Synthesis and biological activity of novel barbituric and thiobarbituric acid derivatives against non-alcoholic fatty liver disease

Article abstract of DOI:10.1016/j.ejmech.2011.02.033

Forty-four barbituric acid or thiobarbituric acid derivatives were synthesized and evaluated for their effects on adipogenesis of 3T3-L1 adipocytes by measuring the expression of adiponectin in vitro. Four compounds (3a, 3o, 3s, 4t) were found to increase the expression of adiponectin and lower the leptin level in 3T3-L1 adipocytes at respective concentration of 10 μM. Among them, 3s showed the most efficacious. Oral administration of 3s effectively reduced body weight, liver weight, and visceral fat and regulated serum levels of biochemical markers in the high-fat/diet-induced Wistar rats. Histopathological evaluation of liver sections by Oil Red O and H&E staining confirmed 3s as a potent, orally active molecule for reducing fat deposition against non-alcoholic fatty liver disease.

Full text of DOI:10.1016/j.ejmech.2011.02.033

European Journal of Medicinal Chemistry 46 (2011) 2003e2010
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of novel barbituric and thiobarbituric acid
derivatives against non-alcoholic fatty liver disease
,
,
Liang Ma a 1, Shilin Li a 1, Hao Zheng a, Jinying Chen a, Lin Lin b, Xia Ye a, Zhizhi Chen a, Qinyuan Xu a,
Tao Chen a, Jincheng Yang a, Neng Qiu a, Guangcheng Wang a, Aihua Peng a, Yi Ding b, Yuquan Wei a,
Lijuan Chen a
,
*
a State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Gaopeng Street, Chengdu 610041, China
b State Key Laboratory of Oral Disease, West China College of Stomatology, Sichuan University, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Forty-four barbituric acid or thiobarbituric acid derivatives were synthesized and evaluated for their
effects on adipogenesis of 3T3-L1 adipocytes by measuring the expression of adiponectin in vitro. Four
compounds (3a, 3o, 3s, 4t) were found to increase the expression of adiponectin and lower the leptin
Received 28 August 2010
Received in revised form
19 December 2010
Accepted 14 February 2011
Available online 22 February 2011
level in 3T3-L1 adipocytes at respective concentration of 10
mM. Among them, 3s showed the most
efficacious. Oral administration of 3s effectively reduced body weight, liver weight, and visceral fat and
regulated serum levels of biochemical markers in the high-fat/diet-induced Wistar rats. Histopatho-
logical evaluation of liver sections by Oil Red O and H&E staining confirmed 3s as a potent, orally active
molecule for reducing fat deposition against non-alcoholic fatty liver disease.
Keywords:
NAFLD
Insulin resistance
Adiponectin expression
Barbituric acid
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
body mass index (BMI), plasma glucose, insulin and triglycerides but
positively correlated with the plasma levels of high-density lipo-
Non-alcoholic fatty liver disease (NAFLD), defined as fatty
infiltration of the liver exceeding 5%e10% by weight, is a clinic
pathological syndrome characterized by hepatic steatosis without
excess alcohol intake. It has become one of the leading causes of
chronic liver disease in many countries [1e3]. NAFLD comprises
a wide spectrum ranging from simple fatty liver to non-alcoholic
steatohepatitis (NASH), followed by progression to fibrosis,
cirrhosis and hepatocellular carcinoma [4,5]. This disease is closely
associated with insulin resistance (IR) and metabolic syndromes
including obesity, type II diabetes, dyslipidemia, hypertension and
atherosclerosis [6e9].
The adipose tissue secretes many physiologically active adipo-
cytokines such as adiponectin and leptin, which modulate hepatic
and peripheral lipid and glucose metabolism [10,11]. Adiponectin is
a 30-kDa adipocyte protein exclusively expressed in adipose tissue
of normal humans [12]. The plasma adiponectin level, directly
associated with insulin sensitivity, is negatively correlated with
protein (HDL)-cholesterol [13e15]. Leptin, the product of the obese
gene, is an adipocyte-secreted protein hormone with an important
role in energy homeostasis, including appetite, body weight and
metabolism functions. The amount of leptin secreted and expressed
by adipocytes is positively correlated with the lipid content and
adipocyte size [16]. Therefore, the roles of adiponectin and leptin on
regulating energy balance and metabolism make them as potential
therapeutic implications for humans and animals of NAFLD.
As insulin resistance is very common in patients with non-alco-
holic fatty liver disease, considerable studies developed novel
insulin sensitizers as logical treatment strategies. Biguanides and
thiazolidinediones (TZDs) are representatives insulin sensitizers
widely used in clinic (Fig. 1). Metformin, whose mechanisms of
action are not well understood, effectively improves hepatic stea-
tosis in animal model of fatty liver [17] and aminotransaminase
levels in human liver tissue [18] and increases insulin sensitivity by
facilitating glucose consumption and utilization [19]. TZDs, such as
rosiglitazone, are a kind of oral anti-diabetic medications that
significantly ameliorate insulin resistance by acting as selective
peroxisome proliferitor-activated receptor
g (PPAR g) agonists [20].
In addition, TZDs increase circulating levels of adiponectin in
adipose tissue [21e23] and has insulin sensitizing properties to slow
* Corresponding author. Tel.: þ86 28 85164063; fax: þ86 28 85164060.
E-mail address: lijuan17@hotmail.com (L. Chen).
These authors contributed equally.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.033

2004
L. Ma et al. / European Journal of Medicinal Chemistry 46 (2011) 2003e2010
derivatives, 4-hydroxybenzaldehyde, potassium carbonate as base
and potassium iodide as catalyst according to Scheme 1, respectively.
The reaction mixture was refluxed in a solvent of acetone for 24 h and
monitored by the thin-layer chromatography.
And then, to obtain the forty-four targeted compounds varying
with the western substituted group quickly, the Knoevenagel
reaction which accomplished by condensation aldehyde inter-
mediate with babituric acid or thiobarbituric acid and employed
water and ethanol as solvent was performed in parallel using
EYELA Personal Organic Synthesizer (Tokyo, Rikakikai) with a 12-
well liquid-phase reaction block. Through 4 or 5 h’ reaction, the
obtained crude was precipitated in the solvent, filtered and
washed with ethyl ether and water because the babituric acid or
thiobarbituric acid was dissolved into ethyl ether. The advantages
of the Knoevenagel synthetic protocol were generalized [27]: (a)
all starting materials were soluble in the mixture of water and
ethanol; (b) the reaction was clean and quick; (c) the insoluble
targeted compound was precipitated in the course of the reaction,
yielding essentially pure final compound (purities ꢀ97%) and
requiring no further purification. At this stage, the products were
fully analyzed and characterized by hydrogen nuclear magnetic
resonance (1H NMR), Mass spectrum (MS), and High Performance
Liquid Chromatography (HPLC) before entering the biological
tests.
Fig. 1. Structure of Metformin, Rosiglitazone and 3s.
and prevent the progression of NAFLD [24,25]. Recently, a new series
of PPAR ligands based on barbituric acid instead of TZD were
designed and evaluated and six active compounds were found to be
bound to the murine PPAR with IC50 ranging from 0.1 to 2.5
g
g
mM
[26]. Their results provided the impetus to develop novel and potent
therapeutic agents containing barbituric acid or thiobarbituric acid
moiety and investigate their pharmacological functions.
