Green synthesis and anti-infective activities of fluorinated pyrazoline derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.06.072

A new series of fluorinated pyrazoles, 4a-e, were synthesized in good to excellent overall yields (65-82%) from the corresponding chalcones, 3a-e, by ultrasonic irradiation. The newly synthesized compounds were characterized and screened for their in vitr

Full text of DOI:10.1016/j.bmcl.2012.06.072

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5727–5730
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Green synthesis and anti-infective activities of fluorinated
pyrazoline derivatives
Sharad N. Shelke a, , Ganesh R. Mhaske a, Vasco D. B. Bonifácio b, Manoj B. Gawande b,
⇑
⇑
a Department of Chemistry, S.S.G.M. College, Kopargaon, Dist-Ahmednagar (MH) 423601, India
b REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa 2829-516 Caparica, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of fluorinated pyrazoles, 4a–e, were synthesized in good to excellent overall yields (65–82%)
from the corresponding chalcones, 3a–e, by ultrasonic irradiation. The newly synthesized compounds
were characterized and screened for their in vitro anti-bacterial, anti-fungal, and anti-tubercular activi-
ties against Mycobacterium tuberculosis H37Rv.
Received 6 April 2012
Revised 23 June 2012
Accepted 25 June 2012
Available online 6 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Green synthesis
Ultrasonic irradiation
MDR-TB
Anti-infective activity
Pyrazolines
It is well known that introduction of a fluorine atom into a mol-
ecule may lead to significant influences on the biological and phys-
ical properties of compounds, including increased membrane
permeability, changes in hydrophobic bonding, stability against
metabolic oxidation, etc.1 Since, fluorine-containing compounds
possess promising pharmacological activities which originate from
their uniquely high thermal stabilities and lipophilicity,2 the devel-
opment of synthetic methods for fluorine-containing compounds
has been an important field in organofluorine chemistry syntheses.3
Mycobacterium tuberculosis (MTB) still remains the leading
cause of worldwide death among infectious diseases. Statistics
show that 1.7 million people worldwide died from tuberculosis
(TB) in 2009. In addition, an estimated 9.4 million new cases
emerged in 2008, with 35% of these cases occurring in the region
of South-East Asia.4 One-third of the population is infected with
M. tuberculosis and the World Health Organization (WHO) esti-
mates that within the next 20 years approximately 30 million
people will be infected with the bacillus.5 The current frontline
therapy for tuberculosis consists of administering three or more
different drugs (usually rifampin, isoniazid, pyrazinamide and eth-
ambutol) over an extended period of time (6–12 months).6 How-
ever, the evolution of new virulent forms like multidrug resistant
(MDR) and extensively drug resistant (XDR) TB has become a major
threat, and it is urgent to develop new therapeutic agents to over-
come this serious problem.7
Considerable attention has been focused on pyrazoline deriva-
tives, due to their interesting biological activities. They have been
found to possess anti-oxidant, anti-cancer, anti-HIV, anti-malarial,
anti-fungal, anti-bacterial, anti-amoebic, and anti-mycobacterial
activities.8,9 Pyrazole chalcones are useful intermediates for the
synthesis of pyrazolines, and their preparation has been reported
using conventional conditions10 or grinding under solvent free
conditions.11 Ultrasonic irradiation is a ‘green’ alternative method-
ology that offers many advantages over conventional synthesis,
since it provides uniform and noncontact heating, faster reaction
times, and minimal side reactions.12
In a continuation of our efforts in green organic synthesis and its
applications in medicinal chemistry,13–17 herein we report the syn-
thesis and biological assessment of fluorinated pyrazolines using
ultrasonic irradiation. To the best of our knowledge this is the first
report on the synthesis of fluorinated pyrazolines using ultrasonic
irradiation and its applications. For comparison, the conventional
synthesis of fluorinated pyrazolines was also investigated.
A conventional one-pot Claisen–Schmidt condensation was
used for the synthesis of targeted pyrazoline derivatives (4a–e)
using chalcones (3a–e) by a fast, mild, and high yielding ultrasonic
irradiation methodology with good overall yields (Scheme 1).
