Structure-Activity Relationship Studies of Tolfenpyrad Reveal Subnanomolar Inhibitors of Haemonchus contortus Development

Article abstract of DOI:10.1021/acs.jmedchem.8b01789

Recently, we have discovered that the registered pesticide, tolfenpyrad, unexpectedly and potently inhibits the development of the L4 larval stage of the parasitic nematode Haemonchus contortus with an IC₅₀ value of 0.03 μM while displaying good selectivity, with an IC₅₀ of 37.9 μM for cytotoxicity. As a promising molecular template for medicinal chemistry optimization, we undertook anthelmintic structure-activity relationships for this chemical. Modifications of the left-hand side (LHS), right-hand side (RHS), and middle section of the scaffold were explored to produce a set of 57 analogues. Analogues 25, 29, and 33 were shown to be the most potent compounds of the series, with IC₅₀ values at a subnanomolar level of potency against the chemotherapeutically relevant fourth larval (L4) stage of H. contortus. Selected compounds from the series also showed promising activity against a panel of other different parasitic nematodes, such as hookworms and whipworms.

Full text of DOI:10.1021/acs.jmedchem.8b01789

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Article
Structure-activity relationship studies of tolfenpyrad reveal sub-
nanomolar inhibitors of Haemonchus contortus development
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,
Timothy N.C. Wells, Michael J Palmer, Abdul Jabbar, Robin B. Gasser, and Jonathan B. Baell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01789 • Publication Date (Web): 20 Dec 2018
Downloaded from http://pubs.acs.org on December 22, 2018
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Structure-activity relationship studies of tolfenpyrad
reveal sub-nanomolar inhibitors of Haemonchus
contortus development
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‡


‡
#,■
Thuy G. Le, Abhijit Kundu, Atanu Ghoshal, Nghi H. Nguyen, Sarah Preston, Yaqing
#
‡, §
†
†
€,¥
Andreas Hofmann,
Jiao, Banfeng Ruan,
Lian Xue, Fei Huang, Jennifer Keiser,
#
#
£
£
Bill C. H. Chang, Jose Garcia-Bustos, Timothy N. C. Wells, Michael J. Palmer, Abdul
#
#*
†,‡*
Jabbar, Robin B. Gasser, Jonathan B. Baell
†School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu
Road, Nanjing 211816, People’s Republic of China.
‡Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University,
Parkville, Victoria 3052, Australia.
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.
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#Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of
Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria
3010, Australia.
■School of Health and Life Sciences, Federation University, Ballarat, Victoria 3353,
Australia.
§School of Food and Biological Engineering, Hefei University of Technology, Hefei
230009, PR China.
€Swiss Tropical and Public Health Institute, 4051 Basel, Switzerland.
¥University of Basel, 4001 Basel, Switzerland.
£Medicines for Malaria Venture, 1215 Geneva, Switzerland.
ABSTRACT
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Recently, we discovered that the registered pesticide, tolfenpyrad (TFP),
unexpectedly and potently inhibits the development of L4 larval stages of the parasitic
nematode Haemonchus contortus with an IC value of 0.03 µM while displaying good
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selectivity, with an IC of 37.9 µM for cytotoxicity. As a promising molecular template for
50
medicinal chemistry optimization, we undertook anthelmintic structure-activity
relationships (SAR) for this chemical. Modifications of the left hand side (LHS), right hand
side (RHS), and middle section of the scaffold were explored to produce a set of 57
analogues. Analogues 25, 29 and 33 were shown to be the most potent compounds of
the series, with IC50 values at a sub-nanomolar levels of potency against the
chemotherapeutically-relevant fourth larval (L4) stages of H. contortus. Selected
compounds from the series also showed promising activity against a panel of other
different parasitic nematodes such as hookworms and whipworms.
