Small P-gp modulating molecules: SAR studies on tetrahydroisoquinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2007.09.039

The development of small molecules as P-gp modulating agents and SAR studies on these ligands represented the aim of the present work. A series of 6,7-dimethoxytetrahydroisoquinoline derivatives was prepared and their ability to inhibit P-gp activity has been evaluated. The basic nucleus of these compounds, common to the best P-gp inhibitors such as Tariquidar and Elacridar, has been functionalized with no-basic moiety from our studied sigma receptor ligands displaying potent P-gp inhibition. The best results were obtained for compounds 3c and 3a (EC₅₀ = 1.64 and 4.86 μM, respectively) and these results were remarkable because Elacridar showed in the same biological evaluation similar inhibitory activity (EC₅₀ = 2 μM). SAR studies displayed that the removal of double bond on the spacer or its shifting into tetraline ring decreased the P-gp inhibiting activity. Moreover, the P-gp inhibition mechanism of tested compounds was investigated by three selected biological experiments. The results displayed that only compound 3c was P-gp inhibitor as Elacridar, while compound 3a and reference compounds Cyclosporin A and Verapamil modulated P-gp activity saturating the efflux pump as substrates. Flow cytometry studies carried out in Doxorubicin resistant breast cancer cell line (MCF7/Adr) confirmed that compound 3c increased Doxorubicin cell accumulation 5.7-fold. In addition, in MCF7/Adr, antiproliferative effect of 5 μM Doxorubicin shifted from 5% to 95% when co-administered with compound 3c (20 μM). The present study suggested a new class of small molecules displaying P-gp inhibitor activity differing from reference compounds Elacridar and Tariquidar for a simplified, and in the meantime, efficacious no-basic moiety.

Full text of DOI:10.1016/j.bmc.2007.09.039

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 362–373
Small P-gp modulating molecules:
SAR studies on tetrahydroisoquinoline derivatives
Nicola Antonio Colabufo,a,* Francesco Berardi,a Mariangela Cantore,a
Maria Grazia Perrone,a Marialessandra Contino,a Carmela Inglese,a Mauro Niso,a
Roberto Perrone,a Amalia Azzariti,b Grazia Maria Simone,b
Letizia Porcellib and Angelo Paradisob
a
`
Dipartimento Farmacochimico, Universita degli Studi di Bari, via Orabona, 4, 70125 Bari, Italy
bNational Cancer Institute Giovanni Paolo II, Via Hahnemann, 10, 70126 Bari, Italy
Received 23 May 2007; revised 11 September 2007; accepted 19 September 2007
Available online 25 September 2007
Abstract—The development of small molecules as P-gp modulating agents and SAR studies on these ligands represented the aim of
the present work. A series of 6,7-dimethoxytetrahydroisoquinoline derivatives was prepared and their ability to inhibit P-gp activity
has been evaluated. The basic nucleus of these compounds, common to the best P-gp inhibitors such as Tariquidar and Elacridar,
has been functionalized with no-basic moiety from our studied sigma receptor ligands displaying potent P-gp inhibition. The best
results were obtained for compounds 3c and 3a (EC50 = 1.64 and 4.86 lM, respectively) and these results were remarkable because
Elacridar showed in the same biological evaluation similar inhibitory activity (EC50 = 2 lM). SAR studies displayed that the
removal of double bond on the spacer or its shifting into tetraline ring decreased the P-gp inhibiting activity. Moreover, the P-
gp inhibition mechanism of tested compounds was investigated by three selected biological experiments. The results displayed that
only compound 3c was P-gp inhibitor as Elacridar, while compound 3a and reference compounds Cyclosporin A and Verapamil
modulated P-gp activity saturating the efflux pump as substrates. Flow cytometry studies carried out in Doxorubicin resistant breast
cancer cell line (MCF7/Adr) confirmed that compound 3c increased Doxorubicin cell accumulation 5.7-fold. In addition, in MCF7/
Adr, antiproliferative effect of 5 lM Doxorubicin shifted from 5% to 95% when co-administered with compound 3c (20 lM). The
present study suggested a new class of small molecules displaying P-gp inhibitor activity differing from reference compounds Elacr-
idar and Tariquidar for a simplified, and in the meantime, efficacious no-basic moiety.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
some.4 In humans, 49 ABC genes that are organized into
seven subfamilies (A–G) have been described.5 Many
ABC genes are involved in human genetic diseases6–9
and other ABC genes display an important role in mul-
tidrug resistance (MDR).10–13 The most supported
mechanism of MDR is the overexpression of ABC
transporters localized in the cell membrane, which deter-
mines this pharmacological effect by effluxing a variety
of chemotherapeutic drugs from tumor cells.14,15 The
mostly involved ABC transporter in MDR is ABCB1
(ABCB subfamily) well known as P-glycoprotein (P-
gp).16 P-gp is localized in several biological compart-
ments such as on the apical membrane of hepatocytes,
on the mucosal cells in the gastrointestinal tract, on
the apical lumen of gastrointestinal enterocytes, on the
canalicular membrane of hepatocytes, the proximal
tubular cells of the kidney, and the luminal membrane
of brain capillary endothelial cells.17–20 P-gp both mod-
ulates the efflux of many structurally different drugs and
The ATP-binding cassette (ABC) transporters represent
the largest family of transmembrane (TM) proteins.1,2
These proteins bind ATP and use the hydrolysis energy
to extrude various molecules across all cell membranes.
In bacteria, these transporters are predominantly in-
volved in the import of essential compounds that cannot
be obtained by diffusion (sugars, vitamins, metal ions,
etc.) into the cell.3,4 In eukaryotes, most ABC pumps
transport compounds from the cytoplasm to the outside
of the cell or into an intracellular compartment such as
endoplasmic reticulum (ER), mitochondria, and peroxi-
Keywords: P-Glycoprotein; P-gp inhibitors; Tetrahydroisoquinoline
derivatives; Caco-2 cells; Vinblastine transport; MCF-7/Adr cells.
*
Corresponding author. Tel.: +39 080 5442727; fax: +39 080
5442231; e-mail: colabufo@farmchim.uniba.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.039

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
363
controls their intestinal absorption.21 Other ABC trans-
First generation
O
porters related to MDR are ABCC1-7 proteins (ABCC
subfamily) well known as MRP1-7 (multidrug resistance
protein) and ABCG2 (ABCG subfamily) known as
BCRP (Breast Cancer Resistance Protein).16,22 During
the treatment of human cancer with chemotherapeutic
agents, MDR is frequently observed and P-gp has been
the first ABC transporter investigated for resistance
induction.23,24 Traditional P-gp substrates include, e.g.,
cytotoxic agents, anthracyclines (Doxorubicin, Dauno-
rubicin), vinca alkaloids (Vinblastine and Vincristine),
taxanes (Paclitaxel, Docetaxel), colchicines, epipodo-
phyllotoxins (Etoposide, Teniposide), and also some no-
vel antitumor drugs, such as Imatinib, ET-743, and
Gentuzumab are effluxed out of tumor cells by P-gp
activity.25–28
O
O
O
N
N
O
Verapamil
Tamoxifen
Second generation
O
O
O
O
O
O
N
N
O
N
Biricodar
O
Many drugs have been investigated in the last years for
modulating the P-gp transport and among them a cal-
cium channel blocker such as Verapamil and the antis-
teroid Tamoxifen (Fig. 1).29–31 These compound, were
not specifically developed to block P-gp since many of
them were substrates for other transporters and enzyme
system, resulting in unpredictable pharmacokinetic
interaction.24,31 Furthermore, most of these inhibitors
presented low affinity for P-gp and were administered
in high doses which displayed unacceptably high level
of toxicity that, in turn, caused severe side effects.32
O
O
H
N
H
N
O
N
N
N
N
O
O
O
O
O
O
H
N
N
N
O
N
H
N
H
O
Valspodar
Third generation
O
O
The second generation of P-gp modulators has been
developed and the mostly studied compounds are Biric-
odar, Valspodar (PSC-833)32–34 (Fig. 1), dexverapamil,
N
O
O
O
N
H
dexniguldipine
(SR33557),
and
triazinoaminopiperidine derivative
indolizilsulfone
derivative
NH
a
O
(S9788). These compounds are more potent and less
toxic than first generation compounds but some limita-
tions have been evidenced.35 For example, Biricodar
and PSC-833 inhibited cytochrome P450A4-mediated
metabolism of paclitaxel, while vinblastine has made it
difficult to establish a safe but efficient dose of co-
administered chemotherapy agents, which has led to
the restricted use of this second generation of modula-
tors in the treatment of MDR cancers.36,37
N
Tariquidar
O
O
N
O
O
N
H
N
H
O
Elacridar
F
F
The third generation of P-gp modulators includes Zos-
uquidar38,39 (Fig. 1) and other compounds bearing
dimethoxytetrahydroisoquinoline moiety such as Elacri-
dar (Phase I)40,41 and Tariquidar (Phase III).42–45 These
inhibitors have displayed no pharmacokinetic interaction
with chemotherapeutic drugs and showed high potency
and specificity for P-gp. Nevertheless, the results of clini-
cal trials on Tariquidar showed that it displayed limited
clinical activity to restore sensitivity to anthracycline or
taxane chemotherapy.44 Moreover, in these complex mol-
ecules, structure–activity relationship (SAR) studies are
not so easy to carry out and to date the contribution of
each portion to reverse MDR is not clarified.46
H
H
N
N
N
O
OH
LY-335979
Figure 1. P-gp modulators.
