Exploration of the linkage elements of porcupine antagonists led to potent Wnt signaling pathway inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.09.048

The Wnt signaling pathway is a pivotal developmental pathway. It operates through control of cellular functions such as proliferation, differentiation, migration and polarity. Aberrant Wnt signaling has been implicated in the formation and metastasis of tumors. Porcupine is a component of the Wnt signaling pathway. It is a member of the membrane-bound O-acyltransferase family of proteins. Porcupine catalyzes the palmitoylation of Wnt proteins, a process which is essential to their secretion and activity. Here we report a novel series of compounds obtained by a scaffold hybridization strategy from two known porcupine inhibitor classes. The leading compound 62 demonstrated subnanomolar (IC₅₀ 0.11 nM) inhibition of Wnt signaling in a paracrine cellular reporter gene assay. Compound 62 also potently inhibited Wnt secretion into culture medium, an indication of direct inhibition of the porcupine protein. Furthermore, compound 62 showed excellent chemical, plasma and liver microsomal stabilities. Collectively, these results strongly support further optimization of this novel scaffold to develop better Wnt pathway inhibitors.

Full text of DOI:10.1016/j.bmc.2015.09.048

Accepted Manuscript
Exploration of the Linkage Elements of Porcupine Antagonists Led to Potent
Wnt Signaling Pathway Inhibitors
Yan Dong, Kehuang Li, Zhixiang Xu, Haikuo Ma, Jiyue Zheng, Zhilin Hu,
Sudan He, Yiyuan Wu, Zhijian Sun, Lusong Luo, Jiajun Li, Hongjian Zhang,
Xiaohu Zhang
PII:
S0968-0896(15)30073-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.09.048
BMC 12595
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 September 2015
29 September 2015
30 September 2015
Please cite this article as: Dong, Y., Li, K., Xu, Z., Ma, H., Zheng, J., Hu, Z., He, S., Wu, Y., Sun, Z., Luo, L., Li,
J., Zhang, H., Zhang, X., Exploration of the Linkage Elements of Porcupine Antagonists Led to Potent Wnt Signaling
Pathway Inhibitors, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.09.048
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Exploration of the Linkage Elements of Porcupine Antagonists Led to Potent Wnt Signaling
Pathway Inhibitors
Yan Dong a,1, Kehuang Li a,1, Zhixiang Xu a, Haikuo Ma a, Jiyue Zheng a, *, Zhilin Hu b, Sudan He b,
Yiyuan Wu c, Zhijian Sun c, Lusong Luo c,*, Jiajun Li a, Hongjian Zhang a, and Xiaohu Zhang a,*
Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and
a
College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215021, P. R. China
b Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, and
Collaborative Innovation Center of Hematology, Soochow University, Suzhou 215123, P.R. China
c BeiGene (Beijing) Co., Ltd., No. 30 Science Park Road, Zhongguancun Life Science Park, Beijing
102206, P.R.China
1 these authors contributed equally to this paper
*
Corresponding author. Tel.: +86 512 65880380; fax: +86 512 65880380; email:
xiaohuzhang@suda.edu.cn (X.H. Zhang); lusong.luo@beigene.com (L.S. Luo); jyzheng@suda.edu.cn
(J.Y. Zheng)
Abstract: The Wnt signaling pathway is a pivotal developmental pathway. It operates through control
of cellular functions such as proliferation, differentiation, migration and polarity. Aberrant Wnt
signaling has been implicated in the formation and metastasis of tumors. Porcupine is a component of
the Wnt signaling pathway. It is a member of the membrane-bound O-acyltransferase family of
proteins. Porcupine catalyzes the palmitoylation of Wnt proteins, a process which is essential to their
secretion and activity. Here we report a novel series of compounds obtained by a scaffold hybridization
strategy from two known porcupine inhibitor classes. The leading compound 62 demonstrated
subnanomolar (IC50 0.11 nM) inhibition of Wnt signaling in a paracrine cellular reporter gene assay.
Compound 62 also potently inhibited Wnt secretion into culture medium, an indication of direct
inhibition of the porcupine protein. Furthermore, compound 62 showed excellent chemical, plasma and
liver microsomal stabilities. Collectively, these results strongly support further optimization of this
novel scaffold to develop better Wnt pathway inhibitors.
Keywords: Wnt signaling pathway, porcupine, antagonist, scaffold hybridization, cancer therapy
Abbreviations: CYP, cytochrome P450; DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine;
DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DSH, Dishevelled; HPLC, high
performance liquid chromatography; m.p., melting point; N-G-L, NIH3T3-GRE-Luc reporter gene
assay; PBST, PBS buffer supplied with 0.05% Tween-20; P.K., pharmacokinetics; PPA,
polyphosphoricacid; PPACA, propylphosphonic anhydride; SAR, structure-activity relationship; SEM,
standard error measurement; SGF, simulated gastric fluid; sHh, sonic hedgehog; Smo, Smoothened;
STF, super-top flash.
1

1. Introduction
The Wnt signaling pathway is an essential developmental pathway which functions through
regulation of cell patterning, growth and migration during embryonic development. In canonical Wnt
signaling, β-catenin is degraded by a β-catenin destruction complex in cells without Wnt ligands
stimulation, resulting in the inhibition of β-catenin mediated gene expressions. In contrast, in cells with
Wnt ligands stimulation, binding of Wnt ligands to the Frizzled and LRP families of cell surface
receptors initiates a series of signaling events with the help from the cytoplasmic protein Dishevelled
(DSH), leading to the accumulation of cytoplasmic β-catenin. Ultimately, β-catenin translocates to the
nucleus, where it associates with the LEF/TCF family of DNA-binding proteins, resulting in the
activation of β-catenin mediated gene expressions. Aberrant Wnt signaling has been linked to the
formation and metastasis of numerous tumors.1-3 Porcupine, a member of the membrane-bound
O-acyltransferase family of proteins, is an important component of the Wnt signaling pathway by
catalyzing the palmitoylation of Wnt proteins, a process which is essential for their secretion and
activity. Inhibition of porcupine can lead to the inhibition of aberrant Wnt signaling, and therefore
represents a promising approach for anticancer therapy.4-6
The first porcupine inhibitors reported in the literature7,8 were the IWP series of compounds
(Figure 1). Since then, other porcupine inhibitors have also been developed.4-6 The breakthrough was
made by Novartis in 2012 when LGK974, a potent porcupine inhibitor (Figure 1), was advanced into a
phase I clinical trial.9 Furthermore, recent research activities including the publications of a
pharmacophoric model10 and a lead optimization campaign11 greatly enhanced the knowledge of
porcupine antagonist field; underscore the heightened interests in pursuit of potent Wnt signaling
pathway inhibitors (Figure 1).
Figure 1. Reported porcupine inhibitors in the literature
2. Design
Scientists from the Genomics Institute of the Novartis Research Foundation detailed their
2

porcupine inhibitor optimization campaigns in two recently published patents.12,13 The results are
summarized in Figure 2. Compound GNF-1 is the phenotypical lead and the optimization campaign
culminated in the identification of LGK974, a compound which entered clinical development.9 IWP-2
was among the first reported phenotypical porcupine inhibitors by scientists from the University of
Texas Southwestern Medical Center.8 The porcupine inhibition activity was further improved when the
biphenyl element was introduced on the right hand side (Figure 2, pink color).6 We surmised that the
biphenyl element on the left hand side (blue color) might bind to porcupine in the same area where the
fatty acid chain of palmitic acid binds. The central amide, whether it consists of a 4-atom unit (such as
in IWP-2, red color), or a 3-atom unit (such as in LGK974), serves as a linkage element. With this
assumption in mind, we hypothesized that compounds based on the phenotypical structure shown in the
green frame in Figure 2 should also be potent porcupine inhibitors. Here we report the synthesis and
structure-activity-relationship of novel potent porcupine inhibitors featuring a framework as shown in
Figure 2. We were encouraged that our hypothesis was validated by two very recently published papers,
albeit the same conclusion was drawn based on a high throughput screen hit followed by an
optimization campaign (Figure 1).10,11
Figure 2. The evolution of LGK974 and the hybridization strategy for the novel porcupine inhibitors
3. Chemistry
Synthesis of the target compounds 13-15 is outlined in Scheme 1. Commercially available
4-bromophenol was reacted with dimethoxymethane in the presence of P2O5 to give intermediate 2.
Borylation of compound 3 afforded aryl boronate 4, which was coupled with bromide 2 to give
intermediate 5. Removal of MOM of intermediate 5 with 1 N HCl provided phenol 6. The amide
coupling was carried out by treatment of aromatic amines 7-9 with chloroacetic chloride in the
presence of Et3N in DCM, which led to compounds 10-12. Displacement of the chloro group of
compounds 10-12 with phenol 6 provided the desired compounds 13-15, respectively.
3

Scheme 1. Reagents and conditions. (i) CH2(OCH3)2, P2O5, DCM, r. t., 12 h; (ii) bis(pinacolato)diboron,
KOAc, Pd(dppf)Cl2, THF, 80°C, 15 h; (iii) Pd(PPh3)4, Cs2CO3, THF, 75°C, 12 h; (iv) 1 N HCl, r. t., 16
h; (v) ClCH2COCl, Et3N, DCM, r. t., overnight; (vi) 6, KI, K2CO3, DMF, 60°C, overnight.
Scheme 2 shows the synthetic approaches to the target compounds 19-21. Nucleophilic
substitution of intermediate 12 with 3-bromophenol, 3-bromoaniline or 3-bromobenzenethiol afforded
compounds 16-18, respectively. Suzuki coupling of compounds 16-18 with aryl boronate 4 gave the
desired compounds 19-21.
Scheme 2. Reagents and conditions. (i) 3-bromophenol, KI, K2CO3, DMF, 60°C, overnight; (ii)
3-bromoaniline, Bu4NI, DIPEA, toluene, 110°C, overnight; (iii) 3-bromobenzenethiol, K2CO3, acetone,
60°C, overnight; (iv) 4, Pd(dppf)Cl2CH2Cl2, dppf, K3PO4, 1,4-dioxane, 105°C, overnight.
The synthetic pathways used to prepare the key intermediates 26, 27, 31 and 37 are summarized in
Scheme 3. Methylation of indazole 22 with CH3I provided intermediate 23. Compounds 22 and 23
were reduced with H2 and Pd/C to give aromatic amines 24 and 25, which were converted to
intermediates 26 and 27 in the presence of NaNO2 and H2SO4 at 120°C. Commercially available
phenol 28 was protected with THP to give intermediate 29. Cyclization of intermediate 29 with
N-hydroxyacetamide gave compound 30. Deprotection of 30 using 1 N HCl yielded intermediate 31.
Reaction of amine 32 with ethyl chlorocarbonate gave compound 33. Cyclization of 33 with PPA at
120°C afforded amide 34, which was then reduced with LiAlH4 in THF to give amine 35. Phenol 36
was achieved as an HCl salt by demethylation with boron tribromide of amine 35. Compound 36 was
reacted with (CH3CO)2O in the presence of NaHCO3 to give intermediate 37.
4

