Discovery of Benzenesulfonamides with Potent Human Carbonic Anhydrase Inhibitory and Effective Anticonvulsant Action: Design, Synthesis, and Pharmacological Assessment

Article abstract of DOI:10.1021/acs.jmedchem.6b01804

We report two series of novel benzenesulfonamide derivatives acting as effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The synthesized compounds were tested against human (h) isoforms hCA I, hCA II, hCA VII, and hCA XII. The first series of compounds, 4-(3-(2-(4-substitued piperazin-1-yl)ethyl)ureido)benzenesulfonamides, showed low nanomolar inhibitory action against hCA II, being less effective against the other isoforms. The second series, 2-(4-substitued piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide derivatives, showed low nanomolar inhibitory activity against hCA II and hCA VII, isoforms involved in epileptogenesis. Some of these derivatives were evaluated for their anticonvulsant activity and displayed effective seizure protection against MES and scPTZ induced seizures in Swiss Albino mice. These sulfonamides were also found effective upon oral administration to Wistar rats and inhibited MES induced seizure episodes in this animal model of the disease. Some of the new compounds showed a long duration of action in the performed time course anticonvulsant studies, being nontoxic in subacute toxicity studies.

Full text of DOI:10.1021/acs.jmedchem.6b01804

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Article
Discovery of Benzenesulfonamides with Potent Human
Carbonic Anhydrase Inhibitory and Effective Anticonvulsant
Action: Design, Synthesis and Pharmacological Assessment
Chandra Bhushan Mishra, Shikha Kumari, Andrea Angeli, Simona Maria
Monti, Martina Buonanno, Manisha Tiwari, and Claudiu T Supuran
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01804 • Publication Date (Web): 02 Mar 2017
Downloaded from http://pubs.acs.org on March 3, 2017
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Discovery of Benzenesulfonamides with Potent Human Carbonic Anhydrase Inhibitory
and Effective Anticonvulsant Action: Design, Synthesis and Pharmacological Assessment
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Chandra Bhushan Mishra , Shikha Kumari , Andrea Angeli , Simona Maria Monti , Martina
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Buonanno , Manisha Tiwari* and Claudiu T. Supuran*
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BioꢀOrganic Chemistry Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research,
University of Delhi, Delhi, India
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Dipartimento Neurofarba, Universita` degli Studi di Firenze, Sezione di Scienze Farmaceutiche
e Nutraceutiche, Florence, Italy
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Istituto di Biostrutture e Bioimmagini (IBB) CNR,via Mezzocannone, Naples, Italy
Abstract. We report two series of novel benzenesulfonamide derivatives acting as effective
carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The synthesized compounds were tested against
human (h) isoforms hCA I, hCA II, hCA VII and hCA XII. The first series of compounds, 4ꢀ(3ꢀ
(
2ꢀ(4ꢀsubstitued piperazinꢀ1ꢀyl)ethyl)ureido)benzenesulfonamides, showed low nanomolar
inhibitory action against hCA II, being less effective against the other isoforms. The second
series, 2ꢀ(4ꢀsubstitued piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀsulfamoylphenyl)acetamide derivatives, showed low
nanomolar inhibitory activity against hCA II and hCA VII, isoforms involved in epileptogenesis.
Some of these derivatives were evaluated for their anticonvulsant activity and displayed effective
seizure protection against MES and scPTZ induced seizures in Swiss Albino mice. These
sulfonamides were also found effective upon their oral administration to Wistar rats and
inhibited MES induced seizure episodes in this animal model of the disease. Some of the new
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compounds showed a long duration of action in the performed time course anticonvulsant
studies, being nonꢀtoxic in subacute toxicity studies.
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Introduction
.
Epilepsy is one of the most common neurological disorders worldwide, being characterized by
recurrent unprovoked seizure episodes. Globally, 50 million people are affected by epilepsy
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which may cause momentous disability as well as mortality, if untreated. Although various
antiepileptic drugs (AEDs) are used clinically to prevent or suppress seizure episode, about 25ꢀ
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0% of seizure patients suffer of uncontrolled seizures due to nonꢀresponsiveness to the AEDs.
A seizure may lead to an abnormal, excessive, synchronous or paroxysmal discharge in a mass of
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+
ꢀ
2+
neurons.
There are several targets for AEDs, such as GABA, Na , K , Cl , and/or Ca ion
channel, AMPA receptors, etc., with some approved drugs for all of them which are able to
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control seizure episodes in many epileptic patients. Apart from these wellꢀknown molecular
targets for epilepsy, brain carbonic anhydrases (CAs, EC 4.2.1.1) also emerged as interesting yet
controversial targets to control seizures, because they are recognized to modulate several
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ꢀ9
neuronal signaling mechanisms. Several studies have showed that epileptic seizures are related
to rapid changes in the ionic composition within some brain compartments, including the
enhancement of extracellular potassium concentration and pH shifts within the brain. pH directly
affects seizure activity, as for example the alkalosis potentiates seizure spreading, whereas
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acidosis generates the opposite effects. The pH buffering of extracellular and intracellular
ꢀ
spaces is governed by the CO /HCO3 buffer system and CAs prominently regulate this
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,9
equilibrium by catalyzing the interconversion of the two species with high efficacy. Thus, CA
are considered as an interesting target for the development of potent anticonvulsant agents, also
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considering that some of the clinically used AEDs show powerful CA inhibitory activity.
There are 15 different CA isoforms (hCA IꢀhCA XIV, hCA VA and VB) reported in humans,
which are classified into cytosolic (hCA IꢀIII, hCA VII and hCA XIII) membraneꢀbound (hCA
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IV, hCA IX, hCA XII and hCA XIV) and mitochondrial (hCA VA and hCA VB) isoforms.
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These isozyme contain a Zn ion within the active site, which is crucial for the catalytic activity.
The zinc ion is situated at the bottom of a half hydrophilic and half hydrophobic cleft and
tetrahedrally coordinated to the three histidine residues through their imidazole nitrogens and one
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water molecule/hydroxide ion. They are involved in many physiological processes associated
with CO /bicarbonate equilibrium, and proficiently catalyze the interconversion between CO2
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and bicarbonate. In the mammalian brain many CA isoforms are being present: CA II is widely
expressed in the choroid plexus, oligodendrocytes, myelinated tracts, astrocytes as well as
myelin sheaths, being involved in the physiology/pathophysiology of central nervous system
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(CNS), including epileptogenesis.
For example, the expression of CA II was induced in the
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CA1 cells after 3ꢀ12 h of exposure to kainic acid for producing status epilepticus. Furthermore,
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some studies showed that CA II deficient mice are more resistant to seizures, which is in
agreement with using CA inhibition as an antiepileptic mechanism. In the past years, many
studies have proved that CA VII is an isoform mainly present in the brain where it promotes
ꢀ
depolarizing and excitatory GABAergic transmission through HCO3
currents. Several
investigations also suggested the role of CA VII in the generation of seizures through
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GABAergic excitation.