In brief, chalcones were prepared by reacting 3-(4-fluorophenyl)-
1-phenyl-1H-pyrazole-4-carbaldehyde (1)18 with 4-substituted
acetophenones (2a–e) in the presence of a base (KOH). The target
compounds were synthesized both by a conventional method and
with ultrasonic irradiation19 (each experiment was repeated three
times to confirm the consistency of the results). Higher yields (up
to 83%) and faster reaction times (10–25 min), at room tempera-
⇑
Corresponding authors.
E-mail addresses: mbgawande@yahoo.co.in, m.gawande@fct.unl.pt (M.B. Ga-
wande).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.072

5728
S. N. Shelke et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5727–5730
O
F
F
F
R
H
N
R
N
O
H
O
N
N
N
R
N
N
N
2a-e
NH2NH2..H2O
EtOH, KOH 40%
20-25 min, RT, US )))
EtOH, CH3CO2H cat.
10-15 min, RT, US )))
1
4a-e
3a-e
2a R= H
2b R= CH3
2c
R= F
2d R= Cl
2e R= Br
Scheme 1. Synthesis of fluorinated pyrazoline derivatives (4a–e) using ultrasonic irradiation.
Table 2
ture, were obtained when ultrasonic irradiation was applied (see
Table 1). The reaction conditions were optimized by varying the
concentration of base (20 and 40 wt %). Notably, when the weight
percentage of KOH was increased from 20% to 40%, the yield of 3a
increased from 53% to 75% and the time of reaction was reduced
from 30 to 24 h in the conventional method. Using the ultrasonic
irradiation method, the yield of 3a increased from 59% to 82% and
the reaction time was reduced from 60 to 25 min. Reaction between
the newly synthesized chalcones (3a–e) and hydrazine hydrate in
ethanol led to novel pyrazolines (4a–e) with 72–82% yields after
recrystallization from glacial acetic acid.20
Both the chalcones (3a–e) and their pyrazoline derivatives (4a–
e) were tested for their in vitro anti-bacterial activity against Esch-
erichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and
Streptococcus pyogenus strains (Table 2). Their anti-fungal activity
was tested against the Candida albicans, Aspergillus niger, and Asper-
gillus clavatus strains (Table 3). Anti-tubercular activity was
screened for with the M. tuberculosis H37Rv strain using the con-
ventional Lowenstein–Jensen slope method. In all cases, the mini-
mal inhibitory concentration (MIC) values (the highest dilution
showing at least 99% inhibition) were compared with standard
drugs. All 10 newly synthesized compounds (chalcones and pyraz-
olines) were screened for their biological activities.21
Minimum inhibitory concentrations of chalcone (3a–e) and pyrazoline (4a–e)
derivatives against E. coli, P. aeruginosa, S. aureus, and S. pyogenus microbial strains
Compound
MICa
(
l
g/mL)
E. coli
MTCC 443
B. subtilis
MTCC 441
S. aureus
MTCC 96
S. pyogenus
MTCC 442
3a
3b
3c
3d
3e
4a
4b
4c
20
12.5
25
6.25
20
10
50
20
25
6.25
100
50
25
10
25
20
20
12.5
20
20
50
25
25
10
100
50
25
10
1
10
20
6.25
25
25
50
12.5
25
12.5
25
10
20
20
50
10
25
20
10
100
50
50
10
0.5
4d
4e
20
6.25
250
50
50
10
Ampicillinb
Chloramphenicolb
Ciprofloxacinb
Norfloxacinb
Gentamycinb
0.05
0.25
a
MIC = Minimum inhibitory concentration, the lowest concentration of the
compound which inhibits the growth of the bacterium by at least 99%.
b
Standard drug.
