Optimization of Novel 1-Methyl-1 H-Pyrazole-5-carboxamides Leads to High Potency Larval Development Inhibitors of the Barber's Pole Worm

Article abstract of DOI:10.1021/acs.jmedchem.8b01544

A phenotypic screen of a diverse library of small molecules for inhibition of the development of larvae of the parasitic nematode Haemonchus contortus led to the identification of a 1-methyl-1H-pyrazole-5-carboxamide derivative with an IC₅₀ of 0.29 μM. Medicinal chemistry optimization targeted modifications on the left-hand side (LHS), middle section, and right-hand side (RHS) of the scaffold in order to elucidate the structure-activity relationship (SAR). Strong SAR allowed for the iterative and directed assembly of a focus set of 64 analogues, from which compound 60 was identified as the most potent compound, inhibiting the development of the fourth larval (L4) stage with an IC₅₀ of 0.01 μM. In contrast, only 18% inhibition of the mammary epithelial cell line MCF10A viability was observed, even at concentrations as high as 50 μM.

Full text of DOI:10.1021/acs.jmedchem.8b01544

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Article
Optimization of Novel 1-Methyl-1H-Pyrazole-5-carboxamides Leads to
High Potency Larval Development Inhibitors of the Barber’s Pole Worm
Thuy Le, Abhijit Kundu, Atanu Ghoshal, Nghi Nguyen, Sarah Preston, Yaqing Jiao, Banfeng
Ruan, Lian Xue, Fei Huang, Jennifer Keiser, Andreas Hofmann, Bill Chang, Jose Garcia-Bustos,
Abdul Jabbar, Timothy N.C. Wells, Michael J Palmer, Robin B. Gasser, and Jonathan B. Baell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01544 • Publication Date (Web): 07 Nov 2018
Downloaded from http://pubs.acs.org on November 9, 2018
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Optimization of Novel 1-Methyl-1H-Pyrazole-5-
carboxamides Leads to High Potency Larval
Development Inhibitors of the Barber’s Pole
Worm
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‡


‡
#
Thuy G. Le, Abhijit Kundu, Atanu Ghoshal, Nghi H. Nguyen, Sarah Preston,
#
‡, §
†
†
€,¥
Yaqing Jiao, Banfeng Ruan, Lian Xue, Fei Huang, Jennifer Keiser,

#
#
#
Andreas Hofmann, Bill C. H. Chang, Jose Garcia-Bustos, Abdul Jabbar,
£
£
#*
†,‡*
Timothy N. C. Wells, Michael J. Palmer, Robin B. Gasser, Jonathan B. Baell
†School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing
11816, People’s Republic of China.
2
‡Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University,
Parkville, Victoria 3052, Australia.
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TCG Lifesciences Private Limited, Block BN, Plot 7, Salt-lake Electronics Complex,
Sector V, Kolkata 700091, West Bengal, India.
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
Griffith Institute for Drug Discovery, Griffith University, Nathan, Queensland 4111,
Australia.
#Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of
Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria
3010, Australia.
§School of Medical Engineering, Hefei University of Technology, Hefei, PR China.
€Swiss Tropical and Public Health Institute, Basel, Switzerland.
¥University of Basel, Basel, Switzerland.
£Medicines for Malaria Venture, 1215 Geneva, Switzerland.
ABSTRACT
A phenotypic screen of a diverse library of small molecules for inhibition of the
development of larvae of the parasitic nematode Haemonchus contortus led to the
identification of a 1-methyl-1H-pyrazole-5-carboxamide derivative with an IC of 0.29 µM.
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Medicinal chemistry optimization targeted modifications on the left-hand side (LHS),
middle section and right-hand side (RHS) of the scaffold in order to elucidate the
structure-activity relationship (SAR). Strong SAR allowed for the iterative and directed
assembly of a focus set of 64 analogues, from which compound 60 was identified as the
most potent compound, inhibiting the development of the fourth larval (L4) stage with an
IC50 of 0.01 µM. In contrast, only 18% inhibition of the mammary epithelial cell line
MCF10A viability was observed, even at concentrations as high as 50 µM.
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KEYWORDS Haemonchus, Nematodes, Anthelmintics, Pyrazole-5-carboxamide.
