Design, synthesis, in silico and biological evaluation of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazine carboxamides

Article abstract of DOI:10.1007/s11030-016-9714-7

A series of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazinecarboxamide derivatives has been successfully designed and synthesized to evaluate their potential as carbonic anhydrase (CA) inhibitors. The inhibitory potential of synthesized com

Full text of DOI:10.1007/s11030-016-9714-7

Mol Divers (2017) 21:163–174
DOI 10.1007/s11030-016-9714-7
ORIGINAL ARTICLE
Design, synthesis, in silico and biological evaluation of novel
2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazine
carboxamides
Shikha Kumari1 · Chandra Bhushan Mishra1 · Danish Idrees2 ·
Amresh Prakash2 · Rajesh Yadav1 · Md. Imtaiyaz Hassan2 · Manisha Tiwari1
Received: 17 June 2016 / Accepted: 16 December 2016 / Published online: 30 December 2016
© Springer International Publishing Switzerland 2016
Abstract A series of novel 2-(4-(4-substituted piperazin-1-
yl)benzylidene)hydrazinecarboxamide derivatives has been
successfully designed and synthesized to evaluate their
potentialascarbonicanhydrase(CA)inhibitors. Theinhibitory
potential of synthesized compounds against human CAI and
CAII was evaluated. Compounds 3a–n exhibited IC50 values
between 1.89−415.1 μM against CAI and 0.62−66.9 μM
against CAII. Compound 3g was the most active inhibitor,
with an IC50 value of 0.62 μM against CAII. Molecular
docking studies of compound 3g with CAII showed this com-
pound fits nicely in the active site of CAII and it interacts
with the zinc ion (Zn2+) along with three histidine residues
in the active site. Molecular dynamics simulation studies of
compound 3g complexed with CAII also showed essential
interactions which were maintained up to 40ns of simula-
tion. In vivo sub-acute toxicity study using 3g (300mg/kg)
was found non-toxic in adult Wistar rats.
Keywords CAII inhibitors · Docking · Drug design and
discovery · Piperazine-hydrazine carboxamide derivatives ·
MD simulation
Introduction
Carbonic anhydrases (CAs) constitute a large group of metal-
loenzymes expressed in higher vertebrates including humans
[1,2]. CAs catalyze the reversible hydration reaction of CO2
and thus regulate many physiological functions such as pH
and CO2 homeostasis, respiration, bone resorption, and elec-
trolyte secretion in various tissues/organs [3]. CA isozymes
have been established as an important therapeutic target for
the treatment of a wide range of disorders, such as glaucoma,
cancer, obesity, epilepsy, edema and osteoporosis [4,5].
CAs are classified into mitochondrial, membrane bound
and cytosolic isoforms. CAI and CAII are two major CA
isozymes and are present at high concentrations in the cytosol
of erythrocytes [6]. CAII is the most active isozyme and
is found in the eye, inner ear, kidney and central nervous
system (CNS). The catalytic domain of human CAs pos-
sesses a common motif along with a zinc ion (Zn2+) which
is crucial for catalysis. Literature reports show that sul-
fonamides, sulfamates, sulfamides, phenols and coumarins
are important classes of pharmacophores that can inhibit
CAs isoforms efficiently [7–9]. Acetazolamide (AZM; 1)
is a potent CA inhibitor that inhibits CAs with an affin-
ity in the nanomolar range [10]. Moreover, compound 2, a
coumarin analog, is also a potent CAI and CAII inhibitor
with nanomolar range affinity [11]. Recently, other classes of
pharmacophores such as 5,10-dihydroindeno[1,2-b]indoles
[12] (compound 3), thiourea [13] (compound 4) and 1,4-
bis(indolin-1-ylmethyl)benzenes (compound 5) also showed
promising activity toward CAs (Fig. 1) [14].
Shikha Kumari, Chandra Bhushan Mishra, Danish Idrees and Amresh
Prakash have contributed equally.
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-016-9714-7) contains supplementary
material, which is available to authorized users.
Md. Imtaiyaz Hassan
mihassan@jmi.ac.in
B
Manisha Tiwari
mtiwari07@gmail.com
B
1
Bio-Organic Chemistry Laboratory, Dr. B.R. Ambedkar
Centre for Biomedical Research, University of Delhi, North
Campus, New Delhi, Delhi 110007, India
2
Centre for Interdisciplinary Research in Basic Sciences,
Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
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Fig. 1 Structure of reported
CA inhibitors
F
OH
O
N
N
O
S
O
HN
NH2
S
N
H
O
O
O
3
1
2
S
O
H
N
N
H
N
H
N
N
O
OH
4
5
In past decades, various novel and potent CAs inhibitors
have been discovered, but selective CA isozyme inhibition
remains a challenging task in drug design and discovery [15–
17]. Piperazine is an important heterocyclic building block
which has been extensively used to develop various types of
biologically active agents, including CAs inhibitors [18–21].
Considering the broad biological activity of the piperazine
scaffold, we aimed to design and synthesize a novel semicar-
bazide group coupled to a piperazine pharmacophore to study
its CAI and CAII inhibitory potential. CA inhibition of the
synthesized compounds was performed on human erythro-
cyte CAI and CAII isozymes followed by molecular docking
studies to understand the binding interactions of these com-
pounds at the active site of CAII. A molecular dynamics
(MD) simulation study of the most active compound 3g was
also performed to explore its detailed binding paradigm at the
CAII active site. Additionally, the sub-acute toxicity study of
compound 3g was also assessed in rats by oral administration
for 14days.
In vitro carbonic anhydrase inhibitory activity
In vitro inhibition studies of compounds 3a–n against CAI
and CAII were conducted by measuring the esterase activity
of both enzymes using nitrophenol acetate (4-NPA) as a sub-
strate (Table 1). The observed structure activity relationship
(SAR) shows that phenyl-substituted piperazine derivative
(3a) showedhighinhibitiontowardCAII (IC50 = 27.70 μM)
compared to CAI (IC50 = 217.70 μM). A similar inhibitory
trend was also observed with other electron donating sub-
stituted phenyl analogs (compounds 3b and 3c) as well
as halogen-substituted analogs (compounds 3d-3f). How-
ever, the electron-deficient p−nitrophenyl derivative (3g)
showed more potent CA inhibition activity against CAII
(IC50 = 0.62 μM) and CAI (IC50 = 1.89 μM).
