Synthesis and biological evaluation of novel anti-hepatitis C virus (HCV) agents: 2-hydroxylphenethyl sulfanyl-oxopyrimidines

Article abstract of DOI:10.1007/s00044-017-1815-z

A novel series of dihydro-hydroxyl-phene-thylsulfanyl-ω-cyclohexyl/phenyl-oxopyrimidine derivatives have been synthesized and their in vitro anti-hepatitis C virus activities have been evaluated using Huh 7.5.1 cells. Some of the compounds showed moderate

Full text of DOI:10.1007/s00044-017-1815-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1815-z
ORIGINAL RESEARCH
Synthesis and biological evaluation of novel anti-hepatitis C virus
(HCV) agents: 2-hydroxylphenethyl sulfanyl-oxopyrimidines
Daochun Wu1 Yue Feng2 Hua Wang1 Junfeng Yang1 Xian Chen1
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Yueping Wang2 Christopher Cong Lai3 Yufang Zhang1 Cong Li1 Xueshan Xia2
Yanping He1
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Received: 14 October 2016 / Accepted: 16 February 2017
© Springer Science+Business Media New York 2017
Abstract A novel series of dihydro-hydroxyl-phene-thyl-
sulfanyl-ω-cyclohexyl/phenyl-oxopyrimidine derivatives
Introduction
have been synthesized and their in vitro anti-hepatitis C
virus activities have been evaluated using Huh 7.5.1 cells.
Some of the compounds showed moderate anti-hepatitis C
virus activities, with EC50 range from 7.53 to 0.13 μM.
Among all the compounds, 6-(cyclohexylmethyl)-5-ethyl-2-
((2-hydroxy-2-phenylethyl)thio)-pyrimidin-4 (3H)-one (3a)
had the most promising potential in inhibiting hepatitis C
virus with an EC50 value of 0.13 μM and SI value of 121. It
was noticed that some of these compounds are both active
on hepatitis C virus and human immunodeficiency virus. In
addition to experimental evaluation, structure-activity rela-
tionships and the molecular modeling analysis of these new
congeners are also discussed.
The hepatitis C virus (HCV), first characterized in
1989, is responsible for non-A, non-B hepatitis (Choo et al.
1989). This blood-borne pathogen could be transmitted by
blood transfusions, hemodialysis and intravenous
drug use. It is estimated that about 180 million people
worldwide are infected with HCV (Sofia et al. 2012), and
approximately 80% of the infected patients developed
chronic hepatitis, and subsequently liver cirrhosis and
hepatocellular carcinoma (Lavanchy 2011). These compli-
cations are particularly life-threatening for patients with co-
morbid infections such as human immunodeficiency virus
(HIV).
Currently, there are no vaccines or therapies with effec-
tive action against all genotypes of HCV due to its biolo-
gical characteristics (Fauvelle et al. 2013). For a long time,
the standard of care treatment consists of a combination
therapy using pegylated α-interferon with ribavirin (Ghany
et al. 2009). However, the efficacy of this treatment is
limited in patients infected with the HCV genotype 1
(<50% sustained virologic response), the most common
strain of infection in the globally infected population.
However, ribavirin therapy can be associated with severe
side effects or contraindicated in certain patient populations
(Ferenci et al. 2005; Arataki et al. 2006). These limitations
have intensified interest in the discovery and development
of new compounds that target the virus and its host proteins.
Based on the success of small molecule inhibitors for the
treatment of HIV, the development of similar agents, which
target specific viral enzymes of HCV has become an
attractive topic of research.
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Keywords Anti-HCV activity Drug Discovery
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Pyrimidone HCV NS5B SAR
Daochun Wu and Yue Feng contributed equally to this work.
* Xueshan Xia
Oliverxia2000@aliyun.com
* Yanping He
yphe@ynu.edu.cn
1
Key Laboratory of Medicinal Chemistry for Natural Resources,
Ministry of Education, School of Chemical Science and
Technology, Yunnan University, Kunming 650091, China
2
College of Life Science and Technology, Kunming University of
In the past several years, a number of direct-acting
antivirals entering clinical development have emerged as
the advancement of drug discovery efforts. To date, five
Science and Technology, Kunming 650224, China
3
Chemical Biology Laboratory, Center for Cancer Research,
National Cancer Institute, Frederick, MD 21702, USA

Med Chem Res
protease inhibitors (telaprevir, boceprevir, paritaprevir,
ritonavir or simeprevir), a nucleoside NS5B inhibitor
(sofosbuvir), and two NS5A inhibitors (ledipasvir, ombi-
tasvir) have been approved by the FDA (Lim et al. 2014;
Alexandra and Peter 2015). However, these new inhibitors
lack efficacy against all HCV genotypes and most of them
must be used in combination with ribavirin and pegylated
interferon, which imparts several drug-related toxicities.
Therefore, there is still an urgent need for novel, efficacious,
broad spectrum anti-HCV inhibitors with minimal effect on
the host, as well as efficacious agents for special popula-
tions such as HIV-HCV co-infected patients.
sulfanyl–6ω-cyclohexyl/phenyl-oxopyrimidines (compound
3, Fig. 1) was prepared. It was characterized with insertion
of a 2-hydroxyl-2-[(substituted)phenyl]ethyl sulfanyl chain
at C2, a cyclohexyl, cyclohexylmethyl, phenyl or sub-
stituted benzyl group at C6, and a methyl or ethyl sub-
stituent at C5 positions, with the purpose of acquiring new
type of anti-HCV agents as well as their SAR information.
In this paper, we report the synthesis, anti-HCV activity
evaluation and preliminary SAR studies of these new
scaffolds, and discussion of the molecular modeling ana-
lyses of these new derivatives.
In an effort to develop effective HIV Reverse Tran-
scriptase (HIV RT) inhibitors, we reported a series of pyr-
imidone analogs as high active non-nucleoside HIV-1
reverse transcriptase inhibitors (He et al. 2011; Wu et al.
