Synthesis, biological evaluation and docking studies of 4-aryloxymethyl coumarins derived from substructures and degradation products of vancomycin

Article abstract of DOI:10.1016/j.ejmech.2013.10.047

Two series of 4-aryloxymethyl coumarins derived from the reaction of 4-bromomethyl coumarins with ethyl gallate and ethyl ester of N-Benzoyl tyrosine have been synthesized. Gallate ethers 3a-3g and tyrosine derivatives 4e-4j were most effective against Entercoccusfaecalis. They were also found to be effective against Aspergillusniger and Candidaalbicans. Comparative docking studies with novobiocin have indicated better binding ability and higher 'C' score values than novobiocin.

Full text of DOI:10.1016/j.ejmech.2013.10.047

European Journal of Medicinal Chemistry 70 (2013) 750e757
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation and docking studies of
4-aryloxymethyl coumarins derived from substructures
and degradation products of vancomycin
,
b
b
Hrishikesh M. Revankar a, Manohar V. Kulkarni a , Shrinivas D. Joshi , Uttam A. More
*
a Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad 580003, Karnataka, India
b Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry, S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad
580002, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of 4-aryloxymethyl coumarins derived from the reaction of 4-bromomethyl coumarins with
ethyl gallate and ethyl ester of N-Benzoyl tyrosine have been synthesized. Gallate ethers 3ae3g and
tyrosine derivatives 4ee4j were most effective against Entercoccus faecalis. They were also found to be
effective against Aspergillus niger and Candida albicans. Comparative docking studies with novobiocin
have indicated better binding ability and higher ‘C’ score values than novobiocin.
Received 19 June 2013
Received in revised form
12 October 2013
Accepted 16 October 2013
Available online 25 October 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Ethyl gallate
N-Benzoyl tyrosine ethyl ester
Surflex-Dock
Antimicrobial activity
DNA gyrase subunit A
1. Introduction
alkoxy and chloro substituents were found to be effective against E.
coli [19].
Coumarins are a group of bioactive molecules, found extensively
in nature with a wide range of structural modifications [1]. They
exhibit antiviral [2], anti-cancer [3,4], anti-fungal [5], anti-
inflammatory [6,7], anti-HIV [8] properties. They have been
known to be particularly effective against Gram-positive species
[9]. Coumarin based anti-biotics viz. novobiocin and clorobiocin
affect the functioning of DNA gyrase, which is the basis for their
broad spectrum antibacterial activity [10].
Incorporation of bio-compatible fragments like vanillin and
paracetamol has resulted in 4-aryloxymethyl coumarins exhibiting
anti inflammatory activity (Fig. 1) [20].
In view of the importance associated with the above cited
moieties in their potent anti-microbial activity, it was thought of
considerable interest to employ derivatives of gallic acid and N-
Benzoyl tyrosine for the generation of new 4-aryloxymethyl cou-
marins which are represented in the Scheme 1.
Hydroxy coumarins like scopoletin and gallic acid have been
found to occur in Pelargonium sidoides, Pelargonium reniforme [14]
and other plant species, exhibiting a range of biological activities
[11e14]. Ester conjugates of 7-hydroxy coumarin with gallic acid
have been found to be anti-proliferative against human cancer cell
lines [15], methyl gallates with bis-aryl ether linkage and tyrosine
moiety are common substructures to the anti-biotics of vancomycin
family [16,17]. Naturally occurring bromotyrosine derivatives have
been found to possess anti-microbial effect on the methicillin
resistant S. aureus (MRSA) [18]. 4-aryloxymethyl coumarins with
2. Chemistry
4-Bromoethylacetoacetate obtained from bromination of eth-
ylacetoacetate, was treated with various substituted phenols under
Pechmann cyclisation conditions using neat sulphuric acid as
condensing agent. The reaction resulted in the formation of
substituted 4-bromomethyl-coumarins 1 [21]. The allylic nucleo-
philic displacement was brought about by the reaction of 1 with
ethyl gallate 2 (prepared from gallic acid and ethanol) to obtain
compounds 3ae3j.
4-Bromomethyl coumarins 1 were also condensed with N-
benzoyl tyrosine ethyl ester, to obtain compounds 4ae4j. In both
* Corresponding author.
E-mail address: manohar274@gmail.com (M.V. Kulkarni).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.047

H.M. Revankar et al. / European Journal of Medicinal Chemistry 70 (2013) 750e757
751
O
O
R1
R2
O
O
O
O
H
N
O
CHO
N
H
O
HO
HO
N
H
O
O
O
O
OCH3
R3
R
NH2
OH
O
O
Anti proliferative
Anti-inflammatory
Anti-bacterial
Fig. 1. Biologically active structurally related molecules.
the cases reactions were carried out in dry acetone in presence of
anhydrous potassium carbonate at room temperature. Anhydrous
potassium carbonate abstracts proton from phenolic eOH to give
resonance stabilized phenoxide anion, which then reacts with 4-
bromomethyl-coumarins to give desired compounds.
NMR, wherein the OeCH2 protons appear as a singlet at 5.25 ppm
which is characteristic of 4-aryloxymethyl coumarins [19]. The C3e
H of coumarin was observed at 6.73 ppm and the C6eCH3 protons
appear as singlet at 2.34 ppm. Two aromatic protons resonating at
7.02 corresponded to the protons on gallate moiety [24], C5eH of
coumarin resonated as singlet at 7.65, whereas C7eH and C8eH
resonated as doublets with J ¼ 8.0 Hz at 7.32 and 7.44 ppm
respectively. Ethoxy protons appeared as triplet quartet pattern at
1.26 and 4.23 ppm with J ¼ 7.0 Hz. The downfield D2O exchangeable
3. Results and discussion
Formation of products 3 and 4 is well supported by spectro-
scopic analysis. In case of compound 3a, (R ¼ 6-CH3) IR spectrum
exhibited two bands at 1711 cmꢀ1 and 3444 cmꢀ1 due to lactone
and eOH stretching bands of coumarin and gallate moiety
respectively. Formation of ethers 3a was further confirmed by 1H
signal at 9.83 ppm corresponded to the phenolic eOH proton. 13
C
NMR provides additional support for structure of the compounds.