In heteroaryl subseries, pyrimidine derivative (3i) dis-
played better inhibition toward CAI (IC50 = 83.85 μM) and
CAII (IC50 = 13.88 μM) as compared to pyridine derivative
(3h). Next, the introduction of flexible moiety such as ben-
zyl (compound 3j) as well as diphenyl (compound 3l) was
unable to produce potent inhibitory activity against CAI and
CAII. Interestingly, the benzoyl moiety (3k) on this phar-
macophore showed encouraging inhibitory activity against
CAII isoform with an IC50 value of 5.20 μM. Overall, SAR
results clearly indicate that compounds which contains polar
moieties (compound 3g and 3k) at the piperazine ring are
well suitable to generate considerable inhibitory activity for
both CAI and CAII isoforms. The obtained SAR analysis can
also be well correlated with the standard drug acetazolamide,
which also contains two polar ends (one acetamide and other
sulfonamide). The CA inhibition studies clearly revealed
that compound 3g contains the most suitable polar group
(p−nitrophenyl) for producing good CA inhibitory activity.
Results and discussion
Chemistry
We have successfully synthesized novel piperazine-
hydrazinocarboxamide derivatives as described in Scheme 1.
In brief, an equimolar ratio of various substituted piper-
azines and p−fluorobenzaldehyde was heated in dimethyl-
formamide (DMF) in the presence of anhydrous potassium
carbonate (K2CO3, equimolar ratio) at the temperature
of 80−100 ◦C to give formyl intermediates 2a–n. These
formyl derivatives 2a–n were then stirred with semicarbazide
hydrochloride in absolute ethanol for 10–12h at room tem-
perature to afford crude piperazine-hydrazinocarboxamide
derivatives (3a–n). The final products were purified by col-
umn chromatography using chloroform–methanol (95:5) and
characterized by 1H NMR, 13C NMR and mass spectroscopy.
The purity of the target compounds was evaluated by elemen-
tal analysis.
Docking studies
The in vitro CA inhibition studies showed that majority of
targetedcompounds displayed considerableinhibitionpoten-
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165
A
+
F
NH
R
N
N
H
R
N
CHO
C
O
2a-n
a-n
1
B
O
NH2
N
N
H
N
R
N
a-n
3
Compound Number
1a, 2a, 3a
R- Group
Compound Number
1h, 2h, 3h
R- Group
N
N
N
1b, 2b, 3b
1i, 2i, 3i
H3C
1c, 2c, 3c
1d, 2d, 3d
1e, 2e, 3e
1j, 2j, 3j
1k, 2k, 3k
1l, 2l, 3l
O
F
O
Cl
O
1f, 2f, 3f
1m, 2m, 3m
1n, 2n, 3n
F
3C
O
O
1g, 2g, 3g
O2N
O
Scheme 1 Synthesis of piperazine-hydrazinocarboxamide derivatives. Reagents and conditions A: dimethylformamide (DMF), K2CO3, 80−100 ◦C,
15–24h; B: absolute ethanol, semicarbazide hydrochloride, RT, 10–12h
tial toward CAII versus CAI. Therefore, docking analysis of
synthesized compounds (3a–n) was carried against the CAII
isozyme to elucidate the binding mode as well as molecu-
lar interactions within the active site of CAII. The docking
results show that all derivatives fit nicely into the active site
of CAII. However, among all derivatives 3g showed best
binding energy toward CAII (Supplementary Table S1). The
spatial interaction of 3g in the active site is stabilized by
four H-bond interactions with His94, His96, Thr199 and
Thr200 (Fig. 2a, b). Residues in vicinity of the active site,
such as Leu98, Val121, Val143, Leu198 and Trp209, are
involved in hydrophobic interactions, and Trp5 and Trp16
stabilize the nitrophenyl moiety via π − π interactions
(Fig. 2a). The semicarbazide moiety fits well in the active
site and was coordinated with Zn2+ along with three histidine
residues, which is peculiar for isozyme activity. Compound
3g displayed similar spatial arrangement (Fig. 2b) within the
active site of CA II as observed in X-ray structure of CAII
co-crystallized with standard inhibitor acetazolamide [22].
Furthermore, the outer surface of CAII is well partitioned
as hydrophobic and hydrophilic [23]. Compound 3g trans-
versely fits into the active site, flattened from the hydrophilic
core to the hydrophobic outer rim of CAII isozyme. The
surface view of isozyme CAII docked with compound 3g
clearlydepicts thespatial orientationofthenitrophenylgroup
toward the hydrophobic domain of the active site, whereas
the hydrazinecarboxamide is involved in polar interactions
with electron-rich residues (Fig. 2c).
Molecular dynamics (MD) simulation
The molecular interaction of CAII-compound 3g complex
is also investigated using molecular dynamics (MD simula-
tion) which was carried out for 40ns using GROMACS 4.6.5
Biomolecular software package [25]. The dynamic stability
of isozyme-inhibitor complex is analyzed in terms of root
mean square deviation (RMSD). Plot of time versus RMSD
of Cα atoms is calculated to examine the correct conforma-
tion of ligand in the binding pocket (Fig. 3a). As expected, the
RMSD trajectory of CAII remains stable throughout the sim-
ulation time with a small drift of 0.02nm at 20 and 35ns. The
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Table 1 CA I and CA II
inhibition data for the
synthesized compounds (3a–n)
O
NH2
N
N
H
N
R
N
Compounds
R groups
IC50 SD (µM)
CAI CAII
CAI/CAII
3a
3b
3c
217.70 1.51
126.60 1.13
391.2 1.73
27.70 1.20
25.49 1.01
60.14 1.36
7.85
4.96
6.50
H3C
O
F
Cl
F
3d
3e
3f
286.7 1.47
249.8 1.26
217.7 1.50
1.89 0.68
45.70 0.77
44.25 0.95
37.67 1.04
0.620 0.14
6.27
5.64
5.77
3.04
3C
3g
O2N
N
3h
3i
415.1 0.81
83.85 1.11
66.91 1.2
6.20
6.04
N
N
13.88 1.41
3j
126.6 + 1.08
71.89 + 1.52
18.35 + 1.53
5.20 + 0.45
6.89
O
3k
13.82
3l
92.68 1.33
391.20 1.56
18.76 1.40
38.81 1.40
4.94
O
3m
10.07
O
O
3n
-
607.10 1.1
0.248 0.04
71.11 0.81
0.0709 0.01
8.53
3.49
O
AZM
Fig. 2 a Cartoon view of CAII docked with compound 3g, b CAII binding site environment and residual interaction with compound 3g, c surface
representation of CAII docked with compound 3g. Active site residues are colored based on element
result shows that the RMSD trajectory for CAII-compound
3g complex quickly achieved equilibrium in the initial 2.5ns
of simulation. At 7.5ns, a gradual transition of 0.15nm is
observed, and the trajectory is optimized after 12ns. This
substantial drift in RMSD of 0.15nm is demonstrated by
the protein–inhibitor interactions within complex. Trajec-
tory attains the equilibrium with a drop down of ∼0.05 nm
at 12ns. However, the simulation ends with a total drift of
0.2nm from the native structure. The small but continuous
rise in the trajectory (12–40ns) represents the various inter-
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Mol Divers (2017) 21:163–174
167
Fig. 3 a RMSD plot of CAII (blue) and CAII complex with compound
3g (red), b time evolution of radius of gyration (Rg) values during
40,000ps (40ns) of MD simulation. The Rg plot for isozyme CAII is
shown with color blue and CAII-compound 3g complex is shown with
color red, c RMSF plot for CAII (blue) and complex (red). Loop region
and terminus residues show a high degree of fluctuation in CAII, d
potential energy versus time plot for CAII (blue) and complex (red),
average regression is shown with bold lines for both systems
Fig. 4 Secondary structure analysis plot for a CAII and b CA II-compound 3g
actions with ligand. A significant difference in RMSD of
Furthermore, the radiation of gyration (Rg) analysis was
CAII and complex with compound 3g clearly indicates the
conformational change in the protein has occurred to fit the
ligand in the binding pocket of CAII.
