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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5406_Библиотеки_им_академика_М_И_Перельмана.pdf
182 Pharmaceutical Chemistry
Capsomere
Envelope
Nucleocapsid
Nucleic
acid
Capsid
(entire coat)
Naked virus
Fig. 8.1: Structure of virion or flu virus
Capsomere
Nucleic
acid
Enveloped virus
8.3 LIFE CYCLE OF VIRUSES
There are five steps in the life cycle of a virus (Fig. 8.2):
(a) Binding/attachment/adsorption: The virus initially binds to a receptor on the
surface of the host cell using a specific molecule on its outer coat, which is usually
a glycoprotein. The interaction is often referred to as a key-in-lock interaction. Once
bound, the virus particle, or virion, can enter the next stage in the cycle, introduction
of viral nucleic acid into the host cell.
(b) Penetration: Some viruses enter the cell intact and are then uncoated. Others inject
their nucleic acid through the cell membrane. The net result is the release of viral
nucleic acid into the cell, which is then ready to start the process of viral replication.
(c) Replication and transcription: Viral nucleic acid gets integrated into the host
genome which synthesizes viral nucleic acid, mRNA and viral proteins. The exact
mechanism varies from virus to virus. A general sequence of events can be observed
during viral replication:
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The viral proteins are synthesised once the viral genome is integrated into the host
genome and expressed have three main functions:
0RGLI\KRVWFHOOPHWDEROLVPWRHQVXUHYLUDOSURGXFWLRQ
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Maturation
HIV-AIDS: Antiviral Agents 183
Attachment
Receptor
Replication
Penetration
Uncoating
Nucleus
Regulatory
proteins
Gene expression
Release
Membrane
Genome
Assembly
Structural
proteins
Cytoplasm
Fig. 8.2: Infection cycle: Virus enters a host cell and utilises host cellular machinery to reproduce itself
Thus, there are three types of viruses that commonly cause infection in humans:
(i) DNA viruses: The viral genome is coded by the same nucleic acids as the host
(DNA). After integration of the viral DNA into the host DNA by the viral integrase
enzyme, transcription occurs in the nucleus and translation in the cytoplasm.
Generally, transcription of the viral DNA into mRNA is by the host RNA polymerase
systems. The translation of the viral mRNA of early proteins is the key initial step
in viral DNA replication to establish the replicatory process. After DNA synthesis,
the remainder of the genome is transcribed into late messengers. Regulation is
carried out by proteins present in the virions or specified by viral genes transcribed
and translated by the cellular host. Examples of DNA viruses include the poxvirus
(smallpox, cowpox), herpesvirus (chicken pox, shingles, genital, oral herpes and the
first successful antiviral acyclovir is a nucleoside analogue, and is effective against
herpesvirus infections), adenovirus (conjunctivitis, sore throat), hepadnavirus
(hepatitis B), and papillomavirus (warts) (Fig. 8.3). Some other examples of
nucleoside reverse transcriptase inhibitors (NARTIs or NRTIs) are:
184 Pharmaceutical Chemistry
HO
HO
O
NH
NNO
OP
HO
Cidofovir
2
HO
O
I
NH
N
O
O
HO
HO
Idoxuridine Trifluridine
Fig. 8.3: Nucleoside reverse transcriptase inhibitors
F
HO
O
C
3
NH
N
O
O
HO
O
HO
Vidarabine
N
N
OH
O
NH
N
NH
2
HO
N
N
O
Acyclovir
OH
N
N
NH
2
(ii) RNA viruses: In order to replicate the RNA viral genome, the viral RNA can act
directly as the mRNA and be translated to form viral proteins. Alternatively, in the
case of retroviruses, which are unique in that their genomes are transcribed into
DNA from RNA by an RNA-directed DNA polymerase or reverse transcriptase,
so-called because information is going from RNA into DNA, which is the opposite
to the conventional process of transcription (DNA into mRNA). Once transcribed
into DNA, it is integrated into the cellular DNA by the viral integrase enzyme.
