Ultrasound assisted rapid synthesis, biological evaluation, and molecular docking study of new 1,2,3-triazolyl pyrano[2,3-c]pyrazoles as antifungal and antioxidant agent

Article abstract of DOI:10.1080/00397911.2019.1631849

In search of new generation of triazole based antifungal agents, synthesis of series of new 1,2,3-triazolyl pyrano[2,3-c]pyrazoles under ultrasonic irradiation using NaHCO₃ has been reported. The bioevaluation results indicate that, the compoun

Full text of DOI:10.1080/00397911.2019.1631849

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Ultrasound assisted rapid synthesis, biological
evaluation, and molecular docking study of new
1,2,3-triazolyl pyrano[2,3-c]pyrazoles as antifungal
and antioxidant agent
Smita P. Khare, Tejshri R. Deshmukh, Jaiprakash N. Sangshetti, Vijay M.
Khedkar & Bapurao B. Shingate
To cite this article: Smita P. Khare, Tejshri R. Deshmukh, Jaiprakash N. Sangshetti, Vijay M.
Khedkar & Bapurao B. Shingate (2019): Ultrasound assisted rapid synthesis, biological evaluation,
and molecular docking study of new 1,2,3-triazolyl pyrano[2,3-c]pyrazoles as antifungal and
antioxidant agent, Synthetic Communications, DOI: 10.1080/00397911.2019.1631849
To link to this article: https://doi.org/10.1080/00397911.2019.1631849
Published online: 10 Jul 2019.
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https://doi.org/10.1080/00397911.2019.1631849
Ultrasound assisted rapid synthesis, biological evaluation,
and molecular docking study of new 1,2,3-triazolyl
pyrano[2,3-c]pyrazoles as antifungal and antioxidant agent
Smita P. Kharea, Tejshri R. Deshmukha, Jaiprakash N. Sangshettib,
Vijay M. Khedkarc, and Bapurao B. Shingatea
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, MH, India;
bDepartment of Pharmaceutical Chemistry, Y. B. Chavan College of Pharmacy, Aurangabad, MH, India;
cDepartment of Pharmaceutical Chemistry, Shri Vile Parle Kelavani Mandal’s Institute of Pharmacy,
Dhule, MH, India
ABSTRACT
ARTICLE HISTORY
Received 11 March 2019
In search of new generation of triazole based antifungal agents,
synthesis of series of new 1,2,3-triazolyl pyrano[2,3-c]pyrazoles
under ultrasonic irradiation using NaHCO3 has been reported. The
bioevaluation results indicate that, the compounds 7c, 7d, 7e, 7f,
and 7i displayed excellent antifungal activity with lower MIC
ꢀ 25 mg/mL. Most of the compounds from the series showed potent
KEYWORDS
Antifungal activity;
molecular docking study;
multicomponent reactions;
pyrano[2,3-c]pyrazole;
1,2,3-triazole
antioxidant
activity with a lower IC50 value in the range
09.39 0.42–14.97 0.24 mg/mL, in comparison to butylated hydroxyl
toluene (BHT). Molecular docking studies against potential target
sterol 14a-demethylase (CYP51) was also performed and showed
excellent binding affinity with the target enzyme. Moreover, in silico
ADME study shows that the derivatives could serve as drug like mol-
ecules for further drug development in clinical research.
GRAPHICAL ABSTRACT
Introduction
Over the past few decades, invasive fungal infections among the immunocompromised
and critically ill patients are growing rapidly and is becoming a leading cause of
CONTACT Bapurao B. Shingate
bapushingate@gmail.com
Department of Chemistry, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, 431 004, MH, India.
Supplemental data for this article is available online at on the publisher’s website.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsyc.
ß 2019 Taylor & Francis Group, LLC

