A one-pot process for high-yield regioselective synthesis of 9-[(2-hydroxyethoxy) methyl]guanine (acyclovir),  an antiherpetic agent, was achieved from guanine via the steps of reacting of N2,N9 -diprotected guanine with 1-acetyl-2-acetylmethoxy-ethyleneglycol in presence of phosphoric acid or polyphosphoric acid. Total yield of product was 59%. The obtained acyclovir meets the standards in the British Pharmacopoeia 2007 (BP2007). Its activity as inhibitor of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) was evaluated according to the plaque reduction assay method and compared with the standard compound. The synthesized acyclovir showed a similar activity as the control. Keywords: Acyclovir, Herpes simplex virus, Nucleoside, One- pot synthesis

Hung, Tran Quang

Sung, Tran Van

Thuong, Nguyen Thi

Vietnam Academy of Science and Technology: Journals Online

 523
Journal of Chemistry, Vol. 47 (4), P. 523 - 528, 2009 
 
SYNTHESIS OF ACYCLOVIR AS AN ANTIHERPES-VIRUS DRUG  
Received 4 February 2009  
Tran Quang Hung1, Nguyen Thi Thuong2, Tran Van Sung1 
1Laboratory of Organic Synthesis, Institute of Chemistry, VAST 
2Laboratory of Herpes viruses, Faculty of Virus, National Institute of Hygiene and Epidemiology 
 
Abstract 
A one-pot process for high-yield regioselective synthesis of 9-[(2-hydroxyethoxy) 
methyl]guanine (acyclovir),  an antiherpetic agent, was achieved from guanine via the steps of 
reacting of N2,N9 -diprotected guanine with 1-acetyl-2-acetylmethoxy-ethyleneglycol in presence 
of phosphoric acid or polyphosphoric acid. Total yield of product was 59%. The obtained 
acyclovir meets the standards in the British Pharmacopoeia 2007 (BP2007). Its activity as 
inhibitor of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) was evaluated according to 
the plaque reduction assay method and compared with the standard compound. The synthesized 
acyclovir showed a similar activity as the control. 
Keywords: Acyclovir, Herpes simplex virus, Nucleoside, One- pot synthesis. 
 
I - Introduction 
Virus was a cause of many dangerous 
diseases and variety of epidemic diseases in 
large area. It is important that antibiotics have 
no or almost no effect on viruses. Although the 
production of herpes vaccine is desired by many 
countries but it still stays on a testing level. The 
prevention of herpes infection is a special 
problem because the disease often recurred in 
present of antibody. Therefore, development of 
anti-virus drugs is important, necessary and still 
be a leading interest.  
 
1: Acyclovir  
Acyclovir, {9-[(2-hydroxyethoxy)methyl] 
guanine} (ACV 1), was first reported by 
Schaeffer and co-workers [1] and is a nucleoside 
analogue. It acts selectively on herpes-infected 
cells. It has specific inhibition effects on 
replication of herpes virus simplex type 1 and 
type 2 (HSV1 and HSV2) and Varicella zoster 
virus that cause almost no intoxication to the 
host cells. From nearly three decades ago, ACV 
was used as a primary drug to treat herpes virus, 
especially genital herpes and herpes simplex 
encephalitis and herpes in immunity deficient 
people [2].  
ACV has no activity against viral pathogens 
until it is converted to an active form acyclovir 
triphosphate. Its mechanism of action is initiated 
by the viral enzyme Thymidine kinase; 
subsequently, human Cellular kinase perform 
the second and the third phosphorylation steps 
to complete the process (figure 1). Acyclovir 
triphosphate, the active form of acyclovir, is 
present in 40- to 100-fold higher concentrations 
in herpes simplex virus (HSV)-infected cells 
than in uninfected cells. As a result, the 
selectivity of ACV is significant [3, 4]. 
 524
Acyclovir triphosphate has a two-pronged 
mechanism of action: (1) it competes with 2-
deoxyguanosine triphosphate (dGTP) as a 
substrate for viral DNA polymerase and (2) 
once it becomes incorporated into the 
replicating viral DNA, it acts as a chain 
terminator because it does not have a terminal 
3-hydroxyl group [3].  
 
 
 
Figure 1: Acyclovir mechanism of action 
 
ACV was prepared in different forms such 
as tablets (200 mg, 400 mg, 800 mg), capsules 
200 mg, powder for injection, oral suspension, 
creams for skin 5% and for eyes 3%. After oral 
administration, ACV is absorbed slowly and not 
completely, bioavailability of only 10-20% [9].  
ACV rapidly attracted attention of chemist, 
as well as pharmacist by virtue of its selective 
anti-virus activity and effect. There were many 
efforts for synthesis of ACV by different ways 
reported. Among divert orientations of 
synthesizing ACV, it could be accounted 5 
common and most effective pathways. The most 
common pathway departed from guanine with 
effectiveness and simplicity where guanine was 
acylated [5]. The important problem is to 
decrease the N7 substituted isomer and 
selectively akylating into N9 position. Our 
synthesis of ACV is showed in figure 2.
 
 
 
Figure 2: Pathway for synthesis of acyclovir 
 
II - Experimental 
Preparation of dioxolane-diacetate (1-acetyl-
2-acetylmethoxy-ethyleneglycol) 
Dioxolane 5 (224 g, 99%, 211.6 ml, 3 mol) 
was added drop wise to a solution of p-
toluenesulfonic acid monohydrate (10 g, 0.053 
mol) in acetic anhydride (306 g, 283.3 ml,3 
mol) and acetic acid (45 g, 42.9 ml, 0.75 mol) 
precooled to below 10oC, at the rate that 
maintained the reaction temperature under 30oC. 
The solution was then stirred for an additional 
hour at a temperature under 30oC, and then 
distilled. The first portion of distillate was 
collected at a temperature of 40 - 80oC/1.5 
mmHg and contained acetic acid, acetic 
anhydride, and other materials. The second 
portion of distillate was collected at 80 - 95oC. 
at 0.75 mmHg and redistilled to give pure 7 in a 
yield of 431.5 g (81.7%). 1H-NMR (CD3OD, 
500 MHz, δ ppm): 2.06, 2.08 (s, 6H, 2xCH3), 
3.85, 4.21 (s, 4H, OCH2CH2O), 5.28 (s, 2H, 
OCH2O). 
 525
 
 
Figure 3: Preparation of Dioxolane-diacetate  
 
Preparation of acyclovir 
A mixture of guanine (3, 32.13 g, 213 
mmol), acetic anhydride (320 ml) and acetic 
acid (480 ml) was refluxed overnight, during 
which time the mixture became almost clear. 
The liquid was removed by evaporation in 
reduced pressure. To the residue was added 
toluene (300 ml), 7 (75 g, 426 mmol), and 
phosphoric acid (85.5%, 1.5 ml) with stirring. 
The resulting mixture was refluxed for 6 - 7 
hours with vigorous stirring.  
Toluene and acetic anhydride were then 
removed under reduced pressure and the residue 
was heated at 80oC (oil bath) under reduced 
pressure (20 mm Hg) for another 2 hours, 
cooled to room temperature, triturated with 
ethyl acetate (100 ml), and stirred at room 
temperature overnight.  
The resulting solid was collected by 
filtration, washed twice with ethyl acetate (25 
ml), dissolved in ammonium hydroxide (30%, 
300 ml). The solution became clear and solution 
was stirred at room temperature overnight. The 
mixture was concentrated to dryness under 
reduced pressure. The residue was treated with 
methanol (150 ml) and the resulting mixture 
was heated at 80oC for 1 hour, cooled, and 
allowed to stand at room temperature overnight. 
The resulting solid was collected by filtration 
and recrystallized from water (1700 ml) using 1 
gram of activated carbon. The filtrate was 
cooled to room temperature, and stored in the 
refrigerator overnight. The resulting solid was 
collected by filtration, washed twice with 
methanol (20 ml), and dried to give pure 
acyclovir 1 (30 g, 63%). mp. 242 - 244oC (H2O). 
UV λmax (MeOH-H2O; 1-1) nm: 251, 276 (sh); 
1H-NMR (DMSO-d6, 500 MHz, δ ppm): δ 3.34, 
3.46 (s, 4H, CH2CH2), 4.65 (br, 1H, OH), 5.34 
(s, 2H, OCH2O), 6.48 (s 2H, NH2), 7.80 (s, 1H, 
H-8), 10.62 (s, 1H, NH). EI-MS m/e (%) 226 
[M+] (100), 152 [M+-C3H6O2] (42). The mother 
liquor was concentrated to give a second crop 
(2.6 g) which contained the N7-isomer.  
Antiherpetic activity evaluation 
a) Cells and viruses 
Vero cells (Green Africa Monkey Kidney) 
were propagated in culture medium (Medium 
Essential Medium (MEM, GIBCO), 
supplemented with 3% fetal bovine serum (FBS, 
Sigma), glutamine, non-essential amino acid, 
and antibiotics). 
The 5 clinical HSV isolates used in this 
study included 2 HSV-1 and 3 HSV-2. 
b) Method of plaque reduction assay (PRA) 
Confluent Vero cell monolayer in 24-well 
culture plates were infected with 200 μl of 10 
fold dilutions from 10-5 to 10-2 of virus stocks 
per well. After 1 hour incubation at 37oC x 
5%CO2 with gently rocking, inoculates were 
discarded and cells were incubated with culture 
medium and 1.0% methylcellulose (Sigma) with 
the presence of increasing antiviral 
concentrations of 0, 1, 5, 15, and 50 μM. 
Plaques were counted at 72 h post inoculation 
after cells were treated with formaldehyde and 
stained with crystal violet. IC50s were 
determined on log papers and defined as the 
minimum concentration of ACV that reduced 
the numbers of plaque by 50% compared to the 
cell controls (absence of ACV). IC50s ≥ 7 μM 
were considered resistant to ACV. 
III - Results and discussion 
1. Preparation of acyclovir 
A method for the preparation of ACV is 
 526
provided that is suitable for the commercial 
manufacture of the product. In the first step, 
guanine is acetylated using acetic anhydride, 
acetic acid, and phosphoric acid. In the second 
step, diacetyl guanine is alkylated at the N9-
position using CH3COOCH2O(CH2)2OCOCH3, 
acetic anhydride and phosphoric acid or 
polyphosphoric acid. The acetyl groups are then 
removed as desired. 
IC50
 
Figure 4: Plot for determination of IC50 
2. Acetylation of guanine 
In the first step of the reaction, the 2-amino 
group of guanine is protected to prevent it from 
being alkylated in the second step of the 
reaction. The choice of protecting group may 
effect the ultimate yield of product, in that 
protecting groups are removed with varying 
degrees of difficulty. In general, in the process 
of protecting the 2-amino group, the N9 group 
usually also reacts with the protecting group. 
Acylation, and in particular, acetylation, 
activates the N9-position toward alkylation in 
the second step, and therefore, is desirable. 
Diacetylguanine, which has been used as an 
intermediate in the production of acyclovir, has 
been prepared using several methods. Guanine 
has been acetylated using acetic anhydride in 
N,N-dimethylacetamide to give diacetylguanine 
in 90.5% yield [6]. This reaction produces a 
product which is grey in color due to the high 
reaction temperature used (160oC for 7 hours). 
In general, it is preferred to use a symmetrical 
anhydride in the reaction scheme that 
corresponds to the acid used. For example, 
acetic acid is preferably used in combination 
with acetic anhydride, and propionic acid is 
preferably used in combination with propionic 
anhydride. 
Guanine has also been acetylated in acetic 
anhydride and acetic acid to give different 
products depending on the work-up conditions 
[7]. For example, after the reaction mixture 
becomes an almost clear solution, if solvents are 
removed by distillation, only diacetyl guanine is 
obtained in 95% yield. However, the addition of 
water at 60oC. followed by stirring at room 
temperature overnight produces N2-
acetylguanine in 94.4% yield. If the reaction 
mixture is merely cooled down, a mixture of 
mono- and di-acetylguanine is produced.  
3. Preparation of dioxolane-diacetate (7) 
The dioxolane ring is opened using acetic 
anhydride as the ring-opening reagent in the 
presence of catalytic amounts of p-
toluenesulfonic acid. This provided dioxolane-
diacetate in 80% yield after distillation by the 
method of Chen et al. [8]. 
4. Alkylation of diacetylguanine 
In this step of the synthesis, the diacetylated 
guanine from previous step is alkylated in the 
N9-position to produce 2-acetamido-9-(2-
acetylethoxymethyl)guanine.  
Matsumoto, et al., have studied the effect of 
solvent, acid catalyst and reaction temperature 
on the yield of the alkylation of diacetylguanine 
with 2-oxo-1,4-butanediol diacetate ("dioxolane 
diacetate") to produce N2 ,O-diacetylacyclovir 
[7]. Among the acid catalysts tested (p-
toluenesulfonic acid, sulfanilic acid, p-
nitrobenzenesulfonic acid, 2,4-dinitrobenzene 
sulfonic acid, iron(II) sulfate, and zinc chloride), 
Matsumoto reported that p-toluenesulfonic acid 
and sulfanilic acid exhibited the highest 
catalytic activity. In a preferred reaction 
scheme, a combination of an acid and anhydride 
are used.  
5. Deacetylation of acyclovir diacetate 
Both methylamine and ammonium 
hydroxide proved to be good deacetylating 
agents. Although the working-up is easier using 
methylamine than it is using ammonium 
hydroxide, on an industrial scale, the smell of 
methylamine may dictate the preferred use of 
ammonium hydroxide as a deacetylation agent. 
 527
The synthesized ACV has been analyzed in 
the National Institute for Drug Quality Control. 
It meets all the standards of BP 2007. 
6. Biological activity 
ACV was evaluated against HSV-1 and HSV-
2. The effects of standard ACV and tested ACV 
on 5 clinical HSV isolates included HSV-1 and 
HSV-2 were tested in parallel with their antiviral 
activity. The results are given in table 1. 
 
Table 1: The antiviral activity of ACV compared to the standard ACV 
IC50  
Order 
Clinical HSV 
isolate Type Standard Tested 
Correlated 
coefficient
1 06003 HSV-2 1.70 S 1.20 S 
2 07069 HSV-1 0.53 S 0.57 S 
3 07089 HSV-2 0.91 S 0.10 S 
4 07092 HSV-2 2.80 S 2.60 S 
5 07094 HSV-1 0.89 S 0.53 S 
0.95 
S: Sensitive. 
.
The tested ACV showed the anti-HSV 
activity at low concentration and very 
corresponds with standard ACV. The correlated 
factor is 0.95. 
 
‘  
Figure 5: Antiherpestic activity of standard 
(left) and tested (right) acyclovir 
 
IV - Conclusion 
This report describes a one-pot process for 
the preparation of acyclovir from guanine. 
Reacting N2,N9-diprotected guanine with 1-
acetyl-2-acetylmethoxy-ethyleneglycol in 
present of phosphoric acid as catalyst provide 
ACV with high yield and good regioselectivity. 
Therefore, the purification was simple, 
inexpensive and conforming to pilot scale. The 
chemicals and the solvents were common and 
friendly with environment. The antiherpetic 
activity of the synthesized ACV was show to be 
comparable (95%) to the standard ACV. This 
synthesized ACV was approved by National 
Institute of Drug Quality Control to meet the 
quality standard of BP 2007. 
Acknowledgements: This research was 
supported by a research grant from Vietnam 
Academy of Science and Technology (VAST). 
 
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7. H. Matsumoto, C. Kaneko, K. Yamada, T. 
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Corresponding author: Tran Van Sung 
            Laboratory of Organic Synthesis, Institute of Chemistry, VAST. 


Synthesis of acyclovir as an antiherpes-virus drug

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Synthesis of acyclovir as an antiherpes-virus drug

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