Medicinal chemistry optimization of antiplasmodial imidazopyridazine hits from high throughput screening of a softfocus kinase library: Part 2

Article abstract of DOI:10.1021/jm500887k

On the basis of our recent results on a novel series of imidazopyridazine-based antimalarials, we focused on identifying compounds with improved aqueous solubility and hERG profile while maintaining metabolic stability and in vitro potency. Toward this objective, 41 compounds were synthesized and evaluated for antiplasmodial activity against NF54 (sensitive) and K1 (multidrug resistant) strains of the malaria parasite Plasmodium falciparum and evaluated for both aqueous solubility and metabolic stability. Selected compounds were tested for in vitro hERG activity and in vivo efficacy in the P. berghei mouse model. Several compounds were identified with significantly improved aqueous solubility, good metabolic stability, and a clean hERG profile relative to a previous frontrunner lead compound. A sulfoxide-based imidazopyridazine analog 45, arising from a prodrug-like strategy, was completely curative in the Plasmodium berghei mouse model at 4 × 50 mg/kg po.

Full text of DOI:10.1021/jm500887k

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Article
Medicinal Chemistry Optimization of Antiplasmodial Imidazopyridazine Hits
from High Throughput Screening of a SoftFocus Kinase Library: Part 2.
Claire Le Manach, Tanya Paquet, Diego Gonzalez Cabrera, Yassir Younis,
Dale Taylor, Lubbe Wiesner, Nina Lawrence, Sylva Schwager, David
Waterson, Michael J. Witty, Sergio Wittlin, Leslie J. Street, and Kelly Chibale
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Oct 2014
Downloaded from http://pubs.acs.org on October 14, 2014
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Medicinal Chemistry Optimization of
Antiplasmodial Imidazopyridazine Hits from
High Throughput Screening of a SoftFocus
Kinase Library: Part 2.
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⊥
Claire Le Manach†, , Tanya Paquet†, Diego Gonzàlez Cabrera†, Yassir Younis†, Dale
Taylor ∞, Lubbe Wiesner ∞, Nina Lawrence ∞, Sylva Schwager #, David Waterson‡,
⊥
Michael J. Witty‡, Sergio Wittlin◊,∫, Leslie J. Street† and Kelly Chibale*,† ,#,
†Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa;
∞Division of Clinical Pharmacology, Department of Medicine, University of Cape Town,
Observatory, 7925, South Africa;
‡Medicines for Malaria 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;
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#Institute of Infectious Disease and Molecular Medicine, University of Cape Town,
Rondebosch 7701, South Africa;
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⊥South African Medical Research Council Drug Discovery and Development Research
Unit, University of Cape Town, Rondebosch 7701, South Africa.
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ABSTRACT: Based on our recent results on a novel series of imidazopyridazineꢀbased
antimalarials, we focused on identifying compounds with improved aqueous solubility
and hERG profile, while maintaining metabolic stability and in vitro potency. Towards
this objective, 41 compounds were synthesized and evaluated for antiplasmodial activity
against NF54 (sensitive) and K1 (multiꢀdrug resistant) strains of the malaria parasite
Plasmodium falciparum, and evaluated for both aqueous solubility and metabolic
stability. Selected compounds were tested for in vitro hERG activity and in vivo efficacy
in the P. berghei mouse model. Several compounds were identified with significantly
improved aqueous solubility, good metabolic stability and a clean hERG profile relative
to a previous frontrunner lead compound. A sulfoxideꢀbased imidazopyridazine analog
45, arising from a prodrugꢀlike strategy, was completely curative in the Plasmodium
berghei mouse model at 4 x 50 mg/kg p.o.
KEYWORDS: antiplasmodial activity, imidazopyridazines, hERG, solubility, in vivo
efficacy, SoftFocus kinase library, structure activity relationships
According to the 2013 World Health Organization report1, malaria affects 207 million
people worldwide and is responsible for over 627 000 deaths a year, especially among
young children and pregnant women. The disease is transmitted by female Anopheles
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mosquitoes and is caused by 5 different species of the protozoan Plasmodium parasite. Of
these falciparum is the most lethal and the most prevalent in sub Saharan Africa.
The rapid development of antimalarial drug resistance has compromised the use of
previously effective drugs such as chloroquine, making malaria impossible to treat in
some areas. Signs of artemisinin resistance emerging in southeast Asia pose an
immediate and urgent challenge2,3. Various types of drug combinations with independent
modes of action have been gradually introduced to prevent and/or slow down the
emergence of resistant strains,4 yet the need for new molecules with novel mechanisms of
action remains.
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Although there are several compounds in clinical or preclinical stages of development,
it is noteworthy that successful development of these compounds is not guaranteed. In
order to maintain a sustained pipeline of antimalarial drugs to stem the tide of drug
resistance, a natural consequence of chemotherapy, the discovery and development of
new molecules with novel mechanisms of action and able to circumvent antimalarial drug
resistance is warranted.
We recently identified a series of imidazopyridazine derivatives5 as a new class of
antimalarial agents with in vivo activity in both the P. berghei and P. falciparum mouse
models. The early lead and frontrunner compound, the 3,6ꢀdiarylimidazopyridazine 1
(Figure 1), showed high antiplasmodial activity in vitro (IC50 K1 = 6.3/ NF54 = 7.3 nM)
and good oral efficacy (98% at 4 x 50 mg/kg p.o.) in the in vivo mouse P. berghei model.
This compound has also shown activity against other lifecycle stages of the parasite
(unpublished data). However, this compound did not produce an impressive in vivo
efficacy at lower doses (4 x 10 mg/kg and 4 x 3 mg/kg), with only a mouse mean survival
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of 7 days at all three doses. Moreover, compound 1 and the vast majority of the analogues
in the same series displayed poor solubility (<5 ꢀM at pH 6.5) and were found to have a
serious hERG liability. The human Etherꢀàꢀgoꢀgo Related Gene (hERG) is a potassium
channel associated with prolongation of the length of time between the start of the Q
wave and the T wave on an electrocardiogram. Potentially lifeꢀthreatening arrhythmia
may result from drugꢀinduced blockade of the hERG channel. Thus in order to address
the aforementioned shortcomings and identify compounds with an improved profile, we
embarked on further exploration of structureꢀactivity relationships.
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In this paper we report the strategies that were adopted in order to address both poor
aqueous solubility and high hERG activity whilst retaining antiꢀmalarial activity. In
addition, a prodrugꢀlike strategy that was successfully used to improve the antiꢀmalarial
activity in vivo is presented.
>>Figure 1<<
Chemistry: Most of the boronic acids used in the synthesis are commercially available.
For those not commercially available, boronic esters were prepared in house and their
synthesis is described in the Supporting Information.
Compounds (6ꢀ9, 16ꢀ18, 21, 22) were prepared following a previously described
synthetic route5. A Suzuki coupling reaction was performed with the corresponding
boronic acid on 6ꢀchloroꢀ3ꢀ(4ꢀsulfonylmethyl)phenylꢀpyridazine to give the desired
compounds (Scheme 1). An additional deprotection on Bocꢀprotected compound 22ꢀa in
the presence of TFA was required to obtain final compound 22.
