2-[(3-Methoxyphenylethyl)phenoxy]-based ABCB1 inhibitors: Effect of different basic side-chains on their biological properties

Article abstract of DOI:10.1021/jm800928j

Recently, 2-[(3-methoxyphenylethyl)phenoxy]-moiety has been selected for the design and synthesis of new small ABCB1 inhibitors. In the present paper, this moiety has been linked through a spacer of 2-5 carbon atoms to the nitrogen of three different basic nuclei such as: (i) N-4-arylpiperazine, (ii) N-4-methylpiperazine, and (iii) 6,7-dimethoxytetrahydroisoquinoline. The results demonstrated that all the selected basic nuclei were well tolerated and that, globally, the best inhibitory activity for each series was obtained when the spacer between the 2-[(3-methoxyphenylethyl)phenoxy] moiety and the basic nucleus consisted of a four-carbon chain. Among the synthesized compounds, N-4-methylpiperazine- 10c (IC₅₀ = 0-15 μM) and tetrahydroisoquinoline-derivatives 11c (IC₅₀ = 0.08 μM) with the spacer n = 4 for both series, displayed the best potency to inhibit ABCB1 activity. Moreover, for each compound, the ABCB1 interacting mechanism has been evaluated by three combined biological assays. N-4-methylpiperazine- (10a-d) and tetrahydroisoquinoline- (11a-d) derivatives were Cyclosporin A-like ABCB1 nontransported substrates.

Full text of DOI:10.1021/jm800928j

7602
J. Med. Chem. 2008, 51, 7602–7613
2-[(3-Methoxyphenylethyl)phenoxy]-Based ABCB1 Inhibitors: Effect of Different Basic
Side-Chains on Their Biological Properties
Nicola Antonio Colabufo,*,† Francesco Berardi,† Roberto Perrone,† Simona Rapposelli,*,‡ Maria Digiacomo,‡ Michael Vanni,‡
and Aldo Balsamo‡
Dipartimento Farmacochimico, UniVersita´ degli Studi di Bari, Via Orabona, 4, 70125 Bari, Italy, Dipartimento di Scienze Farmaceutiche,
UniVersita` di Pisa, Via Bonanno, 6, 56126 Pisa, Italy
ReceiVed July 24, 2008
Recently, 2-[(3-methoxyphenylethyl)phenoxy]-moiety has been selected for the design and synthesis of new
small ABCB1 inhibitors. In the present paper, this moiety has been linked through a spacer of 2-5 carbon
atoms to the nitrogen of three different basic nuclei such as: (i) N-4-arylpiperazine, (ii) N-4-methylpiperazine,
and (iii) 6,7-dimethoxytetrahydroisoquinoline. The results demonstrated that all the selected basic nuclei
were well tolerated and that, globally, the best inhibitory activity for each series was obtained when the
spacer between the 2-[(3-methoxyphenylethyl)phenoxy] moiety and the basic nucleus consisted of a four-
carbon chain. Among the synthesized compounds, N-4-methylpiperazine- 10c (IC50 ) 0.15 µM) and
tetrahydroisoquinoline-derivatives 11c (IC50 ) 0.08 µM) with the spacer n ) 4 for both series, displayed
the best potency to inhibit ABCB1 activity. Moreover, for each compound, the ABCB1 interacting mechanism
has been evaluated by three combined biological assays. N-4-methylpiperazine- (10a-d) and tetrahydroiso-
quinoline- (11a-d) derivatives were Cyclosporin A-like ABCB1 nontransported substrates.
Introduction
related to the specific pharmacologic activities of compounds
mentioned above. Both valspodar and biricodar, although
displaying high potency and low toxicity, significantly inhibited
the metabolism and excretion of cytotoxic agents. On the basis
of these pharmacokinetic limits, a new generation of ABCB1
modulators (zosuquidar, elacridar, and tariquidar, ONT-093,
MS-209) has been designed (Chart 1).8-11 These inhibitors did
not display any pharmacokinetic interaction with the metabolism
of chemotherapeutic drugs and showed high potency to inhibit
ABCB1. However, these inhibitors failed in clinical trials,
showing only a moderate in vivo efficacy to block ABCB1
pump.12 In addition, one of the most interesting compounds,
elacridar, displayed similar potency to also block ABCG2
transporter, thus indicating a lack of selectivity toward ABCB1
pump.13
Multidrug resistance (MDRa) is the most ascertained cause
of failure in cancer therapy. MDR is due to the contribution of
several factors among which pharmacokinetic and tumor mi-
croenvironmental and cancer-cell-specific factors such as the
overexpression of several ATP-dependent efflux pumps, known
as ATP binding cassette (ABC) family.1 The ABC transporters
involved in MDR are: multiresistant proteins (MRPs), P-
glycoprotein (ABCB1), and breast cancer resistant protein
(BCRP/ABCG2).2,3 Among these transporters, ABCB1 is the
most studied pump, and several modulating ligands have been
designed as potential MDR reverting agents.
The first attempts to modulate ABCB1 have been carried out
by testing compounds displaying known pharmacological activ-
ity such as the calcium channel blocker verapamil and the
antisteroid tamoxifen.4,5 Unfortunately, these compounds in-
terfered with several enzyme systems, resulting in unpredictable
pharmacokinetic interaction.
Recently, we described arylmethyloxy- and arylmethylamino-
phenyl derivatives (1-4) depicted in Figure 1, as potent ABCB1
modulating agents.14 Among them, the arylmethylamino-phenyl
derivatives (3, 4) exhibited both high ABCB1 and ABCG2
inhibition activity, while the corresponding oxygen-analogues
(1, 2), although displaying reduced potency with respect to
compounds 3, 4, they turned out to be inactive against ABCG2
pump. These results led us to consider the 2-[(3-methoxyphe-
nylethyl)phenoxy] moiety, present in the more selective com-
pounds previously synthesized, the pivotal molecular basis to
design new and potentially selective ABCB1 inhibitors.15-18
Starting from these limits, compounds such as valspodar and
biricodar6,7 have been developed to reduce the toxic effects
* To whom correspondence should be addressed. For N.A.C.: phone,
+39-080-5442727; fax, +39-0805442231; E-mail, colabufo@
farmchim.uniba.it. For S.R.: Phone, +39-050-2219582; fax, +39-050-
2219577; E-mail, rappsi@farm.unipi.it.
†
Dipartimento Farmacochimico, Universita´ degli Studi di Bari.
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
‡
a Abbreviations: ABCB1, ATP binding cassette B1 family; ABCG2, ATP
binding cassette G2 family; MDR: multidrug resistance; MRPs, multidrug
resistant proteins; 5-HT1A, serotonergic receptors 1A-subtype; D2, dopam-
inergic receptor 2-subtype; Papp, apparent permeability; Caco-2, human colon
adenocarcinoma cells; IC50, half-maximal inhibitory concentration; r2,
statistical parameter; MDCK-ABCB1, Madin Darby canine kidney cells
overexpressing ABCB1 protein; BCRP, breast cancer resistant protein; Ki,
inhibition constant to define affinity value; MCF-7/Adr, human breast
adenocarcinoma cells resistant to adriamycin; RPMI, Roswell Park Memorial
Institute; DMEM, Dulbecco’s minimum essential medium; Calcein-AM,
calcein-acethoxy methyl ester; TEER, transepithelial electrical resistance;
HBSS, Hank’s balanced salt solution; BL, basolateral; AP, apical; PBS,
phosphate buffered saline; HeLa, Henrietta Lacks cervical cancer cells; C6,
rat glioma cells; Kd, inhibition constant of radioligand at the equilibrium.
Many computational approaches to modeling the ABCB1
transporter suggested a general pharmacophore in which both
a planar aromatic domain and a basic nitrogen atom within an
extended side chain constitute general features of a substrate/
inhibitor of this transporter.19,20 Because in compounds 1, 2 is
lacking the basic nitrogen that is present in almost all the known
ABCB1 inhibitors, we designed and synthesized new com-
pounds in which the 2-[(3-methoxyphenylethyl)phenoxy] moiety
of 1, 2 is joined by an aliphatic spacers of 2-5 methylene
carbons to the nitrogen of different basic nucleus such as N-4-
aryl- (5-8), N-4-heteroaryl- (9a-d) or N-4-methyl-substituted
10.1021/jm800928j CCC: $40.75
2008 American Chemical Society
Published on Web 11/17/2008

2-[(3-Methoxyphenylethyl)phenoxy]-Based ABCB1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7603
Figure 1. Structures and ABCB1 and ABCG2 inhibition activities of arylmethyloxy- and arylmethylamine-phenyl derivatives.
Chart 1. Third Generation ABCB1 Inhibitors
(10a-d) piperazine, or to an unsubstituted- (11a-d) or 6,7-
dimethoxy-substituted-tetrahydroisoquinoline (12a-d) (Figure
2). The choice of tetrahydroisoquinoline nucleus has been made
on the basis of the data reported in literature for compounds
such as tariquidar and other related derivatives that possess this
kind of pharmacophoric moiety. Regarding the other N-
containing moieties, also in this case, on the basis of some
published theoretical studies on the pharmacophoric group
pivotal for P-gp activity, we planned to link some N-aryl-
piperazine-nucleus to the 2-[(3-methoxyphenylethyl)phenoxy]
moiety. Among the piperazine derivatives as N-4-substituents
were selected the 3-Cl-Ph, 3-CF3-Ph, 2-OCH3-Ph or 4-F-Ph,
and 2-pyrimidine groups.
the new compounds of molecular features able to interact with
serotonergic (5-HT1A) and dopaminergic (D2) receptors, the in
vitro affinity for these receptors was also determined.21,22
Moreover, on the basis of the marked overlap in the spectrum
of activity of several compounds3 to inhibit both ABCB1 and
ABCG2 pumps, we investigated the affinity of the new
compounds displaying the best ABCB1 inhibition effect also
toward this latter transporter.
Chemistry
Final compounds 5-12 were synthesized by following the
procedures described in Scheme 1. The reaction of phenol 1323
with the appropriate N-1-(2-chloroethyl)-N-4-arylpiperazine and
N-1-(3-bromopropyl)-N-4-(3′-chlorophenyl)piperazine in the
presence of KOH afforded the compounds 5a-8a, and 5b
The new compounds were evaluated in vitro for their
inhibitory activity toward ABCB1 and for elucidating their
interacting mechanism. In addition, because of the presence in

