Hydroxamic acid based histone deacetylase inhibitors with confirmed activity against the malaria parasite

Article abstract of DOI:10.1016/j.bmcl.2014.12.051

Recent studies have highlighted a key role in regulating gene transcription, in both eukaryotes and prokaryotes, by enzymes that control the acetylation and deacetylation of histones. In particular, inhibitors of histone deacetylases (HDAC-Is) have been s

Full text of DOI:10.1016/j.bmcl.2014.12.051

Bioorganic & Medicinal Chemistry Letters 25 (2015) 459–461
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hydroxamic acid based histone deacetylase inhibitors
with confirmed activity against the malaria parasite
⇑
Giuseppe Giannini , Gianfranco Battistuzzi, Davide Vignola
R&D Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30,400, I-00040 Pomezia, Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Recent studies have highlighted a key role in regulating gene transcription, in both eukaryotes and
prokaryotes, by enzymes that control the acetylation and deacetylation of histones. In particular,
inhibitors of histone deacetylases (HDAC-Is) have been shown effective in controlling the development
of many parasites, such as the plasmodium of malaria. Here we report the results of a study aimed at
evaluating antiparasitic effect of two classes of HDAC-Is bearing different zinc binding group (hydroxamic
acid vs thiol). The study showed that only the hydroxamic acid based HDAC inhibitors were active, with
Plasmodium falciparum being the most sensitive parasite, having from low double-digit to single-digit
nanomolar range in vitro activities. Among three derivatives evaluated also in vivo, ST8086AA1 (8)
effectively inhibited 88% of the development of Plasmodium falciparum.
Received 18 November 2014
Revised 10 December 2014
Accepted 12 December 2014
Available online 19 December 2014
Keywords:
Hydroxamic acid
HDAC inhibitors
Antimalarial activity
Plasmodium falciparum
Antiprotozoan drug
Ó 2014 Elsevier Ltd. All rights reserved.
The problem of parasitic infections is far from being defeated, so
diseases caused by protozoan parasites are still an important
health problem with remarkable social and economic impact on
human societies. Some parasites, if untreated, could cause a wide
spectrum of diseases. Problem of drug resistance is also always
lurking.1 Researchers around the world, working on this front,
are facing the problem following two different approaches: screen-
ing on the protozoa old molecules already approved for other ther-
apeutic indications, for drug repurposing,1 or trying to investigate
on new biological targets such as histone deacetylases (HDACs)
(Fig. 1).2–4
HDACs are a group of Zn-dependent enzymes found in several
organisms such as bacteria, fungi, plants, and animals. In the latter
group they play crucial roles in modulating mammalian cell chro-
matin structure, transcription, and gene expression. They belong to
the huge class of so-called ‘lysine-deacetylase’, a class of enzymes
additional indication, for peripheral T-cell lymphoma (PTCL)
together with Belinostat, the third HDAC-I recently approved by FDA.
From a medicinal chemistry point of view, most of the HDAC
inhibitors falls into a pharmacophoric model widely accepted,
which consists of a capping group (CAP), able to interact with
the rim of the catalytic tunnel of the enzyme, opposite of a zinc-
binding group (ZBG), able to complex the Zn2+ ion at the bottom
of the catalytic cavity, and hydrophobic linker connecting the
two parts (Fig. 2). Results from our researches in this field, along
with others, have highlighted the positive contribution that some
substituents on the kink atom—connection unit—(C.U.) may give
to the activity of this class of compounds.6,7
Beyond the well-known cellular effects in various human
cancer cells line, like growth arrest, pro-differentiation and pro-
apoptosis, other fields of application, such as the antiprotozoan
role, are possible for HDAC inhibitors according to recent studies.
In 2008 Andrews, Fairlie et al. reported the results of a screen-
ing, with different classes of HDAC inhibitors, on the malaria para-
site Plasmodium falciparum, showing that hydroxamate-based
HDAC-Is (i.e., TSA and SAHA) were the most powerful as antimalar-
ial agents. Benzamide analogues (i.e., MS-275) were less potent
and, even less were the thiol-based HDAC inhibitor derivatives.8
Recently, other researchers have re-proposed these data enriched
with other examples, such as the activity of the Pracinostat
(SB939).9a
that work removing acetyl groups from e-amino-lysine residues on
many different substrates, not only histones but non-histone
nuclear and cytoplasmic proteins too.5
Inhibition of histone deacetylase is one of the last biological
targets investigated successfully. Two HDAC inhibitor drugs, the
first-in-class synthetic hydroxamic acid derivative (Vorinostat)
and a natural thiol derivative (Romidepsin), have been approved
by the US Food and Drug Administration (FDA) to treat cutaneous
T-cell lymphoma (CTCL). The latter was also approved for
Over the last years, our research group has carried out an
important project aimed at identifying HDAC inhibitors,5 leading
to the selection of a drug candidate, ST7612AA1 (3), currently in
⇑
Corresponding author. Tel.: +39 069 139 3640.
E-mail address: giuseppe.giannini@sigma-tau.it (G. Giannini).
http://dx.doi.org/10.1016/j.bmcl.2014.12.051
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

