Discovery of acrylonitrile-based small molecules active against Haemonchus contortus

Article abstract of DOI:10.1039/c3md00255a

We report the discovery of a series of acrylonitrile-containing molecules and α-amino amides which cause 99-100% lethality in H. contortus. Of the 22 acrylonitrile analogues investigated, the most active were 2-cyano-3-[1-(3-dimethylaminopropyl)-2-methyl-

Full text of DOI:10.1039/c3md00255a

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Discovery of acrylonitrile-based small molecules
active against Haemonchus contortus†
Cite this: Med. Chem. Commun., 2014,
5, 159
Christopher P. Gordon,a Lacey Hizartzidis,a Mark Tarleton,a Jennette A. Sakoff,b
b
c
c
a
*
Jayne Gilbert, Bronwyn E. Campbell, Robin B. Gasser and Adam McCluskey
We report the discovery of a series of acrylonitrile-containing molecules and a-amino amides which cause
99–100% lethality in H. contortus. Of the 22 acrylonitrile analogues investigated, the most active were
2-cyano-3-[1-(3-dimethylaminopropyl)-2-methyl-1H-indol-3-yl]-N-hexylacrylamide (13a), 2-cyano-3-[1(2-
dimethylaminoethyl)-2-methyl-1H-indol-3-yl]-N-hexylacrylamide (13b), 2-cyano-3-{4-[3-(dimethylamino)-
propoxy]phenyl}-N-octylacrylamide (21), and 2-cyano-3-{1-[3-(dimethylamino)propyl]-1H-pyrrol-2-yl}-N-
octylacrylamide (22) with each displaying LD50 values <15 mM whilst the a-amino amide methyl-2-[2-(2-
benzoylphenylamino)-2-(4-methoxyphenyl)acetamido]acetate (12a) had an LD50 value of 10 mM.
A
cytotoxicity screen of the acrylonitrile analogues (13a, 13b, 21 and 22) against nine cancer cell lines
indicated modest to high cytotoxicity. In contrast, the a-amino amide 12a displayed very low cytotoxicity,
with a maximum of ꢀ30% cell death at 25 mM (A2780, an ovarian carcinoma derived cell line) and with a
mean of 11% cell death across all cell lines evaluated. Thus, 12a is considered a promising lead candidate
for the development of a new anthelmintic.
Received 9th September 2013
Accepted 2nd December 2013
DOI: 10.1039/c3md00255a
www.rsc.org/medchemcomm
Whilst these agents display potent anthelmintic activity,
even with optimally timed and strategic treatments, control is
Introduction
only partially effective. In addition, the excessive and uncon-
trolled use of some of these compounds has resulted in the
emergence of anthelmintic resistance in parasitic nematodes of
livestock.5–7 Therefore, the on-going development of novel and
improved control strategies are crucial to the future sustain-
ability of the livestock and associated industries.8,9 There is a
serious need and signicant global interest in the development
Parasites of livestock and other animals are responsible for
diseases of major socio-economic importance worldwide. The
current nancial losses caused by parasites to agriculture
(sheep and cattle) in Australia alone has been estimated at
ꢀ1 billion dollars annually.1 The barber's pole worm, Haemon-
chus contortus (Nematoda: Strongylida), is the most important
nematode of small ruminants globally and causes major
morbidity and mortality (anaemia and associated complica-
tions), accounting for 15% of gastrointestinal infections
worldwide.2 Thus, there are major economic gains to be made
in agriculture by enhancing the control of this and other
parasites.
For over two decades anthelmintic chemotherapy was
limited to a small number of major compound classes,
including the structurally complex macrocyclic lactones, such
as ivermectin (1), the nicotinic antagonists, such as levamisole
(2), and the benzimidazoles, such as albendazole (3) (Fig. 1).3,4
aChemistry, Centre for Chemical Biology, School of Environmental and Life Science,
The University of Newcastle, University Drive, Callaghan, New South Wales 2308,
Australia. E-mail: Adam.McCluskey@newcastle.edu.au; Fax: +61 249 215472; Tel:
+61 249 216486
bDepartment of Medical Oncology, Calvary Mater Hospital, Newcastle, New South
Wales 2308, Australia
Fig. 1 Structure and dosages of ivermectin (1) which is representative
of the macrocyclic lactone class of anthelmintics along with levami-
sole (2), and albendazole (3) which are representative examples of the
nicotinic antagonists and the benzimidazole classes, respectively. Oral
dosages against H. contortus were reported in ref. 3.
cDepartment of Veterinary Science, The University of Melbourne, Parkville, Victoria
3010, Australia
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3md00255a
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accessed via efficient synthetic pathways which are amenable to
scale-up.16 Furthermore, by and large, the scaffold displays
negligible toxicity against a range of human cancer cell lines.17
Herein we further investigate the acrylonitrile scaffold to
determine crucial features required for anthelmintic activity
and establish an expanded pharmacophore that is lethal to
H. contortus.
Results and discussion
In terms of H. contortus activity our preliminary structure–
activity relationship (SAR) studies of the 2-phenyl-3-(1H-pyrrol-2-
yl)acrylonitrile analogues primarily focused on “Region A” and
“Region B” modications (Fig. 3).16–19 Consequently, in this
work, our rst series of modications examined the requirement
for the acrylonitrile linking moiety. To investigate this aspect, we
envisaged the development of a small library of 1H-pyrrole-2-
propanenitrile analogues (compound 8a–d, Table 1), which we
anticipated could be accessed by ow reduction approaches
(ThalesNano H-cube™) of the parent acrylonitriles 7a–d.16
In a typical synthesis, a 0.05 M solution of each 1H-pyrrol-2-
ylacrylonitrile analogue (7a–d) was hydrogenated at 50 ꢁC,
under 50 bar of H2 pressure, utilising a 10% Pd/C catalyst at 1
mL minꢂ1 ow rate (Scheme 1). Maintaining this ow rate for
10 min afforded sufficient material (5–15 mg) for screening
against H. contortus (Table 1).
As outlined in Table 1, whilst the parent analogues 7a–d
possessed activity against H. contortus,14,15 removal of system
conjugation renders these analogues inactive with compounds
8a–d displaying no activity up to 100 mM.
Given these data, the ensuing series of analogues (9a–e)
possessed an extended aromatic linker, which was achieved via
the insertion of a carbonyl moiety between Region A while
retaining the acrylonitrile pharmacophore. To access this
Fig. 2 Structure of the current generation anthelmintics, derquantel
(4), emodepside (5) and monepantel (6). Derquantel is co-adminis-
tered the existing anthelmintic adamectin, is traded as Startect®, and
was registered for sheep in the UK during 2012. Emodepside is co-
administered with praziquantel, is traded as Profender®, and was
registered for cats in the EU and US during 2007. Monepantel is traded
as Zolvix® and has been registered for sheep since 2009.
of new and safe compounds for controlling multi-resistant
nematodes. Consequently substantial interest has been
directed towards developing next generation agents and as a
result three new classes of anthelmintics have recently become
commercially available, derquantel (4, Fig. 2), emodepside (5),
and monepantel (6). Each of these display activity against
H. contortus as well as against the adult and immature L4 stages
of all major gastrointestinal nematodes.3
Thus in a ve year period three new classes of anthelmintics
have entered the marketplace, however history suggests that the
development of nematode resistance, particularly in sheep, is
inevitable.5,10–13 Further whilst these new classes of anthelmin-
tics address the control of gastrointestinal nematodes in sheep,
cats, and dogs, the problem with treating multi-resistant
infection in livestock remains. Consequently there is a present
and on-going need to identify and develop further classes of
anthelmintic agents.
Table
1 Structure and biological activities of the acrylonitrile
analogues (7a–d) and the propanenitrile analogues (8a–d) tested
against H. contortus in a larval development assay
To this end, we have turned our attention to a series of
pyrrolacrylonitriles (exemplied by 7a, Fig. 3) which potentially
represent privileged anthelmintic molecular scaffolds.14,15 We
have focused on the pyrrolacrylonitriles scaffold, primarily due
to the absence of a stereocentre and that analogues can be
Compound
R1
R2
LD50a (mM)
LD99a (mM)
7ab
7bb
7cb
7db
8a
8b
8c
8d
–H
–H
–H
–Cl
–H
–H
–H
–Cl
–H
–F
–Cl
–Cl
–H
–F
6.0
10.0
10.0
100
50
25
12.5
>100
>100
>100
>100
7.0
>100
>100
>100
>100
–Cl
–Cl
Fig. 3 The structure and biological activity of compound 7a which
exemplifies the generic structure of the 2-phenyl-3-(1H-pyrrol-2-yl)-
acrylonitrile.14,15 LD50 value represent activities against H. contortus in a
larval development assay.
a
LD50 and LD99 determinations are the mean ꢃ95% condence interval
b
(CI) of a single experiment performed in triplicate. LD50 and LD99
values previously reported in ref. 15.
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apparent the acrylonitrile moiety plays a critical in activity
against H. contortus; however, as illustrated by compound 7f,
instillation of this moiety did not automatically bestow
activity. Intrigued by this, we examined our in-house
compound libraries for alternative acrylonitrile containing
analogues. In this ‘screening’, we sought a readily accessible
series of compounds that would allow rapid scaffold decora-
tion, but also access to a series of essentially identical
compounds that only lacked the acrylonitrile moiety. We
rationalised that this would allow direct interrogation with
respect to the acrylonitrile requirement for anthelmintic
activity.
Scheme 1 Reagents and conditions (i) ThalesNano H-cube™, 7a–d
acetone (conc. 0.05 M), 50 ꢁC, 50 bar H2, 1.0 mL minꢂ1 10% Pd/C.
Scheme 2 Reagents and conditions (i) Ac2O, 75 ꢁC, 35 min; (ii) RCHO
(see Table 2 for details), piperidine (cat), EtOH, reflux, 2 h.
To this end, the quninolin-2-(1H)-ones (exemplied by
compound 11a, Fig. 4)24–26 were identied, along with the cor-
responding acrylonitrile-free analogues (exemplied by 12a,
potentially an acrylonitrile-free control). Both compound
series, pyrrole was treated with cyanoacetic acid to afford classes were readily accessed via highly efficient and robust
synthetic routes.24
3-oxo-3-(1H-pyrrol-2-yl)propanenitrile (10) in excellent yield
As outlined in Scheme 3, the quninolin-2-(1H)-ones were
accessed via one-pot sequential Ugi-four-component
condensation and intramolecular Knoevenagel cyclisation
pathway (Scheme 3).24,26 As reported previously,24 an addi-
tional by-product afforded in this Ugi-Knoevenagel sequence
(Scheme 2). The introduction of the propanenitrile moiety
allowed rapid access to the desired series of linker-modied
analogues via a Knoevenangel condensation with a suite of
substituted benzaldehydes (Table 2 for details).18,20–23 Concur-
rent with the development of these 1H-pyrrole-b-oxo-2-pro-
a
panenitrile analogues (9a–e), we also re-examined the original is an a-amino amide adduct (10a–l, Fig. 4). Again these
acrylonitrile compound series, particularly analogues contain- analogues allowed direct validation of our acrylonitrile
ing additional moieties in Region B (7e–g).14 The results of
H. contortus screening for 7e–g and 9a–e are presented in
Table 2.
As noted with analogues 8a–d, disruption of the direct
linking of the two terminal aromatic rings by the acrylonitrile
moiety resulted in all 1H-pyrrole-b-oxo-2-propanenitrile
hypothesis.
The activity of the quninolin-2-(1H)-ones against H. contortus
was investigated through the synthesis of a focused library of
twelve analogues of 11a. Of these analogues, none displayed
signicant activity in the larval development assay (dened
herein as LD50 > 100 mM; ESI†). Serendipitously, one of the
analogues 9a–e being devoid of activity. Consequently, it was twelve acrylonitrile-free control analogues, the a-amino amide
12a, was essentially equipotent with the lead 2-phenyl-3-(1H-
pyrrol-2-yl)acrylonitrile (7a), with an LD50 value of 10 mM. As
outlined in Fig. 5, the preliminary structure–activity relation-
ship data for this series of compounds (ESI†) indicate that an
Table
2 Structure and biological activities of the acylonitrile
analogues (7c–g) and the 1H-pyrrole-b-oxo-2-propanenitrile
analogues (9a–d) tested against H. contortus in a larval development
assay
R1
R2
LD50 (mM)
LD99a (mM)
a
Compound
Fig. 4 Compounds exemplifying the general chemical structure of the
quninoline-2-(1H)-ones (11a). Additionally compound (12a), which
exemplifies the generic structure of the a-amino amides, which are a
resulting by-product formed in the synthesis of the quninolins-2-(1H)-
ones.24
7cb
7db
7ec
7fc
7gc
9a
9b
9c
9d
9e
–H
–Cl
–H
–H
–H
–H
–Cl
–H
–H
–H
–Cl
–Cl
–OCH3
–NO2
–CF3
–Cl
10.0
7.0
25
12.5
6.7
>63
3.1
>100
>100
>100
>100
>100
NDd
NDd
NDd
>100
>100
>100
>100
>100
–Cl
–OCH3
–NO2
–CF3
a
LD50 and LD99 determinations are the mean ꢃ95% condence interval
b
(CI) of a single experiment performed in triplicate. LD50 and LD99
c
values previously reported in ref. 15. LD50 values previously reported
in ref. 14. d ND not determined.
Scheme 3 Reagents and conditions (i) MeOH, rt, 2 h.
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In a bid to further investigate the acrylonitrile hypothesis, the
reduced analogues 16–18 (see Table 3) were also prepared via a
previously reported ow-reductive amination protocol (ESI†).27
Gratifyingly, the initial examination of the activity of this
more exible acrylonitrile analogue 13a against H. contortus
returned an LD50 value of 12.5 mM, which was near equipotent
to the parent acrylonitrile (7a, LD50 ¼ 6 mM). Encouraged by this
Table 3 Structure and biological activities of the a-amino amide 12a,
the cyanoamides 13a–c, 21 and 22, the thiazole-4(5H)-one analogue
19, along with the reduced analogues 16–18 against H. contortus in a
larval development assay
Fig. 5 Structural features of the a-amino amides required to afford
anti-nematode activity. Preliminary data indicate that an aryl moiety
bearing an electron-donating group is required in Region A, whilst an
aromatic moiety and an ester unit is required in Regions B and C,
respectively.
Entry
Structure
LD50a (mM)
LD99a (mM)
aryl moiety bearing an electron-donating group is required in
Region A, whilst an aromatic moiety and an ester unit are
required in Regions B and C, respectively (Fig. 5).
12a
10
30
Whist the lack of activity associated with the quninolin-2-
(1H)-ones was initially disappointing and contrary to our acry-
lonitrile hypothesise, we do note that acrylonitrile moiety found
in 11a and related analogues is constrained within a bicyclic
system; it was possible that this constraint adversely affected
activity against H. contortus. Accordingly we reinvestigated our
in-house libraries and examined the anthelmintic activity of a
series of less conformationally restricted acrylonitrile-contain-
ing compounds (e.g. 13, Fig. 6). Once again, these analogues
were rapidly accessed via Knoevenagel condensation of an
aldehyde (14a) with a cyanoamide (15a–c, Scheme 4).27,28 The
cyanoamide precursors were afforded via the solvent-free
addition of ethyl, or methyl cyanoacetate with an amine. The
subsequent addition of an aldehyde to a cyanoamide afforded
facile access to a diverse array of acrylonitrile containing
compounds, which are more conformationally exible than 11a.
13a
13b
13c
12.5
15
60
100
>100
>100
16
17
18
19
>100
>100
>100
>100
>100
>100
>100
>100
Fig. 6 Our archetypal acrylonitrile indole analogue, Dynole 34-2™
compound (13).28
20
21
>100
10
>100
30
22
10
60
Scheme 4 Reagents and conditions (i) MeOH, rt, 2 h (ii) EtOH,
microwave, 120 ꢁC, 150 W, 20 min. (Note; for conditions to access the
aldehyde precursors see ESI.†)
a
LD50 and LD99 determinations are the mean ꢃ95% condence interval
(CI) of one experiment performed in triplicate.
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Table 4 Growth inhibition (GI50) data for analogues 12a, 13a, 13b, 21 and 22 against a panel of cancer cell lines. Values in parentheses and italics
are the % growth inhibition at a drug concentration of 25 mM
Compound
HT29a
SW480a
MCF-7b
A2780c
H460d
A431e
DU145f
BE2-Cg
SJ-G2h
12a
13a
13b
21
(11 ꢃ 7)
15 ꢃ 1.0
19 ꢃ 1.2
14 ꢃ 0.0
4.5 ꢃ 0.4
(2 ꢃ 5)
8.1 ꢃ 0.3
15 ꢃ 0.7
14 ꢃ 0.3
4.0 ꢃ 0.3
(20 ꢃ 5)
16 ꢃ 0.3
25 ꢃ 3.0
14 ꢃ 0.6
5.8 ꢃ 0.3
(27 ꢃ 3)
15 ꢃ 1.3
24 ꢃ 5.0
15 ꢃ 0.3
7.1 ꢃ 0.1
(4 ꢃ 4)
16 ꢃ 0.7
29 ꢃ 4.7
21 ꢃ 2.7
10 ꢃ 1
(15 ꢃ 1)
15 ꢃ 0.7
26 ꢃ 2.0
20 ꢃ 2.3
12.0 ꢃ 3
(<0)
(8 ꢃ 3)
14 ꢃ 0.0
20 ꢃ 2.8
14 ꢃ 0.3
6.2 ꢃ 0.1
(13 ꢃ 7)
16 ꢃ 0.3
23 ꢃ 3.5
16 ꢃ 0.3
7.8 ꢃ 0.3
24 ꢃ 2.9
37 ꢃ 0.9
27 ꢃ 0.9
14.0 ꢃ 1
22
a
Colon. b Breast. c Ovarian. d Lung. e Skin. f Prostate. g Neuroblastoma. h Glioblastoma cell lines.
nding, we explored additional indole-based nitrile analogues against our panel of cancer cell lines at a concentration of
(13b and c, Table 3). 25 mM. Analogues displaying noteworthy growth inhibition were
As outlined in Table 3, the hexyl substituted 13b retained subject to full dose response evaluation (Table 4).
activity (LD50 ¼ 15 mM), showing that the shortening of the The a-amino amide (12a) is the sole anthelmintic active
indole N-alkyl substituent had limited effect on activity. Addi- analogue that displays negligible levels of toxicity in these cell
tionally, as observed previously for our norcantharidin anthel- models with a maximum of ꢀ30% cell death at 25 mM (A2780,
mintic analogues,15 an extended aliphatic chain appears to play an ovary-derived cell line), and with an average of 11% cell death
a signicant role as the incorporation of heteroatoms (i.e. 13c) across all cell lines evaluated. In contrast, the cyanoamide-
generates compounds devoid of activity against H. contortus. As containing analogues 13a, 13b, 21 and 22 displayed more robust
aforementioned, to further investigate the requirement of the levels of cytotoxicity across the cell lines evaluated.
acrylonitrile moiety in this series, we utilised our previously
However at this early stage of anti-nematode drug develop-
reported ow reduction methodologies to prepare compounds ment, it is important to recognise that this in vitro toxicity data
16–18, in addition to replacing the cyanoamide with the thia- generated from human cancer cell lines should not be viewed as
zole-4(5H)-one isostere (19) (ESI†).27 As outlined in Table 3, each a conclusive predictor of in vivo toxicity.29 Moreover, the current
of these alterations resulted in a signicant loss of activity class of anti-nematode display species dependant toxicity
against H. contortus, once again highlighting the requirement of proles, for example Startect® is currently only registered for
the acrylonitrile moiety. Further within this series of indole sheep, it is well tolerated in dogs but is essentially lethal to
analogues, it appears the dimethylamino tail is crucial as bio- horses.3 Thus toxicity against human cancer cell lines may not
isosteric replacement to –CH(CH3)2 renders the analogue inac- necessarily be equated with ruminant toxicity. Further, while
tive (20). While this effect is consistent with our related efforts compound 13b displays a LD50 value of 15 mM against H. con-
with dynamin GTPase inhibitors, it does not preclude the tortus it displays and average GI50 value of ꢀ26 mM across the
change in activity being a function of a change in the pharma- panel of cancer cells investigated. We cannot preclude differ-
cokinetic properties of 20 relative to 13a. While both analogues ences in cell membrane permeability for affecting the degree of
are soluble in aqueous DMSO at 30 mM (100% DMSO diluted to observed toxicity of 13b against the panel of cancer cells and
a nal DMSO # 1%) there is a considerable difference in c log P H. contortus. This provides initial evidence that with continued
values between 13a (c log P ¼ 4.62) and 20 (c log P ¼ 7.55) which compound development, scope exists within this series to ‘tune’
may also account for the change in observed activity.27,28 Thus, activity towards H. controtus and away from human cell
as with the 2-phenylacrylonitirle series (Table 2), whilst the cytotoxicity.
acrylonitrile is crucial, its inclusion within a scaffold does not
automatically bestow activity against H. contortus. Finally we
developed an additional two simple analogues that retained the
Conclusions
dimethylamino moiety of 13a and the 2-cyanooctylamide chain By building on our initial acrylonitrile hypothesis, we have
of 20. Accordingly, phenoxy (21) and pyrrole (22) analogues were identied two additional scaffolds, the cyanoamides and the
synthesised (ESI†). As shown in Table 3, screening of these a-amino amides as potential leads for next generation
analogues indicated excellent levels of H. contortus kill with compounds active against H. contortus. Of the analogues
LD99 values of 30 and 60 mM, respectively. Therefore, the nature investigated 12a displays the greatest potential displaying an
of the aromatic moiety appears to be of little consequence to the LD99 of 30 mM against H. contortus, and negligible toxicity
observed level of activity whereas the dimethylamino tail and against the panel of cancer cell lines. Currently this analogue is
acrylonitrile linker appear to be crucial.
being subjected to in vivo studies (sheep) and the results of
At this early stage of the program we have engaged in these investigations will be reported in due course.
preliminary evaluation of potential off-target effects. In these
studies analogues were screened against a panel of cancer cell
lines of colon, breast, ovarian, lung, skin, prostate, neuroblas-
Acknowledgements
toma and glioblastoma origin (Table 4). In this study only This work was supported by the Australian Research Council,
compounds displaying anthelmintic activity were screened The Australian Cancer Research Foundation and the Ramaciotti
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Foundation. MT gratefully acknowledges the receipt of a 15 B. E. Campbell, M. Tarleton, C. P. Gordon, J. A. Sakoff,
research scholarship from the University of Newcastle.
J. Gilbert, A. McCluskey and R. B. Gasser, Bioorg. Med.
Chem. Lett., 2011, 21, 3277–3281.
16 M. Tarleton and A. McCluskey, Tetrahedron Lett., 2011, 52,
1583–1586.
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