P-Toluenesulfonic acid-catalyzed solvent-free synthesis and biological evaluation of new 1-(4′,6′-dimethylpyrimidin-2′-yl)-5-amino- 4H-3-arylpyrazole derivatives

Article abstract of DOI:10.1007/s00044-013-0751-9

A solvent-free quick synthesis of 1-(4′,6′-dimethylpyrimidin- 2′-yl)-5-amino-4H-3-arylpyrazoles (3) was accomplished by grinding 2-hydrazino-4,6-dimethylpyrimidine (1) and β-ketonitriles (2) in the presence of p-Toluenesulfonic acid as a catalyst. Subsequently, 5-aminopyrazoles (3) were converted to their corresponding N-acetamide (4) and N-trifluoroacetamide (5) derivatives by treating (3) with acetic anhydride/acetic acid and trifluoroacetic anhydride/trifluoroacetic acid, respectively. The newly synthesized compounds were fully characterized using IR, NMR (¹H, ¹³C, and ¹⁹F), mass spectral studies, and elemental analyses. All of the fifteen compounds were screened for their in vitro antibacterial activity against two Gram-positive and two Gram-negative pathogenic bacteria such as Bacillus pumilus, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, respectively. Additionally, nine of these compounds were screened for their cytotoxicity against the human Caucasian promyelocytic leukemia (HL-60) cell line using the alamarBlue assay. Preliminary results reveal that compounds 3a, 3b, 3d, 5a, and 5d are showing moderate-to-significant cytotoxic and antibacterial activity.

Full text of DOI:10.1007/s00044-013-0751-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1454–1464
DOI 10.1007/s00044-013-0751-9
ORIGINAL RESEARCH
p-Toluenesulfonic acid-catalyzed solvent-free synthesis
and biological evaluation of new 1-(40,60-dimethylpyrimidin-
20-yl)-5-amino-4H-3-arylpyrazole derivatives
•
Ranjana Aggarwal Anshul Bansal Isabel Rozas
•
•
•
•
•
Elena Diez-Cecilia Amanjot Kaur Ritu Mahajan
Jitender Sharma
Received: 29 April 2013 / Accepted: 19 August 2013 / Published online: 1 September 2013
Ó Springer Science+Business Media New York 2013
Abstract A solvent-free quick synthesis of 1-(40,60-dim-
ethylpyrimidin-20-yl)-5-amino-4H-3-arylpyrazoles (3) was
accomplished by grinding 2-hydrazino-4,6-dimethylpyr-
imidine (1) and b-ketonitriles (2) in the presence of p-
Toluenesulfonic acid as a catalyst. Subsequently, 5-amin-
opyrazoles (3) were converted to their corresponding N-
acetamide (4) and N-trifluoroacetamide (5) derivatives by
treating (3) with acetic anhydride/acetic acid and trifluo-
roacetic anhydride/trifluoroacetic acid, respectively. The
newly synthesized compounds were fully characterized
using IR, NMR (1H, 13C, and 19F), mass spectral studies,
and elemental analyses. All of the fifteen compounds were
screened for their in vitro antibacterial activity against two
Gram-positive and two Gram-negative pathogenic bacteria
such as Bacillus pumilus, Staphylococcus aureus, Pseu-
domonas aeruginosa, and Escherichia coli, respectively.
Additionally, nine of these compounds were screened for
their cytotoxicity against the human Caucasian promyelo-
cytic leukemia (HL-60) cell line using the alamarBlueÒ
assay. Preliminary results reveal that compounds 3a, 3b,
3d, 5a, and 5d are showing moderate-to-significant cyto-
toxic and antibacterial activity.
Keywords Solvent-free synthesis ꢀ
1-(40,60-Dimethylpyrimidin-20-yl)-5-amino-4H-3-
arylpyrazoles ꢀ N-acetamide/trifluoroacetamide
derivatives ꢀ p-Toluenesulfonic acid ꢀ
Antibacterial activity ꢀ Cytotoxicity
Introduction
Pyrazole is an important structural motif, which is found as
an integral part of numerous pharmaceutically interesting
compounds displaying antimicrobial (Liu et al., 2008; Gil-
bert et al., 2006), anti-inflammatory (Farag et al., 2008),
antileukemic (Manetti et al., 2008), antidiabetic (Soliman
and Darwish 1983), and antitumor (Benaamane et al. 2008)
activities. Among pyrazole derivatives, 5-aminopyrazoles
have attracted considerable attention because of their enor-
mous applications as pharmaceuticals (Goldstein et al.,
2009) and agrochemicals (Lee et al., 2003) as they have been
found to exhibit antibacterial (Kane et al., 2003; Kumar
et al., 2005; Kumar et al., 2006), anticonvulsant (Michon
et al., 1995), antidepressant (Abdel-Aziz et al. 2009), or
herbicidal (Jung et al., 2002) activities. For example, several
5-aminopyrazoles have been reported to be potent and
selective inhibitors of the p38a kinase (Das et al., 2010).
Moreover, the structurally simple 1-(4-methylphenyl)-5-
aminopyrazole (Fig. 1a) has been described as an antagonist
of the Neuropeptide Y receptor type 5 (NPY5) (Kordik
et al., 2001), whereas 5-amino-4-(3-methoxyphenyl)-3-
thiomethyl-1-(2,6-dichloro-4-trifluoromethylphenyl)pyra-
zole (Fig. 1b), a potential insecticide, exerts potent GABA
inhibition with selectivity toward the insect versus the
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0751-9) contains supplementary
material, which is available to authorized users.