Scheme 3. Reagents and conditions. (i) NaH, DMF, 0°C, 1 h, CH3I, DMF, r. t., 18 h; (ii) Pd/C, H2,
MeOH, r. t., overnight; (iii) NaNO2, H2SO4/H2O, 120°C, 2 h; (iv) DHP, PPTS, DCM, r. t., 16 h; (v)
N-hydroxyacetamide, t-BuOK, DMF, r. t., 24 h; (vi) 1 N HCl, MeOH, r. t., 1 h; (vii) ClCOOEt, Et3N,
DCM, r. t., 1.5 h; (viii) PPA, 120°C, 1.5 h; (ix) LiAlH4, THF, 60°C, overnight; (x) BBr3, DCM, -78°C -
r. t., overnight; (xi) (CH3CO)2O, NaHCO3, H2O, 70°C, 4 h.
Nucleophilic substitution of intermediate 12 with the synthetic intermediates 26, 27, 31, 37 or
other commercially available phenols (ArOHs) gave the final compounds 38-49, respectively (Scheme
4).
Scheme 4. Reagents and conditions. (i) ROH, KI, K2CO3, DMF, 60°C, overnight; (ii) RNH2, Bu4NI,
DIPEA, DMF, 75°C, overnight.
Target compounds 54, 55 and 58 were prepared via the synthetic route depicted in Scheme 5.
Commercially available 1-bromo-4-nitrobenzene was coupled with aryl boronate 4 to give
nitrobenzene 51, which was subsequently reduced with H2 and Pd/C to give amine 52. Methylation of
amine 52 with Me2SO4 provided compound 53. Compounds 52 and 53 were reacted with intermediate
12 in the presence of DIPEA and Bu4NI to give the desired compounds 54 and 55. Suzuki coupling of
compound 3 with 4-(methylthio)phenylboronic acid gave compound 56. Thiophenol 57 was prepared
through oxidation of compound 56 with m-CPBA, followed by a Pummerer rearrangement. Finally,
treatment of thiophenol 57 with intermediate 12 under standard substitution conditions produced the
5

desired compound 58.
Scheme 5. Reagents and conditions. (i) 4, Pd(dppf)Cl2, K2CO3, dioxane/H2O, 100°C, 12 h; (ii) Pd/C,
H2, MeOH, r. t., overnight; (iii) TFAA, DCM, 0°C, 45 min, Bu4NBr, Me2SO4, 50% NaOH, r. t., 16 h;
(iv) 12, Bu4NI, DIPEA, DMF, 75°C, overnight; (v) 4-(methylthio)phenylboronic acid, KOH, Bu4NBr,
Pd(PPh3)4, DME/H2O, 80°C, overnight; (vi) a) m-CPBA, DCM, r. t., 3 h; b) TFAA, DCM, 0°C, 2 h; c)
Et3N, EtOH, r. t., 1 h; (iv) 12, Na2CO3, CH3CN, reflux, 6 h.
As shown in Scheme 6, target compounds 60 and 62 were prepared from aromatic amine 9.
Condensation of 3-(4-bromophenyl)propanoic acid with the aromatic amine 9 in the presence of
PPACA and Et3N in THF gave intermediate 59, which was coupled with aryl boronate 4 to afford the
desired compound 60. The amide coupling was carried out by treatment of the aromatic amine 9 with
2-chloropropionyl chloride in the presence of Et3N, which led to intermediate 61. Subsequently, the
chlorine atom in 61 was displaced by phenol 6 to provide the desired compound 62.
Scheme 6. Reagents and conditions. (i) 3-(4-bromophenyl)propanoic acid, PPACA, Et3N, THF, 60°C,
overnight; (ii) 4, Pd(dppf)Cl2, dppf, K3PO4, dioxane/H2O, 108°C, overnight; (iii) CH3CHClCOCl, Et3N,
DCM, r. t., overnight; (iv) 6, KI, K2CO3, DMF, 60°C, overnight.
4. Results and discussion
4.1. Evaluation of pharmacological activity
A cell based super-top flash (STF) reporter gene assay was used to test Wnt signaling inhibition of
the synthesized compounds. This assay was based on a two-cell paracrine signaling system: Wnt
generating L Wnt3A cells were co-cultured with HEK293 cells which were stably transfected with
“super-top flash” TCF-luciferase reporter plasmid (the Wnt responding cells). The inhibition of the
reporter gene luciferase signaling was used to evaluate the potency of the compound. This is the
primary assay we used to screen compounds.14 We first confirmed that LGK974 was active in our assay,
6

and the IC50 (0.57 nM, Table 2) matched well with the number (0.38 nM) reported in the literature.5,12
LGK974 was used as a control in all of our assays. The structure-activity relationship (SAR) is
summarized in Table 1 and Table 2.
When the 3-atom phenotypical GNF-1 linkage was replaced by the 4-atom oxygen containing
linkage element, a potent Wnt inhibitor was obtained (compound 13, 5.0 nM). Replacement of a carbon
with a nitrogen on the terminal ring of the right hand side further improved potency (compound 14, 2.2
nM). When the optimized LGK974 element was installed on the right hand side, compound 15 (0.23
nM) was achieved. Compound 15 was twice as potent as LGK974 in the same assay. Encouraged, we
kept the LGK974 element on the right hand side and started to explore the structural requirements on
the left hand side. Spatial alterations were not well tolerated, as the meta substitutions of the biphenyl
elements on the left led to significant potency loss, albeit the linkage elements were changed from
oxygen to nitrogen and sulfur (compounds 19, 20, 21; 188, 164, 40 nM, respectively). Fusion of the
biphenyl to bicyclic element was not well tolerated either, as demonstrated by compound 38 (30 nM).
Removal of the key hydrogen bond acceptor completely abrogated activity (compound 39, >1000 nM),
which was consistent with the conclusion drawn by the recently published pharmacophoric model.10
The orientation of the hydrogen bond acceptor seemed very important, as compounds 40-45 were
completely inactive, while a nitrile group brought the activity back to nano molar range (compound 46,
2.7 nM). Nitrile is a versatile functional group in medicinal chemistry. It can be utilized to reduce clogP,
remove metabolic liability, and most importantly, as demonstrated in this case, serve as a hydrogen
bond acceptor.15 The length of the elements on the left hand side also contributed to the Wnt signaling
inhibition, as the shorter single ring was 100 fold less active (compound 47, 485 nM). The aliphatic
rings were tolerated but compared unfavorably with the aromatic rings, as demonstrated by compounds
48 and 49 (24 and 483 nM, respectively, Table 1). Although compound 48 was less potent compared
with the biphenyl counterpart, it represented a breakaway from the conjugated aromatic system and
may provide more desirable physicochemical properties, and therefore, warrants further investigation.
We then focused our structure-activity exploration on the central linkage element. When the O was
replaced by NH, NCH3, S, and CH2, Wnt signaling inhibition activity was all well preserved, as
compounds 54, 55, 58 and 60 (0.96, 0.67, 0.53 and 0.19 nM) all exhibited potencies under 1 nM.
Substitution on the alpha carbon of the amide with a methyl group gave the most potent compound in
this SAR exercise, as demonstrated by compound 62 (0.11 nM, Table 2) which was five times more
potent than LGK974 in the same assay. Again, these results were consistent with the recently published
series of compounds that displayed parallel SAR of the linker.11 Although compound 62 was a racemic
mixture of a pair of enantiomers, based on the pharmacophoric model10 and the molecular similarity of
the two series of compounds; we speculate that the S-isomer would be more active.
The STF report gene assay is a cell based assay suitable for high through put screening. However,
as compounds with common cell toxicity can also inhibit the reporter gene and therefore cause false
positive results, we used a hedgehog reporter gene assay as a counter screening assay to minimize the
possibility for false positive results. The representative compounds were tested using a cell based
NIH3T3-GRE-Luc reporter gene assay with 10 nM SAG as the Hh pathway agonist16-18, and non
specific cell toxicity was confirmed not to be the source of their Wnt inhibition activity (data not
shown).
Table 1. SAR of designed compounds
7

Compound
R1
X
O
R2
IC50 (nM) ± SEMa
5.0 ± 2.9
13
O
O
2.2 ± 0.9
0.23 ± 0.08
188 ± 20
164 ± 54
40 ± 15
30 ± 1
14
15
19
20
21
38
39
40
41
42
43
44
O
NH
S
O
>1000
O
O
>1000
NH
O
>1000
>1000
NH
O
>1000
>1000
8

O
O
O
O
>1000
2.7 ± 0.4
485 ± 24
24 ± 13
45
46
47
48
O
483 ± 79
49
a Inhibition of luminescence signaling in a Wnt generating L Wnt3A cells and Wnt responding HEK293
cells co-cultured system. Data are expressed as geometric mean values of at least two runs ± the
standard error measurement (SEM).
Table 2. SAR of central linkage elements
Compound
X
O
Y
CH2
IC50 (nM) ± SEMa
0.23 ± 0.08
0.96 ± 0.44
0.67 ± 0.06
0.53 ± 0.13
0.19 ± 0.02
0.11 ± 0.01
0.57 ± 0.06
15
NH
NCH3
S
CH2
54
CH2
55
58
CH2
CH2
O
CH2
60
CHCH3
62
LGK974b
a Inhibition of luminescence signaling in a Wnt generating L Wnt3A cells and Wnt responding HEK293
cells co-cultured system. Data are expressed as geometric mean values of at least two runs ± the
standard error measurement (SEM).
b
LGK974 was run as standard in each assay. Data are expressed as geometric mean values of four runs
± the standard error measurement (SEM).
Porcupine is a member of the membrane-bound O-acyltransferase family of proteins. It performs
the important function of catalyzing the palmitoylation of Wnt proteins, a process needed for their
9

