R. Martꢁnez-MꢂÇez et al.
a mesylation of 2-methoxyethanol (1), followed by a nucleophilic substi-
tution reaction of the mesylated derivative (2) with the disodium salt of
N,N-phenyldiethanolamine (3). These reactions yielded aniline derivative
4, which was further coupled with 4-aminopyridine (5) by employing
sodium nitrite.
the solution with dye, thus avoiding the dye leaving the inlets of the
MCM-41 pores. The mixture was stirred and heated at reflux (1108C) for
72 h in a nitrogen atmosphere. The solid (Rh-Azo-S) was isolated as a
dark violet solid by filtration, washed with acetonitrile (150 mL) and
dried at 408C for 12 h.
Synthesis of Azo-S: In a typical synthesis, template-free MCM-41 (0.1 g)
was suspended in dry acetonitrile (20 mL). Then, excess alkoxysilane de-
rivative 3-iodopropyltrimethoxysilane (130 mL, 0.66 mmol) was added to
the solution with the MCM-41 material and the final mixture was stirred
for 24 h at room temperature. Azo (33 mg, 0.082 mmol) was dissolved in
anhydrous acetonitrile (2 mL) and was added to MCM-41 externally
functionalised with 3-iodopropyltrimethoxysilane. The mixture was stir-
red and heated at reflux (1108C) for 72 h in a nitrogen atmosphere. The
solid (Azo-S) was isolated as a dark violet solid by filtration, washed
with acetonitrile (50 mL) and dried at 408C for 12 h.
Synthesis of 2: 2-Methoxyethanol (1) (2 mL, 0.025 mol) was dissolved in
dichloromethane (34 mL) in a 100 mL round-bottomed flask. The solu-
tion was kept in an ice bath for 15 min, then triethylamine (6.9 mL,
0.05 mol) was added to the crude reaction. Mesyl chloride (3.1 mL,
0.025 mol), dissolved in dichloromethane (6 mL), was added drop-wise
through a compensated addition funnel to the crude reaction for 30 min.
After this addition, the crude reaction was stirred at room temperature
for another 60 min period. Then, the crude reaction was poured onto a
water/ice mixture containing concentrated hydrochloric acid (10 mL),
and the organic layer was separated, washed three times with brine and
dried with anhydrous MgSO4. Dichloromethane was eliminated in a
rotary evaporator to give the final product as yellow oil (3.6 g, 0.024 mol,
96%). Spectroscopic data were coincident with those reported in the lit-
erature.
Synthesis of CPT-Azo-S: In a typical synthesis, template-free MCM-41
(100 mg) and chemotherapeutic agent camptothecin (56 mg, 0.16 mmol)
were suspended in acetonitrile/anhydrous methanol (4:1, 25 mL) in a
round-bottomed flask in an inert atmosphere. Excess alkoxysilane deriva-
tive 3-iodopropyltrimethoxysilane (130 mL, 0.66 mmol) was added to the
solution with the MCM-41 material and the final mixture was stirred for
24 h at room temperature. The Azo compound (33 mg, 0.082 mmol) was
dissolved in anhydrous acetonitrile (2 mL) and was added to the MCM-
41 externally functionalised with 3-iodopropyltrimethoxysilane. The mix-
ture was stirred and heated at reflux (1108C) for 72 h in a nitrogen at-
mosphere. The solid (CPT-Azo-S) was isolated as a dark violet solid by
filtration and washed with chloroform/methanol mixture (3:1, 200 mL)
overnight. The final solid (CPT-Azo-S) was filtered and washed with
more of the chloroform/methanol mixture (3:1, 100 mL). It was finally
dried at 408C for 12 h.
Synthesis of 4: N,N-Phenyldiethanolamine (3) (1.80 g, 0.01 mol) was dis-
solved in dry acetonitrile (60 mL) and then the flask was purged several
times with argon to remove oxygen and water from the atmosphere of
the reaction. Sodium hydride (0.48 g, 0.02 mol) was gradually added at
room temperature, after which a white precipitate appeared. Compound
2 (3.62 g, 0.024 mol) was dissolved in anhydrous acetonitrile (10 mL) and
then added drop-wise to the crude reaction using a compensated addition
funnel. After this addition, the crude reaction was heated at reflux for
24 h. The crude reaction was filtered off and the organic solvent was
eliminated using a rotary evaporator, yielding yellow oil containing the
final product 4 together with other secondary compounds. Pure 4 (0.54 g,
1.80 mmol, 20%) was isolated as a yellow oil through column chromatog-
raphy with aluminium oxide and hexane/ethyl acetate (5:1 v/v) as the
eluent. Spectroscopic data were consistent with those reported in the lit-
erature.
Dye release studies: In
a typical experiment, compound Rh-Azo-S
(6.8 mg) was suspended in 17 mL of enzyme solution (17 mg of nitrate re-
ductase in 17 mL of water at pH 7.5) and, at certain time intervals, an ali-
quot was separated and filtered. For the release studies with Rh-Azo-S
without enzyme (blank), solid Rh-Azo-S (6.8 mg) was placed in water
(17 mL, pH 7.5) and, at a certain time, an aliquot was separated and fil-
tered. The same quantities of Rh-Azo-S solid and enzyme were used in
the esterase dye release studies, but at pH 8, which is the optimal for this
enzyme. In both cases, the dye delivery from the pore voids to the aque-
ous solution was followed by using dye fluorescence (Rhodamine B) at
580 nm emission (excitation at 554 nm).
