Design and synthesis of a series of melamine-based nitroheterocycles with activity against trypanosomatid parasites

Article abstract of DOI:10.1021/jm050177+

The parasites that give rise to human African trypanosomiasis (HAT) are auxotrophs for various nutrients from the human host, including purines. They have specialist nucleoside transporters to import these metabolites. In addition to uptake of purine nucleobases and purine nucleosides, one of these transporters, the P2 transporter, can carry melamine derivatives; these derivatives are not substrates for the corresponding mammalian transporters. In this paper, we report the coupling of the melamine moiety to selected nitro heterocycles with the aim of selectively delivering these compounds to the parasites. Some compounds prepared have similar in vitro trypanocidal activities as melarsoprol, the principal drug used against late-stage HAT, with 50% growth inhibitory concentrations in the submicromolar range. Selected compounds were also evaluated in vivo in rodent models infected with Trypanosoma brucei brucei and T. brucei rhodesiense and showed pronounced activity and in two cases were curative without overt signs of toxicity. Compounds were also tested against other trypanosomatid pathogens, Leishmania donovani and Trypanosoma cruzi, and significant activity in vitro was noted for T. cruzi against which various nitro heterocycles are already registered for use.

Full text of DOI:10.1021/jm050177+

5
570
J. Med. Chem. 2005, 48, 5570-5579
Design and Synthesis of a Series of Melamine-based Nitroheterocycles with
Activity against Trypanosomatid Parasites
†
†
‡
§
|
Alessandro Baliani, Gorka Jimenez Bueno, Mhairi L. Stewart, Vanessa Yardley, Reto Brun,
‡
,†
Michael P. Barrett, and Ian H. Gilbert*
Welsh School of Pharmacy, Redwood Building, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF,
United Kingdom; Institute of Biomedical and Life Sciences, Division of Infection & Immunity, University of Glasgow,
Glasgow G12 8QQ, United Kingdom; London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT,
United Kingdom; and Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
Received February 24, 2005
The parasites that give rise to human African trypanosomiasis (HAT) are auxotrophs for various
nutrients from the human host, including purines. They have specialist nucleoside transporters
to import these metabolites. In addition to uptake of purine nucleobases and purine nucleosides,
one of these transporters, the P2 transporter, can carry melamine derivatives; these derivatives
are not substrates for the corresponding mammalian transporters. In this paper, we report
the coupling of the melamine moiety to selected nitro heterocycles with the aim of selectively
delivering these compounds to the parasites. Some compounds prepared have similar in vitro
trypanocidal activities as melarsoprol, the principal drug used against late-stage HAT, with
5
0% growth inhibitory concentrations in the submicromolar range. Selected compounds were
also evaluated in vivo in rodent models infected with Trypanosoma brucei brucei and T. brucei
rhodesiense and showed pronounced activity and in two cases were curative without overt signs
of toxicity. Compounds were also tested against other trypanosomatid pathogens, Leishmania
donovani and Trypanosoma cruzi, and significant activity in vitro was noted for T. cruzi against
which various nitro heterocycles are already registered for use.
Introduction
the corresponding mammalian transporters. The para-
sites also contain a more general purine uptake system,
designated the P1 transporter, which in fact comprises
Parasitic trypanosomatids cause a number of impor-
tant diseases, including human African trypanosomiasis
8
the activities of a number of different transporters. The
(HAT), Chagas disease, and the leishmaniases. HAT is
normal substrates for the P2 transporter are adenosine
and adenine. However, in addition to this, other motifs
such as melamines and benzamidines (Figure 1) have
been shown to be substrates for this transporter, which
differs from the case of mammalian transporters.2
Results of structure-activity relationship studies have
indicated the following requirements for uptake through
the P2 transporter: an amidine moiety, an aromatic
ring, and an electronegative heteroatom. These features
can be seen in substrates for the P2 transporter shown
in Figure 1.2 Melarsoprol and pentamidine have been
shown to be substrates of the P2 transporter, and this
is one of the mechanisms by which these trypanocidal
caused by the protozoan parasites Trypanosoma brucei
gambiense and T. brucei rhodesiense and is endemic in1
sub-Saharan Africa, where it is a major health problem.
The current drugs used for the treatment of the infection
are unsatisfactory, due to poor blood-brain barrier
permeability, toxicity, and increasing problems due to
,7
2,3
resistance. Chagas disease is caused by Trypanosoma
cruzi, and no drugs are currently registered that are
totally active against these parasites, particularly at the
4
chronic stage of infection. The leishmaniases represent
,9
a spectrum of diseases which are caused by several
5
species of leishmania and for which chemotherapy is
6
also difficult.
3
agents are concentrated in the parasite. Loss or muta-
tion of the P2 transporter has been implicated in
When present in the human host, the parasites
require nutrients from the host. To achieve this,
T. brucei encodes a number of transporters for uptake
of essential nutrients, which are expressed in the
clinically relevant bloodstream form of the parasite.
resistance to melarsoprol and also some diamidines in
laboratory studies.2
,3,10
It is possible that the increased
11
incidence of treatment failure in the field is related
to the emergence of drug resistance in this setting too.
One of the nutrient classes that the parasites sequester
from the host are the purines. Carter and Fairlamb7
Recently it has been shown that the situation is more
complex than originally thought. Loss of the P2 trans-
porter only causes a small (50%) decrease in susceptibil-
ity of parasites to melarsoprol, although this small
decrease may be sufficient to explain the clinical resis-
tance of this drug. The small decrease in susceptibility
to melarsoprol of the parasite indicates that there are
additional modes of uptake of melarsoprol into the
parasite, and it has been shown that there are other
transporters involved in uptake of melamine and ben-
first described an unusual purine transporter from
T. brucei that has been designated the P2 transporter.
This purine transporter shows significant differences to
*
Corresponding author. Tel: +44 29 2087 5800. Fax:
+
44 29 2087 4149. E-mail: gilbertih@cf.ac.uk.
†
Cardiff University.
‡
University of Glasgow.
§
London School of Hygiene and Tropical Medicine.
|
Swiss Tropical Institute.
1
0.1021/jm050177+ CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/29/2005

Melamine-Based Nitroheterocycles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5571
Figure 3. Structure of some known nitro heterocycles having
activity against T. brucei and or T. cruzi.
Figure 1. Substrates of the P2 transporter. The amidine and
electronegative heteroatom are highlighted on each structure.
Figure 4. The structure of the lead compound.
relevance, nifurtimox and benznidazole (Figure 3) are
nitro heterocycles used for treatment of Chagas disease,
which is caused by the related organism T. cruzi. In
addition, nifurtimox is being considered as a potential
treatment for melarsoprol refractory African trypano-
somiasis.1
8,19
A key problem with nitroaromatics is that
some, but not all, compounds in this class are muta-
genic.2
0,21
We decided to couple nitroaromatics to the
Figure 2. The known routes of uptake of diamines and
melaminophenyl arsenicals into T. brucei. The P2 transporter
is also represented interacting with a melamine-nitrofuran
compound (6a), whose transport through the membrane is
probably facilitated by the transporter and passive diffusion.
melamine motif, with the aim of selectively delivering
these compounds to the parasites. Rapid and selective
delivery of these compounds to the parasites should give
selective accumulation in the parasite and minimize
side effects, possibly allowing reduced doses. We have
recently described the activity of some compounds in
which we have linked a nitro heterocycle to a melamine
delivery motif.12 In this current paper we report the
preparation of these initial compounds and some new
compounds plus a detailed analysis of the structure-
activity relationships.