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The synthesis of target compounds 10ꢀ15 and 25ꢀ34, 36, 37 and 39ꢀ45 was achieved
following a relatively straightforward 4ꢀstep synthetic route from the commercially
available 3ꢀaminoꢀ6ꢀchloropyridazine 2 (Scheme 2).
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Briefly, a quantitative ring closure using bromoacetaldehyde diethylacetal and HBr was
performed on 2 to give 3. A Suzuki cross coupling reaction6,7 with suitable boronic acid
gave the desired intermediates 4aꢀe. Bromination of 4aꢀe with NBS in DMF then led to
5aꢀe in high yield. Finally, a second Suzuki cross coupling reaction with appropriate
boronic acids gave the desired compounds (10ꢀ12, 25ꢀ34, 36, 37, 39ꢀ45).
Tetrazole 20 was obtained from 19 using 1,4 cycloaddition with sodium azide. Amides
23 and 24 were prepared from the carboxylic acid derivatives using EDCI coupling with
the corresponding amine (hydroxyꢀpiperidine and –azetidine), Scheme 3.
Amides 35 and 38 were prepared from the corresponding acids using EDCI coupling as
shown on Scheme 4.
In vitro Antiplasmodial activity and solubility: Previously disclosed antiplasmodial
imidazopyridazine derivatives showed poor solubility profiles5, which can potentially
negatively impact their in vivo efficacy. In order to address this issue, we investigated
various strategies to improve solubility in the imidazopyridazine series while retaining
potency. To this end, several analogues were synthesized and evaluated for solubility and
in vitro antiplasmodial activity against the sensitive (NF54) strain with selected
compounds also being tested against the multiꢀdrug resistant (K1) strain of P. falciparum.
Chloroquine and artesunate were used as the reference drugs in all experiments. The in
vitro antiplasmodial activities as indicated by their IC50 values as well as the aqueous
solubility data are summarized in Table 1 and Table 2. In general, when tested in both
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strains, all analogues were equipotent. The lack of cross resistance in the K1 strain
suggests a distinct and novel mechanism of action of these compounds.
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Selected compounds (8, 22, 33, 36, 44 and 45) showing good antiplasmodial activity
were tested for cytotoxicity and found not to be cytotoxic at the highest concentration
tested. They all showed a selectivity index of >1000. Cytotoxicity data are summarized in
the Supporting Information (Table ST1). Compound 1 and its 3ꢀ(trifluoromethyl)
analogue, both previously published5, did also not show any cytotoxicity at the highest
concentration tested (IC50 CHO cells: >200 µM, SI: >1000).
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Our first strategy towards addressing the solubility issue involved reducing
lipophilicity of the compounds by replacing phenyl rings with pyridyl rings. In this
context, compounds 6ꢀ15 were prepared. Antiplasmodial activity and aqueous solubility
were assessed and the results are listed in Table 1. Unfortunately most of the pyridine
derivatives showed little improvement in solubility, not unexpected given the weak
basicity of the methylsulfonylꢀ, and trifluoromethyl substituted pyridines, and in some
cases antiplasmodial activity decreased dramatically. Compounds 7, 13ꢀ15 turned out to
be poorly active with IC50’s ≥ 470 nM. Compound 6 was moderately active (IC50 ≈ 100
nM) but its solubility was still poor. Compounds 8 and 11 remained active (IC50 ≈ 50
nM) but solubility still remained an issue. The more basic aminoꢀpyridine compounds 9
and 12 retained high potency and showed improved solubility ≥ 100 µM at pH 2 although
solubility at pH 6.5 remained poor.
A second strategy, which involved introducing waterꢀsolubilizing Hꢀbonding groups
onto the phenyl and pyridine rings was also implemented. In this regard, analogues based
on piperazine amides, sulfonamides, tetrazoles, benzotriazoles, hydroxyꢀpyrrolidine, ꢀ
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piperidine or –azetidine were synthesized accordingly. The structures, in vitro
antiplasmodial activities and solubility values for the best derivatives are shown in Table
2. The rest of the data is in the Supporting Information (Tables ST2). An improvement in
solubility was observed for the hydroxylꢀcontaining amides 17, 35 and 23 and the
piperazine sulfone 26 at both pH 2 and pH 6.5. In all cases solubility appeared to be close
to or above 100 µM, whilst compounds remained highly active with NF54 IC50‘s below
35 nM.
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In a third strategy, the sulfonylmethyl substituent was also replaced by a more strongly
Hꢀbonding sulfinylmethyl group at position 6 and/or 3 (16, 32, 37 and 40) or by various
alkylsulfonyl chains substituted with terminal amine (22, 36) or alcohol groups (21).
Replacement of the sulfone group with sulfoxide led to a significant increase in aqueous
solubility from <5 µM for reference compound 1 to >100 µM for the four sulfoxide
derivatives (16, 32, 37 and 40). It was pleasing to observe that all compounds proved to
be highly potent (IC50‘s ≤ 35 nM), except compound 37, which showed moderate activity
(NF54 IC50 = 103 nM). Regarding compounds with alkylꢀsulfones, the two compounds
with a terminal amino group 22 and 36 also proved to be highly soluble and active (NF54
IC50 = 3.0 nM and 10 nM respectively). The exception was compound 37, which showed
moderate activity (NF54 IC50 = 103 nM).
Additional compounds containing a cyclopropylsulfone instead of a methylsulfone at
the 6ꢀposition were also prepared and tested (Table 3). Interestingly, the cyclopropyl
substituent imparted a significant improvement in potency. Compounds 42, 44 and 45
were about 5 times more active than their sulfonylmethyl analogues 10, 29 and 32.
Tetrazole derivative 43 was 13 times more active than its corresponding sulfone whereas
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41 was only slightly more active than 11 (1.6 times). Solubility remained unchanged for
compounds 41ꢀ43 compared to their methylsulfonyl analogues. For compounds 44 and 45
a drop in solubility was observed.
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hERG activity: The activity against the hERG potassium channel was determined
using in vitro IonWorks patchꢀclamp electrophysiology8. Sulfoxides 32, 16 and 40,
hydroxylꢀcycloalkyl amides 35 and 22, and tetrazoles 43 and 45 were evaluated for
hERG activity. The metabolized form of sulfoxide 45, sulfone 46, proved to be highly
active in the hERG assay with an IC50 of 0.4 (0.3 – 0.5) µM. On the other hand
compounds 32 and 16 were found to have ꢀ hERG IC50’s of 4.0 (2.6 ꢀ 6.2) µM and 4.2
(3.3 – 5.2) µM respectively, while activity was moderate for 40 with an IC50 of 12.0 (8.0
– 18.0) µMꢀ. Gratifyingly, the introduction of hydrophilic groups such as amines,
tetrazoles and hydroxylꢀcycloalkyl amides improves selectivity over hERG and the
analogues with improved solubility (35, 22 and 43) lacked activity against hERG at the
highest (IC50’s > 33 µM) concentration tested. Unfortunately, all of the sulfones and
consequently the proꢀdrug sulfoxides retained a hERG risk due to anticipated oxidation to
the corresponding sulfones in vivo.