2
S. P. KHARE ET AL.
Figure 1. Azole based antifungal drugs.
morbidity and mortality.[1] The high mortality rate was observed due to fungal
infections caused by Candida albicans and Cryptococcus neoformans.[2] The treatment of
fungal infections becomes more difficult due to the decrease in efficiency of present
azole-based drugs and developed resistance in most of the fungal strains. Clinically
approved azole-based first-line drugs for the treatment of invasive fungal infections
includes Fluconazole, Voriconazole, Posaconazole, and Itraconazole, etc. (Figure 1).[3]
Azole based antifungal agents target lanosterol 14a-demethylase (CYP51) which is the
key enzyme for the biosynthesis of ergosterol. The CYP51 enzyme catalyzes oxidative
removal of 14a-methyl group of lanosterol by monooxygenase activity. Azoles disrupt
this oxidative removal through binding of azole N-4 to iron protoporphyrin unit of
CYP51 which results in depletion of ergosterol and accumulation of 14a- methylated
sterol. This affects the membrane fluidity and activity of membrane enzymes which
inhibits fungal growth.[4]
1,2,3-Triazole nucleus has been reported as an important pharmacophoric scaffold,
which is known to possess diverse biological activities.[5] Along with the pharmaco-
logical profile, triazole nucleus possesses interesting physicochemical properties such as
triazole moiety stable to acid–base hydrolysis, oxidation-reduction, capable to form
H–bonding interaction, which helps to bind with biological targets. There are several
reports on the synthesis of triazole-based molecules as antifungal,[6] antitubercular,[7]
anticancer,[8] antimicrobial,[9] antiproliferative,[10] anti-coronavirus,[11] antibacterial,[12]
and neuroprotective agents.[13] Some of the triazole-based drugs available in the market
are Tazobactam, Cefatrizine and some are in the phases of clinical trials like CAI
(Carboxyamidotriazole) (Figure 2).[14]
On the other hand, pyrazole is one of the privileged N-heterocyclic scaffolds in medi-
cinal research.[15] Pyrazole and its derivatives possess a broad spectrum of biological
activities, for instance, antifungal,[16] anti-inflammatory,[17] antihyperglycemic,[18] anti-
microbial,[19] and antimalarial activities.[20] There are a number of pyrazole containing
drugs such as Celecoxib, Phenylbutazone, Pyrazomycine, Rimonabant, Apixaban, and
Pyrazofurin available in the market (Figure 3).[21]
4H-Pyran and pyran annulated heterocycles are observed in both nature and synthetic
origin. Many pyran scaffolds are known to exhibit several biological activities[22] and
some of the biologically active synthetic 4H-pyran derivatives are shown in Figure 4.
4H-Pyran and its derivatives have been synthesized with much attention due to its
biological significance. Several methods have been reported in the literature for the
synthesis of pyrano[2,3-c]pyrazole[23] through multicomponent reactions (MCRs).

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Figure 2. 1,2,3-Triazole based marketed drugs.
Figure 3. Medicinally important pyrazole based drug molecules.
Figure 4. Biologically active 4 H-Pyran derivatives.
In addition, their synthesis has also been reported by non-conventional techniques such
as microwave irradiation, ultrasonic irradiation, and ball milling technique.[24] However,
most of these methods are useful with their own merits and demerits; yet, there is
major scope for the development of newer green and efficient strategies for the synthe-
sis of 4H-pyran and its derivatives.
Multicomponent reactions (MCRs) are one of the advantageous tools for the gener-
ation of diversified heterocyclic scaffolds in a single transformation.[25] In addition to
this, water-mediated MCRs is one of the greener approaches which helps in the devel-
opment of economic and eco-friendly methodologies.[26] Furthermore, ultrasonic irradi-
ation (USI) is a green energy source which offers high yields in shorter reaction
time.[27] The mechanism of reaction under USI is based on acoustic cavitation effect
with the formation and collapse of bubbles. This process generates high temperature
and pressure in microseconds that lead to rate acceleration of reaction as compared to
the traditional methods.
Impressed by the binding efficiency of 1,2,3-triazoles, pharmacological profile of pyra-
zole and pyran moieties discussed above, and in continuation of our research work on
development of green synthetic methods, synthesis and evaluation of new heterocyclic

