Facile synthesis of benzonitrile/nicotinonitrile based s-triazines as new potential antimycobacterial agents

Article abstract of DOI:10.1016/j.ejmech.2014.03.085

A common strategy to synthesize 4/6-(4-(4-methylpiperazin-1-yl)-6-(4-(4- oxo-2-phenylthiazolidin-3-yl)phenyl)-1,3,5-triazin-2-yloxy)benzonitriles/ nicotinonitriles was developed by applying an efficient palladium-catalyzed C-C Suzuki coupling. Moreover, the synthesized compounds were also tested for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H ₃₇Rv using BACTEC MGIT and Lowenstein-Jensen MIC methods. Several compounds displayed profound antimycobacterial activity in combination with low toxicity towards mammalian cells. The best results were observed amongst the nicotinonitrile substituted s-triazine analogs and it could be a potential starting point to develop new lead compounds in the fight against M. tuberculosis H₃₇Rv. The newly synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR, MS and elemental analysis.

Full text of DOI:10.1016/j.ejmech.2014.03.085

European Journal of Medicinal Chemistry 79 (2014) 57e65
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Facile synthesis of benzonitrile/nicotinonitrile based s-triazines
as new potential antimycobacterial agents
,
a,
*
Amit B. Patel a, Kishor H. Chikhalia b , Premlata Kumari
*
a Department of Applied Chemistry, S. V. National Institute of Technology, Surat 395007, India
b Department of Chemistry, School of Science, Gujarat University, Ahmedabad 380009, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A common strategy to synthesize 4/6-(4-(4-methylpiperazin-1-yl)-6-(4-(4-oxo-2-phenylthiazolidin-3-
yl)phenyl)-1,3,5-triazin-2-yloxy)benzonitriles/nicotinonitriles was developed by applying an efficient
palladium-catalyzed CeC Suzuki coupling. Moreover, the synthesized compounds were also tested for
their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using BACTEC MGIT and
LowensteineJensen MIC methods. Several compounds displayed profound antimycobacterial activity in
combination with low toxicity towards mammalian cells. The best results were observed amongst the
nicotinonitrile substituted s-triazine analogs and it could be a potential starting point to develop new
lead compounds in the fight against M. tuberculosis H37Rv. The newly synthesized compounds were
characterized by IR, 1H NMR, 13C NMR, MS and elemental analysis.
Received 1 November 2013
Received in revised form
28 March 2014
Accepted 31 March 2014
Available online 1 April 2014
Keywords:
s-Triazine
Thiazolidin-4-one
Palladium-catalyzed
Antimycobacterial activity
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
synthesized, and their pharmacological activity has consequently
been investigated. It has been reported that, s-triazine ring skeleton
In order to minimize the rapid multidrug-resistance in patho-
genic microbes, there is an urgent need to discover and develop
new drugs that acts through a novel mechanism of action [1]. In the
existing circumstances, Tuberculosis (TB) is a serious global public
health problem and a leading cause of death in most of the
developing regions of the world. According to the recent WHO
global TB report, 1.4 million people have been died from TB,
including almost one million deaths among HIV-negative in-
dividuals and 0.43 million HIV-positive people in 2011 [2a]. Current
control efforts are severely hampered due to Mycobacterium
tuberculosis (MTB) being a leading opportunistic infection in pa-
tients with acquired immune deficiency syndrome and the
spreading of multidrug-resistant tuberculosis (MDR-TB) [2b]. Pre-
vention of drug resistance depends on appropriate treatment of TB
patients with combination drug regimens and early detection of
resistance followed by tailored treatment with second line anti-
mycobacterial agents. These resistant strains are alarming because
only a few effective drugs available for the treatment of MDR-TB [3].
Since last two decades, several heterocyclic compounds from
the series of s-triazine and thiazolidin-4-one have been
possess a broad spectrum of biological and pharmaceutical activ-
ities [4e6]. Moreover, thiazolidin-4-one derivatives are also re-
ported to have important biological activities such as anti-
inflammatory [7a], antituberculosis [7b], anticancer [8a,7b], anti-
tumor [8c], anti-HIV [8d], antibacterial [9a], antifungal [9b], anti-
oxidant [9c], antiviral [9d], anticonvulsant [10a], diuretics [10b],
nematicidal [10c], antihistaminic [10d] activity etc. Recently, Pathak
et al. have synthesized substituted thiazolidin-4-one derivatives
(Fig. 1a) and checked for their biological activity. They reported that
most of the synthesized compounds possessed significant anti-
mycobacterial activity (0.35e1 mg/mL) against M. tuberculosis H37Rv
[11]. In our previous study, we introduced various piperazine and
piperidine derivatives to the s-triazine core (Fig. 1b) and identified
the compounds with remarkable antimycobacterial activity [12]. In
our continuous effort towards synthesis of new heterocyclic
bioactive agents, two series of thiazolidin-4-one fused s-triazines
were synthesized by applying an efficient palladium catalyzed CeC
Suzuki coupling using catalyst system Pd(OAC)2, Xphos and K3PO4
as a base in toluene solvent. Synthesized analogs were further
tested for antimycobacterial activity against M. tuberculosis H37Rv
to observe the variation in biological activity. All the active com-
pounds were further analyzed for their cytotoxicity against
mammalian cells from the VERO line.
* Corresponding authors.
E-mail addresses: chikhalia_kh@yahoo.com (K.H. Chikhalia), premlatakumari1@
gmail.com (P. Kumari).
http://dx.doi.org/10.1016/j.ejmech.2014.03.085
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

58
A.B. Patel et al. / European Journal of Medicinal Chemistry 79 (2014) 57e65
Fig. 1. Lead compounds among s-triazine and thiazolidin-4-one derivatives with antimycobacterial activity.
2. Results and discussion
2.2. Pharmacology
2.1. Chemistry
2.2.1. In vitro antimycobacterial activity
The investigation of in vitro antimycobacterial screening
The first step comprised formation of intermediate 2 by the
nucleophilic displacement of one chlorine atom of s-triazine ring by
N-methylpiperazine. The desired coupling agents 3a and 3b were
synthesized by condensation of 4-hydroxybenzonitrile and 6-
hydroxynicotinonitrile with intermediate 2 (Scheme 1). Synthesis
of compound 4a/4b involved the Suzuki cross-coupling reaction to
facilitate the CeC bond formation between the s-triazine ring and
phenyl ring. To optimize the reaction conditions for the synthesis of
benzonitrile incorporated s-triazines, a series of ligands (Fig. 2),
palladium sources, bases and solvents were screened for viability of
coupling approach. The results of reaction optimization are sum-
marized in Table 1.
To ensure the potency of the CeC coupling reaction, we have
preliminary examined the coupling reaction with catalyst system
Pd(OAC)2, Xantphos L1, K3PO4 in DME at reflux temperature. The
desired coupling product was obtained in 69% yield (Table 1, entry
1). The replacement of K3PO4 by K2CO3 decreased the yield drasti-
cally (entry 2). When the reaction was carried out in the presence of
KOtBu or Cs2CO3 instead of K3PO4, moderate yields of 57% and 63%
were obtained respectively (entry 3, 4). Variations on the palladium
source Pd2(dba)3, [PdCl(allyl)]2 and Pd(PPh3)4 gave lower yields,
even with longer reaction times (entries 5e7). However 1,4-dioxane
gave almost similar yield to DME (entry 8). When switching to
toluene, the product was obtained in higher yield (entries 9) as
compared to the polar solvents (entry 10, 11). An assorted set of
additional bidentate phosphine ligands such as BINAP L2, Dppf L4 or
DPEphos L5 and sterically hindered monodentate ligand such as
Xphos L3 were examined under these conditions to determine the
efficacy of ligand in supporting a catalytic system for this trans-
formation. As shown in entry (9,11e15), among all the tested ligands
(L1eL5), Xphos L3 proved to be the most effective with the yield of
82% (entry 13). Interestingly, increasing the catalyst loading from
1.5 mol % Pd(OAC)2 to 3.0 mol % reduced the reaction time from 16 h
to 13 h (entry 16). Finally, compound 3b on Suzuki coupling with 4-
aminophenylboronic acid pinacol ester with similar reaction con-
ditions gave 86% yield (entry 17).