secretion and activity. Without palmitoylation, Wnt proteins cannot be secreted outside of cells. To
confirm that the compounds obtained from the aforementioned SAR were indeed porcupine inhibitors,
a cell based secretion assay was developed. HEK293T cells were transfected with pLinbin-Wnt3A
plasmid or vehicle control and treated with or without compounds. After 48 hours, both the culture
medium and the cell lysis were checked by Western Blot, and the inhibition of Wnt3A secretion from
the cell was a direct indication of porcupine inhibition. Compounds 15, 54, 62, as well as LGK974 all
potently inhibited Wnt3A secretion into cell culture medium, while the amount of Wnt3A inside of
cells was not affected (Figure 3). These results were consistent with the inhibition of porcupine
activity.
Medium
WNT3A
Cell lysate
WNT3A
WNT3A
b-actin
Figure 3. The Wnt secretion assay. Compound concentration was at 0.1 μM
Even though porcupine is an important post-translational modification protein for the Wnt family,
little is known about its structure. Only very recently have mutational data shed some light on the key
residues and regions important for porcupine-mediated Wnt acylation.19 Despite the lack of structural
information of the protein, a pharmacophoric model was developed based on the limited information of
existing porcupine inhibitors.10 Our data are consistent with the pharmacophoric model and could
provide additional structural variety for the currently available porcupine inhibitors. Furthermore,
compound 62 demonstrated an IC50 of 0.11 nM in the reporter gene assay, which was 5 times more
potent than that of LGK974, putting it among one of the most potent porcupine inhibitors discovered so
far. In view of the similar potencies exhibited by these novel porcupine inhibitors, it is likely that the
pharmacokinetic profiles will dominate further research for and selection of a clinical candidate.
4.2. Evaluation of chemical stability, rat plasma stability, liver microsomal stability and CYP
inhibition
An amide functional group was present in most of the reported porcupine inhibitors. In spite of
being a key feature of the pharmacophore, the amide group was reported to show varied stability in
saline, plasma and under the treatment of liver miscrosomal enzymes.6 Our novel template also
contained an amide functional group, and we therefore decided to test the representative compounds 15,
54, 58, 60 and 62 in simulated gastric fluid (SGF), rat plasma and under the treatment of liver
microsomal enzymes. Although these compounds demonstrated small variation in their stabilities in
SGF, they were largely stable in simulated gastric fluid after 8 hours. The same conclusion can be
10

drawn for their stability in rat plasma after 8 hours (Table 3). However, while compounds 54 and 62
demonstrated moderate clearance under the treatment of human and mouse microsomes (45, 440 and
43, 321 mL/min/kg, respectively), compounds 15, 58 and 60 exhibited much faster clearance under the
same conditions (135, 263, 84 and 1430, 1790, 1260 mL/min/kg, respectively, Table 3). These results
suggested that the nitrogen linkage and the alpha carbon substitution helped to maintain the overall
metabolic stability, while sulfur in the linkage was detrimental to the overall metabolic stability.
A
similar trend was observed in a recently published paper, but the clearance of those compounds were
moderate to low.11 Compounds 15, 54, 58, 60 and 62 were then subjected to the standard CYP
inhibition test, and they showed weak or no CYP inhibition at concentration of 10 μM (Table 3).
Table 3. Chemical Stability, Plasma Stability and Metabolic Stability of Designed Compounds
% Remaining, in
SGFa
% Remaining, in rat
plasmab
Clint
t1/2
CYP
inhibitione
(mL/min/kg)
(min)
Compd
0 h
4 h
8 h
0.5 h
97%
1 h
8 h
HLMc MLMd HLM MLM 3A4
2D6
19%
53%
45%
47%
53%
96%
95%
98%
81% 67%
90%
84%
98%
83%
90%
100%
135
45
1430
440
13
39
6.8
21
41
4.3
14
47%
50%
60%
45%
67%
15
54
58
60
62
88% 85% 100% 100%
89% 83%
93%
98%
91%
94%
263
84
1790
1260
321
3.4
4.8
19
100% 97% 93%
97%
85% 71% 100% 100%
43
a All compounds were tested at 200 μM concentration in simulated gastric fluid (SGF) at 40°C for 0, 4,
8 h.
b All compounds were tested at 1 μM concentration.
c HLM = human liver microsomes. All compounds were tested at 1 μM concentration.
d MLM = mouse liver microsomes. All compounds were tested at 1 μM concentration.
e All compounds were tested at 10 μM concentration.
5. Conclusion
We have utilized a scaffold morphing strategy to design and synthesize a new series of Wnt
signaling pathway inhibitors. This novel scaffold provided numerous compounds (e.g. 15, 54, 55, 58,
60 and 62; 0.23, 0.96, 0.67, 0.53, 0.19 and 0.11 nM, respectively) with Wnt signaling inhibition
activities comparable to or better than that of the clinical candidate LGK974 (0.57 nM). The leading
compound 62 also potently inhibited Wnt secretion into culture medium, an indication of direct
inhibition of the porcupine protein. Furthermore, compound 62 showed excellent chemical, plasma and
liver microsomal stabilities. Collectively, these results strongly support further optimization of this
novel scaffold to develop better Wnt pathway inhibitors.
6. Experimental section
6.1. Chemistry
All reagents and solvents obtained from commercial suppliers were used without any further
purification. Reactions were monitored by thin-layer chromatography performed on silica gel
1
HSGF254 pre-coated plates. H and 13C NMR spectra were recorded on a Varian 300 or Varian 400
11

MHz spectrometer with CDCl3 or DMSO-d6 as the solvent. Chemical shifts were expressed in δ (ppm)
values with tetramethylsilane as an internal reference. Mass spectra were recorded on an Agilent 1100
LC/MSD Trap SL version Mass Spectrometer. HRMS analysis was performed on an Agilent 6540
UHD Accurate-Mass Q-TOF LC/MS. Compounds 820 and 912 were prepared according to the literature
procedure.
6.1.1. 1-Bromo-4-(methoxymethoxy)benzene (2)
To a solution of 4-bromophenol (1) (1.7 g, 10 mmol) and CH2(OCH3)2 (7.6 g, 100 mmol) in
dichloromethane (200 mL) was added P2O5 (5.0 g, 35 mmol). The reaction mixture was stirred at room
temperature for 12 h and the insoluble precipitate was filtered off. Excess solid Na2CO3 was added to
the filtrate. The suspension was stirred for an additional 10 min and filtered. The filtrate was
1
concentrated under reduced pressure to give 2 (1.9 g, 87%) as a colorless oil. H NMR (400 MHz,
CDCl3): δ 7.38 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 5.15 (s, 2H), 3.47 (s, 3H).
6.1.2. 2-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (4)
To a solution of 4-bromo-2-methylpyridine (3) (1.5 g, 8.72 mmol), bis(pinacolato)diboron (2.4 g,
9.42 mmol) and Pd(dppf)Cl2 (142 mg, 0.17 mmol) in THF (20 mL) was added KOAc (1.7 g, 17.4
mmol). The reaction mixture was stirred at 80°C under N2 for 15 h. After cooling to room temperature,
the mixture was diluted with dichloromethane (200 mL) and then filtered. The filtrate was concentrated
to give 4 (3.3 g, crude) as a black oil, which was used directly in the next step without further
purification.
6.1.3. 4-(4-(Methoxymethoxy)phenyl)-2-methylpyridine (5)
To a solution of the above crude oil 4 (3.3 g), 2 (1.5 g, 6.91 mmol) and Pd(PPh3)4 (635 mg, 0.55
mmol) in THF (10 mL) was added Cs2CO3 (2.7 g, 8.29 mmol). The mixture was stirred at 75°C under
N2 for 12 h. After cooling to room temperature, the solvent was evaporated and the residue was
purified by silica gel column chromatography (petroleum ether/ethyl acetate = 5/1) to give 5 (1.3 g,
1
82%) as a colorless oil. H NMR (400 MHz, CDCl3): δ 8.48 (d, J = 5.2 Hz, 1H), 7.56 (d, J = 8.8 Hz,
2H), 7.32 (s, 1H), 7.27 (d, J = 5.2 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 5.22 (s, 2H), 3.49 (s, 3H), 2.60 (s,
3H).
6.1.4. 4-(2-Methylpyridin-4-yl)phenol (6)
The solution of 5 (1.0 g, 4.37 mmol) in 1 N aqueous HCl (10 mL) was stirred at room temperature
for 16 h. The pH was adjusted to 7 with a saturated aqueous NaHCO3. The suspension was filtered to
give 6 (808 mg, 80%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 8.50 (d, J = 5.2 Hz, 1H), 7.54 (d,
J = 8.8 Hz, 2H), 7.34 (s, 1H), 7.29 (d, J = 5.2 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 2.61 (s, 3H).
6.1.5. General procedure for the synthesis of 10-12
To a solution of corresponding amine (1 eq) and Et3N (1.2 eq) in dry dichloromethane (5 mL) was
added chloroacetyl chloride (1.1 eq) slowly at 0°C under N2. After stirred at room temperature
overnight, the mixture was diluted with water (30 mL) and extracted with dichloromethane (30 mL x 3).
The combined organic layer was dried over Na2SO4 and concentrated. The residue was purified by
silica gel column chromatography (petroleum ether/ethyl acetate) to afford the desired products 10-12.
12

6.1.5.1. N-([1,1'-Biphenyl]-4-yl)-2-chloroacetamide (10)
1
Light yellow solid (yield: 67%). H NMR (400 MHz, CDCl3): δ 8.29 (s, 1H), 7.66-7.56 (m, 6H),
7.44 (t, J = 7.6 Hz, 2H), 7.36 (d, J = 7.6 Hz, 1H ), 4.23 ( s, 2H).
6.1.5.2. 2-Chloro-N-(4-(pyridin-3-yl)phenyl)acetamide (11)
Yellow solid (yield: 67%). 1H NMR (400 MHz, DMSO-d6): δ 10.46 (s, 1H), 8.88 (s, 1H), 8.54 (d,
J = 4.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.73 (s, 4H), 7.47 (dd, J = 8.0, 5.2 Hz, 1H), 4.29 (s, 2H).
6.1.5.3. 2-Chloro-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (12)
1
Brown solid (yield: 49%). H NMR (400 MHz, CDCl3): δ 9.06 (s, 1H), 9.00 (d, J = 1.2 Hz, 1H),
8.98 (s, 1H), 8.68 (s, 1H), 8.58 (d, J = 2.4 Hz, 1H), 8.44-8.35 (m, 2H), 4.26 (s, 2H).
6.1.6. General procedure for the synthesis of 13-16
To a suspension of corresponding phenol (1.2 eq), corresponding chloride (1 eq) and K2CO3 (1.2
eq) in DMF (1 mL) was added KI (0.12 eq), and the mixture was stirred at 60°C overnight. After
cooling to room temperature, the mixture was diluted with water (30 mL) and extracted with ethyl
acetate (30 mL x 3). The combined organic layer was washed with brine (30 mL x 3), dried over
Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (petroleum
ether/dichloromethane) to afford the desired products 13-16.
6.1.6.1. N-([1,1'-Biphenyl]-4-yl)-2-(4-(2-methylpyridin-4-yl)phenoxy)acetamide (13)
1
White solid (yield: 52%). H NMR (400 MHz, CDCl3): δ 8.53 (d, J = 5.2 Hz, 1H), 8.31 (s, 1H),
7.69-7.64 (m, 4H), 7.62-7.58 (m, 4H), 7.44 (t, J = 9.8 Hz, 2H), 7.35 (s, 2H), 7.29 (d, J = 2.8 Hz, 1H),
7.12 (d, J = 7.6 Hz, 2H), 4.70 (s, 2H), 2.62 (s, 3H). ESI-MS (m/z): 395.0 [M+H]+.
6.1.6.2. 2-(4-(2-Methylpyridin-4-yl)phenoxy)-N-(4-(pyridin-3-yl)phenyl)acetamide (14)
Light yellow solid (yield: 42%). 1H NMR (400 MHz, DMSO-d6): δ 10.30 (s, 1H), 8.88 (d, J = 1.6
Hz, 1H), 8.53 (dd, J = 4.8, 1.2 Hz, 1H), 8.44 (d, J = 5.2 Hz, 1H), 8.08-8.02 (m, 1H), 7.81-7.76 (m, 4H),
7.72 (d, J = 8.8 Hz, 2H), 7.55 (s, 1H), 7.49-7.44 (m, 2H), 7.14 (d, J = 8.8 Hz, 2H), 4.81 (s, 2H), 2.50 (s,
3H). ESI-MS (m/z): 396.0 [M+H]+.
6.1.6.3. 2-(4-(2-Methylpyridin-4-yl)phenoxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (15)
White solid (yield: 20%). 1H NMR (400 MHz, DMSO-d6): δ 10.89 (s, 1H), 9.31 (s, 1H), 9.12 (d, J
= 2.4 Hz, 1H), 8.72 (s, 1H), 8.63 (d, J = 2.4 Hz, 1H), 8.54 (dd, J = 8.8, 2.4 Hz, 1H), 8.44 (d, J = 5.2 Hz,
1H), 8.22 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.54 (s, 1H), 7.45 (d, J = 4.8 Hz, 1H), 7.11 (d, J
= 8.8 Hz, 2H), 4.93 (s, 2H), 2.51 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 166.9, 158.1, 157.8, 151.8,
148.8, 148.6, 146.1, 146.0, 143.8, 143.0, 141.2, 136.0, 129.5, 127.5, 126.9, 119.3, 117.3, 114.5, 112.75,
66.0, 23.6. HRMS (ESI) calcd for C23H19N5O2 [M+H]+: 398.1612, found: 398.1610.
6.1.6.4. 2-(3-Bromophenoxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (16)
1
White solid (yield: 53%). H NMR (400 MHz, CDCl3): δ 9.05-9.04 (m, 1H), 9.00-8.97 (m, 2H),
8.68-8.64 (m, 1H), 8.56 (d, J = 2.0 Hz, 1H), 8.46-8.39 (m, 2H), 7.24-7.18 (m, 3H), 6.98-6.95 (m, 1H),
4.66 (s, 2H).
13

