Synthesis and biological evaluation of (hetero)arylmethyloxy- and arylmethylamine-phenyl derivatives as potent P-glycoprotein modulating agents

Article abstract of DOI:10.1021/jm701267q

Starting from lead compounds 12b and 28b, previously characterized as P-glycoprotein (P-gp) modulating agents, two series of new compounds were investigated. Compounds 14a,b and 15a,b displayed high P-gp modulating activity in the submicromolar range (EC<

Full text of DOI:10.1021/jm701267q

J. Med. Chem. 2008, 51, 1415–1422
1415
Synthesis and Biological Evaluation of (Hetero)Arylmethyloxy- and Arylmethylamine-phenyl
Derivatives as Potent P-glycoprotein Modulating Agents
Nicola Antonio Colabufo,*,† Francesco Berardi,† Roberto Perrone,† Simona Rapposelli,*,‡ Maria Digiacomo,‡
Michael Vanni,‡ and Aldo Balsamo‡
Dipartimento Farmacochimico, UniVersitá degli Studi di Bari, Via Orabona, 4, 70125 Bari, Italy, and Dipartimento di Scienze Farmaceutiche,
UniVersità di Pisa, Via Bonanno, 6, 56126 Pisa, Italy
ReceiVed October 8, 2007
Starting from lead compounds 12b and 28b, previously characterized as P-glycoprotein (P-gp) modulating
agents, two series of new compounds were investigated. Compounds 14a,b and 15a,b displayed high P-gp
modulating activity in the submicromolar range (EC50 values from 0.25 to 0.80 µM). Moreover, amino
derivatives 23–27 showed EC50 values ranging from 0.085 to 0.90 µM. In the pyridyl series, the best result
has been obtained for 4-pyridyl derivative 17b (EC50 ) 0.85 µM). The best P-gp modulating agents 14a,b,
15a,b, and 23–27 also have been studied for determining their breast cancer resistance protein (BCRP)
inhibition activity. The results demonstrated that only the amino derivatives 23–27 displayed moderate BCRP
inhibition activity.
Chart 1. Third Generation P-gp Inhibitors
Introduction
The ATP-binding cassette (ABC) transporters are a trans-
membrane (TM) family of proteins1,2 that extrudes various
molecules across all cell membranes by using the ATP hy-
drolysis energy. These transporters efflux compounds from the
cytoplasm to the outside of the cell or into an intracellular
compartment such as the endoplasmic reticulum (ER), the
mitochondria, or the peroxisome.3
In humans, 49 ABC genes, organized into seven subfamilies
(A-G), have been reported,4 and many of these genes are
involved in human genetic diseases5–8 and in multidrug resis-
tance (MDR).9–12
MDR is an important cause of failure of cancer treatment
with chemotherapeutic agents.10,13 The mechanism of MDR is
mainly the cell overexpression of some ABC transporters
localized in the cell membrane such as P-glycoprotein (P-gp),
breast cancer resistance protein (BCRP), multidrug resistance
protein 1-7 (MRP1-7), and lung resistance protein (LRP). These
transporters, involved in MDR, determine this pharmacological
effect by effluxing a variety of chemotherapeutic drugs from
tumor cells.14
A potential strategy to reverse MDR could be the coadmin-
istration of a chemotherapeutic agent with an efflux pump
inhibitor, although such reversal agents might actually increase
the side effects of chemotherapy by blocking physiological
anticancer drug efflux from normal cells.15–17 In recent years,
many drugs have been investigated for reversing MDR,18,19 such
as the calcium channel blocker verapamil and the antisteroids
tamoxifen (first generation), biricodar (second generation), and
tariquidar, elacridar, and zosuquidar (third generation), as
depicted in Chart 1.
At the present, clinical trials reported elacridar20 and zosu-
quidar21 in phase I while tariquidar reached phase III, even if
recently it was terminated because of an increased incidence of
side effects.22
Recently, we developed a new class of small molecules
having arylmethyloxyphenyl structure that displayed moderate
P-gp modulating activity.23 Among these previously studied
derivatives, compounds 12b (EC50 ) 17.2 µM) and 28b (EC50
) 27.5 µM), depicted in Figure 1, have shown the best results
in modulating P-gp activity. Previous studies evidenced that the
P-gp inhibition activity was influenced by the presence of a
* To whom correspondence should be addressed. (N.A.C.) Phone: +39-
0805442727. Fax: +39-0805442231. E-mail: colabufo@farmchim.uniba.it.
(S.R.) Phone: +39-0502219582. Fax: +39-0502219605. E-mail: rappsi@
farm.unipi.it.
†
Universitá degli Studi di Bari.
Università di Pisa.
‡
10.1021/jm701267q CCC: $40.75
2008 American Chemical Society
Published on Web 02/08/2008

1416 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Colabufo et al.
Scheme 2a
Figure 1. Lead compounds 12b and 28b.
Scheme 1a
a Reagents: (A) DBU (1,8-diazabicyclic[5.4.0]undec-7-ene), CH3CN; (B)
H2, Pd/C 10%, EtOH; (C) appropriate methoxybenzylchloride, KI, CH3CN,
microwave; (D) (i) appropriate dimethoxybenzaldehyde, EtOH, reflux, (ii)
NaBH4, EtOH/H2O.
methoxybenzylchloride in the presence of a catalytic amount
of KI,25 while compounds 25–27 were obtained by reaction of
21 with the corresponding dimethoxy-benzaldehydes and sub-
sequent reduction with NaBH4. The synthesis of compounds
10a,b, 12a,b, 28a,b, and 29a,b was reported in our previous
work.23
a Reagents: (A) KOH, ∆, DMSO. b Physical properties reported in ref
23.
Results and Discussion
methoxy group on the C-ring. In the present work, compounds
11a,b, 13a,b-17a,b, and 22–27 have been synthesized in order
to investigate the following: (i) the influence of the extent of
methoxylation on the C-ring for P-gp modulating activity
(compounds 11a,b and 13a,b-16a,b), (ii) the effect of the
methoxy substituent on the A-ring (11b, 13b-16b, and 17b),
(iii) the effect of the position of the pyridyl nitrogen on the
P-gp activity (17a,b), and (iv) the effect of the isosteric
substitution of the oxygen in the spacer between the B-ring and
C-ring with a NH group (compounds 22–27).
The P-gp inhibition activity for each compound was studied
by three combined biological assays: (i) inhibition of [3H]-
vinblastine transport, (ii) ATP cell depletion, and (iii) apparent
permeability (Papp) in a Caco-2 cells monolayer. By these assays,
it was possible to determine the structural requirements that
favor P-gp inhibition and the mechanism involved in P-gp
modulation.24 Moreover, the best P-gp modulators have been
tested for both their BCRP activity and cytotoxicity.
