Small molecules inhibit STAT3 activation, autophagy, and cancer cell anchorage-independent growth

Article abstract of DOI:10.1016/j.bmc.2017.03.048

Triple-negative breast cancers (TNBCs) lack the signature targets of other breast tumors, such as HER2, estrogen receptor, and progesterone receptor. These aggressive basal-like tumors are driven by a complex array of signaling pathways that are activated by multiple driver mutations. Here we report the discovery of 6 (KIN-281), a small molecule that inhibits multiple kinases including maternal leucine zipper kinase (MELK) and the non-receptor tyrosine kinase bone marrow X-linked (BMX) with single-digit micromolar IC₅₀s. Several derivatives of 6 were synthesized to gain insight into the binding mode of the compound to the ATP binding pocket. Compound 6 was tested for its effect on anchorage-dependent and independent growth of MDA-MB-231 and MDA-MB-468 breast cancer cells. The effect of 6 on BMX prompted us to evaluate its effect on STAT3 phosphorylation and DNA binding. The compound's inhibition of cell growth led to measurements of survivin, Bcl-X_L, p21^WAF1/CIP1, and cyclin A2 levels. Finally, LC3B-II levels were quantified following treatment of cells with 6 to determine whether the compound affected autophagy, a process that is known to be activated by STAT3. Compound 6 provides a starting point for the development of small molecules with polypharmacology that can suppress TNBC growth and metastasis.

Full text of DOI:10.1016/j.bmc.2017.03.048

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small molecules inhibit STAT3 activation, autophagy, and cancer cell
anchorage-independent growth
Donghui Zhou a, Maya Z. Springer b, David Xu c,d, Degang Liu a, Andy Hudmon a, Kay F. Macleod b,
Samy O. Meroueh a,c,
⇑
a Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, United States
b The Ben May Department for Cancer Research, University of Chicago, United States
c Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, United States
d Department of BioHealth Informatics, Indiana University School of Informatics and Computing, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Triple-negative breast cancers (TNBCs) lack the signature targets of other breast tumors, such as HER2,
estrogen receptor, and progesterone receptor. These aggressive basal-like tumors are driven by a complex
array of signaling pathways that are activated by multiple driver mutations. Here we report the discovery
of 6 (KIN-281), a small molecule that inhibits multiple kinases including maternal leucine zipper kinase
(MELK) and the non-receptor tyrosine kinase bone marrow X-linked (BMX) with single-digit micromolar
IC50s. Several derivatives of 6 were synthesized to gain insight into the binding mode of the compound to
the ATP binding pocket. Compound 6 was tested for its effect on anchorage-dependent and independent
growth of MDA-MB-231 and MDA-MB-468 breast cancer cells. The effect of 6 on BMX prompted us to
evaluate its effect on STAT3 phosphorylation and DNA binding. The compound’s inhibition of cell growth
led to measurements of survivin, Bcl-XL, p21WAF1/CIP1, and cyclin A2 levels. Finally, LC3B-II levels were
quantified following treatment of cells with 6 to determine whether the compound affected autophagy,
a process that is known to be activated by STAT3. Compound 6 provides a starting point for the develop-
ment of small molecules with polypharmacology that can suppress TNBC growth and metastasis.
Ó 2017 Published by Elsevier Ltd.
Received 24 January 2017
Revised 18 March 2017
Accepted 21 March 2017
Available online xxxx
1. Introduction
needed for the development of effective therapeutic agents for
the treatment of TNBC.1
Triple-negative breast cancer (TNBC) has taken on added impor-
tance in recent years as it is characterized by poor prognosis,
aggressive behavior, and a lack of specific targets such as ER,
HER2, or progesterone receptor. Although chemotherapy is effec-
tive for TNBC patients in the adjuvant setting, chemotherapy-resis-
tant TNBC is quickly fatal given its rapid growth and lack of
targeted therapies. TNBC accounts for approximately 15% of breast
cancer cases in the United States. Despite its lower incidence, TNBC
contributes disproportionately to breast cancer morbidity and
mortality compared to other breast cancer subtypes. This can be
attributed in part to its intrinsic aggressiveness, but much of the
excess mortality is due to the absence of effective targeted thera-
pies. New targets and small molecules to probe these targets are
Unlike other subtypes of cancer, hormone therapy or biologics
such as trastuzumab have little effect on TNBC tumors. There is
currently an intense search for vulnerabilities of this breast cancer
sub-type that can be exploited for the development of targeted
therapeutic agents. To identify new targets that suppress growth
and metastasis of TNBC tumors, we previously screened a commer-
cial library of small molecules using structure-based molecular
docking. The focus was on members of the CaMK superfamily of
kinases, which are relatively unexplored in cancer. We identified
1, a small molecule that inhibited CaMKIId with single-digit micro-
molar IC50s. Kinome-wide profiling against 337 kinases confirmed
that CaMKIId was among the top 10 targets of the compound, along
with other members of the CaMK family, such as the maternal
embryonic zipper leucine kinase (MELK), and the non-receptor tyr-
osine kinase bone marrow X-linked (BMX).
The inhibition of multiple kinases by 1 prompted us to explore
the compound and several of its derivatives in TNBC breast cancer
cell lines MDA-MB-468 and MDA-MB-231. We focused on 6 (KIN-
281). The compound was tested for activity in a panel of 25
⇑
Corresponding author at: Department of Biochemistry and Molecular Biology,
Indiana University School of Medicine, Van Nuys Medical Science Building, MS
4023, 635 Barnhill Drive, Indianapolis, IN 46202-5122, United States.
E-mail address: smeroueh@iu.edu (S.O. Meroueh).
http://dx.doi.org/10.1016/j.bmc.2017.03.048
0968-0896/Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048

2
D. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
kinases. We explored the effects of the compound on cell viability
and colony formation in anchorage-dependent and independent
assays. We further explored the effect of compounds on pro-apop-
totic survivin and Bcl-XL, as well as cell cycle protein cyclin-depen-
dent kinase inhibitor 1 (p21WAF1/CIP1). Considering the effect on
BMX and Bcl-XL we examined the effects of the compound on sig-
nal transducer and activator of transcription 3 (STAT3) as well as
autophagy, which is activated by STAT3.
2.5. Electrophoresis mobility shift assay (EMSA)
STAT3 consensus oligonucleotides (Santa Cruz, Dallas, TX) were
first labeled with [
Aldrich, St. Louis, MO). A total of 2
bated with 32P-labeled STAT3 probe in binding buffer (10 mM Tris,
50 mM KCl, 1 mM DTT, pH 7.5, 50 ng/ L Poly (dIdC), 50 mM EDTA
c
32P]-ATP by using polynucleotide kinase (Sigma
l
g of nuclear protein was incu-
l
pH 7.5) for 30 min on ice. After incubation, the samples were sep-
arated on 4% native polyacrylamide gel containing 50 mM Tris, pH
7.5, 0.38 M glycine and 2 mM EDTA. The gel was transferred to
Whatman paper, covered with saran wrap and dried at 80 °C in
vacuum dryer for 1–2 h. 32P-labeled bands were visualized by
autoradiography.
2. Materials and methods
2.1. Cell culture
MDA-MB-231 or MDA-MB-468 cells were cultured in Dul-
becco’s Modified Eagle Medium (Cellgro, Manassas, VA) supple-
mented with 10% FBS, 1% penicillin/streptomycin in a 5% CO2
atmosphere at 37 °C.
2.6. Autophagy assays
Bafilomycin A1 (Enzo) was added at 100 nM for 4 h before har-
vest of cells. Cells were lysed in NP40 buffer (150 mM NaCl, 50 mM
Tris-HCl pH 7.5, 1 mM EDTA, 1% NP40) with protease and phos-
phatase inhibitors. Lysates were centrifuged at 16,300g for
15 min at 4 °C; supernatants were separated by SDS-PAGE. After
transfer to PVDF, membranes were blocked in 5% milk/PBS for
1 h at RT and incubated at 4 °C overnight with primary anti-LC3B
antibody from Novus (cat# NB600-1384) at 1:3000 in 5% milk/
PBS. Membranes were washed 3Â in PBS-T 0.05% Tween and then
incubated for 2 h with secondary (anti-rabbit HRP, 1:10,000) at RT.
