1,2,3,4-Tetrahydroisoquinoline/2H-chromen-2-one conjugates as nanomolar P-glycoprotein inhibitors: Molecular determinants for affinity and selectivity over multidrug resistance associated protein 1

Article abstract of DOI:10.1016/j.ejmech.2018.10.043

A series of coniugates bearing a 1,2,3,4-tetrahydroisoquinoline motif linked to substituted 7-hydroxy-2H-chromen-2-ones was synthesized and assayed through calcein-AM test in Madin-Darby Canine Kidney (MDCK) cells overexpressing P-glycoprotein (P-gp) and closely related multidrug resistance associated protein 1 (MRP1) to probe the interference with efflux mechanisms mediated by P-gp and MRP1, respectively. A number of substituents at C3 and C4 of coumarin nucleus along with differently sized and shaped spacers was enrolled to investigate the effects of focused structural modifications over affinity and selectivity. Linker length and flexibility played a key role in enhancing P-gp affinity as proved by the most potent P-gp modulator (3h, IC₅₀ = 70 nM). A phenyl ring within the spacer (3k, 3l, 3o) and bulkier groups (Br in 3r, Ph in 3u) at coumarin C3 led to derivatives showing nanomolar activity (160 nM < IC₅₀ < 280 nM) along with outstanding selectivity over MRP1 (SI > 350). Molecular docking calculations carried out on a human MDR1 homology model structure contributed to gain insights into the ligands’ binding modes. Some compounds (3d, 3h, 3l, 3r, 3t, 3u) reversed MDR thereby restoring doxorubicin cytotoxicity when co-administered with the drug into MDCK-MDR1 cells.

Full text of DOI:10.1016/j.ejmech.2018.10.043

Accepted Manuscript
1,2,3,4-Tetrahydroisoquinoline/2H-chromen-2-one conjugates as nanomolar P-
glycoprotein inhibitors: Molecular determinants for affinity and selectivity over
multidrug resistance associated protein 1
Mariagrazia Rullo, Mauro Niso, Leonardo Pisani, Antonio Carrieri, Nicola Antonio
Colabufo, Saverio Cellamare, Cosimo Damiano Altomare
PII:
S0223-5234(18)30912-7
10.1016/j.ejmech.2018.10.043
EJMECH 10829
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 August 2018
Revised Date: 15 October 2018
Accepted Date: 16 October 2018
Please cite this article as: M. Rullo, M. Niso, L. Pisani, A. Carrieri, N.A. Colabufo, S. Cellamare, C.D.
Altomare, 1,2,3,4-Tetrahydroisoquinoline/2H-chromen-2-one conjugates as nanomolar P-glycoprotein
inhibitors: Molecular determinants for affinity and selectivity over multidrug resistance associated protein
1, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.10.043.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1,2,3,4-Tetrahydroisoquinoline/2H-chromen-2-one conjugates as nanomolar P-
glycoprotein inhibitors: molecular determinants for affinity and selectivity over
multidrug resistance associated protein 1
a
a
a
Mariagrazia Rullo , Mauro Niso , Leonardo Pisani *,a, Antonio Carrieri , Nicola Antonio
Colabufo a, Saverio Cellamare a, Cosimo Damiano Altomare a
a Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, via
E. Orabona 4, 70125 Bari, Italy
*Corresponding author:
Dr. Leonardo Pisani; tel: +390805442803; fax: +390805442230; email: leonardo.pisani@uniba.it
Abstract
A series of coniugates bearing a 1,2,3,4-tetrahydroisoquinoline motif linked to substituted 7-
hydroxy-2H-chromen-2-ones was synthesized and assayed through calcein-AM test in Madin-
Darby Canine Kidney (MDCK) cells overexpressing P-glycoprotein (P-gp) and closely related
multidrug resistance associated protein 1 (MRP1) to probe the interference with efflux mechanisms
mediated by P-gp and MRP1, respectively. A number of substituents at C3 and C4 of coumarin
nucleus along with differently sized and shaped spacers was enrolled to investigate the effects of
focused structural modifications over affinity and selectivity. Linker length and flexibility played a
key role in enhancing P-gp affinity as proved by the most potent P-gp modulator (3h, IC50 = 70
nM). A phenyl ring within the spacer (3k, 3l, 3o) and bulkier groups (Br in 3r, Ph in 3u) at
coumarin C3 led to derivatives showing nanomolar activity (160 nM < IC50 < 280 nM) along with
outstanding selectivity over MRP1 (SI > 350). Molecular docking calculations carried out on a
human MDR1 homology model structure contributed to gain insight into the ligands’ binding
modes. Some compounds (3d, 3h, 3l, 3r, 3t, 3u) reversed MDR thereby restoring doxorubicin
cytotoxicity when co-administered with the drug into MDCK-MDR1 cells.
Keywords
multidrug resistance; coumarin; structure-activity relationships; P-glycoprotein; multidrug
resistance associated protein 1; molecular docking
Abbreviations
ATP-binding cassette (ABC); Alzheimer’s disease (AD); blood-brain barrier (BBB); calcein
acetoxymethyl ester (Calcein-AM); cytochrome P450 3A (CYP450 3A); diisopropylethylamine
(DIPEA); Dulbecco's modified Eagle's medium (DMEM); dimethyl sulfoxide (DMSO);
deoxyribonucleic acid (DNA); fetal bovine serum (FBS); free energy of binding (FEB); ligand
efficiency (LE); lipophilic ligand efficiency (LLE); Madin-Darby Canine Kidney (MDCK);
multidrug resistance (MDR); multidrug resistance protein 1 (MDR1); multidrug resistance-
associated protein 1 (MRP1); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT);
microwave (MW); N-bromosuccinimide (NBS); phosphate buffered saline (PBS); Parkinson’s
disease (PD); polyethylene glycol (PEG); P-glycoprotein (P-gp); standard error mean (SEM);
selectivity index (SI); 1,2,3,4-tetrahydroisoquinoline (THIQ).
1. Introduction

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Cancer cells may become unresponsive to chemotherapeutics through a phenomenon termed
multidrug-resistance (MDR) affecting agents structurally and mechanistically unrelated.[1] Both
genetic and epigenetic alterations can modulate drug sensitivity by activating different mechanisms
[2]: i) reduced drug uptake; ii) up-regulation of defensive machineries (DNA repair and/or
cytochrome P450 mixed-function oxidases); iii) interruption of apoptosis signalling. In addition, a
major role is featured by the overexpression of energy-dependent ATP-binding cassette (ABC)
transporter proteins,[3] that detoxify cancer cells and lower intracellular concentration under the
therapeutic threshold by pumping drugs out at the expense of ATP hydrolysis. The human ABC
superfamily counts 48 efflux pumps that are expressed at basal levels in normal tissues where serve
cells as shuttles for a variety of xenobiotics and endogenous substances (e.g., lipids, peptides, and
ions).[4] Several members have been clearly associated with MDR, including P-glycoprotein (P-gp,
also called multidrug resistance protein 1, MDR1, or ABCB1) and multidrug resistance-associated
protein 1 (alternatively termed MRP1 or ABCC1).
P-gp is by far the most studied protein,[5] that has been found in epithelial cells membranes of
different districts, such as kidney and liver. Furthermore, P-gp protects brain by extruding toxins
into blood at the luminal interface of blood-brain barrier (BBB). MRP1 is ubiquitously expressed
and transports preferentially anionic substrates and glutathione conjugates.[6] Both P-gp and MRP1
are widely expressed in many leukaemias and solid tumors.
Chart 1. Chemical structures of known P-gp ligands.
The inhibition of ABC pumps, especially P-gp, has long been considered a viable target to
circumvent resistance and ameliorate the clinical outcome of chemotherapeutic agents.
Unfortunately, tested MDR reversers did not score relevant efficacy in clinical trials because of
impeding challenges represented by toxicity/selectivity issues and pharmacokinetic interactions
arising from CYP450 3A inhibition. Therefore, alternative strategies to evade MDR have been
envisaged through innovative drug delivery approaches that bypass ABC proteins inhibition.[7]
However, small molecule P-gp binders are still needed to better understand molecular recognition
interactions and provide safer tools to co-adjuvate chemotherapy.[8] Moreover, P-gp
overexpression at the BBB level has been observed in the early stage of neurodegenerative
syndromes such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) and some ligands are
under evaluation as imaging tracers.[9]

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Figure 1. Rational design of THIQ-coumarin conjugates. Schematic representation of structural
modifications addressing the linker and the two heterocyclic scaffolds.
With the aim of gaining further knowledge about P-gp binding interactions and exploring molecular
determinants for selectivity over another ABC pump (e.g., MRP1), herein we report the design,
synthesis and biological evaluation of novel conjugates where a 1,2,3,4-tetrahydroisoquinoline
(THIQ) moiety was linked to a 7-hydroxy-2H-chromen-2-one (coumarin) scaffold through
differently shaped and sized spacers. On one hand, the protonatable THIQ nucleus is recurrent in
several selective P-gp inhibitors (e.g., tariquidar) as well as non-selective compounds (e.g.,
elacridar). On the other hand, the coumarin motif is a nature-friendly, easy-to-handle chemical
building block that has been exploited in several medicinal chemistry programs (e.g., addressing
cancer [10] and neurodegeneration [11,12]) and in few studies focused on P-gp ligands bearing
lamellarin-type alkaloid skeleton.[13] Polymethylene and ortho-, meta-, para-xylyl chains were
explored as the spacers connecting the two heterocyclic cores. Moreover, the effect of methyl
groups, halogens (Cl, Br), and phenyl rings at position 3 and 4 of the coumarin was investigated, as
well as few modifications on the basic moiety regarding the presence of 6,7-dimethoxy substitution.
All the title compounds were tested in the calcein-AM (calcein acetoxymethyl ester) uptake assay
across MDCK-MDR1 and MDCK-MRP1 cells to evaluate the interference with the pro-fluorescent
substrate transport by MDR1 and MRP1 pumps, respectively. The binding contacts within P-gp
framework were investigated by means of computer-aided molecular docking simulations.
Moreover, cell-based co-incubation experiments with a well-known anticancer drug (doxorubicin)
were supplied to probe the MDR reverting skills for a number of highly potent P-gp inhibitors.
2. Methods
2.1 Chemistry
The synthetic route for the preparation of novel compounds 3a-v is shown in Scheme 1. Starting 7-
hydrocoumarins were commercially available (1b, 1d) or prepared on a laboratory scale by
following literature procedures with slight modifications. The well-known von Pechmann
condensation was employed to synthesize 7-hydroxy-3,4-dimethyl-2H-chromen-2-one [14] (1a) and
7-hydroxy-4-methyl-2H-chromen-2-one,[15] whose regioselective NBS-mediated aromatic
bromination [16] in PEG-400 furnished 1c. Cyclo-condensation of the suitable 2-
hydroxyacetophenone or salicylaldehyde with phenacetylchloride or phenylacetic acid, respectively,
furnished 3-phenylcoumarins 1e-f.[17,18] Methyl-ether cleavage promoted by a Lewis acid (AlCl3)
provided 1g from 3-methoxy-6H-benzo[c]chromen-6-one.[15] A microwave-assisted nucleophilic
substitution between the appropriate hydroxycoumarin 1a-g and excess dihalide (1,ω-

