key: cord-0016679-ofu3p95s
authors: Odi, Reem; Bibi, David; Shusterman, Bella; Erenburg, Natalia; Shaul, Chanan; Supuran, Claudiu T.; Nocentini, Alessio; Bialer, Meir
title: Synthesis and Enantioselective Pharmacokinetic/Pharmacodynamic Analysis of New CNS-Active Sulfamoylphenyl Carbamate Derivatives
date: 2021-03-25
journal: Int J Mol Sci
DOI: 10.3390/ijms22073361
sha: 35e1de4b07ea83918c3eb06deecad2379a5321f4
doc_id: 16679
cord_uid: ofu3p95s

We recently reported a new class of carbamate derivatives as anticonvulsants. Among these, 3-methylpentyl(4-sulfamoylphenyl)carbamate (MSPC) stood out as the most potent compound with ED(50) values of 13 mg/kg (i.p.) and 28 mg/kg (p.o.) in the rat maximal electroshock test (MES). 3-Methylpropyl(4-sulfamoylphenyl)carbamate (MBPC), reported and characterized here, is an MSPC analogous compound with two less aliphatic carbon atoms in its structure. As both MSPC and MBPC are chiral compounds, here, we studied the carbonic anhydrase inhibitory and anticonvulsant action of both MBPC enantiomers in comparison to those of MSPC as well as their pharmacokinetic properties. Racemic-MBPC and its enantiomers showed anticonvulsant activity in the rat maximal electroshock (MES) test with ED(50) values in the range of 19–39 mg/kg. (R)-MBPC had a 65% higher clearance than its enantiomer and, consequently, a lower plasma exposure (AUC) than (S)-MSBC and racemic-MSBC. Nevertheless, (S)-MBPC had a slightly better brain permeability than (R)-MBPC with a brain-to-plasma (AUC) ratio of 1.32 (S-enantiomer), 1.49 (racemate), and 1.27 (R-enantiomer). This may contribute to its better anticonvulsant-ED(50) value. The clearance of MBPC enantiomers was more enantioselective than the brain permeability and MES-ED(50) values, suggesting that their anticonvulsant activity might be due to multiple mechanisms of action.

Out of 21 antiepileptic drugs (AEDs) introduced into the global market between 1989 and 2021, eleven are chiral molecules and nine of these AEDs were introduced into the market as a single enantiomer (or diastereomer). Only two chiral drugs (i.e., vigabatrin and fenfluramine) were introduced as racemates [1] .

Two of the 21 AEDs contain a carbamate moiety (felbamate and cenobamate) in their chemical structure and two contain a sulfonamide (zonisamide) or a sulfamate (topiramate) moiety [2] . In addition, some new AEDs in development such as NBI-921352 (formerly XEN901), a selective inhibitor of Nav1.6 sodium channels, CVL-865, a selective positive allosteric modulator of GABAA, and bumetanide derivatives contain a sulfonamide in their chemical structure [3] [4] [5] [6] . AND-287 is a chiral CNS-active sulfonamide derivative currently in development that its (R)-enantiomer demonstrated a more potent anticonvulsant activity than its (S)-enantiomer [1, 7] . AND-287 has the following three structural elements: (a) an aryl portion that modulates potency, stability, and efficacy; (b) a benzyl substituent to increase the neuroprotective effect and bioavailability, and (c) a sulfamide moiety necessary to produce antiseizure and neuroprotective activity [7] . The anticonvulsant retigabine, currently being developed in a new oral formulation (XEN496), also contains a carbamate moiety in its molecular structure [3, 4] . All the above shows that sulfonamide and carbamate moieties are of relevance in the development and design of new AEDs [8] .

Lately, we reported a new class of (4-sulfamoylphenyl)carbamates as anticonvulsant derivatives. Among these, 3-methylpentyl(4-sulfamoylphenyl)carbamate (MSPC) showed the most potent effects in a maximal electroshock (MES) test with ED 50 values of 13 mg/kg (i.p.) and 28 mg/kg (p.o.) in rats [9, 10] . 3-Methylpropyl(4-sulfamoylphenyl)carbamate (MBPC) is an MSPC analogue with two less aliphatic carbons in its chemical structure ( Figure 1 ). Both MSPC and MBPC have a stereogenic center, making it necessary to study both individual enantiomers for their anticonvulsant activity. Indeed, drug chirality is a major issue in the design and development of new active pharmacological entities, since stereoisomers that possess the same chemical formula but different 3D conformations generally produce diverse pharmacological responses [11] [12] [13] [14] [15] .

