Synthesis and biological evaluation of novel urea-and guanidine-based derivatives for the treatment of obesity-related hepatic steatosis

Article abstract of DOI:10.3390/molecules19056163

Leptin, the product of the obese gene, is an adipocyte-secreted protein hormone playing a key role in the progression of obesity and hepatic steatosis. In this study, 28 novel (thio)urea and guanidine-based analogues have been synthesized and N-(1-(4-(3-(

Full text of DOI:10.3390/molecules19056163

Molecules 2014, 19, 6163-6183; doi:10.3390/molecules19056163
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of Novel Urea- and
Guanidine-Based Derivatives for the Treatment of
Obesity-Related Hepatic Steatosis
Xiaolin Liang †, Heying Pei †, Liang Ma, Yan Ran, Jinying Chen, Guangcheng Wang 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;
E-Mails: liang_11@hsit.edu.cn (X.L.); pei_2014@hsit.edu.cn (H.P.); liang_m@scu.edu.cn (L.M.);
ran_2014@hsit.edu.cn (Y.R.); chen_2011@hsit.edu.cn (J.C.); wang_gc12@hsit.edu.cn (G.W.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: chenlijuan125@163.com;
Tel.: +86-28-8516-4063; Fax: +86-28-8516-4060.
Received: 3 March 2014; in revised form: 14 April 2014 / Accepted: 15 April 2014 /
Published: 15 May 2014
Abstract: Leptin, the product of the obese gene, is an adipocyte-secreted protein hormone
playing a key role in the progression of obesity and hepatic steatosis. In this study,
28 novel (thio)urea and guanidine-based analogues have been synthesized and N-(1-(4-(3-
(2-chloroethyl)ureido)benzyl)piperidin-4-yl)-3-(trifluoromethyl) benzamide (7i) was found
to be a potent regulator of leptin expression in 3T3-L1 adipocytes. Treatment with 7i at a
dose of 50 mg/kg/day for 35 days reduced the body weight and liver weight of
diet-induced obesity mice by 13.5% and 18.4%, respectively, while also improving the
serum levels of triglyceride, total cholesterol, leptin, adiponectin, LDL-c, HDL-c.
Hematoxylin-eosin (H&E) and Oil Red O staining also confirmed that 7i ameliorated fat
deposition in liver tissue and restricted the size of adipocytes in obesity-related fatty
liver disease.
Keywords: leptin; hepatic steatosis; urea and guanidine-based; diet-induced obesity

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1. Introduction
Obesity, with an increasing prevalence around the world, has been cited by the World Health
Organization as one of the greatest public health challenges [1,2]. It is considered a serious health
problem because of the high incidence of other metabolic syndromes such as type 2 diabetes,
cardiovascular disease, and osteoarthritis seen in obese individuals [3,4]. In gastroenterology, obesity-
related complications are also frequent, and the consequences of obesity for the liver have attracted
more attention because of its crucial role in metabolism [5].
Hepatic steatosis, which manifests at a high rate in overweight or obese individuals as fat infiltration
and deposition, is a precursor of advanced non-alcoholic fatty liver diseases (NAFLDs) [6,7]. Currently,
34% of the general population and over 75% of obese individuals are estimated to have fatty liver
diseases [8–10]. The high association between obesity and fatty liver disease may result from the
serum concentration changes of several important adipokines and one such adipokine is the 16 kDa
protein hormone leptin [11]. Leptin, a cytokine secreted by adipose tissue, plays a central role in the
regulation of energy balance via the activation of leptin receptors, particularly within the central
nervous system [12]. It has been demonstrated that the serum leptin concentration in obese subjects is
higher than in non-obese subjects, with a significant correlation between body weight and circulating
serum leptin levels [13,14].
Although the actual mechanisms leading to hepatic steatosis in obese individuals remain unclear,
insulin resistance (IR) and abnormal lipid metabolism are generally accepted as the essential risk
factors of obesity-related fatty liver disease [15]. In fact, elevated leptin levels in obesity inhibit insulin
signaling and enhance the IR effect, which leads to an increase in intracellular fatty acids and
deposition of triglycerides (TG) in the hepatocytes [16–20]. Recent studies also showed
that obesity-induced leptin functions directly in promoting the progression of non-alcoholic
steatohepatitis [21–23]. These compelling evidences suggested that leptin could be a potential
therapeutic target of obesity-related fatty liver disease.
As insulin sensitizers, metformin and rosiglitazone have been proved effective in the clinic to
reduce the serum leptin levels and improve the situation of hepatic steatosis [24–26]. However, it was
generally accepted that the use of TZDs would cause hepatotoxicity and increase the risk of heart
failure [27,28], which has provided the impetus to develop a series of novel compounds integrating the
structural characteristics of metformin and rosiglitazone and investigate their pharmaceutical functions
against obesity-related fatty liver disease. As shown in Figure 1, the long chain motif of rosiglitazone
has been kept with the changed 4-formamide-N-methylpiperidine or 4-hydroxy-N-ethanonepiperidine
bridges between two benzene rings, and then guanidine was introduced to the para-position of ring B
where a substituted thiazolidine was originally. Furthermore, as guanidine’s isosteric urea core has also
shown capacity in decreasing liver TG [29], this encouraged us to try to replace guanidine with
(thio)urea moieties. As a result compound 7i was discovered to be a good leptin regulator and to
improve hepatic steatosis symptoms in DIO mice.

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Figure1. Rationale for the design of compounds 7a–l, 8a–c, 14a–d, 17a–i.
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2. Results and Discussion
2.1. Chemistry
These urea derivatives 7 and thiourea derivatives 8 were prepared using the protocol outlined in
Scheme 1. Compound 3 was synthesized via the reaction of 4-nitrobenzyl bromide and tert-butyl
piperidin-4-ylcarbamate in the presence of K2CO3 as base and acetone as solvent. Then, the
tert-butyloxycarbonyl (Boc) protection was removed in TFA at 0 °C and treatment of the resulting
trifluoroacetate with various substituted benzoic acids in the presence of EDCI and DMAP yielded
compounds 5. The chemical reduction was conducted in anhydrous EtOH under reflux in the presence
of SnCl2 to give the corresponding amines 6. Finally, treatment of various amines 6 with the
corresponding iso(thio)cyanates in DCM at 0 °C afforded the desired (thio)urea derivatives 7 and 8
(Scheme 1 and Table 1).
Scheme 1. Synthesis of compounds 7a–l and 8a–c.
Reagents and conditions: (a) K2CO3, acetone, 70 °C, 4 h, 95.2%; (b) TFA, DCM, 0 °C to room temperature,
3 h, 96.8%; (c) R1COOH, EDCI, DMAP, DCM, room temperature, overnight, 65%–75%; (d) SnCl2, EtOH,
2 h, 80 °C, 45%–75%; (e) method A for 7a–7i, 8: substituted iso(thio)cyanate, DCM, 0 °C to room
temperature, 30%–70%; method B for 7j–7l: NaH, correspond 7, THF, 0 °C to room temperature, 30%–70%.

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Table 1. Structural and Inhibition Effects of Leptin Expression on 3T3-L1 of 7a–l, 8a–c.
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7a–l: X=O
8a–c: X=S
No.
7a
n
R1
R2
Inhibition of Leptin expression in 3T3-L1 (%) a
0
84.3
67.4
7b
7c
0
0
67.5
7d
7e
7f
0
0
0
0
54.3
73.9
68.1
77.1
7g
7h
0
74.5
7i
7j
0
2
89.0
54.1
H
H
H
7k
2
69.2
7l
2
0
78.4
61.4
8a

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Table 1. Cont.
No.
8b
n
R1
R2
Inhibition of Leptin expression in 3T3-L1 (%) a
0
48.1
8c
0
45.0
39.3
Metformin
a the result of inhibition effect were results were recorded from three independent experiments.
Intermediates 6 were treated with N,N'-di(tert-butoxycarbonyl)thiourea in the presence of
triethylamine (TEA) and mercury (II) chloride to give 16, as described in Scheme 2. Deprotection of
16 yielded the desired guanidine analogues 17 (Scheme 2 and Table 2).
Scheme 2. Synthesis of compounds 17a–i.
Reagents and conditions: (a) N,N'-Bis-tert-butoxycarbonylthiourea(or imidazolidinethione), HgCl2, TEA,
EtOH, DCM, 0 °C to room temperature, overnight, 30%–50%; (b) TFA, DCM, 0 °C to room temperature,
3 h, 20%–40%.
Table 2. Structural and Inhibition Effects of Leptin Expression on 3T3-L1 of 17a–i.
17a–i
No.
17a
n
0
R1
Inhibition of Leptin expression in 3T3-L1(%) a
53.0
17b
17c
0
0
42.5
42.3
17d
17e
0
0
22.8
15.1

