Synthesis and structure-activity relationship study of pyrrolidine-oxadiazoles as anthelmintics against Haemonchus contortus

Article abstract of DOI:10.1016/j.ejmech.2020.112100

Parasitic roundworms (nematodes) are significant pathogens of humans and animals and cause substantive socioeconomic losses due to the diseases that they cause. The control of nematodes in livestock animals relies heavily on the use of anthelmintic drugs. However, their extensive use has led to a widespread problem of drug resistance in these worms. Thus, the discovery and development of novel chemical entities for the treatment of parasitic worms of humans and animals is needed. Herein, we describe our medicinal chemistry optimization efforts of a phenotypic hit against Haemonchus contortus based on a pyrrolidine-oxadiazole scaffold. This led to the identification of compounds with potent inhibitory activities (IC₅₀ = 0.78–22.4 μM) on the motility and development of parasitic stages of H. contortus, and which were found to be highly selective in a mammalian cell counter-screen. These compounds could be used as suitable chemical tools for drug target identification or as lead compounds for further optimization.

Full text of DOI:10.1016/j.ejmech.2020.112100

Journal Pre-proof
Synthesis and structure-activity relationship study of pyrrolidine-oxadiazoles as
anthelmintics against Haemonchus contortus
Banfeng Ruan, Yuezhou Zhang, Solomon Tadesse, Sarah Preston, Aya C. Taki,
Abdul Jabbar, Andreas Hofmann, Yaqing Jiao, Jose Garcia-Bustos, Jitendra Harjani,
Thuy Giang Le, Swapna Varghese, Silvia Teguh, Yiyue Xie, Jephthah Odiba, Min Hu,
Robin B. Gasser, Jonathan Baell
PII:
S0223-5234(20)30067-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112100
EJMECH 112100
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 December 2019
Revised Date: 22 January 2020
Accepted Date: 22 January 2020
Please cite this article as: B. Ruan, Y. Zhang, S. Tadesse, S. Preston, A.C. Taki, A. Jabbar, A. Hofmann,
Y. Jiao, J. Garcia-Bustos, J. Harjani, T.G. Le, S. Varghese, S. Teguh, Y. Xie, J. Odiba, M. Hu, R.B.
Gasser, J. Baell, Synthesis and structure-activity relationship study of pyrrolidine-oxadiazoles as
anthelmintics against Haemonchus contortus, European Journal of Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.ejmech.2020.112100.
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Cl
Cl
HN
HN
N
N
N
N
O
N
O
N
HTS
O
O
O
4a
147
Cl
HN
O
xL3 motility IC50 = 0.78 µM
xL3 motility IC50 = 5.3 µM
L4 development IC50 = 21.0 µM
L4 development IC50 = 22.4 µM
N
N
N
O
Cl
HN
N
O
SN00797439
N
O
N
4b
xL3 motility IC50 >100 µM
L4 development IC50 >100 µM

Synthesis and Structure-Activity Relationship Study of
Pyrrolidine-oxadiazoles as Anthelmintics against
Haemonchus contortus
Banfeng Ruan,a, b, † Yuezhou Zhang,a, c, d, † Solomon Tadesse,e, † Sarah Preston,c, f
Aya C. Taki,c Abdul Jabbar,c Andreas Hofmann,c Yaqing Jiao,c Jose Garcia-Bustos,c
Jitendra Harjani,a Thuy Giang Le,a Swapna Varghese,a Silvia Teguh,a Yiyue Xie,a
Jephthah Odiba,a Min Hu,d Robin B. Gasserc and Jonathan Baella, g, h, i *
g School of Pharmaceutical Sciences, Nanjing Tech University, No. 30, South Puzhu Road, Nanjing
211816, P. R. China.
aMedicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville,
Melbourne, Victoria 3052, Australia.
bKey Lab of Biofabrication of Anhui Higher Education, Institution Centre for Advanced
Biofabrication, Hefei University, Hefei 230601, P. R. China.
cFaculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC
3010, Australia.
dState Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong
Agricultural University, Wuhan, Hubei 430070, China.
eDepartment of Pharmaceutical Chemistry and Pharmacognosy, School of Pharmacy, College of
Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia.
f School of Health and Life Sciences, Federation University, Ballarat, Victoria 3353, Australia.
hARC Centre for Fragment-Based Design, Monash University, Parkville, VIC 3052, Australia
iAustralian Translational Medicinal Chemistry Facility (ATMCF), Monash University, Parkville,
Victoria 3052, Australia
†Authors contributed equally
*Corresponding author: Jonathan Baell
E-mail addresses: jonathan.baell@monash.edu
Abstract
Parasitic roundworms (nematodes) are significant pathogens of humans and
animals and cause substantive socioeconomic losses due to the diseases that
they cause. The control of nematodes in livestock animals relies heavily on the
use of anthelmintic drugs. However, their extensive use has led to a widespread
1