In vitro metabolic stability: Metabolic stability of the most active compounds was
assessed in vitro in human, rat (and mouse) microsomal preparations9. A quick (1 point)
assay determines the percentage remaining after 30 minutes incubation in the presence of
liver microsomes. Most of our compounds were assayed for metabolic stability using this
method and the results are shown in Table ST3 of the Supporting Information. The
microsomeꢀpredicted hepatic extraction ratios (EH) were determined using a more precise
assay (5 points – 60 minutes) and the results of the few compounds that have been
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evaluated for metabolic stability using this assay are shown in Table ST4 of the
Supporting Information. In general there is a good correlation between the results
obtained with the oneꢀpoint assay and those resulting from the fiveꢀpoint assay. The
metabolic stability values were most often consistent across rat and human microsomes.
However, in some cases species differences were observed.
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Compounds were generally designed to provide good metabolic stability by ensuring
electron deficient aryl rings and avoiding metabolically labile substituents. An exception
are the compounds with sulfoxides side chains which were expected to behave as proꢀ
drugs and to be rapidly metabolized to the corresponding active sulfones even in the in
vitro microsomal assays.
Amongst the compounds showing good solubility and high potency against P.
falciparum, 22, 35 and 36 showed good in vitro metabolic stability in liver microsomes.
On the other hand, compounds 23, 34 and 32 exhibited moderate metabolic stability
while 16, 17, 40 and 26 had poor to moderate stability with species differences being
observed. All cyclopropyl derivatives proved stable across the 3 species except the bisꢀ
sulfoxide 45.
In vivo efficacy studies: Compounds that displayed good in vitro antiplasmodial
activity and metabolic stability together with improved solubility were tested for in vivo
efficacy in P. berghei infected mice. A few compounds with less metabolic stability, such
as the sulfoxide “proꢀdrugs” were also evaluated in vivo. The in vivo activity was
determined following oral administration (p.o.) of 50 mg/kg/day for 4 days. The results
are summarized in Table 4. Compounds 22, 34, 35 and 43 showed poor (<40% reduction
in parasitemia) despite showing good in vitro potency, solubility and microsomal
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metabolic stability. The three monoꢀ or bissulfoxide derivatives 16, 32 and 40 showed
improved in vivo efficacy at 4 x 50 mg/kg p.o. relative to the previous frontrunner
compound 1. The mean survival days (MSD) improved from 7 to 14 days for 32 and 16.
An encouraging result (25 MSD) was delivered by compound 40 with 2 out of 3 mice
treated with this compound experiencing a complete cure. The most impressive activity,
however, was demonstrated by compound 45, the cyclopropylsulfoxide analog of 32.
This compound was completely curative at 4 x 50 mg/kg p.o. It is noteworthy that this is
the first compound in the imidazopyridazine series to demonstrate a complete cure. It
remains unclear if the very last parasites were actually eliminated by the drug itself or
removed by the host immune system. However, at 4 x 10 mg/kg and 4 x 3 mg/kg a fast
recrudescence of parasites was observed, the number of MSD were 8 and 7 days
respectively. Such fast recrudescence of parasites indicates that the immune system was
at least during that time window not having an influence on parasite proliferation. In
addition a fast recrudescence was also observed with the reference compound artesunate,
which is known to have a short halfꢀlife. The in vivo activity of 45 remained high with
99.6 % inhibition of parasitemia at 4 x 10 mg/kg and 98% at 4 x 3 mg/kg.
Disappointingly, none of the potent compounds with water solubilizing sideꢀchains in the
amide or sulfone series showed in vivo activity, and poor absorption or high nonꢀ
microsomal clearance was considered the likely cause.
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In vivo pharmacokinetic studies: Mouse snapshot pharmacokinetics were performed
in parallel to P. berghei in vivo efficacy studies in an attempt to rationalize the observed
efficacy. Infected mice were treated with the various compounds at 50 mg/kg (p.o.) for 4
days. Blood samples were taken 1 hour, 4 hours and 24 hours after the first dose. These
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samples were then analyzed by HPLC in order to determine the pharmacokinetic profiles
(Table 5). Profiles of 22 and 35 showed that very little compound was present after 4
hours and unusual plasma profiles, suggesting poor absorption. Compounds 16 and 40
were metabolized into 1. The solubility of the initial compounds 16 and 40 was improved
compared to our original compound 1. The increase in in vivo potency can be explained
by the increased plasma exposure of compound 1 at t = 4h. As expected 40 gave better in
vivo efficacy results than 16, which in turn gave better results than 1. Considering all the
data, it is most likely that 32 behaves similarly to 16.
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Similar studies were conducted with compound 45 and similar behavior was observed,
in which both the parent compound and metabolite are active. The sulfoxide was rapidly
metabolized to the very active sulfone 46 (IC50 K1/NF54 = 0.5/0.9 nM) (Figure 2). The
superior solubility of the sulfoxide allowed the sulfone biotransformation product to
achieve a high circulating concentration in vivo. This is analogous to a prodrug approach
albeit the sulfoxide is strictly speaking not a classical prodrug.
Further to the mouse snapshot studies, pharmacokinetic studies with 45 and 46 were
performed in male SpragueꢀDawley rats for comparison (Table 6). Sulfone 46 achieved a
maximum plasma concentration of 1.5 µM with a bioavailability of 24%. As expected,
sulfoxide 45 had a short halfꢀlife (0.55 h) and high clearance (46 mL/min/kg), only
reaching a plasma concentration of 0.04 µM. Yet, an oral bioavailability of 11% was
observed. On the other hand, the plasma concentration of 46, achieved through an
equivalent dose of 45, was 2.3 µM with a proportional increase in bioavailability to 36%.
Presumably the increased exposure of sulfone 46 achieved through administering
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sulfoxide 45, together with the superior potencies of both parent and metabolite
compounds, contributes to the in vivo efficacy observed.
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Conclusion: In our quest for compounds with better solubility and an improved hERG
profile, we have identified two molecules (22 and 35) with improved properties. In
addition, sulfoxide derivatives 16, 32 and 40 were shown to have greatly improved
solubility compared to the original early lead sulfone compound 1 but retain the hERG
liability. This improvement in solubility was accompanied by improvement in in vivo
efficacy. Cyclopropyl analog 45 was completely curative in the P. berghei mouse model
at 4 x 50 mg/kg and becomes only the first compound in this series to achieve this feat.
The curative action is in contrast with artesunate and chloroquine, which, when given
orally at 4 x 30 mg/kg, showed mean survival times of 10 and 24 days, respectively.
Following the same dosing regimen, mefloquine prolonged survival up to 29 days5. For
the 3 reference compounds to achieve cure in the P. berghei mouse model, four daily oral
doses of 100 mg/kg of artesunate or chloroquine are required, whereas mefloquine
induces strong acute toxicity symptoms at this high dose.
