.
4.4.18. 2-Bromo-N-(2-methylbenzyl) acetamide (Vo)
4.4.24. General procedure for preparation of compounds XI (a–p)
Yield: 83%; brown solid; mp = 161–163 °C; 1H NMR (400 MHz,
CDCl3): δ 8.79 (s, 1H), 7.38 (t, J = 6.62 Hz, 1H), 7.36 (d, J = 7.7 Hz,
1H), 7.28 (d, J = 7.7 Hz,1H), 7.22 (m, 1H), 4.24 (d, J = 5.6 Hz, 2H),
3.82 (s, 2H), 2.34 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 171.6, 141.4,
134.2, 130.6, 126.3, 125.6, 125.1, 41.8, 28.2, 18.2. MS (ESI) Calc. for
[C10H12BrNO + H]+ = 243.01, found 243.00.
To a solution of compound 3 (3.37 mmol) in DMF (10 mL), charged
respective compound V (a–p) (4.04 mmol) and heated to 80 °C. After
3 h, TLC analysis showed complete conversion of starting materials. The
reaction mixture was cooled to room temperature, charged water
(30 mL) and extracted with EtOAc (2 × 20 mL). The combined organic
layer was washed with water (20 mL) followed by brine and con-
centrated under reduced pressure to obtain gummy liquid. This crude
material was triturated with MTBE to afford desired product as solid
and which used as such in next step without any further purification.
4.4.19. 2-Bromo-N-(3-chlorobenzyl)acetamide (Vp)
Yield: 74%; mp = 176–178 °C; white solid; 1H NMR (400 MHz,
CDCl3):
δ 8.82 (s, 1H), 7.46 (s, 1H), 7.37 (m, 2H), 7.21 (d,
J = 8.2 Hz,1H), 4.28 (d, J = 5.8 Hz, 2H), 3.89 (s, 2H). 13C NMR
(100 MHz, CDCl3): δ 173.3, 144.1, 134.1, 130.6, 130.6, 127.8, 127.2,
42.3, 28.2. MS (ESI) Calc. for [C9H9BrClNO + H]+ = 262.95, found
263.09.
4.4.25. General procedure for preparation of Lead-1 and compounds
(1–15)
To a solution of compound 4-amino-3-carbamoylisothiazole-5-car-
boxylic acid X (100 mg, 0.534 mmol) and triethylamine (0.15 mL,
1.068 mmol) in EtOAc (8 mL) was added 2-(1H-Benzotriazole-1-yl)
1,2,3,3-tetramethyluronium tetrafluoroborate (TBTU: 1 equiv), and the
mixture was stirred at room temperature for 10 min. Then corre-
sponding amine 11 (a- p) (2 equiv) was added and the reaction was
stirred at room temperature for 3 h (monitored by TLC). The mixture
was washed with water and extracted with EtOAc. The organic layer
was separated, dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure to afford crude material. The crude was purified
by column chromatography (silica gel 100–200 mesh) using EtOAc:
Heptane (2:1) as eluent to afford compounds (1–15) respectively.
Analytical data of compounds Lead-1 and compounds (1–15):
4.4.20. Procedure for synthesis of 2-amino-N-hydroxy-2-oxoacetimidoyl
cyanide (VII)
A solution of 2-cyanoacetamide (VI) (1 mol) in AcOH (200 mL) was
cooled to 0 °C and charged drop wise an aqueous solution of pre-dis-
solved sodium nitrite (2.5 mol). The reaction mixture was stirred at
15 °C for 8 h. The resulting solids were filtered, dried under reduced
pressure to furnish 2-amino-N-hydroxy-2-oxoacetimidoyl cyanide (VII)
with 70% yield and the crude material was used as such in next step
without any further purification.
