Antimalarial histone deacetylase inhibitors containing cinnamate or NSAID components

Article abstract of DOI:10.1016/j.bmcl.2010.09.096

Malaria is the most lethal parasite-mediated tropical infectious disease, killing 1-2 million people each year. An emerging drug target is the enzyme Plasmodium falciparum histone deacetylase 1 (PfHDAC1). We report 26 compounds designed to bind the zinc and exterior surface around the entrance to the active site of PfHDAC1, 16 displaying potent in vitro antimalarial activity (IC ₅₀ <100 nM) against P. falciparum. Selected compounds were shown to cause hyperacetylation of P. falciparum histones and be >10-fold more cytotoxic towards P. falciparum than a normal human cell type (NFF). Twenty-two inhibitors feature cinnamic acid derivatives or non-steroidal anti-inflammatory drugs (NSAIDs) as HDAC-binding components. A homology model of PfHDAC1 enzyme gives new insights to interactions likely made by some of these inhibitors. Results support PfHDAC1 as a promising new antimalarial drug target.

Full text of DOI:10.1016/j.bmcl.2010.09.096

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7080–7084
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antimalarial histone deacetylase inhibitors containing cinnamate
or NSAID components
Nicole C. Wheatley a, Katherine T. Andrews b,c, Truc L. Tran b,c, Andrew J. Lucke a,
Robert C. Reid a, David P. Fairlie a,
⇑
a Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Queensland 4072, Australia
b Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Queensland, Australia
c Queensland Institute of Medical Research, Herston, Queensland, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2010
Revised 15 September 2010
Accepted 16 September 2010
Available online 22 September 2010
Malaria is the most lethal parasite-mediated tropical infectious disease, killing 1–2 million people each
year. An emerging drug target is the enzyme Plasmodium falciparum histone deacetylase 1 (PfHDAC1).
We report 26 compounds designed to bind the zinc and exterior surface around the entrance to the active
site of PfHDAC1, 16 displaying potent in vitro antimalarial activity (IC50 <100 nM) against P. falciparum.
Selected compounds were shown to cause hyperacetylation of P. falciparum histones and be >10-fold
more cytotoxic towards P. falciparum than a normal human cell type (NFF). Twenty-two inhibitors feature
cinnamic acid derivatives or non-steroidal anti-inflammatory drugs (NSAIDs) as HDAC-binding compo-
nents. A homology model of PfHDAC1 enzyme gives new insights to interactions likely made by some
of these inhibitors. Results support PfHDAC1 as a promising new antimalarial drug target.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
HDAC inhibitor
Plasmodium falciparum
NSAID
Malaria
Histone
Malaria is a mosquito-borne infectious disease caused by proto-
zoan parasites. It is widespread in tropical and subtropical regions
and each year there are approximately 500 million clinical cases of
malaria. An estimated 1–2 million people die each year of malaria
related disease and most are children under the age of five.1 There
is currently no licensed malaria vaccine available. Effective treat-
ment and control of malaria continues to be challenged by parasite
resistance to antimalarial drugs,2,3 prompting renewed efforts to
discover compounds with new mechanisms of action on novel tar-
gets in the parasite.
O
O
O
O
N
NHOH
N
O
NH
N
Trichostatin 2
HN
O
N
H
O
HN
H
N
O
H
N
N
N
O
O
NHOH
H
O
NHOH
O
O
4
Apicidin 1
SAHA 3
Figure 1. Examples of HDAC inhibitors with antimalarial activity.
Histone deacetylase enzymes in malaria parasites may repre-
sent potential new targets for antimalarial drug development.
HDACs are Zn-dependent enzymes that play crucial roles in mod-
ulating mammalian cell chromatin structure, transcription, and
gene expression.4,5 HDAC inhibitors can arrest growth and induce
differentiation and/or apoptotic cell death in various human cancer
cells lines6,7 and some HDAC inhibitors also possess antimalarial
activity.8–12 Compounds like apicidin 1, TSA 2, SAHA 3, and com-
Apicidin is known to cause dramatic transcriptional changes at all
stages of the P. falciparum intraerythrocytic developmental cycle.16
Of five known P. falciparum HDACs (PfHDACs), PfHDAC1 was found
localized mainly in the parasite nucleus,15 with sequence homology
to human HDAC1, and is inhibited by 2 and 3.15,17 Homology
models9,18 of PfHDAC1 have been compared to crystal structures of
human HDAC8. Subtle differences in amino acids that line the bind-
ing pocket, especially the exterior surface at the entrance to the
tubular zinc-bound active site, might be exploited to develop selec-
tive PfHDAC1 inhibitors.
We have found that compounds derived from Asu, like 4
and Asu-9, have potent antimalarial activity9 and also inhibit
P. falciparum HDAC nuclear extracts in a dose-dependent manner
(IC50 78 nM and 87 nM, respectively, unpublished results). Asu-9
was a lead compound with a cinnamic acid component. To
pound 4 derived from L-2-aminosuberic acid (Asu) (Fig. 1), inhibit
HDAC enzymes by binding in the zinc-containing active site.9,13
Plasmodium falciparum contains at least five putative HDAC en-
zymes as well as homologs to other histone modifying proteins such
as acetylase enzymes, although little is known about them.14,15
⇑
Corresponding author. Tel.: +61 733461989; fax: +61 733461990.
E-mail address: d.fairlie@imb.uq.edu.au (D.P. Fairlie).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.096