R. Aggarwal (&) ꢀ A. Bansal
Department of Chemistry, Kurukshetra University,
Kurukshetra 136 119, India
e-mail: ranjana67in@yahoo.com; ranjanaaggarwal67@gmail.com
I. Rozas ꢀ E. Diez-Cecilia
Trinity Biomedical Sciences Institute, School of Chemistry,
Trinity College Dublin, 154-160 Pearse St., Dublin 2, Ireland
A. Kaur ꢀ R. Mahajan ꢀ J. Sharma
Department of Biotechnology, Kurukshetra University,
Kurukshetra 136 119, India
123

Med Chem Res (2014) 23:1454–1464
1455
Fig. 1 Structures of
H
3C
5-aminopyrazoles (a and b) and
pyrimidines (c and d) of
pharmaceutical relevance
F
3C
Cl
N
N
N
N
H2N
SCH3
Cl
H2N
OCH3
HN
S
O
O
(b)
(a)
S
O
N
O
N
S
N
N
N
NH
N
N
N
N
N
H
3C
H
3C
H
3C
N
N
N
S
NH2
S
O
O
O
O
(c)
(d)
mammalian receptor (Meegalla et al., 2004). Besides their
biological importance, they are also useful synthons for the
preparation of many biologically important fused hetero-
cycles such as pyrazolo[3,4-d]pyrimidines, pyrazolo[3,4-
b]pyridines or imidazopyrazoles (An et al., 2008; Abu
Almaati and El-Taweel 2004; Sellery et al., 2005; Gopals-
amy et al. 2005). Furthermore, many pyrazole derivatives,
especially those bearing aryl groups, are important for the
creation of promising new antitumor agents (Barril et al.,
2006; Sherif 2006; Dymock et al., 2005). For example, some
N-arylpyrazoles have remarkable activities as tumor necro-
sis inhibitors (Mercep et al., 2004), and also it has been
reported that pyrazole derivatives showed significant activ-
ity toward Ehrlich ascites carcinoma tumor cells (Farag
et al., 2008). It has also been found that some 3-arylpyrazoles
suppressed A549 lung cancer cell growth (Wei et al., 2006).
Pyrimidine derivatives, the fundamental building blocks
for DNA and RNA, also occupy a prominent place in the
pharmaceutical area (Zoltewicz and Uray 1994). They have
attracted a great deal of attention over many years due to
their broad bioactivities, including antitumor (Lindvall
et al., 2011; Heffron et al., 2010), antimicrobial (Shetty
et al., 2009), or anti-inflammatory (El-gazzar et al., 2007).
In particular, 4-morpholinothieno[3,2-d]pyrimidines such
as GDC-0941 and GNE-477 (Fig. 1c and d, respectively)
have shown marked antitumor activities (Heffron et al.,
2010; Hayakawa et al., 2006). For example, GDC-0941,
which is a potent, selective, and orally bioavailable
inhibitor of PI3 K, exerts antiproliferative effects against
an array of human tumor cell lines and is currently
undergoing Phase I clinical trials (Junttila et al., 2009).
Taking all this into account, we present the synthesis of
a number of pyrazoles carrying both a pyrimidine and an
amino group at positions 1 and 5 of the pyrazole ring,
respectively. Our final aim is to find new and more potent
antibacterial and cytotoxic agents.
A recent overview of current methods for the synthesis of
5-aminopyrazoles (Aggarwal et al., 2011) reveals that
common routes involve the reactions of b-ketonitriles,
malononitrile, alkylidenemalononitriles, and their deriva-
tives with hydrazines in the presence of hydrochloric or
acetic acid using ethanol as a solvent. Though these reac-
tions afford 5-aminopyrazoles, they take 8–10 h for
completion and the workup is cumbersome. Recently, sol-
vent-free synthesis has gained much attention due to
advantage over the conventional methods in terms of
enhanced selectivity, efficiency, cleaner reaction profiles,
ease of manipulation, and relatively benign conditions
compared to their solution counterparts. In view of these
observations and continuing with our work on solvent-free
synthesis of heterocyclic compounds (Aggarwal et al.,
2007), we have developed a solvent-free protocol for the
preparation of the mentioned 5-amino-1-pyrimidinopyr-
azole derivatives 3 using p-Toluenesulfonic acid (PTSA) as
a catalyst. Further, the synthesis of their N-acetamide 4 and
N-trifluoroacetamide 5 derivatives was considered as it has
been observed that the acyl moiety, an integral part of many
drugs, enhances the biological activity, e.g., acylation
123

1456
Med Chem Res (2014) 23:1454–1464
converts the natural painkiller morphine into the far more
compounds 4a–e confirmed the incorporation of acetamide
by showing the strong absorption band at 1,690–
1,700 cm-1 due to C=O stretch and only one band at about
3,160–3,170 cm-1 due to N–H stretch. Finally, the corre-
sponding bands for the trifluoroacetamide derivatives
appeared at 1,620–1,640 and 3,110 cm-1 in the IR spectra
of 5a–e.
potent heroin (Kaiko et al., 1981) (diacetylmorphine).
Chemistry
The synthesis of 1-(40,60-dimethylpyrimidin-20-yl)-5-
amino-3-arylpyrazoles 3a–e, 1-(40,60-dimethylpyrimidin-
20-yl)-5-(N-acetamide)-3-arylpyrazoles 4a–e and 1-(40,60-
dimethylpyrimidin-20-yl)-5-(2,2,2-trifluoro-N-acetamide)-
3-arylpyrazoles 5a–e is outlined in Scheme 1.
1
The H NMR spectra of compounds 3a–e displayed a
characteristic sharp singlet integrating for one proton at d
5.9 ppm due to the H-4 of the pyrazole ring, besides two
singlets integrating for six and one protons at about d 2.5
and 6.8 ppm corresponding to the two –CH3 groups present
on the pyrimidine ring and the H-5 of this ring, respec-
tively. The corresponding signal for H-4 pyrazole for
compounds 4a–e and 5a–e appeared at d 7.30 and
6.10 ppm, respectively, as a sharp singlet. The significant
downfield shift of d 1.40 ppm in compounds 4a–e as
compared to 3a–e may be attributed to the weak hydrogen
bonding between the oxygen of the acetyl C=O group and
the H-4 of the pyrazole ring. Formation of such weak
hydrogen bonds has been previously reported (Burley and
Petsko 1988; Banks et al., 1999).
The corresponding starting compounds, the b-ketonitr-
iles 2a–e, were obtained by treating a-bromoacetophe-
nones with potassium cyanide (Gakhar et al., 1971). Then,
grinding 2a–e with 2-hydrazino-4,6-dimethylpyrimidine 1,
in the presence of PTSA as a catalyst, provided 1-(40,60-
dimethylpyrimidin-20-yl)-5-amino-3-arylpyrazoles 3a–e as
an exclusive product in excellent yields in just 2 h fol-
lowing a simple workup. Synthesis of the N-acetamide 4a–
e and N-trifluoroacetamide 5a–e derivatives was accom-
plished by treating the corresponding 5-aminopyrazoles
3a–e with acetic anhydride in the presence of acetic acid
and trifluoroacetic anhydride in the presence of trifluoro-
acetic acid, respectively. The structure and purity of all the
products 3a–e, 4a–e, and 5a–e was confirmed by the cor-
Further, the structure of compounds 3a–e was supported
by 13C NMR spectroscopy that showed signals at d 150, 87,
and 152–153 ppm due to C-3, C-4, and C-5 of the pyrazole
ring, respectively. These values are in accordance with the
literature (Kumar et al., 2006). The 13C NMR spectra of
4a–e displayed signals at d 151, 95, and 152–154 ppm due
to C-3, C-4, and C-5, respectively, whereas the corre-
sponding signals appeared at d 157, 93, and 160–162 ppm
for compounds 5a–e. The downfield shift in the value of
1
responding spectral data (IR, MS, H, 13C, and 19F NMR)
and elemental microanalysis.
