Discovery of HDAC inhibitors with potent activity against multiple malaria parasite life cycle stages Dedicated to Prof. Dr. Alan R. Katritzky, in memoriam.

Article abstract of DOI:10.1016/j.ejmech.2014.05.050

In this work we investigated the antiplasmodial activity of a series of HDAC inhibitors containing an alkoxyamide connecting-unit linker region. HDAC inhibitor 1a (LMK235), previously shown to be a novel and specific inhibitor of human HDAC4 and 5, was used as a starting point to rapidly construct a mini-library of HDAC inhibitors using a straightforward solid-phase supported synthesis. Several of these novel HDAC inhibitors were found to have potent in vitro activity against asexual stage Plasmodium falciparum malaria parasites. Representative compounds were shown to hyperacetylate P. falciparum histones and to inhibit deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts. All compounds were also screened in vitro for activity against Plasmodium berghei exo-erythrocytic stages and selected compounds were further tested against late stage (IV and V) P. falciparum gametocytes. Of note, some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages) and several compounds displayed significantly increased parasite selectivity compared to the reference HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These data suggest that it may be possible to develop HDAC inhibitors that target multiple malaria parasite life cycle stages.

Full text of DOI:10.1016/j.ejmech.2014.05.050

European Journal of Medicinal Chemistry 82 (2014) 204e213
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of HDAC inhibitors with potent activity against multiple
malaria parasite life cycle stages
Finn K. Hansen a, Subathdrage D.M. Sumanadasa b, Katharina Stenzel a, Sandra Duffy b,
Stephan Meister c, Linda Marek a, Rebekka Schmetter a, Krystina Kuna a,
Alexandra Hamacher a, Benjamin Mordmüller d e, Matthias U. Kassack a,
,
,
*
,
, *, 1
Elizabeth A. Winzeler c, Vicky M. Avery b, Katherine T. Andrews b 1, Thomas Kurz a
a
€
€
Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universitat Düsseldorf, Universitatsstr. 1, 40225 Düsseldorf, Germany
b Eskitis Institute for Drug Discovery, Don Young Road, Nathan Campus, Griffith University, QLD 4111, Australia
c Department of Pediatrics, University of California, San Diego, School of Medicine, 9500 Gilman Drive 0741, La Jolla, CA 92093, USA
d
€
Institut für Tropenmedizin, Eberhard Karls Universitat Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany
e
ꢀ ꢀ
Medical Research Laboratory, Albert Schweitzer Hospital, Lambarene, Gabon
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work we investigated the antiplasmodial activity of a series of HDAC inhibitors containing an
alkoxyamide connecting-unit linker region. HDAC inhibitor 1a (LMK235), previously shown to be a novel
and specific inhibitor of human HDAC4 and 5, was used as a starting point to rapidly construct a mini-
library of HDAC inhibitors using a straightforward solid-phase supported synthesis. Several of these
novel HDAC inhibitors were found to have potent in vitro activity against asexual stage Plasmodium
falciparum malaria parasites. Representative compounds were shown to hyperacetylate P. falciparum
histones and to inhibit deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts.
All compounds were also screened in vitro for activity against Plasmodium berghei exo-erythrocytic stages
and selected compounds were further tested against late stage (IV and V) P. falciparum gametocytes. Of
note, some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-
erythrocytic and gametocyte stages) and several compounds displayed significantly increased parasite
selectivity compared to the reference HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These
data suggest that it may be possible to develop HDAC inhibitors that target multiple malaria parasite life
cycle stages.
Received 30 January 2014
Received in revised form
12 May 2014
Accepted 21 May 2014
Available online 22 May 2014
Dedicated to Prof. Dr. Alan R. Katritzky, in
memoriam.
Keywords:
Histone deacetylase inhibitor
Malaria
Liver stages
Asexual blood stages
Gametocyte stages
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
approximately half of the world's population [1]. In 2010 alone it is
estimated that there were ~1.2 million malaria related deaths [2],
Despite decades of research on its prevention and treatment,
malaria remains a significant disease in tropical and subtropical
regions of the world. As reported by the World Health Organization
(WHO), 3.3 billion people were at risk of malaria in 2011, which is
the vast majority of which were due to infection with Plasmodium
falciparum parasites. There is currently no licensed malaria vaccine
and recent clinical trials in African children with the most advanced
candidate, RTS,S/AS02D, were disappointing, with only ~30% pro-
tection being achieved over 18 months follow-up [3,4]. Hence,
antimalarial drugs currently remain the most effective tool for
malaria treatment and, together with vector control strategies, for
malaria prophylaxis. Unfortunately, the rapid spread of drug-
resistant P. falciparum parasites is compromising antimalarial
drug efficacy in a clinical setting [5]. Alarming signs of emerging
resistance to artemisinin derivatives [6,7] could threaten the now
widely-used artemisinin combination therapies (ACTs) and high-
light the urgent need to discover and develop new antimalarials
with novel modes of action. Drugs that target different, or
Abbreviations: DIC, N,N0-diisopropylcarbodiimide; DMF, N,N-dimethylforma-
mide; EEF, exo-erythrocytic form; HDAC, histone deacetylase; Pf, Plasmodium fal-
ciparum; Pf LSG, P. falciparum NF54 late stage gametocytes (IVeV); Pb, Plasmodium
berghei; RT, room temperature; SAHA, suberoylanilide hydroxamic acid; TFA, tri-
fluoroacetic acid; THF, tetrahydrofuran; TSA, trichostatin A.
* Corresponding authors.
E-mail addresses: k.andrews@griffith.edu.au (K.T. Andrews), Thomas.kurz@uni-
duesseldorf.de (T. Kurz).
1
TK and KTA contributed equally to this work as senior authors.
http://dx.doi.org/10.1016/j.ejmech.2014.05.050
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

F.K. Hansen et al. / European Journal of Medicinal Chemistry 82 (2014) 204e213
205
Table 1
Structures and properties of selected hydroxamate-based HDAC inhibitors.
Name
Structure
MW
264
P. falciparum
IC50 M]
Mammalian cell
cytotoxicity IC50 [mM]
SIa
[
m
Vorinostat (SAHA)b
0.109e0.309
2.200e>20
7e>183
Trichostatin A (TSA)b
302
461
0.008e0.011
0.200
18e25
32e82
2-ASA-9b
0.015e0.039
1.240
WR301801 (YC-II-88)c
362
358
0.0006e0.0018
0.080e0.150
0.600
333e1000
4e>1250
SB939d
0.800e>100
a
SI ¼ (mammalian cell IC50)/(P. falciparum IC50) e larger values indicate greater malaria parasite selectivity.
b
c
Data from Ref. [25].
Data from Ref. [28].
Data from Ref. [29].
d
preferably multiple, parasite life cycle stages are also a high priority.
prodrug, romidepsin (FK228), have been approved for treatment of
cutaneous T-cell lymphoma (CTCL) [15e21]. The P. falciparum
genome contains at least five putative HDACs [22] and the enzyme
P. falciparum histone deacetylase 1 (PfHDAC1) has been identified as
Most current antimalarials are active against the asexual blood
stages of the parasite, which are responsible for the clinical
symptoms of malaria [1]. However, recent drug discovery efforts
have moved towards eradication of malaria [8], and seek to addi-
tionally target exo-eryothrocytic liver stages and gametocyte
(transmission) stage parasites [9]. Plasmodium liver stages are
clinically silent pre-erythrocytic life cycle stages that are promising
targets for new drugs as inhibition of this stage leads to a true
causal prophylaxis [10]. The transmission of malaria parasites to the
female Anopheles mosquito vector occurs when sexual stage ga-
metocytes are taken up in the blood of an infected individual during
a blood meal. Following fertilization, meiosis and sporogony in the
mosquito, progeny parasites can then be transmitted to another
host when the female mosquito feeds again. A considerable number
of drugs, which kill asexual parasites and alleviate symptoms, do
not kill late stage gametocytes, allowing the infected individual to
continue to spread the disease even after symptoms have dis-
appeared [11]. Therefore, therapeutically blocking transmission is
also a high priority for the malaria elimination agenda [11,12].
