Potent galloyl-based selective modulators targeting multidrug resistance associated protein 1 and P-glycoprotein

Article abstract of DOI:10.1021/jm201305y

The multifactorial nature of chemotherapy failure in controlling cancer is often associated with the occurrence of multidrug resistance (MDR), a phenomenon likely related to the increased expression of members of the ATP binding cassette (ABC) transporter superfamily. In this respect, the most extensively characterized MDR transporters include ABCB1 (also known as MDR1 or P-glycoprotein) and ABCC1 (also known as MRP1) whose inhibition remains a priority to circumvent drug resistance. Herein, we report how the simple galloyl benzamide scaffold can be easily and properly decorated for the preparation of either MRP1 or P-gp highly selective inhibitors. In particular, some gallamides and pyrogallol-1-monomethyl ethers showed remarkable affinity and selectivity toward MRP1. On the other hand, trimethyl ether galloyl anilides, with few exceptions, exhibited moderate to very high and selective P-gp inhibition.

Full text of DOI:10.1021/jm201305y

Article
pubs.acs.org/jmc
Potent Galloyl-Based Selective Modulators Targeting Multidrug
Resistance Associated Protein 1 and P-glycoprotein
Raffaella Zoe Pellicani, Angela Stefanachi, Mauro Niso, Angelo Carotti, Francesco Leonetti,
Orazio Nicolotti, Roberto Perrone, Francesco Berardi, Saverio Cellamare,* and Nicola Antonio Colabufo*
̀
Dipartimento Farmaco-Chimico, Universita degli Studi di Bari “Aldo Moro”, Via Orabona 4, 70125, Bari, Italy
S
* Supporting Information
ABSTRACT: The multifactorial nature of chemotherapy
failure in controlling cancer is often associated with the
occurrence of multidrug resistance (MDR), a phenomenon
likely related to the increased expression of members of the
ATP binding cassette (ABC) transporter superfamily. In this
respect, the most extensively characterized MDR transporters
include ABCB1 (also known as MDR1 or P-glycoprotein) and
ABCC1 (also known as MRP1) whose inhibition remains a
priority to circumvent drug resistance. Herein, we report how
the simple galloyl benzamide scaffold can be easily and
properly decorated for the preparation of either MRP1 or P-gp
highly selective inhibitors. In particular, some gallamides and
pyrogallol-1-monomethyl ethers showed remarkable affinity
and selectivity toward MRP1. On the other hand, trimethyl
ether galloyl anilides, with few exceptions, exhibited moderate to
very high and selective P-gp inhibition.
Many compounds, termed modulators, reversers, inhibitors,
or chemosensitizers able to reverse MDR in intact cells in vitro
by interfering with the ability of the transporter to efflux drugs,
have been identified.10 Although the discovery of the first MDR
modulators was serendipitous, in recent years, molecular
design, combinatorial chemistry, and the availability of more
protein structural information have all played increasingly
important roles in identifying more effective compounds.11
While considerable effort has been devoted to developing new
P-gp modulators and to deriving convincing pharmacophore
patterns, MRP1 inhibitors have not yet been studied to the
same extent.12,13
INTRODUCTION
■
A well-established cause of multidrug resistance (MDR)
involves the increased expression of members of the ATP
binding cassette (ABC) transporter superfamily, many of which
efflux various chemotherapeutic drugs from cells. The most
extensively characterized MDR transporters include ABCB1
(also known as MDR1 or P-glycoprotein), ABCC1 (also
known as MRP1), and BCRP (also known as ABCG2 or
MXR).1 Multidrug resistance associated proteins (MRPs), in
particular, play a key role in cellular protection by effluxing
xenobiotics, metabolites, and endogenous substrates that can
accumulate in tissues and lead to toxicity. In this respect, MRPs
could be considered a double-edged sword: on one hand they
play an important role in maintaining cellular homeostasis,
while on the other hand, they greatly reduce the intracellular
levels of many anticancer drugs.2−5
So far, only limited high-resolution structural information on
intact mammalian ABC proteins has been published, mainly
related to P-glycoprotein (P-gp) transporter, while no
structure-based information on the entire MRP1 has been
deposited.6 By use of electron microscopy, the structure of
MRP1 showed monomers possessing a putative central pore
that assembles to form dimers.7 Unfortunately, these low-
resolution structures have contributed little to the under-
standing of the complex MRP1’s biological machinery. As a
result, 3D models for P-gp, MRP1, and BCRP have been
created via homology modeling based on a few bacterial ABC
proteins.8,9
Since the discovery of MRP1 in 1992 by Cole,14 it had been
clear that, except for cyclosporine and verapamil, most P-gp
inhibitors (e.g., 1, elacridar; 2, tariquidar; 3, XR9577; Figure 1)
did not affect MRP1.15,16 Hence, many MRP1 modulators have
been studied extensively in recent years, spreading from
nonspecific inhibitors of organic anion transporters (e.g.,
indomethacin and probenecid)17,18 and leukotriene receptor
antagonists (e.g., 4 (MK571) and 5 (ONO-1078), also termed
verlukast and pranlukast, respectively; Figure 1),19,20 to tricyclic
isoxazoles, which are among the most potent and specific
MRP1 inhibitors known to date (e.g., 6 (LY402913) and 7
(LY465803); Figure 1).21,22 Additionally, some polyphenolic
compounds, including naturally occurring flavonoids such as
Received: September 30, 2011
Published: November 23, 2011
© 2011 American Chemical Society
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Journal of Medicinal Chemistry
Article
Figure 1. Selected MRP1 inhibitors.
dehydrosilybin (8; Figure 1), have been investigated for their
ability to modulate MRP1.23,24 Despite their molecular
similarity, there is still no satisfactory interpretation of the
structural requirements of flavonoids necessary for inhibition of
MRPs, although some studies, regarding MRP1/2 mediated
calcein efflux inhibition, have demonstrated the relevance of
having a given ratio between methoxy and hydroxy aromatic
substituents.25 Recently, SAR analyses of a tariquidar-based
library provided insights on the relevance of selected molecular
fragments (i.e., a bulky amine) for the inhibition of breast
cancer resistance protein (BCRP), P-gp, and MRP1.26,27 There
is evidence that a variable alkylphenyl linker incorporating an
amide function would be particularly important in MRP1
inhibition. Interestingly, some of these structural motifs are also
shared by many P-gp and MRP1 inhibitors, as highlighted in
red in Figure 1.
inhibitors, such as 3,4,5-trihydroxybenzoyl (i.e., galloyl) and
3,4- and 3,5-dihydroxybenzoyl moieties.28
First, N-biphenyl-4-yl-3,4,5-trimethoxybenzamide derivatives
bearing on the B ring (Chart 1) R substituents with different
stereoelectronic properties were prepared and tested. With the
exception of the poor but still appreciable MRP1 inhibition
provided by 10e (R = NO2), such compounds (i.e., 10a, 10c,
10f) resulted in selective inhibition of P-gp (Table 1). These
data led us to explore further R1 substituents at the 4-position
both maintaining the 2-nitro group and preserving the 3,4,5-
trimethoxyphenyl moiety (11a−i), thereby providing many
very active and selective P-gp inhibitors that, only in three
cases, were also active against MRP1. Finally, the partial or
complete removal of methyl groups from the 3,4,5-trimethoxy
fragment, affording pyrogallol-1-monomethyl ethers (15a−n)
and 3,4,5-trihydroxy (14a−c) congeners, respectively, permit-
ted us to gain moderate to good inhibitory activity toward
MRP1. Similar reactions were performed in the case of 3,4- and
3,5-dimethoxy congeners (11l,m) which gave the catechol
(14d), 3,5-dihydroxy (14e), and isovanillic (15o) derivatives.
