Antischistosomal Activity of Pyrido[1,2-a]benzimidazole Derivatives and Correlation with Inhibition of β-Hematin Formation

Article abstract of DOI:10.1021/acsinfecdis.6b00205

The extensive use of praziquantel against schistosomiasis raises concerns about drug resistance. New therapeutic alternatives targeting critical pathways within the parasite are therefore urgently needed. Hemozoin formation in Schistosoma presents one such target. We assessed the in vitro antischistosomal activity of pyrido[1,2-a]benzimidazoles (PBIs) and investigated correlations with their ability to inhibit β-hematin formation. We further evaluated the in vivo efficacy of representative compounds in experimental mice and conducted pharmacokinetic analysis on the most potent. At 10 μM, 48/57 compounds resulted in >70% mortality of newly transformed schistosomula, whereas 37 of these maintained >60% mortality of adult S. mansoni. No correlations were observed between β-hematin inhibitory and antischistosomal activities against both larval and adult parasites, suggesting possible presence of other target(s) or a mode of inhibition of crystal formation that is not adequately modeled by the assay. The most active compound in vivo showed 58.7 and 61.3% total and female worm burden reduction, respectively. Pharmacokinetic analysis suggested solubility-limited absorption and high hepatic clearance as possible contributors to the modest efficacy despite good in vitro activity. The PBIs evaluated in this report thus merit further optimization to improve their efficacy and to elucidate their possible mode of action.

Full text of DOI:10.1021/acsinfecdis.6b00205

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Article
Antischistosomal Activity of Pyrido[1,2-a]benzimidazole
Derivatives and Correlation with Inhibition of #-haematin Formation
John Okombo, Kawaljit Singh, Godfrey Mayoka, Ferdinand Ndubi, Linley
Barnard, Peter M. Njogu, Mathew Njoroge, Liezl Gibhard, Christel Brunschwig,
Mireille Vargas, Jennifer Keiser, Timothy J Egan, and Kelly Chibale
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.6b00205 • Publication Date (Web): 25 Apr 2017
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Antischistosomal Activity of Pyrido[1,2ꢀa]benzimidazole Derivatives and
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Correlation with Inhibition of βꢀhaematin Formation
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John Okombo1, Kawaljit Singh1, Godfrey Mayoka1, Ferdinand Ndubi1, Linley Barnard1, Peter M.
Njogu2, Mathew Njoroge3, Liezl Gibhard3, Christel Brunschwig3 Mireille Vargas4,5, Jennifer Keiser4,5,
Timothy J. Egan1 and Kelly Chibale1,6,7
*
1 Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
2 Department of Pharmaceutical Chemistry, University of Nairobi, P.O. Box 19676, Nairobi,
00202, Kenya
3Drug Discovery and Development Centre (H3D), Division of Clinical Pharmacology,
Department of Medicine, University of Cape Town, Observatory, 7925, South Africa
4Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public
Health Institute, Basel, Switzerland
5University of Basel, Basel, Switzerland
6South African Medical Research Council, Drug Discovery and Development Research Unit,
University of Cape Town, Rondebosch 7701, South Africa
7Institute of Infectious Disease and Molecular Medicine, University of Cape Town,
Rondebosch 7701, South Africa
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ABSTRACT
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The extensive use of praziquantel against schistosomiasis raises concerns about drug resistance. New
therapeutic alternatives targeting critical pathways within the parasite are therefore urgently needed.
Haemozoin formation in Schistosoma presents one such target. We assessed in vitro antischistosomal
activity of pyrido[1,2ꢀa]benzimidazoles (PBIs), and investigated correlations with their ability to
inhibit βꢀhaematin formation. We further evaluated the in vivo efficacy of representative compounds
in experimental mice and conducted pharmacokinetic analysis on the most potent. At 10 µM, 48/57
compounds resulted in >70% mortality of newly transformed schistosomula, while 37 of these
maintained > 60% mortality of adult S. mansoni. No correlations were observed between βꢀhaematin
inhibitory and antischistosomal activities against both larval and adult parasites, suggesting possible
presence of other target(s) or a mode of inhibition of crystal formation that is not adequately modelled
by the assay. The most active compound in vivo showed 58.7% and 61.3% total and female worm
burden reduction, respectively, and further pharmacokinetic analysis hinted at solubilityꢀlimited
absorption and high hepatic clearance as possible contributors to the modest efficacy despite good in
vitro activity. The PBIs evaluated in this report thus merit further optimization to improve their
efficacy and to elucidate their possible mode of action.
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Keywords
Schistosoma mansoni, Pyrido[1,2ꢀa]benzimidazoles, Praziquantel, Pharmacokinetics, Haemozoin,
Betaꢀhaematin
Schistosomiasis is a major public health concern, with ~700 million people at risk in 74 countries and
over 200 million infected.1 One of the three main etiological agents of this infection is the parasitic
digenetic trematode Schistosoma mansoni, known to digest large amounts of host blood, in its adult
stage, for nutritional supplementation and completion of its sexual development.2 This process is
characterised by multiꢀenzymatic degradation of hostꢀderived haemoglobin,3 leading to formation of
peptides, essential amino acids and haem.4
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Haem is an amphiphilic molecule that serves as the prosthetic group of numerous haemoproteins and
is essential in such biological processes as control of protein synthesis, cell differentiation and
respiration as well as drug detoxification.5 In its free state, however, haem acts as a proꢀoxidant able
to induce free radical formation 6 as well as compromise phospholipid membrane integrity.7 Bloodꢀ
feeding parasites, such as S. mansoni and Plasmodium falciparum, have consequently evolved various
detoxification mechanisms to rid themselves of the toxicity of free haem, one of the most efficient of
which involves biocrystallization of haem molecules into inert and insoluble haemozoin (Hz).8ꢀ10
Indeed, in S. mansoni, haem crystallization as a preventive antioxidant defence has been documented
11 and evidence of large amounts of Hz within the gut of adult female worms has been reported.12
Furthermore, earlier findings where treatment of Schistosomaꢀinfected mice with chloroquine (CQ)
inhibited Hz formation and significantly reduced both worm burden and egg deposition in the mouse
liver 13 hint at Hz formation as the main haem detoxification pathway. As in P. falciparum, Hz
formation therefore presents a vulnerable druggable target in S. mansoni since it is exclusive to the
parasite, indispensable for its survival and not genomeꢀencoded (hence immutable). This concept has
been extensively explored against P. falciparum through classical aminoquinoline compounds known
to interact with free haem molecules, consequently impeding their crystallization into Hz.14
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While literature is scant on the process of Hz formation in S. mansoni, a recent study reported
fundamental differences to that in P. falciparum, in particular with respect to the extracellular
localization of the process and structural organization of the subsequent Hz crystals.15 In spite of these
contrasts, the resulting Hz crystals themselves are structurally identical and therefore it would not be
unreasonable to postulate similar inhibition of crystal formation in the two organisms. In malaria
parasites, this is thought to involve docking of drugs or of the haemꢀdrug complexes onto a growing
crystal face through πꢀπ stacking and specific weak hydrogen bonding thus blocking its growth 16 and
allowing for interaction of the unbound monomeric/dimeric haem molecules with membranes leading
to severe toxicity, ultimately halting parasite proliferation through oxidative stress.17 However, Xue et
al. recently showed that though the canonical Hz inhibitors CQ, mefloquine (MQ) and quinine (QN)
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exhibited excellent antischistosomal activity, this did not correlate with their Hz inhibition
potencies,18 possibly suggesting a unique mechanism in S. mansoni of Hz inhibition dissimilar to that
in Plasmodium or differences in haemꢀdrug complex formation in the two parasite species.
Praziquantel (PZQ), which constitutes the current mainstay of schistosomiasis control and therapy, is
thought to act through disruption of Ca2+ ion homeostasis by modification of membrane fluidity and
composition thus altering its permeability to ions.19 Though safe, wellꢀtolerated and effective in a
single oral dose against the adult stages of all forms of schistosomiasis, the lack of more therapeutic
alternatives to PZQ presents a genuine concern and warrants the search for more potent and novel
antischistosomal drugs. Towards this aim, organometallic complexes, natural products and a number
of repositioned antimalarial agents have variously been explored as antischistosomal agents and
recently reviewed.20ꢀ22
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In this report, we sought to examine the in vitro and in vivo antischistosomal activity of pyrido[1,2ꢀ
a]benzimidazoles (PBIs), a chemotype that has previously been investigated for antibacterial,
antifungal, antiviral and antitumor activity.23 The antimalarial efficacy of PBIs was first reported by
our group in 2011,24 and recently confirmed with a set of derivatives exhibiting a more diverse range
in scaffold substitutions.25 Though the exact mechanism of action of PBIs remains unknown,
inhibition of βꢀ1, 6ꢀglucan synthase in fungi,26 the lytic activity of poreꢀforming perforin,27 and
cytochrome b in trypanosomes,28 by various PBIs has been reported. Earlier, we hypothesised that
PBIs can potentially inhibit formation of βꢀhaematin/Hz due to their planar heterocyclic architecture,
which would make them candidates for πꢀπ interactions, a hallmark feature of haemꢀdrug complex
formation in inhibition of Hz crystal growth. This hypothesis was corroborated by our recent data in
support of inhibition of Hz formation as a contributing mechanism to the antimalarial effect of PBIs.25
In the current study, we present the in vitro and in vivo activity of 57 PBIs. While some members of
the library have already been reported in a previous paper,25 a hitherto unexplored new set of 3ꢀ
trifluoromethyl pyrido[1, 2ꢀa]benzimidazole derivatives was synthesised in order to increase the
number and diversity of derivatives for structureꢀactivity relationship (SAR) studies (Scheme 1).
Furthermore, we have examined the metabolic profiles of the compounds in liver microsomes and
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present pharmacokinetic analysis of the most active analogues in the in vivo set up. Finally, as a
mechanistic evaluation, we report analyses on the correlations between the antischistosomal activities
of these compounds and their ability to inhibit formation of βꢀhaematin (βH) in a pyridineꢀbased
detergentꢀmediated assay.
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Chemistry: The synthesis of target pyrido[1,2ꢀa]benzimidazole derivatives a is shown in Scheme 1
O
O
O
O
a
EtO
R
EtO
Me
R = Me
R1
2a (R1 = PhCH2)
1a (R = Me)
1b (R = CF3)
2b (R1 = 3ꢀClPhCH2)
2c (R1 = 4ꢀClPhCH2)
2d (R1 = 2ꢀClPhCH2)
X
X
OH
O
O
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N
b
c
d
e
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S
S
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NH
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NH2
N
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O2N
O2N
H2N
4
5
6a: X = NMe2
7a: X = NMe2
3
N
N
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7b: X =
R2
OH
H
Cl
CN
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R
R1
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g
R1
R
f
h
N
N
N
N
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N
N
N
CN
9
CN
CN
10
8
48ꢀ67
a: R1 = PhCH2; R = CH3
b: R1 = 3ꢀClPhCH2; R = CH3
c: R1 = 4ꢀClPhCH2; R = CH3
d: R1 = 2ꢀClPhCH2; R = CH3
e: R1 = H; R = CF3
a Reagents and reaction conditions: (a) appropriate benzyl halide, tBuOK, tBuOH, THF, 70°C, 8ꢀ24 h;
(b) CS2, KOH, EtOH, 80°C, 48 h; (c) MeI, CH3CN, 25°C, 2 h; (d) XCH2CH2NH2, CH3CN, MW,
120°C, 20ꢀ30 min; (e) SnCl2, HCl, THF, 60°C, 24 h; (f) NH4OAc, 150οC, 1 h; (g) POCl3, 130οC, 2 h;
(h) 7a – b or amine, Et3N, THF, 80οC, MW, 20ꢀ40 min (48 ꢀ 59, 61 ꢀ 62) and Pd2(dba)3, BINAP,
K2CO3, Toluene, 120˚C, 12ꢀ16 h (60, 63 ꢀ 67).
