Synthesis and studies of novel 2-(4-cyano-3-trifluoromethylphenyl amino)-4-(quinoline-4-yloxy)-6-(piperazinyl/piperidinyl)-s-triazines as potential antimicrobial, antimycobacterial and anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2011.07.006

A series of novel s-triazine analogs were synthesized and characterized by IR, ¹H NMR, ¹³C NMR, ¹⁹F NMR spectroscopy and elemental analysis. Preliminary screening of target compounds against eight bacteria (Staphylococcus

Full text of DOI:10.1016/j.ejmech.2011.07.006

European Journal of Medicinal Chemistry 46 (2011) 4354e4365
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and studies of novel 2-(4-cyano-3-trifluoromethylphenyl amino)-4-
(quinoline-4-yloxy)-6-(piperazinyl/piperidinyl)-s-triazines as potential
antimicrobial, antimycobacterial and anticancer agents
,
b
c
Rahul V. Patel a, Premlata Kumari a , Dhanji P. Rajani , Kishor H. Chikhalia
*
a Department of Applied Chemistry, S.V. National Institute of Technology, Surat 395 007, Gujarat, India
b Microcare Laboratory, Surat 395 001, Gujarat, India
c Department of Chemistry, School of Science, Gujarat University, Ahmedabad 380 009, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
A series of novel s-triazine analogs were synthesized and characterized by IR, 1H NMR, 13C NMR, 19F NMR
spectroscopy and elemental analysis. Preliminary screening of target compounds against eight bacteria
(Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae,
Salmonella typhi, Proteus vulgaris, Shigella flexneria), four fungi (Aspergillus niger, Aspergillus fumigatus,
Aspergillus clavatus, Candida albicans) and Mycobacterium tuberculosis H37Rv indicated that 5d, 5h, 5n,
5p, 5q, 5r, 5s, 5t and 5u were the most active compounds among twenty one studied. Thus, they were
further subjected to in vitro biological evaluation against human prostate cancer cell line (DU-145) and
the results indicate that two compounds 5n and 5s were markedly active.
Article history:
Received 31 May 2011
Received in revised form
1 July 2011
Accepted 4 July 2011
Available online 8 July 2011
Keywords:
2,4,6-Trichloro-1,3,5-triazine
4-Hydroxyquinoline
Piperazine
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antimicrobial activity
Antimycobacterial activity
Anticancer activity
1. Introduction
among HIV-positive people [5]. The continual emergence of
multidrug resistance to clinically available drugs has lent additional
Control of deadly infectious diseases is seriously threatened by
multidrug emergence and dissemination of resistant pathogenic
microbes. The issue is of serious consideration in the developing
countries. Patients with AIDS are immune suppressed, and very
susceptible to the opportunistic microbial infections [1]. On the
other hand, Mycobacterium tuberculosis remains a leading infec-
tious cause of death in the world today, with an estimated two
million deaths each year. Due to the quiescent form of M. tubercu-
losis strains, many of the currently available antimycobacterial
drugs have become ineffective by the imminent exigency of
multidrug-resistant [2e4]. The WHO has estimated that, according
to the stop TB partnership’s global plan to stop TB, 2006e2015, 1.3
million MDR-TB cases will need to be treated in the 27 high MDR-TB
burden countries between 2010 and 2015. In 2008, there were an
estimated 8.9e9.9 million incident cases of TB, 9.6e13.3 million
prevalent cases of TB, 1.1e1.7 million deaths from TB among HIV-
negative people and an additional 0.45e0.62 million TB deaths
urgency to develop new antimicrobial agents likely to be unaffected
by existing resistance mechanisms.
In chemotherapeutic point of view, the search for a molecule
having multiple targets has always been a very attractive strategy
for medicinal chemists. Since the fluorine containing compounds
often show significant biological activity profiles being their
superior metabolic stability and the lipophilicity [6], we are inter-
ested to have 4-amino-2-trifluoromethyl-benzonitrile substitution
to the s-triazine core. Moreover, 4-amino-2-trifluoromethyl-ben-
zonitrile is also a useful pharmacophore found in the anti prostate
cancer drug bicalutamide as a structural unit [7]. Therefore, in the
present study an attempt has been made to identify the efficacy of
the newly synthesized compounds against DU-145 prostate cancer
cell line. In addition, some piperazine substituted analogs were also
found to display activity against prostate cancer cell line DU-145
[8e10].
As a part of our ongoing research aiming the synthesis of novel
biologically active s-triazine derivatives [11e13], herein we report
the synthesis and pharmacological activities of novel 2-(4-cyano-3-
trifluoromethylphenyl amino)-4-(quinoline-4-yloxy)-6-(piperazinyl
/piperidinyl)-s-triazines. 1,3,5-Triazine nucleus has gathered an
* Corresponding author. Tel.: þ91 9904003863.
E-mail address: premlatakumari1@gmail.com (P. Kumari).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.006

R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
4355
immense attention among chemists due to its diverse biological
3. Results and discussion
3.1. Pharmacology
activities such as antimicrobial [14e16], anticancer [17], antimalarial
[18] and antiviral [19] activities. Profound medicinal applications
associated with piperazine heterocycle render them as useful
structural units in drug research [20e24]. Recent studies have
confirmed that several s-triazine derivatives bearing morpholine,
piperidine and some piperazine moieties are effective against
M. tuberculosis H37RV strain [25], while the piperazineequinoline
combination is found in the structure of many well known antimi-
crobial drugs like ciprofloxacin, norfloxacin, ofloxacin, pefloxacin,
rufloxacin, enrofloxacin etc. Prompted by these observations it was
contemplated to envisage the combination of above mentioned bio-
labile components of significant activities in a compact system to
identify new candidates that may be value in designing new bio-
logically active agents.
3.1.1. Antimicrobial activity
Investigation on antibacterial screening data (Table 1) revealed
that some of the compounds showed a good deal of activity against
all the mentioned bacteria. Final s-triazinyl derivatives with
piperazine moiety bearing halogen substituent in the form of single
chlorine atom (5c and 5p) displayed promising activity against
Klebsiella pneumoniae at 12.5 mg/mL of MIC, 26 mm and 25 mm of
zone of inhibition, respectively. The said compound (5p) was found
to exhibit good activity against Proteus vulgaris at the low MIC value
of 6.25 mg/mL and inhibition zone of 24 mm. Compound 5d with
substitution of two chlorine atoms at the 2nd and 3rd position of
the phenyl ring of the piperazine base displayed strong inhibitory
action against both of the Gram-positive bacteria Staphylococcus
2. Chemistry
aureus and Bacillus cereus at 6.25 mg/mL of MIC and 28 mm of
growth inhibitory diameter. In addition, the same compound (5d)
was also appeared with substantial activity against K. pneumoniae,
Salmonella typhi and Shigella flexneria at 12.5 mg/mL of MIC and
24e25 mm of inhibition zones. Final s-triazine derivatives (5q, 5r)
with single fluorine atom substitution at the 2nd and 4th position
of the piperazinyl phenyl ring, respectively, indicated remarkable
Synthesis of intermediates and target compounds was accom-
plished according to the steps illustrated in Schemes 1 and 2. The
first step comprises formation of intermediate 1 in very good yield
by the nucleophilic displacement of one chlorine atom of s-triazine
ring by 4-amino-2-trifluoromethyl-benzonitrile. Compound 1 dis-
played an absorption band at 2223 cmꢀ1 confirming the presence
of a C^N group, and a strong band near 3282 cmꢀ1 further
confirmed the presence of an eNH group. The synthesis of disub-
stituted s-triazine intermediate 3 was achieved in 80% of yield by
the reaction between 4-(4,6-dichloro-1,3,5-triazin-2-ylamino)-2-
trifluoromethyl-benzonitrile (1) and 4-hydroxyquinoline in the
presence of 60% NaH at 45e50 ꢁC. A characteristic band appeared at
1256 cmꢀ1 corresponded to the CeOeC linkage in the IR spectra of
compound 3. Subsequent coupling of the so formed compound 3
with the desired piperazines and piperidines under basic condi-
tions in 1,4-dioxane solvent at 70e80 ꢁC formed the corresponding
2-(4-cyano-3-trifluoromethylphenyl amino)-4-(quinoline-4-yloxy)
-6-(piperazinyl/piperidinyl)-s-triazines (5aeu). This reaction pro-
ceeded in good yields and is general for different substituted
piperazines and piperidines. The correct synthesis of 5aeu was
confirmed on the basis of IR, 1H NMR, 13C NMR, 19F NMR [26]
spectral analysis and the purity was ascertained by elemental
analysis.
activity against B. cereus at 6.25
26 mm of inhibition zone, respectively. Furthermore, compound 5q
showed appreciable activity at 12.5 g/mL of MIC against Escher-
ichia coli and K. pneumoniae by exhibiting 26 mm of inhibition zone,
whereas, potently inhibited P. vulgaris at 6.25 g/mL of MIC and
mg/mL of MIC and 27 mm and
m
m
25 mm of inhibitory zone. Final derivative (5s) with highly elec-
tronegative trifluoromethyl group at the 3rd position of phenyl ring
of piperazine moiety was found to contribute excellent activity
against six bacteria (S. aureus, B. cereus, E. coli, Pseudomonas aeru-
ginosa, S. typhi and S. flexneria) among eight studied at the MIC level
6.25 and 12.5 mg/mL and 25e28 mm of inhibition zones. Compound
5n with functionalization of two methyl groups at the 3rd and 5th
position of the piperidine ring displayed excellent activity at
6.25
mg/mL of MIC against S. aureus and P. vulgaris as well as against
E. coli, S. typhi and S. flexneria at 12.5
mg/mL of MIC. This compound
indicated 25e27 mm of growth inhibitory zones against the above
mentioned bacterial strains. Final derivative (5h) bearing pipera-
zine ring with the aliphatic acetyl linkage appeared with good
inhibitory potential toward E. coli at 12.5
mg/mL of MIC and 24 mm
of inhibition zone as well as against P. vulgaris at 6.25
m
g/mL of MIC
Cl
N
C
N
Cl
N
C
and 27 mm of inhibition zone. Final s-triazine derivatives with
methoxy functional group(s) in the form of 2,3,4-trimethoxy benzyl
piperazine (5t) and single methoxy group functionalization at the
4th position of the phenyl ring of piperazine base (5u) displayed
strong inhibitory potential against B. cereus and P. aeruginosa at
CF3
THF
Et3N
N
N
N
N
CF3
Cl
N
H
Cl
N
Cl
NH2
2,4,6-Trichloro-1,3,5-
triazine
4-Amino-2-trifluoromethyl-
benzonitrile
1
6.25
against E. coli and S. flexneria at 12.5
of growth inhibitory diameter. Moreover, compound 5t was able to
indicate highest inhibition of S. aureus at 6.25 g/mL of MIC and
28 mm of inhibition zone as well as inhibition of K. pneumoniae,
m
g/mL of MIC and 26e28 mm of inhibitory zones as well as
mg/mL of MIC and 23e26 mm
Cl
OH
N
C
N
N
THF
NaH
m
1
CF3
N
N
H
O
N
N
S. typhi and P. vulgaris at 12.5 mg/mL of MIC and 23e26 mm of
inhibition zones.
