Potent in vitro and in vivo antitubercular activity of certain newly synthesized indophenazine 1,3,5-trisubstituted pyrazoline derivatives bearing benzofuran

Article abstract of DOI:10.1007/s00044-010-9322-5

Fourteen newly synthesized derivatives of indophenazine 1,3,5-trisubstituted pyrazoline bearing benzofuran were prepared from benzofuran chalcones with indophenazine hydrazide through cycloaddition reaction. All the compounds were screened for their in vitro and in vivo antitubercular activity against drug resistant and multidrug-resistant Mycobacterium tuberculosis H37RV. The MIC50 and MIC90 were estimated and compared with rifampicin and gatifloxacin standard drugs. Nitro group containing at ortho 5j, meta 5e, furan ring containing 5m and ortho 5i, para 5h chloro containing compounds were exhibited significant in vitro, in vivo antitubercular activity against standard drugs. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-010-9322-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:300–306
DOI 10.1007/s00044-010-9322-5
ORIGINAL RESEARCH
Potent in vitro and in vivo antitubercular activity of certain newly
synthesized indophenazine 1,3,5-trisubstituted pyrazoline
derivatives bearing benzofuran
•
Kuntal Manna Yadvendra K. Agrawal
Received: 29 May 2009 / Accepted: 12 February 2010 / Published online: 27 February 2010
Ó Springer Science+Business Media, LLC 2011
Abstract Fourteen newly synthesized derivatives of in-
dophenazine 1,3,5-trisubstituted pyrazoline bearing ben-
zofuran were prepared from benzofuran chalcones with
indophenazine hydrazide through cycloaddition reaction.
All the compounds were screened for their in vitro and in
vivo antitubercular activity against drug resistant and
multidrug-resistant Mycobacterium tuberculosis H37RV.
The MIC50 and MIC90 were estimated and compared with
rifampicin and gatifloxacin standard drugs. Nitro group
containing at ortho 5j, meta 5e, furan ring containing 5m
and ortho 5i, para 5h chloro containing compounds were
exhibited significant in vitro, in vivo antitubercular activity
against standard drugs.
Improved therapy for tuberculosis is recognized as a major
need for the developing countries as well as developed
countries (Albalak et al., 2007). Current chemotherapy of
tuberculosis is not likely to be successful in retroviral
infected patients access to the world. Multidrug-resistant
Mycobacterium tuberculosis (MDR-TB) is another serious
threat to treatment and control of tuberculosis. The drug
resistance may be caused by mycobacterium unique cell
wall structure, which is rich in long-chain fatty acids such
as C60 to C90 mycolic acid, which covalently linked with
peptidoglycan-associated polysaccharide arabinogalactan
(Trias and Benz, 1993). The cell wall barrier alone is not
sufficient to explain the intrinsic drug resistance. Resis-
tance of M. tuberculosis strains by existing antitubercular
agents is also increasing problem worldwide (Corbett et al.,
2003). Therefore, powerful novel antitubercular agents
being new mechanism of action is needed to develop for
the treatment of complex tuberculosis cases. In spite of
drug resistance, complex tuberculosis cases severe and
toxicity on repeated dosing of isoniazid (INH), thought to
synthesize a novel series of indophenazine 1,3,5-trisubsti-
tuted pyrazoline containing benzofuran. According to the
past researcher reports, pyrazoline derivatives are highly
active against many mycobacteria (Ali et al., 2007; Kini
et al., 2009). The incorporation of heterocyclic rings
into the pyrazoline ring was reported as potent antibacte-
rial agents (Kirilmis et al., 2008), antiamoebic agents
(Husain et al., 2008), and antifungal agents (Prakash et al.,
2008). The present investigation focus on the synthesis of
1-[3-(5-hydroxybenzo[b]furan-2-yl)-5-substituted phenyl-
4,5-dihydro-1H-1-pyrazolyl]-2-(5H-indolo[2,3-b]quinoxalin-
5-yl)-1-ethanone derivatives 5a–n from 2-(5,8-dihydro-
quinoxalino[2,3-b]indol-5-yl)acetohydrazide 4 with differ-
ent benzofuran chalcones 3a–n and tested for in vitro and in
vivo antitubercular activity (Lenaerts et al., 2005, 2008;
Keywords In vitro and in vivo antitubercular activity Á
Multidrug-resistant M. tuberculosis Á
Indophenazine pyrazoline Á Benzofuran
Introduction
A recent survey indicates the leading mortality cause by
HIV/AIDS is closely associated with tuberculosis.
K. Manna (&)
Department of Pharmaceutical Chemistry, Institute of Pharmacy,
Nirma University, Sarkhej-Gandhinagar Highway,
Ahmedabad 382481, Gujarat, India
e-mail: k_manna2002@yahoo.com
Y. K. Agrawal
Institute of Research and Development, Gujarat Forensic
Sciences University, Sector 18-A, Near Police Bhavan,
Gandhinagar 382007, Gujarat, India
e-mail: drykagrawal@yahoo.com
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Med Chem Res (2011) 20:300–306
301
Stover et al., 2000; Tyagi et al., 2005) against M.
tuberculosis.
