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202 Pharmaceutical Chemistry
Another method for the synthesis of AZT
Zidovudine was the first drug approved for the treatment of AIDS and HIV infection.
Jerome Horwitz finished finalizing the method of synthesis of AZT in 1964. This method
of synthesis is inexpensive and easy to perform. It is synthesized by thymine analogue
when treated with methansulfonyl chloride in pyridine to make the corresponding
mesylate intermediate which on hydrolysis followed by another methansulfonyl chloride
gives us another mesylated intermediate. This mesylated intermediate reacts with lithium
azide in dimethylformamide that gives the product zidovudine.
HO
F
O
H
O
O
OH
F
CO
3
O
Me Me
N
N
1.F
CCl , DMF
3
4
2. MeSO
Cl,
2
pyridine
O
Me
N
N
O
F
CO
3
H
O
2
OH
CO
F
3
OSO2Me
O
H
O
Me
N
N
O
O
H
O
O
3
CO
H
O
O
Me
N
N
HCl, CHCl
ΗO
3
O
H
N
O
N
O
MeSO2Cl
Pyridine, 0°C
O
Me
N
N
Na OH, 100°C
F
CO
3
(Water)
-MeSO
H
O
OSO2Me
O
3
O
Me
N
N
Li N
3
0°C
N
3
N
3
Zidovudine
Synthesis of AZT by Tomasz Dolinowski in 2010
It is synthesized from reaction between thymine analogue and p-methoxy benzoic acid
that gives O-protected intermediate, which on reaction with lithium azide followed by the
hydrolysis gives zidovudine.
HIV-AIDS: Antiviral Agents 203
HO
MeO
OH
O
HN
O
O
O
Me
N
O
N
DIAD, PPh
p-MeOC
DMF, 45 min, 20°C
O
HN
O
N
O
3
6H4
Me
COOH
MeO
,
3
MeONa, MeOH
12 h, 20°C
O
O
O
HO
N
O
HN
O
O
N
3
Zidovudine
O
Me
N
LiN3, DMF
5 h, 125°C
O
Me
N
Thus, it is concluded that the structure of zidovudine looks similar to thymidine
nucleoside analogue. The only difference: thymidine nucleoside analogue contains cyclic
sugar residue—a 2-hydroxyethoxymethyl acyclic side chain—while zidovudine contains
cyclic sugar residue having azide group. Therefore, zidovudine may be a perfect substitute
for the thymidine nucleoside analogues (Fig. 8.14).
Me
O
NH
O
N
O
O
Me
H
O
NH
HO
O
N
O
N
3
Zidovudine 2'-Deoxythymidine
Fig. 8.14: Zidovudine a similar analogue of 2’-deoxythymidine
OH
8.6.1.3 Mechanism of Zidovudine
During infection with HIV, the HIV virus multiplies within the body’s cells. The newlyformed viruses then are released from the cells and spread throughout the body where
they infect other cells. In this manner, the infection continually spreads to new, uninfected
cells that the body is continually producing, and HIV infection is perpetuated. When
producing new viruses, the HIV virus must manufacture new DNA for each virus. Reverse
transcription is necessary for production of HIV’s double-stranded DNA, which would be
subsequently integrated into the genetic material of the infected cell (where it is called a
provirus). Cellular enzymes convert zidovudine into the effective zidovudine-triphosphate
(it is similar to the compound thymidine triphosphate, a chemical that is required by the
HIV virus to make new DNA) form where it may also inhibit DNA polymerase (the reverse
204 Pharmaceutical Chemistry
transcriptase uses zidovudine triphosphate instead of thymidine triphosphate for making
DNA, and it is the zidovudine triphosphate that interferes with the reverse transcriptase)
used by human cells to undergo cell division, and later the cell’s ability to quickly repair
its own DNA chain if it is broken by zidovudine during its formation, whereas the HIV
virus lacks that ability. Thus, zidovudine inhibits HIV replication without affecting the
function of uninfected cells. Zidovudine with high doses begins to inhibit the cellular DNA
polymerase used by mitochondria to replicate, accounting for its potentially toxic but
reversible effects on cardiac and skeletal muscles, causing myositis. Mechanism is
completed in two steps:
(a) Conversion of zidovudine to its active triphosphate form: In the host cell,
zidovudine is converted into zidovudine triphosphate by cellular thymidine kinase
enzymes which makes it a highly selective and relatively non-toxic antiviral agent
(Fig. 8.15).
