Design, synthesis and biological evaluation of anthranilamide derivatives as potent SMO inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115354

A series of anthranilamide derivatives were designed and synthesized as novel smoothened (SMO) inhibitors based on the SMO inhibitor taladegib (LY2940680), which can also inhibit the SMO-D473H mutant, via a ring-opening strategy. The phthalazine core in LY2940680 was replaced with anthranilamide, which retained the inhibitory activity towards the hedgehog (Hh) signaling pathway as evidenced by a dual luciferase reporter gene assay. Compound 12a displayed the best inhibitory activity against the Hh signaling pathway with IC₅₀ value of 34.09 nM, and exhibited better proliferation inhibitory activity towards the Daoy cell line (IC₅₀ = 0.48 μM) than LY2940680 (IC₅₀ = 0.79 μM).

Full text of DOI:10.1016/j.bmc.2020.115354

Journal Pre-proofs
Design, synthesis and biological evaluation of anthranilamide derivatives as
potent SMO inhibitors
Dezhong Ji, Wanwan Zhang, Yungen Xu, Jing-Jing Zhang
PII:
S0968-0896(20)30145-0
https://doi.org/10.1016/j.bmc.2020.115354
BMC 115354
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
9 December 2019
17 January 2020
31 January 2020
Please cite this article as: D. Ji, W. Zhang, Y. Xu, J-J. Zhang, Design, synthesis and biological evaluation of
anthranilamide derivatives as potent SMO inhibitors, Bioorganic & Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.bmc.2020.115354
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Design, synthesis and biological evaluation of anthranilamide
derivatives as potent SMO inhibitors
Dezhong Ji , Wanwan Zhang , Yungen Xu1,2,*, Jing-Jing Zhang1,2,*
1
,a
1,a
1Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing
2
10009, China
2Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical
University, Nanjing 210009, China
aThese authors contributed equally to this work.
*
Corresponding author: Jing-Jing Zhang (Email:jjzhangnj@163.com; Tel:
86-025-83271244).
+
Keywords: Smoothened inhibitor; Hedgehog signaling pathway; ring-opening;
anthranilamide; molecular dynamics

Highlights

SMO inhibitors with novel structural features were developed by replacing the
phthalazine core in LY2940680 with anthranilamide via a ring-opening strategy.

The anthranilamide structure could mimic the phthalazine ring in LY2940680 by
forming a pseudo-ring structure resulting from the intra-molecular hydrogen bond
interaction between the oxygen atom of carbonyl group and hydrogen atom of the
amino group.

The pseudo-ring structure formed by anthranilamide and the hydrogen bond
interaction between its carbonyl group and R400 of SMO, as evidenced by Glide
docking study, suggested that the new compounds can mimic the binding
conformation of LY2940680 to SMO.

Difference in the inhibitory activity between the new compounds and LY2940680
were studied by molecular dynamic analysis.
Graphic abstract

Abstract
A series of anthranilamide derivatives were designed and synthesized as
novel smoothened (SMO) inhibitors based on the SMO inhibitor
taladegib (LY2940680), which can also inhibit the SMO-D473H mutant,
via a ring-opening strategy. The phthalazine core in LY2940680 was
replaced with anthranilamide, which retained the inhibitory activity
towards the hedgehog (Hh) signaling pathway as evidenced by a dual
luciferase reporter gene assay. Compound12a displayed the best
inhibitory activity against the Hh signaling pathway with IC value of
5
0
3
4.09nM, and exhibited better proliferation inhibitory activity towards the
Daoy cell line (IC = 0.48 μM) than LY2940680 (IC = 0.79 μM).
5
0
50
1
. Introduction
The Hedgehog (Hh) signaling pathway plays an important role in
[1]
embryonic and postnatal development. It involves several components
including the Hh ligands, the 12-pass transmembrane receptor Patched 1
(
PTCH1), the 7-pass membrane G-protein-coupled receptor smoothened
SMO), suppressor of fused (SUFU), and the zinc-finger transcription
(
factors GLI proteins.[2] In adults, with the exception of stem cell
homeostasis maintenance and tissue repair, it is mostly inactive as SMO
and GLI are inhibited by PTCH1 and SUFU, respectively.[1, 3]Aberrant
activation of the Hh signaling pathway is involved in the initiation and
progression of various cancers, and also associated with cancer stem cell

[4]
biology. Therefore, the inhibition of the Hh pathway is an attractive
approach for treating cancer.[4]
The Hh pathway can be activated by the binding of the Hh ligands to
PTCH1, which relives SMO from PTCH1. The activated SMO further
represses SUFU, which leads to the release and nuclear translocation of
the GLI proteins. These GLI proteins can promote some cancer-related
[5]
gene expression. Moreover, mutations of the components like PTCH1
and SUFU can also result in activation.
Strategies to target the Hh pathway focus on inhibiting the
components that initiate the pathway including the Hh ligands, SMO, and
[5]
the GLI proteins. To date, targeting SMO with small molecules has
been the most successful strategy. Two SMO inhibitors, vismodegib
(GDC-0449) and sonidegib (LDE225),have been approved by the Food
and Drug Administration(FDA) for the treatment of locally advanced
[5]
basal cell carcinoma (BCC) in 2012 and 2015, respectively. More
recently, another SMO inhibitor, glasdegib (PF-04449913), was also
[6]
approved by FDA for the treatment of acute myeloid leukemia. They are
currently the only drugs used clinically targeting the Hh pathway.
However, drug resistance resulting from SMO mutations like
SMO-D473H mutation was discovered in clinic. To overcome this
problem, novel SMO inhibitors with diverse chemical structures have
been developed.

