A Potent and Selective P-gp Modulator for Altering Multidrug Resistance Due to Pump Overexpression

Article abstract of DOI:10.1002/cmdc.201500538

P-glycoprotein (P-gp) is a membrane protein responsible for the active transport of several endogenous and exogenous substances. It constitutes a defense mechanism and, at the same time, it severely compromises the success rate of antitumor chemotherapy. In this study a small library of alkyl/oxyalkyl derivatives of MC70 [4′-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-ylmethyl)biphenyl-4-ol], a well-known P-gp inhibitor, was synthesized through straightforward functionalization of the phenolic group present in the structure of MC70. All compounds were characterized for their effect on P-gp, proving capable of blocking P-gp-mediated calcein-AM efflux with micromolar potency, following their ability to act as high-affinity substrates of this transporter. Excitingly, compound 4 [6,7-dimethoxy-2-((4′-butoxybiphen-4-yl)methyl)-1,2,3,4-tetrahydroisoquinoline] exhibited low nanomolar potency (5.2 nm) and had a peculiar activity profile, acting both as a positive allosteric modulator and as a substrate of the transporter. A new and more efficient synthesis of MC70 is also described. Simple chemical modification of MC70, a well-known but rather nonselective P-glycoprotein (P-gp) inhibitor, led to a very potent and selective P-gp ligand (EC₅₀=5.2 nm). The derivative displayed an intriguing double-faced mechanism of action, acting as both substrate and modulator. The synthesis of MC70 was also greatly improved, using a straightforward and protecting-group-free Suzuki-Miyaura coupling of halophenol.

Full text of DOI:10.1002/cmdc.201500538

DOI: 10.1002/cmdc.201500538
Communications
A Potent and Selective P-gp Modulator for Altering
Multidrug Resistance Due to Pump Overexpression
[a]
[b]
[a]
[b]
Stefano Guglielmo,* Marialessandra Contino, Loretta Lazzarato, Maria Grazia Perrone,
[a]
[a]
[b, c]
Marco Blangetti, Roberta Fruttero, and Nicola Antonio Colabufo*
P-glycoprotein (P-gp) is a membrane protein responsible for
the active transport of several endogenous and exogenous
substances. It constitutes a defense mechanism and, at the
same time, it severely compromises the success rate of antitu-
mor chemotherapy. In this study a small library of alkyl/oxyalk-
yl derivatives of MC70 [4’-(6,7-dimethoxy-3,4-dihydro-1H-iso-
quinolin-2-ylmethyl)biphenyl-4-ol], a well-known P-gp inhibitor,
was synthesized through straightforward functionalization of
the phenolic group present in the structure of MC70. All com-
pounds were characterized for their effect on P-gp, proving ca-
pable of blocking P-gp-mediated calcein-AM efflux with micro-
molar potency, following their ability to act as high-affinity
substrates of this transporter. Excitingly, compound 4 [6,7-di-
methoxy-2-((4’-butoxybiphen-4-yl)methyl)-1,2,3,4-tetrahydroiso-
quinoline] exhibited low nanomolar potency (5.2 nm) and had
a peculiar activity profile, acting both as a positive allosteric
modulator and as a substrate of the transporter. A new and
more efficient synthesis of MC70 is also described.
been suggested for reversing MDR is to co-administer the anti-
tumor agent with a product that is able to inhibit drug efflux
[
4]
by the pump.
Most of these products have been studied for their effect on
P-gp, and according to their mechanism of action, can be clas-
sified into three groups: substrates, inhibitors, and modulators.
The substrates are actively transported molecules, and high
concentrations of these products saturate the binding sites of
the protein, thus blocking it. The inhibitors block the transloca-
tion activity of the pump by inhibiting ATP binding to the pro-
tein’s two nucleotide binding domains (NBD), and consequent-
ly inhibiting its hydrolysis. The modulators decrease the availa-
bility of substrate binding to its site through a negative alloste-
[
5]
ric interaction. Biphenyl derivatives bearing a tetrahydroiso-
quinoline moiety have emerged as an interesting class of
products displaying P-gp-inhibitory activity, and some of them
[
6]
also show selectivity toward the BCRP transporter. The most
active member of this series is compound 1 (MC70), which
bears a hydroxy group at the 4’ position of the biphenyl scaf-
[
6,7]
fold.
This product is unable to activate ATPase, thus it must
The major limit to the full success of cancer chemotherapy is
the progressive unresponsiveness of the tumor toward anti-
cancer drugs differing in structure and mechanism of action.
