Inhibitors of human histone deacetylase with potent activity against the African trypanosome Trypanosoma brucei

Article abstract of DOI:10.1016/j.bmcl.2012.01.072

A number of hydroxamic acid derivatives which inhibit human histone deacetylases were investigated for efficacy against cultured bloodstream form Trypanosoma brucei. Three out of the four classes tested displayed significant activity. The majority of compounds blocked parasite growth in the submicromolar range. The most potent was a member of the sulphonepiperazine series with an IC₅₀ of 34 nM. These results identify lead compounds with potential for the development of a novel class of trypanocidal agent.

Full text of DOI:10.1016/j.bmcl.2012.01.072

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1886–1890
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
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Inhibitors of human histone deacetylase with potent activity against
the African trypanosome Trypanosoma brucei
John M. Kelly a, Martin C. Taylor a, David Horn a, Einars Loza b, Ivars Kalvinsh b, Fredrik Björkling c,d,
⇑
a Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
b Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia
c TopoTarget A/S, Symbion, Fruebjergvej 3, DK-2100 Copenhagen, Denmark
d Department of Molecular Drug Research, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of hydroxamic acid derivatives which inhibit human histone deacetylases were investigated
for efficacy against cultured bloodstream form Trypanosoma brucei. Three out of the four classes tested
displayed significant activity. The majority of compounds blocked parasite growth in the submicromolar
range. The most potent was a member of the sulphonepiperazine series with an IC50 of 34 nM. These
results identify lead compounds with potential for the development of a novel class of trypanocidal agent.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 1 December 2011
Revised 17 January 2012
Accepted 20 January 2012
Available online 28 January 2012
Keywords:
Anti-parasitic activity
Trypanosoma brucei
Histone deacetylase inhibitors
Hydroxamic acids
Tsetse fly transmitted protozoa of the Trypanosoma brucei spe-
cies complex are the causative agents of human African trypanoso-
miasis (HAT). There are currently around 30,000 HAT cases
annually, although during epidemics, such as in the late 1990s, this
level can increase by more than 10-fold.1–3 In domesticated
animals these parasites also cause nagana, a disease which has a
major impact on agricultural output throughout sub-Saharan
Africa. T. brucei is an extracellular parasite that avoids immune
destruction by a complex process of antigenic variation. This is
mediated by periodic switching of the single variant surface glyco-
protein (VSG) that covers the parasite cell surface to another anti-
genically distinct type, encoded by the large repertoire of VSG
genes.4 As a consequence, vaccines are not a realistic option. Drug
development is therefore of major importance, and is a WHO pri-
ority. Existing therapies for HAT are unsatisfactory for reasons that
include severe toxic side effects, increasing resistance, the need for
hospitalisation during administration and cost.5,6 In the absence of
treatment, late stage disease is almost invariably fatal.5
acetylation/deacetylation reactions, carried out by histone
acetyltransferases and histone deacetylases (HDAC), respectively,
are widespread within eukaryotes and act as regulators of numer-
ous cellular events.7,8 Aberrant HDAC activity has been associated
with a number of different diseases and enzyme inhibitors have
broad therapeutic potential.9,10 This is particularly the case with
cancer, where anti-proliferative effects can result from induction
of cell cycle arrest and apoptosis.11 In T. brucei, there are four puta-
tive non-sirtuin HDAC isoforms, of which two (DAC1 and DAC3)
are essential in bloodstream form parasites12 and play distinct
roles in the telomeric silencing of non-expressed VSG genes.13 Evi-
dence also suggests a role for histone acetylation in epigenetic reg-
ulation of RNA polymerase II polycistronic transcription.14 Previous
studies have demonstrated that inhibitors of HDAC can have signif-
icant anti-malarial and anti-leishmanial properties,15–17 in addi-
tion to activity against a range of other parasites. For instance,
the HDAC inhibitor Trichostatin A suppresses growth of blood-
stream form T. brucei,18 and is an inhibitor of DAC1 and DAC3 activ-
ity.13 Here, we report the efficient growth inhibition of cultured
bloodstream form T. brucei using a series of inhibitors of human
HDACs.
Histone modifications play a central role in transcription, chro-
matin assembly, replication, DNA repair and other regulatory pro-
cesses central to chromosome biology. For example, coupled
A representative set of HDAC inhibitors was selected by screen-
ing our large compound library. These compounds were originally
prepared as part of a programme to identify HDAC inhibitors with
anti-cancer properties. This resulted in the discovery of the drug
candidate BelinostatÒ, currently in phase III clinical trials. The se-
lected compounds were all hydroxamic acid derivatives, a common
Abbreviations: HAT, human African trypanosomiasis; VSG, variant surface
glycoprotein; HDAC, histone deacetylases; DAC, deacetylases; SAR, Structure–
activity relationship.
⇑
Corresponding author. Tel.: +45 35 33 62 31.
E-mail address: fb@farma.ku.dk (F. Björkling).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.072

