Synthesis and biological evaluation of 7-trifluoromethylpyrazolo[1,5-a]pyrimidines as anti-inflammatory and antimicrobial agents

Article abstract of DOI:10.1016/j.jfluchem.2014.08.017

A series of 2-H/methyl-3-phenyl-5-alkyl/aryl/heteroaryl-7-trifluoromethylpyrazolo[1,5-a]pyrimidines (4a-l) were synthesized by refluxing 3(5)-amino-4-phenyl-5(3)-H/methyl-1H-pyrazoles (1-2) with trifluoromethyl-?2-diketones (3a-f) in ethanol for 6 h. The

Full text of DOI:10.1016/j.jfluchem.2014.08.017

Accepted Manuscript
Title: Synthesis and biological evaluation of
7-trifluoromethylpyrazolo[1,5-a]pyrimidines as
anti-inflammatory and antimicrobial agents
Author: Ranjana Aggarwal Eakta Masan Pawan Kaushik
Dhirender Kaushik Chetan Sharma K.R. Aneja
PII:
S0022-1139(14)00244-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jfluchem.2014.08.017
FLUOR 8409
To appear in:
FLUOR
Received date:
Revised date:
Accepted date:
23-5-2014
22-8-2014
26-8-2014
Please cite this article as: R. Aggarwal, E. Masan, P. Kaushik, D. Kaushik, C. Sharma,
K.R. Aneja, Synthesis and biological evaluation of 7-trifluoromethylpyrazolo[1,5-
a]pyrimidines as anti-inflammatory and antimicrobial agents., Journal of Fluorine
Chemistry (2014), http://dx.doi.org/10.1016/j.jfluchem.2014.08.017
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

1
Synthesis and biological evaluation of 7-trifluoromethylpyrazolo[1,5-
a]pyrimidines as anti-inflammatory and antimicrobial agents
Ranjana Aggarwala*, Eakta Masana, Pawan Kaushikb, Dhirender Kaushikb, Chetan
Sharmac and K. R. Anejac
a Department of Chemistry, Kurukshetra University, Kurukshetra-136 119, India
b Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra-136 119, India
cDepartment of Microbiology, Kurukshetra University, Kurukshetra- 136 119, India
*Corresponding author: Ranjana Aggarwal, Department of Chemistry, Kurukshetra University, Kurukshetra- 136
119, India. E-mail: ranjana67in@yahoo.com; Fax: +91-1744-238277
Abstract
A
series
of
2-H/methyl-3-phenyl-5-alkyl/aryl/heteroaryl-7-trifluoromethylpyrazolo[1,5-
a]pyrimidines (4a-l) were synthesized by refluxing 3(5)-amino-4-phenyl-5(3)-H/methyl-1H-
pyrazoles (1-2) with trifluoromethyl-β-diketones (3a-f) in ethanol for 6 hrs. The structure of the
1
compounds was assigned on the basis of H, 13C , 19F NMR and IR spectral data. The
intermediate,
5-methyl-4-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-5,7-diol
(5b),
involved in the reaction was also isolated and characterized in one case by performing the
reaction in DCM at -15 °C. A total of nine compounds 4a-f, 4h-i, 4k were tested for their anti-
inflammatory activity by Carrageenan-induced rat paw edema assay. Compound 4e exhibited the
comparable anti-inflammatory activity (83.4 %) to the standard drug Indomethacin (84.2 %).To
rationalize the anti-inflammatoy activity, docking experiments were performed to study the
ability of these compounds to bind into the active site of COX-2 enzyme. All the twelve
compounds synthesized (4a-l) were screened for their antimicrobial activity in vitro against two
Page 1 of 37

2
Gram +ve, two Gram -ve bacteria and two fungi. Preliminary results reveal that some of the
synthesized compounds revealed promising antimicrobial activity against Gram +ve bacteria and
pathogenic fungi used in this study.
Keywords
2-H/methyl-3-phenyl-5-alkyl/aryl/heteroaryl-7-trifluoromethylpyrazolo[1,5-a]pyrimidines;
Trifluoromethyl-β-diketone; Anti-inflammatory activity; Antifungal activity; Antibacterial
activity.
1. Introduction
Pyrazolo[1,5-a]pyrimidines and their derivatives constitute a class of heterocyclic compounds
currently employed in the field of medicinal chemistry for demonstrating antimicrobial [1-5],
antifungal [6], antitumour [7,8], anticancer [9] and anti-trichromonal [10] activities. Different
pyrazolo[1,5-a]pyrimidine containing compounds possess antischistosomal [11], hypnotic [12]
and anti-inflammatory [13,14] activity and some are known to be used as pharmaceutical as well
as agrochemical products. Zaleplon [15-17] and Indiplon [18] are some of the well known
hypnotic drugs which belong to this class of compounds. Furthermore, pyrazolo[1,5-
a]pyrimidine derivatives act as potent inhibitors of various enzymes such as adenokinase [19],
CHK 1 [20,21], C-Src kinase [22], human cyclin-dependent kinase 2 [23], DPP-IV [24], B-
RafV600E kinase [25] and estrogen receptors ligands [26]. In addition to this, it has been observed
that the addition of trifluoromethyl group, due to its unique stereoelectronic properties, increases
Page 2 of 37

3
the lipophilicity when present in the biologically active molecules [27]. Hence, the introduction
of trifluoromethyl group into bioactive molecules becomes an important strategy in perfecting
pharmaceuticals.
Several methods have been described in the literature for the synthesis of pyrazolo[1,5-
a]pyrimidines. Most of them involve the reaction between 5-amino-1H-pyrazoles with 1,3-
bielectrophilic reagents, such as β-dicarbonyl, α,β-unsaturated carbonyl, alkoxymethylene-β-
dicarbonyl and β-enaminone compounds [28-31]. Reaction of various unsymmetrical 1,3-bis-
electrophiles with 5-amino-4-substituted-1H-pyrazoles usually affords a mixture of two
regioisomers, however, in some of the cases regiospecific/regioselective synthesis of
pyrazolo[1,5-a]pyrimidines has been reported [32-34]. Recently a chemo and regioselective
synthesis of some new pyrazol-1'-ylpyrazolo[1,5-a]pyrimidines has been achieved by us
involving reaction between 3(5)-amino-5(3)-hydrazinopyrazole dihydrochloride and several β-
diketones using water as a solvent [3]. Prompted by these investigations and in continuation of
our efforts to synthesize trifluoromethylated bioactive molecules [35-39], we considered to
synthesize
a
series
of
2-H/methyl-3-phenyl-5-alkyl/aryl/heteroaryl-7-
trifluoromethylpyrazolo[1,5-a]pyrimidines with an aim to find more potent anti-inflammatory
and antimicrobial agents.
Page 3 of 37