Synthesis of Azo: 4-Aminopyridine (5) (36.7 mg, 0.4 mmol) was dissolved
in a mixture of concentrated phosphoric acid (0.25 mL) and concentrated
nitric acid (0.12 mL). This crude was slowly added to a solution contain-
ing sodium nitrite (33.4 mg, 0.48 mmol) and water (0.8 mL) at À58C
(using an ice bath). The generated diazonium salt of 4-aminopyridine was
immediately added to
a solution containing compound 4 (120 mg,
0.403 mmol) and 30% phosphoric acid (2 mL). The crude reaction was
allowed to react for 30 min at À58C and for 60 min at room temperature.
The final dark red crude was neutralised with a saturated sodium carbo-
Cell culture conditions: The HeLa human cervix adenocarcinoma and
the MCF-7 breast cancer cells were purchased from the German Re-
source Centre for Biological Materials (DSMZ) and were grown in
DMEM supplemented with 10% of FCS. Cells were maintained at 378C
in an atmosphere of 5% carbon dioxide and 95% air, and they under-
went passage twice a week.
nate solution and the organic product was extracted with dichloro
ACHTUNGTNERmNUNG eth-
ACHTUNGTRENNUNG
was eliminated in a rotary evaporator. Product Azo (41 mg, 0.17 mmol,
24%) was isolated as a dark red solid through column chromatography
with aluminium oxide and hexane/ethyl acetate (1:1 v/v) as the eluent.
1H NMR (300 MHz, CDCl3): d=8.7 (d, 2H), 7.9 (d, 2H), 7.7 (d, 2H), 6.8
(d, 2H), 3.8–3.5 (m, 16H), 3.2 ppm (s, 6H); 13C: d=52.5, 59.8, 70.1, 73.2,
75.5, 114.6, 119.1, 126.2, 151.8 ppm.
WST-1 cell viability assay: HeLa and MCF-7 cells were cultured in sterile
96-well microtiter plates at a seeding density of 2500 and 3500 cells per
well, respectively, and they were allowed to settle for 24 h. Rh-Azo-S in
DMSO was added to cells at a final concentration of 200, 100 and
50 mgmLÀ1. After 23 h, WST-1 (7 mL of a 5 mgmLÀ1 solution) was added
to each well. Cells were further incubated for 1 h (a total of 24 h of incu-
bation was therefore studied), and absorbance was measured at 450 nm
and normalised versus absorbance at 690 nm.
Synthesis of Rh-S: In a typical synthesis, 100 mg of template-free MCM-
41 and Rhodamine B dye (39 mg, 0.8 mmol Rhodamine B/gMCM-41)
were suspended in dry acetonitrile (25 mL) inside a round-bottomed
amber flask in an inert atmosphere. The mixture was then stirred for 24 h
at room temperature to achieve maximum loading in the MCM-41 scaf-
folding pores. Finally, the solid (Rh-S) was filtered off, washed and dried
at 378C for 12 h.
Live confocal microscopy Rh-Azo-S, CPT-Azo-S and Azo-S cellular in-
ternalisation assays: HeLa cells were seeded in 24 mm f glass coverslips
in 6-well plates at a seeding density of 50000 cells per well for 24 h.
Then, cells were treated when indicated with Rh-Azo-S, CPT-Azo-S or
Azo-S at concentrations of 100 and 50 mgmLÀ1. Next, cells were incubat-
ed for 48 h prior to the confocal microscopy studies. For this purpose,
cells were stained when indicated with Hoechst 33342 (10 ngmLÀ1) and
of WGA Alexa Fluor 647 (5 mgmLÀ1) for 30 min in PBS containing 10%
FCS or by keeping the medium in case of CPT-Azo-S and Azo-S treat-
ments. Slides were visualised under a confocal microscope.
Synthesis of Rh-I-S: Excess alkoxysilane derivative 3-iodopropyltri
ACHTUNGTNERmNUNG eth-
ACHTUNGTRENNUNGoxysilane (137 mL, 0.7 mmol) was added to the solution with the MCM-
41 material loaded with Rhodamine B (Rh-S) and the final mixture was
stirred for 24 h at room temperature. Finally, the solid (Rh-I-S) was fil-
tered off, washed and dried at 378C for 12 h.
Synthesis of Rh-Azo-S: The Azo compound (41 mg, 0.1 mmol) was dis-
solved in anhydrous acetonitrile (2 mL) and was added to MCM-41
(100 mg) loaded with Rhodamine B and externally functionalised with 3-
iodopropyltrimethoxysilane (Rh-I-S) suspended in dry acetonitrile
(20 mL). Excess Rhodamine B was also added to the mixture to saturate
Cytofluorometry studies employing CPT-Azo-S: To perform the cyto-
fluorometry studies, HeLa cells were seeded at 12500 cells per well in a
24-well. After 24 h, cells were treated with CPT-Azo-S or Azo-S at con-
1354
ꢅ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 1346 – 1356
Mas, Núria
