Synthesis, structural activity relationship and anti-tubercular activity of novel pyrazoline derivatives

Article abstract of DOI:10.1016/j.ejmech.2006.08.004

In the present investigation, a series of 5-(-4-(substituted) phenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-2-toluidino methane thione and 5-(substituted) phenyl-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-2-methoxyanilino me

Full text of DOI:10.1016/j.ejmech.2006.08.004

European Journal of Medicinal Chemistry 42 (2007) 268e275
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, structural activity relationship and anti-tubercular
activity of novel pyrazoline derivatives
*
Mohamed Ashraf Ali, Mohammad Shaharyar , Anees Ahamed Siddiqui
Faculty of Pharmacy, Jamia Hamdard University, Department of Pharmaceutical Chemistry, Hamdard Nagar, New Delhi 110062, India
Received 22 February 2006; received in revised form 22 July 2006; accepted 11 August 2006
Available online 26 September 2006
Abstract
In the present investigation, a series of 5-(-4-(substituted) phenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-2-toluidino
methane thione and 5-(substituted) phenyl-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-2-methoxyanilino methane thione were
synthesized by the reaction between hydrazine hydrate and chalcones (3aek) followed by condensation with appropriate aryl isothiocyanate
which yielded N-substituted pyrazoline derivatives. Newly synthesized compounds were tested for their in vitro anti-tubercular activity against
Mycobacterium tuberculosis H37Rv using the BACTEC 460 radiometric system. Among the synthesized compounds, compound anilino-3-(4-
hydroxy-3-methylphenyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-1-pyrazolylmethanethione (6i) was found to be more active agent against
M. tuberculosis H37Rv with minimum inhibitory concentration of 0.0034 mM.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Pyrazoline; Isothiocyanate; Anti-tubercular; Mycobacterium tuberculosis
1. Introduction
[7e10].Thecurrentworkdescribesthesynthesisofnovelpyrazo-
line moiety with encouraging anti-mycobacterial activity against
M. tuberculosis H37Rv.
Tuberculosis, an infectious disease caused by Mycobacterium
tuberculosis, is the primary cause of mortality in the world.
Mycobacteria are ubiquitous organisms that are becoming
increasingly important intracellular pathogens that establish an
infection in oxygen-rich macrophage of the lung [1]. The emer-
genceofAIDS,declineofsocioeconomicstandardsandareduced
emphasis on tuberculosis control programs contribute to the dis-
ease’s resurgence in industrialized countries [2]. Resistance of M.
tuberculosis strains to anti-mycobacterial agents is an increasing
problem worldwide [3e5]. However, powerful new anti-TB
drugs with new mechanism of action have not been developed
in the last 40 years. In spite of severe toxicity on repeated dosing
of isoniazid (INH), it is still considered to be a first line drug for
chemotherapy of tuberculosis [6]. Literature survey reveals that
pyrazoline derivatives are active against many mycobacteria
2. Results and discussion
2.1. Chemistry
5-(-4-(Substituted) phenyl)-3-(4-hydroxy-3-methylphenyl)-
4,5-dihydro-1H-1-pyrazolyl-2-toluidino methane thione and
5-(substituted) phenyl-3-(4-hydroxy-3-methylphenyl)-4,5-di-
hydro-1H-1-pyrazolyl-2-methoxyanilino methane thione
(5aek) and (6aek) described in this study are shown in
Tables 1, 2 and 3, and a reaction sequence for the preparation
is outlined in Scheme 1. The chalcones were prepared by re-
acting 3-methyl-4-hydroxy acetophenone with appropriate
aldehyde in the presence of a base by conventional Claisene
Schmidt condensation. Reaction between newly synthesized
chalcones and hydrazine hydrate in ethanol led to synthesis
of novel pyrazolines (4aek), which on treatment with various
* Corresponding author. Tel.: þ91 9899452373; fax: þ91 11 26059666.
E-mail address: yarmsy@yahoo.co.in (M. Shaharyar).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.08.004

M.A. Ali et al. / European Journal of Medicinal Chemistry 42 (2007) 268e275
269
Table 1
Physical constants of the synthesized compounds
OMe
CH3
S
R
CH3
S
OH
OH
N
NH
N
N
NH
CH3
N
R
5a-k
6a-k
Yield (%)
Compound
R
Melting point (ꢂC)
Molecular formula
25H25N3O2S
Molecular weight
5a
5b
5c
5d
5e
5f
4-Methoxy phenyl-
4-Chloro phenyl-
74
70
72
80
82
85
92
85
77
82
90
82
80
75
80
77
72
82
72
70
44
80
144
131
104
121
102
103
196
115
164
104
205
124
153
115
234
197
106
142
156
172
194
169
C
431.55
435.97
444.59
401.52
461.57
491.60
420.52
435.97
470.41
446.52
392.48
447.55
451.96
460.59
417.52
477.57
507.60
436.52
451.96
486.41
462.52
407.48
C24H22N3OSCl
C26H28N4OS
C24H23N3OS
4-Dimethylamino phenyl-
Phenyl-
3,4-Dimethoxy phenyl-
3,4,5-Trimethoxy phenyl-
4-Fluoro phenyl-
2-Chloro phenyl-
2,6-Dichloro phenyl-
3-Nitro phenyl-
C26H27N3O3S
C27H29N3O4S
C24H23N3OSF
C24H22N3OSCl
C24H21N3OSCl2
5g
5h
5i
5j
C
24H22N4O3S
C22H21N3O2S
25H25N3O3S
C24H22N3O2SCl
26H28N4O2S
C24H23N3O2S
26H27N3O4S
C27H29N3O5S
23H20N3O2S
C24H22N3O2SCl
24H21N3O2SCl2
5k
6a
6b
6c
6d
6e
6f
Furfuryl-
4-Methoxy phenyl-
4-Chloro phenyl-
4-Dimethylamino phenyl-
Phenyl-
C
C
3,4-Dimethoxy phenyl-
3,4,5-Trimethoxy phenyl-
4-Fluoro phenyl-
2-Chloro phenyl-
2,6-Dichloro phenyl-
3-Nitro phenyl-
C
6g
6h
6i
C
C
6j
6k
C24H22N4O4S
C22H21N3O3S
Furfuryl-
Recrystallization: Ethanol, acetic acid.