4
2. Results and discussion
The synthetic pathway to the title compounds is summarized in Scheme-1.
H
R
R
N
CF3
O
O
EtOH, reflux
6 hrs
N
N
N
+
N
R'
CF3
NH2
3a-f
R'
4a-l
1-2
CF
3Ha
Hb
HO
O
O
N
N
DCM, stir
O
OH
CH3
NH
-15°C, 6 hrs
reflux, 0.5 hrs
-2H2O
5b
4
R'
R
R'
3
a
R
1
2
a
b
c
H
H
H
H
H
CF3
CH3
C6H5
CF3
CH3
CH3
b
C6H5
c
4-CH3C6H4
4-BrC6H4
d
e
f
d
4-CH3C6H4
4-BrC6H4
2-thienyl
H
H
e
f
2-thienyl
CF3
g
CH3
h
i
CH3
CH3
CH3
C6H5
j
CH3
CH3
CH3
4-CH3C6H4
k
l
4-BrC6H4
2-thienyl
Scheme-1 Synthetic route to the synthesis of 7-trifluoromethylpyrazolo[1,5-a]pyrimidines (4a-l)
Page 4 of 37

5
The starting compounds, 3(5)-amino-4-phenyl-1H-pyrazole (1) & 3(5)-amino-4-phenyl-5(3)-
methyl-1H-pyrazole (2) were obtained by the condensation of α-phenylformylacetonitrile and α-
phenylacetylacetonitrile with hydrazine hydrate, respectively [48]. The reaction of 1-2 with
equimolar amount of trifluoromethyl-β-diketones (3a-f) in refluxing ethanol afforded the target
compounds,
2-H/methyl-3-phenyl-5-alkyl/aryl/heteroaryl-7-trifluoromethylpyrazolo[1,5-
a]pyrimidines (4a-l) regioselectively in good yield. The reaction between binucleophilic centres
in 1-2 and bielectrophilic centres in 3a-f, in principle, may yield two regioisomers, however, in
the present study due to differential reactivities of nucleophilic and electrophilic sites, only a
single isomer was isolated.
1
The compounds were characterized by a combined application of H, 13C and 19F NMR
spectroscopy.
The 1H NMR spectra of 4a-f exhibited a singlet of one proton intensity at about δ 8.64 due to the
pyrazolopyrimidine 2-H while in 4g-l this singlet was replaced by a sharp singlet of three proton
intensity at ~ δ 2.65 due to methyl protons at C-2. For 4a-l another singlet of one proton
intensity was observed situated at ~ δ 7.55 corresponding to the proton present at position-6 of
the pyrazolo[1,5-a]pyrimidine ring. Compounds 4b and 4h displayed a singlet of three proton
intensity at δ 2.76 and δ 2.68 ppm, respectively which was assigned to methyl protons at
position-5. Had the methyl group been on the position-7 of the pyazolopyimidine ring it would
have appeared as a doublet. This argument is based on our previous observation where 1H NMR
and 2-Dimensional NMR spectrum of 2-(3',5'-dimethylpyrazol-1'-yl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidine [40] showed a singlet of three proton intensity at δ 2.54 corresponding to the C-5
methyl and a doublet of three proton intensity at δ 2.70 having coupling constant 4J= 1.0 Hz for
the C-7 methyl protons due to coupling with the proton at position-6 of pyrazolopyrimidine. This
Page 5 of 37

6
coupling split the signal for the proton at position-6 into a quartet at δ 6.55 (4J= 1.0 Hz ) (CH3-7,
H-6). (Fig-1)
4
2.70 ppm (d, 3H, J= 1.0 Hz)
4
6.55 ppm (q, 1H, J= 1.0 Hz)
H3C
N
H
H3C
N
2.54 ppm (s, 3H)
CH3
N
N
N
H3C
Fig-11H NMR data of 2-(3',5'-dimethylpyrazol-1'-yl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine
[40]
The structure of regioisomers 4a-f can further be established on the basis of 13C NMR. The
methyl group in compounds 4b and 4h appear as a sharp signal at δ 25.26 and 25.12 ppm
respectively, which is characteristic for a methyl group at carbon-5 of the pyrazolopyrimidine
ring [32,33]. Moreover, in all the compounds, while C-5 appears as a singlet at a ~ 150 ppm, C-6
exhibits a quartet at ~ 102 ppm (s 3J=4.0 Hz (C-6, CF3)) and C-7 exhibits a quartet at ~ 133 ppm
(2J=37.0 Hz (C-7, CF3)) due to coupling with the CF3 carbon. This data is concordant with our
earlier reports [41]. The signal for the CF3 carbon appears as a doublet at about ~ 118 ppm (2J=
273.0 Hz) in agreement with the literature value [32,41].
Further, in the 19F NMR spectra of 4a-l, signals in the range δ -68.79 to -69.55 ppm, values
typical to the CF3 at position-7 suggested the isomer as 2-H/methyl-3-phenyl-5-
alkyl/aryl/heteroaryl-7-trifluoromethylpyrazolo[1,5-a]pyrimidine in agreement with literature
values [41] (Table-1).
Page 6 of 37

7
Table 1 13C and 19F NMR data of 4a, 4d-e, 4g, 4j-l (in ppm)
CF3
7
1
N
7a
N
6
4"
3" 4"
2"
2
5"
6"
5
3"
2"
R
R'
N
4
3
3a
5"
1'
S
2'
3'
1"
6'
5'
R'= C6H5
R'=2-thienyl
4'
R= H, CH3
Compounds
4a
4d
4e
4g
4j
4k
4l
C-2
C-3
C-3a
C-5
157.27
110.00
142.59
144.35a
106.25b
132.52
154.25
110.70
141.50
154.84
102.84c
133.56
153.89
112.43
144.24
145.46
103.26c
132.45
156.08
113.35
144.97
145.52a
101.64b
135.00
154.23
110.68
146.91
154.81
102.77c
133.78
153.57
111.20
146.79
154.64
102.54c
132.32
154.06
110.46
146.43
150.15
102.23c
133.64
C-6
C-7a
Aryl carbons
C-1'
C-2',6'
C-3',5'
C-4'
130.19
125.54
125.74
127.75
131.78
129.09
126.66
127.18
134.96
127.03
128.90
126.08
130.11
127.62
129.14
128.77
133.41
128.53
129.07
126.65
131.50
128.61
128.69
129.14
130.87
126.68
128.95
126.82
Thienyl
carbons
142.47
(C-2")
128.52
(C-3")
128.61
(C-4")
131.60
(C-5")
C-1"
-
-
-
-
136.11
129.82
128.54
133.98
134.84
131.09
128.75
126.66
-
-
-
-
133.54
127.15
129.80
131.79
135.15
126.88
129.10
125.76
C-2",6"
C-3",5"
C-4"
Other
carbons
2-CH3
5-CH3
-
-
-
-
-
-
-
14.54
14.58
14.55
14.69
-
-
-
-
-
d
5-CF3e
114.35
119.80
-
-
118.14
-
117.25
118.43
-
-
118.75
-
-
118.19
-
7-CF3
118.32
21.48
118.35
21.46
C-5"-CH3
19F Data
7-CF3
5-CF3
-69.25
-68.21
-68.92
-
-68.94
-
-69.20
-68.18
-68.86
-
-68.88
-
-68.79
-
Page 7 of 37

