Trimethoxybenzanilide-based P-glycoprotein modulators: An interesting case of lipophilicity tuning by intramolecular hydrogen bonding

Article abstract of DOI:10.1021/jm500697c

One of the principal reasons for the chemotherapy failure is the overexpression of drug efflux pumps, ABCB1 (also known as MDR1 or P-gp) and ABCC1 (also known as MRP1), whose inhibition remains a priority to circumvent drug resistance. We have recently sh

Full text of DOI:10.1021/jm500697c

Article
pubs.acs.org/jmc
Trimethoxybenzanilide-Based P‑Glycoprotein Modulators: An
Interesting Case of Lipophilicity Tuning by Intramolecular Hydrogen
Bonding
Piero Tardia,† Angela Stefanachi,† Mauro Niso,† Diana Antonella Stolfa,† Giuseppe Felice Mangiatordi,†
Domenico Alberga,‡ Orazio Nicolotti,† Gianluca Lattanzi,‡ Angelo Carotti,† Francesco Leonetti,†
Roberto Perrone,† Francesco Berardi,† Amalia Azzariti,§ Nicola Antonio Colabufo,*,†
and Saverio Cellamare*,†
†Dipartimento di FarmaciaScienze del Farmaco, Universita
̀
di Bari ”Aldo Moro”, Via Orabona 4, 70125 Bari, Italy
‡Dipartimento di Fisica, Universita
̀
di Bari ”Aldo Moro”, INFN & TIRES, Via Amendola 173, 70126 Bari, Italy
§Clinical and Preclinical Pharmacology Laboratory, National Cancer Research Centre, Istituto Tumori Giovanni Paolo II, Viale O.
Flacco 65, 70124 Bari, Italy
ABSTRACT: One of the principal reasons for the chemo-
therapy failure is the overexpression of drug efflux pumps,
ABCB1 (also known as MDR1 or P-gp) and ABCC1 (also
known as MRP1), whose inhibition remains a priority to
circumvent drug resistance. We have recently shown a clear
trend between lipophilicity and P-glycoprotein inhibitory
activity for a class of galloyl-based modulators targeting P-
glycoprotein and MRP1. Herein we report a new series of
polymethoxy benzamides, whose lipophilicity was modulated
through the establishment of an intramolecular hydrogen bond
(IMHB) which allows reaching of P-gp inhibitory activity at
the submicromolar IC50 level. The present study provides a
strong rationale for candidates in the presence of IMHB as a key element for a high P-gp inhibitory activity.
the P-gp transporter activity is profoundly affected by the ability
of the ligands to cross the cellular membrane depending in turn
by their physicochemical properties. Despite the recent advances
in the ADME-tox optimization, further efforts are needed to
address the P-gp activity of new potential drug candidates for
CNS pathologies.9 In the past two decades, a number of P-gp
modulators spanning a wide range of chemical diversity have
been designed and studied in depth.10 Some of them have
entered phase III clinical trials, but none of them has been
approved so far for clinical use because of their serious side effects
and lack of sufficiently proved efficacy.3
The compelling goal, of covering those gaps, is challenging the
scientific community and new strategies are being exploited.11
Notably, the attrition toward an expedite progression is
determined by the lack of adequate structural information on
the target(s), which is necessary for rationally tuning molecular
activity and selectivity and reducing side effects.12 However, a
firm notion based on structure−activity relationship (SAR) and
quantitative structure−activity relationship (QSAR) studies is
that high lipophilicity is necessary to achieve significant P-gp
activity.13 This concept was highlighted in some recent studies,
indicating an entry pathway through the membrane bilayer.4
INTRODUCTION
■
The multidrug resistance (MDR) of cancer consists in an
acquired lack of sensitivity to anticancer drugs. It is translatable to
a “self-defense” mechanism occurring in numerous kinds of
malignant cells, of blood and solid tumors, after their exposure to
antineoplastic treatment.1
ABCB1 (P-gp, p-glycoprotein) and ABCC1 (multidrug
resistance associate protein 1, MRP1) are members of the
ubiquitous ATP-binding cassette (ABC) superfamily, physiolog-
ically expressed at the blood−brain barrier (BBB), the blood−
cerebrospinal fluid barrier (B-CSF), and the intestinal barrier,
thus modulating the uptake and the elimination of drugs and
toxins.2,3
One of the best characterized ABC proteins is P-gp, an ∼170
kDa mammalian glycoprotein that is a polyspecific multidrug
efflux pump4,5 and whose overexpression in the plasma
membrane of tumor cells contributes to MDR.6,7
P-gp substrates are typically amphipathic, and there is a
considerable evidence that their interaction with the protein
occurs within the plasma membrane either at the extracellular or
cytoplasmic side.2,4
A recently solved X-ray crystal structure of P-gp shows an
open, easily accessible region at the cytoplasmic side. This
suggests that drugs may enter into that pocket through the inner
membrane leaflet.8 As a consequence, it may be postulated that
Received: February 21, 2014
Published: July 20, 2014
© 2014 American Chemical Society
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Figure 1.
Indeed, in our recent work on galloyl-based selective
modulators of MDR1 and P-gp,14 we envisaged a certain
correlation between lipophilicity and the ability of modulating P-
gp. For instance, compound 1a (Figure 1), having the highest
dual P-gp/MRP1 inhibitory potency, was also the most lipophilic
of the series, even if, unexpectedly, its pyridyl congener 1b was
only three times less active despite its much higher water
solubility, as assessed by turbidimetric assay15 (1a, 0.005 0.003
mg/L; 1b, 10 2 mg/L).
Building on this work, we planned the design, synthesis, and
biological evaluation of a new series of dual P-gp/MRP1 N-
biphenyl-benzamide inhibitors with an improved physicochem-
ical profile (solubility, lipophilicity, etc.) and lacking the liable
nitro group. In particular, the lipophilicity was modulated also by
synthesizing a number of trimethoxyphenyl regioisomers with
diverse propensity to form intramolecular hydrogen bond.16−18
The new molecular panel was built by assembling the three
aromatic rings depicted in Figure 1 (rings A, B, and C), namely a
(di)fluorophenyl, a pyridyl, and a (di)trimethoxyphenyl moiety,
respectively, through a carboxamide or a sulfonamide group
linking B to C.
To assess the importance of intramolecular hydrogen bond
formation between ring B and ring C of the molecule, we
performed the N-methylation of amides 4a and 4b by treatment
with sodium hydride and methyl iodide, affording compounds
10a and 10b, respectively. The sulfonamide 7j was prepared to
study the effect of nonclassic bioisosteric replacement on
compound 4e.
Lipophilicity Studies. There is good agreement that P-gp
activity is strongly influenced by lipophilicity as recently reported
for a series of benzophenone-type inhibitors.12 On the basis of
this evidence, we first tried to correlate the calculated log P values
with pIC50 values (P-gp inhibition) of our compounds. To this
purpose, different programs (BioLoom version 1.5, ACD/
Laboratories version 6.00 and ALOGPS v. 2.1) were used. For
the sake of clarity, the log P values shown in Table 1 and Figure 2
are those given by ALOGPS.20
As shown in Figure 2, our expectations were not met as no
correlation was observed between activity at P-gp and
lipophilicity. It is worth noting that the lack of correlation may
be due also to the poor ability of the used software to detect
different lipophilicity for the differently substituted methoxy
regioisomers and also a different lipophilicity arising from the
possible formation of intramolecular hydrogen bonding for some
of the examined compounds.
To overcome these drawbacks, we have thus experimentally
measured both log D values by octanol/pH 7.4 phosphate buffer
partitioning21 and log k′ as an additional lipophilicity index,
determined by HPLC using a reversed stationary phase in
isocratic conditions.22
As expected, poor correlations between calculated log P and
log D/log k′ (parts a and b of Figure 3, respectively) were found
while a higher correlation was seen between the two
experimental parameters log D and log k′ (Figure 3c).