problem of drug resistance in these worms. Thus, the discovery and
development of novel chemical entities for the treatment of parasitic worms of
humans and animals is needed. Herein, we describe our medicinal chemistry
optimization efforts of a phenotypic hit against Haemonchus contortus based on a
pyrrolidine-oxadiazole scaffold. This led to the identification of compounds with
potent inhibitory activities (IC50 = 0.78 – 22.4 μM) on the motility and
development of parasitic stages of H. contortus, and which were found to be
highly selective in a mammalian cell counter-screen. These compounds could be
used as suitable chemical tools for drug target identification or as lead
compounds for further optimization.
Keywords: Anthelmintic, pyrrolidine-oxadiazoles, Haemonchus contortus, drug
discovery, structure-activity relationship
1. Introduction
Helminth infestations of humans and animals pose a public health threat and
result in economic losses around the globe [1, 2]. In humans, helminths cause
significant morbidity and mortality [3]. In animals, their economic impact on
worldwide livestock production is substantial and often devastating [4]. The
ubiquitous blood-feeding nematode, Haemonchus contortus – also known as the
barber’s pole worm – is one of the most important pathogens of ruminant
livestock (including sheep and goats). H. contortus worms attach to abomasal
mucosa and feed on the blood, causing anaemia, oedema, and often death of
infected animals [5, 6]. The control of H. contortus and numerous other related
nematodes relies heavily on the use of anthelmintic drugs. However, the
extensive use of anthelmintics, particularly in livestock animals, has led to a
widespread problem of drug resistance in nematode populations [7].
Efforts towards developing novel anthelmintics are relatively limited, despite the
increased need to reduce the subclinical, clinical and economic burden
2

associated with helminth infections [8, 9]. Existing anthelmintic drugs include
the chemical classes benzimidazoles, imidazothiazoles and macrocyclic lactones
[10]. Over the past decades, only few new classes of anthelmintics have been
discovered and approved, and only three new scaffolds have made it into the
market since 2007 [10]: cyclodepsipeptides (e.g. emodepside), which are
currently restricted to use in companion animals; amino-acetonitrile derivatives
(e.g. monepantel) and spiroindoles (e.g. derquantel) (Figure 1). Unfortunately,
resistance has arisen to most of these drugs too [7, 8], further driving the
urgency for the discovery of new chemical entities to control nematode
infections of livestock animals.
Figure 1. Structures of emodepside, monepantel, and derquantel.
In a previous high-throughput screening (HTS) campaign on the ‘Open Scaffolds’
collection from Compounds Australia [11], we identified SN00797439 (4) as a
‘hit’ compound with broad-spectrum anthelmintic activity against biologically
and genetically distinct nematodes (Figure 2, unknown absolute stereochemical
configuration). In particular, SN00797439 (4) induced a characteristic “coiled”
exsheathed third-stage larvae (xL3) phenotype (IC50 = 3.46 – 5.93 µM) in H.
contortus, inhibited motility of fourth-stage larvae (L4; IC50 = 0.31 – 12.5 µM) and
caused considerable cuticular damage to L4s in vitro. In the absence of prior
literature
describing
the
anthelmintic
activity
of
1,2,4-oxadiazol-5-pyrrolidine-1-carboxamide derivatives, we undertook further
3

development of SN00797439 (4) by means of a hit-to-lead and a lead
optimization campaign. We aimed at improving the in vitro potency, selectivity
and absorption, distribution, metabolism, excretion and toxicity (ADMET)
properties, so as to identify a compound suitable for anthelmintic efficacy and
safety evaluations in animals.
Figure 2. Chemical structure of SN00797439 (4). *unknown absolute
stereochemical configuration. NE -Northeast, SE -Southeast.
Here, we describe the synthesis and optimization efforts toward the discovery of
a novel class of anthelmintics. The initial hit SN00797439 (4) was progressed
through systematic structure-activity relationship (SAR) explorations around the
northeast (NE) and southeast (SE) phenyl rings (Figure 2). In vitro cytotoxicity
evaluation of the active compounds, along with ADME studies, performed on a
lead compound are also reported.
2. Results and Discussion
To determine the absolute stereochemistry of the initial hit SN00797439 (4), we
first synthesized pure R and S enantiomers using commercially available
Boc-protected L- and D-proline (5a and 5b respectively) as shown in Scheme 1.
Amide coupling of the respective prolines with benzamidoxime (6), followed by
subsequent cyclization afforded the key intermediate 7 [12, 13]. Boc
4

deprotection of 7 gave 8 [14], which upon subsequent treatment with
4-chlorophenyl isocyanate (44), yielded the enantiomerically pure target
compounds 4a and 4b [15].
Scheme 1. General synthetic route for the preparation of enantiomerically pure
4a and 4b, and NE substituted pyrrolidine-oxadiazole analogues 79–112.
Reagents and reaction conditions: (a) 1) EDCI, HOAT, RT, overnight; 2)
1,4-dioxane, 100 °C, overnight; (b) TFA, DCM, RT, 4 h; (c) Triphosgene, Et3N, DCE,
0 °C to RT for 0.5 h, then 85 °C for 8 h; (d) 1) DPPA, Et3N, 1,4-dioxane, RT, 1 h; 2)
1,4-dioxane, 100 °C, 2 h; (e) Et3N, DCM, RT, 2 h.
To understand the influence of chirality on the potency of SN00797439 (4), the
enantiomerically pure 4a and 4b were evaluated for their anthelmintic activity
(Figure 3). A significant difference in potency between the enantiomers was
observed. The S enantiomer 4a displayed moderate activity against both xL3
motility and L4 development with an IC50 of 5.3 and 21.0 µM, respectively,
whereas the R enantiomer 4b was inactive (IC50 >100 µM). Thus, it was
ascertained that the initial hit SN00797439 (4) had S configuration and that the
R enantiomer was inactive. Therefore, all subsequent SAR studies were
5

performed using the S enantiomer only.
Figure 3. Enantiomerically pure S (4a) and R (4b) isomers of SN00797439 (4)
and their activity against xL3 and L4.
For
the
present
SAR
studies,
we
kept
the
1,2,4-oxadiazol-5-pyrrolidine-1-carboxamide core constant, and investigated the
impact of modification at the NE and SE phenyl rings, keeping compound 4a as
the reference.
The NE substituted analogues were synthesized using Scheme 1, starting with
Boc, protected L-proline 5a. Intermediate 8a was synthesized as described
earlier and was then reacted with various aromatic isocyanates 44–78 [16, 17]
to obtain NE substituted analogues 79–112.
For the synthesis of SE substituted analogues, the synthetic route was slightly
modified, in order to facilitate late-stage diversification (Scheme 2). All SE
substituted analogues were synthesized with 4-chlorophenyl as the NE ring, the
same as the reference compound 4a. Upon treatment with thionyl chloride in
methanol, Boc-L-proline (5a) gave the ester 113, which was then treated with
4-chlorophenyl isocyanate (44) to obtain compound 114. Ester hydrolysis,
6