The development of small molecules as P-gp modulating
agents and SAR studies on these ligands similarly repre-
sented our target of the present work. Recently, we stud-
ied PB28 (Fig. 2), a cyclohexylpiperazine derivative
Obviously, these studies can be easily performed starting
from small molecules. Indeed, in our previous work, we
carried out SAR studies on a new class of arylmethyl-
oxyphenyl derivatives (Fig. 2, formula I) as small mole-
cules displaying P-gp inhibition activity.47
displaying
a potent P-gp modulating activity in
MCF7/Adr cells.48,49 In order to improve its P-gp mod-
ulating activity, we replaced cyclohexylpiperazine with a

364
N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
Cl
O
R1
R1
R2
N
A
B
3a,c
4
N
O
O
R2
R3
C
R3
PB28
1a-d
2a,c
R
O
O
Ar
N
R3
R2
D
R1
Br
Br
I
II
R1
R2
R1
R2
E
Figure 2. PB28 and general formula of synthesized compounds.
R3
7a-d
R3
dimethoxytetrahydroisoquinoline moiety (Fig. 2, for-
mula II) such as in Tariquidar and Elacridar where it
is functionalized with a bulking and planar substituent.
R2
R3
R1
a: OCH3
5a-d
H
OCH3
H
H
H
OCH3
H
b:
c:
H
H
B
B
d: OCH3 OCH3
The set of compounds presented in this work was charac-
terized by 6,7-dimethoxytetrahydroisoquinoline nucleus
bearing as substituent the same no-basic portion of PB28.
6a-d
8a-d
Cl
O
Since this portion can be easily modified, we investigated
the conformational restriction by inserting a double
bond in the spacer and into tetraline ring. Aims of the
present work were to investigate the effect of the replace-
ment of basic nucleus of PB28 with 6,7-dimethoxytetra-
hydroisoquinoline on MDR activity and also the
influence of the modifications on the no-basic portion
of these compounds.
D
B
11
10
9
Scheme 1. Reagents: (A) cyclopropylMgBr, 3 N HCl; (B) 6,7-dimeth-
oxytetrahydroisoquinoline; (C) piperidine; (D) cyclopropylMgBr, 48%
HBr; (E) H2/Pd (5%).
2. Results and discussion
2.1. Chemistry
for compounds 3c and 3a bearing a (E)-double bond on
the spacer (EC50 = 1.64 and 4.86 lM, respectively). The
corresponding saturated derivatives 8c and 8a displayed
EC50 = 6.35 and 10.2 lM, respectively. Compounds 6c
and 6a having a double bond in tetraline ring showed with
respect to compounds 3c and 3a high decrease of P-gp
inhibition activity (EC50 = 15 and 35.5 lM, respectively).
The preparation of the final compounds 3a, 3c, 4, 6a–d,
8a–d, and 11 is depicted in Scheme 1. The key interme-
diates E-2a, E-2c, 5a–d, and 10 were prepared by Grig-
nard’s reaction starting from the appropriate ketone
(1a–d for intermediates E-2a, E-2c, and 5a–d, while 9
for intermediate 10). Key intermediates 2a, 2c, and 10
have been obtained by kinetic control that failed starting
from tetralones 1b, 1d. The preparation of intermediate
5a–c, E-2c has been previously reported.50–52
The position of methoxy substituent influenced the
activity. In fact, the best results were obtained for com-
pounds 3c, 8c, and 6c for each studied series where
R1 = R2 = H and R3 = OCH3. The shifting of methoxy
in other positions of tetraline ring decreased the activity
in all series (3c vs 3a, 6c vs 6a, and 6b, 8c vs 8a and 8b).
The dimethoxy derivatives 6d and 8d displayed moder-
ate P-gp inhibition activity (EC50 = 31.6 and 17.9 lM,
respectively). These findings led to consider compound
3c as lead compound and starting from it we prepared
compound 4 where the aromatic portion on the basic
tetrahydroisoquinoline moiety has been removed. The
results displayed that this modification moderately de-
creased P-gp inhibition activity with respect to com-
pound 3c (EC50 = 8.2 vs 1.64 lM). Moreover, from 3c,
compound 11 has been prepared where the aromatic
portion of the tetraline ring was removed. The results
displayed that this modification decreased the inhibition
activity ten fold with respect to lead compound 3c
(EC50 = 15.2 vs 1.64 lM).
The intermediates 7a–d have been obtained by catalyti-
cally reducing double bond of corresponding unsatu-
rated derivatives 5a–d under hydrogen atmosphere.50,52
Final compounds 3a, 3c, 6a–d, 8a–d, and 11 were ob-
tained by alkylating commercially available amine 6,7-
dimethoxytetrahydroisoquinoline. Final compound 4
was prepared by reaction between intermediate E-2c
and piperidine.
2.2. Structure–activity relationships studies
For all compounds the ability to inhibit [3H]vinblastine
transport in Caco-2 cell monolayer has been determined.
With respect to PB28 displaying EC50 = 0.55 lM, all
tested compounds 3a, 3c, 4, 6a–d, 8a–d, and 11 were less
potent. The best results were found, as listed in Table 2,
Nevertheless lead compound 3c was found to be two
fold less potent than PB28, it displayed higher P-gp inhi-

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
365
bition activity than reference compounds Cyclosporin A
and Verapamil (EC50 = 80 and 20 lM, respectively). In
addition, compound 3c showed the same P-gp inhibition
value as that of Elacridar (EC50 = 2.0 lM) one of the
most studied P-gp inhibitors.
[3H]vinblastine. In addition, a substrate could be trans-
ported, it could activate ATPase but it could be unable
to compete with [3H]vinblastine (category IIB2) such as
Taxol. Cyclosporin A is a substrate example of category
IIB3, where the substrate is transported and competes
with [3H]vinblastine but it is unable to activate
ATPase.
