Synthesis and comparative study of anti-mycobacterium activity of a novel series of fluoronitrobenzothiazolopyrazoline regioisomers

Article abstract of DOI:10.1002/ardp.201100072

In an attempt to find a new and a safer drug for tuberculosis, we have synthesized a series of fluoronitrobenzothiazolopyrazolines for antitubercular activity. The series comprises three subclasses: fluorobenzothiazolopyrazolines (11a-f), fluoronitrobenzo

Full text of DOI:10.1002/ardp.201100072

Arch. Pharm. Chem. Life Sci. 2012, 345, 137–146
137
Full Paper
Synthesis and Comparative Study of Anti-Mycobacterium
Activity of a Novel Series of Fluoronitrobenzothiazolo-
pyrazoline Regioisomers
Kuntal Hazra1, L. V. G. Nargund1, P. Rashmi1, J. N. Narendra Sharath Chandra1, B. Nandha2,
and M. S. Harish3
1 Department of Pharmaceutical Chemistry, Nargund College of Pharmacy, Bangalore, Karnataka, India
2 Department of Pharmaceutical Chemistry, Vivekananda College of Pharmacy, Bangalore Karnataka, India
3 Department of Pharmacology, Nargund College of Pharmacy, Bangalore, Karnataka, India
In an attempt to find a new and a safer drug for tuberculosis, we have synthesized a series of
fluoronitrobenzothiazolopyrazolines for antitubercular activity. The series comprises three
subclasses: fluorobenzothiazolopyrazolines (11a–f), fluoronitrobenzothiazolopyrazoline, nitro
group at 5th position (12a–f) and 4th position (13a–f). All compounds were screened for their
in-vitro antitubercular activity against Mycobacterium tuberculosis H37Rv strain by using Middlebrook
7H-9 broth. An introduction of –NO2 group at 5th position of benzothiazole ring (12a–f) increased the
antitubercular activity whereas introduction of –NO2 group at 4th position (13a–f) was found to
decrease the activity remarkably. Two compounds from each series showing good antitubercular
activity were tested for cytotoxicity on THP-1 cell lines and they showed low cytotoxicity.
Keywords: Antitubercular activity / ATP phosphoribosyl transferase / Benzothiazole / Pyrazoline / Regioisomers
Received: February 23, 2011; Revised: June 23, 2011; Accepted: June 24, 2011
DOI 10.1002/ardp.201100072
Introduction
course (DOTS) supervision; (b) it should improve the treat-
ment of multidrug-resistant tuberculosis (MDR-TB), which
cannot be treated with Isoniazid (INH) and Rifampin (RIF)
and/or (c) it should provide more effective treatment of
latent/dormant TB infection, which is essential for eliminat-
ing the TB.
There were an estimated 11.1 million (range 9.6–13.3 million)
prevalent cases of Tubercle bacillus (TB) in 2008, equivalent to
164 cases per 100 000 population [1]. India and China alone
account for an estimated 35% of TB cases worldwide. Of the
9.4 million incident cases in 2008, an estimated 1.2–1.6
million (13–16%) were HIV-positive, as their immune systems
are compromised by immunosuppressive drugs, substance
abuse, or AIDS [2–5].
In the recent years nitrobenzothiazole has been discovered
as an inhibitor for Mycobacterium tuberculosis ATP phosphori-
bosyl transferase (HisG) [6]. HisG is an ATP phosphoribosyl
transferase (ATP-PRTase) that catalyzes the first step in the
biosynthetic pathway for histidine, which also leads to inter-
mediates that play a significant role in purine biosynthesis.
HisG condenses ATP with phosphoribosyl pyrophosphate
(PRPP) to produce phosphoribosyl ATP (PR-ATP) and inorganic
pyrophosphate (PPi). Histidine biosynthesis is metabolically
expensive, requiring 10 enzymatic reactions and consuming
an estimated 41 ATP molecules. Therefore the pathway is
tightly regulated through negative feedback via allosteric
inhibition of HisG by histidine. Partly due to its central role
in metabolism and its uniqueness to prokaryotes and lower
eukaryotes, HisG represents a potential drug target for com-
bating tuberculosis. HisG in Mycobacterium tuberculosis has an
a/b-fold containing three domains: two N-terminal catalytic
As a result there is a dire need to develop novel, faster
acting chemotherapeutics with a lower toxicity for the treat-
ment of tuberculosis. A new TB treatment should offer at
least one of three improvements over the existing regimens:
(a) It should shorten the total duration of effective treatment
and/or significantly reduce the total number of doses
needed to be taken under Directly Observed Therapy Short-
Correspondence: Kuntal Hazra, Department of Pharmaceutical
Chemistry, Nargund College of Pharmacy, 2nd Main, Dattatreyanagar,
100 Ft. Ring Road, B.S.K. III Stage, Bangalore-560085, India.
E-mail: kuntalhazra@gmail.com
Fax: þ91-080-26421903
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138
K. Hazra et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 137–146
domains, with a large, relatively solvent exposed active site
formed between them, and a C-terminal regulatory domain
[7]. The active site is formed by residues contributed
by both domains I and II and contains putative binding
sites for both ATP and PRPP, inferred based on co-crystal
structures of complexes with AMP, PRPP and PR-ATP (product)
[7–9]. Domain II contains a 13-residue signature sequence
(residues 149–161) associated with binding PRPP, whereas
conserved residues for ATP-binding are primarily found in
domain I on the opposing face of the cleft. Reaction follows
ordered bi-bi kinetics, with sequential binding of ATP
followed by PRPP, condensation, and then release of
products PPi and PR-ATP [10]. The C-terminal domain (III)
J ¼ 7.60 Hz, NH at d 9.16 ppm singlet 1 proton and CH3 at
d 2.34 ppm singlet 3 protons. Whereas compound 7 showed
(in DMSO-d6) absorption of 5th proton at d 8.40–8.38 ppm as
doublet due to C–F coupling accounting for 1 proton,
J ¼ 9.44 Hz, NH at d 13.21 ppm singlet 1 proton and CH3
at d 2.27 ppm singlet 3 protons. Compounds 5, 6 & 7 were
treated with different aromatic aldehydes in basic medium as
in Claisen-Schmidt condensation yielding 8a–f, 9a–f & 10a–f,
respectively (chalcones). The presence of aromatic peak in the
aromatic region, vinylic peak in d 4.5 to 5.5 ppm and absence
of CH3 peak in between d 2 to 3 ppm showed the formation of
chalcones. The synthesis of target compounds 11a–f, 12a–f &
13a–f was carried out by refluxing 8a–f, 9a–f & 10a–f with
hydrazino benzothiazole (4) in alcohol. Presence of singlet in
between d 3 to 5.5 ppm for pyrazoline hydrogen showed the
formation of target compounds. This was further confirmed
by the mass spectra and CHN elemental analysis.
˚
binds histidine at a site approximately 40 A away from the
active site, which causes a rotation between domains and
a conformational shift in packing within the hexameric
complex [7], which ultimately inhibits catalytic activity by
reducing kcat [11].
The pyrazoline derivatives constitute an interesting class
of organic compounds, which are associated with diverse
chemical and pharmacological properties [12–13].
Fluorine has been incorporated into the molecule [14].
Fluorine, being the second smallest substituent next to
hydrogen, closely mimics hydrogen in enzyme receptor inter-
actions. The substitutions of fluorine by hydrogen increases
lipid solubility which in turn increases the transport and
absorption of drug in vivo, and a strong electron withdrawing
inductive effect of fluorine can significantly influence reac-
tivity and stability of functional groups and the reactivity of
neighboring reaction centers.
Antitubercular activity
The results of the in-vitro evaluation of antituberculosis
activity are reported in Table 1. The compounds were eval-
uated against M. tuberculosis H37Rv using Middlebrook 7H-9
broth. The ability of compounds to inhibit the growth of
Mycobacterium species was determined by Ziehl-Neelsen stain-
ing. Compounds 12b, 12c and 12d have shown 100% inhi-
bition at 25 mg/mL, 11b, 11c and 11d at 50 mg/mL and 13b,
13c and 13d at 100 mg/mL, whereas the standard drug pyr-
azinamide and streptomycin showed 100% inhibition at
7.5 mg/mL. Compounds having a nitro group in 5th position
(12a–f ) of the benzothiazole ring show a better antitubercular
activity than they do without any nitro group (11a–f ) in the
benzothiazole ring. The nitro group at the 4th position (13a–
f ) showed a reduced antitubercular activity than 5th position
nitro compound as well as without a nitro group in the
structure. The antitubercular activity of compounds with
presence of nitro group in 5th position (12a–f ) may be
because this series of compounds packs in the part of the
active site responsible for binding the PRPP substrate [6]. This
may bind with the Tyr116 in the 1NH8 receptor, which is
thought to interact with ATP, along with Glu141 and Asp154,
which are expected to interact with PRPP. Compounds with-
out any nitro group (11a–f ) and a nitro group at the 4th
position (13a–f ) in the benzothiazole ring probably missed
the binding site. Electron donating substituent in the
Drawing on structural knowledge obtained from our past
work we herein synthesized fluorobenzothiazolopyrazolines,
and fluoronitrobenzothiazolopyrazolines (11a–f, 12a–f and
13a–f ) for their antitubercular activity study and cytotoxicity
study of some of the compounds.