As nitro heterocycles are the only drugs currently
registered against Chagas disease, we considered it of
interest to test the new compounds against T. cruzi.
Leishmania parasites too share substantial similarity
to the other trypanosomatids belonging to the same
taxonomic order and here too new drugs are required,
thus we also tested for activity against Leishmania.
zamidine motifs into parasites (Figure 2).9,12 In particu-
lar at least two other transporters have been identified
for pentamidine uptake, designated “HAPT1” (high-
affinity pentamidine transporter) and “LAPT1” (low-
affinity pentamidine transporter). The transporter
HAPT1 is possibly also responsible for uptake of
melarsoprol. The normal physiological roles of LAPT1
and HAPT1 are certainly unknown. It is possible that
other transporters are also involved in uptake of these
motifs as well; for example, another purine nucleoside
transporter, an S-adenosylmethionine transporter, has
1
3
been reported. However it is clear that these motifs
are selectively taken up into parasites.
The presence of the P2 transporter with its particular
substrate specificity presents an opportunity in the field
of drug design. This could be achieved by attaching
trypanocidal compounds to motifs that are substrates
Chemistry
The general structure of our lead compound, 6a, is
shown in Figure 4.12 In this compound the melamine
motif is linked to the nitro heterocycle via a hydrazone.
We proposed to investigate the following structure-
activity relationships: (i) addition of methyl substi-
tutents to the melamine, to alter the lipophilic proper-
ties of the molecules; (ii) replacement of the oxygen in
the nitro heterocycle with sulfur, to modify the electronic
properties of the nitro heterocycle and lipophilic proper-
ties of the molecule; (iii) investigation of the necessity
of the nitro group, by replacement of the nitro group
with a hydrogen or another electron-withdrawing group.
The triazines described were synthesized starting
from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) (1)
and different nucleophiles. The chlorine atoms of cya-
nuric chloride can be replaced successively by substi-
tuted or nonsubstituted amino groups. The nucleophiles
1
4,15
of the P2 transporter.
Then both the P2 recognition
motif and trypanocidal agent should be selectively taken
up into the parasite. To study this, we have investigated
the coupling of the P2 recognition motif, melamine, to
the polyamine analogues. Polyamine analogues are toxic
1
6,17
to T. brucei.
We have reported the coupling of
polyamine analogues to the melamine moiety and
prepared compounds that showed potent trypanocidal
activity and good selectivity on a cellular level.14 How-
ever, the compounds turned out to be toxic in animal
models. Therefore, we have selected an alternate try-
panocidal moiety for attachment to the melamine and
benzamidine delivery moiety, nitroaromatics. Nitro-
aromatic compounds have been shown to have potent
activity against a variety of microbes. Of particular

5
572 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Baliani et al.
Scheme 1a
a
(
i) R1R2NH, acetone/H2O, rt; (ii) R2R3NH, 2 N NaOH, acetone/H2O, rt; (iii) NH2NH2, H2O, reflux; (iv) aryl aldehyde, MeOH, rt. The
yields are shown in brackets.
Scheme 2a
can selectively displace the different chlorines by con-
trolling the reaction temperature.22 In general, the first
chlorine can be displaced while the temperature is
maintained below 0 °C, the second between 25 and
2
3
5
0 °C, and the third substitution at 67 °C. Another
a
important factor that has to be considered for the
preparation of the different derivatives is the nature of
the reactive group and the order of entry of the group.
When different amino groups were introduced, the less
reactive amine was introduced before the more reactive
one.
(i) I2, NH3, THF/H2O, rt; (ii) oxalyl chloride, DMSO, NEt3
CH2Cl2, -78 °C.
Amended data is presented for compound 6b to that
originally presented;12 this compound was remade and
repurified.
With the purpose of improving the solubility and
evaluating the pharmacological importance of the redox
potentials of the heteroaromatic rings, the hydrazine
The reactions, in most cases, were carried out in
aqueous suspensions, since the products precipitate
2
4
from solution, simplifying their isolation. To increase
the reactivity and the yield, the cyanuric chloride was
previously dissolved in acetone and then poured into
ice-water to get a very fine suspension. The direct
reaction of cyanuric chloride with ammonia, methyl-
amine, and dimethylamine gave the 2-substituted-4,
(
4a) was coupled with 5-nitro-2-thiophenecarboxalde-
hyde to prepare the thiophene analogue (6h).
To investigate the pharmacological role of the nitro
group in these compounds, compounds without a nitro
group were prepared. Replacement of the nitro with a
hydrogen was achieved by coupling the hydrazine 4a
with 2-furaldehyde or 2-thiophenecarboxaldehyde to
give the related hydrazones (6f, 6i). The solubility
observed for all these compounds was slightly improved
and the recrystallization was achieved using the condi-
tions described for the previous hydrazones.
We were also interested to replace the nitro with a
cyano group, as this is electron-withdrawing, like the
nitro. To do this, we required the 5-cyano-substituted
2-furaldehyde. This compound was synthesized as
showed in Scheme 2. 5-Hydroxymethyl-2-furaldehyde
(5a) was used as starting material. The aldehyde
function was successfully converted in nitrile by using
iodine in aqueous ammonia. The reaction seems to
proceed via oxidation of aldimine with iodine to give an
N-iodo aldimine intermediate, which eliminates an HI
2
5,26
6
-dichloro-1,3,5-triazines (2a-c) (Scheme 1).
The 2,4-disubstituted-6-chloro-1,3,5-triazines (3a-e)
were obtained by reaction of a further amine with the
-substituted-4,6-dichloro-1,3,5-triazine in the presence
2
of base (NaOH or NaHCO3). The displacement of the
last chlorine by the hydrazine was achieved by further
increasing the temperature to 85 °C, affording the
2
7
hydrazine derivatives (4) in good yields. The first set
of hydrazones (6a-i) were prepared by reaction of the
hydrazine derivatives (4) with 5-nitro-2-furaldeyde,
affording the corresponding 4,6-disubstituted-2-(5-nitro-
furfurylidenehydrazino)-1,3,5-triazines (6a-e). The prod-
ucts had a low solubility in most organic solvents, except
DMSO. However purification was achieved by recrystal-
lization from methanolic and ethanolic water solutions.

Melamine-Based Nitroheterocycles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5573
Scheme 3a
T. brucei brucei AT1 wild type and a TbAT1 knockout
line were compared to study the effects of the P2
transporter on the activity of the compounds (the TbAT1
gene encodes the P2 transporter). A strain of T. brucei
rhodesiense, the causative agent of acute HAT, was also
studied. Compounds were also assayed against mam-
malian L6 cells as a measure of cellular toxicity toward
mammalian cells.
Very similar results were seen with compounds
against both the wild type and P2 knockout T. brucei
brucei. There was a slight reduction in activity for the
knockout line, on the order of 2-fold less active, for many
compounds. This was observed for melarsoprol as well
as our compounds. This reduction in activity is not very
significant and suggests that there are routes other than
the P2 transporter for uptake of these compounds.