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Experimental section:
All commercially available chemicals were purchased from either SigmaꢀAldrich or
CombiꢀBlocks. All solvents were dried by appropriate techniques. Unless otherwise
1
stated, all solvents used were anhydrous. H 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
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internal standard. Highꢀresolution mass spectra were recorded on a VG70 SEQ
micromass spectrometer. 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).
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Purity was determined by HPLC and all compounds were confirmed to have > 95%
purity.
6ꢀChloroimidazo[1,2ꢀb]pyridazine (3): To a solution of 3ꢀAminoꢀ6ꢀChloropyridazine 2
(1 g, 7.7 mmol, 1 eq) in EtOH (15 mL) and water (10 mL) was added bromoacetaldehyde
diethylacetal (2.2 mL, 14.1 mmol, 2 eq) 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
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the next step. δ (ppm): H NMR (300 MHz, CDCl3), 7.91, 7.96 (m, 3H); 7.75 (s, 1H);
7.03 (d, 1H: J= 9.2).
6ꢀ(3ꢀ(Methylsulfonyl)phenyl)imidazo[1,2ꢀb]pyridazine (4a): Compound 3 (2 g, 13
mmol, 1 eq) was dissolved in DMF (15 mL) with 3ꢀ(methylsulfonyl)phenyl boronic acid
(2.8 g, 14.3 mmol, 1.1 eq) and Pd(PPh3)2Cl2 (456 mg, 0.65 mmol, 0.05 eq). The resulting
mixture was flushed with nitrogen for 15 min, after which aqueous K2CO3 (1M) (13 mL,
13.7 mmol, 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
three times. The combined organic phases were rinsed with brine and dried over Na2SO4 .
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The solvents were removed in vacuo, the residue was purified by column
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chromatography (DCM/MeOH 98:2, 95:5) and cristallized in ethyl acetate to give the
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desired product 4 in 50 % yield. δ (ppm): H NMR (300 MHz, CDCl3), 8.59 (t, 1H; J =
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1.8); 8.26 (d, 2H; J = 8.1); 8.10, 8.07 (m, 3H); 7.85 (d, 1H; J = 1.2); 7.77 (t, 1H; J = 7.8);
7.53 (d, 1H; J = 9.6); 3.14 (s, 3H).
Compounds 4b, 4c, 4d and 4e were obtained following the same procedure using 3ꢀ
trifluomethylphenyl boronic acid, 3ꢀ(cyclopropylsulfonyl)phenyl boronic acid, 3ꢀ
(methylsulfonyl)phenyl boronic acid and (5ꢀ(methylsulfonyl)pyridinꢀ3ꢀyl)boronic acid
respectively.
6ꢀ(3ꢀ(Trifluoromethyl)phenyl)imidazo[1,2ꢀb]pyridazine (4b): 58%, δ (ppm): 1H NMR
(400 MHz, CDCl3), 8.21 (s, 1H); 8.13, 7.94 (m, 3H); 7.80 (s, 1H); 7.71 (d, 1H; J = 8.8);
7.61 (t, 1H; J = 8.0); 7.48 (d, 1H; J = 9.6).
1
6ꢀ(3ꢀ(Cyclopropylsulfonyl)phenyl)imidazo[1,2ꢀb]pyridazine (4c): 51%, δ (ppm): H
NMR (400 MHz, CDCl3), 8.52 (t, 1H; J = 1.8); 8.24 (d, 2H; J = 8.0); 8.10, 8.01 (m, 3H);
7.85 (s, 1H); 7.73 (t, 1H; J = 7.8); 7.54 (d, 1H; J = 9.6); 2,57, 2.51 (m, 1H); 1.44, 1.41 (m,
2H); 1.09 1.06 (m, 2H).
6ꢀ(3ꢀ(Methylsulfinyl)phenyl)imidazo[1,2ꢀb]pyridazine (4d): 89%, MS (EI+): m/z
257.1 (exact Mass = 257.0623).
6ꢀ(5ꢀ(Methylsulfonyl)pyridinꢀ3ꢀyl)imidazo[1,2ꢀb]pyridazine (4e): 42%, MS (EI+): m/z
274.1 (exact Mass = 274.0525).
3ꢀBromoꢀ6ꢀ(3ꢀ(methylsulfonyl)phenyl)imidazo[1,2ꢀb]pyridazine (5a): Compound 4a
(2 g, 7.8 mmol, 1 eq) was dissolved in DMF (5 mL) and the resulting mixture was
flushed with nitrogen. NBS (1.5 g, 8.3 mmol, 1.1 eq) was added in one portion and the
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solution was stirred at r.t. for 1 hour, after which the compound crashed out. DMF was
removed in vacuo. The residue was cristallized with EtOAc and the resulting solid was
filtered off and rinsed with hexane to give 5a. R = SO2Me, 85%, δ (ppm): 1H NMR (300
MHz, CDCl3), 8.57 (t, 1H; J = 1.8); 8.39 (dd, 1H; J = 1.5; J = 8.1); 8.09, 8.05 (m, 2H);
7.84 (d, 1H; J = 1.2); 7.79 (t, 1H; J = 7.8); 7.59 (d, 1H; J = 9.6); 3.14 (s, 3H).
Compounds 5b, 5c, 5d and 5e were obtained following the same procedure using 4b, 4c,
4d and 4e, respectively.
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3ꢀBromoꢀ6ꢀ(3ꢀ(trifluoromethyl)phenyl)imidazo[1,2ꢀb]pyridazine (5b): R = CF3, 85%,
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δ (ppm): H NMR (300 MHz, CDCl3), 8.33, 8.22 (m, 2H); 8.07 (d, 1H; J = 9.6); 7.85 (s,
1H); 7.80 (d, 1H; J = 7.8); 7.70 (t, 1H; J = 7.8); 7.59 (d, 1H; J = 9.6).
3ꢀBromoꢀ6ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)imidazo[1,2ꢀb]pyridazine (5c):
R
=
SO2(cyclopropyl), 86%, δ (ppm): 1H NMR (300 MHz, CDCl3), 8.57 (t, 1H; J = 1.8); 8.39
(dd, 1H; J = 1.5; J = 8.0); 8.09, 8.05 (m, 2H); 7.84 (d, 1H; J = 1.2); 7.79 (t, 1H; J = 7.8);
7.59 (d, 1H; J = 9.6); 3.14 (s, 3H); 2,57, 2.50 (m, 1H); 1.44, 1.41 (m, 2H); 1.10 1.07 (m,
2H).
3ꢀBromoꢀ6ꢀ(3ꢀ(methylsulfinyl)phenyl)imidazo[1,2ꢀb]pyridazine (5d): R = SOMe,
92%, δ (ppm): 1H NMR (400 MHz, DMSOꢀd6, 8.32 (t, 1H; J = 1.6); 8.25 (d, 1H; J = 9.6);
8.20 (d, 1H; J = 7.6); 7.96, 7.87 (m, 2H); 7.81 (d, 1H; J = 7.6); 7.72 (t, 1H; J = 7.6); 2.78
(s, 3H).
3ꢀBromoꢀ6ꢀ(5ꢀ(methylsulfonyl)pyridinꢀ3ꢀyl)imidazo[1,2ꢀb]pyridazine (5e): 92%, δ
(ppm): 1H NMR (400 MHz, DMSOꢀd6), 9.60 (d, 1H; J = 2.0); 9.25 (d, 1H; J = 2.4); 8.93
(dd, 1H, J = 2.0, J = 2.4); 8.39 (d, 1H; J = 9.6); 8.13 (d, 1H; J = 9.6); 8.04 (s, 1H); 3.44 (s,
3H).
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General procedure for the first Suzuki crossꢀcoupling reaction: Compounds 5aꢀe (1
eq) were dissolved in DMF (1 mL/100mg of 5aꢀe) 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 (up to 120°C for compound 33) 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
recristallized in an adequate solvent system to give the desired product in 30 to 68 %
yield.
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6ꢀ(3ꢀ(Methylsulfonyl)phenyl)ꢀ3ꢀ(4ꢀ(methylsulfonyl)phenyl)imidazo[1,2ꢀb]pyridazine
1
(16): Column DCM/MeOH (98:2, 95:5), crystallization in AcOEt, 30%. H (400 MHz,
CDCl3), 8.38 (d, 2H; J = 8.8); 8.32, 8.24 (m, 3H); 8.21, 8.17 (m, 1H); 8.11 (d, 2H; J =
13
8.8); 7.77, 7.73 (m; 2H); 3.13 (s, 3H); 2.82 (s, 3H); C NMR (100 MHz, DMSOꢀd6):
150.6, 147.7, 140.1, 139.2, 135.9, 135.4, 133.2, 130.2, 129.2, 127.5, 126.8, 126.4, 126.1,
125.3, 122.2, 116.9, 43.5, 43.3. MS (EI+): m/z = 411.0 (exact Mass = 411.0711).
3ꢀ((3ꢀ(3ꢀ(4ꢀ(Methylsulfonyl)phenyl)imidazo[1,2ꢀb]pyridazinꢀ6ꢀ
yl)phenyl)sulfonyl)propanꢀ1ꢀamine (22): The suzuki coupling was performed with the
boronic
ester
tertꢀbutyl
(3ꢀ((3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀ
yl)phenyl)sulfonyl)propyl)carbamate, which synthesis is described in the Supporting
Information. The desired compound was obtained after purification on silica gel with
DCM/MeOH (98:2) to (95:5) in 40% yield. This compound was then deprotected with
TFA (general procedure in the Supporting Information). After evaporation of the solvent,
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the product was crystallized in MeOH/Et2O. Filtration gave the desired product in 79%
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yield. H
(
400 MHz, dmsoꢀd6), 8.61 (s, 1H); 8.60, 8.56 (m, 4H); 8.46 (d, 1H; J = 9.6);
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8.11, 8.07 (m, 3H); 7.95 (t, 1H; J = 7.8); 7.66 (s, 2H); 3.58 (t, 2H; J = 7.4); 3.35 (s, 3H);
2.91 (t, 2H; J = 7.4); 1.91, 1.68 (m, 2H); 13C NMR (100 MHz, DMSOꢀd6): 150.0,
140.1,139.7, 139.3, 136.3, 135.6, 133.1, 132.4, 130.7, 129.2, 127.4, 126.9, 126.4, 126.1,
116.8, 51.6, 41.5, 374, 20.8. MS (EI+): m/z = 470.1 (exact Mass = 470.1082).
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(4ꢀHydroxypiperidinꢀ1ꢀyl)(3ꢀ(3ꢀ(4ꢀ(methylsulfonyl)phenyl)imidazo[1,2ꢀb]pyridazinꢀ
6ꢀyl)phenyl)methanone (23): 6ꢀchloroꢀ3ꢀ(4ꢀSulfonylmethyl)phenylꢀchloropyridazine
was reacted with 3ꢀcarboxyphenylboronic acid following the general Suzuki procedure
described above. The isolated carboxylic acid derivative (40 mg, 01.102 mmol, 1eq) was
then dissolved in DMF (1 mL) with EDCI.HCl (30 mg, 0.143 mmol, 1.4 eq), Et3N (57
µL, 0.408 mmol, 4 eq) and HOBt (2 mg, 0.01 mmol, 0.1 eq). After stirring at r.t. for 1h,
hydroxypiperidine (11 mg, 0.112 mmol, 1.1 eq) was added. The solution was stirred at r.t.
overnight. Extra hydropiperidine (1.1 eq), EDCI.HCl (1.4 eq), Et3N (4 eq) and HOBt (0.1
eq) were added and the resulting mixture was stirred for an extra 48h. DMF was then
removed and the residue was purified on silica gel DCM/MeOH (100:0, 98:2, 95:5,
1
90:10). White powder, 37%. δ (ppm): H NMR (400 MHz, dmsoꢀd6), 8.55 (d, 2H; J =
8.4); 8.50 (s, 1H); 8.36 (d, 1H ; J = 9.2); 8.23 (d, 1H; J = 7.6); 8.11, 8.06 (m, 3H); 8.00 (d,
1H; J = 9.2); 7.69 (t, 1H; J = 7.6); 7.56 (d, 1H; J = 7.6 ); 4.81 (d, 1H; J = 4.0); 3.84, 3.75
(m, 4H); 3.25 (s, 3H), 3.19, 3.14 (m, 4H); 13C NMR (100 MHz, DMSOꢀd6): 168.0, 150.7,
139.8, 139.0, 137.1, 135.1, 134.9, 133.0, 129.2, 128.2, 127.7, 127.2, 126.4, 126.2, 125.9,
124.9, 116.7, 65.1, 43.3. MS (EI+): m/z = 476.1 (exact Mass = 476.1518).
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3ꢀ(4ꢀ(Methylsulfinyl)phenyl)ꢀ6ꢀ(3ꢀ(methylsulfonyl)phenyl)imidazo[1,2ꢀb]pyridazine
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6
7
1
(32): Column DCM/MeOH (98:2, 96:4), crystallization in AcOEt, 68%. δ (ppm): H
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NMR (400 MHz, CDCl3), 8.56 (t, 1H; J = 2.0); 8.37, 8.31 (m, 3H); 8.21 (s, 1H); 8.18 (d,
1H; J = 9.6); 8.10 (dd, 1H; J = 1.6, J = 7.2); 7.83 (d, 2H; J = 8.8); 7.79 (t; 1H; J = 8.0);
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13
7.64 (d, 1H; J = 9.6); 3.13 (s, 3H); 2.81 (s, 3H); C NMR (100 MHz, CDCl3): 150.1,
145.3, 141.9, 139.7, 137.1, 134.5, 132.0, 131.2, 130.5, 128.8, 127.4, 126.8, 126.0, 124.1,
115.6, 44.5, 44.0. MS (EI+): m/z = 411.0 (exact Mass = 411.0711).