followed by amide coupling with various substituted benzamidoximes (116–
136), led to one-pot cyclization to afford the target compounds 137–157.
Scheme 2. Synthetic route for the preparation of SE pyrrolidine-oxadiazole
analogues 137–157. Reagents and reaction conditions: (a) thionyl chloride,
methanol, 0 °C to 75 °C, 2 h; (b) Et3N, DCM, RT, 4 h to overnight; (c) LiOH,
methanol, RT, overnight; (d) 1) EDCI, HOAT, RT, overnight; 2) 1,4-dioxane, 100 °C,
overnight.
2.1 Anthelminthic Structure Activity Relationship Studies
To determine anthelmintic efficacies in vitro, all of the compounds synthesized
were evaluated for inhibition of exsheathed third-stage larvae (xL3s) motility
and development to the fourth-stage larvae (L4s) of H. contortus. Monepantel and
moxidectin were used as positive controls [18]. Selected compounds were also
tested for cytotoxicity in non-tumorigenic breast epithelial MCF10A cells to
determine their in vitro toxicity to mammals.
NE phenyl ring modifications. To understand the importance of 4-chloro
7

substitution on the NE phenyl ring (as in reference compound 4a), we
synthesized the des-chloro analogue (79). Compound 79 exhibited a complete
loss of activity, thereby confirming that substitution at the 4-position was crucial
for activity (Table 1). Replacement of the electron-withdrawing chlorine atom
with small and long chain electron donating alkyl chains (80, 81) also led to a
complete loss of activity. Therefore, we continued SAR studies with
electron-withdrawing substituents at the 4-position. Of the various halogens
probed, fluoro substitution was least favored, while bromo and iodo substitution
led to a boost in potency. In particular, bromo substitution (83) resulted in a
3-fold increase in activity against xL3s, while maintaining moderate potency
against L4s. Iodo (84) and CF3 (85) substituents, on the other hand, had similar
potencies to the 4-chloro analogue on xL3s, but showed 6-fold and 3-fold
increased potencies, respectively, against L4s. Of the numerous other
electron-withdrawing groups explored, only the 4-OCF3 analogue (88) exhibited
comparable activity to 4a, while all of the other substituents led to a complete
loss of activity.
In summary, among the substituents explored at the 4-position of NE phenyl ring,
the Cl, Br, I, CF3 or OCF3 groups were regarded as favored, illustrating the
importance of the degree of electronegativity and hydrophobicity for the
observed SAR.
Next, the 2- and 3-positions of the NE phenyl ring were explored with similar
substituents as in the 4-position. Unfortunately, none of the substituents at the 2-
and 3- positions (95–112) were tolerated (Table 1). In an attempt to explore SAR
with decreased lipophilicity, the phenyl ring was replaced by a pyridine moiety
(compounds 94 and 103). Akin to their phenyl analogue 79, the pyridine
derivatives were also not active. To understand whether the potency of
compounds 4a, 83–85 and 88 was due to an acidic NH proton in the urea or the
presence of a 4-halophenyl group, we synthesized and tested selected carboxylic
8

acid isosteres (Schemes S1 and S2, and Table S1, Supporting Information). It was
found that the carboxylic acid isosteres were inactive, thereby suggesting that
the activity was greatly determined by the presence of a 4-halophenyl group.
Table 1. SAR of the NE phenyl ring and cytotoxicity data for selected active
compounds on the human breast epithelial MCF10A cells.
Compound
R
xL3
L4
Cytotoxicity
at 50 µM
motility development
IC50 (µM)
± SD
IC50 (µM) ±
SD
4a
79
80
81
82
83
84
85
4-Cl
H
5.3 ± 1.8
>100
21.0 ± 7.0
>100
7.5%
n.d.
4-CH3
4-CH(CH3)2
4-F
>100
>100
n.d.
>100
>100
n.d.
>100
>100
n.d.
4-Br
1.8 ± 0.7
5.6 ± 2.5
9.5 ± 4.5
18.0 ±
15.0
12.0 ± 3.0
3.2 ± 0.2
7.0 ± 6.0
20%
8%
4-I
4-CF3
33%
86
4-CN
>100
27%
87
88
89
4-OCH3
4-OCF3
4-OH
>100
>100
13.0 ± 10.0
>100
n.d.
18%
n.d.
~4.2
>100
9