Antibacterial activity: In Table 2, it should be noted that chal-
cones 3c and 3d exhibited the highest inhibitory anti-bacterial
activities against S. aureus and E. coli, respectively. Chalcone 3c
was only active against S. aureus and S. pyogenus, while chalcone
3d was more active against E-coli and Bacillus subtilis. Poor inhibi-
tory activity was shown by Chalcone 3a against B. subtilis, 3b
against S. pyogenus, 3c against E. Coli, and 3d and 3e against S. aur-
Table 3
Minimum inhibitory concentrations of chalcone (3a–e) and pyrazoline (4a–e)
derivatives for C. albicans, A. niger, and A. clavatus fungal strains
Compound
MICa
(lg/mL)
C. albicans
MTCC 227
A. niger
MTCC 282
A. clavatus
MTCC 1323
3a
25
50
50
3b
3c
3d
3e
50
100
20
>100
>100
50
100
20
>100
>100
50
100
100
10
Table 1
Synthesis of chalcone (3a–e) and pyrazoline (4a–e) derivatives using conventional
and ultrasonic irradiation methods
4a
50
4b
4c
4d
4e
25
20
100
10
500
100
100
50
50
10
100
100
100
50
50
10
100
100
Compound
Conventional
Time (h)
Yielda (%)
Ultrasonic Irradiation
Time (min)
Yield (%)
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
24
24
24
24
24
3
75
70
69
71
68
66
68
65
70
72
25
20
25
25
25
10
10
15
15
15
82a
78a
81a
83a
80a
79
75
72
81
82
Greseofulvinb
Nystatinb
a
MIC = Minimum inhibitory concentration, the lowest concentration of the
compound which inhibits the growth of the fungus by at least 99%.
b
Standard drug.
3
3
3
3
eus. All MIC values of chalcones 3a–3e showed markedly higher
inhibitory anti-bacterial activities compared to the reference
compounds Ampicillin, Chloramphenicol and Ciprofloxacin, while
a
Using KOH 40 wt %

S. N. Shelke et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5727–5730
5729
Table 4
(HOD), S.S.G.M. College, Kopargaon, Ahmednagar (MH) for provid-
ing research facilities and constant encouragement.
Minimum inhibitory concentrations of chalcone (3a–e)
and pyrazoline (4a–e) derivatives for M. tuberculosis
H37Rv strain
References and notes
Compound
MICa
M. tuberculosis
37Rv
(lg/mL)
1. Kuznetsova, L.; Ungureanu, M. I.; Pepe, A. J. Fluorine Chem. 2004, 125, 415.
2. Haga, T.; Fujikawa, K.; Koyanag, T.; Nakajima, T.; Hayashi, K. Heterocycles 1984,
22, 117.
3. Shelke, S. N.; Dalvi, N. R.; Kale, B. K.; More, M. S.; Gill, C. H.; Karale, B. K. Ind. J.
Chem. 2007, 46B, 1174.
4. Tuberculosis, WHO Fact sheet No. 104, 2010..
5. Global Tuberculosis Control: Surveillance, Planning, and Financing, WHO
Report, 2008..
H
3a
3b
3c
3d
3e
4a
4b
4c
50
6.25
25
10
50
25
10
25
6. Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479.
7. Goldman, R. C.; Plumley, K. V.; Laughon, B. E. Infect. Disord. Drug Targets 2007, 7,
73.
4d
6.25
50
0.20
8. Kumar, S.; Bawa, S.; Drabu, S.; Kumar, R.; Gupta, H. Recent Pat. Antiinfect Drug
Discov. 2009, 4, 154.
9. Taj, T.; Kamble, R. R.; Gireesh, T. M.; Hunnur, R. K.; Margankop, S. B. Eur. J. Med.
Chem. 2011, 46, 4366.
4e
Isoniazidb
a
MIC = Minimum inhibitory concentration, the
10. Sharma, P. K.; Kumar, S.; Kumar, P.; Kaushik, P.; Kaushik, D.; Dhingra, Y.; Aneja,
K. R. Eur. J. Med. Chem. 2010, 45, 2650.
lowest concentration of the compound which inhibits
at least 99% of the growth of the mycobacterium.
