4-Methyl-7-Amino/Amido Coumarin Derivatives as Potential Antimicrobials and Antioxidants

Article abstract of DOI:10.1007/s10600-020-03106-y

An array of previously synthesized 4-methyl-7-amino and amido coumarins 4a–u has been screened for their antimicrobial and antioxidant properties. Some of the compounds exhibited promising antibacterial and antifungal activities (MIC ranging from 4–64 μg/

Full text of DOI:10.1007/s10600-020-03106-y

DOI 10.1007/s10600-020-03106-y
Chemistry of Natural Compounds, Vol. 56, No. 4, July, 2020
4-METHYL-7-AMINO/AMIDO COUMARIN DERIVATIVES
AS POTENTIAL ANTIMICROBIALS AND ANTIOXIDANTS
1,2*
2*
Muthipeedika Nibin Joy,
and Sandeep Telkar
Yadav D. Bodke,
3
An array of previously synthesized 4-methyl-7-amino and amido coumarins 4a–u has been screened
for their antimicrobial and antioxidant properties. Some of the compounds exhibited promising antibacterial
and antifungal activities (MIC ranging from 4–64 μg/mL) when compared to the respective standards.
Compound 4u showed comparable antibacterial activity with the standard, ciprofloxacin, whereas compounds
4u and 4t displayed promising antifungal activity when compared to the standard, fluconazole. The in silico
docking studies against gyrase enzyme revealed the fact that 4u possessed hydrogen bonding and significant
hydrophobic interactions, which may be the reason for its superior antibacterial activity as compared to the
other compounds. Compounds 4c and 4m showed comparable antioxidant activity with the standard, BHT,
which can be attributed to the presence of electron-donating substituents.
Keywords: coumarin, antimicrobial, antioxidant.
Coumarins belong to an important class of heterocyclic compounds that are found in green plants and exhibit a broad
spectrum of pharmacological activities [1]. Several natural and synthetic coumarin derivatives are reported to be potent as
antibacterial [2], anti-inflammatory [3], and antiviral agents [4], and the various therapeutic applications of coumarin analogues
include photo chemotherapy, antitumor therapy, and anti-HIV therapy [5]. Some of the commercially available drugs that contain
the coumarin moiety include warfarin, acenocoumarol, carbochromen, etc., and antibiotics such as novobiocin, clorobiocin, and
coumermycinA1 [6]. Owing to these interesting biological properties, the investigation of coumarin derivatives for their applicability
as drugs has attracted medicinal chemists for many decades. On the other hand, diversely substituted amines and amides linked
with heterocyclic compounds are reported to possess various important pharmacological activities [7, 8].
These observations of coumarins and amines inspired us to synthesize a variety of coumarins coupled with various
amines and amides and to evaluate their pharmacological potential. The synthetic methodology for the palladium catalyzed
Buchwald cross-coupling reaction of 4-methyl-7-nonafluorobutylsulfonyloxy coumarins with different aromatic amines and
pyridones was previously reported by us [9]. As an extension of our ongoing research on the biological evaluation of the
previously synthesized molecules [10, 11], it has been planned to examine the antimicrobial and antioxidant properties of the
synthesized 4-methyl-7-amino/amido coumarins. In this article, we report our initial results of the evaluation of antimicrobial
and antioxidant activity as well as the in silico docking studies of the aforementioned compounds.
As mentioned earlier, the synthesis of various 4-methyl-7-amino/amido coumarins 4a–u was achieved by our
previously reported protocol [9]. The typical synthetic methodology starts from the synthesis of the parent coumarin
compound 2 using the modified Pechmann cyclization reaction (Scheme 1) of resorcinol 1 with ethyl acetoacetate [12].
1) Innovation Center for Chemical and Pharmaceutical Technologies, Ural Federal University, 19 Mira Street, 620002,
Yekaterinburg, Russia, e-mail: mnibinjoy@gmail.com; 2) Department of P.G Studies and Research in Industrial Chemistry,
Kuvempu University, Jnana Sahyadri, Shankaraghatta, Shimoga, 577451, Karnataka, India; 3) Department of P.G Studies
and Research in Biotechnology, Kuvempu University, Jnana Sahyadri, Shankaraghatta, Shimoga, 577451, Karnataka, India.
