Accepted Manuscript

One-pot, multi-component synthesis and structure-activity relationships of peptoid-

based histone deacetylase (HDAC) inhibitors targeting malaria parasites

Daniela Diedrich, Katharina Stenzel, Eva Hesping, Yevgeniya Antonova-Koch,

Tamirat Gebru, Sandra Duffy, Gillian Fisher, Andrea Schöler, Stephan Meister,
Thomas Kurz, Vicky M. Avery, Elizabeth A. Winzeler, Jana Held, Katherine T.
Andrews, Finn K. Hansen

PII: S0223-5234(18)30790-6
DOI: 10.1016/j.ejmech.2018.09.018
Reference: EJMECH 10729

To appear in: European Journal of Medicinal Chemistry

Received Date: 10 April 2018

Revised Date: 5 September 2018
Accepted Date: 6 September 2018

Please cite this article as: D. Diedrich, K. Stenzel, E. Hesping, Y. Antonova-Koch, T. Gebru, S. Duffy, G.
Fisher, A. Schöler, S. Meister, T. Kurz, V.M. Avery, E.A. Winzeler, J. Held, K.T. Andrews, F.K. Hansen,
One-pot, multi-component synthesis and structure-activity relationships of peptoid-based histone
deacetylase (HDAC) inhibitors targeting malaria parasites, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.09.018.

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 ACCEPTED MANUSCRIPT
One-pot, multi-component synthesis

CN

O Et3N,
O HN O
NaOMe
+ HO
H H 4Å MS, MeOH H
H N
H
45°C, 150W, ~2h N
H2N O OH
·HCl O O
H2NOH
·HCl O
1u
Pf3D7: IC50 = 4 nM (SI: 2496)
PfDd2: IC50 = 1 nM (SI: 9990)
PbEEF: IC50 = 25 nM (SI: 400)

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 ACCEPTED MANUSCRIPT

One-pot, multi-component synthesis and structure-activity

relationships of peptoid-based histone deacetylase (HDAC)

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inhibitors targeting malaria parasites

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Daniela Diedrich,a Katharina Stenzel,a,b Eva Hesping,b Yevgeniya Antonova-Koch,c Tamirat

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Gebru,d Sandra Duffy,b Gillian Fisher,b Andrea Schöler,e Stephan Meister,c Thomas Kurz,a Vicky

M. Avery,b Elizabeth A. Winzeler,c Jana Held,d Katherine T. Andrews,*,b,# Finn K. Hansen*,a,e,#

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a
Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf,

Universitätsstr. 1, 40225 Düsseldorf, Germany.

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b
Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan Campus,
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QLD 4111, Australia.

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c
Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla,

California 92093, United States.

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d
Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstraße 27, 72074
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Tübingen, Germany.
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e
Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Leipzig University, Brüderstraße

34, 04103 Leipzig, Germany.

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*corresponding authors:

Prof. Dr. Finn K. Hansen, Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Leipzig

University, Brüderstraße 34, 04103 Leipzig, Germany. Tel.: +49 (0)3419736801 Fax: (+49) 341

97 36889. E-mail: finn.hansen@uni-leipzig.de.

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Prof. Dr. Katherine T. Andrews, Griffith Institute for Drug Discovery, Don Young Road, Nathan

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Campus, Griffith University, QLD 4111, Australia. Tel.: (+61) 73735 4420. Fax: (+61) 73735

6001. E-mail: k.andrews@griffith.edu.au.

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#
FKH and KTA contributed equally to this work as senior authors.

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Abstract
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Malaria drug discovery has shifted from a focus on targeting asexual blood stage parasites, to the

development of drugs that can also target exo-erythrocytic forms and/or gametocytes in order to
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prevent malaria and/or parasite transmission. In this work, we aimed to develop parasite-

selective histone deacetylase inhibitors (HDACi) with activity against the disease-causing
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asexual blood stages of Plasmodium malaria parasites as well as with causal prophylactic and/or
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transmission blocking properties. An optimized one-pot, multi-component protocol via a

sequential Ugi four-component reaction and hydroxylaminolysis was used for the preparation of
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a panel of peptoid-based HDACi. Several compounds displayed potent activity against drug-
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sensitive and drug-resistant P. falciparum asexual blood stages, high parasite-selectivity and
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submicromolar activity against exo-erythrocytic forms of P. berghei. Our optimization study

resulted in the discovery of the hit compound 1u which combines high activity against asexual

blood stage parasites (Pf 3D7 IC50: 4 nM; Pf Dd2 IC50: 1 nM) and P. berghei exo-erythrocytic

forms (Pb EEF IC50: 25 nM) with promising parasite-specific activity (SIPf 3D7/HepG2
: 2496, SIPf
Dd2/HepG2
: 9990, and SIPb EEF/HepG2: 400).

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Keywords

Peptoid, HDAC inhibitor, histone deacetylase, malaria, Plasmodium falciparum

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Abbreviations
EEF, exo-erythrocytic form; Et3N, triethylamin; HDAC, histone deacetylase; HDACi, histone

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deacetylase inhibitor; h, hour; MeOH, methanol; MS, molecular sieve; SI, Selectivity Index

(mammalian cell IC50/P. falciparum IC50); Pf, Plasmodium falciparum; PfGAPDH, Plasmodium

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falciparum Glyceraldehyde-3-phosphate dehydrogenase; PfESG, P. falciparum early stage
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gametocytes; PfLSG, P. falciparum late stage gametocytes; Pb, Plasmodium berghei; RT, room

temperature; SAHA, suberoylanilide hydroxamic acid.

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1. Introduction

According to the World Health Organization (WHO), there were an estimated 216 million cases
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of malaria worldwide in 2017, and approximately 445,000 deaths due to this disease [1]. Six
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species of Plasmodium cause malaria in humans: P. falciparum, P. vivax, P. ovale curtisi, P.

ovale wallikeri, P. malariae and the zoonotic simian parasite, P. knowlesi. P. falciparum causes
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the highest mortality, predominantly in Sub-Saharan Africa, with P. vivax also being responsible
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for significant global morbidity [1]. Considerable research activities have been devoted to the

development of vaccines, however it is unlikely that a highly effective and broadly applicable

malaria vaccine will be deployed in the near future [2]. Although the GlaxoSmithKlein (GSK)

RTS,S malaria vaccine has been approved by the European Medicines Agency (EMA) and the

WHO supports pilot studies [2], the efficacy of this vaccine against malaria in children in

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endemic areas was only moderate [3]. Vector control and antimalarial drugs therefore continue to

be a mainstay for malaria prevention and treatment. While highly effective malaria treatment

drug combinations are available, a major limitation is reduced clinical efficacy and/or

development of drug-resistant Plasmodium parasites [4]. Parasite resistance has been reported to

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all currently used antimalarial drugs, including artemisinin combination therapies (ACTs), the

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current gold standard treatment for uncomplicated P. falciparum malaria [5]. Spread of ACT-

resistance to sub-Saharan Africa would be devastating, potentially impacting the significant

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improvements in malaria morbidity and mortality that have been made over the past decade.

Alternatives to ACTs are urgently needed to prime the malaria drug discovery pipeline and to

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ensure that new treatment options are available in the event that ACTs fail. Next generation
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drugs for malaria also need to address the global agenda that is striving for elimination and

ultimately eradication of this disease. This includes development of new drug candidates that are
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not only active against disease-causing asexual intraerythrocytic stage malaria parasites, but also
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against liver and sexual gametocyte stage parasites to prevent malaria symptoms from
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developing and to interrupt transmission [5–7].

Histone deacetylases (HDACs) are emerging drug targets for various diseases and some
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HDAC inhibitors (HDACi) are already FDA approved for cancer therapy (vorinostat,

romidepsin, belinostat, and panobinostat) [8–11]. There is also growing evidence that HDACi
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may have therapeutic potential in a variety of non-cancer diseases including inflammation,

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immune disorders, HIV/AIDS, neurodegenerative diseases, and parasitic diseases [12–15].

Interestingly, among parasitic diseases some parasites appear to be more sensitive to HDACi

than others [16,17]. Notably, P. falciparum parasites have been shown to be highly sensitive to

pan-reactive HDACi (Table 1) [14,15].

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Table 1. In vitro activity profiles of selected HDAC inhibitors against malaria parasites.

HDACi P. falciparum Cytotoxicity Selectivity

Name Structure
Type IC50 [nM] IC50 [nM] Indexa

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H H
N O
N
class I O
O
HN O

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Apicidinb HN 200 50 – 100 <1
selective O

N
O

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class I
Entinostatb 7,800 – 8,300 >20,000 >2
selective

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O
NH O
class I HN
S
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c
Romidepsin O S NH 90 – 130 1 – <5 <1
HN
selective O
O

O
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Trichostatin A
pan 8 – 11 200 18 – 25
(TSA)d
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Vorinostat
pan 120 – 190 5,170 – 5,500 27 – 46
(SAHA)c
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Belinostatc pan 60 – 130 1,420 – 2,370 11 – 40

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Panobinostatc pan 10 – 30 70 – 180 2 – 18

a
Selectivity index - mammalian cell IC50/IC50 P. falciparum – larger values indicate greater
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malaria parasite selectivity; bData from reference [15]. cData from reference [18]. dData from
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reference [19].

Human HDACs (hHDACs) are divided into different classes according to their co-factor

dependence and homology: class I (hHDAC1, 2, 3 and 8), class II (subdivided into class IIa:

HDAC4, 5, 7 and 9; and class IIb: HDAC6 and 10) and class IV (HDAC11) are zinc-dependent

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isotypes, whereas class III HDACs are NAD+-dependent enzymes [20]. Thus far, five HDAC

isoforms (PfHDACs) have been identified in P. falciparum parasites [15]. Three of these

PfHDACs are homologues of zinc-dependent human class I (PfHDAC1) or class II HDACs

(PfHDAC2 and 3), whereas two are class III homologues (PfSir2A and B) [15,21–23]. P.

