New Analogues of Acyclovir – Synthesis and Biological Activity
Ivanka Stankovaa,*, Stoyan Schichkovb, Kalina Kostovab, and Angel Galabovc
a Department of Chemistry, South-West University "Neoﬁ t Rilski'', Ivan Michailov Str. 66,
Blagoevgrad 2700, Bulgaria. Fax: ++359 73 88 55 16. E-mail: [email protected]
b St. Kl. Ohridski Soﬁ a University, Faculty of Biology, Laboratory of Virology, Soﬁ a 1164,
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Soﬁ a 1113, Bulgaria
* Author for correspondence and reprint requests
Z. Naturforsch. 65 c
, 29 – 33 (2010); received September 21/October 19, 2009
New acyclovir esters with peptidomimetics were synthesized and evaluated in vitro
their antiviral activity against the replication of Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The inﬂ uence of peptidomimetics containing oxazole and thiazolyl-thiazole moieties on the antiviral activity is also reported. The esters were synthesized using the coupling reagents N
(EDC) and N
-dimethyl-4-aminopyridine (DMAP) as a catalyst.
Peptidomimetics, Acyclovir, HSV
peptide transporter-mediated transport of valacy-clovir. The compound is recognized as a peptidyl
The discovery of acyclovir, 9-[(2-hydroxy-
derivative and absorbed by peptide transporters,
ethoxy)methyl] guanine (ACV) as a selective even though there is no peptide bond in its struc-
antiherpes agent heralded a new era in antiviral
ture (Spruance et al.,
2002; Painter and Hostetler,
chemotherapy (Elion et al
., 1977). ACV is an acy-
2004; Field et al.,
clic nucleoside analogue of guanosine. The prob-
Modiﬁ cation of anti-herpes agents like ACV
lem with ACV is its high lipophilicity and, from
by peptidomimetics, whose chemical structures
this, its low bioavailability. Its limited absorption
are different from those of the natural peptides
(15% – 20%) in humans after oral administration
but have the same ability to interact with speciﬁ c
prompted the search for prodrugs (De Clercq et
receptors, is of deﬁ nite interest (Field et al.
2006; Balzarini et al.
2004). A possible way to
Vabeno et al
., 2004a, b).
increase the bioavailability is by modifying the
Considering all these facts, we have been in-
known antiviral drugs with various amino acids terested in looking for other esters of ACV. Here
(Beauchamp et al
., 1992; Zacchigna et al
., 2002; we report the synthesis of oxazole- and thiazolyl-
Anand et al
., 2003, 2004a; Nashed and Mitra, thiazole-containing amino acid esters of ACV and
2003). Amino acid ester prodrugs of nucleoside exploration of their activity on the Herpes sim-
antiviral drugs have been employed to increase plex virus type 1 (HSV-1) and type 2 (HSV-2).
the oral bioavailability of the parent drugs.
The L-valyl ester of acyclovir (valacyclovir) is Results and Discussion
obtained in this manner (Beauchamp and Kren-itsky, 1993). Valacyclovir is such a prodrug, which
In the last two decades, inprecedented biologi-
is derived from ACV by esterifying ACV with cally active natural products containing directly L-valine. Upon administration valacyclovir is rap-
linked azoles have been isolated from natural
idly and completely converted to ACV, the active
sources. Many of these compounds are candi-
parent drug, by enzymatic hydrolysis (Anand et
dates for drug development. In particular thia-
2004a, b; Anand and Mitra, 2002). The prod-
zole, oxazole and imidazole amino acids that may
rug increases the oral bioavailability of ACV in play a key role in biological activities of unusual humans three- to ﬁ ve-fold. Enhanced oral absorp-
peptides are important intermediates for natural
tion of ACV has been attributed to the human product synthesis and peptidomimetics.
0939 – 5075/2010/0100 – 0029 $ 06.00 2010 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D
I. Stankova et al.
· New Acyclovir Analogues
ACV modiﬁ ed with amino acids and peptides is
the same mode (Fig. 2a). Applied in the maximal
found, but ACV containing peptidomimetics is not
tested dose (100 μ
g/ml) they suppressed the virus
know till now. In order to obtain analogues with
by 60% and 49%. Their effects at 10 μ
more desirable characteristics, we synthesized new
same – within 20% and 5%. The ED value of 3b
esters of ACV containing Boc-2-aminomethyl-ox-
was 78.4 μ
g/ml, whereas the ED value of ACV,
azole-4-carboxylic acid and Fmoc-2-(2'-aminome-
g/ml, differed considerably. The referent drug
in same dose inhibited completely the replication (Golankiewicz et al
., 2001). The inﬂ uence of these
Synthesis of esters of ACV
esters on the replication of HSV-2 were analogi-
A mixture of Boc-2-aminomethyl-oxazole-4-
cal (Fig. 2b). The established activities were cor-
carboxylic acid (1a
) or Fmoc-2-(2'-aminomethyl-
relative with our results for application of similar
thiazol-4'-yl)-thiazole-4-carboxylic acid (1b
) and prodrugs against the replication on these viral N
strains (Stankova et al.
ide hydrochloride (EDC) in dimethylformamide
In conclusion, in this study we extended the
(DMF) with the EDC/amino acid ratio 1:2 was scope on modiﬁ cation of ACV with various pep-stirred for 1 h at 0 °C under nitrogen atmosphere
(Nakajima and Ikada, 1995).
First, two novel esters with peptidomimetics of
A solution of ACV (2
) (Fig. 1) and N
ACV were synthesized. One oxazole-containing
methyl-4-aminopyridine (DMAP) was added to dipeptide mimetic and one tripeptide mimetic the reaction mixture and stirred for 24 h. Then with two fused 5-ring heterocycles derived from DMF was evaporated in vacuo,
and the residue glycine were used. The ESI-MS and NMR analy-was chromatographed on silica gel, using MeOH/
ses proved the identity of the ﬁ nal products 3a
CH Cl (1:4). The 1H, 13C NMR and mass spectra
of the compounds were consistent with the de-
Second, the results of the antiviral activity test
showed that compounds 3a
the replication of HSV-1 and HSV-2.
Third, the results of our investigations showed
The two esters of ACV, 3a
, were ex-
that modiﬁ cation of ACV with amino acids con-
plored against HSV-1 and HSV-2. They were taining oxazole and thiazolyl-thiazole reduce the tested in the following concentrations: 100, 40, antiviral effect in comparison with modiﬁ cations 20, 10, 5 and 1 μ
g/ml. The two modiﬁ cations of
of ACV with natural amino acids (Beauchamp et
ACV slightly affected the replication of HSV-1 in
I. Stankova et al.
· New Acyclovir Analogues
Fig. 2. In vitro
antiviral activity of 3a
(a) on the replication on HSV-1 and (b) on the replication on
Material and Methods
Yield: 0.187 g (40%). – 1H NMR ([D ]-DMSO):
= 1.36 (s, 9H, 3CH), 3.47 (m, 2H, CH O, ACV),
4.22 [m, 2H, CH OC(O), ACV)], 4.34 (d, 2H, CH ),
The amino acids were purchased from Sigma,
5.33 (s, 2H, N-CH -O, ACV), 5.36 (br t, 1H, NH),
DMAP and EDC were purchased from Merck.
6.83 (s, 2H, 2-NH , ACV), 7.94 (s, 1H, H-8, ACV),
TLC analysis was performed on aluminium sil-
), 10.62 (s, 1H, ACV-NH). – ESI-
ica gel 60 F plates (Merck) and detection was
= 468 [M+H]+.
performed using an UV lamp at 254 nm.
NMR spectroscopy: Bruker Avance DRX-600 thiazol-4-yl-acyclovir (3b)
spectrometer; chemical shifts referenced to the
A mixture of 1b
(0.371 g, 8 mmol) and EDC
solvent peaks [δ
(1H, [D ]-DMSO) = 2.49 and δ
(0.764 g, 8 mmol) in DMF was stirred for 1 h at
(13C, [D ]-DMSO) = 39.5].
0 °C under nitrogen atmosphere. A solution of
Mass spectrometry: API III triple quadrupole ACV (2
) (0.900 g, 4 mmol) and DMAP (0.976 g,
mass spectrometer equipped with an electrospray
8 mmol) was added to the reaction mixture and
ion source at atmospheric pressure (Sciex, Thorn-
stirred for 24 h. DMF was evaporated in vacuo
hill, Canada); electrospray ionization (EI) mass and the residue was chromatographed on silica spectra were recorded in the positive ion mode.
gel, using MeOH/CH Cl (1:4).
Yield: 0.081 g (30%). – 1H NMR ([D ]-DMSO):
Synthesis of 1a and 1b
= 3.44 (t, H-Fmoc), 3.48 (d, 2H-Fmoc), 3.51 (m, 2H, CH O, ACV), 3.81 [m, 2H, CH OC(O), ACV],
were prepared according to Videnov
4.46 (d, 2H, CH ), 5.34 (s, 2H, N-CH -O, ACV),
(1996) and Stankova et al
6.50 (s, 2H, 2-NH , ACV), 7.29 (t, 2H-Fmoc), 7.39
(t, 2H-Fmoc), 7.55 (br m, 2H-Fmoc), 7.75 (d, 2H-
Fmoc), 7.81 (s, 1H, H-8, ACV), 7.94 (t, 1H, NH), 8.12, 8.11 (CH
), 10.8 (s, 1H, ACV-NH). – 13C
A mixture of 1a
(0.480 g, 2 mmol) and EDC
NMR ([D ]-DMSO): δ
= 42.0 (CH ), 47.86 (CH -
(0.191 g, 2 mmol) in DMF was stirred for 1 h at
CH O, Fmoc), 47.86 (CH -CH O, Fmoc), 64.13
0 °C under nitrogen atmosphere. A solution of (CH OCO, ACV), 66.28 (CH O, ACV), 67.60
) (0.225 g, 1 mmol) and DMAP (0.244 g,
(Fmoc), 71.68 (NCH O), 116.84 (C-5, ACV), 117.9
2 mmol) was added to the reaction mixture and
5'), 120.46 (2C, Fmoc), 125.60 (2C, Fmoc),
stirred for 24 h. Then DMF was evaporated in
127.72 (2C, Fmoc), 128.21 (2C, Fmoc), 128.9
and the residue was chromatographed on
5), 137.55 (C-8, ACV), 141.87 (2C, Fmoc),
silica gel, using MeOH/ CH Cl (1:4).
144.68 (2C, Fmoc), 147.3 (C
4'), 148.2 (C 4),
I. Stankova et al.
· New Acyclovir Analogues
151.08 (C-4, 151.08), 156.63 (C-6, ACV), 157.05 The maximal concentration, which did not alter (C-O, Fmoc), 162.0 (C
2'), 162.2 (C 2), 168.71 neither the morphology nor viability of the cells,
(C=O, ACV). – ESI-MS: m/z
= 671 [M+H]+.
was recognized as MTC.
Antiviral activity of 3a and 3b against HSV-1
Experiments were done under multicycle
growth conditions. Conﬂ uent cell monolayers were
Viruses and cells
washed and infected with 320 cell culture infec-
The two laboratory strains, DA (HSV-1) and Bja
tious doses (CCID ) per 0.1 ml of the appropri-
(HSV-2), were kindly provided by Prof. S. Dunda-
ate virus strain. After 1 h, cells were covered with
rov (National Center of Infectious and Parasitic
maintenance media including test drugs at tested
Diseases, NCIPD, Bulgaria). Madin-Darby bovine
concentrations. The effect on viral replication was
kidney (MDBK) cells were cultured at 37 °C as
determined after 48 h (for strains DA and Bja) of
monolayers in RPMI-1640 medium (Flow Labo-
culturing at 37 °C by reduction of infectious virus
ratories, USA) supplemented with antibiotics titres as compared to that of the untreated viral
(penicillin and streptomycin) and 10% bovine se-
control. The 50% inhibitory concentration (IC )
rum (NCIPD). Serum concentration was reduced
for virus-induced cytopathic effect (CPE) was de-
to 5% for growth of viruses and for testing the termined by a dose-response curve. To calculate
the standard deviation of IC , each experiment
was done in triplicate (for HSV-1 strain DA) or
Cytotoxicity assay – determination of the maxi-
duplicate (for HSV-2 strain Bja).
mal tolerate concentration (MTC)
To compare the MTC values of substances to
that of ACV, conﬂ uent monolayers were covered
with media containing different concentrations of
Partial support of this work by the National
compounds or reference substance (ACV) and Found for Scientiﬁ c Research of Bulgaria (VUL-cultured at 37 °C for 96 h. Samples of cells grown
304/07 and DVU 01/0197, DO 02/162/16.12.08) is
in test prodrug-free medium served as a control.
Anand B. S. and Mitra A. K. (2002), Mechanism of cor-
Beauchamp L. M., Orr G. F., de Miranda P., Burnette
neal permeation of L-valyl ester of acyclovir: target-
T., and Krenitsky T. A. (1992), Amino acid ester
ing the oligopeptide transporter on the rabbit cornea.
prodrugs of acyclovir. Antiv. Chem. Chemoth. 3
Pharm. Res. 19
, 1194 – 1202.
157 – 164.
Anand B. S., Nashed Y. N., and Mitra A. K. (2003), Novel
De Clercq E., Field J., and Hugh V. (2006), Antiviral
dipeptide prodrugs of acyclovir for ocular herpes in-
prodrugs – the development of successful prodrug
fection: Bioreversion, antiviral activity and transport
strategies for antiviral chemotherapy. Br. J. Pharma-
across rabbit cornea. Curr. Eye Res. 26
, 151 – 163.
, 1 – 11.
Anand B. S., Katragadda S., Nashed Y. E., and Mitra A.
Elion G. B., Furman P. A., Fyfe J. A., de Miranda P.,
K. (2004a), Amino acid prodrugs of acyclovir as pos-
Beauchamp L., and Schaeffer H. J. (1977), Selectiv-
sible antiviral agents against ocular HSV-1 infection:
ity of action of an antiherpetic agent, 9-(2-hydrox-
interaction with the neutral and cationic amino acid
yethoxymethyl)guanine. Proc. Natl. Acad. Sci. USA
transporter on the cornel epithelium. Curr. Eye Res.
, 5716 – 5720.
, 153 – 166.
Field H. J., Dejesus E., Wald A., Warren T., Schacker T.
Anand B. S., Katragadda S., and Mitra A. K. (2004b),
W., Trottier S., Shahmanesh M., Hill J. L., and Bren-
Pharmacokinetics of novel dipeptide ester prodrugs
nan C. A. (2003), Valacyclovir for the suppression of
of acyclovir after oral administration: intestinal ab-
recurrent genital herpes inhuman immunodeﬁ ciency
sorption and liver metabolism. J. Pharmacol. Exp.
virus-infected subjects. J. Infect. Dis. 188
, 1009 – 1016.
, 659 – 667.
Golankiewicz B. T., Ostrowski T., Goslinski P., Januszc-
Balzarini J., Schols D., Baba I., Field H. J., and de Clercq
zyk J., Zeidler D., Baranowski S., and de Clercq E.
E. (2004), Antiviral drugs – a short history of their
(2001), Fluorescent tricyclic analogues of acyclovir
discovery and development. Microbiol. Today 31
and gancyclovir. A structure-antiviral activity study.
J. Med. Chem. 44
, 4284 – 4287.
Beauchamp L. M. and Krenitsky T. A. (1993), Acyclovir
Nakajima N. and Ikada Y. (1995), Mechanism of amide
prodrugs: the road to valacyclovir. Drugs Future 18
formation by carbodiimide for bioconjugation in
619 – 628.
aqueous media. Bioconjug. Chem. 6
, 123 – 130.
I. Stankova et al.
· New Acyclovir Analogues
Nashed Y. E. and Mitra A. K. (2003), Synthesis and
Symposium (Rolka K., Rekowski P., and Silberring J.,
characterization of novel dipeptide ester prodrugs of
eds.). Escom, Leiden, pp. 226 – 227.
acyclovir. Spectrocim. Acta A 59
, 2033 – 2039.
Vabeno J., Lejon T., Nielsen C. U., Steffansen B., Chen
Painter G. R. and Hostetler K.Y. (2004), Design and
W., Quyang H., Borchard R., and Luthman K. (2004a),
development of oral drugs for the prophylaxis and
Phe-Gly dipeptidomimetics designed for di/tri trans-
treatment of smallpox infection.
porters PEPT1 and PEPT 2; synthesis and biological
, 423 – 427.
J. Med. Chem. 47
, 1060 – 1069.
Spruance S. L., Jones T. M., Blatter M. M., Vargas-Cortes
Vabeno J., Nielsen C. U., Ingebrigtsen T., Lejon T., Stef-
M., Barber J., Hill J., Goldstein D., and Schultz M.
fansen B., and Luthman K. (2004b), Dipeptidomi-
(2002), Valacyclovir cold sore study group. High-
metics ketomethylene isosters as pro-moieties for drugs transport via the human intestinal di-/tripep-
dose, short duration, early valacyclovir therapy for
tide transporter hPEPT1: design, synthesis, stabil-
episodic treatment of cold sores: results of two rand-
ity and biological investigation. J. Med. Chem. 47
omized, placebo-controlled, multicenter studies. An-
4755 – 4765.
timicrob. Agents Chemother. 47
, 1072 – 1080.
Videnov G., Kaiser D., Kempter C., and Jung G. (1996),
Stankova I. G., Videnov G. I., Golovinsky E. V., and
Synthesis of naturally occurring conformationally
Jung G. (1999), Synthesis of thiazole, imidazole and
restricted oxazole and thiazole containing di- and
oxazole containing amino acids for peptide backbone
tripeptide mimetics. Angew. Chem. 108
, 1604 – 1607;
modiﬁ cation. J. Peptide Sci. 5
, 392 – 398.
Angew. Chem. Int. Ed. Engl. 35
, 1503 – 1506.
Stankova I. G., Dzimbova T., Shishkov St., Kostova K.,
Zacchigna M., Di Luca, Maurich G. V., and Boccu E.
and Galabov A. (2007), Synthesis and biological ac-
(2002), Syntheses, chemical and enzymatic stability
tivity of amino acid ester prodrugs of acyclovir. Pep-
of new poly(ethyleneglycol)-acyclovir prodrugs. Far-
tides 2006, Proceedings of the 29th European Peptide
, 207 – 214.
La Gazette de l' N° 22 Mensuel - Avril 2009 Bonjour à tous et à toutes !!! Voici le traditionnel mot du CEI qui accompagne chaque numéro de votre gazette préférée. Tout d'abord bravo à tous nos mi-thésards pour leur exposé de mi-thèse. En espérant que la dernière moitié de la thèse soit fructueuse. Ça y est, c'est le printemps ! Le soleil n'est pas encore vraiment au rendez-vous, mais ça ne saurait tarder. En avril
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