Potential of nafimidone derivatives against co-morbidities of epilepsy: In vitro, in vivo, and in silico investigations.

Nafimidone is known for its clinical antiepileptic effects and alcohol derivatives of nafimidone were reported be potent anticonvulsants. These compounds are structurally similar to miconazole, which is known to inhibit cholinesterases, protect neurons, and ameliorate cognitive decline. Herein, we aimed to reveal the potential of three nafimidone alcohol esters (5 g, 5i, and 5 k), which were previously reported for their anticonvulsant effects, against co-morbidities of epilepsy such as inflammatory and neuropathic pain, cognitive and behavioral deficits, and neuron death, and understand their roles in related pathways such as γ-butyric acid type A (GABAA ) receptor and cholinesterases using in vitro, in vivo and in silico methods. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for cytotoxicity evaluation, hippocampal slice culture assay for neuroprotection, formalin test for acute and inflammatory pain, sciatic ligation for neuropathic pain, Morris water maze and open field locomotor tasks for cognitive and behavioral deficits, radioligand binding for GABAA receptor affinity, spectrophotometric methods for cholinesterase inhibition in vitro, and molecular docking in silico. The compounds were non-toxic to fibroblast cells. 5 k was neuroprotective against kainic acid-induced neuron death. 5i reduced pain response of mice in both the acute and the inflammatory phases. 5i improved survival upon status epilepticus. The compounds showed no affinity to GABAA receptor but inhibited acetylcholinesterase, 5 k also inhibited butyrylcholinesterase. The compounds were predicted to interact mainly with the peripheric anionic site of cholinesterase enzymes. The title compounds showed neuroprotective, analgesic, and cholinesterase inhibitory effects, thus they bear promise against certain co-morbidities of epilepsy with neurological insults.

Synthesis, in vivo anticonvulsant testing, and molecular modeling studies of new nafimidone derivatives.

An estimated 50 million people suffer epilepsy worldwide and 30% of the cases do not respond to current antiepileptic drugs (AEDs). Here, we report synthesis and anticonvulsant screening of new derivatives of nafimidone, a well-known member of (arylakyl)azole anticonvulsants. The compounds showed promising protection against maximal electroshock (MES)-induced seizures in mice and rats when administered via intraperitoneal (ip) and oral route. Especially, 5b, 5c, and 5i displayed outstanding activity in rats in MES test when given ip (ED50 : 16.0, 15.8, and 11.8 mg/kg, respectively). Additionally, 5l was active against 6 Hz and corneal-kindled mice models. Behavioral toxicity of the compounds was very low and their therapeutic indexes were high compared to some currently available AEDs. A number of pharmaceutically relevant descriptors and properties were predicted for the compounds in silico in comparison with a set of known drugs. Favorable results were obtained such as good blood-brain barrier permeability and high oral absorption, as well as drug-likeness. 5l was found to show affinity to the benzodiazepine binding site of A-type GABA receptor via molecular docking simulations.

Synthesis, anticonvulsant and antimicrobial activities of some new 2-acetylnaphthalene derivatives.

In this study, as a continuation of our research for new (arylalkyl)imidazole anticonvulsant compounds, the design, synthesis and anticonvulsant/antimicrobial activity evaluation of a series of 2-acetylnaphthalene derivatives have been described. Molecular design of the compounds has been based on the modification of nafimidone [1-(2-naphthyl)-2-(imidazol-1-yl)ethanone], which is a representative of the (arylalkyl)imidazole anticonvulsant compounds as well as its active metabolite, nafimidone alcohol (3, 4). In general, these compounds were variously substituted at the alkyl chain between naphthalene and imidazole rings and subjected to some other modifications to evaluate additional structure-activity relationships. The anticonvulsant activity profile of those compounds was determined by maximal electroshock seizure (MES) and subcutaneous metrazol (scM) seizure tests, whereas their neurotoxicity was examined using rotarod test. All the ester derivatives of nafimidone alcohol (5a-h), which were designed as prodrugs, showed anticonvulsant activity against MES-induced seizure model. Four of the most active compounds were chosen for further anticonvulsant evaluations. Quantification of anticonvulsant protection was calculated via the ip route (ED(50) and TD(50)) for the most active candidate (5d). Observed protection in the MES model was 38.46mgkg(-1) and 123.83mgkg(-1) in mice and 20.44mgkg(-1), 56.36mgkg(-1) in rats, respectively. Most of the compounds with imidazole ring also showed antibacterial and/or antifungal activities to a certain extent in addition to their anticonvulsant activity.

