Discovery of a low-systemic-exposure DGAT-1 inhibitor with a picolinoylpyrrolidine-2-carboxylic acid moiety

Article abstract of DOI:10.1016/j.bmc.2017.07.007

A series of diacylglycerol O-acyltransferase 1 (DGAT-1) inhibitors with a picolinoylpyrrolidine-2-carboxylic acid moiety were designed and synthesized. Of these compounds, compound 22 exhibited excellent DGAT-1-inhibitory activity (hDGAT-1 enzyme assay, 50% inhibitory concentration [IC₅₀]=3.5±0.9nM) and effectively reduced the intracellular triglyceride contents in 3T3-L1, HepG2 and Caco-2 cells. A preliminary study of the plasma and tissue distributions of compound 22 in mice revealed low plasma exposure and high concentrations in different segments of the intestine and liver, which may facilitate targeting DGAT-1. Furthermore, in an acute lipid challenge test, compound 22 showed a dose-dependent inhibitory effect on high-serum triglycerides in C57/KSJ mice induced by olive oil (1, 3, and 10mg/kg, i.g.).

Full text of DOI:10.1016/j.bmc.2017.07.007

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of a low-systemic-exposure DGAT-1 inhibitor
with a picolinoylpyrrolidine-2-carboxylic acid moiety
Jianwei Yan a,b,c, Gaihong Wang a,c, Xiangyu Dang a, Binbin Guo a, Wuhong Chen a, Ting Wang a,
Limin Zeng a, Heyao Wang a, , Youhong Hu a,
⇑
⇑
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 ZuChongZhi Road, Shanghai 201203, China
b School of Pharmacy, Xinxiang Medical University, 601 Jisui Avenue, Xinxiang, Henan 453003, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of diacylglycerol O-acyltransferase 1 (DGAT-1) inhibitors with a picolinoylpyrrolidine-
Received 20 May 2017
Revised 1 July 2017
Accepted 6 July 2017
Available online xxxx
2-carboxylic acid moiety were designed and synthesized. Of these compounds, compound 22
exhibited excellent DGAT-1-inhibitory activity (hDGAT-1 enzyme assay, 50% inhibitory concentration
[IC50] = 3.5 0.9 nM) and effectively reduced the intracellular triglyceride contents in 3T3-L1, HepG2
and Caco-2 cells. A preliminary study of the plasma and tissue distributions of compound 22 in mice
revealed low plasma exposure and high concentrations in different segments of the intestine and liver,
which may facilitate targeting DGAT-1. Furthermore, in an acute lipid challenge test, compound 22
showed a dose-dependent inhibitory effect on high-serum triglycerides in C57/KSJ mice induced by olive
oil (1, 3, and 10 mg/kg, i.g.).
Keywords:
DGAT-1
Inhibitor
Triglyceride
Hyperlipidemia
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
heteroaryl-linker-acid, which is based on compounds 1 and 2
described in the early patents of Japan Tobacco/Tularik and
Diacylglycerol O-acyltransferase (DGAT) is a key enzyme that
catalyzes the final committed step in triglyceride synthesis and is
highly expressed in the small intestine, adipose tissue, liver and
mammary gland.1–4 Its imbalance could lead to the excessive accu-
mulation of triglycerides, which is frequently associated with
metabolic diseases, such as obesity, insulin resistance and hepatic
steatosis.5,6 Although the DGAT family contains two isozymes
DGAT-1 and DGAT-2, their sequence homology is limited.7 A previ-
Bayer.16,17 Several inhibitors containing the phenylcyclohexy-
lacetic acid functional group from compound 1 have entered into
clinical trials,18–24 and compound 3 (LCQ908) was included in a
phase III study for the treatment of familial chylomicronemia syn-
drome (FCS) and showed high plasma exposure after oral dosing.25
Based on compound 2 with a keto acid moiety that was originally
developed by Abbott, the terminal benzothiazole group was
opened, creating lead compound 8 (A-922500), which showed
highly potent inhibition of DGAT-1 (50% inhibitory concentration
[IC50] = 7 nM) with oral efficacy.26 This compound was extensively
applied in the study of DGAT-1, but it is poorly soluble. To increase
the solubility of this lead compound, researchers attempted to
exchange the keto acid moiety of compound 8 with a simple amino
acid while retaining the functional acid group to reduce its LogP
property and avoid possible isomerization (Fig. 2, 9 and 10).27–31
In our efforts to discover novel potent DGAT-1 inhibitors, we
designed a series of compounds based on compound 8 in which
the phenyl linker was replaced by a six-membered heteroaryl
group with a five-membered amino acid scaffold. Here, we report
the discovery of a new low-systemic-exposure DGAT-1 inhibitor
22, which bears a picolinoylpyrrolidine-2-carboxylic acid moiety
targeting DGAT-1 specifically.32,33 We found that this compound
exhibited dose-dependent efficacy in an oral triglyceride uptake
study in mice.
ous investigation revealed that DGAT-2 knockout (DGAT-2ꢀ/ꢀ
)
mice suffer from lipopenia and die soon after birth.8 However,
DGAT-1ꢀ/ꢀ mice are healthy and significantly resistant to diet-
induced obesity (DIO), hyperlipidemia and hepatic steatosis when
fed a high-fat diet.9–12 Thus, DGAT-1 could be a potential target for
the modulation of triglycerides to treat hyperlipidemia and other
metabolic disorders.
Over the past decade, an increasing number of small-molecule
DGAT-1 inhibitors had been developed with variable structural
types.13–15 Most (Fig. 1) share a privileged structure with the
⇑
Corresponding authors.
E-mail addresses: hywang@simm.ac.cn (H. Wang), yhhu@simm.ac.cn (Y. Hu).
J. Y. and G. W. contributed equally to this work.
c
http://dx.doi.org/10.1016/j.bmc.2017.07.007
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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2
J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
O
COOH
O
H
N
N
OH
NH2
N
S
N
O
F
N
2 (BAY-744113)
1 (T863)
F
OH
COOH
O
NH
2O
F3C
N
4 (PF-04620110)
N
N
N
3 (LCQ908)
N
N
H
O
OH
OH
O
O
NH2
O
N
N
N
H2N
6 (AZD-7687)
5 (ABT-046)
N
N
OH
O
O
NH2
HN
O
OH
O
N
HN
N
F3C
N
O
8 (A-922500)
7 (JTT-553)
Fig. 1. Selected examples of DGAT-1 inhibitors.
H
N
N
O
O
S
H
N
OH
HN
S
OH
O
F
N
HN
N
H
Cl
O
N
O
10
N
9 (P-7435)
O
O
O
O
O
O
HN
O
HN
HN
HN
OH
OH
N
F3C
22
low plasma exposure
8
Fig. 2. The development of a low-systemic-exposure DGAT-1 inhibitor.
arylboronic ester 38. The title product 40 was prepared through
the Suzuki coupling of 12b with 38 and the subsequent hydrolysis
of carboxylic ester 39 (Scheme 3).
2. Results and discussion
2.1. Chemistry
The condensation of methyl L/D-prolinate and halogenated het-
2.2. SAR study and lead generation
eroaromatic acids (11a–d) afforded halogenated heteroaromatic
amides (12a–e), following cross-coupling with 4-nitrophenylboro-
nic acid pinacol ester and the reduction of nitro compounds (13a–e)
to generate the key intermediate biphenyl amines (14a–e). Reac-
tion with isocyanate generated urea compounds (15a–q), which
were hydrolyzed under basic conditions to give compounds
16–32 (Scheme 1). The addition reaction of 14b with phenyl
isothiocyanate and the subsequent hydrolysis of 33 afforded com-
pound 34. Compound 36 was obtained through the condensation
of 14b with m-(trifluoromethyl) phenylacetic acid and the hydroly-
sis of 35 (Scheme 2).
Initially, L-pyrrolidine-2-carboxylic acid was selected as the ter-
minal moiety. Several different aromatic acyl linker compounds
were synthesized and analyzed for their effects on DGAT-1 activity,
and the results are listed in Table 1. Compound 16, which has a
benzoyl linker, caused a certain degree of hDGAT-1 inhibition. This
result indicates that the keto acid moiety of compound 8 could be
replaced with an N-acylpyrrolidine, which represents a rational
optimization strategy for maintaining the functional acid group.
When the benzoyl linker was replaced by 2-acyl pyridine, com-
pound 17 displayed strong DGAT-1 inhibition activity, with an
IC50 of 23.4 3.4 nM for the human DGAT-1 enzyme. Compounds
bearing 3-acyl pyridine (18) and 2-acyl pyrimidine (19) linkers
exhibited significantly decreased hDGAT-1 inhibition. Changing
Amide intermediate 37 was synthesized via the condensation of
4-bromophenylacetic acid and 3-(trifluoromethyl) aniline, and
then, cross-coupling with bis(pinacolato)diboron afforded the
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J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
O
CO2Me
O
CO2Me
c
O
CO2Me
Z
Z
Z
CO2H
H
N
Y
N
Y
N
O
a
Y
N
Z
b
CO2Me
+
Y
N
W
W
W
X
R
W
X
11
13a~e
14a~e
12a~e
H2N
O2N
O
CO2Me
CO2H
∗
Z
Y
C
d
Z
N
O
R
Y
N
e
W
W
O
O
R
N
H
N
N
H
N
H
15a~q
H
16
R=Ph, Y,Z,W=CH; *:S
25
R=3-MeOC6H4, Y,Z=CH, W=N; *:S
R=4-MeOC6H4, Y,Z=CH, W=N; *:S
17 R=Ph, Y,Z=CH, W=N; *:S
26
18
19
R=Ph, Z,W=CH, Y=N; *:S
R=Ph, Y=CH, Z,W=N; *:S
27 R=2-ClC6H4, Y,Z=CH, W=N; *:S
28
29
R=3-ClC6H4, Y,Z=CH, W=N; *:S
R=4-ClC6H4, Y,Z=CH, W=N; *:S
20 R=Ph, Y,Z=CH, W=N; *:R
21
22
R=2-CF3C6H4, Y,Z=CH, W=N; *:S
R=3-CF3C6H4, Y,Z=CH, W=N; *:S
30 R=3-FC6H4, Y,Z=CH, W=N; *:S
31
32
R=n-Bu, Y,Z=CH, W=N; *:S
R=cyclohexyl, Y,Z=CH, W=N; *:S
23 R=4-CF3C6H4, Y,Z=CH, W=N; *:S
24
R=2-MeOC6H4, Y,Z=CH, W=N; *:S
Scheme 1. Synthesis of 16–32. Reagents and conditions: (a) for 12a: DIC, HOBT, DIPEA, DCM, room temperature (rt), 95%; for 12b–12e: TBTU, DIPEA, DMF, rt, 88–96%; (b) 4-
nitrophenylboronic acid pinacol ester, KF, Pd(PPh3)4, PhMe/EtOH/H2O, N2, 80 °C, 71–87%; (c) for 13a: Fe, NH4Cl, EtOH/H2O, 90 °C, 70%; for 13b–13e: Pd/C, NaBH4, EtOH, rt, 75–
82%; (d) TEA, THF, 40 °C, 88–98%; and (e) LiOH, CH3CN/H2O, rt, 88–98%.
O
CO2Me
O
CO2Me
O
CO2H
N
N
N
N
N
a
c
N
S
S
14b
H2N
N
H
N
H
33
N
H
N
H
34
b
O
O
O
N
O
N
N
c
CO2H
HN
CO2Me
HN
N
F3C
F3C
36
35
Scheme 2. Synthesis of 34 and 36. Reagents and conditions: (a) phenyl isothiocyanate, TEA, THF, 40 °C, 90%; (b) m-(trifluoromethyl) phenylacetic acid, TBTU, DIPEA, DMF, rt,
88%; and (c) LiOH, CH3CN/H2O, rt, 89–93%.
