Synthesis and biological evaluation of 3-(4-chlorophenyl)-4-substituted pyrazole derivatives

Article abstract of DOI:10.1039/c3ob27290g

3-(4-Chlorophenyl)-4-substituted pyrazole derivatives were synthesised and tested for their in vitro antifungal activity. Some compounds showed very good antifungal activity against four pathogenic strains of fungi. The same compounds exhibited an interesting activity against the tested strain of Mycobacterium tuberculosis H37Rv. The results suggest that 1,3,4-oxadiazoles and 5-pyrazolinones bearing a core pyrazole scaffold may be promising antifungal and antitubercular agents.

Full text of DOI:10.1039/c3ob27290g

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Synthesis and biological evaluation of 3-(4-chloro-
phenyl)-4-substituted pyrazole derivatives
Cite this: DOI: 10.1039/c3ob27290g
P. Horrocks,a M. R. Pickard,a H. H. Parekh,b S. P. Patelb,c and Ravindra B. Pathak*a,b
3-(4-Chlorophenyl)-4-substituted pyrazole derivatives were synthesised and tested for their in vitro anti-
fungal activity. Some compounds showed very good antifungal activity against four pathogenic strains of
fungi. The same compounds exhibited an interesting activity against the tested strain of Mycobacterium
tuberculosis H37Rv. The results suggest that 1,3,4-oxadiazoles and 5-pyrazolinones bearing a core
pyrazole scaffold may be promising antifungal and antitubercular agents.
Received 26th November 2012,
Accepted 31st May 2013
DOI: 10.1039/c3ob27290g
www.rsc.org/obc
Review of the literature suggests that14,15 azole derivatives
show potent antimycobacterial activity often associated with
Introduction
There have been numerous reports on the synthesis of pyrazole good antifungal activity. Moreover, it was also found that some
derivatives owing to their wide ranging biological activities,1,2 azoles were effective inhibitors for enoyl acyl carrier protein
and their associated therapeutic potential as anti-HIV, anti- reductase (FabI), a novel antibacterial target,16 while some
inflammatory and anti-microbial agents.3–8
azole compounds showed a remarkable antibacterial efficacy
Considering the increased incidence of severe opportunistic against MRSA.17 However, the emergence of azole resistant
fungal infections in immunocompromised patients together strains has spurred the search for new antifungal compounds.
with the development of resistance among pathogenic Candida With the aim of obtaining new antifungal compounds, we
spp., there is a great need for new antifungal compounds. On synthesised the series of pyrazole derivatives 2a–j and 3a–j
the other hand, tuberculosis (TB), caused by Mycobacterium (Table 1). All the compounds were evaluated for in vitro anti-
tuberculosis (MTB), has become an important worldwide fungal activity against pathogenic fungi. The MIC (minimum
public health problem with one-third of the world’s population inhibitory concentration) values were determined by a com-
infected by the TB bacillus. The pathogenic synergy of tuber- parison with fluconazole and 5-fluorocytosine for antifungal
culosis with HIV9 enhances the overall incidence of TB in HIV-- activity as standard agents. The activities reported as MICs
positive patients by 50 times relative to the rate for HIV- were determined according to the NCCLS method.18–20
negative individuals.10 Single agent TB therapy readily leads to
All the compounds were tested for antitubercular activity
drug-resistant organisms,11 while multi-drug treatment needs against M. tuberculosis H37Rv strain. Employing the BACTEC
to be prolonged as MTB divides slowly and it is metabolically 460 TB system, all compounds underwent an initial in vitro
capable of becoming drug insensitive and/or bacilli may screening to determine their activities against the M. tuber-
become sequestered.12 The emergence of multidrug and exten- culosis H37Rv strain. The MIC values were determined by a
sively drug resistant tuberculosis (MDR-TB and XDR-TB) further comparison with rifampin (RIP) as a standard agent.
aggravates the problems associated with TB treatment, as
patients could become virtually untreatable using currently BACTEC 460 system in which a stock solution as test molecules
available anti-TB drugs.
was prepared in dimethylsulfoxide (DMSO) at 1 mg mL−1 con-
Antitubercular activity was determined using the modified
Furthermore, no new drugs have been introduced over the centration and sterilised by passage through 0.22 μm PTFE
last four decades, except for the recently introduced fluoro- filters.21,22
quinolones,13 which testifies to the lack of significant research
in this area in the pharmaceutical industry. Hence the develop-
ment of new drugs capable of overcoming MDR- and XDR-TB
is imperative to efficiently treat this disease.
Results and discussion
Synthesis
aInstitute for Sciences and Technology in Medicine, Keele University,
Our efforts focused on the introduction of chemical diversity
in the molecular framework in order to synthesise pharmaco-
logically interesting compounds of different compositions.
KeeleStaffordshire, ST5 5BG, UK. E-mail: r.pathak@keele.ac.uk
bDepartment of Chemistry, Saurashtra University, Rajkot, Gujarat, India
cAptuit LLC, 10245, Hickman Mills Drive, Kansas City, MO 64134-9724, USA
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Table 1 In vitro antifungal and antitubercular activity for 1,3,4-oxadiazoles and 5-pyrazolinones
Antitubercular activity
Antifungal activity MICa,c (μg mL−1
)
MICc (μg mL−1
)
Compound
R/R′
C. kruseib
C. neoformansb
A. nigerb
A. flavusb
H37Rv
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
3a
3b
3c
3d
3e
3f
C6H5–
12.5
100.0
100.0
50.0
100.0
12.5
12.5
50.0
25.0
12.5
12.5
100.0
12.5
3.2
25.0
12.5
12.5
3.2
100.0
25.0
25.0
6.3
50.0
50.0
25.0
50.0
50.0
1.6
100.0
25.0
50.0
50.0
50.0
3.2
12.5
25.0
12.5
3.2
100.0
25.0
3.2
3.2
6.3
12.5
12.5
3.2
50.0
12.5
6.3
50.0
100.0
50.0
100.0
100.0
25.0
12.5
50.0
50.0
12.5
50.0
100.0
12.5
25.0
12.5
12.5
50.0
12.5
100.0
50.0
25
1.0
1.8
1.4
1.3
>5
4-OCH3–C6H4–
2-OH–C6H4–
4-OH–C6H4–
2,4-(OH)2–C6H3–
2-Cl–C6H4–
2-NO2–C6H4–
4-NH2–C6H4–
–CHvCH–C6H5
2-C5H4N–
H–
0.6
0.5
1.1
1.1
0.42
1.4
1.6
1.4
0.6
0.95
0.42
0.9
0.35
1.4
2.0
—
1.6
12.5
12.5
1.6
25.0
50.0
12.5
1.6
12.5
6.3
6.3
4-OCH3–
2-Cl–
4-Cl–
4-NO2–
3-Cl–4-F–
3,4-(Cl)2–
4-F–
2-CH3–
4-COOH–
—
3g
3h
3i
3j
1.6
25.0
25.0
6.3
6.3
—
Fluconazole
5-FCc
—
—
6.3
—
25
—
—
0.125
RIPc
—
a The MIC value is defined as the 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 × 105 cells per mL.