7604 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Colabufo et al.
Scheme 1a
a Reagents and conditions: (i) KOH, DMSO, reflux; (ii) (a) KOH, t-BuOH, mw 10 min, 120 °C,150 W; (b) KOH, t-BuOH, rt; (iii) K2CO3, CH3CN, 80
°C.
of 14 with the N-methylpiperazine afforded compound 10, while
the same reaction with 1,2,3,4,-tetrahydroisoquinoline or with
6,7-dimethoxytetrahydroisoquinoline led to final products 11 and
12, respectively. The intermediate N-1-(2-chloroethyl)-arylpip-
erazines 23-26 used in the synthesis of 5a-8a were prepared,
as described in Scheme 2, by acylation of the appropriate
arylpiperazine (15-18) with chloroacetylchloride, and subse-
quent reduction of the amide-derivative (19-22) with LiAlH4.
Results and Discussion
ABCB1 Inhibition Activity. The ABCB1 inhibition activity
and the interacting mechanism of the synthesized compounds
were investigated as previously described.23,24 This study was
highlighted combining the results of the [3H]vinblastine transport
inhibition with those obtained by the evaluation of ATPase
activation and of the apparent permeability (Papp) in Caco-2 cell
monolayer. This latter parameter (Papp) was determined through
the ratio between the basolateral to apical versus flux (B f A)
and the apical to basolateral versus flux (A f B).
Figure 2. General formulas of compounds 5-12.
respectively. The same type of reaction carried out using
3-(chloropropyl)-N-4-methylpiperazine or N-(2-chloroethyl)-
tetrahydroisoquinoline gave compounds 10b and 11a, respec-
tively. The reaction of the phenol 13 with the appropriate 1,2-
1,3- 1,4-, and 1,5-alkyl-dihalide in the presence of KOH afforded
the intermediates 14a-d, which by reaction with the appropriate
arylpiperazine yielded the final compounds 5-9. The reaction
In the [3H]vinblastine transport inhibition test (Table 1), all
N-4-aryl- and N-4-heteroaromatic-piperazine derivatives dis-
played from moderate to appreciable ABCB1 inhibitory activity,

2-[(3-Methoxyphenylethyl)phenoxy]-Based ABCB1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7605
Scheme 2a
a Reagents and conditions: (i) KOH, CH2Cl2-H2O, rt; (ii) LiAlH4, Et2O, 40 °C.
Table 1. ABCB1 Reverting Activity Evaluation of Compounds 5-10
Table 2. ABCB1 Reverting Activity Evaluation of Compounds 11-12
[3H]vinblastine
[3H]vinblastine transport
Papp (BA)a ATP-ase transport inhibition
Papp (BA)a ATP-ase inhibition IC50 ( SEM
compd
n
R
Papp (AB)a activationb IC50 ( SEM (µM)c
c
compd
11a
n
R′ Papp (AB)a activationb
(µM)
5a
5b
5c
5d
6a
6b
6c
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
2
3
4
5
2
3
4
2
3
4
5
2
3
4
5
2
3
4
5
2
3
4
5
3-Cl-Ph
3-Cl-Ph
3-Cl-Ph
3-Cl-Ph
3-CF3-Ph
3-CF3-Ph
3-CF3-Ph
2-OMe-Ph
2-OMe-Ph
2-OMe-Ph
2-OMe-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
2-Pym
2-Pym
2-Pym
2-Pym
Me
Me
Me
Me
3.6
6.8
5.1
6.2
2.6
4.4
4.4
6.8
8.1
3.9
5.2
2.2
2.5
3.5
5.1
6.6
N
N
N
N
N
N
N
3.2 ( 0.3
2.5 ( 0.3
1.8 ( 0.3
2.65 ( 0.5
40 ( 2.5
6.5 ( 0.3
0.75 ( 0.05
25 ( 2.5
14 ( 1.2
5.8 ( 0.80
1.27 ( 0.02
10 ( 1.2
5.3 ( 0.80
1.2 ( 0.40
2.12 ( 0.30
2.0 ( 0.30
1.2 ( 0.40
0.2 ( 0.05
1.72 ( 0.08
0.80 ( 0.10
2.5 ( 0.50
0.15 ( 0.05
6.82 ( 1.2
2 H
3 H
4 H
5 H
2 OMe
3 OMe
4 OMe
5 OMe
7.2
6.3
6.9
7.3
6.4
4.3
5.8
4.9
N
N
N
N
12 ( 1.5
0.50 ( 0.02
0.08 ( 0.01
3.17 ( 0.2
0.75 ( 0.02
0.14 ( 0.03
0.19 ( 0.05
0.35 ( 0.04
11b
11c
11d
12a
12b
12c
12d
Y (55%)
N
N
Y (10%)
Y (20%)
Y (54%)
Y (15%)
Y (10%)
Y (38%)
Y (25%)
Y (40%)
N
N
N
N
N
N
N
N
Y (20%)
CyclosporinA
Verapamil
Elacridar
9.6d
1.2d
Nd
Yd
Ne
80 ( 7.5d
20 ( 1.0d
2.0e
<2e
a The experimental conditions (solvent and spectroscopic methods) to
detect the concentration of each tested compound are reported in Experi-
mental Section. b All compounds were tested at 100 µM and for positive
response are reported the percentage value given in parentheses. c The values
are the mean of three independent experiments, samples in triplicate. d See
ref 14. e See ref 19.
131
8.2
7.1
6.8
4.7
6.3
2.5
10a
10b
10c
10d
series, the compound having a linker of four carbon atoms (10c),
displayed the highest ABCB1 inhibition activity with IC50 in
the submicromolar range (0.15 µM).
CyclosporinA
Verapamil
Elacridar
9.6d
1.2d
Nd
Yd
Ne
80 ( 7.5d
20 ( 1.0d
2.0e
<2e
For compounds of type 11 and 12, the N-4-substituted-
piperazine moiety of 5-10 has been replaced by a tetrahy-
droisoquinoline nucleus also present in the chemical structures
of other classes of ABCB1 inhibitors such as elacridar and
tariquidar (Table 2). Analogously to the N-4-arylpiperazine
derivatives, also within the tetrahydroisoquinoline derivatives
(11a-d), the spacer elongation improved ABCB1 inhibitory
activity with the best result obtained for compound 11c (n ) 4,
IC50 ) 0.08 µM); on the contrary, the inhibition properties of
the 6,7-dimethoxytetrahydroisoquinoline-derivatives (12a-d)
appeared only slightly influenced by the elongation of the spacer
(Table 2).
The evaluation of the correlation between the pIC50 values
of compounds 5 and 7-12 and the spacer length (Figure 3)
indicates that globally the linker of four-methylene groups
confers the best ABCB1 inhibition activity. For each set of
compounds bearing from 2 to 4 methylene groups r2 has been
found from 0.90 to 0.99 excepting for N-methylpiperazine and
for 6,7-dimethoxytetrahydroisoquinoline derivatives displaying
r2 ) 0.34 and 0.59, respectively.
a The experimental conditions (solvent and spectroscopic methods) to
detect the concentration of each tested compound are reported in Experi-
mental Section. b All compounds were tested at 100 µM and for positive
response are reported the percentage value given in parentheses. c The values
are the mean of three independent experiments, samples in triplicate. d See
ref 14. e See ref 19.
with IC50 values ranging from 40 µM for 6a to 0.20 µM for 9c,
indicating a competition for the ABCB1 binding site between
the radiolabeled vinblastine (known ABCB1 substrate) and the
tested compounds. The similar results obtained among both the
meta-chlorophenyl (5) and the pyrimidyl- (9) derivatives seem
to indicate that for these series of compounds the spacer length
has not a marked influence on the ABCB1 activity; indeed, in
the other series of compounds, the spacer length seems to play
an inhomogeneous role: the best affinity toward ABCB1 was
observed for a spacer length of four carbon atoms (n ) 4) in
derivatives 6 (6c: IC50 ) 0.75 µM) and 8 (8c: IC50 ) 1.2 µM),
and for n ) 5 in compounds of type 7 (7d: IC50 ) 1.27 µM).
Anyway, in all the series, the linker of two carbon atom (n )
2) seems to be detrimental.
Also the N-4-methyl-piperazine derivatives (10a-d) showed
From the study on the ATPase activation, it appears that only
the N-4-substituted-piperazine derivatives 7a-d and 8a-d and
a good degree of [3H]vinblastine transport inhibition. In this

7606 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Colabufo et al.
Table 3. Affinity Binding Values of Compounds 5-12
Ki ( SEMc
a
b
D2
5-HT1A
5a
5b
5c
5d
6a
6b
6c
7a
7b
7c
7d
8a
8b
8c
8d
9a
>1000 (43%)d
298 ( 20
>1000 (35%)
>1000 (25%)
>1000 (31%)
1412 ( 100
>1000 (27%)
427 ( 12
6.3 ( 0.8
67 ( 1.5
>1000 (35%)
>1000 (38%)
655 ( 30
686 ( 50
1130 ( 200
1308 ( 150
>5000 (14%)
1409 ( 155
2874 ( 150
>5000 (36%)
1241 ( 250
756 ( 60
1092 ( 90
>5000 (14%)
783 ( 15
800 ( 25
1324 ( 250
847 ( 20
>5000 (47%)
>5000 (27%)
656 ( 100
629 ( 80
9b
9c
9d
10a
10b
10c
10d
11a
11b
11c
11d
12a
12b
12c
12d
Haloperidol
8-OH-DPAT
20 ( 3.0
331 ( 12
558 ( 25
854 ( 10
>1000 (24%)
178
>5000 (38%)
>5000 (48%)
>5000 (40%)
>5000 (38%)
>5000 (24%)
1310 ( 120
1124 ( 180
1331 ( 150
>5000 (15%)
>5000 (26%)
>5000 (36%)
>5000 (18%)
>5000 (22%)
565
>1000 (14%)
>1000 (26%)
>1000 (35%)
114 ( 15
>1000 (48%)
>1000 (16%)
>1000 (12%)
>1000 (35%)
>1000 (40%)
>1000 (18%)
6.4 ( 0.8
3.3 ( 0.5
a Human cloned D2 receptors in C6 rat glioma cells; [3H]spiroperidol,
Kd ) 0.2 nM; nonspecific binding determined in the presence of 10 µM
haloperidol. b Human cloned 5-HT1A receptors in HeLa cells; [3H]-8-OH-
DPAT, Kd ) 8.8 nM; nonspecific binding determined in the presence of 10
µM 8-OH-DPAT. c The value is the mean of three independent experiments,
samples in triplicate. d Ki not obtained, the inhibition percentage at 10 µM
given in parentheses.
Figure 3. (A) Spacer elongation effect (n ) 2-5) vs ABCB1 inhibition
activity (pIC50). (B) Graphic of pIC50 mean related to the spacer carbon
numbers.
the 6,7-dimethoxytetrahydroisoquinoline compounds 12a,d were
able to activate this enzyme, thus indicating their nature as
ABCB1 substrates. On the other hands, the BA/AB ratio, which
defines the intrinsic biological properties of a modulator, permits
the classification of these compounds as unambiguous ABCB1
substrates. As regards all the other compounds, they were unable
to activate the ATPase but exhibited a BA/AB ratio >2, thus
allowing us to consider them as ABCB1 Cyclosporin A-like
transported substrates.
Cell Calcein Accumulation. Taking into account that Caco-2
cell lines express also other drug efflux proteins such as MRP224
and BCRP/ABCG2,25 making interpretation of results difficult,
the compounds displaying submicromolar activity (6c, 9c, 10a,c,
11b,c, 12a-d) in the [3H]vinblastine transport inhibition were
also tested in transfected MDCK-ABCB1 cell lines. In this
assay, the calcein cell accumulation depends on ABCB1
inhibition so that it may be considered as a rapid and direct
biological method to evaluate the inhibition activity of com-
pounds. Regarding the tetrahydroisoquinoline derivatives 11b,c
and 12a-d, the results listed in Table 4 overlapped those
obtained in [3H]vinblastine transport inhibition assay, excepting
for compound 11c, which was found almost 20-fold less active
(IC50 ) 1.78 µM vs 0.08 µM). On the contrary, among the
piperazine derivatives (6c, 9c, 10a,c), only compound 6c showed
a result (IC50 ) 1.79 µM) almost superimposed to that obtained
on Caco-2 cell lines (IC50 ) 0.75 µM), while the other three
compounds resulted in being 10-fold less active (IC50 ) 1.17
µM vs 0.2 µM, IC50 ) 7.76 µM vs 0.80 µM, IC50 ) 4.78 µM
Table 4. Calcein Cell Accumulation in MDCK-MDR1 Cells
compd
IC50 ( SEM (µM)a
6c
9c
1.79 ( 0.45
1.17 ( 0.20
7.76 ( 0.90
4.78 ( 0.20
1.14 ( 0.60
1.78 ( 0.80
0.78 ( 0.08
0.09 ( 0.001
0.16 ( 0.04
0.11 ( 0.02
10a
10c
11b
11c
12a)
12b
12c
12d
a The value is the mean of three independent experiments, samples in
triplicate.
vs 0.15 µM, respectively). These apparent discrepancies between
the results obtained in Caco-2 cell lines and in MDCK cell lines
for compounds 6c, 9c, 10a,c, and 11c could be due to the
expression of several different efflux proteins in both type of
cell lines and to the different sensitivity of the two method
used.26
ABCG2 Inhibition Activity. Compounds displaying ABCB1
inhibition activity e1 µM (6c, 9c, 10a, 10c, 11b,c, and 12a-d)
were also evaluated for their ABCG2 inhibition properties using
[3H]mithoxantrone as a specific substrate. All these compounds
at 100 µM were unable to interfere with [3H]mithoxantrone
efflux, with the only exception of compounds 6c and 9c, which
slightly reduced the [3H]mithoxantrone efflux by ABCG2 pump
(24% and 16% of inhibition effect at 100 µM, respectively).