Interestingly, the differences between regioisomers of the
trimethoxy phenyl ring in terms of lipophilicity such as 2a vs
2b, 3a vs 3b, 4a vs 4b, 5a vs 5b, and 10a vs 10b (highlighted in
red in the plot) can be easily appreciated.
Finally, a significant increase of the determination coefficient
(r2) was observed when considering the relationship between log
D and pIC50. This clearly indicates that also in the series of our N-
biphenyl-benzamides, P-gp inhibition increases along with the
molecular lipophilicity (Figure 3d).
In doing so, we prepared a new series of 21 biologically active
P-gp inhibitors having activity values reaching submicromolar
IC50 level (Table 1). Eleven out of 21 compounds acted as
inhibitors on both P-gp and MDR1 with some very interesting
IC50 values.
CHEMISTRY
■
The synthetic pathways chosen for the preparation of the
designed compounds are shown in Scheme 1, and the obtained
compounds are listed in Table 1. In the molecular design, a
number of substituents were selected to explore a wide range of
stereoelectronic and lipophilic properties. In brief, the starting
(hetero)arylanilines (1−6, Scheme 1) (“A + B” nuclei) were
obtained by Suzuki coupling between the appropriate (hetero)-
aniline and the corresponding arylboronic acids. The end
products (“A + B + C”) were prepared by coupling the suitable
“A + B” nuclei with the appropriate (hetero)aroyl- (or sulfonyl-)
chlorides, affording the corresponding compounds 2a, 2b, 3a, 3b,
4a−i, 5a, 5b, 6b, and 7j (Scheme 1).
Each compound is identified by a number, referring to the
biphenyl system (“A + B” moiety), and a letter corresponding to
a specific (hetero)aryl (C). For instance, 2a and 2b are 3,4,5- and
2,4,5-trimethoxybenzoyl regioisomers, respectively, as well as 3a
and 3b and so on.
Starting from 1a, the reduction of the nitro group provided the
amino congener 8a that was coupled with N-acetyl-L-alanine to
afford 9a.
Interestingly, three compounds (4b, 5b, and 3b), all bearing
2,4,5-trimethoxyphenyl nucleus (C ring), were the most active
ones against P-gp although they did not show the highest
lipophilicity. This result could be interpreted by considering the
presence of an intramolecular hydrogen bond between the 2-
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Table 1. Chemical Structure, Lipophilicity, and Biological Activity of Compounds 2a,b, 3a,b, 4a−i, 5a,b, 6b, 7j, 8a, 9a, and 10a,b
(The PBS Buffer Solubility Measurements Are Given Only for the Compounds 4a−i)
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Table 1. continued
a
b
The IC50 data (Calcein-AM assay), reported as μM SEM, are averaged over two independent experiments, sampled in triplicate. Calculated by
ALOGPS 2.1.19,20 log D pH 7.4 measurements by Analiza Inc.21 Logarithm of the capacity factors measured by RP-chromatography using
c
d
methanol/pH 7.4 phosphate buffer 70:30 as an eluent.22 Solubility measured by turbidimetric assay.15 Reference compounds.28
e
f
methoxy group on the ring C of compounds 4b, 5b, and 3b and
the NH group on the ring B.
compared in both regioisomers, nicely confirming our expect-
ations (Figure 4a).
As already described in literature, changes in 1H chemical shift
of exchangeable protons with temperature can be used to
determine the presence of IMHB in small molecules.23
The temperature coefficient TC was used to evaluate the
temperature effect. TC value corresponds to the slope of the line
obtained plotting the chemical shift versus the temperature of the
experiment (Figure 4b); this value is generally negative and
compounds with IMHB exhibit a less negative TC than
compounds unable to form IMHB (control, compound 4a), as
in our experiments (Figure 4c).
Intramolecular Hydrogen Bonds Detection by NMR
Studies. To validate our hypothesis on the intramolecular
hydrogen bond (IMHB) as a key element for a high P-gp
1
inhibitory activity, a spectrometric analysis by H NMR was
carried out by measuring the chemical shift of one exchangeable
NH proton at a single and at variable temperature.
Generally, an exchangeable NH proton that is involved in a HB
interaction is more deshielded (higher δ chemical shift value,
Figure 4a) than a similar, not hydrogen bonded, exchangeable
NH proton. As a consequence, a comparative analysis of the
chemical shift between two model compounds, namely a sample
(3,4,5-trimethoxybenzamide, 4a) and a control (2,4,5-trimethox-
ybenzamide, 4b), was carried out to demonstrate the existence of
an IMHB between the NH and the oxygen of the 2-methoxy
group in 4b and not in 4a.
This means that the Δδ of the benzamide NH for compound
4b is less pronounced than for 4a, accordingly to temperature
increase. Evidence of IMHB were also obtained with 2D NOESY
experiments, as shown in Figure 5a,b.
We focused our attention on the interactions between the NH
proton and the trimethoxyphenyl moiety of both compounds 4a
and 4b. In the case of compound 4a, the distance between the
amidic proton (Ha, s, 10.32 ppm) and the meta methoxy
substituents (Hc, s, 3.86 ppm) does not allow the NOE, therefore
the only detectable cross pick is the one from the interaction with
the in ortho position protons (Hb, s, 7.36 ppm). In the spectrum
of 4b, as expected, is present the cross pick of the NH (Ha′, s,
10.42 ppm) and the ortho methoxy protons (Hf, s, 4.06 ppm).
To confirm the presence of such a IMHB, two different series
of experiments were performed on the two selected
regioisomers: (i) an eight point 1H NMR chemical shift
determination of the NH exchangeable protons as a function
of temperature boost (Figure 4b−c), and (ii) a 2D-1H-NOESY
assessment (Figures 5a,b).
Because the amide hydrogen involved in IMHB (4b) might be
more deshielded than the analogue proton unable to form IMHB
1
(4a), the H chemical shifts of the NH were measured and
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Scheme 1. Synthesis of (Hetero)arylanilines 1−6, Dimethoxybenzenesulfonamide 7j, Benzamide 4h, 4-Methoxybenzamide 4g,
Dimethoxybenzamides 4e,f Trimethoxybenzamide Derivatives 1a, 2a,b, 3a,b, 4a−d, 5a,b, 6b, 8a, 9a, 10a,b, and Nicotinamide 4i
a
a
Reagents and conditions: (i) Pd(PPh3)4, aq K2CO3 (2M), 1,4-dioxane, reflux, Y: 2 (45%), 3 (50%), 4 (90%), 5 (40%), 6 (30%); (ii) THF dry,
room temp, overnight, Y: 2a (23%), 2b (37%), 3a (10%), 3b (68%), 4a (81%), 4b (79%), 4c (56%), 4d (10%), 4e (84%), 4f (98%), 4g (69%), 4h
(98%), 4i (77%), 5a (54%), 5b (58%), 6b (87%), 7j (61%); (iii) NaH, CH3I, DMF dry, 0 °C → room temp, 3 h, 10a (68%), 10b (19%); (iv) Pd/C
(5% w/w) under H2 atmosphere, absolute ethanol, room temp, 2 h, 8a (91%); (v) N-acetyl-L-alanine, EDC, HOBt, TEA, THF dry, room temp, 72 h,
9a (32%). a = 3,4,5-trimethoxybenzoyl chloride; b = 2,4,5-trimethoxybenzoyl chloride; c = 2,3,4-trimethoxybenzoyl chloride; d = 2,4,6-
trimethoxybenzoyl chloride; e = 3,4-dimethoxybenzoyl chloride; f = 3,5-dimethoxybenzoyl chloride; g = 4-methoxybenzoyl chloride; h = benzoyl
chloride; i = nicotinoyl chloride.
= 150°) only one, planar conformation, is observed for 4b (φ =
180°). Importantly, such difference is more evident when the
solvent is chloroform rather than water, thus suggesting that the
effect of the H-bond on the planarity is enhanced when the
molecule permeates through the membrane. We found an
increased stability of the intramolecular hydrogen bond in
chloroform when compared to water. This effect is significant
because it contributes to the planarity of the molecule in a
lipophilic environment, which facilitates the passage through
membrane.
