Norcantharidin analogues with nematocidal activity in Haemonchus contortus

Article abstract of DOI:10.1016/j.bmcl.2011.04.031

With the major problems with resistance in parasitic nematodes of livestock to anthelmintic drugs, there is an urgent need to develop new nematocides. In the present study, we employed a targeted approach for the design of a series of norcantharidin analogues (n = 54) for activity testing against the barber's pole worm (Haemonchus contortus) of small ruminants in a larval development assay (LDA) and also for toxicity testing on nine distinct human cell lines. Although none of the 54 analogues synthesized were toxic to any of these cell lines, three of them (N-octyl-7-oxabicyclo(2.2.1)heptane-2,3-dicarboximide (B2), N-decyl-7-oxabicyclo(2.2.1)heptane-2,3-dicarboximide (B3) and 4-[(4-methyl)-3-ethyl-2-methyl-5-phenylfuran-10-oxa-4-azatricyclo[5.2.1] decane-3,5-dione (B21) reproducibly displayed 99-100% lethality to H. contortus in LDA, with LD_50s of 25-40 μM. The high 'hit rate' (5.6%) indicates that the approach taken here has advantages over conventional drug screening methods. A major advantage of norcantharidin analogues over some other currently available anthelmintics is that they can be produced in one to two steps in large amounts at low cost and high purity, and do not require any additional steps for the isolation of the active isomer. This positions them well for commercial development.

Full text of DOI:10.1016/j.bmcl.2011.04.031

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3277–3281
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Norcantharidin analogues with nematocidal activity in Haemonchus contortus
Bronwyn E. Campbell a, Mark Tarleton b, Christopher P. Gordon b, Jennette A. Sakoff c, Jayne Gilbert c,
Adam McCluskey b, Robin B. Gasser a,
⇑
a Department of Veterinary Science, The University of Melbourne, Parkville, Vic. 3052, Australia
b Department of Chemistry, School of Environmental and Life Sciences, The University of Newcastle, NSW 2308, Australia
c Department of Medical Oncology, Calvary Mater Hospital, Newcastle, NSW 2308, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
With the major problems with resistance in parasitic nematodes of livestock to anthelmintic drugs,
there is an urgent need to develop new nematocides. In the present study, we employed a targeted
approach for the design of a series of norcantharidin analogues (n = 54) for activity testing against the
barber’s pole worm (Haemonchus contortus) of small ruminants in a larval development assay (LDA)
and also for toxicity testing on nine distinct human cell lines. Although none of the 54 analogues
synthesized were toxic to any of these cell lines, three of them (N-octyl-7-oxabicyclo(2.2.1)heptane-
2,3-dicarboximide (B2), N-decyl-7-oxabicyclo(2.2.1)heptane-2,3-dicarboximide (B3) and 4-[(4-methyl)-
3-ethyl-2-methyl-5-phenylfuran-10-oxa-4-azatricyclo[5.2.1]decane-3,5-dione (B21) reproducibly displayed
Received 6 March 2011
Accepted 7 April 2011
Available online 13 April 2011
Keywords:
Haemonchus contortus
Nematocidal drugs
Norcantharidin analogues
99–100% lethality to H. contortus in LDA, with LD50s of 25–40 lM. The high ‘hit rate’ (5.6%) indicates
that the approach taken here has advantages over conventional drug screening methods. A major
advantage of norcantharidin analogues over some other currently available anthelmintics is that they
can be produced in one to two steps in large amounts at low cost and high purity, and do not require
any additional steps for the isolation of the active isomer. This positions them well for commercial
development.
Ó 2011 Elsevier Ltd. All rights reserved.
In spite of their massive impact, parasites of humans and other
animals are seriously neglected in terms of funding for research
and development (R&D) of drugs, vaccines and diagnostics. The
current production losses caused by parasites to agriculture world-
wide have a major adverse impact on farm profitability and exac-
erbate the global food shortage. For instance, nematodes of
livestock cause major production losses to farmers due to poor pro-
ductivity, failure to thrive and deaths.1–5 In particular, strongylid
nematodes are of paramount importance as pathogens of sheep,
goats, cattle and pigs, causing gastrointestinal diseases and associ-
ated complications, often leading to death in severely affected ani-
mals.5 Currently, these nematodes are controlled predominantly
through the use of anthelmintics, but widespread resistance
against a range of compounds (of three main classes) has compro-
mised their efficacy.6–11 Thus, there is an urgent need to work to-
ward identifying new drug targets and developing new
nematocides.
encoding protein phosphatases (PPs) for selected strongylid nema-
todes. These studies have shown that selected serine–threonine
phosphatases (STPs) are quite conserved between parasitic and
free-living nematodes and have inferred that they play key roles
in pathways required for the growth, development, survival and/
or reproduction.25 In addition, phylogenetic analysis has indicated
that such STPs are specific to nematodes, clustering, with strong
support, to the exclusion of related molecules in other inverte-
brates and vertebrates.24
Current literature indicates that inhibitors, such as cantharidin
(1) (from the blister beetle, Mylabris)32–36 and a number of ana-
logues with the same pharmacophoric units, most notably some
derived from norcantharidin (2), have no adverse toxic effects on
well-defined, cultured human cells34,35,37–39 but were considered
to have unique potential for the development of nematocides
( Fig. 1).25 The former characteristic is important, as the focus
should be on identifying compounds that have no adverse effect
on mammalian cells (representing the host animal) but are lethal
to parasitic nematodes or block their reproduction. Some norcant-
haridin analogues are known to display excellent STP (i.e., PP1 and
PP2A) inhibitory activity.34,35,40,41 Preliminary work conducted by
us showed that some norcantharidin analogues, which had no toxic
effect on human cell lines, killed larvae of the trichostrongylid
nematodes Trichostrongylus vitrinus and/or Haemonchus contortus.25
Homology modelling and in silico docking suggested that that
We have been pursuing the molecular characterization of a
number of gender- and/or stage-enriched molecules in parasitic
nematodes (Strongylida) using C. elegans as a reference organism,
with a perspective on predicting novel drug targets.12–30 Through
a number of studies,16,24,31 we have provided insights into genes
⇑
Corresponding author. Tel.: +61 3 97312283; fax: +61 3 97312366.
E-mail address: robinbg@unimelb.edu.au (R.B. Gasser).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.031

