Design, synthesis, and evaluation of novel porcupine inhibitors featuring a fused 3-ring system based on the ‘reversed’ amide scaffold

Article abstract of DOI:10.1016/j.bmc.2016.09.041

The Wnt signaling pathway is an essential signal transduction pathway which leads to the regulation of cellular processes such as proliferation, differentiation and migration. Aberrant Wnt signaling is known to have an association with multiple cancers. Porcupine is an enzyme that catalyses the addition of palmitoleate to a serine residue in Wnt proteins, a process which is required for the secretion of Wnt proteins. Here we report the synthesis and structure–activity-relationship of the novel porcupine inhibitors based on a ‘reversed’ amide scaffold. The leading compound 53 was as potent as the clinical compound LGK974 in a cell based STF reporter gene assay. Compound 53 potently inhibited the secretion of Wnt3A, therefore was confirmed to be a porcupine inhibitor. Furthermore, compound 53 showed excellent chemical and plasma stabilities. However, the clearance of compound 53 in liver microsomal tests was moderate to high, and the solubility of compound 53 was suboptimal. Collective efforts toward further optimization of this novel tricyclic template to develop better porcupine inhibitors will be subsequently undertaken and reported in due course.

Full text of DOI:10.1016/j.bmc.2016.09.041

Accepted Manuscript
Design, Synthesis, and Evaluation of Novel Porcupine Inhibitors Featuring a
Fused 3-ring System Based on the “Reversed” Amide Scaffold
Zhixiang Xu, Xiangxiang Xu, Ruadhan O’Laoi, Haikuo Ma, Jiyue Zheng,
Shuaishuai Chen, Lusong Luo, Zhilin Hu, Sudan He, Jiajun Li, Hongjian Zhang,
Xiaohu Zhang
PII:
S0968-0896(16)30736-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.041
BMC 13294
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 July 2016
1 September 2016
16 September 2016
Please cite this article as: Xu, Z., Xu, X., O’Laoi, R., Ma, H., Zheng, J., Chen, S., Luo, L., Hu, Z., He, S., Li, J.,
Zhang, H., Zhang, X., Design, Synthesis, and Evaluation of Novel Porcupine Inhibitors Featuring a Fused 3-ring
System Based on the “Reversed” Amide Scaffold, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.09.041
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Design, Synthesis, and Evaluation of Novel Porcupine Inhibitors Featuring a Fused 3-ring
System Based on the “Reversed” Amide Scaffold
Zhixiang Xu a,1, Xiangxiang Xu a,1, Ruadhan O'Laoi b, Haikuo Ma a, Jiyue Zheng a,*, Shuaishuai Chen c,
Lusong Luo c,*, Zhilin Hu d, Sudan He d, Jiajun Li a, Hongjian Zhang a, and Xiaohu Zhang a,*
a
Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psychiatric-Diseases and
College of Pharmaceutical Sciences, Soochow University, Su Zhou, Jiangsu 215021, P. R. China
b Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland
c BeiGene (Beijing) Co., Ltd., No. 30 Science Park Road, Zhongguancun Life Science Park, Beijing
102206, P.R.China
d
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; lusong.luo@beigene.com; jyzheng@suda.edu.cn
Abstract: The Wnt signaling pathway is an essential signal transduction pathway which leads to the
regulation of cellular processes such as proliferation, differentiation and migration. Aberrant Wnt
signaling is known to have an association with multiple cancers. Porcupine is an enzyme that catalyses
the addition of palmitoleate to a serine residue in Wnt proteins, a process which is required for the
secretion of Wnt proteins. Here we report the synthesis and structure-activity-relationship of the novel
porcupine inhibitors based on a “reversed” amide scaffold. The leading compound 53 was as potent as
the clinical compound LGK974 in a cell based STF reporter gene assay. Compound 53 potently
inhibited the secretion of Wnt3A, therefore was confirmed to be a porcupine inhibitor. Furthermore,
compound 53 showed excellent chemical and plasma stabilities. However, the clearance of compound
53 in liver microsomal tests was moderate to high, and the solubility of compound 53 was suboptimal.
Collective efforts toward further optimization of this novel tricyclic template to develop better
porcupine inhibitors will be subsequently undertaken and reported in due course.
Keywords: Wnt signaling pathway, porcupine, antagonist, cancer therapy, scaffold hybridization
Abbreviations: BPO, benzoyl peroxide; DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine;
DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; FaSSIF, fasted state simulated intestinal
fluid;
HATU,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid
hexafluorophosphate; SEM, standard error measurement; SGF, simulated gastric fluid; STF, super-top
flash, TFA, trifluoroacetic acid.
1

1 Introduction
The Wnt signaling pathway plays a critical role in the regulation of cellular processes such as
proliferation, differentiation and migration1-3. The canonical Wnt signaling pathway begins when Wnt
ligands bind to the Frizzled and LRP families of cell surface receptors via the cytoplasmic protein
Dishevelled (DSH), leading to an accumulation of cytoplasmic β-catenin and its translocation into the
nucleus. Ultimately, β-catenin associates with the TCF/LEF family of DNA-binding proteins and
activates the expression of β-catenin mediated genes downstream. In contrast, in the absence of Wnt
ligand stimulation, β-catenin is phosphorylated and degraded by an intracellular β-catenin destruction
complex, resulting in the inhibition of downstream gene expression4. Overexpression of Wnt ligands
has been associated with numerous cancers5,6. Porcupine, a member of the membrane-bound
O-acyltransferase family of proteins, adds palmitoleate to a serine residue in Wnt proteins - a process
which is required for the secretion of Wnt proteins7. Porcupine inhibitors can thus block aberrant Wnt
signaling and inhibit tumor growth8. Therefore, porcupine has emerged as a potential target for the
treatment of cancer.
The IWP series of compounds (Fig. 1) identified in a high throughput screen were the first small
molecule porcupine inhibitors reported by Chen et al9. Since then, other classes of porcupine inhibitors
have also been investigated. LGK974, developed by Novartis in 2012, is a potent porcupine inhibitor
which has been advanced into a phase I/II clinical trial10,11. Recently, Virshup and co-workers reported
their work on porcupine inhibitors.12,13 Among them, ETC-159 has been advanced into a phase I
clinical trial12 (Fig.1).
Figure 1. Reported porcupine inhibitors in the literature
2 Design
We have investigated a novel series of porcupine inhibitors by a scaffold hopping strategy from a
known porcupine antagonist LGK97414. DC-9 was the result of optimization campaigns in a recently
published Novartis patent15. Although the central amide bonds were reversed, both LGK974 and DC-9
showed excellent potency. Encouraged by this result, we decided to introduce the tricyclic element into
the “reversed” amide porcupine inhibitor framework. Here we report the synthesis and
structure-activity-relationship of the novel porcupine inhibitors based on the “reversed” amide scaffold
as shown in Fig. 2.
2

Figure 2. The design strategy for the novel porcupine inhibitors
3 Chemistry
The synthetic route used to prepare compounds 5, 6 and 10 is outlined in Scheme 1. The synthesis of
compounds 3 and 7 was described in our previously published paper14. Commercially available
4-boronobenzoic acid was coupled with 2-chloropyrazine to produce aromatic acid 2. Nitrile 3 was
reduced with H2 and Pd/C to give amine 4, which was reacted with corresponding aromatic acids in the
presence of HATU in DMF to give the final compounds 5 and 6, respectively. Compound 7 was
converted to nitrile 8 in the presence of Zn(CN)2 and Pd(Ph3P)4. Nitrile 8 was reduced with LiAlH4 to
give amine 9, which was reacted with aromatic acid 2 to give the final compound 10.
Scheme 1. Reagents and conditions. (i) 2-chloropyrazine, Pd(Ph3P)4, Na2CO3, CH3CN, H2O, 80°C, 12
h; (ii) Pd/C, 37% HCl, H2, EtOH, r. t., 24 h; (iii) 4-(pyridin-3-yl)benzoic acid or 2, HATU, DIPEA,
DMF, r. t., overnight; (iv) Zn(CN)2, Pd(Ph3P)4, DMF, 90°C, 2 h; (v) LiAlH4, THF, r. t., 8 h; (vi) 2,
HATU, DIPEA, DMF, r. t., overnight.
Target compounds 19-24 were prepared via the synthetic route depicted in Scheme 2. The synthesis
of aryl iodide 11, 9,9-difluoro-9H-fluorene-2-carboxylic acid, 9H-carbazole-2-carboxylic acid and
9-methyl-9H-carbazole-2-carboxylic acid was also described in our previously published paper14.
Compound 11 was converted to nitrile 12 in the presence of Zn(CN)2 and Pd(Ph3P)4. Nitrile 12, 3 and 8
were hydrolyzed to give carboxylic acids 13-15, respectively. Commercially available
4-bromo-2-methylpyridine was borylated to provide aryl boronate 17, which was coupled with
(4-bromophenyl)methanamine to give intermediate 18. Condensation of amine 18 with corresponding
aromatic acids in the presence of HATU in DMF afforded the target compounds 19-24, respectively.
3

