Design, synthesis, and evaluation of potent Wnt signaling inhibitors featuring a fused 3-ring system

Article abstract of DOI:10.1016/j.ejmech.2015.11.026

The Wnt signaling pathway is a critical developmental pathway which operates through control of cellular functions such as proliferation and differentiation. Aberrant Wnt signaling has been linked to the formation and metastasis of tumors. Porcupine, a member of the membrane-bound O-acyltransferase family of proteins, is an important component of the Wnt pathway. Porcupine catalyzes the palmitoylation of Wnt proteins, a process needed for their secretion and activity. Here we report a novel series of compounds obtained by a scaffold hybridization strategy from a known porcupine inhibitor class. The leading compound 59 demonstrated subnanomolar inhibition of Wnt signaling in a paracrine cellular assay. Compound 59 also showed excellent chemical, plasma and liver microsomal stabilities. Furthermore, compound 59 exhibited good pharmacokinetic profiles with 30% oral bioavailability in rat. Collectively, these results strongly support further optimization of this novel scaffold to develop better Wnt pathway inhibitors.

Full text of DOI:10.1016/j.ejmech.2015.11.026

Accepted Manuscript
Design, synthesis, and evaluation of potent Wnt signaling inhibitors featuring a fused
3-ring system
Zhixiang Xu, Jiajun Li, Yiyuan Wu, Zhijian Sun, Lusong Luo, Zhilin Hu, Sudan He,
Jiyue Zheng, Hongjian Zhang, Xiaohu Zhang
PII:
S0223-5234(15)30363-9
10.1016/j.ejmech.2015.11.026
EJMECH 8216
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 July 2015
Revised Date: 13 November 2015
Accepted Date: 17 November 2015
Please cite this article as: Z. Xu, J. Li, Y. Wu, Z. Sun, L. Luo, Z. Hu, S. He, J. Zheng, H. Zhang, X.
Zhang, Design, synthesis, and evaluation of potent Wnt signaling inhibitors featuring a fused 3-ring
system, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.11.026.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, Synthesis, and Evaluation of Potent Wnt Signaling Inhibitors Featuring a Fused 3-ring
System
Zhixiang Xu, Jiajun Li, Yiyuan Wu, Zhijian Sun, Lusong Luo, Zhilin Hu, Sudan He, Jiyue Zheng,
Hongjian Zhang, and Xiaohu Zhang
1

ACCEPTED MANUSCRIPT
Design, Synthesis, and Evaluation of Potent Wnt Signaling Inhibitors Featuring a Fused 3-ring
System
Zhixiang Xu a,1, Jiajun Li a,1, Yiyuan Wu b, Zhijian Sun b, Lusong Luo b,*, Zhilin Hu c, Sudan He c, Jiyue
Zheng 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, Su Zhou, Jiangsu 215021, P. R. China
b BeiGene (Beijing) Co., Ltd., No. 30 Science Park Road, Zhongguancun Life Science Park, Beijing
102206, P.R.China
c
Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, and
Collaborative Innovation Center of Hematology, Soochow University, Suzhou 215123, 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; jyzheng@suda.edu.cn; lusong.luo@beigene.com
Abstract: The Wnt signaling pathway is a critical developmental pathway which operates through
control of cellular functions such as proliferation and differentiation. Aberrant Wnt signaling has been
linked to the formation and metastasis of tumors. Porcupine, a member of the membrane-bound
O-acyltransferase family of proteins, is an important component of the Wnt pathway. Porcupine
catalyzes the palmitoylation of Wnt proteins, a process needed for their secretion and activity. Here we
report a novel series of compounds obtained by a scaffold hybridization strategy from a known
porcupine inhibitor class. The leading compound 59 demonstrated subnanomolar inhibition of Wnt
signaling in a paracrine cellular assay. Compound 59 also showed excellent chemical, plasma and liver
microsomal stabilities. Furthermore, compound 59 exhibited good pharmacokinetic profiles with 30%
oral bioavailability in rat. 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: AUC，area under curve; CE, collision energy; DCM, dichloromethane; DIPEA,
N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DP,
declustering potential; DPPA, diphenylphosphoryl azide; DSH, Dishevelled; EtOAc, ethyl acetate;
FaSSIF, Fasted State Simulated Intestinal Fluid; FBS, fetal bovine serum; HATU,
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HLM, human liver
microsomes;
HPLC,
high
performance
liquid
chromatography;
LiHMDS,
lithium
bis(trimethylsilyl)amide; MLM, mouse liver microsomes; MRM, multiple reaction-monitoring; NFSI,
N-fluorobenzenesulfonimide; NMP, N-methyl-2-pyrrolidone; P.K., pharmacokinetics; SAR,
structure-activity relationship; SEM, standard error measurement; SGF, simulated gastric fluid; TFA
trifluoroacetic acid; THF, tetrahydrofuran; TMS, tetramethylsilane; TMSCN, trimethylsilyl cyanide.
,
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1. Introduction
The Wnt signaling pathway is a pivotal developmental pathway which regulates patterning, growth
and cell migration during embryonic development. In the absence of Wnt ligands stimulation,β-catenin
is degraded by aβ-catenin destruction complex, resulting in the inhibition ofβ-catenin-mediated gene
expressions. Conversely, binding of Wnt ligands to the Frizzled and LRP families of cell surface
receptors initiates a series of signaling events with help from the cytoplasmic protein Dishevelled
(DSH), leading to the accumulation of cytoplasmicβ-catenin. Ultimately,β-catenin translocates to the
nucleus, where it associates with the LEF/TCF family of DNA-binding proteins, resulting in the
activation ofβ-catenin-mediated gene expressions. Aberrant Wnt signaling has been linked to the
formation and metastasis of numerous tumors [1-3]. Multiple approaches e.g. monoclonal antibody
targeting Frizzled receptors, tankyrase inhibitors, have been attempted to modulate the aberrant Wnt
signaling [2,4]. Porcupine, a member of the membrane-bound O-acyltransferase family of proteins, is
an important component of the Wnt pathway. Porcupine catalyzes the palmitoylation of Wnt proteins, a
process needed for their secretion and activity. Thus, inhibition of porcupine can lead to the inhibition
of the aberrant Wnt signaling, therefore represents a promising approach for anticancer therapy [5-7].
The IWP series of compounds (Figure 1) were the first porcupine inhibitors reported in the
literature [8-10]. Since then, other porcupine inhibitors have also been developed [5-7,11,12]. The most
advanced porcupine inhibitor, LGK974, is currently in a phase I clinical trial (Figure 1) [13]. The
development of porcupine inhibitors is still at its infancy, and there is much room for improvement
over the currently available porcupine inhibitors.
2. Design
The evolution of LGK974 is shown in Figure 2. Scientists from the Genomics Institute of the
Novartis Research Foundation detailed their optimization campaign in two recently published patents
[14,15]. Compound GNF-1 is the phenotypical lead and the optimization culminated in the
identification of LGK974, a compound which entered clinical development. Ledipasvir, an extremely
potent NS5A inhibitor developed by Gilead Science for the treatment of hepatitis C virus infection, is a
component of the highly successful drug Harvoni. The central structural element of ledipasvir is
difluoro fluorene, which was selected from a group of tricyclic fused ring classes (Figure 2) [16].
Encouraged by the two aforementioned successful programs, we decided to hybridize the tricyclic
element into the porcupine inhibitor framework. Here we report the design, synthesis and
structure-activity-relationship of the novel potent Wnt inhibitors featuring a tricyclic fused ring system
as shown in Figure 2.
3. Chemistry
The syntheses of the target compounds 16-21 were summarized in Scheme 1. Suzuki coupling of
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1-bromo-4-nitrobenzene with pyridin-3-ylboronic acid afforded nitrobenzene 2, which was
subsequently reduced with H2 and Pd/C to give amine 3. Compound 6 was prepared through Suzuki
coupling of 2-chloropyrazine with aryl boronate 5, which was produced by treating
5-bromopyridin-2-amine with bis(pinacolato)diboron in the presence of Pd(dppf)Cl2 and KOAc.
Commercially available amines 7-9 were converted to the corresponding aryl iodides 10-12 by classic
diazotization-iodination chemistry. Treatment of aryl iodides 10-12 with acetic acid in the presence of
PdCl2, AgOAc and NaOAc using a procedure reported in literature [17] afforded the key intermediates
13-15. Condensation of carboxylic acids 13-15 with the aromatic amine 3 or 6 in the presence of
HATU in DMF gave the target compounds 16-18 and 19-21, respectively.
The key intermediates 25, 32 and 33 were prepared according to Scheme 2. Compound 10 was
converted to nitrile 22 in the presence of Zn(CN)2 and Pd(Ph3P)4. Difluorination of compound 22 with
NFSI and LiHMDS in THF at -78°C provided compound 23, which was subsequently hydrolyzed to
give carboxylic acid 24. The key intermediate 25 was prepared via a Curtius rearrangement by
treatment of carboxylic acid 24 with DPPA in DMF, followed by hydrolysis in DMF and H2O at 100°C.
