A new class of 2-(4-cyanophenyl amino)-4-(6-bromo-4-quinolinyloxy)-6- piperazinyl (piperidinyl)-1,3,5-triazine analogues with antimicrobial/ antimycobacterial activity

Article abstract of DOI:10.3109/14756366.2011.592491

This study presents the synthesis and in vitro pharmacological evaluations of novel 2-(4-cyanophenyl amino)-4-(6-bromo-4-quinolinyloxy)-6-piperazinyl (piperidinyl)-1,3,5-triazines. The title compounds were assayed for their in vitro antimicrobial activity

Full text of DOI:10.3109/14756366.2011.592491

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(3): 370–379
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.592491
ReseaRch aRtIcle
A new class of 2-(4-cyanophenyl amino)-4-(6-bromo-4-
quinolinyloxy)-6-piperazinyl (piperidinyl)-1,3,5-triazine
analogues with antimicrobial/antimycobacterial activity
Rahul V. Patel1, Premlata Kumari1, Dhanji P. Rajani2, and Kishor H. Chikhalia3
1Department of Applied Chemistry, S. V. National Institute of Technology, Surat 395 007, Gujarat, India, 2Microcare
Laboratory, Surat 395 001, Gujarat, India, and 3Department of Chemistry, School of Science, Gujarat University,
Ahmedabad 380 009, Gujarat, India
abstract
This study presents the synthesis and in vitro pharmacological evaluations of novel 2-(4-cyanophenyl amino)-4-(6-
bromo-4-quinolinyloxy)-6-piperazinyl (piperidinyl)-1,3,5-triazines. The title compounds were assayed for their in vitro
antimicrobial activity against eight bacteria (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas
aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, Shigella flexneria) and four fungi (Aspergillus
niger, Aspergillus fumigatus, Aspergillus clavatus, Candida albicans) using paper disc diffusion and agar streak dilution
method as well as against Mycobacterium tuberculosis H37Rv strain using BACTEC MGIT and Lowenstein-Jensen MIC
method. The bioassay results indicate that nine compounds namely 5d, 5h, 5n, 5p, 5q, 5r, 5s, 5t and 5u could be
considered as possible potential agents with dual antimicrobial and antimycobacterial activities. The structures of the
compounds were elucidated with the aid of IR, 1H NMR, 13C NMR, 19F NMR spectroscopy and CHN analysis.
Keywords: 2,4,6-trichloro-1,3,5-triazin, 6-bromo-4-hydroxyquinoline, piperazine, antimicrobial activity,
antimycobacterial activity
Introduction
burden countries between 2010 and 2015. In 2008, there
were an estimated 8.9–9.9 million incident cases of TB,
9.6–13.3 million prevalent cases of TB, 1.1–1.7 million
deaths from TB among HIV-negative people and an addi-
tional 0.45–0.62 million TB deaths among HIV-positive
people (classified as HIV deaths in the International
Statistical Classification of Diseases), with best estimates
of 9.4 million, 11.1 million, 1.3 million and 0.52 million,
respectively5. Hence, there is an urgent need to identify
new regimens involving novel mechanism of action and
synthetically feasible to address resistance crisis as well
as the global health emergency.
As part of our ongoing studies in developing new
active s-triazinyl analogues6–8, here in we report the
synthesis and pharmacological activities of novel 2-(4-
cyanophenyl amino)-4-(6-bromo-4-quinolinyloxy)-6-
piperazinyl (piperidinyl)-1,3,5-triazines. 1,3,5-Triazine
Bacterial infections have been most deleterious to the
human health due to pathogenic microbes which con-
tinuously evolve resistance to currently used antibacte-
rial agents. In the developing countries, this problem is
especially alarming. Furthermore, increased numbers of
patientswithimpairedimmunitymayfurtherincreasethe
burden of antimicrobial resistance1. us development of
novel antimicrobial drugs is crucial need to combat with
the multidrug-resistant infections. However, another
most worrisome trend in recent years is the increase in
multidrug-resistant tuberculosis strains due to the qui-
escent form of mycobacterium tuberculosis strains with
an estimated 2 million deaths each year2–4. e WHO has
estimated that, According to the stop TB partnership’s
global plan to stop TB, 2006–2015, 1.3 million MDR-TB
cases will need to be treated in the 27 high MDR-TB
Address for Correspondence: Dr. K. H. Chikhalia, Department of Chemistry, School of Science, Gujarat University, Ahmedabad-380 009,
Gujarat, India. Tel: +91–9427155529. E-mail: chikhalia_kh@yahoo.com
(Received 27 April 2011; revised 18 May 2011; accepted 18 May 2011)
370

s-Triazines as antibacterial agents 371
analogues constitute an important class of the realm of
heterocycles, which has attracted much synthetic inter-
est due to their wide range of biological activities such as
antimicrobial9–11, anticancer12, 13, antimalarial14, and anti-
viral15 activity. A literature survey revealed that pipera-
zines and substituted piperazines are an important family
of heterocyclic compounds attracting significant interest
in medicinal chemistry16–20. Recently several s-triazine
derivatives bearing morpholine, piperidine and some
piperazine moieties are reported to possess potent anti-
mycobacterial activity21, while quinoline-based anti-TB
compound TMC207 is currently in Phase II clinical trials
withverypromisingactivityagainstMDR-TB22,23.Recently,
several scaffolds-based on bromo-substituted quinoline
moiety and some piperazine derivatives are reported to
possess potent pharmacological activities24–26. is prior
literature survey encouraged us to envisage the combina-
tion of these separate pharmacophoric groups of similar
activity in a compact system to identify new candidates
that may be of value in designing new, potent, selective
and less toxic biologically active agents.
toluene: acetone (7:3) as eluent. e mixture was treated
with crushed ice, filtered and dried to afford 328. M.P.
282–283°C, yield 80%, IR (KBr) cm−1, 2221 (C≡N), 1261
(C-O-C). (see Figures 1 and 2).
General procedure for preparation of compounds 5a-u
To a solution of 3 (0.01 mol) in 1,4-dioxane (30 mL), the
respective substituted piperazine derivative was added
and the reaction mixture was refluxed for 13–25 h.
Potassium carbonate (0.01 mol) was used for neutraliza-
tion of the reaction mixture. Progress of the reaction was
monitored by TLC using toluene: acetone (8:2) as eluent.
e mixture was then treated with crushed ice and neu-
tralized by dilute HCl. e precipitate thus obtained was
filtered off, dried and recrystallized from THF to afford
the desired compounds 5a-u.
5a M.P. 242–244°C, yield 88% (found: C 55.59, H 3.96,
N 21.61, C24H21BrN8O, calc: C 55.71, H 4.09, N 21.66%). IR
(KBr) cm−1, 3280 (-NH), 2224 (C≡N), 1247 (C-O-C), 839
(s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.13 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.78 (d, J= 1.8 Hz, 1H, C5 proton of quinoline),
8.58 (d, J= 8.3 Hz, 1H, -N=CH-, C2 proton of quinoline),
8.46 (s, 1H, C3 proton of quinoline), 8.26 (d, J= 7.5 Hz,
1H, C8 proton of quinoline), 8.02 (dd, J= 7.7, 1.8 Hz, 1H),
7.54–7.35 (m, 6H, Ar-H), 3.79 (br s, 4H, piperazine), 3.56
(br s, 4H, piperazine), 1.98 (s, 1H, N-CH3); 13C-NMR
(100 MHz, DMSO-d6, δ) ppm, 177.1 (1C, C-6, s-triazine,
C-N at piperazine linkage), 165.9 (1C, C-4, s-triazine,
C-O-C at quinoline linkage), 164.4 (1C, C-2, s-triazine,
C-NH at benzonitrile moiety), 154.2, 151.1 (2C, 1C of
-C2H=N-C9 and 1C of -C2H=N-C9, quinoline), 146.1–120.7
(12C, aromatic carbon atoms), 105.1 (1C, C≡N), 99.1 (1C,
-C-C≡N), 48.7, 44.1 (4C, piperazine), 21.3 (1C, -CH3).