(Table 2) revealed that all the newly synthesized compounds
showed moderate to good inhibition at 3.12e25
tuberculosis H37Rv. The primary screening was conducted at a
concentration of 6.25 g/mL using BACTEC MGIT method only for
mg/mL against M.
m
the first selection of active compounds. The results observed from
BACTEC MGIT method indicated that benzonitrile based s-triazine
derivative 8f and nicotinonitrile based derivative 9d, 9f and 9g
exhibited highest inhibition (99%) at a constant concentration level
(6.25 mg/mL). These compounds were considered as most potent
analogs against mycobacteria and were found to indicate equiva-
lence antituberculosis potency as that of standard drug pyr-
azinamide. However, the results of secondary biological screening
using Lowenstein-Jensen MIC method revealed that nicotinonitrile
based s-triazine analog 9f with hydroxy group to thiazolidin-4-one
showed inhibition against M. tuberculosis H37Rv at 3.12 mg/mL MIC,
equipotent to Ethambutol. In addition, two analogs 9d with fluoro
and 9g with methoxy substituent to thiazolidin-4-one displayed
6.25
exhibit moderate to poor activity at MIC ranging from 12.5 to
100 g/mL. From the above results it can be stated that nic-
mg/mL of MIC. All the remaining derivatives were found to
m
otinonitrile based s-triazine derivatives were more active against
M. tuberculosis H37Rv as compared with the benzonitrile de-
rivatives. A close examination of the structures of the active com-
pounds revealed that, their antimycobacterial activity is also bound
to the nature of the substituent at the para position of the phenyl
ring, linked to the thiazolidin-4-one ring. With respect to the
cytotoxicity (IC50) in VERO cells at different concentrations, most of
the active compounds were found to be non-toxic (IC50 > 50 mg/
mL).
3. Conclusion
In summary, we have developed an efficient catalytic system for
the synthesis of various benzonitrile/nicotinonitrile and thiazoli-
din-4-one incorporated s-triazine analogs by applying palladium-
catalyzed CeC Suzuki coupling using an efficient catalyst system
Pd(OAC)2, Xphos and K3PO4 as base in toluene. Several compounds
have displayed remarkable in vitro antimycobacterial activity (MIC,
Finally, the Schiff base derivatives 6aeg/7aeg were further
synthesized by condensing the intermediates 4a/4b with various
aldehydes 5aeg. Cyclization of these Schiff bases with thioglycolic
acid, gave desired thiazolidin-4-one derivatives 8aeg/9aeg
(Scheme 2). The purity of the desired compounds was checked by
TLC and elemental analysis. Spectral data IR, 1H NMR, 13C NMR and
MS of the newly synthesized analogs 8aeg and 9aeg were in full
agreement with their proposed structures.
3.12e25 mg/mL) against M. tuberculosis H37Rv in combination with
low toxicity towards mammalian cells. The bioassay results also
reveals that, nicotinonitrile ring is essential for bioactivities in most
of the cases. Finally, these compounds represent new structure
scaffolds that could be further optimized for future development of
more potent and selective antimycobacterial agents.

A.B. Patel et al. / European Journal of Medicinal Chemistry 79 (2014) 57e65
59
Scheme 1. Schematic diagram for the synthesis of analogs 4a/4b, Reagents and Conditions: (a) N-methylpiperazine, K2CO3, acetone, 0e5 ꢀC; (b) 4-hydroxybenzonitrile/6-
hydroxynicotinonitrile, K2CO3, acetone, 30 ꢀC; (c) 4eaminophenylboronic acid pinacol ester, Pd(OAC)2, Xphos, K3PO4, toluene, 100 ꢀC.
Fig. 2. Phosphine ligands.
4. Experimental
water, and the resulting precipitate was filtered off [13]. White
solid, Recrystallization from MeCN (Yield 89%), mp 193e195 ꢀC; IR
(KBr, cmꢁ1): 2942, 2889 (CH2), 1642 (C]N), 742 (CeCl). 1H NMR
4.1. Chemistry
(400 MHz, DMSOed6):
d 3.57 (br s, 4H, piperazine), 3.31 (br s, 4H,
All reactions were carried out under a nitrogen atmosphere. Air-
and moisture-sensitive solvents and solutions were transferred via
syringe or stainless steel cannula. All chemicals were purchased
from Sigma Aldrich, Merck and Fluka. Solvents used were of
analytical grade. Anhydrous potassium carbonate was stored in a
nitrogen-filled glovebox, ground and was taken out in small
quantities and stored in a desiccator. All reactions were routinely
checked by TLC. TLC was performed on aluminum-backed silica gel
plates (silica gel 60 F254 grade, Merck DC) with spots visualized by
UV light. Column chromatography was performed on silica gel LC
60A (70e200 micron). Melting points were determined in open
capillaries on a Veego electronic apparatus VMPeD (Veego In-
strument Corporation, Mumbai, India) and are uncorrected. 1H
NMR and 13C NMR spectra were recorded on a Bruker 400 MHz
model spectrometer using DMSO-d6 as a solvent and TMS as in-
ternal standard with 1H resonant frequency of 400 MHz and 13C
resonant frequency of 100 MHz. The 1H NMR, 13C NMR chemical
shifts were reported as parts per million (ppm) downfield from
TMS (Me4Si). The splitting patterns are designated as follows; s,
singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet.
Elemental analyses (C, H, N) were performed using a Heraeus Carlo
Erba 1180 CHN analyzer (Hanau, Germany). The mass spectra were
measured with Waters Micromass Q-Tof Micro instrument (time of
flight (TOF) mass spectrometer). Column chromatography was
performed on 1½ feet (2.5 cm diameter) glass column using silica
gel LC 60A (70e200 micron).
piperazine), 2.29 (s, 3H, CH3). 13C NMR (100 MHz, DMSOed6):
d
172.63 (2C, CtriazineeCl) 168.30 (1C, CtriazineeNpiperazine), 56.15 (2C,
piperazine), 46.76 (2C, Cpiperazine), 40.24 (1C, CH3). MS (ESI), m/z 249
C
[M þ 1]þ. Anal. calcd. for C8H11Cl2N5: C, 38.73; H, 4.47; N, 28.23.
Found C, 39.02; H, 4.39; N, 28.12.
4.1.2. 4-(4-Chloro-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
yloxy)benzonitrile (3a)/6-(4-chloro-6-(4-methylpiperazin-1-yl)-
1,3,5-triazin-2-yloxy)nicotinonitrile (3b)
An ovenedried Schlenk flask was charged with a magnetic stir
bar, intermediate 2 (10 mmol), 4ehydroxybenzonitrile/6ehydrox-
ynicotinonitrile (11 mmol), potassium carbonate (15 mmol) and
anhydrous acetone (35 mL). The reaction slurry was stirred vigor-
ously at 30 ꢀC for 12 h. After the completion of the reaction, the
slurry was treated with 200 mL ice cold water, and the resulting
precipitate was filtered off [14]. 3a. Off-white solid, Recrystalliza-
tion from MeCN (Yield 82%), mp 217e219 ꢀC; IR (KBr, cmꢁ1): 2942,
2889 (CH2), 2237 (C^N), 1239 (CeOeC), 758 (CeCl). 1H NMR
(400 MHz, DMSOed6):
d
7.68 (dd, J ¼ 7.2,1.5 Hz, 2H, AreH), 7.38 (dd,
J ¼ 6.8, 1.8 Hz, 2H, AreH), 3.77 (br s, 4H, piperazine), 3.54 (br s, 4H,
piperazine), 2.43 (s, 3H, CH3). 13C NMR (100 MHz, DMSOed6):
d
171.73 (1C, CtriazineeO), 170.08 (1C, CtriazineeCl), 167.15 (1C, Ctria-
zineeNpiperazine), 149.21 (1C, CbenzonitrileeO), 133.96 (2C), 125.44 (2C),
119.82 (1C, CN), 106.51 (1C, CeCN), 58.16 (2C, Cpiperazine), 47.93 (2C,
C
calcd. for C15H15ClN6O: C, 54.47; H, 4.57; N, 25.41. Found C, 54.52; H,
4.40; N, 25.29. 3b. White solid, Recrystallization from MeCN (Yield
85%), mp 188e189 ꢀC; IR (KBr, cmꢁ1): 2930, 2852 (CH2), 2220
(C^N), 1198 (CeOeC), 742 (CeCl). 1H NMR (400 MHz, DMSOed6):
piperazine), 39.34 (1C, CH3). MS (ESI), m/z 332.2 [M þ 1]þ. Anal.
4.1.1. 2,4-Dichloro-6-(4-methylpiperazin-1-yl)-1,3,5-triazine (2)
An oven-dried Schlenk flask was charged with a magnetic stir
bar, 2,4,6-trichloro-1,3,5-triazine 1 (10 mmol), N-methylpiperazine
(11 mmol), potassium carbonate (11 mmol) and anhydrous acetone
(30 mL). The flask was placed in an ice bath at 0e5 ꢀC temperature
and the reaction slurry was stirred vigorously for 6 h. At the end of
the reaction, the reaction mixture was treated with 200 mL ice cold
d
8.31 (s, 1H, CHeN of benzonitrile), 7.94 (dd, J ¼ 7.5, 2.0 Hz, 1H, Are
H), 7.06 (dd, J ¼ 6.8, 1.6 Hz, 1H, AreH), 3.74 (br s, 4H, piperazine),
3.49 (br s, 4H, piperazine), 2.33 (s, 3H, CH3). 13C NMR (100 MHz,
DMSOed6):
d 172.32 (1C, CtriazineeO), 169.60 (1C, CtriazineeCl),
165.26 (1C, CtriazineeNpiperazine), 158.43 (1C, CbenzonitrileeO), 152.71,

60
A.B. Patel et al. / European Journal of Medicinal Chemistry 79 (2014) 57e65
Table 1
138.30, 120.63 (1C, CN), 114.36, 108.39 (1C, CeCN), 56.25 (2C, Cpi-
perazine), 46.82 (2C, Cpiperazine), 41.05 (1C, CH3). MS (ESI), m/z 333.6
[M þ 1]þ. Anal. calcd. for C14H14ClN7O: C, 50.68; H, 4.25; N, 29.55.
