An efficient and convenient approach for the synthesis of novel pyrazolo[1,2-a]triazole-triones and evaluation of their antimicrobial activities

Article abstract of DOI:10.1071/CH13586

A new protocol for the efficient synthesis of novel pyrazolo[1,2-a] triazole-1,3,5-triones has been developed. The synthesis was achieved by reaction of aromatic aldehydes, Meldrum's acid, and 4-phenylurazole in the presence of a catalytic amount of glacial AcOH at 80°C. The structural assignment has been unambiguously confirmed by X-ray analysis. The present finding provides an environmentally benign, efficient, and promising synthetic strategy for the synthesis of libraries with diversity. These compounds were tested in vitro for their antibacterial activity against four strains, namely Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa and antifungal activity against two fungal strains, namely Aspergillus niger and Aspergillus flavus. CSIRO 2014.

Full text of DOI:10.1071/CH13586

CSIRO PUBLISHING
Aust. J. Chem. 2014, 67, 867–874
Full Paper
http://dx.doi.org/10.1071/CH13586
An Efficient and Convenient Approach for the Synthesis
of Novel Pyrazolo[1,2-a]triazole-triones and Evaluation
of their Antimicrobial Activities
A
A C
,
B
Pooja Saluja, Jitender M. Khurana,
Chetan Sharma,
B
and Kamal R. Aneja
A
Department of Chemistry, University of Delhi, Delhi 110007, India.
Department of Microbiology, Kurukshetra University,
Kurukshetra 136119, Haryana, India.
B
C
Corresponding author. Email: jmkhurana1@yahoo.co.in
A new protocol for the efficient synthesis of novel pyrazolo[1,2-a]triazole-1,3,5-triones has been developed. The synthesis
was achieved by reaction of aromatic aldehydes, Meldrum’s acid, and 4-phenylurazole in the presence of a catalytic
amount of glacial AcOH at 808C. The structural assignment has been unambiguously confirmed by X-ray analysis. The
present finding provides an environmentally benign, efficient, and promising synthetic strategy for the synthesis of
libraries with diversity. These compounds were tested in vitro for their antibacterial activity against four strains, namely
Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa and antifungal activity against
two fungal strains, namely Aspergillus niger and Aspergillus flavus.
Manuscript received: 30 October 2013.
Manuscript accepted: 24 January 2014.
Published online: 19 February 2014.
Introduction
report concerning the isocyanate-free synthesis of pyrazolo
[1,2-a]triazole-1,3,5-triones by an MCR methodology. This
encouraged us to develop an environmentally benign synthetic
methodology for pyrazolo[1,2-a]triazole-trione derivatives.
We envisaged that a one-pot three-component synthesis of
pyrazolo[1,2-a]triazole-triones from aromatic aldehydes,
4-phenyl urazole, and a component containing a –CH2CO–
moiety could be handy in the present case. In order to main-
tain the pyrazole ring of pyrazolo[1,2-a]triazole-trione, the
Meldrum’s acid appeared as an appropriate third component
that would eventually allow the construction of the pyrazolo
[1,2-a]triazole-trione framework.
Green chemistry is recognised as a pioneering concept, which
widely reports intrinsic atom economy, energy savings, waste
reduction, easy work ups, and the avoidance of hazardous
chemicals.[1] The development of a simple, eco-friendly reac-
tion protocol for the synthesis of highly functionalized com-
pound libraries of medicinal motifs is an attractive area of
research in both academia and the pharmaceutical industry.[2] In
this context, multicomponent reactions (MCRs)[3] have
emerged as a powerful tool to achieve synthetic efficiency, as
they have unique advantages such as convergence, operational
simplicity, facile automation, one pot reactions, atom and step
economy (PASE),[4] in addition to diminished waste generation.
Heterocyclic compounds occur very widely in nature and are
essential to life. Among a large variety of nitrogen containing
heterocyclic compounds, heterocycles containing a urazole
(1,2,4-triazolidine-3,5-dione) moiety are of interest because
they constitute an important class of natural and non-natural
products, many of which have a myriad of biological functions
such as anticonvulsant,[5] antifungal,[6] herbicidal,[7] hypolipi-
demic,[8] and insecticidal activities.[9] They are also utilised in
the production of anti-tumour drugs[10] and in polymeric mate-
rials.[11] Urazoles have been used as laboratory reagents for the
preparation of novel heterocyclic compounds.[12]
Results and Discussion
Chemistry
We report herein a new, convenient, diversity oriented, and
highly efficient green protocol for the synthesis of novel pyr-
azolo[1,2-a]triazole-1,3,5-triones by the three component con-
densation of aldehydes 1, Meldrum’s acid, and 4-phenylurazole
in the presence of a catalytic amount of glacial AcOH at 808C
under solvent free conditions. The reaction conditions were
optimized using 4-fluorobenzaldehyde, Meldrum’s acid, and
4-phenylurazole in equimolar amounts as a model system. Ini-
tially a control experiment confirmed that the reaction did not
proceed in the absence of a catalyst (entries 1 and 2, Table 1).
The model reaction was then performed in the presence of
different catalysts, such as p-toluene sulfonic acid (p-TSA),
H2SO4, ceric ammonium nitrate (CAN), Et3N, L-proline, La
(OTf)3, and glacial AcOH (10 mol-% each) in refluxing ethanol.
The reported methods for the preparation of pyrazolo
[1,2-a]triazole-1,3,5-trione ring systems involve: (i) reaction
of pyrazolonecarboxylates with isocyanate in the presence
of an equimolar amount of a tertiary amine[13] and (ii) an
ene-type reaction of 4-phenyl-1,2,4-triazoline-3,5-dione and
5-methylfuran-2(3H)-one.[14] As far as we know, there is no
Journal compilation Ó CSIRO 2014
www.publish.csiro.au/journals/ajc

868
P. Saluja et al.
The reactions performed using 10 mol-% of p-TSA, H2SO4,
and CAN as catalysts were found to be incomplete and gave
complex reaction mixtures (entries 3–5, Table 1). React-
ions using Et3N and L-proline led to the formation of
5-(4-fluorobenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione
by Knoevenagel condensation of an aldehyde and Meldrum’s
acid (entries 6 and 7, Table 1). The reaction using La(OTf)3 as
catalyst furnished the desired product 7-(4-fluorophenyl)-2-
phenyldihydropyrazolo[1,2-a][1,2,4]triazole-1,3,5(2H)-trione
(2a) albeit in low yield (entry 8, Table 1). To our delight, glacial
AcOH gave the desired 2a in 66 % yield in 5 h (entry 9, Table 1).
When the reaction was attempted under solvent free conditions,
using glacial AcOH (10 mol-%) as catalyst at 808C, the reaction
was complete in 4 h and yielded 75 % of desired 2a after a simple
work up (entry 10, Table 1). When the reaction was conducted
using 20 mol-% of the glacial AcOH, it required a shorter time
for completion (3.5 h) and yielded 92 % of 2a while upon
increasing the amount of the catalyst to 30 mol-%, no significant
improvement in the time or yield of the product 2a was observed
(entries 11 and 12, Table 1).
The optimized reaction conditions of employing 20 mol-% of
glacial AcOH at 808C was utilised for a variety of substrates to
explore the synthetic scope and generality of this cascade
reaction and to generate a small library of functionalized
pyrazolo[1,2-a]triazole-trione derivatives. Notably, a wide
range of aromatic aldehydes bearing electron-withdrawing as
well as electron-donating groups underwent the reactions
smoothly under the optimized reaction conditions to give
the desired pyrazolo[1,2-a]triazole-triones 2a–q in high
yields (Scheme 1).
Reactions employing aromatic aldehydes with electron-
withdrawing groups were faster and gave higher yields of the
products compared with reactions of aldehydes with electron-
donating groups as the presence of electron-withdrawing
groups on the aryl ring make the arylidene intermediate A
more electrophilic, thereby allowing a facile attack of the
4-phenylurazole on A (Table 2, entries 1–17). Interestingly,
when the catalytic system was applied to terephthaldehyde
(1.0 mmol), Meldrum’s acid (2.0 mmol), and 4-phenylurazole
(2.0 mmol), it led to the synthesis of 2r (Scheme 2) (Table 2,
entry 18). All the products were identified on the basis of IR,
1H NMR, 13C NMR, and mass spectra.
In addition, the structures of the synthesized novel pyrazolo
[1,2-a]triazole-trione derivatives 2 have been confirmed by the
single crystal X-ray diffraction analysis of compound 2m
(Fig. 1). A single crystal of 2m suitable for X-ray diffraction
was obtained by evaporation of CHCl3/hexane solutions at room
temperature. The calculated structure provides accurate molec-
ular shapes, especially in terms of the pyramidal geometry at N1
and N3 and planar geometry at N2. The angle between the two
planes containing the triazole ring and phenyl ring is 83.758
whereas that between the planes of pyrazole and phenyl rings is
61.178. The two five-membered rings deviate very slightly from
planarity as evidenced by the angle between the planes contain-
ing them, i.e. 32.478. All bond lengths and angles are normal and
in agreement with similar compounds.[15]
A mechanistic explanation for the formation of pyrazolo[1,2-
a]triazole-trione derivatives is provided in Scheme 3. The
formation of pyrazolo[1,2-a]triazole-trione derivatives 2 can
be rationalized by initial formation of arylidene Meldrum’s acid
A by the Knoevenagel condensation between an aldehyde 1 and
Table 1. Optimization of reaction conditions for the synthesis of
7-(4-fluorophenyl)-2- phenyldihydropyrazolo[1,2-a][1,2,4]triazole-1,3,5
(2H)-trione (2a)A
Table 2. Synthesis of pyrazolo[1,2-a]triazole-triones 2a r using
]
glacial AcOH at 808C
Entry Catalyst
Catalyst
[mol-%]
Solvent Time Temp. Yield 2a
[h]
[8C]
[%]
Entry
Product
Ar [ArCHO]
Time [h]
Yield [%]
B
1
None
–
H2O
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
–
24
100
80
80
80
80
80
80
80
80
80
80
80
–
1
2a
2b
2c
2d
2e
2f
4-FC6H4
3.5
4
92
85
80
87
83
79
76
81
78
90
86
81
82
85
79
77
79
84
B
2
None
–
24
8
–
2
4-ClC6H4
C
3
p-TSA
10
10
10
10
10
10
10
10
20
30
–
3
3,4,5-(CH3O)3C6H2
4-(CN)C6H4
3-ClC6H4
4
C
4
H2SO4
8
–
4
4.5
4
C
5
CAN
8
–
5
D
6
Et3N
24
24
5
–
6
2,4-Cl2C6H3
3-(NO2)C6H4
C6H5
4
D
7
L-Proline
La(OTf)3
Gl. AcOH
Gl. AcOH
Gl. AcOH
Gl. AcOH
–
7
2g
2h
2i
5
8
43
66
75
92
93
8
4
9
5
9
4-CH3C6H4
3-FC6H4
5
10
11
12
4
10
11
12
13
14
15
16
17
18
2j
3.5
4
–
3.5
3.5
2k
2l
2-FC6H4
–
2-(F3C)C6H4
2-ClC6H4
4
2m
2n
2o
2p
2q
2r
4
AReaction of 4-fluorobenzaldehyde (2a, 1.0 mmol), Meldrum’s acid
(1.0 mmol), and 4-phenylurazole (1.0 mmol).
3-(F3C)C6H4
4-(F3C)C6H4
4-(Me2CH)C6H4
2-(Br),4-(F)C6H3
4-(OHC)C6H4
3.5
3.5
4.5
3.5
4.5
BNo reaction.
CIncomplete reaction with a number of spots observed by TLC including
arylidene Meldrum’s acid.
DThe product formed was identified as arylidene Meldrum’s acid.
O
O
O
O
O
N
N
Gl. AcOH, 80°C
3.5–5h
HN
HN
O
N
N
ϩ
ϩ
ArCHO
O
Ar
O
O
2a–q
Scheme 1.

Antimicrobial Pyrazolotriazoletriones
869
O
O
CHO
O
O
O
N
N
N
N
N
N
Gl. AcOH, 80°C
4.5h
HN
HN
O
2
2
ϩ
O
ϩ
1
O
O
N
O
O
O
CHO
2r
Scheme 2.
C15
C16
C17
among all the pyrazolo[1,2-a]triazole-triones, e.g. 2,4-dichloro,
4-F, 2-F, 4-CF3, 2-Br, 4-F, and 4-H.
C8
C7
O1
C1
Of the 18 chemical compounds screened for their antifungal
activity, two compounds, namely 2f and 2h showed more than
50 % inhibition of mycelial growth against A. niger and four
compounds, 2f, 2h, 2o, and 2q showed more than 50 % inhi-
bition of mycelial growth against A. flavus (Table 5). The
compound with an unsubstituted phenyl ring, 2h, was found
to be most effective with a maximum growth inhibition index,
although compounds with halogen substitution at the 2 or 4
position also exhibited higher activity, e.g. 2,4-dichloro, 4-F3C,
2-Br, and 4-F.
C9
C14
N1
C10
C6
C13
C12
C11
N2
C5
C2
Cl1
N3
O2
C4
C3
O3
Fig. 1. X-ray crystallographic structure of 2m.
Among all the tested chemical compounds, compound 2f
showed good antibacterial and antifungal activity. Therefore,
this compound can be further explored for its toxicity.
Meldrum’s acid. Subsequently 4-phenylurazole undergoes a
Michael-type addition to arylidene Meldrum’s acid A to give
intermediate B which undergoes cyclization with concomitant
loss of acetone and carbon dioxide to afford the desired
compound 2. The proposed reaction pathway was confirmed
by preparing 4-chlorobenzylidene Meldrum’s acid indepen-
dently and then carrying out its reaction with 4-phenylurazole
in the presence of glacial acetic acid which led to the formation
of the desired product 2b, thus supporting the pathway.
Conclusion
In conclusion, we have reported a new multicomponent reaction
for the synthesis of novel pyrazolo[1,2-a]triazole-triones, by
reaction of aldehydes, Meldrum’s acid, and 4-phenylurazole.
Mild reaction conditions and high isolated yields are the
remarkable advantages of this protocol. These derivatives
were evaluated for their antimicrobial activities, of which, some
have shown good antibacterial and antifungal activities.
Experimental
Pharmacology
All the chemicals were purchased from Sigma–Aldrich or
Merck and were used as received. Thin-layer chromatography
(GF254) was used to monitor reaction progress. Melting points
were determined on a Tropical labequip apparatus and were
uncorrected. IR (KBr) spectra were recorded on a Perkin–Elmer
FTIR spectrophotometer and the values are expressed as nmax in
cmꢀ1. Mass spectra were recorded on a Waters LCT Micromass
spectrometer. The 1H, 19F, and 13C NMR spectra were recorded
on a Jeol JNM ECX-400P spectrometer at 400, 376, and
100 MHz, respectively, using TMS as an internal standard. The
chemical shift values are recorded on a d scale and the coupling
constants (J) are in Hz. X-Ray intensity data were collected on
an Oxford Diffraction Xcalibur CCD diffractometer with
A total of 18 novel pyrazolo[1,2-a]triazole-triones 2a–r were
screened for their antibacterial and antifungal activity. The
tested chemical compounds possessed variable antibacterial
activity against Gram-positive (Staphylococcus aureus, Bacil-
lus subtilis) bacteria and antifungal activity against Aspergillus
niger and A. flavus. However, the tested chemical compounds
did not exhibit any activity against Gram-negative bacteria.
Positive controls produced significantly sized inhibition zones
against the tested bacteria and fungi. However, the negative
control produced no observable inhibitory effect against any of
the test organisms.
All the tested compounds showed a zone of inhibition
ranging between 14 and 20 mm against the bacteria. On the
basis of the zone of inhibition produced against the test
bacterium, compound 2f was found to be most effective against
B.subtilis and S. aureus, with a zone of inhibition of 20.3 and
18.3 mm, respectively. However other tested compounds
showed moderate antibacterial activity (Table 3).
˚
graphite monochromated MoKa radiation (l 0.71073 A).
Data Collection and Refinement
The intensity data for compound 2m was collected on an Oxford
Xcalibur CCD diffractometer equipped with graphite mono-
´
˚
chromatic MoKa radiation (l 0.71073 A) at 298(2) K. A multi-
The minimal inhibitory concentration (MIC) of all the
compounds 2a–r ranged between 64 and 256 mg mLꢀ1 against
the tested bacteria. Compound 2f, having 2,4-dichloro substitu-
tion, was found to be best as it exhibited the lowest MIC of
64 mg mLꢀ1 against S. aureus. Six compounds, namely 2a, 2f,
2h, 2k, 2o, and 2q showed the lowest MICs of 64 mg mLꢀ1
against B.subtilis (Table 4). A further perusal of activity data
revealed that compounds with halogen substitution at the 2 or 4
positions and an unsubstituted phenyl ring were more active
scan absorption correction was applied. The structure was
solved by direct methods and refined by full-matrix least-
squares refinement techniques on F2 using SHELXL-97.[16] The
coordinates of non-hydrogen atoms were refined anisotropically
using SHELXL-97. The positions of hydrogen atoms were
obtained from difference Fourier maps and were included in
the final cycles of refinement. All calculations were done
using the Wingx software package.[17] Complete crystallo-
graphic data (excluding factors) of 2m have been deposited at

870
P. Saluja et al.
O
N
HN
HN
Ph
ϩ
OH
O
Ar
H
O
OH
Ar
Ar
O
H
O
O
O
O
ϪH2O
O
O
Gl. AcOH
O
O
O
O
OH
O
O
O
A
O
Ar
Ar
O
N
O
O
N
N
O
N
N
Ph
Ph
N
H
ϪCH3COCH3
ϪCO2
O
O
O
O
H
2
ϪHϩ
B
Scheme 3. Proposed reaction pathway for the synthesis of 2.
Table 3. Antibacterial activity of compounds 2a r through agar well
]
diffusion method
Table 4. Minimum inhibitory concentration of compounds 2a r by
]
using a modified agar well diffusion method
Minimum inhibitory concentration [mg mLꢀ1
]
Compound
Diameter of growth of inhibition zoneA [mm]
Compound
Staphylococcus aureus
Bacillus subtilis
Staphylococcus aureus
Bacillus subtilis
2a
16.3
15.0
16.3
15.3
16.3
18.3
15.3
17.6
14.6
14.3
14.6
14.6
14.3
15.6
17.6
15.3
16.6
15.3
26.6
17.6
15.6
17.0
16.3
17.6
20.3
17.3
18.6
15.6
15.3
15.6
15.3
15.6
16.6
18.6
16.3
18.6
17.3
24.0
2a
128
256
128
128
128
64
64
128
128
128
128
64
2b
2b
2c
2c
2d
2d
2e
2e
2f
2f
2g
2g
128
128
256
256
256
256
256
128
64
128
64
2h
2h
2i
2i
128
128
64
2j
2j
2k
2k
2l
2l
128
128
128
64
2m
2m
2n
2n
2o
2o
2p
2p
128
128
128
6.25
128
64
2q
2q
2r
2r
128
Ciprofloxacin
Ciprofloxacin
6.25
AValues, including diameter of the well (8 mm), are means of three
replicates.
(20 mol-%) was placed in a 25 mL round-bottomed flask and the
contents were stirred magnetically in an oil-bath maintained
at 808C for an appropriate time as indicated in Table 2.
The progress of the reaction was monitored by TLC (eluent:
methanol–chloroform, 10 : 90 v/v). After completion of the
reaction, the reaction mixture was allowed to cool at room
temperature and diluted with cold H2O. A solid eventually
separated out. The solid was filtered under vacuum, and washed
with water to remove residual acetic acid. The crude product so
obtained was recrystallized from CHCl3/hexane (80 : 20, v/v) to
afford the pure product 2. The products were characterized by
the Cambridge Crystallographic Data Centre under number
CCDC 936196.
Crystal Data
C17H12ClN3O3, M 341.75, orthorhombic, space group P
21 21 21, a 6.0132(4), b 11.8864(7), c 23.1834(17), V 1657.04
´
3
(19) A , Z 4, F(000) 704, DC 1.370 mg mꢀ3, l 0.71073 A,
absorption coefficient 0.250 mmꢀ1. Selected bond angles: C(1)–
N(1)–N(3), 107.8(4); C(1)–N(1)–C(5), 124.2(4); N(3)–N(1)–C
(5), 110.3(4); C(2)–N(2)–C(1), 112.3(4); C(2)–N(2)–C(6),
123.1(4); C(1)–N(2)–C(6), 122.9(4); C(2)–N(3)–C(3), 126.5(4);
C(2)–N(3)–N(1), 108.1(3); and C(3)–N(3)–N(1), 110.4(4).
˚
˚
1
IR, H NMR, 13C NMR, 19F NMR, and mass spectra and an
X-ray crystallographic study.
Reaction of terephthaldehyde (1r, 1.0 equiv.) required
2.0 equiv. of Meldrum’s acid and 2.0 equiv. of 4-phenylurazole
for completion to give the bis-condensation product, 7,70-(1,4-
phenylene)bis(2-phenyldihydropyrazolo[1,2-a][1,2,4]triazole-
1,3,5(2H)-trione) (2r).
General Procedure for the Synthesis of Pyrazolo[1,2-a]
triazole-trione Derivatives 2a–q
A mixture of aryl aldehyde 1 (1.0 mmol), Meldrum’s acid
(1.0 mmol), 4-phenylurazole (1.0 mmol), and glacial AcOH

Antimicrobial Pyrazolotriazoletriones
871
Table 5. Antifungal activity of compounds 2a r through poisoned
]
food method
4-(1,3,7-Trioxo-2-phenylhexahydropyrazolo[1,2-a]
[1,2,4]triazol-5-yl)benzonitrile (2d)
White solid. Yield: 87 %. Mp 165–1668C. nmax (KBr)/cmꢀ1
2925, 1752, 1720. dH (400 MHz, CDCl3) 7.73 (d, J 8.08, 2H,
ArH), 7.56 (d, J 7.36, 2H, ArH), 7.45–7.42 (m, 5H, ArH), 5.51
(t, J 8.80, 1H, CH), 3.61 (dd, J 9.52, 18.32, 1H, CHHCO), 3.14
(dd, J 8.04, 18.32, 1H, CHHCO). dC (100 MHz, CDCl3) 162.74,
150.93, 144.03, 141.95, 133.19, 129.43, 129.12, 126.97, 125.54,
118.04, 113.27, 57.96, 42.71. m/z (ESI) 355.14 ([M þ Na]þ).
Anal. Calc. for C18H12N4O3 332.09.
Compound
Mycelial growth inhibition [%]
Aspergillus niger
Aspergillus flavus
2a
47.7
35.5
44.4
41.1
43.3
51.1
41.1
53.3
43.3
38.8
35.5
37.7
34.4
35.5
46.6
38.8
48.8
42.2
81.1
48.8
37.7
48.8
43.3
45.5
53.3
43.3
57.7
45.5
41.1
37.7
42.2
37.7
38.8
51.1
42.2
53.3
46.6
77.7
2b
2c
2d
2e
2f
2g
7-(3-Chlorophenyl)-2-phenyldihydropyrazolo[1,2-a]
[1,2,4]triazole-1,3,5(2H)-trione (2e)
2h
2i
White solid. Yield: 83 %. Mp 115–1168C. nmax (KBr)/cmꢀ1
2929, 1749, 1718. dH (400 MHz, CDCl3) 7.43 (s, 5H, ArH), 7.36
(s, 3H, ArH), 7.30 (s, 1H, ArH), 5.42 (t, J 8.40, 1H, CH), 3.57
(dd, J 9.52, 17.60, 1H, CHHCO), 3.15 (dd, J 6.60, 17.60, 1H,
CHHCO). dC (100 MHz, CDCl3) 163.36, 150.80, 143.99,
134.72, 131.82, 130.35, 130.27, 129.33, 128.91, 127.77,
127.23, 125.60, 55.98, 41.91. m/z (ESI) 364.12 ([M þ Na]þ),
366.11 ([Mþ Na þ 2]þ). Anal. Calc. for C17H12ClN3O3 341.06.
2j
2k
2l
2m
2n
2o
2p
2q
2r
Fluconazole
7-(2,4-Dichlorophenyl)-2-phenyldihydropyrazolo[1,2-
a][1,2,4]triazole-1,3,5(2H)-trione (2f)
White solid. Yield: 79 %. Mp 128–1308C. nmax (KBr)/cmꢀ1
2942, 1736, 1717. dH (400 MHz, CDCl3) 7.53 (d, J 8.08, 1H,
ArH), 7.47–7.44 (m, 5H, ArH), 7.42–7.40 (m, 1H, ArH), 7.37–
7.34 (m, 1H, ArH), 5.73 (t, J 8.04, 1H, CH), 3.76 (dd, J 9.52,
18.32, 1H, CHHCO), 3.06 (dd, J 8.04, 18.32, 1H, CHHCO).
dC (100 MHz, CDCl3) 162.91, 150.98, 143.83, 135.71, 133.33,
132.58, 130.25, 130.17, 129.40, 129.04, 128.21, 128.14,
125.57, 55.71, 41.72. m/z (ESI) 398.00 ([M þ Na]þ), 400.00
([Mþ Na þ 2]þ). Anal. Calc. for C17H11Cl2N3O3 375.02.
Spectroscopic Data of Compounds 2a–r
7-(4-Fluorophenyl)-2-phenyldihydropyrazolo[1,2-a]
[1,2,4]triazole-1,3,5(2H)-trione (2a)
White solid. Yield: 92 %. Mp 171–1738C. nmax (KBr)/cmꢀ1
2929, 1751, 1719. dH (400 MHz, CDCl3) 7.46–7.40 (m, 7H,
ArH), 7.14 (t, J 8.8, 2H, ArH), 5.45 (t, J 8.04, 1H, CH), 3.58
(dd, J 9.52, 18.32, 1H, CHHCO), 3.17 (dd, J 7.32, 17.56, 1H,
CHHCO). dF (376 MHz, DMSO) ꢀ114.13 (s, 1F, ArF).
dC (100 MHz, CDCl3) 163.05, 151.09, 143.66, 132.56, 130.23,
129.35, 128.95, 128.13 (d, 3JC–F 7.63), 125.56, 116.46 (d, 2JC–F
21.93), 58.01, 43.08. m/z (ESI) 348.50 ([M þ Na]þ). Anal. Calc.
for C17H12FN3O3 325.09.
7-(3-Nitrophenyl)-2-phenyldihydropyrazolo[1,2-a]
[1,2,4]triazole-1,3,5(2H)-trione (2g)
White solid. Yield: 76 %. Mp 188–1908C. nmax (KBr)/cmꢀ1
2927, 1760, 1723, 1417. dH (400 MHz, CDCl3) 8.28 (s, 1H,
ArH), 8.22 (d, J 8.24, 1H, ArH), 7.76 (d, J 7.8, 1H, ArH), 7.63
(t, J 8.24, 1H, ArH), 7.41 (m, 5H, ArH), 5.55 (t, J 8.92, 1H, CH),
3.57 (dd, J 9.64, 17.88, 1H, CHHCO), 3.11 (dd, J 8.68, 18.32,
1H, CHHCO). dC (100 MHz, CDCl3) 163.39, 152.06, 148.67,
144.30, 139.37, 132.21, 130.51, 130.10, 129.39, 129.08, 125.63,
124.00, 121.44, 57.79, 42.87. m/z (ESI) 375.09 ([M þ Na]þ).
Anal. Calc. for C17H12N4O5 352.08.
7-(4-Chlorophenyl)-2-phenyldihydropyrazolo[1,2-a]
[1,2,4]triazole-1,3,5(2H)-trione (2b)
White solid. Yield: 85 %. Mp 151–1538C. nmax (KBr)/cmꢀ1
2929, 1752, 1685. dH (400 MHz, CDCl3) 8.01 (d, J 8.04, 1H,
ArH), 7.45–7.38 (m, 8H, ArH), 5.44 (t, J 8.8, 1H, CH), 3.60
(dd, J 9.52, 18.32, 1H, CHHCO), 3.18 (dd, J 8.08, 18.32, 1H,
CHHCO). dC (100 MHz, CDCl3 þ DMSO) 171.03, 152.47,
135.34, 133.35, 130.93, 128.54, 128.23, 128.03, 127.17, 125.14,
124.90, 55.61, 35.89. m/z (ESI) 364.12 ([M þ Na]þ), 366.12
([M þ Na þ 2]þ). Anal. Calc. for C17H12ClN3O3 341.06.
2,7-Diphenyldihydropyrazolo[1,2-a][1,2,4]triazole-
1,3,5(2H)-trione (2h)
White solid. Yield: 81 %. Mp 166–1688C. nmax (KBr)/cmꢀ1
2940, 1739, 1696. dH (400 MHz, CDCl3) 7.39 (s, 10H, ArH),
5.40 (s, 1H, CH), 3.56 (s, 1H, CHHCO), 3.16 (s, 1H, CHHCO).
dC (100 MHz, CDCl3 þ DMSO) 164.42, 152.47, 144.52, 137.61,
129.74, 127.77, 127.68, 127.52, 127.26, 127.12, 126.24, 125.02,
124.73, 124.37, 57.05, 41.91. m/z (ESI) 330.23 ([M þ Na]þ).
Anal. Calc. for C17H13N3O3 307.10.
2-Phenyl-7-(3,4,5-trimethoxyphenyl)dihydropyrazolo
[1,2-a][1,2,4]triazole-1,3,5(2H)-trione (2c)
White solid. Yield: 80 %. Mp 230–2328C. nmax (KBr)/cmꢀ1
2937, 1746, 1720. dH (400 MHz, CDCl3) 7.41 (s, 5H, ArH), 6.60
(s, 2H, ArH), 5.39 (t, J 8.04, 1H, CH), 3.85 (s, 6H, OCH3), 3.82
(s, 3H, OCH3), 3.55 (dd, J 9.52, 18.32, 1H, CHHCO), 3.15 (dd,
J 7.32, 17.56, 1H, CHHCO). dC (100 MHz, CDCl3 þ DMSO)
166.00, 153.21, 153.05, 145.58, 137.10, 134.78, 130.95, 129.00,
128.50, 126.42, 103.95, 59.91, 58.39, 55.95, 42.73. m/z (ESI)
420.05 ([M þ Na]þ). Anal. Calc. for C20H19N3O6 397.13.
2-Phenyl-7-(4-methylphenyl)dihydropyrazolo[1,2-a]
[1,2,4]triazole-1,3,5(2H)-trione (2i)
White solid. Yield: 78 %. Mp 200–2028C. nmax (KBr)/cmꢀ1
2925, 1752, 1720, 759. dH (400 MHz, CDCl3) 7.45 (s, 4H, ArH),
7.40–7.31 (m, 1H, ArH), 7.25 (s, 4H, ArH), 5.44 (s, 1H, CH),
3.57 (br s, 1H, CHHCO), 3.2 (br s, 1H, CHHCO) 2.34 (s, 3H,

872
P. Saluja et al.
CH3). dC (100 MHz, CDCl3 þ DMSO) 163.85, 152.92, 151.24,
138.98, 134.08, 129.87, 129.30, 129.13, 128.84, 128.64, 127.40,
126.00, 125.51, 58.27, 43.07, 21.06. m/z (ESI) 344.37 ([M þ
Na]þ). Anal. Calc. for C18H15N3O3 321.11.
2-Phenyl-7-(4-(trifluoromethyl)phenyl)dihydropyrazolo
[1,2-a][1,2,4]triazole-1,3,5(2H)-trione (2o)
White solid. Yield: 79 %. Mp 138–1408C. nmax (KBr)/cmꢀ1
2925, 1669, 772. dH (400 MHz, CDCl3) 7.68 (s, 2H, ArH), 7.56
(s, 2H, ArH), 7.42 (s, 5H, ArH), 5.59 (s, 1H, CH), 3.65 (s, 1H,
CHHCO), 3.11 (s, 1H, CHHCO). dF (376 MHz, DMSO) ꢀ62.66
(s, 3F, ArCF3). dC (100 MHz, CDCl3) 163.39, 151.53, 144.07,
141.05, 130.18, 129.39, 129.00, 126.78, 126.42, 125.85,
58.40, 43.57. m/z (ESI) 398.46 ([M þ Na]þ). Anal. Calc. for
C18H12F3N3O3 375.08.
7-(3-Fluorophenyl)-2-phenyldihydropyrazolo[1,2-a]
[1,2,4]triazole-1,3,5(2H)-trione (2j)
White solid. Yield: 90 %. Mp 153–1558C. nmax (KBr)/cmꢀ1
2929, 1758, 1718. dH (400 MHz, CDCl3) 7.43–7.32 (m, 6H,
ArH), 7.16–7.09 (m, 2H, ArH), 7.06–7.02 (m, 1H, ArH), 5.41
(t, J 8.08, 1H, CH), 3.56 (dd, J 9.52, 18.32, 1H, CHHCO), 3.13
(dd, J 8.08, 18.32, 1H, CHHCO). dC (100 MHz, CDCl3) 162.90,
7-(4-Isopropylphenyl)-2-phenyldihydropyrazolo[1,2-a]
[1,2,4]triazole-1,3,5(2H)-trione (2p)
3
3
151.13, 143.69, 139.30 (d, JC–F 6.68), 131.18 (d, JC–F 8.59),
130.21, 129.37, 128.99, 125.58, 121.67, 116.32 (d, 2JC–F 20.98),
113.38 (d, 2JC–F 22.89), 57.84, 42.99. m/z (ESI) 348.17 ([M þ
Na]þ). Anal. Calc. for C17H12FN3O3 325.09.
White solid. Yield: 77 %. Mp 175–1778C. nmax (KBr)/cmꢀ1
2961, 1706, 1677. dH (400 MHz, CDCl3) 7.46–7.43 (m, 4H,
ArH), 7.37–7.32 (m, 2H, ArH), 7.20 (s, 3H, ArH), 5.74 (t, J 5.16,
1H, CH), 3.32 (dd, J 6.60, 17.56, 1H, CHHCO), 3.12 (dd, J 5.12,
17.60, 1H, CHHCO), 2.92–2.85 (m, 1H, CH(CH3)2), 1.23 (d, J
7.32, 6H, CH3). dC (100 MHz, CDCl3) 175.57, 153.74, 152.13,
149.41, 132.68, 130.91, 129.20, 128.49, 127.31, 127.07, 125.72,
55.38, 36.84, 33.75, 23.84. m/z (ESI) 350.39 ([M þ H]þ). Anal.
Calc. for C20H19N3O3 349.14.
7-(2-Fluorophenyl)-2-phenyldihydropyrazolo[1,2-a]
[1,2,4]triazole-1,3,5(2H)-trione (2k)
White solid. Yield: 86 %. Mp 178–1808C. nmax (KBr)/cmꢀ1
2925, 1752, 1720. dH (400 MHz, DMSO) 7.70 (t, J 7.32, 1H,
ArH), 7.51 (d, J 6.88, 2H, ArH), 7.46–7.44 (m, 4H, ArH), 7.29
(t, J 8.24, 2H, ArH), 5.62 (t, J 8.72, 1H, CH), 3.62 (dd, J 10.08,
18.32, 1H, CHHCO), 3.24 (dd, J 8.24, 17.88, 1H, CHHCO).
dC (100 MHz, DMSO) 166.05, 158.41, 153.28, 145.63, 130.84,
130.50 (d, 3JC–F 7.62), 129.11, 128.81 (d, 2JC–F 20.97), 126.57,
126.07 (d, 3JC–F 11.45), 124.89, 115.75 (d, 2JC–F 20.02) 52.96,
41.25. m/z (ESI) 348.17 ([M þ Na]þ). Anal. Calc. for
C17H12FN3O3 325.09.
7-(2-Bromo-4-fluorophenyl)-2-phenyldihydropyrazolo
[1,2-a][1,2,4]triazole-1,3,5(2H)-trione (2q)
White solid. Yield: 79 %. Mp 224–2268C. nmax (KBr)/cmꢀ1
2926, 1764, 1720. dH (400 MHz, CDCl3) 7.58 (s, 1H, ArH), 7.48
(s, 4H, ArH), 7.42 (s, 1H, ArH), 7.36–7.34 (m, 1H, ArH), 7.70
(s, 1H, ArH), 5.71 (t, J 8.08, 1H, CH), 3.82 (dd, J 9.52, 18.32,
1H, CHHCO), 3.04 (dd, J 8.08, 18.32, 1H, CHHCO). dC
(100 MHz, CDCl3) 162.76, 161.10, 151.17, 143.87, 135.01
2-Phenyl-7-(2-(trifluoromethyl)phenyl)dihydropyrazolo
[1,2-a][1,2,4]triazole-1,3,5(2H)-trione (2l)
3
White solid. Yield: 81 %. Mp 232–2348C. nmax (KBr)/cmꢀ1
2928, 1752, 1728. dH (400 MHz, CDCl3) 7.84–7.82 (m, 1H,
ArH), 7.72–7.69 (m, 2H, ArH), 7.57–7.47 (m, 6H, ArH), 5.86
(t, J 8.04, 1H, CH), 3.65 (dd, J 9.52, 18.32, 1H, CHHCO), 3.07
(dd, J 7.32, 18.32, 1H, CHHCO). dC (100 MHz, CDCl3) 162.70,
151.01, 143.75, 142.21, 136.14, 133.32, 132.15, 130.20, 129.37,
129.14, 129.00, 128.02, 126.61, 125.53, 54.84, 44.04. m/z (ESI)
398.21 ([M þ Na]þ). Anal. Calc. for C18H12F3N3O3 375.08.
(d, JC–F 8.59), 130.14, 129.43, 129.10, 125.61, 117.75
2
(d, JC–F 22.88), 114.85, 114.54, 57.66, 41.81. m/z (ESI)
425.96 ([M þ Na]þ). Anal. Calc. for C17H11BrFN3O3 403.00.
7,70-(1,4-Phenylene)bis(2-phenyldihydropyrazolo
[1,2-a][1,2,4]triazole-1,3,5(2H)-trione) (2r)
White solid. Yield: 84 %. Mp 154–1568C. nmax (KBr)/cmꢀ1
2929, 1752, 1720. dH (400 MHz, CDCl3) 7.53 (s, 2H, ArH),
7.48–7.38 (m, 12H, ArH), 5.51 (t, J 9.52, 2H, CH), 3.61–3.54
(m, 2H, CHHCO), 3.21–3.14 (m, 2H, CHHCO). dC (100 MHz,
CDCl3 þ DMSO) 171.02, 152.25, 136.95, 130.96, 129.17,
128.11, 127.13, 128.84, 55.64, 35.80. m/z (ESI) 559.87 ([M þ
Na]þ). Anal. Calc. for C28H20N6O6 536.14.
7-(2-Chlorophenyl)-2-phenyldihydropyrazolo[1,2-a]
[1,2,4]triazole-1,3,5(2H)-trione (2m)
White solid. Yield: 82 %. Mp 170–1728C. nmax (KBr)/cmꢀ1
2927, 1752, 1719. dH (400 MHz, CDCl3) 7.56 (d, J 7.32, 1H,
ArH), 7.46–7.31 (m, 8H, ArH), 5.78 (t, J 8.24, 1H, CH), 3.78
(dd, J 10.08, 18.76, 1H, CHHCO), 3.09 (dd, J 7.80, 18.80, 1H,
CHHCO). dC (100 MHz, CDCl3) 163.22, 150.69, 143.88,
134.66, 131.84, 130.39, 130.30, 129.35, 128.94, 127.80,
127.23, 125.60, 55.98, 41.91. m/z (ESI) 364.18 ([M þ Na]þ),
366.18 ([M þ Na þ 2]þ). Anal. Calc. for C17H12ClN3O3 341.06.
Antimicrobial Assay
Test Microorganisms
A total of six microbial strains were selected on the basis of
their clinical importance in causing diseases in humans. Two
Gram-positive bacteria (Staphylococcus aureus MTCC 96 and
Bacillus subtilis MTCC 121), two Gram-negative bacteria
(Escherichia coli MTCC 1652 and Pseudomonas aeruginosa
MTCC 741), and two fungi, Aspergillus niger and A. flavus, the
ear pathogens isolated from the patients of Kurukshetra,[18] were
used in the present study for evaluation of antimicrobial activity
of the chemical compounds. All the bacterial cultures were
procured from Microbial Type Culture Collection (MTCC),
IMTECH, Chandigarh. The bacteria were subcultured on
Nutrient agar whereas fungi were grown on Sabouraud’s
dextrose agar.
2-Phenyl-7-(3-(trifluoromethyl)phenyl)dihydropyrazolo
[1,2-a][1,2,4]triazole-1,3,5(2H)-trione (2n)
White solid. Yield: 85 %. Mp 154–1568C. nmax (KBr)/cmꢀ1
2927, 1762. dH (400 MHz, CDCl3) 7.67–7.54 (m, 4H, ArH),
7.43–7.38 (m, 5H, ArH), 5.51 (t, J 8.92, 1H, CH), 3.59 (dd, J
9.64, 18.36, 1H, CHHCO), 3.14 (dd, J 8.24, 18.32, 1H,
CHHCO). dC (100 MHz, CDCl3) 163.13, 151.63, 143.96,
138.17, 130.17, 130.02, 129.49, 129.38, 129.01, 126.07,
125.59, 123.11, 58.04, 43.01. m/z (ESI) 398.09 ([M þ Na]þ).
Anal. Calc. for C18H12F3N3O3 375.08.

Antimicrobial Pyrazolotriazoletriones
873
Antibacterial Activity
Supplementary Material
1
Copies of H, 19F, and 13C NMR spectra are available on the
Journal’s website.
The antibacterial activity of 18 chemical compounds
was evaluated by the agar well diffusion method. All the
microbial cultures were adjusted to 0.5 McFarland standard,
which is visually comparable to a microbial suspension of
,1.5 ꢁ 106 cfu mLꢀ1. Mueller Hinton agar medium (20 mL)
was poured into each Petri plate and plates were swabbed with a
100 mL inocula of the test microorganisms and kept for 15 min
for adsorption. Using a sterile cork borer of 8 mm diameter,
wells were bored into the seeded agar plates and these were
loaded with 100 mL of 2.0 mg mLꢀ1 of each compound recon-
stituted in dimethyl sulfoxide (DMSO). All the plates were
incubated at 378C for 24 h. The antibacterial activity of each
compound was evaluated by measuring the zone of growth
inhibition against the test organisms with a zone reader
(HiAntibiotic zone scale). DMSO was used as a negative control
whereas Ciprofloxacin was used as a positive control. This
procedure was performed in three replicate plates for each
organism and the mean values of the diameter of inhibition
zones ꢂ standard deviations were calculated.[19]
Acknowledgements
P.S. is thankful to UGC, New Delhi, India for the award of Junior and
Senior Research Fellowship.
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