4.4.21. Procedure for synthesis of 2-amino-2-oxo-N-(tosyloxy)acetimidoyl
cyanide (VIII)
4.4.26. 4-Amino-N5-(2-((4-fluorobenzyl)amino)-2-oxoethyl)-N5-(2-
methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide (Lead-1)
Yield: 56%; off white solid; mp = 273–275 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.62 (t, J = 5.8 Hz, 1H), 7.93 (d, J = 2.0 Hz, 1H),
7.84–7.78 (m, 2H), 7.62–7.54 (m, 2H), 7.29–7.25 (m, 2H), 7.17–7.09
(m, 4H), 4.42 (s, 2H), 4.29 (d, J = 6.0 Hz, 2H), 2.66 (s, 3H); 13C NMR
(100 MHz, DMSO-d6): δ 167.72, 166.31, 163.75, 162.42, 151.21,
150.41, 146.70, 142.18, 136.98, 135.37, 129.15, 129.07, 126.97,
122.11, 119.87, 115.0, 114.79, 113.20, 53.10, 41.42, 14.24. MS (ESI)
Calc. for [C22H19FN6O4S + H]+ = 483.12, found 483.31. HPLC
showed 96.1% of purity.
To a solution of 2-amino-N-hydroxy-2-oxoacetimidoyl cyanide (VII)
(0.25 mol) in anhydrous pyridine (100 mL) was added 4-methylben-
zenesulphonyl chloride (0.27 mol) drop wise at 0–5 °C, slowly allowed
to warm to room temperature and stirred for 12 h. After completion of
the reaction by TLC analysis, it was charged with ethanol (300 mL) and
resulting solid was filtered, washed with cold ethanol and dried under
reduced to afford 2-amino-2-oxo-N-(tosyloxy)acetimidoyl cyanide
(VIII) with 75% yield. Thus obtained crude material was used as such in
next step without any further purification.
4.4.22. Procedure for synthesis of ethyl 4-amino-3-carbamoylisothiazole-5-
carboxylate (IX)
4.4.27. 4-Amino-N5-(2-((2,6-dimethoxybenzyl)amino)-2-oxoethyl)-N5-
(2-ethylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide (1)
A solution of 2-amino-2-oxo-N-(tosyloxy)acetimidoyl cyanide (VIII)
(0.2 mol) and ethyl 2-mercaptoacetate (0.21 mol) in ethanol (250 mL)
was charged Piperidine (0.1 mol) at 0–5 °C. The reaction mixture was
slowly warmed to room temperature and stirred for 12 h. After com-
pletion of the reaction by TLC analysis, it was cooled to −10 °C, the
resulting precipitate formed was filtered, washed with cold ethanol
(100 mL) and dried under reduced pressure to afford ethyl 4-amino-3-
carbamoylisothiazole-5-carboxylate (IX) with 50% yield which was
used as such in next step without any further purification.
Yield 42%; brown solid; mp = 269–271 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.61 (t, J = 5.8 Hz, 1H), 7.93 (d, J = 2.0 Hz, 1H),
7.82–7.76 (m, 2H), 7.64–7.56 (m, 2H), 7.19–7.13 (m, 1H), 7.11–7.04
(m, 4H), 4.38 (d, J = 6.0 Hz, 2H), 3.74 (s, 2H), 2.56 (s, 3H), 2.41 (s,
6H); 13C NMR (100 MHz, DMSO-d6): δ 167.72, 166.31, 163.75, 162.42,
159.6, 151.21, 150.41, 146.70, 142.18, 136.98, 135.37, 126.82,
126.97, 122.11, 119.87, 115.0, 114.79, 113.20, 53.47, 52.71, 41.88,
14.27. MS (ESI) Calc. for [C24H24N6O6S + H]+ = 525.15, found
8

S. Jogula, et al.
BioorganicChemistry100(2020)103905
525.28. HPLC showed 97.5% of purity.
4.4.33. 4-Amino-N5-(2-((4-bromobenzyl)amino)-2-oxoethyl)-N5-(2-
methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide (7)
Yield 38%; pale yellow solid; mp = 286–288 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.52 (t, J = 5.6 Hz, 1H), 8.09 (s, 1H), 7.72 (s, 1H), 7.45 (d,
J = 8.40 Hz, 2H), 7.34 (d, J = 8.40 Hz, 1H), 7.17 (d, J = 8.40 Hz, 2H),
6.64 (s, 1H), 6.62 (dd, J = 1.60 Hz, J = 8.80 Hz, 1H), 6.21 (bs, 1H),
4.26 (d, J = 6.0 Hz, 2H), 3.73 (s, 2H), 2.51 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 170.74, 164.17, 163.17, 160.91, 152.36, 149.65, 149.43,
147.19, 139.48, 132.35, 131.45, 129.91, 123.07, 120.15, 119.27,
111.25, 93.15, 47.81, 41.91, 14.39. MS (ESI) Calc. for
[C22H19BrN6O4S + H]+ = 544.04, found 545.43. HPLC showed
95.90% of purity.
