A novel pyrazolopyridine with in vivo activity in Plasmodium berghei- and Plasmodium falciparum-infected mouse models from structure-activity relationship studies around the core of recently identified antimalarial imidazopyridazines

Article abstract of DOI:10.1021/acs.jmedchem.5b01605

Toward improving pharmacokinetics, in vivo efficacy, and selectivity over hERG, structure-activity relationship studies around the central core of antimalarial imidazopyridazines were conducted. This study led to the identification of potent pyrazolopyridines, which showed good in vivo efficacy and pharmacokinetics profiles. The lead compounds also proved to be very potent in the parasite liver and gametocyte stages, which makes them of high interest.

Full text of DOI:10.1021/acs.jmedchem.5b01605

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Article
A novel pyrazolopyridine with in vivo activity in Plasmodium
berghei and Plasmodium falciparum-infected mouse models
from structure-activity relationship studies around the
core of recently identified antimalarial imidazopyridazines.
Claire Le Manach, Tanya Paquet, Christel Brunschwig, Mathew Njoroge, Ze Han, Diego Gonzalez
Cabrera, Sridevi Bashyam, Rajkumar Dhinakaran, Dale Taylor, Janette Reader, Mariette Botha,
Alisje Churchyard, Sonja Lauterbach, Theresa Coetzer, Lyn-Marie Birkholtz, Stephan Meister,
Elizabeth A Winzeler, David Waterson, Michael J. Witty, Sergio Wittlin, Maria-Belen Jimenez-Diaz,
Maria Santos Martinez, Santiago Ferrer, Inigo Angulo-Barturen, Leslie J. Street, and Kelly Chibale
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01605 • Publication Date (Web): 26 Oct 2015
Downloaded from http://pubs.acs.org on October 31, 2015
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A novel pyrazolopyridine with in vivo activity in
Plasmodium berghei and Plasmodium
falciparumꢀinfected mouse models from
structureꢀactivity relationship studies around the
core of recently identified antimalarial
imidazopyridazines
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Claire Le Manach†, Tanya Paquet†, Christel Brunschwig∞, Mathew Njoroge∞, Ze Han†,
Diego Gonzàlez Cabrera†, Sridevi Bashyamζ,, Rajkumar Dhinakaranζ, Dale Taylor∞,
Janette Reader¶, Mariette Botha¶, Alisje Churchyard¶, Sonja Lauterbach¶, Theresa L
Coetzer§, LynꢀMarie Birkholtz¶, Stephan Meister*, Elizabeth A. Winzeler*, David
Waterson‡, Michael J. Witty‡, Sergio Wittlin◊,∫, MaríaꢀBelén JiménezꢀDíaz¥, María Santos
Martínez¥, Santiago Ferrer¥, Iñigo AnguloꢀBarturen¥, Leslie J. Street† and Kelly
Chibale*,† ,#
†DrugDiscovery and Development Center (H3ꢀD), University of Cape Town,
ζ
Rondebosch 7701, South Africa; Syngene International Ltd., Biocon Park, Plot No. 2 &
∞
3, Bommasandra IV Phase, Jigani Link Road, Bangalore 560099, India; Division of
Clinical Pharmacology, Department of Medicine, University of Cape Town, Observatory,
¶
7925, South Africa; Department of Biochemistry, Centre for Sustainable Malaria
Control, University of Pretoria, Private bag x20, Hatfield, 0028, South Africa;
§Department of Molecular Medicine and Haematology. Wits Research Institute for
Malaria, Wits Medical School, Johannesburg, South Africa; *University of California,
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San Diego (UCSD), School of Medicine, Dept. Pediatrics, Pharmacology & Drug
‡
Discovery, 9500 Gilman Drive, La Jolla, CA 92093, USA; Medicines for Malaria
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Venture, ICC, Route de PréꢀBois 20, PO Box 1826, 1215 Geneva, Switzerland; ◊Swiss
Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland;
∫University of Basel, 4003 Basel, Switzerland; ¥GlaxoSmithKline, Tres Cantos Medicines
Development Campus, Severo Ochoa, 2, 28760 Tres Cantos, Madrid, Spain; #Institute of
Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch
7701, South Africa.
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Supporting information
ABSTRACT: Towards improving pharmacokinetics, in vivo efficacy and selectivity over
hERG, structureꢀactivity relationship studies around the central core of antimalarial
imidazopyridazines were conducted. This study led to the identification of potent
pyrazolopyridines, which showed good in vivo efficacy and pharmacokinetics profiles.
The lead compounds also proved to be very potent in the parasite liver and gametocyte
stages, which make them of high interest.
KEYWORDS: antiplasmodial activity, imidazopyridazines, pyrazolopyridines, in vivo
efficacy, SCID mouse
Despite a notable decrease in the burden of disease between 2000 and 2012, malaria
remains a major global health issue, which still accounted for about 584 000 deaths in
20131. Better prevention and diagnosis, along with the use of artemisininꢀbased
combination therapies (ACTs) have contributed to reduced malariaꢀrelated deaths around
the world in the last 12 years by more than 40%. However, signs of resistance to
artemisinin have recently emerged in SouthꢀEast Asia2–4, highlighting that the global
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efforts against malaria should be maintained, if not strengthened. In this context,
identifying new drugs with new mechanisms of action and activity against resistant
strains of the parasite remains an urgent need.
