Design, synthesis and docking studies of some novel isocoumarin analogues as antimicrobial agents

Article abstract of DOI:10.1039/c4ra07223e

A number of novel isocoumarin analogues have been synthesized by the condensation of homophthalic acid anhydride with different non-steroidal anti-inflammatory drugs (NSAIDs). To investigate the antimicrobial data on a structural basis, in silico docking studies of the synthesized compounds (4a-4g) into the crystal structure of UDP-N-acetylmuramate-l-alanine ligase using an Autodock PyRx virtual screening program were performed in order to predict the affinity and orientation of the synthesized compounds at the activities. UDP-N-acetylmuramate-l-alanine ligase is essential for d-glutamate metabolism and peptidoglycan biosynthesis in bacteria. R2 values showed good agreement with predicted binding affinities obtained by molecular docking studies. The results indicate that the basic nucleic portion of the (4c), (4g), (4f) and (4a) binds into the specificity pocket. In this pocket, the isocoumarin nucleus of these compounds interacts with the amino acid residue of the target. Moreover, it is verified by in vitro antimicrobial screening, in which all of the compounds were active against tested bacterial strains. Among these compounds (4c), (4g), (4f) and (4a) showed good bacterial zone inhibition. This journal is

Full text of DOI:10.1039/c4ra07223e

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Design, synthesis and docking studies of some
novel isocoumarin analogues as antimicrobial
agents
Cite this: RSC Adv., 2014, 4, 53842
Zaman Ashraf,*ab Aamer Saeed and Humaira Nadeemd
c
A number of novel isocoumarin analogues have been synthesized by the condensation of homophthalic
acid anhydride with different non-steroidal anti-inflammatory drugs (NSAIDs). To investigate the
antimicrobial data on a structural basis, in silico docking studies of the synthesized compounds (4a–4g)
into the crystal structure of UDP-N-acetylmuramate-L-alanine ligase using an Autodock PyRx virtual
screening program were performed in order to predict the affinity and orientation of the synthesized
compounds at the activities. UDP-N-acetylmuramate-L-alanine ligase is essential for D-glutamate
metabolism and peptidoglycan biosynthesis in bacteria. R2 values showed good agreement with
predicted binding affinities obtained by molecular docking studies. The results indicate that the basic
nucleic portion of the (4c), (4g), (4f) and (4a) binds into the specificity pocket. In this pocket, the
isocoumarin nucleus of these compounds interacts with the amino acid residue of the target. Moreover,
it is verified by in vitro antimicrobial screening, in which all of the compounds were active against tested
bacterial strains. Among these compounds (4c), (4g), (4f) and (4a) showed good bacterial zone inhibition.
Received 17th July 2014
Accepted 29th September 2014
DOI: 10.1039/c4ra07223e
www.rsc.org/advances
Molecular docking plays an important role in the rational
design of drugs. In the eld of molecular modeling, docking is a
Introduction
Isocoumarins and 3,4-dihydroisocoumarins (3,4-dihydro-1H-2- method that predicts the preferred orientation of one molecule
benzopyran-ones) are natural lactones, which are isolated from to another when bound to each other to form a stable complex.
a wide range of natural sources (microbes, plants and insects), Molecular docking can be dened as an optimization problem
and encompass an array of biological activities, including that would describe the “best-t” orientation of a ligand that
13,14
nephrotoxic, hepatotoxic, cytotoxic, immunomodulatory, algi- binds to a particular protein of interest.
3-Substituted iso-
cidal, gastroprotective, protease inhibition, antifungal and coumarins exhibited valuable pharmacological activities;
1
–6
15–17
antimalarial activities. They also display anti-inammatory, therefore, in continuation of our work,
efforts have been
7–9
antiangiogenic and antiallergic activities. Isocoumarins are directed towards the synthesis of new isocoumarin analogues as
also used as a lead compound for the identication of insecti- excellent antimicrobial agents.
10
cides that selectively bind at the insect GABA receptor.
-Substituted isocoumarins also exhibit anti HIV activity in
3
vitro, have diuretic, antihypertensive, antiarrhythmics, b-
a
Table 1 Antibacterial activity results of isocoumarins (4a–g)
sympatholytics, anticorrosive, laxative, asthmolytic, phytotoxic
11
properties, and are useful in the treatment of emphysema.
Isocoumarin derivatives are potential inhibitors of endothelial
cell proliferation, migration, sprouting, tube formation in vitro, 4a
Codes
P.m. B.s. E.c. S.a. P.p. P.a. S.t. M.l. S. f. K.p.
23
10
24
—
06
24
26
11
08
—
—
05
16
18
20
20
13
26
—
11
18
19
30
15
07
—
11
—
17
20
25
19
—
26
—
—
23
26
30
16
—
19
—
07
21
20
28
19 20
14
—
24
05
13
26
19
30
17
02
17
—
06
20
22
25
12
4
4
4
4
4
b
c
d
e
f
—
25
—
04
—
07
and tumor growth in vivo.
10 12
25 19
24 16
30 25
a
Department of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan.