Culture media tested were the modified Sabouraud dextrose broth. The final concentration of antifungal agents was between 0.2 and
100 μg mL−1 b Fungi tested: C. krusei Berkout KCCM 11655, Cryptococcus neoformans KCCM 50564, Aspergillus niger KCTC 1231, and Aspergillus
.
flavus KCCM 11899. c 5-FC: 5-fluorocytosine; RIP: rifampin; MIC: minimum inhibitory concentration.
This motivated us to design and synthesise new oxadiazoles glacial acetic acid. The yields varied from 48% to 80% in the
and pyrazolinones (Scheme 1) bearing a core pyrazole scaffold. case of oxadiazoles (2); for the pyrazolinones (3) the yields
A convenient method for the synthesis of the pyrazole com- varied from 53% to 79%. All the compound structures were
pound (1) was achieved by a previously reported method.23,24 confirmed by 1H, 13C, HR-MS and IR. Compounds were con-
The 1,3,4-oxadiazole (2) was prepared by the reaction of hydra- firmed to be greater than 95% pure via LC-MS prior to their
zide (1) with the appropriate aromatic acid in freshly distilled use in the evaluation of their biological efficacies.
POCl3 under reflux (Scheme 1).25 The 5-pyrazolinones (3) were
Biological evaluation
synthesised by treating hydrazide (1) with the corresponding
ethyl-2-(arylhydrazono)-3-oxobutyrate26,27 in the presence of
In vitro evaluation of antifungal activity. All the compounds
were evaluated for in vitro antifungal activity against four
pathogenic strains of fungi (C. krusei Berkout KCCM 11655,
Cryptococcus neoformans KCCM 50564, Aspergillus niger KCTC
1231, and Aspergillus flavus KCCM 11899). The MIC (minimum
inhibitory concentration) values were compared with flucon-
azole and 5-fluorocytosine as standard agents. The activities
reported as MICs were determined according to the NCCLS
method. The MIC value is defined as the lowest concentration
of the antifungal agent exhibiting no fungal growth.
MIC values were read after 1 day for C. krusei and C. neo-
formans, and 2 days for A. niger, and A. flavus at 37 °C. The
inoculum sizes contained approximately 1 × 105 cells per mL.
Culture media tested were the modified Sabouraud dextrose
broth. The final concentration of antifungal agents was
between 0.2 and 100 μg mL−1
.
As indicated in Table 1, most of the synthesised com-
pounds generally showed potent antifungal activity against
all the pathogenic fungal strains tested. Compounds (2f, 2g, 2j,
Scheme 1 Reagents and Conditions: (a) Aryl acid (1.05 equiv.), POCl3, reflux,
6–8 h; (b) ethyl-2-(arylhydrazono)-3-oxobutyrate (1.0 equiv.), glacial acetic acid,
reflux, 3–6 h.
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amino (2h) resulted in poor activity, although the dihydroxy
(2e) substituted phenyl ring compound is devoid of such
activity. However, the compounds (2f, 2g and 2j) possess MIC
values under 1 μg mL−1
.
In series 3, the best activity against M. tuberculosis H37Rv
was observed for the (4-fluoro) substituted pyrazolinone deriva-
tive (3h), which is only slightly less potent than the standard
drug rifampin, followed by the (dihalogen) substituted pyr-
azolinone derivative (3f), the (4-chloro, 3,4-dichloro and 4-nitro)
substituted pyrazolinone derivatives (3d, 3g and 3e), which all
possess MIC values under 1 μg mL−1. In contrast, the introduc-
tion of electron-releasing groups such as a methyl (3i) or
methoxy (3b) group in the phenyl ring in pyrazolinone series
resulted in very poor activity. However, the compounds (3a, 3c
and 3j) also show some activity.
Fig. 1 Structure of reference compounds.
In general, the electron-withdrawing groups in the phenyl
ring are important for biological potency. Thus, the substitu-
ents appear to be an important factor in their antimycobacter-
ial activity.
3c, 3d, 3e, 3f, 3g and 3h) showed excellent activity against
some strains of fungi. In contrast, compounds (2c, 2d, 2e, 3b,
and 3i) did not show significant antifungal activity against any
pathogenic fungal strains tested (Fig. 1).
According to the literature survey,28,29 a study of the azole
compounds was found to interact with the target enzyme cyto-
chrome P450-dependent sterol 14-α demethylase in the ergos-
terol biosynthesis pathway, through computer modelling of
drug–enzyme complexes. Keeping this in mind, we rational-
ised that the pyrazole ring (compounds 2a–j and 3a–j) may
take a position perpendicular 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 for forming a complex.
