Anal. Chem. 2003, 75, 7036-7039
Partition Coefficients of Ionizable Compounds in o-Nitrophenyl Octyl Ether/Water Measured by the Potentiometric Method Xiangli Liu,† Ge´raldine Bouchard,† Hubert H. Girault,‡ Bernard Testa,† and Pierre-Alain Carrupt*,†
Institut de Chimie The´ rapeutique, Section de Pharmacie, Universite´ de Lausanne, BEP, CH-1015 Lausanne, Switzerland, and Laboratoire d’Electrochimie Physique et Analytique, EÄ cole Polytechnique Fe´ de´ rale de Lausanne, CH-1015 Lausanne, Switzerland
The objective of this study was to investigate the reliability of potentiometric measurements of partition coefficients (log P) in the o-NPOE/water system, o-nitrophenyl octyl ether being an organic solvent widely used for the voltammetric determination of the partition coefficient of ions. Three sets of ionizable model compounds were explored in this study. The results showed that the potentiometric technique gave precise and reliable log Pnpoe values in the explored range of 0.1-4.3. Lipophilicity, traditionally expressed by the partition coefficient of solutes in the 1-octanol/water system (noted log Poct), is a key parameter correlated with the pharmacokinetic behavior of drugs, as exemplified by numerous relationships established between log Poct and intestinal absorption or blood-brain barrier permeation.1 However, this parameter sometimes fails to give a good estimate of the absorption or permeation of drugs, and other solvent systems are needed to obtain additional information on intermolecular forces. Indeed, four classes of solvent systems were necessary to adequately model the partitioning of solutes into membranes,2,3 namely, (1) an amphiprotic solvent such as 1-octanol, (2) a hydrogen bond acceptor solvent such as di-n-butyl ether, (3) a hydrogen bond donor solvent such as chloroform, and (4) an aprotic inert solvent such as n-alkane. The benefits of derived lipophilicity parameters such as ∆log Poct-alk (log Poct/water - log Pn-alkane/water) to model blood-brain barrier permeation has also been demonstrated.4 For the study of the partitioning of ions, electrochemistry at the interface between two immiscible electrolyte solutions is the choice method. Most experimental work in this field has used nitrobenzene/water and 1,2-dichloroethane (DCE)/water interfaces. However, the polarizable interface can also be formed with * To whom correspondence should be addressed. Fax: +41 (0) 21 692 45 25. E-mail:
[email protected]. † Universite´ de Lausanne. ‡ EÄ cole Polytechnique Fe´de´rale de Lausanne. (1) Testa, B.; Caron, G.; Crivori, P.; Rey, S.; Reist, M.; Carrupt, P. A. Chimia 2000, 54, 672-677. (2) Leahy, D. E.; Morris, J. J.; Taylor, P. J.; Wait, A. R. J. Chem. Soc., Perkin Trans. 2 1992, 723-731. (3) Leahy, D. E.; Morris, J. J.; Taylor, P. J.; Wait, A. R. J. Chem. Soc., Perkin Trans. 2 1992, 705-722. (4) Caron, G.; Reymond, F.; Carrupt, P. A.; Girault, H. H.; Testa, B. Pharm. Sci. Technol. Today 1999, 2, 327-335.
7036 Analytical Chemistry, Vol. 75, No. 24, December 15, 2003
Table 1. Comparison of Some Physicochemical Properties of o-Nitrophenyl Octyl Ether (o-NPOE) and 1,2-Dichloroethane (DCE) at 298 Ka properties
o-NPOE
DCE
molar mass (g‚mol-1) density (g‚cm-3) molar volume (cm3‚mol-1) viscosity (10-3 Pa‚s) solubility of the solvent in water (mol‚L-1) solubility of water in the solvent (mol‚L-1) effective solvent radius (nm) relative permittivity
251.33 1.041b 241.4 13.800 2.01 × 10-6
98.96 1.246 79.4 0.779 8.50 × 10-2
4.06 × 10-2
0.11
0.368 24.20
0.254 10.36
a
Taken from ref 12. b At 293 K.
