Case Study: A Quantitative Structure-Activity Relationship (QSAR) Study of an Antiadrenergic Amine

Transcription

1 Case Study: A Quantitative Structure-Activity Relationship (QSAR) Study of an Antiadrenergic Amine Introductory Reading: The Biology of Anti-adrenergic Drugs Antiadrenergic drugs are a very important class of pharmaceuticals. They are more typically known as beta-blockers, and are used routinely for patients with hypertension (high blood pressure), heart failure, angina (chest pain caused by poor cardiac functioning), and abnormal heart rhythms. How does this work? In the nervous system, nerves transmit electrical signals, or impulses, down the nerve. There are a variety of chemicals stored in the nerve endings, known as neurotransmitters. These neurotransmitters have the task of carrying the electrical signal across a very short gap from one nerve to another. This gap is known as a synapse. After crossing the synapse, the neurotransmitter binds, or connects, to one or more neuroreceptors. You can think of these receptors roughly as locks, and the neurotransmitters as keys. The key-lock theory says that the key has to have the correct shape to fit into the lock. Key wrong shape? Then it won t fit into the lock. The neurotransmitters are the keys and the neuroreceptors, or nerve receptors, are the keys. In this particular example, there are two variations on the nerve receptors, and alpha version and a beta version (alpha and beta are letters in the Greek alphabet). These receptors are known as adrenergic receptors, and were discovered in Alpha and beta receptors are activated by neurotransmitters from the adrenaline family. Epinephrine is another name for adrenaline, and a closely related neurotransmitter called norepinephrine is also present. Synapse Both epinephrine and norepinephrine are natural neurotransmitters produced by the body. In an emergency situation, where your body needs to either fight or flee (be able to respond quickly to some situation), a lot of epinephrine is released from the nerve ending, flooding the receptors. When this happens, your heart beats faster, pumps more blood, constricts the blood vessels, and makes your lungs work more efficiently to deal with the emergency. At such time as the emergency is no longer present, normal amounts of epinephrine and norepinephrine are transmitted to the receptors, and your heart rate and other body functions return to normal. In some people, these neurotransmitters are released at higher than normal levels in nonemergency, every day situations. This results in a situation where the heart is working too { Alpha receptor Electrical impulse Norephinephrine H NH 2 Nerve receptors Beta receptor Nerve ending

2 hard and/or beating too fast. This condition is known as tachycardia, and is potentially life threatening. ther conditions include that might be caused by an over-release of neurotransmitters are hypertension, angina, and heart failure. In these patients, how can a physician prevent this situation? The answer is in a family of drugs known as beta blockers, or, more formally, antiadrenergics. These drugs work by serving as antagonists, drugs that block the biological behavior of a chemical that occurs naturally in the body. In this case, the beta-blocker shown in the diagram binds to the beta-receptor better and/or faster than does the natural chemical norepinephrine. This prevents norepinephrine from stimulating the nerve endings, particularly the beta-receptors that are responsible for an overly active heart. As a result, the patient s heart rate returns to normal, blood pressure is reduced, chest pains go away, and the patient is able to live a more normal life. The beta-blocker, however, does cause other side effects, such as diarrhea, headaches, and other unpleasant conditions. Compared with chest pain and high blood pressure, however, these side effects are acceptable consequences of taking these types of drugs. Chemistry of Anti-adrenergics The table below shows the chemical structure of the naturally occurring (endogenous) neurotransmitter norepinephrine, which binds well to the beta-receptor. Structurally, it is classified as an amine. An amine is an organic compound with the generic structure R-NH 2. You should notice the NH 2 group on the right side of the molecule. The R part of the amine is everything to the left of the NH 2, which includes the six-carbon benzene ring, and three hydroxyl () groups. Norepinephrine Synapse { Alpha receptor Electrical impulse Norephinephrine Nerve receptors Propanolol (beta blocker) H NH 2 Beta receptor Nerve ending Beta blocker H 3 C NH CH 3 H NH 2 NH H 3 C CH 3 Propanolol, a well-known beta-blocker, has a similar structure, with nitrogen located near the end of the molecule. This drug has two connected benzene rings and two singly bonded oxygen atoms. The nitrogen atom has two methyl groups (-CH 3 ) attached to it. As you can see, it is a larger molecule than norepinephrine.

