New: N -phenylacetamide-incorporated 1,2,3-triazoles: [Et3NH][OAc]-mediated efficient synthesis and biological evaluation

Article abstract of DOI:10.1039/c9ra03425k

A facile, highly efficient, and greener method for the synthesis of new 1,4-disubstituted-1,2,3-triazoles was conducted using [Et₃NH][OAc] as a medium by the implementation of ultrasound irradiation via click chemistry, affording excellent yiel

Full text of DOI:10.1039/c9ra03425k

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New N-phenylacetamide-incorporated 1,2,3-
triazoles: [Et3NH][OAc]-mediated efficient
Cite this: RSC Adv., 2019, 9, 22080
synthesis and biological evaluation†
a
Satish V. Akolkar,
Amol A. Nagargoje,a Vagolu S. Krishna,b Dharmarajan Sriram,b
c
d
a
*
Jaiprakash N. Sangshetti,
Manoj Damale and Bapurao B. Shingate
A facile, highly efficient, and greener method for the synthesis of new 1,4-disubstituted-1,2,3-triazoles was
conducted using [Et3NH][OAc] as a medium by the implementation of ultrasound irradiation via click
chemistry, affording excellent yields. The present synthetic method exhibited numerous advantages such
as mild reaction conditions, excellent product yields, minimal chemical waste, operational simplicity,
shorter reaction time, and a wide range of substrate scope. The synthesized compounds were further
evaluated for in vitro antifungal activity against five fungal strains, and some of the compounds displayed
equivalent or greater potency than the standard drug. A molecular docking study against the modelled
three-dimensional structure of cytochrome P450 lanosterol 14a-demethylase was also performed to
understand the binding affinity and binding interactions of the enzyme. Furthermore, the synthesized
compounds were evaluated for DPPH radical scavenging activity and antitubercular activity against
Mycobacterium tuberculosis H37Rv strain.
Received 7th May 2019
Accepted 27th June 2019
DOI: 10.1039/c9ra03425k
rsc.li/rsc-advances
chemotherapy.13 Some well-known antifungal agents including
uconazole, voriconazole, ravuconazole, and itraconazole
1. Introduction
contain a 1,2,4-triazole ring in their structure, as shown in
Fig. 2. However, their clinical uses have been restricted by their
relatively high risk of toxicity, pharmacokinetic deciencies,
emergence of drug resistance, and undesirable side effects.
These antifungal drugs inhibit CYP51, a key enzyme in the
biosynthesis of ergosterol, through a mechanism in which the
antifungal agent having a triazole scaffold is positioned
perpendicular to the porphyrin plane with a ring nitrogen atom
(N-4 of triazole) coordinated with a heme iron atom.14 Over
a couple of decades, the incidence of systemic fungal infections
has increased due to cancer chemotherapy, organ trans-
plantation, tuberculosis, and immunodeciency virus (HIV)
infection.15 However, the extensive use of antifungal drugs has
led to an increase in the resistance to these drugs.16 Hence,
there is an urgent need for developing new antifungal agents
with effective activities, low toxicity, and minimum side effects.
Ionic liquids (ILs) are environment-friendly solvents because
of their interesting properties and they can be used as alterna-
tives to harmful organic solvents. Furthermore, they are useful in
catalytic reactions17 and organic synthesis18 because of their
1,2,3-Triazole, a ve-membered heterocyclic ring system, is
a very well-known biologically active pharmacophore con-
structed by the copper-catalyzed azide–alkyne cycloaddition
(CuAAC) reaction, which is popular as a click chemistry reac-
tion.1 Over the last decade, 1,2,3-triazole has become one of the
key structural motifs and is used in numerous elds including
polymer chemistry,2 material science,3 and drug discovery.4
1,2,3-triazole-based molecules display various biological activi-
ties such as anti-fungal,5,6 anti-bacterial,6 anti-tubercular,7 anti-
inammatory,8 anti-allergic,9 anti-HIV,10 anti-cancer,11 and anti-
phytopathogenic.12 Some marketed drugs have the 1,2,3-triazole
unit in their structure, and these include Cefatrizine (a broad-
spectrum antibiotic), Tazobactam (an antibiotic), and Carbox-
yamidotriazole (CAI) (a calcium channel blocker) (Fig. 1).
Azole drugs have broad-spectrum activities against most
yeasts and lamentous fungi and are mostly used in antifungal
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad 431 004, India. E-mail: bapushingate@gmail.com; Fax: +91-240-
2403113; Tel: +91-240-2403313
bDepartment of Pharmacy, Birla Institute of Technology & Science-Hyderabad Campus,
Jawahar Nagar, Hyderabad 500 078, India
cDepartment of Pharmaceutical Chemistry, Y. B. Chavan College of Pharmacy, Dr.
Raq Zakaria Campus, Aurangabad 431 001, India
dDepartment of Pharmaceutical Chemistry, Srinath College of Pharmacy, Aurangabad
431136, MS, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra03425k
Fig. 1 Structures of drugs containing the 1,2,3-triazole unit.
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1,2,3-triazoles with amide linkage in their structures. Initially,
the starting materials, i.e., (prop-2-yn-1-yloxy)benzenes 2a–c
were prepared by a previously reported method.7d 2-Azido-N-
phenylacetamides 5a–g were synthesized from their corre-
sponding anilines via chloroacetylation using chloroacetyl
chloride, followed by nucleophilic substitution with sodium
azide in excellent yields (Scheme 1).
Aer the synthesis of alkynes 2a–c and azides 5a–g, we
considered the model substrates (prop-2-yn-1-yloxy)benzenes 2a
(1.0 mmol) and 2-azido-N-phenylacetamide 5a (1.0 mmol) for
the click reaction (Scheme 2).
The literature survey reveals the use of t-BuOH–H2O, THF–
H2O, and DMF–H2O as solvents in combination with copper
salts for the click reaction. Accordingly, we performed the
model reactions of 2a and 5a by using CuSO4$5H2O,
Cu(OAc)2$H2O, CuI, and CuCl with or without sodium ascorbate
as a reducing agent in solvents such as t-BuOH and THF (Table
1). In our initial attempt, the model reaction was carried out by
using the conventional method, i.e., the reaction mixture was
stirred at room temperature for 10 h using various copper salts
in t-BuOH or THF as a solvent (Table 1, entries 2–7). The
product 2-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)-N-phenyl-
acetamide 6a was obtained in 28–72% yield. Furthermore, the
model reaction was carried out using copper salts and t-BuOH/
Fig. 2 1,2,4-Triazole-based marketed antifungal drugs.
unique properties, making them superior media for increasing
selectivity, reactivity, and recyclability. Various ionic liquids have
been signicantly used in heterocyclic synthesis as solvents or
catalysts.19 The copper-catalyzed 1,3-dipolar cycloaddition of
organic azides and terminal alkynes has been reported by using
ionic liquids such as [bmim][BF4],20a,b [Bmim]OH,20c (SNIL-
Cu(II)),20d ([Hmim]TFA),20e and [C8dabco][N(CN)2],20f and DBU-
based ionic liquids.20g The ionic liquid [Et3NH][OAc] has been
used extensively in various organic transformations.21
ꢀ
THF as a solvent under ultrasound irradiation (40 kHz, 30 C)
Considering the importance of ionic liquids in organic
synthesis and in continuation of our work7d,22 as well as for the
development of new synthetic methodologies using ionic liquids,23
herein, we reported the efficient synthesis of new 1,4-
disubstituted-1,2,3-triazoles via the click chemistry approach using
[Et3NH][OAc] as the medium under ultrasonic irradiation.
