Download L20 Medicinal Ch 28.07.2015 Metabolism

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Medicinal
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L20
L20.Medicinal Ch. 28.07.2015
Yara Zreigat
Metabolism, continued...
We know by now that metabolism is the chemical alteration of a drug with the principal
purpose of eliminating it from the system. In other words the aim of the metabolism
process is ‘detoxification’ (by making the compound mere hydrophilic).
In this lecture we will continue talking about Phase I of the metabolism process.
As said earlier, in Phase I there are 3 prime reactions: Oxidation, Reduction and
Hydrolysis; oxidation being the most important followed by hydrolysis and finally
reduction.
(1) Microsomal oxidases: this solely means ‘liver enzymes. ‘Microsomes’ are
particles produced from the liver (Active liver particles, and the name indicate that
oxidation happens only in the liver).
(2) Mixed Function oxidases: which refers to their ability to oxidize many different
substrates in the same way; they’re not ‘substrate-specific’. This property is due
to having large catalytic pockets (no hindrance) and they can attach or handle their
substrate in a single attachment (having multiple attachment sites means the
enzyme can act on some substrates but not others). However, due to their mixed
function properties these enzymes tend to be low in affinity and have slow
turnover- it requires hours to produce a significant yield of metabolites.
Because the reaction speed is slow, the drug need to remain in the body more than
2 hours to see metabolic products (more than 2 hours indecates failure of renal
elimination), if it remain less than 2 hours, quick renal elimination happen and no
metabolic products appear.
(3) Cytochrome
P450
isozymes.
Isozymes are multiple forms of enzymes that have different chemical structures
(they differ in the amino acid sequence), but catalyze the same reaction (perform
similar oxidation reaction). They are categorized into family and subfamily
groups according to structure similarity however they all have similar functions
and are called ‘isozymes’. So, we’re not dealing with a single enzyme system
(1oxidase) we’re dealing with multiple oxidases (a group of these enzymes is
specialized in steroid oxidation, another group for aromatic rings … etc).
L20.Medicinal Ch. 28.07.2015.Metabolism
The Oxidation process involves a group of enzymes to which 3 names were given to
describe their properties:
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Why are they called Cytochrome P450 enzymes?
Cytochrome P40 isozymes are large proteins with a
heme cofactor. Notice in the figure the large protein
catalytic part; this is the part that differs in different
oxidases (different amino acid sequence). However,
they all share the non-protein part (heme cofactor), As
you can also see the porphyrin ring in the catalytic ring;
made up of 4 pyrrole rings in a highly conjugated
system with other appendices coming out of the rings
(not so important right now). There’s an Iron in the
middle of the ring that forms coordinate bonds with
Nitrogen atoms [Coordinate bond: a bond between an
electron donor (Nitrogen in this case) that gives the
electors to the empty orbitals of a metal (Iron in this
case)]. The Iron in the porphyrin ring forms the heme;
the Iron is ferric Fe3+. A similar molecule that contains
a heme group is hemoglobin but the Iron in hemoglobin
is a ferrous Fe2+.
- One of the two bonds (the 5th bond) is formed with a Nitrogen atom in the imidazole
group found in the amino acid histidine; this bond is responsible for holding this
porphyrin ring structure in the catalytic pocket of the oxidase enzyme.
- The 6th bond is formed between the Iron and Oxygen molecule (O2). This oxygen is
very important since it’s the oxygen atom that is donated in the oxidation reaction
(act as oxidizing agent). Under the slightly acidic condition of the liver, the oxygen
molecule is in the form of (—O—OH) and this –OH is donated in the oxidation
reaction to the metabolized compounds to form (R—OH). So the source of the –OH
is the oxygen bound to the Iron in cytochrome P450 (CYP-450).