2. Chemistry
3. Biological results and discussion
Various amines inthisstudy wereconverted into chloroacetamide
derivatives (1aev) with excellent yields via a reaction employed 2-
chloroacetyl chloride agent in the presence of triethylamine as base
and dichloromethane as solvent. The similar reactions with the high
yield were dangerous and toxic to organic synthetic operators
becauseamountofhydrogenchloridegaswasreleased. Therefore, the
agent of 2-chloroacetyl chloride must be added slowly in the ice-bath
with the application of absorption solution for tail gas. The pivotal
aldehyde intermediates which contained 2-(4-formyl-phenoxy)-N-
substituted-phenylacetamide (2aep) and special aldehyde interme-
diates (2qev) were synthesized through the use of chloroacetamide
3.1. In vitro assay for adiponectin expression in 3T3-L1 adipocytes
In order to quickly discover the potential therapeutic agents, the
expression of adiponectin was evaluated by measuring the
promoting rate of the adiponection in cultured 3T3-L1 adipocytes.
Rosiglitazone (Fig. 1) was selected as a positive control. Forty-four
compounds based on barbituric acid and thiobarbituric acid moiety
with respective concentration of 10
mM were investigated the
effects on differentiation of 3T3-L1 preadipocytes into adipocytes
by measuring the promoting rates of adiponectin expression. Their
results were depicted in Fig. 2.
Scheme 1. General synthesis of 3, 4aev. Reagents and conditions: (a) 2-chloroacetyl chloride, Et3N, CH2Cl2, 0 ꢁCe25 ꢁC, 20 h; (b) KI, K2CO3, 4-hydroxybenzaldehyde, reflux,
24 h; (c) barbituric acid or thiobarbituric acid, ethanol, H2O, 60 ꢁC, 3e4 h.

L. Ma et al. / European Journal of Medicinal Chemistry 46 (2011) 2003e2010
2005
Fig. 2. Expression of adiponectin under the actions of 3, 4aev in 3T3-L1 adipocytes. Rosiglitazone (RSG) and 3, 4aev were at a same concentration of 10
three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs. vehicle (as baseline).
m
M. Results were at least
As shown in Fig. 2, the initial screening data obtained from the
compounds indicated that promoting rates of adiponectin expres-
sion of compounds (3a, 3o, 3s and 4t) were up-regulated by 135.18%,
143.80%, 180.32% and 146.94%, respectively (The promoting rate of
vehicle was defined as a 100%). Among them, 3s exhibited the most
efficacious compared with positive compound of rosiglitazone
which the promoting rate of adiponectin expression was 157.31%.
As for 3aep and 4aep, the substituents of electron-donating
group (methyl, methoxyl and trifluromethyl group) and electron-
withdrawing group (acetyl group, halogen including fluorine, chlo-
rine and bromine atom) were introduced in the western substituted
part and these compounds were observed no or much lower adipo-
nectin expressions, with the only exception being the 3, 4-difluro
group which significantly promoted the expression of adiponectin.
Additionally, varying with cyclohexyl, benzyl and naphthyl group in
the western group was no remarkable increment of adiponectin
expression. By inspection, one explanation about the strong activities
of 3s and 4t argued that the pyridine group possessed perfect
hydrophilic property in comparison to the remaining compounds
whose western parts were all hydrophobic groups.
weight and visceral fat reached respectively 16.4%, 25.3% and 19.63%
compared with the untreated HF/HC diet group and further the 3s-
treated rats were nearly equal-weight to those of the normal group,
as depicted in Table 2. Throughout the in vivo experiment, decre-
ments in levels of FBS, TG and ALT were 48.77%, 57.23% and 30.43%,
respectively. The insulin sensitivity was assessed by HOMA-IR values
and increased significantly through treatment with 3s, as indicated
by a 54.08% reduction in HOMA-IR. Consistent with our in vitro
results, the serum levels of adiponectin and leptin had achieved to
normal levels. The levels of low-density lipoprotein (LDL)-choles-
terol were down-regulated from 0.97 mM in HF/HC diet model to
0.175 mM in 3s-treated model. However, the serum levels of high-
density lipoprotein (HDL)-cholesterol were unexpected to ascend
after induced by HF/HC diet. The possible reason was that the
amount of cholesterol was elevated in serum of Wistar rats access to
HF/HC diet, and then the HDL tended to carry cholesterol away from
the arteries and back to the liver for excretion or reutilization.
Nevertheless, no abnormal behavioral or physiological signs were
observed during the experiment. In general, 3s significantly allevi-
ated the progression of NAFLD in male Wistar rats.
3.2. Expression of adiponectin and leptin in 3T3-L1 adipocytes in
vitro
3.4. Histopathological evaluation
To evaluate whether 3s improved fat deposition and recruit-
ment of liver cells in Wistar rat model with NAFLD, liver tissues
from 3s-treated and vehicle-treated rats were processed routinely
for Oil Red O and H&E staining assay (Fig. 3). As shown in Oil Red O
staining, we found the distinctly increased number of fat deposition
(the red sections) in the liver tissue from Wistar rats access to HF/
HC diet versus the normal ones, whereas administration of 3s
decreased efficaciously the fat accumulation of liver sections.
In order to evaluate the effects of 3a, 3o, 3s and 4t on adipo-
genesis of 3T3-L1 adipocytes, we further determined the expres-
sion of adiponectin and leptin in 3T3-L1 adipocytes by commercial
kits (Table 1). Consistent with the results obtained from promoting
rates of adiponectin expression at a concentration of 10
mM, the
four compounds significantly down-regulated the expression of
leptin in contrast to rosiglitazone and the 3s was the most potent,
worthy of further biological assays as a potential agent for non-
alcoholic fatty liver disease. Additionally, the four compounds were
no distinct cytotoxicity compared to vehicle in 3T3-L1 adipocytes
(S2 in Supplementary data).
Table 1
Expression values of adiponectin and leptin in 3T3-L1 adipocytes.
a
Compound
Adiponectin (ng/mL)
Leptin (pg/mL)
Vehicle
RSG
3a
3o
3s
843.78 ꢂ 20.61
1324.73 ꢂ 42.47 **
1111.73 ꢂ 91.60
1322.02 ꢂ 62.84 *
1639.61 ꢂ 30.25 **
1239.44 ꢂ 74.05*
3089.21 ꢂ 33.37
1773.67 ꢂ 182.89
**
3.3. Measurement of biochemical markers in treated and untreated
Wistar rat model with NAFLD
1856.32 ꢂ 158.25*
1571.98 ꢂ 95.13
**
1271.98 ꢂ 40.75
Further in vivo studies in male Wistar rats with NAFLD induced by
high-fat/high calorie diet (HF/HC) confirmed our in vitro screening
results. After oral administration of 3s once a day at a dose of
50 mg/kg for four weeks, the average decrease in body weight, liver
4t
1514.81 ꢂ 80.95
*P < 0.05; **P < 0.01 vs. vehicle.
a
Gosiglitazone (RSG) and 3a, 3o, 3s and 4t were at a concentration of 10
Results were means ꢂ SD of at least three independent experiments.
mM.

2006
L. Ma et al. / European Journal of Medicinal Chemistry 46 (2011) 2003e2010
Table 2
agent for the treatment of HF/HC diet-induced non-alcoholic fatty
liver disease in male Wistar rats. In addition, we adopted a feasible
and credible drug screening method on adiponectin expression for
our forty-four synthetic compounds in vitro, and then followed by
serum assays of ALT, TG, FBS, insulin, adiponectin, leptin, LDL-
cholesterol and histopathological evaluation in vivo. Our results
indicated that 3s based on barbituric acid at a concentration of
10 mM significantly up-regulated the expression of adiponectin as
well as down-regulated the expression of leptin in 3T3-L1 adipo-
cytes and further promoted glucose consumption with insulin
Biochemical characteristics of untreated and 3s-treated NAFLD rat model access to
HF/HC diet for 10 weeks.
