Synthesis, characterization and pharmacological activities of 2-[4-cyano-(3-trifluoromethyl)phenyl amino)]-4-(4-quinoline/coumarin-4-yloxy)-6- (fluoropiperazinyl)-s-triazines

Article abstract of DOI:10.1016/j.jfluchem.2011.06.021

A series of 2-[4-cyano-(3-trifluoromethyl)phenyl amino)]-4-(4-quinoline/ coumarin-4-yloxy)-6-(fluoropiperazinyl)-s-triazines has been synthesized by a simple and efficient synthetic protocol. The antimicrobial activity of the compounds was studied against

Full text of DOI:10.1016/j.jfluchem.2011.06.021

Journal of Fluorine Chemistry 132 (2011) 617–627
Contents lists available at ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Synthesis, characterization and pharmacological activities of
2-[4-cyano-(3-trifluoromethyl)phenyl amino)]
-4-(4-quinoline/coumarin-4-yloxy)-6-(fluoropiperazinyl)-s-triazines
a
b
c
Rahul V. Patel a, , Premlata Kumari , Dhanji. P. Rajani , Kishor H. Chikhalia
*
a Department of Applied Chemistry, S.V. National Institute of Technology, Surat 395007, Gujarat, India
b Microcare Laboratory, Surat 395001, Gujarat, India
c Department of Chemistry, School of Science, Gujarat University, Ahmedabad 380009, Gujarat, India
A R T I C L E I N F O
A B S T R A C T
Article history:
A series of 2-[4-cyano-(3-trifluoromethyl)phenyl amino)]-4-(4-quinoline/coumarin-4-yloxy)-6-(fluor-
opiperazinyl)-s-triazines has been synthesized by a simple and efficient synthetic protocol. The
antimicrobial activity of the compounds was studied against several bacteria (Staphylococcus aureus
MTCC 96, Bacillus cereus MTCC 619, Escherichia coli MTCC 739, Pseudomonas aeruginosa MTCC 741,
Klebsiella pneumoniae MTCC 109, Salmonella typhi MTCC 733, Proteus vulgaris MTCC 1771, Shigella
flexneria MTCC 1457) and fungi (Aspergillus niger MTCC 282, Aspergillus fumigatus MTCC 343, Aspergillus
clavatus MTCC 1323, Candida albicans MTCC 183) using paper disc diffusion technique and agar streak
dilution method. Newly synthesized compounds were also tested for their in vitro antimycobacterial
activity against Mycobacterium tuberculosis H37Rv using BACTEC MGIT and Lowenstein–Jensen MIC
method.
Received 13 May 2011
Received in revised form 16 June 2011
Accepted 17 June 2011
Available online 24 June 2011
Keywords:
2,4,6-Trichloro-1,3,5-triazine
4-Hydroxy-1-methyl-2(1H)-quinolone
4-Hydroxy-chromen-2-one
Fluoropiperazine
ß 2011 Elsevier B.V. All rights reserved.
Antimicrobial activity
Antimycobacterial activity
1. Introduction
improved biological profiles [7–9], here in we report the
synthesis and pharmacological activities of novel 2-[4-cyano-
Fluorine has played a pivotal role in novel drug discovery for
modulating physical and biological properties of the molecule.
Due to its higher electonegativity, incorporation of fluorine
atom(s) within the molecule can enhance their biopotency,
bioavailability, metabolic stability and lipophilicity. Trifluoro-
methylation is one of the most significant strategies to improve
pharmacological activities of the molecule due to its high
lipophilicity, thereby enhancing in vivo uptake and transport of
the candidate [1].
The treatment of opportunistic microbial infections has
become an important and challenging problem due to the
emergence of multiple-drug-resistant organisms [2–6]. Hence,
there is an urgent need to develop new classes of agents likely to
be unaffected by existing resistance mechanisms. As a part of our
ongoing studies to establish new s-triazine candidates with
(3-trifluoromethyl)phenyl amino)]-4-(4-quinoline/coumarin-4-
yloxy)-6-(fluoropiperazinyl)-s-triazines. 1,3,5-Triazine analo-
gues have gathered an immense attention among chemists due
to their wide range of biological activities such as antimicrobial
[10,11], antiprotozoal [12], anticancer [13], antimalarial [14] and
antiviral [15] activity. The literature survey revealed that
s-triazinyl analogues with insertion of N-fluorophenyl piperazine
bases have been reported to demonstrate a wide range of
pharmacological activities [16–22]. In addition, quinolones such
as ciprofloxacin, ofloxacin, lomefloxacin, and enoxacin are
established synthetic antibacterial agents [23] and are widely
prescribed for the treatment of infections in humans. The
presence of 4-hydroxy-chromen-2-one is also found to enhance
the various biological activities [24–26]. It is worth to mention
that the intermediate 4-amino-2-trifluoromethyl benzonitrile is
a structural unit of anti cancer agent bicalutamide, used in the
present study to provide essential F-atom(s) to the basic core
[27]. Prompted by these observations it was contemplated to
* Corresponding author. Tel.: +91 9712755525.
E-mail addresses: rahul.svnit11@gmail.com (R.V. Patel),
premlatakumari@gmail.com (P. Kumari), microcaresurat@yahoo.co.in
(Dhanji. P. Rajani), chikhalia_kh@yahoo.com (K.H. Chikhalia).
synthesize
a novel series of s-triazine analogues bearing
fluorinated moieties to identify new candidates that may be
0022-1139/$ – see front matter ß 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2011.06.021

618
R.V. Patel et al. / Journal of Fluorine Chemistry 132 (2011) 617–627
value in designing new potent derivatives endowed with various
biological activities (Schemes 1 and 2).
dichloro-1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzonitrile
(2b) in good yield by the nucleophilic displacement of one chlorine
atom of s-triazine ring by 4-amino-benzonitrile and 4-amino-2-
trifluoromethyl benzonitrile using triethyl amine. The synthesis of
disubstituted s-triazine intermediates 4a, 4b, 5a and 5b was
achieved in good yields by the reaction between 2a and 2b with 4-
hydroxy-1-methyl-2(1H)-quinolone and 4-hydroxy-chromen-2-
one, respectively, in the presence of 60% NaH at 45–50 8C.
Subsequent coupling of the so formed intermediates with the
2. Results and discussion
2.1. Chemistry
The first step comprises formation of intermediate 4-(4,6-
dichloro-1,3,5-triazin-2-ylamino)-benzonitrile (2a) and 4-(4,6-
Cl
CN
THF
Et3N
N
N
N
N
H2N
CN
Cl
Cl
N
N
H
2a
Cl
N
1a: 4-Amino-benzonitrile
N
N
Cl
+
CN
Cl
Cl
THF
Et3N
NC
F3C
NH2
N
N
H
CF3
2,4,6-Trichloro-
1,3,5-triazine
1b: 4-Amino-2-trif luoromethyl-
2b
benzonitrile
Cl
CN
N
N
O
N
N
H
OH
X
4a: X=N-CH3
4b: X=O
THF
O
2a, 2b
+
NaH
Cl
X
O
CN
CF3
N
N
3a: X=N-CH3
3b: X=O
O
N
N
H
X
5a: X=N-CH3
5b: X=O
O
R
CN
N
N
O
N
N
H
1,4-Dioxane
K2CO3
X
4a, 4b, 5a, 5b
+
6a-d
7a-d: X=N-CH3
8a-d: X=O
O
X
R
CN
N
N
O
N
N
H
CF3
9a-d: X=N-CH3
10a-d: X=O
O
Scheme 1. Synthesis of final s-triazinyl fluoro-piperazines.

R.V. Patel et al. / Journal of Fluorine Chemistry 132 (2011) 617–627
619
attributable to the C-6 proton of the coumarin ring. Proton atoms
corresponding to the C-7 and C-8 carbon atoms found to resonate
in the form of multiplets in the range either at 7.55–7.59 or at
7.29–7.33 ppm in the 1H NMR of 10b. 13C NMR spectra of the
compound 8c displayed signals at 178.7 ppm and 165.9 ppm
assigned due to the C-6 and C-4 carbon atoms of triazinyl ring.