J. M. Kelly et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1886–1890
1887
1) 1N NaOH
NH2
or
R1
N
2) (COCl)2
DMF
Base
Compound
1-8, 10-15,
17-19
O
O
O
R1
S
O
+
S
O
NH
3)NH2OH
NaHCO3
N
Cl
O
O
H
O
R1
Scheme 1. Preparation of hydroxamic acids with a sulphoneamide or sulphonepiperazine linker.
Table 1
Prepared HDAC inhibitors and biological activity23,24,29,30
O
H
N
H
N
R2
O
S
R3
OH
OH
OH
N
H
R1
O
O
O
21-26
9 16, 20
Comp.
Comp,
R3
,
Comp. 1-8, 10-15, 17-19
Compound
R1
R2
Trypanosoma brucei (BSF)
IC50 SD ( M) IC90 SD (
HDAC (FDL_HELA)
IC50 SD ( M)
l
l
M)
l
N
1
0.034 0.002
0.064 0.005
0.086 0.009
0.066 0.004
0.130 0.005
0.201 0.005
0.084 0.053
0.149 0.006
0.123 0.076
N
N
N
Cl
2
3
N
N
N
N
N
4
5
6
0.123 0.006
0.137 0.007
0.141 0.009
0.169 0.008
0.22 0.007
0.224 0.014
0.187 0.082
Cl
N
N
H
N
nd
F3C
O
0.191 0.036
N
N
F
N
N
7
0.154 0.013
0.281 0.019
0.068 0.036
Cl
H
N
8
9
0.154 0.013
0.155 0.018
0.306 0.061
0.310 0.040
0.01 0.002
F2HC
O
N
0.028 0.004
O
O
10
11
0.156 0.003
0.167 0.012
0.224 0.003
0.228 0.002
0.212 0.112
0.132 0.103
N
N
(continued on next page)

1888
J. M. Kelly et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1886–1890
Table 1 (continued)
Compound
R1
R2
R3
Trypanosoma brucei (BSF)
IC50 SD ( M) IC90 SD (
HDAC (FDL_HELA)
IC50 SD ( M)
l
l
M)
l
N
N
N
N
12
0.198 0.005
0.265 0.005
0.022 0.002
O
H
N
O
13
14
15
16
0.198 0.021
0.261 0.038
0.343 0.017
0.353 0.016
0.333 0.028
0.485 0.043
0.710 0.017
0.648 0.020
0.022 0.002
0.269 0.087
0.02 0.004
0.022
O
N
N
O
H
N
F
N
H
N
17
18
0.368 0.023
0.606 0.081
0.651 0.083
0.956 0.035
0.042 0.011
0.013 0.005
Cl
H
N
O
H
N
O
O
19
20
21
0.927 0.057
1.54 0.06
>10
2.15 0.14
2.18 0.18
0.023 0.005
0.048 0.028
0.029 0.016
N
O
O
O
O
N
H
O
22
23
>10
>10
0.02 0.002
N
H
O
0.026 0.036
N
H
O
24
25
>10
>10
nd
N
0.018 0.006
structural feature of HDAC inhibitors due to the high affinity of this
group for the Zn(II) ion in the metalloenzyme. Four hydroxamic
acid compound subclasses, belonging to separate patent series,19–
the sulphoneamides, sulphonepiperazines, long chain amides
and a heterocyclic series, were chosen for screening against
trypanosomes.
cule, were prepared according to previously described proce-
dures.19,20 Briefly, they were synthesized from the corresponding
sulphonyl chloride and amide or piperazine, followed by the trans-
formation of the methyl ester to the desired hydroxamic acid
(Scheme 1, Table 1). Hydroxamic acid derivatives with a long chain
amide linker, compounds 21–26, were synthesized by amide cou-
pling between the methyl 6-aminohexanoate and the respective
acid, followed by transformation of the ester to the corresponding
22
Compounds 1–8, 10–15 and 17–19, which are hydroxamic acids
with a sulphoneamide or sulphonepiperazine linker in the mole-

