4-Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoline derivatives as potent P-gp modulators

Article abstract of DOI:10.1016/j.bmc.2008.01.055

Starting from lead compound 1 (EC₅₀ = 1.64 μM), its non-basic nucleus has been conformationally restricted by 4-biphenyl and 2-naphthyl moieties. In each series we investigated if the presence of H-bond donor or acceptor substituents, the basic

Full text of DOI:10.1016/j.bmc.2008.01.055

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3732–3743
4-Biphenyl and 2-naphthyl substituted
6,7-dimethoxytetrahydroisoquinoline derivatives as potent
P-gp modulators
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 Simoneb 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 24 October 2007; revised 23 January 2008; accepted 30 January 2008
Available online 2 February 2008
Abstract—Starting from lead compound 1 (EC50 = 1.64 lM), its non-basic nucleus has been conformationally restricted by 4-biphe-
nyl and 2-naphthyl moieties. In each series we investigated if the presence of H-bond donor or acceptor substituents, the basicity and
the lipophilicity (clogP) were correlated with the P-gp inhibiting activity of tested compounds. In the biphenyl series, derivative 4d
displayed the best results (EC50 = 0.05 lM). The corresponding amide 3d was found less active (EC50 = 3.5 lM) ascertaining the
importance of basicity in this series whilst the presence of hydroxy or methoxy substituents seems to be negligible. In the naphthyl
series, both the basicity and the presence of H-bond donor or acceptor groups seem to be negligible. Moreover, the lipophilicity did
not influence the P-gp inhibition activity of each series. Specific biological assays have been carried out to establish the P-gp inter-
acting mechanism of tested compounds discriminating between substrates and inhibitors. Moreover, compound 4d displayed a
potent P-gp inhibition activity with good selectivity towards BCRP pump.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
P-glycoprotein (P-gp) and Breast Cancer Resistant Pro-
tein (BCRP) are mainly involved in MDR.3
Drug resistance is an obstacle that limits the efficacy of
chemotherapeutic treatment. Some tumours are intrinsi-
cally resistant to pharmacological therapy, while others,
initially sensitive to chemotherapy, become resistant
during the treatment. Resistance to anticancer drugs is
due to the contribution of several factors such as phar-
macokinetic, tumour micro-environmental or cancer
cell-specific factors.1
Several strategies were suggested for reversing MDR
and among them the co-administration of an efflux
pump inhibitors was the most probed, although such
reversal agents might actually increase the side effects
of chemotherapy by blocking physiological anticancer
drug efflux from normal cells.4–7
Many drugs have been tested for modulating the P-gp
activity and among them the calcium channel blocker
Verapamil8 and the antisteroid Tamoxifen,9 termed first
generation P-gp inhibitors. Unfortunately, compounds
belonging to this class displayed side effects such as
interferences with several enzyme systems resulting in
unpredictable pharmacokinetic interaction.
The acquisition of resistance to anticancer drugs differ-
ing in chemical structure and mechanism of action is de-
fined MultiDrug Resistance (MDR). MDR decreases
cellular accumulation of drugs causing their efflux out
of cells by several ATP-dependent efflux pumps, known
as ATP binding cassette (ABC) family, overexpressed in
tumour cells.2 Among ABC transporters, MRPs (1–6),
Then, compounds more potent and less toxic than first
generation drugs, such as Valspodar10 and Biricodar,
have been developed.11 These compounds, claimed mod-
ulators of second generation, although more potent and
less toxic with respect to drugs of first generation,
Keywords: P-glycoprotein; BCRP; 4-Biphenyl-6,7-dimethoxytetrahy-
droisoquinoline; 2-Naphthyl-6,7-dimethoxytetrahydroisoquinoline.
*
Corresponding author. E-mail: colabufo@farmchim.uniba.it
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.055

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
3733
evidenced some limits because significantly inhibited the
metabolism and excretion of cytotoxic agents.12
tion. From these assays can be hypothesised three P-gp
interacting profiles: substrate, inhibitor, and non-trans-
ported substrate.22,23 We summarized these categories
in Table 1 considering the response to the three biolog-
ical assays above mentioned. In Category I unambigu-
ous substrates, non-substrates and inhibitors are
reported. In Category II are reported substrates differing
for the response in one of the three biological assays.
The most studied third generation P-gp modulators are
Zosuquidar,13 Elacridar14,15 and Tariquidar16–18 (Chart
1). These inhibitors displayed no pharmacokinetic inter-
action with chemotherapeutic drugs and showed high
potency and specificity for P-gp. Although these com-
pounds are in different phases of clinical trials, prelimin-
ary results are moderately satisfactory.19
In order to better evaluate the profile of P-gp inhibitors,
we preliminary determined both cytotoxic and antipro-
liferative effects for compounds 4c and 4d. The reason
of this investigation is related to the following assay
where we evaluated the antiproliferative effect of the
chemotherapeutic agent in combination with our com-
pounds in resistant tumour cells.
Starting from PB28, a sigma-2 receptor agonist display-
ing also a good P-gp modulating activity20 (EC50
=
0.55 lM), we developed compound 1 as P-gp inhibitor21
bearing two structural requirements: 6,7-dimethoxytet-
rahydroisoquinoline, the same of Elacridar and
Tariquidar as basic moiety and alkyltetraline nucleus
as non-basic fragment, the same of PB28 (Chart 2).
Moreover, for compound 4d the selectivity towards
BCRP transporter has been also investigated to estab-
lish the selectivity to inhibit P-gp pump because in
Caco-2 cells also this pump is highly expressed.24
Although this compound displayed moderate P-gp inhi-
bition activity (EC50 = 1.64 lM), SAR studies indicated
that the conformational restriction of non-basic moiety
is a requirement for improving P-gp inhibition activity.
In order to restrict the conformation of non-basic nu-
cleus, two different fragments, biphenyl and 2-naphthyl
moieties, as depicted in Chart 2, were inserted.
2. Chemistry
The preparation of amides and amines of biphenyl and
2-naphthyl series are reported in Scheme 1. The amides
of biphenyl series 3a and 3c–e were synthesized by reac-
tion between 6,7-dimethoxytetrahydroisoquinoline and
the acylchlorides of the corresponding carboxylic acids
2a and 2c–e.25,26 The obtained compounds were reduced
to the corresponding amines 4a and 4c–e by LiAlH4.
Amine 4b was prepared by alkylating the basic tetrahy-
droisoquinoline nucleus with bromoderivative 2b. The
final compounds of 2-naphthyl series 7f–h were prepared
by condensing appropriate 2-naphthyl carboxilic acids
5f–h with isoquinoline moiety in the presence of carbon-
yldiimidazole.27 By reducing amides 6f–h with LiAlH4
were obtained final compounds 7f–h.
Moreover, P-gp interacting mechanism of tested com-
pounds was investigated by combining three biological
assays: Apparent Permeability (Papp) determination,
[3H]vinblastine transport inhibition and ATP-ase activa-
HO
N
H
F
N
N
H
O
F
H
Zosuquidar
3. Results and discussion
O
O
N
3.1. Biphenyl derivatives 3c–e and 4a–e
O
O
The biphenyl (Table 2) and 2-naphthyl (Table 3) deriv-
atives have been investigated for their P-gp inhibiting
activity and selectivity towards BCRP transporter. The
importance of the biphenyl linkage position (from 2 to
4 position, compounds 4a–c) has been investigated.
N
H
N
H
O
Elacridar
O
O
The 4-biphenyl derivative 4c displayed the best activity
(EC50 = 0.10 lM) while 3-biphenyl 4b and 2-biphenyl
4a derivatives showed a strong decrease in P-gp inhibi-
tion activity (EC50 = 6.5 lM and 15 lM, respectively).
N
O
O
O
N
H
NH
Moreover, the influence of hydroxy or methoxy substi-
tuent in 40-position (compounds 4d and 4e, respectively)
was studied in order to investigate the effect of hydro-
gen-bond donor or acceptor. Unfortunately, the meth-
oxy derivative 4e presented poor solubility in the
experimental conditions so that it was not tested in the
three biological assays.
O
N
Tariquidar
Chart 1. Third generation P-gp inhibitors.