Another two signals observed at 165.6 and 165.1 appeared due to
either C-2 atom of triazine ring or C55O belonging to coumarin
nucleus. Two peaks observed at 126.3 and 131.1 ppm indicated
the presence of trifluoromethyl functional group in the benzoni-
trile moiety of 8c, while cyano group indicated the corresponding
signals at 105.1 ppm and 98.6 ppm. The carbon atoms of the
F
HN
HN
N
N
N
N
6a
piperazine ring gave signals at 50.2 ppm and 44.9 ppm in the 13
C
F
6b
6c
NMR spectra of 8c. Final analogues with single fluorine
substituted piperazine bases gave a peak in the region 150–
152 ppm corresponding to the carbon atom at which fluorine
atom is introduced. The determined values of 19F NMR chemical
shifts being in compliance with the reported literature [28]. 19F
NMR spectra obtained for compounds 9a and 9b indicated the
confirmation of the presence of single fluorine atom to the ortho
(2) and para (4) position of the phenyl ring attached to the amino
nitrogen of piperazine base by giving the corresponding
resonating peaks at ꢀ120.60 ppm and ꢀ118.09 ppm, respective-
ly. Whereas, the trifluoromethyl group of amino-benzonitrile
moiety found to reveal a singlet at ꢀ64.02 and ꢀ63.89 ppm in the
19F NMR spectra of compounds 10c and 10d, respectively.
Additionally, trifluoromethyl group present at the meta (3) and
para (4) position of the phenyl ring of piperazine base found to
reveal singlet peaks at ꢀ63.60 ppm and ꢀ65.73 ppm, respective-
ly. Assignments of the structure of final scaffolds are based on
correct elemental analysis which was found to be within ꢁ0.2%
limit (Table 1).
CF3
HN
HN
6d
CF3
Scheme 2. Where 6a–d, R = piperazine bases coupled to compounds 4a, 4b, 5a
and 5b.
2.2. Pharmacology
Investigation on antibacterial screening data (Table 2) showed
some of the compounds were active against all the mentioned
bacteria. Final s-triazinyl compounds 9c and 9d have the highest
desired fluoro-piperazines under basic conditions in 1,4-dioxane
solvent at 70–80 8C produced the corresponding 2-[4-cyano-
(3-trifluoromethyl)phenyl amino)]-4-(4-quinoline/coumarin-4-
yloxy) -6-(fluoropiperazinyl)-s-triazines. Compounds 2a and 2b
displayed absorption bands at 2223 cmꢀ1 confirming the presence
of a cyano group and strong bands at 3288 cmꢀ1 and 3282 cmꢀ1
appearing due to the presence of an –NH functional group. A C3N3
stretching frequency in s-triazine ring was observed at 827–
839 cmꢀ1 in the IR spectra of final compounds. Moreover,
absorption bands ranging from 1249 to 1259 cmꢀ1 corresponded
with the C–O–C linkage formed between s-triazine ring and
quinoline or coumarin moieties by disappearing stretching peak
at 3610 cmꢀ1 of O–H gave correction to the formation of
intermediates 4a, 4b, 5a and 5b. The absence of absorption of
C–Cl in the region 700–760 cmꢀ1 indicated the formation of final
compounds. In the 1H NMR spectra, the synthesis of aimed
compounds was confirmed on the basis of the fact that the proton
ability (MIC, 6.25
mg/mL and 27 mm of zone of inhibition) to
inhibit Staphylococcus aureus. Compound 7d exhibited similar
minimum inhibitory concentration against S. aureus with quite
reduced inhibition zone (26 mm). Final analogues 7d and 9b
showed higher effectiveness (26 mm of zone of inhibition) at
12.5
m
g/mL against Bacillus cereus with similar MIC of compound
g/mL)
9c (inhibition zone: 25 mm). The best activity (MIC, 12.5
m
was observed with compound 7a against Escherichia coli,
whereas, MIC values exhibited by 7b and 8b were equal to that
of 7a with a slight enhancement in the inhibition zones (25 mm).
Compounds 7b, 9b and 10c exhibited the greatest activity against
Pseudomonas aeruginosa at 6.25
mg/mL of MIC. Besides, com-
pound 9d was found to have similar inhibitory effect in terms of
MIC against P. aeruginosa with slight reduced inhibitory zone
(23 mm). Inhibition of Klebsiella pneumoniae was also noted for s-
atoms of piperazine ring appeared as two doublets at
d
, 3.41–3.55
triazine derivative 10d at 25
inhibition). Compounds 9a and 9c had an effective action
(MIC, 25 g/mL) against Salmonella typhi along with similar
MIC profile of compound 9d (inhibition zone: 23 mm). Com-
pound 9d was superior in inhibiting the growth (25 mm of zone
mg/mL (23 mm of zone of
and , 3.79–3.89. Signal varying from values 9.14 to 9.28 ppm
d
d
attributable to an –NH group. The 1H NMR spectra of 7b displayed
two doublets at 8.13 and 7.56 ppm attributed to the C-8 and C-5
protons, respectively of the quinoline ring, whereas, another two
signals observed at 7.73 and 7.49 ppm in the form of triplate
assigned due to the C-7 and C-6 protons of the quinoline ring,
respectively. A sharp singlet at 7.41 ppm was due the C-3 proton
of the quinoline ring for 7b. The protons corresponding to the
methyl group appeared as singlet at 3.69 ppm. The 1H NMR
spectra of the compound 10b showed a singlet at 9.15 ppm of –NH
group, set of doublet of doublets at 8.06 ppm due to the C-5
proton of the coumarin ring system as well as a triplet at 7.46 ppm
m
of inhibition) of Proteus vulgaris (MIC, 12.5
inhibitory effect was shown by final derivatives 7d and 9c at
MIC, 12.5 g/mL (26 mm of zone of inhibition) against Shigella
flexneria along with similar efficacy of compounds 7c and 9a in
terms of MIC (MIC, 12.5 g/mL) against the same bacteria with
mg/mL). Strong
m
m
slight reduced diameter of inhibition zone (25 mm). All the
remaining final s-triazine derivatives exerted good to moderate
activity.

620
R.V. Patel et al. / Journal of Fluorine Chemistry 132 (2011) 617–627
Table 1
Physical and analytical data of newly synthesized compounds.
Entry
Yield (%)
m.p. (8C)
Mol. formula
Elemental analysis
Found%
Cal.%
C
C
H
N
H
N
7a
79
84
72
80
86
85
85
80
79
85
72
80
86
85
85
82
247–249
269–271
280–282
>300
C30H25FN8O2
C30H25FN8O2
C31H25F3N8O2
C31H25F3N8O2
C29H22FN7O3
C29H22FN7O3
C30H22F3N7O3
C30H22F3N7O3
C31H24F4N8O2
C31H24F4N8O2
C32H24F6N8O2
C32H24F6N8O2
C30H21F4N7O3
C30H21F4N7O3
C31H21F6N7O3
C31H21F6N7O3
65.52
65.56
62.38
61.98
64.87
64.89
61.33
61.42
60.21
61.24
57.55
57.49
59.89
61.83
57.19
56.89
4.47
4.39
4.08
4.13
4.27
3.99
4.01
3.63
3.78
4.07
3.48
3.42
3.58
3.66
3.48
3.33
20.61
20.31
18.53
18.61
18.51
18.22
16.53
16.64
18.02
18.36
16.60
17.94
16.10
16.41
14.81
15.09
65.68
65.68
62.20
62.20
65.04
65.04
61.54
61.54
60.39
60.39
57.66
57.66
59.70
59.70
56.97
56.97
4.59
4.59
4.21
4.21
4.14
4.14
3.79
3.79
3.92
3.92
3.63
3.63
3.51
3.51
3.24
3.24
20.43
20.43
18.72
18.72
18.31
18.31
16.74
16.74
18.17
18.17
16.81
16.81
16.25
16.25
15.00
15.00
7b
7c
7d
8a
247–250
288–290
280–282
>300
8b
8c
8d
9a
219–222
250–253
258–260
260–261
219–223
250–254
258–261
260–262
9b
9c
9d
10a
10b
10c
10d
Table 2
In vitro antimicrobial activity.
R
N
N
CN
R'
N
N
O
N
N
H
X
O
Entry
R
R0
X
Zone of inhibition [mm (MIC in
Gram-positive bacteriaa
m
g/mL)]
Gram-negative bacteriab
S.a.
B.c.
E.c.
P.a.
K.p.
S.t.
P.v.
S.f.
7a
2-F
H
N-CH3
N-CH3
N-CH3
N-CH3
O
21 (100)
20 (100)
22 (50)
22 (50)
21 (100)
21 (100)
26 (12.5)
18 (100)
18 (100)
20 (100)
19 (100)
24 (25)
26 (12.5)
25 (12.5)
24 (25)
21 (100)
18 (100)
22 (50)
23 (25)
31 (ꢂ3.12)
–
26 (12.5)
25 (12.5)
22 (50)
23 (50)
24 (25)
25 (12.5)
22 (50)
21 (100)
22 (50)
22 (50)
20 (100)
21 (100)
19 (100)
17 (100)
22 (50)
19 (100)
32 (ꢂ3.12)
–
20 (50)
<10 (100)
17 (100)
19 (100)
20 (100)
17 (100)
17 (100)
<10 (100)
<10 (100)
22 (50)
20 (100)
22 (50)
23 (25)
22 (50)
16 (100)
19 (100)
19 (100)
20 (100)
24 (25)
22 (50)
24 (25)
23 (25)
19 (100)
20 (100)
20 (100)
21 (50)
30 (ꢂ3.12)
–
21 (100)
19 (100)
24 (25)
22 (50)
19 (100)
17 (100)
25 (12.5)
26 (12.5)
18 (100)
20 (100)
22 (50)
7b
4-F
H
24 (6.25)
19 (100)
22 (12.5)
19 (100)
21 (25)
7c
3-CF3
4-CF3
2-F
H
7d
H
26 (6.25)
17 (100)
18 (100)
21 (100)
23 (25)
8a
H
<10 (100)
15 (100)
18 (100)
20 (100)
22 (50)
8b
4-F
H
O
8c
3-CF3
4-CF3
2-F
H
O
20 (50)
8d
H
O
17 (100)
20 (50)
21 (100)
25 (12.5)
22 (50)
9a
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
N-CH3
N-CH3
N-CH3
N-CH3
O
22 (50)
9b
4-F
20 (100)
27 (6.25)
27 (6.25)
20 (100)
19 (100)
23 (25)
24 (6.25)
21 (25)
18 (100)
22 (50)
21 (100)
23 (50)
9c
3-CF3
4-CF3
2-F
26 (12.5)
25 (25)
21 (100)
20 (100)
23 (50)
9d
23 (6.25)
20 (50)
21 (100)
<10 (100)
20 (100)
22 (50)
25 (12.5)
18 (100)
18 (100)
21 (100)
23 (50)
10a
10b
10c
10d
Cip.
DMSO
4-F
O
19 (100)
24 (6.25)
21 (25)
3-CF3
4-CF3
O
O
25 (12.5)
30 (ꢂ3.12)
–
23 (25)
22 (50)
33 (ꢂ3.12)
33 (ꢂ3.12)
–
31 (ꢂ3.12)
–
32 (ꢂ3.12)
–
–
Cip. – ciprofloxacin.
Bold values refer to the higher activities in terms of lowest MICs.
a
S.a. – Staphylococcus aureus and B.c. – Bacillus cereus.
b
E.c. – Escherichia coli, P.a. – Pseudomonas aeruginosa, K.p. – Klebsiella pneumoniae, S.t. – Salmonella typhi, P.v. – Proteus vulgaris, and S.f. – Shigella flexneria.

R.V. Patel et al. / Journal of Fluorine Chemistry 132 (2011) 617–627
621
Table 3
In vitro antifungal activity.
R
N
N
CN
N
N
O
N
N
H
R'
X
O
Entry
R
R0
X
Zone of inhibition [mm (MIC in
Fungal strainsa
mg/mL)]
A.n.
A.f.
A.c.
C.a.
7a
2-F
H
N-CH3
N-CH3
N-CH3
N-CH3
O
13 (100)
<10 (100)
<10 (100)
18 (100)
21 (50)
18 (100)
22 (50)
23 (25)
20 (100)
15 (100)
16 (100)
19 (100)
19 (100)
24 (25)
22 (50)
24 (25)
22 (50)
20 (100)
17 (100)
16 (100)
24 (25)
31 (ꢂ3.12)
–
13 (100)
14 (100)
24 (25)
24 (25)
12 (100)
16 (100)
22 (50)
16 (100)
20 (100)
22 (50)
24 (25)
22 (50)
16 (100)
16 (100)
23 (25)
22 (50)
7b
4-F
H
17 (100)
22 (50)
7c
3-CF3
4-CF3
2-F
H
7d
H
23 (25)
8a
H
<10 (100)
14 (100)
17 (100)
18 (100)
20 (100)
19 (100)
25 (12.5)
24 (12.5)
19 (100)
20 (100)
23 (25)
<10 (100)
<10 (100)
14 (100)
16 (100)
16 (100)
22 (25)
8b
4-F
H
O
8c
3-CF3
4-CF3
2-F
H
O
8d
H
O
9a
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
N-CH3
N-CH3
N-CH3
N-CH3
O
9b
4-F
9c
3-CF3
4-CF3
2-F
21 (50)
9d
23 (25)
10a
10b
10c
10d
Kit.
DMSO
<10 (100)
16 (100)
18 (100)
19 (100)
29 (ꢂ3.12)
–
4-F
O
3-CF3
4-CF3
O
O
22 (50)
30 (ꢂ3.12)
–
33 (ꢂ3.12)
–
Kit. – ketoconazole.
Bold values refer to the higher activities in terms of lowest MICs.
a
A.n. – Aspergillus niger, A.f. – Aspergillus fumigates, A.c. – Aspergillus clavatus, and C.a. – Candida albicans.
The antifungal results data (Table 3) revealed that, the
synthesized compounds showed variable degree of inhibition
against the tested fungi. Final s-triazine derivative 9c displayed
preliminary results observed from BACTEC MGIT method
indicated that compounds 7d, 9c and 9d exhibited highest
inhibition (99%) at a constant concentration level (6.25
mg/mL)
antigrowth activity (MIC, 12.5
m
g/mL) against Aspergillus niger.
against M. tuberculosis H37Rv. The primary BACTEC MGIT
bioassay results have driven us to examine the real potency
(MIC) of the title compounds against M. tuberculosis H37Rv. The
secondary biological screening was performed using Lowen-
stein–Jensen MIC method and it is worthwhile to note that
compounds 7d and 9c were the compounds displaying inhibition
Compound 9d displayed activity same as 9c in terms of MIC
against the same fungi with one mm of reduced growth
inhibitory diameter (24 mm). A significant inhibition was also
observed for compounds 9d against Aspergillus fumigatus at
25
m
g/mL of MIC (23 mm of zone of inhibition). Similar minimum
g/mL) observed against the
inhibitory concentration level (25
m
of M. tuberculosis H37Rv completely (99%) at the MIC of 3.12 mg/
same fungi for compound 9b with slightly reduced inhibition
zone (22 mm). Compounds 9a, 9c and 10d act as the most potent
mL. These compound were considered to display even better
efficacy, than the standard drug, pyrazinamide, whereas the MIC
inhibitors of the growth of Aspergillus clavatus fungi (MIC, 25
mL), while compound 7c was found to display similar MIC of
25 g/mL and quite reduced inhibition zone (23 mm). Com-
pounds 7c, 7d and 9c possessed the highest antifungal activity
against Candida albicans at 25 g/mL (24 mm of zone of
inhibition) as well as compound 10c with similar MIC and
one mm of lesser inhibition zone (23 mm). All the remaining
final s-triazine derivatives exerted good to moderate activity
(Table 4).
m
g/
of compound 9d was found (6.25
MGIT test. Compound 9b appeared with good inhibition effect in
the term of MIC at 12.5 g/mL, while Compound 9a demonstrat-
ed 25 g/mL of MIC against M. tuberculosis H37Rv. Final s-
triazine derivative 7c diminishes good activity at MIC level of
50 g/mL, whereas, compound 7b indicated inhibition of M.
tuberculosis H37Rv at 62.5 g/mL. Final compounds 10c as well
as 10d were able to produce moderate inhibitory activity against
M. tuberculosis H37RV at the MIC level of 100 g/mL. All the
remaining derivatives were found to exert higher MIC at 200–
1000 g/mL.
mg/mL) as same as BACTEC
m
m
m
m
m
m
m
In vitro antimycobacterial activities of compounds 5a–u were
assessed against Mycobacterium tuberculosis H37Rv strain. The
m

622
R.V. Patel et al. / Journal of Fluorine Chemistry 132 (2011) 617–627
Table 4
In vitro antimycobacterial activity.
R
N
N
CN
N
N
O
N
N
H
R'
X
O
Entry
R
R0
X
BACTEC MGIT method
MIC ( g/mL)
L. J. MIC method
m
% Inhibition
MIC (
mg/mL)
% Inhibition
7a
2-F
H
N-CH3
N-CH3
N-CH3
N-CH3
O
>6.25
>6.25
>6.25
6.25
–
–
200
95
97
98
99
96
95
97
95
98
97
99
99
96
95
96
95
7b
4-F
H
62.5
50
3.12
500
7c
3-CF3
4-CF3
2-F
H
–
7d
H
99
–
8a
H
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
6.25
8b
4-F
H
O
–
500
250
1000
25
8c
3-CF3
4-CF3
2-F
H
O
–
8d
H
O
–
9a
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
N-CH3
N-CH3
N-CH3
N-CH3
O
–
9b
4-F
–
12.5
3.12
6.25
500
9c
3-CF3
4-CF3
2-F
99
99
–
9d
6.25
10a
>6.25
>6.25
>6.25
>6.25
0.20
10b
4-F
O
–
500
100
100
10c
3-CF3
4-CF3
O
–
10d
O
–
Isoniazid
Refampicin
Ethambutol
Pyrazinamide
99
99
99
99
0.25
3.12
6.25
Bold values refer to the higher activities in terms of lowest MICs.
3. Conclusion
4. Experimental
In conclusion, it is clear that the antimicrobial activity of the
title compounds is strongly bound to the nature of the piperazine
substituent inserted at C-6 position of s-triazine ring, together
with the fluorinated substituent linked to C-2 position of the
s-triazine core. Bioassay results revealed that seven compounds
(7b, 7c, 7d, 9a, 9b, 9c, and 9d) out of the 16 studied displayed
variable in vitro antibacterial, antifungal and antimycobacterial
inhibitory effects. The presence of 4-hydroxy-1-methyl-2(1H)-
quinolone ring in the title compounds significantly increase the in
vitro biological potency of the resultant compounds compared to
that of with 4-hydroxy-chromen-2-one. Furthermore, final
derivatives with piperazine bases containing trifluoromethyl
functional group were found more active than that of containing
single fluorine atom substitution. In addition, the greater
biological profiles were observed for the compounds containing
fluorinated amino-benzonitrile component incorporated to the
nucleus than non-fluorinated amino-benzonitrile insertion.
Therefore, it is concluded that there exists ample scope for
further study in this class of compounds in order to discover varied
biological profiles such as anticancer activity or anti-HIV activity.
The study is currently under investigation and the results will be
published in due course.
4.1. Chemistry
4.1.1. General experimental procedures
2,4,6-Trichloro-1,3,5-triazine,
4-hydroxy-1-methyl-2(1H)-
quinolone and 4-trifluoromethyl phenyl piperazine were pur-
chased from Sigma Aldrich Chemicals Pvt. Ltd., Mumbai, India.
4-Hydroxy-chromen-2-one was a gift from Ami Organics Pvt. Ltd.,
Sachin, Surat, India. 4-Amino-benzonitrile was
a gift from
Yashashvi Rasayan Pvt. Ltd., Maroli, India and 4-amino-2-
trifluoromrthyl-benzonitrile was a gift from Ramdev Chemicals
Pvt. Ltd., Boisar, India. 3-Trifluoromethylphenyl piperazine, 2-
fluorophenyl piperazine and 4-fluorophenyl piperazine were gifts
from Dr. Prem’s Molecules Pvt. Ltd., Vadodara, India. Acetone,
Tetrahydrofuran and 1,4-dioxane of HPLC grade were purchased
from Rankem, Surat, India. The TLC plates (silica gel 60 F254 grade)
were obtained from Merck, Germany.
Melting points were determined in open capillaries on a Veego
electronic apparatus VMP-D (Veego Instrument Corporation,
Mumbai, India) and are uncorrected. IR spectra (4000–
400 cmꢀ1
) of synthesized compounds were recorded on a
Shimadzu 8400-S FT-IR spectrophotometer (Shimadzu India Pvt.
Ltd., Mumbai, India) using KBr pellets. Thin layer chromatography

R.V. Patel et al. / Journal of Fluorine Chemistry 132 (2011) 617–627
623
was performed on object glass slides (2 cm ꢃ 7.5 cm) coated with
silica gel-G and spots were visualized under UV irradiation. 1H
NMR and 13C NMR spectra were recorded on a Varian 400 MHz
model spectrometer (Varian India Pvt. Ltd., Mumbai, India) using
DMSO as a solvent and TMS as internal standard with 1H resonant
4.1.5. General procedure for synthesis of compounds (7a–d, 8a–d, 9a–
d, and 10a–d)
To a solution of 4a, 4b, 5a and 5b (0.01 mol) in 1,4-dioxane
(20 mL), the respective substituted piperazine derivative (6a–d)
was added and the reaction mixture was refluxed for 16–22 h.
Potassium carbonate was used for the neutralization of the
reaction mixture. Progress of the reaction was monitored by TLC
using toluene:acetone (8:2) as eluent. After the completion of the
reaction, it was treated with crushed ice, neutralized by dilute HCl.
The precipitates thus obtained was filtered off, dried and
recrystallized from THF to afford desired compounds 7a–d, 8a–
d, 9a–d and 10a–d.
frequency of 400 MHz and 13C resonant frequency of 100 MHz. 19
F
NMR spectra were obtained on the same spectrometer using CDCl3
as a solvent and CFCl3 as an external standard with 19F resonant
frequency of 400 MHz. The 1H NMR, 13C NMR and 19F NMR
chemical shifts were reported as parts per million (ppm) downfield
from TMS (Me4Si) and CFCl3 and were performed at centre for
excellence, Vapi, India. The splitting patterns are designated as
follows; s, singlet; br s, broad singlet; d, doublet; m, multiplet.
Elemental analyses (C, H, N) were performed using a Heraeus Carlo
Erba 1180 CHN analyzer (Hanau, Germany).
4.2. Characterization data of synthesized compounds (6a–e, 7a–e, 8a–
e, 9a–e)
4-(4,6-Dichloro-1,3,5-triazin-2-ylamino)-benzonitrile (2a) was
synthesized according to the reported literature [29].
4.2.1. 4-[4-[4-(2-Fluoro-phenyl)-piperazin-1-yl]-6-(1-methyl-2-
oxo-1,2-dihydro-quinolin-4-yloxy)-[1,3,5]triazin-2-ylamino]-
benzonitrile (7a)
4.1.2. General procedure for the synthesis of 4-(4,6-dichloro-1,3,5-
triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (2b)
Dark yellow solid; IR (KBr, cmꢀ1):
v
3290 (–NH), 3069–3080
(–CH Str.), 2223 (CN), 1255 (C–O–C), 831 (s-triazine C–N str.), 743
(C–F). 1H NMR (400 MHz, Me2SO-d6):
9.21 (s, 1H, –NH, s-triazine
To a stirred solution of 2,4,6-trichloro-1,3,5-triazine (10 g,
0.054 mol) in anhydrous THF (150 mL) was added 4-amino-2-
trifluoromethyl-benzonitrile 1b (10.09 g in THF, 0.054 mol) drop
wise at 0–5 8C. The resulting reaction mixture was stirred at this
temperature for 3 h, then triethyl amine (5.48 g, 0.054 mol) was
added in the reaction mixture and stirring was continued for
another 6 h. Progress of the reaction was monitored by TLC using
toluene:acetone (9:1) as eluent. The resulted reaction mixture was
then treated with crushed ice, followed by neutralization by dilute
HCl and then filtered, dried and recrystallized from acetone to
afford 2b. Yield: 90%, m.p. 259–261 8C (dec.), FT-IR (KBr, cmꢀ1):
3282 (–NH), 2223 (CN).
d
to amino-benzonitrile linkage), 8.08 (d, J = 7.3 Hz, 1H, C8 proton of
quinoline), 7.67 (t, J = 7.5 Hz, 1H, C7 proton of quinoline), 7.59 (d,
J = 8.1 Hz, 1H, C5 proton of quinoline), 7.51 (t, J = 7.9 Hz, 1H, C6
proton of quinoline), 7.40 (s, 1H, C3 proton of quinoline), 7.31–6.88
(8H, m, Ar-H), 3.89 (4H, br s, piperazine), 3.73 (s, 3H, N-CH3), 3.51
(4H, br s, piperazine). 13C NMR (100 MHz, Me2SO-d6):
d 178.2 (1C,
C-6, s-triazine, C–N at piperazine linkage), 168.7 (1C, C-4,
s-triazine, C–O–C at quinoline linkage), 164.5, 163.5 (2C, 1C at
C-2, s-triazine, C–NH at benzonitrile moiety and 1C of C55O), 151.2
(1C, C–F), 147.3–118.1 (16C, Ar. C), 105.1 (1C, CBB N), 96.5 (1C, –C–
CBB N), 49.5, 46.3 (4C, piperazine ring carbon atoms), 30.2 (1C,
4.1.3. General procedure for the synthesis of 4-(4-chloro-6-(1-
methyl-2-oxo-1,2-dihydroquinolin-4-yloxy)-1,3,5-triazin-2-
ylamino)benzonitrile (4a) and 4-(4-chloro-6-(1-methyl-2-oxo-1,2-
dihydroquinolin-4-yloxy)-1,3,5-triazin-2-ylamino)-2-
N-CH3). 19F NMR (400 MHz, CDCl3):
d
ꢀ120.39 (1F, s, 2-F).