Structure-activity relationships of (arylalkyl)imidazole anticonvulsants: comparison of the (fluorenylalkyl)imidazoles with nafimidone and denzimol.

A recently discovered and structurally distinct class of antiepileptic drugs is the (arylalkyl)imidazoles. Two independently discovered representatives of this class, denzimol (alpha-[4-(2-phenylethyl)phenyl]-1H-imidazole-1-ethanol) and nafimidone (2-(1H-imidazol-1-yl)-1-(2-naphthalenyl)ethanone), are undergoing clinical evaluation. Our structure-activity relationship (SAR) studies revealed that in addition to the naphthalenyl and phenethylphenyl aryl moieties of nafimidone and denzimol, respectively, fluorenyl, benzo[b]thienyl, and benzofuranyl aryl groups provided several highly active (arylalkyl)imidazole anticonvulsants. These structurally diverse aryl moieties, and comparable anticonvulsant activities, lend credence to the hypothesis that the pharmacophore of this class of anticonvulsants is the alkylimidazole portion of the molecule, with the lipophilic aryl portion enabling penetration of the blood-brain barrier. We focused our SAR studies on the (fluorenylalkyl)imidazole series. A representative compound from this series is 1-(9H-fluoren-2-yl)-2-(1H-imidazol-1-yl)ethanone. This agent was twice as potent as nafimidone in inhibiting maximal electroshock seizures in mice (po ED50's = 25 and 56 mg/kg, respectively) and considerably less toxic in the rat (po LD50's = 4550 and 504 mg/kg, respectively). The tertiary alcohol alpha-(9H-fluoren-2-yl)-alpha-methyl-1H-imidazole-1-ethanol was as potent as denzimol in mice (po ED50's = 10 and 12 mg/kg, respectively). This series of imidazole anticonvulsants was highly selective; while many compounds displayed potent antielectroshock activity, little or not activity was observed against pentylenetetrazole-induced clonic seizures or in the horizontal screen test for ataxia. All active compounds that we tested in this series, as well as denzimol and nafimidone, potentiated hexobarbital-induced sleeping time in mice, probably by imidazole-mediated inhibition of cytochrome P-450. The SAR's for the anticonvulsant activity and the sleeping time potentiation were similar. The propensity of these (arylalkyl)imidazole anticonvulsants to interact strongly with cytochrome P-450 and thereby impair the metabolism of other antiepileptic drugs may severely limit their clinical utility as anticonvulsants.

Stereochemical studies of adrenergic drugs. Optically active derivatives of imidazolines.

The synthesis of (R)-(+)-4-methyl-2-(1-naphthylmethyl)imidazoline hydrochloride (2) and (S)-(-)-4-methyl-2-(1-naphthylmethyl)imidazoline hydrochloride (3) is presented. The synthesis involves the preparation of (R)-(+)- and (S)-(-)-1,2-diaminopropane dihydrochloride and then allowing the appropriate diaminopropane to react with ethyl 1-naphthyliminoacetate hydrochloride in the presence of triethylamine. The parent compound, naphazoline, is a potent alpha-adrenoreceptor agonist (-log ED50 = 7.22), whereas the methylated derivatives, 2 and 3, were moderately potent antagonists (pA2 = 5.6 and 5.8, respectively) of the alpha-adrenoreceptor. Compounds 2 and 3 also produced blockade of the response to histamine on the rabbit aorta, but at concentrations approximately 20 times higher than necessary to produce equal blockade of the alpha-adrenoreceptor.

