In the Laboratory
Determination of Lipophilicity Constants of Sulfonamide Substituents
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Maria Auxiliadôra Fontes Prado Departamento de Produtos Farmacêuticos, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 30180-112, Brazil;
[email protected] Background
Experimental Procedure
The partition coefficient (P) is one of the most important factors controlling drug action in biological systems (1). Hansch and coworkers (2) derived constants for the contribution of individual atoms or groups to the partition coefficient, the lipophilicity (or hydrophobicity) constant (π) (1, 2). This is defined as log PRX – log PRH (eq 1), where PRX and PRH are the partition coefficients, determined in the system 1-octanol/ water, of substituted and unsubstituted compounds, respectively (1–5). P πx = log RX (1) P RH
Glass plates measuring 9 × 4.5 cm are coated with silica gel G in the usual manner (for a dozen 0.25-mm-thick plates prepare a suspension of 6 g of silica gel G and 15 mL of water, shake, coat the plates, dry at room temperature, heat in an oven at 120 °C for 30 min, and cool to room temperature). A stationary nonpolar phase is obtained by impregnating the silica gel G layer with a 5% 1-octanol solution in ethyl ether. The impregnation is carried out by instantaneous and careful immersion to avoid damage to the thin layer. The silica gel layer is removed to 3 mm from each lateral edge and 5 mm from the higher edge. The mobile phase is an aqueous buffer at pH 7.4 (mixture of 50 mL of 0.1 mol/L potassium dihydrogen phosphate solution and 39.5 mL of 0.1 mol/L sodium hydroxide solution). The chromatographic chamber (diameter, 8 cm; length, 11 cm) is saturated with the mobile-phase vapor (25 mL). The sulfonamides (sulfanilamide, sulfathiazole, sulfamethazine, and sulfamethoxypyridazine) are dissolved in acetone (10 mg in 2.5 mL). The solutions of sulfonamides are spotted on a line about 1 cm from the lower edge of the plate using a glass capillary. The plate is left in the chromatographic chamber until the mobile phase moves approximately 8 cm away from the origin. The developed plate is dried with hot air and the sulfonamides are visualized with iodine vapor or by spraying with a 99:1 v/v solution of p-dimethylaminobenzaldehyde (0.1% w/v in ethanol) and concentrated hydrochloric acid.
Thus the π constant indicates the change in the logarithm of the partition coefficient resulting from the introduction of a substituent group X (4 ). The π values for many substituent groups were directly measured for a variety of drugs and used to calculate the partition coefficient of other compounds (4). However, the direct determination of partition coefficient is tedious and often presents practical difficulties, particularly when the compound is highly insoluble in either of the solvent phases. To avoid these difficulties, Boyce and Milborrow proposed the chromatographic Rm value as an expression of the lipophilic character of molecules (5). R m is calculated using the R f value determined by means of reversed-phase thin-layer chromatography. Equation 2 is used to calculate the R m value for neutral substances (3–5) and eq 3 for acids and bases (3):
R m = log 1 – 1 Rf
(2)
K + H+ R m = log 1 – 1 + log a + Rf H
(3)
where Ka is the dissociation constant of the sulfonamide and [H+] is the hydrogen ion concentration of the mobile phase in mol/L. The change in Rm (∆Rm) for substituted and unsubstituted compounds has the same meaning as π (eq 4) (3–5). πx = R mRX – R mRH
(4)
In this article is described a simple laboratory activity: the reversed-phase thin-layer chromatography of the sulfonamide antibacterial drugs. It offers an opportunity to demonstrate the differences between the straight and reversed-phase thinlayer chromatography and introduces the chromatographic parameter R m. By means of this experiment it is also possible to show how chromatography can be used to determine a very important physicochemical parameter, the partition coefficient, which is often used in quantitative studies of structure–activity relationships of drugs.
Hazards Prolonged inhalation of silica gel dust can cause fibrosis of the lung. Wear a mask when preparing the silica gel suspension and coating the glass plates. Sodium hydroxide is corrosive; avoid skin contact. Ethyl ether is flammable. Spraying with p-dimethylaminobenzaldehyde solution must be done in an efficient fume cupboard. Calculations 1. Calculate the R f value of each sulfonamide. 2. Calculate the R m value of each sulfonamide from eq 3. 3. Calculate the lipophilicity substituent constant (π) of the substituent groups of sulfathiazole, sulfamethazine, and sulfamethoxypyridazine from eq 4. 4. The partition coefficient of sulfanilamide is 0.15 (6 ). Calculate the partition coefficients of sulfathiazole, sulfamethazine, and sulfamethoxypyridazine from eq 1.
JChemEd.chem.wisc.edu • Vol. 78 No. 4 April 2001 • Journal of Chemical Education
533
In the Laboratory
Results
Table 1. Student Values for Sulfonamides R
π
R
f m After the experiment and calculations, Compound H L MC H L MC H L MC class data are gathered and discussed. The ᎑0.51 ᎑0.28 ᎑0.42 Sulfanilamide 0.76 0.66 0.73 — — — highest, lowest, and most common R f, R m, and Sulfamethazine 0.44 0.30 0.38 0.75 0.29 0.39 0.95 0.69 0.84 π values found by nearly 300 students are presented in Table 1. R m and π values from Sulfametoxypyridazine 0.47 0.36 0.41 0.76 0.57 0.57 1.17 0.99 1.11 the literature are presented in Table 2. Sulfathiazole 0.63 0.51 0.54 0.46 0.25 0.33 0.84 0.65 0.76 Although the student data exhibit variations, Note: H is the highest, L the lowest, MC the most common value. the sequence of values for the four sulfonamides in each experiment does not change and is in agreement with the literature. The π values are analyzed to Table 2. Literature Values for Sulfonamides (3) demonstrate the extent of the substituent effect on the H2N SO2NHR lipophilicity/hydrophobicity ratio (P) of the substances. The ratio increases as the π value increases.
Compound
Conclusion This experiment is very simple to carry out. It is suitable to be performed by classes of up to 20 students and is, on average, completed in three hours, including the calculations and discussion, if the plates coated with silica gel and the solutions were previously prepared. In addition, the experiment requires only common reagents (except the sulfonamides) and glassware. The mass of sulfonamide necessary is very little and can be obtained from any pharmaceutical laboratory.
Sulfanilamide
R Group H
pKa
Rm
π
10.45
᎑0.48
—
7.7
0.49
0.76
7.05
0.64
1.02
7.1
0.43
0.82
CH3 N
Sulfamethazine N CH3
Sulfametoxypyridazine
OCH3
S
Sulfathiazole N
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Supplemental Material
The written material provided to students is available in this issue of JCE Online. Literature Cited 1. Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action; Academic: San Diego, 1992; pp 26–30. 2. Hansch, C.; Maloney, P. P.; Fujita, T. Nature 1962, 194, 178–180.
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3. Biagi, G. L.; Barbaro, A. M.; Guerra, M. C.; Forti, G. C.; Fracasso, M. E. J. Med. Chem. 1974, 17, 28–33. 4. Biagi, G. L.; Barbaro, A. M.; Gamba, M. F.; Guerra, M. C. J. Chromatogr. 1969, 41, 371–379. 5. Boyce, C. B. C.; Milborrow, B. V. Nature 1965, 208, 537–539. 6. Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71, 525– 615.
Journal of Chemical Education • Vol. 78 No. 4 April 2001 • JChemEd.chem.wisc.edu