Me
HO
O
N
3
Zidovudine
Cellular
Thymidylate
Kinase
O
NH
O
N
Viral
thymidine
Kinase
Zidovudine monophosphate
Me
O
O
O
O
O
P
P
O
O
Zidovudine triphosphate
O
O
P
O
Fig. 8.15: Phosphorylation of zidovudine
O
O
O
P
O
O
N
O
N
3
Me
NH
O
O
DNA
Cellular
Thymidylate
Kinase
Zidovudine
phosphate
incorporated
into viral DNA
O
O
O
P
O
Zidovudine diphosphate
mono-
O
P
O
NH NH
N
O
N
3
Polymerases
O
Me
O
O
N
3
Viral DNA
synthesis
inhibited
O
O
N
(b) Zidovudine mimic for guanine and other nucleoside: Zidovudine triphosphate is
a good mimic for thymidine analogue and more potent and selective substrate for
the viral DNA polymerase. This results in zidovudine being incorporated in a
growing viral DNA chain in place of thymidine analogues. Zidovudine contains
non-nucleophilic azide group (no 3’-OH group unlike guanosine) thus, cannot
undergo polymerization resulting in viral DNA chain termination. Thus, it has
been used since last few decades as a potent antiviral drug (Fig. 8.16).
HIV-AIDS: Antiviral Agents 205
5' end
O
Base
O
NH
O
O
P
O
O
O
O
OO
OH
2
N
N
O
O
O
P
O
e
M
O
NH
N
O
O
Zidovudine contains
N
3
non-nucleophilic azide
group. It lacks 3'-OH
group thus leads to DNA
chain termination.
O
O
P
O
O
O
O O
O
O
P
O
5' end
Base
O
NH
O
P
O
O
O
O
P
P
OO
2
N
N
O
O
OH
O
Me
O
Fig. 8.16: DNA Chain reaction with zidovudine leads to DNA chain termination
NH
O
N
O
N
3
O
O
P
O
POP
OHOO
Diphosphate
8.6.1.4 Metabolic Pathways of Zidovudine
AZT is metabolized primarily by three separate pathways, and about 95% of a total dose
is recovered in the urine, with 15–20% as unchanged drug. The major pathway is first-pass
glucuronidation with renal excretion and results in the elimination of about 65–75% of the
WRWDOGRVH7KHXULQDU\JOXFXURQLGHPHWDEROLWHĻD]LGRĻGHR[\Ļ2D-D-glucopyranosyl-
WK\PLGLQH LV IRUPHG E\ WKH DFWLRQ RI XULGLQH ĻGLSKRVSKRJOXFXURQ\O WUDQVIHUDVH $
second pathway involves the action of various hepatic CYP450 oxidases and reductases,
UHVXOWLQJLQWKHSURGXFWLRQRIDWR[LFPHWDEROLWHVXFKDVĻDPLQRĻGHR[\WK\PLGLQH7KLV
metabolite is formed to varying extents in different tissues and represents about 2% of the
WRWDOGRVHLQWKHXULQH,WKDVDORQJHUSODVPDKDOIOLIHWKDQHLWKHU]LGRYXGLQHRUĻD]LGR
ĻGHR[\Ļ2D-D-glucopyranosyl-thymidine (Fig. 8.17).
Phosphorylation of AZT is the crucial stage responsible for the metabolic pathway. The
mono-, di- and tri-phosphates of AZT are formed rapidly through the action of thymidine
kinase, thymidylate kinase and pyrimidine nucleoside diphosphate kinase, respectively.