Furthermore, the newly synthesized compounds were investigated
for their antifungal, antioxidant, and antitubercular activities.
for 7 h, which resulted in the product 6a in 32–83% yield (Table
1, entries 2–7). These results indicate a longer reaction time
with a lower yield of the product 6a.
Ionic liquids have unique properties as solvents and as
media; herein, we attempted to use various ionic liquids for the
synthesis of new amide-linked 1,4-disubstituted-1,2,3-triazole
6a via the click chemistry approach. Freshly synthesized
appropriate ionic liquids such as [Et3NH][OAc], [Et3NH][Cl],
[Et3NH][HSO4], [HDBU][OAc], and [HDBU][HSO4] in combina-
tion with copper salts such as CuSO4$5H2O and Cu(OAc)2$H2O
were used along with sodium ascorbate as a reducing agent to
carry out the model reaction (Table 1, entries 9–16) at room
temperature for 7 h. The maximum yield (90%) of the product
6a was obtained by using Cu(OAc)2$H2O as the catalyst and
[Et3NH][OAc] as the solvent (Table 1, entries 9 and 10). In our
further attempt, the model reaction was carried out under
ultrasound irradiation for appropriate times (35 and 45 min)
(Table 1, entries 9–16). The product 6a was obtained in 95%
yield under ultrasound irradiation using Cu(OAc)2$H2O as the
catalyst and [Et3NH][OAc] as the solvent (Table 1, entries 9 and
10). In short, it was observed from the optimization of the best
reaction conditions that the Cu(OAc)2$H2O-catalyzed click
reaction in [Et3NH][OAc] ionic liquid under ultrasound
2. Results and discussion
2.1 Chemistry
There are several reports on the synthesis of 1,4-disubstituted-
1,2,3-triazoles bearing amide functionality and displaying
various biological activities;24 recently, Ferroni et al. developed
triazoles as non-steroidal anti-androgens for prostate cancer
treatment.25 On the basis of these ndings, we designed small
Scheme
1 Synthesis of alkynes 2a–c and 2-azido-N-phenyl-
acetamides 5a–g. Reagents and conditions: (a) propargyl bromide,
K2CO3, DMF, 2 h, 90–95%; (b) chloroacetyl chloride, NEt3, CH2Cl2,
0 ꢀC to rt, 3–5 h, 85–95%; (c) NaN3, toluene, reflux, 5–7 h, 88–96%.
Scheme 2 Standard model reaction.
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Table 1 Optimization of the reaction conditions for the synthesis of 6a
Conventional method
US method
Timea
Entry
Copper salt
Solvent/medium
Timea
Yieldb (%)
Yieldb (%)
1
2
3
4
5
6
7
8
—
t-BuOH : H2O
t-BuOH : H2O
t-BuOH : H2O
t-BuOH : H2O
t-BuOH : H2O
THF
10 h
10 h
10 h
10 h
10 h
10 h
10 h
10 h
7 h
7 h
7 h
7 h
7 h
0
28
70
72
72
70
60
0
90
90
81
Trace
Trace
84
81
10
6 h
6 h
6 h
6 h
6 h
6 h
6 h
6 h
35 min
35 min
35 min
35 min
35 min
45 min
45 min
45 min
0
32
78
82
83
80
65
0
95
95
84
10
12
88
84
14
CuSO4$5H2O
CuSO4$5H2O + Na ascorbate
Cu(OAc)2$H2O
Cu(OAc)2$H2O + Na ascorbate
CuI
CuCl
THF
—
[Et3NH][OAc]
[Et3NH][OAc]
[Et3NH][OAc]
[Et3NH][OAc]
[Et3NH][Cl]
[Et3NH][HSO4]
[HDBU][OAc]
[HDBU][OAc]
[HDBU][HSO4]
9
Cu(OAc)2$H2O
10
11
12
13
14
15
16
Cu(OAc)2$H2O + Na ascorbate
CuSO4$5H2O + Na ascorbate
Cu(OAc)2$H2O
Cu(OAc)2$H2O
Cu(OAc)2$H2O
7 h
7 h
7 h
CuSO4$5H2O + Na ascorbate
Cu(OAc)2$H2O
a
Reaction conditions: 2a (1 mmol), 5a (1 mmol), and 10 mol% [Cu], 1 mL solvent, 30 ꢀC; reaction progress monitored by TLC. b Isolated yield, US:
ultrasonication.
irradiation at 30 ꢀC efficiently gives the desired 1,4- 92%, 91%, and 88% aer the successful runs (Fig. 3). The result
disubstituted-1,2,3-triazole derivative 6a as compared to the demonstrates the high stability of the ionic liquid [Et3NH][OAc]
conventional method with respect to the reaction time and yield under the reaction conditions, which could be recycled and
(Table 1, entry 9).
reused without a signicant loss in the product yield.
In order to evaluate the amount of the Cu(OAc)2$H2O catalyst
In a further attempt, we also examined a one-pot reaction for
for the model reaction for different concentrations such as 5, the selected model reaction with 2a (1.0 mmol), 4a (1.0 mmol),
10, 15, and 20 mol% (Table 2, entries 1–4), the reaction was and sodium azide (1.5 mmol) for the synthesis of 6a, which was
carried out in the presence of 1 mL of [Et3NH][OAc] as a solvent optimized for different copper sources (Table 3) for the one-pot,
at 30 ꢀC under ultrasound irradiation. It was observed that three-component click reaction in [Et3NH][OAc] as the solvent,
10 mol% of Cu(OAc)2$H2O was sufficient to achieve an excellent thus producing 2-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)-N-
yield of the product 6a in the shortest reaction time (Table 2, phenylacetamide 6a (Scheme 3). The optimization result of this
entry 2), while an excess amount of the catalyst (up to 20 mol%) one-pot model reaction revealed that 15 mol% of Cu(OAc)2$H2O
did not increase the yield of the product. The above results in [Et3NH][OAc] as the solvent was a sufficient catalyst loading
reveal that 10 mol% of Cu(OAc)2$H2O in [Et3NH][OAc] is the amount to obtain an excellent yield. However, it required
sufficient catalyst amount in terms of an excellent yield and a longer reaction time and higher concentration of the Cu
short reaction time (Table 2, entry 2).
catalyst (Table 3, entry 2) than the two-component reaction, as
The recyclability of the [Et3NH][OAc] solvent was also discussed earlier.
explored for the model reaction. The reaction was conducted for
Aer optimization of the reaction parameters and to dene
four successive cycles on the same recycled solvent. Aer the present methodology, as discussed earlier, the reaction was
completion of the rst reaction with 95% yield, the reaction assessed with a variety of substituted alkynes 2a–c and azides
mass was poured into ice cold water to get the solid product, 5a–g in the presence of Cu(OAc)2$H2O in [Et3NH][OAc] under
which was isolated by ltration. The ltrate was subjected to ultrasound irradiation. The reaction conditions were favorable
evaporation of water to get a viscous liquid, which on cooling for the electron-donating and electron-withdrawing groups on
gave the ionic liquid. Then, the recovered [Et3NH][OAc] was alkynes 2a–c and azides 5a–g, resulting in the corresponding
reused for four more successive cycles without signicant loss 1,4-disubstituted-1,2,3-triazole derivatives 6a–g, 7a–g, and 8a–g
in the yield of the product 6a. The yields achieved were 94%, in excellent yields (Scheme 4).
