Discovery of Encequidar, First-in-Class Intestine Specific P-glycoprotein Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.0c01826

Many chemotherapeutics, such as paclitaxel, are administered intravenously as they suffer from poor oral bioavailability, partly because of efflux mechanism of P-glycoprotein in the intestinal epithelium. To date, no drug has been approved by the U.S. Foo

Full text of DOI:10.1021/acs.jmedchem.0c01826

pubs.acs.org/jmc
Drug Annotation
Discovery of Encequidar, First-in-Class Intestine Specific
P‑glycoprotein Inhibitor
Michael P. Smolinski,* Sameer Urgaonkar, Laura Pitzonka, Murray Cutler, GwanSun Lee,
Kwee Hyun Suh, and Johnson Y. N. Lau
Cite This: J. Med. Chem. 2021, 64, 3677−3693
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ABSTRACT: Many chemotherapeutics, such as paclitaxel, are administered
intravenously as they suffer from poor oral bioavailability, partly because of efflux
mechanism of P-glycoprotein in the intestinal epithelium. To date, no drug has
been approved by the U.S. Food and Drug Administration (FDA) that selectively
blocks this efflux pump. We sought to identify a compound that selectively inhibits
P-glycoprotein in the gastrointestinal mucosa with poor oral bioavailability, thus
eliminating the issues such as bone marrow toxicity associated with systemic
inhibition of P-glycoprotein. Here, we describe the discovery of highly potent, selective, and poorly orally bioavailable P-glycoprotein
inhibitor 14 (encequidar). Clinically, encequidar was found to be well tolerated and minimally absorbed; and importantly, it enabled
the oral delivery of paclitaxel.
Clinically, systemic exposure of these excipients can result in
serious side effects such as hypersensitivity reactions, hyper-
lipidemia, sometimes irreversible sensory neuropathy, and
cumulative fluid retention.12 To reduce these risks, often
patients are premedicated with corticosteroids and antihist-
amines. Oral administration of these drugs is expected to
eliminate or significantly reduce these side effects without
necessitating the use of premedications.
The poor oral absorption of paclitaxel and docetaxel can be
attributed to three important underlying factors: (a) when
given orally these drugs are recognized by P-gp in the intestinal
cells of the gastrointestinal tract and undergo drug efflux back
out into the intestinal lumen; (b) these drugs have poor
aqueous solubility, and (c) these drugs undergo cytochrome
P450-mediated (specifically CYP3A4/5) intestinal metabo-
lism.13 Because of the direct role of intestinal P-gp in oral
absorption, it has been the subject of much interest as a target
for boosting oral bioavailability of substrates of P-gp. One of
the early papers describing the potential of intestinal P-gp in
oral bioavailability enhancement of a substrate drug appeared
in 1993 wherein quinidine was used as a P-gp inhibitor to
improve the absorption of etoposide, a P-gp substrate, in rats.14
This finding led to a renaissance in the development of
inhibitors targeting P-gp.
INTRODUCTION
■
P-glycoprotein (P-gp), a 170 kDa membrane protein encoded
by the mdr1 gene in humans and belonging to the class of ABC
(ATP-binding cassette) transporters, was identified by Victor
Ling in 1976 as a permeability alteration protein in colchicine-
resistant Chinese hamster ovary cells.1,2 Since then, P-gp has
shown to be expressed in various healthy and malignant tissues
where it serves a diversity of functions.3−6 The presence of P-
gp on the apical surface of epithelial cells of the small intestine
promotes the efflux of both endogenous and xenobiotic
substrates. Similarly, localization of P-gp on the biliary
canalicular membrane of hepatocytes and on the epithelial
cells of proximal convoluted tubules of nephrons facilitates
biliary and renal excretion of its substrates, respectively. Its
existence in the endothelial cells of the blood-brain barrier
provides protection to the brain against circulating toxins in
the blood. Moreover, overexpression of P-gp in cancer cells is
associated with drug resistance phenotype compromising the
efficacy of anticancer drugs which are substrates of P-gp.7−9
Oral dosing is a preferred route of administration,10 more so
during a global pandemic, for patients due to ease of
administration and reduction in hospital/clinic visits. The
oral route enables development of novel dosing regimens, such
as metronomic, and expands treatment options when used
alone or in combination with other anticancer drugs. One of
the most widely used classes of chemotherapeutic drugs,
taxanes, such as paclitaxel and docetaxel, are administered
intravenously as they suffer from poor oral bioavailability.11
While providing transformative tumor shrinkage, these drugs
are formulated in vehicles containing nonionic surfactants such
as polyoxyl-35 castor oil (for paclitaxel) and tween 80 (for
docetaxel) to allow them to be administered intravenously.
Received: December 4, 2020
Published: March 17, 2021
https://doi.org/10.1021/acs.jmedchem.0c01826
© 2021 American Chemical Society
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Drug Annotation
Figure 1. Representative structures of third generation P-gp inhibitors.
Figure 2. Layout of structural modification proposed to limit the systemic absorption of P-gp inhibitor tariquidar.
The discovery of first-generation P-gp inhibitors was
serendipitous and included the calcium channel blocker
verapamil and the immunosuppressive agent cyclosporin A.15
Particularly, the use of oral cyclosporin A, in the landmark
proof of concept study conducted by Terwogt and co-
workers,16,17 improved the oral bioavailability of paclitaxel up
to 10-fold (47% versus 4% without cyclosporin A) in a clinical
setting. This study clinically validated intestinal P-gp as a target
for improving oral bioavailability of paclitaxel.
Paclitaxel, a well characterized P-gp substrate, is listed by the
World Health Organization (WHO) as an essential medicine
for the treatment of cancer. Remarkably, since its approval by
the U.S. Food and Drug Administration (FDA) in 1992 for
ovarian cancer, it has grown leaps and bounds and has been
established as one of the mainstay drugs in the fight against
cancer. While paclitaxel is administered intravenously either as
Taxol or as nanoparticle albumin-bound paclitaxel, also known
as nab-paclitaxel (Abraxane), transforming this mode of
administration into oral has remained a challenge in the
oncology community. However, the breakthrough result
discussed above paved the path to discover a more selective
P-gp inhibitor suitable for facilitating the oral absorption of P-
gp substrates, such as paclitaxel.
The initial promising result with cyclosporin A was
tempered knowing that the original intended pharmacology
of cyclosporin A being immunosuppressive was undesirable in
a paclitaxel-based cancer treatment regimen, as this could
compromise the immune system when needed most. Second-
generation P-gp inhibitors, for example, dexverapamil (the less
potent calcium antagonist R-enantiomer of verapamil)18 and
valspodar19,20 (a nonimmunosuppressive derivative of cyclo-
sporin A), provided compounds with P-gp inhibition but
lacked selectivity among transporters and metabolic enzymes.
The potential drug−drug interactions of these less selective P-
gp inhibitors would significantly complicate their use with
paclitaxel to facilitate oral absorption.21,22 Third-generation P-
gp inhibitors, such as elacridar23,24 (GF120918), tariquidar25
(XR9576), OC144-09326,27 (ONT-093), zosuquidar28,29
(LY335979), and laniquidar30,31 (R101933) have excellent
potency against P-gp and demonstrated selectivity; however,
they were developed primarily for combatting P-gp-mediated
cancer drug resistance and were orally bioavailable with
significant systemic exposure (Figure 1). Not surprisingly,
many of the adverse events recorded for these P-gp inhibitors,
following administration of P-gp substrate anticancer drugs
(for example, paclitaxel), were due to on-target systemic P-gp
inhibition in sensitive tissues, including the bone marrow and
the central nervous system (CNS), resulting in an increased
exposure of these tissues to the anticancer drug leading to
augmented toxicity.
In our quest to develop a novel P-gp inhibitor to improve
the oral bioavailability of its substrate drugs including taxanes,
we reasoned that specific targeting of intestinal P-gp by a
selective small molecule inhibitor with low systemic exposure
would represent an attractive approach. A tissue and target
specific inhibitor would have the potential to improve oral
bioavailability of a substrate drug while avoiding undesirable
metabolism21 or clearance22 of substrate drugs that may
exacerbate side effects. This approach of ours, if successful and
safe, could serve as a platform technology allowing for oral
absorption of several drugs with P-gp mediated efflux
liability.32 In this drug annotation paper, we describe our
journey of bringing an intestine specific P-gp inhibitor,
encequidar, from conception to the clinic.
RESULTS AND DISCUSSION
■
Discovery of Encequidar. Our drug discovery strategy
entailed identifying a compound with the following attributes:
(a) high selectivity and potency for P-gp, (b) poor systemic
absorption, and (c) ability to improve the oral bioavailability of
P-gp substrates, such as paclitaxel. At the onset, it was apparent
that several conventional rules dictating physicochemical
properties desired in an orally bioavailable drug, such as MW
< 500, log P < 5, HBD < 5, HBA < 10, i.e., Lipinski’s rule of 5
(Ro5),33 would need to be violated in successfully designing an
inhibitor that is restricted to the gastrointestinal tract. P-gp is a
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Drug Annotation
Table 1. SAR Optimization of Tetrazole Series
a
P-gp activity was measured via a cell growth inhibition assay with 200 nM paclitaxel in MES-SA/DX5, a P-gp overexpressing cell line, by varying
the concentrations of the P-gp inhibitor. While 200 nM paclitaxel inhibited 100% cell growth in the parental cell line MES-SA, this concentration of
b
paclitaxel showed minimal inhibition in MES-SA/DX5. Paclitaxel cytotoxicity enhancement represents the ratio of paclitaxel EC50’s in MCF7/Dox
(drug-resistant cell line) in the absence (control) and in the presence of an inhibitor (50 nM) with higher values corresponding to greater P-gp
inhibition by an inhibitor.
transmembrane protein, expressed on the apical side of
polarized enterocytes lining the walls of the small intestine, a
major site for absorption of small molecules into the
bloodstream (systemic absorption). For an inhibitor of P-gp
to have a pharmacological effect, it has to either cross the
upper leaflet of a lipid bilayer and bind to the internal cavity of
P-gp or travel across the bilayer to the cytosolic end to enter
the drug binding domain in the transmembrane region or bind
to the nucleotide binding domain.34−38 In either case,
maintaining a balance between cellular permeability and
absorption would be critical in designing a site-specific P-gp
inhibitor.
