Synthesis, antimicrobial and chitinase inhibitory activities of 3-amidocoumarins

Article abstract of DOI:10.1016/j.bioorg.2020.103700

A series of 3-amidocoumarins has been synthesized and tested in vitro for their anitimicrobial and chitinase inhibitory activities. Among these, compounds 5k, 5l, 8b–8d, 8f and 8g exhibited good antibacterial activity with MIC values in the range of 6.25–25 μg/mL against some of the tested strains while compounds 5l, 8b, 8c and 8f showed good activity against at least one or two fungal strains. Some of the assayed compounds 5d, 5k, 5l, 8b and 8c displayed significant chitinase inhibitory activity with IC₅₀ values in the range of 3.74–5.6 μM. Among them, 5l proved to be potent chitinase inhibitor with IC₅₀ value of 3.74 μM. To better understand the enzyme-inhibitor interactions molecular docking study of all the synthesized compounds was carried out on Aspergillus fumigatus chitinase 1W9U. The compound 5l showed high binding affinity with the receptor with binding energy value of ?8.44 Kcal/mol. This study also provides structure activity relationship (SAR) of synthesized compounds.

Full text of DOI:10.1016/j.bioorg.2020.103700

Journal Pre-proofs
Synthesis, antimicrobial and chitinase inhibitory activities of 3-amidocoumar-
ins
Rajesh Kumar Sharma, Vineeta Singh, Neha Tiwari, R.J. Butcher, Diksha
Katiyar
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S0045-2068(19)31651-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103700
YBIOO 103700
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Bioorganic Chemistry
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30 January 2020
24 February 2020
Please cite this article as: R. Kumar Sharma, V. Singh, N. Tiwari, R.J. Butcher, D. Katiyar, Synthesis,
antimicrobial and chitinase inhibitory activities of 3-amidocoumarins, Bioorganic Chemistry (2020), doi: https://
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Synthesis, antimicrobial and chitinase inhibitory activities of 3-
amidocoumarins
Rajesh Kumar Sharma,a Vineeta Singh,b Neha Tiwari,a R. J. Butcher,c Diksha Katiyara*
aDepartment of Chemistry, MMV, Banaras Hindu University, Varanasi-221005, India
bDepartment of Biotechnology, Institute of Engineering and Technology, Sitapur Road,
Lucknow 226021
cDepartment of Chemistry, Howard University, 525 College Street NW, Washington DC 20059,
USA
*Corresponding author: E-mail: dikshakatiyar@gmail.com
Abstract
A series of 3-amido coumarins has been synthesized and tested in vitro for their
anitimicrobial and chitinase inhibitory activities. Among these, compounds 5k, 5l, 8b-8d, 8f and
8g exhibited good antibacterial activity with MIC values in the range of 6.25-25 µg/mL against
some of the tested strains while compounds 5l, 8c and 8f showed good activity against at least
one or two fungal strains. Some of the assayed compounds 5d, 5k, 5l, 8b and 8c displayed
significant chitinase inhibitory activity with IC50 values in the range of 3.74-5.6 µM. Among
them, 5l proved to be potent chitinase inhibitor with IC50 value of 3.74 µM. To better understand
the enzyme-inhibitor interactions molecular docking study of all the synthesized compounds was
carried out on Aspergillus fumigatus chitinase 1W9U. The compound 5l showed high binding
affinity with the receptor with binding energy -8.44 Kcal/mol. This study also provides structure
activity relationship (SAR) of synthesized compounds.
Keywords: Coumarin, 3-amidocoumarin, Antimicrobial, Antifungal, Chitinase inhibitors,
Molecular docking.
1

1. Introduction
Microbial diseases, especially bacterial and fungal, are plaguing the world’s population since
ancient times.1 In spite of new treatment approaches and development of novel effective drugs
from both natural and synthetic sources, these diseases continue to affect billions of people
worldwide leading to enormous loss of human life to the tune of millions of deaths annually.2-4
The mortality rate is significantly higher in immunosuppressed patients, those undergoing organ
transplants, recipients of cancer chemotherapy and patients undergoing AIDS therapy. Current
drugs of choice for the treatment of these infections are associated with toxicity, low efficacy and
emerging antimicrobial resistance.5-8 Therefore, development of newer drugs involving new
targets is of priority in antimicrobial drug research.
Chitin is N-acetylglucosamine polymer present as main structural component in
exoskeletons of arthropods and fungal cell walls. Chitinase and chitin synthase are the two major
enzymes which are involved in chitin biosynthesis. Chitinases play crucial role in exogenous
chitin decomposition, fungal cell wall degradation and remodeling.9-11 Chitin is essential for
development and survival of fungi. Because of the absence in humans, chitin is considered as
important target for antifungal drug development. Therefore, inhibition of fungal chitinases has
been proposed as important approach for the development of novel antifungals. Over recent
years, several natural and non-natural molecules have been identified and characterized as
chitinase inhibitors (Fig. 1). The most important natural inhibitors are pseudotrisaccharide
allosamidin12,13 and the cyclic peptides such as argifin14 and argadin.15 Allosamidine was isolated
from the mycelia extract of Streptomyces spp. Argifin and argadin were isolated from fungi. The
former was extracted from mycelia of Gliocladium sp., while the latter was extracted from
mycelia of Clonostachys sp. However, because of complex chemical structures and cumbersome
synthesis these molecules are not considered suitable antifungal therapeutic candidates.16-18
Several small designed and/or synthesized molecules such as xanthine19 and purine20 derivatives
as the fungal chitinase AfChiB1 inhibitors and acetazolamide21 as an AfChiA1 inhibitor, have
also been reported. In a quest to discover potent chitinase inhibitors, Jiang et al. had also recently
screened a library of over 4 million chemicals by two rounds structure-based virtual screening
and identified seventeen 1-thia-7-aza-2-indene based carboxamides.22 Recently, the chitinase
inhibitory activity of closantel,23 belonging to salicylanilide class of antihelmintic drugs, has also
2

been reported. Albeit low potency of the molecules all these researches led to renewed interest in
the rationale design of more efficient chitinase inhibitors.24
Coumarins constitute a biologically active class of oxygen-containing 2H-1-benzopyran-
2-one derivatives. They are found widely as secondary metabolite in several plant families,
bacteria and fungi.25 The nature and pattern of substitution on coumarin moiety play important
role in the pharmacological and therapeutic profile of coumarin compounds.26 Coumarins
substituted with amido group at C-3 position are known to display a wide range of biological and
pharmacological properties.27-31 The 3-benzamidocoumarin derivatives, novobiocin and
clorobiocin, are the coumarin based antibiotics which target DNA gyrase.32 Inspired by the
bioactivities of coumarin derivatives especially their anti-chitin synthase activity, Batran et al.
recently synthesized 4-hydroxycoumarins containing Schiff's base motif at the C-3 position as
antifungal agents and studied their binding interactions with modelled structures of some fungal
chitinases.33 Exploiting these ongoing development and in continuation of our work on the
development of antimicrobial agents,34,35 we envisaged to develop some novel bioactive
coumarin derivatives. Inspired by the importance of amide linkage at C-3 position of coumarin
nucleus for biological activity and also by the fact that most of the known fungal chitinase
inhibitors bear amide linkage in their chemical structures, we synthesized a series of 3-amido
coumarin derivatives and examined their antibacterial, antifungal and anti-chitinase activities.
The structure activity relationship (SAR) study of amidocoumarins was also performed to
explore the influence of length, lipophilic or electronic characteristics of the amide moiety with
various substituents. Molecular docking study was also performed to investigate the binding
interactions of synthetic compounds with the active site of A. fumigates chitinase 1W9U.
3