2.3. Pharmacology
The target compounds 3a, 3c, 4, 6a–d, 8a–d, and 11 as
hydrochloride salts were evaluated for determining the
Apparent Permeability (Papp) both in basolateral–apical
(B–A) and apical–basolateral (A–B) transports.47,53
Monolayer Caco-2 cells grown on permeable filters ex-
press human P-gp and are commonly employed to char-
acterize P-gp substrate. In this assay, substrates
generally displayed BA/AB ratio > 2 while nonsubstrate
or inhibitors displayed BA/AB < 2.54 Moreover, the
ATPase activation monitored by ATP cell depletion
due to each compound has been evaluated. In this assay,
only substrates caused ATP cell depletion. The cell ATP
was unchanged for treating in the presence of inhibitor
and nonsubstrate.55 Furthermore, specific radiolabeled
P-gp substrate [3H]vinblastine has been employed and
in this assay both substrate and inhibitor displayed
interacting effect with the radioligand transport, while
nonsubstrates are unable to compete with radiolabeled
substrate.56,57 By comparing the results of these three as-
says, two categories of P-gp ligands are elicited (Table
1). In category I, unambiguous nonsubstrates, unambig-
uous substrates, and inhibitors are listed. A compound
is defined as nonsubstrate when it is non transported,
ATPase is not activated by it, and it is unable to modu-
late [3H]vinblastine transport. A substrate is trans-
ported, it activates ATPase, and displays competition
toward [3H]vinblastine transport. Finally, an inhibitor
differs from a nonsubstrate because it competes with
radioligand substrate transport. This experimental sub-
classification is an efficacy characterization for an inhib-
itor and a nonsubstrate but it is limitative for substrate
identification. Actually, the substrate characterization
presents several deviations by typical substrate defini-
tion as reported in category I. In fact, a substrate could
be a non transported substrate (category IIA) such as
Verapamil, Ketoconazole, and Nifedipine, if it is non
transported. Moreover, it could be classified as a
substrate of category IIB1, such as Daunorubicin, if it
is unable both to activate ATPase and to compete with
2.4. P-gp inhibition mechanism investigation
In monolayer efflux assay the Apparent Permeability
(Papp) both in basolateral–apical (Papp, B–A) and api-
cal–basolateral (Papp, A–B) directions was determined
for each compound. The BA/AB ratio is <2 for com-
pounds 3c and 6d and reference compound Verapamil
(BA/AB = 1.6, 1.5, and 1.2, respectively) while the other
compounds displayed BA/AB > 3.1.
As regards ATPase activation, several compounds were
unable to deplete ATP (3a, 3c, 4, 6b, 11). All other com-
pounds activated ATPase pump at 100 lM with differ-
ent percentage as displayed in Table 2. Moreover, all
compounds inhibited [3H]vinblastine transport as re-
ported in Section 2.2. Combining the results of these
three biological assays, only compound 3c and reference
compound Elacridar are ‘‘claimed’’ P-gp inhibitors. On
the contrary, tetraline derivatives 8a–d were unambigu-
ous substrates. All the other compounds were substrates
but differently characterized: 3a, 4, 6b, 11 were trans-
ported substrates Cyclosporin A–like, while 6d was a
nontransported substrate as Verapamil.
As data standing, we better characterized, by flow
cytometry, the compounds 3c, 3a and 8c and reference
compounds Cyclosporin A and Verapamil displaying
the best P-gp modulating activity although with a differ-
ent mechanism.
As depicted in Figure 3a–e, in MCF7/Adr cells, over-
expressing P-gp, the simultaneous treatment with Doxo-
rubicin and each compound increased Doxorubicin
accumulation, as shown by 20 lM compound 3c
(Fig. 3b) which increased Doxorubicin accumulation
5.7-fold, in the cells (Table 3). In the same flow cytom-
etry experiment, compounds 3a (Fig. 3a) and 8c
(Fig. 3c) displayed an increased Doxorubicin cell accu-
mulation of 4.1- and 4.8-fold, respectively (Table 3).
Table 1. Evaluation of drug interaction with P-glycoprotein by three specific biological assays54
Categories of P-gp ligands
Methods of measurements in Caco-2 cell line
Monolayer efflux
ATPase activation
[3H]-Substrate transport inhibition
Category I
Unambiguous nonsubstrates
Unambiguous substrates
Inhibitors
No
Yes
No
No
Yes
No
No
Yes
Yes
Category II
IIA
IIB1
Nontransported substrates
Transported substrates
Transported substrates
Transported substrates
No
Yes
No
Yes
No
Yes
No
No
Yes
Yes
Yes
Yes
IIB2
IIB3

366
N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
Table 2. Biological in vitro assays
R1
OCH3
H
R2
R3
H
O
O
Y:
a:
b:
c:
d:
H
OCH3
H
X:
N
N
H
H
OCH3
OCH3
H
OCH3
c
Papp (B–A)
c
Papp (A–B)
c
Compound
P-gp inhibition
ATPase
activation
Papp
nm/s
nm/s
(BA/AB)
EC50 SEM (lM)a
X
3a
3c
4.86 1.10
1.64 0.20
No
No
6567
386
1663
237
3.9
1.6
R1
R2
R3
Y
4
8.2 0.5
No
2564
584
4.4
O
X
X
6a
6b
6c
6d
35.5 2.5
83.7 5.2
15 2.5
Yes (20)b
No
3316
1506
1140
1030
610
407
160
672
5.4
3.7
7.1
1.5
R1
Yes (30)
Yes (15)
R2
31.6 2.7
R3
R3
8a
8b
8c
8d
10.2 0.80
8.15 0.30
6.35 0.25
17.9 1.5
Yes (15)
Yes (27)
Yes (70)
Yes (62)
1703
3510
1038
4399
460
828
56
3.7
4.2
18
R1
R2
868
5.1
X
11
15.2 1.5
No
2403
774
3.1
PB28
0.55 0.02
80 7.5
20 1.0
2.0
No
No
4358
153d
687d
2496
15.9d
591d
1.7
Cyclosporin A
Verapamil
Elacridar
9.6d
1.2d
<2d
Yes (52)
Nod
a Data SEM are the mean of three independent determinations (samples in triplicate).
b Percentage activation at 100 lM shown given in parentheses. Data are the mean of three independent determinations with samples in triplicate.
c Data are the mean of three independent determinations (samples in triplicate) each with SEM <10%.
d See Ref. 54.
At 20 lM the reference compounds Cyclosporin A
(Fig. 3d) and Verapamil (Fig. 3e) displayed an increase
of Doxorubicin cell accumulation of 5- and 3.3-fold,
respectively.
increased Doxorubicin accumulation; i.e. 20 lM com-
pound 3c together with Doxorubicin inhibited cell
growth of about 95% . Moreover, high antiproliferative
effect was induced by Doxorubicin in the presence of
compounds 3a and 8c and Cyclosporin A (77%, 85%
and 83%, respectively). In the presence of Verapamil,
Doxorubicin displayed moderate increase of antiprolif-
erative effect (50%).
In graphs (Fig. 3a–e) this accumulation can be appreci-
ated by the shift to right of Doxorubicin curve. The fol-
lowing step was to verify if Doxorubicin cell
accumulation, induced by our compounds, improved
the antiproliferative effect of the chemotherapeutic. In
fact, at 5 lM, Doxorubicin itself displayed a low anti-
proliferative effect (5%) as listed in Table 3.
Moreover, compounds 3a, 3c, and 8c were studied in the
absence of Doxorubicin for the cytotoxicity and anti-
proliferative effects at 24 and 48 h, respectively. The
cytotoxicity of tested compounds at 100 lM, determined
by LDH measurement in cell medium after treatment,48
was negligible (< 20% for each studied compound).
Analogously, in the antiproliferative assay48 at 48 h
The results of Doxorubicin antiproliferative effect as
drugs combination with studied compounds are shown
in Table 3. The results are well correlate with the

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
367
Figure 3. Flow cytometry analysis with MCF7/Adr cells. Autofluorescence (black), 50 lM Doxorubicin (red), 20 lM of tested compounds in the
presence of 50 lM Doxorubicin (blue). (a) Compound 3a. (b) Compound 3c. (c) Compound 8c. (d) Cyclosporin A. (e) Verapamil.
