Selective delivery of 2-hydroxy APA to Trypanosoma brucei using the melamine motif

Article abstract of DOI:10.1016/j.bmcl.2010.06.070

Trypanosoma brucei, the parasite that causes human African trypanosomiasis, is auxotrophic for purines and has specialist nucleoside transporters to import these metabolites. In particular, the P2 aminopurine transporter can also selectively accumulate melamine derivatives. In this Letter, we report the coupling of the melamine moiety to 2-hydroxy APA, a potent ornithine decarboxylase inhibitor, with the aim of selectively delivering this compound to the parasite. The best compound described here shows an increased in vitro trypanocidal activity compared with the parent.

Full text of DOI:10.1016/j.bmcl.2010.06.070

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4364–4366
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Selective delivery of 2-hydroxy APA to Trypanosoma brucei using
the melamine motif
Nina Klee a, Pui Ee Wong b, Beatriz Baragaña a, Farah El Mazouni c, Margaret A. Phillips c, Michael P. Barrett b,
Ian H. Gilbert a,
*
a Division of Biological Chemistry and Drug Discovery, College of Life Science, University of Dundee, Sir James Black Centre, Dundee DD1 5EH, UK
b Division of Infection and Immunity and Wellcome Trust Centre for Molecular Parasitology, Glasgow Biomedical Research Centre, University of Glasgow G12 8TA, UK
c UT Southwestern Medical Center at Dallas 6001 Forest Park, Dallas, TX 75390-9041, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Trypanosoma brucei, the parasite that causes human African trypanosomiasis, is auxotrophic for purines
and has specialist nucleoside transporters to import these metabolites. In particular, the P2 aminopurine
transporter can also selectively accumulate melamine derivatives. In this Letter, we report the coupling of
the melamine moiety to 2-hydroxy APA, a potent ornithine decarboxylase inhibitor, with the aim of
selectively delivering this compound to the parasite. The best compound described here shows an
increased in vitro trypanocidal activity compared with the parent.
Received 12 May 2010
Revised 10 June 2010
Accepted 11 June 2010
Available online 17 June 2010
Keywords:
Trypanosoma brucei
P2 transporter
Ó 2010 Elsevier Ltd. All rights reserved.
Ornithine decarboxylase
Human African trypanosomiasis (HAT), caused by two subspe-
cies of the protozoa parasite Trypanosoma brucei, is an endemic dis-
ease in sub-Saharan Africa, where 60 million people are at risk.
There is an urgent need for safer and more effective new drugs
as current treatments of the infection show toxicity, poor clinical
efficacy and an increase in drug resistance.1 T. brucei cannot syn-
thesise purines and imports them through different nucleoside
and nucleobase transporters.2,3 In particular, the P2 amino purine
transporter is an unusual adenosine/adenine transporter unique
to T. brucei that has a substrate recognition profile that enables it
to carry molecules containing other motifs including the melamine
and benzamidine moieties (Scheme 1).4–7 Thus, melarsoprol,
diminazene and pentamidine are currently used trypanocides that
are substrates of the P2 and other trypanosome transporters lead-
ing to a selectively high concentration of these trypanocidal drugs
into the parasite.7–10
amidine/
amidine-like
moiety
NH2
N
N
N
aromatic
ring
N
HO
O
electronegative
atom
OH OH
Scheme 1. Requirements for uptake through the P2 transporter.
Ornithine decarboxylase (ODC) catalyzes a key step in the bio-
blocking the proliferation of various tumour cells and para-
sites.14–16 A similar degree of enzyme inhibition can be expected
for T. brucei ODC due to the highly conserved active site between
the mammalian and the trypanosome ODC. In spite of this similar-
ity in structure, the selectivity of ODC inhibitors like DFMO arises
from differences in turnover rate between host and parasite en-
zyme and the limited capacity of T. brucei to scavenge the low lev-
els of polyamines from serum.17 Surprisingly APA has not yet been
tested for its ability to inhibit the trypanosome’s ODC, nor for its
trypanocidal activity.
synthesis of polyamines, the conversion of L-ornithine to putres-
cine.11 Polyamines are required for cell proliferation12 and
polyamine metabolism has been exploited successfully for the
development of antiparasitic drugs. Thus,
a-difluoromethylorni-
thine (DFMO), an irreversible ODC inhibitor,13 is an approved drug
for the treatment of African trypanosomiasis. 3-(Aminooxy)prop-
anamine (APA), an isosteric analogue of putrescine, is another po-
tent inhibitor of ODC and has been reported to be effective in
We have an interest in selectively delivering drugs to the try-
panosome’s interior by exploiting specific transporters in the
* Corresponding author. Tel.: +44 1382 386 240; fax: +44 1382 386 373.
E-mail address: i.h.gilbert@dundee.ac.uk (I.H. Gilbert).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.070

N. Klee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4364–4366
4365
parasites plasma membrane. Several transporter systems, which
recognise melamine and benzamidine motifs, have been exploited
to selectively deliver toxic agents to T. brucei. Previously, we have
reported a series of structural analogues of polyamines that cou-
pled to melamine showed potent activity against T. brucei and good
selectivity on a cellular level.18,19 More recently, we have expanded
the range of trypanocidal moieties delivered to the parasite by cou-
pling a P2 motif to nitrofurans,20–22 DFMO, fluoroquinolones and
artesunate.23 In particular, the use of nitrofurans led to the identi-
fication of some very potent compounds that were curative of the
trypanosome infections in mice.20–22
Here, we have selected 2-hydroxy APA, a close analogue of APA,
for coupling to a P2 recognition unit, melamine. Both enantiomers
of 2-hydroxy APA show similar enzyme-inhibition effects against
mammalian ODC and the 2-hydroxy group is apparently not in-
volved in any specific interaction.14 Therefore, we designed com-
pounds where the melamine motif was attached to the 2-hydroxyl
group of 2-hydroxy APA through an ester. The ester bond should
be liable to hydrolysis by esterases thus releasing 2-hydroxy APA in-
side the parasites.
carried out with 2 M HCl in ether to afford 2-hydroxy APA hydro-
chloride 7 in 80% yield (Scheme 2).
We synthesised four melamine derivatives carrying an acid
group (9a–d) by SNAr reactions. Thus, derivatives 9a, 9b and 9d
were obtained in good yields by reaction of glycine, b-alanine
and c-amino butanoic acid, respectively, with 2-chloro-4,6-amino-
triazine 8 at 80 °C for 2 days. Displacement of the chloro group of 8
by the more hindered D,L-alanine required microwave treatment
at 150 °C for 4 h to obtain the melamine derivative 9c in 95% yield.
Coupling of the melamine derivatives 9a–d with BOC protected
2-hydroxy APA 6 gave rise to the 2-hydroxy APA–melamine
conjugates 10a-d. This coupling step was performed via the acid
chlorides obtained by treatment of acids 9a–d with thionylchloride
in DCM followed by esterification with 6 in the presence of base in
acetonitrile at 50 °C. Unfortunately this step was low yielding and
significant amounts of the starting material 6 were recovered. As
10a–d contained the BOC-protected form of 2-hydroxy APA, their
BOC deprotection was carried out with HCl in ether, to afford the
unprotected 2-hydroxy APA–melamine conjugates 11a–d as
hydrochloride salts (Scheme 3). These were kept as their hydro-
chloride salts, to prevent intramolecular cyclisation by attack of
the amine on the carbonyl of the ester.
Firstly, the APA 3 and 2-hydroxy APA 7 were tested for inhibi-
tion of T. brucei ODC using a previously described assay26 showing
that both are effective inhibitors of the enzyme with IC50 of 22 and
48 nM, respectively. As expected due to the highly conserved ac-
tive site of ODC across species, the activities are in line with previ-
ously reported inhibition of rat liver ODC (35 and 39 nM,
respectively).14
First, we synthesised APA (3-(aminooxy)propanamine)24 to
determine its activity against the T. brucei target enzyme and as
reference point for the in vitro activity of the corresponding mela-
mine–APA conjugates. APA (3-(aminooxy)propanamine) was pre-
pared following a new and shorter synthetic route. Starting from
commercially available tert-butyl N-(3-bromopropyl)carbamate 1
and tert-butyl N-hydroxycarbamate in the presence of sodium
hydride, BOC protected APA 2 was obtained in 42% yield. BOC
deprotection was carried out with HCl in ether to afford APA
hydrochloride 3 in 82% yield (Scheme 2).
Compounds were tested against two T. brucei lines in vitro. The
T. brucei brucei wild type that is proficient in the P2 transporter and
a TbAT1 knockout line lacking the P2 transporter. This TbAT1
2-Hydroxy APA14 was also prepared following a new synthetic
route. Commercially available N-allylcarbamate 4 was oxidised
with mCPBA to give the corresponding epoxide 5.25 Selective open-
ing of this epoxide using tert-butyl N-hydroxycarbamate in the
presence of triethylamine in ethanol resulted in the formation of
BOC protected 2-hydroxy APA 6. As before, BOC deprotection was
NH2
NH2
N
N
R
N
N
a.
OH
H2N
N
N
H
n
O
H2N
N
Cl
O
8
9a, n=1, R=H, 95%
a.
NHBoc
9b
9c
, n=2, R=H, 67%
O
b.
N
H
Br
BocHN
O
, n=1, R=Me, 95%
2
9d, n=3, R=H, 90%
1
NH2
N
NHBoc
NH2
N
R
O
H2N
O
b.
*2HCl
c.
O
H2N
N
N
H
3 (APA)
n
O
NHBoc
10a, n=1, R=H, 3%
10b
10c
, n=2, R=H, 18%
, n=1, R=Me, 5%
O
O
10d, n=3, R=H, 2%
NHBoc
c.
O
e.
d.
O
HN
NH2
5
NH2
4
N
N
R
O
O
*2HCl
H2N
N
N
H
n
NH2
*2HCl
O
NHBoc
H2N
NH2
BocHN
O
11a, n=1, R=H
OH
OH
11b
, n=2, R=H
11c,
n=1, R=Me
6
7 (2-Hydroxy APA)
11d, n=3, R=H
Scheme 2. Reagents and conditions: (a) tBuCO2NHOH, NaH, THF, 0 °C, 42%; (b) 2 M
HCl in Et2O, 82%; (c) mCPBA, DCM, 99%; (d) tBuCO2NHOH, Et3N, EtOH, 80 °C, 39%;
(e) 2 M HCl in Et2O, 80%.
Scheme 3. Reagents and conditions: (a) amino acid, NaHCO3, EtOH/water; 80 °C;
(b) (i) SOCl2, DCM, (ii) 6, DMAP, Et3N, CH3CN, 50 °C; (c) 2 M HCl in Et2O, 99%.