3278
B. E. Campbell et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3277–3281
As a series of internal assay-validation standards were also syn-
thesized, using a phase transfer catalyst approach, selected (Z)-2-
phenyl-3-(1H-pyrrol-2-yl)acrylonitrile derivatives, which had been
reported previously to be active against both H. contortus and the
cat flea, Ctenocephalides felis.47,48 The library compounds listed
are those inhibitors that passed our toxicity pre-filter by displaying
Figure 1. The chemical structures of cantharidin (1) and norcantharidin (2).
low levels, defined as a mean GI50 value >75 lM, of cell death in
nine distinct human cancer cell lines (HT29, SW480, MCF-7,
A2780, H460, A431, DU145, BE2-C and SJ-G2).32,34,35,40,41 As re-
ported previously,34,35 the ring-opened acid amine analogues dis-
some prototype molecules designed bind specifically to active
sites in selected STPs of these parasites.25 These initial findings
indicated an opportunity for the discovery of a novel class of
nematocides and significant biotechnological outcomes. In the
present study, we designed a series of norcantharidin analogues
and then tested their nematocidal effect on H. contortus in a larval
development assay (LDA).
Three families of norcantharidin analogues were synthesized:
the ring-opened acid amides (library A, A1–A24), the ring-closed
norcantharimides (library B, B1–B22) and the tetrahydroepoxyiso-
indole carboxamides (library C). These compounds were accessed
through a series of robust in house-developed and generic ap-
proaches reported previously.34,35,42–45 These approaches facili-
played higher levels of cytotoxicity (10–60
corresponding ring-closed norcantharimides (>75
l
M) than the
l
M). All 54 ana-
logues that passed the ‘toxicity filter’ were evaluated for nemato-
cidal activity against H. contortus in LDA.
Based on our previous studies,34–37,40,41 we anticipated our li-
brary A analogues to be the most potent (as we had shown protein
phosphatase inhibition by some of these analogues). To our sur-
prise, none of the analogues from library A returned notable para-
site lethality. Analogues were tested over a drug concentration
range of 12.5–100 lM.
Subsequently, we examined the library B analogues representing
the norcantharimide that we have shown previously to be devoid
of protein phosphatase inhibition. To our surprise, three analogues
(library B; B2, B3 and B21; Fig. 2) from this library achieved 99–
100% lethality of H. contortus in LDA (Table 1). All three compounds
were retested on four separate occasions, achieving the same re-
sult. Progression to a full-dose response evaluation, at concentra-
tated the rapid installation of
a range of substituents for
structure–activity relationship (SAR) studies.
All chemicals were tested for cytotoxicity in vitro against nine
different human cancer cell lines: HT29 (colon), SW480 (colon),
MCF-7 (breast), A2780 (ovarian), H460 (lung), A431 (skin),
DU145 (prostate), BE2-C (neuronal) and SJ-G2 (brain), as previ-
ously described.32,35,40,41 Following cytotoxicity testing, chemicals
were tested in a larval development assay (LDA). H. contortus (Hae-
con 5 strain) was raised in helminth-free lambs (Merino crosses;
24 weeks of age), as described by Nikolaou et al.46
The present study employed a targeted approach for the de-
sign of a series of norcantharimide analogues for toxicity testing
on nine different human cancer cell lines and for subsequent test-
ing for nematocidal activity in LDA against H. contortus. A total of
54 analogues was synthesized (see Supplementary data). For both
the ring-opened acid amide (library A) and ring-closed norcantha-
rimide analogues (library B), the ease of synthesis typically re-
lated to the nucleophilicity of the amine used to develop each
focused library member. The more nucleophilic amines favoured
ring-closing to the corresponding norcantharimide, non-nucleo-
philic amines required prolonged heating to effect this transfor-
mation (path B in Scheme 1). Most notable was the reaction of
N-methyl-1-(5-phenylfuran-3-yl)methanamine which, instead of
tions between 10 and 100
lM, revealed that each of these
analogues had LD50 values in the range of 25–40
l
M (Table 1).
Interestingly, both B2 and B3 possess a long alkyl chain, which
may enhance their bioavailability and assist their transport across
the parasite cuticle and into tissues and cells, allowing access to
the target in the parasite. Given our previous findings in relation
to protein phosphatase inhibition by this class of norcantharidin
analogue, we believe that it is unlikely that the ultimate target is
a serine–threonine protein phosphatase. The highly hydrophobic
nature of the phenylfuran moiety of B21 most likely also assists
transport through cell membranes. We had hoped that further
elongation of the alkyl tail of B2 and B3 may improve uptake,
but the dodecyl B4, tetradecyl B5 and octadecyl B6 were inactive.
Presumably, this lack of activity is a consequence of poor water
solubility.
Given the activity of B2 and B3, we specifically tailored the syn-
thesis of library C to include hydrophobic groups. However no
compound from library C displayed any noteworthy nematocidal
affording the expected ring-opened analogue (path
A in
activity at the initial screening doses (12.5–100 lM).
Scheme 1), underwent a very facile ring-closing to the quaternary
ammonium norcantharimide (B21).
The synthesis of the tetrahydroepoxyisoindole carboxyamides
was more challenging, but the products typically represented good
yields (Scheme 2, library C).
Libraries A and B are related via a simple ring closing which
effectively removes an acid and an amide moiety from the inactive
pharmacophore. This, in turn, suggested that either the ultimate
protein target disfavours the presence of these hydrogen bond
Scheme 1. Reagents and conditions: (i) Et2O, rt, 48 h; (ii) acetone, 10% Pd–C, H2 (g) 50 psi, 18 h; (iii) RNH2, PhCH3,
D, 24–36 h.