The presence of the amino group in compounds 3a–
e was confirmed with the help of IR, which showed two
sharp absorption bands at 3,250 (symm.) and 3,400 cm-1
(asymm.) due to NH2 stretching. The IR spectra of
CH3
N
CH3
O
PTSA, Grinding
70-80 oC
N
+
N
CN
H3C
3a-e
N
N
R
R
H3C
N
NHNH2
H2N
1a-e
2a-e
(CF3CO)2O/
CF3COOH
(CH3CO)2O/
CH3COOH
CH3
CH3
N
2, 3, 4, 5
R
a
b
c
d
e
C6H5
p-(CH3)C6H4
p-FC6H4
p-ClC6H4
p-BrC6H4
N
N
R
N
H3C
N
N
H3C
N
N
R
HN
HN
O
O
H3C
F3C
4a-e
5a-e
Scheme 1 Synthesis of 5-aminopyrazoles 3a–e and their corresponding N-acetamide 4a–e and N-trifluoroacetamide 5a–e derivatives
123

Med Chem Res (2014) 23:1454–1464
1457
13
Table 1 C NMR d (ppm) data for 1-(40,60-dimethylpyrimidin-20-
Table 3 19F NMR d (ppm) data of compounds 5a–e
yl)-5-amino-3-arylpyrazoles 3a–e
Compound
CF3
5a
5b
5c
5d
5e
-75.63
4'
N
-75.11
-75.76
-75.92
-75.43
5'
3'
3''
2''
6''
2
1
N
N
1''
R
N
1'
2'
6'
4''
5''
C-4 in 4a–e as compared to 3a–e supported the existence of
the mentioned hydrogen bond between C–H and O atoms.
The complete assignment of the signals in the 13C NMR
spectra of the compounds 3a–e, 4a–e, and 5a–e is given in
Tables 1 and 2, respectively.
3
5
4
H2N
Compounds
3a
3b
3c
3d
3e
19F NMR spectroscopy helped in establishing the pres-
ence of a trifluoroacetyl group (COCF3) in compounds
5a–e. A sharp singlet at about d -75 ppm confirmed the
presence of CF3 group in these compounds (Table 3). (See
supplementary information).
C-3
150.3
87.5
150.2
87.4
150.4
87.3
150.4
87.3
150.5
87.3
C-4
C-5
C-20
C-40, 60
C-50
153.5
168.4
157.7
116.4
128.2
127.4
129.1
132.9
24.2
153.6
168.4
157.7
116.3
130.0
126.3
129.0
130.2
24.2
152.6
168.5
157.6
115.3
128.1
128.0
129.1
141.4
24.1
152.4
168.5
157.7
116.6
128.4
127.6
131.4
134.1
24.2
152.4
168.5
157.7
116.5
127.9
122.3
131.4
131.9
24.1
C-100
Biological results and discussion
C-200, 600
C-300, 500
C-400
Antibacterial activity
Pyrimidine –CH3
R (–CH3)
All the fifteen compounds were screened in vitro for their
antibacterial activity against four pathogenic bacterial
21.3
13
Table 2 C NMR d (ppm) data for 1-(40,60-dimethylpyrimidin-20-yl)-5-(N-acetamide)-3-arylpyrazoles 4a–e and 1-(40,60-dimethylpyrimidin-20-
yl)-5-(2,2,2-trifluoro-N-acetamide)-3-arylpyrazoles 5a–e
4'
N
3'
5'
3''
2
2''
6''
1
N
1''
R
6' N
1'
N
2'
4''
5''
3
5
4
5
X= H
X= F
4
HN
O
X
3C
Cmpd.
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
C-3
151.3
95.5
151.2
95.4
151.3
95.4
151.4
95.4
151.4
95.4
157.3
93.3
157.2
93.3
157.3
93.6
157.4
93.1
157.4
93.2
C-4
C-5
C-20
C-40/60
C-50
154.0
168.7
157.5
117.0
128.4
126.6
128.8
132.3
24.6
154.1
168.7
157.4
116.9
129.1
126.5
128.6
130.7
24.6
153.1
168.7
157.4
115.2
130.3
128.2
132.7
137.4
24.2
152.9
168.8
157.4
117.1
128.6
127.8
130.8
134.6
24.2
152.9
168.7
157.6
117.2
128.1
122.9
131.2
131.6
24.2
160.8
168.9
158.1
117.8
128.7
126.6
129.4
131.3
23.7
161.8
169.2
158.1
117.8
128.2
126.6
129.4
130.7
23.6
162.0
168.9
158.2
115.7
127.4
128.9
129.0
136.5
23.7
161.6
169.4
158.2
118.4
128.6
125.1
128.9
132.6
23.5
161.4
169.3
158.1
118.4
126.1
124.5
128.7
131.9
23.5
C-100
C-200/600
C-300/500
C-400
PyCH3
R(CH3)
CX3
–
20.6
–
–
–
21.4
–
–
24.2
24.2
23.5
23.6
23.7
124.6
170.2
124.2
169.8
124.5
170.4
124.4
170.6
124.3
170.0
CO
166.2
166.3
166.4
166.4
166.4
123

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Med Chem Res (2014) 23:1454–1464
Table 4 MIC values determined for compounds 3a–e, 4a–e, and
5a–e using agar well diffusion method
Table 5 Percentage cell viability for compounds 3a, b, d, 4a, b, d,
and 5a, b, d at 50 and 100 lM
Compound
MIC (lM)a
Compound
Conc. (lM)
% cell viability
SEM
B. pumilus S. aureus P. aeruginosa E. coli
3a
100
50
2.3
3.6
0.3
0.6
0.1
0.3
0.5
0.9
2.7
3.6
9.1
3a
0.377
1.79
3.53
3.34
2.91
NA
0.377
NA
0.018
0.018
3.53
3.77
NA
3b
3d
4a
4b
4d
5a
5b
5d
100
50
0.5
3b
2.4
3c
NA
NA
100
50
1.1
3d
NA
1.67
NA
7.7
3e
NA
1.16
2.91
NA
100
50
35.5
52.8
72.5
71.9
51.2
68.9
6.3
4a
NA
3.25
4b
NA
NA
3.11
NA
100
50
4c
3.07
2.93
1.29
2.77
1.33
1.31
2.53
2.27
0.002
NA
3.07
NA
10.1
4d
NA
2.19
NA
100
50
1.4
2.8
1.2
4.5
9.2
3.2
0.6
5.0
4e
NA
1.29
2.59
NA
5a
0.554
0.066
0.066
0.063
0.056
0.002
0.003
1.38
100
50
5b
0.533
0.527
0.506
0.022
0.004
0.003
NA
40.4
33.9
47.9
1.3
5c
NA
100
50
5d
NA
5e
NA
100
50
Ampicillin
0.002
0.003
11.4
Chloramphenicol 0.003
NA no activity
a
Mean of triplicates
of CH3, F, Cl, and Br on the phenyl ring of 5-(N-trifluo-
roacetamide)-3-phenylpyrazole 5a increases the antibacte-
rial activity to a significant extent.