3734
N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
N
O
N
PB28
O
O
N
O
1
2-naphthyl series
biphenyl series
O
O
O
O
R
N
X
N
X
R
O
N
X
O
O
O
N
X
X = CO, CH2; R = H, OH, OCH3
Chart 2. Strategy of lead compound modification.
The 40-biphenyl-hydroxy derivative 4d displayed a potent
P-gp inhibition activity but with respect to the unsubsti-
tuted derivative 4c lower difference in P-gp inhibition
activity has been observed (EC50 = 0.05 lM vs 0.10 lM).
3.2. P-gp interacting mechanism of biphenyl derivatives
3c–e and 4a–e
The [3H]vinblastine transport inhibition defined the
competition for the P-gp site between the radiolabeled
vinblastine (known P-gp substrate) and tested com-
pound. Moreover, ATP cell depletion is linked to P-
gp activity so that P-gp transported compounds de-
creased ATP in the cell. Finally, for each compound
the Apparent Permeability (Papp) in Caco-2 cell mono-
layer has been determined both considering the flux
from basolater to apical versus (B–A) and from apical
to basolateral versus (A–B). Compounds displaying
BA/AB ratio 62 are considered non-transported com-
pounds whilst derivatives with BA/AB ratio >2 are eff-
luxed by P-gp.
In order to establish the influence of the basicity for the
P-gp inhibiting activity, compounds 3c–e were prepared.
Amides 3c and 3d were less active than the correspond-
ing amines 4c and 4d (EC50 = 28 lM vs 0.10 lM and
3.5 lM vs 0.05 lM, respectively). Amide 3e was not
tested because of its poor solubility in the experimental
conditions.
Regarding the mechanism involved in P-gp–drug inter-
action, three biological assays, reported in Table 2, have
been carried out.

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
3735
Table 1. Categories of P-gp ligands
Biological assays in Caco-2 cell line
[3H]Substrate ATP-ase Monolayer
Compounds 4c and 4d displayed BA/AB ratio <2 (1.8
and 1.3, respectively) and were unable to activate
ATP-ase so that they could be considered P-gp inhibi-
tors Elacridar-like. Moreover, amide 3c was an unam-
biguous P-gp substrate because it activated ATP-ase
and had BA/AB ratio >2. The other compounds 3d,
4a, 4b were unable to activate ATP-ase and displayed
BA/AB ratio >2 so that they could be termed P-gp
transported substrates Cyclosporin A-like.
transport
inhibition
activation efflux
Category I
Unambiguous
non-substrates
Unambiguous substrates Yes
No
No
No
Yes
No
Yes
No
Inhibitors
Elacridar
Yes
3.3. Naphthyl derivatives 6f–h and 7f–7h
Category II
Non-trans. substrates
(IIA)
Verapamil
Yes
Yes
No
In the 2-naphthyl series, amines 7f–h were found more
potent than the corresponding amides 6f–h as displayed
comparing 6f–7f (EC50 = 20 lM vs 0.80 lM), 6g–7g
(EC50 = 0.65 lM vs 0.45 lM), and 6h–7h (EC50 = 18 lM
vs 0.30 lM). Also in this series the influence of hydrogen-
bond donor or acceptor group was investigated. Com-
paring the unsubstituted amine 7f (EC50 = 0.80 lM) to
6-hydroxy- or 6-methoxy-substituted derivatives (7g
and 7h, respectively) little differences were found
Transported
substrates (IIB1)
Transported
No
No
Yes
No
Yes
No
Yes
Yes
Yes
substrates (IIB2)
Transported
substrates (IIB3)
Cyclosporin A
O
4
O
R'
A
Ar
N
3
2
O
R
3a and 3c-e
2a-e
R
B
C
R'
H
H
H
2-COOH
3-CH2Br
4-COOH
4-COOH
a
b
c
d
e
O
O
Ar
N
OH
OCH3 4-COOH
4a-e
O
O
O
O
D
OH
N
R
R
5f-h
6f-h
R
C
H
f
O
O
OH
OCH3
g
h
N
R
7f-h
Scheme 1. Reagents: SOCl2 and Et3N; CH2Cl2 and 1.2% NaOH; 6,7-dimethoxytetrahydroisoquinoline (A); Na2CO3, DMF and 6,7-dimethoxy-
tetrahydroisoquinoline for the preparation of compound 4b (B); LiAlH4, THF (C); carbonyldiimidazole, THF and 6,7-dimethoxytetrahydroiso-
quinoline (D).