Parameters
Normal
HF/HC
HF/HCþ3sa
Body weight (g)
Liver weight (g)
Liver/body weight (%)
Visceral fat (g/100g BW)
FBS (mM)
349.8 ꢂ 15.9
15.0 ꢂ 2.7
4.29
431.0 ꢂ 19.4
21.4 ꢂ 3.1
360.5 ꢂ 11.9*
16.0 ꢂ 2.0
4.95
4.34*
3.84 ꢂ 0.31
4.77 ꢂ 0.42
29.03 ꢂ 2.68
6.15 ꢂ 0.05
1.18 ꢂ 0.12
30.12 ꢂ 3.48
12.87 ꢂ 2.50
5.77 ꢂ 1.84
0.44 ꢂ 0.23
0.69 ꢂ 0.12
4.95 ꢂ 0.95
9.23 ꢂ 1.68
36.32 ꢂ 2.97
15.31 ꢂ 0.22
2.38 ꢂ 1.20
47.52 ꢂ 4.98
10.49 ꢂ 2.59
7.59 ꢂ 2.36
0.97 ꢂ 0.31
1.20 ꢂ 0.43
3.98 ꢂ 0.39
4.73 ꢂ 0.86**
33.43 ꢂ 2.11*
7.03 ꢂ 0.08**
1.02 ꢂ 0.98*
33.06 ꢂ 2.69**
12.53 ꢂ 1.27*
6.00 ꢂ 1.01*
0.18 ꢂ 0.09*
1.11 ꢂ 0.91
Insulin (mU/mL)
HOMA-IRb
TG (mM)
(0.1 mM) in HepG2 cell lines (see S3 in supplementary data), which
ALT (U/L)
Adiponectin (mg/mL)
Leptin (ng/mL)
LDL-cholesterol (mM)
HDL-cholesterol (mM)
exceeded the potency of known insulin sensitizers, rosiglitazone
and metformin. Serum assay and histopathological evaluation
further validated that 3s remarkably reduced lipid accumulation in
the liver tissue correlated with NAFLD. However, additional studies
are still warranted on the pharmacology of the described
compounds for an evaluation of their mechanism and pharmaco-
kinetic properties and for a deeper investigation of their activity
profile against NAFLD.
*P < 0.05; **P < 0.01 vs. normal.
a
3s was orally administrated once a day at a dose of 50 mg/kg for four weeks.
Results were means ꢂ SD.
b
HOMA-IR [31] was defined as follows. HOMA-IR ¼ [FBS (mM) ꢃ Insulin (
mU/
mL)]/22.5.
In addition, fat deposition ruined severely the liver histology pre-
senting as ballooning degeneration, and numerous lipid droplets as
evidenced by H&E staining. After the rats were treated with 3s,
recruitment and restoration of liver cells versus the normal ones
were improved remarkably. The results suggested that 3s reduced
fat deposition effectively and possessed liver protective effects with
non-alcoholic fatty liver disease.
5. Experimental section
5.1. Biological methods
5.1.1. 3T3-L1 Adipocytes differentiation
Marine 3T3-L1 preadipocytes (American Type Culture Collec-
tion, Rockville, MD) were plated and grown to two days post-
confluence in 96 or 6 well culture plates in DMEM containing 10%
fetal bovine serum; Preadipocytes were induced to differentiate by
replacing the medium with serum-containing DMEM containing
4. Conclusion
In this present study, we discovered a novel compound 3s and
0.5 mM methyl-3-isobutylxantine (IBMX), 0.25
mM dexamethasone
investigated its pharmacological effects as a potential therapeutic
(DEX) and 1 g/mL insulin. Two days later, the medium was again
m
Fig. 3. Oil Red O and H&E staining of liver sections of untreated and 3s-treated NAFLD rat model access to HF/HC diet. Oil Red O staining: (A) Normal diet; (B) Model access to HF/HC
diet; (C) 3s-treated model access to HF/HC diet. H&E staining: (D) Normal diet; (E) Model access to HF/HC diet; (F) 3s-treated model access to HF/HC diet.

L. Ma et al. / European Journal of Medicinal Chemistry 46 (2011) 2003e2010
2007
changed to serum-containing DMEM that contained insulin but no
IBMX or DEX. Two days later, the medium was again changed to the
original DMEM containing 10% fetal bovine serum in the absence of
any differentiating reagents and was replaced every two days. Full
differentiation is usually achieved by 8e12 days.
Ocean Chemical Factory, Shangdong, China). The purity of
compound screened in biological assays was determined to be
ꢀ97% by HPLC analysis with a phodediode array detector (Waters,
Milford, MA, USA) and the chromatographic column was an atlantis
C18 (150 mm ꢃ 4.6 mm, i.d. 5
mm) (Waters, Milford, Ireland).
Hydrogen Nuclear magnetic resonance spectra (1H NMR) were
recorded at 400 MHz on a Varian spectrometer (Varian, Palo Alto,
CA, USA) model Gemini 400 and reported in parts per million.
5.1.2. Expression levels of adiponectin and leptin in 3T3-L1
adipocytes
The twelfth day after differentiation, the culture medium was
replaced by DMEM supplemented with chemical series of 3 and 4
Chemical shifts (
(TMS) as an internal standard, where (
d
) are quoted in ppm relative to tetramethylsilane
) TMS ¼ 0.00 ppm. The
d
(10
mM) and rosiglitazone (SigmaeAldrich, St. Louis, MO, USA) (10
mM).
multiplicity of the signal is indicated as s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet, defined as all multipeak signals
where overlap or complex coupling of signals makes definitive
descriptions of peaks difficult. Mass Spectra (MS) were measured
by Q-TOF Priemier mass spectrometer utilizing electrospray ioni-
zation (ESI) (Micromass, Manchester, UK). Room temperature (RT)
is within the range 20e25 ꢁC.
After 24 h, the expression levels of adiponectin and leptin were
measured by commercial kits (Linco Research, St. Charles, MO, USA).
5.1.3. Animal model and treatment of NAFLD
Normal male Wistar rats weighing 140e180 g were purchased
from Western China Experimental Animal Center. Every five rats
were placed in one cage on a 12 h day/night cycle and quarantined
for 1 week. All animals, which were maintained under controlled
conditions and had free access to standard laboratory chow and
water, received human care according to National Institutes of
Health Guidelines before and during experiment. After acclimati-
zation for 1 week, animals were assigned to four groups randomly
consisting of 10 rats each (G1eG3). In this study, G1eG2 groups
were given access to high-fat diet and G3 group access to normal
diet throughout the experimental period, respectively. The normal
diet was purchased from Shuangshi experimental animal diet
center (Jiangshu province, PR China). The high-fat/high calorie diet
consisted of 20% lard stearin (wt/wt), 10% sucrose and 0.1% bile salt
were added into normal diet. The rats received a normal diet with
18.94% of energy derived from fat, 31.67% from protein and 49.39%
from carbohydrates, which received a high-fat diet with 50.55% of
energy derived from fat, 15.72% from protein and 33.73% from
carbohydrates. In addition to the daily diet, compound 3s sus-
pended in Tween 80 and 5% saline (SigmaeAldrich, St. Louis, MO),
were administered correspondingly to G1 group orally once a day at
a dose of 50 mg/kg. After the experimental period completed, all
rats were anesthetized and samples were collected. Meanwhile,
body weight and liver weight of rats was recorded.