Transcription of the viral genome by the cellular RNA polymerases yields the viral
molecules that end up in virions. Examples of RNA viruses include rubella (German
measles), rhabdovirus (rabies), picornavirus (polio, meningitis, colds), arenavirus
(meningitis, Lassa fever), arbovirus (yellow fever, tick encephalitis), orthomyxovirus
(influenza), paramyxovirus (measles, mumps), and retrovirus (HIV, HTLV or human
lymphocyte leukaemia virus causing viral mediated leukaemia) (Fig. 8.4). Here are
some examples of nucleoside reverse transcriptase inhibitors (NARTIs or NRTIs):
O
N
HO
R
Adefovir dipivoxil (R = H, R
Tenofovir disoproxil
R
1
1
O
O
N
O
Abacavir
O
O
O
N
OO
P
N
NH
O
(R = Me, R
O
Me
2
N
N
R
=CMe3)
1
1
Fig. 8.4: Nucleoside reverse transcriptase inhibitors
HO
NH
2
N
N
= OCHMe2)
Stavudine
NH
O
N
O
R
HO
O
S
Lamivudine (R = H)
Emtricitabine (R = F)
HO
NH
N
O
Zalcitabine
2
N
O
NH
N
Me
O
N
3
Zidovudine
N
N
O
NH
O
N
O
NH
N
2
N
HO
O
HO
O
Didanosine
HIV-AIDS: Antiviral Agents 185
(d) Synthesis/assembly: Viral proteins and viral nucleic acid are assembled into new
‘naked’ virions called nucleocapsids. These are then released from the cell as fully
developed virions in two possible ways.
(e) Release: Naked virions which lack any outer layer around the nucleocapsid are
released by cell lysis, in which the host cell is destroyed. Viruses that contain an
outer envelope are released by a process known as ‘budding’. In the latter, viral
outer coat proteins are first incorporated into the host cell’s membrane. The
nucleocapsid then binds to the inner surface of the host cell membrane and,
simultaneously, viral proteins collect at the site and host cell proteins are excluded.
The plasma membrane containing viral proteins then encases the nucleocapsid, and
the newly formed virion is then pinched off from the cell.
Viruses vs bacteria
Viruses are much simpler organisms than bacteria, and they are made from protein
substances and nucleic acid. A single nucleoprotein molecule formed from molecules of
nucleic acid that are chemically bound to a bulky protein molecule can be considered a
simple viral particle. The protein molecule plays the role of a protective membrane.
Thus the virus can be schematically described as a nucleic acid insert that is protected
by a protein covering. A virus can contain either ribonucleic acid or deoxyribonucleic
acid, but it never contains both of them together. The type of nucleic acid is the basis of
one of the classifications of viruses. Viruses are obligatory intracellular parasites, which,
upon entering a cell (i.e., after being infected) use many biochemical systems of the
host cell.
8.4 ANTIVIRAL DRUG TARGETS
The major challenge facing medicinal chemists attempting to treat viral infections is the
fact that these pathogens reside inside the host cells, utilizing their host’s biochemical
mechanisms to multiply. Therefore, the number of possible drug targets that are unique to
the virus is less in comparison with microbes. The development of effective antiviral drugs
has proved to be much more challenging than in the case of antibiotics. The targets must
be viral proteins that are critical to the viral life cycle, especially in the early stages. These
must be distinct from host proteins in order to obtain good selectivity with minimal side
effects. Ideally, these viral proteins should be common to a wide range of viruses
as this increases the chances of developing a drug with a broad spectrum of antiviral
activity.
Antiviral drugs are a class of medication used specifically for treating viral infections.
Viruses cause blindness, deafness, paralysis, mental retardation, various birth defects, and
in at least a few plants and animals, cancer. Some common viral diseases, are measles,
poliomyelitis, mumps, smallpox, chicken pox, influenza, and yellow fever. There is
suspicion that viruses are the cause of multiple sclerosis, Hodgkin’s disease, Down’s
186 Pharmaceutical Chemistry
syndrome, and possibly even schizophrenia. However, along with the significant progress
made in the area of treating bacterial infections, the development of chemotherapy of viral
diseases has been relatively modest. There is only a small selection of attainable drugs for
treating a limited number of viral infections.
The first antiviral agent appeared in late 1960s and the only three clinically useful firstgeneration antiviral drugs were in use during the 1980s, viz., Idoxuridine and vidarabine
for herpes infections and amantadine for influenza-A. They have limited clinical use
because of their narrow therapeutic index (ratio of effective and lethal doses). These drugs
have a direct effect on viral replication; however, they also inhibit certain host cell functions.
