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2-[2–(4-Methoxybenzoyl)hydrazinyl]-2-oxoethyl(6-nitro-1,3-ben-
zothiazol-2-yl)carbamodithioate (5d)
a negative control while ciprofloxacin and gentamicin were
used as control drugs (positive) in sterile DMSO. After incu-
bation at 37 ꢁC for 24 h, the 96 well plates were read for the
MIC. MIC values are summarized in Table 1 and expressed as
microgram per milliliter (mg/ml).
Yield 56%. Solvent crystallization: ethanol. m.p.: 272–275 ꢁC;
Rf ¼ 0.51; FT-IR (cmꢀ1): 3245, 3215 (NH), 3081 (ArH), 2924,
2854 (CH2), 1685, 1658 (>C¼O), 1620 (>C¼N), 1564 (>C¼S),
1509(–C¼C–), 1337 (NO2), 844 (C-N), 1259,1048 (C-O-C); 1H-
NMR (DMSO-d6): dppm 10.38 (s, 1H, NH), 10.22 (s, 1H, NH),
10.14 (s, 1H, NH), 8.69–8.09 (m, 3 H, benzothiazole), 7.89–7.84
(m, 4 H, ArH), 4.80 (s, 2 H, SCH2), 3.86 (s, 3 H, OCH3); 13C-NMR
spectrum (DMSO-d6): dppm 172.26 (>C¼O), 167.88 (>C¼O),
167.27 (>C¼S), 165.32, 165.32, 162.52, 159.07, 141.15, 132.06,
130.48, 130.39, 129.87, 122.46, 118.17, 67.46 (SCH2), 55.86
(OCH3). GC-MS (EI-TOF) m/z calculated for C18H15N5O5S3
477.53 (Mþ). Found: m/z 481.65 (Mþþ4).
2.3.2. Enzyme inhibitory activity of S. aureus MurD ligase
The S. aureus MurD enzyme (ProFoldin, USA) inhibition assay
was performed using the malachite green assay with slight
modifications [38,39]. All of the experiments were performed
in duplicate. The mixture with final volume of 50 mL con-
tained: 500 mM TrisHCl, pH 8.0, 400 mM KCl, 10 mM DTT,
0.05% Triton X-100, 10 mM MgCl2, 1 mM UDP-MurNAc-L-Ala,
5 mM D-Glu, 10 mM ATP, 5000 nM purified S. aureus MurD
and 0.6 mL of different concentrations of each tested com-
pound solution in dimethylsulfoxide (DMSO). The final con-
centration of DMSO was 5% (v/v) in all cases. The mixture
was then incubated for 60 min at 37 ꢁC and then quenched
with Dye MPA3000. Incubated for 5 min and absorbance was
measured at 650 nm and percentage inhibition was calcu-
lated. Percent inhibitions were calculated without the tested
compounds and with 5% DMSO. IC50 values were deter-
mined using the GraphPad PRISM.
2-f2-[(4-Methylphenyl) sulfonyl] hydrazinylg-2-oxoethyl-1,3-ben-
zothiazol-2-yl carbamodithioate(5e)
Yield 69%. Solvent crystallization: ethanol. m.p.:180–184 ꢁC; Rf
¼ 0.51; FT-IR (cmꢀ1): 3214, 3162 (NH), 3010 (Ar C–H), 2917,
2846 (CH2), 1680, 1658 (>C¼O), 1630 (>C¼N), 1548 (>C¼S),
1509 (NH amide), 1337, 1165 (>S¼O). 1H-NMR (DMSO-d6):
dppm 10.58 (s, 1H, NH), 10.54 (s, 1H, NH), 10.32 (s, 1H, NH),
8.39–8.11 (m, 4 H, ArH), 7.72–7.38 (m, 4 H, ArH), 4.57 (s, 2 H,
SCH2), 2.49 (s, 3 H, CH3); GC-MS (EI-TOF) m/z calculated for
C17H16N4O3S4 (452.59). Found: m/z 452.14 (Mþ).