On this rationale, we approached the problem of the intrinsic
MRP1 modulatory activity and P-gp selectivity of this common
molecular scaffold by designing and testing a small library of
polymethoxy-/hydroxy-N-phenylbenzamides (Chart 1). As
CHEMISTRY
■
Chart 1. General Structure of the Designed Compounds
(See Table 1)
The synthetic pathways, leading to compounds with the general
structures depicted in Table 1, are shown in Schemes 1−4.
Variations of R, R1, and R2 were accomplished with the choice
of substituents by exploring a wide range of stereoelectronic
and lipophilic properties. The starting (hetero)arylanilines 9a−j
(Scheme 1) and 3-nitrobiphenyl-4-carboxylic acid (9k, Scheme 4)
were obtained by Suzuky coupling between the corresponding
aniline/carboxylic acid and appropriate arylboronic acids
(general procedure in Supporting Information). The phys-
icochemical and spectroscopic data of the known compounds
9a−g and of trimethoxybenzamides 10b and 11b (Scheme 1)
fully agreed with those reported in the literature (Tables S1 and
S2, respectively, in Supporting Information).29,30 The novel
synthesized polymethoxy derivatives were prepared by coupling
shown in Chart 1, these compounds retains the trimethoxy
or polyphenolic functions seen in many transporter protein
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Journal of Medicinal Chemistry
Article
a
Table 1. MRP1 and P-gp Inhibitory Activities of Compounds 10a−19
b
b
compd
X
Y
R
R1
R2
R3
R4
R5
MRP1
P-gp
a
First Subset
10a
NH
NH
NH
NH
NH
NH
CO
CO
CO
CO
CO
CO
H
Br
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
>100
20.0
3
10b
10c
10d
10e
10f
H
Ph
Ph
Ph
Ph
Ph
>100
>100
>100
>100
7.2
NH2
F
1
1.4 0.5
>100
NO2
80 10
>100
NHCOCH3
1.9 0.8
a
Second Subset
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
12
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CH2
NH
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
Br
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
>100
>100
OCH3
H
H
Ph
H
H
H
>100
>100
>100
0.57 0.1
2-thienyl
3-pyridyl
>100
17.3
6.8
2
6.1 0.8
2.5 0.9
>100
1
3,5-difluorophenyl
2.6 0.7
0.2 0.1
0.6 0.1
benzo[1,3]dioxol-5-yl
3-acetyphenyl
H
H
H
H
H
H
H
H
>100
3,4,5-(OCH3)3Ph
11.2 1.5
>100
24.3
0.4 0.1
34.0
2
3-nitrophenyl
NO2
NO2
NO2
NO2
NO2
3-methylpyridinium iodide
>100
5
Ph
Ph
Ph
Ph
>100
1.3 0.5
6.6 0.7
1.5 0.5
2.0 0.5
OCH3
OCH3
OCH3
>100
OCH3
OCH3
>100
13
>100
a
Third Subset
14a
14b
14c
14d
14e
NH
NH
NH
NH
NH
CO
CO
CO
CO
CO
NO2
NO2
NO2
NO2
NO2
Br
H
H
H
H
H
OH
OH
OH
H
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
>100
>100
>100
Ph
45.8 10
40.6 18
3-acetyphenyl
39.1
40.3
72.0
7
8
9
Ph
Ph
38.8
46.6
9
6
OH
a
Fourth Subset
15a
15b
15c
15d
15e
15f
15g
15h
15i
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CH2
NH
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
Br
H
H
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OCH3
OH
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
44.5
16.3
5
2
38.4
65.0
40.2
31.2
67.0
16.8
>100
9.1
4
5
4
6
9
1
OCH3
H
44.0 3.0
32.2 1.5
8.0 1.4
22.0 0.3
2-thienyl
Ph
3,5-difluorophenyl
3,4-(OH)2Ph
9.5
1
3-acetyphenyl
7.7 1.3
74.5 10
2
3,4-(OH)2-5-OCH3-Ph
3.1 0.6
15j
3-nitrophenyl
40.6
75.8
5
8
32.5
2
15k
15l
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
>100
>100
14.1
NH2
F
37.9 3.5
30.0 1.5
28.0 5.1
>100
15m
15n
15o
16
1
NHCOCH3
NO2
NO2
NO2
>100
3.5 1.5
OH
OH
OCH3
OCH3
23.6 3.1
>100
72.0
9
17
>100
a
Fifth Subset
18
NH
CO
NO2
Ph
H
o-quinone
OCH3
23.3
6.8
4
3
>100
Ver
19
3
0.5 0.1
31.7
0.8 0.1
17.2
5
c
MC18
6.7 0.8
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Table 1. continued
b
b
compd
X
Y
R
R1
R2
R3
R4
R5
MRP1
P-gp
a
Fifth Subset
d
MK571
2.8 0.5
a
Compounds have been assigned to five different structural subsets (see infra) and accordingly listed. The first and second subsets present variation
at R and R1. The third subset contains polyhydroxy derivatives. The fourth subset collects pyrogallol 1-methyl ethers, and the fifth one comprises
reference compounds, including verapamil (Ver). IC50 data, reported as μM SEM, are the mean of two independent experiments, sampled in
triplicate. See ref 37. See ref 27.
b
c
d
suitable 4-substituted- or 2,4-disubstituted anilines with 3,4-
and 3,5-dimethoxy- and 3,4,5-trimethoxybenzoyl chlorides,
affording the corresponding benzamides 10a,b and 10d,e
(Schemes 1 and 2) and 11a−j and 11l−m (Schemes 1−3) in
good to excellent yields (Table S2 in Supporting Information).