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INTRODUCTION
Parasitic worms (helminths) are major causes of diseases of humans, other
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animals and plants worldwide. Besides the socioeconomic impact on humans, parasitic
helminths of livestock animals cause substantial productivity losses, reaching billions of
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dollars per annum. For instance, gastrointestinal roundworms (nematodes) are the main
cause of reduced weight gain, weight loss, poor meat and milk production and mortality
in farm animals. In this context, nematodes of the order Strongylida are of paramount
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importance as pathogens, and in particular the barber’s pole worm Haemonchus
contortus (H. contortus) represents this order and is one of the most pathogenic
members, affecting hundreds of millions of small ruminants (including sheep and goats)
worldwide.2,3
The control of H. contortus and other strongylid nematodes has relied heavily on
the treatment of ruminants with a small number of anthelmintic drugs. These broad-
spectrum anthelmintics work mainly by inhibiting muscle contraction, leading to a
4
paralysis of nematodes or via the disruption of microtubule function. The excessive and
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uncontrolled use of these anthelmintics has provoked the development of drug resistant
worms.5,6 The two newer groups of anthelmintics, namely amino-acetonitrile derivatives
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(e.g. monepantel) and derquantel (Figure 1), have been effective at combating multi-
resistant nematodes, but emerging resistance is proving problematic.7–10 Recent reports
disclose selenophene and thiophene derivatives, acrylonitrile-based compounds, and 2-
phenylimidazo[1,2-b]pyridazine derivatives, each with potent in vitro activity against H.
contortus, but drug development has yet to be undertaken.11–13 Thus, given the expansion
of resistance to existing anthelmintics, the search for new chemical entities with
nematocidal or nematostatic activity remains crucial.
R
CN
H
O
SCF3
O
O
HO
O
O
O
O
O
H
O
H
N
OH
N
O
O
O
NC
O
HN
N
H
CF3
O
O
OH
*
Derquantel (10 mg/ml)
Abamectin (1 mg/ml)
Monepantel
(ZolvixTM
)
*
Avermectin B1a (>80%)
O
H
2
R = CH CH3
OH
STARTECT®
Avermectin B1b (<20%)
R = CH3
Figure 1. Some current anthelmintic drugs used to treat nematodiases of livestock
animals.
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Our ultimate goal is to discover an orally administered anthelmintic for livestock.
With this focus in mind, our team established a phenotypic screening platform14 that
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assesses the motility and development of parasitic larvae of H. contortus; the use of this
platform has yielded a number of compounds with anthelmintic activity in vitro.13–18 For
instance, we have screened the ‘Pathogen Box’ (www.pathogenbox.org) from the
Medicines for Malaria Venture (MMV; www.mmv.org) to discover that the approved
16
pesticide, tolfenpyrad, possesses potent anthelmintic activity against H. contortus. We
also screened a high quality, diverse library of ~13,500 synthetic small molecules to
discover a proline derivative with similarly interesting anthelmintic activity.18 From the
latter screen, we also identified a 1-methyl-1H-pyrazole-5-carboxamide hit, designated
SN00799639 (1), which we have not yet reported on. Here, were describe medicinal
chemistry optimization and elucidation of the structure-activity relationship (SAR) for 1,
iteratively guided by results from our phenotypic assay for H. contortus.
RESULTS AND DISCUSSION
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The construction of the LHS, middle section, and RHS of the scaffold is depicted
in Scheme 1. For late-stage RHS derivatisation, from Scheme 1, the thiazole halide
building block for the LHS was subjected to a Suzuki coupling reaction for a C-C bond
formation with the middle ring section harbouring a nitrile group, which was subsequently
reduced to give the free amine that served as the precursor for subsequent RHS amide
coupling reaction. The same synthetic principle was applied when varying LHS or middle
section.
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R
N
O
X
S
CN
(b)
NH2
(c)
N
H
R'
(a)
N
N
N
R
R
R
S
S
S
CN
8 to 19
(Table 2)
HO
X = Br or Cl
R = Et or Me
R' = Substituted pyrazole
B
OH
Scheme 1 (a) Pd(dppf)Cl , K CO , dioxane/H O, 110 °C or Pd(PPh ) , toluene, 80 °C; (b)
2
2
3
2
3 4
®
LiAlH , THF; (c) HOAt, EDCI, ACN, 80 °C or HATU, DIPEA, DMF or T P , DIPEA, THF.
