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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5406_Библиотеки_им_академика_М_И_Перельмана.pdf
192 Pharmaceutical Chemistry
strand that has the base sequence as A-T-G-C-C-T-T-A-T-C, then the newly synthesized
strand will have the base sequence T-A-C-G-G-A-A-T-A-G. The process is catalyzed by an
enzyme called DNA polymerase. The new strand is synthesized using nucleoside 5’-Otriphosphates as building blocks. Nucleoside 5’-O-triphosphates are biosynthesized by
three successive phosphorylations of the relevant nucleosides by enzymes known as
kinases (Fig. 8.9).
When a cytidine base is present in the template strand, the DNA polymerase enzyme
brings a deoxyguanosine-5’-O-triphosphate molecule into the active site, where it is
positioned opposite to its Watson-Crick partner. The 3’-OH of the growing synthesized
strand is now primed by the enzyme for a nucleophilic attack on the first phosphorus atom
of the triphosphate group. This results in loss of diphosphate and the incorporation of
deoxyguanosine-5’-O-phosphate into the growing strand. The presence of a free 3’-OH
group enables further chain extension to occur using the requisite nucleoside-5’-Otriphosphate.
HIV-AIDS: Antiviral Agents 193
Fig. 8.9: Extension of DNA helix chain by addition of nucleoside (DNA building blocks)
8.4.1.5 Synthesis of Acyclovir Nucleoside
The D-herpes viruses (HSV-1 and HSV-2) have their own specific viral thymidine kinase
enzyme which is more than 100 times better as phosphorylating acyclovir is the host cell
kinase. So acyclovir is effectively converted first to the 5’-O-monophosphate and then to
the 5’-Otriphosphate (followed by 2
nd
and 3rd phosphorylation) only in a herpesvirus
infected cell (Fig. 8.10). This makes acyclovir a highly selective and relatively non-toxic
antiviral agent.
194 Pharmaceutical Chemistry
O
N
HO
N
O
Acyclovir Acyclovir monophosphate Acyclovir diphosphate
Cellular
thymidylate
Kinase
NH
N
O
Fig. 8.10: Synthesis of 5’-O-triphosphate Zidovudine nucleoside (DNA building blocks)
Viral
thymidine
Kinase
NH
2
O
O
O
O
P
O
O
O
O
P
P
O
Acyclovir triphosphate
O
O
O
P
O
O
O
N
N
O
N
N
N
NH
NH
O
N
2
Cellular
thymidylate
NH
Kinase
NH
2
DNA
polymerases
O
O
O
P
O
Acyclovir
mono-
phosphate
incorporated
into viral DNA
O
O
P
O
N
O
N
O
NH
N
Viral DNA
synthesis
inhibited
NH
2
8.4.1.6 Acyclovir Mimic for Guanine Nucleoside
Acyclovir 5’-O-triphosphate is a good mimic for 2’-deoxyguanosine 5’-O-triphosphate,
and more than 50 times selective as substrate for the viral DNA polymerase than it is for
the host cell DNA polymerase. This results in acyclovir being incorporated in a growing
viral DNA chain in place of guanosine. Because the acyclovir lacks a 3’-OH (unlike
guanosine) no further DNA extension can occur and so viral DNA chain termination
results, inhibiting the proliferation of the herpes viruses. Thus, it has been used since a few
decades as a potent antiviral drug.
Therefore, we can say acyclovir is a nucleoside analogue that selectively inhibits
replication of herpes simplex virus types 1 and 2 (HSV-1, HSV-2) and varicella-zoster virus
(VZV). After intracellular uptake, it is converted to acyclovir monophosphate by virallyencoded thymidine kinase. This step does not occur to any significant degree in uninfected
cells and thereby lends specificity to the drug’s activity. The monophosphate derivative is
subsequently converted to acyclovir triphosphate by cellular enzymes. Acyclovir
triphosphate competitively inhibits viral DNA polymerase by acting as an analogue to
deoxyguanosine triphosphate (dGTP). Incorporation of acyclovir triphosphate into DNA
results in chain termination since the absence of a 3’-OH group prevents attachment of
additional nucleosides. Acyclovir triphosphate has a much higher affinity for viral DNA
polymerase than for the cellular homolog, yielding a high therapeutic ratio (Fig. 8.11).
