Synthesis and biological evaluation of 5-benzylidenepyrimidine-2,4,6(1H,3H, 5H)-trione derivatives for the treatment of obesity-related nonalcoholic fatty liver disease

Article abstract of DOI:10.1021/jm301164y

Nonalcoholic fatty liver disease (NAFLD), one of chronic liver diseases, seems to be rising as the obesity epidemic continues. In this study, 54 novel (thio)barbituric acid derivatives have been synthesized and evaluated for pharmacological activity. 7h exhibited potent glucose-lowering effects on insulin-resistant HepG2 cells and regulated adiponectin and leptin expression in 3T3-L1 adipocytes. Oral administration of 7h at 25 mg kg^-1 day ^-1 for 4 weeks improved the progression of high fat diet-induced NAFLD by reducing the weight of body, liver, and fat, as well as modulating serum levels of fasting glucose, insulin, triglycerides, LDL-c, ALT, adiponectin and hepatic contents of triglycerides, total cholesterol. H&E stainings revealed that 7h blocked fat deposition in liver and the increase of adipocyte number and size in adipose tissues from NAFLD. Furthermore, treatment with 7h alleviated the obese clinical symptoms, recovered serum biomarkers to appropriate ranges, and improved glucose tolerance by OGTT and IGTT in DIO mice.

Full text of DOI:10.1021/jm301164y

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of 5‑Benzylidenepyrimidine-
2,4,6(1H,3H,5H)‑trione Derivatives for the Treatment of Obesity-
Related Nonalcoholic Fatty Liver Disease
Liang Ma, Caifeng Xie, Yan Ran, Xiaolin Liang, Li Huang, Heying Pei, Jinying Chen, Juan Liu, Yun Sang,
Huijun Lai, Aihua Peng, Mingli Xiang, Yuquan Wei, and Lijuan Chen*
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Gaopeng
Street, Chengdu 610041, China
S
* Supporting Information
ABSTRACT: Nonalcoholic fatty liver disease (NAFLD), one
of chronic liver diseases, seems to be rising as the obesity
epidemic continues. In this study, 54 novel (thio)barbituric
acid derivatives have been synthesized and evaluated for
pharmacological activity. 7h exhibited potent glucose-lowering
effects on insulin-resistant HepG2 cells and regulated
adiponectin and leptin expression in 3T3-L1 adipocytes. Oral
administration of 7h at 25 mg kg−1 day−1 for 4 weeks
improved the progression of high fat diet-induced NAFLD by
reducing the weight of body, liver, and fat, as well as
modulating serum levels of fasting glucose, insulin, triglycerides, LDL-c, ALT, adiponectin and hepatic contents of triglycerides,
total cholesterol. H&E stainings revealed that 7h blocked fat deposition in liver and the increase of adipocyte number and size in
adipose tissues from NAFLD. Furthermore, treatment with 7h alleviated the obese clinical symptoms, recovered serum
biomarkers to appropriate ranges, and improved glucose tolerance by OGTT and IGTT in DIO mice.
correlated positively with HDL-c.12−14 Adiponectin possesses a
hepatoprotective effect, as it increases the viability of liver cells
INTRODUCTION
■
Nonalcoholic fatty liver disease (NAFLD), one of the leading
and insulin sensitivity, and attenuates the lipid inflation in liver
causes of chronic liver diseases, affects approximately 20−30%
of NAFLD.2 Similarly, leptin, the product of the obese gene, is
of the general population in Western countries, and the
an adipocyte-secreted protein hormone with a key role in food
incidence seems to be rising as the obesity epidemic
intake, energy expenditure, and glucose homeostasis.10,15
continues.1−3 NAFLD, defined by fat infiltration and
accumulation exceeding 5−10% per liver weight, is a
clinicopathological hepatic steatosis without a history of excess
Therefore, the functions of adiponectin and leptin in adipocytes
or adipose tissues for regulating insulin as well as glucose and
alcohol consumption.4 The histological spectrum of the disease
fat metabolism make them potential therapeutic targets for
NAFLD.
comprises primary fatty liver, nonalcoholic steatohepatitis
Although the pathogenesis of NAFLD is not fully elucidated,
insulin resistance is a common occurrence in obese NAFLD
(NASH), and NAFLD-associated fibrosis, cirrhosis, and
hepatocellular carcinoma (HCC).5 The advanced forms of
patients and characterized by reducing insulin sensitivity.
NAFLD are more common in obese patients who exhibit
Insulin increases glucose uptake in fat and muscle and inhibits
several features of metabolic syndromecentral obesity, type 2
the output and release of hepatic glucose, thus serving as the
diabetes, dyslipidemia, and atherosclerosis, arising from insulin
resistance and abnormal levels of adipokines.6,7 As yet, there is
primary modulator of blood glucose level. Insulin also
promotes the storage of substrates in liver and fat tissues by
no convincingly effective treatment for NAFLD, but a
multimodal therapeutic plan that targets obesity, insulin
stimulating lipogenesis and inhibiting lipolysis.16 Insulin
resistance or deficiency results in profound dysregulation of
these processes and further interferes with the metabolism of
glucose and lipid. Lifestyle modifications with diet and exercise
could improve insulin sensitivity and are the cornerstone of
therapy but are often difficult to maintain long-term. Not
surprisingly, considerable studies have been focused on
resistance, and adipokines might be the optimal choice for
these patients.8
A series of adipokines, such as adiponectin and leptin, play
key roles in modulating insulin-related glucose homeostasis and
lipid metabolism in adipose tissues of obese NAFLD
patients.9,10 Adiponectin, a 247-amino acid collagen-like
polypeptide, is the most abundant and adipose-specific
adipokine.11 The level of adiponectin is correlated negatively
Received: August 7, 2012
with insulin, blood glucose, triglycerides, and LDL-c but
Published: October 1, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structure of metformin, rosiglitazone, 1, 7h, and 7q.
a
Scheme 1. Synthesis of 7 and 8
a
Reagents and conditions: (a) TsCl, pyridine, 0 °C to room temperature, overnight; (b) K2CO3, DMF, 100 °C, 8 h; (c) TFA, CH2Cl2, 4 h. (d)
Procedure A: R2COOH, EDCI, DMAP, CH2Cl2, room temperature, overnight. Procedure B: R2Cl, K2CO3, DMF, 100 °C. (e) (Thio)barbituric acid,
ethanol, water, 60 °C, 3−4 h.
exploring and developing potent and novel insulin regulators as
therapeutic potential for the treatment of obesity-related
NAFLD.
chloride (TsCl) at 0 °C in the solvent of pyridine afforded 2 in
a good yield. The pivotal aldehyde intermediates 4 were
synthesized via a reaction of 2 and appropriate p-hydrox-
ybenzaldehyde in dimethyl formamide (DMF) using anhydrous
potassium carbonate (K2CO3) as a base. After the deprotection
in a solution of TFA/CH2Cl2 (v/v, 1/1) in an ice bath, the 5
compounds were treated with corresponding benzoic acids
followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCI) as an efficient condensing agent
(procedure A) or appropriate 2-chloropyridines in the presence
of anhydrous K2CO3 as a base (procedure B) to give the N-
substituted 6. Finally, the Knoevenagel reaction of 6 with
barbituric acid or thiobarbituric acid in a mixture of ethanol and
water as valid solvent was heated at 60 °C for 3−4 h to afford
the targeted compounds 7 (X = O) and 8 (X = S), summarized
in Table 1.
Next, to explore the study of structure−activity relationship
(SAR), these novel barbituric acid derivatives 12 were
synthesized as outlined in Scheme 2. Compounds 9 were
prepared according to the same synthetic procedures reported
for 6 starting from commercially available tert-butyl piperidin-4-
ylcarbamate. After the deprotection of 9 in TFA/CH2Cl2
solution, the intermediate salts were basified by stirring in a
mixture of K2CO3 for 1 h and then reacted with 4-
(diethoxymethyl)benzaldehyde at 80 °C overnight and further
In our recent publication, we described our research efforts
that began with an adiponectin and leptin screening from our
libraries of (thio)barbituric acid derivatives17,18 and thiazolidi-
nediones (TZDs),19 which culminated in the identification of 5-
benzylidenepyrimidine-2,4,6(1H,3H,5H)-trione derivative 1
(SKLB016, Figure 1) as a potent drug lead for the treatment
of NAFLD by regulating blood glucose level and adipokine
expressions to improve insulin resistance. With the objective of
developing therapeutic agents for obesity-related NAFLD with
optimal biopharmaceutical properties, the structural modifica-
tions on the basis of 1 led to two novel and potent agents
against the animal model of obesity-related NAFLD, which
were designated as barbituric acid derivatives 7h and 7q (Figure
1).
CHEMISTRY
■
The preparation of a library of 5-benzylidenepyrimidine-
2,4,6(1H,3H,5H)-trione and 5-benzylidene-2-thioxodihydro-
pyrimidine-4,6(1H,5H)-dione derivatives (7 and 8) has been
carried out in a tandem five-step sequence from commercially
available tert-butyl 4-hydroxypiperidine-1-carboxylate (1a) as
described in Scheme 1. Treatment of 1a with p-toluenesulfonyl
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Table 1. Structural Information of 7 and 8 for Pharmacological Profiling
were reduced using sodium borohydride (NaBH4) in
anhydrous methanol to give 10 in a good yield. The
condensation of 10 with different aryl(sulfonyl) chlorides
followed by triethylamine (Et3N) as a base (procedure C) or
iso(thio)cyanates and butyric anhydride in the presence of
pyridine solvent (procedure D) afforded the corresponding
aldehydes 11. The desired derivatives 12 were also obtained by
the condensation of 11 with barbituric acid in a solvent of
ethanol/water overnight at 60 °C, and their structural
information of 12 is summarized in Table 2.
Similarly, 14a−c (Scheme 3) and 17a,b (Scheme 4) have
been obtained by the condensation of aldehydes with barbituric
acid according to the previous procedures in moderate overall
yields and in relatively few synthetic steps. The advantages of
the Knoevenagel reaction were numerous in this study: all
starting aldehydes and (thio)barbituric acid were soluble in hot
water/ethanol; the reaction was clean and quick; the insoluble
final compound was precipitated with a purity of more than
95%.20 At this stage, all final products were fully analyzed and
characterized by NMR, MS, and HPLC before being submitted
to the biological screening.
RESULTS AND DISCUSSION
■
Liver tissue plays an important role in glucose and fat
metabolism. The HepG2 cell (hepatocellular carcinoma cell)
is similar to human hepatocyte and is used to evaluate whether
54 novel (thio)barbituric acid derivatives are capable of
regulating glucose consumption (GC) in insulin-resistant
HepG2 cells.21 Simultaneously, the MTT (3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was per-
formed to investigate whether the glucose-lowering effects are
not due to the cytotoxicity of compounds.22 Insulin sensitizers
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a
Scheme 2. Synthesis of 12
a
Reagents and conditions. (a) Procedure A: R1COOH, EDCI, DMAP, CH2Cl2, room temperature, overnight. Procedure B: R1Cl, K2CO3, DMF, 70
°C. (b) (i) TFA, CH2Cl2, 0 °C to room temperature, 3 h; (ii) 4-(diethoxymethyl)benzaldehyde, K2CO3, water, EtOH, 80 °C, overnight; (iii)
NaBH4, methanol, 60 °C, 0.5 h; (iv) 1 N HCl, acetone, 0.5 h. (c) Procedure C: R2COCl or R2SO2Cl, Et3N, CH2Cl2, room temperature, overnight.
Procedure D: (R2CO)2O or iso(thio)cyanate, pyridine, room temperature, overnight. (d) Barbituric acid, ethanol, water, 60 °C, overnight.
Table 2. Structural Information of 12 for Pharmacological Profiling
(metformin and rosiglitazone) were selected as positive
controls in this study.