E-mail: mzchem@yahoo.com; Fax: +92 512891471; Tel: +92 3215194461; +92
4g
Standard 30
9
057182
a
Activity is presented in millimeter (mm), (—) no activity Proteus
mirabilis (P.m.), Bacillus subtilis (B.s.), Escherichia coli (E.c.),
Staphylococcus aureus (S.a.), Pseudomonas putida (P p.), Pseudomonas
aeruginosa (P.a.), Salmonella typhi (S.t.), Micrococcus luteus (M.l.),
Shigella exineri (S.f.) and Klebsiella pneumonae (K.p.).
b
Department of Biology, College of Natural Sciences, Kongju National University,
Kongju 314-701, South Korea
c
Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
d
Riphah Institute of Pharmaceutical Sciences, Riphah International University,
Islamabad, 44000, Pakistan
53842 | RSC Adv., 2014, 4, 53842–53853
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Table 2 AutoGrid calculations of around 8043 receptor atoms of UDP-N-acetylmuramate-L-alanine ligase with minimum and maximum
interacting energies
Minimum energy
ꢀ
1
ꢀ1
Ligand codes
Grid map
Atom type
(kcal mol
)
Maximum energy (kcal mol
)
Setting parameters in PyRx
5
4
c
1
2
3
4
1
2
3
4
5
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
7
1
2
3
4
1
2
3
4
A
OA
e
d
A
OA
F
e
d
A
Cl
OA
N
e
d
A
HD
OA
N
e
d
A
Cl
HD
OA
N
e
d
A
OA
e
d
ꢀ0.88
ꢀ1.78
ꢀ32.71
0.00
ꢀ0.88
ꢀ1.78
ꢀ0.66
ꢀ32.71
0.00
ꢀ0.88
ꢀ1.29
ꢀ1.78
ꢀ1.04
ꢀ32.71
0.00
ꢀ0.88
ꢀ0.72
ꢀ1.78
ꢀ1.04
ꢀ32.71
0.00
ꢀ0.88
ꢀ1.29
ꢀ0.72
ꢀ1.78
ꢀ1.04
ꢀ32.71
0.00
ꢀ0.88
ꢀ1.78
ꢀ32.71
0.00
2.03 ꢁ 10
—
—
5
2.00 ꢁ 10
1
3.33 ꢁ 10
Electrostatic potential
Desolvation potential
—
—
0
1.45 ꢁ 10
5
4
b
2.03 ꢁ 10
5
2.00 ꢁ 10
5
2.00 ꢁ 10
—
1
3.33 ꢁ 10
Electrostatic potential
Desolvation potential
0
1.45 ꢁ 10
5
4
4
4
g
f
2.03 ꢁ 10
5
2.04 ꢁ 10
5
2.00 ꢁ 10
5
2.01 ꢁ 10
1
3.33 ꢁ 10
Electrostatic potential
Desolvation potential
0
1.45 ꢁ 10
5
2.03 ꢁ 10
5
1.13 ꢁ 10
5
2.00 ꢁ 10
5
2.01 ꢁ 10
1
3.33 ꢁ 10
Electrostatic potential
Desolvation potential
0
1.45 ꢁ 10
5
e
2.03 ꢁ 10
5
2.04 ꢁ 10
5
1.13 ꢁ 10
5
2.00 ꢁ 10
5
2.01 ꢁ 10
1
3.33 ꢁ 10
Electrostatic potential
Desolvation potential
0
1.45 ꢁ 10
5
4
4
a
2.03 ꢁ 10
5
2.00 ꢁ 10
1
3.33 ꢁ 10
Electrostatic potential
Desolvation potential
0
1.45 ꢁ 10
5
d
A
OA
e
ꢀ0.88
ꢀ1.78
ꢀ32.71
0.00
2.03 ꢁ 10
5
2.00 ꢁ 10
1
3.33 ꢁ 10
Electrostatic potential
Desolvation potential
0
d
1.45 ꢁ 10
Peptidoglycans are the main constituents of the bacterial cell
In this paper, we report the design, synthesis, antimicrobial
wall, which imparts the structural strength to the bacterial cell activity and docking studies of novel isocoumarin derivatives,
wall and performs different functions as it counteracts the planned as UDP-N-acetylmuramyl-L-alanine ligase (MurC)
osmotic pressure of the cytoplasm. UDP-N-acetylmuramate-L- inhibitor candidates. The synthesis of the nal compounds was
0
0
alanine ligase, also known as MurC synthetase, is essential, and accomplished by using the bases N,N,N ,N -tetramethylguani-
this unique enzyme participates in D-glutamine and D-glutamate dine (TMG) and triethylamine with high yield. In silico docking
metabolism and intracellular pathway of bacterial peptido- studies of the synthesized compounds into the crystal structure
18
glycan biosynthesis. The MurC synthetase with D-alanine- of MurC synthetase were performed using an Autodock PyRx
D-alanine ligase (EC 6.3.2.4), UDP-N-acetylmuramoyl-L-alanyl- virtual screening program. The synthesized compounds were
D-glutamate-L-lysine ligase (EC 6.3.2.7) or UDP-N-acetylmur- then tested against ten different Gram positive and Gram
amoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase (EC negative bacteria.
6.3.2.13), UDP-N-acetylmuramoyl-L-alanine-D-glutamate ligase
(EC 6.3.2.9) and UDP-N-acetylmuramoyl-tripeptide-D-alanyl-
D-alanine ligase (EC 6.3.2.10) are involved in the synthesis of
bacterial cell-wall peptide. In 2004, Ehmann et al. reported that
UDP-N-acetylmuramyl-L-alanine ligase (MurC) is an important
target for the discovery of novel antibacterial agents as it
Experimental
Melting points were recorded using a digital Gallenkamp
1
(
SANYO) model MPD BM 3.5 apparatus and are uncorrected. H
1
3
19
NMR and the C NMR spectra were determined using CDCl3
solutions at 300 MHz and 100 MHz, respectively, on a Bruker
AM-300 machine. FTIR spectra were recorded using an FTS 3000
involved in peptidoglycan biosynthesis.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 53842–53853 | 53843

View Article Online
RSC Advances
Paper
Fig. 1 Amino acid residues on UDP-N-acetylmuramate-L-alanine ligase with shaded receptor binding cavities defined by using Discovery Studio
.0 Visualizer (PDB ID: 1GQQ).