Results and discussion
All the synthesized compounds were found satisfactory
antitubercular agents against both MTB and MDR-TB with
less cytotoxicity (IC50) than standard drugs (Table 1). Out
of fourteen compounds 5j, 5e, 5m, 5i, 5h, 5l, 5f were found
potent against both MTB and MDR-TB, MIC50 of 0.16,
0.62, 1.1, 1.75, 2.45, 3.8, 4.56 lg/ml, respectively. Among
all the active compounds 5j and 5e were found more active
than rifampicin and gatifloxacin (MIC50 = 0.5 lg/ml and
MIC90 = 0.12 lg/ml) against MTB. The N–H, one proton
Chemistry
2,5-Dihydroxy benzaldehyde 1 was utilized for the synthesis
of 5-hydroxy, 2-acetylbenzofuran 2 in presence of chloro-
acetone and K2CO3. A new series of (2E)-1-(5-hydroxy-1-
benzofuran-2-yl)-3-phenylprop-2-en-1-one derivatives 3a–
n were prepared from 5-hydroxy, 2-acetylbenzofuran with
different aromatic aldehydes in presence of strong alkaline
medium. Fourteen new 1-[3-(5-hydroxybenzo[b]furan-2-
yl)-5-substituted phenyl-4,5-dihydro-1H-1-pyrazolyl]-2-
(5H-indolo[2,3-b]quinoxalin-5-yl)-1-ethanone derivatives
5a–n were synthesized from 2-(5,8-dihydroquinoxalino[2,
3-b]indol-5-yl)acetohydrazide 4 with different benzofuran
chalcones 3a–n in presence of glacial acetic acid
(Scheme 1).
1
in indophenazine ring was established by sharp H NMR
peak at 9.834 ppm (s, 1H) and 3402, 3169, 3130 cm-1 in
FTIR. Oxazolidine ring of indophenazine was established
1
by H NMR peak at 8.42–7.65 ppm, by IR (cm-1): 3402,
3169 m (NH), 1686 m (CH), 1617 m (C=N). 6-Carbeth-
oxymethyl indophenazine structure was characterized by
sharp 1H NMR peak at 5.064–1.25 ppm (s, N–CH2), where
N–H peak was absent and additional 4.18 ppm (d, 2H,
J = 7.12 Hz, O–CH2–CH3), 1.25 ppm (d, 3H, J = 9.32
Hz, O–CH2–CH3) peaks were obtained. The IR peaks 1738
and 1748 cm-1 (C=O), 1678 (CH), 1614 (C=C) and (C=N)
were supported for the establishment of the structure. In-
dophenazine-6-acetic acid hydrazide structure was char-
Biology
The in vitro and in vivo antitubercular activity was carried
out with MTB and MDR-TB using H37Rv stains (Interleid,
1991). The clinically isolated stains were resistant to isoni-
azid (INH), rifampicin, ethambutol, and gatifloxacin were
procured from Tuberculosis Research Centre, Chennai,
India. Antitubercular activity was evaluated to determine
the MIC50 and MIC90 using 10% oleic acid–albumin–
dextrose–catalase-enriched 7H11 agar medium (Collins and
Franzblau, 1997). The toxicity (IC50) of the compounds was
examined using mammalian VERO cell line.
1
acterized by sharp H NMR peak at 8.92 (s, 1H) NHNH2
and 5.78 (s, 2H) NHNH2. The IR peaks 1614 cm-1 (C=C)
and (C=N), 1546 cm-1 (NH), 1488, 1467 cm-1 (C=N) and
(C=C) were reconfirmed the structure. Compound 5a
1
structure was elucidated by H NMR, IR, Mass, and ele-
mental analysis data, where NHNH2 peaks were found
1
absent and a sharp H NMR peak at 3.6 (s, 1H) was found
for one proton in position 5 in pyrazoline ring and 2.3 (s,
2H) for two protons at position 4 in pyrazoline ring were
Scheme 1 General synthesis of
3a–n and 5a–n
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Med Chem Res (2011) 20:300–306
Table 1 In vitro and in vivo antitubercular screening oft novel indophenazine 1,3,5-trisubstituted pyrazoline bearing benzofuran derivatives
against Mycobacterium tuberculosis H37RV
Name of comp.