carried to measure the stability and conformational change
of isozyme and compound 3g-CAII complex. We plotted the
time evolution of Rg for both systems. Figure 3b shows that
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in the presence of inhibitor, the average Rg score is decreased
from the native structure of CAII isozyme. The average Rg
value for CAII and complex with inhibitor are recorded as
1.77 and 1.74nm, respectively. The trajectory of Rg for both
systems remains almost stable during the entire simulation
time. The result shows no any significant change in overall
topology isozyme even after formation of a complex with
compound 3g. The stable backbone RMSD and decrease
in Rg score from the native structure strongly indicate that
inhibitor (compound 3g) preferentially fit into the active site
of CAII and essential interactions was retained up to 40ns of
simulation.
Table 2 Hematological parameters after oral administration of vehicle
and compound 3g for 14days in rats
Hb (g/dl)
14.6 0.88
7.1 0.48
26.6 2.06
69.3 3.32
14.7 0.51
7.3 + 0.69
25.5 3.27
70.8 4.35
3.5 1.04
3.1 1.72
0
TLC (103/ml)
Neutrophils (%)
Lymphocytes (%)
Eosinophils (%)
Monocytes (%)
Basophils (%)
4
1.41
3.5 1.64
0
RBC (106/ml)
Platelet count (103/ml)
6.68 0.40
880 21.20
7.05 0.47
831 23.11
To comprehend the progression in dynamic adaptability
of isozyme active site after binding of compound 3g, the
average position fluctuation of residues was analyzed with
RMSF of all Cα atoms. Figure 3c represents that secondary
structure, conformation (α/β sheet) is maintained during
simulation and has stable fluctuation. The residues having
higher fluctuations belong to loops. The result shows bind-
ing of compound 3g led to increase in the overall fluctuation
of isozyme residues. The analysis shows a remarkable peak
of residues His94, His96, His119, Thr199 and Thr200, which
plays an important role in CA inhibitors recognition. A sig-
nificant change in peak of Trp5, Trp16, His64, Asn62, Asn67
and Pro201 suggests that in addition to histidine residues the
loop region also plays a critical role to anchor the inhibitor
at the CAII active site.
The potential energy analysis of CAII and CAII-3g com-
plex showed an average fluctuation around −6.66 × 105 and
−6.7 × 105 kJ/mol, respectively. The relative lower poten-
tial energy of complex from native also confirms the stable
interaction of compound 3g with CAII (Fig. 3d). Our dock-
ing studies, followed by MD simulation, indicate a stable
complex of compound 3g to CAII in aqueous solution. A
consecutive secondary structure analysis is shown in Fig. 4a,
b. Results show the fractional interchange of β-sheet and
coil. This is probably due to direct and indirect constraints
on the residues present at the junction of the loop and β-sheet
and involve in interaction with CAII inhibitor. The thermo-
dynamic stability of trajectory was analyzed by total energy
calculation for both isozyme and complex with inhibitor.
Table 3 Liver function test after oral administration of vehicle and
compound 3g for 14days in rats
Parameters
Compound 3g
Control (vehicle,
(300 mg/kg, po) po) SD
SD
SGOT (U/L)
SGPT (U/L)
66.6 0.90
70.0 3.89
64.1 1.68
69.5 4.19
162.2 2.39
0.37 0.05
6.81 0.40
Alkaline phosphatase (U/L) 161.5 1.79
Total billirubin (mg/dl)
Total protein (g/dl)
0.32 0.04
6.64 0.63
ters were investigated. During the experimental period, all
treated animals were found healthy and no signs of toxicity as
well as no deaths were observed. Results have shown that the
hematological values of the animals treated with compound
3g were not significantly different from those of the control
group (Table 2). In case of liver injury, numerous reports
suggest the elevation of serum biomarkers such as serum
glutamate oxaloacetate transaminase (SGOT), serum gluta-
mate pyruvate transaminase (SGPT), alkaline phosphatase
and bilirubin (Table 3). Treatment with compound 3g does
not alter the values of serum SGOT, SGPT, alkaline phos-
phatase, total protein and total bilirubin as compared with
the control group. The values of renal function tests (Table 4)
such as blood urea, creatinine and uric acid suggested that
compound 3g had not shown any noteworthy increase or
decrease as compared to control group. As per the results,
compound 3g did not induce any pathological changes in
blood composition and any malfunctioning or toxicity of the
liver as well as kidney.
Acute in vivo toxicity studies
The perception that chemicals can cause toxicity is not new.
More than a 1000 drugs of the modern pharmacopoeia can
cause severe liver toxicity with numerous clinical presenta-
tions [24]. Therefore, it is compulsory to evaluate toxicity,
as well as the safety profile of new chemical entities before
their further development for therapeutic use. In the present
study, compound 3g was administered chronically at the dose
of 300 mg/kg for 14 days in adult Wistar rats and various
hematological and biochemical (liver and kidney) parame-
Conclusion
The present study reports the design and synthesis of novel 2-
(4-(4-substituted piperazin-1-yl)benzylidene)hydrazinecar
boxamide derivatives as CA inhibitors. CA inhibition stud-
ies (CAI and CAII isoforms) have shown that compound
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169
Table 4 Renal function test after oral administration of vehicle and
compound 3g for 14days in rats
Equimolar amounts of substituted piperazine and p-
fluorobenzaldehyde were dissolved in DMF, and the reaction
mixture was heated at 80−100 ◦C in the presence of K2CO3
for 12–15h. The reaction mixture was poured into ice-cold
water, and the resulting precipitate was filtered, and washed
with hot water and petroleum ether to achieve pure products
2a–n in excellent yields.
