A regioselective synthesis of some new pyrazol-1′-ylpyrazolo[1,5-a] pyrimidines in aqueous medium and their evaluation as antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2011.04.041

An efficient and environmental benign regioselective synthesis of some new pyrazol-1′-ylpyrazolo[1,5-a]pyrimidines (7b-h) has been accomplished via treatment of 3(5)-amino-5(3)-hydrazinopyrazole dihydrochloride (5) with several unsymmetrical 1,3-diketones (6b-h) using water as a solvent without any catalysts or additives. The structure of 7b-h was established on the basis of rigorous analysis of ¹H, ¹³C NMR, IR spectral data and MS. Eight compounds (7a-h) were screened for their antibacterial activity against two gram-positive and two gram-negative bacteria and compounds (7a, b, d and e) for antifungal activity against four phytopathogenic fungi. Compounds 7c and 7e manifest rather broad antibacterial activity than standard antibiotics. One lead compound, 7a (10 mg/ml and 200 mg/ml) exhibited equipotent or more potent antifungal activity against all tested microorganisms than standard drug.

Full text of DOI:10.1016/j.ejmech.2011.04.041

European Journal of Medicinal Chemistry 46 (2011) 3038e3046
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A regioselective synthesis of some new pyrazol-10-ylpyrazolo[1,5-a]pyrimidines
in aqueous medium and their evaluation as antimicrobial agents
,
a,
Ranjana Aggarwal a , Garima Sumran 1, Neelam Garg b, Ashok Aggarwal c
*
a Department of Chemistry, Kurukshetra University, Kurukshetra 136 119, India
b Department of Microbiology, Kurukshetra University, Kurukshetra 136 119, India
c Department of Botany, Kurukshetra University, Kurukshetra 136 119, India
a r t i c l e i n f o
a b s t r a c t
An efficient and environmental benign regioselective synthesis of some new pyrazol-10-ylpyrazolo[1,5-a]
pyrimidines (7beh) has been accomplished via treatment of 3(5)-amino-5(3)-hydrazinopyrazole dihy-
drochloride (5) with several unsymmetrical 1,3-diketones (6beh) using water as a solvent without any
Article history:
Received 12 April 2011
Accepted 13 April 2011
Available online 22 April 2011
catalysts or additives. The structure of 7beh was established on the basis of rigorous analysis of 1H, 13
C
NMR, IR spectral data and MS. Eight compounds (7aeh) were screened for their antibacterial activity
against two gram-positive and two gram-negative bacteria and compounds (7a, b, d and e) for antifungal
activity against four phytopathogenic fungi. Compounds 7c and 7e manifest rather broad antibacterial
activity than standard antibiotics. One lead compound, 7a (10 mg/ml and 200 mg/ml) exhibited equi-
potent or more potent antifungal activity against all tested microorganisms than standard drug.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Pyrazol-10-ylpyrazolo[1,5-a]pyrimidines
Aquoeus medium synthesis
Regioisoselective synthesis
Antibacterial activity
Antifungal activity
1. Introduction
pyrimidines (3) and/or their structural isomers 1H-pyrazolo[3,4-b]
pyridines (4) (Scheme 1) [15,16] depending upon the nature of
Pyrazolo[1,5-a]pyrimidines are purine analogues and are used as
antimetabolites in purine biochemical reactions [1]. They act as
potent inhibitors of enzymes e 30,50-cyclic AMP phosphodiesterase
[2], cyclooxygenase-2 (COX-2) [3], cyclin-dependent kinase 2 (CDK2)
[4] and c-Src kinase [5]. They also possess affinity for estrogen [6],
GABA, GABAA/peripheral benzodiazepine receptors [7] besides being
corticotropin-releasing factor (CRF) receptor antagonists [8]. The
recent discovery of N-[3-(3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)
phenyl-N-ethylacetamide, Zaleplon, as an ideal hypnotic drug has
stimulated further interest in the pyrazolo[1,5-a]pyrimidine chem-
istry [9]. A large number of pyrazolo[1,5-a]pyrimidine derivatives are
reported to exhibit a broad spectrum of biological activities such as
antitumor [10], anxiolytics [11] and antimicrobial [12].
The most versatile approach available for the synthesis of pyr-
azolo[1,5-a]pyrimidines consists of the condensation of bifunctional
nucleophile 3-amino-1H-pyrazoles with bifunctional electrophiles
[13] such as 1,3-diketones [6,14]. However, condensation of various
symmetrical b-diketones (2) with a series of 5-amino-1H-pyrazoles
(1) has been reported to result in the formation of pyrazolo[1,5-a]
substituents on b-diketones [15]. Solvent also plays a crucial role in
controlling the structure of the product. For instance, heating 1 and
2 in acetic acid leads tothe formation of a mixture of 3 and 4 whereas
pyrazolopyrimidine 3 was formed as the only product when DMSO
was used as solvent and reaction in ethanol in presence of trie-
thylamine (TEA) afforded pyrazolopyridine 4 as the predominant
product [16].
Very recently, we have reported the reaction of 3(5)-amino-5(3)-
hydrazinopyrazole dihydrochloride (5) with pentane-2,4-dione (6a)
in aqueous system which resulted in the exclusive formation of
2-(30,50-dimethylpyrazol-10-yl)-5,7-dimethylpyrazolo[1,5-a]pyrim-
idine (7a) without
a
trace of 3-(30,50-dimethylpyrazol-10-yl)-
4,6-dimethyl-1H-pyrazolo[3,4-b]pyridine isomer (8) (Scheme 2)
[17]. The structure of 7awas unambiguouslyestablished on the basis
of elemental analysis and spectral data (IR, high resolution 1H NMR,
MS). The characteristic chemical shifts of the methyl substituents at
0
0
C5, C7 of pyrazolopyrimidine ring and C3 and C5 of pyrazole ring
were established utilizing (1He13C) HMQC as well as (1He13C) and
(1He15N) HMBC measurements, thus solving the discrepancy
existing in the literature about the position of methyl protons on
pyrazolopyrimidine ring [2,3a].
Encouraged by these observations and in continuation with the
work related to the synthesis, spectral studies and biological
properties of pyrazolo[1,5-a]pyrimidines [18], we report herein the
regioselective synthesis of some new substituted pyrazol-
* Corresponding author. Tel.: þ91 1744238734; fax: þ911744238277.
E-mail address: ranjana67in@yahoo.com (R. Aggarwal).