2
)
3
4-Hydroxyquinoline (
The antifungal results (Table 2) revealed that, the synthesized
compounds showed variable degree of inhibition against the
tested fungi. Final halogen(s) substituted s-triazine derivatives 5d
and 5p displayed higher inhibitory effect against Aspergillus
fumigatus at 25 mg/mL of MIC, 25 mm and 26 mm of inhibition
zones, respectively. Compound 5h with acetyl linkage to the
piperazine ring exhibited substantial activity against Aspergillus
R
N
C
N
N
1,4-Dioxane
K2CO3
4a-u
3
N
N
H
CF3
O
N
5a-u
clavatus at 12.5
mg/mL of MIC and 27 mm of inhibition zone.
Scheme 1. Synthetic route of novel s-triazine derivatives 5aeu.
Derivative (5q) bearing fluorine atom at the 2nd position of phenyl

4356
R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
O
4h HN
N C
HN
N
CH3
HN
HN
N
N
CH
4a
4o
4p
CH3
CH3
CH3
CH
4i
N
HC
N
4b HN
N
CH2 CH3
HN
Cl
F
Cl
4q
4j
HN
HN
N
N
HN
HN
HN
N
N
4c HN
Cl
Cl
N
N
4k
4r
4s
N
F
4d
4e
4f
HN
N
CF3
4l
HN
HN
N CH2
HN
HN
N
OCH3
H3CO
CH2
4m
O
CH2
4t HN
N
OCH3
CH3
4g
4u
HN
N
4n
HN
HN
N
OCH3
CH3
Scheme 2. 4aeu, R ¼ Piperazine and piperidine bases coupled to compound 3.
ring of piperazine moiety showed strong inhibition of A. clavatus
and Candida albicans at 25 g/mL of MIC and 25e26 mm of inhi-
was the only compound displaying inhibition of M. tuberculosis
H37Rv completely (99%) at the MIC of 3.12 g/mL. This compound
m
m
bition zone, while compound 5r with fluorine atom substitution at
the 4th position of phenyl ring indicated excellent inhibition of
Aspergillus niger at 12.5 mg/mL and 26 mm of inhibition zone.
was considered to display even better efficacy, than the standard
drug, pyrazinamide. Final s-triazinyl compounds with chlorine or
fluorine atom(s) substitution (5d and 5q, 5r, 5p) were found to
Moreover, two compounds, trifluoromethylated 5s and trimethoxy
functionalized 5t displayed inhibition of all the fungi studied in
demonstrate MIC level of 12.5, 25 and 50 mg/mL, respectively
against M. tuberculosis H37Rv.
the assay at the MIC range of 12.5e25 mg/mL and inhibition zone
range of 25e28 mm. Finally, compound 5u was appeared with
good inhibitory activity against A. fumigatus (27 mm of inhibition
zone) and A. clavatus (28 mm of inhibition zone) at MIC level
3.1.3. Anticancer activity
The activity of new compounds against prostate (DU-145)
cancer cell proliferation reported in Table 3 suggests that the best
active compound was 5n with 3,5-dimethyl piperazine substituent
25 mg/mL and 12.5 mg/mL, respectively. All the remaining final s-
triazine derivatives exerted good to moderate activity profiles.
with the GI50 value of 14.1
trifluoromethylphenyl piperazine condensation to the nucleus
with the GI50 value of 24.6 g/mL. The said two derivatives were
also displayed total inhibition of DU-145 cell growth (TGI) at the
concentration level of 35.3 g/mL and 47.1 g/mL, respectively and
mg/mL, followed by 5s with 3-
3.1.2. Antimycobacterial activity
m
In vitro tuberculosis activities of compounds 5aeu were
assessed against M. tuberculosis H37Rv. The results observed from
BACTEC MGIT indicated that final s-triazines, 5n with substitution
of two methyl groups at the 3rd and 5th position of the piperidine
ring, 5s and 5t bearing trifluoromethylation and trimethoxy
substitution to the phenyl ring of piperazine base coupled to the
nucleus exhibited highest inhibition (99%) at a constant concen-
m
m
found potent than that of standard drug (ADR: TGI > 100 mg/mL) in
terms of TGI.
4. Conclusion
tration level (6.25
secondary biological screening was performed using Low-
ensteineJensen MIC method and it is worthwhile to note that 5n
m
g/mL) against M. tuberculosis H37Rv. The
This work focused on the development of new potentially active
s-triazines. Bioassay results revealed that nine compounds (5d, 5h,
5n, 5p, 5q, 5r, 5s, 5t and 5u) out of the 21 studied displayed variable

R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
4357
Table 1
In vitro antimicrobial activity.
R
X
N
CN
N
N
O
N
N
H
CF3
N
Entry
R
X
Zone of inhibition [mm (MIC in mg/mL)]
S.a
B.c
E.c
P.a
K.p
S.t
P.v
S.f
11 (100)
13 (100)
5a
5b
CH3
C2H5
N
N
<10 (100)
13 (100)
<10 (100)
14 (100)
<10 (100)
13 (100)
14 (100)
16 (100)
<10 (100)
<10 (100)
13 (100)
15 (100)
<10 (100)
<10 (100)
Cl
5c
N
N
25 (12.5)
28 (6.25)
24 (12.5)
28 (6.25)
22 (50)
23 (50)
23 (50)
23 (25)
26 (12.5)
24 (12.5)
23 (50)
23 (100)
25 (12.5)
21 (50)
Cl
Cl
5d
25 (12.5)
24 (12.5)
5e
5f
H
O
11 (100)
14 (100)
14 (100)
<10 (100)
<10 (100)
<10 (100)
<10 (100)
<10 (100)
15 (100)
14 (100)
12 (100)
13 (100)
14 (100)
16 (100)
<10 (100)
<10 (100)
5g
5h
N
N
14 (100)
26 (12.5)
<10 (100)
<10 (100)
<10 (100)
<10 (100)
13 (100)
23 (100)
14 (100)
27 (6.25)
<10 (100)
COCH3
24 (12.5)
24 (12.5)
24 (12.5)
23 (50)
23 (25)
CH3
HC
5i
5j
N
N
23 (50)
22 (50)
22 (50)
22 (25)
21 (100)
17 (100)
21 (100)
15 (100)
21 (100)
16 (100)
21 (50)
CH3
21 (100)
19 (100)
19 (100)
16 (100)
16 (100)
N
N
5k
5l
N
N
21 (100)
22 (100)
21 (50)
18 (100)
14 (100)
16 (100)
15 (100)
17 (100)
16 (100)
14 (100)
18 (100)
16 (100)
17 (100)
11 (100)
N
22 (100)
<10 (100)
H2C
5m
5n
H
H
24 (100)
27 (6.25)
22 (50)
14 (100)
25 (12.5)
19 (100)
24 (12.5)
16 (100)
23 (25)
16 (100)
27 (12.5)
17 (100)
27 (6.25)
14 (100)
25 (12.5)
H2C
3,5-CH3
25 (12.5)
5o
N
21 (50)
22 (25)
19 (100)
19 (100)
17 (100)
19 (100)
19 (100)
21 (100)
CH
CH
5p
5q
N
N
25 (12.5)
24 (25)
25 (12.5)
27 (6.25)
23 (50)
24 (12.5)
22 (50)
25 (12.5)
26 (12.5)
25 (25)
24 (25)
27 (6.25)
25 (6.25)
22 (25)
23 (25)
Cl
F
26 (12.5)
(continued on next page)

4358
R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
Table 1 (continued )
Entry
R
X
N
Zone of inhibition [mm (MIC in
S.a B.c
m
g/mL)]
E.c
P.a
23 (25)
K.p
23 (25)
S.t
P.v
24 (25)
S.f
21 (50)
5r
25 (25)
26 (6.25)
22 (50)
24 (25)
F
CF3
5s
N
28 (6.25)
28 (6.25)
28 (6.25)
26 (6.25)
26 (12.5)
26 (12.5)
25 (6.25)
26 (6.25)
23 (25)
27 (12.5)
25 (12.5)
26 (6.25)
25 (12.5)
25 (12.5)
H3CO
H2C
OCH3
OCH3
5t
N
N
23 (12.5)
26 (12.5)
24 (25)
5u
24 (25)
28 (6.25)
25 (12.5)
26 (6.25)
23 (25)
24 (12.5)
23 (12.5)
OCH3
Cip.