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Table 2 The influence of the amount of Cu(OAc)2$H2O on the Table 3 The effect of the amount of copper salts for the one-pot
synthesis of 6a
synthesis of 6a
Entry Catalyst
mol% Time (h) Isolated yield (%)
1
2
3
4
5
6
7
8
9
Cu(OAc)2$H2O
Cu(OAc)2$H2O
Cu(OAc)2$H2O
CuSO4$5H2O + Na ascorbate 10
CuSO4$5H2O + Na ascorbate 15
CuI
CuI
CuCl
CuCl
10
15
20
17
16
16
18
17
18
17
17
17
90
95
95
81
87
50
61
40
45
10
15
10
15
Entry
mol%
Time (min)
Isolated yield (%)
1
2
3
4
5
10
15
20
55
35
35
35
70
95
95
95
12.5 mg mLꢁ1 was more potent than Miconazole against the
fungal strain F. oxysporum. Compounds 8e and 8g with MIC of
All the synthesized compounds, namely, 6a–g, 7a–g, and 8a– 12.5 mg mLꢁ1 exhibited equivalent activities as compared to
g were conrmed by physical data and spectral analysis.
Miconazole against A. avus. For the fungal strain A. niger, the
compounds 8b, 8d, 8e, and 8g with MIC of 25 mg mLꢁ1 displayed
equivalent antifungal activities as compared to Miconazole.
However, 8f having MIC of 12.5 mg mLꢁ1 was more active than
Miconazole. For the fungal strain C. neoformans, the
compounds 8e, 8f, and 8g with MIC of 12.5 mg mLꢁ1 were more
potent than the standard drug and 8c with MIC of 25 mg mLꢁ1
was equivalent to Miconazole.
The remaining compounds displayed moderate to low anti-
fungal activities against all the tested fungal strains. The anti-
fungal activity depends on the various substituents present on
phenyl rings. Among the series, the compounds with nitro
groups (8a–g) were the most active against all the tested fungal
strains. Compounds 8f and 8g were the most active in the series
as they possessed a nitro group at R and chloro groups at R2 and
R3; the chloro group at the R1 position for 8e resulted in
less activity against one fungal strain. The methyl group at the
R1/R2/R3 position and the nitro group at the R position in 8b, 8c,
and 8d resulted in good activity than that of the others in the
series.
2.2 Biological activity
2.2.1. Antifungal activity. All the newly synthesized
compounds were screened for their in vitro antifungal activities
against ve different fungal strains, namely, Candida albicans,
Fusarium oxysporum, Aspergillus avus, Aspergillus niger, and
Cryptococcus neoformans. The minimum inhibitory concentra-
tion (MIC, mg mLꢁ1) values of all the newly synthesized
compounds were determined by the standard agar dilution
method as per the CLSI (formerly, NCCLS) guidelines26 (Table
4). DMSO was used as the negative control and Miconazole was
used as the standard antifungal drug for the comparison of
antifungal activities. Among the series, the triazole derivatives 6
and 7 derived from alkynes 2a and 2b (particularly from phenol
and 4-methoxyphenol) did not show any activity against all the
fungal strains. However, the triazoles derived from alkyne 2c
(from 4-nitrophenol) displayed excellent antifungal activity
against all the strains. The compounds 8b, 8c, and 8f having
MIC of 12.5 mg mLꢁ1 were found to be more potent; compounds
8a, 8d, and 8g having MIC of 25 mg mLꢁ1 were found to be
equipotent as compared to the standard drug Miconazole
against the fungal strain Candida albicans. The compounds 8b,
8c, 8d, 8e, and 8g with MIC of 25 mg mLꢁ1 were found to be
equivalent to the standard drug Miconazole and 8f with MIC of
2.2.2. Antitubercular activity. All the newly synthesized 1,4-
disubstituted-1,2,3-triazoles (6a–g, 7a–g, and 8a–g) were
screened for their in vitro antitubercular activities against the
MTB H37Rv (ATCC 27294) strain using the microplate Alamar
Blue assay method with the determination of MIC in triplicate;
the results are mentioned in Table 4. The newly synthesized
1,2,3-triazoles exhibited MICs ranging from 25 to $25 mg mLꢁ1
.
Among the series, compounds 6a, 6c, and 6f exhibited good
antitubercular activities with MIC of 25 mg mLꢁ1 as compared to
other compounds.
Scheme 3 Synthesis of 1,4-disubstituted-1,2,3-triazole 6a via the
Fig. 3 Reusability of [Et3NH][OAc].
one-pot three-component click reaction.
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Scheme 4 Synthesis of 1,4-disubstituted-1,2,3-triazole derivatives 6a–g, 7a–g, and 8a–g.
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Table 4 In vitro biological evaluation of the synthesized compounds 6a–g, 7a–g, and 8a–g MIC (mg mLꢁ1 a
)
Antifungal activity
Anti TB MTB
Entry
CA
FO
AF
AN
CN
H37Rv
DPPH IC50 (mg mLꢁ1
)
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
8d
8e
8f
87.5
150
175
187.5
200
150
162.5
137.5
175
*
100
125
200
162.5
*
175
150
150
187.5
*
125
150
175
200
187.5
200
187.5
137.5
175
*
187.5
*
25
>25
25
>25
>25
25
55.37
26.64
20.54
*
200
*
175
*
29.64
74.28
74.41
11.44
16.79
12.30
19.46
70.33
*
175
175
125
200
*
162.5
175
100
100
200
150
200
100
87.5
25
150
125
200
175
200
*
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
NA
200
175
100
75
175
175
150
100
37.5
25
25
25
25
12.5
25
150
*
100
100
75
25
50
25
25
12.5
25
25
NA
NA
NA
NA
NA
75
200
75
37.5
25
50
12.5
12.5
12.5
25
NA
NA
NA
NA
NA
49.60
97.29
*
25
12.5
12.5
25
50
12.5
25
25
NA
NA
NA
25
25
48.02
22.17
62.01
*
37.5
12.5
25
12.5
12.5
NA
NA
NA
NA
NA
8g
16.99
NA
NA
NA
NA
MA
INH
RIF
EMB
CIP
BHT
25
NA
NA
NA
NA
NA
0.1
0.2
1.56
1.56
NA
NA
NA
NA
16.47
a
CA: Candida albicans; FO: Fusarium oxysporum; AF: Aspergillus avus; AN: Aspergillus niger and CN: Cryptococcus neoformans. MA: Miconazole,
DPPH: 2,2-diphenyl-1-picrylhydrazyl; INH: Isoniazid; RIF: Rifampicin; EMB: Ethambutol; CIP: Ciprooxacin; BHT: butylated hydroxytoluene; *no
activity was observed, NA: not applicable.