Lactone carbonyl resonates at 160 ppm and ester carbonyl of gallate
resonates at 165 ppm, OeCH2 resonates at 69 ppm, the methylene
Br
R
O
OH
O
O
H2SO4
Br
R
O
O
1
COOC2H5
O
HO
HO
OH
OH
NH
Acetone/K2CO3
O
2
O
COOC2H5
O
HO
OH
O
O
NH
O
O
R
R
O
O
O
O
4a-j
3a-j
R= 6-Me; 7-Me; 6-Cl; 7-Cl; 6-OMe; 7-OMe; 5,7-Me; 7,8-Me; 5,6 Benzo; 7,8 Benzo;
Scheme 1. Synthesis of gallate and tyrosinate ethers.

752
H.M. Revankar et al. / European Journal of Medicinal Chemistry 70 (2013) 750e757
and methyl group of ester resonated at 60 and 14 ppm respectively,
aromatic carbons were observed in accordance with expected
values in the range of 112e151 ppm. The assignments are in
agreement with the values reported for ethyl gallate (24). Molec-
ular ion peak at 370 m/z (80%) in the EI-MS confirmed the proposed
structure.
E. faecalis with compounds 4e, 4f, 4he4j showing activity of 0.2 mg/
mL which is significantly lower than standard drug used (Table 2).
Even in these compounds the same trend was observed as in
compounds 3, with the efficacy decreasing in the order eCl (4i), e
OCH3 (4c) and eCH3 (4a) when located at C6 positions. The activity
were similar for eCH3 (4b) and eOCH3 (4d) at C7 positions, how-
ever 7-Cl showed better activity than the other two. However,
compounds of both the series, i.e 3 and 4 were inactive against
Gram-negative bacteria.
Compound 4a (R ¼ 6-CH3) exhibited three prominent bands in
the IR spectra at 3320 cm-1, 1658 cm-1 and 1722 cm-1 due to the NH,
amide carbonyl and lactone carbonyl frequencies respectively. In 1H
NMR spectrum, the signals observed at 5.25 (s); 4.09 (s) J ¼ 7. 2 Hz
and 3.7 ppm (d) are due to OeCH2eAr, OeCH2 (ethoxy) and Are
CH2 protons respectively. The three proton signals at 1.14 (q)
J ¼ 7.2 Hz and 2.39 ppm (s) correspond to the CH3 of ethoxy group
and C6eCH3 respectively. Aromatic protons appear in the range of
7.02e7.83 ppm. The eNH proton appear as a doublet at 8.82 ppm
with a coupling constant of J ¼ 7.6 Hz, whereas the vicinal CeH
proton appear at 4.63 ppm as multiplet. In the 13C NMR lactone,
ester and amide carbonyl resonates at 159, 166 and 171 ppm
respectively, the methylene and methyl group of ester resonated at
60 and 13 ppm respectively, OeCH2 was observed at 64 ppm, the e
CH group flanked by Ester and amide functionalities resonates at
54, in addition the methyl group on coumarin resonates at 20 ppm.
The molecular ion peak in EI-MS at 485 was not observed,
instead a peak at m/z 364 (15%) indicated the loss of C6H5eCONH
moiety. Various compounds synthesized with their data are indi-
cated in the Experimental section.
4.2. Antifungal screening
The compounds of series 3 and 4 were screened for their anti-
fungal activity against Candida albicans and Aspergillus niger by
Broth dilution method [22]. All the gallate ethers 3ae3j were
inactive against both the fungal strains, with MIC as high as 100
mL.
mg/
Compounds 4, were highly active against A. niger with all the
compounds showing activity 40 times better than the standard
used. However in the case of C. albicans, eCH3 and eOCH3 attached
at C6 (4a, 4b) and C7 (4c, 4d) respectively showed significantly
higher growth inhibition than the standard used, almost 80 times
better. Di substituted compounds 4e and 4f, along with benzo
substituted compounds 4g and 4h showed appreciable activity
while the eCl substituted compounds at both C6 and C7 positions
showed poor activity growth inhibition.
4. Biological screening
5. Computational studies
4.1. Anti-bacterial screening
To understand the mechanism of anti-microbial activity of the
compounds synthesized, molecular modelling and docking studies
were performed on X-ray crystal structure of E. coli 24 kDa domain
in complex with clorobiocin (PDB code: 1KZN; resolution 2.30 A)
using Surflex-Dock programme of Sybyl-X software. Clorobiocin
All the newly synthesized compounds were evaluated for their
in-vitro antibacterial activity against Enteroccocus faecalis (MTCC
3382), Staphylococcus aureus (MTCC 3160), Pseudomonas aeruginosa
(MTCC 1034), Escherichia coli (MTCC 1089) by Broth microdilution
method [22]. Table 1 revealed that the compounds 3ae3j were
highly active against S. faecalis and S. aureus. In the activity against
S. aureus, the efficacy of compounds decreased in the order of eCl
(3i), eOCH3 (3g) and eCH3 (3a) when located at C6 positions. In
case of groups at C7 positions, the activity was least with 7-CH3 (3b)
and 7-Cl (3j) groups, the 7-OCH3 group (3h) was ten times more
active than the other two. All the gallate ethers 3ae3j with
exception of 3h were most effective against E. faecalis, showing the
ꢀ
was found to have hydrogen bonding interactions with Asp73
ꢀ
ꢀ
ꢀ
ꢀ
(1.911 A), Thr165 (2.109 A), Asn46 (2.034 A) and Arg136 (2.071 A).
3c forms six hydrogen bonding interactions, phenol containing
two OH group having four hydrogen bonding interactions second
ꢀ
position OH; H interact with Asp49 (1.908 A) and O interact with
Asn46 (2.660 A). In case of sixth position OH; H interact with
ꢀ
ꢀ
ꢀ
Glu50 (1.812 A) and O interact with Arg76 (2.524 A) and two
hydrogen bonding interaction with CO of coumarin moiety Gly77
ꢀ
ꢀ
(2.016 A), Thr165 (2.549 A). 4c forms three hydrogen bonds, two
ꢀ
least MIC value of 0.2
mg/mL. However they were inactive against
hydrogen bonds with CO of coumarin moiety Arg136 (2.014 A) and
Arg136 (1.996 A) and one hydrogen bond with ester CO and H of
Thr165 (1.826 A). All compounds have shown good C-score 8.56e
ꢀ
P. aeruginosa and E. coli.
The anti-bacterial screening of compounds 4ae4j revealed that
the N-benzoyl tyrosine ethyl ester ethers were only active against
ꢀ
6.02 kcal/mol (Tables 3 and 4). Compounds 3aej and 4aej were
Table 1
Antibacterial and antifungal activity of compounds 3.
