Antimalarial Pyrido[1,2-a]benzimidazoles: Lead Optimization, Parasite Life Cycle Stage Profile, Mechanistic Evaluation, Killing Kinetics, and in Vivo Oral Efficacy in a Mouse Model

Article abstract of DOI:10.1021/acs.jmedchem.6b01641

Further structure-activity relationship (SAR) studies on the recently identified pyrido[1,2-a]benzimidazole (PBI) antimalarials have led to the identification of potent, metabolically stable compounds with improved in vivo oral efficacy in the P. berghei mouse model and additional activity against parasite liver and gametocyte stages, making them potential candidates for preclinical development. Inhibition of hemozoin formation possibly contributes to the mechanism of action.

Full text of DOI:10.1021/acs.jmedchem.6b01641

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Article
Antimalarial Pyrido[1,2-a]benzimidazoles: Lead Optimization,
Parasite Life Cycle Stage Profile, Mechanistic Evaluation,
Killing Kinetics and In Vivo Oral Efficacy in a Mouse Model
Kawaljit Singh, John Okombo, Christel Brunschwig, Ferdinand Ndubi, Linley Barnard,
Chad Wilkinson, Peter M. Njogu, Mathew Njoroge, Lizahn Laing, Marta Machado, Miguel
Prudêncio, Janette Reader, Mariette E. Botha, Sindisiwe H. Nondaba, Lyn-Marie Birkholtz,
Sonja Lauterbach, Alisje Churchyard, Theresa L. Coetzer, Jeremy N. Burrows, Clive Leonard
Yeates, Paolo Denti, Lubbe Wiesner, Timothy J Egan, Sergio Wittlin, and Kelly Chibale
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01641 • Publication Date (Web): 17 Jan 2017
Downloaded from http://pubs.acs.org on January 17, 2017
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Antimalarial Pyrido[1,2ꢀa]benzimidazoles: Lead
Optimization, Parasite Life Cycle Stage Profile,
Mechanistic Evaluation, Killing Kinetics and In Vivo
Oral Efficacy in a Mouse Model
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Kawaljit Singh†,#, John Okombo†, Christel Brunschwig∞, Ferdinand Ndubi†, Linley Barnard†,
Chad Wilkinson†, Peter M. Njogu¥, Mathew Njoroge∞,Lizahn Laing∞, Marta MachadoΨ, Miguel
PrudêncioΨ, Janette Reader‡, Mariette Botha‡, Sindisiwe Nondaba‡, LynꢀMarie Birkholtz‡, Sonja
Lauterbach§, Alisje Churchyard§, Theresa L. Coetzer§,Jeremy N. BurrowsÞ, Clive Yeatesζ, Paolo
Denti∞, Lubbe Wiesner∞, Timothy J. Egan†, Sergio Wittlin◊,∫, and Kelly Chibale*,† ,#
†Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa; # South
African Medical Research Council, Drug Discovery and Development Research Unit,
Department of Chemistry and Institute of Infectious Disease and Molecular Medicine, University
of Cape Town, Rondebosch 7701, South Africa; ¥Department of Pharmaceutical Chemistry,
University of Nairobi, P.O. Box 19676, Nairobi, 00202, Kenya; ∞Division of Clinical
Pharmacology, Department of Medicine, University of Cape Town, Observatory, 7925, South
Africa; Ψ Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av.
Prof. Egas Moniz, 1649ꢀ028 Lisboa, Portugal; ‡Department of Biochemistry, Institute for
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Sustainable Malaria Control, University of Pretoria, Private bag X20, Hatfield 0028, South
Africa;§Wits Research Institute for Malaria, Faculty of Health Sciences, University of the
Witwatersrand and National Health Laboratory Service, Johannesburg 2193, South Africa;
ÞMedicines for Malaria Venture, ICC, Route de PréꢀBois 20, P.O. Box 1826, 1215 Geneva,
Switzerland; ζInpharma Consultancy, 6 Dudley Hill Close, Welwyn, Herts AL60QQ, UK; ◊Swiss
Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland; ∫University of
Basel, 4003 Basel, Switzerland
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ABSTRACT: Further structure activity relationship (SAR) studies on the recently identified
pyrido[1,2ꢀa]benzimidazole (PBI) antimalarials, have led to the identification of potent,
metabolically stable compounds with improved in vivo oral efficacy in the P. berghei mouse
model and additional activity against parasite liver and gametocyte stages, making them potential
candidates for preclinical development. Inhibition of haemozoin formation possibly contributes
to the mechanism of action.
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INTRODUCTION
Malaria,a parasitic disease caused by parasites of the Plasmodium genus,remains a global
public health concern due to its morbidity and mortality, with approximately 214 million new
clinical cases and about 438,000 deaths reported in 2015.1Mammalian infection is initiated by
the bite of Plasmodiumꢀinfected female Anopheles mosquitoes. An asymptomatic but obligatory
developmental phase in the liver ensues, leading to the release of newly formed parasites into the
bloodstream, where they infect red blood cells and cause disease symptoms.2 Transmission of the
parasite to the mosquito vector occurs when the latter ingests circulating gametocytes, initiating
the sexual stage of the parasite’s lifecycle. Chemotherapy represents one of the most effective
control measures to mitigate the malaria burden, with the World Health Organization (WHO)
presently recommending the use of artemisinin combination therapies (ACT) for treatment of
uncomplicated malaria. However, there is compelling evidence from Southeast Asia describing
the emergence and spread of ACT tolerance, which is characterised by reduced clearance rates of
Plasmodium falciparum parasites.3, 4 This underscores the urgent need to expand the antimalarial
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drug arsenal by exploring and developing new compound classes, preferably with a combination
of novel modes of action, multiꢀstage activity, good safety profile and efficacy at low doses.