Thus, in conclusion, antiviral drugs are those which are used for the treatment of herpes
simplex virus, varicella-zoster virus, and/or human cytomegalovirus through bioactivation.
These include acyclovir, valacyclovir, penciclovir, famciclovir, and ganciclovir, which
must be phosphorylated on the pentose like side chain to the triphosphate derivative to be
effective for inhibiting the enzyme DNA polymerase. The nucleoside antivirals that are
used in treating acquired immunodeficiency syndrome/human immunodeficiency virus
(AIDS/HIV) must also undergo a similar metabolic conversion to the triphosphate
metabolite. The triphosphate derivative acts as a competitive inhibitor of the enzyme,
reverse transcriptase, which normally uses the triphosphorylated form of nucleic acids.
Examples include zidovudine, stavudine, zalcitabine, lamivudine, and didanosine.
HIV-AIDS: Antiviral Agents 195
Fig. 8.11: Acyclovir 5’-O-triphosphate is a good mimic for 2’-deoxyguanosine-5’-O-triphosphate, and is selectively
incorporated by viral DNA polymerase into the growing viral DNA chain.
8.4.1.7 Metabolic Pathways of Acyclovir
Acyclovir is excreted through the kidney by both glomerulus filtration and tubular
secretion. The terminal or E-phase half-life reported to be about 2-3 hours for adults
without renal impairment. Most of the drug is excreted unchanged in the urine and about
10% excreted as the inactive metabolite 9-carboxymethoxymethylguanine. Protein binding
is reported as in the range from 9–33%. About 15–30% of a dose of acyclovir given oral is
considered to be absorbed from the gastrointestinal tract. The orally active prodrugs
descyclovir and valacyclovir have been developed to overcome this poor absorption.
Acyclovir crosses the placenta and is distributed into breast milk in concentrations
approximately three times higher than those in maternal serum. Acyclovir is absorbed
following application of a 3% ointment to the eye giving a relatively high concentration of
1.7 Pg/mL in the aqueous humor but negligible amounts in the blood.
196 Pharmaceutical Chemistry
8.4.1.8 Side Effects
The common side effects include nausea, dizziness, diarrhoea, rash, headache, and rarely
nephrotoxicity and neurotoxicity (sensory disturbances, tremors, extrapyramidal side
effects, delirium, seizures, and myoclonus), extreme tiredness, irregular heartbeat,
bleeding, fever, bloody/dark urine, severe stomach/abdominal pain, yellow eyes/skin,
sudden vision changes, loss of consciousness, drowsiness, signs of kidney problems (viz.,
change in the amount of urine, unusual back/side pain), mental changes (such as agitation,
confusion, hallucinations), shaky/unsteady movement, trouble speaking, weak immune
system (viz., bone marrow transplant, kidney transplant), serious allergic reaction,
including rash, itching/swelling (face/tongue/throat), and troubled breathing.
8.5 ANTIVIRAL DRUGS ACTING AGAINST RNA VIRUSES: HIV
Since the early 1980s, acquired immunodeficiency syndrome (AIDS) has evolved into a
worldwide epidemic. Death is not caused directly by the virus, the human immunodeficiency
virus (HIV), but the severe impairment of the immune system that is consequence of
opportunistic pathogens which ultimately kill the host. The virus responsible for HIV was
first isolated in 1983 by Robert Gallo of the US and French scientist, Luc Montagnier.
HIV is a lentivirus (a subgroup of retrovirus) that causes HIV infection and acquired
immunodeficiency syndrome (AIDS). AIDS is a condition in humans in which progressive
failure of the immune system allows life-threatening opportunistic infections and cancers
to thrive. Without treatment, average survival time after infection with HIV is estimated
to be 9 to 11 years, depending on the HIV subtype. HIV is an example of a group of viruses
known as the retroviruses. There are two types of HIV found in the world, HIV-1 and
HIV-2. HIV-1, mostly found in America, Europe and Asian patients, is responsible for
acquired immune deficiency virus (AIDS) in the host while HIV-2 is found in Western
Africa.
The genome of retroviruses is made of RNA, and each virus has two single chains of
RNA; and for replication, the virus needs a host cell, and the RNA must first be transcribed
into DNA, which is done by the enzyme reverse transcriptase. HIV infects mainly CD4+
lymphocytes (T cells), and somehow monocytes, macrophages, and dendritic cells (known
as CD4+ cells). Once infected, the cell turns into an HIV-replicating cell and loses its
function in the human immune system.