Synthesis and alpha-adrenergic activities of 2- and 4-substituted imidazoline and imidazole analogues.

Seven analogues of medetomidine and naphazoline were synthesized and evaluated for their alpha 1 (aorta) and alpha 2 (platelet) activities. The analogues were composed of 2- and 4-substituted imidazoles and imidazolines attached through a methylene bridge to either the 1- or 2-naphthalene ring system. In general the 1-naphthalene analogues were the most potent inhibitors of epinephrine-induced platelet aggregation. Of considerable interest was the fact that the 1-naphthalene analogues (2, 5-7) were partial agonists while the 2-naphthalene analogues (3, 8, 9) were antagonists in an alpha 1-adrenergic system (aorta). Thus, appropriately substituted naphthalene analogues of medetomidine and naphthazoline provide a spectrum of alpha 1-agonist, alpha 1-antagonist, and alpha 2-antagonist activity.

Pharmacokinetics of nafimidone in patients with chronic intractable epilepsy.

Nafimidone is a new antiepileptic drug which may be effective in partial onset seizures. We studied the pharmacokinetics of nafimidone and its metabolite, nafimidone alcohol, in 12 patients already taking phenytoin and/or carbamazepine. The half-life of nafimidone was 1.34 +/- 0.48 hours after a 100mg single dose and 1.69 +/- 0.91 hours after a 300mg single dose. However, the half-life of nafimidone alcohol increased from 2.84 +/- 0.72 hours after a 100mg single dose to 4.22 +/- 1.09 hours after a 300mg single dose (p less than 0.02). The clearance of nafimidone was 43.56 +/- 22.11 L/h/kg after a 100mg single dose and 35.51 +/- 28.93 L/h/kg after the 300mg single dose. The respective apparent volumes of distribution of nafimidone after single 100 and 300mg doses were 80.78 +/- 46.11 L/kg and 71.01 +/- 36.86 L/kg. After short term (9 to 10 weeks) and long term (127 to 152 weeks) maintenance therapy on nafimidone 600mg per day the half-life of nafimidone alcohol was 2.23 +/- 0.36 hours and 2.16 +/- 0.60 hours, respectively. No nafimidone could be detected in urine but from 4 to 7% of the daily nafimidone dose was recovered as nafimidone alcohol. Thus, it appears that over 90% of the administered dose of nafimidone is metabolised by pathways other than glucuronidation of nafimidone alcohol and urinary excretion.

The anticonvulsant action of nafimidone on kindled amygdaloid seizures in rats.

The anticonvulsant effectiveness of nafimidone (1-[2-naphthoylmethyl]imidazole hydrochloride) was evaluated in the kindled amygdaloid seizure model in rats. Nafimidone (3.1-120 mg/kg i.p.) was evaluated at 30 min in previously kindled rats using both threshold (20 microA increments) and supranthreshold (400 microA) paradigms. Nafimidone (25-50 mg/kg) significantly reduced supranthreshold elicited afterdischarge length and seizure severity only at doses with some prestimulation toxicity. The maximum anticonvulsant effectiveness for the 25 mg/kg i.p. dose of nafimidone was seen between 15 and 30 min utilizing a suprathreshold kindling paradigm. Nafimidone did not significantly elevate seizure thresholds at the doses tested; however, nafimidone (3.1-50 mg/kg) reduced the severity and afterdischarge duration of threshold elicited seizures in a non-dose response manner. Drug-induced electroencephalographic spikes were seen in both cortex and amygdala in most kindled rats receiving 100-120 mg/kg i.p. within 30 min of dosing before electrical stimulation. The frequency of spike and wave complexes increased in most of these animals leading to drug-induced spontaneous seizures and death in approximately 25% before electrical stimulation. This study has demonstrated that although nafimidone can modify both threshold and suprathreshold elicited kindled amygdaloid seizures, it lacks significant specificity in this model of epilepsy.

Synthesis of some 1-(2-naphthyl)-2-(imidazole-1-yl)ethanone oxime and oxime ether derivatives and their anticonvulsant and antimicrobial activities.