INTRODUCTION
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Parasitic worms, particularly gastrointestinal roundworms (nematodes), are major
pathogens of livestock animals and cause diseases that, through productivity losses,
adversely impact the agricultural, meat and dairy industries.1,2 The control of these
worms relies heavily on the use of anthelmintic chemotherapy. However, the
effectiveness of many anthelmintics around the world has significantly decreased due to
widespread drug resistance in such worms resulting from the excessive and uncontrolled
use of these drugs.3–6 Therefore, the discovery of new anthelmintics with novel modes of
action and that are active against drug-resistant parasites is in high demand.7
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Recently, we identified tolfenpyrad (TFP, Figure 1) to be potently inhibitory of the
8,9
motility and development of parasitic larvae of Haemonchus contortus , a parasitic
nematode of major economic importance in ruminants. TFP is a registered pesticide used
10
in many countries to control arthropod pests on infested crops. Along with the closely
related tebufenpyrad, it belongs to the pyrazole-5-carboxamide class of complex I
inhibitors, which interrupt electron transport through inhibiting NADH:ubiquinone
oxoreductase.11 Despite being reported in 1996,12 published SAR interrogation of
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tolfenpyrad is relatively limited, even though marked insecticidal, fungicidal or miticidal
activities have been observed for this chemical.12–15 Furthermore, nothing is known about
its SAR against parasitic nematodes. Herein, by utilising a well-established but proprietary
and sophisticated phenotypic drug screening platform for H. contortus,16,17 we report, for
the first time, comprehensive SAR of TFP for inhibition of L3 motility and L4 development
of H. contortus larvae and reveal novel modifications that reach sub-nM levels of L4 larval
development inhibition.
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O
Cl
N
N
H
N
O
Tolfenpyrad (TFP)
IC xL3: 2.9 µM
50
IC L4: 0.03 µM
50
MCF10A cytotoxicity: 37.9 µM
clogP: 4.6
Figure 1 Structure, activity and cLogP of the tolfenpyrad hit.
It can be seen that TFP harbours a large, hydrophobic, electron-rich p-
methylphenoxybenzyloxy group. From a medicinal chemistry perspective, this chemical
property of TFP would be considered to be sub-optimal for a drug candidate that is
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proposed to be administered, for example, orally to a vertebrate animal affected by
worms, as opposed to the application (as pesticide) to the surface of arthropod-affected
plants. Therefore, the predominant focus of this study was to explore TFP SAR with a
view to maintaining or even increasing potency of TFP, while moving the scaffold into a
more drug-like physicochemical ‘space’ that might impart improved solubility and
metabolic stability properties.
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RESULTS AND DISCUSSION
The structure of the TFP scaffold can usefully be considered as divided into two
main
components,
namely
the
pyrazole-5-carboxamide
and
the
p-
methylphenoxybenzyloxy parts. For late-stage derivatization of the RHS pyrazole, the p-
methylphenoxybenzyloxy group was obtained via a nucleophilic aromatic substitution
reaction of p-cresol and 4-fluorobenzonitrile, followed by a reduction of the nitrile group,
to yield the benzyl amine, which was then reacted with the pyrazole-5-carboxylic acid via
an amide coupling reaction (Scheme 1). Likewise, in some cases the LHS derivatisation
process could be performed in reverse order, so that the RHS pyrazole was to be installed
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first to form a 4-halobenzylamide intermediate, which was then subjected to an Ullmann-
type coupling with a phenoxy species (Scheme 2). The CuI/N,N-dimethylglycine was an
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efficient catalytic system for the Ullmann-type coupling, as reported by Ma et al.18
CN
CN
a
OH
F
O
1
O
b
R
O
HO
R
N
N
N
NH2
H
N
N
O
O
c
2
3
-10 (Table 1)
Scheme 1 Synthetic pathway of the tolfenpyrad scaffold, a) K CO , DMF; b) LiAlH , THF;
2
3
4
c) HOAt, EDCI.HCl, ACN, or HATU, DIPEA, DMF or T3P®, DIPEA, THF.
O
Cl
O
Cl
N
O
Cl
N
HO
R
N
N
N
OH
N
NH2
H
H
N
R
N
O
Br
Br
b
a
11-12 (Table 2)
Scheme 2 A synthetic pathway used for LHS derivatisation, a) HOAt, EDCI.HCl, ACN, or
HATU, DIPEA, DMF or T3P®, DIPEA, THF, b) CuI, Cs CO , N,N-dimethylglycine, 1,4-
2
3
dioxane.