Suzuki coupling of methyl 4-chloro-3-nitrobenzoate with phenylboronic acid afforded compound 27.
Cyclization of compound 27 with triphenylphosphine in 1,2-dichlorobenzene according to a procedure
reported in literature [18] gave amine 28. Methylation of 28 with CH3I provided compound 29.
Compounds 28 and 29 were hydrolyzed to give carboxylic acids 30 and 31, respectively. The key
intermediates 32 and 33 were achieved by a Curtius rearrangement of the corresponding carboxylic
acids 30 and 31 with DPPA in t-BuOH, followed by de-BOC with TFA/DCM, while the initial attempts
of reacting corresponding carboxylic acids with DPPA in DMF failed.
The synthetic route used to prepare compounds 48-53 and 56-61 was outlined in Scheme 3.
Reaction of 4-bromo-2-methylpyridine with Bu3SnCl in the presence of n-BuLi provided the
corresponding organotin 35. Commercially available nitrile 36 was treated with POCl3 in DMF at
110°C to yield aldehyde 37, which was subsequently reduced with NaBH4 in MeOH to give alcohol 38.
Chlorination of 38 using SOCl2 in CH2Cl2 provided intermediate 39, which was converted to nitrile 40
by the treatment of TMSCN. Compound 40 was hydrolyzed with H2SO4 to give carboxylic acid 41.
Condensation of 41 with corresponding aromatic amines gave intermediates 42-47. The target
compounds 48-53 were synthesized through Stille coupling of compounds 42-47 with organotin 35.
Compound 34 was boronylated and then coupled with ethyl 2-(4-bromophenyl)acetate to give
intermediate 54. Compound 54 was hydrolyzed with NaOH to give the key intermediate 55. The target
compounds 56-61 were achieved using the standard amide condensation conditions by the treatment of
carboxylic acid 55 with corresponding aromatic amines in the presence of HATU in DMF.
4. Results and discussion
4.1. Evaluation of pharmacological activity
The Wnt signaling inhibition of the synthesized compounds was tested using a cell based
super-top flash (STF) reporter gene assay. In this assay, a two-cell paracrine signaling system was used:
HEK293 cells which were stably transfected with “super-top flash” TCF-luciferase reporter plasmid
(the Wnt responding cells) were co-cultured with Wnt generating L Wnt3A cells. The inhibition of the
reporter gene luciferase signaling is an indication of the potency of the compound being evaluated. This
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is the primary assay we used to screen compounds [19]. We first confirmed that both GNF-1 and
LGK974 were active in our assay, and their IC50 numbers matched well with the numbers reported in
the literature [15]. LGK974 was used as a control in our assays. The structure-activity relationship
(SAR) was summarized in Table 1.
Cyclization of the left hand-side rings led to only moderately active compounds, as exemplified
by compounds 16, 18, 19, and 21 (231, 544, 454, and 136 nM, respectively), while compounds 17 and
20 were inactive. This was not unexpected, as the existing SAR indicated that a key hydrogen bond
acceptor was required at this region [14, 15]. We were pleased to see that when this key interaction was
fulfilled, the cyclization of the right hand-side rings provided almost uniformly active compounds.
Here we applied the two most successful bi-aryl pieces from the Novartis optimization campaign to
explore the tricyclic structure-activity relationship on the right hand-side. When R1 was the same as
LGK974, the tricyclic R2 elements fluorene, difluoro-fluorene, fluorene-9-one, and carbazole all
provided very active compounds (compounds 48, 49, 50, 51; 2.5, 2.7, 2.8, 2.6 nM, respectively). The
dibenzonfuran showed slightly decreased activity, while methylation of the carbazole was detrimental
to potency (compounds, 53 and 52; 13 and 49 nM, respectively). When R1 was the same as GNF-1, the
trend was almost the same: the tricyclic R2 elements fluorene, difluoro-fluorene, fluorene-9-one, and
carbazole all provided very active compounds (compounds 56, 57, 58, 59; 1.4, 3.5, 0.45, 2.3 nM,
respectively). The dibenzonfuran showed slightly decreased activity, while methylation of the
carbazole was detrimental to potency (compounds, 61 and 60; 9.0 and 191 nM, respectively). The
addition of the methyl group might have protruded into the narrow space of the porcupine protein
which was evolved to adapt to the long carbon chain of the palmitoyl acid. We were encouraged to see
compound 58 (0.45 nM) was 2-fold more potent than LGK974, while compound 59 and others were
only slightly less potent than LGK974 in our assay. As compounds with common cell toxicity can also
block the reporter gene and therefore cause false positive results, we used a hedgehog reporter gene
assay as a counter screening assay. The representative compounds were tested using a cell based
NIH3T3-GRE-Luc reporter gene assay with 10 nM SAG as the Hh pathway agonist [20-22], and their
Wnt inhibition activity was confirmed not to be from unspecific cell toxicity (data not shown).
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
secretion and activity. Without this critical modification, Wnt proteins cannot be secreted outside of
cells. To confirm that the new compounds 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 h, 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 58, 59, 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.
Even though porcupine is an important post-translational modification protein for the Wnt family,
little is known about porcupine’s structure. Only very recently have mutational data shed some light on
the key residues and regions important for porcupine-mediated Wnt acylation [23]. In spite of the lack
of structural information of the protein, a pharmacophore model was developed based on the limited
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information of existing porcupine inhibitors [24]. Our data are consistent with the pharmacophore
model and could provide additional structural variety for the currently available porcupine inhibitors.
Furthermore, compound 58 was among one of the most potent porcupine inhibitors discovered so far.
Since many of the novel compounds demonstrated very similar potency, it is likely that the
pharmacokinetic profiles and the toxicological profiles will dominate further research for a clinical
candidate.
4.2. Evaluation of chemical stability, rat plasma stability, liver microsomal stability and solubility
Most of the reported porcupine inhibitors featured an amide functionality. It was reported that the
amide group showed varied stability in saline, plasma and under the treatment of liver miscrosomal
enzymes [7]. Our novel template also contained an amide functional group, and we therefore decided to
test the representative compounds 58 and 59 in simulated gastric fluid (SGF), rat plasma and under the
treatment of liver microsomal enzymes. Both compounds 58 and 59 were stable in simulated gastric
fluid after 24 h. Both compounds 58 and 59 were also stable in rat plasma after 8 h. However, while
compound 59 demonstrated moderate clearance under the treatment of human and mouse microsomes
(41 and 67 mL/min/kg, respectively), compound 58 exhibited much faster clearance under the same
conditions (149 and 508 mL/min/kg, respectively). In addition, compound 59 showed better solubility
than compound 58. Based on these results (Table 2), we decided to evaluate compound 59 in rat
pharmacokinetic experiments.
4.3. Evaluation of pharmacokinetic properties
Assessment of the pharmacokinetic properties of a new series at an early stage is advantageous: it
can help with prioritization of multiple templates, identification of potential metabolic hotspots, and
therefore, influence directions of optimization campaigns. We were eager to see if this tricyclic
template could provide compounds which would be metabolically stable to support lead optimization.
Based on the aforementioned reasons, compound 59 was selected to be evaluated in rat P.K. experiment.
When dosed orally at 10mg/kg, compound 59 was quickly absorbed, with a Tmax of 1.0 h. Compound
59 displayed a moderate clearance (22.4 mL/min/kg), a good steady state volume of 2.7 L/kg, and an
excellent half-life of 3.1 h. The Cmax of the oral dosing was 2880 ng/mL. The oral exposure of
compound 59 was AUC = 11293 ng·h/mL, resulting in an estimated bioavailability of 30% (Table 3).
The P.K. results suggested that compound 59 was able to support pharmacodynamic studies, and the
tricyclic template was amenable to lead optimization.
5. Conclusion
We have utilized a scaffold hybridization strategy to design and synthesize a new series of Wnt
inhibitors. This novel scaffold provided numerous compounds (e.g. 48, 58, and 59; 2.5 nM, 0.45 nM,
and 2.3 nM, respectively) with Wnt inhibition activities comparable to or better than that of the clinical
compound LGK974 (0.9 nM). Compound 59 showed good pharmacokinetic profiles with 30% oral
bioavailability in rat. More importantly, the pharmacokinetic profiles of compound 59 suggested that it
could support in vivo efficacy studies and the tricyclic template could support lead optimization
campaign. Structure-activity relationship study of this novel scaffold is ongoing, and results will be
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reported in due course.
6. Experimental protocols
6.1. Chemistry
The progress of the reaction was monitored by analytical thin layer chromatography performed on
silica gel HSGF254 pre-coated plates. All the solvents were dried before usage, and the solvents were
1
removed under reduced pressure. H NMR spectra of the compounds were recorded using Varian 400
MHz NMR spectrometers. 13C NMR spectra of the compounds were recorded using Varian 100 MHz
NMR spectrometers. Chemical shifts were given in ppm using tetramethylsilane (TMS) as internal
standard. Mass spectra were obtained using an Agilent 1100 LC/MSD Trap SL version Mass
Spectrometer. Final compounds were purified by column chromatography with silica gel 100-200
mesh.