N. C. Wheatley et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7080–7084
7081
O
O
EtO
HN
O
O
a, b
c
O
OEt
Br
I
OH
OMe
OMe
O
6
5
7
O
d, e, f
O
OH
O
OMe
O
OMe
O
O
+
g
CbzHN
OMe
CbzHN
OMe
CbzHN
OMe
10
8
9
h, i
R1
O
O
R1
O
R1
j
k
O
O
O
CbzHN
OMe
HN
R2
OMe
HN
R2
NHOH
11
12
13a-z
Scheme 1. Synthesis and resolution of antimalarial compounds. Reagents and conditions: (a) MeOH, cat. H2SO4,
D
;
(b) acetone, NaI,
D
;
(c) DMF, NaH, diethyl
acetamidomalonate, rt; (d) HCl/H2O,
D; (e) MeOH, HCl, D
; (f) EtOAc, Cbz-OSu, pH 8, rt; (g) DMF, 0.2 M NaOAc, Cys. HCl, Carica Papaya; (h) DMF, BOP, DIPEA, R1 (amine) (16), rt;
(i) MeOH/EtOAc, H2, 10% Pd/C, cat. TFA, rt; (j) DMF, BOP, DIPEA, R2 (acid), rt; (k) MeOH, KO(CH3)3, hydroxylamine hydrochloride, rt.
Table 1
explore the active-site surface of PfHDAC1, a series of syntheti-
Antimalarial activity of cinnamic acid derived-inhibitors 13a–m (R1 = 8-
aminoquinoline)
cally accessible derivatives of cinnamic acids, non-steroidal
anti-inflammatory drugs (NSAIDs), and amines were synthesized.
Inhibitor synthesis was performed according to Scheme 1.
Compound 5 was protected as a methyl ester followed by iodide
substitution forming 6. Alkylation of diethyl acetamidomalonate
with 6 gave 7, which was hydrolyzed under acidic conditions
followed by protection of the acid as the methyl ester, and con-
version of the amine to the benzylcarbamate 8. Chiral resolution
of 8 was achieved using the cysteine protease, Carica papaya19,20
and stopped at ꢀ40% conversion to free acid 10 and ester 9.
Optical purity of 10 was determined by derivatizing the amino
acid with a chiral auxiliary and examining the dipeptide with
NMR experiments (Supplementary data). Functionalizing side
chain groups R1 and R2 (Scheme 1) using standard coupling pro-
cedures gave a series of inhibitors.
Synthetic compounds had C log P values between 1.5 and 5.5
and were thus expected to be cell permeable. In vitro P. falciparum
growth inhibition assays were performed as described.9 Initially,
cinnamic acid derivatives (13a–m, Table 1) were synthesized as
putative HDAC inhibitors. Smaller compounds like 13a–c with hy-
droxy/methoxy substituents on the cinnamyl aromatic ring were
potent inhibitors (IC50 14, 34, 52 nM, respectively), as were deriv-
atives with m-bromo (13e, IC50 50 nM) or furylacryl (13f, IC50
15 nM) substituents. Cinnamic acid derivatives 13g–m Table 1,
with longer extended meta/para substituents showed P10-fold
decreased killing potency.
Since extended cinnamates were less potent, we decided to syn-
thesize more compact rigid compounds. A series of conveniently
available carboxylic acids were NSAIDs that were readily coupled
to 8-aminoquinoline derived compounds (NSAIDs: Flubiprofen,
Naproxen, Mefenamic acid, Diclofenac, Indomethacin, Aspirin,
Tolmetin, Sulindac, and Indoprofen compounds; 13n–u, Table 2,
respectively). Partial hydrolysis of indomethacin and aspirin
derivatives occurred, producing 13r and 13s, respectively. All but
one of the NSAID derivatives had potent antimalarial activity
(IC50 665 nM), none being too bulky or too elongated to prevent
parasite killing.
Previously, we reported Asu-13 as a potent antimalarial com-
pound.9 To explore active-site space occupied by the 8-aminoquin-
oline group, four p-dimethylaminobenzoic acid substituted
inhibitors were synthesized with differing amines (13w–z, Table 3).
Compounds 13w–z were all potent inhibitors (IC50 6102 nM), but
13z was the most potent and may be a valuable lead. Compounds
13o, 13r, Asu-13 were also P10 times more cytoselective for
a
a
No.
R2
C log P
IC50 (nM)
IC90 (nM)
O
Asu-9
2.9
15
14
nd
26
OMe
HO
13a
1.8
MeO
O
O
O
MeO
MeO
13b
13c
13d
2.5
2.2
4.9
34
52b
61
nd
nd
MeO
MeO
OMe
O
156b
O
13e
13f
13g
3.7
2.0
4.8
50b
15
nd
Br
O
O
27
O
O
>1000
>1000
O
13h
5.2
400
805
O
O
Br
O
13i
13j
13k
5.4
4.8
5.0
357
217
153
687
536
375
O
O
O
O
O
O
13l
4.4
5.2
184
453
373
827
O
O
13m
O
a
Mean values n = 3.
Duplicate experiments, nd = not determined.
b