-R
Results against MTBa
Results against MDR-TBb
IC50 (lM)c
MIC50 (lg/ml)
MIC90 (lg/ml)
MIC50 (lg/ml)
MIC90 (lg/ml)
5a
–OH (o)
9.81
11.80
5.56
10.20
0.62
4.56
10.53
2.45
1.75
0.16
12.11
3.8
10.84
14.20
9.21
6.87
1.24
8.32
7.52
4.20
2.56
0.42
14.3
1.5
10.34
10.67
5.00
9.56
1.20
7.80
9.86
5.23
4.68
3.24
11.5
10.56
6.40
5.6
12.56
13.64
8.98
7.45
2.00
13.10
11.21
8.66
8.61
7.2
[226.8
[225.6
[216.2
[220.5
[180.2
[206.5
[245.2
[205.4
[195.6
[144.3
[225.5
[196.7
[198.2
[206.5
[77.4
5b
–OCH3 (o)
–N(CH3)2 (p)
–COOH (o)
–NO2 (m)
–OH (o), OCH3 (p)
–OH (p)
5c
5d
5e
5f
5g
5h
–Cl (p)
5i
–Cl (o)
5j
–NO2 (o)
5k
–OCH3 (p)
–H
15.4
6.12
9.78
11.34
7.37
28.46
5l
5m
Furan ring
–CH=CH–Ar
1.1
3.12
3.67
2.0
5n
0.98
0.5
Rifampicin
Gatifloxacin
a
4.21
14.73
0.12
0.5
[159.5
Mycobacterium tuberculosis H37RV
b
c
Multidrug-resistant Mycobacterium tuberculosis
Cytotoxicity in VERO cell line
ensuring the formation of pyrazoline ring. In mass spectra,
m/z: 553.75 (molecular ion peak) was recorded same with
actual molecular weight of the compound and 100% base
peak of the important fragment was also found at m/z:
145.20. The IR data, 3373 (O–H str.), 1345, 1169 (C–O
str.) and the data of elemental analysis were reestablished
the structure of 5a by 0.4% different in calculated C, H,
N values with founded values. Similar way we interpreted
and characterized all the synthesized final compounds.
produced moderate activity. The other substitution in
phenyl ring at position 5 in pyrazoline produced less active
against M. tuberculosis.
Conclusion
It could be concluded that, the screening of antitubercular
activity of the novel series of 1,3,5-trisubstituted indo-
phenazine pyrazoline containing benzofuran derivatives as
a new lead compounds endowed with high antitubercular
activity toward MTB and MDR-TB, exhibiting MICs val-
ues between 0.16 and 29.0 lg/ml. The potency, selectivity,
and low cytotoxicity of the synthesized compounds make
them valid lead for synthesizing a new compound that can
produce better activity.
Structure activity relationships
Structure activity relationship study recommended that
substituted phenyl ring (5a–k) at 5 positions by –OH
(o) and –OCH3 (m) in 4,5-dihydro pyrazole posses a var-
iable in vitro and in vivo antitubercular activity against
MTB and MDR-TB listed in Table 1. The ortho and meta
substituted phenyl ring with –NO2 (5e and 5j) produced
very good antitubercular activity. Where, unsubstituted
phenyl ring (5l) found moderate activity. When phenyl ring
was replaced by five-membered ring (5m) at 5 position of
pyrazole ring created good antitubercular activity. The
single C–C bond between pyrazole ring and 5-phenyl ring
can be replaced by ethylene bridge in compound 5n, results
potent inhibitor of M. tuberculosis. Ortho and para posi-
tions substituted with –Cl (5i and 5h) in phenyl ring
Experimental protocols
Chemistry
All the chemicals were procured from Sigma-Aldrich
(USA), E. Merck (Germany), and SD Fine Chemicals
(India). Melting points were determined using one end
open capillary tubes on a Buchi-530 melting point appa-
ratus and are uncorrected. Infrared spectrophotometry
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303
General procedure for the synthesis of 1-[3-(5-
hydroxybenzo[b]furan-2-yl)-5-substituted phenyl-4,5-
(FTIR) was recorded for all the compounds on Jasco-FTIR-
6100 using KBr. 1H Nuclear Magnetic Resonance (1H
NMR) was recorded in Advance bruker (300 MHz).
Chemical shift was recorded in parts per million (ppm)
using tetramethylsilane (TMS) as an internal standard.
Parkin–Elmer 2400 Series II analyzer carried out the ele-
mental analysis. The purity of the compounds was con-
firmed by thin layer chromatography (TLC) using silica gel
G glass plates and solvent system of benzene:ethyl acetate.
The spots were visualized in iodine vapor.
dihydro-1H-1-pyrazolyl]-2-(5H-Indolo[2,3-b]quinoxalin-
5-yl)-1-ethanone derivatives 5a–n
(2E)-1-(5-Hydroxy-1-benzofuran-2-yl)-3-phenylprop-2-en-
1-one derivatives (0.01 mol) and 2-(5,8-dihydroquinoxali-
no[2,3-b]indol-5yl)acetohydrazide (0.02 mol) were dis-
solved in 20 ml of glacial acetic acid into a 100 ml of flat
bottom flask and then refluxed for a period of 10 h at
175°C. Cool the reaction mixture at room temperature and
removed excess solvent under reduce pressure. The reac-
tion mixture was poured into the 250 ml of ice-cold water
with stirring. The solid product was obtained by filtration
and washed with cold water. Similar way we synthesized
other compounds in this series.
