Synthesis and Anti-HBV Activity of Novel Substituted Pyrimidine Glycosides and Their Acyclic Analogues

Article abstract of DOI:10.1134/S1070363218080285

New aryl substituted uracil and thiouracil glycosides are synthesized by glycosylation at N¹ in the pyrimidine nucleus using glycopyranosyl halides in basic medium. In addition C-linked hydrazinyl acyclic sugar derivatives exhibiting different

Full text of DOI:10.1134/S1070363218080285

ISSN 1070-3632, Russian Journal of General Chemistry, 2018, Vol. 88, No. 8, pp. 1734–1744. © Pleiades Publishing, Ltd., 2018.
Synthesis and Anti-HBV Activity of Novel Substituted Pyrimidine
Glycosides and Their Acyclic Analogues1
M. A. Hawataa, W. A. El-Sayedb*, and Adel A.-H. Abdel-Rahmana**
a Chemistry Department, Faculty of Science, Menoufia University, Shebin El-Kom, 32511 Egypt
*e-mail: waelshendy@gmail.com
b Photochemistry Department, National Research Centre, Dokki, Cairo, 12622 Egypt
**e-mail: adelnassar63@yahoo.com
Received June 28, 2018
Abstract—New aryl substituted uracil and thiouracil glycosides are synthesized by glycosylation at N1 in the
pyrimidine nucleus using glycopyranosyl halides in basic medium. In addition C-linked hydrazinyl acyclic
sugar derivatives exhibiting different sugar moieties, attached at C6, are also prepared. Antiviral activity of the
newly synthesized compounds is studied against Hepatitis B virus (HBV). The antiviral tests data indicated
high activity of compounds 6b, 6c and 12a–12c with mild cytotoxic effects. The influence of glycosyl moieties
attached to the substituted pyrimidine system in addition to the substitution at the aryl fragment on activity is
discussed.
Keywords: pyrimidines, glycosides, acyclic sugars, nucleosides, antiviral, HBV
DOI: 10.1134/S1070363218080285
INTRODUCTION
A variety of nucleosides and their analogs constitute
an interesting class of biologically active compounds
[23] that demonstrate antitumor [24, 25] and antiviral
[26, 27] activities. Pyrimidine nucleosides and their
derivatives are important molecular templates in the
development of new and improved antiproliferative
agents. Several structural changes in both the
nitrogenated base and the furanose ring have been
proposed for developing leading compounds or
enhancing the antitumor activity of existing ones. As a
result of these studies, a series of DNA bases and
nucleoside analogues have been developed and used in
treatment of various types of cancer. Among those are
5-fluorouracil, 6-mercaptopurine and cytarabine
[28–30]. The above facts initiated our study of
synthesis and antiviral activity study of functionalized
pyrimidine glycosides and their hydrzainyl sugar deriva-
tives as acyclic analogues of the glycoside skeletons.
A variety of methods of synthesis of pyrimidine
and 4(3H)-pyrimidinone rings containing compounds
and their diverse properties supported their applica-
tions in medicinal chemistry, including anticancer
[1–6] and many other types of activity [7–12]. Sulfur
modification of pyrimidine nucleosides by replacement
of the natural carbonyl group with the thiocarbonyl is
of high influence upon their biological activity [13–15]
and practical applications [16–19]. It is recognized that
the 2-thiocarbonyl group of 2-thiouracil nucleosides
strongly influences their conformation properties and
plays a key role in the modulation of base pair recogni-
tion [14]. On the other hand, thiated nucleosides
exhibit antineoplastic properties: 4-thiouridine selec-
tively inhibits growth of Ehrlich ascites and mouse
L1210 leukemia cells, and 5-fluoro-4-thio-2'-deoxy-
uridine inhibits growth of L1210 cells and various
hematopoietic human leukemia cells [20]. 2',3'-Di-
deoxy-3'-fluoro-4-thiopyrimidine [21] is determined to
be a potent and selective inhibitor of the retrovirus
HIV. Diarylpyrimidine (DAPYs) was among the earliest
discovered NNRTI-s [22].
RESULTS AND DISCUSSION
In the present study two types of substituted
pyrimidine sugar derivatives, incorporating glycosyl
and acyclic sugar chains, were synthesized. 6-Aryl-5-
cyano-2-(methylsulfanyl)uracils (2a–2c) [31] were
synthesized by treatment of 6-aryl-5-cyano-2-thio-
uracils 1a–1c with methyl iodide and sodium hyd-
roxide in water and ethanol at 60°C according to the
1 The text was submitted by the authors in English.
1734

SYNTHESIS AND ANTI-HBV ACTIVITY OF NOVEL SUBSTITUTED PYRIMIDINE
1735
Scheme 1. Synthesis of N1-thiopyrimidine glycosides.
O
O
CN
MeI/NaOH
CN
Ar
HN
S
HN
MeS
EtOH/H2O
60oC
Ar
1a−1c
N
N
H
2a−2c
(1) NaH/DMF
R1
OAc
O
R2
(2)
AcO
OAc
Br
Ar
N
Ar
NC
NC
N
N
NH3/MeOH
room temperature
O
OH
SMe
O
OAc
O
SMe
N
R1
R2
HO
R1
R2
AcO
O
OH
6a−6c, 7a−6c
OAc
4a−4c, 5a−5c
Ar = C6H5 (1a, 2a, 4a, 5a, 6a, 7a), p-MeC6H4 (1b, 2b, 4b, 5b, 6b, 7b), p-MeOC6H4 (1c, 2c, 4c, 5c, 6c, 7c); R1 = H (3b, 4a–
4c, 6a–6c), OAc (3a, 5a–5c), OH (7a–7c); R2 = OAc (3b, 4a–4c), H (3a, 5a–5c, 7a–7c), OH (6a–6c).
1
earlier developed method [32]. Condensation of sodium
salts of 6-aryl-5-cyano-2-(methylsulfanyl)uracils (2a–
2c) with glycosyl halides, 2,3,4,6-tetra-O-acetyl-α-D-
gluco- or galactopyranosyl bromide (3a, 3b), in dry
DMF at room temprature afforded 1-(2,3,4,6-tetra-O-
acetyl-β-D-glycopyranosyl)-6-aryl-5-cyano-2-(methyl-
sulfanyl) pyrimidin-4(1H)-ones 4a–4c and 5a–5c with
high yields (Scheme 1).
in IR spectra of the latter free nucleoside analogs. H
NMR spectra of the deprotcted nucleosides 6a–6c and
7a–7c demonstrated the signals assigned to the
anomeric protons as doublets in the range of 5.69–5.78
ppm (J = 7.5–7.7 Hz), clearly indicating that these
compounds were also fromed in the β-configuration.
