Dual protonophore-chitinase inhibitors dramatically affect O. Volvulus molting

Article abstract of DOI:10.1021/jm5006435

The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the causative agent of onchocerciasis. Closantel, a known anthelmintic drug, was previously discovered as a potent and specific OvCHT1 inhibitor. As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the structural features that are relevant for its individual or dual biochemical roles. Furthermore, we present that either OvCHT1 inhibition or protonophoric activity was capable of affecting O. volvulus L3 molting and that the presence of both activities in a single molecule yielded more potent inhibition of the nematode's developmental process.

Full text of DOI:10.1021/jm5006435

Article
pubs.acs.org/jmc
Dual Protonophore−Chitinase Inhibitors Dramatically Affect O.
volvulus Molting
Major Gooyit,† Nancy Tricoche,‡ Sara Lustigman,‡ and Kim D. Janda*,†
†Departments of Chemistry and Immunology and Microbial Science, The Skaggs Institute for Chemical Biology, and The Worm
Institute of Research and Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,
United States
‡Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10065, United States
S
* Supporting Information
ABSTRACT: The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the
causative agent of onchocerciasis. Closantel, a known anthelmintic drug, was previously discovered as a potent and specific
OvCHT1 inhibitor. As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the
structural features that are relevant for its individual or dual biochemical roles. Furthermore, we present that either OvCHT1
inhibition or protonophoric activity was capable of affecting O. volvulus L3 molting and that the presence of both activities in a
single molecule yielded more potent inhibition of the nematode’s developmental process.
remodeling processes during filarial molting and egg hatch-
ing.8,9 In the rodent filarial nematode Acanthocheilonema viteae,
RNA interference studies targeting the chitinase gene
significantly inhibited the molting of L3 worms.10 A chitinase
from O. volvulus, OvCHT1, was recently identified and found
to be solely expressed within the glandular esophagus of the
infective L3 larvae and is secreted during postinfective
development.11 OvCHT1 was hypothesized to play roles in
molting and parasite transmission and, as such, presents a
potential target for pharmacological intervention. From our
previous screening efforts, we had disclosed a known veterinary
anthelmintic drug, closantel (1), which exhibits potent and
selective inhibition toward OvCHT1 (IC50 = 1.60 0.08 μM,
Ki = 0.47 0.08 μM).12 As closantel is a known uncoupler of
oxidative phosphorylation,13 our recent findings implicated a
potential bimodal action of the drug. Furthermore, closantel
was shown to inhibit the molting of O. volvulus L3 to L4 larvae
at 100 μM;12 however, this profound effect on filarial molting
was unclear as to whether it was due to inhibition of OvCHT1
or uncoupling of mitochondrial activity. In the same study, a
“retro-fragment”-based approach was conducted to unravel the
INTRODUCTION
■
Onchocerciasis, or river blindness, has plagued over 37 million
people worldwide, occurring mostly in the sub-Saharan Africa,
Yemen, and isolated areas of Central and South America.1 The
culprit for the infection is the filarial nematode Onchocerca
volvulus, which is transmitted to humans through bites of
infected blackflies of the genus Simulium. Initial pathological
manifestations of the disease include inflammation of the skin
and eyes caused by dead or dying microfilariae that eventually
results to skin lesions and blindness. Currently, the antiparasitic
agent ivermectin is the only drug available for mass treatment.2
While the drug has proven to be effective in reducing morbidity
by killing the microfilariae, it does not target the adult worms
and thus does not completely eliminate parasite transmission
and infection.3,4 Additionally, the emergence of ivermectin-
resistant O. volvulus5,6 further necessitates the need to develop
therapeutic strategies for the treatment of onchocerchiasis.
Molting is a crucial feature in the life cycle of O. volvulus. The
shedding of the L3 cuticle during ecdysis and the proteolytic
remodeling of the new L4 cuticle is particularly important for
the transition between hosts and infection process of the
parasitic nematode. Chitinases act in concert with chitin
synthases to facilitate chitin metabolism, a significant process
for nematode development.7 Chitinases have been implicated in
Received: April 24, 2014
© XXXX American Chemical Society
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molecular frameworks that are required for closantel’s chitinase
inhibitory activity, in which compound 2 was identified as the
relevant structural fragment with a comparable potency as that
of closantel (IC50 = 5.8 0.3 μM).12
Table 1. IC50 of O. volvulus Chitinase Inhibition
compd
R1
R2
R3
R4
R5
IC50 (μM)
1
1.60 0.08
1.98 0.13
1.28 0.19
1.81 0.18
3.71 0.14
1.06 0.08
1.11 0.03
1.57 0.09
0.70 0.02
0.60 0.07
0.87 0.10
1.74 0.09
1.20 0.17
0.34 0.04
0.94 0.05
0.89 0.10
1.29 0.03
0.47 0.03
0.99 0.16
0.37 0.03
3a
3b
3c
3d
3e
3f
OH
OMe
OH
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
Me
Me
H
H
H
H
OH
OH
OH
OMe
H
H
4-Me
4-F
4-Cl
4-Cl
4-Cl
3-Cl
2-Cl
4-Cl
3g
3h
3i
H
H
H
In this study, we constructed a series of closantel analogues
to deduce the molecular features that are critical for chitinase
inhibition and mitochondrial-uncoupling activity. Because the
3,5-diiodosalicylate moiety is key to closantel’s binding
specificity by anchoring it within the OvCHT1 active site,14
we retained this fragment and focused on scaffold expansion of
2 to generate analogues 3, 4, and 5 (which were easily accessed
through amide coupling of carboxylic acids 6 and amine
reactants, Figure 1). Simple structural modifications led to the
3j
H
3k
3l
OH
OH
OH
OH
OMe
H
H
H
3m
4a
4b
4c
5a
5b
5c
H
H
Me
Cl
profile as closantel, indicating that the phenolic or amide
proton is not necessary for chitinase inhibition. However,
simultaneous substitution of both hydrogens with methyl
groups (as in the case of compound 3d) resulted to a 2-fold
decrease in potency. Exclusion of the hydroxyl moiety
(compound 3e) led to a slight increase in inhibitory activity
with an IC50 value of 1.06 0.08 μM.
