Novel 1,3,5-triphenyl-2-pyrazolines as anti-infective agents

Article abstract of DOI:10.1016/j.bmcl.2010.03.083

Sixteen 1,3,5-triphenyl-2-pyrazolines were synthesized and their anti-infective activities (against Mycobacterium tuberculosis H₃₇Rv, six bacterial and four fungal strains) were tested. Only compound with SO₂CH₃ in the para position of the A-ring was active against the tubercular strain at 100 μg/ml concentration. All compounds showed good anti infective activity against Escherichia coli and poor activity against Staphylococcus aureus. Compounds 4, 12, 13 and 14 exhibited reasonable activity against all the organisms tested (<0.309 μM except against S. aureus. The activity of these compounds correlated with their lipophilic/hydrophilic nature. Compounds 4, 10 and 16 showed very good activity (>88% reduction) against four fungi studied at 2 mg/ml. All these compounds possess halogen substitutions. Compound 11 showed very high activity (>90%) against three fungi. Majority of the compounds showed more than 90% inhibition against one or two fungi. Since pyrazolines are reported to inhibit the activity of p-glycoprotein, they may prevent drug resistance developed by microorganism.

Full text of DOI:10.1016/j.bmcl.2010.03.083

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3169–3172
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 1,3,5-triphenyl-2-pyrazolines as anti-infective agents
P. M. Sivakumar a, S. Prabhu Seenivasan b, Vanaja Kumar b, Mukesh Doble a,
*
a Department of Biotechnology, Indian Institute of Technology Madras, Adyar, Chennai 600 036, India
b Tuberculosis Research Centre (ICMR), Chetpet, Chennai, Tamil Nadu 600 031, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixteen 1,3,5-triphenyl-2-pyrazolines were synthesized and their anti-infective activities (against Myco-
bacterium tuberculosis H37Rv, six bacterial and four fungal strains) were tested. Only compound with
SO2CH3 in the para position of the A-ring was active against the tubercular strain at 100 lg/ml concen-
tration. All compounds showed good anti infective activity against Escherichia coli and poor activity
against Staphylococcus aureus. Compounds 4, 12, 13 and 14 exhibited reasonable activity against all
Received 15 September 2009
Revised 3 March 2010
Accepted 25 March 2010
Available online 29 March 2010
the organisms tested (<0.309 lM except against S. aureus. The activity of these compounds correlated
Keywords:
1,3,5-Triphenyl-2-pyrazolines
Alog P
Mycobacterium tuberculosis H37Rv
High resolution mass spectrum (HR-MS)
with their lipophilic/hydrophilic nature. Compounds 4, 10 and 16 showed very good activity (>88% reduc-
tion) against four fungi studied at 2 mg/ml. All these compounds possess halogen substitutions. Com-
pound 11 showed very high activity (>90%) against three fungi. Majority of the compounds showed
more than 90% inhibition against one or two fungi. Since pyrazolines are reported to inhibit the activity
of p-glycoprotein, they may prevent drug resistance developed by microorganism.
Ó 2010 Elsevier Ltd. All rights reserved.
Tuberculosis is one amongst the three highly infectious diseases
apart from HIV/AIDS. It is caused by Mycobacterium tuberculosis.1 It
is estimated that between 2002 and 2020, approximately a billion
people will be newly infected, more than 150 million people will
get sick, and 36 million will die of TB.2
above said infectious diseases.6 It also plays a role in expelling
the anticancer agents and make the cancerous cells resistant.
American Society for Microbiology (ASM) in association with the
Society for Healthcare Epidemiology of America (SHEA) together
have launched a program known as Antimicrobial Resistance Pre-
vention Initiative (ARPI) and it is dedicated to explore the epidemi-
ology and mechanisms of resistance by the microorganisms.5
So studies that contribute to the design of new potential drugs
with diverse mechanism of action and broad spectrum of activity
than the current ones that are available for the treatment of infec-
tious diseases and at the same time counteract the resistance
developed by bacteria are gaining importance. Since drug resis-
tance due to the over expression of P-glycoprotein is associated
with tuberculosis and infectious diseases, designing molecules
which posses both P-glycoprotein inhibitory activity and antimi-
crobial activity will be useful to combat infectious diseases. Hence
our study was focused towards the synthesis of novel 1,3,5-tri-
phenyl-2-pyrazolines and to determine their activity against M.
tuberculosis, Gram positive and Gram negative bacteria and fungi.
Pyrazolines are useful lead molecules that are found to act as
antibacterial, antifungal7–9 and antitubercular agents.10,11 More-
over pyrazolines are also tested for their inhibitory activity against
P-glycoprotein.12
Gram +ve and Gram ꢀve bacterial strains are involved in major-
ity of the infectious diseases. Prolonged use of antibiotics is limited
due to their side effects and the resistance developed by the micro-
organism.3 Among the two bacterial strains, Gram ꢀve have rich
lipopolysaccharides in their outer leaflet preventing the penetra-
tion of majority of the drugs through this membrane. This makes
them more drug resistant when compared to Gram +ve organisms.
Moreover, they also have the ability to exchange their genetic
material (DNA) with same as well as different species leading to
genetic change (mutation) thereby developing drug resistance.
Extended use of chemotherapeutic agents for the treatment of
cancer, antibacterial agents for diseases and the use of immuno-
suppressive agents after transplantation leads to fungal infection.
Individuals with AIDS or malignancies are also susceptible to fun-
gal infection.4 Resistance has been observed against triazoles and
polyenes by fungi including Candida albicans and Aspergillus
species.5
Resistance developed by these microbial strains delineates the
therapy against tuberculosis and bacterial and fungal diseases.
P-Glycoprotein is an ABC transporter family of proteins that is
responsible for multidrug resistance. It has an impact on all the
All the chemicals for the synthesis were purchased from Sigma
chemicals, Bangalore, India. Control drugs used for the experi-
ments were purchased from Himedia Laboratories India. The syn-
thesized compounds were tested against six bacterial strains
namely, Staphylococcus aureus NCIM 5021, Bacillus subtilis NCIM
2718, Proteus vulgaris NCIM 2813, Escherichia coli NCIM 2931,
* Corresponding author. Tel.: +91 44 2257 4107; fax: +91 44 2257 4102.
E-mail address: mukeshd@iitm.ac.in (M. Doble).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.083