4
3
To examine SAR, compounds were first subjected to a primary biological assay
that assessed the motility of H. contortus at the exsheathed L3 (xL3) stage, using
monepantel and moxidectin as positive control anthelmintics. Only compounds that
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resulted in ≥ 70% motility inhibition of xL3 larvae at 100 µM concentration were then
subjected to subsequent dose-response experiments to obtain IC50 values and then
further assessed in the L4 development assay.
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Prior to the synthesis of carboxamide derivatives, initial mining of the high
throughput deck for analogues led to the purchase and screening of broadly related
analogues, but none of them was active (see Supporting Information). A set of analogues
with closely related structures of 1 was also obtained from commercial sources to assess
SAR; results are summarized in Table 1. This set mainly probed the substitution pattern
of the RHS pyrazole and the results clearly indicate the importance of the 1-alkyl group,
which was crucial to maintain activity against H contortus, as neither the des-alkyl
pyrazole nor the 2-alkyl pyrazole were tolerated (4-7). Replacing the ethyl group on the
LHS thiazole with either a methyl (2) or iso-propyl (3) group had minor effects, and
resulted in comparable IC values of 10.7 and 12.0 µM respectively. In relation to the
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measured cytotoxicity, the methyl substituents on the thiazole ring exerted only 1%
inhibition of mammal epithelial cell viability at 50 µM. Therefore, thiazole with the methyl
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substituent from 2 was considered as interchangeable with the original thiazole from 1 in
this SAR study.
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Table 1 SAR of commercially resourced compounds closely related to the initial hit
compound 1
O
4
R'
N
3
H
N
HN
1
N
R
2
S
1 to 7
Entry
R
R’
IC (µM) ± SD in IC (µM) ± SD in L4
MCF10A
50
50
#
xL3 motility assay development assay Cytotoxicity*
1
2
3
4
5
6
7
Et
1-Me
1-Me
8.40 ± 1.40
10.7 ± 5.43
12.0 ± 3.38
> 100
0.29 ± 0.10
0.37 ± 0.13
0.33 ± 0.16
24.4%
1%
Me
Iso-prop 1-Me
44%
Et
Et
2-Me
2-Et
2-Et
1-H
>100
Me
Me
>100
>100
#Compounds purchased from ChemDiv (www.chemdiv.com).
*Inhibition at 50 µM.
Tight SAR was observed from the diverse modifications on the RHS pyrazole
(Table 2). In order to first explore the chemical space of the 1-position of the pyrazole, the
methyl group was replaced with ethyl and iso-propyl to produce analogues 8 and 9,
respectively. However, this replacement led to a complete loss of activity. For the 3-
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position of the pyrazole, we experimented with electron withdrawing groups, such as CF3
and CN as seen for analogues 10 and 11; however, none of these groups was active. In
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contrast, when adding an ethyl group also at the 3-position to give 12, activity against
both xL3 motility and L4 development was regained. When CF and CN groups were each
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probed at the 4-position of the pyrazole (compounds 13 and 14), activity could not be
retained. A 4-F substituent gave rise to analogue 15, exhibited comparable activity to the
original hit 1, in terms of inhibition of both xL3 motility and L4 development. Similar to the
observation with 1, compound 15 had low cytotoxicity (< 25%) against mammal epithelial
cell proliferation when tested at a concentration of 50 µM. Interestingly, when Cl in place
of F was probed at the same position (16), activity against xL3 motility was completely
lost. Although having an additional CF group at the 3-postion (17) did not retrieve any
3
activity, activity was ‘recovered’ when the methoxy group was evaluated (18). A bulky
aliphatic group, such as cyclopentane (19), was not tolerated. In summary, among the
substituents explored for position 4, a fluoro in the 4-position of the pyrazole group was
regarded as the most favored.
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Table 2 SAR of the RHS pyrazole.