6.1.1. 3-(4-Nitrophenyl)pyridine (2)
To a suspension of 1-bromo-4-nitrobenzene (2.0 g, 10 mmol), pyridin-3-ylboronic acid (1.2 g, 10
mmol) and Pd(dppf)Cl2 (73 mg, 0.1 mmol) in 1,4-dioxane (30 mL) and H2O (10 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 treated with water (50 mL) and extracted with CH2Cl2 (50 mL x 3).
The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/1) to give
1
the desired product (1.6 g, 80%) as a yellow solid. H NMR (400 MHz, CDCl3) δ 8.90 (d, J = 2.0 Hz,
1H), 8.70 (d, J = 4.8 Hz, 1H), 8.35 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.8 Hz,
2H), 7.44 (dd, J = 7.6, 4.8 Hz, 1H).
6.1.2. 4-(Pyridin-3-yl)aniline (3)
To a solution of 2 (920 mg, 4.6 mmol) in methanol (20 mL) was added Pd/C (90 mg), and the
reaction mixture was stirred at room temperature under H2 overnight. The mixture was filtered, and the
1
filtrate was concentrated to give the desired product (740 mg, 97%) as a yellow solid. H NMR (400
MHz, CDCl3) δ 8.79 (d, J = 2.0 Hz, 1H), 8.50 (dd, J = 4.8, 1.2 Hz, 1H), 7.84-7.76 (m, 1H), 7.40 (d, J =
8.4 Hz, 2H), 7.31 (dd, J = 8.0, 4.8 Hz, 1H), 6.78 (d, J = 8.4 Hz, 2H), 3.81 (s, 2H).
6.1.3. 5-(Pyrazin-2-yl)pyridin-2-amine (6)
To a suspension of 5-bromopyridin-2-amine (5.0 g, 28.9 mmol), bis(pinacolato)diboron (8.1 g, 31.8
mmol) and Pd(dppf)Cl2 (635 mg, 0.87 mmol) in 1,4-dioxane (20 mL) was added KOAc (8.5 g, 86.7
mmol). The reaction mixture was stirred at 120°C under N2 overnight. After cooling to room
temperature, the mixture was concentrated under reduced pressure. The mixture was diluted with
dichloromethane (200 mL) and then filtered. The filtration was concentrated to give a brown oil (6.39 g,
crude) without any further purification for the next step.
To a solution of the above crude oil (6.39 g), Pd(PPh3)4 (1.0 g, 0.86 mmol) and 2-chloropyrazine
(2.90 g, 25.4 mmol) in 1,4-dioxane (40 mL) and H2O (10 mL) was added K3PO4 (15.5 g, 57.8 mmol).
The mixture was stirred at 120°C under N2 overnight. After cooling to room temperature, the mixture
was evaporated and the residue was purified by silica gel column chromatography (petroleum
ether/ethyl acetate = 1/2) to give the desired product (3.3 g, 66%) as a brown solid. 1H NMR (400 MHz,
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DMSO-d6) δ 9.11 (s, 1H), 8.72 (s, 1H), 8.58 (s, 1H), 8.45 (s, 1H), 8.11 (d, J = 8.8 Hz, 1H), 6.55 (d, J =
8.4 Hz, 1H), 6.43 (s, 2H).
6.1.4. General procedure for the synthesis of compounds 10-12
To a solution of corresponding amine (1 eq) in 18% HCl (12 mL) was added NaNO2 (1 M in H2O,
1.5 eq) at 0°C. The mixture was stirred at 0°C for 1 h, and then NaI (2 M in H2O, 2 eq) was added.
After the mixture was stirred at room temperature for 4 h, Na2SO3 (4 eq) was added. The suspension
was filtered to give the desired products 10-12.
6.1.4.1. 2-Iodo-9H-fluorene (10). Yellow solid (43%); 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.76
(d, J = 7.6 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 8.0 Hz, 2H), 7.41-7.31 (m, 2H), 3.88 (s, 2H).
6.1.4.2. 2-Iodo-9H-fluoren-9-one (11). Yellow solid (52%); 1H NMR (400 MHz, CDCl3) δ 7.97 (s, 1H),
7.82 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.53-7.48 (m, 2H), 7.37-7.31 (m, 1H), 7.29 (d, J =
7.6 Hz, 1H).
1
6.1.4.3. 3-Iododibenzo[b,d]furan (12). Yellow solid (57%); H NMR (400 MHz, CDCl3) δ 7.97-7.10
(m, 2H), 7.72-7.64 (m, 2H), 7.57 (d, J = 8.0 Hz, 1H), 7.52-7.46 (m, 1H), 7.39-7.33 (m, 1H).
6.1.5. General procedure for the synthesis of compounds 13-15
To a solution of corresponding iodide (1 eq), NaOAc (5 eq) and PdCl2 (0.1 eq) in CH3COOH (10
mL/mmol) was added AgOAc (1.5 eq). The mixture was stirred at 130°C under N2 for 12 h. After
cooling to room temperature, the mixture was evaporated and the residue was purified by silica gel
column chromatography (dichloromethane/methanol) to give the desired products 13-15.
1
6.1.5.1. 2-(9H-Fluoren-2-yl)acetic acid (13). Yellow solid (61%); H NMR (400 MHz, DMSO-d6) δ
12.35 (s, 1H), 7.87 (d, J = 7.2 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 7.2 Hz, 1H), 7.47 (s, 1H),
7.40-7.35 (m, 1H), 7.29 (dd, J = 14.8, 6.8 Hz, 2H), 3.90 (s, 2H), 3.64 (s, 2H).
6.1.5.2. 2-(9-Oxo-9H-fluoren-2-yl)acetic acid (14). Yellow solid (60%); 1H NMR (400 MHz, CDCl3) δ
7.64 (d, J = 7.2 Hz, 1H), 7.58 (s, 1H), 7.52-7.45(m, 3H), 7.40 (d, J = 7.2 Hz, 1H), 7.29 (d, J = 6.8 Hz,
1H), 3.69 (s, 2H). ESI-MS (m/z): 239.1 [M+H]+.
6.1.5.3. 2-(Dibenzo[b,d]furan-3-yl)acetic acid (15). Yellow solid (65%); 1H NMR (400 MHz,
DMSO-d6) δ 12.41 (s, 1H), 8.12 (d, J = 7.6 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H),
7.61 (s, 1H), 7.54-7.48 (m, 1H), 7.42-7.36 (m, 1H), 7.30 (d, J = 8.0 Hz, 1H), 3.77 (s, 2H).
6.1.6. General procedure for the synthesis of compounds 16-19 and 21
To a solution of 3 or 6 (1 eq), corresponding acid (1 eq) and N,N-diisopropylethylamine (5 eq) in
DMF (1.5 mL) was added HATU (1 eq). After stirring at room temperature for 12 h, the mixture was
diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers
were washed with brine (50 mL x 3), dried over Na2SO4 and concentrated. The residue was purified by
silica gel column chromatography (dichloromethane/methanol) to give the desired products 16-19 and
21.
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1
6.1.6.1. 2-(9H-Fluoren-2-yl)-N-(4-(pyridin-3-yl)phenyl)acetamide (16). White solid (60%); H NMR
(400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.87 (d, J = 2.4 Hz, 1H), 8.52 (dd, J = 4.4, 0.8 Hz, 1H), 8.04 (d,
J = 8.4 Hz, 1H), 7.89-7.84 (m, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 7.60-7.56 (m,
2H), 7.45 (dd, J = 8.0, 4.8 Hz, 1H), 7.39-7.35 (m, 2H), 7.32-7.28 (m, 1H), 3.92 (s, 2H), 3.75 (s, 2H).
ESI-MS (m/z): 377.2 [M+H]+.
1
6.1.6.2. 2-(9-Oxo-9H-fluoren-2-yl)-N-(4-(pyridin-3-yl)phenyl)acetamide (17). Yellow solid (61%); H
NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.87 (d, J = 2.0 Hz, 1H), 8.52 (dd, J = 4.4, 1.2 Hz, 1H),
8.07-8.03 (m, 1H), 7.79-7.69 (m, 6H), 7.63-7.56 (m, 4H), 7.45 (dd, J = 7.2, 4.8 Hz, 1H), 7.39-7.34 (m,
1H), 3.76 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 193.1, 168.8, 148.0, 147.3, 143.9, 142.4, 139.2,
137.5, 136.1, 135.4, 135.0, 133.6, 133.5, 133.4, 131.8, 129.3, 127.2, 124.8, 123.9, 123.8, 121.1, 121.1,
119.6, 42.8. HRMS (ESI): calcd for C26H19N2O2 [M+H]+ 391.1441, found 391.1445.