The substituents (chloro, nitro and pyridyl) for the series 2
contribute significantly toward biological potency; thus, an
electron-withdrawing group in the phenyl ring appears to be
an important factor for antifungal activity. Similarly, in series
3, compounds with an electron-withdrawing group in the
phenyl ring, i.e. halogen substituents, show higher activity
than those bearing electron-releasing groups like methyl and
methoxy. In contrast, the introduction of a hydroxy (2c, 2d,
and 2e) and a methoxy (2b) group in the phenyl ring in the
oxadiazole series resulted in poor antifungal activity. However,
the introduction of an amino group in the phenyl ring led to
some good antifungal activity. In addition, the introduction of
a methoxy (3b) or methyl (3i) group in the phenyl ring in
pyrazolinone series resulted in a lack of antifungal activity.
However, many of the compounds showed potent antifungal
activity.
In vitro evaluation of antimycobacterial activity. Antituber-
cular activity was determined using the modified BACTEC 460
system in which stock solutions as test molecules were pre-
pared in dimethylsulfoxide (DMSO) at 1 mg mL−1 concen-
tration and sterilized by passage through 0.22 μm PFTE filters.
All compounds underwent an initial in vitro screening to deter-
mine their activities against the M. tuberculosis H37Rv strain.
The MIC values were determined by a comparison with rifam-
pin (RIP) as the standard agent.
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 residues. The phenyl ring may be large enough to
extend the number of contacts in the binding site. The 4-chloro-
phenyl ring, common to all inhibitors, is stabilised by favour-
able 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 do not have any computational
approach/molecular modelling results to validate our suggest-
ed mode of action.
Finally, it is noteworthy that both 3d and 3h demonstrated
low toxicity (IC50 of 306 μM and >500 μM, respectively) against
the human hepatocyte cell line, HepG2. This can be contrasted
with their potent antifungal and antimycobacterial activities,
ruling out any mechanism of non-specific cytotoxicity. Indeed,
low toxicity for mammalian cells is a key feature of other
azoles which act via inhibition of CYP51,30 and this has
allowed their widespread use as antifungal and antimyco-
bacterial agents.
From the point of view of the establishment of structure–
MTB activity relationships, the data in Table 1 show that most
of the synthesised compounds possess very good antimyco-
bacterial activity, with MIC values in the range of 0.35–2.0 μg
mL−1, although 2e and 3i are devoid of such activity.
In series 2, the introduction of electron-withdrawing groups
like pyridine (2j), nitro (2g) or chloro (2f) group in the phenyl
ring resulted in very good antimycobacterial activity, while the
electron-releasing groups like hydroxy (2c, 2d), methoxy (2b) or
Conclusions
In conclusion, the synthesis and biological evaluation of a
series of 3-(4-cholorophenyl)-4-substituted pyrazole derivatives
as antifungal and antitubercular agents have been achieved.
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The 1,3,4-oxadiazole and 5-pyrazolinone substituted pyrazole reduction in daily change in GI, which was less than that
scaffolds lead to molecules with potent antifungal and antimyco- observed with a 1 : 100 dilute control culture on the next day,
bacterial activities. Critically, our two leading candidates, 3d reached a GI of at least 30.
and 3h, display limited toxicity against the hepatocyte cell line
HepG2 and modest antimalarial activity (IC50 of 10.4 μM and
16.3 μM, respectively). Our results suggest that the fluoro,
dichloro, chloro, and 3-chloro-4-fluorophenyl pyrazolinone
series and the chloro, pyridyl and nitro oxadiazole series
would be promising leads for the further development of anti-
fungal and antitubercular agents.
Antifungal studies
The MIC values were determined by the standard broth
dilution method.31 The antifungal activities were tested in
modified Sabouraud dextrose broth against C. krusei Berkout
KCCM 11655, C. neoformans KCCM 50564, A. niger KCTC 1231
and A. flavus KCCM 11899 fungal strains. Fluconazole and
5-fluorocytosine were used as antifungal standard agents. The
compounds were tested in the 0.1–100 μg mL−1 range. They
were added to the modified Sabouraud dextrose broth for
fungi over a final concentration range of 0.1–100 μg mL−1. The
Experimental
Antimycobacterial studies
Antitubercular activity was determined using the modified
BACTEC 460 system in which stock solutions as test molecules
were prepared in dimethylsulfoxide (DMSO) at 1 mg mL−1 con-
centration and sterilized by passage through 0.22 μm PFTE
filters (Millex-FG, Millipore, Bedford, MA). To 50 mL of this
solution, 4 mL radiometric 7H12 broth was added (BACTEC
12B; Becton Dickinson Diagnostic Instrument system, Sparks,
MD, USA) to achieve final concentrations of 12.5 μg mL−1 and
6.25 μg mL−1. Controls received 50 μL DMSO. Rifampin (Sigma
Chemicals Co., St. Louis, MO, USA) was included as a positive
drug control.
inoculum sizes contained approximately 1 × 105 CFU mL−1
.
They were incubated at 37 °C for appropriate periods of time
that sufficed to show clearly visible growth on drug-free
control broths. The MIC value was defined as the lowest con-
centration of the antifungal agent at which they showed opti-
cally clear (100% inhibition). 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.