3 The molecule we wish to evaluate in this case study has a very similar structure to both of these molecules. Its general structural name is dimethylbromophenethylamine, meaning it has two methyl groups, a bromine atom, a phenyl group (another name for a benzene ring), then two carbons (an ethyl group), then the nitrogen amine part. The molecule looks like this: X Br CH 3 N CH3 Y Notice the X and Y notation attached to the left of the benzene, or phenyl, part of the molecule. The X and Y refer to substituents, or other atoms or groups of atoms that we can attach at those locations. For example, the notation: X=Br Y=F would suggest to the reader that we substitute a bromine atom (Br) where the X is, and a fluorine atom (F) where the Y is. We can substitute thousands of different chemical entities for the X and Y, such as individual atoms hydrogen (H), iodine (I), chlorine (Cl) or groups of atoms, such as a methyl group, another phenyl group, and the like. Every time we substitute something for the X and/or Y, we have a new chemical, and, in this case, a new drug. The substitution(s) may improve the ability of the drug to act as a beta-blocker, or it may reduce its effectiveness. Measuring Drug Effectiveness There are a number of ways to measure the effectiveness of a drug. The most common, however, is measuring the smallest concentration of a drug needed to cause some biological activity. Hopefully this biological activity is curing whatever it is we want the drug to cure! The measure of the minimum concentration is labeled C. For a variety of reasons, the biological activity is expressed as 1 over the concentration, or 1/C. An increase in the activity of a drug is indicated by an increase in the 1/C value. In order to further standardize these measurements, we take the logarithm of 1/C. For most evaluations of drug effectiveness, therefore, we look at the log(1/c) values. The value of log(1/c) depends on a number of parameters, or variables, that we can measure about the drug. ne of the goals of quantitative structure-activity relationships (QSAR) is to measure what we can measure, and then predict some variable that is harder to measure. In this case, measuring log(1/c) is pretty hard, but it s relatively easy to measure some characteristics of the drug that can then be used to predict the biological activity (log(1/c)). We ll introduce three of them for this case study. Lipophilicity

4 Lipophilicity is a measure of the ability of a drug to move through a lipid membrane. A lipid membrane is composed of fat molecules, and typically has two layers. For a drug to get absorbed from a location such as the stomach, and get into the bloodstream, it has to move through the lipid membrane (such as the lining of the stomach) and get into the blood. If a drug can t get into the bloodstream, it s not going to be very effective. We can avoid the need for this step by injecting the drug directly into a vein, but most people much prefer to take a pill than an injection. Most drugs, therefore, need to be designed with lipophilicity in mind. Hammett Constant The Hammett Constant, named after a scientist by that name, is a little more difficult to understand, at least as compared with lipophilicity. From general chemistry, you should remember that most molecules can dissociate, or break apart into several parts. For example, water can dissociate to form a positive hydrogen ion (H+) and a negative hydroxyl ion (-). We write this equation as follows: H H The arrows indicate that the reaction is in equilibrium. Sometimes we have the water molecule H, and sometimes we have the two ions. Depending on what else is happening, at times we have more H molecules than we have ions. If you can think of this reaction as two sides of a seesaw, sometimes the H is high in the air (and the ions are on the ground), sometimes you have the opposite, and sometimes they are both equally and straight. We call this condition being in equilibrium. Here is a slightly more complicated reaction: X X + H + In this case, this carboxylic acid is dissociating to form a negatively charged ion and a positive hydrogen ion (one definition of an acid, if you remember that class!). In any solution containing this molecule, which part of the reaction is favored? Does the reaction go to the right (favoring the charged parts) or to the left (favoring the complete molecule)? The answer depends on a number of factors, but in this case it depends on what X is. The Hammett constant is a measure of the effect that the substituent has on the reaction. For example, the Hammett constant for the substituent CH 3 (a methyl group) is 0.07, but for the substituent (hydroxyl group) the Hammett constant is What s the significance of these numbers? If the constant is positive, that means that the substituent is an electron-withdrawing group. Conversely, a negative constant represents an electron-donor group. With an electron-withdrawing group, the reaction is forced to the right, producing more charged ions than might normally be the case. An