Later, successful nucleoside analogue antiviral amantadine, acyclovir, ribavirin, and
zidovudine were suggested where specially acyclovir was effective against herpes virus
infections.
Thus, the discovery of antiviral drugs has been accelerated for two principal reasons:
first the fight against acquired immune deficiency syndrome (AIDS) pandemic and the
second to understand the viral infectious mechanism.
Currently, vidarabine, amantadine, idoxuridine, acyclovir, ribavirin, trifluridine, and
zidovudine are used as antiviral drugs. On the basis of analysis of the mechanisms of
action they can increase resistance of the cell to a virus (interferons), suppress adsorption
of the virus in the cell or its diffusion into the cell, and the process of its “deproteinization”
in the cell (amantadine); as well as antimetabolites that inhibit the synthesis of nucleic
acids. The clinical “usefulness” of these pyrimidine and purine drugs depends directly on
their ability to selectively block synthesis of viral nucleic acids while not stopping the
synthesis of “host” cell nucleic acid.
Antiviral drugs are used in the combination therapy or fixed dose combinations and fall
in the following categories based on their mechanisms of action:
Nucleoside reverse transcriptase inhibitors (NARTIs or NRTIs): In order to be
incorporated into the viral DNA, NRTIs must be activated in the cell by the addition
of three phosphate groups to their deoxyribose moiety, to form NRTI triphosphates.
This phosphorylation step is carried out by cellular kinase enzymes that terminate
further growth of the DNA. Examples: Zidovudine (AZT), Didanosine (Videx),
Zalcitabine (ddC, dideoxycytidine or Hivid), Stavudine (d4T and Zerit), Lamivudine
(3TC and Epivir), Abacavir (ABC and Ziagen), Emtricitabine (FTC and Emtriva or
Coviracil), Entecavir (INN and Baraclude), Apricitabine (ATC), Emtricitabine,
Tenofovir (Tenofovir disoproxil fumarate and Viread) and Adefovir (bis-POM
PMPA, and Preveon or Hepsera) are used as NtRTIs.
Non-nucleoside reverse transcriptase inhibitors (NNRTI): They inhibit reverse
transcriptase directly by binding to the enzyme and interfering with its function.
Examples: Efavirenz (Sustiva, and Stocrin), Nevirapine (Viramune), Delavirdine
(Rescriptor) and Etravirine (Intelence) are used as NNRTIs (Fig. 8.5).
HIV-AIDS: Antiviral Agents 187
Me
Cl
O
HN
N
N
Nevirapine
N
S
Me
Cl
Me
Capravirine
HN
N
N
O
N
Fig. 8.5: Non-nucleoside reverse transcriptase inhibitors
O
NH
O
2
O
N
OMe
Emivirine
O
HN
Me
O
N
Me
Me Me
O
SJ 3366
Cl
Efavirenz
Me
F3C
Me
F
C
O
N
O
H
O
O
S
N
H
Cl
H
N
3
DP C083
NNHN
N
O
Delavirdine
NH
N
O
H
Protease inhibitors (PIs): They target viral assembly by inhibiting the activity of
protease, an enzyme used by HIV to cleave nascent proteins for the final assembly
of new virions. Examples: Amprenavir, Emtricitabine, Nelfinavir, Indinavir,
Tenofovir, Ritonavir, Lopinavir, Darunavir and Atazanavir are used as PIs (Fig. 8.6).
S
Me
N
Me
HN
N
O
Me
Me
H2N
S
O
Amprenavir (R = H)
Fosamprenavir (R =
Me
O
O
Me
Ph
Me
N
N
H
Ph
OR
O
Me
HO
H
N
Ritonavir
OH
Lopinavir
H
N
O
O
P
OH
O
Me
O
Ph
OH
H
N
Ph
Me
O
O
N
H
Me
H
N
O
O
Ph
O
)
Fig. 8.6: Protease inhibitors
N
S
Me
Me Me
NHO
N
NN
MeO
OH
HO
Nelfinavir
O
Me
Atazanavir
Me
Me
Me
N
H
O
O
N
H
Ph
H
N
O
OH O
N
N
H
Indinavir
OH
Me
OH
N
CONHCMe
S
Ph
Me
Me
N
H
N
OMe
O
MeMe
3
Me
Me
188 Pharmaceutical Chemistry
Integrase inhibitors: They inhibit the enzyme integrase, which is responsible for
integration of viral DNA into the DNA of the infected cell. Examples: Emtricitabine
and Tenofovir are used as integrase inhibitors while Raltegravir is under clinical trial.