1
6.1.6.3. 2-(Dibenzo[b,d]furan-3-yl)-N-(4-(pyridin-3-yl)phenyl)acetamide (18). Yellow solid (40%); H
NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 8.87 (d, J = 1.6 Hz, 1H), 8.52 (dd, J = 4.4, 1.2 Hz, 1H),
8.15-8.08 (m, 2H), 8.06-8.02 (m, 1H), 7.77-7.74 (m, 2H), 7.70 (d, J = 8.8 Hz, 4H), 7.54-7.48 (m, 1H),
7.47-7.43 (m, 1H), 7.42-7.36 (m, 2H), 3.87 (s, 2H). ESI-MS (m/z): 379.2 [M+H]+.
6.1.6.4. 2-(9H-Fluoren-2-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (19). Off white solid (35%);1H
NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 9.30 (s, 1H), 9.10 (d, J = 2.0 Hz, 1H), 8.71 (s, 1H), 8.62
(d, J = 2.4 Hz, 1H), 8.51 (dd, J = 8.8, 2.4 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.88-7.84 (m, 2H),
7.59-7.55 (m, 2H), 7.40-7.34 (m, 2H), 7.32-7.27 (m, 1H), 3.92 (s, 2H), 3.84 (s, 2H). ESI-MS (m/z):
379.2 [M+H]+.
6.1.6.5. 2-(Dibenzo[b,d]furan-3-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (21). Light yellow solid
1
(30%); H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 9.30 (s, 1H), 9.11 (s, 1H), 8.71 (s, 1H),
8.63-8.61 (m, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.22 (d, J = 8.8 Hz, 1H), 8.14-8.06 (m, 2H), 7.73-7.67 (m,
2H), 7.55-7.47 (m, 1H), 7.43-7.37 (m, 2H), 3.96 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 170.2,
155.6, 155.6, 153.0, 149.2, 146.5, 144.4, 143.5, 141.8, 136.4, 135.5, 127.4, 127.2, 124.5, 123.4, 123.1,
122.2, 121.0, 120.8, 113.1, 112.4, 111.6, 43.2. HRMS (ESI): calcd for C23H17N4O2 [M+H]+ 381.1346,
found 381.1340.
6.1.7. 2-(9-Oxo-9H-fluoren-2-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (20)
To a solution of 6 (42 mg, 0.24 mmol), 14 (60 mg, 0.25 mmol) and 4-dimethylaminopyridine (6
mg, 0.048 mmol) in DMF (2 mL) was added dicyclohexylcarbodiimide (60 mg, 0.29 mmol). After
stirring at room temperature for 12 h, the mixture was treated with water (50 mL) and extracted with
ethyl acetate (50 mL x 3). The combined organic layers were washed with brine (50 mL x 3), dried over
Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography
1
(dichloromethane/methanol = 80/1) to give the desired product (30 mg, 31%) as a yellow solid. H
NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 9.29 (d, J = 1.6 Hz, 1H), 9.10 (d, J = 2.0 Hz, 1H), 8.71
(dd, J = 2.4, 1.6 Hz, 1H), 8.62 (d, J = 2.4 Hz, 1H), 8.51 (dd, J = 8.8, 2.4 Hz, 1H), 8.21 (d, J = 8.4 Hz,
1H), 7.77 (dd, J = 7.6, 3.2 Hz, 2H), 7.63-7.57 (m, 4H), 7.39-7.34 (m, 1H), 3.84 (s, 2H). ESI-MS (m/z):
393.2 [M+H]+.
8

ACCEPTED MANUSCRIPT
6.1.8. 9H-Fluorene-2-carbonitrile (22)
To a suspension of 10 (900 mg, 3.08 mmol) and Pd(PPh3)4 (358 mg, 0.31 mmol) in
dimethylformamide (15 mL) was added Zn(CN)2 (216 mg, 1.85 mmol), and the reaction mixture was
stirred at 90°C under N2 for 2 h. After cooling to room temperature, the mixture was treated with water
(50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with
brine (50 mL x 3), dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel
column chromatography (petroleum ether/ethyl acetate = 25/1) to give the desired product (400 mg,
67%) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.88-7.80 (m, 3H), 7.68 (d, J = 7.6 Hz, 1H),
7.60 (d, J = 7.2 Hz, 1H), 7.47-7.38 (m, 2H), 3.96 (s, 2H).
6.1.9. 9,9-Difluoro-9H-fluorene-2-carbonitrile (23)
To a solution of 22 (200 mg, 1.05 mmol) and N-fluorobenzenesulfonimide (992 mg, 3.15 mmol)
in THF (5 mL) was added LiHMDS (3.1 mL, 1 M in THF, 3.1 mmol) at -78°C under N2, and the
reaction mixture was stirred at -78°C under N2 for 4 h. The reaction was quenched by the addition of
NH3/MeOH, and then the mixture was treated with water (50 mL) and extracted with ethyl acetate (50
mL x 3). The combined organic layers were washed with brine (50 mL x 3), dried over Na2SO4 and
concentrated. The resulting residue was purified by silica gel column chromatography (petroleum
ether/ethyl acetate = 20/1) to give the desired product (160 mg, 67%) as a light yellow solid. 1H NMR
(400 MHz, CDCl3) δ 7.89 (s, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.72-7.62 (m, 4H), 7.57-7.52 (m, 1H),
7.49-7.44 (m, 1H).
6.1.10. 9,9-Difluoro-9H-fluorene-2-carboxylic acid (24)
To a solution of 23 (110 mg, 0.48 mmol) in CH3OH (3.5 mL) and H2O (3.5 mL) was added KOH
(806 mg, 14.4 mmol), and the reaction mixture was stirred at 110°C for 5 h. After cooling to room
temperature, the mixture was concentrated under reduced pressure. The pH was adjusted to 3 with 1 N
HCl, and then the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic layers
were dried over Na2SO4, filtered and concentrated to give the desired product (105 mg, 89%) as a white
1
solid. H NMR (400 MHz, DMSO-d6) δ 8.20-8.11 (m, 2H), 8.00 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 7.6
Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.69-7.62 (m, 1H), 7.55-7.49 (m, 1H).
6.1.11. 9,9-Difluoro-9H-fluoren-2-amine (25)
To a solution of 24 (120 mg, 0.49 mmol) and Et3N (75 mg, 0.74 mmol) in dimethylformamide (4
mL) was added diphenylphosphoryl azide (204 mg, 0.74 mmol). The mixture was stirred at room
temperature for 2 h and then stirred at 80°C for 4 h. H2O (0.5 mL) was added. The mixture was stirred
at 100°C for another 1 h. After cooling to room temperature, the mixture was treated with water (50 mL)
and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine (50
mL x 3), dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 5/1) to give the desired product (40 mg, 38%) as a
1
yellow solid. H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 7.6 Hz, 1H), 7.44-7.39 (m, 2H), 7.34 (d, J =
8.4 Hz, 1H), 7.24-7.19 (m, 1H), 6.96-6.92 (m, 1H), 6.73 (d, J = 8.0 Hz, 1H), 3.89 (s, 2H).
6.1.12. Methyl 2-nitro-[1,1'-biphenyl]-4-carboxylate (27)
9

ACCEPTED MANUSCRIPT
To a suspension of 26 (4.32 g, 20 mmol), PhB(OH)2 (2.68 g, 22 mmol) and Pd(PPh3)4 (460 mg,
0.4 mmol) in toluene (30 mL) and H2O (20 mL) was added K2CO3 (5.52 g, 40 mmol). The reaction
mixture was stirred at 110°C under N2 for 18 h. After cooling to room temperature, the mixture was
treated with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers
were washed with brine (50 mL x 3), dried over Na2SO4 and concentrated. The resulting residue was
purified by silica gel column chromatography (petroleum ether/ethyl acetate = 20/1) to give the desired
product (4.5 g, 88%) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 1.2 Hz, 1H),
8.26 (dd, J = 8.0, 1.6 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.48-7.42 (m, 3H), 7.36-7.30 (m, 2H), 3.99 (s,
3H).
6.1.13. Methyl 9H-carbazole-2-carboxylate (28)
To a solution of 27 (4.5 g, 17.5 mmol) in 1,2-dichlorobenzene (50 mL) was added PPh3 (11.5 g,
403.8 mmol). The reaction mixture was stirred at 180°C under N2 for 20 h. After cooling to room
temperature, the resulting residue was purified by silica gel column chromatography (petroleum
1
ether/ethyl acetate = 10/1) to give the desired product (3.5 g, 90%) as a white solid. H NMR (400
MHz, CDCl3) δ 8.27 (br s, 1H), 8.18 (s, 1H), 8.12 (d, J = 8.4 Hz, 2H), 7.95 (dd, J = 8.4, 1.2 Hz, 1H),
7.51-7.46 (m, 2H), 7.31-7.26 (m, 1H), 3.98 (s, 3H).
