Chimeric derivatives of functionalized amino acids and α-aminoamides: compounds with anticonvulsant activity in seizure models and inhibitory actions on central, peripheral, and cardiac isoforms of voltage-gated sodium channels.

Six novel 3″-substituted (R)-N-(phenoxybenzyl) 2-N-acetamido-3-methoxypropionamides were prepared and then assessed using whole-cell, patch-clamp electrophysiology for their anticonvulsant activities in animal seizure models and for their sodium channel activities. We found compounds with various substituents at the terminal aromatic ring that had excellent anticonvulsant activity. Of these compounds, (R)-N-4'-((3″-chloro)phenoxy)benzyl 2-N-acetamido-3-methoxypropionamide ((R)-5) and (R)-N-4'-((3″-trifluoromethoxy)phenoxy)benzyl 2-N-acetamido-3-methoxypropionamide ((R)-9) exhibited high protective indices (PI=TD50/ED50) comparable with many antiseizure drugs when tested in the maximal electroshock seizure test to mice (intraperitoneally) and rats (intraperitoneally, orally). Most compounds potently transitioned sodium channels to the slow-inactivated state when evaluated in rat embryonic cortical neurons. Treating HEK293 recombinant cells that expressed hNaV1.1, rNaV1.3, hNaV1.5, or hNaV1.7 with (R)-9 recapitulated the high levels of sodium channel slow inactivation.

Chimeric agents derived from the functionalized amino acid, lacosamide, and the α-aminoamide, safinamide: evaluation of their inhibitory actions on voltage-gated sodium channels, and antiseizure and antinociception activities and comparison with lacosamide and safinamide.

The functionalized amino acid, lacosamide ((R)-2), and the α-aminoamide, safinamide ((S)-3), are neurological agents that have been extensively investigated and have displayed potent anticonvulsant activities in seizure models. Both compounds have been reported to modulate voltage-gated sodium channel activity. We have prepared a series of chimeric compounds, (R)-7-(R)-10, by merging key structural units in these two clinical agents, and then compared their activities with (R)-2 and (S)-3. Compounds were assessed for their ability to alter sodium channel kinetics for inactivation, frequency (use)-dependence, and steady-state activation and fast inactivation. We report that chimeric compounds (R)-7-(R)-10 in catecholamine A-differentiated (CAD) cells and embryonic rat cortical neurons robustly enhanced sodium channel inactivation at concentrations far lower than those required for (R)-2 and (S)-3, and that (R)-9 and (R)-10, unlike (R)-2 and (S)-3, produce sodium channel frequency (use)-dependence at low micromolar concentrations. We further show that (R)-7-(R)-10 displayed excellent anticonvulsant activities and pain-attenuating properties in the animal formalin model. Of these compounds, only (R)-7 reversed mechanical hypersensitivity in the tibial-nerve injury model for neuropathic pain in rats.

Substituted N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamides: potent anticonvulsants that affect frequency (use) dependence and slow inactivation of sodium channels.

We prepared 13 derivatives of N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound's whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI=TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed.

A mentoring program to help junior faculty members achieve scholarship success.

The University of North Carolina Eshelman School of Pharmacy launched the Bill and Karen Campbell Faculty Mentoring Program (CMP) in 2006 to support scholarship-intensive junior faculty members. This report describes the origin, expectations, principles, and best practices that led to the introduction of the program, reviews the operational methods chosen for its implementation, provides information about its successes, and analyzes its strengths and limitations.

Benzyloxybenzylammonium chlorides: Simple amine salts that display anticonvulsant activity.

Several antiepileptic drugs exert their activities by inhibiting Na(+) currents. Recent studies demonstrated that compounds containing a biaryl-linked motif (Ar-X-Ar') modulate Na(+) currents. We, and others, have reported that compounds with an embedded benzyloxyphenyl unit (ArOCH2Ar', OCH2=X) exhibit potent anticonvulsant activities. Here, we show that benzyloxybenzylammonium chlorides ((+)H3NCH2C6H4OCH2Ar' Cl(-)) displayed notable activities in animal seizure models. Electrophysiological studies of 4-(2'-trifluoromethoxybenzyloxy)benzylammonium chloride (9) using embryonic cortical neurons demonstrated that 9 promoted both fast and slow inactivation of Na(+) channels. These findings suggest that the potent anticonvulsant activities of the earlier compounds were due, in part, to the benzyloxyphenyl motif and provide support for the use of the biaryl-linked pharmacophore in future drug design efforts.

(Biphenyl-4-yl)methylammonium chlorides: potent anticonvulsants that modulate Na+ currents.

