Synthesis and anti-hepatitis B virus activity of new pyrimidine peptide nucleic acid analogs

Article abstract of DOI:10.1007/s10593-012-0905-6

A series of 4-methylsulfanylpyrimidin-2(1H)-one peptide nucleic acid analogs were synthesized and tested for their antiviral activity against hepatitis B virus. Plaque reduction infectivity assay was used to determine the virus count reduction as a result of treatment with tested compounds. 0009-3122/12/4710-12902012 Springer Science+Business Media, Inc.

Full text of DOI:10.1007/s10593-012-0905-6

Chemistry of Heterocyclic Compounds, Vol. 47, No. 10, January, 2012 (Russian Original Vol. 47, No. 10, October, 2011)
SYNTHESIS AND ANTI-HEPATITIS B VIRUS ACTIVITY OF
NEW PYRIMIDINE PEPTIDE NUCLEIC ACID ANALOGS
R. A. K. Al-Harbi1 and A. A.-H. Abdel-Rahman2*
A series of 4-methylsulfanylpyrimidin-2(1H)-one peptide nucleic acid analogs were synthesized and
tested for their antiviral activity against hepatitis B virus. Plaque reduction infectivity assay was used to
determine the virus count reduction as a result of treatment with tested compounds.
Keywords: peptide nucleic acid analogs, pyrimidine nucleobases, anti-hepatitis B virus activity.
Synthetic compounds that specifically recognize and bind to a specific DNA or RNA sequence of
interest are potentially useful as antisense and antigene drugs or molecular probes, which have numerous
applications in the field of molecular and experimental medicine [1-3]. Particularly successful DNA binding
agents are found in a recently developed class of DNA analogs, the peptide nucleic acids (PNA) [4-9]. PNA are
oligonucleotide analogs, in which the phosphodiester pentose backbone of DNA or RNA is replaced by a
polyamide or peptide backbone [10]. For example, the phosphoribose backbone of natural oligonucleotide has
been replaced by N-(2-aminoethyl)glycine whereby the purine/pirimidine base pair is attached to the glycine
nitrogen through a methylene carbonyl linker [11]. The complete replacement of the ribose phosphate backbone
with an artificial pseudopeptide backbone results in a remarkably improved binding to complementary nucleic
acid sequences occurring with both high affinity and selectivity [12-19]. The hybridization properties of PNA
have attracted widespread interest in this class of compounds [20-22]. Several reviews have covered the
literature concerning new chemically modified PNA [23]. Their biological and chemical stability and their
superior hybridization properties relative to natural oligonucleotides make them attracttive as potential
therapeutic and biomolecular tools. Owing to the described significance of PNA and in connection with our
work on the synthesis of new α-amino acid derivatives [24–28] and investigating their antiviral potential [29],
we report here the synthesis and anti-hepatitis B virus (HBV) activity of new PNA with 4-methylsulfanyl-
pyrimidin-2(1H)-one as the heterocyclic nucleobase.
The coupling reaction at one of the nitrogen atoms of the heterocyclic base is the most effective method
for introducing certain substituents with desired functionalities attached to the heterocycle. Thus, reaction of
4-(methylsulfanyl)uracils 1a-c [30] with ethyl chloroacetate in the presence of potassium carbonate afforded the
1
corresponding nucleobase-substituted acetates 2a-c in 71-75% yields. The H NMR spectra of compounds 2a-c
showed the characteristic signals of the ester ethyl group and a singlet peak for the remaining CH2 group at
_______
*To whom correspondence should be addressed, e-mail: adelnassar63@yahoo.com.
1Department of Applied Chemistry, Faculty of Applied Science, Taibah University, Madinah P.O. 3193, Saudi
Arabia; Remal94@hotmail.com.
2Department of Chemistry, Faculty of Science, Northern Border University, P.O. 1321 Arar, Saudi Arabia.
__________________________________________________________________________________________
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1560-1567, October, 2011.
Original article submitted March 3, 2011.
1290
0009-3122/12/4710-1290©2012 Springer Science+Business Media, Inc.

4.96−4.99 ppm. Treatment of compounds 2a–c with hydrazine hydrate gave the corresponding acid hydrazides
1
3a–c in quantitative yields. The structures were proved by means of IR, H NMR, and mass spectra, which all
agreed with the assigned structure. These hydrazides were selected as starting materials for the coupling reaction
with the appropriate acylated amino acides via the azide coupling method [31]. Thus, treatment of compounds
3a–c at -5 °C in AcOH and 1 N HC1 with NaNO2 afforded the corresponding azide derivatives as an inseparable
mixture. The yellow syrupy azide compound was then treated, in situ, with the corresponding amino acid methyl
esters in ethyl acetate containing Et3N at 0°C to give, after neutralization, the desired peptides 4-15 in 80–90%
yields. For this study, the amino acids were selected according to the amino acid side chain: a) glycine (without
side chain, peptides 4, 8, 12); b) L-valine (branched side chain, peptides 5, 9, 13); c) L-leucine (long side
chain, peptides 6, 10, 14); d) L-phenylglycine (aromatic side chain, peptides 7, 11, 15). The structures of 4−15
were assigned from their 1H NMR and mass spectra.