90
91
4-COCH3
4-SO2CH3
4-SO2N(CH
>100
>100
>100
>100
n.d.
n.d.
92
>100
>100
n.d.
)
3 2
93
94
4-NO2
4-[N]
3-CH3
3-F
~50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
~50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
22%
n.d.
95
n.d.
96
n.d.
97
3-Cl
n.d.
98
3-Br
n.d.
99
3-CN
3-OCH3
3-OH
3-COCH3
3-[N]
2-CH3
2-F
n.d.
100
101
102
103
104
105
106
107
108
109
110
111
112
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2-Cl
n.d.
2-Br
n.d.
2-I
n.d.
2-CN
2-OCH3
2-OH
2-COCH3
n.d.
n.d.
n.d.
n.d.
0.16 ±
99%
(IC50=26.9)
100%
(IC50 < 1uM)
Monepantel
Moxidectin
0.075 ± 0.04
3.45 ± 0.75
0.008
0.08 ±
0.04
n.d. = not determined
10

SE phenyl ring modifications. The reference compound 4a carried an
unsubstituted SE phenyl ring; SAR studies around this region therefore probed
each position of the SE ring with substituents of varying electronic and
hydrophobic properties. The results obtained from this exploration are shown in
Table 2. At the 2-position, a methyl group (137) was highly favored with an
impressive submicromolar (IC50 = 0.78 µM) inhibitory activity against xL3
motility (7-fold improved potency compared with 4a). Chloro (139) and bromo
(140) substituents were also tolerated at the 2-position, with xL3 IC50 values of
9.2 µM and 12 µM, respectively. At the 3-position, a methoxy group (147) was
most potent (xL3 IC50 = 0.78 µM), while other hydrophobic substituents such as
methyl (143), fluoro (144), chloro (145) and bromo (146) also yielded active
compounds with IC50 values ranging from 1.1 – 8 µM in the xL3 motility assay. At
the 4-position, a variety of substituents were explored, of which only fluoro (151)
and chloro (152) substituents were favored with xL3 IC50 values of 1.3 µM and
1.5 µM, respectively.
Table 2. SAR of the phenyl group attached to the oxadiazole (SE) and cytotoxicity
data for selected active compounds on the human breast epithelial MCF10A cells.
Compound
R
xL3
L4
Cytotoxicity
at 50 µM
motilit development
y
IC50 (µM) ±
SD
IC50
(µM) ±
SD
137
2-CH3
~0.78
~50
36%
11

None
but
138
139
2-F
>100
30%
40%
coiled
9.2 ±
2.9
2-Cl
~20.0
140
141
142
2-Br
2-OCH3
2-OH
~12.0
>100
>100
1.9 ±
0.9
22.0 ± 20.0
>100
30%
n.d.
>100
n.d.
143
144
145
146
3-CH3
3-F
>100
18.0 ± 3.0
7.6 ± 4.0
>100
29%
22%
17%
20%
8.1 ±
0.8
3.0 ±
1.3
3-Cl
3-Br
1.1 ±
0.6
147
148
149
3-OCH3
3-OH
~0.78
>100
>100
None
but
~22.4
>100
>100
32%
n.d.
3-CN
16%
150
4-CH3
50-100
47%
coiled
1.3 ±
1.4
151
152
4-F
19.0 ± 5.1
~20.0
22%
22%
1.5 ±
0.8
4-Cl
None
but
153
4-Br
>100
30%
coiled
12

None
but
154
155
4-I
~50
23%
13%
coiled
50 –
4-OCH3
>100
100
156
157
4-OH
4-CN
>100
>100
0.16 ±
0.008
0.08 ±
0.04
>100
>100
n.d.
11%
99%
Monepantel
Moxidectin
0.075 ± 0.04
3.45 ± 0.75
(IC50=26.9)
100%
(IC50 < 1uM)
n.d. = not determined
2.2 Cytotoxicity studies
Cytotoxicity of the active compounds in non-tumorigenic human breast epithelial
MCF10A cells was also measured (Tables 1 and 2). All active compounds from
this series displayed a high level of selectivity towards the parasite, with
cytotoxicity IC50 values of > 50 μM against MCF10A cells.
2.3 Metabolic studies
To assess the drug-likeness of this series of analogues, we measured various
physicochemical and metabolic parameters of compound 4a (Table 3). It was
observed that the compound had poor solubility, however, the polar surface area
(PSA) is within limits preferred for optimum cellular permeability and oral
absorption of drug candidates, and the compound has an intermediate predicted
hepatic clearance.
Table 3. Physicochemical parameters and metabolic stability of compound 4a.
MWa
PSAa
cLogPa
cLogDa
Solubilityb (µg/mL)
13

