Anti-HIV, antimycobacterial and antimicrobial studies of newly synthesized 1,2,4-triazole clubbed benzothiazoles

Article abstract of DOI:10.1007/s00044-012-0129-4

In an attempt to synthesize pharmacologically active molecules, we report here the synthesis and in vitro anti-HIV, antimycobacterial and antimicrobial activity of various series of 3-(3-pyridyl)-5-(4-substitutedphenyl)-4-(N- substituted-1,3-benzothiazol-2-amino)-4H-1,2,4-triazole. The anti-HIV activity of the title compounds was evaluated as selective human immunodeficiency virus type-1 and -2 (HIV-1, HIV-2 RT) inhibitors, antitubercular activity against H₃₇Rv using Lowenstein-Jensen agar method and antimicrobial activity against certain bacterial and fungal strains using the broth microdilution method. The synthesized compounds were characterized by FTIR, ¹H-NMR,¹³C-NMR and mass spectral data together with elemental analysis.

Full text of DOI:10.1007/s00044-012-0129-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1320–1329
DOI 10.1007/s00044-012-0129-4
ORIGINAL RESEARCH
Anti-HIV, antimycobacterial and antimicrobial studies of newly
synthesized 1,2,4-triazole clubbed benzothiazoles
•
•
•
Navin B. Patel Imran H. Khan Christophe Pannecouque
Erik De Clercq
Received: 16 December 2011 / Accepted: 24 May 2012 / Published online: 13 June 2012
Ó Springer Science+Business Media, LLC 2012
Abstract In an attempt to synthesize pharmacologically
active molecules, we report here the synthesis and in vitro
anti-HIV, antimycobacterial and antimicrobial activity
of various series of 3-(3-pyridyl)-5-(4-substitutedphenyl)-
4-(N-substituted-1,3-benzothiazol-2-amino)-4H-1,2,4-tria-
zole. The anti-HIV activity of the title compounds was
evaluated as selective human immunodeficiency virus
type-1 and -2 (HIV-1, HIV-2 RT) inhibitors, antitubercular
activity against H37Rv using Lowenstein-Jensen agar
method and antimicrobial activity against certain bacterial
and fungal strains using the broth microdilution method.
The synthesized compounds were characterized by
FTIR,1H-NMR,13C-NMR and mass spectral data together
with elemental analysis.
developing anti-HIV drugs. Two types of reverse trans-
criptase inhibitors have been developed: (De Clercq, 2004;
Barbaro et al., 2005) nucleoside reverse transcriptase
inhibitors (NRTIs) and non-nucleoside reverse transcrip-
tase inhibitors (NNRTI). Five NNRTIs, nevirapine (Vira-
muneÒ) (Koup et al., 1991), delavirdine (RescriptorÒ)
(Freimuth, 1996), efavirenz (SustivaÒ) (Young et al.,
1995), etravirine (IntelenceÒ) (Andries et al., 2004) and
rilpivirine (EdurantÒ) (Janssen et al., 2005) have been
approved by FDA for the treatment of HIV infection.
However, significant resistance has been developed against
the first-generation NNRTIs, and there is an urgent need to
develop new anti-HIV agents that are effective against
these resistant mutants (Wainberg et al., 2005; Imamichi,
2004) (Fig. 1).
Keywords 1,2,4-Triazole Á Benzothiazoles Á
Anti-HIV activity Á Antimycobaterial activity Á
Antimicrobial activity
Mycobacterium tuberculosis (MTB) leads to a deadly
synergy with human immunodeficiency virus (HIV), and
an impressive number of HIV-infected individuals suc-
cumb to MTB aggression (Morris et al., 1995). Moreover
the emergence of multi-drug-resistant TB (MDR-TB),
defined as resistance to at least isoniazid and rifampin,
and the extensively drug resistant (XDR-TB) strains make
the discovery and the development of new drugs a prior-
ity (Janin 2007; Gutierrez-Lugo and Bewley, 2008). In the
last few years, the determination of the genome sequence
of MTB (Cole et al., 1998; Camus et al., 2002) provided
a much needed boost for research into new drug targets
against this pathogen. In particular, recent promising data
suggest that targeting the lipid metabolism pathways of
MTB may provide an excellent route for attenuating or
killing the bacterium. The genome of MTB encodes for a
relatively large number of cytochrome P450 enzymes.
These data indicate important physiological roles for these
enzymes which given that the substrate preference of the
majority of P450s is for hydrophobic molecules, most are
Introduction
The HIV pandemic remains one of the most serious threats
to public health. HIV-1 reverse transcriptase is a key
enzyme in the HIV replication and has been a key target for
N. B. Patel (&) Á I. H. Khan
Department of Chemistry, Veer Narmad South Gujarat
University, Surat 395007, Gujarat, India
e-mail: drnavin@satyam.net.in
C. Pannecouque Á E. De Clercq
Rega Institute for Medical Research, Katholieke Universiteit
Leuven, 10 Minderbroedersstraat, 3000 Louvain, Belgium
123

Med Chem Res (2013) 22:1320–1329
1321
O
N
O
S
N
N
H
F3C
H
Cl
N
H
O
N
N
N
N
H
O
N
N
N
O
H
O
1: Nevirapine
2: Delavirdine
3: Efavirenz
CN
CN
NH
CN
CN
O
N
NH
HN
N
N
Br
N
NH2
4: Etravirine
5: Rilpivirine
Fig. 1 Examples of anti-HIV agents available
likely to be involved in lipid metabolism. McLean et al.,
2002 cloned and expressed MTB CYP51 and CYP121,
two types of P450 from MTB. They demonstrated that
CYP51 and CYP121 bind azole antifungal drugs tightly
and that azole compounds are potent inhibitor of cell
growth of M. bovis and M. smegmatis, two mycobacterial
species which closely resemble MTB. Azole drugs proved
to have also antitubercular activities in mice (Guardiola-
Diaz et al., 2001). Finally, recent studies demonstrated the
crystal structure of MTB cytochrome P450 CYP121 in
complex with the fluconazole (Ahmad et al., 2006a, b). It
has been found that MTB-CYP121 binds commercially
available azole drugs, such as clotrimazole or econazole,
and that it may be the true target for the antimycobac-
terial activity of the azoles in vivo as suggested by gene-
knock out studies (Munro et al., 2003; Seward et al.,
2006).