After the discovery of the virus, a tremendous amount of research focusing upon the
causative agent of AIDS has been carried out and much has been learnt about the structure
of the virus and its typical course of action. HIV, the virus responsible for AIDS, relies
upon the transcription and translation machinery of its host cell. There are, however, a
number of protein-RNA interactions that are unique and essential to the HIV life cycle.
Inhibition of these key binding interactions may eventually provide a new class of
therapeutic compounds for the treatment of AIDS. At present, most of drug inhibitors are
in action especially for the viral enzymes reverse transcriptase and protease.
HIV-AIDS: Antiviral Agents 197
8.5.1 Structure of the Virus
The human immunodeficiency viruses are approximately 100 nm in diameter. The basic
structure of the virus (Fig. 8.12) is described as follows:
The viral envelope: The outer coat of the virus consists of two layers of lipids; different
proteins are embedded in the viral envelope, forming “spikes” consisting of the outer
glycoprotein (gp) 120 and the trans-membrane gp41. The lipid membrane is borrowed
from the host cell during the budding process (formation of new particles). Gp120 is
needed to attach to the host cell, and gp41 is critical for the cell fusion process.
HIV matrix proteins: It consists of the p17 matrix protein and lies between the envelope
and the core.
gp120
Docking glycoprotein
gp41
Transmembrane
glycoprotein
Integrase
Reverse
transcriptase
RNA
Capsid
Fig. 8.12: Structure of HIV-1 virus
Protease
Liquid
membrane
gag p17
Matrix protein
Nucleocapsid
Vif, Vpr
, Nef and p7
The viral core: It contains the viral capsule core protein p24, p6, and nucleocapsid protein
p7 (bound to the RNA) which surrounds two single strands of HIV RNA and the enzymes
needed for HIV replication, such as reverse transcriptase, protease, ribonuclease, and
integrase. All these proteins are encoded by the viral gag gene. Out of the nine virus genes,
there are three, namely, gag core gene proteins (p55, p24, and p17), pol (polymerase gene
proteins, p66, p51, and p31), and env (envelope proteins gp160, gp120, and gp41) that
contain the information needed to make structural proteins for new virus particles. Within
the viral core, lies two copies of the ~10 kilobase (kb) positive-sense, viral RNA genome
(i.e., it has a diploid RNA genome), together with the protease, integrase and reverse
transcriptase enzymes. These three enzymes are encoded by the viral pol gene. There are
several other proteins coded by both HIV-1 and HIV-2, with various regulatory or
immunomodulatory functions, including vif (viral infectivity protein), vpr (viral protein
R), tat (transactivator of transcription), rev (regulator of viral protein expression) and nef
(negative regulatory factor). An additional protein found in HIV-1 but not HIV-2 is vpu
(viral protein U). Similarly, vpx (viral protein X) is found in HIV-2 and not HIV-1.
198 Pharmaceutical Chemistry
8.5.2 Life Cycle of HIV
The life cycle of HIV completes in seven stages (Fig. 8.13). First and the main attachment
receptor for HIV is the CD4+ molecule or known as cells, that are present on the CD4+
positive T (helper) lymphocyte, macrophages, and microglial cells. The viral gp120 binds
initially to this CD4+ molecule, which then triggers a conformational change in the hostcell envelope that allows binding of the co-receptor (either CCR5 or CXCR4) which is
required for fusion between virus envelope and cell membrane (stage 1 in life cycle).
Macrophages carry the CCR5 co-receptors, hence HIV strains requiring the CCR5
co-receptor for entry, are also referred to as ‘macrophage-tropic’ although they also infect
lymphocytes. These HIV strains are also known, phenotypically, as R5 or non-syncytium
inducing (NSI) strains as they do not form syncytia (cell-fusion) when cultured with CD4+
lymphocytes in vitro. Primary HIV-1 infections tend to involve this R5 NSI macrophage-
tropic phenotype. Uncommonly, individuals may have a homozygous deletion mutation
in the CCR5 gene resulting in the absence of the CCR5 molecule on their macrophages.