Found C, 50.84; H, 4.07; N, 29.43.
Optimization of reaction conditions for CeC Coupling.a
Entry
Pd
Ligand
Base
Solvent
Time
% Yielde
1
2
3
4
5
6
7
8
Pd(OAC)2
Pd(OAC)2
Pd(OAC)2
Pd(OAC)2
Pd2(dba)3
[PdCl(allyl)]2
Pd(PPh3)4
Pd(OAC)2
Pd(OAC)2
Pd(OAC)2
Pd(OAC)2
Pd(OAC)2
Pd(OAC)2
Pd(OAC)2
Pd(OAC)2
Pd(OAC)2
Pd(OAC)2
L1d
L1
L1
L1
L1
L1
L1
L1
L1
K3PO4
K2CO3
KOtBu
Cs2CO3
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
K3PO4
DME
DME
DME
DME
DME
DME
DME
Dioxane
Toluene
THF
17
28
21
19
26
30
24
18
15
19
21
18
16
21
19
13
14
69
34
57
63
42
38
58
67
71
62
55
67
82
46
59
89
84
4.1.3. 4-(4-(4-Aminophenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-
triazin-2-yloxy)benzonitrile (4a)/6-(4-(4-aminophenyl)-6-(4-
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nicotinonitrile (4b)
An oven-dried 250 mL flat bottom flask was charged with a
magnetic stir bar, intermediate 3a/3b (10 mmol), K3PO4 (15 mmol),
ligand L (5 mol %), Pd(OAC)2 (3.0 mol %), 4-aminophenylboronic
acid pinacol ester (15 mmol) and toluene (100 mL). The flask was
placed in a preheated oil bath at refluxed temperature and the
reaction slurry was stirred vigorously for 13 h. The solvent was
recovered at reduced pressure. Then the reaction mixture was
cooled to room temperature. The residue was dissolved in methy-
lene dichloride and filtered through a pad of Celite. The filtrate was
washed with water (2 ꢂ 100 mL), and the organic layer was dried
with sodium sulfate. The filtrate was concentrated and the resulting
residue was purified by column chromatography (20% EA in Hex) to
yield the desired analog 6 [15]. 4a. White solid, Recrystallization
from MeCN (Yield 89%), mp 243e247 ꢀC; IR (KBr, cmꢁ1): 3323
(NH2), 2931, 2847 (CH2), 2224 (C^N), 1256 (CeOeC). 1H NMR
9
10
11
12
13
14
15
16b
17c
L1
L1
DMF
L2d
L3d
L4d
L5d
L3
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
L3
a
Pd: 1.5 mol %, Ligand: 5.0 mol %, 3a: 1.0 mmol, 4-aminophneylboronic acid
pinacol ester: 1.5 mmol, K3PO4: 1.5 mmol, Solvent: 5 mL per mmol.
b
3.0 mol % of Pd catalyst is used.
3b is used.
Xantphos (L1): 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene, BINAP (L2):
c
d
2,20-bis(diphenylphosphino)-1,10
binaphthyl,
Xphos
(L3):
2-
Dicyclohexylphosphino-20,40,60-triisopropylbiphenyl, Dppf (L4): diphenylphosphi-
noferrocene, DPEphos (L5): Bis (2-diphenylphosphinophenyl)ether.
(400 MHz, DMSOed6):
7.56 (m, 4H, AreH), 7.30e7.21 (m, 2H, AreH), 5.02 (br s, 2H, NH2),
d
7.90 (dd, J ¼ 6.8, 1.7 Hz, 2H, AreH), 7.70e
e
Isolated yields.
Scheme 2. Schematic diagram for the synthesis of analogs 8aeg/9aeg, Reagents and Conditions: (a) toluene, reflux; (b) SHCH2COOH, ZnCl2, 1,4-dioxane, reflux.
3.69 (br s, 4H, piperazine), 3.46 (br s, 4H, piperazine), 2.48 (s, 3H,
Table 2
CH3). 13C NMR (100 MHz, DMSOed6):
d 168.53 (1C, CtriazineeO),
Results of in vitro antimycobacterial and cytotoxicity screening of analogs 8aeg/9ae
g.
165.39 (1C, CtriazineeNpiperazine), 163.24 (1C, CtriazineeCPh), 150.78
(1C, CbenzonitrileeO), 146.33 (1C, CeNH2), 134.17 (2C), 131.80 (2C),
127.39, 125.4 (2C), 119.91 (1C, CN), 116.82 (2C), 105.49 (1C, CeCN),
53.72 (2C, Cpiperazine), 46.11 (2C, Cpiperazine), 42.89 (1C, CH3). MS
(ESI), m/z 388.1 [M þ 1]þ. Anal. calcd. for C21H21N7O: C, 65.10; H,
5.46; N, 25.31. Found C, 65.34; H, 5.19; N, 25.04. 4b. White solid,
Recrystallization from MeCN (Yield 84%), mp 221e223 ꢀC; IR (KBr,
cmꢁ1): 3417 (NH2), 2955, 2943, 2847 (CH2), 2227 (C^N), 1228 (Ce
Entrya
X
R
MIC (
m
g/mL)b
Cytotoxicity (mg/mL)
VERO cells (IC50)
8a
8b
8c
8d
8e
8f
8g
9a
9b
9c
9d
9e
9f
9g
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
H
Cl
Br
F
NO2
OH
OCH3
H
Cl
Br
200
62.5
25
100
100
12.5
25
125
100
62.5
6.25
100
3.12
6.25
3.12
6.25
0.25
0.20
e
NDc
ND
>62.5
ND
ND
>62.5
>62.5
ND
ND
ND
OeC). 1H NMR (400 MHz, DMSOed6):
d 8.47 (s, 1H, CHeN of ben-
zonitrile), 8.25 (dd, J ¼ 7.2, 2.2 Hz, 1H, AreH), 7.83e7.47 (m, 5H, Are
H), 7.19e6.98 (m, 2H, AreH), 5.63 (br s, 2H, NH2), 3.78 (br s, 4H,
piperazine), 3.61 (br s, 4H, piperazine), 2.56 (s, 3H, CH3). 13C NMR
F
>62.5
ND
(100 MHz, DMSOed6):
d 168.67 (1C, CtriazineeO), 165.12 (1C, Ctria-
NO2
OH
OCH3
N
N
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
e
zineeNpiperazine), 164.98 (1C, CtriazineeNpiperazine), 159.03 (1C, Cbenzo-
nitrileeO), 155.37, 149.55 (1C, CeNH2), 140.61, 129.26 (2C), 124.51,
119.39 (1C, CN), 117.40 (2C), 104.05 (1C, CeCN), 58.53 (2C, Cpiper-
azine), 47.25 (2C, Cpiperazine), 39.69 (1C, CH3). MS (ESI), m/z 389.8
[M þ 1]þ. Anal. calcd. for C20H20N8O: C, 61.84; H, 5.19; N, 28.85.
Found C, 62.03; H, 4.96; N, 28.93.
Ethambutol
Pyrazinamide
Rifampicin
Isoniazid
DMSO
a
Each value is the mean of three independent experiments using L. J. method.
Minimum Inhibitory Concentration for 99% inhibition of M. tuberculosis.
ND ¼ Not determine.
b
c

A.B. Patel et al. / European Journal of Medicinal Chemistry 79 (2014) 57e65
61
4.1.4. General synthetic procedure for Schiff bases 6aeg/7aeg
An oven-dried Schlenk flask was charged with a magnetic stir
bar, intermediate 4a/4b (2 mmol), various aldehydes 5aeg
(2.5 mmol) and dry toluene (50 mL). The protocol was repeated two
times. The flask was placed in a preheated oil bath at refluxed
temperature and the reaction slurry was stirred vigorously for 6e
8 h. After the completion of the reaction, the solvent was removed
by distillation; solid product obtained was washed with distilled
water, dried and recrystallized from 10% acetone in toluene to
afford intermediate Schiff bases 6aeg/7aeg. The progress of the
reaction was monitored by Thin Layer Chromatography (10%
methanol in chloroform) [16].
White solid, Yield: 78%, mp 219e221 ꢀC; IR (KBr, cmꢁ1): 3027 (CH),
2241 (C^N), 1630 (C]N), 1248 (CeOeC); 1H NMR (400 MHz,
DMSOed6):
d 8.34 (s, 1H, N]CHePh), 7.59e7.30 (m, 7H, AreH),
7.26e6.93 (m, 6H, AreH), 5.37 (1H, OH), 3.74 (br s, 4H, piperazine),
3.52 (br s, 4H, piperazine), 2.45 (s, 3H, CH3). MS (ESI), m/z 492.6
[M þ 1]þ. Anal. calcd. for C28H25N7O2: C, 68.42; H, 5.13; N, 19.95.
