Structure-activity studies of Wnt/β-catenin inhibition in the Niclosamide chemotype: Identification of derivatives with improved drug exposure

Article abstract of DOI:10.1016/j.bmc.2015.07.001

The Wnt signaling pathway plays a key role in regulation of organ development and tissue homeostasis. Dysregulated Wnt activity is one of the major underlying mechanisms responsible for many diseases including cancer. We previously reported the FDA-approved anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. Niclosamide is a multi-functional drug that possesses important biological activity in addition to inhibition of Wnt/β-catenin signaling. Here, we studied the SAR of Wnt signaling inhibition in the anilide and salicylamide region of Niclosamide. We found that the 4′-nitro substituent can be effectively replaced by trifluoromethyl or chlorine and that the potency of inhibition was dependent on the substitution pattern in the anilide ring. Non-anilide, N-methyl amides and reverse amide derivatives lost significant potency, while acylated salicylamide derivatives inhibited signaling with potency similar to non-acyl derivatives. Niclosamide's low systemic exposure when dosed orally may hinder its use to treat systemic disease. To overcome this limitation we identified an acyl derivative of Niclosamide, DK-520 (compound 32), that significantly increased both the plasma concentration and the duration of exposure of Niclosamide when dosed orally. The studies herein provide a medicinal chemical foundation to improve the pharmacokinetic exposure of Niclosamide and Wnt-signaling inhibitors based on the Niclosamide chemotype. The identification of novel derivatives of Niclosamide that metabolize to Niclosamide and increase its drug exposure may provide important research tools for in vivo studies and provide drug candidates for treating cancers with dysregulated Wnt signaling including drug-resistant cancers. Moreover, since Niclosamide is a multi-functional drug, new research tools such as DK520 could directly result in novel treatments against bacterial and viral infection, lupus, and metabolic diseases such as type II diabetes, NASH and NAFLD.

Full text of DOI:10.1016/j.bmc.2015.07.001

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity studies of Wnt/b-catenin inhibition in the
Niclosamide chemotype: Identification of derivatives with improved
drug exposure
Robert A. Mook Jr. a, , , Jiangbo Wang a, , Xiu-Rong Ren a, Minyong Chen a, Ivan Spasojevic a,b
,
⇑
Larry S. Barak c, H. Kim Lyerly d, Wei Chen a,
⇑
a Department of Medicine, Duke University Medical Center, Durham, NC 27710, United States
b Duke Cancer Institute, PK/PD Core Laboratory, Durham, NC 27710, United States
c Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, United States
d Department of Surgery, Duke University Medical Center, Durham, NC 27710, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The Wnt signaling pathway plays a key role in regulation of organ development and tissue homeostasis.
Dysregulated Wnt activity is one of the major underlying mechanisms responsible for many diseases
including cancer. We previously reported the FDA-approved anthelmintic drug Niclosamide inhibits
Wnt/b-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. Niclosamide is a
multi-functional drug that possesses important biological activity in addition to inhibition of Wnt/b-cate-
nin signaling. Here, we studied the SAR of Wnt signaling inhibition in the anilide and salicylamide region
of Niclosamide. We found that the 40-nitro substituent can be effectively replaced by trifluoromethyl or
chlorine and that the potency of inhibition was dependent on the substitution pattern in the anilide ring.
Non-anilide, N-methyl amides and reverse amide derivatives lost significant potency, while acylated sal-
icylamide derivatives inhibited signaling with potency similar to non-acyl derivatives. Niclosamide’s low
systemic exposure when dosed orally may hinder its use to treat systemic disease. To overcome this lim-
itation we identified an acyl derivative of Niclosamide, DK-520 (compound 32), that significantly
increased both the plasma concentration and the duration of exposure of Niclosamide when dosed orally.
The studies herein provide a medicinal chemical foundation to improve the pharmacokinetic exposure of
Niclosamide and Wnt-signaling inhibitors based on the Niclosamide chemotype. The identification of
novel derivatives of Niclosamide that metabolize to Niclosamide and increase its drug exposure may pro-
vide important research tools for in vivo studies and provide drug candidates for treating cancers with
dysregulated Wnt signaling including drug-resistant cancers. Moreover, since Niclosamide is a multi-
functional drug, new research tools such as DK520 could directly result in novel treatments against bac-
terial and viral infection, lupus, and metabolic diseases such as type II diabetes, NASH and NAFLD.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 25 April 2015
Revised 24 June 2015
Accepted 1 July 2015
Available online xxxx
Keywords:
Niclosamide
Wnt signaling inhibitor
Small molecule
b-Catenin
Plasma concentration
Cancer
Disease treatment
1. Introduction
For example in colorectal cancer (CRC) more than 80% of all spo-
radic and hereditary cancers show hyperactivation of the pathway
The Wnt signaling pathway plays a key role in tissue develop-
ment and homeostasis and is dysregulated in many diseases.1–3
due to mutations in the adenomatous polyposis coli (APC) or the b-
catenin gene.1,2 Given the importance of the Wnt signaling activity
underlying tumor formation and metastasis, therapies against the
Wnt signaling pathway are highly sought.1
A mechanistic understanding of the canonical Wnt/b-catenin
signaling pathway has been studied over the past decades.4
Briefly, Wnt proteins are secreted glycoproteins that bind and acti-
vate the seven transmembrane receptor Frizzled and single trans-
membrane receptors LRP5/6. Wnt binding to Frizzled and LRP5/6
results in activation of cytosolic proteins called Dishevelled (Dvl),
leading to internalization of the Frizzled receptor.5 Downstream
Abbreviations: APC, adenomatous polyposis coli; CRC, colorectal cancer; Dvl,
Dishevelled; LEF/TCF, lymphoid enhancer factor/T cell factor; Fzd1, Frizzled1; GFP,
green fluorescent protein; NAFLD, non-alcoholic fatty liver disease; NASH, non-
alcoholic steatohepatitis; SAR, structure–activity relationships; Vss, volume of
distribution steady-state.
⇑
Corresponding authors. Tel.: +1 919 684 6635 (R.A.M.), +1 919 684 4433 (W.C.).
E-mail addresses: robert.mook@duke.edu (R.A. Mook), w.chen@dm.duke.edu (W.
Chen).
Contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2015.07.001
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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R. A. Mook Jr. et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
signaling events resulting from Wnt binding include the stabiliza-
tion and translocation of cytosolic b-catenin proteins into the
nucleus, activation of the transcription factor LEF/TCF and tran-
scription of Wnt/b-catenin target genes. Overall, the Wnt signal
transduction pathway consists of many protein–protein interac-
tions that traditionally have been difficult to target with small
drug-like molecules.1,6
Recent reports, including our own, have begun to provide
insights into mechanisms and chemical starting points for inhibit-
ing the Wnt pathway,7–13 though no drugs designed to target the
pathway have been approved to date.13 Through a high-through-
put drug screen we found Niclosamide (Fig. 1A), a drug approved
by the FDA for human use as an anthelminthic therapy, promotes
Frizzled internalization.7 Subsequent studies found Niclosamide
downregulates Dishevelled and b-catenin and inhibits colon cancer
cell growth in vitro and in vivo.7,9 Subsequently, other laboratories
have confirmed that Niclosamide inhibits Wnt signaling.14–16 Our
initial SAR studies indicated that inhibition of Wnt signaling by
Niclosamide appeared unique among the structurally-related sali-
cylanilide class of anthelmintic agents tested, and that the potency
and Wnt/b-catenin functional response was dependent on small
changes in the chemical structure of Niclosamide.8 In addition to
our SAR studies, a limited SAR study of Niclosamide-mediated inhi-
bition of S100A, a transcriptional target gene of b-catenin, was
Niclosamide improves glycemic control and delays disease pro-
gression reportedly through uncoupling of mitochondria oxidative
phosphorylation.30,32 In recent years, Niclosamide has generated
significant interest as
Niclosamide inhibits the proliferation of tumor cell lines from mul-
tiple tumor types, for example, breast, colon, lung, prostate, ovary,
a
potential anticancer agent.9,14,16,33–39
blood and pancreas, over an IC50 range of 0.13–4 lM that overlaps
with the IC50 of inhibition Wnt/b-catenin signaling,33 and has anti-
cancer activity in drug resistant cancers.9,38,40 Of particular interest
to cancer therapy, Niclosamide has been recently reported to inhi-
bit key oncogenic signaling pathways33 in addition to Wnt,7,9,14,15
such as Notch,41 mTOR,29 NF-kB,34 and STAT-3.42
The increased interest in Niclosamide’s inhibitory activity
against key pathological signaling pathways has stimulated a
search to improve its delivery and drug exposure to treat systemic
disease. Whereas the pharmacokinetic properties of Niclosamide
are appropriate for use in the gut as an anthelmintic agent, its
low solubility, low bioavailability and poor pharmacokinetic pro-
file results in low plasma exposure when dosed orally.9,18,27,43–45
Efforts to improve the solubility include the preparation of salt
forms and derivatives containing water-solubilizing groups.18,46,47
In the search for STAT-3 inhibitors with improved solubility to
treat cancer, amine water-solubilizing groups were added to the
Niclosamide molecule.48 More recently, efforts to identify nanopar-
ticle formulations for use in cancer have been reported.44,49 In one
study in which the pharmacokinetic parameters of nanocrystals of
Niclosamide were evaluated in vivo, the nanocrystal formulation
did not significantly change the plasma concentration vs time pro-
file when administered IV to rat, though increased tissue concen-
tration at 2 h was noted.44 To our knowledge, no formulation of
Niclosamide has been reported that increases its plasma concen-
tration or the duration of exposure.
As part of an effort to discover Wnt/b-catenin inhibitors based
on the Niclosamide chemotype with improved potency, selectivity
and pharmacokinetic properties for clinical evaluation, we contin-
ued our study of the structure–activity relationships of
Niclosamide by focusing on the anilide and salicylamide regions
of the molecule. Consistent with our earlier report, the SAR of inhi-
bition of Wnt signaling was dependent on both the nature and the
location of substitution.8 In particular we found that chloro or tri-
fluoromethyl substituents can replace the nitro substituent, a
major site of metabolism, and retain Wnt/b-catenin inhibitory
activity, and found modifications that can significantly increase
both the plasma concentration and the duration of exposure of
Niclosamide in mice. Whereas dosing mice with Niclosamide
results in low plasma concentrations and a short duration of expo-
sure,9 DK-520 (compound 32), a derivative of Niclosamide had the
unique property that it increased both the plasma concentration
and the duration of exposure of Niclosamide in vivo with no
observable adverse effects over three weeks of oral dosing. The
ability of DK-520 to metabolize to Niclosamide in a manner that
increases the exposure of Niclosamide when dosed orally may pro-
vide a valuable preclinical research tool to study Niclosamide
in vivo in animal models of disease indicated by its known biolog-
ical activity.
reported. Of the six derivatives of Niclosamide tested at 1
lM, none
reduced expression of S100A.14
Niclosamide is a multi-functional drug. It was used initially as
an anthelmintic agent in livestock in the early 1960s before being
approved by the FDA for use in humans in 1982 to treat tapeworm
infections.17,18 Niclosamide is included in the World Health
Organization’s list of essential medicines19 and has been used to
safely treat millions of patients. For such a widely used drug, its
mechanism of action has not been well-delineated, although it
has been reported to involve uncoupling of oxidative phosphoryla-
tion.20–22 In the years since its initial discovery, additional biolog-
ical activities of Niclosamide have been identified that have served
to generate considerable interest for its use to treat other human
diseases. Among the additional biological activities discovered
are antibacterial activity23 including antituberculous activity24
and drug resistant Staphylococcus aureus activity,25 antiviral activ-
ity,26,27 and anti-trypanosomal activity.28 Niclosamide has proto-
nophore activity and has effects on metabolism, including
activation of AMPK.26,29–31 In animal models of type 2 diabetes,
2. Material and methods
2.1. Compound synthesis
Figure 1. Frizzled1-GFP internalization by Niclosamide and related derivatives. (A)
Structure of Niclosamide (B) confocal images of U2OS cells harboring Frizzled1-GFP
treated with DMSO control, Niclosamide, compound 15, compound 24 for 6 h at
Anilide synthesis. General method. To a 100 mL flask equipped
with a reflux condenser was added 5-chloro-2-hydroxybenzoic
acid (1 equiv), the aniline derivative (1 equiv), and dry xylenes
(stored over 3A molecular sieves, 40 mL per gram of 5-chloro-2-
hydroxybenzoic acid) under an Argon atmosphere. The mixture
12.5 lM. Punctuate structures (white arrows) highlight internalized Frizzled1-GFP
vesicles. Compounds 15 and 24 provided as representative examples of scoring.
Scoring: DMSO control = 0; Niclosamide = 5; compound 15 = 1, and compound
24 = 3. See Table 1 for structures of 15 and 24.
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R. A. Mook Jr. et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
was heated to reflux, and PCl3 (0.4 equiv) was added rapidly via
syringe. The mixture was heated at reflux for 1 h and cooled to
room temperature. Water (40 mL per gram of 5-chloro-2-hydroxy-
benzoic acid) was added and the resultant heterogeneous mixture
stirred rapidly for 1 h. Saturated sodium bicarbonate was added to
a final pH of 3–4, and the mixture stirred rapidly overnight. The
solids were filtered and washed sequentially with water, toluene
and hexane. Samples were analyzed by NMR, HPLC/mass spec-
trometry and TLC. Purification by crystallization or column chro-
matography on silica gel was performed when purity was less
than 95% by LC. Additional experimental procedures and analytical
data are provided in Supplemental data.
with compounds from 0.04 to 10
the cells were analyzed by colorimetric MTS assay (Promega,
Madison, WI, USA).
l
M for 72 h, after which point
2.6. Pharmacokinetic analysis
For Niclosamide dosed IV, Niclosamide was dissolved in a sol-
vent of 67% polyethylene glycol 400 and 33% N,N-dimethylac-
etamide at a concentration of 3.27 mg/ml and tail vein injected
at a dose of 2.6 mg/kg of body weight. Blood samples were
obtained at predose and at 0.08, 0.17, 0.33, 0.67, 1.5, 4, 8, 12 h after
drug administration. For compound 34 dosed orally, compound 34
was dissolved in a solvent of 90% polyethylene glycol 300 and 10%
1-methyl-2-pyrrolidinone at a concentration of 20 mg/ml and gav-
aged at a dose of 200 mg/kg of body weight. Blood samples were
obtained at predose and at 0.25, 0.5, 1, 2, 4, 8 h after drug admin-
istration. For DK-520 (compound 32) dosed orally, DK-520 was dis-
solved in corn oil at a concentration of 20 mg/ml and dosed at
200 mg/kg of body weight. Blood samples were obtained at pre-
dose and at 0.25, 0.5, 0.75, 1, 1.5, 4, 8, 12, 24, 48, and 72 h after drug
administration. All the solvent reagents were purchased from
Sigma-Aldrich (St. Louis, MO, USA). CD1 mice were used in the
pharmacokinetic studies. Quantification of Niclosamide in mouse
plasma was done by LC/MS–MS using methods similar to those
previously published.9
2.2. Frizzled internalization assay
The Fzd1-GFP assay was performed following a procedure sim-
ilar to previously published work.7 Briefly, cells stably expressing
Frizzled1-GFP plated in confocal dishes were treated, after 24 h,
with 12.5 lM of test compound or DMSO for 6 h at 37 °C followed
by fixation with 4% paraformaldehyde. The cells were then exam-
ined by microscopy using a LSM 510-Meta confocal microscope
(Carl Zeiss, Thornwood, NY, USA) equipped with 40ꢀ and 100ꢀ
apo chromat objectives. YFP was excited using a 488-nm argon
laser line. Images were processed using the LSM software Image
Browser (CarlZeiss, Thornwood, NY, USA). Plates were read twice
in blinded fashion using a 0–5 point scale. a Punctate similar to con-
trol = 0, trace amount of punctate greater than control = 1, moder-
ate = 3, strong = 5.
2.7. In vivo toxicity study
NOD/SCID mice received oral administration of vehicle (corn
oil) or DK-520 (32) (200 mg/kg in corn oil) 6 days a week for
3 weeks. The animals were observed throughout the period for
any side effect. The body weight was measured the same day of
the first dosing (week 0) and the day after the last dosing (week 3).
2.3. TOPFlash reporter assay
Wnt-3A conditioned medium was prepared using L WNT-3A
cells (ATCCÒ CRL-2647™) purchased from ATCC. Conditioned med-
ium was obtained following published protocols (http://www.atcc.
org/Products/All/CRL-2647.aspx#culturemethod).7 HEK293 cells
were stably transfected with p8xTOPFlash, Renilla luciferase plas-
mid pRL-TK (Promega), and pLKO.1 as previously published.7
3. Results
In our previous studies and the studies herein, we relied on the
Fzd1-GFP confocal microscopy (Fig. 1) and the Wnt-stimulated
TOPFlash assays to interrogate the SAR of Niclosamide and related
derivatives.8 Our previous SAR studies indicted the 40-position of
the nitro anilide portion of Niclosamide had a significant impact
on Wnt-mediated biological activity and that modification at this
position could produce molecules that either increased or
decreased Wnt3A-mediated signaling.8 Given that nitro sub-
stituents often impart poor solubility and poor pharmacokinetic
properties, and that reduction of the nitro group is a major
metabolite of Niclosamide18, we interrogated the SAR in the anilide
to identify inhibitors of Wnt signaling with improved drug
properties.
The SAR of the anilide region could be readily interrogated by
the synthesis of derivatives prepared via the coupling of substi-
tuted anilines with 5-chloro-2-hydroxybenzoic acid (Scheme 1).
Preparation of Niclosamide derivatives in which the nitro group
was replaced with bioisosteric groups such as acetoyl and benzoyl
(compound 4, 7), methyl sulphone (compound 6) or amide (com-
pounds 5, 8) resulted in a significant loss of activity in both the
Fzd1-GFP internalization and Wnt-3A-stimulated TOPFlash assay
(Table 1, Fig. S1, Supplemental information). We then explored
substituents that mimicked the electron-withdrawing characteris-
tics of the nitro group. Substitution of the 40-NO2 group with a 40-
CF3 group (compounds 9 and 10) produced compounds that inter-
nalized Fzd1-GFP and inhibited Wnt3A-stimulated signaling in the
TOPFlash assay with potency similar to Niclosamide and had better
solubility in organic solvents. Moving the CF3 substituent to the 30-
position (compound 11) led to a loss of activity, similar to previous
Briefly, stably transfected cells were seeded in 100 ll of cell growth
medium/well in 96-well plates at 100% confluency. Fifty micro-
liters of Wnt-3A conditioned medium containing the chemical
compounds to be tested or DMSO was added to each well. After
an 8 h treatment, the cells were washed once with PBS and lysed
with 55 ll of Passive Lysis Buffer supplied in the Dual-Luciferase
Reporter Assay kit (Promega, Madison, WI). Twenty-five micro-
liters of cell lysate was used for measuring luciferase activity in a
96-well plate reader (FluoStar Optima, BMG Labtech, Chicago, IL).
2.4. Western blot
Western blots were performed following a procedure similar to
previously published work.9 Briefly, HCT-116 cells were grown to
about 80% confluency in poly-
D-lysine coated six-well plates 48 h
before treatment and followed by 2.0
l
M compounds or DMSO
incubation for 18 h in growth medium. After treatment, the cytoso-
lic fraction was isolated as described.9 Immunoblots using antibod-
ies to b-catenin (E-5, Santa Cruz Biotechnology catalog number sc-
7963) was used to detect b-catenin protein levels in the cytosol,
and immunoblots using antibodies to b-actin (C-4, Santa Cruz
Biotechnology, catalog number SC-47778) was used for loading
controls.
2.5. Cell proliferation assay
The colon cancer cell line HCT116 was used in the cell prolifer-
ation assay. The cells were plated in 100
ll of growing med-
ium/well in 96-well plates at 5000 cells per well and treated
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R. A. Mook Jr. et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
and the b-catenin Western blot assay were also active in the MTS
assay. The IC50 of inhibition of the active compounds,
Niclosamide, compounds 9, 10 and 16 are 0.45 0.05, 0.54 0.08,
0.67 0.09 and 1.18 0.14 lM, respectively.
Given the site of the major metabolite of Niclosamide had been
removed in compound 9 and 16, and the fact that Niclosamide was
efficacious in xenograft models despite exposure well below the
IC50 of inhibition of Wnt signaling in cell culture during the major-
ity of the dosing period,9 we next asked whether these compounds
would have better exposure that could improve the anti-tumor
responses in vivo. Upon searching the literature we found that
the PK properties for both compounds in rat had been reported.27
In these studies, the authors report that the bioavailability of com-
pound 9 and 16 was similar to Niclosamide (%F = 15, 12 and 10,
respectively). In each molecule, compound 9, 16, and
Niclosamide, the clearance was moderate (39, 27, and
20 mL/Kg/min, respectively), the Volumes of Distribution were
low (Vss = 1.1, 0.3, 0.9 L/kg, respectively), and the t1/2 values were
relatively short (3.7, 2.6, 6.7 h, respectively). Based on this data, we
reasoned that factors other than simply metabolism of the nitro
group may need to be addressed to improve the PK properties of
compounds in the Niclosamide chemotype.
It is well known that pharmacokinetic performance, including
oral bioavailability, is influenced by multiple parameters such as
solubility, permeability, metabolic stability, pKa, logP, hydrogen
bond donors, among others.50,51 In light of the published pharma-
cokinetic data we reasoned that the salicylamide group might also
play an important role in limiting the exposure of the inhibitors.
The phenolic–OH group is a potential site of glucuronidation and
clearance. Moreover, the calculated pKa of the ꢁOH group
(pKa = 6.8)52 indicates that the molecule would be substantially
ionized at the pH of intestinal fluid (pH = 4ꢁ8), and the pH of blood
(pH = 7.4). Ionization of the –OH group, would be expected to limit
exposure by reducing the permeability of the molecule and limit
the volume of distribution, both of which could be expected to
reduce exposure. In addition, the salicylamide moiety possesses
two hydrogen bond donors, albeit with the potential for
intramolecular hydrogen bonding, that could be expected to
reduce permeability.51 Based on this reasoning, we next turned
out attention to the SAR of the amide linker and the phenol that
make up the salicylamide functionality.
Molecules to interrogate the SAR of the salicylamide were pre-
pared as outlined in Scheme 2 or purchased from chemical ven-
dors. We found that reversing the orientation of the amide
resulted in the complete loss of activity (compound 26) (Table 2,
Fig. S1, Supplemental information). Insertion of a carbon atom
between the nitrogen atom of the amide and the aromatic ring also
reduced the potency of inhibition (Compounds 28, 29), as did
replacement of the amide NH substituent with an N-Me sub-
stituent (compound 27). Replacing the amide with a sulphonamide
substituent produced a compound (30) that inhibited Wnt signal-
ing in the TOPFlash assay in the low micromolar range and induced
Fzd1-GFP internalization very weakly. Our previous work indicated
that 2-methoxy substituted derivatives were not active inhibitors
of Wnt signaling.8 Thus, in an effort to reduce phase II metabolism
and ionization of the –OH group and reduce the number of hydro-
gen bond donors, we explored 40-CF3 and 40-NO2 anilide deriva-
tives in which the phenol was converted to ester (compound 31,
32) and carbonate (compound 33). In addition, we explored the
activity of compounds in which both –OH and –NH groups were
modified by tying the phenol and the amide together in a 6-mem-
bered ring oxazine-dione motif (compounds 34, 35, and 36), a
modification that removes two hydrogen bond donors. Upon test-
ing these compounds, we were delighted to find that both the 40-
CF3 and the 40-NO2 anilide derivatives produced a robust punctate
pattern in the Fzd1-GFP internalization assay and inhibited Wnt-
Scheme 1. Synthesis of anilide derivatives.
findings with 30-NO2 substitution.8 In contrast to compounds with
40-NO2 substitution (Niclosamide 1 and compound 3), compounds
with 40-CF3 substitution (compounds 9 and 10) containing a 20-Cl
substituent, vs
a
20-H substituent had comparable potency.
Replacement of 20-Cl with 20-F in the 40-CF3 series (compound
12) resulted in similar activity to compounds with 20-Cl, and 20-H
substitution (compounds 9, 10). Substitution of the 40-NO2 group
in Niclosamide with a 40-Cl group (compound 16) resulted in a
compound that internalized Fzd-1-GFP and inhibited Wnt3A-stim-
ulated signaling with potency comparable to Niclosamide. The
potency of compounds with two chlorine substituents was influ-
enced by the substitution pattern. Compound 16 in which the ani-
lide was substituted with chlorine at the 20- and 40-postion had
potency similar to Niclosamide. Compounds with 20,50-dichloro
substitution (compound 17), 30,50-dichloro substitution (com-
pound 18) and 30,40-dichloro substitution (compound 19) were
generally less potent, while 20,60-dichloro substitution (compound
20) produced a compound that was inactive in both assays up
12 lM, the highest concentration tested. Dichloro derivatives
tended to be more potent than mono-chloro derivatives (com-
pounds 13, 14, 15). Compounds in which a chlorine atom was
replaced with a fluorine atom produced compounds that were less
potent than the similarly substituted chloro derivative (com-
pounds 21, 22, 24, 25). Here also, the 20,60-difluoro substituted ani-
lide derivative (compound 23) was not active up to 12 lM,
supporting the notion that the substitution pattern of halogen in
the anilide ring is important for potency. Whereas these studies
did not identify molecules more potent than Niclosamide, they
did identify compounds with similar potency and better overall
handling and solubility properties than Niclosamide in solvents
used in synthesis and purification. In particular, compounds in
which the nitro substituent was replaced with a trifluoromethyl
group (compounds 9, 10) or a chlorine group (compound 16) were
similar in potency to Niclosamide in both the FZD1-GFP internal-
ization and the Wnt3A-stimulated b-catenin TOPFlash transcrip-
tion assays (Table 1 and Fig. 2A). The IC50 of inhibition in the
TOPFlash assays for Niclosamide and compounds 9, 10 and 16
are 0.34 0.08, 0.56 0.21, 0.29 0.06, and 0.42 0.1
respectively.
lM,
To further characterize the Wnt inhibitory and the cancer cell
anti-proliferation activity of these compounds, we evaluated these
compounds along with an inactive derivative (compound 20,
Table 1) in HCT-116 colorectal cancer cell culture. Inhibition of
Wnt signaling was determined by analyzing the reduction of
cytosolic b-catenin by Western blot (Fig. 2B). Upon treating cells
with 2.0 lM of compound for 18 h in culture, b-catenin levels were
significantly decreased only by the compounds that showed activ-
ity in the FZD1-GFP and Wnt-3A stimulated TOPFlash assay. The
amount of cytosolic b-catenin remaining relative to DMSO control
without compound was: Niclosamide: 8%; compound 9: 6%; com-
pound 10: 7%; and compound 16: 7%. The amount of cytosolic b-
catenin remaining for the inactive derivative, compound 20,
equaled 100% of the amount of the DMSO control. Anti-prolifera-
tion activity was also measured by MTS assay (Fig. 2C). In this
assay, the inactive derivative, compound 20, had no effect up to
10
lM. In contrast, the compounds that were active in the FZD1-
GFP internalization assay, the Wnt3A-stimulated TOPFlash assay
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R. A. Mook Jr. et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
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Table 1
Table 1 (continued)
Structure–activity relationships in the anilide ring
Compound Structure
Fzd1-GFP
Inhibition
ClogP pKa
number
R
Internalization Wnt/b-
NHb
OH
O
@12.5
l
Ma
catenin
R
transcription
TopFlash IC50
(lM) SE
Cl
Compound Structure
Fzd1-GFP
Inhibition
ClogP pKa
23
ND
>12
3.9
12.1
number
R
Internalization Wnt/b-
NHb
@12.5
l
Ma
catenin
24
25
3
5
1.41 0.21
1.21 0.17
4.4
4.4
12.2
12.5
transcription
TopFlash IC50
(lM) SE
1
5
0.34 0.08
3.1
11.2
Internalization of Frizzled1-GFP stably expressed in U2OS cells was determined by
confocal microscopy. Cells were treated with compounds for 6 h, fixed, and scored
visually by the amount of punctate observed versus DMSO control. Inhibition of
Wnt3A-stimulated Wnt/b-catenin transcription was determined by TOPFlash nor-
malized to renilla control. ClogP and pKa values were calculated in Maestro 9.3
(Schrodinger, Inc.) using Qikprops and Epik.
2
3
0
5
11.81
3.3
2.7
13.2
12.1
0.99 0.32
a
Punctate similar to control = 0, trace amount of punctate greater than con-
trol = 1, moderate = 3, strong = 5.
b
Calculated pKa values in water are reported. The calculated value of the pKa of
4
5
6
0
0
0
6.42 0.51
>12
3.0
1.9
2.6
12.5
12.7
11.4
the –OH group is 6.8 in each example listed. The calculated pKa of the NH is the
salicylamide NH without ionization of the –OH group. The estimated error of the
pKa calculation for the OH group is 1 log unit, and 2 for the NH group. ND: not
determined.
signaling in the TOPFlash assay (Table 2, Fig 3A). The IC50 of inhibi-
tion in the TOPFlash assay for Niclosamide and compounds 32, 33
7.66
and 34 are: 0.34 0.08, 0.23 0.06, 0.34 0.12 and 0.33 0.1 lM,
respectively.
7
8
9
0
0
5
15.04
4.2
4.1
4.8
12.5
12.6
11.9
Similar to earlier studies, we selected the active derivatives,
compound 32, 33, and 34 and a less active derivative, compound
27 (Table 2), for further evaluation in HCT-116 cells (Fig. 3). As
anticipated by the FZD1-GFP and TOPFlash data, the active com-
pounds inhibited Wnt/b-catenin signaling as measured by the
reduction of cytosolic b-catenin (Fig. 3B). Upon treating cells with
32.89
0.56 0.21
10
11
5
5
0.29 0.06
0.89 0.32
4.5
4.7
12.4
12.5
2.0 lM compound for 18 h in culture, b-catenin levels were signif-
icantly decreased by the active compounds. The amount of cytoso-
lic b-catenin remaining relative to DMSO control without
compound were: Niclosamide: 10%; compound 32: 8%; compound
33: 8%; and compound 34: 9%. The amount of cytosolic b-catenin
remaining for the less active derivative, compound 27, was 96%,
nearly equal to the amount remaining in the DMSO control.
Likewise, only the active compounds inhibited proliferation of
HCT-116 cells in culture by MTS assay, with potency comparable
to Niclosamide (Fig. 3C). The IC50 of inhibition in the MTS assay
for Niclosamide and compounds 32, 33 and 34 are: 0.45 0.05,
12
5
0.55 0.11
4.5
11.8
13
14
15
3
5
1
1.30 0.18
1.65 0.12
3.05 0.87
4.0
4.0
3.9
12.8
12.6
12.3
0.61 0.11, 0.51 0.06 and 0.55 0.07 lM, respectively.
Based on the data in the Wnt/b-catenin signaling assays, the
proliferation assay, and the structural modifications that eliminate
the 40nitro group, ionization of the –OH group and the removal of
two hydrogen bond donors, we next asked whether compound 34
improved exposure in mice compared to Niclosamide at
200 mg/kg, the dose used in previous tumor xenograft studies.9
Upon dosing a solution of compound 34 orally at 200 mg/kg we
were disappointed to find the plasma exposure of the compound
was still comparable to Niclosamide,9 and that exposure was not
significantly improved. Based on the hypothesis compound 34
could be metabolized to an active metabolite (compound 9) that
would reduce plasma levels of compound 34, we also analyzed
plasma for this potential metabolite. Indeed, we could detect com-
pound 9 in the plasma samples from mice dosed with compound
34, but unfortunately, the levels of this active metabolite was still
low and only comparable to the levels obtained by dosing
Niclosamide to mice at 200 mg/kg9 (Fig. S2, Supplemental
information).
16
17
5
5
0.42 0.10
0.75 0.13
4.5
4.4
11.9
11.7
18
19
5
5
0.70 0.13
0.73 0.13
4.5
4.5
12.0
12.2
20
21
22
0
0
2
>12
4.3
3.9
4.0
11.4
13.1
12.6
3.84 0.29
4.02 0.79
Please cite this article in press as: Mook Jr., R. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.001

6
R. A. Mook Jr. et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 2. Niclosamide and its derivatives inhibit Wnt3A/b-catenin signaling and proliferation of HCT-116 cells. (A) Inhibition of Wnt signaling in TOPFlash assay. HEK293
cells stably expressing TOPFlash luciferase reporter and Renilla luciferase were treated with Wnt3A-conditioned medium in the presence of DMSO or compounds from 0.04 to
10
catenin levels. HCT-116 cells were treated with 2.0
and probed with antibodies to b-catenin as described in Section 2. b-Actin was used as a loading control. (C) Inhibition of proliferation of colon cancer cell HCT116. Cells were
treated with DMSO or compounds from 0.04 to 10 M for 72 h, and cell viability was measured using the MTS assay. The cell viability with DMSO treatment was set at 100%.
lM. The TOPFlash reporter activity of Wnt3A with DMSO treatment was set as 100%. Data were fit using GraphPad Prism (mean SEM, n P2). (B) Reduction of cytosolic b-
lM compounds for 18 h, lysed in hypotonic buffer. Lysates were eluted by SDS electrophoresis on acrylamide, transferred,
l
Data were fit using GraphPad Prism (mean SEM, n = 3).
Having replaced the nitro group, and blocked the site of ioniza-
tion and glucuronidation, we reasoned that the parameter now
limiting oral exposure was likely to be poor solubility in aqueous
media. Given these inhibitors are somewhat hydrophobic with
clogPs of 4–5, we questioned whether formulation in oil-based
vehicles, such as corn oil, might be better vehicles for PK stud-
ies.53,54 To test these concepts, we decided to study DK-520 (com-
pound 32), a derivative of Niclosamide because this compound was
freely soluble in corn oil, and because it could metabolize to
Niclosamide, a compound for which we had established and
well-developed methods to accurately measure its plasma concen-
tration. Upon dosing a solution of DK-520 to mice at 200 mg/kg in
corn oil, we were delighted to find DK-520 provided significantly
increased plasma exposure of Niclosamide (Fig. 4) when compared
to published studies of 200 mg/kg Niclosamide dosed orally.9 The
Cmax, the AUC and the duration of exposure of Niclosamide
obtained by dosing DK-520 were all increased (Fig. S3,
Supplemental information), even though on a molar basis, the
amount of DK-520 dosed was approximately 1/3 less than the
amount of Niclosamide dosed. In fact, the plasma levels of
Niclosamide obtained by dosing DK-520 at 200 mg/kg were above
the IC50 of inhibition of Wnt signaling in the TOPFlash assay for
nearly 24 h, whereas the reported plasma levels of Niclosamide
dosed as a solution at 200 mg/kg were only above the IC50 of
Wnt inhibition in the TOPFlash assay for less than 1 h.9
Moreover, DK-520 dosed orally at 200 mg/kg was well tolerated
when administered daily for three weeks, as judged by body
weight and observing the behavior of the mice (Fig. S4,
Supplemental information).
CRC.7,9 In our in vivo tumor models, oral administration of
Niclosamide inhibited tumor growth and decreased levels of Dvl
and b-catenin in tumors implanted subcutaneously despite poor
exposure to Niclosamide during the dosing period. In humans,
low systemic exposure of Niclosamide given orally could mitigate
its utility to treat disseminated metastatic disease.