To examine SAR, compounds were first subjected to a primary screen to assess
their ability to inhibit the motility of H. contortus at the exsheathed L3 (xL3) stage, using
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monepantel and moxidectin as positive control anthelmintics. Only compounds that
resulted in ≥ 70% motility inhibition of xL3 larvae at a concentration of 100 µM were
subjected to subsequent dose-response evaluation, to establish IC50 values, and then
further assessed in the H. contortus L4 development assay. The first aim of the study was
to explore the chemical space on the RHS pyrazole of TFP, and simultaneously to reduce
the overall hydrophobicity by removing some or all of the substituents on the pyrazole,
and to introduce functional groups with differing steric and electronic effects. The results
of this exploration are summarized in Table 1. Loss of both the 3-Et and 4-Cl on the
pyrazole ring (3) led to a moderate decrease in inhibitory potency of xL3 motility and a
slight reduction in L4 development compared to TFP. A similar but diminished loss of
activity was observed when the 3-Et group was maintained but the 4-Cl removed, to give
4, which still exhibited relatively potent L4 larval development inhibition (IC 0.057 µM).
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Hence, comparing 3 with 4 suggests some hydrophobicity on the 3-position is favorable.
Implementing nitrile or trifluoromethyl groups at the 3- or 4-position led to either a
complete or substantial loss of activity, as seen for compounds 5-8. Interestingly, by
keeping the 4-Cl group in place and removing the ethyl group on the pyrazole to give 9,
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we observed a 10-fold improvement in the inhibition of L4 development compared with
TFP, furnishing a single-digit nM IC value of 3 nM. A 3-fold improvement of potency to
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inhibit L4 development was also observed in the case of 10 compared to TFP, where the
4-Cl was replaced with 4-F. The results exhibited by 9 and 10 were encouraging, as both
the aims of increasing potency and partially reducing hydrophobicity were attained. From
our recent SAR study on a broadly related 1-methyl-1H-pyrazole-5-carboxamide derived
from a different screening campaign against H. contortus, we discovered that other 5- or
6-membered rings such as furan, thiophene, substituted phenyl ring or pyridinyl moiety
were all disfavored on the RHS of the scaffold and therefore are not explored in this
study.19
Table 1 SAR of the RHS pyrazole.
O
O
4
R
3
N
N
H
H
N
N N
1
N
O
O
2
3
4-10
Entry
R
IC (µM) ± SD in xL3 IC (µM) ± SD in L4
50
50
motility assay
development assay
3
-
16.3 ± 6.66
0.13 ± 0.10
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3-Et
3-CN
3-CF3
4-CN
4-CF3
4-Cl
10.53 ± 3.44
>100
0.057 ± 0.002
1.69 ± 0.67
6
>100
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50 ± 0.001
>100
8
9a
2.97 ± 2.56
4.37 ± 2.55
2.9 ± 0.58
0.16 ± 0.008
0.08 ± 0.04
0.003 ± 0.004
0.01 ± 0.007
0.03 ± 0.005
0.075 ± 0.04
3.45 ± 0.75
1
0a
4-F
TFP
Monepantel
Moxidectin
acLogP: 9: 3.9; 10: 3.7
The next aim was to explore SAR on the LHS of TFP, again with a focus on not
only improving potency but also enhancing physicochemical properties (Table 2). In order
to increase hydrophilicity within the LHS region of TFP, the phenyl ring was replaced by
the pyridine moiety, as demonstrated by 11, 12 and 13. Gratifyingly, of these three
pyridinyl compounds, while 11 completely lost activity, 12 and 13 maintained potent
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inhibitory activity against L4 development with respective IC values of 0.03 and 0.019
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µM, and a small loss of activity against xL3 motility.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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Table 2 SAR of the LHS region.
O
O
Cl
N
N
N
H
H
R
R
N
N
N
O
O
11-22
23
Entry
R
IC (µM) ± SD in xL3
IC (µM) ± SD in L4
5
0
50
motility assay
development assay
N
1
1
2
>100
N
N
1
4.0 ± 1.84
8.67 ± 4.55
5.2 ± 3.02
0.03 ± 0.01
0.019 ± 0.011
0.14 ± 0.05
F
F
1
3
4
1
F
1
5
13.33 ± 12.0
0.04 ± 0.005
0.04 ± 0.01
F
16
17
8.43 ± 7.53
>100
F3C
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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
8
9
F3C
>100
O
N
1
35.07 ± 17.19
0.45 ± 0.04
0.34 ± 0.32
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
O
N
2
0
1
5.30 ± 3.0
>100
O
2
HO
N3
Cl
2
2
3
50 ± 0
0.16 ± 0.11
2
2.43 ± 1.42
0.08 ± 0.006
TFP
2.9 ± 0.58
0.16 ± 0.008
0.08 ± 0.04
0.03 ± 0.005
0.075 ± 0.04
3.45 ± 0.75
Monepantel
Moxidectin
The roles of fluorine in medicinal chemistry are well established, in terms of
enhancement of metabolic stability, potency and permeability.20–22 Therefore, in addition
to inclusion of a fluorine substituent in pyridinyl compound 13, a “fluorine walk” was
undertaken for TFP itself, as testified by compounds 14-16. Here, it can be seen the L3
activity was slightly weaker compared with TFP but potent inhibitory activity on L4
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development was maintained, in particular for 15 and 16, for which the IC value of both
50
was 0.04 µM. When the trifluoromethyl group was investigated, both the aromatic ring
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
(17) and aliphatic chain (18) variants caused a complete loss of inhibitory activity.
Heterocyclic N-oxides have been successfully used as therapeutic agents.23–25 For this
reason, we synthesized and tested analogue 19, which harbours a pyridine N-oxide
group. However, a substantial loss of activity against H. contortus was observed in
relation to both xL3 motility and L4 development. Benzoxazole species, such as 20, did
not improve the original potency for either xL3 motility or L4 development, while carboxylic
10
acid 21, a reported metabolite of TFP, was not tolerated. Activity was maintained in both
xL3 and L4 when the p-methyl group was replaced with a p-chloro, as seen for 23, which
exhibited a potent L4 development IC value of 0.08 µM. Compound 22 was synthesized
50
as part of the SAR assessment and also for its potential to serve as a probe for target
identification due to the azide-functional group. Click chemistry in activity-based protein
profiling for target identification has been extensively reported in the literature.26–28
Although the azide-tagged analogue 22 resulted in a dramatic motility reduction in xL3, it
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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
still displayed a binding affinity to inhibit L4 development, which suggests that azide-
tagged TFP might find utility for future target identification studies.
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
From the SAR investigation on the RHS of TFP, we identified that the RHS of
compounds 9 and 10, and the LHS of 12 and 23 were optimal for the compounds tested.
Having successfully identified these groups, the focus then was on incorporating them to
develop a new set of analogues to probe the next generation of SAR, whose results are
summarized in Table 3. For LHS with the pyridinyl moiety, compound 24, with the 4-chloro
pyrazole RHS, displayed similar activity in both xL3 and L4 compared to 12, whereas 28,
with the 4-fluoro pyrazole RHS, caused a moderate loss in activity. Excitingly, compounds
25 and 29 showed a substantial improvement in activity, reducing the IC value for xL3
50
motility and L4 development inhibition to sub µM and sub nM ranges, respectively.
Furthermore, we also extended the SAR scope for the LHS by testing the pyrimidine (26
and 30) and 2-chloro pyridinyl (27 and 31) species. However, 26 and 30 caused a
complete loss in activity, whereas no significant improvement in activity was observed for
27 and 31. The loss in activity caused by 26 and 30 suggested a specific binding
interaction exerted by the LHS region.
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Table 3 Next generation SAR on the LHS and RHS regions of TFP.
