Trypanocidal Activity of Melamine-Based Nitroheterocycles

Article abstract of DOI:10.1128/AAC.48.5.1733-1738.2004

A series of nitroheterocyclic compounds were designed with linkages to melamine or benzamidine groups that are known substrates of the P2 aminopurine and other transporters in African trypanosomes of the brucei group. Several compounds showed in vitro trypanotoxicity with 50% inhibitory concentrations in the submicromolar range. Although most compounds interacted with the P2 transporter, as judged by their ability to inhibit adenosine transport via this carrier, uptake through this route was not necessary for activity since TbAT1-null mutant parasites, deficient in this transporter, retained sensitivity to these drugs. One compound, a melamine-linked nitrofuran, also showed pronounced activity against parasites in mice. Studies into the mode of action of this compound indicated that neither reductive, nor oxidative, stress were related to its trypanocidal activity ruling out a genotoxic effect in T. brucei, distinguishing it from some other, mammalian cell toxic, trypanocidal nitroheterocycles.

Full text of DOI:10.1128/AAC.48.5.1733-1738.2004

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2004, p. 1733–1738
0066-4804/04/$08.00ϩ0 DOI: 10.1128/AAC.48.5.1733–1738.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 5
Trypanocidal Activity of Melamine-Based Nitroheterocycles
Mhairi L. Stewart,1 Gorka Jimenez Bueno,2 Alessandro Baliani,2 Burkhard Klenke,2
Reto Brun,3 Janice M. Brock,1 Ian H. Gilbert,2 and Michael P. Barrett1*
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow,
Glasgow G12 8QQ,1 and Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF,2
United Kingdom, and Swiss Tropical Institute, CH-4002 Basel, Switzerland3
Received 24 September 2003/Returned for modification 31 October 2003/Accepted 21 January 2004
A series of nitroheterocyclic compounds were designed with linkages to melamine or benzamidine groups
that are known substrates of the P2 aminopurine and other transporters in African trypanosomes of the brucei
group. Several compounds showed in vitro trypanotoxicity with 50% inhibitory concentrations in the submi-
cromolar range. Although most compounds interacted with the P2 transporter, as judged by their ability to
inhibit adenosine transport via this carrier, uptake through this route was not necessary for activity since
TbAT1-null mutant parasites, deficient in this transporter, retained sensitivity to these drugs. One compound,
a melamine-linked nitrofuran, also showed pronounced activity against parasites in mice. Studies into the
mode of action of this compound indicated that neither reductive, nor oxidative, stress were related to its
trypanocidal activity ruling out a genotoxic effect in T. brucei, distinguishing it from some other, mammalian
cell toxic, trypanocidal nitroheterocycles.
There is an urgent need for the development of new drugs to
treat human African trypanosomiasis, owing to poor clinical
efficacy and toxic side effects of current drugs and a growing
problem of resistance at a time when the disease has become
resurgent (25).
are of particular interest. Nitroheterocycles have long been
known to be effective against trypanosomes. Throughout the
1950s and 1960s a variety of compounds, notably furacin (ni-
trofurazone), were used in clinical trials (1, 19). In spite of
good antitrypanosomal activity, toxicity issues prevented fur-
ther development of nitrofurazone. A nitrofuran, nifurtimox,
however, was licensed for use against Chagas’ disease caused
by Trypanosoma cruzi (36). Nifurtimox is also active against T.
brucei and has been used in treating melarsoprol refractory
trypanosomiasis (25, 33). The World Health Organization and
Bayer are currently engaged in efforts to extend the license for
nifurtimox for routine use against human African trypanoso-
miasis (7). A nitroimidazole, megazol, has recently received
attention for its potent trypanocidal activity (9, 17), although
toxicity issues (34) have stifled further development. Reports
of novel trypanocidal nitroheterocycles continue to appear (8,
30), which emphasizes the fact that trypanosomes are particu-
larly sensitive to this class of compound.
Given the potent trypanocidal activity of various nitrohet-
erocycles, but the disadvantageous impact of host toxicity, se-
lectively targeting nitroheterocycles to the trypanosome’s inte-
rior could greatly increase their therapeutic index. We have
therefore set out to produce a number of nitroheterocycles
carrying either a melamine ring or a benzamidine moiety aim-
ing to facilitate selective uptake into trypanosomes via the P2
and other transporters and circumvent host toxicity. Trypano-
cidal nitroheterocycles appear to be optimally active if at-
tached to a second moiety, possibly because of steric require-
ments in binding to particular enzymes with nitroreductase
activity. Consequently, both delivery across the plasma mem-
brane and targeting to the active sites of particular enzymes
could be bestowed by coupling nitroheterocycles to melamine
or benzamidine moieties.
In order to exert trypanocidal activity, drugs must first enter
the parasites or else act on essential plasma membrane-asso-
ciated targets. An unusual aminopurine transporter, termed
the P2 transporter, is one of several that can carry purine
nucleosides and bases into these cells (27, 37). The P2 trans-
porter recognizes adenine and adenosine as substrates, but it
can also transport melamine (triazine) derivatives and benza-
midine derivatives that include several known drugs used
against the African trypanosomiases (Fig. 1) (4, 11, 14).
Other transporters can also carry drugs into trypanosomes
and contribute to their selectivity (15). A better understanding
of these routes of uptake and the design of agents that can be
delivered to trypanosomes via these portals offers one route to
urgently needed trypanocidal drugs. We have previously re-
ported approaches to selectively deliver compounds to try-
panosomes using the P2 transporter (6, 23, 38). The principal
of this approach was to attach P2 recognition motifs (in par-
ticular the melamine unit) to cytotoxic agents. The cytotoxic
agents first selected were polyamine analogues, which are
known to be cytotoxic to trypanosomes (26, 32). Several series
of compounds were prepared and some of the compounds
were shown to have high activity against Trypanosoma brucei
trypomastigotes (23). However, they were too toxic in mam-
mals for use in vivo.
Alternate cytotoxic moieties to couple to the P2 recognition
motifs have now been considered. Nitroaromatic compounds
* Corresponding author. Mailing address: Institute of Biomedical
and Life Sciences, Division of Infection and Immunity, University of
Glasgow, The Joseph Black Building, Glasgow G12 8QQ, United
Kingdom. Phone: (44) 141-330-6904. Fax: (44) 141-330-6904. E-mail:
m.barrett@bio.gla.ac.uk.
MATERIALS AND METHODS
Chemistry. The compounds were prepared from the 2,4-diamino-6-chlorotria-
zines by displacement of the chlorine by hydrazine, followed by condensation
1733

1734
STEWART ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Uptake of triazines and benzamidines into T. brucei. The left-hand side of the diagram shows the known routes of uptake of diamidines
and melaminophenyl arsenicals into T. brucei. P2 is the product of the TbAT1 gene and encodes an unusual transporter capable of nucleoside and
nucleobase uptake. It recognizes a motif also present on diamidine- and melamine-based drugs. These are shown on the right hand side of the
diagram. Pentamidine enters via two additional transporters: HAPT1 and LAPT1. The uptake routes of selected triazine- and benzamidine-derived
drugs is depicted.
with the appropriate nitrofuraldehyde (Fig. 2). Synthesis was handicapped by the
insolubility of the reagents and products. For control purposes, nitrofurans 4 and
5 were also screened.
bleomycin (2 ␮g/ml), which select for the respective genes giving resistance to
these antibiotics targeted to the RAD51 loci of the T. brucei genome.
N-Acetylcysteine (NAC) has been used frequently as an antagonist of oxida-
tive stress in different cellular systems since it reacts readily with a number of the
reduced oxygen species produced during oxidative stress (2, 12). When admin-
istered at 0.5 mM, no detrimental effect on trypanosomes was induced by this
compound in in vitro assays.
For uptake analysis bloodstream-form parasites were grown to peak para-
sitemia (109 cells per ml of blood) in Wistar rats and then purified from blood by
using DEAE anion-exchange chromatography (24).
(ii) P2 transporter affinity measurements. Parasites purified from blood were
stored on ice in Carter’s buffered saline solution (11). Transport assays used the
centrifugation through oil technique, which is routinely used in analyses (5, 11,
14, 15, 23, 38). Radiolabeled adenosine (0.05 ␮M) uptake via P2 was measured
in the presence of 1 mM inosine that blocks the P1 transporter (11). Compounds
The synthesis of benzamidine derivatives of the imidazoles are shown in Fig.
3. The starting point was 4-aminobenzamide (compound 6). The amidine func-
tionality was protected with a BOC protecting group, which allowed derivatiza-
tion of the aniline position with chloroacetyl chloride. The intermediate com-
pound 8 was then coupled with either 2-nitroimidazole or 4-nitroimidazole,
which followed by deprotection gave the required products 11 and 12.
Biological assays. (i) Cultivation of parasites. Bloodstream-form T. brucei
brucei (strain 427) (13) was cultivated in HMI-9 medium containing 20% fetal
calf serum (22) at 37°C in a humidified CO2 environment. The bloodstream-form
RAD51Ϫ/Ϫ deletion mutant (29) derived from strain 427 was a gift from R.
McCulloch (University of Glasgow), and these cells were cultured in HMI-9
medium with 20% fetal calf serum supplemented with puromycin (1 mg/ml) and
FIG. 2. Preparation of compounds from 2,4-diamino-6-chlorotriazines by displacement of the chlorine by hydrazine, followed by condensation
with the appropriate nitrofuraldehyde.

VOL. 48, 2004
TRYPANOCIDAL ACTIVITY OF NITROHETEROCYCLES
1735
FIG. 3. Synthesis of benzamidine derivatives of the imidazoles. Lettered arrows: a, (BOC)2O, NaOH, THF/H2O; b, chloroacetyl chloride, Et3N,
MeCN; c, 2-nitroimidazole, Et3N, MeCN; d, 4-nitroimidazole, Et3N, MeCN; e, TFA, anisole, DCM.
were assayed for affinity for the P2 transporter by using three separate concen-
trations of adenosine and a range of inhibitor concentrations. The data were
plotted by using the competitive inhibition algorithm of the Grafit 4.0 software
(Erithacus). Plots were viewed by eye to verify inhibition type (Table 1).
(iii) In vitro activities against parasites and cytotoxicity. Activity of com-
pounds was determined for T. brucei rhodesiense trypomastigotes of STIB 900.
This stock was isolated in 1982 from a human patient in Tanzania. Minimum
essential medium (50 ␮l) supplemented with 2-mercaptoethanol and 15% heat-
inactivated horse serum (3) was added to each well of a 96-well microtiter plate.
Serial drug dilutions were prepared covering a range from 90 to 0.123 ␮g/ml.
Then, 50 ␮l of a trypanosome suspension was added to each well, and the plate
incubated at 37°C under a 5% CO2 atmosphere for 72 h. Alamar Blue (10 ␮l) was
then added to each well, and incubation continued for a further 2 to 4 h. The
plate was then read in a Spectramax Gemini XS microplate fluorometer (Mo-
lecular Devices Corp., Sunnyvale, Calif.) by using an excitation wavelength of 536
nm and an emission wavelength of 588 nm (35). Fluorescence development was
expressed as a percentage of the control, and the 50% inhibitory concentration
(IC50) values were determined. Cytotoxicity was assessed by using the same assay
and rat skeletal myoblasts (L-6 cells).
To investigate whether transport of these compounds through the P2 trans-
TABLE 1. Activities of compounds against the P2 transporter and against various strains of T. brucei trypomastigotesa
T. brucei brucei AT1
wild type (IC50
[␮M])
T. brucei brucei AT1
knockout (IC50
[␮M])
L-6 cells
(IC50
[␮M])
Ki, P2
uptake (␮M)
T. brucei rhodesiense
Compound
(IC50 [␮M])
2a
11.9
59.3
22.9
1.9
Ͼ200
Ͼ200
0.23
0.85
16.5
89
Ͼ200
Ͼ200
0.38
1.52
29.3
170
ND
ND
ND
ND
2b
3a
0.025
0.24
12.9
10.2
0.25
0.003
0.68
2.3
183
3b
11.8
Ͼ400
78.2
18.8
18.7
40.4
20.0
ND
3c
15.9
4.9
3d
3e
4.6
11.9
0.2
1.1
14.8
0.3
1.2
3f
129
b
4
–
5
404
23.5
111
Ͼ200
Ͼ200
Ͼ200
0.053
5.6
13.7
119
Ͼ200
191
Ͼ200
0.12
ND
9
13.09
3.65
1.58
2.88
1.2
ND
10
ND
ND
11
6.2
Ͼ300
Ͼ300
7.8
12
79.8
0.006
1.5
Melarsoprol
Nifurtimox
ND
68
a T. brucei AT1 Knockout is a mutant with a nonfunctional P2 transporter. ND, not determined.
b –, No inhibition.

1736
STEWART ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Activities of compounds against RAD51Ϫ/Ϫ mutant T.
brucei in the presence or absence of NACa
porter is necessary for activity, compounds were assayed against the T. brucei
brucei trypomastigotes using either the wild type or P2 knockout mutants
(TbAT1Ϫ/Ϫ) (28). The Alamar Blue assay (35) was also used to determine IC50
values against wild-type lines and the TbAT1Ϫ/Ϫ derivative. To determine
whether DNA damage was associated with trypanocidal activity the Alamar Blue
assay was also used to determine IC50 values against the RAD51Ϫ/Ϫ deletion
mutant.
(iv) In vivo activities. Female NMRI mice weighing 22 to 25 g were infected
with cryopreserved stabilates of T. brucei brucei STIB 795 (derivative of strain
427 (13) or T. brucei rhodesiense STIB 900. Each mouse was infected intraperi-
toneally with 2 to 4 ϫ 104 bloodstream forms. Melarsoprol (Arsobal; Aventis)
acted as a standard drug and was diluted with sterile distilled water to an
appropriate concentration. Groups of four mice were treated on days 3, 4, 5, and
6 intraperitoneally with 20 mg/kg. A control group remained untreated. The
parasitemia of all animals was checked on day 7 and 10 postinfection and every
second day thereafter until day 60. Death of animals was recorded to calculate
the mean survival time. Surviving and aparasitemic mice were considered cured
at 60 days and then euthanized.
Mean IC50 (␮M) Ϯ SD (n ϭ 3)
Compound(s)
Wild type
RAD51Ϫ/Ϫ mutant
Megazol
0.18 Ϯ 0.012
0.20 Ϯ 0.018
5.6 Ϯ 0.48
14.35 Ϯ 2.1
0.32 Ϯ 0.03
0.34 Ϯ 0.02
0.034 Ϯ 0.004
0.032 Ϯ 0.001
4.8 Ϯ 0.67
Megazol ϩ NAC
Nifurtimox
Nifurtimox ϩ NAC
3a
12.3 Ϯ 2.2
0.36 Ϯ 0.06
0.34 Ϯ 0.04
3a ϩ NAC
a The effect of 0.5 mM NAC was assessed by adding this to wild-type cells only
prior to the addition of drug.
In vivo activity in mice. Selected compounds were evaluated
in several rodent models of trypanosome infection. Com-
pounds 3a, 3e, and 3f were examined in rodent models infected
with T. brucei brucei STIB 795 and T. brucei rhodesiense STIB
900. Neither compound 3e nor compound 3f was effective in
vivo at 20 mg/kg. Compound 3a, however, cured the STIB 795
T. brucei brucei model mice (four of four mice all cured, as
defined by no infection at 60 days) at a dose of 20 mg/kg given
for 4 days (days 2 to 5). No overt signs of toxicity were observed
in these mice. Having successfully treated an acute stage
model, the more stringent T. brucei rhodesiense STIB 900
model was then tested in rodents. This model is not cured by
pentamidine, suramin, or any other drugs active against early
stage disease, although it does respond to melarsoprol, which
is used in late-stage disease. It is thought that parasites may
leave the vasculature early in a STIB 900 infection so that
drugs must penetrate extravascular compartments to effect
radical cure. As such, STIB 900 infection is perceived to rep-
resent a good model for determining likely outcomes of drugs
in late-stage models, while providing the advantage of enabling
experiments to be conducted within 2, rather than 6, months.