The 3-pyridyl congener 11e was quaternized to form the
corresponding methyl iodide salt 11k (Scheme 3). The N-
(3,4,5-trimethoxybenzyl)-N-(3-nitro-1,1′-biphenyl-4-yl)amine
12 was obtained by a Mitsunobu protocol from 3-nitro-
biphenyl-4-ylamine 9e and 3,4,5-trimethoxybenzyl alcohol
(Scheme 1). The reduction of the nitro group in 10e provided
the amino congener 10c that, in turn, was acetylated to give 10f
(Scheme 2). The N-(3,4,5-trimethoxyphenyl)-3-nitro-1,1′-bi-
phenyl-4-carboxamide 13, which is the reverse-amide-bond
isomer of 10e, was prepared from 4-bromo-2-nitrobenzoic acid,
phenylated to yield 3-nitro-1,1′-biphenyl-4-carboxylic acid 9k,
which was then coupled, via its acid chloride, with 3,4,5-
trimethoxyaniline (Scheme 4). The final demethylation
reactions, affording the corresponding phenols only in low
yields, were carried out in two different ways: (1) by using 1
equiv of BBr3 (1 M in CH2Cl2) per methoxy group together
with an additional equivalent for the amide function31 and (2)
by using an excess of BBr3 (up to 3 equiv per methoxy
group).32 With the first protocol, the partial ether cleavage
gave, and only in modest yield, 3,4-dihydroxy-5-methoxy
derivatives 15a−n, 16, 17 as confirmed by 2D NOESY spectra
(Figures S1−S7 in Supporting Information). The same
protocol furnished the 3-hydroxy-4-methoxy derivatives (iso-
vanilamide) 15o from the corresponding 3,4-dimethoxybenza-
mide 11l (Scheme 2), and no trace was found for the presence
of the 4-hydroxy-3-methoxybenzamide regioisomer (vanila-
mide). The use of the second protocol was necessary to obtain
the gallamide derivatives 14a−c from 11a, 10e, and 11h
(Schemes 1−3) and the 3,4- and 3,5-dihydroxybenzamides
14d−e from 11l−m, respectively (Scheme 2). The preparation
of 19, a new 3,4-dihydroxy racemic analogue (ring A) of
verapamil, was carried out by using a similar demethylation reaction
as shown in Scheme 4. To our knowledge, this regiospecific
transformation, as confirmed by MS spectrum, was not covered by
previous studies.33−35 Finally, oxidation of 15e, by using
orthochloranil, gave the o-quinone derivative 18 (Scheme 2).
MRP1 substrate, and it cannot cross the cell membrane via
passive diffusion because it is too hydrophilic. Thus, a rapid
increase in the fluorescence of cytoplasmic calcein can be
monitored. P-gp and MRP1 transporters, present in the cell
membrane, rapidly efflux the calcein-AM before its entrance
into the cytosol, resulting in a reduction of the fluorescent
signal due to a decrease in the accumulation of calcein.
Evaluation of P-gp or MRP1 activity in the presence of pump
inhibitors can be performed in a competitive manner.
Compounds that block P-gp and MRP1 pumps inhibit
calcein-AM efflux, increasing intracellular accumulation of
fluorescent calcein. Values of IC50 for calcein-AM uptake
(Table 1) were determined by measuring relative fluorescence
values obtained after 30 min of incubation at 37 °C. Typical
sigmoidal dose−response curves, regarding MRP1 activity, are
shown in Figure 2 in the presence of various concentrations of
selected MRP1 and P-gp inhibitors such as 11f (a
trimethoxybenzamide), 14b (a gallamide), and 15e (a
pyrogallol 1-monomethyl ether). Verapamil and MC1837
were used as reference compounds for MRP1 and P-gp
inhibition, respectively. The accuracy and reproducibility of the
estimated IC50 values are in accordance with the most recent
guidelines.38
COMPOUND STABILITY ASSAY
■
Several lines of evidence indicate that catechol type
polyphenols can be easily oxidized in a cell culture medium.39
Therefore, we applied an analytical method to test the stability
of these compounds, particularly the gallamides and pyrogallol
1-monomethyl ethers, by electron spray ionization/mass
spectrometry (ESI-MS). Because of its greater stability in the
cell medium at 37 °C, the corresponding trimethoxy congener
of each polyphenol derivative (e.g., 10e vs 14b or 15e) was
selected as internal standard (IS). First, a linear relation-
ship between inhibitor/internal standard concentration and
the molecular ion intensity ratios was established in the
1.0−100 μM range (Figures S8 and S9a−e, Supporting
Information). Next, a solution of test compounds and the
corresponding IS were added to a stirring culture medium
(0.5 mL) at 37 °C to yield the final concentration in the
desired range, and sampling at 15, 30, 60, and 90 min after
start was made. The relative concentrations of polyphenols
were ultimately measured by comparing the ratio of the
respective molecular ion intensities and that of the IS
(Figure S10a−d, Supporting Information). All tested phenols
proved to be stable for at least 1 h in the assay conditions
(Figure S11, Supporting Information).
BIOCHEMICAL STUDIES
■
P-gp and MRP1 modulating activities of tested compounds
were determined by fluorescence measurements, using calcein-
AM fluorescent probes, in MDCK-MDR1 and MDCK-MRP1
cell lines.36 These cells overexpress only P-gp or MRP1
transporters, respectively, so that the observed biological effects
can be ascribed to the specific inhibition of these pumps.
Calcein-AM is a lipophilic substrate of both P-gp and MRP1
able to cross the cell membrane. Inside the cell compartment, it
is hydrolyzed by endogenous cytoplasmic esterases, yielding
highly fluorescent calcein. This compound is not a P-gp or
RESULTS AND DISCUSSION
■
In the search for novel and selective transporter protein
inhibitors, a small series of 3,4,5-trimethoxy-N-phenylbenza-
mide derivatives (i.e., 10a−f, first subset of Table 1), whose
general structure is depicted in Chart 1, was designed and
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Scheme 1. Synthesis of Trimethoxybenzamides 10a,b,d, 11a,b,d,g, Trimethoxybenzyl Analogue 12, Galloyl Derivative 14a,
a
Pyrogallol 1-Monomethyl Ether Congeners 15a,b,g,k,m and 3-Methoxybenzene-1,2-Diol Derivative 16
a
Reagents and conditions: (a) appropriate boronic acid, Pd(Ph3)4, aq K2CO3 (2 M), DMF, reflux, 3 h; (b) (DDQ), 3,4,5-trimethoxybenzyl alcohol,
(Ph)3P in DCM, room temp, 22 h; (c) 3,4,5-trimethoxybenzoyl chloride, dry 1,4-dioxane and DMF, microwave irradiation (25 min, 130 °C, Emax
=
500 W); (d) BBr3 (1 M in DCM), 4 equiv, dry DCM, −40 °C → room temp, 2 h; (e) BBr3 (1 M in DCM), 6 equiv, dry DCM, −40 °C → room
temp, 6 h; (f) BBr3 (1 M in DCM), 6−9 equiv, dry DCM, −40 °C → room temp, 2 h.
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Scheme 2. Synthesis of Trimethoxybenzamides 10,c,e,f, 11l,m, 3,4- and 3,5-Dihydroxybenzoyl Derivatives 14d−e, Isovanilloyl
a
Derivative 15o, Galloyl Derivative 14b, Pyrogallol 1-Monomethyl Ether Congeners 15e,l,n, and o-Quinone 19
a
Reagents and conditions: (a) appropriate benzoyl chloride, dry 1,4-dioxane and DMF, microwave irradiation (25 min, 130 °C, Emax = 500 W);
(b) Pd/C (10% w/w) under H2 atmosphere, absolute ethanol, room temp, 12 h; (c) acetic anhydride, DMAP, 1,4-dioxane, room temp, 24 h;
(d) BBr3 (1 M in DCM), 4 equiv, dry DCM, −40 °C → room temp, 2 h; (e) BBr3 (1 M in DCM), 6−9 equiv, dry DCM, −40 °C → room temp, 6 h;
(f) orthochloranil, THF, −30 °C, 2 h.
