Medicinal chemistry optimization of antiplasmodial imidazopyridazine hits from high throughput screening of a SoftFocus kinase library: Part 1

Article abstract of DOI:10.1021/jm500098s

A novel class of imidazopyridazines identified from whole cell screening of a SoftFocus kinase library was synthesized and evaluated for antiplasmodial activity against K1 (multidrug resistant strain) and NF54 (sensitive strain). Structure-activity relationship studies led to the identification of highly potent compounds against both strains. Compound 35 was highly active (IC ₅₀: K1 = 6.3 nM, NF54 = 7.3 nM) and comparable in potency to artesunate, and 35 exhibited 98% activity in the in vivo P. berghei mouse model (4-day test by Peters) at 4 × 50 mg/kg po. Compound 35 was also assessed against P. falciparum in the in vivo SCID mouse model where the efficacy was found to be more consistent with the in vitro activity. Furthermore, 35 displayed high (78%) rat oral bioavailability with good oral exposure and plasma half-life. Mice exposure at the same dose was 10-fold lower than in rat, suggesting lower oral absorption and/or higher metabolic clearance in mice.

Full text of DOI:10.1021/jm500098s

Article
pubs.acs.org/jmc
Medicinal Chemistry Optimization of Antiplasmodial
Imidazopyridazine Hits from High Throughput Screening of a
SoftFocus Kinase Library: Part 1
Claire Le Manach,†,⊥ Diego Gonzalez Cabrera,† Frederic Douelle,† Aloysius T. Nchinda,† Yassir Younis,†
̀
Dale Taylor,∞ Lubbe Wiesner,∞ Karen L. White,§ Eileen Ryan,§ Corinne March,§ Sandra Duffy,ø
Vicky M. Avery,ø David Waterson,‡ Michael J. Witty,‡ Sergio Wittlin,◊,∫ Susan A. Charman,§
Leslie J. Street,† and Kelly Chibale*,†,#,⊥
†Department of Chemistry, University of Cape Town, Rondebosch, 7701 Cape Town, South Africa
∞Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Observatory, 7925 Cape Town, South
Africa
øDiscovery Biology, Eskitis Institute, Griffith University (Nathan Campus), Don Young Road, Brisbane Qld 4111, Australia
§Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville campus), 381
Royal Parade, Parkville VIC 3052, Australia
‡Medicines for Malaria Venture, ICC, Route de Pre-
́
Bois 20, PO Box 1826, 1215 Geneva, Switzerland
◊Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland
∫ University of Basel, Socinstrasse 57, 4002 Basel, Switzerland
#Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701 Cape Town, South Africa
⊥South African Medical Research Council Drug Discovery and Development Research Unit, University of Cape Town, Rondebosch,
7701 Cape Town, South Africa
S
* Supporting Information
ABSTRACT: A novel class of imidazopyridazines identified from whole cell screening of a
SoftFocus kinase library was synthesized and evaluated for antiplasmodial activity against K1
(multidrug resistant strain) and NF54 (sensitive strain). Structure−activity relationship studies led
to the identification of highly potent compounds against both strains. Compound 35 was highly
active (IC50: K1 = 6.3 nM, NF54 = 7.3 nM) and comparable in potency to artesunate, and 35
exhibited 98% activity in the in vivo P. berghei mouse model (4-day test by Peters) at 4 × 50 mg/
kg po. Compound 35 was also assessed against P. falciparum in the in vivo SCID mouse model
where the efficacy was found to be more consistent with the in vitro activity. Furthermore, 35
displayed high (78%) rat oral bioavailability with good oral exposure and plasma half-life. Mice
exposure at the same dose was 10-fold lower than in rat, suggesting lower oral absorption and/or
higher metabolic clearance in mice.
alaria remains a major concern for public health,
especially in tropical and subtropical areas and affects
signs of artemisinin resistance have started to emerge in
southeast Asia.2,3 To overcome this, various types of drug
combinations with independent modes of action have been
gradually introduced; however, these present only a temporary
solution.4 The development of new antimalarial agents is thus
urgently needed to counter the ever-increasing spread of drug-
resistant malaria. In this regard, phenotypic whole cell high
throughput screening (HTS) has been a powerful tool for
identifying novel antimalarial chemotypes.5
M
207 million people worldwide.1 The disease is transmitted by
female mosquitoes and is caused by five different species of the
protozoan Plasmodium parasite, namely, falciparum, vivax,
malariae, ovale, and knowlesi that infect and destroy red blood
cells leading to high fever, anemia, cerebral malaria, and
possibly death.1 Of these, falciparum is the most prevalent
species in sub Saharan Africa and the most lethal, being
responsible for over 627 000 deaths a year,1 especially among
young children and pregnant women.