Table 3. Capability of the newly synthesized P-gp modulating agents to increase Doxorubicin intracellular accumulation in MCF7/Adr cell line (left
column)
Compound
-Folds of increased 50 lM
Antiproliferative effectb (%)
Doxorubicin intracellular accumulationa
(5 lM Doxorubicin)
Doxorubicin
1
5
1.0
Inhibitor
20 lM compound 3a + Doxorubicin
20 lM compound 3 c + Doxorubicin
20 lM compound 8c + Doxorubicin
20 lM Cyclosporin A + Doxorubicin
20 lM Verapamil + Doxorubicin
4.1 1.2
5.7 1.0
4.8 0.5
5.0 0.7
3.3 0.8
77 2.3
95 1.8
85 2.1
83 2.0
50 2.5
Doxorubicin antiproliferative effect of drugs combination (right column).
a Data SEM are the mean of three independent determinations samples in triplicate.
b Data are the mean of three independent determinations samples in triplicate.

368
N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
the tested compounds, at 100 lM, displayed low effect.
Only compound 3c showed at this concentration 25%
of antiproliferative effect while compounds 3a and 8c
displayed an effect < 20%.
compounds E-2c, 5a, 5c, 7a–c has been reported in our
previous works.50–52
4.2. General procedure for the synthesis of amines 3a, 3c,
4, 6a–d, 8a–d, and 11
These findings demonstrated that the drugs combina-
tion, Doxorubicin with newly synthesized P-gp modulat-
ing agents, improved Doxorubicin antiproliferative
effect in MCF7/Adr tumor cells.
A mixture of haloalkylderivatives E-2a, 2c or 5a–d or
7a–d or 10 (0.50 mmol) and amine, 6,7-dimethoxytetra-
hydroisoquinoline or piperidine (1.2 mmol), and anhy-
drous Na2CO3 (0.50 mmol) in 20 mL DMF was stirred
overnight. The solvent was evaporated and the residue
was partitioned between H2O (20 mL) and CHCl3
(30 mL). The organic layer was dried (Na2SO4) and con-
centrated in vacuo. The crude residue was chromato-
graphed as following reported for each final compound.
3. Conclusions
In this paper, we identified among several newly synthe-
sized compounds derivative 3c as a P-gp modulating
agent. It inhibited the drug efflux pump and strongly en-
hanced anthracyclines’ effectiveness. This finding
encouraged us to patent this pharmaceutical class as
new small molecules with P-gp modulating activity.58
4.3. 6,7-Dimethoxy-2-{3-[7-methoxy-3,4-dihydro-2H-
naphthalen-(1E)-ylidene]-propyl}-1,2,3,4-tetrahydro-iso-
quinoline (3a)
Another aspect of the evaluation of P-gp modulating
agents is the mechanism involved in the pump efflux
inhibition and it has been investigated combining
three biological assays. The mechanism of P-gp inhi-
bition is a critical pharmacokinetic step. In fact at
high concentrations both substrate and inhibitor effi-
caciously blocked pump efflux but at low concentra-
tions only inhibitor displayed this effect.59 In the
future we will perform the full biological characteriza-
tion of these compounds, determining their selectivity
toward other ABC transporters involved in MDR and
identifying other hypothetic targets with which they
could interfere, by expression microarray technologies.
Moreover, on the basis of compound 3c, other semi-
rigid congeners might be prepared in order to investi-
gate the requirements for obtaining compounds acting
as P-gp inhibitors.
Yellow oil, 90% yield from column chromatography
(eluent CHCl3/MeOH 19:1). GC–MS m/z 394 (M+ + 1,
1
4), 393 (M+ 11), 206 (100), 165 (6). H NMR d 1.78–
1.85 (m, 2H, CH2CH2CH2), 2.50–2.89 (mm, 12H,
CH2CH2CH2,
NCH2CH2
tetrahydroisoquinoline
CHCH2CH2N), 3.49–3.88 (mm, 11H, NCH2 tetrahy-
droisoquinoline, 9H, CH3), 6.00 (t, 1H, J = 7 Hz,
C@CH), 6.50–7.09 (m, 5H aromatic). UV–vis:
k
max = 260 nm; e = 2235 Mꢀ1 cmꢀ1 (PBS). Anal.
(C25H31NO3ÆHCl) C, H, N (hydrochloride salt melted
at 232–235 ꢁC).
4.4. 6,7-Dimethoxy-2-{3-[4-methoxy-3,4-dihydro-2H-
naphthalen-(1E)-ylidene]-propyl}-1,2,3,4-tetrahydro-iso-
quinoline (3c)
Yellow oil, 58% yield from column chromatography
(eluent CHCl3/MeOH 19:1). GC–MS m/z 395 (M+ + 2,
1), 394 (M+ + 1, 3), 393 (M+ 9), 207 (16), 206 (100).
1H NMR d 1.79–1.87 (m, 2H, CH2CH2CH2CH), 2.47–
2.93 (mm, 12H, CH2CH2CH2CH, NCH2CH2 tetrahy-
droisoquinoline, CHCH2CH2N), 3.63 (s, 2H, NCH2 tet-
rahydroisoquinoline), 3.82–3.87 (3s, 9H, CH3), 6.03 (t,
1H, J = 7 Hz, C@CH), 6.60–7.26 (m, 5H, aromatic).
4. Experimental
4.1. Chemistry
Column chromatography was performed with 1:30
˚
Merck silica gel 60A (63–200 lm) as the stationary
UV–vis:
k
max = 260 nm; e = 8300 Mꢀ1 cmꢀ1 (PBS).
phase. Melting points were determined in open capillar-
ies on a Gallenkamp electrothermal apparatus. Elemen-
tal analyses (C, H, N) were performed on Eurovector
Euro EA 3000 analyzer; the analytical results were with-
in 0.4% of the theoretical values for the formula given.
1H NMR spectra were recorded in CDCl3 at 300 MHz
on a Varian Mercury-VX spectrometer. All spectra were
recorded on the free bases. All chemical shift values are
reported in ppm (d). Recording of mass spectra was
done on an HP6890-5973 MSD gas chromatograph/
mass spectrometer; only significant m/z peaks, with their
percentage of relative intensity in parentheses, are re-
ported. All spectra were in accordance with the assigned
structures. ESI-MS analyses were performed on an Agi-
lent 1100 LC/MSD trap system VL. The UV/vis spectra
were recorded with LAMBDA BIO20 spectrophotome-
ter PerkinElmer. The spectroscopic characterization of
Anal. (C25H31NO3ÆHCl) C, H, N (hydrochloride salt
melted at 237–240 ꢁC).
4.5. 1-{3-[4-Methoxy-3,4-dihydro-2H-naphthalen-(1E)-
ylidene]-propyl}-piperidine (4)
White solid, 88% yield from column chromatography
(eluent CH2Cl2/MeOH 19:1). GC–MS m/z 286
(M+ + 1, 1), 285 (M+ 1), 98 (100). 1H NMR d
1.25–1.85 (m, 10H,
3
of CH2 piperidine,
CH2CH2CH2C tetraline), 2.43–2.47 (m, 8H, 2 of
CH2 piperidine, CHCH2CH2N, CH2CH2CH2C tetra-
line), 2.70 (t, 2H, J = 7.0 CH2CH2CH2C tetraline),
3.81 (s, 3H, CH3), 6.67–7.19 (m, 3H, aromatic).
UV–vis: kmax = 258 nm; e = 12440 Mꢀ1 cmꢀ1 (PBS).
Anal. (C19H27NOÆHCl) C, H, N (hydrochloride salt
melted at 203–205 ꢁC).

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
369
4.6. 6,7-Dimethoxy-2-[3-(7-methoxy-3,4-dihydro-naph-
thalen-1-yl)-propyl]-1,2,3,4-tetrahydro-isoquinoline (6a)
4.10. 6,7-Dimethoxy-2-[3-(7-methoxy-1,2,3,4-tetrahydro-
naphthalen-1-yl)-propyl]-1,2,3,4-tetrahydro-isoquinoline
(8a)
Yellow oil, 53% yield from column chromatography
(eluent CH2Cl2/EtAc 7:3). ESI+/MS m/z 394 (MH+).
