Design, synthesis, and evaluation of cyanopyridines as anti-colorectal cancer agents via inhibiting STAT3 pathway

Article abstract of DOI:10.2147/DDDT.S217800

Background: Colorectal cancer is one of the common malignant tumors. Cyanopyridine and aminocyanopyridine having a carbon-nitrogen bond have been shown to have significant anticancer effects. STAT3 is a promising therapeutic target in multiple cancers. Ho

Full text of DOI:10.2147/DDDT.S217800

Drug Design, Development and Therapy
Dovepress
open access to scientific and medical research
Open Access Full Text Article
O R I G I N A L R E S E A R C H
Design, Synthesis, And Evaluation Of
Cyanopyridines As Anti-Colorectal Cancer
Agents Via Inhibiting STAT3 Pathway
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
Lingyuan Xu,1–3,* Lingxi
Background: Colorectal cancer is one of the common malignant tumors. Cyanopyridine
Shi,1–3,* Sensen Qiu,4,* Siyu
and aminocyanopyridine having a carbon-nitrogen bond have been shown to have significant
Chen,1–3 Mengsha Lin,1–3
anticancer effects. STAT3 is a promising therapeutic target in multiple cancers. However,
Youqun Xiang,1 Chengguang
there are currently no effective STAT3 inhibitors in clinical practice for the treatment of
colorectal cancer.
3
Zhao,
Jiandong Zhu,2
Materials and methods: We screened 27 cyanopyridines for their anticancer activity by
cell viability. The HCT-116, RKO, and DLD-1 cell lines were used to evaluate the anti-
colorectal cancer effect of 3n. Scratch experiments and colony formation assays were used
for the assessment of cell migration and proliferation capacity. Phosphorylated STAT3,
STAT3, MCL-1, and Survivin levels were assessed by Western blot analysis.
Results: In this study, we synthesized 27 cyanopyridines and screened their anticancer
activities in three human tumor cells, HCT-116, Hela229, and A375. We found that 2-
amino-3-cyanopyridine 3n has better anticancer activity with IC50 values in the low micro-
molar range. Furthermore, 3n significantly inhibited the migration and colony formation of
colorectal cancer cells. Mechanistically, 3n inhibited the expression of STAT3 phosphoryla-
tion in a dose- and time-dependent manner.
Liqun Shen,4 Zhigui Zuo1,2
1Department of Colorectal Surgery, The
First Affiliated Hospital, Wenzhou
Medical University, Wenzhou, Zhejiang
325000, People’s Republic of China;
2Department of Surgical Oncology,
Affiliated Yueqing Hospital, Wenzhou
Medical University, Wenzhou, Zhejiang
325600, People’s Republic of China;
3Cancer and Anticancer Drug Research
Center, School of Pharmaceutical
Sciences, Wenzhou Medical University,
Wenzhou, Zhejiang 325035, People’s
Republic of China; 4Department of
Pharmaceutical Sciences, College of
Chemistry and Chemical Engineering,
Guangxi University for Nationalities,
Nanning 530006, People’s Republic of
China
Conclusion: 3n is worth of further investigations toward the discovery of STAT3 inhibitor
as a drug candidate for cancer therapy.
Keywords: design, cyanopyridine, colorectal cancer, STAT3, inhibitor
*These authors contributed equally to
this work
Introduction
Colorectal cancer is one of the common malignant tumors in our digestive system,
and the morbidity and mortality rate are increasing year by year.1–3 At present, the
main treatment for colorectal cancer is surgical treatment, combined with radio-
therapy and chemotherapy, but surgery alone may not be cured.4 Drug treatment of
colorectal cancer often has drug resistance, leading to a reduction in colorectal cancer
survival rate for cancer treatment.5 Signal transduction and transcriptional activator
(STAT3) is defined as an essential oncogene that regulates cancer cell proliferation,
apoptosis, and metastasis.6,7 To a large extent, STAT3 is considered to be one of the
key oncogenes and a key therapeutic target, so inhibition of aberrant activation of
STAT3 in colorectal cancer may be a promising strategy.5,8,9 However, to date there
have been no clinically useful STAT3 inhibitors for the treatment of cancer.1,10,11
There are many reports on the anti-tumor activity of 2-amino-3-cyanopyridines,
but the results are not satisfactory.12 So far, none of these compounds have entered
Correspondence: Liqun Shen
Department of Pharmaceutical Sciences,
College of Chemistry and Chemical
Engineering, Guangxi University for
Nationalities, Nanning 530006, People’s
Republic of China
Tel +86 771 3267019
Email liqunshen@126.com
Zhigui Zuo
Department of Colorectal Surgery, The
First Affiliated Hospital, Wenzhou Medical
University, Wenzhou, Zhejiang 325000,
People’s Republic of China
Tel +86 577 86699057
Email zuozg007@126.com
submit your manuscript | www.dovepress.com
DovePress
Drug Design, Development and Therapy 2019:13 3369–3381
3369
© 2019 Xu et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php
and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work
you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For
permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
http://doi.org/10.2147/DDDT.S217800

Xu et al
Dovepress
clinical research, and the structure–activity relationship of spectrometer MAGNA-IR 550 (Thermo Electron
the system has not been given.13–15 There are relatively Corporation, MMAS, USA) using KBr (υ cm−1). High
few reports on 3-amino-3-cyanopyridine compounds com- resolution mass spectrometry (HRMS) were determined
pared to 2-amino-3-cyanopyridine compounds, and there on LC-MS QE Focus (Thermo Fisher Scientific, MMAS,
are few studies on the anti-tumor activity of such USA). Column chromatography was performed on silica
compounds.16 Our group has previously demonstrated gel (200–300 mesh) from Qingdao Ocean Chemicals
that the aminocyanopyridines exhibit significant antitumor (Qingdao, Shandong, People’s Republic of China) using
activity against lung cancers in vitro and in vivo.17 So we distilled petroleum ether and ethyl acetate as mobile
continued our previous studies to further design, synthe- phase. Analytical HPLC analyses were performed on
size and evaluate the in vitro anticancer activity of a series Agilent 1260 liquid chromatograph fitted with an Inertex-
of aminocyanopyridines.
C18 column, with the purity of compounds >95%. All the
materials were obtained from commercially available
sources and used without further purification, unless other-
wise specified. Yields were not optimized.
Materials And Methods
Antibodies And Reagents
The antibodies against P-STAT3, STAT3, and GAPDH were
purchased from Cell Signal Technology (Danvers, MA,
USA). The horseradish peroxidase (HRP)-conjugated don-
key anti-rabbit IgG and HRP-conjugated goat anti-mouse
IgG were purchased from Santa Cruz Biotechnology Inc.
(Dallas, TX, USA). Methylthiazolyldiphenyl-tetrazolium
bromide (MTT) and dimethyl sulfoxide (DMSO) were pur-
chased from Sigma-Aldrich Co. (St Louis, MO, USA). The
caspase-3 colorimetric assay kit was purchased from
Abcam Co. (Cambridge, MA, USA). A Bradford protein-
assay kit, polyvinylidene fluoride membrane, and enhanced
chemiluminescence kit were obtained from Bio-Rad
Laboratories (Hercules, CA, USA). A protease phospha-
tase-inhibitor mixture was obtained from Applygen
Technologies (Beijing, People’s Republic of China).
Acrylamide (30%), coomassie brilliant blue, tetramethy-
lethylenediamine, tris-glycine, sodium dodecyl sulfate,
prestained protein marker, and nonfat dry milk were from
Bio-Rad Laboratories.
Cell Culture
Cell culture and human colorectal cell lines HCT-116,
RKO, and DLD-1 were purchased from Shanghai cell
bank. All of the above cells were cultured in Roswell
Park Memorial Institute (RPMI)-1640 media (Thermo
Fisher Scientific, Waltham, MA, USA), containing 10%
FBS (Gibco, NY, USA) and were maintained at 37°C in a
humidified chamber with 5% CO2.
Cell Viability Assay In Vitro
Approximately 1×103 cells, suspended in 1640 medium,
were plated onto each well of a 96-well plate and incu-
bated in 5% CO2 at 37°C for 24 hrs. The test compounds
at indicated final concentrations were added to the culture
medium and the cell cultures were continued for 72 hrs.
Fresh MTT was added to each well at a terminal concen-
tration of 5 mg/mL and incubated with cells at 37°C for 4
hrs. The formazan crystals were dissolved in 150 mL
DMSO each well, and the absorbency at 490 nm (for
absorbance of MTT formazan) and 630 nm (for the refer-
ence wavelength) was measured with the ELISA reader.
All the compounds were tested three times in each of the
cell lines. The results expressed as IC50 (inhibitory con-
centration 50%) were the averages of three determinations
and calculated by using the GraphPad Prism 6 software.
Chemistry
Reactions were monitored by thin-layer chromatography on
silica gel plates (HSGF 254), visualizing with ultraviolet.
All the melting points are obtained uncorrected and were
taken in a Microscope Melting Point X-6 (Beijing Tech,
1
Beijing, People’s Republic of China). H NMR and 13C
NMR spectroscopy were determined in Bruker ACANCE
III HD-400 and 100 spectrometers (Bruker, Karlsruhe, Colony Formation Assay
Germany), respectively. Using DMSO-d6, acetone and The untreated human colorectal cancer cells were plated in
CDCl3 solution with TMS as an internal standard. The 6 well plates at a density of 1000 cells per well. After 7–10
following abbreviations mean spin multiplicities: singlet days, the cells were fixed with 4% paraformaldehyde for
(S), doublet (D), triplet (T), and multiplet (M). IR spectra 10–15 mins, colonies were stained with crystal violet for
were recorded on a Fourier transform infrared (FTIR) 15 mins at room temperature.
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3370
DovePress

