Nucleic Acids in Chemistry and Biology

dNTPs required for DNA synthesis. However, the mechanisms by which these drugs achieve selectivity
for their target cells are not clearly understood.
Fludarabine in combination with the alkylating agent cyclophosphamide (see Section 3.7.1.2) has been
highly successful in clinical treatments for CLL. Clinical trials of the nucleoside analogue pentostatin (Figure
3.85) together with cyclophosphamideare currently in progress. Cytarabine (cytosine arabinoside or ara-
C) is an analogue of 2-deoxycytidine in which the 2-hydroxyl is sterically inverted (Figure 3.85). It is used
primarily for the treatment of acute myelocytic leukaemia. Ara-C is first converted into its monophosphate
(araCMP) by deoxycytidine kinase. The monophosphate then reacts with appropriate kinases to form the
di- and triphosphate nucleotides. AraCTP is believed to be the key active component and its accumulation
causes potent inhibition of DNA synthesis in many cells. While this nucleotide is a competitive inhibitor
of many DNA polymerases, it is also a substrate for some DNA polymerases. It thereby becomes incorp-
orated into DNA, leading to inhibition of chain elongation. Unlike many antimetabolites, the effects of ara-
C are directed almost exclusively towards DNA and it has little or no effect on RNA synthesis or function.
Some evidence indicates that the inhibition of synthesis is secondary to incorporation of araCMP into DNA.

3.7.1.1.3 Fluorouracil. 5-Fluorouracil (5FU) must first be converted into the nucleotide to be active

as a cytotoxic agent. The 5ribonucleotide (5FUMP) is formed viaseveral different pathways. 5FUMP is
then incorporated into RNA and is also converted into the deoxynucleotide (FdUMP) by ribonucleotide
reductase. FdUMP is also be formed by the direct phosphorylation of FdUrd by thymidine kinase.
The formation of FdUMP is crucial for the cytotoxicity of 5FU. This is because FdUMP inhibits the enzyme
thymidylate synthetase and so blockades the formation of dTTP, one of the four essential constituents of DNA.
Thymidylate synthetase catalyses the methylation of dUMP in a multi-step process that involves formation
of a ternary complex between dUMP, methylenetetrahydrofolate and the enzyme. This complex reacts further
by loss of a proton from position-5 of the uracil ring. FdUMP (Figure 3.86) also forms such a ternary complex
but its breakdown would require loss of a fluorine cation. Thus the complex is sufficiently stable so that the
enzyme cannot turnover. DNA synthesis is thus inhibited until the drug is removed and de novoenzyme syn-
thesis begins. dFUMP is thus a suicide inhibitor. Dan Santi’s extensive work on this intermediate has shown that
a cysteine residue in the enzyme is the nucleophile that adds to position-6 of the pyrimidine ring (Figure 3.86).
As FdUrd is converted into FdUMP directly in a single step, it is a potent inhibitor of dTMP synthetase and
is often effective in the low nanomolar concentration range. On the other hand 5FU, though less expensive,
is only effective at micromolar concentration where further active metabolites are formed including 5FUTP.
This can be incorporated into RNA in place of UTP and it affects the function of both rRNA and mRNA.
Although 5FU and some of its pro-drugs are widely used in the treatment of common solid tumours, they
show little selectivity and are therefore toxic, causing suppression of the immune system.

3.7.1.1.4 Antifolates^124 – Methotrexate. The importance of folates in tumor cell growth was demon-

strated by Farber in 1948, when aminopterin was shown to produce remissions in leukaemia. Antifolates
produced both the first striking remissions in leukaemia and the first cure of a solid tumour, choriocarcinoma.

Nucleosides and Nucleotides 127

O

HO

O P O

O

O

N

NH

O

O

F

O

HO

O P O

O

O

N

NH

O

O

ENZ S

F

CH 2

N

H

FdUMP

co

fa

cto

r

Figure 3.86 Structure of FdUMP (left) and its suicide complex with thymidylate synthetase (right)