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We recently reported the activity of imidazopyridazine derivatives against Plasmodium
falciparum5,6, of which 1 and 2 (figure 1) showed promising properties both in vitro and
in vivo. Related antimalarial imidazopyrazines have recently been identified7. The
solubility, in vivo efficacy, and exposure of the previously reported imidazopyridazine 15
were improved with 26, which was the first compound of the imidazopyridazine series to
cure mice from Plasmodium berghei infection at 4 x 50 mg/kg/day. However, better in
vivo efficacy at lower doses was yet to be achieved and some offꢀtarget activities, such as
hERG, remained an issue. In order to extend the previous structureꢀactivity relationship
(SAR) studies and to improve pharmacokinetics (PK), in vivo efficacy and selectivity
over hERG, we focused on changes to the imidazopyridazine heterocycle. Our strategy
was recently reinforced by other studies, in which imidazopyridines and
imidazolopiperazines8,9 that showed good PK properties and in vivo efficacy were
identified. Herein we describe how SAR investigations around the central core of our
imidazopyridazines combined with a sulfoxide/sulfone proꢀdrugꢀlike strategy, we have
previously adopted, led to compound 31, which displays good physicoꢀchemical
properties, in vivo efficacy, in both P. berghei and P. falciparum SCID mouse models
and good oral pharmacokinetics. In addition to these studies, lead compounds were also
profiled for activity against liver and gametocyte life cycle stages in order to get a
broader view of the potential of this series of compounds to contribute the malaria
eradication agenda.
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Chemistry: Compounds 1, 2, 3 and 4 were prepared as previously described5,6.
Triazolopyridazine 8, imidazopyrimidine 9, imidazopyrazine 10, imidazopyridines 11
and 12, pyrazolopyridines 17, 18 and 19, and pyrazolopyrimidines 20, 21 and 22 were
synthesized in 3 (4 for 20) steps from the corresponding 6ꢀhalogenated heterocycles
(Scheme 1). The scaffolds were either commercially available (5a, 13a, 13b) or prepared
inꢀhouse (5bꢀd). The preparation of those that were not commercially available is
described in the Supporting Information (SI). Briefly, from the halogenated 5,6ꢀbicyclic
heterocycle, a Suzuki crossꢀcoupling reaction was first performed with the appropriate
boronic acid10. The resulting product was then reacted with NBS in DMF at room
temperature to give the bromo derivative in high yield. Finally, a second Suzuki crossꢀ
coupling reaction with 4ꢀ(methylsulfonyl)phenyl boronic acid was performed to give the
desired compounds. Compound 20 was prepared from 4ꢀ methylsulfoxide intermediate
20a, which was then oxidized with mCPBA. Compound 20 and 22 were reduced using
NaBH4 and a catalytic amount of Pd(PPh3)Cl2 to give their saturated analogues 23 and 24
respectively.
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Compounds 29 and 30 were prepared following the synthetic route depicted in scheme
2. The 2ꢀchloroꢀ5ꢀiodopyridine 25 was reacted with hydrazine to give the 2ꢀhydrazinylꢀ5ꢀ
iodopyridine 26, which was then reacted with 4ꢀ(methylsulfonyl)benzaldehyde to give
intermediate 27. Iodo compound 28 was formed after a ring closure with
(diacetoxy)iodobenzene11. Finally, a Suzuki crossꢀcoupling reaction was carried out with
3ꢀtrifluoromethylphenyl boronic acid, 3ꢀ(methylsulfonyl)phenyl boronic acid, and 3ꢀ
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(methylsulfinyl)phenylboronic acid, respectively, to give the desired products 29, 30 and
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In vitro Antiplasmodial activity: All compounds were evaluated for in vitro
antiplasmodial activity against a sensitive strain (NF54), and selected compounds (12 out
of 19) were tested against a multidrug resistant (K1) strain of P. falciparum. Chloroquine,
artesunate and 1 were used as the reference drugs in all experiments. The in vitro
antiplasmodial activities of these compounds, as indicated by their IC50 values, are
summarized in table 1.
In general, all analogues were equipotent against both strains (K1 and NF54).
Representative compounds of each series have been tested for cytotoxicity, including 1,
3, 12, 18, 19, 22, 24, and 31 (Table D1 in the SI). Except for compound 12 (Selectivity
Index = 700), all compounds tested had a selectivity index of >1000. Therefore,
cytotoxicity was not considered an issue for this series.
Interestingly, the previously observed SAR trends in the imidazopyridazine series were
conserved across the different scaffolds. Cyclopropylsulfone derivatives tend to be more
potent than their methylsulfone analogues, which are in turn more potent than the
trifluoromethyl derivatives. Moving from the imidazopyridazine to the
pyrazolopyrimidine scaffold improved the activity about two fold (IC50 K1/NF54 (nM),
3: 0.53/0.88, 22: 0.37/0.42). A decrease in potency was observed when the nitrogen at the
5ꢀposition was removed, yet the compounds retained good activity with IC50 values in the
50ꢀ150 nM range for the imidazopyridines (IC50 K1/NF54 (nM), 11: 113/150; 12: 38/46),
and IC50 <10 nM for the pyrazolopyridines (IC50 NF54 (nM), 18: 8, 19: 2). As before,
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moving the nitrogen from position 4 to position 9 resulted in an increase in activity, as
the comparison between the direct analogues 12 and 19 shows (IC50 NF54 (nM): 46 and 2
respectively). Compound 31, the sulfoxide analog of 19, also showed potent activity (IC50
NF54 = 6nM). Tetrahydroꢀpyrazolopyrimidines 23 and 24 were also tolerated with IC50
<100nM.
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On the other hand, adding a nitrogen at the 2 position appeared to be detrimental for
activity as 8, 29, and 30 all displayed IC50 >1000 nM. Equally, the introduction of a
nitrogen at the 7 (compound 10) or 8 (compound 9) position proved to be ineffective
(IC50 NF54 >800 nM)
In vitro ADME parameters: Metabolic stability of the most active compounds was
assessed in vitro in human, rat and mouse liver microsomal preparations. All active
compounds were submitted for a single readout assay that determines the percentage
compound remaining after a 30ꢀminute incubation in the presence of liver microsomes.
The microsomeꢀpredicted hepatic extraction ratios (EH) were determined for selected
compounds using a 5ꢀpoint assay (60 minutes). All the results are summarized in the
Supporting Information (Table E1). All compounds proved to be stable across the 3
species (human, rat, and mouse).
The solubility of most compounds remained poor. Compounds 10 and 24 had an
improved solubility at pH 2, but generally none of these core changes improved
solubility.