Found C, 68.57; H, 4.95; N, 20.13.
4.1.4.7. (Z)-4-(4-(4-(4-methoxybenzylideneamino)phenyl)-6-(4-
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)benzonitrile
(6g).
White solid, Yield: 72%, mp 219e221 ꢀC; IR (KBr, cmꢁ1): 3054 (CH),
2225 (C^N), 1621 (C]N), 1248 (CeOeC); 1H NMR (400 MHz,
DMSOed6):
d 8.11 (s, 1H, N]CHePh), 7.54e7.32 (m, 7H, AreH),
4.1. 4.1. (Z)-4-(4-(4-(benzylideneamino)phenyl)-6-(4-
7.16e7.01 (m, 6H, AreH), 3.97 (1H, OCH3), 3.63 (br s, 4H, pipera-
zine), 3.50 (br s, 4H, piperazine), 2.29 (s, 3H, CH3). MS (ESI), m/z 490
[M þ 1]þ. Anal. calcd. for C29H27N7O2: C, 68.89; H, 5.38; N, 19.39.
Found C, 68.71; H, 4.20; N, 19.45.
methylpiperazin-1-yl)-1,3,5-triazin-2
yloxy)benzonitrile
(6a).
White solid, Yield: 83%, mp 196e198 ꢀC; IR (KBr, cmꢁ1): 3028 (CH),
2223 (C^N), 1628 (C]N), 1244 (CeOeC); 1H NMR (400 MHz,
DMSOed6):
d 8.01 (s, 1H, N]CHePh), 7.53e7.27 (m, 7H, AreH),
7.15e6.73 (m, 6H, AreH), 3.89 (br s, 4H, piperazine), 3.47 (br s, 4H,
piperazine), 2.31 (s, 3H, CH3). MS (ESI), m/z 476.5 [M þ 1]þ. Anal.
calcd. for C28H25N7O: C, 70.72; H, 5.30; N, 20.62. Found C, 70.57; H,
5.18; N, 20.55.
4.1.4.8. (Z)-6-(4-(4-(benzylideneamino)phenyl)-6-(4-
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nicotinonitrile
(7a).
White solid, Yield: 71%, mp 202e203 ꢀC; IR (KBr, cmꢁ1): 3038 (CH),
2229 (C^N), 1628 (C]N), 1231 (CeOeC). 1H NMR (400 MHz,
DMSOed6):
d 8.48 (s, 1H, N]CHePh), 8.19 (s, 1H, CHeN of benzo-
4.1.4.2. (Z)-4-(4-(4-(4-chlorobenzylideneamino)phenyl)-6-(4-
nitrile), 8.03 (dd, J ¼ 6.8,1.2 Hz,1H, AreH), 7.72e7.41 (m, 5H, AreH),
7.25e6.88 (m, 2H, AreH), 3.73 (br s, 4H, piperazine), 3.57 (br s, 4H,
piperazine), 2.53 (s, 3H, CH3). MS (ESI), m/z 477.1 [M þ 1]þ. Anal.
calcd. for C27H24N8O: C, 68.05; H, 5.08; N, 23.51. Found C, 67.88; H,
5.14; N, 23.33.
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)benzonitrile
(6b).
Greenish white solid, Yield: 79%, mp 218 ꢀC; IR (KBr, cmꢁ1): 2983
(CH), 2238 (C^N), 1632 (C]N), 1230 (CeOeC); 1H NMR (400 MHz,
DMSOed6):
d 8.14 (s, 1H, N]CHePh), 7.67e7.32 (m, 7H, AreH),
7.09e6.85 (m, 6H, AreH), 3.76 (br s, 4H, piperazine), 3.51 (br s, 4H,
piperazine), 2.39 (s, 3H, CH3). MS (ESI), m/z 511 [M þ 1]þ. Anal.
calcd. for C28H24ClN7O: C, 65.94; H, 4.74; N,19.23. Found C, 66.11; H,
4.80; N, 18.98.
4.1.4.9. (Z)-6-(4-(4-(4-chlorobenzylideneamino)phenyl)-6-(4-
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nicotinonitrile
(7b).
White solid, Yield: 67%, mp 213 ꢀC; IR (KBr, cmꢁ1): 3113 (CH), 2234
(C^N),1642 (C]N),1240 (CeOeC). 1H NMR (400 MHz, DMSOed6):
4.1.4.3. (Z)-4-(4-(4-(4-bromobenzylideneamino)phenyl)-6-(4-
d 8.32 (s, 1H, N]CHePh), 8.14 (s, 1H, CHeN of benzonitrile), 7.89
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)benzonitrile
(6c).
(dd, J ¼ 7.2, 1.8 Hz, 1H, AreH), 7.61e7.47 (m, 5H, AreH), 7.19e6.92
(m, 2H, AreH), 3.78 (br s, 4H, piperazine), 3.53 (br s, 4H, pipera-
zine), 2.39 (s, 3H, CH3). MS (ESI), m/z 512 [M þ 1]þ. Anal. calcd. for
White solid, Yield: 70%, mp 243e245 ꢀC; IR (KBr, cmꢁ1): 3021 (CH),
2214 (C^N), 1619 (C]N), 1239 (CeOeC); 1H NMR (400 MHz,
DMSOed6):
d
8.09 (s, 1H, N]CHePh), 7.69e7.52 (m, 7H, AreH),
C27H23ClN8O: C, 63.46; H, 4.54; N, 21.93. Found C, 63.31; H, 4.38; N,
7.29e6.97 (m, 6H, AreH), 3.80 (br s, 4H, piperazine), 3.58 (br s, 4H,
piperazine), 2.28 (s, 3H, CH3). MS (ESI), m/z 555.1 [M þ 1]þ. Anal.
calcd. for Chemical Formula: C28H24FN7O: C, 60.66; H, 4.36; N,
17.68. Found C, 60.39; H, 4.40; N, 17.52.
22.05.
4.1.4.10. (Z)-6-(4-(4-(4-bromobenzylideneamino)phenyl)-6-(4-
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nicotinonitrile
(7c).
White solid, Yield: 67%, mp 229e233 ꢀC; IR (KBr, cmꢁ1): 3052 (CH),
4.1.4.4. (Z)-4-(4-(4-(4-fluorobenzylideneamino)phenyl)-6-(4-
2245 (C^N), 1622 (C]N), 1257 (CeOeC). 1H NMR (400 MHz,
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)benzonitrile
(6d).
DMSOed6): d 8.49 (s, 1H, N]CHePh), 8.27 (s, 1H, CHeN of ben-
White solid, Yield: 75%, mp 211e212 ꢀC; IR (KBr, cmꢁ1): 3024 (CH),
zonitrile), 8.11 (dd, J ¼ 7.8, 2.1 Hz, 1H, AreH), 7.69e7.53 (m, 5H, Are
H), 7.04e6.76 (m, 2H, AreH), 3.84 (br s, 4H, piperazine), 3.58 (br s,
4H, piperazine), 2.42 (s, 3H, CH3). MS (ESI), m/z 554.3 [M þ 1]þ.
Anal. calcd. for C27H23BrN8O: C, 58.39; H, 4.17; N, 20.17. Found C,
58.21; H, 4.03; N, 20.29.
2228 (C^N), 1628 (C]N), 1248 (CeOeC); 1H NMR (400 MHz,
DMSOed6):
d 7.93 (s, 1H, N]CHePh), 7.46e7.19 (m, 7H, AreH),
7.04e6.68 (m, 6H, AreH), 3.77 (br s, 4H, piperazine), 3.50 (br s, 4H,
piperazine), 2.19 (s, 3H, CH3). MS (ESI), m/z 494.7 [M þ 1]þ. Anal.
calcd. for C28H24FN7O: C, 68.14; H, 4.90; N, 19.87. Found C, 68.21; H,
5.02; N, 19.76.
4.1.4.11. (Z)-6-(4-(4-(4-fluorobenzylideneamino)phenyl)-6-(4-
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nicotinonitrile
(7d).
4 .1. 4 . 5 . ( Z ) - 4 - ( 4 - ( 4 - m e t hyl p i p e ra z i n - 1 - yl ) - 6 - ( 4 - ( 4 -
nitrobenzylideneamino)phenyl)-1,3,5-triazin-2-yloxy)benzonitrile
(6e). White solid, Yield: 83%, mp 219e221 ꢀC; IR (KBr, cmꢁ1): 3083
(CH), 2230 (C^N), 1643 (C]N), 1248 (CeOeC); 1H NMR (400 MHz,
White solid, Yield: 73%, mp 192e193 ꢀC; IR (KBr, cmꢁ1): 3107 (CH),
2219 (C^N), 1610 (C]N), 1234 (CeOeC). 1H NMR (400 MHz,
DMSOed6):
d 8.35 (s, 1H, N]CHePh), 8.18 (s, 1H, CHeN of benzo-
nitrile), 7.92 (dd, J ¼ 7.2, 2.3 Hz,1H, AreH), 7.55e7.31 (m, 5H, AreH),
7.13e6.89 (m, 2H, AreH), 3.78 (br s, 4H, piperazine), 3.50 (br s, 4H,
piperazine), 2.38 (s, 3H, CH3). MS (ESI), m/z 495.7 [M þ 1]þ. Anal.
calcd. for C27H23FN8O: C, 65.58; H, 4.69; N, 22.66. Found C, 65.34; H,
4.72; N, 22.58.