3170
P. M. Sivakumar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3169–3172
Other researchers found the importance of sulfonylmethyl substi-
tution towards antibacterial activity.18 The log P (hydrophobicity
of the compound) plays an important role in the penetration
through M. tuberculosis cell wall and its relationship with antitu-
bercular activity is observed by several researchers.19–21 In this
current study only compound 3 has log P in between 3 and 5 which
is ideal for penetration through mycobacterial cell, while other
compounds have more than 5.
Salmonella typhi 2501, and Enterobacter aerogenes NCIM 5139 (Na-
tional Chemical Laboratory, Pune, India), four fungi namely, Asper-
gillus flavus NCIM 594, Fusarium proliferatum NCIM1105, Aspergillus
niger NCIM 596 and Candida tropicalis NCIM 3556 (National Chem-
ical Laboratory (NCL), Pune, India) and M. tuberculosis H37Rv
(Tuberculosis Research Center, Chetpet, Chennai, India). The inter-
nal energy, log P and Alog P values of the compounds were calcu-
lated using Cerius 2Ò software.
Compounds 4, 12, 13 and 14 were found to be active against al-
most all the bacterial strains tested except S. aureus. Most active
compounds generally have a halogen in para and/or meta position
in the B-ring (except compound 12) which is also observed by
Ansari et al.22 They also showed that halogen substitution is more
active than methoxy, nitro and hydroxyl substitution in 30-hydro-
xyl chalcones. The most active compounds (4, 12, 13 and 14) have
good correlation between their antibacterial activities and thermo-
dynamic internal energy (correlation coefficient = 0.99) and Alog P
(correlation coefficient = ꢀ0.87 to ꢀ0.99). The later term repre-
sents the hydrophobic/lipophilic nature of the molecule. Ansari
et al. also observed a correlation between the conformational en-
ergy and the lipophilicity of the molecule towards the antibacterial
activity.22 Our previous research communications also give suffi-
cient detail about the hydrophilic/lipophilic balance required for
small molecules to penetrate the bacterial cell membrane.13,14,23
In the case of a slime producing organism (myxobacteria which
delineate the antibacterial therapy further), the small molecule
has to cross the exopolysacchride which is hydrophobic in nature
and then it has to cross the bacterial cell membrane which has a
high LPS (lipopolysacchride) content which is hydrophilic in nat-
ure. Hence both the hydrophobicity of the compounds and the
hydrophobicity of the organism cell surface have to balance for
exhibiting antibacterial activity.
Compounds 4, 10 and 16 were found to be the most active
against all the four fungal strains (>88% inhibition). All the three
compounds have a halogen (chlorine or fluorine) substitution in
the ortho or para position in their B ring. Earlier Karthikeyan et
al. also found that the chloro and fluoro substitution in B-ring fa-
vors antibacterial and antifungal activity of 2-pyrzolines.7
In conclusion, sixteen 1,3,5 triphenyl-2-pyrazolines were syn-
thesized, characterized and their in vitro anti-infective activities
(antimycobacterial, antibacterial and antifungal) were evaluated.
Compared to the thiomethyl substitution, methylsulfonyl substitu-
tion in the A-ring leads to higher activity, namely compound 3
Chalcones were used as starting materials for the synthesis of
1,3,5-triphenyl-2-pyrazolines. The preparation of these chalcones
and their characterization were given in our previous research
communications.13,14 The general procedure for the synthesis of
sixteen 1,3,5-triphenyl-2-pyrazolines is given in Scheme 1 and it
is explained at the Supplementary data.
The antitubercular activity of the synthesized compounds was
evaluated against the M. tuberculosis H37Rv by LRP (Luciferase re-
porter phage assay) assay.13
Antibacterial activity of these 1,3,5-triphenyl-2-pyrazoline
derivatives were determined against the six Gram +ve and Gram
ꢀve strains listed before using the microdilution method reported
by Sarker et al. using 96 well plates.15
The antifungal activity of these compounds was tested against
four fungi mentioned before as per the macrodilution method re-
ported by Kumar et al.16
Table 1 lists the sixteen 1,3,5-triphenyl-2-pyrazolines synthe-
sized as per the procedure mentioned before. They were character-
ized by FT-IR, NMR and Mass spectrum. The FTIR spectra for all the
compounds have a C@N stretching band at 1590 cmꢀ1 and the
NMR spectra have three doublet of doublets for HX, HA and HB.
Spectral data for compounds 6, 9 and 16 are given in Ref. 17 and
their 1H and 13C NMR spectrum, HR-mass spectrum are given in
the Supplementary data (see Figs. S1–S9).
The anti tubercular activity at 50 and 100 lg/ml, anti bacterial
activity represented as MIC (minimum inhibitory concentration)
and antifungal activity (as % inhibition) of all the compounds are
given in Table 1.
Among the 16 compounds tested compound 3 alone was found
to be active against M. tuberculosis H37Rv strain (more than 50%
reduction in RLU at 100 lg/ml concentration). Other compounds
showed less than 50% reduction in RLU and hence they can be con-
sidered as inactive at these two concentrations tested. All the 16
compounds exhibited poor activity at 50
lg/ml concentration.
The active compound 3, has a methylsulfonyl group in the A-ring.
O
O
CHO
S
2.6 m-CPBA
COCH3
R
R
EtOH
NaOH
O
R
THF
S
S
O
C6H5NHNH2
CH3COOH
C6H5NHNH2
CH3COOH
N
R
N
N
R
N
O
S
S
O
1-2
3-4
5-16
Scheme 1. Synthesis of 1,3,5-triphenyl-2-pyrazoline derivatives.

