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Brief Articles Anesthetic Potency of Two Novel Synthetic Polyhydric Alkanols Longer than the n-Alkanol Cutoff: Evidence for a Bilayer-Mediated Mechanism of Anesthesia? Justin T. Mohr,† Gordon W. Gribble,† Susan S. Lin,§ Roderic G. Eckenhoff,§ and Robert S. Cantor*,†,‡ Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, Department of Anesthesia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, and MEMPHYSsCenter for Biomembrane Physics, Physics Department, University of Southern Denmark, DK-5230 Odense M, Denmark Received July 7, 2004
The polyhydroxyalkanes 1,6,11,16-hexadecanetetraol (1) and 2,7,12,17-octadecanetetraol (2) were synthesized utilizing the thiophene ring as a scaffold to affix the hydroxyalkyl chains by lithiation of the acidic R-hydrogens and subsequent desulfurization. Both compounds exhibited significant anesthetic potency, individually and in additivity studies with hexanol, using immobility in tadpoles as the phenotypic endpoint. These results, which contradict a proteinbinding mechanism in which cutoff results from steric hindrance, are consistent with recent predictions of a membrane-mediated mechanism involving the lateral pressure profile. The Meyer-Overton rule, expressed as the correlation between anesthetic potency and the partitioning of anesthetics between oil and the gas or aqueous phase, is remarkably accurate for a broad range of general anesthetics, including alkanols, volatile agents, and barbiturates.1 This relationship predicts that although clinical partial pressures or aqueous concentrations of general anesthetics vary widely, their corresponding concentration in oil (whether olive oil, octanol, or a fully hydrated lipid bilayer) is remarkably uniform; for a lipid bilayer, the corresponding anesthetic concentration is typically a few mole percent. This correlation would support the presumption that anesthetics modulate the activity of those membrane proteins responsible for anesthesia through an indirect, bilayer-mediated mechanism, to a degree determined by the membrane concentration of the anesthetic. However, there are wellknown exceptions to this rule. In particular, the anomalous lack of anesthetic potency of n-alkanols above a “cutoff” chain length has commonly been interpreted as evidence that anesthetics act instead by binding directly to protein sites of well-defined volumes, the molecular volume of the alkanol at the cutoff length thus providing a measure of the volume of the putative binding site. The search for such sites on ligand-gated ion channels has been guided in part by the presumption that mutations that alter the volume of any such site should correlate to changes in the cutoff in potency of n-alkanols.2,3 Although this interpretation of steric hindrance as an explanation for cutoff is quite common, it has been argued4 that other ligand-binding mechanisms can explain the existence of a cutoff in potency, * To whom correspondence should be addressed. Phone: (603) 6462504. Fax: (603) 646-3946. E-mail:
[email protected]. † Dartmouth College. § University of Pennsylvania School of Medicine. ‡ University of Southern Denmark.
as is evident from the effect of binding of alkanols on the destabilization of soluble proteins such as BSA. It has recently been suggested4 that another interpretation of these exceptions to the Meyer-Overton correlation is possible within the context of an indirect mechanism in which the anesthetic is solubilized in the bilayer, causing a redistribution of membrane lateral pressures, which shifts the conformational equilibrium of integral proteins such as ligand-gated ion channels that are known to be affected by clinical concentrations of anesthetics. In the context of this putative mechanism, the anesthetic acts as a solute within the lipid solvent that