Membranes were washed 2Â in PBS-T 0.05% Tween. Detection was
performed by enhanced chemiluminescence after 2-h incubation
with HRP-conjugated secondary antibodies (Dako).
2.2. Cell viability assay
MDA-MB-231 (104 cells) were plated overnight in 100 ml in each
well of 96-well plates, and then treated with 1% DMSO (control) or
compounds at the indicated concentrations for 3 d. 20 ml of 5 mg/
mL MTT (Sigma-Aldrich, St. Louis, Missouri) were added to each
well, and cells were incubated at 37 °C in 5% CO2 for 2 h. Viable
cells were quantified at absorbance of 570 and 630 nm (reference
background). Glioma cells were seeded into 96-well plates and
treated with KIN compounds. After 5 d of incubation, cell growth
was determined by methylene blue staining. Each experiment
was conducted in triplicate and repeated three times.
2.7. Virtual screening
2.3. Western blot analysis
The program Glide from Schrödinger (Schrödinger LLC, New
York, NY, 2016) was used for molecular docking of 6 to the MELK
ATP binding pocket. Glide is a robust docking program that has
been extensively validated in previous studies. The structures of
6 was prepared using LigPrep in Schrödinger. Compound was pro-
tonated at pH 7. The structure of MELK (PDB ID: 4CQG, 2.57 Å) was
prepared for docking. A grid box was generated for the MELK struc-
ture for docking in Glide, with a box size of 21 Å and an inner box
of 14 Å. All other parameters were set to default values. A series of
binding poses were generated by docking 6 and its derivatives
against the ATP binding site of MELK using Glide in standard pre-
cision (SP) mode. All other parameters were left to default values.
Anti-b-actin was obtained from Santa Cruz Biotechnology (CA,
USA). Anti-LC3B was obtained from Sigma chemicals Co (St. Louis,
MO, USA). Anti-p21, cyclin A2, anti-p53, anti-phospho p53 (Ser15),
MDMX, anti-phosphor STAT3 (Tyr705), anti-STAT3, anti-Bcl-XL,
anti-survivin, anti-b-catenin, anti-phospho Akt, anti-E-cadherin,
were obtained from Cell Signaling Technology (Beverly, MA,
USA). Total cell lysates were prepared in standard RIPA extraction
buffer containing protease and phosphatase inhibitors (Sigma-
Aldrich, St. Louis, Missouri). Thirty micrograms of protein were
separated by 10% SDS-PAGE and transferred to nitrocellulose
membranes (Amersham, Arlington Heights, IL). The membranes
were immunoprobed with primary antibodies at 4 °C overnight
separately, followed by secondary antibody IRDye 800-conjugated
goat anti-mouse IgG (Rockland, Immunochemicals, Gibertsville,
PA) or Alexa Fluor 680 goat anti-rabbit IgG (Life Technologies,
Grand Island, NY) for 1 h at room temperature. Bands were
detected using Li-Cor Odyssey Imaging System (Li-Cor, Lincoln,
NE).
2.8. General synthesis methods
All chemicals were purchased from either Sigma-Aldrich or
Acros Organics. Column chromatography was carried out with sil-
ica gel G (25–63 lm). Mass Spectra were measured on an Agilent
6520 Mass Q-TOF instrument. 1H NMR and 13C NMR spectra were
recorded on BRUKER 500 MHz or 400 MHz spectrometer, using
TMS as an internal standard and CDCl3 or DMSO-d6 as solvents.
Chemical shifts (d values) and coupling constants (J values) are
reported in ppm and hertz, respectively. Anhydrous solvent and
reagents were all analytically pure and dried through routine pro-
tocols. All compounds that were evaluated in biological essays had
>95% purity verified by HPLC.
2.4. Soft agar assay for colony formation
0.5 mL base layer of 0.5% agar in DMEM medium containing 10%
fetal bovine serum was placed in 24-well plates and permitted to
solidify at room temperature. Cells to be tested for colony forma-
tion were suspended in the top layer (0.5 mL) of 0.3% agar in
DMEM medium with 10% fetal bovine serum. The agar was permit-
ted to solidify at room temperature. 0.25 mL feeding medium was
added on the top layer with 1% DMSO (control) or compounds at
the indicated concentrations for 10 d at 37 °C in a humidified incu-
bator in an atmosphere of 5% CO2. The feeding medium was change
twice a week. Cell colonies were stained with 0.005% Crystal Violet
and inspected with a dissecting microscope.
2.8.1. 2-Methyl-6-nitroquinazolin-4(3H)-one (6a)
2-Amino-5-nitro benzoic acid (1.821 g, 10.0 mmol) was added
to acetic anhydride (60 mL). The mixture was stirred at 110 °C
for 5 h. The mixture was evaporated to dryness under reduced
pressure. The residue was mixed with ammonium hydroxide
solution (20–28% NH3 in H2O, 60 mL) and the mixture was stirred
at 55 °C for 10 h. The suspension was cooled to 5 °C, and the
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048

D. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
precipitate was filtered and washed with water. The filter cake was
dried under vacuum pressure to give the desired product as a bright
yellow solid (1.560 g, 76% yield). 1H NMR (500 MHz, DMSO-d6) d
8.74 (d, J = 2.5 Hz, 1H), 8.48–8.50 (dd, J = 2.5, 9 Hz, 1H), 7.73 (d,
J = 9 Hz, 1H), 2.41 (s, 3H); 13C NMR (125 MHz, DMSO-d6) d 160.9,
158.6, 153.1, 144.3, 128.2, 128.2, 121.8, 120.5; HRMS (ESI) m/z:
calcd for C9H8N3O3Na [M+Na]+ 228.0385, found 228.0372.
(d, J = 6.5 Hz, 3H), 1.16 (t, J = 7.0 Hz, 6H); 13C NMR (125 MHz,
DMSO-d6) d 163.2, 159.9, 154.4, 144.2, 137.1, 135.6, 134.0, 132.4,
132.0, 129.7, 128.9, 126.4, 119.7, 113.7, 52.7, 51.0, 47.5, 34.1,
22.9, 20.3, 10.4; HRMS (ESI) m/z: calcd for C25H28Cl3N5O2 [M+H]+
536.1381, found 536.1404.
2.8.6. (E)-N4-(5-(diethylamino)pentan-2-yl)-2-(2,4,6-trichlorostyryl)
quinazoline-4,6-diamine (6)
2.8.2. 2, 4, 6-Trichlorobenzaldehyde (6b)
To a solution of 6e (310 mg, 0.58 mmol) in MeOH/H2O (1:1,
22 mL) was added ammonium chloride (310 mg, 5.8 mmol) and
Fe powder (167 mg, 2.99 mmol). The resulting solution was
reluxed for 4 h and monitored (by LCMS) for the conversion to
the corresponding aniline. The reaction was then filtered and the
solvent was removed in vacuo. After cooling to room temperature,
the mixture was filtered through a pad of Celite which was washed
with MeOH and the filtrate was concentrated in vacuo. The crude
residue was adjusted to pH 7 by saturated NaHCO3 solution and
extracted with CHCl3. The combined CHCl3 layer was then washed
with brine, dried over anhydrous MgSO4 and solvent was removed
in vacuo. The resulting crude residue was purified by flash column
chromatography (loaded using DCM/Hexane, 1:5 then eluted with
CHCl3(NH4OH saturated)/MeOH, 10:1) and dried under high vac-
uum to afford the desired product 6 (205 mg, 70%) as red solid.