Compounds
R
Antibacterial
Gram positive
S. aureus
Antifungal
Gram negative
E. faecalis
E. coli
P. aeruginosa
C. albicans
A. niger
3a
3b
3c
3d
3e
3f
3g
3h
6-CH3
7-CH3
1.6
12.5
0.8
0.2
0.4
1.6
0.8
1.6
0.4
12.5
2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.8
0.2
3.125
2
100
100
100
100
100
100
100
100
100
100
2
100
100
100
100
100
100
100
100
100
100
2
100
100
100
100
100
100
100
100
100
100
e
100
100
100
100
100
100
100
100
100
100
e
5,7-CH3
7,8-CH3
5,6 benzo
7,8 benzo
6-OCH3
7-OCH3
6-Cl
3i
3j
7-Cl
Ciprofloxacin
Fluconazole
e
e
e
e
16
8

H.M. Revankar et al. / European Journal of Medicinal Chemistry 70 (2013) 750e757
753
Table 2
Antibacterial and antifungal activity of compounds 4.
Compounds
R
Antibacterial
Gram positive
S. aureus
Antifungal
Gram negative
E. faecalis
E. coli
P. aeruginosa
C. albicans
A. niger
4a
4b
4c
4d
4e
4f
4g
4h
6-CH3
7-CH3
100
e
0.8
0.8
0.4
0.8
0.2
0.2
0.4
0.2
0.2
0.2
2
100
100
100
100
100
100
100
100
e
100
100
100
100
100
100
e
0.2
0.2
0.2
0.2
1.6
0.8
1.6
12.5
50
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
e
6-OCH3
7-OCH3
5,7-CH3
7,8-CH3
5,6 benzo
7,8 benzo
6-Cl
100
100
e
100
e
50
100
e
100
100
100
2
4i
4j
7-Cl
e
50
e
Ciprofloxacin
Fluconazole
2
2
e
e
e
e
16
8
having better hydrogen bonding, complex (ligand-protein), and
internal (ligandeligand) energies than novobiocin. Helmholtz free
energies of interactions for protein-ligand atom pairs for all
compounds showed less than chlorobiocin and novobiocin. Charge
and van der Waals interactions between the protein and the ligand
suggest that 3be3d, 4be4d and 4fe4i are superior ligands than
novobiocin to bind with DNA gyrase. Scoring of compounds with
respect to the reward for hydrogen bonding, lipophilic contact,
and rotational entropy, along with an intercept terms revealed
that compounds 3ce3e, 4ae4b, 4e and 4ge4i have more in-
teractions with the protein than novobiocin and other compounds.
The docking models of the compounds 3c and 4c along with
clorobiocin are given below (Figs. 2 and 3).
on Bruker 400 MHz Spectrometer using DMSO d6 as solvents and
TMS as internal standard. The Chemical shifts are expressed in
d
ppm. Mass spectra were recorded using Shimadzu GCMS-
QP2010S. The elemental analyses were carried out using Hereaus
CHN rapid analyser. Purity of the compound was checked by TLC.
All the chemicals purchased were of analytical grade and were used
without further purification unless otherwise stated.
6.1.1. Synthesis of substituted 4-bromomethyl-7-methyl coumarin
(1)
The required substituted 4-bromomethyl coumarin have been
synthesized by the Pechmann cyclization of various substituted
phenols with 4-bromoethylacetoacetate [23].
6.1.2. Synthesis of ethyl gallate (2)
6. Experimental
It was synthesized using known procedure [24].
6.1. Chemistry
6.1.2.1. Synthesis of ethyl 3,5-dihydroxy-4-((6-methyl-2-oxo-2H-
chromen-4-yl)methoxy) benzoate (3a). A mixture of 0.216 g of ethyl
gallate (2) (0.001 M) and 0.138 g of anhydrous potassium car-
bonate (0.001 M) were stirred for 30 min in dry acetone (30 mL),
The melting points were determined by open capillary method
and are uncorrected. IR spectra (KBr disc) were recorded on
Nicolet ꢀ5700 FT-IR spectrometer. 1H NMR spectra were recorded
Table 3
Table 4
Surflex-Dock score (kcal/mol) of compounds 3.
Surflex-Dock score (kcal/mol) of compounds 4.
Compound C-Scorea Crash Polar G scored PMF
scoreb scorec scoree
D scoref
Chem
scoreg
Compound C-Scorea Crash Polar G scored PMF
D scoref Chem
scoreg
scoreb scorec
scoree
Clorobiocin 11.49
Novobiocin 7.41
ꢀ1.50 3.77 ꢀ285.755 ꢀ81.590 ꢀ166.382 ꢀ32.321
ꢀ1.72 3.84 ꢀ163.279 ꢀ77.294 ꢀ134.332 ꢀ23.450
ꢀ1.42 1.61 ꢀ247.259 ꢀ28.464 ꢀ159.034 ꢀ23.846
ꢀ1.10 1.00 ꢀ277.349 ꢀ30.339 ꢀ157.048 ꢀ22.652
ꢀ1.04 3.55 ꢀ208.220 ꢀ45.019 ꢀ123.775 ꢀ22.064
ꢀ1.46 1.49 ꢀ216.957 ꢀ36.187 ꢀ131.762 ꢀ21.255
ꢀ1.14 1.29 ꢀ237.866 ꢀ39.442 ꢀ159.241 ꢀ27.880
ꢀ1.48 2.57 ꢀ214.684 ꢀ31.019 ꢀ127.611 ꢀ22.393
ꢀ0.94 1.75 ꢀ165.384 ꢀ37.433 ꢀ127.515 ꢀ18.688
ꢀ1.48 2.57 ꢀ214.684 ꢀ31.019 ꢀ127.611 ꢀ22.393
ꢀ1.27 3.41 ꢀ177.806 ꢀ35.186 ꢀ121.226 ꢀ22.467
ꢀ1.27 3.29 ꢀ186.516 ꢀ24.328 ꢀ127.040 ꢀ23.724
Clorobiocin 11.49
Novobiocin 7.41
ꢀ1.50 3.77
ꢀ1.72 3.84
ꢀ1.53 1.34
ꢀ1.60 0.87
ꢀ1.39 3.05
ꢀ1.35 1.73
ꢀ2.04 1.02
ꢀ2.81 1.69
ꢀ1.88 1.10
ꢀ1.28 3.66
ꢀ2.59 2.17
ꢀ1.35 1.73
ꢀ285.755 ꢀ81.590 ꢀ166.382 ꢀ32.321
ꢀ163.279 ꢀ77.294 ꢀ134.332 ꢀ23.450
ꢀ245.947 ꢀ46.486 ꢀ139.192 ꢀ22.158
ꢀ258.828 ꢀ33.347 ꢀ151.901 ꢀ23.115
ꢀ222.002 ꢀ47.348 ꢀ121.572 ꢀ25.220
ꢀ183.994 ꢀ46.655 ꢀ122.156 ꢀ21.714
ꢀ220.515 ꢀ42.005 ꢀ136.690 ꢀ24.939
ꢀ245.009 ꢀ26.412 ꢀ145.345 ꢀ23.863
ꢀ286.362 ꢀ28.531 ꢀ153.314 ꢀ22.241
ꢀ195.059 ꢀ65.163 ꢀ114.734 ꢀ24.672
3a
3b
3c
3d
3e
3f
3g
3h
3i
7.88
8.53
8.56
7.01
8.03
6.82
7.01
6.82
6.02
7.31
4a
4b
4c
4d
4e
4f
4g
4h
4i
7.98
7.87
8.35
6.75
7.04
7.03
7.08
7.13
8.21
6.75
ꢀ345.424
1.079 ꢀ178.360 ꢀ27.819
3j
4j
ꢀ183.994 ꢀ46.655 ꢀ122.156 ꢀ21.714
a
a
C-Score (Consensus-score) reports the output of total score.