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dibromoalkane or α,α’-dibromo(chloro)-xylene) yielded the intermediates 2a-g and 2i-r. In a
similar way, alkylation of 1a with 2-[4-(bromomethyl)phenyl]ethanol furnished alcohol 1h that in
turn underwent an Appel-type bromination to 2h. The mono-halo derivatives 2a-r were reacted with
the suitable THIQ in refluxing acetonitrile in the presence of DIPEA to prepare final compounds
3a-v.
Scheme 1. Synthesis of compounds 3a-va
HO
HO
O
O
O
a)
Cl
O
Br
O
HO
O
O
HO
O
1b
1c
1d
b)
b)
b)
R2
O
R2
R1
O
R1
O
R3
R3
b)
g)
N
X
O
HO
O
O
(Cl)Br
X
O
O
1a
2a r
-
3a v
-
b)
f)
b)
Me(H)
O
O
O
HO
O
O
HO
O
O
HO
1g
1e 1f
,
1h
c)
d)
e)
O
Cl(OH)
+
Br
Me(H)
OH
1a
+
O
HO
(H)MeO
O
O
O
a Reagents and conditions: a) N-bromosuccinimide, PEG-400, room temperature, 3 h; b) for 2a-g,
2i-r: an. K2CO3, suitable di-halide (α,α′-dibromo(chloro)-xylenes, trans-1,4-dibromo-2-butene or
1,ω-dibromoalkanes), KI (cat.), anhydrous acetone, 130 °C, 30 min, MW; c) for 1e from
phenylacetic acid and 2-hydroxy-4-methoxybenzaldehyde see ref. [17]; for
1f: 2′,4′-
dihydroxyacetophenone, phenylacetylchloride, an. K2CO3, an. acetone, reflux, 4 h; d) an. AlCl3, o-
xylene, reflux, 9 h; e) an. K2CO3, KI (cat.), anhydrous acetone, 130 °C, 30 min, MW; f) CBr4, PPh3,
an. dichloromethane, 0 °C to room temperature, 4 h; g) suitable 1,2,3,4-tetrahydroisoquinoline
hydrochloride, DIPEA, KI (cat.), anhydrous acetonitrile, reflux, 1 h.
2.2 Biological activity evaluation
2.2.1 Calcein-AM assay
Compounds 3a-v were screened as inhibitors of P-gp and MRP1 mediated transport in the
fluorescence-based Calcein-AM assay by using MDCK cell lines overexpressing MDR1 and
MRP1, respectively. Data are reported in Table 1 and IC50 values (µM) are compared with MC18
[19] and verapamil taken as reference MDR1 and MRP1 inhibitors, respectively. In order to
evaluate the selectivity between the two transporters, the selectivity index (SI) was calculated as the
ratio IC50 (MRP1) / IC50 (P-gp).
2.2.2 Antiproliferative assay

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [20] was used to
measure the percentage of viable MDCK-MDR1 cells after incubation with tested compounds (3d,
3h, 3l, 3r, 3t, 3u) at three different concentrations (0.1, 1 and 10 µM) alone and in co-
administration with doxorubicin (10 µM), in order to evaluate the capacity of compounds to
circumvent resistance to the chemotherapeutic drug and to re-establish the anti-proliferative
activity.
2.3 Computational studies
Conformational sampling in aqueous environment of the most active compound 3h was first
performed throughout molecular dynamics followed by dockings to the MDR1 binding site. The
previously published human P-gp homology model [21] was chosen as biomolecular target to
investigate plausible binding modes of the most potent inhibitor 3h and to gain valuable insights
into the protein sites where the two heterocyclic moieties (THIQ and coumarin) most likely settle.
3. Results and discussion
3.1 Structure-activity relationships (SARs)
Noticeably, all compounds showed submicromolar P-gp inhibition with only three exceptions (3a,
3b and 3j). Conversely, only a moderate modulation of MRP1 efflux was always observed. The
presence of 6,7-dimethoxy substitution pattern at the THIQ basic moiety enhanced P-gp affinity in
comparison to the unsubstituted THIQs (3e > 3a, 3f > 3b, 3g > 3c, 3h > 3d). The distance between
the two bicycles appeared as a critical structural determinant and the length of the alkyl spacer
strongly affected the P-gp mediated efflux inhibition. Among the conjugates 3a-i bearing a
polymethylene alkyl chain, the inhibition potency progressed up to 5 units in the two series of
analogues (3a < 3b < 3c < 3d and 3e < 3f < 3g < 3h, from 2 to 5 methylene groups). The inhibition
data clearly indicate that a pentamethylene linker optimized the interactions of both heterocyclic
cores into hydrophobic binding pockets, which led to the most potent inhibitor of the series (3h)
endowed with nanomolar P-gp activity (IC50 = 70 nM) and high selectivity over MRP1 (SI = 184).
Further elongation reduced the inhibitory potency (3i < 3h). A more rigid geometry due to a trans
double bond in the spacer resulted detrimental, as can be inferred from the comparison of 3j with 3f
as well as 3g. The introduction of a phenyl ring in the linker provided 3,4-dimethylcoumarins 3k,
3m, and 3o that resulted equipotent or less active than their open-chain saturated analogue 3h, 3g,
3f. This clue might rule out additional interactions (e.g., π-π stacking) involving the linker, which
essentially works by placing the two heterocyclic heads in the best binding topology and/or
produces different desolvation penalties. Interestingly, an outstanding selectivity increase was
returned by both the most elongated (para, 3k, SI > 526) and the most constrained (orto, 3o, SI >
625) regioisomer, that resulted inactive towards MRP1. Conversely, it is worth noting that the meta-
isomer 3m proved to be the most potent MRP1 inhibitor of the whole series (IC50 = 3.6 µM). The
homologation of the bridge between the basic nitrogen and the phenyl ring of the spacer (3l vs. 3k)
did not affect neither P-gp affinity nor selectivity over MRP1 while potentially improving solubility
at physiological pH depending upon pKa modulation.
Focused modifications were also probed at the position 3 and 4 of the coumarin template where
dimethyl substitution performed the best fitting into P-gp binding cavities. In fact, the removal of
both methyl groups reduced P-gp inhibition (3n < 3m) without affecting MRP1 transport.
Moreover, the replacement of methyl at the position 3 in 3p-q with Cl, having close van der Waals
volume but opposite electronic effect, decreased the activitytowards P-gp (3p < 3g, 3q < 3h).
An interesting sensitivity to steric hindrance discriminated the two transporters upon binding
compounds bearing a bulky substituent linked to coumarin C3 (3r-u) or fused to positions 3 and 4

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Table 1.
Biological activity of compounds 3a-v towards the inhibition of MDR1- and MRP1-mediated
calcein-AM efflux
R1
X
R2
R3
IC50 (µM)a
SIf
MDR1b
MRP1c
cmpd
MC18d
1.2±0.32
1.3±0.24
verapamile
4.5±0.51
>100
15±1.5
2.7±0.52
> 7
27
44
56
53
218
221
184
200
17
> 526
> 526
23
H
H
H
H
MeO
MeO
MeO
MeO
MeO
-
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
73±5.2
40±3.3
31±3.7
20±1.9
41±4.5
33±2.8
12.9±1.9
44±3.7
18±2.1
>100
CH2
0.91±0.21
0.55±0.10
0.38±0.081
0.19±0.017
0.15±0.016
0.070±0.011
0.22±0.038
1.0±0.31
0.19±0.027
0.19±0.018
0.16±0.010
0.77±0.081
0.16±0.018
0.56±0.056
0.46±0.037
0.28±0.026
0.41±0.027
0.39±0.036
0.22±0.029
0.33±0.047
(CH2)2
(CH2)3
-
CH2
(CH2)2
(CH2)3
(CH2)4
MeO trans-CH=CH Me
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
p-C6H4
p-CH2C6H4
m-C6H4
m-C6H4
o-C6H4
(CH2)2
Me
Me
Me
H
Me
Cl
Cl
Br
Ph
Ph
Ph
>100
3.6±0.52
3.7±0.64
>100
53±6.5
18±0.63
>100
>100
>100
>100
>100
5
> 625
95
38
357
244
256
455
Me
Me
Me
Me
H
(CH2)3
(CH2)3
(CH2)2
(CH2)3
(CH2)3
(CH2)3
H
Me
3t
3u
3v
303
Ph
a
b
Values are the mean ± SEM of two independent experiments performed in triplicate. IC50
determined in MDCK-MDR1 cell lines. c IC50 determined in MDCK-MRP1 cells. d P-gp inhibition
e
f
positive control. MRP1 inhibition positive control. Selectivity index (SI) calculated from the
equation: IC50 (MRP1) / IC50 (P-gp).
(3v). P-gp succeeded in accommodating both derivatives bearing a Br-atom (3r, IC50 = 0.28 µM) or
a phenyl group at position 3 (3s, IC50 = 0.41 µM; 3t, IC50 = 0.39 µM; 3u, IC50 = 0.22 µM), and the
more flat condensed congener 3v (IC50 = 0.33 µM), whereas MRP1 did not tolerate these
substituents and the compounds 3r-v were ineffective in modulating its transport activity. This
molecular discrimination resulted in excellent selectivities towards P-gp (for 3r-v, 244 < SI < 455).
Moreover, the presence at position 3 of a flag methyl, forcing out-of-the-plane phenyl rotation,
improved P-gp modulation and selectivity (3u > 3s-t).
3.2 MDR reverting effect
Among the most active modulators, diverse active compounds (3d, 3h, 3l, 3r, 3t, 3u) were assayed
for their ability to revert resistance to doxorubicin affecting MDCK-MDR1 cells that overexpress P-