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Compound 2a – d were synthesised by reacting methyl acetoacetate with corresponding benzyl
bromide.29 Benzoxazole analogues 7a – b were obtained from 2ꢀaminoꢀ4ꢀnitro phenol using literature
a method,30 while the target PBI derivatives 48 ꢀ 67 were synthesised as previously described.25
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RESULTS AND DISCUSSION
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Effect of Pyrido[1,2-a]benzimidazoles (PBIs) on Newly Transformed Schistosomula (NTS) and
Adult S. mansoni
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Of the 57 compounds screened against newly transformed schistosomula (NTS) at an initial
concentration of 10 µM, 48 showed >70% inhibition of worm viability ꢀ a threshold that was selected
for subsequent investigation against adult S. mansoni at the same drug concentration where 37 of
them caused >60% worm mortality (Supporting Information Table S1 and S2). IC50 determination
against adult worms was also carried out for this set of compounds (exhibiting >60% worm mortality)
and ranged between 0.21 ꢀ 39.4 ꢁM while that of PZQ was 0.1 µM (Table 1 and Supplementary
Information Table S1). Compounds 14, 30, 36, 38 and 41 were the most potent (IC50 range: 1.20 ꢀ
2.08 ꢁM) against the adult worms, which could suggest that the presence of chloroꢀ group(s) on either
side of the ring system, as seen in four of these five compounds, is favoured for activity. Interestingly,
14, which bears the same side chain as CQ, was one of the most potent against adult worms (IC50:
1.20 ꢁM). Among the anilineꢀtype series, with no modifications except on the aromatic side chain, the
cyanoꢀ (56) and sulfonylmethylꢀsubstituted (60) derivatives were the most active (IC50 = 0.21 µM and
0.97 µM, respectively). Additionally, replacement with heteroaromatic side chains also seemed to
preserve antischistosomal activity as exemplified by 63, 65, 66 and 67 (IC50 range 0.4 – 3.14 µM).
Notably, derivatives based on piperidine (15, 17, 19, 20) and sulphonamide substitution (24) in place
of the ethylamine group were not active against adult S. mansoni (< 60% at 10 µM), implying these
modifications are not favoured for antischistosomal activity (Supporting Information Table 1 and 2).
Basicity of the nitrogen linker in the aniline series appeared necessary for activity as potency was lost
with the Nꢀmethylated 62 (26.1% NTS mortality) compared to the nonꢀmethylated analogue 57
(95.7% NTS mortality). Some compounds (19, 20, 24, 47, 50 and 55) seemed very potent against the
juvenile parasites (73.9 – 100% NTS mortality range) but activity significantly diminished (0 ꢀ 42%)
against adult S. mansoni parasites (Supporting Information Table S2).
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Incubation of the compounds with Chinese hamster ovarian (CHO) cells helped ascertain their
potential in vitro cytotoxicity. Activity against S. mansoni was greater than in CHO cells, with the
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anilineꢀbased analogues exhibiting better selectivity in general. Emetine, a potent inhibitor of protein
synthesis in eukaryotic cells was used as control and as expected, exhibited cellular toxicity at
extremely low concentrations (Table 1and Supplementary Information Table S2)
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Beta-haematin Inhibition Activity (BHIA) and Correlation with Antischistosomal Activity
In this assay an IC50 cutꢀoff of < 100 µM was invoked to discriminate active from inactive
compounds, and 27/57 compounds were classified as active against formation of βH. There was no
clear trend in activity that could be correlated to the substituents on the PBI core. Thus the abilities of
these compounds to inhibit Hz formation appear to depend on the presence of a planar ring system,
aliphatic hydrophobicity and the availability of a protonation site. However, the anilineꢀsubstituted
derivatives were generally poor βH inhibitors (Table 1 and Supplementary Information Table S2).
We further attempted to correlate in vitro βH inhibition activity with antischistosomal activity and
observed no associations in either the NTS or the adult S. mansoni (Figure 1). The lack of association
in the juvenile worms is likely reflective of the observation that blood ingestion and consequent
detoxification of haemoglobin degradation products predominantly occurs in the mature S. mansoni
worms.2 The absence of correlation in the adult worms seems driven, at least in part, by the inordinate
βH inhibition IC50s of the anilineꢀsubstituted derivatives (Table 1 and Supplementary Information
Table S2). Expectedly, a winnowed analysis excluding these analogues (n = 9) yields a weak but
significant positive correlation between βH inhibition and activity against adult worms (R2 = 0.1388; p
< 0.05; n = 29). This weak correlation implies that inhibition of Hz formation cannot be discounted as
a contributing mechanism in the activity of these compounds, and that there could be additional (and
possibly more dominant) mechanism(s) involved in the antischistosomal activity of these PBIs, or
factors influencing the localisation of these compounds in the worm gut. Also, the lack of association
is consistent with recent findings involving the antimalarials CQ, MQ and QN, which reported a lack
of correlation between antischistosomal activity and their Hz inhibition properties,18 with MQ in fact
demonstrated to interfere with glycolysis in NTS.31 It is noteworthy that unlike in P. falciparum, Hz
formation proceeds extracellularly in S. mansoni and is thought to be possibly initiated by, or involve
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proteins, lipids and other hydrophobic components in the gut of the parasite.15 It is therefore
conceivable that these PBIs inhibit Hz formation in Schistosoma in a manner dissimilar to
Plasmodium by interacting with any of these targets, resulting in the perturbation of the crystal growth
microenvironment. It is instructive to note that the βH inhibition assay discussed in this report
constitutes an in vitro cellꢀfree setup that can only capture to a limited extent the physiological
landscape involved in intracellular drug activity. Therefore, inꢀorganism mechanistic investigations
that take into account the localization of Hz formation in lipidꢀlike droplet particles 15 would be more
informative. Indeed, using a pyridineꢀlabelled haem fractionation assay that spectroscopically
determines the amounts of haemoglobin, “free haem” (which can be associated with neutral lipids)
and Hz present in trophozoites treated with increasing doses of a drug, we recently showed that
inhibition of Hz formation could be a contributing mechanism in the activity of these compounds,
albeit in P. falciparum.25 Though no similar assay presently exists for S. mansoni, such a model would
be invaluable for investigating drug action in this parasite.
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In vivo Activity against adult S. mansoni
We further examined the efficacy, in experimental mice, of 10 compounds (11, 28, 30, 38, 39, 56, 60,
63, 66 and 67) which had shown the best in vitro activity against NTS and adult S. mansoni (IC50
range: 0.21 – 2.44 ꢁM), moderate to excellent metabolic stability and favourable cytotoxicity profiles.
Treatment of mice with 400 mg/kg of 11 failed to induce any in vivo activity (Table 2) as there was
no observed reduction in total worm burden, and one of the mice died 10 days postꢀtreatment.
