Design, Synthesis, and Characterization of Some Hybridized Pyrazolone Pharmacophore Analogs against Mycobacterium tuberculosis

Article abstract of DOI:10.1002/ardp.201600019

Twenty-seven hybridized pyrazolone analogs were designed, docked, synthesized in two series and evaluated for their in vitro antimycobacterial properties. In the first series, four Schiff base derivatives, 6b, 7b, 7h, and 7i, show good antitubercular activity with minimum inhibition concentration (MIC) values in the range of 32.56-42.55 μM. In the second series, two compounds, 8b and 8c, possessed significant antitubercular activity with MIC <0.37 and <0.44 μM, respectively; they were even more potent than the standards pyrazinamide (12.99 μM), ciprofloxacin (4.82 μM), and streptomycin (5.36 μM), with a selectivity index of >630. Compounds 8b and 8c showed shikimate kinase inhibition activity at 5.84 and 6.93 μM, respectively. The activity and docking results lead to the conclusion that the compounds without double bond in the imine side chain and hydrophobic clashes at the pyrazolone end are necessary for good accommodation in the binding pocket and for imparting flexibility. All the compounds were also tested for antimicrobial activity (antibacterial and antifungal) and show highly significant activities against all the microorganisms tested. Hybridized pyrazolone analogs were designed, docked, and synthesized in two series. Both compound series were evaluated for their in vitro antimycobacterial properties, showing highly significant activities against all microorganisms tested. No double bond in the imine side chain and hydrophobic clashes at the pyrazolone end were necessary for good accommodation in the binding pocket of shikimate kinase.

Full text of DOI:10.1002/ardp.201600019

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Arch. Pharm. Chem. Life Sci. 2016, 349, 383–397
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Full Paper
Design, Synthesis, and Characterization of Some Hybridized
Pyrazolone Pharmacophore Analogs against Mycobacterium
tuberculosis
1
2
1
Sivakumar Kullampalayam Krishnasamy , Vigneshwaran Namasivayam , Sincy Mathew ,
1
1
3
Ragavendran S. Eakambaram , Ibrahim A. Ibrahim , Adhirajan Natarajan ,
1
and Senthilkumar Palaniappan
1
Department of Pharmaceutical Chemistry, Medicinal Chemistry Research, KMCH College of Pharmacy,
Kovai Estate, Coimbatore, India
PharmaCenter Bonn, Pharmaceutical Chemistry I, Pharmaceutical Institute, Rheinische Friedrich-
2
Wilhelms-University Bonn, Bonn, Germany
Department of Pharmaceutical Biotechnology, KMCH College of Pharmacy, Kovai Estate, Coimbatore,
3
India
Twenty-seven hybridized pyrazolone analogs were designed, docked, synthesized in two series
and evaluated for their in vitro antimycobacterial properties. In the first series, four Schiff base
derivatives, 6b, 7b, 7h, and 7i, show good antitubercular activity with minimum inhibition
concentration (MIC) values in the range of 32.56–42.55 mM. In the second series, two compounds,
8
b and 8c, possessed significant antitubercular activity with MIC <0.37 and <0.44 mM, respectively;
they were even more potent than the standards pyrazinamide (12.99 mM), ciprofloxacin (4.82 mM),
and streptomycin (5.36 mM), with a selectivity index of >630. Compounds 8b and 8c showed
shikimate kinase inhibition activity at 5.84 and 6.93 mM, respectively. The activity and docking
results lead to the conclusion that the compounds without double bond in the imine side chain and
hydrophobic clashes at the pyrazolone end are necessary for good accommodation in the binding
pocket and for imparting flexibility. All the compounds were also tested for antimicrobial activity
(
antibacterial and antifungal) and show highly significant activities against all the microorganisms
tested.
Keywords: Antimycobacterial / Mycobacterium tuberculosis / Pyrazolone / Shikimate kinase inhibitor
Received: January 21, 2016; Revised: March 18, 2016; Accepted: March 18, 2016
DOI 10.1002/ardp.201600019
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
threats around the world [1]. Microbial infection has
exacerbated because of the presence of various complicating
factors such as emergence of multidrug resistance, uncon-
trolled and inappropriate use of antibiotics, human immuno-
deficiency virus (HIV) co-infection, lack of patient compliance,
long-term multiple-regimen chemotherapy, variable efficacy
of vaccines, and newly discovered micro-organisms [2].
Tuberculosis (TB), caused by Mycobacterium tuberculosis
New and re-emerging microbial infections continue to be a
growing global health problem and potential bioterrorism
Correspondence: Dr. Senthilkumar Palaniappan, Department of
Pharmaceutical Chemistry, Medicinal Chemistry Research, KMCH
College of Pharmacy, Kovai Estate, Kalapatti Road, Coimbatore
(
MTB), is declared as a global emergency by World Health
Organization (WHO). Some harbor the bacterium without
symptoms (latent TB), but few develop into active disease [3].
Of the new TB cases reported, 95% occur in developing
6
41 048, India.
E-mail: drsenthilkumarp@gmail.com
Fax: þ91422-2628645
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S. K. Krishnasamy et al.
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countries every year. Currently, among the infected individu-
als, approximately eight million develop active TB, and almost
two million die from this disease [4]. The recommended
standard chemotherapeutic regimen for TB treatment is
prescribed under directly observed treatment short-course
(
DOTS), lasting for minimum 6 months. Poor patient compli-
ance can promote the emergence of drug resistance [5]. New
chemical entities with novel mechanism of actions will possess
potent sterilizing activity that will lead to shortening of the
duration of chemotherapy [6].
Among the different mechanisms targeted for the devel-
opment of novel TB drugs and other chemotherapeutic drugs,
shikimate kinase is one of the potential targets. Shikimate
kinase belongs to the family of nucleoside monophosphate
(
NMP) kinases. The NMP family is an important group of
enzymes which catalyzes reversible phosphoryl transfer from
nucleoside triphosphate to a specific nucleoside mono/
diphosphate, and the product of the reaction is subsequently
phosphorylated [7]. Shikimate kinase catalyzes adenosine
triphosphate (ATP)-dependent phosphorylation of shikimate
to form shikimate-3-phosphate. The shikimate pathway is
present only in bacteria, fungi (in cytoplasm), and in plants, to
synthesize the common precursors of essential aromatic
amino acids (phenylalanine, tyrosine, and tryptophan) and
secondary metabolites (phenyl propanoids and alkaloids) [8].
The absence of the pathway in all other genera has rendered
the enzyme a useful target for the development of new
chemotherapeutic agents.
Figure 1. Designed hybridized common pyrazolone structure.
Results and discussion
There are various approaches to develop a new chemo-
therapeutic agent [9] and to create compound library.
Besides the exploitation of new targets, there is another
approach of merging two or more pharmacophores into a
single molecule. These “merged” pharmacophores may be
addressing the active site effectively and offer the possibility
to overcome drug resistance. To achieve the target com-
pound library, pyrazolone moiety was selected since
pyrazolone derivatives are key structures for the develop-
ment of new chemical class of chemotherapeutic agents.
Keeping in view the antimicrobial potential of pyrazolone
derivatives and as part of our ongoing development of new
class of antitubercular and antimicrobial agents [10, 11],
the pyrazolone was merged with another pharmacophore
having potent antimicrobial properties. Literature showed
that hydrazone (–NHN –– C–) and amide (–CONH–) pharmaco-
Library of hybridized pyrazolone compounds was designed
based on literature [12, 13] and our past research experience
on pyrazolone nucleus [10, 11]. The designed hybridized
pyrazolone compounds were screened against shikimate
kinase [14, 15]. The mechanism of transferring phosphate
group from ATP to shikimate by shikimate kinase is reported
as three-dimensional structure in apo and complex state [16].
The shikimate kinase consists of three domains including
SB (shikimate binding domain), a core domain containing
highly conserved phosphate binding loop (P-loop), and the
“lid”, a highly flexible domain in open and closed conforma-
tion [17]. From the reported structures, the complex with
ADP and shikimate was selected for the docking study
because the conformations of SB domain in both the states
were similar [18]. Among these three domains, the SB
is responsible for large conformational changes and the
analogs have shown inhibition of shikimate pathway [14, 19].
Shikimate is stabilized within the binding site through
H-bond interactions with residues Asp 34, Arg 58, Gly 80,
and Arg 136. Furthermore, residues Pro 11, Ile 45, Phe 49, Phe
57, Glu 61, Gly 79, Gly 81, Pro 118, and Leu 119 contributed
within to form the binding site for shikimate (Fig. 2). Initial
goal of the current study was to discover specific shikimate
kinase inhibitors by in silico virtual screening of designed
compound libraries. A library of designed compounds was
docked onto the binding pocket (SB) of the shikimate kinase.
The lowest free binding energy values of the designed
molecules obtained from the docking studies are given in
phores (compounds I and II; Fig. 1) demonstrated significant
antimicrobial activity against various microorganism at low
concentration levels [12, 13]. Consequently, the combination
of pyrazolone ring containing aromatic-substituted group
2
through –NH–CO–CH a promising
–NH–N –– C linkage is
approach in drug-like molecule design (compound III;
Fig. 1) in the current study. We report herein docking,
synthesis of some pyrazolone derivatives by ecofriendly
(
microwave irradiation) method, which have been found to
possess an interesting profile of antitubercular (TB) and
antimicrobial activity as shikimate kinase inhibitor, with
significant reduction in their cytotoxicity potency.
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Hybridized Pyrazolone against Mycobacterium tuberculosis
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Figure 2. (A) The crystal structure of shikimate on the shikimate kinase along with ADP are shown in stick represenation. The amino
acids within the binding pocket are shown in line representation and carbon atoms are colored in green. (B) Binding pose
comparison of docked and crystal structure of shikimate on the shikimate kinase along with ADP. The crystal structure of shikimate
kinase is represented in ribbon and colored in gray. Carbon atoms of AMP is colored in green, crystal structure of shikimate and
docked pose are colored in yellow and orange, respectively. Oxygen atoms are colored in red, nitrogen atoms in blue, hydrogen in
silver white, and phosphor atoms in orange.