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The benzimidazole motif is a recognized privileged scaffold in medicinal chemistry due to its
capacity to interact with numerous biological systems,5 leading to a wide variety of biological
activities, including antimalarial activity.6ꢀ9 Pyrido[1,2ꢀa]benzimidazoles (PBIs), previously
investigated for antibacterial, antifungal, antiviral and antitumor activities,10 were recently shown
to be a novel antimalarial chemotype.11 The lead compound from the previous studies 8 (Figure
1), showed high antiplasmodial activity in vitro (IC50 (PfNF54) = 0.11µM; IC50 (PfK1) =
0.12µM) and promising oral efficacy (95% at 4×50 mg/kg p.o) in vivo in the rodent P. berghei
model. However, pharmacokinetic (PK) studies indicated saturation in oral absorption at low
doses, presumably due to poor dissolution or solubility, which may be a factor in limiting oral
efficacy. Herein, we describe further structure activity relationship (SAR) investigations (SAR1ꢀ
4) around the scaffold of compound 8 (Figure 2), partly to explore the potential for generating
derivatives with an improved PK profile but also to explore the effect on activity of changes
around parts of the scaffold not previously investigated, with the goal of improving oral activity
in the mouse model to confirm if this series has potential for further development.
The SAR studies can be broken down according to Fig. 2:
• SAR1ꢀ replacement of the alkylamino side chain with various alkyl, cycloalkyl and
heterocyclic moieties containing waterꢀsolubilizing Hꢀbonding groups, encompassing
hydroxypyrrolidine, azetidinol, hydroxypiperidine, piperazine or sulfonamide
substituents.
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• SAR2ꢀ introduction of small hydrophobic substituents at the Cꢀ2 position to favor non
coꢀplanar conformations of the Cꢀ3 aryl, lowering the crystal packing energies and
consequently improving aqueous solubility.12
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• SAR3ꢀ replacement of the 4ꢀtrifluoromethylphenyl (4ꢀCF3Ph) at the Cꢀ3 position with
substituted or unsubstituted phenyl rings and with saturated systems or cycloalkyl
groups and introduction of waterꢀsolubilizing Hꢀbonding groups including ester,
sulfinyl, sulfone and sulfoxide substituents on the Cꢀ3 phenyl ring.
• SAR4ꢀ introduction of substituents on the previously11 unexplored left aromatic ring,
including replacement with the less lipophilic pyridyl ring.
The in vitro antiplasmodial activities of synthesized derivatives against chloroquineꢀsensitive
(CQꢀS) and multidrug resistant P. falciparum strains as well as in vivo efficacy in P. bergheiꢀ
infected mice are reported. Additionally, we present metabolic stability, killing kinetics,
gametocytocidal and liver stage activity data on a subset of the compounds. Some mechanistic
studies were done, focusing on the potential for inhibition of haemozoin (Hz) formation, on the
hypothesis that the ability of PBIs to adopt flat conformations would allow for πꢀπ stacking
interactions with ferriprotoporphyrin IX (Fe(III)PPIX), a toxic byꢀproduct of host haemoglobin
degradation, thus leading to inhibition of Hz crystal formation and consequent parasite death.
Chemistry: The synthesis of target PBI derivatives was relatively straightforward using
published procedures (Scheme 1).11
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Biology: In vitro antiplasmodial activity and cytotoxicity: All compounds were evaluated
for in vitro antiplasmodial activity against the CQS PfNF54 strain; those with submicromolar
activity (34/62 compounds) were further tested against the multidrug resistant PfK1 strain and
for mammalian cytotoxicity against Chinese hamster ovary (CHO) cell lines. The results are
summarized in Tables 1ꢀ3. In general, activity of the compounds ranged from 0.02ꢀ0.95 µM and
0.02ꢀ1.07 µM against PfNF54 and PfK1, respectively, with 28/34 exhibiting submicromolar
potency against both strains.
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With regard to SAR:
1. SAR1 – variation in the aminoalkyl side chain, with a heterocyclic, cyclic and alkyl
substituents containing Hꢀbonding groups (9–38; Table 1).
• The only improvement in vitro was found in replacement of the Nꢀethyl in 8 with a 3ꢀ
hydroxy pyrrolidine (11), showing ~ 3x reduction in IC50 relative to 8, with better
selectivity.
• Introduction of the same side chain present in chloroquine (16, 17) resulted in a
significant drop in activity and poor selectivity.
2. SAR2 – Introduction of ꢀMe or –F substituents at Cꢀ2 (66 – 69; Table 3).
• Retaining the Nꢀethyl ethylenediamine side chain of 8 and introducing a Cꢀ2 Me (66)
or F (67) resulted in a significant loss in activity, with a 10ꢀ50x increase in PfNF54
IC50.
• Activity of the 2ꢀMe and 2ꢀF derivatives was improved by introduction of the 3ꢀ
hydroxypyrrolidinyl side chain (68, 69), consistent with the previous observations from
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SAR1 (see 11; Table 1), with the 2ꢀF analogue (69) now showing comparable activity
to 8 with significantly better selectivity.
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3. SAR3 – Replacement of the 4ꢀCF3Ph in 8 with unsubstituted as well as substituted phenyl
rings and cycloalkyl groups (39–53; Table 2).
• Varying the position of the ꢀCF3 group from the para (8) to the metaꢀposition (40)
had only a slight effect on activity. The unsubstituted phenyl and a range of ortho
(43), meta (44), para (41, 42, 46, 50) and diꢀsubstituted derivatives (45) all
retained significant activity (PfNF54 IC50 ˂1 µM), with only 50 showing a
significant improvement in selectivity.
• Substitution with a para –COOMe (47), ꢀSMe (48), or ꢀSOMe (49) significantly
lowered activity, as did replacing the 4ꢀCF3Ph with a ꢀCF3 (51).
• Cycloalkyl groups at Cꢀ3, including cyclohexyl (52) and cyclopentyl (53), were
well tolerated but showed no improvement in selectivity.