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Table 2. Cont.
No.
17f
n
0
R1
Inhibition of Leptin expression in 3T3-L1(%) a
27.5
34.5
33.6
17g
17h
0
2
2
17i
18.7
39.3
Metformin
a the result of inhibition effect were results were recorded from three independent experiments.
Then, to explore the structure activity relationship (SAR) further, 4-piperidinol-substituted urea
analogues have been synthesized as described in Scheme 3.
Scheme 3. Synthesis of compounds 17a–i.
Reagents and conditions: (a) TsCl, pyridine, 0 °C to room temperature, overnight, 95.2%; (b) (i) K2CO3,
DMF, 100 °C, 89.7%, 8 h; (ii) TFA, DCM, 0 °C to room temperature, 3 h, 97.1%; (c) R1COOH, EDCI,
DMAP, DCM, room temperature, overnight, 65%–75%; (d) Pd/C, HCOONH4, MeOH, 2 h, 70 °C, 45%–75%;
(e) method A for 14a, 14b, 14d: substituted iso(thio)cyanate, DCM, 0 °C to room temperature, 30%–70%;
method B for 14c: NaH, 14b, THF, 0 °C to room temperature, 30%–70%.

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The preparation of 4-piperidinol-substituted urea analogues was carried out via treatment of
tert-butyl 4-hydroxypiperidine-1-carboxylate with p-toluenesulfonyl chloride (TsCl) at 0 °C in
pyridine as solvent giving 9 in a good yield. The reaction of 9 and 4-nitrophenol using K2CO3 as a
base, and subsequent deprotection of the Boc group yielded 11. The pivotal amines 13 were obtained
via the Pd/C reduction of 12 in MeOH at reflux. Compounds 14 were obtained by the same methods as
7and 8 (Scheme 3 and Table 3).
Table 3. Structural and Inhibition Effects of Leptin Expression on 3T3-L1 of 14a–c.
14a–c
No.
14a
R1
R2
Inhibition of Leptin expression in 3T3-L1 (%) a
42.5
14b
14c
45.0
50.3
14d
24.8
39.3
Metformin
a The result of inhibition effect were results were recorded from three independent experiments.
2.2. Expression of Leptin in 3T3-L1 Adipocytes
During the in vitro screening, these compounds containing (thio)urea and guanidine were evaluated
at 10 μM for their activity in decreasing leptin expression in 3T3-L1 adipocytes. Metformin was used
as positive control and all tests were performed three times using each agent (Tables 1–3).
As shown in Table 1, leptin expressions was all inhibited after treatment with (thio)urea analogues
at 10 μM. Compound 7i exhibited the most significant activity and reduced the leptin level by 89.0%
compared to metformin which decreased leptin by 39.3%. Further inspection of their structural features
showed that the potencies of the thiourea analogues 8 were in general inferior to those of the urea
analogues 7. As for the compounds 7, the introduction of chloroethyl as a tail group in the R2 position
(7g–i) led to better efficacy than ethyl or allyl groups (7a–f). Cyclizations of 7g–i were also carried
out, which only caused the loss rather than an increase of bioactivity (7j–k).
In Table 2, the analogues substituting (thio)urea for guanidine all displayed weak capability in
down-regulating leptin level in 3T3-L1 adipocytes, which further demonstrated that the urea moiety
was very important for the pharmaceutical activity. Moreover, two bridges, more specifically, 4-
aminopiperidine and 4-hydroxypiperidine (14a–d, Table 3) were compared and the 4-aminopiperidine
linker turned out to be better than 4-hydroxypiperidine for the bioactivity.

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Finally, regarding the role of substituent groups in the phenyl ring R1 position, it seemed that the
compounds with a trifluoromethyl group always displayed better activities than those with other
substituents, which might because of the special effect contributed by this group on compounds’
physicochemical and conformational properties [30].
2.3. Activity in Diet-Induced Obesity (DIO) Mice
Given the excellent in vitro leptin level lowering capability of 7i, it was chosen to evaluate the
in vivo activity in a diet-induced obesity (DIO) model. DIO is associated with hyperleptinemia and
hepatic steatosis in C57Bl/6J mice [31,32]. After oral administration with 7i once daily at 50 mg/kg for
five weeks, the average body weight and liver weight was reduced by 13.5% and 18.4%, respectively,
despite the consistent food intake compared to the untreated high-fat diet (HFD) group (Figure 2).
Subsequently, corresponding with the screening result in 3T3-L1 adipocytes, 7i notably reduced the
serum level of leptin by 34.6%, as well as TG, total cholesterol (TC), LDL-c and HDL-c which were
respectively reduced by 17.3%, 25.0%, 57.7%, and 20.9 %. Adiponectin, which counteracted the effect
of leptin in aggravating IR, is considered ia protector in hepatic steatosis and NASH [33–35]. The
in vivo test showed that the serum level of adiponectin was remarkably elevated by 93.0% after
treatment with 7i. Nevertheless the changes of aspartate aminotransferase (AST), alanine transaminase
(ALT) and total protein (TP) remained in a normal range, which suggested 7i scarcely causes any
serious hepatotoxicity (Figure 3, Table 4).
Figure 2. (a) Body weights of DIO mice, either HFD group (n = 5) or treated groups (n = 5),
were monitored during the oral administration of metformin (150 mg/kg/day) and 7i
(50 mg/kg/day) for 5 weeks; (b) Average body weight gain; (c) liver weight, and (d) Food
intake: *, p < 0.05, **, p < 0.01, vs. HFD.

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Figure 3. (a) Serum leptin level of DIO mice; (b) Serum adiponectin level of DIO mice;
(c) Serum triglyceride; (d) total cholesterol: *, p < 0.05, **, p < 0.01, vs. HFD.
Table 4. Parameters of Serum Biochemical Markers from DIO mice a
.
Parameter
LDL-c (mM)
HDL-c (mM)
ALT (U/L)
AST (U/L)
TP (g/L)
Normal
0.2
HFD
0.3
HFD + Met
0. 2 ± 0. 1
3.0 ± 0.9
HFD + 7i
0.1 **
1.6 ± 0.4
35.0 ± 12.7
96.0 ± 30.1
51.9 ± 3.3
2.8 ± 0.6
44.0 ± 9.6
105.0 ± 10.0
53.1 ± 3.4
2.2 *
34.0 ± 17.8
121.0 ± 30.9
57.1 ± 6.3
36.0 ± 9.6
126.0 ± 23.6
55.8 ± 3.7
a The HFD-fed (n = 5) or treated groups (n = 5) were respectively monitored during the oral administration of
Met (150 mg/kg/day) and 7i (50 mg/kg/day) for 5 weeks. The results were expressed as the mean ± SD:
*, p < 0.05, **, p < 0.01, vs. HFD.
2.4. Histopathological Evaluation
The effect of 7i in protecting the liver from the progression of obesity-related fat accumulation and
hepatic steatosis was further verified by hematoxylin-eosin (H&E) and Oil Red O staining assay [36,37].
As shown in Figure 4, DIO in C57/Bl6 mice is accompanied by an obvious fat deposition (steatosis)
and a remarkable increase in adipocyte size in the liver tissues compared to the normal group, while a
remarkable reduction of lipid droplets (black arrowheads) was observed, which means that there was
an obvious restriction of the fat accumulation in the section of liver tissue after treatment with 7i
(Figure 4a,b). Meanwhile, the adipocytes were smaller in size than those of the model group and
tended to normal levels (Figure 4c). The metformin-treated group showed weaker potency in
ameliorating both the fat deposition and the size of adipocytes compared to 7i.

