Synthesis, antitubercular and antimicrobial evaluation of 3-(4-chlorophenyl)-4-substituted pyrazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.05.063

As a part of our research to develop novel antitubercular and antimicrobial agents, a series of 3-(4-chlorophenyl)-4-substituted pyrazoles have been synthesised. These compounds were tested for antitubercular activity in vitro against Mycobacterium tuberc

Full text of DOI:10.1016/j.bmcl.2012.05.063

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5129–5133
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, antitubercular and antimicrobial evaluation of 3-(4-chlorophenyl)-
4-substituted pyrazole derivatives
Ravindra B. Pathak a,b, , P. T. Chovatia a,c, H. H. Parekh a,
⇑
⇑
a Department of Chemistry, Saurashtra University, Rajkot, Gujarat, India
b Faculty of Natural Sciences, Keele University, Keele, Staffordshire ST5 5BG, UK
c Evotech ltd, Milton Park, Abingdon, Oxfordshire OX14 4SA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
As a part of our research to develop novel antitubercular and antimicrobial agents, a series of 3-(4-chlo-
rophenyl)-4-substituted pyrazoles have been synthesised. These compounds were tested for antitubercu-
lar activity in vitro against Mycobacterium tuberculosis H37Rv strain using the BACTEC 460 radiometric
system, antifungal activity against a pathogenic strain of fungi and antibacterial activity against gram-
positive and gram-negative organisms. Among them tested, many compounds showed good to excellent
antimicrobial and antitubercular activity. The results suggest that hydrazones, 2-azetidinones and 4-thia-
zolidinones bearing a core pyrazole scaffold would be potent antimicrobial and antitubercular agents.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 7 April 2012
Revised 13 May 2012
Accepted 15 May 2012
Available online 24 May 2012
Keywords:
Antimicrobial
Antibacterial
Antitubercular
Antifungal
Hydrazone
Pyrazole
Azetidinone
Thiazolidinone
Pyrazoles and their derivatives have attracted much attention
due to diverse biological activities.1,2 Enormous interest in the
chemistry of pyrazoles is reflected by the design of new synthetic
approaches due to their significant biological and therapeutic va-
lue. There has been overwhelming literature reports reflecting
the immense biological potential of pyrazoles derivatives as anti-
tumor, anti-HIV, anti-inflammatory and anti-microbial activities.3
Over the past few years continuous interest in the synthesis of
C2-substituted thiazolidinone derivatives for their significant activ-
ity,4,5 thiazolidinones derivatives posses verities of bilogical activ-
ities like antibacterial,6 antituberculosis,7 antifungal,8 and
anticancer9 has been observed. Azetidinones known10 as a b-lac-
tams since 1907, possess a four member ring system bearing inter-
nal amide linkage. The discovery of an azitidin-2-one ring in
penicillin led to development of the compounds bearing this ring
system. The powerful activity of penicillin11,12 and the clinically
more useful cephalosporin13 led chemists and medicinal chemists
to investigate the chemistry of the azetidinone ring system. The
basic skeleton commonly encountered in b-lactam antibiotics are
the penam and cepham and the high electrophilicity of the b-lac-
tam ring system is responsible for the antibiotic activity of these
compounds.
After a comprehensive literature survey our focus was to syn-
thesise thiazolidinone and azetidinone derivatives as less toxic,
more active antimicrobial and antituberculosis agents. (See Fig. 1)
Our efforts focused on the introduction of chemical diversity in
the molecular framework in order to synthesise pharmacologically
interesting compounds of different composition. This motivated us
to design and synthesise new hydrazone, 2-azetidinones and 4-
thiazolidinone templates bearing pyrazole moieties (Scheme 1).
A convenient method for the synthesis of the pyrazole com-
pound was achieved starting from p-chloro acetophenone. Accord-
ing to a previously reported method14 the p-chloro acetophenone
upon tretment with diethyl oxalate in presence of base afforded
ethyl 4-(4-chlorophenyl)-2,4-dioxobutanoate (2). 3-(4-chloro-
phenyl)-1H-pyrazole-5-carbohydrazide (3) is previously reported
compound,15 was synthesised by the reaction of (2) with hydrazine
hydrate under reflux condition. The hydrazone (4) was prepared by
the condensation of hydrazide (3) and the appropriate aromatic
aldehyde under reflex.16 The Staudinger [2+2] cycloaddition reac-
tion17,18 of the hydrazone with chloroacetyl chloride to afford b-
lactam (5) with a trans-configuration (On the basis of 1H NMR,
the coupling constant of the the protons on C3 and C4 is in the
range of 2.0–2.4 Hz, indicating trans-configuration of the b-lac-
tam). The reaction of the hydrazone (4) with mercaptoacetic acid
in presence of sodium acetate and galcial acetic acid afforded race-
mic mixture of the thiazolidin-4-one (6). The yields varied from
⇑
Corresponding authors.
E-mail addresses: dryash_271177@yahoo.co.uk, r.pathak@epsam.keele.ac.uk
(R.B. Pathak).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.063

