Arylmethyloxyphenyl DeriVatiVes
-(chloromethyl)pyridine hydrochloride (144 mg, 0.88 mmol)
following the same procedure as described above for the preparation
of 5a. The crude product was transformed in hydrochloride and
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6611
3
Biological Section. Materials and Methods. Caco-2 cells were
obtained from Dr. Aldo Cavallini and Dr. Caterina Messa from
Laboratory of Biochemistry, National Institute for Digestive
Diseases, “S. de Bellis”, Bari, Italy. MultiScreen Caco-2 plate
system with 96 wells was purchased from Millipore Corporation,
2
crystallized from i-PrOH/i-Pr O to give 6b (125 mg, 0.35 mmol,
+
7
9
CH
6
9% yield) as a white solid: mp 147-149 ˚C. MS (m/z) 319 (M ,
+
1
3
8), 212 (M - OCH
CH ), 3.71 (s, 3H, OCH
.65-6.70 (m, 2H, Ar), 6.82 (d, 1H, J ) 8.4 Hz, Ar), 7.00 (t, 1H,
J ) 7.2 Hz, Ar), 7.10-7.24 (m, 3H, Ar), 7.91-7.97 (m, 1H, Py),
.38 (d, 1H, J ) 8.2 Hz, Py), 8.75 (d, 1H, J ) 5.4 Hz, Py), 8.83
s, 1H, Py). 13C NMR: δ 159.61, 155.24, 143.47, 139.41, 138.61,
2
Py, 100). H NMR: δ 2.88-2.98 (m, 4H,
Life Science Division, Danvers, MA. [ H]Vinblastine was purchased
2
2
3
), 5.16 (s, 2H, CH ), 6.58 (s, 1H, Ar),
2
from Amersham Life Sciences (Arlington Heights, IL). Verapamil
hydrochloride was purchased from Tocris Bioscience, Bristol, U.K.
[ H]Vinblastine Transport Inhibition. Caco-2 cells were seeded
3
8
(
1
1
onto Multiscreen plates with 20 000 cells/well for 10 days,
measuring the integrity of the cell monolayers by trans-epithelial
2
30.69, 130.58, 129.36, 127.60, 126.87, 122.35, 121.04, 114.45,
11.90, 111.32, 65.94, 55.29, 36.77, 31.89. Anal. (C21 ‚HCl)
electrical resistance (TEER, Ω‚cm ) with an epithelial voltohmmeter
H21NO
2
(Millicell-ERS; Millipore, Billerica, MA). Mature Caco-2 cell
monolayer exhibited a TEER of >800 Ω‚cm2 prior to use in
transport experiments. Transport experiments for tested compounds
were carried out as described by Taub et al.28
C, H, N.
-(Benzyloxy)-2-(2-phenylethyl)benzene (7a). A solution of
-(2-phenylethyl)phenol 4a (200 mg, 1.01 mmol) in a small amount
of DMSO (2 mL) was added to a solution of KOH (174.4 mg,
.11 mmol) in DMSO (2.5 mL). Stirring was continued over 15
1
2
To the basolateral (BL) compartment in each well, in the absence
and in the presence of P-gp inhibitors (from 200 nM to 400 µM),
3
3
min at room temperature. Then a solution of benzyl chloride (128
mg, 1.01mmol) in DMSO (2 mL) was added dropwise to the
solution of potassium 2-(2-phenylethyl)benzenolate. The suspension
was stirred at room temperature for 12 h. Then it was diluted with
AcOEt and washed with water and brine. The organic layer was
dried and concentrated. The resulting residue was purified by
chromatography on a silica gel column, eluting with hexane/AcOEt
was added 20 nM [ H]vinblastine for 120 min at 37 °C, and its
appearance in the apical (AP) compartment was monitored. At 120
min, a 20 µL sample was taken from the donor compartment to
determine the concentration of radioligand remaining in the donor
chamber at the end of the experiment. Samples were analyzed using
3
a LS6500 Beckman counter. For each compound, [ H]vinblastine
transport inhibition was calculated as the radioactivity difference
between the radioligand in the presence and absence of compound.
These differences were expressed as an inhibition percentage at a
single drug concentration.
Cell ATP availabIlity Assay. This experiment was performed
as reported in the technical sheet of the ATPlite 1step kit for
luminescence ATP detection using Victor3, from PerkinElmer Life
Sciences.32
(
8:2) to give 7a (87 mg, 0.31 mmol, 30% yield) as a colorless oil.
+
+
1
MS m/z 288 (M , 20), 197 (M - CH
3
Ar), 7.16-7.52 (m, 12H, Ar). C NMR: δ 157.42, 143.20, 138.23,
1
1
2
Ph, 73). H NMR: δ 2.88-
2
.07 (m, 4H, CH CH ), 5.13 (s, 2H, CH
2
2
O), 6.89-6.98 (m, 2H,
13
31.44, 130.82, 129.25, 128.96, 128.51, 127.92, 126.45, 121.48,
12.50, 70.75, 37.25, 33.71. Anal. (C21 20O) C, H.
-(Benzyloxy)-2-[2-(3-methoxyphenyl)ethyl]benzene (7b). Com-
H
1
pound 7b was synthesized from 4b (200 mg, 0.88 mmol) and benzyl
chloride (110 mg, 0.88 mmol) following the same procedure as
described above for the preparation of 7a. The crude product was
purified by column chromatography, eluting with hexane/AcOEt
Intracellular Doxorubicin Accumulation. The modulation of
doxorubicin intracellular accumulation by 5b and 8b was evaluated
by flow cytometry, utilizing verapamil as the standard of P-gp
inhibition.29 In all experiments, the various drug solvents (ethanol,
DMSO) were added to each control to evaluate the solvent
influence. In MCF7/Adr cells, doxorubicin was utilized at IC50
concentration (50 µM) for 1 day of exposure, and the standard and
the newly synthesized P-gp compounds were utilized at 20 µM for
2 h. The schedule of drug administration was the P-gp inhibitors
for 2 h, followed, after two wash steps with complete medium, by
doxorubicin for 1 day. After incubation, the cell media were
removed and trypsin-EDTA was used to detach the cells from the
plates. Cells were harvested, washed twice in ice-cold PBS (pH
7.4), and placed on ice (less than 1 h) until analysis. Fluorescence
measurements of individual cells were performed with a Becton-
Dickinson FACScan equipped with an ultraviolet argon laser.
Analysis was gated to include single cells on the basis of forward
and side light scatter and was based on the acquisition of data from
5000 cells. The log of the fluorescence was collected and displayed
as single-parameter histograms. The mean fluorescence intensity
of doxorubicin in the doxorubicin-treated cells, arbitrarily estab-
(
9:1) to afford 7b (137 mg, 0.43 mmol, 49% yield) as an oil. MS
+
+
1
m/z 318 (M , 85), 212 (M - CH
2
Ph, 33). H NMR: δ 2.94-3.09
(m, 4H, CH
2
CH
2
), 3.80 (s, 3H, OCH ), 5.16 (s, 2H, CH O), 6.79-
3
2
6
7
1
1
(
.88 (m, 3H, Ar), 6.95-7.02 (m, 2H, Ar), 7.22-7.30 (m, 1H, Ar),
.40-7.56 (m, 7H, Ar). 13C NMR: δ 159.61, 156.67, 154.31, 144.15,
37.50, 130.64, 130.16, 129.27, 128.62, 127.87, 127.30, 121.0,
20.79, 114.09, 111.68, 111.43, 69.97, 55.22, 36.66, 33.10. Anal.
C
22
H O
22 2
) C, H.
-[(3-Methoxybenzyl)oxy]-2-(2-phenylethyl)benzene (8a). Com-
pound 8a was synthesized from 4a (200 mg, 1.01 mmol) and
-methoxybenzyl chloride (157 mg, 1.01 mmol) following the same
1
3
procedure as described above for the preparation of 7a. The crude
product was chromatographed on a silica gel column, eluting with
hexane/AcOEt (9:1) to give 8a (213 mg, 0.67 mmol, 66% yield)
+
+
1
as an oil. MS m/z 318 (M , 96), 211 (M - CH
NMR: δ 2.90-2.98 (m, 4H, CH CH ), 3.80 (s, 3H, OCH
s, 2H, CH ), 6.86-6.94 (m, 3H, Ar), 7.02-7.09 (m, 2H, Ar), 7.12-
2
Ar, 55). H
2
2
3
), 5.08
(
2
13
lished as 100%, represented the positive control (MF ). The
7
1
1
.31 (m, 8H, Ar). C NMR: δ 159.22, 155.98, 141.85, 138.50,
29.51, 129.02, 128.07, 127.69, 126.65, 125.17, 120.17, 118.75,
12.76, 111.97, 111.08, 69.20, 54.69, 35.92, 32.49. Anal. (C22
PC
autofluorescence of untreated cells, arbitrarily established as 0%,
was the negative control (MFNC). The doxorubicin mean fluores-
cence intensity of doxorubicin in the verapamil or of other newly
synthesized agents plus doxorubicin-treated cells was MF . The
S
amount of doxorubicin in the samples was obtained by the following
formula:
H
22
O
2
)
C, H.
1-[(3-Methoxybenzyl)oxy]-2-[2-(3-methoxyphenyl)ethyl]ben-
zene (8b). Compound 8b was synthesized from 4b (200 mg, 0.88
mmol) and 3-methoxybenzyl chloride (136 mg, 0.88 mmol)
following the same procedure as described above for the preparation
of 7a. The crude product was chromatographed on a silica gel
MF - MF
MFPC - MFNC
S
NC
%
doxorubicin in samples )
× 100
column, eluting with hexane/Et
mmol, 65% yield) as an oil. MS m/z 348 (M , 25). H NMR: δ
.85-3.01 (m, 4H, CH CH ), 3.74 (s, 3H, OCH ), 3.79 (s, 3H,
OCH ), 5.07 (s, 2H, CH ), 6.71-6.80 (m, 2H, Ar), 6.85-6.93 (m,
2
O (8:2) to give 8b (198 mg, 0.57
+
1
2
2
2
3
Cytotoxicity Assay. The assay was performed using the Cyto-
Tox-One kit from the Promega Corp. (Madison, WI) as reported
in a previuous paper.34 Cell death was determined as the release of
lactate dehydrogenase (LDH) into the culture medium. The percent-
age of cytotoxicity was calculated relative to the LDH release from
total lysis of cells in untreated control. It is assumed here that the
drug-treated wells and the control wells contained the same total
3
2
3
7
1
1
3
H, Ar), 7.02-7.05 (m, 2H, Ar), 7.13-7.22 (m, 3H, Ar), 7.25-
.35 (m, 2H, Ar). 1 C NMR: δ 160.59, 160.33, 157.35, 144.78,
39.83, 131.37, 130.84, 130.29, 129.89, 127.94, 121.68, 121.51,
20.08, 114.81, 114.09, 113.38, 112.50, 112.12, 70.62, 55.98, 55.87,
3
24 3
7.30, 33.62. Anal. (C23H O ) C, H.