Melarsoprol was the most active compound against
T. brucei brucei (IC50 ) 53 nM). However, some of the
nitro heterocycles prepared showed only slightly reduced
activity against T. brucei brucei, notably 6a, 6c, 6d, 6h
a
(
iv) Methanol, rt.
molecule in ammonia solution to afford the nitrile
product (5b) in good yield (80%).28 The hydroxyl group
was then oxidized to the aldehyde 5c via Swern oxida-
2
9
tion.
(IC50 ) 230, 200, 130, and 850, respectively).
The three isomers of nitrobenzaldehyde (7a-c) have
also been used to synthesize hydrazones where the
heterocyclic ring has been replaced with a nonhetero-
cyclic structure.
Compounds were more potent against the T. brucei
rhodesiense line studied here than against the T. brucei
brucei line, although very similar trends were seen
within the different species. These probably reflect a
range of activities against different strains of the
T. brucei trypanosomes in general rather than a par-
ticular difference between the nonhuman pathogenic
T. brucei brucei line and the human infectious T. brucei
rhodesiense line. Thus, melarsoprol had an IC50 against
T. brucei rhodesiense of 6 nM, while 6c had an IC50 of
Biology
The activity of the compounds was evaluated in a
variety of different models.
Affinity for the P2 Transporter. Compounds were
evaluated for their ability to interact with the P2 amino-
purine transporter of T. brucei (Table 1). This was
achieved by measuring their ability to antagonize the
uptake of radiolabeled adenosine. In addition to
uptake through the P2 transporter, the other main route
of adenosine uptake is through the P1 system. To
differentiate transport through the P1 and P2 systems,
the experiments were conducted in the presence of a
large excess of inosine, which saturates the P1 trans-
porter. Thus, adenosine uptake will only occur through
the P2 transporter. It is important to note that this
experiment does not actually measure uptake of com-
pounds through the P2 transporter, but it does give a
measure of the affinity of compounds for the P2 trans-
porter.
3
nM. A significant number of our compounds had IC50
values in the nanomolar range (6a, 6b, 6c, 6d, 6e, and
1
0
6
h). Compound 6c was the most active compound in our
series in cellular assays.
In comparing all activities the following structure-
activity relationships can be observed:
(
1) Compounds without a melamine structure (8 and
) showed weak activity (IC50 ) 2.3 µM) and also weak
selectivity compared to the mammalian L6 cells.
2) Compounds with a nitrofuran ring joined to a
melamine ring showed potent in vitro activity (6a-e).
3) Replacement of the oxygen with a sulfur led to a
0-fold loss in activity and a small increase in toxicity
to L6 cells.
9
(
(
1
The results suggest that compounds without a
melamine group have relatively low affinity for the P2
transporter (compound 9 has IC50 ) 404 µM), as would
be anticipated. Also compound 6c in which all the
nitrogens on the melamine ring were methylated showed
poor affinity (IC50 ) 129 µM). The other compounds
assayed showed IC50 values all within about a log range
of each other that were of a similar order as melarsoprol.
Changes to the nitrofuran ring gave very small changes
to the apparent affinity for the P2 transporter: thus,
removal of the nitro group (compare 6a/6f and 6h/6i)
gave virtually no change in affinity, while replacement
of the oxygen with a sulfur gave a small increase in
affinity (compare 6a/6h and 6f/6i). This suggests that
the primary determinant of affinity for the P2 trans-
porter is the melamine ring and the nitro heterocycle
has a limited effect on affinity. This is consistent with
(
4) Compounds in which the nitro group was replaced
by a hydrogen had significant loss in activity (compare
6
a/6f and 6h/6I).
5) Replacement of the nitro group with another
(
electron-withdrawing group, the nitrile group, led to loss
of activity (6g), implying that more than an electron-
withdrawing effect is necessary for activity.
(6) Complete removal of the nitro heterocycle caused
loss of activity (4a).
(7) Replacement of the nitrofuran with a nitrophenol
also led to loss of activity (7a-c).
(8) A number of the nitro heterocycles were markedly
less toxic against mammalian cells than melarsoprol;
of particular note are 6a, 6d, and 6e.
Activities against Trypanosoma cruzi and Leish-
mania donovani. An interesting point noted from the
data on in vitro activities against T. brucei brucei was
the fact that compounds retained activity despite the
loss of the P2 transporter. This indicates that routes
2
-15
previous reports in the literature.
Activities against T. brucei in Vitro. Compounds
were tested against several T. brucei lines in vitro. The

5
574 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Baliani et al.
Table 1. In Vitro Activities of Compounds against the P2 Transporter, Bloodstream T. brucei, and Mammalian Cells as a Measure of
Toxicityd
IC50, µm
T. brucei brucei
AT1 wild type
T. brucei brucei
T. brucei
rhodesiense
P2 uptakea
AT1 knockout
b
L6 cellsc
compd
MW
4
6
6
6
6
6
6
6
6
6
7
7
7
8
9
a
a
b
c
d
e
f
g
h
i
141.1
264.2
292.2
320.3
278.2
292.2
216.2
244.2
280.2
235.3
274.2
274.2
274.2
155.1
156.1
11.9
22.9
ND
129
ND
ND
15.9
ND
1.9
>200
0.23
ND
>200
0.38
ND
ND
ND
183
44.1
18.7
48.9
109.5
>400
ND
11.8
78.2
ND
ND
ND
20
0.025
0.010
0.003
0.018
0.053
12.9
0.2
0.3
0.13
5.36
16.5
ND
0.85
89
>75
>75
>75
23.5
1.18
0.053
5.6
0.06
3.09
29.3
ND
1.52
170
>75
>75
>75
13.5
1.26
0.12
ND
46.27
0.24
10.2
29.9
52.88
36.47
2.3
0.68
0.006
1.5
4.9
a
b
c
ND
ND
ND
404
300
1.2
40
melarsoprol
nifurtimox
7.8
68
ND
a
b
Inhibition of adenosine uptake by the P2 transporter in T. brucei brucei 427. T. brucei brucei AT1 knockout is a mutant with a non
c
functional P2 transporter. L-6 cells are rat skeletal myoblasts and are used as a measure of cytotoxicity to mammalian cells. ND, not
determined. d Data for compounds 4a, 6a, 6c, 6f, 6h, 6i has been reported previously, but is included here for comparative purposes.
Amended data for 6b is presented.
12
Table 2. In Vitro Activities of Compounds against
Intracellular Amastigotes of T. cruzi and L. donovani
Table 3. In Vivo Activities of Compounds against T. brucei
a
a
brucei Model and T. b. rhodesiense Model
T. cruzi
(IC50 [µM])
Leishmania donovani
T. brucei brucei
T. b. rhodesiense
(
STIB 795 model) (STIB 900 model)
Compound
MW
(IC50 [µM])
Survival
Survival
4
a
a
b
c
d
e
f
g
h
i
141.1
264.2
292.2
320.3
278.2
292.2
216.2
244.2
280.2
235.3
274.2
274.2
274.2
156.1
156.1
ND
ND
>12.5
3.14
tox
0.75
tox
tox
ND
tox
tox
Cured/ average Cured/ average
6
6
6
6
6
6
6
6
6
7
7
7
8
9
2.1
Compound
Dose mg/Kg Infected (days) Infected (days)
0.24
0.38
0.39
0.29
>400
122
b
6a
4 × 20 i.p.