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Figure 4. (a), (b) H&E staining and Oil Red O of liver tissue (scale, 200×). (c) H&E staining
of fat tissue from DIO rats (scale, 100×). Lipid droplets are marked using black arrowheads.
3. Experimental
3.1. General Information
Chemistry reagents of analytical grade were purchased from Changzheng Chemical Factory,
(Chengdu, China). TLC was performed on 0.20 mm Silica Gel 60 F254 plates (Qingdao Ocean
1
Chemical Factory, Shandong, China). H- and 13C-NMR spectra were recorded at 400 MHz and
100 MHz, respectively, on a Varian model Gemini 400 spectrometer (Varian, Palo Alto, CA, USA).
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; 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 on a
Q-TOF Premier mass spectrometer (Micromass, Manchester, UK) electrospray ionization (ESI).
3.2. Chemistry
3.2.1. Synthesis of tert-Butyl (1-(4-nitrobenzyl)piperidin-4-yl)carbamate (3)
4-(tert-Butoxycarbonylamino)piperidine (10.5 mmol), nitrobenzyl bromide (10.0 mmol), and potassium
carbonate (30.0 mmol) in acetone (60 mL) were stirred at 60 °C for 4 h. After the reaction was
complete, the mixture was concentrated under reduced pressure, and the residue was poured into water
(100 mL) to afford a pure light yellow solid which was collected by filtration, washed with water, and
then dried for the further use without any additional purification. Yield 95.2%; Yellow solid; 1H-NMR
(DMSO-d6): δ 8.18 (d, 2H, J = 8.4 Hz), 7.51 (d, 2H, J = 8.0 Hz), 4.44 (m, 1H), 3.58–3.49 (m, 3H),
2.77 (m, 2H), 2.15 (m, 2H), 1.95–1.92 (m, 2H), 1.44–1.42 (m, 11H); MS (ESI), m/z: 336.18 [M + H]+
.

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3.2.2. General Procedure for the Synthesis of Compounds 4
To a solution of 3 (1.0 mmol) in DCM (1 mL) was added TFA (1 mL) at 0 °C. The resulting
brownish orange solution is warmed to room temperature and stirred for 3 h and then concentrated
in vacuo to afford a brown oil. The residue was treated with diethyl ether (50 mL) to yield a white
solid which was isolated by filtration.
3.2.3. General Procedure for the Synthesis of Compounds 5 and 12
To a solution of substituted benzoic acid (2.0 mmol), EDCI (1.5 mmol) and DMAP (1.0 mmol) in
DCM (10 mL), 4 (2.0 mmol) was added and the mixture stirred at room temperature for 14 h. The
reaction mixture was then extracted with DCM and water, and the organic layer was washed with brine
and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The
residue was purified by column chromatography on silica gel to afford the title compounds.
3.2.4. General Procedure for the Synthesis of Compounds 6 and 13
A solution of the 5 (1 mmol) in anhydrous EtOH was treated with solid SnCl2·2H2O (7 mmol). The
mixture was stirred at 80 °C for 1.5 h and then poured cautiously into saturated aqueous K2CO3. The
resulting gelatinous emulsion was filtered through a pad of Celite, washed with DCM and the biphasic
filtrate was extracted with DCM. The combined organic extracts were dried (Na2SO4) and concentrated
in vacuo, affording the anilines without further purification.
N-(1-(4-Aminobenzyl)piperidin-4-yl)-3-(trifluoromethyl)benzamide (6a). Yield 75.2%; Yellow solid;
1H-NMR (DMSO-d6): δ 8.02 (s, 1H), 7.93 (d, 1H, J = 7.6 Hz), 7.75 (d, 1H, J = 7.6 Hz), 7.57 (t, 1H,
J = 8.0 Hz), 7.12 (d, 2H, J = 8.0 Hz), 6.66 (d, 2H, J = 8.0 Hz), 6.17 (d, 2H, J = 7.6 Hz), 4.05–4.02 (m,
1H), 3.50 (s, 2H), 2.95 (m, 2H), 2.22 (m, 2H), 2.03 (m, 2H), 1.71–1.65 (m, 2H); MS (ESI), m/z: 378.17
[M + H]+.
N-(1-(4-Aminobenzyl)piperidin-4-yl)-3-fluoro-4-(trifluoromethyl)benzamide (6b). Yield 66.8%;
1
Yellow solid; H-NMR (DMSO-d6): δ 7.69–7.58 (m, 3H), 7.12 (d, 2H, J = 8.0 Hz), 7.66 (d, 2H,
J = 8.4 Hz), 6.13 (m, 1H), 4.03–4.01 (m, 1H), 3.67 (s, 2H), 2.94 (m, 2H), 2.12 (m, 2H), 2.03 (m, 2H),
1.68 (m, 2H); MS (ESI), m/z: 396.16 [M + H]+.
N-(1-(4-Aminobenzyl)piperidin-4-yl)-4-chloro-2-methoxybenzamide (6c). Yield 65.2%; Yellow solid;
1H-NMR (DMSO-d6): δ 8.16 (d, 2H, J = 2.8 Hz), 7.77 (d, 1H, J = 7.6 Hz), 7.37 (dd, 1H, J = 8.8 Hz,
J = 2.8 Hz), 7.85–7.81 (m, 1H), 7.10 (d, 1H, J = 8.0 Hz), 6.90 (d, 1H, J = 8.8 Hz), 6.65 (d, 2H,
J = 8.4 Hz), 4.05–4.03 (m, 1H), 3.91 (s, 3H), 3.64 (brs, 2H), 3.42 (s, 2H), 2.78 (m, 2H), 2.18 (m, 2H),
2.02–1.98 (m, 2H), 1.57–1.55 (m, 2H); MS (ESI), m/z: 374.16 [M + H]+.
N-(1-(4-Aminobenzyl)piperidin-4-yl)-2,2-difluorobenzo[d][1,3]dioxole-4-carboxamide (6d). Yield
1
63.0%; Yellow solid; H-NMR (DMSO-d6): δ 7.67 (dd, 1H, J = 6.4 Hz, J = 2.8 Hz), 7.11–7.07 (m,
4H), 6.59 (d, 2H, J = 8.0 Hz), 6.50 (d, 2H, J = 7.6 Hz), 4.02 (m, 1H), 3.50 (s, 2H), 2.92 (d, 2H, J = 9.6 Hz),
2.25 (t, 2H, J = 10.8 Hz), 2.00 (d, 2H, J = 10.4 Hz), 1.77–1.72 (m, 2H); MS (ESI), m/z: 390.16 [M + H]+.

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N-(1-(4-Aminobenzyl)piperidin-4-yl)-3-(1,1,2,2-tetrafluoroethyl)benzamide (6e). Yield 65.2%; Yellow
1
solid; H-NMR (DMSO-d6): δ 7.64–7.62 (m, 2H), 7.45 (t, 1H, J = 8.0 Hz), 7.13 (d, 2H, J = 7.6 Hz),
6.66 (d, 2H, J = 7.6 Hz), 6.09–5.80 (m, 2H), 4.02 (m, 1H), 3.68 (s, 2H), 2.97 (d, 2H, J = 10.0 Hz), 2.25
(t, 2H, J = 12.0 Hz), 2.04 (d, 2H, J = 11.2 Hz), 1.74–1.72 (m, 2H); MS (ESI), m/z: 410.18 [M + H]+.
N-(1-(4-Aminobenzyl)piperidin-4-yl)-4-(difluoromethoxy)benzamide (6f). Yield 76.2%; Yellow solid;
1H-NMR (DMSO-d6): δ 7.91 (d, 2H, J = 8.4 Hz), 7.45 (d, 2H, J = 6.4 Hz), 7.20 (d, 1H, J = 8.0 Hz),
7.13 (d, 2H, J = 8.4 Hz), 6.83–6.47 (m, 3H), 4.06 (brs, 2H), 3.73 (s, 2H), 2.92 (d, 2H, J = 10.8 Hz),
2.48 (m, 2H), 2.03–1.99 (m, 24H); MS (ESI), m/z: 376.18 [M + H]+.
N-(1-(4-Aminobenzyl)piperidin-4-yl)nicotinamide (6g). Yield 45.2%; Yellow solid; 1H-NMR (DMSO-d6):
δ 8.55 (d, 2H, J = 4.4 Hz), 8.17 (d, 1H, J = 8.0 Hz), 8.03 (d, 1H, J = 7.2 Hz), 7.85–7.81 (m, 1H),
7.43–7,20 (m, 1H), 7.20 (d, 2H, J = 7.2 Hz), 6.69 (d, 2H, J = 8.4 Hz), 5.99 (m, 1H), 4.04 (m, 1H), 3.65
(m, 4H), 3.07 (m, 2H), 2.41–2.39 (m, 2H); MS (ESI), m/z: 310.18 [M + H]+.
N-(1-(4-Aminobenzyl)piperidin-4-yl)-5-methylpyrazine-2-carboxamide (6h). Yield 75.2%; Yellow
1
solid; H-NMR (DMSO-d6): δ 9.25 (s, 1H), 8.37 (s, 1H), 7.66 (d, 2H, J = 8.0 Hz), 7.10 (d, 2H,
J = 8.4 Hz), 6.65 (d, 2H, J = 8.4 Hz), 4.04–3.96 (m, 1H), 3.65 (brs, 2H), 3.43 (s, 2H), 2.86 (d, 2H,
J = 11.2 Hz), 2.64 (s, 2H), 2.17 (t, 2H, J = 11.2 Hz), 1.99 (d, 2H, J = 10.8 Hz), 1.67–1.58 (m, 2H); MS
(ESI), m/z: 326.19 [M + H]+.
(4-(4-Aminophenoxy)piperidin-1-yl)(3-fluoro-4-(trifluoromethyl)phenyl) methanone (13a). Yield
55.2%; Yellow solid; 1H-NMR (DMSO-d6): δ 7.67 (t, 1H, J = 7.6 Hz), 7.29–7.24 (m, 2H), 6.77 (d, 2H,
J = 8.4 Hz), 6.64 (d, 2H, J = 8.4 Hz), 4.44–4.39 (m, 1H), 3.85 (s, 2H), 3.61 (m, 1H), 3.31 (m, 1H),
1.94–1.80 (m, 4H); MS (ESI), m/z: 383.13 [M + H]+.
4-((1-((4-Fluorophenyl)sulfonyl)piperidin-4-yl)oxy)aniline (13b). Yield 60.2%; Yellow solid;
1H-NMR (DMSO-d6): δ 7.81–7.77 (m, 2H), 7.62–7.53 (m, 1H), 7.25–7.21 (m, 1H), 6.66–6.63 (m, 2H),
6.61–6,57 (m, 4H) 4.22–4.18 (m, 1H), 3.17–3.10 (m, 1H), 1.97–1.87 (m, 4H); MS (ESI), m/z: 351.11
[M + H]+.
3.2.5. General Procedure for the Synthesis of Compounds 7a–7i, 8, 14a, 14b, 14d
Ethyl isocyanate (3.6 mmol) was added dropwise to a stirred solution of the relevant aniline 6 or 13
(3 mmol) in DCM (15 mL). The reaction mixture was stirred overnight at ambient temperature. The
resulting crude precipitate was filtered, washed with cold ether, and purified by recrystallization from
ethanol and water.
N-(1-(4-(3-Ethylureido)benzyl)piperidin-4-yl)-3-(trifluoromethyl)benzamide (7a). Yield 55.2%; White
1
solid; H-NMR (DMSO-d6): δ 8.52 (d, 1H, J = 8.0 Hz), 8.35 (m, 2H), 8.15 (s, 1H), 7.99 (d, 1H,
J = 8.0 Hz), 7.71 (t, 2H, J = 8.0 Hz), 7.32 (d, 2H, J = 8.0 Hz), 7.13 (d, 2H, J = 8.0 Hz), 6.05 (t, 1H,
J = 4.0 Hz), 3.77 (m, 1H), 3.37 (s, 2H), 3.06 (m, 2H), 2.82 (d, 2H, J = 12.0 Hz), 1.98 (t, 2H, J = 12.0 Hz),
1.77 (m, 2H), 1.55 (m, 2H), 1.04 (t, 2H, J = 8.0 Hz); HRMSMS (ESI), m/z: 449.2086 [M + H]+
.