2013). We also identified a compound (1, Fig. 1), which has
potential as an anti-HCV agent (EC50 = 2.04 µM), via a
cell-based HCV replication assay. Previously, other related
pyrimidines have also been identified as potential HCV
inhibitors. For example, some pyrimidine diketo acids
derivatives capable of inhibiting both HCV NS5B RdRp
and HIV RT with IC50 values in the low micromolar range
have been reported (e.g., 2a, Fig. 1) (Summa et al. 2004).
Ding et al. have reported 5-cyano-6-aryl-2-thiouracil (2b,
Fig. 1), a compound with similar structure to compound 1,
as a potent inhibitor of HCV NS5B (EC50 = 3.8 μM) (Ding
et al. 2006). In addition, structure–activity relationship
(SAR) study of 5-cyano-6-aryl-2-thiouracil suggested that a
free NH of the uracil ring is necessary for the inhibitory
activity, while a combination of the para-substitution on the
C-6 aryl group and an ortho-substitution on the S-benzyl
group could enhance this activity. Based on the above
observations, a series of dihydro-hydroxyl phenethyl-
Methods and materials
Chemistry
Melting points (m.p.) were determined by a WRS-1 digital
1
melting point instrument. H NMR and 13C NMR spectra
were obtained on a Bruker AM 400 MHz spectrometer
using dimethyl sulfoxide-d6 (DMSO-d6) as solvent. Che-
mical shifts are reported in δ (ppm) units relative to the
internal reference tetramethylsilane (TMS). Mass spectra
were obtained on an Agilent LC/MSD TOF mass spectro-
meter. The regents and solvents used were all of analytical
grades, and were purified and dried by standard methods
before use. All air-sensitive reactions were run under pro-
tection of N2. All the reactions were monitored by thin-layer
chromatography (TLC) on pre-coated silica gel G plates at
254 nm under a UV lamp using ethyl acetate/petroleum
ether as eluents. Flash chromatography separations were
obtained on silica gel (300–400 mesh; Qingdao Marine
Chemical Factory, Qingdao, China).
Fig. 1 Pyrimidine compounds
active against HCV targets and
scaffolds 3
HCV NS5B IC50=29 µM
EC50=3.8 µM
EC50=2.4 µM
HIV-1RT IC50=23 µM

Med Chem Res
General procedure for the preparation of target compounds
3a–w
5-Ethyl-6-(4-fluorobenzyl)-2-((2-hydroxy-2-phenylethyl)
thio)pyrimidin-4(3H)-one (3j). White crystal, yield: 35%.
m.p.: 147.8–148.1 °C. 1HNMR (DMSO-d6, 400 MHz) δ:
0.99–1.03 (t, J = 9.17 Hz, 3H), 2.48–2.53 (q, J = 9.26 Hz,
2H), 3.16–3.22 (m, 2H), 3.33–3.38 (m, 1H), 3.74–3.90 (m,
2 H), 4.86–4.88 (m, 1H), 6.90–6.95 (t, J = 11.25 Hz, 2H),
To a stirred solution of 7a–w (3.37 mmol) in anhydrous
tetrahydrofuran (THF) and MeOH (5 mL), NaBH4 was
added in ice water bath. The reaction was continued for
3–4 h. After TLC (petroleum ether/ethyl acetate) revealed
the disappearance of the starting material, 3 mL H2O was
added to terminate the reaction. After the solvent was
removed under reduced pressure, 5 mL H2O was added to
dilute the residue, and the solution was acidified by adding
13%HCl to pH 6. This solution was extracted with ethyl
acetate, dried with anhydrous Na2SO4, filtered, evaporated
to give crude solid, which was purified by flash chromato-
graphy to obtain the pure target compounds 3a–w.
7.15–7.18 (m, 2H), 7.22–7.30 (m, 5H), 12.45 (br s, 1H). 13
C
NMR (DMSO-d6, 100 MHz) δ: 13.02, 18.68, 39.08, 39.91,
73.59, 115.16, 115.38, 122.93, 125.63, 127.77, 128.36,
130.22, 133.30, 133.53, 133.56, 142.62, 158.20, 160.52,
162.80, 164.24. ESI-MS: m/z 383.3(M–H), C21H21FN2O2S
(384.13).
5-Ethyl-6-(4-fluorobenzyl)-2-((2-hydroxy-2-(4-methoxyphe-
nyl)ethyl)thio)pyrimidin-4(3H)-one (3k). Yellow crystal,
1
yield: 33%. m.p.: 206.6–207 °C. HNMR (DMSO-d6, 400
MHz) δ: 1.01–1.04 (t, J = 7.50 Hz, 3H), 2.50–2.52 (q, J =
7.45 Hz, 2H), 2.69(s, 1H), 3.17–3.21 (m, 1H), 3.31–3.34
(m, 1H), 3.76 (s, 3H), 3.81–3.89 (m, 2H), 4.82–4.84 (dd, J1
= 3.05 Hz, J2 = 5.25 Hz, 1H), 6.81–6.83 (d, J = 7.00 Hz,
2H), 6.83–6.95 (m, 2H), 7.14–7.18 (m, 4H), 12.50 (br s,
1H). 13C NMR (DMSO-d6, 100 MHz) δ: 12.95, 18.63,
39.06, 39.92, 73.12, 113.71, 115.14, 115.31, 123.04,
126.82, 130.2, 130.27, 133.47, 134.70, 157.72, 159.13,
160.60, 162.54, 163.72. ESI-MS: m/z 413.5(M–H),
C22H23FN2O3S (414.14).
6-benzyl-2-((2-hydroxy-2-phenylethyl)thio)-5-methyl-pyri-
midin-4(3H)-one (3g). White crystal, yield: 33%. m.p.:
152–153 °C. 1HNMR (DMSO-d6, 400 MHz) δ: 1.95 (s,
3H), 3.14–3.18 (m, 1H), 3.31–3.36(br s,1H), 3.48–3.51 (m,
1H), 3.84–3.90 (m, 2H,), 4.72–4.74 (m, 1H), 7.19–7.31 (m,
10H), 12.51 (br s, 1H); 13C NMR(DMSO-d6, 100 MHz)δ:
10.83, 39.05, 40.45, 71.56, 115.43, 126.23, 126.60, 127.53,
128.40, 128.72, 129.03, 129.36, 138.59, 144.54, 157.67,
158.22, 160.54, 163.88. ESI-MS: m/z 351.2(M–H),
C20H20N2O2S (352.12).