comprises the fluid bilayer, and it is largely the positional and orientational distribution within the bilayer of the segments and bonds of the anesthetic molecule that determines its potency. In brief, for short alkanols, the localization of the hydroxyl group near the aqueous interfacial region requires the flexible hydrocarbon chain segments to reside close thereto, within the hydrocarbon domain, resulting in a significant redistribution of lateral stresses from the bilayer interior toward the aqueous interfaces. However, for longer chain length, the tails of the alkanols penetrate increasingly deeply into the bilayer, reducing the magnitude of the effect until, for sufficiently long chains, no pressure redistribution is predicted and anesthetic potency is lost. In the context of this indirect mechanism, it was suggested5 that the chain segments of long alkane chains of length n, with multiple hydroxyl or other hydrophilic groups tethered to the backbone at regular intervals of L methylene groups, are distributed spatially and orientationally in the bilayer similarly to 1-alkanols of length n* ≈ (L - 1)/2 and might thus be expected to perturb structural and thermodynamic properties of the bilayer as do the corresponding shortchain 1-alkanols of length n*. Statistical thermodynamic
10.1021/jm049459k CCC: $30.25 © 2005 American Chemical Society Published on Web 04/30/2005
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Scheme 1. Synthesis of 1,6,11,16-Hexadecanetetraol (1) and 2,7,12,17-Octadecanetetraol (2)
ment in response to a provocation as the phenotypic endpoint. Because the EC50 values were found to exceed slightly the aqueous solubility limit, greater sensitivity was obtained using an additivity design in which lower concentrations of the test compounds were added to a soluble alcohol, n-hexanol, at its EC50 concentration. Experimental Section
calculations predicted this to be the case for redistributions of the lateral pressure profile. For example, a 1-alkanol of length ∼n and bilayer concentration c is predicted to perturb the bilayer similarly to 1,ω-diols of length 2n, at a bilayer concentration c/2. A molecule such as 1,6,11,16-hexadecanetetraol should thus have an effect roughly similar to that of 1-propanol or 1-butanol but requiring only roughly 1/6 the molar bilayer concentration of the 1-alkanol. Such polyhydric alkanols thus serve to distinguish between this bilayermediated mechanism and a direct protein-binding mechanism in which cutoff arises from steric hindrance, because such alkanols would only be predicted to be potent anesthetics in the context of the indirect mechanism, whereas they would far exceed the volume of a proteinaceous site. Evidence in support of the indirect mechanism is provided by recent studies on the inhibition of NMDA receptors by 1,ω-diols.6 Although the 1-alkanols exhibit a cutoff in potency at 1-nonanol, no cutoff of potency was observed for diols from 1,3-propanediol through 1,14-tetradecanediol. It would thus be of interest to test the potency of long-chain polyhydroxyalkanes of a range of lengths and hydroxyl spacings. However, such alkanols are not available commercially. We have therefore developed a general synthetic approach that has provided a pair of such compounds: 1,6,11,16-hexadecanetetraol and 2,7,12,17-octadecanetetraol. Our synthetic approach to the target polyhydroxyalkanes involves utilizing the heterocycle thiophene as a scaffold to affix the requisite hydroxyalkyl chains by lithiation of the acidic R-hydrogens. This strategy is a standard method for functionalizing thiophenes.7 Subsequent reductive desulfurization and concomitant double bond reduction with Raney Ni provides the alkanol with the desired four-carbon spacer.7-9 Our syntheses of 1,6,11,16-hexadecanetetraol (1) and 2,7,12,17-octadecanetetraol (2) are shown in Scheme 1. It was necessary to protect the hydroxyl groups in diol 4 as trimethylsilyl ethers prior to lithiation of the thiophene rings. No attempt was made to separate the diastereomers at any stage. Thus, tetraol 1 having two chiral carbons exists as two diastereomers (a pair of enantiomers and a meso compound), and tetraol 2 having four chiral carbons exists as six diastereomers (four pairs of enantiomers and two meso compounds). All compounds prepared in this study were characterized spectrally, and the purity of the 1 and 2 diastereomers was confirmed by HPLC analysis of the perbenzoyl ester derivatives. These two compounds were tested for anesthetic potency on tadpoles in pond water using lack of move-
1. Alkanol Synthesis. All chemicals were purchased from Aldrich or Acros Organics and used without further purification unless otherwise noted. All reactions were run in dried glassware under a dry nitrogen atmosphere unless otherwise noted. In all uses of tetrahydrofuran the solvent was freshly distilled from sodium/benzophenone. Triethylamine was distilled from calcium hydride prior to use in all cases. Brine solutions are saturated aqueous sodium chloride solutions. Raney nickel catalyst was of the W-2 type and was purchased from Acros Organics as a 50% slurry in water and stored as such. Melting points were determined using a Mel-Temp apparatus with open capillary tubes and are uncorrected. NMR spectra were recorded with a 300 MHz Varian Unity Plus NMR spectrometer or, where noted, a 500 MHz Varian Unity Plus NMR spectrometer, each using tetramethylsilane as an internal standard. Low- and high-resolution mass spectra were measured by the group of Dr. Steven Mullen at the University of Illinois at Urbana-Champaign. Flash chromatography was performed using 230-400 mesh silica gel purchased from Fisher. Thin-layer chromatography was performed with silicacoated plastic, aluminum, or glass plates purchased from Whatman. Analytical forward-phase HPLC was performed with an Agilent 1100 series HPLC utilizing Zorbax Sil or RXSil columns (4.6 mm × 25 cm) with visualization at 254 nm. Analytical reverse-phase HPLC was performed with a Waters 717 Plus series HPLC utilizing a Deltapak 300 mm × 3.9 mm, 15 µm C18 column equipped with solvent mixtures 95:5:0.05 water/acetonitrile/trifluoroacetic acid and 5:95:0.01 water/ acetonitrile/trifluoroacetic acid with a constant flow rate of 1 mL/min. 1,6-(2,2′-Dithienyl)hexane-1,6-diol (4). Magnesium turnings (8.1 g, 0.33 mol) were weighed into a dry Erlenmeyer flask containing freshly distilled tetrahydrofuran (100 mL). The turnings were pressed while submerged in the solvent to expose interior surfaces of the metal. The turnings and the solvent were transferred to a dry round-bottom flask, additional THF (350 mL) was added, and the mixture was stirred under dry nitrogen at room temperature for 10 min. A solution of 1,2-dibromoethane (0.3 mL) in THF (10 mL) was slowly added to the mixture. To the resulting mixture a solution of 1,4-dibromobutane (32.5 g, 0.151 mol) in THF (30 mL) was added dropwise via syringe. The resulting solution was heated at reflux for 4.5 h. The resulting mixture was cooled to 0 °C, and a solution of 2-thiophenecarboxaldehyde (3) (18.2 g, 0.163 mol) dissolved in THF (60 mL) was added dropwise. The solution was warmed to room temperature and stirred for 18 h. The mixture was cooled to 0 °C, saturated aqueous ammonium chloride solution (400 mL) was added very slowly over 2 h, and the mixture was stirred at room temperature for 18 h. Ethyl acetate and water were added to dissolve the white solid in the reaction mixture. The aqueous layer was extracted with ethyl acetate (3 × 200 mL), and the combined organic extract was dried (MgSO4), filtered, and concentrated in vacuo to yield a yellow oil and a white solid (29.5 g, 69%). The mixture was filtered and washed with a small amount of dichloromethane to yield white solid: 11.2 g (26%), mp 114.7115.3 °C (lit. mp 110-115 °C).10 The filtrate from above was concentrated under reduced pressure and the resulting solid was isolated in the same fashion, bringing the total yield of 4 as a white solid to 13.2 g (31%): 1H NMR (500 MHz, acetoned6) δ 1.33-1.43 (4H, m, 3,4-position CH2), 1.46-1.56 (4H, m, 2,5-position CH2), 4.47 (2H, d, OH), 4.89 (2H, dt, CH), 6.93 (4H, m, aromatic 3,4-positions), 7.29 (2H, dd, aromatic 5-position); 13C NMR (125 MHz, acetone-d6) δ 26.2, 40.6, 70.1, 123.6, 124.4, 127.1, 151.6; IR (KBr) 3330 (OH), 2945 (CH), 1399
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(aromatic), 1282 (aromatic), 1178, 1009, 849, 832, 703 cm-1; MS (EI+) 264, 246, 197, 180, 152, 113, 85; MS (FAB+, Na) 304, 283 (M+), 279, 265, 247, 235, 220, 195, 177, 155, 133, 119. 1,6-(2,2′-Dithienyl)-1,6-bis(trimethylsilyloxy)hexane (5). Diol 4 (3.50 g, 12.4 mmol) was dissolved in a mixture triethylamine (8.9 mL, 63.8 mmol) and dichloromethane (62 mL). The resulting solution was cooled to 0 °C, and trimethylsilyl chloride (3.93 mL, 31.0 mmol) was added dropwise over 10 min. The mixture was warmed to room temperature and stirred for 1 h, and then the reaction was quenched by the addition of saturated aqueous sodium bicarbonate (10 mL). Additional water was added to dissolve all the solids, and the phases were then separated. The aqueous phase was extracted with dichloromethane (3 × 15 mL). The organic extracts were combined, washed with 5% aqueous sodium citrate (3 × 15 mL) and then brine (1 × 25 mL), dried over MgSO4, filtered, and concentrated in vacuo. The resulting yellow oil was dissolved in ether (20 mL) and again washed with 5% aqueous sodium citrate (3 × 10 mL), followed by brine (1 × 10 mL). The organic phase was dried over MgSO4, filtered, and concentrated in vacuo to yield 5.27 g (100%) of 5 as a yellow oil that showed no impurities by NMR. This material was used without further purification: 1H NMR (500 MHz, CDCl3) δ 0.07 (18H, m, CH3), 1.10-1.50 (4H, m, CH2), 1.50-1.88 (4H, m, CH2), 4.86 (2H, t, CH), 6.86 (2H, m, aromatic), 6.92 (2H, m, aromatic), 7.19 (2H, dd, aromatic); 13C NMR (125 MHz, C6D6) δ 0.50, 26.17, 41.82, 71.65, 123.45, 124.44, 126.77, 150.83; MS (FAB+, Na) 425 (M-), 411, 371, 335, 287, 265, 237, 211, 185, 163, 147, 123. MS high resolution: calcd for C20H33O2Si2S2, 425.1461; found 425.1461. 1,6-(5,5′-Bis(hydroxymethyl)-2,2′-dithienyl)hexane-1,6diol (6). TMS ether 5 (5.27 g, 12.3 mmol) was dissolved in tetrahydrofuran (62 mL) and cooled to -78 °C. To this solution was added N,N,N′,N′-tetramethylethylenediamine (TMEDA, 4.47 mL, 29.6 mmol) that had been freshly distilled from sodium metal. To this solution was added n-butyllithium (2.5 M in hexanes, 10.9 mL, 27.2 mmol) dropwise over 20 min. The solution was stirred at -78 °C for 15 min and then slowly warmed to 0 °C over 45 min. The reaction was quenched by cracking paraformaldehyde (4.45 g, 148.2 mmol) and by passing the resultant gas through the mixture using dry argon as a carrier gas. Following the addition, the mixture was warmed to room temperature and stirred for 2 h. Saturated aqueous ammonium chloride (25 mL) was added to the mixture, and the phases were separated. The aqueous layer was extracted with ethyl acetate (3 × 15 mL), and the combined organics were washed with a 5% aqueous citric acid solution (3 × 15 mL) and brine (1 × 30 mL). The organic phase was dried over MgSO4, filtered, and concentrated in vacuo to a yellow oil. 1H NMR indicated the persistence of the TMS protecting group. The oil was dissolved in dry methanol (20 mL), and anhydrous potassium carbonate (1.5 g) was added. The resulting slurry was stirred at room temperature for 30 min when TLC indicated completion of the deprotection. The methanol was removed in vacuo, and the residue was dissolved in a mixture of ethyl acetate (20 mL) and saturated aqueous ammonium chloride (20 mL). The aqueous phase was brought to neutral pH by addition of additional ammonium chloride solution and was subsequently extracted with ethyl acetate (3 × 25 mL). The combined organic extracts were washed with brine (1 × 25 mL), dried over MgSO4, filtered, and concentrated in vacuo to a yellow oil. Flash chromatography on silica gel (1:1 EtOAc/hexane f 2:1 EtOAc/hexane f 100% ethyl acetate) yielded 2.32 g (55%) of 6 as a yellow oil: 1H NMR (acetone-d6) δ 1.29-1.58 (4H, m, CH2), 1.64-1.88 (4H, m, CH2), 4.31 (2H, t, CH2OH), 4.38 (2H, d, CHOH), 4.68 (4H, s, CH2OH), 4.81 (2H, t, CHOH), 6.76 (2H, d, aromatic), 6.78 (2H, d, aromatic); 13C NMR (125 MHz, acetone-d6) δ 26.68, 40.89, 60.42, 70.69, 123.56, 124.81, 145.44, 151.20. 1,6-(5,5′-Bis(1-hydroxyethyl)-2,2′-dithienyl)hexane-1,6diol (7). A solution of 5 (212 mg, 0.50 mmol) in tetrahydrofuran (44 mL) was stirred under dry nitrogen at -78 °C for 15 min. To the mixture was added dropwise n-butyllithium (2.5 M in hexanes, 1.07 mL, 2.68 mmol). The resulting mixture
was warmed to room temperature and stirred for 1 h. The mixture was again cooled to -78 °C and stirred for 10 min. Acetaldehyde (0.60 g, 0.013 mol) was added dropwise via syringe. The mixture was then warmed to 0 °C and stirred for 30 min. The reaction was quenched with saturated aqueous ammonium chloride solution (60 mL), and the mixture was stirred for 5 min. The aqueous layer was extracted with dichloromethane (3 × 75 mL). The combined organic layers were dried (MgSO4), filtered, and concentrated under reduced pressure to yield a brown oil. Flash chromatography with ethyl acetate yielded 7 as a colorless oil: 152 mg (82%); 1H NMR (acetone-d6) δ 1.10-1.43 (4H, m, CH2), 1.46 (6H, d, CH3), 1.601.93 (4H, m, CH2), 4.04 (2H, q, exterior CH), 4.13 (2H, t, interior CH), 4.38 (2H, br s, OH), 4.79 (2H, br s, OH), 6.73 (4H, m, aromatic). Hexadecane-1,6,11,16-tetraol (1). A solution of 6 (2.30 g, 6.72 mmol) in absolute ethanol (50 mL) was stirred at room temperature to afford a faintly yellow solution. Raney nickel activated catalyst (46.0 g, 20 wt equiv) was added, and the resulting slurry was heated to reflux for 2.5 h when TLC indicated consumption of the starting material. The mixture was cooled to room temperature and then filtered through a bed of Celite and washed thoroughly with 95% ethanol. The filtrate was concentrated by rotary evaporation, the resultant oily residue was then dissolved in absolute ethanol (20 mL), and the solvent was evaporated again. Residual ethanol and water in the gummy solid residue were azeotropically removed by evaporation from dry benzene (3 × 15 mL). 1 was recovered as a white solid (1.28 g, 66%, mp 230 °C, dec): 1H NMR (CD3OD) δ 1.18-1.67 (24H, m, CH2), 1.78 (2H, br s, OH), 1.90 (2H, br s, OH), 2.48 (2H, m, CH), 3.54 (4H, t, CH2OH); 13C NMR (125 MHz, CD3OD) δ 26.76, 27.03, 27.15, 33.77, 38.26, 38.54, 63.08, 72.44; IR (KBr) 3322 (OH), 2926 (CH), 1579, 1421, 1062, 850, 652 cm-1; MS (EI+) 290 (M), 289, 271, 261, 253, 193, 165, 163, 149, 137, 127, 111; MS (FAB+, Na) 313 (M + Na), 291 (M + 1), 283, 261, 225, 207, 185, 137, 114, 93. MS high resolution (EI+): calcd for C16H35O4, 291.2535; found 291.2566. High resolution (ES+): calcd for C16H34O4Na, 313.2355; found 313.2352. For HPLC visualization, 1 was converted to the corresponding tetrabenzoyl ester derivative with benzoyl chloride (triethylamine, 4-(dimethylamino)pyridine, dichloromethane). This derivative exhibited a single peak on three different analytical HPLC columns (forward-phase Zorbax Sil, forward-phase Zorbax RX-Sil, and reverse-phase Deltapak C18) with visualization at 254 nm and using two to four different solvent systems. Octadecane-2,7,12,17-tetraol (2). A solution of 7 (152 mg, 0.41 mmol) in ethanol (75 mL) was stirred at room temperature for 10 min. To the solution was added Raney nickel activated catalyst (3.0 g, 20 wt equiv), and the resulting mixture was refluxed for 4 h. The resulting mixture was filtered through a bed of Celite and the filtrate was concentrated in vacuo to give 53 mg (41% yield) of 2 as a white solid (mixture of diastereomers): 1H NMR (500 MHz, pyridine-d5) δ 1.36 (6H, d, CH3), 1.44-1.90 (24H, m, CH2), 3.84 (2H, m, CH), 4.00 (2H, m, CH), 5.70 (4H, d, OH); 13C NMR (125 MHz, pyridine-d5) δ 24.9, 26.4, 27.2, 38,7, 39.1, 40.8, 44.1, 67.5, 71.3; IR (KBr) 3329 (OH), 2925 (CH), 1710, 1462, 1070, 840, 654 cm-1; MS (EI+) 319 (M+), 299, 281, 263, 247, 199, 181, 163, 149, 123, 109, 95, 81, 69. MS high resolution (EI+): calcd for C18H39O4, 319.2848; found 319.2848. For HPLC visualization, 2 was converted to the corresponding tetrabenzoyl ester derivative with benzoyl chloride (triethylamine, 4-(dimethylamino)pyridine, dichloromethane). This derivative exhibited a single peak on three different analytical HPLC columns (forward-phase Zorbax Sil, forward-phase Zorbax RX-Sil, and reverse-phase Deltapak C18) with visualization at 254 nm and using two to four different solvent systems. 2. Immobilization Potency. Polyalcohols were initially dissolved directly in the tadpole pond water in 20 mL glass vials. Vials were vigorously shaken, vortexed, and then sonicated (bath-type sonicator) over a period of about 5 min. The
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Figure 1. Change in 1.0 mM n-hexanol immobility effects by each of the two compounds at 0.2 mM. Two groups of 10 tadpoles were scored for each compound. In both cases, P < 0.05 compared to 1-hexanol with ANOVA and post hoc Schefe. solution/suspension was then passed through a 0.22 µm cameo syringe filter to remove undissolved particles. Pond water in the Petri dish was replaced with 10 mL of these pond water alcohol solutions, and tadpole responses were assessed periodically (see below). Prelimb Xenopus larvae were ordered through NASCO (Fort Atkinson, WI). Upon arrival, tadpoles were acclimated in the facility overnight and then divided into groups of 10 into 3 in. sterile Petri dishes with 15 mL of the original pond water. To determine alcohol potency, we used a standard approach that looks for movement in response to a provocation. Thus, after 5, 10, 20, and 30 min of exposure, the tadpoles were touched gently on the eye spot with a blunt probe and also prodded against their side with the same probe. (Thirty minute exposures appeared to be adequate to produce stable responses to these compounds, and therefore, the system was considered equilibrated.) Any movement within 10 s after the provocation was scored as a response. The percentage of responses in each group of 10 was recorded. After 30 min, tadpoles were washed twice with fresh pond water and then allowed to recover in 15 mL of pond water. Tadpole recovery was monitored at 5, 10, 20, 40, and 60 min and overnight. It was possible to achieve ∼1 mM solutions of 1 and 2 in pond water, which produced a loss of response in 40% and 30% of tadpoles, respectively. For the additivity studies, independent experiments showed that 1-hexanol had an EC50 for our endpoint in tadpoles of ∼0.7 mM. In combination with the polyhydric alcohol at a reduced concentration of 0.2 mM, a hexanol concentration of 1 mM was chosen to sensitively assess effects of the compounds in either direction. Figure 1 shows that 1 and 2 produced a significant additional immobilizing effect, with 1 having a somewhat greater effect than 2 at this aqueous concentration (0.2 mM). The recovery times mimicked these data in that tadpoles exposed to hexanol and 2 required 20 min for full recovery and those exposed to hexanol plus 1 required 30 min.