(3ꢀHydroxypyrrolidinꢀ1ꢀyl)(4ꢀ(6ꢀ(3ꢀ(methylsulfonyl)phenyl)imidazo[1,2ꢀb]pyridazinꢀ
3ꢀyl)phenyl)methanone (35): Column DCM/MeOH (98:2, 95:5), crystallization in
AcOEt, 33%. δ (ppm): (400 MHz, CDCl3), 8.51 (s, 1H); 8.27 (t, 1H; J = 2.0); 8.17, 7.96
(m, 5H); 7.77, 7.69 (m, 1H); 7.68,7.60 (m, 2H); 7.59, 7.53 (m, 1H); 4.58 (s, 0.5H); 4.47
13
(s, 0.5H); 3.97, 3.47 (m, 4H); 3.11 (s, 3H); 2.15, 1.96 (m, 2H); C NMR (100 MHz,
CDCl3): 169.5, 149.7, 141.8, 137.0, 136.1, 134.1, 134.0, 132.0, 130.4, 130.0, 129.8,
128.7, 128.2, 127.8, 126.5, 125.9, 115.3, 70.8, 69.5, 57.5, 55.0, 47.4, 44.5, 34.8, 32.9,
29.7, 24.8. MS (EI+): m/z = 461.9 (exact Mass = 462.1362).
6ꢀ(3ꢀ(Methylsulfinyl)phenyl)ꢀ3ꢀ(4ꢀ(methylsulfinyl)phenyl)imidazo[1,2ꢀb]pyridazine
1
(40): Column DCM/MeOH (98:2, 95:5), crystallization in AcOEt, 38%. δ (ppm): H
(400 MHz, CDCl3), 8.34, 8.28 (m, 3H); 8.19, 8.12 (m, 3H); 7.80 (d, 2H; J = 8.4); 7.76,
13
7.68 (m; 2H); 7.65 (d, 1H; J = 9.6); 2.80 (s, 3H); 2.79 (s, 3H); C NMR (100 MHz,
DMSOꢀd6): 150.4, 147.7, 145.3, 139.6, 136.1, 134.6, 130.6, 130.1, 129.1, 126.6, 125.2,
124.1, 122.1, 116.4, 43.2. MS (EI+): m/z = 395.1 (exact Mass = 395.0762).
3ꢀ(4ꢀ(1HꢀTetrazolꢀ5ꢀyl)phenyl)ꢀ6ꢀ(3ꢀ(cyclopropylsulfonyl)phenyl)imidazo[1,2ꢀ
b]pyridazine (43): Column DCM/MeOH (98:2) (95:5) (90:10) (85:15), 30%. δ (ppm):
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1H NMR (300 MHz, CDCl3), 8.50 (d, 2H; J = 8.7); 8.34 (s, 1H); 8.28, 8.20 (m, 3H); 8.10
(d, 2H; J = 8.7); 7.92 (d, 1H; J = 9.6); 7.73 (t; 1H; J = 7.2); 7.70, 7.66 (m, 1H); 2.82 (s,
3
4
5
6
7
8
9
13
3H); C NMR (100 MHz, DMSOꢀd6): 155.3, 149.7, 141.5, 139.5, 136.2, 134.5, 131.7,
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130.4, 128.4, 127.0; 126.5, 126.4, 125.3, 123.6, 116.1, 31.7, 5.3. MS (EI+): m/z 443.0755
(exact Mass = 443.1164).
6ꢀ(3ꢀ(Cyclopropylsulfonyl)phenyl)ꢀ3ꢀ(4ꢀ(methylsulfinyl)phenyl)imidazo[1,2ꢀ
b]pyridazine (45): Column: DCM/MeOH (100:0) (98:2) (95:5), 44%. δ (ppm): 1H NMR
(400 MHz, CDCl3), 8.49 (t, 1H; J = 2.0); 8.32, 8.29 (m, 3H); 8.18 (s, 1H); 8.15 (d, 1H; J
= 9.6); 8.05, 8.00 (m, 1H); 7.80 (d, 2H; J = 8.8); 7.75 (t, 1H; J = 8.0); 7.62 (d, 1H; J =
13
9.6); 2.79 (s, 3H); 2.57, 2.47 (m, 1H); 1.43, 1.37 (m, 2H); 1.12, 1.06 (m, 2H); C NMR
(100 MHz, CDCl3), 150.3, 145.3, 142.2, 140.0, 137.0, 134.7, 131.8, 131.4, 130.4, 129.1,
128.4, 127.8, 127.5, 126.9, 126.3, 124.2, 115.7, 44.1, 33.1, 21.1, 6.3. MS (EI+): m/z
437.0 (exact Mass = 437.0868).
6ꢀ(3ꢀ(Cyclopropylsulfonyl)phenyl)ꢀ3ꢀ(4ꢀ(methylsulfonyl)phenyl)imidazo[1,2ꢀ
1
b]pyridazine (46): H NMR (300 MHz, CDCl3), δ (ppm): 8.51 (t, 1H; J = 1.6); 8.40 (d,
2H; J = 8.4); 8.32 (dd, 1H; J = 7.8); 8.26 (s, 1H); 8.20 (d, 1H; J = 9.6); 8.13, 8.04 (m,
3H); 7.79 (t, 1H; J = 7.8); 7.66 (d, 1H; J = 9.6); 3.13 (s, 3H); 2.60, 2.48 (m, 1H); 1.48,
1.40 (m, 2H); 1.16, 1.07 (m, 2H). MS (EI+): m/z 453.1 (exact Mass = 453.0817).
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
assay10. In vivo efficacy was conducted as previously described11, with the modification
that mice (n = 3) were infected with a GFPꢀtransfected P. berghei ANKA strain (donated
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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 orally administered once per day on
four consecutive days (4, 24, 48 and 72 h after infection). Blood samples for the
quadrupleꢀdose regimens were collected on day 4 (96 h after infection).
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Pharmacokinetic studies in Male SpragueꢀDawley Rats
Adult male SpragueꢀDawley rats were starved overnight prior to exposure with the test
compounds. Compounds were freshly prepared to 3.6 mg/kg body weight immediately
prior to dosing. Compounds were dissolved in a mixture of water, 0.01 M hydrochloric
acid solution, propylene glycol, ethanol and Tween 80 (1:2:1:0.1:0.1). All compounds
were dosed orally in 1mL volumes. For the intravenous administration, compounds were
dosed in 350 µL. Water was provided ad libitum throughout the exposure and food was
returned to the animals 5 hours postꢀdose.
Blood samples were collected into heparinized microcentrifugation tubes at specific
timeꢀpoints and spun down at 12000 G for three minutes. Plasma was removed and held
at ꢀ80°C until analysis by LC/MS/MS methods.
Compounds were extracted and quantified by transferring 20 µL of plasma to a
separate vessel and adding 100 µL of cold acetonitrile to precipitate plasma proteins.
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Samples were vortexed vigorously for 30s and then pelleted at 12000 G for three
minutes. A volume of 50 µL of the supernatant was transferred to a 96ꢀwell plate and 50
µL of 0.1% (vol/vol) formic acid solution added to each well. 5µL from each well was
used for LC/MS/MS analysis.