DMSOed6):
d 8.23 (s, 1H, N]CHePh), 7.38e7.21 (m, 7H, AreH),
7.12e6.85 (m, 6H, AreH), 3.69 (br s, 4H, piperazine), 3.48 (br s, 4H,
piperazine), 2.36 (s, 3H, CH3). MS (ESI), m/z 521.3 [M þ 1]þ. Anal.
calcd. for C28H24N8O3: C, 64.61; H, 4.65; N, 21.53. Found C, 64.72; H,
4.43; N, 21.50.
4.1. 4.12. (Z)-6-(4-(4-methyl piperazin-1-yl)-6- (4-(4-
nitrobenzylideneamino)phenyl)-1,3,5-triazin-2-yloxy)nicotinonitrile
(7e). White solid, Yield: 78%, mp 239e242 ꢀC; IR (KBr, cmꢁ1): 2991
4.1.4.6. (Z)-4-(4-(4-(4-hydroxybenzylideneamino)phenyl)-
6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)benzonitrile
(6f).

62
A.B. Patel et al. / European Journal of Medicinal Chemistry 79 (2014) 57e65
(CH), 2224 (C^N), 1629 (C]N), 1224 (CeOeC). 1H NMR (400 MHz,
DMSOed6): 8.49 (s, 1H, N]CHePh), 8.33 (s, 1H, CHeN of ben-
zonitrile), 8.19 (dd, J ¼ 6.9, 1.8 Hz, 1H, AreH), 7.67e7.42 (m, 5H, Are
H), 7.21e7.03 (m, 2H, AreH), 3.89 (br s, 4H, piperazine), 3.62 (br s,
4H, piperazine), 2.31 (s, 3H, CH3). MS (ESI), m/z 522.8 [M þ 1]þ.
Anal. calcd. for C27H23N9O3: C, 62.18; H, 4.45; N, 24.17. Found C,
62.26; H, 4.61; N, 24.08.
2H, AreH), 7.89e7.61 (m, 9H, AreH), 7.19e7.08 (m, 2H, AreH), 6.21
(s, 1H, SeCHeN), 3.98 (d, J ¼ 2.8 Hz, 1H, SeCH2), 3.81 (d, J ¼ 4.0 Hz,
1H, SeCH2), 3.70 (br s, 4H, piperazine), 3.49 (br s, 4H, piperazine),
d
2.48 (s, 3H, CH3). 13C NMR (100 MHz, DMSOed6):
d 171.78 (1C, C]
O), 169.51 (1C, CtriazineeO), 166.04 (1C, CtriazineeNpiperazine), 161.17
(1C, CtriazineeCPh), 149.72 (1C, CbenzonitrileeO), 140.56, 140.03, 135.37
(CeCl), 133.26, 132.50 (2C), 130.84 (2C), 128.89 (2C), 128.35 (2C),
125.17 (2C), 120.41 (2C), 119.76 (1C, CN), 108.18 (1C, CeCN), 66.85
(1C, Cthiazolidin-4-oneeCPh), 56.22 (2C, Cpiperazine), 48.34 (2C, Cpiper-
azine), 40.31 (1C, CH3), 37.48 (1C, CH2). MS (ESI), m/z 584.2 [M þ 1]þ.
Anal. calcd. for C30H26ClN7O2S: C, 61.69; H, 4.49; N, 16.79. Found C,
61.73; H, 4.34; N, 16.43.
4.1.4.13. (Z)-6-(4-(4-(4-hydroxybenzylideneamino)phenyl)-6-(4-
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nicotinonitrile
(7f).
White solid, Yield: 80%, mp 215e217 ꢀC; IR (KBr, cmꢁ1): 3048 (CH),
2235 (C^N), 1642 (C]N), 1242 (CeOeC). 1H NMR (400 MHz,
DMSOed6):
d 8.28 (s, 1H, N]CHePh), 8.09 (s, 1H, CHeN of ben-
zonitrile), 7.98 (dd, J ¼ 7.2, 2.0 Hz, 1H, AreH), 7.61e7.23 (m, 5H, Are
H), 7.06e6.75 (m, 2H, AreH), 5.20 (1H, OH), 3.81 (br s, 4H, piper-
azine), 3.48 (br s, 4H, piperazine), 2.46 (s, 3H, CH3). MS (ESI), m/z
493.1 [M þ 1]þ. Anal. calcd. for C27H23N8O: C, 65.84; H, 4.91; N,
22.75. Found C, 65.78; H, 4.80; N, 22.60.
4.1.5.3. 4-(4-(4-(2-(4-Bromophenyl)-4-oxothiazolidin-3-yl)phenyl)-
6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)benzonitrile
(8c).
White solid, Recrystallization from MeCN (Yield 72%), mp 264e
267 ꢀC; IR (KBr, cmꢁ1): 3089 (CH), 2220 (C^N), 1725 (C]O), 1235
(CeOeC). 1H NMR (400 MHz, DMSOed6):
d
8.13 (dd, J ¼ 6.8, 1.8 Hz,
2H, AreH), 7.67e7.42 (m, 9H, AreH), 7.25e7.12 (m, 2H, AreH), 6.17
(s, 1H, SeCHeN), 3.90 (d, J ¼ 4.2 Hz, 1H, SeCH2), 3.78 (d, J ¼ 6.0 Hz,
1H, SeCH2), 3.67 (br s, 4H, piperazine), 3.53 (br s, 4H, piperazine),
4.1.4.14. (Z)-6-(4-(4-(4-methoxybenzylideneamino)phenyl)-6-(4-
methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nicotinonitrile
(7g).
White solid, Yield: 75%, mp 197 ꢀC; IR (KBr, cmꢁ1): 3059 (eCH),
2.59 (s, 3H, CH3). 13C NMR (100 MHz, DMSOed6):
d
172.07 (1C, C]
O), 168.73 (1C, CtriazineeO), 166.79 (1C, CtriazineeNpiperazine), 160.56
(1C, triazineeCPh), 149.24 (1C, benzonitrileeO), 139.88, 139.17,
2220 (C^N), 1618 (C]N), 1230 (CeOeC). 1H NMR (400 MHz,
DMSOed6):
d 8.37 (s, 1H, N]CHePh), 8.16 (s, 1H, CHeN of benzo-
C
C
nitrile), 7.84 (dd, J ¼ 8.2, 1.9 Hz, 1H, AreH), 7.43e7.16 (m, 5H, AreH),
7.01e6.82 (m, 2H, AreH), 3.97 (1H, OCH3), 3.77 (br s, 4H, pipera-
zine), 3.54 (br s, 4H, piperazine), 2.38 (s, 3H, CH3). MS (ESI), m/z
507.3 [M þ 1]þ. Anal. calcd. for C28H26N8O2: C, 66.39; H, 5.17; N,
22.12. Found C, 66.17; H, 4.95; N, 22.15.
133.875, 133.02 (2C), 131.21 (2C), 129.55 (2C), 129.06 (2C), 126.58
(2C), 122.75 (CeBr), 121.87 (2C), 120.14 (1C, CN), 108.81 (1C, CeCN),
66.16 (1C, Cthiazolidin-4-oneeCPh), 55.78 (2C, Cpiperazine), 48.08 (2C,
C
piperazine), 40.56 (1C, CH3), 38.35 (1C, CH2). MS (ESI), m/z 629.2
[M þ 1]þ. Anal. calcd. for C30H26BrN7O2S: C, 57.33; H, 4.17; N, 15.60.
Found C, 57.52; H, 3.98; N, 15.55.
4.1.5. General synthetic procedure for final analogs (8aeg/9aeg)
An oven-dried Schlenk flask was charged with a magnetic stir
bar, analogs 6aeg/7aeg (2 mmol), thioglycolic acid (3 mmol), a
pinch of anhydrous zinc chloride and 1,4-dioxane (30 mL). The flask
was placed in a preheated oil bath at refluxed temperature and the
reaction slurry was stirred vigorously for 12e14 h. After the
completion of the reaction, the reaction mass was poured in
crushed ice and the resulting precipitate was filtered, washed with
water, dried and crystallized with a suitable solvent to obtain 8aeg/
9aeg. The progress of the reaction was monitored by Thin Layer
Chromatography (20% EA in Hex) [17].