3
Fig. 2 The Ramachandran plot indicates low energy conformations for f (phi) and j (psi), providing the graphical representation of the local
backbone conformation of each residue of our target protein. Points on plot represent the f and j torsion angles of a residue. They also
represent the favorable and unfavorable regions for residues.
53844 | RSC Adv., 2014, 4, 53842–53853
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Scheme 1 Synthesis of 3-alkyl/aryl substituted isocoumarins (4a–g).
Fig. 3 Complexity of protein–ligand interactions. The figure shows a schematic illustration of various interaction components that need to be
considered to predict the structure and binding energetics of two compounds within the active site, in this case, the UDP-N-acetylmuramate-L-
alanine ligase (MurC) (PDB code: 1GQQ) with isocoumarin 4a.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 53842–53853 | 53845

View Article Online
RSC Advances
Paper
MX spectrophotometer; mass spectra (EI, 70 eV) were obtained General procedure for 3-alkyl/aryl isocoumarins (4a–g)
on a GC-MS instrument and elemental analyses were performed
with a LECO-183 CHNS analyzer. The analytical TLC was carried
out using recoated plates from Merck and thick layer chroma-
tography using silica gel from Merck.
A mixture of aliphatic/aromatic carboxylic acids (2a–j) (1 mmol)
and thionyl chloride (1.2 mmol) was reuxed for 1 h in the
presence of a drop of DMF. The completion of the reaction was
determined by the stoppage of the evolution of gas. Excess of
thionyl chloride was rotary evaporated to afford acid chlorides
(3a–j).
Synthesis of homophthalic acid anhydride (1)
A solution of homophthalic acid anhydride (1) (2.00 mmol)
in acetonitrile (12 mL) was added to a solution of N,N,N ,N -
0
0
A solution of homophthalic acid (2.0 g, 12.34 mmol) in dry
toluene (35 mL) was treated with acetic anhydride (1.1 g, 10
mL, 10.8 mmol). The reaction mixture was reuxed for 1 h and
then poured into ice cold water. The organic layer was sepa-
rated, dried over anhydrous sodium sulfate and toluene was
rotary evaporated to obtain homophthalic acid anhydride (1).
tetramethylguanidine (TMG) (2.20 mmol) in acetonitrile (5 mL)
ꢂ
over 36 min maintaining an internal temperature of 0 C. Tri-
ethylamine (4.0 mmol) was added in one portion. Acid chlorides
3a–j) (3.20 mmol) were added over 3 min, and the mixture was
(
stirred for an additional 20 min. Aer the completion of the
reaction, the cooling bath was removed, and the reaction was
allowed to warm to room temperature. The reaction mixture was
quenched by the addition of HCl (1 M, 5 mL). The two phases
were separated, and the organic layer was washed with satu-
rated sodium chloride solution, and then dried (Na SO ) prior
Yield 82%; R
f
: 0.7 (petroleum ether and ethyl acetate, 4 : 1); m.
ꢂ
p. 140–142 C; IR (KBr): 3011 (C–H), 1735 (C]O), 1590 (C]C)
ꢀ
1 1
cm ; H NMR (CDCl , d ppm): 7.85 (1H, d, J ¼ 3.7 Hz, H-8),
3
7
.3–7.4 (2H, m, H-6, H-7), 6.97 (1H, d, J ¼ 3.4 Hz, H-5), 3.47
13
2
4
(2H, s, H-4); C NMR (CDCl
3
, d ppm): 165.5 (C3), 147.1 (C1),
to the removal of the solvent under reduced pressure to dryness.
Isocoumarins (4a–j) were then puried by preparative thin-layer
chromatography using petroleum ether and ethyl acetate (7 : 3)
as an eluent.
1
1
37.2 (C4a), 134.4 (C6), 131.5 (C8a), 130.7 (C8), 129.7 (C5),
+
27.5 (C7), 38.2 (C4); MS (70 eV): m/z (%); 162 [M ] (25), 134
: C, 66.66 H, 3.70;
(43), 118 (100), 90 (32); anal. calcd for C
9
H
6
O
3
found: C, 66.53 H, 3.59.
Fig. 4 Complexity of protein–ligand interactions. The figure shows a schematic illustration of various interaction components that need to be
considered to predict the structure and binding energetics of two compounds within the active site, in this case, the UDP-N-acetylmuramate-L-
alanine ligase (MurC) (PDB code: 1GQQ) with isocoumarin 4b.
53846 | RSC Adv., 2014, 4, 53842–53853
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Fig. 5 Complexity of protein–ligand interactions. The figure shows a schematic illustration of various interaction components that need to be
considered to predict the structure and binding energetics of two compounds within the active site, in this case, the UDP-N-acetylmuramate-L-
alanine ligase (MurC) (PDB code: 1GQQ) with isocoumarin 4c.