O
CO2Me
N
O
B
N
NH2
F3C
Br
Br
O
O
12b
O
O
b
Br
a
c
N
H
F3C
N
H
F3C
OH
38
37
O
CO2H
O
CO2Me
N
N
N
N
d
O
O
N
H
F3C
39
N
H
F3C
40
Scheme 3. Synthesis of 40. Reagents and conditions: (a) TBTU, DIPEA, DMF, rt, 97%; (b) bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, DMF, 80 °C, 71%; (c) KF, Pd(PPh3)4, PhMe/
EtOH/H2O, 62%; and (d) LiOH, CH3CN/H2O, rt, 93%.
L
-proline into R-proline on the acid terminal resulted in a slight
Interestingly, the meta-CF3 substitution significantly improved
the compound’s potency, resulting in an IC50 value of 3.5 nM
(22). The incorporation of the methoxy group at different positions
on the benzene ring resulted in the retention of DGAT-1 inhibitory
activity (compounds 24–26). The introduction of Cl at different
positions on the benzene ring of urea significantly enhanced
hDGAT-1 inhibition, resulting in one-digit IC50 values in the
nanomolar range (27–29). The 3-fluoro-substituted compound
(30) exhibited decreased inhibitory activity compared with the
decline in the hDGAT-1 inhibition observed (20). Based on these
results, the picolinoylpyrrolidine-2-carboxylic acid moiety was
used as the end structure.
The substituent effect of the phenyl ring of the left urea termi-
nal was subsequently investigated (Table 2). Introducing CF3 at the
ortho-, meta- and para-positions resulted in mixed results. The
ortho-CF3 substitution slightly decreased the activity (21), whereas
for the para-CF3 substitution, the activity was maintained (23).
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J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
Structure-activity relationships of the acyl pyrrolidine-2-carboxylic acid moiety.
O
OH
O
Z
Y
N
W
O
N
H
N
H
Compd
Structure
ahDGAT-1 (% inhibition at 1
79.6 4.4
lM)
bhDGAT-1 (% inhibition at 0.1
39.8 2.1
lM)
chDGAT-1 IC50 (nM)
NT
16
O
OH
OH
OH
OH
O
O
O
O
N
17
18
19
20
8
72.2 7.8
48.9 0.8
51.7 3.0
88.1 1.2
86.4 3.2
61.8 2.6
36.7 2.4
42.0 2.9
59.0 7.0
85.7 0.5
23.4 3.4
O
N
N
NT
O
N
N
NT
O
N
N
N
39.9 0.3
O
OH
O
N
N
7.8 1.3
Mb. The compounds with inhibitory activities >50% at 0.1
lM were further
The compounds were primarily tested for their DGAT-1 inhibitory activities at 1
l
Ma and 0.1
l
investigated to determine their respective IC50 (half of the maximal inhibitory concentration) valuesc. All data are shown as the mean standard deviation (SD) from three
independent experiments performed in duplicate. NT indicates not tested.
3-CF3- (22) and 3-Cl-substituted ones (28). The fatty chain (31) and
saturated carbon ring substitutions (32) caused a loss of the inhibi-
tory activity. Changing the urea group to a thiocarbamide (34) and
an amide (36, 40) led to an obvious decline in or loss of the DGAT-1
inhibitory activity.
Compounds 22, 27 and 28 showed the good solubility in the dif-
ferent Buffers (Table 3). Permeability tests of compounds 22, 27
and 28 were performed using the bidirectional Caco-2 monolayer
assay model (Table 3).34 The data revealed that compound 22
exhibited a lower efflux ratio than compounds 27 and 28.
After differentiating for 4 days, the 3T3-L1 cells were main-
tained in the presence of the DGAT-1 inhibitors (compound 8,
The effects of compound 22 on the triglyceride contents in dif-
ferentiated 3T3-L1 adipocytes, enterocyte-like Caco-2 cells and
hepatoma-derived HepG2 cells, in which DGAT-1 is highly
expressed, were investigated. Lipid accumulation was examined
by Oil Red O staining and quantitative analysis of the cellular
triglyceride content (Fig. 3 A and B). The treatment of differenti-
ated 3T3-L1 adipocytes with compound 22 for 4 days significantly
decreased the triglyceride contents. Additionally, after incubation
with exogenous OA, Caco-2 cells and HepG2 cells exhibited signif-
icantly increased triglyceride generation, which was abolished by
compound 8, LCQ908 and compound 22 in a dose-dependent man-
ner (Fig. 3 C–G).
LCQ908 or compound 22 at 20
in each group were subjected to Oil Red O staining (A) and cel-
lular triglyceride content determination (B). Scale bar, 200 m.
l
M) for 4 days; then, the cells
The pharmacokinetic profile of compound 22 was evaluated in
Sprague-Dawley rats (Table 4). The results showed that compound
22 exhibited lower plasma clearance (CL: 0.33 L/h/kg) and lower
tissue distribution (Vss: 0.18 L/kg) after intravenous administra-
tion. After intragastric administration, the plasma peak time was
1.8 h, with low plasma exposure and only 0.28% of the absolute
bioavailability. Based on these results, compound 22 might
distribute specifically to some tissues. To validate this hypothesis,
the tissue distribution of compound 22 was also determined
(Table 5). After a 10-mg/kg oral dose of compound 22 in C57 mice,
the drug levels in the plasma, liver and different segments of
the intestine (duodenum, jejunum, and ileum) were determined
at three time points (1, 2, and 5 h) and are provided in Table 5.
As expected, compound 22 showed higher concentrations
l
Asterisks indicate significant differences relative to the control
group (⁄⁄⁄p < 0.001). Following treatment with the test com-
pounds for 2 h, Caco-2 and HepG2 cells were incubated in the
absence or presence of oleic acid (OA) for 48 h. Then, the cellular
triglyceride contents of each group were determined (C-G).
Asterisks indicate significant differences relative to the group
treated with OA only (⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001). The fig-
ures shown are representative of at least two independent exper-
iments, each of which was performed in triplicate. A one-way
analysis of variance (ANOVA) was performed to determine the
p values.
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J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Table 2
Structure-activity relationships of the urea groups.
O
OH
O
N
N
lin
k
e
r
R
Compd
Structure
ahDGAT-1 (% inhibition 1
65.3 1.6
lM)
bhDGAT-1 (% inhibition 0.1
59.9 1.6
l
M)
chDGAT-1 IC50 (nM)
59.2 6.8
21
CF3
H
H
N
N
O
22
23
90.5 2.6
68.3 3.9
80.5 1.3
55.8 1.1
3.5 0.9
H
N
H
N
F3C
O
O
30.3 8.6
H
N
H
N
F3C
O
24
84.2 1.7
73.2 4.3
15.9 3.2
H
N
H
N
O
25
26
74.8 6.5
78.0 0.2
62.4 2.0
68.3 0.6
32.1 1.8
18.4 3.0
H
N
H
O
N
O
H
N
H
N
O
O
27
98.4 1.2
70.7 0.7
8.5 0.6
Cl
H
N
H
N
O
28
29
84.0 1.6
78.9 8.5
73.6 1.0
56.9 5.8
3.7 0.6
8.8 1.5
H
H
Cl
N
N
O
H
H
N
N
O
Cl
F
30
31
76.0 1.9
29.0 5.0
68.0 4.0
13.4 2.6
37.7 6.9
NT
H
N
H
N
O
H
N
H
N
O
32
34
59.4 4.8
70.3 2.3
40.5 1.7
40.8 1.3
NT
NT
H
N
H
N
O
S
N
H
N
H
36
68.6 1.3
36.7 0.5
NT
O
O
N
H
F3C
F3C
40
8
23.1 1.7
86.4 3.2
7.9 4.0
NT
N
H
85.7 0.5
7.8 1.3
The compounds were primarily tested for their DGAT-1 enzyme inhibitory activities at 1 l l lM were
Ma and 0.1 Mb. The compounds with inhibitory activities >50% at 0.1
further investigated to determine their IC50 (half of the maximal inhibitory concentration) valuesc. All data are shown as the mean SD from three independent experiments
performed in duplicate. NT indicates not tested.
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6
J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 3
Permeability profiles of compounds 22, 27 and 28.
Assay
Parameters
22
27
28
Solubility (
l
M)a
Buffer A
50
50
50
Buffer B
50
50
50
b
b
Caco-2 Permeability
A ? B Papp (10ꢀ6 cmꢂsꢀ1
B ? A Papp (10ꢀ6 cmꢂsꢀ1
Efflux ratio
)
)
0.23
3.82
16.4
0.07
1.75
26.9
0.04
2.01
46.1
a
Determined using nephelometry. Buffer A: Hank’s balanced salt solution (HBSS)/10-mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/0.1% bovine serum
albumin (BSA), pH 6.8; Buffer B: HBSS/10-mM HEPES/0.1% BSA, pH 7.4; Buffer.
b
Papp: apparent permeability rate coefficient.
(45- to 1380-fold) in the liver and different segments of the intes-
tine than in the plasma at all time points. The preferential distribu-
tion in the liver and intestine over the plasma indicated that
compound 22 is a novel, low-systemic-exposure DGAT-1 inhibitor
that could act in certain locations to target DGAT-1.
4. Experimental section
4.1. General information
1H NMR and 13C NMR spectral data were recorded using CDCl3,
DMSO-d6 or MeOH-d4 solutions with a Bruker Avance III 600, 500,
400 or 300 NMR spectrometer. Chemical shifts (d) are reported in
parts per million (ppm), and the signals are described as brs (broad
singlet), s (singlet), d (doublet), dd (doublet of doublets), t (triplet),
q (quartet), and m (multiplet). Coupling constants (J values) are
given in Hz. The mass spectra were obtained using liquid chro-
matography mass spectrometry (LC-MS) on an Agilent 6120 instru-
ment using electrospray ionization (ESI). Column chromatography
was conducted using silica gel (200–300 mesh). All reactions were
monitored using thin-layer chromatography (TLC) on silica gel
plates. The purity (>95%) of final products prepared in this study
was determined using chromatographic analysis with an Agilent
1200 series LC system (Agilent ChemStation Rev. B.03.01); column,
The in vivo lipid-lowering effects of compound 22 were also
evaluated using an acute lipid challenge test (Fig. 4). First, a model
of an acute lipid challenge test was established (Fig. 4A). Male C57/
KSJ mice were dosed with compound 8 or vehicle an hour prior to
the administration of the olive oil bolus. After 2 h, the serum
triglyceride levels of the vehicle-treated animals were maximized,
and the increase in the serum triglyceride level was significantly
suppressed by the administration of compound 8. Thus, this model
was appropriate for the evaluation of the in vivo triglyceride-low-
ering effects of these compounds. Second, the optimal time point
for the administration of compound 22 was investigated (Fig. 4B).
The data showed that the lipid-lowering effects of compound 22
were maximized when the compound and olive oil bolus were
administered simultaneously (0 h). Finally, the dose-dependent
activities of compound 22 were studied and compared with those
of compounds 8 and LCQ908 (1, 3, and 10 mg/kg). Compound 22
was shown to significantly inhibit the acute high-serum triglyc-
eride level induced by olive oil in a dose-dependent manner, and
its effects were less potent than those of 8 but slightly more potent
than those of LCQ908.