Compounds 30, 38, 39, 60, 63, 66 and 67 only showed modest activity with < 50% reduction in both
total and female worm burden. Significant reduction in worm burden was, however, observed
following treatment with 28 and 56, and although comparatively lower than parasite burden reduction
reported for PZQ,32 these were superior to previously reported ferroquineꢀderivatives,33 repurposed
anticancer drugs,34 PZQ ꢀ endoperoxide conjugates 35 and some Malaria Box Medicine for Malaria
Venture (MMV) compounds.36 There was a discernible trend of increasing worm burden reduction
with compound metabolic stability in mouse liver microsomes (MLMs) and in vitro potency in adult
worms. This suggests that poor metabolic stability most likely contributed to the low in vivo efficacy
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of 63 and 66 while the role of solubility, which would result in low systemic exposure due to limited
absorption, could explain the average in vivo activities observed with 56 and 60 despite their excellent
in vitro potencies and stability in MLMs.
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Pharmacokinetic Analysis in mouse
To obtain more insight on the efficacy of 28, we investigated its pharmacokinetic (PK) properties by
dosing the compound orally (20 mg/kg) and intravenously (2 mg/kg) in mice. Blood samples were
collected at various time points over 24 hours and the compound concentrations measured by LCꢀ
MS/MS. The PK parameters derived from nonꢀcompartmental analysis of the data are summarized in
Table 3 and the PK profiles shown in Figure 2.
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Compound 28 was slowly and incompletely absorbed, with large interꢀindividual variations in oral
bioavailability. This observation is likely due to poor solubility of the compound and suggests a
likelihood of solubilityꢀlimited absorption. This would result in plateauing of the Cmax and
consequently efficacy despite an increase in dose, and has previously been observed with this series.24
The oral halfꢀlife of this compound was also lower than that of 11, 38 and 39 reported in our previous
analyses24ꢀ25 and is, at least in part, due to a relatively higher clearance. This is further supported by
the observation of the Nꢀdeethylation metabolite in blood for this compound, but not for 11, 38 or
39,24ꢀ25 suggesting a higher in vivo hepatic clearance. The in vivo efficacy of 28 is therefore tied to its
low exposure on oral dosing, due to solubilityꢀlimited absorption, and a relatively short halfꢀlife due
to high hepatic clearance.
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In summary, our results reveal an antischistosomal property of PBIs that further complements the
broad antimicrobial potency of this class of compounds. Though their activity in S. mansoniꢀinfected
mice was generally low, encouraging efficacy was noted in two of the compounds suggesting further
lead optimization efforts can afford derivatives with improved in vivo activity. In addition, our
preliminary data suggests that inhibition of Hz formation cannot be discounted as a contributing
mechanism through which these compounds exert their antischistosomal effect, especially in the adult
parasites. Evaluation of more PBI derivatives is needed in order to delineate meaningful SARs.
Furthermore, direct correlation of antiparasitic activity and inhibition of Hz formation in the worm
needs to be established as do any other additional mechanisms involved.
Experimental Procedures
General Method. All commercially available chemicals were purchased from either SigmaꢀAldrich
or Combi Blocks. All solvents were dried by appropriate techniques. Unless otherwise stated, all
solvents used were anhydrous. Purification of compounds was carried out by either column
chromatography on silica gel 60 (Fluka), particle size = 0.063−0.2 mm (70−230 mesh), as the
1
stationary phase. All target compounds and intermediates were characterized by H NMR and LCꢀ
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MS. NMR spectra were recorded on either a Varian Mercuryꢀ300 (1H 300.1 MHz, 13C 75.5 MHz) or a
Brukerꢀ400 (1H 400.2 MHz, 13C 100.6 MHz) instrument using CDCl3, CD3OD, and DMSOꢀd6 as
solvents. Liquid chromatograph with mass spectrometer (LCꢀMS) analysis was performed using an
Agilent 1260 Infinity binary pump, an Agilent 1260 Infinity diode array detector (DAD), an Agilent
1290 Infinity column compartment, an Agilent 1260 Infinity standard autosampler, and an Agilent
6120 quadrupole (single) mass spectrometer, equipped with APCI and ESI multimode ionization
source. Purities were determined by Agilent LCꢀMS using a Kinetex Core C18 2.6 ꢁm column (50
mm × 3 mm). Mobile phase B consisted of 0.4% acetic acid, 10 mM ammonium acetate in a 9:1 ratio
of HPLC grade methanol, and type 1 water. Mobile phase A consisted of 0.4% acetic acid in 10 mM
ammonium acetate in HPLC grade (type 1) water, with flow rate = 0.9 mL/min; detection was by
diode array (DAD), and all final compounds were confirmed to have ≥95% purity.
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General procedure for the synthesis of compound 48ꢀ55, 57ꢀ59, 61ꢀ62 and 64
Respective amine (2.0 equiv.) [Nꢀethyl ethylenediamine (48ꢀ51), 7a (52), 7b (53), aniline
(54), 4ꢀmethylaniline (55), 4ꢀmethoxyaniline (57), N,Nꢀdimethylbenzeneꢀ1,4ꢀdiamine (58), 4ꢀ
aminoꢀNꢀmethylbenzamide (59), 4ꢀ(morpholinomethyl)aniline (61), 4ꢀmethoxyꢀNꢀ
methylaniline (62), pyrimidinꢀ2ꢀamine (64)] was added to a stirred mixture of compound 10
(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.
2ꢀbenzylꢀ1ꢀ((2ꢀ(ethylamino)ethyl)amino)ꢀ3ꢀmethylbenzo[4,5]imidazoꢀ[1,2
ꢀa]pyridineꢀ4
carbonitrile (48). Yield: 0.0023 g, 7%, as an orange solid.
1H NMR (400 MHz, DMSOꢀd6) δ
8.28 (dt, J = 8.4, 1.0 Hz, 1H), 7.83 (dt, J = 8.2, 1.0 Hz, 1H), 7.53 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H),
7.33 (m, 3H), 7.20 (m, 3H), 4.27 (s, 2H), 3.21 (t, J = 5.9 Hz, 2H), 2.66 (t, J = 5.9 Hz, 2H), 2.49 (s,
3H), 2.45 (q, J = 7.1 Hz, 2H), 0.97 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ
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151.0, 149.0, 148.3, 145.1, 139.7, 129.3, 129.1 (3C), 128.1, 126.7, 126.0, 120.8, 119.0, 116.9, 116.5,
4
5
6
110.3, 55.3, 48.9, 47.1, 43.4, 32.0, 19.8, 15.6. MS (EI+) m/z calculated for C24H25N5: 383.21; found,
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384.2 (M+1). HPLC purity: 99%.
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2ꢀ(4ꢀchlorobenzyl)ꢀ1ꢀ((2ꢀ(ethylamino)ethyl)amino)ꢀ3ꢀmethylbenzoꢀ[4,5]imidazoꢀ[1,2ꢀ
1
a]pyridineꢀ4ꢀcarbonitrile (50). Yield: 0.137 g, 20 %, as a yellow solid. H NMR (400 MHz,
DMSOꢀd6) δ 8.30 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.33
(m, 3H), 7.20 (d, J = 8.4 Hz, 2H), 4.26 (s, 2H), 3.20 (m, 2H), 2.67 (t, J = 5.9 Hz, 2H), 2.45
13
(m, 5H), 0.97 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, DMSOꢀd6) δ 155.9, 150.8, 149.6,
148.1, 138.6, 131.6, 131.2, 129.8, 129.1, 127.3, 122.2, 120.9, 119.1, 115.8, 115.7, 102.4,
101.0, 89.6, 48.7, 45.9, 44.1, 31.2, 18.7, 13.7. MS (EI+) m/z calculated for C24H24ClN5:
417.17; found, 418.1 (M+1). HPLC purity: 99%.
1ꢀ((2ꢀ((2ꢀ(dimethylamino)ethyl)amino)benzo[d]oxazolꢀ6ꢀyl)amino)ꢀ3ꢀ
(trifluoromethyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile (52). Yield: 0.07 g, 23%, as
a yellow solid. 1H NMR (400 MHz, DMSOꢀd6) δ 8.81 (d, J = 8.1 Hz, 1H), 8.03 (br t, J = 5.6
Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.35 (m, 2H), 7.13 (t, J = 7.1 Hz, 1H), 6.87 (d, J = 2.0 Hz,
1H), 6.62 (dd, J = 8.3, 2.1 Hz, 1H), 5.78 (s, 1H), 3.66 (q, J = 10.4 Hz, 2H), 3.29 (t, J = 7.2
13
Hz, 2H), 2.82 (s, 6H). C NMR (100 MHz, DMSOꢀd6) δ 162.9 (2C), 151.7, 150.6, 147.3,
144.5, 144.2 (2C), 130.8 (2C), 124.6, 122.4, 119.3, 118.5, 117.0, 116.0, 114.9, 109.5, 109.4,
89.2, 56.4, 43.3, 38.3 (2C). MS (EI+) m/z calculated for C24H20F3N7O: 479.47; found, 480.2
(M+1). HPLC purity: 99%.
1ꢀ((2ꢀ((2ꢀ(Pyrrolidinꢀ1ꢀyl)ethyl)amino)benzo[d]oxazolꢀ6ꢀyl)amino)ꢀ3ꢀ
(trifluoromethyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile (53). Yield: 0.13 g, 14%, as
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a yellow solid. 1H NMR (400 MHz, DMSOꢀd6) δ 8.81 (d, J = 7.7 Hz, 1H), 8.03 (br t, J = 5.0
Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.38 (m, 2H), 7.17 (t, J = 8.3 Hz, 1H), 6.90 (d, J = 2.0 Hz,
1H), 6.65 (dd, J = 8.3, 2.1 Hz, 1H), 5.84 (s, 1H), 3.68 (q, J = 10.4 Hz, 2H), 3.42 (t, J = 5.9
Hz, 2H), 3.30 (br s, 4H), 1.95 (br s, 4H). 13C NMR (100 MHz, DMSOꢀd6) δ 162.9 (2C),
151.3, 150.4, 146.4, 144.7, 144.3, 134.5, 130.5, 125.1, 124.9, 122.2, 119.7, 118.1, 117.1,
115.8, 115.0, 109.6, 109.5, 89.8, 55.3 (2C), 53.9, 53.4, 23.0 (2C). MS (EI+) m/z calculated
for C26H22F3N7O: 505.51; found, 506.2 (M+1). HPLC purity: 99%.
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1ꢀ(phenylamino)ꢀ3ꢀ(trifluoromethyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile
(54).
Yield: 0.08 g, 23%, as a yellow powder. 1H NMR (300 MHz, DMSOꢀd6) δ 8.80 (d, J = 8.2
Hz, 1H), 7.70 (d, J = 7.0 Hz, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.45 (m, 3H), 7.15(m, 3H), 6.22
(s, 1H). 13C NMR (101 MHz, DMSOꢀd6) δ 148.5, 148.4, 148.2, 130.1 (4C), 128.7, 127.1
(2C), 124.5, 124.0, 122.7 (2C), 122.5, 117.5 (2C), 115.0, 114.4. MS (EI+) m/z calculated for
C19H11F3N4: 352.09; found, 353.1 (M+1). HPLC Purity: 99%.
General method for the synthesis of 56, 60, 63 and 65ꢀ67
Compound 10 (1.0 equiv.), the appropriate amine (1.2 equiv.) [4ꢀcyanoaniline (56), 4ꢀ
(methylsulfonyl)aniline (60), 2ꢀfluoropyridinꢀ4ꢀamine (63), 5,6ꢀdimethoxypyrimidinꢀ4ꢀamine
(65), pyrazinꢀ2ꢀamine (66), 4ꢀmethylthiazolꢀ2ꢀamine (67)], Pd2(dba)3 (0.03 equiv.), BINAP
(0.06 equiv.) and K2CO3 (20 equiv.) in toluene (5ml) were heated in a sealed tube at 120˚C
for 4ꢀ17 hours. The cooled reaction mixture was stirred in ethyl acetate (50ml) and water
(100ml) for 30 minutes. The separated organic layer was washed with water (2 x 50ml),
followed by saturated NaCl (2 × 50ml), dried over magnesium sulphate and filtered over
celite. The organic fraction was concentrated in vacuo and ethanol used to precipitate the
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crude product which was filtered to furnish the final product. Occasionally, purification by
recrystallization from ethanol was done.
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1ꢀ((4ꢀcyanophenyl)amino)ꢀ3ꢀ(trifluoromethyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀ
carbonitrile (56). Yield: 0.102g, 27%, as a pale yellow powder. 1H NMR (300 MHz, DMSOꢀ
d6) δ 8.80 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 7.3 Hz, 1H), 7.59 (m,
1H), 7.41 (m, 1H), 7.21 (d, J = 8.5 Hz, 2H), 6.22 (s, 1H). 13C NMR (101 MHz, DMSOꢀd6) δ
148.3, 148.1, 135.3, 135.0, 134.4 (3C), 128.6, 127.4 (2C), 123.1 (2C), 122.9 (2C), 119.8,
117.9 (2C), 115.2, 105.5, 96.6. MS (EI+) m/z calculated for C20H10F3N5: 377.09; found,
378.1 (M+1). HPLC Purity: 99%.