4.2.2. 4-[4-[4-(4-Fluoro-phenyl)-piperazin-1-yl]-6-(1-methyl-2-
oxo-1,2-dihydro-quinolin-4-yloxy)-[1,3,5]triazin-2-ylamino]-
benzonitrile (7b)
trifluoromethyl-benzonitrile (5a)
To a stirred solution of 4-hydroxy-1-methyl-2(1H)-quinolone
(3a) (8 g, 0.045 mol) in anhydrous THF (150 mL) was added 60%
NaH (1.08 g, 0.055 mol) at room temperature for 1 h and 2a
(12.15 g, 0.045 mol) and 2b (15.03 g, 0.045 mol) was added into
the reaction mixture. Stirring was continued for another 8 h (2a)
and 12 h (2b) at 45 8C. Progress of the reaction was monitored
by TLC using toluene:acetone (7:3) as eluent. After the
completion of the reaction, it was treated with crushed ice,
filtered and dried by THF to afford 4a and 5a, 4a: Yield: 80%, m.p.
Darkyellow solid;IR (KBr, cmꢀ1):
v
3268(–NH), 3058–3073 (–CH
Str.), 2224 (CN), 1258 (C–O–C), 829 (s-triazine C–N str.), 758 (C–F).
1H NMR (400 MHz, Me2SO-d6):
9.19 (s, 1H, –NH, s-triazine to
d
amino-benzonitrile linkage), 8.13 (d, J = 7.7 Hz, 1H, C8 proton of
quinoline), 7.73 (t, J = 7.8 Hz, 1H, C7 proton of quinoline), 7.56
(d, J = 8.3 Hz, 1H, C5 proton of quinoline), 7.49 (t, J = 7.5 Hz, 1H, C6
proton of quinoline), 7.41 (s, 1H, C3 proton of quinoline), 7.24–6.83
(8H, m, Ar-H), 3.86 (4H, br s, piperazine), 3.69 (s, 3H, N-CH3), 3.45
(4H, br s, piperazine). 13C NMR (100 MHz, Me2SO-d6):
d 177.7 (1C,
277–229 8C (dec.). IR (KBr, cmꢀ1):
v
2219 (CN), 1256 (C–O–C);
C-6, s-triazine, C–N at piperazine linkage), 167.4 (1C, C-4, s-triazine,
C–O–C at quinoline linkage), 165.1, 163.7 (2C, 1C at C-2, s-triazine,
C–NH at benzonitrile moiety and 1C of C55O), 150.8 (1C, C–F),
145.7–117.9 (16C, Ar. C), 106.3 (1C, CBB N), 98.5 (1C, –C–CBB N), 47.7,
43.3 (4C, piperazine ring carbon atoms), 29.8 (1C, N-CH3). 19F NMR
5a: Yield: 83%, m.p. 229–231 8C (dec.). IR (KBr, cmꢀ1):
v 2222
(CN), 1259 (C–O–C).
4.1.4. General procedure for the synthesis of 4-[4-chloro-6-
(quinazolin-4-yloxy)-1,3,5-triazine-2-ylamino]-benzonitrile (4b) and
4-[4-chloro-6-(quinazolin-4-yloxy)-1,3,5-triazine-2-ylamino]-2-
trifluoromethyl-benzonitrile (5b)
(400 MHz, CDCl3):
d
ꢀ117.82 (1F, s, 4-F).
4.2.3. 4-{4-(1-Methyl-2-oxo-1,2-dihydro-quinolin-4-yloxy)-6-[4-(3-
trifluoromethyl-phenyl)-piperazin-1-yl]-[1,3,5]triazin-2-ylamino}-
benzonitrile (7c)
To a stirred solution of 4-hydroxy-chromen-2-one (3b) (8 g,
0.049 mol) in anhydrous THF (150 mL) was added 60% NaH (1.11 g,
0.049 mol) at room temperature for 1 h and 2a (13.03 g, 0.049 mol)
and 2b (16.36 g, 0.049 mol) was added into the reaction mixture.
Stirring was continued for another 6 h (2a) and 8 h (2b) at 45 8C.
Progress of the reaction was monitored by TLC using toluene:
acetone (97:3, v/v) as eluent. After the completion of the reaction,
it was treated with crushed ice, filtered and dried by THF to afford
4b and 5b [30]. 4b: Yield: 74%, m.p. 285–287 8C (dec.). IR (KBr,
Brown solid; IR (KBr, cmꢀ1):
v
3288 (–NH), 3060–3068 (–CH
Str.), 2223 (CN), 1257 (C–O–C), 830 (s-triazine C–N str.), 752 (C–F);
1H NMR (400 MHz, Me2SO-d6):
9.15 (s, 1H, –NH, s-triazine to
d
amino-benzonitrile linkage), 8.17 (d, J = 7.6 Hz, 1H, C8 proton of
quinoline), 7.62 (t, J = 7.5 Hz, 1H, C7 proton of quinoline), 7.51 (d,
J = 8.3 Hz, 1H, C5 proton of quinoline), 7.44 (t, J = 7.7 Hz, 1H, C6
proton of quinoline), 7.38 (s, 1H, C3 proton of quinoline), 7.29–6.91
(8H, m, Ar-H), 3.81 (4H, br s, piperazine), 3.77 (s, 3H, N-CH3), 3.46
cmꢀ1):
(dec.). IR (KBr, cmꢀ1):
v
2218 (CN), 1249 (C–O–C). 5b: Yield: 80%, m.p. 209–213 8C
2223 (CN), 1253 (C–O–C).
v
(4H, br s, piperazine). 13C NMR (100 MHz, Me2SO-d6):
d 176.4 (1C,

624
R.V. Patel et al. / Journal of Fluorine Chemistry 132 (2011) 617–627
C-6, s-triazine, C–N at piperazine linkage), 166.2 (1C, C-4, s-
triazine, C–O–C at quinoline linkage), 164.6, 164.1 (2C, 1C at C-2, s-
triazine, C–NH at benzonitrile moiety and 1C of C55O), 145.9–116.4
(20C, Ar. C including C–CF3 at 131.4 and CF3 at 125.9), 104.9 (1C,
CBB N), 97.8 (1C, –C–CBB N), 48.3, 45.2 (4C, piperazine ring carbon
1H NMR (400 MHz, Me2SO-d6):
d 9.27 (s, 1H, –NH, s-triazine to
amino-benzonitrile linkage), 8.05 (dd, J = 1.4, 1.2 Hz, 1H, C5 proton
of coumarin), 7.57–7.61 (m, 1H, coumarin), 7.49 (t, J = 8.7 Hz, 1H,
C6 proton of coumarin), 7.29–32 (m, 1H, coumarin), 7.23–6.87 (9H,
m, Ar-H), 3.88 (4H, br s, piperazine), 3.53 (4H, br s, piperazine). 13
C
atoms), 29.3 (1C, N-CH3). 19F NMR (400 MHz, CDCl3):
s, 3-CF3).
d
ꢀ63.95 (3F,
NMR (100 MHz, Me2SO-d6): d 178.7 (1C, C-6, s-triazine, C–N at
piperazine linkage), 165.9 (1C, C-4, s-triazine, C–O–C at quinoline
linkage), 165.6, 165.1 (2C, 1C at C-2, s-triazine, C–NH at
benzonitrile moiety and 1C of C55O), 153.3 (1C of C-9, coumarin),
147.1–117.7 (19C, Ar. C including C–CF3 at 131.1 and CF3 at 126.3),
105.1 (1C, CBB N), 98.6 (1C, –C–CBB N), 50.2, 44.9 (4C, piperazine ring
4.2.4. 4-{4-(1-Methyl-2-oxo-1,2-dihydro-quinolin-4-yloxy)-6-[4-(4-
trifluoromethyl-phenyl)-piperazin-1-yl]-[1,3,5]triazin-2-ylamino}-
benzonitrile (7d)
Dark brown solid; IR (KBr, cmꢀ1):
v
3279 (–NH), 3066–3074 (–
CH Str.), 2224 (CN), 1253 (C–O–C), 831 (s-triazine C–N str.), 760 (C–
F). 1H NMR (400 MHz, Me2SO-d6):
9.24 (s, 1H, –NH, s-triazine to
carbon atoms). 19F NMR (400 MHz, CDCl3):
d
ꢀ63.42 (3F, s, 3-CF3).
d
4.2.8. 4-{4-(2-Oxo-2H-chromen-4-yloxy)-6-[4-(4-trifluoromethyl-
amino-benzonitrile linkage), 8.14 (d, J = 7.4 Hz, 1H, C8 proton of
quinoline), 7.78 (t, J = 7.9 Hz, 1H, C7 proton of quinoline), 7.55 (d,
J = 8.5 Hz, 1H, C5 proton of quinoline), 7.50 (t, J = 7.2 Hz, 1H, C6
proton of quinoline), 7.41 (s, 1H, C3 proton of quinoline), 7.33–6.86
(8H, m, Ar-H), 3.84 (4H, br s, piperazine), 3.71 (s, 3H, N-CH3), 3.42
phenyl)-piperazin-1-yl]-[1,3,5]triazin-2-ylamino}-benzonitrile (8d)
Yellow solid; IR (KBr, cmꢀ1):
v
3284 (–NH), 3069–3078 (–CH
str.), 2223 (CN), 1255 (C–O–C), 836 (s-triazine C–N str.), 749 (C–F).