Taladegib (LY2940680) (Figure 1), a phthalazine-based SMO
inhibitor, is one of the few successful examples that can inhibit both the
[7]
wild-type and the D473H mutant SMO in clinical trials. In humans,
SMO comprises three main domains including an N-terminal
extracellular domain (ECD), a heptahelical membrane spanning (7-TM)
[8]
domain (TMD), and a C-terminal cytoplasmic domain. LY2940680
binds to a thin pocket constituted by extracellular loop protrusions and
[2]
the extracellular stretch of TMD. It showed weak interaction with D473,
and D743H does not interfere with its binding to SMO, which contributes
to its inhibitory activity towards SMO-D473H mutant.[9, 10]
Based on the binding mode of LY2940680 and SMO, compound1
was designed and synthesized by replacing the phthalazine ring in
LY2940680with anthranilamide via the ring-opening strategy (Figure 1).
It displayed moderate Hh pathway inhibitory activity with IC value of
5
0
1
23.40 nM.
In order to improve the activity, ring A and ring B of 1 were
modified using 1 as a lead compound. An initial structural modification
was also made to ring C and the linker of 1. The structures of all the new
1
13
compounds were confirmed by H-NMR, C-NMR, and HRMS. All of
these compounds were evaluated for their Hh pathway inhibitory activity.
Only a few compounds showed detectable activity, and they were chosen
to be evaluated for their in vitro proliferation inhibitory activities against

the Daoy cell line in which the Hh pathway is active. Compound 12a
exhibited the best inhibitory activity against the Hh signaling pathway
(
IC =34.09nM) and displayed better proliferation inhibitory activity than
5
0
LY2940680.
Figure 1 Use of the ring-opening strategy to obtain compound 1.
2
. Results and Discussion
2
.1 Identification of the lead compound
Current understanding of the mechanism of drug resistance in SMO
inhibitors suggests that mutation of aspartic acid at position 473 to
histidine (D473H) is the key to the resistance of small-molecule
inhibitors. To explain why the activity of LY2940680 is not affected by
the D473H mutation, a re-docking of the crystal complex of LY2940680
(Figure 2A) (PDB ID: 4jkv) after virtual mutation of the amino acid was
performed. The result showed that the distance between LY2940680 and
the histidine at position 473 was far (4.8 Å) (Figure 2B). Therefore, the
D473H mutation exhibited a weak effect on LY2940680, and this may

explain why LY2940680 is not affected by SMO (D473H) mutation. The
carbonyl group of the amide in LY2940680 forms a key hydrogen bond
with asparagine at position 219 (N219) in SMO to anchor LY2940680
firmly in SMO. The phenylalanine at position 484 can form a π-πstacking
interaction with the 2-trifluoromethyl-4-fluoro-substituted benzene ring to
maintain a stable conformation of LY2940680 in the protein. The
nitrogen atom on the phthalazine ring can also form a hydrogen bond
with arginine at position 400 (R400).
Figure 2 A: Binding mode of LY2940680 and SMO; B: D473H mutation exhibited weak effect on
LY2940680 binding mode
It was assumed that the inhibitory activity towards the Hh pathway
could be reserved if the molecular binding conformation was maintained.
In this work, according to the principle of scaffold hopping, the
phthalazine ring in LY2940680 was transformed into an anthranilamide
structure by the ring-opening strategy (Figure 1).[11, 12] It was expected
that the intra-molecular hydrogen bond between the carbonyl group and

the amino group could be utilized to form a pseudo-ring structure[13, 14]
thus mimicking the binding conformation of LY2940680 to SMO.
Moreover, the nitrogen atom in the phthalazine ring was replaced with a
carbonyl group as a hydrogen bond acceptor for R400.
Changes in the structure of small molecules can change the electric
properties and conformation of the entire molecule, which can affect the
binding efficiency of the ligand to the receptor. Therefore, the surface
electrostatic potential (ESP) of the ring-opened compound 1was
calculated using Gaussian 9.0. The results showed that the ring-opened
structure in 1 had a similar ESP to the phthalazine region of LY2940680
(Figure 3) indicating that the anthranilamide structure can efficiently
simulate the structure and charge distribution of the phthalazine ring.
Figure 3 Surface ESP distribution (A: LY2940680; B: 1).