460
G. Giannini et al. / Bioorg. Med. Chem. Lett. 25 (2015) 459–461
H
N
O
O
antiparasitic activity. Thio derivatives were investigated as such
in binding experiments, meanwhile their thio-acetyl prodrugs
were used in cell cytotoxicity assays (Table 1).
O
OH
OH
N
H
N
H
O
N
Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Plasmodium
falciparum and Giardia lamblia were the four protozoan parasites
selected for this study. Cytotoxicity was evaluated on L6 rat
skeletal muscle derived myoblasts. A set of compounds tested
against Leishmania donovani, showed weak activity.
Under these experimental conditions, thio derivatives either as
free drug (2, 4) or as their corresponding prodrugs (3, 5) displayed
poor antiparasitic activity. Then, we investigated the hydroxamic
acid analogous (drug 6–9), which showed to be very powerful with
a favorable range of activity compared to the cytotoxicity of L6
(Table 2).
SAHA(2a)
TSA(8)
O
O
OH
N
N
N
N
N
H
R
OH
N
( )n
N
H
N
SB939(9a)
HDAC-Is with triazole-linked cap group(9b)
Figure 1. Examples of HDAC inhibitors with antimalarial activity.
Excluding the b-lactam derivative (9), slightly soluble and less
stable than c-lactam analogs, these compounds have been studied
in an in vivo model, with a non-optimized schedule. The results in
Table 3, demonstrated that compound ST8086AA1 (8) is a good
inhibitor of Plasmodium falciparum, with a 87.5% activity versus
99.8% of DHA (DiHydroArtemisinine).
H
N
R
ZBG
O
CU
Linker
CAP
According to the 2013 World Health Organization report,12
malaria is responsible for over 627 thousand deaths a year, espe-
cially among young children and pregnant women, on 207 million
people affected worldwide. The disease can be caused by 5 different
species of the protozoan Plasmodium parasite but of these P. falcipa-
rum is the most lethal and the most prevalent in Sub-Saharan Africa.
Our preliminary data of 8 on Plasmodium represent a very inter-
esting starting point for an in-depth analysis. Since the most criti-
cal aspect of the traditional antimalarial drugs is rapid
development of resistance, it would be interesting to assess this
parameter on 8.
Figure 2. Pharmacophoric model of HDAC inhibitors.
O
N
H
2
3
4
5
6
7
R=H;
R= H
R1 = SH
H
O
R1 = SAc
;
H
N
N
O
R=CF3; R1 = SH
1
R
R=CF3; R1 = SAc
R=CH3; R1= CONHOH
R=CF3; R1= CONHOH
R
This study confirmed that hydroxamic derivatives are active on
Plasmodium but it also showed their higher activity towards the
corresponding thiols, contrary to what was observed in terms of
cytotoxicity. Moreover, placing a methyl or a trifluoromethyl group
as substituent in meta position of phenyl moiety of CAP, was asso-
ciated with the well-known enhancement of the cytotoxic activ-
ity13 but also with an enhanced antiparasitic activity.
O
O
N
O
N
H
H
H
O
H
O
H
N
O
H
N
N
N
OH
OH
N
H
N
H
O
O
8
9
Although several compounds are currently in preclinical and
clinical phase of development, it is noteworthy that successful
development of these compounds is not guaranteed. Discovery
and development of new molecules with novel mechanisms of
action able to circumvent antimalarial drug resistance is strongly
needed.
In conclusion, although not being exhaustive but a contribution
to the advancement of knowledge in this field, this study showed
that: (a) thio-based HDAC inhibitors showed only weak antipara-
sitic activity; (b) hydroxamate-based histone deacetylase inhibi-
tors, having an amide-lactam moiety on kink atom are more
powerful compared to linear counterpart (i.e., suberoylanilide
hydroxamic acid; SAHA). Among the protozoa studied, Plasmodium
falciparum was the most sensitive to this class of drugs.
Figure 3. Lactam-carboxyamides thiol- and hydroxamate-based HDAC inhibitors.
preclinical phase.6 According to the above anticipation, it is known
that HDAC inhibitors are effective on various protozoa, in
particular against Plasmodium falciparum.9 Other authors have also
associated to HDAC inhibitors a potent activity against the African
protozoa of the Trypanosoma brucei.10 This was the context when
this study was inspired (Fig. 3).
Influence of various groups (i.e., substitution in position
a to the
amide CAP, and nature of ZBG) on the antiparasitic activity of a few
SAHA analogues has been looked at. After screening each single
derivative on all know HDAC isoform, and further experiments
on H460 tumor cell line, a few derivatives were selected for their
Table 1
In vitro screening on class I, IIb and IV HDAC isoforms, and cytotoxic activity against non-small cell lung cancer cell line (NCI-H460)6,7
ID
Name or lab. code
Class I (1-3,8), IIb (6,10) and IV HDAC isoforms; IC50 (nM)
Cytotoxicity; IC50 (nM)
NCI-H460
HDAC1
HDAC2
HDAC3
HDAC6
HDAC8
HDAC10
HDAC11
1
2
3
4
5
6
7
8
9
SAHA
258
13
921
78
350
5
29
3
243
281
456
11
362
14
3400
ST7464AA1
ST7612AA1
ST8081AA1
ST8039AA1
ST8074AA1
ST8078AA1
ST8086AA1
ST8087AA1
66
5
12
11
5
316
7
44
30
7
2
34
59
27
8
89
143
11
2
62
88
3
2
3
6
52
33
93
137
30
11
113
125
15
55
13
37
500
500
2200
5400