Presently at Technology Education & Research Integrated Institutions, Kuruk-
1
shetra 136 119, India.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.041

R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 3038e3046
3039
Table 1
H
R
N
N
N
Optimization of reaction conditions for the reaction of 3(5)-amino-5(3)-hydrazi-
AcOH/
DMSO/
nopyrazole dihydrochloride (5) with phenyl-1,3-butanedione (6b).a
H
N
O
O
and/or
R
R
N
N
N
N
+
R
EtOH-TEA
Entry Solvent
5
6b
NaOAc Yield of Yield of Yield of
R
R
R
H2N
R
R
(equiv) (equiv) (equiv) 7b (%)b 9b (%)b 10b (%)b
2
3
4
1
c
1
Acetonitrile/
H2O
1
1
e
9
91
R = CH3, C2H5, C6H5, 4-FC6H4, CF3
Scheme 1. Synthesis of compounds 3 and/or 4 in different solvents.
2
3
THF/H2O
EtOH/H2O
(3:2 v/v)
EtOH/H2O
(3:2 v/v)
EtOH/H2O
(3:2 v/v)
EtOH/H2O
(3:2 v/v)
H2O
1
1
1
1
e
e
8
35
82
65
10
c
10-ylpyrazolo[1,5-a]pyrimidines
(7beh)
based
on
cyclo-
c
c
c
4
5
6
1
1
1
1
1
2
1
2
2
35
35
35
85
65
65
65
condensation of 3(5)-amino-5(3)-hydrazinopyrazole dihydro-
chloride (5) with unsymmetrical 1,3-diketones (6beh) in water,
with an aim to find more potent antimicrobial agents.
2. Chemistry
7
8
1
1
1
1
e
e
10
5
c
H2O þ acetoned
(2:5 v/v)
8d
32d
2.1. Synthesis
a
b
c
Reaction conditions: reflux for 4 h.
Analysis of crude reaction product based on 1H NMR.
Yield not determined.
The starting compound, 3(5)-amino-5(3)-hydrazinopyrazole
dihydrochloride (5) obtained by the condensation of hydrazine
hydrate and malononitrile [19], is quite polar and encountered
solubility problem in most organic solvents such as ethanol, chloro-
form, dichloromethane, tetrahydrofuran and acetonitrile. Attempts
were made to react 5 with phenyl-1,3-butanedione (6b) in THF,
CH3CN and EtOH with or without NaOAc but no product could be
isolated even when the reaction time was extended to 48 h and the
starting material was recovered as such. Recently, aqueous mediated
reactions have received considerable attention in organic synthesis
due to environmental safety reasons. Keeping this in view, a series of
experiments was accomplished with an objective to ascertain the
d
Hydrazone of acetone is obtained in 60% yield as another product of this reaction
through CeN bond cleavage.
similar conditions to obtain corresponding pyrazolo[1,5-a]pyrimi-
dines (7bef) in excellent yields. It is worthy to note that CeN bond
cleavage could be suppressed significantly when water was
employed as the solvent (9b only 10%) and a minute amount of
substituted benzoic acids (10bef) (3e5%) was observed (Scheme 3).
Thus, this route provides an effective, economic and environmentally
friendly method to synthesize these fused pyrazoles.
All the products were purified by column chromatography using
petroleum ether/chloroform (0e50% gradient) as eluent and ratio of
different products was measured by 1H NMR spectroscopy of the
crude reaction mixture. To further widen the scope of this regiose-
lective synthesis, 1-(200-thienyl)-1,3-butanedione (6g) and 2-ace-
tylcyclopentanone (6h) were made to react with 5 under the same
experimental conditions. While the reaction with 6g afforded 2-(30-
methyl-50-(thien-200-yl)pyrazol-10-yl)-5-methyl-7-(thien-200-yl)pyr-
azolo[1,5-a]pyrimidine (7g) and 3-methyl-5-(2-thienyl)-1H-pyrazole
(9g); reaction with 6h afforded 2-(30-methyl-50,60-dihydro-4H-
cyclopenta[d]pyrazol-10-yl)-5-methyl-7,8-dihydro-6H-cyclopenta[g]
pyrazolo[1,5-a]pyrimidine (7h) as an exclusive product without any
trace of cleaved pyrazole and carboxylic acid (Scheme 3).
reaction conditions: temperature, ratio of b-diketone to hydrazine
dihydrochloride, amount of NaOAc and the solvent system (water as
a solvent (entry 7) or co-solvent in conjunction with other organic
solvents, e.g., THF, CH3CN and EtOH (entries 1e6)) with phenyl-
1,3-butanedione 6b as a model compound (Table 1). TLC and 1H
NMR examination of the crude reaction mixture indicated the
formation of three products namely: fused pyrazol-10-ylpyr-
azolopyrimidine (7b), 3(5)-methyl-5(3)-phenyl-1H-pyrazole (9b)
obtained by the CeN bond cleavage of 5 and benzoic acid (10b).
As evident from the Table 1, the optimum condition for the
formation of fused pyrazol-10-ylpyrazolopyrimidine (7b) was estab-
lished as refluxing equimolar amounts of phenyl-1,3-butanedione
with 5 in H2O. Other
b-diketones were also made to react under
H
O
O
N N
+
H3C
CH3
.
HCl:H2NHN
NH2 HCl
6a
5
EtOH:H2O (1:1)
or H2O
Reflux
H
2
N
1
N
CH3
CH3
7
6
'
5
CH3
5'
1
N
'
3
1
7a
N 7
7a
N
4'
N
4'
3a
N
N2'
'
2
3
1'
H3C
N
3a
3'
5
N
CH3
6
4
H3C
3
H3C
CH3
2'
4
5
7a
8
Scheme 2. Synthesis of 7a.

3040
R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 3038e3046
H
O
O
N N
+
R
CH3
.
.
HCl H2NHN
NH2 HCl
R'
6b-h
5
(reflux)
N N
H2O
H
R
7
R
5'
1
N
7a
N
R'
R'
R-COOH
+
6
+
4'
3'
N
H3C
R
2
10b-f
1'
N
3a
5
N
CH3
3
H3C
2'
R'
4
9b-g
7b-h
R'
H
H
where,
R
b = C6H5
c = 4"-CH3C6H4
d = 4"-OCH3C6H4
e = 4"-ClC6H4
H
H
H
H
f = 4"-BrC6H4
g = 2''-thienyl
CH2
h =
3
Scheme 3. Synthesis of title compounds 7beh.
2.2. Results and discussion
4J ¼ 0.8 Hz (CH3-50, H-40) [24], respectively, due to allylic coupling
between them as it has been observed in the case of 7a [17]. On the
contrary, the methyl groups at position-30 and position-5 does not
show coupling to H-40 and H-6, respectively.