Additionally, recent studies evoked significant role of CA VII in the
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generation of febrile seizures through the activation of GABAA receptors. Intraneuronal
−
generation of HCO3 by CA VII influenced GABA response, which probably plays a crucial
18ꢀ20
role in hippocampal epileptiform activity.
CA XII expression has been also observed in brain
besides, its overexpression in various tumors, and high levels of CA XII mRNA were found in
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granule cells and media amygdala. A well known CA inhibitor, acetazolamide (AAZ) was
approved for the treatment of epilepsy in 1954 and it is still used in partial, absence, myoclonic
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and primary generalized tonicꢀclonic seizures in refractory epilepsy alone, or in combination
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with other AEDs. Some other CA inhibitors such as zonisamide (ZNS), topiramate (TPM) and
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methazolamide (MZA) are also used clinically for therapy of epileptic disorders (Chart 1).
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Thus, the development of potent CA inhibitors, which also possess anticonvulsant activity along
with enduring effectiveness is required.
Chart 1: CAIs showing AED behavior.
Primary sulfonamide (ꢀSO NH ) group containing molecules have an incredible history in the
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field of CA research and many of them are successfully used clinically as antiglaucoma agents,
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diuretics as well as AEDs.
The XꢀRay crystallographic studies have revealed that primary
2+
sulfonamide group present in these CA inhibitors binds as anion to the Zn ion within the
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enzyme active site, blocking catalysis efficiently.
Therefore, sulfonamide derivatives are
highly investigated for the inhibition of various CA isoforms. By incorporating this functional
group in various scaffolds, many CA inhibitor classes have been developed in the last 70 years,
which displayed excellent inhibitory activity and various pharmacological applications, as
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mentioned above.
In the present study we extend this research trend and report 2ꢀ(4ꢀ
substitued piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀsulfamoylphenyl)acetamides and 4ꢀ(3ꢀ(2ꢀ(4ꢀsubstitued piperazinꢀ
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ꢀyl)ethyl)ureido) benzenesulfonamides as novel CA inhibitors endowed with good
anticonvulsant activity.
Results and discussion
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Drug Design and Synthesis. The benzenesulfonamide is a versatile scaffold to develop potent
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and selective CA inhibitors. Numerous derivatives of benzenesulfonamide were synthesized
by many investigators via appending a wide range of alkyl, aromatic, heterocycle or sugar
scaffolds, or by including various linkers (such as carboxamide, urea, thiourea, sulfoxide,
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secondary sulfonamide etc) between the aromatic and tail fragments of the molecule.
These
derivatizations bestowed number of potent CA inhibitors including 4ꢀ(4ꢀ
a
3
6c
fluorophenylureido)benzenesulfonamide (Chart 2), in which the benzenesulfonamide group is
linked to a pꢀfluorophenyl moiety via the urea linkage and this compound successfully
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completed Phase I clinical trials as an antiꢀcancer agent targeting hypoxic, metastatic tumors.
Xꢀray crystallographic studies have disclosed that the urea linker of this compound facilitates the
achievement of the desired selectivity against several CA isoforms, by providing a great
36c, 38
flexibility in order to adopt various orientations within the enzyme active site
. Similar
findings were observed for other benzenesulfonamide derivatives in which urea was present as a
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flexible linker. Piperazine is another important heterocyclic building block which has been
extensively used to develop various clinically useful drug molecules with diverse biological
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activities. Literature survey revealed that piperazine moiety is also widely used to develop
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potent CA inhibitors . Recently, Congiu et al. synthesized a series of sulfonamide containing
piperazinylꢀureido derivatives to evaluate their CA inhibitory effect. The results of this study
provide potent piperazine containing CA inhibitors, which possessed nanomolar range affinity
39
towards CA I, CA II, CA IX, CA XII isoform.
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By considering these facts along with keeping the pharmacophoric frame picture of
36c
ureidoꢀcontaining sulfonamides in mind, we aimed to design novel CA inhibitors of the 4ꢀ(3ꢀ
2ꢀ(4ꢀsubstitued piperazinꢀ1ꢀyl)ethyl)ureido)benzenesulfonamide type, in which the
(
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benzenesulfonamide moiety is linked with various substituted piperazines via
ethylurea/acetamide linkers (Chart 2).
Chart 2: Designed novel CA inhibitors reported in the paper.
The introduction of the Nꢀsubstituted piperazine as tail along with the ethylureido linker is
thought to give better flexibility to adjust within the CAs active site. Furthermore, we have
replaced the ethylureido linker with another flexible linker, the acetamide one, in order to obtain
2ꢀ(4ꢀsubstitued piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀsulfamoylphenyl)acetamide derivatives and study the
impact of the acetamide versus ethyl ureidolinker for the CA inhibitory properties (Chart 2).
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Chemistry. The general synthesis of the novel benzenesulfonamide derivatives is depicted in
Scheme
1
and 2. To obtain 4ꢀ(3ꢀ(2ꢀ(4ꢀsubstituedpiperazinꢀ1ꢀyl)ethyl) ureido)
benzenesulfonamide derivatives (Scheme 1), the commercially available sulfanilamide was
reacted with 2ꢀchloroethylisocyanate to afford chloroethylurea derivative (3). Thereafter, the
chloroethylurea derivative was reacted with Nꢀsubstituted piperazines in the presence of sodium
iodide and sodium bicarbonate to yield target compounds 4-15.
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Compound No
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R-Group
Compound No
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Scheme 1. Preparation of compounds 4-15. Reagent and conditions: A. Dried THF, RT, 2ꢀ3 h; B.
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Dried THF, NaI, NaHCO reflux 8ꢀ10 h
3
Next, 2ꢀ(substituted piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀsulfamoylphenyl)acetamides (Scheme 2) were
synthesized by utilizing sulfanilamide as a starting material. In due course, sulfanilamide and 2ꢀ
chloroacetyl chloride were reacted to provide 2ꢀchloroꢀNꢀ(4ꢀsulfamoylphenyl)acetamide
(
compound 17). Compound 17 was coupled with Nꢀsubstituted piperazines in a basic medium
Na CO ) to provide the desired compounds 18-29. The synthesized compounds were purified by
(
2
3
column chromatography followed by reꢀcrystallization. Compounds were characterized by using
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H & C nuclear magnetic resonance (NMR) and mass spectroscopy. The purity of the target
compounds was assessed by reverse phase HPLC.