4.4.28. 4-Amino-N5-(2-methylbenzo[d]oxazol-6-yl)-N5-(2-oxo-2-((1-
phenylethyl)amino) ethyl)isothiazole-3,5-dicarboxamide (2)
Yield 32%; pale yellow solid; mp = 268–270 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.64 (t, J = 5.8 Hz, 1H), 7.85 (d, J = 2.0 Hz, 1H),
7.81–7.75 (m, 2H), 7.62–7.56 (m, 2H), 7.51–7.22 (m, 5H), 7.17 (s, 2H),
5.19–5.08 (m, 1H), 4.39 (s, J = 6.0 Hz, 2H), 2.55 (s, 3H), 1.59 (d, 3H);
13C NMR (100 MHz, DMSO-d6): δ 164.89, 162.21, 161.72, 159.82,
151.29, 150.47, 144.70, 139.18, 136.58, 135.31, 129.25, 129.13,
126.02, 125.87, 122.11, 119.87, 115.0, 114.79, 113.20, 53.47, 49.63,
24.68, 14.24. MS (ESI) Calc. for [C23H22N6O4S + H]+ = 478.14, found
479.15. HPLC showed 95.4% of purity.
4.4.34. 4-Amino-N5-(2-methylbenzo[d]oxazol-6-yl)-N5-(2-oxo-2-((3-
(trifluoromethyl)benzyl) amino)ethyl) isothiazole-3,5-dicarboxamide (8)
Yield 38%; white solid; mp = 271–273 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.68 (t, J = 5.6 Hz, 1H), 7.96 (d, J = 2.0 Hz. 1H),
7.82–7.76 (m, 2H), 7.69–7.58 (m, 2H), 7.30–7.28 (m, 2H), 7.19–7.11
(m, 4H), 4.39 (d, J = 6.0 Hz, 2H), 3.76 (s, 2H), 2.56 (s, 3H); 13C NMR
(100 MHz, DMSO-d6): δ 167.82, 166.39, 163.65, 162.47, 151.24,
150.41, 146.70, 142.18, 136.98, 135.37, 131.28, 130.64, 129.89,
128.34, 126.43, 124.25, 120.75, 115.41, 114.86, 113.71, 54.35, 41.86,
14.69. MS (ESI) Calc. for [C23H19F3N6O4S + H]+ = 533.12, found
533.29. HPLC showed 95.9% of purity.
4.4.29. 4-Amino-N5-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-N5-(2-
methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide (3)
Yield 51%; off-white solid; mp = 272–274 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.21 (t, J = 6.0 Hz, 1H), 8.07 (s, 1H), 7.71 (s, 1H), 7.34 (d,
J = 8.8 Hz, 1H), 7.22 (t, J = 7.2 Hz, 1H), 7.09 (d, J = 6.4 Hz, 1H),
7.01 (d, J = 8.4 Hz, 1H), 6.83 (t, J = 7.6 Hz, 1H), 6.68 (s, 1H), 6.64
(dd, J = 2.0 Hz, J = 8.4 Hz, 1H), 6.19 (bs, 1H), 4.26 (d, J = 5.6 Hz,
2H), 3.748 (s, 2H), 3.74 (s, 3H), 2.55 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 170.56, 164.17, 163.18, 160.89, 157.09, 152.38, 149.65,
149.43, 147.19, 132.32, 128.50, 128.14, 127.07, 123.09, 120.43,
119.27, 111.26, 110.86, 93.12, 55.69, 47.71, 37.79, 14.39. MS (ESI)
Calc. for [C23H22N6O5S + H]+ = 495.14, found 495.05. HPLC showed
97.18% of purity.