Six-week-old male mice (n = 6) were fasted for 16 h and orally
dosed with either vehicle (0.1% w/v Tween 80) or the tested com-
pound (10 mg/kg in A and B; 1, 3, and 10 mg/kg in C and D). One
hour (A); 0, 1, and 3 h (B); or 0 h (C and D) after dosing, an olive
oil bolus (15 mL/kg) was administered, and the mice in the control
group were given an equal volume of water. Then, 1, 2, 3, and 4 h
(A); or 2 h (B–D) later, blood samples were collected, and the
serum triglyceride levels were measured. Asterisks indicate signif-
icant differences relative to the vehicle group (⁄p < 0.05; ⁄⁄p < 0.01;
⁄⁄⁄p < 0.001). A one-way ANOVA was performed to determine the p
values.
ZORBAX Eclipse XDB-C18, 4.6 mm * 50 mm, 5
lm, or Nova Pak C18
3.9 mm * 150 mm, 4 m; mobile phase, MeCN/H2O (0.2% triethy-
l
lamine); flow rate, 1.0 mL/min; UV wavelength, maximal absor-
bance at 254 nm; temperature, ambient; and injection volume,
5 lL.
4.2. Procedure for the preparation of (S)-1-(40-(3-phenylureido)-[1,10-
biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid (16)
A mixture of 4-iodobenzoic acid (11a, 1.0 g, 4.0 mmol), (S)-
methyl pyrrolidine-2-carboxylate (0.52 g, 4.0 mmol), DIC (0.56 g,
4.4 mmol), HOBt (0.60 g, 4.4 mmol) and DIPEA (1.32 mL, 8.0 mmol)
in 30 mL of DCM was stirred at room temperature for 8 h, then
40 mL of water was added to reaction solution. The mixture was
extracted with DCM (3 ꢁ 30 mL). The extracts were dried over Na2-
SO4 and concentrated in vacuo to give a residue that was subjected
to silica gel chromatography to afford 12a as a white powder
(1.19 g, 95% yield). 1H NMR (300 MHz, CDCl3) d 7.76 (d, J = 8.2 Hz,
2H), 7.32 (d, J = 8.2 Hz, 2H), 4.66 (brs, 1H), 3.78 (s, 3H), 3.68–3.46
(m, 2H), 2.40–2.25 (m, 1H), 2.13–1.84 (m, 3H).
3. Conclusions
A solution of 12a (1.2 g, 3.3 mmol), 4-nitrophenylboronic acid
pinacol ester (1.0 g, 4.0 mmol), KF (0.58 g, 9.9 mmol), and tetrakis
(triphenylphosphine)palladium (92 mg, 0.08 mmol) in toluene/
ethanol/H2O (4/2/1 ratio, 20 mL) was heated to 80 °C for 8 h, cooled
to room temperature, filtered through Celite, washed with ethyl
acetate (3 ꢁ 20 mL), concentrated and purified on a silica gel chro-
matography to afford 13a (0.99 g, 85% yield). 1H NMR (300 MHz,
CDCl3) d 8.31 (d, J = 8.6 Hz, 2H), 7.79–7.60 (m, 6H), 4.70 (dd,
J = 8.3, 4.9 Hz, 1H), 3.79 (s, 3H), 3.76–3.65 (m, 1H), 3.60 (s, 1H),
2.40–2.25 (m, 1H), 2.11–1.90 (m, 3H).
A mixture of 13a (0.90 g, 2.54 mmol), iron powder (0.43 g,
7.6 mmol), and NH4Cl (136 mg, 2.54 mmol) in a mixture of sol-
vents (75 mL ethanol and 25 mL water) was heated to 90 °C under
N2 for 8 h. The reaction mixture was filtered through Celite, treated
with aqueous saturated sodium bicarbonate (30 mL) and extracted
Herein, we described the design and synthesis of a series of
DGAT-1 inhibitors with a picolinoylpyrrolidine-2-carboxylic acid
moiety based on compound 8 in which the phenyl linker was
replaced by a six-membered heteroaryl group with a five-mem-
bered amino acid scaffold. Among them, compound 22 exhibited
good inhibitory activity for hDGAT-1 and effectively reduced the
intracellular triglyceride contents in the 3T3-L1, Caco-2 and HepG2
cell lines. Furthermore, an acute lipid challenge test of compound
22 revealed that it exerts dose-dependent lipid-lowering effects
in mice. DGAT-1 inhibitor 22, as an attractive lead candidate, war-
rants further investigation for the treatment of hyperlipidemia and
other metabolic disorders because of its preferential distributions
in the intestine and liver, which correlates perfectly with the loca-
tion of DGAT-1.
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J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Fig. 3. Effects of the tested compounds on the intracellular triglyceride contents of 3T3-L1 cells, Caco-2 cells and HepG2 cells.
Table 4
Pharmacokinetic properties of compound 22.a
AUC0?1 (ngꢂh/mL)
MRT (h)
t1/2 (h)
Tmax (h)
Cmax (ng/mL)
CL (L/h/kg)
Vss (L/kg)
F (%)
i.g.
i.v.
215
30075
3.4
0.53
2.0
0.91
1.8
/
51.6
/
/
/
0.28
/
0.33
0.18
a
Determined using LC/MS/MS. AUC0-1: the area under the concentration time curve; MRT: mean residence time; t1/2: elimination half-life; Tmax: the peak time; Cmax: the
peak concentration; CL: clearance; Vss: steady-state distribution volume; F: absolute bioavailability.
with ethyl acetate. The organic layer was washed with brine, dried
over Na2SO4, filtered, concentrated and purified on a silica gel chro-
matography to afford 14a (0.58 g, 70% yield). To a solution of 14a
(250 mg, 0.77 mmol) and phenyl isocyanate (110 mg, 0.85 mmol)
in dry THF (20 mL), triethylamine (0.28 mL, 1.54 mmol) was added.
The mixture was stirred at 40 °C for 2 h, concentrated and purified
by a flash column (20–50% EtOAc in petroleum ether) to provide
15a (334 mg, 98% yield). Into a 100 mL round bottom flask, 15a
Please cite this article in press as: Yan J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.07.007

8
J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 5
The mice plasma and tissue concentrations of compound 22.a
Time (h)
Plasma (ng/ml)
Liver (ng/g)
Duodenum (ng/g)
Jejunum (ng/g)
36425 5959
Ileum (ng/g)
1
2
5
26.3 5.8
26.6 9.3
17.6 15.0
7752 3373
4729 1812
3290 1546
8043 2052
16535 15208
2623 2800
1245 738
1262 403
5516 2681
9169 1733
5480 3082
a
Determined using LC/MS/MS. All data are shown as the mean SD from three independent experiment.
Fig. 4. The in vivo effects of compounds 22 in the acute lipid challenge test.
(195 mg, 0.44 mmol), LiOH (53 mg, 2.2 mmol), and 20 mL of 1:1
acetonitrile/water were added. The reaction mixtures were stirred
at room temperature overnight. The pH-value of the solution was
adjusted to between 4 and 7 by adding 1 M HCl. The precipitated
solid was filtered and washed with a small amount of ethyl acetate
to provide the title product 16 (334 mg, 96% yield). As a white
solid: m.p. 173–174 °C. 1H NMR (400 MHz, DMSO-D6) d 12.61 (s,
1H), 9.02 (s, 1H), 8.91 (s, 1H), 7.75–7.45 (m, 10H), 7.28 (t,
J = 7.7 Hz, 2H), 6.97 (t, J = 7.2 Hz, 1H), 4.45–4.34 (m, 1H), 3.63–
3.51 (m, 2H), 2.32–2.20 (m, 1H), 2.02–1.78 (m, 3H); 13C NMR
(101 MHz, DMSO-D6) d 173.5, 167.9, 152.5, 141.4, 140.7, 139.9,
139.7, 134.5, 132.4, 128.8, 127.9, 127.4, 127.2, 127.1, 125.8,
125.6, 121.9, 118.5, 118.2, 59.1, 49.6, 31.1, 29.0, 25.1, 22.3; LC-
MS calcd. for C25H24N3O+4 (m/e), 430.18, obsd. 430.2 (M+H). The
purity of the compound 16 was 99.0% by High Performance Liquid
Chromatography (HPLC).
(d, J = 2.3 Hz, 0.45H), 8.52 (d, J = 2.3 Hz, 0.55H), 7.99 (d, J = 8.4 Hz,
0.55H), 7.94–7.90 (m, 1H), 7.85 (d, J = 8.4 Hz, 0.45H), 5.11 (dd,
J = 8.6, 3.1 Hz, 0.55H), 4.67 (dd, J = 8.5, 4.1 Hz, 0.45H), 4.07–3.94
(m, 1H), 3.93–3.73 (m, 2.35H), 3.66 (s, 1.65H), 2.35–2.22 (m, 1H),
2.21–1.90 (m, 3H).
A solution of 12b (3.2 g, 10.2 mmol), 4-nitrophenylboronic acid
pinacol ester (2.67 g, 10.7 mmol), KF (1.18 g, 20.4 mmol), and tet-
rakis(triphenylphosphine) palladium (230 mg, 0.2 mmol) in
toluene/ethanol/H2O (4/2/1 ratio, 70 mL) was heated to 80 °C for
12 h, cooled to room temperature, filtered through Celite, washed
with ethyl acetate (3 ꢁ 50 mL), concentrated and purified on a sil-
ica gel chromatography to afford 13b (3.15 g, 87% yield). 1H NMR
(400 MHz, CDCl3) d 8.86 (d, J = 2.2 Hz, 0.44H), 8.72 (d, J = 2.3 Hz,
0.56H), 8.36 (d, J = 8.6, 0.88H), 8.35 (d, J = 8.6, 1.12H), 8.22 (d,
J = 8.2 Hz, 0.56H), 8.07 (s, 0.44H), 8.03 (dd, J = 8.3, 2.3 Hz, 1H),
7.78 (d, J = 8.5, 0.88H), 7.76 (d, J = 8.5, 1.12H), 5.19 (dd, J = 8.7,
3.0 Hz, 0.56H), 4.71 (dd, J = 8.5, 4.1 Hz, 0.44H), 4.13–4.00 (m, 1H),
3.98–3.81 (m, 1H), 3.79 (s, 1.32H), 3.68 (s, 1.68H), 2.38–2.24 (m,
1H), 2.24–1.93 (m, 3H).
4.3. Procedure for Preparation of (S)-1-(5-(4-(3-phenylureido)phenyl)
picolinoyl)pyrrolidine-2-carboxylic acid (17)
To a solution of 13b (3.05 g, 8.6 mmol) in 50 mL ethanol at 0 °C
under N2, 10% Pd/C (0.30 g) and NaBH4 (1.30 g, 34.4 mmol, portion-
wise) were added. Then it was warmed to room temperature and
stirred for another 6 h. To the reaction mixture, 1 M HCl was added
until no bubbles come out. Ethanol was removed through rotary
evaporation, 50 mL water and 50 mL ethyl acetate were added to
residue, extracted with ethyl acetate (50 mL ꢁ 3). The extracts
were dried over Na2SO4 and concentrated in vacuo to give a residue
that was subjected to silica gel chromatography to afford 14b
(2.10 g, 75% yield). 1H NMR (300 MHz, CDCl3) d 8.77 (s, 0.45H),
8.63 (s, 0.55H), 8.09 (d, J = 8.2 Hz, 0.55H), 7.96 (d, J = 8.1 Hz,
A mixture of 5-bromopyridine-2-carboxylic acid (11b, 2.02 g,
10.0 mmol),
(S)-methyl
pyrrolidine-2-carboxylate
(1.42 g,
11.0 mmol), TBTU (3.53 g, 11.0 mmol) and DIPEA (3.31 mL,
20.0 mmol) in 20 mL of DMF was stirred at room temperature for
12 h, then DMF was removed by rotary evaporation. To the residue,
50 mL of water and 50 mL of ethyl acetate was added, extracted
with ethyl acetate (50 mL ꢁ 3). The extracts were dried over
Na2SO4 and concentrated in vacuo to give a residue that was
subjected to silica gel chromatography to afford 12b as a white
powder (3.31 g, 96% yield). 1H NMR (400 MHz, CDCl3) d 8.65
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J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
0.45H), 7.89 (d, J = 8.5 Hz, 1H), 7.46–7.37 (m, 2H), 6.83–6.72 (m,
2H), 5.24–5.16 (m, 1H), 4.70 (dd, J = 7.9, 3.2 Hz, 1H), 4.16–4.00
(m, 1H), 3.98–3.80 (m, 2H), 3.78 (s, 1.35H), 3.67 (s, 1.65H), 2.36–
2.24 (m, 1H), 2.20–1.93 (m, 3H).
0.34 mmol) in dry THF (10 mL), triethylamine (0.12 mL,
0.68 mmol) was added. The mixture was stirred at 40 °C for 2 h,
concentrated and purified by a flash column (20–50% EtOAc in pet-
roleum ether) to provide 15c (131 mg, 95% yield). Into a 100 mL
round bottom flask, 15c (100 mg, 0.22 mmol), LiOH (27 mg,
1.1 mmol), and 30 mL of 1:1 acetonitrile/water were added. It
was stirred at room temperature overnight. The pH-value of the
solution was adjusted to between 4 and 7 by adding 1 M HCl.
The precipitated solid was filtered and washed with a small
amount of ethyl acetate to provide the title product 18 (90 mg,
93% yield). A light yellow solid as a 1:3 mixture of rotamers: m.
p. 212–213 °C. 1H NMR (400 MHz, DMSO-D6) d 9.54 (s, 1H), 9.29
(s, 1H), 8.80 (s, 0.72H), 8.68 (s, 0.26H), 8.19–7.97 (m, 4H), 7.65
(d, J = 8.2 Hz, 2H), 7.49 (d, J = 7.8 Hz, 2H), 7.29 (t, J = 7.6 Hz, 2H),
6.98 (t, J = 7.2 Hz, 1H), 4.55–4.51 (m, 0.26H), 4.48–4.41 (m,
0.72H), 3.62 (s, 2H), 2.36–2.24 (m, 1H), 2.06–1.79 (m, 3H); 13C
NMR (101 MHz, DMSO-D6) d 173.6, 173.1, 166.6, 165.4, 156.0,
155.5, 152.5, 146.5, 142.2, 139.7, 137.9, 137.2, 130.1, 129.2,
129.1, 128.9, 128.0, 127.9, 122.0, 120.0, 118.2, 117.9, 60.7, 59.3,
49.5, 46.8, 31.2, 29.0, 25.1, 22.4; LC-MS calcd. for C24H23N4O4+
(m/e), 431.17, obsd. 431.2 (M+H). The purity of the compound 18
was 99.6% by HPLC.
To a solution of 14b (100 mg, 0.31 mmol) and phenyl isocyanate
(40 mg, 0.34 mmol) in dry THF (10 mL), triethylamine (0.12 mL,
0.68 mmol) was added. The mixture was stirred at 40 °C for 2 h,
concentrated and purified by a flash column (20–50% EtOAc in pet-
roleum ether) to provide 15b (131 mg, 95% yield). A 1:1 mixture of
rotamers, 1H NMR (300 MHz, CDCl3) d 8.67 (d, J = 1.7 Hz, 0.5H),
8.59 (d, J = 1.9 Hz, 0.50H), 8.01 (d, J = 8.2 Hz, 0.50H), 7.93 (d,
J = 4.2 Hz, 1H), 7.88 (d, J = 5.3 Hz, 1H), 7.82 (d, J = 8.2 Hz, 0.50H),
7.78–7.70 (m, 1H), 7.41 (t, J = 8.3 Hz, 2H), 7.37–7.23 (m, 6H), 7.03
(t, J = 7.3 Hz, 1H), 5.18 (dd, J = 8.6, 3.1 Hz, 0.50H), 4.75 (dd, J = 8.3,
4.3 Hz, 0.50H), 4.04–3.83 (m, 2H), 3.76 (s, 1.50H), 3.69 (s, 1.50H),
2.42–2.26 (m, 1H), 2.25–1.94 (m, 3H).
Into a 100 mL round bottom flask, 15b (100 mg, 0.22 mmol),
LiOH (27 mg, 1.1 mmol), and 30 mL of 1:1 acetonitrile/water were
added. It was stirred at room temperature overnight. The pH-value
of the solution was adjusted to between 4 and 7 by adding 1 M HCl.
The precipitated solid was filtered and washed with a small
amount of ethyl acetate to provide the title product 17 (95 mg,
98% yield). An orange solid as a 4:6 mixture of rotamers: m.p.
168–169 °C. 1H NMR (600 MHz, CD3OD) d 9.04 (d, J = 1.5 Hz,
0.60H), 8.89 (d, J = 1.6 Hz, 0.40H), 8.62 (dd, J = 8.3, 2.0 Hz, 1H),
8.33 (dd, J = 8.3, 2.1 Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H), 8.07
(d, J = 8.2 Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.71–7.60 (m,, 3H), 7.46
(d, J = 8.2 Hz, 2H), 7.33–7.28(m, 2H), 7.05 (t, J = 7.3 Hz, 1H), 5.14
(dd, J = 8.5, 3.3 Hz, 0.4H), 4.68 (dd, J = 8.4, 4.9 Hz, 0.6H), 3.92
(t, J = 6.4 Hz, 1H), 3.88–3.72 (m, 1H), 2.47–2.37 (m, 1H),
2.26–2.00 (m, 3H); 13C NMR (151 MHz, CD3OD) d 175.8, 174.9,
166.2, 163.8, 155.0, 154.9, 149.8, 146.5, 144.9, 143.6, 142.9,
142.1, 141.3, 140.9, 140.3, 140.3, 139.9, 138.1, 130.6, 129.9,
129.2, 129.1, 128.8, 126.6, 125.7, 124.1, 124.0, 120.6, 120.5,
120.4, 62.9, 61.7, 51.1, 32.7, 30.2, 26.5, 23.2; LC-MS calcd. for
4.5. Procedure for the preparation of (S)-1-(5-(4-(3-phenylureido)
phenyl)pyrimidine-2-carbonyl)pyrrolidine-2-carboxylic acid (19)
A
mixture
of
5-bromopyrimidine-2-carboxylic
acid
(11d, 0.51 g, 2.5 mmol), (S)-methyl pyrrolidine-2-carboxylate
(0.36 g, 2.75 mmol), TBTU (0.89 g, 2.75 mmol) and DIPEA
(0.83 mL, 5.0 mmol) in 10mL of DMF was stirred at room temper-
ature for 12 h, then DMF was removed by rotary evaporation. To
the residue, 40 mL of water and 40 mL of ethyl acetate were added,
extracted with ethyl acetate (40 mL ꢁ 3). The extracts were dried
over Na2SO4 and concentrated in vacuo to give a residue that was
subjected to silica gel chromatography to afford 12d (691 mg,
88% yield). 1H NMR (400 MHz, CDCl3) d 8.92 (s, 1H), 8.87 (s, 1H),
4.87 (dd, J = 8.5, 2.8 Hz, 0.5H), 4.75 (dd, J = 8.5, 3.8 Hz, 0.5H),
3.98–3.72 (m, 3.5H), 3.68 (s, 1.5H), 3.36–2.26 (m, 1H), 2.24–1.95
(m, 3H).
C
24H23N4O+4 (m/e), 431.17, obsd. 431.2 (M+H). The purity of the
compound 17 was 99.6% by HPLC.
4.4. Procedure for the preparation of (S)-1-(6-(4-(3-phenylureido)
phenyl)nicotinoyl)pyrrolidine-2-carboxylic acid (18)
A solution of 12d (600 mg, 1.9 mmol), 4-nitrophenylboronic
acid pinacol ester (523 mg, 2.1 mmol), KF (0.22 g, 3.8 mmol), and
tetrakis(triphenylphosphine) palladium (46 mg, 0.04 mmol) in
toluene/ethanol/H2O (4/2/1 ratio, 30 mL) was heated to 80 °C for
12 h, cooled to room temperature, filtered through Celite, washed
with ethyl acetate (3 ꢁ 40 mL), concentrated and purified on a
silica gel chromatography to afford 13d (534 mg, 79% yield). To a
solution of 13d (500 mg, 1.4 mmol)in 20 mL of ethanol at 0 °C
under N2, 10% Pd/C (50 mg) and NaBH4 (212 mg, 5.6 mmol, por-
tion-wise) were added. Then it was warmed to room temperature
and stirred for another 6 h. To the reaction mixture, 1 M HCl was
added until no bubbles come out. Ethanol was removed through
rotary evaporation, 30 mL of water and 30 mL of ethyl acetate
was added to residue, extracted with ethyl acetate (30 mL ꢁ 3).
The extracts were dried over Na2SO4 and concentrated in vacuo
to give a residue that was subjected to silica gel chromatography
to afford 14d (324 mg, 71% yield). 1H NMR (400 MHz, CDCl3) d
8.96 (s, 1H), 8.92 (s, 1H), 7.43 (dd, J = 8.5, 1.9 Hz, 2H), 6.80 (dd,
J = 8.4, 3.5 Hz, 2H), 4.93 (dd, J = 8.5, 2.9 Hz, 0.5H), 4.76 (dd, J = 8.5,
3.9 Hz, 0.5H), 4.02–3.76 (m, 5.5H), 3.68 (s, 1.5H), 2.36–2.23 (m,
1H), 2.21–1.97 (m, 3H).
A mixture of 6-bromonicotinic acid (11c, 1.01 g, 5.0 mmol), (S)-
methyl pyrrolidine-2-carboxylate (0.71 g,5.5 mmol), TBTU (1.77 g,
5.5 mmol) and DIPEA (1.65 mL, 10.0 mmol) in 15 mL of DMF were
stirred at room temperature for 12 h, then DMF was removed by
rotary evaporation. To the residue, 50 mL of water and 50 mL of
ethyl acetate were added, extracted with ethyl acetate
(50 mL ꢁ 3). The extracts were dried over Na2SO4 and concentrated
in vacuo to give a residue that was subjected to silica gel chro-
matography to afford 12c as a white powder (1.61 g, 93% yield).
tetrakis(triphenylphosphine) palladium1H NMR (300 MHz, CDCl3)
d 8.97 (s, 1H), 8.35 (d, J = 8.5 Hz, 2H), 8.22 (d, J = 8.5 Hz, 2H), 8.07
(d, J = 8.2 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 4.72 (dd, J = 7.9, 4.4 Hz,
1H), 3.81 (s, 3H), 3.77–3.60 (m, 2H), 2.44–2.32 (m, 1H),
2.14–1.94 (m, 3H).