4
S. P. KHARE ET AL.
Figure 5. Designing of 1,2,3-triazolyl pyrano[2,3-c]pyrazoles by molecular hybridization approach.
compounds,[28] we report herein the design and synthesis of a series of 1,2,3-triazolyl
pyrano[2,3-c]pyrazoles which showed potent antifungal and antioxidant activities. The
designing of the target scaffold is based on molecular hybridization strategy.[29] We
assembled the three biologically active scaffolds, i.e. 1,2,3-triazole, pyrazole, and
4H-pyran into a single molecular framework hopefully enhance the biological activity of
the target scaffold (Figure 5).
Results and discussion
Chemistry
We have carried out the one-pot three-component synthesis of new 1,2,3-triazolyl pyr-
ano[2,3-c]pyrazoles on the basis of molecular hybridization approach. Initially, the tria-
zolyl aldehydes (4a–j) were prepared according to the reported method,[30a] from the
commercially available anilines (1a–j) by click chemistry approach (Scheme 1).
On the basis of successful applications of triazolyl aldehydes for the synthesis of vari-
ous biologically active heterocyclic scaffolds,[30] we have attempted the one-pot three-
component synthesis of triazolyl pyrano[2,3-c]pyrazoles. At first, to optimize reaction
conditions for the synthesis of triazolyl pyrano[2,3-c]pyrazole derivatives, the reaction of
triazolyl aldehyde (4a), malononitrile (5) and pyrazolone (6) were chosen as a model
reaction (Scheme 2). For this, pyrazolone 6 was prepared in situ by the condensation
reaction of ethyl acetoacetate and hydrazine hydrate without any catalyst.
In our initial attempt, we have performed the model reaction by using piperidine
(10 mol%) as a base in water and the reaction was carried at room temperature for
16 min (Scheme 2) (as per the reported method). The corresponding 1,2,3-triazolyl pyr-
ano[2,3-c]pyrazole, 7a was obtained in 84% yield. Furthermore, under the similar reac-
tion conditions, the reaction of various triazolyl aldehydes (4b–j) was performed and
obtained the corresponding 1,2,3-triazolyl pyrano[2,3-c] pyrazole derivatives (7a–j) in
80–92% yield (Scheme 3, Table 1, Method A). It is observed that the results were not
satisfactory. Despite the reaction rate being fast, the yields were 80–92%.

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Scheme 1. Synthetic route for triazolyl aldehydes (4a–j). Reagents and conditions: (i) NaNO2, HCl
(10%); NaN3, 1–2 h, 0 ꢁC; (ii) propargyl alcohol, CuSO4.5H2O, sodium ascorbate, tert-BuOH-H2O (1:1),
24–48 h, rt; (iii) Collins reagent (CrO3.2Py, CH2Cl2), 3–6 h, rt.
Scheme 2. Model reaction.
Scheme 3. Synthesis of 1,2,3-triazolyl pyrano[2,3-c]pyrazole derivatives (7a–j).
To elaborate greener synthetic protocol and to optimize reaction conditions for the
synthesis of same 1,2,3-triazolyl pyrano[2,3-c]pyrazole derivatives 7a–j, the model reac-
tion (Scheme 2) was performed using various inorganic bases such as K2CO3, Na2CO3
and NaHCO3 in water as a solvent under ultrasonic irradiation (40 kHz) at 30 ꢁC and
the results were summarized in Table 2. At the preliminary stage, the model reaction
was carried out in the absence of catalyst under the same reaction parameters; it was
observed that the desired product 7a was not obtained even after prolonged reaction
time (Table 2, entry 1). In our next attempt, the model reaction was performed by using

