Discovery of AdipoRon analogues as novel AMPK activators without inhibiting mitochondrial complex I

Article abstract of DOI:10.1016/j.ejmech.2020.112466

Activation of AMPK emerges as a potential therapeutic approach to metabolic diseases. AdipoRon is claimed to be an adiponectin receptor agonist that activates AMPK through adiponectin receptor 1 (AdipoR1). However, AdipoRon also exhibits moderate inhibition of mitochondrial complex I, leading to increased risk of lactic acidosis. In order to find novel AdipoRon analogues that activate AMPK without inhibition of complex I, 27 analogues of AdipoRon were designed, synthesized and biologically evaluated. As results, benzyloxy arylamide B10 was identified as a potent AMPK activator without inhibition of complex I. B10 dose-dependently improved glucose tolerance in normal mice, and significantly lowered fasting blood glucose level and ameliorated insulin resistance in db/db diabetic mice. More importantly, unlike the pan-AMPK activator MK-8722, B10 did not cause cardiac hypertrophy, probably owing to its selective activation of AMPK in the muscle tissue but not in the heart tissue. Together, B10 represents a novel class of AMPK activators with promising therapeutic potential against metabolic disease.

Full text of DOI:10.1016/j.ejmech.2020.112466

Journal Pre-proof
Discovery of AdipoRon analogues as novel AMPK activators without inhibiting
mitochondrial complex I
Geng Sun, Yanping You, Haobin Li, Yalong Cheng, Ming Qian, Xinyu Zhou, Haoliang
Yuan, Qing-Long Xu, Liang Dai, Pengfei Wang, Keguang Cheng, Xiaoan Wen,
Caiping Chen
PII:
S0223-5234(20)30437-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112466
EJMECH 112466
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 January 2020
Revised Date: 12 May 2020
Accepted Date: 13 May 2020
Please cite this article as: G. Sun, Y. You, H. Li, Y. Cheng, M. Qian, X. Zhou, H. Yuan, Q.-L. Xu, L. Dai,
P. Wang, K. Cheng, X. Wen, C. Chen, Discovery of AdipoRon analogues as novel AMPK activators
without inhibiting mitochondrial complex I, European Journal of Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.ejmech.2020.112466.
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Discovery of AdipoRon analogues as novel AMPK activators without inhibiting
mitochondrial complex I
a,
a,
a
a
a
†
†
Geng Sun , Yanping You , Haobin Li , Yalong Cheng , Ming Qian , Xinyu Zhou
a
a, Haoliang Yuan a, Qing-Long Xu a, Liang Dai , Pengfei Wang a, Keguang Cheng b,
Xiaoan Wen a,*, Caiping Chen a,*
a
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, State Key
Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009,
China
b State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources, School of Chemistry and Pharmacy of Guangxi Normal University, Guilin
541004, China
† These authors contributed equally to this article.
*
Corresponding
authors:
Tel/fax:
+86-25-83271150.
E-mail:
caiping.chen@cpu.edu.cn (C. P. Chen); wxagj@126.com (X. A. Wen)
Keywords
AMPK, activator, AdipoRon, Complex I, hypoglycemic action

Abstract
Activation of AMPK emerges as a potential therapeutic approach to metabolic
diseases. AdipoRon is claimed to be an adiponectin receptor agonist that activates
AMPK through adiponectin receptor 1 (AdipoR1). However, AdipoRon also exhibits
moderate inhibition of mitochondrial complex I, leading to increased risk of lactic
acidosis. In order to find novel AdipoRon analogues that activate AMPK without
inhibition of complex I, 27 analogues of AdipoRon were designed, synthesized and
biologically evaluated. As results, benzyloxy arylamide B10 was identified as a potent
AMPK activator without inhibition of complex I. B10 dose-dependently improved
glucose tolerance in normal mice, and significantly lowered fasting blood glucose
level and ameliorated insulin resistance in db/db diabetic mice. More importantly,
unlike the pan-AMPK activator MK-8722, B10 did not cause cardiac hypertrophy,
probably owing to its selective activation of AMPK in the muscle tissue but not in the
heart tissue. Together, B10 represents a novel class of AMPK activators with
promising therapeutic potential against metabolic disease.
1. Introduction
Adenosine monophosphate-activated protein kinase (AMPK) is a master sensor of
energy state and nutrient supply [1, 2]. It serves as a heterotrimeric serine/threonine
protein kinase. Composed of a catalytic subunit α (α1 or α2) and two regulatory
subunits β (β1 or β2) and γ (γ1, γ2 or γ3), AMPK theoretically creates 12
heterotrimeric isoforms that exhibit distinct tissue distribution and function in
mammals [3]. Physiologically, AMPK is mainly activated through α-subunit Thr172
phosphorylation by the upstream kinases liver kinase B1 (LKB1) and
Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) [4-6]. It has been
reported that the Thr172 phosphorylation increased the kinase activity of AMPK
by >100 fold [7, 8]. In addition, allosteric activation also plays an important role in
AMPK activation during energy stress [7, 9].
AMPK activation functions allowing for modulating energy balance, turning on
catabolic pathways for ATP generation and switching off biosynthetic pathways for

energy conservation by phosphorylation of downstream target proteins. For instance,
AMPK activation promotes insulin-independent glucose uptake to generate ATP in
skeletal muscle, decreases gluconeogenesis and fatty acid synthesis in liver, and
reduces fatty acid synthesis in adipose tissue [10, 11]. Therefore, AMPK emerges as
an attractive target for treatment of type 2 diabetes mellitus (T2DM) and other
metabolic diseases. Two types of anti-diabetic drugs, metformin [12, 13] and
thiazolidinediones (TZDs) [14], have been proved to exert hypoglycemic effect at
least partly through indirect activation of AMPK.
Various structural types of small molecule direct activators have been reported,
including AICAR (Fig. 1) that entered clinical trials but was abandoned due to side
effects. Several bottleneck problems arise with the current direct activators. For
examples, AICAR as an AMP analogue in vivo could affect other AMP-dependent
enzymes in addition to targeting AMPK, thus generating AMPK-independent effects
[15]. PF-249 (Fig. 1), specifically acting on AMPK β1 isoform, did not result in acute
glucose-lowering effect since the skeletal muscle mainly expresses AMPK β2 isoform
[16]. As a potent pan-AMPK activator, MK-8722 (Fig. 1) could cause cardiac
hypertrophy, although it greatly improved glucose homeostasis [17].
On the other hand, many indirect AMPK activators have been identified. For
example, R419 (Fig. 1) is one of the most potent indirect AMPK activators that works
through inhibiting mitochondrial respiratory complex I to elevate cellular AMP:ATP
ratio [18, 19]. AMPK activation by metformin [20], TZDs [21] and berberine [11, 22]
is also believed to be achieved through inhibition of complex I. Together, indirect
AMPK activation through inhibiting mitochondrial complex I would increase the risk
of lactic acidosis risk [10, 23]. This is evidenced by the result from a phase I trial of
R118 (a close derivative of R419), showing that inhibition of mitochondrial complex I
by R118 led to severe lactic acidosis in all the subjects at the highest dose [24].