One promising strategy to identify new antimalarial agents is the
“piggyback” approach, which focuses on drug targets that have been
validated for other diseases. Using this approach, we, and others,
have previously investigated the antimalarial potential of com-
pounds that target histone deacetylase (HDAC) enzymes [13,14].
While no HDAC inhibitor has yet been used clinically for malaria, this
class of compound has been progressed to clinical use for cancer.
Both the hydroxamate-based pan-HDAC inhibitor, vorinostat (sub-
eroylanilide hydroxamic acid (SAHA)), and the class I selective
a
target of antimalarial HDAC inhibitors [23]. Treatment of
P. falciparum parasites with HDAC inhibitors results in genome wide
transcriptional alterations [24e26] and altered PfHDAC1 expression
has been found in P. falciparum parasite lines with reduced clinical
susceptibility to artemisinin [27]. Together these findings under-
score PfHDACs' potential as novel parasite drug targets. The struc-
tures and properties of selected hydroxamate-based HDAC
inhibitors with antimalarial activity are summarized in Table 1.
Despite some progress in recent years, there are still a number of
challenges in the rational development of HDAC inhibitors as
antimalarial drug leads. Next generation compounds should retain
potent antiplasmodial activity and low host cell toxicity, but they
also require improved pharmacokinetic properties relative to cur-
rent generation compounds. In addition, while most work to date
has focused on asexual stage parasites [13], we recently showed
that two HDAC inhibitors (SAHA and SB939; see Table 1 for struc-
tures) have potent activity (IC50 ~150 nM) against exo-erythrocytic
stage Plasmodium parasites [29]. This raises the possibility that
HDAC inhibitors could be developed as causal prophylactic and/or
transmission blocking agents.
In this work we investigated the antimalarial activity of a new type
of HDAC inhibitor, containing an alkoxyamide connecting-unit linker
region [30], against different parasite life cycle stages. Previous work
onthecytotoxicityand HDAC inhibitoryactivityof thesealkoxyamide-
based HDAC inhibitors against different human cisplatin sensitive and

206
F.K. Hansen et al. / European Journal of Medicinal Chemistry 82 (2014) 204e213
resistant cancer cell lines revealed potent cytotoxic properties and led
tothe discoveryof1a (LMK235). Compound1a(LMK235)hasa unique
selectivity toward human HDAC4 and 5, which are inhibited with low
nanomolar IC50 values [30]. To determine if this new type of HDAC
inhibitor has antimalarial potential, we investigated the antimalarial
activity of 1a (LMK235) and a panel of analogues generated via solid-
phase supported synthesis. All compounds were tested for in vitro
activity against asexual stage P. falciparum parasites, exo-erythrocytic
Plasmodium parasites cultured in HepG2 livercells and forcytotoxicity
against HepG2 liver cells. Selected compounds were then examined
for gametocytocidal properties as well as inhibition of PfHDAC1
enzyme activity and the ability to hyperacetylate histones in asexual
stage P. falciparum parasites.
using a tritiated hypoxanthine incorporation assay. Based on
calculated physicochemical properties, all compounds were within
the limits of Lipinski's rule of five [35] and were thus expected to be
cell permeable (see Table 2 for calculated log P values). When tested
for in vitro activity against the chloroquine-sensitive P. falciparum
3D7 line, 50% inhibitory concentration values (IC50s) were in the
range of 0.09e1.12
m
M (Table 2). Compound 1i was the most active
M).
compound in this series (R ¼ 4-t-Bu-Ph, Pf 3D7 IC50: 0.09
m
Apparently, derivatives bearing alkyl groups in m- or p-position of
the phenyl core exhibited more potent activity compared to the o-
methyl derivative 1h (IC50
:
1.12
mM). Interestingly, the
dimethylphenyl-substituted compounds were more active than
their respective mono-methyl counterparts. The potency increases
with the bulkiness of the alkyl group in p-position (4-t-Bu-Ph
(1i) > 4-i-Pr-Ph (1c) > 4-CH3-Ph (1e); Table 2). A similar pattern is
observed for the alkoxy-substituted compounds, where the 4-
2. Results and discussion
isopropoxyphenyl derivative 1j (IC50: 0.15 mM) showed the high-
2.1. Synthesis
est activity against P. falciparum. It is worth noting that introducing
a (1,10-biphenyl)-4-yl cap group, as in 1k, does not significantly
The conventional synthesis of compounds 1aeh was published
previously [30]. In order to obtain a more rapid and convenient ac-
cess to alkoxyamide-based HDAC inhibitors of type 1, we developed
the 5-step solid-phase synthesis illustrated in Scheme 1. The
commercially available Fmoc-hydroxylamine-2-chlorotrityl resin 2
was chosen as suitable solid support. First, the polymer-bound
phthaloyl-protected linker 3 was prepared in two steps by depro-
tection of the Fmoc group and subsequent acylation of the free hy-
droxylamine moiety with 6-((1,3-dioxoisoindolin-2-yl)oxy)hexanoic
acid (4 equiv.) using DIC/Oxyma (4 equiv.) as coupling agents. Sub-
sequently, the phthaloyl protection was cleaved with methylhy-
drazine to provide the resin-bound key intermediate 4, which
allowed the solid-phase synthesis of the alkoxyamide-based HDAC
inhibitors 1ieu. Our mini library of HDAC inhibitors was prepared
using resin 4 and 13 different benzoic acid derivatives. First, the
desired capgroup (3 equiv.) was introduced by DIC/Oxyma-mediated
(3 equiv.) amide coupling reaction to give the resin-bound HDAC
inhibitors 5ieu. The DIC/Oxyma system in the absence of a base was
chosen to prevent any possible N-overacylation of the NHeO func-
tion, which is a well-known side reaction in the synthesis of
aminoxy-based compounds particularly observed under solid-phase
reaction conditions [31e34]. Finally, HDAC inhibitors 1ieu were
conveniently released from the resin by treatment of 5ieu with 5%
trifluoroacetic acid (TFA) in dichloromethane. Purification by flash
column chromatography provided the target compounds 1ieu in
14e36% overall yields (over five steps).
alter the activity (IC50: 0.16
stituents are well tolerated.
mM), which indicates that bulky sub-
2.3. Cytotoxicity and selectivity indices
To assess the selectivity of alkoxyamide-based HDAC inhibitors
for the parasite versus mammalian cells, cytotoxicity was evaluated
against HepG2 liver cells. The results and selectivity indices (SIs) are
outlined in Table 2. All compounds with a methyl group in
m-position of the phenyl ring showed only moderate parasite
selectivity (SI: 13e27). However, several other compounds revealed
significantly increased selectivity for the parasite compared to the
reference HDAC inhibitor SAHA (SI: 11, Table 2). As already
observed for the activity against asexual blood stages, the parasite
selectivity increases with the bulkiness of the alkyl group in p-
position (4-t-Bu-Ph (1i, SI: 139) > 4-i-Pr-Ph (1c, SI: 73) > 4-CH3-Ph
(1e, SI: 55); Table 2). In addition, the bulky biphenyl-based com-
pound 1k (SI: 64) showed increased selectivity for the parasite
compared to SAHA (SI: 11). The selectivity indices of the alkoxy-
substituted compounds are particularly interesting. The 2- and 3-
propoxy-substituted compounds (1p and 1q, SI: 34 and 29,
respectively) are significantly less parasite selective than the 4-
propoxy analogue 1m (SI: 80). The truncated 4-ethoxy derivative
1o (SI: 33) displayed a decreased selectivity index compared to 1m.
Remarkably, all elongated alkoxy compounds showed no cytotox-
icity at the highest concentration tested (50 mM) and the butoxy-
2.2. In vitro activity against P. falciparum asexual blood stages
(1l), pentoxy- (1n) and hexoxy-substituted (1s) HDAC inhibitors
revealed promising selectivity indices with >294, >238 and >143,
respectively. These data indicate that the introduction of bulky- and
alkoxy-substituted cap groups on HDAC inhibitors of type 1 might
The antimalarial activity of alkoxyamide-based HDAC inhibitors
1aeu was first tested against asexual-stage P. falciparum parasites
Scheme 1. Solid-phase synthesis of alkoxyamide-based HDAC inhibitors 1ieu. Reagents and conditions: a) (i) piperidine, DMF, RT, 1 h; (ii) 6-((1,3-dioxoisoindolin-2-yl)oxy)hex-
anoic acid, DIC, Oxyma, CH2Cl2, RT, 24 h; b) methylhydrazine, ethanol, THF, RT, 3 h; c) RCOOH, DIC, Oxyma, CH2Cl2, RT, 24 h; d) TFA, CH2Cl2, RT, 1 h.