Since AZT is a good substrate for thymidine kinase, thus AZT monophosphate accumulates
quickly. AZT, di- and tri-phosphates are present in equal proportions of about ~5%. There
is evidence that this balance is shifted in HIV-positive patients with CD4+ counts between
300 and 500/mm
3
, in which AZT monophosphate constitutes about 74% of the total
phosphates and AZT di- and tri-phosphate each account for about 13%. The difference
may be due to the viral infection, since short-term and long-term exposure to AZT produces
206 Pharmaceutical Chemistry
similar results. In patients, the zidovudine monophosphorylation pathway saturates after
each dose of 100 mg or more, suggesting that monophosphorylation is largely independent
of current clinical doses. Conversion to di- and tri-phosphates is more closely related to
individual phosphorylation capacity. AZT triphosphate is eventually incorporated into
DNA, resulting in the termination of replication. The antiviral activity of the drug is
thought to result from direct inhibition of viral reverse transcriptase and truncation of
proviral DNA replication. Although critical to the mechanism of antiviral activity, AZT
phosphorylation is responsible for only a small fraction (~1%) of the total disposition of
the drug.
HN
O
O
3
Thymidine
Pyrimidine
nucleoside
Diphosphate
(<1%)
O
N
kinase
kinase
CH
3
O
CH
HN
HO
NH
3'-Amino-3'-deoxythimidine
HOOC
HO
HO
N
O
O
2
OH
3
or P450 reductase
O
N
3
O
HN
HO
CYP450
Glucuronyl
transferase
O
CH
HN
O
N
O
3
5'-Glucuronyl AZT
inactive (60-70%)
Fig. 8.17: Metabolic pathway of zidovudine
O
N
O
N
3
AZT
CH
3
Thymidine
Excreted
in urine
kinase
O
P
O
3
N
AZT mono-phosphate
AZT di-phosphate
AZT tri-
phosphate active
8.6.1.5 Side Effects
Severe allergic reactions (rash, hives, itching, difficulty in breathing, tightness in the chest,
swelling of the mouth, face, lips, or tongue), muscle pain, aches, cramps, or weakness,
severe or persistent tiredness or weakness, symptoms of infection (fever, chills, persistent
cough or sore throat, decreased or painful urination), symptoms of lactic acidosis (fast
breathing, irregular heartbeat, severe or unusual nausea, drowsiness, vomiting,
sluggishness, dizziness or light-headedness, feeling of being unusually cold), symptoms
of the liver problems (yellowing of the skin or eyes, dark urine, pale stools, severe or
persistent loss of appetite, nausea, or stomach pain), symptoms of pancreatitis (severe
stomach or back pain, with or without nausea or vomiting). Less serious side effects may
include sleeping problems (insomnia), mild nausea, constipation, joint pain, headache,
and change in the shape or location of body fat (especially in arms, legs, face, neck, breasts,
and trunk).
HIV-AIDS: Antiviral Agents 207
QUESTIONS
1. What are antiviral agents? Explain in detail.
2. Explain nucleocapsid and virions in detail.
3. How does life cycle of viruses work? Explain.
4. How are viruses different from bacteria?
5. What are the full forms of AIDS and HIV?
6. Write short notes on
(a) Acyclovir
(b) AIDS
(c) Zidovudine (AZT)
(d) Metabolic pathway of AZT
7. How can antiviral drugs be categorised on the basis of their mechanism of action?
Explain.
8. Write the synthesis of Acyclovir.
9. What is the alternate way to synthesize acyclovir?
10. How can DNA building block 5’-O-triphosphate Zidovudine nucleoside be
synthesized?
11. How acyclovir is mimicking of guanine nucleoside? Explain the mechanism.
12. Explain the bio-mechanism of acyclovir.
13. Explain the metabolic pathway of acyclovir.
14. What is HIV? Explain the life cycle of HIV.
15. How is AZT synthesized?
16. How AZT is mimicking of thymine nucleoside? Explain the mechanism.
17. What are the side effects of antiviral drugs?
9
Antimalarial Agents
9.1 INTRODUCTION
Malaria is a major endemic worldwide and especially spreads in South Asian and African
regions. It is one of the most common infectious diseases that causes million deaths around
the globe. About three billion people are at risk of infection in 109 countries and most of
them are living or travelling to sub-Saharan Africa. According to WHO, 214 million cases
of malaria and 4,38,000 deaths due to malaria have been reported till the end of 2015
worldwide while most deaths under five years of age were reported.
