Design, synthesis and molecular modelling of phenoxyacetohydrazide derivatives as Staphylococcus aureus MurD inhibitors

Article abstract of DOI:10.1007/s11696-020-01380-2

In the present work we synthesized a new series of phenoxyacetohydrazide functional compounds 4a-k and characterized by spectral data. Synthesized compounds were screened in vitro for their antibacterial activity. Compounds 4a, 4j and 4k exhibited inhibitory activity against S. aureus NCIM 5022 with MIC value of 64?μg/ml These compounds also exhibited activity against methicillin resistant S. aureus ATCC 43300 with MIC of 128?μg/ml. Among all the tested compounds 4c and 4j showed highest activity, respectively against B. subtilis NCIM 2545 and K. pneumoniae NCIM 2706. Only one compound i.e. 4d showed activity against another Gram-negative bacteria P. aeruginosa NCIM 2036 with MIC value of 64?μg/ml. Among three tested compounds, 4k exhibited highest inhibitory activity against S. aureus MurD enzyme with IC₅₀ value of 35.80?μM. Further binding interactions of 4a-k with the modelled S. aureus MurD catalytic pocket residues is investigated with the extra-precision molecular docking and binding free energy calculation by MM-GBSA approach. The van der Waals energy term was observed to be the driving force for binding. Further, 50?ns molecular dynamics simulations were performed to validate the stabilities of 4j- and 4k-modelled S. aureus MurD. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11696-020-01380-2

Chemical Papers
https://doi.org/10.1007/s11696-020-01380-2
ORIGINAL PAPER
Design, synthesis and molecular modelling of phenoxyacetohydrazide
derivatives as Staphylococcus aureus MurD inhibitors
Srikanth Jupudi1 · Mohammed Afzal Azam1 · Ashish Wadhwani2
Received: 2 March 2020 / Accepted: 27 September 2020
© Institute of Chemistry, Slovak Academy of Sciences 2020
Abstract
In the present work we synthesized a new series of phenoxyacetohydrazide functional compounds 4a-k and characterized
by spectral data. Synthesized compounds were screened in vitro for their antibacterial activity. Compounds 4a, 4j and 4k
exhibited inhibitory activity against S. aureus NCIM 5022 with MIC value of 64 µg/ml These compounds also exhibited
activity against methicillin resistant S. aureus ATCC 43300 with MIC of 128 µg/ml. Among all the tested compounds 4c and
4j showed highest activity, respectively against B. subtilis NCIM 2545 and K. pneumoniae NCIM 2706. Only one compound
i.e. 4d showed activity against another Gram-negative bacteria P. aeruginosa NCIM 2036 with MIC value of 64 µg/ml.
Among three tested compounds, 4k exhibited highest inhibitory activity against S. aureus MurD enzyme with IC50 value of
35.80 µM. Further binding interactions of 4a-k with the modelled S. aureus MurD catalytic pocket residues is investigated
with the extra-precision molecular docking and binding free energy calculation by MM-GBSA approach. The van der Waals
energy term was observed to be the driving force for binding. Further, 50 ns molecular dynamics simulations were performed
to validate the stabilities of 4j- and 4k-modelled S. aureus MurD.
Graphic abstract
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11696-020-01380-2) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
Vol.:(0123456789)
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Chemical Papers
Keywords Phenoxyacetohydrazides · Minimum inhibitory concentration · MurD · Staphylococcus aureus · IC50
Abbreviations
et al. 2006). The carboxyl group of the nucleotide precur-
sor is activated by ATP leading to the formation of an acyl
phosphate intermediate and adenosine diphosphate (ADP).
The acyl phosphate in turn is attacked by the amino group
of the incoming amino acid resulting in the formation of a
high-energy tetrahedral intermediate. This intermediate then
breaks down into the product and inorganic phosphate (Ber-
trand et al. 1997; Perdih et al. 2007). Mur ligases catalytic
pocket binding residues among diferent bacterial strains
are well conserved (Gordon et al. 2001; Ikeda et al. 1990;
Bouhss et al. 1999). N-Acetylmuramoyl-L-alanine:D-gluta-
mate ligase (MurD), second in the series catalyses the addi-
tion of D‐Glu to UDP‐MurNAc‐L‐Ala resulting in the for-
mation of UDP‐Me urNAc‐L‐Ala‐D‐Glu. The cloned MurD
from S. aureus showed similarity to the MurD proteins from
Streptococcus pyogenes (66%), Haemophilus infuenzae
(55%), Escherichia coli (54%) and Bacillus subtilis (65%)
(El-Sherbeini et al. 1998). Due to the high specifcity for
D-amino acid, MurD is considered an attractive target for the
development of new antibacterial agents (Walsh et al. 1999;
Gegnas et al. 1998). This enzyme consists of three distinct
globular domains; the N-terminal domain binds UDP moiety
of the UDP-N-acetylmuramoyl-L-alanine (UMA) substrate.
The central domain is responsible for the binding of ATP,
while the C-terminal domain play an important role in the
fxation of the condensing amino acid or dipeptide residue.
During last few decades several phosphinates,
α-aminophosphonates and N-acetylmuramic acids have
been investigated as tetrahedral-like transition state ana-
logs against E. coli MurD (Gobec et al. 2001; Strancar et al.
2006; Auger et al. 1995; Sova et al. 2009). Phosphorylated
and non-phosphorylated hydroxyethylamines have also been
developed as inhibitors of the whole cascade of intracellular
Mur ligases (MurC to MurF) (Sova et al. 2009) with IC50
values in the low micromolar range. In further development,
D-glutamic acid analogs and substituted naphthalene-N-sul-
fonyl-D-Glutamic acids were tested as transition-state ana-
logs of MurD from E. coli (Pratviel-Sosa et al. 1994; Kotnik
et al. 2007; Humljan et al. 2008; Perdih et al. 2009). The
binding mode analysis of these inhibitors paved the way for
leads optimization. Virtual screening approach has also been
used to identify novel MurD ligase inhibitors (Zidar et al.
2010). Virtual hit was further optimized by incorporating the
D-Glu moiety into the thiazolidin-4-one scafold or its surro-
gate resulting in high potency against MurD. Some of these
designed inhibitors exhibited weak in vitro antibacterial
activity. Discovery of 5-benzylidenethiazolidine-2,4-dione
and 5-benzylidenerhodanine-based E. coli MurD inhibitors
(Zidar et al. 2011) unveiled inhibitors binding mode which
was used successfully for the design of new generation
DAP
2,6-Diaminopimelic acid
D-Glu
MurC
MRSA
MIC
D-glutamic acid
UDP-N-acetylmuramoyl-L-alanine ligase
Methicillin-resistant Staphylococcus aureus
Minimum inhibitory concentration
Minimum bactericidal concentration
UDP-N-acetylmuramoyl-L-alanine:D-gluta-
mate ligase
MBC
MurD
MurE ligase UDP-N-acetylmuramoyl-L-alaninyl-D-glu-
tamate-2,6-diaminopimelate (or L-lysine)
ligase
MurF ligase UDP-N-acetylmuramoyl-L-alanine-D-glu-
tamyl-lysine-D-alanyl-D-alanine ligase
MurNAc
N-Acetyl muramic acid; UDP:
uridine-5′-diphosphate
UMA
Uridine-5′-diphosphate-N-acetylmueamoyl-
l-alanine
RMSD
UMAG
Root mean square deviation
UDP-N-acetylmuramoyl-L-alanine-D-glu-
tamate
Introduction
Infections caused by methicillin-resistant Staphylococcus
aureus (MRSA) are major public health threat worldwide.
MRSA is responsible for morbidity and mortality (Kong
et al. 2016; Hamdy et al. 2017) with an alarming rise of
resistance to most of the available antibiotics (Macmorran
et al. 2017). This instigated new research efort to develop
new class of antibacterial agents acting via novel mecha-
nisms of action. Peptidoglycan (PG) an integral part of S.
aureus cell envelope and is critical for the maintenance of
cell wall structural integrity. Its biosynthetic pathway in bac-
teria is well understood and considered as an attractive target
for the development of novel antibacterial drugs (Bugg et al.