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4. SAR4– Substitution on the benzimidazole (54–65; Table 3)
• Substitution at Cꢀ7, Cꢀ8 or Cꢀ9 with Cl or Br, including multiple substitutions, was well
tolerated, providing in some cases a significant improvement in activity (54, 56, 59,
and 60) and selectivity (54, 59, and 60).
• Replacing the phenyl with a pyridyl (64, 65) had either no effect or lowered
antiplasmodial activity, depending on the position of the ring nitrogen, and was
accompanied by poor selectivity.
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• Replacing the Nꢀethyl ethylenediamine side chain in some of the more active
compounds (59, 60) with various cycloalkylamino side chains did not give any
improvement in activity and generally lowered selectivity (61–63), consistent with the
previous observations in SAR1.
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Physicochemical properties (Table 4): From the range of analogues evaluated the following
observations can be made:
• Permeability generally ranged from moderate to high at pH 6.5, indicative that
permeability is unlikely to be a limiting factor in the oral absorption of these
compounds from the gastrointestinal tract.
• Some improvement in solubility was achieved through introduction of a 2ꢀF (67, 69),
although the corresponding 2ꢀMe derivatives (66, 68) were poorly soluble, despite the
anticipation that distortion in the planarity of resulting from Cꢀ2 substitution would
reduce the crystal packing energy thus helping dissolution in water.
• Introduction of water solubilizing groups like sulfone (50) did improve solubility but
this proved detrimental to permeability.
• Replacement of the 4ꢀCF3Ph with a saturated ring (52) resulted in a slight improvement
in solubility but at the expense of reduced microsomal stability.
• Predicted lipophilicity was relatively high (logP >3 for most compounds). For SAR1
and SAR3 analogues, increased lipophilicity was associated with better permeability
while lower lipophilicity to better solubility, except for 17 with high logP but good
solubility. There was no apparent relationship between lipophilicity and metabolic
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stability for this series of compounds, suggesting that structural changes had a greater
contribution to these properties than lipophilicity.
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Metabolic stability in liver microsomes (Table 4): The most active compounds in the in
vitro antiplasmodial assay were assessed for metabolic stability in mouse and human liver
microsomes (MLMs and HLMs, respectively). Most compounds showed no significant
interspecies difference in microsomal stability and the following trends were observed:
• Introduction of a piperidinyl (20, 26) or CQꢀlike side chain (17) led to a significant
decrease in microsomal stability, with the exception of 36, where the piperidinyl ring
was directly linked to the PBI scaffold.
• 2ꢀMethyl substitution (SAR2) was not detrimental to microsomal stability (66), but
surprisingly the 2ꢀF analogue (67) was significantly less stable.
• Variation in the substitution on the Cꢀ3 aryl (SAR3) had a significant effect on stability,
with the dichloro derivative (45) showing significantly poorer stability compared to 8.
• The more active compounds with halogen substitution on the benzimidazole (SAR4;
54, 57, 59, 60) did not show any significant difference in microsomal stability relative
to 8.
In vivo efficacy in the mouse P. berghei model (Table 5): Compounds with good
antiplasmodial activity, reasonable solubility and high microsomal stability were assessed for in
vivo efficacy in P. bergheiꢀinfected mice, dosing orally at 4 x 50 mg/kg; if mice were cured
lower doses were evaluated (4 x 30 mg/kg, 4 x 10 mg/kg and 4 x 3 mg/kg). Parasitemia reduction
in P. berghei in vivo efficacy testing helped to efficiently prioritize compounds in the knowledge
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that those with good efficacy must have exposure. PK was followed up in detail on those that
were most interesting. In summary:
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• Compound 44, less active than 8 in vitro but with slightly superior microsomal
stability, showed slightly better in vivo efficacy compared to 8, with a single cure at 4
x 50 mg/kg.
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• Compound 57, with comparable in vitro activity and microsomal stability to 8, was not
curative at 4 x 50 mg/kg.
• Compounds 59 and 60, with significantly better in vitro activity and comparable
microsomal stability to 8, were completely curative at 4 x 50 mg/kg; 59 proving better
at 4 x 30 mg/kg. At lower doses (4 x 10 mg/kg and 4 x 3 mg/kg), the mice were not
cured (MSD of 16 – 18) although parasitemia was still reduced by > 99%.
Pharmacokinetic studies (Table 6). Studies were performed in mice dosed orally (p.o) at 10
mg/kg and intravenously (i.v.) at 2.5 mg/kg with compounds 59 and 60. Since the elimination
phase was not captured in the standard 24 h sampling period and nonꢀcompartmental analysis
could not provide reliable parameter estimates, the data were interpreted with nonlinear mixedꢀ
effects modeling where a twoꢀcompartment disposition model was used, with firstꢀorder
absorption and elimination. Both IV and oral data were fit in the same model, and bioavailability
fixed to 100% for the IV infusion and estimated for the oral formulation. Betweenꢀmouse
variability was included in the absorption rate constant, plasma clearance, and oral
bioavailability. The PK parameters from the model were used to derive the exposure metrics in
Table 6.
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Both 59 and 60 were slowly absorbed when dosed orally (Tmax of ≥10 h) and were slowly
eliminated (i.e. CL<30 mL/min/kg). Considered in relation to the previous PK studies on 8,11
which showed broadly similar pharmacokinetics to 59 and 60, it would appear that the
significant improvement in efficacy of 59 and 60 in P. bergheiꢀinfected mice is likely to be due
more to their better inherent antimalarial activity than improved pharmacokinetics.
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Gametocytocidal and liver stage activity (Table 7). The potential of this compound class to
act as dual or tripleꢀaction antimalarials was determined by evaluating their gametocytocidal
potency13 and liver stage activity. Compound 8 and a range of 17 new analogues were tested
against early (EG; >90% stage II/III) and late (LG; >95% stage IV/V) gametocytes. The potential
for inhibition of liver stage Plasmodium infection was assessed using a model with a P. berghei
infected human hepatoma cell line (HuH 7).14 Compounds 54 and 59, with good in vitro blood
stage activity, also showed comparable activity against both EG and LG, whilst others (8, 42, 52)
showed a degree of stageꢀspecific potency for LG gametocytes, indicated by a >2ꢀfold change in
IC50.