4.4.35. 4-Amino-N5-(2-((2,5-dichlorobenzyl)amino)-2-oxoethyl)-N5-(2-
methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide (9)
Yield 39%; brown solid; mp = 264–266 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.74 (t, J = 5.9 Hz, 1H), 7.89 (d, J = 2.0 Hz, 1H),
7.83–7.78 (m, 2H), 7.61–7.56 (m, 2H), 7.49 (s, 1H), 7.42 (s, 2H) 7.34
(d, J = 6.8 Hz, 1H), 7.24 (d, J = 7.1 Hz, 1H), 4.36 (d, J = 6.0 Hz, 2H),
3.71 (s, 2H), 2.52 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 167.79,
166.73, 163.42, 162.41, 151.91, 150.43, 146.50, 142.18, 136.98,
135.37, 129.15, 129.07, 126.82, 126.97, 122.11, 119.87, 116.0,
114.79, 114.60, 53.84, 41.89, 14.36. MS (ESI) Calc. for
[C22H18Cl2N6O4S + H]+ = 534.05, found 534.09. HPLC showed
96.1% of purity.
4.4.30. 4-Amino-N5-(2-((3,4-dichlorobenzyl)amino)-2-oxoethyl)-N5-(2-
methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide (4)
Yield 39%; brown solid; mp = 265–267 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.55 (t, J = 5.6 Hz, 1H), 8.07 (s, 1H), 7.71 (s, 1H), 7.52 (d,
J = 8.40 Hz, 1H), 7.39 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.21 (dd,
J = 1.60 Hz, J = 8.0 Hz, 1H), 6.62 (s, 1H) 6.60 (s, 1H), 6.25 (bs, 1H),
4.28 (d, J = 6.0 Hz, 2H), 3.74 (s, 2H), 2.51 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 170.52, 163.69, 162.70, 160.42, 151.88, 149.16, 148.94,
146.63, 140.85, 131.92, 130.81, 130.24, 129.15, 129.0, 127.54,
122.65, 118.82, 110.78, 92.56, 47.36, 40.96, 13.90. MS (ESI) Calc. for
[C22H18Cl2N6O4S + H]+ = 534.05, found 533.39. HPLC showed
95.72% of purity.
4.4.36. 4-Amino-N5-(2-methylbenzo[d]oxazol-6-yl)-N5-(2-((3-
methylbenzyl)amino)-2-oxoethyl)isothiazole-3,5-dicarboxamide (10)
Yield 44%; white solid; mp = 264–266 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.54 (t, J = 5.8 Hz, 1H), 7.86 (d, J = 6.0 Hz, 1H),