4 × 20 i.p.
4 × 20 i.p.
4 × 20 i.p.
4 × 20 i.p.
4/4
ND
0/4
4/4
0/4
0/4
2/4
ND
4/4
ND
>60
ND
5.25
>60
18.3
7
>60
ND
>60
ND
1/4
0/4
ND
2/4
ND
0/4
0/4
4/4
ND
0/4
35.25
7.25
ND
>38.5
ND
6.5
b
6
6
6
6
c
b
d
e
Control (avrg)
Melarsoprol
2.6
4 × 1 i.p.
21.25
>60
ND
85.84
91.54
328
4
× 8 i.p.
a
b
c
ND
ND
ND
tox
Pentamidine 4 × 5 i.p.
4
× 20
42.75
328
a
ND
The control represents the average of the controls for each
experiments performed. These data was presented previously,
but is included here for comparative purposes.
b
12
ND
tox
Melarsoprol
Nifurtimox
0.006
1.5
7.8
68
a
mice. Compounds 6b and 6e were not curative in vivo
at a dose of 20 mg/kg.
Standards: For T. cruzi, Benznidazole, IC50 ) 1.435 µM;
L. donovani: Miltefosine, IC50 ) 1.16 µM: tox - toxic to mac-
rophages thus precluding measurement of leishmanicidal activity.
The compounds were also tested with T. brucei
rhodesiense STIB 900 model in rodents, which is a more
stringent test. For example, pentamidine, one of the
drugs currently used for early stage HAT, was not able
to cure this model at this dose. However, this model
responds to melarsoprol, a drug currently in use for the
treatment of the late stage. In the STIB 900 model,
parasites appear to leave the vasculature system early
so that drugs must penetrate extravascular compart-
ments to effect radical cure. Therefore, this in vivo test
represents a good model for evaluating likely outcomes
of drugs in late-stage models.
other than the T. brucei specific transporter clearly exist
for these compounds. This prompted us to also test for
activity against the related pathogens T. cruzi and L.
donovani.
As shown in Table 2 activities against T. cruzi were
observed for compounds 6b-e, with activities in the
submicromolar range. None of the compounds showed
sub-micromolar activities in vitro against L. donovani,
except for compound 6d, with an IC50 of 0.75 µM.
In Vivo Activity in Rodent Models of Infection.
Selected compounds were evaluated in several rodent
models of T. brucei group infection. Compounds 6a, 6b,
As already described in our previous work,12 com-
pound 6a was given intraperitoneally at 20 mg/kg for 4
days and cured only one mouse of four. However, the
compound caused a significant increase in life span of
the mice to 35 days compare to the 7 days for the un-
treated control animals. Pentamidine did not cure any
of the four mice but increased the life span to 43 days.
Some compounds were also investigated for their in
vivo activities in mice model infected with T. cruzi.
6
d, and 6e were examined in rodent models infected
with T. brucei brucei STIB 795 at a dose of 20 mg/kg
for 4 days ip (days 3-6). The mice are considered cured
when no infection was found at 60 days. Compounds
6
a and 6d were able to cure the STIB 795 T. brucei
brucei model mice, where four mice of four were all
cured. No overt signs of toxicity were observed in these

Melamine-Based Nitroheterocycles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5575
Table 4. In Vivo Activities against a Rodent Model of Chagas
uptake of compounds into T. brucei using nucleoside
transporters. The compounds prepared were investi-
gated for their ability to antagonize uptake of radio-
labeled adenosine through the P2 transporter. This
assay does not give proof that the compounds are
internalized through the transporter, but does indicate
that many of the compounds showed good affinity for
the P2 transporter, as they compete with adenosine in
binding to the transporter. However, there was no
correlation between affinity for the P2 transporter and
activity in vitro against intact parasites. In addition to
this, there are no remarkable changes in the sensitivity
when the in vitro activities against the T. brucei brucei
model are compared with the activities against the
T. brucei brucei knockout line (i.e., deficient in P2
transporter activity). Therefore, it is not possible to
directly correlate the affinity of the P2 transporter and
the trypanocidal activity. Passive diffusion or other
transporters may be involved in the uptake of the
compounds.33 However, compound 8, which lacked a
melamine group and had poor affinity to the P2 trans-
porter, showed weak activity against the parasites,
suggesting that the melamine group is required for
uptake, or else in interactions with enzymes involved
in either activation or directly in the mode of action of
the compounds. Future research should focus on learn-
ing more about roles of the melamine moiety.
Disease for Some Selected Compounds
Dose
No of
Survival
Time
%
Compound
mg/Kg doses Route
inhibition
6
6
6
6
c
a
d
e
15
25
50
50
-
5
5
5
5
i.p.
i.p.
i.p.
i.p.
12.8
13
N. D.
N. D.
27.75
44.95
0.00
13
14
13
>30
Control
Benznidazole
45
5
p.o.
100.00
Although none of the compounds evaluated was able to
cure the model mice, compounds 6d and 6e were able
to reduce parasitaemia, the latter by 45%. Compound
6
d was investigated in a rodent model of leishmaniasis
(
L. donovani-infected mice). The compound had a mod-
erate effect. When dosed at 40 mg/kg intraperitoneally
for 5 days, there was 28% reduction in parasitaemia,
compared to the control. With the standard drug,
pentostam, there was a 62% reduction in parasitaemia
compared to the control when dosed at 15 mg/kg for 5
days.
Mode of Action Studies. We have previously re-
12
ported mode of action studies and have repeated these
studies now also including 6d and the nitroimidazole
benznidazole, too. Two possible mechanisms of action
of the compounds are damage to DNA and oxidative
damage.
Interestingly, 6c showed poor affinity for the P2
transporter but marked in vitro activity against
T. brucei brucei and T. brucei rhodesiense. In compound
First, to investigate damage to DNA, experiments
were undertaken with a T. brucei mutant deficient in a
DNA repair enzyme (RAD51). This mutant is compro-
mised in its ability to repair double-stranded DNA
breaks; thus, agents that induce such damage are more
active against these mutants than against wild-type
cells. Megazol (see Figure 3 for structure) is a hetero-
cycle that exerts its mode of action by damage to DNA.
6
c, all the hydrogen atoms on the amino group have
been replaced by methyl groups. The lack of affinity of
this compound for the P2 transporter could possibly be
explained by a requirement for substrates of the P2
transporter to have an H-bond donor. Compound 6c
showed much higher solubility in a variety of solvents
than other compounds, and this may allow rapid passive
diffusion, which may account for high activity against
the parasites. It is clear that the melamine moiety (with
at least some NH bonds) is selectively concentrated
within the parasite, but the route of uptake is not
exclusively through the P2 transporter. This is impor-
tant, since loss of the P2 transporter can induce
resistance to drugs that enter cells exclusively via this
route. The fact that other routes of uptake exist for the
melamine nitroheterocycles means that resistance re-
sulting from simple loss of the P2 transporter should
not be an issue.