In principle, the reaction of 3(5)-amino-5(3)-hydrazinopyrazole
dihydrochloride (5) with unsymmetrical -diketones (6beh) might
b
result in the formation of four regioisomeric pyrazol-10-ylpyrazolo
[1,5-a]pyrimidines e (30-(R)-50-methylpyrazol-10-yl-5-(R)-7-met-
hyl-) (I), (30-methyl-50-(R)pyrazol-10-yl-5-(R)-7-methyl-) (II), (30-
methyl-50-(R)pyrazol-10-yl-5-methyl-7-(R)-) (III) and (30-(R)-
50-methylpyrazol-10-yl-5-methyl-7-(R)-) (IV) due to competitive
reactivities of different nucleophilic sites (A, B, C and D) and elec-
trophilic centers (x and y) through the pathways depicted in
Scheme 4, however, only one isomer was obtained during the
present investigation.
Furthermore, the presence of methyl groups at position-30 and
position-5 finds support by an inspection of 13C NMR spectra of
7beh which display CH3-30 signal at
d
11.4e13.7 and CH3-5 at
d
22.0e24.8 ppm in close agreement with 7a spectral results [17].
Also, the location of phenyl group at position-7 of the pyrimidine
ring in regioisomer 7b was determined on the basis of the 13C NMR
data, taking into account characteristic chemical shift of ipso atom
in phenyl ring 7-Ci at
[14a.b]). Had the phenyl group been located at position-5, signal for
5-Ci would have appeared at 136.1 ppm (Ref. [18]). Compounds
7beh displayed C-30 and C-50 signals of pyrazole ring resonated at
148.5e151.2 and 138.1e145.0 ppm, respectively, in complete
d 131.3 ppm (C7eCipso at d 131 ppm Refs.
The fused product was unambiguously characterized as struc-
ture III by a combined application of 1H and 13C NMR spectral data.
High resolution 1H NMR spectra of the isolated compounds 7beh
d
d
d
exhibited two sharp singlets of three protons intensity at
and 2.46e2.64 ppm corresponding to C3 eCH3 and 5-CH3 and three
d
2.2e2.4
agreement with literature data [21,25]. The complete assignment of
the signals in 13C NMR spectra of the compounds is given in Table 2.
It is noteworthy to mention that in 13C NMR spectra of 7bef, the
0
sharp singlets signals of one proton intensity in the range
d
6.1e6.4 ppm, 6.4e6.7 ppm and
H-3 [20] and H-6 [20], respectively. Chemical shift of C3 eCH3 at
2.2e2.4 eliminates the possibility of structures I and IV as it is well
d
6.6e7.0 ppm due to H-40 [20],
C-50 appeared at
d 143.0e145.0 ppm instead of 140.9 ppm [17] in
0
case of 7a. This downfield shift of signal of C-50 by about 3e5 ppm
may be due to the replacement of methyl by an aryl group [21].
Finally, a conclusive evidence for the proposed structure III was
received through (1He13C) HMQC, (1He13C) HMBC and (1He15N)
HMBC experiments on 7b as a representative example. Based on
these 2D correlations, a complete and unambiguous assignment of
1H, 13C, 15N of 7b are given in our paper in Journal of Molecular
Structure [17].
d
established by us [21] and the other workers [22] that the methyl
group located at position-5 of pyrazole moiety resonates downfield
(w2.7 ppm) as compared to 3-CH3 (
d 2.3) in case of 2-(pyrazol-1-yl)
heterocycles. Moreover, had the structure been I, II and IV then
0
0
C4 eH and/or C6eH and C5 eCH3 and/or 7-CH3 signals would have
splitted into quartet 4J ¼ 1.0 Hz (CH3-7, H-6) [23] and doublet

R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 3038e3046
3041
D
..
H
O
O
N N
.. ..
..
+
y
x
R
CH3
.
.
HCl H2NHN
NH2 HCl
C
A
B
6b-h
5
Reflux H2O
CH3
7
CH3
7
R
5'
CH3
5'
N
N
N
N
N
N
N
5
3'
N
R
N
5
3'
H3C
N
R
R
7b-h
7b-h
II (Bx+Cx or Ay+Dy)
I (Ax+Cx or By+Dy)
R
7
R
7
CH3
5'
R
5'
N
N
N
N
N
N
N
3'
5
N
N
CH3
3'
5
N
CH3
R
H3C
7b-h
IV (Ax+Cy or By+Dx)
7b-h
III (Bx+Cy or Ay+Dx)
where, R = aryl, heteroaryl, cyclopentyl
Scheme 4. Synthesis of regioisomers (framed the real structure).
Besides the expected substituted pyrazol-10-ylpyrazolo[1,5-a]
pyrimidines (7beg), an unexpected product 3(5)-methyl-5(3)-aryl-
1H-pyrazole (9beg) was also separated from the reaction mixture
by fractional crystallization or column chromatography. The
structure was confirmed by comparing its physical and spectral
data (m.p., mixed m.p., TLC, IR, NMR and mass spectra) with an
authentic sample [26e28] which was independently synthesized
via reacting equimolar amounts of hydrazine hydrate with 6beg in
ethanol under reflux.
Formation of 3(5)-methyl-5(3)-substitutedphenyl pyrazoles
(9) suggests an unusual CeN pivot bond cleavage [29] either after
the synthesis of pyrazol-10-ylpyrazolo[1,5-a]pyrimidines (7) or
through the self decomposition of hydrazine (5) under the reac-
tion conditions. The former possibility was ruled out by the fact
reactivity of aryl-1,3-diketones towards hydrazine permits the self
decomposition of 5 to afford hydrazone of acetone. In the absence
of acetone, reaction of aryl-1,3-diketones with 5 as well as its
decomposition product (hydrazine) proceeds in a competitive
manner to give pyrazol-10-ylpyrazolo[1,5-a]pyrimidines (7) and
cleaved pyrazoles 9.
Formation of 10bef may be rationalized by the aqueous acidic
hydrolysis of 1,3-diketones. Such acidic hydrolysis and the relation
of structure to the proportion of cleavage products and to the mode
of cleavage (water, alcohol and hydrogen) of 1,3-diketones under
a variety of conditions has already been reported previously [31].