O
O
F
Cl
N
N
H
N
H
R
N
R
N
N
O
O
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
24-27
28-31
R
Entr IC (µM) ± IC (µM) ± Entry IC (µM) ± IC (µM) ±
50
50
50
50
y
SD in xL3
motility
assay
SD in L4
SD in xL3
SD in L4
developmen
t assay
motility
assay
development
assay
N
14.47 ±
24
3.70 ± 1.84 0.04 ± 0.03
28
0.19 ± 0.15
7.45
Cl
0
.0007 ±
0.70 ±
0.24
0.0008 ±
0.0001
25
26
27
0.38 ± 0.10
>100
29
30
31
0.0001
N
>100
N
Cl
N
2.80 ± 2.26 0.02 ± 0.01
2.9 ± 0.58 0.03 ± 0.005
14.8 ± 9.6
0.17 ± 0.09
TFP
Monepantel
0.16 ±
.008
0.075 ± 0.04
0
Moxidectin
0.08 ± 0.04 3.45 ± 0.75
From Table 3, the LHS groups that resulted in active compounds were selected
for the development of a similar set of analogues, but with the 3-methyl-4-chloro and 3-
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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
methyl-4-fluoro pyrazole RHS, as a complement to the 4-chloro and 4-fluoro pyrazole
RHS set (results summarized in Table 4). We included a 3-methyl group based on
evidence already discussed for Table 1 that suggested some hydrophobicity at this
position might be favourable. Overall, similar activities against H. contortus xL3 motility
and L4 development were observed within the two sets of analogues. In particular,
compounds 32, 35 and 37 maintained the activity originally observed for TFP.
Compounds 33, 34 and 36 achieved an IC50 value in the sub-nM range for L4
development inhibition.
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 4 Next generation SAR with 3-methyl-4-chloropyrazole and 3-methyl-4-
fluoropyrazole RHS.
O
O
F
Cl
N
N
H
N
H
R
N
R
N
N
O
O
3
2-34
35-37
R
Entr IC (µM) ±
IC (µM) ±
Entry IC (µM) ±
IC (µM) ±
5
0
50
50
50
y
SD in xL3
motility
assay
SD in L4
development
assay
SD in xL3
motility
assay
SD in L4
development
assay
N
32
3.33 ± 1.89
0.01 ± 0.01
35
7.73 ± 4.41 0.05 ± 0.03
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8
9
Cl
Cl
0
.0008 ±
0.004 ±
0.004
3
3
4
2.03 ± 1.82
36
37
2.63 ± 1.60
0
.0001
N
3
1.8 ± 0.49 0.008 ± 0.009
2.56 ± 1.74 0.03 ± 0.02
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
TFP
2.9 ± 0.58
0.16 ±
0.03 ± 0.005
0.075 ± 0.04
Monepantel
Moxidectin
0.008
0.08 ± 0.04
3.45 ± 0.75
These encouraging results for the LHS and RHS of TFP paved the way to explore
SAR on the middle ring by testing the fluoro substituent and the pyridinyl moiety. The
results are summarized in Table 5. At the 2-position, the pyridinyl group in compounds 38
produced a complete loss of activity, whereas the fluoro substituent in 39 maintained the
level of potency observed for TFP against both xL3 motility and L4 development. Similar
results were observed for the 3-position (40 and 41).
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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
Table 5 SAR on the middle region of TFP.
O
Cl
N
2
5
O
R
Cl
N
3
N
1
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
H
R
N
H
N
O
O
6
N
4
38, 40
39, 41
Entry
R
IC (µM) ± SD in xL3
IC (µM) ± SD in L4
5
0
50
motility assay
development assay
N
3
8
9
>100
3
2-F
1.87 ± 1.27
>100
0.08 ± 0.03
N
40
41
3-F
2.73 ± 1.59
2.9 ± 0.58
0.06 ± 0.03
0.03 ± 0.005
0.075 ± 0.04
3.45 ± 0.75
TFP
Monepantel
Moxidectin
0.16 ± 0.008
0.08 ± 0.04
From these results, it was decided to incorporate the fluoro-substituted prototype,
with the two previously identified optimal LHS and the original RHS of TFP being kept
constant. This evaluation resulted in a set of analogues summarized in Table 6. When
fluorine substitution was explored at the 2-position, a 10-fold improvement in the inhibition
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of L4 development was achieved for compound 42 compared to TFP, while 43 displayed
the original L4 development activity. The originally observed levels of activity against xL3
by TFP was maintained for both 42 and 43. There was no significant improvement in the
original activity against both xL3 and L4 when the same LHS groups were experimented
upon with fluorine substitution at the 3-position of the middle ring, as seen for 44 and 45.