Table 1
The structure and observed anti-infective activities of 1, 3, 5-triphenyl-2-pyrazolines
R2
R3
N
N
R4
R1
Compound
No
Substitutions
in phenyl ring
Antitubercular activity
(% reduction in RLU)
Antibacterial activity (MIC in
lM)
Antifungal activity (percentage inhibition
at 2 mg/ml concentration)
R1
R2
R3
R4
50
g/ml
100
E. coli
P. vulgaris
E. aerogenes S. aureus
S. typhi
B. subtilis
F. proliferatum C. tropicalis
NCIM 3556
A. flavus
NCIM 594
A. niger
NCIM 596
l
l
g/ml
NCIM 2931 NCIM 2813 NCIM 5139
NCIM 5021 NCIM 2501 NCIM 2718 NCIM1105
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
SCH3
SCH3
SO2CH3
SO2CH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
0.00
17.65
17.43
50.71
15.46
27.81
19.26
5.70
48.35
24.87
4.75
26.85
30.30
20.17
18.55
13.79
0.00
0.045
0.082
0.166
0.152
0.080
0.174
0.160
0.161
0.083
0.022
0.167
0.155
0.295
0.148
0.173
0.165
0.726
0.660
0.332
0.304
0.322
0.349
0.321
0.322
0.334
0.345
0.334
0.309
0.295
0.295
0.347
0.330
0.363
0.330
0.332
0.304
0.322
0.349
0.321
0.322
0.334
0.345
0.334
0.155
0.295
0.295
0.347
0.165
0.726
0.660
0.664
0.608
0.644
0.697
0.642
0.643
0.668
0.690
0.668
0.618
0.591
0.591
0.694
0.330
0.726
0.330
0.332
0.304
0.322
0.349
0.321
0.322
0.334
0.690
0.334
0.309
0.295
0.295
0.347
0.330
0.363
0.330
0.332
0.304
0.322
0.349
0.321
0.322
0.334
0.172
0.334
0.309
0.295
0.295
0.347
0.330
94.13 0.00
72.94 1.79
97.61 0.47
88.26 0.90
81.35 0.06
83.79 1.10
56.86 0.80
94.35 0.35
65.87 0.76
98.71 0.16
96.09 0.51
93.19 0.57
90.11 2.34
34.31 3.97
93.56 0.13
93.57 0.73
73.11 0.91 30.50 6.13 93.52 0.42
94.43 0.99 13.11 1.21 92.99 0.34
30.64 0.30 65.85 1.82 47.20 5.03
95.58 1.05 88.44 1.92 97.54 0.07
17.06 0.23 31.19 0.54 71.37 0.00
95.30 0.20 38.63 0.24 92.98 2.98
45.87 0.00 18.98 1.29 65.16 0.77
95.38 1.10 38.25 1.86 85.29 1.20
92.86 0.41 48.37 0.51 81.09 0.82
97.41 1.40 94.82 0.16 97.28 1.31
98.66 0.82 51.53 0.06 90.14 0.41
Cl
0.00
1.70
0.76
10.59
0.00
0.00
36.44
0.00
0.00
7.45
Cl
–O–CH2–O–
NO2
CH3
OEt
OMe
F
OMe
OMe
Br
OMe 32.66
8.55
9.12 0.78 63.83 5.72
9.65 0.08
65.14 0.00 91.36 1.01 64.62 0.61
88.13 0.25 28.20 4.05 88.54 1.17
86.11 1.00 24.97 3.82 84.37 0.00
90.96 0.82 88.22 2.12 98.33 1.22
Br
0.00
0.00
0.00
NH2
Cl

