Design, synthesis and biological evaluation of benzyloxyphenyl-methylaminophenol derivatives as STAT3 signaling pathway inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.04.022

STAT3 signaling pathway has been validated as a vital therapeutic target for cancer therapy. Based on the novel STAT3 inhibitor of a benzyloxyphenyl-methylaminophenol scaffold hit (1) discovered through virtual screening, a series of analogues had been designed and synthesized for more potent inhibitors. The preliminary SAR had been discussed and the unique binding site in SH2 domain was predicted by molecular docking. Among them, compounds 4a and 4b exhibited superior activities than hit compound (1) against IL-6/STAT3 signaling pathway with IC₅₀ values as low as 7.71 μM and 1.38 μM, respectively. Compound 4a also displayed potent antiproliferative activity against MDA-MB-468 cell line with an IC₅₀ value of 9.61 μM. We believe that these benzyloxyphenyl-methylaminophenol derivatives represent a unique mechanism for interrogating STAT3 as well as a potential structure type for further exploration.

Full text of DOI:10.1016/j.bmc.2016.04.022

Bioorganic & Medicinal Chemistry 24 (2016) 2549–2558
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of benzyloxyphenyl-
methylaminophenol derivatives as STAT3 signaling pathway
inhibitors
⇑
⇑
Dingding Gao, Qiang Xiao, Mingming Zhang , Yingxia Li
School of Pharmacy, Fudan University, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
STAT3 signaling pathway has been validated as a vital therapeutic target for cancer therapy. Based on the
novel STAT3 inhibitor of a benzyloxyphenyl-methylaminophenol scaffold hit (1) discovered through
virtual screening, a series of analogues had been designed and synthesized for more potent inhibitors.
The preliminary SAR had been discussed and the unique binding site in SH2 domain was predicted by
molecular docking. Among them, compounds 4a and 4b exhibited superior activities than hit compound
Received 12 January 2016
Revised 20 March 2016
Accepted 11 April 2016
Available online 12 April 2016
(1) against IL-6/STAT3 signaling pathway with IC50 values as low as 7.71
Compound 4a also displayed potent antiproliferative activity against MDA-MB-468 cell line with an IC50
value of 9.61 M. We believe that these benzyloxyphenyl-methylaminophenol derivatives represent a
lM and 1.38 lM, respectively.
Keywords:
STAT3 signaling pathway
Inhibitors
Benzyloxyphenyl-methylaminophenol
derivatives
l
unique mechanism for interrogating STAT3 as well as a potential structure type for further exploration.
Ó 2016 Elsevier Ltd. All rights reserved.
Structure–activity relationship
1. Introduction
in human cancer cells, such as ovarian, cervical, breast, head and
neck squamous cell carcinoma, prostate, and leukemias.17–19
Signal transducers and activators of transcription (STATs) pro-
tein comprise seven members (STAT1–STAT4, STAT5A, STAT5B,
and STAT6) that regulate cell proliferation, differentiation, apopto-
sis, immune and inflammatory response, and angiogenesis.1–6
Among all of these STAT proteins, STAT3 is the most studied mem-
ber for its important function in cell proliferation, differentiation,
apoptosis, angiogenesis, immune responses and metastasis.7–10
Furthermore, newly identified cancer-promoting functions of
STAT3 (mitochondria, epigenetic regulation, obesity, and pre-
metastatic niches) further highlight the importance of targeting
STAT3.11–14 STAT3 is activated by receptor tyrosine kinases (EGFR,
VEGFR, PDGFR, FGFR, etc), receptor associated kinases (JAK) and
non-receptor tyrosine kinases (Src and Abl) at tyrosine residue
(Tyr705).15 Upon Tyr705 phosphorylation, STAT3 forms homo-
dimerization, then translocates into the nucleus and binds to
STAT3-specific DNA binding element, activating the gene tran-
scription.16 Persistent activation of STAT3 signaling has been
demonstrated to induce cell proliferation and prevent apoptosis
Hence STAT3 signaling is an attractive target for cancer therapy.
Our previous work by using virtual screening approach had dis-
covered several benzyloxyphenyl-methylaminophenol scaffolds
STAT3 inhibitors as promising anticancer agents. The hit com-
pound (1) was identified as an inhibitor of IL-6/STAT3 signaling
pathway with an IC50 value of 26.68
on closely related IFN- /STAT1, TNF-
l
M and no obvious effects
c
a
/NF- B signaling pathway,
j
and upstream JAK2 and Src kinases.20 In order to obtain more
potent inhibitors, several series of hit 1 derivatives were designed,
synthesized and tested against STAT3 signaling pathway and
breast cancer MDA-MB-468 cell line. As shown in Figure 1, we first
focused on the optimization on the ring A, B (4a–r) and then the
linker (8t and 10u) among these rings in hit 1. Conformational
restriction strategy was also used to reduce the molecular flexibility
(16v and 16w).
2. Results and discussion
2.1. Chemistry
Abbreviations: STAT3, signal transducers and activators of transcription 3; IL-6,
interleukin-6; JAK, Janus kinase; SAR, structure–activity relationships.
Corresponding authors. Tel.: +1 808 333 0514 (M.Z.), +86 21 51980127 (Y.L.).
E-mail addresses: zmmworld@gmail.com (M. Zhang), liyx417@fudan.edu.cn
(Y. Li).
The benzyloxyphenyl-methylaminophenol derivatives were
prepared according to the synthetic route as outlined in
Scheme 1.21 By reacting with 4-chlorobenzyl chloride in the pres-
ence of potassium carbonate, substituted salicylaldehydes 2a–k
⇑
http://dx.doi.org/10.1016/j.bmc.2016.04.022
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

2550
D. Gao et al. / Bioorg. Med. Chem. 24 (2016) 2549–2558
O
X
OH
R
N
H
A
Y
HN
HN
O
Conformational
restriction
Cl
SAR
HN
N
B
N
O
H
C
N
Cl
Cl
O
Hit (1)
Figure 1. Design of target compounds.
5'
4'
Y
3'
6'
HN
CHO
O
2'
6
CHO
OH
X
5
a
b
X
X
2a: X=H
3a: X=H
4a
: X=H
Y=4'-OH
Y=4'-OH
Y=4'-OH
4
O
2b
3b
: X=3-Cl
: X=3-Cl
: X=4-Cl
3
4b: X=3-Cl
4c: X=4-Cl
2c
3c
: X=4-Cl
2d: X=5-Cl
3d: X=5-Cl
Cl
4d(1)
: X=5-Cl
2a-k
Y=4'-OH
3a-k
4a-r
Cl
2e
3e
: X=6-Cl
: X=5-F
: X=6-Cl
3f
: X=5-F
4e: X=6-Cl
4f: X=5-F
Y=4'-OH
Y=4'-OH
Y=4'-OH
2f
c
OH 2g: X=5-Br
3g: X=5-Br
4g
: X=5-Br
2h
3h
: X=5-CH3
: X=5-CH3
4h: X=5-CH3 Y=4'-OH
4i: X=4-OCH3 Y=4'-OH
2i
3i
: X=4-OCH3
: X=4-OCH3
HN
d
CHO
O
2j: X=3,5-t-Bu 3j: X=3,5-t-Bu
4j
: X=3,5-t-Bu Y=4'-OH
S
2k
: X=5-NO2
3k
: X=5-NO2
4k: X=5-NO2
4l: X=5-Cl
Y=4'-OH
S
Y=4'-SO2NH2
Y=4'-OCH3
Y=4'-OC2H5
Y=3',5'-CH3
Y=4'-OCF3
Y=3'-CF3
4m
O
: X=5-Cl
4n: X=5-Cl
4o: X=5-Cl
4p
: X=5-Cl
Cl
4s
3s
Cl
4q: X=5-Cl
4r: X=5-Cl
Y=3'-CN
Scheme 1. Reagents and conditions: (a) 4-chlorobenzyl chloride, K2CO3, DMF, 120 °C, 2 h, 75.0–99.0%; (b) substituted anilines, NaBH3CN, AcOH, CH3OH, rt, 5 h, 70.0–100%.
(c) 3g, 2-thiopheneboronic acid, Cs2CO3, Pd(PPh3)4, dioxane/H2O (3:1), 120 °C, microwave, 2 h; (d) 4-aminophenol, NaBH3CN, AcOH, CH3OH, rt, 5 h, 77.9%.
were transformed to intermediates 3a–k. In addition, compound
3g was coupled with 2-thiopheneboronic acid via Suzuki reaction
to afford 3s. Subsequently, in the presence of NaBH3CN and substi-
tuted anilines, 3a–k and 3s in methanol were transformed into target
compounds 4a–4r and 4s via reductive amination (Scheme 1).