O
N
R'
H
N
R
1
1
1
1
1
1
1
1
1
1
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S
8 to 19
Entry
R
R’
IC (µM) ± SD in IC (µM) ± SD in L4
MCF10A
50
50
xL3 motility assay development assay Cytotoxicity*
>100
8
9
Me
Me
N
N
N
N
>100
10
11
12
13
Et
Et
>100
>100
CF3
N
N
N
CN
N
N
Me
Et
27.70 ± 18.75
>100
0.71 ± 0.29
26.9%
21.7%
N
CF3
N
N
CN
1
4
Et
Me
Me
Et
>100
14.50 ± 1.22
>100
N
N
F
15
0.53 ± 0.24
N
N
Cl
1
6
7
N
N
Cl
1
>100
CF3
N
N
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2
3
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cl
1
8
Me
Me
24.7 ± 17.9
>100
0.24 ± 0.12
N/A#
O
N
N
19
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
*
Inhibition at 50 µM.
#Not assessed.
To explore SAR of the LHS of the scaffold (Table 3), we first probed different
substitutions of the thiazole, such as the des-methyl analogue (20), 4,5-dimethyl (21),
benzothiazole (22) or the regioisomer 23, as well as a methylene insertion between the
middle phenyl ring section and the thiazole (compound 24). However, none of these
analogues affected H. contortus xL3 motility or L4 development. The incorporation of
hydrophobic electron-withdrawing species to the thiazole ring, such as 4-Cl (compound
2
5) or fluoroalkyl (26-28), resulted in moderate inhibitory activity on L4 development, with
IC values ranging from 0.73 to 3.25 µM. Next, we investigated other 5-membered ring
5
0
isosteres of the thiazole, such as the substituted imidazole 29, pyrazoles 30 and 31,
triazoles 32 to 34, and oxazoles 35 and 36. Of these compounds, 31 displayed moderate
activity, while the rest were completely inactive. In contrast, potent activity was observed
with a set of oxadiazoles (37-39). For the 1,2,4-oxadiazole 37 an IC value of 0.18 µM
50
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was observed for the inhibition of L4 development, which was a clear improvement from
the original hit 1. The regioisomeric 1,2,4-oxadiazole 38 was also quite potent, with an
IC value of 0.69 µM for inhibition of L4 development. Interestingly, the 1,3,4-oxadiazole
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
39 was inactive. In addition to 5-membered rings, we also explored 6-membered rings
such as pyridine, pyridazine and pyrazine (40-42), but did not observe any activity with
those. Overall, from the investigation of the LHS SAR, and in light of the observed
selectivity towards H. contortus over mammalian cells, the 1,2,4-oxadiazole moiety was
considered the best LHS scaffold.
Table 3 SAR of the LHS region
O
N
H
N
N
R
20 to 42
Entry
R
IC (µM) ± SD in IC (µM) ± SD in L4
MCF10A
50
50
xL3 motility assay development assay Cytotoxicity*
>100
N
2
0
1
S
N
2
>100
>100
S
N
22
S
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
2
3
4
>100
>100
S
2
N
S
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
25
26
27
28
65.3 ± 13.2
50 ± 0.00
3.25 ± 1.27
3.27 ± 2.01
0.73 ± 0.23
1.50 ± 0.56
34.8%
22.5%
29%
Cl
S
N
F3C
S
N
14.3 ± 5.40
23.7 ± 1.48
F CH C
3
2
S
S
N
H CF C
2
31.1%
3
N
2
9
0
>100
N
N
3
>100
N
N
31
Et
28.5 ± 4.95
4.87 ± 0.51
23.5%
N
N
N
32
33
34
>100
>100
Et
Et
N
N
NH
N
N
>100
>100
>100
Et
N
N
O
N
35
Et
O
N
36
37
38
39
40
Et
N
N
5.53 ± 4.10
16.5 ± 10.64
>100
0.18 ± 0.04
0.69 ± 0.38
21%
Et
Et
O
N
25.5%
N O
O
Et
N
N
N
>100
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9
N
4
1
2
N
N
>100
>100
4
N
*
Inhibition at 50 µM.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The SAR for the middle section of 1 was probed next, and the results are shown
in Table 4. On the 2-position of the ring, the F substituent (43) displayed moderate activity
against both xL3 motility and L4 development, while substituents such as the methoxy
(
44), methyl (45) or pyridinyl group (46) all led to a total loss of activity against xL3 motility.