2011; Gautam et al. 2011). Adenosine triphosphate (ATP)-
dependent bacterial Mur ligases (MurC-F) catalyze the suc-
cessive addition of L-alanine, D-glutamic acid (D-Glu) and
L-lysine in Gram-positive or meso-2,6-diaminopimelic acid
(meso-DAP) in Gram-negative bacteria, and D-alanine-D-
alanine to the growing UDP-N-acetyl muramic acid (UDP-
MurNAc) (Barreteau et al. 2008; El Zoeiby et al. 2003).
Because of their functional essentiality for bacterial survival
and absence in eukaryotic cells, Mur ligases are considered
as excellent targets for the development of antibacterial
agents (Smith et al. 2006).
The MurC to MurF ligases share a common three-domain
structure and act by the same reaction mechanism (Smith
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Chemical Papers
MurD inhibitors. Second generations sulfonamide inhibi-
tors with rigidifed D-Glu mimetics have been synthesized
and tested (Farlan et al. 2008; Sosic et al. 2011; Perdih et al.
2014). These rigidifed analogs of D-Glu displayed improved
inhibitory activity against E. coli MurD. Moreover, some of
these analogs exhibited promising inhibitory activity against
the whole cascade of Mur ligases (MurD-MurF).
heteroaryl groups to improve the binding afnity. After
several structure optimization cycles, phenoxyacetohy-
drazide derivatives (4a-k) with favorable binding afnity
for the modelled SaMurD active site (Azam et al. 2019b)
were designed for further study. The target compounds 4a-
k was synthesized as depicted in Scheme 1 and character-
ized by spectral data. All the synthesized compounds were
tested for their in vitro antibacterial activity against vari-
ous pathogenic bacterial strains. Few selected compounds
were assayed for their in vitro SaMurD enzyme inhibitory
activity to determine their IC50 values. The interaction of
designed compounds with modelled SaMurD protein was
studied by the extra-precision molecular docking, binding
free calculation and molecular dynamics (MD) studies.
The high resolution MurD crystal structures in complex
with D-Glu derivatives provided further insights into inhibi-
tors binding modes (Kotnik et al. 2007; Humljan et al. 2008).
Binding free energy calculation results clearly indicated
non-polar van der Waals interactions as driving force for
inhibitors binding with MurD (Perdih et al. 2007; Perdih
et al. 2009; Zidar et al. 2011; Perdih et al. 2013. Result from
these studies was used as structural basis for the optimisation
and improvement in activity against MurD enzyme (Tomasic
et al. 2012). Novel MurD inhibitors have also been discov-
ered by the structure-based virtual screenings approaches
(Perdih et al. 2009; Turk et al. 2009; Tomasc et al. 2012;
Simcic et al. 2014). However, the most of these eforts not
yielded MurD inhibitors possessing potent activity against
both Gram-positive and Gram-negative bacteria.
Experimental
Chemistry
The required chemicals for synthesis were of reagent grade
and purifed when required. All reactions were monitored
by thin layer chromatography (TLC) using Silica Gel F254
plates (Merck, Ltd., Germany). Melting points of synthe-
sized compounds were measured in open capillaries and
are uncorrected. The infrared spectra were performed by
means of a Perkin Elmer-Spectrum Two (United Kingdom)
Fourier transform spectrometer and band positions are pre-
sented in units of reciprocal centimetres (cm–1). Proton and
13C-NMR spectra were obtained using a Bruker AV-III 400
spectrometer (Germany) operating at 500 and 125 MHz,
respectively. Dimethyl sulfoxide (DMSO-d6) was used
as solvent and the chemical shifts are expressed in parts
per million (ppm) using DMSO-d6 as internal standard.
Mass spectra were recorded either on electrospray ioni-
zation source Shimadzu LC-MS2020 (Japan) spectrom-
eter (Japan) or on electron impact (EI) JEOL GCMATE
II GC–MS spectrometer (USA) operating at 70 eV. Syn-
thesis of ethyl (2/4-substituted phenoxy)acetates 2a: b.p.
112–114 °C, Lit. b.p. 113 °C (Holla et al. 1992); 2b:
b.p. 150–154 °C, Lit. 149–155 °C (Horner et al. 1974);
2c: m.p. 219–221 °C, Lit. m.p. 220–222 °C (Amer et al.
2018); 2d: b.p. 270 °C, Lit. b.p. 272 °C (Liu et al. 2012)
and 2-(2/4-disubstituted phenoxy)acetohydrazides 3a: m.p.
108–110 °C; Lit. m.p. 110–111 °C (Fun et al. 2010); 3b:
m.p. 155–158 °C, Lit. m.p. 155–157 °C (Chao et al. 1949);
3c: m.p. 190–193 °C (Lit. m.p. 190–192 °C (Amer et al.
2018); 3d: m.p. 178–180 °C, Lit. m.p.180–181 °C (Liu
et al. 2012) were carried out by literature methods. Syn-
thetic routes for the titled compounds 4a-k is represented
in Scheme 1.
In our earlier in silico virtual screening campaign, com-
pound A (Enamine T6806127, CACPD2011a-0001827917)
(Fig. 1) exhibited inhibitory activity against S. aureus
MurD (SaMurD) with an IC50 of 7 µM (Azam et al.
2019a). This compound also exhibited activity against S.
aureus NCIM 5021, S. aureus NCIM 5022 and B. subti-
lis NCIM 2545 with minimum inhibitory concentration
(MIC) of 128 µg/ml for all three tested strains. In this
study compound A was selected as the starting point to
design new and potent SaMurD inhibitors by introducing
diferent structural modifcations. Based on the molecular
dynamics result we sought to substitute appropriate groups
for linker 2-methylphenyl as it did not show any interac-
tions with the catalytic pocket residues. Further, one of
the 1-[1-(3-hydroxyphenyl)ethyl]urea moiety exhibited
less interactions during 30 ns of the MD trajectory. As a
frst step we replaced one of the 1-[1-(3-hydroxyphenyl)
ethyl]urea moiety and the linker 2-methylphenyl with dif-
ferent substituted aryl/heteroaryl-2-carbohydrazide groups
to improve the binding afnity with SaMurD protein. We
also tried diferent substituted aryl/heteroaryl rings in
place of another 1-[1-(3-hydroxyphenyl)ethyl]urea moi-
ety and diferent linkers between hydrazinyl and aryl/
Fig. 1 Chemical structure of compound A as SaMurD inhibitor
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Chemical Papers
Scheme 1 Synthesis and struc-
tures of titled compounds 4a-k
(DMSO-d6): δppm 10.19 (s, 2H, 2NH), 7.91 (dd, J=8.32,
1.81 Hz, 1H), 7.44 (dt, J = 7.79, 1.36 Hz, 1H), 7.32 (dt,
J = 8.12, 1.76 Hz, 1H), 7.22 (dd, J = 7.97, 1.63 Hz, 1H),
7.06 (dd, J=7.97, 1.45 Hz, 1H), 6.85 (dd, J=7.90, 2.35 Hz,
1H), 6.68 (t, J=8.3, 2.61 Hz, 1H), 4.76 (s, 2H, OCH2); 13C-
NMR (DMSO-d6): δppm 167.44 (>C=O), 157.68 (>C=O),
153.95, 146.56, 146.45, 130.67, 128.83, 122.93, 122.12,
115.39, 114.92, 112.53, 67.16 (OCH2). MS (ESI) m/z: cal-
culated for C13H11ClN2O4 (294.69). Found: 295.15 (M+ +1).
General procedure for the synthesis
of N‑(2/4‑substituted phenoxyacetyl)
benzohydrazides(4a‑k)
To a solution of appropriate 2-(2/4-substituted phenoxy)
acetohydrazides (3a-d) (0.008 mol) in acetonitrile (40 ml),
substituted benzoyl chlorides/substituted benzene sulfonyl
chlorides/tetrahydrofuran-2-carbonyl chloride (0.008 mol)
was added drop-wise with continuous stirring at 0 °C over a
period of 15 min. Stirring was further continued overnight
at room temperature. The progress of reaction was routinely
monitored by TLC. After completion of reaction, mixture
was poured into crushed ice and the solid thus obtained was
fltered, washed thoroughly with water, dried and recrystal-
lized with suitable solvent to yield titled compounds 4a-k.