O
H
OH
OH
OH
O
N
NH
O
NH
O
O
HO
O
O
O
HO
O
HO
NHAc
N
H
N
N
N
NHAc
H
O
H
NMe2
OH
OH
HN
O
N
Allosamidin
N
H
O
HO
HO
O
O
Argifin
O
NH
O
O
O
NH
HN
N
O
Br
S
S
Br
N
N
HN
N
H
N
H
H
H
O
O
HO
OH
HO
OH
N
N
HO
Psammaplin
N
H
O
Argadin
CH3
O
N
O
H
N
N
O
O
Cl
O
H
S
H2N
N
S
N
N
N
N
HO
I
O
N
N
O
N
N
O
CN
Closantel
Cl
I
Acetazolamide
C2-dicaffeine
Fig.1. Chemical structures of some chitinase inhibitors
2. Results and dicussion
2.1. Chemistry
The synthetic routes to target coumarin compounds are depicted in Scheme 1. 3-nitrocoumarin 2
was prepared via L-proline catalyzed condensation of salicylaldehyde 1 and ethyl nitroacetate
according to our previously reported method.36 Reduction of 3-nitrocoumarin to 3-
aminocoumarin 3 using acidified SnCl2 followed by acylation reaction with suitable carboxylic
acids 4a-p using PCl3 in acetonitrile under reflux for 5-6 h afforded the 3-amidocoumarins 5a-p
in good yields. It is worth mentioning that acylation of 3-aminocoumarin with acid chloride,
obtained by treatment of 4a with SOCl2 in dry DCM under reflux condition for 6 hours, in
presence of triethylamine in DCM at room temperature for more than 6 hours was also
attempted. But the former method proved more efficient in respect of yield, reaction time and
work up procedure and more importantly afforded 3-amidocoumarins in one step.
4

For the preparation of coumarins derivatives 8a-g, first the commercially available
salicylaldehyde was converted into the coumarin-3-carboxylic acid 6 by knoevenagel
condensation with malonic acid in the presence of catalytic amount of L-proline. The
condensation of carboxylic acid
6
with suitable amines 7a-g using N, N'-
dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) in dry DCM at room
temperature afforded corresponding 3-carboxamide-coumarins 8a-g in good yields (ranging from
68% to 88%). The final step was also tried with PCl3 in acetonitrile but this route gave low yield.
1
13
The chemical structures of synthesized compounds were characterized by H NMR, C NMR,
IR and MS spectral data. Suitable crystals of compound 5e could be obtained by slow
evaporation of acetone solvent.37 The crystallographic data of compound 5e is summarized in
Table 1 and ORTEP plot is shown in Fig. 2. The 2D packing arrangement of crystal has shown
intra and inter molecular H-bonding interactions between N(1)-H(1) and O(1) with bond length
of 2.63 Å (blue) and between O(1)H(10) and O(2) with bond length of 2.65 Å (pink),
respectively (Fig. 3). Crystallographic data for the structure reported in this paper have been
deposited in the Cambridge Crystallographic Data Centre as supplementary publication number
CCDC-1950376. The data can be obtained free of charge from http://www.ccdc.ac.uk/structures.
5
8
4
CHO
OH
COOH
O
NO2
O
3
6
7
Malonic acid
Ethylnitroacetate
30 mol% L- Proline
EtOH, rt
10 mol% L- Proline
80-90 ºC
O
1
O
2
1
6
2
DCC, DMAP
DCM, rt, 4 h
NHRR'
7a-g
SnCl , rt
HCl
2
O
H
R COOH
N
R
R
NH2
4a-p
N
H
PCl3, CH3CN
Reflux
O
O
5a-p
O
O
O
O
O
8a-g
3
Scheme 1. Synthesis of 3-amidocoumarins
5

Table 1. Crystallographic data collection and structure refinement information of compound 5e
5e
Empirical formula
Fw
C16H11NO4
281.26
100(2)
Temp [K]
λ [Å]
Crystal system
Space group
a [Å]
b [Å]
c [Å]
α [º]
0.71073
Orthorhombic
Pbca
12.7766(3)
10.8204(2)
17.8796(4)
90
β [º]
90
γ [º]
90
V [Å3]
Z
2471.82(9)
8
3
1.512
Density [mg/m ]
F(000)
1168
1.045
Goodness-of-fit on F2
R indices (all data)
R1 = 0.0710, wR2 = 0.1327
Fig. 2. ORTEP plot of the X-ray structure of 5e
6

Fig. 3. 2D packing diagram of 5e viewed along a axis (left) and b axis (right), showing
hydrogen-bonding interactions (dotted lines).
2.2. Biology
The minimum inhibitory concentrations (MICs) of amido-coumarins 5a-p and 8a-g against four
Gram-negative bacterial strains Klebsiella pneumoniae MTCC 661, Salmonella typhi MTCC
537, Pseudomonas aeruginosa MTCC 424 and Escherichia coli MTCC 64 and two Gram-
positive bacterial strains Staphylococcus aureus MTCC 96, Bacillus pumilus MTCC 1607, and
three fungi Candida albicans MTCC 3017, A. fumigatus MTCC 2483 and C. tropicalis MTCC
184 were determined by two-fold serial dilution method using antibacterial drugs novobiocin and
chloramphenicol and antifungal drug amphotericin B and fluconazole as standards.38 The results
are summarized in Table 2.
Table 2: Antimicrobial activity and chitinase inhibitory (expressed in IC50 µM) activities of
synthetic compounds
Minimum Inhibitory Concentration (MIC in µg/mL)a
St Pa Ec Sa Bp Ca Af
Compound
R (5a-5p) and
IC50
µM
NRR' (8a-8g)
Kp
Ct
5a
5b
CH3
200
50
200
100
100
200
200
>200
100
8.6
8.6
CH3
100
50
200
50
100
200
100
100
CH3
5c
-C11H23
25
25
50
50
100
100
25
50
100
100
200
200
100
50
100
50
100
200
6.2
(CH2)16CH3
5d
4.68
HO
5e
50
50
>200
50
100
100
>200
200
200
100
>200
100
100
200
100
200
200
200
7.0
6.68
7.2
Cl
5f
100
>200
50
50
>200
50
>200 >200
H3C
5g
5h
>200 >200
100
50
200
100
O
200
200
>200 >200
6.78
H3C
HO
CH3
5i
5j
200
200
12.5
25
25
100
100
100
25
200
50
50
100
200
200
6.76
HO
>200
200
>200
10.14
NH2
7

HO
5k
5l
100
200
50
12.5
50
12.5
6.25
25
200
25
25
50
25
200
100
5.38
3.74
NO2
I
12.5
12.5
12.5
HO
I
OH
5m
200
25
100
25
50
200
50
100
100
6.4
OH
OH
HN
5n
5o
5p
200
25
100
100
25
>200
200
50
50
25
100
200
50
200
200
100
25
200
25
50
200
50
100
200
100
11.61
7.6
N
100
200
7.5
N
N
8a
8b
200
200
100
200
100
25
>200
12.5
200
100
100
50
100
200
8.2
5.6
NH
NH
6.25
12.5
>200
12.5
8c
200
25
200
12.5
25
25
>200
>200
25
12.5
100
200
4.6
8.8
NH
8d
>200
12.5
>200
12.5
>200
>200
NH
8e
>200
>200 200
25
>200
25
25
50
100
7.4
NH
8f
>200
>200
6.25
50
6.25
25
12.5
6.25
25
200
6.25
25
12.5
50
50
11.7
7.4
N
8g
12.5
>200
100
N
N
Novobiocin
Chloramphenicol
Amphotericin B
Fluconazole
25
>25
Rt
30
0.15
<0.78
-
-
-
-
-
-
-
12.5
12.5
12.5
12.5
12.5
12.5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12.5
12.5
12.5
12.5
6.25
12.5
‘–’: not determined; ‘Rt’: resistant; aK. pneumoniae (Kp), S. typhi (St), P. aeruginosa (Pa), E. coli (Ec), S. aureus
(Sa), B. pumilus (Bp), C. albicans (Ca), A. fumigates (af), C. tropicalis (Ct).
By analyzing the data, it can be seen that substitution of small acyclic or cyclic aliphatic
side chain at amidic nitrogen appears to be unfavorable as compounds 5a, 5b and 5n bearing
butyl isopropyl and pyrrolidene substituents, respectively, displayed poor activity toward the
tested strains. However, the presence of a bulky aliphatic group such as dodecyl 5c and stearyl
5d led to some enhancement in activity. These compounds exhibited activity in the range of 25-
50 μg/mL against three bacterial strains. Interesting results were obtained with the introduction
of aromatic group in place of aliphatic group. Compounds 5e, 5f, 5g and 5h bearing hydroxyl,
8

