New N-phenylacetamide-linked 1,2,3-triazole-tethered coumarin conjugates: Synthesis, bioevaluation, and molecular docking study

Article abstract of DOI:10.1002/ardp.202000164

A series of new 1,2,3-triazole-tethered coumarin conjugates linked by N-phenylacetamide was efficiently synthesized via the click chemistry approach in excellent yields. The synthesized conjugates were evaluated for their in vitro antifungal and antioxida

Full text of DOI:10.1002/ardp.202000164

Received: 22 May 2020
Revised: 22 June 2020
Accepted: 11 July 2020
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DOI: 10.1002/ardp.202000164
F U L L P A P E R
New N‐phenylacetamide‐linked 1,2,3‐triazole‐tethered
coumarin conjugates: Synthesis, bioevaluation, and molecular
docking study
Satish V. Akolkar1
Murad Z. A. Warshagha1 | Jaiprakash N. Sangshetti4
| Amol A. Nagargoje1,2 | Mubarak H. Shaikh1,3
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| Manoj G. Damale5
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Bapurao B. Shingate1
1Department of Chemistry, Dr. Babasaheb
Ambedkar Marathwada University,
Abstract
Aurangabad, India
A
series of new 1,2,3‐triazole‐tethered coumarin conjugates linked by
2Department of Chemistry, Khopoli Municipal
Council College, Khopoli, India
N‐phenylacetamide was efficiently synthesized via the click chemistry approach in
excellent yields. The synthesized conjugates were evaluated for their in vitro
antifungal and antioxidant activities. Antifungal activity determination was carried out
against fungal strains such as Candida albicans, Fusarium oxysporum, Aspergillus flavus,
Aspergillus niger and Cryptococcus neoformans. Compounds 7b, 7d, 7e, 8b and 8e
displayed higher potency than the standard drug miconazole, with lower minimum
inhibitory concentration values. Also, compound 7a exhibited potential radical
scavenging activity as compared with the standard antioxidant butylated hydro-
xytoluene. In addition, a molecular docking study of the newly synthesized compounds
was carried out, and the results showed a good binding mode at the active site of the
fungal (C. albicans) P450 cytochrome lanosterol 14α‐demethylase enzyme. Furthermore,
the synthesized compounds were also tested for ADME properties, and they demon-
strated potential as good candidates for oral drugs.
3Department of Chemistry, Radhabai Kale
Mahila Mahavidyalaya, Ahmednagar, India
4Department of Pharmaceutical Chemistry,
Y. B. Chavan College of Pharmacy, Dr. Rafiq
Zakaria Campus, Aurangabad, India
5Department of Pharmaceutical Chemistry,
Srinath College of Pharmacy,
Aurangabad, India
Correspondence
Bapurao B. Shingate, Department of
Chemistry, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad 431 004,
India.
Email: bapushingate@gmail.com
K E Y W O R D S
ADME properties, antifungal activity, antioxidant activity, molecular docking,
triazole–coumarin conjugates
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1
INTRODUCTION
widely used for the prevention and treatment of fungal infections
(Figure 1). These antifungal drugs inhibit CYP51 (P450 cytochrome
The incidence of systemic fungal infection has increased dramatically in
recent years due to an increase in the number of patients undergoing
anticancer chemotherapy and organ transplantation, and AIDS patients.[1]
Fungi are vital opportunistic human pathogens that have become drug‐
resistant to many approved compounds, especially Cryptococcus neofor-
mans, Candida, and Aspergillus species, with serious potential con-
sequences. The commonly used azole‐based antifungal agents are
miconazole, fluconazole, voriconazole, and itraconazole, which showed a
wide range of antifungal activity.[2] Azoles, especially triazole‐based
antifungal agents (e.g., voriconazole, fluconazole, and posaconazole) are
lanosterol 14α‐demethylase), a key enzyme in ergosterol biosynthesis.[3]
However, extensive use of these drugs has resulted in severe drug
resistance.[4] Therefore, the development of more potent, broad‐
spectrum antifungal agents with fewer side effects and improved
efficiency to cure fungal infections is urgently required.
To counteract the harmful effects of free radicals and other
oxidants, the human body has a complex system of natural enzymatic
and nonenzymatic antioxidants. Free radicals are unstable chemical
species having unpaired electrons that are extremely reactive toward
other species. The action of reactive oxygen species (ROS) results in
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Arch Pharm. 2020;e2000164.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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AKOLKAR ET AL.
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FIGURE 1 1,2,4‐Triazole‐based antifungal drugs
key biomolecules being altered and modulated in function. There is a
delicate balance between producing and removing free radicals in
healthy organisms. Triazole–coumarin conjugates have unique po-
tential to scavenge ROS such as hydroxyl and superoxide radicals.[5]
Therefore, the synthesis and development of new antioxidants hav-
ing the triazole–coumarin pharmacophore have enormous sig-
nificance in medicinal chemistry.
display potential biological activities.[25–29] In recent years, coumarin‐
based hybrid molecules have attracted intense interest due to their
diverse biological properties.[30,31] Different nitrogen‐containing
heterocycles (e.g., triazole, thiazolidine, thiazole, etc.) in conjunction
with coumarin backbone significantly increase the antimicrobial ef-
ficiency and also broaden antimicrobial spectrum of these
compounds.[32–34]
Diversely functionalized 1,2,3‐triazole derivatives have attracted
great attention due to their extensive biological properties such
as antioxidant,[5] antifungal,[6,7] antitubercular,[8] antibacterial,[9]
anticancer,[10] anti‐HIV,[11] antimicrobial,[12] and antimalarial activity.[13]
The 1,4‐disubstituted‐1,2,3‐triazoles were synthesized via copper‐
catalyzed azide–alkyne cycloaddition reaction, well known as a click
chemistry reaction.[14] In the field of medicinal chemistry and drug
discovery, 1,2,3‐triazoles have received[15–19] increasing attention since
Sharpless group introduced the “click chemistry” concept. The promising
properties of the 1,2,3‐triazole ring, such as hydrogen bonding cap-
ability, rigidity, stability under in vivo conditions, and moderate dipole
character, could be helpful for binding of biomolecular targets and
increasing solubility.[15,20,21] Moreover, 1,2,3‐triazoles have become
increasingly useful and important in the construction of bioactive and
functional molecules as attractive linker units that could connect two
pharmacophores to provide innovative bifunctional drugs.[22–24] Many
drugs available in the market contain 1,2,3‐triazole core in their struc-
ture, such as cefatrizine (antibiotic), carboxyamidotriazole (anticancer
agent), rufinamide (anticonvulsant) and tazobactam (antibacterial agent;
Figure 2).