The ease of accessibility and the biological signi-
ficance of 2-acetamido-2-deoxy-D-glucose have prompted
us to use this aminosugar as a starting material in the
glycosylation reaction according to the developed
earlier method [33]. 6-Aryl-5-cyanouracils 8a–8c [33]
were synthesized by base-catalyzed condensation
cyclization of ethyl cyanoacetate with urea and
aromatic aldehydes with 40-45% yields. Glycosylation
of sodium salts of the substituted cyanouracil deri-
vatives 2a–2c with 2-acetamido-1-chloro-3,4,6-tri-O-
acetyl-2-deoxy-D-glucose (9) in dry DMF at 90°C
gave the desired 1-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-β-D-glucopyranosyl)-6-aryl-5-cyanopyrimidin-
4(1H)-ones (10a–10c) with 75–80% yields (Scheme 2).
1H NMR spectra of 10a–10c demonstrated the signals
of anomeric protons as doublets at 5.89, 5.88 and
5.90 ppm (J = 9.3, 9.4 and 9.2 Hz, respectively),
assigned to the diaxial orientation of H1' and H2'
1
In H NMR spectra of 4a–4c and 5a–5c signals of
the anomeric protons were recorded as doublets (5.33–
6.44 ppm, J = 7.4–8.4 Hz), that corresponded to a
diaxial orientation of H1' proton which was indicative
of the β-configuration of the glycone part. For com-
pounds 5a–5c containing the galactopyranosyl
moeitey, the axial protons (H4') of the galactose moieties
were recorded as doublets of doublets at 5.30, 5.45 and
5.48 ppm. 13C NMR spectra demonstrated the signals
assigned to sugar moietey carbons in addition to the
acetyl, aryl groups and pyrimidyl ring carbons.
Deacetylation of the glycosides 4a–4c and 5a–5c in
a mixture with methanol and ammonium hydroxide
(25%) (1 : 1) at room temperature gave free nucleo-
sides 6a–6c and 7a–7c, respectively, with high yields
(Scheme 1). The hydroxyl group bands were recorded
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 8 2018

1736
HAWATA et al.
Scheme 2. Synthesis of N1-pyrimidine aminoglycosides.
Ar
O
NC
O
CN
Ar
(1) NaH/DMF
AcO
NH
HN
O
N
O
O
N
H
(2)
AcO
AcO
AcO
AcO
AcO
O
8a−8c
NHAc
Cl
9
NHAc
10a−10c
NH3/MeOH
Ar
NC
NH
O
N
O
HO
HO
HO
O
NHAc
11a−11c
Ar = C6H5 (8a, 10a, 11a), p-MeC6H4 (8b, 10b, 11b), p-MeOC6H4 (8c, 10c, 11c).
protons that were indicative of the β-configuration. 13C
NMR spectra of 10b and 10c demonstrated the
anomeric carbons at 97.3 and 95.1 ppm.
absorption bands attributed to the hydroxy groups in
the region of 3377–3428 cm–1. 1H NMR spectra
contained signals assigned to sugar chain protons in
addition to the sugar hydroxyl groups. The H1 methine
proton (originally the aldehydic hydrogen) was
recorded as a doublet in the range of 7.30–7.70 ppm.
The observed relatively high chemical shifts assigned
to H1 provided an evidence for the acyclic structure of
the sugar moieties incorporated in sugar hydrazinyl
uracil products. For compounds containing the cyclic
sugar structures, the anomeric proton (H1 in the
glycosyl moiety, an sp3 hydrogen) should be recorded
at a lower chemical shift.
Deacetylation of the modified nucleoside analogues
10a–10c in a mixture of methanol and ammonium
hydroxide (25%) (1 : 1) at room temperature afforded
free nucleosides 11a–1c in 83–90% yields (Scheme 2).
In 1H NMR spectra of free tetrahydropyrimidine
nucleosides 11a–1c the signals corresponging to the
anomeric protons were recorded as doublets at 5.76,
5.78 and 5.80 (J = 9.4, 9.6 and 9.5 Hz, respectively)
indicating their β-conformation as well.
The substituted thiouracil derivatives 1a, 1b were
alkylated with formation of the corresponding
alkylthio analogues. The latter were refluxed with
hydrazine hydrate in ethanol to give the corresponding
hydrazine derivatives 12a, 12b [34]. The resulting
hydrazinyl pyrimidines reacted with D-xylose, D-
glucose or D-galactose in an aqueous ethanol solution
in presence of the catalytic amount of acetic acid. The
corresponding sugar hydrazinyl uracil derivatives 13–
18 were obtained with 70–78% yield (Scheme 3).
Antiviral activity. Hepatitis B virus (HBV) is a
DNA virus that causes acute hepatitis and leads to
chronic hepatitis, liver cirrhosis and hepatocellular
carcinoma [35]. The potential target for antiviral
chemotherapy is the reverse transcription step in HBV
life cycle. The minus strand of HBV is synthesized by
reverse transcription of the pregenome using the
endogenous viral reverse transcriptase. It is determined
that reverse transcriptase enzyme leads to incorporated
nucleotide analogues more efficiency than cellular
DNA polymerase [36]. These nucleotide analogues are
competitive inhibitors of the reverse transcriptase with
The IR spectra of the latter products, that contained
acyclic sugar chains, demonstrated the characteristic
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SYNTHESIS AND ANTI-HBV ACTIVITY OF NOVEL SUBSTITUTED PYRIMIDINE
1737
Scheme 3. Synthesis of pyrimidine hydrazinyl acyclic sugars derivatives.
O
O
CN
Ar
CN
Ar
.
HN
S
N2H4 H2O
HN
EtOH/Reflux
H2N
N
H
N
H
N
1a, 1b
12a, 12b
OHC(CHOH)nCH2OH
n = 3, D-xylose
n = 4, D-glucose
n = 4, D-galactose
EtOH/Reflux
O
N
CN
Ar
HN
N
N
H
(CH-OH)n
OH
13−18
OH
HO
OH
OH
(14, 17);
(15, 18).
(13, 16);
HO
HO
HO
OH
OH
OH
OH
OH
OH
OH
Ar = C6H5 (1a, 12a, 13–15), p-MeC6H4 (1b, 12b, 16–18).
the nucleosides pool in the cells cytoplasm in minus
strand synthesis. The recent development of hetero-
cyclic analogues initiated the research in selective
antiviral activities. Among these agents, Lamivudine
acts as a retroviral inhibitor [37]. It demonstrated
activity against HBV replication both in vitro and
in vivo.
glycosyl moities to the substituted pyrimidine nucleus
demonstrated high inhibition activity. The activity of
N-glycosides of pyrimidine was higher than that of
compounds containing the pyrimidine system attached
to acyclic sugar moities via the hydrazinyl linkage.
Table 1. Cytotoxic effect (CC50), inhibitory concentration
(IC50) and selective index (SI) for selected compounds
The results of the viral screening against HBV of
selected compounds indicated that the products 6b, 6c,
11a–11c demonstrated viral replication inhibition and
mild cytotoxicity with selective index > 151 > 625 >
370 > 500, and > 400, respectively. On the other hand,
compounds 6a, 7a–7c exhibited very low inhibition
and high cytotoxicity with selective index > 80 > 64 >
73 and > 84, respectively (Tables 1, 2).