SMe
SMe
R
R
NH2NH2 . H2O
EtOH
ClCH2COOEt
N
N
K2CO3 / acetone
1
R
O
N
R1
O
O
N
H
OEt
2a–c
1a–c
SMe
SMe
N
R
R
N
H2NCH(R2)COOMe
NaNO2 / HCl / Ac2O
N
R1
O
N
O
O
R1
O
H
N
OMe
O
R2
NHNH2
3a–c
4–15
1–3a, 4–7 R = R1 = H; 1–3b, 8–11 R = H, R1 = Me; 1–3c, 12–15 R = Me, R1 = H;
4, 8, 12 R2 = H; 5, 9, 13 R2 = CHMe2; 6, 10, 14 R2 = CH2CHMe2; 7, 11, 15 R2 = Ph
TABLE 1. Antiviral Activity and Cytotoxicity of Compounds 4–15
HBV DNA
in supernatant, µM
Hep G2
viable cells *
HBV DNA
IC50, µM
Compound
SI
Lamivudine
0.25
0.28
0.17
0.13
0.28
0.19
0.19
0.13
0.83
0.19
0.21
0.29
0.19
1.00
0.35
0.56
0.19
0.35
0.33
0.33
0.19
0.90
0.33
0.63
0.44
0.33
0.1
0.7
0.6
0.5
0.7
0.6
0.6
0.5
0.2
0.6
0.3
0.2
0.6
1000.0
142.8
166.6
200.0
142.8
166.6
166.6
200.0
500.0
166.6
333.3
500.0
166.6
4
5
6
7
8
9
10
11
12
13
14
15
_______
* At 0.1 μM concentration of the test compounds.
1291

The synthesized compounds were tested for their antiviral activity and cytotoxicity against HBV using
the HepG2.2.2.15 cell line [32], a human hepatoma cell line producing HBV viral particles [31, 32]. The drug
lamivudine (4-amino-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]pyrimidin-2(1H)-one), which is a potent
selective inhibitor of HBV replication [33], has been used as a standard positive control. The 50% inhibitory
concentration (IC50) of the antiviral drug was determined by plotting the DNA content in the cell culture
supernatant versus the concentration of the test compound. The 50% cytotoxic effect (CC50) was calculated from
the average viability of the cells in proportion to the concentration of the drug; for all the tested compounds its
value was 100 µM. The selectivity index (SI) was calculated as CC50/IC50 [34].
The results of the antiviral activity measurements against HBV are shown in Table 1. L-valine derivative
5, L-phenylglycine derivative 11, and L-valine derivative 13 showed the highest activity against HBV and mild
cytotoxicity with effective concentration of 0.2 µM and selectivity index 166.6−500.0. L-Leucine derivative 14
showed the highest antiviral activity against HBV and high cytotoxicity.
EXPERIMENTAL
1
IR spectra were recorded with a Bruker Vector 22 instrument in KBr. H and 13C NMR spectra were
recorded with a Varian Gemini spectrometer at 300 and 75 MHz, respectively, in DMSO-d6 with TMS as
internal standard. EI MS spectra were recorded with a Hewlett-Packard D5988 A instrument. Elemental analyses
were carried out at the Microanalytical Center of Cairo University, Giza, Egypt. Melting points were determined
using a Buchi apparatus. Antiviral activity against HBV was tested at the Liver Institute, Menoufia University,
Egypt. The progress of the reactions was monitored by TLC using aluminum silica gel plates.
Ethyl [4-(Methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetates 2a–c (General Method). A mixture of
an corresponding 4-methylsulfanylpyrimidin-2(1H)-one 1a–c [30] (0.1 mol), ethyl chloroacetate (14.7 g, 0.12 mol),
and anhydrous K2CO3 (13.8 g, 0.1 mol) in dry acetone (50 ml) was refluxed for 4-5 h (TLC). The solvent was
removed in vacuo, and the residue was diluted with water and extracted with dichloromethane (3×50 ml). The
combined organic layers were dried over Na2SO4, and the solvent was evaporated. The residue was purified by
silica gel column chromatography (dichloromethane–methanol, 98:2) to afford compounds 2a-c.
Ethyl [4-(Methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetate (2a). White needles, yield 75%; mp
89−91°C. Rf = 0.66 (3% MeOH in CH2Cl2). IR spectrum, ν, cm−1: 1755 (CO2Et). 1H NMR spectrum, δ, ppm (J,
Hz): 1.18 (3H, t, J = 8.0, CH3CH2); 2.46 (3H, s, SCH3); 4.15 (2H, J = 8.0 Hz, CH3CH2); 4.96 (2H, s, CH2); 6.78
(1H, d, J = 5.5, H-5); 8.40 (1H, d, J = 5.5, H-6). 13C NMR spectrum, δ, ppm: 12.5 (SCH3); 13.9 (CH3CH2O);
50.4 (NCH2); 61.5 (CH3CH2O); 103.5 (C-5); 138.3 (C-6); 153.9 (C-2); 167.9 (C=O); 178.0 (C-4). Mass
spectrum, m/z (Irel, %): 228 [M]+ (40), 189 (17), 155 (66), 141 (100), 126 (39). Found, %: C 47.22; H 5.12;
N 12.17. C9H12N2O3S. Calculated, %: C 47.35; H 5.30; N 12.27.