F.K. Hansen et al. / European Journal of Medicinal Chemistry 82 (2014) 204e213
207
Table 2
Calculated log P values, in vitro activity against asexual blood stages of P. falciparum 3D7 parasites, cytotoxicity and selectivity indices of 1ae1u.
Compound
R
Log Pa
Pf 3D7b IC50
[m
M]
HepG2 IC50
[m
M]
95% CI for HepG2 IC50
SIc
1a
1b
1c
1d
1e
1f
1g
1h
1i
3,5-CH3-Ph
3,4-CH3-Ph
4-i-Pr-Ph
3-CH3-Ph
4-CH3-Ph
4-CF3-Ph
4-F-Ph
2-CH3-Ph
4-t-Bu-Ph
4-i-PrO-Ph
4-Ph0-Ph
4-BuO-Ph
4-PrO-Ph
4-C5H11O-Ph
4-C2H5O-Ph
2-PrO-Ph
3-PrO-Ph
4-Bu-Ph
4-C6H13O-Ph
furan-2-yl
2-F-Ph
2.05 ( 0.57)
0.10 ( 0.02)
0.12 ( 0.03)
0.12 ( 0.02)
0.19 ( 0.06)
0.22 ( 0.02)
0.26 ( 0.03)
0.58 ( 0.14)
1.12 ( 0.19)
0.09 ( 0.03)
0.15 ( 0.03)
0.16 ( 0.03)
0.17 ( 0.03)
0.21 ( 0.02)
0.21 ( 0.04)
0.23 ( 0.05)
0.24 ( 0.02)
0.24 ( 0.04)
0.26 ( 0.08)
0.35 ( 0.05)
0.62 ( 0.16)
0.73 ( 0.05)
1.26
3.24
8.76
1.06e1.51
2.50e4.19
5.68e13.51
3.83e5.73
8.49e17.28
10.33e16.27
8.33e12.81
10.19e15.94
7.24e21.47
7.00e13.61
8.66e12.10
13
2.05 ( 0.57)
2.47 ( 0.57)
1.59 ( 0.56)
1.59 ( 0.56)
2.10 ( 0.58)
1.35 ( 0.59)
1.59 ( 0.56)
2.82 ( 0.57)
2.17 ( 0.57)
2.77 ( 0.58)
2.89 ( 0.57)
2.36 ( 0.57)
3.42 ( 0.57)
1.82 ( 0.57)
1.92 ( 0.57)
2.36 ( 0.57)
3.18 ( 0.56)
3.95 ( 0.57)
ꢀ0.08 ( 0.58)
0.76 ( 0.59)
27
73
25
55
50
18
11
139
65
64
>294
80
>238
33
34
29
4.69
12.11
12.96
10.33
12.74
12.47
9.76
10.24
>50
16.86
>50
7.68
8.12
7.04
10.77
>50
1j
1k
1l
1m
1n
1o
1p
1q
1r
1s
1t
10.92e26.06
4.68e12.61
4.64e14.23
4.27e11.59
7.71e15.03
41
>143
13
8.03
9.10
6.04e10.69
6.96e11.89
1u
12
SAHA
Chloroquine
0.86 ( 0.21)
4.69 ( 0.32)
0.13 ( 0.02)d
0.01 ( 0.006)
1.49
>10
0.97e2.29
11
>1000
a
LogP values were calculated using ACD/ChemSketch freeware version 12.01.
Three independent assays, each carried out in triplicate wells.
b
c
SI ¼ (mammalian cell IC50)/(P. falciparum IC50) e larger values indicate greater malaria parasite selectivity.
d
Data from Ref. [36]. CI, confidence interval.
be a useful tool for the development of antimalarial lead com-
pounds with potent and selective action against P. falciparum.
2.4. Mode of action of alkoxyamide-based HDAC inhibitors in
asexual-stage P. falciparum parasites
The mode of action of eight representative alkoxyamide-based
HDAC inhibitors in asexual-stage parasites was assessed by exam-
ining in situ hyperacetylation of P. falciparum histones and in vitro
activity against P. falciparum nuclear lysates and recombinant
PfHDAC1. Like the control compound TSA, 1ae1d and 1ie1l caused
hyperacetylation of P. falciparum histone H4 (Fig.1). Histone H3 was
also hyperacetylated by all eight compounds, although apparently
to a lesser extent by 1b (Fig. 1). The reason for this is not yet know,
however the effect was not due to unequal loading, as the same
membrane was also probed with control antisera (anti-IC3/94) to
confirm equivalent protein levels were present.
Fig. 1. Hyperacetylation of P. falciparum histones. Trophozoite-stage P. falciparum 3D7
parasites were treated for 3 h with 500 nM chloroquine (CQ), TSA, or compounds
1ae1d or 1ie1l. Parasites treated with vehicle only (0.05% DMSO) served as negative
controls and were taken at the start of the treatment (C-0) and after 3 h (C-3). Protein
lysates were analysed by Western blot with antisera that recognize tetra-acetylated
histone H4 (anti-acetyl H4) or N-terminally acetylated histone H3 (anti-acetyl H3).
In each case the same membranes were also probed with antisera that recognizes
P. falciparum RAP2 (anti-IC3/94) to show equivalent loading between lanes.
Compounds 1aed and 1ie1l were also tested for their ability to
inhibit deacetylase activity in P. falciparum nuclear lysates and the
activity of recombinant PfHDAC1. All compounds displayed >50%
activity of our novel HDAC inhibitors against this life cycle stage.
Five of 21 compounds tested (1a, 1b, 1d, 1p and 1q) displayed
potent activity against P. berghei exo-erythrocytic stages with IC50
inhibition of PfHDAC1 activity at 1
mM and all but 1j > ~50% inhi-
values ranging from 0.16 to 0.66 mM, 13 had moderate activity with
bition of P. falciparum nuclear extract at 1
mM (Table 3). The reason
for a lack of deacetylase inhibition by 1j is not yet known, however
compound stability can be ruled out, as this compound was still
active in the other mode of action assays (Fig. 1; Table 3). Once
additional recombinant PfHDAC proteins become available it will be
important to profile this compound and other HDAC inhibitors
against different malaria HDAC enzymes.
Table 3
Inhibition of P. falciparum nuclear lysates and recombinant PfHDAC1.
Compound
R
Pf nuclear lysate %
Pf HDAC1%
inhibition at 1
m
Ma
inhibition at 1 m
Ma
1a
1b
1c
1d
1i
1j
1k
1l
3,5-CH3-Ph
3,4-CH3-Ph
4-i-Pr-Ph
3-CH3-Ph
4-t-Bu-Ph
4-i-PrO-Ph
4-Ph0-Ph
58.6 ( 12.5)
93.3 ( 2.4)
72.9 ( 10.4)
73.9 ( 16.3)
48.3 ( 7.3)
9.3 ( 4.6)
89.3 ( 0.2)
50.3 ( 11.5)
78.1 ( 12.6)
73.8 ( 7.5)
82.6 ( 4.2)
67.2 ( 0.5)
75.5 (17.8)
68.6 ( 1.2)
62.4 ( 10.1)
64.2 ( 19.4)
72.1 ( 5.0)
93.3 ( 10.9)
2.5. In vitro activity against tissue schizontocidal stages of
P. berghei
We showed previously that two commercially available HDAC
inhibitors, SAHA and SB939, can potently inhibit exo-erythrocytic
stage Plasmodium berghei parasite development ex vivo in HepG2
4-BuO-Ph
TSA
a
liver cells (IC50 ~ 0.15
mM) [29]. This prompted us to assess the
Average ( SD) of two independent assays, each carried out in duplicate wells.

208
F.K. Hansen et al. / European Journal of Medicinal Chemistry 82 (2014) 204e213
Table 4
Activity of alkoxyamide-based HDAC inhibitors against P. berghei exo-erythrocytic stages.