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hERG activity: The activity against the hERG potassium channel was determined using
in vitro IonWorks patchꢀclamp electrophysiology12. Selected compounds were evaluated
for hERG activity in order to determine if any of the core changes would facilitate
mitigating cardiotoxicity risks. Generally, hERG liability remains high, with IC50’s below
10 µM. Pyrazolopyrimidine 22 (hERG IC50: 0.5 µM) showed similar activity against
hERG as its imidazopyridazine analogue 3 (hERG IC50: 0.4 µM). In the imidazopyridine
series, 11 (hERG IC50: 4.8 µM) showed a slight improvement compared to 1 (hERG IC50:
0.9 µM). Excitingly, compound 18 proved to be devoid of hERG activity with an IC50 >
33 µM as compared to 0.9 for the analogous imidazopyridazine 1. The lack of hERG
activity in the imidazopyridine series, however, appears to only hold for the
methylsulphone since the more active cyclopropylsulphone analogue 19 has an IC50 for
hERG of 1.4 µM. We reasoned that the nitrogen at the 5ꢀposition of the
imidazopyridazine ring might be involved in an interaction with the hERG channel, but
the activity for hERG appears to be more complex given the difference between the
IC50’s of 18 and 19.
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In vivo efficacy, pharmacokinetic studies and metabolite identification: A
representative compound of each series with good in vitro antiplasmodial activity and
microsomal metabolic stability e.g. imidazopyridine 12, pyrazolopyridine 18,
pyrazolopyrimidine 22 and tetrahydroꢀpyrazolopyrimidine 24, was evaluated for in vivo
activity in P. berghei infected mice. The in vivo activity was determined following oral
administration (p.o.) of 50 mg/kg/day for 4 consecutive days. If the mice were cured at 4
x 50 mg/kg, further studies were carried out at lower doses of 4 x 10 mg/kg and 4 x 3
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mg/kg. The efficacy results are summarized in Table 2. Previously published results for 1
and 2 are also included in the table for comparison. For each compound, a mouse
snapshot PK was performed using blood samples from the in vivo efficacy experiments
and a complementary rat PK was independently carried out. Generally, there was a good
correlation between the observed in vivo efficacy and the pharmacokinetic parameters.
Even though 22 displayed similar physicoꢀchemical properties as 1 and was 10 times
more active in vitro, the potency did not translate to in vivo efficacy, providing only 86%
activity and 7 mean survival days (MSD). The reduced analogue of the
pyrazolopyrimidine 22 (the tetrahydropyrazolopyrimidine 24) did not show good activity
in vivo either with only 78% of the parasitemia cleared after treatment. Compound 12
showed a similar activity in vivo to 1 with 99.5% activity but with no major
improvement, albeit the MSD was slightly better, increasing from 7 to 10 days.
Pyrazolopyridines 18 and 19 performed poorly, with 60% and 71% of parasitemia cleared
after treatment respectively, and less than 7 days survival (4 days for 18/ 7 days for 19).
In all cases, plasma exposure was poor following oral dosing and bioavailability very
low, even close to null in the case of 18 and 22 (Tables 3 and 4).
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On the other hand, 31 displayed 99.8% parasitemia reduction at 4 x 50 mg/kg with >30
survival days and 3 out of 3 mice cured. This compound also displayed good activity at
the lower multiple doses of 4 x 10 mg/kg (99.9% activity) and 4 x 3 mg/kg (75% activity)
albeit there was no improvement in mean survival days with 8 MSD at 4 x 10 mg/kg and
7 MSD at 4 x 3mg/kg.
Pharmacokinetic and metabolite identification studies in vitro after incubation with liver
microsomes, and in vivo using blood samples from the rat PK experiment, to indeed
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confirm that 19 was the major metabolite of 31 validated our strategy as they clearly
showed that 31 was mainly metabolized to the active sulfone 19 (figure 3). In rats, further
oxidation of the sulfone (as a metabolite of 31 or as direct parent) occurred on the
pyridine core or phenyl rings (M1, M3, M4), and more unexpectedly on the pyrazole part
of the core (M2). Metabolically liable sites of pyrazolo[1,5ꢀa]pyridines are known to
mainly be on the pyridine core (positions 5, 7 and 8)13, which are electron rich due to
electronꢀdonating resonance effects of bridgeꢀhead nitrogens, or on alkyl side chains
when present at positions 2 and 314 (see supporting information for chromatograms and
fragmentation patterns).
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The mouse snapshot PK from the in vivo experiment of 19 made it clear that exposure
was poor following oral administration, which correlates with the poor in vivo efficacy of
19. On the other hand, the mouse snapshot PK from the in vivo efficacy study of 31
showed a very good exposure of 19 following administration of 31, with a significant
amount of the drug still present in the blood after 24h (3 µM). These findings confirm the
advantage of using the sulfoxide over the sulfone to improve solubility and
bioavailability.
In addition to the mouse snapshot PK, full rat PK studies of both 31 and 19 were carried
out. The parent sulfoxide 31 was rapidly absorbed and converted to the metabolite 19,
therefore yielding a low oral bioavailability for the sulfoxide of 14%. The bioavailability
of the sulfone metabolite 19 was calculated to be 25% in this experiment. PK studies with
19 showed a bioavailability of 61%, which does not correlate with the poor in vivo
efficacy. Moreover, it does not seem to reflect the benefits of the sulfoxide for in vivo
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activity. The disconnect between the results may be due to species differences between
rats and mice.
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The therapeutic efficacy of 31 was also assessed against P. falciparum in a SCID mouse
model15 following administration of one oral dose per day for four consecutive days and
measuring the effect on blood parasitemia. Blood samples were also collected for a
corresponding oral exposure evaluation. Blood levels of both 31 and its metabolite 19
were measured, since these compounds are equipotent against P. falciparum and levels of
the metabolite 19 were observed to be higher than that of the parent compound 31. Under
these conditions, 31 (considered as the sum of the parent compound 31 and the main
metabolite 19) was efficacious against P. falciparum upon onceꢀaꢀday for 4 consecutive
days administration with a 90% effective dose of ED90 = 0.5 mg/kg and exposure at ED90
of AUCED90 = 1 ꢁg·h/ml per day, better than the standard marketed antimalarials
artesunate (11.1 mg/kg), chloroquine (4.3 mg/kg), pyrimethamine (0.9 mg/kg)16 and
mefloquine (7.7 mg/kg)17 in the same model. Exposure data is summarized in table 5.
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In vitro evaluation in the P. berghei (Pb.) liver and gametocyte malaria parasite life
cycle stages: Our lead compounds 1, 18, 19 and 22 were also profiled against different
parasite life cycle stages to evaluate their potential as dual and/or triple action
antimalarials. Activities are reported in table 6. IC50s were obtained for the Pb. liver stage
and the early and late stage gametocytes (EG and LG respectively). Excitingly, all
compounds proved to be very potent with activities below 20 nM for the liver stage and
below 100 nM for the gametocytes, both in the early and late stages. The
pyrazolopyrimidine 22 stands out as the lead compound with activities in the single digit
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nanomolar range (targeting EG and LG equally at ~2 nM). 31 was also potent against EG
(IC50 = 72 nM) and LG (IC50 = 163 nM), which makes it very interesting as a proꢀdrug as
this activity will be then reinforced by the activity of 19.
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Conclusion: The SAR investigations around the central core of imidazopyridazine 1
led to the identification of highly potent compounds against Plasmodium falciparum,
with in vitro activities <50 nM and as low as 0.5 nM for imidazopyrimidine 22 and 2 nM
for pyrazolopyridine 19. In vitro potency did not translate to in vivo efficacy due to the
poor pharmacokinetic profiles of compounds 18 and 22. However, a proꢀdrug approach
applied to methylsulfone 19 led to the preparation of the methylsulfoxide 31, which cured
mice infected with P. berghei parasites at 4 x 50 mg/kg. Metabolite identification studies
showed that 31 rapidly generates 19 in vivo, leading to higher plasma exposure compared
to the direct use of 19. The lack of hERG liability for 18 was gratifying, but unfortunately
it did not translate across all compounds in the pyrazolopyridine series. Our lead
compounds also proved to be highly potent against Pb. liver stage as well as early and
late stage gametocytes. This finding makes them highly desirable in the antimalarial
pipeline. Of particular interest is the activity of 31 against both blood and gametocyte
stages, which will be enhanced by the activity of its metabolite 19.
Experimental section:
All commercially available chemicals were purchased from either SigmaꢀAldrich or
CombiꢀBlocks. All solvents were dried by appropriate techniques. Unless otherwise
stated, all solvents used were anhydrous. Infrared (IR) absorptions were measured on a
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PerkinꢀElmer Spectrum One FTꢀIR spectrometer. H NMR spectra were recorded on a
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Varian Mercury Spectrometer at 300 MHz or a Varian Unity Spectrometer at 400 MHz
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with Me4Si as internal standard. C NMR spectra were recorded at 75 MHz on a Varian
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Mercury Spectrometer or at 100 MHz on Varian Unity Spectrometer with Me4Si as
internal standard. Highꢀresolution mass spectra were recorded on a VG70 SEQ
micromass spectrometer. Melting points were determined using a ReichertꢀJung
Thermovar hotꢀstage microscope and are uncorrected. Analytical thinꢀlayer
chromatography (TLC) was performed on aluminiumꢀbacked silicaꢀgel 60 F254 (70ꢀ230
mesh) plates. Column chromatography was performed with Merck silicaꢀgel 60 (70ꢀ230
mesh). Chemical shifts (δ) are given in ppm downfield from TMS as the internal
standard. Coupling constants, J, are recorded in Hertz (Hz).
Purity was determined by HPLC and all compounds were confirmed to have > 95%
purity.
The data that is not shown below is supplied in the Supporting Information.
General procedure for the first Suzuki crossꢀcoupling reaction: Compound 5aꢀd
(respectively 13aꢀb) (1 eq) was dissolved in DMF with the corresponding boronic acid
(1.1 eq) and Pd(PPh3)2Cl2 (0.05 eq). The resulting mixture was flushed with nitrogen for
15 min after which aqueous K2CO3 (1M) (1.05 eq) was added. The solution was heated to
90°C and stirred for 12 h at this temperature. After dilution in DCM and water, the
solution was extracted with DCM 3 times. The combined organic phases were rinsed with
brine and dried over Na2SO4. The solvents were removed in vacuo, the residue was
purified by column chromatography and recristallized in an adequate solvent system to
give the desired intermediate 6aꢀe (respectively 14aꢀf).
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6ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)imidazo[1,2ꢀa]pyridine (6e): 35%. H NMR (400
MHz, CDCl3): δ, 8.41 (s, 1H); 8.10 (t, 1H; J = 2.0); 7.95ꢀ7.90 (m, 1H); 7.85, 7.82 (m,
1H); 7.76 (d, 1H; J = 9.2 Hz); 7.73ꢀ7.65 (m, 3H); 7.45 (dd, 1H; J = 2.0, J= 9.2 Hz); 2.56ꢀ
2.48 (m, 1H); 1.44ꢀ1.37 (m, 2H); 1.12ꢀ1.04 (m, 2H).
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5ꢀ(3ꢀ(methylsulfonyl)phenyl)pyrazolo[1,5ꢀa]pyridine (14b): 77%. 1H NMR (400 MHz,
CDCl3): δ, 8.45 (d, 1H; J = 7.6 Hz); 8.10 (t, 1H; J = 2.0 Hz); 7.88 (d, 1H; J = 2.4 Hz);
7.85 (ddd, 1H; J = 2.0, J = 2.0, J = 8.0 Hz); 7.80 (ddd, 1H; J = 2.0, J = 2.0, J = 8.0 Hz);
7.68 (dd, 1H; J = 0.8, J = 2.0 Hz); 7.57 (t, 1H; J = 8.0 Hz); 6.91 (dd, 1H; J = 7.6; J = 2.0
Hz); 6.50 (dd, 1H; J = 0.8, J = 2.4 Hz); 3.02 (s, 3H).