We have reported that compounds containing a biaryl linked unit (Ar-X-Ar') modulated Na(+) currents by promoting slow inactivation and fast inactivation processes and by inducing frequency (use)-dependent inhibition of Na(+) currents. These electrophysiological properties have been associated with the mode of action of several antiepileptic drugs. In this study, we demonstrate that the readily accessible (biphenyl-4-yl)methylammonium chlorides (compound class B) exhibited a broad range of anticonvulsant activities in animal models, and in the maximal electroshock seizure test the activity of (3'-trifluoromethoxybiphenyl-4-yl)methylammonium chloride (8) exceeded that of phenobarbital and phenytoin upon oral administration to rats. Electrophysiological studies of 8 using mouse catecholamine A-differentiated cells and rat embryonic cortical neurons confirmed that 8 promoted slow and fast inactivation in both cell types but did not affect the frequency (use)-dependent block of Na(+) currents.

Discovery of lacosamide affinity bait agents that exhibit potent voltage-gated sodium channel blocking properties.

Lacosamide ((R)-1) is a recently marketed, first-in-class, antiepileptic drug. Patch-clamp electrophysiology studies are consistent with the notion that (R)-1 modulates voltage-gated Na(+) channel function by increasing and stabilizing the slow inactivation state without affecting fast inactivation. The molecular pathway(s) that regulate slow inactivation are poorly understood. Affinity baits are chemical reactive units, which when appended to a ligand (drug) can lead to irreversible, covalent modification of the receptor thus permitting drug binding site identification including, possibly, the site of ligand function. We describe, herein, the synthesis of four (R)-1 affinity baits, (R)-N-(4″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-8), (S)-N-(4″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((S)-8), (R)-N-(3″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-9), and (R)-N-(3″-acrylamidobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-10). The affinity bait compounds were designed to interact with the receptor(s) responsible for (R)-1-mediated slow inactivation. We show that (R)-8 and (R)-9 are potent inhibitors of Na(+) channel function and function by a pathway similar to that observed for (R)-1. We further demonstrate that (R)-8 function is stereospecific. The calculated IC50 values determined for Na(+) channel slow inactivation for (R)-1, (R)-8, and (R)-9 were 85.1, 0.1, and 0.2 µM, respectively. Incubating (R)-9 with the neuronal-like CAD cells led to appreciable levels of Na(+) channel slow inactivation after cellular wash, and the level of slow inactivation only modestly decreased with further incubation and washing. Collectively, these findings have identified a promising structural template to investigate the voltage-gated Na(+) channel slow inactivation process.

Identification of the benzyloxyphenyl pharmacophore: a structural unit that promotes sodium channel slow inactivation.

Four compounds that contained the N-benzyl 2-amino-3-methoxypropionamide unit were evaluated for their ability to modulate Na(+) currents in catecholamine A differentiated CAD neuronal cells. The compounds differed by the absence or presence of either a terminal N-acetyl group or a (3-fluoro)benzyloxy moiety positioned at the 4'-benzylamide site. Analysis of whole-cell patch-clamp electrophysiology data showed that the incorporation of the (3-fluoro)benzyloxy unit, to give the (3-fluoro)benzyloxyphenyl pharmacophore, dramatically enhanced the magnitude of Na(+) channel slow inactivation. In addition, N-acetylation markedly increased the stereoselectivity for Na(+) channel slow inactivation. Furthermore, we observed that Na(+) channel frequency (use)-dependent block was maintained upon inclusion of this pharmacophore. Confirmation of the importance of the (3-fluoro)benzyloxyphenyl pharmacophore was shown by examining compounds where the N-benzyl 2-amino-3-methoxypropionamide unit was replaced by a N-benzyl 2-amino-3-methylpropionamide moiety, as well as examining a series of compounds that did not contain an amino acid group but retained the pharmacophore unit. Collectively, the data indicated that the (3-fluoro)benzyloxyphenyl unit is a novel pharmacophore for the modulation of Na(+) currents.

Synthesis, anticonvulsant activity, and neuropathic pain-attenuating activity of N-benzyl 2-amino-2-(hetero)aromatic acetamides.

N-Benzyl 2-acetamido-2-substituted acetamides, where the 2-substituent is a (hetero)aromatic moiety, are potent anticonvulsants. We report the synthesis and whole animal pharmacological evaluation of 16 analogues where the terminal 2-acetyl group was removed to give the corresponding primary amino acid derivatives (PAADs). Conversion to the PAAD structure led to a substantial drop in seizure protection in animal tests, demonstrating the importance of the N-acetyl moiety for anticonvulsant activity. However, several of the PAADs displayed notable pain-attenuating activities in a mouse model.

Primary amino acid derivatives: substitution of the 4'-N'-benzylamide site in (R)-N'-benzyl 2-amino-3-methylbutanamide, (R)-N'-benzyl 2-amino-3,3-dimethylbutanamide, and (R)-N'-benzyl 2-amino-3-methoxypropionamide provides potent anticonvulsants with pain-attenuating properties.

Recently, we reported that select N'-benzyl 2-substituted 2-amino acetamides (primary amino acid derivatives (PAADs)) exhibited pronounced activities in established whole animal anticonvulsant (i.e., maximal electroshock seizure (MES)) and neuropathic pain (i.e., formalin) models. The anticonvulsant activities of C(2)-hydrocarbon N'-benzyl 2-amino acetamides (MES ED(50) = 13-21 mg/kg) exceeded those of phenobarbital (ED(50) = 22 mg/kg). Two additional studies defining the structure-activity relationship of PAADs are presented in this issue of the journal. In this study, we demonstrated that the anticonvulsant activities of (R)-N'-benzyl 2-amino-3-methylbutanamide and (R)-N'-benzyl 2-amino-3,3-dimethylbutanamide were sensitive to substituents at the 4'-N'-benzylamide site; electron-withdrawing groups retained activity, electron-donating groups led to a loss of activity, and incorporating either a 3-fluorobenzyloxy or 3-fluorophenoxymethyl group using a rationally designed multiple ligand approach improved activity. Additionally, we showed that substituents at the 4'-N'-benzylamide site of (R)-N'-benzyl 2-amino-3-methoxypropionamide also improved anticonvulsant activity, with the 3-fluorophenoxymethyl group providing the largest (∼4-fold) increase in activity (ED(50) = 8.9 mg/kg), a value that surpassed phenytoin (ED(50) = 9.5 mg/kg). Collectively, the pharmacological findings provided new information that C(2)-hydrocarbon PAADs represent a novel class of anticonvulsants.

Defining the structural parameters that confer anticonvulsant activity by the site-by-site modification of (R)-N'-benzyl 2-amino-3-methylbutanamide.

Primary amino acid derivatives (PAADs) (N'-benzyl 2-substituted 2-amino acetamides) are structurally related to functionalized amino acids (FAAs) (N'-benzyl 2-substituted 2-acetamido acetamides) but differ by the absence of the terminal N-acetyl group. Both classes exhibit potent anticonvulsant activities in the maximal electroshock seizure animal model, and the reported structure-activity relationships (SARs) of PAADs and FAAs differ in significant ways. Recently, we documented that PAAD efficacy was associated with a hydrocarbon moiety at the C(2)-carbon, while in the FAAs, a substituted heteroatom one atom removed from the C(2)-center was optimal. Previously in this issue, we showed that PAAD activity was dependent upon the electronic properties of the 4'-N'-benzylamide substituent, while FAA activity was insensitive to electronic changes at this site. In this study, we prepared analogues of (R)-N'-benzyl 2-amino-3-methylbutanamide to identify the structural components for maximal anticonvulsant activity. We demonstrated that the SAR of PAADs and FAAs diverged at the terminal amide site and that PAADs had considerably more structural latitude in the types of units that could be incorporated at this position, suggesting that these compounds function according to different mechanism(s).

Merging Structural Motifs of Functionalized Amino Acids and α-Aminoamides Results in Novel Anticonvulsant Compounds with Significant Effects on Slow and Fast Inactivation of Voltage-gated Sodium Channels and in the Treatment of Neuropathic Pain.

We recently reported that merging key structural pharmacophores of the anticonvulsant drugs lacosamide (a functionalized amino acid) with safinamide (an α-aminoamide) resulted in novel compounds with anticonvulsant activities superior to that of either drug alone. Here, we examined the effects of six such chimeric compounds on Na(+)-channel function in central nervous system catecholaminergic (CAD) cells. Using whole-cell patch clamp electrophysiology, we demonstrated that these compounds affected Na(+) channel fast and slow inactivation processes. Detailed electrophysiological characterization of two of these chimeric compounds that contained either an oxymethylene ((R)-7) or a chemical bond ((R)-11) between the two aromatic rings showed comparable effects on slow inactivation, use-dependence of block, development of slow inactivation, and recovery of Na(+) channels from inactivation. Both compounds were equally effective at inducing slow inactivation; (R)-7 shifted the fast inactivation curve in the hyperpolarizing direction greater than (R)-11, suggesting that in the presence of (R)-7, a larger fraction of the channels are in an inactivated state. None of the chimeric compounds affected veratridine- or KCl-induced glutamate release in neonatal cortical neurons. There was modest inhibition of KCl-induced calcium influx in cortical neurons. Finally, a single intraperitoneal administration of (R)-7, but not (R)-11, completely reversed mechanical hypersensitivity in a tibial-nerve injury model of neuropathic pain. The strong effects of (R)-7 on slow and fast inactivation of Na(+) channels may contribute to its efficacy and provide a promising novel therapy for neuropathic pain, in addition to its antiepileptic potential.