Compound
R
Pb EEFa IC50
[
mM]
95% CI for Pb EEF IC50
HepG2 IC50
[
m
M]
95% CI for HepG2 IC50
EEF SIb
1a
1b
1c
1d
1e
1f
1g
1h
1i
3,5-CH3-Ph
3,4-CH3-Ph
4-i-Pr-Ph
3-CH3-Ph
4-CH3-Ph
4-CF3-Ph
4-F-Ph
2-CH3-Ph
4-t-Bu-Ph
4-i-PrO-Ph
4-Ph0-Ph
4-BuO-Ph
4-PrO-Ph
4-C5H11O-Ph
4-C2H5O-Ph
2-PrO-Ph
3-PrO-Ph
4-Bu-Ph
4-C6H13O-Ph
furan-2-yl
2-F-Ph
0.16
0.31
1.34
0.66
1.14
1.90
2.31
2.54
1.87
1.36
1.83
2.14
~5.29
~5.14
1.37
0.48
0.45
~1.79
7.91
1.13
1.25
0.12e0.21
0.23e0.42
0.68e2.62
0.50e0.87
0.78e1.69
1.22e2.96
1.46e3.64
1.68e3.86
1.65e2.12
0.86e2.13
1.51e2.22
1.14e4.01
(Very wide)
(Very wide)
0.86e2.18
0.32e0.72
0.30e0.68
(Very wide)
4.60e13.62
0.72e1.78
0.68e2.28
1.26
3.24
8.76
1.06e1.51
2.50e4.19
5.68e13.51
3.83e5.73
8.49e17.28
10.33e16.27
8.33e12.81
10.19e15.94
7.24e21.47
7.00e13.61
8.66e12.10
8
10
7
7
11
7
4
5
7
7
4.69
12.11
12.96
10.33
12.74
12.47
9.76
10.24
>50
16.86
>50
7.68
8.12
7.04
10.77
>50
1j
1k
1l
6
>23
~3
>10
6
17
16
~6
>6
7
1m
1n
1o
1p
1q
1r
1s
1t
10.92e26.06
4.68e12.61
4.64e14.23
4.27e11.59
7.71e15.03
8.03
9.10
6.04e10.69
6.96e11.89
1u
7
SAHA
Chloroquine
Atovaquone
0.14
>10
0.00016
0.11e0.20
1.49
>10
>10
0.97e2.29
11
e
0.000085e0.00028
>64,102
a
P. berghei exo-erythrocytic forms (EEF).
SI ¼ (mammalian cell IC50)/(Pb EEF IC50) e larger values indicate greater malaria parasite selectivity. CI, confidence interval.
b
IC50s between 1.13 and 2.54
showed no activity with IC50 values > 5
m
M, while three (1m, 1n and 1s)
based HDAC inhibitors appear to be interesting scaffolds for the
development of antimalarials with activity against sexual (game-
tocyte) stages.
A comparison of the IC50 values of the four most potent com-
pounds with those obtained for HepG2 cells (Table 2) indicates that
1a is cytotoxic (SI: 4), while 1b has around 13 fold, 1d 11 fold and 1k
20 fold more activity against late stage gametocytes versus
mammalian cells. It is interesting to note that 1k showed only
mM (Table 4). Remarkably,
the most active compounds all bear a methyl group in the 3-
position of the phenyl ring (1a, 1b, and 1d) or a propoxy group in
2- or 3-position (1p and 1q).
A comparison of the IC50 values for P. berghei exo-erythrocytic
stages with those obtained for HepG2 liver cells indicate a mod-
erate parasite-specific selectivity for compounds with sub-mM IC50s
(SI: ~7 ꢀ17; Table 4). These selectivity indices are comparable with
the selectivity of the reference compound SAHA (SI: 11, Table 4).
Some of the alkoxysubstituted derivatives in this study were not
moderate activity against exo-erythrocytic stages (Pb EEF IC50
:
1.83 M, Table 4). As we do not yet know which PfHDACs are
m
expressed in different life cycle stages, or their relative abundance,
such differences in activity could be due to differences in HDAC
isoform expression. Alternatively, differences could also be due to
the use of different species of parasites in the respective assays
(P. berghei versus P. falciparum). It is also possible that the differ-
ences we see are due to interference with the regulation of HDAC-
cytotoxic at the highest concentration tested (50 mM) indicating
that it should be possible to develop parasite-specific HDAC in-
hibitors that selectively target exo-erythrocytic stage parasites. In
particular compound 1l (SI: > 23) will be the subject of future work
to determine if this compound has potential as chemical starting
point for exo-erythrocytic stage drug development.
Table 5
2.6. In vitro activity against late stage gametocytes
Activity of alkoxyamide-based HDAC inhibitors against stage IVeV P. falciparum
gametocytes.
There is an increasing focus on the elimination, rather than just
prevention and treatment, of malaria and to achieve this it will be
important to identify approaches to block the transmission of
sexual (gametocyte) stage Plasmodium parasites to the mosquito
vector. When we examined the activity of a sub-set of our
alkoxyamide-based HDAC inhibitors against late-stage gameto-
cytes (see Table 5), we found that five of nine compounds tested
Compound
R
Pf LSGa IC50
[mM]
1a
1b
1d
1g
1i
1k
1r
1t
3,5-CH3-Ph
3,4-CH3-Ph
3-CH3-Ph
4-F-Ph
4-t-Bu-Ph
4-Ph0-Ph
4-Bu-Ph
0.33b
0.25b
0.43b
(
(
(
0.01)
0.10)
0.1)
2.0b
(
0.01)
>120c,d
0.50b
5.3c
( 0.14)
(1a, 1b, 1d, 1k and 1t) had IC50 values ꢁ1
activity with IC50 values between 2.0 and 5.3
two (1i and 1u) were not active (IC50 > 120
m
M, two had less potent
M (1g and 1r) while
M). As already
furan-2-yl
2-F-Ph
1.0b
(
0.2)
m
1u
>120c,d
m
Artesunate
Pyronaridine
Chloroquine
0.0038 ( 0.0003)b
2.7 ( 0.87)e
>120b
observed for P. berghei exo-erythrocytic stages, a 3-methylphenyl
substitution emerges as beneficial, as indicated by the potent ac-
tivities of compounds 1a, 1b, and 1d with IC50s between 0.25 and
a
P. falciparum NF54 late stage gametocytes (IVeV).
n ¼ 2, each in duplicate wells.
0.43
p-position, was the most active compound of this series (IC50
0.25 M). Interestingly, compound 1k displayed good activity
against late-stage gametocytes (IC50 0.50 M). Hence, biphenyl-
mM. Notably, compound 1b, bearing a methyl group in m- and
b
c
n ¼ 1, each in duplicate wells.
d
e
m
No activity at 120 mM.
n ¼ 7, each in duplicate wells. nd, not determined.
m

F.K. Hansen et al. / European Journal of Medicinal Chemistry 82 (2014) 204e213
209
associated stage-specific proteins or pathways, or even to action
against alternative molecular targets.
dioxoisoindolin-2-yl) oxy) hexanoic acid (443 mg, 1.6 mmol),
Oxyma (227 mg, 1.6 mmol) and DIC (202 mg, 1.6 mmol) in CH2Cl2
(10 mL) was added and the resin was shaken for 24 h at room
temperature. Afterward, the resin was drained and washed with
CH2Cl2 (3 ꢂ 5 mL), MeOH (3 ꢂ 5 mL), CH2Cl2 (3 ꢂ 5 mL).
3. Conclusions
Starting from 1a (LMK235), a specific inhibitor of human HDAC4
and 5, we have now extended our studies to examine the activity of
1a and novel structural analogues against malaria parasites. For this
purpose, we developed a straightforward solid-phase synthesis
allowing the rapid construction of mini libraries of HDAC inhibitors
with an alkoxyamide connecting-unit linker region. Biological
evaluation of a panel of 21 alkoxyamide-based HDAC inhibitors
against the chloroquine-sensitive 3D7 line of P. falciparum revealed
4.2.2. General procedure for the removal of the phthaloyl-
protection group
The resin was suspended in EtOH:THF (1:1, 10 mL) and meth-
ylhydrazine (1.5 mL, 28.7 mmol) was added. Subsequently, the resin
was shaken at room temperature (3 h). The resin was drained and
washed with MeOH (3 ꢂ 5 mL), THF (3 ꢂ 5 mL), CH2Cl2 (3 ꢂ 5 mL).
IC50 values in the range of 0.09e1.12
m
M. Modification of the cap
4.2.3. General procedure for the coupling of benzoic acid derivatives
A solution of the respective carboxylic acid (1.2 mmol), Oxyma
(171 mg, 1.2 mmol) and DIC (151 mg, 1.2 mmol) in CH2Cl2 (10 mL)
was added to the resin and shaken for 24 h. The resin was washed
with CH2Cl2 (3 ꢂ 5 mL), MeOH (3 ꢂ 5 mL), CH2Cl2 (3 ꢂ 5 mL).
group showed that the nature of the substitution on the phenyl ring
is crucial for the parasite selectivity. All compounds were tested
in vitro for their activity against tissue schizontocidal stages of
P. berghei (exo-erythrocytic forms) and selected compounds were
further tested against sexual stage P. falciparum parasites (stage IV/
V gametocytes). Notably, compounds 1a (LMK235), 1b and 1d
showed nanomolar activity against all three life cycle stages, while
other compounds revealed increased parasite selectivity in com-
bination with at least dual-stage activity. Thus, this series of com-
pounds represents a valuable starting point for the development of
novel antimalarial drug leads with potent antiplasmodial activity
against multiple malaria parasite life cycle stages, low host cell
toxicity, and causal prophylactic and/or transmission blocking
properties.
4.2.4. General procedure for the cleavage of resin-bound HDAC
inhibitors
The resin was treated with 5% TFA in CH2Cl2 (1 ꢂ10 mL) at room
temperature (1 h). The resin was drained and washed with CH2Cl2
(3 ꢂ 5 mL). The filtrates were combined and evaporated to dryness
under reduced pressure. The crude products were purified by col-
umn chromatography using hexane/ethyl acetate (gradient) as
eluent to afford the desired HDAC inhibitors.
4.3. Experimental data
4. Experimental section
4.3.1. 4-(Tert-butyl)-N-((6-(hydroxyamino)-6-oxohexyl)oxy)
benzamide (1i)
4.1. Synthesis
White solid; yield 22%; mp 46 ꢃC. 1H NMR (500.13 MHz, DMSO-
All solvents and chemicals were used as purchased without
further purification. Column chromatography was performed with
MachereyeNagel silica gel 60 M (0.04e0.063 mm). TLC analysis
was performed on Merck precoated silica gel plates (with fluores-
cence indicator UV254). Spots were visualized by irradiation with
ultraviolet light (254 nm). Melting points (mp) were taken in open
capillaries on a Mettler FP 5 melting-point apparatus and are un-
corrected. Proton (1H) and carbon (13C) NMR spectra were recorded
on a Bruker Avance 500 (500.13 MHz for 1H; 125.76 MHz for 13C)
using DMSO-d6 as solvent. Chemical shifts are given in parts per
d6):
d
¼ 11.54 (s, 1H), 10.36 (s, 1H), 8.68 (s, 1H), 7.68 (d, J ¼ 8.4 Hz,
2H), 7.48 (d, J ¼ 8.4 Hz, 2H), 3.85 (t, J ¼ 6.4 Hz, 2H), 1.96 (t, J ¼ 7.3 Hz,
2H), 1.64e1.48 (m, 4H), 1.41e1.22 (m, 11H) ppm. 13C NMR
(125.76 MHz, DMSO-d6):
d
¼ 168.89, 164.01, 154.22, 129.54, 126.78,
125.08, 74.96, 34.52, 32.08, 30.79, 27.34, 24.98, 24.81 ppm. IR (KBr):
ṽ ¼ 3200 (NH), 2954, 2873 (CH2), 1642 (C]O) cmꢀ1. HRMS (ESI)
[M þ H]þ: 323.19656, Calcd. for C17H27N2O4: 323.19653. HPLC
analysis: retention time ¼ 14.18 min; peak area: 97.86%. Eluent A:
5 mM NH4OAc solution; eluent B: CH3CN, gradient (95:5e> 5:95)
over 20 min at a flow rate of 1.0 mL minꢀ1
.
million (ppm), (d
relative to residual solvent peak for 1H and 13C or
to tetramethylsilane). Elemental analysis was performed on a Per-
kin Elmer PE 2400 CHN elemental analyzer. IR spectra were
recorded on a Varian 800 FT-IR Scimitar series. HRMS analysis was
performed on a UHR-TOF maXis 4G, Bruker Daltonics, Bremen.
HPLC purity determination was carried out using a Phenomenex
4.3.2. N-((6-(Hydroxyamino)-6-oxohexyl)oxy)-4-
isopropoxybenzamide (1j)
White solid; yield 23%; mp 125 ꢃC. 1H NMR (500.13 MHz, DMSO-
d6):
d
¼ 11.45 (s, 1H), 10.36 (s, 1H), 8.68 (s, 1H), 7.69 (d, J ¼ 8.7 Hz,
2H), 6.96 (d, J ¼ 8.7 Hz, 2H), 4.76e4.64 (m, 1H), 3.84 (t, J ¼ 6.4 Hz,
2H), 1.96 (t, J ¼ 7.3 Hz, 2H), 1.65e1.48 (m, 4H), 1.44e1.30 (m, 2H),
1.27 (d, J ¼ 6.0 Hz, 6H) ppm. 13C NMR (125.76 MHz, DMSO-d6):
Luna C-18(2) 1.8
m
m particle (250 mm ꢂ 4.6 mm) column, sup-
ported by Phenomenex Security Guard Cartridge Kit C18
(4.0 mm ꢂ 3.0 mm). The purity of all final compounds was 95% or
higher. Fmoc-hydroxylamine-2-chlorotrityl resin was purchased
from Iris Biotech, Germany. All solid phase reactions were carried
d
¼ 168.92,163.81, 159.96, 128.81, 124.00, 114.87, 74.96, 69.25, 32.10,
27.36, 25.00, 24.82, 21.59 ppm. IR (KBr): ṽ ¼ 3358, 3181 (NH), 2938,
2868 (CH2), 1666 (C]O) cmꢀ1. Anal. Calcd. for C16H24N2O5: C 59.24,
H 7.46, N 8.64. Found: C 59.30, H 7.74, N 8.36.
out in
a
Heidolph Synthesis
1
parallel synthesizer. 3-
Propoxybenzoic acid [37] and 6-((1,3-dioxoisoindolin-2-yl)oxy)
hexanoic acid [30] were prepared according to literature.
4.3.3. N-((6-(Hydroxyamino)-6-oxohexyl)oxy)-[1,10-biphenyl]-4-
carboxamide (1k)
4.2. General procedures for solid-phase synthesis
White solid; yield 27%; mp 155 ꢃC. 1H NMR (500.13 MHz, DMSO-
d6):
d
¼ 11.69 (s, 1H), 10.37 (s, 1H), 8.69 (s, 1H), 7.89e7.82 (m, 2H),
4.2.1. General procedure for the coupling of 6-((1, 3-
dioxoisoindolin-2-yl) oxy) hexanoic acid to the resin
7.80e7.75 (m, 2H), 7.75e7.69 (m, 2H), 7.53e7.47 (m, 2H), 7.44e7.38
(m, 1H), 3.89 (t, J ¼ 6.4 Hz, 2H), 1.98 (t, J ¼ 7.3 Hz, 2H), 1.66e1.50 (m,
4H), 1.43e1.31 (m, 2H) ppm. 13C NMR (125.76 MHz, DMSO-d6):
Fmoc-hydroxylamine-2-chlorotrityl resin (500 mg, 0.4 mmol,
Iris Biotech) was treated with 20% piperidine in DMF (1 ꢂ 10 mL,
1 h) and washed with CH2Cl2 (3 ꢂ 10 mL). A solution of 6-((1, 3-
d
¼ 168.91, 163.73, 142.95, 138.96, 131.08, 128.93, 128.01, 127.62,
126.75, 126.53, 75.04, 32.10, 27.36, 24.99, 24.82 ppm. IR (KBr):

210
F.K. Hansen et al. / European Journal of Medicinal Chemistry 82 (2014) 204e213
ṽ ¼ 3222 (NH), 2942, 2862 (CH2), 1648 (C]O) cmꢀ1. Anal. Calcd. for
Calcd. for C16H25N2O5: 325.17580. HPLC analysis: retention
C
19H22N2O4: C 66.65, H 6.48, N 8.18. Found: C 66.50, H 6.33, N 8.09.
time ¼ 9.46 min; peak area: 97.52%. Eluent A: water; eluent B:
methanol, gradient (50:50e>0:100) over 20 min at a flow rate of
4.3.4. 4-Butoxy-N-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide
(1l)
1.0 mL minꢀ1
.