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S. P. KHARE ET AL.
Table 1. Three component synthesis of 1,2,3-triazolyl pyrano[2,3-c]pyrazole derivatives (7a–j)a.
Method A
Method B
Product
R
Time (min)
Yieldb (%)
Time (min)
Yieldb (%)
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
4-Me
4-OMe
3-OMe
2-OMe
4-Cl
16
15
13
14
15
14
15
17
19
15
84
86
90
88
92
88
85
82
80
84
7
5
5
4
5
4
6
4
5
5
98
97
95
92
94
98
94
93
96
92
3-Cl
4-NO2
3-NO2
2-NO2
7j
aReaction conditions: Method A: 4(a–j) (1 mmol), 5 (1 mmol) and 6 (1 mmol), Piperidine (10 mol%), water (5 mL), rt.
Method B: 4(a–j) (1 mmol), 5 (1 mmol) and 6 (1 mmol), NaHCO3 (20 mol%), water (5 mL), Ultrasonic irradiation
(40 kHz), 30 ꢁC;
bIsolated yield.
Table 2. Optimization of the reaction conditions for the preparation of 7aa.
Entry
Catalyst
Amount of catalyst (mol%)
Time (min)
Yield (%)b
1
2
3
4
5
6
7
8
None
K2CO3
K2CO3
-
60
30
30
30
30
10
10
10
10
10
20
20
10
-
80
84
82
88
88
84
96
99
10
20
10
20
10
5
15
20
25
20
25
20
Na2CO3
Na2CO3
NaHCO3
NaHCO3
NaHCO3
NaHCO3
NaHCO3
NaHCO3
NaHCO3
NaHCO3
9
10
11
12
13
99
99
99
Tracec
aReaction conditions: 4a (1 mmol),
5 (1 mmol), and 6 (1 mmol), catalyst, water (5 mL), Ultrasonic irradiation
(40 kHz), 30 ꢁC;
bIsolated yields;
cWithout ultrasonic irradiation.
K2CO3 (10 and 20 mol%) under the similar reaction parameters, the desired product 7a
was obtained in 80 and 84% yield, respectively (Table 2, entries 2–3). Furthermore, the
product 7a was obtained in 82 and 88% yield with the use of 10 and 20 mol% of
Na2CO3, respectively (Table 2, entry 4–5).
With the use of NaHCO3 (10 mol%), the desired product 7a was obtained in excellent
yield (88%) in shorter reaction time (Table 2, entry 6). Therefore, it was observed that,
among the screened inorganic bases, NaHCO3 has been found to be an efficient catalyst
in terms of time and yield. Further, to determine the optimal amount of NaHCO3, the
model reaction was subsequently tested for different concentrations, viz. 5, 15, 20 and
25 mol% (Table 2, entries 7–12). Using less than 20 mol% of NaHCO3, the product 7a
was obtained in good yield (88–96%) (Table 2, entries 6–8). It was evident that 20 mol%
of NaHCO3 was sufficient to carry out the reaction in short reaction time with 99%
yield (Table 2, entry 9). No further improvement in the yield was observed with
25 mol% of NaHCO3 (Table 2, entry 10). However, no further increase in the yield was
observed by using 20 and 25 mol% of the catalyst with extended reaction time (Table 1,
entries 11–12). Finally, to examine the role of ultrasound, the model reaction was

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performed without ultrasonic irradiation in presence of 20 mol% NaHCO3 at 30 ꢁC
(Table 2, entry 13) and it was observed that the trace amount of desired product was
obtained. In summary, the best results were obtained with 20 mol% of NaHCO3 in
water under ultrasonic irradiation (40 kHz) at 30 ꢁC. These results signify that along
with the catalyst, ultrasonic irradiation helps to improve the yield of the product in
short reaction time.
1
The formation of the compound 7a was confirmed by IR, H NMR, 13C NMR, and
mass analysis. The IR spectrum of 7a displayed the absorption band at 3203 and
1
2189 cmꢂ1 due to the NH2 and CN groups. The H NMR spectrum of compound 7a
exhibited singlet peaks at d 4.94, 7.00, 8.67, and 12.17 ppm due to the corresponding
methine, –NH2, triazolyl, and pyrazolyl protons. The 13C NMR spectrum of compound
7a showed a peak at d 28 ppm due to the steriogenic methine carbon of the 4H-pyran
ring. In the mass spectrum of the compound 7a, molecular ion peak observed at
320 corresponding to the calculated for C16H15N7O ([M þ H]þ 320.1260).
With these optimized reaction conditions in hand, the three component reaction of
various substituted triazolyl aldehydes (4b–j), malononitrile (5), and pyrazolone (6) was
investigated for the preparation of 1,2,3-triazolyl pyrano[2,3-c]pyrazole derivatives under
the optimized reaction conditions (Scheme 3) and the obtained results were summarized
in Table 1 (Method B). The reaction conditions work smoothly for electron donating
and withdrawing groups (–Me, –OMe, –Cl, and –NO2) on triazolyl aldehydes and cor-
responding products were obtained in good to excellent yields within shorter reac-
tion time.
Biological assay
In vitro antifungal activity
In vitro antifungal activity of all the compounds, 7a–j were evaluated against five
human pathogenic fungal strains namely Candida albicans (NCIM 3471), Fusarium oxy-
sporum (NCIM 1332), Aspergillus flavus (NCIM 539), Aspergillus niger (NCIM 1196),
and Cryptococcus neoformans (NCIM 576). Minimum inhibitory concentration (MIC)
values were determined by the standard agar dilution method. DMSO was used as solv-
ent control. The results are compared with standard antifungal drug Miconazole and
are illustrated in Table 3. It is observed from Table 3 that, most of the newly synthe-
sized hybrids exhibited good to excellent antifungal activity against nearly all the tested
fungal strains.
Among the 1,2,3-triazolyl pyrano[2,3-c]pyrazole derivatives (7a–j), compounds 7c, 7d,
7e, 7f, and 7i display prominent antifungal activity against most of the tested fungal
strains. Compound 7i exhibited excellent antifungal activity with MIC ¼ 12.5 mg/mL
and found to be more potent than Miconazole against C. albicans. The compounds 7c
and 7e were found to be equipotent to Miconazole against C. albicans with MIC ¼
25 mg/mL. Compounds 7d and 7f were exhibited equivalent activity compared to the
standard drug against F. oxysporum with MIC ¼ 25 mg/mL. It is promising to note that
only the compound 7i exhibited equivalent activity as compared to Miconazole with
MIC ¼ 25 mg/mL against A. niger. The compound 7f exhibited equipotent activity com-
pared to Miconazole against C. neoformans with MIC ¼ 25 mg/mL. From Table 3, it is

