Design and synthesis of 6-oxo-1,4,5,6-tetrahydropyrimidine-5-carboxylate derivatives as neuraminidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.06.059

A series of 6-oxo-1,4,5,6-tetrahydropyrimidine-5-carboxylate derivatives were prepared to evaluate their ability of inhibiting neuraminidase (NA) of influenza A virus. All the compounds were synthesized in good yields starting from aldehyde by using a suitable synthetic strategy, which showed moderate inhibitory activity against influenza A NA. Compound 6g exhibited the strongest inhibitory activity against influenza virus A NA (IC₅₀ = 17.64 μM), which indicated pyrimidine ring could be used as a core structure to design novel influenza NA inhibitors.

Full text of DOI:10.1016/j.ejmech.2014.06.059

European Journal of Medicinal Chemistry 83 (2014) 466e473
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of 6-oxo-1,4,5,6-tetrahydropyrimidine-5-
carboxylate derivatives as neuraminidase inhibitors
Jun Lou, Xiaoyan Yang, Zhigang Rao, Wenwen Qi, Jinhui Li, Haiyu Wang, Yuxi Li, Jinping Li,
Zhiming Wang, Xianming Hu, Peng Liu*, Xuechuan Hong*
State Key Laboratory of Virology, Ministry of Education, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University School of
Pharmaceutical Sciences, 185# Donghu Rd, Wuhan 430071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 6-oxo-1,4,5,6-tetrahydropyrimidine-5-carboxylate derivatives were prepared to evaluate their
ability of inhibiting neuraminidase (NA) of influenza A virus. All the compounds were synthesized in
good yields starting from aldehyde by using a suitable synthetic strategy, which showed moderate
inhibitory activity against influenza A NA. Compound 6g exhibited the strongest inhibitory activity
Received 28 February 2014
Received in revised form
4 June 2014
Accepted 25 June 2014
Available online 26 June 2014
against influenza virus A NA (IC50 ¼ 17.64
mM), which indicated pyrimidine ring could be used as a core
structure to design novel influenza NA inhibitors.
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Neuraminidase inhibitors
Anti-influenza
6-oxo-1,4,5,6-tetrahydropyrimidine-5-
carboxylate derivatives
SAR
1. Introduction
However, the generation and circulation of oseltamivir-resistant
seasonal influenza mutants such as H5N1 avian influenza, require
the development of new NAIs to fight against potential human
influenza pandemic [13e15].
The influenza virus, avian influenza A (H5N1) virus in particular,
is a great threat to human health potentially causing serious public
health and economic problems [1]. Person to person transmission is
very rare, however genetic reassortment resulting in new virulent
subtype of avian influenza virus has made the prospect of a new
pandemic particularly alarming [2e5]. Influenza viruses contain
two major membrane glycoproteins, hemagglutinin (HA) and
neuraminidase (NA). NA is one of the surface glycoproteins of the
influenza virus, which catalyzes the release of newly formed virions
from infected cells, and is considered as an important target for
anti-influenza drugs design [6e8].
Until now three neuraminidase inhibitors have been approved
for the treatment of influenza infections: zanamivir1, oseltamivir2
and peramivir3 (Fig. 1) [9]. Zanamivir1 and Peramivir3 are rarely
used because of their low bioavailability and rapid elimination.
Currently oseltamivir2 is the only orally available drug effective
against influenza virus which is world widely used [10e12].
According to the “air plane” model of NA active site (Fig. 2)
proposed by Wang et al. [16], there are ten conserved residues and
four water molecules in the NA active site, which is divided into
four binding sites. Site 1 consists of Arg118, Arg292, and Arg371
which are positively charged to interact e with the carboxylic acid
of e NA inhibitors. Site 2 consists of Glu119, Glu227 and Asp151
forming a negatively charged area to interact with base groups such
as guanidine or amino. Site 3 is a hydrophobic pocket formed by the
side chains of Trp178 and Ile222, which mainly interacts with acetyl
group. Sited 4 consists of Glu276 and Glu277, which bind to hy-
drophobic side chains.
Pyrimidine is an important heterocyclic compound, which
shows a wide variety of pharmacological properties. Many pyrim-
idine derivatives have been reported to have the properties of anti-
HCV (hepatitis
example, 2-amino-6-hydroxy-3H-pyrimidine-4-one has been
demonstrated to be wide spectrum anti-bacterial reagent
C virus), anti-bacterial, and anti-cancer. For
a
[17e19]. This inspired us to explore novel pyrimidine derivatives as
* Corresponding authors.
NA inhibitors.
E-mail address: liupengwhu@whu.edu.cn (P. Liu).
http://dx.doi.org/10.1016/j.ejmech.2014.06.059
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

J. Lou et al. / European Journal of Medicinal Chemistry 83 (2014) 466e473
467
Fig. 1. The chemical structure of three NA inhibitors (NAIs) approved by FDA: zanamivir, oseltamivir, peramivir.
Based on the structureeactivity relationship (SAR) of NA we
devised and synthesized two series of pyrimidine analogs, 2-
amino-6-oxo-1,4,5,6-tetrahydropyrimidine-5-carboxylate (5ae5p)
and 2-acetamino-6-oxo-1,4,5,6-tetrahydropyrimidine-5-carboxy
late (6ae6r). All the newly synthesized compounds were charac-
terized by 1H NMR, 13C NMR, and MS spectrometry. The overall
strategies for the synthesis of 5ae5p and 6ae6r are outlined in
Scheme 1. The NA inhibitory activity was evaluated in vitro.
trans) illustrated by the 1H NMR characterization. Differentiation
between the signals of trans/cis is clear (trans J5, 6 ¼ 10.8 Hz, cis J5,
¼ 4.0 Hz). Usually, a para-substituted and small group in ortho
6
compound was obtained stereo selectively as the trans isomer. The
stereo selectively of all the compounds were trans isomer [28].
Compound 6 was produced in excellent yield through compound 5
further reacting with acetic anhydride in dichloromethane at room
temperature, using DMAP as catalyst.
2. Chemistry
3. Results and discussion
The procedure of synthesizing compounds 6ae6r was outlined
as Scheme 1: A simple procedure that achieves a high compound
yield. Piperidine and acetic acid were used as a catalyst and toluene
as a water removed reagent. Diethyl malonate reacted with
different aldehydes under the refluxing condition, affording com-
pound 4. This reaction was favorable due to the withdraw-electron
group in the benzene ring. The desired product 5, with yield of
61%e84%, was obtained by treating compound 4 with guanidine
hydrochloride in the presence of sodium ethoxide under the con-
dition of refluxing ethanol for 10 h. The cyclization appears to be
initiated via Michael addition of guanidine to a double bond and
then to complete the cyclization with an ester group. Substituents
on the phenyl ring influenced the trans-cis relationship between
the 5-proton and the 6-proton of the dihydropyrimidone skeleton,
the ratio of trans/cis increased from ortho substitution to para (only
The neuraminidase inhibitory activities of target compounds
were evaluated in vitro. As depicted in Table 1, thirty four com-
pounds displayed the NA inhibitory activity with IC50 value from
17.64 to more than 200
better inhibitory activities (IC50 ¼ 17.64e36.67
(Table 1). Compound 6g (IC50 ¼ 17.64 M) had the best perfor-
mM. Compound 6c, 6d, 6e and 6g exhibited
mM) than others
m
mance. In Table 1, we show that the compound 6 series with
acetamide group in C-2 had better inhibitory activities than the
series of compound 5 with amino group. According to SAR acet-
amide group possesses stronger affinity than the amino group
when binding to the hydrophobic pocket.
