D. Desai, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
methylthiazol-4-yl)thiophene-2-carbaldehyde (90 mg, 0.43 mmol) were
dissolved in THF (8 mL) and a catalytic amount of PTSA (6 mg,
0.04 mmol) was added. The reaction mixture was refluxed overnight.
After evaporation to dryness, the crude mixture was purified by flash
chromatography on silica gel using hexane : Ethyl acetate (6:4) to yield
DABL-N (4) as pale yellow powder (101 mg, 58%). 1H NMR (CDCl3) δ
(ppm) 8.88 (d, 1H, J = 3.0 Hz), 8.16 and 8.14 (dd, 1H, J = 2.5 Hz and
J = 9.0 Hz), 7.38–7.34(m, 2H), 7.29–7.26 (m, 3H), 7.15 (s, 1H), 7.11
(d, 1H, J = 3.5 Hz), 6.80 (d, 1H, J = 4.0 Hz), 6.65 (d, 1H, 9.0 Hz), 6.31
(d, 1H, J = 2.5 Hz), 2.70 (s, 3H, CH3); HRMS (M+H+) calculated for
C22H16N4O3S2: 449.5196; found: 449.1263. Normal phase HPLC Rt:
3.20 min, Reverse phase HPLC Rt: 20.5 min
C23H16N4OS2: 429.5315; found: 429.1397. Normal phase HPLC Rt:
3.44 min; Reverse phase HPLC Rt: 20.26 min
4.1.6. 6-Cyano-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-
phenyl-2,3-dihydro-quinazolin-4(1H)-one (DABL-CM, 10)
6-Cyano-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-
dihydroquinazolin-4(1H)-one (9) (120 mg, 0.28 mmol) was carefully
added to a suspension of NaH (14 mg, 0.58 mmol, 23 mg of 60% in
mineral oil, prewashed with hexanes) in anhydrous THF (10 mL). The
resulting mixture was stirred at 0 °C for 1 h. MeI (63 mg, 0.44 mmol)
was added dropwise and the reaction was allowed to proceed overnight
at room temperature. The reaction flask was cooled down to 0 °C, and
the reaction mixture was quenched with saturated NaHCO3. The aqu-
eous layer was extracted with CH2Cl2 (3 × 30 mL), washed with 1 N
HCl (2 × 10 mL), dried over MgSO4, filtered, and evaporated to dry-
ness. The crude product was purified over silica gel column by eluting
with hexane: Ethyl acetate (6:4) to give DABL-CM (10) in 92% yield. 1H
NMR (CDCl3) δ (ppm) 8.42 (d, IH, J = 1.8 Hz), 7.69 and 7.67 (dd, IH,
J = 2.4 Hz and J = 9.0 Hz), 7.43–7.40 (m, 2H), 7.35–7.29 (m, 3H),
7.20 (d, IH, J = 4.2 Hz), 7.19 (s, IH), 6.84 (d, IH, J = 3.6 Hz), 6.73 (d,
IH, J = 9.0 Hz), 6.07 (s, IH), 3.11 (s, 3H, N-CH3), 2.74 (s, 3H, CH3);
4.1.3. 6-Nitro-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-
phenyl-2,3-dihydro-quinazolin-4(1H)-one (DABL-NM; 5)
6-Nitro-2-(5-(2-methylthiazol-4-yl) thiophen-2-yl)-3-phenyl-2,3- di-
hydroquinazolin-4(1H)-one (4) (50 mg, 0.11 mmol) was carefully
added to a suspension of NaH (6 mg, 0.22 mmol, 10 mg of 60% in mi-
neral oil, prewashed with hexanes) in anhydrous THF (10 mL). The
resulting mixture was stirred at 0 °C for 1 h, followed by MeI (31 mg,
0.22 mmol) dropwise added, and the reaction was allowed to proceed
overnight at room temperature. The reaction flask was cooled down to
0 °C, and the reaction mixture was quenched with saturated NaHCO3.
The aqueous layer was extracted with CH2Cl2 (3 × 30 mL), washed
with 1 N HCl (2 × 10 mL), dried over MgSO4, filtered, and evaporated
to dryness. The crude product was purified over silica gel column by
eluting with hexane: Ethyl acetate (6:4) to give DABL-NM (5) in 71%
yield. 1H NMR (CDCl3) δ (ppm) 9.0 (d, 1H, J = 2.5 Hz), 8.33 and 8.31
(dd, 1H, J = 2.5 Hz and J = 9.0 Hz), 7.43–7.39 (m, 2H), 7.35–7.28 (m,
3H), 7.19 (d, 1H, J = 3.5 Hz), 7.18 (ds, 1H), 6.85 (d, 1H, J = 3.5 Hz),
6.72 (d, 1H, J = 9.0 Hz), 6.09 (s, 1H), 3.16 (s, 3H, NCH3), 2.72 (s, 3H,
HRMS(M+H+
) calculated for C24H18N4OS2: 443.5581; found:
443.1668. Normal phase HPLC Rt: 3.21 min; Reverse phase HPLC Rt:
21 min.
4.2. Cell culture
Normal human dermal fibroblasts (HDFs) (Cell Applications Inc.,
106–05 N), normal human lung fibroblasts (MRC-5) (ATCC, CCL-171),
BALB/3T3 clone A31 mouse fibroblasts (A31) (ATCC, CCL-163) and
African green monkey kidney epithelial cells (VERO) were maintained
in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza). Human ret-
inal pigmented epithelial cells (ARPE-19) were maintained in DMEM/
Ham’s F12 (1:1 mix) (Lonza). All media was supplemented with 10%
fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin,
and 2 mM GlutaMAX (Gibco). Cells were maintained at 37 °C with 5%
CO2.
CH3); HRMS (M+H+
) calculated for C23H18N4O3S2: 463.5462;
found: 463.1108. Normal phase HPLC Rt: 3.10 min, Reverse phase
HPLC Rt: 20.76 min.
4.1.4. 5-Cyano-N-phenylbenzamide (8)
5-cyano-2-aminobenzoic acid (6) (100 mg, 0.62 mmol) was mixed
with aniline (7) (68 mg, 0.75 mmol), DMSO (0.5 mL), and a catalytic
amount of DMAP (3 mg) in a microwave glass vial that was irradiated in
a microwave apparatus (LABMAT) at 130 °C, 250 W for 30 min. After
the reaction mixture was cooled to ambient temperature, the purple
brown reaction mixture was diluted with ethyl acetate and sonicated to
dissolve the entire residue. The organic fraction was washed with water
(3 × 10 mL), dried over MgSO4, filtered, and evaporated to give the
crude product. Flash chromatography [eluted with hexane: Ethyl
acetate (8:2)] afforded 8 (130 mg, 89%) as pale yellow powder. 1H
NMR (D4-MeOH) δ (ppm) 8.04 (d, 1H, J = 1.8 Hz), 7.66 (d, 2H,
J = 8.4 Hz), 7.50 and 7.48 (dd, 1H, J = 1.8 Hz and J = 9.0 Hz), 7.37 (t,
2H, J = 7.2 Hz and J = 7.8 Hz), 7.17 (t, 1H, J = 8.4 Hz, J = 7.2 Hz),
6.86 (d, 1H, J = 9.0 Hz).
4.3. Cell viability assays
Cells used for viability assays in a 96 well plate were treated with
DMSO or analogue compound (100 nm and 10 μM) for 96 h. For the
XTT assay, media was removed from all wells and replaced with fresh
media. XTT and activating reagents (Biotium) were added to each well
according to manufacturer’s instructions. Cells were incubated for 2 h at
37 °C, and absorbance was measured using a Synergy H1 Hybrid Reader
(BioTek). For CellTiter-Glo Luminescent Cell Viability Assay (Promega),
the media was replaced with fresh media and reagents were added to
each well according to manufacturer’s instructions. Luminescence was
measured using the Synergy H1 Hybrid Reader.
4.1.5. 6-Cyano-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-
dihydroquinazolin-4(1H)-one (DABL-C, 9)
4.4. Immunofluorescence staining and confocal microscopy
5-Cyano-N-phenylbenzamide (8) (200 mg, 0.84 mmol) and 5-(2-
methylthiazol-4-yl)thiophene-2-carbaldehyde (90 mg, 0.43 mmol) were
dissolved in THF (8 mL) and a catalytic amount of PTSA (6 mg,
0.04 mmol) was added. The reaction mixture was refluxed overnight.
After evaporation to dryness, the crude mixture was purified by flash
chromatography on silica gel [eluted with hexane : Ethyl acetate (6:4)]
to yield 6-Cyano-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-
2,3- dihydroquinazolin-4(1H)-one (DABL-C) (9) as pale yellow powder
(293 mg, 81%). 1H NMR (CDCl3) δ (ppm) 8.37 (d, 1H, J = 1.8 Hz), 7.60
and 7.59 (dd, J = 1.8 Hz and J = 8.4 Hz), 7.42–7.39(m, 2H), 7.33–7.29
(m, 3H), 7.19 (s, 1H), 7.17 (d, 1H, J = 3.6 Hz), 6.86 (d, 1H, J = 3.6 Hz),
6.77 (d, 1H, J = 8.4 Hz), 6.37 (d, 1H, J = 3.0 Hz), 5.37 (d, 1H,
Coverslips containing either HDFs were washed in phosphate-buf-
fered saline (PBS) and fixed in 2% paraformaldehyde for 15 min at
room temperature. Cells were blocked in PBS containing 10% human
serum, 0.5% Tween-20, and 5% glycine. 0.1% Triton-X 100 was added
for permeabilization. Primary antibodies used were against STX5 (Santa
Cruz, sc-365124) and p115 (Proteintech, 13509-1-AP) as a Golgi
marker. Secondary antibodies were Alexa Fluor 488 or 568
(ThermoFisher Scientific). Coverslips were mounted with ProLong
Diamond Antifade Mountant containing DAPI (ThermoScientific). All
images were acquired using
a C2 + Confocal Microscope System
(Nikon). Images were processed using NIS elements software. Images
shown are volume renderings of Z-stacks.
J = 2.4 Hz), 2.75 (s, 3H, CH3); HRMS (M + H+
) calculated for
8