Discovery of Benzenesulfonamide Derivatives as Carbonic Anhydrase

Article Cite This: J. Med. Chem. 2018, 61, 3151−3165

pubs.acs.org/jmc

Discovery of Benzenesulfonamide Derivatives as Carbonic Anhydrase Inhibitors with Effective Anticonvulsant Action: Design, Synthesis, and Pharmacological Evaluation Chandra Bhushan Mishra,† Shikha Kumari,† Andrea Angeli,‡ Silvia Bua,‡ Manisha Tiwari,*,† and Claudiu T. Supuran*,‡ †

Bio-Organic Chemistry Laboratory, Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, 110007 Delhi, India Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di Firenze, 50019 Florence, Italy

‡

S Supporting Information *

ABSTRACT: Two series of novel benzenesulfonamide derivatives were synthesized and evaluated for their human carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity against four isoforms, hCA I, hCA II, hCA VII, and hCA IX. It was found that compounds of both series showed low to medium nanomolar inhibitory potential against all isoforms. Some of these derivatives displayed selective inhibition against the epileptogenesis related isoforms hCA II and VII, within the nanomolar range. These potent hCA II and VII inhibitors were evaluated as anticonvulsant agents against MES and sc-PTZ induced convulsions. These sulfonamides effectively abolished induced seizures in both models. Furthermore, time dependent seizure protection capability of the most potent compound was also evaluated. A long duration of action was displayed, with efficacy up to 6 h after drug administration. The compound appeared as an orally active anticonvulsant agent without showing neurotoxicity in a rotarod test, a nontoxic chemical profile being observed in subacute toxicity study.

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to handle epileptic seizures.9 Seizures are escorted by rapid alterations in ionic composition in brain compartments which include pH shifts and increase in extracellular potassium concentration. It is well studied that alkalosis generally potentiates seizures spreading by increasing neuronal excitability, though acidosis produces just opposite effects.9 Hence, brain pH has a crucial role in seizure initiation and progression.10,11 The CO2/HCO3− buffer system mainly controls maintenance of a suitable pH in the brain, and CAs actively control this balance by catalyzing the interconversion of these two molecules/ions.12,13 Thus, CAs are promising targets to control seizures as some of the clinically, successful antiepileptic drugs (AEDs) have shown effective CA inhibitory activity.14 In the brain several CA isoforms exist and are actively engaged in various neurophysiologic/neuropathophysiologic processes. CA II is predominantly expressed in the oligodendrocytes, choroid plexus, astrocytes, myelinated tracts, and myelin sheaths and overexpressed in numerous CNS disorder including epilepsy.15,16 Halmi et al. have reported that expression of CA II was significantly increased in the CA1 cells after 3−12 h of kainic acid exposure in status epilepticus model.17 Interestingly, it was also observed that CA II knockout

INTRODUCTION Carbonic anhydrases (CAs, EC 4.2.1.1) belong to a superfamily of metalloenzymes that reversibly catalyze hydration of carbon dioxide to give bicarbonate (HCO3−) and a proton (H+). This reaction controls a wide range of physiological functions which include CO2/bicarbonate transportation, electrolyte secretion, lipogenesis, respiration, gluoconeogenesis, ureagenesis, bone resorption, tumorigenicity, and neuronal excitability.1,2 In humans, 16 different isoforms of CA have been discovered: hCA I−III, hCA VII, and hCA XIII are cylosolic, hCA IV, hCA IX, hCA XII, and hCA XIV are membrane bound, and hCA VA and hCA VB are membrane bound isoforms.3,4 These CAs plays a crucial role in many pathological conditions such as glaucoma, high altitude sickness, kidney dysfunction, migraine, osteoporosis, cancer, and epilepsy.5 In this particular context, epilepsy is a complex brain disorder that affects people of all ages and is characterized by recurrent, unprovoked seizure episodes in affected persons.6 Hypersynchronous neuronal firing and neuronal hyperexcitability in the brain are key factors to originate seizure in epileptic patients.7 Thus, epilepsy is considered as a multifactorial neurological disorder in which numerous receptors, neurotransmitters, enzymes, and ion channels are engaged in generating epileptic symptoms.8 Several studies have shown that brain CAs are also actively involved to generate seizure episodes and are attractive targets © 2018 American Chemical Society

Received: February 7, 2018 Published: March 22, 2018 3151

DOI: 10.1021/acs.jmedchem.8b00208 J. Med. Chem. 2018, 61, 3151−3165

Journal of Medicinal Chemistry

Article

development of potent CA inhibitors, and up until now, several primary sulfonamide containing molecules have been synthesized that have shown an excellent inhibitory profile against CA isoforms with significant pharmacological actions.32,33 To extend our research on CA inhibitors, herein, we report 2-(4substituted piperazin-1-yl)-N-(4-sulfamoylbenzyl)acetamides and 3-(4-substituted piperazin-1-yl)-N-(4-sulfamoylbenzyl)propanamides as novel CA inhibitors (CAIs) endowed with an effective anticonvulsant activity in animal models of epilepsy.

mice are more seizures resistant, which strongly validates the antiepileptic mechanism through inhibition of this CA isoform.18 Another isoform, CA VII, is chiefly present in the brain and widely expressed in the cortex and hippocampus regions, which are the most affected regions during the epileptic activity.19 It was found that CA VII influences GABAergic transmission through HCO3− currents which are also associated with epileptiform activity. During seizure, GABAergic excitation increases extracellular potassium concentration and GABAA mediated responses turn into depolarization during extreme neuronal firing, which are associated with bicarbonate efflux through the GABAA receptors.20,21 Ruusuvuori et al. indicated that this electrophysiological behavior is mainly governed by CA VII in the brain.22 Several reports also indicated the role of CA VII in the initiation of seizure via GABAergic excitation, and studies carried out by Ruusuvuori et al. also revealed that CA VII imparts a crucial role in febrile seizures generation by activating GABAA receptors.23 Immunoblot from glial or neuronal/glial cultures indicated that CA II is present in both types of cells, whereas CA VII expression is restricted to only neurons. It was found that after postnatal day 10, these two isoforms are equally efficient in promoting GABAergic excitation in the brain.23 Several CA inhibitors such as acetazolamide (AAZ), zonisamide (ZNS), methazolamide (MZA), and topiramate (TPM) are clinically used in epilepsy therapy (Chart 1).24,25

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RESULTS AND DISCUSSION Drug Design and Synthesis. Benzenesulfoamide is a widely used scaffold to build up potent CAIs, including SLC0111 (1), which has fruitfully completed phase 1 clinical trials as an anticancer/antimetastatic agent targeting hypoxic, metastatic tumors (Chart 2).34,35 Several reports disclosed that benzenesulfonamide derivatives, which were synthesized by appending alkyl, aryl, heteroaryl, and sugar scaffold on the benzenesulfonamide core with various linkers such as urea, thiourea, sulfoxide, carboxamide, etc., have shown excellent inhibitory property against various CA isoforms.36,37 The carboxamide linker appeared to be suitable for producing excellent inhibitory potency against many CA isoforms, and numerous potent CAIs have been synthesized by using this linker (Chart 2, compounds 2−6).38 Previously we have also developed potent benzenesulfonamide based novel CAIs with a carboxamide linker which have shown subnanomolar range inhibitory action against hCA II and hCA VII isoforms.38 Flexibility of the linker is also considered as an important feature for adopting numerous orientations within the active site of which may provide isoform selective inhibitors.39 Piperazine is an imperative heterocyclic scaffold which has given numerous biologically active molecules, including various clinically active drugs.40 Piperazine derivatives have also shown good CA inhibition, and many of them possessed nanomolar range activity for CA I, CA II, CA IX, CA XII isoforms (Chart 2, compounds 5 and 6).41 By considering these interesting properties of the benzenesulfonamide, piperazine, and carboxamide linkers, we herein report the design of 2-(4-substituted piperazin-1-yl)-N-(4sulfamoylbenzyl)acetamides type of compounds in which benzenesulfonamide was tethered with substituted piperazines by an N-methylacetamide linker. It was thought that the Nmethylacetamide linker along with the piperazine tail will bestow good flexibility to adjust into CA active site, which may appear beneficial for achieving selective and potent CAIs. Furthermore, we extended the linker size with the insertion of one CH2 group, which led to the development of Nmethylpropanamide linkers between benzenesulfonamide and piperazine moieties. This strategy may be helpful to study the impact of the N-methylacetamide linker versus slightly longer N-methylpropanamide linker on CA isoform inhibitory activity and selectivity properties. Chemistry. The synthetic route for the designed benzenesulfonamide derivatives (series 1, 4−16) is shown in Scheme 1. Commercially available homosulfanilamide hydrochloride 1 was reacted with 2-chloroacetyl chloride 2 in 30% NaOH, leading to 2-chloro-N-(4-sulfamoylbenzyl)acetamide 3 in high yield. Intermediate 3 was coupled with various substituted piperazines by performing the reaction in dry DMF and using Na2CO3 as a base to afford the target 2-(4-substituted piperazin-1-yl)-N-(4sulfamoylbenzyl)acetamides 4−16.

Chart 1. Clinically Used CA Inhibitors with Antiepileptic Activity

These CA inhibitors successfully abolish partial, myoclonic, generalized tonic−clonic, and absence seizures in epileptic patients.26 Thus, with this crucial evidence in mind, the development of potent CA inhibitors with long lasting antiepileptic effect may prove to be an interesting strategy in the management of forms of epilepsy reluctant to the treatment with clinically available drugs. Primary sulfonamide (−SONH2) bearing aryl/heteroaryl molecules have proven to be successful CA inhibitors over the past several decades and are widely used clinically as antiglaucoma agents, diuretics, anti-high-altitude sickness agents, and as mentioned above, also antiepileptic drugs (AEDs).27−29 It is a well-known fact that primary sulfonamide is recognized as a zinc binding group inside the CA active site and coordinates to the Zn(II) ion in deprotonated form.30,31 Therefore, this class of molecules is always a first choice for 3152



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Chart 2. Designed Novel Inhibitor and Potent CAIs Reported in Literature

(24), piperonyl (26), diphenylmethyl (27), p-fluorophenyl (20), pyridyl (29), and pyrimidinyl (30) side chains with propanamide linker appeared to be a weak inhibitors for hCA I, and these compounds have shown higher Ki values (263−4140 nM) as compared to standard AAZ (Ki = 250 nM). Thus, compounds having propanamide linker displayed weak inhibition as compared to compounds with acetamide linker against hCA I, generally an off target isoform for antiepileptics. However, acetamide linker bearing p-fluorophenyl (5), benzyl (10), and bis-p-fluorophenyl (13) and propanamide containing p-chlorophenyl (21) derivative showed satisfactory inhibition toward hCA I with Ki values under 90 nM. (ii) Most of the synthesized derivatives in both series showed satisfactory nanomolar range inhibition against the physiologically dominant hCA II isoform. In acetamide series, it appears that substitution on the phenyl ring led to a loss of the efficacy against hCA II except p-methyl substituted phenyl scaffold (7) which has shown a Ki value of 90.3 nM as compared to unsubstituted phenyl (4) which displayed a Ki value of 94.7 nM. In the same series, compounds incorporating benzyl (10), diphenyl (12), and pyrimidinyl (15) moieties showed a satisfactory inhibitory pattern against hCA II, with nanomolar range Ki values (80−96.7 nM). However, compounds having piperonyl (11) and benzoate (16) substitution patterns and an acetamide linker showed better inhibitory property with a Ki values of 75.5 and 80.4 nM. In the second series, which had a propanamide linker, the p-fluoro (20) and p-methoxy (24) substitution on the phenyl ring appeared of interest and showed good inhibitory action against hCA II with Ki values of

3-(4-Substituted piperazin-1-yl)-N-(4-sulfamoylbenzyl)propanamide has been synthesized in two steps (series 2, 19−31) (Scheme 2). Reaction of homosulfanilamide hydrochloride and 3-chloropropionyl chloride in 30% aqueous NaOH solution yielded 3-chloro-N-(4-sulfamoylbenzyl)propanamide 18. The substituted piperazines were linked to the propanamide intermediate 18 by performing the reaction in basic medium (Na2CO3) to offer the target compounds 3-(4substituted piperazin-1-yl)-N-(4-sulfamoylbenzyl)propanamides 19−31. The synthesized compounds of both series were purified by using column chromatography followed by recrystallization. The compounds were well characterized by using 1H, 13C nuclear magnetic resonance (NMR) and mass spectroscopy. The purity of the all the final compounds was examined by reverse phase HPLC and was >97%. In Vitro CA Inhibition Studies. The synthesized novel benzenesulfonamide derivatives (4−16, 19−31) were tested for their inhibition potential against four CA isoforms of human origin, i.e., hCA I, hCA II, hCA VII, and hCA IX; some of them are well studied for their CNS function as well as epileptogenesis.18,19 On the basis of obtained results (Table 1), the following structure−activity relationship (SAR) results were observed. (i) All synthesized compounds (4−31) showed an inhibitory property against ubiquitous cytosolic hCA I beetwen medium nanomolar range (Ki = 64.5−91.8 nM) to micromolar range (Ki = 163−4140 nM). Compounds having toluene (7) as well as bulkier diphenyl side chain (12) with acetamide linker and ptrifluoromethylphenyl (22), p-tolyl (23), p-methoxy phenyl 3153



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Scheme 1a

a Reagents and conditions: (A) 30% NaOH, double distilled H2O, diethyl ether, rt, 5 h; (B) substituted piperazines, Na2CO3, dry DMF, reflux (80− 100 °C), 8−12 h.

16 showed micromolar range (Ki = 1096−2438 nM) inhibitory activity, whereas the phenyl (4), p-fluorophenyl (5), and benzyl (10) derivatives displayed medium potency, nanomolar range inhibition (Ki = 220−395 nM). Compounds of series 2 mostly showed medium inhibitory potential for isoform hCA IX, with Ki values ranging between 98 and 454 nM. Two compounds of this series, having phenyl (19) and p-methylphenyl (23) ring at the piperazine terminal end, displayed effective inhibition against hCA IX with Ki values of 23.7 nM and 78.9 nM, respectively. Hence, it is visualized that the propanamide linker bestowed a better inhibitory potency as compared to the acetamide linker in terms of CA IX inhibition. In Vivo Studies. Anticonvulsant Activity. Several studies have confirmed the presence of isoform CA I, II, III, IV, VB, VII, VIII, X, XI, XII, and XIV in the brain.15−19 hCA I isoform is generally considered as an off target for antiepileptics; hCA IX and hCA XII are well established target for management of hypoxia-induced tumor. Among all, the decisive roles of CA II and CA VII in epileptiogenesis and seizure activity have been well studied in numerous reports It is also well documented that inhibitors, which inhibit CA II and CA VII prominently, showed effective anticonvulsant property.16−18 CA inhibition data explained that compounds

69.9 and 60.7 nM, respectively. The compounds containing piperonyl (26) and benzoate (31) moieties showed better inhibitory property for hCA II as compared to other derivatives in this series and showed a Ki value of 33.2 nM and 41.5 nM, respectively, along with good selectivity over hCA I, hCAVII, and hCA IX. The remaining derivatives in both series, such as compounds 5−7, 14, 21−23, 27, and 28, were medium potency inhibitors against hCA II (Table 1). (iii) Most of the derivatives of series 1, with an acetamide linker, have demonstrated medium inhibitory potency (Ki = 171−547 nM) for hCA VII. However, compounds bearing pchlorophenyl (6), pyrimidinyl (15), and benzoate (16) groups at the terminal end were bestowed with reasonable inhibition profile hCA VII, showing Ki values of 83.9, 56.9, and 27.1 nM, respectively. The majority of compounds in the other series, with propanamide as linker, also displayed medium range nanomolar (Ki = 170−672 nM) inhibitory action for hCA VII. However, compounds holding unsubstituted phenyl (19) and trifluoromethylphenyl (22) were effective inhibitors, with Ki values of 89.6 and 61.3 nM, respectively. (iv) Our results indicated that most of the synthesized derivatives in the acetamide series did not effectively inhibit the tumor associated isoform hCA IX. Compounds 6−9 and 11− 3154



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Scheme 2a

Reagents and conditions: (A) 30% NaOH, H2O, diethyl ether, rt, 5 h; (B) substituted piperazines, Na2CO3, dry MeCN, reflux (80−100 °C), 18− 20 h.

a

MES test at the doses of 30 and 100 mg/kg after 0.5 and 3 h of drug administration is summarized in Table 2. Results of the MES test indicate that compound 15 (30 and 100 mg/kg bwt) showed 50% seizure protection at 0.5 h. At the 3 h time interval this compound provided 67% and 83% protection from MES induced seizures, which indicates its fast with long duration of action. Although compound 16 (30 mg/ kg) indicated slow action at 0.5 h and displayed only 17% seizure protection, its protection percentage was increased at 3 h and showed 67% protection at 30 mg/kg dose. At the dose of 100 mg/kg this compound protected 67% animals and activity was decreased after 3 h of drug administration (50% protection). Thus, compound 16 was sluggish with long duration action at the dose 30 mg/kg and fast with short duration action at 100 mg/kg dose. Interestingly, compound 26 excellently protected 67% of the mice from MES induced seizures at both time intervals (0.5 and 3 h) and with a lower dose (30 mg/kg). At the higher dose (100 mg/kg) compound 26 also showed 83% protection at 0.5 h and 67% protection at 3 h. Thus, our data clearly indicate that compound 26 has a fast action with longer duration of seizure protection at both doses used in these experiments. With these results, we may conclude that compound 26 has better potential to protect animals against MES seizures as compared to compounds 15 and 16. Subcutaneous Pentylenetetrazole Test. Pentylenetetrazole (PTZ) is a chemoconvulsant that acts as a GABAA receptor antagonist and prominently induces myoclonic and absence seizure in experimental animals. The PTZ test is widely used as a model for studying myoclonic as well as absence seizures and

15, 16, and 26 appeared to be most effective inhibitors for CA II as well as CA VII as compared to other derivatives (Table 2). Therefore, we decided to evaluate these compounds for their anticonvulsant action in vivo seizure models. In these experiments, two standard anticonvulsant drugs AAZ and TPM, which prominently inhibited hCAs, were also included to compare in vivo efficacy of our synthesized novel CA inhibitors against seizures. The discovery of AEDs is heavily grounded on the characterization of antiseizure activity of novel chemical entities on a kind of epileptic animal models. Although many animal models of epilepsy are being used to assess the anticonvulsant efficacy of novel compounds, the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure tests are the most accepted test and regularly used by a majority of antiepileptic drug discovery (ADD) programs. These two seizure tests are regarded as the “gold standards” to evaluate anticonvulsant potential of novel drug candidates in the early stages. It has been seen that many clinically approved AEDs show protection in either both tests or one test. Hence, the anticonvulsant action of synthesized derivatives 15, 16, and 26 was evaluated by MES and scPTZ seizure tests. Maximal Electroshock Test. The MES test is considered as a preclinical model that envisages drugs effectiveness against generalized tonic−clonic seizures type (grand mal), which is close to human epilepsy disease. This test also establishes correlation between the power of the drug to abolish seizure in rodents and its efficacy in generalized seizures in humans.42 The anticonvulsant efficacy of derivatives 15, 16, and 26 in the 3155



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Table 1. Inhibition of hCA I, II, VII, and IX with Compounds 4−16 and 19−31 and Acetazolamide (AAZ) as Standarda KI (nM) compd

hCA I

hCA II

hCA VII

hCA IX

hCA I/hCA II

hCA I/hCA VII

hCA IX/hCA II

hCA IX/hCA VII

4 5 6 7 8 9 10 11 12 13 14 15 16 19 20 21 22 23 24 25 26 27 28 29 30 31 AAZ

187.9 70.1 91.8 152.4 263.4 215.0 84.4 371.7 446.0 68.9 83.8 139.6 93.3 163.4 373.2 87.8 612.8 658.7 801.4 64.5 799.9 4140.9 711.2 620.5 762.4 91.2 250

94.7 234. 5 165.2 333.1 90.3 171.6 83.9 75.5 96.7 203.5 384.4 90.9 80.4 120.3 69.9 252.1 461.7 252.5 60.7 55.8 33.2 212.7 268.4 88.2 207.7 41.5 12.1

244.4 515.2 83.9 426.8 451.9 403.9 396.9 547.4 296.9 171.3 271.5 56.9 27.1 89.6 514.9 604.2 61.3 356.1 170.1 314.6 337.2 672.3 578.5 398.4 377.4 387.3 2.5

395.9 227.8 1195.7 1774.9 1530.4 1096.9 220.2 1597.7 1679.7 2438.2 1556.4 2230.8 1627.9 23.7 114.6 117.1 136.9 78.9 136.2 115.7 104.2 890.2 98.0 162.3 454.6 257.3 25

1.9 0.3 0.6 0.4 2.9 1.3 1.0 4.9 4.6 0.3 0.2 1.5 1.1 1.3 5.3 0.3 1.3 2.6 13.2 1.1 24.0 19.4 2.6 7.0 3.7 2.1 20.6

0.7 0.1 1.0 0.3 0.5 0.5 0.2 0.6 1.5 0.4 0.3 2.4 3.4 1.8 0.7 0.1 9.9 1.8 4.7 0.2 2.3 6.1 1.2 1.5 2.0 0.2 100

4.1 0.9 7.2 5.3 16.9 6.3 2.6 21.1 17.3 11.9 4.0 24.5 20.2 0.1 1.6 0.4 0.2 0.3 2.2 2.0 3.1 4.1 0.3 1.8 2.1 6.2 2.0

1.6 0.4 14.2 4.1 3.3 2.7 0.5 2.9 5.6 14.2 5.7 39.2 60.0 0.2 0.2 0.1 2.2 0.2 0.8 0.3 0.3 1.3 0.1 0.4 1.2 0.6 10

a Results reperesent the mean value from three different assays performed by a stopped flow technique (errors were in the range of ±5−10% of the reported values, data not shown).

suitable pH.15−18 Therefore, anticonvulsant action of compounds 15, 16, and 26 was evaluated in sc-PTZ induced seizure model, and the obtained results are summarized in Table 2. These results show that 30 mg/kg of 15 protected 33% animals against PTZ induced seizures at 0.5 h and the activity was increased at 3 h, displaying 50% seizure protection. At the dose of 100 mg/kg, this compound demonstrated 50% seizure protection at both time intervals (0.5 and 3 h), indicating sustained activity up to 3 h which was similar to that of the standard drug AAZ (30 mg/kg). Compound 16 showed a good anticonvulsant activity at 0.5 h but its activity significantly decreased at 3 h. This compound at the dose of 30 mg/kg displayed 67% seizure protection at 0.5 h and 17% protection at 3 h. Higher doses (100 mg/kg) of 16 displayed a better activity as compared to lower dose and showed 50% protection at 0.5 h as well as 33% seizure protection at 3 h. Notably, compound 26 has shown good anticonvulsant activity in PTZ induced seizure test and exhibited 83% protection at 0.5 h as well as 67% protection at 3 h when a lower dose (30 mg/kg) was administered. This indicates a fast action of the compound with sustaining seizure protection potential. Additionally, higher dose (100 mg/kg) of 26 was also endowed with good anticonvulsant potential and showed 50% protection at both time intervals. Thus, overall these results indicate that all compounds have shown satisfactory protection against PTZ induced seizures in mice. However, among these three tested compounds, 26 excellently abolished myoclonic jerks against sc-PTZ induced seizures at lower dose and displayed better seizure protection as compared to standard drugs AAZ and

Table 2. In Vivo Anticonvulsant Activity of Compounds 15, 16, and 26 in MES and sc-PTZ Seizure Test MESb screen a

scPTZc screen

compd (dose in mg/kg)

0.5 h

3.0 h

0.5 h

3.0 h

15 (30) 15 (100) 16 (30) 16 (100) 26 (30) 26 (100) TPM (30)d TPM (100)d AAZ (30)d AAZ (100)d

3/6 3/6 1/6 4/6 4/6 5/6 8/8 NT 7/8 8/8

4/6 5/6 4/6 2/6 4/6 4/6 8/8 NT 4/8 4/8

2/6 3/6 4/6 3/6 5/6 3/6 3/6 2/6 3/6 5/6

3/6 3/6 1/6 2/6 4/6 3/6 3/6 3/6 3/6 3/6

a

30 and 100 mg/kg doses were given ip (intraperitoneally), and the animals were observed at 0.5 and 3 h after drug administration. b Maximal electroshock test (MES) (n = 6). NT: not tested. c Subcutaneous pentylenetetrazole test (scPTZ) (n = 6). dData taken from ref 38.

is a beneficial test for identification of new drug candidates which directly or indirectly act via GABA-ergic system.43 It is well studied that neuronal HCO3− influences the excitation of GABAA which is actively regulated by cytosolic CAs. Especially, isoforms CA II and CA VII are involved in neuronal pH regulation and have shown a well-defined role in seizure generation. Administration of CA inhibitors has been proven beneficial for controlling seizure spread, by maintaining a 3156



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seizure up to 6 h after compound treatment. Compound 26 has protected 70% animals against MES seizures at 0.5 h time interval and showed enhanced protection percentage at 1 h with 80% protection. However, at 2 h time interval anticonvulsant activity was slightly decreased and found to be 60% seizure protection. Again, enhanced anticonvulsant activity was observed at 3 h with 70% seizure protection which was sustained up to 4 h of drug administration. It is also noticed that compound 26 showed 50% seizure protection after 6 h of administration. Thus, this time course study evidently indicates that CA inhibitor 26 possesses good anticonvulsant activity along with long duration of action. Neurotoxicity Assessment. Rotarod test is widely used to evaluate motor coordination dysfunction associated with neurotoxicity.40e Neurotoxic effect of compound 26 has been assessed using a rotarod test at various time intervals (30, 60, 90, 120 min) after providing 30 mg/kg dose to animals (Figure 1). To evaluate the neurotoxic effect, compound 26 pretreated

TPM. In both tests, (MES and sc-PTZ) compound 26 appeared as potent anticonvulsant agent and displayed better seizure protection as compared to the other two, 15 and 16. Therefore, compound 26 was selected for its further advance screening to evaluate extensively its anticonvulsant potential. MES Study after Oral Administration of Compound 26. The oral route of administration is the most favorable one for drug delivery, compared to other routes, and by this route the drugs display pharmacological effects by traveling through the gastrointestinal tract.44 Therefore, compound 26 was administrated orally to rats and its efficacy was evaluated against MES induced seizures at 0.25, 0.5, 1, 2, and 4 h time intervals. The obtained results indicated derivative 26 significantly protects seizure stimulated by MES after 0.5 h of drug administration and the action was found to be persisted up to 4 h (Table 3). At Table 3. Anti-MES Activity of Compound 26 (30 mg/kg, bwt) after Oral Administration time (h)