Fig. 1 The structures of some AMPK activators
Adiponectin is one of the most abundant adipokines, which exerts insulin
sensitizing and anti-inflammatory effects through adiponectin receptors 1 and 2
(AdipoR1 and AdipoR2), leading to activation of AMPK and PPARα pathways,
respectively [25-27]. Therefore, agonism of AdipoR1 might represent an indirect
manner to activate AMPK. There are several peptide AdipoR agonists identified [28].
In 2013, Okada-Iwabu et al. reported a small molecule AdipoR agonist AdipoRon,
which significantly ameliorated glucose intolerance and insulin resistance in mice
through activating AMPK (EC50 about 10 μM) [29, 30]. AdipoRon has attracted much
attention, and by far more than 50 articles have been published with using AdipoRon
as a tool compound for biological and therapeutic proof-of-concept studies.
Nevertheless, in 2018, Akimoto et al. disclosed that AdipoRon could also cause
cellular complex I inhibition and mitochondrial dysfunction [31]. During our studies
on the structural modifications on AdipoRon, we found that AdipoRon indeed
inhibited mitochondrial respiratory chain (data not shown). Beyond AdipoRon, very
few other small molecule AdipoR agonists have been reported by far [32].
In this study, we aimed to develop novel AdipoRon analogues as AMPK activators
without inhibiting mitochondrial complex I. As shown in Fig. 2, AdipoRon shares
high structural similarity with the complex I inhibitor R419, and thus we tried to
substantially reduce this similarity. In this regard, two series of AdipoRon analogues
(A1~A15, B1~B12) were designed (Fig. 2). In series A, the direction of
4-aminopiperidinyl moiety was inverted and the 4-amino was derivatized to

phenylureas. In series B, 4-aminophenoxy moiety was used instead of
4-aminopiperidinyl. Moreover, in both series of compounds, chloride was chosen to
replace the phenyl ketone, a potential metabolic site of AdipoRon, and gem-dimethyl
were introduced at the α-position of amide to further add metabolic stability. After
synthesis and biological evaluation of these analogues, several potent AMPK
activators were discovered. Among them, compound B10 showed significant in vitro
and in vivo hypoglycemic effects. More importantly, it had no complex I inhibitory
activity and did not cause cardiac hypertrophy.
Fig. 2 Design of AdipoRon analogues
2. Results and discussion
2.1 Synthesis of AdipoRon analogues
The general synthetic route to analogues A1~A15 is shown in Scheme 1.
2-(4-chlorophenoxy)-2-methylpropanoic acid was successively treated by oxalyl
chloride and tert-butyl piperidin-4-yl carbamate to give the amide 2, which was
Boc-deprotected by HCl to give 4-amino-piperidine salt 3. Phenyl carbonochloridate
was condensed with various anilines to give the carbamates 1a~1k. Finally, 3
underwent nucleophilic substitution reactions with 1a~1k or various
isocyanatobenzenes to afford analogues A1~A15.
Scheme 1. General synthetic route to the analogues A1~A15

H
Cl
Cl
Cl
.
NH2
N
HCl
a
Boc
b
OH
N
N
O
O
O
O
O
O
2
3
R1
R1
H
N
R2
R3
O
Cl
H2N
R2
R3
O
for A1,
A4~A8,
A10~A15
c
d
+
O
O
1a~1k
R1
R1
H
H
Cl
R2
R3
N
N
OCN
R2
R3
e
3
+
N
O
O
for A2, A3, A9
A1~A15
O
o
Reagents and conditions: (a) (COCl)2, DMF, DCM, 0 C to r.t., 3 h; then tert-butyl
piperidin-4-ylcarbamate, DIPEA, 0 oC to r.t., 1 h, 86%; (b) HCl, EtOAc, r.t., overnight,
92%; (c) TEA, DCM, r.t., 3 h, 49-75%; (d) TEA, DMSO, r.t., 4 h, 34-80%; (e) TEA,
DCM, r.t., 4 h, 44-60%.
Scheme 2. General synthetic route to the analogues B1~B12
o
Reagents and conditions: (a) TBSCl, Imidazole, DCM, 0 C to r.t. 1 h; (b)
o
2-(4-chlorophenoxy)-2-methylpropanoic acid, (COCl)2, DMF, DCM, 0 C to r.t., 3 h;
o
then TEA, DCM, 0 C to r.t., 1 h, 56% for two steps; (c) TEA·3HF, DCM, r.t., 10 h,
82%; (d) substituted benzyl halides, K2CO3, DMF, 65 oC, 27-81%.
Analogues B1~B12 were synthesized according to the route shown in Scheme 2.
4-Aminophenol was protected by TBS to form the silicon ether 4.
2-(4-Chlorophenoxy)-2-methylpropanoic acid was successively treated by oxalyl
chloride and aniline 4 to give amide 5, which was then TBS-deprotected by TEA·3HF
and finally etherified with substituted benzyl halides to afford B1~B12.

2.2 Screening for AMPK phosphorylation by the compounds
All the 27 analogues were evaluated by Western blotting for their effects on
phosphorylation of AMPK Thr172. Screening was performed in mouse C2C12
myotube cells at 10 μM of compounds. The results (Fig. 3) showed that 20
compounds increased phosphorylation of AMPK by more than 50%. Among them, six
compounds (A2, A3, A6, B2, B10 and B11) were much more potent than AdipoRon
and comparable to MK-8722.
Fig. 3 Phosphorylation of AMPK in C2C12 myotube cells. (A) p-AMPK and AMPK were
analyzed by Western blot assay in C2C12 myotubes treated for 2 h with compounds (10 μM). (B)
Ratios of p-AMPK/AMPK were quantified. The values were represented as folds of the DMSO
vehicle group.

2.3 Glucose consumption in C2C12 myotube cells
Skeletal muscle is the primary tissue to dispose glucose and lower glucose levels
upon activation of AMPK [16]. Therefore, all the 27 analogues were evaluated for
their effects on glucose consumption in mouse C2C12 myotube cells at a
concentration of 10 μM. As shown in Table 1, among the A-series analogues, A6 (R3
= CF3) and A11 (R3 = OMe) stimulated a 34~37% increase in glucose consumption,
comparable to the effect of AdipoRon, while other compounds in the A-series had
weak or no activity.
Table 1 Glucose consumption stimulated by A1~A15 in C2C12 myotube cells.
Compd.
R1
H
R2
H
R3
H
GC (%) a
A1
118.0±14.7
H
H
F
A2
119.3±6.5
104.1±10.9
127.3±12.9
116.7±16.3
137.2±17.6
91.0±12.4
116.1±18.5
104.2±11.2
93.7±4.3
H
H
Cl
A3
H
H
Br
A4
H
Cl
H
F
A5
H
CF3
H
A6
H
CF3
H
A7
CF3
H
H
A8
H
OCF3
Me
OMe
COOEt
A9
H
H
A10
A11
A12
H
H
134.4±14.1
126.1±6.1
H
H

H
H
H
H
H
H
COOH
CN
A13
117.7±4.7
116.2±14.6
115.4±12.8
A14
NO2
A15
AdipoRon
132.4±13.6
DMSO
100
a Glucose consumption (GC) relative to the DMSO group. All compounds were tested
at 10 μM.
In the B-series of compounds, three compounds (B4, B10 and B11) were
remarkably more potent than AdipoRon and two compounds (B6 and B7) were
comparable to the effect of AdipoRon (Table 2). Several electron-withdrawing groups
were favorable for R4 in stimulating glucose consumption. Compounds B4, B10 and
B11, respectively bearing CF3, CN and SO2Me, showed the best potency with GC
increasing 44~56%. By contrast, compounds B6 (R4 = OMe) and B7 (R4 = OCF3) had
much decreased potency, and compound B5 (R4 = Me) showed almost no activity.
Table 2. Glucose consumption stimulated by B1-B15 in C2C12 myotube cells.
Compd.
R4
F
GC (%)a
B1
107.6±12.2
Cl
B2
B3
B4
B5
129.0±11.4
112.7±10.9
156.1±14.9
102.2±28.3
Br
CF3
Me

