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Nc_9203_silva.indd

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 for 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-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N,N-dimethyl-4-aminopyridine (DMAP) as a catalyst.
Key words: 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., 2003).
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. 2003; 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- al, 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 μg/ml) they suppressed the virus know till now. In order to obtain analogues with by 60% and 49%. Their effects at 10 μg/ml were 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 μg/ml, whereas the ED value of ACV, azole-4-carboxylic acid and Fmoc-2-(2'-aminome- 1.2 μg/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-ethyl-N'-(3-dimethylaminopropyl)carbodiim-
strains (Stankova et al., 2007).
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,N-di-
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 sired structures.
showed that compounds 3a and 3b affect slightly
the replication of HSV-1 and HSV-2.
Third, the results of our investigations showed The two esters of ACV, 3a and 3b, 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 μg/ml. The two modifi cations of of ACV with natural amino acids (Beauchamp et ACV slightly affected the replication of HSV-1 in al., 1992).
R = Boc-NH-H C
I. Stankova et al. · New Acyclovir Analogues Fig. 2. In vitro antiviral activity of 3a and 3b (a) on the replication on HSV-1 and (b) on the replication on
HSV-2.
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 MS: m/z = 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
δ = 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], 1a and 1b were prepared according to Videnov
4.46 (d, 2H, CH ), 5.34 (s, 2H, N-CH -O, ACV), et al. (1996) and Stankova et al. (1999).
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 ACV (2) (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 vacuo, 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,
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