Results and discussion
Chemistry
A series of 1,2,4-triazole analogues have been synthesized
in good yields using the synthetic route as outlined in
Scheme 1. Nicotinic acid as converted to ethyl nicotinoate
1 using ethanol and catalytic amount of sulphuric acid.
Ethyl nicotinoate on treatment with hydrazine hydrate
yielded nicotinoyl hydrazide 2 by a reported method (Saha
et al., 2010), which on intermolecular cyclisation with
4-nitrobenzoic acid 3 and 4-chlorobenzoic acid 4 in pres-
ence of phosphorous oxy chloride afforded 2-(3-pyridyl)-5-
(4-nitrophenyl)-1,3,4-oxadiazole 5 (Patel and Khan, 2011)
and 2-(3-pyridyl)-5-(4-chlorophenyl)-1,3,4-oxadiazole 6,
which on further condensation with substituted 2-hydra-
zino-1,3-benzothiazoles 7a–j (Patel et al., 2010a, 2010b) in
dry pyridine resulted in 3-(3-pyridyl)-5-(4-nitrophenyl)-4-
(N-substituted-1,3-benzothiazol-2-amino)-4H-1,2,4-triazoles
8a–j (Patel and Khan, 2011) and 3-(3-pyridyl)-5-(4-chlo-
rophenyl)-4-(N-substituted-1,3-benzothiazol-2-amino)-
4H-1,2,4-triazoles 9a–j.
In this context, as a continuation of our previous
studies on the discovery of new antimicrobial agents
(Patel et al., 2010a, b; Patel and Khan, 2011), we decided
to focus our attention on the synthesis and preliminary
biological evaluation of novel azole analogues with
polycyclic structure against MTB, HIV, bacterial and
fungal strains.
The structure of all the products was elucidated with the
1
help of IR, H NMR, 13C NMR and mass spectroscopic
123

1322
Med Chem Res (2013) 22:1320–1329
Scheme 1 Synthetic protocol
for the compounds 5, 6, 8a–j
and 9a–j
O
O
OH
NH2
O
CH3
NH
O
i
+
N
N
R
1
2
3, 4
ii
N
N
O
5, 6
N
R
N
N
N
N
S
R1
NH
NH NH2
iii
N
R
+
5, 6
N
S
7a-j
R1
8a-j, 9a-j
i. NH2NH2.H2O, ethanol, refluxed; ii. POCl3 reflux 9h; iii. Dry pyridine, refluxed
R= NO2, Cl
R1 = a. 6-F,
b. 6-Br,
d. 6-CH3,
e. 6-OCH3,
f. 6-Cl,
g. 4-CH3,
h. 4-NO2,
i. 5-Cl,6-Cl,
j. 4-Cl
c. 6-NO2,
1
data. In the IR spectrum of nicotinoyl hydrazide 2 showed
a stretching band around 3,335 and 3,278 cm-1, where due
to amine/amide NH while strong stretching band at
resonances observed with NH2 function in the H NMR of
9 proved that the condensation of 6 and 7 resulted in the
formation of 1,2,4-triazole ring. This was substantiated by
13C NMR data of 9a which showed a peak at d164.10 and d
162.66 due to C3 and C5 of triazole. Mass spectrum of 9a
displayed a molecular ion base peak at m/z 422 which
confirmed its molecular weight.
1
1,610 cm-1 was attributed to amide carbonyl. H NMR
spectrum showed a singlet at d 4.51 and d 9.81 which were
accounted for NH2 and NH that vanished on D2O
1
exchange. Lack of H NMR resonances observed with NH
1
and NH2 functions in the H NMR spectrum of 6 proved
that ring closure starting from 2 resulted in the formation of
1,3,4-oxadiazole ring. This was further substantiated by the
13C NMR data of 6 which showed a peak at d 160.32 and d
159.60 due to C2 and C5 of oxadiazole. Mass spectrum of 6
displayed a molecular ion base peak at m/z 257 which
confirmed its molecular weight. The IR spectra of 7a
showed a broad stretching band around 3,425 and
3,200 cm-1 for NH and NH2. 1H NMR spectrum showed a
singlet at d 4.8 and d 8.9 which were accounted for NH2
and NH that vanished on D2O exchange. Lack of 1H NMR
Biological activity
Antibacterial and antifungal activity
The MICs of synthesized compounds were carried out by
broth microdilution method as described by Rattan (2000).