Compounds 8t and 10u, with more rigid linkers between these
rings, were synthesized as described in Scheme 2 and 3. Treatment
of the methyl 2-amino-5-chlorobenzoate with 4-chlorobenzoyl
chloride and potassium carbonate in dichloromethane at room
temperature resulted in the formation of 5t. Then it was reduced
with lithium aluminumhydride in anhydrous THF, providing the
corresponding alcohol 6t, which followed by oxidization using
active manganese dioxide and formed 7t. Finally, the compound
7t was transformed to 8t using the same reductive amination
procedure as described above. For the synthesis of compound
10u, the intermediate 2d was oxidized to carboxylic acid 9u using
NaClO2–NaH2PO4ꢀ2H2O system, which was then coupled with
4-aminophenol in the presence of HATU/HOAT/DIPEA to give the
target compound.
The synthesis of conformation restricted compounds were
described in Scheme 4. Quinoline-2,4-diol was treated with nitric
acid to afford 3-nitroquinoline-2,4-diol (11v). Compound 11v
was converted into 2,4-dichloro-3-nitroquinoline (12v) with phos-
phorus oxychloride, which subsequently reacted with 28% NH3(aq)
to form 2-chloro-3-nitroquinolin-4-amine (13v). This compound
was treated with iron powder to give the diaminopyridine (14v),
which was cyclized to give the 4-chloro-1H-imidazo[4,5-c]quino-
lones (15v and 15w) in the presence of sodium pyrosulfate. Finally,
OH
HN
Cl
Cl
CH2OH
NH
Cl
CHO
NH
Cl
CO2CH3
NH
Cl
CO2CH3
NH2
NH
a
b
c
d
O
O
O
O
Cl
Cl
Cl
Cl
6t
7t
8t
5t
Scheme 2. Reagents and conditions: (a) 4-chlorobenzoyl chloride, K2CO3, DCM, rt, 2 h, 80.1%; (b) LiAlH4, THF, 0 °C, 0.5–1 h, 67.2%; (c) MnO2, DCM, reflux, 2 h, 82.8%;
(d) 4-aminophenol, NaBH3CN, AcOH, CH3OH, rt, 2 h, 90.0%.

D. Gao et al. / Bioorg. Med. Chem. 24 (2016) 2549–2558
2551
OH
HN
O
Cl
COOH
O
Cl
CHO
O
Cl
O
a
b
Cl
Cl
9u
Cl
10u
2d
Scheme 3. Reagents and conditions: (a) NaClO2, NaH2PO4ꢀ2H2O, 2-methyl-2-butene, tert-butanol, 95.0%; (b) 4-aminophenol, HATU, HOAT, DIPEA, THF, 84.8%.
NH2
N
Cl
N
OH
OH
N
NO2
Cl
NO2
Cl
NO2
OH
a
b
c
N
OH
13v
11v
12v
R
R
NH2
N
NH2
Cl
f
HN
e
d
HN
N
N
N
14v
N
Cl
O
O
15v
S
: R=
16v
S
: R=
O
O
15w: R= -OH
16w: R= -OH
Scheme 4. Reagents and conditions: (a) HNO3, 75 °C, 98.5%; (b) POCl3, 108 °C, 54.0%; (c) NH3ꢀH2O, CH3CN, 50 °C, 87.0%; (d) Fe powder, NH4Cl, CH3CH2OH/H2O, 94.7%; (e) 15v:
4-methylsulfonyl benzaldehyde, Na2S2O7, THF (dry), reflux, 70.5%; 15w: 4-hydroxybenzaldehyde, Na2S2O7, THF (dry), reflux, 65.7%; (f) phenylboronic acid, Pd(PPh3)4, Cs2CO3,
dioxane/H2O (3:1), 120 °C, microwave, 2 h, 89.6–100%.
Suzuki coupling reaction was used to afford 16v and 16w,
respectively.
found that compounds 4a, 4b and 4f showed superior luciferase
activities than hit compound 1.
2.2. Inhibitory activity on STAT3 transcription
2.3. Cellular potency
We firstly tested the inhibitory activity against STAT3 signal-
ing pathway using a STAT3 luciferase reporter method. As shown
in Table 1, the target compounds displayed greatly varied inhibi-
Some selected compounds were further evaluated the
antiproliferative activity in MDA-MB-468 cell line which harbors
constitutively active STAT3. As shown in Table 2, all of these selected
compounds exhibited micromole IC50 values against the tested
cancer cell. Among them, 4a, 4g, 4k displayed further enhanced
anticancer activities when compared with hit compound 1 with
tory activities in HepG2 cell at 10 lM after 24 h incubation. The
IC50 values of some potent compounds were listed in Table 2
and Figure 2. In light of these data, removing the chlorine atom
at C-5 in ring B of hit compound (1) improved the luciferase
IC50 values of 9.61 lM, 9.85 lM and 6.15 lM, respectively. Com-
activity from 26.68
lM to 7.71
lM (1 vs 4a). The replacement
pounds 4b and 4l showed the similar activity to hit compound 1.
of a fluorine atom at C-5 position in ring B was favorable while
the bromine atom was detrimental (1 vs 4f, 4g). Incorporation
of bigger steric substituents (tertiary butyl and thiophen-2-yl)
at C-5 position rendered the compound inactive (4j, 4s). These
indicated that the size of substituents at C-5 position in ring B
affected the activities. Changing the position of chlorine in ring
B from C-5 to C-4 and C-6 led to the loss of inhibitory activity
(4c, 4e), while moving the chlorine from C-5 to C-3 resulted in
a significant elevated luciferase activity (1 vs 4b), and as a result
2.4. Western blot analysis of compounds 4a and 4b
To further investigate the blockage of compounds 4a and 4b
against the activation of STAT3 signaling pathway, we examined
the STAT3 phosphorylation in MDA-MB-468 cell line using
Western blot analysis. As shown in Figure 3, compounds 4a and
4b dose-dependently inhibited Tyr705 phosphorylation, whereas
the expression of total STAT3 remained unchanged after 2 h incu-
bation. This indicates that the decrease of Tyr705 phosphorylated
STAT3 is not due to a constitutional decrease of total STAT3
protein. It is noteworthy that compounds 4a and 4b had no effect
on the level of STAT1 or Tyr701 phosphorylated STAT1 which
suggested that 4a and 4b had a good selectivity against the tumor
suppressor STAT1. In addition, compounds 4a and 4b displayed no
effects on the phosphorylation of upstream tyrosine kinase (Src),
which participates in activating STAT3 signaling pathway. Above
results prove that compounds 4a and 4b could down regulated
STAT3 phosphorylation in an independent manner.
compound 4b had an IC50 value of 1.38
electron-withdrawing nitro group at ring B (4k) displayed similar
activity to hit compound (1) with an IC50 value of 24.09 M.
lM. Introduction of
l
However, electron-donating groups made the activity decrease
or miss (4h, 4i). For ring A moiety, the hydroxyl group at C-40
position was critical for the activities (4m–r). When the hydroxyl
group was replaced by sulfamine (4l), the luciferase activity
IC50 value dropped to 35.67
conformation restricted compounds displayed inhibitory activities
at concentration of 10 M (8t, 10u, 16v, 16w). Taken together, we
lM. Unfortunately, none of these
l

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D. Gao et al. / Bioorg. Med. Chem. 24 (2016) 2549–2558
Table 1
Inhibitory activity on STAT3 transcription
5'
OH
OH
R
OH
4'
Y
6'
A
3'
HN
O
HN
HN
O
HN
O
6
2'
Cl
Cl
S
O
S
5
O
HN
N
B
X
4
16v: R=
N
NH
O
3
16w: R= -OH
O
C
4a-r
4s
10u
8t
Cl
Cl
Cl
Cl
Compound
IL-6/STAT3 pathwaya 10
l
M (%)
Compound
IL-6/STAT3 pathwaya 10
lM (%)
4a
4b
4c
1
4e
4f
4g
4h
4i
24.38
39.51
ꢁ2.98
30.83
ꢁ4.30
37.18
32.54
ꢁ8.42
11.96
ꢁ31.33
33.31
11.96
4m
4n
4o
4p
4q
4r
4s
8t
10u
16v
16w
Curcumin
ꢁ4.30
ꢁ28.07
1.95
3.13
2.61
0.78
ꢁ12.58
ꢁ220.37
ꢁ28.73
7.17
5.28
12.90
4j
4k
4l
a
STAT3-dependent luciferase reporter gene assay in HepG2 cell. Percent inhibition at 10 lM.