5bM.P. 235–236°C, yield 87% (found: C 56.41, H 4.21,
N 20.97, C25H23BrN8O, calc: C 56.50, H 4.36, N 21.09%). IR
(KBr) cm−1, 3301 (-NH), 2222 (C≡N), 1256 (C-O-C), 831
(s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.21 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.74 (d, J= 1.6 Hz, 1H, C5 proton of quinoline),
8.66 (d, J= 8.9 Hz, 1H, -N=CH-, C2 proton of quinoline),
8.53 (s, 1H, C3 proton of quinoline), 8.31 (d, J= 7.7 Hz,
1H, C8 proton of quinoline), 8.11 (dd, J= 7.8, 1.2 Hz, 1H),
7.49–7.36 (m, 4H, Ar-H), 3.77 (br s, 4H, piperazine), 3.51
(br s, 4H, piperazine), 2.35 (q, J= 7.3 Hz, 2H, N-CH2), 1.88
(t, J= 6.7 Hz, 3H, CH2-CH3); 13C-NMR (100 MHz, DMSO-
d6, δ) ppm, 176.6 (1C, C-6, s-triazine, C-N at piperazine
linkage), 167.1 (1C, C-4, s-triazine, C-O-C at quinoline
linkage), 164.1 (1C, C-2, s-triazine, C-NH at benzonitrile
moiety), 153.2, 148.0 (2C, 1C of -C2H=N-C9 and 1C of
-C2H=N-C9, quinoline), 144.8–121.9 (12C, aromatic car-
bon atoms), 105.8 (1C, C≡N), 95.7 (1C, -C-C≡N), 49.8,
42.3, 38.9 (5C, 4C of piperazine and 1C of N-CH2-CH3),
19.9 (1C, CH2-CH3).
Materials and methods
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 cm−1) of synthesized compounds were
recorded on a Shimadzu 8400-S FT-IR spectrophotometer
(Shimadzu India Pvt. Ltd., Mumbai, India) using KBr pel-
lets. in layer chromatography was performed on object
glass slides (2 × 7.5 cm) coated with silica gel-G and spots
1
were visualized under UV irradiation. H 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. e 1H NMR, 13
C
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.
e 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-[4,6-Dichloro-1,3,5-triazin-2-ylamino]-benzonitrile
(1) was synthesized according to reported literature27.
4-(4-(6-bromoquinolin-4-yloxy)-6-chloro-1,3,5-triaz-
in-2-ylamino)-benzonitrile (3).
To a stirred solution of 6-bromo-4-hydroxyquinoline
(8 g, 0.035 mol) in anhydrous THF (150mL) 60% NaH
(0.84 g, 0.035 mol) was added at room temperature dur-
ing 1 h and 1 (9.31 g, 0.035 mol) was then added to the
mixture. Stirring was continued for another 20 h at 45°C.
Progress of the reaction was monitored by TLC using
5c M.P. 265–267°C, yield 82% (found: C 56.87, H 3.49,
N 18.16, C29H22BrClN8O, calc: C 56.74, H 3.61, N 18.25%).
IR (KBr) cm−1, 3280 (-NH), 2221 (C≡N), 1258 (C-O-C),
1
832 (s-triazine C-N str.), 756 (C-Cl); H NMR (400 MHz,
© 2012 Informa UK, Ltd.

372 R. V. Patel et al.
DMSO-d6, δ) ppm, 9.11 (s, 1H, -NH, s-triazine to amino-
benzonitrile linkage), 8.81 (d, J= 2.2, 1H, C5 proton of
quinoline), 8.68 (d, J= 8.3 Hz, 1H, -N=CH-, C2 proton of
quinoline), 8.49 (s, 1H, C3 proton of quinoline), 8.26 (d,
J= 7.5 Hz, 1H, C8 proton of quinoline), 8.16 (dd, J= 7.7,
1.4 Hz, 1H), 7.58–7.35 (m, 8H, Ar-H), 3.83 (br s, 4H, pip-
erazine), 3.46 (br s, 4H, piperazine); 13C-NMR (100 MHz,
DMSO-d6, δ) ppm, 177.1 (1C, C-6, s-triazine, C-N at
piperazine linkage), 166.7 (1C, C-4, s-triazine, C-O-C at
quinoline linkage), 163.5 (1C, C-2, s-triazine, C-NH at
benzonitrile moiety), 152.9, 148.2 (2C, 1C of -C2H=N-C9
and 1C of -C2H=N-C9, quinoline), 146.3–119.9 (18C, aro-
matic carbon atoms), 106.1 (1C, C≡N), 97.7 (1C, -C-C≡N),
49.2, 47.5 (4C, piperazine).
and 1C of -C2H=N-C9, quinoline), 145.3–117.3 (18C, aro-
matic carbon atoms), 105.5 (1C, C≡N), 98.1 (1C, -C-C≡N),
50.2, 42.8 (4C, piperazine).
5e M.P. 206–208°C, yield 77% (found: C 57.45, H 4.09,
N 19.37, C24H20BrN7O, calc: C 57.38, H 4.01, N 19.52%). IR
(KBr) cm−1, 3297 (-NH), 2222 (C≡N), 1256 (C-O-C), 835
(s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.20 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.80 (d, J= 1.7, 1H, C5 proton of quinoline), 8.66
(d, J= 8.2 Hz, 1H, -N=CH-, C2 proton of quinoline), 8.50 (s,
1H, C3 proton of quinoline), 8.33 (d, J= 7.3 Hz, 1H, C8 pro-
ton of quinoline), 8.15 (dd, J= 7.6, 1.4 Hz, 1H), 7.52–7.40
(m, 4H, Ar-H), 3.81 (t, J= 4.9 Hz, 4H, piperidine), 3.63 (t,
J= 5.7 Hz, 4H, piperidine), 1.55–1.51 (m, 2H, piperidine);
13C-NMR (100 MHz, DMSO-d6, δ) ppm, 176.4 (1C, C-6,
s-triazine, C-N at piperidine linkage), 166.7 (1C, C-4,
s-triazine, C-O-C at quinoline linkage), 163.6 (1C, C-2,
s-triazine, C-NH at benzonitrile moiety), 152.5, 151.1 (2C,
1C of -C2H=N-C9 and 1C of -C2H=N-C9, quinoline), 148.2–
125.2 (12C, aromatic carbon atoms), 104.9 (1C, C≡N),
96.4 (1C, -C-C≡N), 54.9, 47.7, 38.6 (5C, piperidine).
5f M.P. 223–224°C, yield 80% (found: C 54.63, H 3.62, N
19.31, C23H18BrN7O2, calc: C 54.77, H 3.60, N 19.44%). IR
(KBr) cm−1, 3288 (-NH), 2221 (C≡N), 1423 (Morpholine
5d M.P. 271–273°C, yield 81% (found: C 53.63, H 3.18,
N 17.34, C29H21BrCl2N8O, calc: C 53.72, H 3.26, N 17.28%).