chloro, methyl and methoxy substituted phenyl ring, respectively, displayed high MIC values in
the range of 50 to >200 µg/mL. Some enhancement in activity was observed upon introduction
of disubstituted phenyl ring in compounds 5i, 5j and 5k. Compound 5i inhibited the growth of S.
typhi and P. aeruginosa at 12.5 and 25µg/mL concentration, respectively, and compound 5j
having amino and hydroxyl substituted phenyl ring resulted in activity against bacterial strains S.
typhi and S. aureus with MIC values 25 µg/mL. The activity increases with the introduction of
electron withdrawing nitro group in place of amino group as compound 5k showed activity
against three bacterial strains with MIC 12.5-25 µg/mL. Whereas compound 5l having diiodo
and hydroxyl groups exhibited significant activity against S. typhi, E. coli, S. aureus and B.
pumilus at lower MIC (6.25-25 µg/mL). However, introduction of three hydroxyl groups at the
phenyl ring led to compound (5m) showing decreased bactericidal activity. Furthermore,
introduction of heteroaromatic substitutents in compounds 5o and 5p also resulted in reduction in
activity profile. Among the 3-carboxycoumarin derivatives, long alkyl chain containing
compounds 8b (dodecyl group) and 8c (oleyl goup) showed better activity against three (S. typhi,
E. coli and S. aureus) bacterial strains with MIC in range of 6.25-25 µg/mL, respectively, than
the short alkyl chain containing compound 8a. Compounds 8d and 8e bearing cyclopropyl and
cyclohexyl residues displayed activity against three and one bacterial strains, respectively.
Interestingly, compound 8f bearing piperidinyl ring displayed very good activity against four
bacterial strains S. typhi, P. aeruginosa, E. coli and S. aureus (MIC = 6.25-25 µg/mL) and 8g
with piperazinyl ring showed activity against two bacterial stains (E. coli and S. aureus) at
concentration 6.25 µg/mL and 12.5 µg/mL, respectively.
As indicated in Table 2, compounds 5l, 5n, 5p, 8b, 8c, 8f and 8g showed activity at
concentrations 6.25-25 μg/mL at least against one fungal strains. Among these, compounds 5l, 8c
and 8f displayed good antifungal activity against C. albicans and A. fumigatus with MIC’s (6.25-
25 μg/mL) comparable to that of standard drugs. However, the other compounds of the series
showed poor activity with MIC’s in the range of 50 to >200 μg/mL against all the fungal strains
tested.
Altogether, in this series of compounds, SAR study revealed that the antimicrobial activity of
compounds depends on the nature of substituents present at the amido component. The synthesized
compounds bearing phenyl ring substituted with hydroxyl group along with electron donating methyl
group 5i, amino group 5j, and electron withdrawing nitro 5k and diiodo 5l substitutents, showed
9

higher antibacterial activity with MIC values ranging from 6.25-25 μg/mL than compounds bearing
monosubstituted phenyl group (5i-5l vs 5e-5h). This may be due to the reason that the di or
trisubstituted phenyl ring probably induces additional intermolecular interactions leading to the better
penetration of the compounds through the bacterial membrane. Next, comparison of activity profile of
compounds 5c and 8b bearing the dodecyl side chain indicated that 3-carboxy coumarin derivative
showed superior activity than 3-amino coumarin derivative. Furthermore, the higher activity of
compounds bearing long alkyl chains (5c, 5d, 8b and 8c) than those bearing short alkyl chains (5a,b
and 8a) also supports the results of previous studies reporting relationship between lipophilicity and
antimicrobial activity of compounds.35,39,40 Among all the tested compounds, compounds 5l, 8b and
8f were the most promising and showed broad spectrum antimicrobial activity.
2.3. Chitinase inhibition effect
All synthesized coumarins were further assessed for their ability to inhibit chitinase in vitro by
Dinitorsalicylic acid (DNS) method as mentioned by Miller41 using colloidal chitin as a
substrate. The activity results indicated that compounds inhibited the chitinase activity with IC50
values ranging from 3.74 µM to 11.7 µM. It can be seen that compound 5l with diiodo hydroxyl
substituent was the most potent chitinase inhibitor with IC50 value of 3.74 µM. Further,
substitution of amidic nitrogens with long alkyl chain (compounds 5d, 8b and 8c) appears to be
favorable. These compounds exhibited IC50 values at 4.68, 5.6 and 4.6 µM, respectively. While
substitution of nitrogen with short alkyl chain and cycloalkyl group resulted in decrease in anti-
chitinase activity (compounds 5a, 5b, 8a, 8d and 8e). It can be seen that the presence of an
electron withdrawing nitro group at N-phenyl moiety in compound 5k improves activity
compared to compounds 5i, 5j and 5m containing electron donating groups. The mono
substitution in N-phenyl moiety (compounds 5e-5h) and introduction of heterocyclic group as
amidic substituents (compounds 5n, 5o, 5p, 8f and 8g) leads to inferior activity towards
chitinase. Interestingly, these amido coumarins showed superior activity compared to recently
reported chitinase inhibitors.33 Sakuda et al. reported that the allosamidin at 10µM or above this
concentration is able to inhibit the chitinase.42
10

2.4 Molecular docking study
The docking studies of amidocoumarins were performed using the AutoDock Tools
(ADT),43 to rationalize their chitinase inhibitory activity. A well categorized chitinase of
Aspergillus fumigatus (PDB code: 1W9U) was used as the template. The binding energies of
docked compounds are shown in Table 3. The active pocket consisted of 16 amino acid residues
including Ala244, Tyr245, Asp246, Phe251, Trp384, Tyr48, Trp137, Phe76, Gly136, Asp175,
Glu177, Tyr176, Ala217, Met243, Met382, Tyr178. The docked molecules showed binding
energy towards the target receptor ranging from -5.54 to -8.44 Kcal/mol. For comparison
purpose, drug allosamidine was also docked into the same binding site and interestingly out of
twenty three docked molecules, twelve showed higher binding affinity i.e. lower binging energy
(-6.52 to -8.44 Kcal/mol) with the receptor than the allosamidine (B. E. = -6.46 Kcal/mol). Here,
we discuss the binding modes of top five ranked amidocoumarins in the binding site of chitinase.
Among the docked molecules, 5l showed the minimum binding energy value of -8.44 Kcal/mol.
This compound exhibited van der Waals interactions with both hydrophobic and polar residues
in the binding pocket. The hydroxyl group of amido substituent showed hydrogen bond with
carboxyl group of Asp246 of length 2.92Å. The amido substituent was stabilized by Trp137,
Gly136, Asp175, Phe 76, Tyr48, Trp384. The binding is further augmented by π stacking
interactions between benzene group of coumarin and Tyr245. Next compound 5n showed
binding energy value of -8.21 Kcal/mol. The coumarin ring of this compound showed
hydrophobic interactions with Pro219, Ala220 and Gly 221. This compound displayed three
hydrogen bonds between: (i) NH of amido group and Asp246 (ii) carbonyl of amido group with
Tyr178 (iii) NH of proline ring with carboxylic group of Glu177. In compound 5e, coumarin ring
was involved in π stacking with Trp137 and the hydroxyl group of amide substituent formed
hydrogen bond with Ala220 with bond length of 2.82Å. Whereas compound 8e, bearing
cyclohexyl substitution at amide showed slightly less effective binding with the receptor (binding
energy=-7.57 kcal/mol) as compared to compound 5e. The NH of the amide linkage of this
compound formed a hydrogen bond with the hydroxyl group of the Tyr178 with the bond length
of 2.10Å. Benzene ring of coumarin moiety formed stacking contacts to Trp384 and Trp137. The
coumarin ring also showed hydrophobic interactions with Trp137, Gly136 and Phe76 and the
substituent cyclohexane formed hydrophobic interactions with Pro219, Ala220, Gly221, Phe 273
and Tyr247. Compounds 5i showed one hydrogen bond interaction between hydroxyl oxygen of
11

amide substituent and Ala220 residue with bond length of 3.07Å. This compound also showed
hydrophobic interactions with Trp137, Ala217, Tyr245, Glu177, Ala244, Gly136 and Tyr48
residues. Coumarin ring showed π stacking with Trp384 residue. Overall, the docking results
showed that the compound 5l which found to be the best in vitro chitinase assay also showed
lowest binding energy. However, it seems that the other compounds 5c, 5d, 5k, 8b and 8c which
displayed good to moderate in vitro chitinase activity did not exactly fit in the active site and fail
to achieve good in silico score.
Fig. 4. Compound 5l docked with in the active site of 1W9U.
Fig. 5. Compound 5n docked with in the active site of 1W9U.
Fig. 6. Compound 5e docked with in the active site of 1W9U.
12