HBV DNA Hep G2 2.2.15
Compound
SI
IC50, μM
CC50, μM
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Lamivudine
>1000
>80
6a
1.25
0.66
0.16
1.56
1.37
1.19
0.27
0.20
0.25
6b
>151
>625
>64
6c
The accumulated data revealed that attachment of a
certain glycosyl moiety or acyclic sugar unit chain to
the substituted pyrimidinone ring system led to more
active compounds. Derivatives incorporating the
glucopyranosyl fragment demonstrated highar activity
than the galactopyranosyl analogue. The results also
indicated that substitution in the phenyl substituent at
the position 5 in the pyrimidinone ring led to more
active derivatives. The attachment of free hydroxy
7a
7b
>73
7c
>84
11a
11b
11c
>370
>500
>400
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HAWATA et al.
Table 2. Results of inhibition of HBV replication by selected compounds
Compound Concentration, μM HBV DNA in supernatant Compound Concentration, μM HBV DNA in supernatant
Lamivudine
1.0
10.0
100.0
1.0
0.25
0.18
0.15
0.21
0.18
0.15
0.37
0.31
0.21
0.80
0.76
0.69
0.15
0.13
0.11
7b
1.0
10.0
100.0
1.0
0.18
0.16
0.12
0.22
0.20
0.17
0.68
0.61
0.56
0.78
0.72
0.70
0.73
0.70
0.63
6a
6b
6c
7a
7c
10.0
100.0
1.0
10.0
100.0
1.0
11a
11b
11c
10.0
100.0
1.0
10.0
100.0
1.0
10.0
100.0
1.0
10.0
100.0
1.0
10.0
100.0
10.0
100.0
EXPERIMENTAL
with water, dried and recrystallized from ethanol to
give the corresponding compound 2a–2c in 88–92%
yields. Physical data of compounds 2a–2c were in full
agreement with the reported ones.
Melting points were determined on a Kofler block
apparatus and are uncorrected. IR spectra were
recorded on a Perkin-Elmer 1720 FTIR spectro-
Synthesis of 1-(2,3,4,6-tetra-O-acetyl-β-D-glycol-
pyranosyl)-6-aryl-5-cyano-2-(methylsulfanyl)pyri-
midin-4(1H)-ones (4a–4c, 5a–5c). 2-(Methylsulfanyl)-
uracil derivatives 2a–2c (5 mmol) were suspended in
25 mL of dry DMF at room temperature. To this
suspension, NaH (50%, 0.26 g, 5 mmol) was added
and the mixture was stirred at room temprature for
0.5 h. 2,3,4,6-Tetra-O-acetyl-α-D-gluco- or galacto-
pyranosyl bromide (3a, 3b) (5.5 mmol) was added, and
the mixture was stirred at room temperature for 4 h
until the starting material was consumed (TLC). The
residue precipitated upon evaporation of the filtrate
under reduced pressure was purified by recrys-
tallization from absolute ethanol to afford a compound
4a–4c or 5a–5c, respectively.
1
photometer using KBr disks. H NMR spectra were
measured on a Varian Gemini spectrometer (300 MHz)
in DMSO-d6 using TMS as the internal standard. Mass
spectra were measured on a CC 2010 Shimadzu Gas
chromatographer (70 eV). The progress of the
reactions was monitored by TLC using aluminum
silica gel plates 60 F245. Microanalysis was carried
out at the Microanalytical unit, Tokyo University,
Japan. Anticancer activity of the synthesized
compounds was tested at the National Cancer Institute
(NCI), Cairo, Egypt. Antiviral activity against HBV
was tested at the Liver Institute, Menoufia University,
Shebin El-Koam Egypt.
Synthesis of 6-aryl-5-cyano-2-(methylsulfanyl)-
uracils (2a–2c). A mixture of substituted 2-thiouracils
1a–1c (0.05 mole) with methyl iodide (3.11 mL,
0.05 mol) and sodium hydroxide (2 g, 0.05 mole) in
water (50 mL) and ethanol (100 mL) was stirred at 60°C
for 1 h. A white solid began to precipitate upon
cooling. The precipitated solid was filtered off, washed
2-(Methylthio)-6-oxo-4-phenyl-1-(2,3,4,6-tetra-O-
acetyl-β-D-glucopyranosyl)-1,6-dihydropyrimidine-
5-carbonitrile (4a). Yield 78%, mp 166–168°C. IR
spectrum, ν, cm–1: 2224 (C≡N), 1738, 1663 (C=O). 1H
NMR spectrum, δ, ppm: 2.02, 2.03, 2.04, 2.07 4s
(12H, 4OAc), 2.47 s (3H, SCH3), 4.09–4.15 m (2H,
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SYNTHESIS AND ANTI-HBV ACTIVITY OF NOVEL SUBSTITUTED PYRIMIDINE
1739
H6,6'), 4.22 m (1H, H5'), 5.06 t (1H, J3',4' = 9.5 Hz, H4'),
C26H27N3O10S (573.57). Calculated, %: C 54.44, H
4.74, N 7.32.
5.16 t (1H, J2',3' = 9.2 Hz, H2'), 5.58 t (1H, J2',3'
=
9.3 Hz, H3'), 6.42 d (1H, J1',2' = 8.2 Hz, H1'), 7.35–7.40
m (5H, Ar-H). Found, %: C 54.23, H 4.61, N 7.25.
C26H27N3O10S. Calculated, %: C 54.44, H 4.74, N 7.32.
2-(Methylthio)-6-oxo-1-(2,3,4,6-tetra-O-acetyl-β-
D-galactopyranosyl)-4-(p-tolyl)-1,6-dihydropyri-
midine-5-carbonitrile (5b). Yield 69%, mp 163–164°C.
IR spectrum, ν, cm–1: 2218 (C≡N), 1736, 1665 (C=O).
1H NMR spectrum, δ, ppm: 2.00, 2.03, 2.05, 2.14 4s
(12H, 4OAc), 2.33 s (3H, CH3), 2.50 s (3H, SCH3),
3.96–4.00 m (1H, H5'), 4.20–4.27 m (2H, H6,6'), 4.75–
2-(Methylthio)-6-oxo-1-(2,3,4,6-tetra-O-acetyl-β-
D-glucopyranosyl)-4-(p-tolyl)-1,6-dihydropyrimidine-
5-carbonitrile (4b). Yield 79%, mp 166–168°C. IR
spectrum, ν, cm–1: 2220 (C≡N), 1738, 1660 (C=O). 1H
NMR spectrum, δ, ppm: 1.99, 2.02, 2.04, 2.06 4s,
(12H, 4OAc), 2.28 s (3H, CH3), 2.53 s (3H, SCH3),
4.11–4.17 m (2H, H6,6'), 4.29–4.32 m (1H, H5'), 5.11–
5.13 m (1H, H4'), 5.25–5.28 m (1H, H2'), 5.61–5.64 m
(1H, H3'), 6.44 d (1H, J1',2' = 8.4 Hz, H1'), 7.20 d (2H,
J = 10.3 Hz, Ar-H), 7.40 d (2H, J = 10.3 Hz, Ar-H).