6.1.14. Methyl 9-methyl-9H-carbazole-2-carboxylate (29)
To a solution of 28 (1.0 g, 4.44 mmol) in dry DMF (15 mL) was added NaH (266 mg, 80%, 8.88
mmol) at 0°C. The mixture was stirred at 0°C under N2 for 1 h, and then CH3I (1.26 g, 8.88 mmol) was
added. The mixture was stirred at room temperature for 5 h. The mixture was quenched with saturated
aqueous ammonium chloride and extracted with ethyl acetate (50 mL x 3). The combined organic
layers were washed with brine (50 mL x 3), dried over Na2SO4 and concentrated. The resulting residue
was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 20/1) to give the
desired product (1 g, 94%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.19-8.08 (m, 3H), 7.94 (d,
J = 8.4 Hz, 1H), 7.58-7.52 (m, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.30-7.26 (m, 1H), 3.99 (s, 3H), 3.92 (s,
3H).
6.1.15. General procedure for the synthesis of compounds 30 and 31
To a solution of corresponding ester (1 eq) in CH3CH2OH (25 mL/mmol) and H2O (5 mL/mmol)
was added NaOH (15 eq), and the reaction mixture was refluxed for 16 h. After cooling to room
temperature, the pH was adjusted to 4 with 6 N HCl and then the ethanol was evaporated. The resulting
precipitate was filtered and washed with H2O (10 mL) to give the desired products 30 and 31.
1
6.1.15.1. 9H-Carbazole-2-carboxylic acid (30). White solid (95%); H NMR (400 MHz, DMSO-d6) δ
11.37 (s, 1H), 8.10 (d, J = 7.6 Hz, 1H), 8.07-8.01 (m, 2H), 7.76 (dd, J = 8.0, 1.2 Hz, 1H), 7.49 (d, J =
8.4 Hz, 1H), 7.40-7.35 (m, 1H), 7.17-7.12 (m, 1H).
1
6.1.15.2. 9-Methyl-9H-carbazole-2-carboxylic acid (31). White solid (86%); H NMR (400 MHz,
DMSO-d6) δ 8.13 (d, J = 7.6 Hz, 1H), 8.08 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.79 (dd, J = 8.0, 0.8 Hz,
1H), 7.59 (d, J = 8.4 Hz, 1H), 7.48-7.42 (m, 1H), 7.22-7.15 (m, 1H), 3.89 (s, 3H).
6.1.16. General procedure for the synthesis of compounds 32 and 33
10

ACCEPTED MANUSCRIPT
To a solution of corresponding acid (1 eq) and Et3N (1.1 eq) in t-BuOH (8 mL/mmol) was added
diphenylphosphoryl azide (1.1 eq), and the reaction mixture was refluxed for 16 h. After cooling to
room temperature, the mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL
x 3). The combined organic layers were washed with brine (50 mL x 3), dried over Na2SO4 and
concentrated. The residue was dissolved in dichloromethane (2 mL) and TFA (1 mL). The mixture was
stirred at room temperature for 2 h, and then the solvent was removed by vacuum. The resulting residue
was portioned between dichloromethane (20 mL) and saturated sodium carbonate solution (5 mL). The
organic layer was separated, dried over Na2SO4 and concentrated. The resulting residue was purified by
silica gel column chromatography (dichloromethane/ methanol) to give the desired products 32 and 33.
1
6.1.16.1. 9H-Carbazol-2-amine (32) Yellow solid (38%); H NMR (400 MHz, CDCl3) δ 7.91 (d, J =
7.2 Hz, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.35-7.28 (m, 2H), 7.20-7.14 (m, 1H), 6.69 (d, J = 1.2 Hz, 1H),
6.62 (dd, J = 8.0, 1.6 Hz, 1H), 3.82 (s, 2H).
1
6.1.16.2. 9-Methyl-9H-carbazol-2-amine (33) Yellow solid (39%); H NMR (400 MHz, CDCl3) δ 7.93
(d, J = 7.6 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.38-7.29 (m, 2H), 7.20-7.15 (m, 1H), 6.68-6.60 (m, 2H),
3.75 (s, 3H).
6.1.17. 2-Methyl-4-(tributylstannyl)pyridine (35)
To a solution of 4-bromo-2-methylpyridine (2.0 g, 11.6 mmol) in ether (50 mL) was added n-BuLi
(5.1 mL, 12.8 mmol, 2.5 M in hexane) at -78°C. The reaction mixture was stirred at -78°C for 30 min
before the addition of Bu3SnCl (4.0 mL, 14.0 mmol). The mixture was then stirred at -78°C for 30 min.
The mixture was warmed to 0°C slowly before the addition of saturated aqueous NaHCO3 and
extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine (50 mL
x 3), dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 100/1) to give the desired product (1.6 g, 36%) as a
light yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 4.4 Hz, 1H), 7.22 (s, 1H), 7.15 (d, J = 4.4
Hz, 1H), 2.52 (s, 3H), 1.56-1.50 (m, 6H), 1.38-1.27 (m, 6H), 1.09-1.05 (m, 6H), 0.89 (t, J = 7.2 Hz,
9H).
6.1.18. 6-Chloro-5-methylnicotinaldehyde (37)
POCl3 (39 mL, 548 mmol) was stirred at 90°C under N2 followed by addition of
2-methyl-2-butennitrile (20.2 g, 70%, 175 mmol). DMF (33 mL, 548 mmol) was added slowly while
keeping the temperature at 100-110°C. The reaction mixture was stirred at 110°C for 15 h, cooled
down to 0°C and carefully quenched with ice water. The mixture was extracted with ethyl acetate (100
mL x 3), and the combined organic layers were washed with brine (100 mL x 3), dried over Na2SO4
and concentrated. The resulting residue was purified by silica gel column chromatography (petroleum
ether/ethyl acetate = 10/1) to give the desired product (4 g, 15%) as a light yellow solid. 1H NMR (400
MHz, CDCl3) δ 10.07 (s, 1H), 8.69 (s, 1H), 8.02 (s, 1H), 2.48 (s, 3H).
6.1.19. (6-Chloro-5-methylpyridin-3-yl)methanol (38)
To a solution of 37 (3.20 g, 20.7 mmol) in MeOH (22 mL) was added NaBH4 (863 mg, 22.7
mmol) slowly at 0°C. The reaction mixture was stirred at room temperature for 2 h. A saturated
aqueous NH4Cl solution (20 mL) was then added and the mixture was stirred for 0.5 h. After the
11

ACCEPTED MANUSCRIPT
solvent was removed by vacuum, the residue was treated with water (50 mL) and extracted with
dichloromethane (50 mL x 3), and the combined organic layers were dried over Na2SO4 and
concentrated. The resulting residue was purified by silica gel column chromatography (petroleum
ether/ethyl acetate = 2/1) to give the desired product (3.2 g, 88%) as a light yellow solid. 1H NMR (400
MHz, CDCl3) δ 8.09 (s, 1H), 7.56 (s, 1H), 4.65 (s, 2H), 2.33 (s, 3H).
6.1.20. 2-Chloro-5-(chloromethyl)-3-methylpyridine (39)
To a solution of 38 (3.20 g, 18.3 mmol) in DCM (20 mL) was added SOCl2 (10.9 g, 91.4 mmol)
slowly at 0°C. The mixture was stirred at 0°C under N2 for 2 h. The mixture was then treated with
water (20 mL) and extracted with dichloromethane (50 mL x 3), and the combined organic layers were
dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 2/1) to give the desired product (3.47 g, 100%) as a
yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.23 (s, 1H), 7.61 (s, 1H), 4.54 (s, 2H), 2.40 (s, 3H).
6.1.21. 2-(6-Chloro-5-methylpyridin-3-yl)acetonitrile (40)
To a suspension of 39 (3.47 g, 19.8 mmol), K2CO3 (5.47 g, 39.7 mmol) and KI (6.58 g, 39.7 mmol)
was added TMSCN (3.93 g, 39.7 mmol) slowly at 0°C. The mixture was stirred at room temperature
for 4 h. The mixture was then treated with a saturated aqueous NaHCO3 solution (20 mL) and extracted
with dichloromethane (50 mL x 3), and the combined organic layers were dried over Na2SO4 and
concentrated. The resulting residue was purified by silica gel column chromatography (petroleum
ether/ethyl acetate = 2/1) to give the desired product (2.4 g, 73%) as a white solid. 1H NMR (400 MHz,
CDCl3) δ 8.17 (s, 1H), 7.57 (s, 1H), 3.73 (s, 2H), 2.40 (s, 3H).
6.1.22. 2-(6-Chloro-5-methylpyridin-3-yl)acetic acid (41)
The mixture of 40 (800 mg, 4.79 mmol) in water (9 mL) was stirred, followed by addition of 98%
H2SO4 (6.77 mL) slowly at 0°C. The mixture was stirred at 100°C under N2 for 4 h. After cooling to
room temperature, the pH was adjusted to 3 with a saturated aqueous NaHCO3, and then the mixture
was extracted with dichloromethane (50 mL x 3). The combined organic layers were dried over Na2SO4
1
and concentrated to give the desired product (746 mg, 84%) as a white solid. H NMR (400 MHz,
CDCl3) δ 9.80 (br s, 1H), 8.18 (s, 1H), 7.55 (s, 1H), 3.63 (s, 2H), 2.37 (s, 3H).