Synthesis of 5-hydroxy, 2-acetylbenzofuran 2
2,5-Dihydroxy benzaldehyde 1 (0.01 mol) and chloroace-
tone (0.01 mol) were dissolved in 150 ml of dry acetone
into a 250 ml round bottom flask, to that added 30 g of
anhydrous K2CO3 and refluxed for 10 h at 75°C on water
bath. The solid was obtained after cooling and filtration.
The solid was washed with cold acetone and recrystallized
with petroleum ether (40–60°C). Yield was found 79% and
m.p.: 85°C; IR (KBr) tmax (cm-1): 1173, 1768 (C–O–C),
1674 (C=O), 1614 (C=C), 1486, 1469 (C=C), 1318 (CH),
5a: White solid was recrystallized from 95% of ethanol;
yield: 48%, m.p.: 155–157°C, Rf = 0.88 [benzene and
ethylacetoacetate (3:1 ratio)], kmax: 236.2 nm; IR (KBr)
t
max (cm-1): 3373 (O–H str.), 1454 (Aromatic), 1345, 1169
(C–O str.); 1H NMR (CDCl3, 300 MHz) d (ppm): 8.56 (dd,
4H, J = 7.41, 4.2 Hz, Ph–H), 7.92 (s, 2H, OH), 7.79 (dd,
2H, J = 3.41, 6.2 Hz, CH), 7.55 (s, 4H, CH), 7.39 (s, 6H,
Ph–H), 3.6 (s, 1H, pyrazoline ring), 2.3 (s, 2H, pyrazoline
ring); MS (FAB) m/z: 553.75 (m?), 145.20 (100%);
C33H23N5O4 (553.56): calcd. C 71.60%, H 4.19%, N
12.65%; found C 71.90%, H 4.18%, N 12.56%.
1
1228 (C–O), 1244 and 880 (C=C); H NMR (300 MHz,
CDCl3) d (ppm): 2.826 (s, 3H, CH3), 7.228–7.48 (s, 1H,
CH), 7.26–7.40 (m, 4H, J = 8.0 Hz, Ph–H), 7.10–7.25 (m,
3H, J = 8.3 Hz) furan ring.
5b: Yellowish white solid was recrystallized from
methanol; m.p.: 71–73°C, yield: 38%, Rf = 0.79 [benzene
and ethylacetoacetate (3:1 ratio)], kmax: 306.6 nm; IR
(KBr) tmax (cm-1): 1169–1026 (C–O–C str.), 2839 (CH3
str.), 1456 (CH3 def.); 1H NMR (CDCl3, 300 MHz) d
(ppm): 8.15 (m, 2H, J = 3.44, 6.32, 7.56 Hz, Ph–H), 7.87
(s, 2H, OH), 7.7 (dd, 2H, J = 3.44, 6.32 Hz, Ph–H), 7.55
(d, 4H, J = 8.7, 3.4 Hz, CH), 7.39 (s, 6H, Ph–H), 6.89 (d,
4H, J = 8.73, 2.65 Hz), 4.01 (s, 3H, OCH3), 3.61 (s, 6H,
Ph–H), 2.4 (s, 2H, pyrazoline ring); MS (FAB) m/z: 567.90
(m?), 145.13 (100%); C34H25N5O4 (567.59): calcd. C
71.95%, H 4.44%, N 12.34%; found C 71.97%, H 4.48%,
N 12.46%.
General procedure for the synthesis of (2E)-1-(5-hydroxy-
1-benzofuran-2-yl)-3-phenylprop-2-en-1-one derivatives
3a–n
5-Hydroxy, 2-acetylbenzofuran 2 (0.05 mol) and different
aromatic aldehydes (0.05 mol) were dissolved in 20 ml of
ethanol (98%) in a 250 ml conical flask, the reaction was
carried out according to previously published articles
(Agrawal et al., 2007; Manna et al., 2004, 2008). 8 ml of
KOH/NaOH solution (20%) was added to the reaction
mixture. Then the flask was kept for stirring at room
temperature for 20 h. After stirring the reaction mixture
was poured into 100 ml ice-cold water, and acidified with
conc. HCl. The obtained solid was filtered and washed with
cold water and purified from ethanol to obtained different
benzofuran chalcones 3a–n.