Parameters
Compound 3g
(300mg/kg,
po) SD
Control (vehi-
cle, po) SD
Blood urea (mg/dl)
Creatinine (mg/dl)
Uric acid (mg/dl)
Calcium (mg/dl)
Phosphorus (mg/dl)
Na+
39.0 2.82
0.75 0.11
5.5 1.05
37.1 2.77
0.78 0.10
6.0 1.01
4-(4-Phenylpiperazin-1-yl)benzaldehyde (2a)
11.5 0.82
6.91 0.53
146.0 2.04
4.9 0.41
10.8 0.85
7.6 + 0.55
1
White solid; yield 85%; mp: 125−127 ◦C; H NMR (CDCl3,
400MHz): δ 3.35 (t, J = 4.9Hz, 4H, piperazine-H), 3.57 (t,
J = 5.3Hz, 4H, piperazine-H), 6.91–6.98 (m, 5H, Ar-H),
7.28–7.32 (m, 2H, Ar-H, J = 8.4Hz), 7.78 (d, J = 8.4Hz,
2H, Ar-H), 9.80 (s, 1H, CH); ESI-MS: m/z 267 [M+H]+.
144.0 1.97
5.1 0.33
K+
4-(4-(p-Tolyl)piperazin-1-yl)benzaldehyde (2b)
3g appeared as most effective inhibitor in the entire series
with an IC50 value 1.89 μM for CAI and 0.62 μM for
CAII. Molecular docking studies indicate that compound 3g
strongly binds within the active site of CAII including Zn2+
and three histidine residues which are crucial interaction for
CA inhibitory action. Furthermore, the binding potential of
compound 3g with CAII was also verified in MD simulation
studies. Compound 3g appeared as the non-toxic chemical
entity in sub-acute toxicity studies in rats. All these studies
clearly indicate that compound 3g could be a new lead for
CAII inhibitor, which may be used to produce a potent CA
inhibitor after desired modifications.
1
White solid; yield 88%; mp: 145−147 ◦C; H NMR (CDCl3,
400MHz): δ 2.29 (s, 3H, CH3), 3.28 (t, J = 4.9Hz, 4H,
piperazine-H), 3.55 (t, 4H, J = 5.3Hz, piperazine-H), 6.89
(d, J = 8.3Hz, 2H, Ar-H), 6.97 (d, J = 9.1Hz, 2H, Ar-H),
7.11 (d, J = 8.4Hz, 2H, Ar-H), 7.78 (d, J = 9.1Hz, 2H,
Ar-H), 9.79 (s, 1H, CH); ESI-MS: m/z 281 [M+H]+.
4-(4-(4-Methoxyphenyl) piperazin-1-yl)benzaldehyde (2c)
1
White solid; yield 82%; mp: 146−147 ◦C; H NMR (CDCl3,
400MHz): δ 3.22 (t, J = 5.3Hz, 4H, piperazine-H), 3.56 (t,
J = 4.9Hz, 4H, piperazine-H), 3.78 (s, 3H, CH3), 6.86–6.98
(m, 6H, Ar-H), 7.78 (d, J = 9.1Hz, 2H, Ar-H), 9.80 (s, 1H,
CH); ESI-MS: m/z 297 [M+H]+.
Experimental protocols
Chemistry
4-(4-(4-Fluorophenyl)piperazin-1-yl)benzaldehyde (2d)
1
White solid; yield 80%; mp: 130−132 ◦C; H NMR (CDCl3,
400MHz): δ 3.25 (t, J = 4.9Hz, 4H, piperazine-H), 3.55 (t,
J = 4.9Hz, 4H, piperazine-H), 6.90–7.02 (m, 6H, Ar-H),
7.78 (d, J = 9.1Hz, 2H, Ar-H), 9.80 (s, 1H, CH); ESI-MS:
m/z 285 [M+H]+.
All chemicals and reagents were obtained from Sigma
Aldrich (St. Louis, MO, USA), Merck (Darmstadt, Ger-
many), Alfa Aesar (Massachusetts) and Rankem (India) and
were used without prior purification. Melting points were
determined in open capillaries using model KSPII, KRUSS
(Germany) and are uncorrected. Nuclear magnetic reso-
nance (NMR) spectra were recorded on a high-resolution
Jeol-400MHz NMR spectrophotometer (USA). CDCl3 or
DMSO-d6 was used as deuterated solvent, and chemical
shifts are reported in ppm (δ) relative to internal stan-
dard tetramethylsilane (TMS, δ 0.00ppm). The splitting
patterns are designed as: s =singlet; brs=broad singlet;
d =doublet; dd= double doublet; t =triplet; q =quatrate;
and m =multiplet. Mass spectra were recorded on an Agi-
lent 6310 mass spectrophotometer, and elemental analysis
(C, H and N) was carried on an Elementar Analysensysteme
system.
4-(4-(4-Chlorophenyl)piperazin-1-yl)benzaldehyde (2e)
1
White solid; yield 85%; mp: 147−149 ◦C; H NMR (CDCl3,
400MHz): δ 3.31 (t, J = 4.6Hz, 4H, piperazine-H), 3.56
(t, 4H, J = 4.9Hz, piperazine-H), 6.86 (d, J = 9.1Hz, 2H,
Ar-H), 6.96 (d, J = 8.4Hz, 2H, Ar-H), 7.22 (d, J = 8.0Hz,
2H, Ar-H), 7.79 (d, J = 9.1Hz, 2H, Ar-H), 9.80 (s, 1H, CH);
ESI-MS: m/z 301 [M+H]+.
4-(4-(Trifluoromethyl)piperazin-1-yl)benzaldehyde (2f)
1
White solid; yield 87%; mp: 185−187 ◦C; H NMR (CDCl3,
400MHz): δ 3.47 (t, J = 4.9Hz, 4H, piperazine-H), 3.60 (t,
J = 4.9Hz, 4H, piperazine-H), 6.96 (dd, J = 8.7Hz, 4H,
Ar-H), 7.52 (d, J = 9.1Hz, 2H, Ar-H), 7.80 (d, J = 9.1Hz,
2H, Ar-H), 9.81 (s, 1H, CH); ESI-MS: m/z 335 [M+H]+.
General procedure for synthesis of 4-(4-substituted piper-
azin-1-yl)benzaldehyde (2a–n)
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4-(4-(4-Nitrophenyl)piperazin-1-yl)benzaldehyde (2g)
4-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl)
benzaldehyde (2n)
1
Yellowsolid;yield90%;mp:190−193 ◦C; HNMR(CDCl3,
1
400MHz): δ 3.65 (s, 8H, piperazine-H), 6.83 (d, J = 9.9Hz,
2H, Ar-H), 6.92 (d, J = 8.3Hz, 2H, Ar-H), 7.80 (d, J =
8.4Hz, 2H, Ar-H), 8.17 (d, J = 9.9Hz, 2H, Ar-H), 9.81 (s,
1H, CH); ESI-MS: m/z 312 [M+H]+.
