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that induce oxidative stress. This design can be adapted to
other inhibitors and for conjugation to biomolecules. Previously
evaluated PI3K inhibitors were detrimental to normal cell types
resulting in serious side effects. Compound 1 warrants further
testing as it has the benefits of acting synergistically with
chemotherapeutics that induce ROS but with selectivity for
cancerous cells.
148.80, 147.15, 142.09, 141.09, 135.75, 133.43, 131.86, 130.42,
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28.55, 128.32, 128.08, 125.67, 121.69, 120.40, 120.01, 116.19,
15.10, 114.79, 67.56, 66.86, 45.72, 38.64. [M+H]:676.2147
Synthesis
[
(
of
3’,2’:4,5]furo[3,2-d]pyrimidin-2-yl)phenoxy)phenyl)acetamide
1). To a room temperature (RT) solution of the benzyl 2-(5-nitro-2-
2-amino-N-(5-amino-2-(3-(4-morpholinopyrido
(3-(4-morpholinopyrido[3’,2’:4,5]-furo-[3,2-d]pyrimidin-2-yl)
phenoxy)phenylamino)-2-oxoethylcarbamate (3, 30 mg, 0.04 mmol)
in MeOH (5.0 mL),10% Pd on carbon (4 mg) was added, and the
solution purged with H for 3 min. The pressure safe vial was sealed
2
Experimental Section
and allowed H added until the pressure was 30 psi. The resulting
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mixture was stirred at RT for 24 hr. The reaction was filtered with
Celite and concentrated in vacuum. The resulting material was
purified by flash chromatography using DCM/MeOH (9:1) and
product was eluted as yellowish solid [20 mg, 95%]. 1H NMR
(400 MHz, DMSO-d6) δ 8.580–8.50 (m, 2H), 7.96 (d, J=2.5 Hz, 1H),
7.63 (dd, J=7.7, 4.8 Hz, 1H), 7.56–7.36 (m, 2H),7.31 (dd, J=8.1,
2.4 Hz, 1H) 6.96 (dd, J=8.1, 2.6 Hz, 1H), 6.76 (d, J=8.6 Hz, 1H), 6.47
(dd, J=8.8, 2.6 Hz, 1H), 4.01 (t, J=4.8 Hz, 4H), 3.78 (t, J=4.8 Hz, 4H)
3.21(s,2H). 13 C NMR (400 MHz, MeOHd6) δ163.78, 160.27, 150.80,
149.96, 147.97, 146.28, 141.02, 139.44, 135.32, 134.58, 133.32,
Synthesis
Pi103 was obtained from MedChem Express. All other chemicals
were obtained from Fisher Scientific. NMR spectrum was generated
at the Nuclear Magnetic Resonance Facility in Department of
Chemistry, University of Cincinnati using Bruker AV 400 MHz
spectrometer. Mass Spectrum was obtained at R. Marshall Wilson
Mass Spectrometry Facility at University of Cincinnati.
Full synthetic details and spectra are within the supporting
1
1
31.60, 130.66, 123.09, 122.67, 121.88, 119.56, 117.35, 116.21,
13.39, 110.81, 107.43, 67.85, 46.98, 38.89 [M+H] 512.2039.
ox
ox
information including the synthesis of 1A , 1B , and 8. The
following protocol was used to synthesize 1 as shown in Scheme 1.
Synthesis of 5-nitro-2-(3-(4-morpholinopyrido[3’,2’:4,5]-furo-[3,2-
d] pyrimidin-2-yl)phenoxy)benzenamine (2). To a solution of 3-(4-
morpholinopyrido[3’,2’:4,5]-furo-[3,2-d] pyrimidin-2-yl) phenol
(50 mg, 0.15 mmol) in DMSO (2 mL), K CO (30 mg, 0.2 mmol) was
Cell Assays
CD34+ selected Human Umbilical Cord Blood (UCB), HL60, Kasumi-
2
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1
and MOLM13 cell lines were generous gift from Dr. Mark
added, followed by addition of 2-fluoro-5-nitrobenzenamine
Wunderlich at CCHMC. AML cells were cultured in IMDM media
supplemented with 20% bovine calf serum. Human Umbilical Cord
Blood (UCB) cells were cultured in IMDM media in 20% bovine calf
serum supplemented with growth factors such as SCF, IL-3, IL-6, Flt-
(
50 mg, 0.3 mmol). The resulting mixture was stirred at 70°C
overnight. After overnight incubation, the reaction mixture was
diluted with 25 mL H O and extracted with ethyl acetate and
2
washed with brine. The organic layer was dried with Na SO and
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3
1
L and TPO. NH-Fibroblast cells were culture in MDME media with
0% bovine calf serum and supplemented with growth factors
concentrated in vacuum. The resulting material was purified by
flash chromatography using ethyl acetate/hexane (1:1) as eluent to
such as Insulin, HC, EGF.