In an effort to increase the potency against OvCHT1, we
constructed a series of derivatives based on compound 3a.
From Table 1, it is evident that introduction of the chloro
substituent at the para-position of the terminal phenyl ring
(compound 3h) enhanced the potency almost 3-fold relative to
3a. The similar IC50 values of compounds 3i, 3j, and 3h further
confirmed that neither the phenolic proton nor the hydroxyl
functionality is important for chitinase inhibition. Inclusion of a
chloro-substituent in the middle phenyl ring (compound 3m,
also known as rafoxanide and a known veterinary anthelmintic
drug)16 further improved the potency with an IC50 value of 0.34
0.03 μM and a competitive inhibitory constant (Ki) of 0.13
0.01 μM. We then prepared naphthyl analogues 4a−c and
biphenyl compounds 5a−c in an attempt to establish favorable
π-stacking and van der Waals interaction with the hydrophobic
residues within the chitinase active site to optimize OvCHT1
inhibition. Biphenyl scaffolds have been previously considered
as privileged substructures that preferentially interact with
diverse proteins.17 Interestingly, we did observe enhanced
potency with the biaryl compounds 4 and 5, with IC50 values
Figure 1. Structures and preparation of closantel analogues.
identification of more potent compounds with dual modes of
action (as an OvCHT1 inhibitor and a protonophore) as well
as analogues acting as chitinase inhibitors only. We also
demonstrate that while either OvCHT1 inhibition or
mitochondrial uncoupling was effective in abrogating the L3-
to-L4 molt, synergistic activities incorporated into a single
molecule afforded a more profound impact on molting.
RESULTS AND DISCUSSION
for OvCHT1 inhibition as low as 0.37
0.03 μM (for
■
O. volvulus Chitinase Inhibition. The presence of
additional (phenolic and amide) protons in closantel may be
key for its dual mode of action. For instance, the hydrogen-
bonding moieties may be important to affect the required acidic
residues of the chitinase catalytic site.15 To elucidate the
relevance of these protons, we initially prepared a simplified
version of closantel using a phenoxyphenyl scaffold (compound
3a) and its analogues containing methyl substituents in place of
the key hydrogens (compounds 3b−d). As shown in Table 1,
compounds 3a, 3b, and 3c have comparable chitinase inhibitory
compound 5c). Select compounds that exhibited good potency
against OvCHT1 were also evaluated for inhibition of
chitinases from other species. Compounds 3h, 3i, 3j, 3m, 5a,
and 5c (each at a final concentration of 10 μM) showed
complete inhibition of OvCHT1 (see Figure S1 in the
Supporting Information) while sparing other chitinases
including those from another filarial nematode Brugia malayi
(BmCHT1) and protozoans Entamoeba histolytica (EhCHT1)
and Plasmodium falciparum (PfCHT1). These results indicate
that the analogues are highly OvCHT1-specific and thus are
B
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amenable for evaluation in chitinase-specific diseases such as
onchocerciasis.
volvulus is a collagenous framework of proteins that are cross-
linked by disulfide bridges.20 As the bioaccumulation of a drug
is correlated to its bioactivity, we deemed it important to
investigate the penetrability of the compounds in the
nematode. The limited availability of O. volvulus, however,
prompted us to consider an alternative model organism, the soil
nematode Caenorhabditis elegans. The cuticle of adult C. elegans
is composed of multiple layers of collagenous extracellular
structures18 and thus resemble the physical barriers of other
nematodes. In fact, due to its easy culturability and rapid life
cycle, it was previously used as a model system of
bioaccumulation to identify small bioactive molecules.21
Using a similar approach to address nematode permeability,
we initially incubated late-stage L4 worms with compounds 1,
3h, 3i, 3j, 3m, 4a, 4b, 5a, 5c, and CCCP (each at 10 μM final
concentration, and equivalent to 2 nmol/mg worm) for 6 h. By
LC-MS analysis of worm homogenates, we determined that all
the aforementioned compounds accumulated in C. elegans. C.
elegans is also equipped with enzymatic xenobiotic defenses that
add to its resistance to exogenous pharmacologicals.22 Similar
to what was observed in cattle and sheep,23 the primary, albeit
minor, metabolic pathway of closantel in C. elegans is reductive
deiodination, resulting in the formation of two monoiodoclo-
santel isomers (see Figure S4 in the Supporting Information).
Sulfation, glycosylation, and glucuronidation of the salicylate
moiety of closantel were not observed in C. elegans
homogenates. We add that no effect on the viability of the
L4 worms was observed upon incubation with the compounds.
We then set out to quantify the concentration of representative
compounds in the nematode after 6 and 12 h of incubation. As
indicated in Figure 3, compound 3m displayed excellent
Evaluation of Protonophoric Activity. To examine
mitochondrial-uncoupling activity, compounds 1−5 were tested
using tetramethylrhodamine ethyl ester (TMRE), a positively
charged, mitochondrion-selective dye that serves as a
membrane potential sensor. In the presence of a protonophore
(e.g., carbonyl cyanide m-chlorophenyl hydrazone or CCCP),
the mitochondrion gets depolarized and thus fails to sequester
TMRE, resulting in decreased fluorescence intensity. A
microplate fluorescence assay was developed to monitor
mitochondrial uncoupling of HEK-293T/17 cells in the
presence of the inhibitors. Following incubation with the
inhibitors at 37 °C, the cells were stained with TMRE and
subsequently analyzed by fluorescence spectrometry. As
depicted in Figure 2, a dissociable proton (i.e., phenolic
Figure 2. Evaluation of mitochondrial-uncoupling activity using a
microplate fluorescence assay. HEK 293T/17 cells were incubated
with compound (50 μM) and subsequently stained with TMRE. Data
shown as mean fluorescence intensity sd (n = 3). Unstained cells
(no TMRE) and DMSO were used as negative (−) and positive (+)
controls, respectively. RFU = relative fluorescence units (λex = 488 nm,
λem = 575 nm).
hydrogen as in 1, 3a, 3h, and 4a) for the salicylanilide class of
compounds is critical for mitochondrial-uncoupling activity. As
expected, removal of the hydroxyl moiety (as in compounds 3e,
3j, and 4c) or masking it with a methyl group (compounds 3b,
3i, 3d, and 4b) resulted to a loss of uncoupling activity. We
note, however, that the amide proton is equally significant, as
substituting it with a methyl group while retaining the hydroxyl
moiety (like in the case of compound 3c) eradicated
protonophoric activity. Results of mitochondrial-uncoupling
activity for the other compounds listed in Table 1 are given in
Supporting Information Figure S2. Separate experiments using
flow cytometry to analyze membrane polarization were also
conducted and the results (see Figure S3 in the Supporting
Information) were in agreement to those obtained from the
microplate fluorescence assay.
From the series of derivatives listed in Table 1, we have
identified more potent analogues with dual biochemical roles
(both as a chitinase inhibitor and a proton ionophore) as well
as compounds with chitinase inhibitory activity only.
Compounds 3m proved to be the most potent OvCHT1
inhibitor with good mitochondrial-uncoupling activity, while
compounds 3i, 3j, and 4b displayed similar chitinase inhibition
profile but devoid of protonophoric activity.
Figure 3. Bioaccumulation of chitinase inhibitors in C. elegans. Late-
stage L4 worms were incubated with 10 μM inhibitor (equivalent to 2
nmol/mg worm) for 6 and 12 h. Data shown as mean concentration
sd (n = 3), expressed in nmol/mg worm.
bioaccumulation in C. elegans, with worm levels of 1.55 0.37
and 1.73 0.11 nmol/mg at 6 and 12 h, respectively, which
were >75% of the initial dose of the compound to worms. The
concentrations of the naphthyl analogues 4a and 4b were a
little less than 50% of the dose at 6 h, and increased to 1.13
0.14 and 1.28 0.08 nmol/mg, respectively, after 12 h. The
inability to accumulate at adequate concentrations within the
worm can render the compound ineffective no matter how
potent it is against the target in vitro. In a survey of over 1000
drug-like small molecules, only less than 10% were shown to
penetrate C. elegans at concentrations >50% of the initial
doses.21 Thus, the findings that we observed for the closantel
analogues are encouraging because they readily accumulate
within the nematode at high levels. We surmise that these
compounds also accumulate in the filarial nematode O. volvulus
Bioaccumulation in the Model Nematode C. elegans.
In general, nematodes are resistant to perturbation of small
molecules due to the thick cuticles that line their exteriors and
oral and rectal cavities.18,19 The highly insoluble cuticle of O.
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and thus increase the likelihood of interaction with the target
protein OvCHT1.
both display similar IC50 values against OvCHT1; however,
compound 4b could be accumulating within O. volvulus at
higher levels, as was observed in the C. elegans model (vide
supra), which might account for its better activity. At a higher
concentration of 100 μM, compound 3i completely hindered
L3 molting. CCCP also fully inhibited the molting of L3 larvae
at 10 μM, suggesting that mitochondrial-uncoupling, by itself,
affects this developmental process. Many of the proteins that
are necessary for O. volvulus L3 molting are stored within the
granules of the esophageal glands.24 Mitochondrial-uncoupling
in the glandular esophagus may, therefore, affect the synthesis
of key proteins (including OvCHT1) that are essential for
larval development. At a final concentration of 1 μM, both 4b
and CCCP did not show any impact on molting (Figure 4B).
As both chitinase inhibitory and protonophoric activities are
likely required for more potent inhibition, we investigated the
effect of incubating L3 larvae with a 1:1 mixture of 4b and
CCCP. Whereas 3h, 3m, and 4a were effective at 1 μM, no
effect on molting was observed using a combination of 4b +
CCCP at 0.5 or 1 μM of each compound (Figure 4C).
Treatment with 2.5 μM each of 4b and CCCP was sufficient to
completely suppress the L3-to-L4 molt.
Inhibition of O. volvulus L3Molting. The target protein
OvCHT1 is expressed predominantly in the infective L3 larvae
and may be critical in the molting and development of the
nematode.11 Filarial molting of O. volvulus L3 larvae is typically
assayed by incubation of the larvae in complete growth medium
in the presence of peripheral blood mononuclear cells
(PBMCs). To evaluate the efficacy of the inhibitors based
solely on their inhibitory properties, the L3 larvae were initially
incubated with select compounds (10 μM) for 24 h (to allow
sufficient time for accumulation) prior to addition of the
growth medium and PBMCs and monitored until day 6 when
molting was assessed. Under this assay condition, compounds
3h, 3m, and 4a (all have dual protonophoric and chitinase
inhibitory activities) completely abolished molting at 10 μM, as
indicated in Figure 4A. Significant effects on molting (up to
Combination therapy, the simultaneous use of two or more
drugs with distinct modes of action and biological targets, has
been exploited as a treatment modality in several diseases
including HIV,25 metastatic melanoma,26 second-stage Trypa-
nosoma brucie gambiense sleeping sickness,27 and malaria.28
Despite its efficacy in improving response to medication and
minimizing drug resistance, combination therapy is resource-
intensive, complicated to administer, and prone to drug−drug
interactions and adverse side effects. The utility of “combi-
molecules” or single agents with polypharmacological targets
has emerged as an alternative strategy, displaying the same
advantages while bypassing the drawbacks of drug combina-
tions. The combi-targeting concept is a growing field of interest
in the search for therapies against cancer29,30 and infectious
diseases.31 This dual inhibition approach is well exemplified by
compounds 3h, 3m, and 4a, which, through concomitant
uncoupling of oxidative phosphorylation and inhibition of
OvCHT1, resulted in a more robust inhibition of O. volvulus
molting. Moreover, our results demonstrate the superior
activity of the dual-targeted single molecules over monotherapy
or a dual drug combination (4b + CCCP).
Figure 4. Molting of O. volvulus L3 larvae in the presence of inhibitors.
Percent molting at (A) 10 μM and (B) 1 μM inhibitor concentration,
and (C) in the presence of 1:1 combination of 4b and CCCP each at
concentrations shown. Data presented as percent molting in a total of
10 wells containing on average 5−10 larvae per well.