Many new chiral small molecule drugs were recently approved by the US Food and Drug Administration (FDA) as a single stereoisomer [13, 16, 17] .

In this study, we report the synthesis of MBPC and assess the anticonvulsant activity, carbonic anhydrase (CA; EC 4.2.1.1) inhibition, and pharmacokinetics in rats of both MBPC enantiomers in comparison to MSPC and its enantiomers [9, 10] .

The synthesis of 3-methylpropyl (4-sulfamoylphenyl) carbamate (MBPC) and its single enantiomers was accomplished according to Scheme 1. The major pharmacokinetic (PK) parameters of racemic-MBPC and its individual enantiomers are depicted in Tables 1 and 2. The mean plasma and brain concentrations of MBPC (racemate), (R)-MBPC, and (S)-MBPC after i.p. (80 mg/kg) administration to rats are illustrated in Figures 2 and 3 , respectively. As a result, the brain-to-plasma (AUC) ratio (BPR) of racemic-MBPC and its two individual enantiomers was as follows:

Racemic-MBPC = AUC brain /AUC plasma = 48/32 = 1.49 (S)-MBPC = AUC brain /AUC plasma = 52/40 = 1.32 (R)-MBPC = AUC brain /AUC plasma = 30/24 = 1.27 Table 1 . Mean pharmacokinetic (PK) parameters of 3-methylpropyl(4-sulfamoylphenyl)carbamate (MBPC) (racemate) and its two individual enantiomers calculated from plasma levels following i.p. (80 mg/kg dose) administration to rats. Table 3 . Table 3 . Quantitative anticonvulsant and minimal neurotoxicity data (ED 50 and TD 50 values) and protective indexes (PI) of 3-methylpropyl(4-sulfamoylphenyl)carbamate (MBPC) and 3-methylpentyl(4sulfamoylphenyl)carbamate (MSPC). in rats dosed p.o. or i.p. b . Table 4 shows the inhibition constants (K I s) of racemic-MBPC and its enantiomers, in comparison to MSPC and its enantiomers, against four human isoforms of carbonic anhydrase (hCA), namely the cytosolic and ubiquitous hCAs I and II, hCA IV (expressed in the CNS), and the brain-associated hCA VII. The compounds' inhibitory potency spanned from a low to medium nanomolar range depending on the target hCA isoform. Racemic-MBPC and its enantiomers were medium potency hCA IV inhibitors but effectively inhibited hCAs I, II, and VII. (R)-MBPC was five times more effective than its (S)-enantiomer against hCA II, whereas (S)-MBPC was 2.9 and 3.7 times more effective than the (R)-enantiomer against hCAs I and VII, respectively. Table 4 . Inhibition data of human carbonic anhydrase (CA) isoforms I, II, IV, and VII for of 3-methylpropyl(4-sulfamoylphenyl)carbamate (MBPC) and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped-flow CO 2 hydrase assay. The inhibition data previously reported for 3-methylpentyl(4-sulfamoylphenyl)carbamate (MSPC). are also shown. 

In 1992, the US Food and Drug Administration (FDA) issued a policy statement containing guidelines for the development of chiral drugs. In this document, the FDA recommends the evaluation of therapeutic utility and/or toxicological effects of each stereoisomer for chiral drug candidates [18] . Thus, there is an incentive to develop a chiral drug as a single individual enantiomer even if it is marketed first as a racemate [12, 15] . In addition, a chiral switch from a racemate to one of its individual enantiomers is utilized to extend the period of patent exclusivity, since secondary pharmaceutical patents protect a range of aspects other than the active pharmaceutical ingredient (API-protected by the primary pharmaceutical patent) [15] . The term "chiral switch", which is a component of secondary pharmaceutical patents, refers to the development of a single enantiomer from a chiral drug that was developed or marketed previously as a racemate. Thus, the essential criterion of a chiral switch is a change in the status of chirality [14] . The potential advantages of chiral switching are (a) improved therapeutic index through increased potency and selectivity; (b) a faster onset of action (e.g., (S)-ibuprofen); (c) lower susceptibility to drugdrug interactions, and (d) lower dosage compared to the racemate. It should be pointed out that a chiral switch is not clinically beneficial for all racemic drugs and sometimes offers little clinical advantage and is used by pharmaceutical manufacturers to perpetuate revenues as the original racemate approaches the end of its market exclusivity [13, 19, 20] .