B. E. Campbell et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3277–3281
3279
Scheme 2. Reagents and conditions: (i) R1NC, alkynoic acid, furan carbaldehyde, R2NH2, CH3OH, rt 30 min; (ii) PhCH3, sealed tube 200 °C, 36 h.
Figure 2. The three compounds (B2, B3 and B21; ring closed norcantharimide analogues) from library B which consistently killed 99–100% of Haemonchus contortus in the
larval development assay (LDA).
Table 1
Of 54 compounds synthesized (see Supplementary data, and Figs. 1 and 2) tested against Haemonchus contortus in a larval development assay (LDA), three norcantharimide
analogues from library B reproducibly killed H. contortus. Included in the LDA were four control compounds (Z)-2-phenyl-3-(1H-pyrrol-2-yl)acrylonitrile, (Z)-2-(4-fluorophenyl)-
3-(1H-pyrrol-2-yl)acrylonitrile, (Z)-2-(4-chlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile, (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile)47,48
Compounds
Concentration range (lM) tested
LD50
(
lM)
LD99 (lM)
Test compounds with nematocidal activity:
10–100
40
80
B2
B3
10–100
10–100
30
25
90
100
B21
Control compounds:
6.25–100
6.25–100
6.25–100
6.25–100
6
10
10
7
100
50
(Z)-2-Phenyl-3-(1H-pyrrol-2-yl)acrylonitrile
(Z)-2-(4-Fluorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile
(Z)-2-(4-Chlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile
25
12.5
(Z)-2-(3,4-Dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile
The LD50 and LD99 values for each compound are indicated.

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B. E. Campbell et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3277–3281
donor–acceptor groups or that these groups prevent the penetra-
tion of the tissues or cuticle of the parasite, or that analogues with
the library A pharmacophore are substrates for an active efflux or
degradation mechanism. Which mechanism is operational is cur-
rently unknown. Library C differs in the relative spatial presenta-
tion of the key pharmacophoric moieties (relative to library B),
suggesting that the position of these groups is crucial to eliciting
the observed lethality of H. contortus for B2, B3 and B21. Although
an ovicidal effect has been reported for some commercially avail-
able anthelmintics, such as benzimidazoles,49,50 there was no evi-
dence of this effect for any of the analogues tested herein.
H. contortus and also for activity in vitro and in vivo against other
parasitic nematodes. In future, compounds that are not toxic to
mammalian cell lines and have failed screens on plant parasites
and/or, for example, cancer cells should be screened for activity
and lethality against parasitic nematodes of animals and humans.
Acknowledgments
This work was supported by the Australian Research Council
(ARC). Animal ethics approval (AEC no. 0707258) was granted by
The University of Melbourne. Thanks to the late Paul Presidente
for kind and consistent support.
Until relatively recently, the search for novel drugs against par-
asites has usually been carried out using approaches which are
decades old, such as the screening of many thousands of chemicals
for inhibition or disruption of parasite growth and/or development
in vitro. Today, genomic, proteomic, bioinformatic and/or chemo-
informatic technologies are increasingly being used to assist the
search for new compounds.30,51–57 A major goal of current genomic
and transcriptomic studies of parasites is the inference of novel
candidate drug targets, guided by essentiality and genetic interac-
tion studies.16–23,27,28 However, the major challenge is not only to
identify potential targets, but, importantly, to prioritize them, such
that available resources can be focused on those most likely to lead
to effective treatments. The length of time and the prohibitive costs
associated with bringing a new drug to market, together with the
knowledge that most lead-compounds fail at some stage in the
development process, have deterred most pharmaceutical compa-
nies from investing in the discovery of entirely novel targets and
classes of anthelmintics using integrated genomic–bioinformatic–
chemoinformatic platforms. However, the recent success in devel-
oping monepantel through to a commercial product58–64 provides
fresh hope for the discovery of novel classes of anthelmintic
compounds.65
In the present investigation, we were guided by a range of pre-
vious studies16,24,25,31 showing that selected serine–threonine
phosphatases (i.e., PP1 and PP2A) might represent suitable targets
for strongylid nematodes, including H. contortus, because they are:
(i) known to be essential for growth, development, survival and/or
reproduction, (ii) conserved between these nematodes and C. ele-
gans but (ii) divergent from related molecules in other inverte-
brates and vertebrates (including mammalian hosts).25 That
some norcantharidin derivatives display exquisite PP1 and PP2A
inhibitor activity34,35,40,41 suggested that a series of analogues, with
no or limited toxicity to mammalian cell lines, could be designed
and produced to specifically inhibit serine–threonine phosphatases
of H. contortus. Three of the 54 analogues synthesized displayed al-
most complete lethality to H. contortus in LDA, achieving a ‘hit rate’
that exceeded (by at least five times) that reported previously for
traditional screening methods.66
Although norcantharidin is known as a phosphatase inhibi-
tor,40,41,34–37,67 some of the novel analogues synthesized and tested
herein (and which no longer closely resemble the original ‘back-
bone molecule’) might have molecular targets other than PP1s
and/or PP2As. Currently, we are exploring new approaches to facil-
itate and determine the target(s) of these compounds. In addition,
further work should also focus on improving the LD50 and bioavail-
ability of the three compounds. A major commercial advantage of
these chemicals over some other currently available anthelmintics
is that they can be produced in one to two steps in large amounts
at low cost and high purity, and do not require any additional steps
for the isolation of the active isomer. By contrast, monepantel (an
aminoacetonitrile derivative),56 for example, needs to be synthe-
sized in multi-step chemical reaction pathways, followed by the
isolation of the active optical isomer. Given that the present nor-
cantharimide analogues display a lack of toxicity to mammalian
cells, they should now be tested directly in vivo (in sheep) against
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.031.
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