Antimetabolites

In other words, since antimetabolites are similar in

structure with metabolites, they can fool the cell into
believing that they are naturally present building blocks
to interact with the normal cellular process but differ in a
manner sufficient to alter the outcome of that pathway.
They do this by the Inhibition of nucleic acids
biosynthesis: enzyme inhibition (involved in S phase of the
cell cycle)
Mimicking DNA building blocks, leading to cell death

Most antimetabolites are prodrugs

Where Purine (6-mercaptopurine) and pyrimidine
(5-fluorouracil) → nucleic acids and nucleic acid
polymers (DNA and RNA)

Antifolates (methotrexate) → folate cofactor sites for

enzymes in nucleic acid bases biosynthesis

These anticancer drugs are based on a pyrimidine

structure. One of the most common examples is the
pyrimidine derivative 5-fluorouracil. It interferes with the
DNA Synthesis.
 Remember the Normal process:
Uracil [Thymidylate synthetase methylation] → thymine or
thymidine → DNA synthesis → Cell proliferation

*thymine → nucleobase
*thymidine → nucleoside

Uracil is directly used for RNA for protein synthesis

While Thymine is a composition of DNA

5-FU MOA:
→ could affect by blocking the methylation process by
Thymidylate synthetase, converting uracil to thymine
→ could mimic Thymine, a building block in DNA/RNA
structure, which alters the cellular process leading to cell
death

Before the in-depth discussion of 5-FUs MOA, remember the three metabolic rules:
A nucleotide cannot be used as a building block without 3 phosphate groups
A nucleotide has to be in deoxygenated form to be inserted into the DNA
A nucleotide has to be in oxygenated form to be inserted in the RNA

binds to the Uracil transporter

converted to nucleotide in oxygenated form
will be phosphorylated to its diphosphate
Two primary pathways:
RNA:
will be converted to triphosphate form
will enter the nucleus
Be inserted in the transcription process of RNA strand

DNA:
The diphosphate will be reduced to deoxygenated form
and then phosphorylated
Inserted into the DNA replication
*In the diagram, 5-FdUMP is a metabolite of
5-Fluorouracil, which is 5-fluoro-2-deoxyuridylic
acid

Diphosphate fluorouracil will be deoxygenated, then it

will be dephosphorylated to monophosphate → inhibits
thymidylate synthase, but before we study the inhibition
process let’s first understand the normal process.

normal process;
Basically, this is the biochemical conversion of uridine to
thymidine, which shows that, dUMP (deoxyuridine
monophodphate), a metabolite of uracil, will bind to the
Thymidylate synthase, following the addition of folate
derivative (5,10-methylenetetrahydrofolate) and the
formation of cofactor N-5-iminium ion. Normally, this
process is followed by proton loss at the 5th position of
dUMP and elimination of the dihydrofolate from the
ternary complex, regeneration of the enzyme, and the
production of thymidine.
So, how does this drug do the inhibition? Based on
Scheme 10.19,
this is actually quite similar to the biochemical conversion
of uridine to thymidine where the sulfhydryl group of the
TS enzyme will first conjugate to the 6th position of the
Fluorouracil moiety. Then, Fluorouracil’s 5th position binds
to the methylene group of folate derivative
(5,10-methylenetetrahydrofolate), followed by the
formation of N-5-iminium ion (cofactor). The formation of
this ternary complex is where the process stops. Some
clinical studies have shown that administration of a
tetrahydrofolate source (leucovorin) prior to treatment
with 5-FU results in greater inhibition of total TS activity.

And because 5-FU is stable to elimination, the folate is

not eliminated at the 5th position. The enzyme, substrate,
and the cofactor are covalently bonded. Thus, no
proton will be lost at 5th position, na kailangan sana for
the formation of uracil species.

Resistance:
The primary metabolizer of 5-FU is the Dihydropyrimidine
dehydrogenase (DPD) which converts the drug to
a-fluoro-beta-alanine
Cancer cells also has the Uracil-DNA glycosylases (UDG)
enzyme that recognizes the false analogs of Thymine
Cancer cells also Increase their concentration of
Thymidylate synthetase enzymes
Purine Antimetabolites
Antimetabolites' structure and design based on purine structures began with an isosteric thiol/sulfhydryl group, replacing
the 6-hydroxyl group of hypoxanthine and guanine. They are a class of drugs that mimic natural purine nucleobases in
order to penetrate and disrupt cellular processes in cancer cells. The antineoplastic activity of purine drugs depends on
their relative enzymatic activation and inactivation rates in tissues and cells.

Examples of commonly used purine antimetabolites with their structural modifications include: (refer nalang po sa
grisvold page 378 figure 10.9 para sa structures)

● Mercaptopurine (6-MP): Replaces the sulfur atom in the 6-position of guanine with a thiol group.
● Thioguanine (6-TG): Replaces the oxygen atom in the 6-position of guanine with a sulfur atom.
● Fludarabine (F-ara-A): A deoxyribose analog of arabinose with a fluorine substitution at the 2' position. Ppl
nucleotide form that can be incorporated into DNA or RNA.
● Incorporation: The activated nucleotide competes with natural purine nucleotides for incorporation into DNA or
RNA by enzymes like DNA polymerases and RNA polymerases.
● Inhibition of DNA synthesis: Once incorporated, the antimetabolite disrupts DNA synthesis by:
- Causing chain termination due to structural differences.
- Interfering with the fidelity of DNA replication.
● Inactivation: Catabolic enzymes like deaminases and phosphorylases can break down and inactivate the
antimetabolites, limiting their cytotoxic effects.

Purine - adenine and guanine

6-mercaptopurine (6-MP) purine antagonist with inhibits DNA and RNA synthesis

Mechanism of 6-MP

https://youtu.be/XZD5bsXqkLs?si=GJv3C-DC5yBz0zMJ

Azathioprine, is a prodrug which is a derivative of 6-mercaptopurine, was developed to prevent its breakdown but
doesn't show significant improvement in antitumor activity. Instead, it's valuable as an immunosuppressive agent for
organ transplants

6-mercaptopurine will be converted to 6- thioinosine monophosphate (TIMP) by the enzyme Hypoxanthine-guanine

phosphoribosyltransferase (HGPRT). This nucleotide inhibits an early step in purine biosynthesis, preventing the conversion
of 5-phosphoribosylpyrophosphate into 5-phosphoribosylamine.
6- thioinosine monophosphate (TIMP) will be further converted into two derivatives: First is 6-thioguanine nucleotide which
will bind to DNA and mutate it, which is rejected and destroyed by the body via apoptosis; Second is
methylmercaptopurine ribonucleotide which blocks purine synthesis. Both pathways result in the inhibition of the
replication or proliferation of DNA cells.

Other pathways of 6-mercaptopurine include its conversion into thio-uric acid and Methyl mercaptopurine. By the
enzyme Xanthine oxidase (XO), it is converted into Thio-uric acid, which is the excretable form of the drug. This inhibition
of the enzymes responsible for the catabolic breakdown of the purine drugs can potentiate the drug’s antineoplastic
activity.

Allopurinol, a potent inhibitor of XO, which is often used in combination with purine drugs, significantly increases both the
potency and toxicity of 6-mercaptopurine by redirecting its metabolism into 6 TGN/ thioguanine nucleotide. The main
importance of allopurinol in adjunct therapy is the prevention of uric acid kidney toxicity caused by the release of
purines from destroyed cancer cells.

The primary antineoplastic action of 6-MP stems from its ability to inhibit purine biosynthesis, depriving cancer cells of
essential building blocks needed for growth and proliferation. While its incorporation into DNA and RNA also contributes
to its antineoplastic activity, the inhibition of purine biosynthesis is considered the major mechanism underlying its
effectiveness against cancer.

Treatment:
Acute lymphocytic leukemia (ALL)
Crohn’s disease

Adverse effects
Hepatotoxicity
NVD
Myelosuppression ( a decrease in bone marrow activity that results in reduced production of blood cells.)

Interaction:
Warfarin
Allopurinol SM2

Note that 6-MP should not take with Allopurinol

Antimetabolites' structure and design based on purine

structures began with an isosteric thiol/sulfhydryl group,
replacing the 6-hydroxyl group of hypoxanthine and
guanine. They are a class of drugs that mimic natural
purine nucleobases in order to penetrate and disrupt
cellular processes in cancer cells. The antineoplastic
activity of purine drugs depends on their relative
enzymatic activation and inactivation rates in tissues and
cells.
Adenine and Guanine is main nucleotide components
The primary antineoplastic action of 6-MP stems from its
ability to inhibit purine biosynthesis, depriving cancer cells
of essential building blocks needed for growth and
proliferation. While its incorporation into DNA and RNA
also contributes to its antineoplastic activity, the inhibition
of purine biosynthesis is considered the major mechanism
underlying its effectiveness against cancer.

Treatment:
Acute lymphocytic leukemia (ALL)
Crohn’s disease

Adverse effects
Hepatotoxicity
NVD
Myelosuppression ( a decrease in bone marrow activity
that results in reduced production of blood cells.)

Interaction:
Warfarin
Allopurinol SM2

Note that 6-MP should not take with Allopurinol

Folate is an essential B vitamin necessary for producing

red and white blood cells in bone marrow, producing
DNA and RNA, and transforming carbohydrates into
energy.
Folic acid, a crucial nutrient, plays a role in making DNA
and other important molecules in the body. However,
certain drugs, called antifolates, are designed to block
the actions of folic acid, particularly in cancer cells.

We have here the Methotrexate, anti metabolite drug

that is derived from folic acid.

Methotrexate is like a superhero against cancer. It's

derived from folic acid but tweaked to target cancer
cells specifically. By blocking an enzyme called DHFR,
Methotrexate stops cancer cells from making DNA, a
process vital for their growth and survival.

Imagine DHFR as a lock, and folic acid as the key that fits
perfectly into it. Methotrexate comes in and jams the
lock, preventing folic acid from doing its job. Without folic
acid, cancer cells can't make DNA properly, which slows
down their growth.

Methotrexate is a broad-spectrum antineoplastic agent

commonly used in the treatment of acute lymphoblastic
and myeloblastic leukemia and other lymphomas and
sarcomas.

While Methotrexate is powerful against cancer, it can

also affect normal cells, leading to side effects like bone
marrow suppression, pulmonary fibrosis, and stomach
problems. To minimize these effects, another drug called
Leucovorin is often given after Methotrexate to protect
normal cells from its harmful effects.

Pemetrexed is like a cousin to Methotrexate, but with a

broader scope. It not only blocks DHFR but also other
enzymes involved in making DNA and other important
molecules in cancer cells.

These drugs are sneaky—they hitch a ride into cancer

cells using special transport systems. Once inside, they're
converted into a form that stays trapped in the cell,
making them more effective against cancer.
PRODUCTS

- The drug is available as a 50-mg tablet for oral use.

*After MOA, proceed to next slide muna

- The drug does not enter the CNS in therapeutic

quantities.

- The methylated product has anticancer activity.

- Oxidation by xanthine oxidase yields inactive

metabolites. The concurrent use of xanthine oxidase
inhibitors such as allopurinol can enhance the potency of
mercaptopurine by inhibiting its catabolic breakdown,
however this may lead to life-threatening adverse effects.

Mercaptopurine is inactive in its parent form; it undergoes

intracellular phosphorylation by the enzyme
hypoxanthine-guanine phosphoribosyltransferase
(HGPRTase) to form a cytotoxic thioinosine
monophosphate (TIMP), which inhibits the first step of de
novo synthesis of purines. TIMP is converted to
thioguanine monophosphate, which, after
phosphorylation, is converted to 6-thioguanine
triphosphate (6-TGTP).

- The drug is available in 40-mg tablets for oral use.

- This is similar to mercaptopurine, which also competes

with hypoxanthine and guanine for the enzyme
hypoxanthine-guanine phosphoribosyltransferase
(HGPRTase).

Difference with Mercaptopurine:

- Also undergoes deamination

- Thioguanine is not a substrate for the enzyme xanthine

oxidase

Resistance tumor cell mechanism include (both

Mercaptopurine and Thioguanine):
- Decrease activity of HGPRTases
- Increase production of alkaline phosphatases
that inactivates toxic nucleotides
- The drug is available in 50-, 100-, 200-, and 1,000-mg
vials for IV use.

- The mechanism of action of methotrexate involves

inhibition of DHFR leading to a depletion of critical
reduced folates. The reduced folates are necessary for
biosynthesis of several purines and pyrimidines.

*After MOA, proceed to next slide muna

- Oral bioavailability varies with dose because of

saturable uptake processes (increases in the
administered dose will not correspond to increases in
amount of drug absorbed into the body), and high doses
are required to reach therapeutic levels in the CNS.

- Carboxylic acid drugs: penicillin, probenecid,

nonsteroidal anti-inflammatory agents, and aspirin.

Tumor cell resistance mechanism include:

- Decrease drug accumulation
- Changes in the drug sensitivity or activity of
dihydrofolate reductase
- Decreased formation of polyglutamates

- Methotrexate (MTX) enters the cell mainly through the

reduced folate carrier (RFC1) and, to a lesser extent,
through a folate receptor (FR). Upon entry, it gets
polyglutamated (MTX(Glu) n ) by folylpolyglutamate
synthase (FPGS). Polyglutamates of MTX are a superior
antifolate agent compared to MTX, capable of highly
potent irreversible inhibition of DHFR.

- Tetrahydrofolate is the active form of folic acid required

for purine and thymidylate synthesis. Folic acid is reduced
to tetrahydrofolate by dihydrofolate reductase (DHFR).

- MTX works in three ways:

a. Inhibition of DHFR
b. Inhibition of thymidylate (by inhibiting Thymidylate
Synthetase (TYMS) and also inhibition of Glycinamide
ribonucleotide transformylase (GRT) and
5-Amino-4-imidazolecarboxamide ribonucleotide
transformylase/IMP cyclohydrolase (ATIC)
c. Alteration of the transport of reduced folates.

- Inhibition of DHFR results in a deficiency of thymidylate

and purines and therefore a decrease in DNA synthesis,
repair and cellular replication. The affinity of DHFR to
methotrexate is far greater than its affinity for folic acid or
dihydrofolic acid, therefore large doses of folic acid
given simultaneously will not reverse the effects of
methotrexate.

*Toxic effects of methotrexate on normal cells may be

 reduced by the administration of folinic acid like
Leucovorin. This strategy is called Leucovorin rescue.

References:
Parker, W. B. (2009). Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chemical
Reviews, 109(7), 2880–2893. https://doi.org/10.1021/cr900028p

Wilson and Grisvold