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1ꢀ((4ꢀ(methylsulfonyl) phenyl) amino)ꢀ3ꢀ (trifluoromethyl) benzo[4,5]imidazo [1,2ꢀ
a]pyridineꢀ4ꢀcarbonitrile (60). Yield: 0.175 g, 40%, as a yellow powder. 1H NMR (300 MHz,
DMSOꢀd6) δ 8.79 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 7.9 Hz, 1H), 7.46
13
(m, 1H), 7.25 (m, 3H), 6.00 (s, 1H), 3.21 (s, 3H). C NMR (101 MHz, DMSOꢀd6) δ 155.1,
149.3, 133.8, 132.8, 130.0, 129.6, 129.0 (2C), 125.5, 122.7 (2C), 121.4, 120.4, 117.2 (2C),
115.0, 106.7, 91.4, 86.0, 44.0. MS (EI+) m/z calculated for C20H13F3N4O2S: 430.07; found,
431.0 (M+1). HPLC Purity: 99%.
1ꢀ((2ꢀfluoropyridinꢀ4ꢀyl)amino)ꢀ3ꢀ(trifluoromethyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀ
1
carbonitrile (63). Yield: 0.021g, 6% as a yellow powder. H NMR (400 MHz, DMSOꢀd6) δ
8.78 (d, J = 8.3 Hz, 1H), 8.13 (d, J = 5.4 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.57 (t, J = 7.6
Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 6.98 (dt, J = 5.4, 1.7 Hz, 1H), 6.72 (s, 1H), 6.23 (s, 1H). 13C
NMR (101 MHz, DMSOꢀd6) δ 166.3, 164.0, 161.0, 148.6 (2C), 135.8, 128.7, 127.2, 124.1,
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122.7, 121.4, 117.9, 116.1, 115.3, 113.3, 101.7, 101.3, 95.7. MS (EI+) m/z calculated for
4
5
6
C18H9F4N5: 371.08; found, 372.1 (M+1). HPLC Purity: 99%.
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9
1ꢀ((5,6ꢀdimethoxypyrimidinꢀ4ꢀyl)amino)ꢀ3ꢀ(trifluoromethyl)
benzo[4,5]
imidazo[1,2ꢀ
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1
a]pyridineꢀ4ꢀcarbonitrile (65). Yield: 0.073g, 17%, as a yellow fluffy powder. H NMR (300
MHz, DMSOꢀd6) δ 9.08 (d, J = 8.58 Hz, 1H), 8.36 (s, 1H), 7.69 (d, J = 8.7 Hz, 1H), 7.63 (m,
1H), 7.47 (m, 1H), 7.40 (s, 1H), 4.00 (s, 3H), 3.80 (s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ
163.2, 162.7, 151.3, 147.5, 141.4, 135.4, 135.0, 128.7, 127.7(2C), 124.1, 123.1 (2C), 121.4,
118.5, 114.9, 100.0, 60.6, 54.0. MS (EI+) m/z calculated for C19H13F3N6O2: 414.11; found,
415.1 (M+1). HPLC Purity: 98%.
1ꢀ(pyrazinꢀ2ꢀylamino)ꢀ3ꢀ(trifluoromethyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀcarbonitrile
1
(66). Yield: 0.041g, 23%, as an orange powder. H NMR (400 MHz, DMSOꢀd6) δ 9.08 (d, J
= 8.4 Hz, 1H), 8.54 (d, J = 1.4 Hz, 1H), 8.41 (dd, J = 2.7, 1.4 Hz, 1H), 8.25 (d, J = 2.7 Hz,
1H), 7.69 (ddd, J = 8.1, 1.2, 0.7 Hz, 1H), 7.63 (ddd, J = 8.2, 7.3, 1.1 Hz, 1H), 7.57 (s, 1H),
7.47 (ddd, J = 8.5, 7.3, 1.3 Hz, 1H). 13C NMR (101 MHz, DMSOꢀd6) δ 161.2, 156.4, 149.8,
147.9, 147.3, 142.5 (2C), 138.5, 135.5, 128.8, 127.5, 123.1, 118.9, 118.5, 114.8, 112.8, 99.3.
MS (EI+) m/z calculated for C17H9F3N6: 354.08; found, 355.1 (M+1). HPLC Purity: 98%.
1ꢀ((4ꢀmethylthiazolꢀ2ꢀyl)amino)ꢀ3ꢀ(trifluoromethyl)benzo[4,5]imidazo[1,2ꢀa]pyridineꢀ4ꢀ
carbonitrile (67). Yield: 0.138g, 37% as a brown powder. 1H NMR (400 MHz, DMSOꢀd6) δ
9.04 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.59 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.41
(ddd, J = 8.4, 7.2, 1.2 Hz, 1H), 6.97 (s, 1H), 6.84 (d, J = 1.2 Hz, 1H), 2.33 (d, J = 1.2 Hz,
3H). 13C NMR (101 MHz, DMSOꢀd6) δ 150.9, 148.0, 140.9, 137.5, 136.7, 136.3, 131.5,
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129.8, 126.7, 121.8, 118.9, 118.1, 117.8, 114.9, 104.8, 100.0, 14.6. MS (EI+) m/z calculated
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5
6
for C17H10F3N5S: 373.06; found, 374.1 (M+1). HPLC Purity: 99%.
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MATERIALS AND METHODS
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Compounds
For in vitro studies, compounds were dissolved in DMSO (Fluka, Buchs, Switzerland) to obtain stock
solutions of 10 mg/ml while in vivo analyses involved suspension of compounds in 7% (v/v) Tween
80 and 3% (v/v) ethanol shortly before oral treatment (10 ml/kg) of mice.
Animals and Parasites
Animal studies were carried out in accordance with Swiss national and cantonal regulations on animal
welfare at the Swiss Tropical and Public Health Institute (Basel, Switzerland). This current study was
approved by the local veterinary agency, based on Swiss cantonal and national regulations
(permission no. 2070). Female mice (NMRI strain; age 3 weeks; weight ca. 14 g)) were purchased
from Charles River, Germany, kept under environmentally controlled conditions (temperature ~ 25°C;
humidity ~70%; 12ꢀhour light and 12ꢀhour dark cycle) with free access to water and rodent diet and
acclimatized for one week before infection. Cercariae of S. mansoni (Liberian strain) were obtained
from infected intermediate host snails, Biomphalaria glabrata, as previously described.37
In Vitro Studies
a) Screening on newly transformed schistosomula (NTS)
S. mansoni cercariae were mechanically transformed to newly transformed schistosomula (NTS) as
described elsewhere.38 Briefly, snails were placed under light to stimulate cercarial shedding and
cercarial suspension collected. The tails were separated from the heads by rinsing three times with
cold HBSS. NTS were then incubated overnight in culture medium and used the next day.
