Development of Pyrazolopyrimidine Anti- Wolbachia Agents for the Treatment of Filariasis

Article abstract of DOI:10.1021/acsmedchemlett.1c00216

Anti-Wolbachia therapy has been identified as a viable treatment for combating filarial diseases. Phenotypic screening revealed a series of pyrazolopyrimidine hits with potent anti-Wolbachia activity. This paper focuses on the exploration of the SAR for this chemotype, with improvement of metabolic stability and solubility profiles using medicinal chemistry approaches. Organic synthesis has enabled functionalization of the pyrazolopyrimidine core at multiple positions, generating a library of compounds of which many analogues possess nanomolar activity against Wolbachia in vitro with improved DMPK parameters. A lead compound, 15f, was selected for in vivo pharmacokinetics (PK) profiling in mice. The combination of potent anti-Wolbachia activity in two in vitro assessments plus the exceptional oral PK profiles in mice puts this lead compound in a strong position for in vivo proof-of-concept pharmacodynamics studies and demonstrates the strong potential for further optimization and development of this series for treatment of filariasis in the future.

Full text of DOI:10.1021/acsmedchemlett.1c00216

pubs.acs.org/acsmedchemlett
Letter
Development of Pyrazolopyrimidine Anti-Wolbachia Agents for the
Treatment of Filariasis
Paul McGillan, Neil G. Berry, Gemma L. Nixon, Suet C. Leung, Peter J. H. Webborn, Mark C. Wenlock,
Stefan Kavanagh, Andrew Cassidy, Rachel H. Clare, Darren A. Cook, Kelly L. Johnston, Louise Ford,
Stephen A. Ward, Mark J. Taylor, W. David Hong,* and Paul M. O’Neill*
Cite This: https://doi.org/10.1021/acsmedchemlett.1c00216
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sı Supporting Information
ABSTRACT: Anti-Wolbachia therapy has been identified as a
viable treatment for combating filarial diseases. Phenotypic
screening revealed a series of pyrazolopyrimidine hits with potent
anti-Wolbachia activity. This paper focuses on the exploration of
the SAR for this chemotype, with improvement of metabolic
stability and solubility profiles using medicinal chemistry
approaches. Organic synthesis has enabled functionalization of
the pyrazolopyrimidine core at multiple positions, generating a
library of compounds of which many analogues possess nanomolar
activity against Wolbachia in vitro with improved DMPK
parameters. A lead compound, 15f, was selected for in vivo
pharmacokinetics (PK) profiling in mice. The combination of
potent anti-Wolbachia activity in two in vitro assessments plus the exceptional oral PK profiles in mice puts this lead compound in a
strong position for in vivo proof-of-concept pharmacodynamics studies and demonstrates the strong potential for further
optimization and development of this series for treatment of filariasis in the future.
KEYWORDS: Wolbachia, Pyrazolopyrimidine, Onchocerciasis, Filariasis
ilarial nematodes are the causative pathogens of the
neglected tropical diseases lymphatic filariasis (LF) and
is driving current research efforts to identify and generate safe
therapeutic alternatives.
5
,6
F
onchocerciasis that affect tens of millions people throughout
the tropics and contribute to serious public health and socio-
economic problems. Onchocerciasis (the cause of river
blindness) is the second leading infectious cause of blindness.
These diseases combined are one of the leading causes of
morbidity worldwide. The main causative agents for these
conditions are the nematodes Onchocerca volvulus (onchocer-
The nematodes which are responsible for causing these two
filarial diseases share an essential endosymbiotic relationship
7
,8
with the bacterium Wolbachia. Although the exact nature of
this relationship is not yet fully understood, anti-Wolbachia
therapy has been proven clinically by an existing antibiotic,
doxycycline, which delivers safe macrofilaricidal activity with
superior therapeutic outcomes compared to current antifilarial
9
−11
ciasis), Wuchereria bancrofti, Brugia malayi, and Brugia timori
drugs.