COMPUTATIONAL STUDIES
■
To evaluate the influence of the IMHB on the conformation of
the investigated molecules, molecular dynamics simulations were
carried out on regioisomers 4a and 4b. The analysis was
performed by considering the molecules fully solvated either in
water or in chloroform, the latter representing one of the organic
solvents used to model the membrane environment in MD
simulations.24,25 The probability distribution of the values of the
dihedral angle φ (P(φ)), indicating the system planarity (see
Figure 6c), were taken into account. As depicted in parts a and b
of Figure 6, at variance with compound 4a displaying two
different stable conformations (corresponding to φ = 30° and φ
The strength of hydrogen bonds ranges energies enabling even
short-lived association and dissociation under ambient con-
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the different activities reported for the regioisomers 4a and 4b.
The strength of such interaction is here proved to be a sensitive
parameter for a soft modulation of the P-gp response. For
instance, the 2,4,5-trimethoxybenzamide derivatives 3b, 4b, and
5b display the highest activity and are also characterized by the
strongest IMHB.
BIOCHEMICAL STUDIES
■
Calcein-AM Experiment. 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.27 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 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, expressed in the
cell membrane, rapidly efflux the calcein-AM before its entrance
into the cytosol, giving 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 8
in the presence of various concentrations of selected MRP1 and
P-gp inhibitors such as Verapamil and MC1828 were used as
reference compounds for MRP1 and P-gp inhibition, respec-
tively. The accuracy and reproducibility of the estimated IC50
values are in accordance with the most recent guidelines.
Anticancer Activity Evaluation through a Co-admin-
istration Assay. To verify doxorubicin activity in resistant
MCF7adr cell line, the same antineoplastic drug has been
administrated with compounds 2b, 4b, 4c, and 5b displaying P-
gp activity in the submicromolar range. The co-administration
assays have been carried out at three different concentrations of
selected compounds (Figure 9). The effect of doxorubicin in co-
administration has been evaluated after 72 h by MTT test and the
results suggest that the block of P-gp ligands, in a dose dependent
manner as depicted in each corresponding graph reported in
Figure 9, permitted a recovery of the doxorubicin effect with
respect to the control.
Figure 2. Relationship between P-gp inhibitory potency of compounds
listed in Table 1 (expressed as pIC50 values) and calculated log P values
(ALOGPS).
ditions. It goes without saying that the role of intramolecular and
intermolecular hydrogen bonding is relevant, not only for the
recognition mechanism and stabilization of a preferred
conformation but also for the improvement of several
physicochemical properties such as the permeability.
In this respect, it has been recently demonstrated18 that the
occurrence of intramolecular hydrogen bonds can affect several
in vitro properties determining higher lipophilicity and cell
permeability.
Another relevant study,26 emphasized how the formation of
intramolecular hydrogen bonds in drug molecules can shield the
polarity and thus improve lipophilicity. Thus, our design of
compounds having the chance of forming an intramolecular
hydrogen bond (IMHB) is important to bias closed planar
conformation shielding the polarity relative to that open with a
gain in lipophilicity and, more importantly, in the permeation of
biological membranes.
Finally, density functional theory (DFT) calculations were also
performed in order to compute the HB breaking energy barrier
required in 4b. A relaxed scan calculation (level of theory
B3LYP/6-31G(d)) was carried out in order to build a complete
picture of the potential energy profile for the torsion around the
dihedral angle φ (Figure 6d).
The obtained data confirm that the IMHB is highly stable,
being the required energy barrier higher than 10 kcal/mol.
Following the same approach, the IMHB breaking energy
barriers were computed for all those compounds for which an
IMHB could be predicted, namely 2b, 3b, 4b, 4c, 4d, 5b, and 6b.
The obtained data (including the energy barrier already
calculated for compound 4b) confirm the occurrence of a stable
IMHB in these compounds and, more importantly, disclose a
pretty correlation with the IC50 values (r2 = 0.960, see Figure 7).
Satisfactorily, DFT energy barrier values are valuable observables
for a fine-tuning molecular design as they allow accurate
discrimination of even small structural variations of highly active
compounds, IC50 ranging from 0.20 to 2.93 μM (see Figure 7 and
Table 1). It is worth noting that a good correlation (r2 = 0.788)
persists even in absence of 4d, a possible influential outlier. In
summary, both MD simulations and DFT calculations suggest
the presence of a unique conformation of 4b characterized by a
very stable IMHB, thus providing a solid molecular rationale for
Bioluminescent ATP Assay. To obtain some details in
terms of P-gp interacting mechanism, cellular ATP depletion was
evaluated in the presence or absence of compounds 2b, 4b, 4c,
and 5b in MDCK-MDR1 (Figure 10). All compounds were
unable to deplete ATP and therefore behave as potential P-gp
inhibitors. This results permit hypothesizing that tested
compounds may be P-gp inhibitors or, alternatively, not detected
by P-gp sites. Moreover, these results should be matched with
apparent permeability evaluation to prove/confirm this mech-
anistic hypothesis. In this assay, for each compound both the flux
from basolateral to apical (B → A) and the flux from apical to
basolateral (A → B) were evaluated. The B → A flux represents
the passive transport, whereas the flux A → B is the active
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Figure 3. (a) Relationships between calculated log P and experimental log D7.4 values; (b) calculated log P and experimental log k′70%MeOH values; (c) log
D7.4 and log k′ values; (d) log D7.4 and P-gp inhibitory potency (pIC50) of compounds listed in Table 1; (e) determination coefficient (r2)of correlation
between studied theoretical and experimental parameters.
transport. For each tested compound was estimated BA/AB
ratio, and compounds displaying BA/AB > 2 are considered P-gp
substrates whereas compounds BA/AB < 2 are estimated P-gp
inhibitors. Unexpectedly, permeability results indicated that
these ligands were P-gp substrates and so this apparent
discrepancy with ATP cell depletion results will be addressed
later, after the discussion of the experimental permeability data.
Permeability Experiment. The apparent permeability
(Papp) in Caco-2 cell monolayer has been determined for
compounds 2b, 4b, 4c, and 5b. In this experiment, the fluxes of
each ligand from basolateral to apical (B → A) and from apical to
basolateral (A → B) compartments have been evaluated. The (B
→ A) flux indicates the role of passive flux of compound, whereas
the (A → B) flux displays the active transport because P-gp is
localized at apical level. Actually (B → A)/(A → B) ratio permits
prediction of whether a ligand could be considered a substrate
(ratio ≫2) or inhibitor (ratio ≤2) of P-gp.29 The tested
compounds displayed the following (B → A)/(A → B) ratio: 14,
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result is given by compound 4b (bearing a 2,4,5-trimethox-
yphenyl as C ring), exhibiting a submicromolar and low
micromolar inhibitory activity on P-gp and MRP1, respectively
(IC50 0.2 μM on P-gp and 3.38 μM on MRP1). As already
discussed, the 2-methoxy group on C ring is able to establish an
intramolecular hydrogen bond with the benzamide NH that
stabilizes a planar conformation. To detect the optimal
substitution pattern needed to ensure a better P-gp inhibition,
two new regioisomers of compound 4b were synthesized, that is,
the 2,3,4-trimethoxyphenyl derivative 4c and 2,4,6-trimethox-
yphenyl isomer 4d. The observed IC50s on P-gp are 0.67 and 2.93
μM, respectively and on MRP1 are both >100 uM.
Indeed, all the 2,4,5-trimethoxybenzoyl derivatives, namely 2b,
3b, 4b, 5b, and 6b, showed good inhibition results: 2b has an IC50
of 0.97 μM on P-gp and >100 μM on MRP1; 3b an IC50 of 0.54
μM on P-gp and 6.26 μM on MRP1; 5b an IC50 of 0.31 μM on P-
gp and >100 μM on MRP1; 6b, has an IC50 of 1.23 μM on P-gp
and >100 μM on MRP1. All these compounds showed higher
activity than the corresponding 3,4,5-trimethoxybenzoyl re-
gioisomers (named as -a series).