The same set of substituents, when investigated at the 3-position of the ring, produced
similar results in terms of activity, such that the 3-F substituent (47) maintained the original
activity against L4 development, while the analogues 48-50 were inactive. Methylation or
cyanation of the benzylic carbon (51 and 52, as racemic mixtures) also resulted in
inactivity.
Table 4 SAR of the middle section
O
R
O
2
3
N
H
N
H
1
4
N
N
N
N
N
N
6
R
5
S
S
43 to 50
51 and 52
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ID
R
IC50 (µM) ± SD in IC (µM) ± SD in L4
MCF10A
50
xL3 motility assay development assay Cytotoxicity*
43
2-F
39.5 ± 21.9
>100
1.8 ± 0.001
29.8%
27.5%
44
2-OCH3
2-Me
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
45
46
47
>100
2-Pyridinyl
3-F
>100
22.1 ± 3.05
>100
0.24 ± 0.20
48
3-OCH3
3-Me
49
50
51
>100
3-Pyridinyl
Me
>100
>100
52
CN
>100
*
Inhibition at 50 µM.
Following our discovery of novel oxadiazoles 37 and 38, we set out to combine all
determined favorable groups of the RHS and middle section, as well as to modify the alkyl
substituent of the oxadiazole (Table 5). Replacing the ethyl group of the oxadiazole with
either methyl, n-propyl or fluoroalkyl groups led to a slight loss of xL3 motility and L4
development inhibition compared with the parent oxadiazoles (compounds 53, 54, 57, 58,
and 59). Substituents such as iso-propyl or cyclo-propyl (compounds 55 and 56) modestly
improved the activity of the original oxadiazole 37. By retaining the ethyl group of the
oxadiazole and incorporating the favorable 4-fluoropyrazole on the RHS, we observed a
10-fold improvement in L4 development inhibitory activity for 60, and 2-fold improvement
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for 61, compared with 37 and 38 respectively. Not only was a significant improvement
observed, these compounds also displayed a further reduction in cytotoxicity compared
with 37 and 38. When probing the reverse amide 62, no activity was observed. This result
might suggest that a specific orientation of the amide moiety is required for binding to the
target. Retaining the 4-fluoropyrazole RHS and replacing the middle phenyl ring with the
favored fluoro-substituted ring afforded 63 and 64. Neither of those compounds showed
an activity at the order of that observed for 60, but rather has activities at the level of the
parent oxadiazole 37.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5 Oxadiazole refinement SAR
O
O
F
N
N
H
H
N
N
N
N
R
R
53 to 59
60 and 61
O
F
H
N
2
5
R
3
N
1
H
O
N
4
N
N
N
N
R
6
Et
N
O
62
63 and 64
Entry
R
IC (µM) ± SD in IC (µM) ± SD in L4
MCF10A
50
50
xL3 motility assay development assay Cytotoxicity*
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
O
5
5
5
5
5
5
5
6
6
6
3
4
5
6
7
8
9
0
1
2
13.9 ± 6.52
11.3 ± 2.40
6.80 ± 1.23
5.57 ± 4.12
10.7 ± 7.99
11.1 ± 7.85
34.5 ± 6.50
5.67 ± 0.470
16.5 ± 5.23
>100
0.32 ± 0.04
24%
31%
N
N
0.24 ± 0.05
0.11 ± 0.02
0.08 ± 0.01
0.2 ± 0.0
N
O
N
31.5%
26.4%
28.3%
25%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
N
N
O
N
F3C
N
O
N
0.96 ± 0.03
0.95 ± 0.85
0.01 ± 0.01
0.35 ± 0.04
H CF C
3
2
N
O
N
27%
O
N
N
18%
Et
Et
Et
N
O
N
O
N
14%
N
N
O
63
2-F
13.1 ± 8.05
17.4 ± 6.12
0.1 ± 0.0
N/A#
N/A#
64
3-F
0.27 ± 0.11
*
#
Inhibition at 50 µM.
Not assessed.