Ethyl [6-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetate (2b). White needles, yield
71%; mp 99−101ºC. Rf = 0.74 (3% MeOH in CH2Cl2). IR spectrum, ν, cm−1: 1750 (CO2Et). 1H NMR spectrum,
δ, ppm (J, Hz): 1.19 (3H, t, J = 8.0, CH3CH2); 2.35 (3H, s, 6-CH3); 2.43 (3H, s, SCH3); 4.16 (2H, q, J = 8.0,
CH3CH2); 4.97 (2H, s, CH2); 6.61 (1H, s, H-5). 13C NMR spectrum, δ, ppm: 13.4 (SCH3); 13.8 (CH3CH2O);
23.4 (CH3); 51.2 (NCH2); 61.8 (CH3CH2O); 108.6 (C-5); 154.5 (C-2); 167.2 (C-6); 167.8 (C=O); 178.2 (C-4).
Mass spectrum, m/z (Irel, %): 242 [M]+ (32), 227 (19), 197 (66), 169 (55), 155 (100). Found, %: C 49.44; H 5.70;
N 11.45. C10H14N2O3S. Calculated, %: C 49.57; H 5.82; N 11.56.
Ethyl [5-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetate (2c). White needles, yield
74%; mp 105−107ºC. Rf = 0.73 (3% MeOH in CH2Cl2). IR spectrum, ν, cm−1: 1735 (CO2Et). 1H NMR spectrum,
δ, ppm (J, Hz): 1.19 (3H, t, J = 8.0, CH3CH2); 2.09 (3H, s, 5-CH3); 2.44 (3H, s, SCH3); 4.11 (2H, q, J = 8.0,
CH3CH2); 4.99 (2H, s, CH2); 8.26 (1H, s, H-6). 13C NMR spectrum, δ, ppm: 12.9 (CH3); 13.6 (CH3CH2O); 14.3
(SCH3); 51.6 (NCH2); 61.4 (CH3CH2O); 111.8 (C-5); 136.5 (C-6); 153.9 (C-2); 167.9 (C=O); 178.9 (C-4). Mass
spectrum, m/z (Irel, %): 242 [M]+ (18), 213 (33), 169 (24), 155 (38), 140 (100), 108 (29). Found, %: C 49.40;
H 5.68; N 11.42. C10H14N2O3S. Calculated, %: C 49.57; H 5.82; N 11.56.
1292

2-[4-(Methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetohydrazides 3a–c (General Method). A mixture
of the corresponding ester 2a–c (10 mmol) and 95% hydrazine hydrate (1.25 g, 25 mmol) in absolute ethanol
(30 ml) was heated under reflux for 2 h. The excess of ethanol was removed under reduced pressure, and the
resulting precipitate was filtered off, washed with ethanol, and recrystallized from ethanol to give compounds
3a−c.
2-[4-(Methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetohydrazide (3a). White needles, yield 99%; mp
190−192ºC. Rf = 0.26 (3% MeOH in CH2Cl2). IR spectrum, ν, cm−1: 3294 (NH2), 3218 (NH), 1675 (CONH). 1H
NMR spectrum, δ, ppm (J, Hz): 2.45 (3H, s, SCH3); 4.33 (2H, br. s, NH2); 4.78 (2H, s, CH2); 6.68 (1H, d,
J = 6.0, H-5); 8.36 (1H, d, J = 6.0, H-6); 9.35 (1H, br. s, NH). Mass spectrum, m/z (Irel, %): 214 [M]+ (54), 198
(27), 155 (55), 141 (41), 126 (100). Found, %: C 39.08; H 4.56; N 26.00. C7H10N4O2S. Calculated, %: C 39.24;
H 4.70; N 26.15.
2-[6-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetohydrazide (3b). White needles, yield
98%; mp 170−172ºC. Rf = 0.16 (3% MeOH in CH2Cl2). IR spectrum, ν, cm−1: 3300 (NH2), 3200 (NH), 1670
1
(CONH). H NMR spectrum, δ, ppm: 2.35 (3H, s, 6-CH3); 2.46 (3H, s, SCH3); 4.29 (2H, s, NH2); 4.78 (2H, s,
CH2); 6.57 (1H, s, H-5); 9.35 (1H, br. s, NH). Mass spectrum, m/z (Irel, %): 228 [M+] (46), 211 (18), 169 (37),
155 (100). Found, %: C 41.93; H 5.17; N 24.33. C8H12N4O2S. Calculated, %: C 42.09; H 5.30; N 24.54.
2-[5-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetohydrazide (3c). White needles, yield
99%; mp 165−167ºC. Rf = 0.18 (3% MeOH in CH2Cl2). IR spectrum, ν, cm−1: 3320 (NH), 3200 (NH2), 1670
1
(CONH). H NMR spectrum, δ, ppm: 2.08 (3H, s, 5-CH3); 2.45 (3H, s, SCH3); 4.28 (2H, s, NH2); 4.82 (2H, s,
CH2); 8.23 (1H, s, H-6); 9.29 (1H, br. s, NH). Mass spectrum, m/z (Irel, %): 228 [M]+ (19), 212 (46), 197 (11),
169 (22), 155 (67), 140 (100). Found, %: C 41.88; H 5.11; N 24.19. C8H12N4O2S. Calculated, %: C 42.09;
H 5.30; N 24.54.
PNA Methyl Esters 4–15 (General Method). A solution of an corresponding hydrazide 3a–c (4 mmol)
in acetic acid (30 ml), 1 N HCl (15 ml), and water (125 ml) was cooled in an ice-bath (-5oC). Sodium nitrite
(4.35 g, 63 mmol) in cold water (15 ml) was added with stirring. After stirring at -5oC for 15 min, a yellow
syrup was formed, which was taken up in cold ethyl acetate (150 ml), washed with NaHCO3 (3%, 150 ml) and
water (150 ml), and dried over Na2SO4. A solution of the corresponding amino acid methyl ester hydrochloride
(4.5 mmol) in ethyl acetate (100 ml) containing triethylamine (1.0 ml) was stirred at 0oC for 20 min and filtered,
and the filtrate was added to the hydrazide solution. The mixture was kept at -5oC for 12 h, then at room
temperature for another 12 h, followed by washing with 0.5 N HC1 (150 ml), 3% NaHCO3 (150 ml), and water
(150 ml), and drying over Na2SO4. The filtrate was evaporated under reduced pressure, and the residue was
purified by silica gel column chromatography (petroleum ether–ethylacetate, 7:1) to afford the corresponding
product 4–15.
N-{[4-(Methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}glycine Methyl Ester (4). White crystals,
1
yield 85%; mp 173−175ºC. Rf = 0.57 (5% MeOH in CH2Cl2). H NMR spectrum, δ, ppm (J, Hz): 2.45 (3H, s,
SCH3); 3.60 (3H, s, OCH3); 4.85 (4H, s, 2CH2); 6.75 (1H, d, J = 5.5, H-5); 8.31 (1H, d, J = 5.5, H-6); 8.34 (1H,
s, NH). 13C NMR spectrum, δ, ppm: 12.6 (SCH3); 51.2 (NCH2); 52.8 (CH2); 54.1 (OCH3); 103.7 (C-5); 139.0
(C-6); 154.1 (C-2); 167.8 and 172.9 (2C=O); 178.2 (C-4). Mass spectrum, m/z (Irel, %): 271 [M]+ (21), 240 (44),
214 (39), 183 (49), 141 (100). Found, %: C 44.13; H 4.66; N 15.33. C10H13N3O4S. Calculated, %: C 44.27;
H 4.83; N 15.49.
N-{[4-(Methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}-L-valine Methyl Ester (5). White crystals,
1
yield 83%; mp 119−121 ºC. Rf = 0.56 (5% MeOH in CH2Cl2). H NMR spectrum, δ, ppm (J, Hz): 0.87 (6H, d,
J = 7.2, (CH3)2CH); 2.02 (1H, m, CH(CH3)2); 2.43 (3H, s, SCH3); 3.63 (3H, s, OCH3); 4.21 (1H, t, J = 6.6,
NHCH); 4.88 (2H, d, J = 3.6, CH2N); 6.67 (1H, d, J = 5.5, H-5); 8.34 (1H, d, J = 5.5, H-6); 8.41 (1H d, J = 5.9,
NH). 13C NMR spectrum, δ, ppm: 12.4 (SCH3); 16.5 and 18.6 (2CH3); 31.6 (CH); 51.0 (CH); 52.8 (NCH2); 54.5
(OCH3); 103.8 (C-5); 138.9 (C-6); 153.8 (C-2); 167.4 and 175.1 (2C=O); 177.8 (C-4). Mass spectrum, m/z (Irel,
%): 313 [M]+ (18), 283 (19), 252 (63), 211 (54), 141 (100), 126 (42). Found, %: C 49.77; H 6.00; N 13.32.
C13H19N3O4S. Calculated, %: C 49.83; H 6.11; N 13.41.
1293

N-{[4-(Methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}-L-leucine Methyl Ester (6). Pale-yellow
1
oil, yield 86%. Rf = 0.59 (5% MeOH in CH2Cl2). H NMR spectrum, δ, ppm (J, Hz): 0.84 (6H, d, J = 5.4,
(CH3)2CH); 1.45 (1H, m, CH(CH3)2); 1.57 (2H, m, CHCH2CH); 2.45 (3H, s, SCH3); 3.62 (3H, s, OCH3); 4.39
(1H, m, NHCH); 4.85 (2H, s, CH2N); 6.65 (1H, d, J = 5.5, H-5); 8.32 (1H, d, J = 5.5, H-6); 8.43 (1H, s, NH).
13C NMR spectrum, δ, ppm: 12.8 (SCH3); 21.6 (2CH3); 25.8 (CH); 39.6 (CH2); 51.6 (CH); 53.7 (NCH2); 54.3
(OCH3); 104.3 (C-5); 139.7 (C-6); 154.4 (C-2); 167.5 and 172.5 (2C=O); 177.7 (C-4). Mass spectrum, m/z (Irel,
%): 327 [M]+ (11), 284 (19), 270 (37), 239 (39), 141 (100). Found, %: C 51.22; H 6.33; N 12.67. C14H21N3O4S.