5.2.1. General procedure for synthesis of 2-chloro-N-substituted-
acetamide (1aev)
2-Chloroacetyl chloride (24 mmol) was slowly added dropwise
to a mixture of R-NH2 (20 mmol) and Et3N (24 mmol, 3.3 mL) in
anhydrous CH2Cl2 (20 mL) at 0 ꢁC. The reaction mixture was
warmed to room temperature and stirred for an additional 20 h.
After the solvent was removed under reduced pressure, the residue
was washed with ice water (3 ꢃ 20 mL) and the precipitate was
separated by filtration. The crude product was purified by crystal-
lization from a mixture solvent of Et2O/petroleum.
5.2.2. General procedure for synthesis of 2-(4-formyl-phenoxy)-N-
substituted-phenyl-acetamide and special aldehyde intermediates
(2aev)
4-hydroxybenzaldehyde (1.34 g, 11 mmol), anhydrous K2CO3
(2.76 g, 20 mmol) and the 2-chloro-N-substituted-acetamide
(10 mmol) were dissolved in anhydrous acetone (30 mL), and then
KI (166 mg, 1 mmol) were added into the solution. The reaction
mixture was refluxed for 24 h and then cooled to room tempera-
ture. Then the K2CO3 solid was filtered and the acetone solution was
removed under reduced pressure to obtain the crude products. The
residue was purified by silica gel column chromatography (eluent:
ethyl acetate/petroleum ¼ 1/1.5) to give the appropriate aldehyde
product.
5.1.4. Assay for serum biochemical markers
Serum ALT levels were measured by an automated enzyme
assay using commercial kits and Roche automated analyzers (Roche
Diagnostics GmbH, Manheim, Germany). Serum levels of TG, LDL-
cholesterol, HDL-cholesterol, FBS and plasma insulin were deter-
mined by radioimmunoassay using commercially available kits
(Linco Research, St. Charles, MO, USA).
5.2.3. General procedure for synthesis of 3, 4aev
The appropriate aldehyde (3.5 mmol), ethanol (10 mL), distilled
water (10 mL), and (thio) barbituric acid (3 mmol) were added in
parallel in 12 test tubes with reflux condensers and stirred at
1000 rpm in tubes at 80 ꢁC for 3e4 h. The formed solids were
collected by sucking filtration and washed with boiling water
(3 ꢃ 50 mL), ethanol (3 ꢃ 50 mL), and ether (3 ꢃ 25 mL). The
colorful solids obtained were dried in vacuum at 40 ꢁC for 12 h.
5.1.5. Histopathological examination
Liver samples were fixed in 4% buffered formalin and embedded
in Tissue-Tek OCT compound (Sakura Finetek USA, California, USA)
and paraffin for histological analysis. Formalin-fixed and paraffin-
embedded section (5
mm) was processed routinely for H&E staining.
5.2.3.1. N-Phenyl-2-(4-((2,4,6-trioxotetrahydropyrimidin-5(6H)-yli-
The OCT-embedded samples were serially sectioned at 4
the evaluation of fat deposition, the liver section was stained with
Oil red O.
m
m. For
dene)methyl)phenoxy)acetamide (3a). Yield: 89.4%; HPLC: 99.6%;
Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.32 (s, 1H), 11.19 (s,
1H), 10.17 (s, 1H), 8.36 (d, 2H, J ¼ 8.8 Hz), 8.25 (s, 1H), 7.63 (d, 2H,
J ¼ 7.6 Hz), 7.33 (t, 2H, J ¼ 8.0 Hz), 7.13e7.07 (m, 2H), 4.86 (s, 2H);
5.2. Chemistry
13C NMR (400 MHz, DMSO-d6):
d 167.69, 162.10, 161.89, 157.31,
154.54, 150.42, 138.14, 130.28, 130.20, 128.49, 128.68, 121.56,
Chemical reagents of analytical grade were purchased from
Chengdu Changzheng Chemical Factory (Sichuan, PR China). The
final compounds were synthesized using an EYELA Personal
Organic Synthesizer with ChemiStation PPS-CTRL and PPW-20A
(Tokyo, Rikakikai) using a 5-well liquid-phase reaction block. TLC
was performed on 0.20 mm silical gel 60 F254 plates (Qingdao
121.60, 119.12, 114.20, 114.15, 65.89; MS (ESI), m/z: 364.18 [M ꢄ H]ꢄ.
5.2.3.2. N-p-Tolyl-2-(4-((2,4,6-trioxotetrahydropyrimidin-5(6H)-yli-
dene)methyl)phenoxy)acetamide (3b). Yield: 78.0%; HPLC: 98.4%;
Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
11.32 (s, 1H), 11.19 (s,
1H), 10.08 (s, 1H), 8.36 (d, 2H, J ¼ 9.2 Hz), 8.25 (s, 1H), 7.51 (d, 2H,

2008
L. Ma et al. / European Journal of Medicinal Chemistry 46 (2011) 2003e2010
J ¼ 8.4 Hz), 7.12 (t, 4H, J ¼ 8.0 Hz), 4.84 (s, 2H), 2.26 (s, 3H); MS (ESI),
m/z: 378.15 [M ꢄ H]ꢄ.
5.2.3.12. N-(2,4-Dichlorophenyl)-2-(4-((2,4,6-trioxotetrahydropyr-
imidin-5(6H)-ylidene)methyl)phenoxy)acetamide (3l). Yield: 87.3%;
HPLC: 99.6%; Light yellow solid; 1H NMR (400 MHz, DMSO-d6):
5.2.3.3. N-(4-Methoxyphenyl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
d
11.33 (s,1H),11.20 (s,1H), 9.84 (s,1H), 8.36 (d, 2H, J ¼ 8.8 Hz), 8.26 (s,
5(6H)-ylidene)methyl)phenoxy)acetamide (3c). Yield: 89.4%; HPLC:
1H), 7.81 (d,1H, J ¼ 8.4 Hz), 7.47e7.44 (m,1H), 7.17e6.98 (m,1H), 7.13
98.5%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.32 (s, 1H),
(d, 2H, J ¼ 8.8 Hz), 4.95 (s, 2H); MS (ESI), m/z: 464.19 [M ꢄ MeOH]ꢄ.
11.19 (s,1H),10.03 (s,1H), 8.36 (d, 2H, J ¼ 10.2 Hz), 8.25 (s,1H), 7.54 (d,
2H, J ¼ 10.2 Hz), 7.11 (d, 2H, J ¼ 8.4 Hz), 6.90 (d, 2H, J ¼ 7.2 Hz), 4.82 (s,
2H), 3.73 (s, 3H); MS (ESI), m/z: 394.20 [M ꢄ H]ꢄ.
