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Y. Lizarme et al. / Bioorg. Med. Chem. 24 (2016) 1480–1487
The mixture was concentrated under reduced pressure, and the
crude product was dissolved in aq HCl (15 mL, 1 N) and ethyl acetate
(25 mL). After partitioning of the two layers, the aqueous layer
was extracted with ethyl acetate (4 ꢂ 25 mL). All organic layers
were combined and washed with water (10 ꢂ 20 mL). Finally the
solvent was dried with MgSO4 and evaporated under reduced
pressure onto silica. The crude product was purified by flash
chromatography eluting with 1:9?3:7 ethyl acetate/hexane
(containing 0.5% acetic acid) to produce N-(2-(hydroxymethyl)phe-
nyl)-2-nitrobenzamide (17) as a white solid (88 mg, 16%); mp 170–
172 °C; IR (neat) mmax (cmꢀ1) 3232, 3071, 2918, 2864, 2712, 2284,
2111, 2069, 1647, 1521; 1H NMR (300 MHz, CD3CN) d 9.17 (s, 1H,
NH), 8.09 (dd, J = 8.0, 0.7 Hz, ArH3), 7.95 (d, J = 8.4, 1.3 Hz, ArH60),
7.83 (m, 2H, ArH5, ArH6), 7.74 (ddd, J = 8.0, 7.0, 2.1 Hz, ArH4),
7.40 (m, 2H, ArH50, ArH30), 7.24 (ddd, J = 8.4, 7.6, 1.1 Hz, ArH40),
4.71 (d, J = 3.5, 2H, CH2), 3.56 (s, 1H, OH); 13C{1H} NMR (76 MHz,
CD3CN) d 165.4 (C@O), 148.1 (C2), 137.2 (C10), 134.9 (C5), 133.7
(C1 or C20), 133.6 (C20 or C1), 132.1 (C4), 129.6 (C6), 129.4 (C30),
129.1 (C50), 126.3 (C40), 125.5 (C3), 124.1 (C60), 63.1 (CH2); HRMS
(ESI,+ve) C14H12N2O4Na+ [MNa+] requires m/z 295.0689, found
295.0672.
(150 MHz, CD3CN) d 161.8 (C4), 149.3 (C10), 148.1 (C2), 140.3
(C10), 137.1 (C20), 135.5 (C7), 130.7 (C40), 129.9 (C30), 129.6 (C50),
129.3 (C60), 128.5 (C8), 128.4 (C6), 127.5 (C5), 123.4 (C9), 61.2
(CH2); HRMS (ESI,+ve) C15H12N2O2H+ [MH+] requires m/z
253.0972, found 253.0968.
4.9. 3-(2-(Fluoromethyl)-4-methoxyphenyl)-6-methoxyquina-
zolin-4(3H)-one (13)
Compound 1 (28 mg, 0.09 mmol) was dissolved in anhydrous
dichloromethane (2 mL) and cooled to ꢀ78 °C. To this solution
Deoxo-FluorTM (25
lL, 1.4 mmol) was added. The reaction mixture
was warmed to room temperature and stirred under N2 atmo-
sphere for 24 h. The mixture was diluted with dichloromethane
(2 mL) and then quenched by dropwise addition of saturated
NaHCO3 solution (10 mL). After partitioning of the two layers, the
aqueous layer was extracted with dichloromethane (4 ꢂ 3 mL).
All organic layers were combined and the solvent was dried with
MgSO4 and evaporated onto silica under reduced pressure. The
residue was purified by flash chromatography eluting with 1:9
ethyl acetate/dichloromethane to yield the title compound as a
light yellow solid (12 mg, 43%); mp 164–170 °C; IR (neat) mmax
(cmꢀ1) 3751, 3328, 3077, 3021, 2922, 2844, 2100, 1983, 1912,
1740, 1667, 1612; 1H NMR (300 MHz, CDCl3) d 7.94 (s, 1H, ArH7),
7.72 (m, 1H, ArH8); 7.70 (m, 1H, ArH5), 7.40 (dd, J = 8.9, 3.0 Hz,
ArH7), 7.23 (dd, J = 8.6, 0.8 Hz, ArH60), 7.13 (d, J = 2.7 Hz, ArH30),
7.04 (dddd, J = 11.5, 8.6, 2.7, 1.2 Hz, ArH50), 5.28 (dd, J = 47.5,
11.5 Hz, CHH), 5.21 (dd, J = 47.2, 11.5, Hz, CHH), 3.93 (s, 3H,
OCH3), 3.89 (s, 3H, OCH3); 13C{1H} NMR (76 MHz, CDCl3) d 160.9
(C4), 160.6 (C40), 159.3 (C6), 144.6 (C2), 142.5 (C9), 135.5 (d,
J = 16.6 Hz, C20), 129.5 (C60), 129.3 (C8), 128.5 (d, J = 4.2 Hz, C10)),
125.0 (C7), 123.0 (C10), 115.5 (d, J = 2.5 Hz, C50)), 114.8 (d,
J = 7.8 Hz, C30)), 106.7 (C5), 81.2 (d, J = 168.0 Hz, CF), 56.0 (OCH3),
55.8 (OCH3); 19F {1H} NMR (76 MHz, CDCl3) d ꢀ212.5 (s, 1F);
19F NMR (76 MHz, CDCl3) d ꢀ212.5 (t, J = 47.5 Hz); HRMS (ESI,+ve)
4.8.2. Step 2
Compound 17 that was synthesised above (68 mg, 0.25 mmol)
was dissolved in methanol (2 mL), and 10% Pd/C (30 mg,
0.03 mmol) was added to this solution. The reaction mixture was
stirred under H2 atmosphere at room temperature for 4 h. The mix-
ture was filtered through a layer of celite and washed with metha-
nol. The filtrate was concentrated under reduced pressure onto