Synthetic route for the synthesized compounds 4a-k is rep-
resented in Scheme 1.
Nʹ‑[2‑(4‑Methoxyphenoxy) acetyl]
furan‑2‑carbohydrazide (4b)
Yield 63%. Solvent crystallization: methanol.
m.p.:148–150 °C; Rf = 0.50 (ethylacetate:n-hexane,
V/V = 1:4.5); FT-IR (cm−1): 3394, 3214 (NHNH), 3034
(Ar C–H), 2838 (CH2), 1712, 1657 (> C=O), 1658 (NH
amide), 1603, 1470 (Ar –C=C–), 1235, 1040 (C–O–C), 802
(p-substituted benzene); 1H-NMR (DMSO-d6): δppm 10.31
(s, 1H, NH), 10.16 (s, 1H, NH), 7.88 (dd, J=8.04, 1.82 Hz,
1H, ArH), 7.23 (dd, J=7.91, 1.84 Hz, 1H), 6.98–6.66 (m,
4H), 6.662 (t, J=7.96 Hz, 1H), 4.56 (s, 2H, OCH2), 3.70 (s,
3H, OCH3); 13C-NMR spectrum (DMSO-d6): δppm 168.09
(>C=O), 157.72 (>C=O), 154.52, 152.36, 146.68, 146.40,
116.42, 115.31, 115.19, 112.52, 67.47 (OCH2), 56.01
Nʹ‑[(2‑Chlorophenoxy)acetyl]‑2‑furohydrazide (4a)
Yield 61% Solvent crystallization: ethylacetate. m.p.:
202–204 °C; Rf=0.47 (ethylacetate/n-hexane, V/V=1:4);
FT-IR (cm−1): 3323, 3214 (NHNH), 3065 (Ar C–H), 2917
(CH2), 1697, 1681 (> C=O), 1493 (Ar –C=C–), 1251,
1
1040 (C–O–C), 720 (o-substituted benzene); H-NMR
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Chemical Papers
(OCH3). LC–MS (ESI): m/z calculated for C14H14N2O5
(290.27). Found: m/z 291.05 (M+ +1).
m/z calculated for C15H11Cl2BrN2O3 (418.07). Found: m/z
416.95 (M+.-1).
2‑Chloro‑Nʹ‑[2‑(2,4‑dichlorophenoxy)acetyl]
benzohydrazide(4c)
4‑Chloro‑Nʹ‑[(2,4‑dichlorophenoxy)acetyl]
benzohydrazide (4f)
Yield 79%. Solvent crystallization: ethanol. m.p.:
184–188 °C; Rf=0.52 (ethylacetate:n-hexane, V/V=1:4);
FT-IR (cm−1): 3290, 3208, (NHNH), 3068, 3037, 3025 (Ar
C-H), 2825 (CH2), 1716, 1661 (> C=O), 1645, 1615 (NH
amide), 1593, 1486 (Ar –C=C–), 1269, 1047 (C–O–C);
1H-NMR (DMSO-d6): δppm 10.45 (s, 2H, 2NH), 7.61 (d,
J=8.34 Hz, 1H), 7.59 (d, J=7.95 Hz, 1H), 7.57–7.45 (m,
3H), 7.44 (dd, J = 8.40, 2.31 Hz, 1H), 7.15 (dd, J = 7.85,
2.22 Hz, 1H), 4.95 (s, 2H, OCH2); 13C-NMR (DMSO-d6):
δppm 166.62 (> C=O), 165.73 (> C=O), 153.06, 134.88,
132.03, 130.90, 130.35, 129.87, 129.84, 128.46, 127.64,
125.66, 123.07, 115.91, 67.10 (OCH2). MS (ESI): m/z cal-
culated for C15H11Cl3N2O3 (373.62). Found: m/z 371.05
(M+.-2).
Yield 66%. Solvent crystallization: methanol. m.p.:
204–206 °C; Rf=0.52 (ethylacetate:n-hexane, V/V=1:5);
FT-IR (cm−1): 3272, 3243 (NHNH), 3066, 3024 (Ar C–H),
2845 (CH2), 1693, 1673 (>C=O), 1651, 1621 (NH amide),
1598, 1488 (Ar –C=C–), 1260, 1078 (C–O–C), 801 (p-sub-
1
stituted benzene); H-NMR (DMSO-d6): δppm 10.60 (s,
1H, NH), 10.31 (s, 1H, NH), 7.93–7.89 (m, 2H), 7.61–7.58
(m, 2H), 7.41 (d, J=8.3 Hz, 1H), 7.39 (dd, J=8.4, 1.9 Hz,
1H), (d, J = 8.04 Hz, 1H), 4.82 (s, 2H, OCH2); 13C-NMR
(DMSO-d6): δppm 166.94 (> C=O), 164.98 (> C=O),
153.04, 137.28, 131.48, 130.05, 129.89, 129.15, 128.47,
125.71, 123.10, 115.96, 67.28 (OCH2); GC–MS (EI-TOF)
m/z calculated for C15H11Cl3N2O3 (373.62). Found: m/z
373.62 (M+).
Nʹ‑[2‑(2,4‑Dichlorophenoxy)
acetyl]‑3,5‑dinitrobenzohydrazide (4d)
2,4‑Dimethoxy‑Nʹ‑[2‑(4‑nitrophenoxy)acetyl]
benzohydrazide (4g)
Yield 76%. Solvent crystallization: ethanol. m.p.:
155–158 °C; Rf=0.54 (ethylacetate:n-hexane, V/V=1:5);
FT-IR (cm−1): 3318, 3217 (NHNH), 3076, 3039 (Ar
C–H), 2920, 2849 (CH2), 1672,1641 (> C=O), 1632 (NH
amide), 1588 (Ar –C=C–), 1480, 1366 (NO2), 1247, 1047
(C–O–C), 866 (C–N); 1H-NMR (DMSO-d6): δppm 9.26 (s,
2H, 2NH), 7.61–7.54 (m, 3H), 7.37 (d, J = 8.13 Hz, 1H),
7.35–6.92 (m, 2H), 4.60 (s, 2H, OCH2); 13C-NMR (DMSO-
d6): δppm 170.74 (> C=O), 166.47 (> C=O), 153.09,
129.85, 129.66, 128.49, 128.19, 125.50, 123.00, 115.73,
115.31, 67.53 (OCH2); GC–MS (EI-TOF) m/z calculated
for C15H10Cl2N4O5 (429.17). Found: m/z 429.17 (M+).
Yield 81%. Solvent crystallization: ethanol. m.p.:
185–187 °C; Rf=0.54 (ethylacetate:n-hexane, V/V=1:4.5);
FT-IR (cm−1): 3324, 3212 (NHNH), 3085, 3073, 3039 (Ar
C–H), 2992, 2848 (CH2), 1698, 1658 (>C=O), 1641 (NH
amide), 1568, 1462 (Ar –C=C–), 1593, 1342 (NO2), 846
(C–N), 1244, 1050 (C–O–C). 1H-NMR (DMSO-d6): δppm
10.54 (s, 2H, 2NH), 8.25–8.19 (m, 2H), 7.81 (d, J= 8.32,
Hz, 1H), 7.22 (d, J = 7.99 Hz, 1H), 7.09 (d, J = 8.4 Hz,
1H), 6.67–6.65 (m, 2H), 4.87 (s, 2H, OCH2), 3.91 (s, 6H,
OCH3); 13C-NMR (DMSO-d6): δppm 164.03 (> C=O),
163.86 (> C=O), 163.45, 159.26, 141.78, 133.01, 132.96,
126.28, 126.23, 115.86, 115.50, 113.44, 98.99 (CH2), 66.82
(OCH3), 55.98 (OCH3); LC–MS (ESI): m/z calculated for
C17H17N3O7 (375.33). Found: m/z 376.00 (M+ +1.).