Results and discussion
Chemistry
The preparation of final compounds 11a–f, 12a–f and 13a–f
was accomplished by synthetic sequence illustrated in
Scheme 1. The compound 3 was synthesized from 4-fluoro-
3-chloro aniline and potassium thiocyanate with bromine in
acetic acid. The starting material then was treated with
hydrazine hydrate and conc. HCl in the presence of ethylene
glycol yielding compound 4. Alternatively the compound 3
was again treated with acetic anhydride to get compound 5.
Upon nitration with conc. HNO3 & conc. H2SO4 afforded 6 & 7
two regioisomers. The NMR spectra (in CDCl3) of compound 6
showed absorption of 4th proton at d 7.75–7.73 ppm as
doublet due to C–F coupling accounting for 1 proton,
aromatic ring showed
a good antitubercular activity.
4-OH-3-OCH3, 4-OCH3, 4-OH showed good antitubercular
activity, whereas 2-OH substituent does not show any anti-
tubercular activity, irrespective of nitro substituent. This may
conclude that nitro group at 5th position of benzothiazole
ring and 4-OH-3-OCH3, 4-OCH3 and 4-OH group in benzene
ring in fluoronitrobenzothiazolopyrazolines moiety enhanced
antitubercular activity.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 137–146
Anti-Mycobacterium Activity of Novel Fluoronitrobenzothiazolopyrazolines
139
NH2
Br2, CH3COOH
KSCN
NH2.NH2.H2O/HCl
Ethylene glycol
N
N
5oC
F
S
N NH2
H
Cl
F
S
NH2
Cl
F
Cl
3
4
1
2
(CH3CO)2O
N
F
S
NHCOCH3
Cl
H2SO4, HNO3
H2SO4, HNO3
5
NO2
O2N
N
N
F
S
NHCOCH3
CHO
F
S
NHCOCH3
CHO
CHO
R
Cl
6
Cl
7
R
O2N
NO2
N
N
R
O
O
N
F
S
NH C CH CH
F
S
8a-f
NH C CH CH
O
Cl
Cl
F
S
NH C CH CH
9a-f
Cl
10a-f
R
R
N
N
N
R
F
S
N
H
NH2
F
S
N
H
NH2
F
S
N NH2
H
Cl
N
Cl
N
Cl
N
N
N
N
NO2
F
S
N
N
NO2
F
F
S
F
S
N
N
S
N
N
Cl
N
H
Cl
Cl
N
H
N
H
S
S
F
F
R
12a-f
R
R
Cl
Cl
Cl
13a-f
11a-f
R = 11-13a = 4-N(CH3)2, 11-13b = 4-OH, 11-13c = 4-OH, 3-OCH3, 11-13d = 4-OCH3, 11-13e = 2-OH, 11-13f
= H.
Scheme 1. Synthesis of 11a–f, 12a–f and 13a–f.
In-vitro cytotoxicity evaluation
The six compounds were tested for cytotoxicity (CTC50
the nitro group, whereas a nitro group at 4th position and
compounds that do not have any nitro group show less
cytotoxicity. All compounds have higher antitubercular
activity against M. tuberculosis as compared to cytotoxic
activity in eukaryotic cell.
)
in THP-1 cell lines at concentrations 1000, 500, 250 and
125 mg/mL. Table 2 shows the cytotoxicity to the host
cells of compounds 11b, 11c, 12b, 12c, 13b and 13c.
Compounds 12b & 12c were found to be cytotoxic at the
CTC50 values 0.324, 0.300 mM, though this concentration Conclusion
is much higher than the concentration used for antituber-
cular activity. Other compounds have shown comparatively
less cytotoxicity. In the compounds 12b & 12c carrying nitro
group at 5th position, the cytotoxicity may be the position of
In the present study, we have synthesized regioisomers of
fluoronitrobenzothiazolopyrazoline, and their activity on
Mycobacterium tuberculosis H37Rv strain was compared to the
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K. Hazra et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 137–146
Table 1. In-vitro antitubercular evaluation.
SL. No.
Comp. No.
R
Antitubercular activity (MIC)
Min. conc. in mM
showed activity
25 mg/mL
50 mg/mL
100 mg/mL
1
2
3
4
5
6
7
8
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
13a
13b
13c
13d
4-N(CH3)2
4-OH
4-OH-3-OCH3
4-OCH3
2-OH
R
R
R
R
R
R
R
S
S
S
R
R
R
R
R
R
R
S
S
S
S
S
S
R
R
S
S
S
S
S
R
R
S
S
S
R
R
0.174
0.091
0.086
0.089
——
S
R
R
S
S
S
-H
——
4-N(CH3)2
4-OH
4-OH-3-OCH3
4-OCH3
2-OH
0.080
0.042
0.040
0.041
0.084
——
9
10
11
12
13
14
15
16
17
18
19
20
S
S
-H
R
R
R
R
R
R
R
4-N(CH3)2
4-OH
4-OH-3-OCH3
4-OCH3
2-OH
——
0.168
0.160
0.165
——
——
0.060
0.014
13e
13f
Pyrazinamide
Streptomycin
R
R
-H
7.5 mg/mL
7.5 mg/mL
R ¼ resistant; S ¼ sensitive
standard drugs streptomycin and pyrazinamide. Two
possible regioisomers of 4th and 5th positions of benzothia-
zole ring were produced during the nitration of N-(7-chloro-6-
fluorobenzo[d]thiazol-2-yl)acetamide (5), and both the com-
pounds were separated. The final compounds fluorobenzo-
thiazolopyrazolines (11a–f), fluoronitrobenzothiazolopyrazoline
in which nitro group at 5th position (12a–f) and fluoronitro-
benzothiazolopyrazoline, nitro group at 4th position (13a–f)
were tested for in-vitro antitubercular activity against
Mycobacterium tuberculosis H37Rv strain. Compounds with a
nitro substituent have shown better activity than those with-
out a nitro group. Nitro group at position 5th of the benzo-
thiazole ring has shown a better activity compared to 4th
position, but nitro group at 5th position is more cytotoxic
than 4th position. Electron donating substituent in the aro-
matic ring shows
a
better antitubercular activity.
Introduction of nitro group at 5th position has shown higher
cytotoxicity where as introduction of nitro group at 4th has
not shown any significant increase in cytotoxicity.
Experimental
Table 2. In-vitro cytotoxicity evaluation on THP-1 cell line.
Chemistry
Sl. No. Comp.
No.
R
CTC50, mg/mL
CTC50, mM
The melting points were determined with an electrothermal
melting point apparatus and are uncorrected. Infrared spectra
(KBr disc) were performed on FTIR-8400 Shimadzu and the fre-
quencies were expressed in cmꢀ1. 1H-NMR spectra were recorded
on Bruker-Avance 400 MHz instrument with TMS (0 ppm) as an
internal standard; the chemical shifts (d) are reported in ppm
and coupling constants (J) are given in Hertz (Hz). Signal multi-
plicities are represented by s (singlet), d (doublet), t (triplet),
ds (double singlet), dd (double doublet), m (multiplet) and
1
2
3
4
5
6
11b
11c
12b
12c
13b
13c
4-OH
4-OH-3-OCH3
4-OH
4-OH-3-OCH3
4-OH
4-OH-3-OCH3
447.00
423.50
192.00
187.50
432.50
412.50
0.817
0.733
0.324
0.300
0.730
0.663
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Arch. Pharm. Chem. Life Sci. 2012, 345, 137–146
Anti-Mycobacterium Activity of Novel Fluoronitrobenzothiazolopyrazolines
141
br s (broad singlet). Mass spectra were recorded on ESI-MS,
Thermo, Finnigan LCQ deca xp max. Elemental analyses were
performed on Perkin-Elmer 2400 CHN elemental analyzer.
Analyses indicated by the symbols of the elements of functions
were within ꢁ0.4% of the theoretical values. The purity of the
compounds was checked on Merck precoated silica gel 60 F-254.
Column chromatography was performed using P.D. fine chem.
silica gel (100–200 mesh). Yields were not optimized. All the
solvents and reagents were used without further purification.
Synthesis of N-(7-chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-
yl)acetamide & N-(7-chloro-6-fluoro-5-nitrobenzo[d]-
thiazol-2-yl)acetamide 6 & 7
A mixture of compound 5, 100 mg (0.000409 mol), and 0.3 mL of
ice-cold conc. H2SO4 was stirred under ice-cooled condition. To
this 0.1 mL conc. HNO3 was added drop-wise, continued to be
stirred at room temperature for 2 h. Then, 0.1 mL conc. HNO3
was further added to the reaction mixture and stirred overnight.
The reaction mixture was poured into a large amount of water.