aryl isothiocyanates afforded respective 3,5-substituted pyra-
zolines (5aek) and (6aek) in 65e92% yield after recrystalli-
zation with glacial acetic acid and ethanol. The purity of the
compounds was checked by TLC and elemental analyses.
Both analytical and spectral data (1H NMR, IR) of all the syn-
thesized compounds were in full agreement with the proposed
structures. Final compounds in general, in the infrared spectra
(IR), revealed OH, NH, C]O, C]N, CeN, and C]S peaks
at 3307, 3220, 1640, 1590, 1320 and 1130 cmꢀ1, respectively.
In the nuclear magnetic resonance spectra (1H NMR) the sig-
nals of the respective protons of the prepared titled compounds
were verified on the basis of their chemical shifts, multiplici-
ties and coupling constants. The spectra showed singlet
at d 2.2 ppm corresponding to C4-methylene group; singlet
at d 2.8 ppm corresponding to methyl group; singlet at
d 3.9 ppm for methoxy proton; singlet at d 5.78 ppm corre-
sponding to C5 proton; multiplet at d range 7.2e8.4 ppm for
aromatic proton; singlet at d range 8.7e9.5 ppm for OH and
singlet at d range 10.0e10.5 ppm for NH proton. The elemen-
tal analysis results were within ꢁ0.4% of the theoretical
values.
1 and 2 with INH, a standard used for comparison. Among the
22 newly synthesized compounds, compound anilino-3-(4-hy-
droxy-3-methylphenyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-
1-pyrazolylmethanethione (6i) produced highest efficacy and
exhibited >90% inhibition at a concentration of 0.0034 mM
followed by (6f), (6a) and (6e) which showed moderate inhib-
itory activity with 0.0084 mM, 0.0092 mM and 0.0118 mM,
respectively. The 2,6-dichloro group substitution (6aek) de-
rivatives (6i) displayed relatively higher inhibitory activity in
general. However, the electron rich groups such as, 4-chloro,
2-chloro, 2,6-dichloro and 3-nitro substituted analogue com-
poundsd5b, 5h, 5i, 5j, 6b, 6h, 6jdproduced significant de-
crease in inhibitory activity against M. tuberculosis H37Rv.
On the other hand pyrazoline analogues with 40-methoxy phenyl
substitution (6a), 30,40-dimethoxy phenyl substitution (6e) and
30,40,50-trimethoxy phenyl substitution (6f) showed relatively
moderate anti-tubercular activity. But among (5aek) derivatives,
compounds with 30,40-dimethoxy phenyl substitution (5e) and
30,40,50-trimethoxy phenyl substitution (5f) exhibited relatively
low inhibitory activity against M. tuberculosis H37Rv. Instead
of (CH3) group, (OCH3) group substitution at N1 phenyl ring
in pyrazoline analogue worsens the anti-mycobacterial activity.
These reports clearly showed that the increases in the presence
of dichloro substitution at 2,6 position pyrazoline (6aek) deriv-
atives (6i) causes remarkable improvement in anti-mycobacte-
rial activity.