Since 1955, CNS-active alkylcarbamates have been developed for the treatment of epilepsy and other CNS disorders. Out of the regulatory approved carbamates, felbamate, retigabine, carisbamate, and cenobamate had different levels of success as AEDs. Carisbamate did not make it due to inconsistent clinical efficacy, and felbamate caused serious and sometimes life-threatening side effects that restricted its clinical use [9, 10, [21] [22] [23] . Cenobamate, approved by the FDA on November 2019, is currently showing potential for refractory patients with seizures that have been difficult to control with other AEDs [24, 25] .

MSPC was the most potent compound of a series of benzenesulfonamide alkylcarbamate derivatives reported lately [9] . MBPC was not included in such a series. A stereoselective PK and PD analysis showed that (R)-MBPC had a 65% higher clearance than its enantiomer and, consequently, a lower plasma exposure (AUC) than both (S)-MBPC and racemic-MBPC. Nevertheless, racemic-MBPC and its (S)-and (R)-enantiomers have the following brain-to-plasma (AUC) ratios (BPR) of 1.49, 1.32, and 1.27, respectively. The slightly better BPR of (S)-MBPC may contribute to its better rat anticonvulsant ED 50 value. MBPC's BPR values were better than that of (S)-MSPC but lower than those of racemicand (R)-MSPC [10] . While (R)-MSPC has lower plasma and brain exposure compared to (S)-MSPC, the two MBPC enantiomers had similar brain permeability, with a slightly better BPR value for (S)-MBPC. The enantiostability of (R)-and (S)-MBPC was verified by extracting them from rat plasma and measuring their optical purity, and no change was observed in their specific optical rotation [α] D value in comparison to their initial value when they were spiked in organic solvent prior to the optical rotation measurement. This shows that no chiral inversion was observed between the individual enantiomers of MBPC. Relevantly, a significant protective index was observed for MBPC and enantiomers as noted from the TD 50 compared to the MES ED 50 values reported in Table 3 . A recently published study on similar benzenesulfonamide CAIs as antiepileptics showed no toxic effect toward the hepatic system associated with the use of this class of compounds [26] .

Unlike MSPC, MBPC demonstrated enantiospecificity in the anticonvulsant activity of its individual enantiomers. The (S)-MBPC rat MES ED 50 value was 19 mg/kg [95%CI = 13-25 mg/kg] compared to 39 mg/kg [95%CI = 27-51 mg/kg] for (R)-MBPC. This does not rule out the possibility that the anticonvulsant efficacy of MBPC enantiomers might be due to multiple mechanisms of action. It also demonstrates that a whole-body (in vivo) PD measure such as MBPC-MES-ED 50 is enantioselective as an MBPC-specific PD measure, such as a CA inhibition K I or the primary PK parameter clearance, which, in the case of MBPC, is mainly metabolic. The better anticonvulsant activity of (S)-MBPC is an incentive that it will be developed as a single individual enantiomer [12, 15] . Nevertheless, this needs to be confirmed by other anticonvulsant models and does not rule out the possibility that both MBPC individual enantiomers might be candidates for further evaluation as potential new AEDs.

As previously suggested by us [9] , the anticonvulsant activity of these classes of derivatives might be, in part, ascribed to the inhibition of human CAs which have been shown to be implicated in seizures. In fact, the human CNS is among the tissues/organs with the highest number of CA isoforms, among which are Cas I, II, III, IV, VA, VII, XII, and XIV [27] . Their inhibition has been exploited since the 1970s with new AEDs. Acetazolamide, topiramate, sulthiame, and zonisamide are CAI-marketed AEDs that contain a sulfonamide (or sulfamate) moiety in their chemical structure. The mechanisms by which CA isoforms possess antiepileptic activity is rather complex and there is no definitive consensus among researchers about this issue [27] .

