Synthesis of some new 3,5-diamino-4-(4′-fluorophenylazo)-1-aryl/ heteroarylpyrazoles as antimicrobial agents

Article abstract of DOI:10.1007/s00044-012-0343-0

Synthesis of some new 3,5-diamino-4-(4′-fluorophenylazo)-1-aryl/ heteroarylpyrazoles (5a-5i) was achieved by the treatment of aryl/heteroarylhydrazines 4a-4i with 2-[(4-fluorophenyl)hydrazono]malononitrile 3 in refluxing ethanol. The structure of the compounds was established on the basis of IR, NMR (¹H and ¹³C) and mass spectral studies. All the nine synthesized compounds were screened for their in vitro antimicrobial activity against two Gram-positive (Staphylococcus aureus, Bacillus subtilis), two Gram-negative bacteria (Escherichia coli, Klebesiella aerogenes) and three pathogenic fungi (Aspergillus niger, Candida albicans, and Saccharomyces cerevisiae).

Full text of DOI:10.1007/s00044-012-0343-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0343-0
ORIGINAL RESEARCH
Synthesis of some new 3,5-diamino-4-(40-fluorophenylazo)-1-aryl/
heteroarylpyrazoles as antimicrobial agents
•
Ranjana Aggarwal Virender Kumar
•
•
Girish Kumar Gupta Vinod Kumar
Received: 8 August 2012 / Accepted: 7 November 2012
ꢀ Springer Science+Business Media New York 2012
Abstract Synthesis of some new 3,5-diamino-4-(40-fluor-
ophenylazo)-1-aryl/heteroarylpyrazoles (5a–5i) was achieved
by the treatment of aryl/heteroarylhydrazines 4a–4i with
2-[(4-fluorophenyl)hydrazono]malononitrile 3 in refluxing
ethanol. Thestructure of the compounds wasestablishedon the
basis of IR, NMR (1H and 13C) and mass spectral studies. All
the nine synthesized compounds were screened for their
in vitro antimicrobial activity against two Gram-positive
(Staphylococcusaureus, Bacillussubtilis), two Gram-negative
bacteria (Escherichia coli, Klebesiella aerogenes) and three
pathogenic fungi (Aspergillus niger, Candida albicans, and
Saccharomyces cerevisiae).
compounds. Aminopyrazole system, in particular, has
received considerable attention in recent years because of
its applications in pharmaceutical and agrochemical
industries. Aminopyrazole derivatives display different
kind of biological activities such as antibacterial (Aggarwal
et al., 2006; Kumar et al., 2005), antitumor (Samanta et al.,
2006) and act as highly potent agonists of GPR109b
(Skinner et al., 2009), selective inhibitors of MK2 (Velc-
icky et al., 2010) and p38a MAP kinase (Das et al., 2010).
Moreover, they have been successfully employed as active
synthons in the synthesis of condensed heterocyclic prod-
ucts such as pyrazolo[3,4-d]pyrimidines, pyrazolo[1,5-
a]pyrimidines, pyrazolo[3,4-b]pyridines, etc. which exhibit
antiproliferative activity (Schenone et al., 2004), antimi-
crobial (Aggarwal et al., 2011), antiviral activity (Johns
et al., 2007) and are found to be inhibitors of protein kinase
B/Akt (Zhu et al., 2007) and PDE4B (Mitchell et al.,
2010). It is well documented that incorporation of azo and
hydrazono group has been reported to enhance the bio-
logical activity of heterocyclic compounds (Raman and
Ravindernath, 1999). Compounds containing azo- and
hydrazo-groups have been found to display many diverse
types of biological properties such as antimicrobial (Shah
et al., 2011), antifungal (Xu and Zeng, 2010), anticonvul-
sant (Kucukguzel et al., 2000), antioxidant and antitumor
activity (Farghaly and Abdalla, 2009). Arylazopyrazoles
are found to possess antioxidant as well as antibacterial
activity (ManojKumar et al., 2009). Recently, a series of
4-arylazo-3,5-diamino-1H-pyrazoles derivatives have been
reported as novel groups of ATP antagonists with moderate
potency against CDK2-Cyclin E (Krystof et al., 2006). In
view of these observations and in continuation of our work
related to the synthesis, spectral and biological studies of
aminopyrazoles (Aggarwal et al., 2006; Kumar et al.,
2005), it was envisaged to undertake the synthesis and
Keywords
2-[(4-Fluorophenyl)hydrazono]malononitrile ꢀ
Aryl/heteroarylhydrazines ꢀ
3,5-Diamino-4-(40-fluorophenylazo)-1-aryl/
heteroarylpyrazoles ꢀ Antimicrobial activity
Introduction
The pyrazole ring has been widely employed in the
investigation of pharmacologically active heterocyclic
R. Aggarwal (&) ꢀ V. Kumar
Department of Chemistry, Kurukshetra University,
Kurukshetra 136119, India
e-mail: ranjana67in@yahoo.com;
ranjanaaggarwal67@gmail.com
G. K. Gupta
Department of Pharmacy, Maharishi Markandeshwar
University, Mullana 133207, India
V. Kumar
Department of Chemistry, Maharishi Markandeshwar
University, Mullana 133207, India
123

Med Chem Res
antimicrobial activity study of 3,5-diamino-4-(40-fluor-
ophenylazo)-1-aryl/heteroarylpyrazoles.
NMR spectra. The 13C NMR spectra of compounds 5a–5i
displayed signal for C3, C4, and C5 at about d 150–153, 114
and 148–151 ppm, respectively. It is well documented in
literature that incorporation of amino group at position-3
and 5 bring a downfield shift of about d 16–17
and 20–24 ppm for C3 and C5, respectively, and an upfield
shift of about 13–17 ppm for C-4 carbon (Frigola et al.,
1989) and the present values are in complete agreement
with earlier report. The complete assignment of the signals
in 13C NMR spectra of these compounds is given in
Table 2.
Chemistry
Synthesis of 3,5-diamino-4-(40-fluorophenylazo)-1-aryl/
heteroarylpyrazoles (summarized in Scheme 1) was
accomplished by a two step reaction. Initially, the key
intermediate, 2-[(4-fluorophenyl)hydrazono]malononitrile
3 was prepared by the diazotization of 4-fluoroaniline 1,
followed by in situ condensation with malononitrile 2 in
the presence of sodium acetate. Then, various aryl and
heteroarylhydrazines 4a–4i were reacted with this inter-
mediate 3 in refluxing ethanol to yield the title compounds
5a–5i as an exclusive product. The yields ranged from 68
to 83 %. The physical properties of the synthesized com-
pounds are presented in a Table 1. The structure of 5a–5i
A plausible mechanism for the formation of 3 from
malononitrile and diazonium salt of 4-fluoroaniline is
outlined in Scheme 2.
Nucleophilic attack of sodium salt of malononitrile 6
(in situ generated in the separate flask by treatment of
malononitrile 2 with sodium acetate), on diazonium salt 7
afforded 3, which further cyclocondensation with binu-
cleophilic hydrazine 4 to yield 5.
1
was established on the basis of analysis of IR, H and 13C
NMR data.
The IR spectra of 5a–5i revealed four characteristic
absorption bands between 3,186 and 3,556 cm-1 due two
NH2 groups and a strong absorption band at about
1,490 cm-1 due to N=N stretch of the azo group and dis-
appearance of absorption bands at 2,222 cm-1 due to
C:N stretch of 3. The 1H NMR spectra of 5a–5i displayed
the two multiplets each of two protons intensity at d
7.29–7.34 and at d 7.74–7.85 for 4-fluorophenylazo group
as well as two broad singlets of two NH2 groups
(exchangeable with D2O) in the range of d 5.22–8.24 ppm.
A singlet of three protons intensity at d 2.42 and 2.74 ppm
appeared due to –CH3 group of 5f and 5i, respectively.
Compound 5h exhibited two sharp singlets: a singlet of six
protons intensity at d 2.45 for two methyl groups and one
proton intensity due to pyrimidine 5-H at d 6.8 ppm. Fur-
ther support to the structure 5a–5i was provided by 13C
Biological results and discussion
In vitro antibacterial activity
All the synthesized compounds 5a–5i were screened in vitro
for their antibacterial activity against two Gram-positive
(Staphylococcus aureus, Bacillus subtilis) and two Gram-
negative bacteria (Escherichia coli, Klebesiella aerogenes)
(Tables 3, 4). The antibacterial activity of these compounds
was compared with Cefixime as a standard drug. In general,
the results revealed that most of the compounds showed
better inhibition against Gram-negative rather than Gram-
positive bacteria. All the compounds 5a–5i showed mini-
mum inhibitory concentration (MIC) ranging 2–32 lg/ml
against Gram-negative bacterial strain K. aerogenes. The
R
N
N
NaNO2 , HCl
RNHNH2, 4a-4i
NC
NC
HN
F
F
NH2
CH2(CN)2 ,NaOAc
EtOH, reflux
NH2
H2N
N
2
3
1
N
N
F
5a-5i
Scheme 1 Synthesis of 3,5-diamino-4-(40-fluorophenylazo)-1-aryl/heteroarylpyrazoles
123

Med Chem Res
Table 1 General structure and physical data of the synthesized compounds 5a–5i
Compounds
R
Mol. formula (m.wt)
Elemental analysis (N) (%)
M.pt (ꢁC)
Yield (%)
70
Found
28.41
Calcd.
28.36
2"
6''
3''
5''
5a
C15H13FN6 (296.3)
184
1''
4''
5b
5c
C15H12ClFN6 (330.15)
25.47
29.09
25.41
29.01
180
198
73
68
Cl
NO2
C15H11FN8O4 (386.3)
O2N
4''
7''
3''
N
5d
5e
C16H12FN7S (353.58)
28.03
25.37
27.75
25.28
262
282
83
81
3a''
7a''
5''
6''
2"
S
1''
C16H11ClFN7S (387.82)
N
S
Cl
N
5f
C17H14FN7S (367.4)
27.05
26.79
26.69
26.40
278
274
75
78
S
H3C
5g
C16H11F2N7S (371.37)
N
S
F
CH3
5h
C15H15FN8 (326.33)
34.39
34.34
298
72
4''
5''
3''
N
2"
6''
H3C
N
1''
CH3
5i
C19H16FN7 (361.38)
27.18
27.13
238
74
5''
4''
4a''
6''
7''
3''
2"
8a''
N
1''
8''
123

Med Chem Res
Table 2 13C NMR data for compounds 5a–5i (ppm)
2
N
NH2
3
R
N
1
4
5
N
H2N
N
2'
1'
6'
3'
4'
5'
F
Carbon-atoms
5a
5b
5c
5d
5e
5f
5g
5h
5i
C-3
152.54
114.80
149.92
122.42
115.84
115.54
164.14
160.85
129.74
122.87
127.19
122.87
129.74
–
153.66
115.01
150.53
122.89
115.87
116.49
163.41
160.17
124.49
129.60
137.83
129.60
124.59
–
152.56
115.21
148.16
122.47
115.32
115.78
163.14
161.88
142.12
119.34
140.34
127.84
129.26
–
151.33
114.17
150.28
123.35
116.28
115.98
163.84
–
151.42
114.08
150.10
122.25
115.95
115.63
163.97
–
150.32
114.15
149.03
121.10
116.28
115.98
163.81
–
150.87
114.08
148.06
122.12
116.03
115.74
163.84
–
152.82
114.48
150.48
122.80
116.23
115.93
163.76
–
153.06
114.62
150.50
122.93
116.76
115.92
163.52
–
C-4
C-5
C-10
C-20, 60
C-30, 50
C-40
C-100
C-200
C-300
C-400
C-500
C-600
C-700
C-800
C-3a00
C-4a00
C-7a00
C-8a00
CH3
161.48
–
161.22
–
160.66
–
161.22
–
168.42
–
160.28
112.65
145.66
123.04
124.45
128.19
130.66
–
123.24
127.06
124.43
121.48
–
123.02
127.07
128.74
121.58
–
123.33
128.30
134.02
122.09
–
123.22
127.98
160.12
122.42
–
124.18
123.00
124.29
–
–
–
–
–
–
–
–
160.59
–
160.74
–
160.56
–
160.45
–
–
–
–
–
–
125.64
–
–
–
–
131.33
–
132.86
–
131.39
–
132.14
–
–
–
–
–
–
148.02
19.22
–
–
–
–
–
21.43
–
23.96
C-100 to C-8a00 are the carbon of R group
In vitro antifungal activity
antibacterial activity of the tested compounds 5a, 5c, and 5i
against E. coli and 5b–5f against K. aerogenes were com-
parable to the reference antibacterial drug Cefixime at MIC
2 lg/ml. The antibacterial activity of the tested compounds
5c, 5g, and 5i against B. subtilis were similar to reference
antibacterial drug Cefixime at MIC 4 lg/ml. Compound 5i
showed good broad spectrum activity against all the tested
strains. Except for compounds 5h and 5i, all tested com-
pounds were found to be inactive against S. aureus and it is
interesting to note that 5i displayed excellent activity as that
of reference drug (Cefixime), however, 5h showed moderate
antibacterial activity.
All the nine compounds 5a–5i were tested for their anti-
fungal activity in vitro against pathogenic fungi: Asper-
gillus niger, Candida albicans and Saccharomyces
cerevisiae (Tables 3, 4) and Fluconazole was used as the
standard antifungal drug. Compounds, 5c and 5g showed
promising inhibition against the A. niger at MIC 2 lg/ml.
Except 5g and 5i, almost all of the compounds showed
potent inhibition against C. albicans with MIC range of
2 lg/ml which is comparable to reference antifungal drug
(Fluconazole). Compound 5a showed antifungal activity
123

Med Chem Res
F
+
. .
_
NC
NC
Cl N
N
F
NC
NC
_
+
+
NaOAc
7
CH2
Na
CH
-AcOH
HN
2
-NaCl
N
6
Malononitrile
C
C
3
N
N
:
:
RNHNH2
4
5
Scheme 2 Mechanism of formation 3,5-diamino-4-(40-fluorophenylazo)-1-aryl/heteroarylpyrazoles
Table 3 In vitro antibacterial
and antifungal activities of
Compounds Diameter of growth of inhibition zone (mm)a
synthesized compounds through
agar well-diffusion method
Antibacterial
Antifungal
S. aureus B. subtilis K. aerogenes E. coli C. albicans A. niger S. cerevisiae
5a
–
10
–
12
12
13
14
10
10.5
11
09
15
15
–
12
–
07
09
11
12
12
11
–
–
12
–
5b
–
–
–, No activity; S. aureus,
Staphylococcus aureus;
5c
–
10
09
–
13
12
–
10
–
12
09
–
5d
–
B. subtilis, Bacillus subtilis;
K. aerogenes, Klebsiella
aerogenes; E. coli, Escherichia
coli; C. albicans, Candida
albicans; A. niger, Aspergillus
niger; S. cerevisiae,
Saccharomyces cerevisiae
a
Values, including diameter of
the well (8 mm), are means of
three replicates
5e
–
–
5f
–
–
–
–
–
5g
–
12
–
–
14
–
–
5h
4.5
2.6
2.9
–
–
09
10
–
10
–
5i
12
2.8
–
15
14
–
–
Cefixime
Fluconazole
–
–
10
13
12
against S. cerevisiae comparable to the reference antifungal
drug Fluconazole at MIC 4 lg/ml. Therefore, we con-
cluded that almost all of the compounds found to possess
good antifungal activity.
activity against A. niger and S. cerevisiae. It is observed
that replacement of aryl and benzothiazole ring at position-
1 of pyrazole by 400,600-dimethylpyrimidin-200-yl (5h) and
by 400-methylquinolin-200-yl (5i) enhances the antibacterial
activity against S. aureus (5a–5g vs 5h–5i). Moreover,
substitution of fluorine at position-6 of benzothiazole
nucleus also results in increase in antifungal activity
against A. niger and complete loss in activity against
C. albicans amongst the benzothiazole series (5g vs 5d–f).
The data reported in Tables 3 and 4 and Fig. 1 revealed
that 5c is the most active compound as compared to the
other compounds. However, all these compounds represent
promising new leads for combating the emerging
pathogens.
Structure–activity relationship
A careful analysis of the MIC data shows that replacement
of the hydrogen in aryl group of compound 5a by chlorine
at postion-4 (5b) increases the antibacterial activity against
K. aerogenes comparable to the reference drug (Cefixime).
Also, incorporation of nitro group at postion-2 and -4 (5c)
also shows a significant level of increase in antibacterial
activity against K. aerogens and B. subtilis and antifungal
123

Med Chem Res
Table 4 MIC of 5a–5i against
test bacteria and fungi using
agar well-diffusion method
Compounds MIC (lg/ml)a
S. aureus B. subtilis K. aerogenes E. coli C. albicans A. niger S. cerevisiae
5a
–
–
–
–
–
–
–
16
2
2
–
8
–
4
6
–
–
4
–
4
4
–
32
2
2
2
2
2
4
8
8
2
–
2
–
2
8
–
–
–
–
2
2
–
2
2
2
2
2
2
–
2
64
–
2
–
–
2
–
–
–
2
–
–
–
2
4
–
8
6
–
–
–
8
–
–
4
5b
5c
5d
5e
5f
5g
5h
5i
Cefixime
Fluconazole
a
Mean of three replicates
cooling was filtered and recrystallized from ethanol to give
35
30
25
20
15
10
5
pure 5a–5i.
S. aureus
B. subtilis
Analytical and spectral data of synthesized compounds
K. aerogenes
E. coli
(5a–5i)
C. albicans
A. niger
3,5-Diamino-4-(40-fluorophenylazo)-1-phenylpyrazole (5a)
S. cerevisiae
0
IR (KBr): 1,497 (N=N), 3,186, 3,294, 3,387, 3,464 (NH2)
1
cm-1. H NMR (CDCl3) d: 5.22 (bs, 2H, exchangeable
with D2O), 6.98–7.05 (m, 2H), 7.19–7.49 (m, 5H),
7.54–7.60 (m, 2H). MS (EI): [M?1]?, m/z, 297.5.
Fig. 1 Comparison of MIC of tested compounds with commercial
antibiotics range MIC 2–32 (lg/ml)
3,5-Diamino-1-(400-chlorophenyl)-4-
(40-fluorophenylazo)pyrazole (5b)
Experimental
IR (KBr): 1,489 (N=N), 3,256, 3,317, 3,425, 3,479 (NH2)
cm-1. 1H NMR (DMSO-d6) d: 6.90 (bs, 2H, exchangeable
with D2O), 7.21–7.27 (m, 2H), 7.51–7.60 (m, 4H),
7.83–7.85 (m, 2H). MS (EI): [M?1]? and [M?3] ?, m/z,
331.2 and 333.2 in the ratio of 3:1 showing the typical
isotopic profile.
Melting points were determined in open capillaries with an
electrical apparatus and are uncorrected. The IR spectra of
the compounds were recorded on Buck Scientific IR M-500
1
spectrophotometer using KBr pellets (mmax in cm-1). H
and 13C NMR spectra on a Bruker instrument at 300 and
75 MHz, respectively.
3,5-Diamino-1-(200,400-dinitrophenyl)-4-
(40-fluorophenylazo)pyrazole (5c)
Arylhydrazines 4a–4c were commercially available.
Heteroarylhydrazines 4d–4i (Potts et al., 1972; Katz, 1951;
Kosolapoff and Roy, 1961) and 2-[(4-fluorophenyl)
hydrazono]malononitrile 3 (Krystof et al., 2006) were
prepared according to the literature procedure.
IR (KBr): 1,492 (N=N), 3,248, 3,344, 3,456, 3,464 (NH2)
cm-1. 1H NMR (DMSO-d6) d: 5.03 (bs, 2H, exchangeable
with D2O), 7.65–7.68 (m, 2H), 8.23–8.26 (m, 2H),
8.80–8.86 (m, 2H), 9.99 (s, 1H). MS (EI): [M?1]?, m/z,
386.3.
General procedure for the preparation of 3,5-diamino-4-
(40-fluorophenylazo)-1-aryl/heteroarylpyrazoles (5a–5i)
3,5-Diamino-1-(benzothiazol-200-yl)-4-
2-[(4-Fluorophenyl)hydrazono]malononitrile
3
(0.94 g,
(40-fluorophenylazo)pyrazole (5d)
5 mmol) was dissolved in 40 ml ethanol and an equivalent
amount of appropriate hydrazines 4a–4i (5 mmol) and 3–4
drops of acetic acid was added to it. The reaction mixture
was refluxed for 2 h. The crude product so obtained on
IR (KBr): 1,489 (N=N), 3,256, 3,310, 3,379, 3,418 (NH2)
cm-1. 1H NMR (DMSO-d6) d: 6.49 (bs, 2H, exchangeable
123

Med Chem Res
with D2O), 7.26–7.37 (m, 3H), 7.46–7.51 (m, 1H),
7.85–7.88 (m, 3H), 8.01–8.03 (d, J = 7.5 Hz, 1H), 8.24
(bs, 2H, exchangeable with D2O). MS (EI): [M?1]?, m/z,
354.1.
In vitro biological assays
Test microorganisms
Pathogenic bacterial strain namely S. aureus (Gram-posi-
tive), B. subtilis (Gram-positive), E. coli (Gram-negative),
and K. aerogenes (Gram-negative) and three fungi,
A. niger, C. albicans, and S. cerevisiae) were isolated from
the patients in Maharishi Markandeshwar Medical College,
Maharishi Markandeshwar University, Mullana, Haryana
and were used in the present study.
3,5-Diamino-1-(600-chlorobenzothiazol-200-yl)-4-
(40-fluorophenylazo)pyrazole (5e)
IR (KBr): 1,497 (N=N), 3,232, 3,410, 3,472, 3,556 (NH2)
cm-1
.
1H NMR (DMSO-d6) d: 7.11–7.16 (m, 2H),
7.40–7.42 (d, J = 7.5 Hz, 1H), 7.74–7.78 (m, 4H), 7.88
(bs, 2H, exchangeable with D2O), 8.04 (bs, exchangeable
with D2O). MS (EI): [M?1]? and [M?3]?, m/z, 388.1 and
390 in the ratio of 3:1 showing the typical isotopic profile.
In vitro antibacterial activity
The in vitro antibacterial activity of nine synthesized
compounds was evaluated by the agar well-diffusion
method (Sadashiva et al., 2004). 25 ml of nutrient agar
medium was poured into each petri plate and the agar
plates were swabbed with 100-ll inocula of each tested
bacterium and kept for 15 min for adsorption. Using sterile
cork borer of 8-mm diameter, wells were bored into the
seeded agar plates and these were loaded with a 50-ll
volume. Solutions of the tested compounds and standards
were prepared in DMSO at concentration of 2,000 lg/ml.
From this stock solution, twofold dilutions of the com-
pounds (2, 4,…64 lg/ml) were inoculated to the corre-
sponding wells. All the plates were incubated at 37 ꢁC for
24 h. In vitro antibacterial activity of each synthesized
compound was evaluated by measuring the zone of growth
inhibition and MIC against the tested organisms with zone
reader (Hi Antibiotic zone scale). MIC was determined as
the lowest concentration of the compound tested that was
able to inhibit visible growth of bacteria or fungi.
Dimethylsulphoxide (DMSO) was used as a negative
control, whereas Cefixime was used as a reference drug.
The experiments were performed in triplicates.
3,5-Diamino-4-(40-fluorophenylazo)-1-
(600-methylbenzothiazol-200-yl)pyrazole (5f)
IR (KBr): 1,497 (N=N), 3,256, 3,317, 3,379, 3,418 (NH2)
cm-1. 1H NMR (DMSO-d6) d: 2.42 (s, 3H, CH3), 6.47 (bs,
2H, exchangeable with D2O), 7.26–7.32 (m, 3H),
7.73–7.76 (d, J = 8.1 Hz, 1H), 7.82 (s, 1H), 7.86–7.90 (m,
2H), 8.20 (bs, 2H, exchangeable with D2O). MS (EI):
[M?1]?, m/z, 368.3.
3,5-Diamino-1-(600-fluorobenzothiazol-200-yl)-4-
(40-fluorophenylazo)pyrazole (5g)
IR (KBr): 1,489 (N=N), 3,256, 3,310, 3,371, 3,410 (NH2)
cm-1. 1H NMR (DMSO-d6) d: 6.50 (bs, 2H, exchangeable
with D2O), 7.29–7.34 (m, 3H), 7.88–7.96 (m, 4H), 8.19 (bs,
2H, exchangeable with D2O). MS (EI): [M?1]?, m/z,
372.5.
3,5-Diamino-4-(40-fluorophenylazo)-1-
(400,600-dimethylpyrimidin-200-yl)pyrazole (5h)
In vitro antifungal activity
IR (KBr): 1,489 (N=N), 3,225, 3,302, 3,391, 3,418 (NH2)
cm-1. 1H NMR (CDCl3) d: 2.47 (s, 6H, CH3), 5.22 (s, 1H,
exchangeable with D2O), 6.73 (s, 1H), 6.99–7.05 (m, 2H),
7.18 (s, 1H, exchangeable with D2O), 7.56–7.60 (m, 2H).
MS (EI): [M?1]?, m/z, 327.2.
The in vitro antifungal activity of titled compounds was
evaluated by the agar well-diffusion method (Sadashiva
et al., 2004). The moulds were grown on Potato dextrose
agar (PDA) at 25 ꢁC for 7 days and used as inocula. 15 ml
of molten PDA (45 ꢁC) was poisoned by the addition of
50-ll volume of each compound, poured into a sterile petri
plate and allowed to solidify at room temperature. Solu-
tions of the tested compounds and standards were prepared
in DMSO at concentration of 2,000 lg/ml. From this stock
solution, twofold dilutions of the compounds (2,
4,…64 lg/ml) were inoculated to the corresponding wells.
The solidified poisoned agar plates were inoculated at the
centre with fungal plugs (8 mm diameter), obtained from
the actively growing colony and incubated at 25 ꢁC for
3,5-Diamino-4-(40-fluorophenylazo)-1-(400-methylquinolin-
200-yl)pyrazole (5i)
IR (KBr): 1,497 (N=N), 3,186, 3,343, 3,418, 3,476 (NH2)
cm-1
.
1H NMR (DMSO-d6) d: 2.74 (s, 3H, CH3),
7.25–7.31 (m, 2H), 7.53–7.58 (m, 1H), 7.74–7.80 (m, 2H)
7.83–7.88 (m, 2H), 8.03–8.07 (m, 2H). MS (EI): [M?1]?,
m/z, 362.2.
123

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This study comprises the synthesis of 3,5-diamino-4-(40-
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against E. coli, 5b–5f against K. aerogenes and 5c, 5g, and
5i against B. subtilis were similar to the reference anti-
bacterial compound (Cefixime). Except 5i, almost all the
compounds found to possess excellent level of antifungal
activity comparable to standard drug Fluconazole.
Acknowledgments We are thankful to Council of Scientific and
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Research Fellowship to Virender Kumar. Thanks are also due to the
Mass Spectrometry and elemental analysis facility, National Institute
of Pharmaceutical Education and Research, Mohali (Punjab) for
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