Initial screening at 1 µM, 5 µM and 10 µM on liver stage infection and cell confluence using
luciferaseꢀexpressing parasites and a luminescenceꢀbased method15 established the nonꢀtoxicity
of the test compounds, with no significant effect on cell proliferation. Compounds 26, 66 and 69
led to the strongest decrease in infection at the lowest concentration tested (1 µM) and a
subsequent doseꢀresponse analysis confirmed their activity. Compound 69, which also had good
blood stage activity (PfK1 IC50: 0.17 µM), was the most potent inhibitor in the P. berghei liver
stage assay (IC50: 1.42 µM), significantly more active than primaquine (PMQ) (IC50: 8.42 µM),
albeit less active than atovaquone (IC50: 1.1 nM).16 It should be noted that PMQ's activity in vivo
is dependent on its oxidation to active metabolites17 and, as such, the compound's IC50 in vitro
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does not necessarily represent its in vivo potency. Compounds 26 (IC50: 3.31 µM) and 66 (IC50:
2.85 µM) also showed a degree of activity in the in vitro liver stage assay, in addition to their
blood stage activity.
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Speed of action and mechanistic evaluation. There was a suspicion from the previous studies
on 8 in the P. berghei mouse model that the compounds might be inherently slow acting. To
investigate this further a cross section of 9 compounds were run through a [3H] hypoxanthine
incorporation assay designed to assess their killing speed between at 24, 48, and 72 h.18 The IC50
values of pyrimethamine, 11 and 69 were respectively 6ꢀ, 18ꢀ and 22ꢀ fold higher at 24 h
compared to 72 h (Figure 4), indicating that these compounds were slow acting; it may be
noteworthy that 11 and 69 have the 3ꢀhydoxypyrrolidinyl side chain. However, 8, 20, 17 and 44
appeared to be faster acting (IC50 (24 h)/IC50 (72 h) < 1.5), comparable to CQ and AS, while 9,
54 and 59 could be classified as neither fast or slow, with (IC50 (24 h)/ IC50 (72 h) 1.5 ꢀ 2).
Overall, albeit based on the relatively few compounds tested, it appears that there was neither a
bias for any particular killing speed nor significant correlation between speed of action and the
previously quoted in vitro efficacy against PfNF54.
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Mechanistic Studies: Due to their structural likeness to CQ, which includes a planar
heterocyclic moiety, a haloꢀsubstitution and a basic amine side group, the mechanistic potential
of these compounds to inhibit Hz formation was investigated by measuring their ability to inhibit
in vitro βꢀhematin (βH) formation. Although a number of these derivatives were able to inhibit
βH formation (IC50 <100 µM) in a detergentꢀmediated Nonidet Pꢀ40 (NPꢀ40) assay (Supporting
Information Table S1), there was only a weak, albeit statistically significant, linear correlation
with wholeꢀcell activity against PfNF54 (Figure 5). It is possible that the cellꢀfree βH inhibition
assay does not mirror the cellular Hz inhibitory activity or that there is only weak drugꢀhaem
complex formation due to poor binding to free haem molecules, or that Hz inhibition is not the
sole target of this class of compounds.
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To confirm if these compounds were bonaꢀfide inhibitors of Hz formation, a cellular
fractionation assay was used to determine free haem and Hz signatures when synchronized ring
stage parasites were treated with increasing doses; for true inhibitors there is a doseꢀdependent
increase in toxic free haem and corresponding decreases in Hz.19 Of the cross section of
compounds studied 8, 9, 54, 45 were βH inhibitors in the NPꢀ40 assay (BHIAIC50 < 45ꢁM),
whilst the inactive 69 (BHIAIC50 = 120ꢁM) represented a negative control. No significant
changes in toxic free haem or Hz levels with increasing drug concentrations were observed for
69 (Figure 6), whilst 8 and 45 showed significant concentrationꢀdependent increases in free
haem and corresponding decreases in haemozoin compared to the untreated control, with the free
haem and parasite survival curves crossing close to the IC50 values of the compounds (Figure 6)
ꢀ a trend not shown by 9 or 54 despite their potency in the NPꢀ40 assay (Supporting Information
Figure S1). In silico simulations of vacuolar accumulation of these compounds showed that they
are likely to accumulate poorly within the digestive vacuole compared to standard haemozoin
inhibitors (Supporting Information Table S1). Taken together, these studies suggest that
inhibition of haemozoin formation is one but perhaps not the sole or primary target of this class
of compounds; their flat conformation possibly allows for weak haemꢀdrug complex formation
through only πꢀπ interactions as was recently reported for certain benzamide analogues.20 This
would be unlike halofantrine and quinine, for instance, which reportedly require hydrogen
bonding and coordination to Fe(III)PPIX in addition to π−π interactions.21, 22 The lack of βH
inhibition by 69 could be ascribed to disrupted planarity (from the 2ꢀF substitution) which
potentially minimizes π−π interaction.
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This leaves the intriguing prospect that the PBIs could act, at least in part, through an as yet
unidentified mechanism. Amongst the diverse activities reported for other pyrido[1,2ꢀ
a]benzimidazoles is the inhibition of the poreꢀforming protein perforin in mammalian cells by 1ꢀ
aminoꢀ2, 4ꢀdicyanopyrido[1,2ꢀa]benzimidazoles.23 This is interesting as the P. falciparum
proteome harbors perforinꢀlike proteins (PLPs) known to be involved in permeabilizing the
erythrocyte membrane during egress of either gametocytes or merozoites.24 An hydroxy
derivative 2ꢀEthylꢀ1ꢀhydroxyꢀ3ꢀmethylꢀpyrido[1,2ꢀa]benzimidazoleꢀ4ꢀcarbonitrile (GNF7686)
with potent in vitro activity against Trypanosoma cruzi, was also recently shown to act through
inhibition of cytochrome b (a component of cytochrome bc1 or complex III),25 and a wellꢀ
established antimalarial target. Whilst the related hydroxy intermediates from our studies did not
show significant antiplasmodial activity (unpublished data), this potential target warrants further
investigation as a possible mode of action and encourages further testing for potential activity
against kinetoplastids, including Leishmania donovani.