The solids obtained were filtered and washed with water
thoroughly and dried under vacuum. The compound obtained
was a mixture of 6 & 7, which was separated by column chroma-
tography employing n-hexane/ethyl acetate (9:1) as an eluent.
Synthesis of 7-chloro-6-fluorobenzo[d]thiazol-2-amine 3
To glacial acetic acid (40 mL) precooled at 58C were added 40 g
(0.4123 mol) potassium thiocyanate and 7.25 g (0.0498 mol) of
4-fluoro-3-chloro aniline. The mixture was stirred, during which
6 mL of bromine in 24 mL of glacial acetic acid was added at such
a rate that the temperature was not allowed to rise beyond 58C,
for a period of 2 h. The stirring was continued for an additional
2 h at the same temperature, and further at room temperature
for 10 h. It was allowed to stand overnight during which an
orange precipitate was settled at the bottom. 30 mL of water was
added and slurry was heated at 858C on a steam bath and filtered
hot. The filtrate was cooled and neutralized with strong ammo-
nia solution to pH 6, a light yellow precipitate obtained was
collected. The resulting product was recrystallized by toluene.
N-(7-Chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-yl)-
acetamide 6
Yield 56%; mp 297–2988C; buff color crystalline solid; IR (nmax
,
cmꢀ1, KBr): 3301 (N–H), 1535 (NO ), 1680 (C O), 1186 (C–F), 661
–
–
2
(C–Cl); 1H-NMR (400 MHz, CDCl3), d (ppm): 2.34 (s, 3H, CH3), 7.75–
7.73 [d, 1H, ArH, J ¼ 7.60 Hz (C–F)], 9.16 (s, 1H, NH); ESI-MS,
m/z: 288.99 [M]þ, 290.97 [Mþ2]þ; anal. calcd. for C9H5ClFN3O3S:
C, 37.32; H, 1.74; N, 14.51. Found: C, 37.30; H, 1.76; N, 14.50.
Yield 76%; slight yellowish crystalline; mp 180–1828C; IR (nmax
,
N-(7-Chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-yl)-
acetamide 7
cmꢀ1, KBr): 3480 (N–H), 1199 (C–F), 681 (C–Cl); 1H-NMR (400 MHz,
DMSO-d6), d (ppm): 4.21 (s, 2H, NH2), 7.62–7.60 (d, 1H, J ¼ 7.22 Hz,
ArH), 7.70–7.68 (d, 1H, ArH, J ¼ 8.59 Hz); ESI-MS, m/z: 201.98 [M]þ,
203.86 [Mþ2]þ; anal. calcd. for C7H4ClFN2S: C, 41.49; H, 1.99;
N, 13.82. Found: C, 41.51; H, 2.03; N, 13.81.
Yield 30%; mp 336–3378C; white crystalline solid; IR (nmax, cmꢀ1
,
–
KBr): 3311 (N–H), 1530 (NO ), 1680 (C O), 1185 (C–F), 665 (C–Cl);
–
2
1H-NMR (400 MHz, DMSO-d6), d (ppm): 2.27 (s, 3H, CH3), 8.40–8.38
(d, 1H, ArH, J ¼ 9.44 Hz (C–F)), 13.21 (s, 1H, NH); ESI-MS,
m/z: 288.95 [M]þ, 290.95 [Mþ2]þ; anal. calcd. for C9H5ClFN3O3S:
C, 37.32; H, 1.74; N, 14.51. Found: C, 37.31; H, 1.76; N, 14.52.
Synthesis of 7-chloro-6-fluoro-2-hydrazinylbenzo[d]-
thiazole 4
10 mL of conc. HCl was added drop-wise with stirring to 10 mL
(0.3 mol) hydrazine hydrate at 5–108C followed by ethylene
glycol 40 mL. To the above solution 2.025 g (0.01 mol) of com-
pound 3 in portion was added and the resulting mixture was
refluxed for 2 h. On cooling solid separated out, was filtered and
washed with water, dried and recrystallized from ethanol.
Yield 66%, mp 217–2198C, light brown coloured needle shaped
crystals; IR (nmax, cmꢀ1, KBr): 3380 (N–H), 1200 (C–F), 683 (C–Cl);
1H-NMR (400 MHz, DMSO-d6), d (ppm): 5.09 (s, 2H, NH2), 7.42–7.41
(d, 1H, ArH, J ¼ 6.64 Hz), 7.81–7.79 (d, 1H, ArH, J ¼ 9.24 Hz), 9.20
(s, 1H, NH); ESI-MS, m/z: 217.02 [M]þ, 219.05 [Mþ2]þ; anal. calcd.
for C7H5ClFN3S: C, 38.63; H, 2.32; N, 19.31. Found: C, 38.61;
H, 2.34, N, 19.31.
Synthesis of N-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-3-
substituted phenyl acrylamide 8a–f
A mixture of compound 5, 2.445 g (0.01 mol), in ethanol (25 mL) and
8 mL of 10% NaOH was stirred at room temperature. To this mixture
equimolar quantity of substituted aromatic aldehyde (0.01 mol) was
added in small portions. Stirring was continued for overnight in
room temperature. The excess of solvent was distilled off, cool, filter
and residue was thoroughly washed with cold water. The product
was then recrystallized from acetone/water mixture.
N-(7-Chloro-6-fluorobenzo[d]thiazol-2-yl)-3-
(4-dimethylamino)phenyl)acrylamide 8a
Yield 89%; mp 140–1428C; yellowish needle shaped crystal; IR
Synthesis of N-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-
acetamide 5
A mixture of compound 3, 2.025 g (0.01 mol), and 10 mL of
acetic anhydride was refluxed for 1 h. After that the reaction
mixture was cooled, the separated out solid was heated with
water, filtered and washed with water. The product was then
recrystallized in ethanol.
(nmax, cmꢀ1, KBr): 3306 (N–H), 1664 (C O), 1170 (C–F), 667 (C–Cl);
–
–
1H-NMR (200 MHz, CDCl3), d (ppm): 3.09 (s, 6H, CH3), 4.58–4.35
(dd, 2H, CH, J ¼ 7.34, 7.39 Hz), 6.73–6.68 (d, 2H, ArH,
J ¼ 8.88 Hz), 7.39–7.35 (d, 1H, ArH, J ¼ 8.35 Hz), 7.56–7.54
(d, 1H, ArH, J ¼ 7.50 Hz), 7.76–7.72 (d, 2H, ArH, J ¼ 8.88 Hz),
9.74 (s, 1H, NH); ESI-MS, m/z: 375.09 [M]þ, 377.09 [Mþ2]þ; anal.
calcd. for C18H15ClFN3OS: C, 57.52; H, 4.02; N, 11.18. Found:
C, 57.53; H, 4.03; N, 11.17.
Yield 98%, mp 232–2338C, whitish needle shaped crystal; IR
(nmax, cmꢀ1, KBr): 3318 (N–H), 1681 (C O), 1189 (C–F), 652 (C–Cl);
–
–
1H-NMR (400 MHz, DMSO-d6), d (ppm): 2.34 (s, 3H, CH3), 7.26–7.24
(d, 1H, ArH, J ¼ 7.57 Hz), 7.75–7.73 (d, 1H, ArH, J ¼ 7.60 Hz), 9.05
(s, 1H, NH); ESI-MS, m/z: 244.03 [M]þ, 246.02 [Mþ2]þ; anal. calcd.
for C9H6ClFN2OS: C, 44.18; H, 2.47; N, 11.45. Found: C, 44.17;
H, 2.49; N, 11.44.
N-(7-Chloro-6-fluorobenzo[d]thiazol-2-yl)-3-
(4-hydroxyphenyl) acrylamide 8b
Yield 83%; mp 227–2298C; buff colored powder; IR (nmax, cmꢀ1
,
KBr): 3480 (N–H), 3152 (O–H), 1647 (C O), 1189 (C–F), 664 (C–Cl);
–
–
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K. Hazra et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 137–146
1H-NMR (400 MHz, DMSO-d6), d (ppm): 4.15 (s, 1H, OH), 4.54–5.52
(d, 1H, CH, J ¼ 7.32 Hz), 4.68–4.66 (d, 1H, CH, J ¼ 7.30 Hz), 7.19–
6.92 (d, 2H, ArH, J ¼ 8.71 Hz), 7.49–7.25 (d, 2H, ArH, J ¼ 8.78 Hz),
7.56–7.52 (d, 1H, ArH, J ¼ 8.50 Hz), 7.78–7.76 (d, 2H, ArH,
J ¼ 8.82 Hz), 9.65 (s, 1H, NH); ESI-MS, m/z: 348.07 [M]þ, 350.05
[Mþ2]þ; anal. calcd. for C16H10ClFN2O2S: C, 55.10; H, 2.89; N, 8.03.
Found: C, 55.12; H, 2.90; N, 8.02.
distilled off, cooled, filtered and the residue was thoroughly
washed with cold water. The product was then recrystallized
from acetone-water mixture.