DMSO
30 (ꢃ1)
31 (ꢃ1)
32 (ꢃ1)
33 (ꢃ1)
33 (ꢃ1)
30 (ꢃ1)
31 (ꢃ1)
32 (ꢃ3)
e
e
e
e
e
e
e
e
Cip. e Ciprofloxacin; S.a e Staphylococcus aureus, B.c e Bacillus cereus, E.c e Escherichia coli, P.a e Pseudomonas aeruginosa, K.p e Klebsiella pneumoniae, S.t e Salmonella typhi, P.v
e Proteus vulgaris, S.f e Shigella flexneria.
in vitro antibacterial, antifungal and antimycobacterial inhibitory
effects. Higher inhibitory effects observed in this study appear to be
dependent on the mono-chloro, di-chloro, acetyl linkage, dimethyl,
mono-fluoro, trifluoromethyl, trimethoxy and mono-methoxy
functionality to the nitrogen atom of piperazine bases condensed
to the nucleus. Interestingly, derivatives 5d, 5p, 5q, 5r, 5t and 5u,
which showed potent antimicrobial as well as antimycobacterial
activities, were devoid of anticancer activity in the present study,
therefore no correlation was noted between the antibacterial and
anticancer activities with the above compounds. However, two
compounds 5n and 5s were found to contribute to a greater extent
to the antimicrobial, antimycobacterial and anticancer activities.
These two compounds can serve as a good starting point for further
drug discovery study. Further structural optimization as well as
studies on the anti-HIV activities of these derivatives is also
underway in our laboratories in order to gain more information
about their pharmacological applications.
India) using KBr pellets. Thin layer chromatography was performed
on object glass slides (2 ꢂ 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 frequency of 400 MHz and 13C
resonant frequency of 100 MHz. 19F NMR spectra were obtained on
the same spectrometer using CDCl3 as a solvent and CFCl3 as an
external standard, positive for downfield shift 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 excel-
lence, 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).
5.1.1. 4-[4,6-Dichloro-1,3,5-triazin-2-ylamino]-2-trifluoromethyl-
benzonitrile (1)
5. Materials and methods
To a stirred solution of 2,4,6-trichloro-1,3,5-triazine (10 g,
0.054 mol) in anhydrous THF (150 mL) 4-amino-2-trifluoromethyl-
benzonitrile (10.09 g, 0.054 mol) was drop wise added at 0e5 ꢁC.
The resulting reaction mixture was stirred at this temperature for
2 h. Then triethyl amine (5.48 g, 0.054 mol) was added and stirring
was continued for another 5 h. The reaction mixture was then
treated with crushed ice, followed by neutralization with dilute
HCl, and filtered, dried, and recrystallized from acetone to afford 1
[27]. Yield 88%, m.p. 259e261 ꢁC (dec.). IR (KBr, cmꢀ1): 3282 (eNH),
2223 (CN).
5.1. Chemistry
2,4,6-Trichloro-1,3,5-triazine was purchased from Sigma Aldrich
Chemicals Pvt. Ltd., Mumbai, India. 4-Hydroxyquinoline was
purchased from Silica Hetero Cyclics, Hyderabad, India. 4-Amino-2-
trifluoromethyl-benzonitrile was a gift from Ramdev Chemicals
Pvt. Ltd., Boisar, India. Acetone, tetrahydrofuran and 1,4-dioxane of
HPLC grade were purchased from Rankem, Surat, India. The TLC
plates (silica gel 60 F254) were obtained from Merck, Germany.
Substituted piperazine derivatives were gifts from Dr. Prem’s
Molecules Pvt. Ltd., Vadodara, India, Modepro India Pvt. Ltd.,
Mumbai, India, Trichem Pvt. Ltd., Mumbai, India, Siddharth Inter-
chem Pvt. Ltd., Ankleshwar, India and Mahrshee Laboratories,
Bharuch, India, Trichem Pvt. Ltd., Mumbai, India.
5.1.2. 4-[4-Chloro-6-(quinolin-4-yloxy)-1,3,5-triazin-2-ylamino]-2-
trifluoromethy-benzonitrile (3)
To a stirred solution of 4-hydroxyquinoline (8 g, 0.055 mol) in
anhydrous THF (150 mL) 60% NaH (1.32 g, 0.055 mol) was added at
room temperature during 1 h and 1 (18.41 g, 0.055 mol) was then
added to the mixture. Stirring was continued for another 14 h at
45 ꢁC. Progress of the reaction was monitored by TLC using tolue-
ne:acetone (7:3) as eluent. The mixture was treated with crushed
ice, filtered, dried and recrystallized from THF to afford 3 [28]. Yield
Melting points were determined in open capillaries on a Veego
electronic apparatus VMP-D (Veego Instrument Corporation,
Mumbai, India) and are uncorrected. IR spectra (4000e400 cmꢀ1
)
of synthesized compounds were recorded on a Shimadzu 8400-S
FT-IR spectrophotometer (Shimadzu India Pvt. Ltd., Mumbai,

R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
4359
Table 2
In vitro antifungal activity.
R
X
N
CN
N
N
O
N
N
CF3
H
N
Entry
R
X
Zone of inhibition [mm (MIC in mg/mL)]
A.n
A.f
A.c
C.a
5a
5b
CH3
C2H5
N
N
<10 (100)
<10 (100)
<10 (100)
<10 (100)
<10 (100)
<10 (100)
<10 (100)
12 (100)
Cl
5c
N
N
21 (100)
24 (25)
21 (100)
25 (25)
20 (100)
21 (50)
20 (100)
23 (50)
Cl
Cl
5d
5e
5f
H
O
12 (100)
13 (100)
<10 (100)
<10 (100)
14 (100)
13 (100)
12 (100)
13 (100)
5g
5h
N
N
<10 (100)
<10 (100)
<10 (100)
<10 (100)
COCH3
21 (100)
21 (100)
27 (12.5)
17 (100)
CH3
HC
5i
5j
5k
5l
N
N
N
N
<10 (100)
<10 (100)
11 (100)
17 (100)
12 (100)
13 (100)
<10 (100)
17 (100)
<10 (100)
11 (100)
12 (100)
18 (100)
13 (100)
13 (100)
<10 (100)
CH3
N
N
N
<10 (100)
H2C
5m
5n
H
H
<10 (100)
<10 (100)
14 (100)
26 (25)
15 (100)
18 (100)
H2C
3,5-CH3
23 (50)
25 (50)
5o
N
14 (100)
20 (100)
16 (100)
15 (100)
CH
CH
5p
N
25 (25)
26 (25)
25 (50)
25 (25)
(continued on next page)
Cl

4360
R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
Table 2 (continued )
Entry
R
X
Zone of inhibition [mm (MIC in
A.n A.f
mg/mL)]
A.c
C.a
F
5q
5r
5s
N
N
N
25 (25)
25 (25)
24 (50)
27 (25)
24 (50)
26 (25)
26 (12.5)
27 (12.5)
25 (50)
24 (50)
26 (25)
F
CF3
28 (12.5)
H3CO
H2C
OCH3
OCH3
5t
N
N
27 (12.5)
27 (25)
28 (12.5)
25 (25)
5u
25 (25)
27 (25)
28 (12.5)
24 (50)
OCH3
Kit.
DMSO
30 (ꢃ 3)
29 (ꢃ1)
31 (ꢃ1)
33 (ꢃ1)
e
e
e
e
Kit e Ketoconazole; A.n e Aspergillus niger, A.f e Aspergillus fumigates, A.c e Aspergillus clavatus, C.a e Candida albicans.
80%, m.p. 278e281 ꢁC (dec.). IR (KBr, cmꢀ1): 3289 (eNH), 2221
(CN), 1256 (CeOeC).
C
trieNHe), 8.85 (d, J ¼ 8.8 Hz, 1H, H2, quinoline), 8.66 (s, 1H, H3,
quinoline), 8.29 (dd, J ¼ 7.7, 2.3 Hz, 1H, H8, quinoline), 8.08 (dd,
J ¼ 7.6, 1.1 Hz, 1H, H5, quinoline), 7.80e7.77 (m, 2H, quinoline),
7.45e7.38 (m, 3H, AreH), 3.88 (br s, 4H, piperazine), 3.48 (br s, 4H,
piperazine), 2.21 (q, J ¼ 7.3 Hz, 2H, N-CH2), 1.97 (t, J ¼ 6.6 Hz, 3H,
5.1.3. General procedure for preparation of compounds (5aeu)
To a solution of 3 (0.01 mol) in 1,4-dioxane (30 mL), the
respective substituted piperazine or piperidine derivative was
added and the reaction mixture was refluxed for 12e20 h. Potas-
sium carbonate (0.01 mol) was used for neutralization of the
reaction mixture. Progress of the reaction was monitored by TLC
using toluene:acetone (8:2) as eluent. The mixture was then
treated with crushed ice and neutralized by dilute HCl. The
precipitate thus obtained was filtered off, dried and recrystallized
from THF to afford the desired compounds 5aeu.
CH2eCH3). 13C NMR (100 MHz, DMSO-d6):
d 176.9 (1C, C-6,
CtrieNpip), 164.6 (1C, C-4, CtrieOeCqui), 164.1 (1C, C-2, CtrieNHe),
151.8, 147.9 (2C, C2 and C9, quinoline), 146.7, 144.3, 142.9, 140.7,
138.2, 137.1, 133.6, 130.7 (CeCF3), 127.3, 124.8 (CF3), 123.9, 121.4,
119.9 (13C, Ar. C), 106.2 (1C, C^N), 99.1 (1C, eCeC^N), 49.8, 45.2,
41.1 (5C, 4C of piperazine and 1C of N-CH2eCH3), 23.1 (1C, N-
CH2eCH3). Anal Calcd. for C26H23F3N8O: C, 59.99; H, 4.45; N, 21.53;
Found: C, 59.90; H, 4.33; N, 21.42.
5.1.3.1. 4-(4-(4-Methylpiperazin-1-yl)-6-(quinolin-4-yloxy)-1,3,5-
triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5a). Recrystalli-
zation from THF (yield 85%), m.p. 245e246 ꢁC. IR (KBr, cmꢀ1):
3290 (eNH), 2218 (C^N), 1251 (CeOeC), 839 (s-triazine CeN
5.1.3.3. 4-(4-(4-(2-Chlorophenyl)piperazin-1-yl)-6-(quinolin-4-
yloxy)-1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5c).