Antimalarial studies
Rifampin was solubilised and diluted in DMSO and added
to BACTEC-12 broth to achieve a range of concentrations for Intraerythrocytic cultures oDd2 f P. falciparum were maintained
determination of minimum inhibitory concentration (MIC, the in a standard continuous culture. Cultures were maintained at
lowest concentration inhibiting 99% of the inoculums, the 37 °C in a 1% O2 : 3% CO2 : 96% N2 environment. When
MIC value of rifampin is 0.125 μg mL−1 at 97% inhibition of required, cultures were synchronised to ring stages using the
H37Rv strain). M. tuberculosis H37Rv strain (ACTT 27294; sorbitol lysis technique. All compounds were solubilised in
American Type Culture Collection, Rockville, MD, USA) was 100% dimethylsulphoxide (DMSO) to a 100 mM stock (stored
cultured at 37 °C on a rotary shaker in middle brook 7H9 at −20 °C), with dilution to an appropriate concentration
broth (Difco Laboratories, Detroit, MI, USA) supplemented made in complete P. falciparum cell culture medium immedi-
with 0.2% (v/v) glycerol and 0.05% (v/v) Tween 80, until the ately prior to use. Assays were carried out in a 96-well micro-
culture turbidity achieved an optical density of 0.45–0.55 at titre plate using an initial 200 μL of 2% haematocrit, 1%
550 nm. Bacteria were pelleted by centrifugation, washed trophozoite stage culture and five-fold dilutions of the drug
twice, and resuspended in one-fifth of the original volume in (600 μM–91.4 nM). 100% growth was established from cultures
Dulbecco’s phosphate buffered saline (PBS, Irvine Scientific, where no drug was added, and 0% from cultures subjected to
and Santa Ana, CA, USA). Large bacterial clumps were removed a supralethal dose (100 nM) of artemether. Following 48 h of
by passage through an 8 μM filter (Nalgene, Rochester, NY, incubation, 100 μL of resuspended P. falciparum culture was
USA) and aliquots were frozen at −80 °C. Cultures were pre- mixed with an equal volume of lysis buffer (20 mM Tris, pH
pared and an appropriate dilution performed such that a 7.5, 5 mM EDTA, 0.008% (w/v) saponin and 0.08% (v/v) Triton
BACTEC 12B vial inoculated with 0.1 mL would reach a growth X-100) containing SYBR green I (1 × final concentration, from
index (GI) of 999 in 5 days. One-tenth of the diluted inoculum 5000× stock supplied by Invitrogen, UK) in a black 96-multi-
was used to inoculate 4 mL of fresh BACTEC 12B broth con- well plate (Greiner, UK). Well contents were homogenized by
taining the test molecules. An additional control vial was repeated pipetting and incubated for one hour in the dark at
included which received a further 1 : 100 diluted inoculum (as room temperature. The fluorescent signal, in RLU, was
well as 50 μL DMSO) for use in calculating the MIC of rifam- measured using the blue fluorescent module (excitation
pin, respectively, by established procedures. Cultures were 490 nm, emission 510–570 nm) of a Glomax Multi Detection
incubated at 37 °C and the growth of inhibition (GI) deter- System (Promega, UK). The % growth was plotted against
mined daily until control cultures achieved a GI of 999. Assays log10-transformed drug concentration and the IC50 determined
were usually completed in 5–8 days. Percent inhibition was using
a nonlinear regression (sigmoidal dose–response/
defined as 1 − (GI of the test sample/GI of the control) × 100. variable slope equation) in GraphPad Prism v5.0 (GraphPad
Minimum inhibitory concentration of a molecule effecting a Software, Inc., San Diego, CA).
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Cytotoxicity studies
353.2205, requires (MH+) 353.0805 C18H13ClN4O2; LRMS m/z
(ES) MH+ (ES) 353.22.
The human hepatocyte cell line, HepG2 (American Type
Culture Collection, USA), was plated (0.1 mL of 5 × 104 cells
per mL) in 96-well cell culture plates (Greiner, UK) in
R-10 medium (RPMI-1640 medium supplemented with L-glut-
amine [2 mM], fetal bovine serum [10% (v/v)] and gentamicin
[50 μg mL−1]; Sigma Aldrich, UK). After overnight cell attach-
ment, the medium was replaced with 0.2 mL of fresh medium
containing five-fold dilutions of the drug (4–500 μM; in tripli-
cate). Drug stocks (100 mM) were prepared in DMSO; controls
received DMSO vehicle (0.5% v/v final concentration). Cells
were cultured (at 37 °C; a humidified atmosphere of 5%
CO2/95% air) for 70 h. The medium was then aspirated and
cells were washed twice with 0.2 mL per well phosphate-
buffered saline (pH 7.2). Fresh medium (0.2 mL) was added,
followed by MTS reagent (CellTiter 96 AQueous One Solution
Cell Proliferation Assay; Promega, UK; 20 μL), and plates were
incubated for a further 3 h. The absorbance at 490 nm was
determined, medium blank readings (no cells) were subtracted
from test readings, and data were averaged. The experiment
was performed a total of three times (different cultures) and
data were expressed as a percentage of control (no drug)
values. The % viability was plotted against log10-transformed
drug concentration and the IC50 determined as described for
‘antimalarial studies’.
2-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-5-(2-hydroxyphenyl)-
1,3,4-oxadiazole (2c). Yield: (58%, 1.95 g); m.p. 177–179 °C; δH
(250 MHz, CDCl3) 6.1 (1H, s, pyrazole-H), 6.8–6.9 (2H, m,
Ar–H), 7.0–7.1 (1H, m, Ar–H), 7.25–7.4 (3H, m, Ar–H), 7.43 (2H,
d, J 8.3, Ar–H), 8.3 (1H, bs, –NH); δC (125 MHz, CDCl3) 101.1,
111.5, 118.1, 123.3, 128.2, 128.9, 129.7, 131.1, 131.9, 134.8,
137.6, 145.1, 153.9, 158.1, 162.1; IR (KBr, cm−1): 3250, 2075,
2850, 1621, 1545, 1479, 1387, 1245, 1100, 1020, 810, 715;
HRMS found MH+ (ES) 339.2504, requires (MH+) 339.0648
C17H11ClN4O2; LRMS m/z (ES) MH+ (ES) 339.25.
2-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-5-(4-hydroxyphenyl)-
1,3,4-oxadiazole (2d). Yield: (68%, 2.25 g); m.p. 195–197 °C; δH
(250 MHz, CDCl3) 6.1 (1H, s, pyrazole-H), 6.75 (2H, d, J 8.3,
Ar–H), 7.3–7.4 (4H, m, Ar–H), 7.5 (2H, d, J 8.3, Ar–H), 8.0 (1H,
bs, –NH); δC (125 MHz, CDCl3) 101.2, 115.3, 118.4, 128.2,
128.9, 129.7, 130.5, 133.2, 135.5, 145.2, 152.3, 157.4, 164.1; IR
(KBr, cm−1): 3260, 2080, 2860, 1620, 1555, 1480, 1375, 1235,
1105, 1025, 805, 700; HRMS Found MH+ (ES) 339.2041,
requires (MH+) 339.0648 C17H11ClN4O2; LRMS m/z (ES) MH+
(ES) 339.20.
2-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-5-(2,4-dihydroxyphenyl)-
1,3,4-oxadiazole (2e). Yield: (70%, 2.45 g); m.p. 267–269 °C; δH
(250 MHz, CDCl3) 4.9 (2H, bs, –OH), 5.8 (1H, s, pyrazole-H),
6.2 (1H, s, Ar–H), 6.3–6.35 (1H, d, Ar–H), 6.6–6.65 (1H, d,
Ar–H), 7.3 (2H, d, J 8.1, Ar–H), 7.45 (2H, d, J 8.1, Ar–H), 8.3
(1H, bs, –NH); δC (125 MHz, CDCl3) 99.2, 105.3, 108.3, 112.2,
128.6, 129.2, 130.4, 131.4, 135.2, 137.2, 145.4, 152.7, 157.2,
157.4, 164.2; IR (KBr, cm−1): 3245, 2082, 2855, 1627, 1548,
1473, 1370, 1251, 1120, 1030, 800, 705; HRMS Found MH+ (ES)
355.2412, requires (MH+) 355.0597 C17H11ClN4O3; LRMS m/z
(ES) MH+ (ES) 355.24.
General methods
For general experimental details, including information on
solvent purifications and the spectrometers used in this
research, see a previous description.23
General procedure for the synthesis of 1,3,4-oxadiazoles (2a–j)
A solution of compound 1 (Scheme 1) (0.01 mol) and an appro-
2-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-5-(2-chlorophenyl)-
priate acid (0.01 mol) in freshly distilled POCl3 (10 mL) was 1,3,4-oxadiazole (2f). Yield: (61%, 2.15 g); m.p. 238–240 °C; δH
heated at reflux for 6–8 h. Excess POCl3 was distilled off and (250 MHz, CDCl3) 5.9 (1H, s, pyrazole-H), 7.1–7.3 (5H, m,
the residue was poured into iced water. The precipitate was Ar–H), 7.4–7.5 (3H, m, Ar–H), 8.1 (1H, bs, –NH); δC (125 MHz,
filtered, washed with water, dried and recrystallised from CDCl3) 104.3, 127.1, 128.2, 128.9, 129.2, 129.7, 130.4, 131.1,
ethanol to give compounds 2a–j in 48–80% yield.
131.8, 135.1, 136.9, 137.5, 144.9, 153.1, 162.1; IR (KBr, cm−1):
2-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-5-phenyl-1,3,4-oxadi- 3270, 2060, 2860, 1620, 1550, 1480, 1390, 1250, 1108, 1013,
azole (2a). Yield: (48%, 1.5 g); m.p. 155–157 °C; δH (250 MHz, 801, 725; HRMS Found MH+ (ES) 357.4012, requires (MH+
CDCl3) 5.9 (1H, s, pyrazole-H), 6.7 (1H, m, Ar–H), 7.3–7.4 (4H, 357.0309 C17H10Cl2N4O; LRMS m/z (ES) MH+ (ES) 357.40.
)
m, Ar–H), 7.5–7.6 (4H, m, Ar–H), 8.4 (1H, bs, –NH); δC
2-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-5-(2-nitrophenyl)-
(125 MHz, CDCl3) 98.2, 124.7, 127.2, 128.1, 128.6, 128.9, 129.7, 1,3,4-oxadiazole (2g). Yield: (65%, 2.4 g); m.p. 220–222 °C; δH
132.1, 137.6, 138.1, 154.3, 162.1; IR (KBr, cm−1) 3250, 3049, (250 MHz, CDCl3) 6.1 (1H, s, pyrazole-H), 7.35 (2H, d, J 8.4,
2837, 1602, 1537, 1442, 1377, 1245, 1149, 1055, 1026, 825, 750; Ar–H), 7.4–7.5 (2H, d, J 8.4, Ar–H), 7.55–7.7 (3H, m, Ar–H), 7.95
HRMS found MH+ (ES) 323.1225, requires (MH+) 322.0699 (1H, d, Ar–H), 8.3 (1H, bs, –NH); δC (125 MHz, CDCl3)
C17H11ClN4O; LRMS m/z (ES) MH+ (ES) 323.12.
103.1, 124.1, 128.2, 128.8, 129.7, 129.9, 130.5, 131.6, 135.1,
2-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-5-(4-methoxyphenyl)- 137.1, 144.9, 149.4, 152.9, 161.3; IR (KBr, cm−1): 3251, 2076,
1,3,4-oxadiazole (2b). Yield: (64%, 2.2 g); m.p. 130–132 °C; δH 2853, 1627, 1549, 1480, 1380, 1250, 1110, 1027, 830, 725;
(250 MHz, CDCl3) 3.8 (3H, s, –OCH3), 5.95 (1H, s, pyrazole-H), HRMS Found MH+ (ES) 368.0122, requires (MH+) 368.0550
6.8 (2H, d, J 8.1, Ar–H), 7.2–7.3 (4H, m, Ar–H), 7.38 (2H, d, C17H10ClN5O3; LRMS m/z (ES) MH+ (ES) 368.01.
J 8.1, Ar–H), 8.1 (1H, bs, –NH); δC (125 MHz, CDCl3) 54.9, 99.8,
2-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-5-(4-aminophenyl)-
114.1, 119.2, 127.9, 128.6, 129.8, 131.6, 134.7, 137.6, 153.9, 1,3,4-oxadiazole (2h). Yield: (80%, 2.65 g); m.p. 178–180 °C; δH
159.1, 163.1; IR (KBr, cm−1): 3260, 2070, 2845, 1611, 1551, (250 MHz, CDCl3) 5.8 (1H, s, pyrazole-H), 6.45 (2H, d, J 8.2,
1473, 1380, 1258, 1110, 1010, 820, 727; HRMS found MH+ (ES) Ar–H), 7.25 (2H, d, J 8.2, Ar–H), 7.35 (2H, d, J 8.4, Ar–H), 7.4
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(2H, d, J 8.4, Ar–H), 8.0 (1H, bs, –NH); δC (125 MHz, CDCl3) CDCl3) 19.2, 53.4, 99.8, 115.5, 117.8, 127.9, 128.7, 129.7, 131.4,
100.1, 116.1, 116.7, 117.1, 127.9, 128.7, 129.7, 131.6, 134.7, 136.2, 144.1, 145.5, 149.1, 161.1, 170.8; IR (KBr, cm−1):
134, 137.1, 145.9, 151.5, 162.1; IR (KBr, cm−1): 3245, 2080, 3037, 2854, 1703, 1668, 1639, 1616, 1492, 1473, 1371, 1205,
2851, 1628, 1549, 1480, 1390, 1250, 1120, 1005, 800, 705; 1093, 1008, 975, 821, 783; HRMS Found MH+ (ES) 437.2011,
HRMS Found MH+ (ES) 338.1122, requires (MH+) 338.0808 requires (MH+) 437.1128 C21H17ClN6O3; LRMS m/z (ES) MH+
C17H12ClN5O; LRMS m/z (ES) MH+ (ES) 338.11.
(ES) 437.20.
cis-2-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-5-styryl-1,3,4-
1-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-3-methyl-4-(2-(2-chloro-
oxadiazole (2i). Yield: (59% 1.95 g); m.p. 205–207 °C; δH phenyl)hydrazono)-1H-pyrazol-5(4H)-one (3c). Yield: (70%,
(250 MHz, CDCl3) 6.1 (1H, s, pyrazole-H), 6.4 (2H, dd, J 8.0, 2.98 g); m.p. 149–151 °C; δH (250 MHz, CDCl3) 1.95 (3H, s,
–CHvCH–), 7.1–7.3 (5H, m, Ar–H), 7.32 (2H, d, J 8.2, Ar–H), –CH3), 5.95 (1H, s, pyrazole-H), 6.4–6.65 (3H, m, Ar–H), 6.9–7.0
7.44 (2H, d, J 8.2, Ar–H), 8.2 (1H, bs, –NH); δC (125 MHz, (2H, m, Ar–H, –NH), 7.32 (2H, d, J 8.3, Ar–H), 7.4–7.5 (2H, d,
CDCl3) 104.1, 124.1, 126.7, 128, 128.7, 128.9, 129.7, 131.5, J 8.3, Ar–H), 8.1 (1H, bs, –NH); δC (125 MHz, CDCl3) 21, 100.2,
134.1, 135.2, 136.1, 137.1, 145.4, 152.1, 162.2; IR (KBr, cm−1): 117.1, 121.2, 125.1, 127.2, 128.5, 129.2, 129.7, 131.1, 134.2,
3240, 2080, 2860, 1631, 1535, 1480, 1390, 1250, 1100, 1010, 144.1, 145.1, 149.1, 150.2, 163.5, 176.2; IR (KBr): 3047, 2851,
800, 728; HRMS Found MH+ (ES) 349.0124, requires (MH+
)
1708, 1671, 1625, 1620, 1490, 1463, 1370, 1215, 1105, 1025,
985, 831, 760; HRMS Found MH+ (ES) 441.0413, requires (MH+
349.0856 C19H13ClN4O; LRMS m/z (ES) MH+ (ES) 349.01.
)
2-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-5-(2-pyridyl)-1,3,4- 441.0633 C20H14Cl2N6O2; LRMS m/z (ES) MH+ (ES) 441.04.
oxadiazole (2j). Yield: (69%, 2.2 g); m.p. 258–260 °C; δH
1-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-3-methyl-4-(2-(4-chloro-
(CDCl3, δ) 6.0 (1H, s, pyrazole-H), 7.25–7.4 (3H, m, Ar–H), 7.4 phenyl)hydrazono)-1H-pyrazol-5(4H)-one (3d). Yield: (78%,
(2H, d, J 8.3, Ar–H), 7.55–7.6 (1H, m, Ar–H), 7.8–7.85 (1H, m, 3.35 g); m.p. 156–158 °C; δH (250 MHz, CDCl3) 1.9 (3H, s,
Ar–H), 8.4 (2H, m, Ar–H, –NH); δC (125 MHz, CDCl3) 102.1, –CH3), 6.0 (1H, s, pyrazole-H), 6.38 (2H, d, J 8.2, Ar–H), 6.9–7.0
121.1, 124.7, 128.7, 129.7, 131.5, 134.9, 137.1, 137.9, 145.9, (3H, m, Ar–H, –NH), 7.35 (2H, d, J 8.3, Ar–H), 7.43–7.53 (2H, d,
149.5, 152.1, 157.2, 162.7; IR (KBr, cm−1): 3253, 2079, 2855, J 8.2, Ar–H), 8.0 (1H, bs, –NH); δC (125 MHz, CDCl3) 21.1,
1618, 1541, 1472, 1381, 1251, 1120, 1015, 804, 721; HRMS 100.5, 117.9, 123.5, 128.4, 128.8, 129.2, 129.7, 131.6, 134.1,
Found MH+ (ES) 324.5205, requires (MH+
)
324.0652 143.9, 144.8, 149.3, 149.9, 163.5, 176.1; IR (KBr): 3040, 2850,
1712, 1681, 1631, 1611, 1490, 1463, 1370, 1200, 10903, 1018,
C16H10ClN5O; LRMS m/z (ES) MH+ (ES) 324.52.
971, 829, 771; HRMS Found MH+ (ES) 441.1403, requires (MH+
)
General procedure for the synthesis of ethyl-2-(arylhydrazono)-
3-oxobutyrates
Spectroscopic data consistent with the literature.22,23
441.0633 C20H14Cl2N6O2; LRMS m/z (ES) MH+ (ES) 441.14.
1-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-3-methyl-4-(2-(4-nitro-
phenyl)hydrazono)-1H-pyrazol-5(4H)-one (3e). Yield: (77%,
3.46 g); m.p. 278–280 °C; δH (250 MHz, CDCl3) 2.25 (3H, s,
–CH3), 5.8 (1H, s, pyrazole-H), 6.63 (2H, d, J 8.3, Ar–H), 7.32
General procedure for the synthesis of 5-pyrazolinones (3a–j)
To a stirred solution of ethyl-2-(arylhydrazono)-3-oxobutyrate (2H, d, J 8.3, Ar–H), 7.48 (2H, d, J 8.3, Ar–H), 7.73–7.83 (2H, d,
(0.01 mol) in glacial acetic acid (10 mL), a solution of com- J 8.3, Ar–H), 8.2 (1H, bs, –NH); δC (125 MHz, CDCl3) 18.4,
pound 1 (Scheme 1) (0.01 mol) in glacial acetic acid (10 mL) 101.3, 117.5, 122.6, 128.1, 128.9, 129.4, 131.1, 135.3, 140.1,
was added, and the mixture was refluxed for 3–6 h. It was then 143.7, 148.1, 149.8, 150.1, 163.1, 174.2; IR (KBr): 3017, 2850,
cooled and allowed to stand overnight. The solids thus separ- 1700, 1671, 1640, 1620, 1485, 1469, 1361, 1200, 1086, 1015,
ated were filtered, washed with water, dried, and recrystallised 981, 829, 771; HRMS Found MH+ (ES) 452.1310, requires (MH+
from ethanol to give compounds 3a–j in 53–79% yield.
)
452.0874 C20H14ClN7O4; LRMS m/z (ES) MH+ (ES) 452.13.
1-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-3-methyl-4-(2-(3-chloro-
1-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-3-methyl-4-(2-phenyl-
hydrazono)-1H-pyrazol-5(4H)-one (3a). Yield: (61%, 2.4 g); m. 4-fluorophenyl)hydrazono)-1H-pyrazol-5(4H)-one (3f). Yield:
p. 165–168 °C; δH (250 MHz, CDCl3) 2.1 (3H, s, –CH3), 5.9 (1H, (70% 3.18 g); m.p. 215–217 °C; δH (250 MHz, CDCl3) 1.9 (3H, s,
s, pyrazole-H), 6.4–6.55 (3H, m, Ar–H), 6.7–6.8 (2H, m, Ar–H), –CH3), 5.84 (1H, s, pyrazole-H), 6.42 (1H, s, Ar–H), 6.58–6.64
7.25 (2H, d, J 8.5, Ar–H), 7.4 (2H, d, J 8.5, Ar–H), 8.1 (1H, bs, (2H, m, Ar–H), 7.28–7.38 (2H, d, J 8.3, Ar–H), 7.4–7.5 (2H, d,
–NH); δC (125 MHz, CDCl3) 19.2, 99.3, 115.2, 115.3, 117.7, J 8.3, Ar–H), 7.95 (1H, bs, –NH); δC (125 MHz, CDCl3) 21.4,
127.9, 128.3, 128.9, 129.4, 131.1, 135.1, 143.1, 145.1, 102.3, 115.9, 117.1, 118.2, 121.9, 127.9, 128.7, 129.6, 130.8,
148.1, 162.1, 172.5; IR (KBr, cm−1): 3040, 2850, 1710, 1678, 134.8, 140.1, 144.3, 148.2, 149.3, 151.1, 161, 172.9; IR (KBr):
1635, 1612, 1490, 1463, 1370, 1200, 1083, 1018, 970, 825, 3025, 2848, 1708, 1678, 1633, 1622, 1489, 1479, 1361, 1204,
760; HRMS Found MH+ (ES) 407.1210, requires (MH+) 407.1023 1091, 1010, 965, 820, 766; HRMS Found MH+ (ES) 459.0123,
C20H15ClN6O2; LRMS m/z (ES) MH+ (ES) 407.12.
requires (MH+) 459.0539 C20H13Cl2FN6O2; LRMS m/z (ES) MH+
1-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-3-methyl-4-(2-(4-methoxy- (ES) 459.01.
phenyl)hydrazono)-1H-pyrazol-5(4H)-one (3b). Yield: (53%,
1-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-3-methyl-4-(2-(3,4-
2.3 g); m.p. 129–131 °C; δH (250 MHz, CDCl3) 2.0 (3H, s, dichlorophenyl)hydrazono)-1H-pyrazol-5(4H)-one (3g). Yield:
–CH3), 3.8 (3H, s, –OCH3), 5.95 (1H, s, pyrazole-H), 6.35 (2H, d, (79%, 3.72 g); m.p. 187–189 °C; δH (250 MHz, CDCl3) 1.9 (3H,
J 8.4, Ar–H), 6.52 (2H, d, J 8.1, Ar–H), 7.3 (2H, d, J 8.4, Ar–H), s, –CH3), 5.84 (1H, s, pyrazole-H), 6.44 (1H, s, Ar–H), 6.95–7.05
7.44 (2H, d, J 8.1, Ar–H), 8.0 (1H, bs, –NH); δC (125 MHz, (3H, m, Ar–H, –NH), 7.3–7.4 (2H, d, J 8.3, Ar–H), 7.43–7.53 (2H,
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d, J 8.3, Ar–H), 7.98 (1H, bs, –NH); δC (125 MHz, CDCl3) 20.4,
104.3, 116.4, 118.6, 124.7, 127.8, 128.6, 129.5, 130.8, 131.2,
134.4, 134.9, 142.1, 144.1, 148.2, 149.1, 163.1, 175.1; IR (KBr):
3039, 2851, 1701, 1669, 1637, 1620, 1491, 1477, 1369, 1200,
1083, 1011, 976, 821, 755; HRMS Found MH+ (ES) 475.1120,
requires (MH+) 475.0243 C20H13Cl3N6O2; LRMS m/z (ES) MH+
(ES) 475.11.
1-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-3-methyl-4-(2-(4-fluoro-
phenyl)hydrazono)-1H-pyrazol-5(4H)-one (3h). Yield: (71%,
2.99 g); m.p. 199–201 °C; δH (250 MHz, CDCl3) 1.95 (3H, s,
–CH3), 6.0 (1H, s, pyrazole-H), 6.39 (2H, d, J 8.1, Ar–H),
6.75–6.85 (3H, m, Ar–H, –NH), 7.31 (2H, d, J 8.2, Ar–H), 7.45
(2H, d, J 8.2, Ar–H), 7.98 (1H, bs, –NH); δC (125 MHz, CDCl3)
20.9, 104.1, 116.1, 118.2, 127.9, 128.6, 129.5, 131.2, 134.5,
139.2, 144.5, 148.5, 149.1, 151.9, 164.2, 174.3; IR (KBr): 3031,
2 E. O. Ronald, J. Pharm. Sci., 1968, 57, 537.
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2850, 1709, 1688, 1631, 1619, 1482, 1463, 1369, 1209, 1088, 10 E. L. Corbett, C. J. Watt, N. Walker, D. Maher,
1020, 966, 827, 761; HRMS Found MH+ (ES) 425.1210, requires
(MH+) 425.0929 C20H14ClFN6O2; LRMS m/z (ES) MH+ (ES)
425.12.
B. G. Williams, M. C. Raviglione and C. Dye, Arch. Intern.
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11 K. Honer zu Bentrup and D. G. Russell, Trends Microbiol.,
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1-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-3-methyl-4-(2-(2-o-tolyl-
hydrazono)-1H-pyrazol-5(4H)-one (3i). Yield: (69%, 2.85 g); m. 12 (a) C. Dye, B. G. Williams, M. A. Espinal and
p. 167–169 °C; δH (250 MHz, CDCl3) 1.85 (3H, s, –CH3), 2.4
(3H, s, –CH3), 5.8 (1H, s, pyrazole-H), 6.35–6.5 (2H, m, Ar–H),
6.75–6.85 (3H, m, Ar–H, –NH), 7.28 (2H, d, J 8.2, Ar–H), 7.42
(2H, d, J 8.2, Ar–H), 8.1 (1H, bs, –NH); δC (125 MHz, CDCl3)
M. C. Raviglione, Science, 2002, 295, 2042; (b) T. B. Agerton,
S. E. Valway, R. J. Blinkhorn, K. L. Shilkret, R. Reves,
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15.2, 19.9, 101.1, 116.5, 118.5, 125.8, 127.9, 128.6, 129, 129.5, 13 (a) P. R. Mohapatra, Am. J. Respir. Crit. Care Med., 2004,
130, 131.5, 134.7, 139.8, 144.2, 148.2, 148.8, 162.8, 172.7; IR 170, 920; (b) H. L. Rieder, Lancet, 2009, 373, 1148.
(KBr): 3027, 2844, 1709, 1675, 1636, 1611, 1485, 14713, 1366, 14 R. Fioravanti, M. Biava, G. C. Porretta, M. Artico,
1215, 1096, 1019, 966, 815, 761; HRMS Found MH+ (ES)
421.5420, requires (MH+) 421.1179 C21H17ClN6O2; LRMS m/z
(ES) MH+ (ES) 421.54.
4-(2-(1-(3-(4-Chlorophenyl)-1H-pyrazole-5-yl)-3-methyl-5-oxo-
1H-pyrazol-4(5H)-ylidene)hydrazinyl)benzoic acid (3j). Yield:
G. Lampis, D. Deidda and R. Pompei, Med. Chem. Res.,
1997, 7, 87.
15 M. Biava, R. Fioravanti, G. C. Porretta, G. Sleiter, A. Ettorre,
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1997, 7, 228.
(68%, 3.0 g); m.p. 115–117 °C; δH (250 MHz, CDCl3) 1.9 (3H, s, 16 D. A. Heerding, G. Chan, W. E. DeWolf, A. P. Fosberry,
–CH3), 5.85 (1H, s, pyrazole-H), 6.55 (2H, d, J 8.3, Ar–H), 7.25
(2H, d, J 8.4, Ar–H), 7.4 (2H, d, J 8.3, Ar–H), 7.7 (2H, d, J 8.4,
Ar–H), 8.3 (1H, bs, –NH), 11.2 (1H, bs, –COOH); δC (125 MHz,
CDCl3) 21.1, 104.1, 116.5, 120.5, 127.8, 128.6, 129.5, 130.8,
131.9, 134.9, 143.8, 147.7, 148.2, 149.2, 161.9, 172.7, 179.1; IR
C. A. Janson, D. D. Jaworski, E. McManus, W. H. Miller,
T. D. Moore, D. J. Payne, X. Y. Qiu, S. F. Rittenhouse,
C. Slater-Radosti, W. Smith, D. T. Takata, K. S. Vaidya,
C. C. K. Yuan and W. F. Huffman, Bioorg. Med. Chem. Lett.,
2001, 11, 2061.
(KBr): 3037, 2851, 1707, 1677, 1634, 1619, 1471, 1370, 1209, 17 S. H. Lee and C. J. Kim, J. Microbiol. Biotechnol., 1999, 9,
1091, 978, 815, 761; HRMS Found MH+ (ES) 451.2145, requires
572.
(MH+) 451.0921 C21H15ClN6O4; LRMS m/z (ES) MH+ (ES) 451.21. 18 The MIC assays were performed in accordance with the
NCCLS guidelines, Methods for Dilution Antimicrobial Sus-
ceptibility Tests for Bacteria that Grow Aerobically, 5th edn,
NCCLS Document M7 A5, NCCLS, January 2000, 20.
19 Performance Standards for Antimicrobial Susceptibility
Acknowledgements
The authors are thankful to the Saurashtra University for infra-
structure facilities and financial support. The authors also
Testing, Eleventh Informational Supplement, NCCLS Docu-
ment M100-S11, NCCLS, January 2001, 21, 1.
thank Dr Cecil Kwong, TAACF, Alabama, USA for antitubercu- 20 D. Amsterdam, Susceptibility testing of antimicrobials
lar screening.
in liquid media, in Antibiotics in Laboratory Medicine,
Williams & Wilkins, Baltimore, MD, 4th edn, 1996, p. 52.
21 L. Collins and S. G. Franzblau, Antimicrob. Agents
Chemother., 1997, 41, 1004.
22 S. H. Siddiqi, BACTEC 460 TB System, Product and Procedure
Manual Revision. D.Becton Dickinson Microbiology System,
Sparks, MD, 1995.
Notes and references
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