GC–MS m/z 395 (M+ + 2, 4), 394 (M+ + 1, 22), 393
(M+ 79), 206 (100), 191 (60), 164 (32). 1H NMR d
1.82–1.90 (m, 2H, CH2CH2CH2N), 2.19–2.25 (m, 2H,
CH2CH2CH@C), 2.46–2.85 (m, 10H, CH2CH2CH@C,
CH2CH2CH2N, NCH2CH2 tetrahydroisoquinoline),
3.57 (s, 2H, NCH2 tetrahydroisoquinoline), 3.82–3.90
(s, 9H, CH3), 5.89 (t, 1H, C@CH, J = 5 Hz), 6.51–7.06
(m, 5H, aromatic). Anal. (C25H31NO3ÆHCl) C, H, N
(hydrochloride salt melted at 212–216 ꢁC).
Yellow oil, 63% yield from column chromatography
(eluent CH2Cl2/EtAc 7:3). ESI+/MS m/z 392 (MH+).
GC–MS m/z 397 (M+ + 2, 2), 396 (M+ + 1, 10),
1
395 (M+ 37), 206 (100), 193 (25), 192 (54). H NMR
d 1.58–1.90 (m, 8H, CH2CH2CH2CH, CH2CH2CH2N),
2.56–2.84 (m, 9H, CH2CH2CH2CH, CH2CH2CH2N,
NCH2CH2 tetrahydroisoquinoline), 3.60 (s, 2H,
NCH2 tetrahydroisoquinoline), 3.76–3.83 (s, 9H,
CH3), 6.52–6.99 (m, 5H, aromatic). Anal. (C25H33NO3
ÆHCl) C, H, N (hydrochloride salt melted at 205–
207 ꢁC).
4.7. 6,7-Dimethoxy-2-[3-(6-methoxy-3,4-dihydro-naph-
thalen-1-yl)-propyl]-1,2,3,4-tetrahydro-isoquinoline (6b)
4.11. 6,7-Dimethoxy-2-[3-(6-methoxy-1,2,3,4-tetrahydro-
naphthalen-1-yl)-propyl]-1,2,3,4-tetrahydro-isoquinoline
(8b)
Yellow oil, 55% yield from column chromatography
(eluent CH2Cl2/EtAc 7:3). GC–MS m/z 395 (M+ + 2,
3), 394 (M+ + 1, 13), 393 (M+ 47), 218 (62), 206 (100),
191 (47). 1H NMR d 1.83–1.88 (m, 2H, CH2CH2CH2N),
2.19–2.26 (m, 2H, CH2CH2CH@C), 2.50–2.82 (m, 10H,
CH2CH2CH@C, CH2CH2CH2N, NCH2CH2 tetrahy-
droisoquinoline), 3.56 (s, 2H, NCH2 tetrahydroisoquin-
oline), 3.79–3.83 (s, 9H, CH3), 5.75 (t, 1H, C@CH,
J = 6.2 Hz), 6.50–7.21 (m, 5H, aromatic); UV–vis:
Yellow oil, 55% yield from column chromatography
(eluent CH2Cl2/EtAc 7:3). ESI+/MS m/z 396 (MH+).
GC–MS m/z 397 (M+ + 2, 3), 396 (M+ + 1, 17), 395
(M+ 60), 206 (100), 192 (41), 164 (30). 1H NMR d
1.57–1.91 (m, 8H, CH2CH2CH2CH, CH2CH2CH2N),
2.54–2.85 (m, 9H, CH2CH2CH2CH, CH2CH2CH2N,
NCH2CH2 tetrahydroisoquinoline), 3.58 (s, 2H,
NCH2 tetrahydroisoquinoline), 3.76–3.84 (s, 9H,
k
max = 280 nm; e = 10119 Mꢀ1 cmꢀ1 (PBS). Anal.
(C25H31NO3ÆHCl) C, H, N (hydrochloride salt melted
at 205–208 ꢁC).
CH3),
6.51–7.11
(m,
5H,
aromatic).
Anal.
(C25H33NO3ÆHCl) C, H, N (hydrochloride salt melted
at 219–223 ꢁC).
4.8. 6,7-Dimethoxy-2-[3-(4-methoxy-3,4-dihydro-naph-
thalen-1-yl)-propyl]-1,2,3,4-tetrahydro-isoquinoline (6c)
4.12. 6,7-Dimethoxy-2-[3-(5-methoxy-1,2,3,4-tetrahydro-
naphthalen-1-yl)-propyl]-1,2,3,4-tetrahydro-isoquinoline
(8c)
Yellow oil, 90% yield from column chromatography
(eluent CH2Cl2/MeOH 19:1). ESI+/MS m/z 392
(MH+). GC–MS m/z 394 (M+ + 1, 20), 393 (M+ 70),
392 (53), 218 (86), 219 (42), 206 (100). 1H NMR d
1.80–1.85 (m, 2H, CH2CH2CH2N), 2.20–2.24 (m, 2H,
CH2CH2CH@C), 2.47–2.84 (m, 10H, CH2CH2CH@C,
CH2CH2CH2N, NCH2CH2 tetrahydroisoquinoline),
3.58 (s, 2H, NCH2 tetrahydroisoquinoline), 3.82 (s,
9H, CH3), 5.89 (t, 1H, C@CH, J = 4 Hz), 6.50–7.26
(m, 5H, aromatic). Anal. (C25H31NO3ÆHCl) C, H, N
(hydrochloride salt melted at 227–229 ꢁC).
Yellow oil, 77% yield from column chromatography
(eluent CH2Cl2/MeOH 19:1). ESI+/MS m/z 396
(MH+). GC–MS m/z 397 (M+ + 2, 2), 396 (M+ + 1,
10), 395 (M+ 39), 206 (100). H NMR d 1.64-1.78 (m,
1
8H, CH2CH2CH2CH, CH2CH2CH2N), 2.54–2.84 (m,
9H, CH2CH2CH2CH, CH2CH2CH2N, NCH2CH2 tetra-
hydroisoquinoline), 3.58 (s, 2H, NCH2 tetrahydroiso-
quinoline), 3.80-3.84 (s, 9H, CH3), 6.52–7.26 (m, 7H,
aromatic). Anal. (C25H33NO3ÆHCl) C, H, N (hydrochlo-
ride salt melted at 207–211 ꢁC).
4.9. 2-[3-(6,7-Dimethoxy-3,4-dihydro-naphthalen-1-yl)-
propyl]-6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline
(6d)
4.13. 2-[3-(6,7-Dimethoxy-1,2,3,4-tetrahydro-naphthalen-
1-yl)-propyl]-6,7-dimethoxy-1,2,3,4-tetrahydro-isoquino-
line (8d)
Yellow oil, 83% yield from column chromatography
(eluent CH2Cl2/MeOH 19:1). GC–MS m/z 425
(M+ + 2, 4), 424 (M+ + 1, 14), 423 (M+ 39), 218 (69),
206 (100), 191 (44), 164 (19). 1H NMR d 1.84–1.94
(m, 2H, CH2CH2CH2N), 2.18–2.26 (m, 2H,
CH2CH2CH@C), 2.46–2.84 (m, 10H, CH2CH2CH@C,
CH2CH2CH2N, NCH2CH2 tetrahydroisoquinoline),
3.60 (s, 2H, NCH2 tetrahydroisoquinoline), 3.82–3.88
(s, 12H, CH3), 5.78 (t, 1H, C@CH, J = 4 Hz), 6.50-6.83
Yellow oil, 55% yield from column chromatography
(eluent CH2Cl2/MeOH 19:1). ESI+/MS m/z 426
(MH+). GC–MS m/z 427 (M+ + 2, 4), 426 (M+ + 1,
16), 425 (M+ 47), 206 (100), 192 (58), 164 (30). 1H
NMR
d
1.53–1.89 (m, 8H, CH2CH2CH2CH,
CH2CH2CH2N), 2.55–2.85 (m, 9H, CH2CH2CH2CH,
CH2CH2CH2N, NCH2CH2 tetrahydroisoquinoline),
3.61 (s, 2H, NCH2 tetrahydroisoquinoline), 3.82–3.90
(s, 12H, CH3), 6.50–7.76 (m, 4H, aromatic). Anal.
