Coumarinyl pyrazole derivatives of INH: Promising antimycobacterial agents

Article abstract of DOI:10.1007/s00044-012-0222-8

The purpose of this study was to evaluate the antimycobacterial activity of various pyrazole derivatives derived from the isoniazid pharmacophore along with coumarin scaffold. The synthesized title compounds (4a-4k) were investigated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv using Resazurin MIC assay. The synthesized compounds exhibited MIC ranging from 0.625 to 2.50 μg/ml. Among the series tested, compound 3-[3-(4-fluorophenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H- chromen-2-one 4i was found to be the most active with MIC of 0.625 μg/ml. Graphical Abstract: Synthesis, spectral studies and antimycobacterial screening of a series of 11 new 2-pyrazole derivatives containing coumarin nucleus are described.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-012-0222-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2279–2283
DOI 10.1007/s00044-012-0222-8
ORIGINAL RESEARCH
Coumarinyl pyrazole derivatives of INH: promising
antimycobacterial agents
•
Prashant Aragade Mahesh Palkar
•
•
Pradeepkumar Ronad Darbhamulla Satyanarayana
Received: 10 April 2012 / Accepted: 24 August 2012 / Published online: 6 September 2012
Ó Springer Science+Business Media, LLC 2012
Abstract The purpose of this study was to evaluate the
antimycobacterial activity of various pyrazole derivatives
derived from the isoniazid pharmacophore along with cou-
marin scaffold. The synthesized title compounds (4a–4k)
were investigated for their in vitro antimycobacterial
activity against Mycobacterium tuberculosis H37Rv using
Resazurin MIC assay. The synthesized compounds exhib-
ited MIC ranging from 0.625 to 2.50 lg/ml. Among the
series tested, compound 3-[3-(4-fluorophenyl)-1-isonicoti-
noyl-1H-pyrazol-5-yl]-2H-chromen-2-one 4i was found to
be the most active with MIC of 0.625 lg/ml.
where it was almost eradicated, has been favored by the
pathogenic synergy with human immunodeficiency virus
(HIV) infection. In fact, TB and other atypical mycobacteri-
oses are now diseases frequently associated with AIDS; HIV
infection significantly increases the risk that new or latent
TB infections will progress to active diseases (Collins,
1989; Graham et al., 1996; Halsey et al., 1998; Inderlied
et al., 1993). The emergence of TB has also been accom-
panied by the appearance of single-drug-resistant (SDR)
and multidrug-resistant (MDR) strains of M. tuberculosis
which are insensitive to one or more of the first-line anti-TB
drugs (isoniazid [INH], rifampin, ethambutol, streptomycin,
and pyrazinamide) (Telzak et al., 1995). Indeed, a great
amount of work has been done in order to acquire useful
knowledge about the mechanisms of action and resistance to
available anti-TB drugs (Dessen et al., 1995). M. tubercu-
losis often becomes drug resistant as a consequence of
spontaneous genetic mutations involving the molecular
targets of drugs. The primary mechanism of multidrug
resistance in TB is the accumulation of mutations in indi-
vidual drug target genes (Morris et al., 1995). However, such
knowledge is not sufficient to rationally overcome drug
resistance in mycobacteria. In fact, currently, combinations
of two or more anti-TB drugs are used to prevent the
development of resistant mycobacteria; sometimes it is also
necessary to resort to second-line drugs (ciprofloxacin,
ethionamide, kanamycin, amino salicylic acid, etc.) (Man-
dell and Petri, 1996; Sensi and Grassi, 1996). Consequently,
the present anti-TB regimen is rather complex and lengthy.
In immunosuppressed patients, it is also unsatisfactory. All
of these serious concerns require particular attention and
stimulate the continuing search for new anti-TB agents and
therapeutic regimens.