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S. P. KHARE ET AL.
Table 3. In vitro antifungal and antioxidant activity of synthesized compounds 7a–j.
Antifungal activity (MIC) in mg/mL
Antioxidant activity
Compound
CA
FO
AF
AN
CN
IC50 (mg/mL)
7a
7b
7c
7d
7e
7f
7g
7h
62.5
50
25
37.5
25
50
175
137.5
12.5
175
25
87.5
75
50
25
50
50
25
25
37.5
50
75
100
75
75
50
100
50
175
150
25
150
25
62.5
50
50
50
50
25
150
125
27.17 0.74
21.08 0.37
14.77 0.57
11.07 0.09
09.39 0.42
12.91 0.43
19.11 0.77
14.97 0.24
11.99 0.87
10.22 0.54
NT
25
175
125
50
200
25
200
200
7i
7j
25
75
ꢃ
ꢃ
Miconazole
BHT
12.5
NT
25
NT
NT
NT
NT
16.47 0.18
CA: Candida albicans; FO: Fusarium oxysporum; AF: Aspergillus flavus; AN: Aspergillus niger; CN: Cryptococcus neoformans;
ꢃ
BHT: butylated hydroxy toluene. No activity up to 200 mg/mL; NT: Not tested.
observed that the antifungal activity varies with the substituent present on an aromatic
unit of 1,2,3-triazolyl pyrano[2,3-c]pyrazole (7a–j). Compounds substituted with R ¼ 4-
OMe, 3-OMe, 2-OMe, 4-Cl, and 3-NO2 exhibited excellent antifungal activity than the
compounds substituted with R ¼ H, 4-Me, 3-Cl, 4-NO2, and 2-NO2.
Antioxidant activity
The newly synthesized 1,2,3-triazolyl pyrano[2,3-c]pyrazole derivatives were evaluated
for their in vitro antioxidant activity. The antioxidant activity results of the derivatives
7a–j were obtained by using DPPH radical scavenging assay. Radical scavenging activity
was measured in terms of IC50 value. Radical scavenging activity of all the compounds
was compared with butylated hydroxyl toluene (BHT) having an IC50 value
16.47 0.18 mg/mL and the results were summarized in Table 3. Among the 1,2,3-tria-
zolyl pyrano[2,3-c]pyrazoles (7a–j), it is promising to note that most of the compounds
are excellent antioxidant agents than BHT. Compound 7e exhibited excellent activity
with appreciably lower IC50 value 09.39 0.42 mg/mL and found to be more potent than
BHT. Compounds 7c, 7d, 7f, 7h, 7i, and 7j were found to be more potent than BHT
with an IC50 values 14.77 0.57, 11.07 0.09, 12.91 0.43, 14.97 0.24, 11.99 0.87, and
10.22 0.54 mg/mL, respectively.
Computational study
Molecular docking
In an effort to elucidate the possible mechanism by which the title compounds can
induce antifungal activity and guide further SAR studies, molecular docking was per-
formed into the active site of a crucial fungal target-sterol 14a-demethylase (CYP51)
inhibition of which prevents the conversion of lanosterol to ergosterol and subsequent
accumulation of 14a-methyl sterols in the cell leading to impaired cell growth in fungi.
This in silico approach has now become an integral part of drug discovery pipeline,
especially in the absence of available resources to carry out the enzymatic assays,
imparting knowledge on binding affinities, binding modes and the associated