Scheme 2. Reagents and conditions. (i) Zn(CN)2, Pd(Ph3P)4, DMF, 90°C, 2 h; (ii) for 13 and 15: KOH,
MeOH, H2O, reflux, 6 h; (iii) for 14: 70% H2SO4, CH3COOH, 100°C, 12 h; (iv) bis(pinacolato)diboron,
KOAc, Pd(dppf)Cl2, THF, 80°C, overnight; (v) (4-bromophenyl)methanamine, Pd(dba)2, K3PO4, Sphos,
t-BuOH, H2O, 100°C, 12 h; (vi) HATU, DIPEA, DMF, r. t., 12 h.
Scheme 3 shows the synthetic approaches to the target compounds 27, 28, 34 and 35. Compound 25
was reacted with (4-bromophenyl)methanamine to give intermediate 26, which was coupled with
corresponding aryl boronic acid to produce the desired compounds 27 and 28, respectively.
Commercially available 4-bromobenzonitrile 29 was coupled with imidazole or 4-methyl-1H-imidazole
to give intermediates 30 and 31, which were reduced with LiAlH4 to yield amines 32 and 33,
respectively. The amide coupling was carried out by treatment of carboxylic acid 25 with aromatic
amines 32 or 33 in the presence of HATU in DMF, which led to the desired compounds 34 and 35,
respectively.
Scheme 3. Reagents and conditions. (i) (4-bromophenyl)methanamine, HATU, DIPEA, DMF, r. t., 12 h;
(ii) pyridin-4-ylboronic acid or 1-methyl-1H-pyrazol-5-ylboronic acid, Pd(dppf)Cl2, dppf, K3PO4, H2O,
dioxane, 100°C, 12 h; (iii) imidazole or 4-methyl-1H-imidazole, CuI, Cs2CO3, DMF, 130°C, 24 h; (iv)
LiAlH4, THF, 80°C, 2 h; (vi) 25, HATU, DIPEA, DMF, r. t., 12 h.
The synthetic pathways used to prepare the target compounds 50-53 are summarized in Scheme 4.
Suzuki coupling of commercially available 5-(aminomethyl)-2-chloropyridine 36 with aryl boronate 17
produced amine 37. Aldehydes 38 and 42 were reacted with tert-butyl carbamate in the presence of
TFA and Et3SiH to give compounds 39 and 43, respectively. Removal of Boc in 39 and 43 by
TFA/DCM provided amines 40 and 44, which were coupled with aryl boronate 17 to give intermediates
41 and 45, respectively. Bromination of 2-chloro-3-fluoro-5-methylpyridine using NBS in CH3CN
provided compound 47, which was reacted with NaN3 and subsequently reduced with PPh3 to give
amine 48. Compound 48 was coupled with aryl boronate 17 to give intermediate 49. Condensation of
aromatic acid 25 with corresponding amines gave the target compounds 50-53, respectively.
4

Scheme 4. Reagents and conditions. (i) 17, Pd(PPh3)4, K2CO3, dioxane, H2O, 100°C, overnight; (ii)
tert-butyl carbamate, TFA, Et3SiH, CH3CN, r. t., 24 h; (iii) TFA, DCM, r. t., 3 h; (iv) NBS, BPO,
CH3CN, reflux, overnight; (v) a) NaN3, DMF, 50°C, overnight; b) PPh3, THF, reflux, 8 h; (vi)
corresponding amine, HATU, DIPEA, DMF, r. t., 12 h.
The synthesis of the target compounds 57 and 61-66 is summarized in Scheme 5. Commercially
available 6-bromoisoquinoline 54 was converted to nitrile 55 in the presence of Zn(CN)2 and Pd(Ph3P)4.
Compound 55 was reduced with H2 and Pd/C to give amine 56, which was reacted with aromatic acid
25 to yield the target compound 57. 6-Hydroxy-2-naphthonitrile 58 was readily converted to the triflate
intermediate 59. Compound 59 was coupled with MeNO2 in the presence of Pd(dba)2, K3PO4 and
Xphos in dioxane and subsequently reduced by Zn in AcOH to give amine 60, which was reacted with
aromatic acid 25 to provide the target compound 61. Condensation of amine 49 with corresponding
aromatic acids gave the target compounds 62-66, respectively.
Scheme 5. Reagents and conditions. (i) Zn(CN)2, Pd(Ph3P)4, DMF, 90°C, 24 h; (ii) Pd/C, 37% HCl, H2,
EtOH, r. t., 24 h; (iii) 25, HATU, DIPEA, DMF, r. t., overnight; (iv) Tf2O, Et3N, DCM, r. t., overnight;
(v) a) Pd(dba)2, Xphos, K3PO4, MeNO2, dioxane, 80°C, 18 h; b) Zn, AcOH, 35°C, 3 h; (vi)
corresponding acid, HATU, DIPEA, DMF, r. t., overnight.
4. Results and Discussion
5

4.1. Evaluation of pharmacological activity
A cell based STF (super-top flash) reporter gene assay was employed to test Wnt signaling inhibition
of the target compounds. We first confirmed that LGK974 was active in this assay (LGK974, 0.9 nM,
Table 1), and its IC50 number was consistent with the number reported in the literature (LGK974, 0.4
nM) 10,11. The structure-activity relationship is summarized in Table 1.
Cyclization of the left-side rings of DC-9 led to inactive compounds, as exemplified by compounds 5
and 6, while compound 10 was weakly active. This was consistent with recently published papers,
which indicated that a hydrogen bond acceptor was needed at this region14,16. We thus decided to keep
the key interaction by maintaining the structural element of DC-9 on the left hand side, and started to
explore the tricyclic structure-activity relationship on the right hand side. When R1 was the same as
DC-9, the tricyclic elements fluorene, difluoro-fluorene and fluorine-9-one provided active compounds
(compounds 19, 20, 21; 35, 60, 85 nM, respectively). The carbazole and dibenzofuran showed
increased activity (compounds 22 and 24; 7.5 and 9.1 nM, respectively), while methylated carbazole
was inactive (compound 23, >1000 nM). The steric hindrance effect completely eliminated activity,
which was consistent with the conclusion drawn by our recently published paper14. Among the above
compounds, carbazole 22 showed the best activity. Encouraged by this result, we kept the element of
compound 22 on the right side and started to explore the structure-activity relationship on the left hand
side. Removal of methyl from the pyridine resulted in slightly decreased activity (compound 27, 18
nM). When the 2-methyl-pyridine was replaced by N-methyl-pyrazole, imidazole and
4-methyl-imidazole, resulting in compounds 28, 34 and 35 respectively, Wnt signaling inhibition
activity was significantly decreased in all three compounds (130, 314 and 119 nM, respectively).
Replacement of a carbon with a nitrogen on the internal ring of the left hand side was comparable to or
slightly less potent than compound 22 (compounds 50 and 51; 6.2 and 18 nM, respectively).
Substitution on the carbon of the internal ring of the left hand side with a methyl group resulted in
slightly deceased activity (compounds 52, 10 nM); while substitution on the same position with a
fluorine significantly improved potency (compound 53, 0.5 nM). Compound 53 was twice as potent as
LGK974 in the same assay. Fusion of the biphenyl to bicyclic element was not well tolerated, as
demonstrated by compound 57 (207 nM), while the addition of a nitrile group restored activity
(compound 61, 13 nM). Nitrile, a versatile functional group in medicinal chemistry17, served as a more
favorable hydrogen bond acceptor in this case. Thus far, compound 53 showed the best activity among
all of the synthesized compounds. Finally, we kept the left side element of compound 53 and explored
the tricyclic structure-activity relationship on the right hand side. Although the fluorene-9-one and
N-methyl-carbazole showed only moderate activity (compounds 64 and 65; 43 and 175 nM,
respectively), the tricyclic elements fluorene, difluoro-fluorene and dibenzofuran provided much more
active compounds (62, 63, 66; 9.2, 16, 3.1 nM, respectively), In summary, through extensive SAR
studies, numerous active compounds (eg. compounds 22, 50, 53, 66) were achieved, among these, the
most promising compound 53 was as potent as LGK974 in our assay.
Table 1. SAR of designed compounds
IC50(nM)
± SEMa
IC50 (nM)
± SEM
No.
R1
R2
No.
R1
R2
6

1.0 ± 0.2
> 1000
> 1000
701
119 ± 13
6.2 ± 2.4
18 ± 0.5
10 ± 0
DC-9
5
35
50
51
52
53
6
10
19
35 ± 10
0.5 ± 0.2
60 ± 16
85 ± 2
207 ± 21
13 ± 5
20
21
22
57
61
62
7.5 ± 4.5
9.2 ± 3.8
> 1000
9.1 ± 0.3
18 ± 4
16 ± 1.5
44 ± 14
23
24
27
28
34
63
64
65
66
175 ± 66
3.1 ± 1.1
0.9 ± 0.1
130 ± 52
314 ± 91
LGK
974b
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).
7

