Antiviral agents

1. BACKGROUND
Viruses are non-cellular, smallest infectious agents containing one kind of nucleic acid,
either RNA or DNA, which codes for a variety of enzymes and other proteins used in
replication and transmission of the organism.

Viral diseases constitute a set of difficult-to-treat conditions. Over the years, many different
antiviral agents have been discovered and synthesized, and these have provided various means
of treatment of viral conditions.
1.1. Structure of viruses
The viral nucleic acid is contained and protected within a protein coat called the capsid. Once
the capsid contains the viral nucleic acid, the whole assembly is known as the nucleocapsid.
Additional membranous layers of carbohydrates and lipids may surround the nucleocapsid,
depending on the virus concerned.

The complete structure is known as a virion and this is the form that the virus takes when it is
outside the host cell.

1.2. Classification
Viruses are classified on the basis of several features:
• Nucleic acid content (DNA or RNA)
• Viral morphology (helical, icosahedral)
• Site of replication in cell (cytoplasm or nucleus)
• Coating (enveloped or nonenveloped)

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• Serological typing (antigenic signatures)
• Cell types infected (B lymphocytes, T lymphocytes,
monocytes)
1.2. Life cycle of viruses
The various stages involved in the life cycle of a virus are as follows
 Adsorption: involves binding of the virion has to first bind to the outer surface of a
host cell. This involves a specific molecule on the outer surface of the virion binding
to a specific protein or carbohydrate present in the host cell membrane.
 Penetration and uncoating: refer to various ways by which different viruses
introduce their nucleic acid into the host cell. Some inject their nucleic acid through
the cell membrane; others enter the cell intact and are then uncoated.
 Replication and transcription: refer to expressing of viral genes.
 Synthesis and assembly of nucleocapsids: the synthesis of capsid proteins and these
self-assembly to form the capsid. Viral nucleic acid is then taken into the capsid to form
the nucleocapsid.
 Virion release: naked virions (those with no outer layers round the nucleocapsid) are
released by cell lysis where the cell is destroyed.

2. CHEMOPROPHYLAXIS

Prevention of viral infections by conferring artificially acquired active immunity with vaccines
is the main approach for preventing most viral diseases. The primary principles of vaccine
development apply: the vaccine must be sufficiently antigenic to induce an effective antibody

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response, and, ideally, the vaccine should produce a lasting form of immunity, with a
minimum requirement for booster doses.
2. ANTIVIRAL DRUGS: GENERAL PRINCIPLES

The principals involved in the design of antiviral agents are similar to those used in the design
of all chemotherapeutic agents. Drugs in this category are targeted to some process in the virus
that is not present in the host cell.
A variety of factors make the design of effective antiviral agents difficult, including their ability
to undergo antigenic changes, the latent period during which there are no symptoms, and their
reliance on host enzymes and other processes.

The currently marketed antiviral compounds that have been designed in eight general
areas:
1. Agents that disrupt virus attachment to host cell receptors, penetration, or uncoating.
2. Agents that inhibit virus-associated enzymes such as DNA polymerases and others.
3. Agents that inhibit viral transcription.
4. Agents that inhibit viral translation.
5. Agents that interfere with viral regulatory proteins.
6. Agents that interfere with glycosylation, phosphorylation, sulfation, and so on.
7. Agents that interfere with the assembly of viral proteins
8. Agents that prohibit the release of viruses from cell surface membranes.

2.1. Antiviral drugs used against DNA viruses: Acyclic Nucleoside Analogs

Most of the drugs which are active against DNA viruses have been developed against herpes
viruses to combat diseases such as cold sores, genital herpes, chicken pox
and eye diseases. Nucleoside analogues act by interfering with viral nucleic acid replication.

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Inhibitors of viral DNA polymerase
Aciclovir was discovered by compound screening having nucleoside-like structure and
contains the same nucleic acid base as deoxyguanosine. However, it lacks the complete sugar
ring.

In virally infected cells, it is phosphorylated in three stages to form a triphosphate which is

the active agent, and so aciclovir is a prodrug.

Mechanism of action:
Nucleotide triphosphates are the building blocks for DNA replication where a new DNA strand
is constructed using a DNA template—a process catalysed by the enzyme DNA polymerase.