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thermodynamic interactions with the target enzyme governing the inhibition of the
causative pathogen. It is observed that all the ligands (7a–7j) showed similar orientation
in the CYP51 active site and their complex formed was stabilized by the formation of
several bonded and non-bonded interactions. Even their binding energies signifying the
binding affinity were observed to be negative (ꢂ48.562 to ꢂ27.249 kcal/mol) while the
average docking score was seen to be ꢂ7.271 kcal/mol. An in-depth investigation of the
per-residue interaction between these compounds and the residues in the active site of
the enzyme has been carried out to identify the most prominently interacting residues
and their type of thermodynamic interactions (bonded and non-bonded interactions)
that is critical in lead optimization. This analysis is discussed in detail for the most
active 1,2,3-triazolyl pyrano[2,3-c]pyrazole derivative (7i) and the results are summar-
ized in Table 4 for the remaining molecules in the series.
Visual inspection and per-residue interaction analysis for the lowest energy docked
conformation of active analogues 7i showed that they could snugly fit into the active
site of CYP51 at the same coordinates as the native ligand with a significantly higher
binding affinity (docking score of ꢂ9.028 and Glide binding energy of ꢂ48.562 kcal/
mol, respectively) engaging in a series of steric and electrostatic interactions (Figure 6).
Compound 7i was observed to be stabilized in the active site of CYP51 through a ser-
ies of significant van der Waals and electrostatic interactions. However, the most notice-
able interactions are the hydrogen bonding interaction observed for 7i with Met358
(2.04Å) through the pyrazole ring. Though 7i did not engage in hydrogen bonding with
Tyr103 (2.262Å) and Tyr116 (2.32Å) but it showed a very prominent p–p stacking
interaction with these residues. The interactions with these three residues –Met358,
Tyr103, and Tyr116 were consistently observed in all the active molecules in the series
signifying their role in lead optimization. Such hydrogen bonding and p–p stacking
interactions serve as an anchor for guiding the orientation of the ligand in the 3D space
of the active site and facilitate the non-bonded (steric and electrostatic) interactions for
stabilizing the enzyme-inhibitor complex. Further, 7i was found to be engaged in a ser-
ies of significant van der Waals interactions with residues lining the active site of
CYP51; for 7i with Hem500 (ꢂ6.375 kcal/mol), Phe290 (ꢂ1.485 kcal/mol), Ala287
(ꢂ1.614 kcal/mol), Met123 (ꢂ1.930 kcal/mol), Tyr116 (ꢂ2.186 kcal/mol), Ala115
(ꢂ1.962 kcal/mol), and Phe110 (ꢂ2.321 kcal/mol) residues through 6-amino-3-methyl-4-
1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl component while the 1,4-dihydropyrano[2,3-c]-
pyrazole-5-carbonitrile component of the molecule interacted with Val461 (ꢂ2.107 kcal/
mol), Met460 (ꢂ2.308 kcal/mol), Val359 (ꢂ2.401 kcal/mol), Leu356 (ꢂ2.151 kcal/mol),
Ala291 (ꢂ2.938 kcal/mol), Tyr103 (ꢂ2.942 kcal/mol) residues. The higher binding
observed for 7i also attributed to a relatively fewer but significant electrostatic interac-
tions as well observed with Hem500 (ꢂ4.406 kcal/mol), Cys422 (ꢂ2.656 kcal/mol),
Arg361 (ꢂ1.038 kcal/mol), Met358 (ꢂ2.382 kcal/mol), Ala291 (ꢂ1.935 kcal/mol), Arg124
(ꢂ1.602 kcal/mol), and Arg100 (ꢂ1.362 kcal/mol) residues for 7i. A similar network of
bonded and non-bonded interactions was involved in stabilizing other molecules of the
series into the active site CYP51 (Figures S1–S9 Electronic Supplementary Information).
Furthermore, it is noteworthy that all the molecules in the series were seen to be
engaged in a very strong van der Waals as well as electrostatic interactions with Heme
moiety present in the active site of CYP51 which is an important observation

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S. P. KHARE ET AL.