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CONCLUSIONS
Further SAR studies, aided by physicochemical evaluation and microsomal stability studies, on
the antimalarial activity of pyrido[1,2ꢀa] benzimidazoles have led to a number of compounds
with improved in vitro activity against P. falciparum, with substitution on the benzimidazole
phenyl being a key factor in improving in vitro activity. Amongst the most active compounds in
vitro, 59 and 60 showed significantly better in vivo activity in the mouse P. berghei model. The
most active compounds also showed good activity against gametocytes, indicative of potential as
dualꢀacting antimalarials. Other analogues have shown activity against the liver stage of P.
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berghei. There is no conclusive evidence for their mode of action, although inhibition of
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haemozoin formation is identified as a potential contributory factor.
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EXPERIMENTAL SECTION
All commercially available chemicals were purchased from either SigmaꢀAldrich or Combiꢀ
Blocks. All solvents were dried by appropriate techniques. Unless otherwise stated, all solvents
used were anhydrous. 1H NMR spectra were recorded on a Varian Mercury Spectrometer at 300
MHz or a Varian Unity Spectrometer at 400 MHz.13C NMR spectra were recorded at 75 MHz on
a Varian Mercury Spectrometer or at 100 MHz on Varian Unity Spectrometer. Chemical shifts
(δ) are given in ppm downfield from TMS as the internal standard. Coupling constants, J, are
recorded in Hertz (Hz). Highꢀresolution mass spectra were recorded on a VG70 SEQ micromass
spectrometer. Melting points (m.pt) were determined by Differential Scanning Calorimetry
(DSC) using TA Q200/Q2000 DSC from TA Instruments. Analytical thinꢀlayer chromatography
(TLC) was performed on aluminiumꢀbacked silicaꢀgel 60 F254 (70ꢀ230 mesh) plates. Column
chromatography was performed with Merck silicaꢀgel 60 (70ꢀ230 mesh). 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 synthesis of compound 4
A mixture of the substituted diaminobenzene 3(10 equiv.) and ethylcyanoacetate (2.0 equiv.)
in DMF (1 mL) was heated in a microwave reactor at 110 °C for 15–45 min. The reaction
mixture was then diluted with ethyl acetate (20 mL) and washed with water (3×15 ml). The
organic phase was separated, dried over MgSO4, filtered, solvent removed under reduced
pressure and purified through column chromatography using MeOH: DCM as eluent to afford
compound 4.
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2ꢀ(4ꢀChloroꢀ1Hꢀbenzo[d]imidazolꢀ2ꢀyl)acetonitrile 4b:
7
8
9
ο
1
Yellow solid (49%). Rf 0.5 (65% EtOAcꢀHexane). mp 190–192 C. H NMR (400 MHz,
DMSOꢀd6) δ 7.21 (m, 3H), 5.20 (br s, 1H), 3.89 (s, 2H). 13C NMR (100 MHz, DMSOꢀd6) δ
146.3, 140.0, 136.1, 127.3, 125.7, 123.8, 121.9, 116.8, 116.4. MS: LCꢀMS: (ESI)+: found m/z
191.2[M+]:193.4[M+2]+(3:1), (calculated for C9H6ClN3: 191.62).
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2ꢀ(3Hꢀimidazo[4,5ꢀb]pyridinꢀ2ꢀyl)acetonitrile 4i:
Brown solid (97%). Rf 0.3 (10% MeOHꢀDCM). mp 172–174 οC. 1H NMR (400 MHz, DMSOꢀ
13
d6) δ 13.17 (br s, 1H), 8.34 (m, 1H), 7.99 (m, 1H), 7.24 (m, 1H), 4.45 (s, 2H). C NMR (100
MHz, DMSOꢀd6) δ 147.6, 144.2 (2C), 127.2, 118.5 (2C), 116.6, 19.2. MS: LCꢀMS: (ESI)+:
found m/z 159.2 [M+H]+, (calculated for C8H6N4: 158.16).
General procedure for the synthesis of compound 5
A mixture of benzimidazole acetonitrile 4 (1.0 equiv.), NH4OAc (2.0 equiv.), and the
appropriate βꢀketo ester 2 (1.20 equiv.) was heated to reflux at 150οC for 1h and then allowed to
ο
cool to 100 C. MeCN (10 mL) was added and the resulting mixture stirred for 15 min, allowed
to cool to room temperature and then cooled on ice. The cold mixture was filtered and the
residue washed with cold MeCN (4×10 mL), dried in vacuo, and used in the next step without
further purification.
3ꢀ(3,4ꢀdichlorophenyl)ꢀ1ꢀhydroxybenzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile 5i:
Silver tan powder (75%). Rf 0.7 (70% EtOAcꢀHexane). mp 249–251 C. 1H NMR (400 MHz,
ο
DMSOꢀd6) δ 13.80 (br s, 1H), 8.61 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 2.1 Hz, 1H), 7.82 (d, J = 8.1
13
Hz, 1H), 7.64 (dd, J = 8.3, 2.2 Hz, 1H), 7.58 (m, 2H), 7.41 (t, J = 8.2 Hz, 1H), 6.10 (s, 1H). C
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NMR (100 MHz, DMSOꢀd6) δ 158.5, 150.2, 147.9, 137.9, 132.8, 132.5 (2C), 131.9, 131.3,
130.5, 129.0, 128.1, 127.3, 123.1, 117.0, 116.7, 112.2, 105.0. MS: LCꢀMS: (ESI)+: found m/z
354.1[M+]:356.2[M+2]+(3:1), (calculated for C18H9Cl2N3O: 354.19).