The reaction was highly regioselective and only the pyrazolo
[1,5-a]pyrimidine isomer III was obtained, where methyl substit-
uents was attached to C5 in contrast with previous studies [16]. The
high regioselectivity of the reaction could be attributed to the
primary attack of more nucleophilic NH2 group (site C) of 5 rather
than ring nitrogen (site D) of the pyrazole ring (hard) on the more
electrophilic and less hindered carbonyl carbon [32] of acetyl group
(site y) rather than the hindered and electronically disfavored
carbonyl carbon (hard) attached to the aryl/heteroaryl group (site
x) to afford pyrimidine ring. Also the unsubstituted nitrogen of
heteroarylhydrazine 5 (site A) preferentially reacts with the
carbonyl carbon of acetyl group (site y) to afford pyrazole ring in
conformity with our earlier observation [21].
that
7 remains largely unchanged under reflux for 4e5 h.
However, refluxing 5 in EtOH/H2O followed by the addition of
aldehydes/ketone to the reaction mixture led to the formation of
corresponding hydrazones or azines (11) [30a] in 50e60% yield
(Scheme 5), thus indicating the formation of hydrazine as a result
of self decomposition of 5. This observation finds further support
by the isolation of hydrazone of acetone [30b] (Table 1, entry 8)
when the reaction of phenyl-1,3-butanedione (6b) was carried
with 3(5)-amino-5(3)-hydrazinopyrazole dihydrochloride (5) in
H2O having excess of acetone. This indicates that the lesser

3042
R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 3038e3046
Table 2
13C NMR chemical shifts of pyrazol-10-ylpyrazolo[1,5-a]pyrimidines 7beh (in ppm).
R
7
R
5'
1
N
7a
N
4'
6
N
2
1'
N
3a
3'
5
N
CH3
3
H3C
2'
4
7b-h
Carbon
7b
7c
7d
7e
7f
7g
7h
C-2
C-3
152.83
89.2
150.68
86.95
152.87
88.76
152.94
88.75
153.0
88.74
152.98
89.14
150.35
83.45
C-5
C-6
C-7
C-3a
159.42
108.43
145.74
149.6
157.06
105.78
143.90
147.16
22.48
148.59
106.50
143.03
11.49
126.72
126.11
125.31
123.67/127.10/
126.86/139.26/
135.85
159.21
107.44
145.43
149.67
24.79
150.73
108.57
144.76
13.7
123.76
122.63
130.91
130.44/113.78/
113.63161.65/
159.60
159.48
108.20
144.96
148.88
24.48
151.11
109.40
143.61
13.59
129.72
128.98
130.52
130.42/128.75/
128.35/137.40/
159.43
108.17
145.27
148.65
24.39
151.22
109.50
143.69
13.59
130.15
128.78
130.83
130.60/131.84/
131.36/126.01/
122.63
e
158.15
104.67
140.58
147.56
23.81
151.15
110.39
138.11
156.52
121.71
145.49
148.78
22.02
149.32
128.23
142.03
5-CH3
24.82
C-30
150.86
108.88
145.02
13.7
131.3
130.4
130.86
129.11/129.06/
128.4/128.16/
128.06
e
C-40
C-50
30-CH3
Others Ci-7
Ci-50
13.62
12.90
Th-2 133.33/133.05
Th-4 131.07/130.70
Th-3128.41/128.14
Th-5 127.24/126.88
Cyclopentyl
Carbons 22.47,
26.38, 29.59, 29.69,
29.86, 30.70
Co
Co, 2Cm, 2Cp
134.34
e
OCH3
CH3
e
55.27, 55.36
e
e
e
e
e
e
19.18, 19.38
e
e
3. Biological results and discussion
control Linezolid employed in this study against E. coli, P. aeruginosa
and B. cereus.
3.1. Antibacterial activity
The antibacterial activity is considerably affected by substituents
at 50 and 7-positions of pyrazol-10-ylpyrazolo[1,5-a]pyrimidines.
The results suggests that substitution of 50 and 7-positions of pyr-
azol-10-ylpyrazolo[1,5-a]pyrimidines with para-substituted phenyl
ring either with CH3 or Cl group (7c and 7e) increased their anti-
bacterial activities against all or some tested bacteria like E. coli (7c
and 7e) and B. cereus (7e) in comparison to their unsubstituted
analogoue 7b. Similarly, replacement of 4-substituent of the phenyl
ring and phenyl ring of pyrazol-10-ylpyrazolo[1,5-a]pyrimidines 7
with OCH3, Br group and 2-thienyl group (7d, f and g), respectively,
diminished their activities. The significant loss in potency was
observed when phenyl moiety attached to 50 and 7-positions of
pyrazol-10-ylpyrazolo[1,5-a]pyrimidines is replaced with alkyl
moieties (7a and 7h).
Eight chemically synthesized compounds (7aeh) were assayed
in vitro for their antibacterial activity against two gram-negative
bacteria (Escherichia coli ATCC 8739 and Pseudomonas aeruginosa
ATCC 25668) and two gram-positive bacteria (Staphylococcus
aureus ATCC 9144 and Bacillus cereus ATCC 11778).
3.1.1. Results
For evaluating antibacterial activity Gentamicin and Linezolid
were used as the standard drugs. The results of antibacterial
activity have been summarized in Table 3. The observed minimum
inhibitory concentrations (MIC) presented in Table 4 were in
accordance with the results obtained in the primary screening.
Compound 7e exhibited excellent activity against Gram-positive
bacteria B. cereus (MIC 50
against Gram-negative bacteria E. coli (MIC 25
m
g/ml), while exhibited high activity
Table 3
In vitro antibacterial activity of 7aeh by using agar diffusion assay technique.
mg/ml) as compared
with the reference drugs (Gentamicin and Linezolid). Thus, this
compound could be used as lead structure in pharmaceutical
industry if it is nontoxic to the human system. Compound 7c
exhibited good activity towards Gram-negative bacteria E. coli, but
compounds 7a, 7b, 7d and 7feh exhibited slight activity towards
Gram-negative and Gram-positive bacteria. As it can be seen in
Table 4, MICs for compounds 7c and 7e are lower than positive
Compound
Diameter of zone of growth inhibition (mm)a
Gram ꢀve bacteria Gram þve bacteria
E. coli
P. aeruginosa
S. aureus
B. cereus
7a
7b
7c
7d
7
8
10.5
8
7
7.5
8
7
8
8
7
8
8.5
8
8
8
7e
7f
7g
13
8
8
10
8
8
9
8
8
13
8
8
EtOH-H2O
H
R
O
R
NNH2
R'
N
N
1 : 1 (v/v)
+
7h
7
7
7
7
or
C
N N C
Δ
Gentamicin
Linezolid
Control
24
12
e
12
8.5
14
18
e
14
10
e
.
.