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gp. As illustrated in Figure 2, all compounds showed a dose-dependent potentiation of doxorubicin
(10 µM) cytotoxicity and were able to restore the anti-proliferative effect of the chemotherapeutic
on resistant cells upon inhibition of P-gp mediated drug efflux. Interestingly, the most potent
inhibitor of the whole series (compound 3h) showed a remarkable reduction of cell viability already
at a concentration (0.1 µM) close to its IC50 value (0.070 µM). Moreover, at the highest
concentration used (10 µM) all the derivatives under investigation showed no inherent cytotoxicity
along with the most significant reverting activity observed.
Figure 2. Dose-dependent effects on in vitro MDCK-MDR1 cells proliferation by compounds 3d,
3h, 3l, 3r, 3t and 3u at three different concentrations (0.1, 1 and 10 µM) alone and co-incubated
with doxorubicin (10 µM). Cell survival is represented as % of control cell growth in cultures
containing no drug and test compounds. Each bar represents the mean ± SEM of two experiments in
triplicate; one-way ANOVA followed by Bonferroni’s post-hoc comparison test: * P < 0.1, ** P <
0.01, *** P < 0.001 vs. doxorubicin alone.
In view of further drug-like optimization program, it is worth noting that compounds 3h and 3r
behaved as potent reversers with improved efficiency metrics (ligand efficiency, LE, and lipophilic
ligand efficiency, LLE) as detailed in Table 2 compared to tariquidar and elacridar, marketed drugs
belonging to the third P-gp non-competitive inhibitors’ generation.
Table 2.
Ligand efficiency metrics
clogPa
6.38
7.19
5.36
5.87
5.60
6.17
5.37
6.62
7.22
b
cmpd
tariquidar [22]
elacridar [32]
MC18 [19]
NHA
47
42
29
29
33
37
33
37
38
LEc
LLEd
0.98
0.66
0.56
0.39
1.55
0.55
1.18
-0.21
-0.56
0.157
0.187
0.204
0.216
0.217
0.182
0.198
0.173
0.175
3d
3h
3l
3r
3t
3u
a LogP values calculated with ACD/Labs 6.00. b Number of heavy (non-hydrogen) atoms. c Ligand
d
efficiency (LE) was calculated as pIC50 / NHA. Lipophilic ligand efficiency (LLE) was calculated
as LLE = pIC50 – cLogP.

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3.3 Molecular docking simulations
A structure-based analysis of the ligand-P-gp interactions was carried on the most potent P-gp
inhibitor 3h, as representative of the novel molecular dataset. A previously reported human P-gp
homology model [21] was used as the protein target. Docking simulations highlighted two distinct
and adjacent lipophilic clefts of P-gp able to adequately accomodate the heterocyclic moieties of the
inhibitor by means of hydrophobic interactions (van der Waals contacts or π-π stacking). In details,
the THIQ head is accommodated in a pocket lined by Tyr310, Phe336, Phe728, Phe732, Leu975,
Phe983 whereas the coumarin tail is merged in a more widened cavity surrounded by Tyr953,
Phe978 and Val982. In addition, the ligand-protein complex was stabilized by polar and hydrogen
bonding contacts involving the hydroxyl of Tyr310, close to the methoxy groups in positions 6 and
7 of the THIQ moiety, and those occurring between the protonated nitrogen and the sulphur atom of
Met69 side chain. The crucial role of the methoxy substituents as chemical fingerprint on the THIQ
ring was corroborated by the lower activity of compounds 3a-d lacking 6,7-dimethoxy substituents.
The proposed binding mode supported the need of well-defined length and flexibility features of the
linker. As a matter of fact, alkyl chain shorter (3e-g) or even longer (3i) than five carbon units
resulted in less active inhibitors. With respect to the coumarin motif, the presence of methyl groups
is tolerated and more hindered substituents (3r, 3s-u) cause unfavorable steric clashes with residues
of the transmembrane domain, namely Val865, Ile868 and Val982.
Figure 3. Binding mode of 3h into the MDR1 binding site. 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 -11.93 kcal/mol, while the contact surface area measures 411
Å2.
4. Conclusion
Herein we described a SAR study on novel derivatives bearing a protonatable THIQ core
conjugated through a linker of different length and chemical nature with a differently substituted
coumarin motif as inhibitors of P-gp and MRP1 mediated efflux. A systematic exploration
addressing the nature of the linker highlighted the pivotal role of length and flexibility in improving
P-gp inhibition. Focused structural variations were introduced at coumarin C3-4 region, where
steric hindrance proved to be a molecular determinant promoting P-gp selectivity. A number of
potent and selective P-gp inhibitors have been identified and the most potent compound of the
series showed nanomolar inhibition potency (3h, IC50 = 70 nM). Docking simulations were
employed to gain insight into the binding modes, suggesting the key role of the linker in

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accommodating the two heterocyclic templates to fit two different lipophilic cages in P-gp in
agreement with the observed SARs. Finally, some of these modulators proved able in reverting
resistance to doxorubicin and reactivating the cytotoxicity of the antibiotic in MDCK-MDR1 cell
lines. These candidates, which showed negligible cytotoxicity, deserve further investigation aimed
at developing novel small molecules as therapeutic adjuvants against chemoresistant tumors.
5. Experimental section
5.1 Chemistry
Starting materials, reagents, and analytical grade solvents were purchased from Sigma-Aldrich
(Europe). The purity of all the intermediates, checked by HPLC, was always better than 95%. All
the newly prepared and tested compounds showed purity higher than 95% (elemental analysis).
Elemental analyses were performed on the EuroEA 3000 analyzer only on the final compounds
tested as efflux pumps inhibitors. Analyses indicated by the symbols of the elements were within ±
0.4 % of the theoretical values. Column chromatography was performed using Merck silica gel 60
(0.063-0.200 mm, 70-230 mesh). Flash chromatographic separations were performed on a Biotage
SP1 purification system using flash cartridges prepacked with KP-Sil 32−63 µm, 60 Å silica. All
reactions were routinely checked by TLC (thin layer chromatography) using Merck Kieselgel 60
F254 aluminum plates and visualized by UV light or iodine. Regarding the reaction requiring the use
of dry solvents, the glassware was flame-dried and then cooled under a stream of dry argon before
the use. Microwave reactions were performed in a Milestone MicroSynth apparatus, setting
temperature and reaction times, fixing maximum irradiation power to 600 W and heating ramp
times to 2 min. Nuclear magnetic resonance spectra were recorded on a Varian Mercury 300
instrument (at 300 MHz) or on a Agilent Technologies 500 apparatus (at 500 MHz) at ambient
temperature in the specified deuterated solvent. Chemical shifts (δ) are quoted in parts per million
(ppm) and are referenced to the residual solvent peak. The coupling constants J are given in Hertz
(Hz). The following abbreviations were used: s (singlet), d (doublet), dd (doublet of doublet), t
(triplet), q (quadruplet), qn (quintuplet), m (multiplet), br s (broad signal); signals due to OH and
NH protons were located by deuterium exchange with D2O. HRMS experiments were performed
with a dual electrospray interface (ESI) and a quadrupole time-of-flight mass spectrometer (Q-TOF,
Agilent 6530 Series Accurate-Mass Quadrupole Time-of-Flight LC/MS, Agilent Technologies Italia
S.p.A., Cernusco sul Naviglio, Italy). Full-scan mass spectra were recorded in the mass/charge
(m/z) range 50-3000 Da. Melting points were determined by the capillary method on a Stuart
Scientific SMP3 electrothermal apparatus and are uncorrected. 7-Hydroxy-2H-chromen-2-one (1b)
and 3-chloro-7-hydroxy-4-methyl-2H-chromen-2-one (1d) were commercially available. The
synthesis of the following intermediates has been already described in the literature: 7-hydroxy-3,4-
dimethyl-2H-chromen-2-one [14] (1a), 7-hydroxy-3-phenyl-2H-chromen-2-one [17] (1e).
5.1.1 3-Bromo-7-hydroxy-4-methyl-2H-chromen-2-one (1c). N-bromosuccinimide (0.98 g, 5.5
mmol) was added slowly to a suspension of 7-hydroxy-4-methyl-2H-chromen-2-one [15] (0.88 g,
5.0 mmol) in PEG-400 (10 g). After stirring at room temperature for 3 h, the reaction mixture was
diluted with brine (100 mL) and extracted with CH2Cl2 (3 x 80 mL). The collected organic phases
were dried over Na2SO4 and concentrated under reduced pressure. The resulting crude was purified
through column chromatography (gradient eluent: ethyl acetate in n-hexane 5% → 30%). Pale
yellow solid; yield: 0.45 g, 35%. Spectroscopic data are in agreement with those reported in the
literature.[16]
5.1.2 7-Hydroxy-4-methyl-3-phenyl-2H-chromen-2-one (1f). By applying slight modifications to a
previously reported method,[18] 2′,4′-dihydroxyacetophenone (0.76 g, 5.0 mmol) was dissolved in
anhydrous acetone (40 mL), before the addition of potassium carbonate (4.1 g, 30 mmol) and
phenylacetylchloride (2.0 mL, 15 mmol). The reaction was refluxed for 4 h and then cooled to room
temperature. The inorganic residue was filtered-off and the solution was concentrated under rotary
evaporation. The crude dark brown solid residue was then purified through column chromatography