G. Giannini et al. / Bioorg. Med. Chem. Lett. 25 (2015) 459–461
461
Table 2
Antiprotozoan activities against different protozoa as well as cytotoxicity against L6 cells, in vitro
ID Name or lab.
code
Trypanosoma brucei
rhodesiense IC50 (nM)
Trypanosoma cruzi
IC50 (nM)
Leishmania donovani Plasmodium
Giardia lamblia
IC50 (nM)
Cytotoxicity L6 cells
IC50 (nM)
IC50 (nM)
falciparum IC50 (nM)
Melarsoprol
6
Benznidazole
Miltefosine
2130
3150
Chloroquine
Metronidazole
Podophyllotoxin
6
580
56
19
Reference HDAC inhibitor
SAHA
a
1
3170
10,100
25
266
(Vorinostat)
Thiol derivatives
ST7464AA1
ST7612AA1
ST8081AA1
ST8039AA1
2
3
4
5
3230
2510
4750
2380
25,800
2100
19,100
3980
>10,000
9660
6570
3260
3240
12,300
8110
64,800
40,900
5250
189
2670
91
a
>3000
a
Hydroxamic acid derivatives
6
7
8
9
ST8074AA1
ST8078AA1
ST8086AA1
ST8087AA1
5515
4155
19,950
6045
849
972
48,350
32,950
N.D.
N.D.
N.D.
N.D.
6
3
19
23
4640
4270
10,720
12,750
1232
638
75,100
7460
Reference drugs commonly used against each parasite were compared to these novel inhibitors. (see Table1-S.I.). SAHA was used as a further reference in all tests.11
a
Tox at 3 lM, inactive at 1 lM. N.D: not determined.
Table 3
Activity of HDAC inhibitors versus DHA, in the Plasmodium F. berghei mouse model
References and notes
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Name or lab. code
% Activity vs. control 50 mg/kg;
DMSO/water; i.p.; qd; 4 days
(after 4 days)
6
7
8
ST8074AA1
ST8078AA1
ST8086AA1
DHA
33.75
63.25
87.53
99.78
In the same model, Artemisinin cures it at 4 Â 50 mg/kg (activity 99.9% and survival
>30 days).14
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Interestingly, all hydroxamic acid derivatives displayed in vitro
activity in the same range of chloroquine (IC50: 3–23 nM) and
slightly more potent than SAHA (IC50: 25 nM). However, it should
be emphasized that this class of amide-lactam hydroxamate-based
HDAC inhibitors showed varied degrees of selectivity versus other
parasites as well as cytotoxicity against L6 cells.
Taken together, these results demonstrated that this class of
HDAC inhibitor could be a new tool against malaria; ST8086AA1
(8) proved to be an effective antimalarial and is therefore a useful
hit compound for further medicinal chemistry optimization.
Acknowledgment
10. Kelly, J. M.; Taylor, M. C.; Horn, D.; Loza, E.; Kalvinsh, I.; Björkling, F. Bioorg.
Med. Chem. Lett. 1886, 2012, 22.
11. Preliminary Tests was performed by Marcel Kaiser—Swiss Tropical and Public
Health Institute, Parasite Chemotherapy. Socinstrasse 57, CH-4002 Basel,
Switzerland.
The authors would like to thank Dr. Gilles Pain for assistance
with the manuscript.
12. World Health Organization. World Malaria Report; Fact Sheet No. 94; World
Health Organization: Geneva, Switzerland, 2013.
13. Oger, F.; Lecorgne, A.; Sala, E.; Nardese, V.; Demay, F.; Chevance, S.; Desravines,
D. C.; Aleksandrova, N.; Le Guével, R.; Lorenzi, S.; Beccari, A. R.; Barath, P.; Hart,
D. J.; Bondon, A.; Carettoni, D.; Simonneaux, G.; Salbert, G. J. Med. Chem. 1937,
2010, 53.
14. In vivo efficacy studies in mice were conducted at the Swiss Tropical & Public
Health Institute (Basel, Switzerland) according to the rules and regulations for
the protection of animal rights. They were approved by the veterinary office of
Canton Basel-Stadt, Switzerland.
Supplementary data
Supplementary data (a table with information on parasites and
on reference drugs used in the screening and two schemes with
synthesis of tested product) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2014.12.051.