The enzyme porcupine, a member of the membrane-bound O-acyltransferase family of proteins,
catalyzes the palmitoylation of Wnt proteins. This process is essential for their secretion and activity.
Without this crucial palmitoylation, Wnt proteins cannot be secreted outside of cells. We thus
performed a second assay to confirm the target of the new compounds was indeed porcupine14,18,19
.
HEK293T cells were transfected with pLinbin-Wnt3A plasmid or vehicle control. The HEK293T cells
were then treated with or without compounds. Western Blot was used after 48 hours to analyze both the
cell lysis and culture medium. We found that both compounds 22 and 53, as well as LGK974 all
potently inhibited Wnt3A secretion into cell culture medium, while the above compounds did not affect
the amount of Wnt3A inside of HEK293T cells. These results suggested that the new compounds were
indeed porcupine inhibitors (Fig. 3).
Figure 3. The Wnt secretion assay. Compound concentration was at 0.1 μM
4.2. Evaluation of chemical stability, rat plasma stability, liver microsomal stability
The majority of reported Porcupine inhibitors contain an amide group as part of their chemical
structure. Similarly, an amide group was also present in our novel template. Amide groups have been
reported as potentially unstable and may be hydrolyzed in saline, plasma and under the treatment of
liver microsomal enzymes9. We thus evaluated the stability of representative compounds 22 and 53.
The stability of these compounds was tested in simulated gastric fluid (SGF), rat plasma and under the
treatment of liver microsomal enzymes. Compounds 22 and 53 both showed good stability in SGF after
24 hours and were both stable after 8 hours in rat plasma. However, despite compounds 22 and 53
demonstrating moderate clearance under the treatment of human liver microsomes (59 and 57
mL/min/kg, respectively) and rat liver microsomes (7 and 24 mL/min/kg, respectively), compounds 22
and 53 exhibited high clearance when treated with mouse microsomes (141 and 109 mL/min/kg,
respectively). This was in contrast to LGK974, which demonstrated excellent metabolic stability cross
all species (Table 2). These results indicate that further optimization might be needed to improve the
metabolic stability, and therefore in vivo bioavailability in mouse of the current compounds.
Table 2. Chemical stability, plasma stability and metabolic stability of compounds 22 and 53
% Remaining
in SGF, 24 ha
100%
% Remaining
in rat plasma, 8 hb
100%
Clint (mL/min/kg)b
HLMc RLMd MLMe
t1/2 (min)
Compd
HLM RLM MLM
59
57
2
7
24
3
141
109
22
30
31
277
80
43
55
22
53
100%
66%
100%
100%
561
770
277
LGK974
a All compounds were tested at 200 μM concentration in simulated gastric fluid (SGF) at 40°C for 24 h.
8

b All compounds were tested at 1 μM concentration.
c HLM = human liver microsomes.
d RLM = rat liver microsomes.
e MLM = mouse liver microsomes.
4.3. Evaluation of CYP inhibition and solubility
The leading compounds 22 and 53 were subjected to the standard CYP inhibition test, and they
showed weak or no CYP inhibition at concentration of 10 μM. Unfortunately, the solubility of
compounds 22 and 53 was poor (Table 3). The poor solubility in combination with the suboptimal
metabolic stability prevented us from testing these compounds in vivo. Collective efforts toward further
optimization of this novel tricyclic template to develop better porcupine inhibitors will be subsequently
undertaken.
Table 3. CYP inhibition profiles and solubility of compounds 22 and 53
CYP inhibition (%) a
Solubility (μM) b
PH 6.5
Compd
3A4
7
2D6
1A2
64
2C9
2C19
8
9
-22
0.16
22
53
69
37
73
31
59
1.24
a These two compounds were tested at 10 μM concentration.
b
4 mg/mL suspension of each tested compound (HCl salt) 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.
5. Conclusion
We have designed and synthesized a series of Wnt compounds based on the “reversed” amide
scaffold. This novel scaffold provided active compounds (e.g. compounds 22, 50, 53 and 66; 7.5, 6.2,
0.5 and 3.1 nM, respectively). The leading compound 53 was as potent as the clinical compound
LGK974 (0.9 nM). Compound 53 was confirmed to be an inhibitor of Porcupine via a cell based
secretion assay and, furthermore, showed excellent chemical and plasma stabilities. However, the
clearance of compound 53 in liver microsomal tests was moderate to high, and the solubility of
compound 53 was suboptimal. Subsequent efforts to further optimize this novel tricyclic template to
improve liver microsomal stability, solubility and develop better porcupine inhibitors will be
undertaken and reported in due course.
6. Experimental section
6.1. Chemistry
Analytical thin layer chromatography was performed on silica gel HSGF254 pre-coated plates
to monitor the general reaction progress. Final compounds were purified by column
1
chromatography with silica gel 100-200 mesh. H NMR and 13C NMR were performed on 300
MHz (Varian) and 400 MHz (Varian) spectrometers. Chemical shifts were given in ppm using
tetramethylsilane as internal standard. Mass spectra were performed on an Agilent 1100 LC/MSD
Trap SL version Mass Spectrometer. HRMS analysis was obtained using an Agilent 6540 UHD
Accurate-Mass Q-TOF LC/MS.
6.1.1. 4-(Pyrazin-2-yl)benzoic acid (2)
To a suspension of 4-boronobenzoic acid (664 mg, 4 mmol), 2-chloropyrazine (458 mg, 4 mmol) and
9

Pd(PPh3)4 (230 mg, 0.2 mmol) in CH3CN (20 mL) and H2O (20 mL) was added Na2CO3 (848 mg, 8
mmol). The reaction mixture was stirred at 80°C under N2 for 12 h. After cooling to room temperature,
the mixture was filtered. The filtrate was washed with DCM (20 mL x 3). The pH was adjusted to 4
with 1 N HCl, and then the mixture was filtered to give the desired product (647 mg, 80%) as a white
1
solid. H NMR (400 MHz, DMSO-d6) δ 9.33 (d, J = 0.8 Hz, 1H), 8.81-8.74 (m, 1H), 8.68 (d, J = 2.4
Hz, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H). ESI-MS (m/z): 201.0 [M-H]-.
6.1.2. (9H-Fluoren-2-yl)methanamine (4)
To a solution of 9H-fluorene-2-carbonitrile (100 mg, 0.5 mmol) in ethanol (10 mL) was added Pd/C
(20 mg) and 37% HCl (0.1 mL), and the reaction mixture was stirred at room temperature for 24 h
under H2. Saturated aqueous Na2CO3 (10 mL) was added. The mixture was filtered, and the filtrate was
extracted with DCM (30 mL x 3). The combined organic layers were dried over Na2SO4 and
concentrated to give the desired product (40 mg, 39%) as a light yellow solid. 1H NMR (400 MHz,
CDCl3) δ 7.80-7.72 (m, 2H), 7.56-7.50 (m, 2H), 7.39-7.35 (m, 1H), 7.33-7.27 (m, 2H), 3.95 (s, 2H),
3.89 (s, 2H).
6.1.3. General procedure for the synthesis of compounds 5 and 6
To a solution of 4 (1 eq.) and corresponding acid (1 eq.) in DMF (1.5 mL) were added HATU (1 eq.)
and DIPEA (5 eq.). After stirring at room temperature overnight, H2O (30 mL) was added and the
mixture was extracted with ethyl acetate (30 mL x 3). The combined organic layers were washed with
brine (30 mL x 3) and dried over Na2SO4. After concentration, the residue was purified by silica gel
column chromatography to give the desired products 5 and 6.
6.1.3.1. N-((9H-Fluoren-2-yl)methyl)-[1,1'-biphenyl]-4-carboxamide (5). White solid (yield: 83%).
1H NMR (400 MHz, DMSO-d6) δ 9.15 (t, J = 6.0 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.86 (dd, J = 7.2,
5.2 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 7.6 Hz, 2H), 7.57 (d, J = 9.2 Hz, 2H), 7.52-7.46 (m,
2H), 7.44-7.34 (m, 3H), 7.32-7.27 (m, 1H), 4.59 (d, J = 6.0 Hz, 2H), 3.91 (s, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 165.7, 143.1, 142.9, 142.7, 140.8, 139.8, 139.1, 138.4, 138.3, 133.1, 128.9, 127.9, 126.7,
126.6, 126.4, 126.0, 125.0, 124.0, 119.7, 119.6, 42.8, 36.2. ESI-MS (m/z): 375.9 [M+H]+.
6.1.3.2. N-((9H-Fluoren-2-yl)methyl)-4-(pyrazin-2-yl)benzamide (6). Off white solid (yield: 35%).
1H NMR (400 MHz, CDCl3) δ 9.07 (s, 1H), 8.67 (s, 1H), 8.56 (s, 1H), 8.12 (d, J = 8.0 Hz, 2H), 7.97 (d,
J = 8.0 Hz, 2H), 7.83-7.75 (m, 2H), 7.61-7.54 (m, 2H), 7.43-7.36 (m, 2H), 7.34-7.29 (m, 1H), 6.50 (s,
1H), 4.76 (d, J = 5.2 Hz, 2H), 3.91 (s, 2H). ESI-MS (m/z): 378.0 [M+H]+.
6.1.4. Dibenzo[b,d]furan-3-carbonitrile (8)
To a suspension of 3-iododibenzo[b,d]furan (500 mg, 1.7 mmol) and Pd(PPh3)4 (196 mg, 0.17 mmol)
in DMF (15 mL) was added Zn(CN)2 (117 mg, 1.0 mmol), and the reaction mixture was stirred at 90°C
under N2 for 2 h. After cooling to room temperature, H2O (40 mL) was added and the mixture was
extracted with ethyl acetate (40 mL x 3). The combined organic layers were washed with brine (30 mL
x 3) and dried over Na2SO4. After concentration and purification by silica gel column chromatography
1
(petroleum ether/ethyl acetate = 50/1), 8 was obtained as a white solid (310 mg, 85%). H NMR (400
MHz, CDCl3) δ 8.03 (dd, J = 12.4, 8.0 Hz, 2H), 7.88 (s, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.60-7.55 (m,
1H), 7.45-7.40 (m, 1H).
10