tested. Despite the consistent spread of the electronic
(Hammett σ) and lipophilic (π) parameters of the explored
R and, to a lesser extent, R1 substituents, the only compound
indicating an appreciable inhibitory activity against MRP1, and
devoid of any detectable P-gp inhibition (Table 1), was the
nitro derivative 10e (IC50 = 80 μM). The other benzamides of
this subset (10a−d and 10f) showed, conversely, moderate to
good P-gp inhibition with IC50 values from 20 to 1.4 μM. These
intriguing and somehow unexpected outcomes led us to pursue
the study with two parallel approaches: (i) keeping fixed the
3,4,5-trimethoxy-N-(2-nitrophenyl)benzamide scaffold and in-
troducing a series of R1 substituents in the 4′-position of the
aniline moiety to afford a second subset of compounds (11a−
m) and (ii) demethylating one or more methoxy groups on the
ring A to afford a third and a fourth subset comprising
gallamide (14a−c) and pyrogallol 1-monomethyl ether
derivatives (15a−n), respectively (Table 1). The biological
screening of the second subset returned the promising,
although nonactive, MRP1 inhibitors 11e, 11f, and 11i,
showing IC50 values equal to 6.1, 2.5, and 11.2 μM,
respectively. These good activities might be ascribed to the
presence in the 4′-position of aryl groups that may act as strong
HB acceptors. Unfortunately, this interpretation did not hold
for the benzo[1,3]dioxol-5-yl derivative 11g that exhibited no
MRP1 inhibition. The quaternization of the 4′-(3-pyridyl)
derivative 11e led to the totally inactive methyl iodide 11k
(MRP1 IC50 > 100 μM). Even though we cannot exclude the
possibility that the significant drop of lipophilicity may limit
cell membrane penetration of 11k, its P-gp inhibitory activity
seemed to be preserved, though moderate (IC50 = 34 μM).
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Scheme 3. Synthesis of Trimethoxybenzamides 11c−f,h−j, Galloyl Derivative 14c, Pyrogallol 1-Monomethyl Ether Congeners
a
15c,d,f,h−j, and the Methylpyridinium Iodide Derivative 11k
a
Reagents and conditions: (a) 3,4,5-trimethoxybenzoyl chloride, dry 1,4-dioxane and DMF, microwave irradiation (25 min, 130 °C, Emax = 500 W);
(b) BBr3 (1 M in DCM), 4 equiv, dry DCM, −40 °C → room temp, 2 h; (c) BBr3 (1 M in DCM), 6−9 equiv, dry DCM, −40 °C → room temp, 6
h; (d) CH3I, 6 equiv, acetonitrile−dry DCM (2/1, v/v), 50 °C, 2 h.
With the exception of the 4′-bromo- and 4′-methoxy congeners
(11a,b) all compounds of this subset were moderate to very
potent and selective P-gp inhibitors. In particular, the IC50
values of 11c, 11g, 11h, and 11j were in the submicromolar
range. Taken together, these data seem to corroborate the
distinctive role of the trimethoxyphenyl moiety recurring in
many MDR modulators.40
The screening of the gallamide derivatives 14a−e (third subset
in Table 1) indicated a moderate inhibitory potency for both
MRP1 and P-gp, irrespective of the number and position of the
phenolic groups. Compound 14a, lacking the 4′-phenyl group, was
completely inactive, while 14b showed a weak MRP1 selectivity.
Finally, the last series of structural modifications implying the
partial cleavage of the methoxy groups afforded the more
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a
Scheme 4. Synthesis of Trimethoxycarboxamide 13, 3,4-Dihydroxy-5-methoxyphenyl Derivative 17, and Cathecol Derivative 19
a
Reagents and conditions: (a) phenylboronic acid, Pd(Ph3)4, aq K2CO3 (2 M), DMF, reflux, 3 h; (b) (1) SOCl2, reflux, 2 h; (2) 3,4,5-
trimethoxyaniline, dry 1,4-dioxane/DMF, microwave irradiation (25 min, 130 °C, Emax = 500 W); (c) BBr3 (1 M in DCM), 4 equiv, dry DCM,
−40 °C → room temp, 3 h.
protein transporter. The replacement of the 4′-phenyl ring of
15e with substituents showing different stereoelectronic and
lipophilic properties (i.e., R1 = Br and OCH3, in 15a,b,
respectively) led to a reduction of activity, more pronounced in
the bromo derivative 15a (IC50 = 44.5 μM).
Taking into account that the most important inhibitory
activity changes were mainly concerned with the structural
modifications on ring B, the role of the amide bond linking the
A and B aromatic rings was investigated. To this end, two
analogues of compound 15e were prepared: the first with a
reverse amide bond (i.e., from NHCO to CONH); the second
with a reduced amide bond (i.e., from NHCO to NHCH2).
Compound 16, bearing the reduced amide bond as a linker,
retained, at least in part, the activity of parent compound 15e
toward MRP1 and was equipotent toward P-gp despite the
drastic change in terms of planarity and conformational
flexibility. Moreover, compound 17 having the reverse amide
bond showed no inhibitory activity toward both MRP1 and
P-gp. The 3,4,5-trimethoxybenzamide analogues 12 and 13,
bearing the NHCH2 and CONH linkers, respectively, were
inactive toward MRP1 while displaying a high and close
inhibitory potency toward P-gp (IC50 equal to 1.5 and 2.0 μM,
respectively).
Since at the cellular level the catechol moiety of 15e might be
oxidized to the corresponding o-quinone derivative 18, this
possible metabolite was prepared and tested. It showed a
significantly lower inhibitory activity toward both protein
transporters.
Finally, the change of activity arising from the presence of a
catechol moiety was further challenged with the selective bis-
demethylation of the verapamil, a well-known golden standard of
P-gp dependent MDR reverters.43 Disappointingly, the new
cathecol derivative 19 (Scheme 4) showed lower inhibitory
activity than the parent compound toward both MRP1 and P-gp.
In Figure 2, representative activity curves, regarding MRP1
activity, of compounds 11f, 14b, and 15e, the best MRP1
ligands with similar P-gp potency (11f), P-gp inactivity (14f),
or moderate selectivity (15e), were inserted.
Figure 2. Representative curves for the MRP1 inhibitory activities of
selected compounds and verapamil (Ver), carried out with calcein-AM
assay in MDCK-MRP1 cell lines: (▼) 11f, IC50 = 2.5 0.9 μM; (●)
14b, IC50 = 45.8 10 μM; (△) 15e, IC50 = 8.0 1.4 μM; (■) Ver,
IC50 = 6.8
3 μM. Each compound has been tested at seven
concentrations (from 0.1 to 100 μM), each concentration in triplicate
in two independent experiments. The y-axis indicates the percentage of
RFU (relative fluorescence unit). Statistical analysis has been
performed with the Friedman test (P = 0.0078, significant; Friedman
statistic value of 11.87).
hydrophilic pyrogallol 1-monomethyl ether derivatives 15a−n
(fourth subset in Table 1), all showing moderate to good
inhibition activity toward both transporters.