Racemic-MBPC showed an improved CA IV and VII inhibition potency compared to racemic-MSPC (K I of 432 vs. 750 nM and 121 vs. 351 nM, respectively), whereas it acts as a weaker CA I and II inhibitor than its longer analogue (K I of 97.1 vs. 77.0 nM and 20.2 vs. 7.6 nM, respectively). Of note, (R)-MBPC, as with (R)-MSPC, is the eutomer against CA II. In contrast, a eutomer swap occurred against CA I, with (S)-MBPC being (K I of 71.8 nM) more active than (R)-MBPC (K I of 203 nM). Additionally, the K I increase in MBPC against CA IV accounts for the (S)-enantiomer as a eutomer, as in the case of MSPC. Interestingly, (S)-MBPC acted as a eutomer against CA VII (K I of 53.3 nM), showing the greatest eutomer K I increase with respect to MSPC.

Irrespective of the outcome of the sulfonamides and carbamates currently in development (e.g., NBI-921352, CVL-865) [28] , alkyl-or aryl sulfonamides as well as sulfamoylphenyl carbamate derivatives will continue to represent an interesting class of compounds exhibiting anticonvulsant activity in animal models, coupled with a potential to help refractory epileptic patients.

All the solvents were of analytical grade or high-performance liquid chromatography (HPLC) grade and were purchased from Sigma-Aldrich, St. Louis, MI, USA.

The general synthesis of of 3-methylpropyl(4-sulfamoylphenyl)carbamate (MBPC)) is described in Scheme 1. A solution of 2-butanol (for the synthesis of MBPC), (R)-2-butanol (for the synthesis of (R)-MBPC), or (S)-2-butanol (for the synthesis of (S)-MBPC) (1 equiv.) in dichloromethane (DCM, 5 mL) was added dropwise to a stirred solution of triphosgene (0.5 equiv.) and triethylamine (1 equiv.) in DCM (10 mL). The reaction mixture was allowed to stir for 2h at room temperature and then evaporated. The residue was dissolved in THF (10 mL). A solution of sulfanilamide (500 mg) in THF (10 mL) was added and stirred overnight. The reaction was quenched with water and extracted with EtOAc (15 mL). The organic layer was washed with water and brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The desired product was obtained following flash chromatography (EtOAac/hexane). The identity of MBPC enantiomers and their purity were assessed by 1 H NMR, HPLC, and elemental analysis. 

The pharmacokinetics (PK) of racemic-MBPC and its two individual enantiomers were studied following i.p. administration (80 mg/kg) to male Sprague Dawley rats weighing approximately 200-250 g, and their major PK parameters were estimated. The PK experiments were approved by the Ethical Committee of the Hebrew University's Faculty of Medicine. The dose was administered to rats in multisol that is a mixture of propylene glycol, alcohol (EtOH), and water for injection at a ratio of 8:1:1. The study details were as described previously [29] . Plasma and brain levels of racemic-MBPC and its individual enantiomers were monitored at 20, 40, 60, 90, 120, 160, 180, 200, 220, and 260 min after dosing. Two rats were sacrificed at each time point.

Plasma and brain concentrations of each compound were quantified by an HPLC assay. The HPLC analysis was performed on a system (2695 Separation Module; Waters, Milford, MA, USA) with a photodiode array UV detector (2996 PDA Detector; Waters, Milford, MA, USA) conditioned as follows: Kinetex, 5 u EVO C18 100 A, 1504.6-mm column (Phe-nomenexVR, Torrance, CA, USA). Linear gradients (5-95% acetonitrile content) with H 2 O (0.1% formic acid) and acetonitrile were used as the eluents with a flow rate of 1 mL/min at 20 • C. The compounds and the internal standard were detected at 250 nm. Plasma and brain concentrations of MBPC and its two individual enantiomers were quantified as previously described, [29] .

The PK parameters of each compound were calculated by non-compartmental analysis based on statistical moment theory using the PK software Phoenix Winnonlin Tripos L.P. (Pharsight Co., Mountain View, CA, USA) as previously described [30] .

The experiments with the MES model were conducted in male Sprague Dawley rats weighing 100-120 g (Charles River Laboratories, Wilmington, MA, USA) as previously described [30] [31] [32] . Eight rats were tested per dose in the dose-response curve in order to determine the ED 50 value of each compound. The experiments were approved by the Ethical Committee of the NIH-ETSP program where the anticonvulsant activity was tested [30] [31] [32] .

The carbonic anhydrase inhibition of MBPC and its individual enantiomers was assessed using a stopped-flow CO 2 hydrase assay as previously described [33] [34] [35] [36] . 

Chirality as an important factor for the development of new antiepileptic drugs

A Perspective into the physicochemical and biopharmaceutic properties of marketed antiepileptic drugs-from phenobarbital to cenobamate and beyond

Progress report on new antiepileptic drugs: A summary of the Fifteen Eilat Conference (EILAT XV). I. Drugs in preclinical and early clinical development

Progress report on new antiepileptic drugs: A summary of the Fifteenth Eilat Conference on New Antiepileptic Drugs and Devices (EILAT XV). II. Drugs in more advanced clinical development

A novel prodrug-based strategy to increase effects of beumetanide in epilepsy

The search for NKCC1-selective drugs for the treatment of epilepsy: Structure-function relationship of bumetanide and various bumetanide derivatives in inhibiting the human cationchloride cotransporter NKCC1A

Small Molecule Anticonvulsant Agents with Potent In Vitro Neuroprotection

Key factors in the discovery and development of new antiepileptic drugs (AEDs)

Design and comparative evaluation of the anticonvulsant profile, carbonic-anhydrate inhibition and teratogenicity of novel carbamate derivatives of branched aliphatic carboxylic acids with 4-aminobenzensulfonamide

Stereoselective pharmacokinetic and pharmacodynamic analysis of a CNS-active sulphamoylphenyl carbamate derivative

Stereoselectivity in pharmacokinetics: A general theory

Putting chirality to work: The strategy of chiral switches

The market of chiral drugs: Chiral switches versus de novo enantiomerically pure compounds

Chiral switches of chloroquine and hydroxychloroquine: Potential drugs to treat Covid-19

In defense of secondary pharmaceutical patents in drug discovery and development

The pharmaceutical industry in 2019. An analysis of FDA drug Approvals from the Perspective of Molecules

An analysis of FDA drug approvals from the perspective of molecules

Guidance of Development of New Stereoisomeric Drugs

Single-Enantiomer Drugs Elegant Science, Disappointing Effects

Assessing the chiral switch: Approval and use of single-enantiomer drugs

Antiepileptic Drugs

The rise and fall of felbamate as a treatment for partial epilepsy-aplastic anemia and hepatic failure to blame?

Drugs in more advanced clinical development

Safety and efficacy of adjunctive cenobamate (YKP3089) in patients with uncontrolled focal seizures: A multicentre, double-blind, randomised, placebo-controlled, dose-response trial

Cenobamate for focal seizures-a game changer

Discovery of Potent Carbonic Anhydrase Inhibitors as Effective Anticonvulsant Agents: Drug Design, Synthesis, and In Vitro and In Vivo Investigations

Human Carbonic Anhydrases: Tissue Distribution, Physiological Role and Druggability

The ups and downs of alkyl-carbamates in epilepsy therapy: How does cenobamate differ? Epilepsia

Pharmacokinetic-pharmacodynamic correlation and brain penetration of sec-butylpropylacetamide, a new CNS drug possessing unique activity against status epilepticus

Stereoselective pharmacodynamic and pharmacokinetic analysis of sec-butylpropylacetamide (SPD), a new CNS-active derivative of valproic acid with unique activity against status epilepticus

Stereoselective anticonvulsant and pharmacokinetic analysis of valnoctamide, a CNS-active derivative of valproic acid with low teratogenic potential

The potential of valnoctamide and sec-butylpropylacetamide (SPD) for acute seizures and status epilepticus (SPD) and valnoctamide and their individual stereoisomers in status epilepticus

The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C

Carbonic anhydrase inhibitors. Zonisamide is an effective inhibitor of the cytosolic isozyme II and mitochondrial isozyme V: Solution and X-ray crystallographic studies

Carbonic anhydrase inhibitors: SAR and X-ray crystallographic study for the interaction of sugar sulfamates/sulfamides with isozymes I, II and IV

The coumarin-binding site in carbonic anhydrase accommodates structurally diverse inhibitors: The antiepileptic lacosamide as an example