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falciparum HDAC1 (PfHDAC1) is the only PfHDAC enzyme available in recombinant form

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[24,25], however its purity is low and catalytic activity in the absence of endogenous cofactors

has been questioned [26]. PfHDAC1 has high amino acid sequence identity to hHDAC1 (61%)

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and hHDAC2 (62%), potentially compromising development of highly parasite-selective HDACi

that target this enzyme with low activity against human class I human enzymes. Homology

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modeling studies suggest that the zinc-coordinating residues in HDACs and the tubular cavity
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between the zinc and the surface of the HDAC enzymes are well conserved between human class

I HDAC enzymes and PfHDAC1 [16,17,27–30]. It is therefore not surprising that several studies
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with pan- and class I-selective human HDACi revealed potent in vitro anti-plasmodial activity in
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combination with varyingly levels of toxicity to human cells (see Table 1) [27–32]. Some
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HDACi have been shown to target multiple malaria parasite species (P. falciparum and P. vivax),

to act against multi-drug resistant P. falciparum lines and, for a limited number, to have in vivo
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efficacy in animal models of malaria [14,15,29,32–35]. While most work to date has focused on

asexual blood stage parasites, this class of compounds also has promising activity against liver-
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stage Plasmodium parasites [29,36,37] and activity against late stage (IV-V) gametocytes [36–
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39]. This raises the possibility of developing HDACi with potent activity against multiple

Plasmodium life cycle stages and these recent findings underscore the potential for HDACi

development for malaria therapy.

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In addition to targeting multiple Plasmodium life cycle stages, other features of next generation

anti-plasmodial HDACi should include potent killing of Plasmodium parasites and low toxicity

to human cells as well as appropriate bioavailability and pharmacokinetic profiles for

advancement to pre-clinical and early clinical studies [24–26]. Of particular relevance to malaria,

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there is growing evidence that there are intrinsic toxic side effects associated with inhibition of

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human class I isoforms and that this prevents the application of broad spectrum and class

I-selective inhibitors to areas outside of oncology because of a small therapeutic window [40,41].

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Due to their low toxicity to normal mammalian cells [40,41] we, and others, have hypothesized

that hHDAC6 inhibitors might be a better starting point for the development of parasite-selective

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anti-plasmodial HDACi [37,42]. In a previous communication we reported the rational design
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and synthesis of a novel type of peptoid-based HDACi that preferentially targets human HDAC6

over representative examples of all other zinc dependent HDAC classes (class I: HDAC2; class
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IIa: HDAC4; and class IV: HDAC11) [43]. In this project we report a systematic and
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comprehensive structure-activity relationship (SAR) study of these peptoid-based HDACi as

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antiplasmodial agents. To this end, we developed a highly efficient one-pot, five-component

approach for the diversity-oriented synthesis of our peptoid-based HDACi. All compounds were
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tested for in vitro activity against asexual intraerythrocytic stage P. falciparum parasites and

cytotoxicity against mammalian cells. Selected compounds were further screened in vitro for
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their activity against P. berghei exo-erythrocytic liver stages as well as against different P.
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falciparum gametocyte stages [44,45]. Furthermore, we investigated the ability of these

compounds to hyperacetylate histones in asexual stage P. falciparum parasites as a marker of

HDAC inhibition. Herein, we present the design, multicomponent synthesis and antimalarial

properties of a new type of parasite-selective antiplasmodial HDACi with dual stage activity.

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2. Results and discussion

2.1 One-pot synthesis of peptoid-based HDACi.

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We previously described an efficient multi-component approach for the preparation of the

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peptoid-based HDACi 1a-l [43]. Briefly, this methodology was performed at room temperature

under conventional reaction conditions using a two-step protocol. First, the Ugi four-component

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reaction (U-4CR) was used to afford the key intermediates of type 6 in 43-85% yield after

purification by flash column chromatography (step I, Table 2). The target compounds were

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subsequently prepared in 42-69% yield by hydroxylaminolysis followed by purification via flash
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column chromatography (step II, Table 2). However, both steps required relatively long reaction

times (step I: 72 h; step II: 16 h) [43].

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In this project, we aimed at improving the overall efficiency of our synthetic strategy by
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developing a one-pot, five-component approach. Furthermore, we utilized microwave irradiation

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in order to shorten reaction times. These modifications should enable higher yields and a more

time efficient synthetic pathway for the preparation of peptoid-based HDACi. For this purpose,
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we initially investigated several reaction conditions independently for both steps using 1o as
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model compound. The results are summarized in Table 2. Based on our optimization previously
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done for the ‘classical’ U-4CR [43,46], we performed all microwave-assisted syntheses in dry

methanol in the presence of 4 Å molecular sieve (4 Å MS) using a slight excess of the amine 2

(1.2 eq) and paraformaldehyde 3 (1.2 eq) compared to the carboxylic acid 4a (0.5 M

concentration) and isocyanide 5a (0.5 M concentration) components.

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Table 2. Optimization of reaction conditions.a

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entry MW T time yieldb

[W] [°C] [min] [%]
step I 1c
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150 45 60 63
2 150 45 120 79
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3 150 45 60 85
4 150 45 30 68
5 150 70 60 71
6 150 55 60 76
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step II 7 150 45 20 63
8 150 45 30 77
9 150 70 20 48
10 150 55 20 75
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11 150 55 30 80
a b
All reactions were monitored by TLC. Isolated yield after flash column chromatography. cAll
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four components were added at once.

We first attempted the microwave-assisted synthesis of the Ugi-product 6 by subjecting all four

Ugi components and triethylamine to microwave irradiation (150 W, 45°C) for 60 min. The

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desired product 6 was isolated in a moderate yield of 63% (Table 2, entry 1). However, since a

high yield in the first step is crucial for a successful one-pot approach, the U-4CR was further

optimized. The imine formation represents a key step in the U-4CR. Thus, to optimize the

reaction conditions, we focused on the pre-formation of the imine under microwave irradiation.

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A mixture of 4-aminomethyl benzoic acid methyl ester hydrochloride (2), paraformaldehyde (3),

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triethylamine and crushed 4 Å molecular sieves (4 Å MS) in dry methanol was irradiated at 45°C

(150 W). The progress of the imine-formation was monitored by thin layer chromatography after

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2, 5, 10 and 20 min. After 20 min of irradiation the imine-formation was completed and all

further reactions were performed by a pre-formation of the imine under microwave irradiation at

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45°C for 20 min before adding the isocyanide and carboxylic acid components. Next, we
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investigated the U-4CR at different temperatures and reaction times under microwave irradiation

(step I, Table 2, entry 2-6). Best results were achieved at 45°C and 60 min (entry 3). These
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conditions provided the desired Ugi product in a very good isolated yield of 85%.
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We next turned our attention towards the hydroxylaminolysis (step II, Table 2). In all cases, a
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purified sample of the methyl ester intermediate 6 was treated with a freshly prepared solution of

hydroxylamine under microwave irradiation (150 W). In an initial attempt, we performed the
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hydroxylaminolysis at 45 °C for 20 min to afford the desired hydroxamic acid 1o in 63% isolated
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yield (entry 7). A slight increase in temperature from 45°C (entry 8) to 55 °C (entry 10, 11) and
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extension of the reaction time from 20 min (entry 10) to 30 min (entry 11) led to increased yields

of 1o. In contrast, performing the reaction at even higher temperature (70°C) resulted in a

decreased yield (48%, entry 9). Best results were achieved at 55°C and a reaction time of 30 min

(80% isolated yield, entry 11).

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In the last step to develop a rapid and diversity-oriented synthesis of peptoid-based HDACi of

type 1, we combined the optimized conditions for both steps in a one-pot approach without

isolation and purification of the Ugi product 6. By performing both optimized steps in the same

reaction vessel in a sequential fashion under microwave irradiation, the desired target compound

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1o could be obtained in less than two hours (Scheme 1). Purification by flash column

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chromatography afforded 1o in 72% yield.

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This optimized one-pot, five-component protocol via a sequential Ugi-reaction and

hydroxylaminolysis was then used for the preparation of the desired peptoid-based HDACi. In all

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cases, the crude products were purified by flash column chromatography and the HDACi 1m-x
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were isolated in 59-82% yield (Scheme 1). Taken together, this one-pot multi-component

approach offers a lot of utility including (1) time efficiency, (2) high atom economy, (3) absence
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of protecting groups, and (4) multiple points of diversity.

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Scheme 1. Microwave-assisted multi-component synthesis of target compounds 1m-x via the

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optimized one-pot approach.a

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a
Reagents and conditions: (a) (i) 2, 3, Et3N, MeOH, 4Å MS, 150 W, 45°C, 20 min; (ii) R1-NC,

R2-COOH, 150 W, 45°C, 60 min; (b) H2NOH·HCl, Na, MeOH dry, 150 W, 55°C, 30 min.

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2.2 In vitro inhibition of P. falciparum asexual intra-erythrocytic parasite growth, cytotoxicity

and parasite selectivity.

All compounds were tested for activity against P. falciparum asexual intra-erythrocytic parasites

using a tritiated hypoxanthine uptake assay. Compounds 1a-x had calculated log P values

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between 0.5 and 3.2 and were thus expected to be cell permeable (see Table 3 for calculated log

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P values). When tested for in vitro activity against the drug sensitive P. falciparum line 3D7,

compounds 1a-x revealed 50% inhibitory concentration values (IC50s) in the range of 4–158 nM

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(Table 3). The activity of all compounds was either not significantly different to the reference

HDACi vorinostat (p > 0.05), or significantly better (1a, 1d, 1g, 1h, 1i, 1k, 1m, 1t, 1v with p <

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0.05). Based on our synthetic protocol the peptoid-based cap group can be further divided into a
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isocyanide (R1) and carboxylic acid (R2) region (Table 3). We could not identify clear structure-

activity relationships (SARs) for the isocyanide region (Table 3). However, the screening
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provided interesting SARs with regards to the carboxylic acid region. Since the highest number
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of compounds (10 examples, 1f-i and 1p-u) were synthesized bearing cyclohexyl in the
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isocyanide region (R1), notable SARs of the carboxylic acid region will be discussed for

compounds with R1 = cyclohexyl. Compound 1f (R2 = Ph) bearing no substitution at the phenyl
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ring in the carboxylic region exhibited an IC50 of 59 nM against the 3D7 line of P. falciparum.