Due to the rapid and effective distribution of privileged systems with
relatively improved biological properties as compared with individual
entities, the molecular hybridization approach has established eminence
over the past few years.[35] With strong drug‐like properties and desirable
binding interactions, these hybrid molecules have emerged for further
chemical modifications as structurally novel chemotypes with several
exploitable sites. In recent years, a library of coumarin–triazole con-
jugates was synthesized and proved to enhance biological activity.[36–39]
There are various reports on the synthesis of coumarin–triazole
conjugates with antifungal activity.[32,38,40,41] Therefore, the design and
synthesis of coumarin–triazole conjugates is crucial for the enhancement
of activity.
In continuation of our previous works[40,42–51] on the synthesis
and biological evaluation of heterocycles, and the significance of
coumarin and 1,2,3‐triazole moieties in a single molecular framework,
herein, we would like to report the design and syntheses of new
N‐phenylacetamide‐linked coumarin–triazole conjugates by using the
molecular hybridization approach (Figure 3).
The 1,2,3‐triazole moiety is good for antifungal activity, and
coumarin derivatives have been well reported for the antioxidant
activity. Thus, we have evaluated the synthesized compounds for
their antifungal and antioxidant activities. The computational
parameters like docking study for antifungal activity and ADME
(absorption, distribution, metabolism, and elimination) prediction of
synthesized coumarin–triazole conjugates were also performed.
Synthesis of new heterocycles with multiple biological activities
remains an interest of the researchers. Among the oxygen hetero-
cycles, coumarins are the privileged structural motifs commonly
found in many natural products. Literature reveals that coumarin and
its derivatives are isolated from plant‐associated endophytes and
FIGURE 2 Marketed drugs containing the 1,2,3‐triazole unit

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FIGURE 3 The design strategy for the synthesis of new N‐phenylacetamide‐linked 1,2,3‐triazole–coumarin conjugates
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2
RESULTS AND DISCUSSION
data and spectroscopic techniques such as Fourier‐transform infrared
spectroscopy (FTIR), 1H nuclear magnetic resonance (NMR), 13C NMR,
and high‐resolution mass spectra (HRMS). According to the FTIR
spectrum of compound 7a, the peaks observed at 3,275 cm−1 indicate
the presence of N–H group, and the peaks observed at 1,698 and
1,670 cm−1 indicate the presence of two carbonyl groups. In the 1H
NMR spectrum of compound 7a, the signal at 2.41 ppm indicates the
methyl group present on the coumarin ring, and signals at 5.32 and
5.37 ppm are for two protons each and they indicate the presence of
two methylene groups attached with nitrogen and oxygen heteroatom,
respectively. In addition to this, the signal appearing at 8.32 ppm for
one proton clearly indicates the formation of the 1,4‐disubstituted
1,2,3‐triazole ring. In the 13C NMR spectrum of compound 7a, the
signal at 18.6 ppm indicates methyl carbon, and the signals at 52.7 and
62.1 ppm indicate the presence of two methylene groups attached to
the nitrogen of triazole and oxygen attached to the coumarin ring,
respectively. Furthermore, the peak observed at 161.6 ppm indicates
amide carbonyl carbon and the peak at 164.6 ppm indicates the pre-
sence of carbonyl carbon (lactone carbon) in the coumarin ring. The
formation of compound 7a has been further confirmed by mass
spectrometry. The calculated [M+Na]+ for compound 7a is at
413.1226, and observed [M+Na]+ in mass spectrum is at 413.1171.
Similarly, compounds 7b–g and 8a–g were characterized by the
spectral analysis. The structures of synthesized triazole–coumarin
conjugates are represented in Figure 4.
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2.1
Chemistry
1,4‐Disubstituted‐1,2,3‐triazoles bearing amide functionality displayed
several biological activities.[52–55] On the basis of these reports and
molecular hybridization concept, we have designed and synthesized
coumarin–triazole conjugates with amide linkage in their structures. A
library of substituted 2‐(4‐{[(4‐methyl‐2‐oxo‐2H‐chromen‐7‐yl)oxy]
methyl}‐1H‐1,2,3‐triazol‐1‐yl)‐N‐phenylacetamides 7a–g and sub-
stituted 2‐(4‐{[(2‐oxo‐2H‐chromen‐4‐yl)oxy]methyl}‐1H‐1,2,3‐triazol‐
1‐yl)‐N‐phenylacetamides 8a–g was synthesized from commercially
available starting materials. These compounds were constructed by the
fusion of coumarin‐based alkynes and substituted 2‐azido‐N‐
phenylacetamides via the click chemistry approach (Scheme 1).
The starting materials, 2‐azido‐N‐phenylacetamides 3a–g, were
prepared by a previously reported method[45,51] from corresponding
anilines in excellent yields (Scheme 1). The 7‐hydroxy‐4‐methyl cou-
marin 5a has been synthesized via acid‐catalyzed Pechmann con-
densation between resorcinol and ethyl acetoacetate in 80% yield
(Scheme 1). Compounds 6a and 6b were prepared by a previously re-
ported method.[44] Compounds 5a and 5b were treated with propargyl
bromide in the presence of K2CO3 as a base in N,N‐dimethylformamide
(DMF) at room temperature, resulting in 4‐methyl‐7‐(prop‐2‐yn‐1‐
yloxy)‐2H‐chromen‐2‐one 6a and 4‐(prop‐2‐yn‐1‐yloxy)‐2H‐chromen‐2‐
one 6b, respectively, in excellent yields (Scheme 1).
Finally, the click reaction of compounds 6a,b with azides 3a–g in
the presence of Cu(OAc)2 in t‐BuOH‐H2O (3:1) at room temperature
for 8–10 hr gave the corresponding 1,4‐disubstituted‐1,2,3‐
triazole–coumarin conjugates 7a–g and 8a–g, respectively, in good‐
to‐excellent yield (88–94%; Scheme 1).
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2.2
Biological activity
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2.2.1
Antifungal activity
Regioselective formation of 1,4‐disubstituted 1,2,3‐triazole–
coumarin conjugates 7a–g and 8a–g has been confirmed by physical
The synthesized compounds were screened for their in vitro anti-
fungal activities against five different fungal strains such as Candida

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SCHEME 1 The synthesis of coumarin–triazole conjugates. Reagents and conditions: (a) Chloroacetyl chloride, NEt3, CH2Cl2, 0°C at
room temperature (rt), 3–5 hr, 85–95%; (b) NaN3, toluene, reflux, 5–7 hr, 88–96%; (c) H2SO4, 0°C, 80%; (d) propargyl bromide, K2CO3,
N,N‐dimethylformamide, 2 hr, 93–95%; (e) Cu(OAc)2 (10 mol%), t‐BuOH/H2O (3:1), rt, 8–10 hr, 88–94%
albicans, Fusarium oxysporum, Aspergillus flavus, Aspergillus niger and
C. neoformans. The minimum inhibitory concentration (MIC, μg/ml)
values of all the newly synthesized compounds were determined by
the standard agar dilution method as per the Clinical & Laboratory
Standards Institute (CLSI; formerly NCCLS) guidelines.[56] Micona-
zole was used as the standard antifungal drug for the comparison of
antifungal activities and dimethyl sulfoxide (DMSO) was used as
negative control. The data on the antifungal activity are presented in
Table 1. Most of the compounds from the series exhibited a good‐to‐
excellent antifungal activity against all the fungal strains with MIC
values ranging from 12.5 to 25 μg/ml.