8
aQuartet, 2JCF = 37.2 Hz
bMultiplet
cQuartet, 2JCF = 4.0 Hz
dQuartet, 1JCF = 274.7 Hz
eQuartet, 1JCF = 274.7 Hz
The assignments are on the basis of data of compound 4c and 4f [41].
To gain an insight of the reaction mechanism, attempts were made to isolate an intermediate of
the reaction. The intermediate, 5-methyl-3-phenyl-7-trifluoromethyl-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-5,7-diol (5b), could successfully be isolated in one case by
performing the reaction in DCM by stirring 3(5)-amino-4-phenyl-1H-pyrazole (1) and 1,1,1-
trifluoromethylpent-2,4-dione (3b) at -15 °C for 6 hrs. Two nitrogens, endo (part of the pyrazole
ring) and exo (amino group) of 1, in principle, may react with two carbonyls of 1,1,1-
trifluoromethylpent-2,4-dione (3b), however, the intermediacy of 5 indicates that the amino
group of 1 reacts with methyl carbonyl of 3b and the carbonyl carbon near to CF3 is attacked by
endo N. (Scheme-1)
1
The intermediate 5b was characterized by NMR & IR spectroscopy. The H NMR spectrum of
compound 5b showed a set of signals at δ 3.03-3.07 & 3.15-3.19 showing two doublets of AB
system (gem-coupling) belonging to diastereotopic methylene protons at position-6 with J = 16.0
Hz. Broad singlets at δ 5.56, 7.29 and 7.98 were assigned to –NH, C7-OH and C5-OH protons
respectively. The IR spectrum showed a broad absorption band at 3124 cm-1 and a sharp band at
3479 cm-1, characteristic for the –OH and –NH groups. The product was identified as 5-methyl-
3-phenyl-7-trifluoromethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-5,7-diol (5b) which upon
dehydration by refluxing with acetic anhydride for 0.5 hr gave 4b. This was confirmed by co-
TLC and mixed m.pt with the standard sample prepared by reaction between 1 and 3b (Scheme-
1).
Page 8 of 37

9
3. Biological results and discussion
3.1 Anti-inflammatory evaluation and docking studies
The anti-inflammatory activity of compounds 4a-f, 4h-i and 4k was studied using Carrageenan
induced paw edema method [42]. The protocol of animal experiments has been approved by the
Institutional Animal Ethics Committee (IAEC). Each test compound was dosed orally (50 mg/kg
body weight) 30 min prior to induction of inflammation by carageenan injection. Indomethacin
was utilized as a reference anti-inflammatory drug at a dose of 10 mg/kg. The anti-inflammatory
activity was then calculated 60-240 min after induction and presented in Table-2 as the mean
paw volume (ml) in addition to the percentage anti-inflammatory activity (AI%).
A careful analysis of Table-2 and Fig-2 reveals that most of the tested compounds showed good
anti-inflammatory activity after the 2nd hour of drug treatment comparable to the standard drug
Indomethacin. After 1 hour of drug treatment, compound 4a was fairly effective with an activity
of 77.4 % in comparison with Indomethacin (94.3). Compounds 4a, 4d, 4h-i and 4k showed
activity in the range 58.6- 77.6 % while compound 3e, showing an activity of 83.4 % was
comparable to the standard drug Indomethacin (84.2 %). Some of the compounds (4a-c),
showing activity in the
range 58.8-71.7 % were more effective after 3 hours. Only one of the compounds, 4a, was found
to be effective after 4 hrs.
Though no general trend can be assigned to the compounds showing good activity in terms of
substituents present at position-2 and 5 of the pyrazolopyrimidine ring, most of the compounds
exhibited good anti-inflammatory activity. Comparative analysis indicates that compound 4a was
Page 9 of 37

10
found to be most potent showing very high activity after 1st, 2nd, 3rd as well as 4th hour of drug
treatment while compound 4e showed excellent activity after 2 hrs as compared to Indomethacin.
Table-2 Anti-inflammatory activity of compounds 4a-f, 4h-i and 4k through Carrageenan-
induced paw edema test
Compound
1 hr
2 hr
3 hr
4 hr
Tween 80 (5 %)
0.71 ± 0.08
1.21 ±.10
1.36 ± .15
1.25 ±0.09
Indomethacin
(10 mg/kg)
0.04 ± 0.02**(94.3) 0.19 ±0.03**(84.2)
0.26±0.02**(80.8) 0.17 ±0.03**(86.4)
4a
4b
4c
4d
4e
4f
0.16 ±0.04**(77.4) 0.27 ±0.09**(77.6)
0.33±0.05**(75.7) 0.48 ±0.11**(61.6)
0.52±0.20**(61.7) 0.73 ±0.11(41.6)
0.56±0.29**(58.8) 0.70 ±0.12(44.0)
0.34 ±0.06(52.1)
0.55 ±0.20(22.5)
0.30 ± 0.03(57.7)
0.67 ±0.18(5.6)
0.53 ± 0.09(23.3)
0.44 ± 0.04(38.0)
0.36 ± 0.09(49.2)
0.51 ± 0.11(28.1)
0.78 ±0.13(35.5)
0.68 ±0.16(43.8)
0.28 ±0.06**(76.8)
0.20 ±0.07**(83.4)
0.68 ± 0.21(43.8)
0.46±0.11**(61.9)
0.50 ±0.22**(58.6)
0.40 ±0.10**(66.9)
0.86 ±0.05(36.6)
0.69±0.11*(49.2)
0.89 ±0.19(34.5)
0.76 ±0.14(44.1)
0.73 ±0.11(46.3)
1.03 ±0.21(24.2)
1.04 ±0.13(16.8)
0.86 ±0.11(31.2)
1.17 ±0.23(6.4)
0.59 ±0.21*(52.8)
1.04 ±0.13(16.8)
0.76 ±0.08(39.2)
4h
4i
4k
All values are expressed as mean ±SEM of five rats in each group.
Statistically significant **p<0.01,*p>0.05 compared to control.
Values in parenthesis represent % inhibition
.
Page 10 of 37