To a solution of 13c (1.3 g, 3.7 mmol) in 30 mL ethanol at 0 °C
under N2, 10% Pd/C (130 mg) and NaBH4 (560 mg, 14.8 mmol, por-
tion-wise) were added. Then it was warmed to room temperature
and stirred for another 6 h. To the reaction mixture, 1 M HCl was
added until no bubbles come out. EtOH was removed through
rotary evaporation, 50 mL of water and 50 mL of ethyl acetate
was added to the residue, extracted with ethyl acetate
(50 mL ꢁ 3). The extracts were dried over Na2SO4 and concentrated
in vacuo to give a residue that was subjected to silica gel chro-
matography to afford 14c (926 mg, 77% yield). To a solution of
14c (100 mg, 0.31 mmol) and phenyl isocyanate (40 mg,
To a solution of 14d (100 mg, 0.31 mmol) and phenyl isocyanate
(40 mg, 0.34 mmol) in dry THF (10 mL), triethylamine (0.12 mL,
0.68 mmol) was added. The mixture was stirred at 40 °C for 2 h,
concentrated and purified by a flash column (20–50% EtOAc in pet-
roleum ether) to provide 15d (121 mg, 88% yield). Into a 100 mL
round bottom flask, 15d (100 mg, 0.22 mmol), LiOH (27 mg,
Please cite this article in press as: Yan J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.07.007

10
J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
1.1 mmol), and 30 mL of 1:1 acetonitrile/water were added. It was
stirred at room temperature overnight. The pH-value of the solu-
tion was adjusted to between 4 and 7 by adding 1 M HCl. The pre-
cipitated solid was filtered and washed with a small amount of
ethyl acetate to provide the title product 19 (84 mg, 89% yield). A
light yellow solid as a 1:1 mixture of rotamers: m.p. 196–197 °C.
1H NMR (400 MHz, CD3OD) d 9.17–9.07 (m, 2H), 7.77–7.61 (m,
4H), 7.45 (d, J = 7.8 Hz, 2H), 7.30 (t, J = 7.7 Hz, 2H), 7.03 (t,
J = 7.6 Hz, 1H), 5.14–5.06 (m, 0.5H), 4.70–4.59 (m, 0.5H), 3.94–
3.54 (m, 2H), 2.37 (s, 1H), 2.26–1.90 (m, 3H); 13C NMR (126 MHz,
DMSO-D6) d 173.5, 173.0, 163.8, 163.7, 159.3, 158.8, 154.2, 153.8,
152.4, 141.0, 139.5, 132.7, 132.5, 128.8, 127.6, 127.5, 126.2,
126.0, 122.0, 118.6, 118.3, 60.3, 58.7, 48.3, 47.1, 31.1, 28.9, 24.7,
22.1; LC-MS calcd. for C23H22N5O+4 (m/e), 432.17, obsd. 430.2 (M
+H). The purity of the compound 19 was 99.3% by HPLC.
31.3, 28.5, 25.1, 21.7; LC-MS calcd. for C24H23N4O+4 (m/e), 431.17,
obsd. 431.2 (M+H). The purity of the compound 20 was 99.9% by
HPLC.
4.7. General procedure for the preparation of 21–30
To a solution of corresponding aniline (0.46 mmol) and triphos-
gene (136 mg, 0.46 mmol) in dry DCM, triethylamine (0.64 mL,
4.6 mmol) was added, and the mixture was stirred for 10 min at
room temperature. Without further purification, 14b (100 mg,
0.31 mmol) was added, the reaction solution was stirred at 40 °C
for 2 h, concentrated and purified by a flash column (20–50% EtOAc
in petroleum ether) to provide 15f–q. Into a 50 mL round bottom
flask, 15f–q (0.23 mmol), LiOH (27 mg, 1.1 mmol), and 20 mL of
1:1 acetonitrile/water were added. It was stirred at room temper-
ature overnight. The pH-value of the solution was adjusted to
between 4 and 7 by adding 1 M HCl. The precipitated solid was fil-
tered and washed with a small amount of ethyl acetate to provide
the title product 21–30. (107 mg, 93% yield).
4.6. Procedure for the preparation of (R)-1-(5-(4-(3-phenylureido)
phenyl)picolinoyl)pyrrolidine-2-carboxylic acid (20)
A mixture of 5-bromopyridine-2-carboxylic acid (11b, 1.01 g,
5.0 mmol),
(R)-methyl
pyrrolidine-2-carboxylate
4.7.1. (S)-1-(5-(4-(3-(2-(Trifluoromethyl)phenyl)ureido)phenyl)
picolinoyl)pyrrolidine-2-carboxylic acid (21)
(0.71 g,5.5 mmol), TBTU (1.77 g, 5.5 mmol) and DIPEA (1.65 mL,
10.0 mmol) in 15mL of DMF was stirred at room temperature for
12 h, then DMF was removed by rotary evaporation. To the residue,
50 mL water and 50 mL ethyl acetate was added, extracted with
ethyl acetate (50 mL ꢁ 3). The extracts were dried over Na2SO4
and concentrated in vacuo to give a residue that was subjected to
silica gel chromatography to afford 12e (1.66 g, 96% yield). A solu-
tion of 12e (1.5 g, 4.2 mmol), 4-nitrophenylboronic acid pinacol
ester (1.14 g, 4.6 mmol), KF (0.49 g, 8.4 mmol), and tetrakis(triph-
enylphosphine) palladium (92 mg, 0.08 mmol) in toluene/etha-
nol/H2O (4/2/1 ratio, 40 mL) was heated to 80 °C for 12 h, cooled
to room temperature, filtered through Celite, washed with ethyl
acetate (3 ꢁ 40 mL), concentrated and purified on a silica gel chro-
matography to afford 13e (1.42 g, 87% yield). To a solution of 13e
(1.3 g, 3.7 mmol) in 30 mL ethanol at 0 °C under N2, 10% Pd/C
(130 mg) and NaBH4 (560 mg, 14.8 mmol, portion-wise) were
added. Then it was warmed to room temperature and stirred for
another 6 h. To the reaction mixture, 1 M HCl was added until no
bubbles come out. EtOH was removed through rotary evaporation,
50 mL of water and 50 mL of ethyl acetate was added to residue,
extracted with ethyl acetate (50 mL ꢁ 3). The extracts were dried
over Na2SO4 and concentrated in vacuo to give a residue that was
subjected to silica gel chromatography to afford 14e (986 mg,
82% yield). To a solution of 14e (100 mg, 0.31 mmol) and phenyl
isocyanate (40 mg, 0.34 mmol) in dry THF (10 mL), trimethylamine
(0.12 mL, 0.68 mmol) was added. The mixture was stirred at 40 °C
for 2 h, concentrated and purified by a flash column (20–50% EtOAc
in petroleum ether) to provide 15e (128 mg, 93% yield). Into a
100 mL round bottom flask, 15e (100 mg, 0.22 mmol), LiOH
(27 mg, 1.1 mmol), and 30 mL of 1:1 acetonitrile/water were
added. It was stirred at room temperature overnight. The pH-value
of the solution was adjusted to between 4 and 7 by adding 1 M HCl.
The precipitated solid was filtered and washed with a small
amount of ethyl acetate to provide the title product 20 (95 mg,
98% yield). A light yellow solid as a 1:1 mixture of rotamers: m.
p. 139–140 °C. 1H NMR (400 MHz, DMSO-D6) d 12.51 (s, 1H), 8.98
(s, 1H), 8.94 (s, 0.5H), 8.83 (s, 1H), 8.80 (s, 0.5H), 8.19 (s, 1H),
7.92 (d, J = 8.1 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.74 (t, J = 8.3 Hz,
2H), 7.61 (d, J = 5.9 Hz, 2H), 7.47 (d, J = 7.6 Hz, 2H), 7.29 (t,
J = 7.3 Hz, 2H), 6.98 (t, J = 6.7 Hz, 1H), 5.24–5.10 (m, 1H), 4.53–
4.37 (m, 1H), 3.86 (d, J = 5.4 Hz, 1H), 3.73–3.59 (m, 1H), 2.36–
2.20 (m, 1H), 2.08–1.76 (m, 3H); 13C NMR (101 MHz, DMSO-D6) d
173.9, 173.3, 165.2, 164.9, 152.5, 151.1, 145.5, 144.6, 140.5,
139.6, 136.3, 136.3, 134.2, 134.1, 129.3, 129.1, 128.8, 127.5,
127.5, 124.3, 124.0, 122.0, 118. 6, 118.3, 60.7, 59.7, 49.4, 47.9,
A light yellow solid as a 1:1 mixture of rotamers: m.p. 141–
142 °C. 1H NMR (500 MHz, DMSO-D6) d 12.53 (s, 1H), 9.59 (s,
1H), 8.94 (d, J = 1.7 Hz, 0.5H), 8.80 (d, J = 1.9 Hz, 0.5H), 8.20 (ddd,
J = 8.0, 5.6, 2.2 Hz, 1H), 8.16 (s, 1H), 7.96 (d, J = 7.1 Hz, 1H), 7.92
(d, J = 8.3 Hz, 0.5H), 7.83 (d, J = 8.2 Hz, 0.5H), 7.77 (dd, J = 10.9,
8.7 Hz, 2H), 7.69 (d, J = 7.9 Hz, 1H), 7.67–7.53 (m, 4H), 7.30 (t,
J = 7.6 Hz, 1H), 5.17 (dd, J = 8.6, 3.4 Hz, 0.5H), 4.51–4.42 (m,
0.5H), 3.86 (t, J = 6.5 Hz, 1H), 3.71–3.63 (m, 1H), 2.34–2.20 (m,
1H), 2.07–1.77 (m, 3H); 13C NMR (126 MHz, DMSO-D6) d 173.9,
173.3, 165.2, 164.9, 152.4, 151.5, 151.2, 145.5, 144.6, 140.2,
136.3, 136.2, 134.3, 134.2, 133.0, 132.1, 132.1,131.5, 131.5, 129.7,
129.5, 128.8, 128.7, 127.6, 127.6,126.0 (q, J = 5.4 Hz), 125.9,
125.1, 124.3, 124.0, 124.0 (q, J = 272.9 Hz), 123.9, 1202, 120.1 (q,
J = 31.6 Hz), 120.0, 118.6, 60.7, 59.7, 49.4, 47.9, 31.3, 28.5, 25.1,
21.7; LC-MS calcd. for C25H22F3N4O4+ (m/e), 499.16, obsd. 499.2
(M+H). The purity of the compound 21 was 95.7% by HPLC.
4.7.2. (S)-1-(5-(4-(3-(3-(Trifluoromethyl)phenyl)ureido)phenyl)
picolinoyl)pyrrolidine-2-carboxylic acid (22)
Yellow solid as a 1:1 mixture of rotamers: m.p. 146–147 °C. 1H
NMR (400 MHz, DMSO-D6) d 12.51 (s, 1H), 9.18 (s, 1H), 9.07 (s, 1H),
8.94 (d, J = 2.1 Hz, 0.5H), 8.80 (d, J = 2.0 Hz, 0.5H), 8.19 (ddd, J = 8.4,
6.1, 2.3 Hz, 1H), 8.04 (s, 1H), 7.92 (d, J = 8.3 Hz, 0.5H), 7.83 (d,
J = 8.2 Hz, 0.5H), 7.79–7.71 (m, 2H), 7.67–7.57 (m, 3H), 7.52 (t,
J = 7.9 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 5.17 (dd, J = 8.6, 3.5 Hz,
0.5H), 4.50–4.42 (m, 0.5H), 3.87 (t, J = 6.5 Hz, 1H), 3.73–3.61 (m,
1H), 2.35–2.20 (m, 1H), 2.08–1.78 (m, 3H); 13C NMR (101 MHz,
DMSO-D6) d 174.0, 173.4, 165.2, 165.0, 152.5, 151.5, 151.3, 145.5,
144.7, 140.5, 140.1, 136.3, 136.2, 134.3, 134.2, 130.0,129.7, 129.6
(q, J = 31.4 Hz), 129.6, 127.5, 127.5, 124.3, 124.3 (q, J = 272.2 Hz),
124.0, 122.0, 118.9, 118.2 (q, J = 3.9 Hz), 114.2 (q, J = 3.9 Hz), 60.8,
59.8, 49.4, 47.9, 31.4, 28.5, 25.1, 21.7; LC-MS calcd. for C25H22F3N4-
O4+ (m/e), 499.16, obsd. 499.2 (M+H). The purity of the compound
22 was 95.4% by HPLC.