6.1.7. 2-((3-Bromophenyl)amino)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (17)
To a solution of 12 (120 mg, 0.48 mmol), 3-bromoaniline (83 mg, 0.48 mmol) and Bu4NI (17 mg,
0.048 mmol) in toluene (1 mL) was added N,N-diisopropylethylamine (93 mg, 0.72 mmol), and the
mixture was stirred at 110°C overnight. After cooling to room temperature, the mixture was diluted
with water (30 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic layer was
washed with brine (30 mL x 3), dried over Na2SO4 and concentrated. The residue was purified by silica
gel column chromatography (dichloromethane/methanol = 80/1) to give 17 (80 mg, 42%) as a yellow
1
solid. H NMR (400 MHz, CDCl3): δ 9.15 (s, 1H), 9.01 (s, 1H), 8.92 (d, J = 2.0 Hz, 1H), 8.64 (s, 1H),
8.55 (d, J = 2.4 Hz, 1H), 8.45 (d, J = 8.4 Hz, 1H), 8.39 (d, J = 8.8 Hz, 1H), 7.12-7.05 (m, 1H), 6.97 (d,
J = 8.8 Hz, 1H), 6.84 (s, 1H), 6.61 (dd, J = 8.0, 2.4 Hz, 1H), 4.49 (s, 1H), 3.99 (s, 2H).
6.1.8. 2-((3-Bromophenyl)thio)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (18)
To a solution of 3-bromobenzenethiol (137 mg, 0.73 mmol) and 12 (150 mg, 0.6 mmol) in acetone
(10 mL) was added K2CO3 (334 mg, 2.42 mmol). The mixture was stirred at 60°C overnight. After
cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue
was purified by silica gel column chromatography (dichloromethane/methanol = 50/1) to give 18 (178
1
mg, 74%) as a yellow solid. H NMR (400 MHz, CDCl3): δ 9.12 (s, 1H), 9.02 (d, J = 1.2 Hz, 1H),
8.97-8.91 (m, 1H), 8.65 (dd, J = 2.4, 1.6 Hz, 1H), 8.55 (d, J = 2.4 Hz, 1H), 8.39-8.30 (m, 2H), 7.54 (t, J
= 1.8 Hz, 1H), 7.38-7.33 (m, 1H), 7.34-7.29 (m, 1H), 7.18 (t, J = 7.8 Hz, 1H), 3.82 (s, 2H).
6.1.9. General procedure for the synthesis of 19-21
To a suspension of corresponding aryl bromide (1 eq), 4 (1.2 eq), Pd(dppf)Cl2 (0.04 eq) and dppf
(0.04 eq) in 1,4-dioxane (10 mL) was added K3PO4 (2 eq). The mixture was stirred at 105°C under N2
overnight. After cooling to room temperature, the mixture was concentrated under reduced pressure,
and the residue was purified by silica gel column chromatography (dichloromethane/methanol) to
afford the desired products 19-21.
6.1.9.1. 2-(3-(2-Methylpyridin-4-yl)phenoxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (19)
1
White solid (yield: 23%). H NMR (400 MHz, CDCl3): δ 9.11 (s, 1H), 9.04 (d, J = 1.6 Hz, 1H),
9.00 (d, J = 2.0 Hz, 1H), 8.67-8.65 (m, 1H), 8.59-8.54 (m, 2H), 8.46 (d, J = 8.8 Hz, 1H), 8.42-8.39 (m,
1H), 7.49-7.44 (m, 1H), 7.37-7.28 (m, 4H), 7.07 (dd, J = 8.0, 2.4 Hz, 1H), 4.74 (s, 2H), 2.64 (s, 3H).
ESI-MS (m/z): 398.2 [M+H]+.
6.1.9.2.
(20)
2-((3-(2-Methylpyridin-4-yl)phenyl)amino)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide
Light yellow solid (yield: 36%). 1H NMR (400 MHz, DMSO-d6): δ 10.73 (s, 1H), 9.30 (d, J = 1.6
Hz, 1H), 9.09 (d, J = 1.6 Hz, 1H), 8.72 (dd, J = 2.4, 1.6 Hz, 1H), 8.62 (d, J = 2.4 Hz, 1H), 8.53 (dd, J =
8.8, 2.4 Hz, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 7.46 (s, 1H), 7.39 (d, J = 5.2 Hz,
1H), 7.26-7.21 (m, 1H), 7.00-6.95 (m, 2H), 6.71 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 6.31-6.26 (m, 1H),
4.09 (d, J = 6.4 Hz, 2H), 2.51 (s, 3H). ESI-MS (m/z): 397.2 [M+H]+.
6.1.9.3. 2-((3-(2-Methylpyridin-4-yl)phenyl)thio)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (21)
Yellow solid (yield: 11%). 1H NMR (400 MHz, CDCl3): δ 9.25 (s, 1H), 9.00 (s, 1H), 8.93 (s, 1H),
8.64 (s, 1H), 8.53 (d, J = 9.6 Hz, 2H), 8.34 (s, 2H), 7.65 (s, 1H), 7.51-7.39 (m, 3H), 7.31 (s, 1H), 7.25
14

(s, 1H), 3.87 (s, 2H), 2.60 (s, 3H). ESI-MS (m/z): 414.2 [M+H]+.
6.1.10. 1-Methyl-6-nitro-1H-indazole (23)
To a suspension of NaH (552 mg, 80%, 18.4 mmol) in dry DMF (5 mL) was added 22 (1.0 g, 6.13
mmol) in dry DMF (10 mL) slowly at 0°C under N2. The mixture was stirred for 1 h, and then CH3I
(1.05 g, 7.36 mmol) was added. The reaction mixture was stirred at room temperature for 18 h. The
mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 3). The combined
organic layer was washed with brine (30 mL x 3), dried over Na2SO4 and concentrated. The residue
was purified by silica gel column chromatography (petroleum ether/dichloromethane = 10/1) to give 23
(264 mg, 24%) as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.39 (s, 1H), 8.11 (s, 1H),
8.04-8.01(m, 1H), 7.84 (d, J = 8.8 Hz, 1H), 4.19 (s, 3H).
6.1.11. General procedure for the synthesis of 24 and 25
To a solution of corresponding nitrobenzene (1 eq) in methanol (20 mL) was added Pd/C (10%),
and the mixture was stirred at room temperature under H2 overnight. The mixture was filtered, and the
filtrate was concentrated to afford the desired products 24 and 25.
6.1.11.1. 1H-Indazol-6-amine (24)
Yellow solid (yield: 92%). 1H NMR (400 MHz, DMSO-d6): δ 12.25 (s, 1H), 7.71 (s, 1H), 7.35 (d,
J = 8.4 Hz, 1H), 6.52-6.43 (m, 2H), 5.21 (s, 2H).
6.1.11.2. 1-Methyl-1H-indazol-6-amine (25)
1
Brown solid (yield: 96%). H NMR (400 MHz, CDCl3): δ 7.80 (s, 1H), 7.48 (d, J = 8.8 Hz, 1H),
6.57 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 3.94 (s, 3H).
6.1.12. General procedure for the synthesis of 26 and 27
To a suspension of corresponding amine (1 eq) in H2O/98% H2SO4 (1/1) was added NaNO2 (1 eq)
slowly at 0°C. After stirred at 120°C for 2 h, the mixture was diluted with water (30 mL) and extracted
with ethyl acetate (30 mL x 3). The combined organic layer was dried over Na2SO4 and concentrated.
The residue was purified by silica gel column chromatography (dichloromethane/methanol) to afford
the desired products 26 and 27.
6.1.12.1. 1H-Indazol-6-ol (26)
Orange solid (yield: 87%). 1H NMR (400 MHz, DMSO-d6): δ 12.55 (s, 1H), 9.56 (s, 1H), 7.85 (s,
1H), 7.51 (d, J = 8.8 Hz, 1H), 6.75 (s, 1H), 6.62 (dd, J = 8.8, 1.6 Hz, 1H).
6.1.12.2. 1-Methyl-1H-indazol-6-ol (27)
Yellow oil (yield: 79%). 1H NMR (400 MHz, DMSO-d6): δ 7.81 (s, 1H), 7.50 (d, J = 8.8 Hz, 1H),
6.80 (s, 1H), 6.71-6.69 (m, 1H), 3.87 (s, 3H).
6.1.13. 2-Fluoro-4-((tetrahydro-2H-pyran-2-yl)oxy)benzonitrile (29)
To a solution of 28 (2 g, 14.6 mmol) and pyridinium 4-toluenesulfonate (73 mg, 0.29 mmol) in
dry dichloromethane (25 mL) was added 3,4-dihydro-2H-pyran (9.8 g, 29.2 mmol) slowly. The mixture
was stirred at room temperature for 16 h. Then the mixture was concentrated under reduced pressure
15