25 µg/ml. Compound 7c with methyl substituent at meta position of
the phenyl ring is equipotent to miconazole against fungal strains
A. niger and C. neoformans, with an MIC value of 25 µg/ml, and it is
two fold more potent than miconazole against C. albicans, with an
MIC value of 12.5 µg/ml. Compound 7d with methyl group at para
position shows two fold potency than the miconazole against
A. niger, with an MIC value of 12.5 µg/ml, and it is equipotent
against C. albicans (MIC: 25 µg/ml), F. oxysporum (MIC: 25 µg/ml), A.
flavus (MIC: 12.5 µg/ml), and C. neoformans (MIC: 25 µg/ml). Com-
pound 7e (chloro group at ortho position) exhibited a two fold ac-
tivity against C. albicans and A. niger (MIC: 12.5 µg/ml). Compound
7e also displayed an equivalent activity against F. oxysporum and
C. neoformans, with an MIC value of 25 µg/ml. Compounds 7f and 7g
displayed an equipotent activity against A. niger, C. neoformans,
C. albicans and F. oxysporum, with an MIC value of 25 µg/ml. In
addition, compounds 8a–g are equivalent or more potent than the
standard miconazole. Among the compounds, 8b, 8c, 8e, and 8f
showed an equivalent or two fold activity against all the fungal
strains, with MIC values of 12.5–25 μg/ml. The activity results
clearly indicate that most of the triazole–coumarin conjugates are
Without substitution on the phenyl ring, compound 7a ex-
hibited a two fold antifungal activity against A. niger and an equi-
potent activity against C. albicans and F. oxysporum, with MIC
values of 12.5, 25, and 25 µg/ml, respectively, as compared with the
standard antifungal drug miconazole. Compound 7b having methyl
group at ortho position of the phenyl ring was two fold more potent
than the standard miconazole against F. oxysporum, A. niger and
C. neoformans, with an MIC value of 12.5 µg/ml; it was also equi-
potent against the fungal strain C. albicans with an MIC value of

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FIGURE 4 Structures of triazole–coumarin conjugates
more potent or equipotent as compared with the miconazole,
as shown in the graphical representation (Figure 5).
findings obtained are summarized in Table 1. In comparison to the
synthetic antioxidant BHT, compound 7a exhibited excellent radical
scavenging activities, with an IC50 value of 15.01 μg/ml, and re-
maining compounds showed good‐to‐moderate activities.
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2.2.2
Antioxidant activity
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Computational study
The 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging assay
was used to screen the antioxidant activities of the synthesized
compounds 7a–g and 8a–g. The DPPH radical scavenging assay is the
most widely used tool for screening the antioxidant activity of var-
ious natural and synthetic compounds. A lower IC50 value indicates
more activity against antioxidants. The IC50 (concentration required
to scavenge 50% of the radicals) values were calculated to assess the
potential antioxidant activities. Butylated hydroxytoluene (BHT) has
been used as the standard drug to compare antioxidant activities; the
2.3
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2.3.1
Comparative modeling
The sequence identity and atomic resolution are two key parameters
while selection of the template structure, which were 44% and 2.8 Å,
respectively, which satisfy the basic criterion for comparative mod-
eling. The final model was subjected to structure validation tool such
as Procheck, ProSA, SPDBV and were found that 99.7 percent of the

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TABLE 1 In vitro biological evaluation of
the synthesized triazole–coumarin
conjugates 7a–g and 8a–g
Antifungal activity (MIC in μg/ml)
DPPH IC50
Molecular
Compound
CA
25
FO
25
AF
AN
CN
50
(μg/ml)
docking score
7a
7b
7c
25
12.5
12.5
25
15.01 0.26
19.24 0.19
21.14 0.97
24.37 0.34
38.25 0.24
29.34 0.19
38.18 0.54
36.59 0.64
27.44 0.31
67.30 0.05
44.31 0.99
89.32 0.76
71.08 0.26
57.16 0.79
NA
−7.0932
−7.3901
−6.8629
−7.7256
−7.9245
−6.8669
−6.7934
−6.2456
−7.1950
−6.2411
−7.4214
−7.2302
−6.6233
−6.7248
−5.26
25
12.5
50
50
12.5
25
12.5
25
25
7d
7e
7f
25
12.5
25
12.5
12.5
25
25
12.5
50
25
25
50
37.5
75
25
7g
8a
8b
8c
25
25
75
50
37.5
12.5
25
25
25
37.5
12.5
12.5
75
25
25
12.5
25
12.5
25
12.5
25
8d
8e
8f
37.5
12.5
25
12.5
12.5
12.5
62.5
12.5
NA
25
12.5
12.5
50
12.5
12.5
37.5
25
25
25
8g
MA
BHT
37.5
25
12.5
25
25
NA
NA
NA
NA
16.47 0.18
NA
Abbreviations: AF, Aspergillus flavus; AN, Aspergillus niger; BHT, butylated hydroxytoluene;
CA, Candida albicans; CN, Cryptococcus neoformans; DPPH, 2,2‐diphenyl‐1‐picrylhydrazyl; FO,
Fusarium oxysporum; MA, miconazole; MIC, minimum inhibitory concentration; NA, not applicable.
residue followed in the allowed region. Also, overall model quality
was assessed using Prosa where Z score is −3.2 and Cα deviation is
0.45 Å, respectively. The validation study of the model suggested
that it was perfect for further computation study.
three‐dimensional structure of cytochrome P450 lanosterol
14α‐demethylase of C. albicans to understand the binding affinity and
binding interactions of enzyme and synthesized derivatives. The syn-
thesized triazole–coumarin conjugates (7a–g, 8a–g) and the standard
drug miconazole were docked in the active site of modeled CACYP51
using the AutoDock Vina docking tool. The results of docking are shown
in Table 1. The analysis of docking interaction revealed that the triazole
ring was mainly responsible for the interaction.
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2.3.2
Molecular docking study
The molecular docking study of all synthesized triazole–coumarin
conjugates, 7a–g and 8a–g, was performed against modeled
The triazole–coumarin conjugates 7b, 7d, 7e, 8b and 8e re-
produced a similar result as that of in vitro activity data. All active
FIGURE 5 A comparison of the
antifungal activities of triazole–coumarin
conjugates with miconazole

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compounds efficiently interact with the active‐site residues like
Tyr105, Phe108, Phe121, Val130, Tyr168, Tyr154, Phe162, Leu412,
Phe246, Met342, Ala343, Cys439, Ser414, Leu412 and Met544. The
triazole–coumarin conjugates having ortho substitution on the phenyl
ring partially replicate in vitro antifungal results, that is, 8b and 8e.
The ortho‐substituted (–CH3) phenyl ring derivative 8b (−7.19)
interacts with polar and nonpolar amino acids of the active site. The
polar amino acid Ser414 interacts with the nitrogen atom of the
triazole ring to form conventional hydrogen bond interactions with a
distance of 2.37 Å. The sulfur atom of polar amino acid Cys439 in-
teracts with π electron cloud of the phenyl ring to form π–sulfur
interactions. The aliphatic and hydrophobic amino acids Leu412,
Tyr105, Phe108, Phe121, and Val130 interact with π electron cloud
of aromatic ring to form π–π T‐shaped, π–π stacked, and π–sulfur
interactions with various distance values shown in Figure 6a.
chlorine atom with a distance of 2.08 and 3.77 Å. However, aliphatic
and hydrophobic amino acids Leu412, Phe264, Phe162, Tyr154, and
Met342 interact with π electron cloud of aromatic phenyl rings to
form π–π stacked, amide–π stacked, alkyl and π–alkyl interactions
shown in Figure 6b.
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2.3.3
In silico ADME prediction
Early prediction of druglikeness properties of lead compounds is an
important task, as it decides the time and cost of drug discovery and
development. Many of the active agents with a significant biological
activity have failed in clinical trials due to inadequate druglikeness
properties.[57] The druglikeness properties were predicted by ana-
lyzing ADME parameters based on Lipinski's rule of five. We had
calculated and analyzed various physical descriptors and pharma-
ceutical relevant properties for ADMET prediction by using
FAFDrugs2, and data are summarized in Table 2.
The ortho‐substituted (–Cl) phenyl ring derivative 8e (−7.23) in-
teracts with aliphatic and hydrophobic amino acid residues Ala342
and Tyr168, where it interacts with carbonyl oxygen atom and ni-
trogen atom of the triazole ring with a distance of 2.21 and 2.08 Å to
form conventional hydrogen bond interactions. The polar amino acid
All the compounds showed significant values for the various
parameters analyzed and showed good drug‐like characteristics
based on Lipinski's rule of five and its variants that characterized
these agents to be likely orally active. The data obtained for all the
residue Met342 and hydrophobic amino acid Phe264 form
a
carbon–hydrogen bond and π interaction with ortho‐substituted
FIGURE 6 Binding pose and molecular
interactions of (a) 8b and (b) 8e in the
active site of cytochrome P450 lanosterol
14α‐demethylase (CYP51)

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TABLE 2 Pharmacokinetic parameters of triazole–coumarin conjugates (7a–g and 8a–g)
Entry
7a
7b
7c
%ABS
74.76
74.76
74.76
74.76
74.76
74.76
74.76
76.11
76.11
76.11
76.11
76.11
76.11
76.11
MW
LogP
2.98
3.29
3.29
3.29
3.64
3.64
3.64
2.56
2.87
2.87
2.87
3.21
3.21
3.21
PSA
RotB
6
RigidB
25
HBD
1
HBA
6
Ratio H/C
0.4
Lipinski violation
Toxicity
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
390.39
404.42
404.42
404.42
424.84
424.84
424.84
378.38
392.41
392.41
392.41
412.83
412.83
412.83
99.25
99.25
99.25
99.25
99.25
99.25
99.25
95.34
95.34
95.34
95.34
95.34
95.34
95.34
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6
25
1
6
0.4
6
25
1
6
0.4
7d
7e
7f
6
25
1
6
0.4
6
25
1
6
0.4
6
25
1
6
0.4
7g
8a
8b
8c
6
25
1
6
0.4
6
25
1
6
0.4
6
25
1
6
0.4
6
25
1
6
0.4
8d
8e
8f
6
25
1
6
0.4
6
25
1
6
0.5
6
25
1
6
0.5
8g
6
25
1
6
0.5
Abbreviations: HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; MW, molecular weight; PSA, polar surface area; RotB, rotatable bonds;
RigidB, rigid bonds; %ABS, percentage absorption.
synthesized compounds were within the range of accepted values.
None of the synthesized compounds had violated the Lipinski's rule
of five. The value of polar surface area, logP, and H/C ratio of syn-
thesized compounds 7a–g and 8a–g indicated good oral bioavail-
ability. The parameters like the number of rotatable bonds and
number of rigid bonds are linked with the intestinal absorption; re-
sults showed that all synthesized compounds had good absorption.
All the synthesized compounds were found to be nontoxic. The in
silico assessment of all the synthetic compounds has shown that they
have very good pharmacokinetic properties, which is reflected in
their physicochemical values, thus ultimately enhancing pharmaco-
logical properties of these molecules.
oral drug candidates. Thus, we suggest that compounds 7a (anti-
oxidant activity), 7b, 7d, 7e, 8b and 8e (antifungal activity) can be
developed as an important lead moiety, as they replicate in vitro ac-
tivity in the inhibition assay and in silico molecular docking study. They
can be used in scaffolds, hoping for the design and development of
new lead compounds as antifungal agents.
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4
EXPERIMENTAL
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4.1
Chemistry
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4.1.1
General
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3
CONCLUSIONS
All the solvents and reagents were purchased from commercial
suppliers, Sigma‐Aldrich, Rankem India Ltd., and Spectrochem Pvt.
Ltd., and were used without further purification. The completion of
the reactions was monitored by thin‐layer chromatography (TLC) on
aluminum plates coated with silica gel 60 F254, with 0.25 mm
thickness (Merck). The detection of the components was done by
exposure to iodine vapors or UV light. Melting points were de-
termined by open capillary methods and were uncorrected. 1H NMR
and 13C NMR spectra were recorded in DMSO‐d6 on a Bruker
DRX‐400 and 500‐MHz spectrometer. Infrared (IR) spectra were
recorded using a Bruker ALPHA Eco‐ATR FTIR spectrometer. High‐
resolution mass spectra (HRMS) were recorded on an Agilent 6520
(QTOF) mass spectrometer.