A likely reason can be found in the occurrence of an IMHB,
which is established from the methoxy group at the 2-position of
the C ring and the benzamide NH. Interestingly, such IMHB is
responsible for the adoption of a molecular conformation whose
polar content is decreased so that they could shed water in the
presence of a low dielectric environment like a hydrophobic
phospholipid bilayer.30,31
To confirm this hypothesis, the synthesis of the N-methylated
congeners of 4a and 4b was carried out, giving compounds 10a
and 10b respectively, which were supposed to be less active,
being unable to establish an IMHB. As expected, while for 4a and
4b the observed IC50s on P-gp are 4.34 and 0.20 μM, respectively,
and on MRP1 4.28 and 3.38 μM, respectively, for compounds
10a and 10b, increased IC50 values on P-gp (6.66 and 21.6 μM,
respectively) and on MRP1 (8.13 and 23.6 μM respectively)
were obtained.
Figure 4. (a) 1H chemical shifts of exchangeable protons belonging to
compounds 2a−9a; (b) plots of the chemical shift vs the temperature;
(c) temperature coefficients (TC).
The bioisosteric replacement of the carboxamide linker “L” in
4e with a sulfonamide one led to compound 7j, displaying a
decreased activity on P-gp (IC50 = 8.92 vs 67.5 μM), likely due to
the strongly lowered lipophilicity determined by the more polar
and partly ionized sulphonamido linker at pH 7.4.
The reduction in 1a of the nitro group to the amino derivative
8a resulted in an increase of activity on P-gp (IC50 2.76 μM). Its
coupling with N-acetyl-L-alanine gave 9a, showing a lower P-gp
inhibition potency (IC50: 18.5 and 11.1 μM on P-gp and MRP1,
respectively).
Disappointingly, no significant correlation was found between
pIC50 at P-gp and the calculated cLogPs, ALogPs, and log Ds for
our series of compounds. The poor correlation is probably due to
the inability of the aforementioned predicting methods of
assessing the occurrence of intramolecular H-bonds and thus its
effect on lipophilicity.
The log D experimental data at pH 7.4, on the other hand,
showed a still low, but definitely better correlation coefficient,
indicating that an increased lipophilicity is generally accom-
panied by an increased activity (see Figure 3d).
8.1, 6.7, and 10.7 for 4b, 5b, 4c, and 2b, respectively. These
results indicated that all tested compounds could be considered
P-glycoprotein substrates. To confirm such a hypothesis, the Papp
was also detected in the presence of MC18, a known P-gp
inhibitor. This allowed us to verify the increase of A → B flux.
Indeed, for compounds 4b, 4c, and 2b, the BA/AB ratio shifted
from 14, 6.7, 10.7 to 8.2, 6.1, and 8.2, respectively, hence
confirming that these compounds are “pure” P-gp substrates. All
these results are the mean of three independent experiments
samples in triplicate. Compound 5b displayed an out-of-trend
result because BA/AB ratio increases from 8.1 to 11.2. This
apparent discrepancy could be the result of the ability of this
compound to be also a breast cancer resistance protein (BCRP)
substrate and so blocks P-gp and stimulates also the BCRP efflux.
RESULTS AND DISCUSSION
■
To obtain higher aqueous solubility, a pyridyl moiety was
introduced, affording very interesting results in terms of P-gp
inhibition.
The IMHB showed by some compounds of our series involves
the methoxy and NH groups in C- and B-rings, respectively,
leading to the formation of a third pseudo-six-membered ring
system. When the occurrence of the IMHB is not allowed, the
lipophilicity drops dramatically as in case of the tertiary amide
10b (compare with 4b). None of the three aforementioned
programs was able to predict the difference of log D found out
For example, compounds 4a−i, which contain the 2-
fluorophenyl as B ring and the 3-pyridyl as A ring, showed
good aqueous solubility, as assessed by turbidimetric assay (for
compounds 4a−4i, are reported in Table 1),15 and P-gp activity
ranging from 0.2 to 9.2 μM; the only exception is due to the
nicotinamide derivative 4i (IC50 60.1 μM on P-gp and >100 μM
on MRP1), probably due to a drop of lipophilicity. The best
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Figure 5. (a) 2D NOESY of compound 4a; (b) 2D NOESY of compound 4b.
experimentally for compounds 10b (1.75) and 4b (2.84). It is
noteworthy to stress that all the best six hits for P-gp (IC50
ranking from 0.20 to 1.23 μM) have a 2-methoxy group on the C-
ring and therefore the possibility to establish an HB seems to be a
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Figure 6. (a−d) Probability distribution of the dihedral angle φ during the MD trajectory in water (a) and in chloroform (b) for 4a and 4b; (c) DFT
optimized molecular structure of 4b: φ indicates the dihedral angle (rotation around the C−C bond) whose value is indicative of the intramolecular H-
bond occurrence (depicted as a dotted line); (d) DFT potential energy profile for the torsion around the dihedral angle φ in 4b.
Figure 8. Representative curves for the MDR1 inhibitory activities of
selected compounds and MC18, carried out with calcein-AM assay in
MDCK-MDR1 cell lines. Each compound has been tested at seven
concentrations (from 0.1 to 100 μM), each concentration in triplicate in
Figure 7. Relationship between P-gp IC50 and IMHB energy barrier
two independent experiments. The y-axis indicates the percentage of
values. All calculations were performed at the B3LYP/6-31G(d) level of
ΔFLU (fluorescence units). Statistical analysis was performed with the
theory.
Friedman test (P = 0.0078, significant; Friedman value equal to 11.87).
molecular determinant for a high affinity. Compound 4d has two
dimethoxy groups on ring C in positions 2 and 6 and the
probable steric hindrance of the methoxy group with the
carbonyl of the carboxamide linker likely induces an out-of-plane
conformation, leading to the lack of H-bonding activity of the
methoxy group with the amide NH.
The two regioisomers 4b and 5b, bearing in the A-ring a 3- or a
4-pyridinyl ring, respectively, resulted close log P values (2.84
and 3.13, respectively) and likewise of P-gp inhibitory activity
(0.20 and 0.31 μM, respectively). On the other hand, 5b
inhibited P-gp, but not MRP1 (IC50 > 100 μM), while 4b was
much more active on MRP1, showing an IC50 of 3.38 μM.
Concerning the inhibitory activity of compounds reported so
far, a dependence from lipophilicity has never been described.
Interestingly, MRP1 inhibitory activities, measurable as IC50 only
for 11 compounds (see Table 1), were poorly correlated with the
experimental log D7.4 values.
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Figure 9. Co-administration of doxorubicin with compounds 2b, 4b, 4c, and 5b. Antiproliferative effect of 10 μM doxorubicin (white bars) at 48 h in
MCF7 adr cell line. In comparison, P-gp active compounds (1, 10, and 25 μM) were administered for 24 h (black bars). After washing, compounds were
co-administered with doxorubicin (10 μM) at the same concentration for 48 h (gray bars).
Figure 10. ATP consumption in MDCK-MDR1 by compounds 2b, 4b, 4c, and 5b. Control of untreated cells (white bars); cell treatment with 1, 10, 50,
and 100 μM of tested compounds (black bars).