2-((2-(4-Fluorophenyl)-2-hydroxyethyl)thio)-6-(4-methox-
6-(4-Fluorobenzyl)-2-((2-(4-fluorophenyl)-2-hydroxyethyl)
thio)-5-methylpyrimidin-4(3H)-one (3h). White crystal,
yield: 35%. m.p.: 194–195 °C. HNMR (DMSO-d6, 400
yenzyl)-5-methyl-pyrimidin-4(3H)-one (3l). White crystal,
1
yield: 35%. m.p.: 139–140 °C. HNMR (DMSO-d6, 400
1
MHz) δ: 1.79 (s, 3H), 3.17–3.23 (m, 1H), 3.24–3.28 (m,
1H), 3.75 (s, 3H), 3.82 (s, 2H), 4.73–4.75 (d, J = 8.4 Hz,
1H), 5.84 (br s, 1H), 6.81–6.93 (m, 2H), 7.13–7.36 (m, 6H),
12.60 (br s, 1H).13C NMR(DMSO-d6, 100 MHz)δ: 10.37,
38.45 39.21, 54.95, 70.41, 113.73, 114.59, 114.84, 117.46,
127.81, 129.60, 130.02, 140.28, 157.68. ESI-MS: m/z 399.3
(M–H), C21H21FN2O3S (400.13).
MHz) δ: 1.79(s, 3H), 2.95–3.00 (m, 1H), 3.20–3.24 (m,
1H), 3.60–3.67 (m, 1H), 4.28 (s, 2H), 4.60–4.62 (dd, J1 =
3.61 Hz, J2 = 4.66 Hz, 1H), 6.68–6.75 (m, 4H), 6.92–6.95
(m, 2H), 6.99–7.02 (m, 2H), 7.28 (s,1H), 12.45 (br s,1H).
13C NMR(DMSO-d6, 100 MHz)δ: 9.96, 38.91, 39.02,
71.93, 114.56, 114.74, 114.93, 116.34, 127.12, 127.19,
129.84, 129.91, 133.05, 138.36, 160.36, 160.98, 162.30,
162.93. ESI-MS: m/z 387.1(M–H), C20H18F2N2O2S
(388.11).
2-((2-hydroxy-2-phenylethyl)thio)-6-(4-methoxybenzyl)-5-
methylpyrimidin-4(3H)-one (3m). White crystal, yield:
1
35%. m.p.: 129–130 °C. HNMR (DMSO-d6, 400 MHz) δ:
5-Ethyl-6-(4-fluorobenzyl)-2-((2-(4-fluorophenyl)-2-hydro-
xyethyl)thio)pyrimidin-4(3H)-one (3i). White crystal,
yield: 35%. m.p.: 131.2–131.5 °C. 1HNMR (DMSO-d6,
400 MHz) δ: 1.06–1.09 (t, J = 7.38 Hz, 3H), 2.04 (s, 1H),
2.55–2.60 (q, J = 7.36 Hz, 2H), 3.19–3.24 (m, 1H),
3.35–3.38 (m, 1H), 3.80–3.95 (q, 2H), 4.92–4.94 (dd, J1 =
2.59 Hz, J2 = 2.60 Hz, 1H), 6.97–7.00 (m, 4H), 7.19–7.26
(m, 4H), 10.65 (br s, 1H). 13C NMR(DMSO-d6, 100 MHz)
δ: 13.07, 18.67, 39.14, 40.15, 73.12, 115.10,115.27,
115.47, 123.19, 127.30, 127.36, 130.27, 130.33, 133.35,
138.37, 158.10, 160.85, 161.25, 162.63, 163.20, 164.59.
ESI-MS: m/z 401.5(M–H), C21H20F2N2O2S (402.12).
1.94 (s, 3H), 3.13–3.19 (m, 1H), 3.47–3.52 (m, 1H), 3.69 (s,
3H), 3.80 (s, 2H), 4.72–4.75 (dd, J1 = 4.00 Hz, J2 = 4.40
Hz, 1H), 5.78 (br s, 1H), 6.81–6.85 (m, 2H), 7.15–7.18 (m,
2H), 7.24–7.26 (m, 1H), 7.27–7.29 (m, 4H), 12.51 (br s,
1H). 13C NMR(DMSO-d6, 100 MHz)δ: 10.39, 39.22, 39.43,
54.92, 71.06, 113.74, 114.84, 118.23, 125.83, 127.18,
128.04, 129.60, 130.02, 143.34, 154.32, 157.70, 164.34,
165.75. ESI-MS: m/z 381.9(M–H), C21H22N2O3S (382.14).
2-((2-Hydroxy-2-(4-methoxyphenyl)ethyl)thio)-6-(4-methox-
ybenzyl)-5-methyl-pyrimidin-4(3H)-one (3n). White crys-
tal; yield: 33%; m.p.: 138–139 °C; 1HNMR (DMSO-d6,

Med Chem Res
400 MHz) δ: 1.92 (s, 3H), 3.13–3.18 (m, 1H), 3.42–3.47
(m, 1H), 3.70 (s, 3H), 3.74 (s, 3H), 3.79 (s, 2H), 4.66–4.69
(dd, J1 = 4.00 Hz, J2 = 4.00 Hz, 1H), 5.67 (br s, 1H),
6.82–6.89 (m, 4H), 7.15–7.33 (m, 4H), 12.58 (br s, 1H);
13C NMR(DMSO-d6, 100 MHz)δ: 10.39, 39.23, 39.44,
54.92, 54.99, 70.65, 113.37, 115.21, 127.01, 129.62,
130.04, 136.15, 157.69, 158.39, 159.92, 164.56; ESI-MS:
m/z 411.6 (M–H), C22H24N2O4S (412.15).
4.79–4.83 (m, 1H), 7.23–7.45 (m,5H), 12.71 (br s,1H); 13C
NMR (DMSO-d6, 100 MHz) δ:14.06, 17.40, 25.55, 25.64,
30.68, 31.00, 38.61, 40.27, 71.42, 118.85, 125.84, 127.16,
128.00, 144.48, 158.34, 164.34, 165.00. ESI-MS: m/z 357.2
(M–H), C20H26N2O2S (358.17).