Given intraperitoneally at 20 mg/kg for 4 days, compound 3a
cured only one of the four mice as defined by their remaining
infection free at 60 days. The compound did, however, pro-
mote a significant increase in life span of the mice from 8 days
for untreated control animals to 35 days for the treated ani-
mals. In comparison, in the assay for the STIB 900 model,
pentamidine was not curative in any of a group of four mice
when given intraperitoneally at 20 mg/kg for 4 days, although
the average life expectancy was extended to 43 days.
Genotoxicity is not associated with trypanocidal activity. It
is important to determine whether DNA damage plays a role in
activity of nitroheterocyclic trypanocides. For example, the
principal stumbling block in the development of another ni-
troheterocycle, megazol, for use in trypanosomiasis therapy
related to its propensity to induce mutations in DNA. Megazol
is positive in Ames tests (20) and mutagenic in mammalian cell
tests (34). Trypanosomes deficient in their own DNA repair
enzymes are also hypersensitive to megazol, indicating that the
genotoxic effects of this drug are also manifest in these cells
(18). A T. brucei mutant deficient in a DNA repair enzyme
(RAD51) was tested for susceptibility to megazol, to nifurti-
mox, and to compound 3a. The RAD51Ϫ/Ϫ line was more
susceptible than wild-type to megazol but not to compound 3a
or to nifurtimox (Table 2), indicating that megazol, but not
nifurtimox or compound 3a, induces DNA damage in trypano-
RESULTS
Activities of melamine nitroheterocycles against T. brucei in
vitro. Table 1 shows the in vitro activities of a number of
nitroheterocyclic compounds versus T. brucei rhodesiense and
T. brucei brucei and a TbAT1Ϫ/Ϫ derivative that is deficient in
P2 transport. It is noteworthy that all compounds are substan-
tially more active against the T. brucei rhodesiense line in vitro
than against T. brucei brucei. This observation has been made
for a number of other compounds. In vitro, compound 3f (see
Fig. 3) is of similar activity as melarsoprol against T. brucei
rhodesiense, whereas in the T. brucei model melarsoprol is
somewhat more active than any of the new compounds. How-
ever, a number of compounds (notably compounds 3a and 3f)
did show pronounced in vitro activity against these parasites.
Role of the P2 transporter in activity. The compounds were
designed with the intention of eliciting selective uptake into
trypanosomes via the P2 transporter. Loss of the P2 trans-
porter can be a key determinant of drug resistance. In order to
determine the ability of these compounds to interact with that
transporter, Ki values (which give an approximation to the
affinity for the compounds) against P2-dependent adenosine
uptake were determined. Most of the compounds did interact
with the transporter, as judged by their ability to inhibit aden-
osine uptake; however, there was no correlation between the
sensitivity of trypanosomes to these compounds and their ap-
parent affinity for the transporter (Table 1). Toxic activities
were also measured against a parasite line that had been ren-
dered deficient in P2 transporter activity through knockout of
the TbAT1 gene that encodes the transporter. In no instance
was an increase in sensitivity to the nitroheterocyclic com-
pounds of Ͼ2-fold observed. This indicates that the activity of
these compounds is, for the most part, not dependent upon the
P2 transporter, although it is possible that other transporters
may be involved in the uptake of compounds.
Compounds 2a and 2b, melamine-based binding units, were
not active against trypanosomes, and they showed moderate
affinity for the P2 transporter. The lack of activity of these
compounds indicates that the nitrofuran group present on 3a
and 3e is necessary for trypanocidal activity when coupled to a
second aromatic group. In general, the benzamidine-coupled
nitroimidazole compounds showed less trypanocidal activity
than did the melamine-coupled nitrofurans.