strains, two Gram-positive [Bacillus pumilus (MTCC
10414) and Staphylococcus aureus (MTCC 96)] and two
Gram-negative [Pseudomonas aeruginosa (MTCC 741)
and Escherichia coli (MTCC 119)]. Different parameters
assessing the effect of compounds 3a–e, 4a–e, and
5a–e against those four bacterial strains were evaluated: the
diameter of zone of growth inhibition (see supplementary
information) and the minimum inhibitory concentration
(MIC), which is shown in Table 4. The antibacterial
activity of these compounds was compared with two ref-
erence antibiotics, ampicillin and chloramphenicol.
All the fifteen compounds showed moderate-to-excellent
activity against Gram-negative bacterium P. aeruginosa
(Table 4). Compounds 3a and 3b showed the best results
with very promising MIC values of 0.018 lM. Incorporation
of acetyl or trifluoroacetyl groups on these compounds
reduces their antibacterial activity; however, trifluoroacetyl
derivatives 5a and 5b (1.38 and 0.533 lM) are still more
active than the corresponding acetyl derivatives 4a and 4b
(3.25 and 3.11 lM). Compound 5e also showed promising
antibacterial activity with a MIC value 0.022 lM. Another
interesting feature that emerged out of the MIC data reveals
that while the incorporation of substituents on phenyl rings
in compounds 3a–e decreases the activity, this activity
increases significantly when the same substituents are
present on the trifluoroacetamide derivatives 5a–e.
A careful analysis of the data in Table 4 shows that all
the compounds, except 4a and 4b, exhibited moderate-to-
significant activity against B. pumilus, with MIC values
ranging from 0.37 to 3.53 lM. Compound 3a was found
to be the most active, whereas the corresponding amide
derivative 4a was found to be completely inactive toward
B. pumilus. However, the antibacterial activity was restored
by incorporating the trifluoroacetamide group as in com-
pound 5a (MIC 2.77 lM). This trend is again visible, when
comparing the MIC values against Gram-positive S. aureus
of 5-amino-3-phenylpyrazole 3a (0.37 lM), 5-(N-acetam-
ide)-3-phenylpyrazole 4a (no activity) and 5-(N-trifluoro-
acetamide)-3-phenylpyrazole 5a (0.55 lM).
Only compounds 3a, 3e, and 4e were found to be active
against E. coli (with the MIC values of 3.77, 2.91, and
2.59 lM, respectively, see Table 4).
Cytotoxicity in HL-60 cancer cells
Nine compounds (3a, b, d, 4a, b, d, and 5a, b, d) were
screened for their cytotoxicity in human Caucasian pro-
myelocytic leukemia (HL-60) cell line using the well-
established alamarBlueÒ assay (details can be found in the
Experimental section). The HL-60 cell line is an efficient
Compounds 5b–e were found to be the most effective
against S. aureus showing MIC values ranging from 0.056
to 0.066 lM (Table 4). This indicates that para substitution
123

Med Chem Res (2014) 23:1454–1464
1459
in vitro model, which has been used in the literature for the
evaluation of cytotoxic activity of different types of che-
motherapeutic agents such as DNA minor groove binders
(Hiraku et al., 2002), protein kinase inhibitors (Zhao et al.,
2011), and caspases inhibitors (Studzinski et al., 1986)
among others. This assay was chosen as a suitable model
for the assessment of the effect on cell viability of the
compounds here tested. For first general screening, the
viability of the compounds was evaluated at two concen-
trations, 100 lM and 50 lM. The results corresponding to
the percentage cell viability are presented in Table 5.
The cell viability data revealed that the toxicity of all the
compounds is directly related to the concentration. In
particular, compounds 3a, 3b, 3d, and 5d are more potent
(both at 100 and 50 lM) than the rest of the compounds in
the different series. For instance, at 100 lM concentration,
compounds 3a, 3b, 3d, and 5d show 2.3, 0.5, 1.1, and
1.3 % of viability, respectively. On the contrary, at the
same concentration, the rest of the compounds show cell
viability between 6.3 and 72.5 %, indicating that their
ability to generate toxicity in cancer cells is scarce. For this
reason, a more exhaustive study of the cytotoxicity of
compounds 3a, 3b, 3d, and 5d was further carried out.
Thus, a broader range of concentrations (100, 50, 40, 35,
30, and 20 lM) was chosen to assess the activity of these
compounds more in detail. In this way, the IC50 (half
maximal inhibitory concentration) of the drugs in the HL-
60 cell line was determined after 72-h treatment. The IC50
values represent the effectiveness of these compounds to
inhibit cell viability by half. The results obtained together
with those of furamidine (a DNA minor groove binder) and
sorafenib (a protein kinases inhibitor), which were used as
control, are shown in Fig. 2.
A careful analysis of the data, given in Fig. 2, shows
some interesting patterns about the cytotoxicity of the
compounds studied. Most of the compounds with the pri-
mary amine moiety (3a, b, d) were found to be very potent
against cancer cell proliferation. However, incorporation of
an amide group decreases their cytotoxicity even though
the introduction of the CF3 group in the amide produces
some improvements. Considering that the target by which
these molecules exert their activity is not known, some
insights into the nature of the active site of this target could
be hypothesized. It could be possible that the putative
active site has a residue with a hydrogen bond (HB)
acceptor group that can interact with the HB donor NH2
group. Additionally, the active site of the target could have
a lipophilic pocket, what could explain the best results
obtained with those compounds carrying a CF3 group.
These results are very encouraging, and thus, further
biological studies need to be carried out to identify the
specific target of these molecules cytotoxic activity; this
will allow the identification of the corresponding pharma-
cophore that will help to synthesize more potent molecules.
Conclusion
A new series of biologically active 1-heteroaryl-5-amino-
4H-3-arylpyrazoles 3a–e and their corresponding N-acet-
amide 4a–e and N-trifluoroacetamide 5a–e derivatives
were synthesized under solvent-free conditions using p-
Toluenesulfonic acid as a catalyst.
The antibacterial activity of all the compounds prepared
was tested against B. pumilus, S. aureus, P. aeruginosa,
and E. coli. The potency observed for these compounds
was mainly influenced by the substituents at position 5
(NH2, NHCOCH3, or NHCOCF3) and 3 (various substi-
tuted phenyl groups) of the pyrazole ring. While the
introduction of an N-acetyl group results in decrease or
complete loss of activity, incorporation of the corre-
sponding N-trifluoroacetyl group restores or increases the
As can be seen in Fig. 2, the IC50 values measured for
compounds 3a, 3b, 3d, and 5d are relatively similar and
around 30 lM. Compare to known drugs such as furami-
dine or sorafenib, the cytotoxicity of these compounds,
even though not outstanding, it is promising and future
derivatives could improve it.
Fig. 2 Graph representing %
cell viability versus logarithm of
the concentration and IC50
values obtained for compounds
3a, 3b, 3d, and 5d as well as for
furamidine and sorafenib
IC50 (µM)
Compound
IC50 value
SEM
100
80
60
40
20
0
3a
3b
3d
5d
3a
3b
42.6 1.3
28.1 1.3
31.3 1.2
32.5 1.2
77.6 1.15
1.68 1.27
3d
5d
Furamidine
Sorafenib
1.2
1.4
1.6
1.8
2.0
2.2
Log of Conc. [µM]
123

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Med Chem Res (2014) 23:1454–1464
activity. Additionally, the substitution on the phenyl ring
decreases the antibacterial activity in 5-aminopyrazoles
3a–e, whereas these substituents cause an increase in the
activity of N-trifluoroacetamide pyrazoles 5a–e.
monitored by tlc. On completion of the reaction, the crude
product was washed with water, filtered, and crystallized
1
using ethanol. The tlc and H NMR spectra showed the
formation of a single product.
The cytotoxicity of some of the compounds prepared
(3a,b, d, 4a, b, d, and 5a, b, d) was evaluated against the
human leukemia HL-60 cell line. First, a general screening
of the effect of these compounds on the viability of the HL-
60 cells was performed and then the IC50 values of the
most interesting compounds (3a, b, d, and 5d) were mea-
sured. A structure activity study showed that the amino
derivatives 3a, b, d are the most cytotoxic; the introduction
of a N-acetyl group decreases cytotoxicity, while intro-
ducing N-trifluoroacetyl groups instead showed some
improvements in the cancer cell toxicity.
1-(40,60-dimethylpyrimidin-20-yl)-5-amino-3-
phenylpyrazole (3a)
Reaction time 1 h 30 min; Yield 83 %; mp 192–196 °C;
IR (KBr, cm-1): 3,268 (symm.) and 3,391 (asymm.) due to
1
NH2; H NMR (300 MHz, CDCl3) d: 2.57 (s, 6H, 40,
60–CH3); 5.91 (s, 1H, 4-H); 6.02 (bs, 2H, –NH2,
exchangeable with D2O); 6.88 (s, 1H, 5–H); 7.37–7.46 (m,
3H, 300, 400, 500-H); 7.91–7.93 (m, 2H, 200, 600-H); 13C NMR
(75 MHz, CDCl3) d: 24.2, 87.5, 116.4, 127.4, 128.2, 129.1,
132.9, 150.3, 153.5, 157.7, 168.4; MS: m/z [M ? 1]?
266.1. Anal. Calcd. for C15H15N5: C, 67.90; H, 5.70; N,
26.40. Found: C, 67.84; H, 5.39; N, 26.12.
Regarding anticancer activity, further derivatives of 3a,
3b, and 3d and additional biochemical studies are required
to improve the cytotoxic potential of these compounds.
Regarding antibacterial activity, the nanomolar inhibition
shown by compounds 5b, 5c, 5d, and 5e against S. aureus
and 3a, 3b, and 5e against P. aeruginosa is very promising
and it will be investigated further.
1-(40,60-dimethylpyrimidin-20-yl)-5-amino-3-
(400-methylphenyl)pyrazole (3b)
Reaction time 1 h 40 min; yield 85 %; mp 130–134; IR (KBr,
1
cm-1): 3,271 (symm.) and 3,373 (asymm.) due to NH2; H
Experimental protocols
NMR(300 MHz, CDCl3) d:2.38(s, 3H, 400–CH3);2.57(s, 6H,
40, 60, –CH3); 5.89 (s, 1H, 4-H); 6.02–6.06 (bs, 2H, –NH2,
exchangeablewithD2O);6.87(s, 1H, 50–H);7.19–7.22(d, 2H,
J = 8.1 Hz, 300, 500-H); 7.80–7.83 (d, 2H, J = 8.1 Hz, 200, 600-
H); 13C NMR (75 MHz, CDCl3) d: 21.3, 24.2, 87.4, 116.3,
126.3, 129.0, 130.0, 130.2, 150.2, 153.6, 157.7, 168.4; MS: m/
z [M ? 1]? 280.1. Anal. Calcd. for C16H17N5: C, 68.79; H,
6.13; N, 25.07. Found: C, 68.84; H, 6.39; N, 24.92.
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 (m max in cm-1); 1H
and 13C NMR spectra for analytical purpose were recorded in
CDCl3 on a Bruker instrument at 300 MHz and 75 MHz,
respectively; chemical shifts are expressed in d-scale down-
field from TMS as an internal standard. 19F NMR spectra were
run on DRX 300 and DPX 400 at 282 and 376 MHz,
respectively, using deuteriochloroform as a solvent. The
internal standard for 19F spectra was fluorotrichloromethane,
setting the CFCl3 signal at d 0.0. Elemental analyses were
performed at Sophisticated Analytical Instrument Facility,
Central Drug Research Institute, Lucknow, India.