6.1.23. General procedure for the synthesis of compounds 42-47
To a solution of 41 (1 eq), corresponding amine (1 eq) and N,N-diisopropylethylamine (5 eq) in
dimethylformamide (1.5 mL) was added HATU (1 eq). After stirring at room temperature for 12 h, the
mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined
organic layers were washed with brine (50 mL x 3), dried over Na2SO4 and concentrated. The resulting
residue was purified by silica gel column chromatography (dichloromethane/ methanol) to give the
desired products 42-47.
1
6.1.23.1. 2-(6-Chloro-5-methylpyridin-3-yl)-N-(9H-fluoren-2-yl)acetamide (42) White solid (87%); H
NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 7.84 (s, 1H), 7.67-7.63 (m, 3H), 7.46 (d, J = 6.8 Hz, 1H), 7.36
(d, J = 7.6 Hz, 1H), 7.31-7.26 (m, 2H), 7.23-7.20 (m, 1H), 3.81 (s, 2H), 3.60 (s, 2H), 2.33 (s, 3H).
6.1.23.2. 2-(6-Chloro-5-methylpyridin-3-yl)-N-(9,9-difluoro-9H-fluoren-2-yl)acetamide (43). Yellow
12

ACCEPTED MANUSCRIPT
solid (68%); 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 7.77 (s, 1H), 7.65-7.57 (m, 3H), 7.52-7.42 (m,
4H), 7.34-7.29 (m, 1H), 3.69 (s, 2H), 2.40 (s, 3H).
6.1.23.3. 2-(6-Chloro-5-methylpyridin-3-yl)-N-(9-oxo-9H-fluoren-2-yl)acetamide (44). Yellow solid
(78%); 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.65 (s, 1H), 7.55 (s, 2H),
7.39 (d, J = 6.4 Hz, 3H), 7.20-7.15 (m, 1H), 3.59 (s, 2H), 2.33 (s, 3H).
6.1.23.4. N-(9H-Carbazol-2-yl)-2-(6-chloro-5-methylpyridin-3-yl)acetamide (45). Off white solid
(63%); 1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 10.34 (s, 1H), 8.22 (s, 1H), 8.04-7.96 (m, 3H),
7.78 (s, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.35-7.29 (m, 1H), 7.21 (dd, J = 8.4, 1.6 Hz, 1H), 7.14-7.09 (m,
1H), 3.74 (s, 2H), 2.34 (s, 3H).
6.1.23.5. 2-(6-Chloro-5-methylpyridin-3-yl)-N-(9-methyl-9H-carbazol-2-yl)acetamide (46). Yellow
solid (40%); 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.22 (s, 1H), 8.08-8.02 (m, 3H), 7.79 (s,
1H), 7.55 (d, J = 8.0 Hz, 1H), 7.43-7.37 (m, 1H), 7.25 (d, J = 8.4 Hz, 1H), 7.20-7.14 (m, 1H), 3.79 (s,
3H), 3.75 (s, 2H), 2.34 (s, 3H).
6.1.23.6. 2-(6-Chloro-5-methylpyridin-3-yl)-N-(dibenzo[b,d]furan-3-yl)acetamide (47). white solid
1
(65%); H NMR (400 MHz, DMSO-d6) δ 10.56 (s, 1H), 8.21 (d, J = 1.6 Hz, 1H), 8.13 (d, J = 1.2 Hz,
1H), 8.05 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 1.2 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.49-7.43 (m, 2H),
7.40-7.35 (m, 1H), 3.76 (s, 2H), 2.34 (s, 3H).
6.1.24. General procedure for the synthesis of compounds 48-53
To a suspension of corresponding chloride (1 eq) and Pd(PPh3)4 (0.1 eq) in DMF (2 mL) was
added 35 (1.8 eq), and the reaction mixture was stirred at 120°C for 20 h. After cooling to room
temperature, the mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 3).
The combined organic layers were washed with brine (50 mL x 3), dried over Na2SO4 and concentrated.
The resulting residue was purified by silica gel column chromatography (dichloromethane/ methanol)
to give the desired products 48-53.
6.1.24.1. 2-(2',3-Dimethyl-[2,4'-bipyridin]-5-yl)-N-(9H-fluoren-2-yl)acetamide (48). White solid
(60 %); 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.52 (d, J = 4.8 Hz, 1H), 8.48 (d, J = 1.2 Hz,
1H), 7.93 (s, 1H), 7.83-7.81 (m, 2H), 7.73 (d, J = 1.2 Hz, 1H), 7.57-7.53 (m, 2H), 7.42 (s, 1H),
7.37-7.33 (m, 2H), 7.28-7.24 (m, 1H), 3.89 (s, 2H), 3.76 (s, 2H), 2.53 (s, 3H), 2.34 (s, 3H).
6.1.24.2.
N-(9,9-Difluoro-9H-fluoren-2-yl)-2-(2',3-dimethyl-[2,4'-bipyridin]-5-yl)acetamide
(49).
White solid (50%); 1H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 5.2 Hz, 1H), 8.50 (s, 1H), 7.78 (s, 1H),
7.70-7.65 (m, 2H), 7.60 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 6.8 Hz, 3H), 7.48-7.42 (m, 1H), 7.36-7.29 (m,
2H), 7.28-7.26 (m, 1H), 3.77 (s, 2H), 2.64 (s, 3H), 2.39 (s, 3H). ESI-MS (m/z): 442.2 [M+H]+.
6.1.24.3. 2-(2',3-Dimethyl-[2,4'-bipyridin]-5-yl)-N-(9-oxo-9H-fluoren-2-yl)acetamide (50). Yellow
solid (57%); 1H NMR (400 MHz, DMSO-d6) δ 10.56 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.47 (d, J = 1.6
Hz, 1H), 7.95 (s, 1H), 7.73-7.72 (m, 3H), 7.69 (s, 1H), 7.60-7.56 (m, 2H), 7.42 (s, 1H), 7.38-7.29 (m,
2H), 3.77 (s, 2H), 2.53 (s, 3H), 2.34 (s, 3H).
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6.1.24.4. N-(9H-Carbazol-2-yl)-2-(2',3-dimethyl-[2,4'-bipyridin]-5-yl)acetamide (51). White solid
(71%); 1H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 10.38 (s, 1H), 8.55-8.48 (m, 2H), 8.04-7.98 (m,
3H), 7.74 (s, 1H), 7.45-7.39 (m, 2H), 7.36 (d, J = 4.8 Hz, 1H), 7.34-7.29 (m, 1H), 7.23 (dd, J = 8.4, 1.2
Hz, 1H), 7.15-7.09 (m, 1H), 3.78 (s, 2H), 2.53 (s, 3H), 2.35 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
168.3, 157.9, 153.7, 148.7, 147.7, 147.6, 140.1, 139.9, 139.5, 137.1, 131.3, 130.5, 124.8, 123.0, 122.5,
120.9, 120.3, 119.6, 118.6, 118.3, 111.1, 110.7, 101.3, 39.9, 24.1, 19.4. HRMS (ESI): calcd for
C26H23N4O [M+H]+ 407.1866, found 407.1871.
6.1.24.5. 2-(2',3-Dimethyl-[2,4'-bipyridin]-5-yl)-N-(9-methyl-9H-carbazol-2-yl)acetamide. (52) White
1
solid (62%); H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.56-8.48 (m, 2H), 8.10-8.04 (m, 3H),
7.75 (s, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.44-7.38 (m, 2H), 7.36 (d, J = 4.8 Hz, 1H), 7.28 (dd, J = 8.4, 1.2
Hz, 1H), 7.20-7.14 (m, 1H), 3.80 (s, 5H), 2.54 (s, 3H), 2.36 (s, 3H). HRMS (ESI):
calcd for C27H25N4O [M+H]+ 421.2023, found 421.2025.
6.1.24.6. N-(Dibenzo[b,d]furan-3-yl)-2-(2',3-dimethyl-[2,4'-bipyridin]-5-yl)acetamide (53). White solid
1
(57%); H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.53 (d, J = 5.2 Hz, 1H), 8.49 (d, J = 1.6 Hz,
1H), 8.16 (d, J = 1.6 Hz, 1H), 8.06 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 1.6 Hz, 1H), 7.66 (d, J = 8.0 Hz,
1H), 7.51-7.42 (m, 3H), 7.40-7.35 (m, 2H), 3.81 (s, 2H), 2.54 (s, 3H), 2.35 (s, 3H). ESI-MS (m/z):
408.2 [M+H]+.