IR (KBr) cm−1, 3291 (-NH), 2224 (C≡N), 1253 (C-O-C),
1
830 (s-triazine C-N str.), 749 (C-Cl); H NMR (400 MHz,
DMSO-d6, δ) ppm, 9.18 (s, 1H, -NH, s-triazine to amino-
benzonitrile linkage), 8.86 (d, J= 1.8, 1H, C5 proton of
quinoline), 8.62 (d, J= 8.2 Hz, 1H, -N=CH-, C2 proton of
quinoline), 8.47 (s, 1H, C3 proton of quinoline), 8.29 (d,
J= 7.5 Hz, 1H, C8 proton of quinoline), 8.09 (dd, J= 7.6,
1.4 Hz, 1H), 7.61–7.37 (m, 7H, Ar-H), 3.85 (br s, 4H, pip-
erazine), 3.51 (br s, 4H, piperazine); 13C-NMR (100 MHz,
DMSO-d6, δ) ppm, 175.9 (1C, C-6, s-triazine, C-N at
piperazine linkage), 165.9 (1C, C-4, s-triazine, C-O-C at
quinoline linkage), 164.6 (1C, C-2, s-triazine, C-NH at
benzonitrile moiety), 153.1, 148.7 (2C, 1C of -C2H=N-C9
1
C-O-C str.), 1261 (C-O-C), 832 (s-triazine C-N str.); H
NMR (400 MHz, DMSO-d6, δ) ppm, 9.17 (s, 1H, -NH,
s-triazine to amino-benzonitrile linkage), 8.82 (d, J = 1.8,
1H, C5 proton of quinoline), 8.59 (d, J = 8.3 Hz, 1H,
-N=CH-, C2 proton of quinoline), 8.53 (s, 1H, C3 proton
Figure 1. Schematic diagram for the synthesis of 2-(4-cyanophenyl amino)-4-(6-bromo-4-quinolinyloxy)-6-piperazinyl (piperidinyl)-
1,3,5-triazines.
Journal of Enzyme Inhibition and Medicinal Chemistry

s-Triazines as antibacterial agents 373
of quinoline), 8.30 (d, J = 7.5 Hz, 1H, C8 proton of qui-
noline), 8.13 (dd, J = 7.8, 1.3 Hz, 1H), 7.55–7.41 (m, 4H,
Ar-H), 2.42–2.35 (m, 4H, -CH2, morpholine), 1.72–1.67
(m, 2H, -CH2, morpholine), 1.24–1.20 (m, 2H, -CH2,
morpholine); 13C-NMR (100 MHz, DMSO-d6, δ) ppm,
177.1 (1C, C-6, s-triazine, C-N at morpholine linkage),
165.7 (1C, C-4, s-triazine, C-O-C at quinoline linkage),
164.4 (1C, C-2, s-triazine, C-NH at benzonitrile moiety),
154.3, 150.9 (2C, 1C of -C2H=N-C9 and 1C of -C2H=N-C9,
quinoline), 149.2–123.1 (12C, aromatic carbon atoms),
105.2 (1C, C≡N), 98.8 (1C, -C-C≡N), 57.1, 53.3 (4C, mor-
pholine ring carbon atoms).
(m, 9H, Ar-H), 3.80 (br s, 4H, piperazine), 3.53 (br s, 4H,
piperazine); 13C-NMR (100 MHz, DMSO-d6, δ) ppm, 176.7
(1C, C-6, s-triazine, C-N at piperazine linkage), 166.9 (1C,
C-4, s-triazine, C-O-C at quinoline linkage), 164.0 (1C,
C-2, s-triazine, C-NH at benzonitrile moiety), 153.3, 149.8
(2C, 1C of -C2H=N-C9 and 1C of -C2H=N-C9, quinoline),
145.9–119.3 (18C, aromatic carbon atoms), 104.9 (1C,
C≡N), 97.2 (1C, -C-C≡N), 52.0, 48.2 (4C, piperazine).
5h M.P. 234–235°C, yield 77% (found: C 55.15, H 3.81,
N 20.43, C25H21BrN8O2, calc: C 55.06, H 3.88, N 20.55%). IR
(KBr) cm−1, 3281 (-NH), 2223 (C≡N), 1709 (-C=O), 1489
1
(-CH3), 1258 (C-O-C), 828 (s-triazine C-N str.); H NMR
5g M.P. 282–283°C, yield 82% (found: C 60.24, H 3.92,
N 19.46, C29H23BrN8O, calc: C 60.11, H 4.00, N 19.34%). IR
(KBr) cm−1, 3285 (-NH), 2221 (C≡N), 1251 (C-O-C), 831
(s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.15 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.77 (d, J= 1.8, 1H, C5 proton of quinoline), 8.64
(d, J= 8.4 Hz, 1H, -N=CH-, C2 proton of quinoline), 8.47 (s,
1H, C3 proton of quinoline), 8.28 (d, J= 7.6 Hz, 1H, C8 pro-
ton of quinoline), 8.16 (dd, J= 7.2, 1.4 Hz, 1H), 7.54–7.30
(400 MHz, DMSO-d6, δ) ppm, 9.22 (s, 1H, -NH, s-triazine
to amino-benzonitrile linkage), 8.79 (d, J= 1.6, 1H, C5
proton of quinoline), 8.65 (d, J= 8.2 Hz, 1H, -N=CH-, C2
proton of quinoline), 8.49 (s, 1H, C3 proton of quinoline),
8.32 (d, J= 7.5 Hz, 1H, C8 proton of quinoline), 8.08 (dd,
J= 7.6, 1.4 Hz, 1H), 7.47–7.34 (m, 4H, Ar-H), 3.84 (br s,
4H, piperazine), 3.48 (br s, 4H, piperazine), 2.39 (s, 3H,
COCH3); 13C-NMR (100 MHz, DMSO-d6, δ) ppm, 176.9
(1C, C-6, s-triazine, C-N at piperazine linkage), 169.1,
Figure 2. [4(a-u), R]=Piperazine and piperidine bases coupled to compound 3.
© 2012 Informa UK, Ltd.

374 R. V. Patel et al.
167.2 (2C, 1C at C-4, s-triazine, C-O-C at quinoline link-
age and 1C at N-COCH3), 163.3 (1C, C-2, s-triazine, C-NH
at benzonitrile moiety), 155.2, 151.2 (2C, 1C of -C2H=N-C9
and 1C of -C2H=N-C9, quinoline), 147.2–128.1 (12C, aro-
matic carbon atoms), 104.9 (1C, C≡N), 99.4 (1C, -C-C≡N),
48.6, 44.6 (4C, piperazine), 23.1 (1C, COCH3).
165.9 (1C, C-4, s-triazine, C-O-C at quinoline linkage),
163.8 (1C, C-2, s-triazine, C-NH at benzonitrile moiety),
161.2, 159.5, 157.6, 152.4 (5C, 2C of -C2H=N-C9 or 1C of
-C2H=N-C9 of Quinoline and 3C of pyrimidyl ring), 148.4–
120.1 (13C, aromatic carbon atoms), 105.4 (1C, C≡N),
98.7 (1C, -C-C≡N), 48.7, 42.1 (4C, piperazine).
5i M.P. 256–257°C, yield 75% (found: C 57.11, H 4.56,
N 20.61, C26H25BrN8O, calc: C 57.25, H 4.62, N 20.54%).
IR (KBr) cm−1, 3282 (-NH), 2221 (C≡N), 1399 (isopro-
pyl), 1255 (C-O-C), 836 (s-triazine C-N str.); 1H NMR
(400 MHz, DMSO-d6, δ) ppm, 9.23 (s, 1H, -NH, s-triazine
to amino-benzonitrile linkage), 8.84 (d, J= 1.8, 1H, C5
proton of quinoline), 8.61 (d, J= 8.6 Hz, 1H, -N=CH-, C2
proton of quinoline), 8.51 (s, 1H, C3 proton of quinoline),
8.34 (d, J= 7.9 Hz, 1H, C8 proton of quinoline), 8.04 (dd,
J= 7.6, 1.4 Hz, 1H), 7.51–7.36 (m, 4H, Ar-H), 3.88 (br s,
4H, piperazine), 3.44 (br s, 4H, piperazine), 2.81–2.83
(m, 1H, N-CH), 1.96 (d, J= 6.9 Hz, 6H, -2CH3); 13C-NMR
(100 MHz, DMSO-d6, δ) ppm, 177.2 (1C, C-6, s-triazine,
C-N at piperazine linkage), 166.4 (1C, C-4, s-triazine,
C-O-C at quinoline linkage), 164.3 (1C, C-2, s-triazine,
C-NH at benzonitrile moiety), 152.7, 148.9 (2C, 1C of
-C2H=N-C9 and 1C of -C2H=N-C9, quinoline), 146.4–119.9
(12C, aromatic carbon atoms), 106.2 (1C, C≡N), 96.7 (1C,
-C-C≡N), 53.2, 50.3, 39.2 (5C, 4C of piperazine and 1C of
N-CH), 23.1 (2C, 2CH3).