Moreover, 1was re-docked back to SMO protein (PDB ID: 4jkv)
using the Glide docking program in Schrödinger2015. As shown in
Figure 4A, the carbonyl group of benzamide in the ring-opened part of
1
could mimic the hydrogen bond between phthalazine ring and R400, and
the carbonyl group of 2-trifluoromethyl-4-fluorobenzamide in 1 could
interact with N219. When 1 overlapped on the conformation of
LY2940680, a pseudo-ring structure formed by the ring-opened portion
of 1 and a good superposition of 1with LY2940680 in the pocket could be
observed (Figure 4B).
Figure 4 A: Figure of 1 docking with SMO protein; B: Plot of superposition of 1 and LY2940680 in the
binding pocket (yellow: 1, pink: LY940680).
Supported by former results, compound 1was synthesized (scheme
1
) and a dual luciferase reporter gene assay (Gli-Luciferase) was
performed to measure the inhibitory activity of the compound against the
Hhpathway. The results showed that 1 had inhibitory activity with an IC50
value of 123.40 nM, which makes it a novel lead compound although the
activity was weaker than that of the positive drug LY2940680 (IC =
5
0

4
.17 nM).
2
.2 Synthesis and inhibitory activity on the Hh pathway
To improve the inhibitory activity, new compounds were designed
and synthesized for three rounds using 1 as a lead compound. The
anthranilamide derivatives were synthesized via four different routes
described in Scheme1. In route 1, 3a and 3b were prepared by CuI
mediated coupling from compound 2. The intermediates 8a-8h were then
synthesized in three steps from compound 4 and the respective benzoic
acids 6a-6h that went through reductive amination, condensation, and
deprotection. Finally, compound 1 and 9a-9g were obtained via
condensation of 3a with 8a-8h. In route 2, compound 10 was prepared via
condensation of compound 2 and 8a. Then compounds12a-12i were
obtained from 10 and respective aniline11a-11i through a Buchward
coupling reaction. In route 3, 16a and 16b were obtained via three steps
from compound 13 with condensation, deprotection, and condensation
reactions. In route 4, compound 17 was first coupled with aniline to
obtain compound 18 followed by a condensation of 18 and 8a to obtain
compound 19.

Scheme 1. Synthetic routes. Reagents and conditions: (i) anilineor1-methyl-5-amino-1H-pyrazole, CuI,
2 3 3
K CO , DMF, 100°C, 8h, 70–72%; (ii) Methylamine, NaBH CN, MeOH, 25°C, 12h, 99%; (iii)
2 3 3
(COCl) , Et N, DCM, 25°C, 1.5h, 52–91.3%; (iv) CF COOH, DCM, 25°C, 1.5h, 85–95%; (v) EDCI,
3 2 3 2
HOBt, Et N, DCM, 25°C, 8h, 51–73%; (vi) (COCl) , Et N, DCM, 25°C, 1.5h, 86%; (vii) Pd(OAc) ,
2 3
BINAP, toluene, 100°C, 12h, 36-67%; (viii) 4-fluoro-2-(trifluoromethyl)benzoicacid, (COCl) , Et N,
3 3
DCM, 25°C, 1.5h, 95%; (ix) CF COOH, DCM, 25°C, 1.5h, 98.4%; (x) EDCI, HOBt, Et N, DCM,
2
2 3 2
5°C, 8h, 62–68%; (xi) Aniline, CuI, K CO , DMF/H O, 100°C, 8h, 94%; and (xii) EDCI, HOBt,
3
Et N, DCM, 25°C, 8h, 59%.
First, the substituents on ring A of 1 were replaced to obtain
compounds 9a-9g, and they were subjected to the dual luciferase reporter
gene assay. The activities of these compounds were greatly reduced, and
some of them even lost the inhibitory activity against the Hh pathway

(Table 1).
Table 1 Inhibitor activity of compounds 9a-9gagainst the Hh pathway (n = 2).
CH3
NH
N
N
N
N
O
O
CH3
R2
Compound
R2
4-NO
Gli-Luciferase reporter IC50(nM)
308.7 ± 73.1
9
a
2
9
b
4-OCH
4-F
3
561.9 ±185.9
>10000
9
c
9
d
4-CN
4365.5 ±1130.7
1328.0 ±131.5
1909.5 ±262.3
>10000
9
e
4-CF
4-COCH
4-SO CH
3
9
f
3
9
g
2
3
LY2940680
Vismodegib
4.56 ±0.33
13.06 ±0.44
The first round of optimization showed that the activity was
considerably reduced or even removed after the substituents of ring A of
1
were replaced. This preliminary result indicated that
-trifluoromethyl-4-fluorophenyl is the key pharmacophore. It can form
2
non-polar interactions with multiple amino acids (M301, K395, W480,
E481, F484, and P513) in the extracellular ligand-binding domain of
SMO, which makes it important to maintain the activity.
In
further
structural
modifications,
the
2
-trifluoromethyl-4-fluorophenyl structure of ring A was retained, and

ring B was replaced with different substituted benzene rings to obtain
compounds 12a-12i. These compounds were also subjected to the dual
luciferase reporter gene assay, and the results showed that the activity
was improved when the benzene ring was not substituted (IC =
5
0
3
4.09nM, which is higher than that of 1by 3-fold). The activities of the
remaining compounds containing other substituents showed a large
decline (Table 2).
Table 2 Inhibitor activity of compounds 12a-12i against the Hh pathway (n = 2).
R3
CH3
N
NH
N
O
O
CF3
F
Compound
2a
2b
2c
2d
2e
2f
2g
2h
2i
R3
Gli-Luciferase reporter IC50 (nM)
34.09 ±1.60
1
H
1
4-CN
6864 ±315.4
1
2-CN
9369.5 ±125.1
754.05 ±17.32
285.25 ±126.6
>10000
1
4-OCH
2-OCH
3
3
1
1
4-pyridyl
2-pyridyl
4-phenyl
4-COOH
1
>10000
1
4858.0 ±557.2
>10000
1
LY2940680
Vismodegib
7.64 ±0.21
21.87±1.98