3736
N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
Table 2. Biological P-gp modulating assays of biphenyl derivatives
O
O
N
Compound
[3H]Vinblastine
ATP-ase
activation (%)b
Papp B–Ac
(nm/s)
Papp A–Bc
(nm/s)
BA/AB
transport inhibition
EC50 SEMa (lM)
1
1.64 0.20
N
386
237
1.6
O
3c
O
28 2.0
Y (35%)
2441
601
4
3d
O
3.5 0.40
N
2769
280
NT
216
301
9.9
ND
9.1
12
HO
3e
O
NT
NT
NT
O
4a
15 2.5
N
1971
4b
6.5 0.30
N
3659

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
3737
Table 2 (continued)
Compound
[3H]Vinblastine
ATP-ase
activation (%)b
Papp B–Ac
(nm/s)
Papp A–Bc
(nm/s)
BA/AB
transport inhibition
EC50 SEMa (lM)
4c
4d
0.10 0.025
0.05 0.001
NT
N
1409
777
476
NT
1.8
1.3
ND
N
609
HO
4e
NT
NT
O
Elacridar
Verapamil
Cyclosporin A
2.0d
20d
80d
Nd
Y (52%)d
Nd
<2d
1.2d
9.6d
687d
153d
591d
15.9d
a Data are means of three-independent determinations, samples in triplicate.
b Effect measured at 100 lM. Data are means of three-independent determinations with samples in triplicate.
c Data are means of three-independent determinations (samples in triplicate).
d See Refs. 21,22.
(EC50 = 0.45 lM and 0.30 lM, respectively). In contrast,
the presence of hydroxy group on amide 6g
(EC50 = 0.65 lM) strongly improved P-gp inhibition
activity with respect to unsubstituted 6f (EC50 = 20 lM)
and methoxy-substituted 6h (EC50 = 18 lM) derivatives.
It is not possible to assert that the basicity is a require-
ment discriminating P-gp inhibition activity of tested
compounds. In fact, amide 6g displayed an inhibition
activity similar to the best amines of this series.
Compounds 4c and 4d were tested for evaluating their
antiproliferative effect at 24 and 48 h and cytotoxic effect
at 24 h in MCF-7/Adr cell line. The cytotoxicity was not
determined at 48 h because at this time the antiprolifer-
ative effect could cause a reduction of total number of
cells so that the cytotoxicity in the assay could be
underestimated.
The results displayed that at 24 h compounds 4c and 4d
were found not active in both assays while compound 4c
showed moderate antiproliferative effect at 48 h (40%,
data not shown). The results of antiproliferative assay
led us to exclude compound 4c from further investiga-
tions. For this reason compound 4d has been studied
in biological assays where the antiproliferative activity
of chemotherapeutic agent is evaluated both in the ab-
sence and in the presence of the P-gp inhibitor. These as-
says are carried out using a cell line displaying resistance
towards the chemotherapeutic agent. As depicted in Fig-
ure 1, the antiproliferative effect at 48 h of compound
4d, in MCF-7/Adr cell line, was negligible at 2 lM
(Fig. 1D) and at 20 lM (Fig. 1F).
3.4. P-gp interacting mechanism of naphthyl derivatives
6f–h and 7f–h
Regarding P-gp interacting mechanism, all derivatives
displayed BA/AB ratio >2. Moreover, compounds 6f,
6g and 7g could be defined as unambiguous P-gp sub-
strates because of their ability to activate ATP-ase. Dif-
ferently, 6h, 7f and 7h were unable to activate ATP-ase
so that they could be termed transported substrates
Cyclosporin A-like.
4. Cytotoxic and antiproliferative effects
Starting from this finding, compound 4d at 2 lM and at
20 lM has been incubated with 5 lM Doxorubicin
(Fig. 1E and G, respectively). After wash out, Doxoru-
bicin was added for 1 day. The antiproliferative effect
of Doxorubicin after pre-treatment with compound 4d
(2 lM) increased from 28% to 42% (Fig. 1D vs E). After
Among studied compounds, only biphenyl derivatives
4c and 4d could be defined P-gp inhibitors while all
the other compounds could be considered P-gp
modulators.

3738
N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
Table 3. Biological P-gp modulating assays of 2-naphthyl derivatives
O
O
N
Compound
[3H]Vinblastine
ATP-ase
activation (%)b
Papp B–Ac
(nm/s)
Papp A–Bc
(nm/s)
BA/AB
transport inhibition
EC50 SEM (lM)a
6f
20 4.0
Y 10%
4150
1900
324
251
13
O
6g
HO
0.65 0.05
Y (40%)
7.6
O
6h
O
18 1.5
N
N
1234
1797
193
216
6.4
8.3
O
7f
0.80 0.02
7g
HO
0.45 0.02
0.30 0.04
Y (10%)
2351
677
353
187
6.7
3.6
7h
O
N
a Data are means of three-independent determinations, samples in triplicate.
b Effect measured at 100 lM. Data are means of three-independent determinations with samples in triplicate.
c Data are means of three-independent determinations (samples in triplicate).
pre-treatment with 20 lM of compound 4d, this effect
markedly increased from 28% to 90% (Fig. 1F vs G).
100
In the same experimental condition, 20 lM Verapamil, im-
proved Doxorubicin-induced cell growth inhibition from
28% to 75% (Fig. 1C). At 2 lM, Verapamil alone and in drug
combination displayed poor activity (data not shown).
50
0
Comparing compound 4d and Verapamil at 20 lM the
same maximal effect has been obtained. This result seems
to be an apparent discrepancy considering their P-gp inhi-
bition values reported in Table 2 (EC50 = 0.05 lM and
20 lM, respectively). This finding could be explained con-
sidering that the biological assays have been carried out in
different cell lines: Caco-2 cells for the permeability assay
and MCF-7/Adr cells for the cytotoxicity assay. InCaco-2
cells it was reported a simultaneous overexpression of P-
gp and BCRP transporters and the efflux activity of
BCRP pump is recognized by [3H]mitoxantrone.
A
B
C
D
E
F
G
Figure 1. Antiproliferative activity of Doxorubicin (5 lM) in MCF-7/
Adr cell line in the absence and in the presence of 2 lM and 20 lM 4d
or 20 lM Verapamil. (A) control (3 days); (B) Doxorubicin (3 days);
(C) 20 lM Verapamil and Doxorubicin (2 days). After wash out
Doxorubicin (1 day); (D) 2 lM 4d; (E) 2 lM 4d and Doxorubicin (2
days). After wash out Doxorubicin (1 day); (F) 20 lM 4d; (G) 20 lM
4d and Doxorubicin (2 days). After wash out Doxorubicin (1 day).