Keywords Antimycobacterial activity ꢀ Coumarin ꢀ
Isonicotinic acid hydrazide ꢀ Pyrazole ꢀ
Mycobacterium tuberculosis
Introduction
Tuberculosis (TB) is still a challenging worldwide health
problem and Mycobacterium tuberculosis remains one of the
single most deadly human pathogens. The resurgence of TB
over the last two decades, even in industrialized countries
Authors sincerely dedicate the article in the name of Late.
Dr. V. S. Maddi.
P. Aragade ꢀ M. Palkar (&) ꢀ P. Ronad
Department of Medicinal Chemistry, KLES’ College
of Pharmacy, Vidyanagar, Hubli, Karnataka, India
e-mail: palkarmahesh4u@rediffmail.com
D. Satyanarayana
Department of Medicinal Chemistry,
NGSM Institute of Pharmaceutical Sciences,
Paneer, Deralakatte,
The chemistry of heterocyclic compounds has been an
interesting field of study for a long time. The synthesis of
Mangalore, Karnataka, India
123

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Med Chem Res (2013) 22:2279–2283
novel pyrazole derivatives and investigation of their chem-
ical and biological behavior has gained more importance
in recent decades for biological, medicinal, and agricultural
reasons. Living organisms find difficulty in construction of
N–N bonds which limits the natural abundance of com-
pounds having such bonds. Pyrazole and their derivatives,
a class of compounds containing the N–N bond exhibits a
wide range of biological activities (Kucukguzel and
Rollas, 2000; Nauduri and Reddy, 1998; Ali et al., 2007;
Shaharyar et al., 2006a, b). Much attention is given to
pyrazoles as antimicrobial agents after the discovery of the
natural pyrazole C-glycoside like pyrazofurin which dem-
onstrated a broad spectrum antimicrobial activity (Genin
et al., 2000). A literature survey has revealed that pyrazole
derivatives are active against many mycobacterias
(Kucukguzel and Rollas, 2002; Shenoy et al., 2001).
mycobacterias. Therefore, such medicinal properties asso-
ciated with these two heterocycles render them as useful
structural units in drug research. Recently, we have reported
the synthesis and antibacterial activity of 3-[3-(substituted
phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-
one derivatives (Aragade et al., 2009). In view of these
observations and in continuation of our research program on
the synthesis of biheterocyclic compounds (Khode et al.,
2009), we report herein the antimycobacterial activity of
title compounds (4a–4k), which mainly describes the impact
of incorporation of INH in a pyrazole along with coumarin
moiety for their antimycobacterial activity against
M. tuberculosis H37Rv.
Results and discussion
Among the standard antimycobacterial agents, in spite of
toxicity on repeated dosing INH is still considered to be a first-
line drug for chemotherapy of TB. INH has very high in vivo
inhibitory activity against M. tuberculosis H37Rv. Enzymatic
acetylation of INH by N-acetyltransferase represents a major
metabolic pathway for INH in human beings. Acetylation
greatly reduces the therapeutic activity of the drug, resulting in
under dosing, decreased bioavailability, and acquired INH
resistance (Kopec and Zwolska, 2002). Chemical modifica-
tion of INH with a functional group that blocks acetylation,
while maintaining a strong antimycobacterial action, may
improve clinical outcomes and facilitate to reduce the rise of
INH resistance. The aim of our research work was to inves-
tigate such chemical modification of INH. On the other
hand, pyrazoline derivatives were active against many
Chemistry
The synthesis of series of 3-[3-(substituted phenyl)-1-isoni-
cotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one (4a–4k) was
achieved through the versatile and efficient synthetic route
outlined in Scheme 1, as reported earlier by our group. It is
apparent from the scheme that the new target molecules
possess both coumarin and pyrazole units. Reaction of 3-[2,
3-dibromo-3-(substituted phenyl)propanoyl]-2H-chromen-
2-one with INH seemed to be a convenient route for the
synthesis of desired molecules. Starting material 3-acetyl-
2H-chromen-2-one (1) was synthesized by the reaction of
salicylaldehyde with ethylacetoacetate in presence of cata-
lytic amount of piperidine at room temperature following the
Scheme 1 Synthesis of 3-[3-
(substituted phenyl)-1-
O
O
isonicotinoyl-1H-pyrazol-5-yl]-
2H-chromen-2-one (4a–4k)
(Aragade et al., 2009). Reagents
and conditions. (a) Ar–CHO,
piperidine/n-butanol, reflux,
4 h; (b) Br2/CHCl3, stirr r.t.
12 h; (c) isonicotinic acid
hydrazide, TEA/ethanol, reflux,
12 h
CH3
(a)
O
O
O
O
(2a-k)
R
(1)
N
(b)
O
O
Br
O
O
N
N
(c)
Br
(3a-k)
R
O
R
O
(4a-k)
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Med Chem Res (2013) 22:2279–2283
2281
literature procedure (Bolakatti et al., 2008; Knoevenagel,
1898). 3-[(2E)-3-Substituted-prop-2-enoyl]-2H-chromen-2-
one (chalcones, 2a–k) were obtained by Claisen–Schmidt
condensation of 3-acetyl-2H-chromen-2-one (1) with vari-
ous substituted benzaldehydes in presence of mixture of
piperidine and n-butanol. Efforts to convert compounds
2a–2k into target molecules (4a–4k) under a variety of
conditions were not successful. Hence an alternative method
was adopted. This involved the bromination of chalcones
(2a–2k) and subsequent ring closure using isonicotinic acid
hydrazide. Bromination of chalcones (2a–2k) was carried
out in chloroform using bromine in chloroform (Karthikeyan
et al., 2007). The resulting dibromo compounds (3a–3k) on
further treatment with isonicotinic acid hydrazide in the
presence of triethylamine in absolute ethanol yielded the
desired compounds 3-[3-(substituted phenyl)-1-isonicoti-
noyl-1H-pyrazol-5-yl]-2H-chromen-2-one (4a–4k). Struc-
tures of the synthesized compounds were established on the
basis of physicochemical, elemental analysis, and spectral
data (IR and 1H NMR), which were reported earlier by our
group (Aragade et al., 2009).
Further, compounds 4a, 4b, 4d, 4j, and 4k showed
respectable antimycobacterial activity (MIC = 2.5 lg/ml)
with 75.39, 66.67, 73.82, 67.83, and 62.63 % growth
inhibition, respectively. Whereas compound 4g displayed
the least activity (MIC = 5.0 lg/ml) with 62.63 % growth
inhibition as compared to the standard drugs, INH, and
rifampicin, respectively. In general, the brief structure–
activity relationship (SAR) studies revealed that the pres-
ence of electron withdrawing groups such as F, Cl, and
NO2 on the phenyl ring of pyrazole moiety at C-3 position
may be attributed for enhanced antimycobacterial activity
in the series. The presence of 4-fluoro group in (4a–4k)
derivatives, i.e., compound 4i displayed relatively higher
inhibitory activity. Similarly, the chloro and nitro substi-
tuted analogs, such as 4-chlorophenyl (4c), 2,4-dichloro-
phenyl (4e), and 3-nitrophenyl (4f) showed moderate
inhibitory activity against M. tuberculosis. These reports
clearly showed that the compound 4i containing a 4-fluoro
group at C-3 phenyl ring of pyrazole nucleus gave better
result and has emerged as promising antimycobacterial
agents.
Antimycobacterial activity
Table 1 In vitro antimycobacterial activity of 3-[3-(substituted
phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one (4a–4k)
The antimycobacterial evaluation of the title compounds
was carried out at the Tuberculosis Antimicrobial Acqui-
stition Co-ordinating Facility (TAACF), USA, antituber-
culosis drug discovery program coordinated by the
Southern Research Institute (Birmingham, USA) under the
direction of the National Institute of Allergy and Infectious
Diseases (NIAID, USA). All the compounds were screened
against M. tuberculosis strain H37Rv in Middlebrrok 7H9
medium using the Resazurin minimum inhibitory concen-
tration (MIC) assay. This methodology is nontoxic, uses a
thermally stable reagent and shows a good correlation as
that of BACTEC radiometric methods (Reis et al., 2004).