CONCLUSION
■
The implications of OvCHT1 in filarial molting has opened an
avenue to develop strategies toward the elimination of
onchocerciasis. Earlier, DNA immunization with OvCHT1
was shown to provide significant protection against the
developing L3 larvae in mice; however, large and frequent
doses of DNA were needed to attain the result.32 The efficacy
of the dual mechanistic drug, closantel, has steered us to
prepare analogues to further probe the individual effects of
OvCHT1 inhibition and protonophoric activity on O. volvulus
L3 molting. By subtle structural modifications, we have
identified potent bifunctional analogues (3h, 3m, and 4a) as
well as OvCHT1 inhibitors only (3i and 4b). These
compounds were also found to readily bioaccumulate in the
model nematode C. elegans at concentrations up to >75% of the
initial dose to worms. Moreover, our results have clearly
established the significance of each biochemical role in
modulating the O. volvulus L3-to-L4 molt and that synergistic
activities (as displayed by 3h, 3m, and 4a) provided a
84% inhibition) were also observed at a lower concentration of
1 μM for compounds 3h, 3m, and 4a (Figure 4B). Of note, no
considerable effect on cell viability was observed when HEK
293T/17 cells were treated with 1 and 10 μM of 3h, 3m, or 4a,
for 24 h (see Figure S5 in the Supporting Information).
Treatment of L3 larvae with compound 6b (neither an
OvCHT1 inhibitor nor a protonophore), showed no inhibition
on molting (Figure 4A). To ascertain which activity is relevant
for inhibition, we also evaluated compounds 3i and 4b
(chitinase inhibitors only) and CCCP (a protonophore only)
for their impact on L3 molting. Compounds 3i and 4b have
differing effects; while 4b inhibited molting at 10 μM, only
marginal inhibition was observed with 3i at the same inhibitor
concentration (Figure 4A). The difference in efficacy between
the two compounds at 10 μM could not be readily explained as
D
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MHz, CDCl3) δ 95.2, 118.9, 119.7, 122.3, 123.5, 129.9, 132.7, 135.4,
138.3, 148.4, 154.5, 157.4, 162.7. HRMS-ESI (m/z): [M + H]+ calcd
for C19H14I2NO2, 541.9109; found, 541.9106.
formidable impact on molting. Although these strategies will
still need to be validated as potential antifilarial therapies, what
we have presented thus far is a notch closer toward the search
for effective treatments to combat onchocerciasis.
2-Hydroxy-3,5-diiodo-N-(4-(p-tolyloxy)phenyl)benzamide (3f).
1
Procedure A, 51% yield. H NMR (600 MHz, CDCl3) δ 2.34 (s,
3H), 6.91−6.95 (m, 2H), 6.98−7.04 (m, 2H), 7.15 (d, J = 8.25 Hz,
2H), 7.44−7.50 (m, 2H), 7.78 (d, J = 1.89 Hz, 1H), 7.87 (s, 1H), 8.18
(d, J = 1.89 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 20.9, 77.2, 80.3,
89.0, 116.7, 118.9, 119.3, 123.5, 130.5, 130.7, 133.5, 134.3, 151.1,
154.5, 156.0, 160.4, 166.4. HRMS-ESI (m/z): [M + H]+ calcd for
C20H16I2NO3, 571.9220; found, 571.9215.
EXPERIMENTAL SECTION
■
Chemistry. General Information. All reactions were performed
under nitrogen atmosphere unless otherwise noted. 1H and 13C NMR
spectra were recorded on a Bruker DRX-500 or Bruker DRX-600
equipped with a 5 mm DCH cryoprobe. All carboxylic acid and amine
starting reactants were obtained from commercial sources except for
compounds 6b (used for the syntheses of compounds 3b, 3d, 3i, and
4b), 6c (prepared as described previously33 and used for syntheses of
compounds 3e, 3j, and 4c) and N-methyl-4-phenoxyaniline 7 (used
for the syntheses of compounds 3c and 3d).
N-(4-(4-Fluorophenoxy)phenyl)-2-hydroxy-3,5-diiodobenzamide
1
(3g). Procedure A, 43% yield. H NMR (600 MHz, CDCl3) δ 6.96−
7.08 (m, 6H), 7.49 (d, J = 8.84 Hz, 2H), 7.79 (d, J = 1.44 Hz, 1H),
7.92 (s, 1H), 8.19 (d, J = 1.72 Hz, 1H). 13C NMR (151 MHz, CDCl3)
δ 80.3, 89.0, 116.6 (d, J = 23.3 Hz), 116.7, 118.9, 120.8 (d, J = 8.26
Hz), 123.6, 131.1, 134.3, 151.1, 152.7 (d, J = 2.49 Hz), 155.7, 159.1 (d,
J = 242.8 Hz), 160.4, 166.5. HRMS-ESI (m/z): [M + H]+ calcd for
C19H13FI2NO3, 575.8963; found, 575.8957.
Compounds 3−5 were prepared according to general procedure A
or B.
General Procedure A. A mixture of 3,5-diiodosalicylic acid (1.0
equiv) and thionyl chloride (5 equiv) was heated under reflux for 7 h.
After concentration under reduced pressure, cold hexanes were added
to precipitate the acid chloride product. The residue was filtered,
washed several times with cold hexanes, and air-dried. A solution of
the amine reactant (1.0 equiv) and DIPEA (3 equiv) in DMF was
added to the acid chloride, and the resulting mixture was stirred at rt
for 1 h. The product was subsequently subjected to preparative HPLC
purification.
N-(4-(4-Chlorophenoxy)phenyl)-2-hydroxy-3,5-diiodobenzamide
1
(3h). Procedure A, 46% yield. H NMR (600 MHz, DMSO-d6) δ
7.01−7.08 (m, 2H), 7.07−7.14 (m, 2H), 7.41−7.50 (m, 2H), 7.65−
7.74 (m, 2H), 8.23 (d, J = 1.80 Hz, 1H), 8.40 (d, J = 1.80 Hz, 1H),
10.69 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 81.8, 89.1, 117.4,
119.3, 120.0, 123.8, 127.1, 129.9, 133.2, 135.8, 149.7, 153.0, 155.9,
159.5, 166.7. HRMS-ESI (m/z): [M + H]+ calcd for C19H13ClI2NO3,
591.8668; found, 591.8660.