1
generate the product as a yellowish solid [70 mg, 95%]. H NMR
(
400 MHz, Chloroform-d) δ 8.62 (dd, J=4.9, 1.8 Hz, 1H), 8.58 (dd, J=
7
1
1
.7, 1.8 Hz, 1H), 8.32 (d, J=7.8, 1H), 8.28(s,1H), 7.72 (d, J=2.7 Hz,
H), 7.58–7.52 (m, 2H), 7.43 (dd, J=7.7, 4.8 Hz, 1H), 7.13–7.06 (m,
H), 6.81 (d, J=8.9 Hz, 1H), 4.27 (s, 2H), 4.22 (t, J=4.8 Hz, 4H), 9.92
Cell Cytotoxicity Assay (MTT)
6
In all assays cells were seeded at a density of 0.4×10 cells/well in a
96-well plate and incubated at 37°C overnight. Then cells were
treated for 72 hr with indicated concentrations of freshly dissolved
compounds. The plates were centrifuged at 1500Xg, washed, and
each well was incubated for an additional 4 hr with 500 μL fresh
medium containing 20 μL of MTT (5 mg/mL). Next, 100 μL of DMSO
was added to each well and the optical densities (ODs) were
determined at 570 nm using spectrophotometer. Cytotoxicity data
were expressed as IC50 values obtained from the fit to a four-
parameter sigmoid using graph pad prism 7 (GraphPad Software,
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(t, J=4.8 Hz, 4H). C NMR (101 MHz, CDCl3) δ 162.70, 158.52,
155.22, 149.74, 148.81, 147.24, 143.55, 140.80, 137.91, 133.44,
1
31.86, 130.14, 124.74, 121.07, 120.34, 119.52, 115.90, 115.23,
114.32, 110.26, 66.89, 45.75. [M+H]: 485.1564
Synthesis
of
Benzyl-2-(5-nitro-2-(3-(4-morpholinopyrido
[3’,2’:4,5]-furo-[3,2-d]pyrimidin-2-yl)phenoxy)phenylamino)-2-ox-
oethylcarbamate (3). To a room temperature (RT) solution of the 2-
(
(
benzyloxycarbonyl) acetic acid (100 mg, 0.5 mmol) in DMF
2.0 mL), Hexafluorophosphate Azabenzotriazole Tetramethyl Uro-
2
Inc., La Jolla, CA). All R values were greater than 0.98 and standard
nium (380 mg, 1 mmol) was added, then 5-nitro-2-(3-(4-morpholi-
nopyrido[3’,2’:4,5]-furo-[3,2-d] pyrimidin-2-yl) phenoxy) benzene
amine (2, 50 mg, 0.1 mmol) was added after 30 mins. Then N,N-
diisopropylethylamine (0.36 mL, 0.2 mmol) was added. The result-
ing mixture was stirred at 40°C for 24 h. The reaction was diluted
errors of the three replicates were less than 20%. All values are
expressed as the mean of biological triplicates relative to DMSO
control together with standard deviation shown as the error bar.
The bar graph was generated using graph pad prism 7 (GraphPad
Software, Inc., La Jolla, CA).
with 25 mL H O and extracted with ethyl acetate, washed with
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brine. The organic layer was dried with Na SO and concentrated in
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vacuum. The resulting material was purified by flash chromatog-
raphy using ethyl acetate/hexane (1:1) and product was eluted as
In Cell Western Blot Analysis
1
In this experiment, Kasumi-1 cells were grown in a 96-well non-
treated plate and treated with different concentration of tested
yellowish solid [30 mg, 40%]. H NMR (400 MHz, Chloroform-d) δ
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.33 (s, 1H), 8.73(s,1H) 8.61 (d, J=7.7 1H), 8.56 (dd, J=7.7, 1.7 Hz,
H), 8.42 (d, J=7.9 Hz, 1H), 8.23 (s, 1H), 7.91 (dd, J=9.1, 2.8 Hz, 1H),
.57 (t, J=7.9 Hz, 1H), 7.47 (dd, J=7.7, 4.8 Hz, 1H), 6.88 (d, J=
.1 Hz, 1H),6.83 (m,5H) 5.11 (d, J=3.9 Hz, 2H), 4.21 (t, J=4.8 Hz, 4H),
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agents for 18 hr. The cells were counted and reset to 0.4×10 cells/
mL. Then the experiment was performed using following steps.
Step1: media was removed from wells and the cells were washed
two times with 1X PBS. Step 2: 150 μL/well of 3.7% formaldehyde
in PBS was added and incubated for 20 min at room temp without
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.12 (d, J=5.9 Hz, 2H), 3.91 (t, J=4.8 Hz,4H). C NMR
(101 MHz,CDCl3)δ167.70, 162.65, 158.14, 154.27, 151.59, 149.80,
ChemMedChem 2019, 14, 1–8
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