In this study, oxime and oxime ether derivatives of anticonvulsant nafimidone [1-(2-naphthyl)-2-(imidozole-1-yl)ethanone] were prepared as potential anticonvulsant compounds. Nafimidone oxime was synthesized by the reaction of nafimidone and hydroxylamine hydrochloride. O-Alkylation of the oxime by various alkyl halides gave the oxime ether derivatives. Anticonvulsant activity of the compounds was determined by maximal electroshock (MES) and subcutaneous metrazole (scMet) tests in mice and rats according to procedures of the Antiepileptic Drug Development (ADD) program of the National Institutes of Health (NIH). In addition to anticonvulsant evaluation, compounds were also screened for possible antibacterial and antifungal activities because of the structural resemblance to the azole antifungals, especially to oxiconazole. All compounds were evaluated against three human pathogenic fungi and four bacteria using the microdilution method. Most of the compounds exhibited both anticonvulsant and antimicrobial activities; the O-alkyl substituted compounds (2, 3, 4 and 5) were found to be more active than the O-arylalkyl substituted compounds in both screening paradigms.

Interaction of the anticonvulsants, denzimol and nafimidone, with liver cytochrome P450 in the rat.

The presence of an imidazole moiety in the chemical structure of denzimol and nafimidone suggested that these new anticonvulsants might interfere with cytochrome P450-mediated mixed function monooxygenase activities. We therefore investigated their ability to bind reversibly to rat liver cytochrome P450. Both drugs displayed a type II spectra. The Ks values of binding were 6.66 and 7.00 mM, respectively, for denzimol and nafimidone. In other in-vitro studies the IC50 of the inhibition caused by denzimol and nafimidone was determined on carbamazepine (CBZ) epoxidation and diazepam C3-hydroxylation and N1-dealkylation. The IC50 values for CBZ epoxidation were 4.46 x 10(-7) and 2.95 x 10(-7) M, respectively, in the presence of denzimol and nafimidone. The IC50 values for diazepam C3-hydroxylation were 1.44 x 10(-6) and 1.00 x 10(-6) M, respectively, and those for N1-dealkylation 6.66 x 10(-7) and 5.95 x 10(-7) M. The inhibition of CBZ metabolism was also investigated ex-vivo and in-vivo after single oral doses (15 and/or 60 mg kg-1) of denzimol or nafimidone. Inhibition of CBZ-10,11-epoxidation by the two drugs was time- and dose-dependent. Further studies in-vivo showed that denzimol and nafimidone prolong pentobarbitone sleeping times indicating that both drugs bind to rat liver microsomes and are potent inhibitors in the rat of mixed function monooxygense activities both in-vitro and in-vivo.

Synthesis of some novel 1-(2-naphthyl)-2-(imidazol-1-yl)ethanone oxime ester derivatives and evaluation of their anticonvulsant activity.

Twenty-three new oxime ester derivatives of nafimidone were synthesized with the prospect of potential anticonvulsant activities. MES and ScM tests were employed for their anticonvulsant activities and rotorod test for neurological deficits. Eighteen compounds were found to be protective against MES seizures. Alkyl (1-8) and arylalkyl (9, 10) oxime ester derivatives were found to be more active than aryl oxime ester derivatives (11-23). Five compounds (2, 3, 7, 9, 10), which were protective at 0.5 h at the doses of 30 mg/kg and higher in MES test, showed the highest activity. Compound 17 was the most active one in ScM test at all dose levels at 4 h.

Development of a stability-indicating assay for nafimidone [1-(2-naphthoylmethyl)imidazole hydrochloride] by high-performance liquid chromatography.

The rational development of a stability-indicating assay for nafimidone [1-(2-naphthoylmethyl)imidazole hydrochloride] is demonstrated. It allows resolution of the decomposition products, obtained both on melting and resulting from decomposition in solution. Ion-pair chromatography was used to elute the solution decomposition products before the parent compound, thus producing maximum sensitivity of detection of these in the presence of undecomposed reactant. Stability data in terms of first-order rate constants were obtained in two different buffer solutions both from conventional integrated-rate equations using reactant concentration measurements, and by the initial rate method using decomposition product concentrations. In the light of this investigation, additional criteria for stability-indicating assays are suggested, which exploit the sensitivity as well as specificity of high-performance liquid chromatography.

