Structure–Activity Relationship Studies on 6,7-Dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline Derivatives as Multidrug Resistance Reversers

Article abstract of DOI:10.1002/cmdc.201700239

A series of derivatives were synthesized and studied with the aim to investigate the structure–activity relationships of the two P-glycoprotein (P-gp) modulators elacridar and tariquidar. Then, different aryl-substituted amides were inserted, and to explore the effects of varying the amide function, the corresponding isosteric ester derivatives and some alkylamine analogues were synthesized. The new compounds were studied to evaluate their P-gp interaction profile and selectivity toward the two other ABC transporters, multidrug-resistance-associated protein-1 (MRP-1) and breast cancer resistance protein (BCRP). Investigation of the chemical stability of the amide and ester derivatives toward spontaneous or enzymatic hydrolysis showed that these compounds were stable in phosphate-buffered saline and human plasma. This study allowed us to evaluate the selectivity of the three series on the three efflux pumps and to propose the structural requirements that define the P-gp interaction profile. We identified two P-gp substrates, a P-gp inhibitor, and three ester derivatives that were active on BCRP, which opens a new scenario in the development of ligands active toward this pump.

Full text of DOI:10.1002/cmdc.201700239

Accepted Article
Title: Structure-Activity Relationship Studies on 6,7-Dimethoxy-2-
phenethyl-1,2,3,4-tetrahydroisoquinoline Derivatives as Multidrug
Resistance (MDR) reversers
Authors: Elisabetta Teodori, Silvia Dei, Gianluca Bartolucci, Maria
Grazia Perrone, Dina Manetti, Maria Novella Romanelli,
Marialessandra Contino, and Nicola Antonio Colabufo
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To be cited as: ChemMedChem 10.1002/cmdc.201700239
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ChemMedChem
Structure-Activity Relationship Studies on 6,7-Dimethoxy-2-phenethyl-1,2,3,4-
tetrahydroisoquinoline Derivatives as Multidrug Resistance (MDR) reversers
Elisabetta Teodori,*[a] Silvia Dei,[a] Gianluca Bartolucci,[a] Maria Grazia Perrone,[b] Dina Manetti,[a]
Maria Novella Romanelli,[a] Marialessandra Contino,[b] Nicola Antonio Colabufo[b]
Abstract
A series of derivatives were synthesized and studied with the aim to investigate the structure-
activity relationships of the two P-glycoprotein (P-gp) modulators elacridar and tariquidar. Then
different aryl substituted amides were inserted and to explore the effects of varying the amide
function, the corresponding isosteric ester derivatives and some alkylamine analogues were
synthesized. The new compounds were studied to evaluate their P-gp interaction profile and
selectivity towards the two other ABC transporters, Multidrug-Resistance-associated Protein-1
(MRP-1) and Breast Cancer Resistance Protein (BCRP). The investigation on the chemical stability
of amide and ester derivatives towards spontaneous or enzymatic hydrolysis, showed that these
compounds resulted stable in phosphate buffer solution and human plasma. This study allowed us to
evaluate the selectivity of the three series on the three efflux pumps and to propose the structural
requirements that define the P-gp interaction profile. We identified two P-gp substrates and a P-gp
inhibitor and three ester derivatives active on BCRP that opens a new scenario in the development
of ligands active towards this pump.
[a] Prof. E. Teodori, Prof. S. Dei, Dr. G. Bartolucci, Dr. D. Manetti, Prof. M. N. Romanelli,
Dipartimento NEUROFARBA-Sezione di Farmaceutica e Nutraceutica
Università di Firenze
via Ugo Schiff 6, 50019 Sesto Fiorentino (FI), Italy
E-mail: elisabetta.teodori@unifi.it
[b] Dr. M. G. Perrone, Dr. M. Contino, Prof. N. A. Colabufo
Dipartimento di Farmacia-Scienze del Farmaco
Università degli Studi di Bari “A. Moro”
via Orabona 4, 70125, Bari, Italy
1
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Introduction
The transmembrane proteins of the ABC (ATP Binding Cassette) transporter family are able to
transport a variety of compounds across the cell membrane using ATP hydrolysis energy. These
proteins are expressed in many tissues where they play important protective roles by regulating the
permeability of biological membranes.[1] Several ABC transporters are overexpressed in cancer
cells thus causing their chemoresistance, as these proteins can bind a large number of hydrophobic
compounds including anticancer drugs.[2-4] Many structurally and mechanistically unrelated
chemotherapeutic drugs are ABC proteins substrates and are actively pumped out by the
transporters located at the plasma membrane of the tumor cells resulting in a decrease of their
cellular concentration below the effective dose.[1] This cancer cells defence mechanism is called
multidrug resistance (MDR) and represents the main obstacle to achieve success with
chemotherapy; since even in cases when the tumor initially responds to chemotherapy, resistance
can rapidly develop as a results of drug treatment.[5]
The ABC transporter family includes structurally related membrane proteins that share a common
feature of being made of two domains: the nucleotide binding domain (NBD) and the
transmembrane spanning domain (TMD).[6] The function of NBD is to hydrolyse ATP and the
resulting energy is used by the TMD to translocate substances through the membrane by
conformational changes.[7]
The human genome encodes 49 ABC transporters classified into seven subfamilies (ABC-A to
ABC-G). Among the transporters involved in drug efflux from human cells, three are mainly
associated with multidrug resistance (MDR): P-glycoprotein (P-gp, ABCB1), the Multidrug-
Resistance-associated Protein-1 (MRP1, ABCC1), and the Breast Cancer Resistance Protein
(BCRP, ABCG2).[8-11]
P-gp is the most studied ABC transporter[12] and is the first efflux transporter discovered to be
involved in drug resistance.[13] In fact, P-gp is overexpressed in many cancer cells as a result of
chemotherapy treatment causing an acquired resistance to a variety of anticancer drugs.[2-4]
Moreover, in addition to the decrease in the intracellular concentration of chemotherapeutic agents,
P-gp might display other mechanisms of conferring resistance. P-gp overexpressing cells are less
sensitive to caspase-dependent apoptosis induced by a range of different stimuli, including Fas
ligand, tumor necrosis factor, UV irradiation, and serum starvation. Thus the cells with high P-gp
expression may have a higher survival rate or less apoptosis induction.[13]
MRP1 was discovered for the first time in not expressing P-gp cancer cells that showed resistance
to chemotherapeutics treatment due to increased efflux of anticancer drugs.[14,15] This transporter has
broad spectrum anticancer resistance activity and for this reason it belongs to the three main MDR
proteins. Overexpression of this efflux pump was shown to be associated with resistance to many
anticancer drugs such as cisplatin, etoposide, doxorubicin, vincristine, methotrexate and purine
analogs.[16]
BCRP is the most recently ABC transporter identified to be involved in multidrug-resistance BCRP.