Chemistry. Compounds 10a,b-17a,b were prepared by
alkylation of phenols 1a,b23 with the appropriate substituted
benzyl halide (2–8) or with 4-chloromethylpyridine (9) in the
presence of KOH, as reported in Scheme 1. The aniline
derivatives 22–27 were synthesized starting from the phospho-
nium salt 18, which was submitted to Wittig’s reaction with
2-nitrobenzaldehyde (19) to give 1-[2-(3-methoxyphenyl)vinyl]-
2-nitrobenzene (20) as a mixture of cis and trans isomers
(Scheme 2). The subsequent catalytic hydrogenation of 20
afforded the aniline derivative 21. The final compounds 22–24
were obtained by a microwave reaction with the appropriate
SAR Studies. The new compounds synthesized (11a,b,
13a,b-17a,b, and 22–27) were evaluated for their ability to
inhibit the P-gp protein. Results are reported in Tables 1 and 2,
together with those already obtained for the previously described
analogues (10a,b, 12a,b, 28a,b, and 29a,b) and the reference
drugs (elacridar, verapamil, and cyclosporin A).
The results of the 12b analogues (Table 1) studied for
determining the importance of the presence, the position, and
the extent of methoxylation of the C-ring displayed that the
unsubstituted derivative 10b23 is a weak P-gp modulating agent
(EC50 ) 92.8 µM) with respect to monosubstituted derivatives
11b-13b (EC50 ) 12, 17.2, and 5.2 µM, respectively).
Compound 13b, which had the best activity, had a 4-OCH3
substituent on the C-ring.
In regards to the C-ring dimethoxy-substituted derivatives
(14b-16b), all isomers were found to be more active than the
monosubstituted ones, in accord with the hypothesis that
substituents able to increase the hydrogen bond formation with
the P-gp active site might constitute an important structural
requirement for improving the P-gp inhibition activity.26–28 The
best results were observed for 2,3- and 3,4-dimethoxy deriva-
tives 14b (EC50 ) 0.25 µM) and 15b (EC50 ) 0.65 µM), while
the 3,5-dimethoxy derivative 16b displayed a slightly lower P-gp
inhibition activity (EC50 ) 2.5 µM) than those of the corre-
sponding isomers 14b and 15b. This trend was also confirmed
by the data concerning their inferior homologues, lacking the
methoxy substituent on the A-ring (10a-16a). In fact, also in
this series, the best results were observed for 2,3- and 3,4-
dimethoxy derivatives 14a and 15a (EC50 ) 0.80 and 0.75 µM,

Potent P-gp Modulating Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1417
Table 1. Biological Results of Derivatives 10a,b-16a,b and 22–27
[3H]-vinblastine
transport inhibition,
ATP-ase
[Papp(B-A)]/
[Papp(A-B)a]
compd
R
X
R1
R2
EC50 ( S.E.M. (µM)a
activationb
10ac
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
H
H
H
H
H
H
H
H
H
H
>1000c
Yc
Nc
N
10bc
11a
92.8 ( 5.5c
20 ( 2.5
2-OCH3
2-OCH3
3-OCH3
3-OCH3
4-OCH3
4-OCH3
2-OCH3
2-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
2-OCH3
3-OCH3
4-OCH3
2-OCH3
3-OCH3
3-OCH3
1.4
8.7
11b
12 ( 1.5
N
12ac
12bc
13a
95.6 ( 10.0c
17.2 ( 0.5c
3.1 ( 0.5
5.2 ( 0.50
0.80 ( 0.05
0.25 ( 0.05
0.75 ( 0.07
0.65 ( 0.08
1.60 ( 0.20
2.5 ( 0.40
N.D.e
Yc
Nc
N
13
5.1
7.6
0.95
7.8
2.1
5.1
5.7
13b
14a
14b
15a
15b
16a
16b
22
23
24
25
26
N
N
Y
N
N
N
N
3-OCH3
3-OCH3
4-OCH3
4-OCH3
5-OCH3
5-OCH3
H
6.7
N.D.e
N
N.D.e
2.9
H
H
0.48 ( 0.002
N.D.e
N.D.e
N
N.D.e
5.5
3-OCH3
4-OCH3
5-OCH3
0.085 ( 0.02
0.18 ( 0.04
0.90 ( 0.004
2.0d
80 ( 7.5
20 ( 1.0
N
5.6
27
N
6.9
elacridar
cyclosporin A
verapamil
Nd
N
<2d
9.6
Y
1.2
a Data are the mean of three independent determinations, samples in triplicate. b Effect measured at 100 µM. Data are the mean of three independent
determinations with samples in triplicate. c See ref 23. d See ref 24. e Not determined (chemically unstable).
Table 2. Biological Properties of Pyridyl Derivatives 28a,b, 29a,b, and 17a,b
[3H]-vinblastine
transport inhibition,
ATP-ase
[Papp(B-A)]/
[Papp(A-B)c]
compd
R
Het
EC50 ( SEM (µM)a
activationb
28ad
28bd
29ad
29bd
17a
H
OCH3
H
OCH3
H
OCH3
2-pyridyl
2-pyridyl
3-pyridyl
3-pyridyl
4-pyridyl
4-pyridyl
100 ( 25d
27.6 ( 0.2d
125 ( 15d
>1000d
Nd
Nd
Yd
Yd
Y
18 ( 2.5
1.3
2.1
17b
0.85 ( 0.05
Y
a Data are the mean of three independent determinations (samples in triplicate), each with SEM < 10%. b Effect measured at 100 µM. Data are the mean
of three independent determinations with samples in triplicate. c Data are the mean of three independent determinations, samples in triplicate. d See ref 23.
respectively), while 3,5-dimethoxy compound 16a displayed
only moderate P-gp inhibition activity (EC50 ) 1.60 µM).
compound 10b is some 10 times more potent than 10a, and
12b is 5.5 times more potent than 12a.
Moreover, with respect to the dimethoxy compounds
14a-16a, the monosubstituted derivatives 11a-13a displayed
lower activity (EC50 ) 20.0, 95.6, and 3.1 µM, respectively),
but the latter was more potent than the unsubstituted 10a (EC50
> 1000 µM). Among monomethoxy derivatives, also in this
series, the compound bearing 4-OCH3 on the C-ring, 13a,
displayed the best activity.
The isosteric substitution of oxygen present on the spacer
linking the B-ring to the C-ring of the lead compound 12b with
NH, as in compound 23, determined an increase in the P-gp
inhibition activity (EC50
) 17.2 vs 0.48 µM, respectively). The
position of the methoxy on the C-ring in this series has been
evaluated. Derivatives 22 and 24 bearing 2-OCH3 and 4-OCH3
on the C-ring, respectively, were chemically unstable when
dissolved in EtOH, DMSO, and phosphate buffered saline
(PBS), which are the solvents employed in the biological assays.
Among the dimethoxy-substituted derivatives 25-27, the best
P-gp inhibition activity was observed for the 2,3-dimethoxy-
substituted compound 25 (EC50 ) 0.085 µM), while the 3,5-
Furthermore, comparing the biological results of compounds
differing by the presence (10b-16b) or the absence (10a-16a)
of the methoxy group on the A-ring, it was evident that this
substituent played a modest role on the P-gp inhibition activity,
with the exception of compounds 10a,b and 12a,b. In fact,

1418 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Colabufo et al.
dimethoxy-substituted derivative 27 displayed a slightly lower
P-gp inhibition activity (EC50 ) 0.90 µM). Compound 26,
bearing a 3,4-dimethoxy substitution on the C-ring, displayed
good P-gp inhibition activity (EC50 ) 0.18 µM). These results
demonstrated the same trend observed for the corresponding
isomers 14b-16b, having an oxygen atom in the spacer between
the B-ring and the C-ring.
Moreover, starting from the lead heteroaromatic compound
28b, having a 2-pyridyl moiety, instead of the C-ring, the
shifting of the pyridyl linkage from the 2 to the 3 and 4 positions
(29b and 17b, respectively) has been investigated. The results
listed in Table 2 displayed that the best P-gp inhibition activity
was obtained for compound 17b, having a 4-pyridyl nucleus
(EC50 ) 0.85 µM).
Moreover, the P-gp modulating activity dramatically de-
creased in compound 29b where the heteroaromatic was a
3-pyridyl (EC50 > 1000 µM). The same trend was observed
also in the corresponding derivatives lacking a methoxy
substituent on the A-ring (28a, 29a, and 17a). In fact, the
4-pyridyl derivative 17a is the best of the series (EC50 ) 18.0
µM), while the 2- and 3-pyridyl derivatives 28a and 29a
displayed weak inhibition activity (EC50 ) 100 and 125 µM,
respectively). This finding indicates a marked influence on the
P-gp inhibitory activity of the position of the nitrogen atom on
the pyridine nucleus.
Comparing 17a with 17b (EC50 ) 18 vs 0.85 µM, respec-
tively) and 28a with 28b (EC50 ) 100 vs 27.6 µM, respectively),
it was evident that the presence of a methoxy substituent on
the A-ring in this series seems to be significantly involved in
the P-gp modulation activity. On the other hand, compounds
29a and 29b were both weakly active.
P-gp Inhibition Mechanism Investigation. In a monolayer
efflux assay, the apparent permeability (Papp) in both basolateral
to apical [Papp(B-A)] and apical to basolateral [Papp(A-B)]
directions was determined for each compound. The BA/AB ratio
is ranging from 0.95 to 2.1 for compounds 11a, 14a, 15a, 17a,b,
and the reference compounds verapamil and elacridar (BA/AB
) 1.2 and 2, respectively), while the other compounds and
cyclosporin A displayed BA/AB >2.9.
Figure 2. Cytotoxic effect of P-gp inhibitors on the MCF-7/Adr cell
line at 48 h. The effect of compound 17b has been determined at 1
µM.
Table 3. BCRP Inhibition Evaluation of Derivatives 14a,b, 15a, 23, 25,
and 26
[3H]-mitoxantrone
transport inhibition,
compd
EC50 ( SEM (µM)a
14a
14b
15a
15b
23
25
26
75 ( 5
120 ( 20
58 ( 3
150 ( 25
7.0 ( 0.5
1.5 ( 0.20
0.50 ( 0.02
a Data are the mean of three independent determinations, samples in
triplicate.
Biological Cell Activity. Since the P-gp modulators should
be devoid of cytotoxic activity, we tested compounds 14a,b,
15a,b, 17b, 23, and 25–27, displaying submicromolar activity
values, to establish their intrinsic cytotoxicity in the MCF-7/
Adr cell line overexpressing P-gp. As depicted in Figure 2, all
tested compounds until 50 µM were devoid of cytotoxicity at
48 h, except compounds 15a and 23, which displayed only
moderate cytotoxicity (10%). In contrast, compound 17b
displayed high cytotoxicity (50%) at 1 µM (Figure 2, last bar
to the right), so that this result discouraged the development of
the pyridyl series as potential MDR reverting agents.
BCRP Activity. Since the limit of P-gp inhibitors such as
elacridar is the poor selectivity toward other ABC transporters,
in particular the BCRP pump, the best P-gp modulators, 14a,b,
15a,b, 23, 25, and 26, were tested for their ability to inhibit
[3H]-mitoxantrone, a specific BCRP substrate. In Table 3 are
reported the EC50 values of these compounds. The results
showed that oxygen derivatives 14a,b and 15a,b were weakly
active to inhibit the BCRP pump (EC50 from 58 to 150 µM). In
contrast, the amino derivatives 23, 25, and 26 showed a good
BCRP inhibition activity. In particular, compounds 25 and 26
showed micromolar BCRP inhibition activity (EC50 ) 1.5 and
0.5 µM, respectively).
The compounds having BA/AB ratio up to 2.1 were not
effluxed by P-gp, while compounds with BA/AB ratio >2.9
were P-gp transported and consequently considered substrates
of this pump.24
As regards ATP-ase activation, all compounds were unable
to deplete ATP from cells with the exception of 10a, 12a, 14b,
17a,b, and 29a,b. The compounds that reduced the cell ATP
level were considered P-gp substrates, while the compounds
with unmodified ATP cell content were not effluxed by P-gp.
Combining the results of the biological assays, compounds
11a, 14a, 15a, and the reference compound elacridar were
claimed “P-gp inhibitors” because they were not transported
by P-gp, they inhibited [3H]-vinblastine transport, and they were
unable to deplete ATP.
Compounds 17a,b as well as verapamil were not transported
by P-gp; they inhibited [3H]-vinblastine transport, but they were
able to promote the depletion of ATP so that they were claimed
“not transported” P-gp substrates.
Compounds 11b, 13a,b, 15b, 16a,b, 23, and 25–27 as well
as cyclosporin A were effluxed by P-gp; they inhibited [3H]-
vinblastine transport, and they were unable to deplete ATP so
that they were considered “saturating transported” P-gp substrates.
Only compound 14b was a P-gp substrate because it was
effluxed by P-gp, it inhibited [3H]-vinblastine transport, and it
depleted ATP.