1
Pyrazolopyrimidine compounds have frequently appeared in
(
LF). The latest recommended treatment for LF in areas
the literature with a variety of different pharmacological
which are not coendemic for onchocerciasis or other filarial
disease, loiasis, is a triple combination of ivermectin,
diethylcarbamazine plus albendazole. Ivermectin is the
1
2,13
14
17
activities such as kinase inhibitors,
antituberculosis,
15
16
2
antimalarial and antiviral agents, and antidepressants.
The original hit for this chemotype (1) which resulted from a
phenotypic high-throughput screen (HTS) of a divergent
chemical library donated by the Medicines for Malaria Venture
recommended treatment for onchocerciasis; however, it cannot
be used in areas co-endemic for loiasis due to potentially fatal
adverse effects. These direct acting antifilarial drugs primarily
target microfilariae, the immature worm stage, and thus can
prevent transmission, but they have little macrofilaricidal
activity against the adult worms. Hence, these drugs require
lengthy treatments that can be as long as 15 years. The
association of current direct acting antifilarial agents with
undesired adverse effects, contraindicated patient groups
combined with a growing concern of resistance development,
Received: April 15, 2021
Accepted: August 16, 2021
3
,4
©
XXXX The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acsmedchemlett.1c00216
A
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18
(
(
MMV) is displayed in Figure 1 with measured in vitro activity
EC ) against Wolbachia (wAlbB) infected insect cells (Aedes
DMPK screening. Compounds showing a good balance of
potency and metabolic stability were then selected to test their
anti-Wolbachia activity against Brugia malayi microfilariae (mf)
in vitro and for in vivo PK profiling in mice. This secondary in
vitro assay was developed to provide a link between the in vitro
insect cell-based assay and the in vivo B. malayi SCID mouse
model. Although the throughput of the mf assay is limited due
to the availability of the B. malayi mf, it enables the assessment
of the anti-Wolbachia activity in one of the targeted human
parasites, providing important evidence of translation between
different species of Wolbachia and hosts. Finally, front runners
identified from the two above tests would be selected for the in
vivo PD study where SCID mice are infected with the human
5
0
19
parasite B. malayi.
Despite the high potency (EC = 21 nM) of the original hit
5
0
1
, DMPK assessments highlighted poor metabolic stability and
low aqueous solubility. (Figure 1) The phenyl ring, allyl
substitution, and methylene linker were some of the positions
within the hit molecule that were predicted to be susceptible to
oxidative CYP metabolism. Hence, the early hit to lead
optimization was focused on enhancing the metabolic stability
while maintaining potency.
Figure 1. Structure of the HTS hit 1 and the pyrazolopyrimidine
scaffold with the areas for SAR studies highlighted.
albopictus, C6/36) and drug metabolism/pharmacokinetic
(
DMPK) properties. According to the HTS data of other
The initial synthetic route (Scheme 1) was developed to
allow for the synthesis of analogues with modifications at the
close pyrazolopyrimidine analogues screened in the same
campaign (data not shown), some preliminary indication of
structure−activity relationship (SAR) was observed and the
areas of focus for SAR studies and optimization are highlighted
in Figure 1. The aim of the work described here was to develop
pyrazolopyrimidine leads with potent anti-Wolbachia activity
and desired DMPK properties that could be further developed
as oral drugs for the treatment of filariasis.
Scheme 1. Synthetic Route for Analogues in the
Pyrazolopyrimidine Template
The cascade depicted in Figure 2 was followed for the
optimization of the compounds described in this work. In vitro
anti-Wolbachia potency was assessed in parallel with in vitro
2
3
R -, R -, and 7-positions of the pyrazolopyrimidine ring. While
1
many necessary 5-amino pyrazoles (1a, 5d, R = Ph, H
respectively) are commercially available, it was also possible to
synthesize them in a two-step reaction from the corresponding
1
nitrile (4a, R = 4-F-Ph) using base (NaOEt or LDA) and
ethyl formate followed by hydrazine mediated cyclization. This
was followed by an acid-catalyzed pyrimidine ring formation
2
0
using the pyrazoles 1a, 5a,d and β-keto esters 1b, 6a−f.