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
3739
6. Conclusions
compound 1
In present work two series of compounds bearing a sim-
plified fragment linked to the basic nucleus have been
evaluated.
100
75
50
25
0
Several elements such as the basicity, the lipophilicity,
the presence of H-bond donor or acceptor groups have
been investigated in both series. The basicity seems to
be an important requirement in 40-biphenyl series be-
cause amides are lower active than the corresponding
amines. In this series the presence of hydroxy or meth-
oxy substituents seems to be negligible.
compound 4d
1
10
100
1
10
100
Different conclusions could be carried out for the basi-
city in naphthyl series. In fact in this series amines and
amides displayed little differences in P-gp activity. More-
over, the presence of H-bond donor or acceptor groups
also in this series seems to be negligible. Furthermore,
the lipophilicity did not influence the P-gp inhibition
activity of each tested series.
[dose], μM
Figure 2. [3H]Mitoxantrone (20 nM) transport inhibition in Caco-2
cells in the presence of compounds 1 or 4d.
The effect on BCRP pump activity of the lead com-
pound 1 and biphenyl compound 4d has been evaluated.
The results displayed that compound 1 inhibited BCRP
pump in dose-dependent manner with maximal effect at
100 lM as depicted in Figure 2. In contrast, compound
4d was unable to interact with BCRP pump (19% of
inhibition at 100 lM). This finding could suggest that
the fragment linked to 6,7-dimethoxytetrahydroisoquin-
oline nucleus is a potential requirement not only for the
P-gp inhibition potency but also for the selectivity to-
wards BCRP pump.
Among studied compounds, 4d displayed a potent P-gp inhi-
bition activity with good selectivity towards BCRP pump.
In order to better establish for each series the effect of
lipophilicity, basicity and H-bond on no-basic fragment
and the selectivity towards other ABC transporters,
other compounds could be evaluated.
7. Experimental
7.1. Biological method
This finding is consistent with Elacridar clinical trials re-
sults where this compound was found as a potent BCRP
modulating agent.24
7.1.1. Cell lines. The breast cancer cell line of human ori-
gin, MCF-7/Adr (resistant to Adriamycin or Doxorubi-
cin), was routinely cultured in RPMI 1640 supplemented
with 10% foetal bovine serum, 2 mM glutamine, 100 U/
mL penicillin, 100 lg/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 foetal bovine serum, 100 U/mL penicillin,
100 lg/mL streptomycin and 2 mM L-glutamine.
5. Lipophilicity effect on P-gp inhibition activity
In order to verify a potential correlation between lipophy-
lic properties of compounds (clogP) and their activities
(pEC50), we plotted these values for each series as re-
ported in Figure 3. The results displayed that in both ser-
ies there is not a correlation between activities and
lipophilicity.
7.1.2. Permeability experiments
7.1.2.1. Preparation of Caco-2 monolayer. This proce-
dure has been previously reported.28 Briefly, Caco-2
cells were harvested with trypsin–EDTA and seeded
onto MultiScreen Caco-2 assay system at a density of
10,000 cells/well. The culture medium was replaced
every 48 h for the first 6 days and every 24 h thereafter,
and after 21 days in culture, the Caco-2 monolayer was
utilized for the permeability experiments. The transepit-
elial electrical resistance (TEER) of the monolayers was
measured daily before and after the experiment using a
epithelial voltohommeter (Millicellꢂ-ERS; Millipore,
Billerica, MA). Generally, TEER values obtained are
greater than 1000 X for a 21-day culture.
8
Biphenyl series
Naphthyl series
7
6
5
4
3
3.5
4.0
cLogP
4.5
7.1.2.2. Drug transport experiment. Apical to basolat-
eral (Papp A–B) and basolateral to apical (Papp B–A)
permeability of drugs were measured at 120 min and
Figure 3. Correlation between lipophilicity and P-gp inhibition activity
of tested compounds.

3740
N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
at various drugs concentrations (1–100 lM).29 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 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 was removed
and drug solutions added to the filter well (75 lL).
HBSS without the drug was added to the receiver plate
(250 lL). 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.
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
or 20 nM [3H]mitoxantrone for 120 min at 37 ꢁC and
its appearance in the apical (AP) compartment was
monitored. At 120 min a 20 lL 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 or [3H]mitoxantrone
transport inhibition was calculated as radioactivity dif-
ference between radioligand in the presence and absence
of compound. These differences were expressed as inhi-
bition percentage at single drug concentration.
7.1.5. Cytotoxicity assay. The assay was performed
using the CytoTox-One kit from the Promega Corp.
(Madison, WI, USA) as reported in previous paper.32
Cell death was determined as the release of lactate
dehydrogenase (LDH) into the culture medium. The
percentage of cytotoxicity was calculated relative to
the LDH release from total lysis of cells in 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 treat-
ment period. Therefore, the cytotoxic effect of tested
compounds was unaffected by any underestimation 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 fluorescent cell-based as-
say 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 atmo-
sphere 5% CO2 at 37 ꢁC and then 100 lL of substrate
mix in assay buffer was added. Ten microliters lysis
solution was added to untreated wells in order to esti-
mate total LDH. Plates were kept protected from light
for 10 min at room temperature and 50 lL of stop
solution was added to all wells. The fluorescence was
recorded using a LS55 Luminescence Spectrometer
PerkinElmer with a 560 nm excitation wavelength and
a 590 nm emission wavelength. The cytotoxicity per-
centage was estimated as follows: 100 · (LDH in med-
ium of treated cells ꢀ culture medium background)/
(total LDH in untreated cells ꢀ culture medium
background).
The concentration of compounds was analyzed using
UV–vis spectroscopy.
The apparent permeability (Papp), in units of nm/s, was
calculated using the following equation:
ꢀ
ꢁ
ꢀ
ꢁ
½drugꢂ
V A
area ꢁ time
acceptor
Papp
¼
ꢁ
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 UV spectroscopy; [drug]initial is the initial
drug concentration (1 · 10ꢀ4 M) in the apical or baso-
lateral wells.
7.1.3. 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.30 Caco-2 cells were seeded
into 96-well microplate in 100 lL 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 lL of com-
plete medium in the presence or absence of different con-
centrations (from 1 to 100 lM) of tested compounds
was added. The plate was incubated for 2 h in a humid-
ified atmosphere 5% CO2 at 37 ꢁC. Then, 50 lL of mam-
malian cell lysis solution was added to all wells and the
plate stirred for 5 min in an orbital shaker. In all wells
50 lL of substrate solution was added, the plate stirred
for 5 min as above reported. The plate was dark adapted
for 10 min and the luminescence was measured in Vic-
tor3, from PerkinElmer Life Sciences.
7.1.6. Evaluation of cell growth. Determination of cell
growth was performed using the 3-[4,5-dimethylthia-
zol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay
at 24 and 48 h.20 On day one, 10,000 cells/well were pla-
ted in 96-well plates in a volume of 200 lL and on day
two, the various drugs alone or in combination were
added. In all the experiments, the various drug-solvents
(ethanol, DMSO) were added in each control to evalu-
ate a possible solvent cytotoxicity. After the established
incubation time with drugs, 0.5 mg/mL MTT was added
to each well and, after 1 h incubation at 37 ꢁC, the
supernatant was removed. The formazan crystals were
solubilized by 100 lL of DMSO and the absorbance val-
7.1.4. [3H]Substrate transport inhibition. Caco-2 cells
were seeded onto MultiScreen Plates 10,000 cells/well
for 21 days measuring the integrity of the cell monolay-
ers by trans-epithelial electrical resistance (TEER,
X · cm2) with an epithelial voltohommeter. Mature
Caco-2
cell
monolayer
exhibited
a
TEER
>800 X · cm2 prior to use in transport experiments.
Transport experiments for tested compounds were car-
ried out as described by Taub et al.31