1, 3, 5-Trichlorobenzene (1.81 g, 10 mmol) was dissolved in
anhydrous THF (36 mL) and the mixture was stirred at À78 °C. n-
BuLi (1.6 M in Hexane) (6.3 mL, 10 mmol) was slowly added into
the solution over 0.5 h under À78 °C. The solution became light
yellow color. After all the n-BuLi was added, the mixture was kept
at À78 °C and stirred for 0.5 h then anhydrous DMF (1.32 mL,
17 mmol) was added into the mixture dropwise and the resulting
light yellow solution was continuously stirred at À78 °C for
another 1.5 h. The reaction was quenched with 3 N HCl solution
(36 mL) while kept the flask at À78 °C and warmed to ambient
temperature to afford a colorless solution. The mixture was
extracted with ethyl acetate (20 mL Â 3) and the organic layer
was then washed with saturated NaHCO3 (aq) and brine, respec-
tively. The organic layer was dried over anhydrous MgSO4 and con-
centrated to afford product 6b as a white needle like solid (1.99 g,
96%). 1H NMR (500 MHz, CDCl3) d 10.43 (s, 1H), 7.42 (s, 2H); 13C
NMR (125 MHz, CDCl3) d 187.7, 139.4, 137.8, 130.0, 128.8. HRMS
(ESI) m/z: calcd for C7H4Cl3O [M+H]+ 208.92, found 208.9.
1H NMR (400 MHz, CDCl3)
d 8.56 (s, 1H), 8.06–8.02 (d,
J = 16.0 Hz, 1H), 7.69–7.66 (d, J = 8.8 Hz, 1H), 7.46–7.42 (m, 1H),
7.39–7.38 (m, 3H), 7.13–7.07 (m, 1H), 4.62 (m, 1H), 3.08–2.94
(m, 6H), 20.2–1.78 (m, 4H), 1.40–1.39 (d, J = 6.4 Hz, 3H), 1.25–
1.22 (t, 6H); 13C NMR (100 MHz, CDCl3): d 168.9, 157.7, 156.9,
144.9, 143.8, 137.8, 135.2, 133.0, 132.9, 129.6, 128.6, 128.1,
122.9, 115.4, 103.2, 55.1, 46.5, 46.4, 33.5, 22.0, 20.8, 9.2. HRMS
(ESI) m/z: calcd for C25H31Cl3N5 [M+H]+ 506.16, found 506.1.
2.8.3. 6-Nitro-2-(2, 4, 6-trichlorostyryl)quinazolin-4(3H)-one (6c)
A solution of 6a (205 mg, 1 mmol) and 6b (210 mg, 1 mmol) in
acetic acid (3 mL) was refluxed for 30 h. The mixture was cooled to
room temperature, and the precipitate was filtered and washed
with methanol (2 mL Â 3). The filter cake was collected to give
the desired product as a yellow solid (329 mg, 83% yield). 1H
NMR (500 MHz, DMSO-d6) d 8.77 (s, 1H), 8.25 (m, 1H), 7.82 (m,
1H), 7.75 (s, 2H), 7.55 (s, 1H), 6.99 (m, 1H). HRMS (ESI) m/z: calcd
for C16H9C13N3O3 [M+H]+ 395.96, found 396.0.
2.8.7. (E)-N1,N1-diethyl-N2-(6-nitro-2-(2,4,6-trichlorostyryl)
quinazolin-4-yl)propane-1,2-diamine (17a)
Compound 17a (95 mg, 59.3%yield) was prepared by the same
method as 6e. HRMS (ESI) m/z: calcd for C23H25Cl3N5O2 [M+H]+
508.10, found 508.1.
2.8.4. N1,N1-diethyl-N4-(6-nitro-2-(2,4,6-trichlorostyryl)quinazolin-
4-yl)pentane-1,4-diamine (6d)
2.8.8. (E)-N4-(1-(diethylamino)propan-2-yl)-2-(2,4,6-trichlorostyryl)
quinazoline-4,6-diamine (17)
A solution of 6c (397 mg, 1.0 mmol) and DIEA (0.4 mL) in POCl3
(10 mL) was refluxed for 3 h. The mixture was cooled to room tem-
perature and concentrated to remove the solvent. The residue was
added ice-water, and adjusted pH to 9–10 with saturated solution
of Na2CO3. The mixture was extracted with EA (10 mL Â 3). The
combined organic phases were dried with anhydrous Na2SO4, fil-
tered and concentrated. The residue was purified by column chro-
matography (PE/EA = 4:1) to give the desired product as a light
brown solid (274 mg, 66% yield). HRMS (ESI) m/z: calcd for C16H8-
Cl4N3O2 [M+H]+ 413.93, found 413.9.
Compound 17 (35 mg, 28% yield) was prepared by the same
method as 6. 1H NMR (400 MHz, CDCl3) d 8.55 (s, 1H), 8.18–8.16
(d, J = 7.6 Hz, 1H), 7.95–7.91 (d, J = 16.4 Hz, 1H), 7.68–7.66 (d,
J = 8.8 Hz, 1H), 7.39 (s, 2H), 7.38–7.34 (d, J = 16.0 Hz, 1H), 7.33–
7.32 (m, 1H), 7.13–7.11 (m, 1H), 5.03 (m, 1H), 3.74–3.68 (m, 1
H), 3.17–3.06 (m, 5 H), 1.52–1.50 (d, J = 6.8 Hz, 3H), 1.31–1.27 (t,
J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3): d 168.5, 157.9, 156.2,
145.3, 137.5, 135.2, 133.1, 132.7, 129.1, 128.7, 127.8, 123.3,
115.9, 104.1, 55.1, 46.6, 42.8, 18.9, 8.5. HRMS (ESI) m/z: calcd for
C
23H27Cl3N5 [M+H]+ 478.13, found 478.1.
2.8.5. (E)-N1,N1-diethyl-N2-(6-nitro-2-(2,4,6-trichlorostyryl)
quinazolin-4-yl)propane-1,2-diamine (6e)
2.8.9. (E)-N1,N1-diethyl-N4-(6-nitro-2-(2,4,6-trichlorostyryl)
quinazolin-4-yl)butane-1,4-diamine (18a)
Compound 6d (415 mg, 1 mmol) was dissolved in THF (15 mL)
and the solution was added N1,N1-diethylpentane-1,4-diamine
(316 mg, 2.0 mmol). The reaction mixture was stirred at room tem-
perature for 3 h. The mixture was diluted with EA (60 mL). It was
washed with water and saturated solution of NH4Cl. Then the
organic layer was dried by anhydrous Na2SO4 and the solvents
were removed at reduced pressure. After purified by flash column
chromatography (30:1, CHCl3/CH3OH), compounds 6e (301 mg,
yield 56%) was got as a golden yellow solid. 1H NMR (500 MHz,
DMSO-d6) 9.01 (s, 1H), 8.42–8.44 (dd, J = 2.0, 9.0 Hz, 1H), 8.19 (d,
J = 16.0 Hz, 1H), 7.88 (d, J = 6.5 Hz, 1H), 7.83 (d, J = 9.5 Hz, 1H),
7.41 (s, 2H), 7.33 (d, J = 16.0 Hz, 1H), 4.59–4.60 (m, 1H), 2.81–
2.82 (m, 4H), 2.69 (m, 3H), 2.03–2.05 (m, 1H), 1.82 (m, 3H), 1.42
Compound 18a (105 mg, 72% yield) was prepared by the same
method as 6e. HRMS (ESI) m/z: calcd for C24H27Cl3N5O2 [M+H]+
522.12, found 522.1.
2.8.10. (E)-N4-(4-(diethylamino)butyl)-2-(2,4,6-trichlorostyryl)
quinazoline-4,6-diamine (18)
Compound 18 (65 mg, 66%yield) was prepared by the same
method as 6. 1H NMR (400 MHz, CDCl3) d 11.49 (br, 1H), 9.16 (br,
1H), 8.11–8.07 (d, J = 16.0 Hz, 1H), 7.50–7.45 (m, 1H), 7.43–7.42
(m, 2H), 7.35 (s, 2H), 6.95–6.94 (d, J = 7.6 Hz, 1H), 3.75 (br, 2H),
3.14 (br, 6H), 1.97 (m, 2H), 1.87 (m, 2H), 1.34 (t, J = 6.8 Hz, 6H);
13C NMR (100 MHz, CDCl3): d 158.6, 152.1, 148.2, 135.9, 135.2,
134.7, 129.8, 129.6, 129.1, 126.8, 124.0, 120.5, 114.1, 104.5, 51.4,
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048

4
D. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
46.5, 40.9, 25.0, 21.3, 8.3. HRMS (ESI) m/z: calcd for C24H29Cl3N5 [M
2.8.16. N1,N1-diethyl-N4-(6-nitro-2-(2,4,6-trichlorophenethyl)
quinazolin-4-yl)pentane-1,4-diamine (21d)
+H]+ 492.14, found 492.1.