(C26H35NO4ÆHCl) C, H, N (hydrochloride salt melted
at 212–215 ꢁC).
(m, 4H, aromatic); UV–vis:
kmax = 280 nm;
e = 9435 Mꢀ1 cmꢀ1 (PBS). Anal. (C26H33NO4ÆHCl) C,
H, N (hydrochloride salt melted at 219–224 ꢁC).

370
N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
4.14. 2-(3-Cyclohexylidene-propyl)-6,7-dimethoxy-
1,2,3,4-tetrahydro-isoquinoline (11)
was removed and drug solutions added to the filter well
(75 lL). HBSS without the drug was added to the recei-
ver plate (250 lL). The plates were incubated at 37 ꢁC
for 120 min. After incubation time, samples were re-
moved from the apical (filter well) and basolateral (recei-
ver plate) side of the monolayer and then were stored in
a freezer (ꢀ20 ꢁC) pending analysis.
Yellow oil, 17% yield from column chromatography
(eluent CH2Cl2/MeOH 19:1). GC–MS m/z 316
1
(M+ + 1, 2), 315 (M+ 10), 206 (100). H NMR d 1.52
(m, 6H, 4 of CH2 cyclohexen), 2.1–2.3 (m, 4H, 2 of
CH2 cyclohexen), 2.32–2.35 (m, 2H, CHCH2CH2N),
2.50–2.54 (m, 2H, NCH2CH2, isoquinoline), 2.75–2.83
(m, 4H, NCH2CH2, isoquinoline and CHCH2CH2N),
3.60 (s, 2H, NCH2Ar, isoquinoline), 3.82 and 3.83 (2s,
6H, 2 of CH3), 5.10 (t, 2H, J = 7.0 Hz CH), 6.51–6.58
Compounds 6a, 6c and 8a–d were lyophilized at ꢀ52 ꢁC
and 0.06 bars for 24 h and desalted by adding 500 lL of
CH3CN (in order to remove phosphate salts coming
from HBSS), then 500 lL of 50 mM of Ammonium
Acetate, pH 5, was added. The resulting solutions were
analyzed by ESI-MS analyses in order to determine the
concentration of drug in the samples. The concentration
of compounds 3a, 3c, 4, 6b and 6d, and 11 was measured
using UV spectroscopy.
(m,
2H,
aromatic).
UV–vis:
kmax = 280 nm;
e = 3238 Mꢀ1 cmꢀ1 (PBS). Anal. (C20H29NO2ÆHCl) C,
H, N (hydrochloride salt dec. at 238–241 ꢁC).
5. Biological methods
5.1. Cell lines
The apparent permeability (Papp), in units of nm per sec-
ond, was calculated using the following equation:
ꢀ
ꢁ
ꢀ
ꢁ
½drugꢂ
V A
The breast cancer cell line of human origin, MCF7/Adr
(resistant to Adriamycin or Doxorubicin), was routinely
cultured in RPMI 1640 supplemented with 10% fetal bo-
vine serum, 2 mM glutamine, 100,000 U/mL penicillin,
and 100 lg/mL streptomycin in a humidified incubator
at 37 ꢁC with a 5 % CO2 atmosphere. Caco-2 cells were
grown in DMEM with 10% heat-inactivated fetal calf
serum, 100 U/mL penicillin, 100 lg/mL streptomycin,
and 2 mM L-glutamine.
acceptor
Papp
¼
ꢁ
Area ꢁ time
½drugꢂ
initial
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 concentration of the drug measured
by ESI-MS analyses or UV spectroscopy; [drug]initial is
the initial drug concentration (1 · 10ꢀ4 M) in the apical
or basolateral wells.
The cells were trypsinized twice a week with trypsin/eth-
ylenediaminetetraacetic acid (EDTA) (0.02%/0.02%)
and the medium was changed twice a week.
5.3. Cell ATP availability assay
This experiment was performed as reported in technical
sheet of ATPlite 1step Kit for luminescence ATP detec-
tion using Victor3, from PerkinElmer Life Sciences.55
Caco-2 cells were seeded into 96-well microplates in
5.2. Permeability experiments
5.2.1. Preparation of Caco-2 monolayer. This procedure
has been previously reported by Leopoldo et al.60 Briefly,
Caco-2 cells were harvested with trypsin-EDTA and
seeded onto MultiScreen Caco-2 assay system at a density
of 10,000 cells per 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 Epi-
thelial Electrical Resistance (TEER) of the monolayers
was measured daily before and after the experiment using
an epithelial voltohommeter (Millicellꢂ-ERS; Millipore,
Billerica, MA). Generally, TEER values obtained are
greater than 1000 X for a 21-day culture.
100 lL of complete medium at
a
density of
2 · 104 cells/well. The plate was incubated O/N in a
humidified atmosphere of 5% CO2 at 37 ꢁC. The med-
ium was removed and 100 lL of complete medium in
the presence or absence of different concentrations rang-
ing from 1 to 100 lM of test compounds was added. The
plate was incubated for 2 h in a humidified atmosphere
of 5% CO2 at 37 ꢁC. Then, 50 lL of mammalian cell ly-
sis solution was added to all wells and the plate stirred
for 5 min in an orbital shaker. In all wells 50 lL of sub-
strate solution was added, the plate stirred for 5 min as
reported above. The plate was dark adapted for 10 min
and the luminescence was measured in Victor3, from
Perkin-Elmer Life Sciences.
5.2.2. Drug transport experiment. Apical to basolateral
(Papp, A–B) and basolateral to apical (Papp, B–A) per-
meability of drugs were measured at 120 min and at var-
ious drug concentrations (1–100 lM).47 Drugs were
dissolved in Hanks’ 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 lL of fresh HBSS buffer and the receiver plate with
250 lL 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
5.4. [3H]Vinblastine transport inhibition
Caco-2 cells were seeded onto MultiScreen Plates at
10,000 cells/well for 21 days measuring the integrity of
the cell monolayers by Trans Epithelial Electrical Resis-
tance (TEER, X · cm2) with an epithelial voltohomme-
ter. Mature Caco-2 cell monolayer exhibited
a
TEER > 800 X · cm2 prior to use in transport experi-
ments. Transport experiments for tested compounds
were carried out as described by Taub et al.56

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
371
In each well to basolateral (BL) compartment in the ab-
sence and in the presence of P-gp inhibitors (from
200 nM to 400 lM) 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, 20 lL
sample was taken from donor compartment to deter-
mine 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 radio-
ligand in the presence and absence of compound. These
differences were expressed as inhibition percentage at
single drug concentration.
ium background)/(total LDH in untreated cells—culture
medium background).
5.7. Intracellular Doxorubicin accumulation
The modulation of intracellular Doxorubicin accumula-
tion by 3a, 3c, and 8c, Cyclosporin A, and Verapamil
was evaluated by flow cytometry, utilizing Verapamil as
standard of P-gp inhibition.49 In all experiments, the var-
ious drug-solvents (EtOH, DMSO) were added in each
control to evaluate the solvent influence. In MCF7/Adr
cells, the standard and the newly synthesized P-gp com-
pounds were utilized at 20 lM for 2 days exposure, and
during the second day Doxorubicin was given at 50 lM
(3 days-drug IC50 concentration). After incubation, the
cell media were removed and trypsin–EDTA was used
to detach the cells from the plates. Cells were harvested,
washed twice in ice-cold PBS (pH 7.4), and were placed
on ice (less than 1 h) until analysis. Fluorescence mea-
surements of individual cells were performed with a Bec-
ton–Dickinson FACScan equipped with an ultraviolet
argon laser. Analysis was gated to include single cells
on the basis of forward and side light scatter and was
based on the acquisition of data from 5000 cells. Log fluo-
rescence was collected and displayed as single parameter
histograms. The mean fluorescence intensity of Doxoru-
bicin in the Doxorubicin-treated cells, arbitrarily estab-
lished as 100%, represented the positive control and the
auto-fluorescence of untreated cells, arbitrarily estab-
lished as 0%, was the negative control.