White solid; yield 18%; mp 125 ꢃC. 1H NMR (500.13 MHz, DMSO-
4.3.9. N-((6-(Hydroxyamino)-6-oxohexyl)oxy)-3-
propoxybenzamide (1q)
d6):
d
¼ 11.46 (s, 1H), 10.36 (s, 1H), 8.67 (s, 1H), 7.70 (d, J ¼ 8.7 Hz,
2H), 6.98 (d, J ¼ 8.8 Hz, 2H), 4.01 (t, J ¼ 6.5 Hz, 2H), 3.84 (t, J ¼ 6.4 Hz,
2H), 1.96 (t, J ¼ 7.3 Hz, 2H), 1.76e1.64 (m, 2H), 1.63e1.48 (m, 4H),
1.48e1.28 (m, 4H), 0.93 (t, J ¼ 7.4 Hz, 3H) ppm. 13C NMR
White solid; yield 14%; mp 101 ꢃC. 1H NMR (500.13 MHz, DMSO-
d6):
d
¼ 11.61 (s, 1H), 10.36 (s, 1H), 8.68 (s, 1H), 7.44e7.22 (m, 3H),
7.09 (d, J ¼ 6.9 Hz,1H), 3.96 (t, J ¼ 6.5 Hz, 2H), 3.86 (t, J ¼ 6.2 Hz, 2H),
1.97 (t, J ¼ 7.3 Hz, 2H), 1.79e1.68 (m, 2H), 1.64e1.49 (m, 4H),
1.42e1.30 (m, 2H), 0.99 (t, J ¼ 7.4 Hz, 3H) ppm. 13C NMR
(125.76 MHz, DMSO-d6):
d
¼ 168.92, 163.80, 161.13, 128.76, 124.22,
113.98, 74.97, 67.27, 32.10, 30.51, 27.37, 25.00, 24.83, 18.59,
13.58 ppm. IR (KBr): ṽ ¼ 3220, 3120 (NH), 2935, 2871 (CH2), 1619
(C]O) cmꢀ1. Anal. Calcd. for C17H26N2O5: C 60.34, H 7.74, N 8.28.
Found: C 60.01, H 7.78, N 8.48.
(125.76 MHz, DMSO-d6):
d
¼ 168.93, 163.74, 158.48, 133.63, 129.51,
119.06, 117.69, 112.60, 75.00, 69.00, 32.10, 27.35, 24.99, 24.82, 21.90,
10.29 ppm. IR (KBr): ṽ ¼ 3210 (NH), 2938, 2874 (CH2), 1670, 1624
(C]O) cmꢀ1. Anal. Calcd. for C16H24N2O5: C 59.24, H 7.46, N 8.64.
Found: C 59.29, H 7.48, N 8.42.
4.3.5. N-((6-(Hydroxyamino)-6-oxohexyl)oxy)-4-
propoxybenzamide (1m)
White solid; yield 32%; mp 112 ꢃC. 1H NMR (500.13 MHz, DMSO-
4.3.10. 4-Butyl-N-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide
(1r)
d6):
d
¼ 11.48 (s, 1H), 10.37 (s, 1H), 8.68 (s, 1H), 7.71 (d, J ¼ 8.7 Hz,
2H), 6.98 (d, J ¼ 8.7 Hz, 2H), 3.97 (t, J ¼ 6.5 Hz, 2H), 3.84 (t,
J ¼ 6.5 Hz, 2H), 1.96 (t, J ¼ 7.4 Hz, 2H), 1.78e1.68 (m, 2H), 1.63e1.48
(m, 4H), 1.40e1.30 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H) ppm. 13C NMR
White solid; yield 20%; mp 119 ꢃC. 1H NMR (500.13 MHz, DMSO-
d6):
d
¼ 11.54 (s, 1H), 10.36 (s, 1H), 8.68 (s, 1H), 7.66 (d, J ¼ 8.1 Hz,
2H), 7.27 (d, J ¼ 7.9 Hz, 2H), 3.85 (t, J ¼ 6.5 Hz, 2H), 2.61 (t, J ¼ 7.7 Hz,
2H), 1.96 (t, J ¼ 7.4 Hz, 2H), 1.64e1.47 (m, 6H), 1.43e1.23 (m, 4H),
0.89 (t, J ¼ 7.4 Hz, 3H) ppm. 13C NMR (125.76 MHz, DMSO-d6):
(125.76 MHz, DMSO-d6):
d
¼ 168.89, 163.77, 161.09, 128.74, 124.19,
113.96, 74.95, 69.01, 32.09, 27.36, 24.99, 24.82, 21.82, 10.23 ppm. IR
(KBr): ṽ ¼ 3214 (NH), 2939, 2869 (CH2), 1669 (C]O) cmꢀ1. Anal.
Calcd. for C16H24N2O5: C 59.24, H 7.46, N 8.64. Found: C 59.45, H
7.56, N 8.42.
d
¼ 168.90,164.04, 146.11, 129.75, 128.19,126.96, 74.97, 34.52, 32.75,
32.09, 27.35, 24.99, 24.82, 21.58,13.64 ppm. IR (KBr): ṽ ¼ 3205 (NH),
2943, 2865 (CH2), 1650 (C]O) cmꢀ1. Anal. Calcd. for C17H26N2O4: C
63.33, H 8.13, N 8.69. Found: C 63.15, H 8.37, N 8.86.
4.3.6. N-((6-(Hydroxyamino)-6-oxohexyl)oxy)-4-(pentyloxy)
benzamide (1n)
4.3.11. 4-(Hexyloxy)-N-((6-(hydroxyamino)-6-oxohexyl)oxy)
benzamide (1s)
White solid; yield 28%; mp 110 ꢃC. 1H NMR (500.13 MHz, DMSO-
d6):
d
¼ 11.46 (s, 1H), 10.35 (s, 1H), 8.67 (s, 1H), 7.70 (d, J ¼ 8.8 Hz,
White solid; yield 36%; mp 105 ꢃC. 1H NMR (500.13 MHz, DMSO-
2H), 6.98 (d, J ¼ 8.8 Hz, 2H), 4.01 (t, J ¼ 6.5 Hz, 2H), 3.84 (t, J ¼ 6.5 Hz,
2H), 1.96 (t, J ¼ 7.3 Hz, 2H), 1.77e1.67 (m, 2H), 1.63e1.48 (m, 4H),
1.46e1.27 (m, 6H), 0.89 (t, J ¼ 7.1 Hz, 3H) ppm. 13C NMR
d6):
d
¼ 11.46 (s, 1H), 10.36 (s, 1H), 8.67 (s, 1H), 7.70 (d, J ¼ 8.7 Hz,
2H), 6.98 (d, J ¼ 8.8 Hz, 2H), 4.01 (t, J ¼ 6.5 Hz, 2H), 3.84 (t, J ¼ 6.4 Hz,
2H), 1.96 (t, J ¼ 7.3 Hz, 2H), 1.76e1.65 (m, 2H), 1.63e1.47 (m, 4H),
1.46e1.23 (m, 8H), 0.88 (t, J ¼ 6.9 Hz, 3H) ppm. 13C NMR
(125.76 MHz, DMSO-d6):
d
¼ 168.90, 163.76, 161.10, 128.74, 124.21,
113.97, 74.94, 67.54, 32.10, 28.15, 27.55, 27.37, 25.00, 24.82, 21.77,
13.81 ppm. IR (KBr): ṽ ¼ 3211 (NH), 2940 (CH2), 1672, 1616 (C]O)
cmꢀ1. Anal. Calcd. for C18H28N2O5: C 61.34, H 8.01, N 7.95. Found: C
61.14, H 8.16, N 7.72.