-/-
Thus the RAD51 line was more susceptible than wild
3
0
type to megazol (Figure 3). However, compound 6a
showed similar activity against both the wild-type and
-
/-
RAD51 , indicating that the mode of action does not
involve damage to DNA in the same manner as megazol,
although other types of DNA damage cannot be ruled
out. Benznidazole has markedly lower activity against
T. brucei in vitro than other compounds and shows no
additional activity in the RAD51 deficient cell line.
Compound 6d shows a very similar profile to 6a.
Nifurtimox (Figure 3) is another nitro heterocycle that
is currently registered for treatment of Chagas disease
and is being investigated for the treatment of melarso-
prol refractory HAT. Nifurtimox is believed to act via
Some of the compounds prepared showed potent
activity against T. brucei rhodesiense, of the same order
as melarsoprol (6a, IC50 ) 25 nM; 6b, IC50 ) 10 nM 6c,
IC50 ) 3 nM; 6d, IC50 ) 18 nM; 6e, IC50 ) 53 nM; 6h,
IC50 ) 240 nM, melarsoprol, IC50 ) 6 nM). In addition,
some of these compounds showed lower toxicity against
mammalian (L6) cells than melarsoprol, although fur-
ther work is required to show that these compounds
would have better selectivity/therapeutic indices. Com-
pound 6a gave an IC50 value of 0.025 µM against
T. brucei rhodesiense, which is 60-fold better than
nifurtimox (1.5 µM) that is clinically in use against
oxidative stress.3
1,32
Thus, culturing the T. brucei in the
presence of N-acetylcysteine, which reduces free radical
damage due to oxidative stress, antagonizes the mode
of action of nifurtimox. This effect was not seen in the
case of 6a nor 6d, suggesting that the main mode of
action is not due to accumulation of high levels of
reactive oxygen species within compartments that are
targeted by N-acetylcysteine. More subtle effects as-
sociated with oxidative damage, however, cannot be
ruled out.
Discussion
34
Chagas’ disease and in trial against melarsoprol
refractory trypanosomiasis.1
8,19
In this paper, we report results on a group of try-
panocidal nitro heterocycles identified during work
aiming to induce selective toxicity through selective
We decided to investigate the roles of both the nitro
group and the furan ring. The nitro group is a strong

5
576 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Baliani et al.
a
-/-
Table 5. Activities of Compounds against Wild Type, Wild-type with NAC and RAD51
Mutant T. brucei
Compound
6d
6a
megazol
nifurtimox
benznidazole
Wild-type (427)
0.08 ( 0.05
0.09 ( 0.04
0.07 ( 0.03
0.08 ( 0.03
0.12 ( 0.05
0.10 ( 0.02
0.12 ( 0.05
0.13 ( 0.06
0.04 ( 0.04
4.1 ( 1.7
11.4 ( 5.2
4.3 ( 1.6
116.3 ( 7.5
116.3 ( 13.6
114.2 ( 32
Wild-type (427) + NAC
-
/-
RAD51
mutant
a
Values are IC50 (µM).
electron-withdrawing group and also can be involved in
the generation of free radicals. Replacement of the nitro
with a hydrogen (6f) or by another electron-withdrawing
group such as the nitrile (6g) led to an inactive
compound, indicating the importance of the nitro group
itself. Replacement of the nitrofuran with a nitrophenyl
gave significant loss of activity. Even replacement of the
oxygen with sulfur (6h) gave reduced activity, suggest-
ing that perhaps the redox potential of the nitrofuran
is important for the mode of action of these compounds.
Nitro heterocycles often work through free-radical
mechanisms, so genotoxicity represents a main issue in
the development of new trypanocidal nitro heterocyclic
compounds. Megazol, a nitro heterocyclic compounds,
The discovery that the nitro heterocycles were active
against T. brucei regardless of the presence of the P2
transporter prompted us to test the compounds against
other pathogenic trypanosomatids that do not possess
this transporter. Considerable activity was noted against
amastigote T. cruzi in vitro within mammalian cells.
Several compounds were several-fold more active than
the registered drug, nifurtimox, in vitro. However, all
failed to cure mice in vivo, although some compounds
also led to a reduction in parasitaemia in vivo, high-
lighting the lead status of these new molecules for use
in Chagas therapy.
In the case of Leishmania, none of the compounds
were particularly active against the intracellular amastig-
otes, except for compound 6d. A modest activity was
found for this compound in a rodent model of disease.
35
was found to be mutagenic in mammalian cell tests
and also positive in the Ames test.36 It has been found
that the trypanocidal activity of megazol was related
to its ability to induce mutations in DNA; trypanosomes
deficient in their own DNA repair enzymes (the
Conclusion
-
/-
We have prepared some melamine-nitrofuran conju-
gates that have potent activity against T. brucei rhod-
esiense on the same order of magnitude as melarsoprol
and that are significantly more active than nifurtimox,
which is currently undergoing trials for HAT. Two of
the compounds were able to cure an animal model of
trypanosomiasis (T. brucei brucei STIB795) and one of
the compounds was able to have a significant effect on
the course of another model of infection (T. brucei
rhodesiense STIB900). The fact that the mode of action
of 6a does not appear to involve DNA damage in
trypanosomes might indicate that it will be not muta-
genic in tests in mammalian cells, thus greatly improv-
ing the chances that this compound or its derivatives
can proceed to clinical trials against trypanosomiasis.
These compounds represent exciting new leads for
further evaluation for HAT. Moreover, some compounds
also showed considerable activity against T. cruzi in
vitro and some reduction in parasitaemia in vivo,
indicating that this class of molecule should also be
considered as a lead in developing novel drugs against
Chagas disease.
RAD51
line) are hypersensitive to this compound.
-
/-
However, the RAD51
line showed susceptibility
similar to that of wild-type parasites to compound 6a.
This suggests that 6a has a different mode of action to
megazol. Another nitro heterocycle, nifurtimox (which
3
7
is also positive in the Ames test ), appears to work by
oxidative stress, as can be seen by antagonizing the
compound with N-acetylcysteine. Compound 6a was not
antagonized by N-acetylcysteine, indicating that its
mode of action is not by oxidative stress. These findings
may indicate that the compounds do not work by
damage to DNA or oxidative stress, both of which are
implicated in positive Ames tests. Further studies are
continuing in this area.
In addition to in vitro activity, several compounds
retained trypanocidal effect in mice. Compounds 6a and
6d were able to cure mice infected with T. brucei brucei
at a dose of 20 mg/kg for 4 days. It is noteworthy that
addition of methyl groups to the melamine NH2 groups
reduces activity both in vitro and in vivo. Addition of
one methyl group to one of the amino groups (6d) yields
a compound with slightly better activity in vitro and
activity is retained in vivo. However, addition of an
extra methyl to the same nitrogen (6e) or a methyl to
the other amino (6b) led to loss of activity in vivo at
the dose tested. The fact that trypanocidal activity in
each case was barely altered by the presence of the P2
transporter indicates that the relative activity of these
compounds is not related to their ability to interact with
that transporter. It is noteworthy that the most active
compound in vitro, 6c, was inactive in vivo at the dose
tested, highlighting the role of pharmacokinetic issues,
beyond simple antiparasite activity, on in vivo trypano-
cidal capability.
