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.
Electrical impulse
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
1948. 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.
Nerve ending
Norephinephrine
OH
NH2
HO
OH
{
Alpha receptor
Beta receptor
Nerve receptors
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
hard and/or beating too fast. This condition is known as tachycardia, and is potentially
life threatening. Other 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,
Electrical impulse
drugs that block the biological behavior of a chemical that
occurs naturally in the body. In this case, the beta-blocker
Nerve ending
shown in the diagram binds to the beta-receptor better and/or
faster than does the natural chemical norepinephrine. This
Norephinephrine
prevents norepinephrine from stimulating the nerve
endings, particularly the beta-receptors that are responsible
Beta blocker
O
for an overly active heart. As a result, the patient’s heart
OH
rate returns to normal, blood pressure is reduced,
Synapse
NH
chest pains go away, and the patient is able to live
HC
CH
a more normal life. The beta-blocker, however,
Alpha receptor
Beta receptor
does cause other side effects, such as diarrhea,
Nerve
receptors
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.
OH
NH2
HO
OH
{
3
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-NH2. You should notice the NH2 group on the right side of the
molecule. The “R” part of the amine is everything to the left of the NH2, which includes
the six-carbon benzene ring, and three hydroxyl (OH) groups.
Norepinephrine
Propanolol (beta blocker)
OH
O
NH2
HO
OH
NH
OH
H3 C
CH3
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 (-CH3) 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:
Br
X
CH3
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. One 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
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 (OH-). We write this equation as follows:
HOH H+ + OHThe arrows indicate that the reaction is in equilibrium. Sometimes we have the water
molecule HOH, and sometimes we have the two ions. Depending on what else is
happening, at times we have more HOH molecules than we have ions. If you can think
of this reaction as two sides of a seesaw, sometimes the HOH 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
OH
X
O
O
O
+
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 –CH3 (a methyl
group) is –0.07, but for the substituent –OH (hydroxyl group) the Hammett constant is
0.12. 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
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 (Es)
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 Es, 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.
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 1.24. 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,
bromine, and one group, a methyl group (CH3). A copy of the test data set is shown at
the back of this case study.
Once 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 10.25. 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 http://chemistry.ncssm.edu/mc/QSARamineDataTest.xls.
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 1st and 3rd quartiles.
You should conduct your QSAR analysis using both the Hansch method and the FreeWilson method. (NOTE: 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.