Recrystallization from DMF (yield 78%), m.p. 218e219 ꢁC. IR (KBr,
cmꢀ1): 3280 (eNH), 2224 (C^N), 1259 (CeOeC), 834 (s-triazine
str.). 1H NMR (400 MHz, DMSO-d6):
d
9.13 (s, 1H, CtrieNHe), 8.79
CeN str.), 759 (CeCl). 11H NMR (400 MHz, DMSO-d6):
d 9.09 (s, 1H,
(d, J ¼ 8.5 Hz, 1H, H2, quinoline), 8.59 (s, 1H, H3, quinoline), 8.30
(dd, J ¼ 8.2, 1.3 Hz, 1H, H8, quinoline), 8.11 (dd, J ¼ 9.3, 1.1 Hz, 1H,
H5, quinoline), 7.81e7.78 (m, 2H, quinoline), 7.54e7.39 (m, 5H,
AreH), 3.79 (br s, 4H, piperazine), 3.50 (br s, 4H, piperazine), 1.94
C
trieNHe), 8.80 (d, J ¼ 8.6 Hz, 1H, H2, quinoline), 8.63 (s, 1H, H3,
quinoline), 8.40 (dd, J ¼ 7.7, 1.4 Hz, 1H, H8, quinoline), 8.19 (dd,
J ¼ 8.7, 1.9 Hz, 1H, H5, quinoline), 7.70e7.66 (m, 2H, quinoline),
7.45e7.32 (m, 6H, AreH), 6.59 (dd, J ¼ 7.4, 1.4 Hz, 1H, eCH), 3.80 (br
s, 4H, piperazine), 3.44 (br s, 4H, piperazine). 13C NMR (100 MHz,
(s, 1H, N-CH3). 13C NMR (100 MHz, DMSO-d6):
d
177.7 (1C, C-6,
C
trieNpip), 167.1 (1C, C-4, CtrieOeCqui), 164.6 (1C, C-2, CtrieNHe),
DMSO-d6): d 177.3 (1C, C-6, CtrieNpip), 166.6 (1C, C-4, CtrieOeCqui),
153.9, 148.2 (2C, C2 and C9, quinoline), 145.1, 143.7, 141.4, 139.9,
137.2, 134.6, 133.8, 131.7, 130.2 (CeCF3), 128.7, 126.2, 125.4 (CF3),
122.3 (13C, Ar. C), 105.9 (1C, C^N), 96.4 (1C, eCeC^N), 51.1, 46.3
(4C, piperazine), 21.1 (1C, N-CH3). Anal Calcd. for C25H21F3N8O: C,
59.28; H, 4.18; N, 22.12; Found: C, 59.18; H, 4.08; N, 22.01.
163.9 (1C, C-2, CtrieNHe), 153.5, 150.2 (2C, C2 and C9, quinoline),
146.1,145.1, 143.6, 142.9, 140.2, 139.5,137.3,136.2, 135.4, 134.9, 133.1,
131.2, 129.6 (CeCF3), 127.5, 125.5 (CF3), 124.5, 123.2, 122.7, 120.5
(19C, Ar. C), 106.8 (1C, C^N), 97.9 (1C, eCeC^N), 47.2, 45.0 (4C,
piperazine). Anal Calcd. for C30H22ClF3N8O: C, 59.76; H, 3.68; N,
18.58; Found: C, 59.68; H, 3.73; N, 18.62.
5.1.3.2. 4-(4-(4-Ethylpiperazin-1-yl)-6-(quinolin-4-yloxy)-1,3,5-tria-
zin-2-ylamino)-2-trifluoromethyl-benzonitrile
5.1.3.4. 4-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-6-(quinolin-4-
yloxy)-1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5d).
Recrystallization from DMF (yield 80%), m.p. 257e258 ꢁC. IR (KBr,
cmꢀ1): 3289 (eNH), 2220 (C^N), 1255 (CeOeC), 830 (s-triazine
(5b). Recrystallization from THF (yield 81%), m.p. 240e242 ꢁC. IR
(KBr, cmꢀ1): 3279 (eNH), 2220 (C^N), 1246 (CeOeC), 836 (s-
triazine CeN str.). 1H NMR (400 MHz, DMSO-d6):
d 9.25 (s, 1H,

R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
Table 3 (continued )
4361
Table 3
In vitro anticancer activity.
Entry
R
X
Anti DU-145 cell line activitya
LC50 TGI TGI GI50
R
X
GI50
g/mL)
(m
M) M) g/mL) M)
(
m
(
m
(
m
(m
N
N
5r
5s
N
N
>100 >100
>100
>100
>100
F
CN
N
N
CF3
O
N
CF3
H
>100
74.1
47.1
38.7
24.6
N
Entry
R
X
Anti DU-145 cell line activitya
LC50 TGI TGI GI50
H3CO
H2C
OCH3
OCH3
GI50
5t
N
N
>100 >100
>100
>100
>100
>100
>100
(
m
M) M) g/mL) M)
(
m
(
m
(
m
(
mg/mL)
5a
5b
CH3
C2H5
N
N
>100 >100
>100 >100
>100
>100
51.2
25.9
45.0
86.6
92.6
Cl
5u
>100 >100
>100 >100
>100
>100
OCH3
5c
N
N
>100 >100
>100 >100
>100
>100
55.8
ADR
<0.1
<0.1
Cl
Cl
LC50 ¼ Concentration of drug causing 50% cell kill; TGI ¼ Concentration of drug
causing total inhibition of cell growth; GI50 ¼ Concentration of drug causing 50%
inhibition of cell growth; ADR ¼ Adriamycin, Positive control compound.
5d
>100
>100
a
Each value is the mean of three independent experiments.
5e
5f
H
O
>100 >100
>100 >100
>100
>100
94.9
68.3
46.6
33.7
5g
5h
5i
N
N
N
>100 >100
>100 >100
>100 >100
>100
>100
>100
>100
>100
CeN str.), 754 (CeCl). 1H NMR (400 MHz, DMSO-d6):
d 8.98 (s, 1H,
CtrieNHe), 8.71 (d, J ¼ 8.1 Hz, 1H, H2, quinoline), 8.58 (s, 1H, H3,
COCH3
75.1
40.1
quinoline), 8.43 (dd, J ¼ 7.9, 1.6 Hz, 1H, H8, quinoline), 8.02 (dd,
J ¼ 7.3, 1.9 Hz, 1H, H5, quinoline), 7.69e7.65 (m, 2H, quinoline),
7.47e7.36 (m, 3H, AreH), 7.08 (dd, J ¼ 8.2, 1.7 Hz, 1H), 6.91 (t,
J ¼ 7.6 Hz, 1H), 6.50 (dd, J ¼ 7.3, 1.4 Hz, 1H), 3.80 (br s, 4H, pipera-
zine), 3.41 (br s, 4H, piperazine). 13C NMR (100 MHz, DMSO-d6):
CH3
HC
>100
>100
CH3
d
176.4 (1C, C-6, CtrieNpip), 165.9 (1C, C-4, CtrieOeCqui), 164.2 (1C, C-
5j
N
N
>100 >100
>100 >100
>100
>100
85.9
85.2
48.9
48.6
2, CtrieNHe), 149.9, 147.5 (2C, C2 and C9, quinoline), 144.1, 142.2,
141.9, 139.8, 137.6, 136.1, 135.3, 133.9, 131.5, 129.9 (C-CF3), 128.7,
127.6, 126.0 (CF3), 124.3, 123.9, 122.6, 120.5, 119.1, 118.4, 117.5 (19C,
Ar. C), 105.8 (1C, C^N), 97.8 (1C, eCeC^N), 50.2, 42.7 (4C, piper-
azine). Anal Calcd. for C30H21Cl2F3N8O: C, 56.53; H, 3.32; N, 17.58;
Found: C, 56.70; H, 3.21; N, 17.49.
N
N
5k
N
5l
N
>100 >100
>100 >100
>100
>100
>100
>100
5.1.3.5. 4-(4-(Piperidin-1-yl)-6-(quinolin-4-yloxy)-1,3,5-triazin-2-
ylamino)-2-trifluoromethyl-benzonitrile (5e). Recrystallization from
THF (yield 83%), m.p. 179e181 ꢁC. IR (KBr, cmꢀ1): 3305 (eNH), 2219
(C^N), 1251 (CeOeC), 835 (s-triazine CeN str.). 1H NMR (400 MHz,
H2C
5m
5n
H
H
>100
>100
H2C
DMSO-d6):
d
8.90 (s, 1H, CtrieNHe), 8.71 (d, J ¼ 7.7 Hz, 1H, H2,
quinoline), 8.69 (s, 1H, H3, quinoline), 8.36 (dd, J ¼ 8.2, 2.6 Hz, 1H,
H8, quinoline), 8.14 (dd, J ¼ 7.9, 1.7 Hz, 1H, H5, quinoline), 7.80e7.76
(m, 2H, quinoline), 7.51e7.42 (m, 3H, AreH), 3.90 (t, J ¼ 5.3 Hz, 4H,
piperidine), 3.74 (t, J ¼ 5.7 Hz, 4H, piperidine), 1.61e1.58 (m, 2H,
3,5-CH3
>100
68.0
35.3
27.2
14.1
piperidine). 13C NMR (100 MHz, DMSO-d6):
d 175.1 (1C, C-6,
5o
N
>100 >100
>100
>100
>100
CH
CtrieNpip), 166.1 (1C, C-4, CtrieOeCqui), 163.5 (1C, C-2, CtrieNHe),
154.2, 149.7 (2C, C2 and C9, quinoline), 144.1, 143.2, 140.8, 137.6,
136.3, 135.9, 133.8, 132.6, 129.7 (CeCF3), 127.9, 126.7, 125.1 (CF3),
123.8 (13C, Ar. C), 105.8 (1C, C^N), 98.2 (1C, eCeC^N), 53.4, 50.6,
41.1 (5C, piperidine). Anal Calcd. for C25H20F3N7O: C, 61.10; H, 4.10;
N, 19.95; Found: C, 60.98; H, 4.19; N, 19.89.