2.2. Anti-mycobacterial activity
Among the ring substituted pyrazoline derivatives (5aek)
and (6aek) were tested for their anti-mycobacterial activity
in vitro against M. tuberculosis H37Rv using the BACTEC
460 radiometric system. The results are summarized in Tables
All the newly synthesized compounds (5aek) and (6aek)
were tested for cytotoxicity (IC50) in VERO cells at

270
M.A. Ali et al. / European Journal of Medicinal Chemistry 42 (2007) 268e275
Table 2
Primary anti-mycobacterial activity of the synthesized compounds against
Table 3
Anti-mycobacterial activityofthe synthesized compounds against M. tuberculosis
H37Rv
M. tuberculosis H37Rv
CH3
OMe
NH
S
R
CH3
CH
S
S
3
CH3
OMe
NH
S
R
OH
OH
OH
OH
N
N
N
NH
CH3
N
N
NH
CH3
N
N
N
R
R
5a-k
6a-k
Actual
5a-k
6a-k
Compound
R
Compound R
Primary
screen
(6.25 mg/ml)
%
Inhibition (mM)
Concentration
MIC (mg/ml)
5a
5b
5c
5d
5e
5f
4-Methoxy phenyl-
4-Chloro phenyl-
e
6.25
6.25
e
5a
5b
5c
5d
5e
5f
4-Methoxy phenyl- >6.25
4-Chloro phenyl- >6.25
4-Dimethylamino phenyl- >6.25
06
44
12
29
72
44
32
33
21
14
26
94
04
28
06
88
92
16
44
98
08
18
0.0403
0.0143
0.0140
0.0354
0.0977
0.1467
0.0148
0.0150
0.0143
0.0132
0.1706
0.0092
0.0138
0.0135
0.0149
0.0118
0.0084
0.0164
0.0202
0.0034
0.2080
0.0201
4-Dimethylamino phenyl-
Phenyl-
3,4-Dimethoxy phenyl-
3,4,5-Trimethoxy phenyl-
4-Fluoro phenyl-
2-Chloro phenyl-
2,6-Dichloro phenyl-
3-Nitro phenyl-
e
Phenyl- >6.25
3,4-Dimethoxy phenyl- >6.25
3,4,5-Trimethoxy phenyl- >6.25
e
5g
5h
5i
6.25
6.25
6.25
6.25
e
5g
5h
5i
4-Fluoro phenyl-
2-Chloro phenyl-
2,6-Dichloro phenyl-
3-Nitro phenyl-
Furfuryl-
6.25
>6.25
>6.25
>6.25
>6.25
<6.25
>6.25
5j
5k
6a
6b
6c
6d
6e
6f
Furfuryl-
5j
4-Methoxy phenyl-
4-Chloro phenyl-
4-Dimethylamino phenyl-
Phenyl-
4.12
6.25
6.25
6.25
5.67
e
5k
6a
6b
6c
6d
6e
6f
4-Methoxy phenyl-
4-Chloro phenyl-
4-Dimethylamino phenyl- 6.25
3,4-Dimethoxy phenyl-
3,4,5-Trimethoxy phenyl-
4-Fluoro phenyl-
2-Chloro phenyl-
2,6-Dichloro phenyl-
3-Nitro phenyl-
Phenyl-
>6.25
<6.25
3,4,5-Trimethoxy phenyl- <6.25
3,4-Dimethoxy phenyl-
6g
6h
6i
e
e
6g
6h
6i
4-Fluoro phenyl-
2-Chloro phenyl-
2,6-Dichloro phenyl-
3-Nitro phenyl-
Furfuryl-
>6.25
>6.25
6.25
1.66
e
e
6j
6k
Furfuryl-
6j
6k
>6.25
>6.25
Isoniazid (0.025e0.05 mg/ml).
Isoniazid (0.025e0.05 mg/ml).
were recorded on a Bruker AC 300 MHz spectrometer using
TMS as internal standard in DMSO/CDCl3, mass spectra
were recorded on a Bruker Esquire LCeMS using ESI and el-
emental analyses were performed on a Carlo Erba 1106 ele-
mental analyzer.
concentrations of 62.5 mg/ml or 10 times. After 72 h of expo-
sure, viability was assessed on the basis of cellular conversion
of MTT into a formazan product using the Promega Cell Titer
96 Non-radioactive Cell proliferation method. Most of the ac-
tive compounds were found to be non-toxic till 62.5 mg/ml.
Among the newer derivatives, it is conceivable that deriva-
tives showing anti-mycobacterial activity can be further mod-
ified to exhibit better potency than the standard drugs. Further
studies to acquire more information about quantitative struc-
tureeactivity relationships (QSAR) are in progress in our lab-
oratory. The pyrazoline derivatives discovered in this study
may provide valuable therapeutic intervention for the treat-
ment of anti-tubercular diseases.
3.1. General procedure
4-Hydroxy-3-methyl acetophenone (0.01 mol) was dis-
solved in ethanol. Then, a solution of potassium hydroxide
(30%, 5 ml) and suitable substituted aldehydes (0.01 mol) in
10 ml of petroleum ether was added to the resulting solution
with continuous stirring. The resulting solution was allowed
to stand overnight. After 4 h of stirring, it was poured into
ice-cold water, then neutralized with hydrochloric acid. The
solid separate was filtered off, dried and purified from ethanol
(3aek).
3. Experimental
The entire chemicals were supplied by E. Merck (Germany)
and SD Fine Chemicals (India). Melting points were deter-
mined by open tube capillary method and are uncorrected.
Purity of the compounds was checked on thin layer chroma-
tography (TLC) plates (silica gel G) in the solvent system tol-
ueneeethyl formateeformic acid (5:4:1) and benzenee
methanol (8:2), the spots were located under iodine vapors
or UV light. IR spectra were obtained on a PerkineElmer
1720 FT-IR spectrometer (KBr pellets). 1H NMR spectra
3.1.1. 1-(40-Hydroxy-30-methylphenyl)-3-[(substituted)
phenyl]-2-propen-1-one
1-(40-Hydroxy-30-methylphenyl)-3-[(substituted) phenyl]-
2-propen-1-one derivatives were synthesized by condensing
4-hydroxy-3-methyl acetophenone with appropriate aromatic
aldehydes according to ClaiseneSchmidt condensation.

M.A. Ali et al. / European Journal of Medicinal Chemistry 42 (2007) 268e275
271
CH3
CH3
R
OH
OH
NH2NH H O
NaOH
2
2
+
R-CHO
C2H5OH
H3C
C2H5OH
2a-k
O
3a-k
O
1
CH3
N
HN
OH
NCS
NCS
R
4a-k
H3C
OCH3
C2H5OH
C2H5OH
OCH3
CH3
S
CH3
S
CH3
N
NH
N
N
NH
N
OH
OH
R
R
5a-k
6a-k
Scheme 1. Protocol for the synthesis.