Supporting Information Table S1. The binding energy of
the compounds was found to be in the range from ꢀ6.06
to ꢀ9.49 kcal/mol. However, the binding poses generated
for each compound were individually visualized for their
interactions with the amino acids in the binding pocket. After
analyzing the binding poses and interactions of the com-
pounds, 22 molecules were selected in the first series for
synthesis and biological testing. In addition to the lowest
binding energy and interaction with amino acids at the
pocket, for initial experiments, the ease of synthesis was also
taken into consideration.
in the presence of ethanol and glacial acetic acid (pH 4–5). A
total of 22 compounds (6a–k and 7a–k) were synthesized
using Scheme 1. In Scheme 2, the free chlorine group at third
position of pyrazolone ring of the intermediate compound 4a
was replaced with various aromatic amine by microwave
irradiation in the presence of tetrahydrofuran and potassium
carbonate to yield 8a–e. A total of five compounds (8a–e)
were synthesized using Scheme 2. Microwave-assisted tech-
niques offer several advantages [10], more effective in
perspective of environment, reaction time, high yields, ease
of work-up, and isolation of products. The reaction progress
and purity of the synthesized compounds was monitored by
thin layer chromatography (TLC). The physicochemical prop-
erties of the synthesized compounds are tabulated in Table 1.
Structures of the synthesized compounds (6a–k, 7a–k, and
8a–e) were established on the basis of physicochemical,
Chemistry
The synthetic pathway for the synthesis of the targeted
compounds is illustrated in Schemes 1 and 2. In Scheme 1, the
pyrazolones 3a, 3b were synthesized by cyclizing ethyl-
cyanoacetate (1) with hydrazine hydrate (2a)/phenyl hydra-
zine hydrate (2b) under microwave-assisted condition.
Compounds 3a and 3b were acetylated with chloroacetyl-
chloride in the presence of triethylamine to yield compounds
1
elemental analysis, and spectral data (IR, H NMR, and mass).
Furthermore, the titled compounds were confirmed by mass
spectra (m/z values) and elemental analyses (C, H, N) were
within ꢁ0.4% of theoretical values (Supporting Information
Table S2).
4
a and 4b. The intermediate hydrazide derivatives 5a and 5b
were synthesized by reacting compounds 4a and 4b with
hydrazine hydrate in the presence of magnesium sulfate
in ethanol at 0–5°C. Schiff bases (6a–k and 7a–k) were
synthesized under microwave irradiation of various aromatic
carbonyl compounds with intermediate compounds 5a and 5b
Pharmacology
Antimycobacterial activity and docking
All the 22 compounds in the first series (6a–k and 7a–k) were
tested for in vitro antitubercular activity by microplate alamar
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Scheme 1. The synthesis of selected compounds (6a–k and 7a–k).
blue assay (MABA). All the tested compounds inhibited MTB
with MIC in the range of 32.56–100.32 mM. Among the
compounds, 6b, 7b, 7h, and 7i inhibited MTB with MIC of
show inhibition of MTB which is evident from the MIC values
(Table2),butwerenotmuchpromising.Toimprovetheactivity,
compounds were further explored with the interactions
obtained from docking which revealed that the compounds
4
2.55, 33.80, 32.56, and 34.02 mM, respectively. The compounds
Scheme 2. The synthesis of selected componds (8a–e).
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Table 1. Physicochemical data of title compounds (6a–k, 7a–k, and 8a–e).
Structure
Mol.
weight
M.p.
(°C)
1
2
3
a)
Code
R
R
R
R
Mol. formula
% Yield
R
f
value
6
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
a
b
c
d
e
f
g
h
i
–H
–H
–H
–H
–H
–H
–H
–CH
–CH
–CH
–CH
–H
–H
–H
–H
–H
–H
–H
–CH
–CH
–CH
–CH
–
-C
4-ClC
2,4-diClC
-Furyl
2-OHC
6
H
5
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
12
12
12
10
12
13
14
13
13
13
13
18
18
18
16
18
19
20
19
19
19
19
11
11
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
13
12
11
11
13
15
17
14
14
15
14
17
16
15
15
17
19
21
18
18
18
18
12
11
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
5
5
5
5
5
5
5
5
5
5
6
5
5
5
5
5
5
5
5
5
5
6
4
4
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
2
2
2
3
3
3
4
2
2
3
4
2
2
2
3
3
3
4
2
2
3
4
2
2
259.22
293.72
328.21
249.18
275.22
289.25
319.27
307.74
291.24
289.25
318.23
335.32
369.81
404.31
325.28
351.32
365.34
395.36
383.84
367.34
364.34
393.32
232.21
266.70
225.20
301.31
277.19
75
74
80
88
79
84
71
68
75
73
78
73
77
81
83
76
79
81
77
72
82
85
86
89
90
82
92
182
201
214
160
206
141
148
172
209
201
240
189
190
204
173
213
153
158
185
220
207
232
102
125
96
0.51
0.55
0.58
0.54
0.52
0.57
0.55
0.63
0.67
0.62
0.59
0.65
0.60
0.64
0.66
0.57
0.51
0.61
0.68
0.55
0.58
0.53
0.61
0.54
0.63
0.51
0.59
6
H
4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Cl
Cl
6
H
3
2
6
H
4
4-OCH
3
C
6
H
4
2,4-diOCH
4-ClC
4-FC
2-OHC
4-NO
-C
4-ClC
3
C
6
H
3
3
3
3
3
6
H
4
Cl
F
6 4
H
j
6 4
H
k
a
b
c
d
e
f
g
h
i
2
C
6
H
4
6
H
5
–C
6
6
6
6
6
6
6
6
6
6
6
H
5
5
5
5
5
5
5
5
5
5
5
6
H
4
–C
–C
–C
–C
–C
–C
–C
–C
–C
–C
H
H
H
H
H
H
H
H
H
H
Cl
Cl
2,4-diClC
-Furyl
6
H
3
2
2-OHC
4-OCH
2,4-diOCH
4-ClC
4-FC
2-OHC
6
H
4
3
C
6
H
4
3
C
6
H
3
3
3
3
3
6
H
4
Cl
F
6 4
H
j
6 4
H
k
a
b
c
d
e
2 6 4
4-NO C H
–
–
–
–
–
–
–
–
–
–
Aniline
–
4-Chloroaniline
Piperazine
Phenyl piperazine
Cl
–
9
H
15
N
5
O
2
–
15
H
H
19
N
5
O
O
2
4
143
140
–
4-Nitroaniline
11
11
N
5
a)
Solvent system: EtOAc/hexane (1:1).
are stabilized through hydrogen bonding interactions with the
residues Lys 15, Asp 34, Ala 46, Arg 58, Gly 80, Gly 81, Arg 117,
and Arg 136. Furthermore, residues Gly 9, Pro 11, Ser 16, Asp 32,
Val35, Glu38,Ser44, Ile45,Phe57, Arg58, Glu61,Gly79,Gly80,
Ala 82, Val 116, Arg 117, and Leu 119 contribute within to form
thebindingsitefor the compounds(Figs3 and4). Re-examining
closely the binding poses generated for the compounds at the
active site exposes that the presence of double bond in the
imine side chain and phenyl group at the end could alter the
orientation of the compounds. Probably, the double bond
imparts rigidity to the compound, reducing the flexibility, and
the phenyl group introduces hydrophobic clashes during the
bindingprocessofcompounds. Thismightbethereasonforlow
activity of the compounds and the aryl ring at the side chain
influences alteration into the interaction and not fitting into
the binding pocket properly.
The above examination of the results suggested us to design
second series of compounds without the double bond in the
side chain and the phenyl group at the end, thereby reducing
the rigidity and hydrophobicity to the compound. Subse-
quently, all the designed compounds were docked into the
SB of shikimate kinase. The compounds showed an improve-
ment in the fit onto the active site pocket wherein aryl ring in
the side chain sliding perfectly into the loop as shown in
Fig. 5, in a much better way than the compounds 6a–k
and 7a–k. The compounds 8a–e were stabilized within the
binding site through hydrogen bonding with the residues
Pro 11, Lys 15, Asp 32, Asp 34, Arg 58, and Arg 136.
Furthermore, residues Leu 10, Gly12, Ser 16, Glu 38, Ile 45,
Phe 57, Leu 78, Gly 79, Gly 80, Gly 81, Val 116, Arg 117, and
Leu 119 contribute within to form the binding site for the
compounds.
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Table 2. In vitro antitubercular activity of the title compounds (6a–k, 7a–k, and 8a–e).
MTB MIC
Code
IC50 (mg/mL)
IC50 (mM)
Cytotoxicity CC50 (mM)
Selectivity index (SI)
6
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
a
b
c
d
e
f
g
h
i
25
12.5
25
25
25
25
25
25
25
25
25
25
12.5
25
25
25
25
25
96.44
42.55
76.17
100.32
90.83
86.43
78.30
81.43
85.83
86.43
78.55
74.55
33.80
61.83
76.85
71.16
68.42
63.23
32.56
34.02
68.61
63.56
48.22
<0.37
<0.44
20.74
11.29
12.99
4.82
NT
>665.25
NT
NT
15.63
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
19.33
NT
NT
NT
10.60
10.99
23.04
22.49
11.24
11.31
5.83
633.35
630.75
10.01
19.97
39.08
39.13
20.05
NT
NT
NT
NT
NT
NT
NT
NT
j
k
a
b
c
d
e
f
g
h
i
NT
>653.39
NT
NT
NT
>725.45
>695.36
>750.25
>765.14
>771.54
>719.45
>280.99
>234.34
>277.53
>207.64
>225.47
>507.66
>188.62
>107.47
12.5
12.5
25
j
k
a
b
c
d
e
25
12.5
<0.1
<0.1
6.25
3.13
1.6
1.6
3.12
PYZ
CFN
SM
5.36
YZ, pyrazinamide; CFN, ciprofloxacin; SM, streptomycin; NT, not tested.
Second series compounds (8a–e) were tested for in vitro
antitubercular activity using MABA assay. As predicted, the
compounds were found to possess excellent activity. The
results are given in Table 2. Compounds 8b and 8c possessed
the maximum activity with IC50 values less than 0.1 mg/mL
necessary for the antitubercular activity and for binding at the
active site of the enzyme. The phenyl group of the pyrazolone
ring (7a–k) does not significantly contribute to the activity
as compared with the one without phenyl group (6a–k and
8a–e). In addition, the molecule interaction with the amino
acid residues of Lys 15, Asp 34, Arg 58, and Arg 136 are
required to stabilize into the binding pocket of SB domain.