4366
N. Klee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4364–4366
knockout line has been described previously.27 The results are
summarised in the Table 1.
APA and 2-hydroxy APA displayed poor activity against the wild
type strain of T. brucei (EC50 >100 lM). This is probably due to the
ity of up to three-fold. These data suggest that these compounds
may have enhanced uptake potential compared to the parent.
The data also indicates that uptake of 11c and 11d may have some
clear dependency on the P2 transporter since activity is reduced in
the P2 knockout lines. Some compounds are highly dependent on
uptake through the P2 transporter, for example berenil. However,
it has been shown that there are other transporters, for example
HAPT1 and LAPT1 that can also take up and concentrate com-
pounds with a melamine or benzamidine motif.8 In spite of the fact
that we have shown that loss of P2 can be readily achieved without
apparent detriment to parasite physiology, it does not necessarily
lead to resistance to melamine and benzamidine containing com-
pounds. Hence, whilst it is important to consider the risk of resis-
tance developing to drugs carrying such a motif, it is possible to
test for this.
poor uptake of these doubly charged diamines. We therefore inves-
tigated whether coupling to a melamine motif might increase
activity through selective delivery via the P2 transporter. Various
analogues with a melamine motif were investigated including dif-
ferent chain lengths separating the P2 motif (melamine) and APA.
In addition, one example (11c) with a methyl group in the linker
which may slow the rate of hydrolysis was included. The analogues
(with the exceptions of 11a and 11b) showed a moderate increase
in potency against T. brucei. This could be suggestive of uptake
through the P2 or related transporters followed by hydrolysis in
the trypanosome by esterases to release 2-hydroxy APA. In addi-
tion, compounds were evaluated against the P2 deficient strain
TbAT1 KO. Compounds 11c and 11d showed a reduction in activity
against this cell line, further suggesting that the P2 transporter
played some role in their uptake. However these compounds all
show relatively weak activity.
Our previous experience with the P2 transporter has indicated
that where a compound has inherent activity against the trypano-
some, attaching a P2 motif can significantly increase the activity
(e.g., the nitroheterocycles),20–22 whilst in cases where the
compound does not have inherent potency against the parasite,
attaching the P2 motif does not have a large effect (e.g., fluoroquin-
olones).23 APA derivatives might be expected to have activity due to
their potent inhibition of ODC, although a key difference between
APA and DFMO relates to the fact that APA is a competitive reversible
inhibitor of the enzyme while DFMO causes irreversible inactivation
after covalently alkylating the enzyme’s active site. In this case,
accumulating levels of ornithine would reach an equilibrium where
APA or derivatives would be displaced from the active site and
putrescine production would continue, albeit at a changed equilib-
rium. Other possible explanations for the small increase in trypano-
cidal activity upon addition of a melamine uptake motif include a
failure of esterases to bioconvert the melamine derivative, degrada-
tion of the compound, for example through intramolecular attack of
one of the amines onto the ester, or inability to use the P2 trans-
porter—possibly because of the high positive charge of these com-
pounds at physiological pH.
Acknowledgements
We would like to acknowledge the following for funding: Stud-
ienstiftung des deutschen Volkes for a studentship (N.K.); the
Wellcome Trust (WT 083481) (Dundee); the National Institutes
of HealthGrant (R01 AI34432) (M.A.P.); and the Welch Foundation
Grant I-1257 (M.A.P.). M.A.P. holds the Carolyn R. Bacon Professor-
ship in Medical Science and Education.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.070.
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ODC is a proven drug target to treat African trypanosomiasis
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to a known P2 recognition motif, melamine, through four different
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IC50 (nM)
T. brucei brucei EC50
(
l
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Resistance factora
Wild type
TbAT1 KO
3
7
22
48
7
9
>100
NA
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11a
10b
11b
11c
11d
>100
77
>100
29
NA
40
NA
NA
288
0.5
>3
2.5
113
a
EC50 KO/EC50 w.t. NA—no activity at 100
l
M.