3. Computational analysis
2-f2-[(2-Chlorophenoxy)acetyl]hydrazinylg-2-oxoethyl-1,3-benzo-
thiazol-2-yl-carbamodithioate (5f)
3.1. Molecular docking and binding free energy
calculation (MM-GBSA)
Yield 65%. Solvent crystallization: ethanol. m.p.: 126–130 ꢁC;
Rf ¼ 0.55; FT-IR (cmꢀ1): 3214, 3182 (NH), 3049 (Ar C–H),
2924, 2854 (CH2), 1679, 1642 (>C¼O), 1618 (>C¼N), 1572
The 3 D-structures of compounds 5a–f were prepared using
the builder panel in Maestro 11.5 (Schrodinger software suite
2018–1, LLC, New York, NY, USA) and subsequently opti-
mized with LigPrep [40]. The molecular docking was per-
formed in extra-precision (XP) mode with Glide without
applying any constraints. The binding free energies were cal-
culated using VSGB 2.0 energy model (MM-GBSA approach)
and prime [41,42] with OPLS3 force field [43]. In this study,
we used the earlier prepared grid box of modeled S. aureus
MurD protein [33]. The low energy conformation of all pre-
pared ligands were docked and their binding free energies
were computed employing the protocols described ear-
lier [33].
1
(>C¼S), 1611, 1486 (Ar –C¼C–), 1235, 1024 (C-O-C); H-NMR
(DMSO-d6): dppm 10.30 (s, 1H, NH), 10.28 (s, 1H, NH), 10.24
(s, 1H, NH), 7.98–7.29 (m, 4 H, ArH), 7.28– 6.97 (m, 4 H, ArH),
3.89 (s, 2 H, SCH2), 3.60 (s, 2 H, OCH2); 13C-NMR spectrum
(DMSO-d6): dppm 185.26 (>C¼O), 167.16 (>C¼O), 166.63
(>C¼S), 153.80,152.36, 130.53, 128.67,127.08, 126.23, 125.23,
124.42, 123.23, 123.12,122.69, 121.93, 114.63, 66.85 (OCH2),
52.23 (SCH2). GC-MS (EI-TOF) m/z calculated for
C18H15ClN4O3S3 (466.98). Found: m/z 466.35 (Mþ).
2.3. In vitro antibacterial screening of
synthesized compounds
Table 1. Antibacterial activity of synthesized compounds 5a–f against Gram-
positive and Gram-negative bacteria.
2.3.1. Determination of minimum inhibitory concentra-
tion (MIC)
ꢂ
Minimum inhibitory concentration (mg/ml)
The antibacterial activity of the synthetic compounds against
Gram-positive S. aureus (NCIM 5021 and NCIM 5022), methi-
cillin resistant S. aureus (MRSA strain 43300), B. subtilis (NCIM
2545) and Gram-negative strains E. coli (NCIM 2567), K. pneu-
moniae (NCIM 2706) and P. aeruginosa (NCIM 2036) was
assessed according to the guidelines of the Clinical
Laboratories Standard Institute [37] to determine the MIC val-
ues. For each of the selected strains triplicate analyses were
performed. MIC values were determined by microdilution
broth technique using Mueller Hinton medium (Hi-media).
Compounds 5a–f was tested for their antibacterial activity in
sterile dimethyl sulfoxide (DMSO). Sterile DMSO was used as
Compound
aS. a
bS.a
cS. a
dB. s
eK. p
fP. a
gE. c
5a
5b
5c
5d
5e
5f
2
128
64
32
128
64
2
32
128
128
32
128
64
64
128
256
64
128
128
32
16
>256
128
64
128
128
2
2
64
16
4
64
32
2
256
128
256
128
128
256
2
16
64
32
16
>256
32
2
1
Ciprofloxacin
Gentamicin
2
8
8
19.5
8
1
1
ꢂaverage of three independent determinations.
aS. a: Staphylococcus aureus NCIM 5021; bS. a: Staphylococcus aureus NCIM
c
5022; S. a: Staphylococcus aureus ATCC 43300 (MRSA); dB. s: Bacillus subtilis
NCIM 2545; eK. p: Klebsiella pneumoniae NCIM 2706; P. a: Pseudomonas aer-
f
uginosa NCIM 2036; gE. c: Escherichia coli NCIM 2567.