5.2.3.13. N-(3,5-Dichlorophenyl)-2-(4-((2,4,6-trioxotetrahydropyr-
imidin-5(6H)-ylidene)methyl)phenoxy)acetamide (3m). Yield: 92.1%;
HPLC: 99.7%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.33 (s,
5.2.3.4. N-(3,4-Dimethoxyphenyl)-2-(4-((2,4,6-trioxotetrahydropyr-
1H), 11.20 (s, 1H), 10.49 (s, 1H), 8.36 (d, 2H, J ¼ 8.8 Hz), 8.26 (s, 1H),
7.74 (d, 2H, J ¼ 1.6 Hz), 7.33 (s, 1H), 7.12 (d, 2H, J ¼ 9.2 Hz), 4.90 (s,
2H); MS (ESI), m/z: 465.94 [M ꢄ MeOH]ꢄ.
imidin-5(6H)-ylidene)methyl)phenoxy)acetamide (3d). Yield: 76.0%;
HPLC: 99.8%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.32 (s,
1H), 11.19 (s, 1H), 10.02 (s, 1H), 8.37 (d, 2H, J ¼ 8.8 Hz), 8.26 (s, 1H),
7.36 (d, 1H, J ¼ 2.0 Hz), 7.17e7.10 (m, 3H), 6.91 (d, 1H, J ¼ 8.8 Hz),
4.83 (s, 2H), 3.72 (s, 6H); MS (ESI), m/z: 424.07 [M ꢄ H]ꢄ.
5.2.3.14. N-(3-Chloro-4-fluorophenyl)-2-(4-((2,4,6-trioxotetrahydropyr-
imidin-5(6H)-ylidene)methyl)phenoxy)acetamide (3n). Yield: 98.7%;
HPLC: 99. 6%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.32 (s,
5.2.3.5. N-(4-(Trifluoromethyl)phenyl)-2-(4-((2,4,6-trioxotetrahydropyr-
1H), 11.19 (s, 1H), 10.38 (s, 1H), 8.35 (d, 2H, J ¼ 8.8 Hz), 8.25 (s, 1H),
7.95e7.93 (m, 1H), 7.58e7.54 (m,1H), 7.40 (d,1H, J ¼ 8.8 Hz), 7.11 (d, 2H,
J ¼ 8.8 Hz), 4.87 (s, 2H); MS (ESI), m/z: 416.09 [M ꢄ H]ꢄ.
imidin-5(6H)-ylidene)methyl)phenoxy)acetamide (3e). Yield: 83.1%;
1
HPLC: 99.5%; Yellow solid; H NMR (400 MHz, DMSO-d6):
d 11.32 (s,
1H), 11.19 (s, 1H), 10.54 (s, 1H), 8.35 (d, 2H, J ¼ 8.8 Hz), 8.25 (s, 1H), 7.85
(d, 2H, J ¼ 8.0 Hz), 7.70 (d, 2H, J ¼ 8.4 Hz), 7.11 (d, 2H, J ¼ 8.8 Hz), 4.91 (s,
2H); MS (ESI), m/z: 464.08 [M ꢄ MeOH]ꢄ.
5.2.3.15. N-(3,4-Difluorophenyl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
5(6H)-ylidene)methyl)phenoxy)acetamide (3o). Yield: 94.4%; HPLC:
99.8%; Light yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.32 (s, 1H),
5.2.3.6. N-(4-Acetylphenyl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
11.19 (s, 1H), 10.39 (s, 1H), 8.35 (d, 2H, J ¼ 8.8 Hz), 8.25 (s, 1H), 7.82e7.77
5(6H)-ylidene)methyl)phenoxy)acetamide (3f). Yield: 97.5%; HPLC:
(m, 1H), 7.47e7.38 (m, 2H), 7.11 (d, 2H, J ¼ 8.4 Hz), 4.87 (s, 2H); 13C NMR
99.4%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
11.32 (s, 1H),
(400 MHz, DMSO-d6): d167.84, 162.33, 162.13, 157.38, 154.56, 150.42,
11.19 (s, 1H), 10.52 (s, 1H), 8.35 (d, 2H, J ¼ 8.8 Hz), 8.25 (s, 1H), 7.95
(d, 2H, J ¼ 8.4 Hz), 7.77 (d, 2H, J ¼ 8.8 Hz), 7.11 (d, 2H, J ¼ 9.2 Hz),
4.91 (s, 2H), 2.53 (s, 3H); MS (ESI), m/z: 438.26 [M ꢄ MeOH]ꢄ.
147.21, 145.11, 135.76, 130.23, 130.25, 125.65, 121.78, 118.59, 114.89,
114.75, 114.13, 111.70, 59.61; MS (ESI), m/z: 432.27 [M ꢄ MeOH]ꢄ.
5.2.3.16. N-Methyl-N-phenyl-2-(4-((2,4,6-trioxotetrahydropyrimidin-
5.2.3.7. N-(3-Fluorophenyl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
5(6H)-ylidene)methyl)phenoxy)acetamide (3p). Yield: 83.8%; HPLC:
5(6H)-ylidene)methyl)phenoxy)acetamide (3g). Yield: 88.7%; HPLC:
99.0%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.30 (s, 1H),
97.3%; Light yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
11.31 (s,
11.17 (s, 1H), 8.30 (d, 2H, J ¼ 8.4 Hz), 8.22 (s, 1H), 7.500e7.41 (m, 5H),
6.89 (s, 2H), 4.59 (s, 2H), 3.20 (s, 3H); MS (ESI), m/z: 410.28
[M ꢄ MeOH]ꢄ.
1H), 11.19 (s, 1H), 10.38 (s, 1H), 8.35 (d, 2H, J ¼ 8.8 Hz), 8.25 (s, 1H),
7.60 (d, 1H, J ¼ 11.6 Hz), 7.37e7.35 (m, 2H), 7.10 (d, 2H, J ¼ 8.8 Hz),
6.92e6.90 (m, 1H), 4.87 (s, 2H); MS (ESI), m/z: 414.11 [M ꢄ MeOH]ꢄ.
5.2.3.17. N-Cyclohexyl-2-(4-((2,4,6-trioxotetrahydropyrimidin-5(6H)-
5.2.3.8. N-(4-Fluorophenyl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
ylidene)methyl)phenoxy)acetamide (3q). Yield: 87.8%; HPLC: 99.1%;
5(6H)-ylidene)methyl)phenoxy)acetamide (3h). Yield: 86.1%; HPLC:
Light yellow solid; 1H NMR (400 MHz, DMSO-d6)
d 11.31 (s, 1H), 11.19
99.4%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
11.32 (s, 1H),
(s, 1H), 8.34 (d, 1H, J ¼ 8.8 Hz), 8.25 (s, 1H), 7.98 (d, 1H, J ¼ 8.0 Hz),
7.05 (d, 2H, J ¼ 9.2 Hz), 4.60 (s, 2H), 3.61 (t, 1H, J ¼ 4.4 Hz), 1.74e1.68
(m, 4H), 1.58e1.55 (m, 1H), 1.32e1.19 (m, 4H), 1.15e1.06 (m, 1H); MS
(ESI), m/z: 402.14 [M ꢄ MeOH]ꢄ.
11.19 (s, 1H), 10.23 (s, 1H), 8.36 (d, 2H, J ¼ 9.2 Hz), 8.25 (s, 1H),
7.67e7.63 (m, 2H), 7.18 (t, 2H, J ¼ 8.8 Hz), 7.11 (d, 2H, J ¼ 8.0 Hz),
4.86 (s, 2H); MS (ESI), m/z: 414.19 [M ꢄ MeOH]ꢄ.