ACCEPTED MANUSCRIPT
1
(gradient eluent: methanol in dichloromethane 0% → 2%). Off white solid; yield: 0.77 g, 61%. H
NMR (300 MHz, DMSO-d6) δ: 2.19 (s, 3H), 6.73 (d, J = 2.2 Hz, 1H), 6.82 (dd, J = 8.7, 2.2 Hz,
1H), 7.21 – 7.31 (m, 2H), 7.31 – 7.49 (m, 3H), 7.65 (d, J = 8.7 Hz, 1H), 10.49 (s, 1H, dis. with
D2O).
5.1.3 3-Hydroxy-6H-benzo[c]chromen-6-one (1g). Anhydrous AlCl3 (2.1 g, 16 mmol) was added to
a suspension of 3-methoxy-6H-benzo[c]chromen-6-one [15] (0.90 g, 4.0 mmol) in o-xylene (20
mL) and the reaction mixture was refluxed for 9 h. After cooling to room temperature, the solvent
was removed under rotary evaporation. The residue was diluted with CH2Cl2 (200 mL) and washed
with 1 N aq. HCl (3 x 40 mL). The organic phase was washed with brine (100 mL) and dried over
Na2SO4. The evaporation of the solvent under reduced pressure yielded a pink solid (0.76 g, 89%)
that was used without further purification. Spectroscopic data are in agreement with those reported
in the literature.[15]
5.1.4 General procedure for the synthesis of intermediates 1h, 2a-g, 2i-r. The appropriate 7-
hydroxycoumarin 1a-g (2.0 mmol for 2a-g and 2i-r; 3.0 mmol for 1h) was suspended in anhydrous
acetone (10 mL) before adding potassium carbonate (for 2a-g and 2i-r: 0.83 g, 6.0 mmol; for 1h:
1.2 g, 9.0 mmol), the suitable mono-halide (for 1h: 2-[4-(bromomethyl)phenyl]ethanol, 3.0 mmol)
or di-halide (for 2a-g and 2i-r: 6.0 mmol of α,α′-dibromo(chloro)-xylenes or trans-1,4-dibromo-2-
butene; 10 mmol of 1,ω-dibromoalkanes) and a catalytic amount of KI in a Pyrex vessel charged
with a magnetic stirring bar and a Weflon bar. The vessel was placed in a microwave apparatus and
irradiated at 130 °C for 30 min. After cooling to room temperature, the inorganic residue was
filtered off after thorough washing with CH2Cl2. The solution was concentrated to dryness and the
resulting crude was purified as detailed below.
5.1.4.1 7-{[4-(2-Hydroxyethyl)benzyl]oxy}-3,4-dimethyl-2H-chromen-2-one (1h). Prepared from 1a
(0.57 g, 3.0 mmol) and 2-[4-(bromomethyl)phenyl]ethanol [23] (0.65 g, 3.0 mmol). Purification
procedure: the crude solid was washed several times with diethyl ether and the remaining residue
was filtered off. The resulting mother liquor was concentrated to dryness, thus yielding the desired
1
product as a white solid (0.92 g, 95%). H NMR (300 MHz, DMSO-d6) δ: 2.03 (s, 3H), 2.33 (s,
3H), 2.70 (t, J = 7.0 Hz, 2H), 3.58 (d, J = 7.0 Hz, 2H), 4.61 (d, J = 7.0 Hz, 1H, dis. with D2O), 5.14
(s, 2H), 6.93 – 7.04 (m, 2H), 7.22 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.7 Hz,
1H).
5.1.4.2 7-(2-Bromoethoxy)-3,4-dimethyl-2H-chromen-2-one (2a). Prepared from 1a (0.38 g, 2.0
mmol) and 1,2-dibromoethane (0.86 mL, 10 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 0% → 50%). White solid; yield: 0.17 g,
28%. 1H NMR (500 MHz, DMSO-d6) δ: 2.06 (s, 3H), 2.35 (s, 3H), 3.82 (t, J = 7.0 Hz, 2H), 4.41 (t,
J = 7.0 Hz, 2H), 6.94 – 7.00 (m, 2H), 7.70 (d, J = 8.5 Hz, 1H).
5.1.4.3 7-(3-Bromopropoxy)-3,4-dimethyl-2H-chromen-2-one (2b).[14] Prepared from 1a (0.38 g,
2.0 mmol) and 1,3-dibromopropane (1.0 mL, 10 mmol). Purification procedure: flash
chromatography (gradient eluent: ethyl acetate in n-hexane 0% → 60%). White solid; yield: 0.49 g,
79%. 1H NMR (500 MHz, DMSO-d6) δ: 2.07 (s, 3H), 2.27 (q, J = 6.4 Hz, 2H), 2.36 (s, 3H), 3.67 (t,
J = 6.4 Hz, 2H), 4.18 (t, J = 6.4 Hz, 2H), 6.95−6.98 (m, 2H), 7.70 (d, J = 8.8 Hz, 1H).
5.1.4.4 7-(4-Bromobutoxy)-3,4-dimethyl-2H-chromen-2-one (2c).[14] Prepared from 1a (0.38 g, 2.0
mmol) and 1,4-dibromobutane (1.2 mL, 10 mmol). Purification procedure: flash chromatography
1
(gradient eluent: ethyl acetate in n-hexane 0% → 50%). White solid; yield: 0.57 g, 88%. H NMR
(500 MHz, DMSO-d6) δ: 1.85 (qn, J = 6.4 Hz, 2H), 1.97 (qn, J = 6.4 Hz, 2H), 2.07 (s, 3H), 2.36 (s,
3H), 3.61 (t, J = 6.4 Hz, 2H), 4.10 (t, J = 6.4 Hz, 2H), 6.92−6.95 (m, 2H), 7.68 (d, J = 8.8 Hz, 1H).
5.1.4.5 7-[(5-Bromopentyl)oxy]-3,4-dimethyl-2H-chromen-2-one (2d). Prepared from 1a (0.38 g,
2.0 mmol) and 1,5-dibromopentane (1.4 mL, 10 mmol). Purification procedure: column
chromatography (eluent: ethyl acetate in n-hexane 10%). White solid; yield: 0.52 g, 76%. 1H NMR
(500 MHz, CDCl3) δ: 1.56 – 1.72 (m, 2H), 1.76 – 1.89 (m, 2H), 1.89 – 2.03 (m, 2H), 2.18 (s, 3H),
2.37 (s, 3H), 3.45 (d, J = 6.3 Hz, 2H), 4.02 (t, J = 6.3 Hz, 2H), 6.78 (d, J = 2.4 Hz, 1H), 6.84 (dd, J
= 8.8, 2.4 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H).

ACCEPTED MANUSCRIPT
5.1.4.6 7-[(6-Bromohexyl)oxy]-3,4-dimethyl-2H-chromen-2-one (2e). Prepared from 1a (0.38 g, 2.0
mmol) and 1,6-dibromohexane (1.5 mL, 10 mmol). Purification procedure: column chromatography
(gradient eluent: ethyl acetate in n-hexane 10% → 30%). White solid; yield: 0.28 g, 39%. 1H NMR
(500 MHz, DMSO-d6) δ: 1.36 – 1.50 (m, 4H), 1.67 – 1.75 (m, 2H), 1.78 – 1.85 (m, 2H), 2.05 (s,
3H), 2.34 (s, 3H), 3.52 (t, J = 6.7 Hz, 2H), 4.04 (t, J = 6.7 Hz, 2H), 6.88 – 6.94 (m, 2H), 7.66 (d, J =
9.6 Hz, 1H).
5.1.4.7 7-{[(2E)-4-Bromobut-2-en-1-yl]oxy}-3,4-dimethyl-2H-chromen-2-one (2f). Prepared from
1a (0.38 g, 2.0 mmol) and trans-1,4-dibromo-2-butene (1.3 g, 6.0 mmol). Purification procedure:
column chromatography (gradient eluent: ethyl acetate in n-hexane 10% → 30%). Off-white solid;
1
yield: 0.45 g, 69%. H NMR (500 MHz, DMSO-d6) δ: 2.06 (s, 3H), 2.39 (s, 3H), 4.18 (d, J = 6.0
Hz, 2H), 4.69 (d, J = 3.7 Hz, 2H), 5.92 – 6.14 (m, 2H), 6.90 – 6.99 (m, 2H), 7.69 (d, J = 8.5 Hz,
1H).
5.1.4.8 7-{[4-(Bromomethyl)benzyl]oxy}-3,4-dimethyl-2H-chromen-2-one (2g). Prepared from 1a
(0.38 g, 2.0 mmol) and α,α′-dibromo-p-xylene (1.6 g, 6.0 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 10% → 60%). Off white solid; yield:
1
0.60 g, 80%. H NMR (300 MHz, DMSO-d6) δ: 2.05 (s, 3H), 2.35 (s, 3H), 4.70 (s, 2H), 5.20 (s,
2H), 6.96– 7.04 (m, 2H), 7.40 – 7.49 (m, 4H), 7.69 (d, J = 8.5 Hz, 1H).
5.1.4.9 7-{[3-(Chloromethyl)benzyl]oxy}-3,4-dimethyl-2H-chromen-2-one (2i). Prepared from 1a
(0.38 g, 2.0 mmol) and α,α′-dichloro-m-xylene (1.1 g, 6.0 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 10% → 30%). White solid; yield: 0.62
1
g, 94%. H NMR (300 MHz, DMSO-d6) δ: 2.06 (s, 3H), 2.35 (s, 3H), 4.77 (s, 2H), 5.20 (s, 2H),
7.01 (dd, J = 8.7, 2.5 Hz, 1H), 7.03 (d, J = 2.5 Hz, 1H), 7.37 – 7.45 (m, 3H), 7.53 (s, 1H), 7.69 (d, J
= 8.7 Hz, 1H).
5.1.4.10 7-{[3-(Chloromethyl)benzyl]oxy}-2H-chromen-2-one (2j). Prepared from 1b (0.32 g, 2.0
mmol) and α,α′-dichloro-m-xylene (1.1 g, 6.0 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 10% → 50%). White solid; yield: 0.43
g, 72%. 1H NMR (500 MHz, DMSO-d6) δ: 4.78 (s, 2H), 5.22 (s, 2H), 6.28 (d, J = 9.5 Hz, 1H), 7.02
(dd, J = 8.6, 2.4 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H), 7.36 – 7.46 (m, 3H), 7.53 (s, 1H), 7.63 (d, J = 8.6
Hz, 1H), 7.98 (d, J = 9.5 Hz, 1H).
5.1.4.11 7-{[2-(Bromomethyl)benzyl]oxy}-3,4-dimethyl-2H-chromen-2-one (2k). Prepared from 1a
(0.38 g, 2.0 mmol) and α,α′-dibromo-o-xylene (1.6 g, 6.0 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 10% → 30%). White solid; yield: 0.73
1
g, 98%. H NMR (500 MHz, DMSO-d6) δ: 2.07 (s, 3H), 2.36 (s, 3H), 4.82 (s, 2H), 5.34 (s, 2H),
7.04 (dd, J = 8.8, 2.5 Hz, 1H), 7.10 (d, J = 2.5 Hz, 1H), 7.34 – 7.38 (m, 1H), 7.48 – 7.52 (m, 1H),
7.52 – 7.56 (m, 1H), 7.57 – 7.62 (m, 1H), 7.72 (d, J = 8.8 Hz, 1H).
5.1.4.12 7-(4-Bromobutoxy)-3-chloro-4-methyl-2H-chromen-2-one (2l). Prepared from 1d (0.42 g,
2.0 mmol) and 1,4-dibromobutane (1.2 mL, 10 mmol). Purification procedure: flash
chromatography (gradient eluent: ethyl acetate in n-hexane 0% → 30%). White solid; yield: 0.35 g,
51%. 1H-NMR (300 MHz, CDCl3) δ: 1.94-2.13 (m, 4H), 2.55 (s, 3H), 3.49 (t, J = 6.1 Hz, 2H), 4.06
(t, J = 6.1 Hz, 2H), 6.81 (d, J = 2.5 Hz, 1H), 6.89 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.52 (d, J = 8.8
Hz, 1H).
5.1.4.13 7-[(5-Bromopentyl)oxy]-3-chloro-4-methyl-2H-chromen-2-one (2m). Prepared from 1d
(0.42 g, 2.0 mmol) and 1,5-dibromopentane (1.4 mL, 10 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 10% → 30%). White solid; yield: 0.47
g, 66%. 1H NMR (300 MHz, CDCl3) δ: 1.57 – 1.73 (m, 2H), 1.79 – 2.00 (m, 4H), 2.55 (s, 3H), 3.45
(t, J = 6.7 Hz, 2H), 4.03 (t, J = 6.3 Hz, 2H), 6.81 (d, J = 2.5 Hz, 1H), 6.89 (dd, J = 8.9, 2.5 Hz, 1H),
7.52 (d, J = 8.9 Hz, 1H).
5.1.4.14 3-Bromo-7-[(5-bromopentyl)oxy]-4-methyl-2H-chromen-2-one (2n). Prepared from 1c
(0.51 g, 2.0 mmol) and 1,5-dibromopentane (1.4 mL, 10 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 10% → 30%). White solid; yield: 0.50
1
g, 62%. H NMR (300 MHz, DMSO-d6) δ: 1.43 – 1.60 (m, 2H), 1.66 – 1.80 (m, 2H), 1.80 – 1.97

