New INH-pyrazole analogs: Design, synthesis and evaluation of antitubercular and antibacterial activity

Article abstract of DOI:10.1016/j.bmcl.2015.10.057

With the aim of developing promising antitubercular and antibacterial leads, we have designed and synthesized a new series of isonicotinohydrazide based pyrazole derivatives (5a-r). All new derivatives (4a-b and 5a-r) were screened for in vitro antimycoba

Full text of DOI:10.1016/j.bmcl.2015.10.057

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5540–5545
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New INH–pyrazole analogs: Design, synthesis and evaluation of
antitubercular and antibacterial activity
⇑
Nagabhushana Nayak, Jurupula Ramprasad, Udayakumar Dalimba
Organic Chemistry Laboratory, Department of Chemistry, National Institute of Technology Karnataka, Surathkal, Srinivasanagar, Mangalore 575025, India
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim of developing promising antitubercular and antibacterial leads, we have designed and
synthesized a new series of isonicotinohydrazide based pyrazole derivatives (5a–r). All new derivatives
(4a–b and 5a–r) were screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis
H37Rv (MTB) strain. Four compounds 5j, 5k, 5l and 4b emerged as promising antitubercular agents with
Received 4 August 2015
Revised 4 October 2015
Accepted 20 October 2015
Available online 21 October 2015
MIC of 64.9 lM which is much lower than the MIC of the first line antitubercular drug, ethambutol. The
3-chlorophenyl substituent at position-3 of the pyrazole ring enhanced the antiTB activity of the
molecules. Three derivatives 5b, 5k and 4b exhibited promising antibacterial activity against the tested
bacterial strains. The active molecules were nontoxic to normal Vero cells and showed high selectivity
index (>160). The structure and antitubercular activity relationship was further supported by in silico
molecular docking study of the active compounds against enoyl acyl carrier protein reductase (InhA)
enzyme of M. tuberculosis.
Keywords:
Isonicotinohydrazide (INH)
3-Aryl-1H-pyrazole-4-carbaldehyde
Antimycobacterial activity
In vitro cytotoxicity
In silico molecular docking
Ó 2015 Published by Elsevier Ltd.
Tuberculosis (TB) is the most dangerous of various microbial
diseases, which causes ill health and mortality of millions of people
each year. It stands second after HIV-AIDS among the various death
causing diseases. The world health organization (WHO) report esti-
mates that there were 9 million new TB cases and 1.3 million
deaths in the year 2013.1 The WHO has recommended a standard
strategy for the treatment of TB by a program called DOTS (Directly
Observed Treatment, Short-course), which includes six month reg-
imen of four first-line drugs: isoniazid, rifampicin, ethambutol and
pyrazinamide.2 Despite the availability of strong and powerful
antitubercular drugs, tuberculosis is still a leading threat for
human life; the recent emergence of multi drug resistant TB
(MDR-TB) and extensively drug resistant TB (XDR-TB) further
cause serious challenges in TB control. It is also clear from the
WHO data that the mortality rate and spread of the disease by
TB infection is very high in Human Immunodeficiency Virus
(HIV) infected people.3 Thus there is an apparent need for the dis-
covery of fast acting TB drugs which cause fewer side effects and
eliminate the infection in short treatment period.