5 electron-donor group forces the reaction to the left, almost ensuring that the molecule will stay intact. What is the significance of this on the effectiveness of a drug? Depending on what we want the drug to do, it may be the case that we don t want the drug to dissociate into two or more charged ions, we want it to stay whole. It might be just the opposite, where biological activity is completely dependent on the molecule separating into charged ions. Regardless, in this case study you will be asked to design a drug, and you ll have to make some decisions about whether you want your substituent(s) to be electron-withdrawing or electron-donating! Taft s Steric Hindrance (E s ) A third very common parameter is Taft s steric hindrance, again named after its originator. All molecules have a shape. As you might suspect from the key-lock theory, some molecular shapes fit better into a receptor than others. Steric hindrance is a measure of the degree to which some substituent changes the shape of a molecule. Some values for steric hindrance are positive while some are negative. There is not a clear rule of thumb for the significance of a positive or negative E s, but it also addresses the ability of the molecule to bind to the receptor that it is supposed to bind to. In this case study, you will determine the direction and degree to which steric hindrance influences the ability of the drug to do its job. Putting it all together The goal of this case study is as follows: Given a test set of biological activity, lipophilicity, Hammett constants, and Taft steric hindrance for a variety of substituents of dimethyl-bromophenethylamine, can you determine the values of lipophilicity, Hammett constants, and steric hindrance that will predict biological activity? Remember, it s relatively easy to measure the three variables, but it s hard to measure biological activity. In this case study, we ll measure the biological activity (log(1/c)) for 22 sample compounds, as well as the three other variables. A sample of the dataset is shown below.

6 By way of example the first line of the data has hydrogens for both the X and Y positions in the drug. Given that, we measure a value of 7.46 for the biological activity, 0.00 for lipophilicity, 0.00 for the Hammett constant, and a steric hindrance of None of these values are particularly good or bad, but it would be nice to increase the value of the biological activity to something closer to 10. The question is: how do we change lipophilicity, Hammett, and steric hindrance to do that? Do we increase lipophilicity or decrease it? What about for the other two variables? QSAR studies allow us to make that determination, and then use the results of that determination to zero in on exact values. How do we do that? We have to perform a regression calculation. You may remember from math class the formula: y=mx+b where y is the value we are trying to predict, m is the slope, x is the variable we know, and b is the y-intercept. Determining the values of m and b is called several things fitting the data, performing a least-squares fit, performing a regression. All three mean the same thing. We ll do some mathematics to try to determine these values. In this case, we are trying to determine this equation: log(1/c) = a*lipophilicity + b*hammett constant + c*steric hindrance + some constant Your job is to determine the values for a, b, c and the constant the regression coefficients. To do that, you will perform a multiple regression on the complete test dataset of 22 substitutions. Substituents include hydrogen, fluorine, iodine, chlorine,

7 bromine, and one group, a methyl group (CH 3 ). A copy of the test data set is shown at the back of this case study. nce you have determined the regression coefficients, you will be asked to use your coefficients to design some drugs! Case Study Scenario 1: You are a medicinal chemist tasked with designing a beta-blocker that has a biological activity, or log(1/c), of Your lead drug for this is dimethylbromophenethylamine, with two sites on the benzene (phenyl group) that can be substituted. The chemistry group has given you a test set of 22 substitutions, with log(1/c), lipophilicity, Hammett constants, and steric hindrance data. The data is located at Preliminary studies have suggested that a lipophilicity of 1.15 will ensure that the drug can get through the lipid membrane and reach the target receptor. These studies have also indicated that the receptor site will not tolerate any steric hindrance. The question becomes: should the new drug have substituents that are electron-withdrawing or electron-donating? In other words, do we want the molecule to remain intact, or will the drug work better if it dissociates into positive and negative ions? If you determine the correct value of Hammett s constant for these conditions, you ll be able to answer this question! You should also determine a Taft steric hindrance value that is within standard range, defined as between the 1 st and 3 rd quartiles. You should conduct your QSAR analysis using both the Hansch method and the Free- Wilson method. (NTE: with the Free-Wilson method, if you get a singularity error, simply ignore that parameter). In your technical memorandum, show the results of both of your studies (Hansch and Free-Wilson). For the Hansch method, show the calculated log(1/c) for each of the 22 compounds, in addition to your results for the new beta-blocker.