In conclusion, we have synthesized new triazole–coumarin conjugates
via the click chemistry approach, which were evaluated for their in
vitro antifungal and antioxidant activity. The synthesized compounds
displayed a promising antifungal activity as compared with the stan-
dard drug miconazole. Compounds 7b, 7d, 7e, 8b, and 8e displayed an
excellent antifungal activity as compared with the standard antifungal
drug miconazole. Compound 7a displayed a potential antioxidant ac-
tivity when compared with standard BHT. In addition, molecular
docking study of these synthesized triazole–coumarin conjugates re-
veals that they have a high affinity toward the active site of enzyme
P450 cytochrome lanosterol 14α‐demethylase, which offers a strong
platform for new structure‐based design efforts. Furthermore, the
analysis of the ADME parameters for the synthesized compounds
predicted good drug‐like properties and potential for development as
The original spectra of the investigated compounds, together
with their InChI codes and some biological activity data, are provided
as Supporting Information Data.

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4.1.2
General procedure for the synthesis of
111.1, 112.4, 113.2, 116.3, 119.6, 124.3, 126.3, 126.4, 128.6, 138.0,
138.1, 141.6, 153.2, 154.5, 160.0, 160.9, and 163.9; HRMS calculated
for C22H21N4O4 [M+H]+: 405.1563 and found: 405.1571.
substituted 2‐(4‐{[(4‐methyl‐2‐oxo‐2H‐chromen‐7‐yl)‐
oxy]methyl}‐1H‐1,2,3‐triazol‐1‐yl)‐N‐phenylacetamide
derivatives 7a–g and 8a–g
2‐(4‐{[(4‐Methyl‐2‐oxo‐2H‐chromen‐7‐yl)oxy]methyl}‐1H‐1,2,3‐
triazol‐1‐yl)‐N‐(p‐tolyl)acetamide (7d)
To the stirred solution of alkyne 6 (1 mmol), azide 3 (1 mmol) and
copper diacetate (Cu(OAc)2; 10 mol%) in t‐BuOH‐H2O (3:1) were added,
and the resulting mixture was stirred at room temperature for 8–10 hr.
The progress of the reaction was monitored by TLC using ethyl acetate/
hexane as a solvent system. The reaction mixture was quenched with
crushed ice, and the obtained solid was filtered and washed with water.
The crude solid was crystallized in ethanol to afford the corresponding
pure product. The synthesized compounds 7a–g and 8a–g were char-
acterized by IR, 1H NMR, 13C NMR, and mass spectroscopy.
Compound 7d was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3d and alkyne 6a in 9 hr as a red solid, mp: 216–218°C;
FTIR (cm−1): 3,248 (N–H stretching), 1,698 and 1,659 (C═O stretch-
ing); 1H NMR (400 MHz, DMSO‐d6, δ ppm): 2.25 (s, 3H, –CH3), 2.40 (s,
3H, –CH3), 5.31 (s, 2H, –NCH2CO–), 5.34 (s, 2H, –OCH2), 6.22 (s, 1H,
Ar‐H), 7.06 (dd, J = 8.0, 4.0 Hz, 1H, Ar‐H), 7.13 (d, J = 8.0 Hz, 2H, Ar‐H),
7.17 (d, J = 4.0 Hz, 1H, Ar‐H), 7.46 (d, J = 8.0 Hz, 2H, Ar‐H), 7.70 (d,
J = 8.0 Hz, 1H, Ar‐H), 8.31 (s, 1H, triazole), and 10.40 (s, 1H, NH); 13
C
NMR (100 MHz, DMSO‐d6, δ ppm): 18.5, 20.8, 52.5, 61.9, 101.9, 111.6,
113.0, 113.7, 119.6, 126.9, 127.0, 129.6, 133.1, 136.2, 142.1, 153.8,
155.0, 160.5, 161.4, and 164.2; HRMS calculated for C22H19N4O4
[M−H]+: 403.1412 and found: 403.1424.
2‐(4‐{[(4‐Methyl‐2‐oxo‐2H‐chromen‐7‐yl)oxy]methyl}‐1H‐1,2,3‐
triazol‐1‐yl)‐N‐phenylacetamide (7a)
Compound 7a was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3a and alkyne 6a in 8.5 hr as a white solid, mp:
149–151°C; FTIR (cm−1): 3,275 (N–H stretching), 1,698 and 1,670 (C═O
stretching); 1H NMR (500 MHz, DMSO‐d6, δ ppm): 2.41 (s, 3H, –CH3),
5.32 (s, 2H, –NCH2CO–), 5.37 (s, 2H, –OCH2), 6.23 (s, 1H, Ar‐H), 7.07 (dd,
J = 8.0, 3.0 Hz, 1H, Ar‐H), 7.10 (d, J = 8.0 Hz, 1H, Ar‐H), 7.18 (s, 1H, Ar‐H),
7.34 (t, J = 8.0 Hz, 2H, Ar‐H), 7.58 (d, J = 8.0 Hz, 2H, Ar‐H), 7.71 (d,
J = 8.0 Hz, 1H, Ar‐H), 8.32 (s, 1H, triazole), and 10.48 (s, 1H, NH); 13C
NMR (125 MHz, DMSO‐d6, δ ppm): 18.6, 52.7, 62.1, 102.1, 111.8, 113.1,
113.9, 119.7, 124.3, 127.0, 127.1, 129.4, 138.9, 142.3, 153.9, 155.2,
160.6, 161.6, and 164.6; mass calculated for C21H18N4O4Na [M+Na]+:
413.1226 and found: 413.1171.
N‐(2‐Chlorophenyl)‐2‐(4‐{[(4‐methyl‐2‐oxo‐2H‐chromen‐7‐yl)oxy]‐
methyl}‐1H‐1,2,3‐triazol‐1‐yl)acetamide (7e)
Compound 7e was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3e and alkyne 6a in 9.5 hr as a white solid, mp:
206–208°C; FTIR (cm−1): 3,118 (N–H stretching), 1,664 and 1,600
(C═O stretching); 1H NMR (400 MHz, DMSO‐d6, δ ppm): 2.40
(s, 3H, –CH3), 5.31 (s, 2H, –NCH2CO–), 5.48 (s, 2H, –OCH2), 6.22
(s, 1H, Ar‐H), 7.05 (d, J = 8.0, Hz, 1H, Ar‐H), 7.17 (s, 1H, Ar‐H), 7.22 (t,
J = 8.0 Hz, 1H, Ar‐H), 7.34 (t, J = 8.0 Hz, 1H, Ar‐H), 7.52 (d, J = 8.0 Hz,
1H, Ar‐H), 7.69 (d, J = 8.0 Hz, 1H, Ar‐H), 7.74 (d, J = 8.0 Hz, 1H, Ar‐H),
8.32 (s, 1H, triazole), and 10.09 (s, 1H, NH); 13C NMR (100 MHz,
DMSO‐d6, δ ppm): 18.1, 52.0, 61.6, 101.6, 111.3, 112.6, 113.4, 125.9,
126.3, 126.5, 126.7, 126.8, 127.6, 129.6, 134.1, 141.8, 153.4, 154.7,
160.1, 161.1, and 164.9; HRMS calculated for C21H16ClN4O4
[M−H]+: 423.0866 and found: 423.0880.