other organic solvents such as chloroform, acetophenone, nitroethane, benzonitrile, o-nitrotoluene, or o-nitrophenyl octyl ether (o-NPOE).5 More recently, a new electrochemical methodology has allowed study of the transfer of ions across nonpolarizable interfaces as in the 1-octanol/water system.6 This methodology has been successfully applied to measure, for the first time, the standard Gibbs energies of transfer of many inorganic and organic ions at the 1-octanol/water interface.7,8 The replacement of an alkane by DCE/water has experimental benefits in structure-pharmacokinetic relationships,9 as illustrated by the interest of log Pdce or ∆log Poct-dce (log Poct/water log Pdce/water) in the prediction of skin permeation.10 However, the high volatility and the toxicity of DCE limits its value and calls for its replacement by a more appropriate organic solvent. Given its promising physicochemical properties (see Table 1) and (5) Samec, Z.; Trojanek, A.; Samcova, E. J. Electroanal. Chem. 1995, 386, 225228. (6) Komorsky-Lovric, S.; Riedl, K.; Gulaboski, R.; Mirceski, V.; Scholz, F. Langmuir 2002, 18, 8000-8005. (7) Gulaboski, R.; Mirceski, V.; Scholz, F. Electrochem. Commun. 2002, 4, 277283. (8) Bouchard, G.; Galland, A.; Carrupt, P. A.; Gulaboski, R.; Mirceski, V.; Scholz, F.; Girault, H. H. J. Phys. Chem. Chem. Phys. 2003, 5, 3748-3751. (9) Steyaert, G.; Lisa, G.; Gaillard, P.; Boss, G.; Reymond, F.; Girault, H. H.; Carrupt, P. A.; Testa, B. J. Chem. Soc., Faraday Trans. 1997, 93, 401406. (10) Geinoz, S.; Rey, S.; Boss, G.; Bunge, A. L.; Guy, R. H.; Carrupt, P. A.; Reist, M.; Testa, B. Pharm. Res. 2002, 19, 1622-1629. 10.1021/ac034972b CCC: $25.00
© 2003 American Chemical Society Published on Web 11/13/2003
Table 2. log Pnpoe Values Obtained by Potentiometry and the Shake-Flask Method solutes
log Pnpoe (shake-flask)a
log Pnpoe (potentiometry)b
C6H5CH2COOH C6H5(CH2)3COOH C6H5OH pyridine 4-NO2C6H4OH C6H5NH2 C6H5NHC2H5 3-ClC6H4OH 4,4′-(NH2)2-biphenyl
0.12 1.20 0.58 0.26 0.94 1.08 2.36 1.48 1.81
0.10 1.28 0.44 0.20 1.01 1.00 2.24 1.38 1.77
a
Table 3. log Pnpoe Values Obtained by Potentiometry for Two Homologous Sets of Compounds
n ) 3; SD e 0.03. b n ) 2 or 3; SD e 0.05.
absence of known toxicity, o-NPOE has recently been introduced in electrochemistry.11-15 Moreover, due to its medium permittivity, high viscosity, low solubility in water, and low vapor pressure, o-NPOE was identified as a suitable plasticizer for liquid membrane ion-selective electrodes,16,17 and its use in supported liquid membranes appears promising.18,19 Recently, the intermolecular forces expressed in o-NPOE/water partitioning were proven similar to the forces encoded in log Pdce/water, suggesting log Pnpoe to offer a convenient alternative to log Pdce.20 In this technical note, the reliability of log P measurements in the o-NPOE/water system was established using the potentiometric method, an experimental technique widely used in medicinal chemistry to measure partition coefficients and lipophilicity profiles of ionizable compounds in both isotropic and anisotropic partition systems such as 1-octanol/water, 1,2-dichloroethane/ water, di-n-butyl ether/water and liposomes/water.9,21 For this purpose, a set of structurally diverse ionizable acids and bases with log Poct values comprised between 0.6 and 5.1 (Tables 2 and 3) was selected. Their lipophilicity in the o-NPOE/water system was determined by both the classical shake-flask and the potentiometric method. MATERIALS AND METHODS Solutes and Reagents. The (p-methylbenzyl)alkylamines were synthesized by known procedures22 and kindly offered by the research group of Professor R. Fruttero (Department of Pharmaceutical Sciences and Technology, University of Turin, Italy). (11) Valent, O.; Koryta, J.; Panoch, M. J. Electroanal. Chem. Interfacial Electrochem. 1987, 226, 21-25. (12) Samec, Z.; Langmaier, J.; Trojanek, A. J. Electroanal. Chem. 1996, 409, 1-7. (13) Samec, Z.; Langmaier, J.; Trojanek, A. J. Electroanal. Chem. 1997, 426, 37-45. (14) Samec, Z.; Langmaier, J.; Trojanek, A. J. Electroanal. Chem. 1999, 463, 232-241. (15) Wilke, S.; Zerihun, T. J. Electroanal. Chem. 2001, 515, 52-60. (16) Langmaier, J.; Stejskalova, K.; Samec, Z. J. Electroanal. Chem. 2002, 521, 81-86. (17) Langmaier, J.; Stejskalova, K.; Samec, Z. J. Electroanal. Chem. 2001, 496, 143-147. (18) Ulmeanu, S.; Jensen, H.; Samec, Z.; Bouchard, G.; Carrupt, P. A.; Girault, H. H. J. Electroanal. Chem. 2002, 530, 10-15. (19) Qin, Y.; Mi, Y.; Bakker, E. Anal. Chim. Acta 2000, 421, 207-220. (20) Liu, X.; Bouchard, G.; Mu ¨ ller, N.; Galland, A.; Girault, H. H.; Testa, B.; Carrupt, P. A. Helv. Chim. Acta, in press. (21) O’Dowd, B. F. J. Neurochem. 1993, 60, 804-816. (22) Meindl, W. R.; von Angerer, E.; Scho ¨nenberger, H.; Ruckdeschel, G. J. Med. Chem. 1984, 27, 1111-1118.