Using an image-based assay,6 HTS of a BioFocus DPI
SoftFocus kinase library7 identified a number of chemotypes
Malaria may be cured if diagnosed in time and treated with
proper medicines. However, the rapid development of drug
resistance has compromised the use of previously effective
drugs such as chloroquine, sulfadoxine/pyrimethamine, and
Received: January 17, 2014
Published: February 25, 2014
© 2014 American Chemical Society
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with antimalarial activity,8 inclusive of the diaryl-imidazopyr-
idazine (SFK52) series (Figure 1).
Figure 1. General structure of SFK52 series.
Figure 3. Structure of scaffolds A and B.
Malaria-related studies based on the imidazopyridazine
scaffold have been recently reported.9 Furthermore, several
groups have been working on imidazopyridazines, focusing on
other biological activities such as kinase inhibition10 or anxiety
treatment.11 The related imidazolopiperazines with promising
blood- and liver-stage activity have been identified and
optimized from the whole-cell screen undertaken by Novartis.12
From 488 diaryl-imidazopyridazines in the SFK52 library
tested in the HTS, 153 compounds displayed >80% inhibition
at the screening concentration of 1.82 μM. Among these were
compounds 1, 2, and 3 (Figure 2). In order to validate them as
hits, these three compounds were first resynthesized and
retested against P. falciparum sensitive and resistant strains
(NF54 and K1 respectively) to confirm activity (IC50 values
between 15 and 25 nM in both strains). When profiled for their
in vitro ADME properties, these compounds were found to
have poor metabolic stability in human liver microsomes with a
predicted human hepatic extraction ratio (EH) > 0.8. Therefore,
they were not expected to perform well in vivo. In order to
identify compounds with both good in vitro potency and
favorable ADME properties, 1 and 2 were used as a starting
point for initial SAR studies. A series of compounds based on
scaffold A (Figure 3) was initially designed, where the aryl
group at the 3-position was fixed as a pyridyl, and variations at
the 6-position were introduced.
Figure 4. Structure of 33 and 35.
Scheme 1. Synthesis of the Imidazopyridazines 1, 2, 8−30,
a
31−55, 56−63
Based on encouraging initial in vitro ADME data with the 4-
methylsulfonylphenyl substituent, we then set the 3 position as
4-methylsulfonylphenyl instead of 4-pyridyl as reflected in
scaffold B (Figure 3), leading to the identification of highly
potent (IC50 <10 nM, K1 and NF54) and metabolically stable
compounds (EH <0.28) 33 and 35 (Figure 4).
a
Reagents and conditions: (i) BrCH2CCH(OEt)2, HBr, EtOH/H2O,
100 °C, quantitative; (ii) NIS, DMF, rt, 4 days, quantitative; (iii) R1-
B(OH)2, Pd(PPh3)2Cl2, aq K2CO3, DMF, 80 °C, 40−60%; (iv) R1-
B(OH)2, Pd(PPh3)2Cl2, aq K2CO3, DMF, 90 °C, 40−70%.
Herein we describe the synthesis of the various libraries and
the results of our SAR exploration, as well as detailed profiling
of the lead compound 35.
Chemistry. Target compounds were prepared following a
relatively straightforward synthetic route involving 4 steps from
the commercially available 3-amino-6-chloropyridazine 4
(Scheme 1). Briefly, a quantitative ring closure using
bromoacetaldehyde diethylacetal and HBr was first performed
on 4 to give 5. Iodination of 5 with NIS in DMF then led to 6
in a quantitative yield. Lastly, two successive Suzuki cross
coupling reactions13,14 with appropriate boronic acids gave us
the desired compounds (1, 2, 8−63).
In Vitro Antiplasmodial Activity. To establish the SAR,
we first explored aryl substitution at positions 3 and 6. All
compounds were evaluated for in vitro antiplasmodial activity
against the sensitive (NF54) and multidrug resistant (K1)
strains of P. falciparum. Chloroquine and artesunate were used
as the reference drugs in all experiments.8c
Figure 2. Structures of the imidazopyridazines 1, 2, and 3 and associated data.
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Table 1. Exploration of the 6-Position with the 3-Position Fixed as a Pyridyl
a
b
Mean from n values of ≥2 independent experiments. Data from Gonzalez Cabrera et al.8c
The in vitro antiplasmodial activities of the compounds, as
Compounds (1, 2, 8, and 9) exhibited high antiplasmodial
activity with IC50 values < 100 nM, whereas the mean value for
CQ in the same experiment was 194 nM; compounds 1 (IC50
16 nM) and 2 (IC50 16 nM) showed comparable activity to
chloroquine (IC50 16 nM) against NF54.
indicated by their IC50 values, are summarized in Table 1. In
general, all analogues were equipotent against both strains (K1
and NF54). Among the 25 evaluated compounds, seven of
them (1, 2, 8−12) showed higher potency than chloroquine
(CQ) against resistant strain K1 with an IC50 < 140 nM.