Dovepress
Xu et al
Cell Migration Assay
2-Amino-6-(4-hydrophenyl)-4-(4-
bromophenyl) nicotinonitrile (3a)
RKO and DLD-1 cells were seeded into 6 well plates at a
density of 2×105 cells per well and cultured to 90% con-
fluence. Then, the culture area was scratched with a sterile
crystal pipette tip to make a linear gap in the confluent cell
monolayer. The suspended cells were washed gently with
PBS. Finally, cells were observed under an inverted micro-
scope, cells were allowed to fill the gap and images of the
culture area were captured at 0 hr, 24 hrs and 48 hrs after
scratches. Three independent assays were performed.
White floccule. mp: 304–307°C. 1H NMR (400 MHz, DMSO,
δ ppm): 12.91 (s, 1H), 7.71–7.86 (m, 8H), 6.90 (s, 1H). 13C
NMR (100MHz, DMSO-d6, δ ppm): 154.03, 149.28, 143.97,
136.83, 133.05, 132.15, 131.24, 123.73, 119.50, 116.15,
115.77, 105.10, 96.30, 95.25. IR (KBr) σ/cm−1: 3456.77,
3349.12, 2205.78, 1620.41, 1594.70, 1574.30, 1545.15,
1519.76, 1492.04, 1456.67, 1363.68, 1231.16, 1172.41,
805.80. HR-ESI-MS (pos. ion mode): m/z=366.0061[M+H]+
(anal. calcd. for C18H12BrN3O=365.0164).
Western Blot Analysis
The colorectal cells were lysed with RIPA lysis buffer with
protease inhibitor (Boster, Wuhan, People’s Republic of
China) and the protein concentration was determined after
centrifugation at 12,000 rpm at 4°C for 10 mins.
Supernatants collected, in order to determine total protein
concentration, and protein concentrations assessed using a
Bradford protein assay. The PVDF membrane was used to
transfer proteins, and the blots were blocked for 90 mins at
room temperature with fresh 5% nonfat milk in TBST,
then the membrane with the specific primary antibody
was incubated at 4°C overnight and at room temperature
for 60 mins with the secondary antibody. Finally, the
ImageJ software were used to quantify the bands.
2-Amino-4, 6-Bis-(4-bromophenyl)
nicotinonitrile (3b)
White cottony. mp: 279–280°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 8.11 (m, 1H), 8.08 (m, 1H), 7.78 (d,
J=2.0 Hz, 1H), 7.76 (m, 1H), 7.72 (m, 1H), 7.69 (m, 1H),
7.66 (m, 1H), 7.64 (d, J=2.0 Hz, 1H), 7.32 (s, 1H, cyano-
pyridine), 7.11 (s, 2H, amidogen). 13C NMR (101 MHz,
DMSO-d6, δ ppm): 161.25, 157.95, 154.37, 137.09,
136.50, 132.17, 132.11, 131.03, 129.77, 124.40, 123.77,
109.47, 87.25. IR (KBr) σ/cm−1: 3494.14, 3389.24,
2205.50, 1607.41, 1580.65, 1543.00, 1489.62, 1454.13,
1392.56, 1431.69, 1364.77, 1073.69, 1009.54, 818.44.
HR-ESI-MS (pos. ion mode): m/z=429.9371[M+H]+
(anal. calcd. for C18H11Br2N3=428.9299).
Statistical Analysis
Statistics were performed using GraphPad Prism software
(GraphPad, San Diego, CA, USA). The differences
between sets of data were made using the Student's t-
test. The results are expressed as mean SE. P<0.05 was
statistically significant.
2-Amino-6-(5-fluoro-2-benzyloxy)-4-(4-
bromophenyl) nicotinonitrile (3c)
Belge power. mp: 235–237°C. 1H NMR (400MHz,
DMSO-d6, δ ppm): 7.71–7.72 (m, 1H), 7.69–7.70 (q, J
=2.2 Hz, 1H), 7.64–7.65 (m, 1H), 7.42–7.44 (dd, J =7.7,
1.5 Hz, 2H), 7.26–7.38 (m, 8H), 7.05 (s, 2H), 5.15 (s, 2H,
benzyloxy). 13C NMR (100MHz, DMSO-d6, δ ppm):
161.07, 156.49, 153.52, 153.01, 136.83, 136.49, 132.16,
130.56, 128.94, 128.69, 128.57, 123.58, 117.68, 117.18,
117.13, 115.47, 114.09, 86.74, 71.30. IR (KBr) σ/cm−1:
3470.51, 3319.99, 2919.08, 2206.72, 1648.36, 1572.28,
1544.44, 1501.00, 1452.65, 1426.09, 1253.56, 823.97,
727.52. HR-ESI-MS (pos. ion mode): m/z=474.0619[M
+H]+ (anal. calcd. for C25H17BrFN3O =473.0539).
Results
General Procedure For The Compounds
3a-3o (Scheme 1)
A mixture of the substituted acetophenone (1 mmol),
together with the respective benzaldehyde (1 mmol), mal-
ononitrile (1 mmol), and ammonium acetate (8 mmol) in
toluene (10 mL) was stirred at reflux for 8–12 hrs. After
cooling to room temperature, the mixture was diluted ethyl
acetate and THF. The organic phase was washed twice
with brine, dried over Na2SO4, the filtrate and concen-
trated under reduced pressure. The residue was suspended
2-Amino-6-(2, 4-bisbenzyloxy)-4-(4-
in absolute ethanol. The precipitate was collected by filtra- bromophenyl) nicotinonitrile (3d)
tion and purified by silica gel column chromatography to White cottony power. mp: 170–171°C. 1H NMR (400 MHz,
give the desired product.
DMSO-d6, δ ppm): 7.92 (d, J=8.6Hz, 1H), 7.64 (d, J=7.7
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3371
DovePress