5.5. Cell antiproliferative effect
The antiproliferative effect was evaluated using the 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide
(MTT) assay as reported by Colabufo et al. with minor
modifications.48 The cells were seeded to 96-well plates
in the absence and presence of known concentrations
of test compound for 48 h. In each well 10 lL of MTT
solution (5 mg/mL) freshly prepared was added and
the plate was incubated in a humidified atmosphere of
5% CO2 at 37 ꢁC for 3–4 h. MTT solution was removed
and 200 lL of EtOH/DMSO (1:1) was added to each
well to dissolve the blue formazan solid crystals. The
optical density was measured at 570 and 650 nm
wavelenghts using Victor3, from Perkin-Elmer Life
Sciences.
5.8. Effect of antiproliferative drug combination48
5.6. Cytotoxicity assay
In MCF7/Adr, each newly synthesized compound was
utilized at 20 lM; Doxorubicin at 5 lM (IC50 after 3 days
drug exposure when the P-gp was not overexpressed);
Verapamil and Cyclosporin A were utilized as reference
compounds. The schedule of drug administration was
the P-gp inhibitors plus Doxorubicin for 2 days followed,
after two wash steps with complete medium, by Doxoru-
bicin for 1 day. The analysis of cell growth inhibition was
performed using the MTT assay. On day one,
10,000 cells/well were plated in 96-well plates in a volume
of 200 lL and on day two, the various drugs alone or in
combination were added. Six control wells (untreated
cells) and six wells for each treatment were used in each
experiment. In all experiments, the various drug-solvents
(EtOH, DMSO) were added in each control to evaluate a
possible solvent cytotoxicity. Then 0.5 mg/mL MTT was
added to each well and, after 1-h incubation at 37 ꢁC, the
supernatant was removed. To solubilize the formazan
crystals, 100 lL of DMSO was added and the absorbance
values at 570 and 630 nm were determined on the micro-
plate reader SpectraCount (Packard—USA). The cell
growth inhibitory activity of drugs was expressed as per-
centage of control (untreated cells).
The assay was performed using the CytoTox-One kit
from the Promega Corp. (Madison, WI, USA) as re-
ported in previous paper.48 Cell death was determined
as the release of lactate dehydrogenase (LDH) into the
culture medium. The percentage of cytotoxicity was cal-
culated relative to the LDH release from total lysis of
cells in untreated control. It is assumed here that the
drug-treated wells and the control wells contained the
same total number of cells (dead plus alive cells) at the
end of the treatment period. Therefore, the cytotoxic ef-
fect of tested compounds was unaffected by any under-
estimation of cytotoxicity that could occur because of
decreased total number of cells in the treated samples
compared to the untreated control. Cells were seeded
into 96-well plates for optical performance in the fluo-
rescent cell-based assay in 100 lL 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 of 5% CO2 at 37 ꢁC and then
100 lL of substrate mix in assay buffer was added. Ten
microliters of lysis solution was added to untreated wells
in order to estimate total LDH. Plates were kept pro-
tected from light for 10 min at room temperature and
50 lL of stop solution was added to all wells. The fluo-
rescence was recorded using a LS55 Luminescence Spec-
6. Statistical analysis
trometer PerkinElmer 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 med-
The EC50 values of the compounds reported in Table 2
were determined by nonlinear curve fitting utilizing the
GraphPad Prism program.61

372
N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
24. Breier, A.; Barancik, M.; Sulova, Z.; Uhrik, B. Curr.
Cancer Drug Targets 2005, 5, 457.
Supplementary materials
25. Nobili, S.; Landini, I.; Giglioni, B.; Mini, E. Curr. Drug
Targets 2006, 7, 861.
26. Mahon, F. X.; Belloc, F.; Lagarde, V.; Chollet, C.;
Moreau-Gaudry, F.; Reiffers, J.; Goldman, J. M.; Melo, J.
V. Blood 2003, 101, 2368.
Elemental Analysis and experimental section for key
intermediates 2a, 5b, 5d, 7d. Supplementary data
associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2007.09.039.
27. Erba, E.; Bergamaschi, D.; Bassano, L.; Ronzoni, S.; Di
Liberti, G.; Muratore, I.; Vignati, S.; Faircloth, G.;
Jimeno, J.; D’Incalci, M. Br. J. Cancer 2000, 82, 1732.
28. Linenberger, M. L.; Hong, T.; Flowers, D.; Sievers, E. L.;
Gooley, T. A.; Bennett, J. M.; Berger, M. S.; Leopold, L.
H.; Appelbaum, F. R.; Bernstein, I. D. Blood 2001, 98,
988.
29. Safa, A. R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7187.
30. Cornwell, M. M.; Pastan, I.; Gottesman, M. M. J. Biol.
Chem. 1987, 262, 2166.
31. Robert, J. Expert Opin. Invest. Drugs 1998, 7, 929.
32. Thomas, H.; Coley, H. M. Cancer Control 2003, 10, 159.
33. Dey, S. Curr. Opin. Invest. Drugs 2000, 3, 818.
34. Rago, R. P.; Einstein, A.; Lush, R.; Beer, T. M.; Ko, Y. J.;
Henner, W. D.; Bubley, G.; Merica, E. A.; Garg, V.; Ette,
E.; Harding, M. V.; Dalton, V. S. Cancer Chemother.
Pharmacol. 2003, 51, 297.
References and notes
1. Klein, I.; Sarkadi, B.; Varadi, A. Biochim. Biophys. Acta
1999, 1461, 237.
2. Ambudkar, S. V.; Dey, S.; Hrycyna, C. A.; Ramachandra,
M.; Pastan, I.; Gottesman, M. M. Annu. Rev. Pharmacol.
Toxicol. 1999, 39, 361.
3. Vaalburg, W.; Hendrikse, N. H.; Elsinga, P. H.; Bart, J.;
van Waarde, A. Toxicol. Appl. Pharmacol. 2005, 20,
7(S257).
4. Ayrton, A.; Morgan, P. Xenobiotica 2001, 31, 469.
5. Dean, M.; Rzhetsky, A.; Allikmets, R. Genome Res. 2001,
11, 1156.
6. Hyde, S. C.; Emsley, P.; Hartshorn, M. J.; Mimmack, M.
M.; Gileadi, U.; Pearce, S. R.; Gallagher, M. P.; Gill, D.
R.; Hubbard, R. E.; Higgins, C. F. Nature 1990, 346, 362.
7. Quinton, P. M. Physiol. Rev. 1999, 79, S3.
8. Remaley, A. T.; Rust, S.; Rosier, M.; Knapper, C.;
Naudin, L.; Broccardo, C.; Peterson, K. M.; Koch, C.;
Arnould, I.; Prades, C.; Duverger, N.; Funke, H.; Assman,
G.; Dinger, M.; Dean, M.; Chimini, G.; Santamarina-
Fojo, S.; Fredrickson, D. S.; Denefle, P.; Bryan, H.;
Brewer, H. B. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
12685.
9. Brooks-Wilson, A.; Marcil, M.; Clee, S. M.; Zhang, L.
H.; Roomp, K.; van Dam, M.; Yu, L.; Brewer, C.;
Collins, J. A.; Molhuizen, H. O.; Loubser, O.; Ouelette,
B. F.; Fichter, K.; Ashbourne-Excoffon, K. J.; Sensen,
C. W.; Scherer, S.; Mott, S.; Denis, M.; Martindale, D.;
Frohlich, J.; Morgan, K.; Koop, B.; Pimstone, S.;
Kastelein, J. J.; Genest, J.; Hayden, M. R. Nat. Genet.
1999, 22, 336.