Calculated, %: C 51.36; H 6.47; N 12.83.
N-{[4-(Methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}-L-phenylglycine Methyl Ester (7). White
foam, yield 88%. Rf = 0.5 (5% MeOH in CH2Cl2). 1H NMR spectrum, δ, ppm (J, Hz): 2.34 (3H, s, SCH3); 3.63
(3H, s, OCH3); 4.81 (2H, d, J = 5.7, CH2N); 5.57 (1H, d, J = 7.2, CHPh); 6.44 (1H, d, J = 5.5, H-5); 7.30 (5H,
m, H Ar); 7.33 (1H, d, J = 7.2, NH); 8.19 (1H, d, J = 5.5, H-6). 13C NMR spectrum, δ, ppm: 12.9 (SCH3); 52.4
(NCH2); 53.9 (OCH3); 56.5 (CH); 104.6 (C-5); 125.5, 127.9, 134.6, and 137.9 (Ph); 139.0 (C-6); 154.8 (C-2);
167.4 and 172.4 (2C=O); 179.2 (C-4). Mass spectrum, m/z (Irel, %): 347 [M]+ (33), 316 (17), 288 (34), 211 (22),
183 (27), 141 (100). Found, %: C 55.17; H 4.88; N 12.01. C16H17N3O4S. Calculated, %: C 55.32; H 4.93;
N 12.10.
N-{[6-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}glycine Methyl Ester (8). Pale-
yellow oil, yield 82%. Rf = 0.7 (5% MeOH in CH2Cl2). 1H NMR spectrum, δ, ppm (J, Hz): 2.32 (3H, s, 6-CH3);
2.44 (3H, s, SCH3); 3.69 (3H, s, OCH3); 4.03 (2H, d, J = 5.1, NHCH2); 4.84 (2H, s, CH2CO); 6.29 (1H, s, H-5);
6.82 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 13.5 (SCH3); 23.5 (CH3); 51.5 (NCH2); 52.9 (CH2); 53.9
(OCH3); 109.1 (C-5); 154.9 (C-6); 167.0 (C-6); 167.6 and 173.2 (2C=O); 179.3 (C4). Mass spectrum, m/z (Irel,
%): 285 [M]+ (32), 270 (22), 226 (44), 197 (27), 169 (43), 155 (100), 140 (23). Found, %: C 46.22; H 5.09;
N 14.66. C11H15N3O4S. Calculated, %: C 46.31; H 5.30; N 14.73.
N-{[6-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}-L-valine Methyl Ester (9). Yellow
oil, yield 82%. Rf = 0.48 (5% MeOH in CH2Cl2). 1H NMR spectrum, δ, ppm (J, Hz): 0.80 (3H, d, J = 6.9) and
0.85 (3H, d, J = 6.9, (CH3)2CH); 2.07 (1H, m, CH(CH3)2); 2.33 (3H, s, 6-CH3); 2.43 (3H, s, SCH3); 3.66 (3H, s,
OCH3); 4.53 (1H, dd, J = 5.1, J = 1.5, NHCH); 4.75 (1H, d, J = 15.3) and 4.88 (1H, d, J = 15.3, CH2N); 6.32
(1H, s, H-5); 6.63 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 13.7 (SCH3); 16.8 and 18.9 (2CH3); 23.9 (CH3);
32.3 (CH); 51.3 (CH); 52.9 (NCH2); 54.9 (OCH3); 109.3 (C5); 155.5 (C-2); 167.1 (C-6); 167.9 and 175.8
(2C=O); 179.5 (C-4). Mass spectrum, m/z (Irel, %): 327 [M]+ (29), 284 (45), 253 (15), 212 (40), 155 (100), 140
(16). Found, %: C 51.23; H 6.33; N 12.62. C14H21N3O4S. Calculated, %: C 51.36; H 6.47; N 12.83.
N-{[6-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}-L-leucine Methyl Ester (10).
1
Pale-yellow oil, yield 80%. Rf = 0.6 (5% MeOH in CH2Cl2). H NMR spectrum, δ, ppm (J, Hz): 0.82 (3H, d,
J = 6.0) and 0.87 (3H, d, J = 6.0, (CH3)2CH); 1.52–1.59 (3H, m, CH(CH3)2, CHCH2CH); 2.31 (3H, s, 6-CH3);
2.44 (3H, s, SCH3); 3.35 (3H, s, OCH3); 4.30 (1H, m, NHCH); 4.82 (2H, s, CH2N); 6.52 (1H, s, H-5); 8.42 (1H,
br. s, NH). 13C NMR spectrum, δ, ppm: 13.8 (SCH3); 21.8 (2CH3); 23.8 (CH3); 25.5 (CH); 39.2 (CH2); 51.3
(CH); 53.4 (NCH2); 54.0 (OCH3); 110.1 (C-5); 155.7 (C-2); 166.9 (C-6); 167.7 and 172.9 (2C=O); 179.9 (C-4).