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7,8ꢀDichloroꢀ1ꢀhydroxyꢀ3ꢀ(4ꢀ(trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀ
carbonitrile 5t:
ο
1
Brownish solid (74%). Rf 0.3 (70% EtOAcꢀHexane). mp 305–307 C. H NMR (600 MHz,
DMSOꢀd6) δ 8.63 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.72 (s, 1H), 6.02
(s, 1H). 13C NMR (150 MHz, DMSOꢀd6) δ 158.5, 151.8, 150.0, 141.7, 130.0, 129.6 (2C), 129.1,
128.1 (2C), 126.0, 125.9, 125.4, 124.0, 123.6, 117.2 (2C), 114.1, 104.2. MS: LCꢀMS: (ESI)+:
found m/z 422.1[M+]:424.3[M+2]+:426.4[M+4]+(9:3:1), (calculated for C19H8Cl2F3N3O: 422.19).
General procedure for the synthesis of compound 6
A mixture of compound 5 (1.0 equiv.) and POCl3 (20 equiv.) was heated to reflux at 130 οC for
2 h. Excess POCl3 was removed under reduced pressure and iceꢀcold water (20 mL) added to the
residue, stirring to yield a precipitate. The mixture was neutralized with saturated NaHCO3 and
filtered. The resultant solid was washed with iceꢀcold water (4×15 mL), dried in vacuo, and used
without further purification.
1ꢀChloroꢀ3ꢀ(3,4ꢀdichlorophenyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile 6i:
ο
1
Yellow solid (98%). Rf 0.6 (30% EtOAcꢀHexane). mp 260–262 C. H NMR (400 MHz,
DMSOꢀd6) δ 8.72 (d, J = 8.1 Hz, 1H), 8.09 (s, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.90 (d, J = 8.1 Hz,
1H), 7.83 (dd, J = 8.3, 2.2 Hz, 1H), 7.73 (t, J = 2.1 Hz, 1H), 7.62 (d, J = 2.1 Hz, 1H), 7.44 (t, J =
13
8.2 Hz, 1H). C NMR (100 MHz, DMSOꢀd6) δ 159.5, 152.2, 148.5, 136.7, 132.7, 132.1 (2C),
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131.7, 131.2, 130.1, 129.5, 128.8, 127.5, 123.9, 118.1, 116.5, 113.2, 106.0. MS: LCꢀMS: (ESI)+:
found m/z 372.4[M+]:374.3[M+2]+:376.4[M+4]+(9:3:1), (calculated for C18H8Cl3N3: 372.63).
1,7,8ꢀTrichloroꢀ3ꢀ(4ꢀ(trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile
6t:
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ο
Brownish solid (97%). Rf 0.8 (70% EtOAcꢀHexane). mp. 253–255 C. 1H NMR (600 MHz,
DMSOꢀd6) δ 8.86 (s, 1H), 8.34 (s, 1H), 8.03 (m, 4H), 7.70 (s, 1H). 13C NMR (150 MHz, DMSOꢀ
d6) δ 149.5, 149.4, 144.3, 139.1, 135.4, 134.7, 130.9, 130.4, 130.2, 128.9, 126.4 (2C), 125.2,
125.1, 123.4, 121.2, 117.5, 115.0, 113.6; MS: LCꢀMS: (ESI)+: found m/z 440 [M]+: 442.6
[M+2]+:444 [M+4]+ (9:3:1), (calculated for C19H7Cl3N3: 440.63).
General procedure for the synthesis of compounds 8 ꢀ 69
Amine (2.0 equiv.) was added to a stirred mixture of compound 6/7aꢀb (1.0 equiv.) and
triethylamine (2.0 equiv.) in THF (10 mL). The mixture was irradiated in microwave reactor at
80οC for 20 min., filtered hot, and allowed to cool. The solvent was removed in vacuo, and the
residue washed with minimum amount of iceꢀcold ethanol. The resulting solid was recrystallized
from acetone or ethanol to afford the target compound.
1ꢀ((2ꢀ(3ꢀHydroxypyrrolidinꢀ1ꢀyl)ethyl)amino)ꢀ3ꢀ(4ꢀ
(trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile 11:
Yellow solid (53%). Rf 0.3 (8% MeOHꢀDCM). mp 267–269 C. 1H NMR (600 MHz, DMSOꢀ
ο
d6) δ 8.39 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 9.1 Hz, 2H), 7.98 (d, J = 9.1 Hz, 2H), 7.87 (d, J = 8.1
Hz, 1H), 7.58 (t, J = 8.1 Hz, 1H), 7.39 (t, J = 8.1 Hz, 1H), 6.28 (s, 1H), 4.80 (br s, 1H), 4.27 (m,
1H), 3.65 (t, J = 6.2 Hz, 2H), 2.90 (m, 2H), 2.84 (m, 2H), 2.58 (m, 2H), 2.07 (m, 1H), 1.64 (m,
1H).13C NMR (150 MHz, DMSOꢀd6) δ 150.6, 149.3, 149.0, 145.4, 141.8, 130.3, 130.0, 128.3,
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126.4, 126.0, 125.4 (2C), 123.6 (2C), 121.2, 119.0, 117.6, 114.8, 90.7, 69.8, 62.8, 53.3, 52.5,
5
6
41.8,34.9. LCꢀMS: (ESI)+: found m/z =466.1 [M+H]+, (calculated for
C25H22F3N5O:
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465.48).HPLC purity 99% (tr = 4.09 min).
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1ꢀ((5ꢀ(Diethylamino)pentanꢀ2ꢀyl)amino)ꢀ3ꢀ(4ꢀ(trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2ꢀ
a]pyridineꢀ4ꢀcarbonitrile 17:
Yellow solid (50%). Rf 0.3 (10% MeOHꢀDCM). mp 220–222 οC. 1H NMR (400 MHz, DMSOꢀ
d6) δ 8.42 (d, J = 8.3 Hz, 1H), 7.96 (m, 4H), 7.85 (d, J = 8.0 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H),
7.39 (t, J = 7.3 Hz, 1H), 6.31 (s, 1H), 4.03 (m, 1H), 2.48 (m, 6H), 1.92 (m, 1H), 1.65 (m, 3H),
1.38 (d, J = 6.3 Hz, 3H), 0.91 (t, J = 7.0 Hz, 6H).13C NMR (100 MHz, DMSOꢀd6) δ 150.5,
149.6, 149.1(2C), 145.4, 142.0, 130.3, 130.0 (2C), 128.6, 126.3, 126.0 (2C), 123.1, 120.6, 118.8,
117.6, 115.8, 91.3, 52.1, 50.0, 46.6 (2C), 33.7, 23.5, 20.1 (2C), 11.5.LCꢀMS: (ESI)+: found m/z
=494.4 [M+H]+, (calculated for C28H30F3N5: 493.58).HPLC purity98% (tr = 3.69 min).
1ꢀ((NꢀMethylpiperidinꢀ4ꢀyl)amino)ꢀ3ꢀ(4ꢀ(trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2ꢀ
a]pyridineꢀ4ꢀcarbonitrile 20:
Yellow solid (51%). Rf 0.4 (10% MeOHꢀDCM). mp 215–217 ºC.1H NMR (400 MHz, DMSO)
δ 8.41 (d, J = 8.3 Hz, 1H), 7.96 (s, 4H), 7.85 (d, J = 8.1 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.40 (t,
J = 7.4 Hz, 1H), 6.34 (s, 1H), 3.83 (m, 1H), 2.84 (m, 2H), 2.24 (s, 3H), 2.17 (t, J = 10.6 Hz, 2H),
2.07 (m, 2H), 1.92 (t, J = 9.6 Hz, 2H).13C NMR (101 MHz, DMSO) δ 150.6, 149.6, 148.9, 145.4,
142.0, 141.9, 130.3, 130.2, 130.0, 128.7, 128.6, 126.4, 126.2, 126.1, 126.0, 120.7, 118.8, 117.7,
117.6, 116.1, 91.6, 54.4, 50.7, 46.3, 31.1. LCꢀMS: (ESI)+: found m/z =450.2 [M+H]+, (calculated
for C25H22F3N5: 449.48).HPLC purity 99% (tr = 4.03 min).
3ꢀ(3,4ꢀDichlorophenyl)ꢀ1ꢀ((2ꢀ(ethylamino)ethyl)amino)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀ
carbonitrile 45:
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Yellow solid (29%). Rf 0.1 (5% MeOH/DCM). mp 235–237 C. 1H NMR (400 MHz, DMSOꢀ
d6) δ 8.52 (d, J = 8.1 Hz, 1H), 7.96 (s, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H),
7.71 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 6.12 (s, 1H), 3.64 (t, J
= 6.2 Hz, 2H), 3.07 (t, J = 5.6 Hz, 2H), 2.80 (q, J = 8.0 Hz, 2H), 1.13 (t, J = 7.0 Hz, 3H).13C
NMR (100 MHz, DMSOꢀd6) δ 150.0, 148.5, 145.4, 138.9, 132.5, 131.8, 131.1, 130.9, 129.3,
129.0 (2C), 125.7, 120.3, 118.5, 118.3 (2C), 115.4, 90.5, 47.2, 43.1, 43.1, 14.3.LCꢀMS: (ESI)+:
found m/z =424.2[M+]:426.5[M+2]+(3:1), (calculated for C22H19Cl2N5: 424.33).HPLC purity
99% (tr = 3.53 min).
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9ꢀChloroꢀ1ꢀ((2ꢀ(ethylamino)ethyl)amino)ꢀ3ꢀ(4ꢀ
(trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile 57:
ο
Yellow solid (36%). Rf 0.2 (6% MeOHꢀDCM). mp 223–225 C. 1H NMR (400 MHz, DMSOꢀ
d6) δ 8.69 (d, J = 8.7 Hz, 1H), 7.90 (m, 4H), 7.44 (d, J = 7.4 Hz, 1H), 7.13 (t, J = 8.0 Hz, 1H),
5.83 (s, 1H), 3.58 (t, J = 6.2 Hz, 2H), 3.18 (t, J = 6.2 Hz, 2H), 2.94 (q, J = 7.2 Hz, 2H), 1.18 (t, J
= 7.2 Hz, 3H). 13C NMR (100 MHz, DMSOꢀd6) δ 152.2, 151.9, 148.6, 143.5, 142.5, 131.5,
129.7, 129.4, 126.0, 125.8, 125.7, 124.2, 123.3, 120.3, 120.1, 119.5, 115.4 (C2), 72.8, 48.0, 44.4,
43.0, 12.9.LCꢀMS: (ESI)+: found m/z =458.3[M+]:460.5[M+2]+(3:1), (calculated for
C23H19ClF3N5: 457.89).HPLC purity 98% (tr = 3.52 min).
7,8ꢀDichloroꢀ1ꢀ((2ꢀ(ethylamino)ethyl)amino)ꢀ3ꢀ(4ꢀ
(trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile 59:
ο
Yellow solid (47%). Rf 0.4 (8% MeOHꢀDCM). mp 266–268 C. 1H NMR (600 MHz, DMSOꢀ
d6) δ 8.92 (s, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.72 (s, 1H), 5.72 (s, 1H),
3.60 (t, J = 6.2 Hz, 2H), 3.23 (t, J = 6.2 Hz, 2H), 3.03 (q, J = 7.3 Hz, 2H), 1.24 (t, J = 7.3 Hz,
3H).13C NMR (150 MHz, DMSOꢀd6) δ 154.1, 152.4, 148.1, 145.5, 143.8, 130.2, 129.6 (2C),
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129.5, 129.3, 126.3, 125.6, 123.7, 120.3, 119.9, 117.6 (2C), 116.7, 91.6, 48.1, 44.7, 42.9,
12.2.LCꢀMS: (ESI)+: found m/z =492.2[M+]:494.4[M+2]+:496.5[M+4]+(9:3:1), (calculated for
C23H18Cl2F3N5: 492.33).HPLC purity 98% (tr = 4.29 min).