The purpose of the screening program is to provide a
resource whereby new experimental compounds can be
tested for their capacity to inhibit the growth of virulent
M. tuberculosis. MIC was recorded as the lowest concen-
tration of a compound that inhibits the growth of tested
microorganism. The antimycobacterial activity data of test
compounds were compared with the standard drug INH and
rifampin which exhibited a MIC value of 0.06 and
0.003 lg/ml, respectively. The results of the in vitro anti-
mycobacterial activity screening of the test compounds are
summarized in Table 1. Among the newly synthesized
compounds, 3-[3-(4-fluorophenyl)-1-isonicotinoyl-1H-pyr-
azol-5-yl]-2H-chromen-2-one (4i) especially showed
excellent activity at MIC 0.625 lg/ml and exhibited 80 %
growth inhibition, while compounds 4c, 4e, 4f, and 4h
showed good activity (MIC = 1.25 lg/ml) with 75.39,
69.09, 65.39, and 65.14 % growth inhibition, respectively.
N
O
N
N
R
O
O
(4a-k)
Compounds
R
MICa IC50 IC90 % growth inhibition
4a
H–
2.5
1.92 2.26 73.53
1.21 1.35 66.67
0.75 1.22 75.39
4b
4-OMe–
4-Cl–
2.5
4c
1.25
4d
4-NMe2– 2.5
2,4-(Cl)2– 1.25
1.21 2.3
73.82
4e
0.74 1.46 69.09
0.89 1.67 65.39
2.46 2.69 62.63
1.17 1.29 65.14
4f
3-NO2–
4-Me–
3-OMe–
4-F–
1.25
5
4g
4h
1.25
4i
0.625 0.41 0.73 80.09
4j
2-NO2–
4-OH–
–
2.5
1.9
2.74 67.83
4k
2.5
1.51 2.85 69.91
INH
0.06
0.003
Rifampicin
–
a
MIC is expressed in lg/ml
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Med Chem Res (2013) 22:2279–2283
Conclusion
(Bolakatti et al., 2008). The synthesis and spectral charac-
terization of a series of 3-[3-(substituted phenyl)-1-isoni-
cotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one derivatives
(4a–4k) were reported earlier by our group (Aragade et al.,
2009).
We have described earlier an efficient synthesis and
antibacterial activity of a novel series of 3-[3-(substituted
phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-
one (4a–4k). In these novel heterocyclic compounds INH
has been converted into a pyrazole nucleus which also
contains of coumarin ring system. Herein, we report the
antimycobacterial activity of title compounds (4a–4k). In
general, the results of the in vitro antimycobacterial
activity tests were encouraging as out of eleven compounds
tested, one compound 4i exhibited significant antimyco-
bacterial activity, which is comparable to the reference
drugs. The MIC values of these novel compounds evi-
denced that the presence of fluorine, chlorine, and nitro
groups in the phenyl ring at C-3 position of the pyrazole
nucleus gave rise to increased antimycobacterial potency.
In conclusion, the compound 4i may serve as a promising
lead molecule for new anti-tubercular drug development
and our findings will have an impact on medicinal chemists
and pharmacists for further investigations in this field in
search of potent anti-tubercular agents.
Antimycobacterial activity
Medium
Middlebrook 7H9 broth medium (7H9 medium) was used
for testing antimycobacterial activity. Middle brook 7H9
broth medium supplemented with 0.2 % (v/v) glycerol,
10 % (v/v) albumin, dextrose, catalase (ADC), and 0.05 %
(v/v) Tween 80.
Test microorganism
Mycobacterium tuberculosis H37Rv obtained from Colo-
rado State University, Fort Collins, CO was used for testing
antimycobacterial activity.
