Discovery of 5,6-Bis(4-methoxy-3-methylphenyl)pyridin-2-amine as a WSB1 Degrader to Inhibit Cancer Cell Metastasis

Article abstract of DOI:10.1021/acs.jmedchem.1c00586

The gain of cell motility is an essential prerequisite for cancer metastasis. The ubiquitin ligase subunit WD repeat and SOCS box-containing 1 (WSB1) has been demonstrated to regulate hypoxia-driven tumor cell migration. However, there is still a lack of methods for discovering inhibitors targeting the WSB1 axis. Here, we employed phenotypic screening models and identified compound4that displayed migration inhibitory activity against WSB1-overexpressing cells. Further studies indicated that it may function as a WSB1 degrader, thus leading to the accumulation of the Rho guanosine diphosphate dissociation inhibitor 2 (RhoGDI2) protein, reversing the expression of downstream F-actin and formation of membrane ruffles, and disturbing the migration capacity of cancer cells. Moreover, compound4exhibited a promisingin vivoanticancer metastatic effects. Our findings show the discovery of a new WSB1 degrader, providing a unique solution for the treatment of cancer metastasis.

Full text of DOI:10.1021/acs.jmedchem.1c00586

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Discovery of 5,6-Bis(4-methoxy-3-methylphenyl)pyridin-2-amine as
a WSB1 Degrader to Inhibit Cancer Cell Metastasis
Jinxin Che, Zegao Jin, Fangjie Yan, Jieqiong You, Jiangfeng Xie, Binhui Chen, Gang Cheng, Hong Zhu,
Qiaojun He, Yongzhou Hu, Bo Yang, Ji Cao,* and Xiaowu Dong*
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ABSTRACT: The gain of cell motility is an essential prerequisite
for cancer metastasis. The ubiquitin ligase subunit WD repeat and
SOCS box-containing 1 (WSB1) has been demonstrated to
regulate hypoxia-driven tumor cell migration. However, there is
still a lack of methods for discovering inhibitors targeting the
WSB1 axis. Here, we employed phenotypic screening models and
identified compound 4 that displayed migration inhibitory activity
against WSB1-overexpressing cells. Further studies indicated that it
may function as a WSB1 degrader, thus leading to the
accumulation of the Rho guanosine diphosphate dissociation inhibitor 2 (RhoGDI2) protein, reversing the expression of
downstream F-actin and formation of membrane ruffles, and disturbing the migration capacity of cancer cells. Moreover, compound
4 exhibited a promising in vivo anticancer metastatic effects. Our findings show the discovery of a new WSB1 degrader, providing a
unique solution for the treatment of cancer metastasis.
organization and cell motility.10−12 We demonstrated that the
hypoxia-driven WSB1 promotes the proteasome-dependent
1. INTRODUCTION
As an important component of cancer development, cancer
degradation of RhoGDI2, thereby leading to an increased
metastasis is characterized by the migration of cancer cells
migration capacity of tumor cells.9 Therefore, HIF-1α-WSB1-
beyond the primary site where the tumor originated and
RhoGDI2 can be a novel branch of the HIF-1α signaling axis
eventually forming tumors in new sites (secondary and tertiary
with anticancer potential by controlling the migration capacity
foci). Metastasis is generally untreatable and detrimental to
of tumor cells (Figure 1). Besides, targeting the WSB1-
cancer patients. Metastasis involves a multistep complex series
RhoGDI2 pathway may overcome the nucleus escape of HIF-
of cell-biological events, termed as “invasion-metastasis
1α, providing new solutions for hypoxia-promoted cancer
cascade”, including angiogenesis, detachment, migration,
metastasis. However, apart from biological approaches such as
invasion, and adhesion.1,2 Cancer metastasis is thought to be
gene mutation or silencing,6−9 few small molecules have been
closely related to intratumoral hypoxia that contributes to the
reported to suppress tumor migration by interrupting the
increased capacity of invasion and migration of tumor cells.3,4
WSB1-RhoGDI2 pathway, which is crucial in clinical studies
The signal pathways involved in the relationships between
and the survival of patients with metastatic cancer.
hypoxia and cell motility remain obscure.
Given that the crystal structure of the WSB1 protein has
WD repeat and SOCS box-containing 1 (WSB1) is a direct
remained unsolved, and there is still a lack of screening models
target of hypoxia-inducible factor 1 (HIF1), which helps tumor
for identifying inhibitors targeting this pathway, it might not be
cells adapt to and survive in hypoxic conditions.5 WSB1 forms
easy to find active compounds modulating the WSB1−
an E3 ubiquitin ligase in complex with elongin B/C-Cullin5-
RhoGDI2 axis. Alternatively, phenotypic screening is a
Rbx1 and thus regulates tumor progression and chemo-
promising strategy for small-molecule drug discovery,
resistance through ubiquitination of multiple proteins such as
especially for those lacking essential information on targets.
the von Hippel-Lindau protein (pVHL),6 ataxia-telangiectasia
To date, cell-based phenotypic screening of structurally diverse
mutated kinase (ATM),7 and homeodomain-interaction
protein kinase 2 (HIPK2).8 In our previous research,9 WSB1
was found to be significantly upregulated under hypoxia and
subsequently drove the metastatic potential in osteosarcoma
Received: March 31, 2021
Published: June 1, 2021
cells. Moreover, the Rho guanosine diphosphate dissociation
inhibitor 2 (RhoGDI2) level can be modulated by WSB1
through direct ubiquitination. RhoGDI2 is a potent inhibitor
of Rho GTPases, which act as regulators of actin cytoskeletal
https://doi.org/10.1021/acs.jmedchem.1c00586
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Figure 1. Function of the WSB1−RhoGDI2 axis.
Figure 2. Discovery process of K1 and K2 with migration inhibitory activity.
compounds has delivered many hit/lead compounds.13−16 As
part of our continued interest in the area of phenotypic
screening17−19 and anticancer drug discovery,20−22 the results
of our further studies implied that the function of WSB1
promoting cell migration may not be cell-line-specific, so we
started with the phenotypic function of compounds using
wound-healing and transwell assay against different cell lines
for the enrichment of compounds with antimigration potential.
This was followed by a mechanism study to demonstrate
whether the active compounds exert their effects through
modulating the WSB1-RhoGDI2 signal pathway. In the
present study, we report a new WSB1 degrader, compound 4
bearing the pyridin-2-amine scaffold, which can inhibit cancer
cell metastasis in vitro and in vivo. This finding provides
evidence that the hypoxia-driven metastasis of cancer can be
inhibited through modulating the pathway and would help
unleash the potential of the WSB1 protein as a therapeutic
target.
wound-healing assay in human non-small-cell lung cancer
(NSCLC) cells (H1299). Among them, the two compounds
bearing scaffold K (furan-2(5H)-one), K1 and K2, showed
potent inhibitory activities against cancer cell migration at the
concentration of 5 μM. The migration rates of cells treated
with K1 and K2 were 0.51 0.17 and 0.44 0.07, respectively
(Figure 2). Previously, only a few studies aimed to evaluate the
antimigration potential of the compounds with the furan-
2(5H)-one scaffold. This motivated us to use this new scaffold
for further development of anticancer metastasis compounds.
Structurally, K1 and K2 are composed of three different parts
(Figure 2). Part A is a heterocycle system, while B1 and B2 are
substituted phenyl rings. Furthermore, we derivatized K1 and
K2 for preparing more potent compounds.
Compounds 1a−f, 2a−g, 3a−c, and 4 were synthesized at
first for a preliminary structure−activity relationship (SAR)
study. The evaluation of the antimigration activity of such
compounds was performed using a wound-healing assay
against KHOS cells. The compounds that displayed good
activity were chosen for further testing against H460 cells.
Unless specifically mentioned, the wound-healing assay was
performed under normoxia. For determining the concentration
of the compounds in the wound-healing assay, the minimum
toxic concentration of the synthesized compounds was
evaluated. The results demonstrated that most of these
compounds exhibit low cytotoxic activities (Table 1), so the
test concentrations of compounds were set to 5 and 10 μM
against KHOS and H460 cells, respectively.
2. RESULTS AND DISCUSSION
2.1. Identification of the Hit Compounds Based on
Phenotypic Screening. First, the compounds from our in-
house compound library were subjected to structural cluster
analysis to avoid choosing compounds that bear the same
scaffolds. After clustering, a total of 42 compounds bearing
scaffolds A−N (Figure S1) were selected randomly for the
following phenotypic screening. Their inhibitory effects against
the migration of cancer cells were evaluated based on the
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Table 1. Effects of Different Aryl Substituents on Cell Migration Activity
KHOS cytotoxicity IC50
KHOS cell migration
H460 cytotoxicity IC50
H460 cell migration
a
b
compound
R1
(μM)
rate
(μM)
rate
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
3-CH3, 4-OCH3
4-F
4-Cl
4-OCH(CH3)2
3-CH3, 4-OCH2CH2CH3
3-CH3, 4-OCH2(CH2)3CH3
4-OCH3
4-OAc
4-F
4-CF3
H
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.47
0.76
0.70
0.74
0.62
N.A.
0.80
0.86
0.83
0.73
N.A.
0.76
0.77
>20
>20
N.T.
N.T.
>20
N.T.
>20
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
0.17
0.91
N.T.
N.T.
0.54
N.T.
0.64
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
c
d
−CH2CH3
2g
a
The migration rate was normalized to the control: migration rate = (C − D)/(A − B) × 100% (A = blank area of the control, 0 h; B = blank area
of the control, 16 h; C = blank area of the compound, 0 h; D = blank area of the compound, 16 h). The tested concentration was 5 μM. The results
are an average of three independent determinations. The migration rate was normalized to the control: migration rate = (C − D)/(A − B) ×
100% (A = blank area of the control, 0 h; B = blank area of the control, 48 h; C = blank area of the compound, 0 h; D = blank area of the
b
c
d
compound, 48 h). The tested concentration was 10 μM. The results are an average of three independent determinations. N.T., not tested. N.A.,
no activity; the average migration rate was >1.00.
Figure 3. Migration rates of compounds 1a−4 against KHOS or H460 cells. The compounds with an average migration rate >1.00 are not shown.
Results are shown as mean (n = 3) SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs the control.
As shown in Table 1 and Figure 3, some of these
compounds such as 1a, 1d, and 1e showed significant activities
in reducing the migration rate of KHOS cells. When the 3-
position of the furan-2(5H)-one scaffold was 2-fluro phenyl
(1a−f), the introduction of electron-withdrawing substituents
on the 4-position aryl ring of the furan-2(5H)-one scaffold can
lead to a decrease in activity (1b, 1c vs 1a). A loss of activity
was observed when larger substituents such as the pentyloxy
group were introduced (1d, 1e vs 1f). The modification of 2-
fluro phenyl at the 3-position of the furan-2(5H)-one scaffold
(2a−d) further reduced the migration inhibitory activity of the
compounds. Moreover, the compounds in which the phenyl
was changed to indol-3-yl (2e and 2f) or naphthalin-1-yl (2g)
failed to exhibit improved efficacy. The activities of compounds
1a, 1d, and 1e against KHOS cells showed statistical
significance (Figure 3). Further, some compounds that showed
potency or no activity were tested and confirmed on H460
cells. The migration of H460 cells was significantly inhibited
after treating with 1a, 1e, or 2a (Figure 3). The combined
results indicated that compound 1a exhibited the most potent
antimigration efficacy in both KHOS and H460 cell lines
(photographs of wound healing are shown in Tables S1 and
S2).
Considering the structure diversity requirement, compounds
bearing similar scaffolds, 1H-pyrrole-2,5-dione and pyridin-2-
amine, were further synthesized and evaluated (Table 2).
Although compound 3b exerted significant migration reduc-
tion activity against KHOS cells, the comparison between the
compounds bearing the furan-2(5H)-one scaffold revealed that
compounds with the 1H-pyrrole-2,5-dione scaffold (3a−c) did
not exert an improved antimigration effect in KHOS and H460
cancer cell lines. Notably, when both R1 and R2 were 3-methyl-
4-methoxyl substituted, compound 4 bearing the pyridine-2-
amine scaffold exhibited the best efficacy, which was
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Table 2. Effects of Different Scaffolds on Cell Migration Inhibitory Activity
KHOS cytotoxicity IC50
KHOS cell migration
H460 cytotoxicity IC50
H460 cell migration
a
b
compound
R1
R2
(μM)
rate
(μM)
rate
d
3a
3b
3c
4
2-F
4-OCH3
2-F
2-F
>50
>50
>50
0.74
0.72
0.74
0.51
>20
>20
N.T.
N.A.
N.A.
N.T.
0.40
c
3-CH3, 4-OCH3 2-F
3-CH3, 4-OCH3 3-CH3, 4-OCH3
a
39.1
>20
a
The migration rate was normalized to the control: migration rate = (C − D)/(A − B) × 100% (A = blank area of the control, 0 h; B = blank area
of the control, 16 h; C = blank area of the compound, 0 h; D = blank area of the compound, 16 h). The tested concentration was 5 μM. The results
are an average of three independent determinations. The migration rate was normalized to the control: migration rate = (C − D)/(A − B) ×
100% (A = blank area of the control, 0 h; B = blank area of the control, 48 h; C = blank area of the compound, 0 h; D = blank area of the
b
c
d
compound, 48 h). The tested concentration was 10 μM. The results are an average of three independent determinations. N.T., not tested. N.A.,
no activity; the average migration rate was >1.00.
Figure 4. Compounds 1a and 4 inhibited cell migration through elevating the levels of RhoGDI2 protein. (A) Transwell assay of compounds 1a
and 4 against KHOS cells under normoxia or hypoxia; the migration rate was normalized to the control: migration rate = A/B (A = number of
migration cells in the dosing group; B = number of migration cells in the control group). (B) Western blotting of WSB1, RhoGDI2 in KHOS cells
treated with compounds 1a and 4 under hypoxia. Results are shown as mean (n = 3) SD. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control.
Figure 5. SAR exploration of compound 4.
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comparable to compound 1a (Figure 3; photographs of wound
healing are shown in Tables S1 and S2). Therefore,
compounds 1a and 4 were chosen for the following
optimization and mechanism study after the initial SAR
exploration.
Table 3. In Vitro Cell Migration Inhibitory Activity of
Compounds 5a−f
2.2. Compounds 1a and 4 May Function through
Regulating the WSB1-RhoGDI2 Signal Pathway. In
addition to the wound-healing assay, the antimigration
activities of compounds 1a and 4 were validated using a
transwell assay. The results showed that both compounds also
displayed good inhibitory potential under normoxia, with
average cell migration rates of 0.38 and 0.54, respectively
(Figure 4A). Moreover, it was found that compounds 1a and 4
also significantly inhibited cancer cell migration under hypoxia
(Figure 4A), with average cell migration rates of 0.77 and 0.82,
respectively. This effect was comparable to their inhibitory
potential under normoxia.
Since we have noticed the critical role of WSB1-RhoGDI2 in
hypoxia-driven cancer metastasis, it motivated us to test
whether compound 1a or 4 could function through modulating
the WSB1-RhoGDI2 pathway. WSB1 can ubiquitylate the
RhoGDI2 protein and promote its proteasomal degradation,
thereby stimulating the tumor cell motility and invasion. Our
previous study demonstrated that the overexpression of
RhoGDI2 could reverse the metastasis of osteosarcoma
cells.9 Therefore, the effects of compounds 1a and 4 on the
WSB1-RhoGDI2 signaling pathway were evaluated. The WSB1
protein quantity was significantly upregulated in KHOS cells
under hypoxia, leading to a decrease in the RhoGDI2 protein
(Figure 4B). On the other hand, the treatment with 1a or 4
elevated the levels of the RhoGDI2 protein in cells. The results
suggest that compounds 1a and 4 may inhibit the migration of
cancer cells through the WSB1−RhoGDI2 axis, which are
worth further exploring.
2.3. In Vitro Antimetastasis Activity Evaluation of
Pyridin-2-amine Derivatives. According to the preliminary
SAR and mechanism study, pyridin-2-amine was selected as a
privileged scaffold for inhibiting the ubiquitin function of
WSB1 to prevent cancer cell metastasis. To fully understand
the SAR of pyridin-2-amine derivatives and find more potent
antimigration compounds that act as WSB1-RhoGDI2
modulators, we designed four series of compounds on the
structural basis of compound 4 (Figure 5). Regarding series I,
the substituents on A and B aryl rings were studied. Series II
and III were designed to improve the metabolic properties of 4
while maintaining its antimigration activity. From the
perspective of structure diversity, compounds with a skeleton
similar to 4 were also investigated, such as series IV. During
the following study, H460 cells were used to evaluate the
migration activity of the compounds. The cytotoxic IC50 of
each compound was tested before determining the tested
concentration of compounds in the wound-healing assay.
As shown in Table 3, compounds with various substituents
on the phenyl rings were synthesized and tested. Most
compounds such as 5b−f showed low cytotoxicity against
H460 cells except for compound 5a, which exhibited an IC50
value of 0.367 μM. The migration inhibitory activities of the
compounds were tested at a concentration of 10 μM;
compound 5a was excluded because of its high cytotoxicity.