Test compounds and controls were dissolved in DMSO (Fluka, Buchs, Switzerland) to a concentration
of 10 mM. A hundred NTS were then incubated in each well of a 96ꢀwell plate with culture medium
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and the test compound for a final well volume of 250 ꢁL. Culture medium was composed of Medium
199 (Invitrogen, Carlsbad, CA) supplemented with 5% foetal calf serum (Lucerne, Switzerland) and
1% penicillin/streptomycin mixture (Lucerne, Switzerland). Compounds were tested at 10 ꢁM in
triplicate with NTS incubated in no more than 1% DMSO serving as control. NTS were kept in an
incubator at 37°C and 5% CO2 for up to 72 hours after which the condition of the NTS was
microscopically evaluated using a scale from 3 (normal activity and morphological alteration) to 0
(dead). IC50 determination assays for the compounds were subsequently performed in the same
manner.
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b) Adult S. mansoni worms
To obtain adult schistosomes, mice were infected subcutaneously with 80 ꢀ 100 cercariae. The mice
were then euthanized after 7 ꢀ 8 weeks with CO2 and the worms collected from the hepatic portal and
mesenteric veins. Two pairs of adult worms were incubated in each well of a 24ꢀwell plate with 2 ml
culture medium and the test compound. Culture medium was composed of RPMI 1640 (Invitrogen,
Carlsbad, CA) supplemented with 5% foetal calf serum (Lucerne, Switzerland) and 1%
penicillin/streptomycin mixture (Lucerne, Switzerland). Only compounds with > 70% activity against
NTS were evaluated against adult worms at 10 ꢁM with incubation at < 1% DMSO used as control.
Worms were kept in an incubator at 37°C and 5% CO2 for up to 72 hours after which their condition
was microscopically evaluated using a scale from 3 (normal activity and no morphological alterations)
to 0 (dead). IC50 determination was conducted for compounds with > 60% activity. For the in vitro
drug sensitivity assays, all viability scores were averaged across replicates and normalized to the
average viability scores of the control wells. IC50 values were calculated using CompuSyn2
(ComboSyn Inc., Paramus, NJ).
Cytotoxicity Testing
Compounds were screened for in vitro cytotoxicity against Chinese Hamster Ovarian (CHO)
mammalian cellꢀlines, using the 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazoliumbromide (MTT)ꢀ
assay. The reference standard, emetine, was prepared to 2 mg/ml in distilled water while stock
solutions of test compounds were prepared to 20 mg/ml in 100% DMSO with the highest
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concentration of solvent to which the cells were exposed having no measurable effect on the cell
viability. The initial concentration of the drugs and control was 100 ꢁg/mL, which was serially diluted
in complete medium with 10ꢀfold dilutions to give 6 concentrations, the lowest being 0.001 ꢁg/mL.
Plates were incubated for 48 h with 100 ꢁL of drug and 100 ꢁL of cell suspension in each well and
developed afterwards by adding 25 ꢁL of sterile MTT (Thermo Fisher Scientific) to each well and
followed by 4 h incubation in the dark. The plates were then centrifuged, medium aspirated off and
100 ꢁL of DMSO added to dissolve crystals before reading absorbance at 540 nm. IC50 values were
then obtained from doseꢀresponse curves, using a nonlinear doseꢀresponse curve fitting analysis via
GraphPad Prism v.4.0 software (La Jolla, USA). The assay was conducted in triplicate and conducted
on two separate occasions
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Beta-haematin Inhibition Activity (BHIA)
Stock solutions of controls and test compounds were made to 20 mM in DMSO (Fluka, Buchs,
Switzerland). A solution containing water/305.5 ꢁM NP40/DMSO at a v/v ratio of 70%/20%/10%,
respectively was added to columns 1ꢀ11 in a 96ꢀwell plate while 140 ꢁL of water and 40 ꢁL of 305.5
ꢁM NP40 were added to column 12 to mediate the formation of βꢀhaematin. Twenty ꢁL of drug (20
mM) was added to column 12 and 100 ꢁL of this solution serially diluted to column 2, with column 1
left as a blank (0 ꢁM compound). In case the compound was coloured (for instance AQ), a preꢀ
reading of the plate was done by measuring absorbance at 405 nm on a SpectraMax plate reader. A
178.8 ꢁL aliquot of hematin stock was suspended in 20 ml of a 1 M acetate buffer, pH 4.9 and 100 ꢁL
of this hematin suspension added into each well. Plates were then incubated for ~5 h at 37 °C after
which 32 ꢁL of pyridine solution (20% water, 20% acetone, 10% 2M HEPES buffer (pH 7.4), 50%
pyridine) was added followed by addition of 60 ꢁL of acetone to all wells. Plates were again read at
405 nm and IC50s plotted in GraphPad Prism v.4.0 software (La Jolla, USA).
In vivo studies in S. mansoni-infected mice
Groups of 3– 4 NMRI mice harbouring chronic schistosomal infection were treated 49 days postꢀ
infection with single oral doses of 400 mg/kg of compounds 11, 28, 30, 38, 39, 56, 60, 63, 66 and 67
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dissolved in 7% Tween 80 and 3% ethanol in water (v/v/v) while untreated mice served as controls.
The drug vehicle (7% Tween 80 and 3% ethanol in water) has no antischistosomal properties in vitro
and in vivo (unpublished observation). At 21 days postꢀtreatment, animals were euthanized by the
CO2 method and dissected. Surviving schistosomes residing in the mesenteric veins and the liver were
counted and sexed as previously described elsewhere.39
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Single point in vitro metabolic stability assay
The metabolic stability assay was performed in duplicate in a 96ꢀwell micro titre plate. The test
compounds (0.1 ꢁM) were incubated individually at 37 °C in a solution containing 0.35 mg/ml mouse
liver microsomes (MLM; male Mouse BALB/c, Xenotech) and metabolic reactions initiated by the
addition of NADPH (1 mM) in phosphate buffer (100 mM, pH 7.4) and incubated for 30 min.