Compound 21d (90 mg, 48% yield) was prepared by the same
method as 6e. HRMS (ESI) m/z: calcd for C25H31Cl3N5O2 [M+H]+
538.14, found 538.1.
2.8.11. (E)-N1,N1-diethyl-N3-(6-nitro-2-(2,4,6-trichlorostyryl)
quinazolin-4-yl)butane-1,3-diamine (19a)
Compound 19a (85 mg, 62% yield) was prepared by the same
method as 6e. HRMS (ESI) m/z: calcd for C24H27Cl3N5O2 [M+H]+
522.12, found 522.1.
2.8.17. N4-(5-(diethylamino)pentan-2-yl)-2-(2,4,6-
trichlorophenethyl)quinazoline-4,6-diamine (21)
Compound 21 (90 mg, 48% yield) was prepared by the same
method as 6. 1H NMR (400 MHz, CDCl3) d 8.70 (s, 1H), 7.62–7.60
(d, J = 8.8 Hz, 1H), 7.30 (s, 2H) 7.26–7.24 (d, 2H), 7.12–7.09 (d,
J = 9.2 Hz, 1H), 6.35 (br, 1H), 4.44 (m, 1H), 3.46–3.04 (m, 2H),
2.94–2.90 (m, 5H), 2.82–2.80 (m, 1H), 1.31–1.29 (d, J = 6.4 Hz,
3H), 1.22–1.18 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3): d
168.9, 161.9, 158.4, 144.5, 142.7, 136.9, 136.0, 132.1, 128.0,
128.0, 123.1, 114.4, 103.2, 51.9, 46.3, 37.1, 33.3, 29.8, 21.6, 20.8,
9.0. HRMS (ESI) m/z: calcd for C25H33Cl3N+5 [M+H]+ 508.17, found
508.1.
2.8.12. (E)-N4-(4-(diethylamino)butan-2-yl)-2-(2,4,6-trichlorostyryl)
quinazoline-4,6-diamine (19)
Compound 19 (25 mg, 31% yield) was prepared by the same
method as 6. 1H NMR (400 MHz, CDCl3)
d 8.06–8.02 (d,
J = 16.0 Hz, 1H), 7.75–7.69 (m, 2H), 7.46–7.42 (d, J = 16.4 Hz, 1H),
7.38 (s, 2H), 7.14–7.15 (d, J = 9.2 Hz, 1H), 4.54–5.52 (m, 1H),
3.43–3.37 (m, 1H), 3.18–3.16 (m, 2H), 3.11–2.98 (m, 4H), 2.45–
2.36 (m, 1H), 2.15–2.10 (m, 1H), 1.46–1.45 (d, J = 6.4 Hz, 3H),
1.33 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 158.4, 155.7,
145.8, 135.6, 135.4, 133.4, 132.3, 129.3, 128.7, 127.5, 123.3,
115.5, 104.2, 49.6, 45.2, 30.7, 20.9, 8.3. HRMS (ESI) m/z: calcd for
2.8.18. (E)-6-Nitro-2-(4-(trifluoromethyl)styryl)quinazolin-4(3H)-one
(22a)
Compound 22a (170 mg, 47% yield) was prepared by the same
method as 6c. HRMS (ESI) m/z: calcd for C17H11F3N3O3 [M+H]+
362.07, found 362.1.
C
24H29Cl3N5 [M+H]+ 492.14, found 492.1.
2.8.13. 3-(2,4,6-Trichlorophenyl)propanoic acid (21a)
A mixture of 6b (0.81 g, 3.87 mmol), 2,2-dimethyl-1,3-dioxane-
4,6-dione (0.61 g, 4.26 mmol), triethylamine (780 mg, 7.72 mmol)
and formic acid (0.87 g, 19.mmol) in DMF (20 mL) was stirred at
100 °C for 3 h. The mixture was cooled to room temperature and
water was added (60 mL). The mixture was extracted with EA
(25 mL Â 2). The combined organic layers were washed with water
and brine. The solution was dried with anhydrous Na2SO4, filtered
and concentrated to give the desired product as a white solid
(0.9 g, 92.4% yield). 1H NMR (400 MHz, CDCl3) d 11.56 (br, 1H),
7.32 (s, 2H), 3.26–3.22 (t, J = 8.4 Hz, 2H), 2.63–2.59 (m, 2H). HRMS
(ESI) m/z: calcd for C9H6Cl3O2 [M-H]- 250.95, found 250.9.
2.8.19. (E)-4-Chloro-6-nitro-2-(4-(trifluoromethyl)styryl)quinazoline
(22b)
Compound 22b (116 mg, 65% yield) was prepared by the same
method as 6d. HRMS (ESI) m/z: calcd for C17H10ClF3N3O2 [M+H]+
380.03, found 380.0.
2.8.20. (E)-N1,N1-diethyl-N4-(6-nitro-2-(4-(trifluoromethyl)styryl)
quinazolin-4-yl)pentane-1,4-diamine (22c)
Compound 22c (crude 100 mg) was prepared by the same
method as 6e.The crude product was used in next step without fur-
ther purification. HRMS (ESI) m/z: calcd for C26H31F3N5O2 [M+H]+
502.24, found 502.2.
2.8.14. 6-Nitro-2-(2,4,6-trichlorophenethyl)quinazolin-4(3H)-one
(21b)
2.8.21. (E)-N4-(5-(diethylamino)pentan-2-yl)-2-(4-(trifluoromethyl)
styryl)quinazoline-4,6-diamine (22)
A mixture of 3-(2,4,6-trichlorophenyl)propanoic acid (0.9 g,
3.6 mmol) in SOCl2 (10 mL) was refluxed for 2 h. The mixture
was concentrated to dryness to give 2-(2,4,6-trichlorophenyl)
acetyl chloride (1.0 g, crude). To the acyl chloride was added drop-
wise the solution of 2-amino-5-nitrobenzoic acid (0.65 g,
3.6 mmol) in pyridine (15 mL). The mixture was stirred at room
temperature for 2 h. LC-MS showed the reaction was completed.
The reaction was concentrated to remove the solvent and the resi-
due was added acetic anhydride (20 mL). The mixture was stirred
at 110 °C for 4 h. The mixture was evaporated to dryness under
reduced pressure. The residue was mixed with ammonium hydrox-
ide solution (20–28% NH3 in H2O) and the mixture was stirred at
60 °C for 2 h. The suspension was cooled to 5 °C, and the precipi-
tate was filtered and washed with water. The filter cake was dried
under vacuum pressure to give the desired product as a yellow
solid (1.13 g, 79.8% yield). 1H NMR (400 MHz, CDCl3) d 8.79–8.18
(d, J = 2.4 Hz, 1H), 8.49–8.46 (m, 1H), 7.75–7.73 (d, J = 8.8 Hz, 1H),
7.66 (s, 2H), 3.23–3.22 9 (m, 2H), 2.92–2.88 (t, J = 7.6 Hz, 2H).
HRMS (ESI) m/z: calcd for C16H11Cl3N3O3 [M+H]+ 397.98, found
398.0.
Compound 22 (70 mg, 75% yield) was prepared by the same
method as 6. 1H NMR (400 MHz, CDCl3) d 8.26 (s, 1H), 7.90–7.86
(d, J = 16.0 Hz, 1H), 7.71–7.60 (m, 5H), 7.39 (m, 1H) 7.32–7.28
(m, 1H), 7.13–7.10 (m, 1H), 6.75–6.74 (d, J = 5.6 Hz, 1H), 4.68 (m,
1H), 3.05–2.99 (m, 5H), 2.976–2.91 (m, 1H), 1.98–1.82 (m, 4H),
1.40–1.38 (d, J = 6.4 Hz, 3H), 1.25–1.21 (t, J = 7.2 Hz, 6H); 13C
NMR (100 MHz, CDCl3) d 168.4, 158.1, 156.7, 145.3, 142.4, 140.3,
133.9,131.2, 130.0, 128.4, 127.4, 125.6, 125.6, 123.1, 115.3, 103.7,
51.7, 46.3, 46.2, 32.9, 21.2, 21.0, 8.3. HRMS (ESI) m/z: calcd for
C
26H33F3N5 [M+H]+ 472.26, found 472.3.