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S. P. KHARE ET AL.
Figure 6. Binding mode of 7i into the active site of sterol 14a-demethylase (CYP51) (on right side:
green lines signify p–p stacking interactions while the pink lines represent the hydrogen bonding
interactions).
Table 5. Pharmacokinetic parameters of all synthesized compounds using in silico ADME prediction.
Lipinski’s
violations
Drug likeness
model score
Entry
% ABS
TPSA
MV
MW
milog p
n-ON
n-OHNH
n-ROTB
Rule
7a
7b
7c
7d
7e
7f
7g
7h
7i
<500
319.33
333.36
349.35
349.35
349.35
353.77
353.77
364.32
364.32
364.32
ꢀ5
ꢀ10
ꢀ5
ꢀ1
68.21
68.21
64.96
64.96
64.96
68.14
68.14
52.33
52.33
52.33
118.45
118.45
127.68
127.68
127.68
118.45
118.45
164.27
164.27
164.27
271.84
288.40
297.39
297.39
297.39
285.38
285.38
295.18
295.18
295.18
0.87
1.32
0.93
1.12
1.09
1.55
1.74
0.83
1.02
0.99
8
3
0
2
2
3
3
3
2
2
3
3
3
ꢂ0.12
ꢂ0.55
ꢂ0.33
ꢂ0.21
ꢂ0.26
ꢂ0.02
ꢂ0.20
ꢂ0.40
ꢂ0.32
ꢂ0.34
8
9
9
9
8
8
11
11
11
3
3
3
3
3
3
3
3
0
0
0
0
0
0
1
1
7j
3
1
considering the fact that Fluconazole is as well coordinated in the active site of CYP51
through the iron metal indicating that the title compounds may also share a similar
mechanism for their anti-fungal action as Fluconazole. Overall, the information derived
from the per-residue ligand interaction analysis could be fruitfully utilized for the struc-
ture-based lead optimization to arrive at potent antifungal agents with this scaffold.
In silico ADME prediction
The success of drug depends upon its good efficacy, oral bioavailability, and ADME
(absorption, distribution, metabolism, and excretion) properties. We have performed a
computational study of all the compounds for the prediction of ADME properties. In
this study, we have calculated molecular volume (MV), molecular weight (MW), log of
partition coefficient (mi log p), number of hydrogen bond donors (n-ON), number of
hydrogen bond acceptors (n-OHNH), topological polar surface area (TPSA), number of
rotatable bonds (n-ROTB) and Lipinski’s rule of five[31] using the molinspiration online
property calculation toolkit (Molinspiration Cheminformatics, Slovak Republic).[32]
Absorption (%ABS) was calculated by the formula %ABS ¼ 109 ꢂ (0.345 ꢄ TPSA).[33]

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SYNTHETIC COMMUNICATIONSV
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The obtained results of ADME prediction are presented in Table 5. It is observed that
the compounds exhibited good absorption (%ABS) ranging between 52–68%. Moreover,
most of the compounds show good agreement with Lipinski’s rule of five. The drug-
likeness model score, the collective property of physicochemical properties was also cal-
culated by using MolSoft software.[34] Thus, most of the derivatives possess the good
potential to be developed as an orally active drug molecule.
Conclusions
In conclusion, novel 1,2,3-triazolyl pyrano[2,3-c]pyrazole derivatives have been synthe-
sized through a one-pot multicomponent reaction (MCR) approach using NaHCO3 as a
catalyst under ultrasonic irradiation in excellent yield. All the compounds were eval-
uated against five pathogenic fungal strains and results showed that the compound 7i
found to be more potent than Miconazole against Candida albicans. Compounds 7c,
7d, 7e, and 7f exhibited excellent antifungal activity and equipotent to Miconazole.
Furthermore, all the compounds were also investigated for their in vitro antioxidant
activity and most of them found to be more potent than BHT with a lower IC50 value
(IC50 <16.49 0.44). Molecular docking studies against a target enzyme sterol 14a-
demethylase (CYP51) showed a significant correlation between binding score and anti-
fungal activity. In silico ADME properties prediction reveals, most of the compounds
can be developed as lead candidates. Thus, the results suggest that 1,2,3-triazolyl pyr-
ano[2,3-c]pyrazoles can open new opportunities for antifungal and antioxidant agents in
further clinical research.
Experimental
General
All the solvents and reagents were purchased from commercial suppliers Spectrochem
Pvt. Ltd., Sigma Aldrich and Rankem India Ltd. and used without further purification.
All the one-pot reactions were performed using citizen (CUB 2.5, Mumbai, India) ultra-
sonic cleaner bath working at 40 kHz (constant frequency, 50 W). The progress of each
reaction was monitored by ascending thin layer chromatography (TLC) using TLC alu-
minum sheets, silica gel 60 F254 precoated, Merck, Germany and locating the spots
using UV light as the visualizing agent or iodine vapors. Melting points were deter-
mined in open capillary method and are uncorrected. Infrared (IR) spectra were
1
recorded on a Bruker FT-IR spectrometer. H NMR spectra were recorded (CDCl3/
DMSO-d6) on Bruker Avance 200 and 500 MHz NMR spectrometer (Bruker, Billerica,
MA). 13C NMR spectra were recorded (CDCl3/DMSO-d6) on Bruker Avance 50 and
125 MHz NMR spectrometer. Mass spectra were recorded on an Agilent 6520 (Q-Tof)
(ESI-HRMS) (Agilent, Santa Clara, CA) and Waters UPLC-TQD (ESI-MS) instrument
(Waters Corporation, Milford, MA). The elemental analysis values were recorded on
Thermofisher FA-2001 CHNS analyzer. Chemical shifts (d) are reported in parts per
million (ppm) using tetramethylsilane (TMS) as an internal standard. The splitting pat-
tern abbreviations are designed as singlet (s); doublet (d); double doublet (dd); triplet
(t) and multiplet (m).