General Procedure B. A solution of 6b or 6c (1.0 equiv), DIPEA
(2.0 equiv) and HBTU (1.1 equiv) in DMF was stirred for 15 min at
rt. The amine reactant (1.0 equiv) was added, and the resulting
mixture was stirred overnight at rt. The product was isolated by
preparative HPLC purification. Purity of the final products are
generally >95% as assessed by HPLC.
2-Hydroxy-3,5-diiodo-N-(4-phenoxyphenyl)benzamide (3a). Pro-
cedure A, 53% yield. 1H NMR (500 MHz, DMSO-d6) δ 6.99−7.10 (m,
4H), 7.14 (t, J = 7.39 Hz, 1H), 7.36−7.44 (m, 2H), 7.64−7.71 (m,
2H), 8.23 (d, J = 1.90 Hz, 1H), 8.40 (d, J = 1.86 Hz, 1H), 10.66 (s,
1H). 13C NMR (151 MHz, DMSO-d6) δ 81.7, 89.1, 117.3, 118.4,
119.0, 123.4, 123.8, 130.1, 132.8, 135.8, 149.7, 153.5, 156.9, 159.5,
166.7. HRMS-ESI (m/z): [M + H]+ calcd for C19H14I2NO3, 557.9058;
found, 557.9058.
3,5-Diiodo-2-methoxy-N-(4-phenoxyphenyl)benzamide (3b).
Procedure B, 60% yield. 1H NMR (600 MHz, CDCl3) δ 3.91 (s,
3H), 6.95−7.05 (m, 4H), 7.10 (t, J = 7.38 Hz, 1H), 7.33 (t, J = 7.94
Hz, 2H), 7.61 (d, J = 8.84 Hz, 2H), 8.24 (d, J = 2.12 Hz, 1H), 8.38 (d,
J = 2.13 Hz, 1H), 9.38 (s, 1H). 13C NMR (151 MHz, CDCl3) δ 62.7,
90.1, 93.8, 118.7, 119.8, 121.9, 123.4, 129.4, 129.9, 133.2, 141.3, 150.5,
154.2, 157.1, 157.5, 160.9. HRMS-ESI (m/z): [M + H]+ calcd for
C20H16I2NO3, 571.9214; found, 571.9213.
2-Hydroxy-3,5-diiodo-N-methyl-N-(4-phenoxyphenyl)benzamide
(3c). Procedure A, 23% yield. 1H NMR (600 MHz, CDCl3) δ 3.47 (s,
3H), 6.92−7.02 (m, 3H), 7.02−7.09 (m, 4H), 7.13 (t, J = 7.38 Hz,
1H), 7.35 (t, J = 7.91 Hz, 2H), 7.92 (d, J = 1.63 Hz, 1H), 11.89 (s,
1H). 13C NMR (151 MHz, CDCl3) δ 39.7, 79.2, 87.6, 117.7, 119.3,
120.3, 124.0, 128.1, 130.1, 139.2, 139.4, 149.2, 156.8, 157.1, 159.5,
169.2. HRMS-ESI (m/z): [M + H]+ calcd for C20H16I2NO3, 571.9214;
found, 571.9205.
N-(4-(4-Chlorophenoxy)phenyl)-3,5-diiodo-2-methoxybenza-
1
mide (3i). Procedure B, 23% yield. H NMR (600 MHz, CDCl3) δ
3.91 (s, 3H), 6.92−6.96 (m, 2H), 7.01−7.05 (m, 2H), 7.27−7.31 (m,
2H), 7.60−7.66 (m, 2H), 8.26 (d, J = 2.20 Hz, 1H), 8.39 (d, J = 2.20
Hz, 1H), 9.40 (s, 1H). 13C NMR (151 MHz, CDCl3) δ 62.7, 90.1,
93.8, 119.9, 119.9, 122.0, 128.4, 129.3, 129.9, 133.6, 141.3, 150.6,
153.7, 156.2, 157.1, 160.9. HRMS-ESI (m/z): [M + H]+ calcd for
C20H15ClI2NO3, 605.8824; found, 605.8817.
N-(4-(4-Chlorophenoxy)phenyl)-3,5-diiodobenzamide (3j). Proce-
dure A, 44% yield. 1H NMR (600 MHz, CDCl3) δ 6.92−6.97 (m, 2H),
6.99−7.04 (m, 2H), 7.28−7.32 (m, 2H), 7.57 (d, J = 8.78 Hz, 2H),
7.71 (s, 1H), 8.10−8.15 (m, 2H), 8.22 (t, J = 1.35 Hz, 1H). 13C NMR
(151 MHz, CDCl3) δ 95.2, 119.8, 120.0, 122.4, 128.5, 129.9, 133.1,
135.4, 138.3, 148.4, 154.0, 156.1, 162.7. HRMS-ESI (m/z): [M − H]−
calcd for C17H10I2NO2, 573.8568; found, 573.8556.
N-(4-(3-Chlorophenoxy)phenyl)-2-hydroxy-3,5-diiodobenzamide
1
(3k). Procedure A, 43% yield. H NMR (600 MHz, CDCl3) δ 6.88−
6.93 (m, 1H), 7.00 (t, J = 2.12 Hz, 1H), 7.04−7.08 (m, 2H), 7.08−
7.12 (m, 1H), 7.23−7.29 (m, 1H), 7.52−7.58 (m, 2H), 7.80 (d, J =
1.83 Hz, 1H), 7.92 (s, 1H), 8.20 (d, J = 1.85 Hz, 1H). 13C NMR (151
MHz, CDCl3) δ 80.4, 89.1, 116.7, 116.9, 119.1, 120.1, 123.5, 123.7,
130.8, 131.9, 134.3, 135.3, 151.2, 154.3, 158.1, 160.4, 166.5. HRMS-
ESI (m/z): [M + H]+ calcd for C19H13ClI2NO3, 591.8668; found,
591.8663.
N-(4-(2-Chlorophenoxy)phenyl)-2-hydroxy-3,5-diiodobenzamide
1
(3l). Procedure A, 35% yield. H NMR (600 MHz, CDCl3) δ 6.98−
7.02 (m, 2H), 7.03 (dd, J = 1.40, 8.13 Hz, 1H), 7.12 (td, J = 1.44, 7.80
Hz, 1H), 7.23−7.25 (m, 1H), 7.48 (dd, J = 1.54, 8.00 Hz, 1H), 7.49−
7.53 (m, 2H), 7.78 (d, J = 1.88 Hz, 1H), 7.87 (s, 1H), 8.19 (d, J = 1.87
Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 80.3, 89.0, 116.7, 118.5,
121.2, 123.5, 125.3, 126.2, 128.2, 131.1, 131.3, 134.3, 151.2, 152.3,
155.0, 160.4, 166.4. HRMS-ESI (m/z): [M + H]+ calcd for
C19H13ClI2NO3, 591.8668; found, 591.8662.
3,5-Diiodo-2-methoxy-N-methyl-N-(4-phenoxyphenyl)-
benzamide (3d). Procedure B, 58% yield. 1H NMR (600 MHz,
CDCl3) δ 3.46 (s, 3H), 3.88 (s, 3H), 6.82 (d, J = 8.78 Hz, 2H), 6.90
(d, J = 7.85 Hz, 2H), 7.01 (d, J = 8.78 Hz, 2H), 7.10 (t, J = 7.40 Hz,
1H), 7.29−7.40 (m, 4H), 7.93 (d, J = 1.98 Hz, 1H). 13C NMR (151
MHz, CDCl3) δ 37.5, 62.7, 87.6, 93.3, 118.9, 119.5, 123.7, 128.3,
130.0, 133.9, 137.4, 138.1, 147.6, 155.9, 156.3, 157.0, 166.7. HRMS-
ESI (m/z): [M + H]+ calcd for C21H18I2NO3, 585.9376; found,
585.9366.
2-Hydroxy-3,5-diiodo-N-(naphthalen-2-yl)benzamide (4a). Pro-
cedure A, 27% yield. 1H NMR (600 MHz, DMSO-d6) δ 7.46−7.51 (m,
1H), 7.51−7.55 (m, 1H), 7.78 (dd, J = 2.08, 8.86 Hz, 1H), 7.91 (d, J =
8.58 Hz, 2H), 7.95 (d, J = 8.90 Hz, 1H), 8.25 (d, J = 1.81 Hz, 1H),
8.30 (d, J = 1.61 Hz, 1H), 8.48 (d, J = 1.80 Hz, 1H), 10.84 (s, 1H). 13C
NMR (151 MHz, DMSO-d6) δ 81.9, 89.2, 117.6, 118.8, 121.7, 125.5,
126.6, 127.5, 127.6, 128.3, 130.6, 133.1, 134.9, 136.0, 149.7, 159.5,
167.0. HRMS-ESI (m/z): [M − H]− calcd for C17H10I2NO2,
513.8807; found, 513.8800.
3,5-Diiodo-N-(4-phenoxyphenyl)benzamide (3e). Procedure B,
1
45% yield. H NMR (600 MHz, CDCl3) δ 6.98−7.05 (m, 4H), 7.11
(t, J = 7.40 Hz, 1H), 7.31−7.37 (m, 2H), 7.56 (d, J = 8.73 Hz, 2H),
7.71 (s, 1H), 8.12 (s, 2H), 8.22 (t, J = 1.39 Hz, 1H). 13C NMR (151
E
dx.doi.org/10.1021/jm5006435 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3,5-Diiodo-2-methoxy-N-(naphthalen-2-yl)benzamide (4b). Pro-
cedure B, 52% yield. H NMR (600 MHz, CDCl3) δ 3.95 (s, 3H),
was added to a stirred solution of crude N-methyl-N-(4-
phenoxyphenyl)acetamide in ethylene glycol (3 mL). The mixture
was heated under reflux overnight, diluted with H2O, and extracted
with EtOAc. The organic layers were combined, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. Purification of the
crude product by silica chromatography gave compound 7 in 56%
yield (0.25 g) over two steps. 1H NMR (600 MHz, CDCl3) δ 2.85 (s,
3H), 6.61−6.65 (m, 2H), 6.94−6.98 (m, 4H), 7.03 (t, J = 7.35 Hz,
1H), 7.30 (t, J = 7.96 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 31.4,
113.5, 117.1, 121.4, 122.0, 129.6, 146.1, 147.6, 159.3. HRMS-ESI (m/
z): [M + H]+ calcd for C13H14NO, 200.1075; found, 200.1069.
1
7.42−7.46 (m, 1H), 7.48−7.52 (m, 1H), 7.57 (dd, J = 2.12, 8.75 Hz,
1H), 7.81 (d, J = 8.11 Hz, 1H), 7.83−7.87 (m, 2H), 8.27 (d, J = 2.19
Hz, 1H), 8.38 (d, J = 1.70 Hz, 1H), 8.44 (d, J = 2.19 Hz, 1H), 9.60 (s,
1H). 13C NMR (151 MHz, CDCl3) δ 62.8, 90.1, 93.9, 117.3, 120.0,
125.5, 126.9, 127.8, 127.9, 129.2, 129.4, 131.1, 134.0, 135.2, 141.4,
150.6, 157.2, 161.1. HRMS-ESI (m/z): [M + H]+ calcd for
C18H14I2NO2, 529.9109; found, 529.9114.