Crash-score revealing the inappropriate penetration into the binding site.
Polar region of the ligand.
C-Score (Consensus-score) reports the output of total score.
Crash-score revealing the inappropriate penetration into the binding site.
Polar region of the ligand.
b
c
b
c
d
d
G-score showing hydrogen bonding, complex (ligandeprotein), and internal
G-score showing hydrogen bonding, complex (ligandeprotein), and internal
(ligandeligand) energies.
(ligandeligand) energies.
e
e
PMF-score indicating the Helmholtz free energies of interactions for proteine
PMF-score indicating the Helmholtz free energies of interactions for proteine
ligand atom pairs (Potential of Mean Force, PMF).
ligand atom pairs (Potential of Mean Force, PMF).
f
f
D-Score for charge and van der Waals interactions between the protein and the
D-score for charge and van der Waals interactions between the protein and the
ligand.
ligand.
g
g
Chem-score points for hydrogen bonding, lipophilic contact, and rotational
Chem-score points for hydrogen bonding, lipophilic contact, and rotational
entropy, along with an intercept term.
entropy, along with an intercept term.

754
H.M. Revankar et al. / European Journal of Medicinal Chemistry 70 (2013) 750e757
6.1.2.3. Ethyl 3,5-dihydroxy-4-((5,7-dimethyl-2-oxo-2H-chromen-4-
yl)methoxy) benzoate (3c). Pale yellow; yield 68%; m.p: 210e
214 ꢁC; IR (KBr) cmꢀ1 1710 (C]O), 3394 (eOH); 1H NMR (DMSO,
400 MHz, TMS):
d
ppm 1.26 (t, 3H, J ¼ 7.2 Hz, OeCH2eCH3), 2.34 (s,
3H,C5eCH3), 2.65 (s, 3H, C5eCH3), 4.31 (q, 2H, OeCH2eCH3,
J ¼ 7.2 Hz), 5.25 (s, 1H, OeCH2), 6.70 (s,1H, C3eH), 7.00 (s, 2H, AreH
gallate) 7.10 (s, 1H, C8eH of coumarin), 7.30 (s, 1H, C6eH of
coumarin), 9.83 (s, 2H, eOH D2O Exchangeable); m/z 384 (20%);
Anal Calcd. For C21H20O7 (%), Calcd: C, 65.62; H, 5.24, found: C,
65.43; H, 5.15.
6.1.2.4. Ethyl 3,5-dihydroxy-4-((7,8-dimethyl-2-oxo-2H-chromen-4-
yl)methoxy) benzoate (3d). Off-white; yield 69%; m.p: 160e
162 ꢁC; IR (KBr) cmꢀ1 1710 (C]O), 3385 (eOH); 1H NMR (DMSO,
400 MHz, TMS):
d
ppm 1.26 (t, 3H, J ¼ 7.2 Hz, OeCH2eCH3), 2.34 (s,
3H, C7eCH3), 2.75 (s, 3H, C8eCH3), 4.21 (q, 2H, OeCH2eCH3,
J ¼ 7.2 Hz), 5.25 (s, 1H, OeCH2), 6.76 (s, 1H, C3eH), 7.00 (s, 2H, AreH
gallate), 7.30 (d, 1H, C6eH of coumarin), 7.40 (d, 1H, C5eH of
coumarin), 9.83 (s, 2H, eOH D2O Exchangeable); m/z 384; Anal
Calcd. For C21H20O7 (%), Calcd: C, 65.62; H, 5.24, found: C, 65.13; H,
5.10.
6.1.2.5. Ethyl
3,5-dihydroxy-4-((3-oxo-3H-benzo[f]chromen-1-yl)
methoxy) benzoate(3e). Yellow; yield 72%; m.p: 220e222 ꢁC; IR
(KBr) cmꢀ1 1700 (C]O), 3416 (eOH); 1H NMR (DMSO, 400 MHz,
TMS):
d
ppm 1.26 (t, 3H, J ¼ 7.2 Hz, OeCH2eCH3), 4.24 (q, 2H, Oe
Fig. 2. Docking study of 24 kDa fragment DNA gyrase subunit A (1KZN) with com-
pound 3c,which shows the consensus score (C-score) of 8.56 and schematic repre-
sentation of compound 3c bound to the DNA subunit.
to this 0.253 g of 6-methyl 4-bromomethyl coumarin (1) was
added and stirring was continued for 24 h, the reaction was
monitored using TLC. After completion, the reaction mixture was
quenched in crushed ice. The separated solid was filtered and
washed with 1:1 HCl. The compound was dried and crystallized
from methanol. Brownish white; yield 73%; m.p: 222e225 ꢁC; IR
(KBr) cmꢀ11711 (C]O), 3444 (eOH); 1H NMR (DMSO, 400 MHz,
TMS): 1.26 (t, 3H, J ¼ 7.2 Hz, OeCH2eCH3),
d ppm 2.34 (s, 3H, C6e
CH3), 4.21 (q, 2H, OeCH2eCH3, J ¼ 7.2 Hz), 5.25 (s, 2H, OeCH2),
6.73 (s, 1H, C3eH), 7.00 (s, 2H, AreH gallate), 7.32 (d, 1H, C7eH of
coumarin); 7.44 (d, 1H, C8eH of coumarin), 7.65 (s, 1H, C5eH
of coumarin), 9.83 (s, 2H, eOH, D2O Exchangeable), 13C
NMR(400 MHz, DMSO d6,
d ppm) 14.1,20.3,60.4,69.0,108.6,
112.5,115.3,116.5,124.5,125.2,132.7,133.1,
138.0,150.6,151.1,151.6,160.0,165.3 m/z 370 (25%); Anal Calcd. For
C
20H18O7 (%) Calcd. C, 64.86; H, 4.90, found: C, 64.26; H, 4.40.
