Lecture 4

Antimetabolite and Natural

Products
 II- Antimetabolite
An antimetabolite is a chemical that
inhibits the use of a metabolite, which is
another chemical that is part of normal
metabolism. Such substances are often
similar in structure to the metabolite that
they interfere with, such as the antifolates
that interfere with the use of folic acid. The
presence of antimetabolites can have toxic
effects on cells, such as halting cell growth
and cell division, so these compounds are
used as chemotherapy for cancer.
 Cancer treatment
Antimetabolites can be used in cancer
treatment, as they interfere with DNA
production and therefore cell division and
tumor growth. Because cancer cells spend
more time dividing than other cells,
inhibiting cell division harms tumor cells
more than other cells. Antimetabolite drugs
are commonly used to treat leukemia,
cancers of the breast, ovary, and the
gastrointestinal tract, as well as other types
of cancers.
 NH2 O O O
H H H
N N N N
N N N N

N N N O N N H2N N N
N
H H H H H

Adenine Hypoxanthine Xanthine Guanine

O
NH2 O
H
H CH3 N
N N
O N
O N O N
H
H H
Cytosine Thymine Uracil
 Glycosidic binding

Nucleotide monophosphate

Nucleotide diphosphate nu

Nucleotide triphosphate
 OH H COOH
N CH2 N CONH COOH Folic acid
N
 1) Folic acid antagonist (Methotrexate)
H2N N N

NH2 CH3 COOH

N CH2 N CONH COOH Methotrexa
N

H2N N N
Folic acid cycle is the source of one
carbon moiety needed to convert uracil to
thymine.
Methotrexate inhibits dihydrofolate
reductase enzyme which converts
dihydrofolic acid to tetrahydrofolic acid
accordingly stops the folic acid cycle and
depletes the cellular system from thymine
which is needed for DNA biosynthesis
 R
O R
O H
N
HN N N
Dihydrofolate HN N
H reductase
H2N N N H
H2N N N
H
H
Dihydrofolic acid THF acid NH2
HOCH2CHCOOH

pyridoxal phosphate
Thymine Thymidylate
Thymidilate
synthase
synthase H2NCH2COOH

Uracil
O N
N
HN

H2N N N
H
 R R

N N

O O
N N
HN HN
oxidation
H2N H2N N N
N N H
H
5,10-methyenyltetrahydrofolic acid 5,10-methyeneltetrahydrofolic acid

hydrolysis R
R CHO
O
O H N
N HN N
HN N
isomerase H
CHO H2N
H2N N N
N N H
H

10-formyltetrahydrofolic acid 5-formyltetrahydrofolic acid

 N-formyltetrahydrofolic acid (Leucovorin,
citrovorum)
Is formyl donner to 5-amino imidazole-4-
carboxamide in biosynthesis of purine
 Carries the formimino group for synthesis of
formiminoglycine a precursor of purine

 SAR:
 Alkylation of amino groups decreased
activity
 Partial reduction, removal or relocation of
heterocyclic nitrogen decrease activity
 Glutamine are donner of nitrogen atoms of
purine and guanine

 Assay: non aqueous titration, UV

Metabolism of methotrexate

(2,4-diamino-N10-methylpteroic acid)
Metabolism:

1- Converted to 7-OHMTX in liver by aldhyde oxidase

2-Converted to diaminomethyl peteroic acid (DAMPA) by bacterial

carboxypeptidase enzyme in GIT
Rational:
- MTX enter the cells through memberane folate binding protein
(MFBP).
-Some tumours lack or have reduced MFBP, therefore, the uptake of
MTX in these tumours is not achieved after normal doses.

- High dose MTX is required to increase the extracellular levels of MTX.

Under these condition, MTX is transported by passive diffusion.

- In high dose MTX therapy, the uptake of MTX into tumour cells is
highly selective because the affinity of MFBP to MTX is higher (1 uM)
than normal cells (90 uM).