3.1.2. 1-(4-Hydroxy-3-methylphenyl-3-(4-methoxy phenyl)-
2-propen-1-one (3a)
IR: (KBr, cmꢀ1) 3210 (OH), 1682 (C]O), 3030 (CH). H
NMR (DMSO-d6, ppm): 2.2 (3H, s, CH3), 3.9 (3H, s, OCH3),
6.8e6.9 (1H ꢃ 2, d, J ¼ 7.5, 8.5 Hz, CH]CH), 7.2e7.9 (7H,
s, aromatic), 9.2 (1H, s, OH).
OCH3 ꢃ 2), 6.9e7.3 (1H ꢃ 2, d, J ¼ 7.45, 7.29 Hz,
CH]CH), 7.6e8.1 (6H, m, aromatic), 9.2 (1H, s, OH).
1
3.1.7. 1-(4-Hydroxy-3-methylphenyl-3-(3,4,5-tri-methoxy
phenyl)-2-propen-1-one (3f)
1
IR: (KBr, cmꢀ1) 3200 (OH), 1680 (C]O), 3040 (CH). H
NMR (DMSO-d6, ppm): 9.2 (1H, s, OH), 2.2 (3H, s, CH3),
7.7e8.1 (5H, m, aromatic), 3.9 (9H, s, OCH3 ꢃ 3), 6.9e7.5
(1H ꢃ 2, d, J ¼ 7.55, 7.27 Hz, CH]CH).
3.1.3. 1-(4-Hydroxy-3-methylphenyl-3-(4-chloro phenyl)-2-
propen-1-one (3b)
IR: (KBr, cmꢀ1) 3210 (OH), 782 (CeCl), 1680 (C]O),
1
3042 (CH). H NMR (DMSO-d6, ppm): 2.2 (3H, s, CH3),
6.7e6.8 (1H ꢃ 2, d, J ¼ 8.34, 6.79 Hz, CH]CH), 7.7e8.0
3.1.8. 1-(4-Hydroxy-3-methylphenyl-3-(4-fluoro phenyl)-2-
propen-1-one (3g)
(7H, m, aromatic), 9.2 (1H, s, OH).
IR: (KBr, cmꢀ1) 3200 (OH), 1680 (C]O), 3040 (CH), 670
1
3.1.4. 1-(4-Hydroxy-3-methylphenyl-3-(4-dimethyl amino
phenyl)-2-propen-1-one (3c)
(CeF). H NMR (DMSO-d6, ppm): 9.2 (1H, s, OH), 2.2 (3H,
s, CH3), 7.7e8.2 (7H, m, aromatic), 6.9e7.5 (1H ꢃ 2, d,
1
IR: (KBr, cmꢀ1) 3200 (OH), 1684 (C]O), 3040 (CH). H
J ¼ 7.24, 7.29 Hz, eCH]CH).
NMR (DMSO-d6, ppm): 2.2 (3H, s, CH3), 2.83 (6H, s, N
(CH3 ꢃ 2)), 6.8e6.9 (1H ꢃ 2, d, J ¼ 7.61, 7.63 Hz,
CH]CH), 7.6e8.1 (7H, m, aromatic), 9.2 (1H, s, OH).
3.1.9. 1-(4-Hydroxy-3-methylphenyl-3-(2-chloro phenyl)-2-
propen-1-one (3h)
IR: (KBr, cmꢀ1) 3200 (OH), 1680 (C]O), 3040 (CH), 770
(CeCl). 1H NMR (DMSO-d6, ppm): 9.2 (1H, s, OH), 2.2 (3H,
s, CH3), 7.6e8.0 (7H, m, aromatic), 6.9e7.5 (1Hx ꢃ 2, d,
J ¼ 8.35, 3.63 Hz, eCH]CH).
3.1.5. 1-(4-Hydroxy-3-methylphenyl-3-phenyl-2-propen-1-
one (3d)
1
IR: (KBr, cmꢀ1) 3210 (OH), 1670 (C]O), 3040 (CH). H
NMR (DMSO-d6, ppm): 2.2 (3H, s, CH3), 6.8e7.4 (1H ꢃ 2, d,
J ¼ 8.28, 6.70 Hz, CH]CH), 7.7e8.2 (8H, m, aromatic), 9.2
(1H, s, OH).
3.1.10. 1-(4-Hydroxy-3-methylphenyl-3-(2,6-dichloro
phenyl)-2-propen-1-one (3i)
3.1.6. 1-(4-Hydroxy-3-methylphenyl-3-(3,4-dimethoxy
phenyl)-2-propen-1-one (3e)
IR: (KBr, cmꢀ1) 3200 (OH), 1680 (C]O), 3040 (CH), 770
(CeCl). 1H NMR (DMSO-d6, ppm): 9.2 (1H, s, OH), 2.2 (3H,
s, CH3), 7.7e8.0 (6H, m, aromatic), 6.9e7.5 (1H ꢃ 2, d,
J ¼ 5.41, 15.68 Hz, CH]CH).