6.1.5. Dibenzo[b,d]furan-3-ylmethanamine (9)
To a solution of 8 (130 mg, 0.67 mmol) in THF (3 mL) was added LiAlH4 (77 mg, 2.02 mmol) at
0°C. The mixture was stirred at room temperature for 8 h. Aqueous NaOH (2 N, 20 mL) was added and
the mixture was extracted with DCM (30 mL x 3). The combined organic layers were dried over
Na2SO4 and concentrated to give a white solid (80 mg, 60%), which was used directly in the next step
without further purification.
6.1.6. N-(Dibenzo[b,d]furan-3-ylmethyl)-4-(pyrazin-2-yl)benzamide (10)
To a solution of 9 (50 mg, 0.25 mmol) and 2 (50 mg, 0.25 mmol) in DMF (2 mL) were added HATU
(95 mg, 0.25 mmol) and DIPEA (161 mg, 1.25 mmol). After stirring at room temperature overnight,
H2O (30 mL) was added and the mixture was extracted with ethyl acetate (30 mL x 3). The combined
organic layers were washed with brine (30 mL x 3) and dried over Na2SO4. After concentration and
purification by column chromatography (dichloromethane/methanol = 100/1), 11 was obtained as a
white solid (15 mg, 15%). 1H NMR (400 MHz, DMSO-d6) δ 9.38-9.28 (m, 2H), 8.77 (s, 1H), 8.69-8.65
(m, 1H), 8.27 (d, J = 7.6 Hz, 2H), 8.15-8.06 (m, 4H), 7.71-7.65 (m, 2H), 7.53-7.47 (m, 1H), 7.43-7.36
(m, 2H), 4.69 (d, J = 6.0 Hz, 2H). ESI-MS (m/z): 380.0 [M+H]+.
6.1.7. 9-Oxo-9H-fluorene-2-carbonitrile (12)
To a suspension of 2-iodo-9H-fluoren-9-one (11) (1.2 g, 3.92 mmol) and Pd(PPh3)4 (452 mg, 0.39
mmol) in DMF (15 mL) was added Zn(CN)2 (275 mg, 2.35 mmol), and the reaction mixture was stirred
at 90°C under N2 for 2 h. After cooling to room temperature, H2O (40 mL) was added and the mixture
was extracted with ethyl acetate (40 mL x 3). The combined organic layers were washed with brine (30
mL x 3) and dried over Na2SO4. After concentration and purification by column chromatography
(petroleum ether/ethyl acetate = 50/1), 12 was obtained as a yellow solid (800 mg, 93%). 1H NMR (400
MHz, CDCl3) δ 7.91 (s, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.68-7.56 (m, 3H),
7.46-7.40 (m, 1H).
6.1.8. 9-Oxo-9H-fluorene-2-carboxylic acid (13)
To a solution of 12 (200 mg, 0.98 mmol) in CH3OH (7 mL) and H2O (7 mL) was added KOH (1.6 g,
29.4 mmol), and the reaction mixture was stirred at 110°C for 6 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 (200 mg, 91%) as a yellow solid. 1H
NMR (400 MHz, DMSO-d6) δ 13.30 (s, 1H), 8.19 (d, J = 7.6 Hz, 1H), 8.03 (s, 1H), 7.97-9.89 (m, 2H),
7.72-7.64 (m, 2H), 7.50-7.44 (m, 1H).
6.1.9. 9H-Fluorene-2-carboxylic acid (14)
To a solution of 3 (170 mg, 0.89 mmol) in CH3COOH (6 mL) was added 70% H2SO4 (6 mL), and
the reaction mixture was stirred at 100°C for 12 h. After cooling to room temperature, the mixture was
diluted with H2O (30 mL). The pH was adjusted to 9 with Na2CO3, and then the mixture was washed
with DCM (30 mL). The pH of the aqueous layer 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 (80 mg, 43%) as a white solid. 1H NMR (400 MHz,
11

DMSO-d6) δ 12.87 (s, 1H), 8.15(s, 1H), 8.02-7.98 (m, 3H), 7.64 (d, J = 6.8 Hz, 1H), 7.45-7.38 (m, 2H),
4.01 (s, 2H).
6.1.10. Dibenzo[b,d]furan-3-carboxylic acid (15)
To a solution of 8 (160 mg, 0.83 mmol) in CH3OH (6.5 mL) and H2O (6.5 mL) was added KOH (1.4
g, 24.8 mmol), and the reaction mixture was stirred at 110°C for 6 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 (160 mg, 91%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) δ 13.19 (s, 1H), 8.30-8.16 (m, 3H), 8.01 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 8.4 Hz,
1H), 7.65-7.58 (m, 1H), 7.49-7.43 (m, 1H).
6.1.11. (4-(2-Methylpyridin-4-yl)phenyl)methanamine (17)
To a suspension of 4-bromo-2-methylpyridine (2.0 g, 11.6 mmol), Pd(dppf)Cl2 (169 mg, 0.23 mmol)
and KOAc (2.27 g, 23.2 mmol) in THF (40 mL) was added bis(pinacolato)diboron (3.18 g, 12.5 mmol).
The mixture was stirred at 80°C under N2 overnight. After cooling to room temperature, the mixture
was diluted with DCM (250 mL) and then filtered. The filtrate was concentrated to give a black oil (5.1
g, crude), which was used directly in the next step without further purification.
6.1.12. (4-(2-Methylpyridin-4-yl)phenyl)methanamine (18)
To a suspension of 17 (5.1 g, crude), (4-bromophenyl)methanamine (1.8 g, 9.68 mmol) and Pd(dba)2
(557 mg, 0.97 mmol) in t-BuOH (40 mL) and H2O (10 mL) were added K3PO4 (4.1 g, 19.4 mmol) and
Xphos (397 mg, 0.97 mmol). The mixture was stirred at 100°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 = 10/1) to give the desired product (800 mg, 42%) as a
yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 5.2 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.43 (d,
J = 8.0 Hz, 2H), 7.37 (s, 1H), 7.31 (d, J = 5.2 Hz, 1H), 3.94 (s, 2H), 2.62 (s, 3H).
6.1.13. General procedure for the synthesis of compounds 19-24
To a solution of 18 (1 eq.) and corresponding acid (1 eq.) in DMF (1.5 mL) were added HATU (1 eq.)
and DIPEA (5 eq.). After stirring at room temperature overnight, H2O (30 mL) was added and the
mixture was extracted with ethyl acetate (30 mL x 3). The combined organic layers were washed with
brine (30 mL x 3) and dried over Na2SO4. After concentration, the residue was purified by silica gel
column chromatography to give the desired products 19-24.
6.1.13.1. N-(4-(2-Methylpyridin-4-yl)benzyl)-9H-fluorene-2-carboxamide (19). White solid (yield:
22%). 1H NMR (400 MHz, DMSO-d6) δ 9.16 (t, J = 6.0 Hz, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.14 (s, 1H),
8.03-7.94 (m, 3H), 7.76 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 7.2 Hz, 1H), 7.57 (s, 1H), 7.51-7.46 (d, J = 8.0
Hz, 3H), 7.45-7.35 (m, 2H), 4.56 (d, J = 6.0 Hz, 2H), 4.00 (s, 2H), 2.52 (s, 3H). ESI-MS (m/z): 391.0
[M+H]+.
6.1.13.2. 9,9-Difluoro-N-(4-(2-methylpyridin-4-yl)benzyl)-9H-fluorene-2-carboxamide (20). White
solid (yield: 39%).1H NMR (400 MHz, DMSO-d6) δ 9.31 (t, J = 6.0 Hz, 1H), 8.47 (d, J = 5.2 Hz, 1H),
8.23 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 7.2 Hz, 1H), 7.76 (d, J =
12