3‑Bromo‑Nʹ‑[2‑(2, 4‑dichlorophenoxy)acetyl]
benzohydrazide (4e)
2,4‑Dichloro‑Nʹ‑[2‑(4‑nitrophenoxy)acetyl]
benzohydrazide (4h)
Yield 84%. Solvent crystallization: ethanol. m.p.:
198–202 °C; Rf=0.47 (ethylacetate:n-hexane, V/V=1:4.5);
FT-IR (cm−1): 3268, 3160 (NHNH), 3073, 3003 (Ar C–H),
2924 (CH2), 1697, 1658 (> C=O), 1645 (NH amide),
1548, 1486 (Ar –C=C–), 1251, 1071 (C–O–C); 1H-NMR
(DMSO-d6): δppm 10.64 (s, 1H, NH), 10.33 (s, 1H, NH),
8.06 (d, J=7.6 Hz, 1H), 7.88 (dd, J=8.2, 1.5 Hz, 1H), 7.69
(dd, J=7.6, 1.4 Hz, 1H), 7.56 (t, J=7.7 Hz, 1H), 7.44 (dd,
J=8.05, 1.8 Hz, 1H), 7.06 (dd, J=8.15, 2.3 Hz, 1H), 6.96
(d, J=8.32 Hz, 1H), 4.83 (s, 2H, OCH2); 13C-NMR (DMSO-
d6): δppm 166.89 (>C=O), 164.51 (>C=O), 153.0, 135.19,
134.82, 131.32, 130.62, 130.57, 129.88, 129.85, 128.44,
127.05, 125.69, 123.07, 67.23 (OCH2); LC–MS (ESI):
Yield, 71%. Solvent crystallization: methanol.
m.p.:218–220 °C; Rf = 0.48 (ethylacetate:n-hexane,
V/V=1:5); FT-IR (cm−1): 3268, 3222 (NHNH), 3089, 3057
(Ar C–H), 2924, 2846 (CH2), 1712, 1665 (> C=O), 1564
(Ar –C=C–), 1595, 1353 (NO2), 1259, 1040 (C–O–C). 1H-
NMR (DMSO-d6): δppm 10.52 (s, 2H, 2NH), 8.25–7.89 (m,
2H), 7.74 (d, J=8.16 Hz 1H), 7.68–7.53 (m, 2H), 7.49 (dd,
J = 7.94, 1.34 Hz, 1H), 7.21 (d, J = 8.16 Hz, 1H), 4.98 (s,
2H, OCH2); 13C-NMR spectrum (DMSO-d6): δppm 166.47
(>C=O), 164.96 (>C=O), 135.83, 133.72, 132.18, 131.17,
129.96, 129.70, 129.25, 127.92, 127.32, 126.24, 66.79
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Chemical Papers
(OCH2). LC–MS (ESI): m/z calculated for C15H11Cl2N3O5
(384.17). Found: m/z 382.00 (M+.-2).
116.23, 116.03, 115.76, 67.26 (OCH2), 55.85 (OCH3);
GC–MS (EI-TOF) m/z calculated for C16H15ClN2O7S
(414.81). Found: m/z 414.81 (M+).
Nʹ‑[2‑(4‑Methoxyphenoxy)
acetyl]‑2‑methylbenzohydrazide (4i)
Determination of minimum inhibitory
concentration (MIC)
Yield 62%. Solvent crystallization: methanol. m.p.:
88–92 °C; Rf = 0.54 (ethylacetate:n-hexane, V/V = 1:5);
FT-IR (cm−1): 3253, 3185 (> NH), 3065 (Ar C–H), 2995,
2831 (CH2), 1689, 1645 (>C=O), 1658 (NH amide), 1509
(Ar –C=C–), 1227, 1040 (C–O–C); 1H-NMR (DMSO-d6):
δppm 10.23 (s, 1H, NH), 10.13 (s, 1H, NH), 7.41–7.29
(m, 2H, ArH), 7.28–7.25 (m, 2H, ArH), 6.90 (dd, J=8.21,
2.74 Hz, 2H), 6.55 (dt, J=7.81, 1.84 Hz, 2H), 4.68 (s, 2H,
OCH2), 3.71 (s, 3H, OCH3), 2.50 (s, 3H, CH3); 13C-NMR
spectrum (DMSO-d6): δppm 168.78 (> C=O), 167.84
(>C=O), 154.46, 152.36, 136.47, 135.30, 131.12, 130.49,
127.94, 126.10, 116.34, 115.14, 67.43 (OCH2), 55.95
(OCH3), 19.86 (CH3). LC–MS (ESI): m/z calculated for
C17H18N2O4 (314.34). Found: m/z 315.25 (M+ +1).
In vitro antibacterial activity of synthetic compounds 4a-
k was assessed according to the guidelines of the Clinical
Laboratories Standard Institute (CLSI 2007) against Gram-
positive and Gram-negative bacterial strains of S. aureus
(NCIM 5021 and NCIM 5022), methicillin resistant S.
aureus (MRSA strain 43300), B. subtilis (NCIM 2545), E.
coli (NCIM 2567), K. pneumoniae (NCIM 2706) and P. aer-
uginosa (NCIM 2036). Triplicate analyses were performed
for each selected strain by microdilution broth technique
using Mueller Hinton medium (Hi-media). Compounds 4a-
k was screened for their antibacterial activity dissolved in
sterile dimethyl sulfoxide (DMSO). Ciprofoxacin and gen-
tamicin were used as positive control in sterile DMSO, while
sterile DMSO was used as a negative control. The 96 well
microtitre plates were incubation at 37 °C for 24 h, agitated
and read for the absorbance. The fnal DMSO concentra-
tion in assays well was<1% and the fnal volume for MIC
protocols was 100 µL. The absorbance was measured at a
wavelength of 600 nm. The MIC values are summarized
Table 1 and expressed as microgram per millilitre (µg/ml).
Nʹ‑(Benzenesulfonyl)‑2‑(2‑chlorophenoxy)
acetohydrazide (4j)
Yield 72%. Solvent crystallization: methanol.
m.p.:114–116 °C; Rf = 0.51(ethylacetate:n-hexane,
V/V = 1:5.5); FT-IR (cm−1): 3339, 3214 (NHNH), 3034,
3003 (Ar C–H), 2940 (CH2), 1704 (> C=O), 1658, 1603
(NH amide), 1486 (Ar –C=C–), 1243, 1048 (C–O–C),
752 (o-substituted benzene); 1H-NMR (DMSO-d6): δppm
10.30 (s, 1H, NH), 10.06 (s, 1H, NH), 7.81 (dt, J= 8.31,
2.45 Hz, 2H), 7.79 (dd, J = 7.84, 1.59 Hz, 1H), 7.77 (dd,
J = 8.08, 2.67 Hz, 1H), 7.63–7.52 (m, 3H), 7.49–7.24 (m,
2H), 4.57 (s, 2H, OCH2); 13C-NMR (DMSO-d6): δppm
166.69 (> C=O), 133.63, 130.61, 130.58, 130.01, 129.48,
128.75, 128.68, 128.59, 128.43, 128.14, 66.94 (OCH2). MS
(ESI) m/z: calculated for C14H13ClN2O4S (340.78). Found:
339.10 (M+.-1).
Enzyme inhibitory activity of S. aureus MurD ligase
Three compounds 4d, 4j and 4k showing promising inhibi-
tory activity against the tested strains of S. aureus are
selected for SaMurD ligase inhibition assay. The inhibitory
activity of three compounds was assessed against Sa MurD
enzyme (ProFoldin, USA) using the malachite green assay
with slight modifcations (Auger et al. 1998; Lanzetta et al.
1979). All of the experiments were carried out in duplicate.
The fnal volume of 50 µL mixture contained: 40 mM KCl,
50 mM TrisHCl, pH 8.0, 0.005% Triton X-100, 0.1 mM
UDP-MurNAc-L-Ala, 0.5 mM D-Glu, 1 mM ATP, 1 mM
DTT, 1 mM MgCl2, 500 nM purifed SaMurD and 0.6 µL
of each test compound diferent concentrations dissolved
in dimethylsulfoxide (DMSO). In all experiments the fnal
concentration of DMSO was 5% (v/v). The prepared mix-
ture was incubated at 37 °C for 60 min and then quenched
using the Dye MPA3000. Mixture was further incubated for
5 min and absorbance at 650 nm was determined and per-
centage inhibition was calculated. Percent inhibitions were
also calculated omitting the test compounds and with 5%
DMSO. GraphPad PRISM was used to determine IC50 val-
ues (Fig. 2).