pH 2.0
pH 6.5
368.8
71.3
4.7
4.7
1.6–3.1
1.6–3.1
Microsome
Species
T1/2
Clint,
Predicted
Clint,
Predicted
Clblood
Predicted
Clearance
c
(min)
in vitro
(µL/min
/mg
EH
in vivo
(µL/min/k
g)
(µL/min/kg)
protein)
Human
Mouse
93
20
19
89
15
9
0.43
0.66
intermediate
intermediate
229
79
aCalculated using ChemAxon JChem software. bKinetic solubility determined by
nephelometry (SolpH). c Predicted hepatic extraction ratio calculated from in vitro data.
MW, molecular weight; PSA, polar surface area; FRB, free rotatable bonds, HBD,
hydrogen bond donor; HBA, hydrogen bond acceptor; Fsp3, fraction of sp3 carbon
atoms; cLogP, partition coefficient; cLogD, distribution coefficient.
2.4 Crystal structure of compound 146
To determine the structural orientation of these target compounds, we used single
crystals of one of the analogues for X-ray crystallography. As shown in Figure 4, the
absolute configuration of compound 146 (chiral carbon, C11) was confirmed to be the
S configuration. The oxadiazole ring, carbamide group and the NE and SE aromatic
rings, were found to be planar, while the pyrrolidine ring adopts an envelope
conformation with C9 lying significantly out of the plane from the other four atoms.
14

The dihedral angle between the NE ring and the carbamide group is 28.3(2)°, while the
dihedral angle between the 1,2,4-oxadiazole ring and the adjacent phenyl ring is
8.9(3)°. The NE ring and the pyrrolidine ring lie almost perpendicular to the plane of
the 1,2,4-oxadiazole ring, as evidenced by C4···C11···C13 (109.12 (10) °).
Figure 4. ORTEP drawing of compound 146 (H2O was omitted for clarity).
3. Conclusions
Structural optimization of hit compound SN00797439 (4), identified through a
high-throughput phenotypic screening using the H. contortus larval motility and
development assays resulted in the discovery of a pyrrolidine-oxadiazole series
with a potential to become a novel class of anthelmintics. Of the various
compounds generated in this series, compounds 137 and 147 were identified as
promising candidates against xL3 motility (IC50 = 0.78 µM for either compound),
whereas compound 84 was identified as an effective inhibitor of L4 development
(IC50 = 3.2 µM). These compounds exhibited high potency with IC50 values in the
low micromolar range, and displayed good selectivity against mammalian
epithelial cell with very limited inhibition of cell proliferation at 50 µM. The
overall profile of these compounds renders them attractive as early lead
15