Table 2
luciferase reporter gene assay in 293 cells after the addition of
IFN- and TNF- respectively. As Table 3 shows, compounds 4a
did not displayed any of IFN- -induced STAT1 and TNF- -induced
NF- activations with concentration as high as 100 M.
Compound 4b, which displayed the most inhibitory activity against
STAT3 signaling pathway, had IC50 values 40.19 M and 77.96
against IFN- /STAT1 and TNF- /NF- B signaling pathway respec-
tively. These results demonstrate that compounds 4a and 4b
suppressive effects are highly selective for the IL-6/STAT3 signaling
pathway over STAT1 and NF-jB signaling pathways.
Inhibitory activity on STAT3 transcription and human breast cancer cell lines of
selected compounds
c
a
c
a
Compounds
IL-6/STAT3 pathwaya (IC50
lM)
MDA-MB-468b (IC50
lM)
j
B
a
l
4a
4b
1
4f
4g
4k
4l
7.71
1.38
9.61
19.70
18.83
57.25
9.85
l
lM
26.68
11.04
168.4
24.09
35.67
c
a
j
6.15
24.34
a
STAT3-dependent luciferase reporter gene assay in HepG2 cell.
Cell viability assay against MDA-MB-468 (MTT).
2.6. Discussion
b
As 4a and 4b exhibited better inhibitory activity on STAT3
transcription, a molecular docking (Schrodinger, Maestro suite)
model of compounds 4a and 4b bound to STAT3-SH2 domain
was generated on the basis of the crystal structure of STAT3b homo
dimer (PDB entry 1BG1). The results reveal that they bind to a
unique site of STAT3-SH2 domain rather than the pTyr705 binding
site surrounded by Lys591, Arg609, and Ser611 as illustrated in
2.5. The selectivity against related IFN-
c/STAT1 and TNF-a/NF-
jB signaling pathway
The selectivity of compounds 4a and 4b against related IFN-
c/
STAT1 and TNF- /NF- B signaling pathway was investigated using
a
j
a STAT1 luciferase reporter gene assay in HepG2 cells and a NF-jB
Compound 4b
Compound 4a
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-3
-2
-1
0
1
2
3
-3
-2
-1
0
1
2
Log 10 concentration (µM)
Log 10 concentration (µM)
IC50/1.382
IC50/7.711
Figure 2. Dose-dependent inhibition of compounds 4a and 4b on the IL-6-induced STAT3-dependant luciferase activity. HepG2/STAT3-luciferase cells were pretreated with
compounds 4a and 4b at the indicated concentrations for 1 h, and then were stimulated with IL-6 (10 ng/ml) for 5.5 h and the luciferase activity was measured following the
5.5 h stimulation.

D. Gao et al. / Bioorg. Med. Chem. 24 (2016) 2549–2558
2553
Figure 3. Western blot analysis of inhibition of STAT3 activity and selective inhibition against STAT1 and Src by compounds 4a (A) and 4b (B). Compounds 4a and 4b showed
inhibition of STAT3 phosphorylation but not STAT1 and Src phosphorylation in MDA-MB-468. Cells were treated with stattic or 4a and 4b for 2 h, levels of pSTAT3, STAT3,
pSTAT1, STAT1, pSrc, Src were probed by specific antibodies. b-Actin was used as the loading control.
Table 3
the hydroxyl group in ring A forms a hydrogen-bonding interaction
Inhibitory activity against related IFN-
c
/STAT1 and TNF-
a/NF-jB signaling pathway
with Cys712, which plays a vital role in activity. Changing the
hydroxyl group to methoxyl group or ethyoxyl group decline
activities (4m, 4n). The activities also lost (4o–4r) after removing
the hydroxyl group. Furthermore, it is predicted that the NH
between ring A and ring B is able to form a hydrogen bond
interaction with residue Gln644. Also, the size of substituent at
C-5 position in ring B is crucial for interaction. Bulky groups, such
as bromo, tertiary butyl and thiophen-2-yl, may have a steric clash
with the surface of the binding pocket. Furthermore, only chlorine
atom at C-3 and C-5 position in ring B is tolerate (4b, 1). Changing
the substitution position of chlorine atom in ring B to C-4 or C-6 led
to possible clashes with the surface of the protein (4c, 4e). More
interestingly, ring B and ring C bound to the target protein like a
Compounds IL-6/STAT3 pathwaya
IFN-
(IC50
c
l
/STAT1b
M)
TNF-
(IC50
a
l
/NF-
M)
j
Bc
(IC50 M)
l
4a
4b
1
7.71
1.38
26.68
>100
40.19
>100
>100
77.96
>100
a
b
c
STAT3-dependent luciferase reporter gene assay in HepG2 cell.
STAT1-dependent luciferase reporter gene assay in HepG2 cell.
NF-jB-dependent luciferase reporter gene assay in 293 cell.
Figure 4. The ring A in 4a and 4b insert into a hydrophobic cleft
which was provided by Tyr640, Ile653. It is a similar manner as
Phe710 of the phosphotyrosine peptide (APpYLKTKF). Additionally,
Figure 4. Predicted binding modes for compound 4a (1–2) and compound 4b (3–4) with STAT3 SH2 domain (PDB entry 1BG1), generated by Schrodinger, Maestro suite. The
figures were generated using Pymol.

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D. Gao et al. / Bioorg. Med. Chem. 24 (2016) 2549–2558
‘clip’. Therefore, the flexible linker was necessary, as the conforma-
tional restricted compounds (8t, 10u, 16v, 16w) could not bind to
the predicted site so that the activities are missing.
the mixture was cooled to room temperature. Water and ethyl
acetate were added and the mixture was extracted with ethyl acet-
ate twice. The combined extracts were washed with water and
brine, dried over Na2SO4. After filtration, the filtrate was removed
in vacuo. The residue was purified by silica gel column chromatog-
raphy to give 3a–3k (75.0–99.0%).
3. Conclusion
Herein, we reported the structure–activity relationship study of
a benzyloxyphenyl-methylaminophenol scaffold as STAT3 signal-
ing pathway inhibitors. Finally, we discovered 4a and 4b potently
inhibit activation and transactivation of STAT3 signaling pathway
and explained the possible reasons combined with the docking
results. Furthermore, 4a could effectively inhibit the viability of
tumor cells MDA-MB-468 harboring constitutively active STAT3
4.2.2.1. 2-((4-Chlorobenzyl)oxy)benzaldehyde (3a). The title
compound was obtained as a white solid, yield: 84.2%. 1H NMR
(400 MHz, CDCl3) d 10.55 (s, 1H), 7.88 (dd, J = 7.7, 1.6 Hz, 1H),
7.59–7.52 (m, 1H), 7.41–7.38 (m, 4H), 7.10–7.05 (m, 1H), 7.03 (d,
J = 8.4 Hz, 1H), 5.18 (s, 2H). ESI-MS: calcd for [M+H]+ m/z: 247.0,
found: 247.0
with an IC50 value of 9.61 lM. The present study provided more
4.2.2.2. 3-Chloro-2-((4-chlorobenzyl)oxy) benzaldehyde (3b). The
title compound was obtained as a white solid, yield: 99.3%. 1H NMR
(400 MHz, CDCl3) d 10.15 (s, 1H), 7.72 (d, J = 7.1 Hz, 1H), 7.66 (d,
J = 7.2 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 5.09 (s, 2H). ESI-MS: calcd
for [M+H]+ m/z: 281.0, found: 281.0.
structural reference for the development of STAT3 signaling path-
way. Additional in vivo antitumor activity studies on these com-
pounds are undergoing and will be reported in due course.
4. Experiment
4.2.2.3. 4-Chloro-2-((4-chlorobenzyl)oxy)benzaldehyde (3c). The
title compound was obtained as a white solid, yield: 98.1%. 1H NMR
(400 MHz, CDCl3) d 10.44 (s, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.44–7.34
(m, 4H), 7.05 (d, J = 8.0 Hz, 1H), 7.04 (s, 1H), 5.14 (s, 2H). ESI-MS:
calcd for [M+H]+ m/z: 281.0, found: 281.0.
4.1. Biological evaluation
4.1.1. Cell lines and culture
HepG2/STAT3 and HepG2/STAT1 cells, gifts of Prof. Xinyuan Fu
(National University of Singapore, Singapore), were HepG2 cells
stably transfected with a STAT-responsive firefly luciferase repor-
4.2.2.4. 5-Chloro-2-((4-chlorobenzyl)oxy)benzaldehyde (3d). The
title compound was obtained as a white solid, yield: 98.7%. 1H
NMR (400 MHz, DMSO-d6) d 10.32 (s, 1H), 7.69 (dd, J = 8.9, 2.8 Hz,
1H), 7.63 (d, J = 2.8 Hz, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.45 (d,
J = 8.4 Hz, 2H), 7.34 (d, J = 9.0 Hz, 1H), 5.28 (s, 2H). ESI-MS: calcd
for [M+H]+ m/z: 281.0, found: 281.0.
ter plasmid. 293/NF-
jB cells were 293 cells stably transfected with
NF- B-responsive firefly luciferase reporter plasmid. All other cell
j
lines were obtained from the American Type Culture Collection
(USA).