NH2 HCl
R
R'
R
HCl H2NHN
R'
R'
5
11
e
R = C6H5, 4-OCH3C6H4
R' = H, CH3
Inhibition zone, 6e10 mm slight activity, 11e15 mm moderate activity, more than
15 mm high activity.
a
Scheme 5. Isolation of hydrazones or azines 11: indication of CeN bond cleavage.
Mean of three replicates.

R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 3038e3046
3043
Table 4
MIC ( g/ml) values of compounds 7aeh and reference drugs against the respective microorganisms.
m
Organism
7a
7b
200
>200
200
7c
50
100
100
100
7d
7e
25
100
100
50
7f
7g
200
200
200
7h
Gentamicin
Linezolid
E. coli
>400
>400
400
>100
>100
>100
>100
>100
>100
>100
>100
>400
>400
400
4
2
2
4
>100
>100
2
P. aeruginosa
S. aureus
B. cereus
>400
200
>200
400
>100
3.2. Antifungal activity
Four organic compounds (7a, 7b, 7d and 7e) were screened for
their antifungal activity in vitro against phytopathogenic Aspergillus
terrus, Alternaria alternata, Fusarium oxysporum and Helmintho-
sporium sp. by poisoned food technique [33].
3.2.1. Results
All these compounds were found to be active and inhibit the
growth of pathogenic fungi significantly as compared to control and
reference drug Mancozeb at different concentrations (Table 5). In
case of compounds 7a, maximum inhibition was seen at 200 mg/ml
on Helminthosporium sp. (79.2 ꢁ 0.06), A. terrus (73.2 ꢁ 0.45),
A. alternata (73.1 ꢁ0.06) followed by F. oxysporum (71.9 ꢁ 0.02).
Similarly, compound 7d exhibited maximum mycelial inhibition in
Helminthosporium sp. (62.2 ꢁ 0.02) at 200
mg/ml followed by
g/ml. However, there was no
F. oxysporum (26.2 ꢁ 0.18) at 100
m
effect of compounds 7b and 7e at any concentration on Helmin-
thosporium sp. and A. alternata, respectively. Compound 7e showed
Fig. 1. In vitro antifungal assay of test compounds/reference at concentration 10 mg/ml.
maximum inhibition at 200 mg/ml with Helminthosporium sp. and
F. oxysporum, respectively (Table 5). It seems that these nitrogen
containing heterocyclic compounds are the reservoir of effective
chemotherapeutants that can be used as pesticides.
The data reported in Table 5 and Fig. 1 revealed that compound
7a is the most active compound as compared to other compounds
and standard drug against all pathogenic fungal strains tested. To
probe the structureeactivity relationship of pyrazol-10-ylpyrazolo
[1,5-a]pyrimidines versus fungi, the preferred substitution of the
position-50 of pyrazole and position-7 of pyrimidine ring is a methyl
group which is the most potent inhibitor (7a). Results indicate that
7a with over 2.0, 2.6 and 3.4-fold potency than Mancozeb at 10 mg/
ml against F. oxysporum, A. alternata and Helminthosporium sp.,
respectively, could be a promising antifungal agent. Replacement of
CH3 group with phenyl ring in 7b causes huge loss of antifungal
activity against all pathogenic fungal strains tested. Inclusion of
a para-substituted phenyl ring (either electron-donating OCH3
group 7d or electron-withdrawing Cl 7e) regardless of the size,
electronic nature or difference in lipophilicity of substituent,
potency further seems to reduce the antifungal activity.
Table 5
Antifungal activity (% inhibition) of pyrazol-10-ylpyrazolo[1,5-a]pyrimidines.
Compound Control
10
m
g/ml
50
mg/ml
100
m
g/ml
200 mg/ml
Aspergillus terreus
7a
7b
7d
7e
0a (ꢁ0.20) 9.87 (ꢁ0.20) 33.7 (ꢁ0.44) 68.5 (ꢁ0.27) 73.2 (ꢁ0.45)
4. Conclusion
0a (ꢁ0.20) 7.4 (ꢁ0.15) 13.5 (ꢁ0.05) 19.2 (ꢁ0.53) 19.7 (ꢁ0.27)
0a (ꢁ0)
0a (ꢁ0)
2.5 (ꢁ0.02) 6.25 (ꢁ0.02) 7.5 (ꢁ0.02) 11.3 (ꢁ0.05)
In summary, a series of new pyrazol-10-ylpyrazolo[1,5-a]pyrim-
idines was synthesized regioselectively in an efficient and envi-
ronmental benign manner using water as solvent without any
additives or catalysts and their antimicrobial properties were eval-
uated. Structureeactivity studies showed that the antimicrobial
potency was mainly influenced by the substituents at 50 and
7-positions of pyrazol-10-ylpyrazolo[1,5-a]pyrimidines. The results
suggest that compound 7a exhibited more potent and pronounced
0 (ꢁ0)
0 (ꢁ0)
2.5 (ꢁ0.02) 12.5 (ꢁ0.02)
48.31 76.40
Mancozeb 0a (ꢁ0)
13.48
32.58
Alternaria alternata
7a
7b
7d
7e
0a (ꢁ0.37) 47.5 (ꢁ0.09) 69.5 (ꢁ0.05) 71.6 (ꢁ0.07) 73.1 (ꢁ0.06)
0a (ꢁ0.37) 18.2 (ꢁ0.35) 20.7 (ꢁ0.33) 19.5 (ꢁ0.17) 20.7 (ꢁ0.53)
0a (ꢁ0)
0a (ꢁ0)
0 (ꢁ0)
0 (ꢁ0)
18.18
0 (ꢁ0)
0 (ꢁ0)
29.24
11.1 (ꢁ0)
0 (ꢁ0)
11.1 (ꢁ0)
0 (ꢁ0)
Mancozeb 0a (ꢁ0)
45.45
65.90
Fusarium oxysporum
antifungal activity in vitro at the concentration of 10 mg/ml with
7a
7b
7d
7e
0a (ꢁ0.12) 56.0 (ꢁ0.11) 67.0 (ꢁ0.03) 67.0 (ꢁ0.04) 71.9 (ꢁ0.02)
respect to reference drug Mancozeb and it may be considered as
a promising lead for further design and development of new agri-
cultural fungicides. Compounds 7c and 7e exhibited promising
antimicrobial activity/lower MIC values comparable to commercial
antibiotics Gentamicin and Linezolid.
0a (ꢁ0.12) 8.53 (ꢁ0.02) 6.09 (ꢁ0.31) 3.65 (ꢁ0.04) 2.40 (ꢁ0.10)
0a (ꢁ0)
0a (ꢁ0)
6.55 (ꢁ0.05) 3.27 (ꢁ0.08) 26.2 (ꢁ0.18) 20.0 (ꢁ0.04)
1.63 (ꢁ0.04) 3.27 (ꢁ0.02) 9.8 (ꢁ0.02) 16.3 (ꢁ0.04)
Mancozeb 0a (ꢁ0)
Helminthosporium sp.
27.77
42.22
62.22
81.11
7a
7b
7d
7e
0a (ꢁ0.02) 42.2 (ꢁ0.17) 66.6 (ꢁ0.29) 79.2 (ꢁ0.16) 79.2 (ꢁ0.06)
0a (ꢁ0.02) 0 (ꢁ0)
0 (ꢁ0)
0 (ꢁ0)
0 (ꢁ0)
0 (ꢁ0)
5. Experimental
0a (ꢁ0)
0a (ꢁ0)
0 (ꢁ0)
27.7 (ꢁ0.25) 62.2 (ꢁ0.02)
11.1 (ꢁ0.04) 15.5 (ꢁ0.06) 17.7 (ꢁ0.02) 36.6 (ꢁ0.21)
Mancozeb 0a (ꢁ0)
12.22
24.44
44.44
70.00
Melting points were determined in open capillaries and are
uncorrected. IR spectra were recorded on a Buck Scientific IR M-500
spectrophotometer in KBr pellets (nmax in cmꢀ1), 1H and 13C NMR
‘ꢁ’ SEM.
a
Average of three replications each.

3044
R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 3038e3046
spectra for analytical purpose were recorded in CDCl3 on a Bruker
instrument at 300 MHz and 75 MHz, respectively. High resolution
NMR spectra were recorded on a Bruker DRX 400 spectrometer at
393 (Mþ). Anal. Calcd. for C25H23N5: C, 76.31; H, 5.89; N, 17.80.
Found: C, 76.76; H, 5.69; N, 17.67.
400.13 MHz for 1H, 100.62 MHz for 13C. Chemical shifts (
d
in ppm)
5.1.4. 3-Methyl-5-(4-methylphenyl)-1H-pyrazole (9c)
are given from internal solvent, CDCl3 7.26 for 1H and 77.0 for 13C.
Coupling constants (J) are given in hertz (Hz). High resolution mass
spectra (HRMS) were measured in EI mode on a Kratos MS-50
spectrometer. Yields are calculated assuming 1 equiv of 6beh is
reacting with 1 equiv of 5.
M.p. 130e132 ꢂC (lit. [26,27] m.p. 118e119 ꢂC); yield 28%.
5.1.5. 2-(30-Methyl-50-(400-methoxyphenyl)pyrazol-10-yl)-5-methyl-
7-(400-methoxyphenyl)pyrazolo[1,5-a]pyrimidine (7d)
M.p.172e174 ꢂC; yield 62%. IR (cmꢀ1): 3131, 2931,1613,1508,1250
(OCH3).1HNMR (CDCl3,
d
): 2.31 (s, 3H, C3 eCH3), 2.52 (s, 3H, C5eCH3),
0
2-Acetylcyclopentanone (6h) is commercially available. Other
aryl/heteroaryl-1,3-diketones (6beg) [34] and 3(5)-amino-5(3)-
hydrazinopyrazole dihydrochloride (5) [19] were prepared
according to the literature procedures.
0
3.73 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 6.16 (s, 1H, C4 eH), 6.48 (s, 1H,
C3eH), 6.66 (s, 1H, C6eH), 6.78e6.84 (m, 4H, PheH), 7.21e7.24 (dd,
2H, Jo ¼ 8.4 Hz, Jm ¼ 2.1 Hz, PheH), 7.63e7.66 (dd, 2H, Jo ¼ 8.1 Hz,
Jm ¼ 2.1 Hz, PheH). HRMS (m/z): 426.1921 (M þ 1)þ (C25H23N5O2
(M þ 1)þ requires 426.193). Anal. Calcd. for C25H23N5O2: C, 70.57; H,
5.45; N, 16.46. Found: C, 70.36; H, 5.76; N, 16.21.
5.1. Synthesis of 2-(30,50-disubstituted pyrazol-10-yl)-5,7-
disubstituted pyrazolo[1,5-a]pyrimidines (7)
5.1.6. 3-Methyl-5-(4-methoxyphenyl)-1H-pyrazole (9d)
M.p. 112e114 ꢂC (lit. [26,27] m.p. 70e73 ꢂC, [36] 115e116 ꢂC, [37]
111 ꢂC); yield 25%.
5.1.1. 2-(30-Methyl-50-phenylpyrazol-10-yl)-5-methyl-7-
phenylpyrazolo[1,5-a]pyrimidine (7b)
To H2O (20 ml) were added 3(5)-amino-5(3)-hydrazinopyrazole
dihydrochloride (5) (0.93 g, 0.005 mol) and CH3COCH2COC6H5 (6b)
(0.81 g, 0.005 mol). This reaction mixture was found to have pH 0.8
at 25 ꢂC. Then the reaction mixture was refluxed for 4 h. After
completion of the reaction (monitored by TLC), the reaction
mixture was cooled to room temperature and extracted using
2 ꢃ 30 ml portions of ethyl acetate. The combined organic layers
were successively washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated to give a residual mass. The
residue obtained on cooling was found to be a mixture of three
products as predicted by TLC and 1H NMR spectrum. The residue
was column chromatographed over silica gel (100e200 mesh)
using petroleum ether followed by petroleum ether/CHCl3 of
increasing polarity as eluent to yield three compoundse benzoic
acid 10b m.p. 120e121 ꢂC (lit. [35] m.p. 121e123 ꢂC) in the first
fraction, followed by 3-methyl-5-phenyl-1H-pyrazole (9b) m.p.
129e130 ꢂC; (lit. [26e28] m.p. 128 ꢂC); yield 8% and finally 2-(30-
methyl-50-phenylpyrazol-10-yl)-5-methyl-7-phenylpyrazolo[1,5-a]
pyrimidine (7b).