VOL. 48, 2004
TRYPANOCIDAL ACTIVITY OF NITROHETEROCYCLES
1737
somes. Culture of T. brucei in the presence of 0.5 mM NAC
reduces free radical damage due to oxidative stress. Although
the activity of nifurtimox was antagonized by NAC, that of
compound 3a was not, indicating that induction of oxidative
stress might not be responsible for the trypanocidal activity of
this compound.
general lipophilic character of the agents could enable uptake
through passive diffusion as is the case for the nitroimidazole
megazol (5). This observation has an important corollary. It
implies that loss of the P2 transporter, an event that appears to
occur relatively easily (4, 11, 27), will not lead to resistance to
this class of compound. However, in the absence of selective
uptake, the reasons for selective activity against the trypano-
some compared to the mammalian host are not certain.
Several of the compounds showed pronounced trypanocidal
activity. The best compound, 3a, showed in vitro IC50 values
against T. brucei of 0.23 ␮M and against T. brucei rhodesiense
of 0.025 ␮M. In comparison, nifurtimox has an in vitro IC50
value of 5.6 ␮M against T. brucei and 1.5 ␮M against T. brucei
rhodesiense. Compound 3a is therefore some 20-fold better in
terms of in vitro efficacy than this rival nitroheterocycle that is
in use clinically (Table 1). Crucially compound 3a was able to
cure mice infected with T. brucei rhodesiense when given at 20
mg/kg for 4 days. In the difficult to cure T. brucei rhodesiense
STIB 900 model, the same treatment schedule resulted in cure
of one of four animals with a mean survival of 35 days (un-
treated controls had a mean survival of 8 days). The good
trypanocidal activities in vitro and in rodent models seen for
this class of compound suggest that further investigations into
novel derivatives might yield even better trypanocidal agents,
and further studies are planned.
Many nitroheterocyclic drugs are believed to exert their ac-
tivity through either reductive stress or oxidative stress. In the
case of reductive stress, single electron reduction of the nitro
group is believed to create a highly reactive radical, derivatives
of which can then form covalent bonds with numerous cellular
macromolecules, including DNA. Oxidative stress is believed
to arise from reduced oxygen intermediates that arise once
oxygen accepts electrons from the reduced nitro group (16).
We have developed assays using trypanosomes that give insight
as to whether reductive or oxidative stress underlies the activity
of these compounds (18). Reductive stress can be assessed by
measuring the susceptibility of DNA repair-deficient trypano-
somes to toxic agents. Oxidative stress can be assessed by
determining the ability of NAC to antagonize an agent’s activ-
ity, since this compound interacts with several reactive oxygen
intermediates.
Using the RAD51 knockout line and NAC assay system, we
have shown that the nitroimidazole-thiadazole megazol exerts
its activity through reductive stress as RAD51 knockout para-
sites, which are deficient in DNA repair and hypersensitive to
this drug (18). These cells are not hypersensitive to the nitro-
furan nifurtimox. The action of nifurtimox, however, is antag-
onized by NAC, whereas that of megazol is not. It therefore
appears that megazol acts through reductive stress, whereas
nifurtimox acts through oxidative stress (16, 18). Compound 3a
does not have any enhanced activity against the RAD51 knock-
out cells, which indicates that this compound may not exert its
activity through reductive stress. Moreover, NAC fails to an-
tagonize the activity of compound 3a. This could indicate that
the agent does not act through oxidative stress. These results
are important, given that the development of the nitroimidaz-
ole megazol was arrested when it became clear that the com-
pound was genotoxic in mammalian cells, an activity also ap-
parent in trypanosomes (18). Nifurtimox is also toxic to
mammals, probably through events related to oxidative stress
DISCUSSION
The chemotherapy of human African trypanosomiasis re-
cently reached the crisis point (25). The lack of surveillance has
led to a resurgence of the disease in sub-Saharan Africa (25).
Treatment failures with melarsoprol, the principal drug used
against late stage disease, have also increased sharply in recent
years (10). New drugs are urgently needed.