Butylated hydroxytoluene (BHT) was used as the standard drug
for the comparison of antioxidant activities; the observed
results are summarized in Table 4. According to the DPPH
assay, compounds 7a and 7c exhibited excellent radical scav-
2.2.3. Antioxidant activity. The antioxidant activities of the
synthesized compounds 6a–g, 7a–g, and 8a–g were screened
using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
assay.27 The DPPH radical scavenging assay is the most
commonly used procedure for screening the antioxidant activ-
ities of different natural and synthetic compounds. A lower IC50
value demonstrates more antioxidant activity. The IC50
(concentration required to scavenge 50% of the radicals) values
were calculated to assess the potential antioxidant activities.
enging activities with IC50 values of 11.44 and 12.30 mg mLꢁ1
,
respectively, compared to the synthetic antioxidant BHT. Simi-
larly, compounds 7b, 7d, and 8g having IC50 values of 16.79,
19.46, and 16.99 mg mLꢁ1, respectively, showed comparable
antioxidant activities with that of BHT; in contrast, the
Table 5 Molecular docking score for the compounds 6a–g, 7a–g, and 8a–g
Molecular docking score
Entry
Total score (ꢁlog Ki)
Crash score
Polar score
Entry
Total score (ꢁlog Ki)
Crash score
Polar score
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7.0377
6.4564
6.8229
6.0468
6.5873
6.7343
7.0332
6.0045
6.6907
6.5748
7.0239
ꢁ0.6933
ꢁ2.9621
ꢁ0.7726
ꢁ1.4517
ꢁ0.9241
ꢁ0.8582
ꢁ1.0463
ꢁ0.7865
ꢁ0.9183
ꢁ2.7834
ꢁ1.2982
1.9115
0.0004
2.0682
1.3586
2.8329
3.0543
1.6968
2.8105
4.5244
0.0090
3.2802
7e
7f
6.4127
7.2492
7.2394
7.0868
7.5441
7.4658
7.3945
7.3314
7.9971
7.5972
6.4895
ꢁ1.2494
ꢁ1.4724
ꢁ1.4033
ꢁ1.1801
ꢁ2.8665
ꢁ1.0993
ꢁ1.5222
ꢁ0.9292
ꢁ1.9946
ꢁ1.3842
ꢁ1.4009
1.4225
2.5093
3.0192
3.6314
0.9019
0.7391
1.9024
0.7808
0.0031
3.0509
1.0311
7g
8a
8b
8c
8d
8e
8f
8g
MA
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alkyl interactions with the chloro-phenyl (–Cl) ring; the p elec-
tron cloud with the phenyl ring and the p electron cloud of the
triazole ring are shown in Fig. 4a. The second most active tri-
azole derivative 8g (7.597) interacted with the active site of
amino acids by forming non-covalent bonded interactions such
as H-bond interaction and p bond interaction. The hydrophobic
amino acid Ala501 contains a non-polar side chain that inter-
acts with the oxygen (–O) atom of the nitro group to form
a conventional hydrogen bond interaction with distance of 2.02
˚
A. The polar aromatic amino acid Tyr168 forms hydrogen bond
interactions with the nitrogen atom of the triazole ring with
˚
a distance of 1.84 A.
The amino acid Gly500 formed a carbon–hydrogen bond
with the oxygen (–O) of the nitro group on the phenyl ring with
˚
a distance of 2.39 A. The hydrophobic amino acid Try154 with
an aromatic ring interacted with the triazole p cloud to form p–
p stacked interactions, while the amino acids Leu412, Met544,
Met413, and His546 interacted with the p cloud of the chloro
group on the phenyl ring (–Cl) and the phenyl ring to form weak
p-alkyl and alkyl interactions, as shown in Fig. 4b.
3. Conclusion
Fig. 4 Binding pose and molecular interactions in the active sites of C.
albicans lanosterol 14a-demethylase: (a) 8f and (b) 8g.
In conclusion, we demonstrated [Et3NH][OAc]- and ultrasound-
mediated synthesis of new N-phenylacetamide-incorporated
1,2,3-triazole derivatives 6(a–g), 7(a–g), and 8(a–g) in excellent
yields via the click chemistry approach for the rst time. This
safer method reduced the use of harmful solvents and reaction
time. We also studied the recovery and reusability of [Et3NH]
[OAc] as a medium. The synthesized triazole derivatives were
evaluated for their antifungal, antioxidant, and antitubercular
activities. Among the series, the compounds 8a, 8b, 8c, 8d, 8e,
8f, and 8g displayed excellent antifungal activities as compared
to the standard drug Miconazole with lower MIC values.
The in silico molecular docking study supported the experi-
mental results and demonstrated that the 1,2,3-triazole deriv-
atives 8f and 8g are the most active and have the potential to
inhibit the survival of fungal species. The predictions of the
pharmacokinetic parameters suggest that the synthesized
derivatives have the potential to have high oral drug
bioavailability.
compounds 6b, 6c, 6e, and 8d showed moderate activities with
IC50 values of 26.64, 20.54, 29.64, and 22.17 mg mLꢁ1
,
respectively.
2.2.4. Molecular docking. The molecular docking study of
all the synthesized triazole derivatives 6a–g, 7a–g, and 8a–g was
performed against the modelled three-dimensional structure of
cytochrome P450 lanosterol 14a-demethylase of C. albicans to
understand the binding affinity and binding interactions of the
enzyme and the synthesized derivatives using the SYBEL 2.1.1
package following the standard procedures.28,29 The molecular
docking results are presented in Table 5.
The docking results indicated that the triazole scaffold of the
synthesized derivatives 6a–g, 7a–g, and 8a–g was incorporated
appropriately in the active site of CYP450 and through various
kinds of non-bonded interactions such as H-bond interactions
and p bond interactions with the active sites of the amino acid
residues. Among all the synthesized derivatives, 8f (7.9971), 8g
(7.597), and 8b (7.544) were the most active triazole derivatives
when compared with the standard Miconazole drug (6.4895).
The most active triazole derivative 8f (7.9971) contained
hydrophilic as well as hydrophobic amino acids in the active
sites such as isoleucine, valine, methionine, cysteine, leucine,
and phenylalanine. The hydrophobic amino acids Cys506 and
Ile507 formed conventional hydrogen bonds with the oxygen
(–O) of the nitro group on the phenyl ring with the distances of
4. Experimental
4.1 General methods
All the solvents and reagents were purchased from commercial
suppliers, namely, Spectrochem Pvt. Ltd., Avra, Sigma Aldrich,
and Alfa Aesar and were used without further purication. The
progress of each reaction was monitored by thin layer chro-
matography (TLC) using silica gel F254 precoated TLC
aluminium sheets (Merck, Germany) and by locating the spots
using a UV light as the visualizing agent or iodine vapours. The
melting points were obtained by the open capillary method and
were uncorrected. The IR spectra were recorded on a Bruker FT-
IR spectrometer. The 1H NMR spectra were recorded on Bruker
AC-200 MHz and Bruker Advance III 500 MHz NMR spectrom-
eters. The 13C NMR spectra were recorded on Bruker AC-50 MHz
˚
1.95 and 2.02 A, respectively. Met336 contains non-polar side
chain forms conventional hydrogen bond interaction with
a hydrogen atom (–H) attached to the nitrogen atom and
ꢀ
methylene group with distance 2.41 and 2.45 A, respectively.
The hydrophobic amino acids such as Val166, Ile167, Ala182,
Leu186, Leu240, Leu316, and Ala185 interacted with the p
electron cloud to form weak p-interactions such as p-alkyl and
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and Bruker Advance III 125 MHz NMR in DMSO-d6, CDCl3
+
4H, Ar–H), 7.08 (d, J ¼ 8.0 Hz 1H), 7.13–7.30 (m, 3H, Ar–H),
DMSO-d6, and CDCl3 + CD3OD using tetramethylsilane (TMS) as 7.34 (s, 1H, Ar–H), 7.85 (s, 1H, triazole), and 9.70 (s, 1H, NH);
the internal standard; the chemical shis (d) were recorded in 13C NMR (125 MHz, CDCl3 + DMSO-d6, d ppm): 21.1, 52.7,
parts per million (ppm). The splitting pattern abbreviations are 61.4, 114.3, 116.7, 120.2, 124.8, 124.9, 128.3, 129.1, 137.3,
designed as singlet (s), doublet (d), double doublet (dd), triplet 138.4, 143.7, 157.8, and 163.0; LC-MS for C18H18N4O2: [M]+
(t), quartet (q), and multiplet (m). High-resolution mass spectra 323.1.