Identification of a lacosamide binding protein using an affinity bait and chemical reporter strategy: 14-3-3 ζ.

We have advanced a useful strategy to elucidate binding partners of ligands (drugs) with modest binding affinity. Key to this strategy is attaching to the ligand an affinity bait (AB) and a chemical reporter (CR) group, where the AB irreversibly attaches the ligand to the receptor upon binding and the CR group is employed for receptor detection and isolation. We have tested this AB&CR strategy using lacosamide ((R)-1), a low-molecular-weight antiepileptic drug. We demonstrate that using a (R)-lacosamide AB&CR agent ((R)-2) 14-3-3 ζ in rodent brain soluble lysates is preferentially adducted, adduction is stereospecific with respect to the AB&CR agent, and adduction depends upon the presence of endogenous levels of the small molecule metabolite xanthine. Substitution of lacosamide AB agent ((R)-5) for (R)-2 led to the identification of the 14-3-3 ζ adduction site (K120) by mass spectrometry. Competition experiments using increasing amounts of (R)-1 in the presence of (R)-2 demonstrated that (R)-1 binds at or near the (R)-2 modification site on 14-3-3 ζ. Structure-activity studies of xanthine derivatives provided information concerning the likely binding interaction between this metabolite and recombinant 14-3-3 ζ. Documentation of the 14-3-3 ζ-xanthine interaction was obtained with isothermal calorimetry using xanthine and the xanthine analogue 1,7-dimethylxanthine.

Primary amino acid derivatives: compounds with anticonvulsant and neuropathic pain protection activities.

Pharmacological management remains the primary method to treat epilepsy and neuropathic pain. We have advanced a novel class of anticonvulsants termed functionalized amino acids (FAAs). In this study, we examine FAA derivatives from which the terminal acetyl moiety was removed and termed these compounds primary amino acid derivatives (PAADs). Twenty-seven PAADs were prepared; the central C(2) R-substituent was varied, including C(2) stereochemistry, and the compounds were tested in rodent models of seizures and neuropathic pain. C(2)-Hydrocarbon N-benzylamide PAADs were potent anticonvulsants and excellent anticonvulsant activity (mice, ip; rat, po) was observed for C(2) R-substituted PAADs in which the R group was ethyl, isopropyl, or tert-butyl, and the C(2) stereochemistry conformed to the d-amino acid configuration ((R)-stereoisomer). These values surpassed the activities of several clinical antiepileptic drugs. The C(2) (R)-ethyl and C(2) (R)-isopropyl PAADs also displayed excellent activities in the mouse (ip) formalin neuropathic pain model. Significantly, unlike the FAA structure-activity relationship, PAAD anticonvulsant activity increased upon substitution of a methylene unit for a heteroatom in the R-substituent that was one atom removed from the C(2) site, suggesting that these PAADs function by a different pathway than FAAs.

Development and characterization of novel derivatives of the antiepileptic drug lacosamide that exhibit far greater enhancement in slow inactivation of voltage-gated sodium channels.

The novel antiepileptic drug, (R)-N-benzyl 2-acetamido-3-methoxypropionamide ((R)-lacosamide, Vimpat(®) ((R)-1)), was recently approved in the US and Europe for adjuvant treatment of partial-onset seizures in adults. (R)-1 preferentially enhances slow inactivation of voltage-gated Na(+) currents, a pharmacological process relevant in the hyperexcitable neuron. We have advanced a strategy to identify lacosamide binding partners by attaching affinity bait (AB) and chemical reporter (CR) groups to (R)-1 to aid receptor detection and isolation. We showed that select lacosamide AB and AB&CR derivatives exhibited excellent activities similar to (R)-1 in the maximal electroshock seizure model in rodents. Here, we examined the effect of these lacosamide AB and AB&CR derivatives and compared them with (R)-1 on Na(+) channel function in CNS catecholaminergic (CAD) cells. Using whole-cell patch clamp electrophysiology, we demonstrated that the test compounds do not affect the Na(+) channel fast inactivation process, that they were far better modulators of slow inactivation than (R)-1, and that modulation of the slow inactivation process was stereospecific. The lacosamide AB agents that contained either an electrophilic isothiocyanate ((R)-5) or a photolabile azide ((R)-8) unit upon AB activation gave modest levels of permanent Na(+) channel slow inactivation, providing initial evidence that these compounds may have covalently reacted with their cognate receptor(s). Our findings support the further use of these agents to delineate the (R)-1-mediated Na(+) channel slow inactivation process.

The structure-activity relationship of the 3-oxy site in the anticonvulsant (R)-N-benzyl 2-acetamido-3-methoxypropionamide.

Lacosamide ((R)-N-benzyl 2-acetamido-3-methoxypropionamide, (R)-1) is a low molecular weight anticonvulsant recently introduced in the United States and Europe for adjuvant treatment of partial-onset seizures in adults. In this study, we define the structure-activity relationship (SAR) for the compound's 3-oxy site. Placement of small nonpolar, nonbulky substituents at the 3-oxy site provided compounds with pronounced seizure protection in the maximal electroshock (MES) seizure test with activities similar to (R)-1. The anticonvulsant activity loss that accompanied introduction of larger moieties at the 3-oxy site in (R)-1 was offset, in part, by including unsaturated groups at this position. Our findings were similar to a recently reported SAR study of the 4'-benzylamide site in (R)-1 ( J. Med. Chem. 2010 , 53 , 1288 - 1305 ). Together, these results indicate that both the 3-oxy and 4'-benzylamide positions in (R)-1 can accommodate nonbulky, hydrophobic groups and still retain pronounced anticonvulsant activities in rodents in the MES seizure model.

Merging the structural motifs of functionalized amino acids and alpha-aminoamides: compounds with significant anticonvulsant activities.

Functional amino acids (FAAs) and alpha-aminoamides (AAAs) are two classes of antiepileptic drugs (AEDs) that exhibit pronounced anticonvulsant activities. We combined key structural pharmacophores present in FAAs and AAAs to generate a new series of compounds and document that select compounds exhibit activity superior to either the prototypical FAA (lacosamide) or the prototypical AAA (safinamide) in the maximal electroshock (MES) seizure model in rats. A representative compound, (R)-N-4'-((3''-fluoro)benzyloxy)benzyl 2-acetamido-3-methoxypropionamide ((R)-10), was tested in the MES (mice, ip), MES (rat, po), psychomotor 6 Hz (32 mA) (mice, ip), and hippocampal kindled (rat, ip) seizure tests providing excellent protection with ED(50) values of 13, 14, approximately 10 mg/kg, and 12 mg/kg, respectively. In the rat sciatic nerve ligation model (ip), (R)-10 (12 mg/kg) provided an 11.2-fold attenuation of mechanical allodynia. In the mouse biphasic formalin pain model (ip), (R)-10 (15 mg/kg) reduced pain responses in the acute and the chronic inflammatory phases.

Proteomic searches comparing two (R)-lacosamide affinity baits: An electrophilic arylisothiocyanate and a photoactivated arylazide group.

We have advanced a novel strategy to search for lacosamide ((R)-1) targets in the brain proteome where protein binding is expected to be modest. Our approach used lacosamide agents containing affinity bait (AB) and chemical reporter (CR) units. The affinity bait moiety is designed to irreversibly react with the target, and the CR group permits protein detection and capture. In this study, we report the preparation and evaluation of (R)-N-(4-azido)benzyl 2-acetamido-3-(prop-2-ynyloxy)propionamide ((R)-3) and show that this compound exhibits potent anticonvulsant activities in the MES seizure model in rodents. We compared the utility of (R)-3 with its isostere, (R)-N-(4-isothiocyanato)benzyl 2-acetamido-3-(prop-2-ynyloxy)propionamide ((R)-2), in proteomic studies designed to identify potential (R)-1 targets. We showed that despite the two-fold improved anticonvulsant activity of (R)-3 compared with (R)-2, (R)-2 was superior in revealing potential binding targets in the mouse brain soluble proteome. The difference in these agents utility has been attributed to the reactivity of the affinity baits (i.e., (R)-2: aryl isothiocyanate moiety; (R)-3: photoactivated aryl azide intermediates) in the irreversible protein modification step, and we conclude that this factor is a critical determinant of successful target detection where ligand (drug) binding is modest. The utility of (R)-2 and (R)-3 in in situ proteome studies is explored.