Species-dependent binding of tocainide analogues to albumin: affinity chromatography and circular dichroism study.

A series of novel tocainide analogues were characterized for their HSA and RSA binding, by using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). In this HPLAC study, HSA and RSA were covalently immobilized to the silica matrix of HPLC columns, with a procedure that maintained unaltered the binding properties of the proteins. The tocainide analogues were ranked for their affinity to HSA and RSA on the basis of their bound fractions measured by the two albumin-based columns. This technique was also applied to characterize the high affinity binding sites of these tocainide analogues to the protein. For this purpose displacement experiments were carried out by means of increasing concentrations in the mobile phase of competitors known to bind selectively to the main binding sites of HSA. The results obtained with the immobilized proteins were confirmed by investigating the same drug-protein systems in solution by circular dichroism. The comparison of the data collected with both methodologies highlighted the dramatic effect of small differences in the amino acidic sequences of the two proteins. In fact, despite their similar primary and secondary structures, a small difference in the amino acidic sequence leads to significant differences in their three-dimensional structure reflecting their different binding capacity and their stereoselectivity. Therefore, this study confirms how it is crucial to consider the significant differences among the animal models when performing pharmacokinetic studies. It is also clear that the knowledge of serum carrier binding parameters at an early stage of drug discovery represents a great advantage that may help to save time and efforts.

Searching for novel anti-myotonic agents: pharmacophore requirement for use-dependent block of skeletal muscle sodium channels by N-benzylated cyclic derivatives of tocainide.

Drug screening on sodium currents of native myofibers by means of voltage-clamp recordings is predictive of pre-clinical anti-myotonic activity in vivo and ex vivo. By this approach we identified the N-benzylated beta-proline derivative of tocainide (To10) as the most potent use-dependent blocker of Nav1.4 so far. We tested novel analogs with modifications on the pharmacophore groups of To10. The substitution of the proline cycle with less planar piperidine or piperazine rings disclosed the importance of a two carbon atom distance and/or an additional nitrogen atom for potency. Structural changes on the xylididic group corroborated the role of a proper electronic cloud for hydrophobic interactions with the binding site. The N-benzylated moiety lead to a stereoselective behavior only in the rigid alpha-proline analog To11 vs. To10 and N-benzylated tocainide (To12). The results confirm the strict structural requirements of Nav1.4 blockers and allow to refine the drug design toward novel anti-myotonic drugs.

2D- and 3D-QSAR of tocainide and mexiletine analogues acting as Na(v)1.4 channel blockers.

Enantiomeric forms of Tocainide, Mexiletine, and structurally related local anaesthetic compounds, were analyzed with respect to their potency in blocking Na(v)1.4 channel. Structure-activity relationships based on in vitro pharmacological assays, suggested that an increase in terms of lipophilicity and/or molecular surface as well as the presence of specific polar spacers might be determinant for receptor interactions. QSAR and pharmacophore models were then used to support at 3D level this hypothesis.

Constrained analogues of tocainide as potent skeletal muscle sodium channel blockers towards the development of antimyotonic agents.

1-Benzyl-N-(2,6-dimethylphenyl)piperidine-3-carboxamide and 4-benzyl-N-(2,6-dimethylphenyl)piperazine-2-carboxamide, two conformationally restricted analogues of tocainide, were designed and synthesized as voltage-gated skeletal muscle sodium channel blockers. They showed, with respect to tocainide, a marked increase in both potency and use-dependent block.

Synthesis, structure and pharmacology of acyl-2,6-xylidines.

L-2-perhydroheterocyclicalkyl acids were condensed with 2,6-xylidine. 8 new optically active acyl-2,6-xylidines were obtained. Absolute configuration of acyl-2,6-xylidines were selected for pharmacological examinations.

Enantiomeric separation of tocainide and its analogues on an optically active crown ether-based stationary phase by liquid chromatography.

Tocainide and its 14 analogues were resolved on a chiral stationary phase (CSP) based on (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 covalently bonded to silica gel. The resolution was quite good, the separation (alpha) and resolution factors (Rs) being 1.84-15.32 and 1.34-13.78, respectively. Especially, the result for the resolution of tocainide on the CSP turns out to be the best one among others reported so far. The chromatographic resolution behaviors were demonstrated to be dependent on the content and the type of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase.

Effect of acidity on the enantiomeric resolution of thyroxine and tocainide by HPLC on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid column.

Enantiomeric resolution of thyroxine and tocainide was achieved on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid column. The mobile phases were methanol/water (4:1, v/v) and methanol/water containing 5 mM sulfuric acid (4:1, v/v) for tocainide and thyroxine respectively. The flow rate was 0.5 ml/min. The effect of the acidity on the chiral resolution of these drugs was studied. Detection was at 220 nm for both drugs. The values of alpha and Rs were 2.08-3.11 and 1.00-2.60, respectively, for thyroxine while the values of alpha and Rs were 1.13-1.26 and 0.10-1.30, respectively, for tocainide.

Increased rigidity of the chiral centre of tocainide favours stereoselectivity and use-dependent block of skeletal muscle Na(+) channels enhancing the antimyotonic activity in vivo.