and purified by silica gel column chromatography (dichloromethane) to give 29 (1.8 g, 56%) as a
colourless oil. 1H NMR (400 MHz, CDCl3): δ 7.50 (t, J = 7.8 Hz, 1H), 6.93-6.86 (m, 2H), 5.47 (s, 1H),
3.81-3.62 (m, 2H), 1.90-1.64 (m, 6H).
6.1.14. 6-((Tetrahydro-2H-pyran-2-yl)oxy)benzo[d]isoxazol-3-amine (30)
To a suspension of t-BuOK (637 mg, 5.69 mmol) in DMF (4 mL) was added N-hydroxyacetamide
(427 mg, 5.69 mmol) under N2, and the mixture was stirred at room temperature for 1 h. A solution of
29 (600 mg, 2.84 mmol) in DMF (1 mL) was added and the mixture was stirred for 24 h. The reaction
mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 3). The combined
organic layer was washed with brine (30 mL x 3), dried over Na2SO4 and concentrated. The residue
was purified by silica gel column chromatography (dichloromethane/methanol = 30/1) to give 30 (252
mg, 38%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 7.41 (s, 1H), 7.12 (s, 1H), 6.96 (s, 1H), 5.49
(s, 1H), 3.89-3.66 (m, 2H), 1.90-1.71 (m, 6H).
6.1.15. 3-Aminobenzo[d]isoxazol-6-ol (31)
To a solution of 30 (100 mg, 0.43 mmol) in methanol (2 mL) was added 1 N HCl (1 mL), and the
mixture was stirred at room temperature for 1 h. Then the mixture was concentrated under reduced
pressure and purified by silica gel column chromatography (dichloromethane/methanol = 50/1) to give
1
31 (60 mg，93%) as a brown solid. H NMR (400 MHz, DMSO-d6): δ 7.57 (d, J =8.8 Hz, 1H),
6.70-6.69 (m, 2H).
6.1.16. Ethyl (3-methoxyphenethyl)carbamate (33)
To a solution of 32 (3 g, 13.1 mmol) and Et3N (1.46 g, 14.4 mmol) in dry dichloromethane (30
mL) was added ClCOOEt (1.56 g, 14.4 mmol) at 0°C under N2, and the mixture was stirred at 0°C for
1.5 h. Then the mixture was treated with water (50 mL) and extracted with dichloromethane (50 mL x
3). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (dichloromethane) to give 33 (2.82 g, 96%)
as a light yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.22 (t, J = 7.8 Hz, 1H), 6.80-6.75 (m, 2H), 6.73
(s, 1H), 4.10 (dd, J = 13.6, 6.8 Hz, 2H), 3.79 (s, 3H), 3.43-3.41 (m, 2H), 2.78 (t, J = 6.8 Hz, 2H), 1.22
(t, J = 6.8 Hz, 3H).
6.1.17. 6-Methoxy-3,4-dihydroisoquinolin-1(2H)-one (34)
A mixture of 33 (2.82 g, 12.6 mmol) in polyphosphoric acid (12 g, 35.5 mmol) was stirred at
120°C under N2 for 1.5 h. After cooling to room temperature, the mixture was quenched with water (30
mL) and extracted with ethyl acetate (30 mL x 3). The combined organic layer was dried over Na2SO4
and concentrated. The residue was purified by silica gel column chromatography
1
(dichloromethane/methanol = 100/1) to give 34 (1.4 g, 64%) as a white solid. H NMR (400 MHz,
CDCl3): δ 8.01 (d, J = 8.8 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.70 (s, 1H), 6.38 (s, 1H), 3.84 (s, 3H),
3.55 (t, J = 5.2 Hz, 2H), 2.96 (t, J = 6.4 Hz, 2H).
6.1.18. 6-Methoxy-1,2,3,4-tetrahydroisoquinoline (35)
To a solution of 34 (1.4 g, 7.9 mmol) in dry THF (20 mL) was added LiAlH4 (601 mg, 15.8 mmol)
slowly under N2, and the mixture was stirred at 60°C overnight. After cooling to room temperature, the
mixture was diluted with water (0.6 mL) and 15% NaOH solution (0.6 mL), then stirred for 30 min.
16

The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (dichloromethane/methanol = 20/1) to give 35 (1.06 g,
77%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 6.94 (d, J = 8.0 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H),
6.63 (s, 1H), 4.00 (s, 2H), 3.78 (s, 3H), 3.16 (t, J = 5.6 Hz, 2H), 2.83 (t, J = 5.2 Hz, 2H).
6.1.19. 1,2,3,4-Tetrahydroisoquinolin-6-ol hydrochloride (36)
To a solution of 35 (205 mg, 1.26 mmol) in dry dichloromethane (10 mL) was added 1 N BBr3 in
dichloromethane (2.6 mL, 2.6 mmol) at -78°C under N2. The mixture was stirred at -78°C for 1 h, and
then stirred at room temperature overnight. Methanol (10 mL) was added. The reaction mixture was
concentrated under reduced pressure, diluted with 1N HCl (30 mL) and washed with ethyl acetate (30
1
mL x 3). The aqueous layer was concentrated to give crude 36 (288 mg) as a yellow solid. H NMR
(400 MHz, D2O): δ 5.53-5.49 (m, 1H), 5.23-5.14 (m, 2H), 2.69 (d, J = 9.6 Hz, 2H), 1.92-1.84 (m, 2H),
1.50-1.46 (m, 2H).
6.1.20. 1-(6-Hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one (37)
To a solution of 36 (103 mg, 81%, 0.45 mmol) and acetic anhydride (69 mg, 0.68 mmol) in water
(10 mL) was added NaHCO3 (38 mg, 0.45 mmol), and the mixture was stirred at 70°C for 4 h. After
cooling to room temperature, the mixture was diluted with water (30 mL) and extracted with ethyl
acetate (30 mL x 3). The combined organic layer was dried over Na2SO4 and concentrated. The
resulting residue was purified by silica gel column chromatography (dichloromethane/methanol = 20/1)
to give 37 (40 mg, 47%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 8.52 (s, 1H), 6.93 (s,1 H), 6.91
(s, 1H), 6.78-6.71 (m, 2H), 6.68 (s, 2H), 4.63 (s, 2H), 4.52 (s, 2H), 3.81-3.71(m, 2H), 3.68-3.57 (m,
2H). 2.81 (s, 2H), 2.75 (s, 2H), 2.19 (s, 3H), 2.17 (s, 3H).
6.1.21. General procedure for the synthesis of 38-40, 42 and 44-49
To a suspension of 12 (1.2 eq), corresponding phenol (1 eq) and K2CO3 (1.2 eq) in DMF (1 mL)
was added KI (0.12 eq), and the mixture was stirred at 60°C overnight. After cooling to room
temperature, the mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 3).
The combined organic layer was washed with brine (30 mL x 3), dried over Na2SO4 and concentrated.
The residue was purified by silica gel column chromatography (petroleum ether /dichloromethane) to
afford the desired products 38-40, 42 and 44-49.
6.1.21.1 2-(Isoquinolin-6-yloxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (38)
1
Yellow solid (yield: 12%). H NMR (400 MHz, CDCl3): δ 9.19 (s, 1H), 9.05 (d, J = 5.6 Hz, 2H),
8.99 (s, 1H), 8.66 (s, 1H), 8.56 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.47 (d, J = 8.8 Hz, 1H), 8.41 (d, J =
8.4 Hz, 1H), 7.99 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 5.6 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.17 (s, 1H),
4.83 (s, 2H). ESI-MS (m/z): 358.0 [M+H]+.
6.1.21.2 2-(Naphthalen-2-yloxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (39)
1
White solid (yield: 26%). H NMR (400 MHz, CDCl3): δ 9.14 (s, 1H), 9.04 (d, J = 1.2 Hz, 1H),
8.99 (d, J = 1.6 Hz, 1H), 8.66 (t, J = 1.8 Hz, 1H), 8.56 (d, J = 2.4 Hz, 1H), 8.48 (d, J = 8.8 Hz, 1H),
8.42-8.29 (m, 1H), 7.83 (t, J = 9.6 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.40 (t, J
= 7.4 Hz, 1H), 7.32-7.30 (m, 1H), 7.22 (d, J = 2.0 Hz, 1H), 4.80 (s, 2H). ESI-MS (m/z): 357.0 [M+H]+.
17