5.2.3.9. N-(3-Chlorophenyl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
5.2.3.18. N-Benzyl-2-(4-((2,4,6-trioxotetrahydropyrimidin-5(6H)-
5(6H)-ylidene)methyl)phenoxy)acetamide (3i). Yield: 95.0%; HPLC:
ylidene)methyl)phenoxy)acetamide (3r). Yield: 85.9%; HPLC:
99.8%; Light yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
11.31 (s,
99.5%; Light yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.31
1H), 11.19 (s, 1H), 10.35 (s, 1H), 8.35 (d, 2H, J ¼ 8.8 Hz), 8.25 (s, 1H),
7.83 (s, 1H), 7.52 (d, 1H, J ¼ 8.0 Hz), 7.36 (t, 1H, J ¼ 8.0 Hz), 7.15e7.10
(m, 3H), 4.87 (s, 2H); MS (ESI), m/z: 430.13 [M ꢄ MeOH]ꢄ.
(s, 1H), 11.19 (s, 1H), 8.73 (t, 1H, J ¼ 6.0 Hz), 8.34 (d, 2H, J ¼ 8.4 Hz),
8.25 (s, 1H), 7.33e7.21 (m, 5H), 7.08 (d, 2H, J ¼ 8.4 Hz), 4.71 (s,
2H), 4.35 (d, 2H, J ¼ 6.0 Hz); MS (ESI), m/z: 410.22 [M ꢄ MeOH]ꢄ.
5.2.3.10. N-(4-Chlorophenyl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
5.2.3.19. N-(Pyridin-2-yl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
5(6H)-ylidene)methyl)phenoxy)acetamide (3s). Yield: 84.9%;
HPLC: 99.1%; Light yellow solid; 1H NMR (400 MHz, DMSO-d6)
5(6H)-ylidene)methyl)phenoxy)acetamide (3j). Yield: 45.0%; HPLC:
99.2%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.32 (s, 1H),
11.19 (s,1H), 10.32 (s, 1H), 8.36 (d, 2H, J ¼ 8.8 Hz), 8.25 (s,1H), 7.67 (d,
2H, J ¼ 8.4 Hz), 7.39 (d, 2H, J ¼ 8.8 Hz), 7.11 (d, 2H, J ¼ 8.8 Hz), 4.87 (s,
2H); MS (ESI), m/z: 398.11 [M ꢄ H]ꢄ.
d
11.32 (s, 1H), 11.19 (s, 1H), 10.66 (s, 1H), 8.35 (d, 3H, J ¼ 8.8 Hz),
8.25 (s, 1H), 8.04 (d, 1H, J ¼ 8.4 Hz), 7.83e7.78 (m, 1H), 7.16e7.13
(m, 1H), 7.09 (d, 2H, J ¼ 8.4 Hz), 4.96 (s, 2H); 13C NMR (400 MHz,
DMSO-d6):
d 169.48, 162.30, 162.15, 157.31, 154.69, 151.49, 150.41,
5.2.3.11. N-(4-Bromophenyl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
146.71, 138.74, 130.21, 130.24, 125.29, 124.49, 118.90, 115.78,
5(6H)-ylidene)methyl)phenoxy)acetamide (3k). Yield: 95.6%; HPLC:
114.28, 114.21, 66.48; MS (ESI), m/z: 397.11 [M ꢄ MeOH]ꢄ.
98.4%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.32 (s, 1H),
11.19 (s, 1H), 10.31 (s, 1H), 8.35 (d, 2H, J ¼ 8.8 Hz), 8.24 (s, 1H), 7.61 (d,
2H, J ¼ 8.8 Hz), 7.51 (s, 2H, J ¼ 8.8 Hz), 7.10 (d, 2H, J ¼ 8.8 Hz), 4.86 (s,
2H); MS (ESI), m/z: 442.02 [M ꢄ H]ꢄ.
5.2.3.20. N-(5-Methylpyridin-2-yl)-2-(4-((2,4,6-trioxotetrahydro-
pyrimidin-5(6H)-ylidene)methyl)phenoxy)acetamide (3t). Yield:
89.4%; HPLC: 98.7%; Light yellow solid; 1H NMR (400 MHz,

L. Ma et al. / European Journal of Medicinal Chemistry 46 (2011) 2003e2010
2009
DMSO-d6)
d
11.32 (s, 1H), 11.19 (s, 1H), 10.56 (s, 1H), 8.35 (d, 2H,
HPLC: 97.9%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
12.40 (s,
J ¼ 8.8 Hz), 8.25 (s, 1H), 8.18 (s, 1H), 7.95 (d, 1H, J ¼ 7.6 Hz), 7.63
(dd, 1H, J ¼ 8.4 Hz), 7.08 (d, 2H, J ¼ 9.2 Hz), 4.94 (s, 2H), 2.26 (s,
3H); MS (ESI), m/z: 411.13 [M ꢄ MeOH]ꢄ.
1H), 12.30 (s, 1H), 10.39 (s, 1H), 8.40 (d, 2H, J ¼ 8.8 Hz), 8.26 (s, 1H),
7.60(d, 1H, J ¼ 10.4 Hz), 7.37e7.35 (m, 2H), 7.12 (d, 2H, J ¼ 8.8 Hz),
6.91e6.90 (m, 1H), 4.89 (s, 2H); MS (ESI), m/z: 430.09 [M ꢄ MeOH]ꢄ.
5.2.3.21. N-(Naphthalen-1-yl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
5.2.3.30. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-yli-
dene)methyl)phenoxy)-N-(4-fluorophenyl)acetamide (4h). Yield:
82.6%; HPLC: 99.6%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
5(6H)-ylidene)methyl)phenoxy)acetamide (3u). Yield: 89.2%; HPLC:
99.1%; Light yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 11.32 (s,
1H), 11.20 (s, 1H), 10.23 (s, 1H), 8.39 (d, 2H, J ¼ 8.8 Hz), 8.27 (s, 1H),
8.01e7.95 (m, 2H), 7.81 (d, 1H, J ¼ 8.0 Hz), 7.67e7.65 (m, 1H),
7.55e7.49 (m, 3H), 7.18 (d, 2H, J ¼ 8.0 Hz), 5.03 (s, 2H); MS (ESI), m/z:
446.16 [M ꢄ MeOH]ꢄ.
d
12.40 (s, 1H), 12.31 (s, 1H), 10.24 (s, 1H), 8.41 (d, 2H, J ¼ 9.2 Hz),
8.27 (s, 1H), 7.67e7.63 (m, 2H), 7.20e7.18 (m, 2H), 7.13 (d, 2H,
J ¼ 8.8 Hz), 4.87 (s, 2H); MS (ESI), m/z: 430.19 [M ꢄ MeOH]ꢄ.
5.2.3.31. N-(3-Chlorophenyl)-2-(4-((4,6-dioxo-2-thioxotetrahydropyr-
5.2.3.22. N-(Naphthalen-2-yl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-
imidin-5(6H)-ylidene)methyl)phenoxy)acetamide (4i). Yield: 84.1%;
5(6H)-ylidene)methyl)phenoxy)acetamide (3v). Yield: 96.3%; HPLC:
HPLC: 99.8%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 12.39 (s,
99.5%; Light yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
11.33 (s,
1H),12.30(s,1H),10.36(s,1H), 8.40(d, 2H, J ¼ 8.8 Hz), 8.26 (s,1H), 7.82
(s,1H), 7.51 (d,1H, J ¼ 8.4 Hz), 7.26 (t,1H, J ¼ 8.0 Hz), 7.16e7.11 (m, 3H),
4.86 (s, 2H); MS (ESI), m/z: 446.09 [M ꢄ MeOH]ꢄ.
1H), 11.20 (s, 1H), 10.40 (s, 1H), 8.37 (d, 2H, J ¼ 8.8 Hz), 8.32 (s, 1H),
8.26 (s, 1H), 7.91e7.82 (m, 3H), 7.67e7.65 (m, 1H), 7.50e7.40 (m, 2H),
7.15 (d, 2H, J ¼ 8.8 Hz), 4.94 (s, 2H); MS( ESI), m/z: [M ꢄ MeOH]ꢄ.