The replacement of the phenyl ring by a bulky naphthyl group (as in 1g) resulted in an increased
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antiplasmodial activity (Pf 3D7 IC50: 25 nM). The introduction of methyl groups in m- and/or p-
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position provided HDACi with somewhat improved activity compared to 1f (see 1i, 1q, 1s, 1t,

Table 3), whereas compound 1r bearing a methyl group in o-position (R2 = 2-CH3-Ph, Pf 3D7

IC50: 76 nM) showed decreased activity. It is worth noting that a similar trend in regards to the

position of methyl groups was observed in our previous report on alkoxyamide-based HDACi

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[36]. Interestingly, compound 1h (R2 = 4-Me2N-Ph, Pf 3D7 IC50: 9 nM) containing a N,N-

dimethylamino group in p-position showed an ~7-fold higher activity than 1f, while the

corresponding m-substituted analogue 1p (R2 = 3-Me2N-Ph, Pf 3D7 IC50: 44 nM) revealed

reduced activity compared to 1h. Notably, the replacement of the N,N-dimethylamino group in

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1h by an isosteric and less polar isopropyl group provided HDACi 1u (R2 = 4-i-Pr-Ph, Pf 3D7

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IC50: 4 nM) which exhibited the highest activity of all synthesized compounds.

Subsequently, we assessed the antiplasmodial activity of 1a-x against the multidrug resistant P.

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falciparum Dd2 line. All compounds displayed nanomolar activity against this line, with IC50

values ranging from 1 – 118 nM (Table 3) and all compounds exceeded the potency of vorinostat

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(SAHA, Pf Dd2 IC50: 146 nM). Overall, we observed similar IC50 values and structure-activity
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relationships against drug resistant (Dd2) and drug sensitive (3D7) asexual P. falciparum

parasites indicating that resistance mechanisms developed in the Dd2 line do not affect the in
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vitro activity of this series of antiplasmodial HDACi. Again, compound 1u (R1 = c-Hex; R2 = 4-
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i-Pr-Ph) was the most active compound from this series (Pf Dd2 IC50: 1 nM).
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To investigate the parasite-specific selectivity of the peptoid-based HDACi, cytotoxicity of 1a-x

was tested against normal human neonatal foreskin fibroblasts (NFFs). The cytotoxicity and
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selectivity indices (SIs) of 1a-x are summarized in Table 3. Interestingly, the substitution in the
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isocyanide region (R1) had little impact on the cytotoxicity, whereas a clear trend was observed
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in the case of the carboxylic acid region (R2). Compounds 1d, 1h, 1l, and 1t-v with a

p-substituted carboxylic acid component (R2) showed cytotoxicity in the single digit micromolar

concentration range. In contrast, all other compounds revealed low cytotoxicity (NFF IC50:

>10,000 nM). Interestingly, compounds with substituents in m- and p-position (see compounds

1a and 1q) did not show cytotoxicity at the highest concentration tested. The reason for the

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increased cytotoxicity of derivatives with mono-substitution in p-position of the carboxylic acid

region is not yet known. However, these data indicate that derivatives bearing substituents in

meta- or ortho-position of the carboxylic acid region might be a good starting point for the

development of analogues with reduced cytotoxicity.

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A comparison of the IC50 values for asexual blood stage P. falciparum parasites with those

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obtained for NFF cells indicates an encouraging parasite-selectivity for this novel class of

HDACi. All compounds, except 1b and 1f, had at least a 100 fold higher activity against asexual

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blood stage parasites vs mammalian cells (Table 3), whereas the reference HDACi vorinostat

showed only modest parasite-selectivity (SI range: 33–35). The highest parasite-selectivity was

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observed in the case of 1u (SI range: >1000 – >4000).
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Table 3. Calculated logP values, in vitro activity against asexual blood stages of P. falciparum
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parasites, cytotoxicity and selectivity indices of 1a – 1x.

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P. falciparumb IC50
1 2 a NFFc
Co. R R logP [nM] SI ranged
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IC50 [nM]
Pf 3D7 Pf Dd2
1a t-Bu 3,4-Me-Ph 1.44(±0.53) 20(±10) 12(±8) >10,000 >500->833
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1b t-Bu Ph 0.52(±0.52) 158(±51) 54(±21) >10,000 >63->185

1c t-Bu 1-Naphthyl 1.75(±0.52) 59(±22) 51(±20) >10,000 >169->196
1d t-Bu 4-Me2N-Ph 0.94(±0.55) 11(±4) 14(±6) 4,480 (±201) 320-407
1e t-Bu 3,5-Me-Ph 1.44(±0.53) 50(±17) 41(±23) >10,000 >200->244
1f c-Hex Ph 1.36(±0.51) 59(±11) 118(±57) >10,000 >85->169
1g c-Hex 1-Naphthyl 2.59(±0.52) 25(±18) 31(±12) >10,000 >323->400
1h c-Hex 4-Me2N-Ph 1.77(±0.54) 9(±3) 15(±6) 5,890 (±1,660) 393-654
1i c-Hex 3,5-Me-Ph 2.28(±0.52) 21(±16) 33(±15) >10,000 >303->476
1j 4-Tolyl Ph 1.89(±0.45) 67(±23) 53(±27) >10,000 >149->189
1k 4-Tolyl 1-Naphthyl 3.12(±0.45) 39(±17) 33(±21) >10,000 >256->303

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1l 4-Tolyl 4-Me2N-Ph 2.31(±0.47) 29(±14) 20(±11) 5,380(±4,990) 186-269

1m 4-Tolyl 3,5-Me-Ph 2.81(±0.45) 33(±11) 27(13) >10,000 >303->370
1n 4-Tolyl 3-Me2N-Ph 2.10(±0.47) 38(±35) 49(±14) >10,000 >204->263
1o t-Bu 3-Me2N-Ph 0.73(±0.54) 47(±21) 29(±18) >10,000 >213->345
1p c-Hex 3-Me2N-Ph 1.56(±0.54) 44(±10) 37(±27) >10,000 >227->270
1q c-Hex 3,4-Me-Ph 2.28(±0.52) 31(±2) 21(±9) >10,000 >323->476
1r c-Hex 2-Me-Ph 1.82(±0.52) 76(±26) 57(±13) >10,000 >132->175

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1s c-Hex 3-Me-Ph 1.82(±0.52) 43(±17) 48(±10) >10,000 >233->208
1t c-Hex 4-Me-Ph 1.82(±0.52) 16(±8) 11(±1) >3,000 >188->273
1u c-Hex 4-i-Pr-Ph 2.69(±0.52) 4(±1) 1(±1) >4,000 >1000->4000
1v n-Bu 4-Me2N-Ph 1.31(±0.54) 14(±4) 14(±4) >2,000 >143

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1w n-Bu 3-Me2N-Ph 1.10(±0.53) 51(±26) 43(±8) >10,000 >233->196
1x n-Bu 3,5-Me-Ph 1.81(±0.52) 47(±27) 56(±16) >10,000 >178->213
SAHA 0.86(±0.21) 139 (±73) 146(±22) 4,900(±1,240)e 33-35

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CQ 4.69(±0.32) 11(±2) 49(±17) 46,608(±14,420) 953-3628
a b
logP values were calculated using ACD/ChemSketch freeware version 12.01. Three

independent assays were carried out, each carried out in triplicate wells. Resistance indices (Ri;

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Dd2 IC50/3D7 IC50) range from 0.3-2.0 for all HDACi while the Ri of chloroquine is 4.6. cTwo
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independent assays were carried out, each carried out in triplicate wells. For compounds where

IC50 was not achieved in one or more assays, the highest common test concentration is given; dSI
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= NFF IC50/ P. falciparum IC50 – larger values indicate greater parasite selectivity. eData
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previously reported [39].

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Table 4. Cytotoxicity against HepG2 cells and selectivity indices of 1a, 1d, 1g-i, 1l, 1m, 1q, and

1t-v.
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Co. R1 R2 HepG2 95% CI SI rangea

IC50 [µM] forHepG2
IC50
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1a t-Bu 3,4-Me-Ph 25.94 19.65 – 34.24 1295-2158

1d t-Bu 4-Me2N-Ph 11.90 8.47 – 16.70 850-1082
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1g c-Hex 1-Naphthyl >50 >1613->2000

1h c-Hex 4-Me2N-Ph 7.80 5.43 – 11.21 520-867
1i c-Hex 3,5-Me-Ph 21.15 17.62 – 25.38 642-1010
1l 4-Tolyl 4-Me2N-Ph 5.81 4.04 – 8.35 200-291
1m 4-Tolyl 3,5-Me-Ph 21.39 16.82 – 27.20 648-793
1q c-Hex 3,4-Me-Ph 21.98 19.11 – 25.28 710-1048
1t c-Hex 4-Me-Ph 12.34 7.96 – 19.14 769-1118
1u c-Hex 4-i-Pr-Ph 9.99 7.14 – 13.99 2496-9990
1v n-Bu 4-Me2N-Ph 6.99 5.21 – 9.39 499
SAHA 2.51 1.63 – 3.86 17-18
a
SI = HepG2 IC50/P. falciparum IC50 – larger values indicate greater parasite selectivity.