5j M.P. 254–256°C, yield 72% (found: C 57.85, H 3.69,
N 21.83, C28H22BrN9O, calc: C 57.94, H 3.82, N 21.72%). IR
(KBr) cm−1, 3309 (-NH), 2218 (C≡N), 1252 (C-O-C), 831
(s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.19 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.82 (d, J= 1.9, 1H, C5 proton of quinoline), 8.69
(d, J= 8.2 Hz, 1H, -N=CH-, C2 proton of quinoline), 8.47 (s,
1H, C3 proton of quinoline), 8.25 (d, J= 7.5 Hz, 1H, C8 pro-
ton of quinoline), 8.14 (dd, J= 7.6, 1.4 Hz, 1H), 8.02 (dd,
J= 7.3, 1.8 Hz, 1H, pyridyl), 7.53–7.34 (m, 7H, Ar-H), 3.83
(br s, 4H, piperazine), 3.46 (br s, 4H, piperazine); 13C-NMR
(100 MHz, DMSO-d6, δ) ppm, 175.4 (1C, C-6, s-triazine,
C-N at piperazine linkage), 165.8 (1C, C-4, s-triazine,
C-O-C at quinoline linkage), 164.2 (1C, C-2, s-triazine,
C-NH at benzonitrile moiety), 158.8, 158.1, 154.7, 151.1
(4C, 2C of -C2H=N-C9 or 1C of -C2H=N-C9 of Quinoline and
2C of Npip-C-Npy-CH or 1C of Npip-C-Npy-CH), 147.2–117.7
(15C, aromatic carbon atoms), 104.9 (1C, C≡N), 97.2 (1C,
-C-C≡N), 52.2, 48.6 (4C, piperazine).
5l M.P. 255–257°C, yield 74% (found: C 60.58, H 4.28,
N 18.74, C30H25BrN8O, calc: C 60.71, H 4.25, N 18.88%). IR
(KBr) cm−1, 3290 (-NH), 2221 (C≡N), 1257 (C-O-C), 833
(s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.17 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.82 (d, J= 1.7, 1H, C5 proton of quinoline), 8.69
(d, J= 8.2 Hz, 1H, -N=CH-, C2 proton of quinoline), 8.48 (s,
1H, C3 proton of quinoline), 8.29 (d, J= 7.6 Hz, 1H, C8 pro-
ton of quinoline), 8.13 (dd, J= 7.8, 1.5 Hz, 1H), 7.51–7.29
(m, 9H, Ar-H), 3.84 (br s, 4H, piperazine), 3.51 (br s, 4H,
piperazine), 2.72 (s, 2H, Npip-CH2); 13C-NMR (100 MHz,
DMSO-d6, δ) ppm, 177.1 (1C, C-6, s-triazine, C-N at
piperazine linkage), 166.9 (1C, C-4, s-triazine, C-O-C at
quinoline linkage), 164.1 (1C, C-2, s-triazine, C-NH at
benzonitrile moiety), 153.1, 148.6 (2C, 1C of -C2H=N-C9
and 1C of -C2H=N-C9, quinoline), 145.1–119.7 (18C, aro-
matic carbon atoms), 105.7 (1C, C≡N), 97.9 (1C, -C-C≡N),
65.3 (1C, N-CH2), 49.1, 46.2 (4C, piperazine).
5m M.P. 239–241°C, yield 75% (found: C 62.80, H 4.53,
N 16.67, C31H26BrN7O, calc: C 62.84, H 4.42, N 16.55%). IR
(KBr) cm−1, 3296 (-NH), 2221 (C≡N), 1247 (C-O-C), 832
(s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.26 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.79 (d, J= 1.8, 1H, C5 proton of quinoline), 8.61
(d, J= 8.4 Hz, 1H, -N=CH-, C2 proton of quinoline), 8.53 (s,
1H, C3 proton of quinoline), 8.26 (d, J= 7.5 Hz, 1H, C8 pro-
ton of quinoline), 8.05 (dd, J= 7.6, 1.4 Hz, 1H), 7.63–7.36
(m, 9H, Ar-H), 3.88 (4H, t, J= 6.8 Hz, piperidine), 3.67 (4H,
t, J= 8.8 Hz, piperidine), 2.43 (2H, s, -CH2), 1.83 (1H, t,
J= 7.4 Hz, -CH, piperidine); 13C-NMR (100 MHz, DMSO-
d6, δ) ppm, 176.5 (1C, C-6, s-triazine, C-N at piperidine
linkage), 166.4 (1C, C-4, s-triazine, C-O-C at quinoline
linkage), 163.9 (1C, C-2, s-triazine, C-NH at benzonitrile
moiety), 151.7, 150.3 (2C, 1C of -C2H=N-C9 and 1C of
-C2H=N-C9, quinoline), 145.9–117.8 (18C, aromatic car-
bon atoms), 104.9 (1C, C≡N), 98.4 (1C, -C-C≡N), 46.3,
40.2, 36.1, 29.9 (6C, 5C of piperidine and 1C of -CH2).
5n M.P. 284–285°C, yield 74% (found: C 58.91, H 4.43,
N 18.36, C26H24BrN7O, calc: C 58.87, H 4.56, N 18.48%). IR
(KBr) cm−1, 3299 (-NH), 2221 (C≡N), 1258 (C-O-C), 828
(s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.19 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.83 (d, J= 1.7, 1H, C5 proton of quinoline), 8.63
(d, J= 8.0 Hz, 1H, -N=CH-, C2 proton of quinoline), 8.50 (s,
1H, C3 proton of quinoline), 8.25 (d, J= 7.7 Hz, 1H, C8 pro-
ton of quinoline), 8.10 (dd, J= 7.6, 1.4 Hz, 1H), 7.47–7.37
(m, 4H, Ar-H), 3.69 (dd, J= 11.9, 7.2 Hz, 2H, piperidine),
2.91 (dd, J= 12.0, 7.7 Hz, 2H, piperidine), 1.79–1.71 (m,
4H, piperidne), 1.41 (d, J= 6.5 Hz, 6H, 2CH3); 13C-NMR
(100 MHz, DMSO-d6, δ) ppm, 175.8 (1C, C-6, s-triazine,
C-N at piperidine linkage), 167.1 (1C, C-4, s-triazine,
C-O-C at quinoline linkage), 164.4 (1C, C-2, s-triazine,
5k M.P. 267–268°C, yield 76% (found: C 55.62, H 3.56,
N 23.95, C27H21BrN10O, calc: C 55.77, H 3.64, N 24.09%).
IR (KBr) cm−1, 3294 (-NH), 2220 (C≡N), 1257 (C-O-C),
840 (s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.22 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.84 (d, J= 2.0, 1H, C5 proton of quinoline), 8.62
(d, J= 8.2 Hz, 1H, -N=CH-, C2 proton of quinoline), 8.51 (s,
1H, C3 proton of quinoline), 8.47–8.43 (m, 2H, pyrimidyl),
8.22 (d, J= 7.5 Hz, 1H, C8 proton of quinoline), 8.19 (dd,
J= 7.7, 1.5 Hz, 1H), 7.47–7.35 (m, 4H, Ar-H), 6.79 (t, J= 6.8
Hz, 1H, pyrimidyl), 3.79 (br s, 4H, piperazine), 3.42 (br s,
4H, piperazine); 13C-NMR (100 MHz, DMSO-d6, δ) ppm,
176.9 (1C, C-6, s-triazine, C-N at piperazine linkage),
Journal of Enzyme Inhibition and Medicinal Chemistry

s-Triazines as antibacterial agents 375
C-NH at benzonitrile moiety), 153.8, 149.5 (2C, 1C of
-C2H=N-C9 and 1C of -C2H=N-C9, quinoline), 147.0–121.4
(12C, aromatic carbon atoms), 105.9 (1C, C≡N), 96.5
(1C, -C-C≡N), 52.1, 36.6, 31.9 (5C, piperidine), 22.7 (2C,
2CH3).
of -C2H=N-C9 and 1C of -C2H=N-C9, quinoline and 1C of
C-F), 146.2–119.5 (17C, aromatic carbon atoms), 104.9
(1C, C≡N), 97.2 (1C, -C-C≡N), 51.1, 42.5 (4C, piperazine);
19F NMR (400 MHz, CDCl3, δ) ppm, -122.1 (1F, s, C-F).