2.8.22. (E)-6-Nitro-2-(3-(trifluoromethyl)styryl)quinazolin-4(3H)-one
(23a)
Compound 23a (286 mg, 40% yield) was prepared by the same
method as 6c. 1H NMR (400 MHz, DMSO-d6) d 12.79 (s, 1H), 8.81
(d, J = 2.4 Hz, 1H), 8.56–8.53 (m, 1H), 8.16–8.12 (d, J = 16.4 Hz,
1H), 8.045–8.00 (m, 2H), 7.86–7.82 (m, 2H), 7.74–7.71 (t,
J = 7.6 Hz, 1H), 7.21–7.17 (d, J = 16.0 Hz, 1H). HRMS (ESI) m/z: calcd
for C17H11F3N3O3 [M+H]+ 362.07, found 362.1.
2.8.15. 4-Chloro-6-nitro-2-(2,4,6-trichlorophenethyl)quinazoline
(21c)
2.8.23. (E)-4-Chloro-6-nitro-2-(3-(trifluoromethyl)styryl)quinazoline
(23b)
Compound 21c (560 mg, 48% yield) was prepared by the same
method as 6d. HRMS (ESI) m/z: calcd for C16H10Cl4N3O2 [M+H]+
415.94, found 415.9.
Compound 23b (210 mg, 70% yield) was prepared by the same
method as 6d. HRMS (ESI) m/z: calcd for C17H10ClF3N3O2 [M+H]+
380.03, found 380.0.
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048

D. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
2.8.24. (E)-N1,N1-diethyl-N4-(6-nitro-2-(3-(trifluoromethyl)styryl)
quinazolin-4-yl)pentane-1,4-diamine (23c)
Compound 23c (132 mg, 48% yield) was prepared by the same
method as 6e. HRMS (ESI) m/z: calcd for C26H31F3N5O2 [M+H]+
502.24, found 502.2.
and the maternal embryonic zipper kinase (MELK). To better
understand how these compounds engage the ATP-binding pocket
of kinases, we selected MELK to pursue a limited structure-activity
study.
We purchased additional analogs of 1 (compounds 2–15) and
tested several compounds for inhibition of MELK (Fig. 1a). Com-
pound 6 showed the most potent activity with more than 98% inhi-
bition at 20 mM. To identify additional targets for 6, we tested
whether the compound inhibited the activity of the top 25 kinase
targets of compound 1 that were identified from a previous profil-
ing of 337 kinases (Fig. 1b). We found that 6 inhibits kinases with
IC50 values ranging from 1.4 0.9 mM for MELK to 42.7 4.6 mM for
FER kinase. Compound 6 inhibited two other kinases with an IC50
less than 2 mM, namely BMX/ETK (1.9 0.6 mM) and TIE2/TEK
(1.8 0.9 mM).
2.8.25. (E)-N4-(5-(diethylamino)pentan-2-yl)-2-(3-(trifluoromethyl)
styryl)quinazoline-4,6-diamine (23)
Compound 23 (68 mg, 54% yield) was prepared by the same
method as 6. 1H NMR (400 MHz, CDCl3) d 8.67 (s, 1H), 7.90–7.86
(d, J = 15.6 Hz, 1H), 7.81(m, 2H), 7.68–7.65 (d, J = 8.8 Hz, 1H),
7.54–7.49 (m, 1H), 7.41–7.07 (m, 1H), 7.32–7.28 (d, J = 15.6 Hz,
1H), 7.12–7.09 (m, 1H), 6.79 (br, 1H), 4.69 (m, 1H), 3.07–3.00 (m,
5H), 2.97–2.92 (m, 1H), 2.00–1.90 (m, 4H), 1.40–1.39 (d,
J = 6.8 Hz, 3H), 1.25–1.22 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz,
CDCl3) d 168.8, 158.1, 156.3, 145.7, 141.6, 137.5, 134.1, 131.2,
130.9, 130.0, 129.2, 127.8, 125.4, 124.8, 124.3, 124.2, 123.2,
122.7, 115.2, 103.6, 51.7, 46.3, 46.1, 33.0, 21.2, 20.9, 8.4. HRMS
(ESI) m/z: calcd for C26H33F3N5 [M+H]+ 472.26, found 472.2.
To explore the role of each substituent of 6 on the inhibition of
MELK activity, we prepared
9 derivatives of the compound
(Scheme 2, 3, and Fig. 1c). The role of the amine at R2 was investi-
gated by replacing it with a methoxy group affording compound
16. The compound was threefold weaker than 6 suggesting that
the amino group was engaged in favorable interactions likely
through hydrogen bonding (Fig. 1d). The role of the R1 group was
explored in compounds 17, 18, and 19. In 17 and 19 the N-alkyl-
mine moiety was shortened resulting in twofold increase in the
IC50 over the parental compound. Removal of the methyl group
at the carbon atom directly attached to the quinazoline ring in
18 led to little effect on the IC50. An introduction of a morpholino
group in 20 led to a twofold reduction in activity. The effect of
the olefin at R3 was explored by the synthesis of 21. The loss of
activity by nearly 3-fold suggests that a planar moiety at R1 is
preferable for optimal binding to the kinase. This group interacts
with sidechains of the hinge loop in the ATP-binding pocket of
the enzyme (Fig. 1e). At R3, a trifluoromethyl moiety was intro-
duced at the para (compound 22), meta (compound 23), ortho
(compound 24) positions of the ring. Both 23 and 24 had 3-fold
weaker activity than the parent compound, except for 22, which
2.8.26. (E)-6-Nitro-2-(2-(trifluoromethyl)styryl)quinazolin-4(3H)-one
(24a)
Compound 24a (306 mg, 42% yield) was prepared by the same
method as 6c. 1H NMR (400 MHz, DMSO-d6) d 12.99 (s, 1H), 8.81
(d, J = 2.8 Hz, 1H), 8.55–8.52 (m, 1H), 8.34–8.31 (d, J = 14.0 Hz,
1H), 7.96–7.95 (d, J = 8.0 Hz, 1H), 7.90–7.80 (m, 3H, 7.69–7.65 (t,
J = 7.6 Hz, 1H), 7.11–7.07 (d, J = 15.6 Hz, 1H). HRMS (ESI) m/z: calcd
for C17H11F3N3O3 [M+H]+ 362.07, found 362.1.
2.8.27. (E)-4-Chloro-6-nitro-2-(2-(trifluoromethyl)styryl)quinazoline
(24b)
Compound 24b (80 mg, 64% yield) was prepared by the same
method as 6d. HRMS (ESI) m/z: calcd for C17H10ClF3N3O2 [M+H]+
380.03, found 380.0.
2.8.28. (E)-N1,N1-diethyl-N4-(6-nitro-2-(2-(trifluoromethyl)styryl)
quinazolin-4-yl)pentane-1,4-diamine (24c)
resulted in a similar IC50
.
Compound 24c (80 mg, 57% yield) was prepared by the same
method as 6e. HRMS (ESI) m/z: calcd for C26H31F3N5O2 [M+H]+
502.24, found 502.2.
3.2. Compounds inhibit cancer cell growth in anchorage-dependent
and independent assays
Considering that the kinases inhibited by the compound are
known to be up-regulated in various basal-like breast tumors, we
explored the effect of 1–15 on breast MDA-MB-231 cell viability
(Fig. 2). Most of the compounds inhibited breast MDA-MB-231 cell
growth with IC50 values that ranged from 2 to 30 mM (Fig. 2a).