4.1.5.4. 4-(4-(4-(2-(4-Fluorophenyl)-4-oxothiazolidin-4-oneolidin-3-
yl)phenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)benzoni-
trile (8d). Pale yellow solid, Recrystallization from MeCN (Yield
77%), mp 279e283 ꢀC; IR (KBr, cmꢁ1): 3092 (CH), 2238 (C^N), 1738
(C]O), 1221 (CeOeC). 1H NMR (400 MHz, DMSOed6):
d 8.22 (dd,
J ¼ 6.6, 1.4 Hz, 2H, AreH), 7.54e7.29 (m, 9H, AreH), 7.11e7.03 (m,
2H, AreH), 6.23 (s, 1H, SeCHeN), 3.92 (d, J ¼ 2.8 Hz, 1H, SeCH2),
3.80 (d, J ¼ 4.2 Hz, 1H, SeCH2), 3.73 (br s, 4H, piperazine), 3.48 (br s,
4H, piperazine), 2.52 (s, 3H, CH3). 13C NMR (100 MHz, DMSOed6):
d
172.38 (1C, C]O), 168.01 (1C, CtriazineeO), 165.08 (1C, Ctriazinee
piperazine), 160.14 (1C, CtriazineeCPh), 157.56 (CeF), 150.41 (1C,
N
4.1.5.1. 4-(4-(4-Methylpiperazin-1-yl)-6-(4-(4-oxo-2-
phenylthiazolidin-3-yl)phenyl)-1,3,5-triazin-2-yloxy)benzonitrile
(8a). White solid, Recrystallization from MeCN (Yield 78%), mp
255e257 ꢀC; IR (KBr, cmꢁ1): 3039 (CH), 2228 (C^N), 1724 (C]O),
CbenzonitrileeO), 139.23, 138.67, 134.59, 132.47 (2C), 131.58 (2C),
129.61 (2C), 126.43 (2C), 120.80 (2C), 119.37 (1C, CN), 114.94 (2C),
109.05 (1C, CeCN), 67.28 (1C, Cthiazolidin-4-oneeCPh), 55.19 (2C, Cpi-
perazine), 48.87 (2C, Cpiperazine), 39.75 (1C, CH3), 38.14 (1C, CH2). MS
(ESI), m/z 566.8 [M þ 1]þ. Anal. calcd. for C30H26FN7O2S: C, 63.48; H,
4.62; N, 17.27. Found C, 63.71; H, 4.87; N, 17.01.
1256 (CeOeC). 1H NMR (400 MHz, DMSOed6):
d
8.18 (dd, J ¼ 8.2,
2.4 Hz, 2H, AreH), 7.71e7.55 (m, 9H, AreH), 7.15e7.13 (m, 2H, Are
H), 6.15 (s, 1H, SeCHeN), 3.82 (d, J ¼ 2.8 Hz, 1H, SeCH2), 3.79 (d,
J ¼ 4.2 Hz, 1H, SeCH2), 3.72 (br s, 4H, piperazine), 3.52 (br s, 4H,
piperazine), 2.71 (s, 3H, CH3). 13C NMR (100 MHz, DMSOed6):
4.1.5.5. 4-(4-(4-Methylpiperazin-1-yl)-6-(4-(2-(4-nitrophenyl)-4-
oxothiazolidin-4-oneolidin-3-yl)phenyl)-1,3,5-triazin-2-yloxy)benzo-
nitrile (8e). Yellow solid, Recrystallization from MeCN (Yield 70%),
mp 287e288 ꢀC; IR (KBr, cmꢁ1): 3109 (CH), 2233 (C^N), 1721 (C]
d
172.29 (1C, C]O), 168.44 (1C, CtriazineeO), 165.73 (1C, Ctriazinee
piperazine), 160.00 (1C, CtriazineeCPh), 150.66 (1C, CbenzonitrileeO),
N
140.35, 139.54, 134.08, 131.79 (2C), 130.08 (2C), 129.67, 129.26 (2C),
129.17 (2C), 126.40 (2C), 121.58 (2C), 121.09 (1C, CN), 109.42 (1C, Ce
CN), 67.75 (1C, Cthiazolidin-4-oneeCPh), 55.44 (2C, Cpiperazine), 47.03
(2C, Cpiperazine), 39.99 (1C, CH3), 37.30 (1C, CH2). MS (ESI), m/z 549.8
[M þ 1]þ. Anal. calcd. for C30H27N7O2S: C, 65.56; H, 4.95; N, 17.84.
Found C, 65.31; H, 4.89; N, 18.15.
O),1226 (CeOeC). 1H NMR (400 MHz, DMSOed6):
d
8.19 (dd, J ¼ 7.8,
2.4 Hz, 2H, AreH), 7.58e7.20 (m, 9H, AreH), 7.07e6.94 (m, 2H, Are
H), 6.19 (s, 1H, SeCHeN), 3.88 (d, J ¼ 4.4 Hz, 1H, SeCH2), 3.82 (d,
J ¼ 5.8 Hz, 1H, SeCH2), 3.69 (br s, 4H, piperazine), 3.55 (br s, 4H,
piperazine), 2.38 (s, 3H, CH3). 13C NMR (100 MHz, DMSOed6):
d
171.58 (1C, C]O), 168.35 (1C, CtriazineeO), 165.49 (1C, Ctriazinee
piperazine), 159.83 (1C, CtriazineeCPh), 150.11 (1C, CbenzonitrileeO),
4.1.5.2. 4-(4-(4-(2-(4-Chlorophenyl)-4-oxothiazolidin-3-yl)phenyl)-
6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)benzonitrile (8b).
Off-white solid, Recrystallization from MeCN (Yield: 75%), mp 250e
251 ꢀC; IR (KBr, cmꢁ1): 3117 (CH), 2223 (C^N), 1741 (C]O), 1190
N
149.13, 146.85, 139.58, 138.1, 131.55 (2C), 130.87 (2C), 129.03 (2C),
128.21 (2C), 125.94 (2C), 121.31 (2C), 119.92 (1C, CN), 109.78 (1C, Ce
CN), 66.86 (1C, Cthiazolidin-4-oneeCPh), 56.31 (2C, Cpiperazine), 48.56
(2C, Cpiperazine), 39.47 (1C, CH3), 38.91 (1C, CH2). MS (ESI), m/z 594
(CeOeC). 1H NMR (400 MHz, DMSOed6):
d
8.26 (dd, J ¼ 7.2, 1.8 Hz,

A.B. Patel et al. / European Journal of Medicinal Chemistry 79 (2014) 57e65
63
[M þ 1]þ. Anal. calcd. for C30H26N8O4S: C, 60.59; H, 4.41; N, 18.84.
H), 7.40e7.28 (m, 8H, AreH), 7.15e7.01 (m, 2H, AreH), 6.14 (s, 1H,
SeCHeN), 3.91 (d, J ¼ 6.2 Hz, 1H, SeCH2), 3.86 (d, J ¼ 4.8 Hz, 1H, Se
CH2), 3.72 (br s, 4H, piperazine), 3.54 (br s, 4H, piperazine), 2.32 (s,
Found C, 60.29; H, 4.27; N, 18.59.
4.1.5.6. 4-(4-(4-(2-(4-Hydroxyphenyl)-4-oxothiazolidin-4-oneolidin-
3-yl)phenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)ben-
zonitrile (8f). White solid, Recrystallization from MeCN (Yield 81%),
mp 239 ꢀC; IR (KBr, cmꢁ1): 3478 (OH), 3111 (CH), 2227 (C^N), 1729
3H, CH3). 13C NMR (100 MHz, DMSOed6):
d
170.25 (1C, C]O),
168.21 (1C, CtriazineeO), 163.94 (1C, CtriazineeNpiperazine), 163.03 (1C,
triazineeNpiperazine), 160.08 (1C, benzonitrileeO), 152.71, 140.93,
C
C
140.01, 139.35, 135.84, 134.71 (CeCl), 129.17 (2C), 128.90 (2C),
128.38 (2C), 119.56 (2C), 116.21 (1C, CN), 113.11, 107.29 (1C, CeCN),
66.87, 55.55 (2C, Cpiperazine), 47.91 (2C, Cpiperazine), 39.73 (1C, CH3),
38.48 (1C, CH2). MS (ESI), m/z 583.9 [M þ 1]þ. Anal. calcd. for
(C]O), 1238 (CeOeC). 1H NMR (400 MHz, DMSOed6):
d 8.31 (dd,
J ¼ 7.2, 2.2 Hz, 2H, AreH), 7.82e7.55 (m, 9H, AreH), 7.43e7.22 (m,
2H, AreH), 6.20 (s, 1H, SeCHeN), 5.28 (1H, OH), 3.85 (d, J ¼ 2.2 Hz,
1H, SeCH2), 3.78 (d, J ¼ 3.8 Hz, 1H, SeCH2), 3.68 (br s, 4H, pipera-
zine), 3.49 (br s, 4H, piperazine), 2.40 (s, 3H, CH3). 13C NMR
C29H25ClN8O2S: C, 59.53; H, 4.31; N, 19.15. Found C, 59.27; H, 4.54;
N, 18.86.