0
00
0
00
00
3
-[(S)-1 -(4 -Isobutylphenyl)ethyl]isocoumarin (4a). Yield
3-[1 -(5 -Methoxy-2 -naphthyl)ethyl]isocoumarin (4c). Yield
ꢀ
1
1
ꢂ
7
5%; light yellow oil; IR (neat): n ¼ 1710, 1597, 1506 cm ; H 62%; m.p. 35 C; IR (KBr): n ¼ 1705 (C]O, lactonic), 1645, 1601,
ꢀ
1 1
NMR (CDCl
3
): d 0.89 (6H, d, J ¼ 6.6 Hz, 2CH
3
of isobutyl), 1.64 1498 cm ; H NMR (CDCl
3
): d 1.57 (3H, d, J ¼ 7.2 Hz, CH
3
), 3.90
0
0
(
3H, d, J ¼ 7.2 Hz, H-2 ), 1.85 (1H, m, CH of isobutyl), 2.45 (2H, (3H, s, OCH ), 4.13 (1H, q, J ¼ 7.2 Hz, H-1 ), 6.75 (1H, s, H-4),
3
0
0
0
0
0
0
0
d, J ¼ 7.1 Hz, CH of isobutyl), 3.92 (1H, q, J ¼ 7.2 Hz, H-1 ), 6.25 6.81–6.89 (2H, m, H-5 , H-7 ), 6.95–7.18 (4H, m, H-1 , 3 , 4 , 8 ),
2
0
0
00
(
8
8
1H, s, H-4), 7.10 (2H, d, J ¼ 8.1 Hz, H-3 , H-5 ), 7.23 (2H; d, J ¼ 7.24 (1H, d, J ¼ 7.5 Hz, H-5), 7.34 (1H, dd, J ¼ 7.4, 1.3 Hz, H-7),
00
00
.1 Hz, H-2 , H-6 ), 7.33 (1H, d, J ¼ 7.9 Hz, H-5), 7.43 (1H, dt, J ¼ 7.41 (1H, dd, J ¼ 7.3, 1.9 Hz, H-6), 8.01 (1H, d, J ¼ 7.6 Hz, H-8);
+
.1, 1.0 Hz, H-7), 7.64 (1H, dt, J ¼ 7.8, 1.3 Hz, H-6), 8.23 (1H, d, EIMS: m/z (%) ¼ 330 (8.4) [M ], 300 (37.1), 213 (6.5), 185 (100),
+
J ¼ 7.7 Hz, H-8); EIMS: m/z (%) ¼ 306 (100) [M ], 264 (53.2), 263 173 (15.5), 157 (17.2), 155 (17.8), 145 (5.9), 117 (9.8), 89 (26.2);
(
(
88.2), 249 (57.1), 235 (23.3), 189 (7.4), 161 (27.5), 145 (94.1), 119 HRMS: 330.1245 (calcd C22
H
18
O
3
, 330.1256).
0
00
23.2), 117 (78.2), 89 (92.9); HRMS: 306.1625 (calcd for
3-[1 -(4 -Isobutylphenyl)ethyl]isocoumarin (4d). Yield 65%;
ꢀ
1 1
C H O , 306.1620).
light yellow oil; IR (neat): n ¼ 1710, 1597, 1506 cm ; H NMR
2
1
22 2
0
00
00
3
-[1 -(3 -Fluoro-4 -biphenyl)ethyl]isocoumarin (4b). Yield (CDCl ): d 0.89 (6H, d, J ¼ 6.6 Hz, 2CH of isobutyl), 1.64 (3H, d, J
3
3
ꢀ1
0
6
8%; light yellow oil; IR (neat): n ¼ 1723, 1647, 1617, 1580 cm
;
¼ 7.2 Hz, H-2 ), 1.85 (1H, m, CH of isobutyl), 2.45 (2H, d, J ¼ 7.1
1
0
H NMR (CDCl
3
): d 1.27 (3H, d, J ¼ 7.5 Hz, CH
3
), 3.69 (1H, q, J ¼ Hz, CH
2
of isobutyl), 3.92 (1H, q, J ¼ 7.2 Hz, H-1 ), 6.25 (1H, s, H-
0
0
0
00
00
7
¼
.5 Hz, H-1 ), 6.70 (1H, s, H-4), 6.93 (2H, m, H-2 , 6 ), 7.1 (1H, d, J 4), 7.10 (2H, d, J ¼ 8.1 Hz, H-3 ,H-5 ), 7.23 (2H, d, J ¼ 8.1 Hz, H-
00
0
00
00
00
00
00
00
8.1 Hz, H-4 ), 7.30–7.41 (5H, m, H-5 , 2 , 3 , 5 , 6 ), 7.49 (1H, 2 , H-6 ), 7.33 (1H, d, J ¼ 7.9 Hz, H-5), 7.43 (1H, dt, J ¼ 8.1, 1.0
dt, J ¼ 7.6, 1.2 Hz, H-7), 7.55 (2H, m, H-5, H-6), 8.10 (1H, d, J ¼ Hz, H-7), 7.64 (1H, dt, J ¼ 7.8, 1.3 Hz, H-6), 8.23 (1H, d, J ¼ 7.7
+
+
7
.6 Hz, H-8); EIMS: m/z (%) ¼ 344 (15.4) [M ], 329 (15.2), 325 Hz, H-8); EIMS: m/z (%) ¼ 306 (100) [M ], 264 (53.2), 263 (88.2),
(
(
(
2.6), 301 (10.2), 267 (7.1), 227 (5.1), 199 (100), 173 (5.3), 171 249 (57.1), 235 (23.3), 189 (7.4), 161 (27.5), 145 (94.1), 119 (23.2),
7.5), 155 (9.5), 145 (20.1), 117 (9.8), 89 (26.2); HRMS: 344.1221 117 (78.2), 89 (92.9); HRMS: 306.1625 (calcd for C H O ,
2
1
22 2
calcd for C H FO , 344.1213).
306.1620).
2
3
17
2
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 53842–53853 | 53847

View Article Online
RSC Advances
Paper
Fig. 6 Complexity of protein–ligand interactions. The figure shows a schematic illustration of various interaction components that need to be
considered to predict the structure and binding energetics of two compounds within the active site, in this case, the UDP-N-acetylmuramate-L-
alanine ligase (MurC) (PDB code: 1GQQ) with isocoumarin 4d.