CH
F
5p
5q
N
N
>100 >100
>100 >100
>100
>100
>100
>100
>100
>100
5.1.3.6. 4-(4-Morpholino-6-(quinolin-4-yloxy)-1,3,5-triazin-2-yla-
mino)-2-trifluoromethyl-benzonitrile (5f). Recrystallization from
THF (yield 82%), m.p. 211e212 ꢁC. IR (KBr, cmꢀ1): 3288 (eNH), 2222
(C^N), 1409 (Morpholine CeOeC str.), 1250 (CeOeC), 828 (s-
Cl
triazine CeN str.). 1H NMR (400 MHz, DMSO-d6):
d
9.18 (s, 1H,
(continued on next page)
CtrieNHe), 8.88 (d, J ¼ 8.8 Hz, 1H, H2, quinoline), 8.60 (s, 1H, H3,

4362
R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
quinoline), 8.39 (dd, J ¼ 7.8, 1.6 Hz, 1H, H8, quinoline), 7.92 (dd,
J ¼ 7.5, 1.2 Hz, 1H, H5, quinoline), 7.69e7.63 (m, 2H, quinoline),
7.39e7.32 (m, 3H, AreH), 2.54e2.41 (m, 4H, CH2, morpholine),
1.70e1.64 (m, 2H, CH2, morpholine), 1.39e1.34 (m, 2H, CH2, mor-
5.1.3.10. 4-(4-(4-(Pyridin-2-yl)piperazin-1-yl)-6-(quinolin-4-yloxy)-
1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5j). Recry-
stallization from DMF (yield 79%), m.p. 278e279 ꢁC. IR (KBr,
cmꢀ1): 3294 (eNH), 2224 (C^N), 1260 (CeOeC), 830 (s-triazine
pholine). 13C NMR (100 MHz, DMSO-d6):
d
177.0 (1C, C-6, CtrieN-
CeN str.). 1H NMR (400 MHz, DMSO-d6):
d 9.11 (s, 1H, CtrieNHe),
mor), 165.9 (1C, C-4, CtrieOeCqui), 164.1 (1C, C-2, CtrieNHe), 155.0,
152.5 (2C, C2 and C9, quinoline), 146.8, 144.5, 142.6, 138.5, 136.3,
135.9, 134.2, 132.8, 129.9 (CeCF3), 127.5, 126.8, 124.7 (CF3), 123.5
(13C, Ar. C), 105.6 (1C, C^N), 98.8 (1C, eCeC^N), 55.7, 51.1 (4C,
morpholine). Anal Calcd. for C24H18F3N7O2: C, 58.42; H, 3.68; N,
19.87; Found: C, 58.31; H, 3.71; N, 19.76.
8.79 (d, J ¼ 7.5 Hz, 1H, H2, quinoline), 8.66 (s, 1H, H3, quinoline),
8.35 (dd, J ¼ 7.9, 1.8 Hz, 1H, H8, quinoline), 8.10 (dd, J ¼ 7.2, 1.4 Hz,
1H, H5, quinoline), 7.81e7.77 (m, 2H, quinoline), 7.44e7.29 (m, 6H,
AreH), 7.18 (dd, J ¼ 7.7, 1.8 Hz, 1H, pyridyl), 3.89 (br s, 4H, piper-
azine), 3.48 (br s, 4H, piperazine). 13C NMR (100 MHz, DMSO-d6):
d
177.2 (1C, C-6, CtrieNpip), 165.3 (1C, C-4, CtrieOeCqui), 163.9 (1C,
C-2, CtrieNHe), 158.9, 158.1, 152.6, 148.3 (4C, 2C of C2 and C9 of
quinoline and 2C of NpipeCeNpyeCH and 1C of NpipeCeNpyeCH),
145.8, 144.2, 142.9, 140.7, 137.6, 135.2, 134.7, 130.2 (CeCF3), 128.9,
127.6, 126.3, 125.0 (CF3), 123.8, 121.9, 118.2, 117.2 (16C, Ar. C), 106.3
(1C, C^N), 96.9 (1C, eCeC^N), 49.8, 43.2 (4C, piperazine). Anal
Calcd. for C29H22F3N9O: C, 61.16; H, 3.89; N, 22.13; Found: C, 61.07;
H, 3.78; N, 22.21.
5.1.3.7. 4-(4-(4-Phenylpiperazin-1-yl)-6-(quinolin-4-yloxy)-1,3,5-tri-
azin-2-ylamino)-2-trifluoromethyl-benzonitrile
(5g). Recrystalli-
zation from DMF (yield 75%), m.p. 278e280 ꢁC. IR (KBr, cmꢀ1): 3280
(eNH), 2219 (C^N), 1255 (CeOeC), 838 (s-triazine CeN str.). 1H
NMR (400 MHz, DMSO-d6):
d 9.97 (s, 1H, CtrieNHe), 8.77 (d,
J ¼ 7.0 Hz, 1H, H2, quinoline), 8.57 (s, 1H, H3, quinoline), 8.37 (dd,
J ¼ 8.9, 2.7 Hz, 1H, H8, quinoline), 8.08 (dd, J ¼ 8.2, 1.5 Hz, 1H, H5,
quinoline), 7.71e7.68 (m, 2H, quinoline), 7.44e7.27 (m, 8H, AreH),
3.89 (br s, 4H, piperazine), 3.52 (br s, 4H, piperazine). 13C NMR
5.1.3.11. 4-(4-(4-(Pyrimidin-2-yl)piperazin-1-yl)-6-(quinolin-4-
yloxy)-1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5k).
Recrystallization from DMF (yield 77%), m.p. 256e258 ꢁC. IR (KBr,
cmꢀ1): 3299 (eNH), 2220 (C^N), 1263 (CeOeC), 829 (s-triazine
(100 MHz, DMSO-d6):
d 176.6 (1C, C-6, CtrieNpip), 164.8 (1C, C-4,
C
trieOeCqui), 163.3 (1C, C-2, CtrieNHe), 153.5, 148.6 (2C, C2 and C9,
quinoline), 145.6, 144.3, 142.9, 140.1, 139.8, 137.6, 136.2, 134.7, 132.9,
131.8, 130.0 (C-CF3), 128.9, 127.2, 126.5, 125.2 (CF3), 123.9, 121.8,
120.5, 119.1 (19C, Ar. C), 106.6 (1C, C^N), 96.2 (1C, eCeC^N), 50.5,
44.1 (4C, piperazine). Anal Calcd. for C30H23F3N8O: C, 63.38; H, 4.08;
N, 19.71; Found: C, 63.23; H, 3.97; N, 19.81.
CeN str.). 1H NMR (400 MHz, DMSO-d6):
d 9.19 (s, 1H, CtrieNHe),
8.80 (d, J ¼ 8.6 Hz, 1H, H2, quinoline), 8.50 (s, 1H, H3, quinoline),
8.47e8.49 (m, 2H, pyrimidyl), 8.26 (dd, J ¼ 7.2, 1.3 Hz, 1H, H8,
quinoline), 8.14 (dd, J ¼ 7.5, 1.9 Hz, 1H, H5, quinoline), 7.74e7.69 (m,
2H, quinoline), 7.42e7.34 (m, 3H, AreH), 6.78 (t, J ¼ 7.0 Hz, 1H,
pyrimidyl), 3.78 (br s, 4H, piperazine), 3.51 (br s, 4H, piperazine).
5.1.3.8. 4-(4-(4-Acetylpiperazin-1-yl)-6-(quinolin-4-yloxy)-1,3,5-tri-
13C NMR (100 MHz, DMSO-d6):
d 176.6 (1C, C-6, CtrieNpip), 165.9
azin-2-ylamino)-2-trifluoromethyl-benzonitrile
(5h). Recrystalli-
(1C, C-4, CtrieOeCqui), 163.3 (1C, C-2, CtrieNHe), 159.7, 157.9, 157.2,
153.1 (5C, 2C of C2 and C9 of quinoline and 3C of pyrimidyl), 147.3,
145.2, 143.8, 140.1, 137.9, 135.2, 133.8, 131.9, 130.4 (CeCF3), 129.4,
128.7, 127.1, 124.9 (CF3), 120.1 (14C, Ar. C), 106.1 (1C, C^N), 98.8
(1C, eCeC^N), 49.1, 41.9 (4C, piperazine). Anal Calcd. for
C28H21F3N10O: C, 58.95; H, 3.71; N, 24.55; Found: C, 59.03; H, 3.62;
N, 24.44.
zation from THF (yield 79%), m.p. 251e253 ꢁC. IR (KBr, cmꢀ1): 3277
(eNH), 2218 (C^N),1714 (eC]O),1479 (eCH3),1260 (CeOeC), 832
(s-triazine CeN str.). 1H NMR (400 MHz, DMSO-d6):
d 9.21 (s, 1H,
C
trieNHe), 8.76 (d, J ¼ 8.5 Hz, 1H, H2, quinoline), 8.59 (s, 1H, H3,
quinoline), 8.30 (dd, J ¼ 7.8, 1.5 Hz, 1H, H8, quinoline), 8.19 (dd,
J ¼ 8.5, 2.1 Hz, 1H, H5, quinoline), 7.72e7.70 (m, 2H, quinoline),
7.45e7.37 (m, 3H, AreH), 3.77 (br s, 4H, piperazine), 3.50 (br s, 4H,
piperazine), 2.23 (s, 3H, COCH3). 13C NMR (100 MHz, DMSO-d6):
5.1.3.12. 4-(4-(4-Benzylpiperazin-1-yl)-6-(quinolin-4-yloxy)-1,3,5-
triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5l). Recrystalli-
zation from DMF (yield 75%), m.p. 268e270 ꢁC. IR (KBr, cmꢀ1): 3292
(eNH), 2220 (C^N), 1254 (CeOeC), 830 (s-triazine CeN str.). 1H
d
177.9 (1C, C-6, CtrieNpip), 167.9, 164.8 (2C, 1C at C-4, CtrieOeCqui
and 1C at N-COCH3), 164.5 (1C, C-2, CtrieNHe), 152.6, 149.9 (2C, C2
and C9, quinoline), 143.6, 142.1, 140.7, 138.6, 137.4, 135.9, 134.7,
132.9, 129.8 (CeCF3), 128.5, 126.7, 125.2 (CF3), 121.4 (13C, Ar. C),
105.9 (1C, C^N), 97.7 (1C, eCeC^N), 48.8, 44.7 (4C, piperazine),
23.3 (COCH3). Anal Calcd. for C26H21F3N8O2: C, 58.43; H, 3.96; N,
20.96; Found: C, 58.52; H, 3.83; N, 20.91.
