Vincenzo Tortorella

Antiproliferative and cytotoxic effects of some σ2 agonists and σ1 antagonists in tumour cell lines

To establish the activity of σ ligands at σ1 and σ2 receptor, we chose two tumour cell lines, the human SK-N-SH neuroblastoma and the rat C6 glioma lines, which express σ2 receptors at a high density and σ1 receptors in their high-affinity or low-affinity state. We tested the σ2 receptor agonist PB28 and the σ2 antagonist AC927, and (+)-pentazocine and NE100 as agonist and antagonist, respectively, at σ1 receptors, with regard to antiproliferative and cytotoxic effects. In addition, 1,3-di(2-tolyl)guanidine (DTG) and haloperidol were tested as reference compounds displaying nearly equipotent σ affinity (σ2>σ1 and σ1>σ2, respectively). In both SK-N-SH and C6 cells, PB28 and NE100 displayed the most potent results both in antiproliferative and cytotoxic assay while AC927 and (+)-pentazocine were inactive in both assays. The cytotoxic and antiproliferative effects of DTG and haloperidol reflected their σ1 antagonist activity and σ2 agonist activity. Moreover, our results in the tumour cell lines correlated well with those for σ2 activity found previously in a functional assay in the guinea-pig bladder. These findings establish a new model for evaluating both σ2 and σ1 receptor activity of σ ligands, which could be useful for developing new ligands having mixed σ2 agonist/σ1 antagonist activity as potential antineoplastic agents.

Enantiomers of clofibric acid analogs have opposite actions on rat skeletal muscle chloride channels

The S-(−) isomers of a series of clofibric acid analogs produced only a block of chloride conductance of rat skeletal muscle fibers with increasing concentrations until block was nearly complete. The R-(+) isomers, on the other hand, at low concentrations increased chloride conductance by as much as 9% to 39% and at higher concentrations decreased chloride conductance, but never by more than 27% of the control value. The actions of the enantiomeric pairs to either produce or inhibit myotonic excitability paralleled their ability to block or increase chloride conductance, respectively.

Activation and Inhibition of Kidney CLC-K Chloride Channels by Fenamates

CLC-K Cl– channels are selectively expressed in kidney and ear, where they are pivotal for salt homeostasis, and loss-of-function mutations of CLC-Kb produce Bartters syndrome type III. The only ligand known for CLC-K channels is a derivative of the 2-p-chlorophenoxypropionic acid (CPP), 3-phenyl-CPP, which blocks CLC-Ka, but not CLC-Kb. Here we show that in addition to this blocking site, CLC-K channels bear an activating binding site that controls channel opening. Using the voltage-clamp technique on channels expressed in Xenopus laevis oocytes, we found that niflumic acid (NFA) increases CLC-Ka and CLC-Kb currents in the 10 to 1000 μM range. Flufenamic acid (FFA) derivatives or high doses of NFA produced instead an inhibitory effect on CLC-Ka, but not on CLC-Kb, and on blocker-insensitive CLC-Ka mutants, indicating that the activating binding site is distinct from the blocker site. Evaluation of the sensitivity of CLC-Ka to derivatives of NFA and FFA together with a modeling study of these ligands allow us to conclude that one major characteristic of activating compounds is the coplanarity of the two rings of the molecules, whereas block requires a noncoplanar configuration. These molecules provide a starting point for identification of diuretics or drugs useful in the treatment of Bartters syndrome.

A multireceptorial binding reinvestigation on an extended class of σ ligands: N-[ω-(indan-1-yl and tetralin-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine reveal high affinities towards σ1 and EBP sites

New 1-[omega-(2,3-dihydro-1H-inden-1-yl)- and (2,3-dihydro-5-methoxy-1H-inden-1-yl)alkyl]- and 1-[omega-(1,2,3,4-tetrahydronaphthalen-1-yl)- and (6-methoxy- or 6-fluoro-1,2,3,4-tetrahydronaphthalen-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine were synthesized, as homologous compounds of an existing series of sigma ligands, in order to carry out sigma receptor subtypes structure-affinity relationships. The new compounds and some of their related analogues, already reported, were tested in new multireceptorial radioligand binding assays. As reference compounds, the known sigma(1) ligands SA 4503, BD 1008 and NE 100 were also prepared and tested. All reported compounds showed high sigma(1) affinity assayed by (+)-[(3)H]-pentazocine on guinea-pig brain (apparent K(i)=1.75-72.2 nM) and moderate or low sigma(2) affinity by [(3)H]-DTG on rat liver, in contrast with previous results. One tertiary amine function spaced by a five-membered chain from a phenyl group is the structural feature shared by the most active compounds 26 and 43 and some reference sigma(1) ligands. The reported sigma(1) ligands, including reference compounds, also demonstrated a high affinity towards EBP (Delta(8)-Delta(7) sterol isomerase) site (apparent K(i)=0.48-14.8 nM) and some of them (37 and 44) were good ligands at L-type Ca(++) channel. 1-[4-(2,3-Dihydro-1H-inden-1-yl)butyl]-3,3-dimethylpiperidine (26) was the best mixed sigma(1) and EBP ligand (apparent K(i)=1.75 and 1.54 nM, respectively) with a good selectivity versus sigma(2) receptor (138- and 157-fold, respectively).

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.

Studies on 1-arylpiperazine derivatives with affinity for rat 5-HT7 and 5-HT1A receptors.

Several 1‐aryl‐4‐(2‐arylethyl)piperazine derivatives were synthesized and tested in‐vitro for their binding affinity for 5‐HT7 and 5‐HT1A receptors. These compounds displayed 5‐HT7 receptor affinity ranging between Ki = 474 nm and Ki = 8.2 nm, besides high affinity for the 5‐HT1A receptor. Intrinsic activity of the most potent compounds was assessed. 4‐[2‐(3‐Methoxyphenyl)ethyl]‐1‐(2‐methoxyphenyl)piperazine (16) and 1‐(1,2‐benzisoxazol‐3‐yl)‐4‐[2‐(3‐methoxyphenyl)ethyl]piperazine (20) (Ki = 24.5 and 8.2 nm, respectively) behaved as partial agonist and full agonist, respectively, when tested for 5‐HT7 receptor‐mediated relaxation of substance P‐induced guinea‐pig ileum contraction.

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.

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.

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.