Xu et al
Dovepress
Scheme 1 The designed route of the 2-amino-cyanopyridines and the chemical structure of the compound 3a-n.
Hz, 2H), 7.49–7.36 (m, 10H), 7.25 (d, J=9.0Hz, 3H), 6.93 131.22, 123.73, 119.51, 116.14, 115.76, 96.34, 95.28. IR
(s, 3H), 6.79 (d, J=8.7Hz, 1H, cyanopyridine), 5.18 (d, (KBr) σ/cm−1: 3458.51, 3360.33, 3238.35, 2217.85,
J=16.3Hz, 4H, benzyloxy). 13C NMR (100 MHz, DMSO- 1643.64, 1593.06, 1512.73, 1286.61, 1102.15, 813.55.
d6, δ ppm): 161.53, 161.05, 158.44, 157.73, 152.50, 137.19, HR-ESI-MS (pos. ion mode): m/z=386.0102[M
136.84, 136.77, 132.43, 132.10, 130.52, 128.97, 128.75, +H]+(anal. calcd. for C18H10BrF2N3: 385.0026).
128.58, 128.47, 128.37, 123.38, 120.00, 113.70, 107.26,
100.96, 85.26, 70.74, 70.00. IR (KBr) σ/cm−1: 3453.09,
2-Amino-6-(4-hydroxyphenyl)-4-(3,
3289.25, 3153.39, 2205.43, 1637.18, 1603.08, 1573.97,
4-dimethoxyphenyl) nicotinonitrile (3f)
1544.55, 1506.81, 1492.87, 1452.29, 1302.11, 1257.08,
Yellow power. mp: 155–156°C. 1H NMR (400 MHz,
1179.88, 1132.98, 1026.70, 822.96, 737.94, 694.70. HR-
ESI-MS (pos. ion mode): m/z=564.1107 [M+H]+ (anal.
DMSO-d6, δ ppm): 9.91 (s, 1H), 8.01 (d, J=8.5 Hz,
2H), 7.28–7.08 (m, 4H), 6.93–6.73 (m, 4H), 3.84 (d,
J=5.5 Hz, 6H, methoxy).13C NMR (100 MHz, DMSO-
calcd. for C32H24BrN3O2: 563.1031 (97.3%)).
d6, δ ppm): 161.35, 159.96, 158.90, 154.75, 150.39,
2-Amino-6-(2, 4-bisfluoro)-4-(4-
bromophenyl) nicotinonitrile (3e)
149.07, 129.88, 129.42, 128.92, 121.46, 115.83, 112.46,
112.12, 108.51, 85.63, 56.11. IR (KBr) σ/cm−1: 3465.71,
Belge power. mp: 235–238°C. 1H NMR (400 MHz, 2923.19, 2201.05, 1640.74, 1610.51, 1575.25, 1546.32,
DMSO-d6, δ ppm): 7.76–7.47 (m, 6H), 7.30~7.26 1513.00, 1260.11, 1207.93, 1207.93, 1170.39, 1130.99,
(m, 1H), 6.99 (d, J=23.6 Hz, 2H), 6.82 (s, 1H, 1021.82, 835.97. HR-ESI-MS (pos. ion mode): m/
cyanopyridine).13C NMR (100 MHz, DMSO-d6, z=346.1202 [M-H]+ (anal. calcd. for C20H17N3O3:
δ ppm): 154.03, 149.26, 143.96, 136.83, 133.00, 132.15, 347.1270).
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3372
DovePress

Dovepress
Xu et al
HR-ESI-MS (pos. ion mode): m/z=544.2241[M
+H]+(anal. calcd. for C34H29N3O4: 543.2158).
2-Amino-6-(4-bromophenyl)-4-(3,
4-dimethoxyphenyl) nicotinonitrile (3g)
Brown power. mp: 125–127°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 8.10 (d, J=6.9 Hz, 2H), 7.71 (d,
J=6.9 Hz, 2H), 7.30 (d, J=16.3 Hz, 3H), 7.14 (s, 1H,
cyanopyridine), 7.02 (s, 2H), 3.79 (d, J=38.2 Hz, 6H,
methoxy).13C NMR (100 MHz, DMSO-d6, δ ppm):
161.38, 157.59, 155.34, 150.54, 149.08, 137.30, 132.06,
129.75, 129.50, 124.20, 121.61, 117.78, 112.51, 112.11,
109.48, 87.31, 56.13, 56.11. IR (KBr) σ/cm−1: 3461.49,
2920.00, 2200.00, 1607.93, 1506.78, 1256.78, 1173.55,
1024.79, 837.36. HR-ESI-MS (pos. ion mode): m/
z=410.0516 [M+H]+ (anal. calcd. for C20H16BrN3O2:
409.0426).
2-Amino-6-(2, 4-difluorophenyl)-4-(3,
4-dimethoxyphenyl) nicotinonitrile (3j)
White power. mp: 217–220°C. 1H NMR (400 MHz, DMSO-
d6, δ ppm): 7.99 (d, J =7.5 Hz, 1H), 7.39 (t, J=10.5 Hz, 2H),
7.25 (dd, J=18.3, 9.5 Hz, 3H), 7.08 (d, J=42.4 Hz, 3H), 3.83
(s, 6H, methoxy). 13C NMR (100 MHz, DMSO-d6, δ ppm):
201.21, 185.06, 161.35, 150.60, 149.14, 139.96, 135.86,
132.99, 129.36, 121.47, 119.46, 112.36, 112.30, 112.27,
100.45, 90.45, 56.11. IR (KBr) σ/cm−1: 3359.21, 2924.70,
2205.84, 1625.63, 1572.65, 1544.24, 1518.05, 1267.17,
1133.89, 1099.48, 1026.41, 851.47, 791.36. HR-ESI-MS
(pos. ion mode): m/z=368.1217[M+H]+ (anal. calcd. for
C20H15F2N3O2: 367.1132).
2-Amino-6-(2-benzyloxy-5-fluorophenyl)-4-
(3, 4-dimethoxyphenyl) nicotinonitrile (3h)
White power. mp: 170–171°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 7.68 (d, J =9.1 Hz, 1H), 7.41–7.31
(m, 8H), 7.15 (s, 1H, cyanopyridine), 6.97 (dd, J =31.7,
13.3 Hz, 4H), 5.17 (s, 2H, amidogen), 3.84 (s, 3H, meth-
oxy), 3.73 (s, 3H, methoxy). 13C NMR (100 MHz,
DMSO-d6, δ ppm): 161.19, 156.27, 154.12, 150.42,
148.98, 136.97, 129.53, 128.88, 128.44, 128.31, 121.27,
117.64, 115.59, 114.19, 112.24, 86.90, 71.20, 59.13,
56.13, 56.03. IR (KBr) σ/cm−1: 3470.41, 3321.65,
3184.79, 2200.00, 1652.56, 1572.23, 1548.43, 1518.63,
1262.81, 1140.83, 1030.74, 807.60, 730.25, 688.60. HR-
ESI-MS (pos. ion mode): m/z=456.1718[M+H]+(anal.
calcd. for C27H22FN3O3: 455.1645).
2-Amino-4-(2-bromophenyl)-6-(4-
bromophenyl) nicotinonitrile (3l)
Faint yellow power. mp: 162–164°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 8.07 (d, J=7.6Hz, 2H), 7.81 (d,
J=7.2Hz, 1H), 7.70 (s, 2H), 7.50 (m, 3H), 7.23 (s, 1H),
7.14 (s, 2H). 13C NMR (100 MHz, DMSO-d6, δ ppm):
160.51, 157.73, 138.62, 136.94, 133.26, 132.31, 132.16,
131.48, 130.96, 129.69, 128.47, 121.71, 116.42, 110.03.
IR (KBr) σ/cm−1: 3483.25, 3362.95, 2214.86, 1632.91,
1572.02, 1546.87, 1446.52, 1270.71, 1007.51, 824.79,
754.01. HR-ESI-MS (pos. ion mode): m/z =429.9376 [M
+H]+ (anal. calcd. for C18H11Br2N3: 428.9299).
2-Amino-6-(2, 4-bis (benzyloxy)phenyl)-4-
(3,4-dimethoxyphenyl) nicotinonitrile (3i)
2-Amino-6-(2-(benzyloxy)-5-
fluorophenyl)-4-(2-bromophenyl)
nicotinonitrile (3m)
1
Belge power. 159–160°C. H NMR (400 MHz, DMSO-
d6, δ ppm): 7.90 (d, J=8.7 Hz, 1H), 7.51–7.47 (m, 2H),
7.42 (t, J=7.4 Hz, 4H), 7.38–7.28 (m, 5H), 7.13 (d,
J=2.1 Hz, 1H), 6.98 (d, J=8.4 Hz, 1H), 6.92 (d, J=2.3
Hz, 1H), 6.88–6.81 (m, 1H), 6.81 (m, 3H), 5.19 (d,
J=2.9 Hz, 4H, benzyloxy), 3.84 (s, 3H, methoxy), 3.72
(s, 3H, methoxy). 13C NMR (100 MHz, DMSO-d6, δ
ppm): 161.32, 161.12, 158.30, 157.49, 153.62, 150.26,
148.91, 137.23, 137.00, 132.40, 129.84, 128.96, 128.91,
128.33, 121.16, 120.49, 118.05, 113.84, 112.20, 107.24,
101.13, 85.56, 70.61, 69.99, 56.10, 56.00. IR (KBr) σ/
cm−1: 3454.83, 3361.04, 2920.39, 2206.18, 1634.03,
1603.86, 1560.54, 1514.22, 1454.06, 1364.47, 1257.66,
Faint yellow power. mp: 146–148°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 7.78 (d, J =7.2 Hz, 1H), 7.72 (d, J=9.6
Hz, 1H), 7.47 (m, 2H), 7.30 (dd, J =16.6, 11.3 Hz, 9H),
7.09 (s, 2H), 5.15 (s, 2H, benzyloxy). 13C NMR (100
MHz, DMSO-d6, δ ppm): 161.52, 160.31, 158.31,
157.64, 153.74, 138.87, 137.18, 136.96, 133.18, 132.48,
131.18, 130.69, 128.96, 128.76, 128.37, 127.72, 121.69,
120.17, 114.32, 107.38, 101.20. IR (KBr) σ/cm−1:
3455.54, 3390.08, 3285.95, 2207.32, 1604.58, 1569.80,
1546.78, 1266.52, 1180.84, 1024.69, 731.73. HR-ESI-MS
(pos. ion mode): m/z =476.0593[M+H]+ (anal. calcd. for
1777.78, 1132.43, 1022.66, 822.00, 729.31, 693.45. C25H17BrFN3O: 475.0519 (97.3%)).
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3373
DovePress

Xu et al
Dovepress
ethylcyanoacetate (1mmol) and ammonium acetate
(8mmol) in toluene (10mL) was stirred at reflux for 8–
12 hrs. After cooling to room temperature, the mixture was
diluted ethyl acetate and THF. The organic phase was
washed twice with brine, dried over Na2SO4, the filtrate
and concentrated under reduced pressure. The residue was
suspended in absolute ethanol. The precipitate was col-
lected by filtration and purified by silica gel column chro-
matography to give the desired product.
2-Amino-6-(2, 4-bis (benzyloxy) Phenyl)-
4-(2-bromophenyl) nicotinonitrile (3n)
White power. mp: 171–173°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 7.93(d, J=8.7 Hz, 1H), 7.77
(s, 1H), 7.41 (m, 9H), 7.23 (d, J=17.5 Hz, 5H), 6.93
(s, 3H), 6.80 (d, J =7.3 Hz, 1H), 5.18 (d, J=11.1 Hz, 4H,
benzyloxy), 4.35 (d, J=3.2 Hz, 2H).13C NMR (100
MHz, DMSO-d6, δ ppm): 160.35, 158.05, 156.39,
155.70, 154.23, 153.40, 138.57, 136.89, 133.23,
131.34, 130.69, 128.76, 128.42, 128.23, 127.82,
121.63, 117.94, 117.71, 117.28, 117.04, 116.41, 115.75,
115.67, 114.71, 88.91,71.09. IR (KBr) σ/cm−1: 3490.49,
3363.26, 2218.47, 1623.93, 1569.13, 1599.12, 1420.92,
1261.12, 1205.33, 1025.16, 780.48, 763.31, 753.71. HR-
ESI-MS (pos. ion mode): m/z=564.1107[M+H]+(calcd.
for C32H24BrN3O2: 563.1031 (97.3%)).
4, 6-bis(4-bromophenyl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile (4a)
Yellow power. mp: 168–170°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 10.44 (s, 1H), 8.08 (d, J=8.0 Hz,
2H), 7.84 (d, J=7.4 Hz, 2H), 7.66 (s, 3H), 6.90 (d, J=8.0
Hz, 2H). 13C NMR (400 MHz, DMSO-d6, δ ppm): 187.47,
162.77, 141.80, 134.69, 132.30, 131.73, 131.10, 129.48,
124.09, 123.41, 115.87. IR (KBr) σ/cm−1: 3215.87,
2368.47, 1654.30, 1598.86, 1573.37, 1513.99, 1486.06,
1334.97, 1279.95, 1225.22, 1167.92, 812.66. HR-ESI-MS
(pos. ion mode): m/z=367.0077[M+H]+(anal. calcd. for
General Procedure For The Compounds
4a-4o (Scheme 2)
A mixture of the substituted acetophenone (1 mmol),
together with the respective benzaldehyde (1mmol), C18H11BrN2O2: 366.0004).
Scheme 2 The design route of 2-oxo-cyanopyridines and the chemical structure of the compound 4a-o.
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3374
DovePress

Dovepress
Xu et al
4-(4-bromophenyl)-6-(4-hydroxyphenyl)-2- 4-(4-bromophenyl)-6-(2, 4-
oxo-1, 2-dihydropyridine-3-carbonitrile (4b) difluorophenyl)-2-oxo-1, 2-
Faint yellow power. mp: 325–327°C. 1H NMR (400 MHz,
dihydropyridine-3-carbonitrile (4e)
Brown power. mp: 258–260°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 12.95 (s, 1H), 7.78 (m, 7H), 6.87 (s,
1H). 13C NMR (100 MHz, DMSO-d6, δ ppm): 161.23,
160.10, 159.25, 153.75, 136.84, 132.14, 130.94, 129.47,
128.70, 123.54, 117.62, 115.88, 108.48, 85.45. IR (KBr) σ/
cm−1: 3457.31, 3350.13, 2206.09, 1620.56, 1574.89,
1544.68, 1491.87, 1456.89, 1363.84, 1231.59, 1172.57,
805.77. HR-ESI-MS (pos. ion mode): m/z=386.9782[M
+H]+ (calcd. for C18H9BrF2N2O: 385.9866).
DMSO-d6, δ ppm): 12.94 (s, 1H, lactam), 7.77 (m, 8H,
aromatic), 6.91 (s, 1H, cyanopyridine). 13C NMR (100
MHz, DMSO-d6, δ ppm): 135.53, 132.34, 132.27,
130.92, 130.32, 125.48, 124.63, 116.72. IR (KBr) σ/
cm−1: 3074.36, 2926.19, 2218.79, 1631.61, 1602.66,
1560.68, 1493.18, 1349.03, 1233.38, 1066.77, 1006.65,
816.63, 556.73, 494.32. HR-ESI-MS (pos. ion mode): m/
z=430.9213[M+H]+ (calcd. for C18H10Br2N2O: 429.9139).
6-(2-(benzyloxy)-5-fluorophenyl)-4-(4-
bromophenyl)-2-oxo-1,2-
6-(4-bromophenyl)-4-(3, 4-
dihydropyridine-3-carbonitrile (4c)
dimethoxyphenyl)-2-oxo-1, 2-
Yellow needle power. mp: 118–120°C. 1H NMR (400
MHz, DMSO-d6, δ ppm): 7.71 (m, 1H), 7.54 (d, J =8.5
Hz, 3H), 7.33 (m, 8H), 7.21 (s, 1H), 6.99 (d, J=8.4 Hz,
2H), 6.73 (s, 2H), 5.14 (s, 2H). 13C NMR (400 MHz,
DMSO-d6, δ ppm): 167.81, 166.22, 158.44, 154.72,
153.41, 150.78, 140.19, 136.03, 131.87, 131.51, 129.89,
128.90, 128.57, 121.60, 117.34, 116.91, 115.59, 106.06,
71.22. IR (KBr) σ/cm−1: 3415.51, 3272.92, 3173.49,
2985.27, 2247.60, 1680.28, 1615.98, 1572.50, 1491.94,
1282.43, 1250.94, 1192.61, 1108.16, 1011.47, 810.47,
735.28. HR-ESI-MS (pos. ion mode): m/z=475.0450[M
+H]+ (calcd. for C25H16BrFN2O2: 474.0379).
dihydropyridine-3-carbonitrile (4g)
Faint yellow power. mp: 309–312°C. 1H NMR (400
MHz, DMSO-d6, δ ppm): 12.75 (s, 1H), 7.80 (d, J
=36.8 Hz, 4H), 7.36 (d, J=8.9 Hz, 2H), 7.14 (d, J=8.1
Hz, 1H), 6.89 (s, 1H), 3.85 (s, 6H). 13C NMR (100
MHz, DMSO-d6, δ ppm): 151.27, 149.08, 132.31,
130.30, 128.45, 15.29, 125.26, 122.01, 112.41, 112.09,
56.20, 56.18. IR (KBr) σ/cm−1: 2933.95, 2838.96,
2220.79, 1632.59, 1598.04, 1515.67, 1494.22, 1421.33,
1261.98, 1217.85, 1148.23, 1021.81, 831.19. HR-ESI-
MS (pos. ion mode): m/z =411.0172[M+H]+ (calcd. for
C20H15BrN2O3: 410.0266).
6-(2, 4-bis (benzyloxy)phenyl)-4-(4-
bromophenyl)-2-oxo-1,2-
6-(2-(benzyloxy)-5-fluorophenyl)-4-(3,4-
dimethoxyphenyl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile (4d)
dihydropyridine-3-carbonitrile (4h)
Faint yellow power. mp: 165–168°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 7.91 (d, J=8.7 Hz, 1H), 7.41 (m, 13H),
7.13 (s, 1H), 6.97 (d, J=8.3 Hz, 2H), 6.70 (s, 2H), 5.17 (d,
J=20.0 Hz, 4H, benzyloxy). 13C NMR (100 MHz, DMSO-
d6, δ ppm): 167.98, 161.14, 158.59, 158.25, 150.78,
140.69, 137.27, 136.94, 132.24, 131.40, 129.87, 128.95,
128.92, 128.58, 128.44, 128.34, 121.35, 115.38, 107.15,
100.98, 70.64, 69.98. IR (KBr) σ/cm−1: 3422.83, 3266.96,
3168.02, 2918.61, 2360.65, 1680.43, 1603.46, 1571.60,
1488.09, 1258.68, 1186.16, 1100.30, 1021.82, 1011.92,
818.95, 735.55, 693.30. HR-ESI-MS (pos. ion mode): m/
z=563.0972[M+H]+(calcd. for C32H23BrN2O3: 562.0892).
Faint yellow power. mp: 205–208°C. 1H NMR (400
MHz, DMSO-d6, δ ppm): 12.57 (s, 1H), 7.52–7.22 (m,
J=30.3 Hz, 10H), 7.10 (d, J=7.7 Hz, 1H), 6.67 (s, 1H),
5.16 (s, 2H), 3.84 (s, 3H), 3.79 (s,3H). 13C NMR (100
MHz, DMSO-d6, δ ppm): 154.24, 149.04, 136.92,
128.90, 128.86, 128.57, 128.42, 128.11, 128.08,
121.83, 115.35, 112.29, 112.28, 112.08, 82.75, 71.03,
71.02, 58.30, 56.16, 56.12. IR (KBr) σ/cm1: 3420.98,
2 927.97, 2838.63, 2221.89, 1654.48, 1603.88, 1518.79,
1262.70, 1146.03, 1032.76, 818.53, 730.35. HR-ESI-MS
(pos. ion mode): m/z=455.1418[M-H]+ (calcd. for
C27H21FN2O4: 456.1485).
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3375
DovePress

Xu et al
Dovepress
6-(2,4-bis(benzyloxy)phenyl)-4-(3,4-
dimethoxyphenyl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile (4i)
4-(2-bromophenyl)-6-(4-bromophenyl)-2-
oxo-1, 2-dihydropyridine-3-carbonitrile (4l)
Faint yellow cottony. mp: 298–300°C. 1H NMR (400
MHz, DMSO-d6, δ ppm): 13.00 (s, 1H), 7.79 (d, J=37.2
Hz, 4H), 7.51 (d, J=27.3 Hz, 3H), 6.81 (s, 1H). 13C NMR
(100 MHz, DMSO-d6, δ ppm): 137.74, 133.41, 132.41,
131.91, 130.52, 130.25, 128.58. IR (KBr) σ/cm−1:
3443.64, 2923.41, 2222.70, 1644.73, 1605.78, 1561.63,
1493.79, 1229.05, 1009.22, 823.69, 751.75. HR-ESI-MS
(pos. ion mode): m/z=430.9051 [M+H]+ (calcd. for
C18H10Br2N2O: 429.9139).
Faint yellow power. mp: 178–180°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 7.87 (d, J=7.8 Hz, 1H), 7.48–7.33 (m,
10H), 7.20 (s, 1H), 6.90 (s, 2H), 6.78–6.73 (m, 2H), 6.57
(d, J=6.0 Hz, 1H), 6.49 (s, 1H), 5.18 (s, 4H), 3.80 (s, 3H),
3.66 (s, 3H). 13C NMR (400 MHz, DMSO-d6, δ ppm):
168.65, 160.85, 158.13, 158.03, 155.71, 148.77, 137.11,
133.64, 128.95, 128.87, 128.41, 128.32, 128.24, 124.80,
124.74, 120.18, 112.11, 101.11, 70.51, 69.95. IR (KBr) σ/
cm−1: 3427.79, 2961.41, 2922.03, 2360.35, 1679.82,
1603.49, 1562.38, 1508.49, 1457.52, 1260.44, 1104.97,
1025.11, 815.30. HR-ESI-MS (pos. ion mode): m/
z=545.2073[M+H]+ (calcd. for C34H28N2O5: 544.1998).
6-(2-(benzyloxy)-5-fluorophenyl)-4-(2-
bromophenyl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile (4m)
Faint yellow power. mp: 167–169°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 7.96 (d, J=8.7 Hz, 1H), 7.64 (dd,
J=7.9, 1.2 Hz, 1H), 7.50 (m, 2H), 7.42 (m, 2H), 7.33 (m,
3H), 7.08 (m, 3H), 6.92 (d, J=2.3 Hz, 1H), 6.80 (dd, J=8.7,
2.3 Hz, 1H), 5.19 (d, J=5.0 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6, δ ppm): 161.26, 159.52, 158.26, 156.99,
151.91, 143.25, 137.23, 128.95, 128.77, 128.37, 128.37,
127.70, 121.64, 120.36, 115.88, 107.23, 103.34,101.12.
70.44. IR (KBr)σ/cm−1: 3424.16, 3263.82, 3164.77,
2924.13, 2357.69, 1677.35, 1603.39, 1569.37, 1261.04,
1186.93, 1109.95, 1025.89, 817.52, 733.30, 693.96. HR-
ESI-MS (pos. ion mode): m/z=475.0450 [M+H]+(calcd.
for C25H16BrFN2O2: 474.0379).
6-(2, 4-difluorophenyl)-4-(3, 4-
dimethoxyphenyl)-2-oxo-1, 2-
dihydropyridine-3-carbonitrile (4j)
Yellow power. mp: 253–255°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 7.85 (s, 1H), 7.42 (t, J=12.5 Hz, 1H),
7.25 (s, 3H), 7.11 (s, 1H), 6.65 (d, J=29.9 Hz, 1H), 5.31 (s,
1H), 3.83 (s, 6H), 3.82 (s, 6H). 13C NMR (100MHz, DMSO-
d6, δ ppm): 150.73, 148.98, 133.00, 132.99, 132.93, 132.89,
121.58, 112.52, 112.30, 112.25, 112.09, 105.11. IR (KBr) σ/
cm−1: 3444.10, 3055.13, 2919.16, 2847.54, 2360.58,
2215.93, 1656.98, 1617.30, 1520.05, 1271.72, 1023.85,
801.99. HR-ESI-MS (pos. ion mode): m/z=369.1045 [M-
2H]+(calcd. for C20H15F2N3O2: 367.1132).
6-(2, 4-bis (benzyloxy) Phenyl)-4-(2-
bromophenyl)-2-oxo-1, 2-
dihydropyridine-3-carbonitrile (4n)
4-(2-bromophenyl)-6-(4-hydroxyphenyl)-
2-oxo-1, 2-dihydropyridine-3-carbonitrile
White power. mp: 170–172°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 7.95 (d, J=8.3 Hz, 1H), 7.64 (d,
J=6.3 Hz, 1H), 7.38 (dd, J=61.3, 24.8 Hz, 12H), 7.07 (s,
4H), 6.91 (s, 1H), 6.80 (d, J=7.9 Hz, 1H), 5.17 (d,
J=19.3 Hz, 4H). 13C NMR (100 MHz, DMSO-d6, δ
ppm): 159.39, 152.13, 142.80, 136.91, 132.26, 129.58,
129.48, 128.90, 128.77, 128.20, 127.81, 121.56, 117.56,
117.34, 117.23, 116.98, 116.20, 115.65, 115.57, 104.85.
IR (KBr) σ/cm−1: 3422.71, 3263.89, 3165.39, 2922.32,
2337.22, 1617.43, 1602.93, 1568.64, 1259.88, 1185.94,
(4k)
Brown power. mp: 132–135°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 9.88 (s, 1H), 7.98 (t, 1H), 7.95 (t,
1H), 7.67–7.55 (m, 1H), 7.46–7.41 (td, J=7.5, 1.2 Hz, 1H),
7.34–7.29 (m, 2H), 7.13 (s, 2H), 6.84 (s, 2H), 6.82 (d,
J=1.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6, δ ppm):
159.83, 158.23, 152.96, 143.30, 132.26, 131.17, 129.83,
129.45, 129.15, 128.79, 127.80, 121.70, 115.86, 110.41,
103.37. IR (KBr) σ/cm−1: 3470.22, 3413.54, 3369.47,
1686.21, 1599.93, 1570.73, 1516.70, 1360.58, 1282.87,
1230.64, 1169.10, 1102.44, 823.99, 755.09. HR-ESI-MS 1107.73, 1024.55, 817.10, 732.12, 693.23.HR-ESI-MS
(pos. ion mode): m/z =367.0089[M+H]+ (calcd. for (pos. ion mode): m/z=563.0972[M+H]+ (calcd. for
C18H11BrN2O2: 366.0004).
C32H23BrN2O3: 562.0892).
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3376
DovePress

Dovepress
Xu et al
Table 2 IC50 of 4a-o for HCT-116, Hela229, and A375 (IC50 in
μM)
4-(2-bromophenyl)-6-(2, 4-
difluorophenyl)-2-oxo-1, 2-
dihydropyridine-3-carbonitrile (4o)
Comp.
Code
HCT-116
Cell line
Hela229
A375
White power. mp: 300–302°C. 1H NMR (400 MHz,
DMSO-d6, δ ppm): 13.09 (s, 1H), 7.80 (d, J=15.4 Hz,
2H), 7.55–7.47 (m, 4H), 7.28 (s, 1H), 6.61 (s, 1H). 13C
NMR (100 MHz, DMSO-d6, δ ppm): 137.52, 133.47,
133.06, 132.96, 132.01, 130.47, 128.62, 120.87, 115.63,
112.95, 105.77, 105.50, 105.24. IR (KBr) σ/cm−1:
3443.52, 2778.95, 2221.46, 1650.66, 1614.21, 1506.77,
1269.52, 1226.79, 1110.34, 949.56, 853.61, 756.74. HR-
ESI-MS (pos. ion mode): m/z=386.9782[M+H]+ (calcd.
for C18H9BrF2N2O: 385.9866).
4a
4b
4c
4d
4e
4g
4h
4i
35.56 0.65
20.17 1.30
>40
12.40 0.68
8.08 0.74
17.53 1.10
29.26 1.03
6.75 0.75
>40
22.17 0.67
8.08 0.56
19.93 1.00
>40
>40
1.67 0.56
37.81 0.65
10.10 0.56
>40
>40
>40
2.48 0.64
>40
>40
20.88 0.65
33.04 0.74
16.79 1.01
15.21 0.72
>40
4j
>40
8.21 0.53
5.00 0.91
31.91 1.04
>40
4k
4l
7.51 0.69
34.86 1.20
>40
4m
4n
4o
16.93 1.01
>40
>40
>40
Screening Of Cyanopyridines Against
17.91 1.20
16.19 0.85
Human Cancer Cells In Vitro
First, all the synthesized cyanopyridines were evaluated
for their antiproliferative activity against a variety of can-
cer cells by the MTT assay in vitro (Tables 1–2). Among
the cyanopyridines 3n exhibited potent inhibitory activity
on all HCT-116, Hela, and A375 cells with the IC50 values
of 10.50, 14.27 and 4.61 μM, respectively, which was the
more potent compound for further study.
activity compared to 3n. Furthermore, the compound 3n
with 2-amino substituent was more active than 2-oxo con-
gener 4n.
Active Compounds 3n Inhibited Migration
And Colony Formation Of Colorectal
From the preliminary structure–activity relationships,
we may conclude that introduction of benzyloxy group to
the 1-position and 2-position of phenyl ring slightly
Cancer Cells
Moreover, DLD-1 and RKO cells were treated with
different concentrations of 3n to evaluate the effect of
enhance the cytotoxic activity, meanwhile bromine substi- 3n on colorectal cancer cells migration (Figure 1A). The
migration ability of cells treated with 3n was signifi-
cantly reduced, which highlighted the critical role of 3n
in inhibition of colorectal cancer cell migration. In addi-
tion, to observe the effect of 3n on the proliferation of
colorectal cancer cells, we performed colony formation
assays. Similar effects were observed in colony forma-
tion assays, 3n remarkably reduced the colony formation
capacity in HCT116, RKO, and DLD-1 cancer cell lines
(Figure 1B).
tuent to the 6-position on phenyl ring even had decreased
Table 1 IC50 of 3a-n for HCT-116, Hela229, and A375 (IC50 in
μM)
Comp.
Code
HCT-116
Cell line
Hela229
A375
3a
3b
3c
3d
3e
3f
>40
>40
>40
0.22 0.12
>40
26.85 0.91
33.53 1.20
>40
>40
>40
>40
>40
The Compounds 3n Inhibited The
Phosphorylation Of STAT3
2.18 0.32
>40
21.69 1.15
5.80 0.94
5.22 0.65
17.46 1.05
6.63 0.84
32.47 0.74
>40
12.69 1.01
30.17 1.30
6.49 0.78
9.41 0.46
24.97 1.20
37.58 1.32
19.54 1.10
>40
3g
3h
3i
35.51 0.85
>40
Aminocyanopyridines can inhibit cell proliferation and
induce cell death in human cancer cells by inhibiting the
STAT3 pathway.17 We next validated the effects of 3n on
STAT3 signaling in three colorectal cancer cells. As shown
in Figure 2A, STAT3 phosphorylation was inhibited in a
time-dependent manner with a 40 μM concentration of 3n,
which significantly inhibited STAT3 phosphorylation at 24
4.74 0.51
8.16 0.63
6.07 0.56
>40
3j
3l
3m
3n
>40
10.50 1.01
14.27 1.00
4.61 0.75
Abbreviation: IC50, 50% inhibiting concentration.
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3377
DovePress

Xu et al
Dovepress
Figure 1 The compound 3n inhibits colony formation and migration of colorectal cancer cells. (A) In cell scratch test, a wound healing assay for migration by scraping cells
with a yellow tip. The migration ability of RKO and DLD-1 cells was evaluated by a scratch test. Compared with the negative control, cells treated with different
concentrations (10, 20, and 40 μM) of 3n showed that the cell migration was significantly slowed down with increasing concentration. Wound healing assay results from RKO
and DLD-1 were calculated. (**P<0.01, ***P<0.001, ****P<0.0001). (B) For the colony formation assay, the number of colonies was counted 6–8 days after colorectal cancer
cells were incubated and treated with various concentrations for 3n, and DMSO (1 μL) was added as a negative control and then stained with crystal violet.
hrs, but had no effect on total STAT3 levels in the three STAT3. As shown in Figure 2C, the results indicate that
cell lines. Moreover, incubation of HCT116 with a con- 3n, like the recognized role of the STAT3 inhibitor napa-
centration gradient of 3n (10, 20, and 40 μM), HCT116, bucasin, blocks IL6-induced activation of the STAT3 sig-
RKO, and DLD-1 cells significantly downregulated phos- naling pathway. We tested downstream protein levels of
phorylated STAT3 expression in a dose-dependent manner STAT3 and found that 3n also inhibits the expression of
(Figure 2B). These results indicate that the anticancer downstream target proteins, such as Survivin and MCL-1
activity of 3n is mediated through the inhibition of (Figure 2D).
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3378
DovePress

Dovepress
Xu et al
Figure 2 The compound 3n inhibited STAT3 phosphorylation in colorectal cancer cells. (A) The three colorectal cells were exposed to 40 μM 3n for different time periods (0, 2, 4,
8,12, and 24 hrs). Total protein was extracted, and the expression levels of P-STAT3, STAT3, and GAPDH proteins were detected by Western blot analysis. (B) Western blot
analysis of STAT3 pathway associated proteins in colorectal cancer cells exposed to various concentrations (10, 20, and 40 μM) of 3n for 24 hrs. Representative pictures from three
independent experiments are shown. (C) Human colon cancer cells were pretreated with 3n (10, 20, and 40 μM) and napabucasin (napa 1 μM) for 24 hrs and then stimulated with
IL6 (25 ng/mL) for 30 mins. (D) The effect of 3n on Survivin and MCL-1 was analyzed by Western blot in RKO cells. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
Abbreviations: DMSO, dimethyl sulfoxide; STAT3, signal transducer and activator of transcription 3; P-STAT3, phosphorylated STAT3; GAPDH, glyceraldehyde
3-phosphate dehydrogenase; MCL-1, myeloid cell leukemia-1.
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3379
DovePress

Xu et al
Dovepress
toward the discovery of STAT3 inhibitor as a drug candi-
date for cancer therapy.
Discussion
Colorectal cancer remains the leading cause of cancer
deaths worldwide, despite extensive research and progress
in screening and treatment.1,18,19 In addition, to date, there
are no convincing agents that can significantly improve the
survival rate of human colorectal cancer.5 STAT3 is con-
sidered to be an essential oncogene in the development of
colorectal cancer and is thought to be carcinogenic in the
development of colorectal cancer.20 In future studies, find-
ing a suitable STAT3 inhibitor to inhibit abnormal activa-
tion of STAT3 in colorectal cancer cells remains a
promising strategy to address this problem.21,22
Acknowledgments
This work was financially supported by the Zhejiang
Provincial Natural Science Fund (LY18H160047),
National Natural Science Funding of China (21762006),
Wenzhou Science and Technology Project (Y20190179),
and Major Science and Technology Projects of Guangxi
Zhuang Autonomous Region (No. AA17204090).
Disclosure
The authors report no conflicts of interest in this work.
In this study, we design of two new series of com-
pounds with 2-amino-3-cyanopyridine chalcone and 3-
cyano-2-pyridinone skeleton chalcone. Three kinds of
human tumor cells HCT-116, Hela229, and A375 were
screened for biological activity against proliferation. The
results showed that 3n can effectively inhibit the prolifera-
tion and migration ability of colorectal cancer cells.
Further studies confirmed that STAT3 phosphorylation
was inhibited in a time- and dose-dependent manner after
exposure to 3n. These results indicate that aminohydrox-
ypyridine compound 3n can act as an inhibitor of STAT3,
thereby inhibiting tumor growth and inducing apoptosis in
colorectal cancer. 3n is a potent STAT3 inhibitor and may
be a potential drug candidate for colorectal cancer therapy.
Also, of course, 3n can also serve as lead compounds for
optimization to speed the development of drugs selectively
targeting the IL-6/STAT3 cancer signaling pathway.
Additionally, the pharmacokinetics, pharmacodynamics,
and toxicity of 3n will be further comprehensively eval-
uated. Combination therapy may enhance the curative
effects and reduce the therapeutic concentration of che-
motherapeutic drugs.23,24 We also plan to investigate the
combination of 3n and other clinical cancer drugs to
improve the antitumor potency of 3n and verify its effect
on colorectal cancer.
References
1. Serrano PE, Carter DN, Li C, et al. Adjuvant chemotherapy with or
without biologics including antiangiogenics and monoclonal antibo-
dies targeting EGFR and EpCAM in colorectal cancer: a systematic
review and meta-analysis. J Surg Res. 2019;239:14–21. doi:10.1016/
j.jss.2019.01.030
2. Valle L, de Voer RM, Goldberg Y, et al. Update on genetic predis-
position to colorectal cancer and polyposis. Mol Aspects Med. 2019.
doi:10.1016/j.mam.2019.03.001
3. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J
Clin. 2019;69(1):7–34. doi:10.3322/caac.21551
4. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J
Clin. 2018;68(1):7–30. doi:10.3322/caac.21442
5. Zhao CG, Li HM, Lin HJ, Yang SL, Lin JY, Liang G. Feedback
activation of STAT3 as a cancer drug-resistance mechanism. Trends
Pharmacol Sci. 2016;37(1):47–61. doi:10.1016/j.tips.2015.10.001
6. Caetano MS, Hassane M, Van HT, et al. Sex specific function of
epithelial STAT3 signaling in pathogenesis of K-ras mutant lung
cancer. Nat Commun. 2018;9(1):4589. doi:10.1038/s41467-018-
07042-y
7. Yu H, Lee H, Herrmann A, Buettner R, Jove R. Revisiting STAT3
signalling in cancer: new and unexpected biological functions. Nat
Rev Cancer. 2014;14(11):736–746. doi:10.1038/nrc3818
8. Weinberg BA, Marshall JL, Salem ME. The growing challenge of
young adults with colorectal cancer. Oncology (Williston Park, NY).
2017;31(5):381–389.
9. Yakubu UM, Morano KA. Roles of the nucleotide exchange factor
and chaperone Hsp110 in cellular proteostasis and diseases of protein
misfolding. Biol Chem. 2018;399(10):1215–1221. doi:10.1515/hsz-
2018-0209
10. Wang XY, Subjeck JR. High molecular weight stress proteins: iden-
tification, cloning and utilisation in cancer immunotherapy. Int J
Hyperthermia. 2013;29(5):364–375. doi:10.3109/02656736.2013.80
3607
Conclusion
11. Berthenet K, Bokhari A, Lagrange A, et al. HSP110 promotes color-
ectal cancer growth through STAT3 activation. Oncogene. 2017;36
(16):2328–2336. doi:10.1038/onc.2016.403
12. Krens SD, Lassche G, Jansman FGA, et al. Dose recommendations
for anticancer drugs in patients with renal or hepatic impairment.
Lancet Oncol. 2019;20(4):e200–e207. doi:10.1016/S1470-2045(19)
30145-7
13. Lauria A, Abbate I, Gentile C, Angileri F, Martorana A, Almerico
AM. Synthesis and biological activities of a new class of heat shock
protein 90 inhibitors, designed by energy-based pharmacophore vir-
tual screening. J Med Chem. 2013;56(8):3424–3428. doi:10.1021/
jm4002023
In summary, a novel series of cyanopyridines and amino-
cyanopyridines were designed, synthesized, and biologi-
cally evaluated. The most potent compound 3n exhibited
remarkable inhibitory activity on HCT116, Hela, and
A375 cells. Furthermore, 3n significantly inhibits cell col-
ony formation and migration of colorectal cancer cells.
Mechanistically, 3n decreased protein expression level of
P-STAT3Y. Together, 3n is worth of further investigations
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3380
DovePress

Dovepress
Xu et al
14. Gomha SM, Abdulla MM, Abou-Seri SM. Identification of novel
aminothiazole and aminothiadiazole conjugated cyanopyridines as
selective CHK1 inhibitors. Eur J Med Chem. 2015;92:459–470.
doi:10.1016/j.ejmech.2015.01.019
15. Kalaria PN, Satasia SP, Avalani JR, Raval DK. Ultrasound-assisted
one-pot four-component synthesis of novel 2-amino-3-cyanopyridine
derivatives bearing 5-imidazopyrazole scaffold and their biological
broadcast. Eur J Med Chem. 2014;83:655–664. doi:10.1016/j.ejmech.
2014.06.071
16. Dev S, Dhaneshwar SR, Mathew B. Virtual combinatorial library
design, synthesis and in vitro anticancer assessment of −2-amino-3-
cyanopyridine derivatives. Comb Chem High Throughput Screen.
2018;21(2):138–148. doi:10.2174/1386207321666180228113925
17. Xu L, Qiu S, Yang L, et al. Aminocyanopyridines as anti-lung cancer
agents by inhibiting the STAT3 pathway. Mol Carcinog. 2019;
58:1512–1525. doi:10.1002/mc.23038
18. ME IJ, Sanz-Pamplona R, Hermitte F, de Miranda N. Colorectal
cancer: a paradigmatic model for cancer immunology and immu-
notherapy. Mol Aspects Med. 2019. doi:10.1016/j.mam.2019.05.
003
19. La Vecchia S, Sebastian C. Metabolic pathways regulating colorectal
cancer initiation and progression. Semin Cell Dev Biol. 2019. doi:10.
1016/j.semcdb.2019.05.018
20. Gozzi GJ, Gonzalez D, Boudesco C, et al. Selecting the first chemical
molecule inhibitor of HSP110 for colorectal cancer therapy. Cell
Death Differ. 2019. doi:10.1038/s41418-019-0343-4
21. Kesselring R, Glaesner J, Hiergeist A, et al. IRAK-M expression in
tumor cells supports colorectal cancer progression through reduction
of antimicrobial defense and stabilization of STAT3. Cancer Cell.
2016;29(5):684–696. doi:10.1016/j.ccell.2016.03.014
22. Wei N, Li J, Fang C, et al. Targeting colon cancer with the novel
STAT3 inhibitor bruceantinol. Oncogene. 2019;38(10):1676–1687.
doi:10.1038/s41388-018-0547-y
23. Yang L, Lin S, Kang Y, et al. Rhein sensitizes human pancreatic
cancer cells to EGFR inhibitors by inhibiting STAT3 pathway. J Exp
Clin Cancer Res. 2019;38(1):31. doi:10.1186/s13046-018-1015-9
24. Zheng H, Yang L, Kang Y, et al. Alantolactone sensitizes human
pancreatic cancer cells to EGFR inhibitors through the inhibition of
STAT3 signaling. Mol Carcinog. 2019;58(4):565–576. doi:10.1002/
mc.22951
Drug Design, Development and Therapy
Dovepress
Publish your work in this journal
Drug Design, Development and Therapy is an international, peer-
reviewed open-access journal that spans the spectrum of drug design
and development through to clinical applications. Clinical outcomes,
patient safety, and programs for the development and effective, safe,
and sustained use of medicines are a feature of the journal, which has also
been accepted for indexing on PubMed Central. The manuscript
management system is completely online and includes a very quick
and fair peer-review system, which is all easy to use. Visit http://www.
dovepress.com/testimonials.php to read real quotes from published
authors.
Submit your manuscript here: https://www.dovepress.com/drug-design-development-and-therapy-journal
submit your manuscript | www.dovepress.com
Drug Design, Development and Therapy 2019:13
3381
DovePress