4.1.2. Luciferase assay
Procedure is similar to published methods.20
4.2.2.5. 2-Chloro-6-((4-chlorobenzyl)oxy)benzaldehyde (3e). The
title compound was obtained as a white solid, yield: 75.8%. 1H NMR
(400 MHz, CDCl3) d 10.55 (s, 1H), 7.42–7.33 (m, 5H), 7.05 (d,
J = 8.0 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 5.14 (s, 2H). ESI-MS: calcd
for [M+H]+ m/z: 281.0, found: 281.0.
4.1.3. MTT assay
Procedure is similar to published methods.22
4.1.4. Western blot analysis
Procedure is similar to published methods.20
4.2.2.6. 2-((4-Chlorobenzyl)oxy)-5-fluorobenzaldehyde (3f). The
title compound was obtained as a white solid, yield: 81.0%. 1H
NMR (400 MHz, CDCl3) d 10.46 (d, J = 3.1 Hz, 1H), 7.52 (dd,
J = 8.2, 3.2 Hz, 1H), 7.40–7.34 (m, 4H), 7.27–7.20 (m, 1H), 6.99
(dd, J = 9.1, 3.8 Hz, 1H), 5.14 (s, 2H). ESI-MS: calcd for [M+H]+
m/z: 265.0, found: 265.0.
4.2. Chemistry
4.2.1. General information
All solvents and chemical used were reagent grade. Purity and
characterization of compounds were established by a combination
of LC–MS and NMR. Flash column chromatography was performed
on silica gel (200–300 mesh, Qingdao, China). 1H NMR spectra were
recorded with a 400 MHz Varian spectrometer and 13C NMR spec-
tra were recorded with a 600 MHz Bruker spectrometer. Chemical
shifts (d) are expressed in ppm, and coupling constants (J) are
expressed in Hz. All reactions were performed under nitrogen
unless otherwise stated. High resolution mass spectrometry data
were given by AB 5600+Q TOF. The purity of all the designed com-
pounds was analyzed using an Agilent 1200 HPLC system with a
G1311B quaternary pump, a G1329B ALS and a G4212B DAD detec-
tor. The HPLC method consisted of the following: Agilent C18 RP
4.2.2.7. 5-Bromo-2-((4-chlorobenzyl)oxy)benzaldehyde (3g). The
title compound was obtained as a white solid, yield: 98.9%. 1H
NMR (400 MHz, CDCl3) d 10.45 (s, 1H), 7.96 (d, J = 2.5 Hz, 1H),
7.62 (dd, J = 8.8, 2.5 Hz, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.37 (d,
J = 8.7 Hz, 2H), 6.93 (d, J = 8.9 Hz, 1H), 5.16 (s, 2H). ESI-MS: calcd
for [M+H]+ m/z: 325.0, found: 325.0.
4.2.2.8. 2-((4-Chlorobenzyl)oxy)-5-methylbenzaldehyde (3h). The
title compound was obtained as a white solid, yield: 77.3%. 1H NMR
(400 MHz, CDCl3) d 10.50 (s, 1H), 7.66 (d, J = 2.1 Hz, 1H), 7.38–7.36
(m, 4H), 7.34 (dd, J = 8.5, 2.3 Hz, 1H), 6.91 (d, J = 8.5 Hz, 1H), 5.13
(s, 2H), 2.32 (s, 3H). ESI-MS: calcd for [M+H]+ m/z: 261.1, found:
261.1.
column (250 mm ꢂ 4.6 mm, 5
lm); column temperature 25 °C;
inject volume 2.0 L; HPLC solvent H2O (0.1% TFA)/CH3OH = 60/40
l
(v/v); flow rate of 1.2 mL/min; detector wavelength of 275 nm.
4.2.2.9.
(3i). The title compound was obtained as a white solid, yield:
94.6%. 1H NMR (400 MHz, CDCl3)
10.35 (s, 1H), 7.83 (d,
J = 8.7 Hz, 1H), 7.38–7.36 (m, 4H), 6.57 (dd, J = 8.7, 2.1 Hz, 1H),
6.47 (d, J = 2.2 Hz, 1H), 5.12 (s, 2H), 3.85 (s, 3H). ESI-MS: calcd for
[M+H]+ m/z: 277.1, found: 277.1.
2-((4-Chlorobenzyl)oxy)-4-methoxybenzaldehyde
4.2.2. General procedure for the synthesis of 3a–3k
To a solution of substituted salicylaldehyde (1.0 mmol) in anhy-
drous DMF was added 4-chlorobenzyl chloride (139 lL, 1.1 mmol),
K2CO3 (553 mg, 4.0 mmol). The reaction mixture was stirred at
120 °C for 2 h and monitored by LC/MS analysis. Upon completion,
d

D. Gao et al. / Bioorg. Med. Chem. 24 (2016) 2549–2558
2555
4.2.2.10.
3,5-Di-tert-butyl-2-((4-chlorobenzyl)oxy)benzalde-
ESI-MS: calcd for [M+H]+ m/z: 374.1, found: 374.1. ESI-HRMS
[M+H]+ calcd for C20H18Cl2NO2: 374.0709, found: 374.0713. HPLC:
tR = 3.463 min.
hyde (3j). The title compound was obtained as a white solid. The
crude product was used directly for the next step without further
purification.
4.2.4.4. 4-((2-Chloro-6-((4-chlorobenzyl)oxy)benzyl)amino)phe-
nol (4e). The title compound was obtained as a white solid, yield:
95.7%. 1H NMR (400 MHz, CDCl3) d 7.37–7.29 (m, 4H), 7.14 (t,
J = 8.2 Hz, 1H), 7.01 (d, J = 8.1 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H),
6.67–6.61 (m, 4H), 5.06 (s, 2H), 4.47 (s, 2H). 13C NMR (150 MHz,
CDCl3) d 157.08, 147.56, 141.46, 134.91, 134.27, 133.40, 128.34,
128.30, 128.01, 125.57, 121.82, 115.38, 114.99, 110.03, 69.32,
41.22. ESI-MS: calcd for [M+H]+ m/z: 374.1, found: 374.1. ESI-
HRMS [M+H]+ calcd for C20H18Cl2NO2: 374.0709, found:
374.0711. HPLC: tR = 3.294 min.
4.2.2.11. 2-((4-Chlorobenzyl)oxy)-5-nitrobenzaldehyde (3k). The
title compound was obtained as a white solid, yield: 90.0%. 1H NMR
(400 MHz, DMSO-d6) d 10.53 (s, 1H), 8.67 (dd, J = 9.2, 2.8 Hz, 1H),
8.61 (d, J = 2.7 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 9.2 Hz,
1H), 7.66 (d, J = 8.3 Hz, 2H), 5.62 (s, 2H). ESI-MS: calcd for [M+H]+
m/z: 292.0, found: 292.0.
4.2.3. 2-((4-Chlorobenzyl)oxy)-5-(thiophen-2-yl)benzaldehyde
(3s)
A mixture of 2g (325 mg, 1 mmol), thiophen-2-ylboronic acid
(192 mg, 1.5 mmol), Pd(PPh3)4 (116 mg, 0.1 mmol) and Cs2CO3
(1.30 g, 4 mmol) in 12 mL dioxane/Water(v/v = 3:1) was stirred at
120 °C by microwave for 2 h, then cooled to room temperature.
After removing the dioxane, water and ethyl acetate were added
and then the mixture was extracted with ethyl acetate twice. The
combined extracts were washed with brine, dried over Na2SO4.
After filtration, the filtrate was removed in vacuo. The crude pro-
duct was used directly for the next step without further
purification.
4.2.4.5. 4-((2-((4-Chlorobenzyl)oxy)-5-fluorobenzyl)amino)phe-
nol (4f). The title compound was obtained as a white solid, yield:
99.8%. 1H NMR (400 MHz, CDCl3) d 7.38–7.31 (m, 4H), 7.07 (dd,
J = 8.9, 2.9 Hz, 1H), 6.91–6.80 (m, 2H), 6.68 (d, J = 8.7 Hz, 2H),
6.50 (d, J = 8.7 Hz, 2H), 5.05 (s, 2H), 4.30 (s, 2H). 13C NMR
(150 MHz, CDCl3)
d 157.61, 156.03, 151.49, 147.27, 141.40,
134.67, 133.28, 129.64, 129.61, 128.24, 127.98, 115.56, 115.04,
114.00, 113.13, 112.04, 69.31, 43.74. ESI-MS: calcd for [M+H]+
m/z: 358.1, found: 358.1. ESI-HRMS [M+H]+ calcd for C20H18ClFNO2:
358.1005, found: 358.1007. HPLC: tR = 3.099 min.