Interestingly, a complete loss of activity against H. contortus was observed for compound
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
46 when an additional fluorine was implemented at the 5-position of the middle ring.
These findings indicated a very tight SAR for this region.
Table 6 Next generation SAR on the LHS and middle region of TFP.
O
Cl
2
R'
4
3
N
1
H
R
N
N
O
6
5
4
2-46
Entry
R
R’
IC (µM) ± SD in xL3 IC (µM) ± SD in L4
50
50
motility assay
development assay
N
4
2
3
2-F
2-F
1.8 ± 1.57
0.003 ± 0.004
0.04 ± 0.01
Cl
4
2.07 ± 0.40
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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
4
4
5
3
-F
-F
9.47 ± 4.29
2.67 ± 0.79
>100
0.03 ±0.02
Cl
4
3
0.035 ± 0.005
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
46
3
,5-diF
TFP
2.9 ± 0.58
0.16 ± 0.008
0.08 ± 0.04
0.03 ± 0.005
0.075 ± 0.04
3.45 ± 0.75
Monepantel
Moxidectin
Alterations to the ether bridge and the benzylic carbon of TFP were also assessed.
Different functional groups were used to replace the oxygen from the ether bridge, such
as a methylene group (compound 47), a carbonyl group (48), a sulfur (49) or a methylated
amine (50) (Table 7). However, none of these replacement groups yielded active
compounds. A similar result was achieved when the benzylic carbon was methylated to
produce 51. Interestingly, when incorporating the methylene prototype into one of the four
most active RHS pyrazoles to produce 52-55 (Table 8), activity was regained, but with no
significant improvement with respect to TFP. From these results, the same RHS
pyrazoles and the methylene bridge were then experimented upon with the active
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pyridinyl LHS, with the hope of achieving some improvement in activity. However, the
original potency could not be maintained (56-59, Table 8).
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 7 SAR on various linking regions of the TFP scaffold.
O
O
Cl
Cl
N
N
N
H
H
N
N
N
R
O
47-50
5
1
Entry
R
IC (µM) ± SD in xL3 motility
50
assay
>100
>100
>100
>100
>100
4
7
8
CH2
4
CO
49
S
N-Me
-
50
51
Table 8 Next generation SAR when incorporating the methylene bridge into the TFP
scaffold.
O
O
N
N
R
N
R
H
H
5
2-55
56-59
Entry IC (µM) ±
IC (µM) ± Entry IC (µM) ±
IC (µM) ±
R
5
0
50
50
50
SD in xL3
SD in L4
SD in xL3
SD in L4
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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
motility
assay
developmen
motility
assay
developmen
t assay
t assay
F
>
100
52
53
54
55
3.5 ± 1.5
3.0 ± 0
0.19 ± 0.15
56
57
58
59
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
N
F
0.07 ± 0.06
49.5 ± 0.5
5.2 ± 1.2
7.2 ± 0.2
0.97 ± 0.46
0.23 ± 0.12
0.19 ± 0.08
N
N
Cl
3.15 ± 2.35 0.04 ± 0.01
3.07 ± 2.07 0.04 ± 0.02
N
N
Cl
N
N
TFP
2.9 ± 0.58 0.03 ± 0.005
0.16 ± 0.008 0.075 ± 0.04
0.08 ± 0.04 3.45 ± 0.75
Monepantel
Moxidectin
Having successfully identified compounds with significant improvement in activity
compared to TFP, we selected 9 compounds with high potency in inhibiting L4
development to test for their cytotoxicity on the MCF10A cell line, and the results are
summarized in Table 9. We were delighted to observe high selectivity for the 9
compounds tested, particularly for compounds 27-29 and 31-34. Despite the low cytotoxic
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IC value of 8.02 µM for one of our most potent compounds 25, it was still a great level
50
of selectivity when compared to the activity for L4 development of 0.7 nM. It was also
encouraging to see our other most potent compounds 29 and 33 were not cytotoxic.