3172
P. M. Sivakumar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3169–3172
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luciferase reporter phage assay only compound 3 was found to be
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17. Spectral data for compound 6: Yield: 81%,1H NMR (500 MHz, CDCl3): d 2.36 (s,
3H), 2.44 (s, 3H), 3.08 (dd, J = 7, 17 Hz, 1H), 3.78 (dd, J = 12, 17 Hz, 1H), 5.19 (dd,
J = 7.5, 12.5 Hz, 1H), 6.76 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.5 Hz, 2H), 7.15–7.24
(m, 8H), 7.60 (d, J = 8 Hz, 2H). 13C NMR (125 MHz, CDCl3): d 15.82, 21.40, 43.65,
64.07, 113.37, 119.03, 125.72, 126.48, 127.26, 128.90, 129.27, 129.93, 137.65,
138.73, 139.60, 144.97, 146.95. HR-MS (m/z) for molecular formula C23H23N2S:
calcd = 359.1582, found = 359.1576. Spectral data for compound 9: Yield: 81%,
1H NMR (500 MHz, CDCl3): d 2.44 (s, 3H), 3.07 (dd, J = 7.5, 20 Hz, 1H), 3.78 (dd,
J = 12.5, 17 Hz, 1H), 3.81 (s, 3H), 5.17 (dd, J = 7.5, 12.5 Hz, 1H), 6.76 (t, J = 7.5 Hz,
1H), 6.90 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8 Hz, 2H), 7.15–7.25 (m, 6H), 7.65 (d,
J = 9 Hz, 2H). 13C NMR (125 MHz, CDCl3): d 15.82, 43.77, 55.35, 64.10, 113.29,
114.03, 118.90, 125.49, 126.48, 127.22, 127.25, 128.88, 137.61, 139.65, 145.14,
146.78, 160.16. HR-MS (m/z) for molecular formula C23H23N2OS:
calcd = 375.1531, found = 375.1541. Spectral data for compound 16: Yield:
81%, 1H NMR (500 MHz, CDCl3): d 2.45 (s, 3H), 3.32 (dd, J = 7.5, 17.5 Hz, 1H),
4.02 (dd, J = 12, 17.5 Hz, 1H), 5.24 (dd, J = 7.5, 12 Hz, 1H), 6.80 (t, J = 7.5 Hz, 1H),
7.05 (d, J = 8.5 Hz, 2H), 7.16–7.29 (m, 8H), 7.38 (d, J = 8 Hz, 1H), 7.83 (dd, J = 1.5,
8 Hz, 1H). 13C NMR (125 MHz, CDCl3): d 15.78, 46.21, 64.59, 113.63, 119.54,
126.48, 126.77, 127.23, 128.94, 129.42, 130.02, 130.84, 131.68, 132.20, 137.78,
139.11, 144.58, 145.97. HR-MS (m/z) for molecular formula C22H20N2SCl:
calcd = 379.1036, found = 379.1040.
Acknowledgements
Authors thank Dr. Moni (SAIF, IIT) for providing spectral
analysis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.083.
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