6.1.2.2. Ethyl 3,5-dihydroxy-4-((7-methyl-2-oxo-2H-chromen-4-yl)
methoxy) benzoate(3b). Pale yellow; yield 70%; m.p: 235e240 ꢁC;
IR (KBr) cmꢀ1 1713 (C]O), 3404 (eOH); 1H NMR (DMSO, 400 MHz,
TMS): 1.26 (t, 3H, J ¼ 7.2 Hz, OeCH2eCH3),
d ppm 2.34 (s, 3H, C7e
CH3), 4.21 (q, 2H, OeCH2eCH3, J ¼ 7.2 Hz), 5.25 (s, 1H, OeCH2), 6.73
(s, 1H, C3eH), 7.00 (s, 2H, AreH gallate), 7.30 (d, 1H, C7eH of
coumarin), 7.40 (d, 1H, C8eH of coumarin), 7.60 (s, 1H, C5eH of
coumarin), 9.84 (s, 2H, eOH, D2O Exchangeable),; m/z 370; Anal
Calcd. For C20H18O7 (%) Calcd.: C, 64.86; H, 4.90, found: C, 64.46; H,
4.80.
Fig. 3. Docking study of 24 kDa fragment DNA gyrase subunit A (1KZN) with com-
pound 4c,which shows the consensus score (C-score) of 8.35 and schematic repre-
sentation of compound 4c bound to the DNA subunit.

H.M. Revankar et al. / European Journal of Medicinal Chemistry 70 (2013) 750e757
755
CH2eCH3, J ¼ 7.2 Hz), 5.67 (s, 2H, OeCH2), 7.00 (s, 2H, AreH gallate),
7.02 (s, 1H, C3eH), 7.04e8.35 (8H, AreH) 9.86 (s, 2H, eOH D2O
Exchangeable); m/z 406 (35%); Anal Calcd. For C23H18O7 (%), Calcd:
C, 67.98; H, 4.46, found: C, 67.75; H, 4.33.
mixture was quenched in crushed ice. The separated solid was
filtered and washed with 1:1 HCl. The compound was dried and
crystallized from ethanol. White coloured; yield 62%; m.p:176e
180 ꢁC; IR (KBr) cmꢀ1 1726 (C]O) 3318 (eNH) 1650 (amide C]O);
1H NMR (DMSO, 400 MHz, TMS): 1.14 (t, 3H, OeCH2eCH3,
6.1.2.6. Ethyl
3,5-dihydroxy-4-((2-oxo-2H-benzo[h]chromen-4-yl)
J ¼ 7.2 Hz),
d ppm 2.33 (s, 3H, AreCH3), 3.09 (m, 2H, Ph-CH2), 4.09
(q, 2H, OeCH2eCH3, J ¼ 7.2 Hz), 4.61 (t,1H, PheCH2eCH, J ¼ 7.2 Hz),
methoxy) benzoate(3f). Buff coloured; yield 75%; m.p: 230e232 ꢁC;
IR (KBr) cm-11704 (C]O), 3401 (eOH); 1H NMR (DMSO, 400 MHz,
5.31 (s, 2H, OeCH2), 6.51(s, 1H, C3eH), 7.04e7.88 (m, 12H, AreH),
8.84 (d, 1H, NHeCO,
NMR(400 MHz,DMSO-d6,
13.1,20.3,30.4,35.40,54.52,60.47,64.92,111.6,114.8,116.3,124.4,127.3,
128.2,130.2,130.4,131.4,132.9,133.6,151.1,151.2,156.2,159.7,166.4,
171.69; m/z 364 (10%); Anal Calcd. For C29H27NO6 (%), Calcd: C,
71.74; H, 5.61; N, 2.88, found: C, 71.44; H, 5.45; N, 2.64.
TMS):
d
ppm 1.26 (t, 3H, J ¼ 7.2 Hz, OeCH2eCH3), 4.24 (q, 2H, Oe
J
¼
7.2 Hz, D2O Exchangeable); 13C
CH2eCH3, J ¼ 7.2 Hz), 5.68 (s, 2H, OeCH2), 7.00 (s, 2H, AreH gallate),
7.05 (s, 1H, C3eH), 7.04e8.35 (8H, AreH) 9.86 (s, 2H, eOH D2O
Exchangeable); m/z 406 (30%); Anal Calcd. For C23H18O7 (%), Calcd:
C, 67.98; H, 4.46, found: C, 67.11; H, 4.23.
d
ppm)
6.1.2.7. Ethyl 3,5-dihydroxy-4-((6-methoxy-2-oxo-2H-chromen-4-yl)
methoxy) benzoate(3g). Pale yellow; Yield 75%; m.p: 230e232 ꢁC;
IR (KBr)cmꢀ1 1704 (C]O),3401 (-OH); 1H NMR (DMSO, 400 MHz,
6.1.2.12. Ethyl 2-benzamido-3-(4-((7-methyl-2-oxo-2H-chromen-4-
yl)methoxy)phenyl)propionate (4b). Buff coloured; Yield 63%; m.p:
117e120 ꢁC; IR (KBr) cmꢀ11722 (C]O) 3320 (eNH) 1658 (amide
TMS):
d
ppm 1.26 (t, 3H, J ¼ 6.8 Hz, OeCH2eCH3), 3.79 (s, 3H, C6e
OCH3), 4.21 (q, 2H, OeCH2eCH3, J ¼ 6.8 Hz), 5.23 (s, 1H, OeCH2),
6.72 (s, 1H, C3eH), 7.00 (s, 2H, AreH gallate), 7.10 (d, 1H, C8eH of
coumarin), 7.20 (d, 1H, C6eH of coumarin), 7.38 (s, 1H, C5eH of
coumarin), 9.83 (s, 2H, eOH, D2O Exchangeable); m/z 386 (20%);
Anal Calcd. For C20H18O8 (%), Calcd: C, 62.17; H, 4.70, found: C, 62.17;
H, 4.51.