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N-(1-(4-(3-Allylureido)benzyl)piperidin-4-yl)-3-(trifluoromethyl)benzamide (7b). Yield 54.9%; White
1
solid; H-NMR (DMSO-d6): δ 8.52 (d, 1H, J = 4.0 Hz), 8.45 (s, 1H), 8.17–8.13 (m, 1H), 7.89 (d, 1H,
J = 8.0 Hz), 7.71–7.59 (m, 1H), 7.33 (d, 2H, J = 8.0 Hz), 7.14 (d, 2H, J = 8.0 Hz), 6.22 (m, 1H),
5.87–5.85 (m, 1H), 5.16 (d, 1H, J = 16.0 Hz), 5.06 (d, 1H, J = 12.0 Hz), 3.77–3.72 (m, 3H), 3.37 (s,
2H), 2.82 (d, 2H, J = 12.0 Hz), 1.99 (t, 2H, J = 12.0 Hz), 1.78 (m, 2H), 1.58 (t, 2H, J = 12.0 Hz);
HRMS (ESI), m/z: 461.2086 [M + H]+
.
N-(1-(4-(3-Ethylureido)benzyl)piperidin-4-yl)-3-fluoro-4-(trifluoromethyl)benzamide
(7c).
Yield
44.2%; White solid; 1H-NMR (DMSO-d6): δ 8.55 (d, 1H, J = 8.0 Hz), 8.37 (s, 1H), 7.91–7.81 (m, 2H),
7.32 (d, 1H, J = 8.0 Hz), 6.07–6.05 (m, 1H), 3.75 (m, 2H), 3.09–3.06 (m, 2H), 2.80 (m, 2H), 1.99 (m,
2H), 1.77 (m, 2H), 1.57–1.54 (m, 2H) 1.04 (t, 2H, J = 8.0 Hz); HRMS (ESI), m/z: 467.1992 [M + H]+
.
N-(1-(4-(3-Ethylureido)benzyl)piperidin-4-yl)-5-methylpyrazine-2-carboxamide (7d). Yield 47.8%;
1
White solid; H-NMR (CD3OD): δ 9.07 (s, 1H), 8.57 (s, 1H), 7.49 (d, 2H, J = 8.4 Hz), 7.39 (d, 2H,
J = 8.4 Hz), 4.23 (m, 3H), 3.48 (m, 3H), 3.23 (q, 2H, J = 7.2 Hz ), 3.13 (m, 2H), 2.63 (s, 3H), 2.20–2.17
(m, 2H), 2.01–1.98 (m, 2H) 1.15 (t, 2H, J = 7.2 Hz); HRMS (ESI), m/z: 397.2274 [M + H]+
.
4-Chloro-N-(1-(4-(3-ethylureido)benzyl)piperidin-4-yl)-2-methoxybenzamide (7e). Yield 39.5%; White
solid; 1H-NMR (DMSO-d6): δ 7.75 (d, 1H, J = 2.4 Hz), 7.45 (dd, 1H, J = 8.8 Hz, J = 2.8 Hz), 7.33 (d,
2H, J = 8.4 Hz), 7.22 (d, 2H, J = 8.4 Hz), 7.12 (d, 1H, J = 8.8 Hz), 3.92 (s, 4H), 3.50 (s, 2H), 3.22 (q,
2H, J = 7.2 Hz), 2.84 (d, 2H, J = 10.0 Hz), 2.24 (m, 2H), 1.96 (d, 2H, J = 10.0 Hz), 1.62 (m, 2H), 1.14
(t, 2H, J = 11.2 Hz); HRMS (ESI), m/z: 445.1928 [M + H]+
.
N-(1-(4-(3-Allylureido)benzyl)piperidin-4-yl)-4-chloro-2-methoxybenzamide (7f). Yield 57.7%; White
1
solid; H-NMR (CD3OD): δ 7.84 (d, 1H, J = 4.0 Hz), 7.44 (dd, 1H, J = 8.8 Hz, J = 2.8 Hz), 7.35 (d,
2H, J = 8.8 Hz), 7.22 (d, 2H, J = 8.8 Hz), 7.08 (d, 1H, J = 8.8 Hz), 5.93–5.87 (m, 1H), 5.22 (dd, 1H,
J = 17.2 Hz, J = 1.6 Hz), 5.11 (dd, 1H, J = 17.2 Hz, J = 1.6 Hz), 3.93 (s, 3H), 3.82 (d, 2H, J = 5.2 Hz),
3.51 (s, 2H), 2.84 (d, 2H, J = 9.6 Hz), 2.24 (t, 2H, J = 9.6 Hz), 1.98 (d, 2H, J = 10.8 Hz), 1.62 (q, 2H,
J = 10.0 Hz); HRMS (ESI), m/z: 457.1928 [M + H]+
.
4-Chloro-N-(1-(4-(3-(2-chloroethyl)ureido)benzyl)piperidin-4-yl)-2-methoxybenzamide (7g). Yield 60.2%;
White solid; 1H-NMR (DMSO-d6): δ 8.62 (s, 1H), 8.06 (d, 1H, J = 7.6 Hz), 7.59 (d, 1H, J = 2.8 Hz), 7.49
(dd, 1H, J = 8.4 Hz, J = 2.8 Hz), 7.34 (d, 2H, J = 8.4 Hz), 7.16–7.14 (m, 3H), 6.39 (t, 1H, J = 5.6 Hz),
3.86 (s, 3H), 3.83–3.77 (m, 1H), 3.65 (t, 2H, J = 6.4 Hz), 2.72 (d, 2H, J = 10.4 Hz), 2.06 (m, 2H), 1.77
(m, 2H), 1.51 (m, 2H); HRMS (ESI), m/z: 479.1538 [M + H]+
.
N-(1-(4-(3-(2-Chloroethyl)ureido)benzyl)piperidin-4-yl)-3-fluoro-4-(trifluoromethyl)benzamide (7h).
1
Yield 44.5%; White solid; H-NMR (DMSO-d6): δ 9.05 (s, 1H), 8.85 (bs, 1H), 7.95–7.90 (m, 2H),
7.48–7.42 (m, 2H), 6.61 (m, 1H), 4.11–3.99 (m, 2H), 3.66 (m, 2H), 3.42 (m, 2H), 3.02 (m, 2H), 1.94
(m, 3H), 1.20 (m, 2H); HRMS (ESI), m/z: 501.1602 [M + H]+
.
N-(1-(4-(3-(2-Chloroethyl)ureido)benzyl)piperidin-4-yl)-3-(trifluoromethyl)benzamide (7i). Yield
50.4%; White solid; 1H-NMR (DMSO-d6): δ 8.66 (s, 1H), 8.54 (d, 1H, J = 8.0 Hz), 8.18–8.14 (m, 2H),
7.90 (d, 1H, J = 4.0 Hz), 7.69 (t, 1H, J = 8.0 Hz), 7.35 (d, 2H, J = 8.0 Hz), 7.17 (d, 2H, J = 8.0 Hz),