5130
R. B. Pathak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5129–5133
Cl
Cl
Cl
S
H
H
Cl
Cl
Cl
S
Cl
Cl
Cl
H
H
HN
N
HN
N
H
H
HN
N
N
HN
N
N
N
O
N
O
NH
O
NH
O
Cl
Cl
N
N
N
O
O
O
O
O
O
NH
NH
NH
NH
6j
6i
5i
5j
HO
HO
OH
OH
H
H
H
Cl
Cl
Cl
H
H
H
S
S
HN
HN
HN
N
HN
N
Cl
Cl
N
O
O
N
N
O
O
O
NH
6f
NH
O
6e
5f
Figure 1. Structures of the most active compounds.
5e
Cl
Cl
Cl
b
a
NHNH2
O
COOC2H5
COCH3
N
O
O
NH
1
2
3
R
Cl
H
Cl
c
d
S
HN
N
HN
N
R
N
O
NH
N
O
NH
O
racemic
6
4
R = H-,
e
R
4-OCH3-,
3,4-(OCH3)2-,
4-OH-3-OCH3-
2-OH-,
Cl
H
H
HN
N
4-OH-,
Cl
2-NO2-,
N
O
O
NH
3-NO2-,
2-Cl-,
trans-racemic
4-Cl-
5
Scheme 1. Reagents and condition: (a) Diethyl oxalate (1.05 equiv), NaOMe (1.05 equiv), THF, rt, 1 h; (b) NH2NH2ꢁH2O (2.0 equiv), EtOH, reflux, 6 h; (c) aryl aldehyde
(1.05 equiv), MeOH, glacial CH3COOH, reflux, 6 h; (d) mercaptoacetic acid (1.05 equiv), NaOAc (2.1 equiv), glacial CH3COOH, reflux, 10 h; (e) chloroacetyl chloride (1.2 equiv),
Et3N (1.2 equiv), DCM, rt, 2 h.
35% to 60% in case of hydrazone (4); for the 2-azetidinones (5) the
yields varied from 40% to 65% and from 25% to 50% for the 4-thia-
zolidinones (6). All the compounds structures were confirmed by
1H, 13C, HR-MS and IR (refer to Supplementary data). All com-
pounds were confirmed to be greater than 95% pure via LC–MS
prior to their use in the evaluation of their biological efficacies.
All the compounds were evaluated for in vitro antibacterial
activity against Escherichia coli, and Staphylococcus aureus and anti-
fungal activity against pathogenic fungi. The MIC (minimum inhib-
itory concentration) values were determined by a comparison with
ciprofloxacin (CIP), moxifloxacin (MFX) for antibacterial activity
and fluconazole, 5-fluorocytosine for antifungal activity as stan-
dard agent. The activities reported as MICs were determined
according to NCCLS method.19–21
strains tested. Actully, the activity of compounds 5b, 5c, 6d, 5e, 6e,
5f, 5i, 5j and 6j was superior or comparable to those of 5-fluorocy-
tosine against some strain of fungai. In contrast, the compounds 5a,
4b, 4f, 5g, 5h and 6h did not show significant antifungal activity
against all pathogenic fungal strain tested.
The substituents (ꢀOH, ꢀCl) for the compounds 5 and 6 con-
tribute significantly toward biological potency, Thus, the substitu-
ents appear to be an important factor in their antifungal activity. In
addition, the compounds containing 2-azetidinone or 4-thiazolidi-
none showed more potent antifungal activity then the compounds
containing hydrazones.
All the compounds were evaluated for in vitro antibacterial
activity against E. coli ATCC 25922 and S. aureus ATCC 29213.
As indicated in Table 2, compounds 4e, 4f, 4i, 4j, 5e, 5f, 5i, 5j, 6e,
6f, 6i and 6j show excellent activity against the strains of bacteria.
The substituents (ꢀOH, ꢀCl) for the compounds 4, 5 and 6 are
As indicated in Table 1, most of sysnthesised compounds gener-
ally showed potent antifungal activity against all pathogenic fungal

R. B. Pathak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5129–5133
5131
Table 1
In vitro antifungal activity for hydrazones, 2-azetidinones and 4-thiazolidinones
Antifungal activity MICa
C. neoformansb
(l
g/mL)
Compound
R
C. kruseib
A. nigerb
A. flavusb
4a
5a
6a
4b
5b
6b
4c
5c
6c
4d
5d
6d
4e
5e
6e
4f
5f
6f
4g
5g
6g
4h
5h
6h
4i
5i
6i
4j
5j
6j
HA
12.5
100.0
50.0
100.0
12.5
50.0
50.0
12.5
12.5
12.5
1.6
1.6
50.0
25.0
25.0
12.5
3.2
3.2
00
50.0
12.5
50.0
1.6
12.5
12.5
3.2
50.0
1.6
12.5
1.6
25.0
1.6
6.3
50.0
3.2
1.6
100.0
12.5
100.0
12.5
25.0
12.5
6.3
12.5
12.5
25.0
12.5
6.3
12.5
12.5
50.0
12.5
12.5
25.0
50.0
50.0
12.5
50.0
100.0
25.0
12.5
50.0
25.0
12.5
6.3
00
4-OCH3A
00
00
3.2
3,4-(OCH3)2A
25.0
12.5
6.3
25.0
3.2
00
00
4-OH-3-OCH3A
00
00
12.5
50.0
6.3
1.6
2-OHA
100.0
12.5
6.3
12.5
1.6
00
00
12.5
100.0
25.0
50.0
100.0
12.5
25.0
25.0
100.0
50.0
12.5
1.6
12.5
12.5
6.3
25.0
25.0
6.3
4-OHA
00
00
1.6
3.2
2-NO2A
1.6
50.0
3.2
00
00
100.0
50.0
25.0
12.5
25.0
12.5
12.5
25.0
12.5
6.3
3-NO2A
1.6
00
00
50.0
100.0
3.2
6.3
1.6
1.6
1.6
12.5
6.3
6.3
2-ClA
00
00
4-ClA
00
00
12.5
6.3
6.3
Fluconazole
—
—
25
25
5-FCc
a
The MIC value is defined as lowest concentration of the antifungal agent exhibiting no fungal growth. MIC values were read after 1 day for C. krusei and C. neoformans, and
2 days for A. niger, and A. flavus in 37 °C. The inoculum sizes contained approximately 1 ꢂ 105 cells/mL. Culture media tested were the modified Sabouraud dextrose broth. The
final concentration of antifungal agents was between 0.2 and 100 lg/mL.
b
Fungi tested: C. krusei Berkout KCCM 11655, Cryptococcus neoformans KCCM 50564, Aspergillus niger KCTC 1231, and Aspergillus flavus KCCM 11899.
5-FC: 5-fluorocytosine.
c
important for biological potency. In contrast, the introduction of
The compounds 5a, 5b, 5c, 6c, 4e, 4f, 4j, 5j and 6j are exhibited
slightly less biological potency. In contrast, 2 or 3-nitro phenyl
substituted compounds exhibited poor or no, if any, antimycobac-
terial activity, while the compounds 4a and 6a exhibited lack of
activity.
nitro phenyl or phenyl group in compounds 4a, 4g, 4h, 5a, 5g
and 5h resulted in poor or no antibacterial activity. Although,
many of the compounds showed potent antibacterial activity.
All the compounds were tested for antitubercular activity
against Mycobacterium tuberculosis H37Rv strain. Employing the
BACTEC 460 TB system, all compounds underwent an initial
in vitro screening to determine their activities against the M.
tuberculosis H37Rv strain. The MIC values were determined by a
comparison with rifampin (RIP) as standard agent. Antitubercular
activity was determined using the modified BACTEC 460 system
in which stock solution as test molecules were prepared in dimeth-
ylsulfoxide (DMSO) at 1 mg/ml concentration and sterilised by
passage through 0.22 wm PFTE filters.22,23
In general, the substituents (ꢀOH, ꢀCl, ꢀOCH3) are important
for biological potency, Thus, the substituents appear to be an
important factor in their antimycobacterial activity.
According to the literature survey,24,25 a study of the azole com-
pounds was found to interact with the target enzyme cytochrome
P450-dependent sterol 14-a demethylase in the ergosterol biosyn-
thesis pathway, through computer modelling of drug-enzyme
complexes. With this in mind, we assume that the pyrazole ring
(compounds 4a–j, 5a–j and 6a–j) may take a position perpendicu-
lar to the porphyrine plane, with a ring nitrogen atom coordinate
to the haem iron. Presumably, the distance between the nitrogen
of the pyrazole ring and the haem iron is most suitable to form a
complex.
Moreover, the substituted phenyl group of the compounds
might orientate itself in a hydrophobic binding cleft, above the
haem ring. This subsite includes the side chains of the ceiling res-
idues. The phenyl ring may be the large enough to extend the num-
ber of contacts in the binding site. The 4-chlorophenyl ring,
common to all inhibitors, is stabilized by favourable dispersion
forces with the range of side chains. However, it should be noted
that this is a speculative mechanism of action of these pyrazoles,
as we don’t have any computational approach/Molecular model-
ling results to validate our suggested mode of action. In conclusion,
As seen from data of Table 2, most of sysnthesised compounds
generally possess very good antimycobacterial activity, with MIC
values in the range of 0.35–3.15 lg/mL, although the 4a, 4g and
4h are devoid of such an activity. Best activity against M. tuber-
culosis H37Rv was observed for the 2-OH substituted azetidinone
derivative 5e, which is only slightly less potent than the standard
drug rifampin, followed by the 3,4-(OCH3)2 substituted hydrazone
derivative 4c, the 2 and 4-OH substituted thiazolidinone deriva-
tives 6e and 6f, the 2-Cl substituted hydrazone and thiazolidinone
derivatives 4i and 6i, as well as the 4-OCH3 substituted hydrazone
and thiazolidinone derivatives 4b and 6b, 4-OH-3-OCH3 substi-
tuted derivatives 4d, 5d and 6d, 4-OH substituted azetidinone
derivative 5f, and 2-Cl substituted azetidinone derivative 5i (which
all possess MIC values under 1 lg/mL).

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R. B. Pathak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5129–5133
Table 2
In vitro antibacterial and antitubercular activity for hydrazones, 2-azetidinones and 4-thiazolidinones
Compound
R
Antibacterial activity MICb
S. aureusa
(l
g/mL)
Antitubercular activity MICb
H37Rv
(lg/mL)
E. colia
4a
5a
6a
4b
5b
6b
4c
HA
4
1
>5
00
0.51
0.91
0.18
0.0025
0.5
0.48
0.28
0.01
0.060
0.04
4
1.25
3.15
0.85
1.2
0.65
0.37
1.15
1.2
0.55
0.6
0.65
1.1
0.35
0.5
1.1
0.6
0.39
>5
1.9
4
>5
1.99
2
0.36
0.55
0.5
1.1
1
00
4-OCH3A
00
00
3,4-(OCH3)2A
0.25
0.05
0.06
0.12
0.125
0.15
0.40
0.125
0.06
0.35
0.04
0.125
8
00
00
5c
6c
0.5
0.2
4d
5d
6d
4e
5e
6e
4f
4-OH-3-OCH3A
0.017
0.025
0.05
0.015
0.019
0.028
0.012
0.010
0.017
8
0.5
0.1
0.13
0.33
0.6
0.014
0.031
0.048
0.012
0.010
0.022
0.02
0.015
—
00
00
2-OHA
00
00
4-OHA
00
00
5f
6f
4g
5g
6g
4h
5h
5h
4i
5i
6i
4j
5j
2-NO2A
00
00
6
2
1.5
1.2
3-NO2A
00
00
8
2-ClA
0.21
0.034
0.02
0.12
0.28
0.048
0.25
0.06
—
00
00
4-ClA
00
00
6j
1.15
—
—
CIPb
MFXb
RIPb
—
—
—
0.125
a
Bacteria tested: Strphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922.
CIP: Ciprofloxacin, MFX: Moxifloxacin, RIP: Rifampin, MIC: minimum inhibitory concentration.
b
the synthesis and biological evaluation of a series of 3-(4-cholor-
ophenyl)-4-substituted pyrazole derivatives as antimicrobial and
antitubercular agent has been achieved. Most importently, the aze-
tidinone and thiazolidinone substituted pyrazole scaffold lead to
molecules with potent antimicrobial and antimycobacerial activ-
ity. Interestingly, the antibacterial evaluation indicates more
promising results vs E.coli than against S. aureus. The results sug-
gest that hydroxy and chlorophenyl azetidinone and thiazolidi-
none series would be promising leads for the further devlopment
of antimicrobial and antitubercular agents. However, because of
the end of funding to this project the most active compounds are
not tested for cytotoxicity, but it is likely some of these compounds
may display cytotoxicity.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.
063.

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