Discovery of potent anti-convulsant carbonic anhydrase inhibitors: Design, synthesis, in vitro and in vivo appraisal

Article abstract of DOI:10.1016/j.ejmech.2018.07.019

We report the design, synthesis and pharmacological assessment of novel benzenesulfonamide derivatives acting as effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. All the synthesized compounds were screened for their CA inhibitory action against four isoforms of human origin (h), i.e. hCA I, hCA II, hCA VII and hCA IX. In-vitro carbonic anhydrase inhibition studies have shown that first series, 4-(2-(4-(4-substitutedpiperazin-1-yl)benzylidene)hydrazinyl)benzenesulfonamides (4a- 4i) bestowed low nanomolar range to medium nanomolar range inhibitors against hCA II and hCA VII, effectively involved in epileptogenesis. Furthermore, compounds belonging to the second series, 4-(2-(4-(4-substitutedpiperazin-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamides (8a-8k) showed effective inhibition against hCA VII, being less effective against other hCA isoforms. Inspiring with obtained CA inhibition results, we have chosen some of the potent hCA II and hCA VII inhibitors (4g, 4i and 8d) to test their anti-convulsant efficacy in MES and sc-PTZ seizure tests in Swiss Albino male mice. In result, these compounds significantly attenuated both electrical (MES) as well as chemical (sc-PTZ) induced seizures. Next, in advance anticonvulsant tests, compound 8d displayed long duration of action in time course study and successfully attenuated MES induced seizure in mice up to 6 h after drug administration without showing neurotoxicity in rotarod test. Moreover, this compound was also found to be orally active and effectively abolished generalized tonic-clonic seizures in male Wistar rats upon oral administration, being non-toxic in sub acute toxicity studies.

Full text of DOI:10.1016/j.ejmech.2018.07.019

Accepted Manuscript
Discovery of potent anti-convulsant carbonic anhydrase inhibitors: Design, synthesis,
in vitro and in vivo appraisal
Chandra Bhushan Mishra, Shikha Kumari, Andrea Angeli, Silvia Bua, Martina
Buonanno, Simona Maria Monti, Manisha Tiwari, Claudiu T. Supuran
PII:
S0223-5234(18)30575-0
10.1016/j.ejmech.2018.07.019
EJMECH 10554
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 May 2018
Revised Date: 5 July 2018
Accepted Date: 7 July 2018
Please cite this article as: C.B. Mishra, S. Kumari, A. Angeli, S. Bua, M. Buonanno, S.M. Monti, M.
Tiwari, C.T. Supuran, Discovery of potent anti-convulsant carbonic anhydrase inhibitors: Design,
synthesis, in vitro and in vivo appraisal, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.07.019.
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ACCEPTED MANUSCRIPT
Graphical Abstract
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Discovery of Potent Anti-convulsant Carbonic Anhydrase Inhibitors: Design, Synthesis, In vitro and In vivo
Appraisal
Chandra Bhushan Mishra1, Shikha Kumari1, Andrea Angeli2, Silvia Bua2, Martina Buonanno,3 Simona Maria Monti, 3
Manisha Tiwari1*, and Claudiu T. Supuran2*
1Bio-Organic Chemistry Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, 110007, Delhi, India
2Dipartimento Neurofarba, Universita` degli Studi di Firenze, Sezione di Scienze Farmaceutiche e Nutraceutiche, 50019 Sesto Fiorentino,
Florence, Italy.
3 Institute of Biostructures and Bioimaging, CNR, via Mezzocannone, 16, 80134, Naples, Italy.
Corresponding Authors. MT, Phone: +91-01127666272; E-mail:: mtiwari07@gmail.com; CTS, Phone: +39-055-
4573729; fax: +39-055-4573385; E-mail: claudiu.supuran@unifi.it
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Abstract
We report the design, synthesis and pharmacological assessment of novel benzensulfonamide derivatives acting as
effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. All the synthesized compounds were screened for their CA
inhibitory action against four isoforms of human origin (h), i.e. hCA I, hCA II, hCA VII and hCA IX. In-vitro
carbonic anhydrase inhibition studies have shown that first series, 4-(2-(4-(4-substitutedpiperazin-1-
yl)benzylidene)hydrazinyl)benzenesulfonamides (4a- 4i) bestowed low nanomolar range to medium nanomolar range
inhibitors against hCA II and hCA VII, effectively involved in epileptogenesis. Furthermore, compounds belonging
to the second series, 4-(2-(4-(4-substitutedpiperazin-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamides (8a-8k)
showed effective inhibition against hCA VII, being less effective against other hCA isoforms. Inspiring with obtained
CA inhibition results, we have chosen some of the potent hCA II and hCA VII inhibitors (4g, 4i and 8d) to test their
anti-convulsant efficacy in MES and sc-PTZ seizure tests in Swiss Albino male mice. In result, these compounds
significantly attenuated both electrical (MES) as well as chemical (sc-PTZ) induced seizures. Next, in advance
anticonvulsant tests, compound 8d displayed long duration of action in time course study and successfully attenuated
MES induced seizure in mice up to 6 h after drug administration without showing neurotoxicity in rotarod test.
Moreover, this compound was also found to be orally active and effectively abolished generalized tonic-clonic
seizures in male Wistar rats upon oral administration, being non-toxic in sub acute toxicity studies.
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1. Introduction
Epilepsy is a life-shortening neurological disorder, characterized by repeated, uncontrolled seizures and
approximately affecting 1% population worldwide [1]. Epilepsy is considered as a complex disorder, consisting over
15 different types of seizure and more than 30 epileptic syndromes, associated with substantial comorbidity
including, cognition deficit, depression and anxiety [2]. During the past decades, several third generation anti-
epileptic drugs (AEDs) have been developed to treat epileptic patients; however, these AEDs are failing to control
seizure in 20-30% of patients [3]. Currently available AEDs are also unable to prevent the development of drug
resistant epilepsy which is considered as a major challenge in epilepsy management [4]. These serious drawbacks of
existing AEDs are still insisting for development of novel and more efficacious drugs for the treatment of epilepsy.
Pathophysiological studies of epilepsy revealed that several receptor, neurotransmitters, enzyme and ion channels are
actively involved in seizure generation of epileptic patients. Therefore, several targets such as GABA (γ-
aminobutyric acid), Cl-, Na+ K+ Ca2+ ion channel, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)
,
,
receptor etc. are well studied to control epileptic seizure [3]. Apart from these targets, brain carbonic anhydrases
(CAs, EC 4.2.1.1) appeared as an emerging target to control epileptic seizure from several past decades and some of
potent CAs inhibitors are used clinically to control various types of seizure [5,6].
There are 15 different human carbonic anhydrases (hCAs) isoforms have been identified till date, CA I-III, CA VII
and CA XIII are found in cytosol; CA IV, CA IX, CA XII and CA XIV are membrane bound; CA VA and CA VB
are present in the mitochondrion and CA VI is found in milk and saliva. These CAs actively catalyze the
interconversion between CO2 and bicarbonate and are involved in numerous physiological functions associated with
CO2/bicarbonate equilibrium [7,8]
It is well established that CAs modulate numerous neuronal signaling mechanism associated with various central
nervous system (CNS) disorders, including epilepsy [5,6]. Several reports have disclosed that epileptic seizures are
associated with changes in the ionic composition within the brain, including enrichment of extracellular potassium
concentration and pH shifts [6]. Brain pH plays an important role in epilepsy and the alteration in pH directly affects
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seizure generation as well as its severity. It is found that generally alkalosis promotes seizure spreading, while
acidosis blocks/halts seizure generation [5]. The pH buffering between extracellular and intracellular spaces is
-
conducted by CO2 and HCO3 . The regulation of both components is under control of CAs which catalyzes the
-
reversible conversion of CO2 and H2O into H+ and HCO3 [9]. Thus, with these vital evidences, brain CA appears as
an important target for management of epileptic seizure syndromes. Several isoforms of CAs are present in
mammalian brain, physiologically dominant hCA II isoform is widely expressed in oligodendrocytes, myelin sheaths
as well as choroid plexus, which is engaged in various neurological disorders including epileptogenesis [10,11].
Halmi et al. showed that expression of CA II is increased in the CA1 cells after 3-12 h kainic acid treatment in status
epileptic model [12]. The study has also displayed that CA deficient mice are more resistant to epileptic seizure and
these mice also showed less mortality in repeated seizures [13]. CA VII isoform also play vital role in the generation
of seizure and widely expressed in the cortex as well as hippocampus region of brain [14]. It is studied that CA VII
promotes seizure generation via GABAergic excitation and Ruusuvuori et al. experimentally proved that this isoform
is also actively involved in febrile seizures onset by activating GABAergic excitation in brain [15,16]. During the
seizure episode, extracellular potassium concentration enhances due to GABAergic depolarization and intense
-
neuronal firing depolarizes GABAA-mediated responses which are linked with HCO3 , efflux induced by GABAA
receptors [16,17]. Rusuvuori et al. have also revealed that these GABA associated electrophysiological activities are
under the control of brain CA VII [16a]. Bicarbonate reflux through GABAA receptor produces external alkalosis,
supplied by intracellular CA VII and alkalosis may influence pathological incidents such as epileptic seizures [18,19].