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5ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)pyrazolo[1,5ꢀa]pyridine (14c): 75%. H NMR (400
MHz, CDCl3): δ, 8.59 (d, 1H; J = 6.4 Hz); 8.16 (s, 1H); 8.00 (s, 1H); 7.96ꢀ7.84 (m, 2H);
7.79 (s, 1H); 7.66 (t, 1H; J = 7.6 Hz); 7.04 (d, 1H; J = 6.4 Hz); 6.62 (s, 1H); 2.59ꢀ2.47 (m,
1H); 1.42ꢀ1.34 (m, 1H); 1.09ꢀ1.03 (m, 1H).
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5ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)pyrazolo[1,5ꢀa]pyrimidine (14f): H NMR (400
MHz, CDCl3): δ, 8.78 (d, 1H; J = 7.6 Hz); 8.63 (t, 1H; J = 1.6 Hz); 8.42ꢀ8.36 (m, 1H);
8.18 (d, 1H; J = 2.4 Hz); 8.03ꢀ8.00 (m, 1H); 7.74 (t, 1H; J = 8.0 Hz); 7.33 (d, 1H; J = 7.2
Hz); 6.77 (dd, 1H; J = 0.8, J = 2.4 Hz); 2,59ꢀ2.51 (m, 3H); 1.44ꢀ1.39 (m, 2H); 1.11ꢀ1.06
(m, 2H).
General procedure for the bromination: Intermediate 6aꢀe (respectively 14aꢀf) (1eq)
were dissolved in DMF at 0°C and NBS (1.1 eq) was added. The resulting mixture was
allowed to reach r.t. and stirred for 2h. After the DMF was removed, the residue was
dissolved in DCM and washed with a saturated solution of bicarbonate. The organic
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phase was dried over Na2SO4 and DCM was removed. The residue was then crystallized
in EtOAc.
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3ꢀbromoꢀ6ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)imidazo[1,2ꢀa]pyridine (7e): 87% yield.
1H NMR (400 MHz, CDCl3): δ, 8.66ꢀ8.54 (m, 1H); 8.19 (s, 1H); 8.00 (s, 1H); 7.98ꢀ7.91
(m, 2H); 7.80ꢀ7.66 (m, 2H); 7.12 (d, 1H; J = 9.6 Hz); 2.58ꢀ2.47 (m, 1H); 1.45ꢀ1.37 (m,
2H); 1.13ꢀ1.04 (m, 2H).
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3ꢀbromoꢀ5ꢀ(3ꢀ(methylsulfonyl)phenyl)pyrazolo[1,5ꢀa]pyridine (15b): 78% yield. H
NMR (400 MHz, CDCl3): δ, 8.52 (d, 1H; J = 7.6 Hz); 8.25 (t, 1H; J = 1.6 Hz); 8.04ꢀ7.91
(m, 3H); 7.77ꢀ7.67 (m, 2H); 7.06 (d, 1H; J = 7.6 Hz); 3.13 (s, 3H).
3ꢀbromoꢀ5ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)pyrazolo[1,5ꢀa]pyridine (15c): 81% yield.
1H NMR (400 MHz, CDCl3): δ, 8.52 (d, 1H; J = 7.6 Hz); 8.19 (t, 1H; J = 1.6 Hz); 7.98
(s, 1H); 7.97ꢀ7.91 (m, 2H); 7.74ꢀ7.66 (m, 2H); 7.08 (d, 1H; J = 7.6 Hz); 2.57ꢀ2.48 (m,
1H); 1.44ꢀ1.38 (m, 1H); 1.11ꢀ1.05 (m, 1H).
3ꢀbromoꢀ5ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)pyrazolo[1,5ꢀa]pyrimidine (15f): 86%
yield. 1H NMR (400 MHz, CDCl3): δ, 8.73 (d, 1H; J = 7.6 Hz); 8.61 (t, 1H; J = 2.0 Hz);
8.51ꢀ8.48 (m, 1H), 8.15 (s, 1H); 8.00ꢀ8.05 (m, 1H); 7.74 (t, 1H; J = 8.0 Hz); 7.37 (d, 1H;
J = 7.6 Hz); 2,60ꢀ2.50 (m, 3H); 1.44ꢀ1.39 (m, 2H); 1.11ꢀ1.06 (m, 2H).
Compounds 8ꢀ12 and 17ꢀ22 were prepared from intermediates 7aꢀe and 15aꢀf following
the same procedure as for intermediates 6aꢀe and 14aꢀf.
6ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)ꢀ3ꢀ(4ꢀ(methylsulfonyl)phenyl)imidazo[1,2ꢀ
a]pyridine (12): Column chromatography: DCM/MeOH (98:2) (95:5). 20% yield after
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crystallization in AcOEt (light yellow powder), H NMR (400 MHz, CDCl3): δ, 8.53 (s,
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1H); 8.11 (d, 2H; J = 8.8 Hz); 8.05 (s, 1H); 7.93 (d, 1H; J =8.0 Hz); 7.86 (s, 1H); 7.85ꢀ
7.78 (m, 3H); 7.68 (t, 1H; J = 8.0 Hz); 7.53 (d, 1H; J = 9.6 Hz); 3.13 (s, 3H); 2.56ꢀ2.46
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(m, 1H); 1.42ꢀ1.34 (m, 2H); 1.12ꢀ1.03 (m, 2H); C NMR (100 MHz, CDCl3): δ, 146.1,
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142.0, 140.2, 138.6, 134.7, 134.5, 131.9, 130.3, 128.7, 127.2, 126.6, 126.0, 125.7, 124.7,
121.0, 118.9, 44.5, 32.9, 6.1; MS (EI+): m/z 452.0 (exact Mass = 452.0864).
