Z. Zhou, Y. Liu, Q. Ren et al.
Journal of Molecular Structure 1235 (2021) 130261
for 6 h. After the reaction had completed, the reaction solution was
cooled to 100 °C, 100 mL of water was added to the reaction so-
lution, and the mixture was stirred at this temperature for 10 min
to dissolve the excess urea. After the reaction solution had cooled
to room temperature (20 - 25 °C), the mixture was filtered un-
der suction filtration. The solid material was dissolved in an aque-
ous sodium hydroxide, and the pH was adjusted to 6 – 7 using
glacial acetic acid. The white solid 3 (103.2 g, yield: 93.8%) was ob-
tained by suction filtration. 1H NMR (400 MHz, DMSO-d6) δ 8.57
(d, J = 2.6 Hz, 1H), 8.31 – 8.10 (m, 1H), 1.70 (s, 2H). m/z: 243.4
[M + H]+.
2.1.6. Synthesis of N-(4-(2,4-dimorpholinopyrido[2,3-d]pyrimidin-6-
yl)phenyl)pyrrolidine-1- carboxamide (1)
Compound
7
(1 g, 3.2 mmol), potassium carbonate
(1.2 g, 3.2 mmol) and
(0.5 g, 3.5 mmol), compound
5
tetrakis(triphenylphosphine)palladium (0.07 g, 0.05 mmol) were
added to a 100 mL three-necked flask at room temperature, fol-
lowed by a 5:5:1 methanol:toluene:water solvent mixture. Under
a nitrogen atmosphere, the reaction was carried out at 80 °C for
5 h. After the reaction had completed, water was added to quench
the reaction, and the mixture was extracted with ethyl acetate
(4 × 5 mL). The organic phases were combined, dried over anhy-
drous Na2SO4, and the solvent was removed by distillation under
reduced pressure to obtain a crude product 1.2 g, and subjected
to silica gel column chromatography (20:1 acetate:methanol) to
obtain the bright yellow fluorescent solid 1 (0.48 g, yield: 31.2%).
m.p. 133.7 – 135.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.02 (d,
J = 2.4 Hz, 1H), 8.23 (s, 2H), 7.65 (s, 4H), 3.79 (d, J = 15.9 Hz,
12H), 3.69 (d, J = 4.8 Hz, 4H), 3.40 (t, J = 6.6 Hz, 4H), 1.87 (t,
J = 6.6 Hz, 4H). m/z: 490.3 [M + H]+.
2.1.2. Synthesis of 6-bromo-2,4-dichloro-3,4-dihydroquinazoline (4)
Compound 3 (80.0 g, 330.5 mmol), chlorobenzene (200 mL) and
N,N-diisopropylethylamine (DIEA) (8.5 g, 66.1 mmol) were added
to a 1 L three-necked flask, and then slowly stirred as POCl3
(308.1 mL, 3305 mmol) was added dropwise. Under a nitrogen at-
mosphere, the reaction was carried out at 110 °C for 12 h, and
then the solvent was removed by distillation under reduced pres-
sure to obtain a residue of 112 g. The residue was poured into
ice water (300 mL) to precipitate a black solid. The mixed solu-
tion was suction filtered, and then the pH of the filtrate was ad-
justed to 8 with saturated NaHCO3 aqueous solution, and finally
extracted with dichloromethane (100 mL × 3). The organic phases
were combined, dried over anhydrous Na2SO4, and the solvent was
removed by distillation under reduced pressure to obtain a residue
95 g. The crude product was separated on a silica gel column
with dichloromethane as the mobile phase. The red solid 4 (68.1 g,
yield: 73.3%) was obtained by separation. m/z: 280.1 [M + H]+.
2.2. X-ray diffraction
Single crystals of compound 1, were obtained by slow evap-
oration from methanol. A colorless crystal with dimensions of
0.12 × 0.08 × 0.19 mm3 was selected and mounted on a glass
fiber for data collection. A D8 VENTURE diffractometer was used to
obtain the X-ray diffraction data for compound 1 using graphite-
˚
monochromatic Mo-Kα radiation (λ = 0.71073 A) at 170 K. A to-
tal of 15,449 reflections were collected over the range 4.438° to
52.92° (index ranges: 12 ≥ h ≥ −12; 15 ≥ k ≥ −15; 15 ≥ l
≥ −15) via application of a ϕ–ω scan mode; there were 3911
ꢀ
2.1.3. Synthesis of 4,4 -(6-bromo-3,4-dihydroquinazoline-2,
4-diyl)dimorpholine (5)
unique reflections (I
> 2σ(I)) [19]. The structure was deter-
Compound 4 (15 g, 53.8 mmol), dichloromethane (30 mL) and
mined by direct methods using SHELXT [20] and refined using
a full-matrix least-squares procedure, using SHELXL-2018/3 [21],
based on F2 using all data. The crystallographic parameters were
R = 0.0617, wR = 0.1823 (w = 1/[σ2 (Fo2) + (0.0867P)2 + 0.9386P],
triethylamine (16.3 g, 161.4 mmol) were added to
a 500 mL
single-necked flask at room temperature, and morpholine (9.4 mL,
107.6 mmol) was slowly added dropwise under continuous stir-
ring. The reaction completed in 2 h. Water (100 mL) was added to
quench the reaction, and the yellow solid 5 (18.7 g, yield: 91.5%)
was obtained by suction filtration. 1H NMR (400 MHz, CDCl3) δ
8.95 (d, J = 1.5 Hz, 1H), 8.37 (d, J = 1.6 Hz, 1H), 3.84 – 3.67 (m,
16H). m/z: 381.5 [M + H]+.
2
2
where
P
=
(Fo
+
2Fc
)
/3)),
(
/σ)max
=
0.000,
S
=
1.025,
3
˚
(
ρ)max = 0.801, and ( ρ)min = –0.474 e/A .
2.3. MTT assay in vitro
The anti-proliferative activity of compound 1 was evaluated
against human malignant melanoma cells (A375) using the stan-
dard MTT assay in vitro. The cancer cell line was cultured in Dul-
becco’s modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum (FBS). Approximate 4 × 103 cells, suspended in
DMEM medium, were plated onto each well of a 96-well plate and
incubated in 5% CO2 at 37 °C for 24 h. The tested compound at the
final concentration of 5 μM was added to the culture medium and
the cell cultures were continued for 72 h. Fresh MTT was added to
each well at a terminal concentration of 5 mg/mL, and incubated
with cells at 37 °C for 4 h. The formazan crystals were dissolved
in 150 mL DMSO for each well, and the absorbancy at 492 nm (for
absorbance of MTT formazan) and 630 nm (for the reference wave-
length) were measured with an ELISA reader. The compound was
tested three times.
2.1.4. Synthesis of 4-(4,4,5,5-tetramethyl-1,3,
2-dioxaborolan-2-yl)aniline (6)
4,4,5,5-Tetramethyl-2-(4-nitrophenyl)−1,3,2-dioxaborolane
(40 g, 160.6 mmol), 10% palladium/carbon (3 g), and methanol
(120 mL) were added to a 500 mL single-necked flask. Under a
hydrogen atmosphere, the reaction was stirred overnight. After the
reaction was completed, the solvent was removed by distillation
under reduced pressure to obtain a black solid 46 g. The crude
product was purified by stirring with n-hexane (2 × 20 mL), and
the off-white solid 6 (29.8 g, yield: 90.1%) was obtained after
suction filtration. m/z: 220.2 [M + H]+.
2.1.5. Synthesis of N-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)pyrrolidine-1- carboxamide (7)
Triphosgene (2.2 g, 7.5 mmol) and dichloromethane (20 mL)
were added to a 100 mL single-necked flask. Compound 6 (1.5 g,
6.9 mmol) was added under continuous stirring, after which tri-
ethylamine (1.5 mL, 10.4 mmol) was added dropwise. After being
allowed to react at room temperature for 2 min, the reaction sol-
vent was removed by distillation under reduced pressure. After the
residue was dissolved in dichloromethane (20 mL), tetrahydropyr-
role (0.7 g, 9.1 mmol) was added dropwise, and after reacting at
room temperature for 1 min, water was added to quench the reac-
tion. The reaction solution was concentrated under reduced pres-
sure to obtain white solid 7 (2.2 g, yield: 85.1%).
3. Results and discussion
3.1. Crystallographic analysis
In order to further study the structural characteristics of com-
pound 1, we carried out X-ray diffraction crystal structure anal-
ysis on the crystal. All crystal data are listed in Table 1. The
hydrogen bonds play an important role in the stability of the
crystal. The crystal packing diagrams of compound
2