As illustrated in Figure 2, the initial screening data from GC/
MTT assay indicated that seven compounds (7c, 7g, 7h, 7m,
7q, 7w, and 14b) at 10 μM exhibited more comparable or
potent glucose-lowering effects on insulin-resistant HepG2 cells
in the presence of insulin (0.1 μM) and high-glucose condition
(22.2 mM glucose) than two marketed drug rosiglitazone and
metformin. However, the other derivatives did not effectively
promote the process of GC/MTT in contrast to positive
control, regardless of the presence of insulin or not, and the
additional results are presented in Supporting Information
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a
Scheme 3. Synthesis of 14
a
Reagents and conditions: (a) (i) TFA, CH2Cl2, 0 °C to room temperature, 3 h. (ii) Procedure A: 4-formylbenzoic acid, EDCI, DMAP, CH2Cl2,
room temperature, overnight. (b) Barbituric acid, ethanol, water, 60 °C, overnight.
a
Scheme 4. Synthesis of 17
a
Reagents and conditions. (a) Procedure B: R1Cl, K2CO3, DMF, 70 °C. (b) (i) TFA, CH2Cl2, 0 °C to room temperature, 3 h. (ii) Procedure A: 4-
formylbenzoic acid, EDCI, DMAP, CH2Cl2, room temperature, overnight. (c) Barbituric acid, ethanol, water, 60 °C, overnight.
Figure 2. Effects on GC/MTT in insulin-resistant HepG2 cells. 1.0 mM metformin (Met), 10 μM rosiglitazone (RSG), and compounds 7a−z, 8a−
g, 12a−p, 14a−c, and 17a,b at 10 μM were tested in the DMEM (22.2 mM glucose) with or without insulin (0.1 μM), using commercial kits at 570
nm. Results were recorded from three independent experiments: ∗, P < 0.05; ∗∗, P < 0.01; and ∗∗∗, P < 0.001 vs the corresponding control group.
(Figure S1). Compared to vehicle and positive control, 7h
showed significant enhancement on GC/MTT with the effect
of insulin, highlighting that 7h could partly improve insulin
sensitivity to further result in marked glucose-lowering effect on
insulin-resistant HepG2 cells induced by high glucose culture.
Similarly, 7q exhibited a comparable effect on regulating the
glucose consumption to the positive control, and the activity of
7q was slightly less potent than one of 7h in vitro.
Inspection of their structural features and activities high-
lighted that the scaffold of barbituric acid was very profitable for
pharmacological activity, and the potencies of thiobarbituric
acid derivatives (8a−g) were distinctly inferior to those of
barbituric acid compounds 7. The 4-hydroxypiperidine linker of
the chemical library of 7 plays critical roles in the activity
compared to the other series of 12, 14, and 17. As for
compounds 7a−n, when the substituted group is a hydrogen
atom at the R1 position, the introduction of different
substituted benzamides at the R2 position improved the
glucose-lowering effect compared to pyridine groups. The
analysis from the data of 7g−h indicated that the trifluor-
omethyl group in the phenyl ring of the R2 position seemed to
be very crucial for the activity.23 However, the analysis for 7o−z
stated that the cyanopyridine group contributed to the glucose-
lowering activity when the fluorine atom was introduced into
the R2 site.
The functions of adipocytes and adipokines, as potential
therapeutic targets, play pivotal roles in obese and obesity-
related NAFLD. In the study, the mature 3T3-L1 adipocytes
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were first obtained by the induction and differentiation of
preadipocytes evidenced by H&E staining during 8−12 days.
After being cocultured with selected novel barbituric acid
derivatives at 10 μM for 24 h, the expression of adiponectin and
leptin in 3T3-L1 adipocytes was measured by commercial
ELISA kits (Figure 3). Consistent with the glucose-lowering
effects on insulin resistant HepG2 cells, we found that 7h also
possessed excellent potency of up-regulating the expression of
adiponectin (122.9% increase from 115.0 pg/mL for vehicle to
256.3 pg/mL for 7h) and down-regulating the secretion of
leptin (386.9% decrease from 632.0 pg/mL for vehicle to 129.8
pg/mL for 7h) in 3T3-L1 adipocytes, which were comparable
or superior to two standard drugs rosiglitazone (236.0 and
118.8 pg/mL for adiponectin and leptin, respectively) and
metformin (125.8 and 360.8 pg/mL). Although 7q exhibited
lower potency of regulating the adiponectin expression than 7h
and rosiglitazone, 7q effectively down-regulated the leptin level
in 3T3-L1 adipocyte with a significant statistic difference.
However, 7c, 7g, and 7m could promote the secretion of
adiponectin and failed to lower the level of adipocyte-secreted
leptin. In contrast, 7h and 7q exhibited more potent activities
to modulate the expression of adipokines (adiponectin and
leptin) in adipocytes than the other derivatives, with 7h being
the most efficacious.
Effects of 7h and 7q on HFD-Induced NAFLD. On the
basis of their excellent in vitro potency of regulating GC and
the expression of adipokines, 7h and 7q were selected for
further in vivo pharmacological evaluation. The two com-
pounds were assessed for their abilities to improve the
progression of high fat diet (HFD, 50.55% of calories derived
from fat) induced NAFLD in male Wistar rats, a chronic liver
disease model that developed obese and obesity-related
metabolic features. As for vehicle rats, the mean body weight
gain by the reduction for 4 weeks was increased with a
statistical difference in contrast to that of normal rats (P <
0.01), indicating that the animal NAFLD model was
successfully established (Figure 4A). In the acute toxicity test,
the male Wistar rats were safe when they were orally
administrated to 7h and 7q at a high dosage of 5 g/kg (not
Figure 3. Determination of adiponectin and leptin in 3T3-L1
adipocytes. Rosiglitazone (RSG) and selected compounds at 10 μM
and 1.0 mM metformin (Met) were tested in 3T3-L1 adipocytes, using
commercial kits (BOSTER, Wuhan, China). Results were recorded
from three independent experiments: ∗, P < 0.05; ∗∗, P < 0.01; and
∗∗∗, P < 0.001 vs vehicle.
Figure 4. 7h and 7q ameliorate metabolic symptoms in HFD-induced NAFLD model. (A) Body weights of NAFLD rats, either HFD-fed or treated
groups (n = 10), were monitored during the oral administration of metformin (Met, 150 mg kg−1 day−1), 7h (25 mg kg−1 day−1), and 7q (25 mg
kg−1 day−1) for 4 weeks. (B) Food intake, (C) liver weight, and (D) fat weight (epididymal and abdominal fat mass): ##, P < 0.01 vs normal group;
∗, P < 0.05; ∗∗, P < 0.01 vs the corresponding model group.
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Figure 5. 7h and 7q improve fasting blood glucose and insulin levels in NAFLD. The HFD-fed and treated groups (n = 10) were monitored during
the oral administration of Met (150 mg kg−1 day−1), 7h (25 mg kg−1 day−1), and 7q (25 mg kg−1 day−1) for 4 weeks. (A) Serum levels of glucose and
(B) insulin were determined using insulin kits and a glucometer (Accu Check, Roche), respectively. The results were expressed as the mean SD: ∗,
P < 0.05; ∗∗, P < 0.01 vs HFD group.
a
Table 3. Parameters of Serum Biochemical Markers from NAFLD Rats
parameter
HFD
HFD + Met
HFD + 7h
**
HFD + 7q
triglycerides (mmol/L)
ALT (U/L)
1.85 0.14
47.0 1.5
1.60 0.15*
39.0 1.9*
89.0 3.5*
1.25 0.05
1.70 0.14*
41.0 3.3*
140.0 18.1
2.02 0.08*
0.16 0.02*
**
33.0 0.8
AST (U/L)
130.0 5.4
2.37 0.03
0.19 0.01
0.33 0.01
110.5 21.3
20.2 1.4
105.0 4.8*
1.98 0.07*
**
total cholesterol (mmol/L)
LDL-c (mmol/L)
HDL-c (mmol/L)
adiponectin (μg/mL)
leptin (ng/mL)
1.99 0.03
**
0.15*
0.12 0.01
0.22 0.01*
213.9 18.4*
14.1 1.0*
**
0.28 0.01*
198.3 10.2
0.25 0.01
163.3 21.9
17.1 1.6
**
13.6 0.9*
a
The HFD-fed (n = 10) or treated groups (n = 10) were respectively monitored during the oral administration of Met (150 mg kg−1 day−1), 7h (25
*
**
mg kg−1 day−1), and 7q (25 mg kg−1 day−1) for 4 weeks. The results were expressed as the mean SD. P < 0.05 vs HFD group. P < 0.01 vs HFD
group.
Figure 6. Effects of 7h and 7q on hepatic biochemical markers and H&E staining of liver and adipose tissue from NAFLD rats: (A) content of
triglycerides; (B) total cholesterol in liver. The results were expressed as the mean SD (n = 10): ∗, P < 0.05; ∗∗, P < 0.01; and ∗∗∗, P < 0.001 vs
HFD group. (C, D) H&E stainings of liver and fat tissue from NAFLD rats (scale, 200×). Lipid droplets were marked using the black arrowheads.
shown). By the combination of the results of in vitro assay and
previous experimental dosage,17 we accepted a dose of 25 mg
kg−1 day−1 as a therapeutic strategy. After oral administration of
HFD + 7h (25 mg kg−1 day−1), HFD + 7q (25 mg kg−1 day−1),
and HFD + Met (150 mg kg−1 day−1) for another 4 weeks, the
mean body weight was reduced by the loss values of 7.8%,
1.2%, and 5.2%, respectively, and the cumulative food intakes of
treated groups were not affected (Figure 4A,B). Importantly,
the mean loss of liver weight by the treatment of 7h, 7q, and
metformin was 5.9%, 0.7%, and 2.2%, respectively, and the
mean fat mass loss (abdominal and epididymal) was 30.9%,
4.0%, and 18.1%, respectively (Figure 4C,D), which indicated
the reduction of fat deposition in liver and the degradation in
adipose tissue. From these data, we summarized that 7q
exhibited lower potency of controlling the weight of body, liver,
and fat than 7h and metformin at the different dosage, with 7h
being the most efficacious at the end of treatment.
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Figure 7. 7h improved the metabolic symptoms in DIO mice. (A) Body weights of DIO mice, either HFD group (n = 10) or treated groups (n =
10), were monitored during the oral administration of metformin (150 mg kg−1 day−1) and 7h (25 mg kg−1 day−1) for 7 weeks. (B) Food intake, (C)
liver weight, and (D) fat weight (epididymal and abdominal fat mass) were analyzed: ##, P < 0.01 vs normal group; ∗, P < 0.05; ∗∗, P < 0.01 vs the
corresponding HFD group.
Because of the fact that blood glucose and insulin played a
key role in glucose homeostasis and fat metabolism, we further
evaluated serum levels of the two parameters in an HFD-
induced NAFLD animal model. As shown in Figure 5, the levels
of glucose and insulin were induced to increase during the high
fat diet which was orally administrated to the rats for 4 weeks.
After the treatment of metformin, 7h, and 7q, the
concentration of fasting blood glucose was reduced by 19.7%,
16.3%, and 23.4%, and the serum levels of insulin were
decreased by 12.8%, 16.7%, and 6.8%, respectively.
steatosis. Fortunately, the fat accumulation from liver tissues of
treated rats (HFD + Met, HFD + 7h, and HFD + 7q) was
distinctly alleviated by H&E stainings, and the remarkable
reduction of the number and size of lipid droplets was observed
(black arrowheads). This result was accorded with a decline in
the content of triglycerides and cholesterol in liver. At the same
time, no obvious histological changes and toxicity were
detected in H&E stainings of liver from normal Wistar rats,
which were orally administrated 7h at 25 mg kg−1 day−1 for 1
week (not shown).
Concurrently, we also measured another six serum levels of
triglycerides, AST, ALT, total cholesterol, LDL-c, and LDL-c,
which are closely related to fat liver and cardiovascular disease.
As exhibited in Table 3, compared to the data of HFD groups,
7h could significantly improve the serum concentrations of
triglycerides, ALT, AST, total cholesterol, LDL-c, and HDL-c
by 32.4%, 29.8%, 19.2%, 16.5%, 36.8%, and 33.3%, respectively.
Similar to the in vitro results, 7h also regulated the serum levels
of adiponectin and leptin. In contrast, 7h showed more potent
ability of regulating HFD-induced abnormal serum biomarkers
(triglycerides, AST, ALT, total cholesterol, LDL-c, LDL-c,
adiponectin, and leptin) than metformin and 7q.