The o-NO2 derivative 15e exhibited higher MRP1 inhibitory
activity (IC50 = 8.0 μM) compared to the unsubstituted
compound 15k (IC50 = 75.8 μM) and the corresponding
o-NH2 15l (IC50 = 37.9 μM) and o-NHCOCH3 15n (IC50
=
28.5 μM) derivatives. With the interesting exception of
compound 15g (IC50 = 9.5 and >100 μM toward MRP1 and
P-gp respectively), the selectivity of these compounds was quite
moderate.
The replacement of the phenyl ring in 15e with the 2-thienyl
isoster gave 15d, whose MRP1 affinity was decreased (IC50
increased from 8.0 to 32 μM) while the corresponding
trimethoxybenzamide (11d) was inactive toward the same
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Figure 3. Square plot of P-gp/MRP1 inhibition data (Table 1): (◆) trimethoxybenzamides (10a, 11f, 11a−k, 12, 13); (■) gallamides and other
polyphenols (14a−e); (▲) pyrogallol 1-monomethyl ethers (15a−o, 16) and 17; (●) dimethoxybenzamides (11l, 11m); (+) reference
compounds and 19. The solid line represents the bisector, while the dotted lines, traced at the 1 log unit distance above and below the bisector, help
to identify selective compounds.
For ease of interpretation, the inhibition data are presented
in Figure 3 as a square plot of P-gp (x-axis) versus MRP1 (y-
axis) pIC50 values. To avoid the lack of valuable information,
inhibition data of inactive compounds (IC50 > 100 μM) were
reported with an arbitrary cut value (pIC50 = 4.0). Compounds
with equal affinities for both transporters lie on the bisector
(y = x) of the graph, whereas selective P-gp and MRP1
inhibitors lie below and above the bisector, respectively. The
distance of their pIC50 values from the bisector is a direct
measure of their degree of selectivity. The plotted data indicate
that the trimethoxybenzamides 11c,g−j are potent and selective
P-gp inhibitors, with pIC50 < 6. These compounds showed no
MRP1 activity that can be successfully recovered but at the
expense of selectivity by introducing strong HB acceptor
groups (11e,f). To gain selectivity for MRP1, phenolic groups
need to be restored as in the pyrogallol 1-monomethyl ethers
(15e, 15g). However, the potency and selectivity of these
compounds did not rise up to the best P-gp inhibitors.
Despite a relatively large variation of several physicochemical
features (such as molecular lipophilicity spanning a 2.3 log unit
range) and structural features (i.e., phenyl substitution patterns,
regioisomerism, and isosterism), no clear SAR emerged from a
thorough analysis of all inhibition data. Nevertheless, a trend
between lipophilicity and P-gp modulation of the most active
compounds was observed as shown in Figure 4. The
modulation of MRP1, on the contrary, appears to be insensitive
to the lipophilicity.
trimethyl ether derivative, can be easily converted in its
polyphenolic congener 15g, a quite selective MRP1 modulator.
Furthermore, a thorough modulation of the inhibition potency
was obtained by means of suitable substitutions on the B ring,
thus biasing P-gp and MRP1 with IC50 values encompassing 2
and 1 order of magnitude, respectively. Finally, from a careful
tuning of the chemical decoration through demethylation
reaction and introduction of selected HB acceptor groups on
the phenyl fragment bonded to the B ring, dual active MDR
modulators were discovered (e.g., 11e,f).
These encouraging results allow us to consider the galloyl
benzamide as a versatile scaffold for designing promising P-gp
and MRP1 modulators. Further biochemical and pharmaco-
logical studies to better define the interaction mechanism
responsible for the observed selectivity and an exploration of
the therapeutic potential of these compounds as MDR reversers
are warranted.
EXPERIMENTAL SECTION
■
Chemistry. High analytical grade chemicals and solvents were
from commercial suppliers. When necessary, solvents were dried by
standard techniques and distilled. After extraction from aqueous layers,
the organic solvents were dried over anhydrous sodium sulfate. Thin
layer chromatography (TLC) was performed on aluminum sheets
precoated with silica gel 60 F254 (0.2 mm) (E. Merck).
Chromatographic spots were visualized by UV light. Purification of
crude compounds was carried out by flash column chromatography on
silica gel 60 (Kieselgel 0.040−0.063 mm, E. Merck) or by preparative
1
TLC on silica gel 60 F254 plates or crystallization. H NMR spectra
CONCLUSIONS
■
were recorded in DMSO-d6 at 300 MHz on a Varian Mercury 300
instrument. Chemical shifts (δ scale) are reported in parts per million
(ppm) relative to the central peak of the solvent. Coupling constant (J
values) are given in hertz (Hz). Spin multiplicities are given as s
(singlet), br s (broad singlet), d (doublet), t (triplet), dd (double
doublet), dt (double triplet), or m (multiplet). LRMS (ESI) was
Our results demonstrate the versatile nature of the galloyl
benzamide scaffold for preparing very active MRP1 and P-gp
inhibitors. Simple chemical modifications were able to switch
over the selectivity while keeping high potency. In particular,
the potent and very selective P-gp inhibitor 11g, a galloyl
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Figure 4. Relationships of the most relevant inhibition data (Table 1) versus clogP data. The pIC50 data relative to MRP1 and P-gp are represented
as empty upside triangles (italic labels) and solid squares (bold labels), respectively. The clogP values have been calculated by using ACD software.
performed with an electrospray interface ion trap mass spectrometer
(1100 series LC/MSD trap system Agylent, Palo Alto, CA). In all
cases, spectroscopic data are in agreement with known compounds
and assigned structures. Combustion analyses were performed by
Eurovector Euro EA 3000 analyzer (Milan, Italy) and gave satisfactory
results (C, H, N within 0.4% of calculated values). HPLC purity
determinations were carried out using a Phenomenex PhenoSphere-
Next C8 150 mm × 4.6 mm column, with 5 μm particle size: isocratic
1 (for polyphenolic derivatives 14a−e, 15a−o, 16, 17), 10 mM
ammonium acetate in H2O/MeOH, 70/30, v/v, room temp; isocratic
2 (for trimethyl ether galloyl anilides and dimethoxy derivatives 10a−f,
11a−m, 12, 13), 10 mM ammonium acetate in H2O/MeOH, 55/45,
v/v, room temp. All test compounds were confirmed to be ≥95% pure
by HPLC.
General procedures for the Suzuki−Miyaura cross-coupling reaction
(9a−k, 11g) and benzoylation reaction (10a,b,d,e, 11a−j,l,m, and 13)
are reported in Supporting Information.
Procedures for the catalytic hydrogenation (10c), acetylation
reaction (10f), quaternization reaction (11k), and Mitsunobu reaction
(12) are reported in Supporting Information.
General Procedure for the Preparation of Phenol Derivatives
14a−e, 15a−o, 16, 17, and 19. Suitable benzamide (0.50 mmol)
was dissolved in dry DCM (30 mL) under argon and cooled at −
40 °C. Boron tribromide (BBr3, 1 M in DCM), 2.0 mL (2.0 mmol for
compounds 15a−o, 16, 17, 19) and 3.0−4.5 mL (3.0−4.5 mmol for
compounds 14a−e), was added dropwise, and the mixture was stirred
for 2 h. After the reaction was quenched with 50 mL of H2O, the
desired phenol was obtained through two different procedures. When
the phenol precipitated from the reaction mixture, it was recovered by
filtration (15a,e,g,h,i,k,n). In all other cases, the aqueous phase was
extracted with DCM and the organic phase was first dried over
anhydrous Na2SO4 and then evaporated under vacuum to dryness
affording the desired product. Except for 15a,b,e,m, all the phenolic
compounds were purified by preparative thin layer chromatography
(TLC) as specified below.