5.2.3.32. N-(4-Chlorophenyl)-2-(4-((4,6-dioxo-2-thioxotetrahydropyr-
5.2.3.23. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-ylidene)
imidin-5(6H)-ylidene)methyl)phenoxy)acetamide (4j). Yield: 45.7%;
methyl)phenoxy)-N-phenylacetamide (4a). Yield: 86.3%; HPLC: 99.2%;
HPLC: 99.7%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 12.40 (s,
Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
12.40 (s, 1H), 12.31 (s,
1H), 12.31 (s, 1H), 10.32 (s, 1H), 8.41 (d, 2H, J ¼ 8.8 Hz), 8.27 (s, 1H),
7.66 (d, 2H, J ¼ 8.8 Hz), 7.39 (d, 2H, J ¼ 8.8 Hz), 7.13 (d, 2H, J ¼ 8.8 Hz),
4.86 (s, 2H); MS (ESI), m/z: 446.09 [M ꢄ MeOH]ꢄ.
1H), 10.18 (s, 1H), 8.41 (d, 2H, J ¼ 7.2 Hz), 8.27 (s, 1H), 7.63 (d, 2H,
J ¼ 8.4 Hz), 7.33 (t, 2H, J ¼ 10.0 Hz), 7.14e7.07 (m, 2H), 4.88 (s, 2H); MS
(ESI), m/z: 380.03 [M ꢄ H]ꢄ.
5.2.3.33. N-(4-Bromophenyl)-2-(4-((4,6-dioxo-2-thioxotetrahydropyr-
5.2.3.24. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-yli-
imidin-5(6H)-ylidene)methyl)phenoxy)acetamide (4k). Yield: 98.9%;
dene)methyl)phenoxy)-N-p-tolylacetamide (4b). Yield: 80.9%;
HPLC: 98.8%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 12.40 (s,
HPLC: 98.2%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
12.40
1H),12.30 (s,1H), 10.31 (s,1H), 8.40 (d, 2H, J ¼ 8.8 Hz), 8.26 (s,1H), 7.61
(d, 2H, J ¼ 8.8 Hz),7.51 (d, 2H, J ¼ 8.4 Hz), 7.12 (d, 2H, J ¼ 8.8 Hz), 4.86 (s,
2H); MS (ESI), m/z: 458.22 [M ꢄ H]ꢄ.
(s, 1H), 12.31 (s, 1H), 10.09 (s, 1H), 8.41 (d, 2H, J ¼ 9.2 Hz), 8.27 (s,
1H), 7.51 (d, 2H, J ¼ 8.4 Hz), 7.14e7.12 (m, 4H), 4.86 (s, 2H), 2.26 (s,
3H); MS (ESI), m/z: 426.17 [M ꢄ MeOH]ꢄ.
5.2.3.34. N-(2,4-Dichlorophenyl)-2-(4-((4,6-dioxo-2-thioxotetrahy-
dropyrimidin-5(6H)-ylidene)methyl)phenoxy)acetamide (4l). Yield:
91.3%; HPLC: 99.6%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
5.2.3.25. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-yli-
dene)methyl)phenoxy)-N-(4-methoxyphenyl)acetamide (4c). Yield:
85.0%; HPLC: 98.7%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
12.41 (s,1H),12.32 (s,1H), 9.86 (s,1H), 8.42 (d, 2H, J ¼ 9.2 Hz), 8.28 (s,
d
12.40 (s, 1H), 12.31 (s, 1H), 10.04 (s, 1H), 8.41 (d, 2H, J ¼ 10.2 Hz),
1H), 7.81 (d, 1H, J ¼ 8.8 Hz), 7.71 (d,1H, J ¼ 2.6 Hz), 7.47e7.44 (m, 1H),
8.27 (s, 1H), 7.55e7.52 (m, 2H), 7.13 (d, 2H, J ¼ 10.2 Hz), 6.92e6.88
(m, 2H), 4.84 (s, 2H), 3.72 (s, 3H); MS (ESI), m/z: 442.10
[M ꢄ MeOH]ꢄ.
7.15 (d, 2H, J¼ 9.2 Hz), 4.94 (s, 2H); MS (ESI), m/z:480.16 [Mꢄ MeOH]ꢄ.
5.2.3.35. N-(3,5-Dichlorophenyl)-2-(4-((4,6-dioxo-2-thioxotetrahy-
dropyrimidin-5(6H)-ylidene)methyl)phenoxy)acetamide (4m). Yield:
99.1%; HPLC: 97.6%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
5.2.3.26. N-(3,4-Dimethoxyphenyl)-2-(4-((4,6-dioxo-2-thioxotetrahy-
dropyrimidin-5(6H)-ylidene)methyl)phenoxy)acetamide
(4d). Yield:
12.40
d
12.41 (s, 1H), 12.31 (s, 1H), 10.50 (s, 1H), 8.41 (d, 2H, J ¼ 8.8 Hz),
77.5%;HPLC:97.9%;Yellowsolid;1H NMR (400 MHz, DMSO-d6):
d
8.27 (s,1H), 7.73 (d, 2H, J ¼ 2.0 Hz), 7.33 (t,1H, J ¼ 2.0 Hz), 7.14 (d, 2H,
(s,1H),12.31(s,1H),10.03(s,1H),8.41(d,2H,J¼ 9.2Hz),8.27(s,1H),7.32
(d,1H, J ¼ 2.4 Hz), 7.17e7.12 (m, 3H), 6.91 (d, 1H, J ¼ 8.8 Hz), 4.84 (s, 2H),
3.73 (s, 3H), 3.72 (s, 3H); MS (ESI), m/z: 440.11 [M ꢄ H]ꢄ.
J ¼ 9.2 Hz), 4.91 (s, 2H); MS (ESI), m/z: 480.12 [M ꢄ MeOH]ꢄ.
5.2.3.36. N-(3-Chloro-4-fluorophenyl)-2-(4-((4, 6-dioxo-2-thio-
xotetrahydropyrimidin-5(6H)-ylidene)methyl)phenoxy)acetamide
(4n). Yield: 86.4%; HPLC: 98.7%; Yellow solid; 1H NMR
5.2.3.27. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-ylidene)
methyl)phenoxy)-N-(4-(trifluoromethyl)phenyl)acetamide (4e). Yield:
80.2%; HPLC: 99.3%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
(400 MHz, DMSO-d6):
d 12.40 (s, 1H), 12.31 (s, 1H), 10.38 (s, 1H),
8.40 (d, 2H, J ¼ 8.8 Hz), 8.27 (s, 1H), 7.95e7.93 (m, 1H), 7.57e7.54
(m, 1H), 7.40 (t, 1H, J ¼ 8.8 Hz), 7.13 (d, 2H, J ¼ 8.8 Hz), 4.89 (s,
2H); MS (ESI), m/z: 464.20 [M ꢄ MeOH]ꢄ.
d
12.40 (s, 1H), 12.30 (s, 1H), 10.55 (s, 1H), 8.40 (d, 2H, J ¼ 8.8 Hz), 8.26
(s, 1H), 7.85 (d, 2H, J ¼ 8.4 Hz), 7.70 (d, 2H, J ¼ 8.4 Hz), 7.13 (d, 2H,
J ¼ 8.4 Hz), 4.93 (s, 2H); MS (ESI), m/z: 480.06 [M ꢄ MeOH]ꢄ.