It is noteworthy to point out that compound 9a is the only
compound of this series showing a slight selectivity for MRP1
(11.10 vs 18.50 μM). On the other hand, its parent compound
8a, despite similar log D7.4 values, showed the usual trend (P-gp
selectivity), with IC50 of 15.8 and 2.76 μM on MRP1 and P-gp,
respectively.
process. The relationship between modulation activity of P-gp
and lipophilicity was observed and confirmed in many series of
effective inhibitors. However, in many cases, high activities were
shown by compounds with prohibitive solubility and log D
values, compromising a possible hit-to-lead development. In this
work, we demonstrate that the careful selection of regioisomers,
able to form IMHBs, can result in very promising hits; 5 of 21
very small molecules, soluble in an aqueous medium at
concentrations 20 10 mg/L (2b) to 250 50 mg/L (3b),
showed submicromolar IC50 values against P-gp, and three of
them are also active toward MRP1 with IC50 in the one-digit
micromolar range. Finally, both MD simulations and DFT
calculations suggest the presence of a unique conformation of the
CONCLUSIONS
■
P-Glycoprotein is an unusual transporter, and various aspects of
its function appear to be modulated by the lipid environment in
novel and complex ways. The lipophilicity of potential inhibitors
should therefore be carefully considered in the molecular design
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Hz), 7.06−6.96 (m, 2H), 6.79−6.70 (m, 1H), 6.57 (d, 1H, J = 8.7 Hz),
4.58 (br s, 2H). LRMS (ESI) m/z 204.8 [M − H]−.
hit 4b characterized by a very stable IMHB, thus providing a
rationale for the different activities reported for the regioisomers
4a and 4b. The strength of such interaction is here proved to be a
sensitive parameter for a soft modulation of the P-gp response.
For instance, the 2,4,5-trimethoxybenzamide derivatives 3b, 4b,
and 5b display the highest activity and are also characterized by
the strongest IMHB.
3,3′-Bipyridin-6-amine (3).33 Purified by chromatography (dichloro-
methane/methanol, 90:10). Yield 50%; mp 118.3−122.5 °C. 1H NMR
(CDCl3) δ: 8.79−8.76 (m, 1H), 8.58−8.54 (m, 1H), 8.33−8.30 (m,
1H), 7.83−7.77 (m, 1H), 7.67 (dd, 1H, J = 8.5, 2.4 Hz), 7.37−7.31 (m,
1H), 6.61 (d, 1H, J = 8.4 Hz), 4.55 (br s, 2H). LRMS (ESI) m/z 169.9
[M − H]−.
2-Fluoro-4-(pyridin-3-yl)aniline (4). Purified by chromatography
(hexane/ethyl acetate, 50:50). Yield 90%; mp 92.5−95.4 °C. 1H NMR
(CDCl3) δ: 8.79−8.76 (m, 1H), 8.54−8.50 (m, 1H), 7.81−7.76 (m,
1H), 7.33−7.16 (m, 3H), 6.90−6.82 (m, 1H), 3.85 (br s, 2H). LRMS
(ESI) m/z 186.9 [M − H]−.
EXPERIMENTAL SECTION
■
Chemistry. High analytical grade chemicals and solvents were
purchased 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
TLC on silica gel 60 F254 plates or crystallization. 1H NMR spectra were
recorded in DMSO-d6 or CDCl3 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
performed with an electrospray interface ion trap mass spectrometer
(1100 series LC/MSD trap system Agilent, Palo Alto, CA). In all cases,
spectroscopic data are in agreement with known compounds and
assigned structures. The known compounds 1a and 1b (Figure 1) were
already published in our previous work.14 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). clogP
values of the data set were computed by using the Bio-Loom software
package version 1.5; ALOGPS values were computed by using software
developed by Virtual Computational Chemistry Laboratory v. 2.1; log P
values were computed using ACD/Laboratories version 6.00; log D
values at pH 7.4 were obtained by octanol/PBS buffer partitioning
analysis using chemiluminescent nitrogen detection method performed
by Analiza Incorporated.
Chromatographic Settings. Log k′ values and purity determi-
nations were carried out using a Zorbax 300SB-C18 4.6 mm × 250 mm,
with 5 μm size particles, built on a Waters double pump HPLC system in
isocratic conditions. Injection volumes were 5 μL, flow rate was 1 mL/
min, and detection was performed with UV (λ = 230 and 254 nm).
Samples were prepared by dissolving 0.1 mg/mL of the solute in 10%
(v/v) DMSO and 90% (v/v) methanol. Retention times (tr) were
measured at least from three separate injections, and dead time (t0) was
the retention time of deuterated water. The mobile phase was filtered
through a Nylon-66 membrane 0.45 μm (Supelco, USA) before use. For
each reference compound, the average retention time (tr) of three
consecutive injections of 5 μL of sample was used to calculate the log k′
values (log k′ = log[(tr − t0)/t0]). The eluent consisted of three different
mixtures of methanol and PBS buffer at pH 7.4: the first measurement
was 80% (v/v) methanol and 20% (v/v) of buffer, 70% (v/v) methanol
and 30% (v/v) of buffer, and 60% (v/v) methanol and 40% (v/v) of
buffer.
2-Fluoro-4-(pyridin-4-yl)aniline (5). Purified by chromatography
1
(ethyl acetate/hexane, 60:40). Yield 40%; mp 157.4−159.5 °C. H
NMR (DMSO-d6) δ: 8.51−8.47 (m, 2H), 7.64−7.50 (m, 3H), 7.41 (dd,
1H, J = 8.1, 1.8 Hz), 6.86−6.80 (m, 1H), 5.56 (br s, 2H). LRMS (ESI)
m/z 186.9 [M − H]−.
3-Fluoro-4′-methoxybiphenyl-4-amine (6).34 Purified by chroma-
tography (chloroform/hexane, 80:20). Yield 30%; mp 134.8−136.5 °C.
1H NMR (CDCl3) δ: 7.47−7.41 (m, 2H), 7.21−7.17 (m, 1H), 7.16−
7.13 (m, 1H), 6.97−6.92 (m, 2H), 6.86−6.78 (m, 1H), 3.84 (s, 3H),
3.73 (br s, 2H). LRMS (ESI) m/z 215.8 [M − H]−.
ii. Procedure for the Preparation of N-(2-Fluoro-4-(pyridin-3-
yl)phenyl)-3,4-dimethoxybenzenesulfonamide (7j) (Scheme 1). The
aniline derivative 4 (0.50 mmol) and 3,4-dimethoxybenzene-1-sulfonyl
chloride (0.75 mmol) were dissolved in a minimum volume of THF dry
and stirred at room temperature for 7 h. The mixture was evaporated
under vacuum to dryness and the residue purified by chromatography
(ethyl acetate/hexane, 60:40) to obtain the desired product.
Yield 61%; mp 173.4−176.3 °C. 1H NMR (DMSO-d6) δ: 10.17 (s,
1H), 8.85 (m, 1H), 8.53 (d, 1H, J = 4.7 Hz), 8.03 (d, 1H, J = 8.2 Hz),
7.59 (d, 1H, J = 12.1 Hz), 7.51−7.40 (m, 2H), 7.38−7.29 (m, 2H), 7.27
(d, 1H, J = 1.8 Hz), 7.05 (d, 1H, J = 8.7 Hz), 3.78 (s, 3H), 3.72 (s, 3H).
LRMS (ESI) m/z 386.8 [M − H]−.
iii. General Procedures for the Preparation of Compounds 2a, 2b,
3a, 3b, 4a−4i, 5a, 5b, and 6b (Scheme 1). For compounds 2a, 3a, 4a,
4e, 4f, 4h, and 5a, the chosen aniline (0.50 mmol) and the appropriate
benzoyl chloride (0.75 mmol) were dissolved in the minimum volume of
THF dry and stirred at room temperature for 6 h. The suspension
obtained was filtered and the residue purified by crystallization, affording
the desired product.
N-(5-(3,5-Difluorophenyl)pyridin-2-yl)-3,4,5-trimethoxybenza-
mide (2a). Crystallized from ethanol. Yield 23%; mp 171.5−173.2 °C.
1H NMR (DMSO-d6) δ: 8.79 (s, 1H), 8.81 (d, 1H, J = 1.9 Hz), 8.31−
8.26 (m, 2H), 7.57 (dd, 2H, J = 7.2, 1.9 Hz), 7.42 (s, 2H), 7.25−7.22 (m,
1H), 3.86 (s, 6H), 3.72 (s, 3H). LRMS (ESI) m/z 398.9 [M − H]−.