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Table 2. Exploration of the 6-Position with the 3-Position Fixed as a 4-(Sulfonylmethyl) Phenyl
a
b
8c
Mean from n values of ≥2 independent experiments. Data from Gonzalez Cabrera et al.
Regarding the SAR, meta-substitution at the R2 position
showed either higher or comparable activity. Fourteen
compounds (31−44) displayed Pf IC50 <100 nM against K1,
and six of them (31, 32, 33, 34, 35, 36) were even comparable
to artesunate in both strains with Pf IC50 values ranging from
0.5 nM to 7.3 nM (Table 2).
As previously observed for the pyridyl series, electron-
withdrawing meta-substituents on the aromatic ring at the 6-
position, with a preference for 3-sulfonyl- and carbonyl
derivatives, were optimal for good potency. Once again, 4-
aryl substitution was generally detrimental to activity (Table 3).
Compounds shown in Table 3 confirmed the importance of 3-
substitution for activity.
appeared to be optimal for potency. Replacement by a para
substituent generally resulted in significant loss of activity:
compounds 8−11 and 16 have Pf IC50 values ≤250 nM,
whereas their 4-substituted analogues 22, 24, 26, 28, and 29
had IC50 values >1000 nM.
In parallel, we explored changes to the 3-position. Following
encouraging initial data, we set the 3-position as a 4-
methylsulfonylphenyl group based on good predicted metabolic
stability of that aryl side-chain versus 4-pyridyl as reflected in
scaffold B (Figure 3). In vitro antiplasmodial activities for this
series are shown in Table 2. In this case, in comparison to
chloroquine in the K1 strain, 21 of the 25 compounds (31−51)
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Table 3
In Vitro Metabolic Stability. Metabolic stability of the
most active compounds was assessed in vitro in human (and
rat) microsomal preparations15 to help in guiding the choice
toward metabolically stable substituents. The microsome-
predicted hepatic extraction ratios (EH) are summarized in
the Supporting Information (Table ST1). In general, the
metabolic stability values were consistent across rat and human
microsomes. The results showed that none of the pyridyl
derivatives of the first library were metabolically stable except
29. This is not surprising because the pyridine nitrogen of these
compounds is likely to be oxidized to the N-oxide in liver
microsomes. Among the second library where the 3-position
was set as a 4-methylsulfonylphenyl, nine compounds (33, 35,
38, 41, 42, 43, 44, 46, 48) were found to have good metabolic
stability (EH values <0.28). Very encouraging were compounds
33, 35, and 38, which showed both high potency (IC50 ≤10
nM) along with good metabolic stability (EH value <0.28).
Physicochemical Properties. Physicochemical properties
of the most active compounds were assessed using a
combination of in silico and experimental techniques. Most
of the compounds displayed poor kinetic solubility at pH 6.5
but increased solubility under acidic conditions, consistent with
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a d
−
Table 4. In Vitro and in Vivo Oral Antimalarial Efficacy of Selected Compounds
a
b
MSD = mean survival time (in days). Artesunate and mefloquine were dissolved or suspended in a nonsolubilizing, 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
c
d
water. Mice were euthanized on day 4 in order to prevent death otherwise occurring at day 6. Mean from n values of 2 independent experiments.
expected ionization behavior. The pyridine derivatives from the
first library displayed high solubility (>100 μg/mL) at pH 2. As
for the library derived from scaffold B, compounds generally
showed poor aqueous solubility at neutral pH, with a slight
improvement under acidic conditions but the solubility still
remained very low. Only compounds 34, 42 and 50 showed
good aqueous solubility at both pH 2.0 and 6.5, with values
ranging between 50 and 100 μg/mL or higher. 38 showed good
solubility (>100 μg/mL) at pH 2.5. Regarding the partition
coefficients, all the values were in the range of 1.9 − 5.3 at pH
7.4. The physicochemical data are summarized in the
Supporting Information (Table ST2).
In Vivo Efficacy Studies. Compounds 33, 40, 38, and 35,
each of which displayed good in vitro antiplasmodial activity
and metabolic stability, were tested for in vivo efficacy in P.
berghei-infected mice. The in vivo activity was evaluated
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following oral administration (p.o.) of 50 mg/kg/day for 4
days. The results are summarized in Table 4.
Compounds 33 and 38 were ineffective in vivo with less than
40% reduction in parasitemia compared to untreated infected
animals. Mouse exposure studies after po dosing showed very
low blood levels for these two compounds suggesting poor oral
absorption and/or high clearance. 40 showed a reduction in
parasitemia of 79% with a mean survival time of 7 days.