To aid the translation of our initial findings toward a clinical
application, we conducted additional SAR studies seeking to iden-
tify agents with increased potency, selectivity, and pharmacoki-
netic exposure. Since bio-reduction of the nitro group is a major
metabolite of Niclosamide, and that nitro groups can impart poor
solubility and reduce oral exposure, we focused initially on the
SAR of the anilide ring. Consistent with our previous report, activ-
ity of these derivatives in the FZD1-GFP internalization assay and
Wnt3A-stimulated b-catenin TOPFlash gene transcription were in
general agreement. Molecules that produced more potent inhibi-
tion in the Wnt3A-stimulated b-catenin TOPFlash gene transcrip-
tion assay also produced stronger responses in the FZD1-GFP
internalization assay. In the anilide ring, we found that the potency
of inhibition was dependent on both the nature and the location of
substitution. Replacement of the electron-withdrawing 40-nitro
group in Niclosamide or in compound 3 with electron-withdraw-
ing substituents such as acetoyl, benzoyl, amide, and sulphonyl
groups were, in general, all inactive in the FZD1-GFP internaliza-
tion assay and were weak inhibitors in the Wnt3A-stimulated
TOPFlash assay. Given reports that Niclosamide uncouples oxida-
tive phosphorylation, perhaps by acting as a protonophore, we cal-
culated the clogP and the pKa of the OH and NH groups, SAR
parameters associated protonophore activity.31 Here, active and
inactive molecules were in a similar clogP range, had identical
OH pKa values, and variable NH pKa values. In contrast, to the above
derivatives, replacement of the 40-nitro group with electron-with-
drawing 40-trifluoromethyl or 40-chloro groups yielded molecules
of similar potency to Niclosamide. Moving the 40-trifluoromethyl
4. Discussion
In our earlier studies, we demonstrated that Niclosamide inhi-
bits Wnt/b-catenin signaling in vitro and in vivo in models of
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R. A. Mook Jr. et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
Scheme 2. Synthesis of salicylamide derivatives. Reagents and conditions: (i) Route A: PCl3, ArNHMe,
D, xylene; route B: (1) (COCl)2, ArCH2NH2, pyr, 0–5 °C; (ii) ArCOCl, pyr,
0–5 °C; (iii) (a) ClSO2H, (b) ArNH2, pyr, 0–5 °C; (c) PhSNa, DMF, . (iv) R3COCl, pyr, THF; (v) triphosgene, NEt3, THF.
D
or -chloro substituent to other positions in the ring generally
yielded compounds of less potency. The clogP values of the triflu-
oromethyl or chloro substituted compounds were values higher
than Niclosamide, the calculated pKa of the OH groups were
identical, and the pKa of the NH protons varied. The impact of
the structural changes and differences in calculated properties on
protonophore activity and uncoupling of oxidative phosphoryla-
tion activity requires further investigation. With respect to inhibi-
tion of Wnt/b-catenin signaling, as anticipated by the FZD1-GFP
internalization and TOPFlash assay data, compounds with similar
activity to Niclosamide provided similar reduction in cytosolic b-
catenin by Western blot and similar reduction in cell proliferation
by MTS assay in HCT-116 cells. In the salicylamide region, revers-
ing the orientation of the amide, inserting an atom between the
amide nitrogen and the aryl ring, methylation of the amide, and
replacing the amide with a sulphonamide group all reduced activ-
ity. Acylation of the phenolic OH group or tying it back to the
amide via a six-membered oxazine-dione ring motif produced
compounds of similar potency to Niclosamide in the FZD1-GFP
internalization and TOPFlash assays and again yielded a similar
reduction in cytosolic b-catenin by Western blot and similar
reduction in cell proliferation by MTS assay in HCT-116 cells. The
cell-based format of these assays could result in the metabolism
of the compounds in the assay to yield compounds with a phenolic
OH group. Additional studies are needed to better understand the
SAR of these derivatives.
Given the need to improve the systemic exposure of
Niclosamide given orally, we also interrogated structural features
in the anilide and salicylamide for their impact on plasma expo-
sure. Using medicinal chemical principles known to influence
absorption and pharmacokinetic properties, we evaluated specific
changes to the chemical structure. From these studies we discov-
ered DK-520 a derivative of Niclosamide, can be metabolized
in vivo and dramatically increase both the plasma concentration
of Niclosamide and its duration of exposure. Functionally, DK-
520 and Niclosamide had similar Wnt/b-catenin inhibitory activity
and effects on cell proliferation in our assays. When DK-520 was
dosed orally to mice, Niclosamide was detected in plasma in con-
centrations that exceeded the IC50 of inhibition of Wnt/b-catenin
signaling in the TOPFlash assay for as long as 24 h after a
200 mg/kg dose. Importantly, this high level of exposure to
Niclosamide did not result in toxicity as judged by changes in body
Please cite this article in press as: Mook Jr., R. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.001

8
R. A. Mook Jr. et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
Structure–activity relationships in the salicylamide motif
Compound number
Structure
Fzd1-GFP internalization
@12.5
Ma
Inhibition Wnt/b-catenin transcription
Clog P
pKa
OH
pKa
l
TopFlash IC50
(
lM) SE
NHb
26
27
ND
0
>12
2.2
4.5
8.3
8.4
12.1
—
>12
28
29
0
0
15.10 1.40
>12
4.6
4.2
8.4
7.4
12.0
11.9
30
1
5
5
5
2.57 0.27
0.32 0.03
0.23 0.06
0.34 0.12
3.8
4.2
4.3
4.8
6.6
—
6.2
9.6
31
32 (DK-520)
33
—
9.4
—
10.5
34
35
36
5
5
0.33 0.10
0.32 0.03
0.37 0.13
4.3
3.9
2.6
—
—
—
—
—
—
ND
Internalization of Frizzled1-GFP stably expressed in U2OS cells was determined by confocal microscopy. Cells were treated with compounds for 6 h, fixed, and scored visually
by the amount of punctate observed versus DMSO control. Inhibition of Wnt3A-stimulated Wnt/b-catenin transcription was determined by TOPFlash normalized to renilla
control. ClogP and pKa values were calculated in Maestro 9.3 (Schrodinger, Inc.) using Qikprops and Epik.
a
Punctate similar to control = 0, trace amount of punctate greater than control = 1, moderate = 3, strong = 5.