J. M. Kelly et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1886–1890
1889
O
1)
N
N
N
HCl
H2N
O
NH2OH
MeONa
O
O
N
H
N
Compound
21-26
R3
+
O
R3
O
OH
2) Et 3N
O
Scheme 2. Preparation of hydroxamic acids with a long chain amide linker.
Table 2
Inhibition of rhHDAC isoformsa
Compound/rhHDAC
ED50(nM SD)
1
2
3
4
6
7
8
9
3
10
12
15
18
20
25
nd
76
53
44 0.1
19
55
36 0.7
6
2.8 0.4
295 290
96 46
nd
870
nd
65 20
33
179 115
107 13
305 19
35 16
100 97
nd
nd
216 133
42 22
581 334
86 61
1179
145 151
109 56
6
3
955 166
328 193
211 42
167 72
573 30
310 28
221 141
105 64
554 44
7.2 1.0
89 32
1
6
23
2
1
36
68 13
14
3
26
3
134
7.3 1.7
97 0.6
9.2 1.9
373 73
6.6 0.7
286
8
278 65
7.4 4.3
9
8.4 2.0
3033 226
nd = not determined.
a
Each compound was assayed in triplicate per plate. EC50 values were determined from the average of a minimum of two plates. Results are means SD.
hydroxamic acid (Scheme 2, Table 1).21,23 Finally, the heterocyclic
hydroxamic acids 9, 16, 20, were similarly prepared using pub-
lished procedures.22
exhibit similar levels of potent activity (IC50 0.034–0.20 lM, Table
1). Treatment of parasites with micromolar levels of these inhib-
itors could result in cell death within 4 h (Fig. 1). In this sul-
phonepiperazine series, the close SAR suggests that further
modification of the scaffold could enhance the trypanocidal prop-
erties. The sulphoneamide analogues 5, 8, 15, 17–19 also dis-
played significant potency against bloodstream form parasites
(IC50 0.14–0.93 lM, Table 1), which showed a clear SAR. Interest-
ingly, although analogues from both classes inhibited HeLa cell
HDACs at a range of submicromolar concentrations (IC50 0.010–
0.19 lM), there was no obvious correlation with the pattern of
The compounds from the different structural subclasses were
all active as HDAC inhibitors, measured in an assay of enzymes
in HeLa cell lysates.23,24 HeLa cells express multiple HDACs, includ-
ing HDAC 1, 2, 3, 4, and 8.25–27 The compounds showed broad
selectivity, with submicromolar activities (Table 1). To further
characterise the compounds with respect to HDAC selectivity, we
determined their inhibitory properties against recombinant ex-
pressed human HDACs (rhHDAC 1–4, 6–9).28 These assays showed
that the compounds had a broad and potent inhibitory activity to-
wards these enzymes which belong to the different HDAC sub-
classes (class I, HDAC 1, 2, 3, 8; class IIA, HDAC 4, 7, 9; class IIB,
HDAC 6) (Table 2).
When we investigated the potency of the inhibitors against
cultured bloodstream form Trypanosoma brucei (strain 427),29,30
we found that three out of the four subclasses had significant
activity, with growth inhibition occurring in the submicromolar
range (Table 1, Fig. 1). The most active compounds were the sul-
phonepiperazines, particularly those with an aromatic substitu-
tion attached to the piperazine moiety. Compounds 1–4, 6, 7,
and 10–12 are examples of the closely related analogues which
activity observed against trypanosomes.
The heterocyclic compounds 9 and 16, which are close quino-
line analogues, displayed similar trypanocidal properties (Table
1). However compound 20, a benzoxazole, thus structurally dis-
tinct from the other two heterocycles was 5- to 10-fold less active.
The amide series, which contains a C5 carbon linker (compounds
21–26), did not show activity against T. brucei (up to 10 lM, Table
1), despite being very potent inhibitors of HeLa cell HDACs. Com-
parison of the trypanosome inhibitory properties of compounds
1–20, which have an aromatic group attached close to the hydroxa-
mic acid, with those of the inactive amide series (compounds 21–
26), suggests that structural differences between human and T.
Figure 1. Bloodstream form T. brucei bloodstream treated with compound 1 (2
lg/ml). At the time points indicated, parasites were fixed methanol, stained with Giemsa and
visualised using a Leica DMRB microscope.