Entry inhibitors (or fusion inhibitors): They interfere with binding, fusion and
entry of HIV-1 to the host cell by blocking one of several targets. Examples: Maraviroc
and Enfuvirtide are the two currently available agents in this class.
CCR5 receptor antagonists: They are the first antiretroviral drugs which do not
target the virus directly. Instead, they bind to the CCR5 receptor on the surface of the
T-Cell and block viral attachment to the cell. Most strains of HIV attach to T-Cells
using the CCR5 receptor. If HIV cannot attach to the cell, it cannot gain entry to
replicate.
Maturation inhibitors: They inhibit the last step in gag processing in which the viral
capsid polyprotein is cleaved, thereby blocking the conversion of the polyprotein
into the mature capsid protein (p24). Because these viral particles have a defective
core, the virions released consist mainly of non-infectious particles. ǂ-Interferon is a
currently available agent in this class. Two additional inhibitors under investigation
are Bevirimat and Vivecon.
8.4.1 Acyclovir
Acyclovir is a highly selective and non-toxic NRTI drug effective
in a series of acyclic purine nucleosides that possess potent
antiviral activity. In contrast with true nucleosides that have a
ribose or a deoxyribose sugar attached to a purine or a pyrimidine
base, the group attached to the base in acyclovir is similar to an
O
O
N
N
NH
N
NH
2
open chain sugar. The clinically useful antiviral spectrum of
acyclovir is limited to herpes viruses. It is most active (in vitro)
against HSV-I and about two times less active against HSV-2 and
HO
Acyclovir
ten times less potent against varicella-zoster virus.
Acyclovir was the first successful antiviral agent in the world and synthesized in 1974
by Howard Schaeffer at Welcome Research Laboratories. After Schaeffer’s discovery,
Gertrude B. Elion and her team worked to understand the mode of action of acyclovir on
the biological system. They discovered that acyclovir remains inert and nontoxic until it
meets the herpes virus. The first report detailing the selective antiviral activity of acyclovir
against herpes viruses was published in 1977.
The ultimate effect of acyclovir is the inhibition of viral DNA synthesis. Acyclovir is
transported into the infected cell and it gets monophosphorylated in the presence of
thymidinc kinase enzyme. Then monophosphorylated acyclovir is further catalysed by
enzymes present in the infected cell that leads to acyclovir triphosphate. Acyclovir has
more affinity than the guanine triphosphate analogue which is the DNA helix analogue,
thus acyclovir triphosphate completely inhibits viral DNA polymerases by chain
termination because acyclovir lacks 3’- hydroxyl group so can’t go for phosphorylation.
HIV-AIDS: Antiviral Agents 189
The acyclovir drug is bound to proteins in plasma between 9-33% and finally excreted
through urine.
Acyclovir can be used to treat infections caused by certain types of viruses. It treats cold
sores around the mouth (caused by herpes simplex), shingles (caused by herpes zoster),
and chickenpox. This medication is also used to treat outbreaks of genital herpes.
8.4.1.1 Specification
Name : [2-amino-1,9-dihydro-9-[(2-hydroxyethoxy)methyl]-6H-purin-6-one]
or 9-[(2-Hydroxyethoxy)methyl] guanine 2-Amino-1,9-dihydro-9 (2-hydoxyethoxymethyl)-6H-purin-6-one.
Molecular formula : C
8H11N5O3
Molecular weight : 225.2 g/mol
Melting point : 256.5–257°C
Physical state : White crystalline
Solubility : Slightly soluble in water, insoluble in ethanol, solubility is increased
by both strong acids and bases.
8.4.1.2 Synthesis of Acyclovir
It is synthesized by alkylation of guanine with 1-benzoyloxy-2-chloromethoxyethane in
the presence of triethylamine which was synthesized by benzoyl chloride and ethylene
glycol. The hydroxyl and amino groups of guanine are previously protected with a
trimethylsilyl group by being treated with hexamethyldisilazane. After hydrolysis, the
resulting product with water gives 9-(2-benzoyloxymethoxymethyl)-guanine. Treating
this with a methanol solution of ammonia removes the benzoyl protecting group from the
hydroxyethoxymethyl fragment, giving acyclovir. Synonyms of this drug are Aovirax,
Cycloviran and Sifiviral, etc.
O
OH
HO
Cl
Et3N
Benzoyl chloride 2-benzoyloxy-ethanol 1-benzoyloxy-2-chloromethoxyethone
1. (Me3Si)2NH
OH
N
N
H
N
N
NH
2.
Cl
2
O
O
O
N 2. tautomerization
Et
3
O
O
OH
OH
N
H2CO
HCl
NH
2
1. NH3/CH3OH
N
O
N
N
O
O
O
HO
O Cl
N
N
O
Acyclovir
O
O
NH
N
NH
2
190 Pharmaceutical Chemistry
Alternate way to synthesize acyclovir
Alkylation of 2,6-dichloropurine with the 1-benzoyloxy-2-chloromethoxyethane in the
presence of triethylamine and dimethylformamide led to 2,6-dichloro-9-(2benzoyloxyethoxymethyl) purine. Treating this with a methanol solution of ammonia
replaces both chlorine atoms with amino groups, and subsequent diazotization using
sodium nitrite in dilute acetic acid selectively replaces one of the two amino groups for
a hydroxyl group, in particular the amino group at position C6 of the purine system.
Finally, treating the product with a methanol solution of ammonia removes the benzoyl
protecting group from the synthesized 9-(2-benzoyloxy ethoxymethyl) guanine leading to
acyclovir.
1. NH
Cl
N
N
N
H
Cl
N
Cl
O
O
O
O
O
O
Cl
N
N
N
N
Cl
3
2. NaNO2/CH3COOH
/CH3OH
3. NH
3
4. tautomerization
HO
N
N
O
Acyclovir
O
NH
N
NH
2
Thus, it is concluded that the structure of acyclovir looks similar to a guanosine
nucleoside analogue (Fig. 8.7). The only difference is side chain, guanosine nucleoside
analogue contains cyclic sugar residue a 2-hydroxyethoxymethyl acyclic side chain while
in acyclovir, it is an open chain. So after several studies on acyclovir, it is also concluded
that acyclovir possesses antiviral activity with respect to ǂ-type herpes viruses including
HIV1 and HIV2 (which cause cold sores and genital warts respectively), Epstein–Barr
virus, cytomegalovirus and varicella zoster virus (VZV) which causes chickenpox and
shingles.
O
N
HO
Fig. 8.7: Acyclovir, a similar analogue of 2’-deoxyguanosine
N
O
Acyclovir 2'-Deoxyguanosine
NH
N
NH
HO
2
O
OH
O
N
N
NH
N
NH
2
8.4.1.3 Mechanism of Acyclovir
The mechanism of antiviral activity consists of its transformation to triphosphate and
subsequent inhibition of viral DNA synthesis. Its action is highly selective. Acyclovir
HIV-AIDS: Antiviral Agents 191
diffuses into the cell infected by a virus and phosphorylates thymidine kinase of herpes
simplex to a monophosphate. Uninfected cells do not use acyclovir as a substrate. The
monophosphate is subsequently transformed to a diphosphate, and then a triphosphate,
which inhibits viral DNA polymerase, as well as viral DNA, where it acts in the process of
breaking the chain, thus preventing further elongation of the DNA chains and
correspondingly, replication of the DNA virus.
Fig. 8.8: Watson-Crick DNA helix along with bases and nucleoside (DNA building blocks)
Before knowing the mechanism, we must know the structure of DNA helix. There are
WZRFRPELQDWLRQVRIEDVHVDFFRUGLQJWR&KDUJRII·VUXOHF\WLGLQHJXDQLQH&ŋ*EDVHSDLU
is held together by three hydrogen bonds, while the adenine-thymine (A=T) base pair is
held together by two hydrogen bonds (Fig. 8.8).
8.4.1.4 DNA Polymerase
DNA replication occurs using a DNA strand as a template for the construction of a new
strand, that is, its Watson-Crick complement. Thus, for example, if we have a template