Fig. 7. Compound 8e docked with in the active site of 1W9U.
Fig. 8. Compound 5i docked with in the active site of 1W9U.
Table 3. Docking results of coumarin derivatives with A. fumigatus chitinase 1W9U
Ligand
Binding Energy
(Kcal/mol)
No. of H-
Bonds
Ligand
Binding Energy
(Kcal/mol)
No. of H-
Bonds
5l
5n
5e
8e
5i
-8.44
-8.21
-7.90
-7.57
-7.36
-7.15
-7.10
-6.8
1
3
1
1
1
3
2
1
6
1
1
3
8a
-6.42
-6.38
-6.36
-6.35
-6.30
-6.08
-6.05
-5.91
-5.57
-5.56
-5.54
-6.46
1
2
2
2
2
1
2
0
1
1
1
3
5h
5p
5m
8f
8b
8g
5f
8d
5c
5b
5j
-6.79
-6.79
-6.74
-6.52
5a
5g
5o
5k
8c
5d
Allosamidine
13

3. Conclusion
In summary, we designed and synthesized a new series of 3-amidocoumarins and evaluated the
compounds for their in vitro antimicrobial and chitinase inhibitory properties. The results show
that the 3-amidocoumarin derivatives 5k, 5l, 8b-8d, 8f and 8g exhibited superior bactericidal
activity among all the tested compounds. These compounds were found to be equipotent or even
displayed better antibacterial activity against some of the bacterial strains than the standard drug
chloramphenicol. Compounds 5k and 8f (MIC = 12.5 µg/ml and 6.25 µg/ml, respectively)
displayed significant activity against novobiocin resistant P. aeruginosa. In case of antifungal
activity, compounds 5l and 8b were equipotent to standard drugs fluconazole and amphotericin B
against C. albicans while compound 8f was two-fold more active. Compound 8f and 8c exhibited
activity at concentration of 12.5 µg/mL against A. fumigatus, which was camparable to
fluconazole. Some of the compounds from this new series such as 5d, 5k, 5l, 8b and 8c also
exhibited good in vitro chitinase inhibitory activity in which compound 5l showed the highest
potency for chitinase inhibition with IC50 value of 3.74 µM. Compound 5l was stabilized into the
active site of receptor through both hydrophobic and polar interactions and also exhibited lowest
binding energy (-8.44 Kcal/mol) with the receptor among all the docked compounds. Overall,
from this study compounds 5l and 8f emerged as promising antimicrobial agents and further
optimization of these compounds is in progress. This work also provides a new structural type
for the design of chitinase inhibitors.
4. Experimental Section
4.1 Chemistry
Unless otherwise stated, all common reagents and solvents were obtained from commercial
suppliers (Sigma-Aldrich and Spectrochem Pvt. Ltd.) and were used without further purification.
Melting points were determined on a Buchi 510 apparatus and are uncorrected. All the reactions
were monitored by thin layer chromatography (TLC) over basic alumina coated TLC plates and
the spots were visualized by UV absorbance (254 nm) and by iodine vapors. The pure
compounds were isolated by column chromatography using silica gel of mesh size 60-120. IR
spectra were recorded on a JASCO FTIR 5300 in KBr from 400 to 4000 cm-1. Electron spray
1
ionization Mass Spectra (ESIMS) were obtained on Micromass quadro II spectrometer. H and
14

13C NMR spectra were recorded on JEOL ECZ 500 MHz spectrometer using the residual solvent
13
13
peaks of CDCl3 (1H δ 7.26; C δ 77.2), and DMSO-d6 (1H δ 2.50; C δ 39.5), as an indirect
reference to TMS (δ = 0 ppm). The chemical shifts (δ scale) are reported in parts per million
1
(ppm). H NMR spectra are reported in the order: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, m = multiplet and brs = broad singlet), coupling constant (J value) in hertz
(Hz), and integration.
3-nitrocoumarin 2
Salicylaldehyde (1 mmol) and ethyl nitroacetate (1 mmol) were dissolved in 3 ml ethanol and L-
proline (30 mol %) was added. The reaction mixture was stirred for four hours at room
temperature and the status of the reaction was monitored by TLC. After completion of the
reaction the solvent was evaporated and the crude product was dissolved in 12 mL CHCl3 and
washed with water (3 x 10 mL). The organic layer was separated and washed with brine (2 x 10
mL), dried over anhydrous NaSO4, and evaporated under reduced pressure. The residue was
1
recrystallized from ethanol. Yield: 84%, mp 142-143 °C (lit.36 141-142 °C); H NMR (CDCl3):
8.78 (s, 1H, H-4), 7.76 (m, 2H, H-5 and H-7), 7.46 (m, 2H, H-6 and H-8).
3-aminocoumarin 3
To a stirred suspension of 37.4 % HCl (8 mL) and stannous chloride dihydrate (7.5 mmol)
compound 3 (1.0 mmol) was added at room temperature in small portions. The resulting mixture
was stirred for 5 h and then poured onto 20 g of ice. The solution was made alkaline by adding
NaOH solution (5 M) in an ice bath. The resulting suspension was then extracted with ethyl
acetate (2 x 25 mL). The organic layer was dried over anhydrous sodium sulfate and
concentrated.44 The pale yellow solid obtained was recrystallized from methanol. Pale yellow;
1
Yield: 69%, mp 136-138 °C. MS (ESI): m/z 162.05 [M + H]+; H NMR (CDCl3): 7.28 (m, 4H,
H-5, H-6, H-7 and H-8), 6.69 (s, 1H, H-4), 4.27 (brs, 2H, NH2).
General Procedure for the synthesis of compounds 5a-p
The 3-aminocompound 4 (1 mmol) and appropriate carboxylic acid 4a-p (1.1 mmol) were
dissolved in 3 mL CH3CN and the mixture was stirred at room temperature for half an hour. The
15