White solid; yield 78%; mp: 115−117 ◦C; H NMR (CDCl3,
400MHz): δ 2.57 (t, J = 4.9Hz, 4H, piperazine-H), 3.40 (t,
J = 4.9Hz, 4H, piperazine), 3.47 (s, 2H, CH2), 5.95 (s, 2H,
CH2), 6.76 (s, 2H, Ar-H), 6.88–6.91 (m, 3H, Ar-H), 7.74 (d,
J = 8.4Hz, 2H, Ar-H), 9.77 (s, 1H, CH); ESI-MS: m/z 325
[M+H]+.
4-(4-(Pyridin-2-yl)piperazin-1-yl)benzaldehyde (2h)
1
White solid; yield 82%; mp: 140−142 ◦C; H NMR (CDCl3,
Generalprocedureforsynthesisof(E)-2-(4-(4-substituted
piperazin-1-yl)benzylidene)hydrazinecarboxamide (3a–n)
An equimolar amount of formyl derivatives 2a–n was
stirred with semicarbazide hydrochloride in ethanol at room
temperature for 10–12h. The resulting precipitate was fil-
tered and washed with hot ethanol and water for 2–3 times.
The crude products were purified by column chromatogra-
phy using chloroform–methanol (95:5) as eluent to afford
target piperazine-hydrazinocarboxamide derivatives 3a–n.
400MHz): δ 2.39 (s, 4H, piperazine-H), 3.36 (s, 4H,
piperazine-H), 6.83 (d, J = 9.9Hz, 2H, Ar-H), 7.24 (s, 2H,
Ar-H), 7.75 (q, J = 9.1Hz, 4H, Ar-H), 10.0 (s, 1H, CH);
ESI-MS: m/z 268 [M+H]+.
4-(4-(Pyrimidin-2-yl)piperazin-1-yl)benzaldehyde (2i)
1
White solid; yield 79%; mp: 138−140 ◦C; H NMR (CDCl3,
400MHz): δ 3.51 (t, J = 5.3Hz, 4H, piperazine-H), 4.00
(t, J = 5.3Hz,4H, piperazine-H), 6.55 (t, J = 4.4Hz, 1H,
Ar-H), 6.95 (d, J = 9.1Hz, 2H, Ar-H), 7.78 (d, J = 8.4Hz,
2H, Ar-H), 8.33 (d, J = 4.5Hz, 2H, Ar-H), 9.79 (s, 1H, CH);
ESI-MS: m/z 269 [M+H]+
(E)-2-(4-(4-Phenylpiperazin-1-yl) benzylidene)
hydrazinecarboxamide (3a)
White solid; yield 81%; mp: 280−282 ◦C; H NMR
1
(DMSO-d6, 400MHz): δ 3.23–3.30 (m, 8H, piperazine-H),
6.37 (brs, 2H, NH2), 6.79 (t, J = 7.6Hz, 1H, Ar-H), 6.98
(d, J = 8.3Hz, 4H, Ar-H), 7.22 (t, J = 7.6Hz, 2H, Ar-H),
7.55 (d, J = 10.2Hz, 2H, Ar-H), 7.73 (s, 1H, CH), 10.0 (s,
1H, NH); 13C NMR (DMSO-d6): δ 47.3, 50.3, 115.7, 118.3,
127.7, 129.5, 139.5, 147.2, 156.9, 157.8. ESI-MS: m/z 324
[M+H]+; Anal. Calcd for C18H21N5O: C, 66.85; H, 6.55; N,
21.66: Found: C, 66.68; H, 6.64; N, 21.52.
4-(4-Benzylpiperazin-1-yl)benzaldehyde (2j)
White solid; yield 92%; mp: 83−85 ◦C; H NMR (CDCl3,
1
400MHz): δ 2.58 (t, J = 5.3Hz, 4H, piperazine-H), 3.40 (t,
J = 4.9Hz, 4H, piperazine-H), 3.56 (s, 2H, CH2), 6.90 (d,
J = 9.1Hz, 2H, Ar-H), 7.29–7.35 (m, 5H, Ar-H), 7.74 (d,
J = 8.4Hz, 2H, Ar-H), 9.77 (s, 1H, CH); ESI-MS: m/z 281
[M+H]+.
4-(4-Benzoylpiperazin-1-yl)benzaldehyde (2k)
(E)-2-(4-(4-(p-Tolyl)piperazin-1-yl)benzylidene)
hydrazinecarboxamide (3b)
1
White solid; yield 78%; mp: 101−103 ◦C; H NMR (CDCl3,
White solid; yield 85%; mp: 266−268 ◦C; H NMR
1
400MHz): δ 3.30–3.84 (m, 8H, piperazine-H), 6.85 (d,
J = 9.1Hz, 2H, Ar-H), 7.32–7.37 (m, 5H, Ar-H), 7.70 (d,
J = 9.1Hz, 2H, Ar-H), 9.72 (s, 1H, CH), ESI-MS: m/z 295
[M+H]+.
(DMSO-d6, 400MHz): δ 2.19 (s, 3H, CH3), 3.18 (t, J =
4.5Hz, 4H, piperazine-H), 3.32 (s, 4H, piperazine-H), 6.38
(brs, 2H, NH2), 6.89 (d, J = 8.4Hz, 2H, Ar-H), 6.97 (d,
J = 8.4Hz, 2H, Ar-H), 7.04 (d, J = 8.4Hz, 2H, Ar-H),
7.55 (d, J = 8.4Hz, 2H, Ar-H), 7.72 (s, 1H, CH), 10.0 (s,
1H, NH); 13C NMR (DMSO-d6): δ 20.0, 47.6, 48.7, 114.9,
116.0, 125.2, 127.6, 128.0, 129.4, 139.7, 148.8, 151.3, 156.9;
ESI-MS: m/z 337 [M]+; Anal. Calcd for C19H23N5O: C,
67.63; H, 6.87; N, 20.76: Found: C, 67.77, H, 6.78; N, 20.89.