Except for 13 (KIN-288) and 12 (KIN-289), all compounds had
IC50 values that were lower than 10 mM. The most potent com-
pound was 6 (KIN-281) with an IC50 of 2 mM. We also explored
the effect of the compounds in anchorage-independent growth
assay in soft agar. Colony formation in soft agar provides a better
representation of oncogenic transformation, tumor growth, as well
the potential for tumors to metastasize.2 MDA-MB-231 cells form
colonies in soft agar as illustrated in Fig. 2b and c. Compounds 1
(KIN-1), 4 (KIN-236), 6 (KIN-281) and 7 (KIN-283) inhibited colony
formation in a concentration-dependent manner with IC50 values
ranging from 3 to 7 mM. Consistent with cell viability studies, 6
(KIN-281) was generally more potent than the other three
compounds.
2.8.29. (E)-N4-(5-(diethylamino)pentan-2-yl)-2-(2-(trifluoromethyl)
styryl)quinazoline-4,6-diamine (24)
Compound 24 (20 mg, 36% yield) was prepared by the same
method as 6. 1H NMR (400 MHz, CDCl3) d 8.69 (s, 1H), 8.39–8.35
(m, 1H), 7.93–7.91 (d, J = 8.0 Hz, 1H), 7.69–7.64 (m, 2H), 7.58–
7.54 (t, J = 7.6 Hz, 1H), 7.40–7.36 (t, J = 7.6 Hz, 1H), 7.23–7.18 (d,
J = 15.6 Hz, 1H), 7.13–7.10 (m, 1H), 6.42 (br, 1H), 4.61 (m, 1H),
3.00–2.94 (m, 4H), 2.88–2.86 (m, 1H), 1.95–1.90 (m, 4H), 1.41–
1.39 (d, J = 6.4 Hz, 3H), 1.22–1.19 (t, J = 7.2 Hz, 6H); 13C NMR
(100 MHz, CDCl3) d 157.8, 157.0, 145.1, 135.7, 132.5, 132.0,
131.0, 128.9, 127.7, 127.2, 126.0, 123.0, 15.4, 103.5, 51.7, 46.5,
46.1, 33.0, 21.3, 20.7, 8.5. HRMS (ESI) m/z: calcd for C26H33F3N5
[M+H]+ 472.26, found 472.2.
3. Results
3.1. Compounds inhibit protein kinases
Previously, structure-based virtual screening against CaMKIId
led to 1 (Scheme 1), which inhibited the enzyme with single-digit
micromolar IC50. Kinome profiling against 337 kinases of 4 (an ana-
log of 1 with identical IC50) revealed that CaMKIId was among the
top 10 targets of the compound. Also, the compound inhibited
other members of CaMK family of proteins that included CaMKI
3.3. Compounds Up-regulate p21WAF1/CIP1 in a p53-dependent manner
Previous studies had shown that MELK contributes to cell cycle
progression by causing G/M arrest,3,4 and reduced the number of
cells at the G2/M transition.4,5 These studies also showed that
knockdown of MELK resulted in up-regulation of p21WAF1/CIP1 in
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048

6
D. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Scheme 1. Chemical structure of active compounds identified from virtual screening.
Fig. 1. Small molecules inhibit MELK kinase. (a) Derivatives of 1 (KIN-1) were tested for inhibition of MELK activity at 20 mM; data shown as mean SD. (b) Compound 6 (KIN-
281) was tested for its inhibition of 25 kinases that were selected from a previous kinome profiling. IC50s were obtained for each kinase; data shown as mean SD. (c)
Chemical structure of derivatives of 6 (KIN-281) that were synthesized along with IC50 for inhibition of MELK activity. (d) The binding mode of 6 (KIN-281, shown as cyan
stick) to MELK (PDB: 4cqg, shown as gray cartoon). MELK is shown in cartoon, with the side chain of key residues shown in stick. The hinge loop and DFG motif of MELK are
colored in red and green, respectively. The glycine-rich loop from Glu15 to Leu27 is removed to visualize the ATP binding site. In the model, the primary amine on the
quinazoline core of the compound forms a hydrogen bond with Glu57 on the aC helix of the MELK (shown as dotted orange line). The N-alkylamine tail of the compound is
stabilized by a salt bridge to Asp150 in the DFG motif of the kinase (shown as dotted green line). (e) A 2D view of the binding mode of (S)-6 to MELK. Residues are shown as
droplets, with the orientation of the droplet indicating the direction of the side chain of the given residue. The hydrogen bond between Glu57 and the quinazoline core is
shown in magenta. The salt bridge between the N-alkylamine tail of the compound and Asp150 is shown as a line colored as a gradient from orange to blue. The ligand
interaction diagram is generated using Schrödinger Maestro and a cutoff of 4 Å.
U87 GBM cell lines.4 We observed the same effect for 1 (KIN-1) and
6 (KIN-281); at 5 mM, the compound up-regulated p21WAF1/CIP1 in
MDA-MB-468 (Fig. 3a and b) and MDA-MB-231 (Fig. 3c and d). A
time-dependent study was conducted with both of these cell lines.
After 4 h, both 1 (KIN-1) and 6 (KIN-281) up-regulate p21WAF1/CIP1
The effect on p21WAF1/CIP1 was sustained over 48 h in both cell
.
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048

D. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Scheme 2. Synthesis of derivatives of 6 (KIN-281).
Scheme 3. Synthesis of 21.
lines. We investigated the effects of p21WAF1/CIP1 on cyclins, which
are inhibitors of p21WAF1/CIP1. We found that an increase in
p21WAF1/CIP1 corresponded closely to a decrease in cyclin A2, which
was gradually down-regulated over the course of 48 h (Fig. 3a–d).
Although p21WAF1/CIP1 is a p53 target gene, treatment of MDA-
MB-468 cells with 1 (KIN-1) and 6 (KIN-281) showed no effect
on p53 levels over 48 h (Fig. 3e). We investigated whether the
compounds had any effects on p53 phosphorylation, particularly
at Ser15, which has been previously found to be affected by MELK
suppression. Phosphorylation at Ser15 inhibits the interaction of
p53 with its antagonists MDM2 or MDMX, which de-stabilize
p53 and inhibit apoptosis. Interestingly, both compounds inhibited
phosphorylation at Ser15 of p53 consistent with a potential effect
on MELK activity (Fig. 3f).
3.4. Compounds block STAT3 and down-regulate pro-apoptotic
markers survivin and Bcl-XL
Both 1 (KIN-1) and 6 (KIN-281) inhibited STAT3 phosphoryla-
tion in MDA-MB-468 (Fig. 4a and b) and MDA-MB-231
(Fig. 4c and d) cells. A time course revealed that STAT3 phosphory-
lation was completely inhibited at 4 h, but phosphorylation levels
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048

8
D. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 2. Compounds inhibits anchorage dependent and independent growth. (a) Effect of 1 (KIN-1) and derivatives on MDA-MB-231 measured by MTT over 3 days; data are
shown as mean SD (n = 3) (b) Inhibition of anchorage-independent growth in MDA-MB-231 cancer cells (c) Quantification of data in (b); each value represents the
mean SE taken from six different fields from two independent samples.
Fig. 3. Compounds up-regulate p21. (a) Detection of cyclin A2 and p21 levels over the course of 48 h as a result of treatment MDA-MB-468 cells. b-actin serves as loading
control. (b) Quantification of data shown in (a). The data are presented as percentage changes (mean SD; n = 2). (c) Detection of cyclin A2 and p21 levels in MDA-MB-231
cells following treatment with 1 (KIN-1) and 6 (KIN-281) over 48 h. Actin is used as loading control. (d) Quantification of data in (c); the data are presented as percentage
changes relative to control (mean SD; n = 2). (e) Detection of p53 phosphorylation and p53 levels following treatment of MDA-MB-468 cells over 48 h. Actin is used as
loading control. (f) Effect of increasing concentration of 6 on MDMX levels detected by immunoblot analysis following treatment of MDA-MB-231.
were restored after 8 h in MDA-MB-468 cells. A similar observation
was made in MDA-MB-231 except that these cells recover STAT3
phosphorylation levels more slowly. The effect on STAT3 phospho-
rylation was confirmed with electrophoresis mobility shift assay
(EMSA), which showed that 1 (KIN-1) inhibited STAT3 dimer bind-
ing to DNA (Fig. 4e). It is of interest to note that JAK kinase activity
was not affected by our compounds as evidenced by the lack of
inhibition of JAK kinase activity by 6 (Fig. 4f). This suggests that
the inhibitors are following a non-canonical pathway to blocking
STAT3. Previous studies have suggested that BMX promotes STAT3
activation through the PI3K pathway in a manner that is indepen-
dent of Akt.6 We find that 1 (KIN-1) or 6 (KIN-281) had no effect on
Akt phosphorylation (Fig. 4g and h).
