New tetrahydroisoquinoline-based P-glycoprotein modulators: Decoration of the biphenyl core gives selective ligands

Article abstract of DOI:10.1039/c8md00075a

P-glycoprotein (P-gp, MDR1) is a membrane transporter expressed in several regions of our body. It plays a crucial defense role as it mediates the efflux of hundreds of potentially toxic substances. However, P-gp is one of the main causes of failure in ca

Full text of DOI:10.1039/c8md00075a

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Riganti, K. A. Zabielska-Koczyws, A. N. Colabufo and R. Fruttero, Med. Chem. Commun., 2018, DOI:
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New tetrahydroisoquinoline based P-glycoprotein modulators:
decoration of the biphenyl core gives selective ligands.
Marialessandra Contino,[a]‡ Stefano Guglielmo,*[b]‡ Maria Grazia Perrone,[a] Roberta
Giampietro,[a] Barbara Rolando,[b] Antonio Carrieri,[a] Daniele Zaccaria,[a] Konstantin
Chegaev,[b] Vanessa Borio,[b] Chiara Riganti,[c] Katarzyna ZabielskaꢀKoczywąs,[d] Nicola A.
Colabufo,*[a, e] and Roberta Fruttero[b]
[a]
Dr. M. Contino, Dr. M. G. Perrone, Dr. R. Giampietro, Prof. A. Carrieri, D. Zaccaria,
Prof. N. A. Colabufo
Dipartimento di FarmaciaꢀScienze del Farmaco Universita' degli Studi di Bari "Aldo Moro"
Via Orabona 4, 70125 Bari, Italy
Eꢀmail: nicolaantonio.colabufo@uniba.it
[b]
Dr. S. Guglielmo, Prof. B. Rolando, Dr. K. Chegaev, V. Borio, Prof. R. Fruttero
Dipartimento di Scienza e Tecnologia del Farmaco Universita' degli Studi di Torino Via P.
Giuria 9, 10125 Torino, Italy
Eꢀmail: stefano.guglielmo@unito.it
[c]
Santena 5/bis, 10126 Torino, Italy
[d] Dr. Katarzyna ZabielskaꢀKoczywąs Department of Small Animal Diseases with
Prof. C. Riganti Dipartimento di Oncologia, Università degli Studi di Torino, via
Clinic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences,
Nowoursynowska 159c, 02ꢀ776, Warsaw, Poland
[e]
Prof. N. A. Colabufo Biofordrug s.r.l., Spinꢀoff dell’Università degli Studi di Bari “A.
Moro” Via Orabona 4, 70125 Bari, Italy
‡The authors contributed equally.
Abstract: Pꢀglycoprotein (Pꢀgp, MDR1) is a membrane transporter expressed in several
districts of our body. It exerts a crucial defense role as it effluxes hundreds of potentially
toxic substances. Hovewer, Pꢀgp is one of the main causes of the failure in cancer
chemotherapy, as a number of chemotherapeutic agents are Pꢀgp substrates. Another
interesting implication regards the correlation between Pꢀgp expression impairment and
the onset of several central nervous system pathologies such as Alzheimer’s and
Parkinson’s diseases. In view of these considerations, in the present study a new series of
Pꢀgp modulators has been designed, synthesized and evaluated for their activity towards
Pꢀgp and other two sister proteins (BCRP and MRP1). The compounds, structurally
correlated to the potent but nonꢀselective Pꢀgp inhibitor MC70 [4’ꢀ(6,7ꢀdimethoxyꢀ3,4ꢀ
dihydroꢀ1Hꢀisoquinolinꢀ2ꢀylmethyl)biphenylꢀ4ꢀol], proved fairly selective towards Pꢀgp, with
a potency in the micromolar range. Compounds 5a, 5d and 12d proved capable to restore
doxorubicin toxicity in resistant cancer cells.
Keywords: Pꢀgp, MDR, tetrahydroisoquinoline, Pꢀgp ligands
Introduction
Pꢀglycoprotein (Pꢀgp, also known as MDR1 or ABCB1) is a membrane protein belonging to
ATPꢀbinding cassettes (ABC) superfamily; it is a complex molecular machinery able to
recognize and efflux hundreds of structurally uncorrelated substances exploiting energy