1
IR: (KBr, cmꢀ1) 3210 (OH), 1686 (C]O), 3030 (CH). H
NMR (DMSO-d6, ppm): 2.2 (3H, s, CH3), 3.9 (6H, s,

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M.A. Ali et al. / European Journal of Medicinal Chemistry 42 (2007) 268e275
3.1.11. 1-(4-Hydroxy-3-methylphenyl-3-(3-nitro phenyl)-2-
propen-1-one (3j)
IR: (KBr, cmꢀ1) 3200 (OH), 1680 (C]O), 3040 (CH). H
NMR (DMSO-d6, ppm): 9.2 (1H, s, OH), 2.2 (3H, s, CH3),
7.7e8.2 (7H, m, aromatic), 6.9e7.5 (1H ꢃ 2, d, J ¼ 5.46,
16.3 Hz, CH]CH).
CH3), 3.7 (6H, s, 2 ꢃ OCH3), 7.2e7.8 (6H, m, aromatic),
5.50 (1H, s, NH), 4.24 (1H, s, CH), 9.2 (1H, s, OH).
1
4.1.6. 4-[5-(30,40,50-Trimethoxy phenyl)-4,5-dihydro-1H-3-
pyrazolyl]-2-methylphenol (4f)
IR: (KBr, cmꢀ1) 3307 (OH), 1596 (C]N), 1320 (CeN).
1H NMR (DMSO-d6, ppm): 2.3 (2H, s, CH2), 3.4 (3H, s,
CH3), 3.6 (9H, s, OCH3), 4.24 (1H, s, CH), 5.48 (1H, s,
NH), 7.3e7.8 (5H, m, aromatic), 9.5 (1H, s, OH).
3.1.12. 1-(4-Hydroxy-3-methylphenyl-3-furfuryl-2-propen-
1-one (3k)
IR: (KBr, cmꢀ1) 3200 (OH), 1680 (C]O), 3040 (CH). H
1
NMR (DMSO-d6, ppm): 9.2 (1H, s, OH), 2.2 (3H, s, CH3),
7.7e8.2 (6H, m, aromatic), 6.4e7.4 (3H, m, furan), 6.8e6.9
(1H ꢃ 2, d J ¼ 3.0, 8.36 Hz, CH]CH).
4.1.7. 4-[5-(40-Fluorophenyl)-4,5-dihydro-1H-3-pyrazolyl]-
2-methylphenol (4g)
IR: (KBr, cmꢀ1) 3312 (OH), 1590 (C]N), 1320 (CeN),
700 (CeF). 1H NMR (DMSO-d6, ppm): 9.4 (1H, s, OH),
7.3e7.8 (7H, m, aromatic), 5.42 (1H, s, NH), 4.24 (1H, s,
CH), 3.4 (3H, s, CH3), 2.3 (2H, s, CH2).
4. General procedure
4.1. 4-[5-(Substituted) phenyl-4,5-dihydro-1H-3-
pyrazolyl]-2-methylphenol (4aek)
4.1.8. 4-[5-(20-Chlorophenyl)-4,5-dihydro-1H-3-pyrazolyl]-
2-methylphenol (4h)
To a solution of chalcone (3aek) in ethanol, hydrazine hy-
drate (99%) was added drop wise. The reaction mixture was
heated under reflux for 7 h and then cooled and poured onto
crushed ice. The solid pyrazoline product was filtered and re-
crystallized from ethanol (4aek).
(KBr, cmꢀ1) 3306 (OH), 1586 (C]N), 1320 (CeN), 774
1
(CeCl). H NMR (DMSO-d6, ppm): 9.5 (1H, s, OH), 7.6e
8.2 (7H, m, aromatic), 5.50 (1H, s, NH), 4.24 (1H, s, CH),
3.4 (3H, s, CH3), 2.3 (2H, s, CH2).
4.1.9. 4-[5-(20,60-Dichlorophenyl)-4,5-dihydro-1H-3-pyra-
zolyl]-2-methylphenol (4i)
4.1.1. 4-[5-(40-Methoxyphenyl)-4,5-dihydro-1H-3-pyrazolyl]-
2-methylphenol (4a)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN).
1H NMR (DMSO-d6, ppm): 2.3 (2H, s, CH2), 3.4 (3H, s,
CH3), 3.9 (3H, s, OCH3), 4.24 (1H, s, CH), 5.52 (1H, s,
NH), 7.3e7.8 (7H, m, aromatic), 9.5 (1H, s, OH).
IR: (KBr, cmꢀ1) 3317 (OH), 1594 (C]N), 1320 (CeN),
1
770 (CeCl). H NMR (DMSO-d6, ppm): 9.5 (1H, s, OH),
7.3e7.8 (6H, m, aromatic), 5.54 (1H, s, NH), 4.24 (1H, s,
CH), 3.4 (3H, s, CH3), 2.3 (2H, s, CH2).
4.1.10. 4-[5-(30-Nitrophenyl)-4,5-dihydro-1H-3-pyrazolyl]-
2-methylpheno (4j)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN).
1H NMR (DMSO-d6, ppm): 9.4 (1H, s, OH), 7.8e8.4 (7H,
m, aromatic), 5.56 (1H, s, NH), 4.20 (1H, s, CH), 3.2 (3H,
s, CH3), 2.7 (2H, s, CH2).
4.1.2. 4-[5-(40-Chlorophenyl)-4,5-dihydro-1H-3-pyrazolyl]-
2-methylphenol (4b)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN),
1
770 (CeCl). H NMR (DMSO-d6, ppm): 2.3 (2H, s, CH2),
3.4 (3H, s, CH3), 4.24 (1H, s, CH), 5.50 (1H, s, NH), 7.2e
7.6 (7H, m, aromatic), 9.5 (1H, s, OH).
