Laboratory Experiment pubs.acs.org/jchemeduc
Determination of log Kow Values for Four Drugs Mark F. Harris* and Jennifer L. Logan Department of Chemistry, Washington & Jefferson College, Washington, Pennsylvania 15301, United States S Supporting Information *
ABSTRACT: Though many undergraduates are interested in medicine, relatively few experiments related to drug design and development are included in introductory chemistry laboratory courses. In this experiment, aqueous solutions of four different drugs (acetaminophen, caffeine, phenacetin, and sulfanilamide) are extracted using 1-octanol, a mimic of the amphiphilic lipids that constitute cell membranes. UV analysis of the aqueous solutions before and after extraction allows determination of each compound’s log Kow value (partition coefficient), a property that influences a drug’s bioavailability. The concepts of equilibrium and hydrophobicity−hydrophilicity are emphasized, and students gain experience with micropipets in making dilutions. The lab has been implemented in a second-year undergraduate analytical chemistry course but may be suitable for organic, general, or medicinal chemistry depending on an institution’s particular curriculum. KEYWORDS: Second-Year Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Drugs/Pharmaceuticals, Equilibrium, Medicinal Chemistry, UV−Vis Spectroscopy
T
he ability of a small organic molecule to function as a medicine depends on its pharmacodynamics, the way in which the molecule interacts with a biological target, such as an enzyme or receptor, and elicits a biological response. Pharmacodynamics is sometimes described as “what the drug does to the body”, and it is this feature that is often emphasized if pharmaceuticals are discussed in undergraduate chemistry and biochemistry courses. However, for a molecule to be successful as a medicine, it must also have favorable pharmacokinetics. This branch of pharmacology addresses “what the body does to the drug”. It includes the molecule’s absorption, distribution, metabolism, and excretion, known as ADME. Particularly for an orally administered medicine, the hurdles a molecule must overcome to reach its ultimate target molecule at an effective concentration can be quite significant. In new drug development programs, a molecule may show promising pharmacodynamics in a test tube but fail as a drug candidate because of poor absorption or high toxicity. As a result, pharmaceutical companies are increasingly addressing potential pharmacokinetic issues earlier in the drug design and development process.1 For an orally administered compound to enter the bloodstream for distribution throughout the body, it must be absorbed through cells lining the intestinal walls. The drug must pass through the hydrophobic lipid bilayer of the cell membrane on one side of the cell and through the hydrophilic aqueous cytosol and then exit through the cell membrane on the other side of the cell into the blood supply (Figure 1). Because of the necessity to travel through both hydrophilic and hydrophobic environments, the relative lipophilicity of the compound is an important physicochemical property that affects absorption, distribution in fatty versus nonfatty tissues, © 2014 American Chemical Society and Division of Chemical Education, Inc.
Figure 1. Representation of a cell layer separating the intestine from the bloodstream. An orally ingested drug must pass through a cell to enter the bloodstream, encountering both hydrophobic and hydrophilic environments in the process.
affinity for serum albumin, and metabolism. The lipophilicity of a compound is commonly reported as a partition coefficient P, defined as the relative distribution of the compound between 1octanol (a mimic of the hydrophobic bilayer) and water.2 This equilibrium constant may also be denoted as Kow (eq 1). Kow =
[Compound] in 1‐octanol [Compound] in water
(1)
For convenience, these values are more typically reported as log P (or log Kow) values, where a more positive log P value Published: May 1, 2014 915
dx.doi.org/10.1021/ed400655b | J. Chem. Educ. 2014, 91, 915−918
Journal of Chemical Education
Laboratory Experiment
indicates greater lipophilicity and a more negative value greater hydrophilicity. The relevance of these values is indicated by inclusion in Lipinski’s rule of five that addresses likely oral bioavailability of drugs.3 Various methods can measure log Kow values4−7 including extraction/titration8 and thin-layer chromatography (TLC).9 Additionally, a number of software programs available to undergraduates, including ChemDraw, will calculate log P values based on molecular structure, and a recent paper described the use of VCCLAB free online software for calculation of log P values in an undergraduate medicinal chemistry course.10 An experiment using liquid−liquid extraction and UV spectroscopy was developed to measure log Kow values for four common drugs. Implemented in a second-year spring term analytical chemistry course, this experiment introduces medicinal chemistry and drug-design concepts to students of whom a majority have prehealth career interests. In addition, the experiment stresses the importance of equilibrium, reinforces the extraction technique students learned in the first-year organic sequence and the use of UV spectroscopy encountered in a second-year inorganic chemistry course, and introduces chemistry students to micropipets.
Figure 2. Student pairs were assigned one of these four medicinally active compounds.
Kow =
(A i ·DFi − A f ·DFf ) ·Vwater A f ·DFf ·Voctanol
(2)
where Ai and Af are the absorbances of the aqueous layer before and after extraction and Vwater and Voctanol are the volumes of the water and octanol phases. DFi and DFf are the dilution factors used to measure Ai and Af, respectively.
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HAZARDS
1-Octanol is combustible and has a strong, thick odor; it should be used in the fume hood and kept away from flames. Caffeine and acetaminophen are toxic by ingestion, with caffeine having the most acute toxicity. Phenacetin is a carcinogen, and allergies to sulfanilamide are common. All drugs are used in very small quantities below harmful levels, but gloves should be worn and caution exercised when the drugs are weighed out.
PRELAB Students must learn how to use micropipets for any dilutions needed during the experiment. To reduce prelab lecture time, students are asked to watch an 8.5 min video made by the University of Leicester and available on YouTube.11 This excellent video describes the do’s and do not’s of micropipet use. To prove they watched the video or already know how to use a micropipet, students are given a brief quiz (see the Supporting Information) that is graded on the spot. Those with 100% can start the lab; those with