Presently, a number of potent CA inhibitors such as acetazolamide (AAZ), zonisamide (ZNS), methazolamide (MZA)
and topiramate (TPM) possess good anti-convulsant activity (Chart-1) and these potent CA inhibitors are still used
by clinicians against numerous forms of seizures such as partial, myoclonic, generalized tonic-clonic and absence
seizures [20,21,22].
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Chart 1: CA Inhibitors with potent anti-epileptic activity
However, it is clinically proven that some of these CA inhibitors do not show full effectiveness in long term therapy
of epilepsy [23]. Therefore, development of potent CA inhibitors is required, which hold reasonable anti-convulsant
activity along with long lasting efficacy. Aromatic sulfonamide moiety (Ar-SO2-NH2) has classical recognition to
bind to the active site of CA, with deprotonated sulfonamide moiety coordinating to the Zn2+ in the active site,
impairing the catalytic acivity of these highly efficient enzymes [24,25,26]. In the discovery of potent CA inhibitors,
the installation of aryl/ heteroaryl sulfonamide moieties has provided various clinically useful therapeutic agents such
as anti-glucoma, diuretics, anti-high altitude sickness drugs, as well as AEDs. Additionally, our research group has
been constantly utilizing this functionality and numerous potent CA inhibitors are being reported, investigated for
their inhibitory action against CA isoforms, with many inhibitors displaying a low nanomolar activity range against
various such enzymes [27,28,29,30]. Several sulfonamide /sulfamate bearing CA inhibitors have been also developed
which are endowed with excellent anti-convulsant activity in vivo model of epilepsy [31]. Continuing our interest in
the design and development of potent CA inhibitors, herein we report synthesis, CA inhibition and in-vivo
anticonvulsant activity of new 4-(2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazinyl)benzenesulfonamides
(series 1; 4a- 4i) and 4-(2-(4-(4-substituted piperazin-1-yl)benzylidene) hydrazinecarbonyl)benzenesulfonamides
(series 2; 8a- 8k).
2. Results and Discussion
2.1. Compound design and synthesis
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We aimed to design two series of novel CA inhibitors by utilizing the well studied tail approach for potent CA
inhibitor development [32]. We chose benzenesulfonamide as a pharmacophoric head, which has attracted much
attention in this field [33, 34]. In due course, several benzenesulfonamide based compounds have been prepared by
installing a wide range of alkyl, aromatic, heterocyclic, or sugar scaffolds and these derivatives displayed excellent
inhibitory action against various CA isoforms of medicinal importance, such as CA II, CA IV, CAVII, CA IX, CA
XII and CAIV [33,34,35,36]. Such category of compounds, mostly containing three key molecular fragments; the
benzenesulfonamide head, spacer/linker and alkyl/aryl/heteroaryl tail region [34,36] (Chart-2). It is also observed
that the introduction of polar spacers/linkers offer numerous potent CA inhibitors including SLC-0111 which contain
urea as a linker between head and tail region, completed Phase I clinical trials for the therapy of metastatic hypoxic
tumors [33,34,37,38]. 4-(4-Phenyl-[1,2,3]triazol-1-yl)-benzenesulfonamide (PTB, chart 2) and its analog 3 have also
shown potent CA inhibition. Piperazine is also a well studied flexible heterocyclic compound in drug discovery
research [39].
Chart: 2. Designing strategy of novel CA inhibitors (series1; 4a-k and series 2; 8a-m)
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Piperazine containing molecules have shown a wide range of biological activity, including CA inhibitory action
[39,40]. Recently, we have also reported novel benzenesulfonamide derivative 4 as a potent CA inhibitor and
anticonvulsant agent [37e]. Several research reports disclosed that piperazine bearing compounds have shown very
effective CA inhibition with nanomolar range affinity [41]. Another aspect of the piperazine ring is its flexible nature
and this scaffold is widely used to enhance molecular flexibility to influence the adjustment of molecule within active
sites of enzymes [41]. Considering these facts, the present study was designed to explore new 4-(2-(4-(4-substituted
piperazin-1-yl)benzylidene)hydrazinyl)benzenesulfonamides
(4a-4i)
and
4-(2-(4-(4-substitutedpiperazin-1-
yl)benzylidene) hydrazinecarbonyl)benzenesulfonamides (8a-8k) as potent CA inhibitors. In the first series, the
benzenesulfonamide head was tethered with substituted piperazine tails by using the benzylidenehydrazine spacer.
Introduction of piperazine with aromatic tail is thought to bestow flexibility to the tail region, which may enhance
activity and selectivity against different CA isofoms. Furthermore, we replaced the benzylidenehydrazine spacer with
the more polar spacer benzylidenehydrazine carbonyl, in order to obtain 4-(2-(4-(4-substitutedpiperazin-1-
yl)benzylidene) hydrazinecarbonyl)benzenesulfonamides (8a-8k) and studied whether this replacement will produce a
better inhibitory activity and selectivity for CA isoforms. Various substituted piperazines were installed as tail region
in both series to achieve conclusive SAR . Some of them such as furoyl, benzoyl, benzyl carbamate, pyridine and
pyrimidine substituents are well used to produce biological active molecules [39b]. To achieve the target compounds
of both series, the following synthetic strategy has been adopted. For the synthesis of 4-(2-(4-(4-substitutedpiperazin-
1-yl)benzylidene)hydrazinyl) benzenesulfonamides (4a-4i, Scheme 1), commercially available p-fluorobenzaldehyde
1 was coupled with substituted piperazines 2a-2i using K2CO3/Na2CO3 as a base, leading to the key intermediates 4-
(4-substituted piperazin-1-yl) benzaldehydes 3a-3i. Compounds 3a-3i were then reacted with 4-
hydrazinobenzenesulfonamide in acidic medium (acetic acid) to yield the target compounds 4a-4i.
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Compound No
R-Group
4a
4b
4c
4d
4e
4f
4g
4h
4i
Scheme 1: Reagent & conditions: A; K2CO3/Na2CO3, DMF, reflux (90-100°C), B; Glacial acetic acid, ethanol, reflux (90°C).
Next, 4-sulfamoylbenzoic acid was used as starting material for the synthesis of 4-(2-(4-(4-substituted piperazin-1-yl)
benzylidene)hydrazinecarbonyl)benzenesulfonamides (Scheme 2). The acid group of 4-sulfamoylbenzoic acid (5)
was activated by converting it into methyl 4-sulfamoylbenzoate (6). Methyl 4-sulfamoylbenzoate (6) was treated with
hydrazine hydrate in order to obtain 4-(hydrazinecarbonyl)benzenesulfonamide (7). Condensation of 7 with the
corresponding formyl derivatives 3a-3k in ethanol provided the target compounds 4-(2-(4-(4-substituted piperazin-1-
yl)benzylidene)hydrazinecarbonyl)benzenesulfonamides 8a-8k in high yields. The synthesized target compounds
1
were purified by column chromatography, followed by re-crystallization and have been fully characterized with H
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13
NMR, C NMR and mass spectroscopy. Purity of all target compounds has been assessed by reverse phase HPLC
and was > 95%.
Compound No
8a
R-Group
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
Scheme 2: Reagent &conditions: A, Dried methanol, sulfuric acid reflux, 8h; B, Hydrazine hydrate, 1, 4 dioxane, reflux
(100°C), 8h; C, Ethanol, glacial acetic acid, reflux (90°C), 8h
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Additionally, synthesized target compounds (4a-4i and 8a-8k) were screened to detect as a pan assay interference
molecules (PAINS) which might be perturb biological activity [42]. According to SwissADME [43] and FAFDrugs4
[44] tools, none of these target compounds restrain substructural element documented as PAINS (supplementary table
ST1).
2.2. Carbonic anhydrase inhibitory activity: The synthesized novel compounds (4a-4i; 8a-8k) were evaluated for
their CA inhibitory activity against four CA isforms of human origin; hCA I, hCA II, hCA VII, and hCA IX. Among
them, hCA II and hCAVII have been well studied for their role in epileptogenesis and seizure associated activity [14,
15, 16]. Obtained results have been resided in Table 1 and following structure activity relationship (SAR) was
observed on the basis of inhibitory pattern:
i) Majority of synthesized compounds in both series did not display potent inhibitory action against hCA I isoform,
generally considered as off target for epilepsy. However, compounds bearing benzyl carbamate (4g) and furoyl group
(4i) were shown a medium range inhibitory potential against hCA I with a Ki value of 919.6 nM and 321.8 nM,
respectively.
ii) Results indicate that synthesized derivatives exhibited medium to high nanomolar range inhibitory activity towards
physiologically dominant hCA II isoform. Although, derivatives containing benzyl carbamate (4g) and furoyl group
(4i) at the terminal end along with benzylidenehydrazine spacer bestowed satisfactory inhibitory action for hCA II
isoform. Benzyl carbamate derivative and furoyl derivative have shown a Ki value of 93.9 nM and 30.8 nM,
respectively. In this benzylidenehydrazine series, compounds with p-fluorophenyl (4b) and o, p-dimethylphenyl (4d)
displayed medium nanomolar range inhibitory activity with a Ki value of 559.1 nM and 552.6 nM, respectively for
hCA II. In benzylidenehydrazine carbonyl series, compounds with trifluoromethyl (8c), benzoyl (8h) and flexible
side chain benzyl (8j) also have appeared as a medium nanomolar range inhibitor for hCA II, showed a Kis value
range 307-555 nM. It was noticed that most of compounds which displayed medium nanomolar range inhibitory
action against hCA II were also appeared to be selective over hCA I and hCA IX.
iii) It was observed that most of the synthesized compounds effectively inhibited hCA VII isoform with low
nanomolar to medium nanomolar range Kis values. In the first series, compounds holding benzyl carbamate and
furoyl tails demonstrated low nanomolar inhibitory activity for hCA VII. Benzyl carbamate piperazine derivative (4g)
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showed a Ki value of 11.4 nM while furoyl piperazine derivative (4i) exhibited an impressive Ki value 6.2 nM.
Interestingly, in benzylidenehydrazine carbonyl series, compound containing o, p-dimethyl piperazine (8d) tail was
found to be a most selective hCA VII inhibitor among whole series. This compound displayed low nanomolar
inhibitory action (17.8 nM) with satisfactory selectivity over other tested isoforms. Compound 8d showed 561, 527
and 93 fold selective over hCA I, hCII, and hCA IX isoform. Compounds with phenyl (4a), p-fluorophenyl (4b) and
o,p- dimethylphenyl (4d) side chain of benzylidenehydrazine series have also shown satisfactory inhibitory action
towards hCA VII isoform. In addition, compound possess benzoyl (8h), furoyl (8i) and benzyl (8j) tails along with
benzylidenehydrazine carbonyl spacer between the head and tail region also bestowed reasonable inhibitory potential
and displayed Kis values below 100 nM. Other derivatives such as 4c, 4e, 4h, 8a-8c, 8e-8g showed moderate hCA
VII inhibitory action with Kis value ranges 118-180 nM. However, pyrimidine (4f) and piperonyl (8k) substituted
derivatives did not appear to be potent inhibitor of hCA VII and these compounds showed high nanomolar range Kis
value (606-750 nM).
iv) Cancer associated isoform hCA IX was poorly inhibited by most of synthesized derivatives. Most of derivatives
showed high nanomolar range inhibitory activity and exhibited Kis value ranges 1046-2491nM. Although,
compounds of benzylidenehydrazine carbonyl series, substituted with furoyl (8i) and piperonyl (8k) hetrocyclic
group were appeared as effective hCA IX inhibitors and showed a Ki value of 30.4 and 95.2 nM, respectively. It was
also noticed that four compounds, 4e, 8a, and 8e, are medium potency inhibitors for hCA IX. Thus, it seems that
benzylidenehydrazine carbonyl spacer bestowed some effective hCA IX inhibitor, while benzylidenehydrazine spacer
failed to produce such action.
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Table 1. Inhibition data of hCA I, II, VII and IX with compounds 4a-4k, 8a-8m & AAZ
Compound no R-group
Ki (nM)
hCA II
hCA I
hCA VII
hCA IX
7862.0
2377.3
9467.2
4105.9
887.5
559.1
7524.5
552.6
98.4
49.6
118.2
72.0
1243.6
1272.6
1931.7
1738.1
4a
4b
4c
4d
>10000
>10000
4590.8
8396.7
135.3
606.7
235.3
4e
4f
2231.8
919.6
93.9
11.4
127.4
6.2
1631.4
2491.8
1788.6
4g
4h
4i
>10000
321.8
2792.7
30.8
>10000
5795.9
8164.3
4131.1
822.3
555.7
180.8
147.1
147.7
275.7
8a
8b
8c
1956.6
1853.6
8d
>10000
9385.9
17.8
1660.9
>10000
2061.1
4557.9
963.1
125.9
132.2
151.5
8e
8f
1841.0
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9638.6
1447.5
121.5
1903.1
8g
5503.9
9489.9
307.9
686.5
94.7
78.8
1046.3
30.4
8h
8i
7101.7
8368.4
413.6
962.2
41.5
162.7
95.2
8j
750.9
8k
AAZ
250
12.1
2.5
25.8
*Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-10% of the reported values).
v) Overall, the SAR investigation revealed that introduction of benzylidenehydrazine spacer between
benzenesulfonamide head and aryl/ heteroaryl tail, provided effective hCA II and hCA VII inhibitors, while more
polar benzylidenehydrazine carbonyl spacer bestowed only effective and selective hCA VII inhibitors. Our results
indicated that compounds 4g as well as 4i were very effective hCA II and hCA VII inhibitors and compound 8d was a
very selective hCA VII inhibitor which also displayed low nanomolar range inhibition against hCA VII. Therefore,
these three compounds were selected to explore their anti-convulsant action in preclinical animal models of epilepsy.
2.3. In vivo studies
2.3.1. Anti-convulsant action: CA isoforms, CAI-CAVIII, X, XI, XII and XIV are generally present in the brain and
associated with numerous neurosignaling/neuromodulatory functions [14,15]. The role of CA II and CA VII in
epileptogenesis is well studied and its inhibition either suppresses or blocks epileptic seizure [15,16]. It is also
evidence that many potent hCA II and hCA VII inhibitors including AAZ and TPM showed excellent anti-epileptic
effects. Compounds 4g, 4i, and 8d reported here displayed potent hCA II and hCA VII inhibitory activity, therefore,
anti-seizure effect of these compounds was evaluated in vivo models of epilepsy. In addition, two standard anti-
epileptic drugs AAZ and TPM were taken as a reference to correlate the findings. The evaluation of new molecules
for their anti-epileptic activity mainly relies on maximal electroshock test (MES) and pentylenetetrazole (PTZ) test.
These two tests are considered as “gold standards” for evaluation of novel anti-epileptic drug as compared to other
test method [45]. Generally, it is observed that most of the AEDs are found to be effective in either both tests or at
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least one test. Thus, in this study, we also included these two gold standard tests to evaluate anti-convulsant effect of
most active hCA II and hCAVII inhibitors i.e. 4g, 4i, and 8d.
2.3.1.1. Maximal electroshock method: MES test is considered to be very authentic method for identifying new
anti-convulsants which block seizure through neuronal tissue [46]. In this test, animals receive an electric stimulus of
adequate intensity to induce tonic-clonic generalized seizures and they are very similar to be human generalized
seizures. In this experiment, anti-convulsant activity of compounds 4g, 4i, and 8d was evaluated upon 30 and 100
mg/kg dose administration at two time intervals of 0.5 h and 3 h. Observed anti-convulsant activity of these three
compounds in this test is depicted in Table 2. Our results indicate that compound 4g at 30 mg/kg dose showed 50%
seizure protection at 0.5 h, the activity was slightly reduced at 3 h and displayed 33% seizure protection. 100 mg/kg
dose of this derivative, also showed 50% protection at 0.5 h time interval similar to 30mg/kg dose. However, at 3 h
time interval compound (4g) exhibited almost 67% seizure protection, which indicate it’s quick and long duration
action against MES induced seizure. Compound 4i (30 mg/kg) had shown stable seizure protection at both time
intervals, displayed 50% protection at 0.5 and 3 h against MES induced seizures. Although, 100 mg/kg dose of this
compound showed excellent protection at 0.5 h and forcefully defend 83% animals against MES provoked seizures.
However, activity of this compound decreased at 3 h time interval and protected only 50% of the animals. This
compound possesses fast onset of action and short duration of seizure protection capability. Interestingly, compound
8d at lower dose (30 mg/kg) demonstrated excellent protection against MES induced seizure and its activity was
found to be sustained up to 3 h after drug treatment. This compound displayed 67% protection at 0.5 h as well as 3 h
time intervals. Higher dose (100 mg/kg) of this compound also showed 50 % seizure protection at 0.5 h and 3 h time
intervals. These findings clearly indicate that all three derivatives have shown reasonable protection against MES
provoked seizures; however, compound 8d was appeared very effective as compared to other two derivatives 4g and
4i.