Antibacterial activity was screened against two gram
positive bacteria (S. aureus MTCC 96, S. pyogenes MTCC
443) and two gram negative bacteria (E. coli MTCC 442,
P. aeruginosa MTCC 441). Ampicillin was used as a
123

Med Chem Res (2013) 22:1320–1329
1323
standard antibacterial agent. Antifungal activity was
screened against three fungal species C. albicans MTCC
227, A. niger MTCC 282 and A. clavatus MTCC 1323.
Griseofulvin was used as a standard antifungal agent. The
antimicrobial screening data are shown in Table 1 and
Table 2.
Table 2 Minimal inhibitory concentrations (MICs, lg/ml)
Compound
Fungal species
C. albicans
MTCC-227
A. niger
MTCC-282
A. clavatus
MTCC-323
6
500
200
100
500
1,000
250
100
200
100
250
200
500
1,000
500
500
500
1,000
500
500
500
500
250
500
100
1,000
500
All MTCC cultures were collected from Institute of
Microbial Technology, Chandigarh and tested against
above mentioned known drugs. Mueller–Hinton broth was
used as nutrient medium to grow and dilute the drug sus-
pension for the test. Inoculum size for test strain was
adjusted to 108 CFU (Colony Forming Unit) per milliliter
by comparing the turbidity. DMSO was used as diluent to
get desired concentration of drugs to test upon standard
bacterial strains.
9a
9b
1,000
[1,000
1,000
500
9c
9d
9e
9f
1,000
500
9g
9h
500
9i
1,000
500
The antibacterial screening results are summarized
in Table 1. The results revealed that 1,3,4-oxadiazole 6
exhibited good activity against S. aureus. Most of 1,2,
4-triazoles showed good activity (250 lg/ml) while com-
pounds 9c and 9g containing 6-nitro and 4-methyl substi-
tuted benzothiazole possessed good activity (100–125 lg/
ml) against S. aureus. Compound 9c having electron
withdrawing 6-nitro benzothiazole showed good activity
(100 lg/ml) against S. pyogenes. Good activity (100 lg/
ml) was observed with compounds 9h and 9j containing
electron withdrawing 4-nitro and 4-chloro substituent
against E. coli. Compounds 9a and 9j having 6-fluoro and
4-methyl substituted benzothiazole showed good activity
(62.5 lg/ml), whereas others displayed moderate activity
against P. aeruginosa. Compound 9c and 9g exhibited a
very good activity against gram positive bacteria, whereas
9a, 9h and 9j showed a very good activity against gram
negative bacteria.
9j
Griseofulvin
100
The results of antifungal activity are summarized in
Table 2. The results showed that 1,3,4-oxa diazole 6
exhibited comparable activity against C. albicans, while
weak activity was observed against A. niger and A. clav-
atus. Compounds 9b, 9f and 9h having electron with-
drawing 4-bromo, 6-chloro and 4-nitro substituent
exhibited better activity (100 lg/ml) whereas other com-
pounds showed good activity (200–500 lg/ml) except for
9d against C. albicans. All the compounds showed weak
activity against A. niger and A. clavatus.
Antitubercular activity
The encouraging results from the antibacterial studies
prompted us to do preliminary screening of synthesized
compounds against M. tuberculosis as summarized in
Table 3. Compounds 9a, 9d and 9g showed good activity
(50–62.5 lg/ml) against M. tuberculosis which is attributed
due to 6-fluoro, 6-methyl and 4-methyl substituents,
whereas compound 9j had better activity (12.5 lg/ml). Due
to the better activity against tested microorganisms and
mycobacteria, compound 9j has been selected for further
development and studies to acquire more information about
the structure–activity relationships are in progress in our
laboratories.
Table 1 Minimal inhibitory concentrations (MICs, lg/ml)
Compound Gram positive bacteria
Gram negative bacteria
S. aureus S. pyogenes E. coli P. aeruginosa
MTCC-96 MTCC-443 MTCC-442 MTCC-2488
6
200
250
500
125
500
500
250
100
200
500
250
250
500
1,000
100
500
500
500
1,000
250
500
500
100
500
500
500
250
500
500
1,000
500
100
500
100
100
500
62.5
500
125
500
250
500
250
125
250
62.5
100
9a
9b
9c
9d
9e
9f
9g
9h
9i
Anti-HIV activity
The synthesized compounds were evaluated for anti-HIV
activity by determining their ability to inhibit the replica-
tion of HIV-1 (IIIB) and HIV-2 (ROD) in MT-4 cells in
comparison with nevirapine (BIRG587) used as the refer-
ence drug. The cytotoxicity of the compound was deter-
mined in parallel. Comparisons of inhibitory concentration
9j
Ampicillin 250
123

1324
Med Chem Res (2013) 22:1320–1329
Table 3 Minimal inhibitory concentrations (MICs, lg/ml)
Table 4 Anti-HIV-1 and HIV-2 activity (mean IC50 lg/ml) and
cytotoxicity (mean CC50 lg/ml) of compounds 5, 6 and 7a–j in MT-4
cells
Compound
MIC values (lg/ml)
of M. tuberculosis H37Rv
% Inhibition
Compound HIV strain IC50 (lg/ml)a CC50 (lg/ml)b SDc SId
6
500
62.5
500
500
50
98
98
96
98
95
98
96
95
97
98
98
96
99
99
99
5
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
[73.50
[73.50
[4.49
73.50
73.50
4.49
3.57 \1
3.57 \1
1.38 \1
1.38 \1
0.29 \1
0.29 \1
3.81 \1
3.81 \1
0.50 \1
0.50 \1
2.71 \1
2.71 \1
1.30 \1
1.30 \1
0.63 \1
0.63 \1
0.92 \1
0.92 \1
0.86 \1
0.86 \1
6.32 \1
6.32 \1
9a
9b
6
9c
[4.49
4.49
9d
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
a
[13.78
[13.78
[8.01
13.78
13.78
8.01
9e
500
250
50
9f
9g
[8.01
8.01
9h
500
250
12.5
20.4
12.5
0.2
[12.80
[12.80
[4.52
12.80
12.80
4.52
9i
9j
Clotrimazole
Econazole
Isoniazid
Rifampicin
[4.52
4.52
[14.78
[14.78
[2.48
14.78
14.78
2.48
40
[2.48
2.48
[9.80
9.80
(IC50), cytotoxic concentration (CC50) and SI (selectivity,
given by the CC50/IC50 ratio) values for different com-
pounds are presented in Tables 4 and 5.