1. Searching for the structural requirements improving the potency and the stereoselectivity of Na(+) channel blockers as antimyotonic agents, new derivatives of tocainide, in which the chiral carbon atom is constrained in a rigid alpha-proline or pyrrolo-imidazolic cycle, were synthesized as pure enantiomers. 2. Their ability to block Na(+) currents, elicited from -100 to -20 mV at 0.3 Hz (tonic block) and 2-10 Hz (use-dependent block) frequencies, was investigated in vitro on single fibres of frog semitendinosus muscle using the vaseline-gap voltage-clamp method. 3. The alpha-proline derivative, To5, was 5 and 21 fold more potent than tocainide in producing tonic and 10 Hz-use-dependent block, respectively. Compared to To5, the presence of one methyl group on the aminic (To6) or amidic (To7) nitrogen atom decreased use-dependence by 2- and 6-times, respectively. When methylene moieties were present on both nitrogen atoms (To8), both tonic and use-dependent block were reduced. 4. Contrarily to tocainide, all proline derivatives were stereoselective in relation to an increased rigidity. A further increase in the molecular rigidity as in pyrrolo-imidazolic derivatives markedly decreased the drug potency with respect to tocainide. 5. Antimyotonic activity, evaluated as the shortening of the time of righting reflexes of myotonic adr/adr mice upon acute drug in vivo administration was 3 fold more effective for R-To5 than for R-Tocainide. 6. Thus, constraining the chiral centre of tocainide in alpha-proline cycle leads to more potent and stereoselective use-dependent Na(+) channel blockers with improved therapeutic potential.

Synthesis of new 2,6-prolylxylidide analogues of tocainide as stereoselective blockers of voltage-gated Na(+) channels with increased potency and improved use-dependent activity.

A series of tocainide chiral analogues were designed, synthesized, and evaluated in vitro, in pure enantiomeric form, as use-dependent blockers of skeletal muscle sodium channels to better understand the structural requirements responsible for the antimyotonic activity. The voltage clamp recordings showed a remarkable increase of both potency and use-dependent behavior with the analogue N-(2, 6-dimethylphenyl)-2-pyrrolidinecarboxamide (1a). In fact (R)-1a was 5-fold more potent than (R)-tocainide in producing the tonic block, i.e., the reduction of peak sodium current in resting conditions after application of the compound, but it was 21-fold more potent in condition of high frequency of stimulation (phasic block). Furthermore, as opposite to tocainide, this compound was also stereoselective, (S)-1a being 2-3-fold less potent than (R)-1a. The introduction in 1a of a methyl group in place of the hydrogen bonded to either the aminic nitrogen atom [N-(2, 6-dimethylphenyl)-1-methyl-2-pyrrolidinecarboxamide (2a)] or the amidic nitrogen atom [N-(2, 6-dimethylphenyl)-N-methyl-2-pyrrolidinecarboxamide (3a)] led unexpectedly to an inversion of stereoselectivity, the (S)-enantiomers being 3-fold more potent than the (R)-ones. The comparison between eutomers showed that (S)-2a and (S)-3a are almost equieffective to (R)-1a in producing a tonic block, the half-maximal concentrations being about 100 microM; however, the use-dependent behavior was remarkably decreased by the presence of the methyl group: i.e., the gain of potency observed at high frequency of stimulation amounted to 3 and 1.6 times for 2a and 3a, respectively. The replacement of both hydrogens bonded to the aminic and amidic nitrogen atoms resulted in N-(2,6-dimethylphenyl)-N, 1-dimethyl-2-pyrrolidinecarboxamide (4a) in which the (S)-isomer was still twice as potent as the (R)-one, but the absolute potency and mostly the use-dependent behavior were strongly reduced, showing therefore no clear advantages with respect to tocainide. The use-dependent behavior, which plays a pivotal role for antimyotonic activity, is strongly reduced by the presence of methyl groups on the nitrogen atoms, likely for modification of pK(a) and/or for constraint of molecular conformation.

Photochemical and chemical oxidation of mexiletine and tocainide. Structure elucidation of the major products.

Mexiletine (mex) and tocainide (toc) are antiarrhythmic drugs of closely related structure. Several degradation products are formed by interaction with both light and oxidizing agents in the case of mex, and only by oxidants with toc. On the basis of the identified structures, the decomposition reactions can be classified into two types (Scheme 1). Type I is an oxidative cyclization reaction producing the oxazepine derivative 1 from mex and the diazepine derivative 3 from toc. In reaction type II side chain oxidized products (2 or 4 and 5) are formed. While tocainide.HCl has proved to be photostable, in solid mexiletine HCl the oxazepine derivative was observed after several days' exposure to direct sunlight. The pharmacopoeias (Ph.Eur.3, USP 23), however, do not prescribe light-protection for mex.HCl.

Ring and link requirements for tocainide binding to the class I antiarrhythmic drug receptor on rat cardiac myocytes.

Our purpose was to assess the structural and physicochemical determinants of the binding of tocainide and several of its homologs to the class I antiarrhythmic drug receptor associated with rat cardiac sodium channels. The homologs were chosen to assess the contributions of substituents of the aryl ring and the arylamine link on drug binding. Drug affinity was measured with a radioligand binding assay using [3H]Batrachotoxin A 20 alpha-Benzoate and freshly isolated cardiac myocytes. The affinities of the homologs were compared to determine the relationship between the affinity for the receptor and the physicochemical and structural properties of the parent drug. The contributions to the free energy of binding were determined with the Gibb's equation delta G = -RT In (1/Ki). Hydrophobic interactions are important at most sites. Meta substituents on the aryl ring and substituents on the link each interact hydrophobically with the receptor and contribute about 0.3 kcal/mol of carbon. The hydrophobic pocket near the link binding site accommodates at least six carbons. A para methoxy substituent reduces the free energy of tocainide binding by 43%. This profound reduction in the free energy of binding might be due to anomolously high aqueous solubility of alkyl aryl ethers. Longer alkoxy chains contribute 1.09 kcal/mol of carbon to the binding energy. Ortho substituents contribute little to binding specificity. These findings support a notion of a complex drug receptor with hydrophilic and hydrophobic domains that recognize specific moieties on class I antiarrhythmic drugs.