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7,9ꢀDichloroꢀ1ꢀ((2ꢀ(ethylamino)ethyl)amino)ꢀ3ꢀ(4ꢀ
(trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile 60:
Yellow solid (42%). Rf 0.3 (8% MeOHꢀDCM). mp 277–279 C. 1H NMR (600 MHz, DMSOꢀ
ο
d6) δ 8.81 (d, J = 2.0 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 2.0
Hz, 1H), 5.67 (s, 1H), 3.57 (t, J = 6.2 Hz, 2H), 3.26 (t, J = 6.2 Hz, 2H), 3.06 (q, J = 7.2 Hz, 2H),
1.24 (t, J = 7.2 Hz, 3H).13C NMR (150 MHz, DMSOꢀd6) δ 153.9, 152.9, 147.7, 144.0, 141.6,
132.2, 129.6 (2C), 129.2, 125.7, 125.6, 123.7 (2C), 123.1, 121.8, 120.8, 119.6, 115.6, 91.9, 48.1,
44.9, 42.7, 11.8.LCꢀMS: (ESI)+: found m/z =492.2[M+]:494.4[M+2]+:496.5[M+4]+(9:3:1),
(calculated for C23H18Cl2F3N5: 492.33).HPLC purity 99% (tr = 4.26 min).
6ꢀ((2ꢀ(Ethylamino)ethyl)amino)ꢀ8ꢀ(4ꢀ(trifluoromethyl)phenyl)imidazo[1,2ꢀa:4,5ꢀb']dipyridineꢀ
9ꢀcarbonitrile 64
:
Yellow solid (32%). Rf 0.3 (10% MeOHꢀDCM). mp 225–227 οC. 1H NMR (400 MHz, DMSOꢀ
d6) δ 8.41 (dd, J = 4.8, 1.4 Hz, 1H), 8.26 (dd, J = 8.2, 1.4 Hz, 1H), 7.95 (m, 4H), 7.64 (dd, J =
8.2, 4.8 Hz, 1H), 6.33 (s, 1H), 3.67 (t, J = 6.0 Hz, 2H), 2.94 (t, J = 6.0 Hz, 2H), 2.65 (q, J = 7.1
Hz, 2H), 1.08 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSOꢀd6) δ 152.3, 149.2, 149.0, 144.1,
141.9, 140.3, 137.8, 130.4, 130.0 (2C), 126.5, 126.0 (2C), 122.2 (3C), 117.3, 90.1, 47.4, 43.5,
42.7, 15.6. LCꢀMS: (ESI)+: found m/z =425.2[M+1]+, (calculated for
424.43).HPLC purity 96% (tr = 3.25 min).
C22H19F3N6:
2ꢀFluoroꢀ1ꢀ((2ꢀ(3ꢀhydroxypyrrolidinꢀ1ꢀyl)ethyl)amino)ꢀ3ꢀ(4ꢀ
(trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile 69:
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Yellow solid (44%). Rf 0.3 (5% MeOHꢀDCM). mp. 265–267 C.1H NMR (400 MHz, DMSOꢀ
d6) δ 8.54 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 8.0 Hz, 2H), 7.82 (d, J = 8.2
Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 4.30 (m, 1H), 3.87 (m, 2H), 2.98 (m,
2H), 2.92 (m, 2H), 2.69 (m, 2H), 2.10 (m, 1H), 1.70 (m, 1H). 13C NMR (100 MHz, DMSOꢀd6) δ
147.2, 145.6, 141.3, 141.0, 136.5, 133.0, 130.9, 130.1, 130.1, 126.1, 125.8, 125.8, 120.6, 118.7,
117.2, 117.1, 115.5, 69.8, 62.5, 55.4, 55.3, 52.4, 44.3, 44.2, 34.7. LCꢀMS: (ESI)+: found m/z =
484.2 [M+H]+, (calculated for C25H21F4N5O: 483.17). HPLC purity 99% (tr = 3.51 min).
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ASSOCIATED CONTENT
Supporting Information
Characterization of all intermediates as well as final compounds, details of assay procedures
and further descriptions of the biological experiments (cytotoxicity analysis, in vitro
determination of antiplasmodial activity, in vivo efficacy experiments, in vitro gametocytocidal
experiments, liver stage assays and killing kinetics), in vitro ADME assays (kinetic solubility
and artificial membrane permeability experiments), metabolic stability experiments and
determination of mechanism of action (in vitro βꢀhaematin, haem fractionation assays and
prediction of vacuolar accumulation) are available in the Supporting Information section at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding author
* Phone: +27ꢀ21ꢀ6502557. Fax: +27ꢀ21ꢀ6505195. Eꢀmail: Kelly.Chibale@uct.ac.za
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ACKNOWLEDGEMENT
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The University of Cape Town, South African Medical Research Council (SAMRC), 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.). The SAMRC is further acknowledged for support (LꢀMB, T.L.C, and T.J.E.).
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ABBREVIATIONS
SAR, structureꢀactivity relationships; ADME, absorption, distribution, metabolism and
excretion; CQ, chloroquine; p.o., oral administration; i.v., intravenous administration; MSD,
mean survival days; PK, pharmacokinetics; PBIs, pyrido[1,2ꢀa]benzimidazoles; BHIA, betaꢀ
haematin inhibition assay; LG, late gametocytes; EG, early gametocytes.
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