N-(7-Chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-yl)-3-
(4-(dimethylamino)phenyl)acrylamide 9a
Yield 71%; mp 286–2888C; light orange crystal; IR (nmax, cmꢀ1
,
–
KBr): 3429 (N–H), 1537 (NO ), 1688 (C O), 1194 (C–F), 667 (C–Cl);
–
2
1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.12 (s, 6H, CH3), 5.01–4.29
(d, 1H, CH, J ¼ 7.65 Hz), 5.28–5.09 (d, 1H, CH, J ¼ 7.68 Hz), 7.72–
7.70 (m, 4H, ArH), 7.76–7.75 (d, 1H, ArH, J ¼ 7.62 Hz), 9.50 (s, 1H,
NH); ESI-MS, m/z: 420.02 [M]þ, 422.02 [Mþ2]þ; anal. calcd.
for C18H14ClFN4O3S: C, 51.37; H, 3.35; N, 13.31. Found:
C, 51.40; H, 3.37; N, 13.30.
N-(7-Chloro-6-fluorobenzo[d]thiazol-2-yl)-3-(4-hydroxy-3-
methoxyphenyl)acryl-amide 8c
Yield 81%; mp 185–1878C; white powder; IR (nmax, cmꢀ1, KBr):
–
3485 (N–H), 3095 (OH), 1685 (C O), 1250 C–O), 1200 (C–F), 685
–
(C–Cl); 1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.02 (s, 3H, OCH3),
4.22 (s, 1H, OH), 5.03–4.90 (d, 1H, J ¼ 7.44 Hz, CH), 5.16 (d, 1H,
CH, J ¼ 7.40Hz), 7.76–7.73 (m, 3H, ArH), 7.96–7.94 (d, 2H, ArH,
J ¼ 8.26 Hz), 9.85 (s, 1H, NH); ESI-MS, m/z: 378.06 [M]þ, 380.07
[Mþ2]þ; anal. calcd. for C17H12ClFN2O3S: C, 53.90; H, 3.19; N, 7.40.
Found: C, 53.92; H, 3.21; N, 7.39.
N-(7-Chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-yl)-3-
(4-hydroxyphenyl)acrylamide 9b
Yield 68%; mp 240–2428C; buff color powder; IR (nmax, cmꢀ1, KBr):
–
–
3450 (N–H), 3198 (O–H), 1688 (C O), 1629 (C C), 1538 (NO ), 1196
–
–
2
(C–F), 640 (C–Cl); 1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.95
(s, 1H, OH), 4.85–4.83 (d, 1H, CH, J ¼ 8.65 Hz), 5.02–4.94
(d, 1H, CH, J ¼ 8.32 Hz), 7.62–7.60 (m, 4H, ArH), 7.87–7.85
(d, 1H, ArH, J ¼ 7.61 Hz), 9.61 (s, 1H, NH); ESI-MS, m/z: 393.04
[M]þ, 395.06 [Mþ2]þ; anal. calcd. for C16H9ClFN3O4S: C, 48.80;
H, 2.30; N, 10.67. Found: C, 48.85; H, 2.33; N, 10.65.
N-(7-Chloro-6-fluorobenzo[d]thiazol-2-yl)-3-
(4-methoxyphenyl)acrylamide 8d
Yield 87%; mp 175–1788C; orange powder; IR (nmax, cmꢀ1, KBr):
–
3485 (N–H), 1680 (C O), 1270 (C–F), 675 (C–Cl), 1260 C–O);
–
1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.20 (s, 3H, OCH3), 4.62–
4.58 (d, 1H, J ¼ 7.93 Hz, CH), 5.28–5.26 (d, 1H, CH, J ¼ 7.49 Hz),
7.78–7.52 (m, 4H, ArH), 7.82–7.80 (d, 2H, ArH, J ¼ 7.08 Hz), 9.87
(s, 1H, NH); ESI-MS, m/z: 362.08 [M]þ, 364.09 [Mþ2]þ; anal. calcd.
for C17H12ClFN2O2S: C, 56.28; H, 3.33; N, 7.72. Found: C, 56.29;
H, 3.35; N, 7.71.
N-(7-Chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-yl)-3-
(4-hydroxy-3-methoxyphenyl) acrylamide 9c
Yield 73%; mp 275–2788C; yellowish crystal; IR (nmax, cmꢀ1, KBr):
–
3452 (N–H), 3191 (O–H), 1695 (C O), 1537 (NO ), 1258 & 1050
–
2
(C–O), 1202 (C–F), 637 (C–Cl); 1H-NMR (400 MHz, DMSO-d6),
d (ppm): 3.52 (s, 3H, OCH3), 3.93 (br s, 1H, OH), 4.81–4.79
(d, 1H, CH, J ¼ 6.61 Hz), 5.02–4.93 (d, 1H, CH, J ¼ 6.24 Hz),
7.64–7.61 (m, 3H, ArH), 7.80–7.79 (d, 1H, ArH, J ¼ 7.62 Hz),
9.69 (s, 1H, NH), ESI-MS, m/z: 423.07 [M]þ, 425.07 [Mþ2]þ; anal.
calcd. for C17H11ClFN3O5S: C, 48.18; H, 2.62; N, 9.92. Found:
C, 48.21; H, 2.66; N, 9.94.
N-(7-Chloro-6-fluorobenzo[d]thiazol-2-yl)-3-
(2-hydroxyphenyl) acrylamide 8e
Yield 56%; mp 178–1818C; ash colored powder; IR (nmax, cmꢀ1
,
–
KBr): 3487 (N–H), 3095 (O–H), 1695 (C O), 1210 (C–F), 680 (C–Cl);
–
1H-NMR (400 MHz, DMSO-d6), d (ppm): 9.25 (s, 1H, NH), 7.86–7.84
(d, 2H, ArH, J ¼ 7.42 Hz), 7.46–7.42 (m, 4H, ArH), 4.77–4.73 (d, 1H,
CH, J ¼ 7.85 Hz), 5.72 (d, 1H, CH, J ¼ 7.48 Hz), 4.09 (s, 1H, OH);
ESI-MS, m/z: 348.09 [M]þ, 350.11 [Mþ2]þ; anal. calcd.
for C16H10ClFN2O2S: C, 55.10; H, 2.89; N, 8.03. Found: C, 55.11;
H, 2.91; N, 8.02.
N-(7-Chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-yl)-3-
(4-methoxyphenyl)acrylamide 9d
Yield 77%; mp 222–2268C; yellow powder; IR (nmax, cmꢀ1, KBr):
N-(7-Chloro-6-fluorobenzo[d]thiazol-2-yl)-3-
phenylacrylamide 8f
–
3442 (N–H), 1668 (C O), 1523 (NO ), 1252 & 1051 (C–O), 1197
–
2
(C–F), 620 (C–Cl); 1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.62
(s, 3H, OCH3), 4.91–4.86 (d, 1H, CH, J ¼ 7.29 Hz), 5.03–5.01
(d, 1H, CH, J ¼ 7.32 Hz), 7.78–7.75 (m, 4H, ArH), 7.90–7.88
(d, 1H, ArH, J ¼ 7.63 Hz), 10.00 (s, 1H, NH); ESI-MS, m/z: 407.02
[M]þ, 409.09 [Mþ2]þ; anal. calcd. for C17H11ClFN3O4S: C, 50.07; H,
2.72; N, 10.30. Found: C, 50.11; H, 2.76; N, 10.33.
Yield 58%; mp 212–2148C; orange crystalline solid; IR
(nmax, cmꢀ1, KBr): 3489 (N–H), 1705 (C O), 1203 (C–F), 676
–
–
(C–Cl); 1H-NMR (400 MHz, DMSO-d6), d (ppm): 4.40–4.36 (d, 1H,
CH, J ¼ 7.31 Hz), 4.49–4.43 (d, 1H, CH, J ¼ 7.29 Hz), 7.56–7.55
(m, 5H, ArH), 7.78–7.77 (d, 2H, ArH, J ¼ 8.22 Hz), 9.64 (s, 1H, NH);
ESI-MS, m/z: 332.08 [M]þ, 334.07 [Mþ2]þ; anal. calcd.
for C16H10ClFN2OS: C, 57.75; H, 3.03; N, 8.42. Found: C, 57.77;
H, 3.06; N, 8.41.