We found that the content of triglycerides, total cholesterol,
and lipid droplets in the liver tissues was significantly increased
after the rats had access to a high fatty diet, suggesting that the
diet feeding led to an excessive fat deposition, accumulation,
and steatosis in liver (Figure 6). In the treated groups, the
reduction of hepatic triglycerides by the administration of
metformin, 7h, and 7q was 48.8%, 87.5%, and 76.3%, and the
decreased content of total cholesterol was 50.0%, 75.0%, and
83.3%, respectively (Figure 6A,B). Interestingly, 7h exhibited
slight potency compared with 7q on the reduction of hepatic
triglycerides, and 7q was slightly superior to 7h in the
regulation of total cholesterol in liver tissue.
The adipocytes play a critical role in obese and obesity-
related diseases. The size of adipocyte elicited from adipose
tissues was also ascertained by H&E staining. It was observed
that NAFLD rat had a remarkable augmentation in adipocyte
size compared to the normal ones, which were significantly
restricted after the treatment with 7h (Figure 6D). Howerver,
metformin and 7q did not exhibit potent ability of controlling
the size of adipocyte and further affected the regulation of the
body weight.
Effects of 7h on Diet-Induced Obesity. With the
promising anti-NAFLD activities in hand, antiobesity effect of
7h at a dose of 25 mg/kg was further evaluated in diet-induced
obesity (DIO) mice. Oral administration of HFD (60.0% of
calories derived from fat) in C57BL/6J mice for 8 weeks
induced marked obesity as evidenced by the gain of body
weight compared to normal ones (BMI as an evaluated
reference, P < 0.01). After the treatment with 7h for 7 weeks,
the mean body weights of HFD-fed mice were significantly
reduced (Figure 7A, the mean weight loss of 8.35% at 25 mg
kg−1 day−1) in contrast to ones of HFD (P < 0.01) without
affecting dramatic changes in accumulative food intake (Figure
7B) and reached similar levels of the metformin-treated group
(the mean weight loss of 9.62% at 150 mg kg−1 day−1). In
addition, the mean mass of liver and fat (epididymal and
abdominal) was decreased (Figure 7C,D).
As shown in Figure 6C, there were nearly no obvious
histological changes and site of steatosis in normal liver
sections. However, numerous lipid droplets in liver tissues from
HFD-induced rats severely ruined liver histology presented as
ballooning degeneration and further led to the formation of
The analysis of serum levels of HFD-administered mice
indicated significant increase in selected biochemical makers,
and the augmentation of these parameters also aggravated the
progression of obesity. Compared to the data from HFD
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a
Table 4. Parameters of Biochemical Markers from DIO Mice
parameter
normal
HFD
HFD + 7h
HFD + Met
b
glucose (mmol/L)
ALT (U/L)
8.96 0.68
20.01 4.10
0.75 0.17
0.18 0.05
0.04 0.01
23.54 0.77
8.90 1.15
85.80 18.21
1.88 0.23
0.36 0.10
0.15 0.05
28.38 1.71
8.02 0.67**
30.83 5.60*
1.04 0.20*
0.27 0.09*
0.09 0.03*
25.16 0.87*
7.98 0.58**
29.20 8.60*
1.33 0.44*
0.20*
triglycerides (mmol/L)
LDL-c (mmol/L)
HDL-c (mmol/L)
FFA (μmol/L)
0.08 0.04
27.33 1.08
a
The HFD-fed group (n = 10) or treated groups (n = 10) were monitored during the oral administration of metformin (150 mg kg−1 day−1), and 7h
b
(25 mg kg−1 day−1) for 7 weeks. The results were expressed as the mean S.D. Fasting blood glucose. *, P < 0.05; **, P < 0.01 vs HFD group.
Figure 8. Effects of 7h on postprandial glucose levels in OGTT and IGTT. The two tests were performed using 7h-treated DIO mice (n = 5) for 6
weeks after 1 night of fasting. Blood was sampled from the tail vein of mice at 0, 15, 30, 60, 90, 120, and 180 min after an oral glucose load of 3.0 g/
kg or intravenous glucose load of 0.5 g/kg of body weight. (A) Blood glucose level in OGTT and (B) blood glucose level in IGTT: #, P < 0.05 vs
normal group; ∗, P < 0.05; ∗∗, P < 0.01 vs HFD group.
groups, treatment of 7h and metformin effectively suppressed
the serum levels of glucose (9.9%, and 10.3%, respectively),
ALT (64.1%, and 66.0%), triglycerides (44.7%, and 29.3%),
LDL-c (25.0%, and 44.4%), HDL-c (40.0%, and 46.7%), and
free fatty acids (FFA, 11.3%, and 3.7%), with 7h being
comparable to metformin at the respective dose (Table 4 and
Table S2). In summary, 7h possessed the potential for weight-
lowing effect on obese symptoms and improved the obesity-
related metabolic features.
On the basis that 7h effectively regulated the levels of blood
glucose in NAFLD and DIO animal model, we next
investigated the effects of 7h on oral glucose tolerance test
(OGTT) and intravenous glucose tolerance test (IGTT).
Treatment with 7h at 25 mg kg−1 day−1 for 6 weeks could
inhibit the increase in postprandial glucose level after glucose
load intake and improve glucose tolerance in OGTT and
IGTT, comparable to that of the standard drug metformin
(Figure 8). Because of similar effects on regulating the levels of
postprandial glucose, we will further evaluate the potential
potency against obesity-related NAFLD by combination
therapy of 7h with metformin in the future.
in the presence of insulin (0.1 μM) and regulated the
expression of adiponectin and leptin in 3T3-L1 adipocytes.
Importantly, oral administration of 7h, at a dose of 25 mg kg−1
day−1 for 4 weeks, improved effectively the progression of
NAFLD by reducing the weight of body, liver, and fat, as well as
modulating serum parameters of fasting glucose, insulin,
triglycerides, LDL-c, HDL-c, ALT, AST, and hepatic contents
of triglycerides, total cholesterol. The results of H&E stainings
also revealed that 7h reduced fat deposition in liver tissues and
suppressed the increase of adipocyte size in adipose tissues
from NAFLD rats. 7h at a single dose of 25 mg kg−1 day−1
alleviated the clinical symptoms of obesity and regulated serum
biomarkers to appropriate ranges, and this compound displayed
similar effects as metformin related to glucose tolerance by
OGTT and IGTT in DIO mice. These compelling pharmaco-
logical profiles might help us to demonstrate that 7h as a
candidate constitutes an attractive approach to the treatment of
obesity-related NAFLD and is valued for further development.
In addition, the detailed pharmacological profiles of 7h and the
combination therapy of 7h with metformin are in progress.
EXPERIMENTAL SECTION
Further, we also investigated the pharmacokinetics of 7h in
Sprague−Dawley rats. After oral administration of 7h at a dose
of 50 mg/kg, t1/2, Cmax, and Tmax were 5.3 h, 31.3 μg/L, and 8.0
h, respectively, with the oral bioavailability being 13.9% (Table
S3 in Supporting Information). However, the low oral
bioavailability may lead to a relative high oral drug dosage in
the animal model of obesity-related NAFLD (25 mg/kg), and
further structural modification of 7h is in progress.
■
Chemistry. Chemical reagents of analytical grade were purchased
from Chengdu Changzheng Chemical Factory (Sichuan, P. R. China).
TLC was performed on 0.20 mm silical gel 60 F254 plates (Qingdao
Ocean Chemical Factory, Shangdong, China). Hydrogen nuclear
magnetic resonance spectra (1H NMR) were recorded at 400 MHz on
a Varian spectrometer (Varian, Palo Alto, CA, U.S.) model Gemini
400 and reported in parts per million. Chemical shifts (δ) are quoted
in ppm relative to tetramethylsilane (TMS) as an internal standard,
where (δ) TMS = 0.00 ppm. The multiplicity of the signal is indicated
as s, singlet; brs, broad 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 ionization (ESI) (Micromass,
Manchester, U.K.). Room temperature (rt) is within the range 20−
CONCLUSION
■
In this study, 54 novel small-molecule (thio)barbituric acid
compounds have been designed, synthesized, and evaluated for
pharmacological activity. At 10 μM, 7h was found to possess
potent glucose-lowering effect on insulin-resistant HepG2 cells
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25 °C. The purity was analyzed by HPLC system (Waters 2695,
separations module) with a photodiode array detector (Waters 2996,
Milford, MA, U.S.), and the chromatographic column was a reversed
phase C18 column (Waters, 150 mm × 4.6 mm, i.d. 5 μm). All
compounds were supplied in HPLC degree methanol with 10 μL,
which was injected on a partial loop, with isocratic elution with 50%
acetonitrile and 50% water (containing 0.1% formic acid) at a flow rate
of 1 mL/min. The purity of all tested compounds was ≥95% according
to our analytical HPLC method.
0.5 h and cooled in an ice bath. The mixture was quenched by the
addition of 1 N HCl and poured into water (50 mL), and the solution
was basified to pH 8−9 with aqueous NaOH. Finally, the formed solid
was collected by filtration without further purification.
General Procedure C for the Synthesis of 11a−k and 11n−p.
R2COCl or R2SO2Cl (2.0 mmol) was added to a solution of 10 (2.0
mmol) and Et3N (834 μL, 6.0 mmol) in CH2Cl2 (10 mL), and the
mixture was stirred at room temperature overnight. Then the solvent
was removed under reduced pressure and the residue was purified by
column chromatography on silica gel to afford the target compounds.
General Procedure D for the Synthesis of 11l and 11m.
Iso(thio)cyanate (2.0 mmol) or (R2CO)2O (24 mmol) was added to a
solution of 10 (2.0 mmol) in pyridine (10 mL) and stirred at room
temperature overnight. Then the solvent was removed under reduced
pressure. The residue was purified by column chromatography on
silica gel to afford the target compounds.
General Procedure for the Synthesis of 7, 8, 12, 14, and 17.
Appropriate aldehydes (0.5 mmol), ethanol (3 mL), distilled water (3
mL), and (thio)barbituric acid (3.0 mmol) were refluxed at 80 °C for
8−24 h. The formed solids were collected by filtration and washed
with boiling water (3 × 15 mL), ethanol (3 × 15 mL), and ether (3 ×
15 mL). The obtained solids were dried in vacuo for 24 h.
tert-Butyl 4-(Tosyloxy)piperidine-1-carboxylate (2). A sol-
ution of tert-butyl 4-hydroxypiperidine-1-carboxylate 1 (1.0 g, 5.0
mmol) in 15 mL of dry pyridine was stirred at 0 °C for 10 min, and a
solution of TsCl (1.4 g, 7.5 mmol) in 20 mL of dry pyridine was added
slowly. The mixture was stirred at room temperature for 6 h and
poured into ice−water. And then the precipitate was collected by
filtration, washed with water, and dried in vacuo to afford 80.7% of
1
compound 2. White solid. H NMR (400 MHz, CDCl3): δ 7.83 (d,
2H, J = 8.4 Hz), 7.38 (d, 2H, J = 8.0 Hz), 4.72−4.67 (m, 1H), 3.64−
3.58 (m, 2H), 3.30−3.24 (m, 2H), 2.47 (s, 3H), 1.82−1.76 (m, 2H),
1.74−1.66 (m, 2H), 1.45 (s, 9H). MS (ESI), m/z: 378.20 [M + Na]+.
General Procedure for the Synthesis of 5. A mixture of 2 (1.78
g, 5.0 mmol), appropriate 4-hydroxybenzaldehydes 3 (5.5 mmol), and
K2CO3 (1.38 g, 10.0 mmol) in 15 mL of DMF was heated to 100 °C
for 10 h. And then the K2CO3 was removed by filtration. The filtrate
was poured into ice−water and extracted with CH2Cl2, saturated
NaHCO3, and brine, and the combined organic layer was dried over
MgSO4. Finally, the solvent was removed under reduced pressure to
give the crude products 4. A solution of 4 (1.0 mmol) in 1 mL of
TFA/CH2Cl2 (v/v = 1/1) at 0 °C was stirred for 3 h and allowed to
warm to room temperature. After the reaction was completed, the
solvent was removed under reduced pressure. The residue was slowly
basified with 2 N NaOH, and the solid was formed. The precipitate
was collected by filtration and washed with water to afford the crude
products 5 without further purification.