5ꢀ(3ꢀ(methylsulfonyl)phenyl)ꢀ3ꢀ(4ꢀ(methylsulfonyl)phenyl)pyrazolo[1,5ꢀa]pyridine
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(18): 50% yield. H NMR (400 MHz, DMSOꢀd6): δ, 8.93 (d, 1H; J = 8.0 Hz); 8.59 (s,
1H); 8.35ꢀ8.32 (m, 2H); 8.27 (d, 1H; J = 8.0 Hz); 8.07 (d, 2H; J = 8.8 Hz); 8.01ꢀ7.96 (m,
3H); 7.81 (t, 1H; J = 8.0 Hz); 7.47 (dd, 1H; J= 2.0, J = 7.6 Hz); 3.33 (s, 3H); 3.24 (s, 3H),
13C NMR (100 MHz, DMSOꢀd6): δ, 149.2, 149.6, 142.3, 139.5, 138.3, 138.2, 137.0,
136.4, 132.7, 130.7, 130.5, 128.3, 127.5, 127.2, 125.8, 115.3, 112.8, 111.8, 44.2, 43.9.
MS (ESI)+: m/z = 427.0 [M+H]+, 465.0 [M+K]+ (exact Mass = 426.0708).
5ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)ꢀ3ꢀ(4ꢀ(methylsulfonyl)phenyl)pyrazolo[1,5ꢀ
a]pyridine (19): 41% yield. 1H NMR (300 MHz, DMSOꢀd6): δ, 8.94 (dd, 1H; J = 0.9, J
= 7.5 Hz); 8.61 (s, 1H); 8.36ꢀ8.25 (m, 3H); 8.09 (d, 2H; J = 8.7 Hz); 8.01ꢀ7.73 (m, 3H);
8.81 (t, 1H; J = 7.5 Hz); 7.46 (dd, 1H; J = 0.9; J = 7.2 Hz); 3.26 (s, 3H); 3.10ꢀ2.99 (m,
13
1H); 1.24ꢀ1.14 (m, 2H); 1.12ꢀ1.02 (m, 1H), C NMR (100 MHz, DMSOꢀd6): δ, 142.6,
142.1, 139.6, 138.34, 133.28, 137.0, 136.4, 132.7, 130.8, 130.5, 128.3, 127.5, 125.9,
115.3, 112.8, 111.8, 44.2, 32.5, 6.0. MS (ESI)+: m/z = 453.1[M+H]+ (exact Mass =
452.0864).
5ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)ꢀ3ꢀ(4ꢀ(methylsulfonyl)phenyl)pyrazolo[1,5ꢀ
1
a]pyrimidine (22): Column chromatography: DCM/MeOH (98:2), 31% yield. H NMR
(400 MHz, CDCl3): δ, 8.83 (d, 1H; J = 7.2 Hz); 8.64 (dd, 1H; J = 1.6, J = 2.0 Hz); 8.57 (s,
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1H); 8.48 (d, 1H; J = 8.4 Hz); 8.37 (d, 2H; J = 8.4 Hz); 8.10ꢀ8.04 (m, 3H); 7.80 (dd, 1H; J
= 8.0 Hz); 7.46 (d, 1H; J = 7.2 Hz); 2.58ꢀ2.52 (m, 1H); 1.47ꢀ1.43 (m, 2H); 1.15ꢀ1.09 (m,
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2H), C NMR (100 MHz, CDCl3): δ, 163.6, 155.4, 144.9, 144.8, 142.2, 138.1, 137.8,
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132.8, 131.0, 129.8, 128.1, 126.3, 108.3, 107.0, 44.2, 32.4, 6.1. MS (EI+): m/z = 453.0
(exact Mass = 453.0817).
5ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)ꢀ3ꢀ(4ꢀ(methylsulfinyl)phenyl)pyrazolo[1,5ꢀ
a]pyridine (31): Column chromatography: DCM/MeOH (100:0) (98:2) (95:5), 39%
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yield. H NMR (400 MHz, DMSOꢀd6): δ , 8.91 (d, 1H; J = 9.6 Hz); 8.54 (s, 1H); 8.33ꢀ
8.24 (m, 3H); 8.02 (d, 2H; J = 8.8 Hz); 7.99ꢀ7.94 (m, 1H); 7.83 (d, 1H; J = 7.6 Hz); 7.79
(d, 2H; J = 8.8 Hz); 7.43 (dd, 1H; J = 2.0, J = 7.6 Hz); 3.08ꢀ2.98 (m, 1H); 2.79 (s, 3H);
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1.26ꢀ1.18 (m, 2H); 1.13ꢀ1.07 (m, 2H), C NMR (100 MHz, CDCl3): δ, 144.1, 142.2,
142.1, 139.7, 136.8, 135.9, 135.5, 132.6, 130.8, 130.4, 127.7, 127.4, 125.8, 124.9, 115.3,
112.6, 112.4, 43.8, 32.5, 6.0. MS (ESI)+: m/z = 437.1 [M+H]+ (exact Mass = 436.0915).
Tetrahydroꢀpyrazolopyrimidines 23 and 24:
Compound 20 (respectively 22) were dissolved in a mixture of DCM/EtOH (3:2) with
Pd(PPh3)2Cl2 (20% wt). NaBH4 (10 eq) was then added and the reaction mixture was
stirred overnight at r.t. or until consumption of the starting material. The solvents were
removed and the residue was purified by column chromatography DCM/MeOH (100:0),
(98:2) to give 23 (respectively 24) in 74% (respectively 79%) yield.
5ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)ꢀ3ꢀ(4ꢀ(methylsulfonyl)phenyl)ꢀ4,5,6,7ꢀ
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tetrahydropyrazolo[1,5ꢀa]pyrimidine (24): H NMR (400 MHz, DMSOꢀd6): δ , 7.93
(s, 1H); 7.85ꢀ7.75 (m, 4H); 7.71ꢀ7.66 (m, 4H); 6.97 (s, 1H); 4.80ꢀ4.71 (m, 1H); 4.23ꢀ4.12
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(m, 1H); 3.97ꢀ3.87 (m, 1H); 3.17 (s, 3H); 2.88ꢀ2.80 (m, 1H); 2.37ꢀ2.28 (m, 1H); 2.27ꢀ
2.16 (m, 1H); 1.16ꢀ1.09 (m, 2H); 1.08ꢀ1.00 (m, 2H), 13C (100 MHz, DMSOꢀd6) : δ,
144.8, 142.7, 140.7, 139.0, 137.1, 135.5, 131.5, 129.6, 127.3, 126.1, 125.1, 124.6, 100.6,
52.5, 43.8, 43.7, 32.0, 29.4, 5.4. MS (EI+): m/z 457.1 (exact Mass = 457.1130).