8.0 Hz, 3H), 7.67-7.61 (m, 1H), 7.57 (s, 1H), 7.52-7.46 (m, 4H), 4.56 (d, J = 6.0 Hz, 2H), 2.51 (s, 3H).
ESI-MS (m/z): 427.0 [M+H]+.
6.1.13.3. N-(4-(2-methylpyridin-4-yl)benzyl)-9-oxo-9H-fluorene-2-carboxamide (21). Light yellow
solid (yield: 44%). 1H NMR (400 MHz, DMSO-d6) δ 9.31 (t, J = 5.6 Hz, 1H), 8.47 (d, J = 5.2 Hz, 1H),
8.20-8.14 (m, 2H), 7.93 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H),
7.70-7.64 (m, 2H), 7.57 (s, 1H), 7.50-7.42 (m, 4H), 4.55 (d, J = 5.7 Hz, 2H), 2.52 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) δ 192.5, 164.9, 158.5, 149.5, 147.0, 146.4, 143.1, 140.6, 135.9, 135.6, 135.0,
134.7, 133.9, 133.4, 130.2, 128.1, 126.8, 124.2, 122.4, 121.9, 121.2, 120.3, 118.3, 42.5, 24.2. HRMS
(ESI): calcd for C27H21N2O2 [M+H]+ 405.1598, found 405.1597.
6.1.13.4. N-(4-(2-Methylpyridin-4-yl)benzyl)-9H-carbazole-2-carboxamide (22). White solid (yield:
1
62%). H NMR (400 MHz, DMSO-d6) δ 11.49 (s, 1H), 9.22-9.15 (m, 1H), 8.48 (d, J = 5.6 Hz, 1H),
8.22-8.15 (m, 2H), 8.05 (s, 1H), 7.79-7.72 (m, 3H), 7.57 (s, 1H), 7.55-7.42 (m, 5H), 7.22-7.16 (m, 1H),
4.58 (d, J = 6.0 Hz, 2H), 2.52 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 166.9, 158.5, 149.5, 147.1,
141.1, 140.8, 139.2, 135.8, 131.4, 128.0, 126.7, 126.5, 124.7, 121.8, 120.8, 120.3, 119.8, 118.9, 118.3,
117.6, 111.2, 110.4, 42.5, 24.2. HRMS (ESI): calcd for C26H22N3O [M+H]+ 392.1757, found 392.1756.
6.1.13.5. 9-Methyl-N-(4-(2-methylpyridin-4-yl)benzyl)-9H-carbazole-2-carboxamide (23). White
solid (yield: 62%). 1H NMR (400 MHz, DMSO-d6) δ 9.20 (t, J = 6.0 Hz, 1H), 8.48 (d, J = 5.2 Hz, 1H),
8.27-8.16 (m, 3H), 7.81-7.75 (m, 3H), 7.65 (d, J = 8.4 Hz, 1H), 7.58-7.48 (m, 5H), 7.27-7.22 (m, 1H),
4.61 (d, J = 6.0 Hz, 2H), 3.94 (s, 3H), 2.52 (s, 3H). ESI-MS (m/z): 406.2 [M+H]+.
6.1.13.6. N-(4-(2-Methylpyridin-4-yl)benzyl)dibenzo[b,d]furan-3-carboxamide (24). Light yellow
solid (yield: 22%). 1H NMR (400 MHz, DMSO-d6) δ 9.30 (t, J = 6.0 Hz, 1H), 8.48 (d, J = 5.2 Hz, 1H),
8.30-8.19 (m, 3H), 7.99 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 3H), 7.63-7.56 (m, 2H), 7.52-7.42 (m,
4H), 4.59 (d, J = 6.0 Hz, 2H), 2.52 (s, 3H). ESI-MS (m/z): 393.0 [M+H]+.
6.1.14. N-(4-Bromobenzyl)-9H-carbazole-2-carboxamide (26)
To
a
solution of 9H-carbazole-2-carboxylic acid (211 mg, 1.0 mmol) and
(4-bromophenyl)methanamine (186 mg, 1.0 mmol) in DMF (2 mL) were added HATU (380 mg, 1.0
mmol) and DIPEA (645 mg, 5.0 mmol). After stirring at room temperature overnight, H2O (30 mL)
was added and the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic layers
were washed with brine (30 mL x 3) and dried over Na2SO4. After concentration, the residue was
purified by silica gel column chromatography (dichloromethane/methanol = 50/1) to give the desired
1
product (285 mg, 75%) as a white solid. H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 9.14 (t, J =
6.0 Hz, 1H), 8.21-8.15 (m, 2H), 8.03 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.56-7.49 (m, 3H), 7.47-7.41 (m,
1H), 7.32 (d, J = 8.0 Hz, 2H), 7.22-7.16 (m, 1H), 4.49 (d, J = 5.6 Hz, 2H).
6.1.15. General procedure for the synthesis of compounds 27 and 28
To a suspension of 26 (1 eq.), corresponding aryl boronate (1.2 eq.), Pd(dppf)Cl2 (0.05 eq.) and dppf
(0.05 eq.) in 1,4-dioxane (2 mL) was added the solution of K3PO4 (2 eq.) in water (0.5 mL). The
mixture was stirred at 100°C under N2 for 12 h. After cooling to room temperature, the mixture was
concentrated under reduced pressure and purified by silica gel column chromatography
13

(dichloromethane/methanol) to give the desired products 27 and 28.
6.1.15.1. N-(4-(Pyridin-4-yl)benzyl)-9H-carbazole-2-carboxamide (27). Off white solid (yield: 46%).
1H NMR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 9.18 (d, J = 6.0 Hz, 1H), 8.67 (s, 1H), 8.23-8.13 (m,
2H), 8.05 (s, 1H), 7.84-7.68 (m, 5H), 7.58-7.38 (m, 5H), 7.22-7.14 (m, 1H), 4.58 (s, 2H). ESI-MS
(m/z): 378.0 [M+H]+.
6.1.15.2. N-(4-(1-Methyl-1H-pyrazol-5-yl)benzyl)-9H-carbazole-2-carboxamide (28). Light yellow
solid (yield: 35%). 1H NMR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 9.18 (t, J = 6.0 Hz, 1H), 8.22-8.16
(m, 2H), 8.05 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.55-7.41 (m, 7H), 7.22-7.17 (m, 1H), 6.37 (d, J = 1.6
Hz, 1H), 4.58 (d, J = 6.0 Hz, 2H), 3.84 (s, 3H). HRMS (ESI): calcd for C24H21N4O [M+H]+ 381.1710,
found 381.1707.
6.1.16. General procedure for the synthesis of compounds 30 and 31
To a suspension of 4-bromobenzonitrile (1 eq.), CuI (0.2 eq.) in DMF (10 mL) were added Cs2CO3
(2 eq.) and imidazole (1.4 eq.). The mixture was stirred at 130°C under N2 for 24 h. After cooling to
room temperature, H2O (50 mL) was added and the mixture was extracted with ethyl acetate (50 mL x
3). The combined organic layers were washed with brine (50 mL x 3) and dried over Na2SO4. After
concentration, the residue was purified by silica gel column chromatography
(dichloromethane/methanol) to give the desired products 30 and 31.
1
6.1.16.1. 4-(1H-Imidazol-1-yl)benzonitrile (30). White solid (yield: 80%). H NMR (400 MHz,
CDCl3) δ 7.94 (s, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.34 (s, 1H), 7.26 (s, 1H).
1
6.1.16.2. 4-(4-Methyl-1H-imidazol-1-yl)benzonitrile (31). White solid (yield: 85%). H NMR (400
MHz, CDCl3) δ 7.84 (s, 1H), 7.81-7.73 (m, 2H), 7.53-7.44 (m, 2H), 7.05 (s, 1H), 2.30 (s, 3H).
6.1.17. General procedure for the synthesis of compounds 32 and 33
To a solution of lithium aluminum hydride (4 eq.) in tetrahydrofuran (5 mL) was added a solution of
30 or 31 (1 eq.) in tetrahydrofuran (2 mL). The reaction was refluxed at 80°C for 2 h under N2. After
cooling to room temperature, excess Na2SO4·10H2O was added. The mixture was filtered and the
filtrate was concentrated to give the desired products 32 and 33, which was used directly in the next
step without further purification.
6.1.18. General procedure for the synthesis of compounds 34 and 35
To a solution of 25 (1 eq.) and corresponding amine (1 eq.) in DMF (1.5 mL) were added HATU (1
eq.) and DIPEA (5 eq.). After stirring at room temperature overnight, H2O (30 mL) was added and the
mixture was extracted with ethyl acetate (30 mL x 3). The combined organic layers were washed with
brine (30 mL x 3) and dried over Na2SO4. After concentration, the residue was purified by silica gel
column chromatography to give the desired products 34 and 35.
6.1.18.1. N-(4-(1H-Imidazol-1-yl)benzyl)-9H-carbazole-2-carboxamide (34). White solid (yield:
85%). 1H NMR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 9.18 (t, J = 5.6 Hz, 1H), 8.33 (s, 1H), 8.22-8.16
(m, 2H), 8.04 (s, 1H), 7.78-7.69 (m, 2H), 7.62 (d, J = 8.4 Hz, 2H), 7.55-7.48 (m, 3H), 7.47-7.41 (m,
14

1H), 7.22-7.17 (m, 1H), 7.15 (s, 1H), 4.56 (d, J = 5.6 Hz, 2H). ESI-MS (m/z): 367.0 [M+H]+.
6.1.18.2. N-(4-(4-Methyl-1H-imidazol-1-yl)benzyl)-9H-carbazole-2-carboxamide (35). White solid
1
(yield: 86%). H NMR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 9.17 (t, J = 5.6 Hz, 1H), 8.28 (s, 1H),
8.21-8.14 (m, 2H), 8.03 (s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.60-7.40 (m, 7H), 7.23-7.16 (m, 1H), 4.55 (d,
J = 6.0 Hz, 2H), 2.17 (s, 3H). ESI-MS (m/z): 381.0 [M+H]+.
6.1.19. (2'-Methyl-[2,4'-bipyridin]-5-yl)methanamine (37)
To a suspension of (6-chloropyridin-3-yl)methanamine (500 mg, 3.50 mmol), 17 (1.68 g, 50%, 3.85
mmol) and Pd(PPh3)4 (202 mg, 0.175 mmol) in dioxane/H2O (4 mL/1 mL) was added K2CO3 (1.93 g,
14.0 mmol). The mixture was stirred at 100°C under N2 overnight. After cooling to room temperature,
H2O (30 mL) was added and the mixture was extracted with DCM (50 mL x 6). The combined organic
layers were dried and concentrated to give the desired product 37 (620 mg, 89%) as a brown solid. 1H
NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.84-7.75 (m, 3H), 7.66 (d, J = 4.8
Hz, 1H), 3.98 (s, 2H), 2.65 (s, 3H).
6.1.20. tert-Butyl ((5-bromopyridin-2-yl)methyl)carbamate (39)
To a solution of 5-bromopicolinaldehyde (1.00 g, 5.38 mmol) and tert-butyl carbamate (1.27 g, 10.8
mmol) in CH3CN (50 mL) were added TFA (1.84 g, 16.1 mmol) and Et3SiH (6.24 g, 53.8 mmol). After
stirring at room temperature for 24 h, the mixture was quenched with saturated aqueous Na2CO3 (30
mL) and extracted with ethyl acetate (100 mL x 3). The combined organic layers were washed with
brine (80 mL x 3), and dried over Na2SO4. After concentration and purification by silica gel column
chromatography (petroleum ether/ethyl acetate = 5/1), 39 was obtained as a colorless oil (1.4 g, 90%).
6.1.21. (5-bromopyridin-2-yl)methanamine (40)
To a solution of 39 (1.40 g, 5.88 mmol) in DCM (10 mL) was added TFA (10 mL). After stirring at
room temperature for 3 h, excess saturated aqueous Na2CO3 was added slowly. The mixture was
extracted with DCM (100 mL x 3). The combined organic layers were dried over Na2SO4 and
concentrated to give the desired product 40 (880 mg, 80%) as a yellow oil, which was used directly in
1
the next step without further purification. H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 7.76 (d, J = 8.4
Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 3.94 (s, 2H).
6.1.22. (2'-Methyl-3,4'-bipyridin-6-yl)methanamine (41)
To a suspension of 40 (800 mg, 4.28 mmol), 17 (2.04 g, 50%, 4.71 mmol) and Pd(PPh3)4 (247 mg,
0.21 mmol) in dioxane/H2O (20 mL/4 mL) was added K2CO3 (1.77 g, 12.8 mmol). The mixture was
stirred at 100°C under N2 overnight. After cooling to room temperature, H2O (30 mL) was added and
the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic layers were washed
with brine (30 mL x 3) and dried over Na2SO4. After concentration and purification by column
chromatography (dichloromethane/methanol = 50/1-20/1), 41 was obtained as a brown solid (300 mg,
35%).
6.1.23. tert-Butyl (6-chloro-5-methylpyridin-3-yl)methylcarbamate (43)
To a solution of 6-chloro-5-methylnicotinaldehyde (1.00 g, 6.41 mmol) and tert-butyl carbamate
(1.50 g, 12.8 mmol) in CH3CN (50 mL) were added TFA (2.20 g, 19.3 mmol) and Et3SiH (7.40 g, 63.8
15