N-([3,3′-Bipyridin]-6-yl)-3,4,5-trimethoxybenzamide (3a). Purified
by preparative TLC (dichloromethane/methanol, 94:6). Yield 10%; mp
134.3−135.9 °C. 1H NMR (DMSO-d6) δ: 10.99 (s, 1H), 8.97 (d, 1H, J =
2.0 Hz), 8.80 (d, 1H, J = 2.0 Hz), 8.59 (d, 1H, J = 6.0 Hz), 8.32 (d, 1H, J
= 8.8 Hz), 8.23 (dd, 1H, J = 8.8, 2.5 Hz), 8.17 (dd, 1H, J = 6.0, 2.0 Hz),
7.52−7.46 (m, 1H), 7.42 (s, 2H), 3.87 (s, 6H), 3.72 (s, 3H). LRMS
(ESI) m/z 363.9 [M − H]−.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)-3,4,5-trimethoxybenzamide
(4a). Crystallized from methanol. Yield 81%; mp 222.4−224.2 °C. 1H
NMR (DMSO-d6) δ: 10.23 (s, 1H), 9.19 (d, 1H, J = 2.5 Hz), 8.78 (dd,
1H, J = 5.4, 1.2 Hz), 8.64 (d, 1H, J = 8.1 Hz), 7.91−7.89 (m, 1H), 7.87 (s,
1H), 7.80−7.71 (m, 2H), 7.34 (s, 2H), 3.89 (s, 6H), 3.73 (s, 3H). LRMS
(ESI) m/z 383.0 [M + H]+.
i. General Procedure for the Suzuki−Miyaura Cross-Coupling
Reaction: Preparation of Compounds 1−6 (Scheme 1). A suspension
of the chosen aniline (5.0 mmol), the appropriated boronic acid (7.5
mmol), and Pd(PPh3)4 (0.50 mmol) in a 2 M aqueous solution of
K2CO3 (7.5 mL) and 1,4-dioxane (30 mL) was heated at 100 °C for 3 h
under stirring. After cooling, the solvent was evaporated under vacuum
to dryness and the obtained residue was treated with dichloromethane
or ethyl acetate. The suspension was filtered on Celite or silica gel, and
the resulting residue was purified by chromatography.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)-3,4-dimethoxybenzamide
1
(4e). Crystallized from methanol. Yield 84%; mp 256.6−259.1 °C. H
NMR (DMSO-d6) δ: 10.10 (s, 1H), 9.12 (s, 1H), 8.72 (d, 1H, J = 5.0
Hz), 8.52−8.49 (m, 1H), 7.87−7.61 (m, 5H), 7.57 (d, 1H, J = 1.8 Hz),
7.09 (d, 1H, J = 8.7 Hz), 3.83 (s, 6H). LRMS (ESI) m/z 351.0 [M −
H]−.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)-3,5-dimethoxybenzamide
5-(3,5-Difluorophenyl)pyridin-2-amine (2).32 Purified by chroma-
tography (hexane/ethyl acetate, 60:40). Yield 45%; mp 107.4−108.9 °C.
1H NMR (CDCl3) δ: 8.29 (d, 1H, J = 2.4 Hz), 7.62 (dd, 1H, J = 8.5, 2.4
(4f). Crystallized from methanol. Yield 98%; mp 235.5−237.5 °C. H
1
NMR (DMSO-d6) δ: 10.28 (s, 1H), 9.23 (s, 1H), 8.81 (d, 1H, J = 5.2
Hz), 8.73 (d, 1H, J = 8.3 Hz), 7.99−7.86 (m, 2H), 7.81−7.71 (m, 2H),
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7.15 (d, 2H, J = 2.1 Hz), 6.74−6.71 (m, 1H), 3.81 (s, 6H). LRMS (ESI)
m/z 353.0 [M + H]+.
10.38 (m, 1H), 8.66−8.59 (m, 1H), 7.83 (s, 1H), 7.55−7.49 (m, 2H),
7.39−7.28 (m, 2H), 7.00−6.95 (m, 2H), 6.59 (s, 1H), 4.08 (s, 3H), 3.98
(s, 3H), 3.94 (s, 3H), 3.85 (s, 3H). LRMS (ESI) m/z 434.2 [M + Na]+.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)nicotinamide (4i). A suspen-
sion of 4 (0.50 mmol), nicotinoyl chloride hydrochloride (0.75 mmol),
DMAP (0.50 mmol), and Et3N (1.0 mmol) in 1,4-dioxane (3.0 mL) was
heated at 100 °C under stirring for 2 h, then the mixture was evaporated
to dryness and the desired compound was purified by chromatography
(dichloromethane/methanol, 95:5). Yield 77%; mp 165.4−166.7 °C. 1H
NMR (DMSO-d6) δ: 10.44 (s, 1H), 9.12 (d, 1H, J = 1.7 Hz), 8.95 (d,
1H, J = 1.7 Hz), 8.77 (dd, 1H, J = 4.7, 1.7 Hz), 8.57 (dd, 1H, J = 4.7, 1.7
Hz), 8.31 (dt, 1H, J = 8.5, 1.7 Hz) 8.16−8.12 (m, 1H), 7.80−7.74 (m,
2H), 7.66−7.54 (m, 2H), 7.54−7.47 (m, 1H). LRMS (ESI) m/z 291.9
[M − H]−.
iv. General Procedure for Preparation of Compounds 10a and 10b
(Scheme 1). The chosen benzamide 4a or 4b (0.30 mmol) and sodium
hydride (0.33 mmol) were suspended in DMF dry (15 mL) at 0 °C and
stirred for 30 min. Methyl iodide (0.33 mmol) was added, and the
reaction was allowed to warm to room temperature. After 3 h, the
reaction was quenched with ice. The organic phase was extracted with
dichloromethane, dried over anhydrous sodium sulfate, and evaporated
under vacuum to dryness. The residue was purified by chromatography,
affording the desired product.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)-3,4,5-trimethoxy-N-methyl-
benzamide (10a). Purified by chromatography (ethyl acetate/hexane,
80:20). Yield 68%; mp 97.8−102.2 °C. 1H NMR (CDCl3) δ: 8.82−8.76
(m, 1H), 8.67−8.61 (m, 1H), 7.85−7.80 (m, 1H), 7.45−7.36 (m, 1H),
7.35−7.25 (m, 2H), 7.22−7.13 (m, 1H), 6.63 (s, 2H), 3.79 (s, 3H), 3.69
(s, 6H), 3.45 (s, 3H). LRMS (ESI) m/z 419.2 [M + Na]+.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)-2,4,5-trimethoxy-N-methyl-
benzamide (10b). Purified by chromatography (ethyl acetate/hexane,
90:10). Yield 19%; mp 102.7−105.3 °C. 1H NMR (CDCl3) δ: 8.76 (s,
1H), 8.60 (d, 1H, J = 4.4 Hz), 7.78 (d, 1H, J = 8.3 Hz), 7.40−7.25 (m,
2H), 7.17−7.06 (m, 2H), 6.91 (s, 1H), 6.20 (s, 1H), 3.80 (s, 6H), 3.61
(s, 3H), 3.44 (s, 3H). LRMS (ESI) m/z 419.2 [M + Na]+.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)benzamide (4h). Crystallized
1
from methanol. Yield 98%; mp 266.2−268.3 °C. H NMR (DMSO-
d6) δ: 10.30 (s, 1H), 9.20 (s, 1H), 8.79 (d, 1H, J = 4.4 Hz), 8.68 (d, 1H, J
= 8.0 Hz), 7.99 (d, 2H, J = 6.2 Hz), 7.94−7.90 (m, 1H), 7.86−7.84 (m,
1H), 7.80 (d, 1H, J = 8.0 Hz), 7.75−7.70 (m, 1H), 7.63−7.50 (m, 3H).
LRMS (ESI) m/z 293.0 [M + H]+.
N-(2-Fluoro-4-(pyridin-4-yl)phenyl)-3,4,5-trimethoxybenzamide.
