Discovery of carbohybrid-based 2-aminopyrimidine analogues as a new class of rapid-acting antimalarial agents using image-based cytological profiling assay

Article abstract of DOI:10.1021/jm5009693

New antimalarial agents that exhibit multistage activities against drug-resistant strains of malaria parasites represent good starting points for developing next-generation antimalarial therapies. To facilitate the progression of such agents into the development phase, we developed an image-based parasitological screening method for defining drug effects on different asexual life cycle stages of Plasmodium falciparum. High-throughput screening of a newly assembled diversity-oriented synthetic library using this approach led to the identification of carbohybrid-based 2-aminopyrimidine compounds with fast-acting growth inhibitory activities against three laboratory strains of multidrug-resistant P. falciparum. Our structure-activity relationship study led to the identification of two derivatives (8aA and 11aA) as the most promising antimalarial candidates (mean EC₅₀of 0.130 and 0.096 μM against all three P. falciparum strains, selectivity indices >600, microsomal stabilities >80%, and mouse malaria ED₅₀values of 0.32 and 0.12 mg/kg/day, respectively), targeting all major blood stages of multidrug-resistant P. falciparum parasites.

Full text of DOI:10.1021/jm5009693

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Article
Discovery of carbohybrid-based 2-aminopyrimidine
analogues as a new class of rapid-acting antimalarial
agents using image-based cytological profiling assay
Sukjun Lee, Donghyun Lim, Eunyoung Lee, Nakyung Lee, Hong-gun Lee, Jonathan Cechetto, Michel Liuzzi,
Lucio H. Freitas-Junior, Jin Sook Song, Myung Ae Bae, Sangmi Oh, Lawrence Ayong, and Seung Bum Park
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5009693 • Publication Date (Web): 19 Aug 2014
Downloaded from http://pubs.acs.org on August 23, 2014
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Discovery of carbohybrid-based 2-aminopyrimidine
analogues as a new class of rapid-acting antimalarial
agents using image-based cytological profiling assay
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Sukjun Lee,a,† Donghyun Lim,b,† Eunyoung Lee,a Nakyung Lee,c Hong-gun Lee,d Jonathan
Cechetto,d Michel Liuzzi,a Lucio H. Freitas-Junior,c Jin Sook Song,e Myung Ae Bae,e Sangmi
Oh,f Lawrence Ayong,a* and Seung Bum Parkb,g*
aEarly Discovery Program, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do 463-400, Korea
bWCU Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-
747, Korea
cCenter for Neglected Diseases Drug Discovery (CND3), Institut Pasteur Korea, Gyeonggi-do
463-400, Korea
dCenter for Core Technologies, Institut Pasteur Korea, Institut Pasteur Korea, Gyeonggi-do 463-
400, Korea
eDrug Discovery Platform Technology Group, Drug Discovery Division, Korea Research
Institute of Chemical Technology, Daejeon 305-600, Korea
fMedicinal Chemistry and Chemical Biology Group, Institut Pasteur Korea, Gyeonggi-do 463-
400, Korea
gDepartment of Chemistry, Seoul National University, Seoul 151-747, Korea
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ABSTRACT
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New antimalarial agents that exhibit multi-stage activities against drug-resistant strains of malaria
parasites represent good starting points for developing next generation antimalarial therapies. To
facilitate the progression of such agents into the development phase, we developed an image-based
parasitological screening method for defining drug effects on different asexual life cycle stages of
P. falciparum. High-throughput screening of a newly assembled diversity-oriented synthetic
library using this approach led to the identification of carbohybrid-based 2-aminopyrimidine
compounds with fast-acting growth inhibitory activities against three laboratory strains of multi-
drug resistant P. falciparum. Our structure-activity relationship study led to the identification of
two derivatives (8aA and 11aA) as the most promising antimalarial candidates (mean EC50 of
0.130 and 0.096 µM against all three P. falciparum strains, selectivity indices >600, microsomal
stabilities >80%, and mouse malaria ED50 values of 0.32 and 0.12 mg/kg/day, respectively)
targeting all major blood stages of multi-drug-resistant P. falciparum parasites.
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INTRODUCTION
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With nearly a million deaths annually and over 219 million hospital cases each year, malaria
remains a serious public health problem, particularly in the high transmission areas of Sub-Sahara
Africa, Southeast Asia, and Latin America.1–3 The disease is caused by mosquito-borne protozoan
parasites of the genus Plasmodium; five species of which are known to infect humans (P.
falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi).4,5 In the absence of vaccines with
operational utilities against the disease, accurate diagnosis and treatment with available
antimalarial regimens alongside effective vector control measures remain the best hope of
controlling the disease globally.6–8
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Plasmodium parasites, however, exhibit a high propensity to rapidly developed resistance
against newly introduced medicines, necessitating as such a continuous search and development
of new antimalarial agents to combat emerging resistant strains.9,10 To stop the spread of multi-
drug resistance worldwide, the World Health Organization recommends the use of artemisinin-
based combination treatments for all uncomplicated malaria cases globally. A key inclusion benefit
of artemisinin in the current combinations lies in its rapid-acting multi-stage activities against most
drug-resistant strains of Plasmodium parasites.11 Unfortunately, parasite resistance to artemisinin
has been inevitable as revealed by the recent emergence and spread of drug-tolerant parasites along
the Thai-Cambodian borders in South East Asia.12,13
This dire situation calls for the immediate development, preferably, of non-artemisinin-based
compounds with fast-acting, long-lasting and multi-stage activities capable of reducing treatment
durations while also improving patient compliances.11 Indeed, compared to slow-acting single-
target drugs such as pyrimethamine (antifolate), sulfadoxine (antifolate), and atovaquone
(cytochrome bc1 complex inhibitor), anti-malarial compounds that exhibit fast-acting and
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pleiotropic activities similar to that of artemisinin and chloroquine are expected to display shorter
parasite clearance times (PCTs), increased useful therapeutic lives (UTLs), and high parasitaemia
reduction ratios (PRR) with net effects being increased cure rates and mitigation of resistance
development.10,14,15
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Discovering new therapeutic agents in malaria requires a combination of new chemical libraries
and innovative screening technologies. Indeed, plausible efforts by both pharmaceutical industries
and academic research institutions recently had led to the identification of several structurally
diverse antimalarial compounds, some of which are under scrutiny by partnerships such as the
Medicine for Malaria Ventures (MMV) for inclusion into the lead discovery chain.