mmol). After stirring at room temperature for 24 h, the mixture was quenched with saturated aqueous
Na2CO3 (30 mL) and extracted with ethyl acetate (100 mL x 3). The combined organic layers were
washed with brine (80 mL x 3), and dried over Na2SO4. After concentration and purification by column
chromatography (petroleum ether/ethyl acetate = 5/1), 43 was obtained as a colorless oil (1.6 g, 97%).
1H NMR (400 MHz, CDCl3) δ 8.15 (s, 1H), 7.51 (s, 1H), 4.96 (br s, 1H), 4.28 (d, J = 4.8 Hz, 1H), 2.38
(s, 3H).
6.1.24. (6-chloro-5-methylpyridin-3-yl)methanamine (44)
To a solution of 43 (1.60 g, 6.23 mmol) in DCM (10 mL) was added TFA (10 mL). After stirring at
room temperature for 3 h, excess saturated aqueous Na2CO3 was added slowly. The mixture was
extracted with DCM (30 mL x 6). The combined organic layers were dried over Na2SO4 and
concentrated to give the desired product 44 (810 mg, 83%) as a yellow oil, which was used directly in
the next step without further purification.
6.1.25. (2',3-dimethyl-2,4'-bipyridin-5-yl)methanamine (45)
To a suspension of 44 (780 mg, 4.97 mmol), 17 (2.37 g, 50%, 5.46 mmol) and Pd(PPh3)4 (287 mg,
0.25 mmol) in dioxane/H2O (20 mL/4 mL) was added K2CO3 (2.74 g, 19.8 mmol). The mixture was
stirred at 100°C under N2 overnight. After cooling to room temperature, H2O (30 mL) was added and
the mixture was extracted with DCM (50 mL x 6). The combined organic layers were dried and
concentrated. After purification by column chromatography (dichloromethane/methanol = 50/1-20/1),
45 was obtained as a brown oil (794 mg, 75%). 1H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 5.2 Hz, 1H),
8.48 (s, 1H), 7.61 (s, 1H), 7.32 (s, 1H), 7.23 (d, J = 4.8 Hz, 1H), 3.94 (s, 2H), 2.62 (s, 3H), 2.37 (s, 3H).
6.1.26. 5-(Bromomethyl)-2-chloro-3-fluoropyridine (47)
To a solution of 2-chloro-3-fluoro-5-methylpyridine (5 g, 34.3 mmol) and NBS (18.3 g, 103 mmol)
in CH3CN (100 mL) was added BPO (829 mg, 3.43 mmol). The mixture was refluxed overnight. After
cooling to room temperature, H2O (200 mL) was added and the mixture was extracted with ethyl
acetate (200 mL x 3). The combined organic layers were washed with brine (100 mL x 3) and dried
over Na2SO4. After concentration and purification by column chromatography (petroleum ether/ethyl
acetate = 100/1), 47 was obtained as a yellow oil (7.45 g, 97%). 1H NMR (400 MHz, CDCl3) δ 8.23 (d,
J = 1.2 Hz, 1H), 7.56-7.53 (m, 1H), 4.45 (s, 2H).
6.1.27. (6-Chloro-5-fluoropyridin-3-yl)methanamine (48)
To a solution of 47 (7.45 g, 33.1 mmol) in DMF (50 mL) was added NaN3 (8.61 g, 132 mmol) at 0°C.
The mixture was stirred at 50°C overnight. After cooling to room temperature, H2O (100 mL) was
added and the mixture was extracted with ethyl acetate (100 mL x 3). The combined organic layers
were washed with brine (100 mL x 3) and dried over Na2SO4. After concentration and purification by
column chromatography (petroleum ether/ethyl acetate = 50/1), a colorless oil (5.6 g, 90%) was
obtained. To a solution of the above oil in THF (50 mL) was added PPh3. The mixture was refluxed and
H2O (10 mL) was added slowly. The mixture was refluxed for another 8 h. After cooling to room
temperature, the mixture was diluted with ethyl acetate (100 mL) and extracted with 0.2 N HCl (100
mL x 2). The combined aqueous layers were washed with ethyl acetate (100 mL). The pH was adjusted
to 9 with 2 N NaOH, and then the mixture was extracted with ethyl acetate (200 mL x 3). The
combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by silica
16

gel column chromatography (dichloromethane/methanol = 10/1) to give the desired product (2.57 g,
1
53%) as a brown oil. H NMR (400 MHz, DMSO-d6) δ 8.23 (s, 1H), 7.89 (d, J = 9.6 Hz, 1H), 3.75 (s,
2H).
6.1.28. (3-Fluoro-2'-methyl-[2,4'-bipyridin]-5-yl)methanamine (49)
To a suspension of 48 (2.36 g, 14.6 mmol), 17 (7.0 g, 50%, 16.1 mmol) and Pd(PPh3)4 (847 mg, 0.73
mmol) in dioxane/H2O (40 mL/10 mL) was added K2CO3 (8.09 g, 58.6 mmol). The mixture was stirred
at 100°C under N2 overnight. After cooling to room temperature, H2O (30 mL) was added and the
mixture was extracted with DCM (100 mL x 6). The combined organic layers were washed with brine
(30 mL x 3) and dried over Na2SO4. After concentration and purification by column chromatography
(dichloromethane/methanol/ ammonium hydroxide = 50/1/0.01-20/1/0.01), 49 was obtained as a brown
solid (2.5 g, 79%). 1H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 4.8 Hz, 1H), 8.50 (s, 1H), 7.77 (s, 1H),
7.69 (d, J = 4.0 Hz, 1H), 7.58 (d, J = 12.0 Hz, 1H), 4.01 (s, 2H), 2.65 (s, 3H).
6.1.29. General procedure for the synthesis of compounds 50-53
To a solution of 25 (1 eq.) and corresponding amine (1 eq.) in DMF (1.5 mL) were added HATU (1
eq.) and DIPEA (5 eq.). After stirring at room temperature overnight, H2O (30 mL) was added and the
mixture was extracted with ethyl acetate (30 mL x 3). The combined organic layers were washed with
brine (30 mL x 3) and dried over Na2SO4. After concentration, the residue was purified by column
chromatography to give the desired products 50-53.
6.1.29.1. N-((2'-Methyl-[2,4'-bipyridin]-5-yl)methyl)-9H-carbazole-2-carboxamide (50). Light
1
yellow solid (yield: 69%). H NMR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 9.23 (t, J = 5.2 Hz, 1H),
8.72 (s, 1H), 8.53 (d, J = 4.8 Hz, 1H), 8.22-8.14 (m, 2H), 8.08-8.01 (m, 2H), 7.91 (s, 2H), 7.82 (d, J =
4.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.48-7.40 (m, 1H), 7.22-7.16 (m, 1H),
4.60 (d, J = 5.2 Hz, 2H), 2.54 (s, 3H). ESI-MS (m/z): 393.0 [M+H]+.
6.1.29.2. N-((2'-Methyl-[3,4'-bipyridin]-6-yl)methyl)-9H-carbazole-2-carboxamide (51). White
1
solid (yield: 65%). H NMR (400 MHz, DMSO-d6) δ 11.50 (s, 1H), 9.25 (t, J = 5.6 Hz, 1H), 8.96 (s,
1H), 8.53 (d, J = 5.2 Hz, 1H), 8.25-8.15 (m, 3H), 8.08 (s, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.67 (s, 1H),
7.59-7.41 (m, 4H), 7.23-7.17 (m, 1H), 4.68 (d, J = 6.0 Hz, 2H), 2.54 (s, 3H). 13C NMR (75 MHz,
DMSO-d6) δ 167.1, 159.9, 158.7, 149.6, 147.1, 144.5, 140.8, 139.2, 135.0, 131.3, 126.5, 126.1, 124.8,
121.8, 121.2, 120.9, 120.5, 119.9, 118.9, 118.5, 117.7, 111.3, 110.5, 44.8, 24.2. HRMS (ESI): calcd for
C25H21N4O [M+H]+ 393.1710, found 393.1705.
6.1.29.3. N-((2',3-Dimethyl-[2,4'-bipyridin]-5-yl)methyl)-9H-carbazole-2-carboxamide (52). White
solid (yield: 44%). 1H NMR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 9.19 (t, J = 6.0 Hz, 1H), 8.52 (d, J
= 5.2 Hz, 2H), 8.21-8.15 (m, 2H), 8.04 (s, 1H), 7.72 (d, J = 4.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 1H), 7.45
(d, J = 7.6 Hz, 1H), 7.41 (s, 1H), 7.35 (d, J = 4.0 Hz, 1H), 7.22-7.16 (m, 1H), 4.57 (d, J = 5.6 Hz, 2H),
2.53 (s, 3H), 2.34 (s, 3H). ESI-MS (m/z): 407.0 [M+H]+.
6.1.29.4. N-((3-Fluoro-2'-methyl-[2,4'-bipyridin]-5-yl)methyl)-9H-carbazole-2-carboxamide (53).
1
White solid (yield: 84%). H NMR (400 MHz, DMSO-d6) δ 11.50 (s, 1H), 9.25 (t, J = 5.2 Hz, 1H),
8.63 (s, 1H), 8.58 (d, J = 5.2 Hz, 1H), 8.19 (dd, J = 10.8, 8.8 Hz, 2H), 8.04 (s, 1H), 7.84 (d, J = 12.0 Hz,
17