Finally, the linker of the most active compounds 1 and 12a were
shortened to obtain compounds16a and 16b, and the benzene ring in ring
C of 12awasreplaced with a pyridine ring to obtain compound 19.
However, the results of the dual luciferase reporter gene assay showed
that the activities of the modified compounds were significantly
decreased (16a and 16b: IC > 10000 nM; 19: IC = 7117 nM).
5
0
50
2
.3 Molecular dynamics simulation analysis
The compounds obtained via the ring-opening strategy showed a
decrease in the inhibitory activities against the Hh pathway comparing
with LY2940680.The causes of the decline in the inhibitory activities
against the Hh pathway was next explained from a molecular perspective.
First, a molecular dynamics simulation (4 ns) of LY2940680 and
1
2awas performed using Discovery studio 2.5. Throughout the
simulation, the root-mean-square deviations (RMSD) of the system
relative to the initial structure were used to investigate the dynamic
stability of the system and to ensure the rationality of the dynamic
sampling. Figure5 shows that both systems reached equilibrium after 2
ns.

Figure 5 RMSD values of the backbone of the LY2940680-SMO complex and the 12a-SMO complex
during molecular dynamics simulations.
The dynamic trajectory in the last 1 ns of the whole simulation
process was subsequently selected and the number of hydrogen bonds
were separately calculated (Figure 6). The results showed that the average
number of hydrogen bonds in the LY2940680-SMOcomplex was 2.60,
which is more than the value of 1.50 in the 12a-SMO complex. A
conformation at every 10 ps in the last 1 ns of the dynamic trajectory was
then selected to calculate the average conformation (Figure 7).
Although12a could form hydrogen bonds with ARG400, the average
distance was 0.31 ± 0.03 nm. LY2940680 could form two hydrogen
bonds with ARG400, and the average distances were 0.21 ± 0.01 nm and
0
.27 ± 0.02 nm. In addition, 12a migrates to the lower part of SMO
causing a large spatial deviation of the entire molecule from LY2940680.
As a result, the segment of 2-trifluoromethyl-4-fluorobenzamide in 12a
cannot form a hydrogen bond with ASN219, and the activity is reduced.

Figure 6 Hydrogen bond numbers of the LY2940680-SMO complex and the 12a-SMO complex
via molecular dynamics simulations.
Figure 7Average conformations of (A) LY2940680-SMO complex and (B) 12a-SMO complex.
The binding free energy ΔG of LY2940680 and 12awasalso
calculated using QM-Polarized Ligand Docking (QPLD) combined with
Prime-MM/GBSA (Table 3).
ΔGbind = Gcomplex − (Gprotein+ Gligand) = ΔGMM + ΔGsol
TΔS,whereΔGMM = ΔG + ΔG , ΔG = ΔG + ΔGnonpolar
−
.
ele
vdW
sol
polar
The calculation results show that the binding free energies of
LY2940680 and 12awere negative (the influence of entropy change was
ignored here). This is due to several reasons. First, whenthe ring in
LY2940680 is opened, there will inevitably be an increase in entropy
depletion for the binding process to compensate for increasing degree of

freedom caused by multiple conformations—this increases the ΔG.[15]
Second, the current entropy change calculation is greatly affected by the
definition of degree of freedom. There are high requirements for the
computing capability of computers—especially when macromolecules are
involved; thus, the precision is relatively low. Therefore, the influence of
entropy change was ignored. This result indicated that both LY2940680
and 12a could interact strongly with SMO. The binding free energy of
LY2940680 was lower than that of 12a indicating that LY2940680 has a
higher affinity for the SMO binding pocket, which also explains the
difference between the activity of 12a and that of LY2940680. This study
may provide additional references for designing SMO inhibitors using
computational principles in the future.
Table 3 Calculation of binding free energy via the MM/GBSA method.
LY2940680(kJ/mol)
-75.416
12a(kJ/mol)
-72.455
-38.807
54.364
ΔEvdw
ΔEele
-43.737
ΔGpolor
ΔGnonpolor
ΔGbind
55.188
11.769
11.910
-52.196
-44.988
2
.4 Proliferation inhibitory activity
Compounds 1, 9a, 12a, and 12e with relatively strong inhibitory
activity against the Hh pathway were further selected to measure their in
vitro proliferation inhibitory activities. Since the Hh pathway is activated