Interestingly, 35 showed a 98% suppression of parasitemia at
this dose level with a mean survival time of 7 days. Efficacy of
35 was evaluated at lower doses in P.berghei-infected mice and
the effective doses where 50% and 90% reduction in
parasitemia was observed were 1.4 and 15 mg/kg, respectively.
Even though 35 displayed high potency in vitro versus P.
falciparum, this did not translate into high potency in vivo
versus P. berghei. This is despite the plasma concentrations
being maintained at quite high levels (Table 5). The in vivo
Table 5. Plasma Concentrations after First Oral
Administration of 50 mg/kg in P.berghei Mouse Model
(Total Dosing Regimen Was 4 × 50 mg/kg)
dose
(mg/kg)
app. AUC (μM·h) app. Cmax (μM) app. Cav (μM)
24
Figure 5. Pharmacokinetics data of 35 in Sprague−Dawley rats after iv
and po administration.
35
33
38
50
50
50
27.8
c.n.c.
c.n.c.
2.2
1.2
0.02
0.01
c.n.c.
c.n.c.
Table 6. Pharmacokinetics Data of 35 in Sprague−Dawley
Rats
efficacy of 35 was also assessed against P. falciparum in a SCID
mouse model.16 The compound was found to be very potent
(ED90 = 1.5 mg/kg) and comparable to marketed antimalarial
drugs (Table ST3 in Supporting Information). This result
appears to be more consistent with the in vitro activity, and
therefore, the P.berghei data are likely to reflect a parasite strain
difference.
In Vivo Pharmacokinetic Studies. The rat pharmacoki-
netic profile of compound 35 was determined following dosing
at 3.6 mg/kg iv and po. The results are shown in Figure 5 and
Table 6. Compound 35 showed good oral bioavailability in rats
(78%) and a relatively long half-life (7 h). The in vivo plasma
clearance in rats after iv dosing was low (5.8 mL/min/kg) with
a volume of distribution of 3.0 L/kg.
CONCLUSION
■
A novel class of compounds has been identified that combines
good in vitro potency against P. falciparum with oral efficacy in
vivo in a P. berghei mouse model. Despite the high rat oral
bioavailability and microsomal stability, in vivo efficacy of the
lead compound 35 in the P. berghei mouse model remains weak
(MSD = 7). However, the in vivo activity was found to be high
against P. falciparum in the SCID mouse model, which is more
consistent with the in vitro activity and suggesting a parasite
strain difference.
The lead optimization campaign is focused on addressing this
disconnect partly through SAR studies aimed at improving
solubility to address low blood levels and in vivo clearance in
the mouse model and partly through exploring other routes of
clearance.
NMR spectra were recorded on a Varian Mercury spectrometer at 300
MHz or a Varian Unity spectrometer at 400 MHz with Me4Si as
internal standard. 13C NMR spectra were recorded at 75 MHz on a
Varian Mercury spectrometer or at 100 MHz on Varian Unity
spectrometer with Me4Si as internal standard. High-resolution mass
spectra were recorded on a VG70 SEQ micromass spectrometer.
EXPERIMENTAL SECTION
All commercially available chemicals were purchased from either
Sigma-Aldrich or Combi-Blocks. All solvents were dried by appropriate
■
1
techniques. Unless otherwise stated, all solvents were anhydrous. H
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Melting points were determined using a Reichert-Jung Thermovar hot-
stage microscope and are uncorrected. Analytical thin-layer chroma-
tography (TLC) was performed on aluminum-backed silica-gel 60 F254
(70−230 mesh) plates. Column chromatography was performed with
Merck silica-gel 60 (70−230 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 HPLC method is described in
the Supporting Information.
6-Chloroimidazo[1,2-b]pyridazine (5). To a solution of 3-
amino-6-chloropyridazine 4 (1 g, 7.7 mmol, 1 equiv) in EtOH (15
mL) and water (10 mL) was added bromoacetaldehyde diethylacetal
(2.2 mL, 14.1 mmol, 2 equiv) and HBr (0.7 mL). The solution cleared
up after the addition of HBr. The resulting mixture was refluxed at 103
°C overnight. After completion of the starting material, the solution
was diluted in EtOAc and washed with saturated Na2CO3. The
solvents were removed in vacuo, and the crude was used as is for the
next step.
6-Chloro-3-iodoimidazo[1,2-b]pyridazine (6). Compound 5
(948 mg, 6.2 mmol, 1 equiv) was dissolved in DMF, and the resulting
mixture was flushed with nitrogen. NIS (1.5 g, 6.8 mmol, 1.1 equiv)
was added in one portion, and the solution was stirred at room
temperature for 4 days. DMF was removed in vacuo. The residue was
dissolved in DCM and washed with a saturated solution of Na2SO2O5.
The organic phase was concentrated, and the resulting compound was
crystallized in Et2O to give 6 in quantitative yield.
General Procedure for the First Suzuki Cross-Coupling
Reaction. Compound 6 (500 mg, 1.79 mmol, 1 equiv) was dissolved
in DMF (5 mL) with the corresponding boronic acid (1.97 mmol, 1.1
equiv) and Pd(PPh3)2Cl2 (63 mg, 0.09 mmol, 0.05 equiv). The
resulting mixture was flushed with nitrogen for 15 min after which
aqueous K2CO3 (1M) (1.9 mL, 1.88 mmol, 1.05 equiv) was added.
The solution was heated to 80 °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, and the residue was purified by column chromatography and
recrystallized in an adequate solvent system to give the desired product
in 52 to 59% yield.
mixture was stirred for 30 min, and the resin was filtered and rinsed
with MEOH. The filtrate was concentrated and the residue purified on
silica gel.
6-(3,4-Dimethoxyphenyl)-3-(pyridin-4-yl)imidazo[1,2-b]-
pyridazine (1). Column DCM/MeOH (95:5, 90:10), recrystalliza-
1
tion in AcOEt/Hex, 47%. H NMR (300 MHz, CDCl3): δ 8.73 (d,
2H; J = 6.3); 8.24 (s, 1H); 8.15 (d, 2H; J = 6.3); 8.08 (d, H; J = 9.6);
7.65, 7.56 (m, 3H); 7.04 (d, 1H; J = 8.4); 4.02−3.99 (2s, 2 × 3H).
13C NMR (100 MHz, CDCl3): 151.9, 151.4, 150.4, 149.8, 140.8, 136.4,
134.8, 128.2, 126.3, 126.0, 120.4, 120.2, 116.6, 111.6, 110.0, 56.3, 56.2.
MS (EI+): m/z = 331.9 (exact Mass =332.1273). mp = 157 °C.
N-(3-(3-(Pyridin-4-yl)imidazo[1,2-b]pyridazin-6-yl)phenyl)-
methanesulfonamide (2). Column DCM/MeOH (95:5, 90:10),
1
recrystallization in EtOH/DCM, 45%. H NMR (300 MHz, CDCl3):
δ 8.71 (d, 2H; J = 6.0); 8.56 (s, 1H); 8.35 (d, 1H; J = 9.6); 8.30 (d,
2H; J = 6.0); 8.12 (s, 1H); 7.90 (d, 1H; J = 9.6); 7.85 (d, 1H; J = 7.8);
7.56 (dd, 1H; J = 7.8, J = 8.4); 7.39 (d, 1H; J = 8.4); 3.10 (s, 3H). 13C
NMR (100 MHz, CDCl3): 151.0, 150.1, 140.5, 139.5, 135.9; 135.6,
135.5, 130.2, 126.6, 124.9, 122.3, 121.2, 119.4, 117.1, 116.8, 40.0. MS
(EI+): m/z = 364.8 (exact mass = 365.0946). mp =253 °C.
6-(3-(Methylsulfonyl)phenyl)-3-(pyridin-4-yl)imidazo[1,2-b]-
pyridazine (8). Column DCM/MeOH (98:2, 95:5), recrystallization
in DCM/AcOEt, 60%. 1H NMR (400 MHz, CDCl3): δ 8.74 (d, 2H; J
= 6.0); 8.53 (d, 1H; J = 1.8); 8.34 (d, 1H; J = 8.8); 8.28 (s, 1H); 8.17
(d, 1H; J = 9.2); 8.10, 8.07 (m, 3H; J = 6.4); 7.79 (t, 1H; J = 8.8);
7.65 (d, 1H; J = 9.2). 13C NMR (100 MHz, CDCl3): 151.3, 141.8,
140.3, 136.9; 135.7, 135.4, 131.0, 130.4, 128.8, 126.8, 125.9, 119.9,
115.9, 44.4. MS (EI+): m/z = 349.9 (exact mass = 350.0837). mp
=240 °C.