2-[(3-Methoxyphenylethyl)phenoxy]-Based ABCB1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7607
(2.5 mL). Stirring was continued over 15 min at room temperature,
then a solution 1-(2-chloroethyl)-4-(3-chlorophenyl)piperazine 23
(116 mg, 0.45 mmol) in DMSO (2 mL) was added dropwise to the
solution of potassium 2-[2-(3-methoxyphenyl)ethyl]benzenolate.
The suspension was stirred at 40 °C for 1 h, then it was diluted
with AcOEt and washed with water and brine. The organic layer
was dried and concentrated. The crude product was transformed
into the hydrochloride salt and crystallized from i-PrOH/i-Pr2O to
give 5a (125 mg, 0.27 mmol, 57% yield) as a white solid: mp
85-87 °C. MS m/z 450 (M+, 5). 1H NMR (CDCl3): δ 2.74-2.82
(m, 4H, piperazine), 2.85-2.93 (m, 6H, CH2CH2, CH2N), 3.17-3.25
(m, 4H, piperazine), 3.78 (s, 3H, OMe), 4.16 (t, 2H, J ) 5.6 Hz,
Serotonergic and Dopaminergic Receptor Affinity. All
compounds were tested for their affinity toward serotonergic
5-HT1A and dopaminergic D2 receptors (Table 3). The results
displayed that all compounds toward serotonergic 5-HT1A
receptors showed poor affinity with Ki values in the micromolar
range. Regarding dopaminergic D2 receptors affinity, 2-meth-
oxyphenyl derivative 7b (n ) 3) and 1-pyrimidyl derivative 9b
(n ) 3) displayed nanomolar receptor affinities (Ki ) 6.3 nM
and Ki ) 20 nM, respectively). All the other compounds
displayed a modest dopaminergic D2 receptor affinity.
These findings indicated that these new compounds, although
contain molecular features typical of serotonergic and dopam-
inergic agents (i.e., N-4-arylpiperazine nucleus), showed neg-
ligible affinity toward 5-HT1A and D2 receptors, with the only
exception of derivatives 7b and 9b on D2 receptor.
OCH2), 6.75-6.90 (m, 7H, Ar), 7.16-7.25 (m, 5H, Ar) ppm. 13
C
NMR (CD3OD): δ 160.98, 156.86, 152.02, 144.76, 136.05, 131.50,
131.36, 131.28, 130.28, 128.46, 122.82, 121.91, 121.78, 117,61,
115.97, 115.35, 112.86, 112.02, 64.15, 57.30, 55.59, 53.88, 47.36,
37.46, 32.41. Anal. C27H31N2O2Cl·HCl. UV-vis (solvent: PBS) λ
) 254 nm, ε ) 12030.
Conclusions
3-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}propyl-4-(3′-chlo-
rophenyl)piperazine (5b). A stirred solution of KOH (440 mg,
3.19 mmol) in DMSO (5 mL) was heated to 80 °C until the
complete dissolution of KOH. After being cooled at room temper-
ature, a solution of 2-[2-(3-methoxyphenyl)]ethyl]phenol 13 (500
mg, 2.19 mmol) in DMSO (3 mL) was added. The mixture was
vigorously stirred for 20 min and then it was added to a solution
of 1-(3-chlorophenyl)-4-(3-chloropropil)piperazine (598 mg, 2.19
mmol) dissolved in DMSO. The resulting mixture was stirred and
refluxed for 20 h. Then it was diluted with AcOEt and washed
with water, a solution 1N of NaOH and brine. The organic layer
was evaporated to give a crude oil, which was purified by
transformation into the hydrochloride salt and crystallization from
i-PrOH to give 5b (610 mg, 1.31 mmol, 60% yield) as a white
solid: mp 135-137 °C; MS m/z 464 (M+, 40). 1H NMR (CDCl3):
δ 2.02-2.12 (m, 2H, CH2), 2.59-2.70 (m, 6H, piperazine, CH2N),
2.85-2.93 (m, 4H, CH2CH2), 3.20 (t, 4H, J ) 4.9 Hz, piperazine),
3.78 (s, 3H, OMe), 4.05 (t, 2H, J ) 6.0 Hz, OCH2), 6.73-6.91
(m, 8H, Ar), 7.11-7.25 (m, 4H, Ar) ppm. 13C NMR (CDCl3): δ
165.34, 159.04, 154.27, 149.72, 147.81, 130.56, 130.53, 130.15,
129.49, 127.58, 121.64, 121.42, 120.90, 117.45, 115.10, 114.81,
111.50, 110.86, 65.01, 55.62, 55.40, 52.00, 46.53, 36.68, 31.97,
24.34. Anal. (C28H33N2O2Cl·HCl) C, H, N. UV-vis (solvent: PBS)
λ ) 230 nm, ε ) 18850.
4-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}butyl-4-(3′-trifluo-
romethylphenyl)piperazine (5c). A solution of 1-(4-chlorobutoxy)-
2-[2-(3-methoxyphenyl)ethyl]benzene 14c (200 mg, 0.63 mmol) in
a small amount of acetonitrile (3 mL) heated at 80 °C was added
to a solution of K2CO3 (105 mg, 0.76 mmol) in acetonitrile (15
mL). Stirring was continued over 15 min at room temperature, then
a solution of 1-(3-chlorophenyl)piperazine hydrochloride 15 (124
mg, 0.63 mmol) in acetonitrile (2 mL) was added dropwise to the
solution of potassium 2-(3-methoxyphenylethyl)benzenolate. The
suspension was stirred at 80 °C for 4 h, then the potassium carbonate
was filtered off and the solvent removed. The crude product was
purified by column chromatography eluting with n-hexane/AcOEt
(3:7) and transformation into the hydrochloride salt to give 5c (90
mg, 0.19 mmol, 30% yield) as a white solid: mp 65-67 °C. MS
m/z 478 (M+, 34). 1H NMR (CDCl3): δ 1.90-2.20 (m, 4H,
CH2CH2), 2.74-2.98 (m, 4H, CH2CH2), 3.10-3.30 (m, 2H, CH2N),
3.41-3.59 (m, 4H, piperazine), 3.78 (s, 3H, OMe), 3.85-4.09 (m,
4H, piperazine), 4.39-4.59 (m, 2H, OCH2), 6.50-6.96 (m, 5H,
Ar), 7.12-7.23 (m, 3H, Ar), 7.34-7.45 (m, 2H, Ar), 7.55-7.65
(m, 1H, Ar), 7.69-7.73 (m, 1H, Ar) ppm. 13C NMR (CDCl3): δ
159.84, 156.47, 146.82, 144.15, 135.54, 130.64, 130.13, 129.93,
129.54, 127.52, 122.33, 121.06, 120.89, 117.82, 115.41, 114.70,
111.43, 110.99, 67.00, 57.51, 55.44, 51.34, 46.83, 36.34, 32.22,
27.12, 21.17. Anal. (C29H35N2O2Cl ·HCl) C, H, N. UV-vis (solvent:
PBS) λ ) 230 nm, ε ) 18870.
The aim of the present work was to verify the influence of
the basic nucleus linked to the 2-[(3-methoxyphenylethyl)phe-
noxy] moiety on the activity toward two efflux pump proteins:
ABCB1 and ABCG2. Globally, the results indicate that this
scaffold appears able to maintain a satisfactory degree of
ABCB1 inhibition activity and that the addition of a basic
nitrogen to this moiety caused only in a few cases a slight
improvement of ABCB1 inhibition activity. In the meantime,
this chemical manipulation leads to more selective compounds
endowed with reduced affinity toward ABCG2 and possessing
negligible or modest affinity for 5-HT1A and D2 receptors.
Moreover, the results obtained in the two different types of
ABCB1 inhibition tests seem to indicate that the tetrahydroiso-
quinoline derivatives are the more active and selective com-
pounds toward ABCB1 protein.
Finally, the effect of the elongation of the linker between
the 2-[(3-methoxyphenylethyl)phenoxy] moiety and the basic
nucleus, it seems not to markedly influence the ABCB1
inhibition activity: it appeared to be detrimental only when the
chain is two carbon atoms length, while globally, it appeared
to furnish the best results when the spacer length is of four
carbon atoms.
Experimental Section
Melting points were determined on a Kofler hot-stage apparatus
and are uncorrected. 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. Mass spectra were obtained on a Hewlett-
Packard 5988 A spectrometer 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. Reac-
tions 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 evapora-
tor). 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 the
final compounds and the corresponding calibration curves were
recorded with LAMBDA BIO-20 spectrophotometer Perkin-
Elmer.
2-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}ethyl-4-(3′-chlorophe-
nyl)piperazine (5a). A solution of 2-[2-(3-methoxyphenyl)eth-
yl]phenol 13 (103 mg, 0.45 mmol) in a small amount of DMSO (2
mL) was added to a solution of KOH (88 mg, 1.60 mmol) in DMSO
5-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}pentyl-4-(3′-chlo-
rophenyl)piperazine (5d). Compound 5d was synthesized from
1-[(5-bromopentyloxy]-2-[2-(3-methoxyphenyl)ethyl]benzene 14d
(150 mg, 0.40 mmol) and from 1-(3-chlorophenyl)piperazine