These results reinforced the potential of the TFP scaffold to be a novel scaffold with
anthelmintic activity.
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 9 Cytotoxicity data for selected active compounds on the MCF10A cell line.
IC (µM) ± SD in
IC (µM) ± SD in
MCF10A
Cytotoxicity IC50
(µM) ± SEM
50
50
Entry xL3 motility assay
L4 development
assay
TFP
2.57 ± 0.58
4.37 ± 2.55
2.43 ± 1.42
0.38 ± 0.10
2.80 ± 2.26
14.47 ± 7.45
0.70 ± 0.24
14.8 ± 9.6
0.025 ± 0.005
0.01 ± 0.007
0.08 ± 0.006
0.0007 ± 0
37.90 ± 3.11
13.13 ± 0.38
8.8 ± 0.34
10
23
25
27
28
29
8.02 ± 0.48
0.02 ± 0.01
>
50
0.19 ± 0.15
> 50
> 50
> 50
> 50
> 50
0.0008 ± 0.0001
0.17 ± 0.09
31
32
3.33 ± 1.89
2.03 ± 1.82
0.01 ± 0.01
33
0.0008 ± 0.0001
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Journal of Medicinal Chemistry
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2
3
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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
3
4
1.8 ± 0.49
0.008 ± 0.009
> 50
To construct a biological activity profile for TFP and its scaffold, we selected 7
compounds, including TFP as a control, to test for activity on three other parasitic
nematodes at various concentrations (Table 10). The panel included Ancylostoma
ceylanicum (hookworm), Heligosomoides polygyrus (rodent nematode) and T. muris
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
(
whipworm). It can be seen that all seven compounds displayed 100% inhibition and >
7
0% inhibition of L3 of A. ceylanicum at 100 µM and 10 µM, respectively. Similar results
were seen for adult H. polygyrus, where all compounds, except 33, showed complete
inhibition at 100 µM and > 70% inhibition at only 1 µM. All compounds exhibited > 90%
inhibition of first larval (L1) stage of T. muris at 100 µM. There was an obvious
improvement in the inhibition of H. polygyrus L3 for the 6 newly developed compounds
compared with TFP.
Table 10 Biological activity profile of selected compounds against a panel of parasitic
nematodes.
Entry
H. polygyrus
Adult
H. polygyrus
L3
A. ceylanicum L3
T. muris L1
(% inhibition)
(% inhibition)
(% inhibition)
(% inhibition)
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7
8
9
1
0 µM
1 µM
100 µM
100
µM
10 µM
100 µM
TFP
10
100
100
100
100
100
73.4
85.9
100
100
90.6
96.9
100
32.4
91.8
89.2
100
100
100
100
100
100
100
100
94.85
100
100
91.97
100
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
23
27
29
98.8
73.15
98.8
81.6
85.4
100
93.7
66.9
99.2
93.45
100
33
96.5
100
34
100
In relations to the SAR for the previously reported pesticidal activity by Okada et
al.12,29, while compounds 17, 48 and 49 were inactive against H. contortus in this study,
they displayed high potency against Nephotettix cincticeps (for 17) and both Myzus
persicae and Plutella xylostella (for 48 and 49). Likewise, compound 4 with moderate
activity against H. contortus was not potent against Myzus persicae.14,29 These results
indicated a non-parallel SAR, in terms of inhibitory activity observed for H. contortus in
this study and the arthropod species studied by Okada et al.14,29
In order to assess the drug-likeliness of the TFP scaffold, we subjected TFP and
9
other representative analogues (Table 11) to different experiments, which determined
key physicochemical and metabolic parameters. The results showed that all of our key
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Journal of Medicinal Chemistry
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7
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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
compounds had reduced hydrophobicity compared with TFP, including key high potency
compounds such as 34, which with a cLogP of 3.0 was 40 times less lipophilic than TFP
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
(cLogP 4.6). This change, in turn, led to an improvement in aqueous solubility at pH 6.5
for all compounds, with some, such as 28, being around 20-fold more soluble. At pH 2.0,
solubility was also improved for most compounds, except 23, 29 and 33. Concomitant
with decreased lipophilicity and improved solubility, all selected compounds displayed a
longer microsomal half-life than that observed for TFP, ranging from 20 for 10 to 84
minutes for 27.
