Hydroxylated Analogs of Mexiletine as Tools for Structural-Requirements Investigation of the Sodium Channel Blocking Activity

Arch. Pharm. Chem. Life Sci. 2010, 343, 325 – 332 A. Catalano et al. 325

Full Paper

Hydroxylated Analogs of Mexiletine as Tools for Structural-Requirements Investigation of the Sodium Channel BlockingActivity

Alessia Catalano1, Alessia Carocci1, Maria M. Cavalluzzi1, Antonia Di Mola1, Giovanni Lentini1,Angelo Lovece1, Antonella Dipalma2, Teresa Costanza2, Jean-Fran�ois Desaphy2, Diana ConteCamerino2, and Carlo Franchini1

1 Department of Medicinal Chemistry, University of Bari, Bari, Italy2 Department of Pharmacology, University of Bari, Bari, Italy

[2-(2-Aminopropoxy)-1,3-phenylene]dimethanol 1 and 4-(2-aminopropoxy)-3-(hydroxymethyl)-5-methylphenol 2, two dihydroxylated analogs of mexiletine – a well known class IB anti-arrhyth-mic drug – were synthesized and used as pharmacological tools to investigate the blocking-activity requirements of human skeletal muscle, voltage-gated sodium channel. The very lowblocking activity shown by newly synthesized compounds corroborates the hypothesis that thepresence of a phenolic group in the para-position to the aromatic moiety and/or benzylichydroxyl groups on the aromatic moiety of local anesthetic-like drugs impairs either the trans-port to or the interaction with the binding site in the pore of Na+ channels.

Keywords: Hydroxylation / Ion channel / Mexiletine /

Received: September 9, 2009; Accepted: November 3, 2009

DOI 10.1002/ardp.200900218

Introduction

Voltage-gated sodium channels [1] are the target of clini-cally important drugs such as anti-arrhythmics [2], anti-myotonics [3], anticonvulsants [4], and local anaesthetics[5]. Mexiletine (Fig. 1) is an orally effective lidocaine ana-log belonging to the IB anti-arrhythmic drug group. Itsclinical usefulness is due to its ability to block voltage-gated sodium channels more potently in situations ofexcessive burst of action potentials (use-dependent orphasic block). This occurrs in diseased tissues, ratherthan under conditions of physiological excitability (tonicblock). A few years ago [6], we reported that the b2-adren-ergic agonist clenbuterol (Fig. 1) exhibited blockingeffects similar to those shown by mexiletine on skeletal

muscle voltage-gated sodium channels. In contrast, theb2-agonist salbutamol (Fig. 1) had no effect on the sodiumcurrents (INa). On the basis of these results it was hypothe-sized that the presence of hydroxyl groups on the aro-matic moiety may impede the hydrophobic interactionbetween the aforementioned aromatic moiety and thelocal anesthetic receptor [6]. In fact, it should be notedthat most of the b2-agonists present two hydroxyl groupson their aromatic moieties, and that the atypical clenbu-terol was the unique b2-agonist able to block sodiumchannels [6]. Similar experiments with the chemicallysimilar b2-antagonists propranolol and nadolol (Fig. 1)suggested the presence of two hydroxyl groups on thearomatic moiety of the drugs as a molecular requisite forimpeding sodium channel block [6]. Thus, we suggestthat a similar modification on mexiletine (i. e., hydroxyla-tion of the aromatic ring) might cause a reduction of thesodium channel blocking activity. The hypotesis is sup-ported by our previous experience that two of the mainmonohydroxylated mexiletine metabolites, the so-calledp-hydroxymexiletine (PHM, Fig. 1) and hydroxymethyl-mexiletine (HMM), are less active than mexiletine in

Correspondence: Catalano Alessia, Dr. Ph.D., Dipartimento Farmaco-Chimico, Facolt� di Farmacia, Universit� di Bari, Via Orabona 4, 70126Bari, Italy.E-mail: [email protected]: +39 080 544-2724

Abbreviations: hydroxymethylmexiletine (HMM); p-hydroxymexiletine(PHM)

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326 A. Catalano et al. Arch. Pharm. Chem. Life Sci. 2010, 343, 325 –332

reducing INa in both tonic and phasic block [7–9]. Herein,we report the synthesis and complete characterization oftwo new dihydroxylated analogs of mexiletine (1 and 2),which present themselves as valuable pharmacologicaltools for the study of sodium channel blocking activity.

Results and discussion

ChemistryOur strategy for the synthesis of compound 1 is shown inScheme 1. 2-[2-(2,6-Dimethylphenoxy)-1-methylethyl]-1H-isoindole-1,3(2H)-dione 6 was prepared as previouslydescribed [7, 9, 10], by protecting 2-amino-1-propanol 3with phthalic anhydride 4 [11], and then by reacting thephthalimido alcohol 5 with 2,6-dimethylphenol, underMitsunobu conditions [12]. Then, compound 6 wasreacted with N-bromosuccinimide and benzoyl peroxidefor 72 h to give the dibromoderivative 7 in 43% yield bymodifying a procedure reported in the literature [9, 13].Compound 7 was refluxed for 19 h in a mixture of diox-ane and water to give the corresponding diol 8 [14]. Thephthalimido diol 8 was deprotected by hydrazinolysis[15] to give the amine 1 which was converted into itshydrochloride salt (1 N HCl) by treatment with a few dropsof 2 N HCl and azeotropically removing water. It is note-worthy that in our efforts to raise the yield in favor of 7,N-bromosuccinimide and benzoyl peroxide were addedtogether, in four portions, to compound 6 at approx. 4 h-intervals and stopping the reaction after three days. Sur-prisingly, we noticed that, instead of rising, yields ofcompound 7, were lowered (32%), because of the forma-tion of a new unknown side-product. Hydrolysis [14] ofthis mixture gave 8 along with aldehyde 10, which was

isolated by column chromatography and fully character-ized. The formation of the latter brought us to hypothe-size the unknown side-product as 9 (Scheme 1).