1890
J. M. Kelly et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1886–1890
5. Kennedy, P. G. Ann. Neurol. 2008, 64, 116.
brucei target enzymes may influence the efficiency of inhibitor
binding. Importantly, the selectivity implies that the trypanocidal
activity results from specific interaction(s), rather than from gen-
eral metal ion chelating properties. Therefore, the disparity in
activity profiles of these compounds against human and parasite
cells could be due, at least in part, to their differential abilities to
inhibit the corresponding HDACs, or other metalloproteins. Inter-
estingly, the two essential T. brucei HDACs (DAC 1 and 3) are diver-
gent, relative to their mammalian counterparts, as are their histone
substrates. The major differences lie in the amino and carboxyl
extensions beyond the more conserved catalytic core. There is also
a large (174 amino acid) insert within the core of the class II en-
zyme, DAC3.12 The structures of these enzymes have yet to be
determined. However, HDAC inhibitors typically function through
chelation of the active site zinc ion, and the DAC3 insert, as well
as the other differences, may have an impact on access to the active
site pocket for both substrates and inhibitors. It is notable here that
highly specific selective inhibitors have been identified for human
HDACs31,32 and the differences between the human and T. brucei
enzymes might also be exploited for the development of trypano-
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24. Histone deacetylase activity was determined in lysed Human cervix epithelial
cancer HeLa cells. To determine the IC50 of test compound a commercially
available fluorescent assay kit was used employing an acetylated enzyme
substrate.23
In conclusion, by screening a library of compounds produced for
anti-cancer studies, we have identified leads with potential for
treating African trypanosomiasis. These data provide a platform
for the definitive identification of the target enzyme(s) within
the parasite and the informed design of more potent compounds
with optimised pharmacokinetic properties.
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Acknowledgements
28. Khan, N.; Jeffers, M.; Kumar, S.; Hackett, C.; Boldog, F.; Khramtsov, N.; Qian, X.;
Mills, E.; Bergs, S. C.; Carey, N.; Finn, P. W.; Collins, L. S.; Tumber, A.; Ritchie, J.
W.; Jensen, P. B.; Lichtenstein, H. S.; Sehested, M. Biochem. J. 2008, 409, 581–
589.
This work was supported by TopoTarget A/S and by a Wellcome
Trust Grant (No. 084175) to J.M.K.
29. Duque, M. D.; Camps, P.; Profire, L.; Montaner, S.; Vázquez, S.; Sureda, F. X.;
Mallol, J.; López-Querol, M.; Naesens, L.; De Clercq, E.; Prathalingam, S. R.;
Kelly, J. M. Bioorg. Med. Chem. 2009, 17, 3198.
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30. Growth inhibitory properties against cultured bloodstream form T. brucei were
determined as described previously.29 Experiments were performed in
triplicate and data are presented as SD.
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