Mass spectrum, m/z (Irel, %): 341 [M]+ (43), 326 (22), 282 (42), 252 (23), 212 (54), 155 (100). Found, %:
C 52.50; H 6.63; N 12.12. C15H23N3O4S. Calculated, %: C 52.77; H 6.79; N 12.31.
N-{[6-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}-L-phenylglycine Methyl Ester
(11). White foam, yield 83%. Rf = 0.66 (5% MeOH in CH2Cl2). 1H NMR spectrum, δ, ppm (J, Hz): 2.40 (3H, s,
6-CH3); 2.41 (3H, s, SCH3); 3.73 (3H, s, OCH3); 4.88 (2H, s, CH2N); 5.63 (1H, d, J = 7.2, CHPh); 6.39 (1H, s,
H-5); 7.30 (5H, m, H Ar); 7.36 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 14.0 (SCH3); 23.5 (CH3); 52.2
(NCH2); 53.7 (OCH3); 56.2 (CH); 109.8 (C-5); 125.6, 127.7, 134.9, and 137.4 (Ph); 155.6 (C-2); 166.7 (C-6);
167.3 and 172.9 (2C=O); 178.5 (C-4). Mass spectrum, m/z (Irel, %): 361 [M]+ (18), 302 (15), 225 (19), 197 (39),
155 (88), 140 (100). Found, %: C 56.34; H 5.19; N 11.49. C17H19N3O4S. Calculated, %: C 56.50; H 5.30;
N 11.63.
1294

N-{[5-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}glycine Methyl Ester (12). White
foam, yield 90%. Rf = 0.54 (5% MeOH in CH2Cl2). 1H NMR spectrum, δ, ppm (J, Hz): 2.17 (3H, s, 5-CH3); 2.50
(3H, s, SCH3); 3.77 (3H, s, OCH3); 4.11 (2H, d, J = 5.1, NHCH2); 4.93 (2H, s, CH2CO); 6.83 (1H, br. s, NH); 8.14
(1H, s, H6). 13C NMR spectrum, δ, ppm: 12.8 (CH3); 14.5 (SCH3); 51.5 (NCH2); 52.7 (CH2); 54.5 (OCH3); 112.2
(C-5); 136.8 (C-6); 154.2 (C-2); 168.1 and 172.5 (2C=O); 179.0 (C4). Mass spectrum, m/z (Irel, %): 285 [M]+
(28), 254 (17), 226 (19), 197 (23), 140 (100). Found, %: C 46.17; H 5.12; N 14.69. C11H15N3O4S. Calculated, %:
C 46.31; H 5.30; N 14.73.
N-{[5-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}-L-valine Methyl Ester (13). Yellow
1
oil, yield 85%. Rf = 0.47 (5% MeOH in CH2Cl2). H NMR spectrum, δ, ppm (J, Hz): 0.85 (3H, d, J = 6.9) and
0.90 (3H, d, J = 6.9, (CH3)2CH); 2.13 (1H, m, CH(CH3)2); 2.17 (3H, s, 5-CH3); 2.47 (3H, s, SCH3); 3.71 (3H, s,
OCH3); 4.57 (1H, dd, J = 4.8, J = 1.9, NHCH); 4.84 (1H, d, J = 15.3) and 4.93 (1H, d, J = 15.3, CH2N); 6.72
(1H, br. s, NH); 8.12 (1H, s, H-6). 13C NMR spectrum, δ, ppm: 12.9 (CH3); 14.1 (SCH3); 16.1 and 18.4 (2CH3);
32.0 (CH); 51.8 (CH); 52.6 (NCH2); 54.3 (OCH3); 111.8 (C-5); 137.4 (C-6); 153.9 (C-2); 167.4 and 175.2
(2C=O); 178.6 (C-4). Mass spectrum, m/z (Irel, %): 327 [M]+ (33), 296 (22), 268 (28), 225 (34), 155 (100), 140
(45). Found, %: C 51.20; H 6.39; N 12.60. C14H21N3O4S. Calculated, %: C 51.36; H 6.47; N 12.83.
N-{[5-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}-L-leucine Methyl Ester (14). Pale-
yellow oil, yield 82%. Rf = 0.48 (5% MeOH in CH2Cl2). 1H NMR spectrum, δ, ppm (J, Hz): 0.88 (6H, d, J = 6.6,
(CH3)2CH); 1.51–1.66 (3H, m, CH(CH3)2, CHCH2CH); 2.13 (3H, s, 5-CH3); 2.45 (3H, s, SCH3); 3.69 (3H, s,
OCH3); 4.65 (1H, m, NHCH); 4.82 (1H, d, J = 14.7) and 4.90 (1H, d, J = 14.7, CH2N); 6.65 (1H, br. s, NH);
8.11 (1H, s, H-6). 13C NMR spectrum, δ, ppm: 12.5 (CH3); 14.1 (SCH3); 21.2 (2CH3); 25.1 (CH); 39.6 (CH2);
51.5 (CH); 53.3 (NCH2); 54.8 (OCH3); 112.1 (C-5); 136.1 (C-6); 153.5 (C2); 167.9, 172.9 (2C=O); 178.4 (C-4).
Mass spectrum, m/z (Irel, %): 341 [M]+ (16), 298 (13), 284 (19), 253 (23), 211 (29), 155 (66), 140 (100). Found,
%: C 52.45; H 6.59; N 12.18. C15H23N3O4S. Calculated, %: C 52.77; H 6.79; N 12.31.
N-{[5-Methyl-4-(methylsulfanyl)-2-oxopyrimidin-1(2H)-yl]acetyl}-L-phenylglycine Methyl Ester
(15). White crystals, yield 90%; mp 129−131ºC. Rf = 0.66 (5% MeOH in CH2Cl2). 1H NMR spectrum, δ, ppm (J,
Hz): 2.21 (3H, s, 5-CH3); 2.43 (3H, s, SCH3); 3.74 (3H, s, OCH3); 4.91 (2H, s, CH2N); 5.61 (1H, d, J = 7.2,
CHPh); 7.30 (5H, m, H Ar); 7.36 (1H, br. s, NH); 8.16 (1H, s, H-6). 13C NMR spectrum, δ, ppm: 12.6 (CH3);
14.0 (SCH3); 52.1 (NCH2); 56.2 (CH); 53.6 (OCH3); 112.3 (C-5); 125.5, 127.4, 134.8, 136.1, and 137.5 (Ph,
C-6); 167.6 and 172.8 (2C=O); 178.2 (C-4). Mass spectrum, m/z (Irel, %): 361 [M]+ (22), 346 (27), 302 (26), 212
(46), 169 (50), 155 (100). Found, %: C 56.29; H 5.21; N 11.51. C17H19N3O4S. Calculated, %: C 56.50; H 5.30;
N 11.63.
Antiviral Activity. The HepG2.2.2.15 cell line, supplied by State Serum Institute, Denmark, was
maintained in RPMI-1640 Glutamax (Gibco BRL Life technologies) [32]. The standard drug lamivudine was
from GlaxoSmithKline. The cell line was maintained in RPMI-1640 (Glutamax) culture medium containing 100
IU/ml nystatin, 380 µg/ml G418 (geneticin) and 10% fetal calf serum (FCS) (Gibco BRL Life Technologies).
The transferred HEPG2.2.2.15 cells were kept in a tissue culture flask at 37oC and 5% CO2. Subcultures were set
up after a week by trypsination (10% versin/trypsin (Biochrome KG)) and transferred to a 96-well tissue culture
plate. Fivefold serial dilutions of the tested compounds with final concentrations ranging from 100 to 0.03 µM
were added to the cell suspension; it was then incubated for 6 days at 37oC and 5% CO2. Each compound was
tested in triplicate. Cells with no compounds added to their culture were used for comparison (blank cells).
DNA extraction was done by incubating 10 µl of diluted supernatant with 10 µl of 0.2 M NaOH at
37ºC for 1 h, then carefully adding 9.6 µl of 0.2 M HCl followed by addition of 90 µl of Tris–EDTA buffer
[(2-amino-2-(hydroxymethyl)-1,3-propanediol–EDTA) (Gibco BRL Life Technologies)].
PCR-ELISA Detection of HBV DNA. The DNA content in the cell culture supernatant was determined by
polymerase chain reaction amplification of the HBV DNA using 1 µmol/1 of each of the following primers: HCID-1
primer (5'-GGAAAGAAGTCAGAAGGCA-3') and HCID-2 primer (5'-TTGGGGGAGGAGATTAGGTT-3'), in a
reaction mixture containing 14 µl extracted supernatant, 4 mmol/l MgCl2, 10 µmol/1 DIG-11-dUTP (Roche,
Germany), 190 µmol/l dTTP, 200 µmol/1 dATP, dGTP, dCTP (Roche), and 1.5 U Taq polymerase (Roche), in a
1295

total volume 50 µl. PCR reaction conditions were: 32 cycles of 10 min at 94ºC, 30 s at 58ºC, and 30 s at 72ºC
with a 3 s increment for each cycle in a Perkin-Elmer 480 thermal cycler (Perkin-Elmer, USA). The PCR
product was detected by DIG-ELISA assay (Roche). The optical density from DNA of the test compound was
compared to that of the blank culture [34].
Cytotoxicity Assay. 3-(3,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma, USA) is a
colorless substrate that is transformed to a colored product by living cells, but not by dead cells. The assay
utilizes this compound to test for the viability of the cells with the test compound added compared to the
viability of the blank cells [35].
The authors thank DANIDA for the support of the HBV testing of the newly synthesized compounds at
National Liver Institute, Menoufia University, through the project "Development of New Drugs Against
Hepatitis".
REFERENCES
1.
2.
3.
A. De Mesmaeker, R. Haner, and P. Martin, H. E. Moser, Acc. Chem. Res., 28, 366 (1995).
J. F. Milligan, M. D. Matteucci, and J. C. Martin, J. Med. Chem., 36, 1923 (1993).
E. Uhlmann and A. Peyman, Chem. Rev., 90, 543 (1990).
4.
5.
6.
