L. Shi, L.-l. Gao, S.-z. Cai et al.
European Journal of Medicinal Chemistry 221 (2021) 113528
purification. Flash column chromatography was carried out on
200e300 mesh silica gel (Qingdao Haiyang Chemical, China). Re-
actions were monitored by thin-layer chromatography (TLC) on
0.25 mm silicagel plates (GF254) and visualized under UV light. 1H
NMR and 13C NMR spectra were recorded with a Bruker AV-300
spectrometer (Bruker Company, Germany) in the indicated sol-
vents (CDCl3, TMS as internal standard): the values of the chemical
4.2. Cell culture and treatment
The human lung cancer cell line (A549), human breast cancer
cell line (MDA-MB-231), human leukemia cell line (K562), human
live cancer cell line (HepG2), human glioma cell lines (U251, U87,
SHG44), rat glioma cell line (C6), and Human umbilical vein
endothelial cells (HUVEC) were purchased from the Type Culture
Collection of the Chinese Academy of Sciences (Shanghai, China).
The glioblastoma cell lines U251 and U87 were exposed to
increasing concentrations of TMZ to generate TMZ-resistant col-
onies (the U251/TMZ and U87/TMZ cell lines) in our lab. Cells were
cultured with DMEM medium (for U251, U87, U251/TMZ, U87/TMZ
and C6), RPMI 1640 (for A549, MDA-MB-231, K562, HepG2, SHG44
and HUVEC), added with 10% fetal bovine serum (FBS) and
penicillin-streptomycin in an atmosphere of 5% CO2 at 37 ꢂC.
shifts are expressed in d values (ppm) and the coupling constants (J)
in Hz. Low-and high-resolution mass spectrums (LRMS and HRMS)
were measured on Finnigan MAT 95 spectrometer (Finnigan, Ger-
many). The purity (ꢁ95%) of the target compound was verified by
the HPLC (Aglilent Technologies 1260 Infinity) study performed on
an Aglilent C18 column (4.6 ꢀ 150 mm, 5
mm) using a mixture of
solvent methanol/water (1:4) at a flow rate of 1.0 mL/min and peak
detection at 230 nm under UV.
4.3. MTT assay
4.1.2. Procedure for the synthesis of intermediate 12
The cytotoxicity of the compounds was detected by the MTT
assay. Briefly, 5 ꢀ 103 cells per well were seeded into 96-well plates
and allowed to grow to the plate for 24 h. Then, cells were treated
with various concentrations of compounds for 72 h. After treat-
The intermediates 10 and 16 were prepared according to the
previous procedure [31]. To a solution of 4-hydroxy benzaldehyde
(11, 0.12 g, 1 mmol) in acetone (30 mL), anhydrous K2CO3 (1.1 g,
8 mmol, 8 eq.) and 1,10-dibromodecane (2.4 g, 8 mmol, 8 eq.) were
added, and the mixture was stirred overnight at room temperature.
The K2CO3 was removed by filtration, and the solvent was evapo-
rated under the vacuum. The crude products were purified by flash
chromatography on silica gel using the eluent petroleum ether/
ethyl acetate (1:1, v:v) to give compound 12 (0.26 g, 76% yield) as a
ment,
20
mL
of
MTT
(3-(4,5-dimethylthiazolyl-2)-2,5-
diphenyltetrazolium bromide) solutions were added and incu-
bated at 37 ꢂC for 3e4 h. After incubation, the supernatants were
replaced with 150 mL of DMSO to dissolve the purple crystals. Then
the absorbance measured at 570 nm was employed to reflect the
cell growth condition. IC50 values were calculated from dose-
dependent curves using GraphPad 5.0. Data are expressed as the
mean SD of three independent experiments.
yellow solid. 1H NMR (300 MHz, Chloroform-d)
d 9.88 (s, 1H),
7.91e7.72 (m, 2H), 7.06e6.92 (m, 2H), 4.04 (t, J ¼ 6.5 Hz, 2H), 3.41 (t,
J ¼ 6.8 Hz, 2H), 1.83 (ddq, J ¼ 12.5, 7.9, 6.6 Hz, 4H), 1.49e1.24 (m,
13H); ESI-MS: m/z 341.3 [M þ H]þ.
4.4. Measurement of mitochondrial uptake of compounds
HPLC method was used to detect and quantify, for this purpose,
U251 cells were grown in 100 mm dishes and then treated with
designed concentrations of compounds 5 and 14 for 12 h. After the
treatment, the cells were washed twice with ice-cold PBS. Mito-
chondrial isolation was then performed using the cell mitochon-
drial isolation kit (Beyotime, Jiangsu, Chian) according to the
instructions of the manufacturer. The mitochondria pellet was
diluted in PBS and extracted twice with ethyl acetate. After the
organic layers were combined and dried, the dry residue was dis-
solved in acetonitrile for HPLC analysis. The HPLC analysis method
was similar to the purification method.
4.1.3. Procedure for the synthesis of target compound 14
Compound 10 (0.47 g, 2 mmol) and boric anhydride (0.12 g,
1.7 mmol, 0.7 eq.) were dissolved in ethyl acetate and stirred at
70 ꢂC for 30 min. Then compound 12 (0.68 g, 2 mmol, 1 eq.) and
tributyl borate (0.46 g, 2.0 mmol) were added, and the mixture was
stirred for 30 min. After that, n-butylamine (0.15 g, 2 mmol) dis-
solved in ethyl acetate was added dropwise. After increasing the
temperature to 85 ꢂC, stirring was continued for 2.5 h. The mixture
was then hydrolyzed by adding aqueous HCl (1.0 M) and stirring at
60 ꢂC for 30 min. The organic layer was separated, and the aqueous
layer was extracted with ethyl acetate for three times. The com-
bined organic layer were washed with water until neutral. The
solvent was removed in vacuo and the crude product (13) was used
for the next step without purification.
4.5. Cell co-localization study
U251 cells were seeded on glass coverslips at a density of
1 ꢀ 105 cells and allowed to adhere overnight. The following day,
cells were treated with compound 14 for 24 h. After that, media was
removed and cells were washed with PBS for two times prior to
fixation with 3.7% formaldehyde for 15 min at room temperature.
Fixed cells were washed with PBS prior and subsequent to per-
meabilization with ice-cold acetone for 5 min at ꢃ20 ꢂC. Coverslips
were blocked for 30 min with 1% bovine serum albumin in PBS to
inhibit nonspecific antibody binding prior to incubation overnight
at 4 ꢂC with immuno-capture antibody raised against complex I or
antisera recognizing the triphenylphosphonium moiety, each
diluted 1:1000 in 1% BSA/PBS. Antibodies were removed, and
coverslips were washed with PBS. Goat-antirabbit Cy5-conjugated
and goat-antimouse Cy3-conjugated antibodies were diluted
1:200 in 1% BSA/PBS and incubated with coverslips for 2 h. Cov-
erslips were again washed with PBS under gentle agitation, air-
dried, and mounted on precleaned glass slides using Prolong Gold
antifade mounting media. Images were obtained using confocal
The mixture of compound 13 (0.50 g, 0.9 mmol) and PPh3
(1.02 g, 4.5 mmol, 5 eq.) in 20 mL acetonitrile was stirred for 2 days
at refluxing. Evaporation of the solvent, the solid was washed with
ethyl acetate, and the crude product was purified by flash chro-
matography on silica gel using the dichloromethane/methanol
(30:1, v:v) to afford the desired product 14 as a red solid (0.34 g, 51%
yield). 1H NMR (300 MHz, Chloroform-d)
d 7.91e7.74 (m, 10H),
7.74e7.63 (m, 6H), 7.58 (dd, J ¼ 15.8, 6.5 Hz, 2H), 7.47 (d, J ¼ 8.5 Hz,
2H), 7.16e7.03 (m, 2H), 6.96 (d, J ¼ 8.1 Hz, 1H), 6.88 (d, J ¼ 8.5 Hz,
2H), 6.48 (dd, J ¼ 15.8, 1.8 Hz, 1H), 6.14 (s, 1H), 5.79 (s, 1H), 3.93 (s,
3H), 3.81 (d, J ¼ 13.8 Hz, 2H), 1.77e1.23 (m, 18H); 13C NMR (75 MHz,
Chloroform-d)
d 183.3, 160.9, 148.3, 147.1, 140.6, 140.2, 135.1, 135.0,
133.7, 133.6, 130.6, 130.5, 129.8, 127.5, 127.4, 123.1, 121.6, 121.6, 118.8,
117.9, 115.1, 114.9, 109.7, 101.4, 68.1, 56.0, 30.5, 30.4, 29.4, 29.2, 29.1,
29.1, 25.9, 23.0, 22.7, 22.7, 22.5; ESI-MS: m/z 739.4 [M]þ; HRMS:
calculated for C48H52O5Pþ [M]þ 739.3547, found 739.3537; HPLC
purity of 96.05%.
9