5r M.P. 272–273°C, yield 79% (Found: C 58.22, H 3.61,
N 18.70, C29H22BrFN8O, calc: C 58.30, H 3.71, N 18.76%).
IR (KBr) cm−1, 3296 (-NH), 2223 (C≡N), 1258 (C-O-C),
832 (s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.20 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.77 (d, J = 2.0, 1H, C5 proton of quinoline), 8.63
(d, J = 8.5 Hz, 1H, -N=CH-, C2 proton of quinoline), 8.53
(s, 1H, C3 proton of quinoline), 8.26 (d, J = 7.5 Hz, 1H,
C8 proton of quinoline), 8.13 (dd, J = 7.6, 1.3 Hz, 1H),
7.57–7.31 (m, 11H, Ar-H), 7.19 (dd, J = 12.8, 7.7 Hz, 2H),
6.68–6.66 (m, 1H), 6.59 (dd, J = 12.7, 6.1 Hz, 1H), 3.89 (br
s, 4H, piperazine), 3.50 (br s, 4H, piperazine); 13C-NMR
(100 MHz, DMSO-d6, δ) ppm, 177.6 (1C, C-6, s-triazine,
C-N at piperazine linkage), 165.9 (1C, C-4, s-triazine,
C-O-C at quinoline linkage), 163.3 (1C, C-2, s-triazine,
C-NH at benzonitrile moiety), 152.2, 150.4, 147.1 (3C,
2C of -C2H=N-C9 and 1C of -C2H=N-C9, quinoline and
1C of C-F), 144.7–120.1 (17C, aromatic carbon atoms),
104.9 (1C, C≡N), 96.8 (1C, -C-C≡N), 47.7, 43.9 (4C, pip-
erazine); 19F NMR (400 MHz, CDCl3, δ) ppm, -117.9 (1F,
s, C-F).
5o M.P. 286–288°C, yield 72% (found: C 64.67, H 4.25,
N 16.68, C36H29BrN8O, calc: C 64.58, H 4.37, N 16.74%). IR
(KBr) cm−1, 3307 (-NH), 2218 (C≡N), 1255 (C-O-C), 836
1
(s-triazine C-N str.); H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.24 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.85 (d, J = 1.8, 1H, C5 proton of quinoline),
8.62 (d, J = 8.3 Hz, 1H, -N=CH-, C2 proton of quinoline),
8.51 (s, 1H, C3 proton of quinoline), 8.33 (d, J = 7.6 Hz,
1H, C8 proton of quinoline), 8.15 (dd, J = 7.7, 1.4 Hz, 1H),
7.62–7.28 (m, 14H, Ar-H), 4.41 (s, 1H, N-CH), 3.83 (br
s, 4H, piperazine), 3.49 (br s, 4H, piperazine); 13C-NMR
(100 MHz, DMSO-d6, δ) ppm, 177.3 (1C, C-6, s-triazine,
C-N at piperazine linkage), 166.6 (1C, C-4, s-triazine,
C-O-C at quinoline linkage), 164.4 (1C, C-2, s-triazine,
C-NH at benzonitrile moiety), 152.6, 150.2 (2C, 1C of
-C2H=N-C9 and 1C of -C2H=N-C9, quinoline), 146.5–
116.9 (24C, aromatic carbon atoms), 105.6 (1C, C≡N),
97.1 (1C, -C-C≡N), 76.3 (1C, -Npip-CH), 49.5, 46.1 (4C,
piperazine).
5p M.P. 274–275°C, yield 74% (found: C 61.34, H 4.14,
N 15.83, C36H28BrClN8O, calc: C 61.42, H 4.01, N 15.92%).
IR (KBr) cm−1, 3294 (-NH), 2224 (C≡N), 1254 (C-O-C),
839 (s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.12 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.84 (d, J= 1.9, 1H, C5 proton of quinoline),
8.59 (d, J= 8.3 Hz, 1H, -N=CH-, C2 proton of quinoline),
8.47 (s, 1H, C3 proton of quinoline), 8.24 (d, J= 7.5 Hz,
1H, C8 proton of quinoline), 8.04 (dd, J= 7.5, 1.5 Hz, 1H),
7.59–7.25 (m, 13H, Ar-H), 4.02 (s, 1H, N-CH), 3.81 (br s,
4H, piperazine), 3.52 (br s, 4H, piperazine); 13C-NMR
(100 MHz, DMSO-d6, δ) ppm, 176.4 (1C, C-6, s-triazine,
C-N at piperazine linkage), 166.9 (1C, C-4, s-triazine,
C-O-C at quinoline linkage), 164.1 (1C, C-2, s-triazine,
C-NH at benzonitrile moiety), 155.1, 150.1 (2C, 1C of
-C2H=N-C9 and 1C of -C2H=N-C9, quinoline), 148.2–118.4
(24C, aromatic carbon atoms), 106.2 (1C, C≡N), 98.3 (1C,
-C-C≡N), 75.6 (1C, -Npip-CH), 47.8, 44.2 (4C, piperazine).
5q M.P. 235–237°C, yield 81% (found: C 58.18, H 3.63,
N 18.64, C29H22BrFN8O, calc: C 58.30, H 3.71, N 18.76%).
IR (KBr) cm−1, 3288 (-NH), 2221 (C≡N), 1253 (C-O-C),
834 (s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.26 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.74 (d, J= 1.7, 1H, C5 proton of quinoline),
8.61 (d, J= 8.5 Hz, 1H, -N=CH-, C2 proton of quinoline),
8.45 (s, 1H, C3 proton of quinoline), 8.23 (d, J= 7.6 Hz,
1H, C8 proton of quinoline), 8.06 (dd, J= 7.7, 1.3 Hz, 1H),
7.51–7.32 (m, 11H, Ar-H), 6.95 (dd, J= 12.5, 7.2 Hz, 2H),
6.77–6.73 (m, 1H), 6.52 (dd, J= 12.7, 6.9 Hz, 1H), 3.85 (br
s, 4H, piperazine), 3.40 (br s, 4H, piperazine); 13C-NMR
(100 MHz, DMSO-d6, δ) ppm, 177.1 (1C, C-6, s-triazine,
C-N at piperazine linkage), 165.8 (1C, C-4, s-triazine,
C-O-C at quinoline linkage), 164.3 (1C, C-2, s-triazine,
C-NH at benzonitrile moiety), 151.6, 150.4, 148.4 (3C, 2C
5s M.P. 294–295°C, yield 76% (found: C 55.72, H 3.31,
N 17.19, C30H22BrF3N8O, calc: C 55.65, H 3.42, N 17.31%).
IR (KBr) cm−1, 3307 (-NH), 2223 (C≡N), 1256 (C-O-C),
837 (s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.21 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.81 (d, J = 1.8, 1H, C5 proton of quinoline), 8.63
(d, J = 8.2 Hz, 1H, -N=CH-, C2 proton of quinoline), 8.46
(s, 1H, C3 proton of quinoline), 8.27 (d, J = 7.5 Hz, 1H,
C8 proton of quinoline), 8.13 (dd, J = 7.6, 1.4 Hz, 1H),
7.83 (t, J = 7.9 Hz, 1H), 7.56–7.31 (m, 7H, Ar-H), 3.82 (br
s, 4H, piperazine), 3.42 (br s, 4H, piperazine); 13C-NMR
(100 MHz, DMSO-d6, δ) ppm, 175.5 (1C, C-6, s-triazine,
C-N at piperazine linkage), 167.1 (1C, C-4, s-triazine,
C-O-C at quinoline linkage), 164.2 (1C, C-2, s-triazine,
C-NH at benzonitrile moiety), 151.8, 150.3 (2C, 1C of
-C2H=N-C9 and 1C of -C2H=N-C9, quinoline), 147.2–
117.7 (19C, aromatic carbon atoms C-CF3 at 130.3 & CF3
at 125.6), 105.8 (1C, C≡N), 95.2 (1C, -C-C≡N), 49.1, 46.4
(4C, piperazine); 19F NMR (400 MHz, CDCl3, δ) ppm,
-63.7 (3F, s, -CF3).
5t M.P. 294–295°C, yield 72% (found: C 58.06, H 4.69,
N 16.32, C33H31BrN8O4, calc: C 57.98, H 4.57, N 16.39%).
IR (KBr) cm−1, 3291 (-NH), 2222 (C≡N), 1490 (-CH2), 1248
(C-O-C), 839 (s-triazine C-N str.); 1H NMR (400 MHz,
DMSO-d6, δ) ppm, 9.16 (s, 1H, -NH, s-triazine to amino-
benzonitrile linkage), 8.77 (d, J= 1.8, 1H, C5 proton of
quinoline), 8.68 (d, J= 8.3 Hz, 1H, -N=CH-, C2 proton of
quinoline), 8.46 (s, 1H, C3 proton of quinoline), 8.33 (d,
J= 7.6 Hz, 1H, C8 proton of quinoline), 8.14 (dd, J= 7.6,
1.4 Hz, 1H), 7.53–7.33 (m, 6H, Ar-H), 6.86 (d, J= 7.5 Hz,
1H), 6.69 (d, J= 7.6 Hz, 1H), 3.89 (br s, 4H, piperazine),
3.71 (s, 9H, 3OCH3), 3.50 (br s, 4H, piperazine); 13C-NMR
(100 MHz, DMSO-d6, δ) ppm, 177.1 (1C, C-6, s-triazine,
© 2012 Informa UK, Ltd.

376 R. V. Patel et al.
MIC of the compound was determined by agar streak
dilution method30. A stock solution of the synthesized
compound (100 µg/mL) in dimethyl sulfoxide was pre-
pared and graded quantities of the test compounds were
incorporated in a specified quantity of molten sterile
agar, that is, nutrient agar for evaluation of antibacte-
rial and sabouraud dextrose agar for antifungal activity,
respectively. e 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 suspen-
sion of the respective microorganism of approximately
105 CFU/mL was prepared and applied to plates with
serially diluted compounds with concentrations in the
range of 3.12–100 µg/mL in dimethyl sulfoxide and incu-
bated at (37 1)°C for 24 h (bacteria) or 48 h (fungi). e
lowest concentration of the substance that prevents the
development of visible growth is considered to be the
MIC value.
C-N at piperazine linkage), 166.1 (1C, C-4, s-triazine,
C-O-C at quinoline linkage), 164.6 (1C, C-2, s-triazine,
C-NH at benzonitrile moiety), 154.2, 149.5 (2C, 1C of
-C2H=N-C9 and 1C of -C2H=N-C9, quinoline), 147.2–116.3
(18C, aromatic carbon atoms), 106.0 (1C, C≡N), 98.4 (1C,
-C-C≡N), 66.8, 63.1, 54.4 (4C, 3C of 3OCH3 and 1C of Npip
-
CH2), 47.9, 41.8 (4C, piperazine).
5u M.P. 258–260°C, yield 78% (found: C 58.98, H 4.17,
N 18.46, C30H25BrN8O2, calc: C 59.12, H 4.13, N 18.39%).
IR (KBr) cm−1, 3293 (-NH), 2224 (C≡N), 1254 (C-O-C),
827 (s-triazine C-N str.); 1H NMR (400 MHz, DMSO-d6, δ)
ppm, 9.22 (s, 1H, -NH, s-triazine to amino-benzonitrile
linkage), 8.80 (d, J= 1.7, 1H, C5 proton of quinoline), 8.67
(d, J= 8.1 Hz, 1H, -N=CH-, C2 proton of quinoline), 8.49 (s,
1H, C3 proton of quinoline), 8.21 (d, J= 7.3 Hz, 1H, C8 pro-
ton of quinoline), 8.15 (dd, J= 7.6, 1.4 Hz, 1H), 7.51–7.35
(m, 6H, Ar-H), 7.23 (d, J= 8.3 Hz, 1H), 6.65 (d, J= 7.2 Hz,
1H), 4.21 (s, 3H, OCH3), 3.79 (br s, 4H, piperazine ring),
3.49 (br s, 4H, piperazine ring); 13C-NMR (100 MHz,
DMSO-d6, δ) ppm, 176.6 (1C, C-6, s-triazine, C-N at
piperazine linkage), 167.2 (1C, C-4, s-triazine, C-O-C at
quinoline linkage), 163.3 (1C, C-2, s-triazine, C-NH at
benzonitrile moiety), 155.0, 152.1 (2C, 1C of -C2H=N-C9
and 1C of -C2H=N-C9, quinoline), 149.0–119.8 (18C, aro-
matic carbon atoms), 105.5 (1C, C≡N), 97.9 (1C, -C-C≡N),
57.2 (1C, -OCH3), 46.2, 44.8 (4C, piperazine).
In vitro evaluation of antimycobacterial activity
e preliminary antimycobacterial assessment for the
final synthesized compounds was carried out using
BACTEC MGIT method. e Mycobacterial Growth
Indicator Tubes (MGIT) containing 4 ml of modified
Middle brook 7H9 Broth Base were numbered as per
the title compounds to be tasted for antimycobacterial
efficacy by means of various concentrations prepared.
e 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 H37
RV M. tuberculosis strain was added in the medium to be
incubated and 0.1 ml of egg-based medium (L. J.) was
also added. e MGIT tubes were then tightly recapped,
mixed well and incubated into BACTEC MGIT instru-
ment at (37 1)°C until positivity is observed. e read-
ings 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 unin-
oculated tube (negative control). Bright fluorescence
detected by the corresponding MGIT tube was noticed
in the form of bright orange colour in the bottom of the
tube and also an orange reflection on the meniscus.31
e primary screening was conducted at concentra-
tion of 6.25 µg/ml against M. tuberculosis H37 Rv 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 Lowenstein-Jensen MIC method. e MIC was
defined as the lowest concentration inhibiting 99% of
the inoculum.
In vitro evaluation of antimicrobial activity
e synthesized s-triazinyl derivatives 5a-u were exam-
ined for antimicrobial activity against several bacte-
ria (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) species using paper disc diffu-
sion technique29. e Mueller-Hinton agar media were
sterilized (autoclaved at 120°C for 30 min), poured at
uniform depth of 5 mm and allowed to solidify. e
microbial suspension (105 CFU/mL; 0.5 McFarland
Nephelometery Standards) was streaked over the sur-
face of media using a sterile cotton swab to ensure
even growth of the organisms. e tested compounds
were dissolved in dimethyl sulfoxide to give solutions
of 3.12–100 µg/mL. Sterile filter paper discs measuring
6.25 mm in diameter (Whatman no. 1 filter paper), pre-
viously soaked in a known concentration of the respec-
tive 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)°C. A control
disc impregnated with an equivalent amount of dim-
ethyl sulfoxide without any sample was also used and
did not produce any inhibition. Ciprofloxacin and keto-
conazole (100 µg/disc) were used as control drugs for
antibacterial and antifungal activity, respectively.
e secondary antimycobacterial screening for test
compounds was obtained for M. tuberculosis H37 Rv,
by using L. J. (Lowenstein and Jensen) MIC method32,33
.