General Procedure A for the Synthesis of Amides 6, 9, 13,
and 16. Compounds 5 (1.0 mmol), appropriate benzoic acid (1.2
mmol), EDCI (219.2 mg, 1.22 mmol), and DMAP (13.5 mg, 0.11
mmol) were dissolved in 5 mL of CH2Cl2. The mixture was stirred at
room temperature overnight. After the reaction was completed, water
was added into the solution. The solution was extracted with CH2Cl2,
washed with water and brine, and dried with anhydrous Na2SO4. Then
the solvent was removed under reduced pressure. The residue was
purified by column chromatography on silica gel to afford the target
compounds 6.
General Procedure B for the Synthesis of N-Substituted 6, 9,
and 15. To a solution of tert-butyl piperidin-4-ylcarbamate (5.0
mmol) and R1Cl (5.0 mmol) in DMF (15 mL) was added K2CO3
(2.07 g, 15.0 mmol). The mixture was heated to 100 °C for 5−18 h.
After the mxiture was cooled to room temperature, the K2CO3 was
removed by filtration. The filtrate was poured into water, extracted
with EtOAc, washed with water and brine, and dried over MgSO4.
Then the solvent was removed under reduced pressure. The residue
was purified by column chromatography on silica gel to afford the
target compounds 9.
General Procedure for the Synthesis of 10. To a solution of 9
(1.0 mmol) in CH2Cl2 (1 mL) was added TFA (1 mL) at 0 °C. The
resulting brownish solution was allowed to warm to room temperature
and stirred for 3 h. And then the mixture was concentrated in vacuo to
afford a brown oil residue. The residue was treated with diethyl ether
(50 mL) to give a white solid which was collected by filtration. A
mixture of the trifluoroacetate solid (5.0 mmol), anhydrous K2CO3
(1.38 g, 10.0 mmol) in 10 mL of water, and 30 mL of ethanol was
stirred at room temperature for 10 min. 4-(Diethoxymethyl)-
benzaldehyde (1.0 mL, 5.0 mmol) dissolved into 15 mL of ethanol
was added dropwise, and then the mixture was heated to 80 °C
overnight. The cooled mixture was extracted with CH2Cl2 and water.
The organic layer was washed with brine and dried over anhydrous
Na2SO4. Then the solvent was removed under reduced pressure. The
crude was treated in 30 mL of absolute methanol at 0 °C with NaBH4
(378 mg, 10.0 mmol) in small portion. The mixture was refluxed for
5-(4-(1-Isonicotinoylpiperidin-4-yioxy)benzylidene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (7a). Yield: 87.9%. HPLC
1
purity: 98.1%. Light yellow solid. H NMR (400 MHz, DMSO-d6):
δ 11.31 (s, 1H), 11.18 (s, 1H), 8.68 (d, 2H, J = 11.2 Hz), 8.38 (d, 2H,
J = 8.8 Hz), 8.24 (s, 1H), 7.44 (d, 2H, J = 9.6 Hz), 7.13 (d, 2H, J = 9.2
Hz), 4.90−4.86 (m, 1H), 4.04−4.01 (m, 1H), 3.48−3.45 (m, 2H),
3.29−3.24 (m, 1H), 2.09−2.07 (m, 1H), 1.97−1.95 (m, 1H), 1.73−
1.65 (m, 2H). MS (ESI), m/z: 421.11 [M + H]+.
5-(4-(1-Nicotinoylpiperidin-4-yloxy)benzylidene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (7b). Yield: 86.7%. HPLC
1
purity: 99.2%. Light yellow solid. H NMR (400 MHz, DMSO-d6):
δ 11.31 (s, 1H), 11.18 (s, 1H), 8.60 (dd, 1H, J = 1.2 Hz, J = 4.0 Hz),
8.38 (d, 2H, J = 9.2 Hz), 8.24 (s, 1H), 7.95 (td, 1H, J = 1.6 Hz, J = 8.0
Hz), 7.60 (d, 1H, J = 7.6 Hz), 7.50 (qd, 1H, J = 0.8 Hz, J = 4.8 Hz),
7.14 (d, 2H, J = 8.8 Hz), 4.91−4.87 (m, 1H), 4.07−4.03 (m, 1H),
3.62−3.59 (m, 1H), 3.52−3.46 (m, 1H), 3.36−3.32 (m, 1H), 2.07−
2.02 (m, 1H), 2.00−1.97 (m, 1H), 1.72−1.64 (m, 2H). MS (ESI), m/
z: 443.14 [M + Na]+.
5-(4-(1-Picolinoylpiperidin-4-yloxy)benzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (7c). Yield: 85.7%. HPLC purity: 97.8%.
1
Light yellow solid. H NMR (400 MHz, DMSO-d6): δ 11.31 (s, 1H),
11.18 (s, 1H), 8.60 (d, 1H, J = 4.8 Hz), 8.38 (d, 2H, J = 8.8 Hz), 8.24
(s, 1H), 7.95 (t, 1H, J = 7.6 Hz), 7.60 (d, 1H, J = 7.6 Hz), 7.50 (q, 1H,
J = 5.2 Hz), 7.14 (d, 2H, J = 8.8 Hz), 4.90−4.87 (m, 1H), 4.07−4.03
(m, 1H), 3.62−3.59 (m, 1H), 3.51−3.43 (m, 1H), 3.35−3.33 (m, 1H),
2.10−2.08 (m, 1H), 1.99−1.97 (m, 1H), 1.71−1.64 (m, 2H). MS
(ESI), m/z: 443.15 [M + Na]+.
5-(4-(1-(5-Chloropicolinoyl)piperidine-4-yloxy)benzylidene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (7d). Yield: 87.7%. HPLC
1
purity: 97.4%. Light yellow solid. H NMR (400 MHz, DMSO-d6):
δ 11.31 (s, 1H), 11.18 (s, 1H), 8.58 (d, 1H, J = 5.2 Hz), 8.37 (d, 2H, J
= 8.8 Hz), 8.24 (s, 1H), 7.75 (d, 1H, J = 2.0 Hz), 7.65 (dd, 1H, J = 2.0
Hz, J = 5.2 Hz), 7.13 (d, 2H, J = 8.8 Hz), 4.89−4.88 (m, 2H), 4.04−
4.01 (m, 1H), 3.59−3.47 (m, 2H), 3.33−3.30 (m, 1H), 2.08−2.06 (m,
1H), 1.99−1.97 (m, 1H), 1.72−1.66 (m, 2H). MS (ESI), m/z: 477.15
[M + Na]+.
5-(4-(1-(2-Chloroisonicotinoyl)piperidin-4-yloxy)-
benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7e). Yield:
87.3%. HPLC purity: 99.5%. Light yellow solid. 1H NMR (400
MHz, DMSO-d6): δ 17.27 (s, 1H), 10.09 (s, 4H), 8.50 (d, 1H, J = 5.2
Hz), 7.60 (s, 1H), 7.46 (dd, 1H, J = 1.2 Hz, J = 5.2 Hz), 6.93 (d, 2H, J
= 8.0 Hz), 6.79 (d, 2H, J = 8.8 Hz), 5.81 (s, 1H), 4.55−4.53 (m, 1H),
3.90−3.88 (m, 1H), 3.51−3.39 (m, 2H), 3.21−3.19 (m, 1H), 2.00−
1.97 (m, 1H), 1.88−1.86 (m, 1H), 1.68−1.63 (m, 2H). MS (ESI), m/
z: 477.14 [M + Na]+.
5-(4-(1-(3-(Trifluoromethyl)benzoyl)piperidin-4-yloxy)-
benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7f). Yield:
84.3%. HPLC purity: 99.0%. Light yellow solid. 1H NMR (400
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MHz, DMSO-d6): δ 11.31 (s, 1H), 11.19 (s, 1H), 8.38 (d, 2H, J = 9.2
Hz), 8.25 (s, 1H), 7.84 (d, 1H, J = 7.6 Hz), 7.80 (s, 1H), 7.77 (d, 1H, J
= 7.6 Hz), 7.71 (d, 1H, J = 8.0 Hz), 7.14 (d, 2H, J = 8.8 Hz), 4.89−
4.87 (m, 1H), 4.04−4.00 (m, 1H), 3.47−3.41 (m, 3H), 2.09−1.97 (m,
2H), 1.74−1.68 (m, 2H). MS (ESI), m/z: 510.09 [M + Na]+.
5-(4-(1-(3-Fluoro-4-(trifluoromethyl)benzoyl)piperidin-4-
yloxy)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7g).
Yield: 89.5%. HPLC purity: 97.6%. Light yellow solid. 1H NMR
(400 MHz, DMSO-d6): δ 11.32 (s, 1H), 11.19 (s, 1H), 8.38 (d, 2H, J
= 8.8 Hz), 8.25 (s, 1H), 7.90 (t, 1H, J = 7.6 Hz), 7.67 (d, 1H, J = 10.8
Hz), 7.49 (d, 1H, J = 8.0 Hz), 7.14 (d, 2H, J = 8.8 Hz), 4.90−4.86 (m,
1H), 4.03−4.01 (m, 1H), 3.49−3.47 (m, 2H), 3.30−3.28 (m, 1H),
2.09−2.07 (m, 1H), 1.98−1.96 (m, 1H), 1.73−1.71 (m, 2H). MS
(ESI), m/z: 506.07 [M + H]+.
5-(4-(1-(4-(Difluoromethoxy)benzoyl)piperidin-4-yloxy)-
benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7h). Yield:
83.4%. HPLC purity: 99.91% (Supporting Information). Light yellow
solid. 1H NMR (400 MHz, DMSO-d6): δ 11.32 (s, 1H), 11.19 (s, 1H),
8.38 (d, 2H, J = 8.8 Hz), 8.25 (s, 1H), 7.52 (d, 2H, J = 8.4 Hz), 7.51 (t,
1H, J = 74.0 Hz), 7.25 (d, 2H, J = 8.0 Hz), 7.14 (d, 2H, J = 8.8 Hz),
4.88−4.86 (m, 1H), 4.02−4.00 (m, 1H), 3.56−3.34 (m,3H), 2.04−
2.02 (m, 2H), 1.69−1.67 (m, 2H). 13C NMR (100 MHz, DMSO-d6):
δ 166.1, 163.9, 161.2, 160.0, 157.4, 154.8, 150.2, 143.1, 143.0, 137.5,
127.8, 125.2, 123.2, 123.1, 115.6, 115.5, 115.4, 115.2, 72.1, 44.0, 38.6,
30.5, 29.8. MS (ESI), m/z: 508.22 [M + Na]+.
7.15 (d, 2H, J = 8.8 Hz), 7.04 (d, 1H, J = 9.2 Hz), 4.92−4.88 (m, 1H),
4.11−4.06 (m, 2H), 3.53−3.48 (m, 2H), 2.09−2.05 (m, 2H), 1.69−
1.62 (m, 2H). MS (ESI), m/z: 461.14 [M + H]+.
5-(3-Fluoro-4-(1-nicotinoylpiperidin-4-yloxy)benzylidene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (7o). Yield: 83.4%. HPLC
1
purity: 97.9%. Light yellow solid. H NMR (400 MHz, DMSO-d6):
δ 17.25 (s, 1H), 10.10 (s, 4H), 8.59 (d, 1H, J = 4.8 Hz), 7.93 (td, 1H, J
= 7.6 Hz, J = 0.8 Hz), 7.58 (d, 1H, J = 7.6 Hz), 7.48 (q, 1H, J = 4.2
Hz), 7.05 (t, 1H, J = 8.8 Hz), 6.76 (d, 2H, J = 10.4 Hz), 5.86 (s, 1H),
4.53−4.51 (m, 1H), 3.98−3.94 (m, 1H), 3.57−3.44 (m, 2H), 3.29−
3.25 (m, 1H), 2.00−1.98 (m, 1H), 1.89−1.87 (m, 1H), 1.67−1.60 (m,
2H). MS (ESI), m/z: 439.25 [M + H]+.
5-(3-Fluoro-4-(1-isonicotinoylpiperidin-4-yloxy)-
benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7p). Yield:
86.2%. HPLC purity: 98.6%. Light yellow solid. 1H NMR (400
MHz, DMSO-d6): δ 11.37 (s, 1H), 11.26 (s, 1H), 8.68 (d, 2H, J = 6.0
Hz), 8.55 (dd, 1H, J = 1.6 Hz, J = 13.2 Hz), 8.22 (s, 1H), 8.02 (d, 1H,
J = 8.8 Hz), 7.45 (d, 3H, J = 6.0 Hz), 4.94−4.93 (m, 1H), 4.03−3.99
(m, 1H), 3.51−3.46 (m, 2H), 3.29−3.26 (m, 1H), 2.09−2.07 (m, 1H),
1.99−1.97 (m, 1H), 1.77−1.72 (m, 2H). MS (ESI), m/z: 439.31 [M +
H]+.
5-(4-(1-(2-Chloronicotinoyl)piperidin-4-yloxy)-3-
fluorobenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7q).
Yield: 82.1%. HPLC purity: 100% (Supporting Information). Light
yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 11.38 (s, 1H), 11.26
(s, 1H), 8.55 (d, 1H, J = 14.0 Hz), 8.49 (d, 1H, J = 4.0 Hz), 8.22 (s,
1H), 8.02 (d, 1H, J = 8.4 Hz), 7.99 (t, 1H, J = 7.2 Hz), 7.55 (t, 1H, J =
5.6 Hz), 7.45 (q, 1H, J = 8.4 Hz), 4.95−4.93 (m, 1H), 4.08−4.05 (m,
1H), 3.56−3.47 (m, 1H), 3.35−3.33 (m, 1H), 3.27−3.22 (m, 1H),
2.12−2.10 (m, 1H), 2.00−1.98 (m, 1H), 1.79−1.71 (m, 2H). 13C
NMR (100 MHz, DMSO-d6): δ 163.6, 162.1, 160.2, 153.5, 152.0,
150.1, 149.2, 149.1, 144.3, 133.8, 125.5, 125.4, 121.1, 120.9, 117.9,
116.9, 115.2, 114.6, 94.4, 73.6, 45.1, 30.1. MS (ESI), m/z: 473.53 [M +
H]+.
5-(3-Fluoro-4-(1-(3-(trifluoromethyl)benzoyl)piperidin-4-
yloxy)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7r).
Yield: 83.7%. HPLC purity: 98.9%. Light yellow solid. 1H NMR
(400 MHz, DMSO-d6): δ 11.37 (s, 1H), 11.26 (s, 1H), 8.56 (dd, 1H, J
= 1.6 Hz, J = 10.0 Hz), 8.22 (s, 1H), 8.02 (d, 1H, J = 8.4 Hz), 7.84 (d,
2H, J = 10.8 Hz), 7.78 (d, 1H, J = 8.0 Hz), 7.71 (t, 1H, J = 7.6 Hz),
7.45 (t, 1H, J = 8.8 Hz), 4.94−4.92 (m, 1H), 4.02−4.00 (m, 1H),
3.50−3.48 (m, 2H), 3.35−3.33 (m, 1H), 2.09−1.99 (m, 2H), 1.78−
1.74 (m, 2H). MS (ESI), m/z: 506.11 [M + H]+.
5-(4-(1-(2-Chloronicotinoyl)piperidin-4-yloxy)benzylidene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (7i). Yield: 86.2%. HPLC
1
purity: 98.6%. Light yellow solid. H NMR (400 MHz, DMSO-d6):
δ 11.31 (s, 1H), 11.19 (s, 1H), 8.50 (dd, 1H, J = 1.6 Hz, J = 4.8 Hz),
8.38 (d, 2H, J = 9.2 Hz), 8.25 (s, 1H), 7.98 (td, 1H, J = 1.2 Hz, J = 8.0
Hz), 7.55 (q, 1H, J = 4.2 Hz), 7.15 (t, 2H, J = 8.4 Hz), 4.90−4.87 (m,
1H), 4.12−4.06 (m, 1H), 3.52−3.48 (m, 1H), 3.34−3.27 (m, 1H),
3.26−3.20 (m, 1H), 2.10−2.08 (m, 1H), 1.98−1.96 (m, 1H), 1.76−
1.64 (m, 2H). MS (ESI), m/z: 477.03 [M + Na]+.
5-(4-(1-(4-Nitropyridin-2-yl)piperidin-4-yloxy)benzylidene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (7j). Yield: 86.2%. HPLC
1
purity: 98.8%. Light yellow solid. H NMR (400 MHz, DMSO-d6):
δ 11.32 (s, 1H), 11.19 (s, 1H), 8.38 (d, 2H, J = 8.4 Hz), 8.25 (s, 1H),
7.95 (d, 1H, J = 5.6 Hz), 7.14 (d, 2H, J = 8.8 Hz), 6.90 (s, 1H), 6.88
(s, 1H), 4.87−4.85 (m, 1H), 3.78−3.74 (m, 2H), 3.31−3.29 (m, 2H),
2.05−2.01 (m, 2H), 1.68−1.65 (m, 2H). MS (ESI), m/z: 438.36 [M +
H]+.
5-(4-(1-(5-Nitropyridin-2-yl)piperidin-4-yloxy)benzylidene)-
pyrimidine-2,4,6(1H,3H,5H)-trione (7k). Yield: 82.1%. HPLC
1
5-(3-Fluoro-4-(1-(3-fluoro-4-(trifluoromethyl)benzoyl)-
purity: 99.8%. Light yellow solid. H NMR (400 MHz, DMSO-d6):
piperidin-4-yloxy)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-tri-
δ 11.32 (s, 1H), 11.20 (s, 1H), 8.99 (d, 1H, J = 2.8 Hz), 8.39 (d, 2H, J
= 8.8 Hz), 8.25 (s, 1H), 8.24 (dd, 1H, J = 2.8 Hz, J = 9.6 Hz), 7.16 (d,
2H, J = 8.8 Hz), 7.03 (d, 1H, J = 9.6 Hz), 4.93−4.92 (m, 1H), 4.17−
4.15 (m, 2H), 3.69−4.64 (m, 2H), 2.10−2.08 (m, 2H), 1.72−1.69 (m,
2 Hz). MS (ESI), m/z: 438.24 [M + H]+.
2-(4-(4-((2,4,6-Trioxotetrahydropyrimidin-5(6H)-ylidene)-
methyl)phenoxy)piperidin-1-yl)nicotinonitrile (7l). Yield: 81.8%.
HPLC purity: 99.3%. Light yellow solid. 1H NMR (400 MHz, DMSO-
d6): δ 11.31 (s, 1H), 11.19 (s, 1H), 8.43 (dd, 1H, J = 2 Hz, J = 4.8 Hz),
8.39 (d, 2H, J = 8.8 Hz), 8.25 (s, 1H), 8.09 (dd, 1H, J = 1.6 Hz, J = 7.6
Hz), 7.15 (d, 2H, J = 9.2 Hz), 6.94 (q, 1H, J = 7.6 Hz), 4.90−4.88 (m,
1H), 4.00−3.96 (m, 2H), 3.54−3.49 (m, 2H), 2.12−2.10 (m, 2H),
1.80−1.73 (m, 2H). MS (ESI), m/z: 440.11 [M + Na]+.
5-(4-(1-(3-(Trifluoromethyl)pyridin-2-yl)piperidin-4-yloxy)-
benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7m). Yield:
86.3%. HPLC purity: 97.9%. Light yellow solid. 1H NMR (400
MHz, DMSO-d6): δ 11.31 (s, 1H), 11.18 (s, 1H), 8.54 (d, 1H, J = 4.4
Hz), 8.39 (d, 2H, J = 9.2 Hz), 8.25 (s, 1H), 8.08 (d, 1H, J = 8.0 Hz),
7.21 (q, 1H, J = 1.2 Hz), 7.15 (d, 2H, J = 8.8 Hz), 4.84−4.82 (m, 1H),
3.48−3.44 (m, 2H), 3.19 (t, 2H, J = 9.6 Hz), 2.10−2.09 (m, 2H),
1.80−1.78 (m, 2H). MS (ESI), m/z: 461.15 [M + H]+.
5-(4-(1-(5-(Trifluoromethyl)pyridin-2-yl)piperidin-4-yloxy)-
benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7n). Yield:
85.1%. HPLC purity: 98.7%. Light yellow solid. 1H NMR (400
MHz, DMSO-d6): δ 11.31 (s, 1H), 11.19 (s, 1H), 8.42 (s, 1H), 8.39
(d, 2H, J = 9.2 Hz), 8.25 (s, 1H), 7.81 (dd, 1H, J = 2.4 Hz, J = 9.2 Hz),
1
one (7s). Yield: 85.7%. HPLC purity: 98.8%. Light yellow solid. H
NMR (400 MHz, DMSO-d6): δ 11.37 (s, 1H), 11.26 (s, 1H), 8.55 (d,
1H, J = 13.6 Hz), 8.22 (s, 1H), 8.02 (d, 1H, J = 8.4 Hz), 7.89 (t, 1H, J
= 7.6 Hz), 7.68 (d, 1H, J = 10.8 Hz), 7.50 (d, 1H, J = 8.4 Hz), 7.45 (t,
1H, J = 8.8 Hz), 4.95−4.93 (m, 1H), 3.41−3.98 (m, 1H), 3.47−3.41
(m, 2H), 3.34−3.28 (m, 1H), 2.10−2.07 (m, 1H), 1.99−1.97 (m, 1H),
1.76−1.74 (m, 2H). MS (ESI), m/z: 524.25 [M + H]+.
5-(4-(1-(4-(Difluoromethoxy)benzoyl)piperidin-4-yloxy)-3-
fluorobenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7t).
Yield: 81.9%. HPLC purity: 98.1%. Light yellow solid. 1H NMR
(400 MHz, DMSO-d6): δ 11.37 (s, 1H), 11.26 (s, 1H), 8.56 (dd, 1H, J
= 2.0 Hz, J = 13.2 Hz), 8.22 (s, 1H), 8.02 (d, 1H, J = 7.6 Hz), 7.53 (d,
2H, J = 8.4 Hz), 7.48 (t, 1H, J = 61.6 Hz), 7.45 (t, 1H, J = 8.8 Hz),
7.25 (d, 2H, J = 8.8 Hz), 4.94−4.92 (m, 1H), 4.02−3.98 (m, 1H),
3.55−3.40 (m, 3H), 2.09−2.04 (m, 2H), 1.73−1.71 (m, 2H). MS
(ESI), m/z: 526.10 [M + Na]+.
5-(3-Fluoro-4-(1-(3-(1,1,2,2-tetrafluoroethoxy)benzoyl)-
piperidin-4-yloxy)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-tri-
1
one (7u). Yield: 82.5%. HPLC purity: 98.4%. Light yellow solid. H
NMR (400 MHz, DMSO-d6): δ 11.37 (s, 1H), 11.26 (s, 1H), 8.56
(dd, 1H, J = 1.6 Hz, J = 14.0 Hz), 8.22 (s, 1H), 8.02 (d, 1H, J = 8.4
Hz), 7.59 (t, 1H, J = 7.6 Hz), 7.45 (d, 2H, J = 8.8 Hz), 7.40 (d, 1H, J =
5.2 Hz), 7.37 (t, 1H, J = 5.2 Hz), 6.96 (tt, 1H, J = 1.6 Hz, J = 51.6 Hz),
4.94−4.92 (m, 1H), 3.42−3.38 (m, 1H), 3.48−3.56 (m, 3H), 2.09−
2.00 (m, 2H), 1.74−1.73 (m, 2H). MS (ESI), m/z: 576.08 [M + Na]+.