1H NMR (400 MHz, Me2SO-d6):
9.22 (s, 1H, –NH, s-triazine to
d
amino-benzonitrile linkage), 8.10 (dd, J = 1.7, 1.1 Hz, 1H, C5 proton
of coumarin), 7.61–7.64 (m, 1H, coumarin), 7.45 (t, J = 8.5 Hz, 1H,
C6 proton of coumarin), 7.33–36 (m, 1H, coumarin), 7.29–6.90
(9H, m, Ar-H), 3.87 (4H, br s, piperazine), 3.48 (4H, br s,
(4H, br s, piperazine). 13C NMR (100 MHz, Me2SO-d6):
d 178.2 (1C,
C-6, s-triazine, C–N at piperazine linkage), 165.7 (1C, C-4, s-
triazine, C–O–C at quinoline linkage), 164.2, 163.6 (2C, 1C at C-2, s-
triazine, C–NH at benzonitrile moiety and 1C of C55O), 146.3–117.1
(20C, Ar. C including C–CF3 at 130.3 and CF3 at 125.5), 105.7 (1C,
CBB N), 98.1 (1C, –C–CBB N), 51.4, 42.8 (4C, piperazine ring carbon
piperazine). 13C NMR (100 MHz, Me2SO-d6):
d 175.4 (1C, C-6, s-
triazine, C–N at piperazine linkage), 167.2 (1C, C-4, s-triazine, C–
O–C at quinoline linkage), 165.9, 163.8 (2C, 1C at C-2, s-triazine, C–
NH at benzonitrile moiety and 1C of C55O), 152.9 (1C of C-9,
coumarin), 148.3–119.6 (19C, Ar. C including C–CF3 at 129.7 and
CF3 at 126.1), 106.1 (1C, CBB N), 97.4 (1C, –C–CBB N), 48.6, 46.2 (4C,
atoms), 31.6 (1C, N-CH3). 19F NMR (400 MHz, CDCl3):
s, 4-CF3).
d
ꢀ66.62 (3F,
4.2.5. 4-[4-[4-(2-Fluoro-phenyl)-piperazin-1-yl]-6-(2-oxo-2H-
piperazine ring carbon atoms). 19F NMR (400 MHz, CDCl3):
d
chromen-4-yloxy)-[1,3,5]triazin-2-ylamino]-benzonitrile (8a)
ꢀ65.33 (3F, s, 4-CF3).
Yellowish white solid; IR (KBr, cmꢀ1):
v
3282 (–NH), 3078–3085
(–CH Str.), 2223 (CN), 1254 (C–O–C), 831 (s-triazine C–N str.), 744
(C–F). 1H NMR (400 MHz, Me2SO-d6):
9.19 (s, 1H, –NH, s-triazine
4.2.9. 4-[4-[4-(2-Fluoro-phenyl)-piperazin-1-yl]-6-(1-methyl-2-
oxo-1,2-dihydro-quinolin-4-yloxy)-[1,3,5]triazin-2-ylamino]-2-
trifluoromethyl-benzonitrile (9a)
d
to amino-benzonitrile linkage), 7.99 (dd, J = 1.8, 1.1 Hz, 1H,
C5 proton of coumarin), 7.59–7.62 (m, 1H, coumarin), 7.53
(t, J = 8.6 Hz, 1H, C6 proton of coumarin), 7.33–38 (m, 1H,
coumarin), 7.27–6.89 (9H, m, Ar-H), 3.86 (4H, br s, piperazine),
Light yellow solid; IR (KBr, cmꢀ1):
v
3287 (–NH), 3059–3072 (–
CH str.), 2223 (CN), 1258 (C–O–C), 829 (s-triazine C–N str.), 742 (C–
F). 1H NMR (400 MHz, Me2SO-d6):
9.21 (s, 1H, –NH, s-triazine to
d
3.49 (4H, br s, piperazine). 13C NMR (100 MHz, Me2SO-d6):
d
176.5
amino-benzonitrile linkage), 8.11 (d, J = 7.6 Hz, 1H, C8 proton of
quinoline), 7.69 (t, J = 7.5 Hz, 1H, C7 proton of quinoline), 7.59 (d,
J = 8.4 Hz, 1H, C5 proton of quinoline), 7.56 (t, J = 7.3 Hz, 1H, C6
proton of quinoline), 7.41 (s, 1H, C3 proton of quinoline), 7.30–6.82
(7H, m, Ar-H), 3.81 (4H, br s, piperazine), 3.67 (s, 3H, N-CH3), 3.44
(1C, C-6, s-triazine, C–N at piperazine linkage), 166.7 (1C, C-4, s-
triazine, C–O–C at quinoline linkage), 166.2, 164.6 (2C, 1C at C-2, s-
triazine, C–NH at benzonitrile moiety and 1C of C55O), 150.8, 153.4
(2C, 1C of C–F and 1C of C-9, coumarin), 145.3–120.5 (17C, Ar. C),
105.3 (1C, CBB N), 97.7 (1C, –C–CBB N), 48.4, 44.8 (4C, piperazine ring
(4H, br s, piperazine). 13C NMR (100 MHz, Me2SO-d6):
d 175.8 (1C,
carbon atoms). 19F NMR (400 MHz, CDCl3):
d
ꢀ121.45 (1F, s, 2-F).
C-6, s-triazine, C–N at piperazine linkage), 166.2 (1C, C-4,
s-triazine, C–O–C at quinoline linkage), 164.4, 162.9 (2C, 1C at
C-2, s-triazine, C–NH at benzonitrile moiety and 1C of C55O), 151.2
(1C, C–F), 146.9–120.1 (19C, Ar. C including C–CF3 at 129.8 and CF3
at 125.2), 105.5 (1C, CBB N), 96.9 (1C, –C–CBB N), 49.4, 44.1 (4C,
piperazine ring carbon atoms), 30.3 (1C, N-CH3). 19F NMR
4.2.6. 4-[4-[4-(4-Fluoro-phenyl)-piperazin-1-yl]-6-(2-oxo-2H-
chromen-4-yloxy)-[1,3,5]triazin-2-ylamino]-benzonitrile (8b)
Yellowish white solid; IR (KBr, cmꢀ1):
v
3281 (–NH), 3070–3081
(–CH Str.), 2223 (CN), 1256 (C–O–C), 830 (s-triazine C–N str.), 747
(C–F). 1H NMR (400 MHz, Me2SO-d6):
9.20 (s, 1H, –NH, s-triazine
d
(400 MHz, CDCl3):
d
ꢀ120.60 (F, s, 2-F), ꢀ63.01 (3F, s, CF3 of
to amino-benzonitrile linkage), 8.02 (dd, J = 1.8, 1.3 Hz, 1H, C5
proton of coumarin), 7.51–7.54 (m, 1H, coumarin), 7.48 (t,
J = 8.3 Hz, 1H, C6 proton of coumarin), 7.32–35 (m, 1H, coumarin),
7.27–6.91 (9H, m, Ar-H), 3.81 (4H, br s, piperazine), 3.44 (4H, br s,
amino-benzonitrile moiety).
4.2.10. 4-[4-[4-(4-Fluoro-phenyl)-piperazin-1-yl]-6-(1-methyl-2-
oxo-1,2-dihydro-quinolin-4-yloxy)-[1,3,5]triazin-2-ylamino]-2-
trifluoromethyl-benzonitrile (9b)
piperazine). 13C NMR (100 MHz, Me2SO-d6):
d 177.3 (1C, C-6, s-
triazine, C–N at piperazine linkage), 167.2 (1C, C-4, s-triazine, C–O–
C at quinoline linkage), 165.5, 164.3 (2C, 1C at C-2, s-triazine, C–NH
at benzonitrile moiety and 1C of C55O), 151.1, 153.8 (2C, 1C of C–F
and 1C of C-9, coumarin), 148.4–118.8 (17C, Ar. C), 104.9 (1C,
CBB N), 98.5 (1C, –C–CBB N), 49.2, 45.1 (4C, piperazine ring carbon
Yellow solid; IR (KBr, cmꢀ1):
v
3290 (–NH), 3071–3079 (–CH str.),
2221 (CN), 1256 (C–O–C), 830 (s-triazine C–N str.), 757 (C–F). 1H
NMR (400 MHz, Me2SO-d6): 9.16 (s, 1H, –NH, s-triazine to amino-
d
benzonitrile linkage), 8.07 (d, J = 7.9 Hz, 1H, C8 proton of quinoline),
7.75 (t, J = 7.6 Hz, 1H, C7 proton of quinoline), 7.58 (d, J = 8.1 Hz, 1H,
C5 protonofquinoline), 7.53(t, J = 7.6 Hz, 1H,C6 protonofquinoline),
7.42 (s, 1H, C3 proton of quinoline), 7.31–6.92 (7H, m, Ar-H), 3.88
(4H, br s, piperazine), 3.73 (s, 3H, N-CH3), 3.52 (4H, br s, piperazine).