The word “malaria” comes from the Italian word “mal’aria” for “bad airs.” Researchers
were unable to identify the malaria parasite and link the transmission of malaria to
mosquitoes until the 1880s and 1890s. Although the understanding of the mosquito cycle
led to a number of new approaches in vector control in the early 20
prophylaxis and therapy continued to draw on earlier remedies.
Malaria is caused by protozoan parasites called Plasmodium (unicellular pathogen,
plasma + eidos, eidos means form), belonging to the parasitic phylum Apicomplexa.
Approximately, >200 species have been identified for the genus Plasmodium, among them
four species of Plasmodium are well studied worldwide which cause human malaria,
namely, Plasmodium falciparum, P. ovale, P. vivax, and P. malariae. The parasite P. falciparum
is one of the most dangerous forms of malaria while a few cases are reported due to P.
brasilianum, P. cynomolgi, P. cynomolgi bastianellii, P. inui, P. schwetzi, P. rhodiani, P. semiovale,
P. eylesi and P. simium. It is believed that malaria parasites infecting humans were probably
transferred from the apes (in case of P. knowlesi, macaques) to the man. More than 30,000–
40,000 years ago in ancestors of South Asian population have found strong evidences that
P. knowlesi has been analysed by the mtDNA data, thus it could be stated that human
infections with P. knowlesi were not new. It is believed that ecological changes and
increasing human population are responsible for parasitic pathogen Plasmodium to be
transmitted to humans as the preferred host.
Malaria parasites are mainly transferred by female Anopheles mosquitoes. They generally
bite humans and suck the blood to the gut of mosquitoes. A female mosquito injects
sporozoites (15–20 in numbers) into the blood stream of human host and these take almost
one hour to reach the liver cells. Then sporozoite develops into a tissue schizont (it contains
th
century, malaria
Antimalarial Agents 209
~10000-30000 merozoites) in the liver cells with the help of Kupffer cells. After 3 to 16 days,
schizont ruptures and releases the merozoites into the blood stream which finally invade
erythrocytes. Some sporozoites turn into hypnozoites especially in the case of P. ovale and
P. Vivax. Hypnozoites can remain dormant in the liver cells even after the initial infection,
that may cause relapse for months or in some cases, years. Parasite develops in the
erythrocyte stage where ruptured schizont spreads into the bloodstream. Schizonts
produced from immature trophozoite (ring stage) are followed by the mature trophozoite
in erythrocyte stage. Generally, the erythrocyte ruptures after 48 hours in case of P.
falciparum, P. ovale and P. vivax and 72 hours in case of P. malariae and releases
new merozoites (approximately 16–32) into the bloodstream some of which invade
erythrocytes. Then some merozoites and trphozoites result in the formation of gametocytes,
which are later transferred to a mosquito midgut during another blood meal. These
ingested gametocytes in the midgut of mosquitoes undergo sexual reproduction that
generate ookinetes and then oocyst. This oocyst later ruptures, producing thousands of
ineffective sporozoites which finally move towards salivary gland, ready for a new
infection cycle.
Clinical symptoms of malaria can be analysed by analysing the amount of erythrocytic
rupture, cell debris and parasite’s waste actually released into the bloodstream of humans.
Some common symptoms are headache, nausea, fever, chills, vomiting, abdominal upset,
joint or back pain and diarrhoea. P. falciparum malaria is also known as malaria tropica. It
can lead to severe malaria with a fatal outcome (~10–40% of all severe cases of malaria)
whereas severe malaria symptoms of P. ovale, P. vivax and P. malariae infections are found
in non-immune persons. In contrast, malaria has common symptoms such as severe
headache, shivering, vomiting, fever, joint pain, convulsions and coma.
9.2 LIFE CYCLE OF THE MALARIA PARASITE
The life cycle of malaria parasite (Fig. 9.1) concludes the following facts:
(a) A female Anopheles mosquito carrying malaria parasites, injects the parasites into
the bloodstream of human host in the form of sporozoites which travel into the liver
cells.
(b) During 3–16 days of injecting sporozoites, they grow up, divide, and finally produce
a huge quantity of merozoites in each liver cell. Some malaria parasite species
remain dormant for a long period of time in the liver thus they can take weeks or
months to degrade.
(c) After merozoites formation in the liver cells, they again enter the bloodstream.
They start a new cycle where disruption of red blood cells (RBCs) and asexual
replication takes place. Thus, they again release newly formed merozoites from the
RBCs continuously for over 1–3 days of infection, which later results in thousands
of infected cells travelling to the bloodstream leading to illness until not treated
with medicine.
210 Pharmaceutical Chemistry
(d) Some of the merozoite-infected blood cells don’t go for further multiplication
instead they are involved in asexual multiplication and develop male and female
gametocytes, these again travel into the bloodstream.
(e) After mosquito bites an infected human (who is already suffering with malaria)
mosquito ingests some of the infected blood cells and gametocytes into the gut.
These infected human blood cells burst and release the gametocytes into the gut
which later develop into fully mature male and female sex cells called gametes.
After maturation, they fuse together and form diploid zygotes, which develop into
actively moving ookinetes that burrow into the mosquito midgut wall and form
oocysts.
Sexual stage:
male or female
gametocytes
form
4
Gametocytes
5
Early
mosquito
stage
Gametes
Zygote
Ookinete
Oocyst
6
Late
mosquito
stage
Human
blood cell
cycle
Human
blood cell
Liver
The mosquito ingests
the parasite during
3
Merozoites
Human
liver cell
Human
liver stage
Fig. 9.1: Life cycle of malaria parasites (Picture taken from NIH)
2
blood feeding.
The mosquito
injects the
parasite when it
bites the human.
1
Sporozoites
(f) In 8–15 days oocyst mature in the midgut and burst to produce thousands of active
haploid forms of sporozoites which later travel into the salivary glands of the
mosquito and finally cycle completes. The cycle restarts when mosquito bites a
person and injects sporozoites from its salivary glands into the human bloodstream.
Antimalarial Agents 211
9.2.1 Plasmodium Uses Haemoglobin as a Nutrient
RBCs contain haemoglobin and food vacuole. The parasite digests the host cell’s
haemoglobin resulting in the release of essential amino acids and also huge amount of
toxic heme. To protect itself, the parasite uses heme polymerase to polymerize the toxic
heme to nontoxic heamozine. Chloroquine is the drug which can act as the same.
9.2.2 Glossary Used in Life Cycle of Malaria
Haploid (cells containing a half set of chromosomes).
Diploid (cells containing a full set of chromosomes).
Gametes (male & female reproductive elements).
Gametocytes (sexual forms of the malaria parasite known as male or female gametes
which actually are released within the midgut or stomach of the mosquito).
Merozoite (form of the malaria parasite that invades red blood cells in erythrocyte).
Zygote (diploid cell resulting from combining of a male and female gamete).
Ookinete (zygote is a malarial organism that penetrates the mosquito midgut to form an
oocyst under the outer gut lining).
Oocyst (male and female gametes combine and form oocyst in the midgut of mosquito).
Sporozoite (infectious form of the malaria parasite, which is injected into the humans by
mosquitoes through salivary gland).
9.3 ANTIMALARIAL DRUGS
Antimalarial drugs are specially used in the treatment and prevention of malaria infection
caused by parasites. Most of these drugs, specially target the erythrocytic stage of malaria
infection that causes symptomatic illness, while antimalarial drugs are not well
characterized for pre-erythrocytic stage (hepatic stage). Basically, acute blood infection
caused by malaria species is necessary for the treatment while the infection due to P. vivax
or P. ovale requires drugs which are active against hypnozoites (dormant sporozoites in the
liver for months and years). Some drugs can be used prophylactically to prevent malaria,
while others are directed towards treating acute attacks. In general, antimalarial drugs are
classified in terms of the action against the different stages of the life cycle of the parasite
as:
(a) Blood schizonticidal agents are used to treat the acute attack. They act on the
erythrocytic forms of the plasmodium. Examples: Artemisinin, Chloroquine, and
Quinine, etc.
(b) Tissue schizonticidal agents have a radical cure effect by acting on the parasites in
the liver, these drugs also destroy gametocytes and thus reduce the spread of
infection. Example: Primaquine, etc.
There are some common antimalarial agents that have been used for decades, such as
quinoline derivatives (viz., chloroquine, quinine, quinidine, mefloquine, amodiaquine,