4.1.11. 4-[5-(20-Furyl)-4,5-dihydro-1H-3-pyrazolyl]-2-
methylphenol (4k)
IR: (KBr, cmꢀ1) 3317 (OH), 1590 (C]N), 1320 (CeN).
1H NMR (DMSO-d6, ppm): 7.3e7.8 (3H, m, aromatic),
6.8e7.9 (3H, m, furan), 5.52 (1H, s, NH), 4.20 (1H, s, CH),
3.42 (3H, s, CH3), 2.3 (2H, s, CH2), 9.2 (1H, s, OH).
4.1.3. 4-[5-(40-Dimethylaminophenyl)-4,5-dihydro-1H-3-
pyrazolyl]-2-methylphenol (4c)
IR: (KBr, cmꢀ1) 3307 (OH), 1580 (C]N), 1324 (CeN).
1H NMR (DMSO-d6, ppm): 2.3 (2H, s, CH2), 2.9 (3H ꢃ 2, s,
N(CH3)2), 3.4 (3H, s, CH3), 4.24 (1H, s, CH), 5.52 (1H, s,
NH), 7.4e8.0 (7H, m, aromatic), 9.5 (1H, s, OH).
5. General procedure
4.1.4. 2-Methyl-4-(50-phenyl-4,5-dihydro-1H-3-pyrazolyl)
phenol (4d)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN).
1H NMR (DMSO-d6, ppm): 2.3 (2H, s, CH2), 3.4 (3H, s,
CH3), 5.54 (1H, s, NH), 4.24 (1H, s, CH), 7.3e7.6 (8H, m, ar-
omatic), 9.5 (1H, s, OH).
5.1. 3-(4-Hydroxy-3-methylphenyl)-5-(substituted)
phenyl-4,5-dihydro-1H-1-pyrazolyl-2-toluidino methane
thione (5aek)
To a solution of pyrazoline (4aek) (0.01 mol) in ethanol
(20 ml) was added 2-methyl aryl isothiocyanate (1.501 ml,
0.01 mol) and the reaction mixture was refluxed for 4 h. The
reaction mixture was cooled and then poured onto crushed
ice. The obtained solid was filtered, washed with water and
purified from ethanol to give compounds (5aek).
4.1.5. 4-[5-(30,40-Dimethoxyphenyl)-4,5-dihydro-1H-3-pyr-
azolyl]-2-methylphenol (4e)
IR: (KBr, cmꢀ1) 3310 (OH), 1590 (C]N), 1320 (CeN)).
1H NMR (DMSO-d6, ppm): 2.3 (2H, s, CH2), 3.4 (3H, s,

M.A. Ali et al. / European Journal of Medicinal Chemistry 42 (2007) 268e275
273
5.1.1. Compound (5a)
5.1.8. Compound (5h)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 770 (CeCl),
IR: (KBr, cmꢀ1) 3307 (OH), 1596 (C]N), 1320 (CeN),
1130 (C]S), 3224 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.8 (6H, s, 2 ꢃ CH3), 3.9 (3H, s, OCH3), 5.3
(1H, s, CH), 7.2e7.4 (11H, m, aromatic), 9.7 (1H, s, OH),
10.0 (1H, s, NH), EISMS: m/z: 431 (Mþ); Calcd/Anal. [C
69.58/69.52, H 5.84/5.84, N 9.74/9.73].
1
1320 (CeN), 1130 (C]S), 3220 (NH). H NMR (DMSO-
d6, ppm): 2.2 (2H, s, CH2), 2.8 (6H, s, 2 ꢃ CH3), 5.2 (1H, s,
CH), 7.2e7.4 (11H, m, aromatic), 9.4 (1H, s, OH), 10.0
(1H, s, NH), EISMS: m/z: 435 (Mþ); Calcd/Anal. [C 66.12/
66.10, H 5.09/5.05, N 9.64/9.64].
5.1.9. Compound (5i)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 770 (CeCl),
5.1.2. Compound (5b)
IR: (KBr, cmꢀ1) 3317 (OH), 1590 (C]N), 770 (CeCl),
1
1320 (CeN), 1130 (C]S), 3220 (NH). H NMR (DMSO-
1
1320 (CeN), 1130 (C]S), 3220 (NH). H NMR (DMSO-
d6, ppm): 2.2 (2H, s, CH2), 2.7 (6H, s, 2 ꢃ CH3), 5.3 (1H, s,
CH), 7.2e7.4 (10H, m, aromatic), 9.7 (1H, s, OH), 10.0
(1H, s, NH): EISMS: m/z: 471 (Mþ1); Calcd/Anal. [C 61.28/
61.26, H 4.50/4.52, N 8.93/8.93].
d6, ppm): 2.2 (2H, s, CH2), 2.6 (6H, s, 2 ꢃ CH3), 5.2 (1H, s,
CH), 7.2e7.4 (11H, m, aromatic), 9.5 (1H, s, OH), 10.0
(1H, s, NH), EISMS: m/z: 436 (Mþ1); Calcd/Anal. [C 66.12/
66.22, H 5.09/5.04, N 9.64/9.60].
5.1.10. Compound (5j)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN),
1130 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2.1
(2H, s, CH2), 2.4 (6H, s, 2 ꢃ CH3), 5.3 (1H, s, CH), 7.2e8.2
(11H, m, aromatic), 8.6 (1H, s, OH), 10.5 (1H, s, NH):
EISMS: m/z: 446 (Mþ); Calcd/Anal. [C 64.56/64.54, H 4.97/
4.95, N 12.55/12.53].