11
Fig-2 Percentage anti-inflammatory activity of compounds 4a-f, 4h-i and 4k / reference after 2
hrs of drug treatment
Significant anti-inflammatory activity of novel synthesised compounds prompted us to perform
molecular docking studies of compounds 4a-f, 4h, 4i and 4k to understand the ligand-protein
interactions and Cyclooxygenase-2 (COX-2) selectivity in detail. Automated docking studies
were carried out using Molegro Virtual Docker 2010.4.1 [43], the scoring functions and
hydrogen bonds formed with the surrounding amino acids are used to predict their binding
modes and their binding affinities at the active site of COX-2 enzyme.
The standard compound Indomethacin showed one hydrogen bond interaction of oxygen of C=O
group with amino acid Lys 56 having hydrogen bond length 2.87 Å. In most of the compounds
(4), N-4 of pyrazolopyrimidine ring showed interaction with amino acid Thr 60 (2.92 Å - 3.19 Å)
, Lys 56 (3.44 Å) and Arg 61 (3.50 Å) except compound 4c and 4d which showed interaction
with amino acid Thr 60 (2.73 Å - 3.43 Å) via N-1 and N-7a. Compounds 4e, 4f, 4i and 4k
Page 11 of 37

12
exhibited relatively similar binding affinity for COX-2 having docking score 102.82, 100.77,
106.26 and 108.82 respectively, which is comparable to the reference drug Indomethacin (the
original ligand) as presented in Table-3 and Fig-3. (For complete docking interactions see
Supplementary data). Though dock score may be used as an index to explain the better activity
exhibited by 4a, 4d, 4e and 4k, however, no correlation can be made between anti-inflammatory
activity and dock score of these four compounds. Further studies need to be carried out to find
the specific target of these compounds.
Table-3 Dock score and bond interactions of reference drug Indomethacin and synthesized
compounds 4a-f, 4h, 4i and 4k with amino acids of COX-2 enzyme
Compound
4a
Dock Score
(-)
No. of
interactions
3
Distance Amino acids
Atoms of ligand
(Ǻ)
involved
Thr 60
Arg 61
Arg 61
Thr 60
Lys 56
Thr 60
Thr 60
Thr 60
Thr 60
Thr 60
Arg 61
Arg 61
Thr 60
Thr 60
Thr 60
pyrazolopyrimidine
89.08
3.09
3.28
3.46
3.26
3.44
2.73
3.43
2.97
3.13
3.27
3.33
3.50
3.01
3.05
2.92
N-4
N-1
N-7a
N-1
N-4
N-1
N-7a
N-1
N-7a
N-7a
N-1
N-4
N-4
N-4
N-4
4b
4c
4d
4e
82.80
92.61
98.59
102.82
2
2
2
3
4f
4h
4i
100.77
87.68
1
1
3
106.26
Page 12 of 37

13
3.36
3.38
3.19
3.35
2.87
Arg 61
Arg 61
Thr 60
Arg 61
Lys 56
N-1
N-7a
N-4
4k
108.82
110.92
2
1
N-1
Indomethacin
Oxygen of =CO
4e
4f
4i
4k
Fig 3 Zoomed images showing bond interactions of compounds 4e, 4f, 4i and 4k with amino
acids of COX-2 enzyme
3.2 Antimicrobial evaluation
Page 13 of 37

14
Twelve chemically synthesized compounds (4a-l) were assayed for their antibacterial activity in
vitro against Staphylococcus aureus (MTCC 96) and Bacillus subtilis (MTCC 121) as examples
of two Gram +ve bacteria and Escherichia coli (MTCC 1652) and Pseudomonas aeruginosa
(MTCC 741) as examples of two Gram -ve bacteria. Compounds 4a-l were also screened for
their antifungal activity against two fungi Aspergillus niger and Aspergillus flavus.
The Agar well diffusion method [44] was used for the determination of antibacterial activity
while antifungal activity was evaluated by poison food technique [45]. DMSO was used as a
negative control whereas Ciprofloxacin was used as positive control in antibacterial activity
while Fluconazole was used as standard drug for antifungal activity. Minimum Inhibitory
Concentaration (MIC) measurements were performed using a micro dilution tube method
[46,47].
3.2.1 Antibacterial activity
Results revealed that in general, all the tested compounds possessed moderate antibacterial
activity against Gram +ve bacteria (Staphylococcus aureus and Bacillus subtilis). However, none
of them was found to be effective against any of the Gram -ve bacteria (E. coli and P.
aeruginosa).
On the basis of diameter of growth of inhibition zone shown against Gram +ve bacteria,
compound 4g was found to be the most effective against S. aureus with zone of inhibition of
17.6 mm and two compounds namely 4g and 4h, against B. subtilis, with zone of inhibition
ranging between 19.3 mm and 18.6 mm comparable to the standard drug Ciprofloxacin.
Moderate antibacterial activity was observed by compounds 4h, 4j and 4l with zone of inhibition
> 15.0 mm and compounds 4b, 4d, 4i and 4k with zone of inhibition ≥ 15.0 mm (Table-3). In
Page 14 of 37

15
the whole series, the MIC of chemical compounds ranged between 64 and 256 µg/ml against
Gram +ve bacteria. Compound 4g was found to be best as it exhibited the lowest MIC of 64
µg/ml against S. aureus and compounds 4g and 4h showed an MIC of 64 µg/ml against B.
subtilis (Table-3).
The general trend in Table-4 reveals that compounds 4a-l exhibited fairly good activity against
B. subtilis than S. aureus as compared to the standard drug Ciprofloxacin. Moreover, the
substituent at position-2 of the pyrazolopyrimidine ring also affects the results. Replacement of –
H group at position-2 by –CH3 group (4g-l) enhances the antibacterial activity against both the
Gram +ve bacteria B. subtilis and S. aureus. Further, compounds 4b and 4g-h having –CH3 and –
CF3 group at position-5 showed better activity as compared to rest of the compounds having
phenyl and substituted phenyl groups. Ineffective nature of the tested compounds against Gram -
ve bacteria E. coli and P. aeruginosa may be attributed to the outer harder, lipopolysaccharide
containing membrane which makes them more resistant against antibiotics. As can be seen from
Table-4, MIC was lowest for compounds 4g and 4h. Thus, in general, it can be concluded that
compounds containing simple substituents like –CH3 and –CF3 at position-5 exhibit better
antibacterial activity than compounds having aryl/heteroaryl substituents.
Table -4 Antibacterial activity of chemical compounds 4a-l through agar well diffusion method
Page 15 of 37