13C NMR spectrum, δ, ppm: 17.5, 20.8, 20.9, 21.1,
22.2 (5CH3), 61.7 (C6), 67.9 (C4), 69.2 (C3), 70.5 (C2),
72.2 (C5), 91.3 (C1), 116.4 (CN), 133.1–160.9 (Ar-C
and pyrimidine C2,5), 163.4, 169.5, 169.7, 170.0, 170.1,
170.5, 171.8 (C=O, pyrimidine C4,6). Found, %: C
54.98, H 4.81, N 7.05. C27H29N3O10S (587.60). Cal-
culated, %: C 55.19, H 4.97, N 7.15.
4.77 m (1H, H3'), 5.45 d.d (1H, J3',4' = 3.3 Hz, J4',5'
=
1.0 Hz, H4'), 5.47 d (1H, J1',2' = 7.5 Hz, H1'), 5.75 t (1H,
J2',1' = 7.5 Hz, H2'), 7.21 d (2H, J = 10.3 Hz, Ar-H),
7.41 d (J = 10.3 Hz, 2H, Ar-H). Found, %: C 54.02, H
4.77, N 7.00. C27H29N3O10S (587.60). Calculated, %: C
55.19, H 4.97, N 7.15.
4-(4-Methoxyphenyl)-2-(methylthio)-6-oxo-1-
(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-1,6-
dihydropyrimidine-5-carbonitrile (5c). Yield 66%,
mp 205–206°C. IR spectrum, ν, cm–1: 2217 (C≡N),
1
1737, 1660 (C=O). H NMR spectrum, δ, ppm: 2.01,
2.03, 2.05, 2.16 4s (12H, 4OAc), 2.49 s (3H, SCH3),
3.50 s (3H, OCH3), 3.98–4.06 m (1H, H5'), 4.20–4.29
m (2H, H6,6'), 4.79–4.82 m (1H, H3'), 5.48 d.d 1H (J3',4'
=
4-(4-Methoxyphenyl)-2-(methylthio)-6-oxo-1-
(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-1,6-di-
hydropyrimidine-5-carbonitrile (4c). Yield 83%, mp
189–191°C. IR spectrum, ν, cm–1: 2221 (C≡N), 1738,
3.5 Hz, J4',5' = 1.2 Hz, H4'), 5.50 d (1H, J1',2' = 7.4 Hz,
H1'), 5.50 m (1H, H2'), 7.22 d (2H, J = 10.3 Hz, Ar-H),
7.40 d (2H, J = 10.3 Hz, Ar-H). 13C NMR spectrum, δ,
ppm: 14.0, 20.7, 20.8, 21.0, 21.5 (5CH3), 56.4 (OCH3),
62.3 (C6), 68.8 (C4), 69.3 (C3), 71.2 (C2), 72.8 (C5),
98.3 (C1), 114.3 (CN), 135.1-156.6 (Ar-C and pyri-
midine C2,5), 162.6, 169.5, 169.8, 170.0, 170.5, 171.2,
172.5 (C=O, pyrimidine C4,6). Found, %: C 53.63, H
4.77, N 6.88. C27H29N3O11S (603.60). Calculated, %: C
53.72, H 4.84, N 6.96.
1
1662 (C=O). H NMR spectrum, δ, ppm: 1.98, 2.02,
2.05, 2.08 4s (12H, 4OAc), 2.53 s (3H, SCH3), 3.48 s
(3H, OCH3), 4.12–4.17 m (2H, H6,6'), 4.32–4.35 m
(1H, H5'), 5.21–5.30 m (2H, H2', H4'), 5.66–5.68 m (1H,
H3'), 6.43 d (1H, J1',2' = 8.4 Hz, H1'), 7.25 d (2H, J =
10.3 Hz, Ar-H), 7.43 d 2H (J = 10.3 Hz, Ar-H). 13C
NMR spectrum, δ, ppm: 14.1, 20.7, 20.9, 21.1, 21.5
(5CH3), 55.4 (OCH3), 62.1 (C6), 68.5 (C4), 68.9 (C3),
71.1 (C2), 73.3 (C5), 98.3 (C1), 114.9 (CN), 132.1–
154.9 (Ar-C and pyrimidine C2,5), 165.8, 169.5, 169.8,
170.0, 170.5, 171.1, 172.5 (C=O, pyrimidine C4,6).
Found, %: C 53.60, H 4.80, N 7.08. C27H29N3O11S
(603.60). Calculated, %: C 53.72, H 4.84, N 6.96.
Synthesis of 1-(β-D-Glycopyranosyl)-6-aryl-5-cyano-
2-(methylsulfanyl)-pyrimidin-4(1H)-ones (6a–6c,
7a–7c). Solution of a compound 4a–4c or 5a–5c (0.5 g)
in a 1 : 1 mixture (50 mL) with methanol was treated
with conc. ammonia (25%) and stirred at room
temperature for 2 h. The reaction mixture was
concentrated under reduced pressure. The residue was
recrystallized from methanol to give a compound 6a–
6c or 7a–7c, respectively.
2-(Methylthio)-6-oxo-4-phenyl-1-(2,3,4,6-tetra-
O-acetyl-β-D-galactopyranosyl)-1,6-dihydropyrimidine-
5-carbonitrile (5a). Yield 71%, mp 143–144°C. IR
spectrum, ν, cm–1: 2218 (C≡N), 1740, 1662 (C=O). 1H
NMR spectrum, δ, ppm: 2.02, 2.03, 2.07, 2.17 4s
(12H, 4OAc), 2.51 s (3H, SCH3), 3.97–4.01 m (1H,
H5'), 4.16–4.23 m (2H, H6,6'), 4.77–4.81 m (1H, H3'),
5.30 d.d (1H, J3',4' = 3.4 Hz, J4',5' = 1.1 Hz, H4'), 5.33 d
(1H, J1',2' = 7.8 Hz, H1'), 5.76 m (1H, H2'), 7.30–7.40 m
(5H, Ar-H). Found, %: C 54.27, H 4.51, N 7.20.
2-(Methylthio)-6-oxo-4-phenyl-1-(β-D-glucopyra-
nosyl)-1,6-dihydropyrimidine-5-carbonitrile (6a).
Yield 92%, mp 170–171°C. IR spectrum, ν, cm–1:
1
3440–3425 (OH), 2225 (C≡N), 1660 (C=O). H NMR
spectrum, δ, ppm: 2.51 s (3H, SCH3), 3.28–3.37 m
(4H, H6'', H6', H5', H4'), 3.50–3.53 m (1H, H3'), 3.84–
3.87 m (1H, H2'), 4.50–4.54 br.s (1H, 6'-OH), 5.00–
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HAWATA et al.
5.02 br.s (1H, 4'-OH), 5.45–5.51 m (2H, 2'-OH, 3'-
OH), 5.74 d (1H, J1',2' = 7.8 Hz, H1'), 7.30–7.40 m (5H,
Ar-H). Found, %: C 53.09, H 4.63, N 10.17.
C18H19N3O6S (405.42). Calculated, %: C 53.32, H
4.72, N 10.36.
(1H, H3'), 3.99–4.01 m (1H, H2'), 4.74 br.s (1H, 6'-
OH), 5.15 br.s (1H, 4'-OH), 5.52–5.57 m (2H, 2'-OH,
3'-OH), 5.69 d (1H, J = 8.2 Hz, H1') 7.24 d (2H, J =
10.3 Hz, Ar-H), 7.44 d (2H, J = 10.3 Hz, Ar-H). 13C
NMR spectrum, δ, ppm: 14.3, 22.8, 23.7 (2CH3), 61.1
(C6), 67.6 (C4), 72.5 (C3), 74.2 (C2), 77.4 (C5), 88.1
(C1), 110.3 (CN), 124.8-156.5 (Ar-C and pyrimidine
C2,5), 164.4, 167.5 (pyrimidine C4,6). Found, %: C
54.32, H 4.95, N 9.94. C19H21N3O6S (419.45).
Calculated, %: C 54.40, H 5.04, N 10.01.
1-(β-D-Glucopyranosyl)-2-(methylthio)-6-oxo-4-
(p-tolyl)-1,6-dihydropyrimidine-5-carbonitrile (6b).
Yield 90 %, mp 198–199°C. IR spectrum, ν, cm–1:
1
3465–3440 (OH), 2220 (C≡N), 1655 (C=O). H NMR
spectrum, δ, ppm: 2.38 s (3H, CH3), 2.50 s (3H,
SCH3), 3.35–3.47 m (5H, H3', H4', H5', H6,6'), 3.79–3.83
m (1H, H2'), 4.52 br.s (1H, 6'-OH), 5.07 br.s (1H, 4'-OH),
1-(β-D-Galactopyranosyl)-4-(4-methoxyphenyl)-
2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carbo-
nitrile (7c). Yield 78%, mp 243–245°C. IR spectrum,
ν, cm–1: 3465–3440 (OH), 2222 (C≡N), 1665 (C=O).
1H NMR spectrum, δ, ppm: 2.51 s (3H, SCH3), 3.50–
3.65 m (4H, H4', H5', H6'',6'), 3.88 s (3H, OCH3), 3.93–
3.95 m (1H, H3'), 3.99–4.02 m (1H, H2'), 4.77 br.s (1H,
6'-OH), 5.19 br.s (1H, 4'-OH), 5.59 m (2H, 2'-OH, 3'-
OH), 5.71 d (1H, J = 7.8 Hz, H1'), 7.21 d (2H, J = 10.3
Hz, Ar-H), 7.45 d (2H, J = 10.3 Hz, Ar-H). Found, %:
C 52.33, H 4.77, N 9.49. C19H21N3O7S (435.45).
Calculated, %: C 52.40, H 4.86, N 9.64.
5.50–5.59 m (2H, 2'-OH, 3'-OH), 5.78 d (1H, J1',2'
=
7.5 Hz, H1'), 7.25 d (2H, J = 10.3 Hz, Ar-H), 7.45 d
(2H, J = 10.3 Hz, Ar-H). Found, %: C 54.28, H 4.89,
N 9.89. C19H21N3O6S (419.45). Calculated, %: C
54.40, H 5.04, N 10.01.
1-(β-D-Glucopyranosyl)-4-(4-methoxyphenyl)-2-
(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carbo-
nitrile (6c). Yield 88%, mp 218–220°C. IR spectrum,
ν, cm–1: 3448-3430 (OH), 2225 (C≡N), 1668 (C=O).
1H NMR spectrum, δ, ppm: 2.50 s (3H, SCH3), 3.39–
3.52 m (7H, H4', H5', H6,6', OCH3), 3.75–3.81 m (2H,
H3', H2'), 4.48 br.s (1H, 6'-OH), 5.05 br.s (1H, 4'-OH),
5.55 m (2H, 2'-OH, 3'-OH), 5.77 d (1H, J1',2' = 7.7 Hz,
H1'), 7.25 d (2H, J = 10.3 Hz, Ar-H), 7.47 d (2H, J =
10.3 Hz, Ar-H). 13C NMR spectrum, δ, ppm: 15.8, 58.1
(OCH3), 62.2 (C6), 66.8 (C4), 70.4 (C3), 74.5 (C2), 78.8
(C5), 98.5 (C1), 108.9 (CN), 132.1–154.8 (Ar-C and
pyrimidine C2,5), 171.9, 172.1 (pyrimidine C4,6).
Found, %: C 52.27, H 4.80, N 9.53. C19H21N3O7S
(435.45). Calculated, %: C 52.40, H 4.86, N 9.64.
Synthesis of 1-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-β-D-glucopyranosyl)-6-aryl-5-cyanopyrimidin-
4(1H)-ones (10a–10c). A mixture of one of the com-
pounds of 6-aryl-5-cyanouracils 8a–8c (5 mmol) with
50% oil-immersed sodium hydride (0.24 g, 5 mmol) in
DMF (30 mL) was stirred at 70–80°C for 1 h and then
cooled to room temperature. α-Chloroacetamido sugar
10 (1.83 g, 5 mmol) was added to the mixture, and it
was stirred at 90°C for 3–5 h. The mixture was
evaporated to dryness under reduced pressure and the
residue was recrystallized from absolute ethanol to
give the corresponding compound 11a–11c.
1-(β-D-Galactopyranosyl)-2-(methylthio)-6-oxo-
4-phenyl-1,6-dihydropyrimidine-5-carbonitrile (7a).
Yield 80%, mp 210–211°C. IR spectrum, ν, cm–1:
3-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-2,4-dioxo-6-phenyl-1,2,3,4-tetra-
hydropyrimidine-5-carbonitrile (10a). Yield 78%,
mp 150–151°C. IR spectrum, ν, cm–1: 3305 (NH),
2218 (C≡N), 1742, 1663 (C=O). 1H NMR spectrum, δ,
ppm: 1.75 s (3H, NHAc), 1.94, 1.99, 2.00 3s (9H,
3OAc), 3.95–4.11 m (3H, H5', H6'',6'), 4.54–4.47 m (1H,
H2'), 5.15–5.17 m (1H, H4'), 5.30–5.32 m (1H, H3'),
5.89 d (1H, J = 9.3 Hz, H1'), 7.77 d (1H, J = 7.5 Hz,
NHAc), 7.37–7.47 m (5H, Ar-H), 9.18 br (1H, NH).
Found, %: C 55.21, H 4.77, N 10.13. C25H26N4O10
(542.50). Calculated, %: C 55.35, H 4.83, N 10.32.
1
3460–3445 (OH), 2225 (C≡N), 1665 (C=O). H NMR
spectrum, δ, ppm: 2.50 s (3H, SCH3), 3.42–3.49 m
(3H, H6', H6'', H5'), 3.65–3.71 m (2H, H3',4'), 3.98–4.01
m (1H, H2'), 4.70 br.s (1H, 6'-OH), 5.05 br.s (1H,
4'-OH), 5.50 m (2H, 2'-OH, 3'-OH), 5.70 d (1H, J =
7.8 Hz, H1'), 7.30–7.40 m (5H, Ar-H). Found, %: C
53.22, H 4.60, N 10.24. C18H19N3O6S (405.42).
Calculated, %: C 53.32, H 4.72, N 10.36.
1-(β-D-Galactopyranosyl)-2-(methylthio)-6-oxo-
4-(p-tolyl)-1,6-dihydropyrimidine-5-carbonitrile (7b).
Yield 80%, mp 237–239°C. IR spectrum, ν, cm–1:
1
3455–3440 (OH), 2220 (C≡N), 1667 (C=O). H NMR
1-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-2,4-dioxo-6-(p-tolyl)-1,2,3,4-tetra-
hydropyrimidine-5-carbonitrile (10b). Yield 80%,
spectrum, δ, ppm: 2.33 s (3H, CH3), 2.52 s (3H,
SCH3), 3.52–3.69 m (4H, H4', H5', H6'',6'), 3.92–3.95 m
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SYNTHESIS AND ANTI-HBV ACTIVITY OF NOVEL SUBSTITUTED PYRIMIDINE
1741
mp 242–243°C. IR spectrum, ν, cm–1: 3312 (NH),
2224 (C≡N), 1740, 1660 (C=O). 1H NMR spectrum, δ,
ppm: 1.76 s (3H, NHAc), 1.97, 2.00, 2.05 3s (9H, 3
OAc), 2.36 s (3H, CH3), 4.25–4.45 m (4H, H6'',6', H5',
H2'), 5.30–5.32 m (1H, H4'), 5.43–5.46 m (1H, H3'),
5.88 d (1H, J = 9.4 Hz, H1'), 7.21 d (2H, J = 10.3 Hz,
Ar-H), 7.39 d (2H, J = 10.3 Hz, Ar-H), 7.80 d (1H, J =
7.5 Hz, NHAc), 9.21 br (1H, NH). 13C NMR spectrum,
δ, ppm: 20.7, 20.8, 20.9, 21.1, 23.6 (6CH3), 62.1 (C6),
67.7 (C4), 71.1 (C3), 72.5 (C2), 73.8 (C5), 97.3 (C1),
116.4 (CN), 133.1-133.2 (Ar-C and pyrimidine C5),
161.0, 163.4, 169.5, 169.7, 170.0, 170.5, 171.6 (C=O,
pyrimidine C2,4,6). Found, %: C 56.00, H 4.89, N 9.97.
C26H28N4O10 (556.52). Calculated, %: C 56.11, H 5.07,
N 10.06.
5.26 m (3H, 3OH), 5.76 d (1H, J = 9.4 Hz, H1'), 7.30–
7.40 m (5H, Ar-H), 7.90 d (1H, J = 8.5 Hz, NHAc),
9.24 br (1H, NH). Found, %: C 54.60, H 4.71, N
13.37. C19H20N4O7 (416.39). Calculated, %: C 54.80,
H 4.84, N 13.45.
1-(2-Acetamido-β-D-glucopyranosyl)-2,4-dioxo-6-
(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
(11b). Yield 88%, mp 288–289°C. IR spectrum, ν, cm–
1
1: 3305 (NH), 2222 (C≡N), 1748, 1665 (C=O). H
NMR spectrum, δ, ppm: 1.69 s (3H, NHAc), 2.38 s
(3H, CH3), 3.19–3.29 m (4H, H6,6'', H5', H4'), 3.70–3.80
m (2H, H2', H3'), 5.15–5.21 m (3H, 3OH), 5.78 d (1H,
J = 9.6 Hz, H1'), 7.22 d (2H, J = 10.3 Hz, Ar-H), 7.41 d
(2H, J = 10.3 Hz, Ar-H), 7.88 d (1H, J = 8.5 Hz,
NHAc), 9.22 br (1H, NH). 13C NMR spectrum, δ, ppm:
21.7 (OCH3), 61.0 (C6), 69.4 (C4), 71.4 (C3), 78.5 (C2),
81.5 (C5), 84.9 (C1), 111.9 (CN), 128.5-155.7 (Ar-C
and pyrimidine C5), 159.9, 160.2, 161.8, 171.5 (C=O,
pyrimidine C2,4,6). Found, %: C 55.68, H 5.04, N
12.92. C20H22N4O7 (430.41). Calculated, %: C 55.81,
H 5.15, N 13.01.
1-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
galactopyranosyl)-2,4-dioxo-6-(p-tolyl)-1,2,3,4-tetra-
hydropyrimidine-5-carbonitrile (10c). Yield 75%,
mp 263–264°C. IR spectrum, ν, cm–1: 3295 (NH),
2220 (C≡N), 1738, 1662 (C=O). 1H NMR spectrum, δ,
ppm: 1.78 s (3H, NHAc), 1.96, 1.99, 2.02 3s (9H,
3OAc), 3.53 s (3H, OCH3), 4.07–4.19 m (3H, H5',
H6'',6'), 4.33–4.35 m (1H, H2'), 5.32–5.35 m (1H, H4'),
5.50–5.58 t (1H, J = 9.5 Hz, H3'), 5.90 d (1H, J =
9.2 Hz, H1'), 7.19 d (2H, J = 10.3 Hz, Ar-H), 7.33 d
(2H, J = 10.3 Hz, Ar-H), 7.79 d (1H, J = 7.5 Hz,
NHAc), 9.24 br (1H, NH). 13C NMR spectrum, δ, ppm:
20.1, 20.2, 20.3, 20.4 (4CH3), 56.2 (OCH3), 62.0 (C6),
68.1 (C4), 70.1 (C3), 70.4 (C2), 71.8 (C5), 95.1 (C1),
104.2 (CN), 129.1–134.5 (Ar-C and pyrimidine C5),
168.8, 169.1, 170.0, 170.1, 170.2, 170.9 (C=O, pyri-
midine C2,4,6). Found, %: C 54.32, H 4.81, N 9.57.
C26H28N4O11 (572.52). Calculated, %: C 54.54, H 4.92,
N 9.78.