[16]
in medulloblastoma , the human medulloblastoma cell line Daoy was
chosen for in vitro anti-proliferative activity tests. The results are shown
in Table 4. The results showed that the tested compounds exhibited strong
proliferation inhibitory activities towards Daoy cells with IC values
5
0
ranging from 0.40 μM to 0.87 μM; compounds 1, 12a, and 12edisplayed
better activity than the positive control LY2940680.
In comparison, the anti-proliferative activities of these compounds
towards the human melanoma cell line A2058 were also investigated.
This cell line has been known for the over-activation of the MAP kinase
pathway resulting from the BRAF V600E mutation [17]. The results
showed that all the tested compounds displayed very poor inhibitory
activities with IC values more than 50 μM. This indicates that these
5
0
potential SMO inhibitors may selectively inhibit the proliferation of the
cancer cells that depend on the Hh pathway.
Table 4Anti-proliferative activities of the tested compounds on the human medulloblastoma cell line
Daoy (n = 3).
Compound
IC50 (μM)
0.51 ± 0.05
0.87 ± 0.09
0.48 ± 0.05
0.40 ± 0.02
0.79 ± 0.06
1
9
a
1
2a
2e
LY2940680
1
3
. Conclusion
Mutations of SMO, like SMO (D473H) mutant, can result in drug

resistance in clinical treatment. LY2940680 is one of the few drugs that
are active to both wild-type and D473H mutant SMO. In this work, a
novel compound 1with similar electrical property to LY2940680 was
designed by a ring-opening strategy based on the binding mode of
LY2940680 and SMO. The phthalazine ring in LY2940680was replaced
with anthranilamide. The Glide docking study suggested that the
anthranilamide in 1 can form a pseudo-ring structure and its carbonyl
group can mimic the hydrogen bond between the phthalazine ring and
R400 of SMO, which supported this design. As expected, compound 1
displayed a moderate inhibitory activity towards the Hh pathway (IC =
5
0
1
23.40 nM). To improve the activity, three rounds of structural
modification have been made, and compound 12a displayed the most
promising activity with an IC value of34.09nM, which is still less active
5
0
than LY2940680 (IC = 7.64 nM).The weaker hydrogen bond with SMO
5
0
and higher binding free energy might be responsible for the lower activity
as supported by the molecular dynamics analysis. Compound 12a
exhibited a higher proliferation inhibitory activity towards the human
medulloblastoma cell line Daoy than LY2940680, indicating an
alternative mechanism of the anticancer action. Studies into the
mechanism are ongoing.
4
. Experimental Section
4
.1 Chemistry

4
.1.1 General
Unless otherwise stated, all reagents were purchased from commercial
1
13
suppliers and used without purifications. H NMR and C NMR spectra
were obtained on a Bruker AV-300 nuclear instrument in CDCl or
3
DMSO-d using TMS as internal standard, operating at 300 MHz and 75
6
MHz, respectively. Chemical shifts (δ) are expressed in ppm and
coupling constants J are given in Hz. Analytical thin layer
chromatography (TLC) was performed on pre-coated, glass-backed silica
gel plates. Visualization of the developed chromatogram was performed
by UV absorbance (254 nm). High resolution mass spectra (HRMS) were
obtained on a Waters Q-TOF micro TM apparatus. Melting points were
measured with an RY-I melting point apparatus.
4
.1.2Synthesis
2
-((1-Methyl-1H-pyrazol-5-yl) amino) benzoic acid (3a)
To a mixture of 2-iodobenzoic acid (5.00 g, 20.00 mmol),
-methyl-5-amino-1H-pyrazole (1.90 g, 20.00 mmol) and Potassium
1
carbonate (5.50 g, 40.00 mmol) in 20 mL DMF were added CuI (0.76 g,
o
4
.00 mmol). The reaction mixture was stirred at 105 C for 8 h. After
cooling to room temperature the mixture was poured into 60 ml water and
the pH was adjusted to 5-6 using 1N HCl. The formed precipitates were
filtrated and dried to obtained black solid. Yield: 70%, m.p. 134~139
o
1
C. H-NMR (300 MHz, CDCl ) δ (ppm):10.77-8.78 (m, 2H, NH, ArH),
3

8
.14-7.30(m, 3H, ArH), 6.89 (s, 1H, ArH), 6.46(s, 1H, ArH), 3.85(s, 3H,
CH3).
-(Phenylamino) benzoic acid (3b)
2
To a mixture of 2-iodobenzoic acid (5.00 g, 20.00 mmol), Aniline (1.86
g, 20.00 mmol) and Potassium carbonate (5.50 g, 40.00 mmol) in 20 mL
DMF were added CuI (0.76 g, 4.00 mmol).The reaction mixture was
o
stirred at 105 C for 8 h. After cooling to room temperature the mixture
was poured into 60 mL water and the pH was adjusted to 4~5 using 1N
HCl.The formed precipitates were filtrated and dried to obtained yellow
o
1
solid. Yield: 72%,m.p. 180~182 C. H-NMR (300 MHz, CDCl ) δ (ppm):
3
9
.31 (brs, 1H, COOH), 8.05 (d, J = 8.10 Hz, 1H, ArH), 7.40-7.33 (m, 3H,
ArH), 7.29-7.21 (m, 3H, ArH), 7.16-7.11 (m, 1H, ArH), 6.78-6.73 (m,
H, ArH).
1
Tert-butyl-4-(methylamino) piperidine-1-carboxylate (5)
A mixture of tert-butyl 4-oxopiperidine-1-carboxylate (1.00 g, 5.00
mmol) Methylamine (10.00 mL) in 5mlMeOH was adjusted to pH=5-6
with acetic acid and stirred under room temperature for 1 h. Then cooling
o
to 0 C and NaBH CN (0.48 g, 7.60 mmol) was added portion wise. The
3
mixture was stirred under room temperature for 12h until the reaction was
complete. The methanol was evaporated under reduced pressure then the
pH was adjusted to 9 with 1N NaOH. The mixture was extracted with
DCM, dried with Sodium sulphate. The solvent was evaporated to obtain