2‐(4‐{[(4‐Methyl‐2‐oxo‐2H‐chromen‐7‐yl)oxy]methyl}‐1H‐1,2,3‐
triazol‐1‐yl)‐N‐(o‐tolyl)acetamide (7b)
Compound 7b was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3b and alkyne 6a in 9.5 hr as an orange solid, mp:
195–197°C; FTIR (cm−1): 3,276 (N–H stretching), 1,690 and 1,670 (C═O
stretching); 1H NMR (400 MHz, DMSO‐d6, δ ppm): 1.95 (s, 3H, –CH3),
2.12 (s, 3H, –CH3), 5.04 (s, 4H, –NCH2CO– and –OCH2), 5.84 (s, 1H,
Ar‐H), 6.67–6.68 (m, 4H, Ar‐H), 7.25 (s, 3H, Ar‐H), 7.75 (s, 1H, triazole),
and 9.07 (s, 1H, NH); mass calculated for C22H20N4O4Na [M+Na]+:
427.1382 and found: 427.1334.
N‐(3‐Chlorophenyl)‐2‐(4‐{[(4‐methyl‐2‐oxo‐2H‐chromen‐7‐yl)oxy]‐
methyl}‐1H‐1,2,3‐triazol‐1‐yl)acetamide (7f)
Compound 7f was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3f and alkyne 6a in 10 hr as a yellow solid, mp:
218–220°C; 1H NMR (400 MHz, DMSO‐d6, δ ppm): 2.39 (s, 3H, –CH3),
5.31 (s, 2H, –NCH2CO–), 5.38 (s, 2H, –OCH2), 6.21 (s, 1H, Ar‐H), 7.05
(dd, J = 8.0, 4.0 Hz, 1H, Ar‐H), 7.15 (d, J = 8.0 Hz, 2H, Ar‐H), 7.36 (t,
J = 8.0 Hz, 1H, Ar‐H), 7.44 (d, J = 8.0 Hz, 1H, Ar‐H), 7.69 (d, J = 8.0 Hz,
1H, Ar‐H), 7.77 (s, 1H, Ar‐H), 8.31 (s, 1H, triazole), and 10.68 (s, 1H,
NH); 13C NMR (100 MHz, DMSO‐d6, δ ppm): 18.2, 52.2, 61.6, 101.6,
111.3, 112.6, 113.4, 117.7, 118.8, 123.6, 126.5, 126.7, 130.7, 133.2,
139.8, 141.8, 154.7, 160.2, 161.1, and 164.7; HRMS calculated for
C21H16ClN4O4 [M−H]+: 423.0866 and found: 423.0872.
2‐(4‐{[(4‐Methyl‐2‐oxo‐2H‐chromen‐7‐yl)oxy]methyl}‐1H‐1,2,3‐
triazol‐1‐yl)‐N‐(m‐tolyl)acetamide (7c)
Compound 7c was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3c and alkyne 6a in 8 hr as a white solid, mp:
190–192°C; FTIR (cm−1): 3,291 (N–H stretching), 1,678 (C═O
stretching); 1H NMR (500 MHz, DMSO‐d6, δ ppm): 2.27 (s, 3H, –CH3),
2.39 (s, 3H, –CH3), 5.31 (s, 2H, –NCH2CO–), 5.36 (s, 2H, –OCH2),
6.21 (s, 1H, Ar‐H), 6.91 (d, J = 8.0, Hz, 1H, Ar‐H), 7.05 (dd, J = 8.0,
4.0 Hz, 1H, Ar‐H), 7.17 (d, J = 4.0 Hz, 1H, Ar‐H), 7.21 (t, J = 8.0 Hz, 1H,
Ar‐H), 7.37 (d, J = 8.0 Hz, 1H, Ar‐H), 7.42 (s, 1H, Ar‐H), 7.69 (d,
J = 8.0 Hz, 1H, Ar‐H), 8.31 (s, 1H, triazole) and 10.40 (s, 1H, NH); 13C
NMR (125 MHz, DMSO‐d6, δ ppm): 17.9, 20.9, 52.1, 61.4, 101.4,
N‐(4‐Chlorophenyl)‐2‐(4‐{[(4‐methyl‐2‐oxo‐2H‐chromen‐7‐yl)oxy]‐
methyl}‐1H‐1,2,3‐triazol‐1‐yl)acetamide (7g)
Compound 7g was obtained via the 1,3‐dipolar cycloaddition re-
action between azide 3g and alkyne 6a in 8.5 hr as a yellow solid,

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mp: 210–212°C; 1H NMR (400 MHz, DMSO‐d6, δ ppm): 2.40
(s, 3H, –CH3), 5.30 (s, 2H, –NCH2CO–), 5.36 (s, 2H, –OCH2), 6.23
(s, 1H, Ar‐H), 7.05 (dd, J = 8.0, 4.0 Hz, 1H, Ar‐H), 7.17 (d, J = 4.0 Hz,
1H, Ar‐H), 7.39 (d, J = 8.0 Hz, 2H, Ar‐H), 7.60 (d, J = 8.0 Hz, 2H,
Ar‐H), 7.70 (d, J = 8.0 Hz, 1H, Ar‐H), 8.31 (s, 1H, triazole), and
10.63 (s, 1H, NH); HRMS calculated for C21H16ClN4O4 [M−H]+:
423.0866 and found: 423.0878.
2‐(4‐{[(2‐Oxo‐2H‐chromen‐4‐yl)oxy]methyl}‐1H‐1,2,3‐triazol‐1‐yl)‐
N‐(p‐tolyl)acetamide (8d)
Compound 8d was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3d and alkyne 6b in 8.5 hr as a white solid, mp:
247–249°C; 1H NMR (400 MHz, DMSO‐d6, δ ppm): 2.25 (s, 3H, –CH3),
5.38 (s, 2H, –NCH2CO–), 5.47 (s, 2H, –OCH2), 6.20 (s, 1H, Ar‐H), 7.13
(d, J = 8.0, Hz, 2H, Ar‐H), 7.34 (t, J = 8.0, Hz, 1H, Ar‐H), 7.41
(d, J = 8.0 Hz, 1H, Ar‐H), 7.47 (d, J = 8.0 Hz, 2H, Ar‐H), 7.66 (t,
J = 8.0 Hz, 1H, Ar‐H), 7.74 (d, J = 8.0 Hz, 1H, Ar‐H), 8.41 (s, 1H, tria-
zole), and 10.41 (s, 1H, NH); 13C NMR (100 MHz, DMSO‐d6, δ ppm):
20.5, 52.3, 62.8, 91.4, 116.5, 119.2, 122.8, 124.2, 127.0, 129.3, 132.8,
135.9, 140.6, 152.8, 161.6, 163.9, and 164.4; HRMS calculated for
C21H17N4O4 [M−H]+: 389.1255 and found: 389.1314.