1-(40,60-dimethylpyrimidin-20-yl)-5-amino-3-
(400-fluorophenyl)pyrazole (3c)
Reaction time 1 h 40 min; yield 79 %; mp 160–164; IR
(KBr, cm-1): 3,298 (symm.) and 3,408 (asymm.) due to
1
NH2; H NMR (300 MHz, CDCl3) d: 2.58 (s, 6H, 40,
2-Hydrazino-4,6-dimethylpyrimidine 1 was synthesized
according to theliterature procedure (Danagulyan etal., 1989).
60–CH3); 5.88 (s, 1H, 4-H); 6.01–6.04 (bs, 2H, –NH2,
exchangeable with D2O); 6.89 (s, 1H, 50–H); 7.05–7.15 (m,
2H, 300, 500-H); 7.87–7.92 (m, 2H, 200, 600-H); 13C NMR
(75 MHz, CDCl3) d: 24.1, 87.3, 115.3, 128.0, 128.1, 129.1,
141.4, 150.4, 152.6, 157.6, 168.5; MS: m/z [M ? 1]?
284.1. Anal. Calcd. for C15H14FN5: C, 63.59; H, 4.98; N,
24.72. Found: C, 63.84; H, 4.93; N, 24.46.
General procedure for the preparation of 1-(40,60-
dimethylpyrimidin-20-yl)-5-amino-3-arylpyrazoles
(3a–e)
Equimolar amounts of b-ketonitriles 2 (5 mmol) and
2-hydrazino-4,6-dimethylpyrimidine
1
(5 mmol) were
1-(40,60-dimethylpyrimidin-20-yl)-5-amino-3-(400-
grinded in the presence of p-Toluenesulfonic acid (PTSA)
(1 mmol) as catalyst in mortar and pestle. Initially, the
reactants melted on grinding and reaction mixture became
hot and then the contents solidified. The reaction mixture
was then heated on water bath for 1–2 h. The reaction was
chlorophenyl)pyrazole (3d)
Reaction time 1 h 50 min; yield 81 %; mp 196–200; IR
(KBr, cm-1): 3,302 (symm.) and 3,418 (asymm.) due to
1
NH2; H NMR (300 MHz, CDCl3) d: 2.58 (s, 6H, 40,
123

Med Chem Res (2014) 23:1454–1464
1461
60–CH3); 5.88 (s, 1H, 4-H); 6.04 (bs, 2H, –NH2, exchangeable
with D2O); 6.90 (s, 1H, 50–H); 7.36–7.39 (d, 2H, J = 8.7 Hz,
300, 500-H); 7.85–7.88 (d, 2H, J = 8.7 Hz, 200, 600-H); 13C NMR
(75 MHz, CDCl3) d: 24.2, 87.3, 116.6, 127.6, 128.4, 131.4,
134.1, 150.4, 152.4, 157.7, 168.5; MS: m/z [M ? 1]? 300.1/
302.1 (3:1). Anal. Calcd. for C15H14ClN5: C, 60.10; H, 4.71;
N, 23.36. Found: C, 60.04; H, 4.63; N, 23.03.
7.23–7.25 (d, 2H, J = 7.8 Hz, 300, 500-H); 7.32 (s, 1H, 4-H);
7.89–7.92 (d, 2H, J = 7.8 Hz, 200, 600-H); 12.18 (s, 1H, –NH);
13C NMR (75 MHz, CDCl3) d: 20.6, 24.2, 24.6, 95.4, 116.9,
126.5, 128.6, 129.1, 130.7, 151.2, 154.1, 157.4, 166.3, 168.7;
MS: m/z [M ? 1]? 322.2. Anal. Calcd. for C18H19N5O: C,
67.27; H, 5.96; N, 21.79. Found: C, 67.34; H, 5.93; N, 21.67.
1-(40,60-dimethylpyrimidin-20-yl)-5-(N-acetamide)-3-
1-(40,60-dimethylpyrimidin-20-yl)-5-amino-3-(400-
(400-fuorophenyl)pyrazole (4c)
bromophenyl)pyrazole (3e)
Yield 71 %; mp 226–230; IR (KBr, cm-1): 1,694 (C=O),
1
Reaction time 2 h; yield 82 %; mp 194–198; IR (KBr,
cm-1): 3,279 (symm.) and 3,418 (asymm.) due to NH2; 1H
NMR (300 MHz, CDCl3) d: 2.58 (s, 6H, 40, 60–CH3); 5.88
(s, 1H, 4-H); 6.05 (bs, 2H, –NH2, exchangeable with D2O);
6.90 (s, 1H, 50–H); 7.51–7.54 (d, 2H, J = 8.1 Hz, 300, 500-
H); 7.78–7.81 (d, 2H, J = 8.1 Hz, 200, 600-H); 13C NMR
(75 MHz, CDCl3) d: 24.1, 87.3, 116.5, 122.3, 127.9, 131.4,
131.9, 150.5, 152.4, 157.7, 168.5; MS: m/z [M ? 1]?
344/346 (1:1). Anal. Calcd. for C15H14BrN5: C, 52.34; H,
4.10; N, 20.35. Found: C, 52.44; H, 4.13; N, 20.18.
3,145 (NH); H NMR (300 MHz, CDCl3) d: 2.31 (s, 3H,
–COCH3), 2.63 (s, 6H, 40, 60–CH3); 6.98 (s, 1H, 50–H);
7.09–7.15 (m, 2H, 300, 500-H); 7.31 (s, 1H, 4-H); 7.96–8.01
(m, 2H, 200, 600-H); 12.19 (s, 1H, –NH); 13C NMR (75 MHz,
CDCl3) d: 23.5, 24.2, 95.4, 115.2, 128.2, 130.3, 132.7,
137.4, 151.3, 153.1, 157.4, 166.4, 168.7; MS: m/z
[M ? 1]? 326.1. Anal. Calcd. for C17H16FN5O: C, 62.76;
H, 4.96; N, 21.53. Found: C, 62.64; H, 4.93; N, 21.39.
1-(40,60-dimethylpyrimidin-20-yl)-5-(N-acetamide)-3-
(400-chlorophenyl)pyrazole (4d)
General procedure for the preparation of 1-
(40,60-dimethylpyrimidin-20-yl)-5-(N-acetamide)-3-
arylpyrazoles (4a–e)
Yield 72 %; mp 190–194; IR (KBr, cm-1): 1,690 (C=O),
1
3,163 (NH); H NMR (300 MHz, CDCl3) d: 2.31 (s, 3H,
–COCH3), 2.63 (s, 6H, 40, 60–CH3); 6.98 (s, 1H, 50–H); 7.33 (s,
1H, 4-H); 7.39–7.42 (d, 2H, J = 7.8 Hz, 300, 500-H); 7.93–7.96
(d, 2H, J = 7.8 Hz, 200, 600-H); 12.18 (s, 1H, –NH); 13C NMR
(75 MHz, CDCl3) d: 23.6, 24.2, 95.4, 117.1, 127.8, 128.6,
130.8, 134.6, 151.4, 152.9, 157.4, 166.4, 168.8; MS: m/z
[M ? 1]? 342.1/344.1 (3:1). Anal. Calcd. for C17H16ClN5O:
C, 59.74;H, 4.72;N, 20.49. Found:C, 59.64;H, 4.83;N, 21.37.
