B. Rubio-Ruiz et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116217
4
.3. Synthetic procedure and characterization of prodrug 2
(40 mg, 0.1 mmol) and 4 (0.2 mmol) were added to a 25 mL round-
distilled water. A fresh solution of 7 at 100 µM in PBS or 10% FBS in PBS
was added to the Au-catalysts, the mixtures shaken at 1,200 rpm and
◦
6
37 C, and fluorescence measured at 16 h as described above. This cycle
bottom flask and partially dissolved in distilled water (1 mL). N-(3-
was repeated 2 times.
′
Dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (0.4 mmol)
was then added to the mixture in one portion. The pH was monitored
and adjusted to 4.5 with a NaOH and / or HCl aqueous solution (1 M).
The reaction was stirred for 6 h at rt and constant pH (4.5). Water was
removed under reduced pressure, and crude suspended in acetonitrile
and vacuum filtered. Solution was purified by semi-preparative TLC
4.6. Au-mediated deprotection of prodrug 2 at physiological conditions
Prodrug 2 (200
μ
M) was dissolved in PBS (1 mL) with 1 mg of Au-
◦
catalysts and shaken at 1,200 rpm and 37 C in a Thermomixer. Reac-
tion crudes were monitored at 24 and 48 h by LCMS/MS (Agilent 1200)
using a micrOTOF II detector. LCMS/MS method: eluent A: water and
formic acid (0.1%); eluent B: acetonitrile and formic acid (0.1%); A/B =
95:5 isocratic 0.5 min, 95:5 to 0:100 in 4.5 min, isocratic 2 min, 0:100 to
95:5 in 0.5 min, and isocratic 2.5 min (flow = 0.2 mL / min).
eluting with DCM:MeOH/7:3 giving rise a light-yellow solid (12 mg,
1
2
7%). H NMR (500 MHz, DMF) δ 11.42 (bs, 1H), 10.69 (s, 1H), 7.54 (d,
J = 7.4 Hz, 2H), 7.49 (d, J = 15.9 Hz, 1H), 7.38 (dd, J = 14.0, 7.9 Hz,
3
6
H), 7.21 (dt, J = 8.0, 0.8 Hz, 1H), 6.96 (ddd, J = 8.1, 7.1, 1.2 Hz, 1H),
.90 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 6.44 (d, J = 15.0 Hz, 1H), 4.54 (d, J
=
2.4 Hz, 2H), 3.85 (s, 2H), 3.61 (t, J = 2.3 Hz, 1H), 2.83 (m, 2H), 2.72
4.7. Cell culture
(
m, 2H), 2.30 (s, 3H). 13C NMR (126 MHz, DMSO) δ 162.9, 139.5, 135.2,
1
33.5, 132.0, 129.0, 128.2 (x2), 127.6 (x2), 119.9, 118.1 (x2), 117.9,
Human lung adenocarcinoma A549 cells and human glioblastoma
U87 cells were cultured in Dulbecco’s Modified Eagle Media (DMEM)
supplemented with serum (10% FBS) and L-glutamine (2 mM) and
1
17.3, 110.4, 107.3, 78.9, 78.6, 62.5, 51.7, 48.9, 23.4, 11.2. HRMS
+
(
ESI) m/z [M + H] calculated for C24
26 2 3
H O N , 388.2019; found,
◦
3
88.2018. UPLC purity: > 95%; Rt: 1.496 min.
incubated in a tissue culture incubator at 37 C and 5% CO .
2
4
4
.4. Synthesis and characterization of Au-catalysts
.4.1. Synthetic procedure
4.8. Biocompatibility of Au-catalysts
Biocompatibility of Au-catalysts was tested by performing dose-
response studies in A549 and U87 cells. Cells were seeded in a 96-well
plate format (at 1,500 cells/well for A549 and 2,000 cells/well for
U87) and incubated for 48 h before treatment. Each well was then
replaced with fresh media containing Au-catalysts (0.8, 1.0, 1.2 and 1.4
mg/mL) and incubated for 5 days. Untreated cells were incubated with
TentaGel® HL NH
m) were added into a 25 mL Biotage microwave vial and suspended in
THF (2.5 mL). A solution of gold(III) chloride hydrate (120 mg, 0.4
mmol) in distilled water (500 L) was basified with a 1 M NaOH aqueous
2
resins (250 mg, 0.4–0.6 mmol/g, particle size 75
μ
μ
solution (11
μ
L). This freshly prepared solution was immediately added
◦
TM
to the suspended resins and heated to 60 C under stirring for 10 min.
The mixture was then stirred at rt for additional 2 h. Subsequently, the
solvents were filtered and the resins washed with DMF (3 × 10 mL),
DCM (3 × 10 mL) and methanol (3 × 10 mL). A solution of trisodium
citrate in distilled water (200 mM, 5 mL) was prepared. This solution
DMEM media. Experiments were performed in triplicates. PrestoBlue
cell viability reagent (10% v/v) was added to each well and the plate
incubated for 90 min. Fluorescence emission was detected using a Per-
kinElmer Victor multilabel reader (excitation filter at 540 nm and
emissions filter at 590 nm). All conditions were normalized to the un-
treated cells (100%).
2
was added to the gold(III)-treated resins and bubbled with a N flow at rt
for 10 min. Then, resins were stirred for 1 h at rt in a rotatory wheel. The
solvents were then filtered off and the resins washed with water (1 × 10
mL), methanol (3 × 10 mL) and DCM (3 × 10 mL). The solvents were
4.9. Cytotoxicity study: Drug vs prodrug
◦
then filtered and resins were dried in an oven at 40 C for 24 h.
Antiproliferative activity of 1 and O-propargylated HDAC derivative
2
were compared by performing dose–response studies in A549 and U87
4
.4.2. Electron microscopy analyses
cells. Cells were seeded as described above and incubated for 48 h before
treatment. Each well was then replaced with fresh media, containing
compounds (100 µM-0.01 µM) and incubated for 5 days. Untreated cells
were incubated with DMSO (0.1% v/v). Experiments were performed in
Morphological analysis of Au-catalysts was carried out by Scanning
Electron Microscopy (SEM, Inspect F50; FEI, Eindhoven, the
Netherlands) at the LMA-INA-Universidad Zaragoza facilities operated
at 10–15 kV. To analyze the cross section of the Au-catalysts, it was
required to infiltrate and embed them in a liquid epoxy resin. After
curing the resin block, it was sectioned with an Ultramicrotome (Leica
EM UC7) equipped with a diamond knife. Sections of 50–70 nm thick-
ness were collected on a copper grid (formvar-200 mesh) coated with
carbon film, and allowed to dry in air. The cross-section of the Au-
catalysts, that was embedded in the resin block, was analyzed to study
the Au nanoparticles dispersion using both Back Scattering Electron and
secondary electron Detectors. Transmission Electron Microscopy ob-
servations were carried out at using a FEI Tecnai F20 operating at 200
kV.
TM
triplicates. PrestoBlue cell viability reagent (10% v/v) was added to
each well and the plate incubated for 90 min. Fluorescence emission was
detected and results normalized as described above.
4.10. Au-mediated activation of prodrug 2 in cell culture
A549 and U87 cells were plated as described above. Each well was
then replaced with fresh media containing: Au-catalysts (0.8 mg/mL for
U87 cells and 1 mg/mL for A549); 1 (positive control: 10
μ
M, 3
M) or a combination of Au-catalysts + 2 (10
M). All experiments, including the untreated cells,
μ
M or 1
μ
M); 2 (10
μ
M, 3
μ
M or 1
μ
μ
M, 3 M or 1
μ
μ
contained 0.1% v/v of DMSO and were performed in triplicates. Cells
TM
4
.5. Au-catalysed deprotection of probe 7 and reusability of Au-catalysts
Prodye 7 was synthesised as previously reported.7 Au-catalysts (1
were incubated with drugs for 5 days. PrestoBlue cell viability reagent
(
10% v/v) was added to each well and the plates were incubated for 90
min. Fluorescence emission was detected and results normalized as
described above.
mg) were added to a 1 mL solution of reagent 7 (100 µM) in PBS or PBS
supplemented with 10% Fetal Bovine Serum (FBS). The mixtures were
◦
shaken at 1,200 rpm and 37 C in a Thermomixer and reactions moni-
4.11. Multidose prodrug activation study in cancer cell culture
tored at 16 h by fluorescence in a PerkinElmer Victor multilabel reader
4
(
excitation filter at 480 nm and emissions filter at 535 nm). Au-catalysts
A549 cells were seeded in a 24-well plate format (at 2 × 10 cells /
were recovered by centrifugation (13,000 rpm, 5 min) and washed with
well) and incubated for 24 h before treatment. Each well was then
5