- Also, high dose MTX therapy improves its penetration into tumour
sanctuary sites such as testis, CNS and blood brain barrier.
 Leucovorin
 Prevent lethal effects of methotrexate on
normal cells by:
 overcoming the blockade of tetrahydrofolic
acid production.
 Inhibit active transport of methotrexate into
cells and stimulate its efflux.
 Thymine deficiency (lethal effects)
 Pemetrexed

It works by inhibiting three enzymes used in purine and

pyrimidine synthesis—thymidylate synthase (TS),
dihydrofolate reductase (DHFR), and glycinamide
ribonucleotide formyltransferase (GARFT). By inhibiting
the formation of precursor purine and pyrimidine
nucleotides, pemetrexed prevents the formation of DNA
and RNA, which are required for the growth and survival of
both normal cells and cancer cells.
should take folic acid and vitamin B12 supplement even if
levels are normal when they are on pemetrexed therapy (In
clinical trials for mesothelioma, folic acid and B12
supplementation reduced the frequency of adverse effects.
Antimetabolite purine and pyrimidine
Anti-metabolites as a purine (azathioprine,
mercaptopurine) or a pyrimidine, chemicals
that become the building-blocks of DNA.
They prevent these substances from
becoming incorporated into DNA during the
S phase (of the cell cycle), stopping normal
development and cell division. Anti-
metabolites also affect RNA synthesis.
However, because thymidine is used in DNA
but not in RNA (where uracil is used
instead), inhibition of thymidine synthesis
via thymidylate synthase selectively inhibits
DNA synthesis over RNA synthesis.
 Due to their efficiency, these drugs
are the most widely used cytostatics.
Competition for the binding sites of
enzymes that participate in essential
biosynthetic processes and
subsequent incorporation of these
biomolecules into nucleic acids,
inhibits their normal tumor cell
function and triggers apoptosis, or
the cell death process. Because of this
mode of action, most antimetabolites
have high cell cycle specificity and
can target arrest of cancer cell DNA
replication
 Purine and pyrimidine antagonists
The purine and pyrimidine antagonists SH
may be incorporated into nucleic acid N N

biosynthesis, forming abnormal nucleic N N H

H
acids.
1- 6- Mercaptopurine (6MP) 6-Mercaptopurine
(6-MP)

immunosuppressive medication. It is used to treat acute

lymphocytic leukemia, Crohn's disease, and ulcerative
colitis. It is a thiopurine. O
N
Must be Converted to HN
N
N
ribonucleotide( 6-thioinosinate) by H2N N
H

Hypothanthine-guanine phosphoribosyl
transferase. Azaguanine
6-thioinosinate is

1- inhibitor of conversion of phosphoribosyl

pyrophosphate into phosphoribosylamine
2- inhibit conversion of inosinic to adenylic

3- inhibit oxidation of inosinic to xanthylic acid

4- act as substrate for methyl transferase

 Azathioprine

 Azathioprine, is an immune suppressive

medication. It is used in rheumatoid
arthritis, Crohn's disease, ulcerative colitis,
and in kidney transplants to prevent
rejection. It is Prodrug of 6-mercatopurine

6-[(1-Methyl-4-nitro-1H-imidazol-5-yl)
sulfanyl]-7H-purine
 Metabolic degradation
 By guanase gives 6-thioxanthine which
is oxidized by xanthine oxidase to 6-
thiouric acid.
Allopurinol is adjuvant to chemotherapy
prevent uric acid toxicity, by inhibition
of xanthine oxidase
Assay: non aqueous titration
Mechanism of action:

1- Incorporation of FUTP (false nucleotide) instead

of UTP into RNA, interferes with RNA synthesis
and function.

2- Inhibition of thymidylate synthase by (FdUMP)

leads to depletion of TMP and TTP.

3- Incorporation of FdUTP into DNA may affect

DNA stability.
 Pyrimidine antagonist
SH SH
H
O
F
N N
N N
1- 5-Flourouracil (5 FU)
N N
H2N
N

N N O N
H H H
5-Fluoro-2,4-(1H,3H)pyrimidine-dione
6-Mercaptopurine 6-thioguanine 5-Fluorouracil

5-FU mimics Uracil biologically with

(6-MP) (6-TG) (5-FU)

stronger binding to thymidylate synthase

O .
CF3
HN
Why we use 5- fluoro?
N
O
O N
HN
HO
Increased acidity of fluorine cause molecule to
N
- N
O
H2N N
bind stongly to Enz. H

OH
Size of fluorine nearlyAzaguanine
- size of H. Trifluorothymidine

so 5-FU prevents the methylation of C5 by

thymidylate synthase causing its inhibition.
Use: neoplasm of breast, G.I. tract, carcenoma
of pancreas, urinary bladder, colon

NH
Conversion of uridylate
NH to thymidylate 2
2

O
HN N
HN N

HN
O NH
O
O NH

O N N
H 2 O 3P N
HN
O
H H SH N
N
O N
H H H 2O 3P
Enzyme S
OH H
O
Enz.
2`-Deoxyuridylate H H

H H
OH H
R
R
 NH2

O
HN N
CH3
HN
O NH

H 2O3P O N N

O
SH
H H HN
Enzyme
H H
OH H

2`-Deoxythymidylate

R
 O NH2
CF3
2- Cytarabine
O (ara c) HN N

Cytarabine
HN
N
N
is cytotoxic
HO
O to
N
HO
O N

O
Ha
N wide variety of actively O
N N HO
2
H
dividing mammalian cells OH OH

interfering
Azaguanine with pyrimidine synthesis Cytarabine
Trifluorothymidine

use: acute myelocytic leukemia

- Gemcitabine: analogue of cytarabine,
gemcitabine triphosphate (active form)
use: solid tumor, first-line treatment alone for
pancreatic cancer

Metabolism:
 Capecitabine
•Colorectal cancer , Breast cancer
Gastric cancer and Oesophageal
cancer

•Prodrug of 5-flurouracil
•Metabolism:
 Antimetabolites may also be antibiotics, such
as sulfanilamide drugs, which inhibit
dihydrofolate synthesis in bacteria by
competing with para-aminobenzoic acid
(PABA). PABA is needed in enzymatic
reactions that produce folic acid, which acts as
a coenzyme in the synthesis of purines and
pyrimidines, the building-blocks of DNA.
Mammals do not synthesize their own folic
acid so they are unaffected by PABA
inhibitors, which selectively kill bacteria.
Sulfanilamide drugs are not like the antibiotics
used to treat infections. Instead, they work by
changing the DNA inside cancer cells to keep
them from growing and multiplying.
 Natural products
1) Antibiotics
 Antitumor antibiotics are a class of
antimetabolite drugs that are cell cycle
nonspecific. They act by binding with DNA
molecules and preventing RNA (ribonucleic
acid) synthesis, a key step in the creation
of proteins, which are necessary for
cancer cell survival. Anthracyclines are
anti-tumor antibiotics that interfere with
enzymes involved in copying DNA during
the cell cycle.
1- Actinomycines
Phenoxazone 1,9-dicarboxylic
acid (actinocin)
Pentapeptide lactones
 Two identical pentapeptide
lactone (isoactinomycins) L MeVal L MeVal
Different lactones are called Sar Sar
anisoactinomycins D O L Pro L Pro O
actinomycin) D Val D Val
 Mechanism of action
- -
L Thr L Thr

 actinomycin D is have the C O C O

ability to inhibit transcription. N NH2
Actinomycin D does this by
binding DNA at the O O
transcription initiation CH3 CH3
complex and preventing
Phenoxazone ring
elongation of RNA chain by
RNA polymerase
 SAR
 Opening of a lactone ring or change the
stereochemistry of an amino acid
abolishes activity.
 Replacement of 2-amino, 4,6-methyl
Reduce
group by other substituents, activity
 Change in substituents influence their
binding , less active
 Assay: Chromatography
2- Anthracyclines
 Produced naturally from O OH O