Stock solutions of primary 1000, 500, 250 and secondary
200, 100, 62.5, 50, 25, 12.5, 6.25, 3.25 µg/mL dilutions of
Journal of Enzyme Inhibition and Medicinal Chemistry

s-Triazines as antibacterial agents 377
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. tuber-
culosis H37 Rv growing on L. J. medium was harvested
in 0.85% saline in bijou bottles. ese tubes were then
incubated at 37°C for 24 h followed by streaking of M.
tuberculosis H37 Rv (5 × 104 bacilli per tube). ese tubes
were then incubated at (37 1)°C. Growth of bacilli was
seen after 12 days, 22 days and finally 28 days of incu-
bation. Tubes having the compounds were compared
with control tubes where medium alone was incubated
with M. tuberculosis H37 Rv. e concentration at which
no development of colonies occurred or <20 colonies
was taken as MIC concentration of test compound. e
standard strain M. tuberculosis H37 Rv was tested with
known drugs rifampicin, isoniazid, ethambutol and
pyrazinamide.
observed for compounds 5d, 5p, 5s, 5t and 5u towards A.
fumigatus. Compounds 5h, 5n, 5s, 5t and 5u act as the
most potent inhibitors of the growth of A. clavatus fungi.
Compounds 5q, 5r, 5s, 5t and 5u possessed the highest
antifungal activity against C. albicans. All the remain-
ing final s-triazine derivatives exerted good to moderate
activity.
In vitro antimycobacterial activities of compounds
5a-u were assessed against M. tuberculosis H37Rv strain.
e preliminary results observed from BACTEC MGIT
method furnished in Table 3 indicated that final s-triaz-
ines 5n, 5t and 5u exhibited highest inhibition (99%) at
a constant concentration level (6.25 μg/ml) against M.
tuberculosis H37Rv. e secondary biological screening
was performed using Lowenstein-Jensen MIC method
and it is worthwhile to note that 5t was the only com-
pound displaying inhibition of M. tuberculosis H37Rv
completely (99%) at the MIC of 3.12 μg/mL. Compounds
5p and 5s appeared with good inhibition effect in the
term of MIC at 12.5 µg/mL, while all the remaining
derivatives were found to exert MIC values ranging from
25–1000 µg/mL.
In conclusion, it is clear that the introduction of
appropriate substituent on the s-triazine ring would
lead to the more bioactive compounds. Bioassay results
revealed that nine compounds (5d, 5h, 5n, 5p, 5q, 5r,
5s, 5t and 5u) out of the 21 studied displayed variable
in vitro antibacterial, antifungal and antimycobacterial
inhibitory effects. e presence of 6-bromo-4-hydroxy-
quinoline heteroatom ring in the title compounds signif-
icantly increase the biological potency of the resultant
compounds compared to the presence of 8-hydroxyqui-
noline in our previous study. 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. erefore, it is concluded that
there exists ample scope for further study in this class of
compounds in order to discover varied biological pro-
files such as anticancer activity or anti-HIV activity. e
Results and discussion
Investigation on antibacterial screening data (Table 1)
showed some of the compounds were active against all
the mentioned bacteria. Final s-triazinyl compounds 5h,
5n, 5p, 5s, 5t and 5u have the highest ability to inhibit S.
aureus. Final analogues 5d, 5h, 5n, 5p, 5r, 5s, 5t showed
highereffectivenessagainstB. cereus. ebestactivitywas
observed with compounds 5h, 5n, 5s, 5t and 5u against E.
coli and P. aeruginosa. Inhibition of K. pneumoniae was
also noted for s-triazine derivatives 5d, 5p, 5q, 5s and 5t.
Compounds 5q, 5s and 5t had an effective action against
S. typhi along with similar MIC profile of compounds
5h, 5n and 5p. Compounds 5d, 5h, 5q, 5s, 5t and 5u were
superior in inhibiting the growth of P. vulgaris. Strong
inhibitory effect was shown by final derivatives 5d, 5h,
5n, 5s and 5t against S. flexneria. All the remaining final
s-triazine derivatives exerted good to moderate activity.
e antifungal results data (Table 2) revealed that,
the synthesized compounds showed variable degree
of inhibition against the tested fungi. Final s-triazine
derivatives 5q, 5r, 5s and 5t displayed good antigrowth
activity against A. niger. A significant inhibition was also
Table 1. In vitro antimicrobial activity of newly synthesized compounds.
MIC (µg/mL)
Gram-positive
Gram-negative
Entry
S. aureus
>6.25
6.25
B. cereus
6.25
6.25
6.25
6.25
>6.25
6.25
6.25
6.25
>6.25
1.0
E. coli
>12.5
12.5
12.5
>12.5
>12.5
>12.5
12.5
12.5
12.5
1.0
P. aeruginosa
>12.5
12.5
K. pneumoniae
S. typhi
>25
25
P. vulgaris S. flexneria
5d
25
>25
>25
25
6.25
6.25
>6.25
>6.25
6.25
>6.25
6.25
6.25
6.25
1.0
12.5
12.5
12.5
>12.5
>12.5
>12.5
12.5
12.5
>12.5
≤3
5h
5n
6.25
12.5
25
5p
6.25
>12.5
>12.5
>12.5
12.5
25
5q
>6.25
>6.25
6.25
25
25
5r
>25
25
>25
25
5s
5t
6.25
12.5
25
25
5u
6.25
12.5
>25
1.0
—
>25
1.0
—
Ciprofloxacin
DMSO
1.0
1.0
—
—
—
—
—
—
© 2012 Informa UK, Ltd.

378 R. V. Patel et al.
for all the newly synthesized compounds are included
within the supplementary information.
Table 2. In vitro antifungal activity of newly synthesized
compounds.
MIC (µg/mL)
Entry
A. niger A. fumigatus A. clavatus C. albicans
References
5d
>12.5
>12.5
>12.5
>12.5
12.5
12.5
12.5
12.5
>12.5
≤3
25
>25
>25
25
>25
25
>25
>25
>25
>25
25
5h
1. Nathan C. Antibiotics at the crossroads. Nature 2004;431:899–902.
2. Raviglione MC. e TB epidemic from 1992 to 2002. Tuberculosis
(Edinb) 2003;83:4–14.
3. NIAID. http://www3.niaid.nih.gov/topics/tuberculosis/.
4. TAACF. http://www.taacf.org/about-TB-background.htm.
5. World Health Organization, Global tuberculosis control: A short
update to the 2009 report. http://www.who.int/tb/publications/
global_report/2009/update/en/index.html.
6. MahajanDH,PannecouqueC,DeClercqE,ChikhaliaKH.Synthesis
and studies of new 2-(coumarin-4-yloxy)-4,6-(substituted)-S-
triazine derivatives as potential anti-HIV agents. Arch Pharm
(Weinheim) 2009;342:281–290.
5n
25
5p
>25
>25
>25
25
5q
>25
>25
25
5r
25
5s
25
5t
25
25
25
5u
25
25
25
Ketoconazole
DMSO
1.0
—
1.0
—
1.0
—
—
7. Chikhalia KH, Patel MJ. Design, synthesis and evaluation of some
1,3,5-triazinyl urea and thiourea derivatives as antimicrobial
agents. J Enzyme Inhib Med Chem 2009;24:960–966.
Table 3. In vitro antimycobacterial activity of newly synthesized
compounds.
8. Patel DH, Chikhalia KH, Shah NK, Patel DP, Kaswala PB, Buha
VM. Synthesis and antimicrobial studies of s-triazine based
heterocycles. J Enzyme Inhib Med Chem 2010;25:121–125.
9. Zhou C, Min J, Liu Z, Young A, Deshazer H, Gao T et al. Synthesis
and biological evaluation of novel 1,3,5-triazine derivatives as
antimicrobial agents. Bioorg Med Chem Lett 2008;18:1308–1311.