PCl3 (1 mmol) was added dropwise in the reaction mixture and the reaction mixture was again
refluxed for 5-6 h but in case of compounds 5c and 5p 8 h refluxing was required. After
completion the reaction was quenched by adding ice cold water (5 mL) and the solvent was
evaporated under reduced pressure. The residue was dissolved in chloroform (10 mL) and
washed twice with water (2 x 20 mL) and then with saturated NaHCO3 solution (10 mL) and
finally with brine (20 mL). The organic layer was dried over Na2SO4 and the solvent was
evaporated under vacuum. The solid products obtained in case of compounds 5a, 5b, 5d, 5f, 5h,
and 5p were recrystallized from acetone while those in case of compounds 5c, 5i and 5l were
recrystallized in acetone and DMSO mixture (3:1). The crude products obtained in case of
compounds 5e, 5g, 5j, 5k and 5m-5o were purified by silica gel column using
chloroform:methanol (9:1) as eluent.
N-(2-Oxo-2H-chromen-3-yl)butyramide 5a:
Yellow solid; Yield: 91%; mp 116-118 °C; Rf = 0.23 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν
1
cm-1: 3333, 3089, 2930, 1714, 1680, 1604, 1535, 752; MS (ESI): m/z 232 [M + H]+; H NMR
(500 MHz, CDCl3): δ 8.69 (s, 1H, H-4), 8.09 (bs, 1H, NH), 7.49 (d, J = 7.5 Hz, 1H, H-5), 7.44
(m, 1H, H-7), 7.30 (m, 2H, H-6 and H-8), 2.40 (t, J = 7.5 Hz, 2H, -CH2CH2), 1.77 (m, 2H, -
13
CH2CH3), 1.01 (t, J = 6.5 Hz, 3H, CH3); C NMR (125 MHz, CDCl3): δ 168.4, 158.9, 149.9,
129.6, 127.8, 125.2, 124.0, 123.2, 119.9, 116.4, 39.6, 18.8, 13.7.
N-(2-Oxo-2H-chromen-3-yl)isobutyramide 5b:
Orange solid; Yield: 90%; mp 125-126 °C; Rf = 0.35 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-
1: 3336, 3083, 2931, 1717, 1681, 1606, 1527, 1361, 1176, 756; MS (ESI): m/z 232.09 [M + H]+;
1H NMR (500 MHz, CDCl3): δ 8.69 (s, 1H, H-4), 8.03 (brs, 1H, NH), 7.47 (d, J = 6.5 Hz, 1H, H-
5), 7.42 (m, 1H, H-7), 7.32 (m, 2H, H-6 and H-8), 2.59 (m, 1H, -CH), 1.26 (d, J = 6.5 Hz, 6H,
13
2CH3); C NMR (125 MHz, CDCl3): δ 176.5, 159.0, 149.9, 129.6, 127.8, 125.2, 124.1, 123.3,
120.0, 116.4, 36.8, 19.4.
N-(2s-Oxo-2H-chromen-3-yl)dodecanamide 5c:
White solid; Yield: 72%; mp143-144 °C; Rf = 0.53 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-1:
3339, 3087, 1715, 1685, 1523, 1367, 1171, 753 cm-1; MS (ESI): m/z 344.21 [M + H]+; 1H NMR
(500 MHz, CDCl3): δ 8.50 (s, 1H, H-4), 7.80 (brs, 1H, NH), 7.45 (d, J = 7.5 Hz, 1H, H-5), 7.42
16

(m, 1H, H-7), 7.32 (m, 2H, H-6 and H-8), 2.43 (m, 2H, -CH2CH2), 1.68 (m, 2H, -CH2CH2), 1.28
13
(m, 16H, 8 x CH2), 0.86 (t, J = 6.5 Hz, 3H, CH3); C NMR (125 MHz, DMSO-d6): δ 173.5,
157.9, 150.0, 129.5, 127.8, 125.1, 123.7, 120.0, 116.0, 110.0, 36.8, 31.8, 29.9, 29.7, 29.7, 29.6,
29.6, 25.5, 22.4, 14.3.
N-(2-Oxo-2H-chromen-3-yl)stearamide 5d:
White solid; Yield: 89%; mp 85-86 °C; Rf = 0.59 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-1:
1
3331, 3083, 1716, 1687, 1529, 1361, 1179, 757 cm-1; MS (ESI): m/z 428.31 [M + H]+; H NMR
(500 MHz, CDCl3): δ 8.69 (s, 1H, H-4), 8.01 (brs, 1H, NH), 7.48 (d, J = 7.5 Hz, 1H, H-5), 7.42
(m, 1H, H-7), 7.32 (m, 2H, H-6 and H-8), 2.45 (m, 2H, COCH2), 1.69 (m, 2H, COCH2CH2), 1.54
13
(m, 2H, COCH2CH2CH2 ), 1.29 (m, 26H, 13 x CH2), 0.87 (t, J = 6.5 Hz, 3H, CH3); C NMR
(125 MHz, CDCl3): δ 172.8, 157.8, 149.9, 129.6, 127.8, 125.2, 124.0, 123.2, 119.9, 116.4, 36.8,
32.6, 32.0, 31.9, 29.8, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.3, 29.2, 27.2, 22.7, 14.1.
2-Hydroxy-N-(2-oxo-2H-chromen-3-yl)benzamide 5e:
Yellow solid; Yield: 68%; mp 196-197 °C; Rf = 0.65 (Hexane-Ethyl acetate, 7:3); (KBr) ν cm-1:
3105, 3078, 1716, 1629, 1556, 1229, 1167, 757 cm-1; MS (ESI): m/z 282.07 [M + H]+; 1H NMR
(500 MHz, DMSO-d6): δ 11.16 (brs, 1H, OH), 8.79 (s, 1H, H-4), 7.98 (dd, J = 8.5 and 1.5 Hz,
1H, ArH), 7.56 (d, J = 7.0 Hz, 1H, H-5), 7.41 (m, 1H, H-7), 7.33 (m, 3H, H-6, H-8 and NH),
13
7.24 (m, 1H, ArH), 6.92 (d, J = 8.5 Hz, 1H, ArH), 6.91 (m, 1H, ArH); C NMR (125 MHz,
DMSO-d6): δ 164.6, 157.9, 155.9, 149.3, 133.3, 130.7, 128.9, 127.4, 125.4, 124.7, 122.3, 119.4,
118.0, 117.3, 116.7, 115.6.
2-Chloro-N-(2-oxo-2H-chromen-3-yl)benzamide 5f:
White solid; Yield: 65%; mp 150-151 °C; Rf = 0.46 (Hexane-Ethyl acetate, 7:3); IR (KBr): V =
1
3379, 3079, 3039, 1714, 1672, 1594, 1368, 740 cm-1; MS (ESI): m/z 300.04 [M + H]+; H NMR
(500 MHz, CDCl3): δ 8.93 (brs, 1H, NH), 8.87 (s, 1H, H-4), 7.77 (d, J = 7.5 Hz, 1H, H-5), 7.57
(d, J = 7.0 Hz, 1H, ArH), 7.48 (m, 3H, ArH and H-7), 7.44 (m, 1H, ArH), 7.33 (m, 2H, H-6 and
13
H-8); C NMR (125 MHz, CDCl3): δ 165.3, 158.7, 150.1, 133.9, 132.4, 131.1, 130.8, 130.3,
130.0, 128.0, 127.4, 125.3, 124.1, 124.0, 119.8, 116.5.
17