13C NMR (100 MHz, Me2SO-d6):
d 178.1 (1C, C-6, s-triazine, C–N at
piperazine linkage), 167.7 (1C, C-4, s-triazine, C–O–C at quinoline
linkage), 165.9, 163.4 (2C, 1C at C-2, s-triazine, C–NH at benzonitrile
atoms). 19F NMR (400 MHz, CDCl3):
d
ꢀ118.38 (1F, s, 4-F).
4.2.7. 4-{4-(2-Oxo-2H-chromen-4-yloxy)-6-[4-(3-trifluoromethyl-
phenyl)-piperazin-1-yl]-[1,3,5]triazin-2-ylamino}-benzonitrile (8c)
Yellow solid; IR (KBr, cmꢀ1):
v
3280 (–NH), 3066–3075 (–CH
str.), 2224 (CN), 1258 (C–O–C), 828 (s-triazine C–N str.), 740 (C–F).

R.V. Patel et al. / Journal of Fluorine Chemistry 132 (2011) 617–627
625
moiety and 1C of C55O), 152.0 (1C, C–F), 147.6–117.4 (19C, Ar. C
4.2.14. 4-[4-[4-(4-Fluoro-phenyl)-piperazin-1-yl]-6-(2-oxo-2H-
chromen-4-yloxy)-[1,3,5]triazin-2-ylamino]-2-trifluoromethyl-
benzonitrile (10b)
including C–CF3 at 130.1andCF3 at 125.4), 106.3 (1C, CBB N), 97.8 (1C,
–C–CBB N), 50.4, 46.3 (4C, piperazine ring carbon atoms), 29.2 (1C, N-
CH3). 19F NMR (400 MHz, CDCl3):
CF3 of amino-benzonitrile moiety).
d
ꢀ118.09 (F, s, 4-F), ꢀ63.22 (3F, s,
Yellowish white solid; IR (KBr, cmꢀ1):
v
3290 (–NH), 3069–3079
(–CH str.), 2222 (CN), 1256 (C–O–C), 832 (s-triazine C–N str.), 745
(C–F). 1H NMR (400 MHz, Me2SO-d6):
9.15 (s, 1H, –NH, s-triazine
d
4.2.11. 4-{4-(1-Methyl-2-oxo-1,2-dihydro-quinolin-4-yloxy)-6-[4-
(3-trifluoromethyl-phenyl)-piperazin-1-yl]-[1,3,5]triazin-2-
ylamino}-2-trifluoromethyl-benzonitrile (9c)
to amino-benzonitrile linkage), 8.06 (dd, J = 1.6, 1 Hz, 1H, C5 proton
of coumarin), 7.55–7.59 (m, 1H, coumarin), 7.46 (t, J = 8.4 Hz, 1H,
C6 proton of coumarin), 7.29–7.33 (m, 1H, coumarin), 7.25–6.83
(8H, m, Ar-H), 3.83 (4H, br s, piperazine), 3.51 (4H, br s, piperazine).
Brown solid; IR (KBr, cmꢀ1):
v
3279 (–NH), 3070–3078 (–CH
str.), 2225 (CN), 1256 (C–O–C), 834 (s-triazine C–N str.), 748 (C–F).
1H NMR (400 MHz, Me2SO-d6):
9.24 (s, 1H, –NH, s-triazine to
13C NMR (100 MHz, Me2SO-d6):
d 176.5 (1C, C-6, s-triazine, C–N at
d
piperazine linkage), 166.9 (1C, C-4, s-triazine, C–O–C at quinoline
linkage), 166.3, 163.9 (2C, 1C at C-2, s-triazine, C–NH at
benzonitrile moiety and 1C of C55O), 152.1, 153.1 (2C, 1C of C–F
and 1C of C-9, coumarin), 149.4–118.2 (18C, Ar. C), 106.3 (1C,
CBB N), 97.6 (1C, –C–CBB N), 50.2, 45.1 (4C, piperazine ring carbon
amino-benzonitrile linkage), 8.19 (d, J = 7.4 Hz, 1H, C8 proton of
quinoline), 7.64 (t, J = 7.9 Hz, 1H, C7 proton of quinoline), 7.51 (d,
J = 8.5 Hz, 1H, C5 proton of quinoline), 7.45 (t, J = 7.3 Hz, 1H, C6
proton of quinoline), 7.41 (s, 1H, C3 proton of quinoline), 7.28–6.86
(7H, m, Ar-H), 3.86 (4H, br s, piperazine), 3.71 (s, 3H, N-CH3), 3.48
atoms). 19F NMR (400 MHz, CDCl3):
(3F, s, CF3 of amino-benzonitrile moiety).
d
ꢀ117.63 (F, s, 4-F), ꢀ63.31
(4H, br s, piperazine). 13C NMR (100 MHz, Me2SO-d6):
d
177.7 (1C,
C-6, s-triazine, C–N at piperazine linkage), 166.9 (1C, C-4, s-
triazine, C–O–C at quinoline linkage), 166.2, 163.1 (2C, 1C at C-2, s-
triazine, C–NH at benzonitrile moiety and 1C of C55O), 147.2–119.2
(22C, Ar. C including 2C–CF3 at 131.2, 130.6 and 2CF3 at 125.9,
124.9), 105.1 (1C, CBB N), 98.3 (1C, –C–CBB N), 46.4, 43.7 (4C,
piperazine ring carbon atoms), 31.4 (1C, N-CH3). 19F NMR
4.2.15. 4-{4-(2-Oxo-2H-chromen-4-yloxy)-6-[4-(3-trifluoromethyl-
phenyl)-piperazin-1-yl]-[1,3,5]triazin-2-ylamino}-2-trifluoromethyl-
benzonitrile (10c)
Yellow solid; IR (KBr, cmꢀ1):
v
3288 (–NH), 3068–3076 (–CH str.),
2223 (CN), 1255 (C–O–C), 834 (s-triazine C–N str.), 760 (C–F). 1H
NMR (400 MHz, Me2SO-d6): 9.22 (s, 1H, –NH, s-triazine to amino-
(400 MHz, CDCl3):
moiety and –CF3 of amino benzonitrile moiety).
d
ꢀ63.39, ꢀ63.13 (6F, s, –CF3 of piperazine
d
benzonitrile linkage), 7.99 (dd, J = 1.5, 1.1 Hz, 1H, C5 proton of
coumarin), 7.58–7.62 (m, 1H, coumarin), 7.50 (t, J = 8.2 Hz, 1H, C6
proton of coumarin), 7.34–37 (m, 1H, coumarin), 7.26–6.92 (8H, m,
Ar-H), 3.81 (4H, br s, piperazine), 3.43 (4H, br s, piperazine). 13C NMR
4.2.12. 4-{4-(1-Methyl-2-oxo-1,2-dihydro-quinolin-4-yloxy)-6-[4-
(4-trifluoromethyl-phenyl)-piperazin-1-yl]-[1,3,5]triazin-2-
ylamino}-2-trifluoromethyl-benzonitrile (9d)
(100 MHz, Me2SO-d6): d 176.2 (1C, C-6, s-triazine, C–N at piperazine
Dark brown solid; IR (KBr, cmꢀ1):
v
3282 (–NH), 3068–3076 (–CH
str.), 2223 (CN), 1256 (C–O–C), 833 (s-triazine C–N str.), 749 (C–F).