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N. C. Wheatley et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7080–7084
Table 2
17 (3D7 vs HeLa cells) have been used to cure Plasmodium berg-
hei-infected mice.12
Antimalarial activity of compounds 13n–v with NSAIDs as HDAC-binding compo-
nents (R1 = 8-aminoquinoline)
To confirm that compounds investigated here act as HDAC
inhibitors in malaria parasites, three representative compounds
(13o, 13r, and 13z) were assessed for their capacity to hyperacet-
ylate P. falciparum histones. All three compounds and TSA (as con-
trol) caused hyperacetylation of histone H4 in 3D7 parasites
(Fig. 2), as determined by Western blot analysis using a commer-
cial polyclonal anti-tetra-acetyl histone H4 antibody (Upstate).
Artemisinin, a control antimalarial compound, did not affect his-
tone H4 acetylation (Fig. 2).
A homology model of the PfHDAC1 sequence (PFI1260c) was
created from crystal structures of a bacterial homolog HDLP
(1C3R.pdb, 2.00 Å resolution)21 for loop1, and human HDAC8
(using 2v5x.pdb, 2.25 Å resolution,22 rather than multiple crystal
structures used previously9) for the remainder of the protein (se-
quence identities 32% and 42%, respectively). The PfHDAC1 homol-
ogy model has the same fold as Class 1 HDACs, consisting of a
No.
R2
C log P
IC50 (nM)
IC90 (nM)
a
a
13n
4.4
42
14
76
24
F
O
13o
13p
3.4
5.1
O
O
N
51
65
89
H
O
Cl
N
13q
13r
5.3
2.5
119
H
Cl
O
O
O
13
22
N
H
single a/b domain. b-Sheets form an interior scaffold of the enzyme
and help support active-site residues surrounding the catalytic Zn.
Active-site tunnel residues are identical to human Class I HDACs
with loop residues near the entrance showing some variability.
As the homology model was based on HDAC8, it is more flexible
around loops 1 and 2 than PfHDAC1. Four pockets were identified
near the active-site entrance (A–D Fig. 3a). GOLD was used to dock
ligands in the active site using distance and H-bond constraints
(Supplementary data).
Top ranked GOLD scoring conformations provided insights to
possible binding orientations of 13a–z (see Fig. 3). The HDAC8
crystal structure shows an Asu derived inhibitor 2V5X and a
peptide interacting with HDAC8 via both backbone amide–NHs
H-bonding in a cis-conformation to Asp97. GOLD docked smaller
cinnamic acid inhibitors 13a–h, k, and l with one or both (cis-con-
formation) Asu amide–NHs H-bonding to Asp97, the 8-aminoquin-
oline group placed into pocket C, and the cinnamate group in
pocket A (inhibitors 13a, 13d; Fig. 3b). Other cinnamic acid deriv-
atives were more extended with one or no H-bonds to Asp97, forc-
ing side chains to access pockets B, C, and D (e.g., 13h and 13i,
Fig. 3b).
O
13s
13t
2.6
2.8
115
47
214
102
OH
N
O
O
O
13u
13v
3.7
3.4
35
32
35
65
F
O
S
O
N
O
a
Mean values n = 3.
Table 3
Antimalarial activity amine-derived inhibitors (R2 = p-dimethylaminobenzoic acid)
R1
C log P
IC50 (nM)
IC90 (nM)
a
a
No.
Asu-13
2.3
19
nd
Docked conformations of NSAID-derived inhibitors 13n–v
clearly showed the importance of the inhibitor backbone amide–
NHs H-bonding to Asp97 with the 8-aminoquinoline group docked
into pockets C and D with the NSAID groups binding in A or D
(inhibitors 13n yellow, 13p orange, 13r lt blue, Fig. 3c). The C-
terminal derivatives 13w–z docked with the p-dimethylamino-
benzoic acid group binding in pocket A and the amine groups in
N
NH
OMe
OMe
13w
13x
13y
1.7
3.3
3.0
101
22
nd
nd
nd
H
N
HN
21
NH
13z
3.0
5
nd
NH
a
Mean values n = 3, nd = not determined.
Table 4
Inhibitors compared against Pf-3D7 and NFF cells
SIb
a
a
No.
3D7 IC50 (nM)
NFFs IC50 (nM)
13o
13r
Asu-13
14
13
19
169
260
570
12
20
30
a
Mean of three experiments.
Selectivity index = NFF IC50/3D7 IC50
Figure 2. P. falciparum infected erythrocytes (3D7) were cultured in the presence of
20 and 100 nM artemisinin (ART), trichostatin A (TSA), 13o, 13r, or 13z for 3.5 h.
Matched controls taken at the start (C1) and at 3.5 h (C2) received vehicle alone
(0.1% DMSO). Total protein was extracted and separated via 15% SDS–PAGE.
Hyperacetylation was determined by Western blot using polyclonal anti-tetraacetyl
histone H4 antisera (Upstate). Coomassie staining of a matched control gel showed
loading.
b
.
P. falciparum malaria parasites versus human normal neonatal
foreskin fibroblast (NFF) cells (Table 4). Selectivity is variable
depending on cell lines used,9,12 but inhibitors with SI as low as