Table 11 Key physicochemical parameters and in vitro metabolic stability of selected
compounds.
b
CLint, in vitroc
ID
cLogPa
Sol (µg/mL)
T1/2
microsome-
(min)
(µL/min/mg protein)
predicted EHd
pH 2.0 pH 6.5
TFP
4.6
3.3
4.1
2.8
3.1-6.3
25-50
< 1.6
14
20
36
84
121
87
N/A
0.65
0.51
0.31
1
0
3
6.3-2.5
2
1.6-3.1 1.6-3.1
48
27
6.3-
2.5
> 100
6.3-
12.5
21
1
2
8
9
1.7
3.4
2.4
25-50
25
57
80
69
30
22
0.60
0.39
0.32
2
3.1-6.3 1.6-3.1
9-18 9-18
31
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3
3
3
2
3
4
2.3
4.0
3.0
> 100 12.5-25
31
80
55
55
22
31
0.54
0.32
0.40
1.6-3.1 3.1-6.3
4.9-9.8 2.4-4.9
a
b
Calculated using ChemAxon JChem software, kinetic solubility determined by
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
c
Nephelometry (Sol ), in vitro intrinsic clearance determined in mouse liver microsomes,
dpredicted hepatic extraction ratio calculated from in vitro data.
pH
CONCLUSIONS
We have discovered that TFP potently inhibits the development of L4 stage larvae
of the ovine parasitic nematode, H. contortus. Herein, we report a systematic SAR
interrogation that has led to the identification of novel modifications that not only improve
drug-like physicochemical properties, such as lipophilicity, aqueous solubility and
microsomal degradation half-life, but that are exquisitely potent. For example, 25, 29 and
3
3 achieved a remarkable improvement in inhibitory activity against both xL3 motility and
L4 development, reducing the original and already impressive IC values of TFP down
50
to the sub-µM or sub-nM ranges, at the same time, maintaining selectivity towards the
parasite. TFP, as a pesticide, was reported to be a complex I inhibitor that disrupts the
respiratory electron transport chain in mitochondria.11,30 However, the specific biological
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1
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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
target(s) of this series of compounds in nematodes is/are unknown. With a moderately
potent inhibitory affinity observed against L4 development, compound 22 might represent
a potential tool for target identification. Having established a comprehensive antiparasitic
SAR herein, we are now embarking on pathway identification and further downstream
efficacy assessment and hope to report on these efforts in due course.
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
EXPERIMENTAL SECTION
The nematode assays and cytotoxicity assay is as described by Le et al.19
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
JChem for Excel software (version 16.4.11). A brief description of each parameter is
provided: cLogP: Partition coefficients, reflecting the lipophilic character of the neutral
structure.
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Kinetic Solubility Estimation using Nephelometry (SolpH)
Compound in DMSO was spiked into either pH 6.5 phosphate buffer or 0.01M HCl
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
(approximately pH 2.0) with the final DMSO concentration being 1%. After 30 minutes
had elapsed, samples were then analyzed via Nephelometry to determine a solubility
range. See Bevan et al.31
In vitro Metabolic Stability
The procedure is as described by Le et al.19
CHEMISTRY EXPERIMENTAL
All solvents and reagents were used directly from commercial suppliers unless
otherwise stated. All of the final compounds had purities greater than 95% based on
1
analytical HPLC, H NMR and LC-MS. General chemistry experimental conditions were
as reported by Le et al.19
General procedure A1: Nucleophilic aromatic substitution reactions
To a stirred solution of arylfluoride (1.0 eq), and nucleophile (1.1 eq) in DMF (20
mL), was added Cs CO (2.0 eq) and the mixture was stirred at 100 °C for 2 h. Upon
2
3
completion, reaction mixture was diluted with EtOAc (50 mL) and organic layer was
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