Journal of Medicinal Chemistry
Article
organic layers were combined, dried over MgSO , filtered, and
mg/mL amphotericin B (Sigma-Aldrich, USA). The culture
4
evaporated under reduced pressure. The residue was purified by
column chromatography (hexane/ethyl acetate 1:1) to yield 6 (0.110
atmosphere was kept at 37 °C humidified with 5% CO (v/v).
2
Calcium Fura 2 Dynamic Assay. To perform the Calcium Fura 2
assay, LN229 and SNB19 were plated in a black, clear-bottom, 96-well
plate (CLS4580-10EA, Corning Sigma-Aldrich) with a density of 5 ×
1
g, 0.455 mmol, 86% yield) as an off-white solid. H NMR (500 MHz,
CDCl + CD OD): δ 7.16−7.35 (m, 7H), 4.18 (s, 2H), 3.29 (t, J =
3
3
1
3
4
8
.3 Hz, 2H), 2.83 (t, J = 8.3 Hz, 2H). C NMR (126 MHz, CDCl +
10 cells/well. The used cell concentration is an appropriate quantity
3
CD OD): δ 157.5, 154.9, 152.8, 138.3, 129.0, 128.3, 127.9, 127.6,
to multiply and grow in the given area. After overnight incubation, the
cells were washed with pre-warmed 1× phosphate-buffered saline
(PBS). The selected compounds were dissolved in dimethyl sulfoxide
(DMSO, Sigma-Aldrich, St. Louis, MO, United States) to obtain a
stock of 100 mM, from which an intermediate dilution was prepared.
The cells were incubated with CHBC and the known agonist MDL at
different concentrations of 100, 75, 50, 25, 10, and 1 μM for 2 h at 37
°C. The cells were further incubated with 5 μM Fura 2 (47989-1MG-
F, Sigma-Aldrich, St. Louis, MO, USA) and 0.1% Pluronic F-127
(P2443-250G, Sigma-Aldrich, St Louis, MO, USA) in 30 min
darkness at RT to maximize the activity of the loading dye. The level
of fluorescence was recorded using a microplate reader (Spark,
Tecan) at dual excitation/emission wavelengths 340/510 and 380/
510. The 340/380 ratio was calculated using the following formula 1.
3
1
27.0, 106.0, 92.7, 88.5, 53.8, 53.4, 24.5. Purity is >95%, as validated
1
by H NMR.
Preparation of 1-Benzylindoline-4,6-diyl bis(phenylcarbamate)
7). Phenyl isocyanate (103 μL, 0.949 mmol, 2.1 equiv) was added
(
dropwise to a stirred solution of 6 (0.109 g, 0.452 mmol) and
triethylamine (132 μL, 0.949 mmol, 2.1 equiv) in dry acetonitrile (2.3
mL) at 0 °C. The reaction was stirred for 10 min and brought to
ambient temperature. After 30 min, volatiles were evaporated under
reduced pressure. The residue was purified by column chromatog-
raphy (gradient from 1 to 1.5% of MeCN in CH Cl ) to yield 7
2
2
1
(
0.184 g, 0.384 mmol, 85% yield) as a white solid. H NMR (500
MHz, CDCl ): δ 8.48 (s, 1H), 8.37 (s, 1H), 7.44−7.45 (m, 4H),
3
7
4
.27−7.34 (m, 9H), 7.05−7.09 (m, 2H), 6.34 (s, 1H), 6.21 (s, 1H),
13
.22 (s, 2H), 3.37 (t, J = 8.3 Hz, 2H), 2.92 (t, J = 8.3 Hz, 2H).
C
raw
340
blank
NMR (126 MHz, CDCl + CD OD): δ 154.4, 151.1, 146.5, 137.7,
Ratio340/380 = F
− F340
3
3
1
9
37.5, 128.8, 128.8, 128.4, 127.6, 127.10, 123.4, 118.8, 118.6, 104.4,
8.4, 53.2, 52.7, 25.2. Purity is >95%, as validated by H NMR.
raw
blank
− F380
F
(1)
1
380
Preparation of Indoline-4,6-diyl bis(phenylcarbamate) (8). 7
0.149 g, 0.310 mmol) in a dry, degassed MeOH/DCM mixture (7.6
where F r3 aw is the fluorescent intensity of the treated sample measured
40
(
raw
at 340/510 nm and F3 is the fluorescent intensity of the treated
80
mL, 4:1) was added to 10% Pd/C (8.2 mg) under argon at ambient
temperature. The reaction flask was then evacuated and filled with
hydrogen five times and then left to stir under a hydrogen balloon.
After 3 days, the hydrogen balloon was removed and argon was
bubbled into the stirring mixture for 15 min. The reaction mixture
was filtered through Celite, and solids were washed with DCM (3 ×
blank
blank
sample measured at 380/510 nm. F340 and F380 are the fluorescent
intensities of the untreated sample measured at 340/510 nm and 380/
510 nm, respectively. To validate the specificity of the ligand binding
to GPR17, the siRNA assay was conducted using the predesigned
siRNA against human GPR17 (cat no. AM16704; Thermo Fisher
Scientific, USA). LN229 and SNB19 cells with the confluence of 60−
1
0 mL). The filtrates were combined and evaporated under reduced
pressure. The residue was purified by column chromatography
gradient from hexane/ethyl acetate 3:2 to 1:1) to yield 8 (61 mg,
70% were transfected with 20 nM of siRNA by Lipofectamine
RNAiMAX Transfection Reagents (cat no. 13778030; Thermo Fisher
Scientific, USA). After 48 h of transfection, the cells were measured to
quantify the changes in the intracellular calcium level.
(
0
1
.156 mmol, 50% yield) as a white solid. H NMR (500 MHz,
CD OD): δ 7.46−7.49 (m, 4H), 7.27−7.31 (m, 4H), 7.03−7.07 (m,
3
cAMP Glo Dynamic Assay. LN229 and SNB19 cells were seeded
2
H), 6.36 (d, J = 1.7 Hz, 1H), 6.31 (d, J = 2.3 Hz, 1H), 3.56 (t, J = 8.6
in a white 96-well plate (136101, NuclonThermoFisher Scientific,
1
3
Hz, 2H), 2.96 (t, J = 8.3 Hz, 2H). C NMR (126 MHz, CD OD): δ
4
3
USA) with an initial density of 5 × 10 cells/well. After overnight
1
1
54.5, 152.8, 151.9, 151.2, 147.1, 138.4, 128.59, 128.57, 123.1, 118.8,
incubation, the cells were washed with PBS prior to incubation with
1
18.6, 104.9, 100.7, 26.4. Purity is >95%, as validated by H NMR.
10 μM Forskolin (F6886-10MG, Sigma-Aldrich, USA) at 37 °C for
15 min. CHBC with varying concentrations of 100, 75, 50, 25, 10, and
1 μM were added to the cells for 2 h The cAMP-Glo Assay (V1501,
Preparation of 1-((3-Chloro-2-hydroxyphenyl)(4-fluorophenyl)-
methyl)indoline-4,6-diylbis(phenylcarbamate) (CHBC). A solution
of 9 (23.6 mg, 0.151 mmol), 10 (21.1 mg, 0.151 mmol), and 8 (58.8
mg, 0.151 mmol) in 1,2-dichloroethane (1.5 mL) was stirred while
heated to 50 °C. After 1.5 h, the solvent was evaporated under
reduced pressure. The residue was purified by column chromatog-
Promega, USA) was conducted following the manufacturer’s
instructions. Briefly, the cells were loaded with 20 μL of cAMP Glo
lysis buffer, followed by a shaking step of 30 min at RT. Then, the
cells were incubated with the cAMP detection solution in 20 min,
followed by incubation with the Kinase-Glo Reagent for 10 min at
RT. The standard curve was generated for each experiment according
to the manufacturer’s protocol. The cAMP level was calculated based
on the linear equation generated from a standard curve. The change in
the luminescence (ΔRLU) of each sample was measured to calculate
the appropriate cAMP concentration. The ΔRLU of each treated
sample was calculated using formula 2.
raphy (CH Cl /MeCN 98:2) to yield CHBC (71 mg, 0.113 mmol,
2
2
1
7
1
(
5% yield) as a white solid. H NMR (500 MHz, CDCl ): δ 7.51 (s,
3
H), 7.40 (dd, J = 24.3, 7.7 Hz, 4H), 7.24−7.33 (m, 8H), 7.05−7.10
m, 3H), 6.97−7.03 (m, 4H), 6.79 (t, J = 8.0 Hz, 1H), 6.47 (d, J = 2.3
Hz, 1H), 5.97 (d, J = 2.3 Hz, 1H), 5.65 (s, 1H), 3.11−3.22 (m, 2H),
1
3
2
1
1
1
.83−2.90 (m, 2H). C NMR (126 MHz, CDCl ): δ 163.2, 161.2,
3
53.2, 151.4, 150.7, 150.6, 150.0, 146.2, 137.3, 134.94, 134.91, 130.2,
30.1, 129.11, 129.05, 128.6, 127.7, 127.5, 123.9, 123.8, 120.9, 120.8,
19.9, 118.6, 115.6, 115.4, 106.3, 100.9, 62.8, 52.2, 25.2. HRMS (ESI/
TOF): m/z calcd for C H ClFN O Na [M + Na] , 646,1515;
found, 646.1520. Purity is >95%, as validated by H NMR.
ΔRLU = RLU(untreated sample) − RLU(treated sample)
(2)
+
+
3
5
27
3
5
1
Cell Viability Evaluation. LN229, SNB19, and MEF cells were
5
Cell Culture. Human glioblastoma cell lines LN229 and SNB19
obtained as a gift from Dr. Kirsi Granberg, Faculty of Medicine and
seeded with a density of 1 × 10 cells/well in a 12-well plate. The used
(
cell concentration is an appropriate quantity to multiply and grow in
the given area. When the cells reached 60−70% confluency, they were
treated with 10 μM and 100 μM of CHBC, MDL29,951, and TMZ
for 48 h. The Trypan Blue assay was performed according to the
manufacture instructor to determine the cell growth inhibitory effect
of CHBC. Briefly, cells were collected by the centrifugation method
prior to dilution with Trypan Blue (Thermo Fisher Scientific) with a
1:1 ratio. The number of live and dead cells was determined using a
Countess II FL Automated Cell Counter (Thermo Fisher Scientific).
The percentage of cell growth inhibition was calculated using formula
3
Health Technology, Tampere. Finland) and the mouse embryonic
fibroblast cell line (MEF) were used to test the efficacy of a novel
ligand targeted to the GPR17 receptor protein. Originally, LN229 was
derived from a right frontal parieto-occipital glioblastoma patient with
the mutation on p52, p16, and p14ARF tumor suppressor genes while
SNB19 was obtained from a left parieto-occipital glioblastoma patient.
These cell lines were cultured and maintained in Dulbecco’s modified
Eagle’s medium (DMEM) (Sigma-Aldrich, USA) supplemented with
1
0% FBS (Biowest, France), 0.1 mg/mL streptomycin (Sigma-
Aldrich, USA), 100 U/mL penicillin (Sigma-Aldrich, USA), and 0.025
H
J. Med. Chem. XXXX, XXX, XXX−XXX