7.81–7.75 (m, 2H), 7.69–7.58 (m, 2H), 7.30–7.28 (m, 2H), 7.19–7.11
(m, 4H), 4.29 (d, J = 6.0 Hz, 2H), 3.73 (s, 2H), 2.59 (s, 3H), 2.38 (s,
3H); 13C NMR (100 MHz, DMSO-d6): δ 167.81, 166.49, 163.69, 162.46,
151.24, 150.41, 146.70, 142.18, 136.98, 131.34, 129.78, 129.22,
128.37, 126.43, 124.25, 120.75, 115.41, 114.86, 113.71, 54.35, 41.86,
23.8, 14.64. MS (ESI) Calc. for [C23H22N6O4S + H]+ = 479.15, found
479.20. HPLC showed 97.3% of purity.
4.4.31. 4-Amino-N5-(2-((4-methoxybenzyl)amino)-2-oxoethyl)-N5-(2-
methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide (5)
Yield 58%; off-white solid; mp = 283–285 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.38 (t, J = 5.6 Hz, 1H), 8.08 (s, 1H), 7.72 (s, 1H), 7.33
(d, J = 8.40 Hz 1H), 7.14 (d, J = 8.80 Hz, 2H), 6.83 (d, J = 8.80 Hz,
2H), 6.65 (d, J = 2.0 Hz, 1H), 6.62 (dd, J = 2.0 Hz, J = 8.80 Hz, 1H),
6.18 (bs, 1H), 4.22 (d, J = 6.0 Hz, 2H), 3.71 (s, 5H), 2.52 (s, 3H); 13C
NMR (100 MHz, DMSO-d6): δ 170.4, 164.17, 163.18, 160.87, 158.63,
152.37, 149.64, 149.43, 147.23, 132.30, 131.85, 129.01, 119.25,
114.04, 111.26, 93.11, 55.50, 47.77, 41.95, 14.38. MS (ESI) Calc. for
[C23H22N6O5S + H]+ = 495.52, found 495.05. HPLC showed 95.79%
of purity.
4.4.37. 4-Amino-N5-(2-((3,5-dimethylbenzyl)amino)-2-oxoethyl)-N5-(2-
methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide (11)
Yield 49%; brown solid; mp = 278–280 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.79 (t, J = 5.6 Hz, 1H), 7.84 (d, J = 2.0 Hz, 1H),
7.81–7.75 (m, 2H), 7.69–7.58 (m, 2H), 7.30 (s, 1H), 7.19–7.11 (m, 4H),
4.42 (d, J = 6.0 Hz, 2H), 3.74 (s, 2H), 2.36 (s, 6H); 13C NMR (100 MHz,
DMSO-d6): δ 167.83, 166.59, 164.02, 162.46, 151.24, 150.41, 146.70,
142.18, 136.98, 131.34, 129.78, 129.22, 128.37, 126.43, 124.25,
120.75, 115.41, 114.86, 113.71, 54.35, 41.86, 23.49, 14.58. MS (ESI)
Calc. for [C24H24N6O4S + H]+ = 493.16, found 493.31. HPLC showed
97.6% of purity.
4.4.32. 4-Amino-N5-(2-((4-chlorobenzyl)amino)-2-oxoethyl)-N5-(2-
methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide (6)
Yield 49%; brown solid; mp = 274–276 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.64 (t, J = 5.8 Hz, 1H), 7.93 (d, J = 2.0 Hz 1H),
7.84–7.78 (m, 2H), 7.69–7.58 (m, 2H), 7.30–7.28 (m, 2H), 7.19–7.11
(m, 4H), 4.40 (d, J = 6.0 Hz, 2H), 3.73 (s, 2H), 2.52 (s, 3H); 13C NMR
(100 MHz, DMSO-d6): δ 167.86, 166.31, 163.75, 162.42, 151.21,
150.41, 146.70, 142.18, 136.98, 135.37, 129.15, 129.07, 129.69,
127.28, 122.11, 119.87, 115.0, 114.79, 113.60, 54.39, 41.87, 14.26.
MS (ESI) Calc. for [C22H19ClN6O4S + H]+ = 500.05, found 500.13.
HPLC showed 96.8% of purity.
4.4.38. 4-Amino-N5-(2-((3-methyl-5-(trifluoromethoxy)benzyl)amino)-2-
oxoethyl)-N5-(2-methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide
(12)
Yield 31%; yellow solid; mp = 289–292 °C; 1H NMR (400 MHz,
9

S. Jogula, et al.
BioorganicChemistry100(2020)103905
DMSO-d6): δ 8.68 (t, J = 5.8 Hz, 1H), 7.92 (d, J = 2.0 Hz, 1H),
7.84–7.77 (m, 2H), 7.71–7.64 (m, 2H), 7.41 (s, 1H), 7.19–7.11 (m, 4H),
4.38 (d, J = 6.0 Hz, 2H), 3.76 (s, 2H), 2.56 (s, 3H), 2.39 (s, 3H); 13C
NMR (100 MHz, DMSO-d6): δ 167.83, 166.59, 164.02, 162.46, 162.23,
151.24, 150.41, 146.70, 142.18, 136.98, 134.18, 132.79, 130.28,
129.36, 127.41, 124.25, 121.81, 116.95, 114.26, 113.74, 54.39, 41.78,
23.38, 14.47. MS (ESI) Calc. for [C24H21F3N6O5S + H]+ = 563.13,
found 563.00.