ACCEPTED MANUSCRIPT
(m, 2H), 2.56 (s, 3H), 3.54 (t, J = 6.6 Hz, 2H), 4.08 (t, J = 6.3 Hz, 2H), 6.97 (dd, J = 8.9, 2.3 Hz,
1H), 7.02 (d, J = 2.3 Hz, 1H), 7.77 (d, J = 8.9 Hz, 1H).
5.1.4.15 7-(4-Bromobutoxy)-3-phenyl-2H-chromen-2-one (2o). Prepared from 1e (0.48 g, 2.0 mmol)
and 1,4-dibromobutane (1.2 mL, 10 mmol). Purification procedure: flash chromatography (gradient
eluent: ethyl acetate in n-hexane 0% → 30%). White solid; yield: 0.69 g, 92%. 1H-NMR (300 MHz,
DMSO-d6) δ: 1.81-2.06 (m, 4H), 3.60 (t, J= 6.6 Hz, 2H), 4.12 (t, J= 6.3 Hz, 2H), 6.97 (dd, J1= 2.2
Hz, J2= 8.8 Hz, 1H), 7.03 (d, J=2.2 Hz, 1H), 7.34-7.46 (m, 3H), 7.66-7.70 (m, 3H), 8.19 (s, 1H).
5.1.4.16 7-[(5-Bromopentyl)oxy]-3-phenyl-2H-chromen-2-one (2p). Prepared from 1e (0.48 g, 2.0
mmol) and 1,5-dibromopentane (1.4 mL, 10 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 10% → 30%). White solid; yield: 0.62
g, 80%. 1H NMR (300 MHz, DMSO-d6) δ: 1.44 – 1.61 (m, 2H), 1.67 – 1.93 (m, 4H), 3.55 (t, J = 6.7
Hz, 2H), 4.09 (t, J = 6.4 Hz, 2H), 6.96 (dd, J = 8.6, 2.4 Hz, 1H), 7.02 (d, J = 2.4 Hz, 1H), 7.33 –
7.47 (m, 3H), 7.62 – 7.73 (m, 3H), 8.19 (s, 1H).
5.1.4.17 7-[(5-Bromopentyl)oxy]-4-methyl-3-phenyl-2H-chromen-2-one (2q). Prepared from 1f
(0.50 g, 2.0 mmol) and 1,5-dibromopentane (1.4 mL, 10 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 10% → 20%). White solid; yield: 0.67
1
g, 83%. H NMR (300 MHz, DMSO-d6) δ: 1.47 – 1.62 (m, 2H), 1.66 – 1.81 (m, 2H), 1.81 – 1.97
(m, 2H), 2.22 (s, 3H), 3.56 (t, J = 6.7 Hz, 2H), 4.10 (t, J = 6.4 Hz, 2H), 6.93 – 7.03 (m, 2H), 7.23 –
7.32 (m, 2H), 7.32 – 7.49 (m, 3H), 7.74 (d, J = 8.7 Hz, 1H).
5.1.4.18 3-[(5-Bromopentyl)oxy]-6H-benzo[c]chromen-6-one (2r). Prepared from 1g (0.42 g, 2.0
mmol) and 1,5-dibromopentane (1.4 mL, 10 mmol). Purification procedure: column
chromatography (eluent: ethyl acetate in n-hexane 70%). White solid; yield: 0.30 g, 41%. 1H NMR
(300 MHz, CDCl3) δ: 1.57 – 1.73 (m, 2H), 1.77 – 2.05 (m, 4H), 3.46 (t, J = 6.7 Hz, 2H), 4.05 (t, J =
6.3 Hz, 2H), 6.85 (d, J = 2.5 Hz, 1H), 6.91 (dd, J = 8.8, 2.5 Hz, 1H), 7.47 – 7.56 (m, 1H), 7.74 –
7.83 (m, 1H), 7.95 (d, J = 8.8 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 8.36 (dd, J = 8.0, 1.3 Hz, 1H).
5.1.5 7-{[4-(2-Bromoethyl)benzyl]oxy}-3,4-dimethyl-2H-chromen-2-one (2h). Carbon tetrabromide
(0.73 g, 2.2 mmol) was added to a solution of 1h (0.65 g, 2.0 mmol) in anhydrous dichloromethane
(10 mL). The mixture was cooled to 0 °C through an external ice bath and then a solution of
triphenylphosphine (0.63 g, 2.4 mmol) in an. dichloromethane (5 mL) was added dropwise. After
warming at room temperature, the reaction was kept under magnetic stirring for 4 h. The mixture
was concentrated under vacuum and the crude product was purified through column
chromatography (gradient eluent: ethyl acetate in n-hexane 20% → 40%). White solid; yield: 0.45
g, 58%. 1H NMR (300 MHz, DMSO-d6) δ: 2.05 (s, 3H), 2.34 (s, 3H), 3.11 (t, J = 7.2 Hz, 2H), 3.71
(t, J = 7.2 Hz, 2H), 5.16 (s, 2H), 6.94– 7.04 (m, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz,
2H), 7.68 (d, J = 8.7 Hz, 1H).
5.1.6 General procedure for the synthesis of final compounds 3a-v. The appropriate intermediate
2a-r (0.50 mmol) was suspended in anhydrous acetonitrile (4 mL) followed by the addition of
DIPEA (0.52 mL, 3.0 mmol), the suitable 1,2,3,4-tetrahydroisoquinoline hydrochloride (0.75
mmol) and a catalytic amount of KI. The mixture was refluxed for 1 h. After cooling to room
temperature, the solvent was removed under rotary evaporation and the residue was purified
through column chromatography as detailed below.
5.1.6.1
7-[2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethoxy]-3,4-dimethyl-2H-chromen-2-one
(3a).
Prepared from 2a (0.15 g, 0.50 mmol) and 1,2,3,4-tetrahydroisoquinoline hydrochloride (0.13 g,
0.75 mmol). Purification procedure: column chromatography (gradient eluent: ethyl acetate in n-
hexane 0% → 40%). Brown solid, yield: 0.10 g, 59%; mp: 141-3 °C. 1H NMR (500 MHz, DMSO-
d6) δ: 2.06 (s, 3H), 2.35 (s, 3H), 2.71 – 2.83 (m, 4H), 2.88 (t, J = 5.7 Hz, 2H), 3.66 (s, 2H), 4.26 (t, J
= 5.7 Hz, 2H), 6.96 (dd, J = 8.8, 2.2 Hz, 1H), 6.99 (d, J = 2.2 Hz, 1H), 7.00 – 7.04 (m, 1H), 7.05 –
7.12 (m, 3H), 7.68 (d, J = 8.8 Hz, 1H). Anal. C22H23NO3 (C, H, N). HRMS (Q-TOF) calcd for
C22H23NO3 [M+H]+ m/z 350.1751, found 350.1747; [M+Na]+ m/z 372.1570, found 372.1568.
5.1.6.2 7-[3-(3,4-Dihydroisoquinolin-2(1H)-yl)propoxy]-3,4-dimethyl-2H-chromen-2-one (3b).
Prepared from 2b (0.16 g, 0.50 mmol) and 1,2,3,4-tetrahydroisoquinoline hydrochloride (0.13 g,

ACCEPTED MANUSCRIPT
0.75 mmol). Purification procedure: column chromatography (gradient eluent: ethyl acetate in n-
hexane 20% → 70%). Off-white solid; yield: 0.13 g, 74%; mp (3b·hydrochloride) [14]: > 250 °C.
1H NMR (300 MHz, DMSO-d6) δ: 1.98 (qn, J = 6.4 Hz, 2H), 2.05 (s, 3H), 2.34 (s, 3H), 2.59 (t, J =
6.4 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.79 (t, J = 5.8 Hz, 2H), 3.55 (s, 2H), 4.13 (t, J = 6.4 Hz, 2H),
6.91−6.94 (m, 2H), 7.01−7.09 (m, 4H), 7.66 (d, J = 9.9 Hz, 1H). Anal. C23H25NO3 (C, H, N).
HRMS (Q-TOF) calcd for C23H25NO3 [M+H]+ m/z 364.1907, found 364.1906.
5.1.6.3
7-[4-(3,4-Dihydroisoquinolin-2(1H)-yl)butoxy]-3,4-dimethyl-2H-chromen-2-one
(3c).
Prepared from 2c (0.16 g, 0.50 mmol) and 1,2,3,4-tetrahydroisoquinoline hydrochloride (0.13 g,
0.75 mmol). Purification procedure: column chromatography (gradient eluent: ethyl acetate in n-
hexane 20% → 70%). White solid; yield: 0.14 g, 72%; mp (3c·hydrochloride) [14]: 206-8 °C. 1H
NMR (500 MHz, DMSO-d6) δ: 1.67 (qn, J = 6.9 Hz, 2H), 1.79 (qn, J = 6.9 Hz, 2H), 2.07 (s, 3H),
2.36 (s, 3H), 2.46−2.48 (m, 2H), 2.64 (t, J = 5.4 Hz, 2H), 2.79 (t, J = 5.4 Hz, 2H), 3.53 (s, 2H), 4.10
(t, J = 6.9 Hz, 2H), 6.92−6.96 (m, 2H), 7.01−7.08 (m, 4H), 7.67 (d, J = 9.8 Hz, 1H). Anal.
C24H27NO3 (C, H, N). HRMS (Q-TOF) calcd for C24H27NO3 [M+H]+ m/z 378.2064, found
378.2056; [M+Na]+ m/z 400.1883, found 400.1885.
5.1.6.4 7-{[5-(3,4-Dihydroisoquinolin-2(1H)-yl)pentyl]oxy}-3,4-dimethyl-2H-chromen-2-one (3d).
Prepared from 2d (0.17 g, 0.50 mmol) and 1,2,3,4-tetrahydroisoquinoline hydrochloride (0.13 g,
0.75 mmol). Purification procedure: column chromatography (gradient eluent: ethyl acetate in n-
1
hexane 30% → 60%). Pale yellow solid; yield: 0.12 g, 62%; mp: 66-8 °C. H NMR (300 MHz,
DMSO-d6) δ: 1.38 – 1.51 (m, 2H), 1.51 – 1.64 (m, 2H), 1.68 – 1.84 (m, 2H), 2.05 (s, 3H), 2.34 (s,
3H), 2.38 – 2.44 (m, 2H), 2.61 (d, J = 5.6 Hz, 2H), 2.77 (t, J = 5.6 Hz, 2H), 3.50 (s, 2H), 4.05 (t, J =
6.4 Hz, 2H), 6.87 – 6.94 (m, 2H), 7.01 (d, J = 3.9 Hz, 1H), 7.03 – 7.12 (m, 3H), 7.66 (d, J = 9.5 Hz,
1H). Anal. C25H29NO3 (C, H, N). HRMS (Q-TOF) calcd for C25H29NO3 [M+H]+ m/z 392.2220,
found 392.2219; [M+Na]+ m/z 414.2040, found 414.2038.
5.1.6.5 7-[2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethoxy]-3,4-dimethyl-2H-chromen-2-
one (3e). Prepared from 2a (0.15 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
hydrochloride (0.17 g, 0.75 mmol). Purification procedure: flash column chromatography (gradient
eluent: ethyl acetate in n-hexane 30% → 80%). Pale yellow solid; yield: 0.16 g, 76%; mp: 107-9 °C
(dec.). 1H NMR (500 MHz, DMSO-d6) δ: 2.07 (s, 3H), 2.36 (s, 3H), 2.67 – 2.76 (m, 4H), 2.87 (t, J
= 5.7 Hz, 2H), 3.58 (s, 2H), 3.68 (s, 3H), 3.69 (s, 3H), 4.26 (t, J = 5.7 Hz, 2H), 6.61 (s, 1H), 6.65 (s,
1H), 6.97 (dd, J = 8.8, 2.5 Hz, 1H), 7.00 (d, J = 2.5 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H). Anal.
C24H27NO5 (C, H, N). HRMS (Q-TOF) calcd for C24H27NO5 [M+H]+ m/z 410.1962, found
410.1962.
5.1.6.6 7-[3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propoxy]-3,4-dimethyl-2H-chromen-
2-one (3f). Prepared from 2b (0.16 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
hydrochloride (0.17 g, 0.75 mmol). Purification procedure: flash column chromatography (gradient
1
eluent: ethyl acetate in n-hexane 30% → 80%). Orange solid; yield: 0.14 g, 68%; mp: 84-6 °C. H
NMR (500 MHz, DMSO-d6) δ: 1.96– 2.02 (m, 2H), 2.07 (s, 3H), 2.36 (s, 3H), 2.58 (t, J = 7.0 Hz,
2H), 2.62 (7, J = 5.7 Hz, 2H), 2.71 (d, J = 5.7 Hz, 2H), 3.47 (s, 2H), 3.68 (s, 3H), 3.69 (s, 3H), 4.14
(t, J = 6.3 Hz, 2H), 6.62 (s, 1H), 6.64 (s, 1H), 6.91 – 6.97 (m, 2H), 7.68 (d, J = 9.5 Hz, 1H). Anal.
C25H29NO5 (C, H, N). HRMS (Q-TOF) calcd for C25H29NO5 [M+H]+ m/z 424.2118, found
424.2110; [M+Na]+ m/z 446.1938, found 446.1935.
5.1.6.7 7-[4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butoxy]-3,4-dimethyl-2H-chromen-2-
one (3g). Prepared from 2c (0.16 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
hydrochloride (0.17 g, 0.75 mmol). Purification procedure: flash column chromatography (gradient
eluent: ethyl acetate in n-hexane 30% → 80%). Off white solid; yield: 0.17 g, 79%; mp: 122-4 °C.
1H NMR (500 MHz, DMSO-d6) δ: 1.67 (qn, J = 6.8 Hz, 2H), 1.78 (qn, J = 6.8 Hz, 2H), 2.07 (s,
3H), 2.35 (s, 3H), 2.47 (t, J = 6.8 Hz, 2H), 2.60 (d, J = 5.6 Hz, 2H), 2.69 (t, J = 5.6 Hz, 2H), 3.44 (s,
2H), 3.67 (s, 3H), 3.68 (s, 3H), 4.10 (t, J = 6.8 Hz, 2H), 6.60 (s, 1H), 6.63 (s, 1H), 6.91 – 6.96 (m,
2H), 7.66 (d, J = 9.6 Hz, 1H). Anal. C26H31NO5 (C, H, N). HRMS (Q-TOF) calcd for C26H31NO5
[M+H]+ m/z 438.2275, found 438.2274; [M+Na]+ m/z 460.2094, found 460.2098.

ACCEPTED MANUSCRIPT
5.1.6.8
7-{[5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl]oxy}-3,4-dimethyl-2H-
chromen-2-one (3h). Prepared from 2d (0.17 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column
chromatography (eluent: methanol in dichloromethane 10%). Pale yellow solid; yield: 0.072 g,
32%; mp: 112-4 °C (dec.). 1H NMR (300 MHz, DMSO-d6) δ: 1.37 – 1.47 (m, 2H). 1.50 – 1.60 (m,
2H), 1.69 – 1.80 (m, 2H), 2.05 (s, 3H), 2.31 (s, 3H), 2.36 – 2.44 (m, 2H), 2.52 – 2.61 (m, 2H), 2.61
– 2.76 (m, 2H), 3.41 (s, 2H), 3.66 (s, 3H), 3.67 (s, 3H), 4.05 (t, J = 6.4 Hz, 2H), 6.59 (s, 1H), 6.62
(s, 1H), 6.86 – 6.99 (m, 2H), 7.66 (d, J = 9.4 Hz, 1H). Anal. C27H33NO5 (C, H, N). HRMS (Q-TOF)
calcd for C27H33NO5 [M+H]+ m/z 452.2431, found 452.2430; [M+Na]+ m/z 474.2251, found
474.2251.
5.1.6.9
7-{[6-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)hexyl]oxy}-3,4-dimethyl-2H-
chromen-2-one (3i). Prepared from 2e (0.18 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 0% → 30%). Yellow oil; yield: 0.21 g,
89%. 1H NMR (500 MHz, CDCl3) δ: 1.43 – 1.52 (m, 2H), 1.52 – 1.61 (m, 2H), 1.78 – 1.88 (m, 2H),
1.95 (br s, 2H), 2.18 (s, 3H), 2.37 (s, 3H), 2.97 (br s, 2H), 3.10 (br s, 2H), 3.27 (br s, 2H), 3.84 (s,
3H), 3.86 (s, 3H), 3.95 – 4.22 (m, 4H), 6.54 (s, 1H), 6.63 (s, 1H), 6.77 (d, J = 2.5 Hz, 1H), 6.83 (dd,
J = 8.9, 2.5 Hz, 1H), 7.49 (d, J = 8.9 Hz, 1H). Anal. C28H35NO5 (C, H, N). HRMS (Q-TOF) calcd
for C28H35NO5 [M+Na]+ m/z 488.2407, found 488.2410.
5.1.6.10
7-{[(2E)-4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)but-2-en-1-yl]oxy}-3,4-
dimethyl-2H-chromen-2-one (3j). Prepared from 2f (0.16 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column
chromatography (gradient eluent: methanol in dichloromethane 5% → 10%). Yellow low-melting
1
solid; yield: 0.17 g, 79%. H NMR (500 MHz, DMSO-d6) δ: 2.06 (s, 3H), 2.35 (s, 3H), 2.83– 3.06
(m, 2H), 3.21 (br s, 1H), 3.60 (br s, 1H), 3.70 (s, 3H), 3.72 (s, 3H), 3.85 – 3.96 (m, 2H), 4.05 – 4.16
(m, 1H), 4.24 – 4.33 (m, 1H), 4.78 (d, J = 4.3 Hz, 2H), 5.90– 6.03 (m, 1H), 6.17 – 6.27 (m, 1H),
6.74 (s, 1H), 6.80 (s, 1H), 6.97 (dd, J = 8.8, 2.5 Hz, 1H), 7.00 (d, J = 2.5 Hz, 1H), 7.70 (d, J = 8.9
Hz, 1H). Anal. C26H29NO5 (C, H, N). HRMS (Q-TOF) calcd for C26H29NO5 [M+H]+ m/z 436.2118,
found 436.2124.
5.1.6.11 7-({4-[(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl]benzyl}oxy)-3,4-dimethyl-
2H-chromen-2-one (3k). Prepared from 2g (0.19 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column
chromatography (eluent: ethyl acetate in n-hexane 70%). Off white solid; yield: 0.10 g, 42%; mp:
182-4 °C (dec.). 1H NMR (500 MHz, DMSO-d6) δ: 2.06 (s, 3H), 2.34 (s, 3H), 2.62 – 2.66 (m, 2H),
2.67 – 2.74 (m, 2H), 3.41 (s, 2H), 3.61 (s, 2H), 3.64 (s, 3H), 3.68 (s, 3H), 5.18 (s, 2H), 6.56 (s, 1H),
6.64 (s, 1H), 7.01 (dd, J = 8.6, 2.4 Hz, 1H), 7.04 (s, 1H), 7.34 – 7.39 (m, 2H), 7.42 (s, 2H), 7.70 (d,
J = 8.6 Hz, 1H). Anal. C30H31NO5 (C, H, N). HRMS (Q-TOF) calcd for C30H31NO5 [M+H]+ m/z
486.2275, found 486.2271; [M+Na]+ m/z 508.2094, found 508.2092.
5.1.6.12 7-({4-[2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl]benzyl}oxy)-3,4-dimethyl-
2H-chromen-2-one (3l). Prepared from 2h (0.19 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column
chromatography (gradient eluent: methanol in dichloromethane 0% → 5%). Pale yellow solid;
yield: 0.14 g, 57%; mp: 177-9 °C. 1H NMR (300 MHz, CDCl3) δ: 2.18 (s, 3H), 2.36 (s, 3H), 2.75 –
2.92 (m, 6H), 2.92 – 3.04 (m, 2H), 3.72 (s, 2H), 3.84 (s, 3H), 3.85 (s, 3H), 5.09 (s, 2H), 6.53 (s,
1H), 6.61 (s, 1H), 6.85 (d, J = 2.4 Hz, 1H), 6.91 (dd, J = 8.8, 2.4 Hz, 1H), 7.27 (d, J = 8.0 Hz, 2H),
7.36 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.8 Hz, 1H). Anal. C31H33NO5 (C, H, N). HRMS (Q-TOF)
calcd for C31H33NO5 [M+H]+ m/z 500.2431, found 500.2423; [M+Na]+ m/z 522.2251, found
522.2254.
5.1.6.13 7-({3-[(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl]benzyl}oxy)-3,4-dimethyl-
2H-chromen-2-one (3m). Prepared from 2i (0.16 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column

ACCEPTED MANUSCRIPT
chromatography (gradient eluent: ethyl acetate in n-hexane 30% → 70%). Yellow solid; yield: 0.18
1
g, 76%; mp: 72-4 °C (dec.). H NMR (500 MHz, DMSO-d6) δ: 2.06 (s, 3H), 2.34 (s, 3H), 2.60 –
2.63 (m, 2H), 2.64 – 2.70 (m, 2H), 3.41 (s, 2H), 3.62 (s, 2H), 3.64 (s, 3H), 3.67 (s, 3H), 5.20 (s,
2H), 6.54 (s, 1H), 6.62 (s, 1H), 7.00 (dd, J = 8.9, 2.5 Hz, 1H), 7.03 (d, J = 2.5 Hz, 1H), 7.28 – 7.33
(m, 1H), 7.34 – 7.38 (m, 2H), 7.44 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H). Anal. C30H31NO5 (C, H, N).
HRMS (Q-TOF) calcd for C30H31NO5 [M+H]+ m/z 486.2275, found 486.2270; [M+Na]+ m/z
508.2094, found 508.2091.
5.1.6.14 7-({3-[(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl]benzyl}oxy)-2H-chromen-
2-one (3n). Prepared from 2j (0.15 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column chromatography (gradient
1
eluent: ethyl acetate in n-hexane 30% → 70%). Dark yellow solid; yield: 0.16 g, 71%. H NMR
(500 MHz, DMSO-d6) δ: 2.62 (t, J = 5.6 Hz, 2H), 2.68 (t, J = 5.6 Hz, 2H), 3.41 (s, 2H), 3.63 (s, 2H),
3.64 (s, 3H), 3.67 (s, 3H), 5.21 (s, 2H), 6.27 (d, J = 9.5 Hz, 1H), 6.55 (s, 1H), 6.63 (s, 1H), 7.01 (dd,
J = 8.7, 2.4 Hz, 1H), 7.07 (d, J = 2.4 Hz, 1H), 7.29 – 7.34 (m, 1H), 7.34 – 7.37 (m, 2H), 7.45 (s,
1H), 7.61 (d, J = 8.7 Hz, 1H), 7.97 (d, J = 9.5 Hz, 1H). Anal. C28H27NO5 (C, H, N). HRMS (Q-
TOF) calcd for C28H27NO5 [M+H]+ m/z 458.1962, found 458.1961; [M+Na]+ m/z 480.1781, found
480.1777.
5.1.6.15 7-({2-[(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl]benzyl}oxy)-3,4-dimethyl-
2H-chromen-2-one (3o). Prepared from 2k (0.19 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column
chromatography (gradient eluent: ethyl acetate in n-hexane 0% → 40%). Yellow solid; yield: 0.10
1
g, 41%; mp: 79-81 °C (dec.). H NMR (500 MHz, DMSO-d6) δ: 2.04 (s, 3H), 2.29 (s, 3H), 2.59 –
2.65 (m, 2H), 2.65 – 2.72 (m, 2H), 3.40 (s, 2H), 3.62 (s, 3H), 3.66 (s, 3H), 3.70 (s, 2H), 5.38 (s,
2H), 6.51 (s, 1H), 6.60 (s, 1H), 6.97 (d, J = 2.5 Hz, 1H), 7.01 (dd, J = 8.9, 2.5 Hz, 1H), 7.26 – 7.34
(m, 2H), 7.36 – 7.41 (m, 1H), 7.43 – 7.47 (m, 1H), 7.49 (d, J = 8.9 Hz, 1H). Anal. C30H31NO5 (C,
H, N). HRMS (Q-TOF) calcd for C30H31NO5 [M+H]+ m/z 486.2275, found 486.2264; [M+Na]+ m/z
508.2094, found 508.2098.
5.1.6.16
3-Chloro-7-[4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butoxy]-4-methyl-2H-
chromen-2-one (3p). Prepared from 2l (0.17 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column
chromatography (eluent: methanol in dichloromethane 0.1%). Yellow solid; yield: 0.12 g, 53%; mp:
116-8 °C (dec.). 1H NMR (300 MHz, CDCl3) δ: 1.87 – 2.03 (m, 4H), 2.54 (s, 3H), 2.80 – 2.89 (m,
2H), 2.93– 3.07 (m, 4H), 3.72 – 3.86 (m, 8H), 4.08 (t, J = 5.7 Hz, 2H), 6.53 (s, 1H), 6.60 (s, 1H),
6.80 (d, J = 2.5 Hz, 1H), 6.89 (dd, J = 8.9, 2.5 Hz, 1H), 7.50 (d, J = 8.9 Hz, 1H). Anal.
C25H28ClNO5 (C, H, N). HRMS (Q-TOF) calcd for C25H28ClNO5 [M+H]+ m/z 458.1729, found
458.1702; [M+Na]+ m/z 480.1548, found 480.1546.
5.1.6.17 3-Chloro-7-{[5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl]oxy}-4-methyl-2H-
chromen-2-one (3q). Prepared from 2m (0.18 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column
chromatography (gradient eluent: methanol in dichloromethane 0% → 2%). Yellow solid; yield:
1
0.19 g, 80%; mp: 189-191 °C (dec.). H NMR (300 MHz, CDCl3) δ: 1.52 – 1.68 (m, 2H), 1.81 –
1.93 (m, 2H), 1.93 – 2.08 (m, 2H), 2.54 (s, 3H), 2.95 – 3.15 (m, 4H), 3.19 – 3.35 (m, 2H), 3.83 (s,
3H), 3.85 (s, 3H), 3.94 – 4.17 (m, 4H), 6.56 (s, 1H), 6.62 (s, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.89 (dd,
J = 8.9, 2.4 Hz, 1H), 7.51 (d, J = 8.9 Hz, 1H). Anal. C26H30ClNO5 (C, H, N). HRMS (Q-TOF) calcd
for C26H30ClNO5 [M+H]+ m/z 472.1885, found 472.1857; [M+Na]+ m/z 494.1705, found 494.1702.
5.1.6.18 3-Bromo-7-{[5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl]oxy}-4-methyl-2H-
chromen-2-one (3r). Prepared from 2n (0.20 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column
chromatography (gradient eluent: methanol in dichloromethane 0% → 2%). Pale yellow solid;
yield: 0.088 g, 34%; mp: 71-3 °C (dec.). 1H NMR (300 MHz, CDCl3) δ: 1.44 – 1.72 (m, 2H), 1.77 –
2.00 (m, 4H), 2.59 (s, 3H), 2.74 – 2.92 (m, 2H), 2.97 – 3.15 (m, 2H), 3.75 – 3.89 (m, 8H), 3.89 –