When the 5-position of the pyrazolopyrimidine is unsub-
stituted (R = H), acid catalyzed cyclization is not suitable for
2
the initial imine formation step. For these analogues, the
pyrimidine ring was formed by reaction of the appropriate
pyrazole and aldehyde to give the imine intermediate which
t
cyclizes upon addition of KO Bu. Chlorination of the 7-
hydroxy pyrimidine intermediates 1c, 7a−f was achieved using
phosphoryl chloride and subsequent substitution of inter-
mediates 1d, 8a−f was achieved by aromatic substitution with
the appropriate amines, 9a,b, to produce the pyrazolopyr-
imidines, 1, 3, and 10a−d.
Figure 2. Screening cascade for the design, synthesis and testing of
anti-Wolbachia compounds.
B
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While it is possible to use the appropriate nitriles as starting
Table 1. In Vitro Potency Data of Key Analogues
material for the synthesis of target compounds with different
1
groups at the R -position, a divergent orientated synthesis at
this position was desirable for the production of a number of
target analogues from a common intermediate. (Scheme 2)
Scheme 2. Optimized Route for the Synthesis of Analogues
a
with Modified 3-Positions
a
Note: THP = Tetrahydropyran.
1
Functionalization at the R -position can also be achieved via
1
iodination of the pyrazolopyrimidines 8d−f (R = H) using N-
2
iodosuccinimide (NIS). This pathway requires Boc-protection
of the free amines 12 to allow for Suzuki-mediated coupling of
intermediates 13 with the corresponding boronic acid. Finally
TFA-mediated Boc-deprotection of compounds 14a−k
afforded the desired targets 15a−k, which contained a variety
of monosubstituted phenyl rings and nitrogen heterocycles.
a
Note: All tested compounds showed no cytotoxicity against the
insect cells at the top concentration (5 μM) in the assay.
Table 2. In Vitro DMPK Data of Pyrazolopyrimidine
Analogues
a
3
Substitutions at the R -Position. Replacement of the
allyl group at this position in the original hit 1 with a methyl
substituent resulted in excellent potency (compound 2, Table
H.
Aq. Sol.
H. Mics. CL.
R. Hep. CL
PPB
(%)
6
LogD7.4
4.2
4.0
4.3
4.3
3.3
3.7
2.0
4.0
4.1
4.5
2.3
3.0
3.3
2.7
2.3
4.3
2.4
4.0
(μM)
(μL/min/mg) (μL/min/10 cells)
1
, EC50 = 19 nM) and improved metabolic stability as shown
by rat hepatocyte clearance of compound 2 (Table 2). The
analogue with ring-fusion of a lipophilic cyclopentyl ring at R
and R (10b) showed good anti-Wolbachia activity (EC = 79
1
2
3
0.07
0.40
0.90
0.30
0.50
0.01
561
0.60
1.00
5.00
11.0
2.00
2.00
43.0
30.0
2.0
63.22
ND
49.19
24.49
48.86
8.76
261.80
83.97
59.33
8.76
74.15
12.08
21.78
44.83
243.60
44.62
23.65
37.48
105.60
59.03
67.66
75.43
41.25
99.9
99.80
99.8
ND
2
3
5
0
nM) and demonstrated that modifications at both positions
could be tolerated. This modification did not improve aqueous
solubility, but the cyclopentyl ring connecting these two
positions increased metabolic stability. Chlorine was also
10a
10b
10c
10d
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
99.0
99.7
77.0
99.9
99.9
97.9
88.0
96.6
99.8
91.8
97.4
99.5
ND
46.48
109.60
113.20
86.16
156.60
>300.00
147.80
27.58
10.31
144.90
26.37
3
tolerated at the R -position in terms of potency and has a
positive effect on metabolic stability in comparison to the
corresponding 6-methyl analogues (15i vs 3). On the other
3
hand, SO Me group at the R -position (10c) reduced anti-
2
Wolbachia activity noticeably.