Trypanosomes have long been known to be highly suscepti-
ble to a number of nitroheterocyclic compounds. The nitrofu-
ran furacin was used in several trials in the 1950s and 1960s (1,
19), but its development halted when it became clear that it
was responsible for severe toxic side effects. Another nitrofu-
ran, nifurtimox, was registered for use against T. cruzi that
causes Chagas’ disease in Latin America (36). Nifurtimox is
also active against T. brucei, and this compound has been used
against melarsoprol refractory sleeping sickness as a mono-
therapy (33) or in combination with melarsoprol (25), and a
license extension to allow its use for this indication is currently
being sought. Nifurtimox, too, has been linked to important
side effects.
Since African trypanosomes live free in the bloodstream and
cerebrospinal fluid, and not intracellularly, it is possible to
exert selectively toxic effects against parasites, but not against
host cells, by selectively targeting compounds to the parasite’s
interior by the use of transporter proteins present in the par-
asite membrane (21). The P2 aminopurine transporter has
been shown to be responsible for the uptake of the melamine-
based arsenicals and diamidine classes of drugs. The reason for
this relates to the substrate recognition motif of this trans-
porter that is capable of recognizing melamine and benzami-
dine rings in addition to 6-aminopurine samples. We have
previously reported a series of toxic polyamine analogues that
possess the P2 transporter recognition motif in the form of
melamine groups and shown some of these to have a high
activity against T. brucei (6, 23, 38).
Given the susceptibility of T. brucei to nitroheterocycles but
the difficulties in taking such compounds through clinical de-
velopment due to toxicity issues, we considered the possibility
of enhancing the therapeutic index of representatives of this
class of molecule by linking nitrofurans and nitroimidazoles to
the melamine and benzamidine P2 recognition motifs, respec-
tively.
A number of such compounds were derived. The activity of
these compounds is not dependent on uptake via the P2 trans-
porter. First, no correlation between affinity for the P2 trans-
porter, as measured by the ability of these molecules to inhibit
uptake of adenosine via this route, and trypanocidal effect was
noted. Moreover, it was demonstrated that parasites lacking
the P2 transporter were not substantially less sensitive to these
compounds than wild-type cells. It is not currently known what
routes the compounds do take into the cell. It is plausible that
other transporters could be involved in uptake or that the

1738
STEWART ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
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soma cruzi drug, is also mutagenic in the Salmonella/microsome assay. Mem.
Inst. Oswaldo-Cruz 81:49–52.
(31). Compound 3a is notable in that it apparently does not act
through either reductive or oxidative stress in trypanosomes.
It is noteworthy that the activity against L6 cells in vitro is 2
to 3 orders of magnitude lower than against trypanosomes.
Moreover, no overt adverse effects became apparent in mice
challenged with the drug up to 100 mg kgϪ1. It is possible that
the action of compound 3a occurs independently of reduction
of the nitro group. In this case, the chances of the compound’s
passing stringent tests on mammalian cell toxicity would be
greatly enhanced. Such testing has yet to be performed but,
based on the data reported here, further studies into the effi-
cacy and toxicology of this important lead compound are jus-
tified.
ACKNOWLEDGMENTS
21. Hasne, M.-P., and M. P. Barrett. 2000. Drug uptake via nutrient transporters
in Trypanosoma brucei. J. Appl. Microbiol. 89:697–701.
This study was funded by a grant from the Wellcome Trust and the
UNDP/World Bank/WHO Special Programme for Research and
Training in Tropical Diseases. We thank the Welsh School of Phar-
macy for funding (A.B.).
We also thank Elke Gobright and Guy Riccio for excellent technical
assistance and Richard McCulloch (University of Glasgow) and Enock
Matovu (LIRI, Uganda) for the generous provision of the RAD51Ϫ/Ϫ
and TbAT1Ϫ/Ϫ T. brucei lines, respectively. The EPSRC National Mass
Spectrometry Centre in Swansea is acknowledged for accurate mass
spectrometry.
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