16
Compound
No.
Diameter of growth of inhibition zone (mm)a
Minimum
concentration (MIC)
(µg/mL)
inhibitory
Staphylococcus Bacillus Escherichia Pseudomonas Staphylococcus Bacillus
aureus
12.3
14.6
13.6
12.0
12.6
13.6
17.6
15.6
12.3
15.3
13.6
15.3
subtilis
13.6
15.3
12.6
15.6
13.6
14.0
19.3
18.6
15.3
14.3
15.0
12.6
24.0
coli
-
-
-
-
-
-
-
-
-
-
-
aeruginosa
-
-
-
-
-
-
-
-
-
-
-
aureus
>256
256
256
>256
>256
256
128
64
>256
128
256
128
5
subtilis
256
128
>256
128
256
256
64
4a
4b
4c
4d
4e
4f
4g
4h
4i
64
128
256
128
>256
5
4j
4k
4l
-
-
Ciprofloxacin 26.6
25.0
22.0
- No activity
a Values, including diameter of the well (8mm), are mean of three replicates
3.2.2 Antifungal activity
As can be observed from Table-5, of the twelve chemical compounds (4a-l) screened for their
antifungal activity against Aspergillus niger and Aspergillus flavus fungal strains, two
compounds 4d and 4j showed more than 56 % inhibition of mycelial growth against Aspergillus
niger whereas compounds 4d, 4h, 4i and 4k showed more than 55 % inhibition against A. flavus
as compared to the standard drug Fluconazole (81.8 % inhibition). Compound 4j showed highest
inhibition of fungal mycelium (61.1%) against A. flavus. Data reported in Table-5 revealed that
compound 4j is most active as compared to other compounds and standard drug.
Page 16 of 37

17
Table-5 Antifungal activity in vitro of synthetic chemical compounds 4a-l through poisoned
food method [45]
Compound No. Mycelial growth inhibition (%)
Aspergillus niger Aspergillus flavus
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
52.2
50
53.3
51.1
50
55.5
50
52.2
51.1
56.6
58.8
61.1
55.5
48.8
77.7
51.1
58.8
53.3
51.1
50
52.2
53.3
56.6
51.1
50
Fluconazole
81.1
6. Conclusion
In conclusion, we have synthesized a series of 2-H/methyl-3-phenyl-5-substituted-7-
trifluoromethylpyrazolo[1,5-a]pyrimidines (4a-l) regioselectively and evaluated them for their
antimicrobial and anti-inflammatory activitites. Most of the tested compounds (4a-l) were
moderately active as Gram +ve antibacterial and the antifungal agents. Compounds 4g and 4h
were most effective in antibacterial activity showing an inhibition zone of 19.3 and 18.6 mm
respectively against B. subtilis as compared to the standard drug Indomethacin (24.0 mm).
Antifungal activity was best shown by compounds 4d and 4h-i while 4j was most effective
against A. flavus. Anti-inflammatory activity results of the tested compounds showed that
compounds 4a, 4d, 4h-i and 4k showed activity in the range 58.6- 77.68 % while compound 4e,
Page 17 of 37

18
showing an activity of 83.47 % was comparable to the standard drug Indomethacin (84.29 %)
after 2 hrs of drug treatment.
7. Experimental
Melting points were determined in open capillaries and are uncorrected. IR spectra were
1
recorded on a Buck Scientific IR M-500 spectrophotometer in KBr pellets (ν max in cm-1), H
and 13C NMR spectra were recorded in CDCl3 on a Bruker instrument at 300 / 400 MHz and 75
MHz, respectively; chemical shifts are expressed in δ-scale downfield 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 δ 0.0. The reactions were monitored by the
TLC carried out on pre-coated silica gel glass plates. Mass spectra were measured in EI mode on
a Kratos MS-50 spectrometer at University of California, San Francisco, USA. Elemental
analyses were performed at Sophisticated Analytical Instrument Facility, Central Drug Research
Institute, Lucknow, India.
The starting materials 3(5)-amino-4-phenyl-1H-pyrazole 1 & 3(5)-amino-4-phenyl-5(3)-methyl-
1H-pyrazole 2 were prepared according to literature procedure [48]. Fluorinated-β-diketone 3a
was purchased from Sigma-Aldich and other 3b-f were prepared according to literature
procedure [49,50].
7.1 Synthesis of 3-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidine (4a)
To a warm solution of 3(5)-amino-4-phenyl-1H-pyrazole 1 (1.0 g, 6.2 mmol) in ethanol (20 ml)
was added 1,1,1,5,5,5-hexafluoropentan-2,4-dione 2a (1.3 g, 6.2 mmol) and the mixture was
Page 18 of 37

19
refluxed for 6 hrs. The reaction was monitored by TLC carried out on pre-coated silica gel glass
plates. The pale yellow solid obtained on cooling was recrystallised from ethanol.
All other compounds, 4b-l, were synthesized according to procedure mentioned for 4a using 1-2
with fluorinated-β-diketones 3a-f.
The characterization data for these compounds is given below:
7.1.1 3-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidine (4a)
o
1
Mp 132-135 C; Yield 88.5%; IR (KBr, cm-1) 3032, 2361, 1582, 1450, 1265, 1211, 1134; H
NMR (300 MHz, CDCl3) δ: 7.38-7.43 (m, 1H, Pha- 4'H), 7.50 (s, 1H, C6-H), 7.53-7.56 (m, 2H,
Pha- 3'H, 5'H), 8.06-8.08 (m, 2H, Pha- 2'H, 6'H), 8.74 (s, 1H, C2-H). MS (EI) m/z: 332.05
[M+1]+; Elemental analysis calcd. for C14H7F6N3: C, 50.77; H, 2.13; N, 12.69. Found: C, 50.73;
H, 2.06; N, 12.76.
7.1.2 5-Methyl-3-phenyl-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4b)
Mp 106-108 °C (Lit [32] mp 116 °C).
7.1.3 3,5-Diphenyl-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4c)
Mp 174-175 °C (Lit [41] mp 176-177 °C).
7.1.4 5-(4"-Methylphenyl)-3-phenyl-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4d)
o
1
Mp 180-182 C; Yield 89%; IR (KBr, cm-1) : 2361, 1605, 1574, 1404; H NMR (300 MHz,
CDCl3) δ: 2.49 (s, 3H, Phb-4"-CH3), 7.35-7.55 (m, 5H, Pha), 7.67 (s, 1H, C6-H), 8.12-8.18 (m,
Page 19 of 37