9968
dx.doi.org/10.1021/jm301164y | J. Med. Chem. 2012, 55, 9958−9972

Journal of Medicinal Chemistry
Article
5-(4-(1-(2,2-Difluorobenzo[d][1,3]dioxole-4-carbonyl)-
piperidin-4-yloxy)-3-fluorobenzylidene)pyrimidine-2,4,6-
(1H,3H,5H)-trione (7v). Yield: 86.1%. HPLC purity: 99.3%. Light
yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 11.38 (s, 1H), 11.27
(s, 1H), 8.56 (dd, 1H, J = 1.6 Hz, J = 14.0 Hz), 8.23 (s, 1H), 8.02 (d,
1H, J = 8.4 Hz), 7.53 (q, 1H, J = 3.6 Hz), 7.45 (t, 1H, J = 8.8 Hz), 7.31
(dd, 2H, J = 1.6 Hz, J = 5.2 Hz), 4.96−4.93 (m, 1H), 4.04−4.01 (m,
1H), 3.52−3.50 (m, 2H), 3.36−3.34 (m, 1H), 2.11−2.09 (m, 1H),
2.02−2.00 (m, 1H), 1.75−1.68 (m, 2H). MS (ESI), m/z: 540.23 [M +
Na]+.
2-(4-(2-Fluoro-4-((2,4,6-trioxotetrahydropyrimidin-5(6H)-
ylidene)methyl)phenoxy)piperidin-1-yl)nicotinonitrile (7w).
Yield: 88.4%. HPLC purity: 98.3%. Light yellow solid. 1H NMR
(400 MHz, DMSO-d6): δ 11.38 (s, 1H), 11.27 (s, 1H), 8.57 (dd, 1H, J
= 2 Hz, J = 13.6 Hz), 8.43 (dd, 1H, J = 2 Hz, J = 4.8 Hz), 8.23 (s, 1H),
8.10 (dd, 1H, J = 2 Hz, J = 8 Hz), 8.04 (d, 1H, J = 8 Hz), 7.47 (t, 1H, J
= 8.8 Hz), 6.95 (q, 1H, J = 7.6 Hz), 4.96 (m, 1H), 4.00−3.95 (m, 2H),
3.54−3.49 (m, 2H), 2.13−2.09 (m, 2H), 1.83−1.76 (m, 2H). MS
(ESI), m/z: 436.23 [M + H]+.
5-(3-Fluoro-4-(1-(3-(trifluoromethyl)pyridin-2-yl)piperidin-
4-yloxy)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7x).
Yield: 83.4%. HPLC purity: 98.7%. Light yellow solid. 1H NMR
(400 MHz, DMSO-d6): δ 11.37 (s, 1H), 11.27 (s, 1H), 8.57 (d, 2H, J
= 14.0 Hz), 8.23 (s, 1H), 8.09 (d, 1H, J = 7.6 Hz), 8.03 (d, 1H, J = 8.4
Hz), 7.46 (t, 1H, J = 8.8 Hz), 7.22 (q, 1H, J = 4.8 Hz), 4.90−4.89 (m,
1H), 3.47−3.44 (m, 2H), 3.19−3.15 (m, 2H), 2.13−2.11 (m, 2H),
1.85−1.79 (m, 2H). MS (ESI), m/z: 479.25 [M + H]+.
5-(3-Fluoro-4-(1-(5-(trifluoromethyl)pyridin-2-yl)piperidin-
4-yloxy)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7y).
Yield: 83.4%. HPLC purity: 99.8%. Light yellow solid. 1H NMR
(400 MHz, DMSO-d6): δ 11.38 (s, 1H), 11.27 (s, 1H), 8.56 (d, 1H, J
= 2.0 Hz, J = 13.6 Hz), 8.42 (s, 1H), 8.22 (s, 1H), 8.04 (d, 1H, J = 8.8
Hz), 7.82 (dd, 1H, J = 2.4 Hz, J = 8.8 Hz), 7.47 (t, 1H, J = 9.2 Hz),
7.04 (d, 1H, J = 9.2 Hz), 4.96−4.95 (m, 1H), 4.10−4.07 (m, 2H),
3.54−3.49 (m, 2H), 2.09−2.06 (m, 2H), 1.72−1.65 (m, 2H). MS
(ESI), m/z: 479.26 [M + H]+.
2-Thioxo-5-(4-(1-(5-(trifluoromethyl)pyridin-2-yl)piperidin-
4-yloxy)benzylidene)dihydropyrimidine-4,6(1H,5H)-dione
1
(8d). Yield: 82.8%. HPLC purity: 99.2%. Light yellow solid. H NMR
(400 MHz, DMSO-d6): δ 12.41 (s, 1H), 12.31 (s, 1H), 8.44−8.42 (m,
3H), 8.27 (s, 1H), 7.81 (dd, 1H, J = 2.4 Hz, J = 9.2 Hz), 7.17 (d, 2H, J
= 9.2 Hz), 7.04 (d, 1H, J = 9.2 Hz), 4.93−4.91 (m, 1H), 4.11−4.08
(m, 2H), 3.53−3.48 (m, 2H), 2.06−2.04 (m, 2H), 1.69−1.62 (m, 2H).
MS (ESI), m/z: 493.25 [M + H]+.
5-(3-Ethoxy-4-(1-(3-fluoro-4-(trifluoromethyl)benzoyl)-
piperidin-4-yloxy)benzylidene)-2-thioxodihydropyrimidine-
4,6(1H,5H)-dione (8e). Yield: 81.6%. HPLC purity: 99.5%. Light
yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 12.42 (s, 1H), 12.30
(s, 1H), 8.45 (d, 1H, J = 1.6 Hz), 8.26 (s, 1H), 7.92 (q, 2H, J = 7.6
Hz), 7.69 (d, 1H, J = 10.8 Hz), 7.50 (d, 1H, J = 8.0 Hz), 7.27 (d, 1H, J
= 8.8 Hz), 4.89−4.87 (m, 1H), 4.12 (q, 2H, J = 7.2 Hz), 4.02−3.99
(m, 1H), 3.49−3.47 (m, 2H), 3.29−3.24 (m, 1H), 2.10−2.08 (m, 1H),
1.99−1.95 (m, 1H), 1.74−1.72 (m, 2H), 1.39 (t, 3H, J = 7.2 Hz). MS
(ESI), m/z: 582.32 [M + H]+.
2-(4-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-
ylidene)methyl)-2-ethoxyphenoxy)piperidin-1-yl)-
nicotinonitrile (8f). Yield: 82.4%. HPLC purity: 99.8%. Light yellow
solid. 1H NMR (400 MHz, DMSO-d6): δ 12.42 (s, 1H), 12.31 (s, 1H),
8.46 (s, 1H), 8.43 (dd, 1H, J = 1.6 Hz, J = 4.8 Hz), 8.27 (s, 1H), 8.10
(d, 1H, J = 8.0 Hz), 7.94 (d, 1H, J = 8.8 Hz), 7.29 (d, 1H, J = 8.8 Hz),
6.94 (q, 1H, J = 4.8 Hz), 4.91−4.89 (m, 1H), 4.12 (q, 2H, J = 7.2 Hz),
3.99−3.95 (m, 2H), 3.54−3.50 (m, 2H), 2.10−2.08 (m, 2H), 1.81−
1.77 (m, 2H), 1.38 (t, 3H, J = 6.8 Hz). MS (ESI), m/z: 494.16 [M +
H]+.
2-(4-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(6H)-
ylidene)methyl)-2,6-dimethoxyphenoxy)piperidin-1-yl)-
nicotinonitrile (8g). Yield: 83.7%. HPLC purity: 98.4%. Light yellow
solid. 1H NMR (400 MHz, DMSO-d6): δ 12.47 (s, 1H), 12.37 (s, 1H),
8.42 (dd, 1H, J = 1.6 Hz, J = 4.8 Hz), 8.30 (s, 1H), 8.08 (dd, 1H, J =
2.0 Hz, J = 7.6 Hz), 7.94 (s, 2H), 6.92 (q, 1H, J = 4.8 Hz), 4.57−4.55
(m, 1H), 3.94−3.90 (m, 2H), 3.84 (s, 6H), 3.55−3.49 (m, 2H), 1.97−
1.92 (m, 2H), 1.79−1.74 (m, 2H). MS (ESI), m/z: 510.23 [M + H]+.
5-(4-(((1-(5-Nitropyridin-2-yl)piperidin-4-yl)amino)methyl)-
benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (12a). Yield:
35.2%. HPLC purity: 98.8%. White solid. 1H NMR (400 MHz,
DMSO-d6): δ 10.01 (s, 2H), 8.97−8.98 (m, 1H), 8.26−8.22 (m, 1H),
7.27 (d, 2H, J = 8.0 Hz), 7.08 (d, 2H, J = 7.6 Hz), 7.01 (d, 2H, J = 6.4
Hz), 5.98 (s, 1H), 4.60−4.57 (m, 2H), 4.38−4.36 (m, 2H), 4.08 (s,
2H), 2.18−2.16 (m, 2H), 1.49−1.47 (m, 2H). MS (ESI), m/z: 451.16
[M − H]−.
Methyl 4-(4-(2-Fluoro-4-((2,4,6-trioxotetrahydropyrimidin-
5(6H)-ylidene)methyl)phenoxy)piperidin-1-ylsulfonyl)-
thiophene-3-carboxylate (7z). Yield: 85.1%. HPLC purity: 99.4%.
1
Light yellow solid. H NMR (400 MHz, DMSO-d6): δ 11.37 (s, 1H),
11.26 (s, 1H), 8.52 (dd, 1H, J = 2 Hz, J = 13.6 Hz), 8.21 (s, 1H), 8.03
(d, 1H, J = 5.2 Hz), 7.99 (d, 1H, J = 8.8 Hz), 7.49 (d, 1H, J = 5.2 Hz),
7.39 (t, 1H, J = 8.8 Hz), 4.81−4.77 (m, 1H), 3.86 (s, 3H), 3.55−3.51
(m, 2H), 3.81−3.14 (m, 2H), 2.06−2.03 (m, 2H), 1.73−1.68 (m, 2H).
MS (ESI), m/z: 560.03 [M + Na]+.
N-(1-(5-Nitropyridin-2-yl)piperidin-4-yl)-N-(4-((2,4,6-trioxo-
tetrahydropyrimidin-5(2H)-ylidene)methyl)benzyl)-
methanesulfonamide (12b). Yield: 65.4%. HPLC purity: 98.7%.
Yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 11.39 (s, 1H), 11.24
(s, 1H), 8.92−8.91 (m, 1H), 8.25 (s, 1H), 8.16 (dd, 1H, J = 9.6 Hz, J =
2.8 Hz), 7.44 (d, 2H, J = 8.4 Hz), 6.93 (d, 2H, J = 9.6 Hz), 4.60 (m,
1H), 4.46 (s, 2H), 4.09−4.03 (m, 2H), 3.09 (s, 3H), 3.04−2.99 (m,
1H), 1.82−1.54 (m, 4H). MS (ESI), m/z: 528.15 [M − H]−.
Methyl 3-(N-(1-(5-Nitropyridin-2-yl)piperidin-4-yl)-N-(4-
((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)sulfamoyl)thiophene-2-carboxylate (12c). Yield: 31.3%.
HPLC purity: 98.9%. Yellow solid. 1H NMR (400 MHz, DMSO-d6): δ
9.98 (s, 2H), 8.90 (d, 1H, J = 2.8 Hz), 8.12 (dd, 1H, J = 9.2 Hz, J = 2.8
Hz), 7.90 (d, 1H, J = 5.2 Hz), 7.37 (d, 1H, J = 5.2 Hz), 7.05 (d, 2H, J
= 8.0 Hz), 6.91−6.89 (m, 3H), 5.90 (s, 1H), 4.53−4.50 (m, 4H), 3.87
(s, 3H), 2.95−2.93 (m, 2H), 1.54 (s, 4H). MS (ESI), m/z: 654.12 [M
− H]−.
2-(4-((4-((2,4,6-Trioxotetrahydropyrimidin-5(2H)-ylidene)-
methyl)benzyl)amino)piperidin-1-yl)nicotinonitrile (12d).
Yield: 40.2%. HPLC purity: 99.1%. Yellow solid. 1H NMR (400
MHz, DMSO-d6): δ 10.01 (s, 2H), 8.75 (s, 2H), 8.42 (dd, 1H, J = 4.8
Hz, J = 2.0 Hz), 8.10 (dd, 1H, J = 7.6 Hz, J = 2.0 Hz), 7.30 (d, 1H, J =
8.0 Hz), 7.10 (d, 2H, J = 8.0 Hz), 6.97 − 6.94 (m, 1H), 6.00 (1H),
4.27 (d, 2H, J = 13.2 Hz), 4.13 (s, 2H), 3.05 (t, 2H, J = 12.0 Hz), 2.19
(d, 2H, J = 12.0 Hz), 1.69−1.61 (m, 2H). MS (ESI), m/z: 431.17 [M
− H]−.