(HRMS) were recorded on an Agilent 6520 (QTOF) ESI-HRMS 4.2.4. 2-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)-N-(p-
instrument and Agilent Technologies 1200 series electron tolyl)acetamide (6d). The compound 6d was obtained via
spray ionization (ESI) was used for the LC-MS data.
a 1,3-dipolar cycloaddition reaction between azide 5d and
alkyne 2a in 36 min as a yellow solid; mp: 202–204 ꢀC; FT-IR
(cmꢁ1): 3271 (N–H stretching), 1665 (C]O stretching).
4.2.5. 2-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)-N-(2-
chlorophenyl)-acetamide (6e). The compound 6e was ob-
tained via a 1,3-dipolar cycloaddition reaction between
4.2 General experimental procedure for the synthesis of N-
phenylacetamide-incorporated 1,2,3-triazole derivatives (6a–g,
7a–g, and 8a–g)
The mixture of appropriate (prop-2-yn-1-yloxy) benzenes 2a–c (1 azide 5e and alkyne 2a in 36 min as a white solid; mp: 178–
mmol), substituted 2-azido-N-phenylacetamide 5a–g (1 mmol) 180 ꢀC; FT-IR (cmꢁ1): 3267 (N–H stretching), 1671 (C]O
and Cu(OAc)2$H2O (10 mol%) in [NEt3H][OAc] (1 mL) was stretching); 1H NMR (500 MHz, CDCl3 + DMSO-d6, d ppm):
placed in a round bottom ask. The mixture was sonicated (40 5.19 (s, 2H, –NCH2CO–), 5.31 (s, 2H, –OCH2), 6.94 (d, J ¼
kHz) at 30 ꢀC for an appropriate time until the completion of the 8.0 Hz, 3H, Ar–H), 7.05 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.16–7.34
reaction. The progress of the reaction was monitored by TLC. (m, 4H, Ar–H), 7.89 (s, 1H, triazole), 8.02 (d, J ¼ 8.0 Hz, 1H,
Aer the completion of the reaction, the mixture was quenched Ar–H), and 9.01 (s, 1H, NH); 13C NMR (125 MHz, CDCl3
+
with crushed ice. The obtained solid was ltered and washed DMSO-d6, d ppm): 52.8, 61.3, 114.3, 120.8, 123.1, 124.3,
with water. The crude solid was crystallized in ethanol to afford 125.5, 127.1, 129.0, 133.7, 141.9, 157.7, and 162.6. LC-MS for
the corresponding pure product. The synthesized compounds
6a–g, 7a–g, and 8a–g were characterized by IR, 1H NMR, 13C
NMR, and mass spectroscopy.
C
17H15ClN4O2: [M]+ 343.
4.2.6. 2-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)-N-(3-
chlorophenyl)-acetamide (6f). The compound 6f was ob-
4.2.1. 2-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)-N-phenyl- tained via a 1,3-dipolar cycloaddition reaction between
acetamide (6a). The compound 6a was obtained via a 1,3- azide 5f and alkyne 2a in 37 min as a white solid; mp: 140–
dipolar cycloaddition reaction between azide 5a and alkyne 2a 142 ꢀC; FT-IR (cmꢁ1): 3257 (N–H stretching), 1672 (C]O
in 35 min as a white solid; mp: 166–168 ꢀC; FT-IR (cmꢁ1): 3263 stretching); 1H NMR (500 MHz, CDCl3 + DMSO-d6, d ppm):
1
(N–H stretching), 1667 (C]O stretching); H NMR (200 MHz, 5.45 (s, 2H, –NCH2CO–), 5.46 (s, 2H, –OCH2), 7.15–7.57 (m,
DMSO-d6, d ppm): 5.17 (s, 2H, –NCH2CO–), 5.36 (s, 2H, –OCH2), 7H Ar–H), 7.67 (s, 1H, Ar–H), 7.92 (s, 1H, triazole), 8.15 (s,
6.96 (t, J ¼ 8.0 Hz, 1H, Ar–H), 7.08 (t, J ¼ 8.0 Hz, 3H, Ar), 7.28– 1H, Ar–H), and 10.16 (s, 1H, NH). 13C NMR (125 MHz, CDCl3
7.38 (m, 4H, Ar–H), 7.59 (d, J ¼ 8.0 Hz, 2H, Ar–H), 8.26 (s, 1H, + DMSO-d6, d ppm): 53.1, 61.7, 114.7, 117.9, 120.0, 121.2,
triazole), and 10.49 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6, 124.5, 129.5, 129.9, 134.4, 138.9, 158.1, and 163.5; LC-MS for
d ppm): 51.8, 60.5, 114.3, 118.9, 120.5, 123.5, 125.9, 128.6, 129.2,
138.0, 142.2, 157.7, and 163.8; HRMS calcd for C17H17N4O2 [M +
H]+: 309.1346 and found 309.1340.
C
17H15ClN4O2: [M]+ 343.1.
4.2.7. 2-(4-((4-Methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)-N-phenylacetamide (7a). The compound 7a was obtained via
4.2.2. 2-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)-N-(o-tolyl) a 1,3-dipolar cycloaddition reaction between azide 5a and
acetamide (6b). The compound 6b was obtained via a 1,3- alkyne 2b in 37 min as a white solid; mp: 180–182 ꢀC; FT-IR
dipolar cycloaddition reaction between azide 5b and alkyne 2a (cmꢁ1): 3271 (N–H stretching), 1674 (C]O stretching); 1H
in 37 min as a white solid; mp: 184–186 ꢀC; FT-IR (cmꢁ1): 3271 NMR (500 MHz, CDCl3, d ppm): 3.76 (s, 3H, OCH3), 5.19 (s, 2H,
1
(N–H stretching), 1665 (C]O stretching); H NMR (200 MHz, –NCH2CO–), 5.20 (s, 2H, –OCH2), 6.83 (d, J ¼ 8.0 Hz, 2H, Ar–H),
DMSO-d6, d ppm): 2.24 (s, 3H, –CH3), 5.17 (s, 2H, –NCH2CO–), 6.92 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.14 (t, J ¼ 8.0 Hz, 1H, Ar–H), 7.31
5.41 (s, 2H, –OCH2), 6.95 (t, J ¼ 8.0 Hz, 1H, Ar–H), 7.05 (d, J ¼ (t, J ¼ 8.0 Hz, 2H, Ar–H), 7.43 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.81 (s,
8.0 Hz, 2H, Ar–H), 7.10–7.35 (m, 5H, Ar–H), 7.43 (d, J ¼ 8.0 Hz, 1H, triazole), and 7.87 (s, 1H, NH); 13C NMR (125 MHz, CDCl3,
1H, Ar–H), 8.26 (s, 1H, triazole), and 9.84 (s, 1H, NH); 13C NMR d ppm): 53.9, 55.7, 62.5, 114.8, 115.9, 120.3, 125.4, 129.1, 137.0,
(50 MHz, DMSO-d6, d ppm): 17.5, 51.3, 60.5, 114.3, 120.4, 124.8, 139.3, 152.1, and 162.3; LC-MS for C18H18N4O3: [M]+ 339.1.