C]O); 1H NMR (DMSO, 400 MHz, TMS):
d ppm 1.19 (t, 3H, OeCH2e
CH3, J ¼ 7.2 Hz), 2.33 (s, 3H, AreCH3), 3.09 (m, 2H, Ph-CH2), 4.09 (q,
2H, OeCH2eCH3, J ¼ 7.2 Hz), 4.61 (t, 1H, PheCH2eCH, J ¼ 7.2 Hz),
5.31 (s, 2H, OeCH2), 6.51(s, 1H, C3eH), 7.06e7.88 (m, 12, AreH),
8.72 (d, 1H, NHeCO, J ¼ 7.2 Hz, D2O Exchangeable); m/z 364 (10%);
Anal Calcd. For C29H27NO6 (%), Calcd: C, 71.74; H, 5.61; N, 2.88,
found: C, 71.23; H, 5.43; N, 2.74.
6.1.2.8. Ethyl 3,5-dihydroxy-4-((7-methoxy-2-oxo-2H-chromen-4-yl)
methoxy) benzoate(3h). Off-white; Yield 72%; m.p: 210e212 ꢁC; IR
(KBr) cmꢀ1 1710 (C]O), 3355 (eOH); 1H NMR (DMSO, 400 MHz,
6.1.2.13. Ethyl2-benzamido-3-(4-((6-methoxy-2-oxo-2H-chromen-4-
yl)methoxy)phenyl)propionate (4c). Pale yellow; Yield 61%; m.p:
140e144 ꢁC; IR (KBr) cmꢀ1 1727 (C]O) 3289 (eNH) 1615 (amide
TMS) :
d
ppm 1.26 (t, 3H, J ¼ 7.2 Hz, OeCH2eCH3), 3.73 (s, 3H, C7e
OCH3), 4.21 (q, 2H, OeCH2eCH3, J ¼ 7.2 Hz), 5.25 (s, 1H, OeCH2),
6.75 (s, 1H, C3eH), 7.00 (s, 2H, AreH gallate) 7.3 (d, 1H, C8eH of
coumarin) 7.4 (d, 1H, C8eH of coumarin), 7.6 (s, 1H, C5eH of
coumarin), 9.83 (s, 2H, eOH, D2O Exchangeable); m/z 386 (20%);
Anal Calcd. For C20H18O8 (%), Calcd: C, 62.17; H, 4.70, found: C, 62.0;
H, 4.50.
C]O); 1H NMR (DMSO, 400 MHz, TMS):
d ppm 1.14 (t, 3H, OeCH2e
CH3, J ¼ 7.2 Hz), 3.09 (m, 2H, Ph-CH2), 3.81 (s, 3H, C6eOCH3), 4.08
(q, 2H, OeCH2eCH3, J ¼ 7.2 Hz), 4.60 (t,1H, PheCH2eCH, J ¼ 7.6 Hz),
5.37 (s, 2H, OeCH2), 6.53 (s, 1H, C3eH), 7.07e7.80 (m, 12, AreH),
8.78 (d, 1H, NHeCO, J ¼ 7.6 Hz, D2O Exchangeable); Anal Calcd. For
C29H27NO7 (%), Calcd: C, 69.45; H, 5.43; N, 2.79, found: C, 69.15; H,
5.23; N, 2.67.
6.1.2.9. Ethyl 3,5-dihydroxy-4-((6-chloro-2-oxo-2H-chromen-4-yl)
methoxy) benzoate (3i). White coloured; yield 76%; m.p: 240e
242 ꢁC; IR (KBr) cmꢀ11711 (C]O),3401 (-OH); 1H NMR (DMSO,
6.1.2.14. Ethyl2-benzamido-3-(4-((7-methoxy-2-oxo-2H-chromen-4-
yl)methoxy)phenyl)propionate (4c). Grey coloured; Yield 56%; m.p:
150e154 ꢁC; IR (KBr) cmꢀ11722 (C]O) 3278 (-NH) 1640 (amide C]
400 MHz, TMS):
d
ppm 1.26 (t, 3H, J ¼ 7.2 Hz, OeCH2eCH3),4.23 (q,
2H, OeCH2eCH3, J ¼ 7.2 Hz), 5.22 (s, 2H, OeCH2), 6.78 (s,1H, C3eH),
7.00 (s, 2H, AreH gallate) 8.06 (s, 1H, C5eH of coumarin), 7.6 (d, 1H,
C7eH of coumarin), 7.4 (d,1H, C8eH of coumarin), 9.86 (s, 2H, eOH,
D2O Exchangeable); m/z 390(M) (35%), 392(M þ 2) (15%); Anal
Calcd. For C19H15ClO7 (%), Calcd: C, 58.40; H, 3.87, found: C, 58.10; H,
3.64.
O); 1H NMR (DMSO, 400 MHz, TMS):
d ppm 1.19(t, 3H, OeCH2eCH3,
J ¼ 7.2 Hz), 3.09 (m, 2H, Ph-CH2), 3.75 (s, 3H, C7eOCH3), 4.09 (q, 2H,
OeCH2eCH3, J ¼ 7.2 Hz), 4.61 (t,1H, PheCH2eCH, J ¼ 7.2 Hz),5.31 (s,
2H, OeCH2), 6.51 (s, 1H, C3eH), 7.10e7.89 (m, 12H, AreH), 8.83 (d,
1H, NHeCO, J ¼ 7.2 Hz, D2O Exchangeable); Anal Calcd. For
C29H27NO7 (%), Calcd: C, 69.45; H, 5.43; N, 2.79, found: C, 69.35; H,
5.17; N, 2.62.
6.1.2.10. Ethyl 3,5-dihydroxy-4-((7-chloro-2-oxo-2H-chromen-4-yl)
methoxy) benzoate (3j). White coloured; yield 70%; m.p: 225e
227 ꢁC; IR (KBr) cmꢀ1 1712 (C]O), 3400 (eOH); 1H NMR (DMSO,
6.1.2.15. Ethyl2-benzamido-3-(4-((5,7-dimethyl-2-oxo-2H-chromen-
4yl)methoxy)phenyl)propionate ꢀ(14e). White coloured; yield 60%;
m.p:167e170 ꢁC; IR (KBr) cm 1726 (C]O) 3286 (eNH) 1642
400 MHz, TMS):
d
ppm 1.27 (t, 3H, J ¼ 7.2 Hz, OeCH2eCH3), 4.24 (q,
2H, OeCH2eCH3, J ¼ 7.05 Hz), 5.27 (s, 2H, OeCH2), 6.79 (s, 1H, C3e
H), 7.00 (s, 2H, AreH gallate) 7.4 (s, 1H, C5eH of coumarin), 7.62 (s,
1H, C8eH of coumarin), 7.9 (d, 1H, C6eH of coumarin); 9.77þ(s, 2H,
eOH, D2O Exchangeable), m/z 390(M)þ (20%), 392(M þ 2) (8%);
Anal Calcd. For C19H15ClO7 (%), Calc: C, 58.40; H, 3.87, found: C,
58.35; H, 3.63.
(amide C]O); 1H NMR (DMSO, 400 MHz, TMS):
d ppm 1.14 (t, 3H,
OeCH2eCH3, J ¼ 5.1 Hz), 2.34 (s, 3H, C5eCH3), 2.65(s, 3H, C7eCH3),
3.07(m, 2H, Ph-CH2),4.14 (q, 2H, OeCH2eCH3, J ¼ 5.1 Hz), 4.61(t, 1H,
PheCH2eCH, J ¼ 7.2 Hz),5.43 (s, 2H, OeCH2), 6.49 (s, 1H, C3eH),
7.01e7.85 (m, 11H, AreH), 8.75 (d, 1H, NHeCO, J ¼ 5.7 Hz, D2O
Exchangeable); Anal Calcd. For C30H29NO6 (%), Calc: C, 72.13; H,
5.85; N, 2.80, found: C, 72.03; H, 5.63; N, 2.72.
6.1.2.11. Synthesis of ethyl 2-benzamido-3-(4-((6-methyl-2-oxo-2H-
chromen-4-yl)methoxy)phenyl)propionate (4a). A mixture of
0.313 g of N-Benzoyl tyrosine ethyl ester (0.001 M) and 0.138 g of
anhydrous potassium carbonate (0.001 M) were stirred for 30 min
in dry acetone (30 ml), 0.253 g of 6-methyl 4-bromomethyl
coumarin (1) was added and stirring was continued for 24 h, the
reaction was monitored using TLC. After completion, the reaction
6.1.2.16. Ethyl
2-benzamido-3-(4-((7,8-dimethyl-2-oxo-2H-chro-
men-4yl)methoxy)phenyl) propionate (4f). Buff coloured; yield 57%;
m.p:90e94 ꢁC; IR (KBr) cmꢀ11725 (C]O) 3290 (-NH) 1648 (amide
C]O); 1H NMR (DMSO, 400 MHz, TMS):
d ppm 1.12 (t, 3H, OeCH2e
CH3, J ¼ 7.2 Hz), 2.24 (s, 3H, AreCH3), 2.46 (s, 3H, AreCH3), 3.07(m,
2H, Ph-CH2, J ¼ 7.6 Hz), 4.07 (q, 2H, OeCH2eCH3, J ¼ 7.2 Hz), 4.58 (t,

756
H.M. Revankar et al. / European Journal of Medicinal Chemistry 70 (2013) 750e757
1H, PheCH2eCH, J ¼ 7.6 Hz), 5.33 (s, 2H, OeCH2), 6.46 (s, 1H, C3e
H), 7.03e7.82 (m, 11, AreH), 8.78 (d,1H, NHeCO, J ¼ 7.6 Hz D2O
Exchangeable); Anal. Calcd. for C30H29NO6 (%), Calcd.: C, 72.13; H,
5.85; N, 2.80, found: C, 72.06; H, 5.71; N, 2.68.
was repeated up to 10ꢀ9 dilution for each drug. From the main-
tained stock cultures of required organisms, 5 L was taken and
added into 2 mL of BHI broth. In each serially diluted tube 200 L of
m
m
above culture suspension was added. The tubes were incubated for
24 h at 37 ꢁC in the incubator and observed for turbidity.
6.1.2.17. Ethyl2-benzamido-3-(4-((3-oxo-3H-benzo[f]chromen-1-yl)
methoxy)phenyl)propionate (4g). Light brown; Yield 56%; m.p:
155e157 ꢁC; IR (KBr) cmꢀ11728 (C]O) 3370 (eNH) 1647 (amide C]
6.3. Computational methods
O); 1H NMR (DMSO, 400 MHz, TMS):
d ppm 1.10 (t, 3H, OeCH2eCH3,
6.3.1. Preparation of the ligands
J ¼ 7.2 Hz), 3.12 (m, 2H, Ph-CH2, J ¼ 6.8 Hz), 4.05 (q, 2H, OeCH2e
CH3, J ¼ 7.2 Hz), 4.61(t, 1H, PheCH2eCH, J ¼ 7.6 Hz), 5.22 (s, 2H, Oe
CH2), 6.81 (s, 1H, C3eH), 7.00e8.54 (m, 15H, AreH), 8.79 (d, 1H,
NHeCO, J ¼ 7.6 Hz, D2O Exchangeable); Anal. Calcd. for C32H27NO6
(%), Calcd: C, 73.69; H, 5.22; N, 2.69, found: C, 73.63; H, 5.19; N, 2.69.
The three-dimensional structures of coumarin derivatives were
constructed by using Sybyl X-2.0 version (Tripos Inc.) [25], running
on Dual-core Intel (R) core (TM) i3-2130 CPU 3.40 GHz, RAM
Memory 2 GB under the Windows 7 system.
6.3.2. Docking assay
6.1.2.18. Ethyl2-benzamido-3-(4-((2-oxo-2H-benzo[h]chromen-4-yl)
methoxy)phenyl)propionate (4h). Light brown; Yield 61%;
m.p:190e194 ꢁC; IR (KBr) cmꢀ1 1727 (C]O) 3361 (eNH) 1647
For the docking of ligands to protein active sites and for esti-
mating the binding affinities of docked compounds, Surflex-Dock
module, a fully automatic docking tool available on Sybyl X-2.0
version (Tripos Inc.) was used in this study. Docking simulations:
The X-ray Crystal Structure of E. coli 24 kDa Domain in Complex
(amide C]O); 1H NMR (DMSO, 400 MHz, TMS):
d ppm 1.12 (t, 3H,
OeCH2eCH3, J ¼ 7.2 Hz), 3.02 (m, 2H, Ph-CH2, 6.8 Hz),4.07 (q, 2H,
OeCH2eCH3, J ¼ 7.2 Hz), 4.61(t,1H, PheCH2eCH, J ¼ 7.6 Hz), 5.24 (s,
2H, OeCH2), 6.81 (s, 1H, C3eH), 7.02e8.62 (m, 15H, AreH), 8.73 (d,
1H, NHeCO, J ¼ 7.6 Hz D2O Exchangeable); Anal. Calcd. for
ꢀ
with clorobiocin (PDB code: 1KZN; resolution 2.30 A; http://www.