Clearly, the potent activity established for the optimized oxadiazole 60 indicates a
successful additive SAR which typically suggests specific target engagement. However,
we also note that interpretation of SAR in cell-based phenotypic assays may be
influenced by factors such as differences in serum protein binding, permeability and/or
efflux mechanisms, and therefore cannot be entirely exclude other contributions to the
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observed SAR. Despite this, we have observed sharp positive and negative SAR
throughout the evaluation of this compound series. These latter observations agree with
the notion of specific target engagement. This might be attributable to the nature of the
SAR set, which is dominated by heterocyclic interchange rather than gross changes in
hydrogen-bond donating properties, lipophilicity and polarity, such that factors outside
specific target engagement are relatively similar across the compound series.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As a representative of the newly discovered bioactive 1,2,4-oxadiazole moiety,
compound 37 was subjected to testing in motility assays against a panel of different
parasitic nematodes at various concentrations (Table 6). This panel included
Heligosomoides polygyrus, a rodent nematode which is related to H. contortus;19
Ancylostoma ceylanicum, commonly known as hookworm, also related to H. contortus;
and Trichuris muris, commonly known as whipworm, belonging to the adenophorean
nematodes. From the results summarized in Table 6, it can be seen that 37 exerted 100%
inhibition on H. polygyrus adults at the concentration of 10 µM, and both third-stage larvae
(L3s) of A. ceylanicum and first-stage larvae (L1s) of T. muris at 100 µM. When tested at
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
µM on H. polygyrus adults, at 100 µM on H. polygyrus L3s, and at 10 µM on
A. ceylanicum L3s, compound 37 displayed >80% inhibition. These promising results
suggested that the oxadiazole scaffold could be a medium to broad-spectrum
nematocidal or nematostatic scaffold.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6 Biological activity profile of the 1,2,4-oxadiazole 37 against a panel of select
parasitic nematodes. Results are given in percent.
Entry H. polygyrus Adult
% inhibition)
H. polygyrus
L3
A. ceylanicum L3
T. muris L1
(
(% inhibition)
(% inhibition)
(% inhibition)
100 µM
92
1
0 µM
1 µM
87.5
100 µM
100
10 µM
84.2
100 µM
100
37
100
To assess the drug-likeliness of this 1-methyl-1H-pyrazole-5-carboxamide series,
we evaluated various physicochemical and metabolic parameters for the original hit 1, as
well as the two oxadiazoles 37 and 38. The results are summarized in Table 7 and show
that moving from the initial thiazole to oxadiazole did not markedly affect the molecular
weight (MW) and cLogD; both values are within Lipinski’s rule-of-520 for bioavailable
compounds. The polar surface area (PSA) increased slightly, but still remains well within
limits preferred for optimum cellular permeability and oral bioavailability of drug
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2
1
candidates. In addition, compound 37 exhibited a 2-fold improved aqueous solubility at
pH 2.0 and 4-fold at pH 6.5, while 38 exhibited a 2-fold improvement at pH 6.5. The greatly
reduced microsomal degradation, going from the original thiazole to oxadiazole, resulted
in longer microsomal half-lifes, ranging from 37 to 73 minutes. All three compounds (1,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7 and 38) displayed an intermediate predicted hepatic clearance.
Table 7 Key physicochemical parameters and in vitro metabolic stability of selected
compounds
Entr MWa PSA cLogDa
Solb
T1/2
CL
c
Predicted
int , in vitro
a
d
y
(pH 7.4) (µg/mL)
pH 2.0 pH 6.5
(min)
(µL/min/mg)
EH
2
(
Å )
1
326 59.8
2.6
25-50
12.5-
25
22
80
0.63
3
7
311 85.8
311 85.8
2.0
2.0
50-100 50-100
25-50 25-50
37
73
47
24
0.50
0.34
38
a
b
Calculated using ChemAxon JChem software; kinetic solubility determined by
c
nephelometry (Sol ); in vitro intrinsic clearance determined in mouse liver microsomes;
dpredicted hepatic extraction ratio calculated from in vitro data.
pH
CONCLUSIONS
In the search for novel anthelmintics, we developed a compound set based on hit
compound 1 which was discovered in a high-throughput screening of proline derivatives
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5
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
using the H. contortus motility assay. The novel series of 1-methyl-1H-pyrazole-5-
carboxamide derivatives reported here resulted from a variety of modifications made to
the original screening hit 1 with the goal of improving potency and establishing SAR.
Members of this compound series show promise as inhibitors of the development of the
parasitic nematode H. contortus in vitro and yielded a very tight SAR. The novel 1,2,4-
oxadiazole 60 exhibited a remarkable improvement in the inhibition of L4 development,
achieving an IC50 value of 10 nM. A hallmark of this chemotype is its very low in vitro
toxicity when tested in a mammalian epithelial cell type. Current efforts are directed
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
toward progressing the best compounds to the next stages of drug discovery.
EXPERIMENTAL SECTION
Nematode assays
H. contortus xL3 motility and L4 development assays
Analogues of SN00799639 were screened firstly at a concentration of 100 µM
against exsheathed third-stage larvae (xL3s) of H. contortus in 96-well microculture plates
(cat. no. 3635; Corning 3650, Life Sciences, USA) using relevant control compounds (i.e.
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moxidectin and monepantel).15 In brief, compounds were dissolved to a stock
concentration of 10 mM in dimethyl sulfoxide (DMSO; cat no. 2225; Ajax Finechem,
Australia). Using these stock solutions, compounds were then individually diluted to a final
concentration of 100 µM into Luria Bertani medium (LB) supplemented with 100 IU/mL of
penicillin, 100 μg/mL of streptomycin and 2.5 μg/mL of amphotericin (LB*). Compounds
were dispensed (in triplicate) into wells of a 96-well microculture plate using a
multichannel pipette. In addition, the negative controls (LB*, LB* + 0.5% solvent; six wells
each), and positive controls (20 µM of monepantel; Zolvix, Novartis Animal Health,
Switzerland and 20 µM of moxidectin; Cydectin, Virbac, France; triplicate wells) and xL3s
(~300/well) were dispensed into wells of the plate using an automated multichannel
pipette (Viaflo Assist/II, Integra Biosciences, Switzerland). After incubation at 38 °C and
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10% CO for 72 h, a video recording (5 sec) was taken of each well of the 96-well
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microculture plate (containing xL3s) using a grayscale camera (Rolera bolt, Q imaging
Scientific Coms, Canada) and a motorized X-Y axis stage (BioPoint 2, Ludl Electronics
Products, USA). Individual videos were processed to calculate a motility index (MI) using
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an algorithm described previously. For compounds with anti-xL3 activity, half maximum
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inhibitory concentration (IC ) values estimated from 19-point dose-response curves.
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Compounds were also tested for their ability to affect the development of xL3s to L4s and
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IC were determined from a 8-point dose response curve.
Both assays (xL3 motility
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and L4 development) were performed at least in duplicates on separate days, three times
per assay (three wells for each concentration). Motility index data from each assay were
converted to a percentage compared with respect to the negative control (LB* + 0.5%
solvent), and IC50 values determined using a variable slope four-parameter equation,
constraining the top value to 100% and using a least squares (ordinary) fit model (v.6
GraphPad Software).
A. ceylanicum L3 motility assay
A. ceylanicum larvae (L3) were obtained by filtering the faeces from infected
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hamsters and cultivating the eggs for 9 days in the dark at 24 °C. L3s were washed in
penicillin and streptomycin-supplemented tap water and kept under refrigeration until
used. For each compound, three worms were placed in each well of a 24-well plate, using
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wells per compound. Levamisole (50 μM) was used as a positive-control compound.
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Worms were incubated at 37 °C and 5% v/v CO for 72 h in the presence of 50 μM of
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each compound, and culture medium, which was composed of Hanks' Balanced Salt
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Solution (HBSS) supplemented with 10% fetal calf serum, 25 μg/ml of amphotericin B,
100 U/ml of penicillin and 100 μg/ml of streptomycin.. Thereafter, the condition of the
worms was microscopically evaluated.
T. muris L1 motility assay
For T. muris, 40 L1s were placed in each well of a 96-well plate and incubated for
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4 h at 37 °C and 5% v/v CO in the presence of 100 μl RPMI-1640 medium with
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amphotericin B (12.5 μg/ml), penicillin (500 U/ml), streptomycin (500 μg/ml) and 100 μM
of the compound to be tested. Levamisole (100 μM) was used as a positive-control
compound. Each compound was tested in duplicate. At 24 h, the total number of L1s per
well was counted. The larvae were then stimulated with 100 μl hot water and motile L1s
were counted as described by Keiser et al.22
H. polygyrus L3 motility assay
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To screen against H. polygyrus L3s, female NMRI mice were infected with 80ꢀH.
polygyrus L3 and following infection patency, H. polygyrus eggs were obtained from
infected feces as described.22 Collected eggs were placed on agar and incubated for
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days at 24 °C (in the dark), to allow the L3 to hatch. To test the compounds, 40 L3s
were placed in each well of a 96-well plate. Worms were incubated in the presence of
100 μl RPMI 1640 medium, supplemented with 0.63 μg/ml amphotericin B, 500 U/ml
penicillin, 500 μg/ml streptomycin, and compound (10 or 100 μM) to be tested. Each
compound was tested in duplicate. 1 % DMSO and culture medium and 100 μM
levamisole served as a negative and positive controls respectively. Following incubation
for 72 h, the total number of L3 larvae per well was counted, the larvae were stimulated
with 100 μl hot water (~80 °C), and the moving L3s were counted.
H. polygyrus adult motility assay
To test compounds on H. polygyrus adults, female mice were infected as described
above and dissected two weeks post-infection to collect adult worms. Three adult worms
were placed in each well of a 24-well plate and incubated with culture medium and 50 μM
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of the test compound in duplicate. Adult worms incubated with medium only, and 50 μM
levamisole and ivermectin served as negative and positive control, respectively. Worms
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were kept in an incubator at 37 °C and 5 % CO for 72 h and, subsequently,
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microscopically evaluated using a viability scale from 3 to 0 as described previously.22
Cytotoxicity assay
Compounds were tested for cytotoxicity on non-cancerous (‘normal’) mammary
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epithelial cell line (MCF10A) as described by Jiao et al. In brief, MCF10A cells (~700
cells/well) were plated into 384-well, black walled plates (Corning, New York, USA) using
a liquid handling dispenser (BioTek, Vermont, USA). Cells were cultured in DMEM-F12
containing 100 ng/ml cholera toxin (Sigma-Aldrich, St Louis, USA), 20 ng/ml human
epidermal growth factor (EGF, Life Technologies, Carsbad, USA), 10 μg/ml insulin
(human; Novo Nordisk Pharmaceuticals Pty Ltd., Bagsværd, Denmark), 5% horse serum
(Life Technologies, Australia) and 0.5 μg/ml hydrocortisone (Sigma-Aldrich, St Louis,
USA). Following a 24 h incubation (37 °C and 5% CO ), the growth medium was aspirated
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and compounds were added starting at 50 μM as well as positive- (monepantel) and
negative- (medium ± 0.5% DMSO) controls. Compounds were titrated to generate a 5-
point dose-response curve (in quadruplicate) and incubated for a further 48 h. To
measure cell proliferation, cells were fixed and stained with 4′,6-diamidino-2-phenylindole
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(
DAPI; 1:1000) and individual wells imaged at 10-times magnification, covering 16 fields
~90% of well) using a high content imager (Cellomics Cell Insight Personal Cell Imager,
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ThermoFisher Scientific, Bartlesville, USA) at a fixed exposure time of 0.12 s. Viable cells
were counted using the Target Activation BioApplication within the Cellomics Scan
software and normalized to the cell density in wells without compound. Experiments were
repeated twice on two different days.
Physicochemical experimental
Calculated physicochemical parameters using ChemAxon JChem software
A range of physicochemical properties evaluating drug-likeliness and likely oral
absorption characteristics were calculated using the ChemAxon chemistry cartridge via
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JChem for Excel software (version 16.4.11). A brief description of each parameter is
provided below:
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MW: Molecular Weight
PSApH 7.4: Polar surface area, also inversely correlates with membrane permeability
cLogDpH 7.4: Distribution coefficient, reflecting the partitioning properties of the ionised
molecule at a specific pH.
Kinetic Solubility Estimation using Nephelometry (SolpH)
Compound in DMSO was spiked into either pH 6.5 phosphate buffer or 0.01M HCl
(approximately pH 2.0) with the final DMSO concentration being 1%. After 30 minutes
had elapsed, samples were then analysed via Nephelometry to determine a solubility
range. See Bevan et al.23
In vitro Metabolic Stability
Incubation:
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