To realize our goal, we turned our focus on tariquidar, a
highly potent P-gp inhibitor first reported by researchers at
Xenova Group Ltd. in 1999,39 for medicinal chemistry
optimization (Figure 2). Structurally, tariquidar possesses a
tetrahydroisoquinoline ring connected via a hydrophobic linker
to an anthranilamide portion equipped with a hydrophobic
heterocyclic ring. The extensive structure−activity relationship
(SAR) of tariquidar clearly established the importance of
tetrahydroisoquinoline ring as well as the lower amide bond
linked to quinoline for P-gp inhibitory potency.40,41 Despite
the high molecular weight, tariquidar is surprisingly orally
bioavailable.42 To understand factor(s) influencing tariquidar’s
oral bioavailability, and which could be manipulated using
medicinal chemistry strategies, we looked at its Ro5 parameters
and recognized them to be mostly on the anomalous side with
regard to the Lipinski rules, considered gold standard for oral
drugs. However, one of the key molecular descriptors beyond
Ro5, the topological polar surface area (tPSA) of tariquidar of
111.26 Å2, falls well within the limit of 140 Å2 set for well
absorbed drugs.43−45 Notably, another orally absorbed P-gp
inhibitor elacridar46 follows a similar pattern as tariquidar and
has a tPSA of 92.90 Å2.
Because tPSA is a high throughput molecular descriptor
accounting for polar 2D fragments in a molecule and has
shown to have good correlation with cellular permeability and
oral absorption47 we employed this parameter as a guiding tool
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Drug Annotation
a
Scheme 1. Synthesis of Encequidar (14)
a
Reagents and conditions: (a) 20, K2CO3, NaI, DMF, 100 °C; (b) H2, Pd/C, THF-MeOH (1:1), 68% yield over two steps; (c) EtOH, 24, 80 °C,
85% yield; (d) 22, NaNO2, conc. HCl, EtOH, H2O, −15 °C then 25, pyridine, 70% yield; (e) H2, Pd/C, EtOH-DCM (1:1), 85% yield; (f) 28,
DCM, rt, 72% yield.
for designing poorly orally bioavailable P-gp inhibitors. We
sought to do subtle but unique modifications in the tariquidar
structure to further increase the tPSA and thereby reduce the
oral absorption.
The subtle architectural modification of tariquidar was
rapidly accomplished and provided the tetrazole analogue 1.
Encouragingly, this bioisosteric switch of amide group with
tetrazole ring retained the capacity to inhibit the P-gp activity
although with diminished potency as compared to tariquidar
(Table 1). Nevertheless, this result undermines the notion that
the hydrogen bond donor of the amide group would be
necessary for P-gp activity. Alternatively, the polar interactions
of the four nitrogen atoms with the P-gp binding site
compensate for any loss in activity due to the absence of the
hydrogen bond donor group in the tetrazole analogue.
Furthermore, tetrazole 1 had a larger tPSA of 125.77 Å2 and
a reduced lipophilicity of 5.02 (cLogP) as compared with
tariquidar (tPSA of 111.26 Å2 and cLogP of 5.55). Having
obtained this promising result, we set out to further improve
the potency and increase the tPSA in our pursuit for a clinical
candidate with a poor absorption profile.
Next, we put our efforts in exploring the hydrophobic core
of the lower amide portion. As can be seen from the examples
presented in Table 1, the SAR around the quinoline group was
quite narrow and clearly favored fused (bicyclic) hetero-
cycles.51 Thus, phenyl, naphthalene, as well as various
substituted aromatics as replacements for the quinoline
group resulted in a considerably reduced P-gp inhibitory
activity. Even nonfused heterocycles including pyridines,
thiophene, and furan were not tolerated in lieu of quinoline.
Notably, the position of the heteroatom (nitrogen) in the
heterocycle was also found to be equally important, with at
least one nitrogen atom meta to the amide carbonyl proving
vital for increased potency. These unique structural require-
Conformationally restrained tetrazoles are well-known
bioisosteres for amides in peptide and medicinal chemistry.48,49
Tetrazoles possess several key traits. First, this structural
moiety increases the tPSA without having a deleterious effect
on lipophilicity. Second, all four nitrogen atoms of tetrazole
can theoretically participate in hydrogen bond interactions
with the active site residues of a protein (P-gp in the present
context) and therefore have the potential to supplement
interactions offered by the amide group. On the other hand,
although not definitive, the SAR studies of tariquidar suggest
the importance of the upper amide group for P-gp potency.39
Thus, loss of this amide group (and its hydrogen bond
acceptor and donor features) could potentially diminish any
advantage offered by the isosteric replacement with tetrazole.
Finally, tetrazoles tend to be metabolically more stable than the
amides, a desirable characteristic to provide adequate exposure
in the intestine, an organ with significant metabolic capacity.50
It is worth noting here that, despite the increased stability, we
anticipated that the tetrazole scaffold would negatively impact
permeability across enterocytes, the absorptive cells of the
intestine, and into the bloodstream due to a significant increase
in tPSA. All these remarkable features presented by tetrazole
led us to explore this heterocycle as a replacement of the amide
bond of tariquidar while keeping the tetrahydroisoquinoline
nucleus as well as the hydrophobic lower amide intact.
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Drug Annotation
ments of a fused heterocycle and the heteroatom position
indicate that favorable π−π interactions (and hydrogen
bonding) may be at play.
Table 2. Comparison of Physicochemical Properties of
Encequidar and Third Generation P-gp Inhibitors
tariquidar elacridar zosuquidar encequidar
Replacement of nitrogen-based heterocycle with chromone
had the greatest overall impact on improving the potency of
this series. Not only the choice of this heterocycle but also the
position of chromone carbonyl with respect to the amide
carbonyl had a profound impact on the P-gp potency displayed
by these inhibitors. Thus, tetrazole 14 with chromone carbonyl
positioned meta to the amide carbonyl was found to be to be
an equally potent inhibitor of P-gp as tariquidar and emerged
as the leading candidate with 5-fold improvement in the P-gp
activity over the quinoline tetrazole 1 (Table 1). Surprisingly,
this gain in potency was completely negated when the
chromone carbonyl was ortho to the amide carbonyl as in
the tetrazole analogue 15. Next, the SAR exploration focused
around the chromone ring led us to synthesize tetrazole
analogues 16−18 with a monosubstituted chromone ring.
Except for tetrazole analogue 18, which contained the Me
substituted chromone, substitutions at the chromone ring did
not improve the potency as compared to 14. The greater
potency of chromone-derived tetrazole analogues as compared
to the quinoline derived tetrazoles suggests that the chromone
is especially well suited to engage in key interactions with P-gp.
Although tetrazole analogue 18 demonstrated greater in vitro
potency than 14, it was not pursued further because it was
deemed that an extra methyl group of 18 could be
metabolically labile in vivo.
Excellent in vitro potency coupled with poor permeability
and aqueous solubility (Table 2) prompted us to evaluate
tetrazole 14 (INN-encequidar) in a rat pharmacokinetic study
with the anticipation that these favorable physicochemical
properties would translate into poor oral bioavailability in
vivo.52 The results are discussed in the preclinical pharmaco-
kinetics section.
Chemistry. The synthesis of encequidar (14) was
performed as described in Scheme 1.51 Two key building
blocks, compounds 22 and 25, were first constructed using
commercially available starting materials via straightforward
chemistry. The key tetrazole ring forming reaction from 22 and
25 proceeded smoothly, as described by Kondo et al.,53 to
afford 26. Reduction of the nitro group in 26 followed by
coupling of resultant aniline 27 with the thioester 28, readily
synthesized from chromone-2-carboxylic acid, afforded 14
(encequidar). The synthetic route to other final tetrazoles is
analogous to 14 except for the last step where the aniline 27
was either coupled with the carboxylic acids using EDCI/
cat.DMAP or with the thioesters derived from the correspond-
ing carboxylic acids.51
molecular weight
646.7
111.26
5.55
2
563.6
92.90
4.21
2
527.6
48.83
4.96
1
688.7
143.09
3.99
1
a
tPSA (Å2)
b
cLogP
hydrogen bond donor
number
hydrogen bond acceptor
number
10
8
7
13
MDCKII permeability (3
μM) A→B, Papp
0.11
0.01
0.11
0.0002
(×10−5 cm/s)
rotatable bond number
aqueous solubility
(μg/mL)
11
2500
8
6
nd
11
0.219
c
d
e
f
0.123
a
b
Calculated using Molinspiration Software.54 Calculated using
ChemDraw Professional 15.1 (PerkinElmer). At pH 5.5.55 Neutral
c
d
pH.56 nd = not determined. At pH 3.8.
e
f
desolvation penalty. It is to be noted that the possibility of
intramolecular hydrogen bond formation in tariquidar,
elacridar, and encequidar could offer an opposing effect of
increased cellular permeability. We next assessed the passive
permeability of all four inhibitors in MDCKII (Madin-Darby
canine kidney) cells. While none of the inhibitors tested
exhibited good permeability in the traditional sense for orally
bioavailable compounds, extremely poor permeability of
encequidar of at least 2 orders of magnitude less than the
other third generation P-gp inhibitors was striking.
Another variable implicated in the poor oral bioavailability is
the molecular flexibility45 as measured by the number of
rotatable bonds which were 11, 8, 6, and 11 for tariquidar,
elacridar, zosuquidar, and encequidar, respectively (assuming
no intramolecular hydrogen bonding). Again, encequidar (and
tariquidar) is above the limit set for number for rotatable
bonds (≤10) in a drug for good oral bioavailability. Overall,
the combination of poor cellular permeability and very low
aqueous solubility (desirable features in the present context)
boded well for continued development of encequidar as P-gp
inhibitor with poor systemic absorption potential.