MES testa

% protectionb

0.25 0.5 1 2 4

1/6 3/6 4/6 4/6 3/6

17 50 67 67 50

a

Number of protected animals versus number of tested animals; n = 6. Percentage seizure protection (number of protected animals versus number of tested animals × 100).

b

0.25 h compound 26 showed only 17% protection which gradually increased with time. After 1 h of oral drug administration this compound displayed 67% protection, and this effect was maintained up to 2 h after administration. Thus, after 1 h of oral administration, compound 26 has displayed its peak effect. Interestingly, this compound demonstrated 50% protection after 4 h of drug administration, which indicate its prolonged activity against MES induced tonic−clonic seizures. These findings clearly point out that compound 26 displayed effective seizure protection against MES seizures upon oral administration, which also verified the worthy oral bioavailability of the compound. Time Course Anticonvulsant Study of Compound 26. To examine the time duration of anticonvulsant activity of compound 26, a time course study has been carried out by applying the MES test (Table 4). The MES induced clonic− tonic seizure protection ability of compound 26 was evaluated up to 6 h after drug administration. The findings of this study indicate that compound 26 effectively protected MES induced

Figure 1. Mean time spent on the rotarod (s) up to 120 min after vehicle and compound 26 (30 mg/kg) administration. Each point denotes the mean ± SEM of values acquired from eight animals (twoway repeated measures ANOVA followed by Bonferroni post-test).

and vehicle pretreated mice were placed on a rotating rod for 180 s. It was found that compound 26 pretreated and vehicle pretreated mice retained almost identical times on rotarod apparatus at time intervals 30, 60, 90, and 120 min after treatment, which indicated non-neurotoxic nature of compound 26. Subacute Toxicity Studies. Safety profile of compound 26 has been evaluated by conducting a subacute toxicity study in Wistar rats at the dose of 100 (mg/kg)/bwt. During 14 days of compound 26 treatment, no sign of toxicity or death was observed and all animals took food and water properly. Results indicated that compound 26 treatment did not significantly alter hematological parameters such as hemoglobin, RBC, WBC, and platelet count as compared to control animals, which indicates this compound did not exert any significant toxicity associated with hematological parameters (Table 5). Liver associated biomarker quantification analysis revealed that treatment of compound 26 has not altered normal level of serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), total bilirubin, and total protein as compared to control group. Thus, this analysis evidently indicated that compound 26 did not produce liver toxic effect on experimental animals (Table 6). Additionally, in the renal function test, there were no significant changes seen in renal biomarkers such as creatinine, urea, and uric acid in compound 26 treated animals and control

Table 4. Time-Course Anticonulsant Activity of Compound 26 (30 mg/kg, ip) in the MES Test time (h)a

MES testb

% protectionc

0.5 1 2 3 4 6

7/10 8/10 6/10 7/10 7/10 5/10

70 80 60 70 70 50

a

Time after drug administration. bMaximal electroshock test (n = 10; number of protected animals versus total number of tested animals). c Percentage seizure protection ((number of protected animals/total number of tested animals) × 100). 3157



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both participate in epileptogenesis. Among all, compounds 15, 16, and 26 were the most potent hCA II and hCA VII inhibitors. These three derivatives displayed effective anticonvulsant activity against MES as well as sc-PTZ induced seizures. Among these three compounds, 26 was a very effective anticonvulsant with excellent antiseizure action in both models at lower dose (30 mg/kg). Compound 26 also possessed long duration of action and was effective up to 6 h after administration without displaying significant neurotoxicity in the rotarod test at effective dose (30 mg/kg). Interestingly, this compound was found to be orally active and has abolished MES stimulated seizures in male Wistar rats after oral administration. Moreover, this compound did not induce any significant toxicity in male Wistar rats upon oral treatment for 14 days. Thus, novel benzenesulfonamide derivative 26 has emerged as a safe and effective CAI inhibitor, which also possesses valuable anticonvulsant action and may be used as a potent lead for development of suitable anticonvulsants that may act through brain CA inhibition.

Table 5. Hematological Analysis after Vehicle and Compound 26 Administration to Wistar Ratsa parameter

control (vehicle, po)

compd 26 (100 mg/kg, po), ±SD

Hb (g/dL) TLC (103/mL) neutrophils (%) lymphocytes (%) eosinophils (%) monocytes (%) basophils (%) RBC (mill/mm3) platelet count (thou/mm3)

12.7 ± 1.5 6.3 ± 1.4 20.8 ± 5.8 74.6 ± 4.3 2.3 ± 1.2 2.5 ± 1.5 0.3 ± 0.5 7.9 ± 0.94 698 ± 156

12.6 ± 1.2 7.5 ± 2.4 22.3 ± 3.6 74 ± 4 2.3 ± 1.9 2.3 ± 1.2 0.16 ± 0.4 7.7 ± 0.8 662.3 ± 111.9

The data are represented as the mean ± standard deviation; n = 6 per group.

a

Table 6. Liver Function Test of Vehicle and Compound 26 Treated Rats in Subacute Toxicity Studya parameter



SGOT (U/L) SGPT (U/L) alkaline phosphatase (U/L) total bilirubin(mg/dL) total protein (g/dL)

59.9 ± 11 59.2 ± 12.5 114.2 ± 8.4 0.32 ± 0.09 6.35 ± 0.46

64.2 ± 10.8 52.9 ± 16.7 113.4 ± 18.2 0.38 ± 0.11 6.2 ± 0.48

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Chemistry. Chemicals, solvents, and buffers were purchased from Sigma-Aldrich (St. Louis, MO, USA), TCI (Tokyo, Japan), Merck (Darmstadt, Germany), and SD Fine Chemicals (India). Reaction monitoring was assessed by thin layer chromatography (TLC), using methanol/chloroform as mobile phase. The visualization of TLC was performed with the help of ultraviolet (UV) light (λ = 254 nm) as well as an iodine indicator. Column chromatography was performed for purification of synthesized compounds using silica gel (100−200 mesh, Merck) as stationary phase and chloroform/methanol as mobile phase. Melting points were taken with open capillary tubes using a Hicon melting point apparatus (Hicon, India). Mass spectra of compounds were taken using ESI-LC/MS system (Agilent 6310 triple quadrupole mass spectrometer). The nuclear magnetic resonance (NMR) spectra were recorded on 400 MHz Jeol NMR spectrophotometer (USA) using DMSO-d6/CDCl3 as solvent. Chemical shifts (δ) were represented in parts per million relative to standard TMS, and the peak patterns were indicated as s, d, t, m, and brs for singlet, doublet, triplet, multiplet, and broad singlet, respectively. The purity level of target compounds was examined using reverse phase HPLC (Shimadzu, Kyoto, Japan) united with C-18 column as well as a PDA detector. Samples were dissolved in methanol and acetonitrile (50:50), and 25 μL injection volume was used. Methanol + acetonitrile gradient was used as a mobile phase with 1 mL/min flow rate. All analyzed compounds displayed >97% purity level. Synthesis of 2-(4-Substituted piperazin-1-yl)-N-(4sulfamoylbenzyl)acetamide (Series 1, 4−16). General Method for Synthesis of 2-Chloro-N-(4-sulfamoylbenzyl)acetamide (3). 4-(Aminomethyl)benzenesulfonamide (1, 10 mmol) was dissolved in distilled water (50 mL) containing 30% NaOH. 2-Acetyl chloride (2, 10 mmol) solution in diethyl ether (10 mL) was added slowly to the reaction mixture and stirred for 5 h. The precipitate was filtered and washed with hot water to achieve 2-chloro-N-(4-sulfamoylbenzyl)acetamide (3) in high yield. 2-Chloro-N-(4-sulfamoylbenzyl)acetamide (3). White solid; yield 92%; mp 144−146 °C. 1H NMR (DMSO-d6,400 MHz): δ 4.13 (s, 2H, CH2), 4.34 (d, 2H, CH2, J = 6.1 Hz), 7.32 (s, 2H, NH2), 7.41 (d, 2H, Ar, J = 8.4 Hz), 7.65 (d, 2H, Ar, J = 8.4 Hz), 8.91(t, 1H, NH, J = 6.4 Hz). LC−MS: m/z. 262 (M+1) General Procedure for Synthesis of 2-(4-Substituted piperazin-1-yl)-N-(4-sulfamoylbenzyl)acetamide (4−16). An equimolar ratio of intermediate 2-chloro-N-(4-sulfamoylbenzyl)acetamide (3) and substituted piperazines was refluxed (100−120 °C) with Na2CO3 (catalytic amount) in dry DMF for 8−12 h. The reaction mixture was diluted in water, and crude products were isolated with ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure. Crude products were purified by column chromatography using

The data are represented as the mean ± standard deviation; n = 6 per group.

a

animals (Table 7). Hence, this compound also has no renal toxicity property upon repeated administration. Overall, these Table 7. Quantification of Kidney Function Associated Biomarkers after Oral Administration of Vehicle and Compound 26a parameter



blood urea (mg/dL) creatinine (mg/dL) uric acid (mg/dL) calcium (mg/dL) phosphorus (mg/dL) Na+ (mequiv/L) K+ (mequiv/L) Cl− (mequiv/L)

34.7 ± 9.9 0.75 ± 0.1 4.33 ± 2.5 10.3 ± 1.8 9.4 ± 2.0 144 ± 2.8 4.82 ± 0.3 105.5 ± 6.5

39.8 ± 11 0.75 ± 0.2 2.73 ± 1.7 9.9 ± 1.4 6.8 ± 1.1 144 ± 3.8 4.9 ± 0.4 104.3 ± 7.4

EXPERIMENTAL SECTION

The data are displayed as the mean ± standard deviation; n = 6 per group.

a

toxicological analysis results clearly indicate that compound 26 appeared as a nontoxic chemical entity that did not produce toxic effects on the liver functions, kidney functions, and normal body functions. These preliminary toxicological studies proved compound 26 to be a safe and potent CA inhibitor, which also has effective anticonvulsant activity.

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CONCLUSION Herein, we developed two series of novel benzenesulfonamide derivatives as potent hCA inhibitors, incorporating Nmethylacetamide and N-methylpropanamide linker along with substituted piperazines as tails. In vitro enzyme inhibition assay indicated that most of the compounds displayed effective nanomolar range inhibition against hCA II and VII isoforms; 3158



Article

Hz), 7.74 (d, 2H, Ar, J = 8.4 Hz), 8.32 (t, 1H, NH, J = 4.9 Hz). 13C NMR (DMSO-d6): 41.5, 52.1, 52.9, 61.2, 61.6, 100.7, 107.8, 109.0, 122.0, 125.6, 127.4, 142.5, 143.8, 146.1, 147.2, 169.5. LC−MS: m/z 447 (M+1). HPLC purity: 96.7%. 2-(4-Benzhydrylpiperazin-1-yl)-N-(4-sulfamoylbenzyl)acetamide (12). White solid; yield 75%; mp138−140 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.29−2.44 (m, 8H, piperazine CH2), 2.97 (s, 2H, CH2), 4.27 (s, 1H, CH), 4.31 (s, 2H, CH2, J = 6.1 Hz), 7.16 (t, 2H, Ar, J = 7.2 Hz), 7.27 (m, 6H, Ar+NH2), 7.39 (t, 6H, Ar, J = 9.1 Hz), 7.72 (d, 2H, Ar, J = 8.3 Hz), 8.30 (t, 1H, NH, J = 6.0 Hz). 13C NMR (DMSO-d6): 41.5, 51.2, 53.1, 61.2, 75.1, 125.6, 126.8, 127.4, 127.5, 128.5, 142.4, 142.7, 143.8, 169.4. LC−MS: m/z 479 (M+1). HPLC purity: 96.3%. 2-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)-N-(4sulfamoylbenzyl)acetamide (13). White solid; yield 84%; mp 113−115 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.28−240 (m, 8H, piperazine, CH2), 2.97 (s, 2H, CH2), 4.31−4.36 (m, 3H, CH2 + CH), 7.11 (t, 4H, Ar, J = 8.4 Hz), 7.28 (s, 2H, NH2), 7.36−7.45 (m, 6H, Ar), 7.73 (d, 2H, Ar, J = 9.1 Hz), 8.30 (t, 1H, NH, J = 6.8 Hz). 13C NMR (DMSO-d6) 41.5, 51.0, 53.1, 61.2, 79.2, 115.2, 115.4, 125.6, 127.4, 129.3, 129.4, 138.6, 142.5, 143.8, 159.8, 162.2. LC−MS: m/z 515 (M+1). HPLC purity: 97.4%. 2-(4-(Pyridin-2-yl)piperazin-1-yl)-N-(4-sulfamoylbenzyl)acetamide (14). White solid; yield 88%; mp 144−146 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.47 (s, 4H, piperazine CH2), 3.00 (s, 2H, CH2), 3.47 (s, 4H, piperazine, CH2), 4.32 (d, 2H, CH2, J = 6.1 Hz), 6.58 (t, 1H, Ar, J = 5.7 Hz), 6.77 (d, 1H, Ar, J = 8.4 Hz), 7.27 (s, 2H, NH2), 7.39 (d, 2H, Ar, J = 8.4 Hz), 7.47 (t, 1H, Ar, J = 7.9 Hz), 7.72 (d, 2H, Ar, J = 8.4 Hz), 8.04 (d, 1H, Ar, J = 4.6 Hz), 8.43 (t, 1H, NH, J = 6.1 Hz). 13C NMR (DMSO-d6): 40.1, 44.5, 52.6, 61.2, 107.0, 112.9, 125.6, 127.5, 137.4, 142.5, 143.8, 147.5, 158.9, 169.4. LC−MS: m/z 390 (M+1). HPLC purity: 96.2%. 2-(4-(Pyrimidin-2-yl)piperazin-1-yl)-N-(4-sulfamoylbenzyl)acetamide (15). White solid; yield 82%; mp 173−175 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.45 (s, 4H, piperazine CH2), 3.03(s, 2H, CH2), 3.76 (t, 4H, piperazine CH2, J = 4.5 Hz), 4.34 (d, 2H, CH2, J = 6.1 Hz), 6.60 (t, 1H, Ar, J = 4.9 Hz), 7.29 (s, 2H, NH2), 7.42 (d, 2H, Ar, J = 8.4 Hz), 7.76 (d, 2H, Ar, J = 7.6 Hz), 8.33 (d, 2H, Ar, J = 4.5 Hz), 8.45 (t, 1H, NH, J = 6.0 Hz). 13C NMR (DMSO-d6):40.1, 41.5, 43.2, 52.6, 110.1, 125.6, 127.5, 142.4, 143.8, 157.9, 161.1, 169.5. LC− MS: m/z 391 (M+1). HPLC purity: 97.2%. Benzyl 4-(2-Oxo-2-((4-sulfamoylbenzyl)amino)ethyl)piperazine-1-carboxylate (16). White solid; yield 85%; mp 121− 123 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.40 (s, 4H, piperazine CH2), 3.01 (s, 2H, CH2), 3.42 (s, 4H, piperazine CH2), 4.34 (d, 2H, CH2, J = 6.1 Hz), 5.07 (s, 2H, CH2), 7.23−7.41 (m, 9H, Ar+NH2), 7.75 (d, 2H, Ar, J = 7.6 Hz), 8.43 (t, 1H, NH, J = 5.7 Hz). 13C NMR (DMSO-d6): δ 41.5, 43.3, 52.5, 60.9, 66.1, 125.6, 127.4, 127.5, 127.8, 128.4, 136.8, 142.5, 143.7, 154.3, 169.3. LC−MS: m/z 447 (M+). HPLC purity: 97.7%. Synthesis of 3-(4-Phenylpiperazin-1-yl)-N-(4sulfamoylbenzyl)propanamide (Series 2, 19−31). General Synthetic Procedure of 3-Chloro-N-(4-sulfamoylbenzyl)propanamide (18). 4-(Aminomethyl)benzenesulfonamide (1, 10 mmol) and 3-chloropropionyl chloride (17, 10 mmol) were stirred in double distilled water (50 mL) containing 30% NaOH at room temperature for 5 h. Precipitate that appeared was filtered and dried to give 3-chloro-N-(4-sulfamoylbenzyl)propanamide (18). 3-Chloro-N-(4-sulfamoylbenzyl)propanamide (18). White solid; yield 90%; mp 135−137 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.65 (t, 2H, CH2, J = 6.0 Hz), 3.82 (t, 2H, CH2, J = 6.0 Hz), 4.34 (d, 2H, CH2, J = 6.0 Hz), 7.32 (s, 2H, NH2), 7.42 (d, 2H, Ar, J = 8.3 Hz), 7.75 (d, 2H, Ar, J = 8.4 Hz), 8.63 (t, 1H, NH, J = 5.7 Hz). LC−MS: m/z 262 (M+1). General Procedure for Synthesis of 3-(4-Phenylpiperazin-1yl)-N-(4-sulfamoylbenzyl)propanamide (19−31). Intermediate 3chloro-N-(4-sulfamoylbenzyl)propanamide (18) and substituted piperazine were taken in equimolar ratio and refluxed (100−120 °C) with Na2CO3 (catalytic amount) in dry acetonitrile for 8−14 h. Reaction mixture was diluted in water, and crude products were isolated with ethyl acetate, washed with brine, dried with anhydrous

chloroform/methanol as mobile phase, recrystallized with appropriate solvent to yield target compounds 4−16. 2-(4-Phenylpiperazin-1-yl)-N-(4-sulfamoylbenzyl)acetamide (4). White solid; yield 89%; mp 186−188 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.47 (t, 4H, piperazine CH2, J = 4.2 Hz), 2.94 (s, 2H, CH2), 3.06 (t, 4H, piperazine CH2, J = 4.5 Hz), 4.25 (d, 2H, CH2, J = 6.1 Hz), 6.65 (t, 1H, Ar, J = 7.2 Hz), 6.80 (d, 2H, Ar, J = 7.6 Hz), 7.09 (t, 2H, Ar, J = 8.0 Hz), 7.20 (s, 2H, NH2), 7.30 (d, 2H, ArH, J = 7.6 Hz), 7.65 (d, 2H, Ar, J = 8.4 Hz), 8.34 (t, 1H, NH, J = 6.0 Hz). 13C NMR (DMSO-d6): δ 41.5, 48.0, 52.8, 61.2, 115.3, 118.7, 125.6, 127.5, 128.9, 142.5, 143.8, 150.9, 169.4. LC−MS: m/z 389 (M+1). HPLC purity: 99.4%. 2-(4-(4-Fluorophenyl)piperazin-1-yl)-N-(4-sulfamoylbenzyl)acetamide (5). White solid; yield 85%; mp 168−170 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.58 (s, 4H, piperazine CH2), 3.04 (s, 2H, CH2), 3.11 (s, 4H, piperazine), 4.36 (d, 2H, CH2, J = 6.1 Hz), 6.92− 6.94 (m, 2H, Ar), 6.95−7.03 (m, 2H, Ar), 7.32 (s, 2H, NH2), 7.40 (d, 2H, J = 8.4 Hz), 7.74 (d, 2H, CH2, J = 7.6 Hz), 8.44 (t, 1H, NH, J = 6.0 Hz). 13C NMR (DMSO-d6): δ 41.6, 48.8, 52.9, 61.1, 115.4, 117.1, 125.7, 127.5, 142.5, 143.9, 147.9, 154.7, 157.1, 169.5. LC−MS: m/z 407 (M+1). HPLC purity: 95.4%. 2-(4-(4-Chlorophenyl)piperazin-1-yl)-N-(4-sulfamoylbenzyl)acetamide (6). White solid; yield 88%; mp 145−147 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.57 (s, 4H, piperazine CH2), 3.04 (s, 2H, CH2), 3.16 (s, 4H, piperazine CH2), 4.34 (d, 2H, CH2, J = 6.1 Hz), 6.93 (d, 2H, Ar, J = 9.1 Hz), 7.21 (d, 2H, Ar, J = 9.1 Hz), 7.32 (s, 2H, NH2), 7.40 (d, 2H, Ar, J = 8.3 Hz), 7.76 (d, 2H,. Ar, J = 8.4 Hz), 8.45(t, 1H, NH, J = 6.0 Hz). 13C NMR (DMSO-d6): δ 41.6, 47.8, 52.7, 61.1, 116.8, 122.2, 125.6, 127.5, 128.6, 142.5, 143.9, 149.7, 169.4. LC− MS: m/z 423 (M+1). HPLC purity: 99.5%. N-(4-Sulfamoylbenzyl)-2-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)acetamide (7). White solid; yield 86%; mp 165−167 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.58 (s, 4H, piperazine CH2), 3.06 (s, CH2, 2H), 3.32 (s, 4H, piperazine CH2), 4.35 (d, 2H, CH2, J = 6.0 Hz), 7.05 (d, 2H, Ar, J = 8.4 Hz), 7.32 (s, 2H, NH2), 7.43 (d, 2H, Ar, J = 8.4 Hz), 7.49 (d, 2H, Ar, J = 8.8 Hz), 7.77 (d, 2H, Ar, J = 8.4 Hz), 8.48 (t, 1H, NH, J = 6.0 Hz). 13C NMR (DMSO-d6): δ 41.5, 46.8, 52.5, 61.0, 114.1, 125.5, 126.1, 127.5, 142.5, 143.8, 153.2, 169.4. LC−MS: m/z 457 (M+1). HPLC purity: 97.5%. N-(4-Sulfamoylbenzyl)-2-(4-(p-tolyl)piperazin-1-yl)acetamide (8). White solid; yield 79%; mp 167−169 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.18 (s, 3H, CH3), 2.57 (s, 4H, piperazine CH2), 3.03 (s, 2H, CH2), 3.10 (s, 4H, piperazine CH2), 4.35 (d, 2H, CH2, J = 6.0 Hz), 6.82 (d, 2H, Ar, J = 8.4 Hz), 7.00 (d, 2H, Ar, J = 8.4 Hz), 7.28(brs, 2H, NH2), 7.41(d, 2H, Ar, J = 7.6 Hz), 7.75(d, 2H, Ar, J = 7.6 Hz), 8.43(t, 1H, NH, J = 6.0 Hz). 13C NMR (DMSO-d6): δ 20.0, 40.1, 41.6, 48.5, 52.9, 115.6, 125.6, 127.5, 129.4, 142.5, 143.9, 148.9, 169.5. LC−MS: m/z 403 (M+1). HPLC purity: 99.8% 2-(4-(4-Methoxyphenyl)piperazin-1-yl)-N-(4sulfamoylbenzyl)acetamide (9). White solid; yield 76%; mp 178− 180 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.58 (s, 4H, piperazine, CH2), 3.04 (s, 7H,piperazine CH2+ OCH3), 3.66 (s, 2H, CH2), 4.36 (d, 2H, CH2, J = 6.0 Hz), 6.79−6.89 (m, 4H, Ar), 7.30 (s, 2H, NH2), 7.42 (d, 2H, Ar, J = 8.4 Hz), 7.75 (d, 2H, Ar, J = 8.4 Hz), 8.42 (t, 1H, NH, J = 6.1 Hz). 13C NMR (DMSO-d6): 39.9, 40.1, 49.4, 53.0, 55.1, 114.2, 117.3, 125.6, 127.5, 142.5, 143.8, 145.3, 152.8, 169.4. LC−MS: m/z 419 (M+1). HPLC purity: 98.9%. 2-(4-Benzylpiperazin-1-yl)-N-(4-sulfamoylbenzyl)acetamide (10). White solid; yield 83%; mp 120−122 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.41−2.48 (m, 8H, piperazine CH2), 2.96 (s, 2H, CH2), 3.45 (s, 2H, CH2), 4.33 (d, 2H, CH2, J = 6.1 Hz), 7.23−7.30 (m, 7H, Ar + NH2), 7.40 (d, 2H, Ar, J = 7.6 Hz), 7.74 (d, 2H, Ar, J = 8.4 Hz), 8.33 (t, 1H, NH, J = 6.1 Hz). 13C NMR (DMSO-d6): 39.7, 52.3, 53.0, 61.2, 62.0, 125.6, 127.4, 128.1, 128.8, 138.0, 142.4, 143.8, 169.5. LC− MS: m/z 403 (M+1). HPLC purity: 97.3%. 2-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl)-N-(4sulfamoylbenzyl)acetamide (11). White solid; yield 89%; mp 174−176 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.31−2.48 (m, 8H, piperazine CH2), 2.95 (s, 2H, CH2), 3.36 (s, 2H, CH2), 4.31 (d, 2H, CH2, J = 6.0 Hz), 5.97 (s, 2H, CH2), 6.72 (d, 1H, Ar, J = 7.6 Hz), 6.81−6.83 (m, 2H, Ar), 7.29 (s, 2H, NH2), 7.39 (d, 2H, Ar, J = 7.6 3159



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6.1 Hz). 13C NMR (DMSO-d6): δ 39.0, 39.9, 52.6, 54.0, 62.1, 125.6, 127.3, 128.1, 128.7, 138.2, 142.5, 143.7, 171.3. LC−MS: m/z 417 (M+1). HPLC purity: 97.2%. 3-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl)-N-(4sulfamoylbenzyl)propanamide (26). White solid; yield 87%; mp 138−140 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.24−2.48 (m, 10H, piperazine CH2 + CH2), 2.53 (t, 2H, CH2, J = 6.8 Hz), 3.34 (s, 2H, CH2), 4.33 (d, 2H, CH2, J = 6.0 Hz), 5.97 (s, 2H, CH2), 6.73 (d, 1H, Ar, J = 8.4 Hz), 6.83 (d, 2H, Ar, J = 6.8 Hz), 7.34 (s, 2H, NH2), 7.45 (d, 2H, Ar, J = 8.4 Hz), 7.77 (d, 2H, Ar, J = 8.4 Hz), 8.49 (t, 1H, NH, J = 5.7 Hz). 13C NMR (DMSO-d6): δ 39.0, 39.9, 52.5, 54.0, 61.8, 100.7, 107.8, 108.9, 121.8, 125.6, 127.3, 132.1, 142.5, 143.7, 146.1, 147.1, 171.3. LC−MS: m/z 461 (M+1). HPLC purity: 98.4%. 3-(4-Benzhydrylpiperazin-1-yl)-N-(4-sulfamoylbenzyl)propanamide (27). White solid; yield 75%; mp 152−154 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.26−2.45 (m, 10H, piperazine + CH2), 2.51−2.55 (m, 2H, CH2), 4.22 (s, 1H, CH), 4.30 (d, 2H, CH2, J = 6.0 Hz), 7.15 (d, 2H, Ar, J = 7.3 Hz), 7.27 (t, 4H, Ar, J = 7.6 Hz), 7.32 (s, 2H, NH2), 7.41 (t, 6H, Ar, J = 7.2 Hz), 7.75(d, 2H, Ar, J = 8.4 Hz), 8.45(t, 1H, NH, J = 6.8 Hz). 13C NMR (DMSO-d6): δ 39.0, 40.1, 51.6, 52.6, 54.0, 75.3, 125.6, 127.5, 128.5, 142.5, 142.9, 143.7, 171.3. LC−MS: m/z 493 (M+1). HPLC purity: 98.1%. 3-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)-N-(4sulfamoylbenzyl)propanamide (28). White solid; yield 77%; mp 164−166 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.19−2.29 (m, 6H, piperazine CH2 + CH2), 2.51−2.57 (m, 6H, piperazine CH2 + CH2), 4.30−4.34 (m, 3H, CH2 + CH), 7.10 (t, 4H, Ar, J = 8.7 Hz), 7.33 (s, 2H, NH2), 7.40−7.47 (m, 6H, Ar), 7.75 (d, 2H, Ar, J = 8.4 Hz), 8.46 (t, 1H, NH, J = 5.7 Hz). 13C NMR (DMSO-d6): δ 33.2, 41.7, 51.4, 52.6, 54.0, 73.4, 115.2, 115.4, 125.7, 127.5, 129.3, 129.4, 138.8, 142.6, 143.8, 159.9, 162.3, 171.4. LC−MS: m/z 529 (M+1). HPLC purity: 96.1%. 3-(4-(Pyridin-2-yl)piperazin-1-yl)-N-(4-sulfamoylbenzyl)propanamide (29). White solid; yield 82%; mp 158−160 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.35 (t, 2H, CH2, J = 6.8 Hz), 2.45− 2.48 (m, 4H, piperazine, CH2), 2.59 (t, 2H, CH2, J = 6.8 Hz), 3.45 (t, 4H, piperazine CH2, J = 4.5 Hz), 4.33 (d, 2H, CH2, J = 5.3 Hz), 6.61 (t, 1H, Ar, J = 5.3 Hz), 6.80 (d, 1H, Ar, J = 8.4 Hz), 7.30 (s, 2H, NH2), 7.44 (d, 2H, Ar, J = 7.6 Hz), 7.50 (t, 1H, Ar, J = 7.8 Hz), 7.73 (d, 2H, Ar, J = 7.6 Hz), 8.09 (t, 1H, Ar, J = 3.8 Hz), 8.51 (t, 1H, NH, J = 5.7 Hz). 13C NMR (DMSO-d6): δ 33.2, 41.6, 44.6, 52.3, 54.2, 107.0, 112.9, 125.6, 126.6, 127.3, 137.5, 142.5, 143.7, 147.5,159.0, 171.3. LC−MS: m/z 404 (M+). HPLC purity: 99.7%. 3-(4-(Pyrimidin-2-yl)piperazin-1-yl)-N-(4-sulfamoylbenzyl)propanamide (30). White solid; yield 84%; mp 115−117 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.36 (t, 2H, CH2, J = 6.8 Hz), 2.43 (t, 4H, piperazine CH2, J = 4.9 Hz), 2.60 (t, 2H, CH2, J = 6.8 Hz), 3.72 (t, 4H, piperazine CH2, J = 4.5 Hz), 4.33 (d, 2H, CH2, J = 6.1 Hz), 6.60 (t, 1H, Ar, J = 4.9 Hz), 7.32 (s, 2H, NH2), 7.45 (d, 2H, Ar, J = 8.4 Hz), 7.75 (d, 2H, Ar, J = 8.4 Hz), 8.33 (d, 2H, Ar, J = 4.6 Hz), 8.52 (t, 1H, NH, J = 6.1 Hz). 13C NMR (DMSO-d6): δ 33.2, 41.6, 43.3, 52.3, 54.2, 110.1, 125.6, 127.3, 142.5, 143.8, 157.9, 161.1, 171.4. LC−MS: m/z 405 (M+). HPLC purity: 100%. Benzyl 4-(3-Oxo-3-((4-sulfamoylbenzyl)amino)propyl)piperazine-1-carboxylate (31). White solid; yield 79%; mp 98− 100 °C. 1H NMR (DMSO-d6, 400 MHz): δ 2.30−2.38 (m, 6H, CH2), 2.57 (t, 2H, CH2, J = 6.8 Hz), 3.38 (s, 4H, CH2), 4.33 (d, 2H, CH2, J = 5.3 Hz), 5.07 (s, 2H, CH2), 7.29−7.39 (m, 7H, Ar + NH2), 7.43 (d, 2H, Ar, J = 8.4 Hz), 7.75 (d, 2H, Ar, J = 8.4 Hz), 8.47 (t, 1H, NH, J = 6.1 Hz).13C NMR (DMSO-d6): δ 33.0, 41.5, 43.5, 52.2, 54.0, 66.1, 125.5, 127.3, 127.5, 127.8, 128.4, 136.9, 142.5, 143.7, 154.3, 171.1. LC−MS: m/z 461 (M+1). HPLC purity: 97.3%. Carbonic Anhydrase Inhibition Assay. A stopped-flow instrument (Applied Photophysics, Oxford, U.K.) has been used for analyzing the CA catalyzed CO2 hydration/inhibition, as described earlier.32−36 All CA isoforms used in this work (hCA I, II, VII, and IX) were recombinant proteins produced in-house as described earlier.32−36 Anticonvulsant Activity. The anticonvulsant potential of the target compounds was examined by using the MES as well as scPTZ

sodium sulfate, and evaporated under reduced pressure. Column chromatography was used to purify crude products using chloroform/ methanol as an eluent, recrystallized with appropriate solvent to confer target compounds 19−31. 3-(4-Phenylpiperazin-1-yl)-N-(4-sulfamoylbenzyl)propanamide (19). White solid; yield 86%; mp 121−123 °C. 1H NMR (DMSO-d6,400 MHz): δ 2.36 (t, 2H, CH2, J = 6.8 Hz), 2.52 (t, 4H, piperazine CH2, J = 4.9 Hz), 2.60 (t, 2H, CH2, J = 6.8 Hz), 3.11 (t, 4H, piperazine CH2, J = 4.5 Hz), 4.33 (d, 2H, CH2, J = 6.1 Hz), 6.76 (t, 1H, Ar, J = 7.2 Hz), 6.92 (d, 2H, Ar, J = 7.6 Hz), 7.17−7.21 (m, 2H, Ar), 7.28 (brs, 2H, NH2), 7.44 (d, 2H, Ar, J = 8.4 Hz), 7.73 (d, 2H, Ar, J = 7.6 Hz), 8.53 (t, 1H, NH, J = 5.5 Hz). 13C NMR (DMSO-d6): δ 33.2, 40.1, 48.2, 52.5, 54.1, 115.3, 118.7, 125.5, 127.3, 128.9, 142.5, 143.7, 150.9, 171.3. LC−MS: m/z 403 (M+1). HPLC purity: 98.9%. 3-(4-(4-Fluorophenyl)piperazin-1-yl)-N-(4-sulfamoylbenzyl)propanamide (20). White solid; yield 79%; mp 134−136 °C. 1H NMR (DMSO-d6,400 MHz): δ 2.35 (t, 2H, CH2, J = 6.8 Hz), 2.53(s, 4H, piperazine CH2), 2.60(t, 2H, CH2, J = 7.0 Hz), 3.05 (t, 4H, piperazine CH2, J = 4.6 Hz), 4.31(d, 2H, CH2, J = 6.0 Hz), 6.90−6.94 (m, 2H, Ar), 7.00−7.04 (m, 2H, Ar), 7.28 (s, 2H, NH2), 7.43 (d, 2H, Ar, J = 8.4 Hz), 7.72 (d, 2H, Ar, J = 8.4 Hz), 8.49 (t, 1H, NH, J = 6.1 Hz). 13C NMR (DMSO-d6): δ 33.1, 41.5, 48.9, 52.4, 54.0, 115.1, 115.3, 117.0, 117.1, 125.5, 127.3, 142.4, 143.6, 147.8, 154.7, 157.1, 171.2. LC−MS: m/z 421 (M+1). HPLC purity: 99.8%. 3-(4-(4-Chlorophenyl)piperazin-1-yl)-N-(4-sulfamoylbenzyl)propanamide (21). White solid; yield 84%; mp 135−137 °C. 1H NMR (DMSO-d6,400 MHz): δ 2.36 (t, 2H, CH2, J = 6.8 Hz), 2.52 (s, 4H, piperazine CH2), 2.61 (t, 2H, CH2, J = 6.8 Hz), 3.11 (t, 4H, piperazine CH2, J = 4.3 Hz), 4.34 (d, 2H, CH2, J = 6.1 Hz), 6.92−6.94 (m, 2H, Ar), 7.19−7.22 (m, 2H, Ar), 7.32 (s, 2H, NH2), 7.44 (d, 2H, Ar, J = 8.4 Hz), 7.75 (d, 2H, Ar, J = 8.4 Hz), 8.52 (t, 1H, NH, J = 6.1 Hz). 13C NMR (DMSO-d6): δ 33.2, 41.6, 48.0, 52.3, 54.0, 116.8, 122.3, 125.6, 127.3, 128.6, 142.5, 143.7, 149.7, 171.3. LC−MS: m/z 437 (M+1). HPLC purity: 99.0%. N-(4-Sulfamoylbenzyl)-3-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)propanamide (22). White solid; yield 80%; mp 145−147 °C. 1H NMR (DMSO-d6,400 MHz): δ 2.36 (t, 2H, CH2, J = 6.8 Hz), 2.50−2.52 (m, 4H, piperazine CH2), 2.60 (t, 2H, CH2, J = 7.2 Hz), 3.25 (t, 4H, piperazine J = 4.5 Hz), 4.33 (d, 2H, CH2, J = 6.1 Hz), 7.05 (d, 2H, Ar, J = 9.1 Hz), 7.30 (s, 2H, NH2), 7.42−7.49 (m, 4H, Ar), 7.73 (d, 2H, Ar, J = 8.4 Hz), 8.51 (t, 1H, NH, J = 6.0 Hz). 13C NMR (DMSO-d6): δ 33.2, 41.6, 47.0, 52.2, 54.0, 114.1, 125.6, 126.1, 127.3, 142.5, 143.7, 153.2, 171.3. LC−MS: m/z 471 (M+1). HPLC purity: 95.5%. N-(4-Sulfamoylbenzyl)-3-(4-(p-tolyl)piperazin-1-yl)propanamide(23). White solid; yield 72%; mp 126−128 °C. 1H NMR (DMSO-d6,400 MHz): δ2.18 (s, 3H, CH3), 2.35 (t, 2H, CH2, J = 6.8 Hz), 2.51 (s, 4H, piperazine CH2), 2.60 (t, 2H, CH2, J = 6.8 Hz), 3.05 (s, 4H, piperazine CH2), 4.43 (d, 2H, CH2, J = 5.3 Hz), 6.81(d, 2H, Ar, J = 8.4 Hz), 7.00 (d, 2H, Ar, J = 7.6 Hz), 7.30 (s, 2H, NH2), 7.43 (d, 2H, Ar, J = 8.4 Hz), 7.73 (d, 2H, Ar, J = 8.4 Hz), 8.51 (t, 1H, NH, J = 5.7 Hz). 13C NMR (DMSO-d6): δ 20.0, 33.1, 41.5, 48.7, 52.5, 54.1, 115.6, 125.5, 127.3, 127.5, 129.3, 142.5, 143.7, 148.8, 171.3. LC− MS: m/z 417 (M+1). HPLC purity: 99.7%. 3-(4-(4-Methoxyphenyl)piperazin-1-yl)-N-(4sulfamoylbenzyl)propanamide (24). White solid; yield 89%; mp 115−117 °C. 1H NMR (DMSO-d6,400 MHz): δ 2.34 (t, 2H, CH2, J = 6.8 Hz), 2.52 (s, 4H, piperazine CH2), 2.59 (t, 2H, CH2, J = 6.8 Hz), 2.99 (s, 4H, piperazine CH2), 3.66 (s, OCH3), 4.32 (d, 2H, CH2, J = 5.3 Hz), 6.78−6.80 (m, 2H, Ar), 6.86−6.88 (m, 2H, Ar), 7.29 (s, 2H, NH2), 7.43 (d, 2H, Ar, J = 8.4 Hz), 7.73 (d, 2H, Ar, J = 8.4 Hz), 8.51 (t, 1H, NH, J = 6.4 Hz). 13C NMR (DMSO-d6): δ 33.2, 41.5, 49.6, 52.6, 54.0, 55.1, 114.2, 117.3, 125.5, 127.3, 142.4, 143.7, 145.3, 152.8, 171.2. LC−MS: m/z 433 (M+1). HPLC purity: 99.7%. 3-(4-Benzylpiperazin-1-yl)-N-(4-sulfamoylbenzyl)propanamide (25). White solid; yield 85%; mp 95−97 °C. 1H NMR (DMSO-d6,400 MHz): δ 2.24−2.41 (m, 10H, piperazine CH2+ CH2), 2.53 (t, 2H, CH2, J = 6.8 Hz), 3.34 (s, 2H, CH2), 3.43 (s, 2H, CH2), 4.32 (d, 2H, CH2, J = 6.1 Hz), 7.22−7.32 (m, 7H, Ar + NH2), 7.43 (d, 2H, Ar, J = 8.4 Hz), 7.75 (d, 2H, Ar, J = 8.4 Hz), 8.47 (t, 1H, NH, J = 3160



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tests. Swiss Albino male mice (25−30 g) and male Wistar rats (100− 150 g) were procured from Disease Free Small Animal House, Lala Lajpat Rai University of Veterinary and Animal Sciences Hisar, Haryana, India. Animals were adapted for a week at animal house of Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, India. Animals were arbitrarily allocated to experimental groups and used only one time for the reported experiments. 1% gum acacia was used to suspend the test compounds, including standard drugs AAZ and TPM, provided a volume 0.01 mL/g and 0.04 mL/10 g body weight to mice (intraperitoneally, ip) and rats (orally), respectively. Convulsant dose of PTZ was dissolved in normal saline solution. Experimental procedure were preapproved by the Institutional Animal Ethics Committee Wide Approval Number IAEC/ACBR/2016/PML/ 016. Maximal Electroshock (MES) Test. This test was performed using six animals in each group. The test compounds and standard drugs (TPM and AAZ) were administered (ip) to mice 0.5 h prior to seizure stimulation at the doses of 30 and 100 mg/kg. Oral bioavailability study of compound 26 has been carried out at 0.25, 0.5, 1, 2, and 4 h time intervals after drug administration. An electroconvulsiometer (Techno Instruments, Lucknow, India) was used to generate seizures in experimental animals. 50 mA and 150 mA electric stimuli were provided transauricularly to mice and rats, respectively, for 0.2 s. Transauricular stimulation triggers the brain stem region that leads to elicitation of severe tonic convulsions in animals, and total elimination of hind limb extension is measured as protection38f. Subcutaneous Pentylenetrazole (sc-PTZ) Test. The sc-PTZ test was conducted using six animals per group, and 85 mg/kg (CD97) convulsive dose of PTZ was provided to Swiss albino mice subcutaneously (sc). The anticonvulsant effect of test compounds and standard drugs was evaluated at the dose 30 and 100 mg/kg according to our standardized procedures as described earlier.38,40b,e Time Course Study. Time course studies performed on Swiss albino mice and 10 (n = 10) mice were taken per group. The test compound was introduced ip at 30 mg/kg dose, and protection against MES-induced seizure was assessed at 0.5, 1, 2, 3, 4, and 6 h time intervals.38 Neurotoxicity Minimal Motor Impairment (MMI). The acute neurological toxicity (NT) was performed after treatment of compound 26 in mice using the standardized rotarod test. The rotarod instrument contains of a rotating rod of 3.2 cm (Techno Rotarod system, Techno Electronics, Lucknow, India). The mice were pretrained on the accelerating rotating rod at 25 rpm, and the mice, which fails to sustain their equilibrium for a prolonged period of 1 min, were discarded from the experiment. Trained animals were provided an ip injection of compound 26 at three doses of 30 mg/kg, and neurotoxicity was evaluated. The neurological mutilation was indicated by the inability of an animal to maintain equilibrium on rotating rod, and the compound was considered to be toxic if the treated animal falls down from rotating rod 3 times during 1 min time period.40e Toxicological Studies. A subacute toxicity study was performed in male Wistar rats (n = 6) by providing continuous treatment of test compound and vehicle orally for 14 days. Mark of toxicity such as inflammation, tumor induction, allergy, and abnormal behavior was noticed up to whole experimental period. After the stipulated period, animals were anesthetized by anesthetic ether, blood was withdrawn through cardiac puncture, and obtained blood sample was analyzed according to our pervious reported method.38 Statistical Analysis. Statistical analysis was done by using the GraphPad Prism 5 software (La Jolla, CA, USA). The neurotoxicity assay data (rotarod test) were expressed as the mean ± SEM and analyzed by two-way repeated measures ANOVA, followed by the Bonferroni post-test. P < 0.05 was considered to be statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00208. Spectral (1H and 13C NMR) and HPLC data of all new compounds (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*M.T.: phone, +91-01127666272; e-mail, mtiwari07@gmail. com. *C.T.S.: phone, +39-055-4573729; fax, +39-055-4573385; email, claudiu.supuran@unifi.it. ORCID

Manisha Tiwari: 0000-0001-8942-0578 Claudiu T. Supuran: 0000-0003-4262-0323 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.B.M. thanks Department of Health Research, Ministry of Health and Family Welfare and S.K. thanks the Department of Biotechnology New Delhi, India, for financial assistance to carry out this research work. M.T. thanks University of Delhi, Delhi, India, for financial support to conduct this work. The University Science Instrumentation Center (USIC), University of Delhi, is deeply acknowledged for recording NMR spectra. Work from the Supuran laboratory was financed by two EU grants (Dynano and Metoxia).

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ABBREVIATIONS USED DMF, dimethylformamide; THF, tetrahydrofuran; AAZ, acetazolamide; KI, inhibition constant; CA, carbonic anhydrase; CAI, carbonic anhydrase inhibitor; MES, maximal electroshock seizure; scPTZ, subcutaneous pentylenetetrazole; po, per oral; ip, intraperitoneal

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Discovery of Benzenesulfonamide Derivatives as Carbonic Anhydrase

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