7608 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Colabufo et al.
hydrochloride 15 (93 mg, 0.40 mmol) following the same procedure
described above for the preparation of 5c. The crude product was
purified by transformation into the hydrochloride to give 5d (88
mg, 0.17 mmol, 41% yield) as a white solid: mp 113-115 °C. MS
m/z 493 (M+, 23). 1H NMR (CDCl3): δ 1.60-1.99 (m, 6H,
CH2CH2CH2), 2.88-3.02 (m, 8H, CH2CH2, piperazine), 3.23-3.62
(s, 6H, CH2N, piperazine), 3.77 (s, 3H, OMe), 3.99 (t, 2H, J ) 5.8
Hz, OCH2), 6.75-6.94 (m, 7H, Ar), 7.11-7.24 (m, 5H, Ar) ppm.
13C NMR (CDCl3): δ 159.84, 156.82, 150.67, 144.27, 135.48,
130.55, 130.38, 130.05, 129.44, 127.43, 121.66, 121.06, 120.79,
117.51, 115.14, 114.76, 111.55, 111.10, 67.40, 57.59, 55.44, 51.67,
46.53, 36.59, 32.49, 29.09, 24.06, 23.54. Anal. (C30H37N2O2Cl ·HCl)
C, H, N. UV-vis (solvent: PBS) λ ) 226 nm, ε ) 41854.
2-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}ethyl-4-(3′-trifluo-
romethyl)piperazine (6a). Compound 6a was synthesized from
2-[2-(3-methoxyphenyl)ethyl]phenol 13 (91 mg, 0.40 mmol) and
from 1-(2-chloroethyl)-4-(3-trifluoromethylphenyl)piperazine 24
(118 mg, 0.40 mmol) following the same procedure described above
for the preparation of 5a. The crude residue was subjected to a
column chromatography eluting with hexane/AcOEt (1:1) and then
it was transformed into the hydrochloride salt to give 6a (73 mg,
0.15 mmol, 37% yield) as a white solid: mp 55-57 °C. MS m/z
484 (M+, 5); 243 (100). 1H NMR (CDCl3): δ 2.78 (t, 4H, J ) 4.7
Hz, piperazine), 2.85-2.94 (m, 6H, CH2CH2, CH2N), 3.24 (t, 4H,
J ) 4.7 Hz, piperazine), 3.78 (s, 3H, OMe), 4.17 (t, 2H, J ) 5.4
Hz, OCH2), 6.73-6.94 (m, 5H, Ar), 7.03-7.24 (m, 6H, Ar), 7.34
(t, 1H, J ) 7.8 Hz, Ar) ppm. 13C NMR (CDCl3): δ 159.90, 155.31,
149.92, 148.19, 146.80, 143.62, 130.40, 130.09, 129.47, 127.78,
122.22, 120.86, 120.15, 118.31, 114.67, 113.94, 111.92, 111.25,
63.16, 56.82, 55.36, 52.71, 46.70, 36.74, 31.60. Anal.
(C28H31N2O2F3 ·HCl) C, H, N. UV-vis (solvent: PBS) λ ) 230
nm, ε ) 21090.
3-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}propyl-4-(3′-trifluo-
romethylphenyl)piperazine (6b). Compound 6b was synthesized
from 1-(3-bromopropoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene
14b (200 mg, 0.57 mmol) and from 1-[3-(trifluoromethyl)phe-
nyl]piperazine 16 (131 mg, 0.57 mmol) following the same
procedure described above for the preparation of 5c. The crude
product was transformed into the hydrochloride salt and crystallized
from i-PrOH to afford 6b (104 mg, 0.19 mmol, 34% yield) as a
white solid: mp 130-132 °C. MS m/z 498 (M+, 18). 1H NMR
(CDCl3): δ 2.40-2.51 (m, 2H, CH2), 2.80-2.90 (m, 4H, CH2CH2),
3.15-3.31 (m, 4H, piperazine), 3.35-3.50 (m, 2H, CH2N),
3.51-3.70 (m, 4H, piperazine), 3.73 (s, 3H, OMe), 4.01-4.12 (m,
2H, OCH2), 6.70-6.87 (m, 4H, Ar), 6.93 (t, 1H, J ) 7.3 Hz, Ar),
7.12-7.24 (m, 5H, Ar), 7.35-7.48 (m, 2H, Ar) ppm. 13C NMR
(CDCl3): δ 159.73, 156.04, 149.83, 147.86, 130.13, 130.00, 129.47,
127.56, 121.41, 120.90, 120.04, 118.20, 118.10, 114.78, 113.85,
113.78, 111.48, 110.83, 64.89, 55.35, 52.09, 46.53, 43.31, 36.63,
31.93, 24.32. Anal. (C29H33F3N2O2 ·HCl) C, H, N. UV-vis (solvent:
PBS) λ ) 245 nm, ε ) 14090.
2{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}ethyl-4-(2′-methox-
yphenyl)piperazine (7a). Compound 7a was synthesized from 2-[2-
(3-methoxyphenyl)ethyl]phenol 13 (107 mg, 0.47 mmol) and from
1-(2-chloroethyl)-4-(2-methoxyphenyl)piperazine 26 (120 mg, 0.47
mmol) following the same procedure described above for the
preparation of 5a. The crude residue was transformed into the
hydrochloride salt and crystallized from i-PrOH to give 7a (163
mg, 0.36 mmol, 77% yield) as a white solid: mp 93-95 °C. MS
1
m/z 446 (M+, 6); 123 (45). H NMR (CDCl3): δ 2.69-2.87 (m,
4H, piperazine), 2.88-3.00 (m, 4H, CH2CH2), 3.05-3.19 (m, 4H,
piperazine), 3.68 (t, 2H, J ) 5.4 Hz, CH2N), 3.77 (s, 3H, OMe),
3.86 (s, 3H, OMe), 4.17 (t, 2H, J ) 5.8 Hz, CH2O), 6.73-6.98
(m, 7H, Ar), 7.05-7.21 (m, 5H, Ar) ppm. 13C NMR (CDCl3): δ
160.01, 156.20, 152.71, 144.32, 131.56, 130.80, 129.77, 128.94,
126.68, 123.95, 122.00, 120.73, 120.22, 115.89, 114.32, 113.13,
111.81, 111.55, 66.24, 57.31, 56.20, 55.90, 52.63, 48.37, 37.11,
30.96. Anal. (C28H34N2O3 ·HCl) C, H, N. UV-vis (solvent: PBS)
λ ) 230 nm, ε ) 15420.
3-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}propyl-4-(2′-meth-
oxyphenyl)piperazine (7b). Compound 7b was synthesized from
1-(3-bromopropoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14b (200
mg, 0.57 mmol) and from 1-[2-(methoxyphenyl)]piperazine hy-
drochloride 18 (110 mg, 0.57 mmol) following the same procedure
described above for the preparation of 5c. The crude residue was
purified by transformation into hydrochloride and crystallization
from i-PrOH to give 7b (136 mg, 0.30 mmol, 52% yield) as a white
solid: mp 114-116 °C. MS (EI, 70 eV) m/z: M+ 460 (66). 1H
NMR (CDCl3): δ 2.39-2.49 (m, 2H, CH2), 2.82-2.94 (m, 4H,
CH2CH2), 3.21-3.35 (m, 2H, CH2N), 3.49-3.61 (m, 4H, pipera-
zine), 3.76 (s, 3H, OMe), 4.01-4.10 (m, 5H, OCH2, OMe),
4.29-4.44 (m, 2H, piperazine), 4.94-5.04 (m, 2H, piperazine),
6.67-6.83 (m, 4H, Ar), 6.92 (t, 1H, J ) 7.0 Hz, Ar), 7.02-7.21
(m, 5H, Ar), 7.46 (t, 1H, J ) 8.5 Hz, Ar), 8.19 (d, 1H, J ) 7.9 Hz,
Ar). 13C NMR (CD3OD) δ: 160.00, 157.59, 156.19, 153.64, 145.03,
143.23, 131.32, 131.08, 130.24, 130.06, 128.31, 122.58, 121.96,
121.90, 115.44, 114.28, 112.71, 112.15, 66.02, 56.72, 55.97, 55.68,
54.88, 51.79, 37.58, 32.96, 25.40. Anal. (C29H36N2O3 HCl) C, H,
N. UV-vis (solvent: PBS) λ ) 230 nm, ε ) 16540.
4{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}butyl-4-(2′-methox-
yphenyl)piperazine (7c). Compound 7c was synthesized from 1-(4-
chlorobutoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14c (200 mg,
0.63 mmol) and from 1-(2-methoxyphenyl)piperazine 18 (121 mg,
0.63 mmol) following the same procedure described above for the
preparation of 5c. The crude product was purified by column
chromatography eluting with n-hexane/AcOEt (3:7) and transforma-
tion into the hydrochloride to give 7c (109 mg, 0.23 mmol, 36%
yield) as a white solid: mp 105-107 °C. MS m/z 474 (M+, 73).
1H NMR (CDCl3): δ 1.65-1.95 (m, 4H, CH2CH2), 2.50 (t, 2H, J
) 7.2 Hz, CH2N), 2.6-2.71 (m, 4H, piperazine), 2.73-2.95 (m,
4H, CH2CH2), 3.01-3.15 (m, 4H, piperazine), 3.78 (s, 3H, OMe),
3.86 (s, 3H, OMe), 4.01 (t, 2H, J ) 5.8 Hz, CH2O), 6.71-7.00
(m, 9H, Ar), 7.09-7.20 (m, 3H, Ar) ppm. 13C NMR (CDCl3): δ
159.72, 156.44, 152.76, 143.98, 130.86, 130.24, 129.95, 129.40,
127.32, 123.35, 121.79, 121.00, 120.84, 114.47, 113.36, 113.39,
111.46, 111.23, 66.89, 57.33, 56.18, 55.36, 49.10, 48.74, 36.32,
32.06, 27.01, 21.06. Anal. (C30H38N2O3 ·HCl) C, H, N. UV-vis
(solvent: PBS) λ ) 230 nm, ε ) 18240.
5-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}pentyl-4-(2′-meth-
oxyphenyl)piperazine (7d). Compound 7d was synthesized from
1-[(5-bromopentyloxy]-2-[2-(3-methoxyphenyl)ethyl]benzene 14d
(150 mg, 0.40 mmol) and from 1-(2-methoxyphenyl)piperazine 18
(77 mg, 0.40 mmol) following the same procedure described above
for the preparation of 5c. The crude product was purified by
transformation into the hydrochloride to give 7d (160 mg, 0.30
mmol, 76% yield) as a white solid: mp 100-102 °C. MS m/z 488
(M+, 53). 1H NMR (CD3OD): δ 1.65-1.73 (m, 2H, CH2),
1.83-1.99 (m, 4H, CH2CH2), 2.82-2.92 (m, 4H, CH2CH2),
3.45-3.54 (m, 8H, piperazine), 3.70-3.76 (m, 2H, CH2N), 3.89
(s, 3H, OMe), 3.93 (s, 3H, OMe), 4.03 (t, 2H, J ) 5.9 Hz, CH2O),
6.71-7.27 (m, 12H, Ar) ppm. 13C NMR (CD3OD): δ 161.00,
158.10, 153.73, 145.18, 130.94, 130.19, 129.53, 128.31, 128.24,
4{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}butyl-4-(3′-trifluo-
romethylphenyl)piperazine (6c). Compound 6c was synthesized
from 1-(4-chlorobutoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene
14c (200 mg, 0.63 mmol) and from 1-(3-trifluoromethylphe-
nyl)piperazine 16 (145 mg, 0.63 mmol) following the same
procedure described above for the preparation of 5c. The crude
product was purified by column chromatography eluting with
n-hexane/AcOEt (3:7) and transformed into the hydrochloride
salt to give 6c (122 mg, 0.24 mmol, 38% yield) as a white solid:
mp 78-80 °C. MS m/z 512 (M+, 6). 1H NMR (CDCl3): δ
1.75-1.95 (m, 4H, CH2CH2), 2.49 (t, 2H, J ) 7.0 Hz, CH2N),
2.58 (t, 4H, J ) 4.9 Hz, piperazine), 2.85-2.95 (m, 4H,
CH2CH2), 3.21 (t, 4H, J ) 4.9 Hz, piperazine), 3.77 (s, 3H,
OMe), 4.01 (t, 2H, J ) 5.8 Hz, OCH2), 6.72-6.90 (m, 6H, Ar),
7.02-7.38 (m, 6H, Ar) ppm. 13C NMR (CDCl3): δ 162.00,
154.25, 149.89, 148.35, 131.69, 130.05, 129.74, 128.49, 124.12,
122.00, 121.71, 120.33, 120.04, 117.56, 114.57, 114.30, 111.82,
111.51, 67.89, 56.94, 54.10, 52.65, 47.00, 37.11, 32.75,
27.41,21.70. Anal. (C30H35N2O2F3 · HCl) C, H, N. UV-vis
(solvent: PBS) λ ) 230 nm, ε ) 18950.