OMe
B6
134.7±8.1
133.4±23.0
124.7±11.4
116.5±6.7
151.8±4.0
144.4±12.9
105.3±19.7
OCF3
COOMe
COOH
CN
B7
B8
B9
B10
SO2Me
t-Bu
B11
B12
AdipoRon
132.4±13.6
DMSO
100
a Glucose consumption (GC) relative to the DMSO group. All compounds were tested at 10 μM.
2.4 Further confirmation of the AMPK activation effects of B10
Since compound B10 showed strong activity both in AMPK activation screening
and glucose consumption assays, further validation of its AMPK activation effects
was performed.
Acetyl-CoA carboxylase (ACC), a key substrate of AMPK, is commonly used as
the marker of AMPK activation. As shown in Fig. 4A, compound B10 induced
dose-dependently phosphorylation of ACC in C2C12 myotube cells, and the
phosphorylation of ACC was highly consistent with the phosphorylation of AMPK.
Time course experiments revealed that AMPK activation was significant after 1 h of
compound B10 treatment, reached to a high level after 6 h and maintained high within
6-24 h (Fig. 4B). Besides, B10 also increased the phosphorylation of AMPK and ACC
in human hepatocarcinoma HepG2 and HuH-7 cells, though the potency was a little
less than that in C2C12 cells (Fig. 4C-F). All these data confirmed that compound
B10 was a potent AMPK activator.
Preliminary mechanism study proposed that B10 activated AMPK probably in an
indirect manner since in the enzymatic tests, B10 had no influence on the enzymatic

activity of AMPK α1β1γ1 (widely distributed) and α2β2γ1 (predominant in skeletal
muscle) (Fig. S1).
Fig. 4 Compound B10 induced phosphorylation of AMPK and its downstream protein ACC.
C2C12 myotubes (A and B), HepG2 (C and D) and HuH-7 (E and F) cells were treated with
different concentrations of B10 for 2 h or with a single dose of 10 μM for different time courses.
The phosphorylation and total protein levels of cellular AMPK and ACC were analyzed by
Western blotting.
2.5 B10 neither inhibits mitochondrial complex I nor elevates cellular AMP and
ADP
It is well-known that elevation of cellular AMP:ATP and ADP:ATP ratios by
complex I inhibition leads to activation of AMPK. Recently, AdipoRon was reported
to inhibit cellular complex I activity and cause mitochondrial dysfunction [31], which
is contradictory to the previous report by Kadowaki’s group [29]. Notably, we also
found that AdipoRon indeed inhibited mitochondrial respiratory chain at 10 µM (data
not shown). Taken together, AdipoRon could increase the risk of lactic acidosis.

To our delight, compounds B10 and A6 did not inhibit complex I at 5~20 μM
concentrations, while B4 showed a significant inhibition of complex I (Fig. 5A).
Effects of B10 on cellular AMP and ADP levels were then examined. Consistently,
compound B10 did not increase the ratios of ADP/ATP and AMP/ATP in HepG2 cell,
while the positive control R419 robustly increased these two ratios (Fig. 5B and C).
Together, these results demonstrated that AMPK activation by B10 was not through
elevating cellular AMP and ADP levels.
Fig. 5 Effects of B10, B4 and A6 on complex I activity or ratios of AMP/ADP to ATP. (A) Effects
of B10, B4 and A6 on mitochondrial complex I activity. Rotenone was served as positive control.
(B) Ratio of cellular ADP:ATP in HepG2 cells treated with B10 (10 μM) or R419 (0.5 μM) or
DMSO control (CTR). (C) Ratio of cellular AMP:ATP in HepG2 cells treated as (B). Data are
expressed as mean ± S.D. for 3 replicates. **P <0.01, ***P <0.001; relative to CTR; as
determined by Student’s t test. n.s., not significant.
2.6 B10-mediated AMPK phosphorylation requires LKB1
We therefore hypothesized that B10 might induce AMPK phosphorylation through
activation of the upstream signaling pathway. LKB1 and CaMKKβ are the two
upstream kinases of AMPK. To clarify whether one or both of these kinases were
required for the AMPK phosphorylation by B10, LKB1 was first knocked down by
siRNA in C2C12 cells (Fig. S2A). As a result, LKB1 knockdown dramatically
blocked the B10-stimulated phosphorylation of AMPK and ACC (Fig. S2B). Then,
two human lung epithelial cell lines, BEAS-2B (LKB1 wild-type) and A549 (LKB1
mutant, CaMKKβ expressing), were subjected to B10 treatment. The results showed
that B10 activated AMPK in the BEAS-2B cells, but not in the A549 cells (Fig. S2C).

As expected, the positive control MK-8722 was still efficient in the A549 cells (Fig.
S2C), because it activated AMPK by both allosteric enhancement of the catalytic
activity and prevention of Thr172 from dephosphorylation [17, 33]. Next, CaMKKβ
in the C2C12 cells was inhibited using STO-609. As a result, the effect of CaMKKβ
inhibition was negligible on the B10-induced AMPK phosphorylation (Fig. S2D). All
of these demonstrated that AMPK activation by B10 was LKB1 dependent. Further
studies are ongoing to reveal the detailed mechanisms.
2.7 Effect of B10 on cell viability
Prior to in vivo studies, cytotoxicity of compound B10 was examined in C2C12
cells, HepG2 cells and A549 cells by MTT assay. As shown in Fig. 6, no significant
influence was observed for B10 on the cell viability of C2C12 cells and A549 cells at
the tested concentrations, and only slight decrease in the viability of HepG2 cells was
detected after 72 h of treatment at 10 μM and 20 μM.
Fig. 6 Effect of B10 on the cell viability. Cell viability was determined by MTT assay in C2C12
(A and B), HepG2 (C) and A549 cells (D) treated with B10 for 24 h (A) or 72 h (B-D). Each group
had 3 replicates. **P <0.001 vs 0 μM group (Student’s t test).
2.8 Metabolic stability profiles of B10 and B4 in liver microsomes
In vitro metabolic stability profiles of compounds B10 and B4 were measured in
human and mouse liver microsomes, with Ketanserin as control to validate the assay.
As shown in Table 3, both B10 and B4 were fairly stable against human liver
microsome metabolism, with 74% and 93% remaining at 45 min, respectively, and
T1/2 values of 97 min and >120 min, respectively. When exposed to mouse liver
microsomes, B4 also displayed a good stability, while B10 showed a moderate
stability with 39% remaining at 45 min and a T1/2 value of 36 min.

Table 3. Metabolic stability profiles of B10 and B4.
Compd.
Human liver microsomes
Remaining at T1/2
Mouse liver microsomes
Remaining at T1/2
Clint
Clint
45 min
93%
(min)
>120
97
(mL/min/kg) 45 min
(min)
>120
36
(mL/min/kg)
N/Aa
B4
N/Aa
91%
39%
21%
B10
74%
18
152
Ketanserin 27%
a not acquired.
25
71
21
258
As derivatives of para-aminophenol, compounds B10 and B4 had a potential to
produce para-iminoquinone metabolites. However, this potential was probably limited
by the steric hindrance of their benzyl groups, as the ethyl group in phenacetin
successfully prevented the formation of para-iminoquinone metabolites [34].
2.9 In vivo hypoglycemic efficacy of B10
In vivo hypoglycemic efficacy of B10 was then examined, since it showed
promising in vitro properties. First, an intraperitoneal glucose tolerance test (ipGTT)
was performed in normal lean C57BL/6 mice. As shown in Fig. 7A and B, compound
B10 dose-dependently improved glucose tolerance, although not as effective as the
positive control MK-8722. Notably, at the same dose of 100 mg/kg, B10 was a little
more effective than AdipoRon in lowering blood glucose, but no statistical
significance was observed (Fig. 7C and D).
Anti-hyperglycemic effect of B10 was further assessed in diabetic db/db mice.
Compared with vehicle control group, once-daily intragastrical administration of B10
(100 mg/kg) for 2~6 weeks significantly lowered the fasting blood glucose in db/db
mice (Fig. 7E). Moreover, B10 treatment for 9 weeks significantly improved insulin
resistance in db/db mice (Fig. 7F). During this long-term of treatment, the body
weight and the serum levels of alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) (two markers of liver injury) were not significantly affected
(Fig. 7G-I), indicating that B10 at the tested dosages was relatively safe. B10
treatment also seemed to have no improvement in lipid homeostasis, since the serum

levels of triglyceride (TG) and total cholesterol (TC) in the treatment group were
comparable to those in the control group (Fig. S3).
Fig. 7 B10 lowered plasma glucose and improved insulin resistance in mice. (A-B) Effects of
acute B10 administration (25, 50 and 100 mg/kg) in an ipGTT in C57BL/6 mice (8-to

10-week-old male, n=6 per group). (B) Net area under curve (AUC) of the data in (A). (C) ipGTT
in C57BL/6 mice receiving B10 (100 mg/kg), AdipoRon (100 mg/kg), MK-8722 (10 mg/kg) or
vehicle. (D) Net area under curve (AUC) of the data in (C). (E) Fasting blood glucose levels were
analyzed in db/db mice receiving B10 (100 mg/kg) (db/db-B10) or vehicle (db/db-veh) and db/m
mice (nondiabetic control) receiving vehicle (db/m-veh) at the indicated weeks of treatment. (F)
Plasma glucose during insulin tolerance test (ITT) (1.5U insulin per kg body weight) in db/db
mice treated with or without B10 (100 mg/kg) for 9 weeks. (G) Body weights of mice at the
indicated weeks of treatment of B10 (100 mg/kg) or vehicle. (H and I) Serum levels of ALT (H)
and AST (I) from mice receiving B10 (100 mg/kg) or vehicle for 10 weeks. Data are expressed as
mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with vehicle group (A-D) or
db/db-veh group (E and F) or db/m-veh group (H and I), two-way ANOVA (A, C, E and F) or
Student’s t test (B, D, H and I).
2.10 B10 does not cause cardiac hypertrophy
One of the major concerns with AMPK activators is the risk of cardiac side effects.
Gain-of-function mutations in PRKAG2 gene (encoding AMPK γ2 subunit) can cause
hypertrophic cardiomyopathy and an increase in cardiac glycogen. Notably, a similar
phenotype appeared in animals treated by the pan-AMPK activator MK-8722 [17].
We therefore examined whether B10 could cause hypertrophic effect in mice heart.
As shown in Fig. 8A-D, unlike MK-8722, 4-week administration of compound B10
did not cause cardiac hypertrophy nor increased cardiac glycogen in normal lean
C57BL/6 mice. No obvious changes were detected in gross morphology of heart,
heart weight/body weight ratio and heart weight/tibia length ratio. B10 treatment for
10 weeks did not either induce cardiac hypertrophy or glycogen accumulation in
diabetic db/db mice (Fig. S4). The heart and skeleton muscle tissues from B10-treated
normal lean C57BL/6 mice were subjected to comparative analysis of AMPK
activation. Interestingly, unlike MK-8722, B10 did not increase the levels of p-AMPK
or p-ACC in heart tissues (Fig. 8E), but elevated skeletal muscle p-ACC, albeit not as
potent as MK-8722 (Fig. 8F). AMPK activation by B10 was further determined in a
rat cardiac cell line H9C2. Similarly, B10 was much less efficient to stimulate the

phosphorylation of AMPK and ACC in cardiac H9C2 cells, compared with its effect
in C2C12 myotube cells (Fig. 8G and H). All these results suggested that compound
B10 might be selective for muscle over heart tissues in term of AMPK activation, thus
avoiding the risk of cardiac hypertrophy.
Fig. 8 Effect of chronic B10 treatment on cardiac parameters in mice. (A) Gross morphology of
hearts from mice administrated with B10 (50, 100 and 200 mg/kg), MK-8722 (10 mg/kg) and
vehicle control for 4 weeks (6 mice each group). (B-D) Effects of B10 administration on heart
weight/body weight ratio (B), heart weight/tibia length ratio (C) and cardiac glycogen (D). Scale

bar: 50 μm. (E and F) p-AMPK and p-ACC in heart (E) and skeletal muscle (F) were analyzed by
Western blot assay after administration with B10 or MK-8722 or vehicle. (G-H) Western blot
analysis of phosphorylation and total amounts of AMPK and ACC in H9C2 cells after treatment
with concentrations of B10 for 2 h or 4 h (G) or with a single concentration at 10 μM for a time
course (H). **p < 0.01 compared with the vehicle group.
3. Conclusion
In this study, 27 analogues of AdipoRon have been designed and synthesized.
Among them, six compounds (A2, A3, A6, B2, B10 and B11) were found to activate
AMPK much more potently than AdipoRon in C2C12 myotubes, and two compounds
(B4 and B10) exhibited superior activity in stimulating glucose consumption (GC >
150%). Cellular AMP/ADP analysis and in vitro enzymatic test demonstrated that B10
did not inhibit mitochondrial complex I. Experiments with LKB1 knockdown and
mutant cells revealed that the B10-mediated AMPK phosphorylation required the
upstream kinase LKB1. In vivo study results showed that oral administration of B10
dose-dependently improved glucose tolerance in normal mice, and significantly
lowered fasting blood glucose level and ameliorated insulin resistance in db/db
diabetic mice at the doses of 25~100 mg/kg/day. More importantly, unlike the
pan-AMPK activator MK-8722, B10 did not cause cardiac hypertrophy and glycogen
accumulation. It was revealed that B10 selectively activated AMPK in the muscle
tissues, but not in the heart tissues. Taken together, B10 represents a novel class of
AMPK activators that hold therapeutic promise for metabolic diseases.
4. Experimental section
4.1 Chemistry
General information General procedures of the synthesis route were described as
below. Commercially available reagents and solvents were used without further
purification. Analytical thin-layer chromatography (TLC) was performed on silica gel
60 F254 plates (Qingdao Ocean Chemical Company, China). Column
chromatography was carried out on silica gel (200–300 mesh, Qingdao Ocean

Chemical Company, China). Nuclear magnetic resonance (NMR) spectra were
performed on an ACF* 300Q Bruker spectrometer. Proton coupling patterns were
described as singlet (s), doublet (d), doublet of doublets (dd), triplet (t), triplet of
doublets (td), quartet (q), multiplet (m), and broad (br). Low- and high-resolution
mass spectra (MS and HRMS) were given with electrospray ionization (ESI)
produced by Waters Q-TOF Micro and Agilent G6230B, respectively. Melting points
(mp) were measured in open capillary tubes, using an RY-1G melting point apparatus
(0 to 300 °C).
All target compounds were confirmed with over 95% purity by Agilent Prime LC
with a detector 1260 DAD HS at 220 nm and a column Agilent Poroshell HPH-C18
(2.1 × 50 mm, 2.7 μm). Water containing 0.1% formic acid (A) and acetonitrile
containing 0.05% formic acid (B) were used to perform elution at a flow rate of 1.2
mL/min, with a gradient from 30 to 95% B (or 50 to 100% B) over 4.5 min and 95%
B (or 100% B) held for 0.5 min (see Supporting Information, Table S1).
General procedures for synthesis of A1~A15 and B1~B12 are examplified by
A1/A2 and B1, respectively. Experimental details for synthesis of 1a~1k and 2~6 are
included in Supporting information.
4.1.1
1-(1-(2-(4-Chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)-3-phenylurea
(A1)
Phenyl phenylcarbamate (1a) (70 mg, 0.33 mmol) was added to a solution of 3 (100
mg, 0.3 mmol) and TEA (83 µL, 0.6 mmol) in DMSO (4 mL). The reaction mixture
was stirred at room temperature for 4 h, poured into ice water with stirring and filtered.
The collected crude product was suspended in anhydrous Et2O (4 mL), stirred and
o
1
filtered to yield compound A1 (100 mg, 80%) as a white solid. mp: 176-178 C. H
NMR (300 MHz, DMSO-d6) δ ppm 8.20 (s, 1 H), 7.39-7.29 (m, 4 H), 7.24-7.14 (m, 2
H), 6.92-6.78 (m, 3 H), 6.06 (d, J = 7.5 Hz, 1 H), 4.39-4.15 (m, 2 H), 3.69-3.56 (m, 1
H), 3.27-3.13 (m, 1 H), 2.98-2.82 (m, 1 H), 1.89-1.61 (m, 2 H), 1.55 (s, 6 H),
1.20-1.09 (m, 1 H), 0.97-0.80 (m, 1 H). 13C NMR (75 MHz, DMSO-d6) δ ppm 169.69,
154.28, 153.98, 140.24, 129.20, 128.48, 125.11, 120.92, 118.58, 117.50, 80.71, 45.79,

43.63, 41.37, 32.18, 31.94, 25.79, 25.35. ESI-MS: m/z 438.2 [M+Na]+. HRMS (ESI):
m/z calcd for C22H27ClN3O3 [M+H]+ 416.1735, found 416.1738. LC tR: 1.991 min,
purity 99.21%.
4.1.2
1-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)-3-(4-fluorophenyl)urea
(A2)
1-fluoro-4-isocyanatobenzene (41 mg, 0.3 mmol) was added to a solution of 3 (100
mg, 0.3 mmol) and TEA (83 µL, 0.6 mmol) in CH2Cl2 (4 mL). The reaction mixture
was stirred at room temperature for 4 h before water was added. The solution was
extracted with CH2Cl2 (2 x 8 mL) and the organic layer was washed with brine, dried
over Na2SO4, filtered, and concentrated. The crude product was purified by column
chromatography (CH2Cl2/ethyl acetate = 5/1) and the residue was suspended in
hexane (4 mL), stirred and filtered to yield compound A2 (70 mg, 54%) as a white
solid. mp: 186-189 oC. 1H NMR (300 MHz, DMSO-d6) δ ppm 8.24 (s, 1 H), 7.39-7.30
(m, 4 H), 7.03 (dd, J = 8.9, 8.9 Hz, 2 H), 6.82 (d, J = 8.9 Hz, 2 H), 6.06 (d, J = 7.5 Hz,
1 H), 4.40-4.15 (m, 2 H), 3.69-3.53 (m, 1 H), 3.28-3.14 (m, 1 H), 3.00-2.81 (m, 1 H),
13
1.89-1.61 (m, 2 H), 1.55 (s, 6 H), 1.26-1.09 (m, 1 H), 0.98-0.77 (m, 1 H). C NMR
(75 MHz, DMSO-d6) δ ppm 169.69, 156.80 (d, J = 237.2 Hz), 154.34, 153.98, 136.60
(d, J = 2.3 Hz), 129.21, 125.11, 119.11 (d, J = 7.6 Hz), 118.58, 114.93 (d, J = 22.1 Hz),
80.71, 45.84, 43.61, 41.34, 32.17, 31.93, 25.85, 25.37. ESI-MS: m/z 456.2 [M+Na]+.
HRMS (ESI): m/z calcd for C22H26ClFN3O3 [M+H]+ 434.1641, found 434.1645. LC tR:
2.105 min, purity 99.96%.
4.1.3
1-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)-3-(4-chlorophenyl)urea
(A3)
o
White solid, yield 44%. mp: 155-157 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
8.35 (s, 1 H), 7.37 (d, J = 8.8 Hz, 2 H), 7.34 (d, J = 8.8 Hz, 2 H),7.23 (d, J = 8.8 Hz, 2
H), 6.82 (d, J = 8.9 Hz, 2 H), 6.12 (d, J = 7.4 Hz, 1 H), 4.40-4.15 (m, 2 H), 3.70-3.59

(m, 1 H), 3.27-3.12 (m, 1 H), 3.01-2.76 (m, 1 H), 1.90-1.62 (m, 2 H), 1.55 (s, 6 H),
1.27-1.09 (m, 1 H), 1.04-0.79 (m, 1 H). 13C NMR (75 MHz, DMSO-d6) δ ppm 169.70,
154.13, 153.98, 139.24, 129.20, 128.31, 125.12, 124.44, 118.99, 118.58, 80.71, 45.86,
43.61, 41.32, 32.10, 31.83, 25.79, 25.36. ESI-MS: m/z 448.1 [M-H]-. HRMS (ESI):
m/z calcd for C22H26Cl2N3O3 [M+H]+ 450.1346, found 450.1353. LC tR: 2.478 min,
purity 99.95%.
4.1.4
1-(4-bromophenyl)-3-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)urea
(A4)
o
White solid, yield 41%. mp: 176-178 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
8.36 (s, 1 H), 7.41-7.27 (m, 6 H), 6.82 (d, J = 9.0 Hz, 2 H), 6.13 (d, J = 7.4 Hz, 1 H),
4.40-4.18 (m, 2 H), 3.69-3.56 (m, 1 H), 3.26-3.15 (m, 1 H), 2.98-2.84 (m, 1 H),
13
1.87-1.63 (m, 2 H), 1.55 (s, 6 H), 1.25-1.09 (m, 1 H), 0.99-0.80 (m, 1 H). C NMR
(75 MHz, DMSO-d6) δ ppm 169.69, 154.08, 153.97, 139.66, 131.21, 129.21, 125.11,
119.42, 118.58, 112.21, 80.71, 45.85, 43.60, 41.40, 32.13, 31.88, 25.80, 25.41.
ESI-MS: m/z 516.1 [M+Na]+. HRMS (ESI): m/z calcd for C22H26BrClN3O3 [M+H]+
494.0841 and 496.0820, found 494.0841 and 496.0819. LC tR: 2.583 min, purity
99.73%.
4.1.5
1-(3-chloro-4-fluorophenyl)-3-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-
4-yl)urea (A5)
o
White solid, yield 70%. mp: 167-169 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
8.42 (s, 1 H), 7.71 (dd, J = 6.8, 2.5 Hz, 1 H), 7.34 (d, J = 8.9 Hz, 2 H), 7.24 (dd, J =
9.0, 9.0 Hz, 1H), 7.20-7.12 (m, 1H), 6.82 (d, J = 8.9 Hz, 2 H), 6.18 (d, J = 7.4 Hz, 1
H), 4.40-4.17 (m, 2 H), 3.69-3.57 (m, 1 H), 3.25-3.15 (m, 1 H), 2.97-2.81 (m, 1 H),
13
1.88-1.63 (m, 2 H), 1.55 (s, 6 H), 1.21-1.09 (m, 1 H), 0.99-0.84 (m, 1 H). C NMR
(75 MHz, DMSO-d6) δ ppm 169.70, 154.13, 153.97, 151.79 (d, J = 240.1 Hz), 137.55
(d, J = 2.7 Hz), 129.20, 125.12, 118.89 (d, J = 18.3 Hz), 118.72, 118.58, 117.63 (d, J