1H NMR (400 MHz, Me2SO-d6):
9.18 (s, 1H, –NH, s-triazine to
linkage), 167.1 (1C, C-4, s-triazine, C–O–C at quinoline linkage),
166.7, 164.8 (2C, 1C at C-2, s-triazine, C–NH at benzonitrile moiety
and 1C of C55O), 152.7 (1C of C-9, coumarin), 146.3–120.5 (22C, Ar. C
including2C–CF3 at130.7, 130.1 and 2CF3 at 126.2, 125.1), 105.8 (1C,
CBB N), 97.8 (1C, –C–CBB N), 47.8, 45.1 (4C, piperazine ring carbon
d
amino-benzonitrile linkage), 8.10 (d, J = 7.4 Hz, 1H, C8 proton of
quinoline), 7.77 (t, J = 7.6 Hz, 1H, C7 proton of quinoline), 7.59 (d,
J = 8.6 Hz, 1H, C5 proton of quinoline), 7.52 (t, J = 7.2 Hz, 1H, C6
proton of quinoline), 7.39 (s, 1H, C3 proton of quinoline), 7.26–6.93
(7H, m, Ar-H), 3.89 (4H, br s, piperazine), 3.77 (s, 3H, N-CH3), 3.51
atoms). 19F NMR (400 MHz, CDCl3):
piperazine moiety and –CF3 of amino benzonitrile moiety).
d
ꢀ63.60, ꢀ64.02 (6F, s, –CF3 of
(4H, br s, piperazine). 13C NMR (100 MHz, Me2SO-d6):
d
175.8 (1C, C-
4.2.16. 4-{4-(2-Oxo-2H-chromen-4-yloxy)-6-[4-(4-trifluoromethyl-
phenyl)-piperazin-1-yl]-[1,3,5]triazin-2-ylamino}-2-trifluoromethyl-
benzonitrile (10d)
6, s-triazine, C–N at piperazine linkage), 167.5 (1C, C-4, s-triazine, C–
O–C at quinoline linkage), 165.5, 163.6 (2C, 1C at C-2, s-triazine, C–
NH at benzonitrile moiety and 1C of C55O), 144.8–116.7 (22C, Ar. C
including 2C–CF3 at129.9, 130.4and 2CF3 at125.2, 125.7), 105.9(1C,
CBB N), 95.8 (1C, –C–CBB N), 48.2, 45.6 (4C, piperazine ring carbon
Yellow solid; IR (KBr, cmꢀ1):
v
3279 (–NH), 3065–3077 (–CH
str.), 2223 (CN), 1257 (C–O–C), 833 (s-triazine C–N str.), 759 (C–F).
1H NMR (400 MHz, Me2SO-d6):
9.26 (s, 1H, –NH, s-triazine to
d
atoms), 30.6 (1C, N-CH3). 19F NMR (400 MHz, CDCl3):
d
ꢀ66.03,
amino-benzonitrile linkage), 8.04 (dd, J = 1.6, 1.3 Hz, 1H, C5 proton
of coumarin), 7.56–7.60 (m, 1H, coumarin), 7.47 (t, J = 8.5 Hz, 1H,
C6 proton of coumarin), 7.35–38 (m, 1H, coumarin), 7.27–6.87 (8H,
ꢀ63.29 (6F, s, –CF3 of piperazine moiety and –CF3 of amino
benzonitrile moiety).
m, Ar-H), 3.86 (4H, br s, piperazine), 3.49 (4H, br s, piperazine). 13
C
4.2.13. 4-[4-[4-(2-Fluoro-phenyl)-piperazin-1-yl]-6-(2-oxo-2H-
chromen-4-yloxy)-[1,3,5]triazin-2-ylamino]-2-trifluoromethyl-
benzonitrile (10a)
NMR (100 MHz, Me2SO-d6): d 178.1 (1C, C-6, s-triazine, C–N at
piperazine linkage), 165.6 (1C, C-4, s-triazine, C–O–C at quinoline
linkage), 165.2, 163.6 (2C, 1C at C-2, s-triazine, C–NH at
benzonitrile moiety and 1C of C55O), 153.3 (1C of C-9, coumarin),
147.8–117.5 (22C, Ar. C including 2C–CF3 at 129.7, 130.4 and 2CF3
at 125.4, 125.9), 106.4 (1C, CBB N), 99.2 (1C, –C–CBB N), 47.1, 46.6 (4C,
Yellowish white solid; IR (KBr, cmꢀ1):
v
3283 (–NH), 3067–3077
(–CH str.), 2224 (CN), 1256 (C–O–C), 830 (s-triazine C–N str.), 760
(C–F). 1H NMR (400 MHz, Me2SO-d6):
9.16 (s, 1H, –NH, s-triazine
d
to amino-benzonitrile linkage), 8.09 (dd, J = 1.4, 1.3 Hz, 1H, C5
proton of coumarin), 7.52–7.55 (m, 1H, coumarin), 7.44 (t,
J = 8.7 Hz, 1H, C6 proton of coumarin), 7.30–34 (m, 1H, coumarin),
7.29–6.92 (8H, m, Ar-H), 3.85 (4H, br s, piperazine), 3.46 (4H, br s,
piperazine ring carbon atoms). 19F NMR (400 MHz, CDCl3):
d
ꢀ65.73, ꢀ63.89 (6F, s, –CF3 of piperazine moiety and –CF3 of amino
benzonitrile moiety).
piperazine). 13C NMR (100 MHz, Me2SO-d6):
d
177.9 (1C, C-6, s-
4.3. Pharmacology
triazine, C–N at piperazine linkage), 166.2 (1C, C-4, s-triazine, C–O–
C at quinoline linkage), 165.7, 164.9 (2C, 1C at C-2, s-triazine, C–NH
at benzonitrile moiety and 1C of C55O), 151.3, 152.5 (2C, 1C of C–F
and 1C of C-9, coumarin), 148.1–116.8 (18C, Ar. C), 104.8 (1C,
CBB N), 96.9 (1C, –C–CBB N), 51.6, 43.9 (4C, piperazine ring carbon
4.3.1. Paper disc diffusion technique (agar streak dilution method)
The synthesized s-triazinyl derivatives were examined for
antimicrobial activity against eight bacteria (S. aureus MTCC 96, B.
cereus MTCC 619, E. coli MTCC 739, P. aeruginosa MTCC 741, K.
pneumoniae MTCC 109, S. typhi MTCC 733, P. vulgaris MTCC 1771)
and four fungi (A. niger MTCC 282, A. fumigatus MTCC 343, A.
atoms). 19F NMR (400 MHz, CDCl3):
(3F, s, CF3 of amino-benzonitrile moiety).
d
ꢀ121.46 (F, s, 2-F), ꢀ63.08

626
R.V. Patel et al. / Journal of Fluorine Chemistry 132 (2011) 617–627
clavatus MTCC 1323, C. albicans MTCC 183) species using paper disc
diffusion technique [31]. The Mueller–Hinton agar media were
sterilized (autoclaved at 120 8C for 30 min), poured at uniform
depth of 5 mm and allowed to solidify. The microbial suspension
(105 CFU/mL) (0.5 McFarland Nephelometery Standards) was
streaked over the surface of media using a sterile cotton swab
to ensure even growth of the organisms. The tested compounds
were dissolved in dimethyl sulfoxide to give solutions of 3.12–
mL dilutions of each test compound in DMSO (dimethyl sulfoxide)
were added in the liquid L. J. medium and then media were
sterilized by inspissation method. A culture of M. tuberculosis H37
Rv growing on L. J. medium was harvested in 0.85% saline in bijou
bottles. These tubes were then incubated at 37 8C for 24 h followed
by streaking of M. tuberculosis H37 Rv (5 ꢃ 104 bacilli per tube).
These tubes were then incubated at 37 ꢁ 1 8C. Growth of bacilli was
seen after 12 days, 22 days and finally 28 days of incubation. Tubes
having the compounds were compared with control tubes where
medium alone was incubated with M. tuberculosis H37 Rv. The
concentration at which no development of colonies occurred or <20
colonies was taken as MIC concentration of test compound. The
standard strain M. tuberculosis H37 Rv was tested with known drugs
rifampicin, isoniazid, ethambutol and pyrazinamide.
100
mg/mL. Sterile filter paper discs measuring 6.25 mm in
diameter (Whatman no. 1 filter paper), previously soaked in a
known concentration of the respective test compound in dimethyl
sulfoxide were placed on the solidified nutrient agar medium that
had been inoculated with the respective microorganism and the
plates were incubated for 24 h at 37 ꢁ 1 8C.
A control disc
impregnated with an equivalent amount of dimethyl sulfoxide
without any sample was also used and did not produce any inhibition.
Ciprofloxacin and ketoconazole (100 mg/disc) were used as control
drugs for antibacterial and antifungal activity, respectively.
Acknowledgements
The authors are thankful to Applied Chemistry Department of
S.V. National Institute of Technology, Surat for the scholarship,
encouragement and facilities. The authors wish to offer their deep
gratitude to Microcare Laboratory, Surat for carrying out the
biological screening. Authors are also thankful to Dr. Prem’s
Molecules Pvt. Ltd., Vadodara for providing valuable fluorinated
piperazine derivatives and Centre of Excellence, Vapi, India for
carrying out spectral analysis.
MIC of the compound was determined by agar streak dilution
method [32]. A stock solution of the synthesized compound
(100
mg/mL) in dimethyl sulfoxide was prepared and graded
quantities of the test compounds were incorporated in a specified
quantity of molten sterile agar, i.e. nutrient agar for evaluation of
antibacterial and sabouraud dextrose agar for antifungal activity,
respectively. The medium containing the test compound was
poured into a Petri dish at a depth of 4–5 mm and allowed to
solidify under aseptic conditions. A suspension of the respective
microorganism of approximately 105 CFU/mL was prepared and
applied to plates with serially diluted compounds with concentra-
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