2‑Chloro‑5‑{[2‑(4‑methoxyphenoxy)acetohydrazido]
sulfonyl}benzoic acid (4k)
Yield 67%. Solvent crystallization: ethanol. m.p.:
196–200 °C; Rf=0.48 (ethylacetate:n-hexane, V/V=1:6);
FT-IR (cm−1): 3315, 3222 (NHNH), 3065, 3018 (Ar C–H),
2838 (CH2), 1704, 1650 (>C=O), 1650 (NH amide), 1439
(Ar –C=C–), 1235, 1032 (C–O–C). 1H-NMR (DMSO-d6):
δppm 10.28 (s, 1H, OH), 10.27 (s, 1H, NH), 10.12 (s, 1H,
NH), 8.19 (d, J=8.24 Hz, 1H), 7.86 (d, J=8.32 Hz, 1H),
7.40 (dd, J= 7.88, 1.98 Hz, 1H), 6.95–6.75 (m, 4H), 4.54
(s, 2H, OCH2), 3.69 (s, 3H, OCH3); 13C-NMR spectrum
(DMSO-d6): δppm 167.64 (> C=O), 167.41 (> C=O),
154.35, 154.33, 152.08, 138.56, 137.14, 132.09, 131.79,
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Chemical Papers
Table 1 Antibacterial
Comp
Minimum inhibitory concentration (µg/ml)*
screening result of synthesized
compounds 4a-k against
selected Gram-positive and
Gram-negative bacteria
aS. a
bS.a
cS. a
dB. s
256 0.95 64 1.12
128 1.24 128 0.18 >256
256 0.91 32 0.86 256 1.08 256 1.27
128 1.51 128 0.78 64 1.61 128 0.96
128 0.87 256 0.82 64 1.04
>256 128 0.95 32 1.18
eK. p
fP. a
128 0.16 128 0.66
128 1.05
gE. c
4a
4b
4c
4d
4e
4f
64 1.19
>256
32 1.21
32 0.97
>256
64 0.64
>256
64 1.15
128 1.04 >256
>256
256 1.01 >256
>256
>256
>256
128 1.09
256 0.24
256 1.47
256 0.94
256 0.74
64 1.25
64 1.13
>256
4g
4h
4i
128 0.56 256 0.87 256 1.04
128 1.01 256 1.67 128 1.38
128 1.17 256 1.59 256 1.28
128 0.55 128 0.24 >256
>256
128 0.85 64 0.54
>256
128 0.84 32 1.88
128 1.46 128 0.97 32 0.96
128 1.44 >256 >256 >256
128 1.08
4j
32 0.61
64 0.92
64 0.56
64 0.54
128 1.29
128 1.58
32 1.01
64 0.89
64 0.99
4k
Compound A$ 128 1.54 128 2.04 256 0.98
Ciprofoxacin
Gentamicin
2
8
0.84
1.21
2
8
1.13
0.79
32 0.96
19.50 0.69
2
8
1.74
1.41
2
1
0.64
1.51
2
1
1.35
0.98
2
1
1.21
0.78
*Values are mean SEM (n=3)
Ciprofoxacin and Gentamicin used as positive control
aS. a: Staphylococcus aureus NCIM 5021; bS. a: Staphylococcus aureus NCIM 5022; cS. a: Staphylococcus
d
e
aureus ATCC 43300 (MRSA); B. s: Bacillus subtilis NCIM 2545; K. p: Klebsiella pneumoniae NCIM
2706; fP. a: Pseudomonas aeruginosa NCIM 2036; gE. c: Escherichia coli NCIM 2567
MIC minimum inhibitory concentration
$Azam et al. (2019a)
strength binding free energy of compounds 4a-k was calcu-
lated by prime MM-GBSA approach (Jacobson et al. 2004)
using VSGB 2.0 energy (Li et al. 2011) and OPLS3e force
feld (Roos et al. 2019). In this study we used the earlier pre-
pared grid box of homolg modelled SaMurD protein (Azam
et al. 2019b). The low energy conformations of all ligands
were docked in the catalytic pocket and their binding free
energies were determined using the protocols described ear-
lier (Azam et al. 2019b).
MD simulation
The extra-precision-docked structure of compound 4k/
Fig. 2 Graphs used for IC50 value determinations of compounds 4d
(red), 4j (green) and 4k (blue) against SaMurD enzyme
SaMurD modelled protein complex was used as starting
structure for MD simulation using the Desmond software
with OPLS3e force feld (Roos et al. 2019). The complex
was solvated in an orthorhombic box with explicit TIP4P
(Jorgensen and Madura 1985; Lawrence and Skinner 2003)
water with a 10 Å bufer region between protein atoms and
box sides. Eleven counter sodium ions were added to neu-
tralize the solvated system. The modelled system contains
approximately 49,403 atoms, 14,095 water molecules with
a fnal box volume of 1,197,217 Å3. The whole system was
then minimized with OPLS3e (Roos et al. 2019) force feld
using 200 steepest descent and 1000 steps of conjugate gra-
dient until 25 kcal/mol/Å gradient threshold was reached.
Particle-mesh-Ewald method (Essmann et al. 1995) at a
Computational analysis
Molecular docking and binding free energy calculation
(MM‑GBSA)
The 3D-structure of compounds 4a-k were sketched in the
builder panel of Maestro 11.5 (Schrodinger software suite
2018-1, LLC, New York, NY) and subsequently optimized
with LigPrep (Sastry et al. 2013). The extra-precision molec-
ular docking was performed using Glide without applying
any constraints. In order to accurately rank the binding
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tolerance of 1e-09 was used for the long range electrostatic
interactions. A cutof radius of 9 Å was applied for van der
Waals and Coulomb interactions. A 50 ns simulation was
performed for this minimized system under an isothermal-
isobaric ensemble (NPT) at a constant temperature of 300 K
and a constant pressure of 1 bar were used to maintain dur-
ing simulation temperature and pressure were maintained,
respectively with Nose–Hoover thermostat (Martyna et al.
1992) and Martyna–Tobias–Klein barostat (Martyna et al.
1994). For the bonded, near nonbonded, and far nonbonded
interactions a multiple time step RESPA integration algo-
rithm was used, with time steps of 2, 2, and 6 fs, respec-
tively. For the overall simulation 2 fs time step was selected
and data were collected every 100 ps. The 3D structures
and MD trajectory were analyzed visually with the Maestro
graphical interface.
patterns like double doublet (dd), triplet of doublet (dt), tri-
plet (t) and doublet (d). In compound 4a the two protons H-3
and H-6 of 2-chlorophenyl ring appeared as double doublets,
respectively at δ 7.22 and 7.06 ppm with average coupling
constant of δ 7.97 and 1.54 Hz. Two triplet of doublet (dt)
signals at δ 7.44 and 7.32 ppm with average J values of 7.95
and 1.56 Hz were attributed, respectively for H-4 and H-5
hydrogens of the same ring. Two hydrogens H-3′ and H-5′
of the furan ring were observed at δ 7.91 and 6.85 ppm,
respectively with average J values of 8.11 and 1.67 Hz. H-4′
hydrogen of furan ring exhibited triplet signal at δ 6.68 ppm
with J values of 8.3 and 2.61 Hz. Other synthesized com-
pounds also exhibited splitting patterns according to the
substitution pattern. In the 13C-NMR spectra of compounds
4a-k the carbonyl carbon signals appeared in the region δ
157.68–170.74 ppm while the signals appeared in the region
δ 66.79–67.53 ppm is due to the -OCH2- fragment carbon.
Aromatic carbon signals appeared at the expected region. In
the mass spectra, compounds 4a-k exhibited molecular ion
peaks which are in agreement with their molecular formula
(see experimental also Supplementary Figs. S1–S8).
Results and discussion
Chemistry
The synthetic routes for title compounds 4a-k is outlined in
Scheme 1. The intermediate ethyl (2/4-substituted phenoxy)
acetates 2a-d is prepared by treatment of 2/4-substituted
phenols (1a-d, Scheme 1) with 2-chloroethylacetate in dry
acetone (Holla et al. 1992; Horner et al. 1974; Amer et al.