The synthesis of compound 2 is shown in Scheme 2.The key step was 4-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propoxy]-3,5-dimethylphenyl acetate 14 which wasobtained following two alternative routes. The first wasperformed following the previously described synthesis[7], by submitting compound 6 to Friedel–Craft acylationand then Baeyer–Villiger oxidation reactions [16]. By thisroute, 14 was obtained in four steps, in 54% overall yield.An alternative route starts from commercially available2,6-dimethylphenol 11, which was converted into itsacetoxy derivative 12 by reaction with iodine and silveracetate [17], which was then submitted to a Mitsunobureaction with 2-(2-hydroxy-1-methylethyl)-1H-isoindole-1,3(2H)-dione 5 [12], to give the acetoxy intermediate 14.By this route, compound 14 was obtained in three stepsof a convergent synthesis, in 70% overall yield; the latterroute is more convenient and efficient than the formerand obviates the use of large amounts of silver acetate, anexpensive reagent. Then, in order to obtain the bromo-derivative 15, compound 14 was reacted with N-bromo-succinimide and benzoyl peroxide [9, 13]. Unfortunately,the yield of this reaction was very low (2%) because of the

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Figure 1. Structures of mexiletine, clenbuterol, salbutamol, andhydroxylated analogs of mexiletine (PHM, HMM, 1 and 2).

Scheme 1. Synthesis of compound 1.

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Arch. Pharm. Chem. Life Sci. 2010, 343, 325 – 332 Novel Hydroxylated Analogs of Mexiletine 327

formation of a dibrominated derivative as a side-com-pound, with a Rf value very close to the one of compound15. Therefore, compound 14 was subjected to an alterna-tive bromination reaction, by modifying a procedureused in the literature for the bromination of xylenes [18]to give 15 in a mixture with 14. This mixture, withoutany further purification, was submitted to hydrolysis inwater and dioxane, and the dihydroxy derivative 16 wasisolated. The first attempt to obtain amine 2 was by sub-mitting compound 16 to hydrazinolysis [15]. The work-upof this reaction was quite difficult due to the fact thatamine 2 and the 2,3-dihydrophthalazine-1,4-dione – by-product of the hydrazinolysis – are both soluble in theaqueous layer. Compound 2 was obtained in an amountjust sufficient for its characterization by 1H-NMR analysisand GC/MS, but not for its conversion into the corre-sponding salt. Thus, unpurified amine 2 was directly sub-mitted to protection with Boc2O giving the correspond-ing carbamate 17, which was deprotected with gaseousHCl, in an ice-bath, to give 2 N HCl as an oil that could notbe recrystallized. On the other hand, deprotection withgaseous HBr afforded a mixture of 2 N HBr and a by-prod-uct resulting from an intramolecular condensation

between the amino group and the hydroxyl group on themethyl in 2-position of the aromatic moiety (3,9-dimethyl-2,3,4,5-tetrahydro-1,4-benzoxazepin-7-ol hydro-bromide). It was assessed by spectroscopic analyses, asalready observed in the synthesis of HMM [9]. Subse-quently, a satisfactory extraction of 2 from the hydrazi-nolysis deprotection of 16 was obtained using a buffersolution at pH 9 to 11 and with product finally obtainedas the acetate salt 2 N CH3COOH.

PharmacologyThe actions of the drugs were tested in vitro on INa cur-rents recorded in HEK293 cells permanently transfectedwith the human skeletal muscle sodium channel,hNav1.4, using the whole-cell patch-clamp method.Sodium currents were elicited by 25 ms-long depolariz-ing test pulses at –30 mV from the holding potential of –120 mV at two stimulation frequencies, 0.1 Hz for deter-mination of the tonic block and 10 Hz for the phasicblock determination [6]. The IC50 values were calculatedby fitting the concentration/effect relationships with afirst-order binding function, and are reported in Table 1with the S. E. of the fit. Formal introduction of hydroxylgroups onto the aromatic ring of mexiletine (a phenolicone in the para-position or a benzylic one) always causeda dramatic fall of potency of blockade under both tonicand phasic conditions. The effect was additive, eachhydroxyl group determining a lowering effect of aboutone order of magnitude. Thus, 1 and 2 were at least onehundred times less potent than mexiletine in theirsodium channel blocking activity. However, this residualactivity was dependent on the frequency of stimulation:a clear-cut dependency of action was found in all thetested compounds. The IC50 values at 10 Hz stimulationfrequency were from 2 to 10 times lower than those at0.1 Hz. The lowering effect on the IC50 values of the mono-hydroxyl derivatives, PHM and HMM, seemed more pro-nounced for the compound with the benzylic carbinol(HMM). However, this trend is reversed when activities ofthe dihydroxyl derivatives 1 and 2 are compared: the ana-log bearing both a para-phenolic and a benzyl carbinolicgroup has shown the most evident lowering effect of theseries. Further studies are necessary in order to clarifythe reasons of the peculiar behavior of mono and dihy-droxyl analogs of mexiletine in lowering the blockingactivity on Na+ channels.

Conclusion

We have described the synthesis, full characterization,and biological evaluation of two novel dihydroxylated


Scheme 2. Synthesis of compound 2.