Pharmacology. Mechanism of Action. P-gp as a primary
molecular target of encequidar was validated by Li et al. using
affinity-based protein profiling combined with the quantitative
proteomics approach of SILAC (stable isotope labeling with
amino acids in cell culture).57 Furthermore, bioimaging
experiments in either paclitaxel-resistant HT1080 cells58 or
doxorubicin-resistant HepG2 cells57 using fluorescently labeled
encequidar probes established that encequidar colocalizes with
P-gp in the cellular membrane, perinuclear space, and
endoplasmic reticulum.
Molecular Docking Studies. Recent advances in the
structural biology have led to determination of cryo-electron
microscopy (EM) structures of human P-gp bound to its
substrate, paclitaxel (3.6 Å resolution), and human-mouse
chimeric P-gp bound to its inhibitor, zosuquidar (3.9 Å
resolution).59−63 These structures have contributed to our
understanding of how a substrate and an inhibitor bind to P-
gp. As revealed by these structures, a highly dynamic P-gp
encompasses large internal binding pocket surrounded by
transmembrane helices within the lipid bilayer. Both the
substrate (paclitaxel) and the inhibitor (zosuquidar) occupy a
similar region within this pocket sharing some of the binding
Encequidar vs Third Generation P-gp Inhibitors
Differences in Physicochemical Properties. Next, we
examined key physicochemical parameters, generally attributed
to oral bioavailability, for the third-generation P-gp inhibitors
and compared them with encequidar to justify our selection of
encequidar for further investigation. The details are presented
in Table 2. As can be seen, metrics that clearly stand out for
encequidar are molecular weight combined with tPSA, and
total hydrogen bond number, all highest among the P-gp
inhibitors surveyed. Thus, the increase in polarity that results
from large tPSA and hydrogen bond count could be associated
with extensive solvation of encequidar in the intestinal lumen
aqueous environment and any passive permeability across the
lipid bilayer would come at the expense of a very large
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Figure 3. Predicted binding mode of encequidar. A 3D representation of human P-gp (PDB code 6QEX) docked with encequidar is shown as a
cartoon with transmembrane domain 1 and 2 helices colored in cyan. Alpha helices 7 and 9, and nucleotide binding domains 1 and 2 are not shown
for clarity. (A) Top ranked binding pose of encequidar (shown as magenta stick model). (B) Close-up view of of active site of encequidar
surrounded by key amino acid residues of human P-gp. Red dashed lines represent hydrogen bonds. The figures were generated using PyMol and
Microsoft Powerpoint. Color code for amino acid residues: Cflex, green; Crigid, cyan; N, blue; O, red. Color code for encequidar: C, magenta; N,
blue; O, red.
residues. We performed flexible molecular docking studies64
using the cryo-EM structure of paclitaxel-bound human P-gp
(PDB code 6QEX) to retrospectively understand the plausible
binding interactions contributing to encequidar’s potency.
Encequidar was docked into the M-site (modulator site) where
other P-gp inhibitors such as zosuquidar59 and cyclic peptide
inhibitors QZ59-RRR and QZ59-SSS63 are known to bind.
According to our docking results, presented in Figure 3, the
binding affinity for the top 10 binding poses of encequidar
ranged from −12.5 to −11.9 kcal mol−1. As per the most
favorable docked pose, encequidar is oriented in the U-shaped
conformation. The chromone ring is sandwiched between
aromatic rings of Trp232 and Phe343, making π−π stacking
interactions. The tetrazole ring partakes in hydrogen-bond
interactions with Gln725 and Tyr307 and is also involved in
π−π interactions with Phe728. The tetrahydroisoquionline
core is mostly surrounded by hydrophobic amino acid residues
such as Ile340, Leu339, and Phe343. Two additional hydrogen-
bond interactions were predicted for two of the four methoxy
groups with Asn842 and Gln347. Collectively, the docking
model predicts that encequidar interacts strongly with the M-
site located in the deep inner cavity of the transmembrane
region of P-gp. Tariquidar was also found to dock favorably in
this region with the top pose adopting a U-shape conformation
(binding affinities ranged from −12.2 to −11.4 kcal mol−1, data
not shown), correlating well with our experimental results and
consistent with the binding site predictions for tariquidar made
by McCormick et al.65 and Ferreira et al.66 via docking studies
and by Loo and Clarke67a through cysteine cross-linking and
arginine mutagenesis studies. Interestingly, the U-shape
conformation of tariquidar was also predicted using 3D-
QSAR analysis by Labrie et al.67b
MDR1 (P-gp)-enriched vesicles, encequidar inhibited P-gp
with high potency (IC50 = 0.6 nM) and was more potent than
known P-gp inhibitors tested (cyclosporin A, tariquidar, and
elacridar). Encequidar also showed high specificity for P-gp
and did not inhibit the ATPase activity of other ABC
transporters tested, MRP1, MRP2, MRP3, or BCRP (Table 3).
Table 3. In Vitro ABC Transporter Selectivity Data for
Encequidar
a
inhibition of ATPase activity, IC50 (nM)
ABC transporter
encequidar
elacridar
tariquidar cyclosporin A
P-gp
0.6
>3717
>27 233
>100 000
>100 000
4.9
252.2
>4128
32.7
183.3
23 780
141.3
>6568
1412
16 270
631.5
BCRP
MRP1
MRP2
MRP3
a
ATPase assay using transporter enriched membrane vesicles.
Activators: paclitaxel (0.05 μM) for P-gp, sulfasalazine (10 μM) for
BCRP, NEM-GS (3 mM) with GSH (2 mM) for MRP1, sulfasalazine
(150 μM) with GSH (2 mM) for MRP2, benzmarone (50 μM) with
GSH (2 mM) for MRP3. Adapted with permission from ref 52.
Copyright 2010 Elsevier.
In a more recent study, the in vitro interaction potential
between encequidar and the human bile salt export pump
(BSEP) efflux transporter and human MATE1, MATE2-K,
OAT1, OAT3, OATP1B1, OATP1B3, OCT1, and OCT2
transporters from the solute carrier (SLC) superfamily was
evaluated to further explore the selectivity of encequidar.
Vesicular transport assays were used to evaluate BSEP, and
cellular uptake and inhibition assays were used to evaluate the
SLC carriers. BSEP-expressing membrane vesicles and
corresponding empty inside-out membrane vesicles were
used in the vesicular transport assays, while Chinese hamster
ovary, HEK293, and MDCKII cells stably expressing the
respective SLC transporters and the corresponding controls
Selectivity. Previously published studies showed encequidar
is a potent and selective P-gp inhibitor, both main goals of the
medicinal chemistry program.52 Because ATP hydrolysis drives
the excretory function of P-gp, we examined encequidar’s effect
on the ATPase activity of P-gp. In an ATPase assay using
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were used in the cellular uptake and inhibition assays. In these
assays, encequidar showed no inhibition on BSEP at
concentrations of up to 10 μM and <50% inhibition on
MATE1, MATE2-K, OAT1, OAT3, OATP1B1, OATP1B3,
OCT1, and OCT2 transporters at concentrations of up to 100
μM. Additionally, encequidar did not inhibit any of the major
human cytochrome P450 isoforms (CYP1A2, CYP2A6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5) at
concentrations up to 50 μM or induce CYP1A2, CYP2B6, and
CYP3A4 at concentrations up to 1 μM.
Together, the in vitro transporter and metabolic enzyme data
highlight a key advantage of the encequidar molecule, its
selectivity. Regulatory authorities, such as the FDA and EMA
(European Medicines Agency), require in vitro studies to
evaluate the interaction potential between investigational drugs
and transporters to help evaluate the risk of the investigational
drug causing potentially dangerous drug−drug interactions.
Therefore, these data provide valuable safety information for
the development of encequidar, as we have shown low drug
interaction potential for encequidar based transporter activity,
beyond its intestine specific pharmacological action on P-gp
following oral dosing.
Inhibition of P-gp-Mediated Intestinal Efflux of Paclitaxel.
Since encequidar is being developed for oral administration as
an intestine specific P-gp inhibitor, the potency of encequidar
inhibition on P-gp in intestinal cells was also examined. To do
this, the effect of encequidar on the intestinal permeability
(efflux and absorption) of paclitaxel was evaluated in Caco-2
cells. The Caco-2 cell line is a human epithelial cell line derived
from colorectal adenocarcinoma that expresses P-gp on the
apical side and is widely used as a model of the intestinal
epithelial barrier. In the absence of P-gp inhibitors, P-gp
substrates, such as paclitaxel, are excluded from the basal
(“absorption”) experimental compartment into the apical
compartment.
In our experiment, coadministration of encequidar with
paclitaxel in the Caco-2 cell assay increased the apical to basal
apparent permeability (Papp a → b, absorption) and decreased
the basal to apical apparent permeability (Papp b → a, efflux) of
paclitaxel. Based on these data, the IC50 of encequidar for
inhibition of P-gp in Caco-2 intestinal cells was determined to
be 53 nM, demonstrating that encequidar potently inhibits P-
gp and prevents the efflux of paclitaxel from intestinal cells in
vitro. This is consistent with the paclitaxel transcellular
transport inhibition data in MDCK-MDR1 cells (an epithelial
cell line of canine kidney origin and with high P-gp
expression), where encequidar potently inhibited basal to
apical transport of paclitaxel with an IC50 of ∼35 nM.52
Preclinical Pharmacokinetics (ADME). Encequidar
Alone. Based on the excellent in vitro profile of encequidar,
its preclinical pharmacokinetics were investigated (Table 4).
To our delight, encequidar was minimally absorbed in both
rats (Cmax ∼10 nM) and dogs (Cmax ∼39 nM) following oral
dosing, and the bioavailability of encequidar after single oral
doses was estimated to be 6.25% in rats and 11.2% in dogs.
These results are in accordance with our observation in the
Caco-2 permeability assay suggesting that encequidar has low
absorption potential. The time to maximum plasma concen-
trations (Tmax) was 8 h in rats and 13.3 h in dogs, indicating
slow but prolonged absorption in the gastrointestinal tract with
gradual excretion following oral dosing. Following daily repeat
dosing (4 and 13 week) in rats and dogs, no significant
systemic accumulation of plasma encequidar was observed,
Table 4. Plasma Pharmacokinetic Profile of Encequidar in
Selected Preclinical Species
male Sprague-Dawley rat
male Beagle dog
PK parameters
(n = 3)
(n = 3)
dose IV/PO
(mg/kg)
3/10
3/10
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUCpo,last
(ng·h/mL)
7.1
8.0
14.8
129.0
26.6
13.3
16.9
723.9
AUCiv,last (ng·h/mL)
F (%)
618.8
6.25
1919.5
11.2
demonstrating that encequidar systemic exposure remains low
even after repeated doses are administered. In the rat,
encequidar appears to be absorbed at similarly low levels
from all portions of the gastrointestinal tract, indicating that
high exposure of encequidar in the gastrointestinal tract is
achieved to allow for a complete P-gp inhibition, while
systemic encequidar exposure is minimized to avoid systemic
side effects.
Distribution studies in rats and dogs showed that the
minimal concentrations of encequidar that are absorbed
distribute to most tissues, except for the brain and spinal
cord. However, the highest concentrations were observed in
the liver and bile which is not surprising given that encequidar
is excreted via the biliary route. In vitro studies show that
encequidar is mainly metabolized by CYP3A4/5 and, to a
lesser degree, by CYP1A2 and CYP2B6.
Encequidar with Oral Paclitaxel. Preclinical proof of
concept pharmacokinetic studies with oral paclitaxel/encequi-
dar were performed to evaluate if encequidar could inhibit P-
gp in the GI tract to allow for the absorption of paclitaxel
following oral dosing. In rats administered oral paclitaxel alone,
low plasma concentrations and exposures of paclitaxel were
achieved (Cmax ∼127 ng/mL and AUClast ∼308 ng·h/mL). In
contrast, oral administration of paclitaxel in combination with
encequidar as oral paclitaxel/encequidar increased both plasma
concentrations and exposures of paclitaxel by approximately
10-fold. While in rats the oral bioavailability (F%) of paclitaxel
was 41.3%, it ranged from 11 to 24% in dogs following oral
dosing of paclitaxel/encequidar (Table 5, PK data in dogs not
shown).52 The bioavailability of paclitaxel achieved in both
species demonstrates that encequidar facilitates the oral
Table 5. Oral Absorption of Paclitaxel Facilitated by
a
Encequidar in Male Sprague-Dawley Rats
paclitaxel alone
paclitaxel + encequidar
PK parameters
(n = 4)
(n = 9)
IV dose (mg/kg)
PO dose (mg/kg)
CL (L/h)
6
20
2.1
20 + 10
Vd (L)
23.6
Cmax PO (ng/mL)
Tmax (h)
T1/2 (h)
AUCpo,last (ng·h/mL)
AUCiv,last (ng·h/mL)
F (%)
127.2
1253.7
PO 1.0
6.8
PO 0.9, IV 0.08
PO 8.0, IV 7.7
308.5
2728.1
3.4
3756.5
41.3
a
Adapted with permission from ref 52. Copyright 2010 Elsevier.
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Table 6. Response of Subcutaneous MDA-MB-231 Mammary Tumor to Treatment with Oral Paclitaxel/Encequidar or IV
a
Paclitaxel
paclitaxel dose
(mg/kg)
encequidar dose
(mg/kg)
dosing
tumor free survival/
total
days to two
doublings
days delay (T/
C)
group (admin route)
saline control (PO)
vehicle control (PO)
schedule
0
0
12
18
20
0
0
0
0
8
Q2D×4
Q2D×4
Q2D×4
Q2D×4
Q2D×4
0/10
2/10
1/10
5/10
1/10
13.1
11.6
26.1
N/A
−1.5
13.0
b
paclitaxel (IV)
c
d
>69.0
>55.9
oral paclitaxel/encequidar
(PO)
15.1
2.0
c
d
30
40
12
16
Q2D×4
Q2D×4
6/10
9/10
>69.0
>55.9
>55.9
c
d
>69.0
a
IV, intravenous; N/A, not applicable; PO, oral gavage; Q2D×4, every other day for 4 doses; T/C, overall delay in growth of median tumor; n =
b
c
10/group. The vehicle control is a combination of paclitaxel vehicle and encequidar vehicle (see the Supporting Information for details). Only
two animals in the IV paclitaxel group survived past day 69; as such, day 69 was determined as the cutoff. Only one animal in the saline control
d
group survived to day 61. As the delay calculation is dependent on the survival of the controls, 55.9 days was selected as the cutoff.
absorption of paclitaxel which results in appreciable systemic
paclitaxel exposure in vivo.
with 20 mg/kg encequidar) suppressed tumor growth by 94%
and induced remission of tumor growth until day 47, an
outcome superior to the IV paclitaxel arm included in the
study. The results of this study demonstrate the ability of
encequidar to inhibit P-gp and facilitate the absorption of
paclitaxel to therapeutically effective plasma concentrations in
vivo. Clinically, paclitaxel is used to treat many different types
of cancers, including breast, lung, and ovarian cancer.
Therefore, we examined additional tumor models to broaden
the therapeutic potential of oral paclitaxel when given in
combination with oral encequidar.
Following oral paclitaxel/encequidar administration, pacli-
taxel demonstrated nonlinear plasma pharmacokinetics.
Paclitaxel did not accumulate in rats or dogs when
administered orally for 4 consecutive weeks as oral
paclitaxel/encequidar. In both rats and dogs, the absorption
of paclitaxel administered as oral paclitaxel/encequidar was
inhibited by food. No significant gender differences were
observed for paclitaxel following oral paclitaxel/encequidar
administration in rats.
In mice and rats, paclitaxel distributed primarily to the liver,
intestine, and kidney following administration of oral
paclitaxel/encequidar. The paclitaxel tissue to plasma ratio in
all organ tissues, excluding brain, was >1, indicating paclitaxel
distribution to tissues is higher versus the plasma. The wide
distribution of paclitaxel following oral paclitaxel/encequidar
administration is similar to the distribution characteristics
observed following IV administration of paclitaxel.68 Based on
the known human metabolism of paclitaxel after IV
administration,69 paclitaxel and its two metabolites (6α-
hydroxypaclitaxel and 3′-p-hydroxypaclitaxel) were measured
in dog plasma following a single oral paclitaxel/encequidar
administration. The metabolite 6α-hydroxypaclitaxel had a
metabolic ratio of 0.76, and 3′-p-hydroxypaclitaxel had a
metabolic ratio of 0.01. Biliary excretion was the primary route
of paclitaxel elimination in both rats and dogs. Both the
metabolism and excretion of paclitaxel following oral
paclitaxel/encequidar are in agreement with what has been
described for IV paclitaxel.68,69
Assessment of the absorption, distribution, metabolism, and
excretion (ADME) of paclitaxel following oral paclitaxel/
encequidar data demonstrates that although encequidar alters
the absorption of paclitaxel, it does not alter the distribution,
metabolism, or excretion of paclitaxel. Thus, from a clinical
safety perspective, the risks of oral paclitaxel/encequidar are
expected to be consistent with other approved IV paclitaxel
therapies.
The first model we tested was a highly aggressive triple
negative breast/mammary tumor cell (MDA-MB-231) mouse
model. In female athymic NCr-nu mice (n = 10/group)
implanted subcutaneously with MDA-MB-231 mammary
tumor xenografts, oral paclitaxel was administered every
other day for a total of four doses of 20, 30, or 40 mg/kg
and encequidar was administered with paclitaxel at doses of 0
(PO vehicle control), 8, 12, or 16 mg/kg (paclitaxel:encequi-
dar ratio 2.5:1). Intravenous (IV) paclitaxel was administered
at doses of 12 or 18 mg/kg every other day for a total of four
doses. Animals were monitored for 70 and 82 days after tumor
implantation for changes in tumor weights and individual body
weights, respectively. The median number of days for the
tumors in each group to reach two tumor mass doublings were
calculated with an evaluation period of 70 total days after the
tumor was implanted. The median time to reach two tumor
mass doublings was used in the calculation of the overall delay
in the growth of the median tumor.
Body weight reduction was not noted at either dose level.
Following oral paclitaxel/encequidar administration, median
tumor growth delay was 2.0, >55.9, and >55.9 days, in the 20,
30, and 40 mg/kg (paclitaxel doses) oral paclitaxel/encequidar
dose groups, respectively (Table 6). An average maximum
weight loss of 5% was noted in both the 30 and 40 mg/kg oral
paclitaxel/encequidar dose groups, respectively; however, no
reduction was noted in the 20 mg/kg oral paclitaxel/
encequidar dose group. Median tumor growth delay was
−1.5 days in the vehicle control group. Compared to this
control group, median tumor growth delay was 13.0 days in the
12 mg/kg IV paclitaxel dose group and >55.9 days in the 18
mg/kg IV paclitaxel dose group, suggesting the 12 mg/kg IV
dosage does not provide adequate paclitaxel exposure, allowing
residual disease to resume exponential growth. These findings
demonstrate that 30 and 40 mg/kg oral paclitaxel/encequidar
In Vivo Efficacy of Oral Paclitaxel/Encequidar. As
mentioned earlier, paclitaxel is a P-gp substrate whose clinical
administration is limited to IV due to poor oral absorption
caused by P-gp drug efflux in the small intestine. A previous
study by Kwak et al. showed that oral coadministration of
paclitaxel and encequidar effectively inhibited the tumor
growth of human colon cancer cells (HT-29) in a xenograft
model.52 In that study, 40 mg/kg paclitaxel (in combination
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Figure 4. In vivo efficacy of oral paclitaxel/encequidar in mice bearing subcutaneous MDA-MB-231 mammary tumor xenografts (n = 10/group).
Treatments were initiated on day 13.
Figure 5. In vivo efficacy of oral paclitaxel/encequidar in mice bearing subcutaneous NCI-H460 human lung cancer xenografts (n = 10/group).
Treatments were initiated on day 8.
reduced MDA-MB-231 tumor growth comparably to 18 mg/
kg IV paclitaxel (Figure 4).
H460 human lung tumors, oral paclitaxel/encequidar per-
formed comparably to IV paclitaxel (Figure 5).