a
solutes
log Pnpoe (potentiometry)a
1a 4-CH3C6H4CH2NHCH3 1b 4-CH3C6H4CH2NHCH2CH3 1c 4-CH3C6H4CH2NH(CH2)2CH3 1d 4-CH3C6H4CH2NH(CH2)3CH3 1e 4-CH3C6H4CH2NH(CH2)4CH3 1f 4-CH3C6H4CH2NH(CH2)5CH3 1g 4-CH3C6H4CH2NH(CH2)6CH3 2a C6H5CH2COOH 2b C6H5(CH2)2COOH 2c C6H5(CH2)3COOH 2d C6H5(CH2)4COOH 2e C6H5(CH2)6COOH 2f C6H5(CH2)7COOH
1.48 1.86 2.36 2.90 3.48 4.07 4.29 0.10 0.51 1.28 1.77 2.52 2.97
n ) 2 or 3; SD e 0.05.
Figure 1. Correlation between log Pnpoe obtained by potentiometry and the shake-flask method.
All other compounds were obtained from commercial sources (Merck, Darmstadt, Germany; Fluka, Buchs, Switzerland; Janssen, Beerse, Belgium; Aldrich, Steinheim, Germany; Sigma Chemie, Buchs, Switzerland; Lancaster Synthesis, Morecambe, England; ICN) and in the highest available purity. Analytical grade onitrophenyl octyl ether was purchased from Fluka. Distilled water was used throughout. No evidence was found from the literature about any toxicity of o-NPOE. However, all precaution was taken to avoid skin contact by using rubber gloves since skin permeation is possible and might conceivably lead to nitro-reduced metabolites. Potentiometric Titration. The partition coefficients in oNPOE/water were determined with a GLpKa apparatus (Sirius Analytical Instruments, Forest Row, East Sussex, U.K.) for some acidic and basic compounds shown in Figure 1. The o-NPOE phase was presaturated with 0.15 M KCl and added manually to the titration vial. At least three titrations of 0.5-1 mmol‚L-1 for each compound, containing various volumes of o-NPOE, were performed in the pH range 2-11.5. All titrations were conducted at 25 ( 0.5 °C and under a slow argon flow to avoid CO2 absorption. The log Pnpoe values were estimated from difference Bjerrum plots and refined by a nonlinear least-squares procedure by including previous determined pKa values as unrefined contributions. The detailed experimental procedures of the potentiometric method can be found elsewhere.23 The volume ratio of the o-NPOE phase to the aqueous phase was between 0.005 and 0.5. A higher ratio was not possible Analytical Chemistry, Vol. 75, No. 24, December 15, 2003
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because of the emulsification of the mixture under stirring. Opposite to the 1-octanol/water emulsions previously described,24 the o-NPOE/water emulsions were not sufficiently mobile and freeflowing to allow the electrode to work well under those conditions. Since o-NPOE is denser than water, the electrode should be adjusted to rest in the water phase. Since o-NPOE is not mentioned in the pKaLOGP software, the option, water-insoluble partition solvent, should be chosen. Shake-Flask Method. The partition coefficients in o-NPOE/ water were measured by the shake-flask method for the nine compounds shown in Table 2. A 0.02 M phosphate buffer containing 0.15 M KCl was used. The phosphate buffer was adjusted to a pH giving a large excess of the neutral form. Both o-NPOE and phosphate buffer were mutually saturated by stirring the two phases vigorously for 6 h. After phase separation in a separation funnel, the two phases were collected and centrifuged at 2500 rpm for 20 min (MSE Mistral 2000 centrifuge) in order to remove the last drops of the other phase. Drug solutions (0.1-1 mM) in o-NPOE-saturated phosphate buffer were prepared. Three volume ratios of drug solution to buffer-saturated o-NPOE were used. After gentle shaking overnight, the two phases were separated by centrifugation at 4000 rpm for 20 min. Sample concentrations were determined in both phases. The concentration measurements were performed by HPLC using a liquid chromatograph Waters 2690 separation module (Waters, Milford, MA) equipped with a Waters 2487 dualwavelength absorbance detector set at the maximum absorption wavelength (λmax) of each compound. The column was a Symmetry C8 3.5 µm (3.0 × 150 mm, Waters). The mobile phase was the mixture of 0.02 M phosphate buffer (adjusted to a pH where the compound was neutral) and acetonitrile. The phosphate buffer was filtered through 0.45-µm HA Millipore filters (Millipore). RESULTS AND DISCUSSION The potentiometric method gave good experimental precision in log Pnpoe values (SD e 0.05) for all the compounds tested (0.1 e log Pnpoe e 4.3), implying that the electrode worked well despite the high viscosity of o-NPOE. No