Recently, we described the development of an automated image-based screening system for the
quantitative detection and classification of P. falciparum life cycle stages in a 384-culture well
format.16 In parallel, we constructed a small molecule library of over 4,000 drug-like compounds
containing 60 distinct core structures using a privileged-substructure-based diversity-oriented
synthetic (pDOS) approach.17 By using the above image-based system first as a growth inhibition
screening assay and then as a cytological profiling tool, we identified carbohybrid-based 2-
aminopyrimidines from our pDOS library as a new class of anti-malarial compounds with potent
activities against three laboratory strains of multiple drug-resistant P. falciparum. In fact, we
recently reported the synthesis of stereochemically enriched acyclic polyols fused with various
privileged heterocycles from naturally abundant carbohydrates and named them as
“carbohybrids”.18 On the basis of structure-activity relationship studies, we identified 2-
aminopyrimidine analogues (8aA and 11aA) of the initial hit compounds as the most promising
candidates given their high in vitro potency, and excellent in vivo PK properties and efficacy in a
mouse malaria model.
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RESULTS AND DISCUSSION
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A Novel Image-based Cytological Profiling Assay. Plasmodium parasites exhibit a complex
life cycle that involves multiple parasite stages and forms, which display variable degrees of
sensitivity to most antimalarial compounds.14,19,20 To facilitate the discovery and development of
new therapies that target existing strains of drug-resistant P. falciparum, we developed a
cytological profiling method using our previously reported image-based screening system.16 This
assay relies on the quantitative detection of each major blood form (early rings, late rings,
trophozoites, and schizonts) of P. falciparum parasites in a 384-well format based on the numbers
and sizes of detectable nuclear condensation spots in infected erythrocytes. In this assay, all host
cells are stained with wheat germ agglutinin-AlexaFluor 488 conjugate whereas individual
parasites in infected erythrocytes are identified using the nuclear stain DAPI. The assay also
employs the fluorescent dye Mitotracker red CMXRos to stain and discriminate functional
mitochondria in live parasites and disrupted mitochondria in dead parasites. A major advantage of
our image-based system is in its ability to quantify drug activities on the basis of total culture
parasitaemia, and to determine each parasite stage proportion in an automated fashion. The
cytological profiling approach as developed in this study involves parasite treatment at well-
defined time-points in the parasite’s intraerythrocytic life cycle, and comparison of the resulting
stage accumulation indices following 36 or 24 h of drug pressure. We confirmed the reliability of
this approach by profiling the stage specificities of four mechanistically known antimalarial agents
using three different drug resistant lines of P. falciparum (Figure 1). Consistent with their known
modes of actions,14,19,21–27 our data show that artemisinin (ART) and chloroquine (CQ) are fast-
acting in inhibiting early and late ring development to the mid-trophozoite stage, but that these
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compounds are only weakly active against trophozoite development to the schizont stages (Figure
1A). Indeed, at clinically relevant concentrations (peak plasma concentrations of ~2 µM for ART
and 1–10 µM for CQ),28–30 both chloroquine and artemisinin are known to rapidly inhibit cell cycle
development in malaria parasites resulting in parasite clearance times of <24 h for both drugs.
Trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64) on its part inhibited early ring
development and schizont maturation, whereas N-acetylglucosamine (GlcNAc) was only weakly
effective at inhibiting early ring development in all three parasite lines (Figure 1A). As shown in
Figure 1B, ART, E64 and GlcNAc also produced significant decreases in culture parasitaemia
when added at the 42 h time-point for 24 h. Indeed, both ART and E64 gave significantly high
schizont accumulation indices, indicating rapid inhibition of schizont maturation and/or merozoite
egress, whereas GlcNAc reduced culture parasitaemia without a significant retention of schizonts.
This later indicates that GlcNAc predominantly acts on extracellular merozoites, inhibiting host
cell entry as reported previously.21 Taken together, the above results demonstrated the ability of
our cytological profiling approach to quantify the stage-specificities of antimalarial agents at
different time-points during the parasite's asexual developmental cycle with high reliability and in
a high-throughput screening format.
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Discovery of Carbohybrid-based Aminopyrimidines as a New Antimalarial Drug Class. To
identify antimalarial compounds with novel modes of action, we screened an in-house 3,980-
member small molecule library using our image-based parasitological assay system and the
chloroquine/pyrimethamine-resistant P. falciparum K1 strain. This initial screen resulted in 89 hit
compounds (hit rate of 2.4%) that inhibited parasite growth by >90% when used at 5 µM final
concentrations and exhibited EC50 values less than 2 µM. Counter-screening of these initial hits
against two other drug-resistant parasite strains (W2 and HB3) identified the carbohybrid
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frameworks containing 2-aminopyrimidine moiety 1 (SNU3701) and 2 (SNU3662)31 that
displayed similar EC50 values (less than 1 µM) against all three P. falciparum strains (Table 1).
Dose-responsive cytotoxicity analyses of the above compounds using three different human cell
lines (HepG2, THP-1, and U2OS) gave high selectivity indices in all three cell lines (Table 1),
indicating in vitro safety towards the these cell lines. Cytological profiling of the compounds using
drug-resistant P. falciparum K1 parasites revealed that carbohybrid-based 2-aminopyrimidine
analogues (1 and 2) exhibit fast-acting antimalarial activities by inhibiting further development of
early rings, late rings, and mid-trophozoite stage parasites when treated at the 6, 18, or 30 h post-
invasion (Figure 2A and 2B). These two compounds, however, did not significantly affect parasite
mitochondrial integrity as assessed by using the mitochondrial membrane potential indicator,
Mitotracker Red CMXRos (Figure S1, Supporting Information).
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Treatment of the parasites at the 42-hpi time-point resulted in a parasitaemia reduction index of
0.52 for 1 and 0.46 for 2, and schizont accumulation index of approximately 2.4 for each compound.
These results indicate that both compounds are fast acting in inhibiting schizont maturation and/or
merozoite egress from the infected erythrocytes leading to accumulation of schizonts in culture.
Collectively, we concluded that carbohybrid-based 2-aminopyrimidine analogues are a novel class
of fast acting blood-stage antimalarial compounds on the basis of their ability to block cell cycle
progression from the treated parasite stages.
Encouraged by these findings, we constructed a focused library of 2-aminopyrimidine-based
carbohybrids using our divergent synthetic strategy.18 As shown in Scheme 1, 6-hydroxyl groups
in D-galactal (3a, R1 = OPMB, R2 = H) and D-glucal (3b, R1 = H, R2 = OPMB) were selectively
protected with trityl group to afford 4a and 4b. p-Methoxybenzyl (PMB) protection of secondary
hydroxyl groups, followed by Vilsmeier-Haack formylation, resulted in the 2-C-formyl glycals,
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6a and 6b. The condensation of these intermediates (6a and 6b) with dinucleophiles and the
subsequent ring opening yielded the carbohybrid scaffolds 7 consisting of diverse heterocyclic
moieties (pyrimidine, pyrazolopyrimidine, and pyrazole). Sequential modifications of functional
groups through mesylation, trityl deprotection, and azide substitution allowed the formation of
carbohybrids 8, 9 and 10, respectively, with different appendices. Finally, the primary hydroxyl
group in 10 was transformed via tritylation, benzylation, and mesylation to afford analogues 11,
12 and 13, respectively. The modification of 7bA with PMB group, instead of trityl group at the
R5 position, and the subsequent O-mesylation yield analogues 14bA and 15bA, respectively
(Scheme S1, Supporting Information).
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Given the occurrence of 2-aminopyrimidine heterocycles in our carbohybrid-based hit
compounds, we investigated the antimalarial activities of carbohybrids that contained pyrimidine
(2, 7aA, 7aC), pyrazolopyrimidine (7aD), and pyrazole (7aE) in their substructures. As shown in
Table 2, carbohybrid analogues containing all three heterocycles exhibited antimalarial activities
(EC50 < 1 µM) against chloroquine/pyrimethamine-resistant P. falciparum strain K1.
Stereochemical modifications at the R1 and R2 positions, which was achieved by using different
glycals (3a or 3b) as starting material, minimally affected their inhibitory activities. However, the
galactal-derived compounds (7aA, 7aE, 8aA, 8aE, and 11aA) exhibited slightly higher
antimalarial activities compared with those of the glucal-based derivatives (7bA, 7bE, 8bA, 8bE,
and 11bA). The modification at the primary hydroxyl group (R5) appeared critical for the
antimalarial activity. For example, the introduction of bulky hydrophobic moieties such as the
trityl group in 8aA and 11aA allowed a more than 50-fold enhancement in antimalarial activities
compared with those of unmodified hydroxyl version in carbohybrid analogues 9aA and 10aA.
Consistent with this observation, the introduction of benzyl group (12aA) enhanced their
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antimalarial activities, albeit not as much as those in trityl-containing carbohybrid derivatives.
Importantly, O-mesylation as examplified in derivative 8aA and azide substitution at the R4
position of the carbohybrid chain (e.g. 11aA) enhanced the class antimalarial activities. On the
basis of their potent in vitro activities (EC50: 104–147 nM and 80–96 nM, respectively, against all
three drug-resistant parasite strains), fast-acting activities against ring, trophozoite and schizont
development (Figure S2, Supporting Information), and high selectivity indices (S.I. >680 for 8aA
and >1000 for 11aA), the above two carbohybrid-based 2-aminopyrimidine derivatives were
selected for further pharmacokinetics analyses.
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Our candidate compounds 8aA and 11aA were examined for their microsomal stability (n = 3).
As shown in Table 3, both compounds showed excellent in vitro metabolic stability, with greater
than 80% compound remaining after 30 min in human and mouse liver microsomes. In addition,
carbohybrid-based 2-aminopyrimidine derivative 8aA at the dose of 1 and 5 mg/kg showed
excellent pharmacokinetic behavior after intraperitoneal (i.p.) injection in SD male rats (n = 5),
with rapid absorption (time to peak concentration [Tmax] of 1 h and maximum concentration [Cmax
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of 1.80±0.40 and 12.9±9.30 g/mL, respectively) and stable plasma concentration of more than 0.10
and 0.15 µg/mL over 24 h (Figure S3, Supporting Information). The area under the curve (AUC)
of 8aA and 11aA confirmed the excellent pharmacokinetic property, which assures their
pharmacological activities in vivo.
Novel In Vivo Antimalarial Activity of 8aA and 11aA in a Mouse Malaria Model. To assess
the in vivo efficacy of carbohybrid-based antimalarial candidates (8aA and 11aA), groups of P.
chabaudi-infected mice were treated by i.p. injection on Day 1 (trophozoite-stage parasites) to Day
3 post-infection using fixed doses of 0.03, 0.3, 3, 10 or 30 mg/kg for each compound. Percent
inhibitions of parasite growth in treated groups relative to the control parasitaemia on Day 4 were
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then used in probit analyses of compounds’ effects. As shown in Figure 3, both candidate
compounds (8aA and 11aA) displayed significant in vivo antimalarial activities in a dose-
dependent manner. Based on the probit 5-response point, the compound's ED50 against P. chabaudi
infection were estimated to be 0.32 mg/kg for 8aA and 0.12 mg/kg for 11aA. As observed by light
microscopy examination of thin smears, mice treatment with either 8aA or 11aA at doses greater
than 3 mg/kg resulted in a significant reduction in blood parasitaemia, and accumulation of ring-
stage parasites in the infected mice unlike trophozoite stage parasites in untreated controls (Figure
S4, Supporting Information). These findings are consistent with the 24 h life cycle of
synchronously developing P. chabaudi in mice, and indicate a fast acting (approximately 12 h
post-i.p. drug administration) in vivo antimalarial activity of the compounds. Collectively, the
above data demonstrate clearly the therapeutic potential of the carbohybrid-based 2-
aminopyrimidine 8aA and 11aA in a mouse malaria model.
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CONCLUSIONS
Herein, we reported the development and application of a novel image-based cytological
profiling assay for the discovery of novel antimalarial agents. Using this screening system, we
identified carbohybrid-based 2-aminopyrimidines from 4,000-membered in-house pDOS small
molecule library as a new class of blood-stage antimalarial agents. Based on our in vitro biological
evaluation of their synthetic analogues, we optimized our initial hit compound to 8aA and 11aA
as antimalarial candidates with excellent potency, high selectivity indices, and rapid acting
activities against all major asexual parasite forms. Finally, these lead compounds exhibited
favorable pharmacokinetic properties and fast-acting in vivo efficacy. We conclude that these
carbohybrid-based 2-aminopyrimidine analogues 8aA and 11aA can be a promising class of
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antimalarial agents with potentials for clinical use against uncomplicated and/or severe malaria
cases.
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EXPERIEMNTAL SECTION
Chemistry. All commercially available reagents and solvents were used without further
purification unless noted otherwise. 1H and 13C NMR spectra were recorded on Varian Inova-500
[Varian Assoc.] or Agilent 400-MR DD2 [Agilent] instruments. Chemical shifts were reported in
ppm relative to tetramethylsilane (TMS) as an internal standard or residual solvent peak (CDCl3;
13
1H: δ = 7.26 ppm; C: δ = 77.23 ppm). Multiplicity was indicated as follows: s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet), dt (doublet of triplet), td
(triplet of doublet), brs (broad singlet). Coupling constants are reported in hertz. HRMS analyses
were conducted at the National Center for Inter-University Research Facilities in Seoul National
University by fast atom bombardment (FAB) method. Routine mass spectrometric analysis was
performed on a Finnigan Surveyor MSQ Plus LC/MS [Thermo] by electrospray ionization (ESI)
method. Purity of the compounds was determined using the Finnigan Surveyor MSQ Plus LC/MS
[Thermo] equipped with a Hypersil Gold column and photodiode array detector, or a Shimadzu
SCL-10AVP HPLC [Shimadzu] equipped with a VP-ODS column and 254 nm detector. All the
final compounds were >95% pure. The conversion of starting materials was monitored by thin-
layer chromatography (TLC) using pre-coated glass-backed plates (silica gel 60; F254 = 0.25 mm),
and the reaction components were visualized by observation under UV light (254 and 365 nm) or
by treatment of TLC plates with visualizing agents such as KMnO4, phosphomolybdic acid and
ceric sulfate followed by heating. Products were purified by flash column chromatography on
silica gel (230–400 mesh) using a mixture of EtOAc/hexane or MeOH/CH2Cl2 as eluents.
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Synthetic methods and spectroscopic data for 2, 6a, 6b, 7aA–7bA and 10aA–10aD were reported
by Lim et al.18
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Synthesis of a Glucal-derived Pyrazole-containing Carbohybrid (7bE). A solution of 6b
(100 mg) in tetrahydrofuran (3 mL) was added to a stirred solution of methylhydrazine sulfate (2
equiv.) and K2CO3 (5 equiv.) in ethanol (3 mL). The reaction mixture was stirred at room
temperature for 15 h. The solvent was removed under reduced pressure and the residue was
partitioned between ethyl acetate and water. The aqueous layer was extracted with ethyl acetate
twice, and the combined organic layer was dried over anhydrous Na2SO4(s). The filtrate was
condensed under reduced pressure and subjected to flash column chromatography. White solid.
Yield: 56%; 1H NMR (400 MHz, CDCl3) δ 7.43–7.40 (m, 7H), 7.29–7.21 (m, 10H), 7.13 (d, J =
8.6 Hz, 2H), 6.98 (d, J = 8.6 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 6.76 (d, J = 8.6 Hz, 2H), 4.52 (d, J
= 3.9 Hz, 1H), 4.43 (d, J = 11.7 Hz, 1H), 4.31 (s, 2H), 4.11 (d, J = 11.7 Hz, 1H), 3.94–3.90 (m,
1H), 3.85 (s, 3H), 3.783 (s, 3H), 3.778 (s, 3H), 3.69 (dd, J = 6.5, 3.7 Hz, 1H), 3.26–3.23 (m, 1H),
3.19–3.15 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 159.4, 159.3, 144.1, 139.3, 130.3, 130.1, 129.9,
129.84, 129.80, 128.9, 128.0, 127.2, 118.9, 114.0, 113.7, 86.6, 80.5, 73.7, 73.2, 70.8, 70.2, 64.8,
55.43, 55.42, 39.2; HRMS (FAB+) m/z calculated for C43H44N2O6 [M+H]+: 685.3278; found:
685.3282.
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General Synthetic Procedure for O-Mesylated Carbohybrids (8). Triethylamine (3 equiv.)
and methanesulfonyl chloride (2 equiv.) were added to a stirred solution of 7 (100 mg) in
anhydrous CH2Cl2 at 0 °C. The mixture was stirred at 0 °C for 2 h, diluted with CH2Cl2 and washed
with brine. The aqueous layer was extracted with CH2Cl2, and the combined organic layer was
dried over anhydrous Na2SO4(s). The filtrate was condensed under reduced pressure and subjected
to flash column chromatography. 8aA: Prepared from 7aA as a white solid. Yield: 99%; 1H NMR
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(400 MHz, CDCl3) δ 8.18 (s, 2H), 7.39–7.37 (m, 6H), 7.31–7.22 (m, 11H), 6.90 (d, J = 8.6 Hz,
2H), 6.80 (d, J = 8.6 Hz, 2H), 6.71 (d, J = 8.6 Hz, 2H), 4.99 (dt, J = 6.7, 3.5, 1H), 4.32–4.22 (m,
4H), 4.10 (d, J = 10.6 Hz, 1H), 3.91 (dd, J = 6.7, 4.3 Hz, 1H), 3.76 (s, 3H), 3.75 (s, 3H), 3.35 (dd,
J = 10.8, 3.3 Hz, 1H), 3.20 (s, 6H), 3.18–3.16 (m, 1H), 2.92 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 162.4, 159.5, 158.2, 143.4, 130.4, 130.1, 129.8, 129.3, 128.7, 128.2, 127.5, 118.0, 114.0, 113.8,
87.4, 81.5, 80.4, 76.0, 75.3, 70.0, 63.5, 55.5, 55.4, 38.9, 37.4, 31.7; HRMS (FAB+) m/z calculated
for C46H49N3O8S [M+H]+: 804.3319; found: 804.3315.
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General Synthetic Procedure for Detritylaed Carbohyrids (9). To a stirred solution of 8 (100
mg) in methanol, p-toluenesulfonic acid (1.5 equiv.) was added. The mixture was stirred at room
temperature for 8 h and the solvent was removed under the reduced pressure. The residue was
partitioned between ethyl acetate and saturated NaHCO3 solution. The aqueous layer was extracted
with ethyl acetate twice, and the combined organic layer was dried over anhydrous Na2SO4(s). The
filtrate was condensed under reduced pressure and subjected to flash column chromatography. 9aA:
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Prepared from 8aA as a white solid. Yield = 94%; H NMR (400 MHz, CDCl3) δ 8.36 (s, 2H),
7.26 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.6 Hz,
2H), 5.08 (dt, J = 6.7, 3.7 Hz, 1H), 4.37–4.27 (m, 3H), 4.14 (s, 2H), 3.85–3.80 (m, 1H), 3.78 (s,
3H), 3.77 (s, 3H), 3.74 (dd, J = 8.2, 3.1 Hz, 1H), 3.71–3.68 (m, 1H), 3.23 (s, 6H), 3.04 (s, 3H),
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2.35 (brs, 1H); C NMR (100 MHz, CDCl3) δ 162.5, 159.7, 159.6, 158.0, 130.4, 130.3, 129.6,
129.0, 118.3, 114.03, 113.99, 82.4, 80.0, 76.5, 75.0, 70.5, 62.7, 55.44, 55.40, 39.0, 37.4; HRMS
(FAB+) m/z calculated for C27H35N3O8S [M+H]+: 562.2223; found: 562.2222.
General Synthetic Procedure for O-Tritylated Azide-containing Carbohybrids (11). To a
stirred solution of 10 (150 mg) and trityl chloride (1.2 equiv.) in anhydrous CH2Cl2,
diisopropylethylamine (1.5 equiv.) and 4-dimethylaminopyridine (0.1 equiv.) was added. After
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stirring at room temperature for 24 h, the mixture was diluted with CH2Cl2 and washed with brine.
The organic layer was dried over anhydrous Na2SO4(s) and the filtrate was condensed under
reduced pressure followed by flash column chromatography. 11aA: Prepared from 10aA as a white
solid. Yield = 88%; 1H NMR (400 MHz, CDCl3) δ 8.23 (s, 2H), 7.42–7.40 (m, 6H), 7.29–7.20 (m,
9H), 7.07 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 2H), 6.69 (d, J = 8.6
Hz, 2H), 4.42–4.33 (m, 2H), 4.28 (dd, J = 8.0, 2.9 Hz, 2H), 4.06 (d, J = 11.0 Hz, 1H), 3.78 (s, 3H),
3.77 (s, 3H), 3.62 (dd, J = 6.7, 5.5 Hz, 1H), 3.50–3.46 (m, 1H), 3.42–3.39 (m, 1H), 3.33–3.28 (m,
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1H), 3.20 (s, 6H); C NMR (100 MHz, CDCl3) δ 162.4, 159.34, 159.29, 158.1, 143.8, 130.1,
129.9, 129.8, 129.5, 128.9, 128.0, 127.2, 117.7, 113.9, 113.7, 87.4, 80.1, 77.8, 74.2, 70.4, 64.1,
63.2, 55.44, 55.38, 37.4; HRMS (FAB+) m/z calculated for C45H46N6O5 [M+H]+: 751.3608; found:
751.3604.
General Synthetic Procedure for O-Benzylated Azide-containing Carbohybrids (12). To a
stirred solution of 10 (40 mg) in anhydrous dimethylformamide, NaH (2 equiv.) was added slowly
at 0 °C. After stirring the mixture for 10 min, benzyl bromide (2 equiv.) was added. The reaction
mixture was stirred for 5 h at room temperature and partitioned between ethyl acetate and water.
The aqueous layer was extracted with ethyl acetate and the combined organic layer was dried over
anhydrous Na2SO4. The filtrate was condensed under reduced pressure and subjected to flash
column chromatography. 12aA: Prepared from 10aA as a white solid. Yield = 71%; 1H NMR (400
MHz, CDCl3) δ 8.27 (s, 2H), 7.37–7.29 (m, 5H), 7.16 (d, J = 8.6 Hz, 2H), 7.02 (d, J = 8.6 Hz, 2H),
6.85 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 8.6 Hz, 2H), 4.49–4.37 (m, 5H), 4.29 (d, J = 5.9 Hz, 1H),
4.15 (d, J = 11.3 Hz, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.75–3.74 (m, 1H), 3.66–3.58 (m, 3H), 3.21
(s, 6H); 13C NMR (100 MHz, CDCl3) δ 162.5, 159.50, 159.45, 158.1, 138.1, 130.2, 129.9, 129.83,
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129.75, 128.6, 127.9, 127.8, 117.9, 114.0, 113.9, 80.5, 77.3, 74.5, 73.5, 70.3, 69.7, 62.1, 55.5, 55.4,
37.4; HRMS (FAB+) m/z calculated for C33H38N6O5 [M+H]+: 599.2982; found: 599.2979.
General Synthetic Procedure for O-Mesylated Azide-containing Carbohybrids (13).
Triethylamine (3 equiv.) and methanesulfonyl chloride (2 equiv.) were added to a stirred solution
of 10 (38 mg) in anhydrous CH2Cl2 at 0 °C. The mixture was stirred at room temperature for 3 h,
diluted with CH2Cl2 and washed with brine. The aqueous layer was extracted with CH2Cl2, and
the combined organic layer was dried over anhydrous Na2SO4(s). The filtrate was condensed under
reduced pressure and subjected to flash column chromatography. 13aA: Prepared from 10aA as a
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clear syrup. Yield = 88%; H NMR (400 MHz, CDCl3) δ 8.24 (s, 2H), 7.18 (d, J = 8.6 Hz, 2H),
7.01 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 6.77 (d, J = 9.0 Hz, 2H), 4.46 (d, J = 11.0 Hz,
1H), 4.41 (d, J = 11.0 Hz, 1H), 4.35–4.25 (m, 3H), 4.23 (d, J = 7.0 Hz, 1H), 4.16 (d, J = 11.0 Hz,
1H), 3.88 (dt, J = 8.0, 3.8 Hz 1H), 3.79 (s, 3H), 3.78 (s, 3H), 3.68 (dd, J = 7.0, 4.3 Hz, 1H), 3.23
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(s, 6H), 2.97 (s, 3H); C NMR (100 MHz, CDCl3) δ 162.5, 159.7, 159.6, 157.9, 130.2, 130.0,
129.2, 129.1, 117.6, 114.2, 114.0, 80.5, 76.4, 74.2, 70.3, 69.0, 61.7, 55.44, 55.38, 37.6, 37.4;
HRMS (FAB+) m/z calculated for C27H34N6O7S [M+H]+: 587.2288; found: 587.2289.
Biological Materials. HepG2 (human hepatocellular carcinoma), THP-1 (human monocytic
leukemia), and U2OS (human osteosar-coma) cells were obtained from American Type Culture
Collection (ATCC) and cultured in Dulbecco’s modified Eagle medium (DMEM) [Gibco]
containing 10% fetal bovine serum (FBS) [Gibco] and 1% antibiotic-antimycotic solution at 37 °C
in an atmosphere of 5% CO2. BALB/c mice were obtained from The Jackson Laboratory [Bar
Harbor, ME]. All mice were bred and maintained in specific pathogen-free conditions at the animal
facility of Institute of Pasteur Korea. All animal experiments were performed with the approval of
the Institutional Animal Care and Use Committee (IACUC) at IPK (authorization no.
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IPK05050203IPK-12009). The following materials were purchased from companies in the
parentheses; hypoxanthine [Sigma-Aldrich], gentamycin [Life Technologies], Albumax [Life
Technologies], Resazurin [Sigma-Aldrich], Mitotracker Red CMXRos [Life Technologies], and
wheat germ agglutinin-AlexaFluor488 conjugate [Life Technologies].
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Parasite Lines and Culture Conditions. P. falciparum strain HB3 (PyrR), K1 (CQR, PyrR), and
W2 (CQR, PyrR, SDXR) were obtained from the Biodefense and Emerging Infections (BEI)
Research Resources [Manassas, VA] and maintained at <10% parasitaemia in human red blood
cells at 3% hematocrit [blood type O+, Gyeonggi Blood Center, Korean Red Cross]. The culture
media consisted of RPMI1640, 25 mM HEPES buffer (pH 7.4), 0.1 mM hypoxanthine, 0.016 mM
thymidine, 0.5% Albumax, and 20 µg/mL gentamycin. Cultures were maintained at 37 °C in
gassed chambers following injection with a mixture of 5% CO2, 1% O2, and 94% N2. When needed,
parasites were synchronized at the ring stage by two sorbitol treatments (8-h intervals), and further
cultivated through one complete cycle prior to the drug activity studies.
Compound Library and Image-based Parasitological Screening. An in-house drug-like
compound library of 3,840 small-molecules that had previously been constructed using a
privileged substructure-based diversity-oriented synthesis (pDOS) approach was subjected to
automated high-throughput screening using our novel image-based parasitaemia detection
method.16,17,27 Briefly, DMSO-solubilized test compounds were diluted in complete media and co-
cultured with P. falciparum (K1)-parasitized erythrocytes at a parasitaemia of 1% and hematocrit
1.5%. The final drug concentrations were 5 µM in a final DMSO amount of 0.5% per well of 50
µL sample. All liquid-handling procedures were completed using a Biomek’s NX robotic station
(compound dilution), or a CyBi® liquid handler (compound transfer), or a Well-Mate dispenser
(dispensation of parasitized cultures into drugged wells). The cultures were then placed in gassed
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chambers (5% CO2, 1% O2, and 94% N2), and grown for 72 h at 37 °C. Five microliters of each
well content was transferred into a staining solution comprising wheat germ agglutinin-
AlexaFluor488 conjugate (RBC membrane stain) and Mitotracker Red CMXRos (mitochondrial
membrane potential indicator), and maintained at 37 °C for 20 min to allow for complete
incorporation of the dyes. The resulting cultures were again diluted in fixing solution comprising
5 µg/mL DAPI and 4%(w/v) paraformaldehyde to a final hematocrit of 0.02% in a 384-well
imaging plate (black glass-bottom plate, Matrical). Following a 10-min incubation period at room
temperature, the plates were gently vortexed (1,700 rpm for 30 seconds) and spun at 1,000 rpm for
1 min to layer the suspended cells onto the well bottom. Five image fields were then acquired from
each well using an Operetta 2.0 microscopy system that was equipped with a robotic arm and a
40× objective lens. The acquired images were then submitted for automated image analysis using
our previously developed image mining algorithms for malaria.16 Raw data were further exported
onto an Excel spreadsheet and normalized using the DMSO-only wells as positive growth controls,
and RBC-only wells as image-mining background.
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Hit Selection and Confirmation. Primary screening hits were defined as reducing the final
culture parasitaemia by more than 90% when compared to the mean culture parasitaemia in DMSO
control wells. These primary hits were subjected to dose-response studies against all three drug
resistant parasite strains to identify those with novel activities. Compounds exhibiting complete
dose response curves within the analyzed drug range of 0 to 40 µM, and an EC50 less than 2 µM
were selected for cluster analyses and in silico activity searches. Further hit confirmation analysis
involved compound re-synthesis and antimalarial activity testing in dose-response assays.
In Vitro Cytotoxicity Studies. Compounds were evaluated for cytotoxicity using three human
cell lines HepG2, THP-1, and U2OS. Cells were seeded at a density of 2,500 cells/well (total
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volume 50 µL) in 384 well plates and incubated for 24 h prior to drug treatment. Two-fold serial
dilutions of the compounds (starting at 100 µM) were added to the plates and incubated under
humidified conditions at 37 °C for an additional 48 h. Resazurin (7-hydroxy-3H-phenoxazin-3-
one-10-oxide) was added to a final concentration of 10 µM and the plates were further incubated
for 12 h at 37 °C. Fluorescence due to the metabolic product of resazurin, resorufin, was measured
at excitation/emission of 531/572 nm using Victor3 V spectrophotometer [Perkin Elmer]. The
percent cell viability in each triplicate well was quantified by normalizing against the solvent-
treated cells using the formula: % Viability = (uFcompound/uFsolvent) × 100, where uFcompound is the
mean resorufin fluorescence in compound-treated wells and uFsolvent is the mean fluorescence in
the solvent-treated wells. Compound concentrations resulting in a 50% (CC50) reduction in cell
viability was determined by non-linear regression curve fitting using GraphPad Prism 5.04. The
cytotoxic control wells consisted of serial dilutions of amphotericin B, starting from a final
concentration of 100 µM and 0.5% final DMSO concentration.
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Cytological Profiling of Antimalarial Drug Effects. The effects of each selected antimalarial
compound on trophic and schizont development, egress and merozoite invasion, and on the para-
site mitochondrial membrane potential were determined by high content imaging using a four-time
point experimental set-up. Synchronized P. falciparum (strain HB3)-infected cultures at 1.5%
hematocrit and 5% parasitaemia were treated for 36 h starting at either the early ring stage (6 hpi),
late ring stage (18 hpi), or at the late trophozoite (30 hpi) stage with either test or reference
compounds at a single maximum effect concentrations of 10 µM (i.e. the concentration at which
no further increase in culture parasitaemia is observed following drug treatment). Drug effects on
egress and invasion were determined following a 24-h treatment with each compound added at the
mid-schizont stage (42-hpi). Cultures were sequentially diluted in staining and fixing solutions as
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described above, and imaged using an Operetta 2.0 microscopy system. Following parasite stage
quantification using our customized image-mining algorithms, stage accumulation index (stage
proportion in test wells relative to same stage proportion in solvent control wells) were calculated
and used to assess the rapid acting effects of each compound on trophozoite and schizont
development in vitro. Meanwhile, following the 24-h drug treatment at the 42-hpi, the resulting
schizont accumulation index was compared against the total parasitaemia reduction index
(decrease in well parasitaemia relative to control parasitaemia) to distinguish between egress
inhibition (high schizont accumulation and parasitaemia reduction indices) and drug effects on
extracellular merozoites (low schizont accumulation index and high parasitaemia reduction index).
The drug effects on parasite mitochondrial activities were also assessed by comparing the
proportion of viable (Mitotracker-positive) parasitaemias in drug-treated wells relative to that in
the solvent controls following a 36-h treatment allowing for early ring development into the
schizont stage.
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Phase I Metabolic Stability Study. Liver metabolic stability was determined in human and
mouse liver microsomes. The compound (1 µM) was mixed with human or rat liver microsomes
(0.5 mg/mL) [Gentest, BD Biosciences] in 100 mM potassium phosphate buffer (pH 7.4) and
incubated at 37 °C for 5 min. The reaction was initiated by NADPH regeneration solution [BD
Biosciences] and terminated by three times volume of ice-cold acetonitrile with imipramine (80
ng/mL) as internal standard at single-time-point 30 min. After pre-treatment of biological samples
with vortex and centrifuge, the samples were analyzed by LC/MS/MS system.
Pharmacokinetic Study. The carbohybrid-based antimalarial candidates (8aA and 11aA) were
administered by intraperitonial injection (1 and 5 mg/kg) in SD male rats (n = 5). Our candidate
compounds were prepared as a solution [DMSO, PEG400, Tween 80 and hydroxylpropyl-β-
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cyclodextrin (HPβCD) solution in PBS (final 10% HPβCD) at 10:10:2.5:77.5, v:v:v:v %]. Blood
samples were taken at 10 and 30 min and at 1, 4, 8, and 24 h post-injection. After the purification
of plasma by centrifugation, the concentration of carbohybrid-based antimalarial candidates was
analyzed using Agilent 6460 LC/MS/MS system [Agilent] using electron spray ionization and a
reverse-phase column [Hypersil GOLD C18, 50 × 2.1 mm, Thermo Scientific]. Pharmacokinetic
parameters were obtained after the analysis of plasma concentration-time plot with WinNonlin
software [Pharsight].
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In Vivo Efficacy Assessment of Antimalarial Drug Candidates 8aA and 11aA. Compounds
8aA and 11aA that exhibited potent in vitro multi-stage anti-plasmodial activities, selectivity
indices >600, good metabolic stability and intraperitoneal PK properties were selected for in vivo
efficacy assessment in Plasmodium chabaudi-infected BALB/c mice. Each group of five mice was
infected by i.p. injection of 2 × 107 parasitized erythrocytes per mouse. Twenty-four hours later
(Day 1), thin smears were prepared from tail blood and used to determine the pre-treatment
parasitaemias. This was immediately followed by i.p. injection of the compounds to final doses of
0.03, 0.3, 3, 10, or 30 mg/kg body weights per group. These treatments were repeated for two
additional days, followed by parasitaemia assessment by light microscopy examination of Giemsa-
stained thin smears on Day 4 post-infection. Probit analyses of the drug effects were then
undertaken as previously described.32–34
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Figure 1. Stage-specific activities of selected antimalarial agents as determined using a novel
image-based cytological profiling method. Tightly synchronized P. falciparum (HB3, W2 or K1
strains) were treated with 10 µM final concentration of each antimalarial agent at (A) the 6 hpi
(hour post-invasion, early ring), 18 hpi (late ring), or 30 hpi (mid-trophozoite) time-points for a
continuous duration of 36 h, or (B) at the 42 hpi time-point (mid-schizont) for 24 h. The parasite
nuclei, mitochondria, and host erythrocytes were then stained with DAPI, Mitotracker Red
CMXRos, and wheat germ agglutinin AlexaFluor-488, respectively, and the proportion of infected
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and viable parasitaemia determined by automated image mining. (A) Heat-map and parasite stage
accumulation index at each treatment time-point. ER: Early rings, LR: late rings, T: trophozoites,
S: schizonts. (B) A plot of parasitaemia reduction index (PRI) against schizont accumulation index
(SAI) indicating the rapid-acting effects of E64 and artemisinin (ART) on schizonts, and the
invasion-specific effect (high PRI and low SAI) of N-acetylglucosamine (GlcNAc) against all
three parasite strains. Accumulation or reduction indices greater than the control's mean plus three
standard deviations (n = 3) were considered significant.
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Figure 2. Cellular modes of action of 2-aminopyrimidine-based carbohybrid compounds. Tightly
synchronized P. falciparum (K1 strain) were treated at the 6 hpi (early ring), 18 hpi (late ring), or
30 hpi (mid-trophozoite) time-points with 10 µM final concentration 1 or 2 for a continuous
duration of 36 h. Parasite nuclei, parasite mitochondrial, and the host erythrocytes were then
stained with DAPI, Mitotracker Red CMXRos, and wheat germ agglutinin AlexaFluor-488,
respectively, and the proportion of infected and viable parasitaemia determined by automated
image analysis. (A) Heat-map and parasite stage accumulation index at each treatment time-point.
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ER: Early rings, LR: late rings, T: trophozoites, S: schizonts. (B) Representative images of
parasitized cultures showing blockage of further development of "Early Ring", late ring ("Ring"),
or "Trophozoite" stage parasites in 1 or 2-treated cultures compared to the DMSO-treated control.
The mitotracker-positive parasitized erythrocytes are indicated as "Live" whereas mitotracker-
negative cells are labeled as "Dead".
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Figure 3. Chemical structures and probit analyses of in vivo parasitaemia reduction effects of
carbohybrid-based 2-aminopyrimidine 8aA and 11aA derivatives. Mice (n = 5 per group) were
infected with 2 × 107 parasitized erythrocytes per mouse followed by a 3 day treatment from day
1 post-infection with variable doses (0.03, 0.3, 3.0, 10.0, or 30.0 mg/kg body weight) of compound
8aA or 11aA. Day 4 parasitaemia were then quantified by light microscopy examination of thin
smears, and used in probit plots to analyze the dose-response effects.
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Scheme 1. Synthesis of carbohybrid-based molecular frameworks.
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Dinucleophile
HetAr
Reagents and conditions: (a) TrtCl, DIPEA, DMAP, DCM, rt; (b) PMBCl, NaH, DMF, 0 °C to rt;
(c) POCl3, DMF, 0 °C to rt; (d) Dinucleophiles, K2CO3, EtOH/THF, rt; (e) MsCl, TEA, DCM,
0 °C to rt; (f) p-TsOH, MeOH, rt; (g) NaN3, DMF, 120 °C; (h) TrtCl, DIPEA, DCM, rt / BnBr,
NaH, DMF, rt / MsCl, TEA, DCM, 0 °C to rt.
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Table 1. Antimalarial and cytotoxicity profiles of 2-aminopyrimidine-based hit compounds
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9
EC50 (M)*
Selectivity
Index**
Compounds
K1
HB3
W2
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>450
>5550
>226
>187
CQ
ART
1
0.2080.015 0.0330.005 0.2210.012
0.0030.001 0.0180.004 0.0110.001
0.2510.042 0.4420.080 0.3200.038
0.5350.048 0.4480.061 0.4060.066
2
* EC50 values are meansSD of two independent experiments each done in duplicate. **Selectivity indices are
calculated as the ratio of 50% cytotoxicity (CC50) values against HepG2 cells to the highest parasitological EC50 values
of the three P. falciparum strains. The CC50 of amphotericin B (positive cytotoxicity control) was 403.2 M using
the resazurin-based assay as described in the “Materials and Methods” section.
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Table 2. Antimalarial and cytotoxicity profiles of 2-aminopyrimidine-based hit compounds
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9
EC50
Cpds.
(µM)
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(K1 strain)
HetAr
R1
R2
R3
R4
R5
B
A
C
D
E
OPMB
OPMB
OPMB
OPMB
OPMB
H
H
OH
OH
OH
OH
OH
OH
OH
OMs
OMs
OMs
OMs
OMs
OMs
OMs
H
H
H
Trt
Trt
Trt
Trt
Trt
Trt
Trt
Trt
Trt
Trt
Trt
Trt
Trt
H
0.351
0.200
0.578
0.352
0.396
0.559
0.478
0.116
0.225
0.160
0.158
0.198
0.237
5.032
5.447
18.01
0.092
0.371
0.167
0.281
1.424
1.534
1.197
6.665
11.72
2.897
1.043
2
H
H
7aA
H
7aC
H
H
7aD
H
H
7aE
A
E
OPMB
OPMB
H
H
7bA
7bE
H
H
A
C
D
E
OPMB
OPMB
OPMB
OPMB
H
H
8aA
H
H
8aC
H
H
8aD
H
H
8aE
A
A
A
A
D
A
E
OPMB
OPMB
H
H
8bA
8bE
H
H
OPMB
OPMB
OPMB
OPMB
OPMB
H
H
9aA
H
N3
N3
N3
N3
N3
N3
N3
N3
N3
N3
N3
H
H
10aA
10aD
11aA
11aE
11bA
11bE
12aA
12aE
12bA
13aA
13bA
14bA
15bA
H
H
H
H
H
Trt
Trt
Trt
Trt
Bn
Bn
Bn
Ms
Ms
PMB
PMB
H
H
A
E
OPMB
OPMB
H
H
H
H
A
E
OPMB
OPMB
H
H
H
H
A
A
A
A
A
OPMB
H
H
OPMB
H
H
OPMB
OPMB
OPMB
H
H
OH
OMs
H
H
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Table 3. Microsomal stability and in vivo pharmacokinetic properties of candidate compounds
8aA and 11aA*
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Microsomal stability
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(% remaining after
30 min, n =3)
PK study (n = 5)
Cmax
Compound
AUC
(g/mL
× h)
Dose
(mg/kg)
Human
Mouse
Tmax (h)
T1/2 (h)
(g/mL)
1
5
1
5
1.000.00 1.800.40 9.791.47 10.34.62
1.000.00 12.99.30 41.326.1 10.612.1
3.401.34 1.030.30 8.201.62 6.360.70
4.000.00 3.071.03 26.68.37 5.781.32
8aA
88.18.01 88.65.43
89.07.46 81.67.48
11aA
*Data represent mean ± SD.
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