4.2.4. General procedure for the synthesis of 4a–4s
To a stirring solution of aldehyde (3a–k, 3s) (1 mmol) and cat-
alytic amount acetic acid (1 drop) in anhydrous CH3OH (25 mL)
was added phenylamine derivatives (1 mmol). After the solution
was stirred at room temperature for 1 h, NaBH3CN (93.7 mg,
1.5 mmol) was added and stirred for another 2 h. After removing
the methanol, water and ethyl acetate were added and then the
organic was washed with saturated sodium bicarbonate, brine,
dried over Na2SO4. After filtration, the filtrate was removed in
vacuo. The residue was purified by silica gel column chromatogra-
phy to give 4a–s.
4.2.4.6.
4-((5-Bromo-2-((4-chlorobenzyl)oxy)benzyl)amino)
phenol (4g). The title compound was obtained as a white solid,
yield: 97.2%. 1H NMR (400 MHz, CDCl3) d 7.45 (d, J = 2.4 Hz, 1H),
7.38–7.28 (m, 5H), 6.78 (d, J = 8.7 Hz, 1H), 6.71–6.65 (m, 2H),
6.54–6.48 (m, 2H), 5.06 (s, 2H), 4.29 (s, 2H). 13C NMR (150 MHz,
CDCl3) d 154.59, 147.32, 141.41, 134.37, 133.36, 131.06, 130.18,
129.89, 128.28, 127.94, 115.58, 114.07, 112.96, 112.76, 68.90,
43.75. ESI-MS: calcd for [M+H]+ m/z: 418.0, found: 418.0. ESI-
HRMS [M+H]+ calcd for C20H18BrClNO2: 420.0183, found:
420.0186. HPLC: tR = 3.426 min.
4.2.4.1. 4-((2-((4-Chlorobenzyl)oxy)benzyl)amino)phenol (4a).
The title compound was obtained as a white solid, yield: 98.0%. 1H
NMR (400 MHz, CDCl3) d 7.38–7.31 (m, 5H), 7.23 (t, J = 7.8 Hz, 1H),
6.97–6.89 (m, 2H), 6.67 (d, J = 8.7 Hz, 2H), 6.54 (d, J = 8.7 Hz, 2H),
5.09 (s, 2H), 4.33 (s, 2H). 13C NMR (150 MHz, CDCl3) d 155.65,
147.25, 141.63, 134.89, 133.13, 128.66, 128.20, 127.91, 127.71,
127.25, 120.49, 115.52, 114.20, 111.07, 68.59, 44.27. ESI-MS: calcd
for [M+H]+ m/z: 340.1, found: 340.1. ESI-HRMS [M+H]+ calcd for
4.2.4.7.
4-((2-((4-Chlorobenzyl)oxy)-5-methylbenzyl)amino)
phenol (4h). The title compound was obtained as a white solid,
yield: 92.1%. 1H NMR (400 MHz, CDCl3) d 7.36–7.32 (m, 4H), 7.14
(s, 1H), 7.01 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.68 (d,
J = 8.7 Hz, 2H), 6.55 (d, J = 8.7 Hz, 2H), 5.05 (s, 2H), 4.28 (s, 2H),
2.26 (s, 3H). 13C NMR (150 MHz, CDCl3) d 153.55, 147.11, 141.97,
135.09, 133.04, 129.80, 129.46, 128.16, 127.89, 127.10, 115.50,
114.19, 114.10, 111.20, 68.78, 44.27, 19.95. ESI-MS: calcd for [M
+H]+ m/z: 354.1, found: 354.1. ESI-HRMS [M+H]+ calcd for
C20H19ClNO2: 340.1099, found: 340.1100. HPLC: tR = 3.113 min.
C21H21ClNO2: 354.1255, found: 354.1263. HPLC: tR = 3.413 min.
4.2.4.2. 4-((3-Chloro-2-((4-chlorobenzyl)oxy)benzyl)amino)phe-
nol (4b). The title compound was obtained as a brown solid, yield:
95.0%. 1H NMR (400 MHz, DMSO-d6) d 8.42 (s, 1H), 7.51 (d,
J = 8.5 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.37 (dd, J = 7.9, 1.3 Hz, 1H),
7.27 (d, J = 7.6 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 6.48 (d, J = 8.7 Hz,
2H), 6.32 (d, J = 8.7 Hz, 2H), 5.52 (t, J = 4.0 Hz, 1H), 4.99 (s, 2H),
4.14 (d, J = 4.0 Hz, 2H). 13C NMR (150 MHz, CDCl3) d 151.89,
147.39, 141.25, 134.55, 134.41, 133.67, 129.17, 128.99, 128.17,
127.36, 127.17, 124.67, 115.60, 113.97, 73.78, 43.98. ESI-MS: calcd
for [M+H]+ m/z: 374.1, found: 374.1. ESI-HRMS [M+H]+ calcd for
4.2.4.8. 4-((2-((4-Chlorobenzyl)oxy)-4-methoxybenzyl)amino)
phenol (4i). The title compound was obtained as a white solid,
yield: 94.3%. 1H NMR (400 MHz, CDCl3) d 7.35–7.31 (m, 4H), 7.21
(d, J = 8.3 Hz, 1H), 6.67 (d, J = 8.6 Hz, 2H), 6.54 (d, J = 8.7 Hz, 2H),
6.49 (d, J = 2.0 Hz, 1H), 6.45 (dd, J = 8.3, 2.1 Hz, 1H), 5.05 (s, 2H),
4.24 (s, 2H), 3.77 (s, 3H). 13C NMR (150 MHz, CDCl3) d 159.41,
156.58, 147.24, 141.81, 134.72, 133.14, 129.37, 128.20, 127.90,
119.68, 115.48, 114.25, 103.85, 99.37, 68.61, 54.78, 43.97. ESI-
MS: calcd for [M+H]+ m/z: 370.1, found: 370.1. ESI-HRMS [M+H]+
calcd for C21H21ClNO3: 370.1204, found: 370.1197. HPLC:
tR = 3.265 min.
C20H18Cl2NO2: 374.0709, found: 374.0707. HPLC: tR = 3.441 min.
4.2.4.3. 4-((4-Chloro-2-((4-chlorobenzyl)oxy)benzyl)amino)phe-
nol (4c). The title compound was obtained as a brown solid, yield:
95.0%. 1H NMR (400 MHz, CDCl3) d 7.39–7.30 (m, 4H), 7.24 (d,
J = 8.5 Hz, 1H), 6.93–6.89 (m, 2H), 6.66 (d, J = 8.7 Hz, 2H), 6.50 (d,
J = 8.7 Hz, 2H), 5.05 (s, 2H), 4.28 (s, 2H). 13C NMR (150 MHz, CDCl3)
d 156.05, 147.23, 141.53, 134.16, 133.42, 132.81, 129.18, 128.30,
127.97, 126.03, 120.44, 115.53, 114.03, 111.72, 68.89, 43.62.
4.2.4.9.
4-((3,5-Di-tert-butyl-2-((4-chlorobenzyl)oxy)benzyl)
amino)phenol (4j). The title compound was obtained as a white
solid, yield: 94.3%. 1H NMR (400 MHz, DMSO-d6) d 8.39 (s, 1H),
7.50–7.43 (m, 4H), 7.34 (d, J = 2.3 Hz, 1H), 7.20 (d, J = 2.4 Hz, 1H),
6.49 (d, J = 8.8 Hz, 2H), 6.44 (d, J = 8.9 Hz, 2H), 5.39 (s, 1H), 4.91
(s, 2H), 4.14 (d, J = 5.2 Hz, 2H), 1.37 (s, 9H), 1.23 (s, 9H). 13C NMR

2556
D. Gao et al. / Bioorg. Med. Chem. 24 (2016) 2549–2558
(150 MHz, CDCl3) d 153.42, 147.21, 145.82, 141.94, 141.67, 135.69,
132.73, 131.19, 128.00, 127.70, 124.85, 123.29, 115.52, 113.81,
74.35, 44.73, 34.90, 33.96, 30.89, 30.63. ESI-MS: calcd for [M+H]+
m/z: 452.2, found: 452.2. ESI-HRMS [M+H]+ calcd for C28H35ClNO2:
452.2351, found: 452.2354. HPLC: tR = 4.646 min.
(m, 5H), 7.18 (d, J = 7.9 Hz, 1H), 7.00 (d, J = 7.6 Hz, 2H), 6.83 (d,
J = 8.3 Hz, 1H), 6.53 (d, J = 8.1 Hz, 2H), 5.05 (s, 2H), 4.32 (s, 2H).
13C NMR (150 MHz, CDCl3) d 154.06, 145.87, 140.14, 134.30,
133.46, 128.65, 128.30, 127.98, 127.44, 125.63, 121.80, 112.72,
112.36, 69.02, 42.84. ESI-MS: calcd for [M+H]+ m/z: 442.1, found:
442.1. ESI-HRMS [M+H]+ calcd for C21H17Cl2F3NO2: 442.0583,
found: 442.0582. HPLC: tR = 5.414 min.
4.2.4.10. 4-((2-((4-Chlorobenzyl)oxy)-5-nitrobenzyl)amino)phe-
nol (4k). The title compound was obtained as a brown solid, yield:
90.3%. 1H NMR (400 MHz, DMSO-d6) d 8.46 (s, 1H), 8.14 (d,
J = 8.6 Hz, 1H), 8.13 (s, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.48 (d,
J = 8.1 Hz, 2H), 7.28 (d, J = 8.6 Hz, 1H), 6.52 (d, J = 8.3 Hz, 2H),
6.39 (d, J = 8.4 Hz, 2H), 5.72 (t, J = 4.8 Hz, 1H), 5.35 (s, 2H), 4.24
(d, J = 4.8 Hz, 2H). 13C NMR (150 MHz, CDCl3) d 160.26, 147.46,
141.20, 141.01, 133.85, 133.32, 129.09, 128.48, 128.03, 123.97,
123.73, 115.64, 113.97, 110.46, 69.38, 43.49. ESI-MS: calcd for
[M+H]+ m/z 385.1, found: 385.1. ESI-HRMS [M+H]+ calcd for
C20H18ClN2O4: 385.0950, found: 385.0954. HPLC: tR = 2.896 min.
4.2.4.16. N-(5-Chloro-2-((4-chlorobenzyl)oxy)benzyl)-3-(trifluo-
romethyl)aniline (4q). The title compound was obtained as a col-
orless oil, yield: 90.3%. 1H NMR (400 MHz, CDCl3) d 7.38–7.26 (m,
5H), 7.17 (d, J = 7.9 Hz, 1H), 7.04–6.90 (m, 2H), 6.88–6.64 (m,
2H), 6.52 (d, J = 7.5 Hz, 1H), 5.05 (s, 2H), 4.33 (s, 2H). 13C NMR
(150 MHz, CD3OD) d 154.17, 148.29, 146.98, 139.20, 135.15,
132.79, 129.38, 128.65, 128.08, 128.01, 127.67, 126.98, 126.61,
125.06, 121.12, 114.67, 112.50, 111.97, 68.62, 41.29, 40.82. ESI-
MS: calcd for [M+H]+ m/z: 426.1, found: 426.1. ESI-HRMS [M+H]+
calcd for C21H17Cl2F3NO: 426.0634, found: 426.0636. HPLC:
tR = 5.405 min.
4.2.4.11.
4-((5-Chloro-2-((4-chlorobenzyl)oxy)benzyl)amino)
benzenesulfonamide (4l). The title compound was obtained as a
white solid, yield: 83.6%. 1H NMR (400 MHz, DMSO-d6) d 7.53–
7.42 (m, 6H), 7.25 (dd, J = 8.7, 2.5 Hz, 1H), 7.15 (d, J = 2.5 Hz, 1H),
7.09 (d, J = 8.7 Hz, 1H), 6.94–6.87 (m, 3H), 6.58 (d, J = 8.7 Hz, 2H),
5.19 (s, 2H), 4.31 (d, J = 6.0 Hz, 2H). 13C NMR (150 MHz, CD3OD) d
154.18, 151.11, 135.15, 132.82, 128.96, 128.78, 128.10, 127.70,
126.96, 126.78, 126.73, 125.01, 112.57, 110.48, 68.64, 40.41. ESI-MS:
calcd for [M+H]+ m/z: 437.0, found: 437.0. ESI-HRMS [M+H]+ calcd
for C20H19Cl2N2O3S: 459.0307, found: 459.0311. HPLC: tR = 4.064 min.
4.2.4.17.
3-((5-Chloro-2-((4-chlorobenzyl)oxy)benzyl)amino)
benzonitrile (4r). The title compound was obtained as a white
solid, yield: 85.3%. 1H NMR (400 MHz, CDCl3) d 7.33 (d, J = 2.9 Hz,
4H), 7.24–7.14 (m, 3H), 6.95 (d, J = 6.8 Hz, 1H), 6.84 (d, J = 8.5 Hz,
1H), 6.75 (s, 2H), 5.06 (s, 2H), 4.33 (s, 2H). 13C NMR (150 MHz,
CDCl3) d 154.05, 147.30, 134.18, 133.54, 129.33, 128.35, 128.06,
127.76, 127.66, 125.65, 120.63, 118.76, 116.88, 114.58, 112.40.
ESI-MS: calcd for [M+H]+ m/z: 383.1, found: 383.1. ESI-HRMS [M
+H]+ calcd for C21H17Cl2N2O: 383.0712, found: 383.0712. HPLC:
tR = 5.060 min.
4.2.4.12. N-(5-Chloro-2-((4-chlorobenzyl)oxy)benzyl)-4-methoxy-
aniline (4m). The title compound was obtained as a white solid,
yield: 97.5%. 1H NMR (400 MHz, CDCl3) d 7.38–7.31 (m, 5H), 7.16
(dd, J = 8.7, 2.3 Hz, 1H), 6.82 (d, J = 8.7 Hz, 1H), 6.76 (d, J = 8.8 Hz,
2H), 6.56 (d, J = 8.8 Hz, 2H), 5.06 (s, 2H), 4.30 (s, 2H), 3.74 (s, 3H).
4.2.4.18. 4-((2-((4-Chlorobenzyl)oxy)-5-(thiophen-2-yl)benzyl)
amino)phenol (4s). The title compound was obtained as a white
solid, yield: 77.9%. 1H NMR (400 MHz, CDCl3) d 7.58 (d, J = 1.8 Hz,
1H), 7.45 (dd, J = 8.4, 2.1 Hz, 1H), 7.35 (s, 4H), 7.23–7.18 (m, 1H),
7.16 (d, J = 2.5 Hz, 1H), 7.03 (dd, J = 5.0, 3.6 Hz, 1H), 6.90 (d,
J = 8.5 Hz, 1H), 6.68 (d, J = 8.6 Hz, 2H), 6.56 (d, J = 8.6 Hz, 2H),
5.10 (s, 2H), 4.34 (s, 2H). 13C NMR (150 MHz, CDCl3) d 155.20,
147.45, 143.46, 134.66, 133.25, 128.24, 127.95, 127.33, 127.07,
126.52, 125.31, 123.45, 121.77, 115.55, 114.37, 111.49, 68.81,
44.36.ESI-MS: calcd for [M+H]+ m/z: 422.1, found: 422.1. ESI-HRMS
[M+H]+ calcd for C24H21ClNO2S: 422.0976, found: 422.0975. HPLC:
tR = 3.823 min.
13C NMR (150 MHz, CDCl3)
d 154.05, 151.71, 141.47, 134.43,
133.34, 129.58, 128.27, 128.15, 127.94, 127.11, 125.58, 114.27,
113.77, 112.26, 68.97, 55.16, 43.63. ESI-MS: calcd for [M+H]+ m/z:
388.1, found: 388.1. ESI-HRMS [M+H]+ calcd for C21H20Cl2NO2:
388.0866, found: 388.871. HPLC: tR = 3.792 min.
4.2.4.1N3-.(5-Chloro-2-((4-chlorobenzyl)oxy)benzyl)-4-ethoxyani-
line (4n). The title compound was obtained as a white solid, yield:
98.1%. 1H NMR (400 MHz, DMSO-d6) d 7.52–7.42 (m, 4H), 7.27–
7.18 (m, 2H), 7.06 (d, J = 8.9 Hz, 1H), 6.65 (d, J = 8.8 Hz, 2H), 6.43
(d, J = 8.8 Hz, 2H), 5.75 (t, J = 6.2 Hz, 1H), 5.17 (s, 2H), 4.18 (d,
J = 6.1 Hz, 2H), 3.83 (q, J = 6.9 Hz, 2H), 1.23 (t, J = 7.0 Hz, 3H). 13C
NMR (150 MHz, CDCl3) d 154.04, 150.98, 141.47, 134.45, 133.33,
129.64, 128.26, 128.15, 127.93, 127.10, 125.58, 115.12, 113.74,
112.25, 68.96, 63.45, 43.62, 14.40. ESI-MS: calcd for [M+H]+ m/z:
402.1, found: 402.1. ESI-HRMS [M+H]+ calcd for C22H22Cl2NO2:
402.1022, found: 402.1027. HPLC: tR = 3.986 min.
4.2.5. Synthesis of 4-chloro-N-(4-chloro-2-(((4-hydroxyphenyl)
amino)methyl)phenyl)benzamide (8t)
4.2.5.1. Methyl 5-chloro-2-(4-chlorobenzamido)benzoate (5t). To a
solution of methyl 2-amino-5-chlorobenzoate (1.0 g, 5.39 mmol) in
dry CH2Cl2 (25 mL) was added 4-chlorobenzoyl chloride (826 lL,
6.47 mmol). Then the K2CO3 (1.86 g, 13.47 mmol) were added.
Upon completion, the solvent was evaporated under reduce pres-
sure. Water and ethyl acetate were added and the mixture was
extracted with ethyl acetate. The combined extracts were washed
with water and brine, dried over Na2SO4. After filtration, the filtrate
was removed in vacuo. The residue was purified by silica gel col-
umn chromatography to give 5t as a white solid (1.40 g, 80.1%).
1H NMR (400 MHz, DMSO-d6) d 11.40 (s, 1H), 8.30 (s, 1H), 7.96
(d, J = 8.5 Hz, 2H), 7.83–7.55 (m, 4H), 3.82 (s, 3H). ESI-MS: calcd
for [M+H]+ m/z: 324.0, found: 324.0.
4.2.4.14. N-(5-Chloro-2-((4-chlorobenzyl)oxy)benzyl)-3,5-dimethyl
aniline (4o). The title compound was obtained as a colorless oil,
yield: 90.5%. 1H NMR (400 MHz, CDCl3) d 7.39–7.28 (m, 5H), 7.19–
7.12 (m, 1H), 6.81 (d, J = 8.7 Hz, 1H), 6.38 (s, 1H), 6.24 (s, 2H), 5.05
(s, 2H), 4.32 (s, 2H), 2.21 (s, 6H). 13C NMR (150 MHz, CDCl3) d
154.03, 147.40, 138.31, 134.48, 133.32, 129.59, 128.27, 128.11,
127.94, 127.13, 125.60, 119.25, 112.23, 110.40, 68.95, 42.71, 20.90.
ESI-MS: calcd for [M+H]+ m/z: 386.1, found: 386.1. ESI-HRMS [M
+H]+ calcd for C22H21Cl2NO: 408.0892, found: 408.0889. HPLC:
tR = 4.808 min.
4.2.5.2. 4-Chloro-N-(4-chloro-2-(hydroxymethyl)phenyl)benza-
mide (6t). To a solution of 5t (400 mg, 1.23 mmol) in anhydrous
THF (10 mL) was added LiAlH4 (93.6 mg, 2.47 mmol) slowly at
0 °C. Stirring was continued for another 15 min before treating
with ice water dropwise. The resulting suspension was filtered
and washed thoroughly with ethyl acetate. The combined organic
4.2.4.15. N-(5-Chloro-2-((4-chlorobenzyl)oxy)benzyl)-4-(trifluo-
romethoxy)aniline (4p). The title compound was obtained as a
colorless oil, yield: 91.8%. 1H NMR (400 MHz, CDCl3) d 7.38–7.26

D. Gao et al. / Bioorg. Med. Chem. 24 (2016) 2549–2558
2557
fractions were dried over Na2SO4. After filtration, the filtrate was
removed in vacuo. The residue was purified by silica gel column
chromatography to give 6t as a white solid (310 mg, 84.8%). 1H
NMR (400 MHz, DMSO-d6) d 10.13 (s, 1H), 7.93 (d, J = 8.1 Hz, 2H),
7.60 (d, J = 8.2 Hz, 3H), 7.48 (s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 5.60
(s, 1H), 4.54 (s, 2H). ESI-MS: calcd for [M+H]+ m/z 296.0, found:
296.0.
7.60 (d, J = 2.5 Hz, 1H), 7.56–7.48 (m, 3H), 7.42 (d, J = 8.3 Hz, 2H),
7.35 (d, J = 8.7 Hz, 2H), 7.24 (d, J = 8.9 Hz, 1H), 6.68 (d, J = 8.7 Hz,
2H), 5.20 (s, 2H). 13C NMR (150 MHz, DMSO-d6) d 162.05, 154.03,
153.52, 135.24, 132.57, 130.93, 130.28, 129.52, 128.88, 128.32,
127.17, 124.54, 120.97, 115.18, 114.93, 69.41. ESI-MS: calcd for
[M+H]+ m/z 388.0, found: 388.0. ESI-HRMS [M+H]+ calcd for
C20H16Cl2NO3: 388.0502, found: 388.0511. HPLC: tR = 4.180 min.
4.2.5.3. 4-Chloro-N-(4-chloro-2-formylphenyl)benzamide (7t). To
a solution of 6t (200 mg, 0.68 mmol) in anhydrous CH2Cl2 (10 mL)
was treated with MnO2 (177 mg, 2.04 mmol) slowly. Stirring was
continued for another 3 h under reflux. The resulting suspension
was filtered and washed thoroughly with CH2Cl2. The combined
organic fractions were dried over Na2SO4, filtered and concentrated
under reduced pressure to give the crude compound 7t. The purity
of 7t was quite sufficient to use for the following synthesis.
4.2.7. Synthesis of 16v and 16w
4.2.7.1. 3-Nitroquinoline-2,4-diol (11v). Quinoline-2,4-diol (5 g,
31.0 mmol) was dissolved in 30 mL of nitric acid and stirred at
room temperature for 10 min, followed by heating the reaction
mixture at 75 °C for another 15 min. The reaction mixture was then
allowed to cool down to room temperature and was added to ice-
water mixture to precipitate the product. The yellow precipitate
was filtered and dried. The crude product was used directly for
the next step without further purification.
4.2.5.4. 4-Chloro-N-(4-chloro-2-(((4-hydroxyphenyl)amino)methyl)
phenyl)benzamide (8t).
Following the preparation protocol
4.2.7.2. 2,4-Dichloro-3-nitroquinoline (12v). Compound 11v (3 g,
14.5 mmol) was dissolved in 20 mL of phenylphosphonyl dichlo-
ride and heated at 135 °C for 3 h. The reaction mixture was poured
into ice-water and stirred vigorously to obtain the precipitate,
which was filtered and dried to afford the compound 12v (1.90 g,
54.0%).1H NMR (400 MHz, CDCl3) d 8.27 (d, J = 8.1 Hz, 1H), 8.11
(d, J = 8.1 Hz, 1H), 7.95 (t, J = 7.2 Hz, 1H), 7.81 (t, J = 7.2 Hz, 1H).
ESI-MS: calcd for [M+H]+ m/z 243.0, found: 243.0.
of compounds 4a–s. To a stirring solution of 7t and catalytic
amount acetic acid (1 drop) in anhydrous CH3OH (25 mL) was
added 4-aminophenol (15.0 mg, 0.14 mmol). After the solution
was stirred at room temperature for 1 h, NaBH3CN (13.0 mg,
0.27 mmol) was added and stirred for another 2 h. After removing
the methanol, water and ethyl acetate were added and then the
organic was washed with saturated sodium bicarbonate, brine,
dried over Na2SO4. After filtration, the filtrate was removed in
vacuo. The residue was purified by silica gel column chromatogra-
phy to give 8t as a white solid (25.0 mg, 47.5%). 1H NMR (400 MHz,
DMSO-d6) d 10.43 (s, 1H), 8.50 (s, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.57
(d, J = 8.4 Hz, 3H), 7.39 (s, 1H), 7.32 (d, J = 8.2 Hz, 1H), 6.51 (d,
J = 8.7 Hz, 2H), 6.41 (d, J = 8.7 Hz, 2H), 5.73 (t, J = 5.2 Hz, 1H), 4.18
4.2.7.3. 2-Chloro-3-nitroquinolin-4-amine (13v). To a solution of
12v (200 mg, 0.82 mmol) in CH3CN (10.0 mL) was added 28%
NH3(aq) (350 lL, 6.59 mmol) at rt and then the mixture was stirred
at 50 °C for 6.5 h. Upon completion, water and ethyl acetate were
added and then the mixture was extracted with ethyl acetate
twice. The combined extracts were washed with water and brine,
dried over Na2SO4. After filtration, the filtrate was removed in
vacuo. The residue was purified by silica gel column chromatogra-
phy to give 13v (160 mg, 87.0%) as a yellow solid. 1H NMR
(400 MHz, DMSO-d6) d 8.42 (d, J = 8.4 Hz, 1H), 8.15 (s, 2H), 7.83–
7.74 (m, 2H), 7.59 (t, J = 7.5 Hz, 1H). ESI-MS: calcd for [M+H]+ m/z
224.0, found: 224.0.
(d, J = 5.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6)
148.85, 140.60, 136.80, 136.47, 134.82, 132.76, 129.68, 129.31,
128.45, 127.13, 126.77, 126.53, 115.56, 113.93, 44.31. ESI-MS:
calcd for [M+H]+ m/z: 387.1, found: 386.8. ESI-HRMS [M+H]+ calcd
d 164.12,
for
C20H17Cl2N2O2: 387.0662, found: 387.0668. HPLC:
tR = 2.942 min.
4.2.6. Synthesis of 5-chloro-2-((4-chlorobenzyl)oxy)-N-(4-hydroxy-
phenyl)benzamide (10u)
4.2.7.4. 2-Chloroquinoline-3,4-diamine (14v). To a solution of
13v (145 mg, 0.65 mmol) in CH3CH2OH/H2O (5 mL/5 mL) was
added Fe powder (109 mg, 1.94 mmol) and NH4Cl (104 mg,
1.94 mmol). Then the mixture was stirred at 75 °C for 2 h. After
removing the ethanol, ethyl acetate was added. The mixture was
extracted with ethyl acetate twice. The combined extracts were
washed with water and brine, dried over Na2SO4. After filtration,
the filtrate was removed in vacuo. The residue was purified by sil-
ica gel column chromatography to give 14v (119 mg, 94.7%). 1H
NMR (400 MHz, DMSO-d6) d 8.00 (d, J = 8.3 Hz, 1H), 7.56 (d,
J = 8.3 Hz, 1H), 7.36 (t, J = 7.4 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 6.31
(s, 2H), 4.76 (s, 2H). ESI-MS: calcd for [M+H]+ m/z 194.0, found:
194.0.
4.2.6.1. 5-Chloro-2-((4-chlorobenzyl)oxy)benzoic acid (9u). To a
solution of 2d (50 mg, 0.18 mmol) in t-BuOH (5 mL) and 2-methyl-
2-butene (2.5 mL) were added NaClO2 (96.6 mg, 1.07 mmol) and
NaH2PO4ꢀ2H2O (83.3 mg, 0.53 mmol) buffer solution (1.07 mL) at
0 °C. Then the mixture was stirred at room temperature. Upon com-
pletion, the solution was extracted with ethyl acetate and the organic
layer was dried over Na2SO4 and concentrated in vacuo. The crude
compound was purified by silica gel column chromatography to give
9u (50.2 mg, 95.0%). 1H NMR (400 MHz, DMSO-d6) d 7.62 (d,
J = 2.6 Hz, 1H), 7.52 (dd, J = 8.9, 2.6 Hz, 1H), 7.50–7.42 (m, 4H), 7.19
(d, J = 8.9 Hz, 1H), 5.18 (s, 2H). ESI-MS: calcd for [MꢁH]ꢁ m/z 295.0,
found: 295.0.
4.2.6.2. 5-Chloro-2-((4-chlorobenzyl)oxy)-N-(4-hydroxyphenyl)
benzamide (10u). To a solution of 9u (50.0 mg, 0.17 mmol) in
dry THF (10 mL) was added HATU (96.0 mg, 0.25 mmol), HOAT
4.2.7.5. 4-Chloro-2-(4-(methylsulfonyl)phenyl)-1H-imidazo[4,5-
c]quinoline (15v). A mixture of 14v (30.0 mg, 0.16 mmol), 4-
(methylsulfonyl)benzaldehyde (43.0 mg, 0.23 mmol), and sodium
pyrosulfate (52.0 mg, 0.23 mmol) in anhydrous THF (5.0 mL) was
stirred at 67 °C for 4 h. After removing the THF, ethyl acetate and
water were added. The mixture was extracted with ethyl acetate
twice. The combined extracts were washed with water and brine,
dried over Na2SO4. After filtration, the solvent was removed in
vacuo. The crude product was used directly for the next step with-
out further purification.
(34.5 mg, 0.25 mmol), DIPEA (89.0 lL, 0.50 mmol) at 0 °C. After
stirred at room temperature for10 min, 4-aminophenol (18.6 mg,
0.17 mmol) was added. Upon completion, the solvent was evapo-
rated under reduce pressure. The residue was dissolved in ethyl
acetate and washed with water and brine, dried over Na2SO4 and
concentrated in vacuo. The crude compound was purified by silica
gel column chromatography to give 10u as a white solid (55.4 mg,
84.8%). 1H NMR (400 MHz, DMSO-d6) d 9.97 (s, 1H), 9.28 (s, 1H),

2558
D. Gao et al. / Bioorg. Med. Chem. 24 (2016) 2549–2558
4.2.7.6. 4-(4-Chloro-1H-imidazo[4,5-c]quinolin-2-yl)phenol
(15w). Following the preparation protocol of compounds 15v.
Starting from 4-hydroxybenzaldehyde (47.0 mg, 0.39 mmol), 14v
(50 mg, 0.26 mmol) and sodium pyrosulfate (86.0 mg, 0.39 mmol),
the compound 15w was obtained (60.0 mg, 65.7%). ESI-MS: calcd
for [M+H]+ m/z 296.1, found: 296.0.
Acknowledgment
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 81473075).
Supplementary data
4.2.7.7. 2-(4-(Methylsulfonyl)phenyl)-4-phenyl-1H-imidazo[4,5-
c]quinoline (16v). A mixture of 15v (50.0 mg, 0.14 mmol), phenyl-
boronic acid (25.6 mg, 0.21 mmol), Pd(PPh3)4 (16.2 mg, 0.01 mmol)
and Cs2CO3 (137 mg, 0.42 mmol) in 4.0 mL Dioxane/Water
(v/v = 3:1) was stirred at 120 °C by microwave for 2 h, then cooled
to room temperature. After removing the dioxane, water and ethyl
acetate were added and then the mixture was extracted with ethyl
acetate twice. The combined extracts were washed with brine,
dried over Na2SO4. After filtration, the filtrate was removed in
vacuo. The residue was purified by silica gel column chromatogra-
phy to give 16v (42.2 mg, 89.6%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) d 8.91 (d, J = 7.4 Hz, 2H), 8.56 (d, J = 8.1 Hz,
2H), 8.50 (d, J = 7.5 Hz, 1H), 8.22–8.13 (m, 3H), 7.77–7.66 (m,
2H), 7.61 (t, J = 7.5 Hz, 2H), 7.57–7.50 (m, 1H), 3.30 (s, 3H). 13C
NMR (150 MHz, DMSO-d6) d 149.20, 149.06, 143.30, 141.53,
137.48, 136.67, 135.48, 133.98, 129.66, 129.45, 129.33, 128.15,
127.85, 127.73, 127.27, 126.14, 121.60, 116.75, 43.33. ESI-MS:
calcd for [M+H]+ m/z 400.1, found: 400.1. ESI-HRMS [M+H]+ calcd
for C23H18N3O2S: 400.1114, found: 400.1117. HPLC: tR = 2.623 min.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.04.022.
References and notes
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4.2.7.8.
4-(4-Phenyl-1H-imidazo[4,5-c]quinolin-2-yl)phenol
(16w). Following the preparation protocol of compound 16v.
Starting from 15w (30.0 mg, 0.16 mmol), the compound 16w was
obtained (57.0 mg, 100%) as a yellow solid. 1H NMR (400 MHz,
DMSO-d6) d 13.71 (s, 1H), 10.06 (s, 1H), 8.94 (d, J = 7.3 Hz, 2H),
8.51–8.42 (m, 1H), 8.16 (d, J = 8.6 Hz, 2H), 8.14–8.09 (m, 1H),
7.70–7.62 (m, 2H), 7.58 (t, J = 7.4 Hz, 2H), 7.50 (t, J = 7.1 Hz, 1H),
6.99 (d, J = 8.6 Hz, 2H). 13C NMR (150 MHz, CDCl3) d 164.06,
156.18, 153.17, 147.63, 142.54, 141.00, 140.22, 134.27, 134.15,
133.94, 133.16, 132.78, 131.89, 130.47, 126.16, 125.21, 121.47,
120.44. ESI-MS: calcd for [M+H]+ m/z 338.1, found: 338.1. ESI-
HRMS [M+H]+ calcd for C22H16N3O: 338.1288, found: 338.1286.
HPLC: tR = 2.944 min.