rcsb.org) [26] was obtained from protein data bank in PDB format
as starting point. Protein structure with all water molecules deleted
was used for docking simulations. Mislabelled atom types from the
pdb file were corrected, subsequently, proline F angles were fixed at
70ꢁ, side chain amides were checked to maximize potential
hydrogen bonding, side chains were checked for close van der
Waals contacts, and essential hydrogens were added. The model
was checked for conformational problems using the module
ProTable from Sybyl. Ramachandran plot [27] of the backbone tor-
sion angles phi and psi, local geometry and the location of buried
polar residues/exposed non-polar residues were examined. The
protein was subjected to energy minimization following the
gradient termination of the Powell method for 3000 iterations
using Kollman united force field with non-bonding cut-off set at 9.0
and the dielectric constant set at 4.0 [28,29] and energy minimi-
zation for synthesized compounds, including clorobiocin and
novobiocin were carried out by Powell method for 3000 iterations
using Tripos force field [30] and Gasteiger [31] charge with non-
bonding cut-off set at 9.0 and the dielectric constant set at 4.0.
The synthesized compounds and the standard compounds tested in
this study were docked to DNA gyrase subunit A (PDB code: 1KZN)
using Surflex-Dock programme in Sybyl software by incremental
construction approach of building the structure in the active site so
as to favour the binding affinity [32,33]. Finally, the docked ligands
were ranked based on a variety of scoring functions that have been
compiled into the single consensus score (C-score) [34].
C
32H27NO6 (%), Anal. Calcd. for C28H24ClNO6 (%), Calcd: C, 73.69; H,
5.22; N, 2.69, found: C, 73.11; H, 5.16; N, 2.49.
6.1.2.19. Ethyl2-benzamido-3-(4-((6-chloro-2-oxo-2H-chromen-4yl)
methoxy)phenyl)propionate (4i). Off white; Yield 57%; m.p:152e
155 ꢁC; IR (KBr) cmꢀ1 1726 (C]O) 3350 (-NH) 1648 (amide C]O);
1H NMR (DMSO, 400 MHz, TMS):
d ppm 1.13 (t, 3H, OeCH2eCH3,
J ¼ 7.2 Hz), 3.09 (m, 2H, Ph-CH2), 4.09 (q, 2H, OeCH2eCH3,
J ¼ 7.2 Hz), 4.60 (t, 1H, PheCH2eCH, J ¼ 7.6 Hz), 5.31 (s, 2H, OeCH2),
6.59 (s, 1H, C3eH), 7.05e8.09 (m, 11H, AreH), 8.78 (d, 1H, NHeCO,
J ¼ 7.6 Hz, D2O Exchangeable); Anal. Calcd. for C28H24ClNO6(%),
Calc: C, 66.47; H, 4.78; N, 2.77, found: C, 66.43; H, 4.65; N, 2.63.
6.1.2.20. Ethyl 2-benzamido-3-(4-((7-chloro-2-oxo-2H-chromen-4yl)
methoxy)phenyl)propionate (4j). Light brown; Yield 62%; m.p:110e
114 ꢁC; IR (KBr) cmꢀ11724 (C]O) 3340 (-NH) 1648 (amide C]O), 1H
NMR (DMSO, 400 MHz, TMS):
d ppm 1.14 (t, 3H, OeCH2eCH3,
J ¼ 7.2 Hz), 3.09 (m, 2H, Ph-CH2), 4.08 (q, 2H, OeCH2eCH3,
J ¼ 7.2 Hz), 4.61(t, 1H, PheCH2eCH, J ¼ 7.2 Hz),5.35 (s, 2H, OeCH2),
6.59 (s, 1H, C3eH), 7.01e8.19 (m, 11H, AreH), 8.86 (d,1H, NHeCO,
J ¼ 7.2 Hz, D2O Exchangeable); Calc: C, 66.47; H, 4.78; N, 2.77,
found: Calc: C, 66.44; H, 4.67; N, 2.65.
6.2. Anti-microbial screening
The antibacterial activity of the synthesized compounds were
performed in-vitro against (i) Gram-positive bacteria: E. faecalis
(ATCC no.35550), S. aureus (ATCC no. 12598) (ii) Gram-negative
bacteria: E. coli (ATCC No. 25922), P. aeruginosa (ATCC No.25619)
by broth dilution methods [22]. The MIC determination of the
tested compounds was carried out in comparison with
Ciprofloxacin.
7. Conclusion
The present investigation has shown that introduction of gallic
acid and tyrosine at the allylic position in the coumarins leads to
molecules with enhanced degree of specificity in their antimicro-
bial activity which is further supported by docking studies also.
The Anti-fungal activity were performed against these standard
strains: C. albicans (ATCC no.2091) and A.niger (ATCC no. 9029). The
MIC determination of the tested compounds was carried out in
comparison with Fluconazole by broth dilution method [22].
Nine dilutions of each drug were prepared with BHI (brain heart
Acknowledgements
The authors thank NMR Research Center, Indian Institute of
Science (IISc), Bangalore and Dr. V. H. Kulkarni, Principal, S.E.T’s
College of Pharmacy, Dharwad, University Sophisticated Instru-
mentation Center (USIC) for spectral analysis. One of the authors
Hrishikesh M. Revankar acknowledges UGC for providing RFSMS
scholarship. Authors thank Prof. Chung Ming Sun of National Chiao
Tung University for gift sample of N-Benzoyl tyrosine ethyl ester.
infusion) for MIC. In the initial tube 20
into the 380 L of BHI broth. Then from the initial tube 200
transferred to the first tube containing 200 L of BHI broth. This was
considered as 10ꢀ1 dilution. From 10ꢀ1 diluted tube 200
L was
transferred to second tube to make 10ꢀ2 dilution. The serial dilution
m
L (mL) of drug was added
m
mL was
m
m

H.M. Revankar et al. / European Journal of Medicinal Chemistry 70 (2013) 750e757
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Appendix A. Supplementary data
[15] E. Hejchman, P. Taciak, S. Kowalski, D. Maciejewska, science24.com/paper/
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[19] M.V. Kulkarni, B.G. Pujar, V.D. Patil, Arch. Pharm. (Weinhiem) 316 (1981)
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.047.
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