In order to better understand the activity of this series of
compounds we used 3D models to simulate the interactions be-
tween compound 6g and N1 (PDB entry code: 2HU0) in an open
form based on the docking stimulation (Fig. 3). As mentioned
before, the active site of NA can be divided into four different
binding areas, which are the potential targets for designing NA
inhibitors. Fig. 3 indicates that compound 6g adopts a similar
binding mode in active site as zanamivir or oseltamivir. The
carboxyl group at C-5 forms tight salt-bridge with three arginine
residues consisting of Arg118, Arg292 and Arg371. The carbonyl
group at C-4 also interacts with two amino residues (Arg371 and
Arg292) by hydrogen-bond. Acetamide group in C-2 can bind with
a small hydrophobic region that was formed from the side chains of
Trp178 and Ile222 adjacent to Arg152. The benzene ring with
different substituent groups at C-6 binds with active sites through
hydrophobic bond.
Compounds 6e, 6g and 6o had better inhibitory activities than
6l, 6h and 6j. This may be attributed to the ortho position sub-
stituent group of benzene which can occupy the hydrophobic
pocket of active site better. Comparing compound 6be6e, we find
that F and Cl in benzene ring have little effects on the NA inhibi-
tory activity. Compound 6m with acetamide group at C-2 has
better inhibitory activity than 6q with propionamide group, since
the hydrophobic cavity is narrow and there is no space for large
groups to fit into this area. Compound 6a, 6f and 6n do not have
good activities because these compounds have no potent hydro-
phobic interaction with proteins. Compared to oseltamivir, the
anti-virus activity of compound 6g without base group at C-6
decreased dramatically. This suggests the base group plays an
important role.
Fig. 2. “Air plane' model of the NA active site”.

468
J. Lou et al. / European Journal of Medicinal Chemistry 83 (2014) 466e473
Scheme 1. Reactions and conditions:(a) piperidine, acetic acid, toluene, 69e82%; (b) sodium ethoxide, guanidine hydrochloride, ethanol, 61%e84%; (c) acetic anhydride, DMAP,
CH2Cl2, 68e89%.
4. Data and molecular modeling
trap mass analyzer. Melting points were obtained on a XT-4
apparatus.
Auto-Dock was applied to study molecular docking. The crystal
structure of NA was retrieved from RCSB Protein Data Bank (PDB
entry code: 2HU0). The protein structure was utilized in subse-
quent docking experiments without energy minimization. All li-
gands and water molecules were removed first, and the polar
hydrogen atoms then added. Automatic docking was employed.
Other parameters were established using the default values in the
software. All molecules were minimized using default parameters
[20e22].
Molecular modeling experiments (Fig. 3a and 3b) indicate that
carboxyl group at C-5 of compound 6g interacts with three arginine
residues (Arg118, Arg292, Arg371) of NA (Fig. 3). Acetamido group
at C-2 and benzene at C-6 interacts with other amino acids (Arg152,
Glu119, Asp151 and Ile222). Carbonyl group at C-4 interacts with
two amino residues (Arg371 and Arg292). The inhibitor activity can
be mainly attributed to the interactions between carboxyl group
and three arginine residues by electrostatic forces.
6.2. Neuraminidase inhibition assay
All the target compounds were tested for their NA inhibitory
activity in vitro. For comparison, oseltamivir as a standard inhibitor
(see in Supporting information Table 2). The influenza virus (H1N1),
which was donated by Chinese Centers for Disease Control was
used as source of NA. The in vitro assay is based on the method
reported by Duand coworkers. The NA from influenza virus (H1N1)
was obtained by the method described by Laver et al. [23]. Values
for the IC50 were measured by a spectrofluorometric technique, and
20-(4-methylumbelliferyl)-
a-D-acetylneuraminic acid (MUNANA)
was used as substrate and cleavage of this substrate by NA produces
a fluorescent product, which can emit an emission wavelength of
450 nm with an excitation wavelength of 360 nm. The intensity of
fluorescence can reflect the activity of NA sensitivity [24,25].
The assay mixture contained 10
concentrations, there were 30 L of the enzyme in 32.5 mM MES
buffer (pH 3.5), 10 L of 4 mM CaCl2, 20 L of MUNANA, and 30
water in a 96-well microplate. After 1 h at 37 ꢀC,150
L of 0.014 mM
mL of inhibitors at various
m
m
m
mL
5. Conclusion
m
sodium hydroxide in 83% ethanol was added to 0.1 mL of the re-
action mixture to terminate the reaction. A blank was run with the
same substrate solution with no enzyme. Fluorescence was read
using an Aminco-Bowman fluorescence spectrophotometer and
substrate blanks were subtracted from the sample readings. The
IC50 was calculated by plotting percent inhibition versus the in-
hibitor concentration (the curve line of IC50 was see in Supporting
information, Fig. 4), and determination of each point was per-
formed in duplicate [24].
In summary, we have designed and synthesized 6-oxo-1,4,5,6-
tetrahydro-pyrimidine-5-carboxylate analogs as NA inhibitors.
Most of the compounds had potent influenza NA inhibitory ac-
tivities. In particular, compounds 6c, 6d, 6g showed inhibitory
activity with IC50 about 30
mM (see in the Supporting
information, Tables 3e5). We also show that 5-carboxylate
group, 2-acetamino group and the substituted benzene of the
compounds are essential for the inhibitory activities. The sub-
stituent group at ortho position of benzene had better NA
inhibitory activities than the substituent group at other position.
In this study, most of the new compounds showed moderate
potent NA inhibitory activity, which can provide attractive
structural bases to find proper NA inhibitors.
6.2.1. General procedures for the synthesis of alkyl-2-benzylidene-
3-oxobutanoate 4
Diethyl malonate(0.06 mol), aromatic aldehydes (0.072 mol),
piperidine (0.9 mL), acetic acid (1.3 mL) and anhydrous toluene
(50.0 mL) were placed in a round-bottomed flask. Generated water
was removed by DeaneStark trap. After the reaction was refluxed
for 16 h, evaporated toluene and diluted with ethyl acetate
(40.0 mL), washed with water, HCl 1 N (2 ꢁ 20 mL), and saturated
NaHCO3 (2 ꢁ 20 mL). Organic phase was dried over MgSO4 and any
solvent was evaporated under reduced pressure. Crude product
was purified by silica gel chromatography eluted with EtOAc/
PE ¼ 6:1 to provide 4 (11.8 g, 80%) [26,27].
6. Experimental
6.1. Chemistry
All the solvents and reagents were purchased from commercial
sources. All reactions were monitored by thin-layer chromatog-
raphy on 0.254 mm silica gel plates (60GF-254) and visualized with
UV light, or iodine vapor. 1H NMR and 13C NMR spectra were
determined on a Varian Mercury VX400 or Bruker Avance 400
apparatus in CDCl3 or d6-DMSO using TMS as internal standard.
Mass spectra of final products were recorded on a LC/MSD Trap SL
Plus spectrometer (Agilent technologies, Waldbronn, Germany)
equipped with electrospray ionization (ESI) interface and an ion
6.2.2. General procedures for the synthesis of 5ae5p
4 (0.01 mol), guanidine hydrochloride (0.012 mol), sodium
ethoxide (0.02 mol) were dissolved in ethanol (100 mL), and
refluxed for 12 h. After completion of the reaction, the solvent was

J. Lou et al. / European Journal of Medicinal Chemistry 83 (2014) 466e473
Table 1 (continued )
469
Table 1
In vitro inhibitory effects of compounds 5ae5p and 6ae6r against influenza A NAs.