3,5-Diiodo-N-(naphthalen-2-yl)benzamide (4c). Procedure B,
45% yield. 1H NMR (600 MHz, CDCl3) δ 7.44 (ddd, J = 1.09,
6.91, 8.05 Hz, 1H), 7.47−7.51 (m, 1H), 7.57 (dd, J = 2.07, 8.74 Hz,
1H), 7.78−7.86 (m, 3H), 7.89 (s, 1H), 8.17 (d, J = 1.37 Hz, 2H), 8.22
(t, J = 1.46 Hz, 1H), 8.27 (s, 1H). 13C NMR (151 MHz, CDCl3) δ
95.2, 117.6, 120.1, 125.6, 126.9, 127.8, 127.9, 129.2, 131.1, 133.9,
134.8, 135.5, 138.4, 148.4, 162.9. HRMS-ESI (m/z): [M − H]− calcd
for C17H10I2NO, 497.8852; found, 497.8847.
N-([1,1′-Biphenyl]-4-yl)-2-hydroxy-3,5-diiodobenzamide (5a).
N-(2-(1H-Indol-3-yl)ethyl)-2-hydroxy-3,5-diiodobenzamide (8).
Procedure A, 65% yield. H NMR (500 MHz, DMSO-d6) δ 2.97 (t,
1
Procedure A, 72% yield. H NMR (600 MHz, DMSO-d6) δ 7.36 (t,
1
J = 7.35 Hz, 1H), 7.47 (t, J = 7.72 Hz, 2H), 7.69 (d, J = 7.31 Hz, 2H),
7.72 (d, J = 8.62 Hz, 2H), 7.78 (d, J = 8.64 Hz, 2H), 8.24 (d, J = 1.75
Hz, 1H), 8.43 (d, J = 1.79 Hz, 1H), 10.73 (s, 1H). 13C NMR (151
MHz, DMSO-d6) δ 81.8, 89.2, 117.5, 122.2, 126.4, 126.9, 127.4, 129.0,
135.9, 136.6, 136.8, 139.5, 149.7, 159.5, 166.9. HRMS-ESI (m/z): [M
+ H]+ calcd for C19H14I2NO2, 541.9114; found, 541.9116.
2-Hydroxy-3,5-diiodo-N-(4′-methyl-[1,1′-biphenyl]-4-yl)-
benzamide (5b). Procedure A, 70% yield. 1H NMR (600 MHz,
DMSO-d6) δ 2.34 (s, 3H), 7.27 (d, J = 7.91 Hz, 2H), 7.58 (d, J = 8.03
Hz, 2H), 7.69 (d, J = 8.62 Hz, 2H), 7.76 (d, J = 8.62 Hz, 2H), 8.24 (d,
J = 1.65 Hz, 1H), 8.43 (d, J = 1.55 Hz, 1H), 10.70 (s, 1H). 13C NMR
(151 MHz, DMSO-d6) δ 20.7, 81.8, 89.2, 117.5, 122.2, 126.3, 126.6,
129.6, 135.9, 136.5, 136.6, 136.6, 136.6, 149.7, 159.5, 166.8. HRMS-
ESI (m/z): [M + H]+ calcd for C20H16I2NO2, 555.9270; found,
555.9275.
J = 7.45 Hz, 2H), 3.57 (q, J = 7.04 Hz, 2H), 6.94−7.02 (m, 1H), 7.03−
7.11 (m, 1H), 7.18 (d, J = 2.21 Hz, 1H), 7.34 (d, J = 8.09 Hz, 1H),
7.55 (d, J = 7.86 Hz, 1H), 8.14−8.23 (m, 2H), 9.28 (t, J = 5.43 Hz,
1H), 10.84 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 24.6, 40.3,
81.4, 88.8, 111.4, 111.5, 116.3, 118.2, 118.3, 121.0, 122.8, 127.1, 135.2,
136.2, 149.3, 159.9, 168.0. HRMS-ESI (m/z): [M + H]+ calcd for
C17H14I2N2O2, 532.9218; found, 532.9208.
N-(4′-Chloro-[1,1′-biphenyl]-4-yl)-2-hydroxy-3,5-diiodobenza-
mide (5c). Procedure A, 68% yield. 1H NMR (600 MHz, DMSO-d6) δ
7.52 (d, J = 8.46 Hz, 2H), 7.68−7.77 (m, 4H), 7.79 (d, J = 8.61 Hz,
2H), 8.24 (d, J = 1.53 Hz, 1H), 8.42 (d, J = 1.57 Hz, 1H), 10.73 (s,
1H). 13C NMR (151 MHz, DMSO-d6) δ 81.8, 89.2, 117.5, 122.2,
126.9, 128.2, 128.9, 132.2, 135.2, 135.9, 137.1, 138.3, 149.7, 159.4,
166.9. HRMS-ESI (m/z): [M + H]+ calcd for C19H13ClI2NO2,
575.8719; found, 575.8735.
Chitinase Inhibition. Chitinase activity assay was performed as
previously described.12
Mitochondrial-Uncoupling Assays. Adherent human epithelial
kidney cells [HEK 293T/17] (ATCC CRL-11268) were trypsinized
and resuspended in 10% FBS in DMEM at a density of 1−2 × 106
cells/mL. Cells were incubated with DMSO (blank control) or 50 μM
compound for 30 min at 37 °C. Tetramethylrhodamine ethyl ester
perchlorate (TMRE, ∼450 nM final concentration) was added,
followed by incubation in the dark for 30 min at 37 °C. Cells were
washed twice with 0.2% BSA in PBS, centrifuged, and resuspended in
0.2% BSA in PBS. For the microplate assay, fluorescence signals were
measured using a SpectraMaxM2e microplate reader (Molecular
Devices, Sunnyvale, CA) (λex = 549 nm, λem = 575 nm). For flow
cytometry, 1−2 × 106 cells per sample were collected using DIVA 6.0
software on an LSRII flow cytometer (BD Biosciences, San Jose, CA).
FACS data was further analyzed using FlowJo software (Tree Star,
Inc., Ashland, OR).
3,5-Diiodo-2-methoxybenzoic Acid (6b). Methyl iodide (0.80 g,
5.6 mmol) was added dropwise to a mixture of 3,5-diiodosalicylic acid
(1.0 g, 5.1 mmol) and K2CO3 (0.90 g, 6.5 mmol) in acetone (10 mL).