(
<0.37 and <0.44 mM, respectively). The compounds 8b and 8c
were also found to be 35 and 30 times more potent than
standard drug pyrazinamide (12.99 mM); 13 and 11 times more
potent than ciprofloxacin (4.82 mM); 15 and 12 times more
potent than streptomycin (5.36 mM), respectively. Compounds
Cytotoxicity
Among the 27 newly synthesized compounds, compounds
showing ꢂ70 mM antitubercular activity were selected for
in vitro cytotoxicity activity and the results are reported in
Table 2. All the compounds showed CC50 values ranging from
207.64 to 780.99 mM. All the compounds tested were found to
be safe up to 62.5 mg/mL, suggesting that their antimicrobial
activity was not as a result of cytotoxicity.
8
e and 8d showed moderate activity with IC50 values of
3
.13 mg/mL (11.29 mM) and 6.25 mg/mL (20.74 mM), respec-
tively. Further, shikimate kinase inhibition activity of the
lead compounds 8b and 8c showed inhibition at 1.56 mg/mL
(
5.84 and 6.93 mM, respectively).
The structure–activity relationship (SAR) suggests the
presence of electron rich group like Cl, NH
2
, etc., at the
Selectivity index [20] (SI ¼ CC50/MIC) was calculated
(Table 2). According to Orme [21], new antimicrobial agents
must have a selectivity index equal or higher than 10,
with minimum inhibition concentration (MIC) lower than
terminal position of the molecule, essential for the antitu-
bercular activity. Pyrazolone and amide carbonyl groups in
the chain of the designed “merged pharmacophore” are
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Arch. Pharm. Chem. Life Sci. 2016, 349, 383–397
Hybridized Pyrazolone against Mycobacterium tuberculosis
ARC HH PHARM
Archiv der Pharmazie
Figure 3. The docked pose and the 2D interaction diagram of compound 6b at the SB of the shikimate kinase. The binding pose
representation and other atoms are colored as described in Fig. 2.
6
.25 mg/mL and a low cytotoxicity [21]. SI is used to estimate
drugs show MIC of 12.99, 4.82, and 5.36 mM; and SI of 39.08,
39.13, and 20.05, respectively. Thus, the identified pyrazo-
lone derivatives are very promising newer antitubercular
drug candidates.
the therapeutic window of a drug and to identify drug
candidates for further studies. In the current study, four
pyrazolone derivatives (8b–e) with SI values of 633.35,
6
30.75, 10.01, and 19.97, respectively, display significant
MIC, low cytotoxicity with good SI compared with standard
first line or second line drugs (pyrazinamide, ciprofloxacin,
and streptomycin) commonly used to treat TB. Standard
Antimicrobial studies
Antimicrobial testing is indicated for any organism that
contributes to an infectious disease warranting antimicrobial
Figure 4. The docked pose and the 2D interaction diagram of compound 7k at the SB of the shikimate kinase. The binding pose
representation and other atoms are colored as described in Fig. 2.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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389

Arch. Pharm. Chem. Life Sci. 2016, 349, 383–397
S. K. Krishnasamy et al.
ARC HH PHARM
Archiv der Pharmazie
Figure 5. The docked pose and the 2D interaction diagram of compound 8c at the SB of the shikimate kinase. The binding pose
representation and other atoms are colored as described in Fig. 2.
chemotherapy, if its susceptibility cannot be reliably predicted
from pre-existing antibiograms. The introduction of a variety
of antimicrobials today makes it a necessity to perform
antimicrobial susceptibility test, the result of which can guide
us to the selection of appropriate antimicrobial drug with
unquestionable benefits and least antimicrobial resistance.
All newly synthesized title compounds were screened for
their preliminary antibacterial activity against three Gram-
positive strains: Micrococcus luteus, Staphylococcus aureus,
and Staphylococcus albus; three Gram-negative strains:
Escherichia coli, Klebsiella pneumoniae, and Vibrio cholerae;
and three fungal strains: Candida albicans, Aspergillus
fumigatus, and Aspergillus parasiticus by disc diffusion
method. The compounds, which exhibited zone of inhibition
spectrum of activity when compared to all the synthesized
compounds in the series.
Among the 11 compounds of series 7a–k, five compounds
(7a, 7d, 7g, 7i, and 7j) inhibited A. fumigates at MIC as low as
3.12 mg/mL, four compounds (7b, 7e, 7f, and 7k) showed
significant antifungal activity against A. fumigates at MIC
6.25 mg/mL. Compounds 7b–e and 7g demonstrated signifi-
cant fungal activity against A. parasiticus with MIC
6.25 mg/mL. Two of the eleven compounds (7e and 7g)
exhibited excellent antifungal activity against C. albicans with
MIC as low as 1.56 mg/mL. Against S. albus, K. pneumoniae,
and V. cholerae strains, five compounds (7d–f, 7i, and 7k)
display significant activity with MIC 6.25 mg/mL. Among all
the tested compounds, compound 7e inhibited almost all
the micro-organisms tested.
Compound 8b demonstrated MIC of 1.56 mg/mL against all
the screened Gram-positive and Gram-negative bacteria.
On the other hand, compound 8b demonstrated MIC of
1.56 mg/mL aganist Gram-positive bacteria. Compound 8a
shows significant antifungal activity against A. fumigates, A.
parasiticus, and C. albicans at MIC 6.25 mg/mL. Compound 8e
also shows significant antifungal activity against A. fumigates
and C. albicans with MIC 6.25 mg/mL.
(
ꢃ8 mm) in the primary screening at 50 mg/disc, were
evaluated for MIC at 50–0.1953 mg/mL concentration level
by twofold serial dilution method. The values of antimicrobial
results are presented in Table 3.
Among the 11 compounds in the series 6a–k, compound 6a
(
unsubstituted aryl ring in the side chain) demonstrated
excellent activity against E. coli with MIC of 1.56 mg/mL, also
inhibits S. aureus, K. pneumoniae, V. cholerae, A. fumigatus,
and A. parasiticus with MIC of 3.12 mg/mL concentration and
inhibits M. luteus, S. albus, and C. albicans at 6.25 mg/mL.
Among all the 11 compounds of series, compounds 6a, 6b, and
The SAR of the molecules shows that the presence of phenyl
2
ring at pyrazolone ring (R ) has increased the antimicrobial
6
i possessed MIC of 3.12 mg/mL against A. fumigatus and
activity of the molecule. The compounds (7a–k) with
phenyl ring inhibited almost all the microorganisms tested
as compared with unsubstituted compounds (6a–k). In the
individual series, the compounds with electronegative ele-
ment or molecules possessing electron rich group demon-
strated significant activity. The order of activity for the groups
attached to phenyl of the pyrazolone side chain can be given
A. parasiticus, and when compared with all other compounds
in the same series, showed better activity. Compound 6f
demonstrated significant activity against S. aureus, S. albus,
and V. cholerae with MIC 6.25 mg/mL. Compounds 6b, 6d, and
6
k exhibited MIC of 6.25 mg/mL against K. pneumoniae
and compounds 6g, 6i, and 6k inhibited V. cholerae at MIC
.25 mg/mL. Overall, compound 6a demonstrated broad
6
3
as Cl, F > NH, OCH > OH > unsubstituted.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 383–397
Hybridized Pyrazolone against Mycobacterium tuberculosis
ARC HH PHARM
Archiv der Pharmazie
Table 3. In vitro antimicrobial evaluation against bacteria and fungi for the synthesized compounds (6a–k, 7a–k, and 8a–e).
Minimum inhibition concentration (mg/mL)
Code
M. luteus
S. albus
S. aureus
E. coli
K. pneumoniae
V. cholerae
A. fumigates
A. parasiticus
C. albicans
6
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
a
b
c
d
e
f
g
h
i
6.25
25.0
50.0
12.5
12.5
50.0
25.0
25.0
50.0
50.0
12.5
25.0
12.5
12.5
12.5
12.5
25.0
25.0
25.0
50.0
50.0
25.0
12.5
1.56
1.56
12.5
25.0
6.25
50.0
25.0
25.0
50.0
6.25
25.0
25.0
12.5
12.5
12.5
12.5
12.5
12.5
6.25
6.25
6.25
25.0
6.25
6.25
12.5
6.25
12.5
1.56
1.56
12.5
50.0
3.12
12.5
25.0
50.0
50.0
6.25
12.5
25.0
12.5
12.5
25.0
25.0
12.5
25.0
12.5
6.25
6.25
6.25
25.0
6.25
12.5
25.0
12.5
1.56
1.56
25.0
25.0
1.56
50.0
25.0
50.0
12.5
12.5
12.5
6.25
12.5
12.5
25.0
25.0
12.5
12.5
6.25
12.5
50.0
6.25
6.25
6.25
12.5
6.25
25.0
1.56
3.13
12.5
25.0
3.12
6.25
12.5
6.25
25.0
25.0
12.5
12.5
12.5
12.5
6.25
25.0
12.5
12.5
6.25
6.25
6.25
25.0
12.5
6.25
12.5
6.25
25.0
1.56
3.13
25.0
50.0
3.12
12.5
25.0
25.0
12.5
6.25
6.25
12.5
6.25
12.5
6.25
25.0
12.5
12.5
6.25
6.25
6.25
6.25
25.0
6.25
12.5
25.0
12.5
1.56
3.13
3.12
25.0
3.12
3.12
12.5
25.0
12.5
25.0
25.0
25.0
3.12
25.0
3.12
3.12
6.25
12.5
3.12
6.25
6.25
3.12
3.12
12.5
3.12
6.25
6.25
3.13
3.13
12.5
6.25
3.12
3.12
6.25
25.0
12.5
25.0
6.25
6.25
3.12
25.0
3.25
12.5
6.25
6.25
6.25
6.25
12.5
6.25
12.5
12.5
25.0
12.5
6.25
1.56
3.13
12.5
12.5
6.25
50.0
25.0
50.0
25.0
25.0
50.0
25.0
25.0
12.5
12.5
12.5
6.25
12.5
50.0
1.56
50.0
1.56
6.25
12.5
12.5
12.5
6.25
3.13
3.13
6.25
6.25
j
k
a
b
c
d
e
f
g
h
i
j
k
a
b
c
d
e
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅꢅ
ꢅꢅ
ꢅꢅ
Std
Solvent
0.50
–
0.20
0.03
0.05
2.00
0.02
0.12
0.20
0.12
–
–
–
–
–
–
–
–
ꢅ
ꢅꢅ
Std: Ampicillin (bacteria), fluconazole (fungi); solvent: DMSO; –: no inhibition.