enhancement of the activity and sometimes may also help in
reducing the toxicity of the molecule. This approach of molecular
modification has become a promising strategy to design and
develop potent antiTB agents. Isoniazid (INH) is one of the most
studied antiTB drugs. It is a potent first-line antiTB drug; a pro-
drug triggered via oxidation that forms an adduct with NAD (+)
to inhibit NADH dependent targets of Mycobacterium tuberculosis
(MTB) bacillus, such as the enoyl-acyl carrier protein reductase
(InhA).4–6 But the major drawback of INH is its failure against the
treatment of MDR-TB, especially among patients infected with
HIV. The recent studies indicate that the incorporation of lipophilic
moieties into the framework of INH can increase permeation of the
drug into the tissues of the mammalian host and into the waxy cell
wall of the bacterium.7–9 The functionalization of the INH is possi-
ble via its reactive hydrazide group which can easily react with car-
bonyl compounds to form hydrazones or undergo cyclization
reactions. LL-3858 (I) (Fig. 1) is one such isoniazid derivative
(developed by Lupin Limited) which shows activity against both
drug sensitive and multidrug resistant TB10,11 and is in the initial
stages of phase II clinical trial for the treatment of tuberculosis.12
Also, a few other hydrazone derivatives of INH (II–IV) showed
promising anti-TB activity.13,14
In recent days, development of new drugs by molecular modifi-
cation of a lead compound or already existing drug with an estab-
lished activity has become an active area of research in medicinal
chemistry. Such a molecular modification can possibly results in
In view of these facts, we envisaged to design new INH hydra-
zone analogs which could possibly mimic the INH–metabolite–
NAD (+) adduct in inhibiting InhA. Accordingly, we have designed
a new class of INH derivatives containing active 3,4-disubstituted
⇑
Corresponding author. Fax: +91 824 2474033.
E-mail
addresses:
udayaravi80@gmail.com,
udayakumar@nitk.ac.in
pyrazole unit; the pyrazole unit as
a replacement for the
(U. Dalimba).
http://dx.doi.org/10.1016/j.bmcl.2015.10.057
0960-894X/Ó 2015 Published by Elsevier Ltd.

N. Nayak et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5540–5545
5541
N
N
H
N
F
F3C
O
N
N
N
N
O
O
N
N
N
NH
Cl
N
Cl
O
O
O
N
N
N
O
II
N
N
N
MIC99: 0.09-0.19 µg/mL
N
O
I
N
LL-3858 (Lupin Ltd)
MIC: 0.25 µg/mL
O
VI
MIC: 0.625 µg/mL
N
CH3
H3C
N
NH
OH
Cl
OMe
V
O
MIC: 3.13 µg/mL
O2N
N
N
N
N
N
F
O
N
H
N
IV
N
NO2
N
N
H
O
MIC: 1.6 µg/mL
O
N
H
III
MIC: 0.2 µg/mL
VII
MIC: 0.35 µg/mL
Target INH analogs
containing pyrazole units
Cl
O
N
N
N
O
N
N
H
Cl
5l
MIC: 0.8 µg/mL
Figure 1. Some representative isoniazid and pyrazole containing potent antiTB analogs. The target isoniazid analog 5l exhibited the highest antiTB activity among the
synthesized compounds.
five-member sugar of NAD (+) in the bioactive adduct. Further, some
literature reports suggested that INH analogs containing pyrazole
unit (V–VII) exhibit promising antitubercular activity.15–17 So, we
emphasized that introduction of the pyrazole unit may improve
the inhibitory potential of the INH hydrazone analogs. In this
direction, we synthesized a new class of INH analogs (5a–r)
containing a pyrazole ring and evaluated their antimycobacterial
activity against Mycobacterium tuberculosis H37Rv (MTB) strain.
Additionally, the compounds were screened against three common
pathogenic bacterial strains as well.
The synthetic route of new isonicotinohydrazide based pyrazole
derivatives (5a–r) is represented in Scheme 1. 3-Aryl-1H-pyrazole-
4-carbaldehydes (3a–b) were synthesized by the Vilsmeier–Haack
reaction of semicarbazones 2a–b, which in turn were synthesized
by the reaction of semicarbazide hydrochloride with substituted
acetophenones (1a–b) in ethanol media.18,19 The hydrazone scaf-
folds (4a–b) were synthesized in good yield by refluxing a mixture
of 3a–b and isonicotinohydrazide in the presence of catalytic
amount of sulfuric acid.20 The target molecules (5a–r) were syn-
thesized by coupling intermediates 4a–b with different aromatic
acids using the typical acid–amine coupling agents, N-(3-dimethy-
laminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC) and
1-hydroxybenzotriazole hydrate (HOBt) in presence of dimethy-
lamino pyridine (DMAP) as the base at room temperature. The
compounds were purified using appropriate methods like recrys-
tallization and column chromatography. The structural details of
the target compounds (4a–b and 5a–r) are presented in Table 1.