Published in Khimiya Prirodnykh Soedinenii, No. 4, July–August, 2020, pp. 531–536. Original article submitted
April 10, 2019.
614
0009-3130/20/5604-0614 ©2020 Springer Science+Business Media, LLC

a
TABLE 1. Determination of Antimicrobial Activity of Synthesized Compounds (MIC, μg/mL)
Bacterial strains
Fungal strains
Entry
S. aureus
E. coli
B. subtilis
C. albicans
A. flavus
C. keratinophilum
Control
4b
4c
00
256
64
32
64
256
64
128
64
32
16
64
64
64
32
16
8
00
128
64
16
128
128
64
128
64
32
32
32
64
128
64
16
4
00
128
128
32
00
128
128
32
128
256
64
256
128
128
32
64
64
128
32
16
00
128
256
32
256
128
64
128
256
64
16
64
64
64
64
16
8
00
256
128
32
256
128
64
256
128
128
32
32
64
128
64
16
4d
64
4f
256
128
128
128
64
32
64
64
128
64
8
8
4h
4i
4j
4l
4n
4o
4p
4q
4r
4s
4t
4u
8
–
8
16
–
16
Ciprofloxacin
Fluconazole
8
–
4
–
16
–
–
8
______
a
MIC values were evaluated at concentrations ranging between 4–256 μg/mL.
6
1
9
4
5
HO
OH
X
O
O
ONf
O
O
HO
O
O
a
b
c
7
3
R
3
1
2
4a-n: X = R R N
2
1
_
O
F
O
S
F
F
F
F
N
OH
4o-u: X = R R R N
ONf =
3
2
1
O
F F
F
F
CN
F
COOCH
3
N
N
NR R
=
1
2
N
N
N
H
N
H
N
H
N
H
N
N
H
H
H
4c
4d
4a
4b
4e
4f
4g
11
OH
O
OCH
F
3
12
O
S
N
11
N
N
N
N
F
NH
N
H
N
N
H
N
12
H
4j
4h
4k
O
4m
4n
4i
4l
NR R R
=
1
2
3
15
N
N
N
N
N
N
N
N
N
O
11
O
O
OCH
3
O
CF
3
O
Cl
4q
4u
4o
4r
4s
4t
4p
a. [bmim]Cl 2AlCl , CH COCH COOEt; b. Nf O, –10°C, Py; c. R R NH, Pd (dba) –Xantphos, TBAF⋅3H O–Cs CO , dioxane, 100°C
3
3
2
2
1
2
2
3
2
2
3
Scheme 1
The obtained hydroxy coumarin 2 was then converted to the corresponding nonaflate 3 by treating it with nonafluorobutane
sulfonic anhydride and pyridine at –10°C. The intermediate 3 was then subjected to the Buchwald–Hartwig coupling reaction
with various amines and pyridones by using Pd (dba) as catalyst along with xantphos as ligand and TBAF·3H O/Cs CO as
2
3
2
2
3
base in dioxane at 100°C under microwave irradiation (Scheme 1) to procure a series of 4-methyl-7-amino/amido coumarins
4a–u of considerable pharmacological relevance.
615

TABLE 2. DPPH Radical Scavenging Assay of Synthesized Compounds
% Inhibition concentration, μg
% Inhibition concentration, μg
Compound
Compound
25
50
75
100
25
50
75
100
11.3
25.6
41.3
8.3
12.4
10.9
50.4
45.8
–
19.8
39.6
52.9
13.9
19.6
21.9
62.5
59.6
5.8
25.6
50.3
66.8
17.3
25.8
30.4
60.3
61.8
10.6
30.3
56.7
75.3
22.1
28.5
36.9
69.7
70.4
18.5
25.9
45.8
22.8
39.2
30.2
14.7
15.4
75.3
33.4
59.6
36.2
47.6
42.8
23.7
26.6
81.6
43.9
68.5
51.7
54.3
50.3
30.5
33.2
85.6
51.3
76.8
64.8
63.5
56.9
37.6
39.9
88.6
4a
4b
4c
4d
4e
4f
4h
4j
4k
4l
4m
4n
4p
4r
4t
4u
Standard (BHT)
TABLE 3. Binding Affinity (kcal/mol), H-bonds, H-bond Length and H-bond Formation of the Synthesized Molecules
and Ciprofloxacin After in silico Docking
Affinity,
kcal/mol
H-Bond
Length (Å)
2.92
Ligand
H-Bonds
H-Bond Between
Hydrophobic Interactions
4b: O2 : Asp437:N
4b: O2 : Ser438:N
–5.9
–6.1
–6.1
–6.4
–6.2
2
0
2
1
2
Asp512, Glu435, Phe1123, Arg1122, Gly436
4d
2.82
Asp437, Gly436, Ile516, Asp512, Lys460,
Glu435, Asp510, Phe1123
–
–
4o
3.05
3.07
5i: N1: Asp437:OD1
5i: O1: Gly459:N
Phe1123, Glu435, Asp512, Arg458
4t
4u
Phe1123, Gly436, Glu435, Ile516, Gly459,
Lys460, Arg1122, Asp437
2.96
5c: O2 : Ser438:N
3.02
2.83
Cipr: O2: His1081:ND1
Cipr: O3: Asp510:OD2
Ciprofloxacin
Gly459, Asp437, Gly436, Phe1123, Asp512
The analysis of antibacterial and antifungal activities (Table 1) of the compounds 4a–u was carried out against two
Gram-positive bacteria (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633), one Gram-negative bacteria
(Escherichia coli ATCC 25922) and three fungi (Aspergillus flavus ATCC 9643, Chrysosporium keratinophilum ATCC 90272
and Candida albicans MTCC 227) by the serial plate dilution method. As evident from Table 1, some of the compounds
displayed promising antibacterial activity (MIC within the range 4–64 μg/mL) when compared to the standard drug ciprofloxacin.
Compounds 4d, 4n, 4o, 4p, 4q, 4s, 4t, and 4u showed good and comparable activity versus the standard, while compounds 4a,
4e, 4g, 4k, and 4m (MIC ≥ 256 mg/mL) failed to show any significant activity towards the tested strains. Compounds 4t and
4u were found to be highly active against all the bacterial strains. Compound 4u exhibited almost the same activity as that of
the standard ciprofloxacin. All the other compounds showed moderate to poor antibacterial activity. In general, it was identified
that compounds 4o–u (amides) possessed better antibacterial activity than compounds 4a–t (amines).
The antifungal activity of the synthesized compounds was determined by the serial plate dilution method using
fluconazole as the standard. To our disappointment, most of the compounds displayed moderate to poor antifungal activity
(Table 1). Nevertheless, compounds 4t and 4u showed good and comparable activity when compared to the standard drug.
Compounds 4d, 4i, and 4o displayed slightly better activity when compared to the other compounds. Alternatively, all the
other compounds failed to show good and comparable activity to that of the standard.
The presence of cyclic amide with electron withdrawing fluorine atom is presumed to be the main reason for the
comparable antimicrobial activity of compound 4u. The presence of a heterocyclic ring having a nitrogen atom is assumed to
be beneficial for the superior activity of compounds 4n and 4t. Compound 4d, having a para fluoro group, was also found to be
more active when compared to other compounds, whereas the corresponding tertiary amine 4k was found to be less active. This
describes the importance of free N–H in increasing the activity profile of the synthesized molecules. The presence of
electron donating groups considerably lowered the activity. In general, lactams were found to be more active than other secondary
and tertiary amines and the presence of electron-withdrawing groups like fluoro and chloro groups increased the activity.
616

Lys460
4d
4o
4u
Ile516
Ile516
Asp512
Asp512
Glu435
Glu435
Gly459
Glu435
Lys460
Asp437
Asp437
Phe1123
Phe1123
Phe1123
Asp510
Arg1122
Arg1122
Gly436
Gly436
Gly436
4t
Ciprofloxacin
His1081
Asp512
Asp512
Asp510
Gly459
Glu435
Arg458
Phe1123
Asp437
Phe1123
Gly459
Gly436
Fig. 1. 2D representation of the interaction of more active compounds and ciprofloxacin with 2XCT (gyrase).
