Synthesis and anti-protozoal activity of C2-substituted polyazamacrocycles

Article abstract of DOI:10.1016/j.bmcl.2008.02.037

A focused library of C2-substituted-1,4,7,10-tetraazacyclododecanes was synthesised and the compounds were tested for their ability to kill trypanosome and malaria parasites. Several compounds showed significant in vitro activity and were selectively active against the parasites over human embryonic kidney cells used as a counter screen.

Full text of DOI:10.1016/j.bmcl.2008.02.037

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 2455–2458
Synthesis and anti-protozoal activity of C2-substituted
polyazamacrocycles
Caroline M. Reid,a Charles Ebikeme,b Michael P. Barrett,b Eva-Maria Patzewitz,b
Sylke Muller,b David J. Robinsa and Andrew Sutherlanda,*
¨
aWestCHEM, Department of Chemistry, The Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK
bDivision of Infection and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8IA, UK
Received 13 January 2008; revised 13 February 2008; accepted 14 February 2008
Available online 19 February 2008
Abstract—A focused library of C2-substituted-1,4,7,10-tetraazacyclododecanes was synthesised and the compounds were tested for
their ability to kill trypanosome and malaria parasites. Several compounds showed significant in vitro activity and were selectively
active against the parasites over human embryonic kidney cells used as a counter screen.
Ó 2008 Elsevier Ltd. All rights reserved.
Human African trypanosomiasis, also known as sleep-
ing sickness in the late stage of the disease, threatened
to run out of control in the final years of the twentieth
century when a series of epidemics swept across Africa.1
The disease is caused by protozoan parasites, transmit-
ted between people by tsetse fly vectors. Two drugs, sur-
amin and pentamidine are registered to treat the disease
during the haemolymphatic stage and a further two,
melarsoprol and eflornithine are licensed to treat the late
stage disease. However, a number of problems are
associated with the administration and use of these
compounds.2 For example, all are administered by injec-
tion, adverse events are common (in the case of melarso-
prol some 5% of patients taking the drug die from a
reactive encephalopathy) and they are expensive. For
these reasons, there is an urgent need for new drugs to
treat human African trypanosomiasis.
CO2H
CHF2
H2N
H2N
1
H
N
H
N
R
R
R
R
O
O
O
O
N
N
N
N
O
O
O
O
N
H
N
H
2 R= Alkyl or Aryl
NH HN
R
NH HN
A mode of action is known for only one of the currently
registered drugs, eflornithine 1 (difluoromethylornithine,
Fig. 1),3 a suicide inhibitor of the enzyme ornithine
decarboxylase that plays a key role in polyamine biosyn-
thesis.4 Since administration of high levels of extracellu-
lar spermidine protects trypanosomes against the toxic
effect of eflornithine,5 polyamine metabolism is consid-
ered as a validated drug target in these cells.6 We
3
Figure 1.
recently reported the synthesis of carbamate-derived
polyazamacrocycles 2, which are compounds that we be-
lieved may interfere with polyamine biosynthesis in par-
asites.7 While biological testing of these compounds
revealed significant anti-parasitic activity against blood-
stream form African trypanosomes, the structural
requirements for their mode of action were unclear.
Keywords: Macrocycles; Anti-protozoal; Trypanosomes; Malaria.
*
Corresponding author. Tel.: +33 141 330 5936; fax: +33 141 330
4888; e-mail: andrews@chem.gla.ac.uk
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.037

2456
C. M. Reid et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2455–2458
In order to probe further the anti-protozoal activity of
1,4,7,10-tetraazacyclododecanes, we now report the syn-
thesis and biological testing of a focused library of C2-
substituted polyazamacrocycles 3, which are compounds
that lack reactive carbamate side-chains but still retain
significant in vivo activity against bloodstream form
African trypanosomes. The toxicity of these compounds
against malaria parasites from Plasmodium falciparum is
also described.
NH
NH
NH2
NH2
R
O
4
a
b
NH
Our first aim in this study was the synthesis of the parent,
unsubstituted 1,4,7,10-tetraazacyclododecane, cyclen 8.
Many different approaches for the synthesis of cyclen
have been reported due to its importance as an interme-
diate for the preparation of diagnostic and therapeutic
pharmaceutical agents.8–10 These methods include high
dilution conditions,11 Richman–Atkins cyclisations,12
as well as phase transfer conditions.13 All these processes
involve the condensation of terminal halides with bis-sul-
fonamide sodium salts. We recently used the phase trans-
fer approach for the synthesis of our carbamate-derived
polyazamacrocycles.7 However, for the preparation of
cyclen 8 on a large scale with reproducibly high yields,
a three-step approach involving bis-imidazoline 6 proved
the most efficient.14 Reaction of triethylenetetraamine 4
with N,N-dimethylformamide dimethyl acetal 5 under
neat conditions gave bis-imidazoline 6 in quantitative
yield (Scheme 1). Macrocyclisation of 6 with 1,2-dibro-
moethane in the presence of potassium carbonate gave
the imidazolinium compound 7 in quantitative yield.
Hydrolysis of 7 using potassium hydroxide gave cyclen
8 in 71% overall yield in only 3 steps.
O
NH
NH
Cl
Cl
R
Fe
NH
+
H
O
9
10-20
Cl
H
H
N
N
N
N
R
Fe
c
Cl
NH HN
R
NH HN
Having synthesised the parent 1,4,7,10-tetraazacyclod-
odecane, a series of C2-substituted analogues were then
prepared using a metal-templated approach involving
condensation of cis-iron dichloride complex 9 with a ser-
ies of glyoxals 10–20, followed by the reductive removal
of the metal template (Scheme 2).15 Glyoxals 10–20 were
easily prepared in one step by selenium dioxide oxida-
tion of the corresponding methyl ketone.16 This gave
glyoxals 10–20 in generally high yield and the majority
21-32
Scheme 2. Reagents and conditions: (a) FeCl3, MeOH; (b) SeO2, 1,4-
dioxane, H2O, R = tBu (10), 100%; 4-NO2–Ph (11), 80%; 4-MeO–Ph
(12), 100%; 4-Cl–Ph (13), 100%; 4-Br–Ph (14), 100%; 4-CF3–Ph (15),
100%; thiophen-2-yl (16), 100%; naphthalen-2-yl (17), 83%; 3,5-
bis(trifluoromethyl)phenyl (18), 100%; 2-fluorenyl (19), 42%; phenan-
thren-2-yl (20), 99%; (c) i—NaBH4, MeOH, D, ii—HCl then NaOH,
R = tBu (21), 63%; Ph (22), 84%; 4-NO2–Ph (23), 67%; 4-MeO–Ph (24),
83%; 4-Cl–Ph (25), 78%; 4-Br–Ph (26), 71%; 4-CF3–Ph (27), 64%;
thiophen-2-yl (28), 79%; naphthalen-2-yl (29), 77%; 3,5-bis(trifluoro-
methyl)phenyl (30), 54%; 2-fluorenyl (31), 100%; phenanthren-2-yl
(32), 79%.
OMe
OMe
N
N
N
N
NH
NH
NH2
NH2
a
+
N
of these were stored as their hydrates prior to the con-
densation reaction. Attempts were made to prepare
these glyoxals using solvent-free microwave condi-
tions.17 However, conversions were generally poor,
and thus the longer solvent-based approach was pre-
ferred. The glyoxals were then treated with cis-iron
dichloride complex 9 which was prepared using triethyl-
enetetraamine 4 and iron(III) chloride. Reduction of the
resulting diimine with sodium borohydride gave the de-
4
6
5
b
N
N
N
N
NH HN
c
sired
C2-substituted-1,4,7,10-tetraazacyclododecanes
21–32 in good overall yield (Scheme 2).18
NH HN
Br
8
7
The library of C2-substituted polyazamacrocycles was
tested for anti-protozoal activity against bloodstream
form Trypanosoma brucei using a derivative of the Alamar
Scheme 1. Reagents and conditions: (a) neat, D, 100%; (b) 1,2-
dibromoethane, K2CO3, MeCN, D, 100%; (c) KOH, H2O, D, 71%.

C. M. Reid et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2455–2458
2457
blue assay.19 The compounds were also tested for their
ability to kill asexual erythrocytic stages of P. falcipa-
rum. The successful development of useful anti-para-
sitic agents requires the compounds to have low
toxicity to human cells, and thus, the C2-substituted
polyazamacrocycles were also tested against human
embryonic kidney (HEK) cells. The results of the bio-
logical testing are shown in Table 1 (also shown are
two positive standards, pentamidine for T. brucei
and chloroquine for P. falciparum). Several of these
compounds including cyclen 8 and aromatic analogues
22–24 show little or no toxicity towards either try-
panosome or malaria parasites tested. However, a
number of other aromatic derivatives (25, 27 and
29–32) show significant anti-protozoal activity, with
less activity against the mammalian HEK cells. In par-
ticular, the p-Cl-phenyl 25 and naphthalene 29 are the
most selective for the parasites and thus, these com-
pounds provide some scope for the future develop-
ment of more potent and selective analogues. The
subtle effect of the structural activity of these com-
pounds should also be noted. For example, changing
the para-substituent on the phenyl group (e.g., 23 or
24 vs 25) has a dramatic effect on the ability of the
compound to kill trypanosomes. The exact mode of
action of these compounds is still unknown and thus,
we are unable to explain these subtle structural effects.
Future work will focus on the preparation of struc-
tural analogues to probe the mechanism of cytotoxic-
ity, including a targeted analysis of metabolites of the
polyamine pathway in these cells.
In summary, we have designed and synthesised a small
library of C2-substituted-1,4,7,10-tetraazacyclodode-
canes with the aim of interfering with polyamine bio-
synthesis in parasites. On testing,
a number of
compounds showed significant, selective toxicity to
both trypanosome and malaria parasites. This second
generation of 1,4,7,10-tetraazacyclododecanes contains
more stable compounds than in our first generation
(carbamate derived analogues) but the new com-
pounds still retain significant activity against these
parasites. These C-2 substituted derivatives should
have greater bioavailability judging from their esti-
mated logP values (Table 1), and thus they possess
greater potential in leading to a useful drug to treat
parasitic infection. Work is currently underway on
the development of more potent and selective ana-
logues of C2-substituted-1,4,7,10-tetraazacyclodode-
canes including the preparation of derivatives tagged
with moieties that are recognised by the P2 aminopu-
rine and other transporters.20
Table 1. Anti-protozoal activity of cyclic polyamines 8, 21–32
NH HN
R
NH HN
Compound
R
Estimated logP
T. brucei EC50 (lM)
P. falciparum EC50 (lM)
HEK EC50 (lM)
Pentamidine
Chloroquine
—
—
—
—
0.006
—
—
—
—
0.008
>100
>100
26.0
13.0
26.0
2.3
8
H
À1.8
À0.1
À0.2
À0.4
À0.1
0.8
>200
133.0
>200
>200
>200
2.8
5.0
21
22
23
24
25
26
27
tBu
Ph
>200
>200
>200
>200
181.2
>200
73
4-NO2–Ph
4-MeO–Ph
4-Cl–Ph
4-Br–Ph
4-CF3–Ph
0.7
0.8
21.8
6.8
5.8
5.0
S
28
À0.4
72.3
46.7
1.7
>200
29
1.0
6.9
>200
CF3
30
1.7
6.8
4.8
61.0
CF3
31
32
1.8
2.2
2.6
1.3
1.3
26.0
24.9
<1.5

2458
C. M. Reid et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2455–2458
8. Bradshaw, J. S.; Krakoviak, K. E.; Izatt, R. M. Aza-
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pounds, thus preventing access to these compounds
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