silica, and the crude product was subjected to flash chromatogra-
phy eluting with 3:1 hexane/ethyl acetate to yield 2-amino-N-(2-
(hydroxymethyl)phenyl)benzamide (18) as a white crystalline
solid (45 mg, 76%); mp 140–143 °C; IR (neat) mmax (cmꢀ1) 3440,
3278, 3025, 2928, 2877, 2343, 2111, 1997, 1812, 1620, 1617,
1582, 1510; 1H NMR (300 MHz, CD3CN) d 9.45 (s, 1H, NH), 8.04
(dd, J = 8.1, 1.2 Hz, ArH60), 7.57 (dd, J = 8.0, 1.5 Hz, ArH6),
7.37ꢀ7.22 (m, 3H, ArH50, ArH30, ArH3), 7.13 (ddd, J = 14.9, 7.4,
1.2 Hz, ArH40), 6.77 (dddd, J = 9.5, 8.2, 1.2, 0.4, Hz, ArH4), 6.67
(dddd, J = 8.2, 8.0, 7.2, 1.2 Hz, ArH5), 5.92 (s, 2H, NH2) 4.69 (s, 2H,
CH2), 3.72 (s, 1H, OH); 13C{1H} NMR (76 MHz, CDCl3) d 168.6
(C@O), 151.0 (C2), 138.7 (C20), 133.6 (C3), 132.5 (C10), 129.4 (C30),
129.0 (C50), 128.4 (C6), 125.0(C40), 123.5 (C60), 118.0 (C4); 116.9
(C5), 116.1 (C1), 63.9 (CH2); HRMS (ESI,+ve) C14H14N2O2Na+
[MNa+] requires m/z 265.0947, found 265.0944.
C
17H15F1N2O4H+ [MH+] requires m/z 315.1139, found 315.1134.
4.10. Bioassay reagents and instrumentation
All bioassay chemicals were purchased from Gibco Life Tech-
nologies and used as received. All procedures were performed
under aseptic conditions, unless stated otherwise. Optical density
values were measured using a BMG FLUOstar OPTIMA microplate
reader.
4.8.3. Step 3
4.11. Cell culture model
A mixture of compound 18 that was synthesised above (16 mg,
0.06 mmol) and formic acid (100
lL, 1.86 mmol) was stirred at
SH-SY5Y neuroblastoma cells (ATCCÒ CRL-2266TM) were main-
tained for between 10 and 18 passages in T-75 flasks in media
(containing 5% FBS, 46% F12 growth media, 46% DMEM, 1% gluta-
max, 1% antibiotic/antimycotic and 1% sodium pyruvate) at 37 °C
in a humidified atmosphere with 5% CO2. Media was changed every
four days. The cells were subsequently subcultured by 1:2 or 1:3
dilution into two or three T-75 flasks at 37 °C in a humidified atmo-
sphere with 5% CO2, until they were used for experimentation.
When the cells achieved 85–95% confluency, 0.05% trypsin was
used to detach the adherent cells from the flask. Once the cells
detached, they were harvested and combined with media contain-
ing non-adherent cells. The cell suspension was then spun down at
700 rpm for 7 minutes at room temperature. Then, the supernatant
was discarded and the cell pellet resuspended with 10 mL growth
reflux (oil bath temperature 95 °C) for 12 h. The reaction mixture
was cooled to room temperature and diluted with methanol
(1 mL) and tetrahydrofuran (1 mL). Potassium carbonate (270 mg,
1.95 mmol) was added, and the mixture was stirred at room tem-
perature under N2 atmosphere for 12 h. The mixture was diluted
with ethyl acetate (10 mL) and filtered through a silica plug with
ethyl acetate washing. The filtrate was evaporated under reduced
pressure onto silica, and the crude product was subjected to flash
chromatography eluting with 1:3 ethyl acetate/hexane, to produce
the title compound (11) as a light yellow powder (13 mg, 83%); mp
154–159 °C; IR (neat) mmax (cmꢀ1) 3290, 3042, 2920, 2644, 2320,
2109, 1982, 1942, 1876, 1846, 1672, 1599, 1561; 1H NMR
(600 MHz, CD3CN) d 8.26 (dd, J = 7.9, 1.5 Hz, ArH5), 8.06 (s, 1H,
ArH2), 7.86 (ddd, J = 15.3, 7.9, 1.5 Hz, ArH7), 7.77 (dd, J = 8.2,
1.2 Hz, ArH8), 7.64 (d, J = 7.6 Hz, ArH30), 7.59 (m, 1H, ArH4), 7.56
(ddd, J = 15.2, 7.6, 1.4 Hz, ArH40), 7.49 (ddd, J = 15.2, 7.6, 1.4 Hz,
ArH50), 7.37 (dd, J = 7.8, 1.4 Hz, ArH60), 4.44 (d, J = 13.9 Hz, CHH),
4.42 (d, J = 13.9 Hz, CHH), 3.24 (s, 1H, OH); 13C{1H} NMR
media. A small aliquot (10 lL) was taken to estimate cell number
using a hemocytometer. Cells were plated into a Costar 96-well cell
culture plate at
incubated at 37 °C in a humidified atmosphere with 5% CO2 for
a
density 5 ꢂ 105 cells/mL, 100
lL/well and
20 h prior to experimentation.