2.3.1.2. Subcutaneous Pentylenetetrazole Test: PTZ is a strong chemoconvulsant and work as a γ-amino butyric
acid (GABA) receptor antagonist that blocks GABAA receptor subtype in the brain. Administration of PTZ induces
strong myoclonic and absence seizure in experimental animals that mimics seizures induced in human [47]. It is well
validated pre-clinical test, being widely used to discover potent anticonvulsant agent which blocks seizure directly or
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indirectly through GABA receptor. Several studies have shown that hCAs influence GABAA activity through HCO3
in the brain, especially hCAII and hCA VII are the main player. It is also seen that potent CA inhibitor AAZ and
TPM also inhibit sc-PTZ induced seizures in experimental animals. Therefore, anti-convulsant effect of 4g, 4i, and 8d
have been also evaluated against PTZ induced seizures test at two doses 30 mg/kg and 100 mg/kg. In sc-PTZ test,
compound 4g (30 mg/kg) was able to protect 50% animals against sc-PTZ induced seizure at 0.5 h. At the same dose
4g displayed almost 67% seizure protection at 3 h time interval, which indicates stable activity of this compound at
lower dose of 30 mg/kg. Whereas, higher dose (100 mg/kg) of this compound showed fast action, but activity was
unfortunately not retained up to 3 h and exhibited 67% and 33% seizure protection at 0.5 and 3 h time interval,
respectively. A stable activity trend was also observed with compound 4i at 30mg/kg dose, although this compound
was less effective against sc-PTZ induced seizure and demonstrated 33% protection at both time intervals (0.5 and 3
h). However, some improvement was observed at 100 mg/kg dose of this compound and it displayed 67% and 50%
seizure protection at 0.5 and 3 h, respectively. Noticeably, compound 8d (30 mg/kg) has shown 50% seizure
protection at 0.5 h, activity was enhanced over the time and exhibited 67% protection at 3 h. 100 mg/kg dose of
compound 8d displayed 50% protection at 0.5 h and 83% protection at 3 h time duration. Thus, compound 8d
appeared as potent anti-convulsant agent which has shown higher protection against sc-PTZ induced seizures as
compared to standard drugs AAZ and TPM. Overall, in vivo anticonvulsant studies revealed that compound 8d has
shown very significant protection against MES as well as sc-PTZ induced seizures at a lower dose of 30 mg/kg. Thus,
this compound was chosen for advance anti-convulsant studies such as oral bioavailability, time course, and
neurotoxicity evaluations.
Table 2: In vivo anticonvulsant activity of compounds 4g, 4i and 8d against MES and sc-PTZ seizure tests
Compounds (aDose
in mg/kg)
MESb screen
scPTZc screen
0.5 h 3.0 h
0.5 h
3.0 h
4g (30)
4g (100)
4i (30)
3/6
3/6
3/6
5/6
4/6
3/6
2/6
4/6
3/6
3/6
4/6
3/6
3/6
4/6
2/6
4/6
3/6
3/6
4/6
2/6
2/6
3/6
4/6
5/6
4i (100)
8d (30)
8d (100)
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TPM (30)d
TPM (100)d
AAZ (30)d
AAZ (100)d
8/8
NT
7/8
8/8
8/8
NT
4/8
4/8
3/6
2/6
3/6
5/6
3/6
3/6
3/6
3/6
a30 and 100 mg/kg doses were administered intraperitoneally (i.p.). The animals were examined at 0.5 and 3.0 h after injections.
bMaximal electroshock test (MES) (n = 6 mice for each tested dose). scPTZ test (n = 6 mice for each tested dose). dData taken
from reference [37e]. NT: not tested.
2.3.1.3. MES seizure protecting capability of compound 8d upon oral administration: Efficacy of compound 8d
against MES induced seizure was also examined by providing orally to rats. Oral efficacy of novel chemical entity is
considered as an important feature to develop as a successful therapeutic agent. In this experiment, anti-MES activity
of compound 8d has been evaluated at five time intervals (0.25- 4 h) after its oral administration (Table 3).
Table 3: Anti-MES potential of compounds 8d (30 mg/kg, bwt) after oral administration.
Time (h)
MES testa
1/6
% Protectionb
0.25
0.5
1
17
84
84
67
50
5/6
5/6
2
4/6
4
3/6
b
aMES test (number of animals protected/number of animals tested; n=6). Percentage seizure protection (number of animals
protected/number of animals tested x 100).
These results indicate that compound 8d has shown only 17% protection after 0.25 h oral treatment as expected,
because oral administration takes more time to display action as compared to intraperitonial (i.p) or intravenous (i.v)
administration. Compound 8d enhanced protection ability after 0.5 h of oral administration and displayed 84%
protection against tonic-clonic seizures. The activity was found to be sustained up to 1 h and similar percentage
protection (84%) was observed at 0.5 h. Slightly less activity was noticed at 2 h time interval and this compound
displayed 67% protection against MES induced seizures. Finally, this compound protected 50% of tested animals
against MES induced seizure after 4 h of oral administration. Thus, it is noticed that compound 8d has shown
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excellent protection against tonic-clonic seizures up to 4h of oral administration. However, 0.5 to 1h after oral
administration may be considered as a best peak time effect of compound 8d where it displayed 84% protection.
Hence, compound 8d appeared as a potent orally active anti-convulsant agent that appreciably abolished MES
induced tonic-clonic seizures upon oral administration to rats.
2.3.1.4. Time course anti-convulsant study: Long duration action of drug molecule is regarded as a useful property
which may reduces the chance of drug intolerance and repeated dose associated side effects. Therefore, a time course
study of anti-convulsant agent 8d was carried out in Swiss Albino male mice at 30 mg/kg (i.p) against MES induced
seizure and anti-convulsant effect was monitored up to 6 h of drug administration (Table 4). Results indicated that the
compound 8d significantly attenuated MES provoked seizure at 0.5 h time interval and displayed 70% seizure
protection. At 1h time point this compound protected 60% animals and activity was sustained up to next 2 h. Further,
protective ability of this compound was increased and demonstrated 70% protection against MES provoked seizure at
3 h. After 4h of drug administration this compound successfully abolished tonic-clonic generalized seizures in 50% of
the animals. Lastly, at 6 h time point compound 8d shielded 60% tested animals against MES induced generalized
seizures. Thus, this time course study evidently supports long duration action of novel CA inhibitor 8d, which
forcefully protected animals against MES provoked seizures up to 6 h of drug administration.
Table 4: Time course study of compound 8d (30 mg/kg, bwt) in the MES test
Time (h)a
MES Testb
7/10
% Protectionc
0.5
1
70
60
60
70
50
60
6/10
2
6/10
3
7/10
4
5/10
6
6/10
aTime after drug treatment.bMES test (n=10; number of protected animals/total number of tested animals). cPercentage protection
(number of protected animals/ total number of tested animals x 100).
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2.3.2. Neurotoxicity assessment: It is well studied that neurotoxicity induced by any drug molecule affects motor co-
ordination system of the body. The rotarod test is considered as a simple and validated test to observe neurotoxicity
associated motor co-ordination failure [40e]. Therefore, we planned to evaluate the neurotoxic effect of compound 8d
(30 mg/kg; i.p.) by using the rotarod test at different time intervals (30, 60, 90, 120 min) after compound
administration (figure 1). To examine neurotoxicity, vehicle treated and compound 8d pre-treated mice were placed
on a rotating rod for 180s. Results indicated that compound 8d and vehicle pre-treated mice retained almost equal
time after 30, 60, 90 and 120 min of treatment. Thus, results signified that compound 8d did not display any
significant neurotoxicity at active dose 30 mg/kg.
Rotarod Test
200
Control group
150
Compound 8f treated group
100
50
0
Time (min) after drug admisnistration
Figure 1: Time spent on the rotarod in second up to 120 min after the vehicle and compound 8d (30mg/kg, i.p.) treatment. Each point
indicates the mean ± S.E.M (n= 8, two-way repeated measures ANOVA followed by Bonferroni post-test).
2.3.3. Sub-acute toxicity study: Toxicity of biologically active agents is considered as a major barrier in the
development of potent therapeutic agents. Numerous potent pre-clinically active pharmacological agents were not
approved for clinical use due to their toxic nature [40d]. Therefore, a sub-acute toxicity study has been performed to
evaluate safety nature of novel compound 8d in normal, healthy Wistar rats at the dose of 100 mg/kg/bwt. During the
whole experimental period, no signs of clinical toxicity or change in the animal’s behavior and no death were
observed. All animals took the normal amount of food and water during the whole experimental period. The results
showed that compound 8d treatment did not induce any noteworthy alteration in hematological parameters such as
WBC, RBC and platelet count as compared to vehicle treated animals (Table 5).