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
3741
ues at 570 and 630 nm were determined on the micro-
plate reader SpectraCount (Packard-USA).
added to a solution containing 6,7-dimethoxytetrahy-
droisoquinoline (1.0 mmol) in CH2Cl2 (30 mL) and
1.2% in H2O NaOH (30 mL). The resulting solution
was stirred at room temperature for 4 h then the organic
layer was separated and washed with 0.5 M Na2CO3
(3 · 20 mL) and 3 N HCl (3· 20 mL). The organic layer
was dried on anhydrous Na2SO4, the solvent was evap-
orated obtaining a crude solid. Compound 3a was not
characterized. Crude 3a has been reduced affording
compound 4a as following reported.
7.1.7. Effect of antiproliferative drug combination. In
MCF7/Adr, compound 4d and Verapamil were utilized
at 2 lM and 20 lM; Doxorubicin at 5 lM (IC50 after
3 days drug exposure when the P-gp was not overexpres-
sed); Verapamil was utilized as reference compound.
The schedule of drugs administration was the P-gp
inhibitors plus Doxorubicin for 2 days followed, after
two wash steps with complete medium, by Doxorubicin
for 1 day. The analysis of cell growth inhibition was per-
formed using the MTT assay as reported above.20
7.3.1. Biphenyl-4-yl-(6,7-dimethoxy-3,4-dihydro-1H-iso-
quinolin-2-yl)-methanone (3c). White solid, 55% yield
from column chromatography (CHCl3). Recrystallized
from CHCl3/petroleum ether. Mp 143–147 ꢁC. Anal.
(C24H23NO3Æ0.25H2O) C, H, N. UV–vis kmax = 266 nm
(PBS) e = 8240. ESI+/MS m/z 374 (ESI M++1, 63), 282
(M+ 100). 1H NMR: d 2.46–2.75 (m, 2H, NCH2CH2 iso-
quinoline), 3.60–3.68 (m, 8H, 2 of CH3 NCH2CH2 iso-
quinoline), 4.52–4.66 (m, 2H, NCH2 isoquinoline),
6.61–7.99 (m, 11 H, aromatic).
7.2. Chemistry
Column chromathography was performed with 1:30
˚
Merck silica gel 60 A (63–200 lm) as the stationary phase.
Melting points were determined in open capillaries on a
Gallenkamp electrothermal apparatus. Elemental analy-
ses (C, H, N) were performed on Eurovector Euro EA
3000 analyzer; the analytical results were within 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 spectrome-
ter; only significant m/z peaks, withtheir percentage ofrel-
ative intensity in parentheses, are reported. All spectra
were in accordance with the assigned structures. ESI-
MS analyses were performed on an Agilent 1100 LC/
MSD trap system VL. The UV–vis spectra of tested com-
pounds and their corresponding calibration curves were
recorded with LAMBDA BIO20 spectrophotometer
PerkinElmer. The spectroscopic properties of each com-
pound are reported in Section 7. Compound 3a has not
been characterized because of its instability.
7.3.2. (6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-
(40-hydroxy-biphenyl-4-yl)-methanone (3d). White solid,
46% yield from column chromatography (CHCl3/EtAc
4:1). Recrystallized from CHCl3/petroleum ether. Mp
129–132 ꢁC. Anal. (C24H23NO4ÆH2O) C, H, N. UV–vis
k
max = 280 nm (PBS) e = 4420. ESI+/MS m/z 390
1
(ESI++1, 100), 193 (M+ 30). H NMR: d 2.49–2.75 (m,
2H, NCH2CH2 isoquinoline), 3.60–3.80 (m, 8H, 2 of
CH3, NCH2CH2 isoquinoline), 4.55–4.65 (m, 2H,
NCH2 isoquinoline), 6.72–7.66 (m, 10H, aromatic),
9.50–9.80 (br s, 1H, OH, D2O exchanged).
7.3.3. (6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-
(40-methoxy-biphenyl-4-yl)-methanone (3e). White solid,
70% yield from column chromatography (CHCl3/EtAc
9:1). Recrystallized from CHCl3/Petroleum ether. Mp
158–160 ꢁC. Anal. (C25H25NO4Æ0.5ÆH2O) C, H, N.
UV–vis kmax = 280 nm (PBS) e = 4420. ESI+/MS m/z
404 (ESI++1, 100), 211 (M+ 20). 1H NMR: d 2.68–
2.86 (m, 2H, NCH2CH2 isoquinoline), 3.62–3.97 (m,
11H, 3 of CH3, NCH2CH2 isoquinoline), 4.31–4.82 (m,
2H, NCH2 isoquinoline), 6.43–7.62 (m, 10H, aromatic).
7.2.1. 40-Methoxy-biphenyl-4-carboxylic acid (2e). In
35 mL MeOH and 1 mL H2SO4 was dissolved 40-hydro-
xy-biphenyl-4-carboxylic acid (1 mmol). The solution
was heated under reflux for 10 h. The solvent was evap-
orated and the crude was washed with H2O 2 · 10 mL
and dried. The obtained methyl ester was dissolved in
CH3CN (20 mL) in the presence of K2CO3 (2.0 mmol)
and CH3I (2.0 mmol). The mixture was refluxed for
6 h, cooled and the solid residue was removed by filter-
ing. The solvent was evaporated and the crude was dis-
solved in 3 M NaOH (20 mL) and heated at 60 ꢁC for
6 h. The mixture was cooled and acidified to pH 1.0 with
6 N HCl. The precipitate was filtered washed with Et2O
(2 · 10 mL) and dried. The title compound was purified
by silica gel chromatography (EtAc/petroleum ether 3:7)
obtaining a white solid (46% yield).
7.4. General procedure for preparing amines 4a–e and 7f–h
To a suspension of LiAlH4 (2 mmol) in anhydrous THF
(30 mL) under N2 atmosphere was added dropwise a
solution of appropriate amide in THF (10 mL). The
mixture was refluxed for 2 h, cooled and H2O (1 mL)
was added dropwise to eliminate LiAlH4 excess. Then,
were added Et2O (20 mL) and H2O (20 mL) and the or-
ganic layer was separated and washed with 3 N HCl (3 ·
10 mL). The aqueous solution was separated and alka-
linized with solid Na2CO3 and extracted with CHCl3
(3 · 20 mL). The organic layers were collected and dried
on anhydrous Na2SO4. The solvent was evaporated and
the crude was purified on silica gel column.
7.3. General procedure for preparing amides 3a and 3c–e
A mixture of appropriate biphenyl carboxylic acid
(2.0 mmol) and an excess of SOCl2 (2.0 mL) and
0.1 mL triethylamine was stirred for 30 min at 70 ꢁC.
Then the excess of SOCl2 was removed under vacuo
and the crude was solubilized in CH2Cl2 (20 mL) and
7.4.1. 2-Biphenyl-2-yl-methyl-6,7-dimethoxy-1,2,3,4-tet-
rahydro-isoquinoline (4a). White solid, 35% yield from
column chromatography (CHCl3). Recrystallized from

3742
N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
MeOH/Et2O. Mp 207–209 ꢁC. Anal. (C24H25NO2ÆH-
ClÆ1.5H2O) C, H, N. UV–vis kmax = 280 nm (PBS)
e = 1600. ESI+/MS m/z 360 (ESI M++1, 100), 167 (M+
(50 mL) was stirred overnight at room temperature un-
der N2 atmosphere. Then was added a solution of 6,7-
dimethoxytetrahydroisoquinoline (1.0 mmol) in THF
(20 mL). The resulting mixture was stirred at room tem-
perature for 8 h. After this time were added ethylacetate
(30 mL) and H2O (30 mL) and the organic layer was
separated and washed with Na2CO3 (s.s. 3 · 30 mL).
The organic layer was dried on anhydrous Na2SO4
and the solvent was evaporated.
1
17). H NMR: d 2.65–2.77 (m, 4H, NCH2CH2 isoquin-
oline), 3.48 (s, 2H, NCH2 biphenyl), 3.60 (s, 2H, NCH2
isoquinoline), 3.80 and 3.83 (2s, 6H, 2 of CH3), 6.45–
7.67 (m, 11H, aromatic).
7.4.2. 2-Biphenyl-3-yl-methyl-6,7-dimethoxy-1,2,3,4-tet-
rahydro-isoquinoline (4b). Yellow oil, 61% yield from col-
umn chromatography (CHCl3/EtAc 1:1). Recrystallized
7.5.1. (6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-
naphthalen-2-yl-methanone (6f). White solid, 80% yield
from column chromatography (CHCl3/EtAc 9:1).
Recrystallized from CHCl3/petroleum ether. Mp 136–
139 ꢁC. Anal. (C22H21NO3) C, H, N. UV–vis
from
MeOH/Et2O.
Mp
126–130 ꢁC.
Anal.
(C24H25NO2ÆHCl) C, H, N. GC–MS: 360 (M++1, 7),
359 (M+ 40), 358 (100), 192 (64). H NMR: d 2.66–2.85
1
(m, 4H, NCH2CH2isoquinoline), 3.50 (s, 2H, NCH2
biphenyl), 3.63 (s, 2H, NCH2 isoquinoline), 3.80–3.83
(s, 6H, 2 of CH3), 6.44–7.69 (m, 11H, aromatic).
k
max = 280 nm (PBS) e = 6920. ESI+/MS m/z 348 (ESI
1
M++1, 100), 155 (M+ 20). H NMR: d 2.83–2.91 (m,
2H, NCH2CH2 isoquinoline), 3.69–3.99 (m, 8H, 2 of
CH3 and NCH2CH2 isoquinoline), 4.57–4.87 (m, 2H,
NCH2 isoquinoline), 7.51–7.95 (m, 9H, aromatic).
7.4.3. 2-Biphenyl-4-yl-methyl-6,7-dimethoxy-1,2,3,4-tet-
A suspension containing
rahydro-isoquinoline (4c).
bromoderivative 2b (1.0 mmol) 6,7-dimethoxytetrahy-
droisoquinoline (2.0 mmol) Na2CO3 (2.0 mmol) in
DMF (20 mL) was stirred for 6 h at 150 ꢁC. Then, the
solvent was evaporated and were added CHCl3 (20 mL)
and H2O (20 mL). The organic layer was separated
and dried on anhydrous Na2SO4. The solvent was evapo-
rated obtaining crude 4b as an oil that was purified on sil-
ica gel column. White solid, 87% yield from column
chromatography (CHCl3/EtAc 4:1). Recrystallized from
MeOH/Et2O. Mp 246–249 ꢁC. Anal. (C24H25NO2ÆH-
ClÆ0.75H2O) C, H, N. UV–vis kmax = 254 nm (PBS)
e = 8600. ESI+/MS m/z 360 (ESI M++1, 100), 167 (M+
42). 1H NMR: d 2.77–2.85 (m, 4H, NCH2CH2 isoquino-
line), 3.60 (s, 2H NCH2 isoquinoline), 3.74 (s, 2H,
NCH2 biphenyl), 3.81 and 3.84 (2s, 6H, 2 of CH3), 6.50–
7.64 (m, 11 H, aromatic).
7.5.2. (6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-
(6-hydroxy-naphthalen-2-yl)-methanone (6g). White so-
lid, 32% yield from column chromatography (CH2Cl2/
AcEt 1:1). Recrystallized from CHCl3/petroleum ether.
