Discovery and structure-activity relationship study of phthalimide-phenylpyridine conjugate as inhibitor of Wnt pathway

Article abstract of DOI:10.1016/j.bmcl.2019.02.009

Aberrant Wnt signaling has been implicated in a variety of disease. Inhibition of the Wnt pathway is an attractive approach for developing new therapeutics for the treatment of various types of fibrosis and cancers. We have discovered the phthalimide-phenylpyridine conjugate as a novel hit compound for the Wnt pathway inhibitors from cellular screening. The structure-activity relationship of these compounds suggested both of the substituent group on the phthalimide fragment and the structure of the linker were critical to the inhibitory activity. The most potent compound was about 10-folds more potent than the hit compound, with IC₅₀ value of 0.28 ± 0.01 μM.

Full text of DOI:10.1016/j.bmcl.2019.02.009

Accepted Manuscript
Discovery and Structure-activity relationship study of phthalimide-phenylpyr-
idine conjugate as inhibitor of Wnt pathway
Hongna Wu, Jun Wu, Wenxuan Zhang, Zhongwen Li, Jinhui Fang, Xu Lian,
Tong Qin, Hao Jie, Qi Zhou, Song Wu
PII:
S0960-894X(19)30083-6
https://doi.org/10.1016/j.bmcl.2019.02.009
BMCL 26289
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 December 2018
28 January 2019
7 February 2019
Please cite this article as: Wu, H., Wu, J., Zhang, W., Li, Z., Fang, J., Lian, X., Qin, T., Jie, H., Zhou, Q., Wu, S.,
Discovery and Structure-activity relationship study of phthalimide-phenylpyridine conjugate as inhibitor of Wnt
pathway, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.02.009
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Discovery and Structure-activity relationship
study of phthalimide-phenylpyridine
conjugate as inhibitor of Wnt pathway
Hongna Wu a,#, Jun Wub,c,#, Wenxuan Zhanga,#,*, Zhongwen Lib,d, Jinhui Fangb,c, Xu
Lian a, Tong Qin a, Hao Jie b,c, Qi Zhou b,c, Song Wua,*
a. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
b. State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing
100101, China
c. Beijing Stem Cell Bank, Institute of Zoology, Chinese Academy of Sciences, Beijing 100192, China
d. University of Chinese Academy of Sciences, Beijing 100049, China
#These three authors contributed equally to this work.
Corresponding Author*email: wxzhang@imm.ac.cn; ws@imm.ac.cn
Abstract
Aberrant Wnt signaling has been implicated in a variety of disease. Inhibition
of the Wnt pathway is an attractive approach for developing new therapeutics for the
treatment of various types of fibrosis and cancers. We have discovered the
phthalimide-phenylpyridine conjugate as a novel hit compound for the Wnt pathway
inhibitors from cellular screening. The structure-activity relationship of these
compounds suggested both of the substituent group on the phthalimide fragment and
the structure of the linker were critical to the inhibitory activity. The most potent
compound was about 10-folds more potent than the hit compound, with IC50 value of
0.28±0.01 M.
Key words
Wnt signaling pathway, Phthalimide-phenylpyridine conjugate, Structure-activity
relationship
The Wnt signaling pathway is a critical developmental pathway which controls
cellular functions such as proliferation and differentiation.1,2,3 Extracellular Wnt can
trigger the canonical Wnt/β-catenin dependent pathway, the noncanonical Wnt/Ca2+
and Planar Cell Polarity pathway (PCP). The β-catenin dependent signaling pathway
1

is activated by the binding of Wnt ligand to the low-density lipoprotein receptor
(LRP-5/6 receptors) and Frizzled receptors. This in turn activates Disheveled protein
(Dvl), which inhibits Axin-mediated β-catenin phosphorylation, resulting in
accumulation of cytoplasmic β-catenin. The β-catenin migrates to the nucleus,
interacting there with T cell-specific factor (TCF)/lymphoid enhancer binding factor
(LEF) and co-activators, to turn on the Wnt target genes such as c-Myc, cyclin D1and
Cdkn1.
Aberrant Wnt signaling has been implicated in a variety of disease, such as
degenerative diseases, metabolic diseases and cancer.4 In several types of malignancy,
Wnt signaling contribute to the maintenance of the cancer stem cell (CSC)
population.5 Inhibition of the Wnt pathway is an attractive approach for developing
new therapeutics for the treatment of various types of fibrosis and cancers.6,7 Several
types of Wnt-signaling inhibitors are under ongoing development as anticancer
therapies, including Porcupine inhibitors8,9, β-Catenin-destruction complex inhibitors
such as Tankyrase inhibitors10,11 and Disheveled inhibitors12, TCF/β-catenin
transcription complex inhibitors13 and CREB-binding protein (CBP)/β-catenin
antagonist14. The Porcupine inhibitors LGK9748 and CBP/β-catenin antagonist PRI-
724 are now in clinical trails for the treatment of cancers (Fig 1).
O
H
N
N
MeO
N
O
N
N
N
O
O
N
N
N
H
OH
LGK974
PRI-724
Figure 1 The structure of LGK974 and PRI-724
Herein, we describe the identification of phthalimide-phenylpyridine conjugate
as a novel Wnt inhibitor from cellular screening. With the L-Wnt3A cell line that
contains a luciferase reporter for β-catenin mediated transcriptional activation
reported by Lum and co-workers,6 we screened the synthetic small-molecule library in
our lab. Fortunately, one compound (1) showed modest inhibitory activity (34%) at 10
 in the assay (Fig 2), which has a phthalimide and phenylpyridine fragment, linked
by a carbon chain. There are few similar compounds and biological activity reported,
so it is worthy to study their structure-activity relationship.
2