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Table 5: Hematological analysis upon oral administration of vehicle and compounds 8d for 14 days in male Wistar rats.
Parameters
Control(vehicle, po)
Compound 8d (100mg/kg,
po)± SDa
Hb (g/dl)
13.1 ± 0.9
7.88 ± 2.5
23.4 ± 2.9
72.3 ± 6.7
2 ± 1.4
12.2 ± 1.5
9 ± 2.3
23.1 ± 3.7
71.5 ± 3.7
2.6 ± 1.7
2 ± 1.2
0.33 ± 0.5
7.2 ± 0.44
622.3 ± 132.1
TLC (103/ml)
Neutrophils (%)
Lymphocytes (%)
Eosinophils (%)
Monocytes (%)
Basophils (%)
2.6 ± 1.5
0.16 ± 0.4
7.9 ± 0.56
760.8 ± 179.9
RBC (mill/mm3)
Platelet count (thou/mm3)
aThe data are presented as the mean ± standard deviation; n = 6 in each group.
Thus, this compound did not display significant toxicity in the hematological system of animals. Further, toxic effect
of compound 8d on liver function was also analyzed and results showed that continuous treatment of compound 8d
restored optimum level of liver associated biomarkers such as serum glutamate pyruvate transaminase (SGPT), serum
glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), total bilirubin, and total protein similar to
control animals. This analysis evidently proved that compound 8d did not induce liver toxicity in experimental
animals (Table 6).
Table 6: Evaluation of Liver function associated biomarkers after compounds 8d treatment for for 14 days in rats.
Parameters
Control(vehicle, po)
Compound 8d (100mg/kg,
po)± SDa
SGOT (U/l)
69.9 ± 7.7
65 ± 13.9
SGPT (U/l)
Alkaline Phosphatase (U/l)
58.8 ± 18.8
113.2 ± 15.1
60.3 ± 15.2
96.5 ± 21.4
Total bilirubin(mg/dl)
Total protein (g/dl)
0.37 ± 0.11
6.43 ± 0.58
0.35 ± 0.10
6.62 ± 0.73
aThe data are presented as the mean ± standard deviation; n = 6 in each group.
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Furthermore, we have also examined several renal toxicity parameters after 14 days treatment of compound 8d. The
result clearly indicated that compound 8d did not produce any significant renal toxicity and associated biomarkers
such as creatinine, urea and uric acid level were found optimum, being similar to control animals. Hence, in this
toxicological study compound 8d appeared as a non-toxic chemical entity which had not shown any significant
toxicity on liver function, renal function and hematological function (Table 7). Finally, compound 8d has been
proven as a safe CA inhibitor, which also endows excellent anti- convulsant action.
Table 7: Assessment of Kidney function associated biomarkers after oral administration of vehicle and compound 8d up to 14 days
in rats.
Parameters
Control (vehicle, po)
Compound 8d (100mg/kg,
po)± SDa
Blood urea (mg/dl)
Creatinine (mg/dl)
Uric acid (mg/dl)
Calcium (mg/dl)
40.7 ± 3.3
0.62 ± 0.3
3.9 ± 2.8
10.5 ± 1.5
6.71 ± 1
39.6 ± 4.1
0.75 ± 0.1
4.33 ± 2.47
10 ± 1.2
Phosphorus (mg/dl)
6.48 ± 1.5
Na+ (mEq/l)
K+ (mEq/l)
Cl - (mEq/l)
143.8 ± 2.3
4.79 ± 0.45
104.1 ± 6.2
142.5 ± 2.1
4.83 ± 0.38
105.6 ± 7.4
a The data are displayed as the mean ± standard deviation; n = 6 in each group.
3. Conclusion
Herein, two series of novel benzenesulphonamide derivatives (4a- 4i; 8a-8k) as potent CAs inhibitors have been
synthesized using benzylidenehydrazine/ benzylidenehydrazine carbonyl spacer between benzesulphonamide head
and substituted piperazine tails. SAR studies revealed that benzylidenehydrazine spacer containing derivatives
showed moderate to good inhibitory action against hCA II and hCA VII, while benzylidenehydrazine carbonyl spacer
bearing derivatives displayed effective and selective inhibition against hCA VII. Most of these derivatives poorly
inhibited hCA I as well as hCA IX. The results exhibited that compounds 4g and 4i inhibited h CA II as well as hCA
VII very effectively and compound 8d was effectively inhibited only hCA VII. These three potent inhibitors
noticeably inhibited MES as well as sc-PTZ induced seizures in mice. Among these three, compound 8d appeared
most potent anti-convulsant agent which bestowed almost similar potency as standard drug AAZ. Additionally,
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Compound 8d appeared as a long duration acting anti-convulsant without exhibiting significant neurotoxicity. This
compound also appeared to be orally active anti-convulsant agent and powerfully abolished MES induced seizure in
rats upon oral administration. In sub-acute toxicity study, this compound showed non-toxic behavior and did not
provoke significant toxicity to liver, kidney and normal physiology.
4. Experimental Section
Chemistry: Reagents were acquired from Sigma-Aldrich (St. Louis, MO, USA) and TCI (Tokyo, Japan) and solvents
were obtained from Merck (Darmstadt, Germany). Thin layer chromatography (TLC) was performed on
commercially accessible silica gel (Kieselgel 60, F254) coated on aluminium sheets (Merck) by using
methanol/chloroform as mobile phase. Purification of synthesized compounds was carried out by column
chromatography using 100−200 mesh (Merck) silica gel as stationary phase and chloroform: methanol as mobile
phase. Hicon melting point apparatus (Hicon, India) was used to determine melting points of all the compounds and
mass spectra of compounds were recorded with ESI-LC/MS (Agilent 6310 mass spectrometer). NMR spectra were
taken by Jeol-400 MHz NMR spectrophotometer (USA), DMSO-d6 used as a solvent and tetramethylsilane (TMS) as
an internal standard. Chemical shifts (δ) were described in parts per million relative to standard TMS, and the
following abbreviations were employed to express obtained peak patterns: s, (singlet); d, (doublet); t, (triplet); m,
(multiplet); brs, (broad singlet). The purity level of test compounds was investigated with reverse phase Shimadzu
HPLC (Kyoto, Japan) coupled with a PDA detector and C-18 column. Samples were prepared in methanol and
acetonitrile (50:50) and an injection volume of 20 ꢀL was injected. Methanol + acetonitrile were employed as the
mobile phase gradient, and the flow rate was fixed at 1 mL/min. All target compounds demonstrated more than 95 %
purity. The target compounds were
also screened for PAINS alerts by using SwissADME
(http://www.swissadme.ch/) and FAFDrugs4 (http://fafdrugs4.mti.univ-paris-diderot.fr/) software tools.
4.1. General procedure for synthesis of 4-(4-substituted piperazin-1-yl)benzaldehyde (3a-k)
Intermediates (3a-k) were synthesized according to our previously reported method and their spectral data were
matched with our earlier publication [40a] and supplementary material.
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4.2.
General
procedure
for
the
synthesis
of
(E)-4-(2-(4-(4-substitutedpiperazin-1-
yl)benzylidene)hydrazinyl)benzenesulfonamide (4a-4i; scheme1)
An equimolar mixture of formyl derivatives (3a-3i) and 4-hydrazinobenzenesulfonamide were refluxed in ethanol
with a catalytic amount of glacial acetic acid for 6-8 h. Reaction was monitored with TLC and appeared precipitate
was filtered, washed with hot ethanol to accomplish target compounds 4a-4i. Target compounds 4a-4i were purified
by column chromatography using CHCl3: MeOH (95: 05) as eluent followed by re-crystallization by using ethanol
4.2.1. (E)-4-(2-(4-(4-phenylpiperazin-1-yl)benzylidene)hydrazinyl)benzenesulfonamide (4a)
1
Yellowish-white solid; yield 88%; mp: 216-218°C; H NMR (DMSO-d6,400 MHz):δ 3.24-3.29 (m, 4H, piperazine,
CH2), 3.33-3.40 (m, 4H, piperazine, CH2), 6.80 (t, 1H, Ar, J= 7.2Hz), 6.97-7.09 (m, 8H, Ar + NH2), 7.23 (t, 2H, Ar,
13
J= 7.6Hz), 7.55 (d, 2H, Ar, J= 8.4Hz), 7.63 (d, 2H, Ar, J= 8.4Hz), 7.85 (s, 1H, CH), 10.55 (s, 1H, NH); C NMR
(DMSO-d6, 100 MHz): δ 47.6, 48.1, 110.7, 115.1, 115.6, 119.1, 125.7, 127.1, 127.4, 128.9, 132.7, 139.5, 148.0, 150.8,
151.0; LC–MS: m/z; 436 (M+1). HPLC purity: 97.9 %.
4.2.2. (E)-4-(2-(4-(4-(4-fluorophenyl)piperazin-1-yl)benzylidene)hydrazinyl)benzenesulfonamide (4b)
1
Creamish solid; yield 78%; mp: 225-227°C; H NMR (DMSO-d6,400 MHz):δ 3.19-3.23 (m, 4H, piperazine CH2),
3.36 (s, 4H, piperazine, CH2), 7.01-7.09 (m, 10H, Ar+ NH2), 7.54 (d, 2H, Ar, J= 9.1Hz), 7.62 (d, 2H, Ar, J= 9.1Hz),
13
7.85 (s, 1H, CH), 10.55 (s, 1H, NH), C NMR (DMSO-d6, 100 MHz): δ 47.6, 49.0, 110.7, 115.2 (d, J= 22Hz), 117.4,
125.7, 127.2, 132.7, 139.5, 147.7, 148.0, 151.0, 156.1 (d, J= 235 Hz); LC–MS: m/z; 454 (M+1). HPLC purity: 98.5 %.
4.2.3. (E)-4-(2-(4-(4-(trifluoromethyl)phenyl)piperazinyl)benzylidene)hydrazinyl)benzenesulfonamide (4c)
1
Creamish solid; yield 68%; mp: 210-212°C ; H NMR (DMSO-d6,400 MHz):δ 3.34-3.47 (m, 8H, piperazine, CH2),
7.12-7.00 (m, 8H, Ar + NH2), 7.51-7.56 (m, 4H, Ar), 7.62 (d, 2H, Ar, J= 9.1Hz), 7.85 (s, 1H, CH), 10.55 (s, 1H, NH),
13C NMR (DMSO-d6, 100 MHz): δ 46.7, 47.2, 110.8, 114.3, 115.0, 118.2, 123.6, 126.2 (q, J=21 Hz), 126.8 (q, J=166 Hz),
132.7, 139.5, 148.0, 150.8, 153.0; LC–MS: m/z; 504(M+1). HPLC purity: 99.9 %.
4.2.4. (E)-4-(2-(4-(4-(2,4-dimethylphenyl)piperazin-1-yl)benzylidene)hydrazinyl)benzenesulfonamide (4d)
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Yellow solid; yield 76%; mp:204-206°C; 1H NMR (DMSO-d6,400 MHz):δ 2.20 (s, 3H, CH3), 2.23 (s, 3H, CH3), 2.93
(t, 4H, piperazine, CH2, J= 4.9Hz), 3.31-3.32 (m, 4H, piperazine, CH2), 6.94-7.09 (m, 9H, Ar+ NH2), 7.54 (d, 2H, Ar,
J= 8.4Hz), 7.62 (d, 2H, Ar, J= 9.1Hz), 7.85 (s, 1H, CH), 10.54 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): δ 17.4,
20.3, 48.1, 51.4, 110.7, 114.9, 118.6, 127.1, 131.5, 131.8, 132.7, 139.6, 148.0, 148.5, 151.2; LC–MS: m/z; 464(M+1).
HPLC purity: 96.7 %.
4.2.5. (E)-4-(2-(4-(4-(pyridin-2-yl)piperazin-1-yl)benzylidene)hydrazinyl)benzenesulfonamide (4e)
1
Creamish solid; yield 82%; mp: 227-229°C; H NMR (DMSO-d6,400 MHz):δ 3.43 (s, 4H, piperazine, CH2), 3.88(s,
4H, piperazine, CH2), 6.92-7.10 (m, 7H, Ar+ NH2), 7.39(d, 1H, Ar, J= 9.1Hz), 7.55 (d, 2H, Ar, J= 8.4Hz), 7.62 (d,
2H, Ar, J= 9.1Hz), 7.89 (s, 1H, CH), 7.98-8.05 (m, 2H, Ar), 10.70 (brs, 1H, NH), 13C NMR (DMSO-d6, 100 MHz): δ
45.4, 46.5, 110.7, 111.8, 112.2, 112.8, 115.0, 126.0, 127.3, 132.7, 137.8, 139.4, 143.1, 148.0, 150.2, 152.1; LC–MS: m/z;
437(M+1). HPLC purity: 99.1 %.
4.2.6. (E)-4-(2-(4-(4-(pyrimidin-2-yl)piperazin-1-yl)benzylidene)hydrazinyl)benzenesulfonamide (4f)
1
Yellowish-white solid; yield 89%; mp: 193-195°C; H NMR (DMSO-d6,400 MHz):δ 3.28 (s, 4H, piperazine CH2),
3.87 (s, 4H, piperazine, CH2), 6.64 (t, 1H, Ar, J= 4.5Hz), 6.99-7.09 (m, 6H, Ar+ NH2), 7.54 (d, 2H, Ar, J= 8.4Hz),
13
7.63 (d, 2H, Ar, J= 9.1Hz), 7.85 (s, 1H, CH), 8.38 (d, 2H, Ar, J= 4.5Hz), 10.54 (s, 1H, NH), C NMR (DMSO-d6,
100 MHz): δ 43.0, 47.5, 110.3, 110.8, 115.2, 125.7, 127.1, 127.4, 132.7, 139.5, 148.0, 151.1, 158.0, 161.1; LC–MS: m/z;
438(M+1). HPLC purity: 96 %.
4.2.7. (E)-benzyl 4-(4-((2-(4-sulfamoylphenyl)hydrazono)methyl)phenyl)piperazine-1-carboxylate (4g)
1
Cream solid; yield 84%; mp:254-256°C; HNMR (DMSO-d6,400 MHz):δ 3.27 (s, 4H, piperazine, CH2), 3.54 (s, 4H,
piperazine), 5.10 (s, 2H, CH2), 7.00 (d, 2H, Ar, J= 9.1Hz), 7.31-7.37 (m, 5H, Ar), 7.51 (s, 2H, NH2), 7.58 (d, 2H, Ar,
13
J= 9.1Hz), 7.93 (d, 2H, Ar, J= 8.4Hz), 8.04 (d, 2H, Ar, J= 8.32Hz), 8.32 (s, 1H, CH), 11.78 (s, 1H, NH); C NMR
(DMSO-d6, 100 MHz): δ 43.0, 47.0, 66.3, 110.7, 115.3, 125.9, 127.1, 127.4, 127.5, 127.8, 128.4, 132.7, 136.8, 139.4,
147.9, 150.9, 154.4; LC–MS: m/z; 494(M+1). HPLC purity: 99.7 %.
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4.2.8. (E)-4-(2-(4-(4-benzoylpiperazin-1-yl)benzylidene)hydrazinyl)benzenesulfonamide (4h)
1
Creamish solid; yield 71%; mp: 225-227°C ; H NMR (DMSO-d6,400 MHz):δ 3.17-3.27(m, 4H, piperazine, CH2),
3.41-3.82 (m, 4H, piperazine, CH2), 6.97 (d, 2H, Ar, J= 8.4Hz), 7.03 (s, 2H, NH2), 7.07 (d, 2H, Ar, J= 9.1Hz), 7.42-
13
7.47 (m, 5H, Ar), 7.54 (d, 2H, Ar, J=8.4Hz), 7.62 (d, 2H, Ar, J= 8.4Hz), 7.84 (s, 1H, CH), 10.54 (s, 1H, NH); C
NMR (DMSO-d6, 100 MHz): 49.3, 110.9, 117.0, 127.0, 127.1, 127.4, 128.5, 129.8, 133.1, 135.4, 138.8, 147.9, 169.1;
LC–MS: m/z; 464(M+1). HPLC purity: 99.8 %.
4.2.9. (E)-4-(2-(4-(4-(furan-2-carbonyl)piperazin-1-yl)benzylidene)hydrazinyl)benzenesulfonamide (4i)
1
Creamish solid; yield 80%; mp: 211-213°C; H NMR (DMSO-d6,400 MHz):δ 3.29 (s, 4H, piperazine, CH2), 3.81 (s,
4H, piperazine, CH2), 6.64 (s, 1H, Ar), 6.97-7.09 (m, 7H, Ar+ NH2), 7.55 (d, 2H, Ar, J= 8.4Hz), 7.62 (d, 2H, Ar, J=
8.4Hz), 7.84 (d, 2H, Ar, J= 4.5Hz ), 10.54 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): δ 47.7, 110.8, 111.4, 115.2,
115.7, 125.9, 127.1, 127.4, 132.7, 139.5, 144.8, 146.9, 147.9, 150.7, 158.2; LC–MS: m/z; 454(M+1). HPLC purity: 99.9
%.
4.3. Synthesis of (E)-4-(2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide
(8a-k; scheme 2)
4.3.1. Synthetic procedure of methyl 4-sulfamoylbenzoate (6)
Commercially available, 4-sulfamoylbenzoic acid 5 (10 mmol) was refluxed in dried methanol with a catalytic
amount of conc.H2SO4 acid for 8h. After completion of the reaction, the mixture was poured into ice cold water and
obtained precipitate was filtered and dried to give methyl 4-sulfamoylbenzoate 6.
4.3.1. Methyl 4-sulfamoylbenzoate (6)
1
White crystalline solid; yield 95%; mp: 148-150°C; H NMR (DMSO-d6,400 MHz):δ 3.87 (s, 3H, OCH3), 7.55 (s,
2H, NH2), 7.94 (d, 2H, Ar, J= 8.4 Hz), 8.12 (d, 2H, Ar, J= 8.4 Hz); LC–MS: m/z; 216 (M+1).
4.3.2. Synthetic procedure of 4-(hydrazinecarbonyl)benzenesulfonamide (7)
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A mixture of methyl 4-sulfamoylbenzoate 6 (10 mmole) and hydrazine hydrate (20 mmole) refluxed (100°C) in 1,4-
dioxane for 8h. Reaction mixture was poured into ice cold water and obtained precipitate was filtered and dried to
achieve 4-(hydrazinecarbonyl)benzenesulfonamide in high yield.
4.3.2. 4-(hydrazinecarbonyl)benzenesulfonamide (7)
White solid; yield 90%; mp: 214-216°C; 1H NMR (DMSO-d6,400 MHz):δ 4.56 (s, 2H, NH2), 7.47 (s, 2H, NH2), 7.86
(d, 2H, Ar, J= 8.4Hz), 7.94 (d, 2H, Ar, J= 8.4Hz), 9.95 (s, 1H, NH); LC–MS: m/z; 216(M+1).
4.3.3.
General
process
for
the
synthesis
of
(E)-4-(2-(4-(4-substitutedpiperazin-1-
yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide (8a-8k)
An equal mole of 4-(hydrazinecarbonyl)benzenesulfonamide (7) and formyl derivatives (3a-3m) were refluxed
(100°C) in ethanol with a catalytic amount of glacial acetic acid for 8h. The precipitate appeared at the end of the
reaction was filtered and washed with hot ethanol to bestow crude compounds and then after, target compounds (E)-
4-(2-(4-(4-substituted piperazin-1-yl) benzylidene)hydrazinecarbonyl)benzenesulfonamide (8a-8k) were purified by
column chromatography using CHCl3/MeOH (90: 10) as eluent and recrystallization was performed using ethanol as
a solvent.
4.3.3.1. (E)-4-(2-(4-(4-phenylpiperazin-1-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide (8a)
1
Light yellow solid; yield 88%; mp: 278-280°C; H NMR (DMSO-d6, 400MHz): δ 3.23 (t, 4H, piperazine, CH2, J=
4.9Hz), 3.37 (t, 4H, piperazine, J= 4.2Hz), 6.76 (t, 1H, Ar, J= 6.8Hz), 6.95 (d, 2H, Ar, J= 8.4Hz), 7.02 (d, 2H, Ar, J=
8.4Hz), 7.19 (t, 2H, Ar, J= 7.6Hz), 7.47 (s, 2H, NH2), 7.56 (d, 2H, Ar, J= 8.4Hz), 7.89 (d, 2H, Ar, J= 8.4Hz), 8.01 (d,
13
2H, Ar, J= 8.4Hz), 8.29 (s, 1H, CH), 11.74 (s, 1H, NH); C NMR (DMSO-d6, 100 MHz): δ 47.2, 48.1, 114.7, 115.7,
119.1, 124.0, 125.6, 128.1, 128.4, 128.9, 136.5, 148.7, 150.7, 152.0, 161.7; LC–MS: m/z; 464(M+1). HPLC purity: 97.8
%.
4.3.3.2. (E)-4-(2-(4-(4-(4-fluorophenyl)piperazin-1-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide (8b)
Cream solid; yield 78%; mp: 265-267°C; 1H NMR (DMSO-d6, 400 MHz): δ 3.21 (t, 4H, piperazine, CH2, J= 4.9Hz),
3.40 (t, 4H, piperazine, J= 4.9Hz), 6.99-7.09 (m, 6H, Ar), 7.50 (s, 2H, NH2), 7.60 (d, 2H, Ar, J= 9.1Hz), 7.94 (d, 2H, Ar,
13
J= 8.4Hz), 8.05 (d, 2H, Ar, J= 8.4Hz), 8.34 (s, 1H, CH), 11.78 (s, 1H, NH); C NMR (DMSO-d6, 100 MHz): δ 47.2,
25

ACCEPTED MANUSCRIPT
48.9, 114.7, 115.3 (d, J=22 Hz), 117.3, 124.1, 125.7, 128.3, 136.5, 146.4, 147.6, 148.7, 152.1, 156.1 (d, J=230 Hz), 161.7;
LC–MS: m/z; 482(M+1). HPLC purity: 97.2 %.
4.3.3.3.
(E)-4-(2-(4-(4-(4-(trifluoromethyl)phenyl)piperazin-1-
yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide (8c)
1
Creamish-white solid; yield 75%; mp: 270-272°C; H NMR (DMSO-d6, 400 MHz): δ 3.43 (s, 8H, piperazine, CH2),
7.05 (d, 2H, Ar, J= 8.7Hz), 7.11 (d, 2H, Ar, J= 8.4Hz), 7.49-7.54 (m, 4H, Ar+NH2), 7.61 (d, 2H, Ar, J= 8.4Hz), 7.94 (d,
2H, Ar, J= 7.6Hz), 8.05 (d, 2H, Ar, J= 8.4Hz), 8.34 (s, 1H, CH), 11.79 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): δ
46.6, 46.8, 114.2, 118.1, 124.1, 126.3 (q, J=20 Hz), 127.0 (q, J=260 Hz), 136.5, 146.4, 148.7, 151.7, 152.9, 161.7; LC–
MS: m/z; 532(M+1). HPLC purity: 96.7 %.
4.3.3.4. (E)-4-(2-(4-(4-(2,4-dimethylphenyl)piperazin-1-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide
(8d)
Yellow solid; yield 79%; mp: 216-218°C; 1H NMR (DMSO-d6, 400 MHz): δ 2.24 (s, 3H, CH3), 2.24 (s, 3H, CH3), 2.93
(s, 4H, piperazine, CH2), 3.39 (s, 4H, piperazine, CH2), 6.95-7.06 (m, 5H, Ar), 7.51 (s, 2H, NH2), 7.60 (d, 2H, Ar, J=
8.4Hz), 7.93 (d, 2H, Ar, J= 8.4 Hz), 8.04 (d, 2H, Ar, J= 8.4 Hz), 8.33 (s, 1H, CH), 11.78 (s, 1H, NH); 13C NMR
(DMSO-d6, 100 MHz): δ 17.4, 20.3, 47.7, 51.4, 114.5, 118.6, 124.0, 125.7, 126.9, 128.2, 128.4, 131.4, 131.8, 136.5,
146.4, 148.4, 148.8, 152.2, 161.7; LC–MS: m/z; 492 (M+1); HPLC purity: 98.6 %.
4.3.3.5. (E)-4-(2-(4-(4-(pyridin-2-yl)piperazin-1-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide (8e)
1
Cream solid; yield 85%; mp: 275-277°C; H NMR (DMSO-d6, 400 MHz): δ 3.37(t, 4H, piperazine, CH2, J= 4.9Hz),
3.63(t, 4H, piperazine, CH2, J= 4.5Hz), 6.66(t, 1H, Ar, J= 3.4Hz), 6.88(d, 1H, Ar, J= 8.3Hz), 7.05(d, 2H, Ar, J= 9.1Hz),
7.50-7.61(m, 5H, Ar+NH2), 7.93(d, 2H, Ar, J= 8.4Hz), 8.04(d, 2H, Ar, J= 8.4Hz), 8.11(d, 1H, Ar, J= 3.0Hz), 8.33(s,
13
1H, NH); C NMR (DMSO-d6, 100 MHz): δ 44.3, 46.9, 107.2, 113.2, 114.6, 125.7, 128.2, 128.4, 136.5, 137.5, 147.5,
148.8, 159.0, 161.7; LC–MS: m/z; 465(M+1); HPLC purity: 98.9 %.