4.7.3. (S)-1-(5-(4-(3-(4-(Trifluoromethyl)phenyl)ureido)phenyl)
picolinoyl)pyrrolidine-2-carboxylic acid (23)
Light yellow solid as a 1:1 mixture of rotamers: m.p. 139–
140 °C. 1H NMR (500 MHz, DMSO-D6) d 9.31 (s, 1H), 9.16 (s, 1H),
8.94 (s, 0.5H), 8.80 (d, J = 1.7 Hz, 0.5H), 8.25–8.16 (m, 1H), 7.92
(d, J = 8.2 Hz, 0.5H), 7.83 (d, J = 8.2 Hz, 0.5H), 7.80–7.73 (m, 2H),
7.71–7.60 (m, 6H), 5.17 (dd, J = 8.6, 3.4 Hz, 0.5H), 4.53–4.37 (m,
0.5H), 3.86 (t, J = 6.0 Hz, 1H), 3.71–3.62 (m, 1H), 2.37–2.21 (m,
1H), 2.08–1.77 (m, 3H); 13C NMR (126 MHz, DMSO-D6) d 173.9,
Please cite this article in press as: Yan J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.07.007

J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
173.3, 165.1, 164.9, 152.3, 151.4, 151.2, 145.5, 144.7, 143.4, 140.1,
136.3, 136.2, 134.3, 134.2, 129.7, 129.6, 127.6, 127.5, 126.1 (q,
J = 3.7 Hz), 124.6 (q, J = 270.7 Hz), 124.3, 124.0, 121.9 (q,
J = 31.9 Hz), 118.8, 117.9, 60.7, 59.7, 49.4, 47.9, 31.3, 28.5, 25.1,
21.7; LC-MS calcd. for C25H22F3N4O4+ (m/e), 499.16, obsd. 499.2
(M+H). The purity of the compound 23 was 99.0% by HPLC.
(t, J = 7.4 Hz, 1H), 5.17 (d, J = 7.3 Hz, 0.5H), 4.46 (s, 0.5H), 3.87 (s,
1H), 3.67–3.55 (m, 1H), 2.38–2.19 (m, 1H), 2.09–1.78 (m, 3H);
13C NMR (126 MHz, DMSO-D6) d 173.9, 173.3, 165.0, 164.9,
152.1, 151.2, 151.1, 145.4, 144.6, 140.3, 136.4, 136.2, 135.9,
134.3, 129.5, 129.4, 129.3, 127.6, 127.6, 124.3, 124.0, 123.5,
122.2, 121.5, 118.6, 67.0, 60.7, 59.7, 49.4, 47.9, 31.3, 28.5, 25.1,
21.7; LC-MS calcd. For C24H22ClN4O+4 (m/e), 465.13, obsd. 465.2
(M+H). The purity of the compound 27 was 95.6% by HPLC.
4.7.4. (S)-1-(5-(4-(3-(2-Methoxyphenyl)ureido)phenyl)picolinoyl)
pyrrolidine-2-carboxylic acid (24)
Light yellow solid as a 1:1 mixture of rotamers: m.p. 149–
150 °C. 1H NMR (400 MHz, DMSO-D6) d 12.62 (s, 1H), 9.72 (s,
1H), 8.93 (s, 1H), 8.79 (s, 1H), 8.38 (s, 1H), 8.22–8.10 (m, 2H),
7.90 (d, J = 8.1 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.78–7.70 (m, 2H),
7.66–7.52 (m, 3H), 7.05–6.87 (m, 3H), 5.15 (d, J = 6.1 Hz, 0.52H),
4.45 (s, 0.46H), 3.92–3.81 (m, 4H), 3.70–3.59 (m, 1H), 2.34–2.19
(m, 1H), 2.08–1.73 (m, 3H); 13C NMR (126 MHz, DMSO-D6) d
174.5, 173.9, 165.7165.6, 152.9, 151.9, 148.3, 145.9, 145.1, 141.2,
136.8, 136.6, 134.5, 129.6, 129.5, 129.1, 128.0, 127.9, 124.6,
124.4, 122.4, 121.0, 119.0, 118.8, 111.3, 61.5, 60.4, 56.2, 49.8,
48.2, 31.8, 29.0, 25.5, 22.2; LC-MS calcd. for C25H25N4O+5 (m/e),
461.18, obsd. 461.2 (M+H). The purity of the compound 24 was
95.2% by HPLC.
4.7.8. (S)-1-(5-(4-(3-(3-Chlorophenyl)ureido)phenyl)picolinoyl)
pyrrolidine-2-carboxylic acid (28)
Light yellow solid as a 1:1 mixture of rotamers: m.p. 137–
138 °C. 1H NMR (500 MHz, DMSO-D6) d 12.51 (s, 1H), 9.03 (s,
1H), 9.02 (s, 1H), 8.94 (d, J = 1.8 Hz, 0.5H), 8.80 (d, J = 1.8 Hz,
0.5H), 8.20 (ddd, J = 8.2, 5.9, 2.3 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H),
7.83 (d, J = 8.2 Hz, 1H), 7.80–7.69 (m, 3H), 7.62 (dd, J = 8.6, 3.7 Hz,
2H), 7.36–7.26 (m, 2H), 7.07–6.99 (m, 1H), 5.17 (dd, J = 8.6,
3.4 Hz, 0.5H), 4.49–4.44 (m, 0.5H), 3.87 (t, J = 5.8 Hz, 1H), 3.71–
3.62 (m, 1H), 2.34–2.22 (m, 1H), 2.07–1.79 (m, 3H); 13C NMR
(126 MHz, DMSO-D6) d 173.9, 173.3, 165.1, 164.9, 152.3, 151.4,
151.2, 145.5, 144.6, 141.2, 140.2, 136.3, 136.2, 134.2, 134.2,
133.2, 130.4, 129.6, 129.5, 127.5, 127.5, 124.3, 124.0, 121.6,
118.8, 117.6, 116.7, 60.7, 59.7, 49.4, 47.9, 31.3, 28.5, 25.1, 21.7;
LC-MS calcd. for C24H22ClN4O4+ (m/e), 465.13, obsd. 465.2 (M+H).
The purity of the compound 28 was 98.7% by HPLC.
4.7.5. (S)-1-(5-(4-(3-(3-Methoxyphenyl)ureido)phenyl)picolinoyl)
pyrrolidine-2-carboxylic acid (25)
Light yellow solid as a 7:13 mixture of rotamers: m.p. 147–
148 °C. 1H NMR (500 MHz, DMSO-D6) d 10.14–9.80 (m, 1.15H),
9.67 (s, 0.34H), 9.54 (s, 0.34H), 8.92 (s, 0.35H), 8.74 (s, 0.65H),
8.17 (dd, J = 8.2, 1.8 Hz, 1H), 8.04 (d, J = 6.7 Hz, 1H), 7.81 (d,
J = 8.2 Hz, 0.35H), 7.78 (d, J = 8.2 Hz, 0.65H),, 7.72 (d, J = 8.5 Hz,
0.75H), 7.62 (d, J = 8.5 Hz, 0.75H), 7.55 (d, J = 8.3 Hz, 1.25H), 7.49
(d, J = 8.2 Hz, 1.25H), 7.24 (s, 1H), 7.19–7.11(m, 1H), 7.05–6.93
(m, 1H), 6.52 (t, J = 9.3 Hz, 1H), 5.09 (dd, J = 8.2, 3.4 Hz, 0.65H),
4.46 (dd, J = 8.1, 3.9 Hz, 0.35H), 3.86 (t, J = 6.1 Hz, 1H), 3.72 (s,
3H), 3.69–3.58 (m, 1H), 2.33–2.15 (m, 1H), 2.11–1.80 (m, 3H);
13C NMR (126 MHz, DMSO-D6) d 175.5, 173.9, 165.9, 165.1,
159.7, 159.6, 152.8, 152.7, 152.1, 151.6, 145.4, 144.6, 141.6,
141.2, 140.9, 136.3, 135.8, 134.0, 133.6, 129.5, 129.4, 129.1,
128.7, 127.4, 127.0, 123.9, 123.9, 118.5, 118.3, 110.6, 107.1,
106.9, 104.0, 103.9, 61.9, 60.2, 54.9, 54.9, 49.4, 47.6, 31.5, 28.7,
25.1, 22.1; LC-MS calcd. for C25H25N4O+5 (m/e), 461.18, obsd.
461.1 (M+H). The purity of the compound 25 was 98.6% by HPLC.
4.7.9. (S)-1-(5-(4-(3-(4-Chlorophenyl)ureido)phenyl)picolinoyl)
pyrrolidine-2-carboxylic acid (29)
Light yellow solid as a 1:1 mixture of rotamers: m.p. 135–
136 °C. 1H NMR (400 MHz, DMSO-D6) d 12.53 (s, 1H), 9.00 (s,
1H), 8.96 (s, 1H), 8.94 (d, J = 1.8 Hz, 0.5H), 8.80 (d, J = 1.9 Hz,
0.5H), 8.19 (ddd, J = 8.0, 4.2, 2.4 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H),
7.83 (d, J = 8.2 Hz, 1H), 7.75 (t, J = 8.7 Hz, 2H), 7.61 (dd, J = 8.7,
2.7 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 5.17
(dd, J = 8.6, 3.4 Hz, 0.5H), 4.51–4.41 (m, 0.5H), 3.86 (t, J = 6.5 Hz,
1H), 3.71–3.60 (m, 1H), 2.35–2.19 (m, 1H), 2.07–1.76 (m, 3H);
13C NMR (101 MHz, DMSO-D6) d 173.9, 173.3, 165.1, 164.9,
152.4, 151.4, 151.4, 151.2, 145.5, 144.6, 140.3, 138.6, 136.3,
136.2, 134.2, 134.1, 129.5, 129.3, 128.7, 127.5, 127.5, 125.5,
124.3, 124.0, 119.8, 118.7, 60.7, 59.7, 49.4, 47.9, 31.3, 28.5, 25.1,
21.7; LC-MS calcd. For C24H22ClN4O+4 (m/e), 465.13, obsd. 465.2
(M+H). The purity of the compound 29 was 99.9% by HPLC.
4.7.6. (S)-1-(5-(4-(3-(4-Methoxyphenyl)ureido)phenyl)picolinoyl)
pyrrolidine-2-carboxylic acid (26)
4.7.10. (S)-1-(5-(4-(3-(3-Fluorophenyl)ureido)phenyl)picolinoyl)
pyrrolidine-2-carboxylic acid (30)
Light yellow solid as a 1:1 mixture of rotamers: m.p. 156–
157 °C. 1H NMR (500 MHz, DMSO-D6) d 12.49 (s, 1H), 8.93 (s,
0.5H), 8.85–8.75 (m, 0.5H), 8.55 (s, 1H), 8.18 (s, 1H), 7.92 (d,
J = 7.9 Hz, 0.5H), 7.82 (d, J = 8.1 Hz, 0.5H), 7.79–7.66 (m, 2H), 7.61
(s, 2H), 7.37 (d, J = 8.0 Hz, 2H), 6.88 (d, J = 8.1 Hz, 2H), 5.17 (d,
J = 5.2 Hz, 0.5H), 4.46 (s, 0.5H), 3.87 (s, 1H), 3.72 (s, 3H), 3.70–
3.68 (m, 1H), 2.38–2.16 (m, 1H), 2.09–1.75 (m, 3H); 13C NMR
(126 MHz, DMSO-D6) d 174.4, 173.8, 165.6, 165.4, 155.1, 153.1,
151.8, 151.6, 145.9, 145.0, 141.1, 136.8, 136.8, 134.6, 134.5,
133.0, 129.6, 129.4, 127.9, 127.9, 124.7, 124.4, 120.6, 119.0,
114.5, 61.2, 60.2, 55.6, 49.9, 48.3, 31.8, 29.0, 25.6, 22.2; LC-MS
calcd. for C25H25N4O+5 (m/e), 461.18, obsd. 461.2 (M+H). The purity
of the compound 26 was 99.9% by HPLC.