Experimental Section
P2 Transporter Affinity Measurements. Parasites puri-
fied from blood were stored on ice in Carter’s buffered saline
7
solution. Transport assays used the centrifugation-through-
9
,14,15,33,38
oil technique, which is routinely used in analyses.
Radiolabeled adenosine (0.5 µM) uptake via P2 was measured
in the presence of 1 mM inosine, which blocks the P1
7
transporter. Compounds were assayed for affinity for the P2
transporter by using labeled adenosine fixed at 0.5 µM and a
range of inhibitor concentrations. IC50 values were calculated
using the Grafit 4.0 Software (Erithacus) by plotting inhibitory
value against concentration of inhibitor.
In Vitro Activities against T. brucei brucei and T.
brucei rhodesiense and Cytotoxicity. The activity of com-
pounds was determined for T. brucei rhodesiense trypomas-
tigotes of STIB900. This stock was isolated in 1982 from a
human patient in Tanzania. Minimum essential medium
(50 µL) supplemented with 2-mercaptoethanol and 15% heat-
Compound 6a was also tested with the more stringent
T. brucei rhodesiense STIB 900; using the same treat-
ment schedule, it cured only one of four animals infected
with a mean survival of 35 days compare to the
untreated controls that had a mean survival of 8 days.

Melamine-Based Nitroheterocycles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5577
inactivated horse serum39 was added to each well of a 96-well
microtiter plate. Serial drug dilutions were prepared covering
a range from 90 to 0.123 µg/mL. Then, 50 µL of a trypanosome
suspension was added to each well, and the plate incubated
Chemistry. General. Chemicals were purchased from
Aldrich and Fluka and were used without further purification.
Dry solvents were generally purchased from Fluka in sure-
seal bottles and stored over molecular sieves. Qualitative thin-
layer chromatography (TLC) was performed on precoated
aluminum sheets silica gel 60F254 from Merck. Melting points
were determined with a Gallenkamp melting point apparatus
at 37 °C under a 5%CO
2
atmosphere for 72 h. Alamar Blue
(10 µL) was then added to each well and incubation continued
for a further 2-4 h. The plate was then read in a Spectramax
Gemini XS microplate fluorometer (Molecular Devices Corp.,
Sunnyvale, CA) by using an excitation wavelength of 536 nm
1
13
and are not corrected. H and C NMR data were recorded
on a Bruker Avance DPX 300 MHz NMR spectrometer, with
tetramethylsilane as the internal standard and deuterated
solvents purchased from Goss unless stated otherwise. Infra-
red (IR) spectra were recorded on a Perkin-Elmer 1600 FT-IR
spectrometer. Mass Spectra were recorded at a Platform II
mass spectrometer (Micromass) from Fisons. Ionization was
achieved in the positive electrospray mode using a mixture of
acetonitrile/water (1:1) or MeOH (HPLC grade) as mobile
phase. High-resolution mass spectra were recorded by the
National Mass Spectrometry Service Centre in Swansea with
a MAT 900 XLT high resolution double focusing mass spec-
trometer from Finnigan using the same ionization procedure.
Combustion analyses were performed by the analytical and
chemical consultancy services MEDAC Ltd. The triazine
40
and an emission wavelength of 588 nm. Fluorescence devel-
opment 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 transporter is necessary for activity, compounds were
assayed against the T. brucei brucei trypomastigotes using
-/- 41
either the wild type or P2 knockout mutants (TbAT1 ). The
4
0
Alamar Blue assay was also used to determine IC50 values.
To determine whether DNA damage was associated with
trypanocidal activity, the Alamar Blue assay was also used to
-/-
42
determine IC50 values against the RAD51
deletion mutant.
44
derivatives, as observed in other work, often present a
In Vitro Activities against Trypanosoma cruzi. Rat
skeletal myoblasts (L-6 cells) were seeded in 96-well microtiter
plates at 2000 cells/well/100 µL in RPMI 1640 medium with
problem in analysis. The problem was mainly found to be for
the microanalysis of the nitrogen content.
5
-Nitro-2-furaldehyde (4,6-Diamino)-[1,3,5]-triazin-2-
10% FBS and 2 mM L-glutamine. After 24 h, 5000 trypo-
ylhydrazone (6a). A mixture of 4a (149.50 mg, 1.06 mmol)
and 5-nitrofuraldehyde (150.00 mg, 1.06 mmol) was dissolved
in methanol (5 mL). The suspension was left stirring overnight
at room temperature. The reaction mixture was filtered and
the yellow precipitate was washed with methanol and dried
under vacuum at 40 °C. The crude product was recrystallized
from water to get 100.5 mg of a pure yellow solid. Yield:
mastigotes of T. cruzi (Tulahuen strain C2C4 containing the
â-galactosidase (Lac Z) gene) were added in 100 µL per well
with 2× of a serial drug dilution. The plates were incubated
2
at 37 °C in 5% CO for 4 days. Then the substrate CPRG/
Nonidet was added to the wells. The color reaction, which
developed during the following 2-4 h, was read photo-
metrically at 540 nm. From the sigmoidal inhibition curve IC50
values were calculated.
-
1
100.5 mg, 36%. mp: >350 °C. IR: νN-H 3313.9 cm , νCdN
-
1
-1
+
1631.9 cm , νC-NO 1537.8 cm , νC-N 809.3. LRMS (ES ):
2
+
+
+
m/z 265 (M + H , 100%), 287 (M + Na , 80%). HRMS (ES ):
calcd for (C
Activities against L. donovani. Mouse peritoneal
macrophages were seeded in RPMI 1640 medium with 10%
heat-inactivated FBS into Lab-tek 16 chamber slides. After
+
1
8
H
9
N
8
O
3
)
265.0792, found 265.0799. H NMR
): δ 6.51 (bs, 4H), 7.05 (d, J ) 3.84) 7.78
(
(
300 MHz, DMSO-d
d, J ) 3.84 Hz), 8.00 (s, 1H), 11.25 (s, 1H). C NMR (75 MHz,
): δ 114.2, 115.6, 129.4, 151.8, 153.2, 164.9, 167.7.
6
13
2
4 h L. donovani amastigotes were added at a ratio of 3:1
DMSO-d
-Nitro-2-furaldehyde (N ,N -Dimethyl-4,6-diamino)-
1,3,5]-triazin-2-ylhydrazone (6b). Compounds 4b (180 mg,
.06 mmol) and 5-nitrofuraldehyde (142.5 mg, 1.06 mmol) were
6
(
amastigotes to macrophages). The medium containing free
4
6
5
amastigotes was replaced by fresh medium 4 h later. The next
day the medium was replaced by fresh medium containing
different drug concentrations. The slides were incubated at
[
1
suspended in MeOH (10 mL). The suspension was left stirring
overnight. The reaction was reduced under vacuum. The yellow
2
2
7 °C under a 5% CO atmosphere for 96 h. When the medium
was removed, the slides were fixed with methanol and stained
with Giemsa. The ratio of infected to noninfected macrophages
was determined microscopically, expressed as percentage of
the control, and the IC50 value was calculated by linear
regression.
2
solid was then recrystallized from H O/MeOH (50/50 mL).