4.6. P. aeruginosa susceptibility test
Pure colonies grown on agar are used to inoculate 3 mL of sterile
broth to an OD of 0.6. One milliliter of this suspension is added to
29 mL of sterile water to provide the inoculum for the microdilution
trays, which will be inoculated with a multipoint inoculator. Each test
compound stock solutions will be diluted in nutrient broth by four-fold
the final highest concentration to be tested. Compounds are diluted
serially in a sterile 96-well microtiter plates using 100 µL of nutrient
broth. All the sampling will be done in duplicates. Plates will be in-
cubated at 35 °C and readings are noted at 18 to 24 and 48 h. The MIC is
considered as the lowest concentration of an antimicrobial agent that
completely inhibited growth of the bacteria. Test carried out in the
absence and presence of efflux pump inhibitor PAβN (0.025 mg/mL)
and conessine (0.02 mg/mL).
4.4.39. 4-Amino-N5-(2-((3-chloro-5-(trifluoromethyl)benzyl)amino)-2-
oxoethyl)-N5-(2-methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide
(13)
Yield 30%; brown solid; mp = 287–289 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.92 (t, J = 5.9 Hz, 1H), 7.84 (d, J = 2.0 Hz, 1H),
7.84–7.77 (m, 2H), 7.71–7.64 (m, 2H), 7.41 (s, 1H), 7.19–7.11 (m, 4H),
4.39 (d, J = 6.0 Hz, 2H), 3.79 (s, 2H), 2.53 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 167.83, 166.59, 164.02, 162.46, 162.23, 151.24, 150.41,
146.70, 142.18, 136.98, 134.18, 132.79, 130.28, 129.36, 127.41,
124.22, 121.84, 116.35, 114.66, 113.69, 54.38, 41.82, 14.54. MS (ESI)
Calc. for [C23H18ClF3N6O4S + H]+ = 568.07, found 568.39. HPLC
showed 95.2% of purity.
4.7. Molecular dynamics simulations
The Desmond module from Schrödinger was used for running MD
simulations with periodic boundary conditions. The receptor & de-
signed inhibitors complex was immersed in an orthorhombic simulation
box, with the TIP3P explicit water model using the system builder panel
with the minimum thickness of a solvent layer, 10 Å. In order to neu-
tralize the system, counter ions were added.
4.4.40. 4-Amino-N5-(2-methylbenzo[d]oxazol-6-yl)-N5-(2-((2-
methylbenzyl)amino)-2-oxoethyl)isothiazole-3,5-dicarboxamide (14)
Yield 51%, white solid; mp = 271–273 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.61 (t, J = 5.6 Hz, 1H), 7.93 (d, J = 2.0 Hz, 1H),