N-(7-Chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-yl)-3-
(2-hydroxyphenyl)acrylamide 9e
Yield 51%; mp 205–2088C; light gray powder; IR (nmax, cmꢀ1, KBr):
Synthesis of N-(7-chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-
yl)-3-(substituted phenyl) acrylamide 9a–f
A mixture of compound 6, 2.895 g (0.01 mol), in ethanol (25 mL)
and 8 mL of 10% NaOH was stirred at room temperature. To this
mixture equimolar quantity of substituted aromatic aldehydes
(0.01 mol) was added in small portions. Stirring was continued
for overnight in room temperature. The excess of solvent was
–
3425 (N–H), 3152 (O–H), 1678 (C O), 1515 (NO ), 1198 (C–F), 670
–
2
(C–Cl); 1H-NMR (400 MHz, DMSO-d6), d (ppm): 4.26 (br s, 1H, OH),
5.30 (d, 1H, CH, J ¼ 7.35 Hz), 5.77 (d, 1H, CH, J ¼ 7.35 Hz), 7.63–
7.61 (m, 4H, ArH), 7.87–7.85 (d, 1H, ArH, J ¼ 7.62 Hz), 9.65 (s, 1H,
NH); ESI-MS, m/z: 393.04 [M]þ, 395.06 [Mþ2]þ; anal. calcd.
for C16H9ClFN3O4S: C, 48.80; H, 2.30; N, 10.67. Found:
C, 48.87; H, 2.34; N, 10.65.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2012, 345, 137–146
Anti-Mycobacterium Activity of Novel Fluoronitrobenzothiazolopyrazolines
143
(C–F), 680 (C–Cl); 1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.42
(s, 3H, OCH3), 4.35–4.31 (d, 1H, CH, J ¼ 7.92 Hz), 4.46–4.40
(d, 1H, CH, J ¼ 7.95 Hz), 7.78–7.75 (m, 4H, ArH), 8.45–8.44
(d, 1H, ArH, J ¼ 9.40 Hz), 9.29 (s, 1H, NH); ESI-MS, m/z: 407.02
[M]þ, 409.03 [Mþ2]þ; anal. calcd. for C17H11ClFN3O4S: C, 50.07; H,
2.72; N, 10.30. Found: C, 50.16; H, 2.67; N, 10.37.
N-(7-Chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-yl)-3-
phenylacrylamide 9f
Yield 56%; mp 234–2378C; yellow crystalline solid; IR (nmax, cmꢀ1
,
–
KBr): 3434 (N–H), 1697 (C O), 1523 (NO ), 1208 (C–F) 637 (C–Cl);
–
2
1H-NMR (400 MHz, DMSO-d6), d (ppm): 4.86–4.84 (d, 1H, CH,
J ¼ 7.39 Hz), 5.12–4.96 (d, 1H, CH, J ¼ 7.40 Hz), 7.46–7.43
(m, 5H, ArH), 7.72–7.70 (d, 1H, ArH, J ¼ 7.61 Hz), 9.55 (s, 1H,
NH); ESI-MS, m/z: 377.01 [M]þ, 379.02 [Mþ2]þ; anal. calcd.
for C16H9ClFN3O3S: C, 50.87; H, 2.40; N, 11.12. Found:
C, 50.92; H, 2.46; N, 11.17.
N-(7-Chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-yl)-3-
(2-hydroxyphenyl)acrylamide 10e
Yield 54%; mp 280–2818C; greenish powder; IR (nmax, cmꢀ1, KBr):
–
3422 (N–H), 3156 (O–H), 1681 (C O), 1523 (NO ), 1192 (C–F), 676
–
2
(C–Cl); 1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.97 (br s, 1H, OH),
4.42–4.37 (dd, 2H, CH, J ¼ 7.38, 7.46 Hz), 7.43–7.38 (m, 4H, ArH),
8.31–8.26 (d, 1H, ArH, J ¼ 9.42 Hz), 9.64 (s, 1H, NH); ESI-MS, m/z:
393.01 [M]þ, 395.03 [Mþ2]þ; anal. calcd. for C16H9ClFN3O4S:
C, 48.80; H, 2.30; N, 10.67. Found: C, 48.74; H, 2.30; N, 10.61.
Synthesis of N-(7-chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-
yl)-3-(substituted phenyl) acrylamide 10a–f
A mixture of compound 6, 2.895 g (0.01 mol), in ethanol (25 mL)
and 8 mL of 10% NaOH was stirred at room temperature. To this
mixture equimolar quantity of substituted aromatic aldehydes
(0.01 mol) was added in small portions. Stirring was continued
for overnight in room temperature. The excess of solvent was
distilled off, cooled, filtered and the residue was thoroughly
washed with cold water. The product was then recrystallized
from acetone-water mixture.
N-(7-Chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-yl)-3-
phenylacrylamide 10f
Yield 52%; mp 263–2658C; yellow crystal; IR (nmax, cmꢀ1, KBr):
–
3431 (N–H), 1691 (C O), 1533 (NO ), 1186 (C-F), 645 (C–Cl);
–
2
1H-NMR (400 MHz, DMSO-d6), d (ppm): 4.86–4.81 (dd, 1H, CH,
J ¼ 7.39, 7.52 Hz), 7.56–7.51 (m, 5H, ArH), 8.67–8.63 (d, 1H,
ArH, J ¼ 9.42 Hz), 9.57 (s, 1H, NH); ESI-MS, m/z: 377.03 [M]þ,
379.04 [Mþ2]þ; anal. calcd. for C16H9ClFN3O3S: C, 50.87; H,
2.40; N, 11.12. Found: C, 50.69; H, 2.48; N, 11.06.
N-(7-Chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-yl)-3-
(4-(dimethylamino)phenyl)acrylamide 10a
Yield 75%; mp 266–2688C; orange crystal; IR (nmax, cmꢀ1, KBr):
–
3402 (N–H), 1698 (C O), 1525 (NO ), 1197 (C–F), 630 (C–Cl);
–
2
1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.12 (s, 6H, CH3), 4.43–
4.36 (dd, 2H, CH, J ¼ 7.32, 7.40 Hz), 7.76–7.72 (m, 4H, ArH,
J ¼ 8.88 Hz), 8.40–8.38 (d, 1H, ArH, J ¼ 9.44 Hz), 9.65 (s, 1H,
NH); ESI-MS, m/z: 420.05 [M]þ, 422.05 [Mþ2]þ; anal. calcd.
for C18H14ClFN4O3S: C, 51.37; H, 3.35; N, 13.31. Found:
C, 51.42; H, 3.39; N, 13.29.
Synthesis of 7-chloro-N-(1-(7-chloro-6-fluorobenzo[d]-
thiazol-2-yl)-5-substituted phenyl-4,5-dihydro-1H-pyrazol-
3-yl)-6-fluorobenzo[d]thiazol-2-amine 11a–f
A mixture of compound 8a–f (0.002 mol) and 7-chloro-6-fluoro-2-
hydrazinylbenzo[d]thiazole 4 (0.002 mol) in ethanol (25 mL)
containing 3–4 drops of glacial acetic acid was heated under
reflux for 8 h. After completion of reaction, the reaction mixture
was concentrated under reduced pressure. On cooling, solid
separated out, was collected and recrystallized by DMF-water
mixture.
N-(7-Chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-yl)-3-
(4-hydroxyphenyl)acrylamide 10b
Yield 70%; mp 270–2728C; yellow crystal IR (nmax, cmꢀ1, KBr):
–
3402 (N–H), 3102 (O–H), 1675 (C O), 1515 (NO ), 1184 (C–F), 670
–
2
(C–Cl); 1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.94 (br s, 1H, OH),
4.46–4.40 (dd, 2H, CH, J ¼ 7.34, 7.41 Hz), 7.82–7.79 (m, 4H, ArH),
8.41–8.40 (d, 1H, ArH, J ¼ 9.43 Hz), 9.61 (s, 1H, NH); ESI-MS, m/z: 393.01
[M]þ, 395.02 [Mþ2]þ; anal. calcd. for C16H9ClFN3O4S: C, 48.80; H, 2.30;
N, 10.67. Found: C, 48.93; H, 2.38; N, 10.82.
7-Chloro-N-(1-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-5-
(4-(dimethylamino) phenyl)-4, 5-dihydro-1H-pyrazol-3-yl)-
6-fluorobenzo[d]thiazol-2-amine 11a
Yield 82%; mp 240–2428C; buff color powder; IR (nmax, cmꢀ1, KBr):
1
3440 (N-H), 1100 (C–F), 640 (C–Cl); H-NMR (400 MHz, DMSO-d6),
N-(7-Chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-yl)-3-
(4-hydroxy-3-methoxyphenyl)acrylamide 10c
d (ppm): 2.05 (s, 6H, CH3), 3.27 (1H, dd, J ¼ 5.8, 18.20 Hz, H-4 trans
(pyrazoline)), 4.04 (1H, dd, J ¼ 12.10, 18.20 Hz, H-4 cis (pyrazo-
line)), 5.06 (s, 1H, NH), 5.22 (1H, dd, J ¼ 5.80, 12.00 Hz, H-5
(pyrazoline)), 7.23–7.19 (m, 4H, ArH), 7.68–7.64 (m, 4H, ArH);
ESI-MS, m/z: 574.06 [M]þ, 576.05 [Mþ2]þ, 578.05 [Mþ4]þ; anal.
calcd. for C25H18Cl2F2N6S2: C, 52.18; H, 3.15; N, 14.60. Found:
C, 52.23; H, 3.21; N, 14.52.
Yield 72%; mp 266–2698C; Yellow crystal; IR (nmax, cmꢀ1, KBr):
–
3400 (N–H), 3090 (O–H), 1672 (C O), 1508 (NO ), 1251 & 1052
–
2
(C–O), 1198 (C–F), 645 (C–Cl); 1H-NMR (400 MHz, DMSO-d6),
d (ppm): 3.22 (s, 3H, OCH3), 3.93 (br s, 1H, OH), 4.52–4.47
(dd, 2H, CH, J ¼ 7.27, 7.33 Hz), 7.34–7.30 (m, 3H, ArH), 8.42–
8.40 (d, 1H, ArH, J ¼ 9.42 Hz), 9.25 (s, 1H, NH); ESI-MS, m/z: 423.03
[M]þ, 425.03 [Mþ2]þ; anal. calcd. for C17H11ClFN3O5S: C, 48.18;
H, 2.62; N, 9.92. Found: C, 48.32; H, 2.74; N, 9.90.
4-(1-(7-Chloro-6-fluorobenzo[d]thiazol-2-yl)-3-(7-chloro-6-
fluorobenzo[d]thiazol-2-yl-amino)-4,5-dihydro-1H-pyrazol-
5-yl)phenol 11b
Yield 78%; mp 170–1718C; yellow crystal; IR (nmax, cmꢀ1, KBr):
3482 (N–H), 3120 (O–H), 1160 (C–F), 638 (C–Cl); 1H-NMR (400 MHz,
DMSO-d6), d (ppm): 3.31 (1H, dd, J ¼ 5.7, 18.4 Hz, H-4 trans
(pyrazoline)), 4.04 (1H, dd, J ¼ 12.3, 18.5 Hz, H-4 cis (pyrazoline)),
N-(7-Chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-yl)-3-
(4-methoxyphenyl)acrylamide 10d
Yield 66%; mp 272–2748C; yellow crystal; IR (nmax, cmꢀ1, KBr):
–
3402 (N–H), 1649 (C O), 1516 (NO ), 1249 & 1051 (C–O), 1202
–
2
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

144
K. Hazra et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 137–146
4.49 (bs, 1H, OH), 5.12 (s, 1H, NH), 5.25 (1H, dd, J ¼ 5.83, 12.07 Hz,
H-5 (pyrazoline)), 7.43–7.40 (m, 4H, ArH), 7.72–7.69 (m, 4H, ArH);
ESI-MS, m/z: 547.01 [M]þ, 549 [Mþ2]þ, 551 [Mþ4]þ; anal. calcd.
for C23H13Cl2F2N5OS2: C, 50.37; H, 2.39; N, 12.77. Found: C, 50.37;
H, 2.39; N, 12.77.
Synthesis of 7-chloro-N-(1-(7-chloro-6-fluorobenzo[d]-
thiazol-2-yl)-5-substituted phenyl-4,5-dihydro-1H-pyrazol-
3-yl)-6-fluoro-5-nitrobenzo[d]thiazol-2-amine 12a–f
A mixture of compound 9a–f (0.002 mol) and 7-chloro-6-fluoro-2-
hydrazinylbenzo[d] thiazole 4 (0.002 mol) in ethanol (25 mL)
containing 3–4 drops of glacial acetic acid was heated under
reflux for 8 h. After completion of reaction, the reaction mixture
was concentrated under reduced pressure. On cooling, solid
was separated out, collected and recrystallized by DMF/water
mixture.
4-(1-(7-Chloro-6-fluorobenzo[d]thiazol-2-yl)-3-(7-chloro-6-
fluorobenzo[d]thiazol-2-ylamino)-4,5-dihydro-1H-pyrazol-
5-yl)-2-methoxyphenol 11c
Yield 72%; mp 185–1868C; buff color powder; IR (nmax, cmꢀ1, KBr):
3490 (N–H), 3037 (O–H), 1251 & 1050 (C–O), 1178 (C–F), 660 (C–Cl);
1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.27 (1H, dd, J ¼ 5.8,
18.2 Hz, H-4 trans (pyrazoline)), 3.63 (s, 3H, OCH3), 4.03 (1H,
dd, J ¼ 12.30, 18.50 Hz, H-4 cis (pyrazoline)), 4.51(bs, 1H OH),
5.03 (s, 1H, NH), 5.23 (1H, dd, J ¼ 5.9, 12.04 Hz, H-5 (pyrazoline)),
7.58–7.55 (m, 3H, ArH), 7.77–7.73 (m, 4H, ArH); ESI-MS,
m/z: 577.02 [M]þ, 579.05 [Mþ2]þ, 581.03 [Mþ4]þ; anal. calcd.
for C24H15Cl2F2N5O2S2: C, 49.83; H, 2.61; N, 12.11. Found:
C, 49.92; H, 2.69; N, 12.16.
7-Chloro-N-(1-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-5-
(4-(dimethylamino) phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-
6-fluoro-5-nitrobenzo[d]thiazol-2-amine 12a
Yield 77%; mp 104–1058C; orange color crystal; IR (nmax, cmꢀ1
,
KBr): 3508 (N–H), 1513 (NO2), 1190 (C–F), 685 (C–Cl); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 2.78 (s, 6H, CH3), 3.26 (1H, dd,
J ¼ 5.6, 18.19 Hz, H-4 trans (pyrazoline)), 4.08 (1H, dd,
J ¼ 12.06, 18.07 Hz, H-4 cis (pyrazoline)), 5.16 (s, 1H, NH), 5.27
(1H, dd, J ¼ 5.73, 11.99 Hz, H-5 (pyrazoline)), 7.39–7.36 (m, 4H,
ArH), 7.81–7.78 (m, 3H, ArH); ESI-MS, m/z: 619.04 [M]þ, 621.04
[Mþ2]þ, 623.02 [Mþ4]þ; anal. calcd. for C25H17Cl2F2N7O2S2:
C, 48.39; H, 2.76; N, 15.80. Found: C, 48.51; H, 2.79; N, 15.87.
7-Chloro-N-(1-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-5-
(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)-6-
fluorobenzo[d]thiazol-2-amine 11d
Yield 80%; mp 180–1818C; light yellow crystal IR (nmax, cmꢀ1
,
KBr): 3489 (N–H), 1252 & 1055 (C–O), 1178 (C–F), 643 (C–Cl);
1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.29 (1H, dd, J ¼ 5.80,
18.2 Hz, H-4 trans (pyrazoline)), 3.64 (s, 3H, OCH3), 4.07
(1H, dd, J ¼ 11.99, 18.0 Hz, H-4 cis (pyrazoline)), 5.04 (s, 1H,
NH), 5.23 (1H, dd, J ¼ 5.7, 11.97 Hz, H-5 (pyrazoline)), 7.48–
7.45 (m, 4H, ArH), 7.73–7.69 (m, 4H, ArH); ESI-MS, m/z: 561.04
[M]þ, 563.06 [Mþ2]þ, 565.01 [Mþ4]þ; anal. calcd. for
C24H15Cl2F2N5OS2: C, 51.25; H, 2.69; N, 12.45. Found: C, 51.31;
H, 2.76; N, 12.41.
4-(3-(7-Chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-ylamino)-
1-(7-chloro-6-fluorobenzo[d] thiazol-2-yl)-4,5-dihydro-1H-
pyrazol-5-yl)phenol 12b
Yield 73%; mp 182–1848C; buff color powder; IR (nmax, cmꢀ1, KBr):
3392 (N–H), 3180 (O–H), 1536 (NO2), 1186 (C–F), 682 (C–Cl);
1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.27 (1H, dd, J ¼ 5.5,
18.17 Hz, H-4 trans (pyrazoline)), 4.06 (1H, dd, J ¼ 12.02,
18.01 Hz, H-4 cis (pyrazoline)), 4.46 (bs, 1H, OH), 5.12 (s, 1H,
NH), 5.22 (1H, dd, J ¼ 5.74, 11.98 Hz, H-5 (pyrazoline)), 7.33–
7.30 (m, 4H, ArH), 7.75–7.71 (m, 3H, ArH); ESI-MS, m/z: 591.99
[M]þ, 594 [Mþ2]þ, 595.97 [Mþ4]þ; anal. calcd. for
C23H12Cl2F2N6O3S2: C, 46.55; H, 2.04; N, 14.16. Found:
C, 46.67; H, 2.11; N, 14.12.
2-(1-(7-Chloro-6-fluorobenzo[d]thiazol-2-yl)-3-(7-chloro-6-
fluorobenzo[d]thiazol-2-ylamino)-4,5-dihydro-1H-pyrazol-
5-yl)phenol 11e
Yield 62%; mp 175–1778C; white crystal; IR (nmax, cmꢀ1, KBr):
3475 (N–H), 3119 (O–H), 1176 (C–F), 641 (C–Cl); 1H-NMR (400 MHz,
DMSO-d6), d (ppm): 3.32 (1H, dd, J ¼ 5.86, 18.19 Hz, H-4 trans
(pyrazoline)), 4.12 (1H, dd, J ¼ 12.03, 18.06 Hz, H-4 cis (pyrazo-
line)), 4.41 (bs, 1H OH), 5.06 (s, 1H, NH), 5.25 (1H, dd, J ¼ 5.70,
12.02 Hz, H-5 (pyrazoline)), 7.38–7.35 (m, 4H, ArH), 7.84–7.80
(m, 4H, ArH); ESI-MS, m/z: 547 [M]þ, 549.02 [Mþ2]þ, 551
[Mþ4]þ; anal. calcd. for C23H13Cl2F2N5OS2: C, 50.37; H, 2.39;
N, 12.77. Found: C, 50.49; H, 2.45; N, 12.81.
4-(3-(7-Chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-ylamino)-
1-(7-chloro-6-fluorobenzo[d] thiazol-2-yl)-4,5-dihydro-1H-
pyrazol-5-yl)-2-methoxyphenol 12c
Yield 70%; mp 210–2138C; buff color powder; IR (nmax, cmꢀ1, KBr):
3431 (N–H), 3128 (O–H) 1521 (NO2), 1255 & 1052 (C–O), 1196
(C–F), 630 (C–Cl); 1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.30 (1H,
dd, J ¼ 5.81, 18.20 Hz, H-4 trans (pyrazoline)), 3.73 (s, 3H, OCH3),
4.12 (1H, dd, J ¼ 12.02, 18.01 Hz, H-4 cis (pyrazoline)), 4.42
(bs, 1H, OH), 5.14 (s, 1H, NH), 5.22 (1H, dd, J ¼ 5.74, 11.98 Hz,
H-5 (pyrazoline)), 7.43–7.39 (m, 3H, ArH), 7.79–7.77 (m, 3H, ArH);
ESI-MS, m/z: 621.98 [M]þ, 623.99 [Mþ2]þ, 626 [Mþ4]þ; anal. calcd.
for C24H14Cl2F2N6O4S2: C, 46.24; H, 2.26; N, 13.48. Found:
C, 46.32; H, 2.21; N, 13.44.
7-Chloro-N-(1-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-5-
phenyl-4,5-dihydro-1H-pyrazol-3-yl)-6-fluorobenzo[d]-
thiazol-2-amine 11f
Yield 69%; mp 190–1928C; gray color powder; IR (nmax, cmꢀ1
,
KBr): 3465 (N–H), 1188 (C–F), 633 (C–Cl); 1H-NMR (400 MHz,
DMSO-d6), d (ppm): 3.27 (1H, dd, J ¼ 5.8, 18.2 Hz, H-4 trans
(pyrazoline)), 4.07 (1H, dd, J ¼ 11.99, 18.0 Hz, H-4 cis (pyrazo-
line)), 4.98 (s, 1H, NH), 5.23 (1H, dd, J ¼ 5.70, 11.97 Hz, H-5
(pyrazoline)), 7.26–7.23 (m, 5H, ArH), 7.73–7.68 (m, 4H, ArH);
ESI-MS, m/z: 531 [M]þ, 533.01 [Mþ2]þ, 535 [Mþ4]þ; anal. calcd.
for C23H13Cl2F2N5S2: C, 51.89; H, 2.46; N, 13.15. Found: C, 51.96;
H, 2.52; N, 13.11.
7-Chloro-N-(1-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-5-
(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)-6-fluoro-
5-nitrobenzo[d]thiazol-2-amine 12d
Yield 75%; mp 180–1828C; light yellow crystal; IR (nmax, cmꢀ1
,
KBr): 3508 (N–H), 1520 (NO2), 1190 (C–F), 656 (C–Cl); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 3.28 (1H, dd, J ¼ 5.79, 18.17 Hz,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2012, 345, 137–146
Anti-Mycobacterium Activity of Novel Fluoronitrobenzothiazolopyrazolines
145
H-4 trans (pyrazoline)), 3.66 (s, 3H, OCH3), 4.07 (1H, dd, J ¼ 12.00,
17.98 Hz, H-4 cis (pyrazoline)), 5.12 (s, 1H, NH), 5.19 (1H, dd,
J ¼ 5.72, 11.99 Hz, H-5 (pyrazoline)), 7.37–7.34 (m, 3H, ArH),
7.76–7.73 (m, 3H, ArH); ESI-MS, m/z: 606.03 [M]þ, 608.03
[Mþ2]þ, 610.01 [Mþ4]þ; anal. calcd. for C24H14Cl2F2N6O3S2: C,
47.45; H, 2.32; N, 13.84. Found: C, 47.52; H, 2.41; N, 13.78.
4-(3-(7-Chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-ylamino)-
1-(7-chloro-6-fluorobenzo[d] thiazol-2-yl)-4,5-dihydro-1H-
pyrazol-5-yl)phenol 13b
Yield 66%; mp 210–2128C; light green crystalline; IR
(nmax, cmꢀ1, KBr): 3401 (N–H), 3098 (O–H), 1525 (NO2), 1190
(C–F), 688 (C–Cl); 1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.26
(1H, dd, J ¼ 5.79, 18.18 Hz, H-4 trans (pyrazoline)), 4.06 (1H, dd,
J ¼ 12.11, 18.12 Hz, H-4 cis (pyrazoline)), 4.21 (br s, 1H, OH),
5.13 (s, 1H, NH), 5.26 (1H, dd, J ¼ 5.78, 12.11 Hz, H-5
(pyrazoline)), 7.54–7.51 (m, 4H, ArH), 7.92–7.87 (dd, 2H, ArH,
J ¼ 7.02, 8.36 Hz), 8.52–8.49 (d, 1H, ArH, J ¼ 9.42 Hz); ESI-MS,
m/z: 592.03 [M]þ, 594 [Mþ2]þ, 596 [Mþ4]þ; anal. calcd.
for C23H12Cl2F2N6O3S2: C, 46.55; H, 2.04; N, 14.16. Found:
C, 46.52; H, 2.12; N, 14.13.
2-(3-(7-Chloro-6-fluoro-5-nitrobenzo[d]thiazol-2-ylamino)-
1-(7-chloro-6-fluorobenzo[d] thiazol-2-yl)-4,5-dihydro-1H-
pyrazol-5-yl)phenol 12e
Yield 65%; mp 166–1688C; light gray; IR (nmax, cmꢀ1, KBr): 3432
(N–H), 3126 (O–H), 1535 (NO2), 1192 (C–F), 642 (C-Cl); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 3.32 (1H, dd, J ¼ 5.92, 18.24 Hz, H-4
trans (pyrazoline)), 4.05 (1H, dd, J ¼ 12.14, 18.25 Hz, H-4 cis
(pyrazoline)), 4.34 (br s, 1H, OH), 5.12 (s, 1H, NH), 5.25 (1H, dd,
J ¼ 5.86, 12.04 Hz, H-5 (pyrazoline)) 7.35–7.32 (m, 4H, ArH), 7.92–
7.88 (m, 3H, ArH); ESI-MS, m/z: 592.02 [M]þ, 594.01 [Mþ2]þ, 596
[Mþ4]þ; anal. calcd. for C23H12Cl2F2N6O3S2: C, 46.55; H, 2.04;
N, 14.16. Found: C, 46.62; H, 2.12; N, 14.19.
4-(3-(7-Chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-ylamino)-
1-(7-chloro-6-fluorobenzo[d] thiazol-2-yl)-4,5-dihydro-1H-
pyrazol-5-yl)-2-methoxyphenol 13c
Yield 68%; mp 218–2218C; green powder; IR (nmax, cmꢀ1, KBr):
3402 (N–H), 3102 (O–H), 1518 (NO2), 1192 (C–F), 666 (C–Cl);
1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.27 (1H, dd, J ¼ 5.81,
18.22 Hz, H-4 trans (pyrazoline)), 3.78 (s, 3H, OCH3), 4.05
(1H, dd, J ¼ 12.14, 18.27 Hz, H-4 cis (pyrazoline)), 4.41 (br s,
1H, OH), 5.13 (s, 1H, NH), 5.24 (1H, dd, J ¼ 5.86, 12.03 Hz, H-5
(pyrazoline)), 7.32–7.29 (s, 1H, ArH), 7.36–7.34 (d, 1H, ArH,
J ¼ 8.23 Hz), 7.89 (s, 1H, CH), 7.99–7.96 (d, 1H, ArH,
J ¼ 8.01 Hz), 8.10–8.07 (d, 1H, ArH, J ¼ 8.73 Hz), 8.59–8.57
(d, 1H, ArH, J ¼ 9.44 Hz); ESI-MS, m/z: 622.02 [M]þ, 624.01
[Mþ2]þ, 626 [Mþ4]þ; anal. calcd. for C24H14Cl2F2N6O4S2:
C, 46.24; H, 2.26; N, 13.48. Found: C, 46.31; H, 2.33; N, 13.51.
7-Chloro-N-(1-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-5-
phenyl-4,5-dihydro-1H-pyrazol-3-yl)-6-fluoro-5-
nitrobenzo[d]thiazol-2-amine 12f
Yield 69%; mp 177–1788C; light yellow powder; IR (nmax, cmꢀ1
,
KBr): 3498 (N–H), 1515 (NO2), 1191 (C–F), 655 (C–Cl); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 3.30 (1H, dd, J ¼ 5.80, 18.21 Hz,
H-4 trans (pyrazoline)), 4.02 (1H, dd, J ¼ 12.12, 18.24 Hz, H-4 cis
(pyrazoline)), 5.13 (s, 1H, NH), 5.23 (1H, dd, J ¼ 5.81, 12.03 Hz, H-5
(pyrazoline)), 7.35–7.32 (m, 5H, ArH), 7.87–7.84 (m, 3H, ArH);
ESI-MS, m/z: 575.99 [M]þ, 578 [Mþ2]þ, 580 [Mþ4]þ; anal. calcd.
for C23H12Cl2F2N6O2S2: C, 47.84; H, 2.09; N, 14.55. Found:
C, 47.96; H, 2.18; N, 14.59.
7-Chloro-N-(1-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-5-
(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)-6-fluoro-
4-nitrobenzo[d]thiazol-2-amine 13d
Synthesis of 7-chloro-N-(1-(7-chloro-6-fluorobenzo[d]-
thiazol-2-yl)-5-substituted phenyl-4,5-dihydro-1H-pyrazol-
Yield 66%; mp 228–2318C; light off color powder; IR (nmax, cmꢀ1
,
KBr): 3422 (N–H), 3123 (O–H), 1515 (NO2), 1189 (C–F), 654 (C–Cl);
1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.27 (1H, dd, J ¼ 5.79,
18.16 Hz, H-4 trans (pyrazoline)), 3.70 (s, 1H, OCH3), 4.03
(1H, dd, J ¼ 12.13, 18.26 Hz, H-4 cis (pyrazoline)), 5.11 (s, 1H,
NH), 5.25 (1H, dd, J ¼ 5.83, 12.07 Hz, H-5 (pyrazoline)), 7.47–
7.44 (m, 4H, ArH), 7.95–7.92 (dd, 2H, ArH, J ¼ 7.13, 9.66 Hz),
8.48–8.45 (d, 1H, ArH, J ¼ 9.44 Hz); ESI-MS, m/z: 606.02 [M]þ,
608.03 [Mþ2]þ, 610 [Mþ4]þ; anal. calcd for C24H14Cl2F2N6O3S2:
C, 47.45; H, 2.32; N, 13.84. Found: C, 47.57; H, 2.39; N, 13.88.
3-yl)-6-fluoro-4-nitrobenzo[d]thiazol-2-amine 13a–f
A mixture of compound 10a–f (0.002 mol) and 7-chloro-6-fluoro-
2-hydrazinylbenzo[d] thiazole 4 (0.002 mol) in ethanol (25 mL)
containing 3–4 drops of glacial acetic acid was heated under
reflux for 8 h. After completion of reaction, the reaction mixture
was concentrated under reduced pressure. On cooling, solid
was separated out, collected and recrystallized by DMF-water
mixture.
7-Chloro-N-(1-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-5-
(4-(dimethylamino) phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-
6-fluoro-4-nitrobenzo[d]thiazol-2-amine 13a
2-(3-(7-Chloro-6-fluoro-4-nitrobenzo[d]thiazol-2-ylamino)-
1-(7-chloro-6-fluorobenzo[d] thiazol-2-yl)-4,5-dihydro-1H-
pyrazol-5-yl)phenol 13e
Yield 70%; mp 258–2608C; light green crystalline; IR (nmax, cmꢀ1
,
Yield 59%; mp 193–1948C; light green crystalline; IR (nmax, cmꢀ1
,
KBr): 3422 (N–H), 1516 (NO2), 1197 (C–F), 662 (C–Cl); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 2.76 (s, 6H, CH3), 3.27 (1H, dd,
J ¼ 5.78, 18.21 Hz, H-4 trans (pyrazoline)), 4.04 (1H, dd,
J ¼ 12.11, 18.21 Hz, H-4 cis (pyrazoline)), 5.06 (s, 1H, NH), 5.24
(1H, dd, J ¼ 5.79, 12.07 Hz, H-5 (pyrazoline)), 7.50–7.47 (m, 4H,
ArH), 7.84–7.81 (d, 1H, J ¼ 8.28 Hz, ArH), 7.92–7.90 (d, 1H,
ArH, J ¼ 8.31 Hz), 8.47–8.45 (d, 1H, ArH, J ¼ 9.44 Hz); ESI-MS,
m/z: 619.04 [M]þ, 621.02 [Mþ2]þ, 623 [Mþ4]þ; anal. calcd.
for C25H17Cl2F2N7O2S2: C, 48.39; H, 2.76; N, 15.80. Found:
C, 48.53; H, 2.82; N, 15.84.
KBr): 3405 (N–H), 3108 (O–H), 1521 (NO2), 1183 (C–F), 674 (C–Cl);
1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.30 (1H, dd, J ¼ 5.85,
18.19 Hz, H-4 trans (pyrazoline)), 4.09 (1H, dd, J ¼ 12.14,
18.26 Hz, H-4 cis (pyrazoline)), 4.37 (br s, 1H, OH), 5.10 (s, 1H,
NH), 5.24 (1H, dd, J ¼ 5.87, 12.03 Hz, H-5 (pyrazoline)), 7.42–7.39
(m, 4H, ArH), 7.98–7.95 (dd, 2H, ArH, J ¼ 7.11, 9.02 Hz), 8.53–8.51
(d, 1H, ArH, J ¼ 9.44 Hz); ESI-MS, m/z: 591.98 [M]þ, 593.99 [Mþ2]þ,
596 [Mþ4]þ; anal. calcd. for C23H12Cl2F2N6O3S2: C, 46.55; H, 2.04;
N, 14.16. Found: C, 46.52; H, 2.14; N, 14.13.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

146
K. Hazra et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 137–146
was removed. 100 mL of propanol was added and the plates were
gently shaken to solubilize the formed formazan. The absorb-
ance was measured using a microplate reader at a wavelength of
540 nm. The percentage growth inhibition was calculated using
the following formula and concentration of test drug needed to
inhibit cell growth by 50% (CTC50) values is generated from the
dose-response curves for each cell line.
7-Chloro-N-(1-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-5-
phenyl-4,5-dihydro-1H-pyrazol-3-yl)-6-fluoro-4-
nitrobenzo[d]thiazol-2-amine 13f
Yield 64%; mp 226–2288C; gray color powder; IR (nmax, cmꢀ1
,
KBr): 3423 (N-H), 3118 (O–H), 1517 (NO2), 1199 (C–F), 685 (C–Cl);
1H-NMR (400 MHz, DMSO-d6), d (ppm): 3.29 (1H, dd, J ¼ 5.80,
18.20 Hz, H-4 trans (pyrazoline)), 4.32 (br s, 1H, OH), 4.00 (1H,
dd, J ¼ 12.1, 18.2 Hz, H-4 cis (pyrazoline)), 5.04 (s, 1H, NH), 5.22
(1H, dd, J ¼ 5.8, 12.0 Hz, H-5 (pyrazoline)), 7.40–7.37 (m, 4H, ArH),
7.90–7.87 (dd, 2H, ArH, J ¼ 7.27, 8.94 Hz), 8.51–8.49 (d, 1H, ArH,
J ¼ 9.44 Hz); ESI-MS, m/z: 576.01 [M]þ, 578 [Mþ2]þ, 589.98
[Mþ4]þ; anal. calcd. for C23H12Cl2F2N6O2S2: C, 47.84; H, 2.09;
N, 14.55. Found: C, 47.88; H, 2.18; N, 14.57.
%Growth Inhibition
Mean OD of individual test group
¼ 100ꢀ
ꢂ 100
Mean OD of control group
Authors are thankful to Director and Principal, Nargund College of
Pharmacy, for providing the necessary facilities for carrying out the
project work and also to Indian Institute of Science, Bangalore, for
providing spectral data.
Antitubercular activity
The ability of compounds to inhibit the growth of Mycobacterium
species was determined by Ziehl-Neelsen stain and they were
grown in Middlebrook 7H-9 broth. The standard strain was used
for Mycobacterium tuberculosis H37Rv (ATCC 27294). The basal
medium was prepared according to manufacturer’s instructions
(Hi-Media) and sterilized by autoclaving. 4.5 mL of broth was
taken in each sterile bottle and to this 0.5 mL of ADC supplement
was added which containing catalase, dextrose and bovine
serum albumin fraction. Then, the compound solution was
transferred to media bottles to achieve the final concentrations
25, 50, 100 mg/mL. Finally a 10-mL suspension of Mycobacterium
tuberculosis strain (100 000 organisms/mL, adjusted by
McFarland’s turbidity standard) was transferred to each of the
tubes which were incubated at 378C for three weeks. A control
without compound as well as drug was also set up and was
inspected for growth twice a week. The appearance of turbidity
was considered as growth and the indicator of the resistance to
the compound. The growth was confirmed by making a smear
from each bottle and performing a Ziehl-Neelsen stain.
The authors have declared no conflict of interest.
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