5c: White solid was recrystallized from methanol; m.p.:
103–105°C, yield: 72%, Rf = 0.94 [benzene and ethy-
lacetoacetate (3:1 ratio)], kmax: 296.4 nm; IR (KBr) tmax
(cm-1): 2925, 2804 (Ar–N–CH3 str.), 1446 (CH3 ban.),
1365, 1317 (C–N str.); 1H NMR (CDCl3, 300 MHz) d
(ppm): 8.05 (d, 1H, J = 5.52 Hz, Ph–H), 7.92 (s, 2H, OH),
7.58 (d, 1H, J = 1.55, 8.5 Hz), 7.48 (d, 6H, J = 7.64,
4.23 Hz), 6.9 (d, 4H, J = 8.74, 6.72 Hz), 5.85 (d, 1H,
J = 8.36, 6.55 Hz, CH), 3.9 (s, 6H), 2.8 (s, 3H, CH3), 2.3
(s, 2H, pyrazoline ring); MS (FAB) m/z: 580.12 (m?),
145.20 (100%); C35H28N6O3 (580.64): calcd. C 72.4%, H
4.86%, N 14.47%; found C 72.96%, H 4.18%, N 14.51%.
Synthesis of 2-(5,8-dihydroquinoxalino[2,3-b]indol-
5yl)acetohydrazide 4
2-(5,8-Dihydroquinoxalino[2,3-b]indol-5-yl)acetohydraz-
ide 4 was synthesized according to published procedure
(Manna and Agrawal, 2009) from indophenazine ester
(0.1 mol) with hydrazine hydrate (99%, 0.4 mol) in pres-
ence of absolute ethanol.
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Med Chem Res (2011) 20:300–306
5d: White solid was recrystallized from 95% of ethanol;
7.23 Hz), 7.82 (dd, 2H, J = 3.40, 8.40 Hz), 7.52 (dd, 4H,
J = 1.42, 8.64 Hz), 7.15 (dd, 4H, J = 2.63, 8.64 Hz), 6.95
(s, 1H, OH), 4.96 (s, 2H), 3.56 (s, 2H, pyrazoline ring); MS
(FAB) m/z: 572.84 (m?), 144.97 (100%); C33H22N5O3Cl
(572.01): calcd. C 69.29%, H 3.88%, N 12.24%; found C
69.72%, H 3.14%, N 12.36%.
m.p.: 198–200°C, yield: 56%, Rf = 0.54 [benzene and
ethylacetoacetate (3:1 ratio)], kmax: 298.2 nm; IR (KBr)
tmax (cm-1): 3402 (O–H str.), 1711, 1697 (C=O str.),
1173–1074 (C–O str.); 1H NMR (CDCl3, 300 MHz) d
(ppm): 11.13 (s, 1H) COOH, 8.21 (dd, 2H, J = 3.6,
6.32 Hz), 8.10 (d, 1H, J = 8.51 Hz), 7.80 (dd, 2H,
J = 3.41, 6.22 Hz), 7.57 (s, 1H, OH), 7.29 (s, 6H), 3.55 (s,
6H), 2.13 (s, 2H, pyrazoline ring); MS (FAB) m/z: 581.87
(m?), 145.20 (100%); C34H23N5O5 (581.58): calcd. C
70.22%, H 3.99%, N 12.04%; found C 70.90%, H 3.88%,
N 12.56%.
5i: White solid was recrystallized from 95% of ethanol;
m.p.: 195–197°C, yield: 32%, Rf = 0.87 [benzene and
ethylacetoacetate (3:1 ratio)], kmax: 216.4 nm; IR (KBr)
tmax (cm-1): 3255 (C–Cl str.), 1042 (C–O–C str.), 2912
(CH3 str.), 1433 (CH3 def.), 1601, 790, 748 (C–Cl str.); 1H
NMR (CDCl3, 300 MHz) d (ppm): 8.31 (dd, 2H, J = 3.44,
6.22 Hz), 7.82 (dd, 2H, J = 3.44, 6.40 Hz), 7.22 (s, 4H),
7.40 (s, 1H, OH), 3.55 (s, 6H), 2.3 (s, 2H, pyrazoline ring);
MS (FAB) m/z: 571.94 (m?), 144.97 (100%);
C33H22N5O3Cl (572.01): calcd. C 69.29%, H 3.88%, N
12.24%; found C 69.55%, H 3.76%, N 11.98%.
5e: White solid was recrystallized from methanol; m.p.:
97–99°C, yield: 48%, Rf = 0.63 [benzene and ethylace-
toacetate (3:1 ratio)], kmax: 265.4 nm; IR (KBr) tmax
(cm-1): 1527 (asy) (NO2 str.), 1346 (sym) (NO2 str.), 883
1
(C–N str.), 877; H NMR (CDCl3, 300 MHz) d (ppm): 9.2
(d, 1H, J = 2.38 Hz), 8.53 (dd, 1H, J = 2.38, 9.10 Hz),
8.39 (d, 1H, J = 9.10 Hz), 7.6 (s, 4H, OH), 7.42 (s, 6H),
3.61 (s, 6H), 2.32 (s, 2H, pyrazoline ring); MS (FAB) m/z:
582.35 (m?), 145.14 (100%); C33H22N6O5 (582.56): calcd.