Streptomyces species OH
CH2R 2

 They have a planar R1 O OH O

anthraquinone nucleus. glycosidic
linkage
O
They chelate with Ca, CH3
amino sugar
ferrous. OH
NH2

 They exhibit biological

effect Similar to Daunorubicin R1 = OCH3, R2 = H
Doxorubicin R1 = OCH3, R2 = OH
actinomycines, Idarubicin R1 = H, R2 = OH
 Light sensitive, should
be handled with care
 H2N O
H NH2
3- Bleomycins
N COR
CONH2
N
N N CH3 H O
S
O HO N H
H2N O N N

Mechanism of action
O
CH3 N
H N CH3 HO CH3 S
H
N
O
They form chelates with ferrous ions.
complex gives rise to OH and super oxide
OH N
HO O O
radicals which cause cleavage of
H

OH
O OH
phosphodiester bonds of DNA,
OH
OH
causes degradation of DNA strand and
CONH2 death of the cell

Bleomycin A2 R = -NH(CH2)3-S+(CH3)2
Bleomycin B 2 R = -NH(CH2)4 -NHC(=NH)NH2

Bleomycins is inactivated by bleomycins hydrolase enz. –

increases pKa of amino group, poor binding to DNA, chelation,

reduce OH radicals. Resistance

Topoisomerase I and Topoisomerase II
2- Plant product
1- Etoposide
Is a semi synthetic derivative of
podophyllotoxin. It induce strand breaks
in DNA mediated by interaction with
topoisomerase II
2- Vinca Alkaloids (Vinblastin,
Vincristine and Vindesine)
They cause mitotic arrest by promoting
the dissolution of microtubules in cells
3- Paclitaxol (Taxol)
It is mitotic inhibitor. Used for breast,
lung and esophagus cancers cancer
 TUBULIN and MICROTUBULES
•Taxoid Site

•Vanca Alkaloid
Domain

Colchicine Site

•Globular Protein •Tubulin Polymers

 MITOTIC SPINDLE

•Composed of Microtubules and associated proteins

•Needed for cellular division
 TAXOL
•Pacific Yew Tree
•Anti-cancer agent used in treatment of
•Ovarian
•Testicular
•Lung
•Breast

Mechanism of action:
Stabilizes microtubules and
prevents disassembly and
re-polymerization
 COLCHICINE
• Meadow saffron or
autumn Crocus
poisonous

NHCOCH3

H3CO
•Mechanism of action :
H3CO O •Tubulin binder
OCH3
Inhibits tubulin polymerization
OCH3
Copyright @ 1997 Alice B. Russell, James W. Hardin, Larry Grand
COLCHICINE
Monoclonal antibodies

Monoclonal antibodies (mAb or moAb) are

antibodies that are made by identical
immune cells that are all clones of a
unique parent cell. Monoclonal antibodies
can have monovalent affinity, in that they
bind to the same epitope (the part of an
antigen that is recognized by the
antibody). Bispecific monoclonal
antibodies can also be engineered, by
increasing the therapeutic targets of one
single monoclonal antibody to two
epitopes.
 Monoclonal antibodies
Trastuzumab:
Selectively binds with high affinity to
extracellular domain of human epidermal
growth factor receptor 2 protein
Use: breast cancer
Do not mix with other medication, use
immediately.
Mabthera:
monoclonal antibodies directed against CD20
antigen found on the surface of normal and
malignant b lymphocyte
Use: ttt of follicular CD20 positive, B cell NHL
( non hodjekin lymphoma ) in combination with
CVP, reumatoid arthritis in combination with
methotrexate
 Acetominophen and antihistamines should be
taken to decrease infusion reaction