2018; Liu et al. 2012). These acetates (2a-d) are reacted
with hydrazine hydrate (99% w/w) in boiling absolute etha-
nol to furnish 2-(2/4-disubstituted phenoxy)acetohydrazides
3a-d (Fun et al. 2010; Chao et al. 1949; Amer et al., 2018;
Liu et al. 2012). The acetohydrazides 3a-d was then stirred
with appropriate substituted acid chlorides in acetonitrile to
furnish titled compounds 4a-k in good yields (Hariskishore
et al. 2013).
Determination of minimum inhibitory
concentration (MIC)
The antibacterial activities of the synthesized compounds
were screened by two-fold broth micro-dilution method, as
per the procedures of the Clinical and Laboratory Standards
Institute (CLSI 2007). The result of antibacterial screening
is represented in Table 1. Tested compounds MIC values
are compared with the standard drugs ciprofoxacin and
gentamicin. In vitro antibacterial screening of tested phe-
noxyacetohydrazides 4a, 4c, 4d, 4f, 4j, and 4k exhibited
promising inhibitory activity against Gram-positive bacte-
ria S. aureus NCIM 5021 with MIC values in the range of
32–64 µg/ml in comparison to the standard drugs ciprofoxa-
cin (MIC, 2 µg/ml) and gentamcin (MIC 8 µg/ml). Tested
compounds 4a, 4j and 4k also exhibited inhibitory activity
against S. aureus NCIM 5022 with MIC value of 64 µg/ml
in each case. Three compounds i.e. 4a, 4j and 4k exhibited
activity against methicillin resistant S. aureus ATCC 43300
with MIC of 128 µg/ml, while all other tested compounds
were observed to be either weakly active (MIC 256 µg/ml) or
inactive (MIC>256 µg/ml) compared to the standard drugs
ciprofoxacin (MIC, 32 µg/ml) and gentamicin (MIC, 20 µg/
ml). The low activity of these compounds is probably due to
the failure of bacterial cell wall penetration.
Synthesized compounds 4a-k structures were confrmed
by FT-IR, 1H-NMR, 13C-NMR and mass spectroscopy. In
the IR spectra of phenoxyacetohydrazides 4a-k the appear-
ance of band in the range 3208–3339 cm−1 indicated the
presence of hydrazide –NHNH– group, while the bands
appeared in the range 1641–1716 cm−1 is due to carbonyl
groups. In these compounds the C–O–C absorption bands
1
appeared in the region 1032–1259 cm−1. In the H-NMR
spectra of compounds 4a-k two singlet signals appeared in
the region δ 9.26–10.64 ppm indicated the presence of two
protons of NHNH function. The singlet signals appeared in
the region δ 4.54–4.98 ppm is ascribed to two protons of
–OCH2 group. Further, the splitting patterns for the aromatic
hydrogens agree with the substitution pattern of respective
compounds. The chemical shift values and coupling constant
(J values) were assigned accordingly with the position of
aromatic hydrogens. Aromatic hydrogens on coupling with
ortho and meta hydrogens exhibited characteristic splitting
Among all the tested compounds, phenoxyacetohy-
drazides 4e, 4f and 4k displayed MIC of 64 µg/ml against
B. subtilis NCIM 2545. Compound 4j exhibited maximum
activity against this strain with an MIC of 32 µg/ml, whereas
other tested compounds were observed to be either less
active (128 to 256 µg/ml) or inactive (MIC > 256 µg/ml).
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Further, compounds 4a, 4c, 4i and 4j displayed promising
inhibitory activity against Gram-negative K. pneumoniae
NCIM 2706 (Table 1, MIC, 64, 32, 64, and 64 µg/ml, respec-
tively). Among all tested compound, 4c exhibited highest
activity against this bacterial strain with MIC value of 32 µg/
ml in comparison to the standard drugs ciprofoxacin (2 µg/
ml) and gentamicin (1 µg/ml). Only one compound, i.e. 4d
showed activity against another Gram-negative bacteria P.
aeruginosa NCIM 2036 (MIC value of 64 µg/ml), while
all other tested compounds were observed to be either less
active (MIC, 128–256 µg/ml) or inactive (MIC > 256 µg/
ml). On the other hand, compounds 4e, 4f, 4j and 4k dis-
played promising activity against E. coli NCIM 2567 (MIC,
32–64 µg/ml). Against this strain the highest activity was
observed for compounds 4f, 4j and 4k with MIC value of
32 µg/ml in all cases. It is evident from Table 1 that com-
pounds 4j and 4k displayed inhibitory activity against all the
tested strains of Gram-positive and Gram-negative bacteria.
Improvement in activity against S. aureus NCIM 5021was
observed in compounds 4a, 4c, 4d, 4f, 4j and 4k (MIC
32–64 µg/ml) in comparison to compound A (MIC 128 µg/
ml). Further, compared to compound A (MIC 128 µg/ml)
improvement in inhibitory activity against S. aureus NCIM
5022 was also observed for compounds 4a, 4j and 4k (MIC
64 µg/ml). Also in comparison to compound A (MIC 256 µg/
ml), these three compounds also exhibited a lower MIC of
128 µg/ml against methicillin resistant S. aureus ATCC
43300.
Enzyme inhibitory activity of S. aureus MurD ligase
In vitro inhibitory potency of three selected compounds 4d,
4j and 4k was carried out using SaMurD enzyme (ProFol-
din, USA) (Table 2). The tested compounds showed IC50
values in the range 35.80–98.66 µM, compound 4k being the
highest active (IC50 35.80 µM). Remaining two compounds
namely 4d and 4j inhibited SaMurD with IC50 values of
37.82 and 98.66 µM, respectively. It is evident from above
result that the presence of an electron withdrawing substitu-
ent on the phenyl ring attached to either –SO2 or > C=O
group is important for the SaMurD inhibitory activity as evi-
dent by the lower activity of compound 4j (IC50, 98.66 µM)
lacking electron withdrawing substituent on this phenyl ring.
Computational analysis
Table 2 IC50 (µM) values of
Interaction energies of compounds 4a-k with the catalytic
pocket residues of modelled S. aureus MurD enzyme were
computed to get insight into their binding modes and afnity.
Analysis of extra-precision docking poses showed hydro-
gen bonding, π–π stacking and π-cation stacking salt bridge
interactions with conserved residues Lys19, Lys26, His48,
Gly144, Gly147, Val149, Trp425 and Phe 431 (Fig. 3 also
Supplementary Fig. S9). The extra-precision docking and
binding free energy calculation results are summarized in
Tables 3 and 4, respectively.
Comp
IC50(µM)
some selected compounds
SaMurD
against S. aureus MurD enzyme
4d
37.82
98.66
35.80
7.00
4j
4k
Compound A$
Data are the mean values of
two independent experiments.