tert-Butyl(3-(3-(pyridin-4-yl)imidazo[1,2-b]pyridazin-6-yl)-
phenyl)methylcarbamate (9). Column DCM/MeOH (98:2, 95:5),
recrystallization in DCM/AcOEt, 67%. 1H NMR (400 MHz, CDCl3):
δ 8.73 (d, 2H; J = 6.4); 8.24 (s, 1H); 8.12 (d, 2H; J = 6.4); 8.09 (d,
1H; J = 9.4); 7.91 (s, 1H); 7.90 (d, 1H; J = 6.8); 7.59 (d, 1H; J = 9.4);
7.57, 7.51 (m, 1H); 7.45 (d, 1H). MS (EI+): m/z = 401.0 (exact mass
= 401.1852)
N-Cyclopropyl-3-(3-(4-(methylsulfonyl)phenyl)imidazo[1,2-
b]pyridazin-6-yl)benzenesulfonamide (31). Column DCM/
MeOH (98:2), recrystallization in DCM/MeOH, 22%. 1H NMR
(400 MHz, dMSO-d6): δ 8.55, 8.49 (m, 4H); 8.43, 8.38 (m, 2H);
8.07, 8.02 (m, 3H); 7.99, 7.95 (m, 2H); 7.83 (dd, 1H; J = 8.0, J =
7.6); 3.25 (s, 3H); 0.53, 0.45 (m, 2H); 0.43, 0.37 (m, 2H); 2.24, 2.11
(m, 1H). 13C NMR (100 MHz, CD3OD/CDCl3): 151.3, 141.4, 136.2,
134.2, 133.7, 130.9, 130.1, 128.8, 127.8, 127.1, 126.4, 125.9, 116.9,
44.2, 24.0, 5.6. MS (EI+): m/z = 467.7 (exact mass = 468.0926). mp
=235 °C.
3-(3-(4-(Methylsulfonyl)phenyl)imidazo[1,2-b]pyridazin-6-
yl)benzenesulfonamide (32). Column DCM/MeOH (9:1, 85:15),
recrystallization in DCM/MeOH, 43%. 1H NMR (400 MHz, CDCl3):
δ 8.59 (t, 1H; J = 1.2); 8.44 (d, 2H; J = 8.8); 8.27 (ddd, 1H; J = 1.6, J
= 7.6, J = 1.2); 8.25 (s, 1H); 8.19 (d, 1H; J = 9.2); 8.09 (d, 2H; J =
8.8); 8.10, 8.05 (m, 1H); 7.84 (d, 1H; J = 9.2); 7.72 (d, 1H; J = 7.6);
3.15 (s, 3H). 13C NMR (100 MHz, CDCl3): 150.8, 146.8, 140.6,
139.7, 136.0, 133.5, 130.8, 130.6, 128.0, 127.6, 126.9, 124.6, 117.1,
44.1. MS (EI+): m/z = 467.9 (exact mass = 428.0613).
6-Chloro-3-(pyridin-4-yl)imidazo[1,2-b]pyridazine (7a). Col-
umn DCM/MeOH (98:2, 95:5, 90:10), recrystallization in Et2O, 59%.
1H NMR (400 MHz, CDCl3): δ 8.72 (d, 2H; J = 6.4); 8.23 (s, 1H);
8.01, 7.99 (m, 3H; J = 6.4); 7.16 (d, 1H; J = 9.2). 13C NMR (100
MHz, CDCl3): δ 150.8, 150.4, 147.5, 140.1, 135.5, 135.2, 127.7, 126.4,
121.5, 120.1, 119.6. MS (EI+): m/z = 230.0
6-Chloro-3-(4-(methylsulfonyl)phenyl)imidazo[1,2-b]-
pyridazine (7b). Column EtOAc/Hex (3:1), (5:1), EtOAc, EtOAc/
1
MeOH (9:1, 8:2), recrystallization AcOEt/Hex, 52%. H NMR (400
MHz, CDCl3): δ 8.29 (d, 2H; J = 8.9); 8.19 (s, 1H); 8.07 (d, 2H; J =
8.9); 8.01 (d, 1H; J = 9.3); 7.17 (d, 1H; J = 9.3). 13C NMR (100
MHz, CDCl3): δ 147.7, 140.1, 134.8, 133.4, 132.0, 128.0, 127.6, 127.0,
125.9, 119.4, 44.6. MS (EI+): m/z = 306.9
General Procedure for the Second Suzuki Cross-Coupling
Reaction. Compound 7 (100 mg, 1 equiv) was dissolved in DMF (2
mL) with the corresponding boronic acid (1.1 equiv) and
Pd(PPh3)2Cl2 (0.05 equiv). The resulting mixture was flushed with
nitrogen for 15 min after which aqueous K2CO3 (1M) (1.05 equiv)
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 three 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
recrystallized in an adequate solvent system to give the desired product
in 22 to 78% yield.