4
Substitutions at the R -Position. In an effort to reduce
the potential metabolism of the methylene linker, an analogue
with a methyl substitution at this position (10a) was
investigated. Methylation of the linker imparts a 2-fold increase
in human microsomal stability, but this modification leads to a
21.0
6.0
71.84
45.53
4
-fold loss in activity (compounds 10a vs 3). According to
99.6
a
HTS results (data not shown here), we found that the SAR
was restricted at the 2′-pyridyl side-chain. Any minor
modifications to this ring significantly reduced compound
Notes: Aq. Sol. = aqueous solubility in pH7.4 PBS; H. Mics. CL =
intrinsic human microsomal clearance measured in vitro; R. Hep. CL
= intrinsic Rat hepatocyte clearance measured in vitro; ND = not
determined.
1
potency. For this reason, we focused more on the R -position
of the pyrazolopyrimidine core since the HTS data suggested
that a wider range of modifications might be tolerated at this
position.
1
1
Substitutions at the R -Position. The R -aryl side chain
is not essential for anti-Wolbachia activity; however, removal of
C
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the aromatic ring resulted in some reduction in potency (10d
EC = 105 nM) coupled with a significant increase in aqueous
Table 3. In Vitro Potency of Key Analogues in the B. malayi
mf Assay
50
solubility. A range of small substitutions at the para-position of
the phenyl ring are generally tolerated, while a 4-fluoro
substitution (3) appears to be optimal for activity (EC50 = 17
anti-Wolbachia
anti-Wolbachia potency from mf assay (6
potency from cell
days at 5 μM % Wolbachia reduction in
a
molecule
assay EC (nM)
wsp:gst )
50
nM). Substitution of the polar SO Me group at the para-
position (15f) resulted in reducing LogD, increasing aqueous
solubility and improving metabolic stability. However, similar
2
DOX
1
3
17
21
17
105
143
51
86.5
75.60
83.20
77.10
80.40
toxic
85
substitution with the SO Me group at the ortho- and meta-
2
10d
positions of the phenyl ring is not tolerated, as shown by
compounds 15d and 15e, where substitution results in a
significant reduction in potency. On the other hand, smaller
substituents, such as fluorine are tolerated in the ortho-position
although these small substitutions offered no significant
improvements to DMPK properties. While compound 3
displayed improved in vitro metabolic stability comparing
with HTS hit 1, it still suffered from poor aqueous solubility.
In an attempt to reduce LogD further and to improve
aqueous solubility, analogues 15g and 15h containing the
heterocyclic aromatic 1-methyl-1H-pyrazolyl and 2-pyridyl
groups were synthesized as more polar/LogD reducing phenyl
ring replacements. These modifications both greatly enhanced
the aqueous solubility and lowered LogD with the 2-pyridyl
analogue 15h maintaining the majority of its potency.
1
1
5f
5i
b
15j
52
a
wsp, Wolbachia surface protein copy number, median % reduction cf.
vehicle (DMSO) control; gst, GST copy number (single copy gene,
worm size biomarker). Significantly reduced motility of the mf after
6 days incubation comparing with vehicle (DMSO) control.
b
vivo PK evaluation. Compound 15f was dosed orally to SCID
mice at 50 and 100 mg/kg using a standard suspended vehicle
(SSV); results from the study are shown in Chart 1 and Table
Chart 1. Mean Plasma Concentration of Compound 15f
Following Dosing to SCID Mice with SSV
Compound 15g proved to be reasonably stable metabol-
ically; however, a near 10-fold drop in activity supported
further investigation into other areas of the scaffold to improve
overall properties. Exploration of saturated heterocyclic ring
systems led to the synthesis of compound 15j containing the
tetrahydropyran (THP) moiety. The THP side-chain is well
tolerated for potency (EC = 52 nM) while improving DMPK
50
properties comparing with HTS hit 1.