In addition to the breast and lung tumor models tested,
other mouse tumor models that we have tested and which have
shown that oral paclitaxel/encequidar is effective at inhibiting
tumor growth include SK-OV-3 ovarian and vaginal
epidermoid cancer cells (data not shown).
Preclinical Safety. To better understand encequidar and
its potential for clinical use in combination with cytotoxic
drugs, such as paclitaxel, for oral administration, the preclinical
safety of encequidar alone and oral paclitaxel/encequidar
combination was evaluated in rats (Sprague-Dawley) and dogs
(Beagle).
Single oral administration of encequidar up to 2000 mg/kg/
day to male and female Sprague−Dawley rats was well
tolerated. No significant toxicity was noted following repeat
encequidar administration in Sprague−Dawley rats or Beagle
dogs. In rats administered encequidar for 4 and 13 weeks at
doses up to 200 mg/kg/day, the only effects noted were in the
mesenteric lymph nodes. Mesenteric lymph nodes were
enlarged at doses ≥ 50 mg/kg, and histiocytosis in these
lymph nodes was observed at doses ≥ 10 and 50 mg/kg,
respectively. However, these findings were reversible and were
considered an adaptive response to test article administration
Another model tested was subcutaneously implanted NCI-
H460 human lung tumor xenografts in mice. In this study,
encequidar and oral paclitaxel were administered concom-
itantly as a single oral (gavage) solution to female athymic
NCr-nu mice implanted subcutaneously with NCI-H460
tumor xenografts every other day for a total of four doses.
Oral paclitaxel was administered at doses of 30, 40, or 50 mg/
kg, and encequidar was administered with paclitaxel at doses of
12, 16, or 20 mg/kg (paclitaxel:encequidar ratio 2.5:1). For
comparison, paclitaxel was also administered via IV on the
same dosing schedule at doses of 18 and 24 mg/kg. The mice
were monitored for 48 days post tumor implantation for
changes in tumor weights and individual body weights.
There were no mortalities noted in the study, and body
weight loss was similar among the IV and oral dosing groups.
Median tumor growth delay was 8.5 and 16.3 days in the 18
and 24 mg/kg paclitaxel IV dose groups, respectively. Similarly,
the median tumor growth delay was 9.9, 8.7, and 12.7 days in
the 30, 40, and 50 mg/kg oral paclitaxel/encequidar dose
groups, respectively. Following both routes of administration,
tumor growth delay was the greatest following the highest dose
of paclitaxel tested, demonstrating that when targeting NCI-
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Figure 6. Measurement of nociceptive threshold in healthy rats treated with oral paclitaxel/encequidar and IV paclitaxel (Taxol). Statistical analysis
was performed in GraphPad Prism version 5.0. If significance was recognized from the one-way analysis of variance results, Dunnett’s test, a
multiple comparison method, was performed to test the significant result of whether there is a difference between control group and administration
group (n = 5/group).
and were not considered to be adverse in nature. Similarly, in
Beagle dogs treated with encequidar for 13 weeks at
encequidar doses up to 200 mg/day, an increase in total
leukocyte (WBC) and eosinophil counts was noted at doses ≥
100 mg/kg. Enlargement of mesenteric lymph nodes
associated with microscopically observed histiocytosis was
also observed at these doses. These findings were reversible
and were considered of little toxicological significance because
no other inflammatory reactions or functional effects were
observed.
Repeat dose administration of oral paclitaxel/encequidar to
rats and dogs resulted in toxicologically significant plasma
exposure levels and caused toxicities characteristic of paclitaxel.
The characteristic findings include leucopenia and anemia,
bone marrow depression/atrophy, lymphoid atrophy, hepatic
necrosis, and villous stunting and epithelial hyperplasia of the
small intestine. Other target organs include the spleen and
thymus. These toxicity findings are typical of those observed
with paclitaxel after IV administration and were generally
reversible or showed a tendency toward recovery following the
4−8 week recovery phases in the respective studies.
A major clinical dose limiting toxicity of paclitaxel is
neuropathy, which correlates with high plasma concentrations
of paclitaxel.70 One of the advantages of oral paclitaxel/
encequidar to patients is that the maximum plasma
concentration (Cmax) of paclitaxel achieved following admin-
istration is much lower than what is achieved following dosing
of intravenous paclitaxel.71 We postulated that the lower Cmax
of paclitaxel following oral paclitaxel/encequidar would result
in less neuropathy compared to IV paclitaxel. To test this
hypothesis, a rat safety study was performed which examined
the neurotoxicity of paclitaxel following administration of oral
paclitaxel/encequidar versus IV paclitaxel (Taxol). In this
study, neurotoxicity was assessed by measuring the nociceptive
threshold as indicated by pressure (mmHg) needed to produce
a response, measured on the back paws of the healthy rats
(Randall−Selitto test).72 Oral paclitaxel was administered at
doses of 12 and 18 mg/kg (cumulative dose 48 and 72 mg/
kg). Encequidar was administered orally immediately prior to
paclitaxel at doses of 6 and 9 mg/kg, respectively (paclitaxel/
encequidar ratio was 2:1). Taxol was administered intra-
venously at doses of 0 (vehicle control), 3, 6 mg/kg
(cumulative dose 12, 24 mg/kg) four times in 2-day intervals.
Following dosing, the nociceptive threshold for the paw
pressure test was statistically significantly reduced on days 5, 7,
11, and 14 following administration of 6 mg/kg IV paclitaxel
versus its respective control group. In contrast, no significant
reduction in the paw pressure test was noted following doses
up to 18 mg/kg oral paclitaxel given in combination with 9
mg/kg encequidar, suggesting that oral paclitaxel/encequidar is
less neurotoxic than IV paclitaxel in rats (Figure 6). Based on
the encouraging overall preclinical data package that included
favorable potency, target selectivity, pharmacokinetic, and
safety profile, encequidar was advanced into clinical trials in
humans.
Clinical Evaluation of Encequidar. The tolerability and
pharmacokinetics of encequidar in humans was first reported
by Kim et al. from a Phase 1, randomized, double-blind,
placebo-controlled, single and multiple oral dosing, dose-
escalation study in healthy male subjects.73 Although absolute
bioavailability was not defined, the results show that systemic
exposure is low after oral administration at a single dose up to
900 mg (Cmax = 6.6 ng/mL) or repeat dosing of five
consecutive doses of up to 360 mg (Cmax = 22.5 ng/mL).
To evaluate the potential effects of encequidar on systemic P-
gp activity and expression, lymphocytes and monocytes were
isolated from subjects at the beginning and end of repeat
dosing. Flow cytometry assays demonstrated that oral
encequidar had minimal impact on ex vivo rhodamine 123
efflux and P-gp expression with a lack of dose−response across
60, 180, and 360 mg dose levels, indicating that oral
encequidar has negligible systemic P-gp inhibition (unpub-
lished results). In contrast, rhodamine 123 accumulation was
enhanced in lymphocytes isolated from subjects post oral
dosing of elacridar74 and tariquidar,75 systemically absorbed P-
gp inhibitors. Exposure was also seen to increase with dose in a
less than proportional manner, supporting that encequidar is
poorly absorbed (unpublished results). The pharmacokinetics
of encequidar can be described as having slow absorption and
long elimination. Principal drug-related adverse events were
diarrhea and abdominal pain, but all adverse events reported
were mild in intensity and spontaneously resolved. The low
grade and reversible adverse events associated with encequidar
demonstrate its amenable safety profile for future combina-
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tions. Furthermore, the nature of toxicities that do occur
support the localized action of encequidar in the gastro-
intestinal tract.
recurrent gastric cancer. No dose limiting toxicity was
reported, and oral paclitaxel and encequidar had a similar
safety profile to IV paclitaxel, but with less peripheral
neuropathy than conventional weekly paclitaxel.81 Again, the
maximum tolerated dose could not be determined for lack of
toxicity, with neutropenia and diarrhea being the most
common drug-related adverse events. At the recommended
Phase II dose of 150 mg/m2 oral paclitaxel with 15 mg oral
encequidar (days 1, 2, 8, 9, 15, and 16 every 4 weeks), median
progression-free survival and overall survival were 2.6 and 10.7
months, respectively, as a second-line therapy for patients with
advanced gastric cancer.
A later, more dose-intense Phase I study of 34 patients with
advance malignancies escalatedthe administration of oral
paclitaxel/encequidar to a flat dose of 270 mg oral paclitaxel
following 15 mg encequidar five consecutive days for 3 weeks
of a 4 week cycle.82 The most common treatment-related
adverse events were nausea, diarrhea, anorexia, and vomiting.
Serious treatment-related adverse events included febrile
neutropenia, pneumonia, and dehydration. Again, the max-
imum tolerated dose was not reached, although two partial
responses were observed in salivary gland and ovarian cancers
at the 5 day dose level.
Evidence of the efficacy of oral paclitaxel/encequidar
continues to be expanded with ongoing pivotal studies in
angiosarcoma83 (NCT03544567) and metastatic breast
cancer83 (NCT02594371). Oral paclitaxel/encequidar has
showed encouraging efficacy and tolerability in the treatment
of unresectable cutaneous angiosarcoma in an elderly
population. Remarkably, all 26 patients showed reduction in
tumor size, with 7 patients becoming eligible for curative
resections after treatment.84 In breast cancer, IV paclitaxel has
been a backbone of chemotherapy treatment of advanced and
metastatic disease but is associated with irreversible dose-
limiting neuropathy and hypersensitivity in approximately half
of all patients (Taxol monograph).85 Oral paclitaxel/
encequidar at a dosage of 15 mg encequidar given 1 h prior
to 205 mg/m2 oral paclitaxel three consecutive days per week
(a dosage bioequivalent to IV) has recently demonstrated
superior confirmed response, progression-free survival, and
overall survival, with minimal clinically meaningful neuropathy
in an ongoing open-label, randomized, multicenter, Phase III
study against IV paclitaxel for metastatic breast cancer.86 By
replacing IV paclitaxel, these promising results open the
potential for oral paclitaxel/encequidar to not only dramati-
cally improve patient quality of life by enabling at-home care
and reduction of irreversible neuropathy but also significantly
improve efficacy over traditional chemotherapy. On the basis
of extensive clinical studies, we have submitted a New Drug
Application (NDA) to the FDA requesting the approval of oral
paclitaxel/encequidar for metastatic breast cancer and the
application has been granted priority review.87
As a drug designed to induce a drug−drug interaction,
multiple clinical interaction studies have been conducted to
characterize the impact of encequidar on exposure and safety
of clinically relevant substrates. These include two completed
studies of the effect of encequidar on the oral pharmacokinetics
of P-gp substrate loperamide76,77 and ongoing interaction
studies with oral P-gp substrates of narrow therapeutic index,
digoxin and dabigatran etexilate, in healthy volunteers.
Encequidar doses of 15 and 60 mg increased loperamide
AUClast by 1.46 and 1.63 times, respectively, and the effect of
encequidar was maintained for 72 h after the administration
but without affecting the CNS effects of loperamide. This is
consistent with the designed localized effect of encequidar on
the small intestine and low systemic concentrations in humans.
The pharmacokinetic aim of adding encequidar to an oral
formulation of paclitaxel is for the combination to provide
equivalent, if not superior, exposure above the minimum
inhibitory concentration of systemic paclitaxel. The ability of
encequidar to make paclitaxel, a highly insoluble and poorly
absorbed P-gp substrate, bioavailable was demonstrated in a
randomized crossover bioequivalence study with weekly
intravenous paclitaxel in patients with advanced solid tumors.71
Although IV paclitaxel was approved at a dosage of 175 mg/m2
Q3W (every 3 weeks), the more widely used dosage of 80 mg/
m2 weekly was chosen as a clinically relevant comparator.78
Based on population pharmacokinetic modeling, a dosing
regimen of 15 mg oral encequidar given 1 h prior to 205 mg/
m2 oral paclitaxel three consecutive days per week was
predicted to provide an equivalent total weekly exposure of
IV paclitaxel. From 35 subjects evaluated for pharmacokinetics,
oral paclitaxel yielded a weekly AUC (area under the curve)
equivalent to 80 mg/m2 IV paclitaxel with a 90% confidence
interval of the geometric mean ratio within 80−125%. Critical
to proving encequidar’s effectiveness, the absolute bioavail-
ability of oral paclitaxel was calculated as 11.8% at this
bioequivalent dose level. The toxicity of oral paclitaxel/
encequidar was primarily limited to diarrhea, vomiting, and
fatigue. Consistent with the reduced neuropathy compared
with IV paclitaxel, oral paclitaxel/encequidar produced an
approximately seven times lower Cmax
.
Use of encequidar in combination with an oral formulation
of paclitaxel has been explored in a variety of advanced
malignancies where IV paclitaxel would previously be
recommended or no approved therapies exist. For all
indications tested, sequential oral administration of encequidar
given 1 h prior to oral paclitaxel has achieved clinically
efficacious paclitaxel levels. To assess the recommended Phase
II dosages of oral paclitaxel/encequidar, Lee et al.79 evaluated
24 patients with advanced cancers and identified that the
effective plasma concentration of paclitaxel was achieved at a
dose of 120 mg/m2 following 60 mg/m2 encequidar. Oral
paclitaxel/encequidar was well tolerated. By obviating the use
of IV paclitaxel excipient polyoxyl-35 castor oil,80 oral
paclitaxel/encequidar does not induce hypersensitivity. Ther-
apeutic levels of paclitaxel (0.01 to 0.1 μM) were achieved
without reaching the maximum tolerated dose; the only dose-
limiting toxicity was a grade 3 neutropenia, observed at 240
mg/m2 oral paclitaxel following 120 mg/m2 oral encequidar.
The maximum tolerated dose of oral paclitaxel/encequidar
was further explored in a Phase I/II with metastatic or
CONCLUSIONS
■
In summary, by applying the fundamental principles of
medicinal chemistry, we have developed the first-in-class
intestine-specific P-gp inhibitor, encequidar (14), which has
enabled the oral delivery of paclitaxel. The clinical
pharmacokinetics of encequidar suggest minimal absorption
with low systemic exposure at the dose of 15 mg broadly in line
with the preclinical data. Oral paclitaxel/encequidar offers the
possibility of improving the treatment outcome with no
hypersensitivity and reduced neuropathy, while providing the
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8.35 (s, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.59 (s, 1H), 7.45 (d, J = 8.4
Hz, 2H), 7.15 (s, 1H), 3.89 (d, J = 3.6 Hz, 6H), 2.38 (s, 3H). Mass
spec. m/z (ESI, +ve ion): 380.08 (M + H)+.
convenience of at-home care for all cancers currently being
treated with IV paclitaxel. The superior efficacy of oral
paclitaxel/encequidar has already been demonstrated in a
pivotal Phase III study of metastatic breast cancer. These
promising clinical results encouraged us to file for the NDA
submission. Future studies aim to broaden the utility of oral
paclitaxel/encequidar to replace IV paclitaxel as a monotherapy
and in a variety of established combination therapies for a
diverse set of cancer types for which the prior generation of
paclitaxel has established clinical impact.
2-(4-(5-(4,5-Dimethoxy-2-nitrophenyl)-2H-tetrazol-2-yl)-
phenethyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
(26). To obtain 26, 7.40 g (23.7 mmol) of compound 22 was added
to 40 mL of 50% EtOH, and the mixture was cooled to 5 °C. Then
6.32 mL of 35% HCl and a solution obtained by dissolving 1.8 g (26.1
mmol) of sodium nitrite in 10 mL of water were added, and the
mixture was cooled to −15 °C. An amount of 9 g (23.7 mmol) of
compound 25 was dissolved in 140 mL of pyridine, by adding it
slowly. The resulting solution was stirred for 14 h and washed with 1
N HCl. The organic layer was separated, dried over MgSO4, filtered,
and distilled under a reduced pressure. The residue was purified by
silica gel column chromatography (0−5% MeOH/DCM) to obtain
EXPERIMENTAL SECTION
■
All reagents and chemicals were purchased from commercial suppliers
and were used without further purification unless otherwise stated. 1H
NMR spectra were recorded at 400 MHz on a Bruker Avance III HD
NMR spectrometer using DMSO-d6 or CDCl3 with TMS as the
internal standard at ambient temperature. A Waters Alliance 2695
HPLC system equipped with a PDA detector (Waters 2998, 800 nm)
was used. HPLC purity was determined by UV absorption at a
wavelength of 254 nm (LC method: column XBridge C18, 5 μm, 150
× 4.6 mm at 25 5 °C with a 0.8 mL/min flow rate; mobile phase A:
0.1% formic acid in water, mobile phase B: 0.1% formic acid in
acetonitrile; 0.0−4.0 min, 90% A; 4.0−10.10, 95% B; 10.10−12.0 min,
90% A), and the mass ion was obtained by electrospray ionization.
Chemical shifts are reported in parts per million (ppm, δ units).
Coupling constants (J-values) are reported in units of hertz (Hz).
Splitting patterns are abbreviated as s for singlet, d for doublet, t for
triplet, q for quartet, m for multiplet, bs for broad singlet, and dd for
doublet of doublet. The chemical yields reported are unoptimized.
1
9.0 g (yield 70%) of the title compound 26. H NMR (400 MHz,
DMSO-d6): δ 8.04 (d, J = 8.4 Hz, 2H), 7.76 (s, 1H), 7.59 (d, J = 8.0
Hz, 2H), 7.46 (s, 1H), 6.66 (d, J = 10.8 Hz, 2H), 3.96 (d, J = 3.2 Hz,
6H), 3.71 (s, 6H), 3.56 (s, 2H), 2.98−2.94 (m, 2H), 2.77−2.71 (m,
6H). Mass spec. m/z (ESI, +ve ion): 547.19 (M + H)+.
2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-
ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyaniline (27). To
obtain 27, 0.25 g (0.46 mmol) of compound 26 was mixed with 3 mL
of EtOH, 3 mL of DCM, and 0.07 g of Pd/C and stirred under a
hydrogen atmosphere for 12 h at room temperature. The reaction
mixture was filtered through a Celite pad, the pad was washed with
EtOH, and the filtrate and wash solution were combined and distilled
under a reduced pressure, to obtain 0.2 g (yield 85%) of the title
1
compound 27. H NMR (400 MHz, CDCl3): δ 8.11 (d, J = 8.4 Hz,
2H), 7.73 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 6.63 (s, 1H), 6.56 (s,
1H), 6.37 (s, 1H), 5.33 (bs, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 3.87 (d, J
= 2.8 Hz, 6H), 3.69 (s, 2H), 3.04−3.01 (m, 2H), 2.88−2.83 (m, 6H).
Mass spec. m/z (ESI, +ve ion): 517.18 (M + H)+.
1
The purity of final test compounds was ≥95% as determined by H
NMR and HPLC. In all in vitro, animal, and clinical studies,
encequidar was used in the methanesulfonate monohydrate form. The
animal studies presented in this paper were reviewed and assessed by
the Institutional Animal Care and Use Committee (IACUC). All
animals used in the studies were cared for in accordance with the
available guidelines for the care and use of laboratory animals
(Association for Assessment and Accreditation of Laboratory Animal
Care (AAALAC)).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
quinoline-3-carboxamide (1). To obtain 1, 0.2 g (0.39 mmol) of
27 and 0.125 g (0.39 mmol) of thioester of quinoline-3-carboxylic
acid (prepared by stirring a mixture of quinoline-3-carboxylic acid,
triphenyl phosphine, 2,2′-benzothiazolyl disulfide, and Et3N in DCM
at room temperature overnight followed by filtering the obtained
solid, washing with acetone, and drying under vacuum to obtain the
desired thioester which was used without further purification) were
added to 5 mL of DCM, and the mixture was stirred at room
temperature for 12 h. After washing with 50 mL of distilled water, the
organic layer was dried over MgSO4, filtered, and distilled under a
reduced pressure. The residue was subjected to silica gel column
chromatography (0−5% MeOH/DCM) to obtain 0.18 g (yield 69%)
of the title compound 1. 1H NMR (400 MHz, DMSO-d6): δ 10.89 (s,
1H), 9.44 (d, J = 2.4 Hz, 1H), 9.01 (d, J = 2.0 Hz, 1H), 8.14 (d, J =
11.2 Hz, 1H), 8.05 (dd, J = 8.4 and 0.8 Hz, 1H), 7.96−7.90 (m, 4H),
7.74−7.70. (m, 2H), 7.44 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 11.2 Hz,
2H), 3.90 (d, J = 1.6 Hz, 6H), 3.70 (d, J = 2.0 Hz, 6H), 3.55 (bs, 2H),
2.93 (t, J = 7.2 Hz, 2H), 2.71 (bs, 6H). Mass spec. m/z (ESI, +ve
ion): 672.20 (M + H)+. HPLC purity: 98.0%.
4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-
ethyl)aniline (22). To obtain 22, 2.30 g (9.99 mmol) of 1-(2-
bromoethyl)-4-nitrobenzene 19 and 2.29 g (9.97 mmol) of 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride 20 were
dissolved in 150 mL of DMF and then 4.15 g (30.0 mmol) of
K2CO3 and 1.80 g (12.0 mmol) of NaI were added, and the mixture
was allowed to react at 100 °C for 12 h. After mixing 150 mL of water,
the reaction mixture was extracted three times with a 200 mL portion
of EtOAc, and the combined organic layer was washed with saturated
brine solution and dried over MgSO4. The resulting solution was
subjected to a reduced pressure to remove the solvent, and the
residues was recrystallized using EtOAc to obtain 2.40 g of a nitro
derivative 21 which was used in the next step without additional
purification. Compound 21 was added to a mixture of THF and
MeOH (1:1, 300 mL) and then 0.24 g of Pd/C was added, and the
mixture was reduced under an atmospheric hydrogen pressure for 18
h. The resulting solution was filtered and concentrated under a
reduced pressure to obtain 2.03 g (65% yield over 2 steps) of the title
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
quinoline-2-carboxamide (2). To obtain 2, 0.15 g (0.29 mmol) of
27 and 0.05 g (0.29 mmol) of quinaldic acid were added to 5 mL of
DCM, and then 0.1 g (0.52 mmol) of EDCI·HCl and 0.005 g (0.041
mmol) of DMAP were added, and the mixture was stirred at room
temperature for 12 h. After washing with 50 mL of distilled water, the
organic layer was separated, dried over MgSO4, filtered, and distilled
under a reduced pressure. The residue was subjected to silica gel
column chromatography (0−5% MeOH/DCM) to obtain 0.14 g
1
compound 22. H NMR (400 MHz, DMSO-d6): δ 6.90 (d, J = 8.0
Hz, 2H), 6.65 (d, J = 8.4 Hz, 2H), 6.51 (d, J = 8.4 Hz, 2H), 4.81 (s,
2H), 3.70 (d, J = 2.0 Hz, 6H), 3.51 (s, 2H), 2.71−2.55 (m, 8H). Mass
spec. m/z (ESI, +ve ion): 313.19 (M + H)+.
(E)-N’(4,5-Dimethoxy-2-nitrobenzylidene)-4-methylbenze-
nesulfonohydrazide (25). To obtain 25, 6.90 g (37.0 mmol) of p-
toluenesulfonyl hydrazide 24 was dissolved in 40 mL of EtOH, and
7.90 g (37.4 mmol) of 6-nitroveratraldehyde 23 dissolved in a small
amount of EtOH was added. The mixture was stirred at 80 °C for 30
min, cooled to room temperature, and mixed with 100 mL of water.
The solid formed therein was filtered, washed with 100 mL of EtOH,
and dried under reduced pressure to obtain 12.0 g (yield 85%) of the
title compound 25. 1H NMR (400 MHz, DMSO-d6): δ 11.73 (s, 1H),
1
(yield 73%) of the title compound 2. H NMR (CDCl3): δ 12.60 (s,
1H), 8.71 (s, 1H), 8.40 (d, 2H), 8.20 (d, 2H), 8.13 (d, 1H), 7.90 (s,
2H), 7.65 (m, 2H), 7.37 (d, 2H), 6.58 (d, 2H), 4.05 (d, 6H), 3.85 (s,
6H), 3.67 (s, 2H), 3.01 (t, 2H), 2.83 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
isoquinoline-3-carboxamide (3). Following the procedure of
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Drug Annotation
compound 2 except 3-isoquinoline carboxylic acid was used instead of
quinaldic acid, 0.12 g (yield 62%) of the title compound 3 was
obtained. H NMR (CDCl3): δ 12.67 (s, 1H), 9.29 (s, 1H), 8.83 (s,
1H), 8.73 (s, 1H), 8.41 (d, 2H), 8.01 (d, 2H), 7.93 (s, 1H), 7.77 (m,
2H), 7.53 (d, 2H), 6.62 (s, 1H), 6.57 (s, 1H), 4.04 (d, 6H), 3.85 (s,
6H), 3.72 (s, 2H), 3.07 (t, 2H), 2.86 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
quinoline-8-carboxamide (4). Following the procedure of
compound 2 except 8-quinoline carboxylic acid hydrate was used
instead of quinaldic acid, 0.13 g (yield 67%) of the title compound 4
was obtained. 1H NMR (CDCl3): δ 13.69 (s, 1H), 8.87 (d, 1H), 8.77
(q, 1H), 8.37 (s, 1H), 8.24 (d, 1H), 8.06 (d, 1H), 8.00 (d, 2H), 7.38
(m, 1H), 7.23 (s, 1H), 6.58 (d, 2H), 4.03 (d, 6H), 3.85 (s, 6H), 3.65
(s, 2H), 2.95 (m, 2H), 2.81 (m, 6H).
quinaldic acid, 0.12 g (yield 63%) of the title compound 11 was
obtained. H NMR (CDCl3): δ 11.53 (s, 1H), 8.65 (s, 1H), 8.10 (d,
2H), 7.98 (m, 1H), 7.90 (m, 1H), 7.84 (s, 1H), 7.49 (d, 2H), 7.35 (d,
1H), 6.62 (s, 1H), 6.55 (s, 1H), 4.03 (d, 6H), 3.85 (s, 6H), 3.68 (s,
2H), 3.04 (t, 2H), 2.85 (m, 6H).
1
1
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
thiophene-3-carboxamide (12). Following the procedure of
compound 2 except 3-thiophene carboxylic acid was used instead of
quinaldic acid, 0.10 g (yield 55%) of the title compound 12 was
1
obtained. H NMR (CDCl3): δ 11.43 (s, 1H), 8.63 (s, 1H), 8.21 (d,
1H), 8.08 (d, 2H), 7.76 (s, 1H), 7.74 (s, 1H), 7.48 (d, 2H), 7.38 (m,
1H), 6.61 (s, 1H), 6.54 (s, 1H), 3.99 (d, 6H), 3.83 (s, 6H), 3.67 (s,
2H), 3.02 (t, 2H), 2.83 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
furan-3-carboxamide (13). Following the procedure of compound
2 except 0.04 g of 3-furoic acid was used instead of 0.05 g of quinaldic
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
isoquinoline-1-carboxamide (5). Following the procedure of
compound 2 except 1-isoquinoline carboxylic acid was used instead
of quinaldic acid, 0.12 g (yield 62%) of the title compound 5 was
1
acid, 0.05 g (yield 62%) of the title compound 13 was obtained. H
NMR (CDCl3): δ 11.32 (s, 1H), 8.64 (s, 1H), 8.22 (s, 1H), 8.11 (d,
2H), 7.78 (s, 1H), 7.51 (m, 3H), 7.03 (d, 1H), 6.62 (s, 1H), 6.55 (s,
1H), 4.01 (d, 6H), 3.85 (s, 6H), 3.68 (s, 2H), 3.04 (t, 2H), 2.85 (m,
6H).
1
obtained. H NMR (CDCl3): δ 12.76 (s, 1H), 9.76 (d, 1H), 8.91 (s,
1H), 8.73 (d, 1H), 8.37 (d, 2H), 8.05 (s, 1H), 8.00 (m, 1H), 7.93 (d,
1H), 7.86 (m, 2H), 7.47 (d, 2H), 6.70 (d, 2H), 4.17 (d, 6H), 3.96 (s,
6H), 3.80 (s, 2H), 3.15 (t, 2H), 2.94 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-4-
oxo-4H-chromene-2-carboxamide (14). To obtain 14, 0.15 g
(0.29 mmol) of 27 and 0.099 g of thioester of chromone-2-carboxylic
acid (0.29 mmol) 28 (prepared by stirring a mixture of chromone-2-
carboxylic acid, triphenyl phosphine, 2,2′-benzothiazolyl disulfide, and
Et3N in DCM at room temperature overnight followed by filtering the
obtained solid, washing with acetone, and drying under vacuum to
obtain the desired thioester which was used without further
purification) were added to 5 mL of anhydrous DCM, and the
mixture was stirred at room temperature for 12 h. After washing with
50 mL of distilled water, the organic layer was separated, dried over
MgSO4, filtered, and distilled under a reduced pressure. The residue
was subjected to silica gel column chromatography (0−5% MeOH/
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
isoquinoline-4-carboxamide (6). Following the procedure of
compound 2 except 4-isoquinoline carboxylic acid was used instead
of quinaldic acid, 0.11 g (yield 57%) of the title compound 6 was
1
obtained. H NMR (CDCl3): δ 11.38 (s, 1H), 9.09 (d, 1H), 8.74 (s,
1H), 8.52 (d, 1H), 8.23 (d, 1H), 7.89 (s, 1H), 7.79 (m, 4H), 7.64 (t,
1H), 7.36 (d, 2H), 6.62 (s, 1H), 6.55 (s, 1H), 4.08 (s, 3H), 4.01 (s,
3H), 3.85 (s, 6H), 3.67 (s, 2H), 2.98 (t, 2H), 2.82 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
nicotinamide (7). Following the procedure of compound 2 except
0.04 g of nicotinic acid was used instead of 0.05 g of quinaldic acid,
1
1
0.12 g (yield 67%) of the title compound 7 was obtained. H NMR
DCM) to obtain 0.144 g (yield 72%) of the title compound 14. H
NMR (400 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.06 (s, 1H), 7.99−
7.96 (m, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.77−7.73 (m, 1H), 7.47−
7.43 (m,5H), 6.80 (s, 1H), 6.67 (d, J = 8.4 Hz, 2H), 3.77 (d, J = 8.0
Hz, 6H), 3.71 (d, J = 1.6 Hz, 6H), 3.57 (s, 2H), 2.94 (t, J = 7.2 Hz,
2H), 2.75−2.72 (m, 6H). Mass spec. m/z (ESI, +ve ion): 689.31 (M
+ H)+. HPLC purity: >99%.