3,4,5-Trihydroxy-N-(3-nitrobiphenyl-4-yl)benzamide (14b). Pu-
rification by preparative TLC yielded 14b (18 mg, 6%): mp 175 °C
dec. H NMR (DMSO-d6) δ: 10.52 (s, 1H), 9.05 (br s, 3H), 8.23 (s,
1
1H), 8.03 (s, 2H), 7.75 (d, J = 7.2 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H),
7.42 (d, J = 7.2 Hz, 1H), 6.95 (s, 2H). LRMS (ESI) m/z 365 [M − H]−.
N-(3′-Acetyl-3-nitrobiphenyl-4-yl)-3,4,5-trihydroxybenza-
mide (14c). Purification by preparative TLC yielded 14c (30 mg,
15%): mp 180 °C dec. 1H NMR (DMSO-d6) δ: 8.33 (s, 1H), 8.28 (s,
1H), 8.12 (s, 2H), 8.02 (d, J = 7.8 Hz, 1H), 7.98 (s, 1H), 7.64 (t,
J = 7.8 Hz, 1H), 7.07 (s, 1H), 7.02 (s, 1H), 5.4 (br s, 1H), 3.6 (br s,
3H), 3.75 (s, 3H). LRMS (ESI) m/z 407 [M − H]−.
3,4-Dihydroxy-N-(3-nitrobiphenyl-4-yl)benzamide (14d). Puri-
fication by preparative TLC yielded 14d (47 mg, 27%): mp 175−
177 °C. 1H NMR (DMSO-d6) δ: 10.54 (s, 1H), 9.50 (br s, 2H), 8.23
(d, J = 1.7 Hz, 1H), 8.05 (dd, J = 1.7, 8.5 Hz, 1H), 7.95 (d, J = 8.5 Hz,
1H), 7.76 (d, J = 7.2 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H), 7.42 (d, J =
7.2 Hz, 1H), 7.37 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz,
1H). LRMS (ESI) m/z 349 [M − H]−.
3,4-Dihydroxy-N-(3-nitrobiphenyl-4-yl)benzamide (14d). Pu-
rification by preparative TLC yielded 14e (26 mg, 15%): mp 250−
252 °C. 1H NMR (DMSO-d6) δ: 10.63 (s, 1H), 9.66 (s, 2H), 8.23 (d,
J = 2.2 Hz, 1H), 8.05 (dd, J = 2.2, 8.5 Hz, 1H), 7.93 (d, J = 8.5 Hz,
1H), 7.76 (d, J = 7.2 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H), 7.43 (d, J = 7.2
Hz, 1H), 6.79 (d, J = 1.9 Hz, 2H), 6.44 (d, J = 1.9 Hz, 1H). LRMS
(ESI) m/z 349 [M − H]−.
N-(4-Bromo-2-nitrophenyl)-3,4-dihydroxy-5-methoxybenza-
mide (15a). Recrystallization from EtOH yielded 15a (124 mg,
1
65%): mp 206−208 °C. H NMR (DMSO-d6) δ: 10.52 (s, 1H), 9.33
(s, 1H), 9.13 (s, 1H), 8.17 (d, J = 2.2 Hz, 1H), 7.93 (dd, J = 2.2, 8.8
Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.08 (s, 2H), 3.82 (s, 3H). LRMS
(ESI) m/z 382 [M − H]−.
3,4-Dihydroxy-5-methoxy-N-(4-methoxy-2-nitrophenyl)-
benzamide (15b). Recrystallization from EtOH yielded 15b
1
(100 mg, 61%): mp 176−178 °C. H NMR (DMSO-d6) δ: 10.25
(s, 1H), 9.26 (s, 1H), 9.03 (s, 1H), 7.63 (d, J = 9.1 Hz, 1H), 7.49 (d,
J = 2.5 Hz, 1H), 7.32 (dd, J = 2.5, 9.1 Hz, 1H), 7.08 (s, 2H), 3.83 (s,
3H), 3.81 (s, 3H). LRMS (ESI) m/z 333 [M − H]−.
N-(4-Bromo-2-nitrophenyl)-3,4,5-trihydroxybenzamide
(14a). Purification by preparative TLC yielded 14a (38 mg, 21%): mp
185 °C dec. 1H NMR (DMSO-d6): δ = 10.49 (s, 1H), 9.08 (br s, 3H),
8.16 (d, J = 2.2 Hz, 1H), 7.91 (dd, J = 2.2 Hz, J = 8.8 Hz, 1H), 7.83 (d,
J = 8.8 Hz, 1H), 6.91 (s, 2H). LRMS (ESI) m/z 368 [M − H]−.
3,4-Dihydroxy-5-methoxy-N-(4-nitrobiphenyl-3-yl)-benz-
amide (15c). Purification by preparative TLC yielded 15c (19 mg,
10%): mp 230−233 °C. 1H NMR (DMSO-d6) δ: 10.62 (s, 1H),
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9.36 (s, 1H), 9.16 (s, 1H), 8.21 (s, 1H), 8.10 (d, J = 8.5 Hz, 1H), 7.75
(d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.5 Hz, 1H), 7.53 (t, J = 8.0 Hz, 2H),
7.48 (d, J = 8.0 Hz, 1H), 7.12 (s, 2H), 3.83 (s, 3H). LRMS (ESI)
m/z 379 [M − H]−.
3,4-Dihydroxy-5-methoxy-N-(2-nitro-4-thiophen-2-ylphen-
yl)benzamide (15d). Purification by preparative TLC yielded 15d
(18 mg, 8%): mp 165 °C dec. 1H NMR (DMSO-d6) δ: 10.54 (s, 1H),
8.27 (d, J = 2.2 Hz, 1H), 8.08 (dd, J = 2.2, 8.5 Hz, 1H), 7.95 (d, J = 8.5
Hz, 1H), 7.75 (d, J = 3.6 Hz, 1H), 7.72 (d, J = 5.0 Hz, 1H), 7.26 (dd,
J = 3.6, 5.0 Hz, 1H), 7.18 (s, 2H), 5.05 (br s, 2H), 3.91 (s, 3H). LRMS
(ESI) m/z 385 [M − H]−.
3,4-Dihydroxy-5-methoxy-N-(3-nitrobiphenyl-4-yl)-benz-
amide (15e). Purification by preparative TLC yielded 15e (95 mg,
50%): mp 159 °C dec. 1H NMR (DMSO-d6) δ: 10.55 (s, 1H), 9.34 (s,
1H), 9.12 (s, 1H), 8.23 (d, J = 2.2 Hz, 1H), 8.05 (dd, J = 2.2, 8.5 Hz,
1H), 7.91 (d, J = 8.5 Hz, 1H), 7.76 (d, J = 7.2 Hz, 2H), 7.50 (t, J = 7.2
Hz, 2H), 7.42 (d, J = 7.2 Hz, 1H), 7.12 (s, 2H), 3.83 (s, 3H). LRMS
(ESI) m/z 379 [M − H]−.