Aciclovir triphosphate prevents DNA replication in two ways. Firstly, it is sufficiently similar
to the normal deoxyguanosine triphosphate building block that it can bind to DNA
polymerase and inhibit it.

Secondly, DNA polymerase can catalyse the attachment of the aciclovir nucleotide to
the growing DNA chain. As the sugar unit is incomplete and lacks the required hydroxyl group
normally present at position 3′ of the sugar ring, the nucleic acid chain cannot be extended any
further. Thus, the drug acts as a chain terminator.

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Acyclovir analogues:
- The ester prodrugs

The oral bioavailability of Acyclovir is quite low (15– 30%). To overcome this, various
prodrugs were developed to increase water solubility. Valaciclovir is an l-valyl ester prodrug
absorbed from the gut far more effectively than aciclovir.

However, the prodrug has similar polarity and ionization to aciclovir, and so the prodrug is no
more able to cross the cell membranes of the gut wall by passive diffusion than aciclovir.

Desciclovir is a prodrug of aciclovir which lacks the carbonyl group at position 6 of the purine
ring and is more water soluble. Once in the blood supply, metabolism by cellular xanthine
oxidase oxidizes the 6-position to give aciclovir.

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 - Hydroxymethyl analogues:
Ganciclovir is an analogue of aciclovir and bears an extra hydroxymethylene group;
valganciclovir acts as a prodrug for this compound.

Penciclovir and its prodrug famciclovir are analogues of ganciclovir.

In famciclovir, the two alcohol groups of penciclovir are masked as esters making the structure
less polar, resulting in better absorption.

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2.2. Antiviral drugs acting against RNA viruses: HIV
2.2.1. Structure and life cycle of HIV
HIV is an example of a group of viruses known as the retroviruses. There are two variants of
HIV: HIV-1 is responsible for AIDS in the USA, Europe, and Asia, whereas HIV-2 occurs
mainly in western Africa.

At present, most clinically useful antiviral drugs act against two targets: the viral
enzymes reverse transcriptase and protease. There is a need to develop effective drugs
against a third target and a good knowledge of the life cycle of HIV is essential in
identifying suitable targets.

2.2.2. Inhibitors of viral reverse transcriptase

A) Nucleoside reverse transcriptase inhibitors
The synthesis of viral DNA under the direction of RT enzyme requires availability of purines
and pyrimidine nucleosides and nucleotides. Therefore, it is not surprising that a variety of
chemical modifications of natural nucleosides have been investigated.

Removal of the ribosyl 3′-hydroxyl group of the deoxynucleosides has given rise to many
active agents

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All of these drugs have similar mechanisms of action in that their incorporation into the
viral DNA will ultimately lead to chain-terminating blockade due to the lack of a 3′-
hydroxyl needed for the DNA propagation.
B) Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
The NNRTIs are generally hydrophobic molecules that bind to an allosteric binding site
which is hydrophobic in nature. Since the allosteric binding site is separate from the substrate
binding site, the NNRTIs are non-competitive, reversible inhibitors.

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Binding of a NNRTI to the allosteric site results in an induced fit which locks the neighboring
substrate-binding site into an inactive conformation. Unfortunately, rapid resistance
emerges as a result of mutations in the NNRTI binding site the most common being the
replacement of Lys-103 with asparagine.

2.2.3. Protease inhibitors

Like the reverse transcriptase inhibitors, protease inhibitors (PIs) have a short-term benefit
when they are used alone, but resistance soon develops. Consequently, combination therapy
is now the accepted method of treating HIV infections.

Unlike the reverse transcriptase inhibitors, the PIs are not prodrugs and do not need to be
activated. Therefore, it is possible to use in vitro assays involving virally infected cells in order
to test their antiviral activity.

Design of HIV protease inhibitors (PIs):

The HIV protease enzyme is an example of an enzyme family called the aspartyl proteases—
enzymes which catalyse the cleavage of peptide bonds and which contain an aspartic acid in
the active site that is crucial to the catalytic mechanism.