It is often the case that STAT3 phosphorylation leads to down-
regulation of survivin.7,8 Survivin is a member of the XIAP (X-
linked inhibitor of apoptosis) group of proteins. Studies have
shown that survivin works in a complex mechanism to promote
cell growth and to inhibit apoptosis. Survivin is a component of
the chromosomal passenger complex (CPC) and hence has a critical
role cell cycle progression and cell proliferation.9–12 Interestingly, 1
(KIN-1) and 6 (KIN-281) led to down-regulation of survivin in
MDA-MB-468 (Fig. 4a and b) and MDA-MB-231 (Fig. 4c and d).
The effect was sustained over the course of 48 h in both MDA-
MB-468 and MDA-MB-231. Interestingly, both 1 and 6 also
reduced expression of the pro-apoptotic protein Bcl-XL (Fig. 4a–d).
3.5. KIN-281 inhibits cell autophagy
A direct physical interaction between the anti-apoptotic pro-
teins Bcl-2 or Bcl-XL and the autophagy regulator Beclin-1 was pre-
viously shown to inhibit autophagy in cancer cells.13 In addition, it
has been shown that STAT3 promotes autophagy through a com-
plex with PKR.14 Since 1 and 6 suppress Bcl-XL levels and inhibit
STAT3 activation, we wondered whether treatment with these
compounds had an effect on autophagic flux. To test this, levels
of the processed LC3B, a widely used marker of autophagy15 were
measured in MDA-MB-231 and MDA-MB-468 cells following treat-
ment with compound. Autophagic flux was evaluated by determin-
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048

D. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
Fig. 4. Compounds impair STAT3 phosphorylation and down-regulate pro-apoptotic Bcl-XL and survivin. (a) Effect of 1 (KIN-1) and 6 (KIN-281) on STAT3, Bcl-XL, and survivin
levels as well as STAT3 phosphorylation analyzed by Western blot analysis at several time intervals over the course of 48 h in MDA-MB-468 cells. b-Actin is used as loading
control. (b) Quantification of data in (a); the data are presented as percentage changes relative to control (mean SD; n = 2). (c) Effect of 1 (KIN-1) and 6 (KIN-281) on STAT3,
Bcl-XL, and survivin levels as well as STAT3 phosphorylation analyzed by Western blot analysis at several time intervals over the course of 48 h in MDA-MB-231 cells. b-Actin
is used as loading control. (d) Quantification of data in (a); the data are presented as percentage changes relative to control (mean SD; n = 2). (e) EMSA analysis to establish
STAT3 binding to DNA; lane 1 is (f) Activity of 6 against JAK1, JAK2 and JAK3 kinases. (g) Western blot analysis of Akt phosphorylation as a result of treatment with 1. (h)
Quantification of data in (g); the data are presented as percentage changes relative to control (mean SD; n = 2).
ing the levels of LC3B-I and LC3B-II by Western blot analysis in the
presence or absence of the lysosomotropic agent bafilomycin A1
(BafA1) that inhibits autophagy at late stages. In both cases, we
found that the compounds led to increased levels of LC3B-II
(Fig. 5). Treatment of MDA-MB-468 cells with BafA1 resulted in
substantial increase in LC3B-II compared to DMSO control consis-
tent with there being robust autophagic flux in these cells at basal
conditions. MDA-MB-231 cells showed a more modest increase in
LC3B-II when treated with BafA1. Growth of cells at 1% oxygen
(hypoxia) was used as a positive control for autophagy induction
(lanes 5 and 6 in Fig. 5a and Fig. 5b). A pronounced increase in
LC3B-II levels were detected in MDA-B-468 cells (lane 3 in Fig. 5a)
and MDA-MB-231 cells (lane 3 in Fig. 5b) treated with compound.
However, this was not further increased by co-treatment with
BafA1 in either MDA-MB-468 or MDA-MB-231 cells suggesting
that the compound was inhibiting autophagic flux and causing
processed LC3B-II to accumulate.
4. Discussion
Here we test previously-identified quinazoline compounds in
TNBC breast cancer cells and found them to inhibit cell viability
and to impair colony formation in an anchorage-independent soft
Fig. 5. Compounds inhibit autophagy. Western blot analysis of LC3B-I and LC3B-II in MDA-MB-468 cells (a) and MDA-MB-231 cells (b) following treatment for 4 h in vehicle
control (DMSO), 5 mM 6 (KIN-281) or at 1% oxygen. Bafilomycin A1 was added at 100 nM for the last 4 h of incubation to lanes 2, 4 and 6 as indicated.
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048

10
D. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 6. Schematic illustrating potential mechanism of action of KIN-281. Inhibition of BMX by the compound may be responsible for suppressing STAT3 activation through the
PI3K pathway without affecting AKT. STAT3 activation leads to eIF2A phosphorylation and autophagy.
agar assay. The most potent among these compounds, namely 6
(KIN-281), inhibits several kinases with IC50s of approximately
1 mM, including maternal embryonic leucine zipper kinase (MELK)
and bone marrow X-linked (BMX). Studies have shown that both
MELK and BMX are overexpressed in a number of tumors and their
overexpression correlates strongly with patient outcome.16–18 BMX
is a member of the Tec non-receptor tyrosine kinase family. MELK
has been identified as an important target for other epithelial
tumors including colorectal, lung, breast, ovarian and brain.3 High
expression of MELK in cancer stem cells has also been found.19,20
MELK is overexpressed in basal-like breast cancer and several stud-
ies have implicated the enzyme with tumor growth and metasta-
sis.4,21 The inhibition of MELK, BMX and other kinases by 6 may
explain its effect in TNBC cancer cells.
Some of the effects that we observed for 6 (KIN-281) may be
partly associated with inhibition of MELK or BMX. MELK plays an
important role in cell cycle.22–25 Previous studies have shown that
knockdown of MELK in U87 led to p21WAF1/CIP1 up-regulation.4
Interestingly, we found that our compounds up-regulated
p21WAF1/CIP1 in a time-dependent manner over 48 h. The down-reg-
ulation of cyclin A2 that is induced by the compounds is consistent
with the effect on p21WAF1/CIP1. Studies that have suggested that
the effect on p21WAF1/CIP1 is a p53-dependent process have been
conflicting. One study has shown that MELK suppression has no
effect on Ser15 phosphorylation in U87 or levels of p53, suggesting
instead that the effect on p53 may be due to MDMX down-regula-
tion rather than an effect on p53.4 Another study found that sup-
pressing MELK led to inhibition of Ser15 phosphorylation and
increased stabilization of p53.26 1 (KIN-1) and 6 (KIN-281) had
no effect on the levels of p53 in agreement with both studies. How-
ever, the compound inhibited Ser15 phosphorylation in a time-
dependent fashion consistent with the results of Seong and co-
workers.26 We investigated the effect of the compounds on MDMX
levels and observed an increase in MDMX, consistent with the
observation of Seong and co-workers with MDM2.26 6 (KIN-281)
does not affect p53 levels in MDA-MB-468 or MDA-MB-231 cells.
BMX is an intracellular non-receptor tyrosine kinase member of
the Tec family of protein kinases. Like other Tec kinases, BMX pos-
sesses a membrane localization pleckstrin homology (PH) module
along with a BH, SH3, SH2, and kinase domains that mediate
protein-protein interactions and downstream signaling. BMX has
been shown to directly associate and phosphorylate STAT3.6 This
process was found to be independent of JAK kinases, which are
generally associated with STAT3 activation.27 The inhibition BMX
by 6 prompted us to explore whether STAT3 phosphorylation is
affected in both MDA-MB-231 and MDA-MB-468. STAT proteins
have dual roles: they transduce signals through the cytoplasm
and function as transcription factors in the nucleus.28,29 Following
phosphorylation from specific tyrosine kinases, STATs form stable
homodimers, sometimes heterodimers, through SRC homology 2
(SH2) domain interactions. In the nucleus, a STAT3 dimer complex
bind to DNA and modulate the transcription of genes that play a
critical role in promoting inflammatory and malignant processes.