2
Substitutions at the R -Position. Substitution at this
position is not essential for anti-Wolbachia activity. It was
demonstrated that the removal of the methyl substituent from
2
the R -position (15k) was tolerated in terms of potency and
resulted in some improvement in DMPK properties when
2
compared to compound 3. This result suggests the R -position
was another area that could be further explored for
optimization of potency and DMPK in future work.
Table 4. In Vivo PK Profile of Compound 15f, Dosing to
SCID Mice Using SSV
Anti-Wolbachia Activity Assessment in B. malayi mf
Assay. The mf assay is an orthogonal in vitro assay that uses B.
malayi microfilariae to confirm the anti-Wolbachia activity of
dosage (oral)
T1/2 (h)
50 mg/kg
2.9
100 mg/kg
2.4
21,22
tested compounds against the human parasitic nematode.
Cmax (μg/L)
Tmax (h)
13 067
1.33
27 800
1.67
After being screened for potency and DMPK properties in
vitro, a number of selected analogues were tested at 5 μM
alongside the gold-standard doxycycline for comparison of
anti-Wolbachia activity in the mf assay. The majority of tested
compounds demonstrated good activity, comparable to
doxycycline, in this assay except for 15i (Table 3). The
secondary readout of the mf assay is the motility of the mf by
the tested compounds comparing with vehicle control. For
anti-Wolbachia drugs, such as doxycycline, they should not
affect the motility of the parasitic worm since this indicates off-
target effects. For this reason, the chloro-substituted analogue
AUC0−t (μg·h/L)
82 209
162 617
4. Despite limited aqueous solubility, this compound
demonstrates good tolerability, excellent in vivo PK profiles
with high exposure, reasonable half-life and dose-proportional
AUC. Based on this data 15f has been selected as a lead for an
in vivo proof-of-concept pharmacodynamics study and further
optimization.
In summary, the initial potent pyrazolopyrimidine hit 1,
which has poor metabolic stability and inadequate aqueous
solubility parameters, has been optimized to provide a number
of highly potent compounds with enhanced DMPK properties
as represented by lead molecules 15f and 15j. A summary of
the key SAR is highlighted in Figure 3.
1
5i was considered unsuitable for further development as an
anti-Wolbachia drug as it had significant effects on worm
motility in the assay.
In Vivo Pharmacokinetic (PK) Profiling in Mice. Taking
all the in vitro results into consideration, compounds 15f and
1
1
5j possessed a suitable balance of high potency, good DMPK
More explorations of the R -position and potential
properties and acceptable preliminary safety profiles (e.g.,
cytotoxicity and hERG inhibition ), and 15f was chosen for in
functionalization of the 2-position of the pyrazolopyrimidine
core could further improve the anti-Wolbachia potency and the
23
D
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Limited, No. 24 Mereside, Alderley Park, Macclesfield,
Cheshire SK10 4TG, United Kingdom
Stefan Kavanagh − Oncology Safety Sciences, Clinical
Pharmacology & Safety Sciences, R&D, AstraZeneca,
Cambridge CB2 0AA, United Kingdom
Andrew Cassidy − Department of Tropical Disease Biology,
Liverpool School of Tropical Medicine, Liverpool L3 5QA,
United Kingdom
Rachel H. Clare − Department of Tropical Disease Biology,
Liverpool School of Tropical Medicine, Liverpool L3 5QA,
United Kingdom; orcid.org/0000-0002-3945-0530
Darren A. Cook − Department of Tropical Disease Biology,
Liverpool School of Tropical Medicine, Liverpool L3 5QA,
United Kingdom
Kelly L. Johnston − Department of Tropical Disease Biology,
Liverpool School of Tropical Medicine, Liverpool L3 5QA,
United Kingdom; Institute of Systems, Molecular &
Integrative Biology, School of Life Sciences, University of
Liverpool, Liverpool L69 7ZB, United Kingdom
Louise Ford − Department of Tropical Disease Biology,
Liverpool School of Tropical Medicine, Liverpool L3 5QA,
United Kingdom
Figure 3. Summary of the SAR of anti-Wolbachia pyrazolopyrimi-
dines.
overall DMPK properties. These future directions will be
determined by the results of in vivo efficacy studies of 15f
which will be reported in due course. In addition, the in vivo
PK and PD studies of the other lead, 15j will be triggered if the
proof-of-concept in vivo efficacy study of 15f is positive.