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There are eight binding subsites in the enzyme—four on each protein unit, located on either
side of the catalytic region. These subsites accept the amino acid side chains of the substrate
and are numbered S1–S4 on one side and S1′–S4′ on the other side. The
relevant side chains on the substrate are numbered P1–P4 and P1′–P4′

PIs are Transition-state inhibitors, designed to mimic the transition state of an enzyme-
catalysed reaction state (resembles the tetrahedral intermediate). The advantage of this
approach is that the transition state is likely to be bound to the active site more strongly than
either the substrate or product.

As such structures are inherently unstable, it is necessary to design an inhibitor which contains
a transition-state isostere. Such an isostere would have a tetrahedral centre to mimic the
tetrahedral centre of the transition state, yet be stable to hydrolysis.

Fortunately, several such isosteres had already been developed in the design of renin inhibitors
and later modelled as HIV protease inhibitors.

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The prototype PI: Saquinavir
Saquinavir was developed by Roche and, as the first PI to reach the market, it serves as the
benchmark for all other PIs.

The design of saquinavir started by considering a viral polypeptide substrate .A

pentapeptide sequence Leu– Asn–Phe–Pro–Ile was identified and served as the basis for
inhibitor design.

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Indinavir
Develoed by Merck, the design of indinavir included an interesting hybridization strategy.
Unfortunately, it suffered from poor bioavailability and liver toxicity.

At this point, the Merck workers concluded that it might be possible to take advantage
of the symmetrical nature of the active site. As the S and S′ subsites are equivalent, it should
be possible to combine half of one PI with half of another to give a structurally distinct hybrid
inhibitor.

Merck team decided to combine the P′ half of L 685434 with the P′ half of saquinavir. The
P′ moiety of saquinavir was chosen for its solubility enhancing potential and the P′ moiety
of L 685434 is attractive for its lack of peptide character. The resulting hybrid structure (L
704,486) was less active as an inhibitor, but was still potent.

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Further modifications were aimed at improving binding interactions, aqueous solubility,
and oral bioavailability. The decahydroisoquinoline ring was replaced by a piperazine ring,
the additional nitrogen helping to improve aqueous solubility and oral bioavailability.

A pyridine substituent was then added to access the S3 subsite and to improve binding.
This resulted in indinavir, which reached the market in 1996.

Reduced molecular weight analogues:

A) Nelfinavir

Structure-based drug design had been used to develop AG1254 ( Fig. below ), which contains
an extended substituent at P1 capable of spanning and binding to both the S1 and S3 subsites
of the enzyme, allowing the design of compounds with a lower molecular weight.

They also designed a new P2 group to replace an asparagine residue which had been present
in their lead compound. This group was designed to bind effectively to the S2 subsite and, as
it was different from any natural amino acid residue, the peptide character of the compound
was reduced.

Unfortunately, the antiviral activity of AG 1254 was not sufficiently high and the compound
had poor aqueous solubility. Incorporation into saquinavir and this led ultimately to nelfinavir.

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B) Palinavir
Palinavir is a highly potent and specific inhibitor of HIV-1 and HIV-2 proteases. The left-hand
or P half of the molecule is similar to saquinavir and the molecule contains the same
hydroxyethylamine transition-state mimic. The right-hand (P′) side is different and was
designed using the same kind of extension strategy used in nelfinavir.

In this case, the P1′ substituent was extended to occupy the S1′ and S3′ subsites. This was
achieved by replacing the original proline group at P1′ with 4-hydroxypipecolinic acid and
adding a pyridine containing substituent to access the S3′ subsite.

Non-peptide analogues:
Tipranavir
Tipranavir is an example of a PI that was designed from a non-peptide lead compound. High
throughput screening led to the discovery that the anticoagulant warfarin was a weak PI with
antiviral activity.

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Various warfarin analogues were then tested leading to the discovery that phenprocoumon
was a more potent competitive enzyme inhibitor with weak antiviral activity.

Phenprocoumon was used as the lead compound for further development and resulted in the
discovery of tipranavir.

2.2.4. Entry (Fusion) Inhibitors

Entry inhibitors, also known as fusion inhibitors, are a new class of drugs for the treatment of
HIV infection, and enfuvirtide is the first compound of this family to be approved for clinical
use.

Enfuvirtide is an oligopeptide consisting of 36 amino acids. It is a synthetic peptide that

mimics an HR2 fragment of gp41, blocking the the fusion of the HIV-1 virion to a CD4-positive
T lymphocyte.