5.1.3. Compound (5c)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN),
1130 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.8 (6H, s, 2 ꢃ CH3), 3.9 (6H, s, eN(CH3)2),
4.9 (1H, s, CH), 7.2e7.8 (11H, m, aromatic), 8.7 (1H, s,
OH), 10.2 (1H, s, NH), EISMS: m/z: 444 (Mþ); Calcd/Anal.
[C 70.24/70.22, H 6.35/6.32, N 12.60/12.64].
5.1.11. Compound (5k)
5.1.4. Compound (5d)
IR: (KBr, cmꢀ1) 3317 (OH), 1580 (C]N), 1310 (CeN),
1136 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.8 (6H, s, 2 ꢃ CH3), 5.3 (1H, s, CH), 7.2e7.4
(7H, m, aromatic), 6.8e7.9 (Furan), 9.2 (1H, s, OH), 10.0
(1H, s, NH): EISMS: m/z: 392 (Mþ1); Calcd/Anal. [C 67.50/
67.51, H 5.41/5.41, N 10.73/10.72].
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN),
1130 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.8 (6H, s, 2 ꢃ CH3), 5.1 (1H, s, CH), 7.2e7.7
(12H, m, aromatic), 9.7 (1H, s, OH), 10.0 (1H, s, NH), EISMS:
m/z: 402 (Mþ1); Calcd/Anal. [C 71.79/71.70, H 5.77/5.77, N
10.46/10.45].
6. General procedure
5.1.5. Compound (5e)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN),
1130 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.8 (6H, s, 2 ꢃ CH3), 3.9 (6H, s, OCH3 ꢃ 2),
5.0 (1H, s, CH), 7.2e7.4 (10H, m, aromatic), 8.7 (1H, s,
OH), 10.2 (1H, s, NH): EISMS: m/z: 462 (Mþ1); Calcd/
Anal. [C 67.66/67.66, H 5.90/5.90, N 9.10/9.12].
6.1. 3-(4-Hydroxy-3-methylphenyl)-5-(substituted)
phenyl-4,5-dihydro-1H-1-pyrazolyl-2-methoxyanilino
methane thione (6aek)
To a solution of pyrazolines (0.01 mol) (4aek) in ethanol
(20 ml) was added 2-methoxy aryl isothiocyanate (1.661 ml,
0.01 mol) and the reaction mixture was refluxed for 4 h. The
reaction mixture was cooled and then poured onto crushed
ice. The obtained solid was filtered, washed with water and
purified from ethanol to give compounds (6aek).
5.1.6. Compound (5f)
IR: (KBr, cmꢀ1) 3317 (OH), 1596 (C]N), 1320 (CeN),
1132 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2
(2H, s, CH2), 2.8 (6H, s, CH3), 3.9 (9H, s, OCH3 ꢃ 3), 5.3
(1H, s, CH), 7.2e7.4 (9H, m, aromatic), 8.7 (1H, s, OH),
10.4 (1H, s, NH): EISMS: m/z: 491 (Mþ); Calcd/Anal.
[C 65.97/65.94, H 5.95/5.94, N 8.55/8.53].
6.1.1. Compound (6a)
IR: (KBr, cmꢀ1) 3307 (OH), 1592 (C]N), 1320 (CeN),
1132 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 1.6
(2H, s, CH2), 2.5 (3H, s, CH3), 3.3 (6H, s, OCH3 ꢃ 2), 5.2
(1H, s, CH), 6.5e8.4 (11H, m, aromatic), 9.7 (1H, s, OH),
10.0 (1H, s, NH), EISMS: m/z: 447 (Mþ); Calcd/Anal.
[C 67.09/67.05, H 5.63/5.63, N 9.39/9.36].
5.1.7. Compound (5g)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 724 (CeF),
1
1320 (CeN), 1130 (C]S), 3220 (NH). H NMR (DMSO-
d6, ppm): 2.2 (2H, s, CH2), 2.8 (6H, s, 2 ꢃ CH3), 5.4 (1H, s,
CH), 7.2e7.9 (11H, m, aromatic), 8.7 (1H, s, OH), 10.0
(1H, s, NH): EISMS: m/z: 420 (Mþ); Calcd/Anal. [C 68.71/
68.70, H 5.29/5.29, N 10.02/10.06].
6.1.2. Compound (6b)
IR: (KBr, cmꢀ1) 3307 (OH), 1598 (C]N), 770 (CeCl),
1
1310 (CeN), 1130 (C]S), 3220 (NH). H NMR (DMSO-
d6, ppm): 2.5 (3H, s, CH3), 2.7 (2H, s, CH2), 3.8 (3H, s,

274
M.A. Ali et al. / European Journal of Medicinal Chemistry 42 (2007) 268e275
OCH3), 5.0 (1H, s, CH), 6.5e8.4 (11H, m, aromatic), 9.9 (1H,
s, OH), 10.0 (1H, s, NH): EISMS: m/z: 451 (Mþ); Calcd/Anal.
[C 63.78/63.75, H 4.91/4.90, N 9.30/9.32].
(1H, s, NH): EISMS: m/z: 486 (Mþ); Calcd/Anal. [C 59.26/
59.25, H 4.35/4.35, N 8.64/8.63].
6.1.10. Compound (6j)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN),
1130 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.8 (6H, s, CH3), 3.9 (3H, s, OCH3), 7.2e8.4
(11H, m, aromatic), 9.7 (1H, s, OH), 10.10 (1H, s, NH):
EISMS: m/z: 463 (Mþ1); Calcd/Anal. [C 62.32/62.32, H
4.79/4.79, N 12.11/12.10].
6.1.3. Compound (6c)
IR: (KBr, cmꢀ1) 3307 (OH), 1596 (C]N), 1320 (CeN),
1140 (C]S), 3222 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.8 (3H, s, CH3), 2.9 (6H, s, eN(CH3)2), 3.9
(3H, s, OCH3), 4.4 (1H, s, CH), 7.2e7.8 (11H, m, aromatic),
9.7 (1H, s, OH), 10.0 (1H, s, NH): EISMS: m/z: 461 (Mþ1);
Calcd/Anal. [C 63.40/63.42, H 5.73/5.76, N 11.37/11.35].
6.1.11. Compound (6k)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN),
1130 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.5 (6H, s, CH3), 3.8 (3H, s, OCH3), 7.0e7.4
(7H, m, aromatic), 6.8e7.9 (Furan), 9.2 (1H, s, OH), 10.0
(1H, s, NH): EISMS: m/z: 408 (Mþ1); Calcd/Anal.
[C 64.85/64.84, H 5.19/5.18, N 10.31/10.30].
6.1.4. Compound (6d)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1324 (CeN),
1130 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.8 (3H, s, CH3), 3.9 (3H, s, OCH3), 4.24 (1H,
s, CH), 7.2e7.4 (12H, m, aromatic), 9.7 (1H, s, OH), 10.10
(1H, s, NH): EISMS: m/z: 417 (Mþ); Calcd/Anal. [C 69.04/
69.00, H 5.55/5.54, N 10.06/10.06].
7. Biology
6.1.5. Compound (6e)
The primary screening was conducted at concentration of
6.25 mg/ml (or molar equivalent of highest molecular weight
compound in a series of congeners) against M. tuberculosis
H37Rv (ATCC27294) in BACTEC 12B medium using the
BACTEC 460 radiometric system [11,12]. Compounds dem-
onstrating at least 90% inhibition in the primary screen were
re-examined at lower concentration (MIC) in broth micro-dilu-
tion assay with Almar Blue. The MIC was defined as the low-
est concentration inhibiting 99% of the inoculum. Concurrent
with the determination of MICs, compounds were tested for
cytotoxicity (IC50) in VERO at concentration equal to and
greater than the MIC for M. tuberculosis H37Rv after 72 h
of exposure, viability is assessed on the basis of cellular con-
version of MTT into a formazan product using the Promega
Cell Titer 96 Non-radioactive Cell proliferation assay.
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1310 (CeN),
1130 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.8 (3H, s, CH3), 3.9 (9H, s, OCH3 ꢃ 3), 4.24
(1H, s, CH), 7.2e7.4 (11H, m, aromatic), 8.7 (1H, s, OH),
10.10 (1H, s, NH): EISMS: m/z: 478 (Mþ1); Calcd/Anal.
[C 65.39/65.29, H 5.70/5.72, N 8.80/8.84].
6.1.6. Compound (6f)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 1320 (CeN),
1130 (C]S), 3220 (NH). 1H NMR (DMSO-d6, ppm): 2.2
(2H, s, CH2), 2.8 (3H, s, CH3), 3.9 (12H, s, OCH3 ꢃ 4), 4.4
(1H, s, CH), 7.2e7.4 (11H, m, aromatic), 9.7 (1H, s, OH),
10.10 (1H, s, NH): EISMS: m/z: 508 (Mþ1); Calcd/Anal.
[C 63.89/63.86, H 5.76/5.75, N 8.28/8.29].
6.1.7. Compound (6g)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 820 (CeF),
Acknowledgements
1
1320 (CeN), 1130 (C]S), 3220 (NH). H NMR (DMSO-
The author (M. Shahar Yar) wishes to express his thanks to
the University Grant Commission, New Delhi, India for the re-
search award. We thank the Tuberculosis Antimicrobial Ac-
quisition and Coordinating Facility (TAACF), National
Institute of Allergy and Infectious Diseases Southern Research
Institute/GW Long Hansen’s Disease Center, Colorado State
University Birmingham, Alabama, USA, for the in vitro
anti-mycobacterial screening and Dr. Kiran Smith, National
Cancer Institute, USA, for valuable suggestion.
d6, ppm): 2.3 (2H, s, CH2), 2.7 (6H, s, CH3), 3.9 (3H, s,
OCH3), 7.2e7.4 (11H, m, aromatic), 9.4 (1H, s, OH), 10.10
(1H, s, NH): EISMS: m/z: 436 (Mþ); Calcd/Anal. [C 66.19/
66.17, H 5.09/5.07, N 9.65/9.63].
6.1.8. Compound (6h)
IR: (KBr, cmꢀ1) 3307 (OH), 1590 (C]N), 770 (CeCl),
1
1320 (CeN), 1130 (C]S), 3220 (NH). H NMR (DMSO-
d6, ppm): 2.2 (2H, s, CH2), 2.8 (3H, s, CH3), 3.8 (9H, s,
OCH3), 7.2e7.5 (11H, m, aromatic), 12.0 (1H, s, OH),
12.10 (1H, s, NH): EISMS: m/z: 452 (Mþ1); Calcd/Anal.
[C 63.78/63.76, H 4.91/4.90, N 9.30/9.31].
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