Inhibition of microsomal phenytoin metabolism by nafimidone and related imidazoles. Potency and structural considerations.

Nafimidone and other 1-imidazoles were shown to be potent inhibitors of phenytoin p-hydroxylation in rat hepatic microsomes, being very effective even at submicromolar concentrations. The inhibitory potency of these 1-imidazoles was similar to that of SKF 525-A and considerably greater than that observed for other imidazoles (4,5-diphenylimidazole, cimetidine, metronidazole), metyrapone, or other anticonvulsants. The effects of structural modification on the inhibitory activity were examined. Except at the 2-position on the imidazole, alkyl substitution increased the inhibitory potency, probably because of increased lipophilicity. Substitution at the 2-position caused marked diminution in inhibitory activity, possibly due to steric hindrance. The hydroxy analogs of nafimidone exhibited greater inhibitory activity than the corresponding keto compounds. Furthermore, pretreatment of the rats with nafimidone resulted in greater Vmax values for both low affinity and high affinity metabolic sites.

Efficacy of nafimidone in the treatment of intractable partial seizures: report of a two-center pilot study.

Nafimidone is a potential new antiepileptic drug with a therapeutic profile in experimental animal seizure models similar to that of phenytoin (PHT). We report here the first clinical trial of nafimidone in epileptic patients. Twelve adult male patients with a mean of four or more medically intractable seizures per month were enrolled in a 14-week pilot study. Patients were stabilized on therapeutic levels of PHT and carbamazepine (CBZ) (nine patients) or on PHT alone (three patients) before entering a 4-week baseline period. Nafimidone, to a maximum dose of 600 mg/day, was added during 2 weeks in hospital. Patients were then evaluated weekly for 8 weeks. Eight patients experienced 33-98% improvement in seizure control. Three others did not show significant change in seizure frequency but experienced sufficient subjective improvement that they continued into long-term follow-up. One patient, who had a 63% improvement in mean weekly seizures during the pilot study, declined to continue. Thus, 10 patients entered long-term follow-up. Six of the 10 sustained 53 to greater than 99% improvement in seizure control compared with baseline over the course of 46-53 weeks of follow-up. Nafimidone had a marked inhibitory effect on the clearance of CBZ and PHT, resulting in higher plasma levels in nine patients. The possible role of the elevated CBZ levels in the apparent efficacy of nafimidone is discussed.

Disposition of nafimidone in rats.

The absorption, distribution, excretion, and metabolism of 14C-nafimidone, a novel anticonvulsant, have been studied in rats. Nafimidone was completely absorbed following single oral doses of 10 and 100 mg/kg. After both iv and oral administration, nafimidone was rapidly eliminated from plasma (t 1/2 about 5 min), with concomitant formation of a pharmacologically active, nonconjugated metabolite, nafimidone alcohol. Systemic clearance of nafimidone from plasma after iv administration was approximately 2 times higher than hepatic blood flow in rats, and the oral bioavailability was 15%. However, the AUC of nafimidone alcohol was 30% higher after oral administration of nafimidone than that after iv administration of nafimidone. It is likely that, given its pharmacological activity, nafimidone alcohol is the more important species pharmacologically. Distribution of nafimidone-related radioactivity was widespread with highest concentrations associated with liver, kidney, adrenals, and the gastrointestinal tract. Elimination of radioactivity from tissues was rapid and complete, except that retention was noted in arterial vessels and in the ocular melanin of pigmented rats. Determination of hepatic and brain levels of nafimidone and nafimidone alcohol showed no detectable levels of nafimidone in either tissue. However, levels of nafimidone alcohol in liver and brain were as much as 13-fold and 2-fold, respectively, higher than levels in plasma. After either iv or oral administration of 14C-nafimidone, approximately two-thirds of the radioactivity was recovered in urine. The major urinary metabolites of nafimidone after a 100 mg/kg dose were characterized and shown to be dihydroxydihydronaphthalene, substituted nafimidone alcohol, and the 1-beta-glucuronide of nafimidone alcohol.

High-performance liquid chromatographic determination of nafimidone and its major metabolite nafimidone alcohol in human plasma and urine.

A rapid, sensitive and selective method for the determination in plasma and urine of nafimidone, a new antiepileptic drug, and its major metabolite, nafimidone alcohol, has been developed which uses a high-performance liquid chromatographic system and a fluorescence detector for nafimidone or ultraviolet detector for nafimidone alcohol. The detection limits for nafimidone and nafimidone alcohol are 5.0 and 12.5 ng/ml, respectively.

The metabolism of nafimidone hydrochloride in the dog, primates and man.

1. The biotransformation of nafimidone, an imidazole-substituted anticonvulsant, has been studied by characterization of urinary metabolites in dogs, cynomolgus monkeys, baboons and man. 2. The biotransformation of nafimidone in these laboratory animals and man is initially very similar, in each case proceeding by reduction to the aliphatic alcohol metabolite, nafimidone alcohol or 1-[2-hydroxy-2-(2-naphthyl)ethyl]imidazole. 3. Further transformation of this metabolite involves oxidation in the naphthyl and imidazole functions, and/or conjugation. 4. The dog differs from the higher primates in that no metabolic modification of the naphthyl group takes place, the major metabolite in the dog being the O-beta-glucuronide of nafimidone alcohol. 5. In higher primates and man two isomers involving dihydroxylation in the naphthyl ring--1-[2-hydroxy-2-(5,6- or 7,8-dihydroxydihydro-2-naphthyl)ethyl]imidazole--were tentatively identified. These species alone showed evidence of an imidazole linked N-glucuronide of nafimidone alcohol. 6. The possible occurrence of stereoselective metabolism by the introduction of a chiral centre at C-9 in nafimidone alcohol was indicated in human urine by the presence of both epimers of the O-beta-glucuronide of nafimidone alcohol in a 2:1 ratio.

Bile acid synthesis: 7 alpha-hydroxylation of intermediates in the sterol 27-hydroxylase metabolic pathway.

The recognition that the 7 alpha-hydroxylation of 27-hydroxycholesterol is catalyzed by an enzyme that is different from cholesterol 7 alpha-hydroxylase raises the question as to the number of similar enzymes that may be present in liver and subserve bile acid synthesis. Thus, both 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid, further oxidation products derived from 27-hydroxycholesterol, are also 7 alpha-hydroxylated during their metabolism to chenodeoxycholic acid. Using a microsomal fraction of hamster liver and competition plot analysis, we found that the 7 alpha-hydroxylase activity for the acid substrates was approximately one-tenth that found for 27-hydroxycholesterol. Mixtures of the different substrates did not depress the total rate of 7 alpha-hydroxylation. The evidence supports the view that these substrates share the same catalytic site on a single enzyme.

Nafimidone, an imidazole anticonvulsant, and its metabolite as potent inhibitors of microsomal metabolism of phenytoin and carbamazepine.

The effects of nafimidone and its metabolite (reduced nafimidone) on p-hydroxylation of phenytoin were examined by using hepatic microsomes from rats pretreated with phenytoin. Both nafimidone and reduced nafimidone inhibited p-hydroxylation of phenytoin in a concentration-dependent manner and at both high and low affinity metabolic sites. Both compounds were effective inhibitors at submicromolar concentrations. The inhibitory pattern at both metabolic sites was consistent with "mixed type" inhibition. The inhibition constants, Ki, calculated for reduced nafimidone, were about 0.2 microM at both sites. Submicromolar concentrations of nafimidone and its metabolite also inhibited carbamazepine epoxidation. Thus, nafimidone and its metabolite were shown to be potent inhibitors of major biotransformation pathways of two important antiepileptic drugs.