This phenomenon is known as multidrug resistance (MDR). A
number of mechanisms underlie MDR; the most important is
the overexpression of integral membrane proteins known as
ATP binding cassette (ABC) transporters, which pump drugs
out of the cell, thereby decreasing their efficacy. These trans-
porters use the hydrolysis of ATP as their source of energy for
the translocation of their substrates. ABC-B1, better known as
P-glycoprotein (P-gp), ABC-C1-6 (MRP1-6 multidrug resistance
associated proteins), and breast cancer resistant protein (BCRP)
be considered a P-gp inhibitor. It acts as a potent inhibitor
(EC =0.69 mm), and is unable to interact effectively with the
50
BCRP pump (19% inhibition at 100 mm). The antiproliferative
activity of doxorubicin (5 mm) on the MCF-7/Adr cell line after
pretreatment with 1 at 2 and 20 mm increased from 28 to 42%
and to 98%, respectively. To shed light on the structure–activi-
ty relationships of this lead compound, herein we report the
results of our study investigating the capacity of a series of
simple alkyl and oxyalkyl derivatives of MC70 (compounds 2–
11) to block P-gp. An improved synthetic route to MC70 is also
proposed.
The target compounds were synthesized by alkylation of
phenolic derivative 1 (MC70) with the appropriate commercial-
ly available iodo (derivatives 2–9) or bromo (derivatives 10 and
[1–3]
are the most significant transporters.
A strategy that has
1
1) reagents, in CH CN at reflux, with K2CO as base
[
a] Dr. S. Guglielmo, Prof. L. Lazzarato, Dr. M. Blangetti, Prof. R. Fruttero
Department of Drug Science and Technology
Università degli Studi di Torino, Via P. Giuria 9, 10125 Turin (Italy)
E-mail: stefano.guglielmo@unito.it
3
3
(Scheme 1). The derivatives were obtained in moderate to
good yields (Supporting Information).
A procedure to synthesize the starting material MC70 has al-
[
b] Dr. M. Contino, Dr. M. G. Perrone, Prof. N. A. Colabufo
Dipartimento di Farmacia-Scienze del Farmaco
Università degli Studi di Bari “A. Moro”
[6]
ready been reported. Although the synthesis is straightfor-
ward (Scheme 2), the two steps lead to a low overall yield
(16%), and the reaction mixtures are difficult to treat and
purify. In view of these drawbacks, a new synthetic route
via Orabona 4, 70125, Bari (Italy)
E-mail: nicolaantonio.colabufo@uniba.it
[
8]
[
c] Prof. N. A. Colabufo
Biofordrug s.r.l., Spin-off dell’Università degli Studi di Bari “A. Moro”
via Orabona 4, 70125, Bari (Italy)
based on the well-known Suzuki–Miyaura coupling reaction
is described herein (Scheme 3). In particular, the biphenyl inter-
mediate 14 was obtained by starting from the commercially
available 4-hydroxymethylphenylboronic acid (12) and 4-iodo-
phenol (13), through a recently reported procedure for the
Supporting information and ORCID(s) from the author(s) for this article
are available on the WWW under http://dx.doi.org/10.1002/
cmdc.201500538.
ChemMedChem 2016, 11, 374 – 376
374
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Communications
[
9]
protecting-group-free coupling of halophenols; the catalyst
used was the inexpensive palladium on activated carbon, and
the reaction was conducted with water as solvent; compound
achieved by heating compound 16 in a 1:1 mixture of CH CN
3
and 1n aqueous NaOH to give MC70 in 66% overall yield.
To evaluate the potency of each synthesized ligand toward
P-gp and the P-gp-interaction mechanism, all derivatives were
tested by three combined assays: 1) apparent permeability de-
1
4 was isolated in high yield after workup without the need of
further purification. In the subsequent step, the activation of
benzylic alcohol was achieved with p-toluenesulfonyl chloride
in basic medium. In this reaction, the experimental conditions
were not controlled, and an excess of p-toluenesulfonyl chlo-
ride was used, leading to the formation of the doubly sulfony-
lated compound 15. This intermediate was immediately react-
ed with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydro-
chloride and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) under
mild conditions; as expected, this reaction afforded the benzyl-
ic amine 16 as sole reaction product in 72% yield over the two
steps. Hydrolysis of the p-toluenesulfonate group was easily
[
10]
termination in Caco-2 cell monolayers: the Papp value is rep-
resentative of the P-gp-interaction mechanism of the given
test compound, and thus the protein binding sites involved in
the interaction; 2) inhibition of the transport of calcein-AM,
a pro-fluorescent P-gp substrate, in cells overexpressing P-gp
[
10]
(MDCK-MDR1 cells); and 3) determination of the consump-
[
10]
tion of ATP in MDCK-MDR1 cells. By the calcein-AM assay,
the potency of the interaction toward P-gp can be defined.
The activity profile (as substrate or inhibitor) of all ligands can
be determined by combining the three above assays. Sub-
strates inhibit transport of calcein-AM, as transported, induce
a decrease in cellular ATP levels, and show a Papp value >2; in-
hibitors block calcein-AM transport, but, not being transported,
do not induce a decrease in ATP levels, and show a Papp value
<
2. This experimental classification can be used to easily dis-
tinguish between an inhibitor and a substrate. There is an ad-
[
11]
ditional particular kind of substrate, known as a modulator.