11. Kumar, P.; Kumar, S.; Husain, K.; Kumar, A. Chin. Chem. Lett. 2011, 22, 37.
12. Handbook on Applications of Ultrasound: Sonochemistry for Sustainability; Chen,
D., Sharma, S. K., Mudhoo, A., Eds.; CRC Press: Boca Raton, 2012.
13. Shelke, S.; Mhaske, G.; Gadakh, S.; Gill, C. Bioorg. Med. Chem. Lett. 2010, 20,
7200.
b
Standard drug.
they showed comparable results with the reference compound
Norfloxacin. It is necessary to mention here that all chalcones
3a–3e exhibited markedly smaller MIC values compared to the ref-
erence compound Gentamycin.
Pyrazoline 4e was active against all four resistant strains, while
4a showed poor inhibitory anti-bacterial activity against S. aureus
and S. pyogenus, as did 4b against E. coli and B. subtilis. The MIC val-
ues of pyrazolines 4a–4e showed higher inhibitory anti-bacterial
activities compared to Ampicillin, Chloramphenicol, Ciprofloxacin
and Norfloxacin, but not compared to Gentamycin.
Antifungal activity: From Table 3, Chalcone 3e showed the high-
est inhibitory anti-fungal activity against C. albicans, while 3d
exhibited the lowest inhibitory antifungal activity against all three
fungal strains. Chalcone 3a exhibited excellent MIC values com-
pared to both reference compounds Greseofulvin and Nystatin
against all three fungal strains, as did 3b against C. albicans, 3c
against A. niger and A. clavatus and 3e against C. albicans. Chalcone
3b showed comparable MIC values compared with both reference
compounds Greseofulvin and Nystatin against A. niger and A. clav-
atus, as did 3c against C. albicans, 3d against all three fungal strains,
and 3e against A. niger and A. clavatus.
Pyrazoline 4e showed markedly high MIC values against all
three fungal strains compared to the reference compounds Greseo-
fulvin and Nystatin. Pyrazolines 4a, 4c and 4b (except against A. ni-
ger and A. clavatus) and 4d (except against C. albicans) also showed
good MIC values compared with the reference compounds.
Anti-tuberculosis activity: As shown in Table 4, chalcone 3b and
pyrazoline 4ddisplayed significantanti-tubercularactivities against
14. Gawande, M. B.; Branco, P. S. Green Chem. 2011, 13, 3355.
15. Pandya, D.; Kim, J.; Kwak, W.; Park, J.; Gawande, M. B.; An, G.; Ryu, E.; Yoo J.
Nucl. Med. Mol. Imaging 2010, 44, 185.
16. Shelke, S.; Salunkhe, N.; Sangale, S.; Bhalerao, S.; Naik, N.; Mhaske, G.; Jadhav,
R.; Karale, B. J. Korean Chem. Soc. 2010, 54, 59.
17. Shelke, S.; Chaudhari, C. S.; Randhavane, P. V.; Nirmal, P. R.; Karale, B. K.; Gill, C.
H. Acta Cienc Indica, Chem. 2008, 34, 503.
18. Kira, M.; Nofal, Z.; Gadalla, K. Z. Tetrahedron Lett. 1970, 48, 4215.
19. Conventional
method:
3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-
4-substituted acetophenone (2a–e)
carbaldehyde (1) (0.01 mol) and
a
(0.01 mol) were dissolved in EtOH. A solution of 40% KOH (5 mL) solution
was added and the resulting mixture allowed to stir for 24 h at rt.
Sonochemical
carbaldehyde (1) (0.01 mol) and
(0.01 mol) were dissolved in 5 mL EtOH.
method:
3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-
4-substituted acetophenone (2a–e)
solution of KOH (40 wt %,
a
A
0.019 mol, 5 mL) was added, and the resulting mixture subjected to
ultrasonic irradiation (Ultrasonicator model EN-20U-S, manufactured by
Enertech Electronica Pvt., Ltd, Mumbai, India, with
a maximum power
output of 100 W and 33 KHz operating frequency) for 20–25 min at rt. After
completion, the resulting mixture was poured into ice-cold water and then
neutralized with acetic acid. The solid obtained was filtered off, dried and
purified by recrystallization from acetic acid or by column chromatography.
The spectral data for the synthesized compounds are given below.
Compound 3a: Mp 140–142 °C; FT-IR (KBr) vmax (cmÀ1): 1659, 1597, 1214; 1H
NMR (400 MHz, DMSO-d6): 8.33 (1H, s, pyrazole H), 7.96-7.94 (4H, m, ArH),
7.83 (1H, d, J = 15.6 Hz), 7.77–7.13 (11H, m, ArH); 13C NMR (100 MHz, DMSO-
d6): 189.94, 164.39, 161.92, 152.86, 139.32, 138.14, 135.04, 132.81, 130.63,
130.55, 130.55, 129.62, 128.65, 128.46, 128.43, 128.39, 127.36, 126.88, 121.51,
119.32, 118.17, 115.95, 115.73; MS, ES + 1 mode (m/z): 369.2 (M+1).
Compound 3b: Mp 176–178 °C; FT-IR (KBr) mmax (cmÀ1): 1660, 1599, 1220; 1H
NMR (400 MHz, DMSO-d6): 9.43 (1H, s, pyrazole H), 8.01–7.93 (4H, m, ArH),
7.87 (1H, d, J = 15.4 Hz), 7.73–7.70 (2H, m, ArH), 7.65 (1H, d, J = 15.4 Hz), 7.60–
7.38 (7H, m, ArH), 2.4 (3H, s, CH3); 13C NMR (100 MHz, DMSO-d6): 189.43,
164.38, 161.91, 152.81, 152.74, 143.66, 139.35, 139.30, 135.54, 134.57, 133.39,
130.63, 130.58, 130.54, 130.50, 129.61, 129.36, 128.53, 128.51, 128.47, 127.37,
127.32, 126.83, 125.23, 121.59, 119.33, 118.25, 118.00, 115.93, 115.72, 21.69;
MS, ES + 1 mode (m/z): 383.8 (M+1).
Compound 3c: Mp 215–217 °C; FT-IR (KBr) mmax (cmÀ1): 1662, 1604, 1213; 1H
NMR (400 MHz, DMSO-d6): 8.96 (1H, s, pyrazole H), 8.13-7.88 (4H, m, ArH)
7.79 (1H, d, J = 15.6 Hz), 7.71–7.69 (2H, m, ArH), 7.65 (1H, d, J = 15.6 Hz), 7.53–
7.18 (7H, m, ArH); 13C NMR (100 MHz, DMSO-d6): 187.33, 166.18, 163.66,
163.64, 161.18, 152.02, 138.82, 134.35, 134.07, 134.04, 130.72, 130.62, 130.12,
130.04, 129.11, 128.18, 128.13, 127.61, 126.75, 120.71, 118.56, 117.59, 115.40,
115.32, 115.18, 115.10; MS, ES + 1 mode (m/z): 387.2 (M+1).
the M. tuberculosis H37Rv strain (MIC = 6.25
hand, chalcone 3a and pyrazoline 4e showed very poor MIC values
compared to the reference compound isoniazid (MIC = 0.20 g/mL).
lg/mL). On the other
l
In summary, ultrasonic irradiation is a green alternative method
to the conventional synthesis of pyrazoline derivatives, for its ease
of operation, lower temperatures, shorter reaction times, and high-
er yields. Novel chalcone and pyrazoline fluorinated derivatives
were screened for their anti-bacterial, anti-fungal and anti-tuber-
cular activities, and the MIC values obtained were compared
against those of standard drugs.
Compound 3d: Mp 220–222 °C; FT-IR (KBr) mmax (cmÀ1): 1659, 1598, 1214; 1H
NMR (400 MHz, DMSO-d6): 8.57 (1H, s, pyrazole H), 7.97–7.18 (15H, m, ArH);
13C NMR (100 MHz, DMSO-d6): 187.84, 163.33, 152.24, 138.65, 138.44, 135.84,
134.81, 130.00, 129.92, 128.98, 128.28, 126.77, 126.69, 120.30, 118.68, 117.41,
115.29, 115.07; MS, ES + 1 mode (m/z): 403.2 (M+1).
Compound 3e: Mp 223–225 °C; FT-IR (KBr) mmax (cmÀ1): 1659, 1598, 1214; 1H
NMR (400 MHz, DMSO-d6): 8.87 (1H, s, pyrazole H), 7.95–7.22 (15H, m, ArH);
13C NMR (100 MHz, DMSO-d6): 188.01, 163.32, 138.83, 136.41, 134.80, 131.35,
130.12, 130.04, 129.58, 129.10, 127.45, 126.81, 120.47, 118.66, 115.39, 115.1;
MS, ES + 1 mode (m/z): 447.2 (M+1)..
Acknowledgments
The authors are thankful to the Director of the Microcare Labo-
ratory (Surat, India) for the tests of anti-microbial activity. We are
also grateful to the Principal Dr. K. H. Shinde and Dr. A. B. Nikumbh,
20. Conventional method: To a solution of chalcone (0.01 mol) (3a–e) in 10 mL of
ethanol, 1.5 mL (0.048 mol) of hydrazine hydrate (99%) and 2–3 drops of glacial
acetic acid were added dropwise. The reaction mixture was heated under
reflux for 6 h and the progress of the reaction monitored by TLC. After

5730
S. N. Shelke et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5727–5730
completion of the reaction, the resulting solution was cooled and poured into
crushed ice. The solid pyrazolines (4a–e) were filtered and recrystallized from
EtOH.
132.40, 128.78, 128.10, 127.87, 127.57, 126.78, 126.79, 126.38, 125.79, 122.63,
114.00, 56.02, 41.01; MS, ES + 1 mode (m/z): 401.2 (M+1).
Compound 4d: Mp 150–152 °C; FT-IR (KBr) mmax (cmÀ1): 3311 (NH), 1598,
1318; 1H NMR (400 MHz, DMSO-d6): 8.61 (1H, s, NH), 8.31 (1H, s, Pyrazole-H),
8.02–6.52 (13H, m, ArH), 5.51 (1H, dd, J = 10.31, 9.76 Hz, pyrazoline H), 3.43
(1H, dd, J = 16.16, 10.52 Hz, pyrazoline H), 3.12 (1H, dd, J = 16.18, 8.92 Hz,
pyrazoline H); 13C NMR (100 MHz, DMSO-d6): 167.00, 157.02, 153.00, 137.10,
134.19, 132.38, 128.88, 128.15, 127.97, 127.67, 126.89, 126.89, 126.48, 125.89,
123.69, 116.00, 54.03, 43.02; MS, ES + 1 mode (m/z): 417.2 (M+1).
Sonochemical method: chalcones (0.01 mol) (3a–e) were placed in a 50 mL
beaker with 10 mL of EtOH. To this solution 1.5 mL (0.048 mol) of hydrazine
hydrate (99%) and 2–3 drops of glacial acetic acid were added dropwise. The
reaction beaker was suspended at the center of the ultrasonic bath and
sonicated for 10–15 min at rt. After completion of the reaction, the separated
solid was collected by filtration, washed with water and recrystallized from
EtOH. The spectral data for the synthesized compounds are given below.
Compound 4a: Mp 154–156 °C; FT-IR (KBr) mmax (cmÀ1): 3317, 1598, 1224; 1H
NMR (400 MHz, DMSO-d6): 8.34 (1H, s, NH), 8.02 (1H, s, Pyrazole-H), 7.83-7.12
(14H, m, ArH), 5.09 (1H, dd, J = 10.49, 9.06 Hz, pyrazoline H), 3.45 (1H, dd,
J = 16.08, 10.32 Hz, pyrazoline H), 3.05 (1H, dd, J = 16.12, 8.64 Hz, pyrazoline
H); 13C NMR (100 MHz, DMSO-d6): 164.03, 152.08, 139.78, 132.56, 129.93,
129.85, 129.63, 129.47, 129.21, 129.18, 129.10, 128.79, 128.63, 128.39, 12816,
126.64, 126.08, 126.03, 122.96, 119.40, 119.07, 118.98, 115.86, 55.74, 40.82;
MS, ES + 1 mode (m/z): 383.2 (M+1).