(5a). Crystallized from methanol. Yield 54%; mp 265.3−267.2 °C. 1H
NMR (DMSO-d6) δ: 10.35 (s, 1H), 8.91 (d, 2H, J = 6.6 Hz), 8.35 (d,
2H, J = 6.6 Hz), 8.08 (dd, 1H, J = 12.1, 1.9 Hz), 7.96−7.83 (m, 2H), 7.34
(s, 2H), 3.86 (s, 6H), 3.73 (s, 3H). LRMS (ESI) m/z 383.0 [M + H]+.
For compounds 2b, 3b, 4b−d, 4g, 5b, and 6b, the starting benzoic
acid (0.75 mmol) was suspended under argon in thionyl chloride (1.0
mL) and stirred for 3 h at room temperature. The unreacted excess of
thionyl chloride was removed under nitrogen flow to afford the
corresponding benzoyl chloride. The solid obtained and the appropriate
aniline (0.50 mmol) were dissolved in the minimum volume of THF dry
and stirred at room temperature for 6 h. The suspension obtained was
filtered and the residue was purified by crystallization, affording the
desired product.
N-(5-(3,5-Difluorophenyl)pyridin-2-yl)-2,4,5-trimethoxybenza-
mide (2b). Crystallized from methanol. Yield 37%; mp 248.2−252.9 °C.
1H NMR (DMSO-d6) δ: 10.54 (s, 1H), 8.76 (d, 1H, J = 2.2 Hz), 8.35 (d,
1H, J = 8.5 Hz), 8.24 (d, 1H, J = 8.5, 2.2 Hz), 7.59−7.51 (m, 3H), 7.30−
7.20 (m, 1H), 6.87 (s, 1H), 4.06 (s, 3H), 3.90 (s, 3H), 3.77 (s, 3H).
LRMS (ESI) m/z 423.1 [M + Na]+.
N-([3,3′-Bipyridin]-6-yl)-2,4,5-trimethoxybenzamide (3b). Crystal-
lized from methanol. Yield 68%; mp 209.6−212.1 °C. 1H NMR
(DMSO-d6) δ: 10.53 (s, 1H), 8.95 (d, 1H, J = 2.3 Hz), 8.73 (d, 1H, J =
2.3 Hz), 8.58 (dd, 1H, J = 5.0, 1.5 Hz), 8.37 (d, 1H, J = 8.8 Hz), 8.23 (dd,
1H, J = 8.8, 2.5 Hz), 8.15−8.11 (m, 1H), 7.56 (s, 1H), 7.53−7.47 (m,
1H), 6.88 (s, 1H), 4.07 (s, 3H), 3.90 (s, 3H), 3.77 (s, 3H). LRMS (ESI)
m/z 388.2 [M + Na]+.
v. Procedure for the Preparation of N-(3-Amino-3′,5′-difluoro-
[1,1′-biphenyl]-4-yl)-3,4,5-trimethoxybenzamide (8a) (Scheme 1). A
suspension of the benzamide 1a (0.20 mmol) in absolute ethanol (25
mL) was stirred at room temperature with a catalytic amount of Pd/C
(5% w/w) under H2 atmosphere. After 2 h, the suspension was filtered
on syringe filter (Filtek RC, pore size 0.20 μm) to remove the catalyst.
The filtrate was evaporated under vacuum to dryness affording the
desired product.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)-2,4,5-trimethoxybenzamide
(4b). Crystallized from methanol. Yield 79%; mp 256.7−260.1 °C. 1H
NMR (DMSO-d6) δ: 10.41 (d, 1H, J = 2.7 Hz), 9.18 (d, 1H, J = 2.5 Hz),
8.76 (d, 1H, J = 5.4 Hz), 8.70 (d, 1H, J = 8.4 Hz), 8.54 (t, 1H, J = 8.4 Hz),
7.93−7.87 (m, 2H), 7.75 (dd, 1H, J = 8.7, 1.5 Hz), 7.56 (s, 1H), 6.88 (s,
1H), 4.07 (s, 3H), 3.90 (s, 3H), 3.77 (s, 3H). LRMS (ESI) m/z 405.0 [M
+ Na]+.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)-2,3,4-trimethoxybenzamide
1
1
Yield 91%; mp 219.8−222.2 °C. H NMR (DMSO-d6) δ: 9.70 (s,
(4c). Crystallized from ethanol. Yield 56%; mp 236.7−239.0 °C. H
1H), 7.39−7.13 (m, 7H), 6.97 (dd, 1H, J = 8.2, 1.9 Hz), 5.04 (br s, 2H),
NMR (DMSO-d6) δ: 10.47 (s, 1H), 9.14 (s, 1H), 8.75−8.73 (m, 1H),
8.59−8.52 (m, 1H), 8.46 (t, 1H, J = 8.4 Hz), 7.91 (d, 1H, J = 12.9 Hz),
7.86−7.80 (m, 1H), 7.75 (d, 1H, J = 8.9 Hz), 7.74−7.72 (m, 1H), 7.02
(d, 1H, J = 8.9 Hz), 4.02 (s, 3H), 3.88 (s, 3H), 3.80 (s, 3H). LRMS (ESI)
m/z 405.1 [M + Na]+.
3.85 (s, 6H), 3.71 (s, 3H). LRMS (ESI) m/z 412.9 [M − H]−.
vi. Procedure for the Preparation of N-(3-(2-Acetamidopropana-
mido)-3′,5′-difluoro-[1,1′-biphenyl]-4-yl)-3,4,5-trimethoxybenza-
mide (9a) (Scheme 1). The benzamide 8a (0.18 mmol) was dissolved in
THF dry and N-acetyl-L-alanine (0.15 mmol), EDC (0.18 mmol), HOBt
(0.18 mmol), and TEA (0.36 mmol) were added. The mixture was
stirred at room temperature for 72 h. The suspension obtained was
filtered and the filtrate evaporated under vacuum to dryness. The desired
product was purified by chromatography (dichloromethane/methanol,
98:2). Yield 32%; mp 196.4−200.9 °C. 1H NMR (DMSO-d6) δ: 9.93 (s,
1H), 9.58 (s, 1H), 8.30 (d, 1H, J = 6.9 Hz), 7.95 (d, 1H, J = 1.9 Hz), 7.70
(d, 1H, J = 8.5 Hz), 7.61 (dd, 1H, J = 6.0 Hz), 7.41 (m, 2H), 7.32 (s, 2H),
7.27−7.21 (m, 1H), 4.37−4.33 (m, 1H), 3.86 (s, 6H), 3.72 (s, 3H), 1.74
(s, 3H), 1.30 (d, 3H, J = 7.2 Hz). LRMS (ESI) m/z 526.2 [M − H]−.
Computational Studies. Classical molecular dynamics (MD)
simulations were carried out with the NAMD 2.9 software.35 For
compounds 4a and 4b, the CHARMM General Force Field (CGenFF)
force field parameters36 were used and the atomic charges were
calculated with the Restrained Electrostatic Potential (RESP)
approach37 using Jaguar 7.6 software.38 TIP3P model39 was used for
explicit water solvent, while chloroform was simulated using the OPLS-
AA40 force field. A time step of 1 fs was used; data were sampled every
100 fs. The investigated systems were first equilibrated for 0.5 ns at 310
K; then, data were sampled over a 5 ns trajectory at the same
temperature. All simulations were performed in the canonical ensemble.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)-2,4,6-trimethoxybenzamide
(4d). Crystallized from methanol. Yield 10%; mp 189.6−191.0 °C. 1H
NMR (DMSO-d6) δ: 9.91 (s, 1H), 8.92 (d, 1H, J = 2.2 Hz), 8.55 (dd,
1H, J = 4.7, 1.6 Hz), 8.14−8.08 (m, 2H), 7.67 (dd, 1H, J = 12.1, 1.6 Hz),
7.57 (d, 1H, J = 8.2 Hz), 7.47 (dd, 1H, J = 8.2, 4.7 Hz), 6.27 (s, 2H), 3.74
(s, 3H), 3.30 (s, 6H). LRMS (ESI) m/z 405.2 [M + Na]+.