0
00 00
+
3
-[2 -(2 ,6 -Dichlorophenylamino)benzyl]isocoumarin (4e). Hz, H-8); EIMS: m/z (%) ¼ 460 (25) [M + 2], 458 (100) [M ], 265
ꢂ
Yield 65%; m.p. 52 C; IR (KBr): n ¼ 3421, 1719, 1607, 1523 (34.2), 145 (62.5), 119 (21.1), 117 (92.2), 89 (54.4). HRMS:
ꢀ
1 1
4
cm ; H NMR (CDCl ): d 2.09 (2H, s, CH ), 4.52 (1H, s, –NH), 457.9050 (calcd for C27H20ClNO , 457.9062).
3
2
0
6
4
.35 (1H, s, H-4), 6.41 (1H, d, J ¼ 6.5 Hz, H-3 ), 6.5–6.7 (3H, m, H-
0
0
00
–H-6 ), 6.81 (1H, dd, J ¼ 7.2, 6.8 Hz, H-4 ), 7.95 (2H, d, J ¼ 7.8 Antibacterial activity
00
00
Hz, H-3 , H-5 ), 7.13 (1H; d, J ¼ 8.1 Hz, H-5), 7.3–7.4 (2H, m, H-
, H-7), 7.43 (1H, d, J ¼ 7.4 Hz, H-8); EIMS: m/z (%) ¼ 396 (100)
In vitro evaluation of antibacterial activity of the 3-substituted
isocoumarins (4a–g) was carried out by agar well diffusion assay
against ten different Gram positive and Gram negative bacteria,
of which seven were gram negative, i.e. Proteus mirabilis (ATCC
9565), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa
ATCC 33347), Pseudomonas putida (ATCC 47054), Salmonalla
typhi (ATCC 19430), Shigella exineri (ATCC 25929) and Klebsi-
ella pneumoniae (ATCC 43816), and three were Gram positive,
i.e. Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC
9213) and Micrococcus luteus (ATCC 9341). Antibacterial
activity was determined by using the Mueller Hinton Agar
MHA). The fresh inoculums of these bacteria were prepared
and diluted by sterilized normal saline. The turbidity of these
cultures was adjusted by using 0.5 Mc-Farland. A homogeneous
bacterial lawn was developed by sterile cotton swabs. The
inoculated plates were bored by a 6 mm-sized borer to make the
wells. The sample dilutions were prepared by dissolving each
sample (1.0 mg) in 1.0 mL of DMSO used as a negative control in
this bioassay. The equimolar concentration of Levooxacin
1.0 mg mL ), a broad spectrum antibiotic (positive control),
was prepared. These plates were incubated at 37 C for 24 hours.
6
[
(
+
M ], 265 (44.5), 251 (33.2), 235 (23.3), 189 (7.4), 161 (27.5), 145
82.1), 119 (19.2), 117 (78.2), 89 (92.9). HRMS: 396.2678 (calcd
for C22
H
15Cl
2
NO
2
, 396.2669).
4
0
00 00
3
-[2 -(2 ,3 -Dimethylphenylamino)phenyl]isocoumarin (4f).
(
ꢂ
Yield 73%; m.p. 45 C; IR (KBr): n ¼ 3354, 1731, 1586, 1503
ꢀ
1 1
cm ; H NMR (CDCl
3
): d 2.27 (3H, s, CH
3 3
), 2.34 (3H, s, CH ),
0
0
00
4
6
(
6
(
.52 (1H, s, –NH), 6.41 (1H, s, H-4), 6.5–6.6 (3H, m, H-4 –H-6 ),
0
0
0
.65 (1H, d, J ¼ 7.2 Hz, H-3 ), 6.7–6.9 (3H, m, H-4 –H-6 ), 7.15
20
2
1H, d, J ¼ 6.5 Hz, H-5), 7.3–7.5(2H, m, H-6, H-7), 7.63 (1H, d, J ¼
+
.2 Hz, H-8); EIMS: m/z (%) ¼ 341 (100) [M ], 265 (44.5), 251
33.2), 235 (23.3), 145 (74.5), 119 (25.2), 117 (81.6), 89 (76.8).
HRMS: 341.4024 (calcd for C H NO , 341.4036).
(
2
3
19
2
0
00
00
00
3
-[(1 -(4%-Chlorophenyl)-5 -methoxy-2 -methyl-1H-indol-3 -
yl)methyl]isocoumarin (4g). Yield 53%; m.p. 140 C; IR (KBr): n
1725, 1645, 1573, 1503 cm ; H NMR (CDCl
CH ), 2.76 (2H, s, CH ), 3.85 (3H, s, OCH ), 6.25 (1H, s, H-4 ),
ꢂ
ꢀ1 1
¼
3
): d 2.31 (3H, s,
0
0
3
2
3
0
0
00
6
.45 (1H, s, H-4), 6.5–6.6 (2H, m, H-6 –H-7 ), 7.15 (1H, d, J ¼ 7.7
Hz, H-5), 7.3–7.5(2H, m, H-6, H-7), 7.65 (2H, d, J ¼ 8.5 Hz, H-3%,
H-5%), 7.71 (2H, d, J ¼ 6.3 Hz, H-2%, H-6%), 7.78 (1H, d, J ¼ 7.6
ꢀ1
(
ꢂ
53848 | RSC Adv., 2014, 4, 53842–53853
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Fig. 7 Complexity of protein–ligand interactions. The figure shows a schematic illustration of various interaction components that need to be
considered to predict the structure and binding energetics of two compounds within the active site, in this case, the UDP-N-acetylmuramate-L-
alanine ligase (MurC) (PDB code: 1GQQ) with isocoumarin 4e.