compounds. In order to identify compounds with the nanomolar potency that
may be required for agricultural utility, future work will explore core
modifications that alter the geometry of this highly rigid system.
4. Experimental Section
4.1. Chemistry
Please refer to Supporting Information.
4.2 Crystallographic studies
Single crystals of compound 146 were obtained by slow evaporation from
dichloromethane from enantiomerically pure samples. Data collection was
carried out on an Oxford Diffraction SuperNova diffractometer using mirror
monochromated Mo-Kα radiation (λ = 0.71073 Å) at 130 K; care was taken to
ensure the data were sufficiently complete to enable the reliable determination
of the absolute configuration. Data were processed with Olex2 [19] and the
structure solved with ShelxT [20] using intrinsic phasing. Structure refinement
using all data and Gaussian absorption correction was carried out with the
ShelxL refinement package by least squares minimization on F2. All non-
hydrogen atoms were refined with anisotropic displacement parameters.
Crystallographic data: C19H16BrClN4O2.H2O, monoclinic, space group P21; a =
5.9257(4), b =12.6071(8), c = 12.9556 (12) (Å); α = γ = 90, β = 98.804(7) (°); V =
956.46(13) (Å3), Z = 2, Dc =1.617 g/cm3, F(000) = 472, Reflections collected:
11630 (Rint = 0.0351), Data/restraints/parameters: 5815/1/262, Goodness-of-fit
on F2 = 1.077; R1, wR2 (all data) = 0.0457/0.0894, R1, wR2 [I > 2
σ (I)] = 0.0389/
0.0854; Larg. peak/hole (e. Å) = 1.026/ -0.461; CCDC. No 1568704.
Crystallographic data (excluding structure factors) for the structures have been
deposited with the Cambridge Crystallographic Data Center.
4.3. Biology
16

H. contortus xL3 motility assay. The whole-organism screening assay
developed by Preston and coworkers was used to evaluate the effect of
compounds on the motility of xL3s of H. contortus [19]. On each 96-well plate, the
positive control compounds (moxidectin and/or monepantel) were arrayed in
triplicate. Six wells were used for each negative control (LB* + 1% DMSO and LB*
alone). Test compounds (9 point two-fold serial dilution starting at 100 µM) were
arrayed in individual wells as triplicates. Following dispensing into the plates,
300 xL3s in 50 µL of LB* were transferred to each well of each plate (with the
exception of perimeter wells). During dispensing, xL3s were kept in a
homogeneous suspension by bubbling air through the solution using an air pump
(Airpump-S100; Aquatrade, Australia). Plates were incubated in a water-jacketed
CO2 incubator at 38 °C and 10% v/v CO2. After 72 h, the plates were agitated (126
rotations per min) using an orbital shaker (model EOM5, Ratek, Australia) for 20
min at 38 °C. In order to capture the motility of xL3s at 72 h, a video recording (5
sec) was taken of each well on each plate as described previously [19]. Each 5
sec-video capture of each well was processed using a custom macro in the
program Image J (1.47v, imagej.nih.gov/ij), which indirectly measured larval
motility by quantifying the changes in light intensity over time.
H. contortus L4 development assay. The motility of L4s was evaluated using the
same protocol as for xL3 [20]. Following the measurement of larval motility,
plates were re-incubated for four more days in a humidified environment
(water-jacketed CO2 incubator) at 38 °C and 10% v/v CO2. Then, worms were
fixed with 50 µL of 1% iodine, and 30 worms from each well were examined at
20-times magnification (DP26 camera, Olympus, Japan) to assess their
development based on the presence of a well-developed mouth/pharynx in H.
contortus L4s [21]. The number of L4s was expressed as a percentage of the total
worm number (n = 30). Compounds were tested in triplicate on three different
days.
4.4 Cytotoxicity assay
17

Cell toxicity was assessed in a mammary epithelial cell line (MCF10A) as
described previously [22]. In brief, MCF10A cells were seeded in black walled,
flat bottom 384 well black walled plates (Corning, USA) at 700 cells/well using a
BioTek 406 automated liquid handling dispenser (BioTek, Vermont, USA) in a
total volume of 40 µL/well. Cells were cultured in DMEM-F12 containing 5%
horse serum (Life Technologies, Australia), 20 ng/mL human epidermal growth
factor (EGF, Life Technologies, Australia), 100 ng/mL cholera toxin (Sigma,
Australia), 0.5 µg/mL hydrocortisone (Sigma, Australia) and 10 µg/mL insulin
(human; Novo Nordisk Pharmaceuticals Pty Ltd, Australia). After an incubation
for 24 h at 37 °C, 5% v/v CO2, the growth medium was aspirated and the cells
were treated with 50, 25, 12.5, 6.3 and 3.125 µM of test compounds or negative
control (media ±0.5% DMSO), and incubated for a further 48 h. To measure cell
proliferation, cells were fixed and stained with 40,6-diamidino-2-phenylindole
(DAPI; 1:1000), and individual wells imaged at 10-times magnification, covering
16 fields (~90% of well) using a high content imager (Cellomics CellInsight
Personal Cell Imager, ThermoFisher Scientific, USA) at a fixed exposure time of
0.12 sec. Viable cells were counted using the Target Activation BioApplication
within the Cellomics Scan software (v.6.5.0, Thermo Scientific, USA) and
normalized to the cell density in wells without compound. Experiments were
repeated twice on two different days using four technical replicates for each
compound.
4.4 In vitro metabolic stability
The metabolic stability assay was performed by incubating the test compound in
mouse or human liver microsomes (XenoTech, lot no. 1510256) at 37 °C and a
protein concentration of 0.4 mg/mL. The metabolic reaction was initiated by the
addition of an NADPH-regenerating system and quenched at various time points
over a 60 min incubation period by the addition of ACN containing diazepam as
internal standard. Control samples (containing no NADPH) were included (and
quenched at 2, 30, and 60 min) to monitor for potential degradation in the
18