20
4H, Phb- 2"H, 3"H, 5"H, 6"H), 8.57 (s, 1H, C2-H). MS (EI) m/z: 354.11 [M+1]+; Elemental
analysis calcd. for C20H14F3N3: C, 67.98; H, 3.99; N, 11.89 Found: C, 67.80; H, 3.89; N, 11.92.
7.1.5 5-(4"-Bromophenyl)-3-phenyl-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4e)
o
1
Mp 174-176 C; Yield 83%; IR (KBr, cm-1) 3063, 2361, 1582, 1396, 1335, 1265, 1196; H
NMR (300 MHz, CDCl3) δ: 7.31-7.35 (m, 1H, Pha-4' H), 7.47-7.50 (m, 2H, Pha-3'H, 5'H), 7.57
(s, 1H, C6-H), 7.67-7.70 (m, 2H, Pha-2'H, 6'H), 8.00 (m, 2H, Phb-3"H, 5"H), 8.06-8.09 (m, 2H,
Phb-2"H, 6"H), 8.54 (s, 1H, C2-H). MS (EI) m/z: 418.01/420.01 (1:1) [M+1]+ [M+1+2]+ ;
/
Elemental analysis calcd. for C19H11BrF3N3: C, 54.57; H, 2.65; N, 10.05. Found: C, 54.53; H,
2.70; N, 10.18.
7.1.6 3-Phenyl-5-(2"-thienyl)-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4f)
Mp 141-142 °C (Lit [41] mp 142-143 °C).
7.1.7 2-Methyl-3-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidine (4g)
o
1
Mp 110-112 C; Yield 86.5%; IR (KBr, cm-1) 2361, 1450, 1512, 1450, 1265, 1180; H NMR
(300 MHz, CDCl3) δ: 2.85 (s, 3H, C2- CH3), 7.42 (s, 1H, C6-H), 7.52- 7.57 (m, 3H, Pha- 3'H, 4'H,
5'H), 7.71-7.74 (m, 2H, Pha- 2'H, 6'H). MS (EI) m/z: 346.07 [M+1]+; Elemental analysis calcd.
for C15H9F6N3: C, 52.18; H, 2.63; N, 12.17. Found: C, 52.27; H, 2.67; N, 12.03.
7.1.8 2,5-Dimethyl-3-phenyl-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4h)
Mp 122-124 °C (Lit [32] mp 126-127 °C).
Page 20 of 37

21
7.1.9 2-Methyl-3,5-diphenyl-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4i)
Mp 128-130 °C (Lit [32] mp 133-135 °C).
7.1.10 2-Methyl-5-(4"-methylphenyl)-3-phenyl-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4j)
o
Mp 138-140 C; Yield 88.4%; IR (KBr, cm-1) : 3433, 2361, 1628, 1566, 1412, 1335, 1211,
1
1149; H NMR (300 MHz, CDCl3) δ: 2.46 (s, 3H, Phb- 4"H), 2.74 (s, 3H, C2- CH3), 7.33- 7.41
(m, 3H, Pha- 3'H, 4'H, 5'H), 7.52-7.57 (m, 2H, Pha- 2'H, 6'H), 7.59 (s, 1H, C6-H), 7.82-7.84 (d,
2H, J= 6.0 Hz, Phb- 3"H, 5"H), 8.05-8.07 (d, 2H, J= 6.0 Hz, Phb- 2"H, 6"H). MS (EI) m/z:
368.13 [M+1]+; Elemental analysis calcd. for C21H16F3N3: C, 68.66; H, 4.39; N, 11.44. Found:
C, 68.70; H, 4.49; N, 11.32.
7.1.11 5-(4"-Bromophenyl)-2-methyl-3-phenyl-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4k)
Mp 180-185 oC; Yield 87%; IR (KBr, cm-1) : 3472, 3425, 1628, 1566, 1528, 1404, 1335, 1211,
1149; 1H NMR (300 MHz, CDCl3) δ: 2.74 (s, 3H, C2-CH3), 7.38-7.43 (m, 1H, Pha- 4'H), 7.52 (s,
1H, C6-H), 7.55-7.57 (m, 2H, Pha- 3'H, 5'H), 7.66- 7.69 (d, 2H, J= 8.7 Hz, Phb- 3"H, 5"H), 7.78-
7.81 (m, 2H, Pha- 2'H, 6'H), 8.02- 8.05 (d, 2H, J= 8.7 Hz, Phb- 2"H, 6"H). MS (EI) m/z:
432.02/434.02 (1:1) [M+1]+/ [M+1+2]+; Elemental analysis calcd. for C20H13BrF3N3: C, 55.57;
H, 3.03; N, 9.72. Found: C, 55.53; H, 3.13; N, 9.76.
7.1.12 2-Methyl-3-phenyl-5-(2"-thienyl)-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4l)
o
1
Mp 120-125 C; Yield 83%; IR (KBr, cm-1) 3464, 3078, 1628, 1528, 1427, 1211, 1157; H
NMR (300 MHz, CDCl3) δ: 2.73 (s, 3H, C2- CH3), 7.17-7.20 (m, 1H, Pha- 4'H), 7.34-7.38 (m,
Page 21 of 37

22
2H, Pha- 3'H, 5'H), 7.44 (m, 1H, C6-H), 7.51-7.56 (m, 2H, Pha-2'H, 6'H), 7.73-7.83 (m, Th-3"H,
4"H, 5"H). MS (EI) m/z: 360.07 [M+1]+; Elemental analysis calcd. for C18H12F3N3S: C, 60.16;
H, 3.37; N, 11.69. Found: C, 60.18; H, 3.27; N, 11.73.
7.2
Synthesis
of
5-Methyl-3-phenyl-7-trifluoromethyl-4,5,6,7-tetrahydropyrazolo[1,5-
a]pyrimidin-5,7-diol (5b)
To a stirred solution of 3(5)-amino-4-phenyl-1H-pyrazole 1 (1.0 g, 6.2 mmol) in DCM (15 ml)
was added 1,1,1-trifluoromethylpent-2,4-dione (3b) (0.7 g, 6.2 mmol) at -15 °C. The reaction
mixture was stirred for 0.5 hr. The reaction was monitored by TLC carried out on pre-coated
silica gel glass plates. The solid mass separated on stirring was filtered off and washed with cold
dichloromethane.
7.2.1 5-Methyl-3-phenyl-7-trifluoromethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-5,7-diol
(5b)
1
Mp 112-115 oC; Yield 56%; IR (KBr, cm-1) 3479 (-NH), 3124 (-OH), 1628, 1428, 1373; H
NMR (400 MHz, CDCl3) δ: 2.34 (s, 3H, C5- CH3), 3.03-3.07 (d, 1H, J= 16.0 Hz, C6-Ha), 3.15-
3.19 (d, 1H, J= 16.0 Hz, C6-Hb), 5.56 (bs, 1H, -NH), 7.26-7.49 (m, 5H, Pha), 7.29 (bs, 1H, C5-
OH), 7.98 (bs, 1H, C7-OH)
7.3
Conversion
of
5-Methyl-3-phenyl-7-trifluoromethyl-4,5,6,7-tetrahydropyrazolo[1,5-
a]pyrimidin-5,7-diol (5b) to 5-Methyl-3-phenyl-7-trifluoromethylpyrazolo[1,5-a]pyrimidine (4b)
5-Methyl-3-phenyl-7-trifluoromethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-5,7-diol 5b (
0.5 g, 1.6 mmol) was refluxed in acetic anhydride (10 ml) for 0.5 hr and the reaction was
Page 22 of 37