The effects on STAT3 went beyond merely inhibition of phosphory-
lation of the transcription factor. We found through EMSA that 1
(KIN-1) inhibited STAT3 DNA-binding. This suggests that the com-
pound is likely also affecting STAT3 transcription factor activity.
However, compound 6 had no effect on JAK kinases suggesting that
inhibition of STAT3 occurs through another mechanism, possibly
through PI3K by activation of BMX. A recent study has shown that
phosphorylation of STAT3 in PI3K-transformed cells is driven by
BMX.6 The study demonstrates that instead of the usual PIP3/
Akt/mTOR pathway, PIP3 leads to phosphorylation of BMX, which
in turn phosphorylates STAT3 and leads to oncogenic phenotype
(Fig. 6). In fact, we tested the effects of the compounds on Akt
phosphorylation and found no effect confirming that the com-
pound does not affect the PI3K/Akt/mTOR pathway.
We found that the compounds also suppressed the levels of
pro-apoptotic survivin and Bcl-XL. Although survivin is often asso-
ciated with apoptosis, it is a cell cycle protein that is highly up-reg-
ulated in cancer versus normal cells. Survivin has multiple
functions. Initially thought to be strictly involved in programmed
cell death, survivin has been shown to play a key role in cell cycle
progression and cell proliferation. In fact, survivin is required for
several stages of cell division. At metaphase, it localizes to kineto-
chores, then transfers to the central spindle mid-zone at anaphase,
and finally accumulates in mid-bodies at telophase.30 It physically
associates with polymerized microtubules, mitotic chromatin-
associated spindle microtubules, and the kinetochore-associated
CPC through protein-protein interactions with Aurora B,9,10 Bore-
alin/Dasra B,11 and Crm1.12 Expression of survivin is controlled
by several transcription factors including p53, FoxM1, and STAT3.
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048

D. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
The effects of 6 on STAT3 levels and Bcl-XL prompted us to
explore the effect of the compounds on cell autophagy. Bcl-XL
forms a physical interaction with the BH3 domain of beclin-1.31
Phosphorylation of beclin-1 by the CaMK member death-associ-
ated protein kinase 1 (DAPK1) leads to the disruption of the
Bcl-XLÁbeclin-1 complex and subsequent increase in autophagy.32
Previous studies have shown that STAT3 promotes autophagy
through a complex with PKR (Fig. 6).14 Autophagy is a highly con-
served self-degradative process that plays a critical role in cellular
stress response and survival mechanisms. Cells use autophagy to
turnover damaged organelles, pathogens and large protein aggre-
gates. This degradation process provides a critical source of amino
acids, nucleotides, and fatty acids, especially for cancer cells, which
are generally unable to acquire sufficient nutrients to sustain the
ATP production required for these cells to survive. A commonly
(SOM), and by the Indiana University School of Medicine Biomed-
ical Research Grant (SOM).
References
1. Jamdade VS, Sethi N, Mundhe NA, Kumar P, Lahkar M, Sinha N. Br J Pharmacol.
2015;172:4228–4237.
2. Mori S, Chang JT, Andrechek ER, et al. Oncogene. 2009;28:2796–2805.
3. Gray D, Jubb AM, Hogue D, et al. Cancer Res. 2005;65:9751–9761.
4. Kig C, Beullens M, Beke L, et al. J Biol Chem. 2013;288:24200–24212.
5. Nakano I, Paucar AA, Bajpai R, et al. J Cell Biol. 2005;170:413–427.
6. Guryanova OA, Wu Q, Cheng L, et al. Cancer Cell. 2011;19:498–511.
7. Yu H, Kortylewski M, Pardoll D. Nat Rev Immunol. 2007;7:41–51.
8. Basseres D, Baldwin A. Oncogene. 2006;25:6817–6830.
9. Honda R, Körner R, Nigg EA. Mol Biol Cell. 2003;14:3325–3341.
10. Wheatley SP, Carvalho A, Vagnarelli P, Earnshaw WC. Curr Biol.
2001;11:886–890.
11. Gassmann R, Carvalho A, Henzing AJ, et al. J Cell Biol. 2004;166:179–191.
12. Knauer SK, Bier C, Habtemichael N, Stauber RH. EMBO Rep. 2006;7:1259–1265.
13. Mariño G, Niso-Santano M, Baehrecke EH, Kroemer G. Nat Rev Mol Cell Biol.
2014;15:81–94.
14. Shen S, Niso-Santano M, Adjemian S, et al. Mol Cell. 2012;48:667–680.
15. Klionsky DJ, Abdalla FC, Abeliovich H, et al. Autophagy. 2012;8:445–544.
16. Nakano I, Masterman-Smith M, Saigusa K, et al. J Neurosci Res. 2008;86:48–60.
17. Marie SK, Okamoto OK, Uno M, et al. Int J Cancer. 2008;122:807–815.
18. Pickard MR, Green AR, Ellis IO, et al. Breast Cancer Res. 2009;11:R60.
19. Ganguly R, Hong CS, Smith LGF, Kornblum HI, Nakano I. Mol Cancer Ther.
2014;13:1393–1398.
used marker for autophagy is LC3B-II.15 We found that
6
(KIN-281) led to a significant increase in LC3-II levels suggesting
that MELK may play a role in suppressing autophagy. The effects
of the compound at 5 mM were as pronounced as bafilomycin A1,
a well-known late-stage autophagy inhibitor. Inhibition of autop-
hagy may have benefits for cancer treatment, particularly breast
cancer where autophagy may help cells evade apoptosis.33,34
Beyond cell survival, accumulating evidence suggests that autop-
hagy is involved in nearly every step of the metastatic cascade.
In fact, 50% of human breast cancers exhibit heterozygous deletion
of beclin-1, which is critical for autophagosome formation.35,36 By
inhibiting autophagy, these compounds may thus have a beneficial
effect for TNBC patients by limiting progression of disease.
20. Gu C, Banasavadi-Siddegowda YK, Joshi K, et al. Stem Cells. 2013;31:870–881.
21. Wang Y, Lee Y-M, Baitsch L. eLife. 2014;e01763.
22. Mirey G, Chartrain I, Froment C, et al. Cell Cycle. 2005;4:806–811.
23. Seong H, Gil M, Kim K, Kim S, Ha H. Biochem J. 2002;361:597–604.
24. Cordes S, Frank CA, Garriga G. Development. 2006;133:2747–2756.
25. Davezac N, Baldin V, Blot J, Ducommun B, Tassan J-P. Oncogene.
2002;21:7630–7641.
26. Seong H-A, Ha H. J Biol Chem. 2012;287:20797–20810.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
27. Rawlings JS, Rosler KM, Harrison DA. J Cell Sci. 2004;117:1281–1283.
28. Yu H, Pardoll D, Jove R. Nat Rev Cancer. 2009;9:798–809.
29. Song L, Turkson J, Karras JG, Jove R, Haura EB. Oncogene. 2003;22:4150–4165.
30. Vagnarelli P, Earnshaw WC. Chromosoma. 2004;113:211–222.
31. Maiuri MC, Le Toumelin G, Criollo A, et al. EMBO J. 2007;26:2527–2539.
32. Zalckvar E, Berissi H, Mizrachy L, et al. EMBO Rep. 2009;10:285–292.
33. Karantza V, White E. Autophagy. 2007;3:610–613.
34. Ruddy SC, Lau R, Cabrita MA, et al. Mol Cancer Ther. 2014;13:1882–1893.
35. Futreal P, Söderkvist P, Marks J, et al. Cancer Res. 1992;52:2624–2627.
36. Saito H, Inazawa J, Saito S, et al. Cancer Res. 1993;53:3382–3385.
The research was supported by the National Institutes of Health
(CA135380) (SOM), the American Cancer Society Research Scholar
Grant RSG-12-092-01-CDD (SOM), by the 100 Voices of Hope
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.048