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In order to identify HDACi with potent and parasite-specific activity, all compounds with an

average activity against asexual blood stages of ≤ 30 nM were further tested for cytotoxicity

against mammalian HepG2 cells (Table 4). In general, the HepG2 line was less sensitive to the

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HDACi of type 1 compared to NFF cells. Four of the eleven compounds tested revealed IC50

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values between 5.81 and 9.99 µM, two had moderate cytotoxicity in the range of 11.90 to 12.34

µM, while five compounds showed low cytotoxicity with IC50s > 20 µM. Notably, compound 1g

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showed no cytotoxicity against HepG2 cells at the highest concentration tested (HepG2 IC50 > 50

µM). Again, we observed the trend that derivatives with a mono-substitution in p-position of the

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carboxylic acid region exhibited higher cytotoxicity against HepG2 cells than the other
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compounds. A comparison of activity against asexual blood stage parasites vs HepG2 cells

showed that all compounds have a selectivity index of at least 200 (Table 4). In contrast,
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vorinostat showed only low parasite-selectivity (SI range: 17–18). The highest parasite-
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selectivity was found for compound 1u (SI range: 2496–9990). In summary, the peptoid-based
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HDACi possess potent activity against asexual blood stage parasites and, compared to other

HDACi with anti-plasmodial activity (see Table 1), an encouraging parasite-specific activity.
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2.3 Inhibition of human HDAC1 and HDAC6.

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The cytotoxicity screening against mammalian cells revealed some differences in the
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cytotoxicity of the peptoid-based HDACi (Table 3 and Table 4). In order to investigate whether

the differences in the toxicity against mammalian cells correlates with different human HDAC

isoform profiles, we decided to screen selected compounds in a biochemical assay against

hHDAC1 and hHDAC6 using ZMAL (Z-(Ac)Lys-AMC) as substrate. hHDAC6 was selected

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because several peptoid-based HDACi were previously identified as preferential hHDAC6

inhibitors [43,47]. Toxic side effects of HDACi have been primarily associated with inhibition of

human class I isoforms [40]. hHDAC1 was therefore chosen as representative class I isoform.

The three compounds with the highest cytotoxicity (1l, 1u and 1v) and the three compounds with

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the lowest toxicity (1a, 1g, and 1q) against HepG2 cells were chosen for the screening against

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hHDAC1 and hHDAC6. The results are presented in Table 5. As expected, all compounds

revealed potent activity against HDAC6 with IC50 values ranging from 0.030 – 0.064 µM.

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Interestingly, we observed some noteworthy differences in the activity against hHDAC1.

Compounds 1a, 1g, and 1q demonstrated only weak activity against hHDAC1 (IC50: 0.304 –

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0.552 µM), whereas 1l, 1u and 1v revealed a more potent inhibition of hHDAC1 (IC50: 0.040 –
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0.086 µM). Most probably, the higher cytotoxicity of 1l, 1u and 1v against human cells is

therefore related to the increased activity against human class I isoforms.

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Table 5. Activities of compounds 1a, 1g, 1l, 1q, 1u, 1v and the reference HDACi SAHA against
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human HDAC1 (hHDAC1) and HDAC6 (hHDAC6).

Co. R1 R2 hHDAC1 hHDAC6

IC50 [µM] IC50 [µM]
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1a t-Bu 3,4-Me-Ph 0.428(±0.015) 0.057(±0.007)

1g c-Hex 1-Naphthyl 0.552(±0.007) 0.031(±0.001)
1l 4-Tolyl 4-Me2N-Ph 0.080(±0.008) 0.030(±0.005)
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1q c-Hex 3,4-Me-Ph 0.304(±0.032) 0.032(±0.002)

1u c-Hex 4-i-Pr-Ph 0.086(±0.018) 0.064(±0.006)
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1v n-Bu 4-Me2N-Ph 0.040(±0.006) 0.035(±0.002)

SAHA 0.079(±0.010) 0.055(±0.011)

2.4 Effect of peptoid-based HDAC inhibitors on P. falciparum histone acetylation

The effect of selected compounds (1a, 1d, 1g, 1h, 1i, 1l, 1m, 1q, 1t, 1u, 1v) on P. falciparum

histone H4 hyperacetylation was assessed via protein hyperacetylation assays using trophozoite

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stage parasites treated for 3 h with 5x IC50 concentrations of each compound. Unlike the HDAC

inhibitor control, SAHA, which showed ~2.5-3.5 fold increased acetylation of bands

corresponding to either H4 (~11 kDa band; Figure 1 light grey) or H4 plus cross reactive bands

(H2B/H2Bv (~13-14 kDa); H2A.Z (~16 kDa); Figure 1 dark grey), the effect of the eleven

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peptoid-based HDAC inhibitors was variable. Three compounds (1d, 1h and 1l) caused ~2-fold

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or greater increase in hyperacetylation, while the remainder of the compounds caused only ~1.0-

1.5- fold increases.

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D
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C EP
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Figure 1. Hyperacetylation of P. falciparum histone H4 by peptoid-based HDAC inhibitors.

Western blot analysis of P. falciparum 3D7 protein lysates prepared from trophozoite-stage

infected erythrocytes treated for 3 h with 5x IC50 of test or control compounds. Negative controls

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were parasites exposed to the antimalarial drug chloroquine (CQ) or compound vehicle (0.2%

DMSO; 3h-C). (A) Western blot was carried out using anti-(tetra) acetyl-histone H4 antibody

and IRDye 680 goat anti-rabbit conjugate secondary antibody. Total protein per lane was

detected using REVERTTM Total Protein Stain on the same membrane. Representative Western

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blot shown. Protein molecular weight marker bands (kDa) are indicated. (B) Graph showing

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mean relative density (+/-SD) for two independent Western blot experiments. Western density

signals were normalized to total protein load in the respective lane and expressed as fold change

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compared to the 3h-C DMSO vehicle control (taken as 1.0; dotted line). Data are mean (±) SD

for two independent experiments. Dark grey bars show total density for all bands (~11 kDa band

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corresponds to the correct size of histone H4, with the doublet likely cross reactivity with
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acetylated forms of H2B/H2Bv (~13-14 kDa) and the ~16 kDa band cross reactivity with

H2A.Z) while light grey bars shows signal corresponding to the ~11 kDa H4 band only.
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2.5 In vitro activity against early-stage and late-stage P. falciparum gametocytes and P. berghei
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exo-erythrocytic forms.

The potential of this compound class to act as antiplasmodials with dual- or multi-stage activity
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was determined by evaluating their gametocytocidal potency and liver stage activity. Compounds

1a, 1d, 1g, 1h, 1i, 1l and 1m were first screened for their inhibition of gametocyte development
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from early stage I to stage III gametocytes (Pf ESG) and from late stage IV to stage V
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gametocytes (Pf LSG) using an imaging-based viability assay [44,45]. The results are

summarized in Table 6. All five compounds were found to have only moderate activity against

early (Pf ESG IC50 >2 µM) and late stage gametocytes (Pf LSG IC50 >1 µM). The best activity

against early and late stage gametocytes was observed for compound 1h (Pf ESG IC50 2.36 µM;

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Pf LSG IC50 1.81 µM). To further investigate the activity of this class of compounds against

mature stage V gametocytes, compounds 1a-x were tested in an ATP bioluminescence assay [48]

at two fixed concentrations of 0.5 and 5 µM. The most promising compounds from this primary

screen were subsequently tested in dose response to determine IC50 values (Table 6). In good

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agreement with the results from the imaging-based viability assay, the compounds showed only

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moderate activity in the ATP-based assay (IC50 >2 µM). Again, compound 1h showed the

highest activity of all tested compounds (IC50 2.46 µM).

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Table 6. Activity of peptoid-based HDAC inhibitors against different P. falciparum gametocyte

stages.
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Co. R1 R2 imaging-based viability assay ATP assay
Pf ESGa Pf LSGb Pf stage V gametocytesc
IC50 [µM]
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IC50 [µM] IC50 [µM]

1a t-Bu 3,4-Me-Ph 86%d 92%d nd
1d t-Bu 4-Me2N-Ph 4.41±0.66 2.72±0.48 7.69±3.26
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1g c-Hex 1-Naphthyl 4.46±0.54 97%d nd

1h c-Hex 4-Me2N-Ph 2.36±0.31 1.81±0.07 2.46±0.89
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1i c-Hex 3,5-Me-Ph 8.37±0.85 5.25±0.20 15.7±1.60

1j 4-Tolyl Ph nd nd 9.41±5.12
1l 4-Tolyl 4-Me2N-Ph 5.90±0.34 3.89±0.44 4.35±2.16
1m 4-Tolyl 3,5-Me-Ph 7.60±0.17 7.28±0.15 10.1±3.72
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1q c-Hex 3,4-Me-Ph nd nd 13.12±2.25

1u c-Hex 4-i-Pr-Ph nd nd 7.74±0.56
SAHA 1.41±0.13e 0.81±0.21e 2.80±0.83
a
P. falciparum NF54 early stage gametocytes (I-III); n=2, each in duplicate wells; artesunate
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(IC50: 0.003±0.001 µM) and pyronaridine (IC50: 0.029±0.001 µM) were used as reference
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compounds. bP. falciparum NF54 late stage gametocytes (IV-V); n=2, each in duplicate wells;

artesunate (IC50: 0.008±0.0008 µM) and pyronaridine (IC50: 1.71±0.33 µM) were used as
c
reference compounds. P. falciparum stage V gametocytes; at least two independent

experiments, each in duplicate wells; epoxomicin (IC50: 0.007±0.007 µM), methylene blue (IC50:

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0.48±0.35 µM) and chlorotonil A (IC50: 0.041±0.021 µM) were used as reference compounds.
d
Inhibition at 40 µM. eData previously reported [39]. nd, not determined.