4-(4-Benzhydrylpiperazin-1-yl)benzaldehyde (2l)
1
White solid; yield 82%; mp: 122−124 ◦C; H NMR (CDCl3,
400MHz): δ 2.54 (t, J = 4.9Hz, 4H, piperazine-H), 3.39
(t, 4H, J = 4.9Hz, piperazine-H), 4.26 (s, 1H, CH), 6.87
(d, J = 8.4Hz, 2H, Ar-H), 7.18–7.31 (m, 6H, Ar-H), 7.44
(d, J = 7.6Hz, 4H, Ar-H), 7.71 (d, J = 9.1Hz, 2H, Ar-H),
9.76 (s, 1H, CH); ESI-MS: m/z 357 [M+H]+.
(E)-2-(4-(4-(4-Methoxyphenyl)piperazin-1-yl)
benzylidene)hydrazinecarboxamide (3c)
4-(4-(Furan-3-carbonyl)piperazin-1-yl)benzaldehyde (2m)
1
White solid; yield 87%; mp: 285−287 ◦C; H NMR
1
White solid; yield 86%; mp: 98−100 ◦C; H NMR (CDCl3,
(DMSO-d6, 400MHz): δ 3.12 (s, 4H, piperazine-H), 3.33 (s,
4H, piperazine-H), 3.67 (s, 3H, CH3), 6.38 (brs, 2H, NH2),
6.83 (d, J = 9.1Hz, 2H, Ar-H), 6.92–6.98 (m, 4H, Ar-H),
7.55 (d, J = 8.4Hz, 2H, Ar-H), 7.73 (s, 1H, CH), 10.0 (s,
1H, NH); 13C NMR (DMSO-d6): δ 47.6, 49.6, 55.1, 114.9,
114.3, 117.7, 125.1, 127.6, 139.6, 145.2, 151.4, 153.2, 156.9;
400MHz): δ 3.51 (t, J = 5.3Hz, 4H, piperazine-H), 4.01 (s,
4H, piperazine-H), 6.52 (dd, J = 3.4Hz, 1H, Ar-H), 6.95
(d, J = 9.1Hz, 2H, Ar-H), 7.08 (d, J = 3.8Hz, 1H, Ar-H),
7.52 (s, 1H, Ar-H), 7.77 (d, J = 9.1Hz, 2H, Ar-H), 9.81 (s.
1H, CH); ESI-MS: m/z 285 [M+H]+.
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171
ESI-MS: m/z 353 [M]+; Anal. Calcd for C19H23N5O2: C,
(E)-2-(4-(4-(Pyridin-2-yl) piperazin-1-yl) benzylidene)
hydrazinecarboxamide (3h)
64.57; H, 6.56; N, 19.82 Found: C, 64.69; H, 6.63; N, 19.73.
1
(E)-2-(4-(4-(4-Fluorophenyl)piperazin-1-yl)benzylidene)
hydrazinecarboxamide (3d)
White solid; yield 79%; mp: 240−242 ◦C; H NMR
(DMSO-d6, 400MHz):δ 3.30(t, J = 4.9Hz, 4H, piperazine-
H), 3.61 (t, J = 5.3Hz, 4H, piperazine-H), 6.38 (brs, 2H,
NH2), 6.65 (t, J = 6.1Hz, 1H, Ar-H), 6.88 (d, J = 8.4Hz,
1H, Ar-H), 6.97 (d, J = 9.1Hz, 2H, Ar-H), 7.55 (d, J =
9.1Hz, 3H, Ar-H,), 7.72 (s, 1H, CH), 8.11 (d, J = 3.0Hz,
1H, Ar-H), 10.0 (s, 1H, NH); 13C NMR (DMSO-d6): δ 44.4,
47.3, 107.2, 113.2, 114.8, 125.2, 127.6, 137.6, 139.6, 147.6,
151.3, 156.9, 158.9; ESI-MS: m/z 325 [M+H]+; Anal. Calcd
for C17H20N6O: C, 62.95; H, 6.21; N, 25.91; Found: C,
62.83; H, 6.15; N, 26.01.
1
White solid; yield 82%; mp: 280−282 ◦C; H NMR
(DMSO-d6, 400MHz):δ 3.20(t, J = 4.9Hz, 4H, piperazine-
H), 3.34–3.36 (m, 4H, piperazine-H), 6.36 (brs, 2H, NH2),
6.95–7.09 (m, 6H, Ar-H), 7.55 (d, J = 9.1Hz, 2H, Ar-H),
7.73 (s, 1H, CH), 10.0 (s, 1H, NH); 13C NMR (DMSO-d6): δ
47.6, 49.02, 114.9, 115.2, 115.4, 117.4, 125.3, 127.6, 139.6,
147.8, 151.2, 156.8. ESI-MS: m/z 342 [M+H]+; Anal. Calcd
for C18H20FN5O: C, 63.33; H, 5.91; N, 20.51; Found: C,
63.52; H, 5.19; N, 20.42.
(E)-2-(4-(4-(Pyrimidin-2-yl) piperazin-1-yl)benzylidene)
hydrazinecarboxamide (3i)
(E)-2-(4-(4-(4-Chlorophenyl)piperazin-1-yl)benzylidene)
hydrazinecarboxamide (3e)
1
White solid; yield 68%; mp: 224−226 ◦C; H NMR
1
White solid; yield 87%; mp: 310−312 ◦C; H NMR
(DMSO-d6, 400MHz):δ 3.28(t, J = 4.5Hz, 4H, piperazine-
H), 3.85 (t, J = 5.6Hz, 4H, piperazine-H), 6.36 (brs, 2H,
NH2), 6.64 (t, J = 5.3Hz 1H, Ar-H), 6.96 (d, J = 8.4Hz,
2H, Ar-H), 7.55 (d, J = 8.4Hz, 2H, Ar-H), 7.72 (s, 1H, CH),
8.36 (d, J = 4.6Hz, 2H, Ar-H), 10.0 (s, 1H, NH); 13C NMR
(DMSO-d6): δ 43.0, 47.4, 110.2, 115.0, 125.3, 127.6, 139.6,
151.2, 156.8, 158.0, 161.2;ESI-MS:m/z 326[M+H]+;Anal.
Calcd for C16H19N7O: C, 59.06; H, 5.89; N, 30.13 Found:
C, 59.25; H, 5.78; N, 30.22.
(DMSO-d6, 400MHz): δ 3.23–3.29 (m, 8H, piperazine-H),
6.37 (brs, 2H, NH2), 6.98 (t, J = 8.7Hz, 4H, Ar-H), 7.24 (d,
J = 7.6Hz, 2H, Ar-H), 7.55 (d, J = 9.1Hz, 2H, Ar-H), 7.72
(s, 1H, CH), 10.0 (s, 1H, NH); 13C NMR (DMSO-d6): δ 47.4,
47.9, 114.9, 117.1, 122.6, 125.3, 127.6, 128.7, 139.5, 149.6,
151.2, 156.8; ESI-MS: m/z 358 [M+H]+; Anal. Calcd for
C18H20ClN5O: C, 60.42; H, 5.63; N, 19.57 Found: C, 60.5;
H, 5.70; N, 19.65.
(E)-2-(4-(4-(4-(Trifluoromethyl)phenyl)piperazin-1-yl)
benzylidene)hydrazinecarboxamide (3f)
(E)-2-(4-(4-Benzylpiperazin-1-yl) benzylidene)
hydrazinecarboxamide (3j)
1
White solid; yield 84%; mp: 260−262 ◦C; H NMR
White solid; yield 77%; mp: 220−222 ◦C; H NMR
1
(DMSO-d6, 400MHz): δ 3.35–3.37 (m, 4H, piperazine-H),
3.41–3.44 (m, 4H, piperazine-H), 6.37 (brs, 2H, NH2), 6.98
(d, J = 9.1Hz, 2H, Ar-H), 7.11 (d, J = 8.4Hz, 2H, Ar-
H), 7.51–7.57 (m, 4H, Ar-H), 7.73 (s, 1H, CH), 10.0 (s,
1H, NH); 13C NMR (DMSO-d6): δ 46.8, 47.2, 114.3, 114.8,
125.3, 126.2, 127.6, 139.6, 151.1, 153.0, 156.8; ESI-MS:
m/z 391[M]+; Anal. Calcd for C19H20F3N5O: C, 58.31; H,
5.15; N, 17.89 Found: C, 58.25; H, 5.21; N, 17.76.
(DMSO-d6, 400MHz): δ 2.45–2.47 (m, 4H, piperazine-H),
3.19 (t, J = 4.5Hz, 4H, piperazine-H), 3.50 (s, 2H, CH2),
6.34 (brs, 2H, NH2), 6.89 (d, J = 8.4Hz, 2H, Ar-H), 7.23–
7.33 (m, 5H, Ar-H), 7.51 (d, J = 8.4Hz, 2H, Ar-H), 7.71 (s,
1H, CH), 10.0 (s, 1H, NH); 13C NMR (DMSO-d6): δ 47.5,
52.4, 62.0, 114.6, 124.9, 127.0, 127.5, 128.2, 128.9, 138.0,
139.6, 151.3, 156.7; ESI-MS: m/z 337 [M]+; Anal. Calcd for
C19H23N5O: C, 67.63; H, 6.87; N, 20.76: Found: C, 67.84;
H, 6.91; N, 20.87.
(E)-2-(4-(4-(4-Nitrophenyl) piperazin-1-yl)benzylidene)
hydrazinecarboxamide (3g)
(E)-2-(4-(4-Benzoylpiperazin-1-yl)benzylidene)
hydrazinecarboxamide (3k)
Yellow solid; yield 89%; mp: 307−309 ◦C; H NMR
1
(DMSO-d6, 400MHz): δ 3.38 (t, J = 4.9Hz 4H, piperazine-
H), 3.62 (t, J = 4.9Hz, 4H, piperazine-H), 6.37 (brs, 2H,
NH2), 6.95 (d, J = 8.4Hz, 2H, Ar-H,), 7.06 (d, J = 9.9Hz,
2H, Ar-H), 7.56 (d, J = 8.4Hz, 2H, Ar-H), 7.72 (s, 1H, CH),
8.07 (d, J = 9.1Hz, 2H, Ar-H), 10.0 (s, 1H, NH); 13C NMR
(DMSO-d6): δ 45.9, 46.8, 112.5, 114.6, 125.1, 125.8, 127.6,
136.9, 139.6, 150.8, 154.2, 156.8; ESI-MS: m/z 368 [M]+;
Anal. Calcd for C18H20N6O3: C, 58.69; H, 5.47; N, 22.81
Found: C, 58.76; H, 5.42; N, 22.92.
White solid; yield 82%; mp: 229−231 ◦C; H NMR
1
(DMSO-d6, 400MHz): δ 3.18–3.25 (m, 4H, piperazine-H),
3.45–3.77 (m, 4H, piperazine-H), 6.36 (brs, 2H, NH2), 6.93
(d, J = 9.9Hz, 2H, Ar-H), 7.38–7.46 (m, 5H, Ar-H), 7.54 (d,
J = 9.1Hz, 2H, Ar-H), 7.72 (s, 1H, CH), 10.0 (s, 1H, NH);
13C NMR (DMSO-d6): δ 46.8, 47.7, 115.1, 125.5, 127.0,
127.6, 128.4, 129.6, 135.8, 139.4, 151.1, 156.8, 169.0; ESI-
MS: m/z 352 [M+H]+; Anal. Calcd for C19H21N5O2: C,
64.94; H, 6.02; N, 19.93; Found: C, 65.04; H, 5.96; N, 20.01.
123

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(E)-2-(4-(4-Benzhydrylpiperazin-1-yl) benzylidene)
hydrazinecarboxamide (3l)
[100 − (Abs of test comp/Abs of control) × 100]. The IC50
values for each test compounds were calculated by using
GraphPad Prism (Version 6.0) software.
1
White solid; yield 75%; mp: 230−232 ◦C; H NMR
(DMSO-d6, 400MHz):δ 2.43(t, J = 5.3Hz, 4H, piperazine-
H), 3.21 (t, J = 6.4Hz, 4H, piperazine-H), 4.31 (s, 1H, CH),
6.35 (brs, 2H, NH2), 6.87 (d, J = 8.4Hz, 1H, Ar-H), 7.18 (t,
J = 6.8Hz, 2H, Ar-H), 7.29(t, J = 7.6Hz, 4H, Ar-H), 7.44–
7.52 (m, 6H, Ar-H), 7.70 (s, 1H, CH), 9.99 (s, 1H, NH); 13C
NMR (DMSO-d6): δ 47.5, 51.3, 75.0, 114.4, 125.1, 126.9,
127.5, 128.6, 139.6, 142.7, 151.3, 156.8; ESI-MS: m/z 414
[M+H]+; Anal. Calcd for C25H27N5O: C, 72.61; H, 6.58; N,
16.94; Found: C, 72.48, H, 6.49; N, 17.06.
Molecular docking
Atomic coordinates of CAII (PDB ID: 3V7X) were taken
from the Protein Data Bank (www.rcsb.org) [26]. Steep-
est descent method in GROMACS 4.6.5 [25] was used to
optimize the coordinates, and both isozymes were saved
in pdbqt format to carry docking analysis. AutoDock 4.2
with standard protocol was used to dock compounds 3a–
n in CAII. The Lamarckian genetic algorithm (LGA) was
applied to deal with protein–ligands interactions [27]. The
most favorable free binding energy and docking orientations
lying within the range of 2.0 Å in root mean square devi-
ation (RMSD) tolerance were used to cluster the molecule
and ranked accordingly. PyMOL [25] was used to visualize
molecular interactions. All calculations were carried out on
workstation machines running Linuxx86 operating systems.
(E)-2-(4-(4-(Furan-3-carbonyl)piperazin-1-yl)
benzylidene)hydrazinecarboxamide (3m)
1
White solid; yield 85%; mp: 219−221 ◦C; H NMR
(DMSO-d6, 400MHz): δ 3.28 (s, 4H, piperazine-H), 3.79
(s, 4H, piperazine-H), 6.37 (brs, 2H, NH2), 6.63 (s, 1H, Ar-
H), 6.94 (d, J = 7.6Hz, 2H, Ar-H), 7.03 (s, 1H, Ar-H),
7.54 (d, J = 7.2Hz, 2H, Ar-H), 7.72 (s, 1H, CH), 7.86 (s,
1H, Ar-H), 10.0 (s, 1H, NH); 13C NMR (DMSO-d6): δ 47.6,
111.3, 114.9, 115.8, 125.4, 127.6, 139.5, 144.8, 146.9, 150.9,
156.8, 158.3; ESI-MS: m/z 342 [M+H]+; Anal. Calcd for
C17H19N5O3: C, 59.81; H, 5.61; N, 20.52 Found: C, 59.88,
H, 5.73; N, 20.46.
Molecular dynamics simulation studies
MD simulations were performed with GROMACS 4.6.5
using the CHARMm force-field and spc216 water molecules.
ThetrajectoryfileswereanalyzedusingGROMACSutilities:
g_gyrate, g_rmsd and g_rmsf to obtain the radius of gyration
(Rg), root mean square deviation (RMSD) and root mean
square fluctuation (RMSF). The coordinates of CAII were
energy minimized with steepest descent, up to a tolerance of
100 kJ mol−1 to remove all bad contacts. Two independent
MD simulations were carried out with standard protocol in
water for CAII and CAII-compound 3g complex, respec-
tively, at 300K to evaluate the structural changes, binding
affinity and stability in course of simulation. The SHAKE
algorithm was used to restrain the covalent bonds con-
taining hydrogen atoms at their equilibrium distances, and
periodic boundary conditions were applied. Particle mesh
Ewald (PME) method was used for computing the long-
range electrostatic interactions. A 1.4-nm cutoff for van der
Waals interactions and a 1.0-nm cutoff for Coulombs interac-
tion were adopted in the simulation, respectively. Analytical
graphs were plotted using Xmgrace, and VMD and PyMol
were used for structure and molecular interaction analysis
[26].
(E)-2-(4-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl)
benzylidene)hydrazinecarboxamide (3n)
1
White solid; yield 88%; mp: 220−222 ◦C; H NMR
(DMSO-d6, 400MHz): δ 2.46 (s, 4H, piperazine-H), 3.18
(s, 4H, piperazine-H), 3.41 (s, 2H, CH2), 5.98 (s, 2H, CH2),
6.33 (brs, 2H, NH2), 6.76 (d, J = 7.6Hz, 1H, Ar-H), 6.83–
6.90 (m, 4H, Ar-H), 7.50 (d, J = 8.4Hz, 2H, Ar-H), 7.70 (s,
1H, CH), 9.97 (s, 1H, NH); 13C NMR (DMSO-d6): δ 47.5,
52.2, 61.6, 100.7, 107.8, 109.2, 114.6, 122.1, 125.0, 127.5,
131.8, 139.7, 146.2, 147.1, 151.4, 156.8; ESI-MS: m/z 382
[M+H]+; Anal. Calcd for C20H23N5O3: C, 62.98; H, 6.08;
N 18.36: Found: C, 63.14, H, 6.01; N, 18.48.
Carbonic anhydrase inhibition assay
CA inhibition was measured according to our previously
reported method [25]. The inhibition assay was done after
standardization of reaction conditions like enzyme concen-
tration, substrate concentration, pH, duration of reaction. The
method is based on spectrophotometric determination of p-
nitrophenol (a yellow color compound) which is formed as
CA catalyzed hydrolysis product of p-nitrophenyl acetate (4-
NPA). The absorbance of each well was measured at 400nm
after 30min with a SpectraMax Plus 384 microplate spec-
trophotometer (Molecular Devices, Sunnyvale, CA). Percent
In vivo sub-acute toxicity studies
The in vivo sub-acute toxicity studies were carried out
according to our previously reported procedure [28]. A total
of 12 rats were taken and divided into two groups where
the control group received only vehicle (1% gum acacia in
inhibition calculated by the formula, Inhibition (%)
=
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Mol Divers (2017) 21:163–174
173
normal saline). The other group was administered with the
test compound 3g at the dose of 300mg/kg (per oral) daily
for two weeks. After the stipulated period, each animal was
anesthetized by anesthetic ether and blood was collected
by cardiac puncture. The blood samples were placed sep-
arately in sterilized heparinized and non-heparinized tubes
for further hematological as well as biochemical evalua-
tions. Hematological parameters were estimated by using
automated hematological analyzer machine (Nihon Kohden,
Tokyo, Japan). The clear serum was separated from the non-
heparinized blood at 37 ◦C and was assayed for biochemical
parameters by biochemical auto-analyzer (Agappe, Kolkata,
India).
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Acknowledgements CBM and AP are thankful to the University
Grants Commission (UGC) for the award of Dr. D.S. Kothari post-
doctoral fellowship. SK and DI are thankful to UGC for the fellowship.
MT thanks University of Delhi for financial assistance. The Univer-
sity Science Instrumentation Center (USIC), University of Delhi, is
acknowledged for the recording NMR spectra of synthesized com-
pounds. We thank Department of Science and Technology, India, for
FIST support (SR/FST/LSI-541/2012). MIH thanks to the Council of
Scientific and Industrial Research, India for the financial support (Grant
No. 37(1665)/15/EMR-II).
15. SupuranCT(2016)Druginteractionconsiderationsinthetherapeu-
tic use of carbonic anhydrase inhibitors. Expert Opin Drug Metab
Toxicol 12:423–431. doi:10.1517/17425255.2016.1154534
16. Karioti A, Ceruso M, Carta F, Bilia AR, Supuran CT (2015) New
natural product carbonic anhydrase inhibitors incorporating phenol
moieties. Bioorg Med Chem 23:7219–7225. doi:10.1016/j.bmc.
2015.10.018
17. Supuran CT, Winum JY (2015) Carbonic anhydrase IX inhibitors in
cancer therapy: an update. Future Med Chem 7:1407–1414. doi:10.
4155/fmc.15.71
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