R1
Compound IC50
(
m
M) R2
Compound IC50 (mM)
R1 Compound IC50 M) R2 Compound IC50 M)
(m
(m
5a
5b
>200
CH3CO
6a
6b
>200
5m
124.62
CH3CO
6m
71.28
129.47
119.76
CH3CO
CH3CO
57.53
28.46
5n
5o
>200
CH3CO
CH3CO
6n
6o
>200
5c
6c
89.04
46.46
5d
5e
5f
121.38
86.87
>200
CH3CO
CH3CO
CH3CO
6d
6e
6f
29.69
36.67
5p
>200
CH3CO
6p
138.96
CH3CH2CO 6q
106.34
69.84
164.52
CF3CO
6r
Oseltamivir
0.00087
5g
5h
75.68
CH3CO
CH3CO
6g
6h
17.64
evaporated by reduced pressure; and the solid obtained by filtra-
tion then, washed with a large amount of water and EtOAc. Crude
product was dried under vacuo, then recrystallized by MeOH to
provide compound ethyl 2-amino-4-isopropyl-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5a [28e31]. A White solid,
yield 70%: m.p. 183e186 ꢀC; ESI-MS m/z (MþH) 228.14; 1H NMR
188.62
144.62
(400 MHz, DMSO-d6)
d
7.29 (s, 1H), 6.44 (s, 2H), 4.09 (q, J ¼ 7.1 Hz,
2H), 3.53 (d, J ¼ 8.4, 1H), 3.13 (d, J ¼ 8.4 Hz, 1H), 1.66 (dd, J ¼ 11.3,
6.7 Hz, 1H), 1.18 (t, J ¼ 7.1 Hz, 3H), 0.91 (d, J ¼ 6.9 Hz, 3H), 0.83 (d,
J ¼ 6.9 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
d 172.56, 168.08,
5i
5j
5k
5l
>200
CH3CO
CH3CO
CH3CO
CH3CO
6i
6j
6k
6l
>200
157.06, 60.47, 55.27, 53.09, 30.43, 18.68, 16.65, 14.23.
6.2.2.1. Ethyl 2-amino-4-phenyl-6-oxo-1,4,5,6-tetrahydropyrimidine-
5-carboxylate 5b. A white solid, yield 79%: m.p. 228e231 ꢀC; ESI-
MS m/z (MþH) 262.14; 1H NMR (400 MHz, DMSO-d6)
d 7.76 (s,
1H), 7.52e7.28 (m, 5H), 6.54 (s, 2H), 4.80 (d, J ¼ 10.8 Hz,1H), 3.98 (q,
144.60
96.45
165.76
89.64
J ¼ 7.1 Hz, 2H), 3.48 (d, J ¼ 10.8 Hz, 1H), 1.04 (t, J ¼ 7.1 Hz, 3H). 13
C
NMR (101 MHz, DMSO-d6)
d 172.73, 168.28, 159.98, 136.07, 129.04,
128.08, 127.02, 60.19, 54.96, 54.41, 13.90.
6.2.2.2. Ethyl 2-amino-4-(2,4-dichlorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrim-dine-5-carboxylate 5c. A white solid, yield 71%:
m.p. 224e227 ꢀC; ESI-MS m/z (MþH) 331.14; 1H NMR (400 MHz,
>200
DMSO-d6)
d
7.97 (s, 1H), 7.66 (s, 1H), 7.42 (d, J ¼ 7.4 Hz, 1H), 7.14 (d,
J ¼ 7.4 Hz, 1H), 5.20 (d, J ¼ 10.8 Hz, 1H), 4.06 (q, J ¼ 7.1 Hz, 1H), 3.57
(d, J ¼ 10.8 Hz,1H),1.11 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-
d6)
d 172.03, 168.53, 161.09, 134.25, 130.08, 122.88, 120.47, 115.29,
112.19, 60.47, 52.83, 52.18, 13.89.
110.02
6.2.2.3. Ethyl 2-amino-4-(4-bromo-2-fluorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5d. A white solid, yield 74%:

470
J. Lou et al. / European Journal of Medicinal Chemistry 83 (2014) 466e473
Fig. 3. Docking result: compound 6g in the active site of NA (PDB ID: 2HU0). (3a) Compound 6g binding with active pocket of NA in stick mode. (3b) Compound 6g binding with
molecular surface of NA.
m.p. 216e219 ꢀC; ESI-MS m/z (MþH) 359.14; 1H NMR
6.2.2.6. Ethyl 2-amino-4-(2-nitrophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5g. A gray solid, yield 76%:
m.p. 246e248 ꢀC; ESI-MS m/z (MþH) 307.14; 1H NMR (400 MHz,
(400 MHz, DMSO-d6)
d 7.86 (s, 1H), 7.69 (s, 1H), 7.52 (d,
J ¼ 8.3 Hz, 1H), 7.42 (d, J ¼ 8.3 Hz, 1H), 6.94 (s, 2H), 5.22 (d,
J ¼ 10.8 Hz, 1H), 4.06 (d, J ¼ 6.9 Hz, 1H), 3.52 (d, J ¼ 10.8 Hz, 1H), 1.11
(t, J ¼ 6.9 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) 172.83, 168.41,
159.99, 133.25, 130.28, 123.88, 121.47, 116.29, 113.19, 60.47, 53.03,
52.48, 14.29.
DMSO-d6)
d
8.02 (d, J ¼ 8.1 Hz, 1H), 7.88 (s, 1H), 7.80 (t, J ¼ 8.1 Hz,
1H), 7.70e7.57 (m, 2H), 6.83 (s, 2H), 5.38 (d, J¼ 10.8 Hz, 1H), 4.05 (q,
J ¼ 7.1 Hz, 1H), 3.52 (d, J ¼ 10.8 Hz, 1H), 1.11 (t, J ¼ 7.1 Hz, 1H). 13
C
NMR (101 MHz, DMSO-d6)
d 170.47, 168.08, 160.84, 149.08, 138.39,
133.96, 130.49, 128.75, 124.90, 60.09, 53.53, 49.02, 13.57.
6.2.2.4. Ethyl 2-amino-4-(2-chlorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5e. A white solid, yield 84%:
m.p. 210e212 ꢀC; ESI-MS m/z (MþH) 296.14; 1H NMR (400 MHz,
6.2.2.7. Ethyl 2-amino-4-(4-nitrophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5h. A gray solid, yield 82%:
m.p. 250e252 ꢀC; ESI-MS m/z (MþH) 307.14; 1H NMR (400 MHz,
DMSO-d6)
d 8.07 (s, 1H), 7.54e7.47 (m, 2H), 7.43 (s, 2H), 6.62 (s, 2H),
5.27 (d, J ¼ 10.8 Hz, J ¼ 5.0 Hz, 1H), 4.06 (q, J ¼ 7.0 Hz, 2H), 3.54 (d,
DMSO-d6)
d
8.02 (s, 1H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.62 (d, J ¼ 8.4 Hz,
J ¼ 10.8 Hz, 1H), 1.10 (t, J ¼ 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO-
2H), 6.80 (s, 2H), 5.36 (d, J ¼ 6.2 Hz, 1H), 4.04 (q, 2H), 3.54 (d,
d6)
d 172.63, 168.18, 159.63, 140.03, 133.57, 130.41, 127.43, 126.08,
J ¼ 6.2 Hz, 1H), 1.09 (t, J ¼ 6.9 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
121.92, 60.28, 53.36, 52.03, 13.66.
d 172.73, 168.28, 159.98, 136.07, 129.04, 128.08, 127.02, 60.19, 54.96,
54.41, 13.90.