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6.41 (s, 1H), 3.79 (s, 2H), 3.66 (m, 2H), 3.42 (m, 3H), 2.85 (s, 2H), 2.01 (s, 2H), 1.80 (m, 2H), 1.61
(m, 2H); HRMS (ESI), m/z: 483.1772 [M + H]+
.
N-(1-(4-(3-Ethylthioureido)benzyl)piperidin-4-yl)-3-(trifluoromethyl)benzamide (8a). Yield 64.2%;
1
White solid; H-NMR (CDCl3): δ 8.01 (s, 1H), 7.94 (d, 1H, J = 8.0 Hz), 7.93 (m, 2H), 7.55 (m, 2H),
7.39 (d, 1H, J = 8.0 Hz), 7.19 (d, 2H, J = 8.0 Hz), 6.12–6.07 (m, 2H), 4.04 (m, 1H), 3.69–3.66 (m,
2H), 3.56 (s, 2H), 2.92 (d, 1H, J = 12.0 Hz), 2.28–2.23 (m, 3H), 2.06 (d, 2H, J = 12.0 Hz), 1.67–1.64
(m, 2H), 1.20 (t, 2H, J = 8.0 Hz); HRMS (ESI), m/z: 465.1858 [M + H]+
.
N-(1-(4-(3-Ethylthioureido)benzyl)piperidin-4-yl)-3-fluoro-4-(trifluoromethyl)benzamide (8b). Yield
35.2%; White solid; 1H-NMR (CDCl3): δ 7.62–7.60 (m, 3H), 7.29 (d, 1H, J = 8.0 Hz), 7.17 (d, 1H, J =
8.0 Hz), 3.93–3.92 (m, 1H), 3.58 (q, 2H, J = 8.0 Hz), 3.50 (s, 2H), 2.90 (d, 1H, J = 12.0 Hz), 2.15 (m,
2H), 1.92 (d, 2H, J = 12.0 Hz), 1.65–1.55 (m, 2H), 1.13 (t, 2H, J = 8.0 Hz); HRMS (ESI), m/z:
483.1763 [M + H]+
.
4-Chloro-N-(1-(4-(3-ethylthioureido)benzyl)piperidin-4-yl)-2-methoxy benzamide (8c). Yield 65.2%;
White solid; 1H-NMR (DMSO-d6): δ 9.46 (bs, 1H), 8.12 (d, 1H, J = 7.2 Hz), 7.75 (s, 1H), 7.65 (s, 1H),
7.54 (d, 1H, J = 7.2 Hz), 7.38 (d, 2H, J = 8.0 Hz), 7.28 (d, 2H, J = 8.0 Hz), 7.20 (d, 1H, J = 8.0 Hz),
3.91–3.82 (m, 4H), 3.47–3.42 (m, 4H), 2.79 (d, 2H, J = 9.6 Hz), 2.13 (m, 2H), 1.87 (m, 2H), 1.57 (m,
2H), 1.16 (t, 2H, J = 6.8 Hz); HRMS (ESI), m/z: 461.1700 [M + H] +
.
1-Ethyl-3-(4-((1-(3-fluoro-4-(trifluoromethyl)benzoyl)piperidin-4-yl)oxy)phenyl)urea (14a). Yield
1
35.2%; White solid; H-NMR (CDCl3): δ 7.67 (t, 1H, J = 6.8 Hz), 7.19 (d, 2H, J = 8.8 Hz), 6.87 (d,
2H, J = 8.8 Hz), 6.26 (s, 1H), 4.65–4.56 (m, 2H), 3.87 (m, 2H), 3.47 (m, 2H), 2.87–2.37 (m, 2H),
1.95–1.84 (m, 4H), 1.13 (t, 3H, J = 7.2 Hz); HRMS (ESI), m/z: 454.1676 [M + H]+
.
1-(2-Chloroethyl)-3-(4-((1-(3-fluoro-4-(trifluoromethyl)benzoyl)piperidin-4-yl)oxy)phenyl)urea (14b).
Yield 35.2%; White solid; 1H-NMR (CDCl3): δ 7.68 (t, 1H, J = 6.8 Hz), 7.18 (d, 2H, J = 8.0 Hz), 6.86
(d, 2H, J = 8.0 Hz), 6.59 (s, 1H), 5.27 (m, 1H), 4.56–4.55 (m, 1H), 3.87 (m, 2H), 3.65–3.63 (m, 2H),
3.59–3.55 (m, 2H), 3.47 (m, 1H), 2.05–1.84 (m, 4H); MS (ESI), m/z: 488.13 [M + H]+
.
1-Ethyl-3-(4-((1-((4-fluorophenyl)sulfonyl)piperidin-4-yl)oxy)phenyl)urea (14d). Yield 55.2%; White
solid; 1H-NMR (DMSO-d6): δ 8.16 (s, 1H), 7.86–7.83 (m, 1H), 7.76–7.73 (m, 1H), 7.69–7.65 (m, 1H),
7.51 (t, 1H, J = 7.2 Hz), 7.21 (dd, 2H, J = 8.8 Hz, J = 2.8 Hz), 6.74 (dd, 2H, J = 8.8 Hz, J = 3.2 Hz),
5.94 (s, 1H), 4.31–2.89 (m, 1H), 3.22 (m, 2H), 3.10–3.03 (m, 2H), 2.84 (t, 2H, J = 8.8 Hz), 1.93 (m,
2H), 1.65–1.63 (m, 2H), 1.02 (t, 3H, J = 7.2 Hz); HRMS (ESI), m/z: 422.1472 [M + H]+
.
3.2.6. General Procedure for the Synthesis of Compounds 7j, 7k, 7l and 14c
Sodium hydride (3 mmol) was added slowly to a cold solution of compound 7g, 7h, 7i or 14b (1 mmol)
in THF (1.5 mL) under a nitrogen atmosphere. The ice bath was then removed after 30 min, and the
mixture was stirred at room temperature for 5 h. After the reaction was completed, the mixture was
diluted with cold water, and the resulting precipitate was filtered, washed with cold water and cold
ether, then dried to afford the target compounds without further purification.