All newly synthesized compounds were characterized using 1H
NMR, 13C NMR, and mass spectral analysis followed by elemental
analysis. In the 1H NMR spectrum of 4a a new singlet signal is
appeared at d 11.79 ppm which corresponds to the ANH of pyra-
zole ring whereas the ANH proton of isonicotinohydrazide
appeared at d 13.49 ppm. The signals due to the aromatic protons
also are in agreement with the structure of 4a. The ESI MS spec-
trum displayed a molecular ion peak at (m/z) 326.1 (M+H)+ which
is in agreement with its molecular formula (C16H12ClN5O). The 1H
NMR spectrum of 5a shows the pyrazole NH signal at d 12.12 ppm
and the signal due to the ANH of isonicotinohydrazide was absent.
Also, the number of signals in the aromatic region is in well agree-
ment with its structure. Furthermore, the ESI MS spectrum shows
the molecular ion peak at (m/z) 448.2 (M+H)+, which confirms its
molecular formula (C23H15ClFN5O2). Similarly, the 13C NMR
spectral data also supports the structure of 5a. The spectral and
elemental data of all the target compounds and some representa-
tive spectra are given in the ESI.
All new compounds were screened against M. tuberculosis
H37Rv (MTB) strain using the microplate alamar blue assay
(MABA).22,23 Isoniazid, ethambutol and pyrazinamide were used
as the positive drug standards. The results of the in vitro antimy-
cobacterial screening are given in Table 1. Four compounds namely
4b, 5j, 5k and 5l displayed significant inhibition activity with a
MIC 6 4.9
l
M. Among these, 5l is the most potent molecule with
M which is comparable with the MIC value of stan-
a MIC of 1.7
l
dard drug INH, whereas the inhibition activity of compounds 4b,
5j and 5k is much greater (in terms of MIC value) than that of stan-
dard drug, ethambutol. The 3-(chlorophenyl)-1H-pyrazole scaf-
folds, 4a and 4b, differ in their structure only in terms of the
position of the chloro substituent on the phenyl ring. However,

5542
N. Nayak et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5540–5545
O
R1
R1
O
O
N
NH2
N
H
H
N
a
c
b
N
NH
O
R1
1a-b
N
N
R1
N
H
3a-b
N
H
2a-b
4a-b
d
1a-4a R1 = 4-Cl
1b-4b R1 = 3-Cl
R1
O
N
N
N
O
N
N
H
R2
5a-r
Scheme 1. Synthetic route of target compounds (5a–r). Reagents and conditions: (a) semicarbazide hydrochloride, sodium acetate, ethanol, 80 °C, 8 h; (b) POCl3, DMF, 80 °C,
2 h; (c) isonicotinohydrazide, ethanol, cat. sulfuric acid, 80 °C, 4 h; (d) aromatic acid derivative, EDC, HOBt, DMAP, dichloromethane, room temperature, 16 h.