5-Aminopyrazoles (2 mmol) were treated with acetic
anhydride (2 mmol) in the presence of acetic acid (20 ml)
as a solvent. The reaction mixture was heated on water bath
for about 15 min and then cooled. Solid separated on
cooling was filtered, washed with water, and crystallized
1
using ethanol. The tlc and H NMR spectra showed the
formation of a single product.
1-(40,60-dimethylpyrimidin-20-yl)-5-(N-acetamide)-3-
1-(40,60-dimethylpyrimidin-20-yl)-5-(N-acetamide)-3-
(400-bromophenyl)pyrazole (4e)
phenylpyrazole (4a)
Yield 71 %; mp 204–208; IR (KBr, cm-1): 1,686 (C=O),
Yield 75 %; mp 220–224; IR (KBr, cm-1): 1,690 (C=O),
3,168 (NH); H NMR (300 MHz, CDCl3) d: 2.30 (s, 3H,
1
1
3,178 (NH); H NMR (300 MHz, CDCl3) d: 2.31 (s, 3H,
–COCH3), 2.62 (s, 6H, 40, 60–CH3); 6.97 (s, 1H, 50–H);
7.32 (s, 1H, 4-H); 7.53–7.56 (d, 2H, J = 7.8 Hz, 300, 500-H);
7.86–7.89 (d, 2H, J = 7.8 Hz, 200,600-H); 12.12–12.16 (s,
1H, –NH); 13C NMR (75 MHz, CDCl3) d: 23.7, 24.2, 95.4,
117.2, 122.9, 128.1, 131.2, 131.6, 151.4, 152.9, 157.6,
166.4, 168.7; MS: m/z [M ? 1]? 386.1/388.1 (1:1). Anal.
Calcd. for C17H16BrN5O: C, 52.86; H, 4.18; N, 18.13.
Found: C, 52.64; H, 4.26; N, 17.97.
–COCH3), 2.63 (s, 6H, 40, 60–CH3); 6.97 (s, 1H, 50–H); 7.36
(s, 1H, 4-H); 7.39–7.46 (m, 3H, 300, 400, 500-H); 8.00–8.02 (m,
2H, 200, 600-H); 12.18 (s, 1H, –NH); 13C NMR (75 MHz,
CDCl3) d: 24.2, 24.6, 95.5, 117.0, 126.6, 128.4, 128.8,
132.3, 151.3, 154.0, 157.5, 166.2, 168.7; MS: m/z [M ? 1]?
308.1. Anal. Calcd. for C17H17N5O: C, 66.43; H, 5.58; N,
22.79. Found: C, 66.34; H, 5.43; N, 22.57.
1-(40,60-dimethylpyrimidin-20-yl)-5-(N-acetamide)-3-
(400-methylphenyl)pyrazole (4b)
General procedure for the preparation of 1-(40,60-
dimethylpyrimidin-20-yl)-5-(2,2,2-trifluoro-N-
acetamide)-3-arylpyrazoles (5a–e)
Yield 73 %; mp 252–256; IR (KBr, cm-1): 1,703 (C=O), 3,170
(NH); 1H NMR (300 MHz, CDCl3) d: 2.30 (s, 3H, –COCH3),
2.39 (s, 3H, 400-H); 2.62 (s, 6H, 40, 60–CH3); 6.95 (s, 1H, 50–H);
5-Aminopyrazoles (2 mmol) were treated with trifluoro-
acetic anhydride (2 mmol) in the presence of trifluoroacetic
123

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Med Chem Res (2014) 23:1454–1464
acid (20 mL) as solvent. The reaction mixture was heated
on water bath for about 15 min and then cooled. Solid
separated on cooling was filtered, washed with water, and
600-H); 12.18 (s, 1H, –NH); 13C NMR (75 MHz, CDCl3) d:
23.5, 93.1, 118.4, 124.4, 125.1, 128.6, 128.9, 132.6, 157.4,
158.2, 161.6, 169.4, 170.6; MS: m/z [M ? 1]? 396.1/398.1
(3:1). Anal. Calcd. for C17H13ClF3N5O: C, 51.59; H, 3.31; N,
17.70. Found: C, 51.64; H, 3.36; N, 17.54.
1
crystallized using ethanol. The tlc and H NMR spectra
showed the formation of a single product.
1-(40,60-dimethylpyrimidin-20-yl)-5-(2,2,2-trifluoro-N-
1-(40,60-dimethylpyrimidin-20-yl)-5-(2,2,2-trifluoro-N-
acetamide)-3-(400-bromophenyl)pyrazole (5e)
acetamide)-3-phenylpyrazole (5a)
Yield 71 %; mp 138–142; IR (KBr, cm-1): 1,609 (C=O),
3,112 (NH); 1H NMR (300 MHz, CDCl3) d: 2.59 (s, 6H, 40,
60–CH3); 6.17 (s, 1H, 4-H); 7.03 (s, 1H, 50–H); 7.44–7.49
(m, 3H, 300, 400, 500-H); 7.95–7.98 (m, 2H, 200, 600-H); 12.18
(s, 1H, –NH); 13C NMR (75 MHz, CDCl3) d: 23.7, 93.3,
117.8, 124.6, 126.6, 128.7, 129.4, 131.3, 157.3, 158.1,
160.8, 168.9, 170.2; MS: m/z [M ? 1]? 362.1. Anal.
Calcd. for C17H14F3N5O: C, 56.51; H, 3.91; N, 19.38.
Found: C, 56.64; H, 3.96; N, 19.47.