Conclusions
In our previous work, we studied arylmethyloxyphenyl
derivatives displaying P-gp modulating activity, and among them
12b and 28b were suggested as lead compounds of this new
class of small P-gp modulators. Starting from 12b, the extent
of methoxylation on the C-ring, the effect of a methoxy
substituent on the A-ring, and the influence of isosteric
substitution have been investigated.
Starting from 28b, the effect of 3- and 4-pyridyl isomers
and the influence of a methoxy substituent on the A-ring have

Potent P-gp Modulating Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1419
chemical manipulations of this new class of P-gp inhibitors may
lead to the synthesis of new derivatives endowed of improved
inhibitory activity and selectivity.
Experimental Section
General Methods. Melting points were determined on a Kofler
hot-stage apparatus and are uncorrected. 1H and 13C NMR spectra
were obtained with a Varian Gemini 200 MHz spectrometer.
Chemical shifts (δ) are reported in parts per million downfield from
tetramethylsilane and referenced from solvent references; coupling
constants J are reported in hertz; 13C NMR spectra were fully
decoupled. The following abbreviations are used: singlet (s), doublet
(d), triplet (t), double-doublet (dd), and multiplet (m).
Mass spectra were obtained on a Hewlett-Packard 5988 A
spectrometer by using a direct injection probe and an electron beam
energy of 70 eV. The elemental compositions of the compounds
agreed to within (0.4% of the calculated value. Chromatographic
separation was performed on silica gel columns by flash (Kieselgel
40, 0.040–0.063 mm; Merck) or gravity column (Kieselgel 60,
0.063–0.200 mm; Merck) chromatography. Reactions were followed
by thin-layer chromatography (TLC) on Merck aluminum silica
gel (60 F254) sheets that were visualized under a UV lamp.
Evaporation was performed in vacuo (rotating evaporator). Sodium
sulfate was always used as the drying agent. The microwave-assisted
procedures were carried out with a CEM Discover LabMate
microwave. Commercially available chemicals were purchased from
Sigma-Aldrich. The UV–vis spectra of compounds 11a,b,
13a,b-17a,b, and 22–27, and the corresponding calibration curves
were recorded with a PerkinElmer LAMBDA BIO-20 spectropho-
tometer.
Figure 3. Comparison between P-gp (gray bars) and BCRP (white
bars) inhibition activity of oxygen derivatives (A) and amino derivatives
(B).
been studied. As expected, the extent of methoxylation on
the C-ring improved the P-gp inhibition activity both in the
series lacking a methoxy group (14a-16a) and in the
corresponding series bearing a methoxy group (14b-16b)
on the A-ring. However, the presence of a methoxy on the
A-ring slightly potentiated P-gp inhibition activity. Among
dimethoxy substitutions on the C-ring, the best results have
been obtained for the 2,3- and 3,4-ortho-disubstituted deriva-
tives, while 3,5-disubstituted derivatives were less active in
all of the series. The effect of the methoxy position on the
C-ring in monosubstituted derivatives has been investigated,
demonstrating that the para-substitution determined the best
activity toward P-gp in all series, although these compounds
were less active than the corresponding disubstituted deriva-
tives. The best results in inhibiting P-gp were obtained for
the amino derivatives bearing a methoxy substituent on the
A-ring, which are always more potent than the corresponding
oxygen derivatives. Also in this series, 2,3- and 3,4-ortho-
disubstituted derivatives on the C-ring showed the highest
P-gp inhibition values in the nanomolar range (EC50 ) 0.085
µM for compound 25). Considering the inhibition activity
profile of some reference compounds, elacridar in particular,
displaying dual inhibition activity toward P-gp and BCRP
pumps, the selectivity of the best P-gp modulators has been
investigated. The results displayed that both the oxygen and
the amino derivatives had similar potency in inhibiting P-gp,
but the oxygen derivatives were more selective toward P-gp
with respect to BCRP.
General Procedure To Synthesize Final Compounds 11a,b
and 13a,b-17a,b. A solution of phenol 1a or 1b23 (1.0 mmol) in
a small amount of DMSO (2 mL) was added to a solution of KOH
(174 mg, 3.11 mmol) in DMSO (2.5 mL). The mixture was stirred
at room temperature for 15 min, and then a solution of the
appropriate arylmethylhalide 2–9 (1.0 mmol) in DMSO (2 mL) was
added dropwise to the solution of potassium 2-(2-phenylethyl)ben-
zenolate. The suspension was stirred at room temperature for 12 h,
and 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.
1-[(2-Methoxybenzyl)oxy]-2-(2-phenylethyl)benzene (11a). Pu-
rified with hexane/AcOEt (95:5) (55% yield) as a colorless oil. MS
1
m/z 318 (M+, 85); 198 (27). H NMR (CDCl3): δ 2.90–3.02 (m,
4H, CH2CH2), 3.90 (s, 3H, OCH3), 5.15 (s, 2H, OCH2), 6.85–7.04
(m, 4H, Ar), 7.12–7.36 (m, 8H, Ar), 7.51–7.55 (m, 1H, Ar). 13C
NMR (CDCl3): δ 156.99, 156.88, 142.71, 130.83, 130.08, 128.79,
128.62, 128.37, 128.31, 127.27, 126.09, 125.78, 120.70, 120.65,
112.05, 110.31, 65.55, 65.37, 36.60, 33.24. Anal. (C22H22O2) C,
H. UV–vis (solvent: PBS) λ ) 230 nm, ꢀ ) 16350.
1-[(2-Methoxybenzyl)oxy]-2-[2-(3-methoxyphenyl)ethyl]ben-
zene (11b). Purified with hexane/AcOEt (95:5) (89% yield) as a
1
colorless oil. MS m/z 348 (M+, 62); 227 (38). H NMR (CDCl3):
δ 2.87–3.02 (m, 4H, CH2CH2), 3.75 (s, 3H, OCH3), 3.86 (s, 3H,
OCH3), 5.14 (s, 2H, OCH2), 6.72–7.35 (m, 11H, Ar), 7.52 (d, 1H,
J ) 7.7 Hz, Ar). 13C NMR (CDCl3): δ 159.66, 156.93, 156.82,
144.31, 130.69, 130.07, 129.25, 128.78, 128.31, 127.29, 125.99,
121.04, 120.62, 114.17, 111.97, 111.43, 110.24, 65.31, 55.46, 55.24,
36.63, 33.19. Anal. (C23H24O3) C, H. UV–vis (solvent: PBS) λ )
230 nm, ꢀ ) 16350.
1-[(4-Methoxybenzyl)oxy]-2-(2-phenylethyl)benzene (13a). Pu-
rified with hexane/Et2O (95:5) to (94% yield) as an oil. MS m/z
318 (M+, 16); 121 (100). 1H NMR (CDCl3): δ 2.83–3.00 (m, 4H,
CH2CH2), 3.83 (s, 3H, OCH3), 5.03 (s, 2H, OCH2), 6.85–6.95 (m,
4H, Ar), 7.11–7.40 (m, 9H, Ar).13C NMR (CDCl3): δ 159.42,
156.82, 142.58, 130.80, 130.12, 129.65, 128.94, 128.62, 128.31,
127.25, 125.79, 120.73, 114.07, 111.86, 69.91, 55.46, 36.57, 33.10.
Anal. (C22H22O2) C, H. UV–vis (solvent: PBS) λ ) 230 nm, ꢀ )
14810.
In Figure 3A, for the best oxygen derivatives, the activity
values (pEC50) toward P-gp and BCRP have been compared.
In Figure 3B, for the best amino derivatives, the same
comparison has been reported.
These findings suggest that the presence of a NH group in
the side chain of the arylmethylamine derivatives increases the
inhibitory activity toward P-gp but in the meantime decreases
the selectivity between the two types of extrusion pumps (P-gp
vs BCRP). In conclusion, the present paper suggests that suitable

1420 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Colabufo et al.
1-[(4-Methoxybenzyl)oxy]-2-[2-(3-methoxyphenyl)ethyl]ben-
(m, 8H, Ar), 7.86 (d, 2H, J ) 6.2 Hz, Py), 8.77 (d, 2H, J ) 6.2
Hz, Py). 13C NMR (CDCl3): δ 157.77, 154.33, 141.11, 140.36,
130.09, 129.91, 127.85, 127.71, 126.89, 125.40, 123.45, 121.71,
111.17, 66.93, 36.08, 31.37. Anal. (C20H19NO·HCl) C, H, N.
UV–vis (solvent: PBS) λ ) 230 nm, ꢀ ) 13260.
4-({2-[2-(3-Methoxyphenyl)ethyl]phenoxy}methyl)pyridine (17b).
The oil 17b was transformed to the hydrochloride salt, a white solid
(158 mg, 0.44 mmol, 51% yield). Mp: 117–119 °C. MS m/z 319
(M+, 100); 240 (35). 1H NMR (CDCl3): δ 2.91–3.10 (m, 4H,
CH2CH2), 3.75 (s, 3H, OCH3), 5.25 (s, 2H, OCH2), 6.67–6.88 (m,
4H, Ar), 7.00–7.29 (m, 4H, Ar), 7.92 (d, 2H, J ) 5.8 Hz, Py), 8.82
(d, 2H, J ) 5.8 Hz, Py). 13C NMR (CDCl3): δ 160.28, 158.90,
155.62, 144.04, 141.65, 131.46, 131.27, 130.02, 128.22, 124.68,
123.15, 121.61, 115.29, 112.41, 111.76, 68.18, 55.93, 37.47, 32.55.
Anal. (C21H21NO2HCl) C, H, N. UV–vis (solvent: PBS) λ ) 230
nm, ꢀ ) 14690.
General Procedure To Synthesize Final Compounds 22–
24. To a solution of 21 (100 mg, 0.44 mmol) in acetonitrile (3
mL) in a microwave vial were added the appropriate methoxyben-
zylchloride (23 mg, 0.15 mmol) and KI (2.4 mg, 0.015 mmol).
The vial was sealed and heated in a microwave at 110 °C, 150 W,
200 PSI for 10 min. The cooled reaction mixture was diluted with
CH2Cl2 (15 mL) and washed successively with H2O. Then, the
organic layer was dried and evaporated to dryness.
N-(2-Methoxybenzyl)-2-[2-(3-methoxyphenyl)ethyl]aniline (22).
The crude product was purified by chromatography, eluting with
hexane/CHCl3 (7:3) to give 22 (44.8 mg, 0.129 mmol, 30% yield).
MS m/z 348 (M+, 84%); 227 (100). 1H NMR (CDCl3): δ 2.72–2.96
(m, 4H, CH2CH2), 3.76 (s, 3H, OMe), 3.84 (s, 3H, OMe), 4.35 (s,
2H, NCH2), 6.65–7.29 (m, 12H, Ar). 13C NMR (CDCl3): δ 159.82,
157.57, 146.0, 143.77, 129.49, 129.13, 128.43, 127.43, 125.83,
120.86, 120.71, 117.31, 114.27, 111.55, 111.10, 110.46, 55.46,
55.31, 43.98, 35.35, 33.53. Anal. (C23H25NO2) C, H, N. UV–vis
(solvent: PBS) λ ) 230 nm, ꢀ ) 16333.
zene (13b). Purified with hexane/Et2O (4:1) (70% yield) as a
1
semisolid. MS m/z 348 (M+, 55); 228 (76). H NMR (CDCl3): δ
2.81–2.97 (m, 4H, CH2CH2), 3.74 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3), 5.02 (s, 2H, OCH2), 6.69–6.77 (m, 3H, Ar), 6.85–6.96 (m,
4H, Ar), 7.13–7.23 (m, 3H, Ar), 7.38 (d, 2H, J ) 8.6 Hz, Ar). 13
C
NMR (CDCl3): δ 159.02, 158.79, 156.18, 143.58, 130.09, 129.49,
129.0, 128.60, 128.34, 126.63, 120.40, 120.09, 113.54, 113.43,
111.21, 110.81, 69.28, 54.80, 54.58, 36.00, 32.40. Anal. (C23H24O3)
C, H. UV–vis (solvent: PBS) λ ) 225 nm, ꢀ ) 22560.
1-[(2,3-Dimethoxybenzyl)oxy]-2-(2-phenylethyl)benzene (14a).
Purified with CHCl3 (25% yield) as an oil. MS m/z 348 (M+, 65).
1H NMR (CDCl3): δ 2.90–2.98 (m, 4H, CH2CH2), 3.89 (s, 3H,
OCH3), 3.90 (s, 3H, OCH3), 5.16 (s, 2H, OCH2), 6.85–7.29 (m,
12H, Ar), 13C NMR (CDCl3): δ 156.84, 152.63, 146.86, 142.56,
131.46, 130.67, 130.05, 128.58, 128.29, 127.29, 125.76, 124.17,
123.24, 120.71, 112.26, 111.94, 65.21, 61.10, 56.00, 36.56, 33.00.
Anal. (C23H24O3) C, H. UV–vis (solvent: PBS) λ ) 230 nm, ꢀ )
22560.
1-[(2,3-Dimethoxybenzyl)oxy]-2-[2-(3-methoxyphenyl)ethyl]-
benzene (14b). Purified by CHCl3 (24% yield) as an oil. MS m/z
1
378 (M+, 71). H NMR (CDCl3): δ 2.83–3.00 (m, 4H, CH2CH2),
3.75 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 5.17
(s, 2H, OCH2), 6.72–7.26 (m, 11H, Ar). 13C NMR (CDCl3): δ
160.33, 157.49, 153.29, 147.52, 144.86, 132.11, 131.29, 130.73,
129.87, 127.96, 127.96, 124.85, 121.70, 121.41, 114.85, 112.90,
112.58, 112.10, 65.87, 61.75, 56.64, 55.88, 37.28, 33.61. Anal.
(C24H26O4) C, H. UV–vis (solvent: PBS) λ ) 230 nm, ꢀ ) 10420.
1-[(3,4-Dimethoxybenzyl)oxy]-2-(2-phenylethyl)benzene (15a).
Purified with hexane/Et2O (95:5) (15% yield) as an oil. MS m/z
1
348 (M+, 25); 210 (76). H NMR (CDCl3): δ 2.88–3.03 (m, 4H,
CH2CH2), 3.84 (s, 3H, OCH3), 3.91(s, 3H, OCH3), 5.04 (s, 2H,
OCH2), 6.86–7.02 (m, 6H, Ar), 7.11–7.31 (m, 6H, Ar). 13C NMR
(CDCl3): δ 156.78, 149.34, 148.94, 142.49, 130.82, 130.20, 130.11,
128.51, 128.31, 127.27, 125.81, 120.84, 119.93, 111.99, 111.43,
110.99, 70.19, 56.15, 56.00, 36.54, 33.00. Anal. (C23H24O3) C, H.
UV–vis (solvent: PBS) λ ) 230 nm, ꢀ ) 15250.
N-(3-Methoxybenzyl)-2-[2-(3-methoxyphenyl)ethyl]aniline (23).
The crude product was purified by chromatography, eluting with
hexane/CHCl3 (1:1) to give 23 (29.8 mg, 0.086 mmol, 20% yield).
1-[(3,4-Dimethoxybenzyl)oxy]-2-[2-(3-methoxyphenyl)ethyl]-
benzene (15b). Purified with CHCl3 (16% yield) as an oil. MS
1
MS m/z 348 (M+, 47); 227 (97). H NMR (CDCl3): δ 2.73–2.98
(m, 4H, CH2CH2), 3.75 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 4.28
1
m/z 378 (M+, 38); 227 (85). H NMR (CDCl3): δ 2.83–3.02 (m,
(s, 2H, NCH2), 6.60–6.96 (m, 8H, Ar), 7.07–7.30 (m, 4H, Ar). 13
C
4H, CH2CH2), 3.74 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.90 (s,
3H, OCH3), 5.03 (s, 2H, OCH2), 6.70–6.78 (m, 3H, Ar), 6.86–7.01
(m, 5H, Ar), 7.13–7.24 (m, 3H, Ar), 13C NMR (CDCl3): δ 160.33,
157.42, 149.96, 149.56, 144.73, 131.37, 130.75, 129.85, 127.89,
121.57, 121.46, 120.57, 114.89, 112.61, 112.06, 111.96, 111.65,
70.81, 56.75, 56.64, 55.80, 37.21, 33.53, 31.57. Anal. (C24H26O4)
C, H. UV–vis (solvent: PBS) λ ) 230 nm, ꢀ ) 11350.
NMR (CDCl3): δ 160.0, 160.50, 145.77, 143.58, 141.34, 129.76,
129.53, 129.05, 127.49, 120.88, 119.86, 117.64, 114.23, 111.30,
112.77, 111.72, 110.88, 55.36, 55.29, 48.65, 35.48, 33.42. Anal.
(C23H25NO2) C, H, N. UV–vis (solvent: PBS) λ ) 230 nm, ꢀ )
16333.
N-(4-Methoxybenzyl)-2-[2-(3-methoxyphenyl)ethyl]aniline (24).
The crude product was purified by chromatography, eluting with
hexane/CHCl3 (4:6) to give an oil 24, which was transformed to
the hydrochloride salt (26.9 mg, 0.07 mmol, 15% yield). MS m/z
1-[(3,5-Dimethoxybenzyl)oxy]-2-(2-phenylethyl)benzene (16a).
Purified with hexane/CHCl3 (1:1) (26% yield) as an oil. MS m/z
1
348 (M+, 50); 198 (67). H NMR (CDCl3): δ 2.89–3.02 (m, 4H,
1
348 (M+, 70%); 227 (100). H NMR (CDCl3): δ 2.75–2.96 (m,
CH2CH2), 3.77 (s, 6H, OCH3), 5.04 (s, 2H, OCH2), 6.42 (t, 1H, J
) 2.1 Hz, Ar), 6.62 (d, 2H, J ) 2.1 Hz, Ar), 6.86–6.93 (m, 2H,
Ar), 7.12–7.30 (m, 7H, Ar). 13C NMR (CDCl3): δ 161.79, 157.35,
143.15, 140.69, 131.42, 130.82, 129.22, 128.96, 127.94, 126.47,
121.53, 112.57, 105.71, 100.61, 70.71, 56.13, 37.21, 33.68. Anal.
(C23H24O3) C, H. UV–vis (solvent: PBS) λ ) 230 nm, ꢀ ) 15377.
1-[(3,5-Dimethoxybenzyl)oxy]-2-[2-(3-methoxyphenyl)ethyl]-
benzene (16b). Purified with hexane/CHCl3 (3:7) (43% yield) as
an oil. MS m/z 378 (M+, 22); 227 (93). 1H NMR (CDCl3): δ
2.86–3.05 (m, 4H, CH2CH2), 3.75 (s, 3H, OCH3), 3.77 (s, 6H,
OCH3), 5.04 (s, 2H, OCH2), 6.42 (t, 1H, J ) 2.3 Hz, Ar), 6.62 (d,
2H, J ) 2.3 Hz, Ar), 6.71–6.93 (m, 4H, Ar), 7.12–7.21 (m, 4H,
Ar). 13C NMR (CDCl3): δ 161.79, 160.39, 157.35, 144.77, 140.67,
131.40, 130.84, 129.89, 127.96, 121.66, 121.57, 114.87, 112.59,
112.14, 105.73, 100.59, 70.75, 56.11, 55.87, 37.28, 33.60. Anal.
(C24H26O4) C, H. UV–vis (solvent: PBS) λ ) 230 nm, ꢀ ) 16350.
4-{[2-(2-Phenylethyl)phenoxy]methyl}pyridine (17a). The oil
17a was transformed to the hydrochloride salt, a white solid (343
mg, 1.1 mmol, 56% yield). Mp: 120–122 °C. MS m/z 289 (M+,
4H, CH2CH2), 3.75 (s, 3H, OMe), 3.79 (s, 3H, OMe), 4.24 (s, 2H,
NCH2), 6.71–6.87 (m, 6H, Ar), 7.09–7.28 (m, 6H, Ar). Anal.
(C23H25NO2 ·HCl) C, H, N.
General Procedure To Synthesize Final Compounds 25–
27. To a solution of 21 (200 mg, 0.88 mmol) in ethanol (10 mL)
was added the appropriate dimethoxybenzylaldehyde (146 mg, 0.88
mmol), and the resulting solution was stirred and refluxed for 12 h
until the disappearance of the aniline 21. The reaction mixture was
then cooled at 0 °C and treated with a solution of NaBH4 (33.3
mg, 0.88 mmol) in H2O (2 mL). The suspension was stirred at room
temperature for 3 h, and then it was concentrated to dryness.
N-[(2,3-Dimethoxy)benzyl]-2-[2-(3-methoxyphenyl)ethyl]an-
iline (25). The crude product was purified by chromatography,
eluting with hexane/AcOEt (8:2) to give 25 as an oil (93.8 mg,
1
0.25 mmol, 28% yield). MS m/z 378 (M+, 65%); 227 (100). H
NMR (CDCl3): δ 2.72–2.97 (m, 4H, CH2CH2), 3.76 (s, 3H, OMe),
3.87 (s, 6H, OMe), 4.36 (s, 2H, NCH2), 6.65–7.25 (m, 11H, Ar).
13C NMR (CDCl3): δ 160.41, 153.50, 147.95, 146.70, 144.31,
133.82, 130.11, 129.60, 128.05, 126.43, 124.83, 121.62, 121.51,
118.04, 114.81, 112.43, 112.26, 111.55, 61.56, 56.58, 55.91, 44.20,
1
37); 196 (40). H NMR (CDCl3): δ 2.89–3.08 (m, 4H, CH2CH2),
5.22 (s, 2H, OCH2), 6.73–6.77 (d, 1H, J ) 7.9 Hz, Ar), 6.97–7.26

Potent P-gp Modulating Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1421
35.99, 34.06. Anal. (C24H27NO3) C, H, N. UV–vis (solvent: PBS)
λ ) 226 nm, ꢀ ) 4437.
the total transport time in seconds (7200 s), [drug]acceptor is the
concentration of the drug measured by UV spectroscopy, and
[drug]initial is the initial drug concentration (1 × 10-4 M) in the
apical or basolateral wells.
N-[(3,4-Dimethoxy)benzyl]-2-[2-(3-methoxyphenyl)ethyl]an-
iline (26). The crude product was purified by chromatography,
eluting with hexane/AcOEt (8:2) to give 26 as an oil (64.2 mg,
Cell ATP Availability Assay. This experiment was performed
as reported in the technical sheet of the ATPlite firstep Kit for
luminescence ATP detection using Victor3, from PerkinElmer Life
Sciences.31 Caco-2 cells were seeded into a 96 well microplate in
100 µL of complete medium at a density of 2 × 104 cells/well.
The plate was incubated O/N in a humidified atmosphere, 5% CO2
at 37 °C. The medium was removed, and 100 µL of complete
medium in the presence or absence of different concentrations of
test compounds was added. The plate was incubated for 2 h in a
humidified atmosphere, 5% CO2 at 37 °C. Then, 50 µL of
mammalian cell lysis solution was added to all wells, and the plate
was stirred for 5 min in an orbital shaker. In all wells, 50 µL of
substrate solution was added, and the plate was stirred for 5 min
as above-reported. The plate was dark adapted for 10 min, and the
luminescence was measured in Victor3.
1
0.20 mmol, 22% yield). MS m/z 378 (M+, 54%); 227 (100). H
NMR (CDCl3): δ 2.73–2.96 (m, 4H, CH2CH2), 3.74 (s, 3H, OMe),
3.86 (s, 3H, OMe), 3.88 (s, 3H, OMe), 4.24 (s, 2H, NCH2),
6.64–6.91 (m, 8H, Ar), 7.08–7.26 (m, 3H, Ar). 13C NMR (CDCl3):
δ 159.95, 149.63, 148.72, 145.93, 143.60, 132.40, 129.53, 129.14,
127.51, 125.87, 120.90, 119.97, 117.71, 114.36, 112.01, 111.74,
111.52, 111.01, 56.30, 56.18, 55.31, 48.63, 35.57, 33.42. Anal.
(C24H27NO3) C, H, N. UV–vis (solvent: PBS) λ ) 226 nm, ꢀ )
47970.
N-[(3,5-Dimethoxy)benzyl]-2-[2-(3-methoxyphenyl)ethyl]an-
iline (27). The crude product was purified by chromatography,
eluting with hexane/AcOEt (9:1) to give 27 as an oil (81.8 mg,
1
0.22 mmol, 24% yield). MS m/z 378 (M+, 54%); 227 (100). H
NMR (CDCl3): δ 2.74–2.99 (m, 4H, CH2CH2), 3.75 (s, 3H, OMe),
3.78 (s, 6H, OMe), 4.25 (s, 2H, NCH2), 6.38 (t, 1H, J ) 2.4 Hz,
Ar), 6.54 (d, 2H, J ) 2.4 Hz, Ar), 6.61–6.84 (m, 4H, Ar), 7.02–7.24
(m, 4H, Ar). 13C NMR (CDCl3): δ 161.81, 160.43, 146.37, 144.18,
142.82, 130.15, 129.65, 128.12, 127.87, 126.32, 121.48, 119.62,
118.26, 116.43, 114.83, 112.30, 111.48, 106.17, 99.90, 56.09, 55.89,
49.45, 36.06, 34.06. Anal. (C24H27NO3) C, H, N.
Biological Method. Cell Lines. The breast cancer cell line of
human origin, MCF-7/Adr (resistant to adriamycin or doxorubicin),
was routinely cultured in RPMI 1640 supplemented with 10% fetal
bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/
mL streptomycin in a humidified incubator at 37 °C with a 5%
CO2 atmosphere. Caco-2 cells were grown in DMEM medium with
10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100
µg/mL streptomycin, and 2 mM L-glutamine.
Permeability Experiments. Preparation of the Caco-2 Mono-
layer. This procedure has been previously reported by Leopoldo
et al.29 Briefly, Caco-2 cells were harvested with trypsin-EDTA
and seeded onto a MultiScreen Caco-2 assay system at a density
of 10 000 cells/well. The culture medium was replaced every 48 h
for the first 6 days and every 24 h thereafter, and after 21 days in
culture, the Caco-2 monolayer was utilized for the permeability
experiments. The trans-epitelial electrical resistance (TEER) of the
monolayers was measured daily before and after the experiment
by using a epithelial voltohommeter (Millicell-ERS; Millipore,
Billerica, MA). Generally, the TEER values obtained are greater
than 1000 Ω for a 21 day culture.
Drug Transport Experiment. The apical to basolateral [Papp(A-
B)] and basolateral to apical [Papp(B-A)] permeabilities of drugs
were measured at 120 min and at various drug concentrations
(1–100 µM).30 Drugs were dissolved in Hank’s balanced salt
solution (HBSS, pH 7.4) and sterile filtered. After 21 days of cell
growth, the medium was removed from filter wells and from the
receiver plate. The filter wells were filled with 75 µL of fresh HBSS
buffer, and the receiver plate was filled with 250 µL per well of
the same buffer. This procedure was repeated twice, and the plates
were incubated at 37 °C for 30 min. After the incubation time, the
HBSS buffer was removed, and drug solutions were added to the
filter well (75 µL). HBSS without the drug was added to the receiver
plate (250 µL). The plates were incubated at 37 °C for 120 min.
After the incubation time, samples were removed from the apical
(filter well) and basolateral (receiver plate) sides of the monolayer
and then were stored in a freezer (-20 °C) pending analysis.
The concentration of compounds was analyzed using UV–vis
spectroscopy.
[3H]-Vinblastine Transport Inhibition. Caco-2 cells were
seeded onto multiscreen plates, 10 000 cells/well for 21 days,
measuring the integrity of the cell monolayers by trans-epithelial
electrical resistance (TEER, Ω × cm2) with an epithelial volto-
hommeter. A mature Caco-2 cell monolayer exhibited a TEER >
800 Ω × cm2 prior to use in transport experiments. Transport
experiments for tested compounds were carried out as described
by Taub et al.32
In each well to basolateral (BL) compartment, in the absence
and in the presence of P-gp inhibitors (from 0.20 to 400 µM) was
added 20 nM [3H]-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 by
using a LS6500 Beckman counter. For each compound, [3H]-
vinblastine transport inhibition was calculated as the radioactivity
difference between radioligand in the presence and absence of
compound. These differences were expressed as a percentage of
inhibition effect for each single drug concentration.
[3H]-Mitoxantrone Transport Inhibition. Caco-2 cells were
seeded onto multiscreen plates, 10 000 cells/well for 21 days,
measuring the integrity of the cell monolayers by trans-epithelial
electrical resistance (TEER, Ω × cm2) with an epithelial volto-
hommeter. A mature Caco-2 cell monolayer exhibited a TEER >
800 Ω × cm2 prior to use in transport experiments. Transport
experiments for tested compounds were carried out as described
by Taub et al.32
In each well to basolateral (BL) compartment, in the absence
and in the presence of P-gp inhibitors (from 0.20 to 400 µM) was
added 20 nM [3H]-mitoxantrone 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 by
using a LS6500 Beckman counter. For each compound, [3H]-
mitoxantrone transport inhibition was calculated as the radioactivity
difference between radioligand in the presence and absence of
compound. These differences were expressed as a percentage of
inhibition effect for each single drug concentration.
Cytotoxicity Assay. The assay was performed by using the
CytoTox-One kit from the Promega Corp. (Madison, WI), as
reported in a previous paper.33 Cell death was determined as the
release of lactate dehydrogenase (LDH) into the culture medium.
The percentage of cytotoxicity was calculated relative to the LDH
release from total lysis of cells in the untreated control. It is assumed
here that the drug-treated wells and the control wells contained the
same total number of cells (dead plus alive cells) at the end of the
treatment period. Therefore, the cytotoxic effect of tested com-
pounds was unaffected by any underestimation of cytotoxicity that
could occur because of the decreased total number of cells in the
treated samples as compared to that of the untreated control. Cells
The apparent permeability (Papp), in units of nanometers per
second, was calculated using the following equation:
VA
[drug]acceptor
[drug]initial
Papp
)
(
)
(
)
area × time
where VA is the volume (in mL) in the acceptor well, area is the
surface area of the membrane (0.11 cm2 of the well), time is

1422 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Colabufo et al.
(13) Krishna, R.; Mayer, L. D. Multidrug resistance (MDR) in cancer.
Mechanisms, reversal using modulators of MDR, and the role of MDR
modulators in influencing the pharmacokinetics of anticancer drugs.
Eur. J. Pharm. Sci. 2000, 11, 265–283.
(14) Leslie, E. M.; Deeley, R. G.; Cole, S. P. C. Multidrug resistance
proteins: role of P-glycoprotein MRP1, MRP2, and BCRP (ABCG2)
in tissue defense. Toxicol. Appl. Pharmacol. 2005, 204, 216–237.
(15) Nobili, S.; Landini, I.; Giglioni, B.; Mini, E. Pharmacological strategies
for overcoming multidrug resistance. Curr. Drug Targets 2006, 7, 861–
879.
(16) Thomas, H.; Coley, H. M. Overcoming multidrug resistance in cancer:
an update on the clinical strategy of inhibiting P-glycoprotein. Cancer
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(17) Pérez-Tomás, R. Multidrug resistance: retrospect and prospects in anti-
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(18) Teodori, E.; Dei, S.; Martelli, S.; Scapecchi, F.; Gualtieri, F. The
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were seeded into 96 well plates for optical performance in the
fluorescent cell-based assay in 100 µL of complete medium in the
presence or absence of different concentrations of test compounds.
The plate was incubated for 24 h in a humidified atmosphere, 5%
CO2 at 37 °C, and then 100 µL of substrate mix in assay buffer
was added. Lysis solution (10 µL) was added to the untreated wells
in order to estimate total LDH. Plates were kept protected from
light for 10 min at room temperature, and 50 µL of stop solution
was added to all wells. The fluorescence was recorded using a
PerkinElmer LS55 luminescence spectrometer with a 560 nm
excitation wavelength and a 590 nm emission wavelength. The
cytotoxicity percentage was estimated as follows:
(
100 LDH in medium of treated cells -
) (
culture medium background / total LDH in untreated cells -
)
culture medium background
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Statistical Analysis. The EC50 values of the compounds reported
in Tables 1 and 2 were determined by nonlinear curve fitting
utilizing the GraphPad Prism program.34
Supporting Information Available: Elemental analysis of
compounds 11a,b, 13a,b, 14a,b, 15a,b, 16a,b, 17a,b, and 22-27
and experimental section for key intermediates 18, 20, and 21. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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