The antibacterial activity of these three series of compounds from the coordinates of the ligand in the original target protein
was determined by measuring the diameter of the zone of sites. The active recognition site of the ensemble has been
˚
inhibition (mm, ꢃstandard deviation) and presented by sub- dened as the collection of residues within 25.0 A of the bound
tracting the activity of the negative control (Table 1).
inhibitor, and it comprised the union of all ligands of the
˚
ensemble. All atoms located less than 25.0 A from any ligand
atom were identied and were considered as active site resi-
dues. The amino acid residues on the MurC synthetase with
Docking studies
Preparation of the ligands. The MOL SDF formats of all shaded receptor binding cavities dened by using Discovery
ligands were prepared and were further translated to the PDBQT Studio 3.0 Visualizer (PDB ID: 1GQQ) Analysis of Protein Model

le using a PyRx virtual screening tool to generate the 3D atomic and AutoGrid of target protein exhibiting active ligand binding
coordinates of a molecule. Discovery Studio 3.0 visualizer was sites by using AutoDock Vina are shown in Fig. 1.
used to label the atoms of the molecule. Finally, these ligand
Next, the biopolymer protein analysis tool was used in a
models were evaluated for the docking procedure. The AutoGrid stepwise process of analysis and correction of geometric
dimensions between ligands and enzymes are Grid Center X: parameters. For each structure, the description of an ensemble
4
5
8.6290, Y: 23.4707, Z: 43.7046 with total number of points X: contains the denition of the protein atoms, the resolution of
0, Y: 50, Z: 50 and spacing (Angstrom): 0.3750. The compounds ambiguities in the PDF le, the location of hydrogen atoms at
were then tested for Lipinski's rule of 5 using the Molinspira- hetero atoms, and the denition of the active site atoms. The
tion server (http://www.molinspiration.com). Table 2 lists the assignment of hydrogen positions has been made on the basis
structural properties of the isocoumarin derivatives (4a–g).
of default rules, except for the denition of the hydrogen posi-
Accession of the target protein. The crystallographic 3D tions inside the histidine side-chain. Water molecules con-
structure of UDP-N-acetylmuramate-L-alanine ligase was tained in the PDF le have been removed. The hydrophobicity
accessed from the Protein Data Bank (PDB ID: 1GQQ). The plot of MurC synthetase was generated by using Discovery
˚
Studio 3.0 Visualizer. The Ramachandran plot indicates low
resolution of the XRD structure of this model enzyme is 3.10 A
˚
ꢂ
energy conformations for f (phi) and j (psi), providing the
with an R-value of 0.241 (obs.). The length (A) and angle ( )
properties of the structure are a ¼ 65.51, b ¼ 99.49, c ¼ 180.59 graphical representation of the local backbone conformation of
and a ¼ 90.00, b ¼ 90.00, g ¼ 90.00. The active site was dened each residue of our target protein (Fig. 2). Points on the plot
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 53842–53853 | 53849

View Article Online
RSC Advances
Paper
Fig. 8 Complexity of protein–ligand interactions. The figure shows a schematic illustration of various interaction components that need to be
considered to predict the structure and binding energetics of two compounds within the active site, in this case, the UDP-N-acetylmuramate-L-
alanine ligase (MurC) (PDB code: 1GQQ) with isocoumarin 4f.
represent the f and j torsion angles of a residue. It is also based on scoring functions. An exhaustive search was per-
representative of the favorable and unfavorable regions for the formed by enabling the “Run AutoGrid” option and then the
residues.
“Run AutoDock” option in the control panel by selecting the
Lipinski's rule of ve. Lipinski's rule of ve was applied to Lamarckian GA docking algorithm. In this approach, the ligand
evaluate in vivo absorption capabilities of the designed mole- performs a random walk around the static protein. The energy
cules. Any of the newly synthesized compounds, if they satisfy of interaction of this single atom with the protein is assigned to
the rule of ve, have good absorption when administered orally. the grid point. Interaction energies are calculated with a free-
A molecule having a molecular mass of less than 500, hydrogen energy based expression comprising terms for dispersion/
bond donors (–OH, NH) fewer than ve, hydrogen bond repulsion energy and directional hydrogen bonding. At each
acceptors (N, O) fewer than 10 and a calculated log P of less than step of the simulation, the energy of the interaction of ligand

ve satises the rule of ve. This principle has been extensively and protein is evaluated using atomic affinity potentials
employed on newly synthesized compounds for their further computed on a grid. The receptor coordinates (maximum and
use as drug candidates. The results of the calculations for the minimum) t within the following volume: 90.031, 55.286,
molecules designed in this study show that all molecules have a 102.112 and 8.870, ꢀ3.517, ꢀ18.845, respectively. The A and OA
potential for good in vivo absorption except for (4b) and (4e). are the ligand atom types docked and involved in interaction
The log P values of compounds (4b) and (4e) are 5.88 and 7.22, with A C NA OA N SA HD target atom types.
respectively, which are beyond the limits of the partition
coefficient.
Docking run. In order to understand the structural basis of
UDP-N-acetylmuramate-L-alanine ligase specicity, structural
Results and discussion
The aforementioned compounds (4a–g) were obtained accord-
ing to the synthetic route showed in Scheme 1. Homophthalic
acid was treated with acetic anhydride to afford its anhydride
complexes of this target enzyme with probable synthetic
inhibitors are determined using a computational docking
approach. The AutoDock (PyRx) suite of programs is used to
determine the binding modes of the synthetic inhibitors.