After stirring for 3 h at rt, the solvent was removed in vacuo, and the
residue was partitioned between H2O and CH2Cl2. The organic layer
was separated, and the aqueous phase was extracted with CH2Cl2. The
organic extracts were concentrated under reduced pressure to give
crude methyl 3,5-diiodo-2-methoxybenzoate. The crude product was
dissolved in hot methanol (10 mL) to which 1 M KOH (10 mL, 10
mmol) was added. The resulting mixture was heated under reflux for 2
h, diluted with water, and neutralized by addition of 1 M HCl. The
product was collected by filtration and recrystallized with ethanol to
C. elegans Culture. C. elegans growth and maintenance were
performed using standard protocols.35
Bioaccumulation in C. elegans. The procedure for the small
molecule accumulation assay was adapted from Burns et al.21 Briefly,
synchronized worms were grown to late-stage L4 worms at 25 °C on
Escherichia coli OP50 for 40 h. Worms were harvested, washed three
times with water, and resuspended in M9 buffer to a final
concentration of ∼5−10 worms/μL. To 200 μL of the worm
suspension was added 2 μL of 1 mM compound in DMSO (equivalent
to ∼2 nmol/mg worm). After incubation for 6 h (or 12 h) at rt, the
worms were washed with 500 μL of 0.1% SDS, followed by three 500
μL washes with water. After washing, the worms were flash frozen in
liquid nitrogen, lyophilized, weighed, and stored at −80 °C until
analyses.
Sample Analysis. Worms were homogenized for 5 min in a 100-
volume equivalent of cold methanol/acetonitrile mixture (1:1)
containing 500 nM internal standard 8, using a bullet blender (Next
Advance, Inc., Averill Park, NY). After centrifugation for 10 min at 4
°C, the supernatants were collected and analyzed by reversed-phase
1
give 6b in 61% yield (0.62 g) over two steps. H NMR (600 MHz,
DMSO-d6) δ 3.76 (s, 3H), 7.95 (d, J = 2.09 Hz, 1H), 8.30 (d, J = 2.08
Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 61.9, 89.2, 96.6, 128.7,
139.2, 149.2, 158.1, 165.1. HRMS-ESI (m/z): [M − H]− calcd for
C8H5I2O3, 402.8328; found, 402.8325.
N-Methyl-4-phenoxyaniline (7). Compound 7 was synthesized
according to the literature procedure.34 To a cooled solution of N-(4-
phenoxyphenyl)acetamide (0.50 g, 2.2 mmol) in anhydrous THF (5
mL) was added NaH (97 mg, 2.4 mmol, 60% dispersion in mineral
oil). After stirring for 5 min, MeI (0.20 mL, 3.2 mmol) was added
dropwise, and the resulting mixture was stirred for 1 h at rt and then
partitioned between H2O and CH2Cl2. The organic phase was
separated and the aqueous layer was extracted with CH2Cl2.
Concentration of the combined organic extracts in vacuo gave the
crude N-methyl-N-(4-phenoxyphenyl)acetamide, which was used in
the next step without further purification. Concentrated HCl (0.5 mL)
F
dx.doi.org/10.1021/jm5006435 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
LC with MS detection (mass filter at m/z = 589.846−589.856 for 3h,
m/z = 605.878−605.888 for 3i, m/z = 623.807−623.817 for 3m, m/z
= 513.875−513.885 for 4a, and m/z = 529.906−529.916 for 4b).
Standard calibration curves were prepared by fortification of blank
worms with compounds 3h, 3i, 3m, 4a, and 4b at concentrations up to
2 nmol/mg worm. The concentrations in worms were quantified using
peak area ratios relative to the internal standard and linear regression
parameters were calculated from the calibration curve standards
prepared in blank worms.
Liquid Chromatography and Mass Spectrometry. The LC
system consisted of Agilent 1200 LC (Agilent Technologies, Santa
Clara, CA) equipped with an autosampler and a column heater. Mass
spectrometric experiments were performed with Agilent TOF 6210
and monitored with Mass Hunter qualitative analysis software, version
B.03.01 (Agilent Technologies, Santa Clara, CA). Mass spectrometry
acquisition was performed using electrospray ionization with the
following parameters: capillary voltage = 3.5 kV, nebulizer pressure =
20 psig, drying gas flow = 7 L/min, and gas temperature = 350 °C.
Samples were analyzed on a Poroshell 120 EC-C8 column (2.7 μm, 2.1
mm i.d. 100 mm, Agilent, USA). The mobile phase consisted of
elution at 0.20 mL/min starting with an 8 min linear gradient from
70% A/30% B to 100% B and then 100% B for 12 min (positive-ESI, A
= 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile;
negative-ESI, A = water, B = acetonitrile).
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O. volvulus L3 Molting Inhibition. Experiments using O. volvulus
L3 larvae in molting assay were the same as described previously12
except that the larvae were incubated with the compounds for 24 h
prior to addition of the complete medium (containing 20% heat
inactivated FCS) and normal PBMCs.
Cell Viability Assay. HEK 293T/17 cells (2 × 104 cells) were
plated in 96-well plates and incubated at 37 °C for 24 h. Cells were
then treated with compounds 3h, 3m, and 4a (at a final concentration
of 1 and 10 μM), and an MTS assay was performed at 0.5 and 24 h
postincubation using the CellTiter 96 aqueous non-radioactive cell
proliferation assay (Promega, Madison, WI) per manufacturer’s
instructions.
ASSOCIATED CONTENT
* Supporting Information
■
S
Activity of chitinases from different species, evaluation of
mitochondrial-uncoupling by fluorescence spectrometry and
flow cytometry, metabolism of closantel in C. elegans, and cell
viability after treatment with compounds 3h, 3m, and 4a. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (858) 785-2515. Fax: (858) 784-2595. E-mail:
kdjanda@scripps.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by NIH grant AI092076. We thank
Greg McElhaney for preparation of compounds 5a−c.
■
ABBREVIATIONS USED
■
O. volvulus, Onchocherca volvulus; OvCHT1, Onchocherca
volvulus chitinase; L3, third larval stage; L4, fourth larval
stage; CCCP, C. elegans, Caenorhabditis elegans; LC, liquid
chromatography; MS, mass spectrometry
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