P. E. Nielsen, M. Egholm, R. H. Berg, and O. Buchardt, Science, 254, 1497 (1991).
M. Egholm, O. Buchardt, P. E. Nielsen, and R. H. Berg, J. Am. Chem. Soc., 114, 1895 (1992).
B. Hyrup and P. E. Nielsen, Bioorg. Med. Chem., 4, 5 (1996).
7.
P. E. Nielsen, Pure Appl. Chem., 70, 105 (1998).
8.
9.
E. Uhlmann, A. Peyman, G. Breipohl, and D. W. Will, Angew. Chem. Int. Ed., 37, 2796 (1998).
K. N. Ganesh and P. E. Nielsen, Curr. Org. Chem., 4, 931(2000).
10.
M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim,
B. Norden, and P. E. Nielsen, Nature, 365, 566 (1993).
11.
12.
F. Debaene and N. Winssinger, Org. Lett., 5, 4445 (2003).
M. Egholm, L. Christensen, K. L. Dueholm, O. Buchardt, J. Coull, and P. E. Nielsen, Nucleic Acids
Res., 23, 217 (1995).
13.
14.
A. Ray and B. Norden, FASEB J., 14, 1041 (2000).
P. Zhou, A. Dragulescu-Andrasi, B. Bhattacharya, H. O’Keefe, P. Vatta, J. J. Hyldig-Nielsen, and
D. H. Ly, Bioorg. Med. Chem. Lett., 16, 4931 (2006).
15.
16.
S. Hyun, K. H. Lee, and J. Yu, Bioorg. Med.Chem. Lett., 16, 4757 (2006).
K. L. Duehohm, M. Egholm, C. Behrens, L. Christensen, H. F. Hansen, T. Vulpius, K. H. Petersen,
R. H. Berg, P. E. Nielsen, and O. Buchardt, J. Org. Chem., 59, 5767 (1994).
J. A. Thomson, S. A. Josey, R. Cadilla, M. D. Gaul, C. F. Hassman, M. J. Luzzio, A. J. Pipe, K. L. Reed,
D. J. Ricca, R. W. Wiethe, and S. A. Noble, Tetrahedron, 51, 6179 (1995).
D. W. Will, G. Breipohl, D. Langner, J. Knolle, and E. M. Uhlmann, Tetrahedron, 51, 12069 (1995).
A. Alahiane, M. Taourirte, A. Rochdi, N. Redwane, S. Sebti, J. W. Engels, and H. B. Lazrek, Nucleos.,
Nucleot., Nucleic Acids, 22, 109 (2003).
17.
18.
19.
20.
P. Zhou, M. Wang, L. Du, G. W. Fisher, A. Waggoner, and D. H. Ly, J. Am. Chem. Soc., 125, 6878
(2003).
21.
22.
23.
24.
P. E. Nielsen, Acc. Chem. Res., 32, 624 (1999).
O. Seitz, Angew. Chem. Int. Ed., 39, 3249 (2000).
K. N. Ganesh and P. E. Nielsen, Curr. Org. Chem., 4, 931 (2000).
I. A. I. Ali, I. A. Al-Masoudi, B. Saeed, N. A. Al-Masoudi, and P. La Colla, Heteroatom Chem., 16, 148
(2005).
24.
I. A. I. Ali, O. M. Ali, and A. A.-H. Abdel-Rahman, Monatsh. Chem., 138, 909 (2007).
1296

25.
26.
27.
O. M. Ali and A. A.-H. Abdel-Rahman, Monatsh. Chem., 139, 53 (2008).
A. A.-H. Abdel-Rahman, Monatsh. Chem., 139, 61 (2008).
A. A.-H. Abdel-Rahman, W. A. El-Sayed, H. M. Abdel-Bary, A. E.-S. Abdel-Megied, and
E. M. I. Morcy, Monatsh. Chem., 139, 1095 (2008).
28.
A. A.-H. Abdel-Rahman, A. E.-S. Abdel-Megied, H. M. Abdel-Bary, A. H. Abdel-Aleem, and
E. M. I. Morcy, M. T. Shabaan, Monatsh. Chem., 140, 559 (2009).
29.
30.
W. A. El-Zayat, W. A. El-Sayed, and A. A.-H. Abdel-Rahman, Z. Naturforsh., 64c, 6 (2009).
T. H. Brown, R. C. Blakemore, G. J. Durant, J. C. Emmett, C. R. Ganellin, M. E. Parson,
D. A. Rawlings, and T. F. Walker, Eur. J. Med. Chem., 23, 53 (1988).
R. Schwyzer and H. Kappeler, Helv. Chim. Acta, 44, 1991 (1961).
M. A. Sells, A. Z. Zelent, M. Shvartsman, and G. Acs, J. Virol., 62, 2836 (1988).
B. E. Korba and J. L. Gerin, Antiviral Res., 19, 55 (1992).
31.
32.
33.
34.
S. L. Doong, C. H. Tsai, R. F. Chinazi, D. C. Liotta, and Y. C. Cheng, Proc. Nat. Acad. Sci. USA, 88,
4895 (1991).
35.
T. Fouad, C. Nielsen, L. Brunn, and E. B. Pederson, Sc. J. Az. Med. Fac. (GIRLS), 19, 1173 (1998).
1297