1-(2-Acetamido-β-D-glucopyranosyl)-2,4-dioxo-
6-(p-methoxyphenyl)-1,2,3,4-tetrahydropyrimidine-
5-carbonitrile (11c). Yield 83%, mp 290–292°C. IR
spectrum, ν, cm–1: 3290 (NH), 2225 (C≡N), 1738,
1
1668 (C=O). H NMR spectrum, δ, ppm: 1.73 s (3H,
NHAc), 3.39–3.58 m (8H, H3', H4', H5', H6', OCH3),
3.88 m (1H, H2'), 5.20–5.30 m (3H, 3OH), 5.80 d (1H,
J = 9.5 Hz, H1'), 7.21 d (2H, J = 10.3 Hz, Ar-H), 7.39 d
(2H, J = 10.3 Hz, Ar-H), 7.90 d (1H, J = 8.5 Hz,
NHAc), 9.23 br (1H, NH). Found, %: C 53.60, H 4.83,
N 12.33. C20H22N4O8 (446.41). Calculated, %: C 53.81,
H 4.96, N 12.55.
Synthesis of hydrazinyl sugar derivatives 13–18.
The mixture of 2-hydrazinyluracils 12a, 12b
(2.79 mmol) in ethanol (70 mL) with the appropriate
pentose or hexose sugar derivative (2.79 mmol) in
water (10 mL) and acetic acid (0.4 mL) was refluxed
for 5–6 h. The excess ethanol was removed under
reduced pressure, and the residue was triturated with
diethyl ether (15 mL), filtered off, washed with ether,
and recrystallized from ethanol to give the corres-
ponding compound 13–18.
Synthesis of 1-(2-acetamido-β-D-glycopyranosyl)-
2,4-dioxo-6-phenyl-1,2,3,4-tetrahydropyrimidine-5-
carbonitrile (11a–11c). Solution of a compound 11a–
11c (0.5 g) in a 1 : 1 mixture (50 mL) of methanol with
conc. ammonia (25 %) was stirred at room temperature
for 8 h. The reaction mixture was concentrated under
reduced pressure. Thus obtained residue was recrys-
tallized from methanol to give one of the corres-
ponding compounds 12a–12c.
1-(2-Acetamido-β-D-glucopyranosyl)-2,4-dioxo-6-
phenyl-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
(11a). Yield 90%, mp 276–277°C. IR spectrum, ν, cm–1:
6-Oxo-4-phenyl-2-[2-(D-xylotetritolylidene)hyd-
razinyl]-1,6-dihydropyrimidine-5-carbonitrile (13).
White powder, yield 75%, mp 210–212°C. IR
spectrum, ν, cm–1: 3445–3411 (OH and NH), 2220
(C≡N), 1663 (C=O). 1H NMR spectrum, δ, ppm: 3.35–
3.42 m (3H, H5',5'', H4'), 3.76–3.80 m (1H, H3'), 4.20–
1
3310 (NH), 2225 (C≡N), 1745, 1662 (C=O). H NMR
spectrum, δ, ppm: 1.68 s (3H, NHAc), 3.32–3.50 m
(4H, H6,6'', H5', H4'), 3.77–4.88 m (2H, H2', H3'), 5.16–
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 8 2018

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HAWATA et al.
4.25 m (2H, H2' and OH), 4.98–5.05 m (2H, 2OH),
5.12–5.14 m (1H, OH), 7.30 d (1H, J = 7.4 Hz, H1'),
7.35–7.72 m (5H, Ar-H), 7.94–8.02 br.s (2H, 2NH).
Found, %: C 53.32; H 4.90; N 19.24. C16H17N5O5
(359.12). Calculated, %: C 53.48; H 4.77; N 19.49.
C 53.32; H 4.98; N 17.27. C18H21N5O6 (403.15).
Calculated, %: C 53.59; H 5.25; N 17.36.
2-[2-(D-Galactopentitolylidene)hydrazinyl]-6-
oxo-4-(p-tolyl)-1,6-dihydropyrimidine-5-carbonitrile
(18). White powder, yield 71%, mp 206–208°C. IR
spectrum, ν, cm–1: 3403, 3221 (OH and NH), 2222
(C≡N), 1672 (C=O). 1H NMR spectrum, δ, ppm: 2.27 s
(3H, CH3), 3.39–3.48 m (3H, H6',6'', H5'), 3.77–3.83 m
(2H, H4', H3'), 4.26–4.32 m (2H, H2' and OH), 5.00–
5.08 m (2H, 2OH), 5.19–5.24 m (2H, 2OH), 7.48 d
(2H, J = 7.2 Hz, Ar-H), 7.68 d (1H, J =7.4 Hz, H1'),
7.75 d (2H, J = 7.2 Hz, Ar-H), 10.05–10.12 br.s (2H,
2NH). Found, %: C 54.31; H 5.18; N 17.49.
C18H21N5O6 (403.15). Calculated, %: C 53.59; H 5.25;
N 17.36.
2-[2-(D-Glucopentitolylidene)hydrazinyl]-6-oxo-
4-phenyl-1,6-dihydropyrimidine-5-carbonitrile (14).
White powder, yield 78%, mp 188–190°C. IR spec-
trum, ν, cm–1: 3418 (OH and NH), 2221 (C≡N), 1664
1
(C=O). H NMR spectrum, δ, ppm: 3.37–3.45 m (3H,
H6',6'', H5'), 3.78–3.84 m (2H, H4', H3'), 4.22–4.28 m
(2H, H2' and OH), 5.02–5.08 m (2H, 2OH), 5.14–5.19
m (2H, 2OH), 7.33 d (H, J = 7.4 Hz, H1'), 7.38–7.70 m
(5H, Ar-H), 7.98–8.05 br.s (2H, 2NH). Found, %: C
52.32; H 4.85; N 18.10. C17H19N5O6 (389.13). Cal-
culated, %: C 52.44; H 4.92; N 17.99.
Antiviral activity. Preparation and culture of Hep
G2 2.2.15 cells. The required cell line was made by
transfection of Hep G2-cells with a plasmid containing
multiple tandem copies of HBV genome (subtype ayw)
[38]. The 2.2.15 cell line was maintained in RPMI-
1640 (Glutamax) culture media containing 100 IU/mL
nystatin and 380 μg/ mL G418 (geneticin). The
transferred HEP G2-2.2.15 cell line was stored in
tissue culture flask at 37°C + 5%CO2. Subcultures
were set up after a week by aspiration of the media
from culture flask and washing the cells twice by PBS.
A 10% versene/trypsin was added and the cells were
incubated for 1 min at 37°C.
2-[2-(D-Galactopentitolylidene)hydrazinyl]-6-oxo-
4-phenyl-1,6-dihydropyrimidine-5-carbonitrile (15).
White powder, yield 75%, mp 218–220°C. IR spec-
trum, ν, cm–1: 3428 (OH and NH), 2220 (C≡N), 1647
1
(C=O). H NMR spectrum, δ, ppm: 3.37–3.50 m (3H,
H6',6'', H5'), 3.77–3.83 m (2H, H4', H3'), 4.27–4.34 m
(2H, H2' and OH), 5.09–5.15 m (2H, 2OH), 5.17–5.25
m (2H, 2OH), 7.35 d (1H, J = 7.6 Hz, H1'), 7.39–7.74
m (5H, Ar-H), 8.12–8.17 br.s (2H, 2NH). Found, %: C
52.28; H 4.71; N 18.12. C17H19N5O6 (389.13).