C 68.04%, H 3.81%, N 14.43%; found C 67.98%, H 3.78%,
N 14.36%.
5j: Light yellow solid was recrystallized from methanol;
m.p.: 105–107°C, yield: 54%, Rf = 0.82 [benzene and
ethylacetoacetate (3:1 ratio)], kmax: 280.6 nm; IR (KBr)
t
max (cm-1): 3089, 1599, 1552 (asy) (NO2 str.), 1344 (sym)
1
(NO2 str.), 815 (C–N str.); H NMR (CDCl3, 300 MHz) d
(ppm): 9.1 (d, 1H, J = 2.41 Hz), 8.56 (dd, 1H, J = 2.32,
9.20 Hz), 8.30 (d, 1H, J = 9.17 Hz), 7.6 (s, 1H, OH), 7.22
(s, 6H), 3.23 (s, 6H), 2.41 (s, 2H, pyrazoline ring); MS
(FAB) m/z: 582.62 (m?), 145.14 (100%); C33H22N6O5
(582.56): calcd. C 68.04%, H 3.81%, N 14.43%; found C
68.48%, H 3.70%, N 14.22%.
5f: Light yellow solid was recrystallized from methanol;
m.p.: 223–225°C, yield: 70%, Rf = 0.54 [benzene and
ethylacetoacetate (3:1 ratio)], kmax: 267.6 nm; IR (KBr)
tmax (cm-1): 3240 (O–H str.), 1383 (O–H ben.),
1170–1080 (C–O str.), 1020 (C–O–C str.), 2927 (CH3 str.),
1
1450 (CH3 def.); H NMR (CDCl3, 300 MHz) d (ppm):
5k: Light brown solid was recrystallized from methanol;
m.p.: 164–166°C, yield: 75%, Rf = 0.64 [benzene and
ethylacetoacetate (3:1 ratio)], kmax: 240.2 nm; IR (KBr)
tmax (cm-1): 3010, 1602, 1170–1026 (C–O–C str.), 2837
(CH3 str.), 1446 (CH3 def.); 1H NMR (CDCl3, 300 MHz) d
(ppm): 8.12 (dd, 2H, J = 3.42, 6.30 Hz), 7.69 (dd, 2H,
J = 3.39, 6.30 Hz), 7.57 (d, 4H, J = 8.72 Hz), 7.41 (s, 1H,
OH), 6.90 (d, 4H, J = 8.63 Hz), 3.60 (s, 6H), 2.35 (s, 2H,
pyrazoline ring); MS (FAB) m/z: 567.33 (m?), 145.13
(100%); C34H25N5O4 (567.59): calcd. C 71.95%, H 4.44%,
N 12.34%; found C 72.11%, H 4.31%, N 11.97%.
8.25 (dd, 2H, J = 3.34, 6.34 Hz), 7.81 (dd, 2H, J = 3.47,
6.36 Hz), 7.35 (d, 4H, J = 8.41 Hz), 7.42 (s, 1H, OH),
7.31 (s, 1H, Ph–OH), 6.82 (d, 4H, J = 8.73 Hz), 3.20
(s, 3H, Ph–OCH3), 2.4 (s, 2H, pyrazoline ring); MS (FAB)
m/z: 583.62 (m?), 145.22 (100%); C34H25N5O5 (583.59):
calcd. C 69.97%, H 4.32%, N 12.00%; found C 70.23%, H
4.18%, N 13.56%.
5g: White solid was recrystallized from methanol; m.p.:
154–156°C, yield: 66%, Rf = 0.78 [benzene and ethy-
lacetoacetate (3:1 ratio)], kmax: 220.3 nm; IR (KBr) tmax
(cm-1): 3367 (O–H str.), 1367–1252 (O–H ben.),
1172–1012 (C–O str.); 1H NMR (CDCl3, 300 MHz) d
(ppm): 8.32 (dd, 2H, J = 3.45, 6.32 Hz), 7.81 (dd, 2H,
J = 3.54, 6.41 Hz), 7.24 (s, 4H), 7.42 (s, 6H), 6.27 (s, 1H,
OH), 3.56 (s, 6H), 2.3 (s, 2H, pyrazoline ring); MS (FAB)
m/z: 553.85 (m?), 145.30 (100%); C33H23N5O4 (553.56):
calcd. C 71.60%, H 4.19%, N 12.65%; found C 71.70%, H
4.38%, N 12.16%.
5l: Light yellowish solid was recrystallized from meth-
anol; m.p.: 215–217°C, yield: 68%, Rf = 0.86 [benzene
and ethylacetoacetate (3:1 ratio)], kmax: 203.2 nm; IR
1
(KBr) tmax (cm-1): 3063, 1660, 1592; H NMR (CDCl3,
300 MHz) d (ppm): 8.10 (d, 1H, J = 8.52 Hz), 7.91 (s, 1H,
OH), 7.63 (dd, 1H, J = 1.72, 8.52 Hz), 7.22 (s, 4H), 7.40
(s, 6H), 3.55 (s, 6H), 2.41 (s, 2H, pyrazoline ring); MS
(FAB) m/z: 537.44 (m?), 145.00 (100%); C33H23N5O3
(537.57): calcd. C 73.73%, H 4.31%, N 13.03%; found C
73.12%, H 4.68%, N 13.59%.
5h: White solid was recrystallized from 95% of ethanol;
m.p.: 213–215°C, yield: 81%, Rf = 0.95 [benzene and
ethylacetoacetate (3:1 ratio)], kmax: 197.6 nm; IR (KBr)
tmax (cm-1): 3240 (C–Cl str.), 1035 (C–O–C str.), 2930
(CH3 str.), 1447 (CH3 def.), 1599, 786, 746 (C–Cl str.); 1H
NMR (CDCl3, 300 MHz) d (ppm): 8.5 (dd, 2H, J = 3.42,
5m: Yellowish white solid was recrystallized from
methanol; m.p.: 185–187°C, yield: 54%, Rf = 0.95 [ben-
zene and ethylacetoacetate (3:1 ratio)], kmax: 358.0 nm;
IR (KBr) tmax (cm-1): 3134 (C–H str.), 1550 (furan), 742
123

Med Chem Res (2011) 20:300–306
305
(C–H ban.); 1H NMR (CDCl3, 300 MHz) d (ppm): 8.14 (d,
1H, J = 8.53 Hz), 7.89 (s, 1H), 7.62 (dd, 1H, J = 1.70,
8.56 Hz), 7.24 (s, 4H), 7.42 (s, 1H, OH), 5.73 (s, 1H), 5.82
(d, 2H, J = 5.23 Hz), 3.58 (s, 4H), 2.17 (s, 2H, pyrazoline
ring); MS (FAB) m/z: 527.81 (m?), 145.00 (100%);
C31H21N5O3 (527.53): calcd. C 70.58%, H 4.01%, N
13.28%; found C 70.40%, H 4.18%, N 13.36%.
MICs determination
The MDR-TB clinical isolate was obtained from the
Tuberculosis Research Centre (Chennai, India) and was
resistant to isoniazid, rifampicin, and gatifloxacin. Mini-
mum inhibitory concentration is defined as the minimum
concentration of compounds required to give complete
inhibition of bacterial growth, the method was perfumed
accordingly to Bryson and Szybalski (1952) published
method. Fresh colonies of M. tuberculosis H37RV (MTB)
were collected and suspended in distilled water; the tur-
bidity of the resulting suspensions was then adjusted with
distilled water to match that of a standard 1 mg/ml sus-
pension of M. bovis BCG (containing approximately
108 CFU/ml), after which the suspensions were further
diluted to 10-1 and 10-2 mg/ml. The MICs were deter-
mined on 10% oleic acid–albumin–dextrose–catalase-
enriched 7H11 agar medium. All the synthesized final
compounds and standard drugs were dissolved in dimethyl
formamide (DMF). One volume of drug solution was
added to 99 volume of culture medium, and serial twofold
dilutions were carried out; final synthesized compounds
concentrations ranged from 4 to 0.12 lg/ml and rifampicin,
gatifloxacin from 4 to 0.12 lg/ml bacterial suspensions.
The bacterial suspensions were plated, in duplicate, on both
drug-free and drug-containing medias. MIC50 was defined
as the lowest drug concentration that inhibited 50% of the
bacterial growth, and the MIC90 was 90% of the bacterial
growth, compared to that on drug-free medium after
incubation at 30°C for 60 days. The MICs was determined
against M. tuberculosis (H37Rv) on 7H11 agar medium.
The MICs of all the synthesized compounds and standard
drugs were reported in Table 1.
5n: Light brown solid was recrystallized from methanol;
m.p.: 173–175°C, yield: 72%, Rf = 0.89 [benzene and
ethylacetoacetate (3:1 ratio)], kmax: 348.7 nm; IR (KBr)
tmax (cm-1): 2933, 1610, 978 (R–CH=CHR); 1H NMR
(CDCl3, 300 MHz) d (ppm): 8.44 (d, 1H, J = 8.60 Hz),
7.78 (s, 1H), 7.55 (dd, 1H, J = 2.70, 8.20 Hz), 7.32 (s,
4H), 7.60 (s, 1H, OH), 3.52 (s, 4H), 2.35 (s, 2H, pyrazoline
ring); MS (FAB) m/z: 563.60 (m?), 145.10 (100%);
C35H25N5O3 (563.60): calcd. C 74.59%, H 4.47%, N
12.43%; found C 76.90%, H 4.13%, N 12.56%.