Mp 116–120 ꢁC. Anal. (CC22H21NO4Æ0.8H2O) C, H,
N. UV–vis kmax = 288 nm (PBS) e = 5650. ESI+/MS m/
z 364 (ESI M++1, 100), 171 (M+ 18). 1H NMR: d
2.87–2.92 (m, 3H, NCH2CH2 isoquinoline and OH,
D2O exchanged), 3.62–4.02 (m, 8H, 2 of CH3 and
NCH2CH2 isoquinoline), 4.62–4.87 (m, 2H, NCH2 iso-
quinoline), 6.64–7.95 (m, 8H, aromatic).
7.5.3. (6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-
(6-methoxy-naphthalen-2-yl)-methanone (6h). White so-
lid, 57% yield from column chromatography (CHCl3/
EtAc 9:1). Recrystallized from CHCl3/petroleum ether.
Mp 166–168 ꢁC. Anal. (C23H23NO4Æ0.5H2O) C, H, N.
UV–vis kmax = 283 nm (PBS) e = 6980. ESI+/MS m/z
7.4.4. 40-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-
yl-methyl)-biphenyl-4-ol (4d). White solid, 35% yield from
column chromatography (CHCl3/EtAc 4:1). Recrystal-
lized from MeOH/Et2O. Mp 265–268 ꢁC. Anal. (C24H25NO3Æ
HClÆ0.5H2O) C, H, N. UV–vis kmax = 270 nm (PBS)
e = 8240. ESI+/MS m/z 376 (ESI M++1, 100), 183 (M+
70). 1H NMR: d 2.65–2.71 (m, 4H, NCH2CH2 isoquino-
line), 3.25–3.41 (m, 4H, NCH2 isoquinoline and NCH2
biphenyl), 3.63–3.67 (s, 6H, 2 of CH3), 6.56–7.53 (m,
10H, aromatic), 9.55 (br s, 1H, OH, D2O exchanged).
1
378 (ESI M++1, 100), 185 (M+ 17). H NMR: d 1.67–
2.86 (m, 2H, NCH2CH2 isoquinoline), 3.62–3.94 (m,
11H, 3 of CH3, NCH2CH2 isoquinoline), 4.62–4.84 (m,
2H, NCH2 isoquinoline), 6.64–7.90 (m, 8H, aromatic).
7.5.4. 6,7-Dimethoxy-2-naphthalen-2-yl-methyl-1,2,3,4-
tetrahydro-isoquinoline (7f). White solid, 26% yield from
column chromatography (CHCl3/EtAc 1:1). Recrystal-
lized from MeOH/Et2O. Mp 234–238 ꢁC. Anal.
7.4.5. 6,7-Dimethoxy-2-(40-methoxy-biphenyl-4-yl-methyl)-
1,2,3,4-tetrahydro-isoquinoline (4e). White solid, 57%
yield from column chromatography (CHCl3/EtAc 9:1).
Recrystallized from MeOH/Et2O. Mp 238–241 ꢁC.
Anal. (C25H27NO3ÆHClÆ0.4H2O) C, H, N. UV–vis
(C22H23NO2ÆHClÆ0.5H2O) C, H, N. UV–vis kmax =
278 nm (PBS) e = 6030. ESI+/MS m/z 334 (ESI M++1,
100), 141 (M+ 17). 1H NMR: d 2.76–2.84 (m, 4H,
NCH2CH2 isoquinoline and NCH2 isoquinoline), 3.58
(s, 2H, NCH2 biphenyl), 3.80–3.86 (m, 8H, 2 of CH3
NCH2CH2 isoquinoline), 6.47–7.85 (m, 9H, aromatic).
k
max = 270 nm (PBS) e = 8240. ESI+/MS m/z 390 (ESI
1
M++1, 100), 197 (M+ 91). H NMR: d 2.77–2.85 (m,
4H, NCH2CH2 isoquinoline), 3.60 (s, 2H NCH2 iso-
quinoline), 3.73 (s, 2H, NCH2 biphenyl), 3.77–3.89 (m,
9H, 3 of CH3), 6.50–7.56 (m, 10H, aromatic).
7.5.5.
6-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-
ylmethyl)-naphthalen-2-ol (7g). White solid, 42% yield
from column chromatography (CH2Cl2/AcEt 1:1).
Recrystallized from MeOH/Et2O. Mp. 207–209 ꢁC.
Anal. (C22H23NO3Æ0.25H2O) C, H, N. UV–vis
7.5. General procedure for preparing amides 6f–h
k
max = 278 nm (PBS) e = 7740. ESI+/MS m/z 350 (ESI
1
A mixture of appropriate 2-naphthalenylcarboxylic acid
(1.0 mmol), carbonyldiimidazole (1.1 mmol) in THF
M++1, 100), 194 (M+ 68). H NMR: d 2.75–2.96 (m,
5H, NCH2CH2 isoquinoline and NCH2 isoquinoline,