Standard
deviations
were
within 10% of these mean val-
ues
Sulfonylhydrazide 4j with IC50 of 98.66 µM against
SaMurD protein occupied all three domains of the
$Azam et al. (2019a)
Fig. 3 Plots represent interactions of catalytic pocket residues of modelled SaMurD with compounds (a) 4j and (b) 4k
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Chemical Papers
Table 3 Extra-precision
docking results of designed
compounds 4a-k in the active
site of SaMurD enzyme (kcal/
mol)
Comp Extra-precision docking scores
agscore bgvdw cgcoul
dgmodel egenergy fgH-bond gg rot.penal hg Lip. EvdW
4a
4b
4c
4d
4e
4f
− 4.91
− 6.45
− 4.75
− 5.45
− 4.81
− 5.21
− 4.62
− 4.90
− 4.48
− 4.48
− 5.54
− 35.06 − 26.93 − 54.31 − 42.00
− 34.71 − 9.01 − 57.70 − 43.72
− 0.975
− 1.415
− 1.108
− 1.506
− 1.718
− 1.85
0.336
0.384
0.384
0.382
0.368
0.379
0.368
0.379
0.313
0.348
0.313
− 3.534
− 3.673
− 2.013
− 3.612
− 3.243
− 3.346
− 3.353
− 3.608
− 3.149
− 3.582
− 3.636
− 38.51 − 17.81 − 59.72 − 46.33
− 46.10 − 23.69 − 71.24 − 49.79
− 38.23 − 41.33 − 69.23 − 49.56
− 37.49 − 55.13 − 57.18 − 42.62
− 44.29 − 25.33 − 73.98 − 54.63
− 38.59 − 27.62 − 69.00 − 46.21
− 33.54 − 49.33 − 63.52 − 42.87
− 31.26 − 31.76 − 56.51 − 43.02
− 40.10 − 11.96 − 74.92 − 52.07
4g
4h
4i
4j
4k
− 1.215
− 0.82
− 1.27
− 1.149
− 1.98
c
e
aglide score, bglide van der Waals energy, glide Coulomb energy, dglide emodel energy, glide energy,
fhydrogen bond, grotational penality, hglide lipophilic contact plus phobic attractive term in the glide score
Table 4 Binding free energy
contribution (MM-GBSA)
of designed compounds 4a-k
in the active site of SaMurD
enzyme (kcal/mol)
Comp
Binding free energy components
aΔGCoul
bΔGLipo
cΔGSolv
dΔGvdW
e∆GH-bond
fΔGbind
4a
4b
4c
4d
4e
4f
4g
4h
4i
− 48.70
− 12.42
− 31.26
− 55.57
− 84.53
− 92.60
− 39.29
− 20.21
− 88.65
− 70.28
− 16.42
− 17.41
− 15.00
− 14.98
− 25.93
− 30.87
− 22.52
− 18.69
− 27.55
− 18.63
− 25.78
− 20.04
18.95
− 14.85
4.83
38.78
73.09
46.24
15.56
4.21
− 53.78
− 30.49
− 28.53
− 70.70
− 76.36
− 27.88
− 52.86
− 38.88
− 51.21
− 57.22
− 52.04
− 3.63
− 3.66
− 2.82
− 4.80
− 3.70
− 2.49
− 2.67
− 3.85
− 3.04
− 3.54
− 6.64
− 77.60
− 68.31
− 77.32
− 112.23
− 94.24
− 80.06
− 92.23
− 82.70
− 67.28
− 83.60
− 69.30
76.86
30.65
− 2.96
4j
4k
aCoulomb energy, bhydrophobic energy (nonpolar contribution estimated by solvent accessible surface
area), celectrostatic solvation energy, dvan der Waals energy, ehydrogen bonding, ffree energy of binding
catalytic pocket and exhibited two hydrogen bonding inter-
actions. Precisely, oxygen atom of carbonyl group attached
to the 2-chlorophenoxymethylene fragment in compound
4j accepted hydrogen bond from the protonated NH2 of
Lys19 (NH⋯O=C < , 1.90 Å), while the –NH– function
attached to this carbonyl group formed hydrogen bond
with the carbonyl oxygen of Gly147 (> C=O⋯HN–,
1.82 Å) (Fig. 3a). The phenyl ring of 4-chlorophenoxym-
ethyle fragment established π-cation interaction with the
protonated NH2 of Lys328. The lower inhibitory activ-
ity of compound 4j against SaMurD may be due to fewer
interactions with the catalytic pocket residues. The high
active sulfonylhydrazide 4k (IC50 35.80 µM) exhibited four
hydrogen bonding interactions, two more than 4j (Fig. 3b).
The NH of -CONHNH- fragment formed hydrogen bond
with carbonyl oxygen of Gly147 (> C=O⋯NH, 2.19 Å),
while > C=O oxygen of the same fragment established
hydrogen bonding interaction with the NH of Lys19
(NH⋯O=C < , 2.47 Å). The oxygen atom attached to
the 4-methoxyphenyl ring also accepted hydrogen bond
from the NH of Lys19 (NH⋯O-C6H4-4-OCH3 2.59 Å),
while the oxygen atom of –SO2– function attached to the
4-chloro-3-carboxy phenyl moiety accepted a hydrogen
bond from the NH of Val149 (NH⋯O=S(=O)–, 2.12 Å).
The carboxylate oxygen of 4-chloro-3-carboxy phenyl
moiety exhibited two salt bridge interaction one each with
Ly26 and Ly152. This compound is further stabilized in
the catalytic pocket by two π–π stacking interactions one
each with Tyr22 and Phe431 residues. Compound 4d (IC50
37.82 µM) belonging to the hydrazide series exhibited
three hydrogen bonding interactions one each with Asn78,
Gly80 and Gly144 (Supplementary Fig. S9). 2,4-Dichloro-
phenyl ring of this compound also established a π–π stack-
ing interaction with the phenyl ring of Phe431. A π-cation
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interaction was also observed between the nitrogen atom
of one of nitro groups and Glu101. It is evident that com-
pounds belonging to the hydrazide series exhibited less
interactions with the catalytic pocket residues compared to
the sulfonylhydrazide 4k (Supplementary Fig. S9).
Glu232-Leu234 present on the loop (Tyr223-Thr238) and
connecting two β-sheets (Leu239-Phe241).
It is evident from analysis of MD trajectory that com-
pound 4k occupied all three domains of the catalytic pocket
and exhibited hydrogen bonding, salt bridge, π–π stacking
and π-cation interactions (Fig. 4c and also Supplementary
Fig. S10). Accurately, one of the –NH– function linked to
the –O–CH2CO–NH–NH– fragment formed a strong hydro-
gen bonding interaction with Gly147 (90% of the MD trajec-
tory), while another NH function linked to the –SO2– group
established a strong hydrogen bonding interaction (82% of
the MD trajectory) with Ser429. A strong hydrogen bonding
interaction (99% of the MD trajectory) was also observed
between carbonyl oxygen of the same fragment and Trp425
located in the C-terminal domain. The phenyl ring of this
residue also exhibited a π–π stacking interaction with the
phenyl ring of 4-chloro-3-carboxyphenyl moiety. One of
the oxygen atoms of SO2 group accepted two direct hydro-
gen bond one each from the central domain residue Val149
(48% of the MD trajectory) and the N-terminal residue
Lys19 (21% of the MD trajectory). Another oxygen atom of
this group accepted water mediated hydrogen bond (38% of
the MD trajectory) from Val149. The carboxylate oxygen
of 4-chloro-3-carboxyphenyl moiety accepted two moder-
ate frequency hydrogen bonds, a direct bond from the side
chain NH of Lys19 (24% of the MD trajectory) and a water
mediated bond from the side chain NH of Glu23 (39% of the
MD trajectory). Glu23 also established moderate frequency
water mediated hydrogen bond (30% of the MD trajectory)
with the carbonyl oxygen of carboxylate function. Com-
pound 4k also exhibited low frequency hydrogen bonding
and salt bridge interactions (10–16% of the MD trajectory)
with Lys152, Asn145, Tyr428 and Phe 431. It is evident
from above result that extensive hydrogen bonding interac-
tions play a crucial role for the stabilization of compound 4k
within the catalytic pocket of SaMurD protein.
To accurately rank the binding strength of compounds
4a-k with modelled SaMurD protein the binding free
energy (ΔGbind) was computed by the MM-GBSA approach
(Table 4). However, in the present investigation ΔGbind did
not exhibit any correlation either with the MIC or IC50 val-
ues. The ΔGbind values are observed to be in range between
− 67.28 to − 112.23 kcal/mol. It is evident from binding
free energy components that van der Waals (ΔGvdW, − 28.53
to − 76.36 kcal/mol) and electrostatic interaction (ΔGCoul
,
− 12.42 to − 92.60 kcal/mol) energy terms are major con-
tributors for the inhibitors binding. Except for 4b and 4k,
electrostatic solvation energy (ΔGSolv, 4.21 to 78.92 kcal/
mol) term disfavors, while non-polar solvation (ΔGLipo
,
− 14.98 to − 30.87 kcal/mol) energy term moderately favors
the binding to the SaMurD protein. Compared to ΔGCoul
,
the high negative value of ΔGvdW indicated it as the driving
force for binding. This is observed to be in agreement with
the earlier reports (Perdih et al. 2007,2009,2014,2013; Zidar
et al. 2011) and also with the value of Glide Emodel (Gemodel
,
− 54.14 to − 94.85 kcal/mol) possessing signifcant weight-
ing of the force feld. s
To validate the stability of docking complex and to get
insight about molecular mechanism of binding, we per-
formed a 50 ns MD simulation for 4k/SaMurD complex.