2-[(3-Methoxyphenylethyl)phenoxy]-Based ABCB1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7609
122.42, 121.87, 121.70, 121.43, 115.33, 114.15, 113.86, 112.53,
112.13, 68.48, 58.09, 56.68, 56.50, 55.68, 52.28, 43.76, 37.56,
33.54, 30.01, 24.73. Anal. (C31H40N2O3 HCl) C, H, N. UV-vis
(solvent: PBS) λ ) 271 nm, ε ) 4696.
6H, piperazine, CH2N), 3.79 (s, 3H, OMe), 3.99 (t, 2H, J ) 5.5
Hz, CH2O), 6.68-6.90 (m, 5H, Ar), 7.06-7.28 (m, 7H, Ar) ppm.
13C NMR (CDCl3): δ 159.86, 156.80, 144.24, 130.70, 130.45,
130.09, 129.47, 127.40, 122.28, 121.06, 120.84, 117.55, 117.04,
114.74, 111.61, 111.23, 67.40, 57.59, 55.49, 50.19, 50.16, 36.59,
32.40, 29.13, 24.01, 23.75. Anal. (C30H37N2O2F · HCl) C, H, N.
UV-vis (solvent: PBS) λ ) 228 nm, ε ) 24918.
2{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}ethyl-4-(4′-fluorophe-
nyl)piperazine (8a). Compound 8a was synthesized from 2-[2-(3-
methoxyphenyl)ethyl]phenol 13 (186 mg, 0.82 mmol) and from
1-(2-chloroethyl)-4-(4-fluorophenyl)piperazine 25 (198 mg, 0.82
mmol) following the same procedure described above for the
preparation of 5a. The crude residue was subjected to a column
chromatography eluting with hexane/AcOEt (1:1) and then it was
transformed into the hydrochloride salt to give 8a (143 mg, 0.33
mmol, 70% yield) as a white solid: mp 72-75 °C. MS m/z 434
(M+, 6); 193 (100). 1H NMR (CDCl3): δ 2.75-2.81 (m, 4H,
piperazine), 2.88-2.94 (m, 6H, CH2N, CH2CH2), 3.09-3.14 (m,
4H, piperazine), 3.77 (s, 3H, OMe), 4.16 (t, 2H, J ) 5.6 Hz, OCH2),
6.73-7.00 (m, 9H, Ar), 7.12-7.22 (m, 3H, Ar) ppm. 13C NMR
(CDCl3) δ: 163.90, 159.92, 155.29, 149.66, 143.67, 130.46, 130.36,
129.47, 127.76, 122.17, 120.84, 119.78, 119.62, 116.33, 115.89,
114.65, 111.88, 111.23, 63.07, 56.69, 55.35, 52.87, 48.03, 36.74,
31.64; Anal. (C27H31N2O2F·HCl) C, H, N. UV-vis (solvent: PBS)
λ ) 230 nm, ε ) 13780.
2-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}ethyl-4-(2′-pyrimi-
dil)piperazine (9a). To a solution of 1-(2-chloroethoxy)-2-[2-(3-
methoxyphenyl)ethyl]benzene 14a (106 mg, 0.37 mmol) in a small
amount of acetonitrile (3 mL) was added K2CO3 (112 mg, 0.81
mmol), KI (7 mg, 0.04 mmol), and 2-piperazinylpyrimidine
dihydrochloride (87 mg, 0.37 mmol). The resulting suspension was
heated for 10 min by microwave irradiation at 160 °C and with a
power of 120 W. Then the potassium carbonate was filtered off
and the solvent removed. The crude product was transformed into
the hydrochloride to give 9a (104 mg, 0.19 mmol, 34% yield) as
1
a white solid: mp 164-166 °C. MS m/z 418 (M+, 42). H NMR
(CDCl3): δ 2.67 (t, 4H, J ) 5.1 Hz, piperazine), 2.86-2.94 (m,
6H, CH2CH2, CH2N), 3.77 (s, 3H, OMe), 3.81-3.87 (m, 4H,
piperazine), 4.15 (t, 2H, J ) 5.7 Hz, CH2O), 6.47 (t, 1H, J ) 4.8
Hz, Pyr), 6.72-6.91 (m, 5H, Ar), 7.10-7.24 (m, 3H, Ar), 8.30 (d,
2H, J ) 4.6 Hz, Pyr) ppm. 13C NMR (CD3OD): δ 161.03, 158.63,
156.90, 150.46, 144.76, 131.48, 131.37, 130.21, 128.44, 122.92,
121.92, 115.40, 113.20, 112.51, 112.29, 64.11, 57.59, 55.68, 53.21,
42.66, 37.55, 32.59. Anal. (C25H30N4O2 ·HCl) C, H, N. UV-vis
(solvent: PBS) λ ) 250 nm, ε ) 15820.
3-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}propyl-4-(4′-fluo-
rophenyl)piperazine (8b). Compound 8b was synthesized from
1-(3-bromopropoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14b (200
mg, 0.57 mmol) and from 1-[4-(fluorophenyl)]piperazine hydro-
chloride 17 (144 mg, 0.57 mmol) following the same procedure
described above for the preparation of 5c. The crude residue was
purified by transformation into the hydrochloride salt and crystal-
lization from i-PrOH to give 8b (77 mg, 0.17 mmol, 30% yield) as
3-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}propyl-4-(2′-pyrim-
idyl)piperazine (9b). Compound 9b was synthesized from 1-(3-
bromopropoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14b (246 mg,
0.70 mmol) and from 2-piperazinylpyrimidine dihydrochloride (273
mg, 0.70 mmol) following the same procedure described above for
the preparation of 5c. The crude residue was purified by transfor-
mation into the hydrochloride to give 9b (107 mg, 0.25 mmol, 35%
yield) as a white solid: mp 143-145 °C. MS m/z 432 (M+, 35);
324 (100). 1H NMR (CDCl3): δ 2.05-2.17 (m, 2H, CH2),
2.54-2.71 (m, 6H, piperazine, CH2N), 2.81-2.90 (m, 4H,
CH2CH2), 3.77 (s, 3H, OMe), 3.87-3.92 (m, 4H, piperazine),
4.03-4.14 (m, 2H, OCH2), 6.50 (t, 1H, J ) 4.8 Hz, Pyr), 6.73-6.91
(m, 5H, Ar), 7.10-7.21 (m, 3H, Ar), 8.31 (d, 2H, J ) 4.8 Hz, Pyr)
ppm. 13C NMR (CD3OD): δ 161.00, 158.45, 156.82, 148.12,
145.01, 131.32, 130.60, 130.23, 128.31, 122.03, 121.92, 115.46,
112.71, 112.36, 112.11, 65.99, 56.03, 55.72, 52.26, 46.92, 37.55,
32.92, 25.40. Anal. (C26H32N4O2 ·HCl) C, H, N.
4-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}butyl-4-(2′-pyrim-
idyl)piperazine (9c). Compound 9c was synthesized from 1-(4-
chlorobutoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14c (180 mg,
0.57 mmol) and from 2-piperazinylpyrimidine dihydrochloride (134
mg, 0.57 mmol) following the same procedure described above for
the preparation of 5c. The crude residue was purified by transfor-
mation into the hydrochloride and crystallization from i-PrOH/i-
Pr2O to give 9c (83 mg, 0.17 mmol, 30% yield) as a white solid:
mp 130-132 °C. MS m/z 446 (M+, 31). 1H NMR (CDCl3): δ
1.72-1.87 (m, 4H, CH2CH2), 2.43-2.51 (m, 6H, CH2CH2, CH2N),
2.86-2.94 (m, 4H, piperazine), 3.77-3.83 (m, 7H, OMe, pipera-
zine), 4.00 (t, 2H, J ) 5.5 Hz, CH2O), 6.47 (t, 1H, J ) 4.7 Hz,
Pyr), 6.71-6.89 (m, 5H, Ar), 7.09-7.20 (m, 3H, Ar), 8.30 (d, 2H,
J ) 4.8 Hz, Pyr) ppm. 13C NMR (CD3OD): δ 160.98, 158.69,
157.87, 145.10, 131.17, 130.90, 130.24, 128.27, 127.13, 121.85,
121.61, 115.37, 112.71, 112.47, 112.00, 68.17, 58.29, 55.74, 52.42,
42.77, 37.44, 33.36, 27.79,22.38. Anal. (C27H34N4O2 ·HCl) C, H,
N. UV-vis (solvent: PBS) λ ) 230 nm, ε ) 17440.
1
a white solid: mp 123-125 °C. MS m/z: M+ 448 (M+, 89). H
NMR (CDCl3): δ 2.32-2.50 (m, 2H, CH2),2.81-2.91 (m, 4H,
CH2CH2), 3.23-3.70 (m, 2H, CH2N), 3.50-3.70 (m, 4H, pipera-
zine), 3.75 (s, 3H, OMe), 3.96-4.20 (m, 4H, piperazine), 4.50-4.64
(m, 2H, OCH2), 6.68-6.97 (m, 5H, Ar), 7.15-7.24 (m, 5H, Ar),
7.77-7.84 (m, 2H, Ar). 13C NMR (CD3OD): δ 162.36, 161.02,
157.57, 146.58, 145.03, 131.32, 131.05, 130.24, 128.31, 122.05,
121.91, 120.78, 120.58, 117.06, 116.61, 115.48, 112.67, 112.07,
66.04, 55.09, 55.68, 53.04, 49.29, 37.55, 32.90, 25.46. Anal.
(C28H33N2O2F·HCl) C, H, N. UV-vis (solvent: PBS) λ ) 230 nm,
ε ) 18350.
4{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}butyl-4-(4′-fluorophe-
nyl)piperazine (8c). Compound 8c was synthesized from 1-(4-
chlorobutoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14c (200 mg,
0.63 mmol) and from 1-(4-fluorophenyl)piperazine hydrochloride
17 (160 mg, 0.63 mmol) following the same procedure described
above for the preparation of 5c. The crude product was purified by
column chromatography eluting with n-hexane/AcOEt (3:7) and
transformation into the hydrochloride salt to give 8c (64 mg, 0.16
mmol, 26% yield) as an white solid: mp 88-90 °C. MS m/z 462
(M+, 13). 1H NMR (CDCl3): δ 1.90-2.20 (m, 4H, CH2CH2),
2.81-2.95 (m, 4H, CH2CH2), 3.15-3.32 (m, 2H, CH2N), 3.42-3.59
(m, 4H, piperazine), 3.78 (s, 3H, OMe), 3.98-4.25 (m, 4H,
piperazine), 4.73 (m, 2H, OCH2), 6.65-6.96 (m, 5H, Ar), 7.11-7.23
(m, 6H, Ar), 7.88-8.00 (m, 1H, Ar) ppm. 13C NMR (CDCl3): δ
160.86, 158.62, 154.40, 149.11, 145.23, 130.57, 129.70, 128.44,
126.68, 122.99, 121.34, 120.30, 120.16, 116.49, 115.95, 114.30,
111.87, 111.58, 64.00, 55.97, 54.91, 52.66, 49.13, 37.07, 31.76,
31.15, 24.73. Anal. (C29H35N2O2F·HCl) C, H, N. UV-vis (solvent:
PBS) λ ) 230 nm, ε ) 16480.
5-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}pentyl-4-(4′-fluo-
rophenyl)piperazine (8d). Compound 8d was synthesized from
1-[(5-bromopentyloxy]-2-[2-(3-methoxyphenyl)ethyl]benzene 14d
(150 mg, 0.40 mmol) and from 1-(4-fluorophenyl)piperazine
hydrochloride 17 (101 mg, 0.40 mmol) following the same
procedure described above for the preparation of 5c. The crude
product was purified by transformation into the hydrochloride to
give 8d (125 mg, 0.24 mmol, 61% yield) as a white solid: mp
123-125 °C. MS m/z 476 (M+, 15). 1H NMR (CDCl3): δ
1.56-1.70 (m, 2H, CH2), 1.82-1.92 (m, 4H, CH2CH2), 2.79-2.92
(m, 4H, CH2CH2), 3.03-3.21 (m, 4H, piperazine), 3.41-3.66 (m,
5-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}pentyl-4-(2′-pyrim-
idyl)piperazine (9d). Compound 9d was synthesized from 1-[(5-
bromopentyloxy]-2-[2-(3-methoxyphenyl)ethyl]benzene 14d (400
mg, 1.06 mmol) and from 2-piperazinylpyrimidine dihydrochloride
(250 mg, 1.06 mmol) following the same procedure described above
for the preparation of 5c. The crude product was transformed into
the hydrochloride to give 9d (146 mg, 0.32 mmol, 30% yield) as
1
a white solid: mp 135-137 °C. H NMR (CDCl3): δ 1.56-1.66
(m, 2H, CH2), 1.82-1.94 (m, 4H, CH2CH2), 2.85-3.05 (m, 8H,