(100 MHz, DMSOed6):
d 170.90 (1C, C]O), 169.53 (1C, CtriazineeO),
166.28 (1C, CtriazineeNpiperazine), 159.36 (1C, CtriazineeCPh), 154.84
(1C, CeOH), 149.82 (1C, CbenzonitrileeO), 140.21, 139.36, 133.86,
132.00 (2C),131.21 (2C),128.66 (2C),125.35 (2C),121.69 (2C),120.34
(1C, CN), 118.47 (2C), 108.71 (1C, CeCN), 67.34 (1C, Cthiazolidin-4-onee
4.1.5.10. 6-(4-(4-(2-(4-Bromophenyl)-4-oxothiazolidin-4-oneolidin-
3-yl)phenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nic-
otinonitrile (9c). White solid, Recrystallization from MeCN (Yield
71%), mp 243e246 ꢀC; IR (KBr, cmꢁ1): 3084 (CH), 2240 (C^N), 1730
C
Ph), 56.78 (2C, Cpiperazine), 48.21 (2C, Cpiperazine), 39.74 (1C, CH3),
(C]O), 1223 (CeOeC). 1H NMR (400 MHz, DMSOed6):
d 8.49 (s, 1H,
37.20 (1C, CH2). MS (ESI), m/z 565.3 [M þ 1]þ. Anal. calcd. for
CHeN of benzonitrile), 8.11 (dd, J ¼ 7.4, 2.2 Hz,1H, AreH), 7.59e7.30
(m, 8H, AreH), 7.09e6.82 (m, 2H, AreH), 6.30 (s, 1H, SeCHeN), 3.83
(d, J ¼ 4.2 Hz, 1H, SeCH2), 3.76 (d, J ¼ 6.8 Hz, 1H, SeCH2), 3.69 (br s,
4H, pip), 3.50 (br s, 4H, piperazine), 2.62 (s, 3H, CH3). 13C NMR
C
30H27N7O3S: C, 63.70; H, 4.81; N, 17.33. Found C, 63.48; H, 4.59; N,
17.64.
4.1.5.7. 4-(4-(4-(2-(4-Methoxyphenyl)-4-oxothiazolidin-4-
oneolidin-3-yl)phenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
yloxy)benzonitrile (8g). White solid, Recrystallization from MeCN
(Yield 73%), mp 212e213 ꢀC; IR (KBr, cmꢁ1): 3085 (CH), 2216
(C^N), 1740 (C]O), 1229 (CeOeC). 1H NMR (400 MHz, DMSOed6):
(100 MHz, DMSOed6): d 171.47 (1C, C]O), 168.68 (1C, CtriazineeO),
164.15 (1C, CtriazineeNpiperazine), 163.49 (1C, CtriazineeNpiperazine),
159.34 (1C, CbenzonitrileeO), 152.11, 141.04, 139.66, 138.83, 133.41
(2C), 129.03 (2C), 128.36 (2C), 122.04 (1C, CeBr), 120.13 (2C), 117.25
(1C, CN), 115.37, 108.05 (1C, CeCN), 66.41, 55.86 (2C, Cpiperazine),
47.55 (2C, Cpiperazine), 39.18 (1C, CH3), 38.87 (1C, CH2). MS (ESI), m/z
628 [M þ 1]þ. Anal. calcd. for C29H25BrN8O2S: C, 55.33; H, 4.00; N,
17.80. Found C, 55.65; H, 4.19; N, 17.54.
d
8.16 (dd, J ¼ 6.9, 2.2 Hz, 2H, AreH), 7.65e7.38 (m, 9H, AreH), 7.23e
7.04 (m, 2H, AreH), 6.27 (s, 1H, SeCHeN), 4.43 (s, 3H, OCH3), 3.97
(d, J ¼ 1.8 Hz, 1H, SeCH2), 3.81 (d, J ¼ 3.2 Hz, 1H, SeCH2), 3.74 (br s,
4H, piperazine), 3.56 (br s, 4H, piperazine), 2.68 (s, 3H, CH3). 13C
NMR (100 MHz, DMSOed6):
d
171.86 (1C, C]O), 169.03 (1C, Ctria-
4.1.5.11. 6-(4-(4-(2-(4-Fluorophenyl)-4-oxothiazolidin-4-oneolidin-
3-yl)phenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nic-
otinonitrile (9d). Pale yellow solid, Recrystallization from MeCN
(Yield 73%), mp 188e190 ꢀC; IR (KBr, cmꢁ1): 3114 (CH), 2221 (C^N),
zineeO), 165.38 (1C, CtriazineeNpiperazine), 162.54 (1C, CeOCH3),
160.79 (1C, CtriazineeCPh), 149.25 (1C, CbenzonitrileeO), 140.71, 139.86,
134.01, 133.14 (2C), 130.03 (2C), 128.71 (2C), 126.00 (2C), 120.54
(2C), 119.69 (1C, CN), 115.31 (2C), 108.24 (1C, CeCN), 68.10 (1C,
1719 (C]O), 1237 (CeOeC). 1H NMR (400 MHz, DMSOed6):
d 8.60
C
thiazolidin-4-oneeCPh), 58.03 (1C, OCH3), 55.86 (2C, Cpiperazine), 47.48
(s, 1H, CHeN of benzonitrile), 8.08 (dd, J ¼ 8.2, 2.8 Hz, 1H, AreH),
7.47e7.22 (m, 8H, AreH), 7.03e6.89 (m, 2H, AreH), 6.19 (s, 1H, Se
CHeN), 3.89 (d, J ¼ 4.0 Hz, 1H, SeCH2), 3.80 (d, J ¼ 6.8 Hz, 1H, Se
CH2), 3.71 (br s, 4H, piperazine), 3.55 (br s, 4H, piperazine), 2.67 (s,
(2C, Cpiperazine), 40.03 (1C, CH3), 37.06 (1C, CH2). MS (ESI), m/z 578.9
[M þ 1]þ. Anal. calcd. for C31H29N7O3S: C, 64.23; H, 5.04; N, 16.91.
Found C, 64.55; H, 4.88; N, 16.60.
3H, CH3). 13C NMR (100 MHz, DMSOed6):
d
170.70 (1C, C]O),
168.34 (1C, CtriazineeO), 164.66 (1C, CtriazineeNpiperazine), 160.94 (1C,
triazineeNpiperazine), 159.62 (1C, CbenzonitrileeO), 155.27 (1C, CeF),
4.1.5.8. 6-(4-(4-Methylpiperazin-1-yl)-6-(4-(4-oxo-2-
phenylthiazolidin-4-oneolidin-3-yl)phenyl)-1,3,5-triazin-2-yloxy)nic-
otinonitrile (9a). Beige solid, Recrystallization from MeCN (Yield
74%), mp 258e261 ꢀC; IR (KBr, cmꢁ1): 3123 (CH), 2229 (C^N), 1738
C
152.81, 141.24, 140.80, 138.33, 136.01, 129.25 (2C), 127.29 (2C),
120.81 (2C), 116.87 (1C, CN), 114.25 (2C), 113.04, 106.36 (1C, CeCN),
66.32, 56.11 (2C, Cpiperazine), 48.28 (2C, Cpiperazine), 39.87 (1C, CH3),
37.21 (1C, CH2). MS (ESI), m/z 568.1 [M þ 1]þ. Anal. calcd. for
(C]O), 1240 (CeOeC). 1H NMR (400 MHz, DMSOed6):
d 8.64 (s, 1H,
CHeN of benzonitrile), 8.09 (dd, J ¼ 7.6, 2.8 Hz, 1H, AreH), 7.65e
7.42 (m, 8H, AreH), 7.28e7.09 (m, 2H, AreH), 6.26 (s, 1H, SeCHeN),
3.94 (d, J ¼ 3.8 Hz, 1H, SeCH2), 3.81 (d, J ¼ 4.0 Hz, 1H, SeCH2), 3.68
(br s, 4H, piperazine), 3.49 (br s, 4H, piperazine), 2.45 (s, 3H, CH3).
C29H25FN8O2S: C, 61.25; H, 4.43; N, 19.71. Found C, 61.13; H, 4.50; N,
19.63.
13C NMR (100 MHz, DMSOed6):
C
N
d
172.76 (1C, C]O), 168.59 (1C,
4.1.5.12. 6-(4-(4-(2-(4-Nitrophenyl)-4-oxothiazolidin-4-oneolidin-3-
yl)phenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nic-
otinonitrile (9e). Yellow solid, Recrystallization from MeCN (Yield
69%), mp 282e285 ꢀC; IR (KBr, cmꢁ1): 3104 (CH), 2232 (C^N), 1729
triazineeO), 164.72 (1C, CtriazineeNpiperazine), 162.14 (1C, Ctriazinee
piperazine),159.70 (1C, CbenzonitrileeO),153.15,140.46,139.91,138.24,
136.11, 130.63 (2C), 130.05, 129.61 (2C), 127.54 (2C), 120.90 (2C),
117.43 (1C, CN), 114.28, 105.62 (1C, CeCN), 67.15, 55.37 (2C, Cpiper-
azine), 47.31 (2C, Cpiperazine), 40.44 (1C, CH3), 38.16 (1C, eCH2). MS
(ESI), m/z 549.5 [M þ 1]þ. Anal. calcd. for C29H26N8O2S: C, 63.26; H,
4.76; N, 20.35. Found C, 63.45; H, 4.53; N, 20.39.