Binding sites and docking runs of target proteins with ligands
were analyzed by using the PyRx and AutoDock Vina options
(1). The acid chlorides of different non-steroidal anti-
inammatory drugs (NSAIDs) (3a–g) were synthesized by react-
ing them with thionyl chloride in the presence of dry benzene as
a solvent. These acid chlorides were then condensed with
53850 | RSC Adv., 2014, 4, 53842–53853
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Fig. 9 Complexity of protein–ligand interactions. The figure shows a schematic illustration of various interaction components that need to be
considered to predict the structure and binding energetics of two compounds within the active site, in this case, the UDP-N-acetylmuramate-L-
alanine ligase (MurC) (PDB code: 1GQQ) with isocoumarin 4g.
homophthalic acid anhydride (1), yielding the target iso- (4c), which may also determine its greater potential against
1
coumarins (4a–g) in good yield. The H NMR spectra of these gram negative bacteria. The compound (4g) displayed excellent
isocoumarins showed a characteristic signal for the H-4 proton antibacterial activity compared to all other derivatives. It
as the singlet in the region between 6.3 and 6.8 ppm. The mass possesses N-substituted indole scaffolds, which play very
spectral results also conrmed the formation of the nal important roles in bacterial growth inhibition. Further studies
compounds.
The in vitro antibacterial activity of synthesized compounds examine the use of these compounds as therapeutic agents.
was performed against ten different gram positive and gram In an attempt to theoretically explain the difference found in
of these derivatives are required, which will enable us to
negative bacteria to evaluate their potential against these the antibacterial activity of the isocoumarins (4a–g), docking
strains. All of the selected bacterial strains in this study are studies using the enzyme MurC synthetase were performed. The
pathogenic. The antibacterial results show that all of the molecular construction and the docking analysis of the
synthesized compounds afforded antibacterial activity with the
greatest inhibition of bacterial growth produced by compounds
(
4c), (4g), (4f) and (4a). The preliminary ndings suggested that
Table 3 Calculation of root mean square deviation (RMSD) using the
AutoDock Vina option of PyRx tool (total runs ¼ 9)
the derivative (4c) exhibited more potential against gram
negative than Gram positive bacteria. The basic nucleus of
isocoumarin remains the same in all of the derivatives, but they
differ as they have different steric bulk than that of the
substituent present at position 3. This is the factor due to which
these compounds have different hydrophobicity and
absorption.
Ligand–target
Ligand code complex
Binding affinity RMSD/ub RMSD/ub
4
4
4c
a
b
1GQQ_4a
1GQQ_4b
1GQQ_4c
1GQQ_4d
1GQQ_4e
1GQQ_4f
1GQQ_4g
ꢀ11.9
ꢀ12.7
ꢀ12
4.273
4.948
3.456
1.081
4.789
4.698
4.772
0.668
1.309
2.834
0.814
1.716
1.36
The derivative (4c) have methoxy-substituted naphthyl 4d
ꢀ5.1
4
4
4
e
f
g
ꢀ13.1
ꢀ12.9
ꢀ13.8
moiety at position 3 of the isocoumarin nucleus, which selec-
tively target Gram negative bacteria. The naphthyl group in this
case increases the hydrophobic character of the isocoumarin
1.98
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 53842–53853 | 53851

View Article Online
RSC Advances
Paper
Table 4 Interacting energy obtained during the docking of the compounds 4a–g (total runs ¼ 10)
Ligand–target
complex
Binding
energy
Intermolecular
energy
Torsional
energy
Ligand code
Internal energy
Unbound energy
4
4
4
4
4
4
4
a
b
c
d
e
f
1GQQ_4a
1GQQ_4b
1GQQ_4c
1GQQ_4d
1GQQ_4e
1GQQ_4f
1GQQ_4g
ꢀ10.33
ꢀ10.52
ꢀ10.05
ꢀ10.31
ꢀ11.09
ꢀ10.63
ꢀ12.26
ꢀ10.92
ꢀ11.42
ꢀ10.95
ꢀ10.91
ꢀ11.99
ꢀ11.53
ꢀ13.75
0
0
0
0
0
0
0
0.6
0
0
0
0
0
0
0
0.89
0.89
0.6
0.89
0.89
1.49
g
isocoumarins were accomplished as described in the subse-
The binding energies of the ligand target complexes during
quent section. Lipinski's rule of ve also veried drug-likeness docking were calculated and presented in Table 4. The higher
properties of the compounds (4c), (4g), (4f) and (4a) because value depicted the stable complex formed between the drug and
their log P values are below 5, whereas the compounds (4b), (4e) macromolecule. The value of the binding energy of the deriva-
and (4d) have log P values beyond partition coefficient limits. tive (4g) calculated during docking studies was ꢀ12.26, whereas
The protein–ligand interaction score values were simulta- the binding energies of the other derivatives (4a), (4c) and (4f)
neously obtained by using the PyRx AutoDock Vina and Auto- were ꢀ10.33, ꢀ10.05 and ꢀ10.63, respectively. These values
Grid options, the binding active sites and docked poses veried that most stable drug receptor complex was formed by
obtained were visualized and have been shown in Fig. 3–9.
compound (4g) with target protein. The isocoumarin nucleus
These inhibitors were docked with MurC synthetase using with nitrogen-substituted indole moiety in compound (4g)
our AutoDock procedure. All of the computationally predicted interacts with the LYS129, ALA172, VAL124 and GLY125 amino
lowest energy complexes of MurC are stabilized by the inter- acid residues of the target protein. The docking results also
molecular hydrogen bonds and stacking interactions. In these support our ndings.
computed complexes of MurC, the specicity pocket residues
LEU182, PHE181, GLY125, THR131, ASN193, PHE327, GLU195,
ASP345, LYS129, ALA172, VAL124, MET194, ASN193, LEU111, Conclusion
THR133 and THR148, ILU114, GLU113, ARG107, MET209,
Isocoumarin
derivatives
bearing
non-steroidal
anti-
LYS215, VAL412, LYS208, PHE117, GLU113, ALA174, and
ASP170 of MurC are involved in hydrogen bonding with the
bound inhibitors.