The results indicate that the cells treated with compounds 5b
and 5c showed a significant decrease in migration rates (Figure
6). The activities of compounds 5b−f were lower than that of
compound 4. Compounds with substituents altered on the B
a
compound
R1
cytotoxicity IC50 (μM) migration rate
b
5a
5b
5c
5d
5e
5f
H
0.367
13.7
26.13
>40
13.08
21.7
24.47
N.T.
0.67
0.60
N.A.
N.A.
0.91
0.48
4-OCH(CH3)2
4-CF3
c
H
4-OCH(CH3)2
4-CF3
4
a
The migration rate was normalized to the control: migration rate =
(C − D)/(A − B) × 100% (A = blank area of the control, 0 h; B =
blank area of the control, 48 h; C = blank area of the compound, 0 h;
D = blank area of the compound, 48 h). The tested concentration was
10 μM. The results are an average of three independent
determinations. N.T., not tested. N.A., no activity; the average
migration rate was >1.00.
b
c
ring showed better activities than that altered on the A ring (5b
vs 5e, 5c vs 5f).
Further, heteroatoms such as nitrogen or oxygen were
introduced between the pyridine scaffold and the B ring, which
indicated that the original B ring was changed to phenoxyl or
phenylamino substitution (Table 4). All of the compounds
displayed low cytotoxicity against H460 cells, exhibiting IC50
values ranging from 2.58 to >40 μM. The migration inhibitory
activities of the compounds were tested at a concentration of
10 μM; compound 6c was excluded because of its high
cytotoxicity. Among the compounds, compounds 6h, 6j, and 6l
displayed statistical significance in inhibiting the migration of
cancer cells (Figure 6). Regarding phenoxyl substitution, all
compounds exhibited a loss of activity (6a, 6b, 6d−f),
irrespective of the substituents on the phenyl. Regarding
phenylamino substitution, removal of substituents on the A
ring resulted in a loss of the activity of compound 6g.
Substituent change of the A ring led to a decreased potency of
the compounds 6h and 6i vs 4, except for compound 6j, in
which R1 is 4-trifluoromethyl and exhibited comparable
migration inhibitory activity to 4. After changing the B ring
to the phenylamino group, 6k showed a dramatic loss in
potency, but the activity of compound 6l improved when
introducing 4-isopropyl to the phenylamino group. Collec-
tively, the introduction of heteroatoms did not show any
improvement in the migration inhibitory activity of the
compounds.
Moreover, the introduction of hydrophilic side chains to the
amino groups caused the cytotoxicity of the compounds to
remain at low levels (Table 5). Most compounds (7a−c, 7e,
and 7f) showed significant migration inhibitory activity against
the cancer cells except for compound 7d (Figure 6). The
migration inhibitory activities of 7b and 7f were comparable to
that of 4.
Furthermore, compounds 8a−i sharing a similar skeleton to
4 were synthesized and evaluated (Table 6). The pyridin-2-
amine scaffold was changed to nitrobenzene or aniline scaffold,
and the original B ring was changed to aliphatic heterocycles
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Figure 6. Migration rates of compounds 5b−8i against H460 cells. The compounds with an average migration rate >1.00 are not shown. Results
are shown as mean (n = 3) SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs control.
Table 4. In Vitro Cell Migration Inhibitory Activity of Compounds 6a−l
a
compound
R1
cytotoxicity IC50 (μM)
migration rate
0.82
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
>40
29.1
2.58
15.9
>40
>40
>40
33.4
>40
11.4
25.4
>40
24.47
b
4-OCH(CH3)2
3-CH3, 4-OCH3
4-CF3
N.A.
N.T.
c
0.91
N.A.
N.A.
N.A.
0.79
0.65
0.55
N.A.
0.77
0.48
H
4-OCH(CH3)2
H
4-OCH(CH3)2
3-CH3, 4-OCH3
4-CF3
6j
6k
6l
H
4-OCH(CH3)2
4
a
The migration rate was normalized to the control: migration rate = (C − D)/(A − B) × 100% (A = blank area of the control, 0 h; B = blank area
of the control, 48 h; C = blank area of the compound, 0 h; D = blank area of the compound, 48 h). The tested concentration was 10 μM. The
b
c
results are an average of three independent determinations. N.A., no activity; the average migration rate was >1.00. N.T., not tested.
such as substituted piperidyl, piperazinyl, or morpholinyl.
Although the compounds showed low cytotoxicity against
H460 cells, the migration inhibitory rates suffered a dramatic
loss. Although compounds 8f and 8i with morpholinyl
substitution at R2 displayed migration inhibitory activity,
they performed worse than 4 (Figure 6).
2.4. Compounds Inhibit WSB1-Mediated Metastasis
of Cancer Cells. To further address the effect of compounds
on WSB1-manipulated cell metastasis, the inhibition of
compounds on the metastasis of WSB1-overexpressing cells
was evaluated. Compared with the empty plasmid pCDH-
transduced KHOS cells (KHOS-pCDH), WSB1-overexpress-
ing KHOS cells (KHOS-WSB1) enhanced the wound-healing
capability of KHOS cells, indicating an efficient migrating
potency of KHOS-WSB1 (Figure 7A). The wound-healing was
not affected when the cells were treated with 7d, but
compound 4 can significantly block WSB1-enhanced wound
healing of KHOS-WSB1 cells, exhibiting a low migration rate
of 0.31
0.10. A similar effect was observed in pCDH-
transduced and WSB1-overexpressing H1299 cells. As shown
in Figure 7B, the wound-healing of H1299-WSB1 cells was
significantly inhibited by treating with compound 1a or 4.
Notably, compound 1a displayed lower migratory inhibitory
potency than compound 4.
Additionally, we further performed the transwell assay. As
shown in Figure 7C, H1299-WSB1 cells migrated more
efficiently into the lower chamber of the Transwell than
H1299-pCDH cells. In line with the results from the wound-
healing assay, the migrated capability of H1299-WSB1 cells
was also blocked by compound 1a or 4 (Figure 7C). To
confirm the role of WSB1 in compound-mediated migration
inhibition effect, we generated WSB1 knockout A2780 cells
(A2780-WSB1/KO) by the crispr-cas9 technology. As
expected, the A2780-WSB1/KO cells displayed a decrease in
the wound-healing capability compared to wild-type A2780
(A2780-WT) cells (Figure 7D). Notably, compound 4 can
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Table 5. In Vitro Cell Migration Inhibitory Activity of Compounds 7a−f
a
The migration rate was normalized to the control: migration rate = (C − D)/(A − B) × 100% (A = blank area of the control, 0 h; B = blank area
of the control, 48 h; C = blank area of the compound, 0 h; D = blank area of the compound, 48 h). The tested concentration was 10 μM. The
b
results are an average of three independent determinations. N.T., not tested.
only block the wound-healing capability of wild-type A2780
(A2780-WT) cells but not the A2780-WSB1/KO cells (Figure
7D). Thus, these results clearly suggested that WSB1 could be
a major knot for the function of compound 4.
observed (Figure 8C). To investigate the mechanism of
compound 4-mediated WSB1 inhibition, we first explored the
change of the mRNA level in H1299-WSB1 cells. The WSB1
mRNA level was 7.5-fold higher in H1299-WSB1 cells than
that in H1299-pCDH cells, while the RhoGDI2 mRNA level
was not influenced (Figure S2). After H1299-WSB1 cells were
treated with compound 4, the mRNA levels of RhoGDI2 and
WSB1 displayed no change compared to that of untreated cells
(Figure 8D), which indicated that the change in protein levels
induced by compound 4 might not function through
manipulating the mRNA level.
Next, we asked whether compound 4 affected the stability of
WSB1. To address this, MG123 (a proteasome degrader) and
Chloroquine (CQ, a lysosomal inhibitor) were utilized (Figure
8C). It was found that MG132 can prevent the compound 4-
induced degradation of WSB1 and lead to a comparable level
of WSB1 to that in untreated cells without affecting the
improvement of the RhoGDI2 level. However, CQ displayed
no influences on the protein levels of WSB1 and RhoGDI2.
These results suggested that compound 4 may induce WSB1
degradation in a proteasome-dependent manner. To confirm
this, a time-dependent degradation effect of compound 4
against WSB1 was investigated (Figure 8E). Cycloheximide
(CHX) was employed as a positive control, and a time-
dependent decreased level of WSB1 was observed when cells
were treated with compound 4, which suggested that
compound 4 may be a degrader of the WSB1 protein. Further,
we evaluated the dose-dependent effect of compound 4. As
shown in Figure 8F, with the concentration of compound 4
decreased, so did the degradation effect. Moreover, we
2.5. Compound 4 Is a WSB1 Degrader Increasing the
RhoGDI2 Level. Next, we tried to explore the mechanism of
compound-mediated migration inhibition. Accordingly, the
effects of active compounds on the WSB1 signal pathway were
evaluated. Some compounds that exhibited good or low
migration inhibitory activity were chosen for incubating with
the WSB1-transfected KHOS cells (KHOS-WSB1). As shown
in Figure 8A, compounds such as 6a−c, 6i, 6k, and 7d that
displayed no significant migration inhibition could not prevent
the degradation of RhoGDI2 in cells. The quantity of
RhoGDI2 protein increased after cells were incubated with
the active compounds 6l and 4. However, compound 7f, which
showed good activities in H460 cells, did not exhibit the
potential of modulating the WSB1-RhoGDI2 pathway, and the
underlying mechanism still needs to be investigated. In this
research, we mainly focused on compound 4.
To confirm the way that the active compounds modulate the
WSB1-RhoGDI2 signaling pathway, the H1299-WSB1 cell line
was used to be treated with compounds 1a and 4. The result
indicated that the WSB1-RhoGDI2 pathway was inhibited with
a subsequent increase in the quantity of the downstream
RhoGDI2 protein (Figure 8B), but compound 1a showed a
weaker activity than that of compound 4. Interestingly, an
obvious decrease in the level of the Myc-tagged WSB1 (Myc-
WSB1) protein was seen after the cells were treated with
compound 4. When WSB1 was Flag-tagged, inhibition was also
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Table 6. In Vitro Cell Migration Inhibitory Activity of Compounds 8a−i
a
The migration rate was normalized to the control: migration rate = (C − D)/(A − B) × 100% (A = blank area of the control, 0 h; B = blank area
of the control, 48 h; C = blank area of the compound, 0 h; D = blank area of the compound, 48 h). The tested concentration was 10 μM. The
b
results are an average of three independent determinations. N.A., no activity; the average migration rate was >1.00.
generated the Flag-tagged green fluorescent protein (Flag-
GFP) stably expressed H1299 cells. Compound 4 did not
affect the protein level of Flag-GFP dose-dependently, which
suggested that compound 4 induced the degradation of WSB1
rather than the Flag-tag.
organization and motility of cells.23 Since compound 4 was
demonstrated as a degrader of WSB1, we examined the effect
of compound 4 on the status of downstream polymerized actin
(F-actin) organization in H1299-WSB1 cells. As illustrated in
Figure 9, overexpression of WSB1 enhanced the fluorescence
intensity of F-actin in cells, and the appearance of membrane
ruffles was observed, indicating abundant F-actin expression
and the cell motility ability was enhanced. Notably, after the
cells were treated with compound 4, the fluorescence intensity
of F-actin and the formation of membrane ruffles decreased.
These results suggest that the WSB1 degrader, compound 4,
can reverse the WSB1-driven expression of downstream F-actin
and the formation of membrane ruffles and consequently
abrogate cell motility and migration.
2.7. Pharmacokinetic Study. Compound 4 was selected
for the in vivo pharmacokinetic studies in rats. After 100 mg/kg
oral dosing or 160 mg/kg intraperitoneal dosing of compound
4, the two ways of administration were observed with quick
absorption (Tmax), but the former dosing displayed a fast
clearance (T1/2). Moreover, Cmax and AUC0−t values of
compound 4 in oral or intraperitoneal dosing groups showed
acceptable blood exposure (Table 7). Therefore, compound 4
was suitable for in vivo efficacy studies.
To determine whether compound 4 can interact with WSB1
directly, we tried efforts to purify the WSB1 protein using the
affinity chromatography method for in vitro binding experi-
ments. However, it was found that the GST- or His-tagged
WSB1 protein showed unexpected physicochemical properties.
They were unstable and easily precipitated out at high
concentrations (Figure S3), thereby being hard to be purified.
Then, the interaction was further investigated by immunopre-
cipitation (Figure 8G). WSB1 and RhoGDI2 were coprecipi-
tated from 293T cells overexpressing both proteins. However,
compound 4 cannot block the coprecipitation of the proteins
at different concentrations. The results indicated that
compound 4 may not interact with the WSB1 protein directly.
Taken together, our results demonstrated that compound 4
may function through inducing the degradation of WSB1 in a
proteasome-dependent manner, thus leading to the accumu-
lation of the RhoGDI2 protein and subsequently lowering the
migration capacity of cancer cells (Figure 8H).
2.6. Compound 4 Decreases the WSB1-Enhanced
Expression of Downstream Polymerized Actin. Our
previous findings indicated that WSB1-triggered RhoGDI2
suppression activates Rho GTPases.9 RhoGDIs modulate the
cycling of Rho GTPases between active or inactive states; Rho
GTPases are known as regulators of actin cytoskeletal
2.8. Compound 4 Can Inhibit Metastasis of Cancer
Cells In Vivo. To test whether the migration inhibitory effect
could be reproduced in vivo, compound 4 was further
evaluated using the Balb/c (nu/nu) mice model bearing
highly metastatic 4T1 breast cancer cells. Paclitaxel was used as
a positive control (10 mg/kg ip twice a week). The treatment
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Figure 7. Compounds inhibited WSB1-mediated metastasis of cancer cells. (A) Images of the wound-healing assay of compounds 7d and 4 against
KHOS-pCDH and KHOS-WSB1 cells. (B) Images of the wound-healing assay and the transwell assay (C) of compounds 1a and 4 against H1299-
pCDH and H1299-WSB1 cells. (D) Images of the wound-healing assay of compound 4 against A2780-WT or A2780-WSB1/KO cells. Results are
shown as mean (n = 3) SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control.
with compound 4 (po, 100 mg/kg/day) showed a comparable
tumor inhibition and safety profile to paclitaxel (Figure 10A−
C). Lungs from the tumor-bearing mice were excised for
assessing metastasis. As shown in Figure 10D, the lungs of the
untreated animals had 14.3 9.0 colonies compared to only
5.7 1.2 colonies in the animals treated with compound 4,
although the result showed no statistical significance. The
results suggest that compound 4 can effectively inhibit the
pulmonary metastasis of cancer cells.
2.9. Chemistry. The synthetic routes for the compounds
1a−f, 2a−g, and 3a−c are outlined in Scheme 1. At first, the
bromoacetophenone derivatives 10a−f were synthesized via
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Figure 8. Compound 4 functions as a WSB1 degrader. (A) Western blotting of Flag-tag and RhoGDI2 in pCDH (P)- or WSB1 (W)-transfected
KHOS cells treated with indicated compounds. (B, C) Western blotting of Myc-tag, Flag-tag, and RhoGDI2 in H1299-WSB1 cells treated with
indicated compounds. (D) Relative mRNA levels of RhoGDI2 and WSB1 in H1299-WSB1 cells treated with compound 4. The mRNA expression
was analyzed by real-time polymerase chain reaction (RT-PCR) and shown as a histogram after normalization; GAPDH was used as a control gene.
(E) Time-dependent and (F) dose-dependent degradation of Flag-WSB1 or Flag-GFP in H1299-WSB1 cells as indicated by Western blotting of
Flag-tag. (G) Interaction between WSB1 and RhoGDI2. 293T cells transfected with Flag-WSB1 and RhoGDI2 plasmids, followed by compound 4
treatment. Cell lysates were immunoprecipitated with the anti-Flag antibody, followed by immunoblotting with anti-Flag and anti-RhoGDI2
antibody. (H) Schematic view of the possible mechanism of compound 4 for inhibiting cancer cell metastasis.
the bromination of acetophenone 9a−f using CuBr2 in EtOAc.
Then, the intermolecular condensation of 6a−f and 2-(2-
fluorophenyl)acetic acid delivered the cyclized products 1a−f.
Besides, 2-flurobromoacetophenone (10) was brominated to
obtain the bromoacetophenone derivative 12, which was
further condensed with corresponding phenylacetic acids 13a−
g to receive cyclized products 2a−g. Furthermore, compounds
K1, 1a, and 2a were treated with a saturated ethanolic solution
of ammonia, affording target compounds 3a−c.
The synthetic routes for compounds 4 and 5a−f are outlined
in Scheme 2. Bromination of compound 14 or 17 using N-
bromosuccinimide (NBS) afforded compound 15 or 18,
respectively. Subsequently, Suzuki coupling reaction with 2-
(substituted phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
was performed in the presence of Pd(PPh3)4 and a base to
obtain compounds 16a−c and 19, which were then further
coupled to afford the target products 4 and 5a−f.
The synthetic routes for compounds 6a−l are outlined in
Scheme 3. A nucleophilic reaction of compound 20 with 2-
iodine propane yielded the phenol intermediate 21. Oxidation
and hydrolysis of compound 22 produced the intermediate 23.