7.84–7.78 (m, 2H), 7.62–7.54 (m, 2H), 7.18–7.12 (m, 1H), 7.10–7.06
(m, 5H), 4.42 (d, J = 6.0 Hz, 2H), 3.74 (s, 2H), 2.54 (s, 3H), 2.28 (s,
3H); 13C NMR (100 MHz, DMSO-d6): δ 167.72, 166.31, 163.75, 162.42,
159.6, 151.21, 150.41, 146.70, 142.18, 136.98, 135.37, 127.15,
126.82, 126.97, 122.11, 119.87, 115.0, 114.79, 113.20, 53.47, 52.71,
21.82, 14.27. MS (ESI) Calc. for [C23H22N6O4S + H]+ = 479.15, found
479.01. HPLC showed 96.7% of purity.
Before equilibration and long production of MD simulations, the
systems were minimized and pre-equilibrated using relaxation routine
implemented in Desmond. Whereas, program ran six steps composed a)
energy minimization was used by hybrid method of steepest descent
and limited-memory Broyden-Fletcher- Goldfarb-Shanno (LBFGS) al-
gorithm with a maximum steps of 2000 including preliminary 10 steps
of steepest descent with solute restrained, b) Energy minimization for
2000 steps without solute restraints, c) 12 ps simulation in NVT en-
semble (temperature 10 K) restraining nonhydrogen solute atoms, d)
12 ps simulation in the NPT ensemble (temperature 10 K) restraining
non-hydrogen solute atoms, e) 24 ps simulation in the NPT ensemble
restrained along with solute non-hydrogen atoms (temperature 300 K)
and f) 24 ps simulation in the NPT ensemble (temperature 300 K) with
no restraints respectively. The temperatures and pressures in the short
initial simulations were checked by applied Berendsen thermostats and
barostats algorithms. The equilibrated system was simulated for 50 ns
with a time step of 2 fs, NPT ensemble was used a Nosé-Hoover ther-
mostat at 300 K and Martyna-Tobias- Klein barostat at 1.01325 pressure
bar. A time step of 1.2 fs was used. Saving energy and structure en-
umerated for every 4.8 ps during simulation, the MD trajectory was
generated. Finally to analyze trajectory simulation, a simulation inter-
action diagram tool was used.
4.4.41. 4-Amino-N5-(2-((3-chlorobenzyl)amino)-2-oxoethyl)-N5-(2-
methylbenzo[d]oxazol-6-yl)isothiazole-3,5-dicarboxamide (15)
Yield 46%; pale yellow solid; mp = 261–263 °C; 1H NMR (400 MHz,
DMSO-d6): δ 8.48 (t, J = 5.2 Hz, 1H), 8.07 (s, 1H), 7.71 (s, 1H), 7.42
(d, J = 7.20 Hz, 1H), 7.35 (d, J = 8.40 Hz, 1H), 7.30–7.19 (m, 6H),
6.70 (s, 1H), 6.64 (dd, J = 2.0 Hz, J = 8.40 Hz, 1H), 6.23 (s, 1H), 4.36
(d, J = 6.0 Hz, 2H), 3.78 (s, 2H), 2.53 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 170.92, 164.17, 163.18, 160.93, 152.39, 149.64, 149.3,
147.19, 136.73, 132.41, 132.36, 129.51, 129.14, 129.02, 127.43,
123.14, 119.28, 111.27, 93.18, 47.70, 14.39. MS (ESI) Calc. for
[C22H19ClN6O4S + H]+ = 500.8, found 499.26. HPLC showed 91.67%
of purity.
Declaration of Competing Interest
4.5. DNA supercoiling assay
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Supercoiling assay was performed using the commercially available
P. aeruginosa DNA gyrase assay kit (Inspiralis Pvt. limited, Norwich,
UK). The assay was performed in 1.5 mL eppendorf tubes at room
temperature. 1 U of DNA gyrase was incubated with 0.5 µg of relaxed
pBR 322 DNA in 30 µL reaction volume at 37 0C for 30 min [18] with
40 mM HEPES. KOH (pH 7.6), 10 mM magnesium acetate, 10 mM DTT,
2 mM ATP, 500 mM potassium glutamate, 0.05 mg/mL albumin (BSA).
Standard compound novobiocin was the positive control and 4% DMSO
was considered as negative control. Subsequently, each reaction was
stopped by the addition of 30 µL of stop dye [40% sucrose, 100 mM
Tris–HCl (pH 7.5), 1 mM EDTA and 0.5 mg/mL bromophenol blue],
briefly centrifuged for 45 s and supernatant was loaded and run in 1%
agarose gel in 1X TAE buffer (40 mM Tris acetate, 2 mM EDTA). Fur-
thermore, concentration of the range of compounds that inhibits 50% of
supercoiling activity (IC50) was determined using densitometry and NIH
image through Bio-Rad GelDoc image viewer.
Acknowledgments
DS is thankful to Department of Biotechnology, Government of India
for the Tata innovation fellowship (BT/HRD/35/01/04/2015). SJ is
thankful to Albany Molecular Research center Hyderabad for providing
financial support. VSK is thankful to Department of Science and
Technology, Government of India for the INSPIRE fellowship
(IF160108).
Appendix A. Supplementary material
Supporting information files include spectral data (1H and 13C NMR,
10

S. Jogula, et al.
BioorganicChemistry100(2020)103905
and MS) of the compounds. Supplementary data to this article can be
found online at https://doi.org/10.1016/j.bioorg.2020.103905.
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11