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4-Chloro-2-methoxy-N-(1-(4-(2-oxoimidazolidin-1-yl)benzyl)piperidin-4-yl)benzamide (7j). Yield
1
55.2%; White solid; H-NMR (CDCl3): δ 8.15 (d, 1H, J = 2.4 Hz), 7.77 (d, 1H, J = 7.2 Hz), 7.48 (d,
1H, J = 8.4 Hz), 7.37 (dd, 1H, J = 8.8 Hz, J = 2.8 Hz), 7.30 (d, 2H, J = 8.4 Hz), 6.90 (d, 2H,
J = 8.8 Hz), 4.03 (m, 1H), 3.96–3.92 (m, 4H), 3.58 (t, 2H, J = 8.0 Hz), 3.50 (s, 2H), 2.79 (m, 2H), 2.22
(t, 2H, J = 10.4 Hz), 2.01 (d, 2H, J = 10.4 Hz), 1.58 (q, 2H, J = 10.4 Hz); HRMS (ESI), m/z: 443.1772
[M + H]+
.
3-Fluoro-N-(1-(4-(2-oxoimidazolidin-1-yl)benzyl)piperidin-4-yl)-4-(trifluoromethyl)benzamide (7k).
Yield 66.4%; White solid; 1H-NMR (CDCl3): δ 8.52 (d, 1H, J = 2.4 Hz), 8.17 (s, 1H), 8.14 (d, 1H,
J = 8.0 Hz), 7.89 (d, 1H, J = 7.6 Hz), 7.71 (t, 1H, J = 7.6 Hz), 7.10 (d, 2H, J = 8.4 Hz), 6.98 (d, 2H,
J = 8.4 Hz), 3.80–3.76 (m, 1H), 3.36 (s, 2H),3.58 (t, 2H, J = 8.0 Hz), 2.79 (m, 2H), 2.84 (d, 2H,
J = 11.6 Hz), 1.99 (t, 2H, J = 10.8 Hz), 1.87–1.77 (m, 2H), 1.61–1.53 (m, 2H); HRMS (ESI), m/z:
465.1835 [M + H]+
.
N-(1-(4-(2-Oxoimidazolidin-1-yl)benzyl)piperidin-4-yl)-3-(trifluoromethyl)benzamide
(7l).
Yield
1
47.9%; White solid; H-NMR (DMSO-d6): δ 8.01 (s, 1H), 7.93 (d, 1H, J = 7.6 Hz), 7.75 (d, 1H,
J = 7.6 Hz), 7.57 (t, 1H, J = 7.6 Hz), 7.50 (d, 2H, J = 8.4 Hz), 7.30 (d, 2H, J = 8.4 Hz), 6.10 (d, 1H,
J = 7.2 Hz), 4.04–4.02 (m, 1H), 3.95 (t, 2H, J = 8.0 Hz), 3.58 (t, 2H, J = 8.0 Hz), 2.90 (m, 2H), 2.20
(m, 2H), 2.02 (m, 2H), 1.61 (m, 2H); HRMS (ESI), m/z: 447.1930 [M + H]+
.
1-(4-((1-(3-Fluoro-4-(trifluoromethyl)benzoyl)piperidin-4-yl)oxy)phenyl)imidazolidin-2-one
(14c).
Yield 35.2%; White solid; 1H-NMR (CDCl3): δ 7.67 (t, 1H, J = 7.6 Hz), 7.54 (d, 2H, J = 9.2 Hz), 6.91
(d, 2H, J = 9.2 Hz), 4.69 (m, 1H), 4.55 (m, 2H), 3.93–3.87 (m, 4H), 3.57 (t, 2H, J = 8.0 Hz), 1.97–1.84
(m, 4H); HRMS (ESI), m/z: 452.1519 [M + H]+
.
3.2.7. General Procedure for the Synthesis of 9
A solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (5.0 mmol) in dry pyridine (15 mL) was
stirred at 0 °C for 10 min, and a solution of TsCl (7.5 mmol) in dry pyridine (20 mL) was added
slowly. The mixture was stirred at room temperature for 6 h. After the reaction was completed, the
mixture was poured into ice water. And then the precipitate was collected by filtration, washed with
water, and dried in vacuo to afford 9 as a white solid.
3.2.8. General Procedure for the Synthesis of 4-(4-Nitrophenoxy)piperidine (11)
A mixture of 9 (5.0 mmol), 4-nitrophenol 10 (5.5 mmol), and K2CO3 (10.0 mmol) in DMF (15 mL)
was heated to 100 °C for 10 h. After the reaction was completed, the K2CO3 was removed by filtration.
The filtrate was poured into ice water and extracted with DCM, saturated NaHCO3, and brine, and the
combined organic layer was dried with MgSO4. Finally, the solvent was removed under reduced
pressure to give the crude. A solution of the above crude solid (1.0 mmol) in 1 mL TFA/DCM
(v/v = 1/1) was stirred for 3 h at 0 °C, then allowed to warm to room temperature. After the reaction
was completed, the solvent was removed under reduced pressure. The residue was slowly made
alkaline with 2 N NaOH, then the precipitate formed was collected by filtration and washed with water
to afford the crude product 11 without further purification. Yield 89.5%; White solid; 1H-NMR (DMSO-

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d6): δ 8.20 (d, 2H, J = 9.2 Hz), 6.96 (d, 2H, J = 8.0 Hz), 4.08 (m, 1H), 3.73–3.67 (m, 2H), 3.41–3.35
(m, 2H), 1.99–1.94 (m, 2H), 1.80–1.77 (m, 2H); MS (ESI), m/z: 323.15 [M + H]+
.
3.2.9. General Procedure for the Synthesis of 16
The corresponding amine (2.0 mmol) in DCM was treated with mercury (II) chloride (2.2 mmol),
N,N'-di(tert-butoxycarbonyl)thiourea (2.0 mmol) and TEA (6.3 mmol) at 0 °C. The ice bath was
removed after 1h, and the resulting mixture was at room temperature overnight. Then, the reaction
mixture was filtered through a pad of Celite to get rid of the mercury sulfide formed. The filter cake
was rinsed with DCM. The organic phase was concentrated under vacuum to give a residue that was
purified by silica gel column chromatography to afford the title compound.
3.2.10. General Procedure for the Synthesis of 17
Each of the corresponding Boc-protected precursors (0.5 mmol) was treated with TFA/ DCM (15 mL,
v/v = 1/1) acid in DCM for 3 h. After the reaction was completed, the solvent was removed under
reduced pressure. The residue was slowly basified with saturated NaHCO3, extracted with EtOAc. The
organic phase was washed with water and brine, then dried over anhydrous Na2SO4 and concentrated
under vacuum to give a residue that was purified by silica gel column chromatography to afford the
title compound.
N-(1-(4-Guanidinobenzyl)piperidin-4-yl)-3-(trifluoromethyl)benzamide (17a). Yield 30.7%; Yellow
1
solid; H-NMR (DMSO-d6): δ 8.64 (d, 1H, J = 7.2 Hz), 8.18 (d, 2H, J = 8.0 Hz), 7.90 (d, 2H,
J = 8.0 Hz), 7.71 (t, 2H, J = 8.0 Hz), 7.59 (bs, 3H), 7.36 (d, 2H, J = 7.6 Hz), 7.18 (d, 2H, J = 7.6 Hz),
3.48 (s, 2H), 2.85 (d, 2H, J = 10.8 Hz), 2.09–1.99 (m, 2H), 1.79 (m, 2H), 1.66–1.57 (m, 2H); HRMS
(ESI), m/z: 420.1933 [M + H]+
.
3-Fluoro-N-(1-(4-guanidinobenzyl)piperidin-4-yl)-4-(trifluoromethyl)benzamide (17b). Yield 45.2%;
1
White solid; H-NMR (DMSO-d6): δ 8.55 (d, 1H, J = 7.6 Hz), 7.93–7.85 (m, 3H), 7.11 (d, 2H,
J = 8.0 Hz), 6.77 (d, 2H, J = 8.0 Hz), 3.77 (m, 1H), 3.37 (s, 2H), 2.84 (d, 2H, J = 11.6 Hz), 1.99 (t, 2H,
J = 11.2 Hz), 1.80–1.78 (m, 2H), 1.60–1.54 (m, 2H); HRMS (ESI), m/z: 438.1839 [M + H]+
.
4-Chloro-N-(1-(4-guanidinobenzyl)piperidin-4-yl)-2-methoxybenzamide (17c). Yield 35.2%; White
1
solid; H-NMR (DMSO-d6): δ 8.08 (d, 1H, J = 7.2 Hz), 7.59 (d, 1H, J = 2.4 Hz), 7.49 (dd, 1H,
J = 8.8 Hz, J = 2.4 Hz), 7.23 (t, 2H, J = 7.6 Hz), 7.15 (d, 1H, J = 8.8 Hz), 6.98 (d, 2H, J = 7.6 Hz),
3.86 (s, 3H), 3.77 (m, 1H), 3.41 (s, 2H), 2.75 (d, 2H, J = 10.0 Hz), 2.06 (t, 2H, J = 10.0 Hz), 1.80–1.77
(m, 2H), 1.04 (q, 2H, J = 10.0 Hz); HRMS (ESI), m/z: 416.1775 [M + H]+
.
2,2-Difluoro-N-(1-(4-guanidinobenzyl)piperidin-4-yl)benzo[d][1,3]dioxole-4-carboxamide
(17d).
Yield 25.9%; Yellow solid; 1H-NMR (CDCl3): δ 7.75–7.72 (m, 1H), 7.41 (d, 2H, J = 7.6 Hz), 7.20–7.15
(m, 4H), 6.55 (d, 2H, J = 7.6 Hz), 4.04 (m, 1H), 3.56 (s, 2H), 2.86 (d, 2H, J = 10.4 Hz), 2.30–2.25 (m,
2H), 1.66 (q, 2H, J = 10.0 Hz), 1.60–1.54 (m, 2H); HRMS (ESI), m/z: 432.1769 [M + H]+
.