Coupling reaction of compound 15 using the CuI catalyst with
different substituted phenols provided 24a−c, which were then
coupled with substituted phenylboronic acid to give the target
compounds 6a−f. The amino group of compound 15 was
protected by condensation with pivaloyl chloride. As described
above, there were two steps of the coupling reaction of
compound 25 to give 26d−j using Pd(OAc)2 and Pd(dppf)-
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Figure 9. Representative images of the F-actin fluorescence intensity and the ruffles of H1299-WSB1 or H1299-pCDH cells treated with the
indicated compounds.
the probability of tumor metastasis. The inhibition of this
pathway may provide a new approach for suppressing hypoxia-
driven cancer metastasis. However, essential structural
information and screening models for WSB1 protein are still
lacking. In this study, we employed a phenotypic screening
method and discovered furan-2(5H)-one derivatives with
potent antimigration efficacy. Further optimization and
bioevaluation identified a series of pyridin-2-amine derivatives
that displayed excellent in vitro efficacy. The migration rates of
cells treated with active compounds range from 0.4 to 0.9. The
compounds displaying significant anticancer cell migration
effects were enriched.
Table 7. Pharmacokinetic Profiles of Compound 4
A preliminary mechanism study disclosed that some of the
active compounds can inhibit the migration capacity of WSB1-
overexpressing cells. Compounds 1a, 4, and 6l exert their
antimigration effects by increasing the level of the RhoGDI2
protein. Further mechanism study indicated that compound 4
may induce WSB1 degradation through a proteasome-
dependent manner without affecting the mRNA levels of
WSB1 and RhoGDI2. Thus, this led to the accumulation of
RhoGDI2 protein, subsequently lowering the migration
capacity of cancer cells. For demonstrating the interaction
between compound 4 and the WSB1 protein, although the
purification of the WSB1 protein using affinity chromatography
for in vitro binding experiments failed, immunoprecipitation
showed that compound 4 had no influences on the WSB1−
RhoGDI2 interaction, indicating that it may not interact with
the WSB1 protein directly. The WSB1-driven expression of the
downstream F-actin and formation of membrane ruffles in
cancer cells can be reversed upon the treatment of compound
4, suggesting that compound 4 displayed efficacies in
modulating this pathway. Moreover, compound 4 showed
promising pharmacokinetic profiles and an excellent effect in
reducing the number of metastatic colonies in vivo. To our
knowledge, compound 4 is the first reported WSB1 degrader,
which would help unleash the potential of the WSB1 protein as
a therapeutic target. In-depth mechanism exploration of the
degrader is being carried out in our laboratory.
parameters
Tmax (h)
T1/2 (h)
Cmax (ng/mL)
AUC0−t (ng/mL·h)
AUC0‑∞ (ng/mL·h)
po
ip
0.50 0.01
1.28 0.31
1543 595
2898 797
2941 827
0.42 0.14
4.97 0.88
3773 120
27 712 4251
28 778 5008
Cl2, which were then deprotected to the target compounds
6g−l.
The synthetic routes for compounds 7a−f are outlined in
Scheme 4. A nucleophilic reaction of compound 4 with allyl
bromide gave compound 27, which was subsequently oxidized
to afford the target compounds 7a and 7b. Compound 28 was
prepared by condensation of compound 4 with 3-chloropro-
pionyl chloride, followed by nucleophilic reaction with various
heterocycles to obtain compounds 7c−e. A reduction reaction
was performed to convert the amide 7d to 7f.
The synthetic routes for compounds 8a−i are outlined in
Scheme 5. Substituted benzoic acids 29a and 29b were
condensed with 2-fluoro-5-nitroaniline, giving amide 31a and
31b. Substitution reaction was performed with different
aliphatic heterocycles such as piperidin-4-ol, 1-methylpiper-
azine, and morpholine to afford target compounds 8a−f. The
nitryl in 8a−c was reduced in the presence of Fe/NH4Cl to get
the target compounds 8g−i.
3. CONCLUSIONS
4. MATERIALS AND METHODS
Hypoxia-induced WSB1 expression can lead to the activation
of the Rho pathway via RhoGDI2 depletion and consequently
promote motility and migration of cancer cells. This increases
4.1. Chemistry. All reagents and solvents were used as purchased
from commercial sources. Chromatography was performed using
silica gel (200−300 mesh). All reactions were monitored by thin-layer
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Figure 10. Treatment with compound 4 leads to decreased metastasis of tumor in the 4T1 xenograft mouse model. Tumor volume (A), body
weight (B), tumor weight (C), and number of metastatic colonies (D) at indicated days for 28 days following treatment with compound 4 at
indicated doses. Data represent the mean (n = 6) SD values.
a
Scheme 1. Synthetic Route for Target Compounds 1a−g, 2a−g, and 3a−c
a
(a) Cu2Br, ethyl acetate; (b) 2-(2-fluorophenyl)acetic acid, dimethylformamide (DMF); (c) i, triethylamine (TEA), MeCN; ii, 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU), dichloromethane (DCM); (d) NH3 saturated EtOH; (e) i, hexamethylenetetramine, DCM; ii, HBr,
MeOH; (f) 2-(2-fluorophenyl)acetic acid, 1-hydroxybenzotriazole (HOBt), EDCI, DCM; (g) t-BuOK, t-BuOH.
chromatography (TLC), using silica gel plates with fluorescence F254
and UV-light visualization. 1H NMR and 13C NMR spectra were
obtained on a Bruker Advance III 500 with the use of CDCl3 or
DMSO-d6 as the solvent. Chemical shifts are referenced to the
residual solvent peak and reported in ppm (d scale), and all coupling
constant (J) values are given in hertz. Multiplicities are recorded by
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b
Scheme 2. Synthetic Route for Target Compounds 4 and 5a−f
b
(a) NBS, DCM; (b) 2-(substituted phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh3)4, Na2CO3, MeOH/H2O/toluene = 1:25:10; (c) 2-
(substituted phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh3)4, Cs2CO3, H2O/1,4-dioxane = 1:2; (d) 2-(4-methoxy-3-methylphenyl)-
4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh3)4, K2CO3, toluene/ethanol = 5:3; (e) 4,4,5,5-tetramethyl-2-(4-(trifluoromethyl)phenyl)-1,3,2-
dioxaborolane, Pd(OAc)2, Cs2CO3, XPhos, n-butyl alcohol.
the following abbreviations: s, singlet; d, double; t, triplet; q, quartet;
m, multiplet; J, coupling constant (Hz). Electrospray ionization mass
spectrometry (ESI-MS) data were recorded on a Shimadzu LC-MS
2020 instrument. Purity of all final compounds was ≥95% as
determined by NMR and high-performance liquid chromatography
(HPLC). The analytical HPLC was performed on an Agilent 1260
Infinity II (LC03) machine and a C18 reversed-phase column
(Agilent Eclipse XDB-C18, 4.6 × 250 mm2, 5 μm), with a flow rate of
1.0 mL/min, detection by UV absorbance at a wavelength of 254 nm,
column temperature of 25 °C, and eluting with water (0.1% H3PO4)
as A phase and acetonitrile as B phase (for 20 min elution time: 0 min,
A phase: 90%, B phase: 10%; 15 min, A phase: 10%, B phase: 90%; 20
min, A phase: 5%, B phase: 95%; for 23 min elution time: 0 min, A
phase: 55%, B phase: 45%; 8 min, A phase: 30%, B phase: 70%; 15
min, A phase: 10%, B phase: 90%; 23 min, A phase: 5%, B phase:
95%; for 25 min elution time: 0 min, A phase: 80%, B phase: 20%; 15
min, A phase: 10%, B phase: 90%; 20 min, A phase: 5%, B phase:
95%).
4.1.1. General Procedure A. To a solution of substituted
acetophenone (compounds 9a−f and 11, 1 equiv) in ethyl acetate
was added CuBr2 (3.05 equiv); then, the mixture was refluxed
overnight. After completion of the reaction, the mixture was filtered
and washed with ethyl acetate. The combined organic layers were
washed with 1 N hydrochloric acid and brine, dried over Na2SO4, and
concentrated. The residue was purified by silica gel chromatography
using 2% ethyl acetate in petroleum ether to afford compounds 10a−f
and 12.
To the solution of substituted phenylacetic acid (compounds 13a−
g, 1 equiv) in MeCN (5 mL) were added substituted α-
bromoacetophenone (compounds 10a−f and 12, 1 equiv) and
triethylamine (0.3 mL). The mixture was stirred at room temperature
for 0.5−4 h. Then, DBU (0.3 mL) was added and stirred at 0 °C for
0.3−8 h. After the solvent was removed, the residue was diluted with
1 N HCl and then extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over Na2SO4, and then
concentrated under vacuum. The residue was purified by silica gel
chromatography using 12% ethyl acetate in petroleum ether to afford
compounds 1a−f and 2a−g.
4.1.2. General Procedure B. To a solution of compound K1, 1a, or
2a (0.36 mmol) was added ammonia saturated ethanol (3 mL) in a
sealed bottle. Then, the mixture was heated to 110 °C for 6 h. After
the reaction was completed, the reaction was cooled to room
temperature and concentrated under vacuum. The residue was
purified by silica gel chromatography using 1% methanol in
dichloromethane to afford compound 3a−c.
4.1.3. General Procedure C. To a solution of compound 15 (2
mmol), 2-(substituted phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lane (2 mmol) and Na2CO3 (4 mmol) in MeOH/H2O/toluene =
10 mL:25 mL:10 mL were added Pd(PPh3)4 (0.06 mmol) under a N2
atmosphere. Then, the mixture was heated to 85 °C for 4 h. After
completion of this reaction, it was cooled to room temperature. The
mixture was poured into 100 mL of cold water and extracted with
ethyl acetate. The combined organic layers were washed with brine,
dried over Na2SO4, and then concentrated under vacuum. The
residue was purified by silica gel chromatography using ethyl acetate
in petroleum ether (1:20) to afford compounds 16a−c.
4.1.4. General Procedure D. To a solution of compounds 16a−c
(2 mmol), 2-(substituted phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxabor-
olane (2.2 mmol) and Cs2CO3 (4 mmol) in H2O/1,4-dioxane = 3
mL:6 mL were added Pd(PPh3)4 (0.06 mmol) under a N2
atmosphere. Then, the mixture was heated to 85 °C for 8 h. After
completion of this reaction, it was cooled to room temperature. The
mixture was poured into 100 mL of cold water and extracted with
ethyl acetate. The combined organic layers were washed with brine,
dried over Na2SO4, and then concentrated under vacuum. The
residue was purified by silica gel chromatography using ethyl acetate
in petroleum ether (1:10−1:4) to afford compounds 4, 5a, 5b, and
5d−f.
4.1.5. General Procedure E. To a solution of compound 15 (9.64
mmol), substituted phenol (14.5 mmol), and TMEDA (0.55 mmol)
in DMSO were added CuI (0.55 mmol) and Cs2CO3 (5.52 mmol)
under a N2 atmosphere. The mixture was heated to 100 °C and
reacted overnight. The mixture was poured into 300 mL of cold water
and extracted with ethyl acetate. The combined organic layers were
washed with brine, dried over Na2SO4, and then concentrated under
vacuum. The residue was purified by silica gel chromatography using
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c
Scheme 3. Synthetic Route for Target Compounds 6a−l
c
(a) 2-Iodine propane, KOH, EtOH; (b) m-3-chloroperoxybenzoic acid (CPBA), DCM; (c) 10% KOH, MeOH; (d) CuI, Cs2CO3, N,N,N′,N′-
tetramethylethylenediamine (TMEDA), dimethylsulfoxide (DMSO); (e) (substituted phenyl)boronic Acid, Pd(PPh3)4, K2CO3, 1,4-dioxane/H2O
= 3:1; (f) TEA, 4-dimethylaminopyridine (DMAP), pivaloyl chloride, DCM; (g) substituted aniline, sodium tert-butoxide, Pd(OAc)2, PPh3, o-
xylene; (h) 2-(substituted phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(dppf)Cl2, Cs2CO3, DMF/H2O = 6:1; (i) 3 N KOH, MeOH.
ethyl acetate in petroleum ether (1:10−1:4) to afford compounds
24a−c.
of water and extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over Na2SO4, and then
concentrated under vacuum. The residue was purified by silica gel
chromatography using ethyl acetate in petroleum ether (1:20−1:10)
to afford compounds 26a−c.
4.1.6. General Procedure F. To a solution of compounds 24a−c
(2.6 mmol), (substituted phenyl)boronic acid (5.2 mmol), and
K2CO3 (5.2 mmol) in the mixed solvent of 1,4-dioxane/H2O = 90
mL:30 mL was added Pd(PPh3)4 (0.26 mmol) under a N2
atmosphere. Then, the mixture was heated to 85 °C for 4 h. After
completion of this reaction, it was cooled to room temperature. The
mixture was poured into 100 mL of cold water and extracted with
ethyl acetate. The combined organic layers were washed with brine,
dried over Na2SO4, and then concentrated under vacuum. The
residue was purified by silica gel chromatography using ethyl acetate
in petroleum ether (1:5−1:2) to afford compounds 6a−f.
4.1.8. General Procedure H. To a solution of compounds 26a−c
(1.2 mmol), 2-(substituted phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxa-
borolane (1.8 mmol), and Cs2CO3 (4.9 mmol) in the mixed solvent
of DMF/H2O = 24 mL:4 mL was added Pd(dppf)Cl2 (0.12 mmol)
under a N2 atmosphere. Then, the mixture was heated to 85 °C for 3
h. After completion of this reaction, it was cooled to room
temperature. The mixture was poured into 200 mL of cold water
and extracted with ethyl acetate. The combined organic layers were
washed with brine, dried over Na2SO4, and then concentrated under
vacuum. The residue was purified by silica gel chromatography using
ethyl acetate in petroleum ether (1:15−1:10) to afford compounds
26d−j.
4.1.7. General Procedure G. To a mixture of compound 25 (3
mmol), sodium tert-butoxide (7.64 mmol), Pd(OAc)2 (0.45 mmol),
and PPh3 (0.6 mmol) in a sealed bottle was added substituted aniline
dissolved in o-xylene (12 mL) under a N2 atmosphere and then
heated to 110 °C and reacted overnight. After completion of this
reaction, the mixture was filtered. The filtrate was poured into 100 mL
4.1.9. General Procedure I. To a solution of compounds 26d−j
(0.88 mmol) in methanol was added 5 mL of 3 N KOH and then
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a
Scheme 4. Synthetic Route for Target Compounds 7a−f
a
(a) Potassium tert-butoxide, allyl bromide, tetrahydrofuran (THF); (b) BH3·THF, 30% H2O2, 3 N NaOH, THF; (c) 3-chloropropionyl chloride,
TEA, DCM; (d) heterocycles, potassium iodide (KI), EtOH; (e) LiAlH4, THF.
Scheme 5. Synthetic Route for Target Compounds 8a−i. (a) SOCl2, DCM; (b) 2-Fluoro-5-nitroaniline, Pyridine, DCM; (c)
Aliphatic Heterocycles, DIPEA, DMF; (d) Fe, NH4Cl, EtOH/H2O = 5:1
heated to reflux for 4 h. After completion of this reaction, the mixture
was concentrated under vacuum. To the residue was added 50 mL of
water and extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over Na2SO4, and then concentrated
under vacuum. The residue was purified by silica gel chromatography
using ethyl acetate in petroleum ether (1:20−1:10) to afford
compounds 6g−l.
at 0 °C. The mixture was moved to room temperature and stirred for
3 h and then concentrated under vacuum to afford the acyl chloride
30a or 30b, which can be directly used in the next step.
To a solution of 2-fluoro-5-nitroaniline (6.3 mmol) in 10 mL of
DCM was added 30a or 30b (8.1 mmol) and pyridine (8.1 mmol)
dropwise. The mixture was stirred at room temperature overnight.
After completion of the reaction, the mixture was concentrated under
vacuum and a yellow solid can be precipitated out. The precipitate
can be purified to afford compound 31a or 31b through stirring in the
mixed solvent of ethyl acetate in petroleum ether (1:20) for 1 h.
4.1.12. General Procedure L. To a solution of compound 31a or
31b (1.64 mmol) in DMF (10 mL) were added 4-hydroxypiperidine
(2.47 mmol) and DIPEA (3.26 mmol). The mixture was stirred at
room temperature for 4 h. After completion of the reaction, the
mixture was poured into 200 mL of water and extracted with ethyl
acetate. The combined organic layers were washed with brine, dried
over Na2SO4, and then concentrated under vacuum. The residue was
purified by silica gel chromatography to afford compounds 8a−f.
4.1.10. General Procedure J. To a solution of compound 28 (0.47
mmol) and the indicated base (4.7 mmol) in 5 mL of ethanol was
added KI (0.47 mmol) and then heated to reflux overnight. After
completion of this reaction, it was cooled to room temperature. The
mixture was poured into 100 mL of cold water and extracted with
ethyl acetate. The combined organic layers were washed with brine,
dried over Na2SO4, and then concentrated under vacuum. The
residue was purified by silica gel chromatography using methanol in
dichloromethane (1:30−1:20) to afford compounds 7c−e.
4.1.11. General Procedure K. To a solution of compound 29a or
29b (16.4 mmol) in dichloromethane was added SOCl2 (19.6 mmol)
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4.1.13. General Procedure M. To a solution of compounds 8a−c
(0.4 mmol) in the mixed solvent of ethanol/H2O = 10 mL:2 mL were
added Fe (1.95 mmol) and NH4Cl (0.8 mmol). The mixture was
heated to reflux and mechanically stirred for 3 h. After completion of
the reaction, the mixture was poured into 200 mL of water and
extracted with ethyl acetate. The combined organic layers were
washed with brine, dried over Na2SO4, and then concentrated under
vacuum. The residue was purified by silica gel chromatography to
afford compounds 8g−i.