2-Methyl-N-(2-oxo-2H-chromen-3-yl)benzamide 5g:
White solid; Yield: 86%; mp 149-150 °C; Rf = 0.64 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-
1: 3380, 3075, 1722, 1683, 1542, 1372, 745 cm-1; MS (ESI): m/z 280.0 [M + H]+; 1H NMR (500
MHz, DMSO-d6): δ 8.92 (brs, 1H, NH), 8.85 (s, 1H, H-4), 7.98 (d, J = 8.5 Hz, 1H, ArH), 7.76 (d,
J = 8.5 Hz, 1H, H-5), 7.41 (m, 2H, H-7, ArH), 7.36 (m, 2H, H-6 and H-8), 7.22 (m, 2H, Ar-H),
13
2.44 (s, 3H, CH3); C NMR (125 MHz, DMSO-d6): δ 169.2, 159.1, 156.0, 147.7, 139.4, 132.6,
128.6, 126.6, 126.3, 125.6, 125.2, 124.5, 121.7, 119.7, 117.1, 116.0, 21.4.
2-Methoxy-N-(2-oxo-2H-chromen-3-yl)benzamide 5h:
White solid; Yield: 56%; mp 178-179 °C; Rf = 0.56 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-
1: 3294, 1716, 1665, 1602, 1539, 1363, 1163, 750 cm-1; MS (ESI): m/z 296.0 [M + H]+; 1H NMR
(500 MHz, CDCl3): δ 10.93 (brs, 1H, NH), 8.88 (s, 1H, H-4), 8.26 (dd, J = 7.5 and 2.0 Hz, 1H,
ArH), 7.53 (m, 2H, H-5 and ArH), 7.42 (m, 1H, H-7), 7.32 (m, 2H, H-6 and H-8), 7.13 (m, 1H,
ArH), 7.05 (d, J = 8.5 Hz, 1H, ArH), 4.11 (s, 3H, OCH3); 13C NMR (125 MHz, CDCl3): δ 164.2,
158.9, 157.6, 149.9, 133.8, 132.2, 129.2, 127.7, 124.9, 123.3, 121.4, 120.7, 120.3, 120.2, 116.2,
112.1, 56.2.
2-Hydroxy-4-methyl-N-(2-oxo-2H-chromen-3-yl)benzamide 5i
Off white solid; Yield: 58%; mp 142-144 °C; Rf = 0.62 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν
1
cm-1: 3110, 2830, 1746, 1619, 1558, 1222, 1049, 761 cm-1; MS (ESI): m/z 318.07 [M + Na]+; H
NMR (300 MHz, DMSO-d6): δ 11.08 (brs, 1H, OH), 8.77 (s, 1H, H-4), 7.86 (m, 1H, ArH), 7.52
(m, 1H, H-5), 7.37 (m, 1H, H-7), 7.25 (m, 2H, H-6 and H-8), 6.75 (m, 2H, ArH), 2.23 (s, 3H,
13
CH3); C NMR (75 MHz, DMSO-d6): δ 164.2, 157.9, 155.8, 149.2, 144.0, 132.1, 130.5, 128.8,
127.2, 125.4, 124.7, 122.1, 121.2, 120.6, 119.6, 116.9, 20.9.
5-Amino-2-hydroxy-N-(2-oxo-2H-chromen-3-yl)benzamide 5j:
Yellow solid; Yield: 80%; mp 217-218 °C; Rf = 0.64 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-
1: 3286, 1705, 1658, 1549, 1365, 1219, 754 cm-1; MS (ESI): m/z 296.08 [M + H]+; 1H NMR (500
MHz, DMSO-d6): δ 11.27 (brs, 1H, OH), 8.81 (s, 1H, H-4), 7.73 (d, J = 7.0 Hz, 1H, H-5), 7.52
(m, 2H, ArH and H-7), 7.38 (m, 2H, H-6 and H-8), 6.97 (dd, J = 8.5 and 3.0 Hz, 1H, ArH), 6.90
18

(d, J = 9.0 Hz, 1H, ArH), 3.5 (brs, 2H, NH2); 13C NMR (125 MHz, DMSO-d6): δ 163.8, 158.0,
150.0, 149.4, 135.4, 128.6, 127.9, 125.0, 124.9, 123.5, 122.2, 119.8, 118.4, 117.8, 115.9.
2-Hydroxy-5-nitro-N-(2-oxo-2H-chromen-3-yl)benzamide 5k:
Sticky solid; Yield: 72%; Rf = 0.52 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-1: 3352, 3016,
1
2745, 1716, 1672, 1529, 1265, 1170, 756 cm-1; MS (ESI): m/z 327.03 [M + H]+; H NMR (500
MHz, DMSO-d6): δ 11.4 (brs, 1H, OH), 8.79 (m, 2H, ArH), 8.53 (s, 1H, H-4), 7.66 (d, J = 7.5
Hz, 1H, H-5), 7.45 (m, 1H, H-7), 7.29 (m, 3H, H-6, H-8 and ArH); 13C NMR (125 MHz, DMSO-
d6): δ 169.1, 164.4, 158.1, 150.0, 141.4, 130.5, 129.8, 128.4, 127.4, 125.7, 125.5, 125.4, 123.1,
122.7, 120.4, 116.3.
2-Hydroxy-3,5-diiodo-N-(2-oxo-2H-chromen-3-yl)benzamide 5l:
Yellow solid; Yield: 75%; mp 190-191 °C; Rf = 0.43 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-
1: 3258, 3021, 2755, 1719, 1685, 1535, 1269, 1168, 759 cm-1; MS (ESI): m/z 533.87 [M + H]+;
1H NMR (500 MHz, DMSO-d6): δ 11.0 (brs, 1H), 8.57 (s, 1H, ArH), 8.24 (s, 1H, ArH), 8.20 (s,
1H, H-4), 7.73 (d, J = 7.5 Hz, 1H, H-5), 7.53 (m, 1H, H-7), 7.41 (d, J = 7.5 Hz, 1H, H-8), 7.34
13
(m, 1H, H-6); C NMR (125 MHz, DMSO-d6): δ 165.3, 158.2, 157.9, 150.8, 150.4, 138.2,
131.0, 128.8, 128.3, 125.6, 124.2, 120.8, 119.7, 116.5, 92.0.
3,4,5-Trihydroxy-N-(2-oxo-2H-chromen-3-yl)benzamide 5m:
Off white solid; Yield: 55%; mp 209-211 °C; Rf = 0.32 (Hexane-Ethyl acetate, 1:1); IR (KBr) ν
1
cm-1: 3345, 3018, 1716, 1688, 1533, 1266, 1165, 756 cm-1; MS (ESI): m/z 314 [M + H]+; H
NMR (500 MHz, DMSO-d6): δ 9.0 (brs, 1H, OH), 8.56 (s, 1H, H-4), 7.71 (d, J = 7.5 Hz, 1H, H-
5), 7.49 (m, 1H, H-7), 7.39 (m, 3H, H-6, H-8 and OH), 7.34 (bs, 1H, OH), 6.89 (m, 2H, ArH);
13C NMR (125 MHz, DMSO-d6): δ 165.8, 158.3, 150.2, 146.3, 132.5, 128.4, 125.6, 124.6,
120.1, 118.7, 116.5, 107.2.
N-(2-Oxo-2H-chromen-3-yl)pyrrolidine-2-carboxamide 5n:
Yellow solid; Yield: 63%; mp 115-117 °C; Rf = 0.23 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-
1
1: 3334, 3081, 1715, 1679, 1525, 1359, 1174, 754 cm-1; MS (ESI): m/z 259.1 [M + H]+; H
NMR (500 MHz, CDCl3): δ 8.67 (s, 1H, H-4), 7.90 (brs, 1H, NH), 7.46 (d, J = 7.5 Hz, 1H, H-5),
19

7.41 (m, 1H, H-7), 7.29 (m, 2H, H-6 and H-8), 4.24 (m, 1H, NCH), 3.47 (m, 2H, pyrrolidene
13
CH2), 2.12 (m, 2H, pyrrolidene CH2), 1.82 (m, 2H, pyrrolidene CH2); C NMR (125 MHz,
CDCl3): δ 169.2, 159.5, 149.1, 129.3, 127.9, 125.1, 124.7, 124.5, 121.2, 119.8, 117.0, 116.2,
61.8, 45.6, 31.5, 24.7.
N-(2-Oxo-2H-chromen-3-yl)picolinamide 5o:
White solid; Yield: 88%; mp 168-169 °C; Rf = 0.36 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-
1: 3335, 3085, 1714, 1682, 1525, 1364, 1175, 758 cm-1; MS (ESI): m/z 267 [M + H]+; 1H NMR
(500 MHz, CDCl3): δ 10.7 (bs, 1H, NH), 8.87 (s, 1H, H-4), 8.68 (d, J = 5.0 Hz, 1H, pyridinyl
proton), 8.25 (d, J = 8.5 Hz, 1H, pyridinyl proton), 7.91 (m, 1H, pyridinyl proton), 7.54 (m, 2H,
H-5 and pyridinyl proton), 7.45 (m, 1H, H-7), 7.33 (m, 2H, H-6 and H-8); 13C NMR (125 MHz,
CDCl3): δ 163.6, 158.2, 150.3, 149.0, 148.6, 137.6, 129.7, 127.9, 127.0, 125.1, 123.5, 122.3,
120.0, 116.5.
N-(2-Oxo-2H-chromen-3-yl)pyrazine-2-carboxamide 5p:
Coffee brown solid; Yield: 92%; mp 198-200°C; Rf = 0.60 (Hexane-Ethyl acetate, 7:3); IR
(KBr) ν cm-1: 3340, 3078, 1712, 1683, 1537, 1371, 1171, 1020, 757 cm-1; MS (ESI): m/z 268 [M
1
+ H]+; H NMR (500 MHz, CDCl3): δ 10.47 (brs, 1H, NH), 9.48 (s, 1H, pyrazinyl proton), 8.89
(s, 1H, H-4), 8.85 (d, J = 3.0 Hz, 1H, Pyrazinyl proton), 8.67 (d, J = 2.0 Hz, 1H, pyrazinyl
proton), 7.59 (dd, J = 7.5 and 2.0 Hz, 1H, H-5), 7.51 (m, 1H, H-7), 7.38 (m, 2H, H-6 and H-8);
13C NMR (125 MHz, CDCl3): δ 162.1, 158.5, 150.4, 148.1, 144.5, 143.7, 143.0, 130.1, 128.0,
125.3, 124.2, 123.7, 119.7, 116.5.
3-Carboxycoumarin 6
To the stirred reaction mixture of malonic acid (1 mmol) and salicylaldehyde (1 mmol), L-
proline (10 mol %) was added under neat conditions. The reaction mixture was refluxed for 6 h
and the completion of reaction was monitored by TLC. After completion of the reaction, the
reaction mixture was cooled and solid obtained was recrystallized from ethanol. Yield: 89%, mp
189-191 °C (lit.45 190-191 °C); 1H NMR (CDCl3): 7.48 (s, 1H, H-4), 6.54 (d, J = 8.0 Hz, 1H, H-
5), 6.45 (m, 1H, H-7), 6.13 (m, 2H, H-6 and H-8).
20

General Procedure for the synthesis of coumarin derivatives 8a-g
3-Carboxycoumarin 6 (1 mmol) was dissolved in 10 mL of dichloromethane and DCC (1 mmol)
was added to the mixture. The resulting reaction mixture was stirred for 1 h at room temperature.
Then appropriate amine 7a-g (1 mmol) and DMAP (10 mol%) were added sand stirring
continued at room temperature for 3-4 hours. The progress of the reaction was monitored by
TLC. After the completion of the reaction, the reaction mixture was diluted with DCM (5 mL)
and solid formed was separated by filtration. The organic layer was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel
(60–120 mesh) column chromatography using 1% MeOH in CHCl3 as eluent.
N-(Butyl)-2-oxo-2H-chromene-3-carboxamide 8a
White solid; Yield: 70%; mp 70-71 °C; Rf = 0.75 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-1:
1
3337, 3084, 1718, 1682, 1528, 1362, 1177, 757 cm-1; MS (ESI): m/z 246.11 [M + H]+; H NMR
(500 MHz, CDCl3): δ 8.89 (s, 1H, H-4), 8.78 (bs, 1H, NH), 7.65 (m, 2H, H-5 and H-7), 7.36 (m,
2H, H-6 and H-8), 3.45 (m, 2H, NCH2), 1.61 (m, 2H, NCH2CH2), 1.41 (m, 2H, CH2CH3), 0.94
13
(t, J = 7.5 Hz, 3H, CH3); C NMR (125 MHz, CDCl3): δ 161.5, 161.4, 154.4, 148.2, 134.0,
129.8, 125.3, 118.7, 118.6, 116.6, 39.7, 31.5, 20.2, 13.8.
N-(Dodecyl)-2-oxo-2H-chromene-3-carboxamide 8b
White solid; Yield: 75%; mp 168-170 °C; Rf = 0.43 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-
1
1: 3331, 3077, 1715, 1677, 1522, 1356, 1171, 751 cm-1; MS (ESI): m/z 358.23 [M + H]+; H
NMR (500 MHz, CDCl3): δ 7.79 (s, 1H, H-4), 7.55 (m, 2H, H-5 and H-7), 7.34 (m, 2H, H-6 and
H-8), 3.35 (t, J = 6.5 Hz, 2H, NCH2), 1.62 (m, 2H, NCH2CH2), 1.29 (m, 18H, 9 x CH2), 0.87 (t, J
13
= 6.5 Hz, 3H, CH3); C NMR (125 MHz, CDCl3): δ 167.3, 159.2, 153.2, 140.9, 132.9, 128.8,
125.8, 125.2, 118.1, 116.8, 39.7, 34.0, 31.9, 29.8, 29.7, 29.6, 29.6, 29.4, 29.3, 26.0, 22.7, 14.1.
N-(Octadec-9-en-1-yl)-2-oxo-2H-chromene-3-carboxamide 8c
White solid; Yield: 68%; mp 55-56°C; Rf = 0.67 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-1:
1
3338, 3085, 1722, 1684, 1529, 1363, 1178, 758 cm-1; MS (ESI): m/z 440 [M + H]+; H NMR
(500 MHz, CDCl3): δ 8.89 (s, 1H, H-4), 8.79 (brs, 1H, NH), 7.66 (m, 2H, H-5 and H-7), 7.37 (m,
2H, H-6 and H-8), 5.32 (m, 2H, CH=CH), 3.43 (t, J = 6.5 Hz, 2H, NCH2), 1.97 (m, 4H,
21

CH2CH=CHCH2), 1.61 (m, 2H, NCH2CH2), 1.30 (m, 22H, 11 x CH2), 0.87 (t, J = 6.5, 3H, CH3);
13C NMR (125 MHz, CDCl3): δ 161.57, 161.4, 154.4, 148.2, 133.9, 129.8, 125.3, 118.7, 118.6,
116.6, 40.0, 32.6, 32.0, 31.9, 29.8, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.3, 29.2, 29.1, 27.2, 27.1,
22.7, 14.1.
N-(Cyclopropyl)-2-oxo-2H-chromene-3-carboxamide 8d
White solid; Yield: 82%; mp 170-172 °C; Rf = 0.46 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν cm-
1
1: 3329, 3074, 1712, 1674, 1519, 1353, 1168, 748 cm-1; MS (ESI): m/z 230 [M + H]+; H NMR
(500 MHz, CDCl3): δ = 8.90 (s, 1H, H-4), 8.80 (brs, 1H, NH), 7.64 (d, J = 7.5 Hz, 1H, H-5), 7.55
(m, 1H, H-7), 7.36 (m, 2H, H-6 and H-8), 2.19 (m, 1H, NCH), 0.45 (m, 2H, Cyclopropyl CH2),
13
0.31 (m, 2H, Cyclopropyl CH2); C NMR (125 MHz, CDCl3): δ = 163.5, 162.8, 153.2, 148.2,
134.1, 129.9, 125.2, 118.7, 118.1, 116.8, 30.7, 6.7.
N-(Cyclohexyl)-2-oxo-2H-chromene-3-carboxamide 8e
White solid; Yield: 88%; mp 170-171 °C (lit.30 176.2–177.3 °C); Rf = 0.52 (Hexane-Ethyl
acetate, 7:3); 1H NMR (500 MHz, CDCl3): δ = 8.89 (s, 1H, H-4), 8.75 (brs, 1H, NH), 7.89 (d, J =
8.0, Hz, 1H, H-5), 7.72 (m, 1H, H-7), 7.42 (m, 2H, H-6 and H-8), 3.76 (m, 1H, NCH), 1.72 (m,
4H, 2 x NCH2), 1.24 (m, 6H, cyclohexyl CH2).
3-(Piperidine-1-carbonyl)-2H-chromen-2-one 8f
Brown solid; Yield: 80%; mp 139-140 °C; Rf = 0.23 (Hexane-Ethyl acetate, 7:3); IR (KBr) ν
1
cm-1: 3337, 3784, 1718, 1682, 1528, 1362, 1177, 757 cm-1; MS (ESI): m/z 258 [M + H]+; H
NMR (500 MHz, CDCl3): δ 7.80 (s, 1H, H-4), 7.51 (m, 2H, H-5 and H-7), 7.27 (m, 2H, H-6 and
H-8), 3.44 (m, 4H, 2 x NCH2), 1.63 (m, 4H, piperidinyl CH2), 1.52 (m, 2H, piperidinyl CH2); 13C
NMR (125 MHz, CDCl3): δ 163.3, 158.1, 154.0, 142.3, 132.9, 128.5, 125.9, 118.6, 118.4, 116.8,
61.6, 49.1, 24.4.
3-(4-Methylpiperazine-1-carbonyl)-2H-chromen-2-one 8g
Light brown solid; Yield: 85%; mp 175-176 °C; Rf = 0.25 (Hexane-Ethyl acetate, 7:3); IR
(KBr) ν cm-1: 3339, 3517, 3786, 1720, 1684, 1530, 1364, 1179, 759 cm-1; MS (ESI): m/z 273.12
1
[M + H]+; H NMR (500 MHz, CDCl3): δ 7.79 (s, 1H, H-4), 7.56 (m, 2H, H-5 and H-7), 7.34
22

(m, 2H, H-6 and H-8), 3.49 (m, 4H, piperazinyl ring protons), 2.42 (m, 7H, NCH3 and
13
piperazinyl ring protons); C NMR (125 MHz, CDCl3): δ 163.4, 159.1, 153.1, 140.8, 132.8,
128.7, 125.7, 125.0, 117.9, 116.7, 60.0, 59.6, 51.2, 49.9, 47.1.
4.2 Biological Studies
4.2.1. In vitro evaluation of antimicrobial activity
All the prepared coumarin derivatives 5a-5p and 8a -8g were evaluated for their antibacterial
activity against K.pneumoniae MTCC 661, S. typhi MTCC 537, P. aeruginosa MTCC 424 and
E. coli MTCC 64 and two Gram-positive bacterial strains Staphylococcus aureus MTCC 96,
Bacillus pumilus MTCC 1607 and antifungal activity against Candida albicans MTCC 3017, A.
fumigatus MTCC and Candida tropicalis MTCC 184. All the test cultures were retrieved from
Institute of Microbial Technology, Chandigarh, India. Antibacterial drugs novobiocin and
chloramphenicol and antifungal drugs amphotericin B and fluconazole were used as standards.
Antimicrobial activities of the coumarins were quantitatively determined in terms of minimum
inhibitory concentration (MIC) values depicting the lowest concentration of the tested compound
at which no growth of the strain was observed in a period of time and under specified
experimental conditions. MIC of compounds was tested by micro broth dilution method using 96
well plates using nutrient broth for bacterial test strain and sabouraud dextrose broth for fungal
test strains as per guidelines of National Committee for Clinical Laboratory Standards. Test
organisms were inoculated in sterile NB/SDB and incubated at 30◦C until the solution turbidity
achieved to 0.5 McFarland standards. Compounds of concentration 1 mg/mL were prepared. In
the first well 100 μL of blank (SDB/NB without test inoculum) as negative control, was added
whereas in the second well 180 μL of SDB/NB with test culture was added. In the rest of the
well of a row 100 μL of the test culture were added. 20 μL of the compound from the 1mg/mL
stock was added in the second well and mixed. From this well, 100 μL of the suspension was
transferred to the third well and the so on upto eleventh well. 100 μL of suspension was
discarded from eleventh well. Last well was taken as positive control. For fungi, the plates were
incubated for 48-72 h at 28 ˚C and for bacteria the plates were incubated for 24 h at 37 ˚C. All
the experiments regarding the activity were performed in triplicate and average value of results
were considered. MIC values were visually read as the lowest concentration of compound that
prevented the microbial growth completely.
23

4.2.2 Chitinase assay for coumarin derivatives
Chitinase inhibitory activity of the coumarin compounds was determined by Dinitorsalicylic acid
(DNS) method as mentioned by Miller using colloidal chitin as a substrate.46 Chitin from crab
shell was used to prepare colloidal chitin. Five grams of chitin powder was gradually mixed with
o
6 mL of concentrated HCl and the mixture was incubated at 27 C for 2 h on the orbital shaker.
Chitin was precipitated by adding ice-cold water to the mixture and was further incubated for
o
overnight. After incubation, the solution was centrifuged at 10000 rpm for 20 min at 4 C. The
pellet was washed repeatedly with distilled water until the pH of the colloidal chitin reached the
pH 7. Finally, the colloidal chitin solution was autoclaved and stored at 4 oC.
The stock solutions of coumarin compounds of 1 mg/mL concentrations were prepared in
DMSO. To examine the anti-chitinase activity in term of IC50 of the coumarin derivatives, in a
reaction mixture different concentration of the coumarin compounds and 20 mU chitinase were
o
taken in test tubes and incubated for 30 min at 37 C in shaking water bath. After that 1ml
colloidal chitin was added to the and further incubated for 30 min at 37oC in shaking water bath.
The reaction was stopped by adding 2 mL DNS reagent and kept in a boiling water bath for 5
min to develop the color. After cooling the test tubes for 10 min; color and optical density were
observed against control and IC50 was calculated. The color concept lies in DNS reaction is the
change in color from yellowish orange to brownish orange.
4.2.3. Molecular docking study
The high resolution crystal structure of A. fumigatus chitinase with PDB ID: 1w9u was retrieved
from the Protein Data Bank (www.rcsb.org). Active site was predicted by supercomputing
facility for bioinformatics
&
computational biology, IIT Delhi through the
link http://www.scfbio-iitd.res.in/dock/ActiveSite_new.jsp. The model showed 16 active site
pockets/cavities. The cavity 10 which was the most prominent (152 Å3 with Grid X-Y-Z
coordinates: 78.669, 70.021, -3.316 Å) was selected as active site. Molecular docking was
compound 5l and allosamidine performed using the Auto- Dock Tools (ADT) version 1.5.6 and
AutoDock version 4.0 docking program. The graphical user interface AUTODOCKTOOLS was
employed to setup the protein: all polar hydrogens were added, Kollman charges and nonpolar
hydrogens were merged to protein structure. The distance between donor and acceptor atoms that
24

form a hydrogen bond was defined as 1.9 Å with a tolerance of 0.5 Å and the acceptor–
hydrogen– donor angle was not less than 120°. The structures were then saved in .pdbqt file
format for further studies in ADT. The structure of ligand and allosamidine were drawn using
ACD/ChemSketch and converted into 3D structures, optimized and saved in MDL Molfiles
[V2000] format. These partial charges of MDL Molfiles were further modified by using the
Open Babel GUI to .pdb format for input to ADT. Further the generated files were saved in
.pdbqt file format to carry out docking in ADT. All docking were carried out with 1.000 Å
spacing and centered on grid box size of 78.669, 70.021, -3.316 points in x, y, and z directions
was built. Ten runs were generated by using Lamarckian genetic algorithm searches. Results
differing by less than 0.5 Å in positional root–mean– square deviation (RMSD) were clustered
together and the results of the most satisfactory free energy of binding were selected as the
resultant complex structures. Docked ligand conformations were evaluated in terms of energy,
hydrogen bonding, and hydrophobic interaction between ligand and receptor protein. Detailed
analysis of the ligand–receptor interactions were carried out and final coordinates of the ligand
and receptor were saved. For display of the receptor with the ligand binding site, Chimera 1.8.1
software was used.
Acknowledgement
DK thanks Department of Science and Technology (DST), New Delhi (Grant No.
EMR/2016/001396) for the funding and RKS thanks Council of Scientific and Industrial
Research (CSIR), New Delhi for Senior Research Fellowship.
Conflict of Interest
The authors declare no conflict of interest.
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28

Highlights
 Novel 3-amidocoumarins were designed and synthesized as antimicrobial and anti-
chitinase agents.
 Antimicrobial activity of the compounds was evaluated against six bacterial and three
fungal strains.
 Compounds 5l and 8f showed significant antimicrobial activity with MIC values ranging
between 6.25 and 25 µg/mL.
 Compound 5l with IC50 value of 3.74 µM was the most potent chitinase inhibitor.
 Compound 5l also exhibited lowest binding energy (-8.44 Kcal/mol) with the active site
of docked receptor chitinase (1W9U) among all the docked compounds.
29