N-(2-Fluoro-4-(pyridin-3-yl)phenyl)-4-methoxybenzamide (4g).
Crystallized from methanol. Yield 69%; mp 271.2−272.7 °C. 1H
NMR (DMSO-d6) δ: 10.12 (s, 1H), 9.17 (s, 1H), 8.77 (d, 1H, J = 5.2
Hz), 8.63 (d, 1H, J = 8.1 Hz), 7.98 (d, 2H, J = 8.5 Hz), 7.92−7.82 (m,
2H), 7.78 (d, 1H, J = 8.1 Hz), 7.70 (d, 1H, J = 8.4 Hz), 7.06 (d, 2H, J =
8.5 Hz), 3.83 (s, 3H). LRMS (ESI) m/z 323.2 [M + H]+.
N-(2-Fluoro-4-(pyridin-4-yl)phenyl)-2,4,5-trimethoxybenzamide
(5b). Crystallized from methanol. Yield 58%; mp 247.7−251.5 °C. 1H
NMR (DMSO-d6) δ: 10.52 (d, 1H, J = 3.0 Hz), 8.84 (d, 2H, J = 6.0 Hz),
8.60 (t, 1H, J = 8.5 Hz), 8.24 (d, 2H, J = 6.0 Hz), 8. 09 (dd, 1H, J = 11.3,
1.9 Hz), 7.93 (d, 1H, J = 11.3 Hz), 7.55 (s, 1H), 6.89 (s, 1H), 4.09 (s,
3H), 3.90 (s, 3H), 3.78 (s, 3H). LRMS (ESI) m/z 405.1 [M + Na]+.
N-(3-Fluoro-4′-methoxy-[1,1′-biphenyl]-4-yl)-2,4,5-trimethoxy-
benzamide (6b). Purified by chromatography (hexane/ethyl acetate,
50:50). Yield 87%; mp 157.8−160.3 °C. 1H NMR (CDCl3) δ: 10.40−
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All DFT calculations were carried out using Jaguar 7.6 software.
Geometry optimizations and relaxed scan calculations were performed
at the B3LYP/6-31G(d)41 level. Harmonic vibrational frequency
analyses were applied in order to confirm the nature of the localized
stationary states.
Biology. Materials. ATPlite 1 step kit and CulturePlate 96-well
plates were purchased from PerkinElmer Life and Analytical Sciences
(Boston, MA, USA). Cell culture reagents were obtained from
EuroClone (Milan, Italy). Calcein-AM, MTT (3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide), and doxorubicin were obtained
from Sigma-Aldrich (Milan, Italy).
Cell Cultures. Human MCF7adr breast adenocarcinoma cells
(resistant to doxorubicin) were kindly provided by Prof. G. Zupi
(IRE, Rome, Italy), and MDCK-MDR1 and MDCK-MRP1 cells were a
gift of Prof. P. Borst (NKI-AVL Institute, Amsterdam, Nederland).
Caco-2 cells were kindly donated by IRCCS “S. De Bellis” (Castellana
Grotte, Bari, Italy). MCF7adr cells were grown in RPMI 1640
supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/
mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at
37 °C with a 5% CO2 atmosphere. MDCK-MDR1, MDCK-MRP1, and
Caco-2 cells were grown in DMEM high glucose supplemented with
10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100
μg/mL streptomycin in a humidified incubator at 37 °C with a 5% CO2
atmosphere.
Co-administration Assay. The co-administration assay with
doxorubicin was performed in MCF7adr cells at 72 h.42 On day 1,
25000 cells/well were seeded into 96-well plates in a volume of 100 μL.
On day 2, three drug concentrations (1, 10, and 25 μM) were added. On
day 3, the medium was removed and the three drugs concentrations
were added alone and in combination with doxorubicin 10 μM. After the
established incubation time with drugs, MTT (0.5 mg/mL) was added
to each well, and after 3−4 h incubation at 37 °C, the supernatant was
removed. The formazan crystals were solubilized using 100 μL of
DMSO/EtOH (1:1), and the absorbance values at 570 and 630 nm were
determined on the microplate reader Victor 3 from PerkinElmer Life
Sciences.
2 × 104 cells/well. The plate was incubated O/N 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 2 h in a
humidified atmosphere 5% CO2 at 37 °C. Then, 50 μL of mammalian
cell lysis solution was added to all wells and the plate stirred for 5 min in
an orbital shaker. In all wells, 50 μL of substrate solution was added and
the plate stirred for 5 min in an orbital shaker. The plate was dark
adapted for 10 min, and the luminescence was measured on the
microplate reader Victor 3 from PerkinElmer Life Sciences.
Permeability Experiment. The preparation of Caco-2 monolayer has
been previously reported by Leopoldo et al.44 Apical to basolateral (Papp
,
A−B) and basolateral to apical (Papp, B−A) permeability of drugs were
measured at 120 min and at various drug concentrations (1−100 μM).
Drugs were dissolved in Hanks’ Balanced Salt Solution (HBSS, pH 7.4)
and sterile filtered. After 21 days of cell growth, the medium was
removed from filter wells and from the receiver plate. The filter wells
were filled with 75 μL of fresh HBSS buffer and the receiver plate with
250 μL per well of the same buffer. 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 added to the filter well
(75 μL). HBSS without the drug was added to the receiver plate (250
μL). The plates were incubated at 37 °C for 120 min. After incubation
time, samples were removed from the apical (filter well) and basolateral
(receiver plate) side of the monolayer and then were stored in a freezer
(−20 °C) pending analysis. The concentration of compounds was
measured using UV spectroscopy. The apparent permeability (Papp), in
units of nm per s, was calculated using the following equation:
⎛
⎝
⎞
⎠
[drug]acceptor
[drug]initial
⎛
⎜
⎞
⎟
VA
⎜
⎜
⎟
⎟
P
=
×
app
⎝
⎠
Area × time
where VA is the volume (in μL) in the acceptor well; Area is the surface
area of the membrane (0.11 cm2 of the well); time is the total transport
time in seconds (7200 s); [drug]acceptor is the concentration of the drug
measured by ESI-MS analyses or UV spectroscopy; [drug]initial is the
initial drug concentration (1 × 10−4 M) in the apical or basolateral wells.
Calcein-AM Experiment. These experiments were carried out as
described by Feng et al. with minor modifications.27 Calcein-AM is a
profluorescent probe and is a P-gp substrate. In cells overexpressing P-
gp or MRP1, Calcein-AM is not able to permeate cell membrane
whereas when the efflux pump is not present or is inhibited, the probe
enters living cells and is converted to fluorescent Calcein by intracellular
esterases. Calcein is not able to diffuse through the membrane because it
is hydrophilic and is not a P-gp or MRP1 substrate; thus, calcein
accumulates in cells when the pump is blocked. Therefore, the
fluorescent signal is directly correlated to the amount of P-gp or
MRP1 interaction. MDCK-MDR1 or MDCK-MRP1 cells (50000 cells
per well) were seeded into black CulturePlate 96-well plates with 100 μL
of medium and allowed to become confluent overnight. Then 100 μL of
test compounds were solubilized in culture medium and added to
monolayers. The 96-well plates 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 the 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 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. IC50 values
were determined by fitting the fluorescence increase percentage versus
log[dose].
AUTHOR INFORMATION
Corresponding Authors
*For S.C.: phone, +39-080-5442090; fax, +39-080-5442230; E-
mail, saverio.cellamare@uniba.it.
*for N.A.C.: phone, +39-080-5442727; fax, +39-080-5442231;
E-mail, nicolaantonio.colabufo@uniba.it.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
MDR, multidrug resistance; ABC, ATP-binding cassette; P-gp,
ABCB1, MDR1, P-glycoprotein; MRP1, ABCC1, human
multidrug resistance associate-protein 1; lipophilicity; IMHB,
intramolecular hydrogen bond; DFT, density functional theory
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