Compounds with activity against liver stages (exo-erythrocytic forms) can potentially prevent

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disease development and be used in chemoprotection. We therefore profiled key compounds of

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this series for their activity against P. berghei exo-erythrocytic forms (Pb EEF) as described

previously [49]. In contrast to the activity against gametocytes, several compounds displayed

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potent activity against P. berghei exo-erythrocytic forms (Table 7). Six out of eleven compounds

tested (1d, 1h, 1l, 1t, 1u and 1v) revealed submicromolar activity with IC50 values ranging from

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0.025 to 0.91 µM. The two most potent compounds 1t (Pb EEF IC50: 0.084 µM) und 1u (Pb EEF
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IC50: 0.025 µM) both bear a cyclohexyl residue in the isocyanide region and a p-alkylsubstituted

phenyl group in the carboxylic acid region. These data indicate that this substitution pattern
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might be important for the development of peptoid-based HDACi with improved activity against
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liver stages.
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When we compared the IC50 values for P. berghei exo-erythrocytic forms with those obtained for

HepG2 cells, we found that most compounds showed only moderate parasite-specific activity
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(SIHepG2/Pb EEF
: 4–33). However, 1t and 1u, the compounds with the best activity against P.

berghei exo-erythrocytic forms, revealed high parasite-selectivity with selectivity indices of >
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100 (1t: SIHepG2/Pb EEF: 147 and 1u: SIHepG2/Pb EEF: 400).
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Table 7. Activity of peptoid-based HDAC inhibitors against P. berghei exo-erythrocytic forms.

Co. R1 R2 Pb EEFa 95% CI for HepG2 95% CI for EEF SIb

IC50 [µM] Pb EEF IC50 IC50 [µM] HepG2 IC50
1a t-Bu 3,4-Me-Ph 1.35 0.65 – 2.80 25.94 19.65 – 34.24 19
1d t-Bu 4-Me2N-Ph 0.91 0.31 – 2.73 11.90 8.47 – 16.70 13
1g c-Hex 1-Naphthyl 10.64 3.26 – 34.74 >50 >5
1h c-Hex 4-Me2N-Ph 0.57 0.17 – 1.86 7.80 5.43 – 11.21 14

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1i c-Hex 3,5-Me-Ph 1.84 (very wide) 21.15 17.62 – 25.38 12

1l 4-Tolyl 4-Me2N-Ph 0.58 (very wide) 5.81 4.04 – 8.35 10
1m 4-Tolyl 3,5-Me-Ph 5.55 (very wide) 21.39 16.82 – 27.20 4
1q c-Hex 3,4-Me-Ph 1.47 0.64 – 3.35 21.98 19.11 – 25.28 15
1t c-Hex 4-Me-Ph 0.084 0.050 – 0.14 12.34 7.96 – 19.14 147
1u c-Hex 4-i-Pr-Ph 0.025 0.013 – 0.048 9.99 7.14 – 13.99 400
1v n-Bu 4-Me2N-Ph 0.21 0.11 – 0.37 6.99 5.21 – 9.39 33

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SAHA 0.0077 0.0055 – 0.011 2.51 1.63 – 3.86 326
ATQ 0.000014 0.000012 – 0.000017 >0.50 n/a >35,000
a
P. berghei exo-erythrocytic forms (EEF). bSI = (mammalian cell IC50)/(Pb EEF IC50) – larger

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values indicate greater malaria parasite selectivity. CI, confidence interval.

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3. Conclusions
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In conclusion, we have developed a highly efficient and diversity-oriented one-pot multi-
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component approach for the synthesis of peptoid-based HDACi. The optimized microwave-

assisted protocol allowed the synthesis of the target compounds in less than two hours via an Ugi
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four-component reaction followed by the introduction of the hydroxamic acid in a post-Ugi

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transformation. A series of 24 peptoid-based HDACi was evaluated for anti-plasmodial activity

and cytotoxicity against mammalian cells. All compounds displayed potent submicromolar
EP

activity against drug sensitive and drug resistant asexual blood stage P. falciparum parasites with

IC50 values ranging from 4–158 nM (3D7 line) and 1–118 nM (Dd2 line). Key compounds were
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further tested for activity against different P. falciparum gametocyte stages as well as against P.
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berghei exo-erythrocytic forms. While only moderate activity was found against gametocytes,

several compounds revealed submicromolar activity against P. berghei exo-erythrocytic forms.

The most promising compound from this series, compound 1u, combines high activity against

asexual blood stage parasites (Pf 3D7 IC50: 4 nM; Pf Dd2 IC50: 1 nM) and P. berghei exo-

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erythrocytic forms (Pb EEF IC50: 25 nM) with promising parasite-specific activity (SIPf 3D7/HepG2:

2496, SIPf Dd2/HepG2: 9990, and SIPb EEF/HepG2: 400).

Taken together, this study provides further evidence that hHDAC6 inhibitors are a promising

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starting point for the development of antiplasmodial drug candidates. Furthermore, the findings

described in this work thus offer a promising gateway for more elaborate studies to further

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optimize the activity of this class of HDACi towards the different malaria parasite life cycle

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stages. The described one-pot synthesis is suitable for various alterations of this scaffold. Future

structural modifications should include the variation of the linker and linker lengths, substitution

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of the α-carbon as well as prodrugs strategies. The suggested modifications are underway and
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will deliver valuable structure-activity relationships and might provide pioneering insights

towards design principles to specifically target the different life cycle stages.
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4. Experimental section

4.1. Chemistry

All chemicals and solvents were obtained from commercial suppliers (Sigma-Aldrich, Acros

Organics, Carbolution Chemicals) and used as purchased without further purification. All

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microwave-assisted reactions were carried out with a CEM Focused Microwave System, Model

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Discover. The progress of all reactions was monitored by thin layer chromatography (TLC) using

Merck precoated silica gel plates (with fluorescence indicator UV254). Components were

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visualized by irradiation with ultraviolet light (254 nm) or staining in potassium permanganate

solution following heating. Flash column chromatography was performed using prepacked silica

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cartridge with the solvent mixtures specified in the corresponding experiment. Melting points
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(mp) were taken in open capillaries on a Mettler FP 5 melting-point apparatus and are

uncorrected. Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker Avance 300,
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500 or 600 using DMSO-d6 or CDCl3 as solvents. Chemical shifts are given in parts per million
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(ppm), relative to residual solvent peak for 1H and 13C. 1H NMR signals marked with an asterisk
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(*) correspond to peaks assigned to the minor rotamer conformation. Elemental analysis was

performed on a Perkin Elmer PE 2400 CHN elemental analyzer. High resolution mass spectra
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(HRMS) analysis was performed on a UHR-TOF maXis 4G, Bruker Daltonics, Bremen by

electrospray ionization (ESI). Analytical HPLC analysis were carried out on a Varian Prostar
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system equipped with a Prostar 410 (autosampler), 210 (pumps) and 330 (UV-detector) using a
AC

Phenomenex Luna 5u C18(2) 1.8 µm particle (250 mm × 4.6 mm) column, supported by

Phenomenex Security Guard Cartridge Kit C18 (4.0 mm × 3.0 mm). UV absorption was detected

at 254 nm with a linear gradient of 10% B to 100% B in 20 min using HPLC-grade water +0.1%

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TFA (solvent A) and HPLC-grade acetonitrile +0.1% TFA (solvent B) for elution at a flow rate

of 1 mL/min. The purity of all final compounds was 95% or higher.

4.1.1. Experimental Data. General procedures for the synthesis of antiplasmodial HDAC

inhibitors and compound characterization data for 1m-x are given below. The syntheses of

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compounds 1a-l have been previously described by our group [43].

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4.1.1.1. Synthesis of antimalarial HDAC inhibitors. General procedure for the preparation of

target compounds 1m-x. A mixture of 4-aminomethyl benzoic acid methyl ester hydrochloride

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(2) (121 mg, 0.6 mmol, 1.2 eq), paraformaldehyde (3) (18 mg, 0.6 mmol, 1.2 eq), triethylamine

(83 µl, 0.6 mmol, 1.2 eq), and 50 mg of crushed molecular sieves (MS) 4 Å in dry methanol (1

U
mL, 0.5 M) was added into a 10 mL glass pressure microwave tube equipped with a magnetic
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stirrer bar. The tube was closed with a silicon septum and the reaction mixture was subjected to

microwave irradiation (Discover mode; power: 150 W; hold time: 20 min; temperature: 45 °C;
M

PowerMax-cooling mode) under medium speed magnetic stirring. Next, the appropriate
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carboxylic acid (4a-h) (0.5 mmol, 1.0 eq) and isocyanide (5a-d) (0.5 mmol, 1.0 eq) components
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were added and the reaction mixture was again irradiated at 45°C for 60 min (150W).

Subsequently, a mixture of hydroxylamine hydrochloride (348 mg, 5.0 mmol. 10 eq) in a sodium
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methanolate solution, freshly prepared from dry methanol (8 mL) and sodium (175 mg, 7.5

mmol, 7.5 eq) was added and the reaction mixture was subjected to microwave irradiation at
C

55°C for 30 min (150W). After completion of the reaction, the reaction mixture was filtered and
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the solvent was removed under reduced pressure. Water (15 mL) was added and the pH was

adjusted to a pH 7-8 using 4 M HCl. The mixture was extracted with ethyl acetate (3 x 20 mL),

the combined organic layers were dried over anhydrous sodium sulfate, filtered, and

concentrated in vacuum. The crude products were purified by flash column chromatography

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(prepacked silica cartridge, dichloromethane-dichloromethane/methanol (70:30), gradient: 90:10

70:30 in 20 min) to yield the desired products 1m-x.

4.1.1.2. Methyl 4-((3,5-dimethyl-N-(2-oxo-2-(p-tolylamino)ethyl)benzamido)methyl)benzoate

(1m). Synthesized from 2, 3, 3,5-dimethylbenzoic acid (4b) and p-tolyl isocyanide (5b)

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according to the general procedure. Colorless solid; yield 76%, mp 183 °C; tR: 14.57 min, purity:

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99.4%. 1H NMR (600 MHz, DMSO-d6): δ = 11.21 (s, 1H), 9.93*/9.77 (2 x s, 1H), 9.03 (s, 1H),

7.84-7.67 (m, 2H), 7.55-7.25 (m, 4H), 7.20-6.93 (m, 5H), 4.72/4.58* (2 x s, 2H), 4.10*/3.92

SC
13
(2 x s, 2H), 2.24 (s, 9H). C NMR (151 MHz, DMSO): δ = 171.7, 166.55, 166.1, 164.0, 140.6,

140.4, 137.7, 137.6, 136.4, 136.0, 135.9, 135.8, 132.5, 132.1, 131.8, 131.7, 130.9, 130.8, 129.15,

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127.7, 127.2, 127.1, 126.9, 124.1, 124.1, 119.3, 119.0, 53.2, 51.7, 48.7, 48.1, 20.8, 20.4. HRMS
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(ESI) Anal. Calcd. for C26H28N3O4: 446.2074 [M+H]+, Found: 446.2077.