Compound No
R-Group
Compound No
R-Group
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Scheme 2. Synthesis of sulfonamides 18-29. Reagent and Conditions: A. Dried Acetone, K CO , RT,
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ꢀ8 h; B. DMF, Na CO , reflux 8ꢀ10 h
2 3
In-Vitro CA Inhibition Studies. The newly synthesized compounds (4-14, 18-29) were tested
for the inhibition against four CA isozymes of human origin, i.e. hCA I, hCA II, hCA VII and
hCA XII (Table 1), some of which, as mentioned earlier, are involved in CNS function and
epileptogenesis. The following structureꢀactivity relationship (SAR) was observed by analyzing
CA inhibition data of Table 1.
i) Most of the synthesized compounds in both series did not display potent inhibitory activity for
8,10
hCA I, normally an offtarget isoform for antiepileptics. Only the phenylpiperazine containing
ethylureidobenzenesulfonamide derivative (4) and the phenyl (18), pꢀbromophenyl (21), 2ꢀ
pyridine (24) as well as benzoylpiperzine (26) bearing sulfamoylphenylacetamide derivatives
showed medium potency inhibitory inhibition (82.1ꢀ138 nM) against hCA I.
ii) The majority of the synthesized compounds in both series strongly inhibited the
physiologically dominant hCA II isoform. Although, compounds 7, 15, 20ꢀ23, 27ꢀ29 exhibited
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satisfactory nanomolar range (22.3ꢀ50.7 nM) inhibitory activity, they were slightly weaker
inhibitors compoared to the standard drug AAZ. However, the rest of the derivatives showed
excellent inhibitory potency in the nanomolar/subnanomolar range (0.69ꢀ12 nM), being superior
to AAZ. Particularly, in the ethylureido benzenesulfonamide series most of the compounds
showed a low nanomolar range activity, except compounds 7 and 15 which contained
trifluorophenyl and acetophenyl moieties in their tails, respectively. SAR studies revealed that
most of the derivatives of this series were found hCA II ꢀ selective over hCA I, hCA VII and
hCA XII. Thus, it may be stated that trifluorophenyl and acetophenyl present at the terminal end
for this pharmacophore is not suitable to generate effective inhibitory potency for hCA II.
Interestingly, thye pꢀfluorophenyl piperazine substituted derivative 5 appeared as an excellent
hCA II inhibitor, which possessed subꢀnanomolar activity (0.69 nM) along with high selectivity
towards hCA I, hCA VII and hCA XII. Derivative 5 displayed 5000ꢀfold selectivity over hCA I
and >7246 fold selectivity over hCA VII as well as hCA XII. In the sulfamoylphenyl acetamide
series, derivatives bearing phenyl (18), pyridine (24), benzyl (25) and benzoyl (26) within the tail
inhibited hCA II better than AAZ.
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iii) The synthesized ethylureido benzenesulfonamides were not able to strongly inhibit hCA VII.
However, some of them showed medium inhibitory potency (58.5ꢀ203 nM) against this isoform
(e.g., 7, 9 and 14 which bear trifluorophenyl, pyrimidine, pꢀfluorodiphenyl group at the terminal
end, respectively). Compounds belonging to the second series, with acetamide linker, showed
better inhibition of hCA VII compared to series 1 compounds (which contains ethyl ureido
linker). In series 2, the majority of the compounds showed good inhibitory activity against hCA
VII, with inhibition constants ranging between 0.74 and 60.5 nM (Table 1). The unsubstituted
phenyl derivative (18) displayed better inhibitory activity for hCA VII compared to AAZ.
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Derivatives which contain pꢀbromophenyl (21), 2ꢀpyridine (24), benzyl (25), benzoyl (26),
diphenyl (27) as well as pipernoyl (29) groups were also highly effective inhibitors (Ki= 0.74ꢀ
10.5 nM) of this isoform. Only two derivatives, 22 and 23 did not display the satisfactory
0
1
2
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6
7
8
9
0
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8
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4
5
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8
9
0
inhibitory potency (Ki= 188ꢀ204 nM) in this series against hCA VII.
iv) Our results revealed that ethylureido benzenesulfonamide derivatives of the first series did
not show an inhibitory effect against hCA XII and displayed Ki >50000 nM. However, two
derivatives of series 2 (sulfamoylphenyl acetamides), compounds 18 and 24, incorporating
phenyl and pyridine tails, respectively, showed good inhibitory potential for hCA XII (Ki of 6.8
–
8.4 nM). Other two derivatives of this series (compound 19 and 26) appeared as weak
inhibitors. Thus, in terms of hCA XII inhibition, sulfamoylphenyl acetamides emerged as better
inhibitors than ethylureido benzenesulfonamides.
Table 1. hCA I, II, VII and XII inhibition with compounds 4-15, 18-29 with AAZ as standard
_____________________________________________________________________
Compound
K (nM)*
I
hCA I
hCA II
hCA VII
hCA XII
_
4
5
6
7
____________________________________________________________________
83.8±7.1
3490±125
909±64
6.2±0.4
310±23
>50000
>50000
58.5±41
>50000
>50000
>50000
>50000
0.69±0.05
8.8±0.6
8130±540
50.7±4.2
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1790±97
3060± 169
860±73
7.9±0.5
7.9±0.3
5.2±0.2
6.1±0.5
4.3±0.3
8.7±0.8
9.1±0.7
26.3±1.4
3.6±0.1
12.0±0.9
22.3±1.7
28.9±1.0
48.1±3.8
27.0±1.5
4.4±0.3
4.9±0.1
9.3±0.7
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
6.8±0.3
49.9±3.3
>50000
>50000
>50000
>50000
8.6±0.4
>50000
91.5±7.6
9
318±21
>50000
360±24
299±18
>50000
203±16
490±35
0.74±0.05
21.0±1.2
161±15
9.4±0.8
188±10.2
404±29
10.5±0.9
6.5±0.4
2.6±0.09
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18
19
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25
26
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60
10
11
12
13
14
3390±245
1730±106
3300±175
5900±342
>50000
15
18
19
20
138±10
437±24
527±40
21
92.3±6.3
2330±189
680±34
22
23
24
25
82.1±6.1
220±17
26
96.4±5.1
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5
5
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2
7
8
1771±107
245±16
8550±653
250±11
250±8
43.2±1.8
16.9±1.3
36.1±1.8
12.1±0.8
10.4 ±0.9
8.9±0.7
>50000
>50000
>50000
5.7±0.3
3.8 ±0.2
2
64.5±4.9
4.0±0.3
0
1
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5
6
7
8
9
0
29
AAZ
TPM
2.5±0.2
0.9 ±0.01
*
Mean ± standard error (from 3 different determinations).
v) The SAR studies clearly evoked that the ethylureido linker containing compounds showed
selective inhibition towards hCA II exclusively, whereas the acetamide linker bearing
compounds bestowed excellent inhibition for both hCA II as well as hCA VII. Thus, for
inhibition of both epilepsy associated isoforms (hCA II and hCA VII), the acetamide linker
bearing derivatives appeared as more appropriate to be investigated in detail in animal models of
the disease.
In-Vivo Studies
Anticonvulsant activity. It is well known that isoform CA I, II, III, IV, VB, VII, VIII, X, XI,
14ꢀ20
XII and XIV are present in the brain.
Several studies highlighted a crucial role of CA II and
CA VII in epileptiogenesis and have shown that their inhibition either blocks or suppresses
epileptic seizure spread. There is sufficient evidence implicating that numerous CA inhibitors
9
,10
(CAIs), which prominently inhibit CA II and CA VII possess good anticonvulsant property.
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Our in vitro CA inhibition study revealed that compounds 5, 18, 24, 25 and 26 appeared as most
potent inhibitors for CA II and CA VII (Table 1). These compounds displayed a (sub)nanomolar
range inhibitory activity against both isoforms (CA II: 0.69ꢀ9.3 nM; CA VII 0.74ꢀ10.5 nM) and
thus were chosen to conduct anticonvulsant activity in-vivo. Both AAZ and TPM are potent
CAIs and are used clinically to treat several forms of epilepsy, as mentioned in the introduction.
Therefore, AAZ and TPM were taken as standard drugs for in vivo experiments.
10
11
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18
19
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23
24
25
26
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59
60
The AED discovery is based on the characterization of antiꢀepileptic activity of the new AED
candidate in a variety of epileptic animal models. Even though there are many animal models of
epilepsy are available, the maximal electroshock (MES) and subcutaneous pentylenetetrazole
(scPTZ) seizure tests are used regularly by most of antiepileptic drug discovery (ADD) programs
4
2
and are considered as the ‘gold standards’ in early stages of anticonvulsant testing . Most of the
clinically used AEDs are protective either in both or at least one of these (primarily MES)
seizure tests. Therefore, anticonvulsant activity of compounds 5, 18, 24, 25 and 26 was
performed using MES and scPTZ seizure tests.
Table 2: Anticonvulsant Activity in MES and scPTZ seizure test in mice
a
b
c
Compounds ( Dose MES screen
scPTZ screen
in mg/kg)
(30)
5 (100)
0.5 h
6/8
6/8
7/8
7/8
6/8
3.0 h
5/8
0.5 h
3/6
4/6
5/6
5/6
0/6
3.0 h
3/6
5
8/8
3/6
1
1
8 (30)
6/8
5/6
8 (100)
6/8
5/6
24 (30)
6/8
1/6
1
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
4 (100)
8/8
8/8
8/8
7/8
5/8
8/8
NT
7/8
8/8
6/8
6/8
5/8
7/8
4/8
8/8
NT
4/8
4/8
1/6
3/6
3/6
4/6
4/6
3/6
2/6
3/6
5/6
1/6
4/6
3/6
4/6
4/6
3/6
3/6
3/6
3/6
25 (30)
2
2
2
5 (100)
6 (30)
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8
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7
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9
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2
3
4
5
6
7
8
9
0
6 (100)
TPM (30)
TPM (100)
AAZ (30)
AAZ (100)
a
Response comments: 30 and 100 mg/kg of doses were administered intraperitoneally (i.p.), The
b
animals were examined at 0.5 h and 3.0 h after injection were administered. Maximal
c
electroshock test (MES) (n=8 mice for each tested dose in the MES test); subcutaneous
pentylenetetrazole test (scPTZ) (n=6 mice for each tested dose in scPTZ test); NT: Not Tested.
Maximal Electroshock Test: The MES test is an excellent animal model for the recognition of
novel agents that block seizure spread and as such are likely to be efficacious for the
4
3
management of generalized tonic–clonic seizures (GTCS) in human . This anticonvulsant
screening test implies the ability of new compound to inhibit the generalized (grandꢀmal)
seizures in rodents. Table 2 summarizes the anticonvulsant activity in the MES test of tested
compounds at the doses of 30 and 100 mg/kg up to 3 hour time duration. It was clearly observed
that compound 5 selectively inhibited CAII (table 1) among all tested CA isoforms. In MES test,
compound 5 showed 75% seizure protection at 0.5 h (30 &100 mg/kg) and 100% seizure
cessation at 3 h (100 mg/kg), which suggests its fast onset and longer duration of action to
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prevent seizure spread from the epileptic focus in the brain. Compound 18 displayed a significant
(87.5%) seizure protection at 0.5 h and this protection was maintained up to 75% at 3 h and in
quantitative evaluation it’s displayed an ED value 18.0 mg/kg. The compound 24 (100 mg/kg)
5
0
10
11
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13
14
15
16
17
18
19
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21
22
23
24
25
26
27
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revealed 100% seizure protection after 0.5 has similar to standard drug AAZ but after 3 h, this
compound was found more effective in blocking seizures where it protected 75% percent of
animals while, AAZ only showed 50% seizure protection Similarly compounds 25 and 26
provided seizure attenuation at the low dose of 30 mg/kg in both time intervals specifying their
fast onset as well as longer duration of action as compare with reference drug TPM. Compound
25 and 26 showed an ED50 13.7mg/kg and 15.0 mg/kg respectively in anticonvulsant
quantification study. Overall, we observed that compounds 5, 18, 24, 25 and 26 selectively
inhibited CA II and CA VII isoform except 5, which is only potent CA II inhibitor. Therefore, it
may be concluded that CA II and CA VII inhibition contribute pointedly to the anticonvulsant
activity by modulation of pH dependent voltage and receptorꢀgated ion channels.
Subcutaneous Pentylenetetrazole Test. Pentylenetetrazole (PTZ) is a γꢀamino butyric acid
(
GABA) receptor antagonist and known to block GABA subtype receptor in the brain. PTZ is a
A
potent chemoconvulsant and systemic injection of PTZ produces severe seizure episodes in both
mice and rats. Therefore, the PTZ test serves as a valid model for studying human generalized
myoclonic and absence and is useful to identify new anticonvulsant candidate which acts by
44
raising the seizure threshold . Table 2 summarizes the anticonvulsant activity of compounds 5,
18, 24, 25 and 26 in the scPTZ test. It was perceived that all tested compounds except 24
displayed antiꢀPTZ effects. It is reported that GABA Rꢀmediated excitation is dependent on the
A
−
continuous replenishment of neuronal HCO3 by cytosolic CA activity. In particular, CA II and
VII isoforms regulate neuronal pH and has a defined role in the generation of seizures. Thus,
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6
administration of CA inhibitor has proven beneficial for the suppression of seizure activity in the
brain. Compound 18 had shown significant protection by raising the seizure threshold at both
time intervals at the lower dose of 30 mg/kg and it was better than TPM as well as AAZ. This
appreciable antiꢀPTZ effect of compound 18 (CA II: K = 3.6nM; CA VII: K = 0.74 nM) could be
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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8
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5
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7
8
9
0
i
i
explained by its good inhibition of CA VII isoform better than AAZ (CA II: K = 12.1 nM ; CA
i
VII: K = 2.5 nM). Other active compound 26 (30 and 100 mg/kg) determined a unique pattern of
i
seizure protection (66%) at 0.5 and 3 h. Rest compounds 5 and 25 have also shown abolition of
myoclonic jerks against scPTZ induced seizures as similar with TPM and AAZ. Three
compounds 18, 25 and 26 presented good anticonvulsant effect in both MES and scPTZ seizure
tests. Hence, these compounds were further evaluated for phase II anticonvulsant screening.
MES study in rats upon oral administration of test compounds. The anticonvulsant potential
of compounds 18, 25 and 26 were also evaluated in rats upon oral administration in MES test at
various time intervals (0.25, 0.5, 1, 2 and 4 h). The results showed that compounds 18
significantly protect MES induce seizures after 1h of drug administration orally to rats and
activity was sustained up to 4h (Table 3). Compound 25 showed significant protection from
seizures up to 1h of drug administration and action was gone reduced after 1h. Thus, 1h after the
compound 18 administration showed best peak time effect in this test. However, Compound 26
displayed satisfactory protection from seizure up to 4h of administration. It displayed 50 %
protection at 0.25 h time interval and 75% at 4h. Studies clearly indicate that these derivatives
showed reasonable protection from MES induced seizures after oral administration and this
finding also proved good oral bioavailability of these compounds.
Table 3: AntiꢀMES activity in Rats dosed orally with test compounds 18, 25 and 26
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Test compounds
Number of protected/tested rats vs. time, after drug
a
administration in the MES test
b
Time intervals (h)
10
11
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14
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17
18
19
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21
22
23
24
25
26
27
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29
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0
.25
0.5
1/4
3/4
2/4
1
2
4
18
1/4
0/4
2/4
3/4
3/4
4/4
4/4
2/4
3/4
3/4
1/4
3/4
2
2
5
6
a
b
Maximal electroshock test (number of animals protected/number of animals tested; n=4), time
after drug administration
Time course anticonvulsant studies. To investigate the duration of anticonvulsant activity of
compounds 18, 25 and 26, a time course study was performed in mice through MES test. In this
test seizure protection capability of these compounds was analyzed up to 6h after compounds
administration. The obtained results indicate these compounds to be highly effective in
protecting mice from MES induced seizures over a long duration of time (Table 4). Compound
1
8 showed 87.5% protection up to 1h and 75.0ꢀ62 % protection in next 2ꢀ6h. Although,
compound 25 sustained the activity 100ꢀ62.5 % in first 3 h and 87.5ꢀ62% in next 3h. However,
compound 26 showed less variable active with progressive time interval and it displayed 75ꢀ77.5
% protection in first 3h and no variation was observed in next 3h and constantly displayed 75%
protection in this time interval. Thus, these CA inhibitors successfully protected MES induced
seizures for longer duration (up to 6h) with an excellent degree of protection.
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4: Time course anticonvulsant activity of compounds 18, 25 and 26 in MES Test with
eight Swiss Albino mice each time
a,b
Test
% Protection in MES seizure test vs. time
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds
0
.5 h
1 h
87
75
87
2 h
62
3 h
75
75
87
4 h
75
87
75
6 h
62
62
75
1
2
8*
5*
87
100
87
62
75
26*
a
b
The test compounds were administered i.p. to Swiss Albino mice (n=8 in each group), time
after drug administration. *p<0.0001 compared to control group [the control group was treated
with vehicle only (1% acacia gum in normal sterile solution) and showed zero percent seizure
protection at all time intervals].
Sub-acute toxicity studies. To assess the safety profile of compounds 18, 25 and 26 a subꢀacute
toxicity study was conducted in normal, healthy Wistar rats at the dose of 200mg/kg/bwt. The
results of this study revealed that animals of the control group and treated groups were found
healthy and no signs of toxicity as well as no deaths were observed during whole experimental
period. Animals preꢀtreated with compounds 18, 25 and 26 did not display any considerable
alteration in hematological parameters as compared to control animals (Table 5). Thus,
compounds 18, 25 and 26 did not produce any toxicity related to hematological alteration. In
addition, treatment of these compounds did not significantly changed the basic level of serum
glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT),
alkaline phosphatase (ALP), total protein and total bilirubin compared to control group (Table 6).
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No alteration of these liver function associated enzymes with compounds 18, 25 and 26 indicates
these new chemical entities did not produce any significant liver toxicity to experimental rats.
Moreover, treatment of these compounds did not sow major changes in renal biomarkers such as
creatinine, uric acid and urea as compared to control group and these results indicative
compounds had not exerted any renal toxicity (Table 7). Overall toxicology data clearly indicate
that these new chemical entities did not have the toxic nature associated with normal body
function, liver function and renal function. These primarily toxicological studies proved these
derivatives as safe and potent CA inhibitors, which endowed with excellent anticonvulsant
activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
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Table 5: Hematological parameters after oral administration of vehicle and compounds 18, 25
and 26 for 14 days in Wistar rats
Parameters
Control(vehicle,
Compound 18
Compound 25
Compound 26
po) ± SD
(200mg/kg, po) ± (200mg/kg, po) ± (200mg/kg, po) ±
SD
SD
SD
Hb (g/dl)
13 ± 1.8
6 ± 0.8
25 ± 3.2
74 ± 3.2
2 ± 1.0
3 ± 1.0
0
13 ± 0.4
7 ± 0.5
25 ± 1.7
71 ± 2.1
2 ± 0.5
2 ± 0.5
0
13 ± 0.6
8 ± 1.2
22 ± 3.7
73 ± 3.3
2 ± 0.5
2 ± 0.5
0
12 ± 0.4
7 ± 0.7
24 ± 3.1
72 ± 3.5
2 ± 0.5
2 ± 0.5
0
3
TLC (10 /ml)
Neutrophils (%)
Lymphocytes (%)
Eosinophils (%)
Monocytes (%)
Basophils (%)
3
RBC (mill/mm )
7 ± 0.2
8 ± 0.6
7 ± 0.4
7 ± 0.1
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Platelet count
1209 ± 97.0
1074 ± 221.8
1065 ± 125.3
1085 ± 127.6
3
(
thou/mm )
The data is represented as Mean ± standard deviation; n=6 in each group.
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9
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2
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8
9
0
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2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6: Liver function test (LFT) after oral administration of vehicle and compounds 18, 25 and
26 for 14 days in rats
Parameters
Control(vehicle,
Compound 18
(200mg/kg, po)
Compound 25
(200mg/kg, po)
± SD
Compound 26
(200mg/kg, po)
± SD
po) ± SD
±
SD
SGOT (U/l)
SGPT (U/l)
79 ± 1.7
61 ± 4.7
122 ± 3.0
78 ± 1.7
59 ± 13.2
125 ± 3.4
79 ± 1.44
78 ± 2.2
55 ± 10.3
60 ± 14.3
Alkaline Phosphatase
(U/l)
122 ± 6.1
123 ± 3.0
Total
0.2 ± 0.01
0.2 ± 0.01
0.2 ± 0.01
0.2 ± 0.01
6 ± 0.3
bilirubin(mg/dl)
Total protein (g/dl)
6 ± 0.3
6 ± 0.29
6 ± 0.1
The data is represented as Mean ± standard deviation; n=6 in each group.
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Table 7: Kidney function test (KFT) after oral administration of vehicle and compounds 18, 25
and 26 for 14 days in rats
Parameters
Control(vehicle,
Compound 18
Compound 25
Compound 26
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po) ± SD
(200mg/kg, po) ± (200mg/kg, po) ± (200mg/kg, po) ±
SD
SD
SD
Blood urea
44 ± 1.6
43 ± 1.3
43 ± 2.7
41 ± 2.0
(mg/dl)
Creatinine (mg/dl)
Uric acid (mg/dl)
Calcium (mg/dl)
Phosphorus
0.5 ± 0.03
2 ± 0.7
0.5 ± 0.01
2 ± 1.3
0.5 ± 0.02
2 ± 0.3
0.5 ± 0.03
2 ± 0.1
9 ± 0.4
9 ± 0.1
9 ± 0.3
9 ± 0.1
8 ± 0.1
8 ± 0.4
8 ± 0.5
7 ± 0.2
(mg/dl)
+
Na (mEq/l)
144 ± 1.5
5 ± 0.3
146 ± 2.0
5 ± 0.3
148 ± 1.2
5 ± 0.2
146 ± 1.7
5 ± 0.2
+
K (mEq/l)
ꢀ
Cl (mEq/l)
110 ± 1.4
107 ± 2.7
111 ± 0.9
107 ± 3.6
The data is represented as Mean ± standard deviation; n=6 in each group.
Conclusions. We have designed and synthesized two novel series of benzenesulfonamide
derivative which behave as potent CA inhibitors, using two diverse linkers and substituted
piperazines as tails. The SAR studies revealed that the acetamide linkerꢀcontaining compounds
(
18-29) showed low nanomolar range inhibitory activity against hCA II and hCA VII, although,
another series of compounds which had ethylurea linker (4-15) strongly inhibited only isoform
hCA II. Compounds 5, 18, 24, 25 and 26 displayed the most potent inhibitory action against hCA
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II and hCA VII isoforms. Among these potent CAIs, compounds 18, 25 and 26 showed excellent
protection against MES and scPTZ induced seizures in mice. In the time course anticonvulsant
studies, these compounds displayed long duration action and successfully opposed MES induced
seizure up to 6 h (62ꢀ75% protection). Additionally, compounds 18, 25 and 26 also inhibited
MES induced seizures in rats upon oral administration. In subꢀacute toxicity, studies these novel
CA inhibitors did not produce any significant toxicity in rats. Thus, overall, the in vivo studies
clearly indicate that the novel benzene sulfonamide derivatives 18, 25 and 26 appeared as potent
and safe CA inhibitors, being endowed with excellent anticonvulsant potential and may be
considered as suitable leads for further development of anticonvulsant with an alternative
mechanism of action, brain CA inhibition.
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Experimental protocol
Chemistry. All the chemicals, reagents and solvents were procured from Sigma Aldrich (St.
Louis, MO, USA), S.D Fine Chemicals (India) and Merck (Darmstadt, Germany). The
homogeneity and purity of the compounds was checked by Thin layer chromatography (TLC),
performed on commercially available silica gel (Kieselgel 60, F ) coated aluminium sheets
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54
(Merck) by using methanol: chloroform (5:95) as the solvent system. The visualization on TLC
was done by both ultraꢀviolet (UV) light (λ= 254 nm) and iodine indicator. All the compounds
were purified by column chromatography by using silica gel 100ꢀ200 mesh (Merck). Melting
points were determined in open capillary tubes in a Hicon melting point apparatus (Hicon, India).
The nuclear magnetic resonance (NMR) spectra were obtained on high resolution Jeolꢀ400MHz
NMR spectrophotometer (USA) DMSOꢀd using Tetramethylsilane (TMS) as the internal
6
reference. Chemical shifts (δ) were expressed in parts per million relative to TMS and the
following abbreviations were used to describe the peak patterns when appropriate: s, (singlet); d,
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(
doublet); t, (triplet); m, (multiplet); brs, (broad singlet); dd, (double doublet). The coupling
constant (J) values are given in hertz (Hz). Mass spectra were recorded using electrospray
ionization liquid chromatography–mass spectrometry (ESIꢀLC/MS) on an Agilent 6310 Triple
Quadrupole Mass Spectrometer instrument and the purity of final compounds was analyzed by
reverse phase Shimadzu HPLC (Kyoto, Japan) coupled with a photodiode array detector (PDA)
and Cꢀ18 column. All compounds reported here showed more than 97% HPLC purity. Samples
were dissolved in acetonitrile and methanol (50:50), and an injection volume of 25 ꢁL was used.
Methanol + acetonitrile used as gradient mobile phase and flow rate was 1mL/min. All
compounds reported here showed more than 97% HPLC purity.
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Synthesis of 4-(3-(2-(4-substituedpiperazin-1-yl)ethyl)ureido)benzene sulfonamide (Series 1)
General procedure for the synthesis of 4-(3-(2-chloroethyl)ureido)benzene sulfonamide (3)
4
ꢀaminobenzenesulfonamide and 2ꢀchloroethylisocyanate were taken in equimolar ratios (10 mM
each) in 25 ml of dried THF, Na CO and NaI were added to the reaction mixture and stirred at
2
3
room temperature for 2ꢀ3 h. Progress of the reaction was monitored by TLC and the obtained
crude solid product at the end of the reaction was filtered and washed twice with petroleum ether.
0
The product was then washed with hot water and dried in oven at 50 C to afford pure white
colored compound 3.
4
-(3-(2-chloroethyl)ureido)benzenesulfonamide (3)
0
1
White solid; yield 92%; mp 170ꢀ172 C; δ H NMR (DMSOꢀd ,400 MHz): 3.42 (q, 2H, CH , J=5.8
6
2
Hz), 3.65 (t, 2H, CH ), 6.54 (t, 1H, NH, ), 7.16 (s, 2H, NH ), 7.53 (d, 2H, ArꢀH, J=9.1 Hz), 7.66 (d,
2
2
+1
2
H, ArꢀH, J=8.4 Hz), 9.05 (s, 1H, NH). LC–MS: m/z; 277 (M ).
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6
General procedure for the synthesis of 4-(3-(2-(4-substituedpiperazin-1-yl)ethyl)
ureido)benzenesulfonamide derivatives (4-15)
The synthesis of target compounds (4-15) was accomplished by reacting compound 3 with various
substituted piperazine in an equimolar ratio of 10 mM each. Firstly, compound 3 was fully
dissolved in dried DMF (20ꢀ25 ml) and then piperazine was added portion wise. Then after,
anhydrous sodium carbonate (15 mM) was added to the reaction mixture and heated at a reflux
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temperature of 90ꢀ100 C for 8ꢀ10 h. After completion, the reaction mixture was diluted with water
and the product was isolated with ethyl acetate. The organic layer was dried over anhydrous
sodium sulphate and concentrated on rotatory evaporator. The obtained crude product was washed
with petroleum and all compounds (4-15) were purified by column chromatography using
chloroform and methanol (98:02) as eluent.
4-{3-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-ureido}-benzenesulfonamide (4)
0
1
White solid; yield 80%; mp 182ꢀ184 C; δ H NMR (DMSOꢀd ,400 MHz): 2.39 (t, 2H, CH , J= 5.6
6
2
Hz), 2.44 (s, 4H, piperazine), 3.07 (s, 4H. piperazine), 3.20 (q, 2H, CH , J=5.8 Hz), 6.20 (t, 1H,
2
NH, J=5.3 Hz), 6.70 (t, 1H, ArꢀH, J=7.2 Hz), 6.86 (d, 2H, ArꢀH, J=7.6 Hz), 7.09ꢀ7.16 (m, 4H, Arꢀ
13
H+NH ), 7.48 (d, 2H, ArꢀH, J=8.4 Hz), 7.60 (d, 2H, ArꢀH, J=9.1 Hz), 9.01 (s, 1H, NH). C NMR
2
(DMSOꢀd ): 36.2, 48.1, 52.6, 57.2, 115.3, 116.7, 118.8, 126.8, 128.9, 135.9, 143.7, 151.0, 154.8.
6
+
LC–MS: m/z; 403 (M ). HPLC purity: 99.8%.
4-(3-{2-[4-(4-Fluoro-phenyl)-piperazin-1-yl]-ethyl}-ureido)-benzenesulfonamide (5)
0
1
White solid; yield 89%; mp 208ꢀ210 C; δ H NMR (DMSOꢀd ,400 MHz): 2.39 (t, 2H, CH , J= 6.0
6
2
Hz), 2.49 (t, 4H, piperazine, J=5.3 Hz), 3.02 (t, 4H. piperazine, J=5.07 Hz), 3.19 (q, 2H, CH2,
J=5.5 Hz), 6.19 (t, 1H, NH, J=5.3 Hz), 6.86ꢀ6.90 (m, 2H, ArꢀH), 6.95ꢀ7.00 (m, 2H, ArꢀH), 7.09 (s,
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H, NH ), 7.47 (d, 2H, ArꢀH, J=8.4 Hz), 7.60 (d, 2H, ArꢀH, J=8.4 Hz), 8.99 (s, 1H, NH). C NMR
2
(
DMSOꢀd ): 36.2, 48.9, 52.6, 57.1, 115.1, 115.3, 116.7, 117.0, 126.7, 135.9, 143.7, 147.9, 154.7,
6
+
157.1. LC–MS: m/z; 421 (M ). HPLC purity: 98.2%.
10
11
12
13
14
15
16
17
18
19
20
21
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23
24
25
26
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60
4
-(3-{2-[4-(4-Chloro-phenyl)-piperazin-1-yl]-ethyl}-ureido)-benzenesulfonamide (6)
0
1
White solid; yield 92%; mp 168ꢀ170 C; δ H NMR (DMSOꢀd ,400 MHz): 2.39 (t, 2H, CH , J= 6.3
6
2
Hz), 2.50 (s, 4H, piperazine), 3.08 (s, 4H. piperazine), 3.20 (q, 2H, CH , J=6.8 Hz), 6.20 (t, 1H,
2
NH, J=5.6 Hz), 6.89 (d, 2H, AꢀH, J=8.4 Hz), 7.10 (s, 2H, NH ), 7.16 (d, 2H, ArꢀH, J=9.1 Hz), 7.48
2
13
(d, 2H, ArꢀH, J=9.1 Hz), 7.60 (d, 2H, ArꢀH, J=8.4 Hz), 9.01 (s, 1H, NH). C NMR (DMSOꢀd ):
6
36.1, 47.9, 52.5, 57.2, 116.6, 116.7, 122.1, 126.7, 128.6, 135.9, 143.7, 149.8, 154.8. LC–MS: m/z;
+
437 (M ). HPLC purity: 98.7%.
4-(3-{2-[4-(4-Trifluoromethyl-phenyl)-piperazin-1-yl]-ethyl}-ureido)-benzene sulfonamide (7)
0
1
White solid; yield 69%; mp 196ꢀ198 C; δ H NMR (DMSOꢀd ,400 MHz): 2.39 (t, 2H, CH , J=6.12
6
2
Hz), 2.49 (s, 4H, piperazine), 3.16ꢀ3.22 (m, 6H, piperazine +CH ), 6.20 (t, 1H, NH, J= 5.3 Hz),
2
7.00 (d, 2H, ArꢀH, J=8.4 Hz), 7.09 (s, 2H, NH ), 7.42ꢀ7.48 (m, 4H, ArꢀH), 7.59 (d, 2H, ArꢀH,
2
13
J=8.4 Hz), 8.99 (s, 1H, NH). C NMR (DMSOꢀd ): 36.1, 46.9, 52.3, 57.1, 114.1, 116.6, 126.1,
6
+
126.7, 135.9, 143.7, 153.2, 154.7. LC–MS: m/z; 471(M ). HPLC purity: 99.8%.
4-{3-[2-(4-p-Tolyl-piperazin-1-yl)-ethyl]-ureido}-benzenesulfonamide (8)
0
1
White solid; yield 77%; mp 204ꢀ206 C; δ H NMR (DMSOꢀd ,400 MHz): 2.18 (s, 3H, CH ), 2.44
6
3
(t, 2H, CH , J= 6.1 Hz), 2.53 (s, 4H, piperazine), 3.07 (s, 4H. piperazine), 3.25 (q, 2H, CH , J=5.5
2
2
Hz), 6.25 (t, 1H, NH, J=4.9 Hz), 6.80 (d, 2H, ArꢀH, J=8.4 Hz), 7.00 (d, 2H, ArꢀH, J=8.4 Hz), 7.15
1
3
(s, 2H, NH ), 7.53 (d, 2H, ArꢀH, J=8.4 Hz), 7.66 (d, 2H, ArꢀH, J=8.4 Hz), 9.06 (s, 1H, NH). C
2
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2
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2
2
2
2
3
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3
3
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4
4
4
4
4
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5
5
5
5
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6
NMR (DMSOꢀd ): 20.0, 36.2, 48.6, 52.6, 57.2, 115.5, 116.6, 126.7, 127.5, 129.3, 135.9, 143.7,
6
+
1
48.9, 154.7. LC–MS: m/z; 417 (M ). HPLC purity: 99.4%.
4-{3-[2-(4-Pyrimidin-2-yl-piperazin-1-yl)-ethyl]-ureido}-benzenesulfonamide (9)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
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5
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7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
1
White solid; yield 58%; mp 214ꢀ216 C; δ H NMR (DMSOꢀd ,400 MHz): 2.41ꢀ2.49 (m, 6H,
6
piperazine+ CH ), 3.25 (q, 2H, CH , J=5.7 Hz), 3.73 (s, 4H, piperazine), 6.23 (t, 1H, NH, J=
2
2
4
.9 Hz), 6.60 (t, 1H, ArꢀH, J=5.1 Hz), 7.15 (s, 2H, NH ), 7.54 (d, 2H, ArꢀH, J=8.3 Hz), 7.66
2
1
3
(d, 2H, ArꢀH, J=9.1 Hz), 8.32 (d, 2, ArꢀH, J= 4.5 Hz), 9.02 (s, 1H, NH). C NMR (DMSOꢀ
d6): 36.2, 43.3, 52.4, 57.2, 110.1, 116.7, 126.7, 135.9, 143.7, 154.7, 157.9, 161.2. LC–MS:
+
m/z; 405 (M ). HPLC purity: 99.5%.
4-{3-[2-(4-Benzyl-piperazin-1-yl)-ethyl]-ureido}-benzenesulfonamide (10)
0
1
White solid; yield 74%; mp 206ꢀ208 C; δ H NMR (DMSOꢀd ,400 MHz): 2.35ꢀ2.42 (m, 9H,
6
piperazine+ CH ), 3.15ꢀ3.20 (m, 3H, piperazine), 3.44 (s, 2H, CH ), 6.17 (t, 1H, NH, J= 4.9 Hz),
2
2
7
.14 (s, 2H, NH ), 7.21ꢀ7.32 (m, 5H, ArꢀH), 7.52 (d, 2H, ArꢀH, J=8.4 Hz), 7.65 (d, 2H, ArꢀH,
2
13
J=8.4 Hz), 9.03 (s, 1H, NH). C NMR (DMSOꢀd ): 36.2, 48.6, 52.5, 57.1, 62.0, 116.7, 126.7,
6
+
1
26.9, 128.2, 128.9, 135.9, 137.9, 143.7, 154.7. LC–MS: m/z; 417 (M ). HPLC purity: 99.4%.
4-{3-[2-(4-Benzo[1,3]dioxol-5-ylmethyl-piperazin-1-yl)-ethyl]-ureido}-benzene sulfonamide
(11)
0
1
White solid; yield 82%; mp 200ꢀ202 C; δ H NMR (DMSOꢀd ,400 MHz): 2.27ꢀ2.42 (s, 10 H,
6
piperazine +CH ), 3.19 (q, 2H, CH , J=5.5 Hz), 3.36 (s, 2H, CH ), 5.97 (s, 2H, CH ), 6.19 (t, 1H,
2
2
2
2
NH, J=4.5 Hz), 6.71ꢀ6.74 (m, 1H, ArꢀH), 6.82ꢀ6.83 (m, 2H, ArꢀH), 7.14 (s, 2H, NH ), 7.51 (d, 2H,
2
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ArꢀH, J=8.3 Hz), 7.64 (d, 2H, ArꢀH, J=7.6 Hz), 9.05 (s, 1H, NH). LC–MS: m/z; 461 (M ). HPLC
purity: 99.2%.
4-{2-[3-(4-Sulfamoyl-phenyl)-ureido]-ethyl}-piperazine-1-carboxylic acid benzyl ester (12)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
1
White solid; yield 73%; mp 176ꢀ178 C; δ H NMR (DMSOꢀd ,400 MHz): 2.34ꢀ2.41 (m, 6H,
6
piperazine+ CH ), 3.21 (q, 2H, CH , J=5.5 Hz), 3.40 (s, 4H, piperazine), 5.06 (s, 2H, CH ),
2
2
2
6
.23 (t, 1H, NH, J= 4.9 Hz), 7.15 (s, 2H, NH ), 7.29ꢀ7.38 (m, 5H, ArꢀH), 7.52 (d, 2H, ArꢀH,
2
1
3
J=9.1 Hz), 7.65 (d, 2H, ArꢀH, J=9.1 Hz), 9.02 (s, 1H, NH). C NMR (DMSOꢀd ): 36.2, 43.5,
6
5
2.3, 57.1, 66.2, 116.7, 126.8, 127.6, 127.8, 128.4, 135.9, 136.9, 143.7, 154.4, 154.7. LC–MS:
+
m/z; 461 (M ). HPLC purity: 99.9%.
4-{3-[2-(4-Benzhydryl-piperazin-1-yl)-ethyl]-ureido}-benzenesulfonamide (13)
0
1
White solid; yield 77%; mp 210ꢀ212 C; δ H NMR (DMSOꢀd ,400 MHz): 2.27ꢀ2.40 (m, 10H,
6
piperazine +CH ), 3.19 (s, 2H, CH ), 4.27 (s, 1H, CH), 6.20 (brs, 1H, NH), 7.14ꢀ7.19 (m, 4H,
2
2
ArꢀH+NH ), 7.28 (t, 4H, ArꢀH, J=7.6 Hz), 7.40 (d, 4H, ArꢀH, J=7.6 Hz), 7.50 (d, 2H, ArꢀH,
2
1
3
J=8.4 Hz), 7.64 (d, 2H, ArꢀH, J=8.4 Hz), 9.05 (s, 1H, NH). C NMR (DMSOꢀd ): 36.1, 51.2,
6
5
4
2.7, 57.1, 75.0, 116.7, 126.7, 126.8, 127.5, 128.5, 135.9, 142.7, 143.7, 154.7. LC–MS: m/z;
+
93 (M ). HPLC purity: 98.3%.
4
-[3-(2-{4-[Bis-(4-fluoro-phenyl)-methyl]-piperazin-1-yl}-ethyl)-ureido]benzene sulfonamide
(14)
0
1
White solid; yield 64%; mp 225ꢀ227 C; δ H NMR (DMSOꢀd ,400 MHz): 2.17ꢀ2.41 (m, 10H,
6
piperazine +CH ), 3.17 (q, 2H, CH , J=6.0 Hz), 4.34 (s, 1H, CH), 6.16 (t, 1H, NH, J=4.9 Hz),
2
2
7
.09ꢀ716 (m, 6H, ArꢀH+NH ), 7.39ꢀ7.42 (m, 4H, ArꢀH), 7.50 (d, 2H, ArꢀH, J=9.1 Hz), 7.64 (d,
2
2
9
ACS Paragon Plus Environment