Calculated, %: C 52.44; H 4.92; N 17.99.
6-Oxo-4-(p-tolyl)-2-[2-(D-xylotetritolylidene)-
hydrazinyl]-1,6-dihydropyrimidine-5-carbonitrile
(16). White powder, yield 70%, mp 200–202°C. IR
spectrum, ν, cm–1: 3378 (OH and NH), 2221 (C≡N),
The drug Lamivudine, which is a potent selective
inhibitor of HBV replication [39], has been used as a
standard for the comparative studies.
1
1678 (C=O). H NMR spectrum, δ, ppm: 2.33 s (3H,
CH3), 3.38–3.60 m (3H, H5',5'', H4'), 3.78–3.83 m (1H,
H3'), 4.53–4.59 m (2H, H2' and OH), 5.09–5.20 br.s
(3H, 3OH), 7.50 d (2H, J = 7.6 Hz, Ar-H), 7.66 d (1H,
J = 7.5 Hz, H1'), 7.76 d (2H, J = 7.6 Hz, Ar-H), 9.95–
10.02 br.s (2H, 2NH). Found, %: C 54.82; H 5.22; N
18.58. C17H19N5O5 (373.14). Calculated, %: C 54.69;
H 5.13; N 18.76.
DNA extraction. HBV-DNA extraction was carried
out by mixing 10 μL of diluted supernatant (1 : 5 with
PBS) in a reaction tube with 10 μL of 0.2 M NaOH
and incubated at 37°C for 1h. Carefully, 9.6 μL of
0.2 M HCl was added followed by addition of 90 μL of
TE buffer solution.
PCR-Ellisa. The PCR reaction mixture contained
14 μL extracted supernatant, 4 mmol/l MgCl2, 10 μmol/L
DIG-11-dUTP, 190 μmol/L dTTP, 200 μmol/L dATP,
dGTP, dCTP, 1.5 U Taq polymerase, 20 mmol/l HCl
(pH 8.4), 50 mmol/l KCl, 1 μmol/L HCID-1 primer
(5'GGA AAG AAG TCA GAA GGC A3') and 1 μmol/
L HCID-2 (5'TTG GGG GAG GAG ATT AGG TT3'),
in total volume 50 μL. PCR reaction conditions were
32 cycles of 1 min at 94°C, 30 s at 58°C and 30 s at
72°C + 3 s for each cycle in a thermal circler as
described in literature [39].
2-[2-(D-Glucopentitolylidene)hydrazinyl]-6-oxo-
4-(p-tolyl)-1,6-dihydropyrimidine-5-carbonitrile (17).
White powder, yield 72%, mp 222–224°C. IR spec-
trum, ν, cm–1: 3377 (OH and NH), 2223 (C≡N), 1673
1
(C=O). H NMR spectrum, δ, ppm: 2.30 s (3H, CH3),
3.38–3.46 m (3H, H6',6'', H5'), 3.78–3.84 m (2H, H4',
H3'), 4.22–4.28 m (2H, H2' and OH), 5.02–5.08 m (2H,
2OH), 5.14–5.19 m (2H, 2OH), 7.50 d (2H, J = 7.2 Hz,
Ar-H), 7.70 d (1H, J = 7.4 Hz, H1'), 7.82 d (2H, J =
7.2 Hz, Ar-H), 10.25–10.31 br.s (2H, 2NH). Found, %:
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 8 2018

SYNTHESIS AND ANTI-HBV ACTIVITY OF NOVEL SUBSTITUTED PYRIMIDINE
1743
Bortolotto, Z.A., Roberts, P.J., Bleakman, D.,
Collingridge, G.L., and Jane, D., J. Med. Chem.
2005, vol. 48, p. 7867. doi 10.1021/jm050584l
Cytotoxicity assay. A colorimetric assay for living
cells utilized the colorless substrate of 3-(3,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) that was modified to a colored product by any
living cells, but not by dead cells or tissue culture
medium. The cytotoxic effect of the compounds was
accessed by culturing the Hep G2-2.2.15 cells in the
presence of compounds using a MTT-assay [40].
8. Gichinga, M.G., Olson, J.P., Butala, E., Navarro, H.A.,
Gilmour, B.P., Mascarella, S.W., and Carroll, F.I., ACS
Med. Chem. Lett., 2011, vol. 2, p. 882. doi 10.1021/
ml200162f
9. Dolman, N.P., More, J.C.A., Alt, A., Knauss, J.L.,
Troop, H.M., Bleakman, D., Collingridge, G.L., and
Jane, D.E., J. Med. Chem., 2006, vol. 49, p. 2579. doi
10.1021/jm051086f
10. Zhang, Z., Wallace, M.B., Feng, J., Stafford, J.A.,
Skene, R.J., Shi, L., Lee, B., Aertgeerts, K., Jennings, A.,
Xu, R., Kassel, D.B., Kaldor, S.W., Navre, M.,
Webb, D.R., and Gwaltney, S.L., J. Med. Chem., 2011,
vol. 54, p. 510. doi 10.1021/jm101016w
Calculation of IC50, CC50, and SI. The 50%
inhibitory concentration of antiviral drugs (IC50) was
determined by interpolation of the plots of amount of
DNA copies versus antiviral drug concentration. The
50% cytotoxic effect (CC50) was calculated from the
average viability of the cells with concentration of
drugs. The selective index (SI) could be calculated as
CC50/IC50 [40].
11. Ruyle W.V. and Shen, T.Y., J. Med. Chem., 1967,
vol. 10, p. 331. doi 10.1021/jm00315a009
12. Romeo, G., Chicchio, U., Corsaro, A., and Merino, P.,
Chem. Rev., 2010, vol. 110, p. 3337. doi 10.1021/
cr800464r
13. Davis, R.D., Biophysical and Conformational
Properties of Modified Nucleosides in RNA, Grosjean, H.
and Benne, R., Eds., Washington: ASM Press, 1998,
p. 85. doi 10.1128/9781555818296ch5
14. Guerra, C.F., Baerends, E.J., and Bickelhaupt, F.M.,
Cent. Eur. J. Chem., 2008, vol. 6, p. 15. doi 10.2478/
s11532-007-0068-y
CONCLUSIONS
New substituted pyrimidine N1-gycosides and C-
linked hydrazinyl acyclic sugars were synthesized and
charactarized. Their bological tests indicated that
incorporation of glycosyl moiety or acyclic sugar unit
in the substituted pyrimidinone ring system afforded
more active antiviral derivatives against HBV.
CONFLICT OF INTERESTS
No conflict of interest was declared by the authors.
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