4.1.1.3. 3-(Dimethylamino)-N-(4-(hydroxycarbamoyl)benzyl)-N-(2-oxo-2-(p-tolylamino)-
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ethyl)benzamide (1n). Synthesized from 2, 3, 3-(dimethylamino)benzoic acid (4a) and p-tolyl

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isocyanide (5b) according to the general procedure. Yellow solid; yield 68%; mp 169°C; tR:
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10.33 min, purity: 96.2%. 1H NMR (300 MHz, DMSO-d6): δ = 11.18 (s, 1H), 9.94*/9.80 (2 x s,

1H), 9.03 (s, 1H), 7.86-7.67 (m, 2H), 7.57-7.31 (m, 4H), 7.30-7.17 (m, 1H), 7.18-7.03 (m, 2H),
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6.85-6.58 (m, 3H), 4.72/4.62* (2 x s, 2H), 4.12*/3.94 (2 x s, 2H), 3.01 (s, 6H), 2.83*/2.25 (2 x s,
13
3H). C NMR (126 MHz, DMSO-d6) δ 171.9, 166.6, 166.1, 163.9, 149.9, 140.5, 136.5, 136.3,
C

135.9, 132.35, 132.0, 131.7, 127.6, 127.1, 127.0, 126.6, 119.2, 119.0, 113.9, 113.8, 113.2, 109.7,
AC

53.15, 51.5, 48.6, 48.1, 39.9, 39.5, 20.3. HRMS (ESI) Anal. Calcd. for C26H29N4O4: 461.2183

[M+H]+, Found: 461.2181.

4.1.1.4. N-(2-(tert-Butylamino)-2-oxoethyl)-3-(dimethylamino)-N-(4-(hydroxycarbamoyl)-

benzyl)benzamide (1o). Synthesized from 2, 3, 3-(dimethylamino)benzoic acid (4a) and

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tert-butyl isocyanide (5a) according to the general procedure. Colorless solid; yield 72%;

mp 138 °C; tR: 8.68 min, purity: 96.4%. 1H NMR (300 MHz, DMSO-d6): δ = 11.20 (s, 1H), 9.02

(s, 1H), 7.85-7.64 (m, 2H), 7.52- 7.41 (m, 1H), 7.41-7.26 (m, 2H), 7.27-7.15 (m, 1H), 6.85-6.57

(m, 3H), 4.61/4.51* (2 x s, 2H), 3.88*/3.67 (2 x s, 2H), 2.89/2.78* (2 x s, 6H), 1.25*/1.21 (2 x s,

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13
9H). C NMR (75 MHz, DMSO-d6): δ = 171.9, 167.1, 166.6, 164.0, 150.0, 140.55, 136.7,

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136.6, 131.7, 129.0, 129.0, 127.7, 127.2, 127.1, 126.7, 114.2, 113.8, 113.2, 110.1, 109.7, 52.9,

51.2, 50.3, 50.2, 48.5, 47.3, 39.9, 28.6, 28.4. Anal. Calcd. for C23H31N4O4: 427.2340 [M+H]+,

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Found: 423.2342.

4.1.1.5. N-(2-(Cyclohexylamino)-2-oxoethyl)-3-(dimethylamino)-N-(4-(hydroxycarbamoyl)-

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benzyl)benzamide (1p). Synthesized from 2, 3, 3-(dimethylamino)benzoic acid (4a) and
AN
cyclohexyl isocyanide (5c) according to the general procedure. Colorless solid; yield 66%; mp

146 °C; tR: 9.53 min, purity: 98.2%. 1H NMR (300 MHz, DMSO-d6): δ = 11.20 (s, 1H), 9.03 (s,
M

1H), 7.86-7.64 (m, 3H), 7.51-7.28 (m, 2H), 7.28-7.12 (m, 1H), 6.89-6.55 (m, 3H), 4.64/4.54*
D

(2 x s, 2H), 3.90*/3.70 (2 x s, 2H), 3.64-3.45 (m, 1H), 2.89/2.79* (2 x s, 6H), 1.81-1.47 (m, 5H),
TE

1.37-0.99 (m, 5H). 13C NMR (75 MHz, DMSO-d6): δ = 171.9, 166.7, 166.4, 164.0, 163.8, 150.0,

140.5, 136.6, 136.5, 131.7, 129.0, 127.7, 127.2, 127.1, 126.7, 114.1, 113.75, 113.2, 110.05,
EP

109.7, 53.0, 50.9, 48.5, 47.7, 47.15, 39.9, 32.4, 32.3, 25.1, 24.4. HRMS (ESI) Anal. Calcd. for

C25H33N4O4: 453.2496 [M+H]+, Found: 453.2502.

C

4.1.1.6. N-(2-(Cyclohexylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-3,4-dimethyl-
AC

benzamide (1q). Synthesized from 2, 3, 3,4-dimethylbenzoic acid (4c) and cyclohexyl isocyanide

(5c) according to the general procedure. Colorless solid; yield 80%; mp 146 °C; tR: 13.67 min,

purity: 98.8%. 1H NMR (300 MHz, DMSO-d6): δ = 11.20 (s, 1H), 9.02 (s, 1H), 7.92-7.63 (m,

3H), 7.51-7.01 (m, 5H), 4.64/4.53* (2 x s, 2H), 3.88*/3.68 (2 x s, 2H), 3.63-3.45 (m, 1H),

27
 ACCEPTED MANUSCRIPT

2.23*/2.22 (2 x s, 6H), 1.81-1.44 (m, 5H), 1.37-0.98 (m, 5H). 13C NMR (126 MHz, DMSO-d6) δ

171.4, 166.6, 163.9, 140.5, 137.8, 136.15, 133.5, 131.65, 129.2, 127.6, 127.0, 126.75, 124.0,

52.9, 51.0, 48.7, 47.55, 47.15, 32.15, 25.1, 24.3, 19.1. Anal. Calcd. for C25H32N3O4: 438.2387

[M+H]+, Found: 438.2391.

PT
4.1.1.7. N-(2-(Cyclohexylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-2-

RI
methylbenzamide (1r). Synthesized from 2, 3, 2-dimethylbenzoic acid (4d) and cyclohexyl

isocyanide (5c) according to the general procedure. Colorless solid; yield 69%; mp 142 °C; tR:

SC
12.71 min, purity: 98.8%. 1H NMR (300 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.03 (s, 1H),

7.85-7.54 (m, 3H), 7.46-7.35 (m, 1H), 7.35-7.12 (m, 5H), 4.68*/4.35 (2 x s, 2H), 3.89*/3.57

U
(2 x s, 2H), 3.59-3.46 (m, 1H), 2.32*/2.23 (2 x s, 3H), 1.91-1.44 (m, 5H), 1.34-0.91 (m, 5H).
AN
13
C NMR (151 MHz, DMSO-d6): δ = 171.1, 171.05, 166.2, 166.2, 163.95, 163.8, 140.6, 139.8,

136.1, 135.95, 134.3, 134.0, 131.9, 131.8, 130.25, 130.2, 128.75, 127.8, 127.2, 127.1, 127.0,
M

125.7, 125.65, 125.55, 125.5, 52.0, 50.3, 48.1, 47.7, 47.6, 46.2, 39.5, 32.4, 32.2, 25.2, 25.1, 24.5,
D

24.4, 18.6, 18.6. Anal. Calcd. for C24H30N3O4: 424.2231 [M+H]+, Found: 424.2228.
TE

4.1.1.8. N-(2-(Cyclohexylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-3-

methylbenzamide (1s). Synthesized from 2, 3, 3-dimethylbenzoic acid (4e) and cyclohexyl

EP

isocyanide (5c) according to the general procedure. Colorless solid; yield 69%; mp 164 °C;

tR: 13.00 min, purity: 99.3%. 1H NMR (600 MHz, DMSO-d6): δ = 11.21 (s, 1H), 9.03 (s, 1H),
C

7.83-7.66 (m, 3H), 7.42-7.37 (m, 1H), 7.34-7.17 (m, 5H), 4.66/4.52* (2 x s, 2H), 3.90*/3.68
AC

(2 x s, 2H), 3.61-3.48 (m, 1H), 2.32/2.30* (2 x s, 3H), 1.81-1.47 (m, 5H), 1.35-0.97 (m, 5H).
13
C NMR (75 MHz, DMSO-d6): δ = 171.5, 166.6, 166.3, 164.1, 164.0, 140.5, 140.2, 137.85,

137.7, 136.1, 135.9, 131.7, 130.1, 128.25, 127.7, 127.25, 127.1, 127.0, 126.85, 123.65, 53.0,

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51.0, 48.7, 47.6, 45.9, 39.5, 32.4, 32.25, 25.1, 24.5, 24.4, 20.9. Anal. Calcd. for C24H30N3O4:

424.2231 [M+H]+, Found: 424.2233.

4.1.1.9. N-(2-(Cyclohexylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-4-

methylbenzamide (1t). Synthesized from 2, 3, 4-dimethylbenzoic acid (4f) and cyclohexyl

PT
isocyanide (5c) according to the general procedure. Colorless solid; yield 82%; mp 187 °C;

RI
tR: 13.00 min, purity: 99.1%. 1H NMR (600 MHz, DMSO-d6): δ = 11.21 (s, 1H), 9.03 (s, 1H),

7.86-7.67 (m, 3H), 7.46-7.26 (m, 4H), 7.27-7.18 (m, 2H), 4.64/4.54* (2 x s, 2H), 3.89*/3.70

SC
(2 x s, 2H), 3.61-3.48 (m, 1H), 2.33/2.31* (2 x s, 3H), 1.77-1.49 (m, 5H), 1.33-1.00 (m, 5H).
13
C NMR (126 MHz, DMSO-d6): δ = 171.35, 166.5, 163.9, 140.4, 139.1, 133.1, 131.6, 128.7,

U
127.6, 127.0, 126.6, 52.9, 51.05, 48.7, 47.5, 47.1, 32.1, 25.05, 24.3, 20.8. Anal. Calcd. for
AN
C24H30N3O4: 424.2231 [M+H]+, Found: 424.2229.