10. Srinivas K, Srinivas U, Bhanuprakash K, Harakishore K, Murthy US,
Rao VJ. Synthesis and antibacterial activity of various substituted
s-triazines. Eur J Med Chem 2006;41:1240–1246.
11. Baliani A, Bueno GJ, Stewart ML, Yardley V, Brun R, Barrett
MP, Gilbert IH. Synthesis and antibacterial activity of various
substituted s-triazines. Eur J Med Chem 2006;41:1240–1246.
12. Menicagli R, Samaritani S, Signore G, Vaglini F, Dalla Via L. In vitro
cytotoxic activities of 2-alkyl-4,6-diheteroalkyl-1,3,5-triazines: new
molecules in anticancer research. J Med Chem 2004;47:4649–4652.
13. Garaj V, Puccetti L, Fasolis G, Winum JY, Montero JL, Scozzafava
BACTEC MGIT methoda
L. J MIC methoda
MIC
(µg/mL) inhibition
MIC
%
%
Entry
(µg/mL)
inhibition
5d
>6.25
>6.25
6.25
—
—
25
98
98
98
99
99
99
99
99
99
5h
62.5
6.25
12.5
50
5n
99
5p
>6.25
>6.25
>6.25
>6.25
6.25
—
5q
—
5r
—
25
5s
—
12.5
3.12
6.25
5t
99
5u
6.25
99
Isoniazid
Refampicin
Ethambutol
Pyrazinamide
0.20
99%
99%
99%
99%
0.25
A
et al. Carbonic anhydrase inhibitors: novel sulfonamides
3.12
incorporating 1,3,5-triazine moieties as inhibitors of the cytosolic
and tumour-associated carbonic anhydrase isozymes I, II and IX.
Bioorg Med Chem Lett 2005;15:3102–3108.
6.25
a: each value is the mean of two independent experiments.
14. Melato S, Prosperi D, Coghi P, Basilico N, Monti D. A combinatorial
approach to 2,4,6-trisubstituted triazines with potent antimalarial
activity: combining conventional synthesis and microwave-
assistance. Chemmedchem 2008;3:873–876.
study is currently under investigation and the results will
be published in due course.
15. Xiong YZ, Chen FE, Balzarini J, De Clercq E, Pannecouque C.
Non-nucleoside HIV-1 reverse transcriptase inhibitors. Part 11:
structural modulations of diaryltriazines with potent anti-HIV
activity. Eur J Med Chem 2008;43:1230–1236.
16. Kerns RJ, Rybak MJ, Kaatz GW, Vaka F, Cha R, Grucz RG et al.
Structural features of piperazinyl-linked ciprofloxacin dimers
requiredforactivityagainstdrug-resistantstrainsofStaphylococcus
aureus. Bioorg Med Chem Lett 2003;13:2109–2112.
17. Singh KK, Joshi SC, Mathela CS. Synthesis and in-vitro antibacterial
activity of N-alkyl and N-aryl piperazine derivatives. Ind J Chem
2011;50(B):196–200.
18. Upadhayaya RS, Sinha N, Jain S, Kishore N, Chandra R, Arora SK.
Optically active antifungal azoles: synthesis and antifungal activity
of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-
ethyl]-tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol. Bioorg
Med Chem 2004;12:2225–2238.
acknowledgments
e authors are thankful to Applied Chemistry
Department of S. V. National Institute of Technology,
Surat for the scholarship, encouragement and facilities.
e authors wish to offer their deep gratitude to Microcare
Laboratory, Surat, India for carrying out the biological
screenings and Centre of Excellence, Vapi, India for car-
rying out 1H NMR, 13C NMR and 19F NMR analysis.
Declaration of interest
e authors report no conflict of interest.
19. Upadhayaya RS, Vandavasi JK, Kardile RA, Lahore SV, Dixit
SS, Deokar HS et al. Novel quinoline and naphthalene
derivatives as potent antimycobacterial agents. Eur J Med Chem
2010;45:1854–1867.
supplementary material
20. Kumar CS, Vinaya K, Chandra JN, immegowda NR, Prasad
SB, Sadashiva CT et al. Synthesis and antimicrobial studies of
novel 1-benzhydryl-piperazine sulfonamide and carboxamide
derivatives. J Enzyme Inhib Med Chem 2008;23:462–469.
Detailed discussion on the analytical and biological char-
acterization is included within the supplementary infor-
mation. Tables containing zone of inhibition and MICs
Journal of Enzyme Inhibition and Medicinal Chemistry

s-Triazines as antibacterial agents 379
21. Sunduru N, Gupta L, Chaturvedi V, Dwivedi R, Sinha S, Chauhan
PM. Discovery of new 1,3,5-triazine scaffolds with potent activity
against Mycobacterium tuberculosis H37Rv. Eur J Med Chem
2010;45:3335–3345.
22. Andries K, Verhasselt P, Guillemont J, Göhlmann HW,
Neefs JM, Winkler H et al. A diarylquinoline drug active on
the ATP synthase of Mycobacterium tuberculosis. Science
2005;307:223–227.
27. Xiong YZ, Chen FE, Balzarini J, De Clercq E, Pannecouque C.
Non-nucleoside HIV-1 reverse transcriptase inhibitors. Part 13:
synthesis of fluorine-containing diaryltriazine derivatives for in
vitro anti-HIV evaluation against wild-type strain. Chem Biodivers
2009;6:561–568.
28. Patel RV, Kumari P, Chikhalia KH. Novel s-triazinyl piperazines:
Design, synthesis, characterization and anti-microbial activity.
Arch Appl Sci Res 2010;2:232–240.
23. Rustomjee R, Diacon AH, Allen J, Venter A, Reddy C, Patientia
RF et al. Early bactericidal activity and pharmacokinetics of the
diarylquinoline TMC207 in treatment of pulmonary tuberculosis.
Antimicrob Agents Chemother 2008;52:2831–2835.
24. Upadhayaya RS, Lahore SV, Sayyed AY, Dixit SS, Shinde PD,
Chattopadhyaya J. Conformationally-constrained indeno[2,1-c]
quinolines—a new class of anti-mycobacterial agents. Org Biomol
Chem 2010;8:2180–2197.
25. Upadhayaya RS, Shinde PD, Sayyed AY, Kadam SA, Bawane AN,
Poddar A et al. Synthesis and structure of azole-fused indeno[2,1-c]
quinolines and their anti-mycobacterial properties. Org Biomol
Chem 2010;8:5661–5673.
29. Gillespie SH. Medical microbiology-Illustrated, Butterworth
Heinemann Ltd., United Kingdom: 1994:234–247.
30. HawkeyPM,LewisDA.Medicalbacteriology—apracticalapproach.
Oxford University Press, United Kingdom: 1994:181–194.
31. Anargyros P, Astill DS, Lim IS. Comparison of improved BACTEC
and Lowenstein-Jensen media for culture of mycobacteria from
clinical specimens. J Clin Microbiol 1990;28:1288–1291.
32. Isenberg HD. Clinical microbiology procedures handbook, vol. 1,
American Society for Microbiology, Washington, D.C. 1992.
33. Patel NB, Khan IH, Rajani SD. Antimycobacterial and antimicrobial
study of new 1,2,4-triazoles with benzothiazoles. Arch Pharm
(Weinheim) 2010;343:692–699.
26. Upadhayaya RS, Shinde PD, Kadam SA, Bawane AN, Sayyed
AY, Kardile RA et al. Synthesis and antimycobacterial activity of
prodrugs of indeno[2,1-c]quinoline derivatives. Eur J Med Chem
2011;46:1306–1324.
34. Dandia A, Arya K, Sati M, Sarawgi P. Green chemical synthesis of
fluorinated 1,3,5-triaryl-s-triazines in aqueous medium under
microwaves as potential antifungal agents.
2004;125:1273–1277.
J Fluorine Chem
© 2012 Informa UK, Ltd.