7610 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Colabufo et al.
CH2CH2, piperazine), 3.63-3.76 (m, 6H, piperazine, CH2N), 3.77
(s, 3H, OMe), 3.97 (t, 2H, J ) 5.7 Hz, CH2O), 6.71-6.90 (m, 6H,
Ar), 7.09-7.25 (m, 3H, Ar), 8.55 (d, 2H, J ) 4.8 Hz, Ar) ppm.
13C NMR (CDCl3): δ 159.81, 157.62, 157.60, 156.80, 144.24,
130.40, 130.04, 129.42, 127.40, 121.04, 120.75, 114.70, 111.57,
111.34, 111.14, 67.38, 57.66, 55.42, 51.65, 41.73, 36.59, 32.48,
29.11, 23.97, 23.54. Anal. (C28H36N4O2 ·HCl) C, H, N. UV-vis
(solvent: PBS) λ ) 227 nm, ε ) 32206.
2-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}ethyl-4-(N-meth-
yl)piperazine (10a). Compound 10a was synthesized from 1-(2-
chloroethoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14a (200 mg,
0.68 mmol) and from N-methylpiperazine (70 mg, 0.68 mmol)
following the same procedure described above for the preparation
of 9a. The crude residue was transformed into the hydrochloride
and crystallized from i-PrOH to give 10a (54 mg, 0.15 mmol, 22%
yield) as a white solid: mp 165-167 °C. 1H NMR (CDCl3): δ 2.28
(s, 3H, Me), 2.30-2.48 (m, 4H, piperazine), 2.50-2.71 (m, 4H,
piperazine), 2.81-2.89 (m, 6H, CH2CH2, CH2N), 3.78 (s, 3H,
OMe), 4.11 (t, 2H, J ) 5.6 Hz, OCH2), 6.73-6.90 (m, 5H,
Ar),7.09-7.25 (m, 3H, Ar) ppm. 13C NMR (CD3OD): δ 161.50,
156.94, 146.56, 131.60, 131.37, 130.24, 128.41, 122.92, 122.00,
115.51, 113.27, 112.25, 64.44, 57.43, 55.70, 51.48, 50.77, 43.45,
37.49, 32.63. Anal. (C22H30N2O2 ·HCl) C, H, N. UV-vis (solvent:
PBS) λ ) 271 nm, ε ) 2620.
3-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}propyl-4-(3′-meth-
yl)piperazine (10b). Compound 10b was synthesized from 2-[2-
(3-methoxyphenyl)]ethyl]phenol 13 (500 mg, 2.19 mmol) and 1-(3-
chloropropyl)-4-methylpiperazine (390 mg, 2.19 mmol) following
the same procedure described above for the preparation of 5b. The
crude residue was purified by transformation into the hydrochloride
and crystallization from EtOH to give 10b (380 mg, 1.03 mmol,
47% yield) as a white solid: mp 194-196 °C. MS m/z 368 (M+,
22). 1H NMR (DMSO): δ 2.10-2.32 (m, 2H, CH2), 2.51-2.95
(m, 7H, CH2CH2, Me), 3.10-3.67 (m, 10H, piperazine, CH2N),
3.71 (s, 3H, OMe), 4.06 (t, 2H, J ) 5.9 Hz, OCH2), 6.70-7.05
(m, 5H, Ar), 7.09-7.35 (m, 3H, Ar) ppm. 13C NMR (CDCl3): δ
159.91, 156.25, 149.53, 130.76, 130.41, 129.75, 128.40, 121.93,
120.51, 114.38, 111.87, 111.54, 66.44, 55.89, 55.70, 55.20, 52.67,
46.94, 43.21, 36.77, 29.27, 24.65. Anal. (C23H32N2O2 ·HCl) C, H,
N. UV-vis (solvent: PBS) λ ) 278 nm, ε ) 4110.
67.29, 57.22, 55.46, 50.10, 48.50, 43.20, 36.50, 32.35, 29.02, 23.72,
23.65. Anal. (C25H36N2O2 ·HCl) C, H, N. UV-vis (solvent: PBS)
λ ) 271 nm, ε ) 3056.
2-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}ethyl-(3,4-dihydroiso-
quinoline) (11a). A stirred solution of KOH (360 mg, 6.40 mmol)
in DMSO (2 mL) was heated to 80 °C until the complete dissolution
of KOH. The solution was treated with a solution of 2-[2-(3-
methoxyphenyl)]ethyl]phenol 13 (420 mg, 1.83 mmol) in DMSO
(3 mL) and the resulting suspension was vigorously stirred for 20
min. Then a solution of 2-(2-chloroethyl)-1,2,3,4-tetrahydroiso-
quinoline (360 mg, 1.83 mmol) dissolved in DMSO (2 mL) was
added and the stirring was continued for 24 h at 40 °C. The mixture
was eluting with AcOEt and washed with water, 1N NaOH, and
brine. The organic layer was evaporated to give a crude oil which
was purified by transformation into the hydrochloride and crystal-
lization from i-PrOH to give 11a (194 mg, 0.46 mmol, 25% yield)
as a white solid: mp: 156.158 °C. MS m/z 387 (M+, 27); 146 (100).
1H NMR (CDCl3): δ 2.86-2.95 (m, 8H), 3.02 (t, 2H, J ) 5.9 Hz,
CH2N), 3.76 (s, 3H, OMe), 3.82 (s, 2H, CH2), 4.22 (t, 2H, J ) 5.9
Hz, CH2O), 6.72-6.92 (m, 5H, Ar), 7.04-7.22 (m, 7H, Ar) ppm.
13C NMR (CDCl3): δ 159.68, 155.33, 143.49, 130.42, 130.11,
129.80, 129.29, 128.91, 128.45, 127.61, 127.47, 126.94, 126.60,
121.90, 120.80, 114.41, 111.77, 111.26, 63.43, 55.26, 54.24, 53.25,
49.70, 36.52, 31.71,24.45. Anal. (C26H29NO2 ·HCl) C, H, N.
UV-vis (solvent: PBS) λ ) 230 nm, ε ) 16040.
3-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}propyl-(3,4-dihy-
droisoquinoline) (11b). A solution of K2CO3 (142 mg, 1.03 mmol)
in THF (5 mL) was heated at 80 °C until the complete dissolution
of K2CO3. Then a solution of 1,2,3,4-tetrahydroisoquinoline (172
mg, 1.29 mmol) in THF (7 mL) was added, and the resulting
solution, cooled at 0 °C, was treated with a solution of 1-(3-
bromopropoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14b (300 mg,
0.86 mmol) in THF (3 mL). The suspension was stirred at room
temperature for 7 h, then KI (35 mg, 0.21 mmol) and DMF (1 mL)
were added and the stirring was continued for 40 h. After this
period, the solvent was evaporated, water was added, and the
solution was extracted with AcOEt. The organic layer was dried
and evaporated. The crude product was purified by column
chromatography eluting with CHCl3/Et2O (8:2) to give 11b (233
mg, 0.58 mmol, 45% yield) as an white solid: mp 199-201 °C.
1
MS m/z 401 (M+, 27). H NMR (CDCl3): δ 2.12-2.19 (m, 2H,
4-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}butyl-4-(N-meth-
yl)piperazine (10c). Compound 10c was synthesized from 1-(4-
chlorobutoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14c (180 mg,
0.57 mmol) and from N-methylpiperazine (57 mg, 0.57 mmol)
following the same procedure described above for the preparation
of 5c. The crude residue was purified by transformation into
hydrochloride and crystallized from i-PrOH/i-Pr2O to give 10c (47
mg, 0.11 mmol, 20% yield) as a white solid: mp 168-170 °C. MS
CH2), 2.75-2.94 (m, 10H), 3.71 (s, 2H, CH2), 3.77 (s, 3H, OMe),
4.07 (t, 2H, J ) 6.0 Hz, CH2O), 6.72-6.89 (m, 4H, Ar), 7.07-7.21
(m, 8H, Ar) ppm. 13C NMR (CDCl3): δ 156.09, 143.87, 141.84,
130.75, 130.20, 130.09, 129.40, 129.38, 128.91, 128.52, 127.52,
126.96, 126.61, 121.31, 120.88, 114.69, 111.99, 110.95, 65.05,
55.29, 53.23, 52.61, 49.26, 36.56, 31.86, 24.74, 24.56. Anal.
(C27H31NO2) C, H, N. UV-vis (solvent: PBS) λ ) 230 nm, ε )
14790.
1
m/z 382 (M+, 30). H NMR (CDCl3): δ 1.66-1.87 (m, 8H), 2.28
(s, 3H, Me), 2.39-2.46 (m, 6H, piperazine, CH2N), 2.81-2.94 (m,
4H, CH2CH2), 3.78 (s, 3H, OMe), 3.98 (t, 2H, J ) 5.8 Hz, CH2O),
6.72-6.89 (m, 5H, Ar), 7.09-7.24 (m, 3H, Ar) ppm. 13C NMR
(CD3OD): δ 158.00, 145.02, 131.82, 130.90, 130.97, 130.19,
128.24, 121.91, 121.63, 115.37, 112.47, 112.11, 68.03, 57.40, 55.66,
51.24, 43.70, 40.70, 37.78, 37.46, 33.34, 27.64, 22.23. Anal.
(C24H34N2O2 ·HCl) C, H, N. UV-vis (solvent: PBS) λ ) 271 nm,
ε ) 2460.
5-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}pentyl-4-(N-meth-
yl)piperazine (10d). Compound 10d was synthesized from 1-[(5-
bromopentyloxy]-2-[2-(3-methoxyphenyl)ethyl]benzene 14d (400
mg, 1.06 mmol) and from N-methylpiperazine (106 mg, 1.06 mmol)
following the same procedure described above for the preparation
of 5c. The crude residue was purified by transformation into the
hydrochloride to give 10d (115 mg, 0.26 mmol, 25% yield) as a
white solid: mp 217-219 °C. MS m/z 396 (M+, 28). 1H NMR
(CDCl3): δ 1.52-1.65 (m, 2H, CH2), 1.81-2.01 (m, 4H, CH2CH2),
2.86-2.90 (m, 7H, CH2CH2, Me), 3.03-3.11 (m, 4H, piperazine),
3.48-3.60 (m, 6H, piperazine, CH2N), 3.79 (s, 3H, OMe),
3.88-4.06 (m, 2H, CH2O), 6.72-6.91 (m, 5H, Ar), 7.10-7.23 (m,
3H, Ar) ppm. 13C NMR (CDCl3): δ 159.75, 156.71, 144.11, 130.36,
130.04, 139.42, 127.30, 121.00, 120.75, 114.60, 111.50, 111.26,
4{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}butyl-(3,4-dihydroiso-
quinoline) (11c). Compound 11c was synthesized from 1-(4-
chlorobutoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14c (200 mg,
0.63 mmol) and from 1,2,3,4-tetrahydroisoquinoline (84 mg, 0.63
mmol) following the same procedure described above for the
preparation of 5c. The crude product was purified by column
chromatography eluting with n-hexane/AcOEt (3:7) and transforma-
tion into the hydrochloride to give 11c (60 mg, 0.13 mmol, 21%
yield) as a white solid: mp: 115-117 °C. MS m/z 415 (M+, 8);
146 (M+, 100). 1H NMR (CDCl3): δ 1.80-1.92 (m, 4H, CH2CH2),
2.60 (t, 2H, J ) 6.8 Hz, CH2), 2.69-2.75 (m, 2H, CH2), 2.83-2.98
(m, 6H, CH2CH2, CH2N), 3.63 (s, 2H, CH2), 3.77 (s, 3H, OMe),
4.02 (t, 2H, J ) 5.6 Hz, CH2O), 6.71-6.90 (m, 5H, Ar),6.96-7.24
(m, 7H, Ar) ppm. 13C NMR (CDCl3): δ 159.90, 156.58, 144.13,
130.82, 130.22, 130.00, 129.45, 128.96, 128.58, 127.60, 127.51,
127.05, 126.69, 121.02, 120.93, 114.67, 111.54, 111.12, 67.16,
55.36, 55.07, 52.27, 48.85, 36.45, 32.35, 27.16, 24.46, 21.66. Anal.
(C28H33NO2 ·HCl) C, H, N. UV-vis (solvent: PBS) λ ) 226 nm,
ε ) 42580.
5-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}pentyl-(3,4-dihy-
droisoquinoline) (11d). Compound 11d was synthesized from
1-[(5-bromopentyloxy]-2-[2-(3-methoxyphenyl)ethyl]benzene 14d

2-[(3-Methoxyphenylethyl)phenoxy]-Based ABCB1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7611
(400 mg, 1.06 mmol) and from 1,2,3,4-tetrahydroisoquinoline (141
mg, 1.06 mmol) following the same procedure described above for
the preparation of 5c. The crude product was purified by transfor-
mation into the hydrochloride and crystallization from EtOH/i-PrO2
to give 11d (160 mg, 0.34 mmol, 32% yield) as a white solid: mp
129-131 °C. MS m/z 429 (M+, 11). 1H NMR (CDCl3): δ
1.52-1.70 (m, 4H, CH2CH2), 1.85-1.92 (m, 2H, CH2), 1.98-2.19
(m, 2H, CH2), 2.80-2.92 (m, 4H, CH2CH2), 2.85-3.17 (m, 4H,
CH2, CH2N), 3.43-3.63 (m, 2H, CH2), 3.74 (s, 3H, OMe), 3.98 (t,
2H, J ) 5.8 Hz, CH2O), 6.68-6.90 (m, 5H, Ar), 7.06-7.28 (m,
7H, Ar) ppm. 13C NMR (CDCl3): δ 159.79, 156.82, 144.22, 130.82,
130.38, 130.02, 129.36, 129.00, 128.60, 127.61, 127.38, 127.03,
126.72, 121.01, 120.71, 114.63, 111.54, 111.14, 67.42, 55.38, 55.11,
52.29, 48.97, 36.57, 32.51, 29.13, 24.50, 24.08, 24.03. Anal.
(C29H35NO2 ·HCl) C, H, N. UV-vis (solvent: PBS) λ ) 227 nm,
ε ) 32206.
2-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}ethyl-(6,7-dimethoxy-
3,4-dihydroisoquinoline) (12a). Compound 12a was synthesized
from 1-(2-chloroethoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14a
(150 mg, 0.44 mmol) and from 6,7-dimethoxy-1,2,3,4-tetrahy-
droisoquinoline (100 mg, 0.44 mmol) following the same procedure
described above for the preparation of 5c. The crude product was
purified by transformation into the hydrochloride and crystallization
from EtOH to give 12a (89 mg, 0.18 mmol, 42% yield) as a white
solid: mp 184-186 °C. MS m/z 447 (M+, 16). 1H NMR (DMSO):
δ 2.83-2.99 (m, 6H, CH2CH2, CH2N), 3.10-3.45 (m, 4H, CH2),
3.63-3.73 (m, 11H, CH2, OMe), 4.06-4.15 (m, 2H, CH2O),
6.65-7.04 (m, 7H, Ar), 7.13-7.25 (m, 3H, Ar) ppm. 13C NMR
(DMSO): δ 159.00, 155.29, 143.09, 129.65, 129.43, 128.90, 127.03,
123.73, 123.04, 120.84, 120.35, 120.25, 113.90, 111.90, 111.61,
110.99, 110.08, 109.62, 62.64, 55.56, 55.49, 54.72, 54.01, 52.12,
49.28, 35.48, 31.18, 24.06. Anal. (C28H33NO4 ·HCl) C, H, N.
UV-vis (solvent: PBS) λ ) 279 nm, ε ) 2426.
3-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}propyl-(6,7-
dimethoxy-3,4-dihydroisoquinoline) (12b). Compound 12b was
synthesized from 1-(3-bromopropoxy)-2-[2-(3-methoxyphenyl)eth-
yl]benzene 14b (150 mg, 0.42 mmol) and from 6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline (96 mg, 0.42 mmol) following the
same procedure described above for the preparation of 5c. The crude
product was purified by transformation into the hydrochloride to
give 12b (158 mg, 0.32 mmol, 76% yield) as a white solid: mp
138-140 °C. MS m/z 461 (M+, 27). 1H NMR (CDCl3): δ
2.04-2.16 (m, 2H, CH2), 2.44-2.58 (m, 2H, CH2), 2.75-2.89 (m,
4H, CH2CH2), 3.18-3.39 (m, 4H, CH2, CH2N), 3.60-3.74 (m, 2H,
CH2), 3.70 (s, 3H, OMe), 3.81 (s, 3H, OMe), 3.82 (s, 3H, OMe),
4.07 (t, 2H, J ) 5.1, CH2O), 6.52 (s, 1H, Ar), 6.58 (s, 1H, Ar),
6.67-6.72 (m, 3H, Ar), 6.79-6.93 (m, 2H, Ar), 7.10-7.21 (m,
3H, Ar) ppm. 13C NMR (CDCl3): δ 159.81, 156.13, 149.68, 149.03,
143.93, 130.24, 130.18, 129.40, 127.56, 122.88, 121.37, 120.93,
118.44, 114.74, 111.76, 111.66, 110.03, 109.90, 65.12, 56.35, 56.26,
55.36, 53.07, 52.49, 49.41, 36.65, 32.00, 24.83, 24.21. Anal.
(C29H35NO4 ·HCl) C, H, N. UV-vis (solvent: PBS) λ ) 278 nm,
ε ) 4112.
24.12, 21.75. Anal. (C30H37NO4 ·HCl) C, H, N. UV-vis (solvent:
PBS) λ ) 228 nm, ε ) 26690.
5-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}propyl-(6,7-
dimethoxy-3,4-dihydroisoquinoline) (12d). Compound 12d was
synthesized from 1-(4-chlorobutoxy)-2-[2-(3-methoxyphenyl)eth-
yl]benzene 14d (150 mg, 0.40 mmol) and from 6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline (92 mg, 0.40 mmol) following the
same procedure described above for the preparation of 5c. The crude
product was purified by transformation into the hydrochloride to
give 12d (67 mg, 0.13 mmol, 32% yield) as a white solid: mp
136-138 °C. MS m/z 489 (M+, 21). 1H NMR (CDCl3): δ
1.55-1.70 (m, 2H, CH2), 1.74-1.82 (m, 2H, CH2), 1.84-1.99 (m,
2H, CH2), 2.00-2.18 (m, 2H, CH2), 2.80-2.94 (m, 4H, CH2CH2),
3.05-3.28 (m, 4H, CH2), 3.30-3.68 (m, 2H, CH2N), 3.75 (s, 3H,
OMe), 3.85 (s, 6H, OMe), 3.93-4.05 (m, 2H, CH2O), 6.41 (s, 1H,
Ar), 6.58 (s, 1H, Ar), 6.70-6.93 (m, 5H, Ar), 7.11-7.23 (m, 3H,
Ar) ppm. 13C NMR (CDCl3): δ 159.88, 156.10, 150.68, 149.00,
144.15, 130.15, 130.02, 129.20, 127.51, 122.50, 121.11, 120.93,
118.42, 114.67, 111.25, 111.14, 110.05, 109.76, 65.30, 56.29, 56.27,
54.91, 52.70, 52.14, 49.01, 36.52, 32.48, 24.75, 24.12, 21.75. Anal.
(C31H39NO4 ·HCl) C, H, N. UV-vis (solvent: PBS) λ ) 228 nm,
ε ) 21840.
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. CulturePlate 96-well
plates were purchased from Perkin-Elmer Life Science and Calcein-
AM from Molecular Probes (Eugene, OR). Verapamil was pur-
chased from Tocris Bioscience (Bristol, United Kingdom).
Permeability Experiments. Preparation of Caco-2 Mono-
layer. This procedure has been previously reported by Colabufo et
al.27 Briefly, Caco-2 cells were harvested with trypsin-EDTA and
seeded onto MultiScreen Caco-2 assay system at a density of 10000
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 transepithelial electrical resistance (TEER) of the monolayers
was measured daily before and after the experiment using a
epithelial voltohommeter (Millicell -ERS; Millipore, Billerica, MA).
Generally, TEER values obtained are greater than 1000 Ω for a 21
day culture.
Drug Transport Experiment. Apical to basolateral (A f B)
and basolateral to apical (B f A) permeability of drugs were
measured at 120 min and at various drugs concentrations (1-100
µM).19 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 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 incubation time, the HBSS buffer was
removed and drug solutions 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 incubation time,
samples were removed from the apical (filter well) and basolateral
(receiver plate) side of the monolayer and then were stored in a
freezer (-20 °C) pending analysis. The concentration of compounds
were analyzed using UV-vis spectroscopy. The apparent perme-
ability (Papp), in units of nm/second, was calculated using the
following equation:
4-{2-[2-(3-Methoxyphenyl)ethyl]phenoxy}butyl-(6,7-dimethoxy-
3,4-dihydroisoquinoline) (12c). Compound 12c was synthesized
from 1-(4-chlorobutoxy)-2-[2-(3-methoxyphenyl)ethyl]benzene 14c
(200 mg, 0.55 mmol) and from 6,7-dimethoxy-1,2,3,4-tetrahy-
droisoquinoline (126 mg, 0.55 mmol) following the same procedure
described above for the preparation of 5c. The crude product was
purified by transformation into the hydrochloride and crystallized
from EtOH/i-Pr2O to give 12c (144 mg, 0.28 mmol, 51% yield) as
1
a white solid: mp 149-151 °C. MS m/z 475 (M+, 44). H NMR
(CDCl3): δ 1.63-1.72 (m, 2H, CH2), 1.89-2.00 (m, 2H, CH2),
2.15-2.28 (m, 2H, CH2), 2.75-2.92 (m, 4H, CH2CH2), 3.05-3.38
(m, 4H, CH2, CH2N), 3.46-3.64 (m, 2H, CH2), 3.74 (s, 3H, OMe),
3.79 (s, 3H, OMe), 3.84 (s, 3H, OMe), 3.98-4.09 (m, 2H, CH2O),
6.41 (s, 1H, Ar), 6.58 (s, 1H, Ar), 6.70-6.93 (m, 5H, Ar),
7.11-7.23 (m, 3H, Ar) ppm. 13C NMR (CDCl3): δ 159.88, 156.10,
150.68, 149.00, 144.15, 130.15, 130.02, 129.20, 127.51, 122.50,
121.11, 120.93, 118.42, 114.67, 111.25, 111.14, 110.05, 109.76,
65.30, 56.29, 56.27, 54.91, 52.70, 52.14, 49.01, 36.52, 32.48, 24.75,
VA
[drug]acceptor
[drug]initial
Papp
)
×
(1)
(
)
(
)
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 the
total transport time in seconds (7200 s), [drug]acceptor is the concent-

7612 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Colabufo et al.
passive diffusion. Thus, a rapid increase in the fluorescence of
cytoplasmic calcein can be monitored. MDR pump, present in the
plasma membrane, rapidly effluxes the calcein-AM before its
entrance into the cytosol, determining a reduction in the fluorescent
signal due to the accumulation of calcein. Evaluation of ABCB1
activity in the presence of pump inhibitors can be performed in a
competitive manner. Compounds that block ABCB1 pump inhibit
Calcein-AM efflux, increasing fluorescent calcein accumulation.
This experiment was carried out as described by Feng et al.29 with
minor modifications. The cells (50000 cells per well) were seeded
into black CulturePlate 96-well plate with 100 µL medium and
allowed to become confluent overnight. Test compounds were
solubilized in 100 µL of culture medium and added to monolayers.
The 96-wells plate was incubated at 37 °C for 30 min. Calcein-
AM was added in 100 µL of phosphate buffered saline (PBS) to
yield a final concentration of 2.5 µM and the plate was incubated
for 30 min. Each well was washed 3 times with ice cold PBS. Saline
buffer was added to each well, and the plate was read to Victor3
fluorometer (PerkinElmer) at excitation and emission wavelengths
of 485 and 535 nm, respectively. In these experimental conditions,
calcein cell accumulation in the absence and in the presence of
tested compounds was evaluated and fluorescence basal level was
estimated by untreated cells. In treated wells, the increase of
fluorescence with respect to basal level was measured. IC50 values
were determined by fitting30 the fluorescence increase percentage
versus log[dose].
ration 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.
Cell ATP Availability Assay. This experiment was performed
as reported in technical sheet of ATPlite Kit for luminescence ATP
detection using Victor3, from PerkinElmer Life Sciences.28 Caco-2
cells were seeded into 96-well microplate in 100 µL of complete
medium at a density 2 × 104 cells/well. The plate was incubated
overnight in a humidified atmosphere 5% CO2 at 37 °C. The
medium was removed and 100 µL of complete medium in the
presence or the absence of different concentrations (from 1 to 100
µM) of tested 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
stirred for 5 min in an orbital shaker. In all wells, 50 µL of substrate
solution was added and the plate stirred for 5 min as above-reported.
The plate was dark adapted for 10 min, and the luminescence was
measured in Victor3, from PerkinElmer Life Sciences.
[3H] Substrate Transport Inhibition. Caco-2 cells were seeded
onto multiscreen plates, 10000 cells/well, for 21 days measuring
the integrity of the cell monolayers by transepithelial electrical
resistance (TEER, Ω × cm2) with an epithelial voltohommeter.
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.26
In each well to basolateral (BL) compartment in the absence
and in the presence of ABCB1 inhibitors (from 200 nM 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 donor compartment to
determine the concentration of radioligand remaining in the donor
chamber at the end of the experiment. Samples were analyzed using
LS6500 Beckman Counter. For each compound, [3H]vinblastine
transport inhibition was calculated as radioactivity difference
between radioligand in the presence and the absence of compound.
These differences were expressed as inhibition percentage at single
drug concentration.
Radioligand Binding Assay at Rat Human Cloned 5-HT1A
Receptor. Human 5-HT1A serotonin receptors stably expressed in
HeLa cells were radiolabeled with 1.0 nM [3H]-8-OH-DPAT (Kd
) 8.8 nM).21 Samples containing 40 µg of membrane protein,
different concentrations of each compound ranging from 0.1 nM
to 10 µM were incubated in a final volume of 500 µL of 50 mM
Tris-HCl, pH 7.4, 5 mM MgSO4 for 120 min at 37 °C. After this
incubation time, samples were filtered through GF/C presoaked in
polyethylenimine 0.5% for at least 30 min prior to use. The filters
were washed twice with 1 mL of ice-cold buffer (50 mM Tris-
HCl, pH 7.4). Nonspecific binding was determined in the presence
of 10 µM 8-OH-DPAT.
[3H] Mithoxantrone Transport Inhibition. Caco-2 cells were
seeded onto multiscreen plates, 10000 cells/well, for 21 days
measuring the integrity of the cell monolayers by transepithelial
electrical resistance (TEER, Ω × cm2) with an epithelial volto-
hommeter. Mature Caco-2 cell monolayer exhibited a TEER > 800
Ω × cm2 prior to use in transport experiments. Transport experi-
ments for tested compounds were carried out as previously
described.14,26
In each well to basolateral (BL) compartment in the absence
and in the presence of ABCB1 inhibitors (100 µM) was added 20
nM [3H]mithoxantrone 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 donor compartment to determine the
concentration of radioligand remaining in the donor chamber at
the end of the experiment. Samples were analyzed using LS6500
Beckman Counter. For each compound, [3H]mithoxantrone transport
inhibition was calculated as radioactivity difference between
radioligand in the presence and absence of compound. These
differences were expressed as percentage of inhibition effect for
each single drug concentration.
Calcein-AM Assay in MDCK-ABCB1 Cells. MDCK-ABCB1
cells (a gift of Prof. P. Borst, NKI-AVL Institute, Amsterdam,
Netherlands) were grown in DMEM high glucose 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. In this assay calcein
accumulation in transfected MDCK cells has been monitored. Intact
cells are incubated with acetoxymethyl (AM) esters of calcein
(Calcein-AM), a lipophilic ABCB1 substrate that diffuses across
the plasma membrane into the cell, where it is hydrolyzed by
endogenous cytoplasmic esterases into highly fluorescent calcein.
The hydrolyzed compound is not a MDR proteins substrate, and
because it is hydrophilic, it cannot cross the cell membrane via
Radioligand Binding Assay at Human Cloned D2 Dopa-
minergic Receptors in C6 Rat Glioma. Binding of [3H]spiro-
peridol at rat cloned D2 receptor was performed according to
Leopoldo et al.22 with minor modifications. The reaction buffer
consisted of 50 mM Tris, 10 mM MgCl2, 1 mM EDTA (pH 7.4),
including 200 µg of dopamine D2 receptor diluted membranes, 0.40
nM [3H]spiroperidol (Kd ) 0.20 nM), and 100 µL of the drug
solution (six to nine concentrations) for a total volume of 1 mL.
Samples were incubated at 27 °C for 60 min, then the incubation
was stopped by rapid filtration through Whatman GF/C glass fiber
filters (presoaked in 0.3% polyethylenimine). The filters were
washed twice with 1 mL of ice-cold buffer (50 mM Tris, pH 7.4).
Nonspecific binding was defined in the presence of 10 µM
haloperidol.
Statistical Analysis. The IC50 values of the compounds reported
in Tables 1, 2, and 4, and Ki values reported in Table 3 were
determined by nonlinear curve fitting utilizing the GraphPad Prism
program.30
The values r2 have been determined by linear regression plotting
the length of the spacer (from 2 to 4 methylene groups) vs pIC50
value for each series calculating the 95% confidence interval of
the regression line (GraphPad Prism program).30
Supporting Information Available: Experimental procedures
for the intermediate compounds, a table reporting the combustion
analysis data of the final products and a table reporting the affinity
binding values toward 5-HT1a and D2 receptors. Flow CytoMetry
(FCM) procedure and the plots for visualizing ABCB1 and ABCG2
in Caco-2 cells and ABCB1 in MDCK-ABCB1 cells. This material
is available free of charge via the Internet at http://pubs.acs.org.

2-[(3-Methoxyphenylethyl)phenoxy]-Based ABCB1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7613
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