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Chromatographic separation was achieved on a Phenomenex HydroꢀRP column using a
varying gradient of 0.1% (vol/vol) formic acid and acetonitrile. Detection was carried out
on an ABSciex API 3200 mass spectrometer. Pharmacokinetic parameters were
determined using nonꢀcompartmental analysis methods in PK Solutions 2.0 (Summit
Research Services, Montrose CO, USA).
ASSOCIATED CONTENT:
Supporting Information. Additional details of the characterization of selected
compounds and the procedures used for metabolism studies. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION:
Corresponding author
* Phone: +27ꢀ21ꢀ6502557. Fax: +27ꢀ21ꢀ6505195. Eꢀmail: Kelly.Chibale@uct.ac.za
ACKNOWLEDGMENT:
We thank MEDICINES FOR MALARIA VENTURES (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
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Department of Science and Technology, administered through the South African National
Research Foundation are gratefully acknowledged for support (K.C). We thank Christian
Scheurer, Sibylle Sax and Ursula Lehmann (Swiss Tropical and Public Health Institute)
for assistance in performing antimalarial assays. We also thank Prof Peter J. Smith and
Dr Carmen de Kock (Division of Pharmacology, University of Cape Town) for
antiplasmodial data as well as Nina Lawrence and Sylva Schwager (Division of
Pharmacology, University of Cape Town) for in vitro microsomal stability data.
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ABBREVIATIONS USED:
CQ, chloroquine; p.o., oral administration; i.v., intraveneous administration; MSD,
mean survival days; PK, pharmacokinetics; NMR, nuclear magnetic resonance; TLS, thin
layer chromatography; MMV, medicines for malaria ventures; r.t., room temperature,
LC/MS/MS, liquid chromatography – tandem mass spectrometry.
REFERENCES:
1. World Health Organization (WHO), World Malaria Report 2013;
http://www.who.int/malaria/publications/world_malaria_report_2013/report/en/.
accessed June 12.2014.
Date
2. Burrows, N. J.; Chibale, K.; Wells, T.N.C. The state of the art in antiꢀmalarial drug
discovery and development. Curr. Top. Med. Chem. 2011, 11, 1226ꢀ1254.
3. Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning, J.; Lwin, K. M.;
Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.; Silamut, K.; Imwong, M.,
Chotivanich, K.; Lim, P.; Herdman, T.; An, S. S.; Yeung, S.; Singhsivanon, P.; Day, N.
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P.; Lingegardh, N.; Socheat, D.; White, N. J. Artemisinin resistance in Plasmodium
falciparum malaria. N. Engl. J. Med. 2009, 361, 455ꢀ467.
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4. Pink, R.; Hudson, A.; Mouries, M. A.; Bendig, M. Opportunities and challenges in
antiparasitic drug discovery. Nat. Rev. Drug Dicovery 2005, 4, 727ꢀ740.
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5. Le Manach, C.; Gonzalez Cabrera, D.; Douelle, F.; Nchinda, A.; Younis, Y.; Taylor,
D.; Wiesner, L.; White, K. L.; Ryan E.; March, C.; Duffy, S.; Avery, V. M.; Waterson,
D.; Witty, M. J.; Wittlin, S.; Charman, S. A.; Street, L. J.; Chibale, K. Medicinal
chemistry optimization of antiplasmodial imidazopyridazines hits from high throughput
screening of a SoftFocus Kinase Library: Part 1. J. Med. Chem. 2014, 57, 2789ꢀ2798.
6. Miyaura, N.; Suzuki, A. Palladiumꢀcatalyzed crossꢀcoupling reactions of organoboron
compounds. Chem. Rev. 1995, 95, 2457ꢀ2483.
7. Thompson, A. E.; Hughes, G.; Batsanov, A. S.; Bryce, M. R.; Parry, P. R.; Tarbit, B.
Palladiumꢀcatalyzed crossꢀcoupling reactions of pyridylboronic acids with heteroaryl
halides bearing a primary amine group: synthesis of highly substituted bipyridines and
pyrazinopyridines. J. Org. Chem. 2005, 70, 388ꢀ390.
8. (a) BridglangꢀTaylor, M. H.; Hargreaves, A. C.; Easter, A.; Orme, A.; Henthorn, D. C.;
Ding, M.; Davis, A. M.; Small, B. G.; Heapy, C. G.; AbiꢀGerges, N.; Persson, F.;
Jacobson, I.; Sullivan, M.; Albertson, N.; Hammond, T. G.; Sullivan, E.; Valentin, J.ꢀP.;
Pollard, C. E. Optimisation and validation of a mediumꢀthroughput electrophysiologyꢀ
based hERG assay using IonWorks TM HT. J. Pharmacol. Toxicol. Methods 2006, 54,
189ꢀ199; (b) Essen BioScience, hERG IC50 IonWorks Assay Example Report;
http://www.essenbioscience.com/media/uploads/files/hERG_10xcpd_Exemplar_Report.p
df. Date accessed April 25.2014
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9. Obach, R. S. Prediction of Human Clearance Data: An examination of in vitro halfꢀlife
approach and nonspecific binding to microsomes. Drug. Metab. Dispos. 1999, 27, 1350ꢀ
1359.
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10. Snyder, C.; Chollet, J.; SantoꢀTomas, J.; Scheurer, C.; Wittlin, S. In vitro and in vivo
interaction of synthetic peroxide RBx11160 (OZ277) with piperaquine in Plasmodium
models. Exp Parasitol. 2007 115, 296ꢀ300.
11. González Cabrera, D.; Douelle, F.; Younis, Y.; Feng, T.ꢀS.; Le Manach, C.; Nchinda,
A. T.; Street, L. J.; Scheurer, C.; Kamber, J.; White, L. K.; Montagnat, O. D.; Ryan, E.;
Katneni, K. M.; Joseph, J. T.; Bashyam, S.; Waterson D.; Witty, M. J.; Wittlin, S.;
Charman, S. A.; Chibale, K. Structureꢀactivity relanshionship studies of orally active
antimalarial 3,5ꢀsubstituted 2ꢀaminopyridines. J. Med. Chem. 2012, 55, 11022ꢀ11030.