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-4-
oxo-4H-chromene-3-carboxamide (15). Following the procedure
of compound 2 except 0.06 g of chromone-3-carboxylic acid was used
instead of 0.05 g of quinaldic acid, 0.08 g (yield 40%) of the title
compound 15 was obtained. 1H NMR (CDCl3): δ 12.15 (s, 1H), 9.04
(s, 1H), 8.89 (d, 1H), 8.50 (d, 2H), 7.60 (m, 3H), 7.49 (m, 3H), 7.04
(s, 1H), 6.55 (s, 1H), 6.54 (s, 1H), 4.04 (d, 6H), 3.84 (s, 6H), 3.67
(s, 2H), 3.03 (m, 2H), 2.84 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-5-
methoxy-4-oxo-4H-chromene-2-carboxamide (16). Following
the procedure of compound 2 except 0.3 g of 27 was used instead of
0.15 g of 27 and 0.19 g of 5-methoxychromone-2-carboxylic acid was
used instead of 0.05 g of quinaldic acid, 0.23 g (yield 55%) of the title
compound 16 was obtained. 1H NMR (CDCl3): δ 12.39 (s, 1H), 8.62
(d, 1H), 8.15 (d, 2H), 7.78 (s, 1H), 7.64 (t, 1H), 7.48 (d, 2H), 7.36
(d, 1H), 7.15 (s, 1H), 6.84 (d, 1H), 6.63 (s, 1H), 6.56 (s, 1H), 4.02
(m, 9H), 3.85 (s, 6H), 3.76 (s, 2H), 3.09 (m, 2H), 2.91 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-6-
fluoro-4-oxo-4H-chromene-2-carboxamide (17). Following the
procedure of compound 2 except 0.3 g of 27 was used instead of 0.15
g of 27 and 0.16 g of 6-fluorochromone-2-carboxylic acid was used
instead of 0.05 g of quinaldic acid, 0.27 g (yield 66%) of the title
compound 17 was obtained. 1H NMR (CDCl3): δ 12.60 (s, 1H), 8.66
(s, 1H), 8.17 (d, 2H), 7.92 (dd, 1H), 7.87 (dd, 1H), 7.82 (s, 1H),
(CDCl3) δ: 11.77 (s, 1H), 9.54 (s, 1H), 8.92 (d, 1H), 8.78 (s, 1H),
8.55 (d, 1H), 8.20 (d, 2H), 7.93 (s, 1H), 7.60 (m, 3H), 6.69 (d, 2H),
4.14 (d, 6H), 3.96 (d, 6H), 3.79 (s, 2H), 3.14 (t, 2H), 2.95 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-2-
naphthamide (8). Following the procedure of compound 2 except
0.06 g of 2-naphthoic acid was used instead of 0.05 g of quinaldic acid,
1
0.15 g (yield 77%) of the title compound 8 was obtained. H NMR
(CDCl3): δ 11.65 (s, 1H), 8.79 (s, 1H), 8.69 (s, 1H), 8.23 (d, 1H),
8.11 (d, 1H), 7.97 (m, 3H), 7.60 (m, 2H), 7.44 (m, 3H), 6.62 (s,
1H), 6.56 (s, 1H), 4.08 (s, 3H), 4.03 (s, 3H), 3.86 (s, 6H), 3.69 (s,
2H), 3.03 (t, 2H), 2.85 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
pyrazine-2-carboxamide (9). Following the procedure of com-
pound 2 except 0.04 g of 2-pyrazine carboxylic acid was used instead
of 0.05 g of quinaldic acid, 0.14 g (yield 78%) of the title compound 9
was obtained. 1H NMR (CDCl3): δ 12.47 (s, 1H), 9.56 (d, 1H), 8.83
(d, 1H), 8.73 (s, 1H), 8.70 (m, 1H), 8.30 (d, 2H), 7.93 (s, 1H), 7.52
(d, 2H), 6.59 (d, 2H), 4.05 (d, 6H), 3.86 (d, 6H), 3.86 (d, 6H), 3.70
(s, 2H), 3.06 (t, 2H), 2.85 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-
benzamide (10). Following the procedure of compound 2 except
benzoic acid was used instead of quinaldic acid, 0.15 g (yield 84%) of
1
the title compound 10 was obtained. H NMR (CDCl3): δ 11.39 (s,
1H), 8.68 (s, 1H), 8.15 (d, 2H), 8.08 (d, 2H), 7.78 (s, 1H), 7.53 (m,
3H), 7.42 (d, 2H), 6.59 (s, 1H), 6.52 (s, 1H), 3.98 (d, 6H), 3.82 (s,
6H), 3.66 (s, 2H), 2.98 (t, 2H), 2.83 (m, 6H).
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-3,4-
difluorobenzamide (11). Following the procedure of compound 2
except 0.06 g of 3,4-difluorobenzoic acid was used instead of 0.05 g of
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Drug Annotation
7.56 (m, 3H), 7.29 (s, 1H), 6.65 (s, 1H), 6.58 (s, 1H), 4.06 (d, 6H),
3.88 (s, 6H), 3.72 (s, 2H), 3.08 (m, 2H), 2.88 (m, 6H).
DEDICATION
■
We gratefully honor the late Sung-Ki Lim, former Chairman of
Hanmi Pharmaceuticals. Sung-Ki Lim’s vision, dedication, and
leadership were imperative to the development of this research
program and his collaborative spirit was critical to the success
of the partnership between Athenex Inc. and Hanmi
Pharmaceuticals.
N-(2-(2-(4-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxyphenyl)-6-
methyl-4-oxo-4H-chromene-2-carboxamide (18). Following the
procedure of compound 2 except 0.08 g of 6-methylchromone-2-
carboxylic acid was used instead of 0.05 g of quinaldic acid, 0.16 g
(yield 79%) of the title compound 18 was obtained. 1H NMR
(CDCl3): δ 12.49 (s, 1H), 8.62 (s, 1H), 8.14 (d, 2H), 8.02 (s, 1H),
7.78 (s, 1H), 7.69 (d, 1H), 7.57 (d, 1H), 7.47 (d, 2H), 6.58 (d, 2H),
4.02 (d, 6H), 3.85 (d, 6H), 3.68 (s, 2H), 3.04 (t, 2H), 2.82 (m, 6H),
2.49 (s, 3H).
ABBREVIATIONS USED
■
P-gp, P-glycoprotein; BCRP, breast cancer resistance protein;
MRP, multidrug resistance protein; BSEP, bile salt export
pump; MATE, multidrug and toxin extrusion; OCT, organic
cation transporter; OATP, organic anion transporting poly-
peptide; CYP, cytochrome P450; AUC, area under the curve;
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01826.
P
app, apparent permeability; IV, intravenous; CNS, central
1H NMR and HPLC chromatograms for 1 and 14;
experimental details for in vitro and in vivo studies
(PDF)
nervous system; tPSA, topological polar surface area; PK,
pharmacokinetic; SAR, structure−activity relationship; EC50,
effective concentration giving 50% target inhibition; IC50,
inhibitory concentration giving 50% of maximal effect; MW,
molecular weight; HBA, hydrogen bond acceptor; HBD,
hydrogen bond donor; MDCK, Madin-Darby canine kidney
cell; SC, subcutaneous; WBC, white blood cells; SILAC, stable
isotope labeling with amino acids in cell culture; PO, per os
(oral); ADME, absorption, distribution, metabolism, and
excretion; ATPase, adenosine triphosphatase; Q3W, every 3
weeks; Q2D, every other day; QSAR, quantitative structure−
activity relationship; NDA, New Drug Application; FDA, U.S.
Food and Drug Administration; EMA, European Medicines
Agency; WHO, World Health Organization; INN, Interna-
tional Nonproprietary Name.
Molecular formula strings and some data (CSV)
AUTHOR INFORMATION
■
Corresponding Author
Michael P. Smolinski − Athenex Inc., Conventus Building,
Buffalo, New York 14203, United States; orcid.org/0000-
0002-5303-2618; Email: msmolinski@athenex.com
Authors
Sameer Urgaonkar − Athenex Inc., Conventus Building,
Buffalo, New York 14203, United States; orcid.org/0000-
0002-6012-0923
Laura Pitzonka − Athenex Inc., Conventus Building, Buffalo,
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Murray Cutler − Athenex Inc., Conventus Building, Buffalo,
New York 14203, United States
GwanSun Lee − Hanmi Pharmaceutical Co. Ltd., Songpa-gu,
Seoul 05545, Korea
Kwee Hyun Suh − Hanmi Pharmaceutical Co. Ltd., Songpa-
gu, Seoul 05545, Korea
Johnson Y. N. Lau − Athenex Inc., Conventus Building,
Buffalo, New York 14203, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01826
Notes
The authors declare the following competing financial
interest(s): M.P.S., S.U., L.P., M.C., and J.Y.N.L. are all
employees of Athenex and hold equity in Athenex. G.L. and
K.H.S. are employees of Hanmi.
ACKNOWLEDGMENTS
■
The authors are grateful to Professor Victor Ling and his team
for discovering P-glycoprotein which form the basis for our
work and thank Professor Ling personally for his encourage-
ment. Additionally, we would like to acknowledge the years of
effort and dedication from our Athenex and Hanmi colleagues
who believed in this program and rallied behind the hope of
bringing this important medication to patients. Special thanks
are extended to David Hangauer, Ahmed Said, and Yahao Bu
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