5.2.3.37. N-(3,4-Difluorophenyl)-2-(4-((4,6-dioxo-2-thioxotetrahy-
dropyrimidin-5(6H)-ylidene)methyl)phenoxy)acetamide (4o). Yield:
72.4%; HPLC: 98.9%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
5.2.3.28. N-(4-Acetylphenyl)-2-(4-((4,6-dioxo-2-thioxotetrahydropyr-
imidin-5(6H)-ylidene)methyl)phenoxy)acetamide (4f). Yield: 99.0%;
HPLC: 98.7%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d
12.40 (s,
d
12.40 (s, 1H), 12.30 (s, 1H), 10.40 (s, 1H), 8.40 (d, 2H, J ¼ 8.8 Hz),
1H), 12.30 (s, 1H), 10.53 (s, 1H), 8.41 (d, 2H, J ¼ 9.2 Hz), 8.27 (s, 1H),
7.95 (d, 2H, J ¼ 8.8 Hz), 7.77 (d, 2H, J ¼ 8.4 Hz), 7.13 (d, 2H, J ¼ 8.8 Hz),
4.91 (s, 2H), 2.53 (s, 3H); MS (ESI), m/z: 454.32 [M ꢄ MeOH]ꢄ.
8.26 (s, 1H), 7.82e7.77 (m, 1H), 7.47e7.38 (m, 2H), 7.12 (d, 2H,
J ¼ 8.8 Hz), 4.88 (s, 2H); MS (ESI), m/z: 448.24 [M ꢄ MeOH]ꢄ.
5.2.3.38. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-yli-
5.2.3.29. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-ylidene)
methyl)phenoxy)-N-(3-fluorophenyl)acetamide (4g). Yield: 86.9%;
dene)methyl)phenoxy)-N-methyl-N-phenylacetamide
(4p). Yield:
79.4%; HPLC: 98.0%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):

2010
L. Ma et al. / European Journal of Medicinal Chemistry 46 (2011) 2003e2010
d
12.38 (s, 1H), 12.29 (s, 1H), 8.35 (d, 2H, J ¼ 8.0 Hz), 8.24 (s, 1H),
References
7.50e7.41 (m, 5H), 6.91 (s, 2H), 4.60 (s, 2H), 3.20 (s, 3H); MS (ESI),
m/z: 426.23 [M ꢄ MeOH]ꢄ.
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5(6H)-ylidene)methyl)phenoxy)acetamide (4q). Yield: 87.8%; HPLC:
99.2%; Yellow solid; 1H NMR (400 MHz, DMSO-d6)
d 12.40 (s, 1H),
12.30 (s, 1H), 8.40 (d, 1H, J ¼ 9.2 Hz), 8.26 (s, 1H), 7.99 (d, 1H,
J ¼ 8.0 Hz), 7.06 (d, 2H, J ¼ 9.2 Hz), 4.62 (s, 2H), 3.62e3.60 (m, 1H),
1.74e1.64 (m, 4H), 1.58e1.55 (m, 1H),1.31e1.19 (m, 4H), 1.15e1.06 (m,
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5.2.3.40. N-Benzyl-2-(4-((4,6-dioxo-2-thioxotetrahydropyrimidin-
5(6H)-ylidene) methyl)phenoxy)acetamide (4r). Yield: 77.4%;
HPLC: 99.3%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 12.40
(s, 1H), 12.30 (s, 1H), 8.73 (t, 1H, J ¼ 5.6 Hz), 8.40 (d, 2H, J ¼ 8.4 Hz),
8.26 (s, 1H), 7.33e7.21 (m, 5H), 7.09 (d, 2H, J ¼ 8.4 Hz), 4.73 (s,
2H), 4.35 (d, 2H, J ¼ 5.6 Hz); MS (ESI), m/z: 426.17 [M ꢄ MeOH]ꢄ.
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5.2.3.41. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-ylidene)
methyl)phenoxy)-N-(pyridin-2-yl)acetamide (4s). Yield: 88.9%; HPLC:
98.6%; Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 12.40 (s, 1H),
12.31 (s, 1H), 10.73 (s, 1H), 8.40 (d, 2H, J ¼ 9.2 Hz), 8.36e8.34 (m, 1H),
8.27 (s, 1H), 8.03 (d, 1H, J ¼ 8.4 Hz), 7.85e7.81 (m, 1H), 7.180e7.15 (m,
1H), 7.11 (d, 2H, J ¼ 9.2 Hz), 4.99 (s, 2H); MS (ESI), m/z: 413.06
[M ꢄ MeOH]ꢄ.
5.2.3.42. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-yli-
dene)methyl)phenoxy)-N-(5-methylpyridin-2-yl)acetamide (4t).
Yield: 78.2%; HPLC: 99.6%; Yellow solid; 1H NMR (400 MHz,
DMSO-d6)
d 12.40 (s, 1H), 12.30 (s, 1H), 10.57 (s, 1H), 8.40 (d, 1H,
J ¼ 8.8 Hz), 8.27 (s, 1H), 8.18 (s, 1H), 7.94 (d, 1H, J ¼ 8.0 Hz), 7.63
(dd, 1H, J ¼ 8.4 Hz), 7.10 (d, 2H, J ¼ 8.8 Hz), 4.95 (s, 2H), 2.26 (s,
3H); 13C NMR (400 MHz, DMSO-d6):
d 175.19, 169.20, 166.69,
166.45, 157.23, 151.10, 149.41, 137.84, 132.32, 132.01, 129.50, 124.51,
124.19, 118.56, 114.89, 114.29, 114.03, 66.97, 21.23; MS (ESI), m/z:
427.15 [M ꢄ MeOH]ꢄ.
5.2.3.43. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-
ylidene)methyl)phenoxy)-N-(naphthalen-1-yl)acetamide (4u).
Yield: 88.6%; HPLC: 99.9%; Yellow solid; 1H NMR (400 MHz, DMSO-
d6)
d
12.40 (s, 1H), 12.31 (s, 1H), 10.24 (s, 1H), 8.44 (d, 2H, J ¼ 8.0 Hz),
8.29 (s,1H), 8.03e7.94 (m, 2H), 7.81 (d,1H, J ¼ 8.0 Hz), 7.67e7.65 (m,
1H), 7.56e7.49 (m, 3H), 7.20 (d, 2H, J ¼ 8.4 Hz), 5.05 (s, 2H); MS
(ESI), m/z: 462.09 [M ꢄ MeOH]ꢄ.
5.2.3.44. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-yli-
dene)methyl)phenoxy)-N-(naphthalen-2-yl)acetamide (4v). Yield:
89.3%; HPLC: 99.3%; Yellow solid; 1H NMR (400 MHz, DMSO-d6)
d
12.41 (s, 1H), 12.31 (s, 1H), 10.41 (s, 1H), 8.43 (d, 2H, J ¼ 9.2 Hz),
8.32 (s, 1H), 8.28 (s, 1H), 7.91e7.82 (m, 3H), 7.67e7.65 (m, 1H),
7.50e7.40 (m, 2H), 7.16 (d, 2H, J ¼ 8.8 Hz), 4.94 (s, 2H); MS(ESI), m/
z: 462.10 [M ꢄ MeOH]ꢄ.
Acknowledgment
We are grateful to the National Key Programs of China during the
11th Five-Year Plan Period (2009ZX09102-045 and 2009ZX09501-
015).
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Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.02.033.
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