ACCEPTED MANUSCRIPT
4.11 (m, 4H), 6.53 (s, 1H), 6.62 (s, 1H), 6.80 (d, J = 2.4 Hz, 1H), 6.88 (dd, J = 8.9, 2.4 Hz, 1H),
7.55 (d, J = 8.9 Hz, 1H). Anal. C26H30BrNO5 (C, H, N). HRMS (Q-TOF) calcd for C26H30BrNO5
[M+Na]+ m/z 538.1200, found 538.1201.
5.1.6.19
7-[4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butoxy]-3-phenyl-2H-chromen-2-
one (3s). Prepared from 2o (0.19 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column chromatography (gradient
eluent: methanol in dichloromethane 0% → 2%). Yellow solid; yield: 0.11 g, 47%; mp: 132-4 °C.
1H NMR (300 MHz, CDCl3) δ: 1.73 – 1.97 (m, 4H), 2.60 (t, J = 7.1 Hz, 2H), 2.74 (t, J = 5.7 Hz,
2H), 2.84 (t, J = 5.7 Hz, 2H), 3.58 (s, 2H), 3.82 (s, 3H), 3.83 (s, 3H), 4.08 (t, J = 6.2 Hz, 2H), 6.52
(s, 1H), 6.59 (s, 1H), 6.81 – 6.88 (m, 2H), 7.33 – 7.48 (m, 4H), 7.63 – 7.72 (m, 2H), 7.75 (s, 1H).
Anal. C30H31NO5 (C, H, N). HRMS (Q-TOF) calcd for C30H31NO5 [M+H]+ m/z 486.2275, found
486.2264; [M+Na]+ m/z 508.2094, found 508.2096.
5.1.6.20 7-{[5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl]oxy}-3-phenyl-2H-chromen-
2-one (3t). Prepared from 2p (0.19 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column chromatography (gradient
eluent: methanol in dichloromethane 1% → 2%). Pale yellow solid; yield: 0.17 g, 70%; mp: 128-
130 °C (dec.). 1H NMR (300 MHz, DMSO-d6) δ: 1.38 – 1.58 (m, 2H), 1.66 – 1.92 (m, 4H), 2.79 –
3.10 (m, 2H), 3.10 – 3.35 (m, 4H), 3.53 – 3.79 (m, 8H), 4.11 (t, J = 6.2 Hz, 2H), 6.76 (s, 1H), 6.80
(s, 1H), 6.96 (dd, J = 8.6, 2.3 Hz, 1H), 7.01 (d, J = 2.3 Hz, 1H), 7.32 – 7.49 (m, 3H), 7.61 – 7.72
(m, 3H), 8.18 (s, 1H). Anal. C31H33NO5 (C, H, N). HRMS (Q-TOF) calcd for C31H33NO5 [M+H]+
m/z 500.2431, found 500.2429; [M+Na]+ m/z 522.2251, found 522.2255.
5.1.6.21 7-{[5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl]oxy}-4-methyl-3-phenyl-2H-
chromen-2-one (3u). Prepared from 2q (0.20 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column
chromatography (gradient eluent: methanol in dichloromethane 0% → 2%). Yellow solid; yield:
1
0.11 g, 44%; mp: 168-170 °C (dec.). H NMR (300 MHz, CDCl3) δ: 1.50 – 1.67 (m, 2H), 1.75 –
1.99 (m, 4H), 2.27 (s, 3H), 2.67 – 2.86 (m, 2H), 2.91 – 3.08 (m, 4H), 3.72 – 3.93 (m, 8H), 4.06 (t, J
= 6.2 Hz, 2H), 6.54 (s, 1H), 6.61 (s, 1H), 6.83 (d, J = 2.4 Hz, 1H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H),
7.25 – 7.32 (m, 2H), 7.33 – 7.51 (m, 3H), 7.56 (d, J = 8.8 Hz, 1H). Anal. C32H35NO5 (C, H, N).
HRMS (Q-TOF) calcd for C32H35NO5 [M+H]+ m/z 514.2588, found 514.2586; [M+Na]+ m/z
536.2407, found 536.2409.
5.1.6.22 3-{[5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl]oxy}-6H-benzo[c]chromen-
6-one (3v). Prepared from 2r (0.18 g, 0.50 mmol) and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
hydrochloride (0.17 g, 0.75 mmol). Purification procedure: column chromatography (eluent:
methanol in dichloromethane 5%). Yellow solid; yield: 0.11 g, 45%; mp: 106-8 °C (dec.). 1H NMR
(300 MHz, CDCl3) δ: 1.64 – 1.49 (m, 2H), 1.65 – 1.79 (m, 2H), 1.82 – 1.96 (m, 2H), 2.53 – 2.71
(m, J = 8.7 Hz, 2H), 2.75 – 2.94 (m, 4H), 3.67 (s, 2H), 3.83 (s, 3H), 3.84 (s, 3H), 4.04 (t, J = 6.4 Hz,
2H), 6.52 (s, 1H), 6.59 (s, 1H), 6.85 (d, J = 2.5 Hz, 1H), 6.90 (dd, J = 8.8, 2.5 Hz, 1H), 7.45 – 7.55
(m, 1H), 7.73 – 7.83 (m, 1H), 7.89 – 8.03 (m, 2H), 8.36 (d, J = 8.0 Hz, 1H). Anal. C29H3 NO5 (C, H,
1
N). HRMS (Q-TOF) calcd for C29H31NO5 [M+H]+ m/z 474.2275, found 474.2267; [M+Na]+ m/z
496.2094, found 496.2090.
5.2 Biological assays
5.2.1 Materials
CulturePlate 96/wells plates were purchased from PerkinElmer Life and Analytical Sciences
(Boston, MA, USA). Calcein-AM, doxorubicin and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazoliumbromide) were purchased from Sigma-Aldrich-RBI s.r.l. (Milan, Italy). Cell
culture medium and reagents were purchased from EuroClone (Milan, Italy) and Sigma-Aldrich.
5.2.2 Cell cultures

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MDCK-MDR1 and MDCK-MRP1 are a gift of Prof. P. Borst, NKI-AVL Institute, Amsterdam,
Netherlands. MDCK-MDR1, MDCK-MRP1 were grown in DMEM high glucose supplemented
with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL
streptomycin, in a humidified incubator at 37 °C with a 5 % CO2 atmosphere.
5.2.3 Calcein-AM assays
According to a previously reported procedure, [24] MDCK-MDR1 or MDCK-MRP1 cell lines
(50,000 cells per well) were seeded into black CulturePlate 96/wells plate with 100 µL medium and
grown overnight to confluence. Aliquots (100 µL) of tested compounds in culture medium, at scalar
concentrations ranging from 0.1 to 100 µM, were added to each well. The plate was incubated at 37
°C for 30 min. Calcein-AM in phosphate buffered saline (PBS, 100 µL) was added to each well at a
final concentration of 2.5 µM, and the plate was incubated for 30 min. The plate was washed 3
times with 100 mL ice cold PBS. Saline buffer (100 µL) was added to each well and the plate was
read by a PerkinElmer Victor3 spectrofluorimeter at excitation and emission wavelengths of 485
nm and 535 nm, respectively. Under these conditions, calcein cell accumulation in the absence and
in the presence of tested compounds was evaluated, and a fluorescence basal level was estimated by
untreated cells. In treated wells, the increase of fluorescence with respect to the basal level was
measured. IC50 values were determined by fitting the fluorescence increase percentage versus
log[dose] with GraphPad Prism Software 5.0 (GraphPad Software, Inc.: San Diego, CA).
5.2.4 Antiproliferative assay
The co-administration assay with doxorubicin was performed in MDCK-MDR1 cells at 72 h. [25]
On day 1, 10,000 cells/well were seeded into 96-well plates in a volume of 100 µL. On day 2, the
compounds under study, each in three concentrations (0.1, 1, and 10 µM), were added. On day 3,
the medium was removed and the tested compounds, each in three concentrations, were added alone
and in combination with 10 µM doxorubicin. In all the experiments, the solvents (EtOH, DMSO)
were added in each control to evaluate their cytotoxicity. After the established incubation time, 0.5
mg/mL MTT was added to each well and after 3 h incubation at 37 °C the supernatant was
removed. The formazan crystals were solubilized using 100 µL of DMSO and the absorbance
values at 570 and 630 nm were determined on the microplate reader Victor 3. Data were analyzed
by one-way ANOVA (GraphPad Prism 5.0) for repeated measures followed by post-hoc Bonferroni’s
multiple comparison test. Results are expressed as mean ± SD of at 2-3 independent experiments in
triplicates. Statistical significance was accepted at a level of P < 0.05.
5.3 Molecular docking studies
The molecular model of the ligand 3h in protonated form, with standard values of bond lengths and
valence angles, was built within Maestro software package [26], while Marsili-Gasteiger charges
were calculated with the molcharge suite of QUACPAC.[27] The THIQ dihedral ring angles were
tailored according to the puckering observed in the previously reported X-ray of the structurally
related P-gp inhibitor MC70.[28] AMBER FF14SB force field charges were assigned to MDR1
protein structure within CHIMERA 1.11.[29] Due to the flexibility of the ligand and a large
extension of the binding surface, in order to assess a reliable bioactive conformation molecular
dynamics sampling followed by docking to MDR1 structure were carried out according to similar
studies performed on the same biomolecular target.[21] In detail, the 3h solvated structure was
assembled using the Desmond system builder tool implemented in Maestro [30], and stochastic
molecular dynamics were performed using the default settings and relaxation protocol of Desmond
on a NVDIA Quadro M4000 GPU at constant temperature (300 K) and pressure (1 bar) for a total
of 500 ns, storing energies and trajectory frames each 250 ps. An initial screening of the sampled
2000 conformations was first carried out with VINA 1.1.2 [31] submitting each structures to rigid
body dockings, increasing the search space size by 30 Å in each of the three dimension from the

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center of its binding site comprising residues Met69, Tyr310, Phe336, Phe728, Ala729, Phe732,
Met949, Ser952, Tyr953, Leu975, Phe978, Ser979, Val982, Phe983, Met986. Afterwards the
refinement of VINA poses was obtained as follows: affinity maps were calculated on a 65×65×65
cubic box 0.375 Å spaced, and a local optimization with AUTODOCK 4.2.6 [32] was used in a
ligand-receptor flexible docking to estimate the free energy of binding (FEB) as scoring function.
The best FEB solution was then selected as representative of the binding mode of compound 3h.
Acknowledgements
This work was supported by University of Bari “Aldo Moro”. L.P. acknowledges financial support
from APQ Research Apulian Region “FutureInResearch (FKY7YJ5) - Regional program for smart
specialization and social and environmental sustainability” Fondo di Sviluppo e Coesione 2007–
2013.
Author Contributions
L.P. designed and supervised the project, contributed to the synthesis, made the literature research
and wrote the paper. M.R. carried out the synthesis and the physico-chemical characterization. M.N.
carried out the in vitro screening and revised the paper. A.C. performed the docking studies and
contributed to write the paper. N.A.C. analyzed and supervised the in vitro screening. S.C.
supervised the synthesis and revised the manuscript. C.D.A. supervised the project and contributed
to write the paper. All authors have given approval to the final version of the manuscript.

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TOC graphic

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1,2,3,4-Tetrahydroisoquinoline/2H-chromen-2-one conjugates as nanomolar P-
glycoprotein inhibitors: molecular determinants for affinity and selectivity over
multidrug resistance associated protein 1
a
a
a
Mariagrazia Rullo , Mauro Niso , Leonardo Pisani *,a, Antonio Carrieri , Nicola Antonio
Colabufo a, Saverio Cellamare a, Cosimo Damiano Altomare a
Highlights:
1. THIQ-coumarin conjugates were identified as nanomolar P-gp inhibitors
2. Linker length and flexibility affected P-gp inhibition potency
3. Bulky groups (Br, Ph) at coumarin C3 improved P-gp / MRP1 selectivity
4. A pentamethylene linker led to the most active compound (3h, IC50 = 70 nM)
5. Six diverse novel compounds reversed resistance of MDCK-MDR1 cells to doxorubicin