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ANTICANCER AGENTS
ALKYLATING AND
METALLATING AGENTS
Patrick: An Introduction
to Medicinal Chemistry 5e Chapter 21
1
Notes
•Contain highly electrophilic groups
•React with several nucleophiles groups in DNA
•Form covalent bonds to nucleophilic groups in DNA
(e.g. 7-N of guanine)
•Prevent replication and transcription
•Useful anti-tumour agents
•Toxic side effects (e.g. alkylation of proteins)
•Used as anticancer agents
•Can cause interstrand and intrastrand crosslinking if two
electrophilic groups are present
•Alkylation of nucleic acid bases can result in miscoding
(Incorrect coding)
Alkylating agents
2
Interstrand crosslinking Intrastrand crosslinking
Crosslinking
Alkylating agents
X XNu
Nu
X X
Nu
Nu
NuNu Nu
Nu
• In genetics , crosslinking of DNA occurs when various exogenous or endogenous
agents react with two different positions in the DNA.
• This can either occur in the same strand (intrastrand crosslink) or in the opposite
strands of the DNA (interstrand crosslink).
• Crosslinks also occur between DNA and protein .
• DNA replication is blocked by crosslinks, which causes replication arrest and cell
death if the crosslink is not repaired. 3
Nucleophilic groups on nucleic acid bases
nucleophil icgroups
n u c l e o p h i l i cg r o u p s
NH2
N
N
N
N
1
3
R
H2N
HN
N N
N
O
7
R
NH2
N
NR
O
3
Cytosine Guanine Adenine
Alkylating agents
4
Miscoding resulting from alkylated nucleic acid bases
Guanine prefers
keto tautomer
Normal base pairing
Alkylated guanine prefers
enol tautomer
Abnormal base pairing
Thymi neAl kyl at ed guani ne
N
NH
O
O
Me
R
N
N
N
NHO
R
H2N
DRUG
Alkylating agents
N
N
NH2
OR
Cytosine Guanine
HN
N
N
NO
H2N
R
5
Chlormethine
Notes
•Used medicinally in 1942
•Example of a nitrogen mustard
•Causes intrastrand and interstrand crosslinking
•Prevents replication
•Monalkylation of guanine also possible
•Analogues with better properties have been prepared
Alkylating agents
H3C N
Cl
Cl
6
Mechlorethamine
H3C N
Cl
Cl Cl
NH3C
Aziridine ion
G = Guanine
DNA
G
N
HN
N
N
N
NNH
N
O
O
NH2
NH2
G
DNA
N
N
N
NN
HN
NH
N
O
NH2
O NH2
H3C N
Cl
DNA
N
N
N
NNH
N
O
NH2
N
HNO NH2
NH3C
DNA
CH3
N
N
N
N
N
N
HN
NH
N
O NH2
O
NH2
Crosslinked DNA
Mechanism of action
Alkylating agents
Chlormethine
7
N
Cl
Cl
•Aromatic ring is electron withdrawing
•Lowers nucleophilic strength of nitrogen
•Less reactive alkylating agent
•Selective for stronger nucleophiles (e.g. guanine)
•Less side reactions and less toxic
•Aromatic ring is present
•Less reactive alkylating agent
•Mimics phenylalanine
•Transported into cells by transport proteins
N
Cl
ClCO2H
H2N
H
Melphalan
N
Cl
Cl
HN
NH
O
O
Uracil mustard
•Uracil ring is electron withdrawing
•Less reactive alkylating agent
•Mimics a nucleic acid base
•Attached to a nucleic acid building block
•Concentrated in fast growing cells (tumours)
•Used vs leukaemia
•Some selectivity
Alkylating agents
Chlormethine analogues
8
•Urethane group is electron withdrawing
•Lowers nucleophilic strength of nitrogen
•Alkylating group is attached to estradiol
•Steroid is hydrophobic
•Capable of crossing cell membranes
OHMe
ON
O
Cl
Cl
H
HH
H
Estramustine
Urethane
Alkylating agents
Chlormethine analogues
9
O
P
H2N O
NR2
O
H
H
O
P
NH O
NR2
HO
Cytochrome
P450 enzymes
H+
HO
P
Cl
Cl
N
OH2N
H
O
Alkylating agent Acrolein
O
P
NH O
NR2
Cyclophosphamide
Notes
•Cyclophosphamide is the most commonly used alkylating agent
•Non-toxic prodrug
•Orally active
•Acrolein is associated with toxicity
Alkylating agents
Cyclophosphamide
10
Side effect which results in inflammation, oedema, bleeding, ulceration,
and cell death. This is caused by the metabolite acrolein and can be
countered by increased fluid intake or by administering mesna