Differently from P-gp and MRP1, BCRP is a half-transporter which requires at least
dimerization,[17] or even tetramerization,[18,19] to be functional. Many evidences suggest that BCRP
transporter is expressed in several hematological and solid tumors, together with P-gp,
compromising the therapeutic effectiveness of several substrate agents.[20,21] Moreover, BCRP is
also expressed in cancer stem cells, thus causing direct protection against chemotherapeutics.[22,23]
2
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10.1002/cmdc.201700239
ChemMedChem
Cancer stem cells are capable of long-term self-renewal and are probably responsible for long-term
failure of many cancer chemotherapies.
BCRP and P-gp are the two dominant ABC transporters located at the blood-brain barrier (BBB).
They are responsible of the extrusion of several drugs that are substrates of these two transporters
including anticancer drugs.[24] For this reason many drugs have a limited capacity to cross the
blood-brain barrier (BBB) and reach therapeutically meaningful concentrations.[25]
Since their discovery and elucidation of the mechanism of action, P-gp, MRP1 and BCRP have
been considered suitable targets for circumventing MDR.[26] This is the main reason prompting the
design and synthesis of transporters modulators to co-administrate with the antineoplastic drugs,
that are substrates of these proteins, and that would restore their efficacy in resistant cancer cells.[27]
A large number of compounds showing P-gp modulating activity has been synthesized and studied
(classified as first, second- or third-generation)[28-30] and several have reached clinical trials.[31]
However, most of these trials have not been successful because of limitations due to the changes in
the pharmacokinetic properties of the co-administered chemotherapeutic drug[32] since some P-gp
modulators are also able of inhibiting CyP3A4 enzymes. Nevertheless the third generation of P-gp
modulators have not shown an appreciable impact on CyP3A4 and do not require a reduction in the
dose of anticancer drug.[33]
The third generation P-gp modulators elacridar and tariquidar are not specific for P-gp because they
are also able to bind the BCRP transporter.[34] The common structural feature of the two compounds
is the presence of a basic 6,7-dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline nucleus linked
to an aryl substituted amide (Figure 1).
Structure-activity relationship studies on these compounds suggested that the 6,7-dimethoxy-2-
phenethyl-1,2,3,4-tetrahydroisoquinoline nucleus is essential for the inhibition of the two
transporter proteins P-gp and BCRP while changes at the aryl substituted amide nucleus cause
variations in the selectivity.[24]
In this study, with the aim to better evaluate the features for selectivity towards these transporters
we decided to deeper explore the structure-activity relationships of these compounds. As a matter of
fact one of the problems of MDR modulators might be the lack of information on the structural
requirements for selectivity against the large family of transporters. Toward this end we designed
and synthesized new series of molecules characterized by the presence of a 6,7-dimethoxy-2-
phenethyl-1,2,3,4-tetrahydroisoquinoline scaffold linked, as the two lead compounds, to an aryl
substituted amide, on which different aryl nucleus have been inserted (compounds 1-6, Figure 1).
To explore the consequences of varying the amide function, we also synthesized the corresponding
isosteric ester derivatives (7-12) and some analogue compounds containing an alkylamine function
(13-16) (Figure 1). The inserted aryl moieties were chosen since they were present in compounds
previously studied and proved to be very potent multidrug resistance reversers.[35-38]
3
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10.1002/cmdc.201700239
ChemMedChem
OCH3
OCH3
OCH3
OCH3
N
N
O
O
N
O
H
H
3CO
N
N
H
H
NH
3CO
Elacridar
Tariquidar
H
OCH3
O
N
OCH3
OCH3
N
Ar
Y
O
O
N
H
N
H
O
I
II
III
1-6
7-12
13-16
=
Ar
OCH3
OCH3
OCH3
A
H
3CO
OCH3
H
H
3CO
H
H
3CO
H
H
3CO
3CO
3CO
3CO
B
D
E
F
C
Figure 1: Structures of lead compounds and designed derivatives.
The synthesized compounds were studied to evaluate their P-gp interaction profile and selectivity
towards the two other ABC transporters, MPR1 and BCRP.
The P-gp interacting-mechanism of each compound was investigated by three combined biological
assays: i) Apparent Permeability (Papp) determination (BA/AB) in Caco-2 cell monolayer; ii) ATP
cell depletion in cells overexpressing the transporter MDCK-MDR1 cells; iii) the inhibition of
Calcein-AM transport in MDCK-MDR1 cells.
The inhibitory activity on MRP1 and BCRP, was evaluated on tumor cell lines overexpressing each
transporter (MDCK-MRP1 and MDCK-BCRP cells). The assay is performed by measuring the
inhibition of the efflux of the pro-fluorescent probe Calcein-AM in in MDCK-MRP1 cells or the
fluorescent probe Hoechst 33342 in MDCK-BCRP cells.
Furthermore, the stability of amide and ester derivatives 1-12, towards spontaneous or enzymatic
hydrolysis was investigated in phosphate buffer solution (PBS) and human plasma, respectively,
and the degradation profiles were evaluated.
Results and Discussion
Chemistry
The reaction pathways used to synthesize derivatives 1-16 are reported in Schemes 1-4. Amide
derivatives were synthesized starting from aniline 17, which in turn was obtained by alkylation of
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride with 1-(2-bromoethyl)-4 nitrobenzene
4
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10.1002/cmdc.201700239
ChemMedChem
followed by catalytic reduction of the nitro group.[39] Amides 1-5 were then obtained by reaction of
17 with the appropriate carboxylic acid chloride (Scheme 1), while amide 6, carrying a triple bond,
was obtained by reaction of 17 with 3-(3,4,5-trimethoxyphenyl)propiolic acid using the activating
agent EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimmide hydrochloride) in the presence of
DMAP (4-dimethylaminopyridine) (Scheme 1). (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid, 3,4,5-
trimethoxybenzoic acid, anthracene-9-carboxylic acid and 9H-fluorene-9-carboxylic acid are
commercially available, while 3-(3,4,5-trimethoxyphenyl)propiolic acid[40] and 2,2-bis(4-
methoxyphenyl)acetic acid[41] were synthesized as described previously. Ester derivatives were
obtained as reported in Scheme 2. The chloroethyl derivative 18 was obtained by the commercially
available 4-(hydroxyethyl)phenol by treatment with SOCl2 in ethanol-free CHCl3 and anhydrous
triethylamine, slightly modifying the procedure described by Smith;[42] 18 was then reacted with
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride in anhydrous acetonitrile in the
presence of K2CO3, leading to phenol 19. As in the case of amides, esters 7-11 were obtained by
reaction of 19 with the appropriate carboxylic acid chloride, while propiolic derivative 12 was
obtained by reaction of 19 with 3-(3,4,5-trimethoxyphenyl)propiolic acid using EDCI and DMAP.