23
monitored by TLC carried out on pre-coated silica gel glass plates. The reaction mixture was
cooled and poured in ice. Orange coloured solid so obtained was filtered, dried in air and
recrystallised
in
chloroform.
It
was
characterized
as
5-Methyl-3-phenyl-7-
trifluoromethylpyrazolo[1,5-a]pyrimidine (4b).
Pha represents phenyl ring at position-3 of the pyrazolopyrimidine ring
Phb represents phenyl ring at position-5 of the pyrazolopyrimidine ring
8. Pharmacological assay
8.1 Anti-inflammatory assay
The anti-inflammatory activity was evaluated by using the Carragenan-induced paw edema test
[42]. Male Wister albino rats weighing 200-250 g were used throughout the study. They were
kept in the animal house under standard conditions of light and temperature with free access to
food and water. Food was withdrawn 12 h before and during experimental hours. The animals
were randomly divided into groups each consisting of six rats. One group of six rats was kept as
control and received tween 80 (95:5). Another group received the standard drug Indomethacin at
a dose of 10 mg/kg body weight, i.p. Other groups of rats were administered the test compounds
at a dose of 50 mg/kg body weight orally. A mark was made on the left hind paw just beyond the
tidiotarsal articulation, so that every time the paw was dipped up to fixed mark and constant paw
volume was ensured. Paw volumes were measured using a plethsymometer (model 7140, Ugo
Basile, Italy). Thirty minutes after administration of test and standard drugs, 0.1 ml of 1% w/v of
carageenan suspension in normal saline was injected into subplanter region of the left hind paw
Page 23 of 37

24
of all the animals. The initial paw volume was measured within 30 sec of the injection and
remeasured again 1 hour, 2 hours, 3 hours and 4 hours after administration of Carrageenan. The
anti-inflammatory effect of ethanolic extract was calculated by the following equation:
Anti-inflammatory activity (%) = (Vc - Vt/ Vc) x 100
Where Vt represents the paw volume in drug treated animals and Vc represents the paw volume
of control group of animals.
8.2 Antimicrobial assay
8.2.1 Antibacterial assay
The antibacterial activity of newly synthesized compounds was evaluated by the agar well
diffusion method [44]. All the microbial cultures were adjusted to 0.5 McFarland standard,
which is visually comparable to a microbial suspension of approximately 1.5×108 cfu/mL
[46,51]. 20 mL of Mueller Hinton agar medium was poured into each Petri plate and the agar
plates were swabbed with 100 mL inocula of each test bacterium and kept for 15 min for
adsorption. Using sterile cork borer of 8 mm diameter, wells were bored into seeded agar plates
and these were loaded with a 100 mL volume with concentration of 4.0 mg/mL of each
compound reconstituted in dimethylsulphoxide (DMSO). All the plates were incubated at 37 °C
for 24 h. Antibacterial activity of 12 compounds was evaluated by measuring the zone of growth
inhibition against the test bacteria with zone reader (Hiantibiotic zone scale). DMSO was used as
a negative control whereas ciprofloxacin was used as a positive control. The experiments were
performed in triplicates. The antibacterial activity of the compounds was compared with
ciprofloxacin as standard. Minimum Inhibitory Concentration (MIC) of newly synthesized
compounds against tested bacteria was determined using macrodilution tube method as
Page 24 of 37

25
recommended by NCCLS [46,47]. MIC is the lowest concentration of an antimicrobial
compound that will inhibit the visible growth of a microorganism after overnight incubation. In
this method, various test concentrations of newly synthesized compounds were prepared from
128 to 0.25 mg/mL in sterile tubes No. 1-10. 100 mL sterile Mueller Hinton Broth (MHB) was
poured in each sterile tube followed by addition of 200 mL test compound in tube 1. Two fold
serial dilutions were carried out from tube 1 to tube 10 and excess broth (100 mL) was discarded
from the last tube No. 10. To each tube, 100 mL of standard inoculums (1.5×108 cfu/mL) was
added. Ciprofloxacin was used as control Turbidity was observed after incubating the inoculated
tubes at 37 °C for 24 h.
8.2.2 Antifungal assay
The antifungal activity of newly synthesized compounds was evaluated by the poisoned food
method [45]. The molds were grown on Saburaud Dextrose Agar (SDA) at 25 °C for 7 days and
used as inocula. 15 mL of molten SDA (45 °C) was poisoned by the addition of 100 mL volume
of each compound having concentration of 4.0 mg/mL, reconstituted in DMSO, poured into a
sterile Petri plate and allowed to solidify at room temperature. 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 7 days. DMSO was used as a negative
control whereas fluconazole was used as a positive control. The experiments were performed in
triplicates. Diameter of the fungal colonies was measured and expressed as percent mycelial
inhibition determined by applying the following formula:
Inhibition of mycelial growth % = (dc - dt)/dc × 100
Page 25 of 37

26
where dc = average diameter of fungal colony in negative control plates; dt = average diameter
of fungal colony in experimental plates.
8.3 Docking methodology
Docking study was carried out for the target compounds using Molegro Virtual Docker version
2010. MolDock scoring function is used by MVD program is defined by:
Escore = Einter + Eintra
where, Escore = MolDock score.
E
inter = ligand - Protein interaction
intra = internal energy of the ligand:
E
The molecules/ligands were built using Marvin Sketch 5.11.0. The 2D structure was then
converted into 3D which was saved as MDL MolFile. Crystal structure of COX-2 (PDB code:
1CX2) was obtained from the Protein Data Bank in order to prepare protein for docking studies.
Compounds were docked into the active sites using Molegro Virtual Docker 2010.4.1 software
using the standard protocol. [43]
Acknowledgement
We are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India for
providing financial assistance to Eakta Masan. Thanks are also due to University of California,
San Francisco for carrying out mass spectra and to Sophisticated Analytical Instrument Facility
Page 26 of 37

27
19
(SAIF), Central Drug Research Institute (CDRI), Lucknow for providing the F NMR spectra
and elemental analysis.
References
[1] S. Bondock , R. Rabie, A. Hassan, A. Etman, A. Fadda, Eur. J. Med. Chem. 43 (2008)
2122-2129.
[2] M. A. Al-Adiwish, M. I. M. Tahir, A. Siti-Noor-Adnalizawati, F. S. Hashim, N. Ibrahim,
W.A. Yaacob, Eur. J. Med. Chem 64 (2013) 464-476.
[3] R. Aggarwal, G. Sumran, N. Garg, A. Aggarwal, Eur. J. Med. Chem. 46 (2011) 3038-
3046.
[4] H. Behbehani, H. M. Ibrahim, S. Makhseed, H. Mahmoud, Eur. J. Med. Chem. 46 (2011)
1813-1820.
[5] S. Bondock, W. Fadaly, M. A. Metwally, Eur. J. Med. Chem. 45 (2010) 3692-3701.
[6] Q. Chen, X. L. Zhu, L. L. Jiang, Z. M. Liu, G. Yang, Eur. J. Med. Chem. 43 (2008) 595-
603.
[7] O. M. Ahmeda, M. A. Mohamed, R. R. Ahmed, R. R. Sayed, S. A. Ahmed, Eur. J. Med.
Chem. 44 (2009) 3519–3523.
[8] M. El-Enany, M. M. Kamel, O. M. Khalil, H. B. El-Nassan Eur. J. Med. Chem 2 (2011)
331-336.
[9] A. Kamal, J. R. Tamboli, V. L. Nayak, S. F. Adil, M. V. P. S. Vishnuvardhan, S.
Ramakrishna, Bioorg. Med. Chem. Lett. 23 (2013) 3208-3215.
Page 27 of 37

28
[10] M. G. Ghagare, D. R. Birari, D. P. Shelar, R. B. Toche, M. N. Jachak, J. Heterocycl.
Chem. 46 (2009) 327-331.
[11] S. Keitaro, T. Novinson, H. R. Wilson, J. Med. Chem. 24 1981 610-613.
[12] S. Q. Wang, L. Fang, X. J. Liu, K. Zhao, Chinese Chemical Letters 15 (2004) 885-888.
[13] R. A. Nugent, S. T. Schlachter, Patent (US) (1995) 5397774.
[14] K. M. Shokat, N. Sepetov, C. Zhang, H. W. Gschwend, E. J. Kunkel, Patent (US) (2009)
20090124638A1.
[15] C. F. P. George, Lancet 358 (2001) 1623-1626.
[16] D. Drover, H. Lemmens, S. Naidu, W. Cevallos, M. Darwish, D. Stanski, Clinical
Therapeutics 22 (2000) 1443-1461.
[17] K. W. Weitzel, J. M. Wickman, S. G. Augustin, J. G. Strom, Clinical Therapeutics 22
(2000) 1254-1267.
[18] R. E. Petroski, J. E. Pomeroy, R. Das, H. Bowman, W. Yang, A. P. Chen, A. C. Foster,
J. Pharmacol. Exp. Ther. 317 (2006) 369-377.
[19] A. Gomtsyan, S. Didomenico, C. Lee, A. O. Stewart, S, S. Bhagwat, E. A. Kowaluk, M.
F. Jarwis, Bioorg. Med. Chem. Lett. 14 (2004) 4165-4168.
[20] M. Labroli, K. Paruch, M. P. Dwyer, C. Alvarez, K. Keetikar, C. Poker, R. Rossman, J.
S. Duca, T. O. Fischmann, V. Madison, D. Parry, N. Davis, W. Seghezzi, D. Wiswell, T. J.
Guzi, Bioorg. Med. Chem. Lett. 21 (2011) 471-474.
[21] M. P. Dwyer, K. Paruch, M. Labroli, C. Alvarez, K. M. Keertikar, C. Poker, R.
Rossman, T. O. Fischmann, J. S. Duca, V. Madison, D. Parry, N. Davis, W. Seghezzi, D.
Wiswell, T. J. Guzi Bioorg. Med. Chem. Lett. 21 (2011) 467-470.
Page 28 of 37

29
[22] P. B. Bandgar, S. S. Gawande, R. G. Bodade, J. V. Totre, C. N. Khobragade, Bioorg.
Med. Chem. 18 (2010) 1364-1370.
[23] D. S. Williamson, M. J. Parratt, J. F. Bower, J. D. Moore, C. M. Richardson, P.
Dokurno, A. D. Cansfield, G. L. Francis, R. J. Hebdon, R. Howes, P. S. Jackson, A. M.
Lockie, J. B. Murray, C. L. Nunns, J. Powles, A. Robertson, A. E. Surgenor, C. J. Torrance,
Bioorg. Med. Chem. Lett. 15 (2005) 863-867.
[24] N. Kato, M. Oka, Murase, M. Yoshida, M. Sakairi, S. Yamashita, Y. Yasuda, A.
Yoshikawa, Y. Hayashi, M. Makino, M. Takeda, Y. Mirensha, T. Kakigami, Bioorg. Med.
Chem. 19 (2011) 7221–7227.
[25] L. Ren, E. R. Laird, A. J. Buckmelter, V. Dinkel, S. L. Gloor, J. Grina, B. Newhouse, K.
Rasor, G. Hastings, S. N. Gradl, J. Rudolph, Bioorg. Med. Chem. Lett. 22 (2012) 1165-
1168.
[26] D. R. Compton, K. E. Carlson, J. A. Katzenellenbogen, Bioorg. Med. Chem. Lett. 14
(2004) 5681-5684.
[27] (a) G. B. Elien, S. Gallawan, H. Nathan,S. Bicher, R. W. Randler, G. H. Hitchings,
Biochem. Pharmacol. 12 (1963) 85-93. (b) F. M. D. Ismail, J. Fluor. Chem. 118 (2002) 27-
33. (c) H.-J. Böhm, D. Banner, S. Bendels, M. Kansy, B. Kuhn, K. Müller, U. Obst-
Sander, M. Stahl, ChemBioChem 5 (2004) 637-643. (d) C. Isanbor, D. O'Hagan, J. Fluor.
Chem. 127 (2006) 303-319. (e) J.-P. Bégué, D. Bonnet-Delpon, J. Fluor. Chem. 127 (2006)
992-1012. (f) K. Müller, C. Faeh, F. Diederich, Science 317 (2007) 1881-1886. (g) S. Purser,
P. R. Moore, S. Swallow, V. Gouverneur, Chem. Soc. Rev. 37 (2008)
320-330. (h) W. K. Hagmann, J. Med. Chem. 51 (2008) 2108-2114.
Page 29 of 37