14
S. P. KHARE ET AL.
General procedure for the synthesis of 1,2,3-triazolyl pyrano[2,3-c]
pyrazoles (7a–j)
Method A
A mixture of 1-aryl-1H-1,2,3-triazole-4-carbaldehyde (4a–j) (1 mmol), malononitrile (5)
(1 mmol), pyrazolone 6 (1 mmol) and piperidine (10 mol%) in water (5 mL) was taken
in 50 mL round bottom flask and the mixture was stirred at room temperature for
appropriate time. After the completion of the reaction (monitored by TLC), the reaction
mixture was poured into 20 mL ice-water and the obtained solid was filtered, washed
thoroughly with water, dried, and recrystallized from ethanol to afford the pure prod-
ucts (7a–j).
Method B
A mixture of 1-aryl-1H-1,2,3-triazole-4-carbaldehyde (4a–j) (1 mmol), malononitrile (5),
(1 mmol) pyrazolone 6 (1 mmol), and NaHCO3 (20 mol%) in water (5 mL) was taken in
50 mL round bottom flask and the mixture was sonicated in an ultrasonic bath working
at 40 kHz (constant frequency) at 30 ꢁC for appropriate time. After the completion of
the reaction (monitored by TLC), the reaction mixture was poured into 20 mL ice-water
and the obtained solid was filtered, washed thoroughly with water, dried and recrystal-
lized from ethanol to afford the pure products (7a–j) in excellent yields.
6-Amino-3-methyl-4-(1-phenyl-1H-1,2,3-triazol-4-yl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (7a)
Mp: 205–207 ꢁC. IR vmax (cmꢂ1): 3319, 3203 (–NH2), 2189 (–CN), 1634 (C ¼ C vinyl
1
nitrile), 1587 (C ¼ C–Aromatic). H NMR (200 MHz, DMSO-d6, d ppm): 1.98 (s, 3H,
–CH3), 4.94 (s, 1H, methine), 7.00 (s, 2H, –NH2), 7.43–7.62 (m, 3H), 7.93 (d, 2H,
J ¼ 8 Hz), 8.67 (s, 1H, triazolyl-H), 12.17 (s, 1H, pyrazolyl-H). 13C NMR (50 MHz,
DMSO-d6, d ppm): 9.8, 28, 54.9, 96, 119.7, 120, 120.7, 128.5, 130, 136, 136.6, 151.3,
154.6, 161.3. ESI-MS: m/z 320 [M þ H]þ. Anal. Calcd. for C16H13N7O: C, 60.18; H,
4.10; N, 30.70%; Found: C, 60.37; H, 4.11; N, 30.67%.
Supporting information PDF file contains an experimental protocol for biological
1
activity and computational studies, and spectral data of compounds 7a–j (FT-IR, H
and 13C NMR, mass and CHNS spectra) of the compounds.
Acknowledgments
One of the authors S. P. K. is grateful to Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad, for the award of University Scholars Fellowship. Authors are also thankful to the
Head, Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad,
431 004, Maharashtra, India, for providing laboratory facility. Authors also acknowledge
Schrodinger Inc. for providing GLIDE software to perform the molecular modeling studies.

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