6.2.2.5. Ethyl 2-amino-4-(4-methylphenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5f. A white solid, yield 64%:
m.p. 224e227 ꢀC; ESI-MS m/z (MþH) 276.14; 1H NMR (400 MHz,
6.2.2.8. Ethyl 2-amino-4-(3,4-dimethoxyphenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5i. A white solid, yield 61%:
m.p. 242e245 ꢀC; ESI-MS m/z (MþH) 322.14; 1H NMR (400 MHz,
DMSO-d6)
d
7.73 (s, 1H), 7.26 (d, J ¼ 7.8 Hz, 2H), 7.19 (d, J ¼ 7.6 Hz,
2H), 4.76 (d, J ¼ 10.9 Hz, 1H), 3.98 (q, J ¼ 7.0 Hz, 2H), 3.45 (d,
DMSO-d6)
d
7.63 (s, 1H), 7.01 (s, 1H), 6.94 (d, J ¼ 8.0 Hz, 1H), 6.88 (d,
J ¼ 10.9 Hz, 1H), 2.33 (s, 3H), 1.06 (t, J ¼ 7.0 Hz, 3H). 13C NMR
J ¼ 8.0 Hz, 1H), 4.73 (d, J ¼ 11.3 Hz, 1H), 3.99 (q, J ¼ 7.0 Hz, 2H), 3.76
(s, 3H), 3.75 (s, 3H), 3.52 (d, J ¼ 11.3 Hz, 1H), 1.07 (t, J ¼ 7.0 Hz, 3H).
(101 MHz, DMSO-d6)
d 172.18, 168.44, 161.13, 139.43, 130.11, 128.33,
127.03, 60.47, 55.65, 54.23, 22.15, 14.24.
13C NMR (101 MHz, DMSO-d6)
d 172.07, 168.64, 159.94, 149.44,

J. Lou et al. / European Journal of Medicinal Chemistry 83 (2014) 466e473
471
139.09, 131.93, 119.81, 111.29, 109.62, 60.42, 56.12, 55.93, 55.88,
54.57, 14.17.
6.2.3. General procedures for the synthesis of 6ae6r
5a (2 mmol) and 2 mL acetic anhydride were dissolved in CH2Cl2
(20 mL), added a catalytic amount of DMAP to solution, stirred for
8 h at room temperature. After that evaporated solvent by reduce
pressure. The residues product was diluted with EtOAc and washed
with water, saturated NaHCO3. Organic phase was dried over
MgSO4 and solvent was evaporated under reduced pressure. Crude
product was purified by silica gel chromatography eluted with
EtOAc/PE ¼ 10:1 to furnish ethyl 2-acetamino-4-iso-propyl-6-oxo-
1,4,5,6-tetrahydropyrimidine-5-carboxylate 6a [29]. A yellow solid,
yield 77%: m.p. 130e132 ꢀC; ESI-MS m/z (MþH) 270.14; 1H NMR
6.2.2.9. Ethyl 2-amino-4-(4-fluorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5j. A white solid, yield 73%:
m.p. 237e240 ꢀC; ESI-MS m/z (MþH) 280.14; 1H NMR (400 MHz,
DMSO-d6)
d
7.79 (s, 1H), 7.46 (d, J ¼ 8.5 Hz, 2H), 7.41 (d, J ¼ 8.5 Hz,
2H), 4.83 (d, J ¼ 10.5 Hz, 1H), 4.00 (q, J ¼ 7.1 Hz, 2H), 3.48 (d,
J ¼ 10.5 Hz, 1H), 1.06 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-
d6)
d 172.24, 168.88, 160.08, 134.52, 130.24, 126.68, 124.02, 61.42,
54.76, 54.21, 14.03.
(400 MHz, CDCl3)
d
9.98 (s, 1H), 4.17 (q, J ¼ 7.1 Hz, 2H), 3.84 (d,
6.2.2.10. Ethyl 2-amino-4-(4-methoxyphenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5k. A white solid, yield 68%:
m.p. 211e214 ꢀC; ESI-MS m/z (MþH) 292.14; 1H NMR (400 MHz,
J ¼ 6.8 Hz, 1H), 3.38 (d, J ¼ 6.8 Hz, 1H), 2.26 (s, 3H), 1.91e1.74 (m,
1H), 1.21 (t, J ¼ 7.1H, 3H), 0.97 (d, J ¼ 6.0 Hz, 3H), 0.90 (d, J ¼ 5.9 Hz,
3H); 13C NMR (101 MHz, CDCl3)
d 176.00, 172.35, 168.06, 158.92,
DMSO-d6)
d
7.66 (s, 1H), 7.28 (d, J ¼ 25.3, 2H), 6.99 (d, J ¼ 25.3, 2H),
61.70, 56.49, 50.11, 30.51, 24.75, 18.93, 17.09, 13.73.
4.74 (d, J ¼ 11.3 Hz, 1H), 3.97 (q, J ¼ 7.1 Hz, 2H), 3.74 (s, 3H), 3.46 (d,
J ¼ 11.3 Hz,1H),1.05 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
6 . 2 . 3 .1. E t h yl 2 - a c e t a m i n o - 4 - p h e n yl - 6 - o x o - 1, 4 , 5 , 6 -
tetrahydropyrimidine-5-carboxylate 6b. A white solid, yield 75%:
m.p. 152e154 ꢀC; ESI-MS m/z (MþH) 304.14; 1H NMR (400 MHz,
d
172.58, 168.74, 159.47, 130.79, 128.41, 127.69, 113.55, 60.09, 55.26,
54.89, 54.22, 13.86.
CDCl3)
d
10.08 (s, 1H), 7.45e7.31 (m, 5H), 5.17 (d, J ¼ 10.8 Hz, 1H),
6.2.2.11. Ethyl 2-amino-4-(4-chlorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5l. A white solid, yield 77%:
m.p. 258e260 ꢀC; ESI-MS m/z (MþH) 296.14; 1H NMR (400 MHz,
4.16 (q, J ¼ 7.0 Hz, 2H), 3.65 (d, J ¼ 10.8 Hz, 1H), 2.35 (s, 3H), 1.16 (t,
J ¼ 7.0 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 175.90, 172.58, 167.42,
158.19, 136.71, 129.36, 126.85, 123.75, 61.69, 55.55, 54.63, 24.71,
14.01.
DMSO-d6)
d
7.76 (s, 1H), 7.59 (d, J ¼ 8.3 Hz, 2H), 7.35 (d, J ¼ 8.3 Hz,
2H), 6.60 (s, 2H), 4.82 (d, J ¼ 10.6 Hz,1H), 4.00 (q, J ¼ 7.1 Hz 2H), 3.46
(d, J ¼ 10.6 Hz, 1H), 1.07 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz,
6.2.3.2. Ethyl 2-acetamino-4-(2,4-dichlorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6c. A white solid, yield 74%:
m.p. 67e70 ꢀC; ESI-MS m/z (MþH) 373.14; 1H NMR (400 MHz,
DMSO-d6)
d 171.93, 168.58, 161.18, 135.47, 130.09, 127.88, 126.42,
60.89, 53.96, 52.41, 13.74.
