A new efficient domino approach for the synthesis of coumarin-pyrazolines as antimicrobial agents targeting bacterial d-alanine-d-alanine ligase

Article abstract of DOI:10.1039/c9nj00703b

The inhibition of d-alanine-d-alanine ligase (Ddl) prevents bacterial growth, which makes this enzyme an attractive and viable target in the urgent search for novel effective antimicrobial drugs. In this work, a series of novel coumarin-linked pyrazoline

Full text of DOI:10.1039/c9nj00703b

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A new efficient domino approach for the synthesis of coumarin-pyrazolines as
antimicrobial agents targeting bacterial D-alanine-D-alanine ligase
Asha V. Chate,a* Ankita A. Redlawar,a Girabala M. Bondle,a Aniket P. Sarkate,b Shailee V.
Tiwari,c Deepak K. Lokwanid
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad-431
004, MS., India.
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bDepartment of Chemical Technology, Dr Babasaheb Ambedkar Marathwada University,
Aurangabad-431004, MS, India.
cDepartment of Pharmaceutical Chemistry, Durgamata Institute of Pharmacy, Dharmapuri,
Parbhani-431401, MS, India.
dR. C. Patel Institute of Pharmaceutical Education & Research, Shirpur-425405, MS, India.
*Corresponding author-chateav@gmail.com
Keywords: Coumarin-pyrazolines, β-Cyclodextrin, Multicomponent reaction, Antimicrobial
agents, D-alanine-D-alanine ligase inhibitors (Ddl), SAR, Molecular docking.
Abstract
Inhibition of D-alanine-D-alanine ligase (Ddl) prevents bacterial growth, which makes
this enzyme an attractive and viable target in the urgent search of novel effective antimicrobial
drugs. In this work, a series of novel coumarin linked pyrazoline inhibitors of D-alanine-D-
alanine ligase were synthesized and evaluated as inhibitors of Escherichia coli DdlB ligase in
order to target resistant strain of bacteria by using environmentally benevolent β-cyclodextrin as
a supramolecular catalyst via one-pot four component synthesis in water as a green reaction
media. All the newly synthesized compounds have been characterized by elemental analysis and
various spectroscopic methods. The new procedure has noteworthy advantages including easy
work-up, short reaction times, high yields of products and column free synthesis. The
synthesized compounds were evaluated in vitro for their antimicrobial activity. Among the
synthesized compounds, namely 3-(5-(4-hydroxy-3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-
yl)-2H-chromen-2-one (5f) was found to be the most potent D-Alanine-D-Alanine ligase enzyme
inhibitor with IC50 value 106 M and the compound 3-(5-(p-tolyl)-4,5-dihydro-1H-pyrazol-3-
yl)-2H-chromen-2-one (5g) was found to be the second most potent inhibitor of the DdlB

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enzyme with IC50 value 111 M against the standard D-Cycloserine. In addition, SAR study
provides the evidence that -OH, -CH3 and –OCH3 group at 4- and 3-position of the coumarins
linked to pyrazolines scaffold is increasing the enzymatic inhibition, followed by the molecular
docking study of most active compounds 5a, 5g, and 5j against DdlB enzyme of E. coli exhibited
good binding properties. This work thus highlights the coumarin linked pyrazoline motif as a
very promising tool for the development of novel antimicrobial compounds acting through an
interesting bactericidal mechanism of action.
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1. Introduction
Over the last decades, the principles of green chemistry have been successfully embraced
by the scientific community and now the awareness of environmental issues regarding chemical
processes is considered mandatory. Several contributions to the green chemistry issue can be
found in the literature, covering different aspects of this topic.1-5 In this scenario, chemists and
chemical engineers are expected to develop safe, sustainable and eco-friendly processes to attend
all social demands as well as economic development, whilst providing environmental protection
and preservation of the natural resources for future generations. Multicomponent reactions
(MCRs) represent highly suitable synthetic tools to provide molecular keys, which fulfilling
these criteria. In MCRs, three or more precursor components are combined to one reaction
product, containing moieties of all precursors.6 The “one-pot strategy” is considered the most
efficient way to accomplish the synthesis of pharmaceutically relevant structures. In contrast to
conventional multi-step reactions, it allows a fast way to build up substance libraries with the
advantage of pot, step, and atom economy.7 They also offer an eloquent tool for the one-pot
synthesis of the distinct and complex molecule as well as small and drug-like heterocycles.
Microbial infections are becoming the most pressing issue for global health and the
economy.8 In recent years, the treatment of bacterial infections has become a major challenge in
the realm of conventional antibiotic therapy.9 The emergence of bacterial resistance to
established antibiotics, as well as hospital-acquired infections, causes a growing concern for the
global community.10 Thus, increasing the resistance of microorganisms to currently available
antimicrobial drugs is the major cause of morbidity and mortality throughout the world.11 In an
era of growing antibiotic resistance, the search for effective molecules with novel mechanisms of
action is a priority.12 Today, the bacterial cell wall and the enzymes involved in peptidoglycan

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biosynthesis, a major cell wall structural component, constitute validated targets of many
antimicrobial agents.13 Searching for molecules acting on still unexploited or ill-exploited targets
represents, therefore, an important strategy for maintaining our capacity to effectively fight
bacteria and avoiding returning to the pre-antibiotic era. Coumarins represent an important
family of precious structural units, largely distributed in a wide range of natural products and
pharmaceutical candidates.11–17 The functionalization of naturally occurring skeletons has gained
significant attention, as interesting and unexpected biological properties would be generated.18-20
Some synthetic analogs of 3-substituted coumarin derivatives (Fig. 1) were reported in the
literature possess antibacterial and anticonvulsant activities respectively.21 In addition, a lot of
coumarin compounds as medicinal candidates for drugs with strong pharmacological activity,
low toxicity and side effects, fewer drug resistance, high bioavailability, broad spectrum, better
curative effects, etc., to treat various types of diseases are being actively studied.22 Alternatively,
the literature survey reveals that pyrazoline shows an integral architectural concept in
heterocyclic chemistry, the importance of pyrazoline ring a scaffold for new anti-neoplastic
agents was widely investigated,23,24 which also represents a common motif in many
pharmaceutically active compounds and demonstrating a wide range of activities.
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It was also envisaged that coumarin and pyrazoline pharmacophores if linked together,
would generate novel molecular templates which are likely to exhibit interesting biological
properties in animal models. In particular, those pyrazoline pharmacophores linked to coumarin
have been reported to possess antitumor, antimalarial and anticancer properties.25 Against this
background, the efforts are concentrated on establishing coumarin scaffold integrated with
pyrazoline frame-work to describe the relevance pharmacological activity. Based on these
interesting biological activity profiles of coumarins and pyrazolines analogs, we are inspired and
made an effort to synthesize some new number of coumarin integrated pyrazolines analogs as
potent antibacterial agents targeting D-alanine-D-alanine ligase (Ddl) in bacteria. Molecular
docking of the drug molecule with the receptor (target) offers important information about drug-
receptor interactions and is commonly employed to identify the binding orientation of drug
molecules to their protein targets in order to predict the affinity and activity.26 Expectedly, the
additive effect of this combination might produce a synergistic effect in enhancing the
bioactivity of the coumarin-pyrazoline derivatives.

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The captivating framework of coumarin linked pyrazoline increased the thirst of chemists
in preparing these compounds by numerous methods.25 Many of these methods, however, engage
synthetic problems associated with difficult separation processes and expensive, hardly available,
or non-recoverable catalysts, expensive reagents, or long reaction time. Therefore, there is a need
to develop a new efficient and green method for the synthesis of these heterocyclic compounds.
Hence, in a quest for a new easy and eco-friendly procedure for the synthesis of coumarins
linked to pyrazoline, we planned our strategy to exploit cyclodextrins as catalysts an aqueous
medium. Cyclodextrin-mediated organic reactions an aqueous medium are very useful both from
economical and environmental point of view. Cyclodextrins apart from being nontoxic are
considered to be metabolically safe. Cyclodextrins are cyclic oligosaccharides of D-(+)-
glucopyranosyl units linked by α-1,4-glycosidic bonds with a hydrophilic outer surface and a
hydrophobic central cavity, of different sizes, and are able to form complexes with the
hydrophobic guest in water.27 They have substrate selective binding ability and catalyze a wide
range of chemical reactions through noncovalent bonding, forming reversible host-guest
complexes just like enzymes. There are several examples in organic chemistry of reactions
catalyzed by cyclodextrins. They have been used to catalyze oxidations,28 reductions,29,30 ring
openings,31 protections,32 deprotections,33,34 and even cycloadditions.35,36 In all of these
examples, the cyclodextrins have always been used in a catalytic amount and always recovered
and reused.
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O
O
HN
N
NH2
HN
N
N
R
HN
O
O
S
S
R1
S
N
Ph
N
H
O
O
O
O
O
O
O
Antibacetrial agents
Anti-inflammatory Anticonvulsant agents
agents
Analgesic agents
Figure 1. Representative examples of pharmacologically active compounds bearing coumarin-
annulated scaffolds

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2. Result and discussion
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8
In our initial study toward the development of this methodology, a model reaction between
salicylaldehyde, 1 (1 mol), ethyl acetoacetate 2 (1 mol), hydrazine hydrate 3 (1 mol), and
benzaldehyde 4 (1 mol) (Scheme 2), using water as solvent, was investigated in detail by varying
the catalyst, in order to develop optimized conditions (Table 1). Then, under the optimized
reaction conditions, we carried out one-pot reaction between salicylaldehyde, ethyl acetoacetate,
hydrazine hydrate, and benzaldehyde in water (in the absence of cyclodextrin) and in water (in
the presence of cyclodextrin) to compare the reported with the new strategy and to investigate
the effects of time and yield (Scheme 2). In all the instances, the reaction did not proceed with as
expected, improvement of yields which resulted in (Table 1, entry 1).
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Then the screening was initiated by using the main cyclodextrins (α-CD, β-CD, and γ-CD) to
determine their catalytic efficiency. The catalytic activity of CD was established on the basis that
coumarin linked pyrazoline formation was not observed in the absence of cyclodextrin in water
at 100 °C for 7-8 hr or longer time (Table 1, entry 1). The α-CD and γ-CD did not show any
catalytic activity in this transformation, maybe the small or large cavity is not able to form
inclusion complexes with the substrates. Superbly good results were obtained with β-CD as
catalyst (Table 1, entry 3). Of course, β-CD is able to include in its cavity of all substrates, but
lowering the amount of β-CD to 5 mol% gave identical results to the former reactions. By
increasing the catalyst of mol% gives an excellent yield of product, which showed that
cyclodextrins play an essential role to catalyze the reaction. Hence β-CD was chosen as a catalyst
for this transformation. The results changed using 15 mol% of cyclodextrin; high yields of
products were observed, in fact, when the quantity of catalyst was increased to over 20 mol%,
the yield was constant, This behavior could be due to the formation of host-guest complexes in
which the reactants are hosted inside its lipophilic cavity by means of non-covalent interactions,
and it provides an indirect proof that the β-CD behaves as an effective chemical reactor. In this
way, the substrates are first solubilized in the aqueous medium, where they would be otherwise
insoluble. Second, the large dimensions of the β-CD cavity would allow the formation of
complexes, which could be either homo- or heterocomplexes. In particular, the formation of
heterocomplexes.

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We also screened different solvents such as EtOH, CH3OH, 1,4-Dioxane, EtOH: H2O,
DMSO, THF, CH3CN, and H2O with cyclodextrin a catalyst (Table 1, entries 5-11). After
several optimizations, we found promising results with water as a solvent due to better solubility
of β-cyclodextrin in water. Subsequently, to verify the general procedure of reaction variously
substituted aldehyde and substituted hydrazine hydrate were tested under optimized reaction
conditions, the results have been summarized in (Table 2).
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OH
H2O
R1
O
O
HO
O
OH
HO
H2O
OH
Hydrophilic medium
H2O
O
OH
N-H
HO
HO
O
O
N
O
OH
CHO
OH
OEt
O
O
O
O
-CD/H2O
80-100 °C, 1-2.5 hr
2a
5 (a-l) & 5 (s-w)
OH
O
OH
1a
CHO
R1
HO
OH
R1
H O
R-NH-NH2
2
3a
O
4 (a-q)
H2O
OH R = H, Ph
N-R
OH
N
OH
O
H2O
O
O
OH
O
H2O
OH
O
O
OH
5 (m-r)
HO
O
-cyclodextrin
OH
Hydrophobic cavity
Scheme 1. General scheme for the synthesis of 3-(5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-2H-
chromen-2-one and 3-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one
derivatives
Temperature has an imperative role on the product yield. To our surprise, at lower
temperature furnished the product in trace to poor yield, but by increasing the temperature upto
ο
80-100 C desired product form in excellent yields within much shorter reaction time. The
reaction was very clean with no side product formation.

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CHO
OH
O
O
NH
N
CHO
-CD/H O

2
NH2-NH2
3a
OEt
Reflux 1-2 hr
O
5a
O
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1
4a
2
Scheme 2. Standard model reaction
The catalyst reusability was studied four times including the use of a fresh catalyst for the
synthesis of compound (5a) and there was an inevitable loss of catalyst during the recovery
process. Besides this, no significant loss in catalytic activity was observed (Fig. 2) the catalyst
was reused the next batch without any treatment.
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90
85
80
75
70
Catalyst recovery
Yield
Fresh
Run 1
Run 2
Run 3
Figure 2. Reuse and recovery of β-CD and its effect on yield
Table 1. Summary of different catalyst and solvent used for the synthesis of 5a
Entry
1.
Catalyst
-
Solvent
Water
Water
Water
Water
Time (hr)
Yielda (%)
7-8
7
1
5
6
7
7
7-8
5
7
NRb
41
90
45
43
20
40
34
56
41
39
2.
3.
4.
5.
6.
7.
8.
9.
α-CD
β-CD
γ-CD
β-CD
β-CD
β-CD
β-CD
β-CD
β-CD
β-CD
EtOH
MeOH
1,4-Dioxane
DMSO
EtOH:H2O
THF
10.
11.
CH3CN
5
aIsolated Yield of product,bNo Reaction

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Table 2. Synthesis of compound coumarins linked to pyrazolines 5 (a-w)
5
6
7
8
Entry Products -R1
-R Timea Yieldb
(%)
M. P. οC M. P. οC
(Found)
189-191
(Reported)37
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
Ph
H
H
1.00
1.05
1.05
1.45
1.00
1.19
1.26
1.20
1.10
1.30
1.49
1.30
1.00
1.45
1.45
1.15
1.45
1.35
1.00
1.45
1.00
1.10
1.15
97
97
96
92
86
83
83
82
92
83
96
84
81
83
83
92
79
93
81
96
86
86
87
-
9
3,4-di- OCH3
3,4,5-tri- OCH3
4-OCH3
2-OCH3-Napthaldehyde
4-OH,3-OCH3
4-CH3
149-152
171-174
182-185
146-148
139-142
169-171
176-179
156-159
128-131
140-142
146-149
132-135
139-141
107-110
169-172
152-155
183-186
196-198
109-111
132-135
176-178
139-142
-
-
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H
H
H
H
H
H
H
H
183-185
-
-
165-167
175-177
158-160
4-Cl
2,6-di-Cl
2-Thiophenealdehyde
Valeraldehyde
Butyraldehyde
Ph
4-CH3
4-OH
N,N-di-CH3
3-Br
Valeraldehyde
4-NO2
-
-
-
-
-
-
-
-
H
H
Ph
Ph
Ph
Ph
Ph
Ph
H
-
199-201
5t
N,N-di-CH3
3-CH3
4-F
H
H
H
H
-
-
5u
5v
5w
174-176
-
4-OH
aTime in hr., bIsolated yield of products
3. Pharmacology
In Vitro Antimicrobial Activity
All the synthesized compounds were screened for their in vitro antifungal and
antibacterial activity. The antibacterial activity was evaluated against three human pathogenic
bacterial strains: Escherichia coli (NCIM-2256), Bacillus subtilis (NCIM-2063) and
Staphylococcus aureus (NCIM-2901). The antifungal activity was evaluated against seven
human pathogenic fungal strains: Candida albicans (NCIM-3471), Candida glabrata (NCYC-
388), Fusarium oxysporum (NCIM-1332), Aspergillus fumigates (NCIM-902), Aspergillus flavus
(NCIM-539), Aspergillus niger (NCIM-1196) and Cryptococcus neoformans (NCIM-576),
which were often encountered clinically. Miconazole was used as standard drug. Minimum

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inhibitory concentration (MIC) values were determined as per CLSI guidelines.38-40 Dimethyl
sulfoxide (DMSO) was used a solvent control.
7
8
In Vitro Antifungal Activity
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Antifungal activity was determined as per CLSI (formerly, NCCLS) guidelines.38-40 The
synthesized compounds 5 (a-r) and the standard drug Miconazole were dissolved in DMSO
solvent. The medium yeast nitrogen base was dissolved in phosphate buffer pH 7 and it was
autoclaved at 110 °C for 10 min. With each set, a growth control without the antifungal agent
and solvent control DMSO were included. The fungal strains were freshly sub cultured on to
Sabouraud dextrose agar (SDA) and incubated at 25 °C for 72 hr. The fungal cells were
suspended in sterile distilled water and diluted to get 105 cells/mL. 10 μL of the standardized
suspension was inoculated onto the control plates and the media incorporated with the antifungal
agents. The inoculated plates were incubated at 25 °C for 48 hr. The readings were taken at the
end of 48 hr and 72 hr. The MIC was the lowest concentration of drug preventing the growth of
macroscopically visible colonies on drug-containing plates when there was visible growth on the
drug-free control plates.
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In Vitro Antibacterial Activity
All the synthesized compounds 5 (a–r) were screened for their in vitro antibacterial activity.
Minimum inhibitory concentration (MIC) values were determined using the method
recommended by the National Committee for Clinical Laboratory Standards (NCCLS). In vitro
antibacterial activities of the synthesized compounds, 5 (a–r) were tested in Nutrient Broth (NB)
for bacteria by the two-fold serial dilution method. Seeded broth (broth containing microbial
spores) was prepared in NB from 24 hr old bacterial cultures on nutrient agar (Hi-media) at 37±
1 °C. The bacterial suspension was adjusted with sterile saline to a concentration of 1× 10−4–10−5
C.F.U. The synthesized compounds and standard drug Ampicillin were prepared by two-fold
serial dilutions to obtain the required concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, and
3.13 μg/mL. The tubes were incubated in BOD incubators at 37± 1 °C for bacteria. The MICs
were recorded by visual observations after 24 hr (for bacteria) of incubation.38-40

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D-alanine-D-alanine ligase (DdlB) enzyme inhibition study
5
6
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8
The D-Ala-adding activity of DdlB ligase was monitored by the detection of orthophosphate
generated during the reaction based on the colorimetric malachite green method described by
Walsh, A. et al.41 Assays were performed at 37 °C in a mixture (final volume: 50 µL) containing
38.5 mMHepes, pH 8.0, 3.25 mMMgCl2, 6.5 mM (NH4)2SO4, 700 µM D-Ala, 500 µM ATP,
purified DdlB (diluted in 20 mM Hepes, pH 7.2, and 1 mM dithiothreitol) and the test compound
(IC50 values were determined for a range of inhibitor concentrations). All the compounds were
soluble in the assay mixture containing 5 % DMSO. After 30 min of incubation, 100 µL Biomol
reagent was added. After 5 min, absorbance was read at 650 nm. Residual activity was calculated
with respect to a similar assay without inhibitor. To exclude possible non-specific (promiscuous)
inhibitors, representative compounds 5 (a-r) were also tested in the presence of Tween (0.003
%), Triton X-114 (0.005 %), and SDS (420 µM), as described by McGovern, S. L. et al.42,43 No
significant differences were found when compared to measurements without detergents.
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In –vitro Antibacterial activity and D-alanine assay
All the synthesized compounds 5 (a-r) were screened for their in vitro antibacterial activity
against Staphylococcus aureus (NCIM-2901), Escherichia coli 1411 (standard laboratory strains)
and Escherichia coli SM1411 (an acrAB deficient derivative of 1411). Minimum inhibitory
concentration (MIC) values were determined using the method recommended by the National
Committee for Clinical Laboratory Standards (NCCLS). Dimethyl sulfoxide (DMSO) was used a
solvent control. The results of the in vitro antibacterial activity are presented in (Table 3). D-
Alanine-d-alanine ligase (Ddl) is one of the key enzymes in peptidoglycan biosynthesis and is an
important target for drug discovery. Inhibition of DdlB essential enzymes in either Gram-
positive or Gram-negative organisms leads to the loss of cell shape and integrity followed by
bacterial death. The synthesized derivatives 5 (a-r) were tested for their inhibitory activity on
DdlB from E.coli. The results are presented as IC50 values.

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Table 3. In-vitro antibacterial activity of the synthesized compounds 5 (a-r)
5
6
7
Compd.
IC50 (M)
MICa (g/ml)
E. coli SM1411 S. aureus NCIM-2901
E. coli 1411
8
9
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
178
129
155
220
335
106
111
124
115
230
245
268
>400
>400
54
35
48
55
58
14
16
30
20
54
32
46
54
>60
14
18
28
20
64
>70
>70
>70
>70
>70
>70
>70
>70
16
110
58
50
120
166
32
40
36
24
112
158
176
>200
>200
185
>200
>200
155
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
68
>70
>70
>70
>70
>70
>70
>70
>70
16
348
>400
>400
288
5p
5q
5r
D-Cycloserine
276
32
a Values are the average of three readings; E.coli 1411: Escherichia coli 1411; E.coli SM1411
Escherichia coli SM 1411; S. aureus NCIM-2901: Staphylococcus aureus (NCIM-2901); MIC:
Minimum inhibitory concentration.
From the results of in-vitro antibacterial activity data, it reveals that the compound 5f was
found to be the most potent antibacterial compound among the synthesized series against the
tested pathogens. The compounds 5f has shown MIC values 14 μg/mL, 14 μg/mL and 32 μg/mL
against E.coli 1411, E.coli SM1411 and S. aureus NCIM-2901 respectively. From the MIC values of
compound 5f, it can be observed that it is more potent than that of standard drug D-cycloserine
against the selected pathogens. The compound 5g was found to be the second most active
antibacterial compound among the synthesized series 5 (a-r). The compounds 5g has shown MIC
values 16 μg/mL, 18 μg/mL and 40 μg/mL against E.coli 1411, E.coli SM1411 and S. aureus
NCIM-2901 respectively. The compounds 5i and 5h were also found to be good antibacterial
compounds among the synthesized series. The other active compounds are 5a, 5b, 5c, 5d, 5e, 5j, 5k,
5l and 5o. The compounds 5m, 5n, 5p, and 5q were almost inactive against selected pathogens.

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The synthesized compounds were also screened for their D-alanine inhibitory activity. The
synthesized derivatives 5 (a-r) were tested for their inhibitory activity on DdlB from E.coli. The
result of D-alanine enzyme assay reveals that most of the synthesized compounds have good D-
alanine enzyme inhibitory activity. The synthesized compound 5f was found as the most potent D-
alanine enzyme inhibitor. The compound 5g was found too be the second most potent inhibitor of
the DdlB enzyme with IC50 value 111 M. The compounds 5a, 5b, 5c, 5d, 5f, 5g, 5h, 5i, 5j, 5k, and
5l were found to be better DdlB enzyme inhibitors than that of standard drug D-cycloserine.
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Structure-activity relationship (SAR) revealed that the derivatives with substitution on the
‘NH’ group of the pyrazole heterocycle were less active antibacterial compounds than those with
no substitution on the ‘NH’ group of the pyrazole heterocycle. The compounds 5 (m-r) consists
of substitution on the ‘NH’ group of the pyrazole heterocycle and are less active antibacterial
compounds than compounds 5 (a-l) with no substitution on the ‘NH’ group of the pyrazole
heterocycle. The most potent enzyme inhibitor compound 5f consists of the 4-hydroxy-3-
methoxy group on the phenyl ring and no substitution on the ‘NH’ group of the pyrazole
heterocycle. The compound 5g was found to be the second most potent inhibitor of the DdlB
enzyme with “CH3” group on the phenyl ring and no substitution on the ‘NH’ group of the
pyrazole heterocycle. The compound 5a with no substitution on the phenyl ring was found to be
less active than compounds with substitution on the phenyl ring. When the phenyl ring was
replaced with the thiophene heterocycle there was a decrease in antibacterial potency of the
synthesized compound 5j. When the phenyl ring was replaced with the acyclic compounds there
was a decrease in antibacterial potency of the synthesized compounds 5p and 5q.
In-vitro antifungal activity
The newly synthesized derivatives 5 (a-r) were screened for their in vitro antifungal activity
against different yeast and filamentous fungal pathogens. Minimum inhibitory concentration (MIC)
values for in vitro antifungal activity were determined the method recommended the National
Committee for Clinical Laboratory Standards (NCCLS). Miconazole was used as standard drug.
Dimethyl sulfoxide (DMSO) was used a solvent control. The MIC (g/mL) of all the tested
compounds and that of the reference drug Miconazole has been listed in the (Table 4). Results from
the (Table 4) indicated that all the synthesized compounds have shown good to moderate antifungal
activity against tested fungal strains.

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Table 4. In-vitro antifungal activity of the synthesized compounds 5 (a-r).
MICaμg/mL
Compd.
Candida Candida Fusarium Aspergillus Aspergillus Aspergillus Cryptococcus
albicans glabrata oxysporum fumigates
flavus
29
16
18
25
14
33
14
26
23
12
28
29
31
52
42
36
37
44
12
niger
30
16
14
18
18
36
14
26
26
14
28
32
32
54
39
33
38
40
12
neoformans
9
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
38
28
28
35
25
48
24
38
36
20
46
40
42
69
60
52
58
60
25
42
34
32
34
28
44
25
38
33
20
41
40
42
58
57
48
55
62
25
36
33
30
34
30
39
28
36
35
22
38
42
48
64
55
49
48
58
25
46
40
38
42
34
49
32
45
46
34
46
46
51
72
58
54
57
60
35
28
27
16
22
20
31
16
28
22
12
33
32
29
48
38
38
35
42
12
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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Miconazole
Among the synthesized compounds the compound 5g was found to be equipotent that of
standard drug Miconazole against the selected pathogens. The compound 5g has shown MIC
values 24 μg/mL, 25 μg/mL, 28 μg/mL, 32 μg/mL, 14 μg/mL, 14 μg/mL and 16 μg/mL against
Candida albicans, Candida glabrata, Fusarium oxysporum, Aspergillus fumigates, Aspergillus
flavus, Aspergillus niger and Cryptococcus neoformans respectively. The compound 5j with
thiophene nucleus in the structure was found to be more potent than the standard drug
Miconazole against Candida albicans, Candida glabrata, Fusarium oxysporum, and Aspergillus
fumigates. The compound 5j has shown MIC values 20 μg/mL, 20 μg/mL, 22 μg/mL, 34 μg/mL,
12 μg/mL, 14 μg/mL and 12 μg/mL against Candida albicans, Candida glabrata, Fusarium
oxysporum, Aspergillus fumigates, Aspergillus flavus, Aspergillus niger, and Cryptococcus
neoformans respectively.
Structure-activity relationship (SAR) revealed that the derivatives with substitution on the
‘NH’ group of the pyrazole heterocycle were less active antibacterial compounds than those with

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no substitution on the ‘NH’ group of the pyrazole heterocycle. The compounds 5 (m-r) consists
of substitution on the ‘NH’ group of the pyrazole heterocycle and are less active antibacterial
compounds than compounds 5 (a-l) with no substitution on the ‘NH’ group of the pyrazole
heterocycle. The compound 5i with di-chloro substitution on phenyl ring was found to be more
potent than compound 5h with p-chloro substitution on the phenyl ring. The compound 5c with
3,4,5, tri-methoxy substitution on phenyl ring was found to be more potent antifungal compound
than the compound 5b with 3,4-dimethoxy substitution on the phenyl ring. The compound 5e i.e.
3-(5-(3-methoxynaphthalen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one was also
found a good antifungal agent among the synthesized series.
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Molecular docking
Docking is an effective and reliable approach to simulate the probable binding mode of
ligands and proteins. Bacterial growth is inhibited if the enzyme D-alanine ligase (Ddl) is been
inhibited by the designed molecule. D-Alanine ligase (Ddl) enzyme inhibition is an attractive and
tenable target in the search for novel and effective antimicrobial drugs.44 Recently, chromene
derivatives have been reported to inhibit E. coli DdlB enzyme.45 Hence, the designed and
synthesized compounds were appraised for their inhibition effect using D-alanine-D-alanine
ligase (DdlB) enzyme assay study. Molecular docking studies into a homology model of the E.
coli DdlB enzyme (PDB entry: 2I80) were performed using Glide v. 6.8 (Schrodinger, LLC,
New York, NY, USA, 2015) to predict its antifungal and antibacterial activity mode of action,
respectively.
Molecular Docking Study into E. coli DdlB Enzyme
Molecular docking study was performed in Maestro 9.1 using Glide v. 6.8 (Schrodinger
LLC). All compounds were built using Maestro build panel and optimized to lower energy
conformers using Ligprep v3.5 (Schrodinger, Inc., New York, NY, USA). The coordinates for E.
coli DdlB enzyme were taken from RCSB Protein Data Bank and prepared for docking using
‘protein preparation wizard’ in Maestro v10.3. The bond orders and formal charges were added
for hetero-groups and hydrogens were added to all atoms in the structure. Side chains that are not
close to the binding cavity and do not participate in salt bridges were neutralized and termini
were capped by adding ACE and NMA residue. After preparation, the structure was refined to
optimize the hydrogen bond network using the OPLS_2005 force field. The minimization was

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terminated when the energy converged or the RMSD reached a maximum cutoff of 0.30 Å. The
extra precision (XP) docking mode for all compounds was performed on the generated grid of
protein structure.47 The final evaluation of ligand-protein binding was done with Glide score.46
Inhibition of Ddl in bacteria by drugs hinders the growth of bacteria, which makes this
enzyme an irresistible and important target for the discovery of novel antimicrobial drug. Hence,
molecular docking was performed against E. coli DdlB (PDB entry: 2I80). The docking results
indicated that the compounds were held in the active pocket by the combination of various
hydrogen and hydrophobic interactions with DdlB enzyme. The docking results revealed that the
highest binding compound to DdlB enzyme was compound 5g with a G-Score of -9.086 when
compared with standard D-Cycloserine.
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All compounds with highest docking scores showed hydrogen bonding with amino acid
residue THR 23 and a Pi-Pi stacking interaction with the amino acid residue PHE 313 which
helped in the smooth attachment of the molecule into the pocket of DdlB enzyme. Also,
compound 5g with the highest G-Score showed additional hydrogen bonding and Pi-Pi
interaction with the amino acid residue HIP 96 which increases the chances of good activity of
the compound.
< Figure 3. The docking pose of compound 5g into the active pocket of DdlB enzyme. (Pink
bond represents the hydrogen bonding between ligand and receptor and Blue bond indicates the
Pi-Pi stacking interaction, Purple colored structure represents the molecule) >
< Figure 4. The docking pose of compound 5a into the active pocket of DdlB enzyme.>
< Figure 5. The docking pose of compound 5j into the active pocket of DdlB enzyme. >