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N-(1-(4-Guanidinobenzyl)piperidin-4-yl)-3-(1,1,2,2-tetrafluoroethoxy)benzamide (17e). Yield 25.0%;
Yellow solid; 1H-NMR (DMSO-d6): δ 7.79 (d, 1H, J = 6.8 Hz), 7.70 (s, 1H), 7.53–7.43 (m, 4H), 7.26
(d, 2H, J = 6.4 Hz), 6.48–6.22 (m, 1H), 3.89 (m, 1H), 3.63 (s, 2H), 3.00 (d, 2H, J = 9.2 Hz), 2.22 (t, 2H,
J = 10.0 Hz), 1.96 (d, 2H, J = 10.8 Hz), 1.73–1.70 (m, 2H); MS (ESI), m/z: 468.1944 [M + H]+
.
4-(Difluoromethoxy)-N-(1-(4-guanidinobenzyl)piperidin-4-yl)benzamide (17f). Yield 22.4%; White
1
solid; H-NMR (CD3OD): δ 7.76 (d, 2H, J = 8.4 Hz), 7.37 (d, 2H, J = 7.6 Hz), 7.16 (d, 2H,
J = 8.4 Hz), 7.10 (d, 2H, J = 8.4 Hz), 7.02–6.65 (m, 1H), 3.84–3.77 (m, 1H), 3.53 (s, 2H), 2.90 (d, 2H,
J = 11.6 Hz), 2.15 (t, 2H, J = 11.6 Hz), 1.85 (d, 2H, J = 9.6 Hz), 1.64–1.56 (m, 2H); HRMS (ESI), m/z:
418.1976 [M + H]+
.
1
N-(1-(4-Guanidinobenzyl)piperidin-4-yl)nicotinamide (17g). Yield 20.7%; White solid; H-NMR
(CDCl3): δ 8.54 (d, 1H, J = 4.4 Hz), 8.15 (d, 1H, J = 7.6 Hz), 8.01 (d, 1H, J = 8.4 Hz), 7.84 (td, 1H,
J = 7.6 Hz, J = 1.6 Hz), 7.44–7.41 (m, 1H), 7.38 (d, 2H, J = 8.4 Hz), 7.15 (d, 2H, J = 8.0 Hz),
3.98–3.96 (m, 1H), 3.50 (s, 2H), 2.82 (d, 2H, J = 11.6 Hz), 2.19 (t, 2H, J = 10.8 Hz), 1.99 (d, 2H,
J = 10.4 Hz), 1.68–1.59 (m, 2H); HRMS (ESI), m/z: 353.2012 [M + H]+
.
N-(1-(4-((4,5-Dihydro-1H-imidazol-2-yl)amino)benzyl)piperidin-4-yl)-3-(trifluoromethyl)benzamide
(17h). Yield 33.2%; White solid; 1H-NMR (CDCl3): δ 8.01 (s, 1H), 7.92 (d, 1H, J = 7.6 Hz), 7.75 (d,
1H, J = 7.6 Hz), 7.56 (t, 1H, J = 7.6 Hz), 7.20 (d, 2H, J = 8.0 Hz), 6.96 (d, 2H, J = 8.0 Hz), 6.08 (d,
1H, J = 7.6 Hz), 4.02–4.00 (m, 1H), 3.54 (m, 4H), 3.46 (s, 2H), 2.87 (d, 2H, J = 11.6 Hz), 2.16 (t, 2H,
J = 11.2 Hz), 2.02 (d, 2H, J = 11.2 Hz), 1.63–1.54 (m, 2H); HRMS (ESI), m/z: 674.1947 [M + H]+
.
N-(1-(4-((4,5-Dihydro-1H-imidazol-2-yl)amino)benzyl)piperidin-4-yl)-3-fluoro-4-(trifluoromethyl)-
1
benzamide (17i). Yield 38.1%; White solid; H-NMR (CDCl3): δ 7.67–7.59 (m, 3H), 7.20 (d, 2H,
J = 8.0 Hz), 6.95 (d, 1H, J = 8.0 Hz), 6.19 (d, 1H, J = 7.6 Hz), 3.99 (m, 1H), 3.45 (s, 2H), 2.18–2.12 (m,
2H), 2.01–1.99 (m, 2H), 1.62–1.54 (m, 2H); HRMS (ESI), m/z: 692.1853 [M + H]+
.
3.3. Biological Assay Methods
3.3.1. 3T3-L1 Adipocyte Differentiation
Marine 3T3-L1 preadipocytes (American Type Culture Collection, Rockville, MD, USA) were plated
and grown to two days post confluence in 96 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 mM dexamethasone (DEX) and
1 mg/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.

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3.3.2. Expression Levels of 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 (10 mM) and rosiglitazone (Sigma-Aldrich, St. Louis, MO, USA)
(10 mM). After 24 h, the expression levels of leptin were measured by commercial kits (Linco
Research, St. Charles, MO, USA).
3.3.3. 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, Nanjing, china) ad libitum for 8 weeks.
Then these animals were randomly assigned to four groups consisting of five mice each. The mice
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/day) and HFD + 7i (50 mg/kg/day) in PEG400
or vehicle were orally administered per day for five weeks. Body weight and food intake were
measured per day. And serum levels of biomarkers were analyzed after all animals were sacrificed.
The percentages were calculated using the formula [(VHFD − Vtreatment)/VHFD] × 100.
3.3.4. Assay for Serum Biochemical Markers
Serum ALT and AST levels were measured by an automated enzymeassay using commercial kits
and Roche automated analyzers (RocheDiagnostics GmbH, Manheim, Germany). Serum levels of TG,
LDL cholesterol, HDL-cholesterol, and plasma insulin were determined by radioimmunoassay using
commercially available kits (Linco Research).
3.3.5. Histopathological Examination
Liver samples were fixed in 4% buffered formalin and embedded in Tissue-Tek OCT compound
(Sakura Finetek USA, Torrance, CA, USA) and paraffin for histological analysis. Formalin-fixed and
paraffin embedded section (5 mm) was processed routinely for H&E staining. The OCT-embedded
samples were serially sectioned at 4 mm. For the evaluation of fat deposition, the frozen liver sections
was stained with Oil red O.
4. Conclusions
In this study, twenty eight novel (thio)urea- and guanidine-based derivatives have been synthesized
by an easy and practicable synthetic method. Subsequently, a credible screening method was adopted
to evaluate their bioactivity and 7i was proved to be a potential agent against diet-induced hepatic
steatosis. The pharmacological evaluation was started with the leptin expression on 3T3-L1
adipocytes, and among the tested compounds, 7i displayed potent capacity in decreasing leptin levels
at 10 μM. The weight of body and liver of high-fat diet induced mice were effectively reduced after
oral administration of 7i at a dose of 50 mg/ kg/day for five weeks. At the same time, 7i modulated

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serum parameters of leptin, adiponectin, TG, TC, LDL-c and HDL-c to appropriate ranges. Consistent
with the reduction of liver weight, further H&E and Oil Red O staining confirmed 7i reduced fat
deposition in the liver tissue and restricted the size of adipocytes against fatty liver disease. In
summary, these compelling results suggested that 7i improved the progression of obesity-related
hepatic dysfunctions and protected the liver tissue in diet-induced obese mice. Research on the detailed
pharmacokinetics and action of mechanism of 7i are in progress.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/5/6163/s1.
Acknowledgments
The authors greatly appreciate the financial support from National Key Programs of China during
the 12th Five-Year Plan Period (Grant 2012ZX09103101-033)
Author Contributions
L.X, P.H and C.L designed research; L.X, and P.H performed research and analyzed the data; M.L,
R.Y, C.J, and W.G helped analyzed the data. L.X wrote the paper. All authors read and approved the
final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Bray, G.A. Obesity: The disease. J. Med. Chem. 2006, 49, 4001–4007.
2. Kelly, T.; Yang, W.; Chen, C.-S.; Reynolds, K.; He, J. Global burden of obesity in 2005 and
projections to 2030. Int. J. Obesity. 2008, 32, 1431–1437.
3. O’Brien, P.E.; Dixon, J.B. The extent of the problem of obesity. Am. J. Surg. 2002, 184, S4–S8.
4. Pi-Sunyer, F.X. Medical hazards of obesity. Ann. Intern. Med. 1993, 119, 655–660.
5. Feneberg, A.; Malfertheiner, P. Epidemic trends of obesity with impact on metabolism and
digestive diseases. Digest. Dis. 2012, 30, 143–147.
6. Schindhelm, R.K.; Diamant, M.; Dekker, J.M.; Tushuizen, M.E.; Teerlink, T.; Heine, R.J. Alanine
aminotransferase as a marker of non-alcoholic fatty liver disease in relation to type 2 diabetes
mellitus and cardiovascular disease. Diabetes. Metab. Res. Rev. 2006, 22, 437–443.
7. Bacon, B.R.; Farahvash, M.J.; Janney, C.G.; Neuschwander-Tetri, B.A. Nonalcoholic
steatohepatitis: An expanded clinical entity. Gastroenterology 1994, 107, 1103–1109.
8. Henao-Mejia, J.; Elinav, E.; Jin, C.; Hao, L.; Mehal, W.Z.; Strowig, T.; Thaiss, C.A.; Kau, A.L.;
Eisenbarth, S.C.; Jurczak, M.J.; et al. Inflammasome-mediated dysbiosis regulates progression of
NAFLD and obesity. Nature 2012, 482, 179–186.
9. Angulo, P. Nonalcoholic fatty liver disease. N. Engl. J. Med. 2002, 346, 1221–1231.

Molecules 2014, 19
6182
10. Browning, J.D.; Horton, J.D. Molecular mediators of hepatic steatosis and liver injury. J. Clin.
Invest. 2004, 114, 147–152.
11. Jéquier, E. Leptin signaling, adiposity, and energy balance. Ann. N. Y. Acad. Sci. 2002, 967, 379–388.
12. Børbaek, C.; Kahn, B.B. Leptin signaling in the central nervous system and the periphery.
Prog. Horm. Res. 2004, 59, 305–331.
13. Dellas, C.; Lankeit, M.; Reiner, C.; Schäfer, K.; Hasenfuß, G.; Konstantinides, S.
BMI-independent inverse relationship of plasma leptin levels with outcome in patients with acute
pulmonary embolism. Int. J. Obes. (Lond.) 2013, 37, 204–210.
14. Considine, R.V.; Sinha, M.K.; Heiman, M.L.; Kriauciunas, A.I.; Stephens, T.W.; Nyce, M.R.;
Ohannesian, J.P.; Marco, C.C.; Mckee, L.J.; Bauer, T.L.; et al. Serum immunoreactive-leptin
concentrations in normal-weight and obese humans. N. Engl. J. Med. 1996, 334, 292–295.
15. Roberts, E.A. Pediatric nonalcoholic fatty liver disease (NAFLD): A “growing” problem?
J. Hepatol. 2007, 46, 1133–1142.
16. Pérez, C.; Fernández-Galaz, C.; Fernández-Agulló, T.; Arribas, C.; Andrés, A.; Ros, M.;
Carrascosa, J.M. Leptin impairs insulin signaling in rat adipocytes. Diabetes 2004, 53, 347–353.
17. Cohen, B.; Novick, D.; Rubinstein, M. Modulation of insulin activities by leptin. Science 1996,
274, 1185–1188.
18. Muller, G.; Ertl, J.; Gerl, M.; Preibish, G. Leptin impairs metabolic actions of insulin in isolated
rat adipocytes. J. Biol. Chem. 1997, 272, 10585–10593.
19. Valerio, N.; Melania, M.; Paolo, C.; Vincenzo, D.; Rita, D.; Fiorella, P.; Roberto, C.; Donatella, G.;
Matilde, M. Leptin, free leptin index, insulin resistance and liver fibrosis in children with
non-alcoholic fatty liver disease. Eur. J. Endocrinol. 2006, 155, 735–743.
20. Lewis, G.F.; Carpentier, A.; Adeli, K.; Giacca, A. Disordered fat storage and mobilization in the
pathogenesis of insulin resistance and type 2 diabetes. Endocr. Rev. 2002, 23, 201–229.
21. Leclercq, I.A.; Farrell, G.C.; Schriemer, R.; Robertson, G.R. Leptin is essential for the hepatic
fibrogenic response to chronic liver injury. J. Hepatol. 2002, 37, 206–213.
22. Imajo, K.; Fujita, K.; Yoneda, M.; Nozaki, Y.; Ogawa, Y.; Shinohara, Y.; Kato, S.; Mawatari, H.;
Shibata, W.; Kitani, H.; et al. Hyperresponsivity to low-dose endotoxin during progression to
nonalcoholic steatohepatitis is regulated by leptin-mediated signaling. Cell. MeTable 2012, 16,
44–54.
23. Patel, K.; Muir, A., McHutchison, J.G.; Patton, H.M. A link between leptin and steatosis in
chronic hepatitis C? Time to weigh up the fats. Am. J. Gastroenterol. 2003, 98, 952–955.
24. Freemark, M.; Bursey, D. The effects of metformin on body mass index and glucose tolerance in
obese adolescents with fasting hyperinsulinemia and a family history of type 2 diabetes.
Pediatrics 2001, 107, E55.
25. Duseja, A.; Das, A.; Dhiman, R.K.; Chawla, Y.K.; Thumburu, K.T.; Bhadada, S.; Bhansali, A.
Metformin is effective in achieving biochemical response in patients with nonalcoholic fatty liver
disease (NAFLD) not responding to lifestyle interventions. Ann. Hepatol. 2007, 6, 222–226.
26. Stein, L.L.; Dong, M.H.; Loomba, R. Insulin Sensitizers in Nonalcoholic Fatty Liver Disease and
Steatohepatitis: Current Status. Adv. Ther. 2009, 26, 893–907.
27. Plosker, GL.; Faulds, D. Troglitazone: A review of its use in themanagement of type 2 diabetes
mellitus. Drugs 1999, 57, 409–438.

Molecules 2014, 19
6183
28. Pereira, C.V.; Moreira, A.C.; Pereira, S.P.; Machado, N.G.; Carvalho, F.S.; Sardao, V.A.;
Oliveira, P.J. Investigating Drug-induced Mitochondrial Toxicity: A Biosensor to Increase Drug
Safety? Curr. Drug Saf. 2009, 4, 34–54.
29. Venier, O.; Pascal, C.; Braun, A.; Namane, C.; Mougenot, P.; Crespin, O.; Pacquet, F.; Mougenot, C.;
Monseau, C.; Onofri, B.; et al. Pyrrolidine-pyrazole ureas as potent and selective inhibitors of
11β-hydroxysteroid-dehydrogenase type 1. Bioorg. Med. Chem. Lett. 2011, 21, 2244–2251.
30. Sophie, P.; Peter, R.M.; Steve, S.; Veronique, G. Fluorine in medicinal chemistry. Chem. Soc. Rev.
2008, 37, 320–330.
31. Hoffler, U.; Hobbie, K.; Wilson, R.; Bai, R.; Rahman, A.; Malarkey, D.; Travlos, G.; Ghanayem, B.I.
Diet-induced obesity is associated with hyperleptinemia, hyperinsulinemia, hepatic steatosis, and
glomerulopathy in C57Bl/6J mice. Endocrine 2009, 36, 311–325.
32. Kang, H.S.; Okamoto, K.; Takeda, Y.; Beak, J.Y.; Gerrish, K.; Bortner, C.D.; DeGraff, L.M.;
Wada, T.; Xie, W.; Jetten, A.M. Transcriptional profiling reveals a role for RORα in regulating
gene expression in obesity-associated inflammation and hepatic steatosis. Physiol. Genomics
2011, 43, 818–828.
33. Adachi, M.; Brenner, D.A. High molecular weight adiponectin inhibits proliferation of hepatic
stellate cells via activation of adenosine monophosphate-activated protein kinase. Hepatology
2008, 47, 677–685.
34. Polyzos, S.A.; Kountouras, J.; Zavos, C.; Tsiaousi, E. The role of adiponectin in the pathogenesis
and treatment of non-alcoholic fatty liver disease. Diabetes Obes. Metab. 2010, 12, 365–383.
35. Wang, Y.; Zhou, M.; Lam, K.S.; Xu, A. Protective roles of adiponectin in obesity-related fatty
liver diseases: Mechanisms and therapeutic implications. Arq. Bras. Endocrinol. Metabol. 2009,
53, 201–212.
36. Hench, J.; Hench, I.B.; Pujol, C.; Ipsen, S.; Brodesser, S.; Mourier, A.; Tolnay, M.; Frank, S.;
Trifunović, A. A tissue-specific approach to the analysis of metabolic changes in Caenorhabditis
elegans. PLoS One 2011, 6, e28417.
37. Cook, H.C. Origins of Tinctorial methods in histology. J. Lin. Pathol. 1997, 50, 716–720.
Sample Availability: Samples of the compounds 7a–l, 8a–c, 14a–d, 17a–i are available from the authors.
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