CDCl3)
d
10.18 (s, 1H), 7.26 (dd, 2H), 6.99 (s, 1H), 5.54 (d, J ¼ 10.9 Hz,
6.2.2.12. Ethyl 2-amino-4-(4-bromophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5m. A white solid, yield 62%:
m.p. 256e258 ꢀC; ESI-MS m/z (MþH) 341.14; 1H NMR (400 MHz,
1H), 4.18 (d, J ¼ 7.1 Hz, 2H), 3.72 (d, J ¼ 10.9 Hz, 1H), 2.19 (s, 3H), 1.12
(d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 175.69, 171.20,
166.97, 158.53, 132.02, 128.61, 123.45, 121.27, 115.54, 114.61, 61.98,
53.75, 51.95, 24.75, 13.70.
DMSO-d6)
d
7.73 (s, 1H), 7.45 (d, J ¼ 8.4, 2H), 7.23 (d, J ¼ 8.8 Hz, 2H),
4.82 (d, J ¼ 10.9 Hz, 1H), 3.98 (q, J ¼ 7.1 Hz, 2H), 3.50 (d, J ¼ 10.9 Hz,
1H), 1.05 (t, J ¼ 7.1 Hz, 1H). 13C NMR (101 MHz, DMSO-d6)
d
172.56,
6.2.3.3. Ethyl 2-acetamino-4-(4-bromo-2-fluoro phenyl)-6-oxo-
1,4,5,6-tetrahydropyrimidine-5-carboxylate 6d. A white solid, yield
68%: m.p. 60e62 ꢀC; ESI-MS m/z (MþH) 401.14; 1H NMR (400 MHz,
168.40, 161.89, 138.76, 131.84, 129.81, 121.54, 60.46, 55.27, 53.92,
13.86.
CDCl3)
d
10.12 (s, 1H), 7.20 (dd, 2H), 6.96 (s, 1H), 5.48 (d, J ¼ 10.9 Hz,
6.2.2.13. Ethyl 2-amino-4-(furan-2-yl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5n. A gray solid, yield 83%:
m.p. 171e173 ꢀC; ESI-MS m/z (MþH) 252.14; 1H NMR (400 MHz,
1H), 4.20 (d, J ¼ 7.1 Hz, 2H), 3.72 (d, J ¼ 10.9 Hz, 1H), 2.17 (s, 3H), 1.14
(d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 175.67, 171.23,
166.45, 158.13, 132.24, 127.68, 123.25, 121.07, 118.04, 116.21, 61.94,
52.71, 50.90, 23.72, 13.73.
DMSO-d6)
d 7.91 (s, 1H), 7.68 (s, 1H), 7.17 (s, 2H), 6.45 (s, 1H), 6.38 (s,
1H), 4.91 (d, J ¼ 9.6 Hz, 1H), 4.06 (q, J ¼ 7.1 Hz, 1H), 3.50 (d,
J ¼ 9.6 Hz, 1H), 1.12 (t, J ¼ 7.1 Hz, 1H). 13C NMR (101 MHz, DMSO-d6)
6.2.3.4. Ethyl 2-acetamino-4-(2-chlorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6e. A white solid, yield 84%:
m.p. 56e58 ꢀC; ESI-MS m/z (MþH) 338.14; 1H NMR (400 MHz,
d
172.15, 168.12, 160.02, 145.31, 141.49, 108.59, 105.47, 62.04, 52.01,
50.19, 13.94.
CDCl3)
d
10.24 (s, 1H), 7.43 (d, 1H), 7.29 (s, 3H), 5.68 (d, J ¼ 4.8 Hz,
6.2.2.14. Ethyl 2-amino-4-(2-fluorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5o. A white solid, yield 71%:
m.p. 224e226 ꢀC; ESI-MS m/z (MþH) 280.14; 1H NMR (400 MHz,
1H), 4.24 (q, J ¼ 7.2 Hz, 2H), 3.84 (d, J ¼ 4.8 Hz, 1H), 2.28 (s, 3H), 1.25
(t, J ¼ 7.2 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 175.71, 171.44,
166.87, 158.21, 134.41, 132.94, 130.75, 130.23, 128.03, 127.11, 62.34,
52.58, 51.59, 24.75, 13.72.
DMSO-d6)
d 8.09 (s, 1H), 7.58e7.49 (m, 2H), 7.45 (s, 2H), 6.63 (s, 2H),
5.28 (d, J ¼ 10.8 Hz, 1H), 4.08 (q, J ¼ 7.0 Hz, 2H), 3.56 (d, J ¼ 10.8 Hz,
1H) 1.09 (t, J ¼ 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
d
172.03,
6.2.3.5. Ethyl 2-acetamino-4-(4-methylphenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6f. A white solid, yield 89%:
m.p. 137e139 ꢀC; ESI-MS m/z (MþH) 318.14; 1H NMR (400 MHz,
168.38, 160.63, 138.03, 134.07, 131.21, 129.93, 128.48, 125.02, 60.18,
53.66, 52.03, 13.64.
CDCl3)
d
9.94 (s, 1H), 7.23 (d, J ¼ 8.0 Hz, 1H), 7.18 (d, J ¼ 8.0 Hz, 2H),
6.2.2.15. Ethyl2-amino-4-(4-(dimethylamino) phenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 5p. A yellow solid, yield 73%:
m.p. 216e218 ꢀC; ESI-MS m/z (MþH) 305.14; 1H NMR (400 MHz,
5.13 (d, J ¼ 10.8 Hz, 1H), 4.14 (q, J ¼ 7.2 Hz, 1H), 3.65 (d, J ¼ 10.8 Hz,
1H), 2.34 (s, 3H), 2.31 (s, 3H), 1.16 (t, J ¼ 7.1 Hz, 3H); 13C NMR
(101 MHz, CDCl3)
d 175.73, 172.35, 167.43, 157.47, 139.05, 133.84,
DMSO-d6)
d
7.63 (s, 1H), 7.17 (d, J ¼ 8.6 Hz, 2H), 6.69 (d, J ¼ 8.6 Hz,
130.19, 126.19, 61.42, 55.59, 54.41, 24.76, 20.75, 13.99.
2H), 4.65 (d, J ¼ 11.5 Hz, 1H), 3.96 (q, J ¼ 7.1 Hz, 2H), 3.41 (d,
J ¼ 11.5 Hz, 1H), 2.88 (s, 6H), 1.06 (t, J ¼ 7.1 Hz, 3H). 13C NMR
6.2.3.6. Ethyl 2-acetamino-4-(2-nitrophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6g. A gray solid, yield 81%:
m.p. 54e57 ꢀC; ESI-MS m/z (MþH) 349.14; 1H NMR (400 MHz,
(101 MHz, DMSO-d6)
d 172.43,168.28,160.48,137.43, 128.16,125.42,
119.64, 60.94, 54.38, 51.59, 43.32, 14.02.

472
J. Lou et al. / European Journal of Medicinal Chemistry 83 (2014) 466e473
CDCl3)
d
10.33 (s, 1H), 8.06 (d, J ¼ 7.9 Hz, 1H), 7.71e7.66 (m, 1H), 7.56
CDCl3)
d
10.18 (s, 1H), 7.37 (d, 1H), 6.34 (s, 2H), 5.30 (d, J ¼ 5.9 Hz,
(t, J ¼ 7.6 Hz, 1H), 7.50 (d, J ¼ 7.7 Hz, 1H), 5.89 (d, J ¼ 10.8 Hz, 1H),
1H), 4.24 (q, J ¼ 7.1 Hz, 2H), 3.82 (d, J ¼ 5.9 Hz, 1H), 2.35 (s, 3H), 1.25
4.23 (q, J ¼ 7.1 Hz 2H), 3.87 (d, J ¼ 10.8 Hz, 1H), 2.28 (s, 3H), 1.26 (t,
(t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 176.00,171.42,167.15,
J ¼ 7.1 Hz, 1H); 13C NMR (101 MHz, CDCl3)
d
175.72, 172.34, 166.88,
158.02, 148.81, 143.89, 110.59, 107.87, 62.00, 51.01, 49.19, 24.75,
13.74.
158.36, 134.13, 132.94, 132.59, 129.83, 127.73, 125.28, 62.91, 53.48,
52.67, 24.75, 13.97.
6.2.3.14. Ethyl 2-acetamino-4-(2-fluorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6o. A white solid, yield 85%:
m.p. 105e108 ꢀC; ESI-MS m/z (MþH) 322.14; 1H NMR (400 MHz,
6.2.3.7. Ethyl 2-acetamino-4-(4-nitrophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6h. A white solid, yield 86%:
m.p. 102e104 ꢀC; ESI-MS m/z (MþH) 349.14; 1H NMR (400 MHz,
CDCl3)
d
10.23 (s, 1H), 7.66 (d, J ¼ 7.9 Hz, 1H), 7.41e7.46 (m, 1H), 7.26
CDCl3)
d
10.03 (s, 1H), 8.18 (d, J ¼ 7.9 Hz, 2H), 7.50 (d, J ¼ 7.9 Hz, 2H),
(t, J ¼ 7.6 Hz,1H), 7.20 (d, J ¼ 7.7 Hz,1H), 5.69 (d, J ¼ 4.8 Hz, 1H), 4.20
(q, J ¼ 7.1 Hz, 2H), 3.87 (d, J ¼ 4.8 Hz, 1H), 2.26 (s, 3H), 1.26 (t,
5.26 (d, J ¼ 10.0 Hz, 1H), 4.11 (q, J ¼ 6.9 Hz, 2H), 3.59 (d, J ¼ 10.0 Hz,
1H), 2.21 (s, 3H), 1.12 (t, J ¼ 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3)
J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 175.91, 172.44, 167.87,
158.16, 133.49, 131.94, 130.45, 130.03, 129.03, 128.11, 61.84, 53.58,
52.59, 26.75, 13.92.
d
175.91, 171.42, 165.93, 158.69, 128.14, 126.92, 123.52, 122.43, 61.27,
60.91, 52.95, 23.53, 13.02.
6.2.3.8. Ethyl 2-acetamino-4-(3,4-dimethoxyphenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6i. A white solid, yield 83%:
m.p. 152e154 ꢀC; ESI-MS m/z (MþH) 364.14; 1H NMR (400 MHz,
6.2.3.15. Ethyl
2-acetamino-4-(4-(dimethylamino)phenyl)-6-oxo-
1,4,5,6-tetrahydropyrimidine-5-carboxylate 6p. A white solid, yield
CDCl3)
d
9.93 (s, 1H), 7.03 (s, 1H), 6.93 (d, J ¼ 8.3 Hz, 1H), 6.85 (d,
73%: m.p. 86e89 ꢀC; ESI-MS m/z (MþH) 347.14; 1H NMR (400 MHz,
J ¼ 8.3 Hz, 1H), 5.08 (d, J ¼ 12.0 Hz, 1H), 4.11 (q, J ¼ 6.9 Hz, 2H), 3.86
(s, 6H), 3.73 (d, J ¼ 12.0 Hz, 1H), 2.34 (s, 3H), 1.13 (t, J ¼ 6.9 Hz, 3H);
CDCl3)
d
9.81 (s, 1H), 7.19 (d, J ¼ 8.7 Hz, 2H), 6.68 (d, J ¼ 8.7 Hz, 2H),
5.05 (d, J ¼ 11.4 Hz, 1H), 4.15 (q, J ¼ 7.1 Hz, 1H), 3.65 (t, J ¼ 11.9 Hz,
13C NMR (101 MHz, CDCl3)
d 175.72, 172.35, 168.07, 158.24, 149.44,
1H), 2.94 (s, 6H), 2.32 (s, 3H), 1.17 (t, J ¼ 7.1 Hz, 3H); 13C NMR
149.09, 128.93, 119.81, 111.29, 109.62, 61.42, 56.12, 55.93, 55.38,
54.57, 24.53, 14.11.
(101 MHz, CDCl3)
d 175.51, 172.44, 167.87, 158.32, 138.43, 128.46,
124.42, 118.94, 60.94, 54.38, 51.59, 43.32, 24.10, 14.02.
6.2.3.9. Ethyl 2-acetamino-4-(4-fluorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6j. A white solid, yield 85%:
m.p. 157e159 ꢀC; ESI-MS m/z (MþH) 322.14; 1H NMR (400 MHz,
6 . 2 . 3 .16 . E t h y l 4 - ( 4 - b r o m o p h e n y l ) - 6 - o x o - 2 - ( 2 , 2 , 2 -
trifluoroacetamido)-1,4,5,6-tetrahydropyrimidine-5-carboxylate 6q.
A white solid, yield 74%: m.p. 134e136 ꢀC; ESI-MS m/z (MþH)
CDCl3)
d
9.88 (s, 1H), 7.35 (d, J ¼ 7.8 Hz, 2H), 7.10 (d, J ¼ 7.8 Hz, 2H),
5.16 (d, J ¼ 11.0 Hz, 1H), 4.16 (q, J ¼ 7.1 Hz, 2H), 3.60 (d, J ¼ 11.2 Hz,
437.14; 1H NMR (400 MHz, DMSO-d6)
d 11.86 (s, 1H), 10.39 (s, 1H),
1H), 2.34 (s, 3H), 1.17 (t, J ¼ 7.1 Hz, 1H); 13C NMR (101 MHz, CDCl3)
7.66 (d, J ¼ 8.4 Hz, 2H), 7.36 (d, J ¼ 8.4 Hz, 2H), 5.34 (d, J ¼ 7.9 Hz,
d
176.31, 172.38, 167.39, 158.39, 132.79, 129.17, 128.50, 127.52, 61.83,
1H), 4.38 (d, J ¼ 7.9 Hz, 1H), 4.12 (q, J ¼ 7.1 Hz, 2H), 1.11 (t, J ¼ 7.1 Hz,
55.02, 54.35, 24.65, 13.76.
3H); 13C NMR (101 MHz, DMSO-d6)
d 175.64, 165.91, 164.93, 158.21,
135.88, 132.00,129.35, 128.97, 121.98, 61.82, 53.85, 52.69, 13.78.
6.2.3.10. Ethyl 2-acetamino-4-(4-methoxyphenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6k. A white solid, yield 71%:
m.p. 67e69 ꢀC; ESI-MS m/z (MþH) 334.14; 1H NMR (400 MHz,
6.2.3.17. Ethyl 4-(4-bromophenyl)-6-oxo-2-propionamido-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6r. A white solid, yield 87%:
m.p. 65e67 ꢀC; ESI-MS m/z (MþH) 397.14; 1H NMR (400 MHz,
CDCl3)
d
9.93 (s, 1H), 7.27 (d, J ¼ 8.7 Hz, 2H), 6.91 (d, J ¼ 8.6 Hz, 2H),
5.11 (d, J ¼ 11.2 Hz, 1H), 4.14 (q, J ¼ 7.1 Hz, 2H), 3.81 (s, 3H), 3.63 (d,
DMSO-d6)
d
10.21 (s, 1H), 10.06 (s, 1H), 7.52 (d, J ¼ 8.1 Hz, 2H), 7.27
J ¼ 11.2 Hz, 1H), 2.32 (s, 3H), 1.16 (t, J ¼ 7.1 Hz, 3H); 13C NMR
(d, J ¼ 8.1 Hz, 2H), 5.15 (d, J ¼ 10.8 Hz, 1H), 4.16 (q, J ¼ 6.8 Hz, 2H),
3.63 (d, J ¼ 10.9 Hz, 1H), 2.73 (dd, J ¼ 16.5, 7.7 Hz, 1H), 2.57 (dd,
J ¼ 16.7, 7.6 Hz, 1H), 1.18 (t, 3H), 1.12 (t, J ¼ 6.8 Hz 3H); 13C NMR
(101 MHz, CDCl3)
d 175.85, 172.16 167.54, 158.09, 128.48, 128.07,
114.99, 114.61, 61.70, 55.41, 54.98, 54.74, 24.61, 13.95.
(101 MHz, DMSO-d6)
d 175.64, 172.22, 166.86, 158,12, 136.31, 132.03,
6.2.3.11. Ethyl 2-acetamino-4-(4-chlorophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6l. A white solid, yield 81%:
m.p. 104e106 ꢀC; ESI-MS m/z (MþH) 338.14; 1H NMR (400 MHz,
128.93, 123.45, 61.43, 55.58, 54.04, 30.24, 13.72, 8.30.
CDCl3)
d
10.05 (s, 1H), 7.38 (d, J ¼ 8.4 Hz, 2H), 7.32 (d, J ¼ 8.4 Hz, 2H),
Acknowledgments
5.14 (d, J ¼ 10.9 Hz, 1H), 4.16 (q, J ¼ 7.1 Hz, 2H), 3.63 (d, J ¼ 10.9 Hz,
1H), 2.33 (s, 3H), 1.18 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
We thank Guofu Qiu (School of Pharmaceutical Sciences, Wuhan
University, Wuhan, China) for 1H NMR and 13C NMR spectrums and
Xulin Chen for the help with testing NA inhibitory activity in vitro.
This work was supported by National Natural Science Foundation of
China (81273363 and 21102107), the National Mega Project on
Major Drug Development (2011ZX09401-302), and the Scientific
and Technological Innovative Research Team of Wuhan
(2013070204020048).
d
176.01, 172.06, 167.40, 158.72, 134.77, 129.85, 128.25, 127.39, 61.99,
55.59, 54.40, 24.75, 14.02.
6.2.3.12. Ethyl 2-acetamino-4-(4-bromophenyl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6m. A white solid, yield 78%:
m.p. 92e94 ꢀC; ESI-MS m/z (MþH) 383.14; 1H NMR (400 MHz,
CDCl3)
d
10.05 (s, 1H), 7.53 (d, J ¼ 8.3 Hz, 2H), 7.26 (d, J ¼ 8.3 Hz, 2H),
5.15 (d, J ¼ 10.8 Hz, 1H), 4.16 (q, J ¼ 7.1 Hz, 2H), 3.63 (d, J ¼ 10.8 Hz,
1H), 2.32 (s, 3H), 1.18 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d
176.01, 172.35, 167.43, 158.32, 135.67, 132.31, 128.64, 127.40, 61.70,
Appendix A. Supporting information
55.31, 53.75, 24.74, 14.37.
Supporting information associated with this article can be found
in the online version, at http://dx.doi.org/10.1016/j.ejmech.2014.06.
059. These data include MOL files and InChiKeys of the most
important compounds described in this article.
6.2.3.13. Ethyl 2-acetamino-4-(furan-2-yl)-6-oxo-1,4,5,6-
tetrahydropyrimidine-5-carboxylate 6n. A gray solid, yield 83%:
m.p. 50e52 ꢀC; ESI-MS m/z (MþH) 294.14; 1H NMR (400 MHz,

J. Lou et al. / European Journal of Medicinal Chemistry 83 (2014) 466e473
473
virus neuraminidase: modifications of essential pyrrolidinone ring sub-
stituents, Bioorg. Med. Chem. 11 (2003) 2739e2749.
References
[16] G.T. Wang, Y. Chen, S. Wang, R. Gentles, T. Sowin, W. Kati, S. Muchmore,
V. Giranda, K. Stewart, H. Sham, D. Kempf, W.G. Laver, Design, synthesis, and
structural analysis of influenza neuraminidase inhibitors containing pyrroli-
dine cores, J. Med. Chem. 44 (2001) 1192e1201.
[17] S. Sasaki, T. Imaeda, Y. Hayase, Y. Shimizu, S. Kasai, N. Cho, M. Harada,
N. Suzuki, S. Furuya, M. Fujino, A new class of potent nonpeptide luteinizing
hormone-releasing hormone (LHRH) antagonists: design and synthesis of 2-
phenylimidazo[1,2-a]pyrimidin-5-ones, Bioorg. Med. Chem. Lett. 12 (2002)
2073e2077.
[1] J. Zhang, Q. Wang, H. Fang, W. Xu, A. Liu, G. Du, Design, synthesis, inhibitory
activity, and SAR studies of hydrophobic p-aminosalicylic acid derivatives as
neuraminidase inhibitors, Bioorg. Med. Chem. 16 (2008) 3839e3847.
[2] Y. Xie, B. Huang, K. Yu, F. Shi, T. Liu, W. Xu, Caffeic acid derivatives: a new type
of influenza neuraminidase inhibitors, Bioorg. Med. Chem. Lett. 23 (2013)
3556e3560.
[3] W. Li, L. Xia, A. Hu, A. Liu, J. Peng, W. Tan, Design and synthesis of 4-alkyl-2-
amino(acetamino)-6-aryl-1,3-thiazine derivatives as influenza neuraminidase
inhibitors, Arch. Pharm. 346 (2013) 635e644.
[18] K.M. Wilcoxen, Y.F. Zhu, P.J. Connors, J. Saunders, T.D. Gross, Y. Gao,
G.J. Reinhart, R.S. Struthers, C. Chen, Synthesis and initial structure-activity
relationships of a novel series of imidazolo[1,2-a]pyrimid-5-ones as potent
GnRH receptor antagonists, Bioorg. Med. Chem. Lett. 12 (2002) 2179e2183.
[19] K.M. Bonger, R.J. van den Berg, L.H. Heitman, I.J. AP, J. Oosterom,
C.M. Timmers, H.S. Overkleeft, G.A. van der Marel, Synthesis and evaluation of
homo-bivalent GnRHR ligands, Bioorg. Med. Chem. 15 (2007) 4841e4856.
[20] R.J. Russell, L.F. Haire, D.J. Stevens, P.J. Collins, Y.P. Lin, G.M. Blackburn, A.J. Hay,
S.J. Gamblin, J.J. Skehel, The structure of H5N1 avian influenza neuraminidase
suggests new opportunities for drug design, Nature 443 (2006) 45e49.
[21] D. Ye, W.J. Shin, N. Li, W. Tang, E. Feng, J. Li, P.L. He, J.P. Zuo, H. Kim, K.Y. Nam,
W. Zhu, B.L. Seong, K.T. No, H. Jiang, H. Liu, Synthesis of C-4-modified zana-
mivir analogs as neuraminidase inhibitors and their anti-AIV activities, Eur. J.
Med. Chem. 54 (2012) 764e770.
[22] Q. Li, J. Qi, W. Zhang, C.J. Vavricka, Y. Shi, J. Wei, E. Feng, J. Shen, J. Chen, D. Liu,
J. He, J. Yan, H. Liu, H. Jiang, M. Teng, X. Li, G.F. Gao, The 2009 pandemic H1N1
neuraminidase N1 lacks the 150-cavity in its active site, Nat. Struct. Mol. Biol.
17 (2010) 1266e1268.
[23] W.G. Laver, P.M. Colman, R.G. Webster, V.S. Hinshaw, G.M. Air, Influenza virus
neuraminidase with hemagglutinin activity, Virology 137 (1984) 314e323.
[24] P. Chand, P.L. Kotian, A. Dehghani, Y. El-Kattan, T.H. Lin, T.L. Hutchison,
Y.S. Babu, S. Bantia, A.J. Elliott, J.A. Montgomery, Systematic structure-based
design and stereoselective synthesis of novel multisubstituted cyclopentane
derivatives with potent antiinfluenza activity, J. Med. Chem. 44 (2001)
4379e4392.
[25] R. Morphy, Z. Rankovic, Designed multiple ligands. An emerging drug dis-
covery paradigm, J. Med. Chem. 48 (2005) 6523e6543.
[26] T. Wang, J. Liu, H. Zhong, H. Chen, Z. Lv, Y. Zhang, M. Zhang, D. Geng, C. Niu,
Y. Li, K. Li, Synthesis and anti-tumor activity of novel ethyl 3-aryl-4-oxo-
3,3a,4,6-tetrahydro-1H-furo[3,4-c]pyran-3a-carboxylates, Bioorg. Med. Chem.
Lett. 21 (2011) 3381e3383.
[27] Y. Moussaoui, R. Ben Salem, Catalyzed Knoevenagel reactions on inorganic
solid sorts: application to the synthesis of coumarine compounds, C. R. Chim.
10 (2007) 1162e1169.
[28] H. Cho, M. Veda, Y. Ohnaka, M. Hayashimatsu, Synthesis of novel 5-
alkoxycarbonyl-5,6-dihydropyrimidin -4(3H)-ones from 3-substituted-20-
alkoxycarbonyl-2-propenoates and amidines, Heterocycles 22 (1984)
1959e1963.
[4] J. Kongkamnerd, L. Cappelletti, A. Prandi, P. Seneci, T. Rungrotmongkol,
N. Jongaroonngamsang, P. Rojsitthisak, V. Frecer, A. Milani, G. Cattoli,
C. Terregino, I. Capua, L. Beneduce, A. Gallotta, P. Pengo, G. Fassina, S. Miertus,
W. De-Eknamkul, Synthesis and in vitro study of novel neuraminidase in-
hibitors against avian influenza virus, Bioorg. Med. Chem. 20 (2012)
2152e2157.
[5] P. Chand, P.L. Kotian, P.E. Morris, S. Bantia, D.A. Walsh, Y.S. Babu, Synthesis and
inhibitory activity of benzoic acid and pyridine derivatives on influenza
neuraminidase, Bioorg. Med. Chem. 13 (2005) 2665e2678.
[6] J. Zhang, Q. Wang, H. Fang, W. Xu, A. Liu, G. Du, Design, synthesis, inhibitory
activity, and SAR studies of pyrrolidine derivatives as neuraminidase in-
hibitors, Bioorg. Med. Chem. 15 (2007) 2749e2758.
[7] C.U. Kim, W. Lew, M.A. Williams, H.T. Liu, L.J. Zhang, S. Swaminathan,
N. Bischofberger, M.S. Chen, D.B. Mendel, C.Y. Tai, W.G. Laver, R.C. Stevens,
Influenza neuraminidase inhibitors possessing a novel hydrophobic interac-
tion in the enzyme active site: design, synthesis, and structural analysis of
carbocyclic sialic acid analogues with potent anti-influenza activity, J. Am.
Chem. Soc. 119 (1997) 681e690.
[8] S. Magesh, N. Sriwilaijaroen, V. Savita, H. Ando, T. Miyagi, Y. Suzuki, H. Ishida,
M. Kiso, Investigation of 2-fluoro benzoic acid derivatives as influenza a viral
sialidase selective inhibitors, Anti Infect. Agents Med. Chem.
9 (2010)
198e204.
[9] A. Meeprasert, W. Khuntawee, K. Kamlungsua, N. Nunthaboot,
T. Rungrotmongkol, S. Hannongbua, Binding pattern of the long acting neur-
aminidase inhibitor laninamivir towards influenza A subtypes H5N1 and
pandemic H1N1, J. Mol. Graph. Model. 38 (2012) 148e154.
[10] T.T. Dao, P.H. Nguyen, H.S. Lee, E. Kim, J. Park, S. Il Lim, W.K. Oh, Chalcones as
novel influenza A (H1N1) neuraminidase inhibitors from Glycyrrhiza inflata,
Bioorg. Med. Chem. Lett. 21 (2011) 294e298.
[11] E. Feng, W.-J. Shin, X. Zhu, J. Li, D. Ye, J. Wang, M. Zheng, J.-P. Zuo, K.T. No,
X. Liu, W. Zhu, W. Tang, B.-L. Seong, H. Jiang, H. Liu, Structure-based design
and synthesis of C-1- and C-4-modified analogs of zanamivir as neuramini-
dase inhibitors, J. Med. Chem. 56 (2013) 671e684.
[12] T. Woodhall, G. Williams, A. Berry, A. Nelson, Synthesis of screening substrates
for the directed evolution of sialic acid aldolase: towards tailored enzymes for
the preparation of influenza A sialidase inhibitor analogues, Org. Biomol.
Chem. 3 (2005) 1795e1800.
[13] K.G. Watson, R. Cameron, R.J. Fenton, D. Gower, S. Hamilton, B. Jin,
G.Y. Krippner, A. Luttick, D. McConnell, S.J.F. MacDonald, A.M. Mason,
V. Nguyen, S.P. Tucker, W.Y. Wu, Highly potent and long-acting trimeric and
tetrameric inhibitors of influenza virus neuraminidase, Bioorg. Med. Chem.
Lett. 14 (2004) 1589e1592.
[14] P. Chand, Y.S. Babu, S. Bantia, S. Rowland, A. Dehghani, P.L. Kotian,
T.L. Hutchison, S. Ali, W. Brouillette, Y. El-Kattan, T.H. Lin, Syntheses and
neuraminidase inhibitory activity of multisubstituted cyclopentane amide
derivatives, J. Med. Chem. 47 (2004) 1919e1929.
[29] A.P. Phillips, J. Mentha, Acetylation of some aminopyrimidines, J. Am. Chem.
Soc. 76 (1954) 6200e6202.
[30] B. Ahmed, R.A. Khan, Habibullah, M. Keshari, An improved synthesis of
Biginelli-type compounds via phase-transfer catalysis, Tetrahedron Lett. 50
(2009) 2889e2892.
[31] S. Senda, K. Hirota, T. Asao, K. Maruhashi, Pyrimidine derivatives and related
compounds. 31.
A new photochemical transformation of 6-azido-1,3-
dimethyluracil to 6-alkylamino-5-amino-1,3-dimethyluracils and its applica-
tion to one-step synthesis of lumazines and fervenulins, J. Am. Chem. Soc. 100
(1978) 7661e7664.
[15] W.J. Brouillette, S.N. Bajpai, S.M. Ali, S.E. Velu, V.R. Atigadda, B.S. Lommer,
J.B. Finley, M. Luo, G.M. Air, Pyrrolidinobenzoic acid inhibitors of influenza