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analogs of mexiletine, [2-(2-aminopropoxy)-1,3-pheny-lene]dimethanol 1 and 4-(2-aminopropoxy)-3-(hydroxy-methyl)-5-methylphenol 2. Our present data demonstratethat structural modifications on the aromatic moiety ofmexiletine (i. e. introduction of two hydroxyl groups)reduce the activity towards voltage-gated sodium chan-nels (Table 1). These findings confirm our previoushypothesis that the introduction of two hydroxyl groupsonto the aromatic moiety of drugs acting on such chan-nels (anti-arrhythmics and/or b-adrenergic receptorligands) impairs either the transport to or the interactionwith the binding site in the pore of the Na channels(Table 1) [6]. Experimental logD values were also deter-

mined for Mex, 1, 2, and for the aforementioned b-adren-ergic receptor ligands, in order to confirm our hypothe-sis that the difference in activity is related to variationsin lipophilicity. Table 1 shows clearly that the lower thelipophilicity, the lower is the activity of compounds onsodium channels. In addition, we previously reportedthat PHM and HMM [7–9], two metabolites of mexiletine,bearing one hydroxyl group (para-phenolic or a benzylcarbinolic, respectively), did not show the same blockingactivity as mexiletine on voltage-gated sodium channels,but only a slight residual activity on such channels, deter-mined in voltage-clamp experiments, and these resultshave been confirmed herein by means of patch-clamp


Table 1. Concentrations for half-maximal tonic and use-dependent block of sodium currents (IC50) and logD of mexiletine, PHM,HMM, 1, 2, and several b2-adrenergic receptor ligands.

Compound Tonic blockIC50 at 0.1 Hz (lM)a

Use-dependent blockIC50 at 10 Hz (lM)a

logDb

Mexiletine 260 € 30 23.9 € 1.7 0.53

PHM 1140 € 220 390 € 80 –0.06

HMM 2320 € 400 800 € 140 –0.59

1 12 100 € 1500 5100 € 700 –0.85

2 >30 000 18 400 € 7000 –1.28

clenbuterol 76 € 5 26 € 5 0.255

propranolol 68.9 € 2.8 7.9 € 0.2 1.12

nadolol >30 000 >30 000 –1.55

salbutamol >30 000 >30 000 –2.01

a The half-maximum inhibitory concentrations (IC50) were calculated from the fit with a first-order binding function of the concen-tration/sodium current block relationships measured at 0.1 Hz and 10 Hz stimulation frequencies. The IC50 values are expressedwith the S.E. of the fit.

b logD values were determined using a Sirius pKa analyzer on the basis of experimentally determined pKa and logP values (seeExperimental).

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recordings (Table 1). A possible explanation for the lackof activity of the dihydroxylated analogs is that the twohydroxyl groups may prevent these compounds fromcrossing the plasma membrane and/or they may hamperthe proposed relevant interaction of the xylyloxy moietyof these compounds with a Tyr residue in the local anes-thetic-like drug binding site receptor [19, 20].

Experimental

Materials and methodsChemicals were purchased from Sigma-Aldrich (Italy) or Lancas-ter (Lanchester Chemical, USA) in the highest quality commer-cially available. Yields refer to purified products and were notoptimized. The structures of the compounds were confirmed byroutine spectrometric and spectroscopic analyses. Compounds5, 6, and 12–14 were prepared as described previously [7, 9, 10,17]. Only spectra for compounds not previously described aregiven. Melting points were determined on a Gallenkamp appara-tus (Weiss-Gallenkamp, London, UK) in open glass capillarytubes and are uncorrected. Infrared spectra were recorded on aPerkin-Elmer (Norwalk, CT, USA) Spectrum One FT spectropho-tometer and band positions are given in reciprocal centimeters(cm–1). 1H-NMR and 13C-NMR spectra were recorded on a VarianVX Mercury spectrometer (Varian Inc., Palo Alto, CA, USA) oper-ating at 300 and 75 MHz for 1H and 13C, respectively, using CDCl3

as solvent unless otherwise indicated. Chemical shifts arereported in parts per million (ppm) relative to solvent reso-nance: CDCl3, d: 7.26 (1H-NMR) and d: 77.3 (13C-NMR). J values aregiven in Hz. EI-MS spectra were recorded on a Hewlett-Packard6890-5973 MSD gas chromatograph/mass spectrometer (Hew-lett-Packard, Palo Alto, CA, USA) at low resolution. ESI+/–/MS/MSanalyses were performed with an Agilent 1100 series LC-MSDtrap system VL Workstation (Agilent, Palo Alto, CA, USA). Ele-mental analyses were performed on a Eurovector Euro EA 3000analyzer. Electrospray ionization (ESI) time-of-flight (TOF) reflec-tron experiments are performed on an Agilent ESI-TOF massspectrometer (Agilent). Samples are electrosprayed into the TOFreflectron analyzer at a ESI voltage of 4000 V and a flow rate of200 lL/min. Chromatographic separations were performed onsilica gel columns by flash chromatography (Kieselgel 60, 0.040–0.063 mm, Merck, Darmstadt, Germany) as described by Still etal. [21]. TLC analyses were performed on precoated silica gel onaluminum sheets (Kieselgel 60 F254, Merck).