(125.76 MHz, DMSO-d6):
d
¼ 168.92, 163.80, 161.11, 128.75, 124.20,
113.98, 74.96, 67.56, 32.09, 30.86, 28.41, 27.54, 27.35, 25.02, 24.81,
21.95, 13.80 ppm. IR (KBr): ṽ ¼ 3237 (NH), 2930 (CH2), 1666, 1619
(C]O) cmꢀ1. Anal. Calcd. for C19H30N2O5: C 62.27, H 8.25, N 7.64.
Found: C 62.38, H 8.40, N 7.35.
4.3.7. 4-Ethoxy-N-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide
(1o)
4.3.12. N-((6-(Hydroxyamino)-6-oxohexyl)oxy)furan-2-
carboxamide (1t)
White solid; yield 25%; mp 125 ꢃC. 1H NMR (500.13 MHz, DMSO-
d6):
d
¼ 11.46 (s, 1H), 10.35 (s, 1H), 8.67 (s, 1H), 7.71 (d, J ¼ 8.8 Hz,
White solid; yield 32%; mp 113 ꢃC. 1H NMR (500.13 MHz, DMSO-
2H), 6.97 (d, J ¼ 8.8 Hz, 2H), 4.07 (q, J ¼ 7.0 Hz, 2H), 3.84 (t,
d6):
d
¼ 11.61 (s, 1H), 10.35 (s, 1H), 8.67 (s, 1H), 7.85 (s, 1H), 7.09 (d,
J ¼ 6.4 Hz, 2H), 1.96 (t, J ¼ 7.3 Hz, 2H), 1.62e1.48 (m, 4H), 1.41e1.29
J ¼ 3.1 Hz, 1H), 6.63 (dd, J ¼ 3.1, 1.6 Hz, 1H), 3.83 (t, J ¼ 6.4 Hz, 2H),
1.95 (t, J ¼ 7.3 Hz, 2H), 1.65e1.45 (m, 4H), 1.39e1.27 (m, 2H) ppm.
(m, 5H) ppm. 13C NMR (125.76 MHz, DMSO-d6):
d
¼ 168.92, 163.79,
160.95, 128.76, 124.23, 113.94, 74.97, 63.20, 32.10, 27.36, 25.00,
24.82, 14.42 ppm. IR (KBr): ṽ ¼ 3170 (NH), 2850 (CH2), 1619 (C]O)
cmꢀ1. Anal. Calcd. for C15H22N2O5: C 58.05, H 7.15, N 9.03. Found: C
57.81, H 7.25, N 8.97.
13C NMR (125.76 MHz, DMSO-d6):
d
¼ 168.95, 155.91, 145.60,
145.30, 113.89, 111.61, 75.36, 32.08, 27.29, 24.93, 24.80 ppm. IR
(KBr): ṽ ¼ 3250, 3137 (NH), 2936, 2867 (CH2), 1666 (C]O) cmꢀ1
.
Anal. Calcd. for C11H16N2O5: C 51.56, H 6.29, N 10.93. Found: C 51.56,
H 6.46, N 10.72.
4.3.8. N-((6-(Hydroxyamino)-6-oxohexyl)oxy)-2-
propoxybenzamide (1p)
4.3.13. 2-Fluoro-N-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide
(1u)
White solid; yield 20%; mp 102 ꢃC. 1H NMR (500.13 MHz, DMSO-
d6):
d
¼ 10.95 (s, 1H), 10.35 (s, 1H), 8.67 (s, 1H), 7.51e7.35 (m, 2H),
Orange solid; yield 26%; mp 122 ꢃC. 1H NMR (500.13 MHz,
7.08 (d, J ¼ 8.3 Hz, 1H), 6.99 (t, J ¼ 7.4 Hz,1H), 3.99 (t, J ¼ 6.4 Hz, 2H),
3.85 (t, J ¼ 6.5 Hz, 2H), 1.96 (t, J ¼ 7.3 Hz, 2H), 1.80e1.68 (m, 2H),
1.66e1.47 (m, 4H), 1.42e1.27 (m, 2H), 0.98 (t, J ¼ 7.4 Hz, 3H) ppm.
DMSO-d6):
d
¼ 11.46 (s, 1H), 10.36 (s, 1H), 8.68 (s, 1H), 7.60e7.50 (m,
2H), 7.34e7.25 (m, 2H), 3.87 (t, J ¼ 6.5 Hz, 2H), 1.96 (t, J ¼ 7.4 Hz,
2H), 1.66e1.47 (m, 4H), 1.42e1.30 (m, 2H) ppm. 13C NMR
13C NMR (125.76 MHz, DMSO-d6):
d
¼ 168.92, 163.20, 155.83,
(125.76 MHz, DMSO-d6):
d
¼
168.92, 160.99, 158.89 (d,
131.65, 129.39, 122.91, 120.16, 112.59, 74.93, 69.54, 32.10, 27.24,
24.94, 24.84, 21.82, 10.32 ppm. IR (KBr): ṽ ¼ 3349, 3230 (NH),
2936(CH2), 1651 (C]O) cmꢀ1. HRMS (ESI) [M þ H]þ: 325.17567,
1JC,F ¼ 249.2 Hz), 132.58 (d, 3JC,F ¼ 8.4 Hz), 128.86 (d, 4JC,F ¼ 3.1 Hz),
3
2
124.47 (d, JC,F ¼ 3.6 Hz), 121.85 (d, JC,F ¼ 15.6 Hz), 116.00 (d,
2JC,F ¼ 21.9 Hz), 75.08, 32.09, 27.23, 24.91, 24.81 ppm. IR (KBr):

F.K. Hansen et al. / European Journal of Medicinal Chemistry 82 (2014) 204e213
211
ṽ ¼ 3297 (NH), 2949, 2878 (CH2), 1673, 1635 (C]O) cmꢀ1. Anal.
4.7. Late stage P. falciparum (IVeV) anti-gametocyte assay
Calcd. for C13H17FN2O4: C 54.92, H 6.03, N 9.85. Found: C 55.05, H
6.31, N 9.65.
Late stage gametocyte viability assays were carried out using
highly synchronous stage IV gametocytes induced from transgenic
NF54 P. falciparum parasites expressing the gametocyte specific
protein, Pfs16, fused to green fluorescent protein (NF-54-pfs16-
GFP), as described previously [41]. Gametocytes were harvested
by magnetic isolation on day 8 post induction then added to 384
well imaging plates at 33,000 gametocytes per well. Following the
addition of test or control compounds, cells were incubated for 72 h
under reduced oxygen tension (5% CO2, 5% O2, 80% N2). Mitotracker
Red CM-H2XRos was then added to each well (0.07 ug/ml) and the
cells were incubated overnight as described above. DMSO (0.4%)
4.4. Asexual stage in vitro antimalarial assays
Activity against asexual stage P. falciparum line 3D7 was deter-
mined using [3H]-hypoxanthine incorporation, essentially as pre-
viously described [29,38]. Serial dilutions of compounds were
prepared in parasite culture media (RPMI 1640 supplemented with
10% heat inactivated human serum), followed by addition of syn-
chronous ring-stage parasites 0.25% parasitaemia and 2.5% hae-
matocrit. Following incubation for 48 h at 37 ꢃC under standard
and puromycin (5
mM) were used as controls in each assay.
parasite culture conditions, [3H]-hypoxanthine (0.5
mCi/well) was
Gametocyte viability was evaluated on an OPERA™ (PerkinElmer)
High Content Screening System. Images acquired for GFP and
Mitotracker Red CM-H2XRos were overlaid and the number of
elongated viable gametocytes per image was determined using a
script based on Acapella™ software, developed for use with the
OPERA™ imaging system. Percent inhibition compared to DMSO
vehicle controls was calculated and mean IC50 values ( SD) deter-
mined by non-linear regression analysis, sigmoidal dose response
(variable slope) fit using Prizm 4.0 for two separate experiments in
duplicate point.
added to each well, and the cultures incubated for a further 24 h.