Alkylamine derivatives were obtained as reported in Scheme 3. Amine 14 was obtained, with a
low yield (15%), by reaction of aniline 17 with 5-(iodomethyl)-1,2,3-trimethoxybenzene 21,
obtained by treatment of the chloro analog 20[43] with NaI in acetone. Therefore, the other desired
amines 13, 15 and 16 were obtained, with better yields, according to the Mattson procedure[44] by
reductive alkylation of the suitable aldehyde with amine 17 using titanium (IV) isopropoxide as
Lewis catalyst and NaBH3CN as reducing agent. The aldehydes are commercially available
(anthracene-9-carbaldehyde) or synthesized by us (Scheme 4). Reduction of (E)-methyl 3-(3,4,5-
trimethoxyphenyl)acrylate, using 2 equiv of DIBAL-H in anhydrous CH2Cl2 at -78°C furnished the
corresponding alcohol 22,[45] which was oxidized to (E)-3-(3,4,5-trimethoxyphenyl)acrylaldehyde
23 by means of pyridinium chlorochromate and celite in anhydrous CH2Cl2. Finally, (2,2-bis(4-
methoxyphenyl)acetaldehyde 24 was obtained by reduction of ethyl 2,2-bis(4-
methoxyphenyl)acetate using a little excess of DIBAL-H in toluene at -78°C.
NH2
H
H
3CO
3CO
N
Ar
HO
O
17
Ar
a
Cl
O
c
b
H
N
Ar
H
N
Ar
H
H
3CO
O
N
H
H
3CO
3CO
O
N
3CO
=
1
2
3 Ar
4
5
Ar
Ar
A
B
C
D
E
=
6 Ar
F
=
=
=
=
Ar
Ar
Scheme 1. Reagents and conditions: a) SOCl2, ethanol-free CHCl3; b) ethanol-free CHCl3; c) EDCI, DMAP,
an. CH2Cl2. For the meaning of Ar, see Table 1.
5
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ChemMedChem
OH
Ar
O
OH
H
H
3CO
N
a
HO
Cl
3CO
Ar
b
19
18
Cl
O
c
d
Ar
O
Ar
O
H
H
3CO
O
N
H
H
3CO
3CO
O
N
3CO
=
=
=
=
=
7
8
9
Ar
Ar
Ar
A
B
C
D
E
=
12
Ar
F
10 Ar
11
Ar
Scheme 2. Reagents and conditions: a) 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline HCl, K2CO3, an.
CH3CN; b) SOCl2, ethanol-free CHCl3; c) ethanol-free CHCl3; d) EDCI, DMAP, an. CH2Cl2. For the
meaning of Ar, see Table 1.
NH2
H
H
3CO
3CO
N
Ar
Cl
a
=
20 Ar
B
17
H
Ar
I
Ar
O
=
21
Ar
B
b
d
c,
H
N
Ar
H
N
Ar
H
3CO
N
H
H
3CO
3CO
N
H
3CO
=
=
=
13 Ar
15 Ar
16 Ar
A
C
D
=
14
Ar
B
Scheme 3. Reagents and conditions: a) NaI, acetone; b) 17, K2CO3, an. CH3CN; c) titanium(IV)
isopropoxide, suitable aldehyde: anthracene-9-carbaldehyde or 23 or 24; d) NaBH3CN, abs. EtOH. For the
meaning of Ar, see Table 1.
6
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10.1002/cmdc.201700239
ChemMedChem
OCH3
OCH3
OCH3
H
H
3CO
H
H
3CO
b
a
H
H
3CO
H
OH
3CO
3CO
O
3CO
O
23
22
O
OCH3
OCH3
c
O
H
O
OCH3
O
OCH3
24
Scheme 4. Reagents and conditions: a) DIBAL-H (2 eq.), an. CH2Cl2, -78°C; b) PCC, celite, an. CH2Cl2; c)
DIBAL-H, toluene, -78°C.
Biological studies
The P-gp interacting mechanism of the compounds was investigated by three combined assays,
previously reported: i) Apparent Permeability (Papp) determination (BA/AB) in Caco-2 cell
monolayer;[46] ii) ATP cell depletion in cells overexpressing the transporter MDCK-MDR1;[47] and
iii) the inhibition of the transport of a pro-fluorescent probe (Calcein-AM) in MDCK-MDR1
cells.[48] The first assay measures the ratio between two fluxes: from the basolateral to apical
compartments (BA, representative of passive diffusion) and from the apical to basolateral
compartments (AB, representative of active transport). If (BA/AB) < 2, the compound can be
considered an inhibitor, also taking into account the results of the ATPase assay and Calcein-AM
modulation. In the same manner, if (BA/AB) > 2, the compound is classified as a substrate.[49,50]
The second assay detects the consumption of ATP due to transport mediated by the pump;
generally, a substrate, being transported by the pump, induces ATP cell depletion (unambiguous
substrate, category I), while a P-gp inhibitor does not induce ATP consumption. However, there is a
third substrate category (known as category IIB3) displaying a Papp value > 2 but not inducing an
ATP cell depletion.[51] The third assay establishes the potency of the interaction between the
compounds and the pump by measuring the transport inhibition of the pro-fluorescent probe
Calcein-AM, that is a P-gp substrate, in a cell line overexpressing P-gp (MDCK-MDR1 cells). The
selectivity of all compounds 1-16 vs P-gp was measured by detecting also the activity of the same
compounds towards the other ABC transporters MRP1 and BCRP. For this purpose, the interaction
with MRP1 was evaluated by measuring the inhibition of the efflux of Calcein-AM (that is also a
MRP1 substrate) in cells overexpressing MRP1 (MDCK-MRP1 cells); the interaction with BCRP
was evaluated by the measure of the inhibition of the efflux of the fluorescent probe Hoechst 33342
(that is a BCRP substrate) in cells overexpressing BCRP (MDCK-BCRP cells).
The results of the three assays on compounds 1-16 are reported in Table 1 together with those on
tariquidar and elacridar used as reference compounds.
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Table 1: Biological results of derivatives 1-16.