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Reaction mechanism
5
6
HO
OH
7
8
9
O
NH
O
O
N
H
HO
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OEt
OH
O
O
5a
-cyclodextrin catalyst
OH
OH
O
O
O
OH
OH
H
OEt
CH3
N
N
OH
OH
O
O
OH
-H2O
OH
OH
OH
HO
OH
CH3
H3COC
O
C
C2H5OOC
NH2
H
N
H
OH
OH
O
O
OH
Cyclization
OH
OH
OH
OH
H
O
O
O
O
CH3
OC2H5
COCH3
-C2H5OH
O
NH2-NH2
OH
OH
Scheme 3. Plausible reaction mechanism of coumarins linked to pyrazoline derivative
4. Conclusion
We have explored a facile construction of biologically active coumarins linked to
pyrazoline by the multicomponent approach. A new, simple, efficient and environmentally
benign method involving the usage of β-cyclodextrin as a supramolecular catalyst in water for
the synthesis of coumarins-pyrazoline was developed. The synthesized novel molecules are
inhibitors of DdlB, with IC50 values in the micromolar range. The result shows that coumarin
attached to pyrazoline moiety is essential for inhibitory activity against DdlB. The designed
compounds represent an important starting point for further optimization and modification, to
improve these inhibitory activities against DdlB. These types of inhibitors have the potential to

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be developed into drugs that would reverse bacterial resistance to D-Cycloserine. The
synthesized compound 5f was found to be the most potent D-alanine-D-alanine ligase enzyme
inhibitor. The compound 5g was found to be the second most potent inhibitor of the DdlB
enzyme with IC50 value 111 M. Furthermore, the potency of these compounds against DdlB
suggests that it will be possible to develop broad-spectrum antimicrobials that target both Gram-
negative and Gram-positive infections.
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5. Acknowledgments
The authors are thankful to Professor Charansingh H. Gill for his invaluable discussions
and guidance. The authors are also thankful to the Department of Chemistry, Dr. Babasaheb
Ambedkar Marathwada University, Aurangabad and this work was supported by Special
Assistance Programme SAP, University Grants Commission, New Delhi, India, intended to
encourage the pursuit of excellence and teamwork in advanced teaching and research to
accelerate the realization of international standards in specific fields. The authors are grateful to
Schrodinger, Inc. for providing the Demo license of the Schrodinger Molecular Modeling Suite
to perform the molecular modeling studies.
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New Journal of Chemistry
Page 20 of 20
View Article Online
DOI: 10.1039/C9NJ00703B
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Graphical Abstract
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A new efficient domino approach for the synthesis of coumarin-pyrazolines as
antimicrobial agents targeting bacterial D-alanine-D-alanine ligase
Asha V. Chate,a* Ankita A. Redlawar,a Girabala M. Bondle,a Aniket P. Sarkate,b Shailee V.
Tiwari,c Deepak K. Lokwanid
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aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad-431
004, MS., India.
bDepartment of Chemical Technology, Dr Babasaheb Ambedkar Marathwada University,
Aurangabad-431004, MS, India.
cDepartment of Pharmaceutical Chemistry, Durgamata Institute of Pharmacy, Dharmapuri,
Parbhani-431401, MS, India.
dR. C. Patel Institute of Pharmaceutical Education & Research, Shirpur-425405, MS, India.
*Corresponding author-chateav@gmail.com
OH
IC50 = 106 M against
D-Cycloserine

OCH3
NH
N
O
O
Ddl
Ddl
D-Ala
D-Ala-D-Ala
UDP-MurNAc
D-Ala
D-Ala-D-Ala
UDP-MurNAc
MurA-F
MurA-F
M
e
m
b
r
a
n
e
Nascent Peptidoglycan
Transpeptidases
Transpeptidases
X
Peptidoglycan
Peptidoglycan
X
Inhibition of cell
wall synthesis