35. Minderman, H.; O’Loughlin, K. L.; Pendyala, L.; Baer,
M. R. Clin. Cancer Res. 2004, 10, 1826.
36. Rowinsky, E. K.; Smith, L.; Wang, Y. M.; Chaturvedi, P.;
Villalona, M.; Campbell, E.; Aylesworth, C.; Eckhardt, S.
G.; Hammond, L.; Kraynak, M.; Drengler, R.; Stephen-
son, J., Jr.; Harding, M. W.; Von Hoff, D. D. J. Clin.
Oncol. 1998, 16, 2964.
37. Wandel, C.; Kim, R. B.; Kajiji, S.; Guengerich, P.;
Wilkinson, G. R.; Wood, A. J. Cancer Res. 1999, 59, 3944.
38. Starling, J. J.; Shepard, R. L.; Cao, J.; Law, K. L.;
Norman, B. H.; Kroin, J. S.; Ehlhardt, W. J.; Baughman,
T. M.; Winter, M. A.; Bell, M. G.; Shih, C.; Gruber, J.;
Elmquist, W. F.; Dantzig, A. H. Adv. Enzyme Regul. 1997,
37, 335.
39. Dantzig, A. H.; Shepard, R. L.; Cao, J.; Law, K. L.;
Ehlhardt, W. J.; Baughman, T. M.; Bumol, T. F.; Starling,
J. J. Cancer Res. 1996, 56, 4171.
10. Bates, E. S.; Robey, R.; Knutsen, T.; Honjo, Y.; Litman,
T.; Dean, M. Emerg. Ther. Targets 2000, 4, 561.
11. Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer
2002, 2, 48.
12. Schneider, E.; Paul, D.; Ivy, P.; Cowan, K. H. Cancer
Chemother. Biol. Response Mod. 1999, 18, 152.
13. Glavinas, H.; Krajcsi, P.; Cserepes, J.; Sarkadi, B. Curr.
Drug Delivery 2004, 1, 27.
40. Traunecker, H. C.; Stevens, M. C.; Kerr, M. C.; Ferry, D.
R. Br. J. Cancer 1999, 81, 942.
41. Sparreboom, A.; Plantings, A. S.; Jewell, R. C.; van der
Burg, M. E.; van der Gaast, A.; de Bruijn, P.; Loos, W. J.;
Nooter, K.; Chandler, L. H.; Paul, E. M.; Wissel, P. S.;
Verweij, J. Anticancer Drugs 1999, 10, 719.
42. Mistry, P.; Stewart, A. J.; Dangerfield, W.; Okiji, S.;
Liddle, C.; Bootle, D.; Plumb, J. A.; Templeton, D.;
Charlton, P. Cancer Res. 2001, 61, 749.
43. Martin, C.; Berridge, G.; Mistry, P.; Higgins, C.; Charl-
ton, P.; Callaghan, R. Br. J. Pharmacol. 1999, 128, 403.
44. Pusztai, L.; Wagner, P.; Ibrahim, N.; Rivera, E.; Theria-
ult, R.; Booser, D.; Symmans, F. W.; Wong, F.; Blu-
menschein, G.; Fleming, D. R.; Rouzier, R.; Boniface, G.;
Hortobagyi, G. N. Cancer 2005, 104, 682.
´
´
14. Perez-Tomas, R. Curr. Med. Chem. 2006, 13, 1859.
15. Teodori, E.; Dei, S.; Martelli, S.; Scapecchi, F.; Gualtieri,
F. Curr. Drug Targets 2006, 7, 893.
16. Leslie, E. M.; Deeley, R. G.; Cole, S. P. C. Toxicol. Appl.
Pharmacol. 2005, 204, 216.
17. Benet, L. Z.; Izumi, T.; Zhang, Y.; Silverman, J. A.;
Wacher, V. J. J. Controlled Release 1999, 62, 25.
18. Chan, L. M. S.; Lowes, S.; Hirst, B. Eur. J. Pharm. Sci.
2004, 21, 25.
45. Walker, J.; Martin, C.; Callaghan, R. Eur. J. Cancer 2004,
40, 594.
19. Kusuhara, H.; Sugiyama, Y. J. Controlled Release 2002,
78, 43.
46. McDevitt, C. A.; Callaghan, R. Pharmacol. Ther. 2007,
113, 429.
20. Borst, P.; Evers, R.; Kool, M.; Wijnholds, J. J. Natl.
Cancer Inst. 2000, 92, 1295.
21. Thiebaut, F.; Tsuruo, T.; Hamada, H.; Gottesman, M.
M.; Pastan, I.; Willingham, M. C. Proc. Natl. Acad. Sci.
U.S.A. 1987, 84, 7735.
22. Tan, B.; Piwnica-Worms, D.; Ratner, R. Curr. Opin.
Oncol. 2000, 12, 450.
23. Fardel, O.; Lecureur, V.; Guillouzo, A. Gen. Pharmacol.
1996, 39, 361.
47. Colabufo, N. A.; Berardi, F.; Perrone, R.; Rapposelli, S.;
Digiacomo, M.; Balsamo, A. J. Med. Chem. 2006, 49,
6607.
48. Colabufo, N. A.; Berardi, F.; Contino, M.; Niso, M.;
Abate, C.; Perrone, R.; Tortorella, V. Naunyn Schmiede-
berg’s Arch. Pharmacol. 2004, 370, 106.
49. Azzariti, A.; Colabufo, N. A.; Berardi, F.; Porcelli, L.;
Niso, M.; Simone, M. G.; Perrone, R.; Paradiso, A. Mol.
Cancer Ther. 2006, 5, 1807.

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 362–373
373
50. Perrone, R.; Berardi, F.; Colabufo, N. A.; Tortorella, V.;
Fiorentini, F.; Olgiati, V.; Vanotti, E.; Govoni, S. J. Med.
Chem. 1994, 37, 99.
51. Perrone, R.; Berardi, F.; Colabufo, N. A.; Leopoldo, M.;
Tortorella, V.; Fiorentini, F.; Olgiati, V.; Ghiglieri, A.;
Govoni, S. J. Med. Chem. 1995, 38, 942.
56. Taub, M. E.; Podila, L.; Almeida, I. Drug. Metab. Dispos.
2005, 33, 1679.
57. Rautio, J.; Humphreys, J. E.; Webster, L. O.; Bala-
krishnan, A.; Keogh, J. P.; Kunta, J. R.; Serabjit-
Singh, J.; Polli, J. W. Drug Metab. Dispos. 2006, 34,
786.
52. Perrone, R.; Berardi, F.; Colabufo, N. A.; Leopoldo, M.;
Tortorella, V.; Fornaretto, M. G.; Caccia, C.; McArthur,
R. A. J. Med. Chem. 1996, 39, 4928.
58. Paradiso, A.; Azzariti, A.; Berardi, F.; Colabufo, N. A.;
Perrone, R. Italian Patent number: RM2006A000443, 11
Agosto, 2006.
53. Berardi F.; Colabufo N. A.; Perrone, R.; Balsamo A.;
Rapposelli S. Italian Patent number RM2006A000217, 14
Aprile, 2006.
59. Acharya, P.; Tran, T. T.; Polli, J. W.; Ayrton,
A.; Ellens, H.; Bentz, J. Biochemistry 2006, 45,
15505.
54. Polli, J. W.; Wring, S. A.; Humphreys, J. E.; Huang, L.;
Morgan, J. B.; Webster, L. O.; Serabjit-Singh, C. S. J.
Pharmacol. Exp. Ther. 2001, 299, 620.
60. Leopoldo, M.; Lacivita, E.; De Giorgio, P.; Colabufo, N.
A.; Niso, M.; Berardi, F.; Perrone, R. J. Med. Chem. 2006,
49, 358.
55. Kangas, L.; Gro¨nroos, M.; Nieminem, A. L. Med. Biol.
1984, 62, 338.
61. GraphPad Prism Software, version for Windows (1998);
GraphPad Software, Inc.: San Diego, CA.