General Procedure for Boc-Deprotection. Boc-protected
compound (1 equiv) was dissolved in DCM. Ten equivalents of
TFA was added, and the resulting mixture was stirred overnight at
room temperature. After evaporation of the solvents, the residue was
dissolved in DCM/MEOH (1:1) and Amberlyst A21 was added. The
6-(3-(Aminosulfonyl)phenyl)-3-(4-(methylsulfonyl)phenyl)-
imidazo[1,2-b]pyridazine (33). Column DCM/MeOH (90:10),
recrystallization in DCM/MeOH, 43%. 1H NMR (300 MHz, CDCl3):
δ 8.59 (t, 1H; J = 2.0); 8.44 (d, 2H; J = 8.4); 8.27 (t, 1H; J = 6.0);
8.25 (s, 1H); 8.19 (d, 1H; J = 9.6); 8.09 (d, 2H; J = 8.7); 8.07 (m,
1H); 7.84 (d, 1H; J = 9.6); 7.72 (d, 1H; J = 8.1); 3.15 (s, 3H). 13C
NMR (100 MHz, CDCl3): 150.8, 146.8, 140.6, 139.7, 136.0, 133.5,
130.8, 130.6, 128.0, 127.6, 126.9, 124.6, 117.1, 44.1. MS (EI+): m/z =
428.1 (exact mass = 428.0613). mp =237 °C
(4-Methylpiperazin-1-yl)(3-(3-(4-(methylsulfonyl)phenyl)-
imidazo[1,2-b]pyridazin-6-yl)phenyl)methanone (34). Column
DCM/MeOH (98:2), recrystallization in DCM/MeOH, 50%. 1H
NMR (300 MHz, CDCl3): δ 8.37 (d, 1, J = 8.7); 8.21 (s, 3H); 8.15
(d, 1H, J = 9.6); 8.10 (d, 2H, J = 8.7); 8.04 (dd, 1H; J = 1.5, J = 9.0);
7.64 (d, 1H, J = 7.2); 7.61 (d, 1H, J = 9.6); 7.56 (dd, 1H, J = 7.8, J =
1.5); 4.06, 3.55 (m, 4H); 3.14 (s, 3H); 2.79, 2.51 (m, 4H); 2.47 (s,
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3H). 13C NMR (100 MHz, CDCl3): 169.4, 151.4, 140.1, 139.0, 136.9,
135.8, 134.9, 134.0, 129.3, 128.7, 128.2, 127.8, 126.8, 126.5, 125.9,
116.3, 55.8, 47.6, 45.9, 44.5, 42.2. MS (EI+): m/z = 475.1 (exact mass
= 475.1678). mp =215 °C.
Gonzal
́
ez Cabrera et al.8c In vivo efficacy was conducted as previously
described,8c with the modification that mice (n = 3) were infected with
a GFP-transfected P. berghei ANKA strain (donated by A. P. Waters
and C. J. Janse, Leiden University, The Netherlands) and parasitemia
determined using standard flow cytometry techniques. Compounds
were dissolved or suspended in a nonsolubilizing, 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). Blood samples for the quadruple-
dose regimens were collected on day 4 (96 h after infection).
6-(3-(Methylsulfonyl)phenyl)-3-(4-(methylsulfonyl)phenyl)-
imidazo[1,2-b]pyridazine (35). Column DCM/MeOH (98:2,
1
95:5), recrystallization in DCM/MeOH, 48%. H NMR (400 MHz,
CDCl3): δ 8.44 (d, 1H; J = 1.6); 8.27 (d, 2H; J = 8.6); 8.24 (d, 1H: J
= 8.4); 8.10 (s, 1H); 8.08 (d, 1H; J = 9.6); 7.99, 7.93 (m, 3H); 7.69
(dd, 1H; J = 8.0, J = 8.4); 7.65 (d, 1H; J = 9.6); 3.04 − 3.02 (2s, 2 ×
3H). 13C NMR (100 MHz, CDCl3): 150.2, 141.9, 140.2, 139.4, 136.9,
135.2, 133.7 131.9, 130.4, 128.8, 127.9, 126.9, 125.9, 115.9, 44.5, 44.4.
MS (EI+): m/z = 427.0 (exact mass = 427.0660). mp =135 °C.
(3-(3-(4-(Methylsulfonyl)phenyl)imidazo[1,2-b]pyridazin-6-
yl)phenyl)(piperidin-1-yl)methanone (36). Column DCM/
MeOH (98:2, 95:5), recrystallization in DCM/MeOH, 48%. 1H
NMR (300 MHz, CD3OD): δ 8.39 (d, 2H; J = 8.7); 8.22 (s, 1H);
8.15(d, 1H; J = 9.3); 8.10, 8.02 (m, H; J = 8.7); 8.03 (m, 4H); 7.63
(dd, 1H; J = 9.3); 7.59, 55 (m, 1H); 3.13 (s, 3H); 3.87, 3.64 (m, 2H);
3.57, 3.29 (m, 2H); 1.84, 1.47 (m, 2H). 13C NMR (100 MHz,
CD3OD/CDCl3): 169.3, 151.5, 140.3, 139.1, 137.5, 135.7, 134.8,
134.0, 129.3, 128.5, 127.9, 127.8, 126.8, 126.5, 125.6, 116.4, 44.5, 43.2,
26.5, 25.6, 24.4. MS (EI+): m/z 460.0 (exact mass = 460.1569).