= 6.6 Hz), 116.52 (d, J = 21.6 Hz), 80.71, 45.97, 43.58, 41.38, 32.13, 31.82, 25.78,
25.41. ESI-MS: m/z 490.1 [M+Na]+. HRMS (ESI): m/z calcd for C22H25Cl2FN3O3
[M+H]+ 468.1252, found 468.1253. LC tR: 2.590 min, purity 99.86%.
4.1.6
1-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)-3-(4-(trifluoromethyl)p
henyl)urea (A6)
o
White solid, yield 45%. mp: 169-171 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
8.66 (s, 1 H), 7.60-7.49 (m, 4 H), 7.35 (d, J = 8.9 Hz, 2 H), 6.82 (d, J = 8.9 Hz, 2 H),
6.25 (d, J = 7.4 Hz, 1 H), 4.43-4.17 (m, 2 H), 3.75-3.60 (m, 1 H), 3.29-3.13 (m, 1 H),
2.99-2.80 (m, 1 H), 1.9-1.62 (m, 2 H), 1.55 (s, 6 H), 1.28-1.09 (m, 1 H), 1.08-0.83 (m,
1 H). 13C NMR (75 MHz, DMSO-d6) δ ppm 169.71, 153.98, 153.91, 143.94, 129.21,
125.79 (q, J = 3.8 Hz), 125.13, 124.51 (q, J = 270.9 Hz), 120.97 (q, J = 31.8 Hz),
118.59, 117.12, 80.71, 45.92, 43.64, 41.35, 32.09, 31.78, 25.79, 25.31. ESI-MS: m/z
506.2 [M+Na]+. HRMS (ESI): m/z calcd for C23H26ClF3N3O3 [M+H]+ 484.1609,
found 484.1607. LC tR: 2.865 min, purity 98.96%.
4.1.7
1-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)-3-(3-(trifluoromethyl)p
henyl)urea (A7)
White solid, yield 34%. mp: 80-83oC. 1H NMR (300 MHz, DMSO-d6) δ ppm 8.60
(s, 1 H), 7.93 (s, 1 H), 7.48-7.40 (m, 2 H), 7.35 (d, J = 8.9 Hz, 2 H), 7.26-7.16 (m, 1
H), 6.82 (d, J = 8.9 Hz, 2 H), 6.23 (d, J = 7.4 Hz, 1 H), 4.41-4.17 (m, 2 H), 3.72-3.57
(m, 1 H), 3.27-3.14 (m, 1 H), 2.97-2.81 (m, 1 H), 1.90-1.63 (m, 2 H), 1.55 (s, 6 H),
1.24-1.10 (m, 1 H), 1.01-0.84 (m, 1 H). 13C NMR (75 MHz, DMSO-d6) δ ppm 169.75,
154.18, 154.00, 141.10, 129.59, 129.39 (q, J = 31.1 Hz), 129.23, 125.16, 124.16 (d, J
= 272.1 Hz), 121.06, 118.62, 117.18 (d, J = 3.6 Hz), 113.50 (q, J = 4.0 Hz), 80.74,
46.01, 43.68, 41.43, 32.04, 31.76, 25.78, 25.37. ESI-MS: m/z 506.2 [M+Na]+. HRMS
(ESI): m/z calcd for C23H26ClF3N3O3 [M+H]+ 484.1609, found 484.1613. LC tR: 2.743
min, purity 99.98%.

4.1.8
1-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)-3-(2-(trifluoromethyl)p
henyl)urea (A8)
o
White solid, yield 61%. mp: 138-140 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
7.96 (d, J = 8.4 Hz, 1 H), 7.65-7.50 (m, 3 H), 7.35 (d, J = 8.9 Hz, 2 H), 7.20-7.11 (m,
1 H), 6.98 (d, J = 7.2 Hz, 1 H), 6.83 (d, J = 8.9 Hz, 2 H), 4.36-4.15 (m, 2 H),
3.70-3.59 (m, 1 H), 3.28-3.17 (m, 1 H), 3.01-2.88 (m, 1 H), 1.89-1.63 (m, 2 H), 1.55
13
(s, 6 H), 1.25-1.11 (m, 1 H), 0.99-0.84 (m, 1 H). C NMR (75 MHz, DMSO-d6) δ
ppm 169.73, 153.99, 137.21, 132.60, 129.24, 125.61 (q, J = 5.1 Hz), 125.14, 124.12,
124.00 (d, J = 272.7 Hz), 122.33, 118.59, 118.23 (q, J = 29.0 Hz), 80.72, 46.08, 43.57,
41.30, 32.11, 31.79, 25.86, 25.37. ESI-MS: m/z 506.2 [M+Na]+. HRMS (ESI): m/z
calcd for C23H26ClF3N3O3 [M+H]+ 484.1609, found 484.1616. LC tR: 2.521 min,
purity 99.85%.
4.1.9
1-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)
piperidin-4-yl)-3-(4-(trifluoromethoxy) phenyl) urea (A9)
o
White solid, yield 60%. mp: 96-98 C. 1H NMR (300 MHz, DMSO-d6) δ ppm 8.43
(s, 1 H), 7.43 (d, J = 9.1 Hz, 2 H), 7.35 (d, J = 9.0 Hz, 2 H), 7.20 (d, J = 8.6 Hz, 2 H),
6.82 (d, J = 9.0 Hz, 2 H), 6.14 (d, J = 7.4 Hz, 1 H), 4.37-4.19 (m, 2 H), 3.70-3.56 (m,
1 H), 3.28-3.15 (m, 1 H), 3.00-2.82 (m, 1 H), 1.87-1.61 (m, 2 H), 1.55 (s, 6 H),
1.20-1.08 (m, 1 H), 0.97-0.82 (m, 1 H). 13C NMR (75 MHz, DMSO-d6) δ ppm 169.89,
154.33, 154.10, 142.14, 139.61, 129.36, 125.26, 121.57, 120.23 (q, J = 255.1 Hz),
118.75, 118.72, 80.84, 46.03, 43.77, 41.48, 32.35, 31.87, 25.98, 25.42. ESI-MS: m/z
522.2 [M+Na]+. HRMS (ESI): m/z calcd for C23H26ClF3N3O4 [M+H]+ 500.1558,
found 500.1557. LC tR: 2.794 min, purity 99.87%.
4.1.10 1-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)-3-(p-tolyl)urea
(A10)
o
White solid, yield 54%. mp: 168-171 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
8.09 (s, 1 H), 7.34 (d, J = 8.4 Hz, 2 H), 7.21 (d, J = 7.8 Hz, 2 H), 6.99 (d, J = 7.8 Hz,

2 H), 6.82 (d, J = 8.5 Hz, 2 H), 6.01 (d, J = 6.9 Hz, 1 H), 4.39-4.15 (m, 2 H),
3.70-3.53 (m, 1 H), 3.28-3.14 (m, 1 H), 2.98-2.82 (m, 1 H), 2.19 (s, 3 H), 1.88-1.62
13
(m, 2 H), 1.55 (s, 6 H), 1.24-1.05 (m, 1 H), 0.97-0.76 (m, 1 H). C NMR (75 MHz,
DMSO-d6) δ ppm 169.69, 154.28, 153.98, 140.24, 129.20, 128.48, 125.11, 120.92,
118.58, 117.50, 80.71, 45.79, 43.63, 41.37, 32.18, 31.94, 25.79, 25.35, 20.20. ESI-MS:
m/z 452.2 [M+Na]+. HRMS (ESI): m/z calcd for C23H29ClN3O3 [M+H]+ 430.1892,
found 430.1893. LC tR: 2.299 min, purity 99.95%.
4.1.11
1-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)-3-(4-methoxyphenyl)ur
ea (A11)
o
White solid, yield 67%. mp: 189-192 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
8.01 (s, 1 H), 7.34 (d, J = 8.8 Hz, 2 H), 7.23 (d, J = 8.8 Hz, 2 H), 6.82 (d, J = 9.5 Hz,
2 H), 6.79 (d, J = 9.4 Hz, 2 H), 5.95 (d, J = 7.3 Hz, 1 H), 4.38-4.17 (m, 2 H), 3.68 (s,
3 H), 3.64-3.53 (m, 1 H), 3.26-3.13 (m, 1 H), 2.98-2.81 (m, 1 H), 1.89-1.62 (m, 2 H),
1.55 (s, 6 H), 1.21-1.08 (m, 1 H), 0.95-0.79 (m, 1 H). 13C NMR (75 MHz, DMSO-d6)
δ ppm 169.68, 154.52, 153.98, 153.88, 133.39, 129.20, 125.11, 119.25, 118.58, 113.80,
80.71, 55.06, 45.81, 43.64, 41.41, 32.30, 32.02, 25.75, 25.45. ESI-MS: m/z 468.2
[M+Na]+. HRMS (ESI): m/z calcd for C23H29ClN3O4 [M+H]+ 446.1841, found
446.1840. LC tR: 1.931 min, purity 99.90%.
4.1.12
ethyl
4-(3-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)ureido)benzoate
(A12)
o
White solid, yield 48%. mp: 127-129 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
8.65 (s, 1 H), 7.81 (d, J = 8.7 Hz, 2 H), 7.46 (d, J = 8.7 Hz, 2 H), 7.34 (d, J = 8.9 Hz,
2 H), 6.82 (d, J = 8.9 Hz, 2 H), 6.25 (d, J = 7.4 Hz, 1 H), 4.39-4.19 (m, 2 H), 4.25 (q,
J = 7.1 Hz, 2 H), 3.74-3.59 (m, 1 H), 3.26-3.14 (m, 1 H), 2.99-2.83 (m, 1 H),
1.88-1.64 (m, 2 H), 1.55 (s, 6 H), 1.29 (t, J = 7.1 Hz, 3 H), 1.23-1.09 (m, 1 H),
13
1.02-0.83 (m, 1 H). C NMR (75 MHz, DMSO-d6) δ ppm 169.72, 165.36, 153.98,

153.82, 144.81, 130.16, 129.21, 125.13, 121.99, 118.59, 116.58, 80.72, 60.02, 45.92,
43.61, 41.37, 32.07, 31.72, 25.82, 25.48, 14.12. ESI-MS: m/z 510.2 [M+Na]+. HRMS
(ESI): m/z calcd for C25H31ClN3O5 [M+H]+ 488.1947, found 488.1953. LC tR: 2.491
min, purity 99.97%.
4.1.13
4-(3-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)ureido)benzoic acid
(A13)
To a solution of A12 (84 mg, 0.158 mmol) in THF (3 mL) and MeOH (2 mL) was
added a solution of KOH (27 mg, 0.474 mmol) in H2O (1 mL). The mixture was
stirred at room temperature overnight and concentrated in vacuum. The residue was
acidified to pH 2-3 by 1 N HCl, and filtered. The collected crude product was washed
with CH2Cl2 and dried to yield compound A13 (40 mg, 55%) as a white solid. mp:
178-180 oC. 1H NMR (300 MHz, DMSO-d6) δ ppm 12.48 (s, 1 H), 8.62 (s, 1 H), 7.79
(d, J = 8.3 Hz, 2 H), 7.44 (d, J = 8.4 Hz, 2 H), 7.35 (d, J = 8.6 Hz, 2 H), 6.82 (d, J =
8.6 Hz, 2 H), 6.25 (d, J = 7.1 Hz, 1 H), 4.40-4.15 (m, 2 H), 3.71-3.57 (m, 1 H),
3.27-3.14 (m, 1 H), 2.96-2.82 (m, 1 H), 1.90-1.62 (m, 2 H), 1.55 (s, 6 H), 1.26-1.08
13
(m, 1 H), 1.02-0.80 (m, 1 H). C NMR (75 MHz, DMSO-d6) δ ppm 169.73, 166.99,
154.01, 153.91, 144.54, 130.40, 129.28, 125.13, 122.86, 118.59, 116.52, 80.73, 45.92,
43.65, 41.35, 32.17, 31.77, 25.92, 25.33. ESI-MS: m/z 458.1 [M-H]-. HRMS (ESI):
m/z calcd for C23H27ClN3O5 [M+H]+ 460.1634, found 460.1638. LC tR: 1.554 min,
purity 99.56%.
4.1.14
1-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)-3-(4-cyanophenyl)urea
(A14)
o
White solid, yield 53%. mp: 183-185 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
8.76 (s, 1 H), 7.64 (d, J = 8.6 Hz, 2 H), 7.51 (d, J = 8.6 Hz, 2 H), 7.34 (d, J = 8.8 Hz,
2 H), 6.82 (d, J = 8.8 Hz, 2 H), 6.33 (d, J = 7.3 Hz, 1 H), 4.40-4.18 (m, 2 H),
3.74-3.59 (m, 1 H), 3.27-3.15 (m, 1 H), 2.98-2.80 (m, 1 H), 1.89-1.62 (m, 2 H), 1.55

13
(s, 6 H), 1.28-1.10 (m, 1 H), 1.04-0.82 (m, 1 H). C NMR (75 MHz, DMSO-d6) δ
ppm 169.71, 153.97, 153.69, 144.67, 133.03, 129.22, 125.12, 119.26, 118.59, 117.34,
102.37, 80.72, 45.94, 43.66, 41.31, 31.97, 31.78, 25.72, 25.33. ESI-MS: m/z 463.2
[M+Na]+. HRMS (ESI): m/z calcd for C23H26ClN4O3 [M+H]+ 441.1688, found
441.1685. LC tR: 2.172 min, purity 99.78%.
4.1.15
1-(1-(2-(4-chlorophenoxy)-2-methylpropanoyl)piperidin-4-yl)-3-(4-nitrophenyl)urea
(A15)
o
White solid, yield 36%. mp: 179-182 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
9.02 (s, 1 H), 8.12 (d, J = 9.0 Hz, 2 H), 7.57 (d, J = 9.0 Hz, 2 H), 7.35 (d, J = 8.7 Hz,
2 H), 6.82 (d, J = 8.7 Hz, 2 H), 6.41 (d, J = 7.2 Hz, 1 H), 4.41-4.17 (m, 2 H),
3.76-3.57 (m, 1 H), 3.28-3.15 (m, 1 H), 3.01-2.82 (m, 1 H), 1.91-1.63 (m, 2 H), 1.55
13
(s, 6 H), 1.28-1.11 (m, 1 H), 1.01-0.84 (m, 1 H). C NMR (75 MHz, DMSO-d6) δ
ppm 169.74, 154.00, 153.55, 146.91, 140.42, 129.28, 125.14, 125.04, 118.59, 116.75,
80.73, 46.08, 43.64, 41.32, 32.01, 31.62, 25.90, 25.32. ESI-MS: m/z 483.2 [M+Na]+.
HRMS (ESI): m/z calcd for C22H26ClN4O5 [M+H]+ 461.1586, found 461.1581. LC tR:
2.371 min, purity 99.82%.
4.1.16
2-(4-chlorophenoxy)-N-(4-((4-fluorobenzyl)oxy)phenyl)-2-methylpropanamide (B1)
A solution of 2-(4-chlorophenoxy)-N-(4-hydroxyphenyl)-2-methylpropanamide (6)
(150
mg,
0.49
mmol),
K2CO3
(136
mg,
0.98
mmol)
and
o
1-(bromomethyl)-4-fluorobenzene (74 µL, 0.59 mmol) in DMF was stirred at 65 C
for 2 h. The reaction mixture was cooled to room temperature and acidified to pH 2-3
by 1 N HCl. The solution was extracted with EtOAc, washed with brine, dried over
Na2SO4 and concentrated. The residue was purified by flash chromatography (petrol
ether/ethyl acetate = 10/1) to give compound B1 (120 mg, 59%) as a white solid. mp:
120-122 oC. 1H NMR (300 MHz, DMSO-d6) δ ppm 9.85 (s, 1 H), 7.54 (d, J = 9.0 Hz,
2 H), 7.48 (dd, J = 8.3, 5.8 Hz, 2 H), 7.34 (d, J = 8.9 Hz, 2 H), 7.20 (dd, J = 8.9, 8.9

Hz, 2 H), 6.95 (d, J = 9.1 Hz, 2 H), 6.94 (d, J = 8.9 Hz, 2 H), 5.05 (s, 2 H), 1.52 (s, 6
H). 13C NMR (75 MHz, DMSO-d6) δ ppm 171.66, 161.62 (d, J = 243.5 Hz), 154.51,
153.60, 133.31 (d, J = 2.9 Hz), 131.73, 129.68 (d, J = 8.3 Hz), 129.03, 126.15, 122.00,
121.38, 115.07 (d, J = 21.4 Hz), 114.64, 80.85, 68.60, 24.68. ESI-MS: m/z 436.2
[M+Na]+. HRMS (ESI): m/z calcd for C23H22ClFNO3 [M+H]+ 414.1267, found
414.1268. LC tR: 1.779 min, purity 99.80%.
4.1.17
N-(4-((4-chlorobenzyl)oxy)phenyl)-2-(4-chlorophenoxy)-2-methylpropanamide (B2)
o
White solid, yield 62%. mp: 120-123 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
9.86 (s, 1 H), 7.54 (d, J = 8.9 Hz, 2 H), 7.49-7.41 (m, 4 H), 7.34 (d, J = 8.8 Hz, 2 H),
13
6.94 (d, J = 9.0 Hz, 2 H), 6.93 (d, J = 8.8 Hz, 2 H), 5.07 (s, 2 H), 1.52 (s, 6 H). C
NMR (75 MHz, DMSO-d6) δ ppm 171.66, 154.41, 153.59, 136.16, 132.23, 131.79,
129.25, 129.03, 128.28, 126.15, 122.00, 121.38, 114.65, 80.85, 68.47, 24.68. ESI-MS:
m/z 452.1 [M+Na]+. HRMS (ESI): m/z calcd for C23H22Cl2NO3 [M+H]+ 430.0971,
found 430.0971. LC tR: 2.215 min, purity 99.48%.
4.1.18
N-(4-((4-bromobenzyl)oxy)phenyl)-2-(4-chlorophenoxy)-2-methylpropanamide (B3)
o
White solid, yield 27%. mp: 141-142 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
9.85 (s, 1 H), 7.58 (d, 8.7 Hz, 2 H), 7.54 (d, 9.7 Hz, 2 H), 7.39 (d, J = 8.2 Hz, 2 H),
13
7.34 (d, J = 8.7 Hz, 2 H), 7.00-6.87 (m, 4 H), 5.06 (s, 2 H), 1.52 (s, 6 H). C NMR
(75 MHz, DMSO-d6) δ ppm 171.65, 154.38, 153.59, 136.59, 131.79, 131.20, 129.55,
129.02, 126.14, 121.99, 121.37, 120.71, 114.65, 80.84, 68.50, 24.68. ESI-MS: m/z
496.1 [M+Na]+. HRMS (ESI): m/z calcd for C23H22BrClNO3 [M+H]+ 474.0466, found
476.0443. LC tR: 2.334 min, purity 100%.
4.1.19
2-(4-chlorophenoxy)-2-methyl-N-(4-((4-(trifluoromethyl)benzyl)oxy)phenyl)propena
mide (B4)
o
White solid, yield 65%. mp: 125-127 C. 1H NMR (300 MHz, DMSO-d6) δ ppm

9.87 (s, 1 H), 7.75 (d, J = 7.9 Hz, 2 H), 7.65 (d, J = 7.8 Hz, 2 H), 7.56 (d, J = 8.7 Hz,
2 H), 7.33 (d, J = 8.7 Hz, 2 H), 6.97 (d, J = 9.0 Hz, 2 H), 6.94 (d, J = 8.9 Hz, 2 H),
13
5.20 (s, 2 H), 1.52 (s, 6 H). C NMR (75 MHz, DMSO-d6) δ ppm 171.73, 154.33,
153.61, 142.04, 131.91, 129.05, 128.21 (q, J = 31.6 Hz), 127.82, 126.17, 125.19 (q, J
= 3.7 Hz), 124.16 (d, J = 272.0 Hz), 122.07, 121.41, 114.68, 80.87, 68.45, 24.70.
ESI-MS: m/z 486.2 [M+Na]+. HRMS (ESI): m/z calcd for C24H22ClF3NO3 [M+H]+
464.1235, found 464.1239. LC tR: 2.415 min, purity 99.50%.
4.1.20
2-(4-chlorophenoxy)-2-methyl-N-(4-((4-methylbenzyl)oxy)phenyl)propenamide (B5)
o
White solid, yield 70%. mp: 100-102 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
9.84 (s, 1 H), 7.53 (d, J = 8.8 Hz, 2 H), 7.34 (d, J = 8.7 Hz, 2 H), 7.31 (d, J = 7.6 Hz,
2 H), 7.18 (d, J = 7.7 Hz, 2 H), 6.98-6.89 (m, 4 H), 5.01 (s, 2 H), 2.30 (s, 3 H), 1.52 (s,
6 H). 13C NMR (75 MHz, DMSO-d6) δ ppm 171.63, 154.65, 153.60, 136.84, 134.05,
131.59, 129.02, 128.81, 127.55, 126.14, 121.98, 121.38, 114.63, 80.85, 69.21, 24.68,
20.63. ESI-MS: m/z 408.2 [M-H]-. HRMS (ESI): m/z calcd for C24H25ClNO3 [M+H]+
410.1517, found 410.1524. LC tR: 2.143 min, purity 99.65%.
4.1.21
2-(4-chlorophenoxy)-N-(4-((4-methoxybenzyl)oxy)phenyl)-2-methylpropanamide
(B6)
o
White solid, yield 76%. mp: 126-129 C. 1H NMR (300 MHz, DMSO-d6) δ ppm
9.84 (s, 1 H), 7.53 (d, J = 8.8 Hz, 2 H), 7.35 (d, J = 8.1 Hz, 2 H), 7.34 (d, J = 8.6 Hz,
13
2 H), 7.00-6.86 (m, 6 H), 4.98 (s, 2 H), 3.75 (s, 3 H), 1.52 (s, 6 H). C NMR (75
MHz, DMSO-d6) δ ppm 171.67, 158.89, 154.69, 153.64, 131.58, 129.31, 129.08,
128.98, 126.13, 121.98, 121.34, 114.65, 113.73, 80.86, 69.08, 55.03, 24.72. ESI-MS:
m/z 424.2 [M-H]-. HRMS (ESI): m/z calcd for C24H25ClNO4 [M+H]+ 426.1467, found
426.1470. LC tR: 1.664 min, purity 99.88%.
4.1.22
2-(4-chlorophenoxy)-2-methyl-N-(4-((4-(trifluoromethoxy)benzyl)oxy)phenyl)propen