Calculated pKa values in water are reported. The calculated pKa of the NH is the salicylamide NH without ionization of the –OH group. The estimated error of the pKa
b
calculation for the OH group is 1 log unit, and 2 for the NH group. ND: not determined.
weight or by observing the mice behavior when dosed at
200 mg/kg over three weeks. Previously we and others9,18,27,30
reported that Niclosamide is cleared rapidly when dosed by the
oral, intraperitoneal (IP), or intravenous (IV) route. To our knowl-
edge, this is the first report to describe the ability to increase the
systemic drug exposure of Niclosamide in plasma and extend its
duration of exposure. Further studies are needed to define the
plasma, tissue and tumor levels of the parent compound DK-520,
tissue and tumor levels of the metabolite Niclosamide, and the
mechanism responsible for its increased absorption and conver-
sion to Niclosamide. Regardless of the mechanism, DK-520 pro-
vides a significant level and duration of plasma exposure to the
drug Niclosamide to enable in vivo assessment of Niclosamide in
animal models of disease. Given the extent of systemic exposure,
DK-520 and related derivatives may provide exposure suitable to
evaluate inhibition of Wnt/b-catenin signaling by Niclosamide in
the clinic.
addition to Wnt/b-catenin signaling, (2) is active in drug-resistant
cancers,9,38,40 and (3) appears well-tolerated in vivo, greater selec-
tivity may not give better anti-tumor effects. In other words, the
current selectivity profile of Niclosamide may be clinically valuable
in its own right such that the ability of Niclosamide to inhibit
important anticancer targets such as mTOR, Notch, NF-kB and
STAT-3 in addition to Wnt, may provide important therapeutic
benefits versus more selective agents. Thus, a benefit of derivatives
of Niclosamide such as DK-520 that increase the exposure of
Niclosamide in vivo, is that they may behave in part, like pro-drugs
that metabolize to produce the multi-functional drug Niclosamide.
As prodrugs of Niclosamide they could be valuable agents to study
clinically to treat a broad range of diseases in which Niclosamide
has demonstrated biological activity in preclinical models.
5. Conclusion
Efforts are underway to identify the target responsible for inhi-
bition of Wnt signaling and to understand the SAR that impacts the
selectivity of Niclosamide. However, given Niclosamide is a multi-
functional drug that (1) inhibits other important oncogenic signal-
ing pathways such as mTOR,29 Notch,41 NF-kB34 and STAT-342 in
SAR studies in the Niclosamide chemotype have identified
structural features that impact inhibition of Wnt/b-catenin signal-
ing and plasma exposure when dosed orally. Through these stud-
ies, we identified multiple active derivatives of Niclosamide
including DK-520 (compound 32) that when dosed orally, can be
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R. A. Mook Jr. et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
Figure 3. Salicylamide derivatives inhibit Wnt3A-stimulated b-catenin signaling and proliferation of HCT-116 cells. (A) Inhibition of Wnt signaling in TOPFlash assay.
HEK293 cells stably expressing TOPFlash luciferase reporter and Renilla luciferase were treated with Wnt3A-conditioned medium in the presence of DMSO or compounds
from 0.04 to 10
cytosolic b-catenin levels. HCT-116 cells were treated with 2.0
transferred, and probed with antibodies to b-catenin as described in Section 2. b-Actin was used as a loading control. (C) Inhibition of proliferation of colon cancer cell
HCT116. Cells were treated with DMSO or compounds from 0.04 to 10 M for 72 h, and cell viability was measured using the MTS assay. The cell viability with DMSO
lM. The TOPFlash reporter activity of Wnt3A with DMSO treatment was set as 100%. Data were fit using GraphPad Prism (mean SEM, n P2). (B) Reduction of
l
M compounds for 18 h, lysed in hypotonic buffer. Lysates were eluted by SDS electrophoresis on acrylamide,
l
treatment was set at 100%. Data were fit using GraphPad Prism (mean SEM, n = 3).
of Niclosamide-based drug candidates to treat disease associated
with its multi-function activity ranging from cancer,33 particularly
cancers with dysregulated Wnt signaling,1,9 drug-resistant
cancers9,38,40 including resistant cancers due to up-regulation of
genes involved in oxidative phosphorylation,55–57 to bacterial and
viral disease,23–27 and lupus58 to metabolic diseases such as type
II diabetes,30,32 non-alcoholic fatty liver disease (NAFLD), and
non-alcoholic steatohepatitis (NASH).59,60
Acknowledgements
This work was funded in part by 5 R01 CA172570 (W.C.),
BC123280 (W.C.), and Clinical Oncology Research Center
Development Grant 5K12-CA100639-08 (R.A.M., M.C.). Wei Chen
is
a V foundation Scholar and an American Cancer Society
Figure 4. Plasma concentration of Niclosamide. CD1 mice were dosed with DK-520
(32) (p.o., 200 mg/kg of body weight) or Niclosamide (IV, 2.6 mg/Kg). For oral dosing
of DK-520, blood samples were drawn serially at predose and at 0.25, 0.5, 0.75, 1,
1.5, 4, 8, 12, and 24 h after drug administration, n = 4 per time point. For IV dosing of
Niclosamide, blood samples were obtained at predose and at 0.08, 0.17, 0.33, 0.67,
1.5, 4, 8, 12 h after drug administration, n = 2 per time point. Quantification of
Niclosamide in mouse plasma was done by LC/MS–MS9 and reported as ng/mL. The
dotted line indicates the IC50 of Niclosamide inhibition of Wnt/b-catenin signaling
in the TOPFlash assay. The data were presented as mean SEM.
Research Scholar. NIH 5P30-CA-014236-36 (I.S.). NMR instrumen-
tation in the Duke NMR Spectroscopy Center was funded by the
NIH, NSF, NC Biotechnology Center and Duke University. The
authors gratefully acknowledge this support and the support of
Anthony Ribeiro in the Duke NMR Center and Professor Eric
Toone and the Duke Small Molecule Synthesis Facility.
Supplementary data
metabolized to Niclosamide in vivo and produce high concentra-
tions of Niclosamide in plasma and extend the duration of expo-
sure to the drug Niclosamide. To our knowledge, this is the first
report to identify derivatives of Niclosamide that increase plasma
exposure to Niclosamide and extend its duration of exposure.
The discovery of Niclosamide derivatives that improve systemic
Niclosamide drug exposure overcomes a significant barrier to the
clinical translation of Niclosamide to treat cancer. Moreover, it also
allows the study of Niclosamide in vivo in other diseases for which
Niclosamide has demonstrated biological activity. Thus, the find-
ings described here may provide a breakthrough to a new class
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.07.001.
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Please cite this article in press as: Mook Jr., R. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.001