+
+
Yield: 220 mg, 71%. LRMS (ES ): m/z 293.1 ((M + H) , 100%),
+
+
3
2
15.2 (M + Na , 20%). HRMS: calcd mass for (C10
H
13
N
8
O
3
)
1
93.1105, found 293.1104. H NMR (300 MHz, DMSO-d
6
):
δ (mixture of the two geometric isomers) 3.36 (m, 6H), 7.05
In Vivo Biological Activity against T. brucei. Female
NMRI mice weighting 22-25 g were infected with cryo-
preserved stabilates of T. brucei brucei STIB 795 (derivate of
(
(
bs, 2H) 7.23 (bs, 1H), 7.78 (m, 1H), 11.06 (s, 1H), 11.20
13
6
s, 1H). C NMR (75 MHz, DMSO-d ): δ (mixture of the two
geometric isomers) 27.4, 27.6*, 113.9, 115.6, 129.0, 129.4*,
51.8, 153.3, 164.1, 166.4, 166.6*. Anal. (C10 ‚0.4H O)
C, H, N.
-Nitro-2-furaldehyde (N ,N ,N ,N -Tetramethyl-4,6-di-
amino)-[1,3,5]-triazin-2-ylhydrazone (6c). A mixture of 4c
201 mg, 1.01 mmol) and 5-nitro-2-furaldehyde (143 mg,
.02 mmol) was dissolved in 10 mL of ethanol. The reaction
4
3
strain 427 ) or T. brucei rhodesiense STIB 900. Each mouse
1
H
12
N
8
O
3
2
4
was infected intraperitoneally with (2-4) × 10 bloodstream
forms. Melarsoprol (Arsobal; Aventis) acted as standard drug
and was diluted with sterile distilled water to an appropriate
concentration. Groups of four mice were treated on days 3, 4,
4
4
6
6
5
(
5
, and 6 intraperitoneally with 20 mg/kg. A control group
1
remained untreated. The parasitemia of all animals was
checked on day 7 and 10 postinfection and every second day
thereafter until 60 days. Death of animals was recorded to
calculate the mean survival time. Surviving and aparasitemic
mice were considered cured at 60 days and then euthanized.
mixture was allowed to stir overnight at room temperature
and the solvent was evaporated under vacuum. The residue
was purified by flash column chromatography (gradient from
1
to 5% MeOH/DCM). Compound 6c was isolated as a clear
white solid. Yield: 187 mg, 58%. HRMS: calcd mass for
+
1
In Vivo Activity for T. cruzi. Female BALB/c mice
17 8 3
(C12H N O ) 321.1424, found 321.1421. H NMR (300 MHz,
weighing 20 g (Charles Rivers Ltd, UK) were infected intra-
6
DMSO-d ): δ 3.21 (s, 12H), 7.04 (m, 1H), 7.44 (m, 1H), 7.92
4
13
peritoneally with 2 × 10 trypomastigotes in 0.2 mL, harvested
6
(s, 1H), 9.05 (bs, 1H). C NMR (75 MHz, DMSO-d ): δ 36.5,
from the blood of a passage mouse. Infected mice were
randomly sorted into groups of five. After 5 days tail blood
was examined for patency of infection: the number of trypo-
mastigotes in 10 microscope field was noted. Dosing com-
menced for five consecutive days. Tail blood was examined 2
days after the end of treatment and at 7 day intervals on any
surviving mice.
111.1, 114.2, 129.4, 153.5, 164.1, 165.8.
4
5-Nitro-2-furaldehyde (N -Methyl-4,6-diamino)-[1,3,5]-
triazin-2-ylhydrazone (6d). 5-nitrofuraldehyde (90 mg,
0.58 mmol) and 3d (81.82 mg, 0.58 mmol) were mixed together
and suspended in MeOH (5 mL), and the suspension was left
stirring overnight. The mixture was reduced under vacuum,
giving 120 mg of brown solid. The solid was recrystallized from

5
578 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
O:MeOH (35 mL:20 mL), giving a dark yellow solid. Yield:
Baliani et al.
8 10 7
((M + H) , 100%). HRMS (ES ): calcd mass for (C H N
S)+
1
+
+
H
2
+
8
3 mg, 51%. Mp: 261-263 °C. LRMS (ES ): m/e 279.2
236.0713, found 236.0717. H NMR (300 MHz, DMSO-d ):
δ 6.38 (br s, 4H), 7.07 (m, 1H), 7.25 (m, 1H), 7.53 (m, 1H),
6
+
+
(
M + H , 50%), 301.1 (M + Na , 100%). HRMS: calcd mass
1
+
13
for (C
9
H
11
8
N O
3
)
6
279.0949, found 279.0951. H NMR (300
8.25 (s, 1H), 10.53 (s, 1H). C NMR (75 MHz, DMSO-d
δ 127.6, 127.9, 129.1, 137.3, 140.2, 164.9, 167.6. Anal. (C
0.7H O) C, H, N, S.
6
):
S‚
MHz, DMSO-d
): δ 2.77 (s, 3H), 6.77 (bs, 3H), 7.07 (s, 1H),
8 9 7
H N
1
3
7
.78 (d, J ) 4.02), 8.01 (s, 1H), 11.10 (s, 1H). C NMR
75 MHz, DMSO-d ): δ 27.6, 114.0, 115.6, 129.5, 151.8, 153.3,
‚0.4H O‚0.02HCl) C, H, Cl; calcd: N,
2
(
6
4-Nitrobenzaldehyde (4,6-diamino)-[1,3,5]-triazin-2-yl-
hydrazone (7a). Compounds 4a (328.23 mg, 2.32 mmol) and
iv-a (357.75 mg, 98%, 2.32 mmol) were suspended in MeOH
(5 mL), and the suspension was left stirring overnight. The
mixture was reduced under vacuum, giving a yellow solid. The
1
66.9. Anal. (C
8.4. Found: N, 37.9.
-Nitro-2-furaldehyde (N, N -Dimethyl-4,6-diamino)-
1,3,5]-triazin-2-ylhydrazone (6e). 5-Nitrofuraldehyde
9
H
10
8
N O
3
2
3
4
4
5
[
2
solid was recrystallized from H O:MeOH (120 mL:200 mL),
(
246 mg, 99%, 1.45 mmol) and 3e (204.56 mg, 1.45 mmol) were
giving a yellow solid. Yield: 493 mg, 77%. Mp: 321-322 °C.
suspended in MeOH (5 mL) and left stirring overnight. The
mixture was reduced under vacuum, giving 120 mg of brown
solid. The solid was recrystallized from H
+
LRMS (EI): m/z 274 (M , 100%). HRMS: calcd mass for
1
(
C
10
H
11
N
6
8
O
2
)+ 275.0999, found 275.1004. H NMR (300 MHz,
2
O:MeOH (30 mL:
DMSO-d
(s, 1H), 8.28 (d, J ) 8.78 Hz, 1H), 10.98 (s, 1H). C NMR
(75 MHz, DMSO-d ) δ 124.4, 127.3, 139.3, 142.0, 147.3, 165.1,
) δ 6.50 (br s, 4H), 7.86 (d, 2H, J ) 8.78), 8.18
2
2
(
0 mL), giving a light brown solid. Yield: 336 mg, 80%. Mp:
13
+
+
64-266 °C. LRMS (ES ): m/z 293.1 ((M + H) , 20%), 315.1
+
+
M + Na , 100%). HRMS: calcd mass for (C10
H
13
N
8
O
3
)
6
6
1
10 10 8 2 2
167.7. Anal. (C H N O ‚1.1H O‚0.03HCl) C, N, Cl calcd:
2
93.1105, found 293.1110. H NMR (300 MHz, DMSO-d
):
H, 4.2. Found: H, 3.4.
δ 3.11 (s, 6H), 6.75 (br s, 3H), 7.11 (d, 1H, J ) 3.84), 7.82
1
3
3-Nitrobenzaldehyde (4,6-Diamino)-[1,3,5]-triazin-2-
ylhydrazone (7b). Compounds 4a (331.6 mg, 2.35 mmol) and
iv-b (358.71 mg, 98%, 2.35 mmol) were suspended in MeOH
(
d, J ) 3.84), 8.08 (s, 1H), 11.21 (s, 1H). C NMR (75 MHz,
DMSO-d ): δ 36.1, 113.8, 115.6, 129.4, 151.8, 153.4, 164.5,
66.0, 167.5. Anal. (C10 ‚0.02HCl) C, H, N, Cl.
-Furaldehyde (4,6-Diamino)-[1,3,5]-triazin-2-ylhydra-
6
1
12 8 3
H N O
(
5 mL), and the suspension was left stirring overnight. The
2
mixture was reduced under vacuum, giving a green solid. The
zone (6f). A mixture of 4a (150 mg, 1.06 mmol) and 2-fur-
aldehyde (102.88 mg, 1.06 mmol) was suspended in MeOH (5
mL). The suspension was left stirring overnight at room
temperature. The mixture was filtered and the brown solid
was washed with methanol and then dried under vacuum at
2
solid was recrystallized from H O:MeOH (170 mL:50 mL),
giving a light green solid. Yield: 83 mg, 13%. Mp: > 345 °C.
+
LRMS (EI): m/z 274 (M , 100%). HRMS: calcd mass for
+
1
10 11 8 2
(C H N O ) 275.0999, found 275.0999. H NMR (300 MHz,
DMSO-d
6
): δ 6.50 (bs, 4H), 7.72 (m, 1H), 8.01 (d, 1H), 8.20
4
0 °C. The crude product was recrystallized from H
2
O/MeOH
13
(
m, 2H), 8.47 (s, 1H), 10.85 (s, 1H). C NMR (75 MHz, DMSO-
(
5%) to give a pure brown solid. Yield: 133 mg, 58%. Mp: 281-
+
+
+
d
6
): δ 120.0, 123.3, 130.7, 133.3, 137.4, 139.4, 148.6, 165.2,
2
83 °C. LRMS (ES ): m/z 219 (M + H) , 241.9 (M + Na) .
+
167,7.
HRMS: calcd mass for (C
H N
8 10 7
O) 220.0941, found 220.0941.
): δ 6.38 (bs, 4H), 6.57 (m, 1H),
.69 (d, J ) 3.29 Hz, 1H), 7.76 (m, 1H), 7.94 (m, 1H), 10.56
1
2-Nitrobenzaldehyde (4,6-Diamino)-[1,3,5]-triazin-2-
H NMR (300 MHz, DMSO-d
6
ylhydrazone (7c). Compounds 4a (328.23 mg, 2.32 mmol) and
iv-c (357.75 mg, 98%, 2.32 mmol) were suspended in MeOH
6
(
1
3
s, 1H). C NMR (75 MHz, DMSO-d
44.3, 150.5, 165.0, 167.7.
-Cyano-2-furaldehyde (4,6-Diamino)-[1,3,5]-triazin-2-
6
): δ 111.5, 112.2, 131.8,
(
5 mL), and the suspension was left stirring overnight. The
1
mixture was reduced under vacuum, giving a yellow solid. The
5
2
solid was recrystallized from H O:MeOH (120 mL:80 mL,)
ylhydrazone (6g). Compounds 4a (98.7 mg, 0.70 mmol) and
crude 5c (85 mg, 0.70 mmol) were mixed together and
suspended with MeOH (3 mL), and the suspension was left
stirring overnight. The mixture was reduced under vacuum,
giving a light brown solid. The solid was recrystallized from
giving a yellow solid. Yield: 107 mg, 17%. Mp: 288-291 °C.
+
LRMS (EI): m/z 274 (M , 100%). HRMS: calcd mass for
)+ 275.0999, found 275.1001. H NMR (300 MHz,
1
(
C
10
H
11
N
6
8
O
2
DMSO-d
): δ 6.45 (bs, 4H), 7.62 (m, 1H), 7.78 (m, 1H), 8.09
13
(
m, 2H), 8.48 (m, 1H), 10.98 (s, 1H). C NMR (75 MHz, DMSO-
2
H O:MeOH (10 mL:15 mL), giving a light brown solid. Yield:
d
6
): δ 124.9, 127.8, 129.8, 129.9, 133.7, 136.5, 147.9, 165.2,
67,7.
-
1
+
2
2 mg, 13%. Mp: >350 °C. IR: νCN 2229.7 cm . LRMS (ES ):
1
+
+
m/z 245 ((M + H) , 100). HRMS: calcd mass for (C
9
H
9
N
8
O)
45.0894, found 245.0893. H NMR (300 MHz, DMSO-d ):
δ 6.52 (bs, 4H), 6.96 (d, J ) 3.76 Hz, 1H) 7.71 (d, J ) 3.76 Hz,
H), 8.04 (s, 1H), 10.95 (s, 1H). 13C NMR (75 MHz, DMSO-
): δ 111.8, 112.3, 124.7, 125.5, 129.7, 155.3, 165.0, 167,7.
-Nitrothiophene-2-carbaldehyde (4,6-Diamino)-[1,3,5]-
1
2
6
Acknowledgment. We would like to acknowledge
the Welsh School of Pharmacy (A.B.), the Wellcome
Trust (G.J., M.S.), and the UNDP/World Bank/WHO
Special Program for Research and Training in Tropical
Diseases (R.B.) for funding. We also would like to
acknowledge the EPSRC National Mass Spectrometry
Service Centre for the accurate mass spectroscopy.
1
d
6
5
triazin-2-ylhydrazone (6h). A mixture of 4a (150 mg,
1
.06 mmol) and 5-nitro-2-thiophenecarboxaldehyde (166.58 mg,
.06 mmol) was suspended in methanol (5 mL). The suspension
1
was left stirring for 2 days at room temperature. The reaction
mixture was filtered and the brown precipitate was washed
with MeOH and then dried under vacuum at 40 °C. The crude
product was recrystallized from water and ethanol (5:1) to give
a pure brown solid. Yield: 75.7 mg, 25%. Mp: >350 °C. LRMS
Supporting Information Available: Experimental pro-
cedures and spectral data for compounds 2-5 and micro-
analytical data. This material is available free of charge via
the Internet at http://pubs.acs.org.
+
+
+
(
ES ): m/z 281 ((M + H) , 100%), 303 ((M + Na) , 10%).
References
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+
+
HRMS (ES ): calcd mass for (C
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H
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N
8
O
2
S) 281.0569, found
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Yield: 95 mg, 38%. Mp: 309-310 °C. LRMS (ES ): m/z 236

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JM050177+