It is evident from Fig. 4a that root mean square deviations
(RMSDs) of protein all Cα, backbone and heavy atoms
increased sharply (3.91, 3.97, 4.18 Å, respectively) during
frst 5 ns of MD simulation and then observed to stable with
average fuctuations of 1.08, 1.05 and 1.03 Å, respectively
during rest of the simulation trajectory. This shows less con-
formational changes in protein structure during simulation.
Ligand–protein interactions were observed in the region
Ala18-Lys52 and Ser327-Glu432, while 4k did not show
contacts in the region Val153-Asp326 (Fig. 4b also Supple-
mentary Figs. S10 and S11). The root mean square fuctua-
tions (RMSF) in Cα, backbone and heavy atoms of ligand
contacting residues was observed in the range 0.65–2.17,
0.5–2.11, 1.01–2.30 Å, indicating less fuctuations in protein
structure during simulation. During simulation the maxi-
mum RMSF in Cα, backbone and heavy atoms was observed
in amino acid stretch Ser44-Gln45 (3.61–3.18, 3.25–3.49
and 3.41–4.29 Å, respectively) present on loop (Asp39-
His48). The values for Cα, backbone and heavy atoms of
binding residues of catalytic pocket was found to be in the
range 0.68 to 1.27 and 0.69 to 1.28 Å, respectively (Fig. 4b).
This indicated no signifcant conformational changes in
these residues. The maximum RMSFs for Cα (2.38–3.17 Å)
and backbone (2.66–3.16 Å) atoms was observed in residues
The rGyr 2.21–3.84 Å of ligand (Supplementary Fig.
S12), which measures the extendedness, indicated the stabil-
ity of compound 4k within the catalytic pocket during 50 ns
MD simulation. The RMSD value of ligand fuctuated within
a narrow range of 2.21–3.84 Å during 25–50 ns of MD simu-
lation, further indicating stability and less conformational
fexibility of 4k. An increase in the solvent accessible sur-
face area (SASA) of inhibitor 4k was observed up to 25 ns
(96–300 Å2) which is then stabilized in the lower window
of 85.3–16.4 Å2 showing no signifcant change in the bind-
ing pocket volume during simulation. Further, polar surface
area (PSA) of 4k stabilized in the narrow range of 125–145
Å2 during last 25 ns of simulation trajectory, indicating the
burial of inhibitor within catalytic pocket. After superposi-
tion similar orientations was observed between conforma-
tions of 4k best docking pose and pose after MD simula-
tion with RMSD of 1.25 Å (Supplementary Fig. S13). This
1 3

Chemical Papers
Fig. 4 Plot represent (a) RMSD
(Å) of the simulated positions of
Cα, backbone and heavy atoms
of 4k/SaMurD complex protein
protein compared to the initial
structure. (b) RMSF profle of
active site residues of SaMurD
complex during MD simulation
of 4k/SaMurD complex (c) dif-
ferent interactions of compound
4k with active site residues of
SaMurD during 50 ns simula-
tion trajectory
1 3

Chemical Papers
indicated the validity and rationality of the extra-precision
docking model. In order to compare the binding modes, we
also performed a 50 ns MD simulation for 4j/SaMurD com-
plex using the protocols as described in experimental sec-
tion. It is evident from supplementary Fig. S14a that RMSDs
of protein Cα and backbone all atoms increased upto 6.25
and 6.24 Å, respectively during frst 12 ns of MD simula-
tion and then fuctuated, respectively in the range 5.54–3.22
and 5.57–3.23 Å during 12–23 ns of the simulation trajec-
tory. After 23 ns RMSDs of protein Cα and backbone all
atoms stabilized in the range 3.18–4.81 Å, indicating slightly
higher fuctuations compared to the fuctuations observed
for Cα and backbone atoms in case of 4k/SaMurD complex
simulation trajectory. During simulation Ala423 from C-ter-
minal domain showed medium frequency hydrogen bond-
ing interactions with both oxygen atoms of the sulphonyl
group (32 and 26% of MD trajectory) (Supplementary Fig.
S14b-d). One of the oxygen atoms of the sulphonyl group
exhibited a low frequency water mediated hydrogen bonding
interaction with Asp354. A strong frequency π-π stacking is
also observed between phenyl rings of the chlorophenoxy
moiety and Trp425. It is evident that interactions observed
in case of compound 4j are comparatively less compared to
4k and probably may be the reason for the lower inhibitory
activity of 4j against SaMurD protein.
bacteria compared to hydrazides 4a-i. The high activity of
sulfonylhydrazides may be attributed to the presence of -SO2
group in these compounds. The inhibitory activity of three
compounds 4d, 4j and 4k against SaMurD was performed
using the malachite green assay. Compound 4k exhibited
highest inhibitory activity with IC50 value of 35.80 µM,
while other two tested compounds 4d and 4j showed IC50
values of 37.82 and 98.66 μM. These three tested com-
pounds exhibited low inhibitory activity against SaMurD
enzyme, however the bacterial cell penetration of these
compounds is better compared to compound A as evident
by their MIC values against the tested strains of S. aureus.
Further molecular mechanism of binding of 4a-k with the
catalytic pocket residues of modelled SaMurD is investi-
gated with the extra-precision molecular docking and bind-
ing free energy calculation by MM-GBSA approach. The
van der Waals (ΔGvdW, − 28.53 to − 76.36 kcal/mol) and
electrostatic (ΔGCoul, − 12.42 to − 92.60 kcal/mol) interac-
tion energy terms are observed to be major contributors for
binding to the SaMurD enzyme. Further, the high negative
value of ΔGvdW indicated it as the driving force for binding.
50 ns molecular dynamics simulations were performed for 4j
and 4k/SaMurD complexes to validate the stability of their
docking poses. Similar orientations was observed between
conformations of 4k best docking pose and pose after MD
simulation indicating the validity and rationality of the extra-
precision docking model.
Conclusion
Acknowledgements We would like to thank the Science and Engineer-
ing Research Board (SERB), Government of India for the fnancial
support (No. EMR/2016/002981).
In the present investigation we synthesized a new series
of phenoxyacetohydrazide derivatives 4a-k and character-
ized them by spectral data. Synthesized compounds were
screened in vitro for their antibacterial activity against
Gram-positive S. aureus (NCIM 5021, NCIM 5022 and
methicillin-resistant isolate 43300), B. subtilis (NCIM 2545)
and Gram-negative E. coli (NCIM 2567), K. pneumoniae
(NCIM 2706) and P. aeruginosa (NCIM 2036) bacteria.
Tested compounds 4a, 4c, 4d, 4f, 4j and 4k displayed prom-
ising inhibitory activity against Gram-positive S. aureus
NCIM 5021 (MIC 32–64 µg/ml), while compounds 4a, 4j
and 4k exhibited inhibitory activity against S. aureus NCIM
5022 with MIC value of 64 µg/ml in each case. These com-
pounds also exhibited activity against methicillin resistant
S. aureus ATCC 43300 with MIC of 128 µg/ml. Among all
the tested compounds 4j showed maximum inhibitory activ-
ity against B. subtilis NCIM 2545 with MIC value 32 µg/
ml. Whereas compound 4c exhibited highest activity against
Gram-negative K. pneumoniae NCIM 2706 with MIC value
of 32 µg/ml. Only compound 4d showed activity against
another Gram-negative bacteria P. aeruginosa NCIM 2036
with MIC 64 µg/ml. Further, it is evident from result that
sulfonylhydrazides (4j and 4k) is more active against the
tested strains of both Gram-positive and Gram-negative
Compliance with ethical standards
Conflict of interest On behalf of all authors, the corresponding author
states that there is no confict of interest.
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Afliations
Srikanth Jupudi1 · Mohammed Afzal Azam1 · Ashish Wadhwani2
2
* Mohammed Afzal Azam
Department of Biotechnology, JSS College of Pharmacy (A
afzal9azam@hotmail.com; afzal@jssuni.edu.in
Constituent College of JSS Academy of Higher Education
and Research), Ooty, Tamil Nadu 643001, India
1
Department of Pharmaceutical Chemistry, JSS College
of Pharmacy, Ooty, Tamil Nadu 643001, India
1 3