NMR (400 MHz, DMSO-d6):
d 9.05 (s, 1H, CtrieNHe), 8.89 (d,
J ¼ 7.8 Hz, 1H, H2, quinoline), 8.53 (s, 1H, H3, quinoline), 8.41 (dd,
J ¼ 8.3, 1.4 Hz, 1H, H8, quinoline), 8.21 (dd, J ¼ 7.9, 1.1 Hz, 1H, H5,
quinoline), 7.81e7.78 (m, 2H, quinoline), 7.43e7.31 (m, 8H, AreH),
3.83 (br s, 4H, piperazine), 3.46 (br s, 4H, piperazine), 2.79 (s, 2H, N-
5.1.3.9. 4-(4-(4-Isopropylpiperazin-1-yl)-6-(quinolin-4-yloxy)-1,3,5-
triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5i). Recrystalli-
zation from DMF (yield 73%), m.p. 250e252 ꢁC. IR (KBr, cmꢀ1):
3283 (eNH), 2221 (C^N), 1390 (isopropyl), 1250 (CeOeC), 838 (s-
CH2). 13C NMR (100 MHz, DMSO-d6):
d 176.8 (1C, C-6, CtrieNpip),
166.7 (1C, C-4, CtrieOeCqui), 163.7 (1C, C-2, CtrieNHe), 153.9, 151.1
(2C, C2 and C9, quinoline), 148.1, 146.3, 144.8, 142.1, 137.7, 135.5,
133.0, 132.2, 130.2 (CeCF3), 128.45, 126.16, 125.8 (CF3), 124.6, 124.0,
123.1, 122.6, 119.2, 117.8, 115.1 (19C, Ar. C), 106.1 (1C, C^N), 97.0 (1C,
eCeC^N), 66.1 (1C, N-CH2), 49.1, 46.4 (4C, piperazine). Anal Calcd.
for C31H25F3N8O: C, 63.91; H, 4.33; N, 19.23; Found: C, 63.83; H,
4.24; N, 19.35.
triazine CeN str.). 1H NMR (400 MHz, DMSO-d6):
d 9.16 (s, 1H,
C
trieNHe), 8.75 (d, J ¼ 7.9 Hz, 1H, H2, quinoline), 8.65 (s, 1H, H3,
quinoline), 8.38 (dd, J ¼ 7.9, 1.9 Hz, 1H, H8, quinoline), 8.14 (dd,
J ¼ 7.3, 1.7 Hz, 1H, H5, quinoline), 7.60e7.57 (m, 2H, quinoline),
7.44e7.32 (m, 3H, AreH), 3.85 (br s, 4H, piperazine), 3.40 (br s, 4H,
piperazine), 2.79e2.76 (m, 1H, N-CH), 2.12 (d, J ¼ 6.8 Hz, 6H,
5.1.3.13. 4-(4-(4-Benzylpiperidin-1-yl)-6-(quinolin-4-yloxy)-1,3,5-
triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5m). Recrystalli-
zation from DMF (yield 73%), m.p. 238e239 ꢁC. IR (KBr, cmꢀ1): 3282
(eNH), 2223 (C^N), 1249 (CeOeC), 836 (s-triazine CeN str.). 1H
e2CH3). 13C NMR (100 MHz, DMSO-d6):
d 176.4 (1C, C-6, CtrieNpip),
167.1 (1C, C-4, CtrieOeCqui), 164.1 (1C, C-2, CtrieNHe), 151.9, 147.3
(2C, C2 and C9, quinoline), 145.1, 143.3, 139.4, 137.4, 135.9, 133.7,
130.5 (CeCF3), 129.1, 127.6, 126.3, 125.9 (CF3), 123.6, 122.4 (13C, Ar.
C), 106.5 (1C, C^N), 97.2 (1C, eCeC^N), 50.3, 46.8, 38.9 (5C,
piperazine and N-CH), 22.9 (2C, 2CH3). Anal Calcd. for
C27H25F3N8O: C, 60.67; H, 4.71; N, 20.96; Found: C, 60.55; H, 4.75;
N, 20.88.
NMR (400 MHz, DMSO-d6):
d 9.20 (s, 1H, CtrieNHe), 8.79 (d,
J ¼ 7.3 Hz, 1H, H2, quinoline), 8.56 (s, 1H, H3, quinoline), 8.36 (dd,
J ¼ 7.9, 2.2 Hz, 1H, H8, quinoline), 8.08 (dd, J ¼ 7.5, 1.0 Hz, 1H, H5,
quinoline), 7.67e7.63 (m, 2H, quinoline), 7.47e7.29 (m, 8H, AreH),
3.75 (4H, t, J ¼ 7.5 Hz, piperidine), 3.57 (4H, t, J ¼ 8.8 Hz, piperidine),

R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
4363
2.48 (2H, s, eCH2), 1.90 (1H, t, J ¼ 6.9 Hz, eCH, piperidine). 13C NMR
5.1.3.17. 4-(4-(4-(2-Fluorophenyl)piperazin-1-yl)-6-(quinolin-4-
(100 MHz, DMSO-d6):
d
175.1 (1C, C-6, CtrieNpip), 166.6 (1C, C-4,
yloxy)-1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5q).
Recrystallization from DMF (yield 82%), m.p. 280e281 ꢁC. IR (KBr,
cmꢀ1): 3285 (eNH), 2220 (C^N), 1259 (CeOeC), 837 (s-triazine
C
trieOeCqui), 163.8 (1C, C-2, CtrieNHe), 151.7, 150.3 (2C, C2 and C9,
quinoline), 145.9, 143.8, 140.6, 139.1, 138.5, 137.6, 135.4, 133.1, 132.8,
131.9, 129.3 (CeCF3), 128.5, 127.1, 126.4, 124.6 (CF3), 123.7, 122.1,
121.9, 121.5 (19C, Ar. C), 105.9 (1C, C^N), 96.2 (1C, eCeC^N), 47.1,
43.6, 36.2, 29.5 (6C, 5C of piperidine and 1C of N-CH2). Anal Calcd.
for C32H26F3N7O: C, 66.08; H, 4.51; N, 16.86; Found: C, 66.21; H,
4.59; N, 16.81.
CeN str.). 1H NMR (400 MHz, DMSO-d6):
d 8.99 (s, 1H, CtrieNHe),
8.83 (d, J ¼ 7.8 Hz,1H, H2, quinoline), 8.53 (s,1H, H3, quinoline), 8.40
(dd, J ¼ 7.1, 1.7 Hz, 1H, H8, quinoline), 8.18 (dd, J ¼ 7.8, 1.5 Hz, 1H, H5,
quinoline), 7.69e7.66 (m, 2H, quinoline), 7.46e7.33 (m, 3H, AreH),
6.88 (dd, J ¼ 12.9, 6.2 Hz, 2H), 6.76e6.71 (m, 1H), 6.50 (dd, J ¼ 12.8,
6.6 Hz, 1H), 3.89 (br s, 4H, piperazine), 3.44 (br s, 4H, piperazine).
5.1.3.14. 4-(4-(3,5-Dimethylpiperidin-1-yl)-6-(quinolin-4-yloxy)-
1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5n). Recry-
stallization from DMF (yield 80%), m.p. 256e258 ꢁC. IR (KBr, cmꢀ1):
3286 (eNH), 2218 (C^N), 1256 (CeOeC), 834 (s-triazine CeN str.).
13C NMR (100 MHz, DMSO-d6):
d 177.8 (1C, C-6, CtrieNpip), 166.0
(1C, C-4, CtrieOeCqui), 163.8 (1C, C-2, CtrieNHe), 154.1, 153.5, 148.1
(3C, C2 and C9 of quinoline and CeF), 146.0, 144.7, 143.2, 141.0, 139.5,
137.2, 136.1, 135.3, 133.9, 132.7, 131.2 (CeCF3), 128.7, 126.4, 124.8
(CF3), 123.6, 122.7, 121.9, 120.3 (18C, Ar. C), 105.8 (1C, C^N), 95.9
(1C, eCeC^N), 51.8, 44.5 (4C, piperazine). 19F NMR (400 MHz,
1H NMR (400 MHz, DMSO-d6):
d 9.04 (s, 1H, CtrieNHe), 8.65 (d,
J ¼ 7.5 Hz, 1H, H2, quinoline), 8.51 (s, 1H, H3, quinoline), 8.30 (dd,
J ¼ 7.4, 1.9 Hz, 1H, H8, quinoline), 7.99 (dd, J ¼ 7.2, 1.5 Hz, 1H, H5,
quinoline), 7.77e7.72 (m, 2H, quinoline), 7.43e7.31 (m, 4H, AreH),
3.78 (dd, J ¼ 12.7, 7.6 Hz, 2H, piperidine), 2.90 (dd, J ¼ 12.8, 7.1 Hz,
2H, piperidine), 1.77e1.71 (m, 3H, piperidine), 1.58 (d, J ¼ 6.5 Hz,
CDCl3):
d
ꢀ122.1 (1F, s, CeF), ꢀ65.1 (3F, eCF3). Anal Calcd. for
C30H22F4N8O: C, 61.43; H, 3.78; N, 19.10; Found: C, 61.50; H, 3.69; N,
19.21.
6H, 2CH3). 13C NMR (100 MHz, DMSO-d6):
d
176.5 (1C, C-6,
5.1.3.18. 4-(4-(4-(4-Fluorophenyl)piperazin-1-yl)-6-(quinolin-4-
yloxy)-1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5r).
Recrystallization from DMF (yield 80%), m.p. 269e270 ꢁC. IR (KBr,
cmꢀ1): 3290 (eNH), 2223 (C^N), 1257 (CeOeC), 831 (s-triazine
C
trieNpip), 167.8 (1C, C-4, CtrieOeCqui), 164.3 (1C, C-2, CtrieNHe),
154.2, 148.6 (2C, C2 and C9, quinoline), 144.1, 142.2, 141.9, 139.7,
136.8, 134.6, 133.2, 131.9, 130.0 (CeF3), 128.4, 126.8, 124.9 (CF3),
121.9 (13C, Ar. C), 106.7 (1C, C^N), 99.1 (1C, eCeC^N), 50.5, 40.7,
31.3 (5C, piperidine), 22.7 (2C, 2CH3). Anal Calcd. for C27H24F3N7O:
C, 62.42; H, 4.66; N, 18.87; Found: C, 62.53; H, 4.61; N, 18.81.
CeN str.). 1H NMR (400 MHz, DMSO-d6):
d 8.98 (s, 1H, CtrieNHe),
8.86 (d, J ¼ 7.7 Hz,1H, H2, quinoline), 8.58 (s, 1H, H3, quinoline), 8.31
(dd, J ¼ 8.9, 1.9 Hz, 1H, H8, quinoline), 8.13 (dd, J ¼ 7.6, 1.7 Hz, 1H, H5,
quinoline), 7.66e7.61 (m, 2H, quinoline), 7.44e7.31 (m, 3H, AreH),
7.18 (dd, J ¼ 13.2, 7.7 Hz, 2H), 6.71e6.66 (m, 1H), 6.53 (dd, J ¼ 12.6,
6.9 Hz, 1H), 3.81 (br s, 4H, piperazine), 3.53 (br s, 4H, piperazine).
5.1.3.15. 4-(4-(4-Benzhydrylpiperazin-1-yl)-6-(quinolin-4-yloxy)-
1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5o). Recry-
stallization from DMF (yield 80%), m.p. 283e284 ꢁC. IR (KBr, cmꢀ1):
3301 (eNH), 2220 (C^N), 1255 (CeOeC), 833 (s-triazine CeN str.).
13C NMR (100 MHz, DMSO-d6):
d
177.9 (C-6, CtrieNpip), 165.7 (C-4,
CtrieOeCqui), 163.6 (C-2, CtrieNHe), 153.2, 151.1, 148.9 (3C, C2 and C9
1H NMR (400 MHz, DMSO-d6):
d
9.15 (s, 1H, CtrieNHe), 8.80 (d,
of quinoline and CeF), 144.1, 142.1, 140.9, 139.4, 138.7, 135.6, 134.2,
133.1, 132.8, 130.2 (CeCF3), 128.7, 127.6, 126.3, 125.4 (CF3), 123.2,
122.9, 120.5, 118.1 (18C, Ar. C), 106.1 (1C, C^N), 99.2 (1C, eCeC^N),
J ¼ 8.1 Hz, 1H, H2, quinoline), 8.61 (s, 1H, H3, quinoline), 8.39 (dd,
J ¼ 7.4, 1.7 Hz, 1H, H8, quinoline), 8.16 (dd, J ¼ 7.6, 1.2 Hz, 1H, H5,
quinoline), 7.70e7.65 (m, 2H, quinoline), 7.53e7.24 (m, 13H, AreH),
4.09 (s, 1H, N-CH), 3.79 (br s, 4H, piperazine), 3.50 (br s, 4H,
49.5, 47.5 (4C, piperazine). 19F NMR (400 MHz, CDCl3):
d
ꢀ117.9 (1F,
s, CeF), ꢀ64.4 (3F, eCF3). Anal Calcd. for C30H22F4N8O: C, 61.43; H,
piperazine). 13C NMR (100 MHz, DMSO-d6):
d
177.5 (1C, C-6,
3.78; N, 19.10; Found: C, 61.31; H, 3.71; N, 18.98.
C
trieNpip), 167.5 (1C, C-4, CtrieOeCqui), 164.4 (1C, C-2, CtrieNHe),
150.9, 149.1 (2C, C2 and C9, quinoline), 148.7, 146.5, 145.2, 143.9,
141.4, 140.7, 139.2, 137.1, 136.0, 134.2, 133.1, 131.8, 130.8 (CeCF3),
129.0, 127.6, 126.8, 125.3 (CF3), 124.1, 123.8, 122.7, 121.0, 119.6, 119.2,
118.7, 117.3 (25C, Ar. C), 105.8 (1C, C^N), 96.1 (1C, eCeC^N), 75.9
(1C, eNpipeCH), 49.2, 46.5 (4C, piperazine). Anal Calcd. for
C37H29F3N8O: C, 67.47; H, 4.44; N, 17.01; Found: C, 67.56; H, 4.31; N,
16.93.
5.1.3.19. 4-(4-(Quinolin-4-yloxy)-6-(4-(3-(trifluoromethyl)phenyl)
piperazin-1-yl)-1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzoni-
trile (5s). Recrystallization from DMF (yield 75%), m.p. 290e292 ꢁC.
IR (KBr, cmꢀ1): 3288 (eNH), 2222 (C^N), 1252 (CeOeC), 835 (s-
triazine CeN str.). 1H NMR (400 MHz, DMSO-d6):
d 9.15 (s, 1H,
C
trieNHe), 8.78 (d, J ¼ 7.3 Hz, 1H, H2, quinoline), 8.55 (s, 1H, H3,
quinoline), 8.32 (dd, J ¼ 7.2, 1.1 Hz, 1H, H8, quinoline), 8.06 (dd,
J ¼ 6.9, 1.3 Hz, 1H, H5, quinoline), 7.74e7.68 (m, 2H, quinoline),
7.51e7.36 (m, 7H, AreH), 3.85 (br s, 4H, piperazine), 3.43 (br s, 4H,
5.1.3.16. 4-(4-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
6-(quinolin-4-yloxy)-1,3,5-triazin-2-ylamino)-2-trifluoromethyl-
benzonitrile (5p). Recrystallization from DMF (yield 83%), m.p.
269e270 ꢁC. IR (KBr, cmꢀ1): 3284 (eNH), 2222 (C^N), 1249
(CeOeC), 839 (s-triazine CeN str.). 1H NMR (400 MHz, DMSO-d6):
piperazine). 13C NMR (100 MHz, DMSO-d6):
d
175.3 (1C, C-6,
CtrieNpip), 166.9 (1C, C-4, CtrieOeCqui), 164.2 (1C, C-2, CtrieNHe),
153.2, 148.8 (2C, C2 and C9, quinoline), 145.1, 144.1, 141.4, 140.2,
138.7, 137.3, 136.0, 134.2, 132.5, 131.9, 130.7 (CeCF3), 129.8 (CeCF3),
127.4, 125.9 (CF3), 125.0 (CF3), 123.6, 122.9, 122.1, 120.3, 119.5 (20C,
Ar. C), 105.9 (1C, C^N), 97.3 (1C, eCeC^N), 49.7, 42.8 (4C, piper-
d
9.18 (s, 1H, CtrieNHe), 8.81 (d, J ¼ 8.5 Hz, 1H, H2, quinoline), 8.60
(s, 1H, H3, quinoline), 8.25 (dd, J ¼ 7.1, 1.5 Hz, 1H, H8, quinoline), 8.11
(dd, J ¼ 8.4, 1.3 Hz, 1H, H5, quinoline), 7.75e7.71 (m, 2H, quinoline),
7.49e7.21 (m, 12H, AreH), 3.97 (s, 1H, N-CH), 3.87 (br s, 4H,
piperazine), 3.54 (br s, 4H, piperazine). 13C NMR (100 MHz, DMSO-
azine). 19F NMR (400 MHz, CDCl3):
Calcd. for C31H22F6N8O: C, 58.49; H, 3.48; N, 17.60; Found: C, 58.57;
H, 3.36; N, 17.72.
d
ꢀ64.8, ꢀ65.9 (6F, s, 2CF3). Anal
d6):
d 176.2 (1C, C-6, CtrieNpip), 165.1 (1C, C-4, CtrieOeCqui), 164.4
(1C, C-2, CtrieNHe), 152.6, 150.9 (2C, C2 and C9, quinoline), 147.1,
146.2,144.1,143.9,141.2,139.8,139.1,138.0,136.7,135.4,134.2,132.9,
131.8, 129.9 (CeCF3), 128.3, 127.1, 126.5, 125.5 (CF3), 123.9, 122.8,
121.0, 120.4, 119.1, 118.6, 118.1 (25C, Ar. C), 106.1 (1C, C^N), 98.2 (1C,
eCeC^N), 76.2 (1C, eNpipeCH), 49.1, 46.4 (4C, piperazine). Anal
Calcd. for C37H28ClF3N8O: C, 64.12; H, 4.07; N, 16.17; Found: C,
63.99; H, 4.16; N, 16.28.
5.1.3.20. 4-(4-(Quinolin-4-yloxy)-6-(4-(2,3,4-trimethoxybenzyl)
piperazin-1-yl)-1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzoni-
trile (5t). Recrystallization from DMF (yield 78%), m.p. 252e254 ꢁC.
IR (KBr, cmꢀ1): 3279 (eNH), 2223 (C^N), 1489 (eCH2), 1258
(CeOeC), 838 (s-triazine CeN str.). 11H NMR (400 MHz, DMSO-d6):
d
9.17 (s, 1H, CtrieNHe), 8.83 (d, J ¼ 9.1 Hz, 1H, H2, quinoline), 8.68 (s,
1H, H3, quinoline), 8.35 (dd, J ¼ 7.9, 2.2 Hz, 1H, H8, quinoline), 8.18

4364
R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
(dd, J ¼ 7.4, 1.7 Hz, 1H, H5, quinoline), 7.69e7.67 (m, 2H, quinoline),
7.43e7.29 (m, 5H, AreH), 6.80 (d, J ¼ 7.5 Hz, 1H), 6.69 (d, J ¼ 7.6 Hz,
1H), 3.80 (br s, 4H, piperazine), 3.64 (s, 9H, 3OCH3), 3.49 (br s, 4H,
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 concen-
piperazine). 13C NMR (100 MHz, DMSO-d6):
d
177.2 (1C, C-6,
trations in the range of 3.12e100 mg/mL in dimethylsulfoxide and
CtrieNpip), 165.3 (1C, C-4, CtrieOeCqui), 164.6 (1C, C-2, CtrieNHe),
incubated at (37 ꢄ 1) ꢁC for 24 h (bacteria) or 48 h (fungi). The
lowest concentration of the substance that prevents the develop-
ment of visible growth is considered to be the MIC value.