absence of cofactor. Microsomal incubations were performed at a substrate
concentration of 0.5 μM. Data analysis. Species scaling factors were used to
convert the in vitro CLint (μL/min/mg) to an in vivo CLint (mL/ min/kg) [23].
Hepatic blood clearance and the corresponding hepatic extraction ratio (EH)
were calculated using the well stirred model of hepatic extraction in each
species, according to the “in vitro T1/2” approach described in Obach et al [24].
The EH was then used to classify compounds as low (<0.3), intermediate (0.3 −
0.7), high (0.7 − 0.95), or very high (>0.95) extraction compounds.
4.5 Physicochemical Experimental
Calculated physicochemical parameters. Physicochemical properties were
calculated using the ChemAxon chemistry cartridge via JChem for Excel software
(version 16.4.11). Kinetic solubility estimation using Nephelometry (SolpH).
Compound 4a in DMSO was spiked into either pH 6.5 phosphate buffer or 0.01 M
HCl (approximately pH 2.0) with the final DMSO concentration being 1%. After
30 min had elapsed, samples were analyzed via Nephelometry to determine a
solubility range [25].
Conflict of interest: The authors declare no conflict of interest.
Author contributions: The manuscript was written through contributions from
all authors. All authors have given approval to the final version of the
manuscript.
Acknowledgements
For research at Monash University, the National Health and Medical Research
Council of Australia (NHMRC) is thanked for Fellowship support for J.B.
(2012-2016 Senior Research Fellowship #1020411, 2017- Principal Research
Fellowship #1117602). The Australian Translational Medicinal Chemistry
Facility (ATMCF) within Monash Institute of Pharmaceutical Sciences (MIPS)
19

acknowledges the support of the Australian Government's National Collaborative
Research Infrastructure Strategy (NCRIS) program via Therapeutic Innovation
Australia (TIA). For research at the University of Melbourne, funding from the
Australian Research Council (ARC; LP160100058), Yourgene Health, the National
Health and Medical Research Council (NHMRC; APP1102322) of Australia is
gratefully acknowledged; YJ held scholarships from the China Scholarship
Council (CSC) and The University of Melbourne; SP was the recipient of a
Pathfinder Fellowship (Wellcome).
Appendix A. Supplementary data
Supplementary data to this article can be found online at XXX
Abbreviations
Boc- tert-butyl carbonate
DPPA- Diphenyl phosphoryl azide
EDCI- 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
HOAt- 1-Hydroxy-7-azabenzotriazole
HTS- High throughput screening
NE- North East
RT- Room temperature
SAR- Structure activity relationship
SE- South East
TFA- Trifluoroacetic acid
TLC- Thin layer chromatography
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Highlights
•
•
•
•
•
Parasitic diseases present a considerable socio-economic impact to society.
A new class of anthelmintic oxadiazol-pyrrolidine derivatives is reported.
Compounds display lowmicromolar activity against Haemonchus contortus larvae.
Compounds exhibit good selectivity against mammalian epithelial cell.
Oxadiazol-pyrrolidine compounds are promising agents against Haemonchus contortus.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: