Giovanni Lentini

Phytochemical, Antioxidant and Anti-α-glucosidase Activity Evaluations of Bergenia cordifolia

The antioxidant, anti‐α‐glucosidase and anticholinesterase activity of the leaves and rhizomatous extract of Bergenia cordifolia were investigated. The rhizomes extract that showed a higher degree of 1,1‐diphenyl‐2‐picrylhydrazyl radical scavenging and anti‐α‐glucosidase activity than reference compounds (rutin and acarbose respectively) were subjected to phytochemical analysis. The study revealed that previously unknown minor constituents from the plant, (+)‐catechin 3‐O‐gallate, (+)‐catechin 3,5‐di‐O‐gallate and 1,2,4,6‐tetra‐O‐galloyl‐β‐ d‐glucopyranoside, were the radical scavenging and anti‐α‐glucosidase principles. These compounds as well as the crude extracts were weak acetylcholienesterase inhibitors, suggesting a higher degree of selectivity against α‐glucosidase enzyme. In comparison with the minor constituents, the previously known major constituents of the plant, bergenin and arbutin, were poor radical scavengers and enzyme inhibitors. Copyright

Stereoselective effects of mexiletine enantiomers on sodium currents and excitability characteristics of adult skeletal muscle fibers

The effects of the enantiomers of mexiletine were tested on sodium currents of frog skeletal muscle fibers recorded by means of the three vaseline gap voltage clamp method and compared with the effects produced by tocainide enantiomers. The R-( − ) mexiletine produced a tonic block of the sodium current, elicited by single depolarizing test pulses from the holding potential of − 100 mV to − 20 mV, with an IC50 of 43.9 ± 1 μM, whereas the corresponding S-( + ) enantiomer produced the same effects at about twofold higher concentrations. A similar stereoselectivity was observed with tocainide enantiomers, but at about 5 fold higher concentrations. Both the R-( − ) and S-( + ) enantiomers of mexiletine and tocainide produced a further use-dependent block of sodium currents when the test pulse was applied repetitively at a frequency of 2 Hz. The use dependent behaviour led to a significant lowering of the IC50 values with respect to the tonic block but the eudismic ratios ([IC50S-( + )]/[IC50R( − )]) and the relative potency between mexiletine and tocainide were maintained. All the tested compounds produced a left shift of the steady state inactivation curves (h∞) , suggesting a high-affinity interaction with the inactivated sodium channels. Again a stronger potency of R-( − ) vs. S-( + ) enantiomers and of mexiletine vs. tocainide was observed. The excitability characteristics recorded from the semitendinosus muscle by the two microelectrode technique were modified by the tested drugs in agreement with their ability to block sodium current. Thus a concentration-related increase in the threshold current required to elicit an action potential was observed along with a decrease in the amplitude and a shortening of the latency of action potential and a decrease in the firing capability of the membrane. Again the R-( − ) isomers were more potent than the S-( + ) ones and mexiletine was more effective than tocainide. These data corroborate the presence of a stereospecific site for these drugs on adult skeletal muscle sodium channels. The constant eudismic ratios between the enantiomers during both tonic and use-dependent block suggest that the increase in the apparent affinity of the receptor during statedependent conformational changes of the channel does not enhance its stereospecificity. The decrease in effective concentration upon high frequency stimulation supports the potential usefulness of low doses of R-( − ) mexiletine in the treatment of the abnormal hyperexcitability of the myotonic muscles, with a likely reduction of unwanted side effects.

Stereospecific synthesis of mexiletine and related compounds: Mitsunobu versus Williamson reaction

Abstract Mexiletine [1-(2,6-dimethylphenoxy)-2-propanamine], a chiral, orally effective antiarrhythmic agent, and several analogues substituted on either the stereogenic centre or the xylyloxy moiety, were prepared in both, highly enriched, optically active forms. According to the ‘chiral pool’ approach, the appropriate amino alcohols, protected as the corresponding phthalimide derivatives, were condensed with the desired phenols under either Mitsunobu (method A) or Williamson (method B) conditions. Generally, method A provided the most efficient route, both in terms of yields and number of steps necessary. Only when an isopropyl group was present on the stereogenic centre, i.e. when 2-amino-3-methylbutanol was used as the starting alcohol, method B proved to be the only available route, method A giving no product other than the starting phthalimide derivative. Regardless of the method used, enantiomeric excesses ranged from 91 to 99%. Given the availability of both variously substituted phenols and optically active amino alcohols, the two methods described herein, taken together, may serve as a versatile approach, useful to meet the needs of new chiral, optically active mexiletine analogues, possibly endowed with higher potency in exerting a use-dependent block on sodium channels and/or more resistant to biotransformations.

INHIBITION OF FROG SKELETAL MUSCLE SODIUM CHANNELS BY NEWLY SYNTHESIZED CHIRAL DERIVATIVES OF MEXILETINE AND TOCAINIDE

To search for potent use-dependent blockers of skeletal muscle sodium channels as potential antimyotonic agents, the actions of newly synthesized chiral analogs of mexiletine and tocainide were tested in vitro on sodium currents of single fibers of frog semitendinosus muscle by vaseline-gap voltage clamp method. The effect of each drug on the maximal peak Na+ transient (INa max) was evaluated as both tonic and use-dependent block by using infrequent depolarizing stimulation and trains of pulses at 2–10 Hz frequency, respectively. The mexiletine analog 3-(2,6-dimethylphenoxy)-2-methylpropanamine (Me2), having an increased distance between the phenyl and the amino groups, was less potent than mexiletine in producing a tonic block but produced a remarkable use-dependent block. In fact, the half-maximal concentration (IC50) for tonic block of S(–)-Me2 was 108 μM vs. 54.5 μM of R(–)-mexiletine, but the IC50 was 6.2 times lowered by the 10 Hz stimulation with respect to the 2.4fold decrease observed with mexiletine. The R(–)-mexiletine and the S(–)-Me2 were about twofold more potent than the corresponding enantiomers in producing a tonic block, but the stereoselectivity attenuated during use-dependent blockade. The more lipophilic 2-(4-chloro-2-methylphenoxy)-1-phenylethylamine (Me1), presently available as raceme, produced a potent and irreversible tonic block of the sodium currents with an IC50 of 29 μM, but had a less pronounced use-dependent inhibition, with a 1.9fold decrease of the IC50 at 10 Hz. The R(–) isomer of 2′,6′-valinoxylidide (To1), a tocainide derivative with an increased hindrance on the chiral carbon atom, was twofold (IC50 = 209 μM) and tenfold (IC50 = 27.4 μM) more potent than R(–)-tocainide in tonic and use-dependent block, respectively. Tocainide was almost devoid of stereoselectivity, whereas the eudismic ratio of To1 [(IC50 S(+)-To1/IC50 R(–)-To1] was 1.7. As for mexiletine and Me2, the stereoselectivity of To1 was the weaker the higher the frequency of stimulation. The cyclic pyrrolo-imidazolonic tocainide analog To2 produced a small tonic block at 500 μM, and 1 min stimulation at 10 Hz was needed to show up a 50% block of INa max. All the compounds produced a left-shift of the steady-state inactivation curve correlated positively with the extent of use-dependent inhibition, with the exception of the cyclic To2 that acted as an open-channel blocker. The highly use-dependent blockers Me2 and To1 might be promising drugs to solve high frequency discharges of action potentials typical of myotonic muscles. Concomitantly the high potency of Me1 and the open-channel block exerted by To2 can represent important features to get selective blockers for skeletal muscle sodium channels.

The therapeutic potential of rutin for diabetes: an update.

Diabetes and its major risk factor, obesity, have become a world-wide epidemic and cause of suffering for millions of people. There is still no drug of cure for diabetes and the currently available drugs suffer from a number of limitations either due to side effects and/or loss of efficacy during prolonged use. Rutin is one of the most abundant polyphenolic compounds belonging to the flavonoid class. In the present communication, its therapeutic potential for diabetes is critically analysed by reviewing its effect on the various targets of diabetes. The multifunctional nature of rutin including action via antioxidant, anti-inflammatory, organoprotection, etc., mechanisms is outlined through review of evidences from in vitro and in vivo studies.

Facile entry to (-)-(R)- and (+)-(S)-mexiletine.

The title compounds, 1a and 1b, have been synthesized in a three-step sequence starting from (-)-(S) and (+)-(R)-propylene oxide, respectively, in acceptable overall yields. The enantiomeric excess values for 1a and 1b were 96% and 93% respectively, as assessed by HPLC analysis on a chiral stationary phase of the corresponding N-acetyl derivatives. The synthetic route herein presented may represent a facile entry to highly enriched mexiletine enantiomers, alternative to those previously reported in the literature.

New N-(phenoxydecyl)phthalimide derivatives displaying potent inhibition activity towards α-glucosidase

Several members of a new family of non-sugar-type alpha-glucosidase inhibitors, bearing a phthalimide moiety connected to a variously substituted phenoxy ring by an alkyl chain, were synthesized and their activities were investigated. The efficacy of the inhibition activity appeared to be governed by the chain length of the substrate. Substrates possessing 10 carbons afforded the highest levels of activity, which were one to two orders of magnitude more potent than the known inhibitor 1-deoxynojirimycin (dNM). Furthermore, structure-activity relationship studies indicated a critical role of electron-withdrawing substituents at the phenoxy group for the activity. Derivatives bearing a chlorine atom along with a strong electron-withdrawing group, such as a nitro group, were the most potent of the series.

Design, synthesis, and pharmacological effects of structurally simple ligands for MT1 and MT2 melatonin receptors

A series of phenoxyalkyl and phenylthioalkyl amides were prepared as melatoninergic ligands. Modulation of affinity of the newly synthesized compound by applying SARs around the terminal amide moiety, the alkyl chain, and the methoxy group on the aromatic ring provides compounds with nanomolar affinity for both melatonin receptor subtypes. Affinity towards MT(1) and MT(2) receptors were modulated also exploiting chirality. The investigation of intrinsic activity revealed that all the tested compounds behave as full or partial agonists.

New potent mexiletine and tocainide analogues evaluated in vivo and in vitro as antimyotonic agents on the myotonic ADR mouse.

The antimyotonic activity of chiral derivatives of mexiletine and tocainide, selected as potent use-dependent blockers of skeletal muscle sodium channels, was evaluated in vivo acutely in myotonic ADR mice. The compounds had either aromatic (Me4 and Me6) or branched isopropyl groups (Me5 and To1) on the asymmetric centre, or had this latter one methylene apart from the amino group (Me2). Therapeutic doses of mexiletine (5-10 mg/kg) and tocainide (7-20 mg/kg) significantly reduced the long time of righting reflex (TRR), typical of ADR mice. Me4, Me5 and Me6 were 2-fold more potent than mexiletine. To1 fully normalised the TRR at 7 mg/kg. The electromyographic analysis confirmed a muscle-based activity for drug effectiveness on TRR. All the compounds reduced the myotonic hyperexcitability of intercostal muscle fibres when tested in vitro by current-clamp recordings, with a potency correlated with their action on sodium channels. On stimulus-evoked firing, the isopropyl analogues were 2-4-fold more potent than parent compounds, while the aromatic analogues were about 10-fold more potent than mexiletine. Patch-clamp recordings confirmed a normal-like pharmacological sensitivity of sodium channels of native ADR muscle fibres. Finally, the in vivo antimyotonic activity is due to the block of sodium channels and divergences with in vitro potency can be related to structure-based changes in drug pharmacokinetics.

Molecular Insights into the Local Anesthetic Receptor within Voltage-Gated Sodium Channels Using Hydroxylated Analogs of Mexiletine

We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003), suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogs by adding one or two hydroxyl groups to the aryloxy moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration–response relationships were constructed using 25-ms-long depolarizing pulses at −30 mV applied from an holding potential of −120 mV at 0.1 Hz (tonic block) and 10 Hz (use-dependent block) stimulation frequencies. The half-maximum inhibitory concentrations (IC50) were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. In particular, replacement of Phe1586 and Tyr1593 by non-aromatic cysteine residues may help in the understanding of the role of π–π or π–cation interactions in mexiletine binding. Alteration of tonic block suggests that the aryloxy moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryloxy moiety may modify high-affinity binding of the drug amine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion, these results confirm our former hypothesis by showing that the presence of hydroxyl groups to the aryloxy moiety of mexiletine greatly reduced sodium channel block, and provide molecular insights into the intimate interaction of local anesthetics with their receptor.