N. C. Wheatley et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7080–7084
7083
Figure 3. Solvent excluded surface of PfHDAC1 homology model colored by electrostatic potential (blue +, red À); (a) transparent surface showing residues that surround
surface pockets A–D; (b) Gold docked cinnamate inhibitors (13a green, 13d magenta, 13h orange, 13i yellow); (c) NSAID-derived inhibitors (13n yellow, 13p orange, 13r lt
blue); (d) varied amine-derived inhibitors (13w yellow, 13x pink, 13y lt blue, 13z green) shown.
pockets B and C (inhibitors 13w, 13x, 13y, and 13z, Fig. 3d). The
model and docking results suggest that binding of Asu derivatives
requires inhibitors to maximize H-bonding to Asp97 and to effi-
ciently fill pockets nearest the active-site entrance. Docking placed
side chains of ligands close to residues Ala95 and Thr96 not pres-
ent in HDAC1, thus posing Thr96 as a polar target for increase
selectivity over HDAC1.
Grant 569735) for partially funding this work, and ARC for a Feder-
ation Fellowship (to D.P.F.) and a Future Fellowship (to K.T.A.). We
thank the Red Cross Blood Service for providing human blood and
sera.
Supplementary data
Described herein is a series of potent antimalarial compounds,
16 with IC50 <100 nM and 5 with IC90 <50 nM against a drug sensi-
tive (3D7) strain of P. falciparum. Five inhibitors were equipotent or
more potent than Asu-9, but selectivity was reduced relative to
Asu-13. Some inhibitors contained NSAIDs as effective HDAC-bind-
ing components. The inhibitors showed selectivity in killing the
malarial parasite over normal cells and they caused hyperacetyla-
Supplementary data (experimental methods, compound syn-
theses, and characterization) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2010.09.096.
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