Yield 69 %; mp 76–80; IR (KBr, cm-1): 1,643 (C=O), 3,108
(NH); H NMR (300 MHz, CDCl3) d: 2.51 (s, 6H, 40,
1
60–CH3); 6.16 (s, 1H, 4-H); 6.93 (s, 1H, 50–H); 7.38–7.41 (d,
2H, J = 8.1 Hz, 300, 500-H); 7.63–7.65 (d, 2H, J = 8.1 Hz, 200,
600-H); 12.18 (s, 1H, –NH); 13C NMR (75 MHz, CDCl3) d:
23.5, 93.2, 118.4, 124.3, 124.5, 126.1, 128.7, 131.9, 157.4,
158.1, 161.4, 169.3, 170.0; MS: m/z [M ? 1]? 440/442
(1:1). Anal. Calcd. for C17H13BrF3N5O: C, 46.38; H, 2.98; N,
15.91. Found: C, 46.54; H, 2.86; N, 15.84.
1-(40,60-dimethylpyrimidin-20-yl)-5-(2,2,2-trifluoro-N-
Evaluation of antibacterial assay: preliminary screening
acetamide)-3-(400-methylphenyl)pyrazole (5b)
The agar well diffusion method (Perez et al., 1990) was
used for the determination of antimicrobial activity.
Overnight broth culture of the respective bacterial strains
was adjusted to approximately 108 colony-forming units
(CFU/mL) with sterile distilled water, and 1 mL of diluted
inoculum was spread over the Petri plates containing
25 mL of nutrient agar media. Six to eight wells (7 mm in
diameter) were made equidistance in each of the plates
using a sterile cork borer. The test compounds were dis-
solved in dimethyl sulfoxide (DMSO) and then antimi-
crobial effect of the test compounds was tested. The wells
were filled with 1 mL of test compound having concen-
tration 1 mg/mL. The plates were incubated at 37 °C for
48 h. The antimicrobial activity was evaluated by mea-
suring the zone of growth inhibition of bacteria surround-
ing the wells after 24 h and 48 h. Ampicillin and
Chloramphenicol (10 lg/mL) served as positive antibac-
terial controls. DMSO was taken as negative control.
Yield 69 %; mp 230–234; IR (KBr, cm-1): 1,630 (C=O),
1
3,126 (NH); H NMR (300 MHz, CDCl3) d: 2.42 (s, 3H,
400–CH3); 2.61 (s, 6H, 40, 60–CH3); 6.12 (s, 1H, 4-H); 7.02
(s, 1H, 50–H); 7.35–7.38 (d, 2H, J = 7.8 Hz, 300, 500-H);
7.83–7.86 (d, 2H, J = 7.8 Hz, 200, 600-H); 12.18 (s, 1H,
–NH); 13C NMR (75 MHz, CDCl3) d: 21.4, 23.6, 93.3,
117.8, 124.2, 126.6, 128.2, 129.4, 130.7, 157.2, 158.1,
161.8, 169.2, 169.8; MS: m/z [M ? 1]? 376.1. Anal.
Calcd. for C18H16F3N5O: C, 57.60; H, 4.30; N, 18.66.
Found: C, 57.64; H, 4.36; N, 18.47.
1-(40,60-dimethylpyrimidin-20-yl)-5-(2,2,2-trifluoro-N-
acetamide)-3-(400-fluorophenyl)pyrazole (5c)
Yield 67 %; mp 128–132; IR (KBr, cm-1): 1,626 (C=O),
3,114 (NH); 1H NMR (300 MHz, CDCl3) d: 2.57 (s, 6H, 40,
60–CH3); 6.05 (s, 1H, 4-H); 6.89 (s, 1H, 50–H); 7.18–7.24
(m, 2H, 300, 500-H); 7.95–7.99 (m, 2H, 200, 600-H); 12.18 (s,
1H, –NH); 13C NMR (75 MHz, CDCl3) d: 23.7, 93.6,
115.7, 124.5, 127.4, 128.9, 129.0, 136.5, 157.3, 158.2,
162.0, 168.9, 170.4; MS: m/z [M ? 1]? 380.1. Anal.
Calcd. for C17H13F4N5O: C, 53.83; H, 3.45; N, 18.46.
Found: C, 53.64; H, 3.36; N, 18.37.
Determination of minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of each
compound giving an inhibitory zone at the concentration of
1 mg/mL was also tested by a modified agar well diffusion
method (Okunji et al., 1990; Rios et al., 1988). Different
concentrations (1,000–1 lg/mL) of a single compound
were applied to number of wells in the agar plates. About
1 mL of each dilution was poured into wells. All test plates
were incubated at 37 °C for 48 h. The minimum concen-
tration of each compound showing a clear zone of inhibi-
tion was considered to be MIC. The determinations were
performed in triplicates and the results averaged.
1-(40,60-dimethylpyrimidin-20-yl)-5-(2,2,2-trifluoro-N-
acetamide)-3-(400-chlorophenyl)pyrazole (5d)
Yield 72 %; mp 106–110; IR (KBr, cm-1): 1,656 (C=O),
3,117 (NH); 1H NMR (300 MHz, CDCl3) d: 2.51 (s, 6H, 40,
60–CH3); 6.15 (s, 1H, 4-H); 6.93 (s, 1H, 50–H); 7.23–7.26 (d,
2H, J = 8.1 Hz, 300, 500-H); 7.70–7.73 (d, 2H, J = 8.1 Hz, 200,
123

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Evaluation of cytotoxic activity
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BlueÒ assay incorporates
a fluorometric/colorimetric
growth indicator based on the detection of metabolic
activity. The system comprises an oxidation–reduction
redox reaction that is reflected in fluorescence and color
change. These changes occur as a result of the cellular
metabolic reduction of the indicator. Only leaving cells
metabolise the indicator, reducing it, and emitting fluo-
rescence. After incubation, the fluorescence of the dye was
read using a plate reader. The reader measures absorption
of the alamarBlueÒ at two wavelengths and produces fig-
ures that are used to calculate the percentage viability of
the cells. The mean values and the standard error of the
mean (SEM) of all the experiments for each compound
were then used to plot graphs using GraphPad Prism and
Excel. The IC50 was calculated using GraphPad Prism.
Acknowledgments We (RA and IR) thank the Department of Sci-
ence and Technology, New Delhi, India, and Trinity College, Dublin,
Ireland, for providing financial assistance under India–Ireland
Cooperative Science Programme. Anshul Bansal is thankful to the
Council of Scientific and Industrial Research, New Delhi, India, for
Senior Research Fellowship. Elena Diez-Cecilia thanks IRCSET
(Ireland) for financial support.
Gopalsamy A, Yang H, Ellingboe JW, Tsou HR, Zhang N, Honores
E, Powell D, Miranda M, McGinnis JP, Rabindran SK (2005)
Pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides as novel anti-
proliferative agents: parallel synthesis for lead optimization of
amide region. Bioorg Med Chem Lett 15:1591–1594
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