Reactions were quenched by adding 300 ꢁL of ice cold acetonitrile containing internal standard
(carbamazepine, 0.0236 ꢁg/mL). Test compounds in the supernatant were centrifuged, filtered and
analysed by means of LCꢀMS/MS (Agilent Rapid Resolution HPLC, AB SCIEX 4000 QTRAP MS).
The incubations of compounds and controls (midazolam and MMV390048) were performed in
triplicates. The relative disappearance of parent compound over the course of the incubation was
monitored by LCꢀMS/MS and results reported as % remaining after 30 min incubation. Metabolite
searches were not conducted during the metabolic stability assay.
Mouse pharmacokinetic (PK) Analysis
Compound 28 was weighed out and prepared freshly immediately prior to dosing. The compound was
prepared in dimethylacetamide, polyethylene glycol, and propylene glycol/ethanol mixture 4:1 at a
ratio of 1:3:6 for intravenous dosing (n = 3) and administered in a volume of 50 ꢁL via the tail vein
after anaesthetising the animals. For the orally dosed group (n = 3), compound 28 was formulated as
suspension in 100% hydroxypropyl methylcellulose and the dose administered by gavage.
Approximately 20ꢁL of blood was drawn from the tail of each animal at a series of predetermined
timeꢀpoints and placed into a microcentrifuge tube coated with lithium heparin S12 to prevent
clotting. Samples were kept on ice and transferred to storage at ꢀ80 °C within one hour. Quantitation
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of the levels of the compounds was carried out using highꢀperformance liquid chromatography and
mass spectrometry (LCꢀMS/MS) method developed inꢀhouse. Animals are housed in temperatureꢀ
controlled rooms and fed a standard diet with water provided ad libitum. The study was conducted
using three animals per dosing route; all animals were killed by inhalation of isoflurane to knock them
out followed by cervical dislocation. Authorisation for the study was obtained from the University of
Cape Town Research Ethics Committee protocol number AEC 013/028.
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Statistics
In vitro activities of the compounds against NTS and adult S. mansoni were calculated from the mean
viability values in relation to the control value using the formula:
Drug effect = 1 − (score drug ÷ score control
)
From the obtained drug effects, IC50 values were calculated using CompuSyn software (version 3.0.1;
ComboSyn) with the linear correlation coefficient (r value) reflecting the conformity of the
experimental data The nonꢀparametric MannꢀWhitney test was applied for in vivo studies, comparing
the medians of the worm counts in the treatment and control groups. A difference in median was
considered to be significant if the pꢀvalue < 0.05 (GraphPad Prism, version 4.0, La Jolla, USA).
Supporting Information
Additional details of the structures of all derivatives assessed as well as tabulated biological activity
data of all compounds evaluated against larval and adult parasites, βꢀhaematin formation, mammalian
cells and mouse liver microsomes. Excel file with the compounds SMILES format is also provided
Author Information
*Corresponding Author: Kelly Chibale
Email: Kelly.Chibale@uct.ac.za: Phone: +27ꢀ21ꢀ6502553. Fax: +27ꢀ21ꢀ6505195.
Acknowledgement
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The University of Cape Town, South African Medical Research Council, and South African Research
Chairs Initiative of the Department of Science and Technology, administered through the South
African National Research Foundation are gratefully acknowledged for support (K.C). TJE also
acknowledges the Medical Research Council for financial support. JK is grateful to the European
Research Council for financial support (ERCꢀ2013ꢀCoG 614739ꢀA_HERO).
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Transparency declaration: None to declare
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Antischistosomal Agents. Future Med. Chem. 7, 713ꢀ725.
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a) Antischistosomal vs BHI Activity (Schistosomula)
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R2 = 0.0321; p > 0.05
1.0
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0.0
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ꢀ0.5
ꢀ1.0
log BHIA IC50
b) Antischistosomal vs BHI Activity (Adult Worms)
R2 = 0.0411; p > 0.05
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ꢀ1
ꢀ2
log BHIA IC50
Figure 1: Correlation between antischistosomal and βH inhibition activity of the PBI compounds.
This analysis was performed on a) 19 PBIs exhibiting a broad spectrum of activity against
schistosomula and b) 38 compounds with IC50 data against adult S. mansoni.
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2.0
1.6
1.2
0.8
0.4
0.0
2.0
Oral Mean 20mpk
Oral1
Oral2
Oral3
1.5
1.0
0.5
0.0
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Time (h)
Time (h)
1.5
1.0
0.5
0.0
IV1
1.5
1.0
0.5
0.0
IV Mean 2mpk
IV2
IV3
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24
0
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24
Time (h)
Time (h)
Figure 2: Systemic exposure of compound 28 following single intravenous and oral dose
administration to healthy mice (n = 3).
Table 1: In vitro effect against adult stages of S. mansoni, βꢀhaematin and CHO cells of PBIs that
were active against both NTS (>70 mortality) and adult parasites (IC50 ≤ 10 µM)
R2
N
R3
N
R
CN
Antischistosomal
Activity
R2
R
R3
Cytotoxicity
Compound
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IC50 Adult
IC50
BHIA
(µM)
IC50
CHO
(µM)
Selective
Index
S.mansoni
(r value)
µM
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14
16
2.41 (0.88)
10.14 (0.88)
10.02 (0.99)
1.20 (0.93)
9.20 (0.95)
40.5
8.0
1.56 ± 0.03
10.5 ± 2.93
6.12 ± 1.20
2.91 ± 0.5
0.6
1.0
0.6
2.4
0.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
85.3
87.1
40.8
4.07 ± 0.21
22
6.96 (0.95)
121
27
28
2.28 (0.90)
2.44 (0.88)
157.1
51.5
5.92 ± 1.84
9.60 ± 1.58
2.6
3.9
30
1.69 (0.92)
19.2
1.55 ± 0.44
0.9
33
34
35
2.62 (0.99)
8.86 (0.92)
2.41 (0.85)
74.5
22,380
14.5
3.19 ± 0.42
3.77 ± 0.50
8.71 ± 0.68
1.2
0.4
3.6
Cl
36
1.62 (0.99)
10.1
1.63 ± 0.24
1.0
Br
37
38
2.89 (0.94)
2.08 (0.90)
14.9
30.8
1.55 ± 0.60
3.39 ± 0.70
0.5
1.6
39
2.32 (0.94)
6.9
11.2 ± 1.1
4.8
ACS Paragon Plus Environment