Table 1
target compounds (5j, 5k and 5l) are the derivatives of scaffold
4b. It is worth mentioning that the inhibition activity of all the tar-
get compounds derived from scaffold 4b, except 5r, is either the
same or higher than that of their respective analogs derived from
scaffold 4a. That is, compounds 5j–q are either equipotent or more
potent than compounds 5a–h, respectively. Among molecules
derived from 4a, compound 5i showed a moderate activity with
Substitution pattern and antitubercular activity of target compounds 4a–b and 5a–r
Compound
R1
R2
LogPa
MIC (
l
g/mL)
MIC (lM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
3-Cl
—
4-F
4-Cl
2-Cl
4-NO2
4-CH3
4-OCH3
H
3,4-Dimethoxy
4-CH2-Cl
4-F
4-Cl
2-Cl
4-NO2
4-CH3
4-OCH3
4.10
4.54
4.42
3.56
4.23
3.92
3.83
3.89
4.44
4.10
4.54
4.42
3.56
4.23
3.92
3.83
3.89
4.44
2.93
2.93
—
50
25
25
12.5
50
50
50
50
6.25
1.6
1.6
0.8
12.5
50
50
50
50
25
111.6
53.8
53.8
26.3
112.6
108.7
116.3
102.1
13.1
3.6
a MIC of 13.1 lM whereas rest of the molecules did not show a
promising activity. The substitution of a halogen (F or Cl) at R2
enhances the inhibition activity of the molecules which is evident
from the observation that all three active target molecules 5j, 5k
and 5l contain a halide substitution at R2. Compound 5l with a 2-
chloro substitution is twofold more potent than 5k which has a
4-chloro substitution at R2. This observation signifies the effect of
the position of the halide substitution on the activity. However
the nature of the halogen (F or Cl) has no effect on the inhibition
activity, both fluoro and chloro substituted derivatives (5j and
5j
5k
5l
3.4
1.7
5m
5n
5o
5p
5q
5r
4a
4b
INH
ETB
PZA
26.3
112.6
108.7
116.3
102.1
52.3
38.4
4.9
H
3,4-Dimethoxy
4-CH2-Cl
5k, respectively) are equipotent (MIC = 3.6 and 3.4 lM, respec-
tively). But this structure–activity trend was not observed in the
case of halogen substituted target molecules (5a–c) derived from
scaffold 4a. The chloro substituted derivative (5b) was more active
than the fluoro substituted one (5a) whereas both 2- and 4-chloro
substituted derivatives (5b and 5c, respectively) showed a similar
—
—
—
—
—
12.5
1.6
0.1
3.13
50
0.7
15.3
406.1
—
—
—
—
a
Obtained from Molsoft.21
activity (MIC = 53.8 lM). The electron releasing groups such as
CH3, AOCH3 and 3,4-dimethoxy at R2 failed to enhance the
inhibition activity of the molecules whereas, molecules 5i and 5r
which contain an electron withdrawing nitro group at R2 showed
moderate activity.
only compound 4b with a 3-Cl substitution showed a significant
activity, whereas compound 4a with a 4-chloro substitution
The results of the antibacterial activity study of all the com-
pounds (5a–r and 4a–b) against Staphylococcus aureus, Escherichia
coli and Pseudomonas aeruginosa strains are presented in Table 2.
displayed a moderate activity with a MIC of 38.4 lM. This result
clearly suggests that the chloro substitution at the meta position
enhances the antitubercular activity (Fig. 2). Further, the active
INH- Active
R1
O
R1
pharmacophore
Anti-TB activity: 3-Cl
N
substitution is favorable
than 4-Cl
N
N
O
Antibacterial activity:
R2
N
both 3-Cl and 4-Cl
Anti-TB and Antibacterial:
N
enhance the activity
Electron withdrawing groups
H
R2
enhance the activity.
Electron donating groups
reduce the activity
Figure 2. The structure–activity relationship in the target compounds.