The presence of electron-withdrawing groups is expected to increase the lipophilicity and thus enhance the cell permeability
of the molecule [13]. In general, it can be summarized that in the present study, the presence of electron-withdrawing group
and heterocyclic group with nitrogen atom in 4-methyl-7-amino/amido coumarins is crucial for the enhanced antimicrobial
potency of the synthesized compounds.
The DPPH procedure is one of the most convenient methods for analyzing the concentration of radical scavenging
materials because it does not have to be generated prior to analysis [14]. DPPH radical scavenging activity evaluation is a
rapid and common assay for screening the antioxidant activity of newly synthesized compounds. Since the newly synthesized
coumarin derivatives possessed an extended p-conjugated system, it was planned to examine their radical scavenging activity
[15]. Accordingly, the synthesized compounds 4a–u were screened for their antioxidant activities by taking butylated
hydroxytoluene (BHT) as the standard (Table 2).
In this assay, the standard BHT exhibited a strong scavenging activity, while compounds 4c (75.3%), 4h (69.7%),
4j (70.4%), and 4m (76.8%) showed comparable activity. Unfortunately, compounds 4g, 4i, 4o, and 4s failed to show any
antioxidant properties. All the other compounds also exhibited considerable scavenging activity, but only at higher concentrations
of the compounds.
The results of radical scavenging activity revealed that the presence of electron-donating groups or atoms attached to
the 7 position of 4-methyl-7-amino/amido coumarins is an indispensable characteristic for their enhanced antioxidant property.
th
The hydrophilic electron-donating groups are presumed to assist the stabilization of the oxygen-centered radical and reduce
the O–H bond dissociation enthalpy (BDE), thereby increasing the radical scavenging activity by hydrogen abstraction [16].
This may be the reason for the better antioxidant activity of compounds 4c, 4h, 4j, and 4m to that of the other synthesized
molecules.
617

Fig. 2. 3D representation of the interaction of 4u and ciprofloxacin with 2XCT (gyrase).
Molecular Docking Studies. Stimulated by the noteworthy antibacterial activity of some of the synthesized compounds
with respect to the standard as per the in vivo results, it was thought to corroborate those results by performing molecular
docking studies or in silico studies. The DNA gyrase is an essential enzyme in all bacteria but absent in higher eukaryotes, and
hence, it is a good antibacterial target [17, 18]. Moreover, the mode of antibacterial action of ciprofloxacin is by significantly
inhibiting the gyrase enzyme. These observations encouraged us to carry out molecular docking studies of the more active
compounds against DNA gyrase enzyme. The 2D representation of the more active compounds and the standard ciprofloxacin
is depicted in Fig. 1 and the docking scores are presented in Table 3.
The comparative docking of receptor gyrase with the compounds 4d, 4o, 4t, 4u, and ciprofloxacin exhibited good
binding affinity (Table 3). They established significant hydrogen bonding and hydrophobic interactions with different amino
acids in the receptor active pocket (Fig. 1). The standard ciprofloxacin represented by hydrophobic contacts with five different
residues and two H-bonds was formed with various amino acids. The compound 4u established hydrogen bonding between
one amino acid and hydrophobic interactions between eight different residues. In all the cases of the 2D representation,
ligands are highlighted. The set of conserved residues that are commonly involved in interaction with the ligands and ciprofloxacin
has been encircled.
Furthermore, the extrapolation of binding conformation of the most active compound 4u and ciprofloxacin was
carried out by 3D protein-ligand interaction analysis. Figures 2A and 2B illustrate the 3D interaction of 4u and ciprofloxacin,
respectively, with gyrase by using the educational version of PyMol. The ligands, H-bonds with their respective distances and
the interacting residues are displayed with ball and stick model representation.
In the current study, 4u was found to be the best antibacterial agent among all the synthesized compounds, which can
be ascribed to the electron withdrawing character of fluorine atoms as well as the ability of the molecule to form significant
hydrogen bonding and hydrophobic interactions.
EXPERIMENTAL
General.All solvents and reagents were obtained from commercial suppliers and used without any further purification.
Microwave reactions were performed in a single mode. Biotage Initiator Microwave Synthesizer and temperature was monitored
1
using infrared. Melting points were determined on an EZ - Melt automated melting point apparatus. H NMR (400 MHz) and
13
C NMR (100 MHz) were recorded on Bruker Avance II spectrometer and chemical shifts were measured in δ (ppm).
1
4-Methyl-7-(piperazin-1-yl)-2H-chromen-2-one (4l), mp 86–88°C. H NMR (400 MHz, CDCl , δ, ppm, J/Hz):
3
2.49 (3H, s, H-10), 2.92–2.96 (4H, m, H-12), 3.59–3.64 (4H, m, H-11), 3.99 (1H, br.s, H-13), 6.44 (1H, s, H-8), 7.02–7.05
13
(2H, m, H-3, 6), 7.32 (1H, d, J = 8.8, H-2). C NMR (100 MHz, CDCl , δ, ppm): 21.2 (C-10), 49.8 (C-12), 50.7 (C-11), 104.9
3
(C-2), 111.8 (C-5), 112.3 (C-8), 113.7 (C-3), 127.5 (C-6), 150.2 (C-1), 152.1 (C-7), 153.1 (C-4), 160.4 (C-9). LC-MS: calcd 244.3,
Observed 245.3; Anal. calcd. for C
H N O , 68.83; H, 6.60; N, 11.47%, found: C, 68.38; H, 6.42; N, 11.47%.
14 16 2 2
1
4-Fluoro-1-(4-methyl-2-oxo-2H-chromen-7-yl)pyridin-2(1H)-one (4u), mp 75–77°C. H NMR (400 MHz, CDCl ,
3
δ, ppm, J/Hz): 2.44 (3H, s, H-10), 6.13 (1H, d, J = 7.8, H-14), 6.23 (1H, s, H-8), 7.19 (1H, s, H-12), 7.30–7.35 (3H, m, H-2, 3, 6),
618

13
8.23 (1H, d, J = 7.8, H-15). C NMR (100 MHz, CDCl , δ, ppm): 22.7 (C-10), 90.5 (C-12), 107.8 (C-2), 112.1 (C-5), 115.6
3
(C-8), 119.5 (C-3), 124.8 (C-6), 130.7 (C-14), 139.2 (C-1), 144.5 (C-15), 147.5 (C-7), 153.9 (C-4), 159.7 (C-11), 161.1 (C-9),
165.7 (C-13). LC-MS: calcd 271.2, Observed 272.2; Anal. calcd for C H FNO , C, 66.42; H, 3.72; N, 5.16%, found:
15 10
3
C, 66.76; H, 3.75; N, 4.94%.
Antibacterial Activity. The synthesized compounds 4a–u were evaluated for their antibacterial potency against
E. coli, S. aureus, and B. subtilis bacterial strains by the serial plate dilution method [19]. Serial dilutions of the drug in
Mueller-Hinton broth were taken in tubes, and their pH was adjusted to 5.0 using phosphate buffer. A standardized suspension
of the test bacterium was incubated and inoculated for 16–18 h at 37°C. The minimum inhibitory concentration (MIC) was
determined by analyzing the lowest concentration of the drug at which there was no visible growth. A number of antimicrobial
discs were placed on agar for the sole purpose of producing the zone of inhibition in the bacterial lawn. Agar media (20 mL)
was poured into each Petri dish, the excess of suspension was decanted, and plates were dried in an incubator at 37°C for an
hour. Using agar punch, wells were made on the seeded agar plates, and the minimum inhibitory concentrations (MIC) of the
test compounds in dimethylsulfoxide (DMSO) were added into each labeled well. A control was also prepared for the plates in
the same way using DMSO. The Petri dishes were prepared in triplicate and maintained at 37°C for 3–4 days and antibacterial
activity was evaluated by measuring the diameter of the inhibition zone. The activity of each compound was compared with
the standard ciprofloxacin. The MIC (μg/mL) and inhibition zone (mm) were determined for all the synthesized compounds,
and the results are illustrated in Table 1.
Antifungal Activity. The synthesized compounds 4a–u were screened for antifungal activity against A. flavus,
C. keratinophilum, and C. albicans in DMSO by the serial plate dilution method [20]. Sabouraud agar media was prepared
by dissolving peptone (1 g), D-glucose (4 g), and agar (2 g) in distilled water (100 mL), and the pH was adjusted to 5.7.