Conclusion
Environment software package (MOE.2013.08). AutoDock 4.2
and AutoDockTools [22, 23] were used for flexible ligand
docking into the protein structure and to add atomic partial
charges. Three-dimensional scoring grids were computed
within a user-specified three dimensional box of 60 ꢄ 60 ꢄ 60
All the designed and synthesized compounds showed
promising antimycobacterial, antibacterial, and antifungal
activities. Compounds 8a–e were accommodating well into
the SB pocket of shikimate kinase and also possessed good
interaction with the amino acid residues at the binding
pocket. Based on our modifications to the structure of the
pyrazolone derivatives (docking interaction studies), thereby
significant improvement in the antitubercular activity and by
the shikimate kinase enzyme inhibition studies, it can be
concluded that the compounds act by the response of the
shikimate kinase enzyme inhibition. Further modifications of
these lead molecules can be a promising candidate for the
treatment of tuberculosis. Against antimicrobials, compounds
̊
grid points with a spacing of 0.375 A centered on the co-
crystallized ligand shikimate. Fifty independent docking runs
for the ligand molecules were run into the binding site of
shikimate kinase using the Lamarckian Genetic Algorithm in
AutoDock 4.2. All the other parameters were set to their
default values. Docking simulations with Autodock 4.2
successfully reproduced the crystallographic pose of shikimate
̊
with an RMSD value of 0.75 A (Fig. 2). On the basis of visual
inspection of their interactions with the enzyme, high-scoring
docking poses were selected as putative binding modes for
the analysis and further designing of molecules.
7
a–k and 8a–e showed better antifungal activity compared
with compounds 6a–k.
Chemistry
Microwave synthesis was carried out using Biotage microwave
initiator. The homogeneity of the compounds was monitored
by ascending thin layer chromatography (TLC) on silica gel-G
(Merck)-coated aluminum plates, visualized by iodine vapor
and UV light. Melting points were determined in open end
capillary tubes on a Buechi 530 m.p. apparatus and were
uncorrected. Infrared (IR) and proton nuclear magnetic
Experimental
Docking studies
The protein-ligand docking studies were performed on the
basis of crystal structure of shikimate kinase in complex with
ADP and shikimate (2IYQ.pdb) [16]. The shikimate and solvent
molecules were removed from the protein and polar
hydrogen atoms were added using Molecular Operating
1
resonance ( H NMR) spectra were recorded for the com-
pounds on JASCO FTIR Report 4100 (KBr) and Bruker Avance
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391

Arch. Pharm. Chem. Life Sci. 2016, 349, 383–397
S. K. Krishnasamy et al.
ARC HH PHARM
Archiv der Pharmazie
1
(
300 MHz) instruments, respectively. Chemical shifts are
H NMR (DMSO-d
able), 6.8–7.6 (5H, m, Ar–H), 4.78 (2H, d, CH
(2H, s, –CH pyrazolone); Anal. (C11 Cl) C, H, N.
6
) d ppm: 10.12 (2H, s, NH
2
, D
2
O exchange-
reported in parts per million (ppm) using tetramethyl silane
(
were confirmed by the addition of D O. C NMR spectra were
2
2
CO), 2.9
TMS) as an internal standard. All exchangeable protons
2
10 3 2
H N O
13
recorded on Bruker AC 200/DPX 400 MHz. The mass spectra
were recorded on JEOL GCMATE instrument. The mass of the
compounds are expressed in m/z values. Elemental analyses
Synthesis of 2-hydrazinyl-N-(5-oxo-4,5-dihydro-1H-
pyrazol-3-yl)acetamide (5a) and 2-hydrazinyl-N-(5-oxo-1-
phenyl-4,5-dihydro-1H-pyrazol-3-yl)acetamide (5b)
The solution of product 4a/4b (0.01 mol) with ethanol (10 mL)
was added to a mixture of hydrazine hydrate (0.1 mol),
magnesium sulfate (0.5 g) and ethanol (10 mL) maintained
at 0°C. The reaction mixture was stirred for 8 h. Ethanol is
then distilled and added to the crushed ice with stirring
and extracted using ethyl acetate. Distilled ethyl acetate to
yield compound 5a/5b.
(
C, H, and N) were undertaken with PerkinElmer model 240C
analyzer and all analyses were consistent with theoretical
values (within ꢁ0.4%) unless indicated.
Please also see the Supporting Information for the InChI
codes of the new compounds.
Synthesis of 5-amino-2,4-dihydro-3H-pyrazol-3-one (3a)
and 5-amino-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (3b)
Ethyl cyanoacetate (1; 0.01 mol) and hydrazine hydrate
2-Hydrazinyl-N-(5-oxo-4,5-dihydro-1H-pyrazol-3-yl)-
(
2a; 0.01 mol)/phenyl hydrazine hydrate (2b; 0.01 mol) was
acetamide (5a)
Yield: 71%; m.p.: 121°C; IR (KBr) cm : 3420, 3325 (NH str),
ꢀ
1
taken in an Erlenmeyer flask and mixed thoroughly. The
mixture was irradiated under microwave for 5 min at 80°C.
The mixture was then poured into ice cold water. The crude
product was filtered, dried, and recrystallized from ethanol.
1
1568 (C–ONH), 1695 (C–O str); H NMR (DMSO-d
11.17 (H, s, NH, D O exchangeable), 9.08 (3H, s, NHNH
O exchangeable), 3.61 (2H, d, COCH ), 2.7 (2H, s, CH
) d ppm:
6
2
2
,
D
2
2
2
1
3
pyrazolone); C NMR (DMSO-d
6
þ
) d ppm: 174.8, 172.1, 156.2,
5
-Amino-2,4-dihydro-3H-pyrazol-3-one (3a)
73.1, 59.6; MS: m/z 171.16 [M] ; Anal. (C
5
H
9
N
5
O
2
) C, H, N.
ꢀ
1
Yield: 82%; m.p.: 85°C; IR (KBr) cm : 3432 (broad, NH), 1708
1
(
C–O ring str); H NMR (DMSO-d
pyrazolone), 9.67 (2H, s, NH , D O exchangeable), 2.8 (2H, s,
CH pyrazolone); Anal. (C O) C, H, N.
6
) d ppm: 10.32 (1H, s, NH–
2-Hydrazinyl-N-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-
3-yl)acetamide (5b)
2
2
ꢀ
1
–
2
3
5
H N
3
Yield: 76%; m.p.: 128°C; IR (KBr) cm : 3420, 3325 (NH str),
112 (Ar C–H str), 1568 (C–ONH), 1690 (C–O str); 1H NMR
(DMSO-d ) d ppm: 11.27 (H, s, NH, D O exchangeable), 9.12
(3H, s, NH–NH , D O exchangeable), 6.6–7.6 (5H, m, Ar–H),
2 2
3.82 (2H, d, COCH ), 2.9 (2H, s, –CH pyrazolone); C NMR
3
5
-Amino-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (3b)
6
2
ꢀ
1
Yield: 79%; m.p.: 92°C; IR (KBr) cm : 3462 (broad, NH), 3116
Ar–H str), 1692 (C–O ring str); 1H NMR (DMSO-d
) d ppm:
O exchangeable), 6.8–7.6 (5H, m, Ar–H),
pyrazolone); Anal. (C O) C, H, N.
2
2
13
(
1
2
6
0.12 (2H, s, NH
.8 (2H, s, –CH
2
, D
2
(DMSO-d
6
) d ppm: 174.3, 173.7, 154.9, 141.1, 130.7, 130.2,
þ
2
H
9 9
N
3
125.9, 121.2, 120.9, 71.1, 53.3; MS: m/z 247.11 [M] ; Anal.
11 13 5 2
(C H N O ) C, H, N.
Synthesis of 2-chloro-N-(5-oxo-4,5-dihydro-1H-pyrazol-3-
yl)acetamide (4a) and 2-chloro-N-(5-oxo-1-phenyl-4,5-
dihydro-1H-pyrazol-3-yl)acetamide (4b)
To a solution of 3a/3b (0.01 mol) in tetrahydrofuran (10 mL),
added triethylamine (0.5 mL). The mixture was stirred
properly and then added dropwise chloroacetylchloride
Synthesis of 2-[2-(substituted)hydrazinyl]-N-(5-oxo-4,5-
dihydro-1H-pyrazol-3-yl)acetamide (6a–k) and 2-[2-
(substituted)hydrazinyl]-N-(5-oxo-1-phenyl-4,5-dihydro-
1H-pyrazol-3-yl)acetamide (7a–k)
To a solution of substituted aldehydes/substituted ketones
(0.01 mol) in ethanol (10 mL), added compound 5a/5b
(0.01 mol). To this added glacial acetic acid (0.2 mL) to
maintain the pH between 4 and 5. The reaction mixture
was irradiated with microwave for 5–7 min at 75°C. The solid
obtained then was filtered and recrystallized from ethanol to
give the compounds 6a–k/7a–k.
(
0.02 mol) and stirred for 5 h. Then poured into crushed ice
with stirring. The solid separated is filtered and dried to
give 4a/4b.
2
-Chloro-N-(5-oxo-4,5-dihydro-1H-pyrazol-3-yl)acetamide
4a)
Yield: 86%; m.p.: 103°C; IR (KBr) cm : 3420 (broad, NH), 1695
C–ONH), 1554 (C–O str), 756 (C–Cl str); 1H NMR (DMSO-d
d ppm: 10.84 (H, s, NH, D O exchangeable), 9.52 (1H, s, NH–
pyrazolone), 3.83 (2H, d, COCH –), 2.6 (2H, s, CH pyrazolone);
Anal. (C Cl) C, H, N.
(
ꢀ
1
(
6
)
2-[2-Benzylidenehydrazinyl]-N-(5-oxo-4,5-dihydro-1H-
2
pyrazol-3-yl)acetamide (6a)
ꢀ
1
IR (KBr) cm : 3520 (broad, NH), 1658 (C–N str), 1567 (C–ONH),
2
2
1
6
H N
3
O
2
1612 (C–O str); H NMR (DMSO-d
6
) d ppm: 10.67 (2H, bs, 2NH,
5
D
2
O exchangeable), 9.12 (1H, s, NH– pyrazolone), 8.5 (s, 1H,
–CH), 6.9–7.5 (5H, m, Ar–H), 3.92 (2H, d, COCH
), 2.7 (2H, s,
) d ppm: 175.3, 173.9,
2
-Chloro-N-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-
acetamide (4b)
Yield: 75%; m.p.: 117°C; IR (KBr) cm : 3435 (broad, NH), 3114
Ar C–H str), 1696 (C –– ONH), 1557 (C–O str), 759 (C–Cl str);
2
1
3
2 6
–CH pyrazolone); C NMR (DMSO-d
ꢀ
1
153.2, 141.2, 132.7, 130.2, 128.9, 127.2, 71.1, 53.3; MS: m/z
þ
(
259.26 [M] ; Anal. (C12
13 5 2
H N O ) C, H, N.