Compound 4e: Mp 161–163 °C; FT-IR (KBr) mmax (cmÀ1): 3313, 1597, 1300; 1H
NMR (400 MHz, DMSO-d6): 8.53 (1H, s, NH), 8.18 (1H, s, Pyrazole-H), 8.12–6.43
(13H, m, ArH), 5.42 (1H, dd, J = 10.39, 9.86 Hz, pyrazoline H), 3.54 (1H, dd,
J = 16.20, 10.25 Hz, pyrazoline H), 3.19 (1H, dd, J = 16.13, 8.99 Hz, pyrazoline
H); 13C NMR (100 MHz, DMSO-d6): 167.00, 157.00, 153.78, 138.12, 134.29,
132.37, 128.78, 128.16, 127.87, 127.77, 126.90, 126.87, 126.58, 125.87, 123.68,
115.20, 53.33, 41.23; MS, ES + 1 mode (m/z): 461.3 (M+1)..
21. The
H37Rv clinical isolate was obtained from the Institute of Microbial
Technology, Surat, India. L. J. was used as nutrient medium to grow and
dilute the testing drug suspensions. The inoculum size for the strain test was
Compound 4b: Mp 198–200 °C; FT-IR (KBr) mmax (cmÀ1): 3317, 1598, 1507,
1224; 1H NMR (400 MHz, DMSO-d6): 8.58 (1H, s, NH), 8.09 (1H, s, Pyrazole-H),
7.90–7.18 (13H, m, ArH), 4.95 (1H, dd, J = 10.72, 9.10 Hz, pyrazoline H), 3.48
(1H, dd, J = 16.16, 10.60 Hz, pyrazoline H), 3.10 (1H, dd, J = 16.16,9.02 Hz,
pyrazoline H), 2.30 (3H, s, CH3); 13C NMR (100 MHz, DMSO-d6): 167.66, 153.47,
139.14, 133.39, 132.68, 132.40, 128.87, 128.52, 128.14, 127.97, 127.78, 127.70,
127.59, 127.30, 126.88, 126.77, 126.29, 125.81, 125.56, 122.84, 118.02, 51.81,
41.90, 21.69; MS, ES + 1 mode (m/z): 396.8 (M+1).
adjusted to 1 mg/mL.
A 2 mg/mL stock solution was prepared for each
synthesized drug. DMSO (1%) was used as diluent/vehicle to obtain the
desired concentration of drug to test upon standard bacterial strains. In a
primary screening, 500, 250, and 125 lg/mL concentrations of the synthesized
drugs were used. The active synthesized drugs identified in this primary
screening were then diluted to obtain 100, 50, 25, 12.5, 6.250, 3.125 and
1.5625 lg/mL concentrations, and further tested in a second set of dilutions
against all microorganisms. The standard strain MTB H37Rv was retested with
each new batch of medium. The minimum inhibitory concentration (MIC) was
defined as the minimum concentration of compound required to inhibit 99% of
bacterial growth. Vehicle and reference agents were used in every test as the
negative and positive controls, and the assays were performed in duplicate..
Compound 4c: Mp 152–154 °C; FT-IR (KBr) mmax (cmÀ1): 3311, 1599, 1224; 1H
NMR (400 MHz, DMSO-d6): 8.54 (1H, s, NH), 8.12 (1H, s, Pyrazole-H), 8.10–7.02
(13H, m, ArH), 5.42 (1H, dd, J = 10.39, 8.96 Hz, pyrazoline H), 3.51 (1H, dd,
J = 16.12, 10.42 Hz, pyrazoline H), 3.05 (1H, dd, J = 16.08, 9.60 Hz, pyrazoline
H); 13C NMR (100 MHz, DMSO-d6): 163.02, 156.01, 152.01, 138.10, 131.09,