Inoculum preparation
Experimental
The mycobacterium was inoculated in 50 ml of 7H9 med-
ium in 1 l bottles that were placed on a roller bottle appa-
ratus in an ambient 37 °C incubator. When the cells were
reached OD600 of 0.150 (equivalent to *1.5 9 107 CFU/
ml), they were diluted 200-fold in 7H9 medium.
Chemistry
All research chemicals were purchased from Sigma-Aldrich
(St. Louis, MO, USA) or Lancaster Co. (Ward Hill, MA,
USA) and used as such for the reactions. Solvents except
laboratory reagent grade were dried and purified, when
necessary, according to the literature. Reactions were mon-
itored by thin-layer chromatography (TLC) on pre-coated
silica gel plates from Merck (Darmstadt, Germany). Melting
points of synthesized compounds were determined in
Thermonik melting point apparatus (Thermonik, Mumbai,
India) and are uncorrected, UV spectra were recorded on
Thermospectronic spectrometer (Rochester, NY, USA) and
IR spectra were recorded on Thermo Nicolet IR200 FT-IR
spectrometer (Madison, WI, USA) by using KBr pellets. The
1H NMR was recorded on Bruker AVANCE 300 (Bruker,
Rheinstetten/Karlsruhe, Germany) using DMSO-d6 as sol-
vent. Chemical shifts are given in d ppm units with respect
to TMS as internal standard. The elemental analyzes (C, H,
and N) of the compounds were performed on Heraus
CHN–O rapid elemental analyzer (Heraeus, Hanau, Ger-
many). Results of elemental analysis were within 0.4 % of
the theoretical values. The purity of compounds was
examined by TLC on silica gel plate using chloroform and
methanol (10:1) as mobile phase and iodine vapors as
visualizing agent. The starting material 3-acetyl coumarin
(1) was synthesized by our earlier reported method
Minimum inhibitory concentration
The in vitro antimycobacterial activity for newly synthe-
sized compounds 4a–4k was evaluated using Resazurin
MIC assay (Collins and Franzblau, 1997). 20 ll of the
3.2 mg/ml test compound is added to 96-well microtiter
plate. Twofold dilutions were made by the addition of
20 ll of diluents. Further progressive double dilution with
dilutent was performed to obtain the required concentra-
tions 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, and 0.078 lg/ml.
Then each dilution was further diluted 1:10 in sterile water.
6.25 ll of each dilution was transferred to duplicate
96-well test plates. The cell suspension (93.75 ll * 104
bacteria) in 7H9 medium was added to the test plate. The
96-well test plates were incubated in an ambient 37 °C
incubator for 6 days. At the end of incubation period,
sterile resazurin solution (5 ll of 0.05 %) was added to
each cell of the 96-well plate and placed in an ambient
37 °C incubator for 2 days. After completion of 2 day
incubation, the MIC (a visual evaluation) and IC50 and IC90
(fluorometric readout) were determined.
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Med Chem Res (2013) 22:2279–2283
2283
Acknowledgments The authors sincerely acknowledge AICTE,
New Delhi (India), for financial support under RPS Scheme (File No.
8023/BOR/RID/RPS-170/2008-09). Authors are thankful to Principal,
KLES’ College of Pharmacy, Hubli, for providing necessary facilities
to carry out this research work. We sincerely express our gratitude to
Dr. M.N.A. Rao, General Manager (R&D), Divi’s Laboratory,
Hyderabad and Dr. L.V.G. Nargund, Professor, Nargund College of
Pharmacy, Bangalore, for their encouragement. We sincerely express
our gratitude to Tuberculosis Antimicrobial Acquisition Co-ordinat-
ing Facility (TAACF, USA) for providing the antimycobacterial
activity profile. We are grateful to The Director, SAIF, Punjab Uni-
versity and The Chairman, USIC, Karnataka University, for providing
elemental and spectral analysis.
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