Stephen A. Ward − Department of Tropical Disease Biology,
Liverpool School of Tropical Medicine, Liverpool L3 5QA,
United Kingdom
Mark J. Taylor − Department of Tropical Disease Biology,
Liverpool School of Tropical Medicine, Liverpool L3 5QA,
United Kingdom
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00216.
■
*
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00216
In vitro biological testing methods, experimental
procedure of the synthesis, and characterization of the
reported compounds (PDF)
Author Contributions
The manuscript was written through contributions of all
authors. W.D.H., M.J.T., S.A.W., and P.M.O. designed
research; P.McG. performed synthesis; P.J.H.W., M.C.W.,
and S.K. performed in silico and in vitro DMPK and safety
studies; A.C., R.H.C., D.A.C., and K.L.J. performed para-
sitology studies; W.D.H., N.G.B. G.L.N., L.F., K.L.J., and
P.M.O. analyzed data.
AUTHOR INFORMATION
Corresponding Authors
■
Paul M. O’Neill − Department of Chemistry, University of
Liverpool, Liverpool L69 7ZD, United Kingdom;
orcid.org/0000-0003-4338-0317; Email: pmoneill@
liverpool.ac.uk
W. David Hong − Department of Chemistry, University of
Liverpool, Liverpool L69 7ZD, United Kingdom; Department
of Tropical Disease Biology, Liverpool School of Tropical
Medicine, Liverpool L3 5QA, United Kingdom; orcid.org/
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
0
000-0002-0030-3007; Email: davidhwq@liverpool.ac.uk
The A·WOL consortium is supported by grants from the Bill &
Melinda Gates Foundation awarded to the Liverpool School of
Tropical Medicine. This work was also supported by a GHIT
grant awarded to M.J.T., S.A.W., and P.O.N. Experiments,
including synthesis and animal PK studies, performed at Wuxi
AppTec, led by Nigel Liverton and Chengkun Huang, were
supported by the Bill & Melinda Gates Foundation financially.
We would also like to thank Mark Timms in AstraZeneca
Compound Management in Alderley Park for the logistics of
arranging the in vitro DMPK and safety studies.
Authors
Paul McGillan − Department of Chemistry, University of
Liverpool, Liverpool L69 7ZD, United Kingdom
Neil G. Berry − Department of Chemistry, University of
Liverpool, Liverpool L69 7ZD, United Kingdom
Gemma L. Nixon − Department of Chemistry, University of
Liverpool, Liverpool L69 7ZD, United Kingdom;
orcid.org/0000-0002-9730-0960
Suet C. Leung − Department of Chemistry, University of
Liverpool, Liverpool L69 7ZD, United Kingdom
Peter J. H. Webborn − Drug Safety & Metabolism, IMED
Biotech Unit, AstraZeneca U.K., Cambridge CB2 0AA,
United Kingdom; Present Address: PJHW Consultancy
Ltd., Macclesfield, Cheshire SK11 9HD, United Kingdom
Mark C. Wenlock − Drug Safety & Metabolism, IMED
Biotech Unit, AstraZeneca U.K., Cambridge CB2 0AA,
United Kingdom; Present Address: Cyprotex Discovery
ABBREVIATIONS
■
LF, lymphatic filariasis; DEC, diethylcarbamazine; HTS, high-
throughput screening; DMPK, drug metabolism pharmacoki-
netics; DOX, doxycycline; PD, Pharmacodynamics; PK,
pharmacokinetics; SAR, structure−activity relationships; B.ma-
layi, Brugia malayi; mf, microfilariae; SCID, severe combined
immunodeficient
E
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antimalarial, and Pf-dihydroorotate dehydrogenase inhibitors. Eur. J.
Med. Chem. 2017, 126, 72−83.
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