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392

Arch. Pharm. Chem. Life Sci. 2016, 349, 383–397
Hybridized Pyrazolone against Mycobacterium tuberculosis
ARC HH PHARM
Archiv der Pharmazie
2
-[2-(4-Chlorobenzylidene)hydrazinyl]-N-(5-oxo-4,5-
10.61 (2H, bs, 2NH, D
pyrazolone), 8.6 (s, 1H, –CH), 6.7–7.9 (4H, m, Ar–H), 3.9 (2H, d,
COCH ), 3.5 (6H, d, OCH ), 2.62 (2H, s, CH pyrazolone);
C NMR (DMSO-d ) d ppm: 175.3, 173.6, 164.9, 161.8, 155.7,
2
O exchangeable), 9.07 (1H, s, NH
dihydro-1H-pyrazol-3-yl)acetamide (6b)
IR (KBr) cm : 3675 (broad, NH), 1733 (C–O str), 1684 (C–N str),
ꢀ
1
2
3
2
1
13
1
1
521 (NHCO–R), 1089 (Ar C–Cl str); H NMR (DMSO-d
0.52 (2H, bs, 2NH, D
6
) d ppm:
6
2
O exchangeable), 9.15 (1H, s, NH
143.5, 129.9, 108.9, 107.2, 101.9, 71.1, 56.3, 53.3; MS: m/z
þ
pyrazolone), 8.7 (s, 1H, –CH), 6.9–7.9 (4H, m, Ar–H), 3.89 (2H,
319.32 [M] ; Anal. (C14
H
17 5
N O
) C, H, N.
4
13
2 2 6
d, COCH ), 2.9 (3H, s, CH pyrazolone); C NMR (DMSO-d )
d ppm: 175.7, 174.1, 153.6, 141.5, 132.4, 129.8, 128.3, 127.5,
2-{2-[1-(4-Chlorophenyl)ethylidene]hydrazinyl}-N-(5-oxo-
þ
7
2.1, 52.9; MS: m/z 293.71 [M] ; Anal. (C12
H
12
5
N O
2
Cl) C, H, N.
4,5-dihydro-1H-pyrazol-3-yl)acetamide (6h)
ꢀ
1
IR (KBr) cm : 3648 (NH str), 1733 (C–O str), 1684 (C–N str),
1
2
-[2-(2,4-Dichlorobenzylidene)hydrazinyl]-N-(5-oxo-4,5-
1601 (C–C str), 1558 (NHCO), 1022 (Ar C–Cl str); H NMR
dihydro-1H-pyrazol-3-yl)acetamide (6c)
(DMSO-d
6 2
) d ppm: 10.61 (2H, bs, 2NH, D O exchangeable),
ꢀ
1
IR (KBr) cm : 3675 (broad, NH), 2923 (Ar C–H str), 1684 (C–N
9.13 (1H, s, NH pyrazolone), 6.6–7.8 (4H, m, Ar–H), 3.82 (2H, d,
1
13
str); 1539 (NHCO); 1090 (Ar C–Cl str); H NMR (DMSO-d
6
) d
COCH
(DMSO-d
131.9, 129.9, 73.1, 53.1.1, 14.3; MS: m/z 307.74 [M] ; Anal.
Cl) C, H, N.
2
), 2.75 (2H, s, CH
2 3
pyrazolone), 2.2 (3H, s, CH ); C NMR
ppm: 10.50 (2H, bs, 2NH, D
2
O exchangeable), 9.09 (1H, s, NH
6
) d ppm: 175.3, 173.9, 169.2, 155.2, 137.7, 132.6,
þ
pyrazolone), 8.5 (s, 1H, –CH), 6.6–7.8 (3H, m, Ar–H), 3.82 (2H,
13
d, COCH
2
), 2.8 (2H, s, CH
2
pyrazolone); C NMR (DMSO-d
6
) d
14 5 2
(C13H N O
ppm: 175.5, 173.7, 156.2, 143.2, 138.3, 136.1, 132.9, 131.6,
þ
1
32.1.2, 127.1, 71.9, 53.2; MS: m/z 328.15 [M] ; Anal.
2-{2-[1-(4-Fluorophenyl)ethylidene]hydrazinyl}-N-(5-oxo-
(
C
12
H
11
N
5
O
2
Cl
2
) C, H, N.
4,5-dihydro-1H-pyrazol-3-yl)acetamide (6i)
ꢀ
1
IR (KBr) cm : 3648 (NH str), 1733 (C–O str), 1683 (C–N str),
1
2
-[2-[1-(Furan-3-yl)ethylidene]hydrazinyl]-N-(5-oxo-4,5-
1558 (CONH), 1071 (C–F str); H NMR (DMSO-d
6
) d ppm: 10.59
dihydro-1H-pyrazol-3-yl)acetamide (6d)
(2H, bs, 2NH, D
6.4–7.58 (4H, m, Ar–H), 3.89 (2H, d, COCH
pyrazolone), 2.1 (3H, s, CH
173.9, 169.2, 167.5, 155.2, 132.6, 131.9, 129.9, 73.1, 53.1.1,
2
O exchangeable), 9.07 (1H, s, NH pyrazolone),
), 2.72 (2H, s, CH
); C NMR (DMSO-d ) d ppm: 175.3,
ꢀ
1
IR (KBr) cm : 3648 (NH str), 3122 (Ar C–H str), 1653 (C–N str),
2
2
1
13
1
2
1
539 (NHCO str); H NMR (DMSO-d
6
) d ppm: 10.55 (2H, bs,
3
6
NH, D
2
O exchangeable), 9.11 (1H, s, NH pyrazolone), 8.7 (s,
), 2.65
) d ppm: 174.8,
þ
H, –CH), 6.5–7.4 (3H, m, furan), 3.89 (2H, d, COCH
14.3; MS: m/z 291.11 [M] ; Anal. (C13
H N
14 5
O
2
F) C, H, N.
2
13
(
2H, s, CH
2
pyrazolone); C NMR (DMSO-d
6
1
73.8, 156.1, 150.2, 143.7, 135.2, 110.2, 109.1, 72.1, 53.8; MS:
2-{2-[1-(2-Hydroxyphenyl)ethylidene]hydrazinyl}-N-(5-
þ
m/z 249.23 [M] ; Anal. (C10
H
11
5
N O
3
) C, H, N.
oxo-4,5-dihydro-1H-pyrazol-3-yl)acetamide (6j)
ꢀ
1
IR (KBr) cm : 3649 (NH str), 3115 (OH bending), 1733 (C–O
1
2
-[2-(2-Hydroxybenzylidene)hydrazinyl]-N-(5-oxo-4,5-
str), 1684 (C–N str), 1559 (CONH); H NMR (DMSO-d
6
) d ppm:
dihydro-1H-pyrazol-3-yl)acetamide (6e)
10.55 (2H, bs, 2NH, D
pyrazolone), 9.2 (1H, s, Ar–OH), 6.5–7.89 (4H, m, Ar–H), 3.79
(2H, d, COCH ), 2.76 (2H, s, CH pyrazolone), 2.2 (3H, s, CH );
C NMR (DMSO-d ) d ppm: 175.6, 173.5, 169.2, 162.2, 155.7,
2
O exchangeable), 9.6 (1H, s, NH
ꢀ1
IR(KBr)cm : 3648 (NH str), 3132(OH bend), 1653 (C –– N str), 1540
1
(
NHCO str); H NMR (DMSO-d
6
) d ppm: 10.61 (2H, bs, 2NH,
2
2
3
1
3
2
D O exchangeable), 9.13 (1H, s, NH pyrazolone), 8.7 (1H, s,
6
Ar–OH), 8.2 (s, 1H, –– CH), 6.4–7.79 (4H, m, Ar–H), 3.79 (2H, d,
133.2, 131.6, 126.9, 117.2, 115.1, 73.1, 53.3, 15.2; MS: m/z
13
þ
COCH
2
),2.62 (2H, s, CH
2
6
pyrazolone); CNMR (DMSO-d ) dppm:
289.12 [M] ; Anal. (C13
H
15 5
N O
3
) C, H, N.
1
7
75.2, 173.5, 161.4, 155.4, 144.1, 133.2, 131.1, 121.9, 119.2, 117.1,
þ
13 5 3
1.7, 53.9; MS: m/z 275.26 [M] ; Anal. (C12H N O ) C, H, N.
2-{2-[1-(4-Nitrophenyl)ethylidene]hydrazinyl}-N-(5-oxo-
4
,5-dihydro-1H-pyrazol-3-yl)acetamide (6k)
ꢀ1
2
-[2-(4-Methoxybenzylidene)hydrazinyl]-N-(5-oxo-4,5-
IR (KBr) cm : 3648 (NH str), 1653 (C–N str), 1558 (amide str),
1
dihydro-1H-pyrazol-3-yl)acetamide (6f)
1521 (NO str); H NMR (DMSO-d
6
) d ppm: 10.55 (2H, bs, 2NH,
ꢀ
1
IR (KBr) cm : 3649 (NH str), 1684 (C–N str), 1603 (C–C str),
D
O exchangeable), 9.11 (1H, s, NH pyrazolone), 6.8–7.7
(4H, m, Ar–H), 3.89 (2H, d, COCH ), 2.65 (2H, s, CH
); C NMR (DMSO-d ) d ppm:
2
1
1
540 (NHCO), 1258 (C–O–C str); H NMR (DMSO-d
0.59 (2H, bs, 2NH, D
6
) d ppm:
2
2
1
3
1
2
O exchangeable), 9.1 (1H, s, NH
pyrazolone), 2.3 (3H, s, CH
3
6
pyrazolone), 8.8 (s, 1H, –CH), 6.4–7.8 (4H, m, Ar–H), 3.8 (2H,
175.7, 173.5, 169.2, 151.2, 155.7, 141.2, 130.9, 121.9, 73.2, 53.3,
þ
d, COCH ), 3.2 (3H, s, OCH ), 2.62 (2H, s, CH pyrazolone);
2
3
2
14.4; MS: m/z 318.11 [M] ; Anal. (C13
H N
14 6
O
4
) C, H, N.
1
3
C NMR (DMSO-d
30.3, 126.7, 117.2, 71.9, 56.7, 54.3; MS: m/z 289.29 [M] ; Anal.
6
) d ppm: 175.3, 173.9, 163.2, 155.2, 143.3,
þ
1
2-[2-Benzylidenehydrazinyl]-N-(5-oxo-1-phenyl-4,5-
(
C
13
H
15
N
5
O
3
) C, H, N.
dihydro-1H-pyrazol-3-yl)acetamide (7a)
ꢀ1
IR (KBr) cm : 3059 (Ar C–H str), 1693 (C–O str), 1522 (CONH);
1
2
4
-[2-(2,4-Dimethoxybenzylidene)hydrazinyl]-N-(5-oxo-
,5-dihydro-1H-pyrazol-3-yl)acetamide (6g)
H
NMR (DMSO-d
O exchangeable), 8.4 (s, 1H, –CH), 6.9–7.5 (10H, m,
Ar–H), 3.89 (2H, d, COCH ), 2.5 (2H, s, CH pyrazolone);
6
)
d
ppm: 10.69 (2H, bs, 2NH,
D
2
ꢀ
1
IR (KBr) cm : 3649 (NH str), 1683 (C–N str), 1623 (C–C str),
2
2
1
13
1
558 (NHCO), 1269 (C–O–C str); H NMR (DMSO-d
6
) d ppm:
C NMR (DMSO-d
6
) d ppm: 175.3, 173.2, 153.7, 142.2, 140.7,
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
393

Arch. Pharm. Chem. Life Sci. 2016, 349, 383–397
S. K. Krishnasamy et al.
ARC HH PHARM
Archiv der Pharmazie
1
32.9, 130.9, 128.9, 128.2, 127.9, 123.8, 121.3, 70.1, 54.3; MS:
2-[2-(2,4-Dimethoxybenzylidene)hydrazinyl]-N-(5-oxo-1-
þ
m/z 335.14 [M] ; Anal. (C18
H
17
5
N O
2
) C, H, N.
phenyl-4,5-dihydro-1H-pyrazol-3-yl)acetamide (7g)
ꢀ
1
IR (KBr) cm : 3437 (NH str), 1653 (C–N str), 1601 (C–C str),
1
2
4
-[2-(4-Chlorobenzylidene)hydrazinyl]-N-(5-oxo-1-phenyl-
1513 (CONH); H NMR (DMSO-d
6
) d ppm: 10.51 (2H, bs, 2NH,
,5-dihydro-1H-pyrazol-3-yl)acetamide (7b)
D
2
O exchangeable), 8.8 (s, 1H, –CH), 6.4–7.9 (8H, m, Ar–H), 3.9
(2H, d, COCH ), 3.5 (6H, d, OCH ), 2.81 (2H, s, CH pyrazolone);
C NMR (DMSO-d ) d ppm: 174.2, 172.8, 162.8, 161.2, 154.2,
ꢀ
1
IR (KBr) cm : 3536 (NH str), 2942 (Ar C–H str), 1559 (NH–C–O
2
3
2
1
13
str), 1089 (C–Cl str); H NMR (DMSO-d
6
) d ppm: 10.61 (2H, bs,
6
2
NH, D
2
O exchangeable), 8.72 (s, 1H, –CH), 6.8–8.2 (9H, m,
), 2.8 (3H, s, CH pyrazolone);
) d ppm: 175.6, 173.1, 153.9, 143.1, 140.1,
143.2, 140.3, 130.9, 130.1, 129.8, 124.3, 121.8, 121.1, 110.1,
107.1, 102.4, 69.1, 56.4, 56.1, 54.1; MS: m/z 395.16 [M] ; Anal.
(C20H N O ) C, H, N.
21 5 4
þ
Ar–H), 3.76 (2H, d, COCH
2
2
13
C NMR (DMSO-d
6
1
1
34.4, 129.8, 129.5, 129.3, 128.6, 128.4, 128.3, 128.0, 124.5,
22.4, 122.1, 71.1, 53.9; MS: m/z 369.10 [M] ; Anal.
þ
2-{2-[1-(4-Chlorophenyl)ethylidene]hydrazinyl}-N-(5-oxo-
(
C
18
H
16ClN
5
O
2
) C, H, N.
1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)acetamide (7h)
ꢀ
1
IR (KBr) cm : 3385 (NH str), 2924 (Ar C–H str), 1607 (C–C str),
1
2
-[2-(2,4-Dichlorobenzylidene)hydrazinyl]-N-(5-oxo-1-
1091 (Ar C–Cl str); H NMR (DMSO-d
6
) d ppm: 10.65 (2H, bs,
phenyl-4,5-dihydro-1H-pyrazol-3-yl)acetamide (7c)
2NH, D
COCH ), 2.85 (2H, s, CH
6
(DMSO-d ) d ppm: 174.4, 172.9, 168.9, 154.3, 141.2, 136.6,
2
O exchangeable), 6.7–8.2 (9H, m, Ar–H), 3.86 (2H, d,
ꢀ1
1
13
IR (KBr) cm : 3588 (NH str), 1601 (C–O str); H NMR (DMSO-
) d ppm: 10.58 (2H, bs, 2NH, D O exchangeable), 8.54 (s, 1H,
CH), 6.6–8.1 (8H, m, Ar–H), 3.62 (2H, d, COCH ), 2.6 (2H, s, CH
3
pyrazolone), 2.1 (3H, s, CH ); C NMR
2
2
d
6
2
–
133.1, 129.5, 129.4, 129.1, 128.8, 128.5, 128.3, 124.6, 121.4,
–
2
2
13
þ
pyrazolone); C NMR (DMSO-d
6
) d ppm: 175.4, 172.9, 154.1,
121.1, 69.1, 54.9, 14.8; MS: m/z 383.11 [M] ; Anal.
1
1
43.3, 140.2, 134.6, 129.8, 129.5, 129.4, 128.7, 128.3, 128.1,
27.9, 125.2, 122.4, 122.1, 71.1, 53.9; MS: m/z 403.06 [M] ;
(C19H N
18 5
O
2
Cl) C, H, N.
þ
15 5 2 2
Anal. (C18H N O Cl ) C, H, N.
2-{2-[1-(4-Fluorophenyl)ethylidene]hydrazinyl}-N-(5-oxo-
1
-phenyl-4,5-dihydro-1H-pyrazol-3-yl)acetamide (7i)
ꢀ1
2
-{2-[1-(Furan-3-yl)ethylidene]hydrazinyl}-N-(5-oxo-1-
IR (KBr) cm : 3437 (NH str), 1595 (NHCO–R), 1496 (C–C ring
1
phenyl-4,5-dihydro-1H-pyrazol-3-yl)acetamide (7d)
str), 964 (C–F str); H NMR (DMSO-d
6
) d ppm: 10.49 (2H, bs,
ꢀ
1
IR (KBr) cm : 3406 (NH str), 2923 (Ar C–H str), 1582 (NHCO–R),
2NH, D
COCH ), 2.76 (2H, s, CH
6
(DMSO-d ) d ppm: 174.9, 172.5 167.8, 154.9, 141.1, 136.2,
2
O exchangeable), 6.6–8.1 (9H, m, Ar–H), 3.83 (2H, d,
1
13
1
138 (C–O str); H NMR (DMSO-d
O exchangeable), 8.7 (s, 1H, –CH), 6.2–7.6 (8H, m, 3H furan
and 5H Ar), 3.89 (2H, d, COCH ), 2.65 (2H, s, CH pyrazolone);
) d ppm: 175.3, 173.9, 153.2, 148.2, 143.4,
6
) d ppm: 10.55 (2H, bs, 2NH,
2
2
3
pyrazolone), 2.1 (3H, s, CH ); C NMR
D
2
2
2
133.6, 129.8, 129.5, 129.2, 128.8, 128.5, 128.2, 124.5, 121.6,
13
þ
C NMR (DMSO-d
6
121.3, 69.5, 55.9, 14.2; MS: m/z 367.14 [M] ; Anal.
1
6
39.7, 135.2, 129.9, 129.6, 124.3, 120.9, 120.5, 111.4, 110.1,
(C19H N
18 5
O
2
F) C, H, N.
þ
9.5, 54.7; MS: m/z 325.12 [M] ; Anal. (C16
H
15
5
N O
3
) C, H, N.
2-{2-[1-(2-Hydroxyphenyl)ethylidene]hydrazinyl}-N-(5-
2
-[2-(2-Hydroxybenzylidene)hydrazinyl]-N-(5-oxo-1-
oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)acetamide (7j)
ꢀ
1
phenyl-4,5-dihydro-1H-pyrazol-3-yl)acetamide (7e)
IR (KBr) cm : 3796 (NH str), 3392 (OH bend), 1607 (C–C str),
IR (KBr) cm : 3400 (NH str), 2923 (OH bend), 1603 (C–C str);
ꢀ
1
1
6 2
H NMR (DMSO-d ) d ppm: 10.69 (2H, bs, 2NH, D O
1
1
507 (C–N str); H NMR (DMSO-d
6
) d ppm: 10.61 (2H, bs, 2NH,
exchangeable), 9.8 (1H, s, Ar–OH), 6.6–7.8 (4H, m, Ar–H),
3.79 (2H, d, COCH ), 2.76 (2H, s, CH pyrazolone), 2.3 (3H, s,
); C NMR (DMSO-d ) d ppm: 174.2, 172.9, 167.2, 162.1,
D
2
O exchangeable), 8.9 (1H, s, Ar–OH), 8.1 (s, 1H, –CH), 6.3–7.7
9H, m, Ar–H), 3.69 (2H, d, COCH ), 2.72 (2H, s, CH
) d ppm: 174.4, 172.9,
2
2
1
3
(
2
2
CH
3
6
13
pyrazolone);
C NMR (DMSO-d
6
155.1, 141.0, 133.2, 130.8, 129.9, 129.5, 124.2, 121.8, 121.5,
þ
1
1
60.9, 154.3, 143.2, 140.6, 133.8, 129.5, 129.4, 129.2, 124.9,
22.1, 121.9, 121.6, 119.4, 115.1, 69.1, 54.9; MS: m/z 351.13
121.2, 119.5, 116.6, 69.3, 55.6, 14.9; MS: m/z 365.15 [M] ; Anal.