[9.80
9.80
[14.48
[14.48
[80.05
[80.05
[117.0
[117.0
14.48
14.48
80.05
80.05
C117.0
C117.0
Some of the compounds showed cytotoxicity. Unfortu-
nately, none of the compounds showed anti-activity at sub
toxic concentrations.
–
–
X1
X1
Concentration required protecting the cell against viral cytopath-
ogenicity by 50 % in MT-4 cells
Conclusion
b
Selectivity index ratio CC50/IC50, a higher SI means a more selec-
tive compound
A new class of azole derivatives has been synthesised. All
the compounds were obtained in good yield and were
tested as new potential anti-HIV, antitubercular and anti-
microbial agents. Modification of substituents on benzo-
thiazole ring with various electron releasing and electron
withdrawing substituents improved the activity. It is
important to point out that activity is not affected by
electronic properties of the substituents. The anti-HIV data
reveals that compounds 6 (IC50 4.49 lg/ml), 7b (IC50
8.01 lg/ml), 7d (IC50 4.52 lg/ml), 7f (IC50 2.48 lg/ml)
and 7g (IC50 9.80 lg/ml) showed cytotoxicity to consid-
erable extent, but unfortunately none of the compounds
proved active to subtoxic concentrations. The antibacterial
data indicate that the analogues with chloro, methyl and
nitro substituents emerged as promising antimicrobials
showing better to moderate activity. It was also observed
that the promising antimicrobials have proved to be better
antituberculars. Specially, compound 9j due to their better
activity against H37Rv strain, is the best choice for the
preparation of new derivatives in order to improve antitu-
bercular activity.
c
Standard deviation
d
All data represent mean values for at least two separate experiments
Experimental
Chemistry
All chemical were of analytical grade and used directly.
Melting points were determined in PMP-DM scientific
melting point apparatus and are uncorrected. The purity of
compounds was checked by TLC using Merck silica gel 60
F and visualized by exposure to iodine vapours or UV light.
IR spectra were recorded on a Perkin-Elmer RX 1 FTIR
spectrophotometer, using potassium bromide pellets, the
1
frequencies are expressed in cm-1. The H NMR and 13C
NMR spectra were recorded with a Bruker Avance II 400
NMR spectrometer, using tetramethylsilane as the internal
reference, with chloroform (CDCl3) as solvent. The
chemical shifts are reported in parts per million (d ppm).
Elemental analyses were performed on a Heraeus Carlo
123

Med Chem Res (2013) 22:1320–1329
1325
Erba 1180 CHN analyzer. The mass spectra were recorded
on micromass Q-T of micro (TOF MS ES?).
Table 5 Anti-HIV-1 and HIV-2 activity (IC lg/ml) and cytotoxicity
of compounds 8a–j and 9a–j in MT-4 cells
Compound HIV strain IC50 (lg/ml)a CC50 (lg/ml)b SDc SId
General procedure for 2-(3-pyridyl)-5-(4-substituted
phenyl)-1,3,4-oxadiazole 5,6
8a
8b
8c
8d
8e
8f
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
III B
ROD
[108.00
[108.00
[13.20
[13.20
[50.80
[50.80
[64.43
[64.43
[12.93
[12.93
[53.68
[53.68
[13.48
[13.48
[13.78
[13.78
[68.65
[68.65
[15.55
[15.55
[92.90
[92.90
[29.63
[29.63
[62.13
[62.13
[75.48
[75.48
[66.85
[66.85
[54.30
[54.30
[36.45
[36.45
[21.33
[21.33
[52.13
[52.13
[27.17
[27.15
0.050
C108.00
C108.00
13.20
13.20
50.80
50.80
64.43
64.43
12.93
12.93
53.68
53.68
13.48
13.48
13.78
13.78
68.65
68.65
15.55
15.55
92.90
92.90
29.63
29.63
62.13
62.13
75.48
75.48
66.85
66.85
54.30
54.30
36.45
36.45
21.33
21.33
52.13
52.13
27.15
27.15
[4.00
–
–
X1
X1
0.67 \1
0.67 \1
3.28 \1
3.28 \1
4.53 \1
4.53 \1
1.32 \1
1.32 \1
6.22 \1
6.22 \1
0.49 \1
0.49 \1
0.14 \1
0.14 \1
5.53 \1
5.53 \1
2.40 \1
2.40 \1
18.53 \1
18.53 \1
20.44 \1
20.44 \1
1.83 \1
1.83 \1
15.66 \1
15.66 \1
1.56 \1
1.56 \1
2.39 \1
2.39 \1
16.24 \1
16.24 \1
12.99 \1
12.99 \1
3.43 \1
3.43 \1
9.64 \1
9.64 \1
A mixture of nicotinoyl hydrazide 1 (5 mmol) and
4-chloro/nitro benzoic acid 2,3 (5 mmol) in phosphorus
oxy chloride (5 ml) was refluxed in a water bath for 9 h.