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derived by ATP hydrolysis. It has a strategic localization in a number of organs and
tissues, thus exerting a crucial defence role against toxic substances, both of endogenous
and exogenous origin, and constituting an essential component of several biological
barriers.[1] Pꢀgp has raised great attention since several years ago, owing to its
involvement in multidrug resistance (MDR), a phenomenon that represents one of the
main causes of cancer chemotherapy failure. In some tumors MDR appears without a
previous exposure to antiꢀcancer drugs, whilst in other cases its onset occurs during
treatment, quite often as a consequence of selection of resistant over sensitive cells in
heterogeneous tumor tissue.[2] Pꢀgp is overexpressed in cancer cells and is responsible of
the efflux of several chemotherapeutic agents, such as taxanes, vinca alkaloids,
doxorubicin, etoposide, topotecan, methotrexate, imatinib, dasatinib, gefitinib among
others.[3] The wide expression of Pꢀgp on the surface of endothelial cells at the bloodꢀbrain
barrier makes it a gatekeeper for central nervous system (CNS), as it prevents potentially
harmful substances from entering, but also hampers many structurally and functionally
uncorrelated drugs, compromising the success of pharmacological treatment of different
CNS disorders and tumours. More recently, an interesting connection has emerged
between Pꢀgp and the onset of Alzheimer’s Disease (AD), Parkinson’s Disease (PD),
epilepsy and other CNS diseases: an impairment in the expression level of the protein has
been observed in the early stages of these neurological disorders.[4,5] Starting from this
background, medicinal chemistry efforts to target Pꢀgp have been undertaken, mainly in
the aim of obtaining inhibitors which should be coꢀadministered with chemotherapeutic
agents subjected to Pꢀgp mediated efflux, thus restoring therapy efficacy. According to this
approach a number of MDR reversal agents have been reported, which are usually
classified in three generations; despite promising in vitro results, most of them failed in the
clinical trial phase for several reasons: in the case of first generation agents which are
drugs in clinical use for different indications, the main drawback was poor selectivity of
action.[6] Modulators of the second generation, supposed to be more selective, proved
toxic.[7] As to third generation agents, despite their high affinity for Pꢀgp, they did not return
satisfying results due to pharmacokinetic and safety concerns.[8] Thus the development of
new agents able to impede Pꢀgp mediated drug efflux is still an unmet need and, as MDR
remains a prominent issue in cancer chemotherapy, it is still a field of interest in medicinal
chemistry. In view of this, an alternative approach is the search of broad spectrum
inhibitors targeting different transporters, which found a very recent exemplification in the
study of Stefan et al. reporting on the design of 9ꢀdeazapurineꢀbased inhibitors able to
counteract drug efflux mediated by Pꢀgp, multidrug resistanceꢀassociated protein 1
(MRP1, ABCC1) and breast caner resistance protein (BCRP, ABCG2).[9]
Besides the design of Pꢀgp modulators to be flanked to chemotherapy agents, recently
different approaches to counteract MDR have been undertaken by several groups. An
important strategy is obviously the development of antiꢀcancer drugs that are not
substrates of Pꢀgp, both through design of new molecules or modification of already known
drugs.[10] Some successful examples have been obtained, among others, with taxanes,
with epothilones (a class of natural compounds structurally correlated to taxaxnes) and
with vinca alkaloids.[11] In the setting of this strategy, given the lack of structural insights
into the mechanism of interaction of antiꢀcancer drugs with Pꢀgp, it is difficult to envisage