Discussion The n-alkanol cutoff for tadpole immobilization has been reported to be approximately dodecanol.11 Thus, the measurement of significant anesthetic potency of 1 and 2, whose van der Waals volumes exceed that of dodecanol by roughly 110 and 150 Å3, respectively, provides strong evidence against the idea that the potency cutoff observed for other homologous series, such as n-alkanols, arises from a steric limitation dictated by the volume of a putative binding pocket on a protein. This suggests that for whichever proteins are involved in anesthesia arising from these polyhydric alkanols, either (a) protein activity is modulated indirectly (mediated by changes in membrane properties) or (b) the mechanism involves direct binding to a protein site, but cutoff does not result from steric hindrance (and would thus provide no information about the size of the
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binding pocket). However, it is not possible to distinguish between these two types of mechanisms in the absence of any direct measurement of binding of these compounds to a putative protein target. The low aqueous solubility of these and similar compounds makes an accurate determination of potency significantly more difficult. The additivity design12 commonly used to address this problem in studies of anesthetic potency (amdin studies of nonspecific aquatic toxicity) relies on the assumption that even if different compounds act via different mechanisms, they nonetheless contribute additively to a given phenotypic endpoint. Although the overwhelming majority of anesthetic compounds do exhibit such additivity, there is always the possibility that for an otherwise untested class of compounds, such as long-chain polyhydric alcohols, it may not hold. Particularly for larger compounds for which the kinetics may be quite slow, it is important to ensure that measurements are taken only after the system has equilibrated. In the present work, the only evidence of equilibration was a stable behavioral response over at least a 10 min period. Also, particularly for the more hydrophobic compounds, it is possible that the equilibrated aqueous concentration is considerably lower than the starting concentration because of partitioning into the tadpole tissues. If these concerns are validated, however, the direction would further strengthen, not weaken, our conclusions. Nevertheless, future work will involve radiolabeling of compounds to determine concentration in the tadpole tissues responsible for the endpoint behavior. Such labeling will have another important consequence. As discussed above,5 if anesthesia arises from a redistribution of lateral stresses in the bilayer, the EC50 molar concentration of the polyol in the membrane will be significantly lower than that of the “equivalent” short-chain alkanol, by roughly a factor of 6 for the two molecules studied here. Labeling will also enable accurate determination of bilayer/ aqueous partition coefficients, which, in conjunction with aqueous EC50 values, will provide an additional test of this mechanism. References (1) Janoff, A. S.; Miller, K. W. A Critical Assessment of the Lipid Theories of General Anaesthetic Action. In Biological Membranes; Chapman, D., Ed.; Academic Press: London, 1982; Vol. 4, pp 417-476. (2) Wick, M. J.; Mihic, S. J.; Ueno, S.; Mascia, M. P.; Trudell, J. R.; Brozowski, S. J.; Ye, Q.; Harrison, N. L.; Harris, R. A. Mutations of γ-aminobutyric acid and glycine receptors change alcohol cutoff: Evidence for an alcohol receptor? Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6504-6509. (3) Jenkins, A.; Greenblatt, E. P.; Faulkner, H. J.; Bertaccini, E.; Light, A.; Lin, A.; Andreasen, A.; Viner, A.; Trudell, J. R.; Harrison, N. L. Evidence for a common binding cavity for three general anesthetics within the GABAA receptor. J. Neurosci. 2001, 21, 1-4. (4) Eckenhoff, R. G.; Tanner, J. W.; Johansson, J. S. Steric hindrance is not required for n-alkanol cutoff in soluble proteins. Mol. Pharmacol. 1999, 56, 414-418. (5) Cantor, R. S. Breaking the Meyer-Overton rule: Predicted effects of varying stiffness and interfacial activity on the intrinsic potency of anesthetics. Biophys. J. 2001, 80, 2284-2297. (6) Peoples, R. W.; Ren, H. Inhibition of N-methyl-D-aspartate receptors by straight-chain diols: Implications for the mechanism of the alcohol cutoff effect. Mol. Pharmacol. 2002, 61, 169176. (7) Meyers, A. I. Heterocycles in Organic Synthesis; John Wiley: New York, 1974; pp 15-20, 228-231.
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(8) Krishna, P. R.; Lavanya, B.; Ilangovan, A.; Sharma, G. V. M. Stereoselective synthesis of C-alkyl and functionalised C-alkyl glycosides using “thiophene” as a masked C-4 synthon. Tetrahedron: Asymmetry 2000, 11, 4463-4472. (9) Yang, S.-M.; Nandy, S. K.; Selvakumar, A. R.; Fang, J.-M. Distant functionalization via incorporation of thiophene moieties in electrophilic reactions promoted by samarium diiodide. Org. Lett. 2000, 2, 3719-3721. (10) Vaitiekunas, A.; Nord, F. F. Studies on the chemistry of heterocyclics. XXV. Investigations on diacetylenes and diacetyl-
Brief Articles enic glycols in the thiophene series. J. Am. Chem. Soc. 1954, 76, 2733-2736. (11) Alifimoff, J. K.; Firestone, L. L.; Miller, K. W. Anaesthetic potencies of primary alkanols: implications for the molecular dimensions of the anaesthetic site. Br. J. Pharmacol. 1989, 96, 9-16. (12) Cole, D.; Kalichman, M.; Shapiro, H.; Eger, E. I., II. Does 1 + 1 ) 2? A continuing debate. Anesth. Analg. 1990, 70, 126-127.
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