4.3.3.6. (E)-4-(2-(4-(4-(pyrimidin-2-yl)piperazin-1-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide (8f)
1
Creamish solid; yield 77%; mp: 274-276°C; H NMR (DMSO-d6, 400 MHz): δ 3.35-3.36 (m, 4H, piperazine, CH2),
3.87 (t, 4H, piperazine, J= 4.9Hz), 6.65 (t, 1H, Ar, J= 4.5Hz), 7.04 (d, 2H, Ar, J= 9.1Hz), 7.51 (s, 2H, NH2), 7.59 (d, 2H,
26

ACCEPTED MANUSCRIPT
Ar, J= 8.4Hz), 7.93 (d, 2H, Ar, J= 8.4Hz), 8.04 (d, 2H, Ar, J= 8.4Hz), 8.33 (s, 1H, CH), 8.37 (d, 2H, Ar, J= 4.5Hz),
11.78 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): δ 42.9, 47.0, 110.3, 114.7, 124.1, 125.7, 128.2, 128.4, 136.5, 146.3,
148.7, 152.0, 157.9, 161.1, 161.7; LC–MS: m/z; 466(M+1); HPLC purity: 95 %.
4.3.3.7. (E)-benzyl 4-(4-((2-(4-sulfamoylbenzoyl)hydrazono)methyl)phenyl)piperazine-1-carboxylate (8g)
Light yellow solid; yield 85%; mp: 251-253°C; 1H NMR (DMSO-d6, 400 MHz): δ 3.26 (s, 4H, piperazine, CH2), 3.53
(s, 4H, piperazine, CH2), 5.10 (s, 2H, CH2), 7.00 (d, 2H, Ar, J= 8.4Hz), 7.31-7.37 (m, 5H, Ar), 7.51 (s, 2H, NH2), 7.59
13
(d, 2H, Ar, J= 8.4Hz), 7.91-7.95 (m, 2H, Ar), 8.05 (d, 2H, Ar, J= 8.4Hz), 8.33 (s, 1H, CH), 11.79 (a, 1H, NH); C
NMR (DMSO-d6, 100 MHz): δ 43.1, 47.1, 66.3, 114.9, 124.3, 125.7, 127.6, 127.9, 128.3, 128.4, 128.5, 136.6, 136.8,
146.5, 148.8, 151.9, 154.4, 161.8; LC–MS: m/z; 522(M+1); HPLC purity: 99.1 %.
4.3.3.8. (E)-4-(2-(4-(4-benzoylpiperazin-1-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide (8h)
Cream solid; yield 89%; mp: 260-262°C; 1H NMR (DMSO-d6, 400 MHz): δ 3.24-3.29 (m, 4H, piperazine, CH2), 3.47-
3.75 (m, 4H, piperazine, CH2), 7.01 (d, 2H, Ar, J= 9.1Hz), 7.44-7.51 (m, 7H, Ar+NH2), 7.59 (d, 2H, Ar, J= 9.1Hz), 7.93
13
(d, 2H, Ar, J= 7.6Hz), 8.04 (d, 2H, Ar, J= 8.4Hz), 8.33 (s, 1H, CH), 11.78 (s, 1H, NH); C NMR (DMSO-d6, 100
MHz): δ 46.6, 47.4, 114.9, 124.3, 125.7, 127.0, 128.2, 128.4, 129.6, 135.7, 146.5, 148.7, 151.8, 161.7, 169.0; LC–MS:
m/z; 492 (M+1); HPLC purity: 99.6 % .
4.3.3.9. (E)-4-(2-(4-(4-(furan-2-carbonyl)piperazin-1-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide
(8i)
Creamish solid; yield 84%; mp: 242-244°C; 1H NMR (DMSO-d6, 400 MHz): δ 3.36 (t, 4H, piperazine, CH2, J= 4.5Hz),
3.81 (s, 4H, piperazine, CH2), 6.64 (t, 1H, Ar, J= 1.5Hz), 7.01-7.04 (m, 3H, Ar), 7.51 (s, 2H, NH2), 7.60 (d, 2H, Ar, J=
9.1 Hz), 7.86 (s, 1H, Ar), 7.93 (d, 2H, Ar, J= 8.4Hz), 8.04 (d, 2H, Ar, J= 7.6Hz), 8.33 (s, 1H, CH), 11.78 (s, 1H, NH),
13C NMR (DMSO-d6, 100 MHz): δ 47.2, 111.3, 114.7, 115.8, 124.2, 125.7, 128.2, 128.5, 136.6, 144.8, 146.5, 146.9,
148.7, 151.7, 158.3, 161.8; LC–MS: m/z; 482(M+1); HPLC purity: 99.8 %.
4.3.3.10. (E)-4-(2-(4-(4-benzylpiperazin-1-yl)benzylidene)hydrazinecarbonyl)benzenesulfonamide (8j)
Yellow solid; yield 86%; mp: 228-230°C; 1H NMR (DMSO-d6, 400 MHz): δ 3.24 (s, 4H, piperazine, CH2), 3.36 (s, 4H,
piperazine, CH2), 3.50 (s, 2H, CH2), 6.97 (d, 2H, Ar, J= 8.4Hz), 7.25-7.33 (m, 5H, Ar), 7.52-7.58 (m, 4H, Ar+NH2),
7.94 (d, 2H, Ar, J= 8.4Hz, Ar), 8.05 (d, 2H, Ar, J= 8.4Hz), 8.32 (s, 1H, CH), 11.77 (s, 1H, NH), 13C NMR (DMSO-d6,
27

ACCEPTED MANUSCRIPT
100 MHz): δ 47.1, 52.3, 62.0, 114.4, 123.8, 125.7, 126.9, 128.2, 128.4, 128.9, 136.6, 137.9, 146.4, 148.8, 152.1, 161.7;
LC–MS: m/z; 478(M+1); HPLC purity: 98.4 %.
4.3.3.11.
(E)-4-(2-(4-(4-(benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl)benzylidene)hydrazinecarbonyl)
benzenesulfonamide (8k)
1
Yellowish-white solid; yield 77%; mp: 230-232°C; H NMR (DMSO-d6, 400 MHz): δ 2.48 (s, 4H, piperazine, CH2),
3.24 (s, 4H, piperazine, CH2), 3.42 (s, 2H, CH2), 5.98 (s, 2H, CH2), 6.77 (d, 1H, Ar, J= 8.4Hz), 6.85 (t, 2H, Ar, J=
8.3Hz), 6.97 (d, 2H, Ar, J= 8.4Hz), 7.50 (s, 2H, NH2), 7.55 (d, 2H, Ar, J= 8.4Hz), 7.92 (d, 2H, Ar, J= 8.4Hz), 8.03 (d,
13
2H, Ar, J= 8.4Hz), 8.31 (s, 1H, CH), 11.75 (s, 1H, NH); C NMR (DMSO-d6, 100 MHz): δ 47.0, 52.1, 61.6, 100.7,
107.8, 109.1, 114.3, 121.8, 123.7, 125.7, 128.1, 128.4, 131.7, 136.6, 146.1, 146.4, 147.2, 148.7, 161.7, LC–MS: m/z;
522(M+1); HPLC purity: 98.9 %.
4.4. Carbonic anhydrase inhibition assay: An applied photophysics stopped-flow instrument has been employed to
examine the CA catalyzed CO2 hydration activity as protocol described in our earlier publications [13,30,33, 34]. The
human isoforms hCA I, II, VII and IX were recombinant enzymes produced in our laboratory as described earlier 13,30,
33,34].
4.5. Anti-convulsant Activity: The anti-convulsant potency of novel CA inhibitors was evaluated in MES as well as
sc-PTZ induced seizure animal models. Healthy adult male Swiss Albino mice (25-30g) as well as male Wistar rats
(100-150g) were obtained from “Disease free Small Animal House, Lala Lajpat Rai University of Veterinary and
Animal Sciences” Hisar, Haryana, India. Animals were housed under standard animal laboratory conditions at animal
house of Dr. B.R Ambedkar Center For Biomedical Research, University of Delhi, India. The test compounds were
freshly prepared in 1% gum acacia, injected intraperitoneally (i.p.) to mice (0.01 ml/g body weight) and orally to rats
(0.04 ml per 10 g of body weight). The pentylenetetrazole was dissolved in normal sterile saline solution and
experimental protocols were earlier approved by the Institutional Animal Ethics Committee (Approval number:
IAEC/ACBR/2016/PML/016) for animal care. Additionally, it was tried to minimize animal suffering during whole
experimental tenure and used only necessary number of animals to acquire reliable scientific data. All animal
experiments performed in the manuscript were conducted in compliance with institutional guidelines.
28