The progress of the reaction was monitored by TLC using
toluene:ethylacetate:methanol (70:20:10) as mobile phase.
After completion of the reaction, it was cooled and poured
onto crushed ice with continuous stirring. The solid mass
separated was neutralized with sodium bicarbonate solu-
tion (10 % w/v). The resulting solid thus obtained was
collected by filtration, washed well with cold water, dried
and crystallized from absolute ethanol.
8g
8h
8i
2-(3-Pyridyl)-5-(4-nitro phenyl)-1,3,4-oxadiazole 5 as
described (Patel and Khan, 2011)
2-(3-Pyridyl)-5-(4-chloro phenyl)-1,3,4-oxadiazole 6.
Yeild 69 %; m.p. 160–162 °C; IR (KBr): 1667 (C=N),
1
1287, 1073 (C–O–C) cm-1; H NMR (CDCl3) d 9.37 (s,
1H, CH), 8.81 (dd, 1H, J = 3.50 Hz, CH), 8.44 (d, 1H,
J = 8.08 Hz, CH), 7.51 (t, 1H, CH), 8.21 (d, 2H,
J = 8.12 Hz, 2CH), 7.65 (d, 2H, J = 8.06 Hz, 2CH); 13C
NMR (CDCl3) d 160.32 (C5-oxadiazole), 159.60 (C2-oxa-
diazole), 148.5, 145.34, 137.50, 131.62, 127.56, 125.01,
123.20, 122.10, 112.97 (aromatic ring) ppm; MS (m/z): 257
(M?), 259 (M ? 2); Anal. Calcd. for C14H11N3O2: C,
60.60; H, 3.13; N, 16.31; Found: C, 60.64; H, 3.17; N,
16.33 %.
8j
9a
9b
9c
9d
9e
9f
General procedure for the synthesis of 3-(3-pyridyl)-5-(4-
nitro/chlorophenyl)-4-(N-substituted-1,3-benzothiazol-2-
amino)-4H-1,2,4-triazole 8a–j and 9a–j
A mixture of 2-(3-pyridyl)-5-(4-nitro/chlorophenyl)-1,3,
4-oxadiazole 5,6 (5 mmol) and substituted 2-hydrazino-
1,3-benzothiazole 7a–j (Patel et al., 2010a, b) (5 mmol) in
dry pyridine (10 ml) was refluxed for 18–24 h. The reac-
tion was monitored by TLC on silica gel using ethyl ace-
tate: toluene (2.5:7.5).It was then cooled and poured onto
crushed ice. The reaction mass was neutralized by dilute
hydrochloric acid and resulting solid was washed with cold
water, dried and crystallized from ethanol.
9g
9h
9i
9j
Nevirapine III B
[80
The other compounds of the series were prepared by a
similar procedure.
ROD
a
3-(3-Pyridyl)-5-(4-nitrophenyl)-4-(N-substituted-1,3-ben-
zothiazol-2-amino)-4H-1,2,4-triazole 8a–j as described
(Patel and Khan, 2011)
Concentration required protecting the cell against viral cytopath-
ogenicity by 50 % in MT-4 cells
b
Selectivity index ratio CC50/IC50, a higher SI means a more selec-
tive compound
3-(3-Pyridyl)-5-(4-chlorophenyl)-4-(N-6-flouro-1,3-ben-
zothiazol-2-amino)-4H-1,2,4-triazole 9. Yield 70 %; m.p.
c
Standard deviation
1
198–200 °C; IR (KBr): 3385 (NH), 1654 (C=N) cm-1; H
d
All data represent mean values for at least two separate experiments
123

1326
Med Chem Res (2013) 22:1320–1329
NMR (DMSO) d 7.87 (s, 1H, NH), 9.31 (s, 1H, CH), 8.80
(dd, 1H, J = 4.0 Hz, CH), 8.44 (d, 1H, J = 8.40 Hz, CH),
7.58 (t, 1H, CH), 8.11 (d, 2H, J = 8.14 Hz, 2CH), 7.31 (d,
2H, J = 8.08 Hz, 2CH), 7.07–7.24 (m, 3H, benzothiazole-
H); 13C NMR (DMSO) d 164.10 (C3-triazole), 162.66 (C5-
triazole), 158.95, 156.58, 152.60, 149.38, 147.73, 137.98,
134.21, 132.30, 129.64, 128.47, 124.17, 122.10, 120.20,
118.56, 112.95, 107.59 (aromatic ring) ppm; MS (m/z): 422
(M?), 424 (M?2); Anal. Calcd. for C20H12N6ClFS: C,
56.81; H, 2.86; N, 19.87. Found: C, 56.78; H, 2.90; N,
19.91 %.
123.47, 121.52 (aromatic ring) ppm; MS (m/z): 418 (M?),
420 (M?2); Anal. Calcd. for C21H15N6ClS: C, 60.21; H,
3.61; N, 20.06. Found: C, 60.18; H, 2.57; N, 20.10 %.