OCH3
OCH3
OCH3
OCH3
OCH3
N
N
N
O
O
OCH3
Ar
II
I
III
Ar
N
Ar
N
H
O
H
Ar
OCH3
OCH3
OCH3
H
3CO
OCH3
H
H
3CO
H
H
3CO
H
H
3CO
3CO
3CO
3CO
a
c
e
b
d
f
EC50 µM[a]
ATP cell
depletion
no
[b]
Compd
Structure Ar
P-gp
MRP1
BCRP
Papp
I
I
I
I
I
A
B
C
D
E
F
0.78±0.12
1.04±0.20
0.66±0.13
0.30±0.06
0.70±0.11
0.70±0.14
1.40±0.20
0.93±0.18
1.23±0.24
0.33±0.07
10±1.80
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
nd
NA
NA
NA
NA
NA
18
5.1
10.6
6.6
5.3
>20
1.7
5.5
>20
4.7
7.9
4.8
5.6
5.6
16.4
4.7
22
1
2
3
4
5
6
7
8
9
no
no
no
no
no
no
no
no
no
no
yes
no
no
no
no
yes[d]
yes[e]
I
NA
NA
II
II
II
II
II
II
III
III
III
III
A
B
C
D
E
F
A
B
C
D
17±2.3
5.9±1.10
NA
NA
10±1.5
NA
10
11
12
13
14
15
16
Tar[c]
Elacr
2±0.40
0.68±0.13
0.73±0.15
1.01±0.20
0.57 ±0.11
0.044±0.001
0.014±0.003
NA
NA
NA
0.010±0.005
10±2.0
NA
>20
[a] Values are the mean ±SEM of two independent experiments, with samples in triplicate. [b]
Apparent permeability estimation: values are from two independent experiments, with samples in
duplicate. [c] See reference 52. [d] Percentage of the effect at a concentration of 50 µM (30%). [e]
Percentage of the effect at a concentration of 10 µM (23%). NA = not active; nd = not determined.
As shown in Table 1, all the compounds displayed a high/moderate activity on P-gp (EC50 ranging
from 0.30 to 10 µM). The amide derivatives of series I were more potent than the corresponding
ester analogues (series II) (EC50 = 0.78 vs 1.40 µM for Ar = A (compounds 1 and 7); EC50 = 0.66 vs
1.23 µM for Ar = C (compounds 3 and 9); EC50 = 0.70 vs 10 µM for Ar = E (compounds 5 and 11);
EC50 = 0.70 vs 2 µM for Ar = F (compounds 6 and 12), respectively) except for compounds 2 and 8,
bearing the 3,4,5-trimethoxy phenyl moiety (B), and compounds 4 and 10, bearing the 2,2-bis(4-
methoxyphenyl) moiety (D), that showed comparable activity (EC50 = 1.04 vs 0.93 µM, and EC50=
0.30 vs 0.33 µM, respectively). Also the alkylamine derivatives (13-16, series III) were more potent
than the corresponding ester derivatives (7-10) and showed activity values (EC50 ranging from 0.57
to 1.01 µM) comparable to those of compounds 1-4 of series I.
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With respect to the reference compounds tariquidar (EC50 = 0.044 µM) and elacridar (EC50 = 0.014
µM), all compounds of the three series were less potent but more selective since they were inactive
towards both MRP1 and BCRP except for compounds 8, 9, 12 belonging to series II. In fact, they
displayed moderate activities on BCRP (EC50 ranging from 5.9 to 17 µM).
The Apparent Permeability determination Papp (BA/AB) in Caco-2 cell monolayer indicated that
only compound 7 had a BA/AB ratio < 2 and therefore can be considered a P-gp inhibitor since it
did not induce ATP cell depletion and inhibited Calcein-AM transport. Compound 12 showing a
BA/AB ratio > 2 and displaying the ability to induce ATP cell depletion is defined as P-gp
unambiguous substrate (category I). All the other compounds were not transported substrates
(category IIB3), since they had a BA/AB ratio > 2 and were not able to induce ATP cell
depletion.[51] Therefore, the esters derivatives 7 and 12 were the only ones displaying a different P-
gp interacting mechanism (inhibitor and unambiguous substrate, respectively) compared to the other
compounds.
These results suggest that in this series of compounds the presence of an amide function is not a
prerequisite for the activity on the transporter proteins P-gp and BCRP. All amide and alkylamine
derivatives appeared to be selective for P-gp efflux pump, whereas three ester derivatives were also
able to bind BCRP. The nature of the aryl moieties does not influence the behaviour of these
compounds within each series, even if, in all the three series, compounds with the 2,2-bis(4-
methoxyphenyl) moiety (D) showed the best activity on P-gp.
Chemical stability tests
An investigation about the chemical stability of compounds 1-12 (series I and II) in phosphate
buffer solution (PBS) and human plasma was performed since the amide and ester groups present in
these compounds may be susceptible to spontaneous or enzymatic hydrolysis.
The degradation analyses were performed by LC-MS/MS method operating in Multiple Reaction
Monitoring (MRM) mode. The half-life values were obtained by monitoring the variation of analyte
concentration at different incubation times in phosphate buffer solution (PBS) or human plasma.
Each set of samples was incubated at 37°C in triplicate at four different times, 0, 30, 60 and 120
min. The human plasma batch was collected from a pool of healthy volunteers and its hydrolytic
activity was checked using ketoprofene ethylester (KEE) as reference compound (half-life < 2 h).
The raw data processing and evaluation of calibration results are reported in Supporting
Information.
Under these experimental conditions all the tested compounds resulted stable in phosphate buffer
solution (PBS), whereas in human plasma were stable or showed a negligible degradation rate.
Conclusions
In these new series of compounds the presence of an amide, ester or alkylamine appears to be an
important structural requirement for defining the P-gp selectivity and interacting mechanism. In
fact, both amide and alkylamine derivatives (series I and III) were P-gp modulators resulting to be
more selective than the reference compounds tariquidar and elacridar, which resulted active also on
the BCRP transporter. The derivatives of these two series appeared to be substrates belonging to the
category IIB3.
The ester derivatives were in general less potent on P-gp than the corresponding amide and
alkylamine analogues but, interestingly, three of them were also able to bind the BCRP transporter.
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Also in the series II most of the compounds appeared to be not transported substrates (category
IIB3), with two interesting exceptions: compound 12 is a P-gp unambiguous substrate and
compound 7, bearing the (E)-3-(3,4,5-trimethoxyphenyl)vinyl fragment, is a P-gp inhibitor
endowed with high selectivity.
The presence of amide or ester groups in the compounds of series I and II does not compromise
their chemical stability to spontaneous or enzymatic hydrolysis. In fact, they resulted stable in PBS
and also in human plasma allowing us to predict the in vivo bioavailability of these compounds.
In summary, the design of the reported three series I, II, III allowed the identification of two potent
and selective P-gp substrates, compounds 4 and 10, and a P-gp inhibitor endowed with high
selectivity, compound 7. It is noteworthy also the identification in the ester series II of new
compounds 8, 9, 12 active on BCRP, a result that opens a new scenario in the development of
ligands active towards this pump.
Experimental part
Chemistry
All melting points were taken on a Büchi apparatus and are uncorrected. NMR spectra were
1
13
recorded on a Bruker Avance 400 spectrometer (400 MHz for H NMR, 100 MHz for C NMR)
using residual solvent such as chloroform (δ=7.26) as internal standard. Chromatographic
separations were performed on a silica gel column by gravity chromatography (Kieselgel 40, 0.063-
0.200 mm; Merck) or flash chromatography (Kieselgel 40, 0.040-0.063 mm; Merck). Yields are
given after purification, unless otherwise stated.
The semi-preparative LC-UV apparatus consisted of a Perkin-Elmer series 200 (Norwalk, CT)
composed by quaternary pump, autosampler, column oven and UV-VIS detector coupled with a
Biologic BioFrac fraction collector (from Bio-Rad, Milan, Italy). The LC-MS analysis was carried
out using a Varian 1200L triple quadrupole system equipped by two Prostar 210 pumps, a Prostar
410 autosampler and a Elettrospray Source (ESI). The mass spectrometer acquired the positive ions
in scan mode in the range between 150-700 m/z. Raw-Data were collected and processed by Varian
Workstation Vers. 6.8 software. The expected ions species are composed by the molecular adducts
with common positive ions present in solution such as [H]+, [NH4]+, [Na]+. In our conditions the
most abundant ion species obtained were the molecular adducts [M+H]+.
The compounds 1-16 were obtained in a purity of no less than 95%. Their combustion analyses are
indicated by symbols, and the analytical results are within ±0.4% of the theoretical values.
Compounds were named following IUPAC rules as applied by ChemBio-Draw Ultra 12.0 software.
When reactions were performed in anhydrous conditions, the mixtures were maintained under
nitrogen. Compounds 1-16 that were in the form of free bases, were transformed into the
hydrochloride by treatment with acetyl chloride in anhydrous CH3OH, or into the oxalate by
treatment with oxalic acid in warm ethyl acetate. The salts were crystallized from abs.
ethanol/petroleum ether.
(E)-N-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (1)
A 0.23 mmol (54.3 mg) portion of (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid was transformed into
the acyl chloride by reaction with 0.07 mL of SOCl2 (0.91 mmol) in 4 mL of CHCl3 (free of
ethanol) at 60°C for 4 h. The reaction mixture was cooled to rt, and the solvent was removed under
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reduced pressure; the mixture was then treated twice with cyclohexane and the solvent was removed
under reduce pressure. The acyl chloride obtained was dissolved in CHCl3 (free of ethanol), and to
this solution 0.04 mL of Et3N (0.27 mmol) and 0.23 mmol (68.0 mg) of 17[39] were added. The
mixture was kept at rt. After 24 h the organic layer was washed twice with Na2CO3 saturated
solution. After drying with Na2SO4, the solvent was removed under reduced pressure and the
residue was purified by flash chromatography using CH2Cl2/diethylether/abs. EtOH/petroleum
ether/NH4OH (180:180:90:450:5) as eluting system. The title compound (47.6 mg, 40%) was
obtained as a pale yellow oil.
Anal. calcd for C31H36NO6: C 69.90; H 6.81; N 5.26; found: C 70.17; H 7.03; N 5.11.
The oily product was transformed into the hydrochloride. Mp (HCl): 250-252°C.
Compounds 2, 3, 4 and 5 were obtained in the same way starting from 3,4,5-trimethoxybenzoic
acid, anthracene-9-carboxylic acid, 2,2-bis(4-methoxyphenyl)acetic acid[41] and 9H-fluorene-
carboxylic acid respectively.
N-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3,4,5-
trimethoxybenzamide (2)
Chromatographic
eluent:
CH2Cl2/diethylether/abs.
EtOH/petroleum
ether/NH4OH
(180:180:45:45:2.5). Yield: 65%.
Anal. calcd for C29H34NO6: C 68.76; H 6.76; N 5.53; found: C 68.47; H 6.41; N 5.75.
The oily product was transformed into the hydrochloride. Mp (HCl): 140-142°C.
N-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)anthracene-9-
carboxamide (3)
Chromatographic eluent: CH2Cl2/MeOH/NH4OH (97:3:0.3). Yield: 58%.
Anal. calcd for C34H32N2O3: C 79.04; H 6.24; N 5.42; found: C 78.79; H 6.51; N 5.28.
The oily product was transformed into the hydrochloride. Mp (HCl): 255-256°C.
N-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-2,2-bis(4-
methoxyphenyl)acetamide (4)
Chromatographic eluent: CH2Cl2/MeOH/NH4OH (98:2:0.2). Yield: 67%.
Anal. calcd for C35H38N2O5: C 74.18; H 6.76; N 4.94; found: C 73.97; H 6.47; N 5.12.
The oily product was transformed into the hydrochloride. Mp (HCl): 146-148°C.
N-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-9H-fluorene-9-
carboxamide (5)
Chromatographic
eluent:
CH2Cl2/diethylether/abs.
EtOH/petroleum
ether/NH4OH
(180:180:45:45:2.5). Yield: 47%.
Anal. calcd for C33H32N2O3: C 78.55; H 6.39; N 5.55; found: C 78.27; H 6.60; N 5.31.
The oily product was transformed into the hydrochloride. Mp (HCl) 188-190°C.
N-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3-(3,4,5-
trimethoxyphenyl)propiolamide (6)
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To a solution of 17[39] (70.5 mg, 0.23 mmol) in 4 mL of anhydrous CH2Cl2 cooled to 0°C were
added in this sequence: 3-(3,4,5-trimethoxyphenyl)propiolic acid[40] (80.0 mg, 0.34 mmol), DMAP
(36.0 mg, 0.29 mmol) and EDCI (99.0 mg, 0.54 mmol). The reaction mixture was stirred at 0°C for
1 h and then at room temperature for 48 h. The mixture was treated with CH2Cl2 and the organic
layer was washed with water and then with a saturated solution of NaHCO3. After drying with
Na2SO4, the solvent was removed under reduced pressure and the residue was purified by flash
chromatography using CH2Cl2/MeOH/NH4OH (98:2:0.2) as eluting system. The title compound
(73.0 mg, 61% yield) was obtained as a pale yellow oil.
Anal. calcd for C31H34N2O6: C 70.17; H 6.46; N 5.28; found: C 70.45; H 6.23; N 5.41.
The oily product was transformed into the oxalate. Mp (oxalate): 188-190°C.
4-(2-Chloroethyl)phenol (18)[42]
To a solution of 200.0 mg (1.45 mmol) of 4-(2-hydroxyethyl)phenol and 0.5 mL (3.60 mmol) of an.
Et3N in CHCl3 (free of ethanol), 0.2 mL (2.90 mmol) of SOCl2 were added. After 24 h the solvent
was removed under reduced pressure, the reaction mixture was treated with CH2Cl2, and the
resulting organic layer was washed twice with a saturated solution of NaHCO3, dried on Na2SO4,
and the solvent was removed under reduced pressure. The crude compound was purified by flash
chromatography using CH2Cl2/MeOH/NH4OH (99:1:0.1) as eluting system. The title compound
(169.7 mg, 75%) was obtained as a pale yellow oil.
4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenol (19)
To a solution of 169.7 mg (1.08 mmol) of 18 in an. CH3CN, 297.7 mg (1.30 mmol) of 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride and 373.2 mg (2.70 mmol) of K2CO3 were
added. After 24 h at 80°C the solvent was removed under reduced pressure, the reaction mixture
was treated with H2O and the aqueous layer was extracted three times with CH2Cl2. The organic
layer was dried over Na2SO4, and the solvent was removed under reduced pressure. The crude
compound was purified by flash chromatography using CH2Cl2/MeOH/NH4OH (95:5:0.5) as
eluting system. The title compound (224.8 mg, 67%) was obtained as a pale yellow oil.
(E)-4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl 3-(3,4,5-
trimethoxyphenyl)acrylate (7)
A 0.19 mmol (45.5 mg portion) of (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid was transformed into
the acyl chloride by reaction with 0.06 mL of SOCl2 (0.78 mmol) in 5 mL of CHCl3 (free of
ethanol) at 60°C for 4 h. The reaction mixture was cooled to room temperature, and the solvent was
removed under reduced pressure; the mixture was then treated twice with cyclohexane and the
solvent removed under reduce pressure. The acyl chloride obtained was dissolved in CHCl3 (free of
ethanol), and a solution of 0.19 mmol (59.8 mg) of 19 in CHCl3 (free of ethanol) was added. The
mixture was kept at room temperature. After 24 h, the reaction mixture was treated with CH2Cl2 and
the organic layer was washed twice with NaHCO3 saturated solution. After drying with Na2SO4, the
solvent was removed under reduced pressure and the residue was purified by flash chromatography
using CH2Cl2/MeOH/NH4OH (97:3:0.3) as eluting system. The title compound (39.3 mg, 40%
yield) was obtained as a pale yellow oil.
Anal. calcd for C31H35NO7: C 69.78; H 6.61; N 2.62; found: C 70.02; H 6.88; N 2.74.
The oily product was transformed into the hydrochloride. Mp (HCl): 215-217°C.
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Compounds 8, 9, 10 and 11 were obtained in the same way starting from 3,4,5-trimethoxybenzoic
acid, anthracene-9-carboxylic acid, 2,2-bis(4-methoxyphenyl)acetic acid[41] and 9H-fluorene-
carboxylic acid respectively.
4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl 3,4,5-trimethoxybenzoate
(8)
Chromatographic eluent: CH2Cl2/MeOH/NH4OH (97:3:0.3). Yield: 56%.
Anal. calcd for C29H33NO7: C 68.62; H 6.55; N 2.76; found: C 68.35; H 6.76; N 2.93.
The oily product was transformed into the hydrochloride. Mp (HCl): 207-208°C.
4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl anthracene-9-carboxylate
(9)
Chromatographic eluent: CH2Cl2/MeOH/NH4OH (97:3:0.3). Yield: 31%.
Anal. calcd for C34H31NO4: C 78.89; H 6.04; N 2.71; found: C 79.05; H 5.91; N 2.60.
The oily product was transformed into the hydrochloride. Mp (HCl): 171-173°C (dec).
4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl 2,2-bis(4-
methoxyphenyl)acetate (10)
Chromatographic eluent: CH2Cl2/MeOH/NH4OH (98:2:0.2). Yield: 47%.
Anal. calcd for C35H37NO6: C 74.05; H 6.57; N 2.47; found: C 73.88; H 6.39; N 2.62.
The oily product was transformed into the hydrochloride. Mp (HCl): 210-213°C.
4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl 9H-fluorene-9-carboxylate
(11)
Chromatographic eluent: CH2Cl2/MeOH/NH4OH (97:3:0.3). Yield: 39%.
Anal. calcd for C33H31NO4: C 78.39; H 6.18; N 2.77; found: C 78.15; H 6.30; N 2.91.
The oily product was transformed into the hydrochloride. Mp (HCl): 180-183°C.
4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl 3-(3,4,5-
trimethoxyphenyl)propiolate (12)
To a solution of 19 (96.0 mg, 0.31 mmol) in 4 mL of anhydrous CH2Cl2 cooled to 0°C were added
in this sequence: 3-(3,4,5-trimethoxyphenyl)propiolic acid[40] (109.0 mg, 0.46 mmol), DMAP (30.0
mg, 0.25 mmol) and EDCI (106.0 mg, 0.55 mmol). The reaction mixture was stirred at 0°C for 1 h
and then was kept at room temperature for 48 h. The mixture was treated with CH2Cl2 and the
organic layer was washed with water and then with a saturated solution of NaHCO3. After drying
with Na2SO4, the solvent was removed under reduced pressure and the residue was purified by flash
chromatography using CH2Cl2/MeOH/NH4OH (98:2:0.2) as eluting system. The title compound
(30.2 mg, 18%) was obtained as a pale yellow oil.
Anal. calcd for C31H33NO7: C 70.04; H 6.26; N 2.63; found: C 70.29; H 6.05; N 2.44.
The oily product was transformed into the oxalate. Mp (oxalate): 161-164°C (dec).
5-(Iodomethyl)-1,2,3-trimethoxybenzene (21)
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To a solution of 97.0 mg (0.45 mmol) of 5-(chloromethyl)-1,2,3-trimethoxybenzene (21)[43] in ethyl
acetate, 268.0 g (1.80 mmol) of NaI were added. The mixture was maintained at reflux in the dark
for 14 h. The organic layer was washed twice with H2O and dried on Na2SO4, and the solvent was
removed under reduced pressure in the dark. The title compound (124.0 mg, 90% yield) was
obtained as a red oil.
(E)-3-(3,4,5-Trimethoxyphenyl)prop-2-en-1-ol (22)[45]
To a solution of (E)-methyl 3-(3,4,5-trimethoxyphenyl)acrylate (1.0 g, 3.96 mmol) in 25 mL of an.
CH2Cl2 at -78 °C, DIBAL-H (5.82 ml, 8.72 mmol, 1.5 M in toluene ) was added dropwise. The
mixture was stirred for 1 h at the same temperature, then a solution of NaOH 10% (10 mL) was
added. The solution was warmed to room temperature and was stirred for 90 min. The aqueous
layer was extracted twice with ethyl acetate and the organic layers were washed with a saturated
aqueous NaCl solution, dried over Na2SO4, and concentrated in vacuo. The residue was purified by
flash chromatography using CH2Cl2/CH3OH (99:1) as eluting system, to give the title compound
(672.0 mg, 76% yield).
(E)-3-(3,4,5-Trimethoxyphenyl)acrylaldehyde (23)
To a suspension of 121.0 mg (0.562 mmol) of pyridinium chlorochromate and 96.0 mg of celite in 5
mL of an. CH2Cl2, a solution of 22 (84.0 g, 0.375 mmol) in 3 mL of an. CH2Cl2 was added. The
mixture was stirred at room temperature for 2 h, then diethyl ether was added, and the mixture was
filtered and concentrated in vacuo. The residue was purified by flash chromatography using
CH2Cl2/n-hexane (90:10) as eluting system. The title compound was obtained in a 90% yield (75.0
mg).
2,2-bis(4-Methoxyphenyl)acetaldehyde (24)
To a solution of ethyl 2,2-bis(4-methoxyphenyl)acetate (223.0 mg, 0.732 mmol) in an. toluene
maintained at -78°C, DIBAL-H (0.6 mL of a solution 1.5 M in toluene, 0.879 mmol) was added.
After 1 h at the same temperature, 3 mL of a solution of NaOH 10% were added, and the mixture
was left to reach rt, extracted with diethyl ether, dried on Na2SO4 and concentrated in vacuo. The
residue was purified by flash chromatography using CH2Cl2/n-hexane (70:30) as eluting system
yielding 78.0 mg (41% yield) of the title compound.
4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-N-(3,4,5-trimethoxybenzyl)aniline
(14)
To a solution of 110.0 mg of 21 (0.360 mmol) in an. CH3CN, 158.0 mg (0.500 mmol) of 17[39] were
added. The reaction mixture was stirred at room temperature in the dark for 3 h, then was treated
with CH2Cl2 and the organic layer was washed twice with a saturated solution of NaHCO3and dried
on Na2SO4. The solvent was removed under reduced pressure and the residue was purified by flash
chromatography using CH2Cl2/CH3OH/NH4OH (98:2:0.2) as eluting system. The title compound
was further purified by semi-preparative LC-UV apparatus using a isocratic elution with 10 mM of
formic acid in milliQ water:acetonitrile 70:30 (v/v). (20.0 mg, 9% yield) was obtained as a pale
yellow oil.
Anal. calcd for C29H36N2O5: C 70.71; H 7.37; N 5.69; found: C 71.00; H 7.62; N 5.51.
The oily product was transformed into the hydrochloride. Mp (HCl): 103-105°C.
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(E)-4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-N-(3-(3,4,5-
trimethoxyphenyl)allyl)aniline (13)
A mixture of aldehyde 23 (63.4 mg, 0.284 mmol), an equimolar amount of 17[39] (88.8 mg) and an
excess of titanium(IV) isopropoxide (0.1 mL, 0.360 mmol) in 1 mL of abs. EtOH was stirred with a
drying tube at room temperature. After 20 h, the IR spectrum of the mixture showed no ketone
band, and the viscous solution was diluted with abs ethanol (2 mL). Sodium cyanoborohydride
(18.0 mg, 0.288 mmol) was added, and the solution was stirred for 6 h. Water (0.5 mL) was then
added, and the mixture was concentrated in vacuo. The crude product was dissolved in CH2Cl2,
filtered to remove the solids, washed with a solution of NaHCO3 and water, dried over Na2SO4 and
concentrated in vacuo. The crude substance was then purified by column chromatography using
CH2Cl2/MeOH/NH4OH (99:1:0.1) as eluting system. The title compound (23.0 mg, 20% yield) was
obtained as a pale yellow oil.
Anal. calcd for C31H38N2O5: C 71.79; H 7.38; N 5.40; found: C 71.42; H 7.63; N 5.18.
The oily product was transformed into the hydrochloride. Mp (HCl): low melting solid.
Compounds 15 and 16 were obtained in the same way starting from anthracene-9-carbaldehyde and
2,2-bis(4-methoxyphenyl)acetaldehyde respectively.
N-(Anthracen-9-ylmethyl)-4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)aniline
(15)
Chromatographic eluent: CH2Cl2/MeOH/NH4OH (99:1:0.1). Yield: 38%.
Anal. calcd for C34H34N2O2: C 81.24; H 6.82; N 5.57; found: C 81.56; H 7.03; N 5.35.
The oily product was transformed into the oxalate. Mp (oxalate): 115-120°C (dec).
N-(2,2-bis(4-Methoxyphenyl)ethyl)-4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)aniline (16)
Chromatographic eluent: CH2Cl2/MeOH/NH4OH (98:2:0.2). Yield: 41%.
Anal. calcd for C35H40N2O4: C 76.06; H 7.29; N 5.07; found: C 75.81; H 7.07; N 4.86.
The oily product was transformed into the hydrochloride. Mp (HCl): 110-112°C.
Biology
Materials. Cell culture reagents were purchased from Celbio s.r.l. (Milano, Italy). CulturePlate
96/wells plates were purchased from PerkinElmer Life Science; Calcein-AM, MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) were obtained from Sigma-Aldrich (Milan,
Italy).
Cell cultures. MDCK-MDR1, MDCK-MRP1 and MDCK-BCRP cells are a gift of Prof. P. Borst,
NKI-AVL Institute, Amsterdam, Nederland. MDCK cells were grown in DMEM high glucose
supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL
streptomycin, in a humidified incubator at 37°C with a 5% CO2 atmosphere. Caco-2 cells were a
gift of Dr. Aldo Cavallini and Dr. Caterina Messa from the Laboratory of Biochemistry, National
Institute for Digestive Diseases, “S. de Bellis”, Bari (Italy).
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Calcein-AM experiment. These experiments were carried out as described by Guglielmo et al.
with minor modifications.[53] Each cell line (30,000 cells per well) was seeded into black
CulturePlate 96/wells plate with 100 μl medium and allowed to become confluent overnight. 100 μl
of test compounds were solubilized in culture medium and added to monolayers. 96/Wells plate was
incubated at 37°C for 30 min. Calcein-AM was added in 100 μl of Phosphate Buffered Saline (PBS)
to yield a final concentration of 2.5 μM and plate was incubated for 30 min. Each well was washed
3 times with ice cold PBS. Saline buffer was added to each well and the plate was read to Victor3
(PerkinElmer) at excitation and emission wavelengths of 485 nm and 535 nm, respectively. In these
experimental conditions Calcein cell accumulation in the absence and in the presence of tested
compounds was evaluated and fluorescence basal level was estimated by untreated cells. In treated
wells the increase of fluorescence with respect to basal level was measured. EC50 values were
determined by fitting the fluorescence increase percentage versus log[dose].
Hoechst 33342 experiment. These experiments were carried out as described by Guglielmo et al.
with modifications.[53] Each cell line (30,000 cells per well) was seeded into black CulturePlate
96/wells plate with 100 μl medium and allowed to become confluent overnight. 100 μl of test
compounds were solubilized in culture medium and added to monolayers. 96/Wells plate was
incubated at 37°C for 30 min. Hoechst 33342 was added in 100 μl of Phosphate Buffered Saline
(PBS) to yield a final concentration of 8 μM and plate was incubated for 30 min. The supernatants
were drained and the cells were fixed for 20 min under light protection using 100 µL per well of a
4% PFA solution. Each well was washed 3 times with ice cold PBS. Saline buffer was added to
each well and the plate was read to Victor3 (PerkinElmer) at excitation and emission wavelengths
of 340/35 nm and 485/20 nm, respectively. In these experimental conditions Hoechst 33342
accumulation in the absence and in the presence of tested compounds was evaluated and
fluorescence basal level was estimated by untreated cells. In treated wells the increase of
fluorescence with respect to basal level was measured. EC50 values were determined by fitting the
fluorescence increase percentage versus log[dose].
ATPlite assay. The MDCK-MDR1 cells were seeded into 96-well microplate in 100 µl of complete
medium at a density 2x104 cells/well.[53] The plate was incubated overnight in a humidified
atmosphere 5% CO2 at 37°C. The medium was removed and 100 µl of complete medium in the
presence or absence of different concentrations of test compounds was added. The plate was
incubated for 2h in a humidified atmosphere 5% CO2 at 37°C. 50 µl of mammalian cell lysis
solution was added to all wells and the plate shacked for five minutes in an orbital shaker. 50 µl of
substrate solution was added to all wells and the plate shacked for five minutes in an orbital shaker.
The plate was dark adapted for ten minutes and the luminescence was measured.
Permeability Experiments.
Preparation of Caco-2 monolayer. Caco-2 cells were seeded onto a Millicell® assay system
(Millipore), where a cell monolayer is set in between a filter cell and a receiver plate, at a density of
20,000 cells/well. The culture medium was replaced every 48 h and the cells kept for 21 days in
culture. The Trans Epithelial Electrical Resistance (TEER) of the monolayers was measured daily,
before and after the experiment, using an epithelial voltohometer (Millicell® -ERS).[53] Generally,
TEER values greater than 1000 Ω for a 21day culture, are considered optimal.
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Drug transport experiment. After 21 days of Caco-2 cell growth, the medium was removed from
filter wells and from the receiver plate, which were filled with fresh HBSS buffer (Invitrogen). This
procedure was repeated twice, and the plates were incubated at 37°C for 30 min. After incubation
time, the HBSS buffer was removed and drug solutions and reference compounds, were added to
the filter well at the concentration of 100 µM, while fresh HBSS was added to the receiver plate.
The plates were incubated at 37°C for 120 min. Afterwards, samples were removed from the apical
(filter well) and basolateral (receiver plate) side of the monolayer to measure the permeability.
The apparent permeability (Papp), in units of nm/second, was calculated using the following
equation:
VA
[drug]acceptor
[drug]initial
x
Papp=
Area x time
VA = the volume (in mL) in the acceptor well;
Area = the surface area of the membrane (0.11 cm2 of the well);
time = the total transport time in seconds (7200 sec);
[drug]acceptor = the concentration of the drug measured by U.V. spectroscopy;
[drug]initial = the initial drug concentration (1 x 10-4 M) in the apical or basolateral wells.
Chemical stability tests
Chemicals
Acetonitrile, ethanol (Chromasolv), formic acid and ammonium formate (MS grade), NaCl, KCl,
Na2HPO4 2H2O, KH2PO4 (Reagent grade), verapamil hydrochloride (used as internal standard) and
ketoprofen (analytical standard) were purchased by Sigma-Aldrich (Milan, Italy). Ketoprofen Ethyl
Ester (KEE) were obtained by Fisher's reaction from ketoprofen and ethanol. MilliQ water 18 MΩ
was obtained from Millipore's Simplicity system (Milan - Italy).
Phosphate buffer solution (PBS) was prepared by adding 8.01 g L-1 of NaCl, 0.2 g L-1 of KCl, 1.78
g L-1 of Na2HPO4 2H2O and 0.27 g L-1 of KH2PO4. Human plasma was collected from healthy
volunteer and was kept at -80°C until use.
Instrumental
The LC-MS/MS analysis was carried out using a Varian 1200L triple quadrupole system (Palo Alto,
CA, USA) equipped by two Prostar 210 pumps, a Prostar 410 autosampler and an Elettrospray
Source (ESI) operating in positive ions. The ion sources and ion optics parameters were optimized
during the calibration of the instrument introducing, via syringe pump at 10 µL min−1, a 1 µg mL-1
tuning solution.
Raw-data were collected an processed by Varian Workstation Vers. 6.8 software.
G-Therm 015 thermostatic oven was used to maintained the samples at 37°C during the test of
degradation.
17
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10.1002/cmdc.201700239
ChemMedChem
Preparation of samples
Each sample was prepared adding 10 μl of Working solution 1 to 100 μL of PBS or human plasma.
The obtained solutions correspond to 1 μM of analyte. Each set of samples was incubated in
triplicate at four different times, 0, 30, 60 and 120 min. at 37°C. Therefore the degradation profile
of each analyte was represented by a batch of 12 samples (4 incubation times x 3 replicates). After
the incubation, the samples were added with 300 μL of ISTD solution and centrifuged. The
supernatants were transferred in autosampler vials and dried under a gentle stream of nitrogen.
The dried samples were dissolved in 1.0 mL of mQ water:acetonitrile 80:20 added with 10 mM of
formic acid. The obtained sample solutions were analyzed by LC-MS/MS method described in
Supporting Information.
Acknowledgements
This work was partially supported by MIUR (FIRB 2012 RBFR12SOQ1_002 and
RBFR12SOQ1_003).
Supporting Information.
Physical and chemical data for compounds 1-16, 18-19, 21-24 and chemical stability data of
compounds 1-12.
Keywords
6,7-Dimethoxytetrahydroisoquinoline derivatives; multidrug resistance reversers; P-gp modulators;
tariquidar and elacridar.
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