6.1.25. Ethyl 2-(4-(2-methylpyridin-4-yl)phenyl)acetate (54)
To a suspension of 4-bromo-2-methylpyridine (500 mg, 2.90 mmol), bis(pinacolato)diboron (795
mg, 3.13 mmol) and Pd(dppf)Cl2 (42 mg, 0.058 mmol) in THF (10 mL) was added KOAc (568 mg,
5.80 mmol). The reaction mixture was stirred at 80°C under N2 overnight. After cooling to room
temperature, the mixture was diluted with dichloromethane (200 mL) and then filtered. The filtration
was concentrated to give the desired product (1.27 g, crude) as a black oil, which was used directly in
the next step without further purification.
To a suspension of the above crude oil (1.27 g), ethyl 2-(4-bromophenyl)acetate (640 mg, 2.63
mmol) and Pd(PPh3)4 (243 mg, 0.21 mmol) in THF (10 mL) was added Cs2CO3 (1.7 g, 5.26 mmol).
The mixture was stirred at 75°C under N2 for 12 h. After cooling to room temperature, the mixture was
evaporated and the residue was purified by silica gel column chromatography (petroleum ether/ethyl
acetate = 5/1) to give the desired product (500 mg, 75%) as a colorless oil. 1H NMR (400 MHz, CDCl3)
δ 8.51 (d, J = 5.2 Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 7.34 (s, 1H), 7.29 (d, J =
5.2 Hz, 1H), 4.16 (q, J = 7.2 Hz, 2H), 3.66 (s, 2H), 2.60 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H).
6.1.26. 2-(4-(2-Methylpyridin-4-yl)phenyl)acetic acid (55)
To a solution of 54 (500 mg, 1.96 mmol) in CH3OH (6 mL) and H2O (2 mL) was added NaOH
(157 mg, 3.92 mmol), and the reaction mixture was stirred at room temperature for 2 h. After the
solvent was removed by vacuum, the pH was adjusted to 6 with 1 N HCl. The resulting precipitate was
1
filtered and washed with H2O (10 mL) to give the desired product (356 mg, 80%) as a white solid. H
NMR (400 MHz, DMSO-d6) δ 8.48 (d, J = 4.8 Hz, 1H), 7.73 (d, J = 8.0 Hz, 2H), 7.58 (s, 1H), 7.49 (d,
J = 5.2 Hz, 1H), 7.39 (d, J = 8.0 Hz, 2H), 3.64 (s, 2H), 2.52 (s, 3H).
14

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6.1.27. General procedure for the synthesis of compounds 56-61
To a solution of 55 (1 eq), corresponding amine (1 eq) and N,N-diisopropylethylamine (5 eq) in
DMF (1.5 mL) was added HATU (1 eq). After stirring at room temperature for 12 h, the mixture was
diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers
were washed with brine (50 mL x 3), dried over Na2SO4 and concentrated. The resulting residue was
purified by silica gel column chromatography (dichloromethane/methanol) to give the desired products
56-61.
6.1.27.1. N-(9H-Fluoren-2-yl)-2-(4-(2-methylpyridin-4-yl)phenyl)acetamide (56). White solid (75%);
1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 7.94 (s, 1H), 7.80 (d, J = 8.4
Hz, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.60-7.47 (m, 6H), 7.37-7.31 (m, 1H), 7.28-7.23 (m, 1H), 3.88 (s,
2H), 3.75 (s, 2H), 2.52 (s, 3H). ESI-MS (m/z): 390.8 [M+H]+.
6.1.27.2. N-(9,9-Difluoro-9H-fluoren-2-yl)-2-(4-(2-methylpyridin-4-yl)phenyl)acetamide (57). White
1
solid (60%); H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.08 (s, 1H),
7.80-7.74 (m, 4H), 7.70-7.65 (m, 2H), 7.59-7.54 (m, 2H), 7.49 (d, J = 8.0 Hz, 3H), 7.40-7.35 (m, 1H),
3.76 (s, 2H), 2.52 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 169.3, 158.6, 149.5, 147.02, 140.2, 138.9
3
2
2
3
(t, JC-F = 4.8 Hz), 137.5 (t, JC-F = 25.0 Hz), 136.8, 136.6 (t, JC-F = 24.5 Hz), 135.8, 133.5 (t, JC-F
=
4.8 Hz), 132.9, 130.0, 128.5, 126.8, 123.7, 123.1 (t, 1JC-F = 242.9 Hz), 122.6, 121.8, 120.7, 120.3, 118.3,
114.2, 43.0, 24.2. HRMS (ESI): calcd for C27H21F2N2O [M+H]+ 427.1616, found 427.1618.
6.1.27.3. 2-(4-(2-Methylpyridin-4-yl)phenyl)-N-(9-oxo-9H-fluoren-2-yl)acetamide (58). Orange solid
1
(69%); H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 7.95 (s, 1H),
7.79-7.67 (m, 5H), 7.61-7.54 (m, 3H), 7.51-7.46 (m, 3H), 7.34-7.29 (m, 1H), 3.74 (s, 2H), 2.52 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ 193.0, 169.2, 158.5, 149.5, 147.0, 144.1, 140.4, 138.5, 136.8, 135.8,
135.5, 134.1, 133.5, 130.0, 128.7, 126.8, 124.7, 124.0, 121.7, 120.6, 120.3, 118.3, 114.7, 43.0,
24.1.ESI-MS (m/z): 405.0 [M+H]+. HRMS (ESI): calcd for C27H21N2O2 [M+H]+ 405.1598, found
405.1598.
6.1.27.4. N-(9H-Carbazol-2-yl)-2-(4-(2-methylpyridin-4-yl)phenyl)acetamide (59). White solid (70%);
1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 10.34 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.07-7.96 (m,
3H), 7.77 (d, J = 8.0 Hz, 2H), 7.58 (s, 1H), 7.54-7.48 (m, 3H), 7.41 (d, J = 8.0 Hz, 1H), 7.34-7.28 (m,
1H), 7.22 (d, J = 8.4 Hz, 1H), 7.14-7.08 (m, 1H), 3.76 (s, 2H), 2.52 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 168.7, 158.5, 149.5, 147.1, 140.1, 139.9, 137.3, 137.2, 135.7, 130.0, 126.8, 124.8, 122.5,
120.3, 120.2, 119.6, 118.5, 118.3, 118.2, 111.1, 110.7, 101.2, 43.2, 24.2. HRMS (ESI): calcd for
C26H22N3O [M+H]+ 392.1757, found 392.1757.
6.1.27.5. N-(9-Methyl-9H-carbazol-2-yl)-2-(4-(2-methylpyridin-4-yl)phenyl)acetamide (60). White
solid (55%); 1H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 4.8 Hz, 1H), 8.12 (s, 1H), 8.01 (d, J = 7.6 Hz,
1H), 7.94 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.47-7.33 (m, 5H),
7.24-7.20 (m, 1H), 6.84 (dd, J = 8.4, 1.6 Hz, 1H), 3.86 (s, 2H), 3.82 (s, 3H), 2.64 (s, 3H). HRMS (ESI):
calcd for C27H24N3O [M+H]+ 406.1914, found 406.1914.
6.1.27.6. N-(Dibenzo[b,d]furan-3-yl)-2-(4-(2-methylpyridin-4-yl)phenyl)acetamide (61). Off white
15

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1
solid (61%); H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.16 (s, 1H),
8.05 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 7.6 Hz, 2H), 7.66 (d, J = 8.4 Hz, 1H), 7.58 (s, 1H), 7.53-7.43 (m,
5H), 7.40-7.34 (m, 1H), 3.78 (s, 2H), 2.52 (s, 3H). ESI-MS (m/z): 393.0 [M+H]+.
6.2. In vitro biological assays
6.2.1. Super-Top Flash (STF) Reporter Gene Assay
L Wnt3A cells (CRL-2647, ATCC) were maintained in DMEM (Gibico) supplemented with 10%
FBS (Hyclone). HEK293 STF stable clones (HEK293 cells stably transfected with "Super-Top Flash"
TCF-luciferase reporter plasmid) were maintained in complete culture medium (DMEM with 4 mM
L-Gln, 1.5 g/L sodium bicarbonate and 4.5 g/L glucose supplemented with 6 ꢀg/mL Blasticidin and
10% FBS). L Wnt3A cells and HEK293 STF cells were harvested when 90% confluence and cell
suspension was mixed with a fixed ratio of 1:1(L Wnt3A: HEK293 STF). 100 ꢀL/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 h before adding compounds.
Compounds were serially diluted in DMSO and further diluted with DMEM medium. 20 ꢀL 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 h.
Following the 48 h incubation, 50 ꢀL luciferase solution (Bright-Glo, Promega) was added to each
well. 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
The detailed experimental procedure has been reported before [17, 18].