Therefore, these individuals cannot be infected by this R5 phenotype. The ‘lymphotrophic’
HIV strains use CXCR4 as the co-receptor. These viruses are also known as X4 viruses and
do produce syncytia (i.e., are phenotypically syncytium-inducing or SI) when cultured in
vitro with CD4+ lymphocytes. X4 viruses tend to appear later in about 50% of HIV-1
subtype B-infected individuals, but seldom with other subtypes, as they progress to AIDS.
So far, CXCR4 deficient individuals have not been found. This attachment and fusion
process allows the HIV viral core to enter the host-cell.
All retroviruses encode a reverse transcriptase enzyme that transcribes its viral RNA
into double-stranded DNA (dsDNA), which is then integrated, via the action of the
integrase enzyme into the host-cell genome (stage 2 in life cycle). The viral integrated
dsDNA or ‘provirus’ then acts as a template for viral genomic and messenger RNA
transcription by the host cell’s nucleic acid replicating machinery. Recombination between
these two RNA strands during viral replication, coupled with the extremely error-prone
action of the RT enzyme, give rise to the extreme genetic diversity of HIV.
Integration of the linear provirus dsDNA into the genome of the host-cell establishes an
infection that lasts for the lifespan of the cell, and all its progeny, which usually means
life-long infection for the organism, in case of the human host. Viral replication occurs
along with cellular replication and is enhanced by various factors, including co-infection
with other organisms, the presence of inflammatory cytokines and cellular activation.
During cellular replication, the provirus is transcribed by the host-cell RNA polymerase II
enzyme, and the viral messenger RNA (vmRNA) and genomic RNA, are carried with the
cellular mRNAs, to be translated into proteins. This vmRNA codes for a gag-pol precursor
polypeptide that is ultimately cleaved by the viral-encoded protease enzyme to produce
the gag and pol viral proteins. In addition, the vmRNA is also spliced to produce
other vmRNAs coding for the viral proteins, tat, rev, vif, vpr, vpu (for HIV-1), and the env
precursor polypeptide. Ultimately, the env precursor polypeptide is cleaved by
cellular (not viral) proteases, producing the envelope glycoproteins gp41 and gp120.
HIV
gp120
CD4
1
Fusion of HIV
to the host cell
surface
HIV-AIDS: Antiviral Agents 199
2
HIV R NA, reverse
transcriptase, integrase,
and other viral proteins
enter the host cell.
Co-receptor
(
CCR5 or CXCR4)
Host cell
4
Mature virion
7
The virus matures
by protease
releasing individual
HIV proteins.
(From: http://www.niaid.nih.gov/topics/HIVAIDS/Understanding/Biology/Pages/hiv ReplicationCycle.aspx)
6
New viral RNA
and proteins move to
the cell surface and a
new, immature, HIV
forms.
Fig. 8.13: HIV replication cycle
3
Viral DNA is
formed by reverse
transcription.
Viral DNA is
transported across the
nucleus and integrates
into the host DNA.
Preintegration
complex
5
New viral RNA is
used as genomic
RNA and to make
viral proteins.
Viral RNA
transcriptase
Integrase
Viral DNA
Host DNA
New viral RNA
Reverse
These viral proteins, together with the replicated diploid viral genomic RNA, are assembled
and enveloped by budding through the host-cell membrane; producing complete HIV
virions (stages 3–7 in life cycle).
200 Pharmaceutical Chemistry
8.6 ACQUIRED IMMUNE DEFICIENCY SYNDROME (AIDS)
AIDS is a disease that progressively destroys the human immune system. It is caused by
the human immunodeficiency virus (HIV), which is a retrovirus. The AIDS virus causes a
chronic disease in which infection persists despite a strong antibody response to the virus
(at least initially, HIV can circumvent the humoral response to infection by attacking and
killing CD4+ T cells). This virus enters and destroys human T4 lymphocyte cells. These T
helper cells are used to upregulate the human immune system. By eradicating the CD4+
cells, the HIV virus effectively destroys the immune system. Cell-mediated immune
responses are critical to the prevention and treatment of HIV infection. Their destruction
reduces the body’s resistance to other infectious diseases, such as pneumonia, and some
rare forms of cancer.
The entry of the virus into the body usually causes an initial period of acute ill health
with the patient suffering from headaches, fevers and rashes, amongst other symptoms.
This is followed by a period of relatively good health where the virus replicates in the
lymph nodes. This relatively healthy period normally lasts a number of years before the
full blown AIDS appears. Full-blown AIDS is characterised by a wide variety of diseases
such as bacterial infections, neurological diseases and cancers. Treatment is more effective
when a mixture of antiviral agents is used.