1H), 7.77-7.70 (m, 2H), 7.68 (d, J = 5.2 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.47-7.41(m, 1H), 7.21-7.17
(m, 1H), 4.64 (d, J = 5.6 Hz, 2H), 2.55 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 167.3, 158.5, 157.6 (d,
J = 260.2 Hz), 149.5, 145.3 (d, J = 4.2 Hz), 142.2 (d, J = 5.6 Hz), 140.9, 140.8 (d, J = 10.2 Hz), 139.2,
138.9 (d, J = 3.7 Hz), 131.1, 126.6, 124.9, 123.9 (d, J = 20.7 Hz), 121.8, 121.7 (d, J = 7.1 Hz), 120.9,
119.9, 119.8 (d, J = 6.4 Hz), 119.0, 117.7, 111.3, 110.5, 40.3, 24.3. HRMS (ESI): calcd for C25H20FN4O
[M+H]+ 411.1616, found 411.1614.
6.1.30. Isoquinoline-6-carbonitrile (55)
To a suspension of 6-bromoisoquinoline (624 mg, 3.0 mmol) and Pd(PPh3)4 (173 mg, 0.15 mmol) in
DMF (6 mL) was added Zn(CN)2 (210 mg, 1.8 mmol), and the reaction mixture was stirred at 90°C
under N2 for 24 h. After cooling to room temperature, H2O (40 mL) was added and the mixture was
extracted with ethyl acetate (40 mL x 3). The combined organic layers were washed with brine (30 mL
x 3) and dried over Na2SO4. After concentration and purification by column chromatography
1
(petroleum ether/ethyl acetate = 3/1), 55 was obtained as a white solid (360 mg, 78%). H NMR (400
MHz, CDCl3) δ 9.37 (s, 1H), 8.70 (d, J = 5.6 Hz, 1H), 8.25 (s, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.77 (d, J
= 8.4 Hz, 1H), 7.73 (d, J = 5.6 Hz, 1H).
6.1.31. Isoquinolin-6-ylmethanamine (56)
To a solution of 55 (20 mg, 0.13 mmol) in ethanol (2 mL) were added Pd/C (4 mg) and 37%HCl
(0.05 mL), and the reaction mixture was stirred at room temperature under H2 for 24 h. Saturated
aqueous Na2CO3 (10 mL) was added. The mixture was filtered, and the filtrate was extracted with
DCM (30 mL x 3). The combined organic layers were dried over Na2SO4 and concentrated to give the
desired product (20 mg, 97%) as a light yellow solid, which was used directly in the next step without
further purification. 1H NMR (400 MHz, CDCl3) δ 9.31 (s, 1H), 8.60 (d, J = 5.6 Hz, 1H), 8.27 (s, 1H),
8.04 (d, J = 8.8 Hz, 1H), 7.96 (d, J = 8.8 Hz, 1H), 7.74 (d, J = 5.6 Hz, 1H), 4.00 (s, 2H).
6.1.32. N-(Isoquinolin-6-ylmethyl)-9H-carbazole-2-carboxamide (57)
To a solution of 25 (26 mg, 0.13 mmol) and 56 (20 mg, 0.13 mmol) in DMF (2 mL) were added
HATU (48 mg, 0.13 mmol) and DIPEA (84 mg, 0.65 mmol). After stirring at room temperature
overnight, H2O (30 mL) was added and the mixture was extracted with ethyl acetate (30 mL x 3). The
combined organic layers were washed with brine (30 mL x 3) and dried over Na2SO4. After
concentration and purification by column chromatography (dichloromethane/methanol = 50/1), 57 was
obtained as a light yellow solid (8 mg, 18%). 1H NMR (400 MHz, DMSO-d6) δ 11.49 (s, 1H), 9.28 (s,
2H), 8.47 (d, J = 5.6 Hz, 1H), 8.19 (dd, J = 10.8, 8.4 Hz, 2H), 8.11 (d, J = 8.4 Hz, 1H), 8.07 (s, 1H),
7.88 (s, 1H), 7.82 (d, J = 5.6 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.53 (d, J =
8.0 Hz, 1H), 7.47-7.41 (m, 1H), 7.22-7.16 (m, 1H), 4.74 (d, J = 5.6 Hz, 2H). ESI-MS (m/z): 352.0
[M+H]+.
6.1.33. 6-Cyanonaphthalen-2-yl trifluoromethanesulfonate (59)
To a solution of 6-hydroxy-2-naphthonitrile (1.0 g, 5.92 mmol) and Et3N (896 mg, 8.88 mmol) in
DCM (1.5 mL) was added Tf2O (2.5 g, 8.88 mmol). After stirring at room temperature overnight, H2O
(30 mL) was added and the mixture was extracted with ethyl acetate (30 mL x 3). The combined
organic layers were washed with brine (30 mL x 3) and dried over Na2SO4. After concentration and
18

purification by column chromatography (petroleum ether/ethyl acetate = 30/1), 59 was obtained as a
white solid (1.65 g, 93%). 1H NMR (400 MHz, CDCl3) δ 8.30 (s, 1H), 8.03 (d, J = 9.2 Hz, 1H), 7.99 (d,
J = 8.4 Hz, 1H), 7.83 (d, J = 1.6 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.52 (dd, J = 9.2, 2.4 Hz, 1H).
6.1.34. 6-(Aminomethyl)-2-naphthonitrile (60)
To a suspension of 59 (1.35 g, 4.49 mmol), Pd(dba)2 (114 mg, 0.20 mmol) and K3PO4 (1.27 g, 5.99
mmol) in dioxane (25 mL) were added XPhos (143 mg, 0.30 mmol) and MeNO2 (4 mL). The mixture
was stirred at 80°C under N2 for 18 h. After cooling to room temperature, CH3COOH (8 mL) and Zn
powder (2.93 g, 45 mmol) were added. The mixture was stirred at 35°C for 3 h and filtered to remove
residual Zn powder. The filtrate was diluted with water (30 mL) and washed with ethyl acetate (30 mL).
The pH was adjusted to 10 with 1 N NaOH, and then the mixture was extracted with ethyl acetate (30
mL x 3). The combined organic layers were washed with brine (30 mL x 3), and dried over Na2SO4.
After concentration and purification by column chromatography (petroleum ether/ethyl acetate = 2/1),
1
60 was obtained as a light yellow solid (280 mg, 34%). H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H),
7.93-7.83 (m, 3H), 7.63-7.55 (m, 2H), 4.10 (s, 2H).
6.1.35. N-((6-Cyanonaphthalen-2-yl)methyl)-9H-carbazole-2-carboxamide (61)
To a solution of 25 (53 mg, 0.25 mmol) and 60 (46 mg, 0.25 mmol) in DMF (2 mL) were added
HATU (95 mg, 0.25 mmol) and DIPEA (161 mg, 1.25 mmol). After stirring at room temperature for 12
h, H2O (30 mL) was added and the mixture was extracted with ethyl acetate (30 mL x 3). The
combined organic layers were washed with brine (30 mL x 3) and dried over Na2SO4. After
concentration and purification by column chromatography (dichloromethane/methanol = 50/1), 61 was
obtained as a light yellow solid (12 mg, 13%). 1H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 9.28 (t,
J = 5.6 Hz, 1H), 8.53 (s, 1H), 8.22-8.14 (m, 2H), 8.10-8.02 (m, 3H), 7.95 (s, 1H), 7.77-7.66 (m, 3H),
7.52 (d, J = 8.0 Hz, 1H), 7.45-7.43 (m, 1H), 7.22-7.16 (m, 1H), 4.72 (d, J = 5.6 Hz, 2H). ESI-MS (m/z):
376.1 [M+H]+.
6.1.36. General procedure for the synthesis of compounds 62-66
To a solution of 49 (1 eq.) and corresponding acid (1 eq.) in DMF (1.5 mL) were added HATU (1 eq.)
and DIPEA (5 eq.). After stirring at room temperature overnight, H2O (30 mL) was added and the
mixture was extracted with ethyl acetate (30 mL x 3). The combined organic layers were washed with
brine (30 mL x 3) and dried over Na2SO4. After concentration, the residue was purified by silica gel
column chromatography to give the desired products 62-66.
6.1.36.1. N-((3-Fluoro-2'-methyl-[2,4'-bipyridin]-5-yl)methyl)-9H-fluorene-2-carboxamide (62).
White solid (yield: 71%). 1H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 5.2 Hz, 1H), 8.57 (s, 1H), 8.03 (s,
1H), 7.83 (s, 3H), 7.75 (s, 1H), 7.67 (d, J = 4.8 Hz, 1H), 7.64-7.55 (m, 2H), 7.45-7.35 (m, 2H), 6.76 (t,
J = 6.0 Hz, 1H), 4.77 (d, J = 6.0 Hz, 2H), 3.95 (s, 2H), 2.64 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ
166.7, 158.4, 157.5 (d, J = 259.8 Hz), 149.4, 145.2 (d, J = 4.3 Hz), 144.1, 144.0, 143.0, 142.2 (d, J =
5.5 Hz), 140.7 (d, J = 9.9 Hz), 140.2, 138.7 (d, J = 3.6 Hz), 132.2, 127.7, 127.0, 126.4, 125.3, 124.2,
123.9 (d, J = 20.7 Hz), 121.7 (d, J = 5.4 Hz), 120.8, 119.8, 119.7, 40.3, 36.4, 24.2. HRMS (ESI): calcd
for C26H21FN3O [M+H]+ 410.1663, found 410.1662.
6.1.36.2.
19

9,9-Difluoro-N-((3-fluoro-2'-methyl-[2,4'-bipyridin]-5-yl)methyl)-9H-fluorene-2-carboxamide
1
(63). White solid (yield: 70%). H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 5.6 Hz, 1H), 8.56 (s, 1H),
8.03-7.99 (m, 2H), 7.76 (s, 1H), 7.70-7.58 (m, 5H), 7.55-7.49 (m, 1H), 7.45-7.39 (m, 1H), 6.75 (t, J =
5.6 Hz, 1H), 4.77 (d, J = 6.0 Hz, 2H), 2.64 (s, 3H). HRMS (ESI): calcd for C26H19F3N3O [M+H]+
446.1475, found 446.1473.
6.1.36.3. N-((3-Fluoro-2'-methyl-[2,4'-bipyridin]-5-yl)methyl)-9-oxo-9H-fluorene-2-carboxamide
1
(64). Yellow solid (yield: 50%). H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 5.2 Hz, 1H), 8.56 (s, 1H),
8.11 (d, J = 8.0 Hz, 1H), 7.99 (s, 1H), 7.75 (s, 1H), 7.68 (d, J = 6.8 Hz, 2H), 7.65-7.51 (m, 4H),
7.40-7.34 (m, 1H), 6.89 (t, J = 5.6 Hz, 1H), 4.76 (d, J = 5.6 Hz, 2H), 2.64 (s, 3H). HRMS (ESI): calcd
for C26H19FN3O2 [M+H]+ 424.1456, found 424.1453.
6.1.36.4.
N-((3-Fluoro-2'-methyl-[2,4'-bipyridin]-5-yl)methyl)-9-methyl-9H-carbazole-2-carboxamide (65).
White solid (yield: 64%). 1H NMR (400 MHz, DMSO-d6) δ 9.27 (t, J = 5.6 Hz, 1H), 8.64 (s, 1H), 8.58
(d, J = 5.2 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 7.6 Hz, 1H), 8.17 (s, 1H), 7.86 (d, J = 12.0 Hz,
1H), 7.80-7.74 (m, 2H), 7.70-7.62 (m, 2H), 7.56-7.50 (m, 1H), 7.27-7.21 (m, 1H), 4.66 (d, J = 5.6 Hz,
2H), 3.94 (s, 3H), 2.55 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 167.1, 158.5, 157.5 (d, J = 259.8 Hz),
149.4, 145.3 (d, J = 4.2 Hz), 142.2 (d, J = 5.5 Hz), 141.7, 140.8 (d, J = 10.3 Hz), 140.1, 138.8 (d, J =
3.7 Hz), 131.1, 126.7, 124.4, 123.9 (d, J = 20.5 Hz), 121.7 (d, J = 5.4 Hz), 121.3, 120.9, 120.0, 119.7 (d,
J = 6.2 Hz), 119.2, 117.9, 109.5, 108.6, 40.3, 29.1, 24.2. HRMS (ESI): calcd for C26H22FN4O [M+H]+
425.1772, found 425.1769.
6.1.36.5. N-((3-Fluoro-2'-methyl-[2,4'-bipyridin]-5-yl)methyl)dibenzo[b,d]furan-3-carboxamide
(66). White solid (yield: 37%). 1H NMR (400 MHz, DMSO-d6) δ 9.36 (t, J = 5.6 Hz, 1H), 8.63 (s, 1H),
8.58 (d, J = 5.6 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 8.24-8.20 (m, 2H), 7.98 (d, J = 8.0 Hz, 1H), 7.86 (d,
J = 12.4 Hz, 1H), 7.80-7.74 (m, 2H), 7.68 (d, J = 4.8 Hz, 1H), 7.63-7.57 (m, 1H), 7.49-7.41 (m, 1H),
4.65 (d, J = 5.6 Hz, 2H), 2.55 (s, 3H). HRMS (ESI): calcd for C25H19FN3O2 [M+H]+ 412.1456, found
412.1453.
6.2. In vitro biological assays
Super-top flash (STF) reporter gene assay and Wnt secretion assay had been reported in our
published papers14, 18
.
6.3. Chemical stability, plasma stability and metabolic stability test
The methods of chemical stability, plasma stability and metabolic stability test had been reported in
our published papers14, 18
.
6.4. CYP inhibition assays
The experimental procedures for the CYP inhibition assays had been reported in our published
papers18.
Acknowledgements
The authors appreciate the supports from National Natural Science Foundation of China (Grant
20

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
References
1.
2.
3.
4.
5.
6.
Clevers, H. Cell 2006, 127, 469.
Reya, T.; Clevers, H. Nature 2005, 434, 843.
Espada, J.; Calvo, M. B.; Diaz-Prado, S.; Medina, V. Clin. Transl. Oncol. 2009, 11, 411.
MacDonald, B. T.; Tamai, K.; He, X. Dev. Cell 2009, 17, 9.
Iozzo, R. V.; Eichstetter, I.; Danielson, K. G. Cancer Res. 1995, 55, 3495.
Yeo, E. J.; Cassetta, L.; Qian, B. Z.; Lewkowich, I.; Li, J. F.; Stefater, J. A., 3rd; Smith, A. N.;
Wiechmann, L. S.; Wang, Y.; Pollard, J. W.; Lang, R. A. Cancer Res. 2014, 74, 2962.
Kurayoshi, M.; Yamamoto, H.; Izumi, S.; Kikuchi, A. Biochem. J. 2007, 402, 515.
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.
7.
8.
9.
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.
10.
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.
11.
12.
13.
14.
Cheng, D.; Liu, J.; Han, D.; Zhang, G.; Gao, W.; Hsieh, M. H.; Ng, N.; Kasibhatla, S.; Tompkins,
C.; Li, J.; Steffy, A.; Sun, F.; Li, C.; Seidel, H. M.; Harris, J. L.; Pan, S. ACS Med. Chem. Lett.
2016, 7, 676.
Madan, B.; Ke, Z.; Harmston, N.; Ho, S. Y.; Frois, A. O.; Alam, J.; Jeyaraj, D. A.; Pendharkar, V.;
Ghosh, K.; Virshup, I. H.; Manoharan, V.; Ong, E. H.; Sangthongpitag, K.; Hill, J.; Petretto, E.;
Keller, T. H.; Lee, M. A.; Matter, A.; Virshup, D. M. Oncogene 2016, 35, 2197.
Duraiswamy, A. J.; Lee, M. A.; Madan, B.; Ang, S. H.; Tan, E. S.; Cheong, 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. 2016, 58, 5889.
Xu, Z.; Li, J.; Wu, Y.; Sun, Z.; Luo, L.; Hu, Z.; He, S.; Zheng, J.; Zhang, H.; Zhang, X. Eur. J.
Med. Chem. 2016, 108, 154.
15.
16.
Cheng, D.; Zhang, G.; Han, D.; Gao, W.; Pan, S.; Shen, L.; Leleti, R. R. WO 2012003189.
Poulsen, A.; Ho, S. Y.; Wang, W.; Alam, J.; Jeyaraj, D. A.; Ang, S. H.; Tan, E. S.; Lin, G. R.;
Cheong, V. W.; Ke, Z.; Lee, M. A.; Keller, T. H. J. Chem. Inf. Model 2015, 55, 1435.
Fleming, F. F.; Yao, L.; Ravikumar, P. C.; Funk, L.; Shook, B. C. J. Med. Chem. 2010, 53, 7902.
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. Bioorg. Med. Chem. 2015, 23, 6855.
17.
18.
21

19.
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.
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For Table of Contents Use Only
Design, Synthesis, and Evaluation of Novel Porcupine Inhibitors Featuring a Fused 3-ring
System Based on the “Reversed” Amide Scaffold
Zhixiang Xu a,1, Xiangxiang Xu a,1, Ruadhan O'Laoi b, Haikuo Ma a, Jiyue Zheng a,*, Shuaishuai
Chen c, Lusong Luo c,*, Zhilin Hu d, Sudan He d, Jiajun Li a, Hongjian Zhang a, and Xiaohu Zhang
a,*
a
Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psychiatric-Diseases and
College of Pharmaceutical Sciences, Soochow University, Su Zhou, Jiangsu 215021, P. R. China
b Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland
c BeiGene (Beijing) Co., Ltd., No. 30 Science Park Road, Zhongguancun Life Science Park, Beijing
102206, P.R.China
d
Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, and
Collaborative Innovation Center of Hematology, Soochow University, Suzhou 215123, P.R. China
23