6.2.3. Wnt secretion assay
HEK293T cells were cultured in 6-well plates. HEK293T cells were transfected with 1ꢀg
pLibin-WNT3A plasmid or vehicle control and treated with 0.1 ꢀM compounds (58, 59, and LGK974)
simultaneously when cells reached 60% confluence. After 48 h, 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 ꢀg 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 58 and 59 were dissolved in 8% DMSO and 92% simulated gastric fluid (SGF) and kept
at 40°C for 24 h. The extent of degradation was quantified by HPLC using the respective UV
absorption peak areas at 254 nm. These two compounds were tested at 200 ꢀM concentration.
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SGF: 3.8 mL of conc. HCl diluted to 1000 mL of water
6.4. Plasma stability test
The stability of test compounds 58 and 59 in rat plasma was evaluated by incubating each compound
(1 ꢀM) with blank rat plasma. After incubation for 8 h, samples were de-proteinized by the addition of
100 ꢀL of acetonitrile with internal standard. The samples were centrifuged at 13,000 rpm for 10 min,
and the supernatants were analyzed by LC-MS/MS system.
6.5. Metabolic stability test
To determine the metabolic stability in vitro, test compounds 58 and 59 were incubated with mouse
and human liver microsomes. The experimental procedures were set as follows: the reaction mixtures
consisted of microsomes (0.2 mg/mL), NADPH (1 mM), phosphate buffer (100 mM, pH 7.4), and
substrate (1 ꢀM). After a 10-min pre-incubation, the reaction was initiated by the addition of NADPH.
The incubations were obtained at three time points (0, 30, 60 min) and terminated with 100 ꢀL 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)
t
1/2
=
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,in vitro
=
×
mgof proteinper mlincubation
(2)
b
gof liver
×
kgof body weight
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, in vitro
+ Clint, in vitro
Clhepatic
=
(3)
Q
h
Where Qh is the hepatic blood flow (90 mL/min for mouse and 20.7 mL/min for human).
6.6 Rat pharmacokinetic experiments
6.6.1. Animal experiments
Healthy male Spraguee-Dawley rats (200-250 g) were purchased from Slac Laboratory Animals
(Shanghai, China). Rats were housed on a 12 h light and dark cycle with access to food and water.
Before dosing, rats were fasted for 12 h. Free access to water and food was allowed approximately 4 h
post dosing. Compound 59 was dissolved in formulation (NMP/Tween80/PEG400/Water, 10/1/50/39,
v/v/v/v) at 1 mg/mL. The dosing solution was administered via tail vein injection at 2 mL/kg or oral
gavage at 10 mL/kg. Blood samples were collected at specified time points, and then centrifuged at
13,000 rpm for 10 min to obtain plasma, which was stored at -80°C until analysis. 25 ꢀL plasma
sample was extracted with 100 ꢀL cold acetonitrile containing internal standard, and then centrifuged at
13,000 rpm for 10 min to precipitate plasma proteins. An aliquot of 10 ꢀL supernatant was injected
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onto the LC-MS/MS system for analysis. Pharmacokinetic parameters were calculated using
noncompartmental methods in WinNolin 6.2 software (Pharsight).
6.6.2. LC-MS/MS analysis
All samples were analyzed by a LC-MS/MS system, which contains an API 4000 Qtrap mass
spectrometer equipped with a turbo-V ionization source (Applied Biosystems, Ontatrio, Canada),
DGU-20A solvent degasser, two LC-20AD pumps with a CBM-20A controller and a SIL-20A
autosampler (Shimadzu, Columbia, MD, USA). Chromatographic separation was achieved with an
Agela Venusil XBP C18 column (50 × 2.1mm; 5ꢀm) and the column temperature was maintained at
40°C. Gradient elution at a flow rate of 0.3 mL/min was performed using the following mobile phase:
A, acetonitrile:water:formic acid (5:95:0.1) and B, acetonitrile:water: formic acid (95:5:0.1).
For MS/MS quantitation, the API 4000 Qtrap mass spectrometer was operated in the ESI positive
mode with multiple reaction-monitoring (MRM). The MS/MS parameters were set as follows: curtain
gas, 30 psi; turbo gas (GS2), 55 psi; ion source temperature, 450°C; nebulizer gas (GS1), 55 psi and ion
spray voltage, 4500 V. All compounds were monitored with dwell time set to 100 ms. Data were
collected using transitions of m/z 405.3→183.2 (58); m/z 392.0→183.2 (59). For the selected ion
transitions, the declustering potential (DP) and the collision energy (CE) were optimized for the best
sensitivity for each analyte, and processed using AB Sciex Analysist 1.5.2 data collection and
integration software.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant No.81273372,
81473090, 81473278), China Postdoctoral Science Foundation (2013M541729), 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).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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H.J. Zhang, S.L. Yang, X.H. Zhang, Design, synthesis, and structure-activity-relationship of
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Figure Captions
Fig.1 Examples of porcupine inhibitors in the literature
Fig.2 The evolution of LGK974 and the hybridization strategy for the novel Wnt inhibitors
Fig.3 Wnt secretion assay. Compound concentration was at 0.1 ꢀM
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Table 1. SAR of designed compounds
Compd
R1
R2
IC50 (nM) Compd
± SEMa
R1
R2
IC50 (nM)
± SEMa
2.9 ± 0.6
2.6 ± 0.5
GNF-1
51
231 ± 67
>1000
49 ± 1
13 ± 4
16
17
18
19
20
21
48
49
50
52
53
56
57
58
59
60
61
544 ±
299
1.4 ± 0
3.5 ± 1.1
454 ±
195
>1000
0.45 ±
0.22
136 ± 32
2.5 ± 0.8
2.7 ± 1.1
2.8 ± 1.5
2.3 ± 1.0
191 ± 75
9.0 ± 4.0
LGK
974
0.90 ±
0.1b
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).
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Table 2. Stability of representative compounds in simulated gastric fluid, rat plasma, liver microsomal
enzymes, and thermodynamic solubility in FaSSIF (pH 6.5)
% Remaining
in SGF, 24 ha
99%
% Remaining
in rat plasma, 8 hb
100%
Clint (mL/min/kg)
t1/2 (min)
Solubility
(µg/mL)e
< 1
HLMc
MLMd
HLM
MLM
12
Compd
58
149
508
12
43
100%
100%
41
67
91
2
59
a These two compounds were tested at 200 ꢀM concentration in simulated gastric fluid (SGF) at 40°C for 24 h.
b These two compounds were tested at 1 ꢀM concentration.
c HLM = human liver microsomes. These two compounds were tested at 1 ꢀM concentration.
d MLM = mouse liver microsomes. These two compounds were tested at 1 ꢀM concentration.
e 4 mg/mL suspension of each tested compound in FaSSIF (pH 6.5) was shaken for 1 h, then equilibrated overnight at room temperature.
Concentrations of the supernatants after centrifugation were determined by LCMS/MS detection.
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Table 3. Rat Pharmacokinetic Profile of compound 59
compound 59a
route
i.v.
2
p.o.
10
dose (mg/kg)
AUC0-24h (ng·h/mL)
CL (mL·min-1·kg-1)
Vd,ss (L/kg)
7442
22.4
2.7
11293
C
max (ng/mL)
1950
2880
1
Tmax (h)
t1/2 (h)
F%
2.5
3.1
30
a Formulated in a solution of 10% NMP, 1% Tween80, 50% PEG40, and 39% Water.
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Figure 1
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Figure 2
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Figure 3
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Scheme 1. Reagents and conditions. (i) pyridin-3-ylboronic acid, Pd(dppf)Cl2, K2CO3, dioxane, H2O,
100°C, 12 h; (ii) Pd/C, MeOH, r. t., overnight; (iii) bis(pinacolato)diboron, KOAc, Pd(dppf)Cl2,
dioxane, 120°C, overnight; (iv) 2-chloropyrazine, Pd(PPh3)4, K3PO4, dioxane, H2O, 120°C, overnight;
(v) HCl, NaNO2, 0°C, 1 h, then NaI, H2O, r. t., 4 h; (vi) PdCl2, AgOAc, NaOAc, AcOH, 130°C, 12 h;
(vii) for 16-19 and 21: HATU, DIPEA, 3 or 6, DMF, r. t., 12 h; (viii) for 20: DCC , DMAP, 6, DMF, r. t.,
12 h.
Scheme 2. Reagents and conditions. (i) Zn(CN)2, Pd(Ph3P)4, DMF, 90°C, 2 h; (ii) NFSI, LiHMDS,
THF, -78°C, 4 h; (iii) KOH, H2O, MeOH, 110°C, 5 h; (iv) DPPA, Et3N, DMF, r. t.-80°C, 4 h, then H2O,
DMF, 100°C, 1 h; (v) PhB(OH)2, Pd(PPh3)4, K2CO3, toluene, 110°C, 18 h; (vi) PPh3,
1,2-dichlorobenzene, 180°C, 20 h; (vii) NaH, CH3I, DMF, 0°C-r. t., 6 h; (viii) NaOH, EtOH, H2O,
reflux, 16 h; (ix) DPPA, Et3N, t-BuOH, reflux, 16 h, and then TFA, DCM, r. t., 2 h.
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