(500 MHz, CDCl3) δ 7.47−7.44 (m, 2H), 7.40 (m, 1H), 7.22 (m,
1H), 7.18−7.14 (m, 1H), 7.12−7.07 (m, 3H), 5.20 (d, J = 2.3 Hz,
2H), 3.30 (s, 3H). ESI-MS: m/z = 314.1 [M + H]+.
4.1.22. 4-(2-Fluorophenyl)-3-(4-fluorophenyl)furan-2(5H)-one
(2c). General procedure A, yield: 35%; 1H NMR (500 MHz,
CDCl3) δ 7.44−7.39 (m, 3H), 7.22−7.14 (m, 2H), 7.10 (m, 1H),
7.04 (m, 2H), 5.19 (d, J = 2.2 Hz, 2H). ESI-MS: m/z = 274.2 [M +
H]+.
4.1.23.10. 4-(2-Fluorophenyl)-3-(4-(trifluoromethyl)phenyl)-
1
furan-2(5H)-one (2d). General procedure A, yield: 28%; H NMR
4.1.14. 3-(2-Fluorophenyl)-4-(4-methoxy-3-methylphenyl)furan-
1
2(5H)-one (1a). General procedure A, yield: 62%; H NMR (500
(500 MHz, CDCl3) δ 7.58 (m, 4H), 7.47−7.42 (m, 1H), 7.20−7.15
(m, 2H), 7.14−7.10 (m, 1H), 5.24 (d, J = 2.2 Hz, 2H). ESI-MS: m/z
= 322.0 [M + H]+.
MHz, CDCl3) δ 7.44−7.38 (m, 2H), 7.26−7.22 (m, 1H), 7.13 (t, J =
9.1 Hz, 2H), 7.07 (d, J = 1.9 Hz, 1H), 6.74 (d, J = 8.6 Hz, 1H), 5.25
(s, 2H), 3.83 (s, 3H), 2.12 (s, 3H). 13C NMR (126 MHz, DMSO-d6)
δ 173.3, 161.0, 160.1, 159.6, 159.0, 132.2, 132.2, 131.5, 131.5, 129.8,
127.3, 126.6, 125.4, 125.4, 122.6, 119.7, 119.6, 117.4, 116.5, 116.3,
111.1, 71.3, 56.0, 16.4. ESI-MS: m/z = 299.2 [M + H]+. High-
resolution mass spectrometry (HRMS): calculated (M + H):
299.1083; found: 299.1079.
4.1.15. 3-(2-Fluorophenyl)-4-(4-fluorophenyl)furan-2(5H)-one
(1b). General procedure A, yield: 43%; 1H NMR (500 MHz,
CDCl3) δ 7.43 (dt, J = 7.2, 6.3 Hz, 2H), 7.32−7.28 (m, 2H), 7.27−
7.24 (m, 1H), 7.14−7.09 (m, 1H), 7.06−7.02 (m, 2H), 5.25 (s, 2H).
13C NMR (126 MHz, DMSO-d6) δ 172.9, 164.8, 162.9, 160.9, 158.9,
158.9, 132.1, 132.1, 131.8, 131.8, 130.3, 130.2, 127.5, 127.4, 125.5,
125.5, 119.7, 118.9, 118.8, 116.8, 116.6, 116.6, 116.4, 71.5. ESI-MS:
m/z = 273.2 [M + H]+.
4.1.16. 4-(4-Chlorophenyl)-3-(2-fluorophenyl)furan-2(5H)-one
(1c). General procedure A, yield: 66%; 1H NMR (500 MHz,
CDCl3) δ 7.42 (m, 2H), 7.34−7.31 (m, 2H), 7.28−7.24 (m, 1H),
7.24−7.21 (m, 2H), 7.14−7.09 (m, 1H), 5.25 (s, 2H). 13C NMR (126
MHz, DMSO-d6) δ 172.7, 160.8, 158.8, 158.8, 136.2, 132.1, 132.1,
131.9, 131.8, 129.8, 129.6, 129.5, 125.5, 125.4, 120.4, 118.8, 118.7,
116.6, 116.5, 71.4. ESI-MS: m/z = 289.1 [M + H]+.
4.1.24. 4-(2-Fluorophenyl)-3-(1H-indol-3-yl)furan-2(5H)-one
(2e). General procedure A, yield: 21%; 1H NMR (500 MHz,
DMSO-d6) δ 12.53 (s, 1H), 7.71 (m, 1H), 7.48−7.43 (m, 2H), 7.39
(d, J = 8.1 Hz, 1H), 7.22 (m, 1H), 7.16 (m, 1H), 7.04−7.00 (m, 1H),
6.73−6.68 (m, 1H), 6.59 (m, 1H), 5.34 (s, 2H). 13C NMR (126
MHz, DMSO-d6) δ 173.6, 160.7, 158.7, 147.5, 136.6, 132.4, 132.3,
130.4, 130.4, 127.7, 125.2, 125.1, 125.0, 122.4, 121.9, 120.9, 120.8,
120.00, 119.7, 116.8, 116.6, 112.4, 105.1, 71.7, 71.6. ESI-MS: m/z =
294.2 [M + H]+.
4.1.25. 3-(1-Ethyl-1H-indol-3-yl)-4-(2-fluorophenyl)furan-2(5H)-
one (2f). General procedure A, yield: 4%; 1H NMR (500 MHz,
CDCl3) δ 7.83 (s, 1H), 7.34 (t, J = 7.8 Hz, 2H), 7.31 (d, J = 6.2 Hz,
1H), 7.29 (s, 1H), 7.15 (dd, J = 14.9, 7.6 Hz, 2H), 6.95 (t, J = 7.6 Hz,
1H), 6.82 (t, J = 7.5 Hz, 1H), 6.66 (d, J = 8.2 Hz, 1H), 5.29 (s, 2H),
4.24 (q, J = 7.3 Hz, 2H), 1.54 (m, 3H). 13C NMR (126 MHz, DMSO-
d6) δ 173.5, 160.7, 158.7, 147.4, 136.1, 132.4, 132.3, 130.3, 130.3,
130.1, 125.6, 125.2, 125.2, 122.0, 122.0, 120.9, 120.8, 120.3, 119.8,
116.8, 116.6, 110.8, 104.5, 71.7, 71.6, 41.1, 15.8. ESI-MS: m/z = 323.2
[M + H]+.
4.1.26. 4-(2-Fluorophenyl)-3-(naphthalen-1-yl)furan-2(5H)-one
(2g). General procedure A, yield: 48%; 1H NMR (500 MHz,
CDCl3) δ 7.89 (m, 2H), 7.65 (d, J = 8.4 Hz, 1H), 7.53−7.46 (m, 2H),
7.44−7.38 (m, 2H), 7.26−7.23 (m, 1H), 7.08 (m, 1H), 6.91 (m, 1H),
6.79−6.75 (m, 1H), 5.53−5.41 (m, 2H). 13C NMR (126 MHz,
DMSO-d6) δ 173.1, 160.7, 158.7, 155.8, 133.7, 133.1, 133.1, 131.1,
130.1, 130.1, 129.5, 129.1, 128.9, 128.1, 127.4, 126.9, 126.7, 126.1,
125.4, 125.2, 125.2, 119.1, 119.0, 117.0, 116.8, 72.2, 72.2. ESI-MS: m/
z = 305.2 [M + H]+.
4.1.17. 3-(2-Fluorophenyl)-4-(4-isopropoxyphenyl)furan-2(5H)-
1
one (1d). General procedure A, yield: 71%; H NMR (500 MHz,
CDCl3) δ 7.44−7.38 (m, 2H), 7.26−7.21 (m, 3H), 7.13 (dd, J = 13.8,
5.1 Hz, 1H), 6.82−6.78 (m, 2H), 5.25 (s, 2H), 4.56 (dt, J = 12.1, 6.1
Hz, 1H), 1.33 (s, 3H), 1.32 (s, 3H). 13C NMR (126 MHz, DMSO-d6)
δ 173.3, 161.0, 160.3, 159.3, 159.0, 132.2, 132.2, 131.6, 131.5, 129.6,
125.5, 125.4, 122.6, 119.7, 119.6, 117.3, 116.6, 116.4, 116.1, 71.3,
70.0, 22.1. ESI-MS: m/z = 289.1 [M + H]+.
4.1.27. 3,4-Bis(2-fluorophenyl)-1H-pyrrole-2,5-dione (3a). Gen-
4.1.18. 3-(2-Fluorophenyl)-4-(3-methyl-4-propoxyphenyl)furan-
1
eral procedure B, yield: 52%; H NMR (500 MHz, DMSO-d6) δ
1
2(5H)-one (1e). General procedure A, yield: 31%; H NMR (500
11.48 (s, 1H), 7.51−7.46 (m, 2H), 7.41 (td, J = 7.5, 1.7 Hz, 2H), 7.26
(td, J = 7.6, 1.0 Hz, 2H), 7.22 (dd, J = 10.4, 8.4 Hz, 2H). 13C NMR
(126 MHz, DMSO-d6) δ 170.8, 160.8, 158.8, 135.7, 132.6, 132.5,
131.9, 131.9, 124.9, 124.8, 117.5, 117.3, 116.4, 116.2. ESI-MS: m/z =
284.0 [M − H]−.
MHz, CDCl3) δ 7.44−7.38 (m, 2H), 7.24 (td, J = 7.7, 1.1 Hz, 1H),
7.15−7.09 (m, 2H), 7.07 (d, J = 1.9 Hz, 1H), 6.71 (d, J = 8.6 Hz,
1H), 5.25 (s, 2H), 3.92 (t, J = 6.4 Hz, 2H), 2.13 (s, 3H), 1.85−1.78
(m, 2H), 1.04 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ
173.3, 161.0, 159.6, 159.5, 159.0, 132.2, 132.2, 131.5, 131.5, 129.9,
127.3, 126.8, 125.4, 125.4, 122.4, 119.8, 119.7, 117.3, 116.5, 116.3,
111.8, 71.3, 69.6, 22.4, 16.4, 10.9. ESI-MS: m/z = 327.1 [M + H]+.
4.1.19. 3-(2-Fluorophenyl)-4-(3-methyl-4-(pentyloxy)phenyl)-
4.1.28. 3-(2-Fluorophenyl)-4-(4-methoxyphenyl)-1H-pyrrole-2,5-
1
dione (3b). General procedure B, yield: 25%; H NMR (500 MHz,
CDCl3) δ 7.60 (s, 1H), 7.46 (m, 4H), 7.24 (s, 1H), 7.12 (d, J = 8.4
Hz, 1H), 6.85 (d, J = 7.6 Hz, 2H), 3.82 (s, 3H). 13C NMR (126 MHz,
DMSO-d6) δ 172.1, 171.4, 161.1, 160.8, 158.8, 138.8, 132.3, 132.3,
132.2, 132.1, 131.3, 130.3, 125.2, 125.1, 121.6, 117.9, 117.8, 116.5,
116.4, 114.5, 55.7. ESI-MS: m/z = 296.1 [M − H]−.
1
furan-2(5H)-one (1f). General procedure A, yield: 47%; H NMR
(500 MHz, CDCl3) δ 7.43−7.38 (m, 2H), 7.24 (td, J = 7.6, 1.1 Hz,
1H), 7.15−7.05 (m, 3H), 6.71 (d, J = 8.6 Hz, 1H), 5.25 (s, 2H), 3.95
(t, J = 6.4 Hz, 2H), 2.12 (s, 3H), 1.82−1.76 (m, 2H), 1.46−1.35 (m,
4H), 0.92 (t, J = 7.2 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ
173.3, 161.00, 159.6, 159.5, 159.0, 132.2, 132.2, 131.5, 131.5, 129.9,
127.3, 126.7, 125.4, 125.4, 122.4, 119.8, 119.6, 117.3, 116.5, 116.3,
111.8, 71.3, 68.2, 28.7, 28.2, 22.3, 16.4, 14.4. ESI-MS: m/z = 355.2
[M + H]+.
4.1.20. 4-(2-Fluorophenyl)-3-(4-methoxyphenyl)furan-2(5H)-one
(2a). General procedure A, yield: 13%; 1H NMR (500 MHz, CDCl3)
δ 7.44−7.39 (m, 3H), 7.26 (dd, J = 7.5, 1.6 Hz, 1H), 7.18 (dd, J =
10.7, 8.5 Hz, 1H), 7.11 (dd, J = 11.1, 4.1 Hz, 1H), 6.91−6.87 (m,
2H), 5.20 (d, J = 1.3 Hz, 2H), 3.84 (s, 3H). 13C NMR (126 MHz,
DMSO-d6) δ 172.9, 160.2, 160.0, 158.3, 151.9, 132.8, 132.7, 130.5,
130.5, 130.1, 126.9, 125.5, 125.5, 122.8, 119.8, 119.7, 117.0, 116.9,
114.4, 71.5, 71.5, 55.6. ESI-MS: m/z = 285.1 [M + H]+.
4.1.29. 3-(2-Fluorophenyl)-4-(4-methoxy-3-methylphenyl)-1H-
1
pyrrole-2,5-dione (3c). General procedure B, yield: 52%; H NMR
(500 MHz, CDCl3) δ 7.55 (s, 1H), 7.47−7.36 (m, 3H), 7.33 (s, 1H),
7.25−7.21 (m, 1H), 7.11 (dt, J = 19.0, 9.5 Hz, 1H), 6.76 (d, J = 8.6
Hz, 1H), 3.83 (s, 3H), 2.12 (s, 3H). 13C NMR (126 MHz, DMSO-d6)
δ 172.1, 171.4, 160.8, 159.3, 158.8, 138.9, 132.3, 132.3, 132.1, 132.1,
131.7, 130.2, 129.2, 126.0, 125.1, 125.1, 121.9, 116.5, 116.3, 110.8,
71.3, 55.9, 16.5. ESI-MS: m/z = 310.1 [M − H]−.
4.1.30. 5,6-Dibromopyridin-2-amine (15). To a solution of 6-
bromopyridin-2-amine (28.7 mmol) in 100 mL of dry dichloro-
methane was added NBS (28.9 mmol) slowly. The mixture was stirred
at room temperature for 6 h. After completion of the reaction, the
mixture was poured into 100 mL of water and extracted with
dichloromethane. The combined organic layers were washed with
brine, dried over Na2SO4, and then concentrated under vacuum. The
4.1.21. 4-(4-(2-Fluorophenyl)-2-oxo-2,5-dihydrofuran-3-yl)-
1
phenyl Acetate (2b). General procedure A, yield: 30%; H NMR
8636
https://doi.org/10.1021/acs.jmedchem.1c00586
J. Med. Chem. 2021, 64, 8621−8643

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
residue was purified by silica gel chromatography using ethyl acetate
in petroleum ether (1:10−1:5) to afford the compound, yield: 73.9%;
1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.5 Hz, 1H), 6.42 (d, J =
8.5 Hz, 1H), 4.48 (s, 2H). ESI-MS: m/z = 250.8 [M + H]+.
4.1.31. 5-Bromo-6-phenylpyridin-2-amine (16a). General proce-
× 2, 109.2, 106.7, 70.0, 55.3, 22.1 × 2, 16.2. ESI-MS: m/z = 348.9 [M
+ H]+. HRMS: calculated (M + H): 349.1916; found: 349.1925.
4.1.39. 6-(4-Methoxy-3-methylphenyl)-5-(4-(trifluoromethyl)-
phenyl)pyridin-2-amine (5f). General procedure D, yield: 82.1%;
melting point: 192−195 °C; retention time: 10.802 min; purity:
99.32%; 1H NMR (400 MHz, CDCl3) δ 7.51 (t, J = 8.6 Hz, 3H), 7.27
(s, 2H), 7.25−7.22 (m, 1H), 7.02 (dd, J = 8.4, 2.2 Hz, 1H), 6.67 (d, J
= 8.4 Hz, 1H), 6.56 (d, J = 8.3 Hz, 1H), 4.72 (s, 2H), 3.83 (s, 3H),
2.18 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 157.7, 157.5, 155.5,
144.6, 140.4, 132.0, 131.8, 129.8 × 2, 128.6, 128.4, 126.3, 125.0 × 2,
124.7, 109.3, 106.7, 55.3, 16.1. ESI-MS: m/z = 358.9 [M + H]+.
HRMS: calculated (M + H): 359.1371; found: 359.1378.
1
dure C, yield: 79.2%; H NMR (400 MHz, CDCl3) δ 7.69−7.56 (m,
3H), 7.47−7.34 (m, 3H), 6.37 (d, J = 8.6 Hz, 1H), 4.59 (s, 2H). ESI-
MS: m/z = 249.0 [M + H]+.
4.1.32. 5-Bromo-6-(4-isopropoxyphenyl)pyridin-2-amine (16b).