6.1.21.3 2-((1H-Indazol-6-yl)oxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (40)
1
Light yellow soild (yield: 19%). H NMR (400 MHz, CDCl3): δ 10.20 (s, 1H), 9.11 (s, 1H), 9.05
(s, 1H), 9.00 (s, 1H), 8.67 (d, J = 1.6 Hz, 1H), 8.57 (d, J = 2.0 Hz, 1H), 8.48-8.40 (m, 2H), 8.03 (s, 1H),
7.72 (d, J = 8.8 Hz, 1H), 7.00 (d, J = 10.8 Hz, 2H), 4.74 (s, 2H). ESI-MS (m/z): 347.0 [M+H]+.
6.1.21.4 2-((1-Methyl-1H-indazol-6-yl)oxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (42)
1
White solid (yield: 21%). H NMR (400 MHz, DMSO-d6): δ 10.81 (s, 1H), 9.31 (d, J = 1.2 Hz,
1H), 9.12 (d, J = 2.0 Hz, 1H), 8.73-8.72 (m, 1H), 8.63 (d, J = 2.4 Hz, 1H), 8.56-8.53 (m, 1H), 8.23 (d, J
= 8.8 Hz, 1H), 7.92 (s, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.14 (s, 1H), 6.91-6.88 (m, 1H), 4.94 (s, 2H), 3.96
(s, 3H). ESI-MS (m/z): 361.0 [M+H]+.
6.1.21.5 2-((3-Aminobenzo[d]isoxazol-6-yl)oxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (44)
Light yellow solid (yield: 5%). 1H NMR (400 MHz, DMSO-d6): δ 10.90 (s, 1H), 9.32 (s, 1H),
9.13 (d, J = 2.0 Hz, 1H), 8.73 (s, 1H), 8.64 (d, J = 2.4 Hz, 1H), 8.56-8.54 (m, 1H), 8.21 (d, J =9.2 Hz,
1H), 7.69 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 1.2 Hz, 1H), 6.96-6.94 (m, 1H), 6.31 (s, 2H), 4.93 (s, 2H).
13C NMR (75 MHz, DMSO-d6): δ 166.6, 162.8, 159.5, 157.7, 151.6, 148.5, 146.0, 143.8, 143.0, 141.1,
136.0, 127.0, 121.6, 112.8, 111.5, 109.9, 93.2, 66.4. ESI-MS (m/z): 363.0 [M+H]+. HRMS (ESI) calcd
for C18H14N6O3 [M+H]+: 363.1200, found: 363.1201.
6.1.21.6 2-((6-Methoxynaphthalen-2-yl)oxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (45)
1
White solid (yield: 14%). H NMR (400 MHz, CDCl3): δ 9.14 (s, 1H), 9.04 (d, J = 1.2 Hz, 1H),
8.99 (d, J = 1.2 Hz, 1H), 8.66 (t, J = 1.8 Hz, 1H), 8.56 (d, J = 2.4 Hz, 1H), 8.47 (d, J = 8.8 Hz, 1H),
8.41-8.39 (m, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.29 (d, J = 2.4 Hz, 1H),
7.19-7.15 (m, 2H), 7.13 (s, 1H), 4.77 (s, 2H), 3.91 (s, 3H). ESI-MS (m/z): 387.0 [M+H]+.
6.1.21.7 2-((6-Cyanonaphthalen-2-yl)oxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (46)
1
White solid (yield: 4%). H NMR (400 MHz, DMSO-d6): δ 10.96 (s, 1H), 9.32 (s, 1H), 9.13 (s,
1H), 8.73 (s, 1H), 8.64 (d, J = 2.0 Hz, 1H), 8.56-8.54 (m, 1H), 8.50 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H),
8.03 (d, J = 9.6 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 6.8 Hz, 2H),
5.04 (s, 2H). ESI-MS (m/z): 382.0 [M+H]+.
6.1.21.8 2-(4-Cyanophenoxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (47)
1
White solid (yield: 38%). H NMR (400 MHz, DMSO-d6): δ 10.97 (s, 1H), 9.31 (d, J = 1.6 Hz,
1H), 9.12 (d, J = 2.0 Hz, 1H), 8.73-8.82 (m, 1H), 8.63 (d, J = 2.4 Hz, 1H), 8.56-8.53 (m, 1H), 8.18 (d, J
= 8.8 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 8.8 Hz, 2H), 4.98 (s, 2H). ESI-MS (m/z): 332.0
[M+H]+.
6.1.21.9
2-((2-Acetyl-1,2,3,4-tetrahydroisoquinolin-6-yl)oxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide
(48)
White solid (yield: 18%). 1H NMR (400 MHz, CDCl3): δ 9.04 (s, 2H), 8.98 (s, 1H), 8.66 (s, 1H),
8.56 (s, 1H), 8.46-8.38 (m, 2H), 7.14-7.08 (m, 1H), 6.88 (d, J = 9.6 Hz, 1H), 6.80 (s, 1H), 4.69 (s, 1H),
4.65 (s, 2H), 4.58 (s, 1H), 3.82 (s, 1H), 3.67 (s, 1H), 2.91 (s, 1H), 2.85 (s, 1H), 2.18 (s, 3H). ESI-MS
(m/z): 404.0 [M+H]+. HRMS (ESI) calcd for C22H21N5O3 [M+H]+: 404.1717, found: 404.1713.
18

6.1.21.10
(49)
2-(4-(4-Acetylpiperazin-1-yl)phenoxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide
1
Light yellow solid (yield: 6%). H NMR (400 MHz, CDCl3): δ 9.08 (s, 1H), 9.04 (s, 1H), 8.98 (s,
1H), 8.66 (s, 1H), 8.56 (s, 1H), 8.47-8.39 (m, 2H), 7.01-6.90 (m, 4H), 4.63 (s, 2H), 3.83-3.73 (m, 2H),
3.65-3.62 (m, 2H), 3.12-3.05 (m, 4H), 2.15 (s, 3H). ESI-MS (m/z): 433.0 [M+H]+.
6.1.22. General procedure for the synthesis of 41 and 43
To a solution of 12 (1 eq), corresponding amine (1 eq) and Bu4NI (0.1 eq) in DMF (1 mL) was
added DIPEA (1.5 eq), and the mixture was stirred at 75°C overnight. After cooling to room
temperature, the mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 3).
The combined organic layer was washed with brine (30 mL x 3), dried over Na2SO4 and concentrated.
The residue was purified by silica gel column chromatography (petroleum ether/dichloromethane) to
afford the desired products 41 and 43.
6.1.22.1 2-((1H-Indazol-6-yl)amino)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (41)
1
Light yellow solid (yield: 26%). H NMR (400 MHz, DMSO-d6): δ 12.41 (s, 1H), 10.66 (s, 1H),
9.29 (s, 1H), 9.08 (d, J = 2.4 Hz, 1H), 8.72 (s, 1H), 8.62 (d, J = 2.4 Hz, 1H), 8.53 (dd, J = 8.8, 2.0 Hz,
1H), 8.25 (d, J = 8.8 Hz, 1H), 7.76 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 6.65 (d, J = 8.8 Hz, 1H), 6.37 (s,
1H), 4.04 (s, 2H). ESI-MS (m/z): 346.1 [M+H]+.
6.1.22.2 2-((1-Methyl-1H-indazol-6-yl)amino)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (43)
Yellow solid (yield: 15%). 1H NMR (400 MHz, DMSO-d6): δ 10.68 (s, 1H), 9.30 (d, J = 1.2 Hz,
1H), 9.09 (d, J = 1.6 Hz, 1H), 8.72-8.71 (m, 1H), 8.62 (d, J = 2.4 Hz, 1H), 8.54-8.52 (m, 1H), 8.25 (d, J
= 8.8 Hz, 1H), 7.73 (s, 1H), 7.43 (d, J = 8.8 Hz, 1H), 6.68-6.65 (m, 1H), 6.45 (s, 1H), 6.39 (t, J = 6.2
Hz, 1H), 4.10 (d, J = 6.0 Hz, 2H), 3.84 (s, 3H). ESI-MS (m/z): 360.0 [M+H]+.
6.1.23. 2-Methyl-4-(4-nitrophenyl)pyridine (51)
To a solution of 1-bromo-4-nitrobenzene (2.0 g, 10 mmol), 4 (3.8 g, 58%, 10 mmol) and
Pd(dppf)Cl2 (73 mg, 0.1 mmol) in 1,4-dioxane (45 mL) and H2O (15 mL) was added K2CO3 (4.1 g, 30
mmol), and the reaction mixture was stirred at 100°C under N2 for 12 h. After cooling to room
temperature, the mixture was diluted with water (50 mL) and extracted with dichloromethane (50 mL x
3). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/1) to give
1
51 (1.8 g, 84%) as a yellow solid. H NMR (400 MHz, CDCl3): δ 8.62 (d, J = 5.2 Hz, 1H), 8.34 (d, J =
8.8 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.39 (s, 1H), 7.33 (d, J = 5.2 Hz, 1H), 2.66 (s, 3H).
6.1.24. 4-(2-Methylpyridin-4-yl)aniline (52)
To a solution of 51 (1.8 g, 8.4 mmol) in methanol (20 mL) was added Pd/C (180 mg), and the
reaction mixture was stirred at room temperature under H2 overnight. The mixture was filtered, and the
filtrate was concentrated to give 52 (1.4 g, 90%) as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.48
(d, J = 5.2 Hz, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.33 (s, 1H), 7.27 (d, J = 5.2 Hz, 1H), 6.77 (d, J = 8.0 Hz,
2H), 3.87 (s, 2H), 2.60 (s, 3H).
19

6.1.25. N-Methyl-4-(2-methylpyridin-4-yl)aniline (53)
To a solution of 52 (50 mg, 0.27 mmol) in dichloromethane (2 mL) was added TFAA (108 mg,
0.51 mmol) at 0°C. The reaction mixture was stirred at 0°C for 45 min. Bu4NBr (43 mg, 0.14 mmol),
Me2SO4 (68 mg, 0.54mmol) and 50% NaOH aqueous (1 mL) were added to the cooled solution. The
mixture was allowed to warm up to room temperature and stirred for 16 h. The mixture was treated
with water (30 mL) and extracted with dichloromethane (30 mL x 3). The combined organic layer was
dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel
column chromatography (dichloromethane/methanol = 30/1) to give 53 (40 mg, 75%) as a yellow solid.
1H NMR (400 MHz, CDCl3): δ 8.45 (d, J = 4.8 Hz, 1H), 7.52 (d, J = 7.2 Hz, 2H), 7.32 (s, 1H),
7.27-7.26 (m, 1H), 6.68 (d, J = 7.6 Hz, 2H), 2.89 (s, 3H), 2.58 (s, 3H).
6.1.26. General procedure for the synthesis of 54 and 55
To a solution of 12 (1 eq), corresponding amine (1 eq) and Bu4NI (0.1 eq) in DMF (1 mL) was
added DIPEA (1.5 eq), and the mixture was stirred at 75°C overnight. After cooling to room
temperature, the mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 3).
The combined organic layer was washed with brine (30 mL x 3), dried over Na2SO4 and concentrated.
The residue was purified by silica gel column chromatography (petroleum ether/dichloromethane) to
afford the desired products 54 and 55.
6.1.26.1.
(54)
2-((4-(2-Methylpyridin-4-yl)phenyl)amino)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide
Yellow solid (yield: 13%). 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 9.31 (s, 1H), 9.10 (d,
J = 2.4 Hz, 1H), 8.72 (s, 1H), 8.63 (d, J = 2.0 Hz, 1H), 8.53 (dd, J = 8.8, 2.0 Hz, 1H), 8.37 (d, J = 5.2
Hz, 1H), 8.24 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.50 (s, 1H), 7.42 (d, J = 5.2 Hz, 1H), 6.73
(d, J = 8.4 Hz, 2H), 6.58-6.52 (m, 1H), 4.08 (d, J = 6.4 Hz, 2H), 2.48 (s, 3H). ESI-MS (m/z): 397.0
[M+H]+.
6.1.26.2.
2-(Methyl(4-(2-methylpyridin-4-yl)phenyl)amino)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (55)
1
Yellow solid (yield: 15%). H NMR (400 MHz, DMSO-d6): δ 10.88 (s, 1H), 9.31 (d, J = 1.6 Hz,
1H), 9.11 (d, J = 2.0 Hz, 1H), 8.72 (dd, J = 2.0, 1.6 Hz, 1H), 8.62 (d, J = 2.4 Hz, 1H), 8.51 (dd, J = 8.8,
2.4 Hz, 1H), 8.37 (d, J = 5.2 Hz, 1H), 8.19 (d, J = 8.8 Hz, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.49 (s, 1H),
7.40 (dd, J = 4.8, 1.2 Hz, 1H), 6.81 (d, J = 8.8 Hz, 2H), 4.39 (s, 2H), 3.13 (s, 3H), 2.47 (s, 3H). HRMS
(ESI) calcd for C24H22N6O [M+H]+: 411.1928, found: 411.1931.
6.1.27. 2-Methyl-4-(4-(methylthio)phenyl)pyridine (56)
To a solution of 4-(methylthio)phenylboronic acid (2.0 g, 11.9 mmol), Bu4NBr (479 mg, 1.49
mmol ), Pd(PPh3)4 (493 mg, 0.43 mmol) and 4-bromo-2-methylpyridine (2.0 g, 11.9 mmol) in ethylene
glycol dimethyl ether (40 mL) and water (20 mL) was added KOH (3.3 g, 59.52 mmol), and the
mixture was stirred at 80°C under N2 overnight. After cooling to room temperature, the mixture was
concentrated under reduced pressure and purified by silica gel column chromatography (petroleum
1
ether/ethyl acetate = 1/1) to give 56 (800 mg, 31%) as a yellow solid. H NMR (400 MHz, CDCl3): δ
8.52 (d, J = 5.2 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.35 (s, 2H), 7.33 (s, 1H), 7.29 (d, J = 5.2 Hz, 1H),
2.61 (s, 3H), 2.53 (s, 3H).
20