6-Cyclohexyl-5-ethyl-2-((2-(4-fluorophenyl)-2-hydro-
xyethyl)thio)pyrimidin-4(3H)-one (3s). White crystal,
1
yield:39%. m.p.: 158.7–160.6 °C. HNMR(DMSO-d6, 400
2-((2-hydroxy-2-phenylethyl)thio)-6-(4-hydroxybenzyl)-5-
methylpyrimidin-4(3H)-one (3o) White crystal, yield:
25%. m.p.: 148.5–149.2 °C. 1HNMR (DMSO-d6, 400
MHz) δ: 1.89 (s, 3 H), 2.09(s, 1H), 3.10–3.15 (m, 1H),
3.44–3.47 (m, 1H), 3.67 (s, 2H), 4.75–4.77 (dd, J1 = 3.60
Hz, J2 = 4.4 Hz, 1H), 6.65–6.67 (d, J = 8.00 Hz, 2H),
7.02–7.04 (d, J = 8.40 Hz, 2H), 7.24–7.26 (m, 1H),
7.30–7.35 (m, 4H), 9.36 (br s, 1H), 12.48 (br s, 1H). 13C
NMR(DMSO-d6, 100 MHz)δ: 11.28, 39.49, 39.91, 72.58,
114.11, 115.52, 126.34, 127.46, 128.46, 129.25, 129.69,
129.91, 145.19, 156.11, 160.78. ESI-MS: m/z 367.3(M–H),
C20H20N2O3S (368.12).
MHz) δ: 0.95–1.00 (t, J = 7.3 Hz, 3H), 1.19–1.35 (m, 3H),
1.56–1.73 (m, 7H), 2.34–2.41 (m, 2H), 2.62–2.68 (m, 1H),
3.14–3.21(m, 1H), 3.46–3.52(dd, J1 = 3.60 Hz, J2 = 3.90
Hz, 1H), 4.33–4.35(br s, 1H), 4.80–4.84 (m, 1H), 7.13–7.19
(t, 2H), 7.42–7.47 (m, 2H), 12.30 (br s, 1H). 13C NMR
(DMSO-d6, 100 MHz) δ: 14.06, 17.42, 25.64, 30.74, 30.98,
38.60, 40.27, 70.84, 114.56, 114.84, 118.82, 127.76,
127.87, 140.61, 159.68, 162.89, 164.86. ESI-MS: m/z 375.6
(M–H), C20H25FN2O2S (376.16).
6-Cyclohexyl-5-ethyl-2-((2-hydroxy-2-(4-methoxyphenyl)
ethyl)thio)pyrimidin-4(3H)-one (3t) White crystal, yield:
42.0%. m.p.: 137.5–139.1 °C. 1H NMR(DMSO-d6, 400
MHz) δ: 0.95–1.00 (t, J = 7.35 Hz, 3H), 1.34–1.38 (m, 3H),
1.60–1.74 (m, 7H), 2.35–2.42 (m, 2H), 2.64–2.71 (t, 1H),
3.11–3.35 (m, 2H), 3.49–3.55 (dd, J1 = 3.90 Hz, J2 = 3.90
Hz, 1H), 3.74 (s, 3H), 4.74–4.77 (m, 1H), 5.66–5.67 (br s,
1H), 6.89–6.92 (d, J = 8.70 Hz, 1H), 7.32–7.35 (d, J =
8.70 Hz, 2H), 12.46 (br s, 1H). 13C NMR (DMSO-d6, 100
MHz) δ: 14.06, 17.40, 25.55, 25.64, 30.68, 31.00, 38.61,
40.27, 49.36, 71.42, 118.85, 125.84, 127.16, 128.00,
144.48, 158.34, 164.34, 165.00. ESI-MS: m/z 387.4(M–H),
C21H28N2O3S (388.18).
2-((2-hydro-2-(4-hydroxyphenyl)ethyl)thio)-6-(4-hydroxy-
benzyl)-5-methyl-pyrimidin-4(3H)-one (3p). White crystal;
1
yield: 18%; m.p.: 249–249.5 °C; HNMR (DMSO-d6, 400
MHz) δ: 2.24 (s, 3H), 3.70–3.75 (m, 2H), 3.96 (s, 1H),
4.05–4.09 (m, 1H), 5.41–5.43 (dd, J1 = 3.82 Hz, J2 = 4.74
Hz, 1H), 7.14–7.26 (m, 4H), 7.39–7.41 (d, J = 3.40 Hz,
2H), 7.56(s, 1 H), 7.69–7.71 (d, J = 8.45 Hz, 2H), 8.71 (s,
1H), 11.48 (br s, 1H). 13C NMR(DMSO-d6, 100 MHz)δ:
11.10, 40.47, 40.65, 72.61, 115.89, 116.19, 116.41, 123.84,
128.09, 129.37, 130.56, 157.60, 158.43, 158.66, 162.03,
164.83. ESI-MS: m/z 383.5(M–H), C20H20N2O4S (384.11).
5-Ethyl-2-((2-hydroxy-2-phenylethyl)thio)-6-phenylpyrimi-
2-((2-hydro-2-(4-methoxyphenyl)ethyl)thio)-6-(4-hydroxy-
din-4(3H)-one (3u). White crystal; yield: 39.46%. m.p.:
benzyl)-5-methyl-pyrimidin -4(3H)-one (3q). White crys-
133.0–134.4 °C.
1HNMR(DMSO-d6,
400 MHz)
1
tal, yield: 23%. m.p.: 230–230.6 °C. HNMR (DMSO-d6,
δ:1.00–1.05 (t, J = 7.32 Hz, 3H), 2.31–2.38 (m, 2H),
3.12–3.19 (m, 1H), 3.50–3.55 (dd, J1 = 3.90 Hz, J2 = 3.60
Hz, 1H), 4.46(br s, 1H), 4.80–4.84 (m, 1H), 7.19–7.33 (m,
5H), 7.46–7.49 (m, 5H), 12.47 (br s, 1H); 13C NMR
(DMSO-d6, 125 MHz) δ: 13.25, 19.30, 38.60, 120.95,
125.79, 127.11, 127.94, 128.14, 128.66, 138.39, 143.12,
144.16, 162.00, 163.25. ESI-MS: m/z 351.3(M–H),
C20H20N2O2S (352.12).