N-(3-Acetylaminobiphenyl-4-yl)-3,4-dihydroxy-5-methoxy-
benzamide (15n). Recrystallization from EtOH yielded 15n (22 mg,
11%): mp 235 °C dec. 1H NMR (DMSO-d6) δ: 9.88 (s, 1H), 9.66 (s,
1H), 9.26 (s, 1H), 9.03 (s, 1H), 7.77 (d, J = 8.3 Hz, 1H), 7.71 (s, 1H),
7.64 (d, J = 7.7 Hz, 2H), 7.57−7.41 (m, 3H), 7.37 (d, J = 7.4 Hz, 1H),
7.09 (s, 2H), 3.83 (s, 3H), 2.21 (s, 3H). LRMS (ESI) m/z 391
[M − H]−.
3-Hydroxy-4-methoxy-N-(3-nitrobiphenyl-4-yl)benzamide
(15o). Recrystallization from EtOH yielded 15o (47 mg, 26%): mp
1
195−198 °C. H NMR (DMSO-d6) δ: 10.60 (s, 1H), 9.45 (s, 1H),
8.23 (d, J = 2.2 Hz, 1H), 8.05 (dd, J = 2.2, 8.5 Hz, 1H), 7.92 (d, J = 8.5
Hz, 1H), 7.76 (d, J = 7.2 Hz, 1H), 7.75−7.40 (m, 5H), 7.07 (d, J = 8.5
Hz, 1H), 5.70 (s, 1H), 3.84 (s, 3H). LRMS (ESI) m/z 363 [M − H]−.
3-Methoxy-5-[(3-nitrobiphenyl-4-ylamino)methyl]benzene-
1,2-diol (16). Purification by preparative TLC yielded 16 (93 mg,
1
51%): mp 135 °C dec. H NMR (DMSO-d6) δ: 8.63 (t, J = 5.8 Hz,
1H), 8.30 (d, J = 2.2 Hz, 1H), 7.83 (d, J = 9.1 Hz, 1H), 7.62 (d, J = 7.2
Hz, 2H), 7.42 (t, J = 7.2 Hz, 2H), 7.31 (d, J = 7.2 Hz, 1H), 7.17 (m,
2H), 7.02 (d, J = 9.1 Hz, 1H), 6.51 (s, 1H), 6.41 (s, 1H), 4.47 (d,
J = 5.8 Hz, 2H), 3.71 (s, 3H). LRMS (ESI) m/z 365 [M − H]−.
3-Nitrobiphenyl-4-carboxylic Acid (3,4-Dihydroxy-5-me-
thoxyphenyl)amide (17). Purification by preparative TLC yielded
17 (42 mg, 22%): mp 182−184 °C. 1H NMR (DMSO-d6) δ: 10.36 (s,
1H), 9.00 (s, 1H), 8.32 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H),
7.83−7.79 (m, 3H), 7.53 (t, J = 6.9 Hz, 2H), 7.48 (d, J = 6.9 Hz, 1H),
6.89 (d, J = 2.2 Hz, 1H), 6.83 (d, J = 2.2 Hz, 1H), 3.71 (s, 3H). LRMS
(ESI) m/z 379 [M − H]−.
N-(3′,5′-Difluoro-3 nitrobiphenyl-4-yl)-3,4-dihydroxy-5-
methoxybenzamide (15f). Purification by preparative TLC yielded
15f (31 mg, 15%): mp 182 °C dec. 1H NMR (DMSO-d6) δ: 10.60 (s,
1H), 8.38 (s, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H),
7.60 (s, 2H), 7.58 (s, 1H), 7.27 (m, 1H), 7.15 (m, 1H), 5.12 (br s,
2H), 3.84 (s, 3H). LRMS (ESI) m/z 415 [M − H]−.
N-(3′,4′-Dihydroxy-3-nitrobiphenyl-4-yl)-3,4-dihydroxy-5-
methoxybenzamide (15g). Purification by preparative TLC
1
yielded 15g (51 mg, 25%): mp 235 °C dec. H NMR (DMSO-d6)
2-(3,4-Dihydroxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl]-
methylamino}-2-isopropylpentanenitrile (19). Purification by
δ: 10.45 (s, 1H), 8.05 (s, 1H), 7.95−7.80 (m, 2H), 7.17−7.02 (m,
4H), 6.83 (d, J = 8.3 Hz, 1H), 6.50 (br s, 2H), 5.45 (br s, 2H), 3.82 (s,
3H). LRMS (ESI) m/z 411 [M − H]−.
1
flash chromatography yielded 19 (53 mg, 25%): mp 135 °C dec. H
NMR (DMSO-d6) δ: 9.06 (s, 1H), 6.98−6.69 (m, 3H), 6.66−6.55 (m,
2H), 5.17 (br s, 2H), 3.74 (s, 3H), 3.70 (s, 3H), 3.14−3.07 (m, 4H),
2.77−2.73 (m, 2H), 2.47 (s, 3H), 2.23−2.08 (m, 4H), 1.43−1.39 (m,
1H), 1.19−1.13 (m, 3H), 0.68 (d, J = 6.3 Hz, 3H). LRMS (ESI) m/z
425 [M − H]−.
N-(3′-Acetyl-3 nitrobiphenyl-4-yl)-3,4-dihydroxy-5-methoxy-
benzamide (15h). Purification by preparative TLC yielded 15h (25
1
mg, 12%): mp 202−205 °C. H NMR (DMSO-d6) δ: 8.33 (s, 1H),
8.28 (s, 1H), 8.12 (s, 2H), 8.02 (d, J = 9.6 Hz, 1H), 7.98 (d, J = 9.6
Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.07 (s,
1H), 7.02 (s, 1H), 5.25 (br s, 2H), 3.75 (s, 3H), 2.67 (s, 3H). LRMS
(ESI) m/z 421 [M − H]−.
5-Methoxy-3,4-dioxocyclohexa-1,5-dienecarboxylic Acid (3-
Nitrobiphenyl-4-yl)amide (18). Compound 15e (0.50 mmol) was
dissolved in dry THF (8 mL) and added dropwise over 4 h via a
dropping funnel to a stirring solution of orthochloranil (0.60 mmol) in
dry diethyl ether (20 mL) at −30 °C. The reaction mixture was stirred
at −30 °C for 2 h and then stored at −20 °C for 12 h. The orange
precipitate was collected by filtration and washed with diethyl ether,
affording the desired ortho-quinone derivate 18 (75 mg, 40%): mp
N-(3′,4′-Dihydroxy-5′-methoxy-3-nitrobiphenyl-4-yl)-3,4-
dihydroxy-5-methoxybenzamide (15i). Purification by prepara-
tive TLC yielded 15i (26 mg, 12%): mp 240 °C dec. 1H NMR
(DMSO-d6) δ: 10.46 (s, 1H), 9.07 (br s, 4H), 8.10 (d, J = 1.9 Hz, 1H),
7.93 (dd, J = 1.9, 8.5 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.10 (s, 2H),
6.83 (d, J = 1.7 Hz, 1H), 6.77 (d, J = 1.7 Hz, 1H), 3.84 (s, 3H), 3.83
(s, 3H). LRMS (ESI) m/z 441 [M − H]−.
3,4-Dihydroxy-5-methoxy-N-(3'-nitrobiphenyl-4-yl)-benz-
amide (15j). Purification by preparative TLC yielded 15j (38 mg,
20%): mp 180 °C dec. 1H NMR (DMSO-d6) δ: 10.08 (s, 1H), 9.11 (s,
2H), 8.43 (s, 1H), 8.18−8.13 (m, 2H), 7.91 (d, J = 8.8 Hz, 2H), 7.78
(d, J = 8.8 Hz, 2H), 7.73 (t, J = 8.3 Hz, 1H), 7.13 (d, J = 2.5 Hz, 2H),
3.84 (s, 3H). LRMS (ESI) m/z 379 [M − H]−.
N-Biphenyl-4-yl-3,4-dihydroxy-5-methoxybenzamide
(15k). Purification by preparative TLC yielded 15k (16 mg, 8%): mp
165 °C dec. 1H NMR (DMSO-d6) δ: 9.42 (s, 1H), 7.94 (d, J = 8.8 Hz,
2H), 7.70−7.61 (m, 4H), 7.45 (t, J = 7.4 Hz, 2H), 7.34 (d, J = 7.4 Hz,
1H), 7.24 (d, J = 1.9 Hz, 2H), 5.70 (br s, 2H), 3.89 (s, 3H). LRMS
(ESI) m/z 334 [M − H]−.
N-(3-Aminobiphenyl-4-yl)-3,4-dihydroxy-5-methoxybenza-
mide (15l). Purification by preparative TLC yielded 15l (93 mg,
53%): mp 192 °C dec. 1H NMR (DMSO-d6) δ: 9.46 (s, 1H), 7.57 (d,
J = 7.3 Hz, 2H), 7.42 (t, J = 7.3 Hz, 2H), 7.32 (d, J = 7.3 Hz, 1H), 7.22
(d, J = 8.1 Hz, 1H), 7.14 (s, 1H), 7.12 (s, 1H), 7.06 (d, J = 1.8 Hz,
1H), 6.05 (br s, 2H), 6.88 (dd, J = 1.8, 8.1 Hz, 1H), 4.96 (s, 2H), 3.82
(s, 3H). LRMS (ESI) m/z 349 [M − H]−.
1
186 °C dec. H NMR (DMSO-d6) δ: 10.91 (s, 1H), 8.24 (d, J = 2.2
Hz, 1H), 8.08 (dd, J = 2.2, 8.5 Hz, 1H), 7.78−7.75 (m, 4H), 7.51 (d,
J = 7.4 Hz, 2H), 7.44 (t, J = 7.2 Hz, 2H), 3.77 (s, 3H). LRMS (ESI)
m/z 377 [M − H]−.
Biology. Cell Culture. MDCK-MDR1 and MDCK-MRP1 cell
lines were a gift from Prof. P. Borst, NKI-AVL Institute, Amsterdam,
The Netherlands. MDCK-MDR1 and MDCK-MRP1 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. Cell culture
reagents were purchased from Celbio s.r.l. (Milano, Italy).
CulturePlate 96-well plates were purchased from PerkinElmer Life
Science. Calcein-AM was obtained from Sigma-Aldrich (Milan, Italy).
Calcein-AM Experiment. The experiments were carried out as
described by Feng et al.36 with minor modifications. Each cell line
(50 000 cells per well) was seeded into black CulturePlate 96-well plates
with 100 μL of medium and allowed to become confluent overnight.
Test compounds were dissolved in 100 μL of culture medium and
were added to the cell monolayers. The plates were then 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 incubation was continued for 30 min. Each well was washed three
times with ice-cold PBS. Saline buffer was added to each well, and the
plates were read with a Victor3 fluorimeter (PerkinElmer) at excitation
and emission wavelengths of 485 and 535 nm, respectively. Under
these experimental conditions, calcein cell accumulation in the absence
and presence of tested compounds was evaluated, and basal-level
N-(3-Fluorobiphenyl-4-yl)-3,4-dihydroxy-5-methoxybenza-
mide (15m). Recrystallization from EtOH yielded 15m (24 mg,
14%): mp 225−228 °C. 1H NMR (DMSO-d6) δ: 9.84 (s, 1H), 9.21 (s,
1H), 8.96 (s, 1H), 7.71 (d, J = 7.2 Hz, 2H), 7.63 (d, J = 8.3 Hz, 1H),
7.58 (d, J = 1.7 Hz, 1H), 7.51 (dd, J = 1.7, 8.3 Hz, 1H), 7.46 (t, J = 7.2
Hz, 2H), 7.38 (d, J = 7.2 Hz, 1H), 7.16 (s, 1H), 7.14 (s, 1H), 3.83 (s,
3H). LRMS (ESI) m/z 352 [M − H]−.
434
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Journal of Medicinal Chemistry
Article
fluorescence was estimated by untreated cell fluorescence. In treated
wells, the increase in fluorescence was measured relative to the basal
level. IC50 values were determined by fitting the percent fluorescence
increase percentage versus log(dose).43
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Compound Stability Assay. The ESI-MS measurements were
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(1100 series LC/MSD trap system Agylent, Palo Alto, CA) operating
in the following regime: electrospray voltage, 4.0 kV; capillary
temperature, 350 °C; sample solution flow rate, 50 μL/h.; dry gas,
5.00 L/min; nebulizer, 15.00 psi. All analyses were performed in the
negative ion detection mode. Pure solvent spectrum was monitored
between each two samples until the analyzed peak intensity decreased
to the initial level observed before sample introduction. All reference
samples were dissolved in methanol. A 50 μL aliquot of a sample solution
was injected into the instrument with a 500 μL Hamilton syringe. After a
stationary ion current level was attained, the final spectrum was obtained
by averaging over 100 sequentially measured spectra.
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ASSOCIATED CONTENT
* Supporting Information
■
S
General procedure for the Suzuki−Miyaura cross-coupling
reaction, nitro group reduction by catalytic hydrogenation,
benzoylation, acetylation, quaternization, and Mitsunobu
reactions; Tables S1 and S2 listing spectroscopic data; Figures
S1−10 showing NOESY results, relationships, mass spectra,
and stability profiles; and ESI-MS method. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For S.C.: phone, +39-080-5442090; fax, +39-080-5442231/0;
e-mail, s.cellamare@farmchim.uniba.it. For N.A.C.: phone, +39-
080-5442727; e-mail, colabufo@farmchim.uniba.it.
ACKNOWLEDGMENTS
■
Credits are due to Dr. Andrea Gissi and Dr. Antonellina
Introcaso for experimental support. The authors gratefully
acknowledge the financial support from European Commission
(“CancerGrid” STREP project, FP VI, Contract LSHC-CT-
2006-03755) and from University of Bari “Aldo Moro”, Italy
(Progetto IDEA Giovani Ricercatori 2008).
ABBREVIATIONS USED
■
MDR, multidrug resistance; ABC, ATP-binding cassette;
MRP1, multidrug resistance associated protein 1; P-gp, P-
glycoprotein; calcein-AM, calcein acetoxymethyl ester
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