N. Nayak et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5540–5545
5543
Table 2
Antibacterial activity of target compounds 4a–b and 5a–r
Compound
Zone of inhibition (mm)
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
Conc. in
Control
lg/mL
75
00
50
00
75
00
50
00
75
00
50
00
Ciprofloxacin
26 0.1
17 0.2
24 0.2
18 0.3
19 0.4
20 0.1
12 0.1
11 0.1
10 0.2
16 0.3
16 0.2
23 0.2
15 0.2
17 0.3
12 0.3
10 0.2
13 0.4
10 0.3
17 0.3
19 0.3
23 0.2
21 0.2
14 0.4
21 0.2
14 0.2
17 0.1
17 0.1
10 0.3
09 0.2
08 0.2
13 0.2
13 0.4
18 0.2
13 0.3
14 0.2
10 0.3
07 0.4
11 0.1
08 0.3
14 0.3
16 0.2
18 0.1
32 0.2
19 0.3
24 0.3
19 0.2
20 0.3
14 0.4
09 0.3
—
09 0.2
19 0.4
14 0.3
25 0.3
18 0.1
15 0.1
11 0.3
11 0.1
—
27 0.2
17 0.3
19 0.3
16 0.4
18 0.3
12 0.2
07 0.2
—
06 0.2
15 0.1
12 0.2
21 0.2
14 0.4
13 0.3
08 0.4
09 0.3
—
21 0.2
17 0.1
21 0.1
19 0.1
17 0.2
15 0.1
13 0.4
12 0.3
11 0.2
20 0.2
17 0.2
21 0.3
16 0.1
14 0.1
10 0.2
14 0.1
—
18 0.1
14 0.1
18 0.2
15 0.3
15 0.3
11 0.2
10 0.4
10 0.3
09 0.1
18 0.2
13 0.2
19 0.3
12 0.1
11 0.2
08 0.3
11 0.4
—
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
—
—
—
—
16 0.4
16 0.4
24 0.2
14 0.3
14 0.2
20 0.1
20 0.3
18 0.4
19 0.3
18 0.3
14 0.3
15 0.2
4a
4b
—: bacterial resistant; control: dimethylsulfoxide.
Table 3
Table 4
Cytotoxicity of active antiTB derivatives
Data obtained from the molecular docking studies: docking scores and details of the
amino acid residues of InhA that interacted with the ligands
Compound
Percentage inhibitiona
IC50
(lg/mL)
SIb
Entry name
Docking score
Amino acids that interacted with ligands
5i
5j
5k
5l
4b
12.01 1.0
17.83 2.4
10.51 2.5
14.94 4.4
26.32 3.9
>1000
>1000
>1000
513.33
>1000
>160
>625
>625
641.6
>625
GEQ (ligand)
5k
5j
5l
ꢀ12.51
ꢀ11.61
ꢀ10.93
ꢀ10.58
ꢀ9.63
Phe 149, Tyr 158
Gly 104
Phe 149, Gly 104, Ala 157
Gly 104, Tyr158
Phe 149, Tyr 158
Phe 149, Tyr 158
Ala 157, Tyr 158
5i
a
Percentage inhibition at 62.5
Selectivity index (in vitro) = IC50 against Vero cells/MIC against M. tuberculosis.
lg/mL.
4b (conf-1)
4b (conf-2)
ꢀ8.69
b
ꢀ8.56
The study was carried out using the well plate method24 by
measuring the zone of inhibition and the commercial antibiotic
Ciprofloxacin was taken as the standard. The zone of inhibition
values were calculated in millimeter (mm) scale at two different
concentrations of compounds, 75 and 50 lg/mL. Most of the tested
compound showed good inhibition against Staphylococcus aureus
The in vitro cytotoxicity studies of the active compounds
with MTB MIC 6 6.25 lg/mL were carried out by MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay25
against a normal Vero cell line. The results of the cytotoxicity study
are presented in Table 3. All active antitubercular compounds (5i,
5j, 5k, 5l and 4b) exhibited low toxicity between 10.51% and
and Pseudomonas aeruginosa strains.
Three compounds 5b, 5k and 4b exhibited significant activity
against all the tested strains whereas other nine compounds 5a,
5c, 5d, 5e, 5i, 5j, 5l, 5m and 5r showed moderate activity. It is
worth mentioning that all these active molecules, except 5e, carry
an electron withdrawing substitution at R2. The variation in the
position of chloro substitution at R1 did not affect the antibacterial
activity of the target compounds. However, 3-chloro substituted
intermediate 4b is more active than its 4-chloro analog 4a, which
is quite similar to the activity trend seen in their anti-TB study. It
is interesting to note that the lead derivatives of the series, 5b
and 5k have a 4-chloro substitution at R2 whereas their 2-chloro
analogs (5c and 5l, respectively) showed only moderate activity
which reveals the significant contribution of the 4-chloro
substituent towards the antibacterial activity. Other electron with-
drawing substituents like NO2 group (in 5d and 5m), also enhanced
the antibacterial activity of the compounds. Thus, derivatives with
a chlorophenyl substitution on the pyrazole ring and an electron
withdrawing group at R2 (a 4-chloro group in particular) are
promising antibacterial agents and are best suited for further lead
optimization.
26.32% at a concentration of 65 lg/mL. Further, the high selectivity
index of these derivatives (>160) clearly implies the suitability of
the compounds for drug development as new anti-TB agents.
In order to further rationalize the biological results, molecular
docking studies were performed on five active compounds of the
series (5i, 5j, 5k, 5l and 4b) with the enzyme, InhA (PDB code:
1P44) of Mycobacterium tuberculosis which has been validated as
an effective anti-microbial target. The docking results were drawn
based on docking score, hydrogen bonding, p–H and p–p interac-
tion of the ligand with the protein. The docking score of the ligands
is in the range ꢀ8.56 to ꢀ11.61 (Table 4) which is comparable with
that of the reference ligand (ꢀ12.51). The ligands showed one or
more hydrogen bonding interaction with the amino acid residues
of InhA along with other type of interactions. The docking score
of the most active TB compound of the series, 5l was found to be
ꢀ10.58 with a H-bond interaction between Gly 104 and nitrogen
atom of the pyrazole ring (Fig. 3). The docking score of 5l is slightly
lower than that of the reference ligand (GEQ). But it is interesting
to note that both 5l and the reference ligand showed two
interactions with the target enzyme. Both molecules interacted

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N. Nayak et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5540–5545
Figure 3. Three dimensional diagrams displaying the binding poses and interactions for the compounds 4b, 5j, 5k and 5l in the active site of InhA.
with Tyr158 residue of InhA. Further, compound 5l interacted with
the residue Gly 104, which could be a reason for its significant
inhibition activity. It is important to note that, the other two active
molecules (5j and 5k) also showed interactions with the same
amino acid residue signifying the contribution of this interaction
towards the inhibition activity.
development of new anti-TB agents. The molecular docking study
against InhA domain further revealed the favorable interactions
of the active molecules with amino acid residues of the enzyme.
Acknowledgments
NB and RP thank National Institute of Technology Karnataka,
India for providing the research fellowship. We thank MIT Manipal
and Dr. Reddy’s Institute of Life Sciences, University of Hyderabad
Campus, Hyderabad for NMR and Mass spectrometer facility. The
authors are thankful to Maratha Mandal’s NGH Institute of Dental
Sciences and Research Centre, Belgaum for providing facility for
the biological screening.
The higher inhibition activity (in terms of MIC values) of
3-chloro (R1) derivatives when compared with their 4-chloro
analogs could be rationalized based on the docking results. For
instance, 4-chloro derivative 5i showed lower docking score
(Table 4) than the active 3-chloro derivatives (5j–l). Further, the
nature of the interactions of 5i with the target enzyme is different
from that of compounds 5j–l. In the case of 5i, the nitrogen atom of
the pyridine ring interacts with Try 158 whereas compounds 5j–l
interact with Gly 104 through the nitrogen atom (@NA) of the
pyrazole ring. In addition, the resonance structures (involving the
mesomeric electron release from N1 of pyrazole ring) of 3-chloro
derivatives are more stable than those of 4-chloro derivatives.
One of the resonance structures of 4-chloro derivatives has a neg-
ative charge on the carbon atom bonded to Cl atom which makes
the structure unstable. No such unstable structure is possible with
3-chloro derivatives. This electronic factor may also contribute
towards the observed activity in these molecules.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.10.
057.
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relationship revealed that the 3-chlorophenyl-1H-pyrazole
derivatives of INH which contain an electron withdrawing halide
substitution at R2 are the promising lead molecules for the
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l
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