Biology
In vitro antitubercular activity
The in vitro antitubercular activity was screened against M.
tuberculosis using egg base Lowenstein–Jensen and Ogawa
¨
media (Tanoue et al., 2002), by measuring the growth of
M. tuberculosis (H37RV) after 3 and 6 weeks. Cultures
were incubated at 37°C in ambient air for up to 6 weeks.
Bacterial counts were measured and compared with stan-
dard drugs (rifampicin, gatifloxacin) and controls (vehicle-
treated).
In vivo antitubercular studies
Cytotoxicity
The in vivo animal model was used for evaluating antitu-
bercular activity against MTB at a dose of 25 mg/kg body
weight in 6-week-old female CD-1 mice, six per group.
The mice were infected intravenously through the caudal
vein with 106 to 107 viable M. tuberculosis ATCC 35801
(Sriram et al., 2005). Test drug treatment was carried out
by the intra-peritoneal route began after 10 days of inoc-
ulation of the animal with the microorganism and contin-
ued for 10 days. Thirty-five days post infection, the spleen
and right lung were aseptically removed and grounded
using tissue homogenizer, and the numbers of viable
organism were determined by serial tenfold dilutions.
Viable cell counts were converted to logarithms, which
were then evaluated by one or two-variable analyses of
variance. Statistically significant effects from the analyses
of variance were further evaluated by Tukey’s honestly
significant difference tests to make pair-wise comparisons
among means.
All the synthesized compounds were examined for in vitro
cytotoxicity (Gundersen et al., 2002; Sriram et al., 2007;
Mustafa et al., 2005) using a mammalian VERO cell
(CCL-81, American Type Culture Collection) by exposing
monolayers in 96-well plates to threefold dilutions of test
compounds for 72 h at concentration of 58.7 mg/l. Cell
viability was measured using the CellTiter96 aqueous non-
radioactive cell proliferation assay (Promega Corp, Madi-
son, WI), which determines the extent of reduction of a
tetrazolium dye by measuring the absorbance of the prod-
uct at 490 nm. Untreated cells and cells lysed with sodium
dodecyl sulfate were used to determine 0 and 100% inhi-
bition, respectively.
Acknowledgments The authors gratefully acknowledge to Depart-
ment of Microbiology, Kasturba Medical College, Manipal, India for
providing laboratory facilities to carryover the in vivo and in vitro
123

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Med Chem Res (2011) 20:300–306
antitubercular activity. We acknowledge to IISc, Bangalore, SPIC
Foundation, Chennai and Tuberculosis Research Centre (Chennai,
Lenaerts AJ, Gruppo V, Marietta KS, Johnson CM, Driscoll DK,
Tompkins NM, Rose JD, Reynolds RC, Orme IM (2005)
Preclinical testing of the nitroimidazopyran PA-824 for activity
against Mycobacterium tuberculosis in a series of in vitro and in
vivo models. Antimicrob Agents Chemother 49:2294–2301
Lenaerts AJ, Bitting C, Woolhisher L, Gruppo V, Marietta KS,
Johnson CM, Orme IM (2008) Evaluation of a 2-pyridone, KRQ-
10018, against Mycobacterium tuberculosis in vitro and in vivo.
Antimicrob Agents Chemother 52:1513–1515
Manna K, Agrawal YK (2009) Microwave assisted synthesis of new
indophenazine 1,3,5-trisubstituted pyrazoline derivatives of
benzofuran and their antimicrobial activity. Bioorg Med Chem
Lett 19:2688–2692
Manna K, Babu VH, Srinivasan KK, Sneha J, Bhatt GV (2004)
Synthesis and biological evaluation of 1,3,5-trisubstituted pyr-
azolines bearing benzofuran. Indian J Heterocycl Chem 13:
253–256
Manna K, Agrawal YK, Srinivasan KK (2008) Synthesis and
biological evaluations of new benzofuran isoxazole derivatives
as potential antitubercular, antibacterial and antifungal agents.
Indian J Heterocycl Chem 17:250–252
Mustafa J, Khan SI, Ma G, Walker LA, Khan IA (2005) Synthesis and
in vitro cytotoxic activity of N-, F-, and S-ether derivatives of
podophyllotoxin fatty acid adducts. Lipids 40:375–382
Prakash O, Kumar R, Prakash V (2008) Synthesis and antifungal
activity of some new 3-hydroxy-2-(1-phenyl-3-aryl-4-pyrazolyl)
chromones. Eur J Med Chem 43:435–440
Sriram D, Yogeeswari P, Basha SJ (2005) Synthesis and antimyco-
bacterial evaluation of various 7-substituted ciprofloxacin deriv-
atives. Bioorg Med Chem 13:5774–5778
1
India) for provided H NMR spectra, mass spectra, elemental analy-
sis, and MDR-TB stain, respectively. Authors heartily acknowledge
to Nirma University, Ahmedabad, Gujarat, India for the financial
support, laboratory facilities, and other requirements.
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