6.1.28. 4-(2-Methylpyridin-4-yl)benzenethiol (57)
To a solution of 56 (700 mg, 3.26 mmol) in dry dichloromethane (20 mL) was added m-CPBA
(696 mg, 3.42 mmol) slowly at 0°C under N2. The mixture was stirred at 0°C for 3 h, then diluted with
water (30 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic layer was dried
over Na2SO4 and concentrated under reduced pressure. To the residue was added TFFA (1.4 g, 652
mmol) at 0°C under N2, and the mixture was stirred at 0°C for 2 h. Et3N (19 mL) and ethanol (19 mL)
were added, and the mixture was stirred at room temperature for 1 h. The mixture was concentrated
under reduced pressure and diluted with water (30 mL), extracted with ethyl acetate (30 mL x 3). The
combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (ethyl acetate) to give 57 (250 mg, 38%) as a light
1
yellow solid. H NMR (400 MHz, CDCl3): δ 8.53 (d, J = 4.8 Hz, 1H), 7.66-7.46 (m, 4H), 7.33 (s, 1H),
7.29-7.26 (m, 1H), 2.61 (s, 3H).
6.1.29. 2-((4-(2-Methylpyridin-4-yl)phenyl)thio)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (58)
To a solution of 57 (80 mg, 0.40 mmol) and 12 (99 mg, 0.40 mmol) in CH3CN (5 mL) was added
Na2CO3 (51 mg, 0.48 mmol), and the mixture was refluxed for 6 h. After cooling to room temperature,
the mixture was concentrated under reduced pressure and diluted with water (30 mL) and extracted
with ethyl acetate (30 mL x 3). The combined organic layer was dried over Na2SO4 and concentrated,
then purified by silica gel column chromatography (dichloromethane/methanol = 25/1) to give 58 (20
mg, 12%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 9.22 (s, 1H), 9.02 (s, 1H), 8.95 (s, 1H), 8.65
(s, 1H), 8.54 (d, J = 2.4 Hz, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.35 (s, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.48
(d, J = 8.4 Hz, 2H), 7.31 (s, 1H), 7.26-7.24 (m, 1H), 3.87 (s, 2H), 2.60 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 168.0, 158.6, 152.7, 149.5, 149.2, 146.6, 146.4, 144.4, 143.6, 141.8, 137.4, 136.6, 134.7,
128.1, 127.4, 127.3, 120.1, 118.1, 113.2, 36.6, 24.1. HRMS (ESI) calcd for C23H19N5OS [M+H]+:
414.1383, found: 414.1363.
6.1.30. 3-(4-Bromophenyl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)propanamide (59)
To a solution of 9 (150 mg, 0.87 mmol), 3-(4-bromophenyl)propanoic acid (300 mg, 1.31 mmol)
and PPACA (2.11 g , 50 wt.% in ethyl acetate, 3.31 mmol) in THF (10 mL) was added Et3N (623 mg,
6.1 mmol). The mixture was stirred at 60°C overnight. After cooling to room temperature, the mixture
was concentrated under reduced pressure and diluted with water (30 mL) and extracted with ethyl
acetate (30 mL x 3). The combined organic layer was dried over Na2SO4 and concentrated. The
resulting residue was purified by silica gel column chromatography (dichloromethane/methanol = 20/1)
1
to give 59 (150 mg, 45%) as a white solid. H NMR (400 MHz, CDCl3): δ 9.00 (s, 1H), 8.89 (s, 1H),
8.68 (s, 1H), 8.64 (s, 1H), 8.54 (s, 1H), 8.44-8.32 (m, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.0 Hz,
2H), 3.03 (t, J = 7.6 Hz, 2H), 2.73 (t, J = 7.6 Hz, 2H).
6.1.31. 3-(4-(2-Methylpyridin-4-yl)phenyl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)propanamide (60)
To a solution of 59 (140 mg, 0.37 mmol), 4 (207 mg, 58%, 0.55 mmol), Pd(dppf)Cl2 (24 mg, 0.03
mmol) and dppf (16 mg, 0.03 mmol) in 1,4-dioxane (8 mL) was added the solution of K3PO4 (155 mg,
0.73 mmol) in water (2 mL). The mixture was stirred at 108°C under N2 overnight. After cooling to
room temperature, the mixture was concentrated under reduced pressure and purified by silica gel
column chromatography (dichloromethane/methanol = 50/1) to give 60 (68 mg, 47%) as a white solid.
21

1H NMR (400 MHz, DMSO-d6): δ 10.80 (s, 1H), 9.29 (d, J = 1.2 Hz, 1H), 9.07 (d, J = 2.0 Hz, 1H),
8.75-8.69 (m, 1H), 8.62 (d, J = 2.4 Hz, 1H), 8.51 (dd, J = 8.8, 2.4 Hz, 1H), 8.46 (d, J = 5.2 Hz, 1H),
8.26 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.56 (s, 1H), 7.47 (d, J = 4.4 Hz, 1H), 7.41 (d, J =
8.4 Hz, 2H), 2.99 (t, J = 7.6 Hz, 2H), 2.80 (t, J = 7.6 Hz, 2H), 2.51 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 171.6, 158.5, 153.1, 149.5, 149.3, 147.1, 146.5, 144.4, 143.5, 142.3, 141.7, 136.4, 135.1,
129.1, 127.0, 126.8, 120.2, 118.2, 113.1, 37.5, 30.3, 24.2. HRMS (ESI) calcd for C24H21N5O [M+H]+:
396.1819, found: 396.1822.
6.1.32. 2-Chloro-N-(5-(pyrazin-2-yl)pyridin-2-yl)propanamide (61)
To a solution of 9 (300 mg, 1.74 mmol) and Et3N (211 mg, 2.09 mmol) in dry dichloromethane (5
mL) was added 2-chloropropanoyl chloride (232 mg, 1.83 mmol) slowly at 0°C under N2. The mixture
was stirred at room temperature overnight, then diluted with water (30 mL) and extracted with
dichloromethane (30 mL x 3). The combined organic layer was dried over Na2SO4 and concentrated.
The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2/1) to
give 61 (200 mg, 44%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 9.04 (s, 1H), 8.98 (s, 1H), 8.96
(s, 1H), 8.66 (s, 1H), 8.56 (s, 1H), 8.42-8.34 (m, 2H), 4.58 (q, J = 7.2 Hz, 1H), 1.85 (d, J = 6.8 Hz, 3H).
6.1.33. 2-(4-(2-Methylpyridin-4-yl)phenoxy)-N-(5-(pyrazin-2-yl)pyridin-2-yl)propanamide (62)
To a suspension of 4-(2-methylpyridin-4-yl)phenol (39 mg, 0.21 mmol), 61 (66 mg, 0.25 mmol)
and K2CO3 (35 mg, 0.25 mmol) in DMF (1 mL) was added KI (4 mg, 0.025 mmol). The mixture was
stirred at 60°C overnight. After cooling to room temperature, the mixture was diluted with water (30
mL) and extracted with ethyl acetate (30 mL x 3). The combined organic layer was washed with brine
(30 mL x 3), dried over Na2SO4 and concentrated. The residue was purified by silica gel column
1
chromatography (dichloromethane/methanol = 20/1) to give 62 (20 mg, 22%) as a white solid. H
NMR (400 MHz, CDCl3): δ 9.02 (d, J = 1.2 Hz, 1H), 8.98-8.93 (m, 2H), 8.67-8.63 (m, 1H), 8.55 (d, J
= 2.8 Hz, 1H), 8.51 (d, J = 4.8 Hz, 1H), 8.44 (d, J = 8.4 Hz, 1H), 8.38 (dd, J = 8.8, 2.4 Hz, 1H), 7.61 (d,
J = 8.8 Hz, 2H), 7.33 (s, 1H), 7.27 (d, J = 1.2 Hz, 1H), 7.10 (d, J = 8.8 Hz, 2H), 4.91 (q, J = 6.8 Hz,
1H), 2.61 (s, 3H), 1.73 (d, J = 6.8 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 170.9, 159.0, 157.4, 151.7,
150.0, 149.7, 147.9, 146.7, 144.5, 143.6, 141.7, 136.9, 132.7, 128.9, 128.7, 120.9, 118.6, 116.2, 114.0,
75.4, 24.7, 18.7. HRMS (ESI) calcd for C24H21N5O2 [M+H]+: 412.1768, found: 412.1770.
6.2. In vitro biological assays
6.2.1. Super-Top Flash (STF) Reporter Gene Assay
HEK293 STF stable clones (HEK293 cells stably transfected with "Super-Top Flash"
TCF-luciferase reporter plasmid) were kept in complete culture medium (DMEM with 4 mM L-Gln,
1.5 g/L sodium bicarbonate and 4.5 g/L glucose containing 6 μg/mL Blasticidin and 10% FBS). L
Wnt3A cells (CRL-2647, ATCC) were kept in DMEM (Gibico) containing 10% FBS (Hyclone).
HEK293 STF and L Wnt3A cells were harvested when 90% confluence and cell suspension was mixed
with a fixed ratio of 1:1 (HEK293 STF: L Wnt3A). 100 μL/well of mixed cells suspension was added
to the 96-well-plate with final cell concentration of 12,000 cells/well and then cultured for an
additional 24 hours before adding compounds.
22

Compounds were diluted in DMSO in sequence and then diluted with DMEM medium. 20 μL
medium containing compound was added to the cells according to the pre-setting dose. Cell plates were
incubated at 37°C for an additional 48 hours.
50 μL luciferase solution (Bright-Glo, Promega) was added to each well during the 48 h
incubation. The plates were incubated at room temperature for 5 min under gentle shaking.
Luminescence signaling was measured with a plate reader (PHERAstar FS, BMG). The IC50 (potency)
of compounds was calculated based on the inhibition of luminescence signaling.
6.2.2. NIH3T3-GRE-Luc reporter gene assay
NIH3T3 cells (CRL-1658, ATCC) were kept in DMEM (Gibico) containing 10% FBS (Hyclone).
GRE-Luc plasmid was established by inserting 8x Gli-1 responsive element (GRE) into the multiple
cloning site of the pGL4.26 vector (Promega). After being transfected with GRE-Luc luciferase
reporter plasmid, NIH3T3-GRE-Luc reporter cell line was generated by hygromycin (Invitrogen)
selection. Single clones were validated by the induction of luciferase by small molecule agonist SAG
(ABIN629346) or sonic hedgehog (sHh) protein. Selected clone was used to monitor the Hh signaling.
6.2.3. Wnt secretion assay
HEK293T cells were cultured in 6-well plates. HEK293T cells were transfected with 1μg
pLibin-WNT3A plasmid or vehicle control and treated with 0.1 μM compounds (15, 54, 62, and
LGK974) simultaneously when cells reached 60% confluence. After 48 hours, cell lysates were
collected with lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1
mM Na3PO4, 25 mM β-glycerol-phosphate, 0.1mM PMSF, Roche complete protease inhibitor set and
phosphatase inhibitor set). The supernatants were collected with 4x SDS. Protein concentrations were
measured using the BCA Protein Assay Kit (Thermo, USA). Cell lysates containing 40-50 μg proteins
and collected supernatants were loaded to 8% or 10% SDS PAGE gel. Separated proteins were then
transferred to a PVDF film. Then the film was incubated first with corresponding primary antibody at
room temperature for 2 h, then with HRP-labeled secondary antibody at room temperature for 1.5 h.
The protein lanes were visualized using Western Lighting® Plus-ECL Kit（PerkinElmer, USA）
according to the manufacture’s instruction.
6.3. Chemical stability test
Compounds 15, 54, 58, 60 and 62 were dissolved in 92% simulated gastric fluid (SGF) and 8%
DMSO. These five compounds were tested at 200 μM concentration at 40°C for 0, 4, 8 h. The extent of
degradation was quantified by HPLC using the respective UV absorption peak areas at 254 nm. SGF:
3.8 mL of conc. HCl diluted to 1000 mL of water.
6.4. Plasma stability test
To test the stability of compounds 15, 54, 58, 60 and 62 in rat plasma, each compound (1 μM) was
incubated with blank rat plasma. Samples were deproteinized by the addition of 100 μL of acetonitrile
with internal standard after incubation for 8 h. The samples were then centrifuged at 13,000 rpm for 10
min, and the supernatants were subjected to LC-MS/MS analysis.
6.5. Metabolic stability test
To test the metabolic stability of compounds 15, 54, 58, 60 and 62 in vitro, each compound was
23

incubated with mouse and human liver microsomes in the presence of NADPH. The reaction mixtures
include NADPH (1 mM), microsomes (0.2 mg/mL), phosphate buffer (100 mM, pH 7.4), and test
compounds (1 μM). The reaction was initiated by the addition of NADPH after a 10 min
pre-incubation,. The incubations were taken at three time points (0, 30, 60 min) and placed into 100 μL
of cold acetonitrile with internal standard. The samples were then centrifuged at 13,000 rpm for 10 min
and the supernatants were subjected to LC-MS/MS analysis. The in vitro kinetic parameters were
calculated using the following equations:
0.693
(1)
t1/2

k
Where k is the slope from linear regression analysis of the natural Ln percent remaining of test
compound as a function of incubation time.
k
mgof proteina
gof liver
Clint,invitro


mgof proteinper mlincubation
(2)
b
gof liver

kg of bodyweight
Where a is 20 mg/g for mouse and human, b is 87.5 g/kg for mouse and 25.7 g/kg for human.
Q
h
Clint,invitro
 Clint,invitro
Clhepatic

(3)
Qh
Where Qh is the hepatic blood flow (90 mL/min for mouse and 20.7 mL/min for human).
6.6. Evaluation of CYP Inhibitory Potency.
Inhibitory potency of test compounds against human P450 enzymes was investigated in human
liver microsomes. The two major CYP isoforms and their respective probe substrates were as follows:
CYP3A4 (midazolam, 1 μM) , and CYP2D6 (dextromethorphan, 5 μM). All probe substrates were used
at concentrations near or below their Kms (Michaelis constant). Triplicate incubations were conducted
in the 200 μL incubation mixtures at 37 °C. The mixture contained HLM (0.2 mg/mL), phosphate
buffer (100 mM, pH 7.4), NADPH (1 mM), tested compounds (10 μM), and individual CYP probe
substrate. The mixtures were preincubated for 10 min to allow inhibitor/enzyme interactions before the
initiation of the reaction with NADPH. After 10 min, reactions were terminated by the addition of 100
μL of cold acetonitrile containing an appropriate internal standard. Samples were then centrifuged and
injected into the LC−MS/MS apparatus to quantify the concentrations of specific metabolites formed
by individual CYP enzymes.
Acknowledgements
The authors appreciate the supports from National Natural Science Foundation of China (Grant
No.81273372, 81473090, 81473278, 21502133), Natural Science Foundation of Jiangsu
Province (BK2012004, BK20140313), BM2013003, and PAPD (A Project Funded by the Priority
Academic Program Development of Jiangsu Higher Education Institutions).
Supplementary data
Supplementary data related to this article can be found at
24

References
1. Clevers, H.; Nusse, R. Cell 2012, 149, 1192.
2. Anastas, J.N.; Moon, R.T. Nat. Rev. Cancer 2013, 13, 11.
3. Baarsma, H.A.; Konigshoff, M.; Gosens, R. Pharmacol. Ther. 2013, 138, 66.
4. Proffitt, K.D.; Madan, B.; Ke, Z.; Pendharkar, V. ; Ding, L.; Lee, M.A.; Hannoush, R.N.; Virshup,
D.M. Cancer Res. 2013, 73, 502.
5. Liu, J.; Pan, S.; Hsieh, M.H.; Ng, N.; Sun, F.; Wang, T.; Kasibhatla, S.; Schuller, A.G.; Li, A.G.;
Cheng, D.; Li, J.; Tompkins, C.; Pferdekamper, A.; Steffy, A.; Cheng, J.; Kowal, C.; Phung, V. ;
Guo, G.; Wang, Y.; Graham, M.P.; Flynn, S.; Brenner, J.C.; Li, C.; Villarroel, M.C.; Schultz, P.G.;
Wu, X.; McNamara, P. ; Sellers, W.R.; Petruzzelli, L.; Boral, A.L.; Seidel, H.M.; McLaughlin,
M.E.; Che, J.; Carey, T.E.; Vanasse, G.; Harris, J.L. Proc. Natl. Acad. Sci. U.S.A. 2013, 110,
20224.
6. Wang, X.; Moon, J.; Dodge, M.E.; Pan, X.; Zhang, L.; Hanson, J.M.; Tuladhar, R.; Ma, Z.; Shi, H.;
Williams, N.S.; Amatruda, J.F.; Carroll, T.J.; Lum, L.; Chen, C. J. Med. Chem. 2013, 56, 2700.
7. Dodge, M.E.; Moon, J.; Tuladhar, R.; Lu, J.; Jacob, L.S.; Zhang, L.S.; Shi, H.; Wang, X.; Moro, E.;
Mongera, A.; Argenton, F.; Karner, C.M.; Carroll, T.J.; Chen, C.; Amatruda, J.F.; Lum, L. J. Biol.
Chem. 2012, 287, 23246.
8. Chen, B.; Dodge, M.E.; Tang, W. ; Lu, J.; Ma, Z.; Fan, C.W.; Wei, S.; Hao, W.; Kilgore, J.;
Williams, N.S.; Roth, M.G.; Amatruda, J.F.; Chen, C.; Lum, L. Nat. Chem. Biol. 2009, 5, 100.
9. Lum, L.; Clevers, H. Science 2012, 337, 922.
10. Poulsen, A.; Ho, S.Y.; Wang, W.; Alam, J.; Jeyaraj, D.A.; Shi, H.A.; Tan, E.S.W.; Lin, G.R.;
Cheong, V.W.W.; Ke, Z. J. Chem. Inf. Model. 2015, 55, 1435.
11. Duraiswamy, A.J.; Lee, M.A.; Madan, B.; Ang, S.H.; Tan, E.S.W.; Cheong, V.W.W.; Ke, Z.;
Pendharkar, V. ; Ding, L.J.; Chew, Y. S.; Manoharan, V.; Sangthongpitag, K.; Alam, J.; Poulsen, A.;
Ho, S.Y.; Virshup, D.M.; Keller, T.H. J. Med. Chem. 2015, 58, 5889.
12. Cheng, D.; Zhang, G.; Han, D.; Gao, W.; Pan, S.; Shen, L.; Leleti, R.R. WO 2010101849 (Sep 10,
2010).
13. Cheng, D.; Zhang, G.; Han, D.; Gao, W.; Pan, S.; Shen, L.; Leleti, R.R. WO 2012003189 (Jan 5,
2012).
14. Xu, Q.; Wang, Y.S.; Dabdoub, A.; Smallwood, P.M.; Williams, J.; Woods, C.; Kelley, M.W.; Jiang,
L.; Tasman, W.; Zhang, K.; Nathans, J. Cell 2004, 116, 883.
15. Fleming, F. F.; Yao, L.; Ravikumar, P. C.; Funk, L.; Shook, B. C. J. Med. Chem. 2010, 53, 7902.
16. Ma, H.K.; Lu, W.F.; Sun, Z.J.; Luo, L.S.; Geng, D.L.; Yang, Z.H. Li, E.Q.; Zheng, J.Y.; Wang,
M.Y.; Zhang, H.J.; Yang, S.L.; Zhang, X.H. Eur. J. Med. Chem. 2015, 89, 721.
17. Lu, W.F.; Geng, D.L.; Sun, Z.J.; Yang, Z.H.; Ma, H.K.; Zheng, J.Y.; Zhang, X.H. Bioorg. Med.
Chem. Lett. 2014, 24, 2300.
18. Yang, Z.; Ma, H.; Sun, Z.; Luo, L.; Tian, S.; Zheng, J.; Zhang, X. Bioorg. Med. Chem. Lett. 2015,
25, 3665.
19. Rios-Esteves, J.; Haugen, B.; Resh, M.D. J. Biol. Chem. 2014, 289, 17009.
20. Ohmori, J.；Sakamoto, S.；Kubota, H.; Shimizu-Sasamata, M.; Okada, M.; Kawasaki, S.; Hidaka,
K.; Togami, J.; Furuya, T.; Murase, K. J. Med. Chem., 1994, 37, 467.
25

For Table of Contents Use Only
Exploration of the Linkage Elements of Porcupine Antagonists Led to Potent Wnt Signaling
Pathway Inhibitors
Yan Dong a,1, Kehuang Li a,1, Zhixiang Xu a, Haikuo Ma a, Jiyue Zheng a, *, Zhilin Hu b, Sudan He
b, Yiyuan Wu c, Zhijian Sun c, Lusong Luo c,*, Jiajun Li a, Hongjian Zhang a, and Xiaohu Zhang a,*
a Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and
College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215021, P. R. China
b Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, and
Collaborative Innovation Center of Hematology, Soochow University, Suzhou 215123, P.R. China
c BeiGene (Beijing) Co., Ltd., No. 30 Science Park Road, Zhongguancun Life Science Park, Beijing
102206, P.R.China
26