Cyclosporin A (CSA) is a substrate/modulator belonging to the
[
11]
category IIB3, as it is transported, competes with a P-gp sub-
strate, but is unable to activate ATPase. Modulators and inhibi-
tors exert the same final biological effect, restoring cell sensi-
tivity to chemotherapeutic agents, and are usually co-adminis-
tered with radiotracers to study the pharmacodynamic profile
in vivo of a new PET radiotracer candidate.
2 3 3
Scheme 1. Reagents and conditions: a) K CO , CH CN, reflux.
The selectivity of all ligands was established by testing their
effect toward MRP1 (calcein-AM assay on MDCK cells overex-
pressing MRP1). As depicted in Table 1, all compounds dis-
played substrate behavior (Papp >2 in all cases) and had poten-
cies similar to that of the lead. Compounds 10 and 11, which
bear oxyalkyl chains, were the most active agents of the series.
Compound 4 is an exception to this trend, showing particularly
high activity (5.2 nm).
To shed light on the reasons for the very high activity of 4,
its behavior was studied in the presence of 30 mm CSA, a well-
[
12]
known product able to bind the modulator site of P-gp. We
selected this dose (30 mm) because we preliminarily probed all
the concentrations of modulator (from 0.5 to 30 mm) and
[
6]
Scheme 2. Reported synthesis for MC70. Reagents and conditions: a) SOCl
2
,
Et N cat., 708C; b) CH Cl , 2n NaOH(aq); c) LiAlH , THF, reflux.
3
2
2
4
Scheme 3. Reagents and conditions: a) K
2
CO
3
, Pd/C cat., H
2
O, 808C, 45 min; b) p-toluenesulfonyl chloride, Et
CN 1:1, 808C, 6 h.
3 2 2
N, DMAP cat., CH Cl , RT, 4 h; c) 6,7-dimethoxy-
1
,2,3,4-tetrahydroisoquinoline hydrochloride, DBU, CH
3
CN, 658C, 2 h; d) 1n NaOH/CH
3
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Table 1. Biochemical characterization of compounds 2–11.
[
a]
[c]
Compd
R
EC50 [mm]
P
app
P-gp
MRP1
[
b]
2
3
4
5
6
7
0.63Æ0.12
1.20Æ0.20
NA
3.96Æ0.20
[
b]
NA >20
[
b]
Figure 1. Representative P-gp activity evaluation of compound 4 at several
concentrations (0.1–100 nm) in the absence (blue bars) and in the presence
of 30 mm CSA (grey bars). Results are the mean of three independent experi-
ments (samples for each concentration in duplicate). Statistical evaluation
gave p=0.001 (*p<0.05, significant; **p<0.005).
0.0052Æ0.001 NA >20
[
b]
0.96Æ0.22
NA >20
[
b]
[b]
NA
NA >20
[
b]
[b]
NA
NA
NA
15Æ1.2
[
[
b]
b]
8
9
0.82Æ0.16
1.25Æ0.25
3.3+0.10
with the immunomodulator, it is the first molecule devoid of
any other pharmacological activities exerting this effect. There-
fore, compound 4 discloses new perspectives in reverting MDR
by the co-administration of chemotherapeutic drugs and a P-
gp modulator.
NA
3.5Æ0.12
[
[
b]
b]
1
0
0.42Æ0.08
0.32Æ0.06
NA
NA
9.8Æ0.15
4.1Æ0.3
11
[
7]
[7]
[6]
1
(MC70)
H
0.69
9.30
1.3
Acknowledgements
[
a] Data are the meanÆSEM of three independent determinations (sam-
ples in triplicate). [b] Not active at 100 mm. [c] Data are the mean with
SEM<10% of three independent determinations (samples in triplicate).
This study was supported by the University of Turin grant “Ricerca
Locale-GUGSRILO13”, and MIUR (FIRB 2012 RBFR12SOQ1_003).
We are grateful to Prof. Alberto Gasco for helpful discussions.
found that CSA exerts the highest effect at 30 mm. In this ex-
periment, compound 4 was used in the concentration range of
Keywords: antitumor agents · CÀC coupling · multidrug
resistance · P-glycoprotein · proteins
0
.1–100 nm. Under these conditions, the compound can only
bind the substrate site, as the modulator site is occupied by
CSA. Figure 1, which give the results of the experiments,
shows that the activity at P-gp decreases markedly in the pres-
ence of CSA, suggesting that binding to the modulator site
positively modulates the P-gp substrate site.
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known P-gp inhibitor. A small library of alkyl/oxyalkyl deriva-
tives of MC70 was synthesized and studied in order to eluci-
date the pharmacological profiles in terms of potency, selectivi-
ty, and mechanism of interaction with P-gp. Most compounds
proved to be selective inhibitors of P-gp-mediated calcein-AM
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potency in the low or sub-micromolar range, similar to that of
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Received: November 17, 2015
Revised: January 8, 2016
Published online on January 21, 2016
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www.chemmedchem.org
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim