Cinnamic esters of acyclovir-synthesis and biological activity

Article abstract of DOI:10.1080/15257770.2010.518185

In the present study we have synthesized esters of acyclovir (2-4) with cinnamic acids (p-coumaric, ferulic, and sinapic acids) and evaluated them for their antiviral and antioxidant potential. The antiviral activity of the newly synthesized compounds has

Full text of DOI:10.1080/15257770.2010.518185

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Cinnamic Esters of Acyclovir-Synthesis
and Biological Activity
Ivanka Stankova a , Kiril Chuchkov a , Peter Wutzler b & Michaela
Schmidtke b
a Department of Chemistry , South-West University “Neofit Rilsky” ,
Blagoevgrad, Bulgaria
b Jena University Hospital, School of Medicine , Department of
Virolgy and Antiviral Therapy , Jena, Germany
Published online: 04 Oct 2010.
To cite this article: Ivanka Stankova , Kiril Chuchkov , Peter Wutzler & Michaela Schmidtke (2010)
Cinnamic Esters of Acyclovir-Synthesis and Biological Activity, Nucleosides, Nucleotides and Nucleic
Acids, 29:10, 760-767, DOI: 10.1080/15257770.2010.518185
To link to this article: http://dx.doi.org/10.1080/15257770.2010.518185
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Nucleosides, Nucleotides and Nucleic Acids, 29:760–767, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.518185
CINNAMIC ESTERS OF ACYCLOVIR-SYNTHESIS
AND BIOLOGICAL ACTIVITY
Ivanka Stankova,1 Kiril Chuchkov,1 Peter Wutzler,2 and Michaela Schmidtke2
1Department of Chemistry, South-West University “Neofit Rilsky,” Blagoevgrad, Bulgaria
2Jena University Hospital, School of Medicine, Department of Virolgy and Antiviral Therapy,
Jena, Germany
In the present study we have synthesized esters of acyclovir (2–4) with cinnamic acids (p-
2
coumaric, ferulic, and sinapic acids) and evaluated them for their antiviral and antioxidant
potential. The antiviral activity of the newly synthesized compounds has been tested against hu-
man herpes virus 1 (HSV-1) in vitro. The results indicate that none of the synthesized compounds
inhibits the tested virus strain. The antioxidant properties have been studied using 2,2-diphenyl-1-
picrylhydrazyl (DPPH)∗ test.
Keywords Acyclovir; cinnamic acids; HSV-1; antioxidant effect; DPPH∗ test
INTRODUCTION
Following the discovery of the first effective antiviral compound
(idoxuridine) in 1959, nucleoside analogues, especially acyclovir, 9-[(2-
hydroxyethoxy)methyl] guanine (ACV), for the treatment of herpesvirus
infections, have dominated antiviral therapy for several decades.[1,2] How-
ever, ACV and similar acyclic nucleosides suffer from low aqueous solubility
and low bioavailability following oral administration. Derivatives of acyclic
nucleosides, typically esters, were developed to overcome this problem, and
valaciclovir (L-valine ester of ACV) was among the first of a new series of com-
pounds that were readily metabolized upon oral administration to produce
the antiviral nucleoside in vivo, thus increasing the bioavailility by several
fold.[3,4]
Valacyclovir represents a prototype prodrug for enhancing the oral
bioavailability of a poorly absorbed drug like acyclovir. The enhancement in
Received 20 June 2010; accepted 19 August 2010.
For the support of this work, the authors are grateful to the National Science Fund, Bulgaria
(Contracts VUL-304/07), DAAD project 03/2007, and South-West University “Neofit Rilski,” Blagoevgrad,
Bulgaria (SRP-C2/2010).
Address correspondence to Ivanka Stankova, Department of Chemistry, South-West University “Ne-
ofit Rilsky,” Blagoevgrad 2700, Bulgaria. E-mail: ivastankova@abv.bg
760

Cinnamic Esters of Acyclovir-Synthesis and Biological Activity
761
oral absorption of this amino acid ester prodrug has since been attributed to
carrier-mediated transport by intestinal oligopeptide transporters, followed
by rapid bioconversion to acyclovir. The successful clinical utility of oral
valacyclovir indicated the potential for amino acids as promoieties for other
potent agents.
Trans-Cinnamic acids, originally isolated from plant sources,[5,6] have
been reported.[7–9] This leads us to investigate a new pharmacological ac-
tivity of trans-cinnamic acid and its derivatives. For example, trans-cinnamic
acid induces cytostasis and a reversal of malignant properties of human tu-
mour cells in vitro. Furthermore, molecular analysis have been shown that
the antitumor activity of cinnamic acid may be due in part to the inhibition
of protein isoprenylation in mitogenic signal transduction.[10] p-Coumaric
acid or 4-hydroxy-trans-cinnamic acid has shown to possess antioxidant ac-
tivity. It minimized the oxidation of low-density lipoprotein (LDL) involving
direct scavenger of reactive oxygen species (ROS).[11] Moreover, the dehy-
drogenated polymers of p-coumaric acid inhibited HIV-1 protease activity.[12]
4-Methoxy-trans-cinnamic acid exhibited a potent hepatoprotective ac-
tivity in rat hepatocytes from toxicity induced by carbon tetrachloride
(CCl4).[13,14] Consequently, a broad range of biological activities of cinnamic
acids have been reported, this leads us to investigate a new pharmacological
activity of trans-cinnamic acid and its derivatives.
The aims of this study include the synthesis and biological character-
ization of acyclovir analogues, derived from two compounds with proven
pharmacological activity-cinnamic esters and acyclovir. The cytotoxic and
antiherpetic activity, as well as the antioxidative effect of the sinapic-, p-
coumaric-, ferulic- esters of acyclovir are reported.
RESULTS AND DISCUSSION
Chemistry
Acyclovir derivatives modified with amino acids and peptides[15–18] and
acyclovir containing peptidomimetics[19,20] have been reported but acyclovir
containing cinnamic acids are not known. In order to obtain analogues
with more desirable characteristics, we synthesized new esters of acyclovir
containing p-coumaric, ferulic, and sinapic acids.
A mixture of cinnamic acid and N,N’-dicyclohexylcarbodiimide (DCC)
in dimethylformamide (DMF) was stirred for 1 hour at 0◦C under nitrogen
atmosphere. A solution of ACV (Scheme 1) and 4-N ,N-(dimethylamino)-
pyridine (DMAP) was added to the reaction mixture and stirred for 24
1
hours. The H, 13CNMR and mass spectra were consistent with the desired
structure.

762
I. Stankova et al.
O
N
N
HN
O
N
N
H2N
N
N
OH
NH
NH2
O
CO
O
R'
O
ii
HO
R
i
COOH
R'
HO
R
SCHEME 1 Synthesis of the compounds 2–4. (i) DCC, DMF, 0◦C, 1 hour; (ii) DMAP, room temperature,
24 hours.
Nuclear magnetic resonance (NMR) data recorded in this research
clearly indicated the structure of the esters (Figure 1). The cinnamic acids
could react with ACV at either CH2–OH or NH2 sites. However, only the es-
ter linkage was formed in this research, which was supported by the change
of chemical shift of the CH2–OR signal from 3.35 in ACV to 4.1 after the re-
action. The values of the proton–proton vicinal coupling constants (3JH/H
about 15.5 Hz) measured for the olefinic protons of feruloyl-, sinapoyl-, and
p-coumaroyl residues define the E configuration of the double bond of all
O
O
N
NH
N
HN
CO
O
N
N
NH2
O
N
H2N
N
OH
O
HO
acyclovir-2'-O-p-coumarate (2)
acyclovir (1)
O
N
O
N
N
N
N
NH
NH2
NH
NH2
CO
O
CO
O
N
O
MeO
HO
O
HO
OMe
acyclovir-2'-O-ferulate (4)
OMe
acyclovir-2'-O-sinapolate (3)
FIGURE 1 Structures of acyclovir (ACV) and its cinnamic esters.

Cinnamic Esters of Acyclovir-Synthesis and Biological Activity
763
TABLE 1 Cytotoxicity and antiherpetic activity of the compounds[21] (2–4) in GMK cells
50% cytotoxic
concentration (µg/ml)
Antiherpetic
activity
Compound
Acyclovir-p-coumarate (2)
Acyclovir-sinaponate (3)
Acyclovir-ferulate (4)
23.4
41.5
>50
not active
not active
not active
studied compounds. The chemical shift of the esters protons established the
ester linkage of the acyclovir with cinnamic acid.
Biological Evaluation and Discussion
Cytotoxicity and antiherpetic activity of the newly synthesized cinnamic
esters of acyclovir (2–4) was tested in GMK cells.
To exclude nonspecific antiviral activities, the derivatives of acyclovir
were tested for cytotoxicity on confluent monolayer’s of host cells. Table 1
summarizes the 50% cytotoxic concentrations of the test compounds in GMK
cells. The results demonstrate a good compatibility of the new acyclovir
analogous for the tested cell lines. None of the compounds inhibited the
HSV-1-induced cytopathic effect in GMK cells (Table 1).
Antioxidant Activity
The antioxidative potential of the synthesized esters was studied against
DPPH∗ (1,1-diphenyl-2-picrylhydrazyl radical). The obtained results are sum-
marized in Table 2. The synthesized cinnamoyl esters of acyclovir were found
TABLE 2 Antioxidative activity of esters of acyclovir with cinnamic acid by DPPH∗
(%) RSA
0.9 mM
1.8 mM
3.6 mM
Reaction time (min)
Compound
10ꢁ
20ꢁ
10ꢁ
20ꢁ
10ꢁ
20ꢁ
Acyclovir 1
1.3
2.1
1.2
16.1
4.1
12.0
2.2
1.4
2.9
1.4
17.2
5.3
1.6
3.7
1.7
26.5
5.7
21.0
3.3
1.7
4.7
1.9
31.9
7.1
25.1
3.7
2.1
4.5
2.0
69.0
8.5
36.7
4.3
2.7
6.1.
2.3
69.6
10.1
44.3
4.9
p-Coumaric acid
Acyclovir-p-coumarate 2
Sinapic acid
Acyclovir-sinaponate 3
Ferulic acid
13.8
2.7
Acyclovir-ferulate 4
% RSA—percentage radical scavenging activity; sinapic-, ferulic-, p-coumaric, acids and acyclovir used
as standards.

764
I. Stankova et al.
to be inefficient radical scavengers. The compounds (2–4) showed lower an-
tioxidative effect than that of the standards: p-coumaric, ferulic, and sinapic
acids. The ester of acyclovir with sinapic acid showed border line activity
compared to the free cinnamic acids in the DPPH∗ test.
In conclusion, p-coumaroyl, sinapoyl-, feruloyl-, esters of acyclovir have
been synthesized by the methods used in the peptide chemistry, and their
antiherpetic activity, as well as their antioxidant potential, has been explored.
The results show that modification of acyclovir with cinnamic acids reduced
its antiviral activity. The new compounds 2–4 showed antioxidant activity,
but lower to the standards, p-coumaric, ferulic, and sinapic acid.
EXPERIMENTAL
Chemistry
3-(4-Hydroxyphenyl)-2-propenoic acid (p-coumaric), 3-methoxy-4-
hydroxy-cinnamic (ferulic) and 3,5-dimethoxy-4-hydroxy-cinnamic (sinapic)
acids were purchased from Fluka (Buchs, Switzerland) and DMAP and N, N’-
dicyclohexylcarbodiimide (DDC) were purchased from Merck (Darmstadt,
Germany).
Thin layer chromatography (TLC) analysis was performed on aluminum
silica gel sheets 60 F254 plates (Merck) and spots were detected using an UV
lamp at 254 nm.
The NMR spectra was obtained on a Bruker Avance DRX-500 spectrom-
eter (Bruker, Germany), operating at 500.13 MHz. The ESI mass spectra was
obtained on Finnigan MAT 8200 (Finnegan, MasCom, Bremen, Germany).
“Agilent 8453” spectrophotometer was used for the measurement of the
reduction of DPPH∗ (1,1-diphenyl-2-picrylhydrazyl radical) absorbance at
516 nm.
General Synthetic Procedure for Preparation of Acyclovir
Cinammic Esters 2–4
The cinnamic acid (p-coumaric-, ferulic-, and sinapic-) (0.8 mM) and
was dissolved in DMF and the solution was cooled at 0◦C and N,N ꢁ-
dicyclohexylcarbodiimide (DDC) (0.165 g, 0.8 mmol) was added. After 1
hour, ACV (0.100 g, 0.4 mmol) and DMAP (0.097 g, 0.8 mmol) were added.
The reaction mixture was kept at 0◦C for 1 hour and was stirred continuously
for 24 hour at room temperature. The urea derivative was removed by filtra-
tion. After DMF was removed in vacuum, 30 ml of ethyl acetate was added
to the residue. This solution was washed with 10 ml of saturated sodium
bicarbonate solution and 20 ml of brine, respectively. The organic layer
was collected and dried over MgSO4. After removal of most of the solvent,
the residue was loaded on a silica gel column and eluted with a mixture of
hexane/ethyl acetate = 1:6.

Cinnamic Esters of Acyclovir-Synthesis and Biological Activity
765
2-(2-Amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)ethyl-p-coumarate 2
Yield: 0.076 g (51%); 1H NMR (500 MHz; CDCl3) δ = 3.69 (t, 1H, CH),
3.73 (s, 6H, OCH3), 4.1 (t, 2H, CH2), 5.28 (s, H), 5.42 (s, 2H,NH2), (6.03,
(s, H), 6.5 (s, H), 6.59 (d, 1H, J = 15.5, CH=), 7.21, 7.68 (d, 1H, J = 15.4 Hz,
CH=), 7.89 (s, 1H). 13C-NMR (500 MHz; CDCl3): δ = 173.3, 164.0, 166.1,
160.1, 147.4, 146.5, 137.2, 131.3, 126.8, 120.8, 119.4, 118.2, 115.3, 59.78.
ESI-MS: 372 ([M + H]+).
2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)ethyl-sinaponate 3
Yield: 0.104 g (60%); 1H NMR (500 MHz; CDCl3) δ = 3.85 (t, 1H, CH),
3.91 (s, 6H, OCH3), 4.10 (t, 2H, CH2), 6.53 (d, 1H, J = 15.5, CH=), 6.67 (s,
H), 6.74 (s, 2H, Ar–H), 7.57 (d, 1H, J = 15.4 Hz, CH=), 7.83 (s, 1H).13C-
NMR (500 MHz; CDCl3): δ = 162.58, 152.65, 114.22, 105.48, 104.64, 60.37,
56.36, 56.12, 55.59. ESI-MS: 432 ([M + H]+).
2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)ethyl-ferulate 4
Yield: 0.094 g (58%); 1H NMR (500 MHz; CDCl3) δ = 3.87 (s, 6H, OCH3),
4.1 (t, 2H, CH2), 5.86 (br. s, H), 5.42 (s, 2H,NH2), 6.3 (d, 1H, J = 15.4 Hz,
CH=), 6.8 (s, H, NH2), 6.9 (d, 1H, J = 8.9, Ar-H (m)), 7.02 (d, 1H, J = 1.7
Hz, Ar–H(o)), 7.6 (d, 1H, J = 15.5 Hz, CH=), 7.8 (s, H), 9.89 (s, H). 13C-
NMR (500 MHz; CDCl3): δ = 206.79, 167.23, 147.93, 146.79, 144.62, 122.99,
115.73, 114.72, 109.38, 60.31, 55.95. ESI-MS: 402 ([M + H]+).
Evaluation of the Biological Activity
Estimation of Radical Scavenging Activity (RSA) by the DPPH∗ Test
∗
1,1-Diphenyl-2-picrylhydrazyl radical (DPPH ) was used as stabile radical.
For each compound and concentration tested (0.9, 1.8, and 3.6 mM), the
reduction of DPPH∗ radical was followed by monitoring the decrease of
absorbance at 516 nm. The absorption was monitored at the start and at 10
and 20 minutes. The results are expressed as% RSA = [Abs516 nm (t = 0)
− Abs516 nm (t = tꢁ) × 100/Abs516 nm (t = 0)], as proposed by Pekkarien
et al.[22]
Estimation of Cytotoxic and Antiviral Activity
Cell Culture and Virus Propagation. Green monkey kidney cells (GMK;
Schaper and Bruemmer, Salzgitter, Germany) were grown in Dulbecco’s
modified MEM/E (Sigma, St. Louis, MO, USA) supplemented with 10%
fetal bovine serum (FBS, Greiner, Germany), 100 U/ml penicillin, and 100
mg/ml streptomycin (CCpro, Germany). The test medium used for the
cytotoxic assays and anti-HSV-1 tests contained only 2% of the appropriate
serum. All cells were proved to be free of mycoplasma contamination.

766
I. Stankova et al.
The acyclovir- and phosphonoformic acid-sensitive laboratory herpes
simplex virus type 1 strain Kupka (HSV-1) was propagated in GMK cells and
50 µl aliquots of the virus stocks were stored at −70◦C until use.
Cytotoxicity Test. Cytotoxicity test was described previously.[21] Briefly, 2-
day-old confluent GMK cell monolayer’s grown in 96-well cell culture platesin
an incubator with 5% CO2, 37◦C, and 95%, were incubated with different
concentrations (100 µl/well, dilution factor of 2, 2 parallels/concentration,
0.2–50 µg/ml)) in culture medium. For the control value determination (six
untreated cell controls) were used. 72 hours after substance application and
incubation, staining of the cells with crystal violet/methanol was carried out.
After extraction of the dye the optical density of individual wells was quan-
tified spectrophotometrically at 5570/630 nm with Dynex Immuno Assay
System (DIAS, Guernsey, GB). Cell viability was evaluated as the percentage
of the mean value of optical density resulting from six mock-treated cell
controls which was set 100%. The 50% cytotoxic concentration (CC50) was
defined as the compound concentration reducing the viability of untreated
cell cultures by 50%.
Antiviral Tests. The cytopathic effect (CPE) inhibitory assay was per-
formed as described previously.[21] HSV-1 replication results in the com-
plete destruction of host cells (CPE) that can be selectively inhibited by the
addition of active antiviral agents (50 µl/well, dilution factor 2, each concen-
tration in triplicate). Compound dilutions were added immediately before
inoculation of a virus dilution (50 µl/well, multiplicity of infection of 0.05)
to 2-day-old confluent GMK. This virus dose leads to a complete CPE in the
6 untreated virus controls 48 hours after infection. At this time point, the
still adherent cells were fixed and stained with a crystal violet/formalin solu-
tion. The inhibition of virus-induced CPE was photometrically determined
by comparing the individual optical density (OD) of compound treated,
virus-infected wells with that of the mean OD of 6 untreated, uninfected cell
control that was set 100%.
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