When they studied the microsomes under exposure to carbon monoxide, they found that
this system has a very strong affinity to carbon monoxide CO (around 200 times stronger
than oxygen), this CO binding gives an intense red color that has an absorption at 450nm
(λ-max) and this is why it’s called cytochrome P450 (cyto= cell, chrome= the red color,
L20.Medicinal Ch. 28.07.2015.Metabolism
The Iron (whether a ferrous or a ferric ion) can form up to 6 coordinate bonds in a
bipyramidal system as you can see in the figure. In the porphyrin ring, the Iron forms 4
bonds with the Nitrogen atoms in the pyrrole ring so that means there are two bonds left
for the Iron to form:
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L20.Medicinal Ch. 28.07.2015
Yara Zreigat
P450= the spectrophotometric peak at the wavelength of the absorption maximum of the
enzyme; 450 nm).This is all due to the porphyrin ring and the iron that has a high affinity
to Carbon Monoxide. Keep in mind that these three names; microsomal, mixed function,
CYP450 refer to the same group of oxidase enzymes.
The oxidation systems
There are 5 groups that are oxidized with CYP450 system: Carbon systems, Nitrogen
systems, Sulphur systems, Oxygen systems and Halogen systems.
I.
Carbon systems,
7 C-systems get oxidized by CYP450 which are:
1.
2.
3.
4.
5.
6.
7.
Aromatic Ring Oxidation
If a drug containing an aromatic
ring gets exposed to liver enzymes
(CYP450 enzymes which are also
mixed function and microsomal
enzymes), the aromatic ring gets
oxidized by replacing a –H with an
–OH producing a phenol.
 Mechanism:
- Step 1:
Recall the porphyrin ring structure where the Iron is bonded
with an oxygen molecule. When two oxygen atoms are bond
to each other (like peroxide also), both are strongly
electrophilic, they both start to pull the electrons from each
other making the bond quite unstable.
L20.Medicinal Ch. 28.07.2015.Metabolism
(1)
aromatic ring,
benzylic carbon,
allylic carbon,
double bond,
alicyclic,
aliphatic
and carbons α to carbonyls oxidations.
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L20.Medicinal Ch. 28.07.2015
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Due to this instability, the O—OH bond tends to break when a nucleophile (with a lone
pair of electrons) is present, such nucleophile attacks one of them and binds to it, so it get
the electrons of the nucleophile. And the other oxygen gets the electrons that were
present in the O—O bond. Therefore, the O—O system is referred to as an oxidizing
system offering oxygen to a nucleophile and oxidizing it due to its instability.
The same applies in the adjacent
figure; the O—OH that is bound to
the ferric is very unstable (reactive)
and oxidizing. The ring electrons
perform nucleophilic attack on the
system, so the –OH takes a pair of
electrons from the aromatic ring
(nucleophile) by breaking the
double bond in the ring; and the
bond O—O is broken giving its electrons to the other O. The breaking of the double
bond and the binding of –OH to the ring gives an unstable positively charged
intermediate (Reaction similar to Friedel-Crafts Acylation).
- The O—OH bond is unstable due to the withdrawing effect of both oxygen
atoms and therefore tends to break.
- Upon breaking, the –OH takes a pair of electrons from the aromatic ring by
breaking one of the double bonds and binding to the ring (oxidizing it).
- The bond O—OH is broken down as well giving the electrons to the O
attached to the Fe.
- An unstable intermediate with a positive charge forms due to breaking the
bond.
- Step 2:
The lone pair of electrons of the oxygen adds to the positive
charge on the neighboring carbon in order to get rid of the charge
and increase the stability a little bit. As a result, an epoxide forms.
This epoxide is highly unstable because it’s highly strained and
therefore tends to break. How did they know that an epoxide
forms? It was isolated from the urine in a small percentage (1%)
when a drug with an aromatic ring was taken.
L20.Medicinal Ch. 28.07.2015.Metabolism
So let’s study the figure again step by step:
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L20.Medicinal Ch. 28.07.2015
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- Step 3:
Protonation of the epoxide due to the
liver’s acidic conditions where the oxygen
gains a hydrogen, followed by the
breaking of the strained bond (in the
circle) and then the breaking of the
hydrogen bonded to the ring to return the
ring to its aromaticity by returning back
the electrons. As a result we get a phenol.
L20.Medicinal Ch. 28.07.2015.Metabolism
Summary:
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Sometimes the epoxide is stable and remains in the body. An
example on this is benzo[a]pyrene which is a compound found in
cigarette. When metabolized, it forms a stable epoxide as seen in
the adjacent figure; this compound is planar and has an
electrophile center which is the epoxide this makes it able to
intercalate with DNA strands (it’s a DNA intercalating agent).
What is meant by intercalation is that this compound can squeeze
and insert itself between the double stranded DNA due to its
planarity and the nucleophile in the DNA can attack the
electrophile center forming a covalent bond. Therefore, this
compound is toxic and a carcinogen. Benzo[a]pyrene is a rare
example on a compound that is initially nontoxic but became toxic
when metabolized (usually detoxification happens but here is a
rare example where metabolism activate a toxin).
NOTE: Some people have a special CYP450 for aromatic ring
which is CYP450-A3, so they are more prone to develop cancer
upon exposure to smoking because they accumulate more of the epoxide.
 Properties of the aromatic ring oxidation reaction
- Moderate speed reaction.
Since the aromatic ring is actually an electron dense system we’d expect the reaction to
be rapid (Full oxidation); it can be easily detected by CYP450 and oxidized. So what
made this reaction of moderate speed?
The intermediates that were formed in the reaction are not aromatic; there is a loss of
aromaticity in the process (Aromatic system is stable and loss of aromaticity mean loss of
stability). This loss gives the intermediate very high energy making the reaction slower
[increases the transition state energy]. So initially this reaction is fast because the
CYP450 can take electrons from the electron rich aromatic ring but since during the
reaction there was a loss of aromaticity this renders the reaction slower than expected.
As a result, the oxidation yield is (40-50%).
So if a drug with an aromatic ring is taken, 40-50% of its metabolites can be found in the
urine and the rest are not metabolized if there is not another place of oxidation.
L20.Medicinal Ch. 28.07.2015.Metabolism
The cytochrome P450 scans the molecule to be metabolized until it detects an electron
rich region and then oxidizes it. The CYP450 is an electron deficient species (due to the
withdrawing effects of the two oxygen atoms) and gets its electrons from the detected
electron rich species.
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L20.Medicinal Ch. 28.07.2015
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- This reaction happens only on aromatic rings that are electron rich.
E.g. on an electron deficient ring is the compound in the
adjacent figure, Clonidine (a biphasic antihypertensive
agent). As you can see there is a guanidine group that is
positively charged under physiologic conditions. As a
result the cationic center has a withdrawing electron
effect. In addition the 2 chlorine atoms are withdrawing as
well. As a result, clonidine is an example of an electron
deficient aromatic ring that cannot be metabolized by
aromatic ring oxidation (it is too resistant to be oxidized at
the aromatic ring but it can be metabolized at other
locations as we will see later). Also, clonidine is a very
water soluble compound due to the presence of a
guanidine; consequently, once it gets filtered in the kidneys it cannot be reabsorbed
(because of +ve charge) and is therefore quickly excreted before its metabolism. (If a
drug resists elimination it can get metabolized on the other hand if it’s eliminated then it
resists metabolism).
- Oxidation happens on para- position only
CYP450 is sterically controlled (sterically driven) and tries to
avoid sterically hindered positions. In the aromatic ring, the
para position is the one with the least crowdedness
(stearically un-hindered). If the para position is already
occupied, it starts to look for the next least crowded position.
For example, in the adjacent structure, If R1 is less hindered,
the –OH goes Meta to R2 or ortho to R1 and vice versa. So it
stays away from steric crowdedness as much as possible.
L20.Medicinal Ch. 28.07.2015.Metabolism
Another example:
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Yara Zreigat
L20.Medicinal Ch. 28.07.2015
- Oxidation at just one ring only (no need for second oxidation reaction)
Oxidation of C-systems happens only one!! There’s no dihydroxy, no other oxidation
reaction is needed. The oxidized compound gets conjugated immediately.
An example: Phenobarbitone
It has two aromatic rings that are rich in electrons (no
electron withdrawing groups attached to them). This
compound is highly lipophilic and is renally reabsorbed so it
remains for around 8 hours in the body. So it therefore gets
metabolized since it resists elimination. What happens is that
only one of the rings can get oxidized at the para position.
Due to the moderate rate of the reaction, 50% gets oxidized
and is found in the urine as either the oxidized form (30-40%
of the 50% oxidized form) or as the glucuronic acid
conjugate form (50-60% of the 50% oxidized form).
NOTE: once you oxidize the aromatic ring, phenol can be
eliminated as such or as conjugant (phase 1 and phase 2
products).