1
General procedure C, yield: 82.5%; H NMR (400 MHz, CDCl3) δ
7.77−7.73 (m, 1H), 7.68−7.52 (m, 2H), 7.03−6.89 (m, 2H), 6.65 (d,
J = 9.0 Hz, 1H), 6.54 (s, 1H), 4.62 (hept, J = 6.1 Hz, 1H), 1.36 (d, J =
6.0 Hz, 6H). ESI-MS: m/z = 307.0 [M + H]+.
4.1.33. 5-Bromo-6-(4-methoxy-3-methylphenyl)pyridin-2-amine
(16c). General procedure C, yield: 83.7%; 1H NMR (400 MHz,
CDCl3) δ 7.69 (d, J = 8.6 Hz, 1H), 7.53 (dd, J = 8.4, 2.3 Hz, 1H),
7.47 (d, J = 2.3 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.40 (d, J = 8.6 Hz,
1H), 4.71 (s, 2H), 3.91 (s, 3H), 2.31 (s, 3H). ESI-MS: m/z = 292.8
[M + H]+.
4.1.34. 5,6-Bis(4-methoxy-3-methylphenyl)pyridin-2-amine (4).
General procedure D, yield: 50%; 1H NMR (400 MHz, DMSO-d6) δ
7.34 (d, J = 8.3 Hz, 1H), 7.22 (d, J = 1.6 Hz, 1H), 6.93 (dd, J = 6.7,
3.7 Hz, 2H), 6.81 (d, J = 2.2 Hz, 2H), 6.73 (d, J = 8.5 Hz, 1H), 6.46
(d, J = 8.3 Hz, 1H), 5.96 (s, 2H), 3.76 (s, 3H), 3.75 (s, 3H), 2.09 (s,
6H). 13C NMR (126 MHz, DMSO-d6) δ 158.6, 156.9, 156.1, 154.0,
140.4, 133.3, 133.3, 132.2, 131.6, 128.8, 128.3, 125.5, 124.7, 123.7,
110.4, 109.4, 106.7, 55.6, 16.6. ESI-MS: m/z = 335.3 [M + H]+.
HRMS: calculated (M + H): 335.1760; found: 335.1754.
4.1.35. 5-(4-Methoxy-3-methylphenyl)-6-phenylpyridin-2-amine
(5a). General procedure D, yield: 94.1%; melting point: 162−164 °C;
retention time: 8.996 min; purity: 99.09%; 1H NMR (400 MHz,
CDCl3) δ 7.53 (dd, J = 8.3, 1.6 Hz, 1H), 7.39 (dh, J = 7.9, 2.4, 1.9 Hz,
2H), 7.27 (dt, J = 6.0, 1.9 Hz, 3H), 6.96 (d, J = 2.2 Hz, 1H), 6.88 (dd,
J = 8.5, 2.2 Hz, 1H), 6.69 (dd, J = 8.4, 1.5 Hz, 1H), 6.56 (dd, J = 8.3,
1.7 Hz, 1H), 4.67 (s, 2H), 3.83 (s, 3H), 2.17 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 156.8, 156.5, 154.9, 140.7, 140.6, 132.2, 131.9, 129.8,
128.1, 127.8, 127.4, 126.4, 126.3, 109.6, 107.3, 55.3, 16.2. ESI-MS: m/
z = 291.0 [M + H]+. HRMS: calculated (M + H): 291.1497; found:
291.1503.
4.1.40. 5-Bromo-6-chloropyridin-2-amine (18). To a solution of
6-chloropyridin-2-amine (15.6 mmol) in 30 mL of dry dichloro-
methane was added NBS (17.1 mmol) slowly. The mixture was stirred
at room temperature for 8 h. After completion of the reaction, the
mixture was poured into 100 mL of water and extracted with
dichloromethane. The combined organic layers were washed with
brine, dried over Na2SO4, and then concentrated under vacuum. The
residue was purified by silica gel chromatography using ethyl acetate
in petroleum ether (1:10−1:5) to afford the compound, yield: 53.5%;
1H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 8.5 Hz, 1H), 6.01 (d, J =
8.5 Hz, 1H), 3.95 (s, 2H). ESI-MS: m/z = 206.9 [M + H]+.
4.1.41. 6-Chloro-5-(4-methoxy-3-methylphenyl) pyridin-2-amine
(19). To the mixture of compound 14 (2.43 mmol), 2-(4-methoxy-3-
methylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.65 mmol),
and K2CO3 (6 mmol) in the mixed solvent of toluene/ethanol = 5
mL:3 mL was added Pd(PPh3)4 (0.24 mmol) under a nitrogen
atmosphere. The reaction mixture was heated to 80 °C for 8 h. After
the solvent was removed under vacuum, to the residue was added
water and extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over Na2SO4, and concentrated to
afford the crude product, and then, recrystallization using methanol
1
was performed to afford the white powder; yield: 67.7%; H NMR
(400 MHz, CDCl3) δ 7.41 (d, J = 8.2 Hz, 1H), 7.22 (dd, J = 8.3, 2.4
Hz, 1H), 7.20−7.15 (m, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.47 (d, J =
8.2 Hz, 1H), 4.57 (s, 2H), 3.87 (s, 3H), 2.26 (s, 3H). ESI-MS: m/z =
249.0 [M + H]+.
4.1.42. 5-(4-Methoxy-3-methylphenyl)-6-(4-(trifluoromethyl)-
phenyl)pyridin-2-amine (5c). To a solution of compound 15
(0.605 mmol), 4,4,5,5-tetramethyl-2-(4-(trifluoromethyl)phenyl)-
1,3,2-dioxaborolane (1.13 mmol), Cs2CO3 (3.02 mmol), and XPhos
(0.072 mmol) in 4 mL of n-butyl alcohol was added Pd(OAc)2 (0.036
mmol) under a N2 atmosphere. The mixture was heated to 100 °C
and stirred overnight. After completion of the reaction, the mixture
was poured into 100 mL of water and extracted with ethyl acetate.
The combined organic layers were washed with brine, dried over
Na2SO4, and then concentrated under vacuum. The residue was
purified by silica gel chromatography using ethyl acetate in petroleum
ether (1:8−1:2) to afford the compound, yield: 11.6%; melting point:
4.1.36. 6-(4-Isopropoxyphenyl)-5-(4-methoxy-3-methylphenyl)-
pyridin-2-amine (5b). General procedure D, yield: 80.2%; melting
1
point: 137−140 °C; retention time: 11.505 min; purity: 99.34%; H
NMR (400 MHz, CDCl3) δ 7.45 (d, J = 8.3 Hz, 1H), 7.28−7.24 (m,
2H), 6.95−6.89 (m, 1H), 6.86 (dd, J = 8.3, 2.3 Hz, 1H), 6.79−6.70
(m, 2H), 6.67 (d, J = 8.4 Hz, 1H), 6.48 (d, J = 8.3 Hz, 1H), 4.61 (s,
2H), 4.51 (h, J = 6.1 Hz, 1H), 3.79 (s, 3H), 2.13 (s, 3H), 1.30 (d, J =
6.1 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 157.4, 156.7, 156.4,
154.5, 140.8, 132.8, 132.4, 131.8, 131.0 × 2, 128.0, 126.2, 126.1, 115.4
× 2, 109.7, 106.8, 69.9, 55.3, 22.0 × 2, 16.1. ESI-MS: m/z = 349.0 [M
+ H]+. HRMS: calculated (M + H): 349.1916; found: 349.1929.
4.1.37. 6-(4-Methoxy-3-methylphenyl)-5-phenylpyridin-2-amine
(5d). General procedure D, yield: 89.2%; melting point: 187−190 °C;
retention time: 9.264 min; purity: 98.40%; 1H NMR (400 MHz,
CDCl3) δ 7.51 (d, J = 8.3 Hz, 1H), 7.31−7.23 (m, 4H), 7.21−7.15
(m, 2H), 7.07 (dd, J = 8.4, 2.3 Hz, 1H), 6.66 (d, J = 8.5 Hz, 1H), 6.54
(d, J = 8.3 Hz, 1H), 4.63 (s, 2H), 3.82 (s, 3H), 2.18 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 157.4, 157.1, 155.2, 140.8, 140.6, 132.4,
132.1, 129.6 × 2, 128.6, 128.1 × 2, 126.3, 126.1, 125.9, 117.2, 109.1,
106.6, 77.4, 77.3, 77.1, 76.7, 55.3, 16.2. ESI-MS: m/z = 291.0 [M +
H]+. HRMS: calculated (M + H): 291.1497; found: 291.1503.
4.1.38. 5-(4-Isopropoxyphenyl)-6-(4-methoxy-3-methylphenyl)-
pyridin-2-amine (5e). General procedure D, yield: 81.6%; melting
1
130−132 °C; retention time: 10.067 min; purity: 99.70%; H NMR
(400 MHz, CDCl3) δ 7.56−7.43 (m, 5H), 6.93−6.87 (m, 1H), 6.81
(dd, J = 8.4, 2.3 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 8.3 Hz,
1H), 4.58 (s, 2H), 3.80 (s, 3H), 2.14 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 157.0, 156.7, 153.4, 144.4, 140.8, 131.8, 131.5, 130.1 × 2,
128.1, 126.7, 126.6, 124.7 × 2, 109.8, 107.9, 55.3, 16.1. ESI-MS: m/z
= 359.2 [M + H]+. HRMS: calculated (M + H): 359.1371; found:
359.1381.
4.1.43. 5-Bromo-6-phenoxypyridin-2-amine (24a). General pro-
1
cedure E, yield: 79.7%; H NMR (400 MHz, CDCl3) δ 7.60 (d, J =
8.3 Hz, 1H), 7.42−7.30 (m, 2H), 7.21−7.14 (m, 1H), 7.13−7.07 (m,
2H), 6.12 (d, J = 8.3 Hz, 1H). ESI-MS: m/z = 365.0 [M + H]+.
4.1.44. 5-Bromo-6-(4-methoxy-3-methylphenoxy)pyridin-2-
amine (24b). General procedure E, yield: 81.1%. ESI-MS: m/z =
309.0 [M + H]+.
4.1.45. 5-Bromo-6-(4-isopropoxyphenoxy)pyridin-2-amine (24c).
General procedure E, yield: 83.5%. ESI-MS: m/z = 323 [M + H]+.
4.1.46. 6-(4-Methoxy-3-methylphenoxy)-5-phenylpyridin-2-
amine (6a). General procedure F, yield: 88.4%; melting point:
127−129 °C; retention time: 18.213 min; purity: 100.00%; 1H NMR
1
point: 157−159 °C; retention time: 11.516 min; purity: 98.86%; H
NMR (400 MHz, CDCl3) δ 7.50 (d, J = 8.3 Hz, 1H), 7.27 (d, J = 2.3
Hz, 1H), 7.10 (dd, J = 8.4, 2.2 Hz, 1H), 7.07 (d, J = 8.2 Hz, 2H), 6.81
(d, J = 8.3 Hz, 2H), 6.68 (d, J = 8.4 Hz, 1H), 6.53 (d, J = 8.3 Hz, 1H),
4.62 (s, 2H), 4.56 (p, J = 6.0 Hz, 1H), 3.83 (s, 3H), 2.19 (s, 3H), 1.37
(d, J = 6.0 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 156.8, 156.4,
154.9, 140.6, 133.0, 132.5, 132.1, 130.6 × 2, 128.6, 126.0, 125.9, 115.7
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J. Med. Chem. 2021, 64, 8621−8643

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Article
1
(400 MHz, CDCl3) δ 7.70−7.57 (m, 3H), 7.44 (t, J = 7.6 Hz, 2H),
7.37−7.31 (m, 1H), 6.96 (d, J = 8.1 Hz, 2H), 6.84 (d, J = 8.3 Hz,
1H), 6.30 (d, J = 8.0 Hz, 1H), 4.26 (s, 2H), 3.87 (s, 3H), 2.27 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 159.2, 156.6, 154.1, 147.9,
141.5, 137.1, 128.8 × 2, 128.2 × 2, 127.5, 126.5, 123.4, 118.6, 114.6,
110.4, 102.7, 55.7, 16.3. ESI-MS: m/z = 307.0 [M + H]+. HRMS:
calculated (M + H): 307.1447; found: 307.1453.
afford the compound, yield: 88.6%; H NMR (400 MHz, CDCl3) δ
8.18 (d, J = 8.6 Hz, 1H), 8.03 (s, 1H), 7.88 (d, J = 8.6 Hz, 1H), 1.35
(s, 9H). ESI-MS: m/z = 334.9 [M + H]+.
4.1.53. N-(5-Bromo-6-(phenylamino)pyridin-2-yl)pivalamide
(26a). General procedure G, yield: 60.1%; 1H NMR (400 MHz,
CDCl3) δ 7.90 (s, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz,
1H), 7.61−7.54 (m, 2H), 7.45−7.36 (m, 2H), 7.13 (tt, J = 7.3, 1.2
Hz, 1H), 6.99 (s, 1H), 1.36 (s, 9H). ESI-MS: m/z = 348.1 [M + H]+.
4.1.54. N-(5-Bromo-6-((4-methoxy-3-methylphenyl)amino)-
pyridin-2-yl)pivalamide (26b). General procedure G, yield: 50.6%;
1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 8.5 Hz, 1H), 7.58 (d, J =
4.1.47. 5-(4-Isopropoxyphenyl)-6-(4-methoxy-3-
methylphenoxy)pyridin-2-amine (6b). General procedure F, yield:
90.9%; melting point: 171−174 °C; retention time: 19.192 min;
1
purity: 99.57%; H NMR (400 MHz, CDCl3) δ 7.56 (t, J = 7.9 Hz,
8.5 Hz, 1H), 7.32 (dd, J = 8.7, 2.7 Hz, 1H), 7.21 (d, J = 2.7 Hz, 1H),
6.83 (d, J = 8.7 Hz, 1H), 6.76 (s, 1H), 3.85 (s, 3H), 2.24 (s, 3H), 1.29
(s, 9H). ESI-MS: m/z = 392.1 [M + H]+.
4.1.55. N-(5-Bromo-6-((4-isopropoxyphenyl)amino)pyridin-2-yl)-
pivalamide (26c). General procedure G, yield: 71.3%. ESI-MS: m/z =
406 [M + H]+.
3H), 6.95 (h, J = 3.1 Hz, 4H), 6.83 (d, J = 8.3 Hz, 1H), 6.28 (d, J =
8.0 Hz, 1H), 4.61 (hept, J = 6.1 Hz, 1H), 4.24 (s, 2H), 3.86 (s, 3H),
2.26 (s, 3H), 1.40 (d, J = 6.2 Hz, 6H). 13C NMR (100 MHz, CDCl3)
δ 159.0, 156.8, 156.1, 154.1, 148.1, 141.3, 129.8 × 2, 129.2, 127.5,
123.3, 118.5, 115.6 × 2, 114.5, 110.4, 102.8, 69.9, 55.7, 22.2 × 2, 16.3.
ESI-MS: m/z = 365.0 [M + H]+. HRMS: calculated (M + H):
365.1865; found: 365.1868.
4.1.56. N-(6-((4-Methoxy-3-methylphenyl)amino)-5-phenylpyri-
1
din-2-yl)pivalamide (26d). General procedure H, yield: 61.9%; H
4.1.48. 6-(4-Methoxy-3-methylphenoxy)-5-(4-methoxy-3-
methylphenyl)pyridin-2-amine (6c). General procedure F, yield:
80.5%; melting point: 130−132 °C; retention time: 16.616 min;
NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.58−7.50 (m, 3H), 7.50−
7.42 (m, 3H), 7.30−7.24 (m, 1H), 7.13 (s, 1H), 6.86 (dd, J = 15.0,
8.3 Hz, 1H), 3.87 (s, 3H), 2.25 (s, 3H), 1.38 (s, 9H). ESI-MS: m/z =
390.0 [M + H]+.
4.1.57 N-(5-(4-Isopropoxyphenyl)-6-((4-methoxy-3-
methylphenyl)amino)pyridin-2-yl)pivalamide (26e). General proce-
dure H, yield: 73.4%. ESI-MS: m/z = 448.0 [M + H]+.
4.1.58. N-(5-(4-Methoxy-3-methylphenyl)-6-((4-methoxy-3-
methylphenyl)amino)pyridin-2-yl)pivalamide (26f). General proce-
dure H, yield: 69.9%; 1H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H),
7.54 (dd, J = 8.8, 2.8 Hz, 1H), 7.31 (d, J = 2.7 Hz, 1H), 7.25 (dd, J =
8.4, 2.3 Hz, 1H), 7.23−7.17 (m, 2H), 7.06 (d, J = 8.4 Hz, 1H), 6.81
(d, J = 8.8 Hz, 1H), 6.62−6.50 (m, 1H), 2.22 (s, 3H), 2.13 (s, 3H),
1.28 (s, 9H). ESI-MS: m/z = 434.2 [M + H]+.
4.1.59. N-(6-((4-Methoxy-3-methylphenyl)amino)-5-(4-
(trifluoromethyl)phenyl)pyridin-2-yl)pivalamide (26g). General pro-
cedure H, yield: 81.5%; 1H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H),
7.76 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz,
1H), 7.23 (s, 1H), 7.11 (s, 1H), 6.82 (d, J = 8.7 Hz, 1H), 6.25 (s,
1H), 3.86 (s, 3H), 2.24 (s, 3H), 1.37 (s, 9H). ESI-MS: m/z = 458.0
[M + H]+.
1
purity: 99.68%; H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.0 Hz,
1H), 7.46 (dd, J = 8.3, 2.3 Hz, 1H), 7.40 (d, J = 2.3 Hz, 1H), 6.98−
6.87 (m, 3H), 6.83 (d, J = 8.3 Hz, 1H), 6.28 (d, J = 8.0 Hz, 1H),
4.77−3.91 (m, 1H), 3.88 (d, J = 11.0 Hz, 6H), 2.28 (d, J = 18.0 Hz,
6H). 13C NMR (100 MHz, CDCl3) δ 159.1, 156.7, 156.1, 154.1,
148.1, 141.3, 131.1, 129.1, 127.5, 127.2, 126.3, 123.3, 118.6, 114.7,
110.4, 109.8, 102.7, 55.7, 55.4, 16.3 × 2. ESI-MS: m/z = 351.0 [M +
H]+. HRMS: calculated (M + H): 351.1709; found: 351.1713.
4.1.49. 6-(4-Methoxy-3-methylphenoxy)-5-(4-(trifluoromethyl)-
phenyl)pyridin-2-amine (6d). General procedure F, yield: 61.8%;
melting point: 146−149 °C; retention time: 15.461 min; purity:
1
100.00%; H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 8.7 Hz, 2H),
7.68 (d, J = 8.2 Hz, 2H), 7.60 (dd, J = 8.1, 0.7 Hz, 1H), 6.98−6.90
(m, 2H), 6.88−6.80 (m, 1H), 6.30 (dd, J = 8.1, 0.7 Hz, 1H), 4.21 (d,
J = 127.2 Hz, 2H), 3.87 (s, 3H), 2.26 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 159.3, 157.1, 154.4, 147.4, 141.4, 140.8, 129.0 × 2, 127.7,
125.0 × 2, 123.4, 118.7, 112.8, 110.4, 102.7, 55.7, 16.3. ESI-MS: m/z
= 375.1 [M + H]+. HRMS: calculated (M + H): 375.1320; found:
375.1323.
4.1.50. 5-(4-Methoxy-3-methylphenyl)-6-phenoxypyridin-2-
amine (6e). General procedure F, yield: 79.8%; melting point:
155−156 °C; retention time: 14.570 min; purity: 100.00%; 1H NMR
(400 MHz, CDCl3) δ 7.59 (d, J = 8.0 Hz, 1H), 7.44 (dd, J = 8.4, 2.3
Hz, 1H), 7.42−7.34 (m, 3H), 7.21−7.10 (m, 3H), 6.89 (d, J = 8.4 Hz,
1H), 6.32 (d, J = 8.1 Hz, 1H), 4.07 (s, 2H), 3.89 (s, 3H), 2.30 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 158.4, 156.8, 156.2, 155.4,
141.5, 131.1, 129.2 × 2, 128.9, 127.2, 126.3, 123.4, 120.5 × 2, 115.2,
109.8, 103.4, 55.4, 16.3. ESI-MS: m/z = 307.1 [M + H]+. HRMS:
calculated (M + H): 307.1447; found: 307.1452.
4.1.51. 6-(4-Isopropoxyphenoxy)-5-(4-methoxy-3-
methylphenyl)pyridin-2-amine (6f). General procedure F, yield:
83.5%; melting point: 144−147 °C; retention time: 17.526 min;
purity: 100.00%; 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.0 Hz,
1H), 7.49−7.37 (m, 2H), 7.05 (d, J = 8.7 Hz, 2H), 6.90 (dd, J = 8.9,
3.6 Hz, 3H), 6.28 (d, J = 8.0 Hz, 1H), 4.53 (hept, J = 6.1 Hz, 1H),
4.45−4.22 (m, 2H), 3.89 (s, 3H), 2.30 (s, 3H), 1.38 (d, J = 6.0 Hz,
6H). 13C NMR (100 MHz, CDCl3) δ 159.0, 156.7, 156.1, 154.1,
148.5, 141.3, 131.1, 129.1, 127.2, 126.3, 121.8 × 2, 116.6 × 2, 114.7,
109.8, 102.7, 70.6, 55.4, 22.2 × 2, 16.3. ESI-MS: m/z = 365.1 [M +
H]+. HRMS: calculated (M + H): 365.1865; found: 365.1871.
4.1.52. N-(5,6-Dibromopyridin-2-yl)pivalamide (25). To a
solution of compound 11 (23.8 mmol), TEA (35.7 mmol), and
DMAP (2.38 mmol) dissolved in anhydrous DCM was added pivaloyl
chloride (35.7 mmol) at 0 °C. The mixture was moved to room
temperature and stirred overnight. After completion of this reaction,
the mixture was concentrated under vacuum. The residue was
dissolved in DCM, washed with brine, dried over Na2SO4, and then
concentrated under vacuum. The residue was purified by silica gel
chromatography using ethyl acetate in petroleum ether (1:20) to
4.1.60. N-(5-(4-Methoxy-3-methylphenyl)-6-(phenylamino)-
pyridin-2-yl)pivalamide (26h). General procedure H, yield: 84.9%.
ESI-MS: m/z = 390.2 [M + H]+.
4.1.61. N-(6-((4-Isopropoxyphenyl)amino)-5-(4-methoxy-3-
methylphenyl)pyridin-2-yl)pivalamide (26i). General procedure H,
yield: 18.1%. ESI-MS: m/z = 448.2 [M + H]+.
4.1.62. N2-(4-Methoxy-3-methylphenyl)-3-phenylpyridine-2,6-di-
amine (6g). General procedure I, yield: 72.3%; Melting point: 132−
1
134 °C; retention time: 9.167 min; purity: 99.64%; H NMR (400
MHz, CDCl3) δ 7.49 (q, J = 5.9, 5.0 Hz, 4H), 7.40 (dt, J = 12.7, 5.2
Hz, 2H), 7.27 (d, J = 7.9 Hz, 1H), 7.18 (d, J = 2.8 Hz, 1H), 6.80 (d, J
= 8.7 Hz, 1H), 6.37 (s, 1H), 6.06 (d, J = 7.9 Hz, 1H), 4.37 (s, 2H),
3.85 (s, 3H), 2.25 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 156.7,
153.2, 152.0, 139.9, 138.6, 133.7, 129.3 × 4, 127.1, 126.8, 123.2,
118.6, 113.0, 110.5, 98.1, 55.8, 16.3. ESI-MS: m/z = 306.1 [M + H]+.
HRMS: calculated (M + H): 306.1606; found: 306.1610.
4.1.63. 3-(4-Isopropoxyphenyl)-N2-(4-methoxy-3-methylphenyl)-
pyridine-2,6-diamine (6h). General procedure I, yield: 91.1%;
melting point: 95−96 °C; retention time: 11.393 min; purity:
1
98.81%; H NMR (400 MHz, CDCl3) δ 7.41 (dd, J = 8.7, 2.8 Hz,
1H), 7.38−7.32 (m, 2H), 7.23 (d, J = 7.9 Hz, 1H), 7.17 (d, J = 2.7
Hz, 1H), 7.00 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.7 Hz, 1H), 6.35 (s,
1H), 6.04 (d, J = 7.8 Hz, 1H), 4.63 (hept, J = 6.1 Hz, 1H), 4.40 (s,
2H), 3.84 (s, 3H), 2.24 (s, 3H), 1.43 (d, J = 6.0 Hz, 6H). 13C NMR
(100 MHz, CDCl3) δ 157.1, 156.3, 153.2, 152.1, 139.9, 133.7, 130.5
× 2, 130.3, 126.8, 123.1, 118.4, 116.4 × 2, 112.9, 110.5, 97.9, 70.0,
55.8, 29.7, 22.1 × 2. ESI-MS: m/z = 364.1 [M + H]+. HRMS:
calculated (M + H): 364.2025; found: 364.2030.
4.1.64. N2,3-Bis(4-methoxy-3-methylphenyl)pyridine-2,6-dia-
mine (6i). General procedure I, yield: 69.5%; melting point: 140−
1
143 °C; retention time: 11.311 min; purity: 93.44%; H NMR (400
8638
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J. Med. Chem. 2021, 64, 8621−8643

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Article
MHz, CDCl3) δ 7.41 (dd, J = 8.7, 2.8 Hz, 1H), 7.29−7.24 (m, 1H),
7.25−7.21 (m, 2H), 7.20−7.17 (m, 1H), 6.94 (d, J = 8.2 Hz, 1H),
6.80 (d, J = 8.7 Hz, 1H), 6.39 (s, 1H), 6.04 (d, J = 7.9 Hz, 1H), 3.89
(d, J = 31.4 Hz, 6H), 2.28 (d, J = 27.7 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ 157.0, 156.3, 153.1, 152.2, 139.9, 133.8, 131.7, 130.2, 127.7,
127.5, 126.8, 123.2, 118.5, 113.0, 110.5 × 2, 97.9, 55.8, 55.4, 16.3 × 2.
ESI-MS: m/z = 350.1 [M + H]+. HRMS: calculated (M + H):
350.1869; found: 350.1879.
For compound 7a, yield: 59.8%; retention time: 9.977 min; purity:
99.28%; H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 8.4 Hz, 1H),
1
7.19 (d, J = 7.2 Hz, 2H), 7.02−6.96 (m, 1H), 6.89 (dd, J = 8.4, 2.3
Hz, 1H), 6.71 (dd, J = 8.8, 2.3 Hz, 2H), 6.42 (d, J = 8.4 Hz, 1H), 4.71
(s, 1H), 3.83 (d, J = 7.3 Hz, 6H), 3.66 (dt, J = 11.1, 6.2 Hz, 4H), 3.31
(s, 1H), 2.17 (d, J = 18.3 Hz, 6H), 1.83−1.75 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 157.5, 157.3, 156.3, 154.3, 140.5, 132.7, 132.4,
132.1, 131.7, 128.4, 128.0, 126.2, 125.7, 124.8, 109.7, 109.2, 106.0,
58.7, 55.3 × 2, 38.0, 16.2 × 2, 14.2. ESI-MS: m/z = 393.1 [M + H]+.
HRMS: calculated (M + H): 393.2178; found: 393.2185.
4.1.65. N2-(4-Methoxy-3-methylphenyl)-3-(4-(trifluoromethyl)-
phenyl)pyridine-2,6-diamine (6j). General procedure I, yield:
75.2%; melting point: 131−132 °C; retention time: 11.041 min;
purity: 100.00%; 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 8.0 Hz,
2H), 7.60 (d, J = 8.0 Hz, 2H), 7.36 (dd, J = 8.7, 2.7 Hz, 1H), 7.26 (d,
J = 8.0 Hz, 1H), 7.15 (d, J = 2.7 Hz, 1H), 6.80 (d, J = 8.7 Hz, 1H),
6.25 (s, 1H), 6.08 (d, J = 8.0 Hz, 1H), 4.44 (s, 2H), 3.85 (s, 3H), 2.24
(s, 3H). 13C NMR (100 MHz, CDCl3) δ 157.2, 153.5, 151.9, 142.5,
140.2, 133.2, 129.5 × 2, 126.9, 126.1 × 2, 123.6, 118.9, 111.3, 110.4,
98.4, 55.7, 16.2. ESI-MS: m/z = 374.1 [M + H]+. HRMS: calculated
(M + H): 374.1480; found: 374.1496.
For compound 7b, yield: 36.5%; retention time: 9.920 min; purity:
1
100.00%; H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 8.4 Hz, 1H),
7.17 (dd, J = 8.8, 1.6 Hz, 2H), 6.98 (dd, J = 2.3, 0.9 Hz, 1H), 6.88
(dd, J = 8.4, 2.3 Hz, 1H), 6.72 (d, J = 4.0 Hz, 1H), 6.70 (d, J = 3.6 Hz,
1H), 6.47 (d, J = 8.4 Hz, 1H), 5.04 (d, J = 6.0 Hz, 1H), 4.05 (pd, J =
6.4, 2.4 Hz, 1H), 3.83 (d, J = 6.3 Hz, 6H), 3.55 (ddd, J = 14.2, 5.6, 2.4
Hz, 1H), 3.36 (ddd, J = 14.0, 6.9, 5.0 Hz, 1H), 2.17 (d, J = 17.9 Hz,
6H), 1.25 (d, J = 6.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 157.3,
156.3, 154.1, 140.7, 132.6, 132.2, 132.1, 131.7, 128.4, 128.0, 126.2,
125.7, 125.3, 109.7, 109.3, 106.3, 68.3, 55.3 × 2, 50.7, 20.9, 16.2 × 2.
ESI-MS: m/z = 393.1 [M + H]+.
4.1.70. N-(5,6-Bis(4-methoxy-3-methylphenyl)pyridin-2-yl)-3-
chloropropanamide (28). To a solution of compound 4 (1.49
mmol) and TEA (4.5 mmol) in 15 mL of DCM was added 3-
chloropropionyl (3 mmol) dropwise at 0 °C. The mixture was moved
to room temperature and reacted for 5 h. After completion of this
reaction, the mixture was poured into 100 mL of water and extracted
with ethyl acetate. The combined organic layers were washed with
brine, dried over Na2SO4, and then concentrated under vacuum. The
residue was purified by silica gel chromatography using ethyl acetate
in petroleum ether (1:10−1:4) to afford the compound, yield: 67.7%;
1H NMR (400 MHz, CDCl3) δ 8.49 (s, 1H), 8.29 (d, J = 8.4 Hz, 1H),
4.1.66. 3-(4-Methoxy-3-methylphenyl)-N2-phenylpyridine-2,6-di-
amine (6k). General procedure I, yield: 93.2%; melting point: 151−
1
153 °C; retention time: 10.604 min; purity: 98.49%; H NMR (400
MHz, CDCl3) δ 7.56 (d, J = 7.9 Hz, 2H), 7.31 (d, J = 7.6 Hz, 2H),
7.28−7.24 (m, 2H), 7.04−6.90 (m, 2H), 6.58 (s, 1H), 6.10 (d, J = 7.9
Hz, 1H), 4.40 (s, 2H), 3.93 (s, 3H), 2.32 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 157.1, 156.2, 151.5, 141.1, 140.0, 131.7, 129.9, 128.7
× 2, 127.7, 127.6, 121.3, 119.0 × 2, 113.6, 110.5, 98.6, 55.4, 16.3. ESI-
MS: m/z = 306.1 [M + H]+. HRMS: calculated (M + H): 306.1606;
found: 306.1614.
4.1.67. N2-(4-Isopropoxyphenyl)-3-(4-methoxy-3-methylphenyl)-
pyridine-2,6-diamine (6l). General procedure I, yield: 67.6%; melting
1
point: 130−133 °C; retention time: 12.060 min, purity: 95.63%; H
NMR (400 MHz, CDCl3) δ 7.49−7.37 (m, 2H), 7.29−7.17 (m, 3H),
6.94 (d, J = 8.2 Hz, 1H), 6.93−6.81 (m, 2H), 6.43 (s, 1H), 6.05 (d, J
= 7.9 Hz, 1H), 4.50 (hept, J = 6.1 Hz, 1H), 4.36 (s, 1H), 3.92 (s, 3H),
2.31 (s, 3H), 1.36 (d, J = 6.1 Hz, 6H). 13C NMR (100 MHz, CDCl3)
δ 157.1, 156.3, 152.9, 152.0, 139.9, 134.4, 131.7, 130.1, 127.7, 127.5,
121.2 × 2, 116.6 × 2, 113.1, 110.5, 97.9, 70.6, 55.4, 29.7, 22.2 × 2.
ESI-MS: m/z = 364.1 [M + H]+. HRMS: calculated (M + H):
364.2025; found: 364.2032.
7.78 (d, J = 8.4 Hz, 1H), 7.29 (dd, J = 2.4, 1.0 Hz, 1H), 7.08 (dd, J =
8.5, 2.3 Hz, 1H), 7.04 (dd, J = 2.3, 1.0 Hz, 1H), 6.95 (dd, J = 8.4, 2.3
Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 8.5 Hz, 1H), 6.52 (dd,
J = 16.9, 1.2 Hz, 1H), 6.31 (dd, J = 16.9, 10.2 Hz, 1H), 5.85 (dd, J =
10.2, 1.2 Hz, 1H), 3.85 (d, J = 7.1 Hz, 6H), 2.21 (d, J = 3.1 Hz, 6H).
ESI-MS: m/z = 425.0 [M + H]+.
4.1.71. N-(5,6-Bis(4-methoxy-3-methylphenyl)pyridin-2-yl)-3-(4-
hydroxypiperidin-1-yl)propenamide (7c). General procedure J,
yield: 87.1%; melting point: 62−64 °C; retention time: 10.123 min;
4.1.68. N-Allyl-5,6-bis(4-methoxy-3-methylphenyl)pyridin-2-
amine (27). To a solution of compound 4 (12 mmol) and potassium
tert-butoxide (17.8 mmol) in 40 mL of anhydrous THF was added
allyl bromide (14 mmol) under a N2 atmosphere. The mixture was
stirred at room temperature for 12 h. After completion of the reaction,
the mixture was concentrated under vacuum. To the residue was
added 300 mL of water and extracted with ethyl acetate. The
combined organic layers were washed with brine, dried over Na2SO4,
and then concentrated under vacuum. The residue was purified by
silica gel chromatography using ethyl acetate in petroleum ether
1
purity: 99.65%; H NMR (400 MHz, CDCl3) δ 11.42 (s, 1H), 8.11
(d, J = 8.3 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.40 (dd, J = 2.3, 1.0 Hz,
1H), 7.10 (dd, J = 8.5, 2.3 Hz, 1H), 7.05 (dd, J = 2.3, 1.0 Hz, 1H),
6.96 (dd, J = 8.4, 2.3 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 6.64 (d, J =
8.5 Hz, 1H), 3.91−3.86 (m, 1H), 3.84 (d, J = 13.2 Hz, 6H), 2.95 (dt,
J = 10.9, 4.5 Hz, 2H), 2.81 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 6.0 Hz,
2H), 2.43 (s, 2H), 2.20 (d, J = 8.7 Hz, 6H), 2.09 (p, J = 3.7 Hz, 2H),
1.84 (dtd, J = 12.5, 8.7, 3.6 Hz, 2H), 1.73 (s, 1H). 13C NMR (100
MHz, CDCl3) δ 171.0, 157.5, 156.8, 154.4, 150.1, 140.9, 132.3, 132.2,
131.9, 131.6, 131.2, 128.8, 128.0, 126.5, 125.7, 111.6, 109.8, 108.9,
67.3, 55.3 × 2, 53.3, 50.0 × 2, 34.3 × 2, 33.4, 16.2 × 2. ESI-MS: m/z =
490.0 [M + H]+.
1
(1:30−1:20) to afford the compound, yield: 40.0%; H NMR (400
MHz, DMSO-d6) δ 7.35 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 2.2, 0.9 Hz,
1H), 7.01 (dd, J = 8.5, 2.2 Hz, 1H), 6.93 (s, 1H), 6.85−6.77 (m, 2H),
6.74 (d, J = 8.5 Hz, 1H), 6.70 (t, J = 5.8 Hz, 1H), 6.49 (d, J = 8.4 Hz,
1H), 5.98 (ddt, J = 17.3, 10.4, 5.3 Hz, 1H), 5.26 (dq, J = 17.2, 1.8 Hz,
1H), 5.11 (dq, J = 10.3, 1.6 Hz, 1H), 3.97 (tt, J = 5.7, 1.6 Hz, 2H),
3.76 (d, J = 2.3 Hz, 6H), 2.08 (d, J = 7.2 Hz, 6H). ESI-MS: m/z =
375.0 [M + H]+.
4.1.72. N-(5,6-Bis(4-methoxy-3-methylphenyl)pyridin-2-yl)-3-
morpholinopropanamide (7d). General procedure J, yield: 89.6%;
melting point: 78−80 °C; retention time: 11.182 min; purity: 99.74%;
1
H NMR (400 MHz, CDCl3) δ 11.09 (s, 1H), 8.12 (d, J = 8.4 Hz,
1H), 7.68 (d, J = 8.4 Hz, 1H), 7.41−7.35 (m, 1H), 7.13 (dd, J = 8.5,
2.3 Hz, 1H), 7.08−7.01 (m, 1H), 6.97 (dd, J = 8.4, 2.3 Hz, 1H), 6.76
(d, J = 8.4 Hz, 1H), 6.67 (d, J = 8.5 Hz, 1H), 3.92 (t, J = 4.6 Hz, 4H),
3.85 (d, J = 8.4 Hz, 6H), 2.82 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 4.4 Hz,
4H), 2.61 (t, J = 6.0 Hz, 2H), 2.21 (d, J = 10.3 Hz, 6H). 13C NMR
(100 MHz, CDCl3) δ 171.0, 157.5, 156.8, 154.4, 150.1, 140.9, 132.3,
132.2, 131.9, 131.6, 131.2, 128.8, 128.0, 126.5, 125.7, 111.6, 109.8,
108.9, 67.3, 55.3 × 2, 53.3, 50.0 × 2, 34.3 × 2, 33.4, 16.2 × 2. ESI-MS:
m/z = 476.0 [M + H]+. HRMS: calculated (M + H): 476.2549;
found: 476.2553.
4.1.69. 3-((5,6-Bis(4-methoxy-3-methylphenyl)pyridin-2-yl)-
amino)propan-1-ol (7a) and 1-((5,6-bis(4-methoxy-3-
methylphenyl)pyridin-2-yl)amino)propan-2-ol (7b). To a solution
of compound 23 (0.53 mmol) in 5 mL of anhydrous THF was added
BH3·THF (5.34 mmol). The mixture was stirred at room temperature
for 1 h; then, 30% H2O2 (0.8 mmol) and 9 mL of 3 N NaOH were
added and stirred overnight. After completion of the reaction, the
mixture was poured into 100 mL of water and extracted with ethyl
acetate. The combined organic layers were washed with brine, dried
over Na2SO4, and then concentrated under vacuum. The residue was
purified by silica gel chromatography using ethyl acetate in petroleum
ether (1:8−1:2) to afford compounds 7a and 7b.
4.1.73. N-(5,6-Bis(4-methoxy-3-methylphenyl)pyridin-2-yl)-3-
(1H-1,2,4-triazol-1-yl)propenamide (7e). General procedure J,
yield: 70.3%; retention time: 15.827 min; purity: 100.00%; 1H
8639
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J. Med. Chem. 2021, 64, 8621−8643

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Article
NMR (400 MHz, CDCl3) δ 8.98 (s, 1H), 8.14 (s, 1H), 8.06 (d, J =
8.4 Hz, 1H), 7.90 (s, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 2.2
Hz, 1H), 7.03−6.95 (m, 2H), 6.89 (dd, J = 8.4, 2.3 Hz, 1H), 6.70 (d, J
= 8.4 Hz, 1H), 6.60 (d, J = 8.5 Hz, 1H), 4.48 (t, J = 6.2 Hz, 2H), 3.79
(d, J = 12.5 Hz, 6H), 2.73 (t, J = 6.2 Hz, 2H), 2.13 (d, J = 22.9 Hz,
6H). 13C NMR (100 MHz, CDCl3) δ 168.1, 157.7, 157.0, 154.8,
152.1, 149.1, 143.9, 141.3, 132.2, 132.0, 131.6, 131.4, 131.3, 128.6,
128.0, 126.6, 126.2, 112.0, 109.7, 109.2, 55.3 × 2, 44.8, 29.7, 16.2 × 2.
ESI-MS: m/z = 458 [M + H]+. HRMS: calculated (M +H): 458.2192;
found: 458.2200.
4.1.74. 5,6-Bis(4-methoxy-3-methylphenyl)-N-(3-
morpholinopropyl)pyridin-2-amine (7f). To a mixture of LiAlH4
(0.63 mmol) in 5 mL anhydrous THF was added compound 7d (0.11
mmol) at 0 °C under a N2 atmosphere. Then, the mixture was moved
to room temperature and stirred for 10 h. The reaction was quenched
by adding 24 μL of H2O, 24 μL of 15% NaOH, and 72 μL of H2O in
sequence. Then, the mixture was filtered and the filtrate was
concentrated under vacuum. The residue was purified through
chromatography to afford the compound, yield: 63.2%; retention
time: 7.380 min; purity: 98.60%; 1H NMR (400 MHz, CDCl3) δ 7.50
(d, J = 8.4 Hz, 1H), 7.34−7.28 (m, 1H), 7.11 (dd, J = 8.4, 2.3 Hz,
1H), 7.00 (dd, J = 2.2, 0.9 Hz, 1H), 6.90 (dd, J = 8.4, 2.3 Hz, 1H),
6.69 (dd, J = 18.6, 8.4 Hz, 2H), 6.44 (d, J = 8.4 Hz, 1H), 3.84 (d, J =
6.4 Hz, 6H), 3.79 (t, J = 4.7 Hz, 4H), 3.46 (t, J = 6.6 Hz, 2H), 2.60−
2.50 (m, 6H), 2.20 (d, J = 5.0 Hz, 6H), 1.90 (p, J = 6.8 Hz, 2H). 13C
NMR (100 MHz, CDCl3) δ 157.3, 156.3, 154.5, 140.6, 132.9, 132.6,
132.1, 131.7, 128.6, 128.0, 126.2, 125.7, 124.8, 109.7, 109.1, 104.2,
67.0 × 2, 56.9, 55.3 × 2, 53.8 × 2, 41.1, 26.2, 16.2 × 2. ESI-MS: m/z =
462.2 [M + H]+. HRMS: calculated (M + H): 462.2757; found:
462.2765.
3H). 13C NMR (100 MHz, CDCl3) δ 164.6, 161.2, 146.6, 145.0,
134.0, 129.4, 127.4, 126.3, 125.7, 120.3, 119.0, 115.0, 109.8, 67.4 × 2,
55.6, 52.0 × 2, 16.5. ESI-MS: m/z = 372.0 [M + H]+. HRMS:
calculated (M + H): 372.1559; found: 372.1564.
4.1.80. N-(2-(4-Hydroxypiperidin-1-yl)-5-nitrophenyl)benzamide
(8d). General procedure L, yield: 93.2%; melting point: 188−190 °C;
1H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 1H), 8.68 (d, J = 2.7 Hz,
1H), 8.03 (dd, J = 9.1, 2.9 Hz, 2H), 7.99 (d, J = 1.5 Hz, 1H), 7.68−
7.61 (m, 1H), 7.57 (dd, J = 11.4, 4.4 Hz, 2H), 7.30 (d, J = 9.0 Hz,
1H), 4.79 (d, J = 3.9 Hz, 1H), 3.67 (dt, J = 11.4, 3.7 Hz, 1H), 3.31−
3.18 (m, 2H), 2.96−2.77 (m, 2H), 1.93−1.78 (m, 2H), 1.65−1.49
(m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 165.4, 152.1, 141.8,
134.4, 132.6, 131.2, 129.2 × 2, 127.8 × 2, 121.5, 120.2, 119.8, 65.8,
48.8 × 2, 34.8 × 2. ESI-MS: m/z = 342.0 [M + H]+. HRMS:
calculated (M + H): 342.1454; found: 342.1460.
4.1.81. N-(2-(4-Methylpiperazin-1-yl)-5-nitrophenyl)benzamide
(8e). General procedure L, yield: 73.6%; melting point: 166−169
°C; 1H NMR (400 MHz, CDCl3) δ 9.40 (d, J = 2.6 Hz, 1H), 9.12 (s,
1H), 7.97 (dd, J = 8.8, 2.7 Hz, 1H), 7.95−7.88 (m, 2H), 7.63−7.58
(m, 1H), 7.55 (t, J = 7.3 Hz, 2H), 7.30−7.26 (m, 1H), 3.03 (t, J = 4.7
Hz, 4H), 2.65 (m, 4H), 2.40 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
165.0, 147.3, 145.0, 134.3, 133.8, 132.5, 129.2 × 2, 127.1 × 2, 120.5,
119.5, 115.1, 55.8 × 2, 51.9 × 2, 46.2. ESI-MS: m/z = 341.0 [M +
H]+. HRMS: calculated (M + H): 341.1614; found: 341.1622.
4.1.82. N-(2-Morpholino-5-nitrophenyl)benzamide (8f). General
1
procedure L, yield: 37.3%; melting point: 218−220 °C; H NMR
(400 MHz, DMSO-d6) δ 9.81 (s, 1H), 8.71 (d, J = 2.7 Hz, 1H), 8.06
(dt, J = 8.2, 4.1 Hz, 1H), 8.01−7.97 (m, 2H), 7.64 (dd, J = 8.4, 6.1
Hz, 1H), 7.57 (dd, J = 15.2, 8.2 Hz, 2H), 7.35 (d, J = 9.0 Hz, 1H),
3.84−3.72 (m, 4H), 3.13−3.01 (m, 4H). 13C NMR (100 MHz,
DMSO-d6) δ 165.0, 150.8, 141.9, 133.8, 132.1, 131.1, 128.8 × 2,
127.4 × 2, 121.1, 120.0, 119.5, 66.1 × 2, 50.6 × 2. ESI-MS: m/z =
328.0 [M + H]+.
4.1.75. N-(2-Fluoro-5-nitrophenyl)benzamide (31a). General
procedure K, yield: 56.8%; 1H NMR (400 MHz, DMSO-d6) δ
10.49 (s, 1H), 8.67 (dd, J = 6.5, 2.9 Hz, 1H), 8.17 (ddd, J = 9.0, 4.0,
3.0 Hz, 1H), 8.03−7.95 (m, 2H), 7.65 (td, J = 5.2, 2.2 Hz, 1H), 7.63−
7.51 (m, 3H). ESI-MS: m/z = 260.8 [M + H]+.
4.1.76. N-(2-Fluoro-5-nitrophenyl)-4-methoxy-3-methylbenza-
mide (31b). General procedure K, yield: 63.8%; 1H NMR (400
MHz, CDCl3) δ 9.46 (dd, J = 6.8, 2.7 Hz, 1H), 8.08 (s, 1H), 7.99
(ddd, J = 7.5, 4.0, 3.1 Hz, 1H), 7.76 (dd, J = 8.5, 2.2 Hz, 1H), 7.69 (s,
1H), 7.27 (t, J = 9.5 Hz, 1H), 6.91 (d, J = 8.5 Hz, 1H), 3.91 (s, 3H),
2.28 (s, 3H). ESI-MS: m/z = 305.0 [M + H]+.
4.1.83. N-(5-Amino-2-(4-hydroxypiperidin-1-yl)phenyl)-4-me-
thoxy-3-methylbenzamide (8g). General procedure M, yield:
1
43.6%; melting point: 181−183 °C; H NMR (400 MHz, DMSO-
d6) δ 9.60 (s, 1H), 7.72 (dd, J = 8.5, 2.0 Hz, 1H), 7.68 (d, J = 8.0 Hz,
1H), 7.66 (d, J = 2.5 Hz, 1H), 7.11 (d, J = 8.6 Hz, 1H), 6.96 (d, J =
8.4 Hz, 1H), 6.26 (dd, J = 8.4, 2.5 Hz, 1H), 5.00 (s, 2H), 4.75 (d, J =
3.9 Hz, 1H), 3.87 (s, 3H), 3.61 (dt, J = 26.9, 13.4 Hz, 1H), 2.82 (d, J
= 11.8 Hz, 2H), 2.63 (t, J = 9.7 Hz, 2H), 2.23 (s, 3H), 1.87 (d, J = 9.9
Hz, 2H), 1.57 (td, J = 12.5, 3.4 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 163.6, 160.6, 146.5, 134.4, 131.9, 129.6, 127.1, 126.5,
126.5, 121.8, 110.7, 109.1, 104.9, 66.2, 56.1 × 2, 51.4, 36.2, 16.7 × 2.
ESI-MS: m/z = 356.0 [M + H]+. HRMS: calculated (M + H):
356.1974; found: 356.1986.
4.1.77. N-(2-(4-Hydroxypiperidin-1-yl)-5-nitrophenyl)-4-me-
thoxy-3-methylbenzamide (8a). General procedure L, yield:
1
94.2%; melting point: 188−190 °C; H NMR (400 MHz, DMSO-
d6) δ 9.45 (s, 1H), 8.72 (d, J = 2.1 Hz, 1H), 7.99 (dd, J = 8.7, 1.9 Hz,
1H), 7.86 (d, J = 8.0 Hz, 1H), 7.79 (s, 1H), 7.30 (d, J = 8.9 Hz, 1H),
7.10 (d, J = 8.5 Hz, 1H), 4.57 (s, 1H), 3.88 (s, 3H), 3.77−3.57 (m,
1H), 3.22 (d, J = 11.6 Hz, 2H), 2.84 (t, J = 9.8 Hz, 2H), 2.22 (s, 3H),
1.87 (d, J = 9.5 Hz, 2H), 1.72−1.48 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 164.9, 160.9, 151.6, 142.1, 131.8, 130.2, 127.3, 126.3,
125.9, 121.0, 120.3, 118.9, 110.6, 65.8, 56.1, 48.9 × 2, 34.9 × 2, 16.6.
ESI-MS: m/z = 386.0 [M + H]+. HRMS: calculated (M + H):
386.1716; found: 386.1719.
4.1.84. N-(5-Amino-2-(4-methylpiperazin-1-yl)phenyl)-4-me-
thoxy-3-methylbenzamide (8h). General procedure M, yield:
1
44.8%; H NMR (400 MHz, CDCl3) δ 9.66 (s, 1H), 8.01 (d, J =
2.6 Hz, 1H), 7.78 (dt, J = 10.6, 5.3 Hz, 1H), 7.73 (d, J = 1.8 Hz, 1H),
7.03 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.38 (dd, J = 8.4,
2.7 Hz, 1H), 3.90 (s, 3H), 3.10−2.87 (m, 4H), 2.86−2.40 (m, 4H),
2.38 (s, 3H), 2.30 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 164.5,
160.6, 144.6, 135.0, 132.4, 129.4, 127.0, 126.9, 126.5, 121.9, 109.6,
109.5, 105.7, 56.5 × 2, 55.5, 52.7 × 2, 46.3, 16.4. ESI-MS: m/z =
355.1 [M + H]+. HRMS: calculated (M + H): 355.2134; found:
355.2136.
4.1.85. N-(5-Amino-2-morpholinophenyl)-4-methoxy-3-methyl-
benzamide (8i). General procedure M, yield: 73.9%; 1H NMR
(400 MHz, CDCl3) δ 9.66 (s, 1H), 8.06 (d, J = 2.6 Hz, 1H), 7.80 (dt,
J = 7.2, 3.6 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 8.4 Hz,
1H), 6.96 (d, J = 8.5 Hz, 1H), 6.44 (dd, J = 8.4, 2.6 Hz, 1H), 3.94 (s,
3H), 3.90 (t, J = 13.0 Hz, 4H), 2.99−2.84 (m, 4H), 2.34 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 164.6, 160.7, 144.9, 135.1, 132.3, 129.5,
127.2, 127.0, 126.3, 122.0, 109.7, 109.7, 105.9, 68.2 × 2, 55.6, 53.3 ×
2, 16.6. ESI-MS: m/z = 342.1 [M + H]+.
4.1.78. 4-Methoxy-3-methyl-N-(2-(4-methylpiperazin-1-yl)-5-
nitrophenyl)benzamide (8b). General procedure L, yield: 56.8%;
melting point: 187−189 °C; 1H NMR (400 MHz, CDCl3) δ 9.40 (d,
J = 2.6 Hz, 1H), 9.07 (s, 1H), 7.96 (dd, J = 8.7, 2.7 Hz, 1H), 7.79 (dd,
J = 8.5, 2.2 Hz, 1H), 7.74 (s, 1H), 7.28 (d, J = 1.8 Hz, 1H), 6.95 (d, J
= 8.5 Hz, 1H), 3.94 (s, 3H), 3.05 (t, J = 4.7 Hz, 4H), 2.96−2.53 (m,
4H), 2.44 (s, 3H), 2.32 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
164.6, 161.1, 146.9, 144.9, 134.0, 129.4, 127.3, 126.5, 125.8, 120.3,
118.9, 114.8, 109.8, 55.7 × 2, 55.6, 51.6 × 2, 46.1, 16.4. ESI-MS: m/z
= 385.0 [M + H]+. HRMS: calculated (M + H): 385.1876; found:
385.1883.
4.1.79. 4-Methoxy-3-methyl-N-(2-morpholino-5-nitrophenyl)-
benzamide (8c). General procedure L, yield: 70.8%; melting point:
1
210−213 °C; H NMR (400 MHz, CDCl3) δ 9.37 (d, J = 2.6 Hz,
4.2. Cell Culture and Plasmids. The NCI-H1299, H460, KHOS,
A2780, and 293T cell lines were purchased from the Cell Bank of
Type Culture Collection of the Chinese Academy of Sciences
(Shanghai, China). All cells were cultured in Dulbecco’s modified
1H), 9.05 (s, 1H), 7.95 (dd, J = 8.7, 2.6 Hz, 1H), 7.75 (dd, J = 8.5, 2.1
Hz, 1H), 7.70 (s, 1H), 7.25 (d, J = 8.8 Hz, 1H), 6.92 (t, J = 7.3 Hz,
1H), 3.97−3.93 (m, 4H), 3.92 (s, 3H), 3.06−2.95 (m, 4H), 2.30 (s,
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Eagle’s medium (DMEM) or RPMI-1640 medium supplemented
with 10% fetal bovine serum (FBS; Gibco) in a humidified
atmosphere of 5% CO2 at 37°C. The full-length coding sequence
for WSB1 and RhoGDI2 was amplified from the U2OS cDNA library
and subsequently subcloned into the pCDH plasmid (Addgene).
4.3. Antiproliferation Assay. H460 or KHOS cells were seeded
in 96-well plates at a density of 5000 cells per well. After 24 h, cells
were incubated with a medium alone or a medium containing test
compounds for 16 h or 48 h. Serial concentrations of both
compounds were added into the wells. Cell viability was determined
by the methyl thiazolyl tetrazolium (MTT) or CCK8 assay. Growth
inhibition was calculated as % = [1 − (A/B)] ×100, where A and B
are the absorbance of treated and untreated cells, respectively.
4.4. Transwell Assay. Under a hypoxic or normoxic environment,
the cell migration assay was performed in a 24-well transwell plate
with 8 μm of polycarbonate sterile membrane (Corning Incorpo-
rated). Cells were plated in the upper chamber at 2 × 104 cells per
insert in 200 μL of serum-free medium. Inserts were placed in wells
containing 600 μL of medium supplemented with 10% FBS. After
being cultured for 24 h, cells on the upper surface were detached with
a cotton swab. Filters were fixed, and cells in the lower filter were
stained with 0.1% crystal violet for 15 min and photographed, under a
100× microscope. Three different visual fields were chosen randomly
to take the count of the number of cells. The migration rate was
normalized to the control: migration rate = A/B (A = number of
migration cells in the dosing group; B = number of migration cells in
the control group)
acid (EDTA), 2 mM ethylene glycol tetraacetic acid (EGTA), 25 mM
NaF, 25 mM glycero-2-phosphate disodium salt, 0.1% Cocktail, pH =
7.5). After centrifuging with the speed of 12 000g for 30 min at 4 °C,
the supernatant was obtained. The cell lysate (2−3 mg from harvested
cells) was incubated with 20 μL of suspension of anti-FLAG beads for
6 h at 4 °C. After centrifugation at 500g for 2 min at 4 °C, the beads
were washed five times with 500 μL of washing buffer (25 mM Tris,
150 mM NaCl, 0.2% Nonidet P-40, pH = 8.0), and proteins were
dissociated from beads by 2.5× loading buffer.
4.9. F-Actin Staining Assay and Confocal Microscopy.
H1299-WSB1 cells were planted into the 24-well plate containing
cell slides and treated with compound 4 (20 μM) for 24 h. After that,
cells were fixed with 4% paraformaldehyde for 15 min at 4°C and then
permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells
were then blocked with 1% bovine serum albumin (BSA) for 20 min
and incubated with Texas Red-X phalloidin (Life Technologies)
diluted according to the dilution ratio of 1:40 in PBS for 20 min. Cells
were stained with 4′,6-diamidino-2-phenylindole (DAPI; 100 mg/
mL) for 3 min. The slides were mounted with antifade reagent. The
cells were observed and photographed under a confocal microscope
(Leica-TCS SP8).
4.10. Animals. Sprague−Dawley (SD) rats (male, 8 weeks old,
250−300 g) were purchased from Zhejiang Laboratory Animal Center
(Hangzhou, China). Balb/c mice (nu/nu, female, 3−4 weeks old, 20−
25 g) were purchased from the National Rodent Laboratory Animal
Resource (Shanghai, China). These animals were bred at the College
of Pharmaceutical Sciences, Zhejiang University. The animals were
maintained under a 12 h light−dark cycle with free access to water
and food. All animal studies were performed in strict accordance with
the institutional guidelines as defined by the Institutional Animal Care
and Use Committee (IUCAC) and approved by the Animal Care and
Use Committee, Zhejiang University Laboratory Animal Center
(Hangzhou, China).
4.11. Pharmacokinetic Studies. SD rats were administered
compound 4 100 mg/kg p.o. or 160 mg/kg ip in the solvent (2%
DMSO + 98% (0.5% methyl cellulosem (MC))). Three rats were
dosed in each group. Venous blood (100 μL) samples were collected
at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h. Plasma was separated from whole
blood by centrifugation and stored at −20°C until analysis.
Compound levels were determined by API4000+ LC/MS. The
Cmax, Tmax, t1/2, and AUC were evaluated using Analyst 1.5.1.
4.12. Measurement of In Vivo Activity. Balb/c (nu/nu) mice
were maintained in a pathogen-free animal facility. Tumors were
established via armpit injection of 4T1 cells (1 × 107 cells/animal)
into the 3- to 4-week-old female Balb/c (nu/nu) mice. When tumors
reached 100−200 mm3, the mice were randomly divided into control
and treatment groups to receive one of the following daily treatments
for 28 days: vehicle (phosphate-buffered saline (PBS), po), 4 (100
mg/kg, po); Taxol (10 mg/kg, ip). Six mice were included in each
group. Tumor volume (V) was calculated as V = (length × width2)/2.
The relative tumor volume (RTV) was calculated as RTV = Vn/V0 (Vn
is tumor volume on days n; V0 is tumor volume on the first day of
treatment). After completion of the treatment, mice were sacrificed.
The lung tissues were dissected, formalin-fixed, and paraffin-
embedded. Then, they were cut into slices and stained with
hematoxylin/eosin. The lung metastasis foci were observed and
counted under a microscope.
4.5. Wound-Healing Assay. Cells (5 × 105 cell/well) were
seeded in a 24-well plate until they attached and formed a monolayer
on plates. A straight band of cells was scraped using a pipette tip. After
being scraped, the cells were washed with phosphate-buffered saline
(PBS) and were subsequently exposed to 1‰ DMSO or tested
compounds. The migration of cells across this artificial wound was
assessed at different time intervals by microscopic observation. The
cell migration rate was calculated as (C − D)/(A − B) (A = blank
area of the control, 0 h; B = blank area of the control, 16, 24, or 48 h;
C = blank area of the compound, 0 h; D = blank area of the
compound, 16, 24, or 48 h).
4.6. Western Blotting. Cells were harvested and lysed in a 2×
sodium dodecyl sulfate (SDS) gel loading buffer [24 mM Tris-HCl
(pH 6.8), 0.02% mercaptoethanol, 4% SDS, 0.4% bromphenol blue,
20% glycerol]. Equal volumes of cell lysates were resolved on 8−12%
SDS-polyacrylamide gel electrophoresis (PAGE) gels and transferred
to poly(vinylidene difluoride) (PVDF) membranes (Pierce Chem-
ical). The blots were incubated for 1 h in a TBS-T solution
supplemented with 5% nonfat dry milk. Membranes were probed with
the specific primary antibodies, followed by horseradish peroxidase-
conjugated secondary antibodies. The bands were visualized by
chemiluminescence (Perkinelmer). The primary antibody for β-actin
was from Santa Cruz Biotechnology (Santa Cruz, CA), Myc-tag and
Flag-tag were obtained from Cell Signaling Technology (Beverly,
MA), and RhoGDI2 was from Spring Bioscience (Pleasanton, CA).
Secondary antibodies were from Fude Biological Technology.
4.7. RT-PCR Analysis. Total RNA was extracted with the
EasyPure RNA kit according to the manufacturer’s protocol
(Transgen Biotech), and cDNA was synthesized with TranScript
One-Step gDNA Removal and cDNA Systhesis SuperMix (Transgen
Biotech). The relative mRNA levels were evaluated by quantitative
RT-PCR using Eppendorfep Gradient Mastercycler (Eppendorf,
Hamburg, Germany) and Bio-Rad SYBR Green Master Mix. Primers
used were as follows: GAPDH, forward primer: 5′-GTCATCCAT-
GACAACTTTGG-3′ and reverse primer: 5′-GAGCTTGA-
CAAAGTGGTCGT-3′; WSB1, forward primer: 5′-CGTACTA-
TAGGTGAACTTTTAGCTCCT-3′ and reverse primer: 5′-
CCAAAGGAAAACTGCTTTACTGG-3′; RhoGDI2, forward pri-
mer: 5′-CCCTGGTTTGTGAGAGTGC-3′ and reverse primer: 5′-
GGCCTGACACAATATCCCTG-3′.
4.13. Statistical Analysis. Statistical analysis was performed using
GraphPad Prism 5 software (GraphPad software, Inc, La Jolla, CA).
Unless otherwise stated, the values for all samples in the different
experimental conditions were averaged, and the standard deviation of
the mean was calculated. Statistical differences between two groups
were assessed by Student’s t-test. A P value of less than 0.05 was
considered statistically significant.
ASSOCIATED CONTENT
■
sı
* Supporting Information
4.8. Immunoprecipitation. The transfected 293T cells were
harvested and lysed by IP buffer (0.5% NP-40, 0.5% Triton X-100,
150 mM NaCl, 50 mM Tris-HCl, 2 mM ethylenediaminetetraacetic
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00586.
8641
https://doi.org/10.1021/acs.jmedchem.1c00586
J. Med. Chem. 2021, 64, 8621−8643

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Research, College of Pharmaceutical Sciences and Cancer
Center, Zhejiang University, Hangzhou 310058, P. R. China
Qiaojun He − Hangzhou Institute of Innovative Medicine,
Institute of Drug Discovery and Design, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, P. R. China; Institute of Pharmacology &
Toxicology, Zhejiang Province Key Laboratory of Anti-
Cancer Drug Research, College of Pharmaceutical Sciences
and Cancer Center, Zhejiang University, Hangzhou 310058,
P. R. China; Innovation Institute for Artificial Intelligence in
Medicine of Zhejiang University, Hangzhou 310018, P. R.
China
Structures of representative compounds from an in-
house structurally diverse library (Figure S1); relative
mRNA levels of RhoGDI2 and WSB1 in H1299-WSB1
cells (Figure S2); purification of WSB1 protein using
whole-cell lysate (Figure S3); representative figures of
the wound-healing assay of compounds 1a and 4 against
KHOS cells (Table S1); representative figures of the
wound-healing assay of compounds 1a and 4 against
H460 cells (Table S2); and NMR spectra and HPLC
results for the target compounds (PDF)
Molecular formula strings (CSV)
Yongzhou Hu − Hangzhou Institute of Innovative Medicine,
Institute of Drug Discovery and Design, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, P. R. China; orcid.org/0000-0001-7349-4062
Bo Yang − Institute of Pharmacology & Toxicology, Zhejiang
Province Key Laboratory of Anti-Cancer Drug Research,
College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, P. R. China; Innovation Institute for
Artificial Intelligence in Medicine of Zhejiang University,
Hangzhou 310018, P. R. China
AUTHOR INFORMATION
■
Corresponding Authors
Xiaowu Dong − Hangzhou Institute of Innovative Medicine,
Institute of Drug Discovery and Design, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, P. R. China; Innovation Institute for Artificial
Intelligence in Medicine of Zhejiang University, Hangzhou
310018, P. R. China; Cancer Center, Zhejiang University,
Hangzhou 310058, P. R. China; orcid.org/0000-0002-
2178-4372; Email: dongxw@zju.edu.cn
Ji Cao − Institute of Pharmacology & Toxicology, Zhejiang
Province Key Laboratory of Anti-Cancer Drug Research,
College of Pharmaceutical Sciences and Cancer Center,
Zhejiang University, Hangzhou 310058, P. R. China;
Innovation Institute for Artificial Intelligence in Medicine of
Zhejiang University, Hangzhou 310018, P. R. China;
Email: caoji88@zju.edu.cn
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00586
Author Contributions
J.C., Z.J., F.Y., and J.Y. contributed equally to this work. J.C.
designed and synthesized the compounds, analyzed the data,
and drafted the manuscript. Z.J., J.X., B.C., and G.C.
synthesized the compounds. F.Y. and J.Y. performed the
biological experiments. H.Z., Q.H., Y.H., B.Y., J.C., and X.D.
conceived the study and helped analyze the data.
Authors
Jinxin Che − Hangzhou Institute of Innovative Medicine,
Institute of Drug Discovery and Design, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, P. R. China
Zegao Jin − Hangzhou Institute of Innovative Medicine,
Institute of Drug Discovery and Design, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, P. R. China
Funding
Key R&D Program of Zhejiang Province; National Natural
Science Foundation of China; National Natural Science
Foundation for Distinguished Young Scholar of China.
Notes
The authors declare no competing financial interest.
Fangjie Yan − Institute of Pharmacology & Toxicology,
Zhejiang Province Key Laboratory of Anti-Cancer Drug
Research, College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou 310058, P. R. China; Innovation
Institute for Artificial Intelligence in Medicine of Zhejiang
University, Hangzhou 310018, P. R. China
Jieqiong You − Institute of Pharmacology & Toxicology,
Zhejiang Province Key Laboratory of Anti-Cancer Drug
Research, College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou 310058, P. R. China
Jiangfeng Xie − Hangzhou Institute of Innovative Medicine,
Institute of Drug Discovery and Design, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, P. R. China
Binhui Chen − Hangzhou Institute of Innovative Medicine,
Institute of Drug Discovery and Design, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, P. R. China
ACKNOWLEDGMENTS
■
The authors thank Jianyang Pan (Research and Service Center,
College of Pharmaceutical Sciences, Zhejiang University) for
performing NMR spectrometry for structure elucidation.
Figure 8H and Table of Contents Graphic were created with
BioRender.com. This work was supported by a grant from the
Key R&D Program of Zhejiang Province (2020C03010), the
National Natural Science Foundation of China (81673294),
and the National Natural Science Foundation for Distin-
guished Young Scholar of China (81625024).
ABBREVIATIONS
■
AUC, area under curve; DBU, 1,8-diazabicyclo[5.4.0]undec-7-
ene; DCM, dichloromethane; DMAP, 4-dimethylaminopyr-
idine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide;
EDCI, 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide hy-
drochloride; HOBt, 1-hydroxybenzotriazole; MC, methyl
cellulose; m-CPBA, 3-chloroperoxybenzoic acid; RT-PCR,
real-time polymerase chain reaction; TMEDA, N,N,N′,N′-
tetramethylethylenediamine; TEA, triethylamine; THF, tetra-
hydrofuran
Gang Cheng − College of Pharmaceutical Science, Zhejiang
Chinese Medical University, Hangzhou 311402, P. R. China;
orcid.org/0000-0002-1760-9539
Hong Zhu − Institute of Pharmacology & Toxicology,
Zhejiang Province Key Laboratory of Anti-Cancer Drug
8642
https://doi.org/10.1021/acs.jmedchem.1c00586
J. Med. Chem. 2021, 64, 8621−8643

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
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