125.8, 129.1, 130.1, 131.2, 135.1, 142.2, 157.7, and 164.0; HRMS
calcd for C18H19N4O2 [M + H]+: 323.1508 and found 323.1510.
4.2.3. 2-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)-N-(m-
4.2.8. 2-(4-((4-Methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)-N-(o-tolyl)acetamide (7b). The compound 7b was obtained
via a 1,3-dipolar cycloaddition reaction between azide 5b and
tolyl)acetamide (6c). The compound 6c was obtained via alkyne 2b in 35 min as a white solid; mp: 176–178 ꢀC; FT-IR
a 1,3-dipolar cycloaddition reaction between azide 5c and (cmꢁ1): 3266 (N–H stretching), 1674 (C]O stretching); 1H
ꢀ
alkyne 2a in 38 min as a yellow solid; mp: 130–132 C; FT-IR NMR (500 MHz, CDCl3, d ppm): 2.10 (s, 3H, CH3), 3.75 (s, 3H,
(cmꢁ1): 3302 (N–H stretching), 1676 (C]O stretching); 1H –OCH3), 5.21 (s, 2H, –NCH2CO–), 5.22 (s, 2H, –OCH2), 6.82 (d, J
NMR (500 MHz, CDCl3 + DMSO-d6, d ppm): 2.21 (s, 3H, –CH3), ¼ 8.0 Hz, 2H, Ar–H), 6.91 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.08 (t, J ¼
5.10 (s, 2H, –NCH2CO–), 5.13 (s, 2H, –OCH2), 6.79–6.93 (m, 8.0 Hz, 1H, Ar–H), 7.15 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.20 (t, J ¼
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8.0 Hz, 1H, Ar–H), 7.80 (s, 1H, triazole), and 7.82 (s, 1H, NH); 13
NMR (125 MHz, CDCl3, d ppm): 17.4, 53.9, 55.7, 62.4, 114.8,
115.9, 122.3, 124.5, 125.9, 126.9, 127.9, 137.6, and 162.8; LC-MS
for C19H20N4O3: [M]+ 353.1.
C
4.2.13. N-(4-Chlorophenyl)-2-(4-((4-methoxyphenoxy)methyl)-
1H-1,2,3-triazol-1-yl)acetamide (7g). The compound 7g was ob-
tained via a 1,3-dipolar cycloaddition reaction between azide 5g
and alkyne 2b in 36 min as a white solid; mp: 192–194 ꢀC; FT-IR
(cmꢁ1): 3266 (N–H stretching), 1672 (C]O stretching).
4.2.9. 2-(4-((4-Methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)-N-(m-tolyl)acetamide (7c). The compound 7c was obtained
via a 1,3-dipolar cycloaddition reaction between azide 5c and
4.2.14. 2-(4-((4-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)-
N-phenylacetamide (8a). The compound 8a was obtained via
a 1,3-dipolar cycloaddition reaction between azide 5a and
alkyne 2c in 40 min as a yellow solid; mp: 180–182 ꢀC; FT-IR
(cmꢁ1): 3250 (N–H stretching), 1682 (C]O stretching); 1H
NMR (500 MHz, CDCl3 + CD3OD, d ppm): 5.18 (s, 2H,
–NCH2CO–), 5.27 (s, 2H, –OCH2), 7.05 (d, J ¼ 8.0 Hz, 3H, Ar–H),
7.27 (t, J ¼ 8.0 Hz, 3H, Ar–H), 7.49 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.98
(s, 1H, triazole), and 8.17 (d, J ¼ 8 Hz, 2H, Ar–H); 13C NMR (125
MHz, CDCl3 + CD3OD, d ppm): 52.7, 61.9, 114.5, 125.1, 125.7,
128.8, 137.5, 141.7, and 162.8; LC-MS for C17H15N5O4: [M]+ 354.
4.2.15. 2-(4-((4-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)-
N-(o-tolyl)acetamide (8b). The compound 8b was obtained via
a 1,3-dipolar cycloaddition reaction between azide 5b and
alkyne 2c in 39 min as a white solid; mp: 178–180 ꢀC; FT-IR
(cmꢁ1): 3257 (N–H stretching), 1668 (C]O stretching); 1H
NMR (500 MHz, CDCl3 + CD3OD, d ppm): 2.15 (s, 3H, CH3), 5.23
(s, 2H, –NCH2CO–), 5.25 (s, 2H, –OCH2), 7.03–7.13 (m, 6H, Ar–
H), 7.47 (s, 1H, NH), 7.98 (s, 1H, triazole), and 8.14 (s, 2H, Ar–H);
13C NMR (125 MHz, CDCl3 + CD3OD, d ppm): 17.2, 52.6, 62.1,
114.9, 124.9, 125.4, 125.9, 126.4, 126.5, 127.4, 130.7, 131.2, and
163.7; LC-MS for C18H17N5O4 [M]+ 368.1.
4.2.16. 2-(4-((4-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)-
N-(m-tolyl)acetamide (8c). The compound 8c was obtained via
a 1,3-dipolar cycloaddition reaction between azide 5c and
alkyne 2c in 38 min as a white solid; mp: 168–170 ꢀC; FT-IR
(cmꢁ1): 3280 (N–H stretching), 1662 (C]O stretching); 1H
NMR (200 MHz, DMSO-d6, d ppm): 2.28 (s, 3H, CH3), 5.36 (s, 4H,
–NCH2CO, –OCH2), 6.91 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.19 (d, J ¼
8.0 Hz, 1H, Ar–H), 7.27 (t, J ¼ 8 Hz, 1H, Ar–H), 7.33 (d, J ¼ 8.0 Hz,
2H, Ar–H), 7.42 (s, 1H, Ar–H), 8.24 (d, J ¼ 8.0 Hz, 2H, Ar–H), 8.32
(s, 1H, triazole), and 10.42 (s, 1H, NH); 13C NMR (50 MHz,
DMSO-d6, d ppm): 21.1, 52.2, 61.9, 115.3, 116.4, 119.8, 124.5,
125.9, 126.7, 128.7, 138.1, 138.3, 141.0, 141.5, 163.3, and 164.0;
HRMS calcd for C18H18N5O4 [M + H]+: 368.1359 and found
368.1364.
ꢀ
alkyne 2b in 35 min as a yellow solid; mp: 140–142 C; FT-IR
(cmꢁ1): 3271 (N–H stretching), 1665 (C]O stretching); 1H
NMR (500 MHz, CDCl3 + DMSO-d6, d ppm): 2.18 (s, 3H, CH3),
3.62 (s, 3H, OCH3), 5.01 (s, 2H, –NCH2CO–), 5.10 (s, 2H, –OCH2),
6.68 (d, J ¼ 6 Hz, 2H, Ar–H), 6.79 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.04
(t, J ¼ 8.0 Hz, 1H, Ar–H), 7.18 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.26 (d, J
¼ 8.0 Hz, 1H, Ar–H), 7.31 (s, 1H, Ar–H), 7.79 (s, 1H, triazole), and
9.73 (s, 1H, NH); 13C NMR (125 MHz, CDCl3 + DMSO-d6, d ppm):
21.3, 52.9, 55.5, 62.4, 114.5, 115.7, 116.8, 120.4, 124.4, 125.1,
128.5, 137.6, 138.5, 144.1, 152.2, 153.9, and 163.3. LC-MS for
C
19H20N4O3: [M]+ 353.1.