[3H]-hypoxanthine incorporation was measured by harvesting onto
1450 MicroBeta filter mats (Wallac) and counting using a 1450
MicroBeta liquid scintillation counter. Percentage inhibition of
growth compared to matched DMSO controls (0.5%) was deter-
mined. Three independent experiments were carried out, each in
triplicate wells. Chloroquine was included in each assay as a posi-
tive control. IC50 values were determined using log-linear inter-
polation of inhibition curves [39] and are presented as mean ( SD)
of the three independent assays.
4.7.1. Principle of script used for image analysis
Maximal fluorescent pixel intensities are identified for the MTR
acquired image and the average intensity for designated objects
calculated. Objects with an average fluorescent MTR intensity,
above an assay specified classification of minimal MTR signal, are
identified as viable objects. GFP positive objects are identified and
overlaid with the MTR positive objects. The GFP objects with a MTR
positive signal are then evaluated for the characteristic of being at
least four times longer than they are wide. Objects that are both
MTR positive and GFP object elongated are identified as viable late
stage gametocytes.
4.5. Histone hyperacetylation assays
The effect of compounds on histone hyperacetylation was
determined essentially as previously described [29]. Briefly, syn-
chronous trophozoite-stage P. falciparum 3D7 parasites were
incubated for 3 h under standard in vitro culture conditions with
500 nM of test compounds or vehicle control (0.05% DMSO). The
antimalarial drug chloroquine (500 nM) was included as a negative
control and TSA was included as a HDAC inhibitor positive control.
Vehicle control samples were taken at the start (t ¼ 0) and end
(t ¼ 3 h) of the treatment. Protein lysates were prepared by lysing
cells with saponin (Sigma, USA) and extensive washing in phos-
phate buffered saline to remove haemoglobin. Parasite pellets were
resuspended in SDS-PAGE loading dye, heat denatured at 96 ꢃC
then separated via 15% SDS-PAGE. Western blot was carried out
using Odyssey reagents (LI-COR Biosciences) according to the
manufacturers' instructions. The following primary antisera were
used: anti-(tetra) acetyl H4 (Millipore) which recognizes lysines 5,
8, 12, and 16 of histone H4; anti-acetyl H3 which recognizes N-
terminally acetylated histone H3; and anti-IC3/94 monoclonal
antisera which was used as a loading control and recognizes
P. falciparum rhoptry associated protein 2 (RAP2) [40]. IRDye®
secondary antibodies (LI-COR Biosciences) were used and mem-
branes imaged using an Odyssey infrared imaging system (LI-COR
biosciences).
4.8. Pb liver stage assay
4.8.1. Parasites
P. berghei Luciferase sporozoites from MR4 (MRA-868) [42] were
obtained by dissection of infected Anopheles stephensi mosquito
salivary glands. Dissected salivary glands were homogenized in a
glass tissue grinder and filtered twice through nylon cell strainers
(40 mm pore size, BD Falcon 087711) and counted using a haemo-
cytometer. The sporozoites were kept on ice until needed.
4.8.2. Cell lines
HepG2-A16-CD81EGFP cells stably transformed to express a
GFP-CD81 fusion protein [43] were cultured at 37 ꢃC in 5% CO2 in
DMEM (Invitrogen, Carlsbad, USA) supplemented with 10% FCS, and
1ꢂ Penicillin-Streptomycin-Glutamine (Gibco, Life Technologies,
USA).
4.6. Deacetylase assays
4.8.3. Sporozoite infection
Recombinant PfHDAC1 (SigmaeAldrich, USA) and P. falciparum
nuclear protein lysates were tested for deacetylase activity using a
fluorometric HDAC Assay Kit (Merck Millipore, USA), as per the
manufacturers' instructions. Preparation of P. falciparum nuclear
lysates from trophozoite stage parasites was carried out using a
NucBuster protein extraction kit (Calbiochem, USA), according to
the manufactures instructions. PfHDAC1 activity was assessed us-
ing Fluorogenic HDAC substrate 3 (BPS Bioscience, USA). Assays
were carried out in duplicate, on two separate occasions and data
presented as mean ( SD) % inhibition compared to controls.
We used P. berghei for this screen since its higher infection rates
of immortal human liver cell lines are more conducive to high
throughput screening than the infection rates of human malaria
parasites. P. berghei is able to infect human hepatocarcinoma
HepG2 cells expressing the tetraspanin CD81 receptor [43,44].
3
ꢂ
103 HepG2-A16-CD81EGFP cells in
5 ml of medium
(2 ꢂ 105 cells/ml, 5% FBS, 5ꢂ Penicillin-Streptomycin-Glutamine
(Gibco, Life Technologies, USA)) were seeded in 1536-well plates
(Greiner BioOne white solid bottom, custom GNF mold) 20e26 h
prior to the actual infection. 18 h prior to infection, 50 nl of

212
F.K. Hansen et al. / European Journal of Medicinal Chemistry 82 (2014) 204e213
[3] S. Abdulla, N. Salim, F. Machera, R. Kamata, O. Juma, M. Shomari, S. Kubhoja,
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transferred with a PinTool (GNF Systems) or Echo liquid handler
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tion starting at 10 mM) and 0.5% DMSO were used as positive and
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K. Marsh, S. Owusu-Agyei, K.P. Asante, K. Osei-Kwakye, O. Boahen, S. Ayamba,
K. Kayan, R. Owusu-Ofori, D. Dosoo, I. Asante, G. Adjei, D. Chandramohan,
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were freshly dissected as described above and their concentration
adjusted to 200 sporozoites per ml (final concentration: 1000 spo-
rozoites per well) with 5ꢂ Penicillin-Streptomycin-Glutamine
(Gibco, Life Technologies, USA) in DMEM (Invitrogen). The
increased antibiotic concentration did not interfere with the para-
site or HepG2-A16-CD81EGFP growth. The HepG2-A16-CD81EGFP
cells were then infected with 1 ꢂ 103 sporozoites per well (5
ml)
with a single tip Bottle Valve liquid handler (GNF Systems), and the
plates spun down at 37 ꢃC for 3 min in an Eppendorf 5810 R
centrifuge with a centrifugal force of 330ꢂ g on lowest acceleration
and brake setting. The plates were then incubated at 37 ꢃC for 48 h
in 5% CO2 with high humidity to minimize media evaporation and
edge effect. After incubation, media was removed by spinning the
inverted plates at 150ꢂ g for 30 s 2
ml BrightGlo luciferase (Prom-
ega) were dispensed with the MicroFlo (BioTek) liquid handler.
Immediately after addition of the luminescence reagent, the plates
were vortexed at median intensity setting for 10 s and read by an
EnVision Multilabel Reader (PerkinElmer). IC50 values were deter-
mined using a non-linear variable slope four parameter regression
curve fitting model in Prism 6 (GraphPad Software Inc).
4.8.4. HepG2 toxicity assay
HepG2 viability after 56 h was used to assess compound toxicity.
Specifically we assayed ATP levels of live HepG2 host cells and
compared them to ATP levels of DMSO-treated control cells. He-
patocytes were cultured as described above, but on the day of
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MicroFlo (BioTek) liquid handler. After the 48 h incubation period,
^
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30 s 2 ml of CellTiterGlo (Promega) diluted 1:1 with ddH2O were
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EnVision Multilabel Reader (PerkinElmer). IC50 values were deter-
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Funding sources
This work was supported by the Australian Research Council
(FT0991213 to KTA; LP120200557 to VMA), Medicines for Malaria
Ventures (MMV) (to SM and VMA) as well as the Bill and Melinda
Gates Foundation, MMV, and NIH (R01AI090141) (to EAW).
Acknowledgements
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mechanisms of action, Oncogene 26 (2007) 5541e5552.
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novel strategies for therapy and prevention, Oncogene 26 (2007) 5310e5318.
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We thank the Australian Red Cross Blood Service for the provision
of human blood and sera. The Deutsche Forschungsgemeinschaft
(DFG) is acknowledged for funds used to purchase the UHR-TOF
maXis 4G, Bruker Daltonics, Bremen HRMS instrument used in this
research.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.050.
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