4.1.1.10. N-(2-(Cyclohexylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-4-
M

isopropylbenzamide (1u). Synthesized from 2, 3, 4-isoproplbenzoic acid (4g) and cyclohexyl

D

isocyanide (5c) according to the general procedure. Colorless solid; yield 60%; mp 167 °C;
TE

tR: 14.76 min, purity: 96.9%. 1H NMR (300 MHz, DMSO-d6): δ = 11.20 (s, 1H), 9.02 (s, 1H),

7.89-7.64 (m, 3H), 7.49-7.16 (m, 6H), 4.63/4.54* (2 x s, 2H), 3.88*/3.70 (2 x s, 2H), 3.61-3.46
EP

13
(m, 1H), 3.06-2.80 (m, 1H), 1.88-1.45 (m, 5H), 1.37-0.91 (m, 5H), 1.20 (d, 6H). C NMR

(75 MHz, DMSO-d6): δ = 171.5, 166.6, 166.4, 164.0, 163.9, 150.0, 140.6, 133.6, 131.7, 127.7,
C

127.3, 127.1, 126.9, 126.8, 126.7, 126.4, 126.2, 51.2, 48.8, 47.6, 33.3, 32.5, 32.25, 25.2, 24.5,
AC

24.4, 23.7. Anal. Calcd. for C26H34N3O4: 452.2544 [M+H]+, Found: 452.2544.

4.1.1.11. N-(2-(Butylamino)-2-oxoethyl)-4-(dimethylamino)-N-(4-(hydroxycarbamoyl)benzyl)-

benzamide (1v). Synthesized from 2, 3, 4-(dimethylamino)benzoic acid (4h) and butyl isocyanide

(5d) according to the general procedure. Colorless solid; yield 73%; mp 169 °C; tR: 9.08 min,

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 ACCEPTED MANUSCRIPT

purity: 98.7%. 1H NMR (300 MHz, DMSO-d6): δ = 11.20 (s, 1H), 9.02 (s, 1H), 7.89 (s, 1H),

7.82-7.64 (m, 2H), 7.49-7.19 (m, 4H), 6.81-6.57 (m, 2H), 4.63 (s, 2H), 3.81 (s, 2H), 3.07 (q, J =

6.4 Hz, 2H), 2.93 (s, 6H), 1.51-1.14 (m, 4H), 0.87 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz,

DMSO): δ = 171.6, 167.7, 163.9, 151.2, 140.7, 131.6, 128.5, 127.0, 122.05, 110.9, 39.6, 38.1,

PT
31.0, 19.4, 13.5. Anal. Calcd. for C23H31N4O4: 427.2340 [M+H]+, Found: 427.2337.

RI
4.1.1.12. N-(2-(Butylamino)-2-oxoethyl)-3-(dimethylamino)-N-(4-(hydroxycarbamoyl)benzyl)-

benzamide (1w). Synthesized from 2, 3, 3-(dimethylamino)benzoic acid (4a) and butyl

SC
isocyanide (5d) according to the general procedure. Colorless solid; yield 59%; mp 116 °C; tR:

8.84 min, purity: 96.3%. 1H NMR (300 MHz, DMSO-d6): δ = 11.00 (s, 1H), 9.07 (s, 1H), 7.94-

U
7.82 (m, 1H), 7.82-7.64 (m, 2H), 7.45-7.11 (m, 3H), 6.90-6.54 (m, 3H), 4.64/4.55* (2 x s, 2H),
AN
3.91*/3.71 (2 x s, 2H), 3.18-2.98 (m, 2H), 2.89/2.79* (2 x s, 6H), 1.47-1.16 (m, 4H), 1.00-0.77

(m, 3H). 13C NMR (75 MHz, DMSO): δ = 171.9, 167.6, 167.3, 163.9, 163.8, 150.0, 140.5, 136.6,
M

136.45, 131.7, 129.0, 127.7, 127.2, 127.1, 126.65, 114.0, 113.8, 113.2, 110.0, 109.8, 53.1, 50.9,
D

48.5, 47.35, 39.9, 39.5, 38.25, 31.2, 31.1, 19.5, 13.6. Anal. Calcd. for C23H31N4O4: 427.2340
TE

[M+H]+, Found: 427.2337.

4.1.1.13. N-(2-(Butylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-3,5-
EP

dimethylbenzamide (1x). Synthesized from 2, 3, 3,5-dimethylbenzoic acid (4b) and butyl

isocyanide (5d) according to the general procedure. Colorless solid; yield 68%; mp 181 °C; tR:
C

12.97 min, purity: 98.9%. 1H NMR (300 MHz, DMSO-d6): δ = 11.20 (s, 1H), 9.03 (s, 1H), 7.94-
AC

7.80 (m, 1H), 7.80-7.66 (m, 2H), 7.51-7.19 (m, 2H), 7.16-6.91 (m, 3H), 4.65/4.51* (2 x s, 2H),

3.89*/3.69 (2 x s, 2H), 3.16-2.95 (m, 2H), 2.27/2.24* (2 x s, 6H), 1.50-1.10 (m, 4H), 0.9-0.75

(m, 3H). 13C NMR (75 MHz, DMSO-d6): δ = 171.6, 167.6, 167.3, 164.0, 140.5, 140.3, 137.64,

137.6, 136.05, 135.9, 131.85, 131.7, 130.9, 127.7, 127.3, 127.15, 126.9, 124.1, 53.1, 51.0, 48.7,

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47.3, 38.25, 31.25, 20.8, 19.6, 19.5, 13.7. Anal. Calcd. for C23H30N3O4: 412.2231 [M+H]+,

Found: 412.2230.

4.2. Biological evaluation

PT
4.2.1. P. falciparum asexual intraerythrocytic culture and growth inhibition assays.

RI
P. falciparum drug-sensitive 3D7 [50] and multi-drug resistant Dd2 parasites [51] were cultured

in vitro in O+ human erythrocytes in RPMI 1640 (Gibco, USA) supplemented with 10% heat

SC
inactivated human serum and 5 µg/ml gentamycin (Sigma Aldrich, USA) . Cultures were

maintained at 37oC in 5% O2 and 5% CO2 in N2 gas mixture. Asexual intraerythrocytic growth

U
inhibition assays were performed using the [3H]-hypoxanthine uptake assays, essentially as
AN
previously described [28]. Briefly, ring-stage parasitized erythrocytes (0.25% parasitemia; 2.5%

hematocrit) were incubated in dose response format with test compounds or controls. Percent
M

inhibition of growth was compared to vehicle controls (0.5% DMSO) and 50% inhibitory
D

concentrations (IC50s) calculated using linear interpolation of inhibition curves. At least three
TE

independent assays were carried out, in triplicate wells, with data presented as mean IC50 (±

standard deviation [SD]).

EP

4.2.2. Protein hyperacetylation assays

C

Protein hyperacetylation assays were carried out essentially as previously described [18]. Briefly,
AC

synchronized trophozoite-stage P. falciparum 3D7 infected erythrocytes (3-5 % parasitemia, 5 %

haematocrit) were incubated with 5x IC50 of test compounds or controls for 3 h. Control included

a matched sample treated with vehicle only (0.18 % DMSO; 3h-C), the antimalarial drug

chloroquine (CQ; 5x IC50) and the HDAC inhibitor vorinostat (SAHA; 5x IC50). Following

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treatment, cells were lysed in 0.15 % saponin, washed with 1x phosphate buffered saline (PBS)

to remove haemoglobin and the resulting parasite pellets resuspended in 1x SDS-PAGE loading

dye. Heat denatured protein (93oC; 3min) was analysed by SDS-PAGE and Western blot using

PVDF membrane and anti-(tetra) acetyl histone H4 primary antibody (1:2000 dilution, Merck) in

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combination with IRDye 680 goat anti-rabbit secondary antibody (1:10,000 dilution, Li-Cor

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Biosciences). Total protein load was assessed by staining PVDF membranes with REVERTTM

Total Protein Stain (Li-Cor Biosciences), as per manufacturer’s instructions, prior to Western

SC
analysis. Membranes were imaged using an Odyssey Classic (Li-Cor Biosciences) and

densitometry analysis carried out using Image Studio Lite Version 5.2 software. Western density

U
signals were normalized to total protein load and expressed as fold change compared to the 3h-C
AN
DMSO vehicle control (taken as 1.0). Data are mean (±) SD for two independent experiments.
M

4.2.3. Gametocytocidal activity (Imaging based viability assay)

D

Highly synchronous gametocytes were prepared as published previously [44]. In brief a

TE

transgenic NF54-Pfs16-GFP parasite culture was, by the application of several conditions identified

to stimulate gametocytogenesis, induced to produce gametocytes. After stimulation gametocytes

EP

were maintained in the presence of complete media, (5% serum (Sigma-Aldrich AU), 2.5mg/ml

Albumax II (Invitrogen), 50µg/ml hypoxanthine (Sigma-Aldrich), 10mM HEPES (Sigma-

C

Aldrich) and 50mM N-acetyl-D-glucosamine (Sigma-Aldrich)) at 5% O2, 5% CO2, 21%N2.

AC

The inhibition of gametocyte development from early stage I gametocytes to stage III

gametocytes and stage IV to stage V gametocytes was performed as previously published [45].

Highly synchronized gametocytes at day 2 or day 8 of development, stage I or IV respectively,

were isolated by magnetic column, adjusted to 10% gametocytes at 0.1%H in complete medium.