Mesna
Phosphoramide mustard
Cl
N2 + HO
Alkylatingagent
ClN N
N
O
O
R
H
Nitrosoureas
Notes
Decompose in the body to form an alkylating agent and a
carbamoylating agent
O
N NH
NO
Cl Cl
Ca r m u s t i ne
O
N NH
NO
Cl
Lom us t i ne
O C N R
ClN
NOH
+
Isocyanate(carbamoylating agent)
H O
Alkylating agents
11
ClDNA
X Y
Cl
X Y
Cr oss-l i nki ng
DNA DNA
Al kyl at i onAl kyl at i ngagent
Notes
•Alkylating agent causes interstrand crosslinking
•Cross linking between G-G or G-C
•Carbamoylating agent reacts with lysine residues on proteins
•May inactivate DNA repair enzymes
O C N R
I s o c y a n a t e
P r o t e i n - L y s -N H2P r o t e i n - L y s -N H
O
HN RC a r b a m o y l a t i o n
Alkylating agents
Nitrosoureas
12
Busulfan
Notes
•Synthetic agent used as an anticancer agent
•Causes interstrand crosslinking
OO
SMe
OO
SMe
OO
Alkylating agents
OO
SMe
OO
SMe
OO
HN
N N
N
O
H2N
Guanine
DNA
N
N
DNA
-MeSO3-
OSO2Me
N
N
DNA
-MeSO3-
N
N
DNA
N
N
DNA
13
Dacarbazine (DTIC-Dome ®)
HN N
CONH2NN
NH3C
CH3
•Prodrug activated by demethylation in liver
•Decomposes to form a methyldiazonium ion
•Alkylates guanine groups
HN N
CONH2NN
NH3C
CH3
Cyt P-450
liver
HN N
CONH2NN
N
CH3OHH
HN N
CONH2NHN
N
CH3
-CH2O
H
HN N
CONH2H2N
N N CH3
AIC
Methyldiazonium ion
N2 + CH3
Alkylating agents
14
Dacarbazine is bioactivated in liver by demethylation to methyl triazeno
imidazole carboxamide "MTIC" and then to diazomethane, which is an
alkylating agent
(Dimethyl triazeno) imidazole carboxamide
DTIC
AIC= Amino imidazole carboxamide
MTIC
15
Synthesis of Dacarbazine
2-Amino imidazole
carboxamide
Nitrous acid Ethylamine
Diazide imidazole
carboxamide (Dimethyl triazeno)
imidazole carboxamide
O
O
N
H2N
Me
CH2OCONH2
NH
OMe
Mitomycin C
•Prodrug activated in the body to form an alkylating agent
•Converted to alkylating agent in the body
•One of the most toxic anticancer drugs in clinical use
Alkylating agents
16
OH
OH
N
H2N
Me
CH2
NH2
NH
NH
HN
N N
N
O
HN
N N
N
O Guanine
Guanine
H2N-DNA
O
O
N
H2N
Me
CH2OCONH2
NH
OMe
O
OH
N
H2N
Me
CH2OCONH2
NH
H
-MeOH
O
OH
N
H2N
Me
CH2OCONH2
NH2
Ring
opening
-H +
Alkylating agent
OH
OH
N
H2N
Me
CH2
NH2
NH-DNA
NH-DNA
-CO2
-NH3
Crosslinked DNA
OH
OH
N
H2N
Me
CH2OCONH2
NH
OMe
Reduction
OH
OH
N
H2N
Me
CH2
NH2
NH-DNA
O
C
O
NH2
H2N-DNA
H
H
H
17
NH
Me
O
N
O
NH
OMe
OH
N
O
NH
OMe
OH
N
O
NH2
Alkylating agents
CC1065
•Naturally occurring agent
•Binds to minor groove of DNA
•Alkylates adenine bases
•1000 times more active than cisplatin in vitro
•CC1065 is the lead compound for Adozelesin
18
NH
Me
O
N
O
NH
HN
O
O
Alkylating agents
Adozelesin
•Simplified synthetic analogue of CC 1065
•Possible use in antibody-drug conjugates
19
CC 1065
Adozelesin
•Neutral inactive molecule acting as a prodrug
•Platinum covalently linked to chloro substituents
•Ammonia molecules act as ligands
•Activated in cells with low chloride ion concentration
•Chloro substituents are replaced with neutral water ligands
•Produces positively charged species that react with DNA
Pt
NH3Cl
NH3Cl
Cisplatin
PtNH3Cl
NH3Cl
H2O
Pt
NH3H2O
NH3Cl
Pt
NH3H2O
NH3H2O
+ 2 +
+
DNAPt
NH3DNA
NH3DNA
C i s p l a t i n
Metallating agents
• Binds to DNA in regions rich in guanine units
• Intrastrand links rather than interstrand
• Inhibits transcription 20
PtH3N
H3NO
O
O
O
Pt
OAc
OAc
ClCl
H3NNH2
H2N
NH2
Pt
O
O O
O PtNH3Cl
NCl
Me
Carboplatin
Less side effects
JM216
First orally
active analogue
Oxaliplatin
Approved in 1999
Picoplatin
Metallating agents
Cisplatin analogues
21
22
KEY POINTS
Alkylating agents contain electrophilic groups that react with
nucleophilic centres on DNA. If two electrophilic groups are present,
interstrand and/or intrastrand cross-linking of the DNA is possible.