5.1.7. 2-(30-Methyl-50-(400-chlorophenyl)pyrazol-10-yl)-5-methyl-7-
(400-chlorophenyl)pyrazolo[1,5-a]pyrimidine (7e)
M.p. 170e172 ꢂC; yield 55%. IR (cmꢀ1): 3150, 2925, 1615, 1540,
1488, 1091. 1H NMR (CDCl3,
d
): 2.32 (s, 3H, C3 eCH3), 2.57 (s, 3H,
0
0
C5eCH3), 6.19 (s, 1H, C4 eH), 6.66 (s, 1H, C3eH), 6.69 (s, 1H, C6eH),
7.19e7.22 (d, 2H, Jo ¼ 8.7 Hz, PheH), 7.23e7.26 (d, 2H, Jo ¼ 8.7 Hz,
PheH), 7.29e7.32 (d, 2H, Jo ¼ 8.4 Hz, PheH), 7.50e7.53 (dd, 2H,
Jo ¼ 8.7 Hz, Jm ¼ 1.8 Hz, PheH). HRMS (m/z): 434.0945 (M þ 1)þ
(C23H17N5Cl2 (M þ 1)þ requires 434.0939). Anal. Calcd. for
C23H17N5Cl2: C, 63.60; H, 3.95; N, 16.12. Found: C, 63.32; H, 3.66; N,
16.27.
5.1.8. 3-Methyl-5-(4-chlorophenyl)-1H-pyrazole (9e)
M.p. 150e160 ꢂC (Lit. [28] m.p. 147e148 ꢂC); yield 32%.
5.1.9. 2-(30-Methyl-50-(400-bromophenyl)pyrazol-10-yl)-5-methyl-7-
(400-bromophenyl)pyrazolo[1,5-a]pyrimidine (7f)
M.p.194 ꢂC; yield 50%. IR (cmꢀ1): 3147, 2918,1617, 1523, 1486. 1H
0
NMR (0CDCl3,
d
): 2.32 (s, 3H, C3 eCH3), 2.59 (s, 3H, C5eCH3), 6.20 (s,
5.1.2. 2-(30-Methyl-50-phenylpyrazol-10-yl)-5-methyl-7-
1H, C4 eH), 6.68 (s, 1H, C3eH), 6.69 (s, 1H, C6eH), 7.14e7.17 (d, 2H,
Jo ¼ 8.4 Hz, PheH), 7.41e7.52 (m, 6H, PheH). MS (m/z): 521 (Mþ).
Anal. Calcd. for C23H17N5Br2: C, 52.80; H, 3.27; N, 13.39. Found: C,
52.42; H, 3.72; N, 13.61.
phenylpyrazolo[1,5-a]pyrimidine (7b)
M.p. 179e180 ꢂC; yield 76%. IR (cmꢀ1): 3038, 2924, 1616, 1519,
1414. 1H NMR0(CDCl3,
d
): 2.39 (s, 3H, C3 eCH3), 2.61 (s, 3H, C5eCH3),
0
6.28 (s, 1H, C4 eH), 6.61 (s, 1H, C3eH), 6.75 (s, 1H, C6eH), 7.31e7.46
(m, 8H, PheH), 7.63 (m, 2H, PheHo). MS (m/z): 365 (Mþ). Anal.
Calcd. for C23H19N5: C, 75.59; H, 5.24; N, 19.16. Found: C, 75.82; H,
5.57; N, 19.51.
5.1.10. 3-Methyl-5-(4-bromophenyl)-1H-pyrazole (9f)
M.p. 132e134 ꢂC; yield 34%. IR (cmꢀ1): 3183, 3101, 2979, 1587,
1445. 1H NMR (CDCl3,
d): 2.26 (s, 3H, CH3), 6.26 (s, 1H, C4eH),
All other compounds (7ceh) were synthesized according to the
procedure mentioned for 7b using 3-amino-5-hydrazinopyrazole
7.41e7.44 (d, 2H, Jo ¼ 8.7 Hz, Ph-20, 60-H), 7.51e7.54 (d, 2H,
Jo ¼ 8.7 Hz, Ph-30, 50-H). 13C NMR (CDCl3,
d): 11.43 (CH3),102.05 (C4),
dihydrochloride (5) and different
b
-diketones (6c-h). After the
121.69 (C-40), 127.16 (C-20, C-60), 128.89 (C-10), 131.77 (C-30, C-50),
142.22 (C5), 149.33 (C3).
reaction was complete (from TLC), the mixture was cooled and
extracted with EtOAc (2 ꢃ 30 ml). The combined organic layers
were dried over anhydrous Na2SO4. The solvent was evaporated
and the crude product was purified by recrystallization or column
chromatography. The characterization data for 7ceh and 9ceg are
presented below:
5.1.11. 2-(30-Methyl-50-(thien-200-yl)pyrazol-10-yl)-5-methyl-7-
(thien-200-yl)pyrazolo[1,5-a]pyrimidine (7g)
M.p. 149e150 ꢂC; yield 58%. IR (cmꢀ1): 2930, 2852, 1687, 1603,
1426. 1H NMR (CDCl3,
d
): 2.40 (s, 3H, C3 eCH3), 2.64 (s, 3H, C5eCH3),
0
0
6.40 (s, 1H, C4 eH), 6.63 (s, 1H, C3eH), 6.98e7.02 (dt, 1H, Jo ¼ 5.1 Hz,
TheH), 7.09 (s, 1H, C6eH), 7.12e7.14 (dd, 1H, Jo ¼ 3.6 Hz, Jo ¼ 1.2 Hz,
TheH), 7.15e7.18 (dt, 1H, Jo ¼ 5.1 Hz, TheH), 7.31e7.33 (dd, 1H,
Jo ¼ 5.1 Hz, Jo ¼ 0.9 Hz, TheH), 7.59e7.61 (dd, 1H, Jo ¼ 5.1 Hz, TheH),
8.15e8.17 (dd, 1H, Jo ¼ 3.9 Hz, Jo ¼ 0.9 Hz, TheH). HRMS (m/z):
378.085 (M þ 1)þ (C19H15N5S2 (M þ 1)þ requires 378.0847). Anal.
Calcd. for C19H15N5S2: C, 60.45; H, 4.01; N, 18.55. Found: C, 63.32; H,
3.71; N, 18.27.
5.1.3. 2-(30-Methyl-50-(400-methylphenyl)pyrazol-10-yl)-5-methyl-
7-(400-methylphenyl)pyrazolo[1,5-a]pyrimidine (7c)
M.p. 171e172 ꢂC; yield 60%.0IR (cmꢀ1): 3038, 2924, 1615, 1518. 1H
NMR (CDCl3,
d
): 2.31 (s, 3H, C3 eCH3), 2.32 (s, 3H, CH3), 2.33 (s, 3H,
0
CH3), 2.57 (s, 3H, C5eCH3), 6.17 (s, 1H, C4 eH), 6.48 (s, 1H, C3eH),
6.67 (s, 1H, C6eH), 7.05e7.13 (m, 4H, PheH), 7.16e7.19 (d, 2H,
Jo ¼ 8.1 Hz, PheH), 7.51e7.54 (d, 2H, Jo ¼ 8.4 Hz, PheH). MS (m/z):

R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 3038e3046
3045
5.1.12. 3-Methyl-5-(2-thienyl)-1H-pyrazole (9g)
compound) and a negative control (medium only, without inoc-
ulum) were also prepared. Appropriately diluted each test sample
was added to 20 ml of warm, melted, autoclaved MHA (separate
flasks were taken for each dilution). After thorough mixing,
medium was poured in sterilized Petri plates. Bacterial inoculums
were spotted in a predefined pattern by aseptically transferring
M.p. 139e140 ꢂC; yield 35%. IR (cmꢀ1): 3179, 3083, 2932, 1584,
1423. 1H NMR (CDCl3,
d): 2.28 (s, 3H, CH3), 6.25 (s, 1H, C4eH),
7.01e7.04 (m, 1H, TheH), 7.21e7.28 (m, 2H, TheH). MS (m/z): 164
(Mþ).
5.1.13. 2-(30-Methyl-50,60-dihydro-4H-cyclopenta[d]pyrazol-10-yl)-
5-methyl-7,8-dihydro-6H-cyclopenta[g]pyrazolo[1,5-a]pyrimidine
(7h)
100 ml of each bacterial culture on the surface of presolidified agar
plates. The plates were incubated in at 37 ꢁ 1 ꢂC and the MICs were
recorded by visual observations after 24 h. MICs values were the
lowest concentration of compounds inhibiting visible growth of
organism and are listed in Table 4.
M.p. 250 ꢂC; yield 75%. IR (cmꢀ1)0: 3113, 2938, 1627, 1552, 1366.
1H NMR (CDCl3,
d): 2.24 (s, 3H, C3 eCH3), 2.46 (s, 3H, C5eCH3),
2.52e2.62 (m, 5H, CyclopentyleH), 2.89e2.98 (m, 5H, Cyclo-
pentyleH), 3.29e3.34 (m, 2H, CyclopentyleH), 6.50 (s, 1H, C3eH).
HRMS (m/z): 294.1714 (M þ 1)þ (C17H19N5 (M þ 1)þ requires
294.1719). Anal. Calcd. for C17H19N5: C, 69.60; H, 6.53, N, 23.87.
Found: C, 69.93; H, 6.77; N, 24.12.
6.2. Evaluation of antifungal activity
The pathogenic fungi were isolated from the soil of Kurukshetra.
Potato dextrose agar (PDA) medium was autoclaved at 15 psi for
15 min. All the fungal isolates were inoculated on PDA plates in
triplicates and incubated at 28 ꢁ 1 ꢂC for 6 days to obtain young,
actively growing colonies of molds. Each of the test compound first
prepared as concentrated suspension or solution (stock solution
1 mg/ml in DMSO) was added to 15e20 ml of molten agar media at
40 ꢁ 2 ꢂC into 9.0 cm diameter sterile Petri plates to achieve desired
6. Biological activity
6.1. Evaluation of antibacterial assay: preliminary screening
The bacterial isolates representing Gram-negative and Gram-
positive bacteria were maintained and stored at 5e8 ꢂC on Brain
Heart Infusion Agar (HI-media). The screening of eight compounds
7aeh was done in vitro using the agar-well diffusion method [38].
The stock solutions (2 mg/ml) of the test compounds were
prepared by dissolving 2 mg of test compound in 1 ml of dimethyl
concentrations (10, 50, 100 and 200 mg/ml) and allowed the media
to solidify. A mycelial disc of diameter 6 mm, cut from the
periphery of 6 day old culture, was aseptically inoculated at the
center of each Petri plate. For each treatment three replicates were
maintained. Mancozeb was used as a standard and PDA medium
without the test compound or with DMSO were served as the
control. The inoculated Petri dishes were incubated in a biochem-
ical oxygen demand (BOD) incubator at 28 ꢁ 1 ꢂC for the growth of
pathogen. The radial growth of the colony was measured in mm
and recorded after 7 days of incubation. Percentage of mycelial
growth inhibition was calculated by using the formula:
sulfoxide (DMSO). All samples were sterilized through a 0.2 mm
membrane filter and stored at 4 ꢂC until further use. Bacterial
inoculums were prepared from 24 h old cultures and turbidity was
adjusted equivalent to 0.5 McFarland turbidity standard, i.e.
1 ꢃ106 CFU/ml [39].
By inoculating 100 ml of each test bacterial culture in 20 ml of
warm, melted, autoclaved Mueller Hinton Agar (HI-media) seed
layers were prepared (separate flasks were used for each bacterial
culture). After mixing, it was poured in sterilized and labeled Petri
plates (150 mm ꢃ 20 mm). The wells of 6 mm were punched in the
solidified Petri plates with the help of a sterile steel borer. With the
Percent inhibition ¼ C ꢀ T ꢃ 100/C
where, C ¼ Average increase in mycelial growth in control, and
T ¼average increase in mycelial growth in the presence of test
compound.
help of micropipette, 100 ml of each test compound (stock 2 mg/ml)
was added aseptically to the individual wells. The loaded plates
were incubated in upright position at 37 ꢁ 1 ꢂC for 24 h. The
diameter of the zone of growth inhibition around each well after
incubation was measured in mm using a Vernier Caliper (Table 3).
Gentamicin (4 mg/ml) and Linezolid (1 mg/ml in water) were used
as standard antibiotic and DMSO as a negative control under similar
conditions for comparison.
Acknowledgments
We are thankful to University Grants Commission, New Delhi,
India for financial assistance and the Mass Spectrometry Facility,
University of California, San Francisco, USA for running the mass
spectra and SAIF, CDRI, Lucknow for providing elemental analysis.
6.1.1. Determination of minimum inhibitory concentration (MIC)
MICs were determined by the broth microdilution method
according to the National Committee for Clinical Laboratory Stan-
dards (NCCLS) recommendations [40]. Mueller Hinton broth
(HI-media) was used as the test medium. Testing was performed at
pH 7.4 ꢁ 0.1. The inoculums were prepared using a 16 h broth
culture of each bacterial strains adjusted to a turbidity equivalent to
a 0.5 McFarland standard, diluted in Mueller Hinton Agar broth
media to give concentration of 1 ꢃ106 CFU/ml for bacteria. For
antibacterial assay the final inoculum size was 105 CFU/ml. The test
compounds were dissolved in DMSO to obtain 4 mg/ml stock
solutions. Serial twofold dilutions of the test compounds in distilled
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