The interactions in these complexes vary depending on the
size, linkage and the functional groups. These stacking inter-
actions have been proposed as the reason for the increased
binding affinities of these larger inhibitors.
inammatory drugs (NSAIDs) moiety have been synthesized
by the condensation of homophthalic acid anhydride. The
synthesized compounds (4a–g) were evaluated for their in vitro
antibacterial activity and in silico docking studies into the
crystal structure of UDP-N-acetylmuramate-L-alanine ligase
using the Autodock PyRx virtual screening program. In the
entire series, the compounds (4c), (4g), (4f) and (4a) showed
excellent bacterial growth inhibition. The docking studies also
veried that these compounds possess high affinity for the
receptor and bind into the specic pocket. R2 values showed
good agreement with predicted binding affinities obtained by
molecular docking studies.
Fig. 3–9 show the most energetically favorable binding mode
of isocoumarins (4a–g) to MurC. From these gures, it can be
seen that the basic nucleic portion of the (4c), (4g), (4f) and (4a)
binds into the specicity pocket. In this pocket, the iso-
coumarin nucleus of these compounds interacts with LEU182,
PHE181, GLY125, and THR131; LYS129, ALA172, VAL124, and
GLY125; MET194, ASN193, LEU111, and THR133; and MET209,
LYS215, VAL412, and LYS208, respectively, amino acid residues
of the MurC target, whereas the rest of the compound showed
poor absorption ability.
The analysis of different choices for combining structures
into a single representative energy grid was performed in our
study. These inhibitors were docked into the generated
combined grids using AutoDock Vina (PyRx tool) and the RMSD
from the native pose, and their binding energies were evaluated
to determine whether the weighed averaged grids performed
greater. Table 2 lists the AutoGrid calculation around 8043
receptor atoms of UDP-N-acetylmuramate-L-alanine ligase with
minimum and maximum interacting energies. The RMSD
values (total run ¼ 9) has been discussed under Table 3.
References
1
2
3
W. Zhang, K. Krohn, S. Draeger and B. Schulz, J. Nat. Prod.,
008, 71, 1078–1081.
K. F. Devienne, G. Raddi, R. G. Coelho and W. Vilegas,
Phytomedicine, 2005, 12, 378–381.
L. C. DiStasi, D. Camuesco, A. Nieto, W. Vilegas, A. Zarzuelo
and J. Galvez, Planta Med., 2004, 70, 315–320.
2
4
5
I. Kostova, Curr. Med. Chem., 2005, 5, 29–46.
K. Krohn, U. Florke, M. S. Rao, K. Steingrover, H. J. Aust,
S. Draeger and B. Schulz, Nat. Prod. Lett., 2001, 15, 353–361.
Y. F. Huang, L. H. Li, L. Tian, L. Qiao, H. M. Hua and
Y. H. Pei, J. Antibiot., 2006, 59, 355–357.
6
53852 | RSC Adv., 2014, 4, 53842–53853
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
7
8
9
N. Koohei, Y. Mikikio, T. Yoshiko, K. Kenichi and N. Shoichi, 14 G. M. Maria, Z. Daniele, V. Luciano, F. Maurizio, F. Marco,
Chem. Pharm. Bull., 1981, 29, 2689–2691.
F. Takuya, F. Yoshiyasu and A. Yoshinori, Phytochemicals,
P. Sabrina, S. Giuditta and B. Elena, Bioorg. Med. Chem.,
2005, 13, 3797–3800.
1
986, 25, 517–520.
15 A. Saeed and Z. Ashraf, J. Heterocycl. Chem., 2008, 45, 679–
682.
H. L. Jeong, J. P. Yun, S. K. Hang, S. H. Young, K. Kyu-Won
and J. L. Jung, J. Antibiot., 2001, 54, 463–466.
0 Y. Ozoe, T. Kuriyama, Y. Tachibana, K. Harimaya,
16 A. Saeed, Z. Ashraf and H. Raque, J. Asian Nat. Prod. Res.,
2011, 13, 97–104.
1
N. Takahashi, T. Yaguchi, E. Suzuki, K. Imamura and 17 A. Saeed and Z. Ashraf, Chem. Heterocycl. Compd., 2008, 44,
K. Oyama, J. Pestic. Sci., 2004, 29, 328. 967–972.
1 J. B. Hudson, E. A. Graham, L. Harris and M. J. Ashwood- 18 J. Humljan, S. Starcevi ´c , V. Car, A. P. Stefanic, D. Kocjan,
Smith, Photochem. Photobiol., 1993, 57, 491–496. B. Jenko and U. Urleb, Pharmazie, 2008, 63, 102–106.
2 L. R. Corinne, A. Naoki, G. T. Jennifer, B. Michael, 19 D. E. Ehmann, J. E. Demeritt, K. G. Hull and S. L. Fisher,
M. D. William, D. K. George, L. R. Susan, M. Michael, Biochim. Biophys. Acta, 2004, 1698, 167–174.
F. Robert, K. Raghu, K. Donald and K. Surender, Cancer 20 Z. Ashraf, A. Muhammad, M. Imran and A. H. Tareq, Int. J.
1
1
Res., 2002, 62, 789–795.
Org. Chem., 2001, 1, 257–261.
1
3 I. A. Hamed, T. Keiichiro, A. Eiichi, K. Hiroto, M. Shinji,
H. Hiroyuki, A. Noriyuki, K. Yutaka and Y. Takehiro,
Bioorg. Med. Chem., 2007, 15, 242–256.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 53842–53853 | 53853