Chemistry2-{2-[2,6-Bis(bromomethyl)phenoxy]-1-methylethyl}-1H-isoindole-1,3(2H)-dione 7To a stirred solution of 6 (0.50 g, 1.62 mmol) in CCl4 (10 mL), N-bromosuccinimide (0.57 g, 3.24 mmol) and benzoyl peroxide (54mg, 0.23 mmol) were added. The reaction mixture was refluxedfor 48 h; then, additional N-bromosuccinimide (0.15 g, 0.81mmol) and benzoyl peroxide (27 mg, 0.11 mmol) were added.The mixture was refluxed for 24 h and then the solid residuewas filtered off. After evaporation of the solvent under vacuum,the residue (1.30 g) was purified by silica gel column chromatog-raphy (EtOAc/petroleum ether, 0.5:9.5) to give 7 (0.32 g, 0.69mmol, 43%) as a white solid: m.p.: 117–1198C; IR (KBr): 1774,

1698 (C=O) cm–1; 1H-NMR d: 1.57 (d, J = 7.1 Hz, 3H, CH3), 4.25 (dd, J= 9.3, 5.2 Hz, 1H, CHHCH), 4.39 (d, J = 10.2 Hz, 2H, CHHBr), 4.57(d, J = 9.9 Hz, 2H, CHHBr), 4.76 (apparent t, J = 9.6 Hz, 1H,CHHCH), 4.90–5.10 (m, 1H, CH), 7.08 (t, J = 7.7 Hz, 1H, ArO), 7.31(d, J = 7.7 Hz, 2H, ArO), 7.65–7.80 (m, 2H, Ar), 7.80–7.95 (m, 2H,Ar); 13C-NMR d: 15.2 (1C), 27.9 (2C), 47.2 (1C), 73.7 (1C), 123.6 (2C),125.5 (1C), 132.3 (4C), 132.5 (2C), 134.2 (2C), 155.1 (1C), 168.9(2C); GC/MS (70 eV) m/z (%): 465 [M+] (a1), 188 (100). Anal. calcd.for C19H17NO3Br2 (467.15): C, 48.85; H, 3.67; N, 3.00. Found: C,48.51; H, 3.63; N, 3.06.

2-{2-[2,6-Bis(hydroxymethyl)phenoxy]-1-methylethyl}-1H-isoindole-1,3(2H)-dione 80.20 g (0.43 mmol) of 7 were dissolved in 4 mL of H2O/dioxane(1:1). The reaction mixture was kept under reflux for 19 h. Diox-ane was removed by evaporation under vacuum and the aqueousphase was extracted several times with EtOAc. The combinedorganic layers were dried (Na2SO4) and concentrated under vac-uum to give 4.27 g of a yellow oil. Purification of the crude resi-due by silica gel column chromatography (EtOAc/petroleumether, 1:1) gave 8 (0.14 g, 0.41 mmol, 95%) as a yellow oil: IR(neat): 3458 (OH), 1773, 1708 (C=O) cm–1; 1H-NMR d: 1.50 (d, J = 7.1Hz, 3H, CH3), 2.47 (br s, 2H, exch D2O, OH), 3.98 (dd, J = 9.5, 5.4 Hz,1H, CHHCH), 4.48–4.65 (m overlapping s at 4.59, 1H, CHHCH),4.59 (br s overlapping m at 4.48–4.65, 4H, CH2OH), 4.78–4.94 (m,1H, CH), 7.05 (t, J = 7.6 Hz, 1H, ArO), 7.26 (d, J = 7.4 Hz, 2H, ArO),7.66–7.76 (m, 2H, Ar), 7.78–7.88 (m, 2H, Ar); 13C-NMR d: 15.2 (1C),47.4 (1C), 60.8 (2C), 74.4 (1C), 123.6 (2C), 125.0 (1C), 129.4 (2C),132.0 (2C), 134.1 (2C), 134.4 (2C), 154.7 (1C), 169.0 (2C); GC/MS (70eV) m/z (%): 341 [M+] (a1), 188 (100).

2-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)propoxy]-3-(hydroxymethyl)benzaldehyde 102.1 g of a mixture of 7 (55%, 1.16 g, 2.49 mmol) and 9 (45%, 0.94g, 1,72 mmol) was reacted as above described for the synthesis of8. Purification of the crude residue (1.50 g) by silica gel columnchromatography (EtOAc/petroleum ether, 1:1) gave 0.9 g of 8and 0.4 g of 10. Compound 10 was recrystallized from CHCl3/hex-ane to give 0.30 g (0.88 mmol, 52%) of 10 as white crystals: m.p.:133–1348C; IR (KBr): 3441 (OH), 1771, 1705 (C=O) cm–1; 1H-NMR d:1.53 (d, J = 7.1 Hz, 3H, CH3), 2.26 (br s, 1H, exch D2O, OH), 4.12 (dd,J = 9.5, 5.1 Hz, 1H, CHHCH), 4.66 (s overlapping apparent t at4.71, 2H, CH2OH), 4.71 (apparent t overlapping s at 4.66, J = 9.6Hz, 1H, CHHCH), 4.84–5.02 (m, 1H, CH), 7.21 (apparent t, J = 7.7Hz, 1H, ArO), 7.62 (dd, 1H, J = 6.9, 1.1 Hz, ArO), 7.69–7.78 (m, 2H,Ar), 7.82–7.91 (m, 2H, Ar + 1H, ArO), 10.3 (s, 1H, CHO); 13C-NMR d:15.2 (1C), 47.2 (1C), 60.2 (1C), 76.9 (1C), 123.6 (3C), 125.0 (1C),129.5 (1C), 132.0 (2C), 134.4 (2C), 135.2 (1C), 135.9 (1C), 159.4(1C), 168.8 (2C), 189.6 (1C); GC/MS (70 eV) m/z (%): 339 [M+] (3), 188(100).

2-[2-(4-Acetyl-2,6-dimethylphenoxy)-1-methylethyl]-1H-isoindole-1,3(2H)-dione 13Prepared as reported in the literature for R- and S-isomers in 78%yield [7]. The solid obtained was recrystallized from EtOAc/petro-leum ether to give 13 as white crystals (61% yield): m.p.: 93–948C(EtOAc/petroleum ether); IR (KBr): 1773, 1708, 1676 (C=O) cm–1;1H-NMR d: 1.55 (d, J = 7.1 Hz, 3H, CH3CH), 2.22 (s, 6H, CH3ArO),2.51 (s, 3H, CH3CO), 3.93 (dd, J = 9.3, 5.6 Hz, 1H, CHHO), 4.43(apparent t, J = 9.2 Hz, 1H, CHHO), 4.80–4.94 (m, 1H, CH), 7.57 (s,


16


2H, ArO), 7.70–7.78 (m, 2H, Ar), 7.82–7.90 (m, 2H, Ar); 13C-NMR d:15.3 (1C), 16.7 (2C), 26.7 (1C), 47.3 (1C), 71.9 (1C), 123.4 (2C), 129.6(2C), 131.2 (2C), 132.2 (2C), 133.2 (2C), 134.2 (1C), 159.7 (1C),168.6 (2C), 197.8 (1C); GC/MS (70 eV) m/z (%): 351 [M+] (2), 188(100). Anal. calcd. for C21H21NO4 (351.39): C, 71.78; H, 6.02; N,3.99. Found: C, 72.22; H, 6.46; N, 4.04.

4-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)propoxy]-3,5-dimethylphenyl acetate 14Prepared as reported in the literature [7] for R- and S-isomersobtaining 14 as a white solid (95% yield): m.p.: 75–778C; IR (KBr):1766, 1706 (C=O) cm–1; 1H-NMR d: 1.54 (d, J = 6.9 Hz, 3H, CH3CH),2.17 (s, 6H, CH3ArO), 2.23 (s, 3H, CH3CO), 3.88 (dd, J = 9.3, 5.8 Hz,1H, CHHO), 4.35 (apparent t, 1H, J = 9.2 Hz, CHHO), 4.76–4.92 (m,1H, CH), 6.66 (s, 2H, ArO), 7.68–7.76 (m, 2H, Ar), 7.80–7.88 (m, 2H,Ar); 13C-NMR d: 15.4 (1C), 16.6 (2C), 21.3 (1C), 47.3 (1C), 71.9 (1C),121.6 (2C), 123.4 (4C), 132.2 (2C), 134.2 (2C), 146.4 (1C), 153.1(1C), 168.7 (2C), 170.1 (1C); GC/MS (70 eV) m/z (%): 367 [M+] (4), 188(100).

3-(Bromomethyl)-4-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propoxy]-5-methylphenyl acetate 15Method APrepared as above described for 7 starting from 14 (1.90 g, 5.18mmol), N-bromosuccinimide (0.92 g, 5.18 mmol) and benzoylperoxide (0.10 g, 0.4 mmol) in CCl4 (77 mL). Purification by col-umn chromatography (toluene/EtOAc, 10:0.5) gave only a smallamount of pure 15 (40 mg, 0.09 mmol, 2%): IR (neat): 1771, 1709(C=O) cm–1; 1H-NMR d: 1.55 (d, J = 6.9 Hz, 3H, CH3CH), 2.20 (s, 3H,CH3ArO), 2.24 (s, 3H, CH3CO), 4.03 (dd, J = 9.3, 5.6 Hz, 1H, CHHCH),4.34 (d, J = 9.9 Hz, 1H, CHHBr), 4.548 (d overlapping t at 4.553, J =10.2 Hz, 1H, CHHBr), 4.553 (apparent t overlapping d at 4.548, J =9.3 Hz, 1H, CHHCH), 4.86–5.02 (m, 1H, CH), 6.82 (d, J = 3.0 Hz, 1H,ArO), 6.91 (d, J = 3.0 Hz, 1H, ArO), 7.66–7.76 (m, 2H, Ar), 7.80–7.90(m, 2H, Ar); 13C-NMR d: 15.3 (1C), 16.7 (1C), 21.3 (1C), 28.0 (1C),47.2 (1C), 72.7 (1C), 121.7 (1C), 123.5 (2C), 125.0 (1C), 128.4 (1C),129.3 (1C), 132.2 (2C), 134.2 (2C), 146.6 (1C), 152.9 (1C), 168.8(2C), 169.6 (1C); GC/MS (70 eV) m/z (%): 445 [M+] (a1), 188 (100).

Method BTo a solution of 14 (0.50 g, 1.36 mmol) in EtOAc (3 mL), a solutionof NaBrO3 (0.61 g, 4.08 mmol) in water (2 mL) was added. Then, asolution of Na2S2O5 (0.39 g, 2.04 mmol) in water (4 mL) was addeddropwise over a period of 15 min. Cyclohexane (3 mL) was thenadded and the reaction mixture was stirred at room tempera-ture for 4 h. The aqueous layer was extracted twice with ethylether and the combined organic layers were dried (Na2SO4) andconcentrated under vacuum to give 0.60 g of a yellow oil. Purifi-cation of the residue by silica gel column chromatography(EtOAc/petroleum ether, 3:7) gave 15 in 71% yield (0.43 g, 0.97mmol) along with 14 (0.14 g, 0.39 mmol, 29%).

2-{2-[4-Hydroxy-2-(hydroxymethyl)-6-methylphenoxy]-1-methylethyl}-1H-isoindole-1,3(2H)-dione 161.43 g of a mixture of 15 (1.01 g, 2.26 mmol, 71%) and 14 (0.42 g,1.14 mmol, 29%) were dissolved in a mixture of H2O (29 mL) anddioxane (29 mL). The reaction mixture was kept under reflux for20 h. Dioxane was removed by evaporation under vacuum andthe aqueous phase was extracted several times with EtOAc. Thecombined organic layers were dried (Na2SO4) and concentrated

under vacuum to give 1.23 g of a yellow oil. Purification of thecrude residue by silica gel column chromatography (EtOAc/petroleum ether, 1:1 fi EtOAc) gave 16 as a yellow oil (0.77 g,2.26 mmol, >99%), which was crystallized from CHCl3/hexane togive 0.68 g (1.99 mmol, 88%) of 16 as white crystals: m.p.: 153–1548C (CHCl3/hexane); IR (KBr): 3396 (OH), 1771, 1705 (C=O) cm–1;1H-NMR d: 1.48 (d, J = 7.1 Hz, 3H, CH3CH), 2.06 (s, 3H, CH3ArO),3.33 (s, 2H, exch. with D2O, OH), 3.81 (dd, J = 9.5, 5.4 Hz, 1H,CHHCH), 4.35 (apparent t, J = 9.5 Hz, 1H, CHHCH), 4.45 (d overlap-ping d at 4.52, J = 12.7 Hz, 1H, AB system, CHHOH), 4.52 (d over-lapping d at 4.45, J = 12.7 Hz, 1H, AB system, CHHOH), 4.72–4.88(m, 1H, CH), 6.47 (d, J = 3.0 Hz, 1H, ArO), 6.58 (d, J = 3.0 Hz, 1H,ArO), 7.64–7.72 (m, 2H, Ar), 7.78–7.86 (m, 2H, Ar); 13C-NMR d: 15.2(1C), 16.3 (1C), 47.4 (1C), 60.9 (1C), 73.2 (1C), 113.5 (1C), 117.5 (1C),123.5 (3C), 132.0 (2C), 132.3 (1C), 134.3 (2C), 148.1 (1C), 152.4(1C), 169.1 (2C); GC/MS (70 eV) m/z (%): 341 [M+] (4), 188 (100).

tert-Butyl {2-[4-hydroxy-2-(hydroxymethyl)-6-methylphenoxy]-1-methylethyl}carbamate 17To a stirred solution of 16 (0.75 g, 2.2 mmol) in MeOH (5 mL), gla-cial AcOH (0.25 mL, 4.38 mmol) and N2H4 . H2O (8.8 mmol) wereadded and the mixture was kept under reflux for 3 h. After evap-oration of the solvent under vacuum, the residue was dissolvedin 3 mL of 1 N NaOH and kept in an ice bath. A solution of Boc2O(1.15 g, 5.26 mmol) in THF (12 mL) was then added dropwise andthe mixture was stirred at room temperature for 24 h. After stir-ring, the solvent was removed under vacuum and the aqueousphase was acidified with 2 N HCl and extracted twice withEtOAc. The combined organic layers were dried (Na2SO4) andconcentrated under vacuum to give an oil consisting of 17 andpoli-Boc derivatives of 2. Purification of the residue by silica gelcolumn chromatography (EtOAc/petroleum ether, 2:8) gave 17(68 mg, 0.22 mmol, 10%) as a yellow oil: IR (neat): 3356 (NH, OH),1688 (C=O) cm–1; 1H-NMR d: 1.26 (d, J = 7.0 Hz, 3H, CH3CH), 1.44 (s,9H, t-Bu), 2.12 (s, 3H, CH3Ar), 3.50–3.75 (m, 2H, CH2CH), 4.48 (doverlapping d at 4.55, J = 12.8 Hz, 1H, AB system, CHHOH), 4.55 (doverlapping d at 4.48, J = 12.8 Hz, 1H, AB system, CHHOH), 4.95–5.20 (br m, 1H, CH), 6.52 (d, J = 2.9 Hz, 1H, Ar), 6.63 (d, J = 2.6 Hz,1H, Ar), 7.62 (br s, 3H, exch. with D2O, OH + NH); 13C NMR: d = 16.3(1C), 17.9 (1C), 28.6 (3C), 46.9 (1C), 60.6 (1C), 75.9 (1C), 80.1 (1C),113.6 (1C), 117.6 (1C), 132.2 (1C), 134.4 (1C), 148.0 (1C), 152.6(1C), 156.1 (1C); ESI+/MS m/z: 334 [M + Na+]; ESI+/MS/MS m/z: 234(100).

[2-(2-Aminopropoxy)-1,3-phenylene]dimethanol 1To a stirred solution of 8 (0.22 g, 0.65 mmol) in absolute EtOH(2.6 mL), glacial AcOH (0.11 mL, 1.93 mmol) and N2H4 N H2O (1.93mmol) were added and the mixture was kept under reflux for2 h. The solid residue was filtered off. After evaporation of thefiltrate, the residue was taken up with EtOAc and extracted with2 N HCl; then, the aqueous phase was treated with 2 N NaOHand extracted several times with EtOAc. The combined organiclayers were dried (Na2SO4) and concentrated under vacuum togive 1 (0.12 g, 0.57 mmol, 87%) as a slightly yellowish oil: 1H-NMRd: 1.19 (d, J = 6.6 Hz, 3H, CH3), 1.76 (br s, 4H, exch. with D2O, OH +NH2), 3.35–3.50 (m, 1H, CH), 3.72 (dd, J = 9.2, 7.8 Hz, 1H, CHHCH),4.06 (dd, J = 9.3, 3.0 Hz, 1H, CHHCH), 4.63 (d, J = 12.6 Hz, 2H,CHHOH), 4.77 (d, J = 12.4 Hz, 2H, CHHOH), 7.09 (t, J = 7.6 Hz, 1H,Ar), 7.20–7.30 (m, 2H, Ar).


17


[2-(2-Aminopropoxy)-1,3-phenylene]dimethanolHydrochloride 1 N HCl0.125 g (0.59 mmol) of 1 was treated with 1 mL of 2 N HCl andthe water was azeotropically removed (toluene/abs. EtOH) givinga solid which was recrystallized from MeOH/Et2O to give 1 N HCl(42 mg, 0.16 mmol, 27%) as white crystals: m.p.: 167–1688C(MeOH/Et2O); IR (KBr): 3363 (OH, NH) cm–1; 1H-NMR (DMSO-d6; d:2.48) d: 1.28 (d, J = 6.6 Hz, 3H, CH3), 3.25–3.70 (m, 3H, exch. withD2O, NH3 + 1H, CH), 3.8–4.0 (m, 2H, CH2CH), 4.54 (s, 4H, CH2OH),7.12 (t, J = 7.4 Hz, 1H, Ar), 7.32 (d, J = 7.7 Hz, 2H, Ar), 8.22 (br s, 2H,exch. with D2O, OH); 1H-NMR (CD3OD; d: 3.31) d: 1.41 (d, J = 6.6 Hz,3H, CH3), 3.65–3.80 (m, 1H, CH), 4.03 (dd, J = 10.2, 7.3 Hz, 1H,CHHCH), 4.15 (dd, J = 10.2, 3.7 Hz, 1H, CHHCH), 4.66 (s, 4H,CH2OH), 7.17 (t, J = 7.7 Hz, 1H, Ar), 7.37 (d, J = 7.7 Hz, 2H, Ar); 13C-NMR (CD3OD, d: 47.8) d: 14.1 (2C), 47.8 (1C), 59.4 (1C), 74.9 (1C),124.7 (1C), 129.6 (2C), 134.2 (2C), 154.6 (1C); ESI+/MS m/z: 234 [M +Na+]; ESI+/MS/MS m/z: 176 (100). Anal. calcd. forC11H17NO3 N HCl N 0.33 H2O (253.72): C, 52.07; H, 7.42; N, 5.52.Found: C, 52.24; H, 7.13; N, 5.87.

4-(2-Aminopropoxy)-3-(hydroxymethyl)-5-methylphenol 2To a stirred solution of 16 (0.34 g, 1.0 mmol) in absolute EtOH (10mL), glacial AcOH (2.0 mmol) and aqueous hydrazine (4.0 mmol)were added and the mixture was kept under reflux for 5 h. Thesolid residue was filtered off. After evaporation of the filtrate,the residue was taken up with EtOAc and extracted with 2 N HCl(3610 mL); then, the aqueous phase was brought to 9 a pH a 11with 2 N NaOH (20 mL) and 2 N Na2CO3 (20 mL) and extracted sev-eral times with EtOAc. The combined organic layers were dried(Na2SO4) and concentrated under vacuum. The final product wasa slightly yellowish oil (147 mg, 0.70 mmol, 70%): IR (neat): 3353(OH, NH) cm–1; 1H-NMR (CD3OD, d: 3.09) d: 0.96 (d, 3H, J = 6.6 Hz,CH3CH), 1.99 (s, 3H, CH3Ar), 3.0–3.15 (m overlapping CH3OH sig-nal, 1H, CH), 3.35 (apparent t, J = 8.1 Hz, 1H, CHHCH), 3.44 (dd, J =9.1, 4.4 Hz, 1H, CHHCH), 4.36 (s, 2H, CH2OH), 6.31 (d, J = 3.0 Hz,1H, Ar), 6.46 (d, J = 3.0 Hz, 1H, Ar); 13C-NMR (CD3OD, d: 47.8) d:15.3 (1C), 18.1 (1C), 47.0 (1C), 59.2 (1C), 78.6 (1C), 113.0 (1C), 116.5(1C), 131.6 (1C), 134.9 (1C), 147.7 (1C), 153.4 (1C); GC/MS (70 eV)m/z (%): 211 [M+] (2), 58 (100).

4-(2-Aminopropoxy)-3-(hydroxymethyl)-5-methylphenolHydrochloride 2 N HClA solution of 17 (70 mg, 0.22 mmol) in Et2O (4 mL) in an ice-bathwas treated with gaseous HCl for a few seconds until the solutionturns into a suspension under formation of a white gum, whichwas filtrated giving 2 N HCl (38 mg, 0.14 mmol, 73%) as a whitegum, when taken at –208C, that became an oil at room temper-ature: 1H-NMR (CD3OD, d: 3.31) d: 1.41 (d, J = 6.9 Hz, 3H, CH3CH),2.23 (s, 3H, CH3Ar), 3.65–3.75 (m, 1H, CH), 3.80–3.90 (m, 1H,CHHCH), 3.93 (dd, J = 10.2, 3.8 Hz, 1H, CHHCH), 4.51 (s, 2H,CH2OH), 6.56 (d, J = 3.0 Hz, 1H, Ar), 6.67 (d, J = 3.0 Hz, 1H, Ar); 13C-NMR (CD3OD, d: 46.3) d: 12.6 (1C), 13.7 (1C), 46.5 (1C), 58.0 (1C),71.7 (1C), 112.1 (1C), 115.3 (1C), 130.1 (1C), 133.1 (1C), 145.7 (1C),152.2 (1C).

4-(2-Aminopropoxy)-3-(hydroxymethyl)-5-methylphenolAcetate 2 N CH3COOHTo a solution of 2 (157 mg, 0.74 mmol) in MeOH (4 mL), a solutionof glacial AcOH (0.88 mmol) in MeOH (2 mL) was added. The sol-vent was removed by evaporation under vacuum to give a brown

oil which was crystallized from abs. EtOH/iPr2O to give2 N CH3COOH (123 mg, 0.45 mmol, 61%) as yellow crystals: m.p.:169–1708C (abs. EtOH/i-Pr2O); 1H-NMR (CD3OD, d: 3.08) d: 1.15 (d, J= 6.6 Hz, 3H, CH3CH), 1.68 (s, 3H, CH3Ar), 2.01 (s, 3H, CH3COO),3.36–3.48 (m, 1H, CH), 3.57 (dd, J = 10.1, 7.0 Hz, 1H, CHHCH), 3.67(dd, J = 10.1, 3.7 Hz, 1H, CHHCH), 4.33 (s, 2H, CH2OH), 6.33 (d, J =2.7 Hz, 1H, Ar), 6.44 (d, J = 2.7 Hz, 1H, Ar); 13C-NMR (CD3OD, d:47.8) d: 14.5 (1C), 15.2 (1C), 22.8 (1C), 47.8 (1C), 59.6 (1C), 73.8(1C), 113.6 (1C), 116.8 (1C), 131.7 (1C), 134.7 (1C), 147.3 (1C),153.7 (1C), 178.8 (1C); ESI+/MS m/z: 234 [M + Na+]; ESI+/MS/MS m/z:177 (100); Anal. calcd. for C11H17NO3 N CH3COOH (271.31): C,57.55; H, 7.80; N, 5.16. Found: C, 57.33; H, 7.88; N, 5.12.

Physicochemical dataPhysicochemical data of the compounds shown in Table 1 wereobtained by a pH-metric technique using a GlpKa apparatus (Sir-ius Analytical Instruments Ltd., Forrest Row, East Sussex, UK)[22–25]. Because of the low solubility of the investigated com-pounds in aqueous medium, methanol was used as a co-solventfor pKa measurements. Three separate solutions of a concentra-tion approximately 10–5 M, in 10–30% w/w (MeOH/H2O), wereprepared. They were acidified with 0.5 M HCl to pH 4. The solu-tions were titrated with 0.5 M KOH to pH 12. Initial pKa values,which are the apparent ionization constants relative to the mix-ture of the solvents, were obtained by Bjerrum Plot i. e., the curveobtained by the difference between the titration curve of the ion-isable substance and that of the blank solution. These valueswere optimized by a weighted nonlinear least-squares procedure(Refinement Pro 1.0 software) to obtain pKa values in the absenceof the cosolvent by extrapolation using the Yasuda–Shedlovskyequation [26]. To obtain logP data, at least three separate titra-tions were performed for each compound. The concentration ofthe analite was approximately 10–5 M, in mixtures of H2O (7.5mL) and n-octanol, (0.1 to 10 mL). The biphasic solutions wereacidified to pH 4 with 0.5 M HCl and then titrated with 0.5 MKOH until pH 12. The obtained data were optimized as describedabove and the average of these data gave the logP value for eachcompound [23, 24]. All titrations were carried out at 25 € 0.18Cunder nitrogen atmosphere to exclude CO2.

PharmacologyIn-vitro drug testing was performed as previously described [6].Permanent transfection of HEK293 cells (human embryonic kid-ney cell line) with the full-length hNav1.4 cDNA, encoding thehuman skeletal muscle sodium channel subtype, was obtainedusing the calcium-phosphate precipitation method followed byclone selection with geneticin (Gibco-Invitrogen, Italy). Morethan 95% of the cloned cells express robust whole-cell INa of 1–4nA amplitude with voltage- and time-dependent properties typi-cal of voltage-gated sodium channels. Whole-cell sodium cur-rents were recorded at room temperature (20 to 228C) using anAxopatch 1D amplifier (Axon Instruments, Union City, CA, USA).Voltage-clamp protocols and data acquisition were performedwith pClamp 6.0 software (Axon Instruments, USA) through a12-bit A-D/D-A Digidata 1200 interface (Axon Instruments). Theexternal solution contained (in mM): 150 NaCl, 4 KCl, 2 CaCl2, 1mg Cl2, 5 Hepes and 5 glucose; the pH was set to 7.4 with NaOH.The pipette solution contained (in mM): 120 CsF, 10 CsCl, 10NaCl, 5 EGTA, and 5 Hepes; The pH was set to 7.2 with CsOH. Cur-rents were low-pass filtered at 2 kHz (–3 dB) by the four-pole Bes-sel filter of the amplifier and digitized at 10–20 kHz. After rup-turing the patch membrane, sodium currents were elicited by a


18


25 ms-long test pulse to –30 mV from a holding potential of –120mV applied at 0.1 Hz frequency, until stabilization of the currentamplitude was achieved (typically 5 min). Then, the drug(directly dissolved in saline solution) was applied at the desiredconcentration around the cell through a plastic capillary, andthe effects of the drug on INa was measured first at 0.1 Hz stimu-lation frequency, then at 10 Hz. Little or no run-down wasobserved during the experiments. Analysis was performed off-line. The mean ratio IDRUG/ICTRL calculated from at least three cellswas reported as a function of drug concentration. At least threedrug concentrations were used to obtain each relationship,which were fitted by a first-order binding function:

IDRUG/ICTRL = 1/(1 + ([DRUG]/IC50))

allowing the calculation of half-maximum inhibitory concentra-tion values. Because of the limited drug quantity available andto avoid drug-solubility problems, the maximum drug concen-tration used was 10 mM. Thus, the IC50 value calculated for mex-iletine derivatives are to be considered merely qualitative.

This work was accomplished thanks to financial support from the Min-istero dell'Istruzione, dell'Universit� e della Ricerca (MIUR, grant num-ber 2005033023) to CF and Telethon-Italy (grant number GGP04140) toDCC.

The authors have declared no conflict of interest

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Documents

Hydroxylated Analogs of Mexiletine as Tools for Structural-Requirements Investigation of the Sodium Channel Blocking Activity