A new era in the treatment of AIDS ushered with the advent of some clinically useful,
potent inhibitors of HIV. For the first time in the history of AIDS, the death rate reversed
itself. There are several different classes of drugs that can be used to treat HIV infection.
These are the NRTIs, the NNRTIs, the HIV PIs, the HIV entry inhibitors, and the HIV
integrase inhibitors (IN). At present, at least more than 20 antiretroviral agents belonging
to three distinct classes (NRTIs, NNRTIs, PIs) have been licensed. All of these agents are
limited by rapid development of resistance and cross-resistance. These drugs can effect
dramatic reductions in viral load, but eventually, as resistance develops, the virus reasserts
itself. Current treatment for HIV infection consists of highly active antiretroviral therapy
(HAART). Examples: Zidovudine, Lamivudine, Emtricitabine, Tenofovir, Lopinavir,
Ritonavir, Efavirenz, etc.
The common adverse effects are abdominal pain (Ritonavir), anemia (AZT), diarrhoea
(Abacavir), headache (overdose), hepatitis, jaundice, liver failure, mental confusion (EVZ),
nausea (AZT), rash and vomiting (AZT), etc.
8.6.1 Zidovudine (AZT)
Zidovudine is a pyrimidine nucleoside analogue of thymidine in which
the 3’ hydroxyl group is replaced by an azido group, synthesized by
Jerome Horwitz in 1964. It is NRTI’s active against human
immunodeficiency virus (HIV-1, HIV-2, HTLV-1), which causes the
acquired immunodeficiency syndrome (AIDS). It is used as an antiviral
medication that prevents HIV cells from multiplying in your body or
in other words AZT is considered a chain terminator of DNA synthesis.
Commercially, it is named Retrovir and Retrovis, having an ingredient
HO
Zidovudine
Me
O
NH
O
N
O
N
3
HIV-AIDS: Antiviral Agents 201
in Combivir and Trizivir. It was the first drug approved for the treatment of AIDS in 1987,
and it was the first drug approved for “preventative” treatment of HIV/AIDS in 1990.
Zidovudine was originally synthesized in the 1960s as a possible anti-cancer agent, but
was found to be ineffective. In 1985, it was found to be active against HIV-1 in vitro. It is
also given during pregnancy to prevent an HIV-infected woman from passing the virus to
her baby. Examples: Zalcitabine (Hivid), Stavudine (Zerit), Didanosine (Videx), and
Lamivudine (Epivir). Zidovudine works by selectively inhibiting HIV’s reverse
transcriptase, the enzyme that the virus uses to make a DNA copy of its RNA.
8.6.1.1 Specification
Name : D]LGRGLGHR[\ǃ'HU\WKURSHQWRIXUDQRV\OPHWK\O
pyrimidine-2,4(1H,3H)-dione; azidothymidine (AZT); Zidovudine;
3’-azido-3’-deoxythymidine
Molecular formula : C
10H13N5O4
Molecular weight : 267.24 g/mol
Melting point : 106–112°C (from petroleum ether); 120–122°C (from water)
Physical state : White to off-white crystals or needles, odourless
Solubility : Soluble in water and ethanol.
8.6.1.2 Synthesis of AZT
AZT can be synthesized by various methods. Here is one method that avoids the use of the
expensive thymidine starting material, and instead uses the readily available alternative
D-mannitol. The reaction in the penultimate step does not proceed with any stereochemical
control. The mixture produced can only be separated cleanly after removal of the silyl
protecting group.
CH2OH
HO
HO
OH
OH
CH2OH
O
HO
di-TMS-thymine,
TMS-trate, EDC
CO, H
1. Me
2
reux
2. Pb(OAc)
1. -Bu(Me)t
Imidazole, DMF
O
2. LiN
AcOH, H
TBDMSO
,THF
3
2
O
2
+
4
SiCl,
HN
O
N
3
O
TBDMSO
O
N
O
O
CHO
N
Me
Ph3P=CHCO2Et
MeOH
O
O
2. Ac
3
n-Bu
NH FH
43
THF
O
1. DIBAL,
DCM, -78°C
O, pyridine
2
HO
O
O
N
HN
O
3
AZT
CO2Et
TBDMSO
O
N
Me
HCl
O
OAc
N
3