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258 12 Enzymatic inhibition: other antibacterial agents
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Scheme 12.17: Representation of 4-quinolone-3-carboxylic acids as 4-hydroxy-3-carboxyquinolinium salts.
12.11 Fundamentals
Sulfonamides
Mechanism of action
Synthesis
Examples of other antimetabolites
Trimethoprim
Antibacterial agents affecting protein synthesis
Aminoglycosides
Tetracyclines
Chloramphenicol
Erythromycin
Aminoacridines
1,8-Naphthyridine and fluoroquinolones
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of nitrogenous bases
13.1 Goals
– Knowledge of the preponderant role of some enzymes in various pathologies
– Knowledge of the various ways of inhibiting enzymes and/or coenzymes
– Use of some inhibitors as antitumor and antiviral agents
13.2 Introduction
In this chapter we will first discuss the anticancer drugs that fall within the category of
drugs that interfere with DNA synthesis. However, other categories of drugs such as
those that interact directly with DNA (alkylating agents, metal complexes that bind to
DNA, derivatives causing DNA degradation and antisense agents) will not be treated because they are not enzymatic inhibitors. A more modern approach, such as tyrosine kinase inhibitors, will be discussed later. Finally, antivirals will also be treated briefly.
13.3 Nucleic acids
Cells contain two types of nucleic acids: RNA and DNA. These complex structures are
essential in the biosynthesis of proteins. DNA is also the genetic material of cells.
Smooth degradation of nucleic acids produces a mixture of acids known as nucleotides: purine and pyrimidine bases, a phosphate, and a pentose moiety (Schemes 13.1
and 13.2). Phosphate group can be selectively removed by careful hydrolysis and the
nucleotide is converted into a nucleoside (Fig. 13.2). In a nucleotide, the C-1 of the sugar
is bound to the N-1 of the pyrimidine or the N-9 atoms of the purine. The phosphoric
acid forms an ester with the C-5ʹ of the sugar (Fig. 13.1).
13.4 The birth of cancer chemotherapy: accident and research
Nitrogen mustards are cytotoxic organic compounds with the bis(2-chloroethyl)amino
functional group. Although originally produced as chemical warfare agents, they
were the first chemotherapeutic agents for treatment of cancer.
During World War II, nitrogen mustards were studied at the Yale School of Medicine, and in December 1942, they started classified human clinical trials of nitrogen
mustards for the treatment of lymphoma. In early December of 1943, an incident dur-
https://doi.org/10.1515/9783111316888-013
260 13 Enzymatic inhibition: inhibitors of biosynthesis of nitrogenous bases
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Fig. 13.1: DNA and RNA degradation.
ing the air raid on Bari, Italy, led to the release of mustard gas that affected several
hundred soldiers and civilians. Medical examination of the survivors showed a decreased number of lymphocytes. After World War II was over, the Bari incident and
the Yale group’s studies eventually converged prompting a search for other similar
compounds. Due to its use in previous studies, the nitrogen mustard known as mustine became the first chemotherapy drug. It is still in clinical use, often in combination
with other drugs (Scheme 13.1).
The reaction of major importance in the cytotoxic effect of nitrogen mustards is the
formation of a covalent bond with the N-7 of a guanine base of DNA. There is a good
correlation between DNA cross-linking and inhibition of cell growth, and cross-links are
generally believed to be responsible for the antitumor activity of bifunctional alkylating
Fig. 13.2: Examples of a nucleoside and a nucleotide.
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13.5 Thymidylate synthase inhibitors 261
Scheme 13.1: Cross-linking of DNA by means of mustine.
agents. The cross linkage prevent separation of the individual strands, thereby mainly
inhibiting DNA replication. DNA replication is the biological process of producing two
identical replicas of DNA from one original DNA molecule.
13.5 Thymidylate synthase inhibitors
Rapidly dividing cells require an abundant supply of deoxythymidylate (dTMP) to synthesize their DNA. The susceptibility of these cells to inhibiting the synthesis of dTMP
has been exploited in cancer chemotherapy. Uracil is not a component of DNA. Instead, DNA contains thymine, the methylated analog of uracil. Deoxyuridylate (dUMP)
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is methylated to dTMP by means of thymidylate synthase. The methyl donor of this
reaction is N5,N10-methylenetetrahydrofolate. In this reaction tetrahydrofolate is oxidized to dihydrofolate. On the other hand, transfers of monocarbon fragments occur
at the level of tetrahydrofolate and not of dihydrofolate. Therefore, tetrahydrofolat e
should be regenerated. This is achieved by means of dihydrofolate reductase, which
uses NADPH as reducing agent. The target enzymes are dihydrofolate reductase and
thymidylate synthase.
13.5.1 Tetrahydrofolic acid
THF is a very versatile carrier of active units of one carbon atom. The monocarbon
fragment is attached to the N-5 and N-10 atoms of THF and comes from the methylene
group of the serine (Scheme 13.2).
Scheme 13.2: Formation of N5,N10-methylenetetrahydrofolic acid (N5,N10-methylene-THF).
Methotrexate has an indirect effect on thymidylate synthase by decreasing the amount
of the required cofactor N5,N10-methylene-THF.
5-Fluorouracil (5-FU, Scheme 13.3) is an anticancer drug that directly inhibits this
enzyme.
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Scheme 13.3: Use of methotrexate and 5-FU as anticancer drugs.
13.5.2 5-Fluorouracil (5-FU)
5-FU, a clinically useful anticancer drug, is converted in vivo into F-dUMP. It acts as
an anticancer prodrug of a suicide substrate. 5-FU transforms in the organism into the
5-fluorinated analog of 2ʹ-deoxyuridylate (F-dUMP) (Scheme 13.4). Under normal conditions, DNA biosynthesis occurs from dTMP.
The mechanism of inhibition of thymidylate synthase is as follows:
dUMP is combined with the enzyme and cofactor (Schemes 13.5 and 13.6). Tetrahydrofolate has formed a covalent bond with the uracil fragment via the methylene
fragment, which is subsequently transferred to uracil. This would be the normal
mechanism.
Scheme 13.6 shows the mechanism of thymine nucleotide formation from uracil
nucleotide.
Under normal conditions, a proton is lost from position 5 of uracil (Scheme 13.6).
However, 5-FU has a fluorine atom in this position instead of a hydrogen atom. It is
not possible for any further reaction to proceed, as this would require fluorine to
+
leave as a positive ion (F
). A fluorine atom is too electronegative since its usual be-
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Scheme 13.4: Biosynthesis of dTMP and inhibition of thymidylate synthase by F-dUMP.
Scheme 13.5: Cofactor obtained from N5,N10-methylene-THF.
havior is to produce the fluorine (F−) anion. As a result, the 5-FU backbone remains
covalently and irreversibly attached to the active site of the enzyme. Synthesis of thymidine is terminated, which halts DNA synthesis (Fig. 13.3). Consequently, replication
and cell division are blocked.
5-FU is administered intravenously for the palliative treatment of colorectal, breast,
stomach, and pancreatic cancers. Patients are treated for 4 consecutive days, followed
by a treatment of odd days up to a maximum of 12 days. Although up to 20% of the dose
is excreted unchanged in the urine, the majority undergoes hepatic catabolism via a
series of enzymes including dihydropyrimidine dehydrogenase (DPYD) (Scheme 13.7).
Patients who are genetically deficient in this enzyme (∼5% of the population) will experience a greater effect of this drug and are at additional risks, unless the doses are adequately adjusted.
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Scheme 13.6: Mechanism of formation of thymine nucleotide from uracil nucleotide. TS is thymidylate
synthase.
Fig. 13.3: A ternary complex formed between THF, thymidylate synthase, and 5-FU mononucleotide.
The most important toxicities are spinal cord depression, stomatitis, esophago pharyngitis, and ulcerations of the gastrointestinal tract. Nausea and vomiting are also
common. Scheme 13.7 shows the metabolism of 5-FU.
Although capecitabine is a cytidine carbamate, the drug is actually a prodrug of
F-dUMP (Scheme 13.8).
Administered orally it is metabolized to 5-FU, which is converted into the previously described active fluorinated deoxyribonucleotide. Uridine phosphorylase, an enzyme involved in its biotransformation, is much more active in tumors than in
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Scheme 13.7: Metabolism of 5-FU.
Scheme 13.8: Activation of capecitabine.
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healthy tissues, which improves the selective generation of 5-FU in tumors. Active
drug levels in the tumor may be 3.5 times greater than in surrounding tissues, which
translates into fewer side effects compared to 5-FU therapy.
Capecitabine is indicated as first-line therapy in patients with colorectal cancer. It
®
is also used alone or in combination with docetaxel (Taxotere
) in patients with meta-
static breast cancer who have experienced a disease progression or recurrence after
®
treatment with anthracycline. Paclitaxel (Taxol
) and its semisynthetic analog docetaxel are important anticancer agents that inhibit depolymerization of tubulin. Paclitaxel was isolated from yew bark and was identified in 1971, following a screening
program conducted by the US National Cancer Institute. The term taxoid is generally
used for paclitaxel and its derivatives (Fig. 13.4).
Fig. 13.4: Paclitaxel (Taxol®) and docetaxel (Taxotere®).
13.6 DHFR inhibitors
Aminopteridine and methotrexate (Fig. 13.5) are competitive inhibitors that bind to the
active site of DHFR, between 3,000 and 100,000 times more strongly than the natural
substrate, by formation of hydrogen bonds between the portion of 2,4-diaminopteridine,
protonated at physiological pH, and the anionic groups of the active site of the enzyme.
Methotrexate is given orally for the treatment of cancers of the breast, head and
neck, and several lung cancers.
13.7 Tyrosine kinase inhibitors
Today it is a widely accepted fact that “there is a growing need for new targets for the
development of anticancer drugs, in addition to DNA”. Traditionally anticancer drugs
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