N. A. Colabufo et al. / Bioorg. Med. Chem. 16 (2008) 3732–3743
3743
13. Morschhauser, F.; Zinzani, P. L.; Burgess, M.; Sloots, L.;
Bouafia, F.; Dumontet, C. Leuk. Lymphoma 2007, 48,
708–715.
OH, D2O exchanged), 3.62 (s, 2H NCH2 naphthalene),
3.78–3.86 (m, 8H, NCH2CH2 isoquinoline 2 of CH3),
7.00–7.70 (m, 8H aromatic).
14. Planting, A. S.; Sonneveld, P.; van der Gaast, A.;
Sparreboom, A.; van der Burg, M. E.; Luyten, G. P.; de
Leeuw, K.; de Boer-Dennert, M.; Wissel, P. S.; Jewell, R.
C.; Paul, E. M.; Purvis, N. B.; Verweij, J. Cancer
Chemother. Pharmacol. 2005, 55, 91–99.
15. 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, 719–728.
16. 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, 682–691.
17. Walker, J.; Martin, C.; Callaghan, R. Eur. J. Cancer 2004,
40, 594–605.
7.5.6. 6,7-Dimethoxy-2-(6-methoxy-naphthalen-2-ylmethyl)
-1,2,3,4-tetrahydro-isoquinoline (7h). White solid, 4%
yield from column chromatography (CHCl3). Recrystal-
lized from MeOH/Et2O. Mp 232–236 ꢁC. Anal.
(C23H25NO3ÆHClÆ0.5H2O) C, H, N. UV–vis kmax
=
278 nm (PBS) e = 5310. ESI+/MS m/z 364 (ESI M++1,
43), 171 (M+ 100). 1H NMR: d 2.78–2.86 (m, 4H,
NCH2CH2 and NCH2 isoquinoline), 3.58 (s, 2H,
NCH2 naphthalene), 3.77–3.92 (m, 11H, 3 of CH3 and
NCH2CH2), 6.46–7.73 (m, 8H, aromatic).
7.6. Statistical analysis
18. Mistry, P.; Stewart, A. J.; Dangerfield, W.; Okiji, S.;
Liddle, C.; Bootle, D.; Plumb, J. A.; Templeton, D.;
Charlton, P. Cancer Res. 2001, 61, 749–758.
19. Fox, E.; Bates, S. E. Expert Rev. Anticancer Ther. 2007, 7,
447–459.
The EC50 values of the compounds reported in Tables 1
and 2 were determined by non-linear curve fitting utiliz-
ing the GraphPad Prism program.33
20. Azzariti, A.; Colabufo, N. A.; Berardi, F.; Porcelli, L.;
Niso, M.; Simone, M. G.; Perrone, R.; Paradiso, A. Mol.
Cancer Ther. 2006, 5, 1807–1816.
Supplementary data
21. Colabufo, N. A.; Berardi, F.; Cantore, M.; Perrone, M.
G.; Contino, M.; Inglese, C.; Niso, M.; Perrone, R.;
Azzariti, A.; Simone, G. M.; Porcelli, L.; Paradiso, A.
Bioorg. Med. Chem. 2008, 16, 362–373.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2008.01.055.
22. Polli, J. W.; Wring, S. A.; Humphreys, J. E.; Huang, L.;
Morgan, J. B.; Webster, L. O.; Serabjit-Singh, C. S.
J. Pharmacol. Exper. Ther. 2001, 299, 620–628.
23. Higgins, C. F. Nature 2007, 446, 749–757.
24. Boumendjel, A.; Macalou, S.; Ahmed-Belkacem, A.;
Blanc, M.; Di Pietro, A. Bioorg. Med. Chem. 2007, 15,
2892–2897.
25. Perrone, R.; Berardi, F.; Colabufo, N. A.; Leopoldo, M.;
Tortorella, V. J. Med. Chem. 1999, 42, 490–496.
26. Bambal, R. B.; Hanzlik, R. P. Arch. Biochem. Biophys.
1996, 334, 59–66.
27. Leopoldo, M.; Lacivita, E.; De Giorgio, P.; Colabufo, N.
A.; Niso, M.; Berardi, F.; Perrone, R. J. Med. Chem. 2006,
49, 358–365.
28. Colabufo, N. A.; Berardi, F.; Perrone, R.; Rapposelli, S.;
Digiacomo, M.; Balsamo, A. J. Med. Chem. 2006, 49,
6607–6613.
References and notes
1. Longley, D. B.; Johnston, P. G. J. Phatol. 2005, 205, 275–292.
2. Altemberg, G. A. Curr. Med. Chem. 2004, 4, 53–62.
3. Borst, P.; Evers, R.; Kool, M.; Wijnholds, J. J. Nat.
Cancer Inst. 2000, 92, 1295–1302.
4. Thomas, H.; Coley, H. M. Cancer Control 2003, 10, 159–
165.
5. Nobili, S.; Landini, I.; Giglioni, B.; Mini, E. Curr. Drug
Target 2006, 7, 861–879.
6. Perez-Tomas, R. Curr. Med. Chem. 2006, 13, 1859–1876.
7. Teodori, E.; Dei, S.; Martelli, S.; Scapecchi, F.; Gualtieri,
F. Curr. Drug Targets 2006, 7, 893–909.
´
´
8. Safa, A. R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7187–
7191.
29. Rautio, J.; Humphreys, J. E.; Webster, L. O.;
Balakrishnan, A.; Keogh, J. P.; Kunta, J. R.; Serabjit-
Singh, J.; Polli, J. W. Drug Metab. Dispos. 2006, 3, 786–
792.
9. Nagaoka, R.; Iwasaki, T.; Rokutanda, N.; Takeshita, A.;
Koibuchi, Y.; Horiguchi, J.; Shimokawa, N.; Iino, Y.;
Morishita, Y.; Koibuchi, N. Endocrine 2006, 30,
261–268.
30. Kangas, L.; Gro¨nroos, M.; Nieminem, A. L. Med. Biol.
1984, 62, 338–343.
31. Taub, M. E.; Podila, L.; Almeida, I. Drug Metab. Dispos.
2005, 33, 1679–1687.
32. Colabufo, N. A.; Berardi, F.; Contino, M.; Niso, M.;
Abate, C.; Perrone, R.; Tortorella, V. Naunyn-Schmiede-
berg’s Arch. Pharmacol. 2004, 370, 106–113.
33. GraphPad Prism Software, version for Windows; Graph-
Pad Software, Inc.: San Diego, CA, 1998.
´
´
´
10. Baekelandt, M.; Lehne, G.; Trope, C. G.; Szanto, I.;
Gustavssson, B.; Kristensen, G. B. J. Clin. Oncol. 2001,
19, 2983–2993.
11. Minderman, H.; O’Loughlin, K. L.; Pendyala, L.; Baer,
M. R. Clin. Cancer Res. 2004, 10, 1826–1834.
12. 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, 297–305.