Light yellow solid as a 1:1 mixture of rotamers: m.p. 169–
170 °C. 1H NMR (400 MHz, DMSO-D6) d 10.13 (s, 1H), 10.05 (s,
1H), 8.93 (s, 0.5H), 8.79 (s, 0.5H), 8.20 (s, 1H), 7.90 (s, 0.5H),
7.86–7.67 (m, 2.5 H), 7.61 (s, 2H), 7.51 (d, J = 11.2 Hz, 1H), 7.29
(s, 1H), 7.11 (s, 1H), 6.75 (s, 1H), 5.16 (s, 0.5 H), 4.45 (s, 0.5H),
3.85 (s, 1H), 3.65 (s, 1H), 2.24 (s, 1H), 2.07–1.75 (m, 3H); 13CNMR
(126 MHz, DMSO-D6) d 173.7, 173.1, 164.9, 164.8, 162.3 (d,
J = 240.1 Hz), 152.5, 151.0, 150.9, 145.2, 144.4, 141.7, 141.6,
140.4, 136.3, 136.2, 134.2, 134.2, 130.3 (d, J = 9.4 Hz), 129.0,
128.9, 127.4 (d, J = 7.7 Hz), 124.1, 123.9, 118.1, 113.4, 107.9 (d,
J = 21.0 Hz), 104.3 (d, J = 26.4 Hz), 60.6, 59.6, 49.3, 47.8, 31.3,
28.4, 25.0, 21.6; LC-MS calcd. For C24H22FN4O+4 (m/e), 449.16, obsd.
449.2 (M+H). The purity of the compound 30 was 96.2% by HPLC.
4.7.7. (S)-1-(5-(4-(3-(2-Chlorophenyl)ureido)phenyl)picolinoyl)
pyrrolidine-2-carboxylic acid (27)
Light yellow solid as a 1:1 mixture of rotamers: m.p. 148–
149 °C. 1H NMR (400 MHz, DMSO-D6) d 9.86 (s, 1H), 8.95 (s, 1H),
8.81 (s, 1H), 8.49 (s, 1H), 8.29–8.10 (m, 2H), 7.93 (d, J = 8.2 Hz,
1H), 7.84 (d, J = 7.7 Hz, 1H), 7.77 (t, J = 8.6 Hz, 2H), 7.65 (d,
J = 7.3 Hz, 2H), 7.47 (d, J = 8.0 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.05
4.8. Procedure for preparation of 31 and 32
Compounds 31 and 32 were prepared by using the same proce-
dure as described for compound 17 substituting butyl isocyanate
for and isocyanatocyclohexane for phenyl isocyanate.
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12
J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.8.1. (S)-1-(5-(4-(3-Butylureido)phenyl)picolinoyl)pyrrolidine-2-
carboxylic acid (31)
solution was adjusted to between 4 and 7 by adding 1 M HCl.
The precipitated solid was filtered and washed with a small
amount of diethyl ether to provide the title product 36 (102 mg,
93% yield). Light yellow solid as a 1:1 mixture of rotamers: m.p.
154–155 °C. 1H NMR (400 MHz, DMSO-D6) d 10.55 (s, 1H), 8.94
(s, 0.5H), 8.80 (s, 0.5H), 8.19 (d, J = 6.4 Hz, 1H), 7.92 (d, J = 8.4 Hz,
0.5H), 7.83 (d, J = 8.2 Hz, 0.5H), 7.81–7.70 (m, 5H), 7.69–7.51 (m,
4H), 5.16 (d, J = 5.6 Hz, 0.5H), 4.46 (d, J = 4.9 Hz, 0.5H), 3.84 (s,
3H), 3.71–3.59 (m, 1H), 2.37–2.16 (m, 1H), 2.11–1.73 (m, 3H);
13C NMR (126 MHz, DMSO-D6) d 173.9, 173.3, 168.8, 165.1,
164.9, 151.5, 151.4, 145.6, 144.7, 139.7, 137.2, 136.2, 136.1,
134.4, 134.4, 133.5, 132.1, 131.5, 131.5, 130.8, 130.7, 129.3, 129.0
(q, J = 31.4 Hz), 128.8, 128.7, 127.5, 127.4, 125.9 (q, J = 2.7 Hz),
124.3 (q, J = 271.6 Hz), 124.3, 124.0, 123.4 (q, J = 3.7 Hz), 123.2,
119.6, 60.7, 59.7, 49.4, 47.9, 42.6, 31.3, 28.5, 25.1, 21.7; LC-MS
calcd. for C26H23F3N3O+4 (m/e), 498.16, obsd. 498.2 (M+H). The pur-
ity of the compound 36 was 95.7% by HPLC.
Light yellow solid as a 1:1 mixture of rotamers: m.p. 183–
184 °C. 1H NMR (400 MHz, DMSO-D6) d 12.51 (s, 1H), 8.91 (s,
0.5H), 8.77 (s, 0.5H), 8.65 (s, 1H), 8.20–8.10 (m, 1H), 7.91 (d,
J = 8.4 Hz, 0.5H), 7.81 (d, J = 8.1 Hz, 0.5H), 7.68 (t, J = 8.8 Hz, 2H),
7.55 (d, J = 6.3 Hz, 2H), 6.22 (t, J = 5.3 Hz, 1H), 5.17 (dd, J = 8.4,
3.1 Hz, 0.5H), 4.48–4.42 (m, 0.5H), 3.87 (t, J = 6.1 Hz, 1H), 3.65 (t,
J = 7.2 Hz, 1H), 3.10 (q, J = 6.3 Hz, 2H), 2.37–2.21 (m, 1H), 2.07–
1.76 (m, 3H), 1.47–1.38 (m, 2H), 1.37–1.27 (m, 2H); 13C NMR
(101 MHz, DMSO-D6) d 174.0, 173.3, 165.2, 165.0, 155.1, 151.2,
151.0, 145.4, 144.5, 141.4, 136.5, 136.4, 134.0, 133.9, 128.4,
128.2, 127.4, 127.3, 124.3, 124.0, 118.0, 60.8, 59.7, 49.4, 47.9,
38.8, 31.9, 31.3, 28.5, 25.2, 21.7, 19.6, 13.7; LC-MS calcd. For
C
22H27N4O+4 (m/e), 411.20, obsd. 411.3 (M+H). The purity of the
compound 31 was 99.9% by HPLC.
4.8.2. (S)-1-(5-(4-(3-Cyclohexylureido)phenyl)picolinoyl)pyrrolidine-
2-carboxylic acid (32)
Light yellow solid as a 1:1 mixture of rotamers: m.p. 141–
142 °C. 1H NMR (400 MHz, DMSO-D6) d 8.91 (s, 0.5H), 8.77 (s,
0.5H), 8.60 (s, 1H), 8.16 (s, 1H), 7.91 (s, 0.5H), 7.82 (s, 0.5H), 7.68
(s, 2H), 7.53 (s, 2H), 6.20 (s, 1H), 5.17 (s, 1H), 4.45 (s, 1H), 3.86
(s, 1H), 3.65 (s, 1H), 3.48 (s, 1H), 2.24 (s, 1H), 2.06–1.52 (m, 9H),
1.38–1.08 (m, 5H); 13C NMR (126 MHz, DMSO-D6) d 173.9, 173.3,
165.1, 164.9, 154.3, 151.1, 150.9, 145.3, 144.5, 141.4, 136.5,
136.4, 134.0, 134.0, 128.3, 128.2, 127.4, 127.3, 124.3, 124.0,
117.9, 60.7, 59.7, 49.4, 47.9, 47.6, 32.9, 31.3, 28.5, 25.3, 25.1,
24.4, 21.7; LC-MS calcd. for C24H29N4O+4 (m/e), 437.22, obsd.
437.2 (M+H). The purity of the compound 32 was 99.9% by HPLC.
4.11. (S)-1-(5-(4-(2-Oxo-2-((3-(trifluoromethyl)phenyl)amino)ethyl)
phenyl)picolinoyl)pyrrolidine-2-carboxylic acid (40)
A solution of 4-bromophenylacetic acid (1.08 g, 5.0 mmol), 3-
(trifluoromethyl)aniline (0.85 g, 5.3 mmol), TBTU (1.70 g,
5.3 mmol) and DIPEA (1.65 mL, 10.0 mmol) in 10 mL of DMF was
stirred at room temperature for 12 h, then DMF was removed by
rotary evaporation. To the residue, 50 mL of water and 50 mL of
ethyl acetate were added, extracted with ethyl acetate
(3 ꢁ 50 mL), concentrated and purified on a silica gel chromatogra-
phy to afford 37. A solution of 37 (1.50 g, 4.2 mmol), bis(pinaco-
lato)diboron (1.12 g, 4.4 mmol), Pd(dppf)Cl2ꢂCH2Cl2 (69 mg,
0.084 mg), and KOAc (0.82 g, 8.4 mmol) in DMF was stirred at
80 °C for 8 h, then DMF was removed by rotary evaporation. To
the residue, 50 mL of water and 50 mL of ethyl acetate was added,
extracted with ethyl acetate (2 ꢁ 50 mL), concentrated and puri-
fied on a silica gel chromatography to afford 38 (1.21 g, 71% yield).
1H NMR (400 MHz, CDCl3) d 7.73 (s, 1H), 7.65 (d, J = 7.7 Hz, 1H),
7.53 (d, J = 8.3 Hz, 2H), 7.41 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.3 Hz,
1H), 7.27 (s, 1H), 7.21 (d, J = 8.3 Hz, 2H), 3.70 (s, 2H), 1.26 (s, 12H).