obvious carbon dioxide absorption was observed in this solvent during the titrations. Even if the electrode does not work well below pH 2.5 and above pH 11.5, the lower volatility of o-NPOE allows the use of a much smaller volume (0.1 mL) with respect of DCE (at least 1 mL), and thus, reliable log P measurements for lipophilic bases with low pKa values (near 3) and lipophilic acids with high pKa values (near 11) can be obtained. Diazepam is a good example of this situation: with a phase ratio of 0.1 mL of o-NPOE/19 mL of 0.15 M KCl aqueous solution, the pKa (3.45) shifted to poKa (2.76), still allowing a reliable determination of its log Pnpoe value (2.96). When the ratio of o-NPOE to 0.15 M KCl was higher than 0.5, the electrode could not function properly due to emulsification. This makes the measurement of log Pnpoe impossible by potentiometry for hydrophilic compounds whose log Pnpoe values are negative. For compounds whose log Pnpoe values fall between 0 (23) Avdeef, A. In Lipophilicity in Drug Action and Toxicology; Pliska, V., Testa, B., van de Waterbeemd, H., Eds.; VCH Publishers: Weinheim, 1996; pp 109-139. (24) Pagliara, A.; Testa, B.; Carrupt, P. A.; Jolliet, P.; Morin, C.; Morin, D.; Urien, S.; Tillement, J. P.; Rihoux, J. P. J. Med. Chem. 1998, 41, 853-863.
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Figure 2. Correlation between log Pnpoe and NCH2.
and 0.5, a longer equilibrium time of solutes in the two phases should be allowed to avoid the effect of emulsification. A comparison was made between the log Pnpoe values obtained by the potentiometric method and the shake-flask method for the nine compounds in Table 2. The slope and intercept of the linear regression (see eq 1) are close to the ideal values of 1 and 0, indicating that there is no significant deviation between the data obtained by the two methods (Figure 1).
log Pnpoe(P) ) 0.95((0.10) log Pnpoe(SF) + 0.05((0.13) (1) n ) 9;
r2 ) 0.99;
s ) 0.09;
F ) 507
In this equation, 95% confidence limits are in parentheses, n is the number of compounds, r2 the squared correlation coefficient, s the standard deviation, and F Fisher’s test. The reliability of the log Pnpoe values by potentiometric titration is thus validated for the compounds whose log Pnpoe lies in the range 0.10-2.24 (see Table 2). Due to the narrow range of log P measurable by the shake-flask method, it was difficult to extend the validation for log Pnpoe > 2.5. Thus, the value’s reliability of log Pnpoe by potentiometric titration was verified beyond this limit by using two homologous sets of compounds, namely, alkylamines 1a1g and carboxylic acids 2a-2f (Table 3). Due to the additivity of hydrophobic interactions observed in log P measurements and, in particular, in the 1-octanol/water system,25 log Pnpoe should increase linearly with the number of methylene groups (noted NCH2) in each of these two sets. The linear relationships between log Pnpoe and NCH2 reported in eqs 2 and 3 and in Figure 2 confirm the quality of the potentiometric approach. For alkylamines 1a-1g
log Pnpoe(P) ) 0.50((0.05)NCH2 + 1.42((0.18) n ) 7;
r2 ) 0.99;
s ) 0.10;
(2)
F ) 643
For carboxylic acids 2a-2f
log Pnpoe(P) ) 0.48((0.08)NCH2 - 0.31((0.33) n ) 6;
r2 ) 0.99;
s ) 0.14;
(3)
F ) 304
(25) Caron, G.; Ermondi, G.; Boschi, D.; Carrupt, P. A.; Fruttero, R.; Testa, B.; Gasco, A. Helv. Chim. Acta 1999, 82, 1630-1639.
It should be noted that the slopes of the two equations are quite similar and represent the fragmental log Pnpoe increment of the CH2 group (0.49). It was thus verified that the potentiometric method can give a reliable log Pnpoe values from 0.1 to 4.3 (Table 3). CONCLUSION The results of this study show that the potentiometric method can provide reliable and accurate log Pnpoe values within the investigated range. A comparison of the log Pnpoe values of nine compounds determined by potentiometry showed a good agreement with the shake-flask method. The reliability of the poten-
tiometric method could also be validated by the good correlations between log Pnpoe values and the number of CH2 for two homologous sets of compounds. ACKNOWLEDGMENT P.-A.C. and H.H.G. are indebted to the Swiss National Science Foundation for support.
Received for review August 20, 2003. Accepted October 10, 2003. AC034972B
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