Figure 1. Structure of compound 1
IC50 (nM): K1 = 6.3 / NF54 = 7.3
Kin. Sol. (µM): pH 2: 78 / pH 6.5: <5
EH (h/r/m): <0.42/<0.30/<0.33
hERG (µM): 0.9
in vivo P. berghei (p.o.) at 4 x 50 mg/kg: 98%, 7 MSD
Figure 2. Structure of 46
N
N
N
O2S
SO2Me
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Scheme 1. Synthetic route for compounds 6ꢀ9, 16ꢀ18, 21, 22
5
6
7
8
N
N
i
N
N
Cl
N
X
Y
N
9
R
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11
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15
16
17
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60
SO2Me
SO2Me
6: X = N, Y = CH, R = SO2Me
7: X = N, Y = CH, R = CF3
8: X = CH, Y = N, R = CF3
9: X = N, Y = CH, R = NH2
16: X = CH, Y = CH, R = SOMe
17: X = CH, Y = CH, R = C(O)-NH-(CH2)2-OH
18: X = CH, Y = CH, R = C(O)-NH2
21: X = CH, Y = CH, R = SO2-(CH2)2-OH
22a: X = CH, Y = CH, R = SO2-(CH2)3-NHBoc
22: X = CH, Y = CH, R = SO2-(CH2)3-NH2
ii
Reagents and conditions: (i) boronic acid (1.1 eq), Pd(PPh3)2Cl2 (0.05 eq), aq. K2CO3
(1.05 eq), DMF, 90°C; (ii) 20% TFA:DCM (v:v), 0°C, 1h
Scheme 2. Synthetic route for compounds 10ꢀ15, 25ꢀ34, 36, 37, 39ꢀ45
NH2
i
ii
iii
N
N
N
N
N
N
N
N
X
N
X
N
Cl
N
Br
Cl
2
R
R
3
4a - R = SO2Me
4b - R = CF3
4c - R =
5a - R = SO2Me
5b - R = CF3
X = CH
X = N
X = CH
X = N
5c - R =
O2S
O2S
4d - R = SOMe
4e - R = SO2Me
5d - R = SOMe
5e - R = SO2Me
iv
N
N
N
X
R'
R
X = CH
R = SO2Me
10-12
25-34
36
R = CF3
R = O2S
39
41-46
37, 40
13-15
R = SOMe
R = SO2Me
X = N
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Reagents and conditions: (i) BrCH2CCH(OEt)2 (1.3 eq), HBr, EtOH/H2O, 100°C,
quantitative; (ii) Boronic acid (1.1 eq), Pd(PPh3)2Cl2 (0.05 eq), aq. K2CO3 (1.05 eq),
DMF, 80°C, 50ꢀ58 %; (iii) NBS (1.1 eq), DMF, r.t., 1h, 85%; (iv) R’ꢀB(OH)2 (1.1 eq),
Pd(PPh3)2Cl2 (0.05 eq), aq. K2CO3 (1.05 eq), DMF, 90°C, 40ꢀ70%
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Scheme 3. Synthetic route for compounds 19, 20, 23 and 24
N
N
N
ii
i
N
N
N
Cl
N
N
N
COOH
O
R
SO2Me
SO2Me
SO2Me
R =
R =
N
23
iii
OH
OH
N
N
24
N
iv
N
N
N
N
CN
HN
N
N
N
SO2Me
SO2Me
19
20
Reagents and conditions: (i) 3ꢀ(COOH)PhꢀB(OH)2, (1.1 eq) Pd(PPh3)2Cl2 (0.05 eq), aq.
K2CO3 (1.05 eq), DMF, 100°C, 71 %; (ii) Hydroxyꢀpiperidine (23) or Hydroxyꢀazetidine
(24) (1.1 eq), EDCI.HCl (1.4 eq), Et3N (4 eq), HOBt (0.1 eq), DMF, r.t. (23) or 90°C
(24), 37ꢀ51%; (iii) 3ꢀ(CN)PhꢀB(OH)2 (1.1 eq), Pd(PPh3)2Cl2 (0.05 eq), aq. K2CO3 (1.05
eq), DMF, 90°C, 54 %; (iv) NaN3 (6 eq), NH4Cl (6 eq), 120°C, 5h, 86%
Scheme 4. Synthetic route for amide compounds 35 and 38
N
N
N
ii-a
i-a
N
N
N
N
N
N
Br
i-b
R
SO2Me
SO2Me
35
COOH
N
O
5a-b
OH
N
N
N
N
ii-b
N
N
CF3
38
CF3
COOH
N
O
OH
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Reagents and conditions: (i) 4ꢀ(COOH)ꢀPhꢀB(OH)2 (1.1 eq), Pd(PPh3)2Cl2 (0.05 eq), aq.
K2CO3 (1.05 eq), DMF, 100°C, 68% (a), 54% (b); (ii) (a) Hydroxyꢀpyrrolidine (1.1 eq),
EDCI.HCl (1.4 eq), Et3N (4 eq), HOBt (0.1 eq), DMF, r.t., 33%; (ii) (b) Hydroxyꢀ
piperidine (1.1 eq), EDCI.HCl (1.4 eq), Et3N (4 eq), HOBt (0.1 eq), DMF, r.t., 30%
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Table 1. Effect of pyridine rings on solubility and antiplasmodial activity
Solubility (µM)
Pf IC50 (nM)a,b
Compd
Structure
pH 2 (STD) pH 6.5 (STD)
K1
82
NF54
96
<5
<5
<5
<5
6
7
<5
ꢀ
1050
45
5.4 (0.1)
26
8
9
174(0.4)
15 (0.2)
55
69
<5
<5
<5
9.9
42
19
12
58
10
11
16 (0.1)
192 (3.6)
52 (0.9)
<5
23
12
13
14
13 (0.3)
53 (1.4)
808
636
610
473
183 (0.6)
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<5
<5
662
15
484
7
8
a
Mean from n values of ≥2 independent experiments with multidrug resistant (K1) and sensitive (NF54)
9
strains of P. falciparum. The majority of the individual values differed less than 2x (maximum 3x).
b Chloroquine and artesunate were used as the reference drugs in all experiments. Against NF54 and K1,
our laboratory standard IC50 values for chloroquine and artesunate are 16/194 nM and 4.0/3.0 nM (mean
from ≥ 10 independent assays). IC50 values, which differed more than 3x from the laboratory standard
values were not included in the analysis.
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19
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21
22
23
24
25
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Table 2. Introduction of water solubilizing groups to improve solubility
Solubility (µM)
Pf IC50 (nM)a,b
Compd
Structure
pH 2 (STD) pH 6.5 (STD)
K1
NF54
188 (2.7)
>200
157 (4.1)
>100
36
39
16
17
78
ꢀ
99
190 (8.2)
>200
8.6 (0.1)
>200
2.2
21
22
23
26
2.6
ꢀ
2.7
6.5
>200
178 (1.1)
166 (8.8)
207 (1.2)
162 (0.4)
98 (1.6)
8.8
ꢀ
14
6.5
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N
N
N
5
6
7
8
191 (0.8)
177 (11)
18
11
35
SO2Me
21
N
O
OH
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
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34
35
36
37
38
39
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43
44
45
46
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60
9.4
>200
180 (7.5)
<5
36
37
112 (7.5)
ꢀ
103
35
205 (1.3)
192 (2.4)
30
40
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 differed less than 2x (maximum 3x).
b Chloroquine and artesunate were used as the reference drugs in all experiments. Against NF54 and K1,
our laboratory standard IC50 values for chloroquine and artesunate are 16/194 nM and 4.0/3.0 nM (mean
from ≥ 10 independent assays). IC50 values, which differed more than 3x from the laboratory standard
values were not included in the analysis.
Table 3. Increasing potency with 3ꢀcyclopropylsulfonylphenyl
Solubility (µM)
Pf IC50 (nM)a,b
Compd
41
R1
pH 2 (STD) pH 6.5 (STD)
K1
31
NF54
37
18 (0.3)
<5
<5
<5
42
2.0
3.3
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