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2.3. Antiviral drugs acting against RNA viruses: flu virus
2.3.1. Structure and life cycle of the influenza virus
Influenza (or flu) is an airborne, respiratory disease caused by an RNA virus which infects the
epithelial cells of the upper respiratory tract.

The nucleocapsid of the flu virus contains (−) ssRNA and a viral enzyme called RNA
polymerase. Surrounding the nucleocapsid there is a membranous envelope derived from host
cells which contains two viral glycoproteins called neuraminidase (NA) and haemagglutinin
(HA).

In order to reach the epithelial host cells, the virus has to negotiate a layer of protective mucus
and it is thought that the viral protein NA is instrumental in achieving this. The mucosal
secretions are rich in glycoproteins and glycolipids which bear a terminal sugar substituent
called sialic acid (also called N-acetylneuraminic acid).

NA (also called sialidase) is an enzyme which cleaves sialic acid from these glycoproteins and
glycolipids, thus degrading the mucus layer and allowing the virus to reach the surface of
epithelial cells.

Once the virus reaches the epithelial cell, adsorption takes place whereby the virus binds to
cellular glycoconjugates that are present in the host cell membrane, and
which have a terminal sialic acid moiety.

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2.3.2. Ion channel disrupters: adamantanes

The adamantanes were discovered by random screening and are the earliest antiviral drugs used
clinically against flu. Amantadine and rimantadineare related adamantanes with similar
mechanisms of action and can inhibit viral infection in two ways.

At low concentration (<1 μg/ml), they inhibit the replication of influenza A viruses by blocking
a viral ion channel protein called matrix (M2) protein. At higher concentrations
(>50 μg/ml), the basic nature of the compounds becomes important and they buffer the pH of
endosomes to prevent the acidic environment needed for HA to fuse the viral membrane
with that of the endosome. These mechanisms inhibit penetration and uncoating of the virus.

2.3.3. Neuraminidase inhibitors

The role played by the surface glycoproteins HA, an enzyme important for viral binding to host
cell receptors via a terminal sialic acid residue, and NA, an enzyme involved in various aspects
of activation of influenza viruses, was discussed earlier.

Freshly shed virus particles are coated with sialic acid residues. NA is found in both
influenza A and B viruses and is thought to be involved in catalytically cleaving glycosidic
bonds between terminal sialic acid residues and adjacent sugars on HA. The cleavage of
sialic acid bonds facilitates the spread of viruses by enhancing adsorption to cell surface
receptors, and thus increases the infective level of the virus.

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The most important interactions involve the carboxylate ion of sialic acid, which is involved in
ionic interactions and hydrogen bonds with three arginine residues, particularly with Arg-371.

It is believed that the hydrolysis of sialic acid proceeds through an oxonium cation–stabilized
carbonium ion. Mimicking the transition state with novel carbocyclic derivatives of sialic
acid has led to the development of transition state–based inhibitors.

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The first of these compounds, 2-deoxy-2,3-dehydroN-acetylneuraminic acid (DANA), was
found to be an active neuraminidase inhibitor but lacked specificity for viral neuraminidase.
Upon determination of the crystal structure of neuraminidase, more sophisticated
measurements of the binding site for sialic acid led to the development of zanamivir and, later,
oseltamivir.

ZANAMIVIR Crystallographic studies of DANA bound to NA defined the receptor site to

which the sialic acid portion of the virus binds. These studies suggested that substitution of the
4-hydroxy with an amino group or the larger guanidino group should increase binding of the
inhibitor to NA.

The 4-guanidino derivative was able to form both a salt bridge to Glu 119 and a charge–
charge interaction with a glutamic acid at position 227.

The result of these substitutions was a dramatic increase in binding capacity of the 4-amino
and 4-guanidino derivatives to NA, leading to effective competitive inhibition of the enzyme.

OSELTAMIVIR X-ray crystallographic studies further demonstrated that additional binding

sites exist between NA and substrate involving the C-5 acetamido carbonyl of DANA and an
arginine (Arg 152).

Structure–activity relationship studies showed that maximum binding occurred to NA when C-

6 (based on DANA numbering) was substituted with the 3-pentyloxy side chain such as the
one found in oseltamivir.

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