Nitrogen mustards react with guanine groups on DNA to produce
cross-linking.
The reactivity of the agents can be lowered by attaching electron-
withdrawing groups to the nitrogen to increase selectivity against
DNA over proteins.
Incorporation of important biosynthetic building blocks aids the
uptake into rapidly dividing cells.
Cisplatin and its analogues are metallating agents which cause
intrastrand cross-linking. They are commonly used for the treatment
of testicular and ovarian cancers.
CC-1065 analogues are highly potent alkylating agents which are
being considered for use in antibody–drug conjugates.
23
Clinical aspects of alkylating and metallating agents
• Chlormethine has been used for the treatment of Hodgkin's lymphoma (cancer
of the lymphatic system) as part of a multidrug regime. The related structure
melphalan is currently used in the treatment of multiple myeloma, as well as
advanced ovarian and breast cancers.
• Uracil mustard has been used successfully in the treatment of chronic
lymphatic leukaemia.
• Estramustine can be given orally and is used predominantly for the treatment
of prostate cancer.
• Chlorambucil is an orally active drug used primarily in the treatment of
chronic lymphocytic leukaemia and Hodgkin's disease
• Bendamustine was approved in 2008 for the treatment of chronic lymphatic
leukaemia and lymphomas. Resistance to alkylating agents can arise
through reaction with cellular thiols and decreased cellular uptake.
Bendamustine Chlorambucil
24
Clinical aspects of alkylating and metallating agents
Cyclophosphamide is given orally or intravenously, and is widely used for
the treatment of leukaemias, lymphomas, soft tissue sarcoma, and solid
tumours.
Haemorrhagic cystitis is a rare, but serious, side effect which results in
inflammation, oedema, bleeding, ulceration, and cell death.
This is caused by the metabolite acrolein and can be countered by increased
fluid intake or by administering mesna .The related drug ifosfamide is given
intravenously along with mesna.
Mesna
Lomustine and carmustine are lipid-soluble and can cross the blood–brain
barrier.
As a result, they have been used in the treatment of brain tumours and
meningeal leukaemia.
Lomustine can be given orally, but carmustine is given intravenously because
it is rapidly metabolized. Carmustine implants have also been approved.
25
Clinical aspects of alkylating and metallating agents
Streptozotocin has been used for the treatment of pancreatic islet cell
carcinoma. There is a specific uptake of the drug into the pancreas where it
carbamoylates proteins.
Busulfan is given orally in the treatment of chronic myeloid leukaemia and may
increase the life expectancy of patients by about a year. It is also administered alongside
cyclophosphamide prior to stem cell transplantation. It acts selectively on the bone
marrow and has little effect on lymphoid tissue or the gastrointestinal tract. However,
excessive use may lead to irreversible damage to the bone marrow. Resistance to
busulfan is related to the rapid removal and repair of the DNA cross-links.
Cisplatin is a very useful antitumour agent which is used alone or in combination with
other drugs for the intravenous treatment of lung, cervical, bladder, head, neck,
testicular, and ovarian tumours. It is also used in various combination therapies to treat
other forms of cancer.
Unfortunately, cisplatin is associated with very severe nausea and vomiting, but the
administration of the 5-HT 3 receptor antagonist ondansetron is effective in combating
this problem.
Streptozotocin
26
Clinical aspects of alkylating and metallating agents
Carboplatin is now preferred over cisplatin for the intravenous treatment of
advanced ovarian tumours, and is also used to treat lung cancers. It is
better tolerated than cisplatin and has less severe side effects.
Oxaliplatin was approved in 1999 for the treatment of colorectal cancer and
shows a better safety profile than cisplatin or carboplatin. It is used in
combination with fluorouracil and folinicacid
Tumour resistance to cisplatin and similar agents has been attributed to a
number of factors.
Cisplatin requires a transporter protein in order to enter the cell and
resistance can occur if there are low levels of the transport protein.
The activated species arising from cisplatin reacts easily with cellular
thiols, such as glutathione, and resistance can occur if these thiols are
present in high concentration.
The agent is ‘mopped up’ before it has a chance to react with DNA.
Finally, resistance may arise because of increased efflux of the drug from
the cell.
Carboplatin
27
Clinical aspects of alkylating and metallating agents
Dacarbazine is used clinically in combination therapies for the treatment
of melanoma and soft tissue sarcomas.
Procarbazine is most often used for the treatment of Hodgkin's disease
and is given orally.
Temozolomide is used for the treatment of certain types of brain tumour
and is administered orally in capsules at least one hour before a meal.
Mitomycin C is used intravenously for the treatment of upper
gastrointestinal and breast cancers.
Mitomycin C can also be used to treat superficial bladder cancers.
Mitomycin C has many side effects and is one of the most toxic
anticancer drugs in clinical use. Prolonged use can lead to permanent
bone marrow damage.
Temozolomide Procarbazine