3-(3-Pyridyl)-5-(4-chlorophenyl)-4-(N-6-methoxy-1,3-ben-
zothiazol-2-amino)-4H-1,2,4-triazole 9e Yield 63 %;
m.p. 185–187 °C; IR (KBr): 3388 (NH), 1664 (C=N)
1
cm-1; H NMR (CDCl3) d 7.86 (s, 1H, NH), 3.84 (s, 1H,
OCH3), 9.31 (s, 1H, CH), 8.80 (dd, 1H, J = 4.0 Hz, CH),
8.45 (d, 1H, J = 8.12 Hz, CH), 7.45 (t, 1H, CH), 8.10 (d,
2H, J = 8.14 Hz, 2CH), 7.35 (d, 2H, J = 8.18 Hz, 2CH),
6.86–7.30 (m, 3H, benzothiazole-H); 13C NMR (CDCl3) d
164.58 (C3-triazole), 162.78 (C5-triazole), 52.69 (OCH3),
154.96, 149.48, 147.83, 139.28, 137.02, 134.16, 132.60,
131.13, 129.48, 128.44, 127.61, 124.23, 122.08, 119.92,
110.98, 108.25 (aromatic ring) ppm; MS (m/z): 434 (M?),
436 (M?2); Anal. Calcd. for C21H15N6OClS: C, 58.00; H,
3.48; N, 19.38. Found: C, 57.96; H, 3.52; N, 19.35 %.
3-(3-Pyridyl)-5-(4-chlorophenyl)-4-(N-6-bromo-1,3-ben-
zothiazol-2-amino)-4H-1,2,4-triazole 9b Yield 65 %;
m.p. 176–178 °C; IR (KBr): 3387 (NH), 1660 (C=N)
1
cm-1; H NMR (CDCl3) d 7.88 (s, 1H, NH), 9.31 (s, 1H,
CH), 8.81 (dd, 1H, J = 4.0 Hz, CH), 8.43 (d, 1H,
J = 8.35 Hz, CH), 7.47 (t, 1H, CH), 8.10 (d, 2H,
J = 8.10 Hz, 2CH), 7.33 (d, 2H, J = 8.12 Hz, 2CH),
7.62–7.74 (m, 3H, benzothiazole-H); 13C NMR (CDCl3) d
164.32 (C3-triazole), 162.72 (C5-triazole), 156.48, 152.61,
149.31, 147.59, 136.69, 134.28, 133.04, 131.98, 129.48,
128.92, 126.78, 124.49, 124.23, 121.09, 114.99, 109.13
(aromatic ring) ppm; MS (m/z): 483 (M?), 485 (M?2), 489
(M?4); Anal. Calcd. for C20H12N6ClBrS: C, 49.65; H,
2.50; N, 17.37. Found: C, 49.69; H, 2.47; N, 17.41 %.
3-(3-Pyridyl)-5-(4-chlorophenyl)-4-(N-6-chloro-1,3-benzo-
thiazol-2-amino)-4H-1,2,4-triazole 9f Yield 66 %; m.p.
1
163–165 °C; IR (KBr): 3382 (NH),1659 (C=N) cm-1; H
NMR (CDCl3) d 7.88 (s, 1H, NH), 9.30 (s, 1H, CH), 8.81
(dd, 1H, J = 4.0 Hz, CH), 8.43 (d, 1H, J = 8.40 Hz, CH),
7.51 (t, 1H, CH), 8.12 (d, 2H, J = 8.16 Hz, 2CH), 7.32 (d,
2H, J = 8.08 Hz, 2CH), 7.60–7.79 (m, 3H, benzothiazole-
H); 13C NMR (CDCl3) d 164.44 (C3-triazole), 162.30 (C5-
triazole), 154.91, 152.97, 149.32, 144.44, 137.08, 134.52,
132.36, 131.05, 128.37, 125.85, 124.33, 123.01, 122.08,
121.95, 121.52, 120.84 (aromatic ring) ppm; MS (m/z): 438
(M?), 440 (M?2), 444 (M?4); Anal. Calcd. for
C20H12N6Cl2S: C, 54.68; H, 2.75; N, 19.13. Found: C,
54.72; H, 2.79; N, 19.09 %.
3-(3-Pyridyl)-5-(4-chlorophenyl)-4-(N-6-nitro-1,3-benzothi-
azol-2-amino)-4H-1,2,4-triazole 9c Yield 72 %, m.p. 168
–170 °C; IR (KBr): 3382 (NH), 1652 (C=N) cm-1
;
1H
NMR (CDCl3) d 7.86 (s, 1H, NH), 9.31 (s, 1H, CH), 8.80
(dd, 1H, J = 3.8 Hz, CH), 8.46 (d, 1H, J = 8.5 Hz, CH),
7.60 (t, 1H, CH), 8.33 (d, 2H, J = 8.12 Hz, 2CH), 7.31 (d,
2H, J = 8.18 Hz, 2CH), 7.58 (d, H, J = 7.86 Hz, CH),
8.09 (d, 1H, J = 8.06 Hz, CH), 8.74 (s, 1H, CH); 13C
NMR (CDCl3) d 164.74 (C3-triazole), 162.66 (C5-triazole),
156.29, 152.31, 148.98, 146.82, 146.61, 141.77, 134.48,
132.86, 132.60, 132.32, 129.53, 128.31, 122.61, 121.97,
120.98, 117.72 (aromatic ring) ppm; MS (m/z): 449 (M?),
451(M?2); Anal. Calcd. for C20H12N7O2ClS: C, 53.40; H,
2.69; N, 21.79. Found: C, 53.38; H, 2.71; N, 21.81 %.
3-(3-Pyridyl)-5-(4-chlorophenyl)-4-(N-4-methyl-1,3-benzo-
thiazol-2-amino)-4H-1,2,4-triazole 9g Yield 61 %; m.p.
1
199–201 °C; IR (KBr): 3385 (NH),1662 (C=N) cm-1; H
NMR (CDCl3) d 2.61 (s, 1H, CH3), 7.84 (s, 1H, NH), 9.31
(s, 1H, CH), 8.80 (dd, 1H, J = 3.8 Hz, CH), 8.43 (d, 1H,
J = 8.36 Hz, CH), 7.57 (t, 1H, CH), 8.09 (d, 2H,
J = 8.14 Hz, 2CH), 7.37(d, 2H, J = 8.18 Hz, 2CH),
7.11–7.31 (m, 3H, benzothiazole-H); 13C NMR (CDCl3) d
164.40 (C3-triazole), 162.35 (C5-triazole), 19.89 (CH3),
154.88, 152.52, 149.58, 147.23, 143.28, 134.10, 132.32,
131.48, 129.78, 128.62, 128.40, 127.23, 124.59, 122.98,
122.04, 119.70 (aromatic ring) ppm; MS (m/z): 418 (M?),
420 (M?2); Anal. Calcd. for C21H15N6ClS: C, 60.21; H,
3.61; N, 20.06. Found: C, 60.18; H, 3.65; N, 20.10 %.
3-(3-Pyridyl)-5-(4-chlorophenyl)-4-(N-6-methyl-1,3-benzo-
thiazol-2-amino)-4H-1,2,4-triazole 9d Yield 58 %; m.p.
1
175–178 °C; IR (KBr): 3378 (NH), 1659 (C=N) cm-1; H
NMR (CDCl3) d 2.38 (s, 1H, CH3), 7.90 (s, 1H, NH), 9.31
(s, 1H, CH), 8.80 (dd, 1H, J = 3.8 Hz, CH), 8.45 (d, 1H,
J = 8.40 Hz, CH), 7.49 (t, 1H, CH), 8.11 (d, 2H,
J = 8.06 Hz, 2CH), 7.62 (d, 2H, J = 8.38 Hz, 2CH),
7.06–7.48 (m, 3H, benzothiazole-H); 13C NMR (CDCl3) d
164.01 (C3-triazole), 162.38 (C5-triazole), 22.91 (CH3),
155.93, 148.85, 146.98, 144.75, 135.28, 134.17, 132.31,
131.15, 129.92, 128.82, 128.52, 124.59, 124.30, 124.02,
3-(3-Pyridyl)-5-(4-chlorophenyl)-4-(N-4-nitro-1,3-benzo-
thiazol-2-amino)-4H-1,2,4-triazole 9h Yield 64 %; m.p.
1
184–186 °C; IR (KBr): 3387 (NH), 1660 (C=N) cm-1; H
NMR (CDCl3) d 7.92 (s, 1H, NH), 9.31 (s, 1H, CH), 8.81
123

Med Chem Res (2013) 22:1320–1329
1327
(dd, 1H, J = 4.0 Hz, CH), 8.44 (d, 1H, J = 8.38 Hz, CH),
7.51 (t, 1H, CH), 8.08 (d, 2H, J = 8.42 Hz, 2CH), 7.39 (d,
2H, J = 8.08 Hz, 2CH), 6.67 (t, 1H, CH), 8.28 (d, 1H,
J = 8.14 Hz, CH), 8.56 (d, 1H, J = 8.14 Hz, CH); 13C
NMR (CDCl3) d 164.52 (C3-triazole), 162.42 (C5-triazole),
156.21, 152.48, 148.83, 146.89, 137.78, 144.36, 139.06,
134.51, 133.32, 131.10, 129.12, 128.60, 128.07, 124.19,
122.55, 120.20 (aromatic ring) ppm; MS (m/z): 449 (M?),
451 (M?2); Anal. Calcd. for C20H12N7O2ClS: C, 53.40; H,
2.69; N, 21.79. Found: C,53.38; H, 2.73; N, 21.81 %.
tubes were then incubated overnight. The MIC of the
control organism was read to check the accuracy of the
drug concentrations. The lowest concentration inhibiting
growth of the organism was recorded as the MIC. All the
tubes not showing visible growth (in the same manner as
control tube described above) were subcultured and incu-
°
bated overnight at 37 C. The amount of growth from the
control tube before incubation (which represents the ori-
ginal inoculum) was compared. Subcultures might show
similar number of colonies indicating bacteriostatic activ-
ity; a reduced number of colonies indicating a partial or
slow bactericidal activity and no growth if the whole
inoculum has been killed. The test must include a second
set of the same dilutions inoculated with an organism of
known sensitivity. Each synthesized drug was diluted
obtaining 2,000 lg/ml concentration, as a stock solution.
In primary screening, 500, 250 and 125 lg/ml concentra-
tions of the synthesized drugs were taken. The active
synthesized drugs found in this primary screening were
further tested in a second set of dilution against all
microorganisms. The drugs found active in primary
screening were similarly diluted to obtain 100, 50, 25,
12.5, 6.250, 3.125 and 1.5625 lg/ml concentrations. The
highest dilution showing at least 99 % inhibition is taken
as MIC.