A solution of 38 (1.1 g, 2.7 mmol), 12b (0.84 g, 2.7 mmol), KF
(0.31 g, 5.4 mmol), and Pd(PPh3)4 (69 mg, 0.06 mmol) in toluene/
ethanol/H2O (4/2/1 ratio, 50 mL) was heated to 80 °C for 12 h,
cooled to room temperature, filtered through Celite, washed with
ethyl acetate (3 ꢁ 40 mL), concentrated and purified on a silica
gel chromatography to afford 39 (0.86 g, 62% yield). Into a 50 mL
round bottom flask, 39 (113 mg, 0.22 mmol), LiOH (27 mg,
1.1 mmol), and 30 mL of 1:1 acetonitrile/water was added. It was
stirred at room temperature overnight. The pH-value of the solu-
tion was adjusted to between 4 and 7 by adding 1 M HCl. The pre-
cipitated solid was filtered and washed with a small amount of
ethyl acetate to provide the product 40 (102 mg, 93% yield). Light
yellow solid as a 1:1 mixture of rotamers: m.p. 173–174 °C. 1H
NMR (400 MHz, DMSO-D6) d 12.57 (s, 1H), 10.60 (s, 1H), 8.93 (s,
1H), 8.79 (s, 1H), 8.19 (t, J = 7.0 Hz, 1H), 8.10 (s, 1H), 7.91 (d,
J = 8.1 Hz, 0.5H), 7.86–7.70 (m, 3.5H), 7.64–7.45 (m, 5H), 7.38 (d,
J = 7.6 Hz, 1H), 5.13 (d, J = 5.5 Hz, 0.5H), 4.44 (d, J = 5.0 Hz, 0.5H),
3.83 (t, J = 7.6 Hz,1H), 3.74 (s, 2H), 3.67–3.58 (m, 1H), 2.31–2.18
(m, 1H), 2.04–1.77 (m, 3H); 13C NMR (126 MHz, DMSO-D6) d
173.9, 173.3, 169.5, 165.1, 165.0, 152.0, 151.9, 146.0, 145.1,
140.0, 136.4, 136.3, 134.9 (q, J = 5.8 Hz), 134.7, 134.6, 133.12,
133.1, 132.3, 132.1, 131.5, 131.4, 130.1, 130.0, 129.5 (q,
J = 31.4 Hz), 128.8, 128.7, 127.1, 127.0, 124.3, 124.1 (q,
J = 272.5 Hz), 124.0, 122.6, 120.86, 119.6, 115.1, 60.8, 59.8, 49.4,
47.8, 42.9, 31.3, 28.5, 25.1, 21.7; LC-MS calcd. for C26H23F3N3O4+
(m/e), 498.16, obsd. 498.2 (M+H). The purity of the compound 40
was 95.1% by HPLC.
4.9. Procedure for preparation of (S)-1-(5-(4-(3-phenylthioureido)
phenyl)picolinoyl)pyrrolidine-2-carboxylic acid (34)
Compound 34 was prepared by using the same procedure as
described for compound 17 substituting phenyl isothiocyanate
for phenyl isocyanate. Light yellow solid as a 1:1 mixture of rota-
mers: m.p. 114–115 °C. 1H NMR (400 MHz, DMSO-D6) d 12.53 (s,
1H), 10.01 (s, 1H), 9.96 (s, 1H), 8.97 (d, J = 2.0 Hz, 1H), 8.83 (d,
J = 2.1 Hz, 1H), 8.27–8.19 (m, 1H), 7.95 (d, J = 8.3 Hz, 0.5H), 7.85
(d, J = 8.2 Hz, 0.5H), 7.79 (t, J = 8.8 Hz, 2H), 7.69 (d, J = 7.5 Hz, 2H),
7.51 (d, J = 8.2 Hz, 2H), 7.36 (t, J = 7.8 Hz, 2H), 7.15 (t, J = 7.4 Hz,
1H), 5.18 (dd, J = 8.7, 3.4 Hz, 0.5H), 4.51–4.42 (m, 0.5H), 3.88 (t,
J = 6.5 Hz, 1H), 3.72–3.61 (m, 1H), 2.36–2.21 (m, 1H), 2.08–1.79
(m, 3H); 13C NMR (126 MHz, DMSO-D6) d 179.5, 173.9, 173.3,
165.1, 164.9, 151.7, 151.5, 145.7, 144.9, 140.2, 140.2,139.4, 136.2,
136.1, 134.6, 134.5, 131.8, 131.6, 128.5, 127.1, 127.1, 124.5,
124.3, 124.0, 123.6, 60.7, 59.7, 54.9, 49.4, 47.9, 31.3, 28.5, 25.1,
21.7; LC-MS calcd. for C24H23N4O3S+ (m/e), 447.15, obsd. 447.2
(M+H). The purity of the compound 34 was 97.4% by HPLC.
4.10. (S)-1-(5-(4-(2-(3-(Trifluoromethyl)phenyl)acetamido)phenyl)
picolinoyl)pyrrolidine-2-carboxylic acid (36)
A solution of 14b (100 mg, 3.1 mmol), m-(trifluoromethyl)
phenylacetic acid (70 mg, 0.34 mmol), TBTU (109 mg, 0.34 mmol)
and DIPEA (1.02 mL, 6.2 mmol) in 10mL of DMF were stirred at
room temperature for 12 h, then DMF was removed by rotary
evaporation. To the reaction mixture, 50 mL of water and 50 mL
of ethyl acetate were added to residue, extracted with ethyl acetate
(50 mL ꢁ 3). The extracts were dried over Na2SO4 and concen-
trated in vacuo to give a residue that was subjected to silica gel
chromatography to afford 35 (140 mg, 88% yield). Into a 50 mL
round bottom flask, 35 (113 mg, 0.22 mmol), LiOH (27 mg,
1.1 mmol), and 20 mL of 1:1 acetonitrile/water were added. It
was stirred at room temperature overnight. The pH-value of the
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J. Yan et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
13
4.12. In vitro DGAT enzyme assay
2 h later, and the mice of control group were given equal volume
water. The blood samples were collected and the serum triglyc-
eride levels were subsequently measured by a commercially
triglyceride assay kit (Shanghai Mind).
The recombinant full length human DGAT-1 protein were
expressed in Sf9 insect cells for 48 h, and the microsome contain-
ing DGAT-1 enzyme was prepared by differential centrifugation1,20
and stored at ꢀ80 °C for DGAT-1 enzyme assay.
4.16. Pharmacokinetic study of 22 in SD rats
The DGAT-1 inhibitory activities of compounds were deter-
mined by Phospholipid FlashPlate assay as described before.26 In
brief, the assay mixture containing 20 mM HEPES (pH 7.5), 2 mM
MgCl2, 0.04% BSA, 50 mM 1, 2-didecanoyl-sn-glycerol (DAG, Chy-
man), 5 mM palmitoyl-1-14C coenzyme A (PerkinElmer) and test
7 Sprague dawley male rats (200–220 g) were randomly divided
into 2 groups, each group 4/3, intragastric or intravenous adminis-
tration of the compounds respectively. The compound 22 were
prepared with 5%DMSO, 5% Tween, and 80/90% physiological sal-
ine. Fasting 12 h before the test, free drinking water, after 2 h feed-
ing. Pharmacokinetic parameters were calculated using a non-
compartmental model of Phoenix 1.3 software (US Pharsight Cor-
poration). The peak concentration Cmax and the peak time Tmax
are measured; the area under the concentration time curve of
AUC0-t value was calculated by the trapezoidal method; AUC0-
compounds (1
a phospholipid FlashPlate (PerkinElmer) in a total volume of
100 L. The reaction was initiated by the addition of DGAT-1
lL) dissolved in DMSO was added to each well of
l
enzyme (1.5 mg/well), followed by incubation for 60 min at 37 °C
and was stopped by the addition of 100 mL isopropanol. Plates were
sealed, incubated for 12 h and read on 1450 microbeta trilux
microplate Scintillation and luminescence Counter (PerkinElmer).
= AUC0-t + Ct/ke, Ct was the last blood concentration that can be
1
measured, ke was elimination rate constant; elimination half-life
1/2 = 0.693/ke; mean residence time MRT = AUMC/AUC; clearance
4.13. Cell-based assays
t
CL = D/AUC0-1; steady-state distribution volume Vss=CL ꢁ MRT;
Mouse 3T3-L1 preadipocytes, human colon carcinoma Caco-2
cells and hepatoma HepG2 cells were maintained in Dulbecco’s
modified Eagle medium (DMEM, Gibco) with 10% fetal
bovine serum (FBS, Hyclone). The 3T3-L1 cells were treated as
follows: two days after confluence, the differentiation of 3T3-L1
preadipocytes were induced by 1 lg/mL insulin (Sigma), 1 lM dex-
amethasone (Sigma), and 0.5 mM 3-isobutyl-1-methyl-xanthine
(IBMX; Sigma) in DMEM with 10% FBS for 2 days. Then, the med-
ium was changed to DMEM with 10% FBS containing 1 lg/mL insu-
lin. Two days later, the medium was changed back to DMEM with
10% FBS and the cells were treated with test compounds for 4 days.
The medium was renewed every 2 days. Then the cells in each
group were subjected to Oil Red O staining and cellular triglyceride
content determination. Following the treatment with test com-
pounds for 2 h, Caco-2 cells and HepG2 cells were incubated in
the absence or presence of oleic acid (OA, Sigma) for 48 h in DMEM
with 10% FBS, respectively. Then oil red O staining and cellular
triglyceride content determination experiments were performed
as previously described.35
absolute bioavailabilityF = (AUCi.g. ꢁ Di.v.)/(AUCi.v. ꢁ Di.g.)ꢁ100%.
4.17. Tissue distribution of compound 22 in C57 mice
9 C57 male mice (18–24 g, 8 weeks old) were randomly divided
into 3 groups. Oral administration, after anesthesia with isoflurane,
0.1 mL blood (from orbit) and tissue (liver, duodenum, jejunum
and ileum) was taken, EDTAK2 anti-freezing, and p.o. group was
collected at 1 h, 2 h, 5 h after giving 22. Blood samples were col-
lected on ice, centrifugal separation of plasma in 1 h. Collected
plasma were stored at ꢀ80 °C; after weighing, adding water
according to 1:10 (m/v), homogenating, tissues were kept at low
temperature. 10 lL to 1.5 mL centrifuge tube, adding 100 lL inter-
nal standard solution (5 ng/mL vera pammy and 50 ng/mL gliben-
clamide acetonitrile solution), vortex 60 s and centrifugating for
3 min; the supernatant (75 lL for plasma, 50 lL tissue samples)
were added to 96-well plates. The concentration of 22 in plasma
was determined by LC/MS/MS (Agilent 1200, API 4000 QTRAP).
The data acquisition and control system software is Analyst1.5.1
(Applied Biosystem). The peak integral mode of the sample is auto-
matic integration; the ratio of the sample peak area and internal
standard peak area is used as the index, and the concentration of
the sample was regressed. Linear regression, the weight coefficient
is 1/X2.
4.14. Permeability assay
The Caco-2 method was used to study permeability. Caco-2 cells
(American Type Culture Collection) were cultured on Millicell-24
cell culture insert plates (Merck Millipore). For basolateral and api-
cal compartment, pH value of transport buffer (HBSS/HEPES
10 mM/0.1% BSA) was adjusted to 7.4 and 6.8 respectively. Per-
meation time lasted 95 min. The kinetics were performed at 35
and 95 min. The test compounds were analysed by LC-MS/MS (Xevo
TQ-S coupled to UPLC HAClass, Waters). The apparent permeability
4.18. Statistical analysis
Values in figures were expressed as mean SEM. The compar-
ison of different groups was assessed by one-way ANOVA followed
by Tukey’s multiple comparison test. It was considered to be statis-
tically significant when a p value < 0.05.
was calculated as follows: Papp (cm/s) =
DQ/Dt * 1/(A * C0), where,
D
Q/Dt is the product quantity which appears in the receiver by time
unit; A is the membrane area (cm2); C0 is the initial concentration in
the donor compartment (mM). The efflux ratio was the quotient of
the Papp from apical side to the basolateral side divided by the Papp
from basolateral side to the apical side.
Acknowledgments
We are grateful for financial support from the National Natural
Science Foundation of China (Grant No. 81225022, 81503124) and
the Institutes for Drug Discovery and Development, Chinese Acad-
emy of Sciences (No. CASIMM0120162025).
4.15. In vivo assay
All animal care and experiments were permitted by Institu-
tional Animal Care and Use Committees of Shanghai Institute of
Materia Medica. For the acute lipid challenge study, 6-week-old
male C57/KSJ mice (SLAC, 20 2 g, n = 6) were fasted 16 h and then
orally dosed with either vehicle (0.1% w/v Tween 80) or test com-
pounds, then an olive oil bolus (15 mL/kg) was orally administered
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