400 MHz) δ: 2.25 (s, 3H), 3.65 (s, 3H), 3.68–3.70 (m, 2H),
3.93 (s, 2H), 4.02–4.06 (m, 1H), 5.39–5.42 (dd, J1 = 3.76
Hz, J2 = 4.74 Hz, 1H), 7.04–7.05 (d, J = 8.55 Hz, 2H),
7.16–7.17 (d, J = 8.38 Hz, 2H), 7.39–7.41 (d, J = 8.31 Hz,
2H), 7.69–7.70 (d, J = 8.52 Hz, 2H), 8.71 (s, 1H), 12.51 (br
s, 1H). 13C NMR(DMSO-d6, 100 MHz)δ: 11.09, 40.47,
40.54, 55.21, 72.37, 114.25, 115.91, 116.41, 127.85,
129.37, 130.56, 137.52, 157.60, 158.60, 159.56, 162.07,
164.83. ESI-MS: m/z 397.5 (M–H), C21H22N2O4S (398.13).
5-Ethyl-2-((2-(4-fluorophenyl)-2-hydroxyethyl)thio)-6-phe-
nylpyrimidin-4(3H)-one (3v). White crystal, yield:47.90%.
m.p.: 168.8–170.1 °C. 1HNMR(DMSO-d6, 400 MHz) δ:
1.00–1.04 (t, J = 7.3 Hz, 3H), 2.30–2.37 (m, 2H),
3.13–3.21 (m, 1H), 3.46–3.51 (dd, J1 = 3.90 Hz, J2 = 3.90
Hz, 1H), 4.47(s, 1H), 4.81–4.85 (m, 1H), 7.04–7.10 (t, 2H),
7.32–7.36 (t, 2H), 7.49 (m,5H), 12.66(br s,1H); 13C NMR
(DMSO-d6, 100 MHz) δ: 13.22,19.28, 38.21, 70.39, 114.47,
6-cyclohexyl-5-ethyl-2-((2-hydroxy-2-phenylethyl)thio)pyri-
midin-4(3H)-one (3r). White crystal, yield: 42%. m.p.:
135.6–137.1 °C. 1HNMR (DMSO-d6, 400 MHz) δ:
0.94–0.99 (t, J = 7.28 Hz, 3H), 1.17–1.35(m, 3H),
1.57–1.73(m, 7H), 2.35–2.42 (m, 2H), 2.63–2.70 (m, 1H),
3.10–3.17 (m, 1H), 3.52–3.58 (m, 1H), 4.33–4.35(br s, 1H),

Med Chem Res
114.76, 127.70, 127.80, 128.31, 128.68, 138.39, 140.28,
159.64, 162.85. ESI-MS: m/z 369.2(M–H), C20H19FN2O2S
(370.12).
brief, the test samples were prepared at different con-
centrations. After Huh7.5.1 cells had been seeded in a 96-
well microplate for 4 h, the samples (20 μL) were placed in
each well and incubated for 3 days at 37 °C; then, 0.1 mL
MTT was added for 4 h. After removal of the MTT med-
ium, DMSO (100 μL/well) was added onto the microplate
for 10 min. The formazan crystals were dissolved, and the
UV absorbance was measured on a microplate reader at 490
nm.
5-Ethyl-2-((2-hydroxy-2-(4-methoxyphenyl)ethyl)thio)-6-
phenylpyrimidin-4(3H)-one (3w). White crystal, yield:
49.65%. m.p.:141.2–142.6 °C. 1HNMR(DMSO-d6, 400
MHz) δ: 0.98–1.08 (t, J = 7.32 Hz, 3H), 2.30–2.37 (m, 2H),
3.12–3.19 (m, 1H), 3.41–3.48 (m, 2H), 3.72 (s, 3H),
4.74–4.78 (m, 1H), 6.81–6.83 (d, J = 8.40 Hz, 2H),
7.22–7.25(d, J = 9.60 Hz, 2H), 7.44–7.50(m, 5H), 12.47(br
s,1H). 13CNMR(D MSO-d6, 100 MHz) δ: 13.28, 19.31,
38.89, 54.96, 70.76, 113.30, 126.98, 128.12, 128.59,
136.21, 137.63, 138.82, 158.35, 160.36 ESI-MS: m/z 381.2
(M–H), C21H22N2O3S (382.14).
Molecular modeling analysis
Molecular docking studies were performed using Auto-
Dock4.2 program (Huey et al. 2007). The X-ray crystal
structure of NS5B polymerase in complex with 1NV (PDB
code: 4JTY), with HJZ (PDB code: 3MF5), with T18 (PDB
code: 3G86), with 1PV (PDB code: 4KAI) downloaded
from the Protein Data Bank (PDB) at the Research Colla-
boration for Structural Bioinformatics (http://www.rcsb.org/
pdb/home/home.do). Auto Dock Tools 1.5.4 were used to
prepare the molecules and proteins for docking. The bound
ligand and all water molecules were removed from the
protein. Gasteiger charges were assigned to both protein and
ligands. A grid with spacing of 0.375 Å and 60 × 60 × 60
points in the x, y and z axes was built, and centered on the
center of mass of the bundling and in the crystal structure.
Energy grid maps for all possible ligand atom types were
calculated before performing docking. While keeping the
protein structure rigid, we selected Lamarckian genetic
algorithm to search the conformational and orientational
space of the ligands. For each small molecule, 100 separate
docking calculations were performed with the following
settings of parameters: population size of individuals at 350,
a maximum number of 25 million energy evaluations, a
maximum number of generations of at 27,000, a mutation
rate of 0.02, a crossover rate of 0.8, and an elitism value
of 1. All other docking parameters were set to default. After
docking, cluster analysis was performed on the results with
the root-mean-square deviations less than1.0 Å. The best-
docked conformation was then selected as the lowest energy
pose in the most populated cluster.