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the nature and the position of the structural modifications required to evade Pꢀgp efflux
and to hold desireable cytotoxicity.
Another strategy that has been undertaken to overcome Pꢀgp mediated efflux consists in
enhancing drug uptake or modifying the mechanism of cellular uptake through nonꢀ
covalent or covalent conjugation of chemotherapy agents with targeting systems such as
nanoꢀparticles, liposomes, micelles, polymeric conjugates, antibodies among others.
Particular attention has been paid to liposome formulation and a number of such systems
is currently under clinical evaluation.[12] A word of caution must be spent regarding this
approach, as the intracellular release of the drugs from these formulations may still result
in Pꢀgp efflux. Regarding covalent derivatives, strategies that gave interesting results
consist in the modification of the drug through the introduction of guanidiniumꢀrich tags,
with consequent higher water solubility and modifyied recognition by Pꢀgp[13,14], and of
more complex cationic oligopeptideꢀbased moieties, as in the case of Taxolꢀoctaarginine
conjugates.[15]
Besides the above reported approaches, based on mainly medicinal chemistry tools, in the
recent past some less explored strategies have been attempted to counteract MDR,
aiming at the downregulation of ABCB1 gene expression or translation, such as antisense
oligonucleotides and small interfering RNA.[16]
In addition to the MDR issue, the potential application in the early diagnosis through
imaging techniques such as Positron Emission Tomography (PET) of several neurological
disorders has been proposed for Pꢀgp ligands, in view of the involvement of the transporter
in the onset of these CNS pathologies: in particular radiolabeled substrates are useful to
measure in vivo function of the transporter at the blood brain barrier (BBB).[17]
Following the heavy effects of Pꢀgp mediated MDR on cancer chemotherapy, despite the
obstacles encountered so far, the development of new Pꢀgp modulators still appears as a
valuable and possibly straightforward route to counteract MDR. In this ongoing effort and
also in view of the application of Pꢀgp ligands in early diagnosis of some CNS
degenerative diseases, in the present study the authors have carried out several
modifications on MC70 (Figure 1), a previously studied Pꢀgp inhibitor.[18,19] MC70 shows a
good Pꢀgp inhibiting potency (EC50 = 0.69 ꢀM), on the other hand displaying a nonꢀ
selective profile towards Pꢀgp.
OH
O
O
N
MC70
EC50 = 0.69 M (Pꢀgp)
ꢀ
EC50 = 9.30 M (MRP1)
ꢀ
Figure 1. Structure and biological activity profile of MC70.
The authors have previously explored structure activity relationships of MC70 derivatives:
in particular they focused on functionalization of phenolic group with alkyl and oxyalkyl
chains,[20] and with more complex moieties containing variously substituted furazan (1,2,5ꢀ

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oxadiazole) rings.[21] In the present investigation the structural modifications involve the
biphenyl core: the aim of the study was to evaluate, in terms of inhibition potency and
selectivity on Pꢀgp, the effect of substituents able to modify electronic properties and
endowed with hydrogen bond donor or acceptor properties. Thus at the four positions of
the biphenyl moiety different substituents were inserted: fluorine, a small strongly
electronegative atom, able to accept hydrogen bond, nitro group, a prototypical electronꢀ
withdrawing group with strong resonance effect and hydrogen bond acceptor properties,
methoxy group, an electronꢀdonating substituent with resonance effect and hydrogen bond
acceptor properties, and amino group, electron donating with hydrogen bond donor and
acceptor properties.
The compounds have been synthesized through an inexpensive and straightforward route;
compared to MC70, potency was slightly decreased, EC50 values being in the micromolar
range for most compounds, but all the ligands display high selectivity towards Pꢀgp, being
essentially inactive against MRP1 and BCRP. Three of them have been further evaluated
in coꢀadministration with doxorubicin and proved able to restore intracellular concentration
of the anthracycline to different extent, without any effects on Pꢀgp expression. A sensible
insight of the binding pose of a selected compound has been gained through a molecular
docking study.
Results and discussion
Synthesis of target compounds
The target compounds were synthesized according to the methods reported in Scheme 1
and 2. In brief, the key step is a Suzuki coupling reaction,[22] carried out with palladium on
activated charcoal in a ligand free fashion in aqueous environment, starting from the 4ꢀ
bromobenzylalcohols 1a-f (Scheme 1), or the 4ꢀbromophenols 8a-e (Scheme 2). The
substrates
were
coupled
with
4ꢀhydroxyphenylboronic
acid
or
4ꢀ
hydroxymethylphenylboronic acid respectively. The coupling products were then reacted
with an excess of pꢀtoluensulfonyl chloride in presence of triethylamine, to give the diꢀ
tosylate derivatives 3a-f (Scheme 1) or 10a-e (Scheme 2). The latter was used without
purification to obtain benzylation of 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline. Finally
the removal of the phenyl tosylate group was achieved through hydrolysis with NaOH in
THF/CH3OH mixture.
The two benzylic alcohols 1c and 1d were synthesized starting from the corresponding
benzoic acids through reduction with BH3ꢀTHF complex (Scheme 1).

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OH
OTs
R
R
O
O
OH
R
b
c
d
N
a
Br
OH
OTs
4a-f
R
3a-f
1a-f
2a-f
OH
R
3' ꢀOMe
3' ꢀF
5a
5b
5c
O
O
OTs
2'
N
3' ꢀNO2
R
3'
5a-f
2' ꢀOMe
2' ꢀF
5d
5e
2' ꢀNO2
5f
3
3
NO2
2
NO2
2
Br
Br
e
O
OH
OH
: 2 ꢀNO
: 3 ꢀNO
6
7
: 2 ꢀNO
2
1c
2
2
: 3 ꢀNO
1f
2
Scheme 1. Synthesis of target compounds 5a-f. Reagents and conditions: a) 4ꢀ
hydroxybenzeneboronic acid, KOH, Pd/C cat., H2O, 130 °C, 3 hours; b) pꢀtoluenesulfonyl
chloride, Et3N, DMAP cat., CH2Cl2, room temperature, 6 hours; c) 6,7ꢀdimethoxyꢀ1,2,3,4ꢀ
tetrahydroisoquinoline hydrochloride, DBU, CH3CN, 70 °C, 18 hours; d) NaOH,
THF/CH3OH 2/1, 65 °C, 1 hour; e) BH3–THF complex, THF, N2, 18 hours, room
temperature.
As for compound 16, the procedure reported above for coupling reaction starting from 1ꢀ
bromoꢀ4ꢀmethoxyꢀ2ꢀnitrobenzene 13 did not afford the desired intermediate, probably due
to the poor solubility of 13. For this reason the Suzuki coupling reaction was carried out in
a more “traditional” manner (Scheme 2) using palladium tetrakis triphenylphosphine as
catalyst in 1,4ꢀdioxane/water mixture; the intermediate 14 was subdued to hydrolysis of
methoxy group with BBr3, obtaining the concomitant bromination of benzylic alcohol. The
bromo derivative 15 was then reacted with 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline,
yielding compound 16.
Finally the two amino substituted compounds 17a and 17b were obtained through catalytic
hydrogenation of the nitro derivatives 12c and 16 (Scheme 2).

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O
O
OH
OTs
N
R
Br
c
b
a
d
R
R
OH
OH
OTs
8a-e
9a-e
10a-e
11a-e
R
R
OH
3 ꢀOMe
3 ꢀF
12a
12b
12c
OTs
O
O
3
R
2
N
3 ꢀNO2
12a-e
2 ꢀOMe
2 ꢀF
12d
12e
O
OH
Br
N
NO2
O
NO2
NO2
Br
c
e
f
O
O
OH
13
14
15
16
NO2
OH
OH
OH
O
O
O
3
3
g
NH2
NO2
2
2
N
N
O
: 3 ꢀNH
17a
12c,16
2
: 2 ꢀNH
17b
2
Scheme 2. Synthesis of target compounds 12aꢀe, 16, 17a,b. Reagents and conditions: a)
4ꢀhydroxymethylbenzeneboronic acid, KOH, Pd/C cat., H2O, 130 °C, 3 hours; b) pꢀ
toluenesulfonyl chloride, Et3N, DMAP cat., CH2Cl2, room temperature, 6h; c) 6,7ꢀ
dimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline hydrochloride, DBU, CH3CN, 70 °C, 18 hours; d)
NaOH, THF/CH3OH 2/1, 65 °C, 1 hour; e) 4ꢀhydroxymethylbenzeneboronic acid, K2CO3,
Pd[(C6H5)3P]4, 1,4ꢀdioxane/water 2/1, N2, 90 °C, 18 hours; f) BBr3, CH2Cl2, 18 hours, room
temperature; g) H2, Pd/C cat., CH3OH, room temperature, 3 hours.
Biological evaluation
All the new synthesized compounds have been tested to establish their Pꢀgp interacting
mechanism as substrate or inhibitor by three assays: 1) the inhibition of the transport of a
fluorescent or a proꢀfluorescent substrate of the transporter;[23] 2) the determination of the
apparent permeability value (Papp);[23] 3) the detection of the ATP cell level depletion;[23] the
results are reported in Table 1.
Table 1. Biological characterization of target compounds.
MRP1
BCRP
EC50
MDR1
ATP
Papp
[b]
Compound Structure
EC50
EC50 (ꢀM) [a]
consumption
(ꢀM) [a] (ꢀM) [a]

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OH
O
O
5a
1.60 ± 0.27
na[c]
na
na
na
NO[c]
4.5
N
O
OH
O
O
5b
5c
5d
4.73 ± 0.90
8.4 ± 1.58
1.51 ± 0.30
na
na
NO
NO
NO
4.1
5.8
4.4
N
F
OH
O
O
N
NO2
OH
O
O
38 ± 7.6 na
N
N
O
OH
O
O
5e
6.2 ± 1.24
15.2 ± 3.00
2.0 ± 0.40
2.8 ± 0.56
12.7 ± 2.50
6.9 ± 1.32
1.86 ± 0.37
2.17 ± 0.43
2.54 ± 0.48
na
na
na
na
28.9
na
62
na
na
na
na
na
na
na
na
na
na
na
NO
NO
NO
NO
NO
NO
NO
NO
NO
3.7
2.9
3.9
4.9
6.3
7.5
3.5
4.5
11.2
F
OH
O
O
5f
N
NO2
OH
O
O
O
12a
12b
12c
17a
12d
12e
16
N
N
OH
F
O
O
OH
O
O
NO2
N
OH
O
O
NH2
N
N
N
N
OH
OH
OH
O
O
O
O
O
F
O
O
NO2

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OH
O
O
17b
5.4 ± 1.00
0.69
na
na
73
NO
NO
4.5
1.3
N
NH2
OH
O
O
MC70
9.30
N
[a] The value is the mean of three independent experiments, sample in duplicate. [b]
Apparent permeability determined as BA/AB ratio (the value is from two independent
experiments). [c] Not active at 100 ꢀM. [d] No significative ATP consumption at the activity
doses.
The first assay is performed in a cell line overexpressing Pꢀgp (MDCKꢀMDR1 cells) and it
detects the ability of the ligands to compete with the efflux of the proꢀfluorescent Pꢀgp
substrate CalceinꢀAM towards Pꢀgp; it is the measure of the potency of each compound
towards the target. The second assay evaluates a ratio basolateralꢀapical vs apicalꢀ
basolateral (BA/AB) representative of two contributions, the passive diffusion (BA) and
active transport (AB) in a system mimicking BBB, such as Caco2 cells; if this ratio is < 2,
the compound can be considered a Pꢀgp inhibitor, otherwise (Papp> 2) it behaves as a
substrate. The third assay measures the consumption of ATP in MDCKꢀMDR1 cells.
Generally, a substrate, since transported by the pump, induces an ATP consumption while
an inhibitor, inhibiting the binding of ATP on its site on Pꢀgp, is not transported and thus it
does not induce a decrease in ATP cell level. The selectivity of all compounds towards the
sister proteins BCRP and MRP1 has been also tested by the measure of the ability of the
compounds to interfere with the transport BCRPꢀ or MRP1ꢀmediated of the fluorescent
BCRP substrate Hoechst33342 or the proꢀfluorescent MRP1 substrate CalceinꢀAM.
As depicted in Table 1, all of the compounds were found less active than the lead
compound MC70 (EC50 = 0.69 ꢀM) displaying EC50 values ranging from 1.51 to 15.2 ꢀM,
and among them 5a (EC50 = 1.60 ꢀM), 5d (EC50 = 1.51 ꢀM), and 12d (EC50 = 1.86 ꢀM),
displayed the best activity values. However, all compounds were more selective towards
Pꢀgp, proving inactive towards BCRP and MRP1. Only compound 5d and 12c showed a
moderate MRP1 activity (EC50 = 38 ꢀM and 28.9 ꢀM, respectively). All the ligands, having
a Papp values > 2 and not inducing an ATP cell depletion, behaved as substrates of the
Category IIB3.[18]
As a whole only slight differences can be observed along the series; nevertheless some
general considerations are worthy of note: the presence of methoxy group, electronꢀ
donating and hydrogen bond acceptor, seems to give a slightly higher activity in the series,
no matter the position on the biphenyl core (EC50 ranging from 1.51 ꢀM for 5d, to 2 ꢀM for
12a). On the contrary the presence of a strong electronꢀwithdrawing substituent, such as
nitro group, determines a loss of activity which is more pronounced for compounds where
the substituent influences to a greater extent the acidity of phenol group (position 2’ and 3
of the biphenyl core) (EC50= 15.2 ꢀM for 5f and EC50 = 12.7 ꢀM for 12c). Fluorine atom
seems not to greatly influence the activity, with a slightly more detrimental effect when
present on the benzylic ring (position 2’ and 3’) (EC50= 6,2 ꢀM for 5e). Finally, the

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presence of amino substituent, able to donate hydrogen bond, bears a significant loss of
activity (EC50= 6,9 ꢀM for 17a).
Molecular docking study
A structure based study was then carried to furnish fresh insights into the binding mode of
the new compounds and to support explication to the EC50 data. To fulfill this topic our
previously published Pꢀgp receptor model[21] served as valuable tool to explain ligand pose
of the most active compound (5d). In our past study we have postulated that the “inwardꢀ
outward facing” of the Pꢀgp scaffold should facilitate the “pulling out” of substrates as soon
as they pass the cell bilayer, while active inhibitors would hamper the Pꢀgp flipping
depending on molecular shape and pharmacophoric features of the molecules; in line with
this view, molecular docking further supports this evidence.
As it might be perceived from Figure 2 reporting the binding mode of 5d, the ligand
scaffold properly fits the binding site space delimited by the two six helicesꢀ
transmembrane domains (TMDs), and deeply locks into the crevice comprising the
intracellular moiety of the same domains, with a kind of “reversed wedge” pose that might
be responsible for the hampering of Pꢀgp flipping upon ligand binding.
Figure 2. Binding mode of 5d into the MDR1 binding site. Water molecules are
represented as red spheres, and the extracellular and intracellular sides are at top and
bottom of the scene respectively. The free energy of binding calculated with hydration
force field of AutoDock is ꢀ8.62 kcal/mol, while the contact surface area measures 440 Å2.
Interestingly the tetrahydroisoquinoline ring is placed close to one of the TMDs, and it
occupies a mainly hydrophobic receptor slot, surrounded by Tyr310, Phe336, Phe728 and
Phe983 generating extensive favourable contacts and π−π stacking, as well as a stable

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binding through two water molecules enlacing hydrogen bonding bridges with Tyr310 and
Ser979. The rest of the aromatic moiety points to the opposite TMD with the phenolic
group making polar contacts with the backbone of Met949 and the methoxy group
interacting with Tyr953 throughout a water molecule coordinating a hydrogen bond, and as
long as this is concerned, the same methoxy substituent should oblige, to some extent, the
biphenyl motif in a nonꢀplanar conformation. Indeed, this steric hindrance, in combination
with an hydrogen bond formation, might clarify, at least within this series of derivatives, the
better EC50 value of 5d.
Co-administration of doxorubicin with 5a, 5d and 12d
Compounds 5a, 5d, and 12d, displaying the best Pꢀgp activity profile, have been evaluated
in coꢀadministration with doxorubicin, in order to study their efficacy as doxorubicinꢀ
rehabilitating agents in the treatment of resistant tumours (Figure 3).
Preliminary data demonstrated that the three compounds were not cytotoxic at 48h and
72h (data not shown) and doxorubicin at 10 ꢀM is not able to induce cell death as effluxed
by Pꢀgp in the resistant tumours cell model overexpressing Pꢀgp (MDCKꢀMDR1 cells).
When MC70, 5a, 5d, and 12d were coꢀadministred with doxorubicin, they restore its
antineoplastic cytotoxicity. In fact, while the lead compound MC70 and 5a, 5d displayed a
moderate ability to restore doxorubicin effect (20% of cytotoxicity increase for MC70 and
15% for 5a, 5d), 12d was able to produce an increase of 60% of doxorubicin citotoxicity,
demonstrating its ability to almost completely rehabilitate the access of the antineoplastic
drug in tumour cells. Considering the structural correlation and the similarity of EC50 of the
three tested compounds, it is difficult to envisage the reason for the efficient restoring of
doxorubicin toxicity elicited by 12d; the hypothesis that can be made is a possible
reinforcement of activity at higher concentration than EC50: this can be due to the
peculiarity of the binding region of Pꢀgp, which is large and comprises multiple and
possibly overlapping binding sites. This feature has led several authors to postulate the
possibility for some small molecules to bind simultaneously to different binding sites and
this has also been exploited through the design of dimeric modulators with enhanced
potency.[24]

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Figure 3. Coꢀadministration of doxorubicin (10 ꢁM) with compounds MC70, 5a, 5d, and
12d (10 ꢀM). Antiproliferative effect of MC70, 5a, 5d, and 12d (10 ꢁM) alone (gray bars)
and coꢀadministred with doxorubicin (10 ꢁM) (black bars) at 48h in MDCKꢀMDR1 cell line.
Ctr bar represents the administration of 10 ꢀM doxorubicin alone.
Immunoblotting experiments on MC70, 5a, 5d and 12d
The same compounds (MC70, 5a, 5d and 12d) which were evaluated in coꢀadministration
with doxorubicin were further studied in immunoblotting experiments to verify a possible
interference with Pꢀgp expression, in the same experimental condition of cell viability tests.
As it can be seen in Figure 4, no differences in Pꢀgp expression were observed in treated
cells compared to the control, thus supporting the hypothesis of a direct effect of the
compounds on the transporter.
Figure 4. Immunoblotting experiment with compounds MC70, 5a, 5d, and 12d (10 ꢀM).
Proteins were extracted from MDCKꢀMDR1 cells after 48 hours of incubation. Actin levels
were used as protein loading control. CTRL bands refer to untreated cells. The figure is
representative of 1 out of 3 experiments with similar results.
Conclusion
A new series of Pꢀgp modulators has been developed through the “decoration” of the
biphenyl moiety of MC70 with substituents endowed with different electronic and hydrogen

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bond donor/acceptor properties. The compounds were synthesized exploiting a
straightforward and inexpensive route. They displayed slightly lower potency than the
parent compound but proved highly selective, thus representing suitable candidates to be
used in imaging techniques for measurement of Pꢀgp function at BBB.
A molecular docking simulation carried out for the most potent compound (5d) highlighted
an additional anchoring for the biphenyl moiety through the methoxy substituent, which
engages a hydrogen bond mediated by a water molecule. Besides the biochemical
characterization aimed to clarify the mechanism of interaction of the ligands with Pꢀgp, the
most potent compounds (5a, 5d, and 12d) and MC70 were evaluated in a coꢀ
administration assay with doxorubicin, and proved efficient to restore drug toxicity against
doxorubicin resistant MDCKꢀMDR1 cells. A possible effect on Pꢀgp expression was ruled
out by performing immunoblotting experiment on the same selected compounds.
The present study thus represents an additional investigation of a previously started
structure activity relationship study on tetrahydroisoquinoline derivatives, and furnishes
additional information about the mode of binding of this class of compounds which can be
useful for future development of potent Pꢀgp modulators.
Along the series, compound 12d deserves particular consideration: it represents a
valuable candidate for further development of Pꢀgp mediated efflux reversal agents as it
displayed an interesting efficacy (60%) in restoring doxorubicin cytotoxicity in resistant
tumour cells. Moreover this data probably reflects a peculiar behavior of the compound,
which may interact with different binding sites simultaneously; this hypothesis deserves
further inꢀdepth analyses and, if confirmed, opens the way to the exploitation of a “dimeric
analogue” approach to design Pꢀgp modulator based on biphenylꢀtetrahydroisoquinoline
substructure.
Acknowledgements
This study was supported by the University of Turin ꢀ „Ricerca Locale“ to SG and by MIUR
(FIRB 2012 RBFR12SOQ1_002, FIRB 2012, grant RBFR12SOQ1). This work was also
supported by My First AIRC GrantꢀMFAG2015 (Project Id.17566). We are thankful to Prof.
A. Gasco for fruitful discussion.
Conflict of interest
The authors declare no conflict of interest.
Contributions
S. Guglielmo and N.Colabufo desigend the study and wrote the manuscript.
S. Guglielmo planned the synthetic strategy.
M. Contino planned and performed the biological assays to define the Pꢀgp interacting
mechanism and participated in the editing of the manuscript.
M.G. Perrone performed the permeability assays.
R.Giampietro performed the coꢀadministration assay.
A. Carrieri and D. Zaccaria performed the molecular docking study.
K. Chegaev and V. Borio carried out the synthesis of compounds.
B. Rolando carried out the structural characterization and purity assessment.
C. Riganti and K. ZabielskaꢀKoczywąs carried out the immunoblotting experiments.

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R. Fruttero carried out literature research and wrote the manuscript.
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A new series of P-gp modulators is reported. All compounds show selectivity with EC50 in the low
micromolar range. One of them displays good efficacy in restoring doxorubicin citotoxicity in resistant
cancer cells.
OH
O
O
12a
O
N