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Ten mM compounds stocks in DMSO were diluted in concentration response curves for

determination of IC50 values. The stock compounds were diluted 1ul in a total of 25µl of water

and 5µl transferred into Poly-D-lysine coated 384 well CellCarrier plates (PerkinElmer). Forty-

five microliters of stage specific gametocytes (I or IV) were then dispensed into the imaging

PT
plates. The plates were incubated under standard conditions for 72 hours before the addition of

RI
5µl of MitoTracker Red CM-H2Xros (Invitrogen) followed by a further 16 hours incubation. The

plates were then imaged on the OPERA confocal imaging system (PerkinElmer), and the number

SC
of gametocytes identified by their GFP morphology and MitoTracker Red CM-H2Xros viability

signal enumerated. Inhibition of gametocyte development and loss of viability was normalized

U
by representing all data as percent inhibition in relation to 0.4%DMSO and 5µM puromycin
AN
assay controls. The IC50 values were then calculated using Graphpad prizm 4 using non-linear

regression, sigmoidal dose response (variable slope) with no constrains utilizing GraphPad Prizm
M

4.0.
D
TE

4.2.4. Gametocytocidal activity (ATP bioluminescence assay)

The gametocytocidal activity against mature stage V gametocytes of the P. falciparum strain
EP

NF54 was evaluated by an ATP bioluminescence assay as described previously [52] with minor

modifications [48]. Gametocyte culture was initiated from synchronized parasites in complete
C

culture medium supplemented with 5% serum, starting with a 6% hematocrit level and a 0.3%
AC

parasitemia level. Culture medium was changed daily without parasite dilution throughout the

entire process. When the parasitemia level reached 5%, the volume of the medium was doubled.

Between days 12 to 15, the cultures were treated with 50 mM N-acetyl-D-glucosamine (MP

Biomedicals GmbH) to remove the asexual stages, and on day 15 or 16, the culture was purified

by a NycoPrep 1.077 cushion density gradient and magnetic column separation in order to

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remove uninfected erythrocytes and enrich the gametocyte population. All tested compounds

were dissolved in DMSO before further dilutions with complete culture medium was done. After

purification, mature gametocytes (50,000 per well) were incubated for 48 hours with the

compound before performance of the ATP based luminescence assay. To assess the

PT
gametocytocidal activity of the compounds a pre-screening based on 2 concentrations (5 µM and

RI
500 nM) together with the control compounds epoxomycin, methylene blue and chlorotonil A

with known gametocytocidal activity was performed. Of the most promising compounds a 2-fold

SC
serial dilution was done to determine the 50% inhibitory concentration (IC50) by analysing the

nonlinear regression of log concentration–response curves using the drc-package v0.9.0 of R

v2.6.1 [53].
U
AN
4.2.5. Activity against P. berghei-Luc EFF and HepG2 viability (P. berghei-Luc Liver stage and
M

HepG2 Cytotoxicity assays)

D

The liver stage activity against P. berghei-Luc (Pb-Luc) and toxicity to HepG2 cells were
TE

evaluated in Pb-Luc and HepG2 cytotoxicity bioluminescence assays as described previously

[49] with minor modification as follows: HepG2-A16-CD81EGFP cells, stably transformed to

EP

express a GFP-tetraspanin receptor CD81 fusion protein, were cultured at 37 °C and 5% CO2 in

culture media (DMEM with phenol red (Life Technology, CA), 10% FBS and 1x Pen Strep
C

Glutamine (Life Technologies, CA)). For both, P. berghei-Luc and HepG2 cytotoxicity assays,
AC

20-26 hour prior to Pb-Luc sporozoites infection, HepG2-A16-CD81EGFP20 cells at

concentration 6x105 cells/mL in assay medium (DMEM without Phenol Red (Life Technologies,

CA), 5% FBS, and 5x Pen Strep Glutamine (Life Technologies, CA)) were seeded in white solid

bottom 1536-well plates (custom GNF mold ref# 789173-F, Greiner Bio-One) at volume 5 µL

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per well (3x103 cells per well). Directly after that, 50 nL of compounds in 1:3 serial dilutions in

DMSO (final DMSO concentration per well 0.5%) were transferred with Acoustic Transfer

System (ATS) (Biosero) into the assay plates. Atovaquone (0.5 µM) and Puromycin (10 µM) in

1:3 serial dilutions in DMSO were used as positive controls for Pb-Luc Liver Stage and HepG2

PT
Cytotoxicity assays respectively. Wells containing 0.5% DMSO were used as negative control

RI
for the both assays. For Pb-Luc infection, P. berghei-ANKA-GFP-Luc-SMCON (Pb-Luc) [54]

sporozoites were freshly isolated from infected Anopheles stephensi mosquitoes (received from

SC
Insectary Core Facility at New York University) as follows: Dissected salivary glands were

homogenized in DMEM media (Life Technology, CA) using a glass tissue grinder and filtered

U
twice through a 20 µM nylon net filter (Steriflip, Millipore). The sporozoites were counted using
AN
a Neubauer hemocytometer (C-Chip, InCyto, Republic of Korea), adjusted to final concentration
M

of 200 sporozoites per 1µL in the assay media, and placed on ice until needed. To infect the

HepG2-A16-CD81EGFP cells with Pb-Luc, 5 uL of the sporozoites suspension were added per
D

well with a single tip Bottle Valve liquid handler (GNF) to a final number of 1,000 sporozoites
TE

per well. The assay plates were spun down at 37 °C for 3 minutes with a centrifugal force of

330xg on normal acceleration and brake setting. For the HepG2 Cytotoxicity assay, 5 uL per
EP

well of additional assay media (but not sporozoites) was added to the HepG2-A16-CD81EGFP

cell to maintain equal concentrations of compounds with Pb-Luc infected plates. The plates were
C

then incubated at 37 °C for 48 h in 5% CO2 with high humidity to minimize media evaporation
AC

and edge effect. After the incubation, the EEF growth and HepG2-A16-CD81EGFP cells

viability were assessed by a bioluminescence measurement as follows: Media was removed by

spinning the inverted plates at 150xg for 30 seconds; 2 µL per well of BrightGlo (Promega) for

quantification of Pb-Luc EEFs or CellTiterGlo (Promega) reagent (diluted 1:2 with deionized

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 ACCEPTED MANUSCRIPT

water) for quantification of HepG2-A16-CD81EGFP cell viability were dispensed with the

MicroFlo (BioTek) liquid handler. Immediately after addition of the luminescence reagent, the

luminescence was measured by the Envision Multilabel Reader (PerkinElmer). For the data

analysis, the background for the EEF inhibition was defined as the average of the five highest

PT
Atovaquone concentrations (20 wells), and the background for the HepG2 cytotoxicity was

RI
defined as the average of the 3 highest Puromycin concentrations (12 wells). IC50 values were

determined using the average normalized bioluminescence intensity of 4 wells per concentration

SC
and plate (96 wells in total for each compound) and a nonlinear variable slope four-parameter

regression curve fitting model in Prism 6 (GraphPad Software Inc.).

U
AN
4.2.6. In vitro testing on hHDAC1 and hHDAC6

The in vitro inhibitory activity of 1a, 1g, 1l, 1q, 1u, 1v and SAHA against two human HDAC
M

isoforms (1 and 6) were measured using a previously published protocol [55]. OptiPlate-96 black
D

microplates (Perkin Elmer) were used with an assay volume of 50 µL. 5 µL test compound or
TE

control, diluted in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM

MgCl2, 0.1 mg/mL BSA), were incubated with 35 µL of the fluorogenic substrate ZMAL (Z-
EP

(Ac)Lys-AMC) [56] (21.43 µM in assay buffer) and 10 µL of human recombinant HDAC1 (BPS

Bioscience, Catalog# 50051) or HDAC6 (BPS Bioscience, Catalog# 50006) at 37 °C. After an
C

incubation time of 90 min, 50 µL of 0.4 mg/mL trypsin in trypsin buffer (50 mM Tris-HCl, pH
AC

8.0, 100 mM NaCl) were added, followed by further incubation at 37 °C for 30 min.

Fluorescence was measured with an excitation wavelength of 355 nm and an emission

wavelength of 460 nm using a Fluoroskan Ascent microplate reader (Thermo Scientific). All

compounds were evaluated in duplicate in at least two independent experiments.

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Funding Sources

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (HA 7783/1-1 to

PT
FKH and HE 7607/1-1 to JH), the Australian National Health and Medical Research Council

(APP1093378 and APP1074016 to KTA), the A-PARADDISE program funded under the

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European Union's Seventh Framework Programme (KTA) and a Griffith University Postdoctoral

SC
Fellowship (GMF). EAW and JA are supported by grants from the Medicines for Malaria

Venture and NIH (5R01AI090141 and R01AI103058); VMA acknowledges the Australian

U
Research Council (LP120200557) and the Medicines for Malaria Venture for their continued
AN
support.
M

Acknowledgment
D

The Deutsche Forschungsgemeinschaft (DFG) is acknowledged for funds used to purchase the
TE

UHR-TOF maXis 4G, Bruker Daltonics, Bremen HRMS instrument used in this research. We

acknowledge the Australian Red Cross Blood Service for the provision of human blood and sera
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for P. falciparum culture.

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References
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[1] World Health Organization, World Malaria Report 2017. WHO Press, Geneva 2017.

[2] World Health Organization. Malaria vaccine: WHO position paper – January 2016, The

Weekly Epidemiological Record (WER) 91 (2016) 33–52.

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[3] RTS,S Clinical Trials Partnership. Efficacy and safety of RTS,S/AS01 malaria vaccine

with or without a booster dose in infants and children in Africa: final results of a phase 3,

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[4] T.N. Wells, R. Hooft van Huijsduijnen, W.C. Van Voorhis, Malaria medicines: a glass

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U
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Highlights

Novel HDACi were synthesized using a one-pot multi-component protocol

All compounds showed nanomolar activity against asexual blood stage parasites

Selected compounds were shown to hyperacetylate P. falciparum histones

Compound 1u revealed potent and selective activity against exo-erythrocytic forms

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This series of HDACi represents a valuable starting point for further optimization

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