(C19H N
19 5
O
3
) C, H, N.
þ
[
M] ; Anal. (C18
H N
17 5
O
3
) C, H, N.
2
-{2-[1-(4-Nitrophenyl)ethylidene]hydrazinyl}-N-(5-
2
-[2-(4-Methoxybenzylidene)hydrazinyl]-N-(5-oxo-1-
oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)acetamide
phenyl-4,5-dihydro-1H-pyrazol-3-yl)acetamide (7f)
(7k)
ꢀ1
ꢀ1
IR (KBr) cm : 3654 (NH str), 1683 (C–N str), 1567 (NHCO–R),
IR (KBr) cm : 3675 (NH str), 1684 (C–N str), 1558 (NHCO–R str),
1
1
1
2
253 (C–O–C str); H NMR (DMSO-d
6
) d ppm: 10.68 (2H, bs,
1521 (NO str); H NMR (DMSO-d
6
) d ppm: 10.76 (2H, bs, 2NH,
NH, D
2
O exchangeable), 8.6 (s, 1H, –CH), 6.3–8.1 (9H, m,
), 3.1 (3H, s, OCH ), 2.62 (2H, s, CH
pyrazolone); C NMR (DMSO-d ) d ppm: 174.2, 172.6, 162.9,
54.2, 143.1, 140.2, 130.8, 130.5, 129.4, 129.2, 126.0, 123.1,
D
2
O exchangeable), 6.8–8.2 (4H, m, Ar–H), 3.75 (2H, d,
COCH ), 2.65 (2H, s, CH pyrazolone), 2.1 (3H, s, CH );
C NMR (DMSO-d ) d ppm: 174.4, 172.3, 168.4, 155.6,
Ar–H), 3.8 (2H, d, COCH
2
3
2
2
2
3
13
13
6
6
1
1
151.1, 141.0, 140.2, 130.4, 129.9, 129.6, 129.0, 124.5, 121.6,
121.1, 120.8, 120.7, 69.5, 55.2, 14.6; MS: m/z 394.14 [M] ; Anal.
(C19H N O ) C, H, N.
18 6 4
þ
21.9, 121.6, 115.4, 115.1, 69.1, 56.4, 54.1; MS: m/z 365.15
þ
[
M] ; Anal. (C19H N
19 5
O
3
) C, H, N.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
394

Arch. Pharm. Chem. Life Sci. 2016, 349, 383–397
Hybridized Pyrazolone against Mycobacterium tuberculosis
ARC HH PHARM
Archiv der Pharmazie
þ
Synthesis of 2-[(substituted)]-N-(5-oxo-4,5-dihydro-1H-
pyrazol-3-yl)acetamide (8a–e)
53.9, 53.6, 50.7, 50.2; MS: m/z 301.34 [M] ; Anal. (C15
C, H, N.
H N
19 5
O
2
)
To a solution of compound 4a (0.01 mol) in tetrahydrofuran
(
10 mL), various substituted aromatic amines (0.01 mol)
2-(4-Nitrophenylamino)-N-(4,5-dihydro-5-oxo-1H-pyrazol-
and potassium carbonate (0.1 g) are added. The reaction
mixture was irradiated with microwave at 80°C for 2–4 min.
The resultant mixture was poured into cold water with
stirring. The solid separated was filtered, washed with water
3-yl)acetamide (8e)
IR (KBr) cm : 3339 (pyrazolone N–H str), 3026 (Ar C–H str),
ꢀ
1
1689 (C–O str), 1620 (C–N str), 1593 (Ar C–C str), 1550 (Ar N–O
1
str); H NMR (DMSO-d
6
) d ppm: 11.12 (1H, s, NH pyrazolone),
O exchangeable), 9.6 (1H, s, Ar–NH,
(
3 ꢄ 20 mL), and dried. The crude compound was recrystallized
10.3 (1H, s, NHCO, D
D O exchangeable), 6.5–7.9 (4H, m, Ar–H), 3.72 (2H, d,
2
COCH ), 2.58 (2H, s, CH pyrazolone); C NMR (DMSO-d )
2 2 6
2
using ethanol to give 8a–e.
1
3
N-(4,5-Dihydro-5-oxo-1H-pyrazol-3-yl)-2-(phenylamino)-
d ppm: 174.4, 172.3, 155.6, 154.1, 138.0, 122.9, 122.4, 116.2,
115.8, 113.9, 113.5, 73.5, 56.2; MS: m/z 277.24 [M] ; Anal.
(C11H N O ) C, H, N.
11 5 4
þ
acetamide (8a)
ꢀ
1
IR (KBr) cm : 3337 (pyrazolone N–H str), 3029 (Ar C–H str),
1
(
694 (C–O str), 1618 (C–N str), 1588 (Ar C–C ring str), 1520
1
–NHCOCH2–); H NMR (DMSO-d
6
) d ppm: 11.12 (1H, s, NH
O exchangeable), 9.2 (1H, s,
Pharmacology
pyrazolone), 10.4 (1H, s, NHCO, D
2
Antimycobacterial activity: Microplate Alamar Blue assay
(MABA)
The antimycobacterial activity of compounds was assessed
against MTB using MABA [24]. This methodology is non-toxic,
uses a thermally stable reagent and shows good correlation
with proportional and BACTEC radiometric method. Briefly,
Ar–NH, D
2
O exchangeable), 6.8–7.5 (5H, m, Ar–H), 3.75 (2H, d,
13
COCH ), 2.65 (2H, s, CH
2
2
pyrazolone); C NMR (DMSO-d
6
) d
ppm: 174.4, 172.3, 156.1, 148.1, 131.4, 131.2, 118.8, 114.5,
þ
1
14.2, 73.8, 55.2; MS: m/z 232.24 [M] ; Anal. (C11
H N
12 4
O
2
)
C, H, N.
2
00 mL of sterile-deionized water was added to all outer
2
-(4-Chlorophenylamino)-N-(4,5-dihydro-5-oxo-1H-
perimeter wells of sterile 96 well plate to minimize evapora-
tion of medium in the test wells during incubation. The 96
well plate received 100 mL of the Middlebrook 7H9 broth
(HiMedia, Mumbai) and serial dilution of compounds. The
final drug concentrations tested were 100–0.2 mg/mL. A
pyrazol-3-yl)acetamide (8b)
IR (KBr) cm : 3332 (pyrazolone N–H str), 3022 (Ar C–H str),
ꢀ
1
1
691 (C–O str), 1615 (C–N str), 1586 (Ar C–C str), 889 (Ar C–Cl
1
str); H NMR (DMSO-d
6
) d ppm: 11.16 (1H, s, NH pyrazolone),
O exchangeable), 9.3 (1H, s, Ar–NH,
O exchangeable), 6.9–7.8 (4H, m, Ar–H), 3.68 (2H, d,
5
1
0.2 (1H, s, NHCO, D
2
100 mL of M. tuberculosis inoculum (2 ꢄ 10 cfu/mL) was
D
2
added to the wells. Plates were covered and sealed with
parafilm and incubated at 37°C for 5 days. Then 50 mL of
freshly prepared 1:1 mixture of Alamar Blue reagent and 10%
Tween 80 was added to the plate and incubated for 24 h.
A blue color in the well was interpreted as no bacterial
growth, and pink color was scored as growth. The MIC was
the lowest drug concentration which prevented the color
change from blue to pink. In addition, a control and a blank,
excluding the test compounds, were also performed with
and without the organisms, respectively. The MIC is defined as
the minimum concentration of compound required to give
complete inhibition of bacterial growth.
13
2 2 6
COCH ), 2.62 (2H, s, CH pyrazolone); C NMR (DMSO-d )
d ppm: 174.3, 172.5, 155.9, 146.5, 130.6, 130.1, 125.1, 114.8,
þ
1
14.5, 73.5, 55.6; MS: m/z 266.68 [M] ; Anal. (C11
11
H N
4
O
2
Cl)
C, H, N.
N-(4,5-Dihydro-5-oxo-1H-pyrazol-3-yl)-2-(piperazin-1-yl)-
acetamide (8c)
IR (KBr) cm : 3339 (pyrazolone N–H str), 3021 (Ar C–H str),
ꢀ
1
1
1
(
689 (C–O str), 1612 (C–N str), 1574 (Ar C–C str); H NMR
DMSO-d ) d ppm: 11.16 (1H, s, NH pyrazolone), 10.9 (1H, s,
O exchangeable), 3.72 (2H, d, COCH ), 2.96 (4H, t,
pyrazolone), 2.34 (2H, t, CH
6
NHCO, D
CH
2
2
2
piperazine), 2.68 (2H, s, CH
piperazine); C NMR (DMSO-d
2
2
13
piperazine), 2.12 (2H, t, CH
d ppm: 174.5, 172.3, 155.1, 73.5, 57.3, 55.8, 55.3, 46.1, 45.9; MS:
m/z 225.25 [M] ; Anal. (C
2
6
)
Antimicrobial activity: Determination of zone of inhibition
8
A standard inoculum (1.5 ꢄ 10 cfu/mL 0.5 OD McFarland
þ
9
H N
15 5
O
2
) C, H, N.
standards) was introduced onto the surface of sterile agar
plates and a sterile cotton swab was used for even distribution
of the inoculum. The discs measuring 6.25 mm in diameter
were prepared from Whatman filter paper no. 1 and sterilized
by dry heat at 140°C for 1 h. The sterile discs previously soaked
in a known concentration (50 mg/10 mL) of the test compounds
were placed on top of the nutrient agar medium. The plates
were kept for diffusion at 4°C for 15 min. Then the plates were
inverted and incubated for 24 h at 37 ꢁ 1°C for bacteria and
72–96 h at 27 ꢁ 1°C for fungi. After the incubation zone of
inhibition was measured. The media used was nutrient agar
medium and Sabouraud dextrose medium for antibacterial
N-(4,5-Dihydro-5-oxo-1H-pyrazol-3-yl)-2-(4-
phenylpiperazin-1-yl)acetamide (8d)
IR (KBr) cm : 3334 (pyrazolone N–H str), 3026 (Ar C–H str),
ꢀ
1
1
1
(
(
691 (C–O str), 1616 (C–N str), 1596 (Ar C–C str); H NMR
DMSO-d ) d ppm: 10.84 (1H, s, NH pyrazolone), 10.2
1H, s, NHCO, D O exchangeable), 6.5–7.8 (5H, m, Ar–H),
.78 (2H, d, COCH
6
2
3
2
), 3.36 (4H, t, CH piperazine), 2.68
2
pyrazolone), 2.41 (2H, t, CH piperazine), 2.25
piperazine); C NMR (DMSO-d ) d ppm: 174.9,
6
2
(
(
2H, s, CH
2H, t, CH
2
13
2
1
73.3, 156.4, 150.6, 131.9, 131.1, 119.2, 115.4, 115.2, 73.2, 59.1,
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Arch. Pharm. Chem. Life Sci. 2016, 349, 383–397
S. K. Krishnasamy et al.
ARC HH PHARM
Archiv der Pharmazie
and antifungal activities, respectively. Ampicillin (5 mg/disc)
and fluconazole (5 mg/disc) were used as standard drugs for
antibacterial and antifungal activities, respectively. Triplicates
were maintained for all tested strains. Activity was deter-
mined by measuring the diameter (in mm) of the zone
showing complete inhibition [25].