Normal saline was utilized to make a suspension of spore of fungal stains for lawning. Aloopful of the particular fungal strain
was transferred to 3 mL saline to obtain a suspension of the corresponding species. Agar media (20 mL) was poured into each
Petri dish, the excess of the suspension was decanted, and the plates were dried in an incubator at 37°C for one hour. Using a
punch, wells were made on the seeded agar plates, and the minimum inhibitory concentrations of the test compounds in
DMSO were added into each labeled well. A control was also prepared for the plates in the same way by using DMSO as
solvent. The Petri dishes were arranged in triplicate and maintained at 37°C for 3–4 days. The antifungal activity of each
compound was analyzed by measuring the diameter of the inhibition zone and comparing it with fluconazole as the standard.
The MIC (μg/mL) and zone of inhibition (mm) were determined for all the synthesized compounds, and the results are
summarized in Table 1.
·
Antioxidant Activity. The DPPH scavenging capacity was determined according to a previously reported colorimetric
laboratory protocol [21]. Four different concentrations (25, 50, 75, and 100 μg) of the sample compounds in methanol were
·
added to 3 mL of 0.004% w/v DPPH solution, and each test tube was made up to 4 mL volume. BHT was used as a reference
standard and dissolved in methanol to get the same concentration as of the remaining extracts. Each mixture was vortexed and
kept in the dark for 10 min at ambient temperature. The absorbance (Ab) of each reaction mixture at 517 nm was measured
·
against a blank of methanol using a UV-visible spectrometer (Shimadzu UV-1800). The amount of DPPH remaining in each
reaction was calculated as
% Scavenging Activity = (Ab of the control – Ab of the test sample/Ab of the control) × 100.
The inhibition curve was plotted for triplicate experiments and characterized as percentage of mean inhibition standard
deviation, and the results are summarized in Table 2.
In silico Studies. An entirely in-house extended drug discovery informatics system OSIRIS was used to perform
ADMET calculations. It is a Java-based library layer that provides reusable cheminformatics functionality that can be used to
calculate the toxicity risks and overall drug score [22]. The structures of the synthesized molecules and the standards were
drawn in ChemBioDraw tool (ChemBioOffice Ultra 14.0), assigned with proper 2D orientation, and checked for drawing
error. The energy of each molecule was decreased using ChemBio3D (ChemBioOffice Ultra 14.0). These energy-minimized
ligand molecules were then utilized as input for AutoDockVina to carry out the docking simulation [23]. The protein databank
(PDB) coordinate file entitled ‘2XCT.pdb’ was used as the receptor (protein) molecule, which is the structure of S. aureus
gyrase in complex with ciprofloxacin and DNA [24]. The water molecules were removed from the receptor, and SPDBV
DeepView was used to rebuild the missing side chains automatically. The graphical user interface program ‘MGL Tools’
was used to set the grid box for docking simulations, and it was set in such a way that it surrounded the region of interest
(active site) in the macromolecule. In the current study, the active site was chosen based on the amino acid residues of 2XCT
that are involved in binding with ciprofloxacin. Therefore, the grid was centered at the region including the two amino acid
619

residues (Arg 458 and Gly 459) and four nitrogenous bases from DNA, i.e., guanine (G), adenine (A), thymine (T), or cytosine
°
(C), as evidenced by Bax et al. [24]. This surrounded the active site. The grid box volume was then set to 8, 14, and 14 A for
x, y, and z dimensions, respectively, and the grid centers was set to 3.194, 43.143 and 69.977 for x, y, and z center that covered
the 2 amino acid residues and four nitrogenous bases in the considered active pocket. The AutoGrid 4.0 Program was used to
generate the grid maps [25]. The docking algorithm provided with AutoDockVina was used to look for the best docked
conformation between the ligand and protein. During the docking procedure, a maximum of 10 conformers was taken into
consideration for each ligand. AutoDockVina was compiled and run under the Windows 8.0 professional operating system.
LigPlot+ [26] and PyMol [27] were used to deduce the pictorial representation of interaction between ligands and the target
protein.
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