(C]O), 1220 (CeOeC). 1H NMR (400 MHz, DMSOed6):
d 8.43 (s, 1H,
CHeN of benzonitrile), 8.15 (dd, J ¼ 6.8, 2.0 Hz,1H, AreH), 7.61e7.39
(m, 8H, AreH), 7.23e7.04 (m, 2H, AreH), 6.21 (s, 1H, SeCHeN), 3.86
(d, J ¼ 2.2 Hz, 1H, SeCH2), 3.78 (d, J ¼ 4.6 Hz, 1H, SeCH2), 3.66 (br s,
4H, piperazine), 3.48 (br s, 4H, piperazine), 2.52 (s, 3H, CH3). 13C
4.1.5.9. 6-(4-(4-(2-(4-Chlorophenyl)-4-oxothiazolidin-4-oneolidin-
3-yl)phenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)nic-
otinonitrile (9b). Off-white solid, Recrystallization from MeCN
(Yield 76%), mp 226e227 ꢀC; IR (KBr, cmꢁ1): 3057 (CH), 2234
(C^N), 1722 (C]O), 1215 (CeOeC). 1H NMR (400 MHz, DMSOed6):
NMR (100 MHz, DMSOed6): d 172.96 (1C, C]O), 167.85 (1C, Ctria-
zineeO), 164.13 (1C, CtriazineeNpiperazine), 163.67 (1C, CtriazineeNpiper-
azine), 160.37 (1C, CbenzonitrileeO), 153.01, 148.04, 146.16, 140.64,
140.25, 139.06, 135.29, 130.44 (2C), 126.40 (2C), 122.47 (2C), 120.35
(2C), 117.61 (1C, CN), 113.83, 107.88 (1C, CeCN), 67.81, 56.70 (2C,
d
8.52 (s, 1H, CHeN of benzonitrile), 8.18 (dd, J ¼ 7.2, 1.8 Hz, 1H, Are
Cpiperazine), 47.54 (2C, Cpiperazine), 40.15 (1C, CH3), 37.86 (1C, eCH2).

64
A.B. Patel et al. / European Journal of Medicinal Chemistry 79 (2014) 57e65
MS (ESI), m/z 594.8 [M þ 1]þ. Anal. calcd. for C29H25N9O4S: C, 58.48;
as most potent compounds. All the other compounds to be tasted
were re-examined for their actual MIC by adopting conventional L.
J. agar dilution method. The MIC was defined as the lowest con-
centration inhibiting 99% of the inoculum.
H, 4.23; N, 21.16. Found C, 58.62; H, 4.27; N, 21.51.
4.1.5.13. 6-(4-(4-(2-(4-Hydroxyphenyl)-4-oxothiazolidin-4-
oneolidin-3-yl)phenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
yloxy)nicotinonitrile (9f). White solid, Recrystallization from MeCN
(Yield 78%), mp 256e257 ꢀC; IR (KBr, cmꢁ1): 3483 (OH), 3119 (CH),
2237 (C^N), 1732 (C]O), 1234 (CeOeC). 1H NMR (400 MHz,
4.2.1.2. Lowenstein and Jensen method. The secondary anti-
mycobacterial screening for test compounds was obtained for
M. Tuberculosis H37Rv, by adopting L. J. (Lowenstein and Jensen)
agar dilution method [19] for the measurement of MIC. Stock so-
lutions of primary 1000, 500, 250, and secondary 200, 100, 62.5, 50,
DMSOed6):
d
8.39 (s, 1H, CHeN of benzonitrile), 8.05 (dd, J ¼ 8.4,
2.5 Hz, 1H, AreH), 7.52e7.21 (m, 8H, AreH), 7.01e6.80 (m, 2H, Are
H), 6.09 (s, 1H, SeCHeN), 5.32 (1H, OH), 3.95 (d, J ¼ 3.4 Hz, 1H, Se
CH2), 3.84 (d, J ¼ 6.8 Hz, 1H, SeCH2), 3.78 (br s, 4H, piperazine), 3.57
(br s, 4H, piperazine), 2.40 (s, 3H, CH3). 13C NMR (100 MHz, DMSOe
25, 12.5, 6.25, 3.12 and 1.56 mg/mL dilutions of each test compound
in DMSO (dimethylsulfoxide) 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 ꢀC for 24 h followed by streaking of M. tuberculosis H37Rv
(5 ꢂ 104 bacilli per tube). These tubes were then incubated at 37 ꢀ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 development
of colonies occurred or <20 colonies was taken as MIC concentra-
tion of test compound. The standard strain M. tuberculosis H37Rv
was tested with known drugs Isoniazid, Rifampicin, Ethambutol
and Pyrazinamide.
d6):
d 172.23 (1C, C]O), 168.05 (1C, CtriazineeO), 163.71 (1C, Ctria-
zineeNpiperazine), 160.94 (1C, CtriazineeNpiperazine), 160.08 (1C, Cbenzo-
nitrileeO), 155.57, 152.87, 141.31, 140.80, 135.06, 131.81, 130.15 (2C),
129.74 (2C), 119.88 (2C), 117.35 (1C, CN), 114.38 (2C), 110.91, 107.27
(1C, CeCN), 67.23, 56.00 (2C, Cpiperazine), 48.14 (2C, Cpiperazine), 40.68
(1C, CH3), 37.61 (1C, eCH2). MS (ESI), m/z 565.4 [M þ 1]þ. Anal.
calcd. for C29H26N8O3S: C, 61.47; H, 4.62; N,19.78. Found C, 61.38; H,
4.49; N, 19.67.
4.1.5.14. 6-(4-(4-(2-(4-Methoxyphenyl)-4-oxothiazolidin-4-
oneolidin-3-yl)phenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
yloxy)nicotinonitrile (9g). White solid, Recrystallization from MeCN
(Yield 75%), mp 213e214 ꢀC; IR (KBr, cmꢁ1): 2998 (CH), 2226
(C^N), 1737 (C]O), 1249 (CeOeC). 1H NMR (400 MHz, DMSOed6):
4.2.2. Cytotoxicity against VERO cells
d
8.55 (s, 1H, CHeN of benzonitrile), 8.19 (dd, J ¼ 7.2, 2.6 Hz, 1H, Are
All the active compounds were tested for cytotoxicity (IC50) in
VERO cells at concentrations 62.5 mg/mL or 10 times. After 72 h of
exposure, viability was assessed on the basis of cellular conversion
of MTT into a formazan product using the Promega Cell Titre 96
non-radioactive cell proliferation method [20].
H), 7.43e7.16 (m, 8H, AreH), 7.04e6.79 (m, 2H, AreH), 6.13 (s, 1H,
SeCHeN), 4.02 (3H, OCH3), 3.90 (d, J ¼ 2.8 Hz, 1H, SeCH2), 3.78 (d,
J ¼ 4.2 Hz, 1H, SeCH2), 3.70 (br s, 4H, piperazine), 3.51 (br s, 4H,
piperazine), 2.37 (s, 3H, CH3). 13C NMR (100 MHz, DMSOed6):
d
171.64 (1C, C]O), 168.35 (1C, CtriazineeO), 164.21 (1C, Ctriazinee
piperazine), 163.28 (1C, CtriazineeNpiperazine), 161.67 (1C, CeOCH3),
N
Acknowledgments
158.25 (1C, CbenzonitrileeO), 152.60, 140.57, 139.30, 136.64, 129.36
(2C), 128.01 (2C), 119.29 (2C), 116.04 (1C, CN), 113.47 (2C), 111.29,
102.35 (1C, CeCN), 66.11, 58.27 (1C, OCH3), 53.83 (2C, Cpiperazine),
47.19 (2C, Cpiperazine), 40.64 (1C, CH3), 37.91 (1C, CH2). MS (ESI), m/z
580 [M þ 1]þ. Anal. calcd. for C30H28N8O3S: C, 62.05; H, 4.86; N,
19.30. Found C, 61.92; H, 4.70; N, 18.56.
The authors are thankful to Director, S. V. National Institute of
Technology, Surat for the scholarship, encouragement and facilities.
We wish to offer our deepest gratitude to Microcare Laboratory,
Surat, India for carrying out the biological screenings. We are also
thankful to SAIF Panjab University and Centre of Excellence, Vapi,
India for spectral analyses.
4.2. Biology
Appendix A. Supplementary data
4.2.1. In vitro evaluation of antimycobacterial activity
4.2.1.1. BACTEC MGIT method. The Mycobacteria 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
antituberculosis efficacy by means of various concentrations pre-
pared. The suspension was allowed to sit for 20 min and the tubes
were centrifuged at 3000 rpm for 15 min. After that 104e107 CFU/
mL of M. tuberculosis H37Rv strain suspension prepared was added
in the medium to be incubated and 0.1 mL of egg-based medium
was also added. The MGIT tubes were then tightly recapped, mixed
well and incubated into BACTEC MGIT instrument at 37 ꢀ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. 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 [18]. The primary
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.085.
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