2‐(4‐{[(2‐Oxo‐2H‐chromen‐4‐yl)oxy]methyl}‐1H‐1,2,3‐triazol‐1‐yl)‐
N‐phenylacetamide (8a)
Compound 8a was obtained via the 1,3‐dipolar cycloaddition re-
action between azide 3a and alkyne 6b in 10 hr as a white solid,
mp: 216–218°C; 1H NMR (400 MHz, DMSO‐d6, δ ppm): 5.40 (s,
2H, –NCH2CO–), 5.47 (s, 2H, –OCH2), 6.20 (s, 1H, Ar‐H), 7.09
(t, J = 8.0, Hz, 1H, Ar‐H), 7.33 (t, J = 8.0 Hz, 3H, Ar‐H), 7.40
(d, J = 8.0 Hz, 1H, Ar‐H), 7.59 (d, J = 8.0 Hz, 2H, Ar‐H), 7.65
(t, J = 8.0 Hz, 1H, Ar‐H), 7.74 (d, J = 8.0 Hz, 1H, Ar‐H), 8.43 (s, 1H,
triazole), and 10.51 (s, 1H, NH); 13C NMR (100 MHz, DMSO‐d6, δ
ppm): 52.3, 62.8, 91.4, 115.1, 116.5, 119.3, 122.8, 123.8, 124.3,
127.1, 128.9, 132.8, 138.4, 152.8, 164.2, and 164.4; HRMS cal-
culated for C20H15N4O4 [M−H]+: 375.1099 and found: 375.1080.
N‐(2‐Chlorophenyl)‐2‐(4‐{[(2‐oxo‐2H‐chromen‐4‐yl)oxy]methyl}‐1H‐
1,2,3‐triazol‐1‐yl)acetamide (8e)
Compound 8e was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3e and alkyne 6b in 9.5 hr as a brown solid, mp:
217–219°C; FTIR (cm−1): 3,254 (N–H stretching), 1,709 and 1,673
(C═O stretching); 1H NMR (400 MHz, DMSO‐d6, δ ppm): 5.47
(s, 2H, –NCH2CO–), 5.51 (s, 2H, –OCH2), 6.19 (s, 1H, Ar‐H),
7.20–7.76 (m, 8H, Ar‐H), 8.43 (s, 1H, triazole), and 10.12 (s, 1H,
NH); 13C NMR (100 MHz, DMSO‐d6, δ ppm): 52.0, 62.7, 91.4, 115.0,
116.5, 122.8, 124.2, 125.9, 126.3, 126.8, 127.1, 127.6, 129.6, 132.8,
134.1, 152.8, 161.5, 164.3, and 164.8; HRMS calculated for
C20H14ClN4O4 [M−H]+: 409.0709 and found: 409.0722.
2‐(4‐{[(2‐Oxo‐2H‐chromen‐4‐yl)oxy]methyl}‐1H‐1,2,3‐triazol‐1‐yl)‐
N‐(o‐tolyl)acetamide (8b)
Compound 8b was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3b and alkyne 6b in 8.5 hr as a white solid, mp:
170–172°C; FTIR (cm−1): 3,248 (N–H stretching), 1,698 and 1,659
(C═O stretching); 1H NMR (400 MHz, DMSO‐d6, δ ppm): 2.24
(s, 3H, –CH3), 5.45 (s, 2H, –NCH2CO–), 5.47 (s, 2H, –OCH2), 6.20
(s, 1H, Ar‐H), 7.11 (t, J = 8.0, Hz, 1H, Ar‐H), 7.17 (t, J = 8.0 Hz, 1H,
Ar‐H), 7.23 (d, J = 8.0 Hz, 1H, Ar‐H), 7.33 (t, J = 8.0 Hz, 1H, Ar‐H), 7.42
(d, J = 8.0 Hz, 2H, Ar‐H), 7.65 (t, J = 8.0 Hz, 1H, Ar‐H), 7.73
(d, J = 8.0 Hz, 1H, Ar‐H), 8.43 (s, 1H, triazole), and 9.83 (s, 1H, NH);
13C NMR (100 MHz, DMSO‐d6, δ ppm): 17.8, 52.0, 62.8, 91.4, 115.1,
116.5, 122.8, 124.2, 124.8, 125.6, 126.1, 127.0, 130.5, 131.6, 132.8,
135.5, 152.8, 161.6, 164.3, and 164.4; HRMS calculated for
C21H17N4O4 [M−H]+: 389.1255 and found: 389.1311.
N‐(3‐Chlorophenyl)‐2‐(4‐{[(2‐oxo‐2H‐chromen‐4‐yl)oxy]methyl}‐1H‐
1,2,3‐triazol‐1‐yl)acetamide (8f)
Compound 8f was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3f and alkyne 6b in 10 hr as a red solid, mp:
218–220°C; 1H NMR (400 MHz, DMSO‐d6, δ ppm): 5.42 (s, 2H,
–NCH2CO–), 5.47 (s, 2H, –OCH2), 6.20 (s, 1H, Ar‐H), 7.15 (d, J = 8.0,
Hz, 1H, Ar‐H), 7.32–7.41 (m, 3H, Ar‐H), 7.45 (d, J = 8.0 Hz, 1H, Ar‐H),
7.65 (t, J = 8.0 Hz, 1H, Ar‐H), 7.73 (d, J = 8.0 Hz, 1H, Ar‐H), 7.78 (s, 1H,
Ar‐H), 8.43 (s, 1H, triazole), and 10.71 (s, 1H, NH); 13C NMR
(100 MHz, DMSO‐d6, δ ppm): 52.3, 62.8, 91.4, 115.1, 116.5, 117.7,
118.8, 122.8, 123.6, 124.2, 127.0, 130.7, 132.8, 133.2, 139.8, 140.8,
152.8, 161.6, 164.4, and 164.6; HRMS calculated for C20H14ClN4O4
[M−H]+: 409.0709 and found: 409.0724.