5-(4-(1-(5-Methylpyrazine-2-carbonyl)piperidin-4-yloxy)-
benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione
1
(8a). Yield: 84.6%. HPLC purity: 97.5%. Light yellow solid. H NMR
(400 MHz, DMSO-d6): δ 12.41 (s, 1H), 12.31 (s, 1H), 8.74 (d, 1H, J
= 1.6 Hz), 8.57 (s, 1H), 8.43 (d, 2H, J = 9.2 Hz), 8.27 (s, 1H), 7.16 (d,
2H, J = 9.2 Hz), 4.94−4.90 (m, 1H), 4.07−4.03 (m, 1H), 3.68−3.65
(m, 1H), 3.53−3.48 (m, 1H), 3.45−3.39 (m, 1H), 2.55 (s, 3H), 2.10−
2.08 (m, 1H), 2.02−2.00 (m, 1H), 1.73−1.67 (m, 2H). MS (ESI), m/
z: 468.32 [M + H]+.
5-(4-(1-(4-Chloro-2-methoxybenzoyl)piperidin-4-yloxy)-
benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione
1
(8b). Yield: 82.3%. HPLC purity: 98.1%. Light yellow solid. H NMR
(400 MHz, DMSO-d6): δ 12.41 (s, 1H), 12.31 (s, 1H), 8.43 (d, 2H, J
= 9.2 Hz), 8.26 (s, 1H), 7.46 (dd, 1H, J = 2.4 Hz, J = 8.8 Hz), 7.32
(dd, 1H, J = 2.4 Hz, J = 9.2 Hz), 7.16−7.11 (m, 3H), 4.88−4.86 (m,
1H), 4.03−3.99 (m, 1H), 3.81 (s, 3H), 3.45−3.32 (m, 2H), 3.18−3.15
(m, 1H), 2.05−1.93 (m, 2H), 1.69−1.58 (m, 2H). MS (ESI), m/z:
516.56 [M + H]+.
5-(4-(1-(4-Fluorophenylsulfonyl)piperidin-4-yloxy)-
benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione
1
(8c). Yield: 85.2%. HPLC purity: 98.9%. Light yellow solid. H NMR
(400 MHz, DMSO-d6): δ 12.40 (s, 1H), 12.30 (s, 1H), 8.37 (d, 2H, J
= 8.8 Hz), 8.23 (s, 1H), 7.88 (q, 2H, J = 5.2 Hz), 7.56 (t, 2H, J = 9.2
Hz), 7.06 (d, 2H, J = 8.8 Hz), 4.66−4.45 (m, 1H), 3.35−3.31 (m, 2H),
2.84−2.80 (m, 2H), 2.06−2.03 (m, 2H), 1.75−1.68 (m, 2H). MS
(ESI), m/z: 506.14 [M + H]+.
9969
dx.doi.org/10.1021/jm301164y | J. Med. Chem. 2012, 55, 9958−9972

Journal of Medicinal Chemistry
Article
N-(1-(3-Cyanopyridin-2-yl)piperidin-4-yl)-N-(4-((2,4,6-trioxo-
tetrahydropyrimidin-5(2H)-ylidene)methyl)benzyl)-
methanesulfonamide (12e). Yield: 65.4%. HPLC purity: 98.9%.
Yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 11.38 (s, 1H), 11.24
(s, 1H), 8.36−8.35 (m, 1H), 8.26 (s, 1H), 8.13 (d, 1H, J = 8.0 Hz),
8.04−8.02 (m, 1H), 7.46 (d, 2H, J = 8.0 Hz), 6.89−6.86 (m, 1H), 4.47
(s, 2H), 4.28−4.25 (m, 2H), 4.00−3.94 (m, 1H), 3.08 (s, 3H), 3.05−
2.97 (m, 2H), 1.83−1.65 (m, 4H). MS (ESI), m/z: 509.15 [M − H]−.
Methyl 3-(N-(1-(3-Cyanopyridin-2-yl)piperidin-4-yl)-N-(4-
((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)sulfamoyl)thiophene-2-carboxylate (12f). Yield: 27.8%.
HPLC purity: 97.9%. White solid. 1H NMR (400 MHz, DMSO-d6): δ
9.99 (s, 2H), 8.34 (m, 1H), 8.25 (s, 1H), 8.00 (dd, 1H, J = 7.6 Hz, J =
2.0 Hz), 7.89 (d, 1H, J = 5.6 Hz), 7.36 (d, 1H, J = 5.6 Hz), 7.04 (d,
2H, J = 8.0 Hz), 6.89−6.83 (m, 3H), 5.92 (s, 1H), 4.76 (s, 2H), 4.21−
4.16 (m, 3H), 3.87 (s, 3H), 2.93 (t, 2H, J = 12.0 Hz), 1.65−1.54 (m,
4H). MS (ESI), m/z: 635.13 [M − H]−.
Hz, J = 10.8 Hz), 2.94 (d, 3H, J = 4 Hz) 1.74−1.62 (m, 4H). MS
(ESI), m/z: 503.17 [M − H]−.
N-(1-(4-(Difluoromethoxy)benzoyl)piperidin-4-yl)-N-(4-
((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)methanesulfonamide (12n). Yield: 20.7%. HPLC purity:
99.1%. Yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 11.40 (s, 1H),
11.25 (s, 1H), 8.27 (s,1H), 8.14 (d, 2H, J = 8.4 Hz), 7.47 (d, 2H, J =
8.0 Hz), 7.21 (d, 2H, J = 7.4 Hz), 7.20 (d, 2H, J = 8.4 Hz), 7.40 (d,
2H, J = 8.0 Hz), 4.51−4.37 (m, 4H), 4.00−3.94 (m, 1H), 3.06 (s, 3H),
1.78−1.61 (m, 4H). MS (ESI), m/z: 577.15 [M − H]−.
N-(1-(3-Fluoro-4-(trifluoromethyl)benzoyl)piperidin-4-yl)-N-
(4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)methanesulfonamide (12o). Yield: 18.2%. HPLC purity:
99.5%. Yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 11.39 (s, 1H),
11.25 (s, 1H), 8.27 (s,1H), 8.14 (d, 2H, J = 8.0 Hz), 7.85 (t, 1H, J =
8.0 Hz), 7.58 (d, 2H, J = 10.8 Hz), 7.47 (d, 2H, J = 8.4 Hz), 7.40 (d,
2H, J = 8.0 Hz), 4.52 (m, 4H), 4.01−4.00 (m, 1H), 3.11−2.78 (m,
5H), 1.82−1.62 (m, 4H). MS (ESI), m/z: 597.14 [M − H]−.
N-(1-(3-(1,1,2,2-Tetrafluoroethoxy)benzoyl)piperidin-4-yl)-
N-(4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)methanesulfonamide (12p). Yield: 45.4%. HPLC purity:
97.8%. Yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 11.39 (s, 1H),
11.24 (s, 1H), 8.27 (s,1H), 8.14 (d, 2H, J = 8.4 Hz), 7.54 (t, 1H, J =
8.0 Hz), 7.48 (d, 2H, J = 8.4 Hz), 7.36−7.34 (m, 2H), 7.27 (s, 1H),
6.81 (t, 1H, J = 5.2 Hz), 4.51−4.36 (m, 4H), 3.98 (m, 1H), 3.05 (s,
3H), 1.78−1.62 (m, 4H). MS (ESI), m/z: 627.15 [M − H]−.
N-(1-(4-Nitropyridin-2-yl)piperidin-4-yl)-4-((2,4,6-trioxote-
trahydropyrimidin-5(2H)-ylidene)methyl)benzamide (14a).
Yield: 35.2%. HPLC purity: 98.6%. Yellow solid. 1H NMR (400
MHz, DMSO-d6): δ 11.44 (s, 1H), 11.28 (s, 1H), 8.98 (d, 1H, J = 2.8
Hz), 8.46 (d, 1H, J = 6.8 Hz), 8.29 (s, 1H), 8.22 (dd, 1H, J = 9.6 Hz, J
= 2.8 Hz), 8.05 (d, 2H, J = 8.4 Hz), 7.89 (d, 2H, J = 8.4 Hz), 7.02 (d,
1H, J = 9.6 Hz), 4.53 (d, 2H, J = 8.8 Hz), 4.20−4.19 (m, 1H), 3.23 (t,
2H, J = 12.0 Hz), 1.97−1.94 (m, 2H), 1.59−1.51 (m, 2H). MS (ESI),
m/z: 465.14 [M − H]−.
N-(1-(5-Nitropyridin-2-yl)piperidin-4-yl)-4-((2,4,6-trioxote-
trahydropyrimidin-5(2H)-ylidene)methyl)benzamide (14b).
Yield: 21.0%. HPLC purity: 99.6%. White solid. 1H NMR (400
MHz, DMSO-d6): δ 10.12 (s, 2H), 8.98−8.97 (m, 1H), 8.24−8.19 (m,
1H), 8.05 (d, 1H, J = 8.4 Hz), 7.87 (d, 1H, J = 8.0 Hz), 7.62 (d, 1H, J
= 8.0 Hz), 7.08 (d, 1H, J = 8.0 Hz), 7.03−7.00 (m, 1H), 5.94 (s, 1H),
4.52 (s, 2H), 4.17 (m, 1H), 3.12−3.19 (m, 2H), 1.96−1.91 (m, 2H),
1.56−1.50 (m, 2H). MS (ESI), m/z: 465.14 [M − H]−.
N-(1-(3-Cyanopyridin-2-yl)piperidin-4-yl)-4-((2,4,6-trioxote-
trahydropyrimidin-5(2H)-ylidene)methyl)benzamide (14c).
Yield: 37.9%. HPLC purity: 99.0%. Yellow solid. 1H NMR (400
MHz, DMSO-d6): δ 10.06 (s, 2H), 8.41 (s, 2H), 8.17 (d, 2H, J = 8.0
Hz), 8.09−8.05 (m, 1H), 7.63 (d, 2H, J = 8.0 Hz), 7.07 (d, 2H, J = 8.0
Hz), 6.93−6.88 (m, 1H), 5.97 (s, 1H), 4.26−4.23 (m, 4H), 4.09 (m,
1H), 3.19−3.14 (m, 2H), 1.91−1.88 (m, 2H), 1.69−1.64 (m, 2H). MS
(ESI), m/z: 445.15 [M − H]−.
5-Chloro-N-(1-(3-cyanopyridin-2-yl)piperidin-4-yl)-N-(4-
((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)thiophene-2-carboxamide (12g). Yield: 26.4%. HPLC
1
purity: 98.9%. White solid. H NMR (400 MHz, DMSO-d6): δ 9.98
(s, 2H), 8.38 (d, 1H, J = 4.0 Hz), 8.02 (d, 1H, J = 4.0 Hz), 7.08−6.99
(m, 6H), 6.89−6.85 (m, 1H), 5.93 (s, 1H), 4.68 (s, 2H), 4.31−4.28
(m, 3H), 2.99 (t, 2H, J = 12.0 Hz), 1.91−1.82 (m, 4H). MS (ESI), m/
z: 575.12 [M − H]−.
N-(1-(3-Cyanopyridin-2-yl)piperidin-4-yl)-3-morpholino-N-
(4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)propane-1-sulfonamide (12h). Yield: 33.6%. HPLC purity:
99.6%. White solid. 1H NMR (400 MHz, DMSO-d6): δ 10.02 (s, 2H),
8.37 (s, 1H), 8.03 (d, 1H, J = 8.0 Hz), 7.11 (d, 2H, J = 8.0 Hz), 6.96
(d, 2H, J = 8.0 Hz), 6.87 (m, 1H), 5.94 (s, 1H), 4.31−4.28 (m, 5H),
3.86−3.64 (m, 6H), 3.00 (m, 8H), 1.94−1.79 (m, 6H). MS (ESI), m/
z: 622.23 [M − H]−.