Negative control experiments were performed in a similar
manner, adding DMSO instead of test compounds. The
concentration of DMSO in the functional assay was kept
lower than 2% (v/v), a concentration that was found not to
influence the enzymatic activity of mycobacterial shikimate
kinase (MtSK). In order to ensure reproducibility, all func-
tional assays were performed in duplicate and analyzed twice.
The inhibition curves were plotted and the IC50 value of each
compound was calculated by fitting the data to a sigmoidal
dose–response curve.
Antimicrobial activity: Determination of minimum
inhibitory concentration
The minimum inhibitory concentration (MIC) in mg/mL of the
title compounds was carried out by twofold serial dilution
method. The test compounds were dissolved in dimethyl
sulfoxide (DMSO) to obtain 0.5 mg/mL stock solution. Seeded
broth (containing microbial spores) was prepared in nutrient
broth (NB) from 24 h old bacterial cultures on nutrient agar at
Cytotoxicity
Some compounds were further examined for toxicity (CC50) in
mammalian Vero cell line till 62.5 mg/mL concentration. After
72 h of exposure, viability was assessed on the basis of cellular
conversion of MTT into a formazan product. MTT is a yellow
water soluble tetrazolium salt. A mitochondrial enzyme in
living cells, succinate dehydrogenase, cleaves the tetrazolium
ring, converting MTT to an insoluble purple formazan.
Therefore, the amount of formazan produced is directly
proportional to the number of viable cells [27, 28]. The % cell
inhibition was determined using the following formula:
3
7 ꢁ 1°C while fungal spores from 1 to 7 days old Sabouraud
agar slant cultures were suspended in Sabouraud dextrose
broth (SDB). The colony forming units (cfu) of the seeded
broth was determined by plating technique and adjusted in
4
5
the range of 10 –10 cfu/mL. The final inoculums size was
5
2
1
0 cfu/mL for antibacterial assay and 1.1–1.5 ꢄ 10 spores/mL
for antifungal assay. Testing was performed at pH 7.4 ꢁ 0.2 for
bacteria and at a pH 5.6 for fungi. Exactly 0.4 mL of the
solution of test compound was added to 1.6 mL of seeded
broth to form the first dilution. One milliliter of this was
diluted with a further 1 mL of seeded broth to give the
second dilution and so on till eight of such dilutions are
obtained. A set of assay tubes containing only seeded broth
was kept as control. The tubes were incubated in BOD
incubators at 37 ꢁ 1°C for bacteria and 28 ꢁ 1°C for fungi.
The MICs were recorded through visual observations after
%
Cell inhibition ¼ 100 ꢀ ½AbsðsampleÞ=AbsðcontrolÞ ꢄ 100ꢆ
The authors are thankful to Dr. Thavamani D. Palaniswami,
Managing Trustee, Kovai Medical Research and Education
Trust, Coimbatore. The authors are grateful to Indian Institute
of Science, Bangalore and Indian Institute of Technology,
Madras for providing NMR and mass spectral data.
2
4 h (for bacteria) and 72–96 h (for fungi) of incubation.
The authors have declared no conflicts of interest.
Ampicillin was used as standard for bacterial studies and
fluconazole was used as standard for fungal studies. The
lowest concentration at which there was no visible growth
was taken as MIC [25, 26].
References
[
1] T. D. Chugh, J. Biosci. 2008, 33, 549–555.
Shikimate kinase enzyme inhibition studies
[2] D. M. Morens, G. K. Folkers, A. S. Fauci, Nature 2004, 430,
242–249.
[3] D. Sriram, P. Yogeeswari, J. T. Patrisha, P. Senthilkumar,
N. Kurre, Y. R. Prasad, Pharmacologyonline 2011, 1,
185–195.
The shikimate kinase enzyme inhibition studies were per-
formed using the method described by Simithy et al. [14], with
a slight modification. Test compounds at different concen-
trations viz., 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20,
0
.10 mg/mL, and 15 nM of shikimate kinase were pre-
incubated for 15 min in a micro-centrifuge tube containing
55 mL of assay buffer (100 mM Tris-HCl pH ¼ 7.5, 50 mM KCl,
and 5 mM MgCl ) at 25°C. The reaction was initiated by the
[4] P. Senthilkumar, M. Dinakaran, Y. Chandraseakaran,
P. Yogeeswari, D. Sriram, Arch. Der Pharmazie 2009, 342,
100–112.
[5] T. G. Benedek, J. Hist. Med. Allied Sci. 2004, 59, 50–89.
[6] P. Senthilkumar, M. Dinakaran, P. Yogeeswari, A. China,
V. Nagaraja, D. Sriram, Biomed. Pharmacother. 2009, 63,
27–35.
[7] A. R. Leonardo, A. B. Luiz, S. S. Diogenes, Mostra de
Pesquisa da P oꢀ s-Gradua cS a~ o (Master thesis), Pontif ꢀı cia
Universidade Cat oꢀ lica do Rio Grande do Sul, 2009,
4
2
addition of an aqueous solution of shikimic acid and ATP (2
and 0.2 mM, final concentrations, respectively) and quenched
after 5 min by the addition of 2 mL of 100% formic acid.
The total volume of the reaction mixture was 500 mL. The
presence of shikimate-3-phosphate after incubation indicates
enzyme activity. The shikimate-3-phosphate in the final
solution was determined by using HPLC (Supporting Informa-
tion S1.1). Inhibitory concentration is the conc. at which the
compounds inhibit the enzyme and the absence or decrease in
the amount of shikimate-3-phosphate in the final solution.
1147–1149.
[8] I. Gientka, Pol. J. Food Nutr. Sci. 2009, 59, 113–118.
[9] D. Sriram, P. Yogeeswari, R. V. Devakaram. Bioorg. Med.
Chem. 2006, 14, 3113–3118.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
396

Arch. Pharm. Chem. Life Sci. 2016, 349, 383–397
Hybridized Pyrazolone against Mycobacterium tuberculosis
ARC HH PHARM
Archiv der Pharmazie
[
[
[
[
[
[
[
10] K. K. Sivakumar, A. Rajasekaran, P. Senthilkumar, P. W.
Prasad. Bioorg. Med. Chem. Lett. 2014, 24, 2940–2944.
11] K. K. Sivakumar, A. Rajasekaran. J. Pharm. Bio. All. Sci.
C. Gonz aꢀ lez-Bello, J. Am. Chem. Soc. 2013, 135(33),
12366–12376.
[20] F. R. Pavan, P. I. da S Maia, S. R. Leite, V. M. Deflon,
A. A. Batista, D. N. Sato, S. G. Franzblau, C. Q. Leite, Eur. J.
Med. Chem. 2010, 45, 1898–1905.
[21] I. Orme, Antimicrob. Agents Chemother. 2001, 45, 1943–
1946.
[22] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,
R. K. Belew, D. S. Goodsell, A. J. Olson, J. Comput. Chem.
2009, 30, 2785–2791.
[23] M. F. Sanner, J. Mol. Graph. Mod. 1999, 17, 57–61.
[24] S. G. Franzblau, R. S. Witzig, J. C. McLaughlin, P. Torres,
G. Madico, A. Hernandez, M. T. Degnan, M. B. Cook,
V. K. Quenzer, R. M. Ferguson, R. H. Gilman, J. Clin.
Microbiol. 1998, 36, 362–366.
2013, 5, 126–135.
12] E. M. Bavin, D. J. Drain, M. Seiler, D. E. Seymour. J. Pharm.
Pharmacol. 1952, 4, 844–855.
13] P. P. Sah, S. A. Peoples. J. Am. Pharm. Assoc. Am. Pharm.
Assoc. 1954, 43, 513–524.
14] J. Simithy, N. Reeve, J. V. Hobrath, R. C. Reynolds,
A. I. Calder oꢀ n. Tuberculosis 2014, 94(2), 152–158.
15] B. S. Bandodkar, S. Schmitt, Patent: PCT/GB2006/003042,
WO/2007/020426 A1.
16] M. D. Hartmann, G. P. Bourenkov, A. Oberschall,
N. Strizhov, H. D. Bartunik, J. Mol. Biol. 2006, 364,
411–423.
[
17] J. H. Pereira, J. S. De-oliveira, F. Canduri, M. V. Dias,
M. S. Palma, L. A. Basso, D. S. Santos, W. F. Jr. de Azevedo,
Acta Crystallogr D Biol Crystallogr. 2004, 60, 2310–
[25] K. K. Sivakumar, A. Rajasekaran, K. Ramalingeswararao,
B. Narasimhan, Indian J. Pharm. Sci. 2013, 75(4), 463–475.
[26] J. M. Andrews, J. Antimicrob. Chemother. 2001, 48, 5–16.
[27] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull,
D. Vistica, C. Hose, J. Langley, P. Cronise, A. Vaigro-Wolff,
M. Gray-Goodrich, H. Campbell, J. Mayo, M. Boyd, J. Natl.
Cancer Inst. 1991, 83, 757–766.
2319.
[
[
18] S. Gordon, J. Simithy, D. C. Goodwin, A. I. Calder oꢀ n,
Perspect Med. Chem. 2015, 7, 9–20.
19] B. Blanco, V. Prado, E. Lence, J. M. Otero, C. Garcia-Doval,
M. J. van Raaij, A. L. Llamas-Saiz, H. Lamb, A. R. Hawkins,
[28] T. Mosmann. J. Immunol. Methods 1983, 65, 55–63.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
397