4.2.10. 2-(4-((4-Methoxyphenoxy)methyl)-1H-1,2,3-triazol-
1-yl)-N-(p-tolyl)acetamide (7d). The compound 7d was obtained
via a 1,3-dipolar cycloaddition reaction between azide 5d and
alkyne 2b in 36 min as a white solid; mp: 196–198 ꢀC; FT-IR
(cmꢁ1): 3273 (N–H stretching), 1675 (C]O stretching); 1H
NMR (500 MHz, CDCl3 + DMSO-d6, d ppm): 2.10 (s, 3H, CH3),
3.55 (s, 3H, OCH3), 4.94 (s, 2H, –NCH2CO–), 5.02 (s, 2H, –OCH2),
6.62 (d, J ¼ 8.0 Hz, 2H, Ar–H), 6.72 (d, J ¼ 8.0 Hz, 2H, Ar–H), 6.89
(d, J ¼ 8.0 Hz, 2H, Ar–H), 7.24 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.73 (s,
1H, triazole), and 9.68 (s, 1H, NH); 13C NMR (125 MHz, CDCl3 +
DMSO-d6, d ppm): 20.4, 52.6, 55.2, 62.1, 114.2, 115.4, 119.5,
124.2, 128.9, 133.5, 134.9, 151.9, 153.6, and 162.8; LC-MS for
C
19H20N4O3: [M]+ 353.1.
4.2.11. N-(2-Chlorophenyl)-2-(4-((4-methoxyphenoxy)methyl)-
1H-1,2,3-triazol-1-yl)acetamide (7e). The compound 7e was ob-
tained via a 1,3-dipolar cycloaddition reaction between azide 5e
and alkyne 2b in 35 min as a white solid; mp: 168–170 ꢀC; FT-IR
1
(cmꢁ1): 3260 (N–H stretching), 1683 (C]O stretching); H NMR
(500 MHz, CDCl3 + DMSO-d6, d ppm): 3.89 (s, 3H, OCH3), 5.32 (s,
2H, –NCH2CO–), 5.47 (s, –OCH2), 6.96 (d, J ¼ 8.0 Hz, 2H, Ar–H),
7.06 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.23 (t, J ¼ 8.0 Hz, 1H, Ar–H), 7.39 (t,
J ¼ 8.0 Hz, 1H, Ar–H), 7.50 (d, J ¼ 8.0 Hz, 1H, Ar–H), 8.03 (s, 1H,
triazole), 8.26 (d, J ¼ 8.0 Hz, 1H, Ar–H), and 8.98 (s, 1H, NH); 13
C
NMR (125 MHz, CDCl3 + DMSO-d6, d ppm): 53.0, 55.4, 62.3, 114.4,
115.6, 122.8, 124.3, 125.6, 127.3, 129.1, 133.4, 144.7, 152.0, 153.9,
and 163.3; LC-MS for C18H17ClN4O3: [M]+ 373.
4.2.17. 2-(4-((4-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)-
N-(p-tolyl)acetamide (8d). The compound 8d was obtained via
a 1,3-dipolar cycloaddition reaction between azide 5d and
alkyne 2c in 40 min as a white solid; mp: 210–212 ꢀC; FT-IR
(cmꢁ1): 3260 (N–H stretching), 1696 (C]O stretching); 1H
NMR (500 MHz, CDCl3 + CD3OD, d ppm): 2.26 (s, 3H, CH3), 5.14
(s, 2H, –NCH2CO–), 5.26 (s, 2H, –OCH2), 7.0 (d, J ¼ 8.0 Hz, 2H,
Ar–H), 7.07 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.35 (d, J ¼ 8.0 Hz, 2H, Ar–
4.2.12. N-(2-Chlorophenyl)-2-(4-((4-methoxyphenoxy)methyl)-
1H-1,2,3-triazol-1-yl)acetamide (7f). The compound 7f was ob-
tained via a 1,3-dipolar cycloaddition reaction between azide 5f
and alkyne 2b in 35 min as a green solid; mp: 160–162 ꢀC; FT-IR
1
(cmꢁ1): 3266 (N–H stretching), 1675 (C]O stretching); H NMR
H), 7.96 (s, 1H, triazole), and 8.16 (d, J ¼ 8.0 Hz, 2H, Ar–H); 13
C
(500 MHz, CDCl3 + DMSO-d6, d ppm): 3.57 (s, 3H, OCH3), 4.96 (s,
2H, –NCH2CO–), 5.05 (s, 2H, –OCH2), 6.63 (d, J ¼ 8.0 Hz, 2H, Ar–H),
6.73 (d, J ¼ 8.0 Hz, 2H, Ar–H), 6.87 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.03 (t,
J ¼ 8.0 Hz, 1H, Ar–H), 7.24 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.53 (s, 1H, Ar–
H), 7.73 (s, 1H, triazole), and 10.04 (s, 1H, NH); 13C NMR (125 MHz,
CDCl3 + DMSO-d6, d ppm): 52.4, 55.1, 62.0, 114.1, 115.27, 117.3,
119.3, 123.7, 124.1, 129.4, 133.8, 138.7, 151.8, 153.5, and 163.2; LC-
MS for C18H17ClN4O3 [M + H]+ 373.
NMR (125 MHz, CDCl3 + CD3OD, d ppm): 17.7, 53.1, 62.6, 115.4,
124.8, 125.9, 126.5, 126.9, 131.2, 143.5, 152.0, and 164.2; LC-MS
for C18H17N5O4: [M + H]+ 368.1.
4.2.18. N-(2-Chlorophenyl)-2-(4-((4-nitrophenoxy)methyl)-
1H-1,2,3-triazol-1-yl)acetamide (8e). The compound 8e was ob-
tained via a 1,3-dipolar cycloaddition reaction between azide 5e
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and alkyne 2c in 39 min as a red solid; mp: 169–171 C; FT-IR concentration of the drug preventing the growth of macroscopi-
(cmꢁ1): 3243 (N–H stretching), 1672 (C]O stretching); 1H cally visible colonies on the drug-containing plates when there is
NMR (500 MHz, CDCl3 + CD3OD, d ppm): 5.31 (s, 2H, visible growth on the drug-free control plates.
–NCH2CO–), 5.33 (s, 2H, –OCH2), 7.08 (d, J ¼ 8.0 Hz, 3H, Ar–H),
4.3.2. In vitro Mtb MABA assay. The newly synthesized
7.29 (d, J ¼ 8.0 Hz, 2H), 7.37 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.96 (s, compounds were screened for their in vitro antitubercular
1H, triazole), 8.15 (s, 1H, NH), and 8.20 (d, J ¼ 8.0 Hz, 2H, Ar–H); activity against Mtb H37Rv (ATCC 27294) by using the Micro-
13C NMR (125 MHz, CDCl3 + CD3OD, d ppm): 53.2, 62.1, 114.8, plate Alamar Blue Assay (MABA)30 for the determination of MIC
125.1, 126.0, 126.1, 127.7, 129.4, 133.5, 136.5, 146.4, 152.3, and in triplicate. The MIC (in mg mLꢁ1) was recorded as the lowest
163.0; LC-MS for C17H14ClN5O4: [M]+ 388.
concentration/highest dilution of the compounds/control drugs
4.2.19. N-(3-Chlorophenyl)-2-(4-((4-nitrophenoxy)methyl)-
that completely inhibited the growth of the Mtb cultures. The
1H-1,2,3-triazol-1-yl)acetamide (8f). The compound 8f was ob- MIC values of the compounds were compared with those of the
tained via a 1,3-dipolar cycloaddition reaction between azide 5f standard drugs (Isoniazid, Rifampicin, Ethambutol, and
and alkyne 2c in 40 min as a white solid; mp: 190–192 ꢀC; FT-IR Ciprooxacin). The experimental method for antitubercular
(cmꢁ1): 3327 (N–H stretching), 1683 (C]O stretching); 1H NMR activity is briey described as follows.
(200 MHz, DMSO-d6, d ppm): 5.36 (s, 2H, –NCH2CO–), 5.40 (s,
Initially, the inoculum was prepared from a fresh LJ medium
2H, –OCH2), 7.15 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.29 (d, J ¼ 8.0 Hz, re-suspended in the 7H9-S medium (7H9 broth, 0.1% casitone,
2H, Ar–H), 7.42 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.78 (s, 1H, Ar–H), 8.23 0.5% glycerol, supplemented oleic acid, albumin, dextrose, and
(d, J ¼ 8.0 Hz, 2H, Ar–H), 8.33 (s, 1H, triazole), and 10.81 (s, 1H, catalase [OADC]), adjusted to a McFarland tube no. 1, and
NH); 13C NMR (50 MHz, DMSO-d6, d ppm): 52.3, 61.9, 115.4, diluted 1 : 20; 100 mL was used as the inoculum. Each drug
117.7, 118.9, 123.6, 125.9, 126.8, 130.7, 133.3, 139.9, 141.1, stock solution was thawed and diluted in 7H9-S at four-fold the
141.7, 163.4, and 164.7; HRMS calcd for C17H15ClN5O4 [M + H]+: nal highest concentration tested. Serial two-fold dilutions of
388.0813 and found 388.0814.
each drug were prepared directly in a sterile 96-well microtiter
4.2.20. N-(4-Chlorophenyl)-2-(4-((4-nitrophenoxy)methyl)-
plate using 100 mL 7H9-S. A growth control containing no
1H-1,2,3-triazol-1-yl)acetamide (8g). The compound 8g was ob- antibiotic and a sterile control was also prepared on each plate.
tained via a 1,3-dipolar cycloaddition reaction between azide 5g Sterile water was added to all perimeter wells to avoid evapo-
and alkyne 2c in 37 min as a white solid; mp: 188–190 ꢀC; FT-IR ration during incubation. The plate was covered, sealed in
(cmꢁ1): 3243 (N–H stretching), 1672 (C]O stretching); 1H NMR plastic bags, and incubated at 37 ꢀC in normal atmosphere.
(200 MHz, DMSO-d6, d ppm): 5.36 (s, 4H, –NCH2CO, –OCH2), Aer 7 days incubation, 30 mL of alamar blue solution was
7.29 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.39 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.61 added to each well, and the plate was re-incubated overnight. A
(d, J ¼ 8.0 Hz, 2H, Ar–H), 8.23 (d, J ¼ 8.0 Hz, 2H, Ar–H), 8.32 (s, change in the colour from blue (oxidised state) to pink (reduced)
1H, triazole), and 10.64 (s, 1H, NH); 13C NMR (50 MHz, DMSO- indicated the growth of bacteria, and the MIC was dened as
d6, d ppm): 52.2, 61.9, 115.3, 120.8, 125.8, 126.7, 127.4, 128.8, the lowest concentration of the drug that prevented this change
137.3, 141.0, 141.6, 163.3, and 164.3; HRMS calcd for in colour.
C
17H15ClN5O4 [M + H]+: 388.0813 and found 388.0819.
4.3.3. Antioxidant activity (DPPH radical scavenging). The
hydrogen atom or electron donation ability of some of the
compounds was measured from the bleaching of the purple col-
oured methanol solution of 1,1-diphenyl-1-picrylhydrazyl (DPPH).
4.3 Experimental protocol for biological activity
4.3.1. Antifungal activity. Antifungal activity was deter- The spectrophotometric assay uses the stable radical DPPH as
mined by the standard agar dilution method as per the CLSI a reagent. One mL of various concentrations of the test compounds
(formerly, NCCLS) guidelines.26 The newly synthesized (5, 10, 25, 50, and 100 mg mLꢁ1) in methanol was added to 4 mL of
compounds were screened for their in vitro antifungal activity 0.004% IJ w/v methanol solution of DPPH. The reaction mixture
against ve human pathogenic fungal strains including Candida was incubated at 37 ꢀC. The scavenging activity on DPPH was
albicans (NCIM 3471), Fusarium oxysporum (NCIM 1332), Asper- determined by measuring the absorbance at 517 nm aer 30 min.
gillus avus (NCIM 539), Aspergillus niger (NCIM 1196), and All tests were performed in triplicate, and the mean values were
Cryptococcus neoformans (NCIM 576). The synthesized obtained. The percent of inhibition of free radical production from
compounds and the standard Miconazole were dissolved in DPPH was calculated by using the following equation:
DMSO. The medium yeast nitrogen base was dissolved in phos-
ꢀ
% of scavenging ¼ [(Acontrol ꢁ Asample)/IJAsample ꢂ 100)],
phate buffer of pH 7 and 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
where Acontrol is the absorbance of the control (DPPH radical
without test sample) and Asample is the absorbance of the test
freshly subcultured onto sabouraud dextrose agar (SDA) and
sample (DPPH radical with test sample). The control contained
incubated at 25 ꢀC for 72 h. The fungal cells were suspended in
all the reagents except the test samples.
sterile distilled water and diluted to get 105 cells per mL. Ten mL
4.3.4. Homology
modeling.
The
three-dimensional
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 h. The
readings were taken at the end of 48 and 72 h. MIC is the lowest
homology model structure of cytochrome P450 lanosterol 14a-
demethylase of C. albicans was predicted using the theoretical
methods of protein structure prediction, i.e., comparative
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modelling using the Biopredicta module of VLifeMDS 4.3 Pro-
Model. The protein sequence of cytochrome P450 lanosterol 14a-
demethylase for C. albicans was retrieved from the protein
knowledge database Universal Protein Resource (UniProtKB)
(http://www.uniprot.org/) (accession code: P10613) and its homol-
ogous template was identied from the protein structural data-
base, i.e., Protein Data Bank (PDB) using Basic Local Alignment
Search Tool for proteins (BLASTp). The homologous template
identied using BLASTp solved the three-dimensional crystal
structure of human lanosterol 14a-demethylase (CYP51) complex
with azole (PDB ID: 3LD6). To construct a basic framework and to
build the loop regions of C. albicans cytochrome P450 lanosterol
14a-demethylase, pairwise sequence alignment and loop rene-
ment of CA-CYP51 (P10613) and human CYP51 (3LD6_B) were
performed. Three-dimensional modelled protein structures were
subjected to structural quality check programs such as PRO-
CHECK and its other structural validation parameters.31
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Conflicts of interest
There are no conicts to declare.
Acknowledgements
One of the authors, SVA, is very much grateful to the Council of
Scientic and Industrial Research (CSIR), New Delhi for the
award of research fellowship. Authors are also thankful to the
Head, Department of Chemistry, Dr Babasaheb Ambedkar
Marathwada University Aurangabad for providing laboratory
facility.
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