30-40% of the
oxidized form is
found in the urine
Phenobarbitone
50%
unmetabolized
and found in urine
50-60% is
eliminated in the
conjugated form
Another
example:
Paracetamol
Paracetamol compound is already hydroxylated; there is no need for further oxidation.
Therefore, it’s eliminated in the urine as it is or as its conjugated form (the most
important conjugate is glucuronic acid).
L20.Medicinal Ch. 28.07.2015.Metabolism
50% oxidized
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L20.Medicinal Ch. 28.07.2015
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Q) When oxidation happens on an aromatic ring, why it won't happen at another ring??
1) Because we add –OH that is ready for conjugation.
2) When conjugation happens, the compound becomes too hydrophilic and
quickly renally eliminated.
3) When conjugation happens, it is too hydrophilic and can't cross the hepatocyte
membrane (remember that for metabolism to happen, compound should cross
the hepatocyte's membrane).
To sum up, what you should know about aromatic ring oxidation is:
(2)
Mechanism of oxidation.
It's a moderate speed reaction.
It only oxidizes electron rich rings.
It happens at para position.
Oxidation happens only at one ring.
Benzylic Carbon Oxidation
The benzylic carbon is the carbon α to aromatic ring as seen above. This carbon is very
electron rich since it’s attached to the aromatic ring. As an electron deficient system,
cytochrome P450, scans the molecule until it detects an electron rich species and oxidizes
it. When there is a benzylic carbon, the CYP450 detects and oxidizes it by replacing an –
H with –OH. The reason why it ‘ignored’ the aromatic ring was because it’s a moderate
rate reaction that forms intermediates that lost their aromaticity. So, if we have aromatic
ring and benzylic carbon, CYP-450 will oxidize the benzylic carbon because it is easier
and its oxidation doesn't involve loss of aromaticity so it's fast (near 100 % yield of
oxidation at this carbon).
 Mechanism
First of all, check if the benzylic carbon is a primary, secondary, tertiary or quaternary
carbon.
L20.Medicinal Ch. 28.07.2015.Metabolism





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L20.Medicinal Ch. 28.07.2015
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1° benzylic carbon
The benzylic carbon gets oxidized as we already
know by replacing an –H with an –OH.
Some of this oxidized product:
- can be found in the urine.
- might get conjugated to glucuronic acid and eliminated in the urine.
- Also, this product might get further oxidized to an aldehyde by the loss of two
hydrogens and this aldehyde is immediately oxidized by the loss of the hydrogen
of the aldehyde with the hydrogen of a water molecule into carboxylic acid. The
carboxylic acid is eliminated in the urine as it is and as its conjugated forms
(glucuronic acid and amino acid conjugation). These reactions are done by the
enzyme system Alcohol Dehydrogenase that takes the hydrogens (as it name
implies) and gives them to NAD+ which is converted to NADH and H+. There’s
a partially reversable reaction that happens by reductases when the alcohol is
converted to an aldehyde (as seen below), a small amount of the aldehyde is
converted back to alcohol.
So what can be found in the urine?
- the oxidized alcohol drug (with an –OH) and also its conjugate form
- NO ALDEHYDE in the urine! They are instable and toxic
- Carboxylic acid is eliminated in the urine ( by renal filtration and secretion by an
anionic pump) and also its conjugated forms (glucuronic acid conjugation and amino
acid conjugation).
1. The first step ( from alcohol to aldehyde ), oxidation is the major route while
reduction is a minor route
2. There is no reduction from carboxylic acid to aldehyde.
3. Alcohol dehydrogenase is an oxidizing system that is not related at all to CYP450.
L20.Medicinal Ch. 28.07.2015.Metabolism
NOTE:
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L20.Medicinal Ch. 28.07.2015
Yara Zreigat
2° benzylic carbon
The same thing happens in the secondary carbon. First it’s oxidized by CYP450 into a 2°
alcohol then it’s further oxidized by Alcohol dehydrogenase enzyme system (it’s a
different system that has nothing to do with CYP450) into a ketone which cannot be
further oxidized. The alcohol is eliminated in the urine as it is and as it’s conjugated
form, as we know. The ketone also is eliminated in the urine. So these three products are
found in the urine (ketone, hydroxyl and conjugated hydroxyl).
3° and 4° benzylic carbon
- 3° alcohol can't be further oxidized so it's eliminated as such with its conjugant.
- 4° carbon, no oxidation because there is no H to replace (remember that H must
exist to replace it with –OH).
L20.Medicinal Ch. 28.07.2015.Metabolism
Any atom to be metabolized in the C-system must have at least one hydrogen attached to
it; in the quaternary benzyl there is no hydrogen so it’s not metabolized.
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 Properties of the reaction
- The reaction is very fast with a 100% yield; in around 3 hours
the drug is fully metabolized.
- The benzylic carbon must have hydrogen bonded to it in order
for it to be replaced by –OH.
E.g. In phenobarbitone mentioned previously the benzylic carbon
had no –H on it, so this benzylic carbon is resistant to oxidation.
- Once the benzylic carbon gets oxidized, the ring doesn’t get
oxidized; C-systems are oxidized only once.
Example: Tolbutamide (sulfonylurea hypoglycemic agent)
What was done is that they replaced the
methyl with a chlorine atom (isosterism);
chlorine resists oxidation because it’s
electron withdrawing. The compound is
called Chlorporpamide and is given
twice a day, once in the morning and the
other in the evening.
NOTE: in Chlorporpamide, the ring also is not metabolized because of its slow reaction
compared to the benzylic carbon oxidation that's very fast and favorable, furthermore
para-position of the ring is occupied by substitution, also sulfone and chlorine
withdrawing effect makes the ring electron deficient.
L20.Medicinal Ch. 28.07.2015.Metabolism
The circled carbon is the benzylic carbon
and the one that gets oxidized (this drug
is found in the urine mostly with 70% in
its carboxylic acid form and its
conjugant; and 30% as alcohol and its
conjugant). A long time ago, this drug
was used in the treatment of diabetes but
because it gets oxidized quickly the
patient had to take a dose every 3 hours.
This is very inconvenient so it’s no
longer used.
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L20.Medicinal Ch. 28.07.2015
(3)
Yara Zreigat
Allylic carbon (α to double bond)
The allylic carbon is the carbon alpha to a double bond.

-
Properties of this reaction:
Like benzylic carbon, it’s very electron rich because it’s attached to double bond.
The oxidation reaction is very fast (but of lesser speed than benzylic).
90% oxidation yield.
Some of the parent compound can be found in the urine un-metabolized (unlike
benzylic that has a 100% oxidation yield).
 Mechanism
Similar to benzylic carbon oxidation, the hydrogen is replaced by a –OH by CYP450.
- If it’s a primary alcohol it will get further oxidized by alcohol dehydrogenase into an
aldehyde which is immediately oxidized into carboxylic acid.
- The secondary allylic gets oxidized by CYP into a secondary alcohol which gets
further oxidized by alcohol dehydrogenase into a ketone.
- The tertiary allylic gets oxidized by CYP450 to a tertiary alcohol but with no further
oxidation.
- The quaternary allylic remains un-metabolized because it lacks a hydrogen atom.
Example: Tetrahydrocannabinol (found in Cannabis)
There are 3 allylic carbons around the double
bond. The easiest one to access, the one that is
sterically exposed is the methyl group
numbered 1 in the figure. Next is the –CH2
and the last one is the carbon attached to
second ring numbered 3.
 The methyl is a primary allylic carbon
that gets oxidized into an alcohol then
further oxidized to an aldehyde and
carboxylic acid (HIGHEST YIELD).
L20.Medicinal Ch. 28.07.2015.Metabolism
So, compound containing allylic carbon gets eliminated in the urine un-metabolized,
alcohol forms, conjugated alcohols, ketone, carboxylic acid and conjugated carboxylic
acid. No aldehyde is found in the urine.
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L20.Medicinal Ch. 28.07.2015
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 The –CH2 is a secondary carbon that gets oxidized into a secondary alcohol which
gets further oxidized into a ketone (SECOND HIGHEST IN YIELD %)
 The tertiary carbon gets oxidized into a tertiary alcohol but with no further
oxidation (LOWEST YIELD).
L20.Medicinal Ch. 28.07.2015.Metabolism
BUT KEEP IN MIND; oxidation can only happen once at one of these positions! It
never happens on two positions or three. So only the metabolites of one of these
carbons can be found.
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