3-(3-Pyridyl)-5-(4-chlorophenyl)-4-(N-(5,6-dichloro)-1,3-ben-
zothiazol-2-amino)-4H-1,2,4-triazole 9i Yield 68 %; m.p.
1
192–194 °C; IR (KBr): 3385 (NH), 1664 (C=N) cm-1; H
NMR (CDCl3) d 7.87 (s, 1H, NH), 9.31 (s, 1H, CH), 8.80
(dd, 1H, J = 3.8 Hz, CH), 8.43 (d, 1H, J = 8.06 Hz, CH),
7.51 (t, 1H, CH), 8.11 (d, 2H, J = 8.48 Hz, 2CH), 7.46 (d,
2H, J = 8.16 Hz, 2CH), 7.61 (s, 1H, CH), 7.67 (s, 1H,
CH); 13C NMR (CDCl3) d 164.54 (C3-triazole), 162.70
(C5-triazole), 155.35, 152.19, 149.14, 147.70, 144.86,
137.49, 134.21, 133.36, 130.08, 131.75, 129.41, 128.82,
124.48, 123.78, 123.90, 121.10 (aromatic ring) ppm; MS
(m/z): 471 (M?), 473 (M?2), 477 (M?4), 483 (M?6);
Anal. Calcd. for C20H11N6Cl3S: C, 50.70; H, 2.34; N,
17.74. Found: C, 50.67; H, 2.37; N, 17.72 %.
3-(3-Pyridyl)-5-(4-chlorophenyl)-4-(N-4-chloro-1,3-benzo-
thiazol-2-amino)-4H-1,2,4-triazole 9j Yield 60 %; m.p.
In vitro evaluation of antitubercular activity
1
160–162 °C; IR (KBr): 3376 (NH), 1649 (C=N) cm-1; H
Drug susceptibility and determination of MIC of the test
compounds against M. tuberculosis H37Rv were per-
formed by Lowenstein-Jensen agar (MIC) method (Rat-
tan, 2000; Castagnolo et al., 2009) where primary 1,000,
500 and 250 lg/ml and secondary 200, 100, 62.5, 50, 25,
12.5, 6.25 and 3.25 lg/ml dilutions of each test com-
pound were added liquid L. J. Medium and then media
were sterilized by inspissations method. A culture of
M. tuberculosis H37Rv growing on Lowenstein-Jensen
medium was harvested in 0.85 % saline in bijou bottles.
All test compounds starting from a stock solution of
2,000 lg/ml concentration of compounds were prepared
in DMSO. These tubes were then incubated at 37 °C for
24 h followed by streaking of M. tuberculosis H37Rv
(5 9 104 bacilli per tube). These tubes were then incu-
bated 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 colo-
nies occurred or \20 colonies noted was taken as MIC
concentration of test compound. The standard strain
M. tuberculosis H37Rv was tested with known drug
rifampicin.
NMR (CDCl3) d 7.86 (s, 1H, NH), 9.31 (s, 1H, CH), 8.80
(dd, 1H, J = 4.0 Hz, CH), 8.45 (d, 1H, J = 8.36 Hz, CH),
7.53 (t, 1H, CH), 8.11 (d, 2H, J = 8.36 Hz, 2CH), 7.37 (d,
2H, J = 8.08 Hz, 2CH), 6.89–7.32 (m, 3H, benzothiazole-
H); 13C NMR (CDCl3) d 164.61 (C3-triazole), 162.60 (C5-
triazole), 155.59, 151.92, 148.94, 146.72, 144.86, 137.44,
134.20, 132.40, 129.62, 131.59, 128.47, 124.17, 123.18,
122.10, 120.29, 119.18 (aromatic ring) ppm; MS (m/z): 438
(M?), 440 (M?2), 444 (M?4); Anal. Calcd. for
C20H12N6Cl2S: C, 54.68; H, 2.75; N, 19.13. Found: C,
54.72; H, 2.72; N, 19.16 %.
In vitro evaluation of antimicrobial activity
The MICs of synthesized compounds were carried out by
the broth microdilution method. DMSO was used as dil-
uents to get desired concentration of drugs to test upon
standard bacterial strains. Serial dilutions were prepared in
primary and secondary screening. The control tube con-
taining no antibiotic was immediately sub cultured (before
inoculation) by spreading a loop evenly over a quarter of
plate of medium suitable for the growth of the test
°
organism and put for incubation at 37 C overnight. The
123

1328
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described (Pauwels et al., 1988). Stock solution (109 final
concentration) of the test compound was added in 25-lL
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HIV-1(IIIB) or HIV-2 (ROD) stock (50 lL) at
100–300 CCID50 (50 % cell culture infectious dose) or
culture medium was added to microtiter tray wells. Mock-
infected cells were used to evaluate the effect of test com-
pound on uninfected cells in order to assess the cytotoxicity
of the test compound. Exponentially growing MT-4 cells
were centrifuged for 5 min at 1,000 rpm, and the supernatant
was discarded. The MT-4 cells were resuspended at
66 9 105 cells/mL, and an amount of 50-lL volume was
transferred to the microtiter tray wells. Five days after
infection, the viability of mock- and HIV-infected cells were
examined spectrophotometrically by the MTT assay.
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Acknowledgments The authors are thankful to Veer Narmad South
Gujarat University for providing necessary facilities and UGC for
providing research fellowship. We also thank SAIF Lucknow for
elemental analysis, SAIF Chandigarh for spectral analysis of the
compounds and Rega Institute for Medical Research for Anti-HIV
screening.
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