154.1, 151.5 (2C, C2 and C9, quinoline),146.3, 145.2, 143.9,141.2, 139.0,
137.5, 136.8, 134.9, 133.2, 132.8, 130.1 (CeCF3), 129.0, 128.7, 127.1,
125.1 (CF3), 124.2, 123.7, 121.9, 120.7, 117.1 (19C, Ar. C), 106.6 (1C,
C^N), 97.7 (1C, eCeC^N), 66.5, 61.9, 56.5 (4C, 3OCH3 and
5.2.2. In vitro evaluation of antimycobacterial activity
N
pipeCH2), 49.4, 41.5 (4C, piperazine). Anal Calcd. for C34H31F3N8O4:
The preliminary antimycobacterial assessment for the final
synthesized compounds was carried out using BACTEC MGIT
method. The Mycobacterial Growth Indicator Tubes (MGIT) con-
taining 4 mL of modified Middle brook 7H9 Broth Base were
numbered as per the title compounds to be tasted for anti-
mycobacterial efficacy by means of various concentrations
prepared. The suspension was allowed to sit for 20 min and the
tubes were centrifuged at 3000 rpm for 15 min. After that
prepared suspension of 104 to 107 CFU/mL of H37Rv M. tuberculosis
strain was added in the medium to be incubated and 0.1 mL of
egg-based medium (L.J) was also added. The MGIT tubes were
then tightly recapped, mixed well and incubated into BACTEC
MGIT instrument at (37 ꢄ 1) ꢁC until positivity is observed. The
readings were measured daily starting from the second day of
incubation. Positive cultures were usually detected within 10 days.
For reading the actual results the MGIT tubes were removed from
incubator and placed on the UV light next to a positive control
tube and an uninoculated tube (negative control). Bright fluores-
cence detected by the corresponding MGIT tube was noticed in the
form of bright orange color in the bottom of the tube and also an
orange reflection on the meniscus [31]. The primary screening was
C, 60.71; H, 4.65; N, 16.66; Found: C, 60.79; H, 4.53; N, 16.53.
5.1.3.21. 4-(4-(4-(4-Methoxyphenyl)piperazin-1-yl)-6-(quinolin-4-
yloxy)-1,3,5-triazin-2-ylamino)-2-trifluoromethyl-benzonitrile (5u).
Recrystallization from DMF (yield 76%), m.p. 227e229 ꢁC. IR (KBr,
cmꢀ1): 3296 (eNH), 2224 (C^N),1252 (CeOeC), 836 (s-triazine CeN
str.). 1H NMR (400 MHz, DMSO-d6):
d 9.09 (s, 1H, CtrieNHe), 8.80 (d,
J ¼ 7.2 Hz, 1H, H2, quinoline), 8.54 (s, 1H, H3, quinoline), 8.42 (dd,
J ¼ 8.3, 1.2 Hz, 1H, H8, quinoline), 8.09 (dd, J ¼ 7.9, 1.7 Hz, 1H, H5,
quinoline), 7.82e7.75 (m, 2H, quinoline), 7.41e7.30 (m, 5H, AreH),
7.23 (d, J ¼ 8.2 Hz,1H), 6.73 (d, J ¼ 7.6 Hz,1H), 4.31 (s, 3H, OCH3), 3.80
(br s, 4H, piperazine ring), 3.55 (br s, 4H, piperazine ring). 13C NMR
(100 MHz, DMSO-d6):
d 177.1 (1C, C-6, CtrieNpip), 165.5 (1C, C-4,
CtrieOeCqui), 164.1 (1C, C-2, CtrieNHe), 156.1, 152.8 (2C, C2 and C9,
quinoline), 144.4, 143.2, 142.8, 139.0, 138.2, 137.4, 136.5, 135.1, 133.9,
132.0,131.6,129.8 (CeCF3),127.5,126.3,124.9 (CF3),122.6,121.9,120.1,
119.4 (19C, Ar. C), 105.6 (1C, C^N), 98.2 (1C, eCeC^N), 56.2 (1C,
eOCH3), 48.9, 47.1 (4C, piperazine). Anal Calcd. for C31H25F3N8O2: C,
62.20; H, 4.21; N, 18.72; Found: C, 62.07; H, 4.14; N, 18.83.
5.2. Biological assay
conducted at concentration of 6.25 mg/ml against M. tuberculosis
H37Rv in BACTEC MGIT system. Compounds demonstrating 99%
inhibition in the primary screen were described as most potent
compounds. All the other compounds to be tasted were re-
examined for their actual MIC by using LowensteineJensen MIC
method. The MIC was defined as the lowest concentration inhib-
iting 99% of the inoculum.
The secondary antimycobacterial screening for test compounds
was performed against M. tuberculosis H37Rv using L.J. (Lowenstein
and Jensen) MIC method [32,33] for the measurement of MIC. Stock
solutions of primary 1000, 500, 250, and secondary 200, 100, 62.5,
5.2.1. In vitro evaluation of antimicrobial activity
The synthesized s-triazinyl derivatives 5aeu were examined
for antimicrobial activity against several 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, S.
flexneria MTCC 1457) and fungi (A. niger MTCC 282, A. fumigatus
MTCC 343, A. clavatus MTCC 1323, C. albicans MTCC 183) species
using paper disc diffusion technique [29]. The MuellereHinton
agar media were sterilized (autoclaved at 120 ꢁC 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 dimethylsulfoxide to give solutions
50, 25, 12.5, 6.25, 3.25 mg/mL dilutions of each test compound in
dimethylsulfoxide (DMSO) were added in the liquid L.J. Medium
and then media were sterilized by inspissation method. A culture of
M. tuberculosis H37Rv growing on L.J. medium was harvested in
0.85% saline in bijou bottles. These tubes were then incubated at
(37 ꢄ 1) ꢁC for 24 h followed by streaking of M. tuberculosis H37Rv
(5 ꢂ104 bacilli per tube). These tubes were then incubated at
(37 ꢄ 1) ꢁC. 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 H37Rv. The concentration at which no devel-
opment of colonies occurred or <20 colonies was taken as MIC
concentration of test compound. The standard strain M. tuberculosis
H37Rv was tested with known drugs rifampicin, isoniazid,
ethambutol and pyrazinamide.
of 3.12e100
diameter (Whatman no. 1 filter paper), previously soaked in
known concentration of the respective test compound in
mg/mL. Sterile filter paper discs measuring 6.25 mm in
a
dimethylsulfoxide were placed on the solidified nutrient agar
medium that had been inoculated with the respective microor-
ganism and the plates were incubated for 24 h at (37 ꢄ 1) ꢁC. A
control disc impregnated with an equivalent amount of dime-
thylsulfoxide 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.
MIC of the compound was determined by agar streak dilution
method [30]. A stock solution of the synthesized compound
5.2.3. In vitro evaluation of anticancer activity
5.2.3.1. SRB assay. The cell lines were grown in RPMI 1640
(100
m
g/mL) in dimethylsulfoxide was prepared and graded quan-
medium containing 10% fetal bovine serum and 2 mM
For present screening experiment, cells were inoculated into 96
well microtiter plates in 90 L at plating densities, depending on
the doubling time of individual cell lines. After cell inoculation, the
microtiter plates were incubated at (37 ꢄ 1) ꢁC, 5% CO2, 95% air and
100% relative humidity for 24 h prior to addition of experimental
L-glutamine.
tities 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 4e5 mm and allowed to
m

R.V. Patel et al. / European Journal of Medicinal Chemistry 46 (2011) 4354e4365
4365
drugs. After 24 h, one plate of each cell line was fixed in situ with
Acknowledgements
TCA, to represent a measurement of the cell population for each
cell line at the time of drug addition (Tz). Experimental drugs were
solubilized in appropriate solvent at 400-fold the desired final
maximum test concentration and stored frozen prior to use. At the
time of drug addition, an aliquot of frozen concentrate was thawed
and diluted to 10 times the desired final maximum test concen-
tration with complete medium containing test article at a concen-
tration of 10ꢀ3. Additional three, 10-fold serial dilutions were made
to provide a total of four drug concentrations plus control. Aliquots
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, India and Tata Memorial
Advanced Centre for Treatment, Research and Education in Cancer,
Mumbai, India for carrying out the biological screenings. We are
also thankful to Centre of Excellence, Vapi, India for carrying out 1H
NMR, 13C NMR and 19F NMR analysis.
of 10
mL of these different drug dilutions were added to the
appropriate microtiter wells already containing 90
resulting in the required final drug concentrations [34].
m
L of medium,
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[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 for concentrations for which Ti ꢅ Tz
and
[(Ti ꢀ Tz)/Tz] ꢂ 100 for concentrations for which Ti < Tz.
The dose response parameters were calculated for each test
article. Growth inhibition of 50% (GI50) was calculated from
[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 ¼ 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as
measured by SRB staining) in control cells during the drug incu-
bation. The drug concentration resulting in total growth inhibition
(TGI) was calculated from Ti ¼ Tz. The LC50 (concentration of drug
resulting in a 50% reduction in the measured protein at the end of
the drug treatment as compared to that at the beginning) indicating
a net loss of cells following treatment is calculated from [(Ti ꢀ Tz)/
Tz] ꢂ 100 ¼ ꢀ50. Values were calculated for each of these three
parameters if the level of activity was reached; however, if the
effect was not reached or was exceeded, the values for that
parameter were expressed as greater or less than the maximum or
minimum concentration tested.
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