In vitro Anti-HCV activity assays
Inhibitor preparation For the inhibitory activity assays, the
obtained compounds 3a–w and 2b were dissolved in
dimethyl sulfoxide (DMSO), followed by serial dilution at
different concentration. The blank DMSO was used as
negative control. The commercial ribavirin was chosen as
the positive control.
Cell line and cell culture
Stocks of infectious HCV J6/JHH-1 viral particles were
generated, as previously described (Lindenbach 2005) and
aliquoted for storage at −80 °C. The HCV load (RNA
copies) in stocks was measured with real-time quantitative
RT-PCR.
Anti-HCV assay in vitro (Li et al. 2012)
Anti-HCV assay was carried out in clear bottom 96-well
plates. Huh7.5.1 cells (Cell Bank of the Chinese Academy
of Sciences) were seeded at a density of 1.5 × 104 cells/well
in 100 μL of Dulbecco's modified Eagle's medium (DMEM)
and incubated overnight, and virus stocks were added into
the wells and incubated for additional 8 h. Before the
samples were added, the supernatants were removed and
cells were washed with completed medium for five times.
The samples or controls were serially diluted in DMEM and
then added to the appropriate wells. After incubation for
3 days, the supernatants were collected to determine their
viral load with real-time quantitative RT-PCR.
Results and discussion
Chemistry
The general synthetic route utilized to obtain the desired
compounds 3a–w is outlined in Scheme 1. Following the
procedure described previously (Wu et al. 2013), the key
intermediate β-ketoesters 5a–i were prepared by exposure
of various acid 4a–g to 1,1′-carbonyl-diimidazole (CDI)
followed by treatment with different ethyl potassium
Cytotoxicity assay
The toxicity of the compounds was assayed by a modified
means 3-(4,5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-
tetrazolium bromide (MTT) method (Mosmann 1983). In

Med Chem Res
Scheme1 Synthesis of
compounds 3a–w. Reagents and
conditions were: (a) (i) CDI,
CH3CN, rt, 1 h; (ii) R2CH
(CO2Et)(CO2K), Et3N,
anhydrous MgCl2, rt, overnight,
then refluxed for 6 h; (iii) 13%
HCl; (b) thiourea, EtONa,
EtOH, refluxed for 6–8 h; (c)
R3PhCOCH2Br, K2CO3, DMF,
rt, 12–24 h; (d) NaBH4, THF/
MeOH, ice water, 3–5 h
malonates in the presence of anhydrous MgCl2 and Et3N
(Ronald et al. 1993). Condensation of 5a–i with thiourea in
the presence of EtONa in refluxing EtOH led to substituted
uracil 6a–I, which was subjected to S-alkylation in anhy-
drous DMF with appropriate halide (R3PhCOCH2Br) in the
presence of K2CO3 to afford 7a–w (Huey et al. 2007).
Treatment of 7a–w with NaBH4 in anhydrous THF and
MeOH under ice water to gain the desirable target com-
pounds 3a–w, respectively. Both analytical and spectral
data of all the designed compounds are in full agreement
with the proposed structures.
anti-HCV activity was about 77-fold higher than that of
Ribavirin. Notably, this compound also exhibited a higher
selectivity index than Ribavirin. In addition to compound
3a, compounds 3b, 3c, 3i and 3j also showed higher anti-
HCV potency (EC50 = 2.02, 0.27, 2.22, 1.64 μM, respec-
tively) and better selectivity index (SI = 97, 22, 52, 56,
respectively) than what found for Ribavirin (SI = 9).
Having identified the leading compound 1, we sought to
explore the potential effect of variation in the position of
C-2, C-5 and C-6 of the pyrimidine ring. As indicated in,
introduction of fluorine atom or hydroxyl group at the para-
position of phenyl of C-2 side chain was disadvantageous
for the activity of inhibitors. For example, substitution of
the hydrogen atom (compound 3a) with fluorine atom
(compound 3b) demonstrated a 15-fold lower activity. This
is in agreement with the compounds 3d/3e, 3i/3j, 3l/3m. As
we can see from Table 1, substitution at this position with
methoxyl group showed two phenomenon: (1) If the C-6
position of pyrimidine ring is the cyclohexyl-methyl or 4′-
fluorine-benzyl, introduction of methoxyl group (com-
pounds 3f, 3k) at the para-position of phenyl of C-2 side
chain resulted a decrease of inhibitory activity (such as
compounds 3f/3e, 3k/3j); (2) If the C-6 position of pyr-
imidine ring is the 4′-methoxyl-benzyl, Cyclohexyl or 4′-
hydroxyl-benzyl, slight enhancement of the activity could
be achieved (3n/3m, 3t/3r, 3q/3o).
Anti-HCV activity
The newly synthesized 2-hydroxylphenethyl sulfanyl-oxo-
pyrimidine analogs 3a–w were evaluated for their cyto-
toxicities andanti-HCV activities in Huh 7.5.1 cells, with
ribavirin as positive control. The result activity data are
listed in Table 1, interpreted in CC50 values (cytotoxicities),
EC50 values (anti-HCV activities) and SI (selectivity index,
given by the CC50/EC50 ratio). In addition, compound 2b
reported by Ding et al. (Ding et al. 2006) was included in
our assays for comparison.
As showed in Table 1, the EC50 value of compound 2b in
our cell assay is 2.4-fold higher than the IC50 value in
enzyme (HCV NS5B RdRp) assay reported by Ding et al.
(Ding et al. 2006). Nine of the tested compounds exhibited
moderate efficiencies in inhibiting the replication of the
HCV with EC50 values in the range from 0.13 to 7.53 μM,
which are better than that of compound 2b (EC50 = 9.04
μM) and Ribavirin (EC50 = 10.35 μM). Among them, 6-
(cyclohexyl-methyl)-5-ethyl-2-((2-hydroxy-2-phenylethyl)
thio)pyrimidin-4(3 H)-one (3a) turned out to be the most
potent inhibitor, with an EC50 of 0.13 μM, against HCV. Its
The substituent at C-5 position of the pyrimidinone core
essentially plays a vital role in the antiviral activity of novel
compounds. The investigation revealed that an ethyl to
methyl group change at the C-5 position would result a drop
in the activity significantly, with 3e being 34-fold less
potent than 3a. This effect can also be found in compounds
3h/3i and 3d/3b. It should to be noted that the substituent at
C-5 position of the pyrimidinone core also strongly

Med Chem Res
Table 1 Antiviral activity of compounds against HCV in Huh 7.5.1cells
Compd
R1
R2
R3
CC50a(µM)
EC50b(µM)
SIc
3a
Cyclohexyl–CH2–
Et
H
15.67 0.73
0.13 0.02
121
3b
3c
Cyclohexyl–CH2-
Cyclohexyl–CH2-
Cyclohexyl–CH2-
Cyclohexyl–CH2-
Cyclohexyl–CH2-
PhenylCH2–
Et
4′-F
195.91 7.37
6.05 0.12
2.02 0.06
0.27 0.05
16.35 0.43
4.43 0.20
11.40 0.58
20.57 1.79
10.51 1.47
2.17 0.076
1.64 0.12
13.41 0.55
11.17 0.88
7.53 0.40
6.28 0.49
18.78 0.81
31.29 2.03
5.65 0.44
22.10 1.03
9.80 1.08
10.52 1.20
21.82 1.12
19.98 1.69
29.65 1.66
9.04 0.10
10.35 0.68
97
22
6
Et
4′-OH
4′-F
3d
3e
Me
Me
Me
Me
Me
Et
103.06 0.44
30.61 0.82
55.30 1.76
72.05 1.58
52.41 205
H
7
3f
4′-OCH3
H
5
3g
4
3h
3i
4′-FphenylCH2–
4′-FphenylCH2–
4′-FphenylCH2–
4′-FphenylCH2–
4′-OCH3 phenylCH2–
4′-OCH3 phenylCH2–
4′-OCH3 phenylCH2–
4′-OH phenylCH2–
4′-OH phenylCH2–
4′-OH phenylCH2–
Cyclohexyl-
4′-F
5
4′-F
112.87 2.95
92.03 1.67
183.99 7.06
127.06 3.21
77.45 1.50
74.94 1.72
48.42 1.07
83.12 1.71
119.13 8.01
261.01 2.66
273.42 1.08
237.59 2.32
316.24 4.31
380.23 1.87
287.37 5.22
312.75 2.69
92.91 2.10
52
56
14
11
10
12
3
3j
Et
H
3k
3l
Et
4′-OCH3
4′-F
Me
Me
Me
Me
Me
Me
Et
3m
3n
3o
H
4′-OCH3
H
3p
3q
3r
4′-OH
4′-OCH3
H
3
21
12
28
23
14
19
10
35
9
3s
Cyclohexyl–
Et
4′-F
3t
Cyclohexyl–
Et
4′-OCH3
H
3u
3v
Phenyl
Et
Phenyl
Et
4′-F
3w
2b
Ribavirin
Phenyl
Et
4′-OCH3
All data represent mean values for three separate experiments
a
Cytostatic concentration required to reduce Huh7.5.1 cell proliferation by 50% tested by MTT method
Effective concentration required to protect Huh7.5.1 cell against the cytopathogenicity of HCV by 50%
Selectivity index: ratio IC50/EC50, a high SI means a more selective compound
b
c
influenced the cytotoxicity. Compounds with a ethyl group
activity (EC50 = 0.006, 0.086 and 0.003 μM, respectively)
without any cytotoxicity (CC50 > 200 μM) (Wu et al. 2013).
This information will provide better guidance in future
structural designs on our research to identify novel, and
more effective agents for HCV or as double targets inhi-
bitors of HCV and HIV.
had lower cytotoxicity than compounds with an methyl
group, which could be clearly reflected in compounds 3b
vs. 3d, and 3i vs. 3h.
As indicated by pervious SAR study of 5-cyano-6-aryl-2-
thiouracil compounds, the substituent at the C-6 position of
the pyrimidine ring plays an important role in their
anti-HCV activities. In this paper, C6-substitutions were
studied and revealed the potency of the cyclohexylmethyl
group present in the most active compound 3a. Substituted
benzyl groups seemed to retain some activities. Conversely,
cyclohexyl or phenyl groups led to a dramatic drop of
activity, for example, 6-cyclohexylderivative 3r and
6-phenylderivative 3u were about 170-fold and 167-
Molecular modeling analysis
Since structurally related 5-cyano-6-aryl-2-thiouracil deri-
vative (e.g., compound 2b) are active against HCV NS5B
RdRp; it is possible that our novel scaffolds could also
target the HCV NS5B RdRp. In order to investigate the
potential binding mode of these new prymidine derivatives
to HCV NS5B, we carried out docking analysis using the
best active compound 3a with different complexes related to
four allosteric binding sites (“Thumb” Pockets I, and II and
“Palm” Pockets I and II/III) of the HCV NS5B RdRp (PDB
fold
less
active
than
the
corresponding
6-
cyclohexylmethylcounterpart 3a. It is interesting to note
that when tested against wt HIV-1, the C6-cyclohex-
ylmethyl derivatives 3a, 3b and 3c displayed nanomolar

Med Chem Res
Fig. 2 Superimposition of the
docked conformations of 3a
(amaranth) and quinolinediones
inhibitor T18 (green) in the
palm I allosteric site. The
docking results are showed by
DS3.5. The amino acid residues
important for binding
interactions are labeled. Dotted
lines show the interactions
between NS5B RdRp and 3a or
T18 (color figure online)
code: 4JTY, 3MF5, 3G86, 4KAI, correspond to the four
allosteric binding sites, respectively). The relatively more
negative binding energy in palm I allosteric site vs. other
pockets indicated a better fit of compound 3a in “Palm”
Pockets I. The docking mode of 3a to the palm I allosteric
site compared with T18 (the bound inhibitor within
cocrystal 3G86) are shown in Fig. 2.
were synthesized and their activities of anti-HCV were
evaluated. Results showed that nine compounds showed
anti-HCV activities with an EC50 range from 7.53 to 0.13
μM, which were more active than the reference drug riba-
virin and HCV NS5B inhibitor 2b reported previously. The
C6-cyclohexyl-methyl derivatives 3a, 3b and 3c were
identified as potent inhibitors of HCV, with EC50 values of
0.13, 2.02 and 0.27 μM respectively. Meanwhile, these
compounds also exhibited an excellent activity of anti-HIV-
1, with EC50 values of 0.006, 0.086 and 0.003 μM,
respectively, thus becoming attractive candidates for further
development of new HCV and HIV-1 specific inhibitors
with a good antiviral profile. Moreover, since structurally
related 5-cyano-6-aryl-2-thiouracil derivatives are active
against HCV NS5B RdRp, it is possible that our novel
scaffolds could also target at the HCV NS5B RdRp. The
molecular modeling studies predicted their binding sites in
pulm I of NS5B. Further mechanism studies on this class of
compounds are undergoing and will be reported upon
accomplishment.
As we can see from Fig. 2, both compounds satisfy the
large hydrophobic pocket of the palm I allosteric site, while
the p-fluorobenzyl moiety for T18 and the cyclohexyl group
for 3a reach the bottom of the hydrophobic pocket, making
close hydrophobic contact with the side chain of Pro197
and Tyr415. Both the pyrimidine 4-carbonyl of 3a and the
quinolinedione oxy anion of T18 made a hydrogen bonding
interaction with the N–H of the backbone residue Tyr448.
Interestingly for 3a, the core prymidine ring engages in a
classic edge-to-face π-stack with the side chain of Tyr448.
In the X-ray co-crystal structure of T18, T18 directly
interacts with the side chain of one of the key catalytic
aspartates, Asp318, in a hydrogen bond via the sulfonamide
NH. Although the key H-binding interaction found between
T18 and the Asp318 is absent from 3a, another H-binding is
observed between the C2ß–OH of compound 3a and the
backbone C=O of Asp319, which is also beneficial to the
combination of 3a and the enzyme.
Acknowledgements This research was supported by the National
Natural Science Foundation of China (Grant No. 21262044 and
81260248).
Compliance with ethical standards
Conclusion
In summary, a series of novel dihydro-hydroxylphene-thyl-
Conflict of interest The authors declare that they have no competing
sulfanyl-6ω-cyclohexyl/phenyl-oxopyrimidine derivatives
interests.

Med Chem Res
Huey R, Morris GM, Olson AJ (2007) A semiempirical free energy
force field with charge-based desolvation. J Comput Chem
28:1145–1152
Lavanchy D (2011) Evolving epidemiology of hepatitis C virus. Clin
Microbiol Infect 17:107–115
Li HM, Tang YL, Zhang ZH, Liu CJ, Li HZ, Li RT, Xia XS (2012)
Compounds from arnebia euchroma and their related anti-HCV
and antibacterial activities. Planta Med 78:39–45
Lim TR, Tan BH, Mutimer DJ (2014) Evolution and emergence of a
new era of antiviral treatment for chronic hepatitis C infection. Int
J Antimicrob Agents 43:17–25
Lindenbach BD (2005) Complete replication of hepatitis C virus in
cell culture. Science 309:623–626
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63
Ronald C, Thomas C, Gregory K, James W (1993) A safe, economical
method of the preparation of β–oxo esters. Synthesis 3:290–292
Sofia MJ, Chang W, Furman PA, Mosley RT, Ross BS (2012)
Nucleoside, nucleotide, and non-nucleoside inhibitors of hepatitis
C virus NS5B RNA-dependent RNA-Polymer-ase. J Med Chem
55:2481–2531
References
Alexandra A, Peter K (2015) Interferon-based combination treatment
for chronic hepatitis C in the era of direct acting antivirals. Ann
Gastroenterol 28:55–65
Arataki K, Kumada H, Toyota K, Ohishi S, Takahashi S, Tazuma S,
Chayama K (2006) Evolution of hepatitis C virus quasispecies
during ribavirin and interferon-alpha-2b monotherapy. Inter-
virology 49:352–361
Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW (1989) Iso-
lation of a cDNA clone derived from a blood-borne non-A, non-B
viral hepatitis genome. Science 244:359–362
Ding YL, Girardet JL, Smith KL, Larson G, Prigaro B, Wu JZ, Yao
NH (2006) Parallel synthesis of 5-cyano-6-aryl-2-thiouracil
derivatives as inhibitors for hepatitis C viral NS5B RNA-
dependent RNA polymerase. Bioorg Chem 34:26–38
Fauvelle C, Lepiller Q, Felmlee DJ, Fofana I, Habersetzer F, Stoll-
Keller F, Baumert TF, Fafi-Kremer S (2013) Hepatitis C virus
vaccines-progress and perspectives. Microb Pathog 58:66–72
Ferenci P, Fried MW, Shiffman ML, Smith CL, Marinos G, Gonca-
lesJr FL, Haussinger D, Daigo M, Carosi G, Dhumeaux D, Craxi
A, Chaneac M, Reddy KR (2005) Predicting sustained virological
responses in chronic hepatitis C patients treated with peginter-
feron alfa-2a (40KD)/ribavirin. J Hepatol 43:425–433
Ghany MG, Strader DB, Thomas DL, Seeff LB (2009) Diagnosis,
management, and treatment of hepatitis C: an update, hepatology.
Hepatology 49:1335–1374
He YP, Long J, Zhang SS, Li C, Lai CC, Zhang CS, Li DX, Zhang
DH, Wang H, Cai QQ, Zheng YT (2011) Synthesis and biolo-
gical evaluation of novel dihydro-aryl/alkyl sulfanyl-cyclohexyl-
methyl-oxopyrimidines (S-DACOs) as high active anti-HIV
agents. Bioorg Med Chem Lett 21:694–697
Summa V, Petrocchi A, Matassa VG, Taliani M, Laufer R, De
Francesco R, Altamura S, Pace
P (2004) HCV NS5b
RNA-dependent RNA polymerase inhibitors: from α,γ-Diketoa-
cids to 4,5-Dihydroxypyrimidine- or 3-Methyl-5- hydro-
xypyrimidinonecarboxylic acids. Design and synthesis. J Med
Chem 47:5336–5339
Wu DC, Zhuang DM, Liu XF, Lu LH, Wang H, Li C, Lai CC, Li JY,
He YP (2013) Synthesis and biological evaluation of novel
hydroxyphenethyl-S-DACOs as high active anti-HIV agents. Lett
Drug Des Discov 10:271–276