5-Bromo-N-(1-(3-cyanopyridin-2-yl)piperidin-4-yl)-N-(4-
((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)thiophene-2-carboxamide (12i). Yield: 21.2%. HPLC
purity: 98.8%. White solid. 1H NMR (400 MHz, DMSO-d6): δ
10.01 (s, 2H), 8.38 (s, 1H), 8.03 (s, 1H), 7.18−6.88 (m, 8H), 4.88 (s,
2H), 4.29 (s, 3H), 3.00 (s, 2H), 1.91−1.82 (m, 4H). MS (ESI), m/z:
619.07 [M − H]−.
(3R,5R,7R)-N-(1-(3-Cyanopyridin-2-yl)piperidin-4-yl)-N-(4-
((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)adamantane-1-carboxamide (12j). Yield: 39.2%. HPLC
purity: 98.8%. White solid. 1H NMR (400 MHz, DMSO-d6): δ 9.96 (s,
2H), 8.37 (d, 2H, J = 4.4 Hz), 8.02 (d, 1H, J = 7.2 Hz), 6.90−6.85 (m,
4H), 5.91 (s, 1H), 4.42−4.27 (m, 4H), 1.98−1.54 (m, 12H). MS
(ESI), m/z: 593.28 [M − H]−.
N-(1-(3-Cyanopyridin-2-yl)piperidin-4-yl)-N-(4-((2,4,6-trioxo-
tetrahydropyrimidin-5(2H)-ylidene)methyl)benzyl)-
pentanamide (12k). Yield: 19.2%. HPLC purity: 98.3%. White solid.
1H NMR (400 MHz, DMSO-d6): δ 9.98 (s, 2H), 8.37 (d, 2H, J = 4.0
Hz), 8.22 (s, 2H), 8.01 (d, 1H, J = 8.0 Hz), 6.98−6.86 (m, 4H), 5.93
(s, 1H), 4.44 (s, 2H), 4.41 (s, 1H), 4.26 (d, 2H, J = 12.0 Hz), 3.05−
2.94 (m, 6H), 2.17 (t, 2H, J = 8.0 Hz), 1.71−0.79 (m, 9H). MS (ESI),
m/z: 515.23 [M − H]−.
1-(1-(3-Cyanopyridin-2-yl)piperidin-4-yl)-3-ethyl-1-(4-
((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)urea (12l). Yield: 44.2%. HPLC purity: 98.7%. Yellow solid.
1H NMR (400 MHz, DMSO-d6): δ 11.38(s, 1H), 11.24 (s, 1H), 8.37−
8.36 (m, 1H), 8.25 (s, 1H), 8.12 (d, 2H, J = 8.4 Hz), 8.04−8.01 (m,
1H), 7.30 (d, 2H, J = 8.4 Hz), 6.89−6.86 (m, 1H), 6.48−6.47 (m, 1H)
4.48 (s, 2H), 4.94 (s, 2H), 4.28−4.25 (m, 3H), 3.13−2.05 (m, 4H),
1.64 (s, 4H), 1.00 (t, 3H, J = 6.8 Hz). MS (ESI), m/z: 502.21 [M −
H]−.
1-(1-(3-Cyanopyridin-2-yl)piperidin-4-yl)-3-methyl-1-(4-
((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-
benzyl)thiourea (12m). Yield: 34.2%. HPLC purity: 99.6%. Yellow
solid. 1H NMR (400 MHz, DMSO-d6): δ 11.39(s, 1H), 11.24 (s, 1H),
8.38−8.36 (m, 1H), 8.25 (s, 1H), 8.13 (d, 2H, J = 8.4 Hz), 8.04−8.02
(m, 1H), 7.66 (brs, 1H), 7.25 (d, 2H, J = 8.4 Hz), 6.94 (m, 1H) 5.18
(brs, 1H), 4.94 (s, 2H), 4.28 (d, 2H, J = 12.4 Hz), 3.02 (t, 2H, J = 12.4
5-(4-(4-((4-Nitropyridin-2-yl)amino)piperidine-1-carbonyl)-
benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (17a). Yield:
22.8%. HPLC purity: 99.1%. White solid. 1H NMR (400 MHz,
DMSO-d6): δ 10.09 (s, 2H), 7.96−7.94 (m, 1H), 7.61 (d, 2H, J = 8.4
Hz), 7.07 (d, 2H, J = 8.4 Hz), 7.63−7.89 (m, 3H), 5.95 (s, 1H), 4.10−
4.08 (m, 1H), 4.02−3.98 (m, 2H), 1.88−1.82 (m, 2H), 1.53 (t, 2H, J =
10.0 Hz). MS (ESI), m/z: 465.14 [M − H]−.
5-(4-(4-((5-Nitropyridin-2-yl)amino)piperidine-1-carbonyl)-
benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (17b). Yield:
27.5%. HPLC purity: 98.3%. White solid. 1H NMR (400 MHz,
DMSO-d6): δ 10.09 (s, 2H), 7.96−7.94 (m, 1H), 7.61 (d, 2H, J = 8.4
Hz), 7.07 (d, 2H, J = 8.4 Hz), 7.63−7.89 (m, 3H), 5.95 (s, 1H), 4.10−
4.08 (m, 1H), 4.02−3.98 (m, 2H), 1.88−1.82 (m, 2H), 1.53 (t, 2H, J =
10.0 Hz). MS (ESI), m/z: 465.14 [M − H]−.
Glucose Consumption/MTT in HepG2 Cell Lines. The HepG2
cells (ATCC, Rockville, MD) were grown in DMEM (22.2 mM
glucose) containing 10% fetal bovine serum. Two days before the
experiment, the cells were plated into 96-well tissue culture plates and
treated with 0.1 μM insulin. When the cells reached confluence, the
medium was removed and the DMEM (22.2 mM glucose) containing
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these compounds (10 μM), metformin (1.0 mM), and rosiglitazone
(10 μM, Sigma-Aldrich, St. Louis, MO) were added to all wells. After
24 h, the culture medium was sampled and its glucose concentrations
were determined by the glucose oxidase method (GENMED
SCIENTIFICS, U.S.). The amount of glucose consumption (GC)
was calculated by the glucose concentration of blank wells minus the
remaining glucose in cell plated wells and divided by corresponding
MTT values. The results were recorded from three independent
experiments.
Expression of Adiponectin and Leptin in 3T3-L1 Adipocytes.
Murine 3T3-L1 preadipocytes (ATCC, Rockville, MD) were plated
and grown to 2 days after confluence in six-well culture plates in
DMEM containing 10% fetal bovine serum. Preadipocytes were
induced to differentiate by replacing the medium with serum-
containing DMEM containing 0.5 mM methyl-3-isobutylxantine
(IBMX), 0.25 μM dexamethasone (DEX), and 1 μg/mL insulin.
Two days later, the medium was again 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
8−12 days. The twelfth day after differentiation, the culture medium
was replaced by DMEM supplemented with compounds (10 μM),
rosiglitazone (10 μM), and metformin (1.0 mM). After 24 h, the
determination of adiponectin and leptin was performed by commercial
ELISA kits (BOSTER, Wuhan, China). The results were recorded for
three independent experiments.
bleeding at 0.05, 0.25, 0.5, 1, 3, 6, 8, and 24 h postdose for the iv group
and at 0.25, 0.5, 1, 3, 6, 8, and 24 h postdose for the po group. At each
time point, three mice were bled resulting in a composite
pharmacokinetic profile. The tubes were inverted several times to
ensure mixing and placed on ice. Plasma was obtained following
centrifugation at 4 °C (1500−2000g). Plasma samples were stored at
−20 °C until analysis by a UPLC system.
Animal Model and Treatment of Diet-Induced Obesity.
C57BL/6J mice were obtained from Western China Experimental
Animal Center and housed individually in a room maintained at 25 °C
on a light/dark schedule. For DIO mice, 3-week-old male C57BL/6J
mice were fed a high fat diet (HFD, Research Diets) ad libitum for 8
weeks. Then these animals were assigned to four groups randomly
consisting of 10 mice each. The rats received a normal diet with
18.94% of energy derived from fat, 31.67% from protein, and 49.39%
from carbohydrates and received a high fat diet with 60.0% of calories
from fat, 20.0% from protein, and 20.0% from carbohydrates. HFD +
Met (150 mg kg−1 day−1) and HFD + 7h (25 mg kg−1 day−1) in
PEG400 or vehicle were orally administered per day for 7 weeks. Body
weight and food intake were measured per day. After all animals were
sacrificed, and serum levels of markers were analyzed according to the
NAFLD procedure. The percentages were calculated using [(VHFD
−
Vtreatment)/VHFD] × 100. Oral glucose tolerance test (OGTT) and
intravenous glucose tolerance test (IGTT) were performed in 7h-
treated DIO mice (n = 5) for 6 weeks after 1 night of fasting. Blood
were sampled from the tail vein of mice at 0, 15, 30, 60, 90, 120, and
180 min after an oral glucose load of 3.0 g/kg or intravenous glucose
load of 0.5 g/kg of body weight.
Animal Model and Treatment of NAFLD. Male Wistar rats
(160−180g) were purchased from Western China Experimental
Animal Center (Chengdu, China). 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 acclimatization for 1 week, animals were
assigned to five groups randomly consisting of 10 rats each (G1-G5).
In this study, G1−G4 groups were given access to high fat diet and G5
group was given access to normal diet throughout the experimental
period. The high fat diet was purchased from Shuangshi Experimental
Animal Diet Centre (Nanjing, China), which consisted of 20% lard
stearin (w/w) and 10% sucrose, and 0.1% bile salt was 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
and 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 high fat diet, metformin (150 mg kg−1 day−1), 7h (25 mg
kg−1 day−1), and 7q (25 mg kg−1 day−1) suspended in Tween 80 and
5% saline were orally administered correspondingly to the G1−G3
group, respectively. After the experimental period was completed, all
rats were anesthetized and samples were collected. Meanwhile, body,
liver, and fat weight of rats were recorded. Serum levels of triglycerides,
LDL-c, HDL-c, AST, ALT, and glucose were measured using a
multifunctional biochemistry analyzer Olympus AU2700 (Olympus,
Tokyo, Japan). The fasting insulin was tested by commercial kits
(R&D Systems, Minneapolis, MN, U.S.). The serum levels of
adiponectin and leptin were determined by commercial ELISA kits
(BOSTER, Wuhan, China). The percentages were calculated using
[(VHFD − Vtreatment)/VHFD] × 100. Liver tissues were homogenized in
50 mM Tris-HCl buffer (pH 7.4), containing 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM EDTA. The liver content of
triglycerides and total cholesterol were analyzed using a diagnostic kit
(Jiancheng, Nanjing, China) according to the manufacturer’s
instruction. Liver and adipose samples were fixed in 4% buffered
formalin and embedded in Tissue-Tek OCT compound (Sakura
Finetek USA, U.S.) and paraffin for H&E staining.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis and analytical data of all intermediates not described
in the Experimental Section; biological studies and details of PK
study in SD rats, and HPLC chromatograms of representative
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +86-28-85164063. E-mail: lijuan17@hotmail.com.
Author Contributions
⊥These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the National Key Programs of China during
the 12th Five-Year Plan Period (Grant 2012ZX09103101-033).
ABBREVIATIONS USED
■
NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic
steatohepatitis; HCC, hepatocellular carcinoma; GC, glucose
consumption; TsCl, p-toluenesulfonyl chloride; EDCI, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;
NaBH4, sodium borohydride; DMF, dimethylformamide;
TZD, thiazolidinedione; H&E, hematoxylin and eosin; HFD,
high fat diet; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide; LDL-c, low-density lipoprotein choles-
terol; HDL-c, high-density lipoprotein cholesterol; FFA, free
fatty acid; ALT, alanine transaminase; AST, aspartate amino-
transferase; DIO, diet-induced obesity; OGTT, oral glucose
tolerance test; IGTT, intravenous glucose tolerance test
Pharmacokinetics of 7h in Sprague−Dawley Rats. Two
groups (n = 5) of male and female Sprague−Dawley rats (180−
200g) were fasted overnight and received 7h as an intravenous (iv)
dose (10 mg/kg) or by oral gavage (50 mg/kg, dissolved in Tween
80). Blood samples (0.5 mL) were obtained from retro-orbital
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