2‐(4‐{[(2‐Oxo‐2H‐chromen‐4‐yl)oxy]methyl}‐1H‐1,2,3‐triazol‐1‐yl)‐
N‐(m‐tolyl)acetamide (8c)
Compound 8c was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3c and alkyne 6b in 9 hr as a yellow solid, mp:
211–213°C; FTIR (cm−1): 3,307 (N–H stretching), 1,703 and 1,666
(C═O stretching); 1H NMR (400 MHz, DMSO‐d6, δ ppm): 2.27
(s, 3H, –CH3), 5.40 (s, 2H, –NCH2CO–), 5.47 (s, 2H, –OCH2), 6.20
(s, 1H, Ar‐H), 6.90 (d, J = 8.0, Hz, 1H, Ar‐H), 7.21 (t, J = 8.0, Hz, 1H,
Ar‐H), 7.30–7.44 (m, 4H, Ar‐H), 7.65 (t, J = 8.0 Hz, 1H, Ar‐H), 7.73
(d, J = 8.0 Hz, 1H, Ar‐H), 8.43 (s, 1H, triazole) and 10.44 (s, 1H, NH);
13C NMR (125 MHz, DMSO‐d6, δ ppm): 21.4, 52.5, 63.0, 91.6, 115.3,
116.6, 116.7, 120.0, 123.3, 124.4, 124.7, 127.3, 129.0, 133.0, 138.4,
138.5, 153.0, 161.8, 164.3, and 164.6; HRMS calculated for
C21H17N4O4 [M−H]+: 389.1255 and found: 389.1346.
N‐(4‐Chlorophenyl)‐2‐(4‐{[(2‐oxo‐2H‐chromen‐4‐yl)oxy]methyl}‐1H‐
1,2,3‐triazol‐1‐yl)acetamide (8g)
Compound 8g was obtained via the 1,3‐dipolar cycloaddition reaction
between azide 3g and alkyne 6b in 8 hr as a white solid, mp: 240–242°C;
1H NMR (400 MHz, DMSO‐d6, δ ppm): 5.40 (s, 2H, –NCH2CO–), 5.46
(s, 2H, –OCH2), 6.19 (s, 1H, Ar‐H), 7.30–7.44 (m, 4H, Ar‐H), 7.57–7.68 (m,
3H, Ar‐H), 7.74 (d, J = 8.0 Hz, 1H, Ar‐H), 8.41 (s, 1H, triazole), and 10.64
(s, 1H, NH); 13C NMR (100 MHz, DMSO‐d6, δ ppm): 52.8, 63.2, 91.8,
115.6, 117.0, 121.3, 123.3, 124.7, 127.5, 127.9, 129.3, 133.3, 137.8,
141.3, 153.3, 162.0, and 164.8; HRMS calculated for C20H14ClN4O4
[M−H]+: 409.0709 and found: 409.0715.

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Molecular modeling study
4.2
Biological activity assays
4.3
|
|
Homology modeling
4.2.1
Antifungal activity
4.3.1
The antifungal activity was determined by the standard agar
dilution method as per the CLSI (formerly NCCLS) guidelines.[56]
The newly synthesized compounds were screened against five
human pathogenic fungal strains, including C. albicans (NCIM
3471), F. oxysporum (NCIM 1332), A. flavus (NCIM 539), A. niger
(NCIM 1196), and C. neoformans (NCIM 576). The standard mico-
nazole and synthesized compounds were dissolved in DMSO. In
phosphate buffer of pH 7, the medium yeast nitrogen base was
dissolved and autoclaved for a duration of 10 min at 110°C. With
each set, a growth control without the antifungal agent and sol-
vent control DMSO were included. The fungal strains were freshly
subcultured on Sabouraud dextrose agar and incubated at 25°C
for 72 hr. The fungal cells were suspended in sterile distilled water
and diluted to get 105 cells/ml. Then, 10 ml of the standardized
suspension was inoculated on the control plates and the media
were 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 and 72 hr. MIC is the lowest concentration of the
drug preventing the growth of macroscopically visible colonies on
the drug‐containing plates when there is visible growth on the
drug‐free control plates.
The homology modeling technique was employed to build a 3D model
structure of cytochrome P450 lanosterol 14α‐demethylase of C. albicans
with the help of VLifeMDS 4.3 Promodel molecular modeling tool.
The protein sequence was retrieved from UniprotKB database
(accession number: P10613). The homologs' template sequence
search was carried out against the protein structure database
(http://www.rcsb.org/) by using BlastP. The appropriate template
crystal structure of human lanosterol 14α‐demethylase (CYP51)
complexes with miconazole (3LD6 B) which was based on default
parameters, identity and positive criteria. The secondary structure
assignment and sequence realignment were carried out to build the
final modeled structure of fungal CYP51.
|
4.3.2
Molecular docking study
The model protein structure and 3D structure of sketched syn-
thesized compound were prepared for molecular docking using
AutoDock Vina docking tool. The molecular docking study of
synthesized compounds 7a–g and 8a–g was carried out using
the final modeled structure of fungal CYP51. To understand
this mechanism of action of inhibitors, molecular interactions
were analyzed.
|
4.2.2
Antioxidant activity
|
ADMET testing
Synthesized triazole–coumarin conjugates were screened for in vitro
radical scavenging potential by using the DPPH radical scavenging
assay. The results were compared with the standard synthetic anti-
oxidant BHT.
4.3.3
The ADMET properties of synthesized compounds 7a–g and 8a–g
and standard drug were tested using FAFDrug2 tool, which is run
on the Linux operating system.[59] The FAFDrug2 tool works on
assumptions of Lipinski's rule of five and Veber's rule, which was
widely followed in filtering lead compounds that would likely be
further developed in drug design programs.[60] In addition to this,
some other parameters were also considered to test ADMET
properties, such as the number of rotatable bonds (>10), the
number of rigid bonds, and percentage absorption (%ABS), which
significantly contribute to good oral bioavailability and good in-
testinal absorption.[61]
The antioxidant activity of the synthesized compounds was
assessed in vitro by the DPPH radical scavenging assay.[58] Results
were compared with the standard antioxidant BHT. The hydrogen
atom or electron‐donating ability of the compounds was measured
from the bleaching of the purple‐colored methanol solution of
DPPH. The spectrophotometric assay uses the stable radical DPPH
as a reagent. Furthermore, 1 ml of various concentrations of the
test compounds (5, 10, 25, 50, and 100 mg/ml) in methanol was
added to 4 ml of 0.004% (w/v) methanol solution of DPPH. After a
30‐min incubation period at room temperature, the absorbance
was measured against blank at 517 nm. The percent inhibition (I%)
of free radical production from DPPH was calculated by the fol-
lowing equation:
ACKNOWLEDGMENTS
S. V. A. is very grateful to the Council of Scientific and Industrial
Research (CSIR), New Delhi, for providing a research fellowship.
The authors are also grateful for providing laboratory facilities to
the Head, Department of Chemistry, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad.
% scavenging = [(Acontrol − Asample)/Ablank] × 100,
where Acontrol is the absorbance of the control reaction (containing all
reagents except the test compound) and Asample is the absorbance of
the test compound. Tests were carried out in triplicate.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.

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How to cite this article: Akolkar SV, Nagargoje AA, Shaikh
MH, et al. New N‐phenylacetamide‐linked 1,2,3‐triazole‐
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