6-(3-(Cyclopropanesulfonamido)phenyl)-3-(4-
(methylsulfonyl)phenyl)imidazo[1,2-b]pyridazine (37). Column
DCM/MeOH (99:1, 98:2), recrystallization in DCM/MeOH, 50%.
1H NMR (300 MHz, CD3OD): δ 8.43 (d, 2H; J = 8.7); 8.23 (s, 1H);
8.15 (d, 1H; J = 9.3); 8.12, 8.09 (m, 3H); 7.79 (ddd, 1H; J = 1.2, J =
1.6, J = 7.8); 7.62 (d, 1H; J = 9.3); 7.53 (t, 1H; J = 7.8); 7.33 (ddd,
1H; J = 1.2, J = 1.8, J = 7.8); 6.62 (s, 1H); 3.13 (s, 3H); 2.64, 2.51 (m,
2H); 1.32, 1.22 (m, 2H); 1.06, 0.96 (m, 2H). 13C NMR (100 MHz,
CDCl3): 151.6, 138.8, 136.0, 133.6, 129.8, 127.5, 126.8, 125.7, 122.6,
122.2, 118.8, 116.8, 43.9, 29.7, 5.0. MS (EI+): m/z = 467.9 (exact mass
= 468.0926).
(3-(3-(4-(Methylsulfonyl)phenyl)imidazo[1,2-b]pyridazin-6-
yl)phenyl)(piperazin-1-yl)methanone (38). Suzuki reaction was
performed with homemade boc-protected [3-(1-piperazinylcarbonyl)-
phenyl]-boronic acid, pinacol ester. After deprotection following the
Boc deprotection procedure, compound 38 was obtained. Column
DCM/MeOH (95:5, 90:10), recrystallization in DCM/MeOH, 90%.
1H NMR (300 MHz, CD3OD): δ 8.52 (d, 2H; J = 8.7); 8.34 (s, 1H);
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional details of the characterization of selected com-
pounds and the procedures used for the in vitro and in vivo
antimalarial studies as well as PK and metabolism studies. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: Kelly.Chibale@uct.ac.za. Fax: +27-21-6505195. Tel.:
+27-21-6502557.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Medicines for Malaria Venture (MMV) 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 Christian Scheurer,
Petros Papastogiannidis and Jolanda Kamber for assistance in
performing antimalarial assays. We also thank GlaxoSmithKline
(Tres Cantos, Spain) for the Plasmodium falciparum SCID
mouse in vivo efficacy studies.
8.26, 8.22 (m, 2H); 8.18 (s, 1H); 8.12 (d, 2H; J = 8.7); 7.95 (d, 1H; J
= 9.6); 7.71 (t, 1H; J = 7.8); 7.63 (d, 1H; J = 7.8); 3.92, 3.49 (m,
4H); 3.21 (s, 3H); 3.06, 2.82 (m, 4H). 13C NMR (100 MHz, dmso-
d6): 169.2, 151.5, 140.6, 139.7, 136.4, 135.9, 133.8, 130.0, 129.4, 129.0,
130.0, 127.2, 126.9, 126.6, 126.4, 117.6, 44.1, 43.1. MS (EI+): m/z =
460.9 (exact Mass =461.1522)
ABBREVIATIONS USED:
■
HTS, high throughput screening; SAR, structure−activity
relashionships; ADME, absorption, distribution, metabolism,
and excretion; CQ, chloroquine; po, oral administration; iv,
intraveneous administration; MSD, mean survival days; PK,
pharmacokinetics; NMR, nuclear magnetic resonance; TLC,
thin layer chromatography; MMV, Medicines for Malaria
Ventures
(3-(3-(4-(Methylsulfonyl)phenyl)imidazo[1,2-b]pyridazin-6-
yl)phenyl)methanesulfonamide (39). Column AcOEt/MeOH
(1:0, 9:1), recrystallization MeOH/AcOEt, 40%. 1H NMR (400
MHz, DMSO-d6): 8.65 (d, 2H; J = 7.6); 8.58 (s, 1H); 8.41 (d, 1H; J =
9.6); 8.14 (s, 1H); 8.12 (d, 2H; J = 7.6); 7.96 (d, 1H; J = 9.6); 7.87
(d, 1H; J = 7.6); 7.59 (t, 1H; J = 7.6); 7.39 (d, 1H; J = 7.6); 3.31 (s,
3H); 3.12 (s, 3H). 13C NMR (100 MHz, CD3OD): 150.9, 140.0,
139.6, 139.0, 135.9, 135.1, 133.2, 130.0, 127.2, 126.4, 126.1, 125.8,
122.0, 121.1, 117.3, 116.4, 43.4, 38.4. MS (EI+): m/z = 441.9 (exact
mass = 442.0769). mp =135 °C.
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