1
the yellow oil, Yield: 100%. H-NMR (300 MHz, CDCl3) δ (ppm): 3.98
(
d, J = 10.20 Hz, 2H, Boc-NCH ), 2.74 (t, J = 12.20 Hz, 2H, BocNCH ),
2
2
2
2
.48-2.43(m, 1H, NHCH), 2.38 (s, 3H, NHCH ), 1.79 (d, J = 12.20 Hz,
3
H, NHCHCH ), 1.39 (s, 9H, Boc-H), 1.17 (dd, J = 20.20, 10.20 Hz, 2H,
2
NHCHCH2).
General procedures for the synthesis of 7a-7h
The purchased acids (14.40 mmol) was added into 5 mL DCM and
o
cooled to 0 C.After the oxalyl chloride (1.50 mL,17.30 mmol) was added
drop wise, the mixture was removed to room temperature and stirred for
3
0min. The solvent was evaporated for use.
To a cooled solution of 5 (3.70 g，17.30 mmol) in 20 mL DCM was added
Et N (3.00 mL，21.60 mmol) then the prepared chloride was added drop
3
wise. The mixture was stirred at room temperature for 1 h. The solvent
was evaporated under reduced pressure and the residue was purification
by column chromatography with dichloromethane/ methanol as eluent on
silica gel to give the derivatives7a-7h.
Tert-butyl-4-(4-fluoro-N-methyl-2-(trifluoromethyl)
benzamido)
piperidine-1-carboxylate (7a)
1
Colorless oil, yield: 91.3%. Major rotamer: H-NMR (300 MHz, CDCl )
3
δ (ppm): 7.44-7.39 (m, 1H, ArH), 7.32-7.29 (m, 2H, ArH), 4.73 (m, 1H,
CH3NCH), 4.25 (m, 2H, BocNCH ), 2.88-2.82 (m, 2H, BocNCH ), 2.63
2
2
(
s, 3H, NCH ), 1.79-1.63 (m, 4H, CH N(CH ) ), 1.46 (s, 9H,
3
3
2 2

1
Boc-H).Minor rotamer: H-NMR (300 MHz, CDCl ) δ (ppm): 7.44-7.39
3
(m, 1H, ArH), 7.32-7.29 (m, 2H, ArH), 4.15-4.11 (m, 2H, BocNCH2),
3
.23 (s, 1H, CH NCH), 2.98 (s, 3H, NCH ), 2.51-2.35 (m, 2H,
3
3
BocNCH ), 1.79-1.63 (m, 4H, CH N(CH ) ), 1.43 (s, 9H, Boc-H).
2
3
2 2
Tert-butyl-4-(N-methyl-4-nitrobenzamido) piperidine-1-carboxylate
7b)
(
o
1
Yellow solid, yield: 82%, m.p. 152~153 C. H-NMR (300 MHz, CDCl )
3
δ (ppm): 8.31 (d, J = 7.90 Hz, 2H, ArH), 7.58 (d, J = 7.20 Hz, 2H, ArH),
4
.72 (s, 1H, CONCH), 4.26 (brs, 2H, CONCHCH ), 3.01 (brs, 2H,
2
CONCHCH ), 1.77 (s, 3H, CONCH ), 1.49 (brs, 4H, BocN(CH ) ),
2
3
2 2
1
.49(s, 9H, Boc-H).
Tert-butyl-4-(4-methoxy-N-methylbenzamido)piperidine-1-carboxyla
te (7c)
o
1
White solid, yield: 88%, m.p. 116~117 C. H-NMR (300 MHz, CDCl ) δ
3
(ppm):7.39 (d, J = 8.50 Hz, 2H, ArH), 6.94 (d, J = 8.60 Hz, 2H, ArH),
4
.62 (brs, 1H, CONCH), 4.25-4.21 (m, 2H, CONCHCH ), 3.87 (s, 3H,
2
OCH ), 2.91 (s, 3H, NCH ), 2.77 (brs, 1H, ONCHCH ), 1.73 (brs, 4H,
3
3
2
BocN(CH ) ), 1.51 (s, 9H, Boc-H).
2
2
Tert-butyl-4-(4-fluoro-N-methylbenzamido) piperidine-1-carboxylate
7d)
Yellow oil, yield: 76%. H-NMR (300 MHz, CDCl ) δ (ppm):7.53-7.34
(
1
3
(m, 2H, ArH), 7.15-7.08 (m, 2H, ArH), 4.67 (s, 1H, CONCH), 4.23 (s,

2
1
H, CONCHCH ), 3.27-3.00 (m, 2H, CONCHCH ), 2.88 (s, 3H, NCH ),
2
2
3
.72 (brs, 4H, ONCHCH ), 1.49 (s, 9H, Boc-H).
2
Tert-butyl-4-(4-cyano-N-methylbenzamido) piperidine-1-carboxylate
7e)
White solid, yield: 71%, m.p. 120~121 C. H-NMR (300 MHz, CDCl ) δ
(
o
1
3
(ppm):7.76 (d, J = 8.00 Hz, 2H, ArH), 7.51 (d, J = 7.30 Hz, 2H, ArH),
4
.71 (s, 1H，CONCH), 4.27 (brs, 2H, CONCHCH ), 3.00-2.89 (m, 1H,
2
CONCHCH ), 2.81 (s, 3H, NCH ), 2.51 (s, 1H, CONCHCH ), 1.77-1.71
2
3
2
(
m, 4H, BocN(CH ) ), 1.50 (s, 9H, Boc-H).
2
2
Tert-butyl-4-(N-methyl-4-(trifluoromethyl)benzamido)
piperidine-1-carboxylate (7f)
o
1
White solid, yield: 70%, m.p. 122~123 C. H-NMR (300 MHz, CDCl ) δ
3
(ppm):7.71 (d, J = 8.00 Hz, 2H, ArH), 7.52 (d, J = 7.60 Hz, 2H, ArH),
4
.73 (s, 1H, CONCH), 4.27 (s, 2H, CONCHCH ), 3.00 (s, 1H,
2
CONCHCH ), 2.81 (s, 3H, NCH ), 2.52 (s, 1H, CONCHCH ), 1.77 (brs,
2
3
2
4
H, BocN(CH ) ), 1.49 (s, 9H, Boc-H).
2
2
Tert-butyl-4-(4-acetyl-N-methylbenzamido) piperidine-1-carboxylate
7g)
(
o
1
Yellow solid, yield: 52%, m.p. 101~102 C. H-NMR (300 MHz, CDCl )
3
δ (ppm):8.02 (d, J = 8.10 Hz, 2H, ArH), 7.48 (d, J = 7.60 Hz, 2H, ArH),
4
.72 (s, 1H, CONCH), 4.25 (brs, 2H, CONCHCH ), 2.94-2.89 (m, 1H,
2
CONCHCH ), 2.80 (s, 3H, NCH ), 2.64 (s, 3H, COCH ), 2.50-2.43 (m,
2
3
3

1
H, CONCHCH ), 1.76 (brs, 4H, BocN(CH ) ), 1.48 (s, 9H, Boc-H).
2
2 2
Tert-butyl-4-(N-methyl-4-(methylsulfonyl)
piperidine-1-carboxylate (7h)
benzamido)
o
1
White solid, yield: 69%, m.p. 196~197 C. H-NMR (300 MHz, CDCl ) δ
3
(ppm):8.01 (d, J = 8.10 Hz, 2H, ArH), 7.57 (d, J = 7.40 Hz, 2H, ArH),
4
.69 (m, 1H, CONCH), 4.44-4.00 (m, 2H, CONCHCH ), 3.07 (s, 3H,
2
SO CH ), 3.03-2.81 (m, 2H, CONCHCH ), 2.77 (s, 3H, NCH ), 1.85-1.60
2
3
2
3
(
m, 4H, BocN(CH ) ), 1.46 (s, 9H, Boc-H).
2
2
General procedures for the synthesis of 8a-8h
To a solution of 7a-7h (10.70 mmol) in 20 mL DCM was added 5 mL
TFA the stirred at room temperature for 1h. The solvent was evaporated
under reduced pressure and the residue was used without further
purification.
Tert-butyl
4-(4-fluoro-2-(trifluoromethyl)
benzoyl)
piperazine-1-carboxylate (14)
4
-Fluoro-2-(trifluoromethyl)benzoic acid (3.00 g, 14.40 mmol) was added
o
into 5 mL DCM and cooled to 0 C.After the oxalyl chloride (1.50 mL,
1
7.30 mmol) was added drop wise, the mixture was removed to room
temperature and stirred for 30min. The solvent was evaporated for use.
To a cooled solution of tert-butyl piperazine-1-carboxylate (3.20 g，17.30
mmol) in 20 mL DCM was added Et N (3.00 Ml，21.60 mmol) then the
3
prepared chloride was added drop wise. The mixture was stirred at room

temperature for 1 h. The solvent was evaporated under reduced pressure
and the residue was purification by column chromatography on silica gel
to give the colorless oil, yield: 95%.
(
4-Fluoro-2-(trifluoromethyl) phenyl) (piperazin-1-yl) methanone
15)
(
To a solution of 14(5.13 g, 13.60 mmol) in 20 mL DCM was added 5 ml
TFA the stirred at room temperature for 1h. The solvent was evaporated
under reduced pressure and the residue was purification by column
chromatography on silica gel to give the yellow oil, yield: 98.4%.
1
H-NMR (300 MHz, CDCl ) δ (ppm):7.37 (dd, J = 8.70, 1.90 Hz, 1H,
3
ArH), 7.33-7.20 (m, 2H, ArH), 3.94-3.49 (m, 2H, piperazine-H), 3.10 (t, J
=
5.00 Hz, 2H, piperazine-H), 2.88 (t, J = 5.10 Hz, 2H, piperazine-H),
2
.80-2.59 (m, 2H, piperazine-H).
4
-Fluoro-N-(1-(2-iodobenzoyl)
piperidin-4-yl)-N-methyl-2-(trifluoromethyl) ben-zamide (10)
2
-Iodobenzoic acid(2.00 g, 8.00 mmol) was added into 10 mL DCM and
o
cooled to 0 C.After the oxalyl chloride (0.80 mL，9.60 mmol) was added
drop wise, the mixture was removed to room temperature and stirred for
3
0min. The solvent was evaporated for use.
To a cooled solution of 8a (2.40 g, 8.00 mmol) in 20 mL DCM was added
Et N (1.70 mL, 12.00 mmol) then the prepared chloride was added drop
3
wise. The mixture was stirred at room temperature for 1 h. The solvent

was evaporated under reduced pressure and the residue was purification
by column chromatography with dichloromethane/ methanolas eluent on
o
silica gel to give the yellow solid, yield: 86%, m.p. 85~86 C. Major
1
rotamer: H-NMR (300 MHz, CDCl ) δ (ppm): 7.91-7.81 (m, 1H, ArH),
3
7
.45-7.36 (m, 2H, ArH), 7.36-7.28 (m, 2H, ArH), 7.24-7.17 (m, 1H,
ArH), 7.14-7.02 (m, 1H, ArH), 5.00-4.75 (m, 1H, CH NCH), 4.23 (brs,
3
2
H, NCHCH ), 3.07 (br, 2H, NCHCH ), 2.58(s, 3H, CH ), 1.75 (m, 4H,
2
2
3
1
CON(CH ) ). Minor rotamer: H-NMR (300 MHz, CDCl ) δ (ppm):
2
2
3
7
.91-7.81 (m, 1H, ArH), 7.45-7.36 (m, 2H, ArH), 7.36-7.28 (m, 2H,
ArH), 7.24-7.17 (m, 1H, ArH), 7.14-7.02 (m, 1H, ArH), 4.23 (brs, 2H,
NCHCH ), 3.38(m, 1H, CH NCH), 3.02 (s, 3H, CH ), 2.41(brs, 2H,
2
3
3
NCHCH ), 1.75 (m, 4H, CON(CH ) ).
2
2 2
2
-(Phenylamino) nicotinic acid (18)
To a mixture of 2-iodonicotinic acid (5.00 g, 20.00 mmol), aniline (1.86
g, 20.00 mmol) and Potassium carbonate (5.50 g, 40.00 mmol) in 20 mL
DMF were added CuI (0.76 g, 4.00 mmol). The reaction mixture was
o
stirred at 105 C for 8 h. After cooling to room temperature the mixture
was poured into 60 mL water and the pH was adjusted to 5~6 using 2N
HCl. The formed precipitates were filtrated and dried to obtained yellow
o
1
solid, yield: 94%, m.p. 183~184 C. H-NMR (300 MHz, CDCl ) δ
3
(ppm):10.08 (s, 1H, COOH), 8.49-8.37 (m, 1H, ArH), 8.33 (d, J = 7.70
Hz, 1H, ArH), 7.64 (d, J = 7.90 Hz, 2H, ArH), 7.36 (t, J = 7.40 Hz, 2H,

ArH), 7.10 (t, J = 7.40 Hz, 1H, ArH), 6.88-6.57 (m, 1H, ArH), 5.38(brs,
H, NH).
1
General procedure for the synthesis of 1, 9a-9g, 16a, 16b, 19
To a mixture of3a(500.00 mg, 2.30 mmol) and 8a-8h, 15 (2.3 mmol) in
2
0 mL DCM was added HoBt (372.00 mg, 2.76 mmol) then EDCI
(530.00 mg, 2.76 mmol) was added portion wise. After the reaction was
complete, solvent was evaporated under reduced pressure and the residue
was purification by column chromatography with dichloromethane/
methanol as eluent on silica gel to give the compounds 1, 9a-9g, 16a,
1
6b, 19.
N-methyl-N-(1-(2-((1-methyl-1H-pyrazol-5-yl)
piperidin-4-yl)-2-trifluoromethyl-4-fluoro-benzamide (1)
amino)
benzoyl)
o
1
Green solid, yield: 64%, m.p. 206~207 C. Major rotamer: H-NMR (300
MHz, CDCl ) δ (ppm): 7.47-7.41(m, 2H, ArH), 7.34-7.29 (m, 3H, ArH),
3
7
5
3
.27-7.12 (m, 2H, ArH, NH), 6.91-6.75 (m, 2H, ArH), 6.06(s, 1H, ArH),
.00-4.81 (m, 1H, CH NCH), 4.80-4.12 (m, 2H, CON(CH ) ), 3.72 (s,
3
2 2
H, CH ), 3.29-3.03 (m, 2H, CON(CH ) ), 2.67(s, 3H, CH ), 1.84 (m, 4H,
3
2 2
3
1
NCH(CH ) ).Minor rotamer: H-NMR (300 MHz, CDCl ) δ (ppm):
2
2
3
7
6
.47-7.41(m, 2H, ArH) 7.34-7.29 (m, 3H, ArH), 7.27-7.12 (m, 2H, ArH),
.91-6.75 (m, 2H, ArH), 6.06(s, 1H, ArH), 4.80-4.12 (m, 2H,
CON(CH ) ), 3.70 (s, 3H, CH ), 3.43-3.60 (m, 1H, CH NCH), 3.02 (s,
2
2
3
3
3
H, CH ), 2.64-2.55 (m, 2H, CON(CH ) ), 1.84 (m, 4H,
3
2 2