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In vitro P. falciparum assay and in vivo antimalarial efficacy studies.
Compounds were screened against multidrug resistant (K1) and sensitive (NF54) strains
of P. falciparum in vitro using the modified [3H]ꢀhypoxanthine incorporation assay18. In
vivo efficacy was conducted as previously described19, with the modification that mice (n
= 3) were infected with a GFPꢀtransfected P. berghei ANKA strain (donated by A. P.
Waters and C. J. Janse, Leiden University, The Netherlands), and parasitemia was
determined using standard flow cytometry techniques. The detection limit was 1 parasite
in 1,000 erythrocytes (that is, 0.1%). Activity was calculated as the difference between
the mean per cent parasitaemia for the control and treated groups expressed as a per cent
relative to the control group. Compounds were dissolved or suspended in a nonꢀ
solubilizing, standard suspension vehicle called HPMC (0.5% [wt/vol]
hydroxypropylmethylcellulose, 0.5% [vol/vol] benzyl alcohol, 0.4% [vol/vol] Tween 80,
and 0.9% [wt/vol] sodium chloride in water), and administered orally as four consecutive
daily doses (4, 24, 48 and 72 h after infection). Blood samples for the quadrupleꢀdose
regimens were collected on day 4 (96 h after infection).
In vitro gametocytocidal activity:
In vitro gametocytocidal activity was determined using luciferase reporter lines
specifically enabling screening against early stage gametocytes (>90% stage II/III) and
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late stage gametocytes (>95% stage IV/V) as previously reported20. All gametocytocidal
activities were additionally confirmed in an orthogonal assay by measuring ATP
production in gametocytes treated with the compounds20 with resultant activities
comparative across assay platforms.
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In vitro P.b. liver assays:
This assay is based on the murine malaria parasite Plasmodium berghei transformed with
Luciferase. Hepatic human transformed cells (HepG2), pretreated for 18 hours with the
compound to investigate, are infected with freshly dissected P. berghei Luciferase sporozoites.
After another 48 hours of incubation with the compound to investigate, the viability of P. berghei
exoerythrocytic forms (EEF) and the HepG2 host cells is measured by bioluminescence.
This assay allows us to identify compounds with an eventual activity against sporozoite infection
of liver cells as well the viability of liver schizonts. More details are provided in the Supporting
Information.
In vitro and in vivo metabolites identification – Methods
Metabolite identification of 31 and 19 was performed by LCꢀESIꢀMS/MS using in vivo
blood samples from rat PK and in vitro rat liver microsomal incubations. Details of the
metabolite identification methods are presented in the supporting information.
ASSOCIATED CONTENT:
Supporting Information Available: Additional details of the characterization of selected
compounds and the procedures used for the in vitro and in vivo antimalarial studies as
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well as PK and metabolism studies. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION:
Corresponding author
* Phone: +27ꢀ21ꢀ6502557. Fax: +27ꢀ21ꢀ6505195. Eꢀmail: Kelly.Chibale@uct.ac.za
ACKNOWLEDGMENT: We thank Medicines for Malaria Ventures (MMV) and the
South African Technology Innovation Agency (TIA) for financial support of this research
(Project MMV09/0002). The University of Cape Town, South African Medical Research
Council, and South African Research Chairs Initiative of the Department of Science and
Technology, administered through the South African National Research Foundation are
gratefully acknowledged for support (K.C). We thank Christoph Fischli, Jolanda Kamber,
Sibylle Sax, Christian Scheurer and Ursula Lehmann for assistance in performing
antimalarial assays. We thank L. D. Shultz and The Jackson Laboratory for providing
access to nonobese diabetic scid IL2Rgc null mice (NSG mice) through their
collaboration with GSK Tres Cantos Medicines Development Campus.
ABBREVIATIONS USED: SAR, structureꢀactivity relationships; ADME, absorption,
distribution, metabolism and excretion; CQ, chloroquine; p.o., oral administration; i.v.,
intraveneous administration; MSD, mean survival days; PK, pharmacokinetics; NMR,
nuclear magnetic resonance; TLC thin layer chromatography; r.t., room temperature;
MMV, Medicines for Malaria Venture.
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Figure 1. Structure of compounds 1 and 2
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Figure 2. Structure of compound 31
Figure 3. Proposed metabolic pathway of 31 and 19 in rats
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Scheme 1: Preparation of compounds 8ꢀ12 and 17ꢀ24
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Reagents and conditions: (i) 3ꢀRꢀPhꢀB(OH)2 (1.1 eq), Pd(PPh3)2Cl2 (0.05 eq), aq. K2CO3
(1M) (1.05 eq), DMF, 90°C; (ii) NBS (1.1 eq), DMF, r.t., 1h; (iii) 3ꢀR’ꢀPhꢀB(OH)2 (1.1
eq), Pd(PPh3)2Cl2 (0.05 eq), aq. K2CO3 (1M) (1.05 eq), DMF, 90°C; (iv) NaBH4 (10 eq),
Pd(PPh3)2Cl2 (20% wt), DCM/EtOH; (v) mCPBA (1.2 eq), DCM, 0°C to r.t., 1h
Scheme 2: Preparation of compounds 29 and 30
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7
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Reagents and conditions: (i) NH2ꢀNH2 (5 eq), EtOH; (ii) 4ꢀSO2ꢀbenzaldehyde (1 eq),
EtOH; (iii) PhI(OAc)2 (1.4 eq), DCM, r.t.; (iv) 3ꢀRꢀPhꢀB(OH)2, (1.2 eq), Pd(PPh3)2Cl2
(0.07 eq), K2CO3 (1) (1.5 eq), Dioxane/water (25:10), 110°C
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Antiplasmodial activities.