O
N
NH
N
O
O
1
Figure 1 The structure of hit compound 1
At first, the modifications on the phthalimide fragment were carried out (Scheme
1). The derivatives of the compound 1 were synthesized via a two–step amide
coupling reactions from the commercially available precursor phthalic anhydrides. In
the first step, the phthalic anhydrides coupled with 1, 4- aminobutyric acid in the
presence of the acetic acid at 120 ℃ to afford the immediate III. This compound was
pre-activated by the HATU, followed by the coupling reaction with 2-amino-5-
phenylpyridine, giving the finial phthalimide-phenylpyridine conjugates in 29~67%
yields.
O
O
O
O
N
a
b
OH
R
O
H2N
NH
R
N
R
OH
N
O
O
O
O
O
III
I
II
1-16
R=H,F,Cl,Br,NO2,
Me,tertyl-,phenyl-,
a:HOAC,120℃；b:
HATU, TEA, DCM, r.t
Scheme 1. The synthetic route of compounds
With the LGK 974 as the positive control agent, these compound were assayed in
the L-Wnt3A-luciferase reporter system.15 Some derivatives showed modest
inhibition ratio at 10  in this assay (Table 1). It is clear that the substitutions and
their positions on the phthalimide fragment had great effects on the biological activity.
The conjugates with halogen and methyl group at 3-position (3, 7, 14) were more
potent than that with substitutions at 4-group (2, 6, 13, 15, 16). By contrast, both of
the derivatives with the strong electron-withdraw nitro group at 3- or 4-position (11 or
12) showed weaker biological activity. Similarly, the extra halogen-substitutions of
the 3- or 4-halogenated compounds (4, 5, 8, 9) decreased the inhibitory activity of the
Wnt signaling pathway. Among these phthalimide-phenylpyridine conjugates, the
electron-donating 3-methyl derivatives (14) showed the best inhibitory activity, with
IC50 value of 2.79±0.01  in the cell assay. These results suggested that the
electrical property of the phthalimide fragment played the important effect on the Wnt
3

pathway inhibitory activity, possibly by the adjustment the electronegativity of the 1-
carbonyl group which may provide a hydrogen-honding acceptor to the target. In
additions, the stereo-hindrance effect also had great influence, thus compound 16 with
Phenylacetylenyl group on 4-position showed weaker inhibitory rate than the methyl
and butyl derivatives 13 and 15.
Table 1. Wnt signaling pathway inhibitory activity of synthesized compounds
Comp
1
R
H
Yield/%
68
Inhibition ratio /%
34
20
44
21
37
22
46
-8
2
4-F
40
3
3-F
40
4
3,4-F
3,6-F
4-Cl
41
5
40
6
55
7
3-Cl
52
8
4,5-Cl
2,3,4,5-Cl
4-Br
68
9
69
-5
10
11
12
13
14
55
18
19
17
20
61
4-NO2
3-NO2
4-Me
3-Me
58
57
50
52
(IC50=2.79±0.01 )
15
16
4-butyl
57
34
23
4-Phenylacetylenyl
7
LGK 974
93
(IC50=0.57±0.28 nM)
The structure of linker between the phthalimide and phenylpyridine fragments
was then explored. With the similar synthetic methods as compounds 1, the modified
derivatives of the lead compound 14 with different linkers were prepared (Scheme 2).
4

As expected, the length of the linker had great influences on the biological activities
(Table 2). By the decrease of the number of the carbon atoms from 3 to 2, the
conjugate 17 showed much less inhibitory activity. By contrast, compound 18 with a
one carbon-linker was about 10-folds more potent than conjugate 14, with IC50 value
of 0.28±0.01 M. However, when the linker was replaced by ethylidene, the
inhibitory rate of the compound 19 was reduced significantly. The difference of the
biological activity of these conjugates with different linker possibly because of the
change of the conformation of the molecules. Assumedly, the compound 18 may
adopt the relatively appropriated conformation to bind with the target, but the
conformation of the compound 17 is different. For compound 19, the linker has a
chirality center, leading to each mesomer adopts the opposite conformation, so they
showed about half inhibitory rate of compound 18.
CH3
O
CH3
CH3
O
O
O
N
b
a
H
O
H2N
N
X
OH
N
X
N
X
OH
O
O
O
O
O
17-19
a:HOAC,120℃；b:
HATU, TEA, DCM, r.t
Scheme 2. The synthetic route of compounds
Table 2. Wnt signaling pathway inhibition activity of synthesized compounds
inhibition
Comp
X
Yield/%
IC50/M
ratio /%
29
17
-CH2CH2-
54
>10
18
19
-CH2-
52
48
76
39
0.28±0.01a
>10
-CH(CH3)-
a
IC50=Mean ± S.E.(n=3)
In conclusion, we have discovered the phthalimide-phenylpyridine conjugate as a
promising new chemotype for the Wnt pathway inhibitors. The structure-activity
relationship of these compounds suggested both of the substituent group on the
phthalimide fragment and the structure of the linker were critical to the inhibitory
5

activity. Derivative 18 was the most potent compound to inhibit the Wnt signaling,
which will be studied and evaluated in the future.
ASSOCIATED CONTENT
Supporting Information
ACKNOWLEDGMENTS
We thank The National Natural Science Fund (81602958), 973 Program
(2015CB964803), The Drug Innovation Major Project (2018ZX09711001-005) and
CAMS Innovation Fund for Medical Sciences (CIFMS 2017-I2M-2-004) for financial
support.
Graphics
O
optimization
O
N
O
N
NH
N
N
N
H
H3C
O
O
O
19
1
Inhibitory activity IC50 =0.28
M

Inhibitory activity IC50 =2.79
M

±0.01
±0.01
Novel Wnt signaling pathway inhibitor
~10 folds more potent than 1
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