Pf IC50 (nM)a,b
Compd
Structure
K1
6.3
NF54
7.3
N
N
N
1
2
3
4
8
9
SO2Me
SO2Me
N
N
N
N
N
N
ꢀ
0.53
46
1.1
0.88
O2S
SOMe
SO2Me
SO2Me
SO2Me
N
N
O2S
N
N
60
CF3
N
N
N
N
ꢀ
>2336
>1000
SO2Me
N
N
>1000
SO2Me
SO2Me
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2
3
N
N
4
N
5
6
7
8
ꢀ
877
150
46
10
11
12
17
18
19
20
21
22
SO2Me
SO2Me
9
N
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
113
SO2Me
SO2Me
N
N
38
O2S
SO2Me
N
N
ꢀ
98
CF3
SO2Me
N
N
ꢀ
8
SO2Me
SO2Me
N
N
2.4
2.7
48
O2S
SO2Me
N
N
N
43
CF3
SO2Me
N
N
N
2.2
2.6
0.42
SO2Me
SO2Me
N
N
N
0.37
O2S
SO2Me
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3
N
N
4
5
6
7
8
9
N
H
ꢀ
818
44
23
24
29
30
31
CF3
SO2Me
N
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
H
46
O2S
SO2Me
N
N
N
>1000
>1000
>1000
6
CF3
SO2Me
SO2Me
SOMe
N
N
N
>1000
SO2Me
N
N
ꢀ
O2S
a
Mean from n values of ≥2 independent experiments with multidrug resistant (K1) and sensitive (NF54)
strains of P. falciparum. The majority of the individual values varied less than 2x (maximum 3x)
b Chloroquine and artesunate were used are reference drugs in all experiments. Against NF54 and K1, our
laboratory standard IC50 values for chloroquine and artesunate are 16 nM / 194 nM and 4.0 nM / 3.0 nM
(mean from ≥10 independent assays). IC50 values that differed more than 3x from laboratory standard
values were not included in the analysis.
Table 2. In vivo antimalarial oral efficacy of selected compounds in the P.berghei mouse
model
% reduction
Oral dose
Compound
Structure
parasitemia
(MSD)b,c,d
(mg/kg)a
N
N
N
4 x 50
99.8 (7)
1
SO2Me
SO2Me
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4
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7
8
9
4 x 50
99.8 (>30)
3 out of 3 mice cured
99.6 (8)
N
N
N
2
4
4 x 10
O2S
SOMe
4 x 3
98 (7)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4 x 50
99.8 (>30)
N
N
N
3 out of 3 mice cured
70(7)
4 x 10
CF3
SO2Me
4 x 3
<40 (4)
N
N
4 x 50
99.5 (10)
60 (4)
12
18
19
22
24
O2S
SO2Me
N
N
4 x 50
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
N
N
N
N
N
N
4 x 50
4 x 50
71 (7)
O2S
O2S
O2S
N
86 (7)
N
H
4 x 50
4 x 50
78 (7)
99.8 (>30)
3 out of 3 mice cured
99.9 (8)
N
N
31
4 x 10
4 x 3
O2S
SOMe
75 (7)
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8
9
Chloroquinee
Artesunatee
Mefloquinee
4 x 30
4 x 30
4 x 30
99.9 (24)
99 (10)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
99.9 (29)
a Once per day on 4 consecutive days (4, 24, 48 and 72 h after infection).
bMSD = mean survival time (in days).
c
Mice with <40 and 60% parasitemia reduction were euthanized on day 4 in order to
prevent death otherwise occurring at day 6.
d Artesunate and mefloquine were dissolved or suspended in a nonꢀsolubilizing, standard
suspension vehicle called SSV (0.5% [wt/vol] carboxymethylcellulose, 0.5% [vol/vol]
benzyl alcohol, 0.4% [vol/vol] Tween 80 and 0.9% [wt/vol] sodium chloride in water).
e data from Le Manach et al.21
Table 3. Mouse snapshot pharmacokinetic profiles for compounds 18, 22, 24 and 31 after
the first oral administration of 50 mg/kg
Wholeꢀblood concentration (µM)
Compound
1h
4h
24h
7.6
0.486
1.061
0.145
0.177
1.78
0.29
18
19
22
24
31
0.697
0.169
0.456
2.96
BLQa
<0.01
BLQa
0.03
metabolized
into
7.9
10.3
3.1
19
a below detection limits
Table 4. Rat PK parameters for compounds 18, 22, 24 and 31
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t1/2
(h)
Plasma Cl
(mL/min/kg) (L/kg)
Vd
Compound
12
Method
Dose
B (%)
p.o.
i.v.
p.o.
i.v.
p.o.
i.v.
p.o.
i.v.
p.o.
i.v.
20
5
3.9
3.0
2.6
4.1
5.7
2.7
7.8
2.8
1.0
2.7
ꢀ
8.7
ꢀ
ꢀ
2.3
ꢀ
51
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15
5
1
ꢀ
18
19
22
31
13.9
ꢀ
4.8
ꢀ
10
3
61
ꢀ
3.5
ꢀ
0.8
ꢀ
20
5
1
1.1
ꢀ
0.3
ꢀ
ꢀ
15
5
14
ꢀ
2.9
6.8
Table 5. Exposure of 31 and 19 in P.falciparumꢀinfected humanized mouse after oral
administration of 31.
Dose
Cmax
AUC (0ꢀt)
Compound
tmax (h)
(mg.kgꢀ1) (µg.mLꢀ1)
(µg.h/mLꢀ1)
0.1
0.5
1.5
10
0.002
0.004
0.030
0.050
0.226
0.25
0.25
0.5
2
0.0021a
0.0121b
0.0405b
0.293b
1.2354c
0.139b
1.012c
3.196c
24.78c
78.28c
31
30
6
0.1
0.5
1.5
10
0.025
2
0.106
0.467
2.600
7.43
2
2
19
4
30
8
at = 2h ; bt = 8h ; ct = 23h
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