Novel functionalized melamine-based nitroheterocycles: Synthesis and activity against trypanosomatid parasites

Article abstract of DOI:10.1039/b813394h

Human African trypanosomiasis (HAT), caused by the protozoan parasite Trypanosoma brucei spp., is a major health problem in sub-Saharan Africa. New drugs are urgently required for the disease. Selective uptake of toxic compounds into trypanosomes has been achieved by exploiting plasma membrane transporters. For example, the P2 aminopurine transporter, along with other transporters, selectively concentrates melamine and benzamidine moieties into trypanosomes. We have previously reported the use of the melamine motif to selectively target nitrofuran to the trypanosome. In this paper we report the further investigation of the structure activity relationships and the effect of the introduction of different functionalized substituents onto the melamine unit. Most of the compounds tested in vitro for their trypanocidal activity showed activities in the submicromolar range against T. b. rhodesiense. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b813394h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Novel functionalized melamine-based nitroheterocycles: synthesis and activity
against trypanosomatid parasites†‡
Alessandro Baliani,a,b Valerie Peal,b Ludovic Gros,a Reto Brun,c Marcel Kaiser,c Michael P. Barrettd and
Ian H. Gilbert*a,b
Received 5th August 2008, Accepted 2nd December 2008
First published as an Advance Article on the web 28th January 2009
DOI: 10.1039/b813394h
Human African trypanosomiasis (HAT), caused by the protozoan parasite Trypanosoma brucei spp., is a
major health problem in sub-Saharan Africa. New drugs are urgently required for the disease. Selective
uptake of toxic compounds into trypanosomes has been achieved by exploiting plasma membrane
transporters. For example, the P2 aminopurine transporter, along with other transporters, selectively
concentrates melamine and benzamidine moieties into trypanosomes. We have previously reported the
use of the melamine motif to selectively target nitrofuran to the trypanosome. In this paper we report
the further investigation of the structure activity relationships and the effect of the introduction of
different functionalized substituents onto the melamine unit. Most of the compounds tested in vitro for
their trypanocidal activity showed activities in the submicromolar range against T. b. rhodesiense.
nucleosides/nucleobases to the P2 transporter which was later
expanded using inhibition data collected from a series of potential
Introduction
substrates (De Koning and Jarvis).9 The recognition motif for the
P2 transporter is: an amidine moiety, an aromatic ring and an
electronegative heteroatom, as found in the so-called melamine
and benzamidine motifs.
Interestingly De Koning revealed the presence of two additional
transporters specific for pentamidine whose physiological roles are
still unknown: the high affinity pentamidine transporter (HAPT1)
and the low affinity pentamidine transporter (LAPT1).10,11 Fur-
thermore, experiments indicate that melamine-arsenicals also have
uptake routes in addition to the P2 transporter,12 with HAPT1
being a likely candidate.13 The novel diamidine, furamidine, also
appears to enter cells via the P2 and other transporters.14
Parasitic organisms belonging to the order Kinetoplastida are
responsible for several diseases affecting humans such as Human
African Trypanosomiasis, Chagas’ disease and leishmaniasis.1
Human African trypanosomiasis (HAT) or sleeping sickness is
a serious public health threat in 36 countries of sub-Saharan
Africa where it is estimated that 60 million people are at risk
for contracting the disease. HAT is caused by subspecies of
Trypanosoma brucei (T. b. gambiense and T. brucei rhodesiense), a
parasitic protozoan transmitted by the tsetse fly. Infection is fatal if
untreated. Current drug therapy is inadequate due to serious side
effects, poor efficacy, undesirable routes of administration and an
increase of drug resistance.2
T. brucei lacks the ability to synthesise important nutrients for
their survival. These include purines which they need to import
from the host. A number of different nucleoside/nucleobase
transporters have been identified in T. brucei.3,4 One of these,
called the P2 transporter, was characterised in T. brucei by Carter
and Fairlamb in 1993.5,6 In addition to uptake of adenosine and
adenine, it also mediates the uptake of other motifs such as
melamines (e.g. melamine-arsenical drugs) and benzamidines. The
P2 transporter differs substantially from the human mammalian
equilibrative nucleoside transporters.7,8 Carter and Fairlamb2
formulated a structure activity relationship for the binding of
It is clear that both the melamine and benzamidine moieties
are selectively concentrated in T. brucei and we have been
trying to exploit this for selective delivery of compounds to
T. brucei by coupling trypanotoxic agents to the P2 motif.15 In
our previous work we have described the trypanocidal activity
of melamine compounds linked to nitroheterocycles such as
nitrofuran. Nitrofurans were chosen because they are known for
potent antimicrobial/trypanocidal activities.16–18 Benznidazole (a
nitroimidazole) and nifurtimox (a nitrofuran) are used for the
treatment of Chagas’ disease and nifurtimox is also under clinical
trial for the treatment of HAT.19,20
Previous work had identified some melamine-nitroheterocycles
which showed good selectivity and caused potent inhibition of
growth of T. b. rhodesiense.21 The lead compound was compound
5a (Fig. 1). This compound and several analogues were also able to
cure the rodent STIB795 model of human African trypanosomi-
asis. These structure activity relationship (SAR) studies indicated
that it was possible to derivatise one of the melamine amino groups
with a methyl and retain in vivo activity. Interestingly, multiple
derivatisation of the melamine amino groups with methyls led to
increased in vitro activity, but reduced in vivo activity. In this paper
we report further SAR studies on the lead compound; in particular
we decided to investigate the substituent on the amino group and
aSchool of Life Sciences, Division of Biological Chemistry and Drug
Discovery, College of Life Sciences, University of Dundee, Sir James Black
Centre, Dundee, DD1 5EH, UK. Fax: +44 (0) 1382 386 373; Tel: +44 (0)
1382 386 240
bWelsh School of Pharmacy, Cardiff University, King Edward VII Avenue,
Cardiff, CF10 3XF, UK
cSwiss Tropical Institute, Socinstrasse 57, CH-4002, Basel, Switzerland
dUniversity of Glasgow, Glasgow Biomedical Research Centre, Institute of
Biomedical and Life Sciences, Glasgow, G12 8TA, UK
† This paper is dedicated to Professor Andrew Holmes on the occasion of
his 65th birthday.
‡ Electronic supplementary information (ESI) available: Further experi-
mental details and NMR spectra. See DOI: 10.1039/b813394h
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most organic solvents and were purified by recrystallisation from
ethanolic or methanolic water solutions. Cleavage of the silyl
protecting group in hydrazones 5e–5f could not be achieved as
decomposition occurred. The solubility of hydrazone 5g in organic
solvents was found to be dramatically improved compared to the
hydrazone lead compound 5a. The compound was purified by
flash column chromatography (CH2Cl2:MeOH gradient to 95/5)
and this represents a big advance as previously the solubility of
some hydrazones made their purification problematic.
The purification of 5h–5j was difficult to achieve and ion
exchange chromatography was found to be the best approach
using Dowex 50WX8.200 resin. Water/HCl/methanol mixtures
as eluent gave rise to formation of the methyl ester of 5h. Replacing
methanol with acetonitrile avoided this problem; however further
by-product formation was observed using larger amounts of
acetonitrile. The first fractions off of the column contained pure
product with the unwanted impurities eluting later and from this
we can conclude that although the method is time consuming, with
care it can work. Compound 5k was purchased from Aldrich. In
this compound both the amino groups of the triazine moiety bear
one ethyl substituent.
Fig. 1 Structure of the lead compound 5a and planned modifications.
also the effect of varying the nitroheterocycle. The aim of these
studies was to alter the physicochemical properties of the lead
(especially solubility) and potency, thus increasing the spectrum of
pharmacological properties associated with these trypanotoxins.
The modifications can be summarised as follows:
- introduction of alkyl substituents to the melamine to alter the
lipophilicity of the molecule;
- introduction of hydroxyl and carboxylic acid and ester
derivatives to the melamine to obtain a series of derivatives with a
wide range of partition coefficient values;
Further information could be obtained by generating ester
derivatives (6a–6h) in order to determine their effects on uptake
and activity. The acetyl ester of 5g was obtained in good yield with
acetic anhydride and DMAP in pyridine. For the esterification
of compounds 5h–5j it was envisaged this could be cleanly
carried out using HCl(g) bubbled through the carboxylic acid
starting material and the desired alcohol. Although this certainly
proved to be the case for the first compound prepared (6b), our
subsequent attempt to prepare compound 6c via this route met
with considerable problems due to the difficulties in removing
the large volume of alcohol used. With these problems in mind,
subsequent esterifications (6c–6h) were carried out in alcohol using
SOCl2 (Scheme 1).
- introduction of small substituents on the hydrazone bridge
and effect of the modification of the geometry of the hydrazone;
- replacement of 5-nitrofuran moiety with nitro-imidazoles;
- effect of the replacement of the nitro group.
Chemistry
The chemical start point was cyanuric chloride (1) which was con-
verted to the key intermediate 2-amino-4,6-dichloro[1,3,5]triazine
(2) (Scheme 1). The first amino group was introduced by adding
ammonia in water solution at 0 ◦C which displaced the first
chlorine affording (2).21
Displacement of one of the chloro groups of 2 with a variety of
amines allowed introduction of different substituents. Thus, 2,4-
bis-substituted-6-chloro-1,3,5-triazines 3c and 3d were obtained
by reaction of 2 with propylamine and butylamine in the presence
of sodium hydroxide. The insertion of the hydroxyl-alkyl chain was
found to be more problematic. When ethanolamine was used with
the conditions above, the outcome of the reaction was not clear;
hence we decided to protect the hydroxyl group with TBDPSiCl
to facilitate regioselectivity and to increase the lipophilicity to
aid with purification. Ethanolamine and propanolamine were
protected and then successfully reacted with 2 to afford 3e
and 3f using the same conditions. The insertion of unprotected
propanolamine to give 3g was less problematic than observed for
ethanolamine in water/acetone. The original yield of 3g (31%)
was improved to 91% by replacing the solvent acetone/water with
DMF at -20 ◦C and using DIPEA as the base. The carboxylic acid
substituted triazine products 3h–3j were obtained in good yields
by displacement with the relevant amino acids.
Another series of modifications we carried out on our lead struc-
ture involved the nitrofuran moiety. The hydrazones synthesised
(9a–9f) are shown on Scheme 3. The nitro group was replaced
with a carboxylic acid function (9a). The carboxylate should be
an isostere for the nitro group and should remove any possible
problems connected with toxicity of a nitroheterocycle. We also
were interested to see the effect of a substituent on the hydrazone—
this is a point of substitution that we have not investigated, that
might be useful as a handle for altering the physicochemical
properties of the molecule (9b–d). Finally we wanted to see the
effect of changing a nitrofuran to a nitroimidazole (9e–f); this
should have an effect on the redox potential of the nitroheterocycle.
Hydrazone 9a was obtained by treatment of 4a with
the commercially available 5-formyl-2-furancarboxylic acid (8a,
Scheme 3). The other nitroheterocycles were prepared as shown in
Scheme 2 (8b–8d).
Nitration of 2-acetylfuran (7b) after different attempts, was
carried out with concentrated nitric acid in acetic anhydride as
a solvent, and a base such as triethylamine.22,23 After the coupling
with 4a using methanol and molecular sieves overnight at room
temperature two stereoisomers E (9c) and Z (9b) were generated,
with an overall yield of 50%. The imidazole-carboxaldehyde
derivatives (7d, 7e Scheme 2) were also nitrated using HNO3 70%
at room temperature for a short period of time (30 minutes) giving
the corresponding nitrated products (8d, 8e) in quantitative yields.
The displacement of the last chlorine is more difficult to
achieve and requires more harsh conditions. Hydrazine-triazines
4a–4j were achieved in good yields by refluxing the triazines
3a–3j with hydrazine hydrate in water/ethanol. For the triazine
derivative 4g, DMF was used at 85 ◦C. Hydrazones 5b–5k
were obtained by reaction of the hydrazine triazines 4a–4k with
5-nitrofuraldehyde. Hydrazones 5a–5d showed low solubility in
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Scheme 1 Reagents and conditions: (i) NH4OH, acetone, 0 ◦C to rt; (ii) 3b–3f. RNH2, NaOH 2 N, acetone/H2O, rt; 3g. R1NH2, -20 ◦C to rt, DIPEA,
DMF; 3h–3j. R1NH2, 2 ◦C to rt, NaHCO3, EtOH; (iii) 4a–4e. NH2NH2, H2O, reflux; 4f, 4a–4j, NH2NH2, EtOH, reflux; 4g.NH2NH2, DMF; (iv) 5a–5k,
nitrofuraldehyde, MeOH, rt; (v) 6a. Ac2O, Py, DMAP, 0 ◦C to rt; 6b, 6d, 6f. SOCl2, EtOH, rt; 6c, 6e, 6g. SOCl2, BnOH, rt; 6 h. SOCl2, BnOH, rt. The
preparation of compound 5a has been reported previously.21
Hydrazones corresponding compounds 9e and 9f were obtained in
methanol with the conditions described above and the purification
was achieved by reverse phase chromatography.
(rat skeletal myoblast cells) to give an indication of selectivity of the
compound for the parasite (Table 1). The following comments can
be made on the structure activity relationships, with comparison
to the lead compound 5a.
Alkyl groups on one of the amines (5b–d) did not affect the
trypanocidal activity compared to the lead compound 5a, al-
though there was a drop in selectivity with substantially increased
toxicity to the mammalian L6 cell line apparent. Substitution of
ethyl onto two of the amines (5k) led to drop in activity of about
10 fold.
Biology
Activities against T. brucei in vitro
Compounds were evaluated for their in vitro activities against
T. brucei rhodesiense lines, with a counterscreen against L6 cells
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the mammalian cells (1700 fold). When the hydroxyl function was
acetylated (6a) both activity and the selectivity were retained. It is
possible that in the conditions of the cellular assay the acetate was
rapidly cleaved by esterases. Derivatisation of the hydroxypropyl
with a tert-butyl diphenylsilyl group also led to retention of
activity. It is conceivable that the silyl group is also labile under
the assay conditions. Alternatively, the molecular target is tolerant
of hydrophobic substituents at this position. Interestingly, the
silylated hydroxyethyl derivative (5e) was about 5-fold less active
that the corresponding silylated hydroxypropyl derivative.
Compounds with a free carboxylic acid (5h–j) were markedly
less active than their alkyl substituted counterparts (IC50 28 mM,
8 mM, 0.8 mM). Interestingly as the alkyl chain length was
increased, the potency increased, with the hexanoyl derivative
being approximately 30x more active than the propionyl derivative.
Esterification of the propionyl derivative (5h) to give the ethyl
ester (6b) led to a significant (400 fold) increase in potency. This
was also found for the ethyl ester (6d) of the butanoyl derivative
(5i), where there was a 17 fold increase in activity. However for
the hexanoyl derivative there was no effect on converting the
free acid (5j) to the ethyl ester (6f). Interestingly, the benzyl
esters of the propionyl (6c) and butanoyl (6e) derivatives were
significantly less active than the corresponding ethyl esters (6b
and 6d). With the hexanoyl series, the free acid (5j), the methyl
ester (6h), the ethyl ester (6f) and the benzyl ester (6g) were all
equipotent and had broadly similar levels of selectivity compared
to L6 cells. Interestingly these 4 compounds are predicted to have
Scheme 2 Reagents and conditions: (i) and (ii): 1) HNO3 70%, Ac2O 0 ◦C,
2) TEA, rt 3 h; (iii), (iv): HNO3, 70%, 30 min, rt.
The introduction of a hydroxypropyl chain (compound 5g)
maintained the activity against T. b. rhodesiense in the nanomolar
range (IC50 49 nM) together with a good selectivity compared to
Scheme 3 Reagents and conditions: (i) MeOH, rt, 6 h; (ii–v) MeOH, mol. sieves, overnight.
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Table 1 In vitro activities of compounds against bloodstream T. brucei, and mammalian cells as a measure of toxicity
Substituents
T. brucei rhodesiense (IC50 [mM])
L6 cells (IC50 [mM])
Selectivity
LogD (pH 7.4)
5a
R1, R2, R3=H
R1= -Pr
0.025
0.023
0.065
0.025
0.17
0.037
0.049
27.7
8.0
0.81
0.19
0.049
0.07
0.4
0.46
6.7
0.93
0.86
1.3
285
61
183
17
5.5
18
0.49
7.4
7320
752
84
733
3
199
1710
10
-1.9
-0.5
-0.7
0.05
4.9
5b
5c
R1= -i-Pr
R1= -Bu
5d
t
5e
R1= -(CH2)2OSiPh2 Bu
t
5f
R1= -(CH2)3OSiPh2 Bu
5.1
5g
R1= -(CH2)3OH
R1= -(CH2)2CO2H
R1= -(CH2)3CO2H
R1= -(CH2)5CO2H
R1= Et, R2= Et
R1= -(CH2)3OAc
R1= -(CH2)2CO2Et
R1= -(CH2)2CO2Bn
R1= -(CH2)3CO2Et
R1= -(CH2)3CO2Bn
R1= -(CH2)5CO2Et
R1= -(CH2)5CO2Bn
R1= -(CH2)5CO2Me
X= 2-carboxyl-furan-5-yl-
R3= -Me (Z)
84
-2.0
-4.6
-4.2
-3.6
-0.2
-1.1
-0.8
0.5
-0.5
0.7
0.2
1.2
-0.5
-5.0
-1.9
-1.9
-0.8
NA
NA
-0.2
0.3
5h
268
257
>238
36
5i
5j
32
294
187
1523
1567
332
162
17
167
>223
145
>1
>5
>3
1292
>3
>21
59
5k
6a
75
6b
110
133
75
6c
6d
6e
116
155
>192
187
>342
>324
>324
96.9
>341
>341
40
6f
6g
6h
9a
9b
9c
R3= -Me (E)
96
9d
R3= -n-Pr (E, Z)
X= 4-nitro-imidazol-2-yl-
X= 2-nitro-imidazol-5-yl-
NA
0.075
118.2
16.32
0.68
2.3
0.006
1.5
9e
9f
10
11
NA
NA
NA
20
7.8
68
9
Melarsoprol
Nifurtimox
1300
45
NA = not applicable, X = 5-nitrofuran-2-yl when not specified, R1, R2 and R3 are H when not specified. LogD values were calculated using the ACD
software. Data for compound 5a have been reported previously—references 21 and 24.
widely varying degrees of lipophilicity; assuming the same mode
of action, their equipotency is consistent with some kind of active
transport.
Replacement of the nitro group with the carboxylic acid (9a)
led to a large reduction of activity. The carboxylate should
be an isostere for the nitro group, in terms of its size, steric
properties and H-bond acceptor properties. However, like other
replacements that we have investigated previously (hydrogen and
nitrile)21 it was inactive. This implies that the redox properties of
the nitro(heterocycle) are critical for anti-trypanosomal activity.
Attachment of a substituent to the hydrazone linker may
provide another point for variation of the activity, selectivity
and physicochemical properties of the compounds. The methyl
substituent was isolated as the two stereoisomers 9b and 9c. The
stereochemistries of these were assigned by NOESY experiments.
With the propyl derivative, the stereoisomers were much more
difficult to separate, and the compound was investigated as
the mixture of stereoisomers (60/40). Compounds 9b and 9c
showed very little activity, although it was noted that they were
both very insoluble. The propyl derivatives, however, were potent
trypanocides (IC50 of 75 nM).
The SAR of this series of compounds does not lend itself to
simple explanation. For the carboxylate derivatives (5h–j) as the
chain length was increased, the activity increased. One possible
explanation for this is that the carboxylate group makes a specific
interaction with a molecular target. Another possibility is that an
increase in chain length could give more lipophilic compounds
with greater cellular permeability. However, interestingly as the
chain length was increased, the calculated logD value did not
increase significantly, suggesting that the increase in activity was
probably not due to a significant increase in passive diffusion.
Indeed the logD values suggested that uptake was probably not
primarily by passive diffusion, but by active transport. When
considering the esters of the carboxylate series, (6b–h) ethyl
esters were generally more potent that the more lipophilic benzyl
esters. The lack of clear correlations probably relates to the
fact that overall activity is multi factorial, involving uptake
at the cellular membrane, possible partitioning into different
cellular compartments, thus crossing additional membranes, and
interaction with cellular targets.
Replacing the nitrofuran with a nitroimidazole (9e and 9f) led
to a drastic loss in activity. Low activity was also observed when
nitroimidazoles were linked to a benzamidine moiety.24 In this
case, the nitroimidazole is conjugated to the melamine ring in a
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Table 2 In vivo activities
STIB 795 (T. b. brucei)
STIB 900 (T. b. rhodesiense)
Compound
Dose (mg/Kg) ip
Cured/infected
Survival average (days)
Dose (mg/Kg) ip
Cured/infected
Survival average (days)
Control
5a
—
0/4
4/4
4/4
4/4
3/4
0/4
4/4
0/4
4/4
ND
6
>60
>30
>30
>30
>27.25
>30
5.75
>30
ND
—
0/4
1/4
0/4
1/4
1/4
ND
ND
ND
ND
0/4
8
4 ¥ 20
4 ¥ 50
4 ¥ 50
4 ¥ 50
4 ¥ 50
4 ¥ 50
4 ¥ 40
4 ¥ 50
ND
4 ¥ 20
4 ¥ 50
4 ¥ 50
4 ¥ 50
ND
ND
ND
35.25
16.75
>26.75
>40.75
ND
ND
ND
5b
5c
5d
5f
5g
5j
6a
ND
4 ¥ 50
ND
6d
8.75
similar fashion to the active nitrofurans. One possible explanation
for the loss in activity is the probable change in redox potential
with nitroimidazoles being more difficult to reduce.
effect and, moreover, be generally cell permeant due to increased
hydrophobicity.
To summarise the SAR, simple substituents led to high cellular
potency. The carboxylic acid derivatives reduced activity. Ester-
ification of the carboxylic acid improved potency, but this was
not as significant when the chain length of the carboxylic acid
increased. There was no marked correlation between lipophilicity
and activity.
Conclusions
We have reported the synthesis of new functionalised melamine
derivatives as novel potential drugs for the treatment of human
African trypanosomiasis. We have also investigated the structure
activity relationship and the effect of the introduction of func-
tionalised groups onto the melamine moiety of the compounds.
Derivatisation at one of the amino groups of the melamine is
possible, creating compounds that retain anti-parasite activity in
cellular and rodent models. Complex structure activity relation-
ships exist, indicative of the multifactorial nature of drug action
(uptake at the plasma membrane, possible crossing of organellar
membranes and interaction with molecular targets all playing
roles). Compounds 5c and 5d are particularly interesting, since
they were potently anti-trypanosomal in cell culture and also
in rodent models of infection, indicating that pharmacological
properties were sufficiently good to allow the compounds to
remain present at trypanocidal concentrations long enough to
be effective in vivo. In one case, substitution of the hydrazone led
to a compound with significant activity in vitro, which indicates a
possible point for modification of the physicochemical properties
of the molecule.
Given their potent in vitro activity against T. brucei, some
selected compounds were tested in two different rodent models
of infection: STIB 795 T. brucei brucei infection model and the
more stringent STIB 900 T. brucei rhodesiense model. Table 2
shows a summary of the in vivo activities for the tested compounds.
Previously published data21,24 for the lead compound 5a are shown,
for comparative purposes. Compounds 5b, 5c, 5g and 6a were
able to cure all the mice infected with the STIB 795 model of
infection when treated at a dose of 50 mg/Kg intraperitoneally
for 4 days. The mice were considered cured when no infection was
found after 30 days. No overt sign of toxicity was found in these
mice. Compound 5d also cured 3 of the 4 mice infected with the
same model of infections. Compounds 5c and 5d were also able to
cure one of the 4 mice infected with the more stringent model of
infection STIB 900 and 5b showed a significant increase in survival
time.
Attempts to replace the nitro group have, to date, failed to yield
compounds with substantial trypanocidal activity, indicating that
the nitro group is necessary for activity. Moreover, we investigated
the possibility of replacing the nitrofuran trypanocidal moiety
with other nitroheterocycles. Straight replacement of the nitrofu-
ran with a nitroimidazole appears to cause a significant drop in
activity. This may well be due to a significant change in the redox
potential of the nitroheterocycle.
The derivatives with one alkyl group retained in vivo activity,
indicating that there is some room for variation, if there is one
substituent on just one of the melamine amino groups. The silyl
derivative was slightly less active. However the acid (5j) and ester
(6d) derivatives lost activity. This is probably due to a combination
of slightly lower activity against the parasites (as shown by the
cellular assays) and pharmacokinetic reasons.
Compounds were also tested for their activities against the other
kinetoplastid parasites T. cruzi, L. donovani and the apicomplexan
Plasmodium falciparum (Table 3). In general the activities were
lower than against T. b. rhodesiense. This is not surprising, as
the compounds were designed for selective uptake into T. brucei
and are quite polar and may not cross cell membranes readily by
passive diffusion. However, several of the compounds showed sub-
micromolar activity against T. cruzi; 5d, 5f and 6d were particularly
potent (IC50 <0.26 mM). This is significant, given the intracellular
location of the parasites, which makes it more difficult to access
and kill them. Compound 5f was also active against L. donovani
and P. falciparum. Given the relatively low IC50 against L6 cells,
this suggests that this compound may have a general cytotoxic
Experimental
Cell assays and in vivo assays
The protocols for these assays have been described previously.21
General
Normal phase tlc was carried out on pre-coated silica plates
(Kieselgel 60 F254, BDH), whilst reverse phase tlc was carried on
Merck RP-18F reverse phase silica coated aluminium plates with
1
visualisation via U.V. light and/or ninhydrin solution. H-NMR
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Table 3 Activities against T.cruzi, Leishmania donovani and Plasmodium falciparum
Compound
MW
T. cruzi (IC50 [mM])
Leishmania donovani (IC50 [mM])
P. falciparum (IC50 [mM])
L6 cells (IC50 [mM])
5a
5b
5c
5d
5e
5f
264.28
306.28
306.28
320.31
546.65
560.68
322.28
336.26
350.29
378.34
320.31
364.32
364.32
426.39
378.34
440.41
406.4
468.19
392.37
263.21
278.23
278.23
306.28
264.20
264.20
156.1
2.1
22.17
0.98
0.26
36.76
0.13
64.85
>89.2
85.6
12.2
0.43
70.54
0.8
0.2
0.59
1.04
1.42
12.3
2.42
>12.5
2.38
2.63
1.25
1.64
0.54
18.37
41.9
38.3
22.6
2.18
11.31
9.6
2.9
2.96
3.59
2.47
1.45
0.24
4.06
>14.9
>14.3
>13.2
6.58
4.72
9.2
183
17.33
5.45
18.32
0.49
7.38
5g
5h
5i
83.77
267.6
256.9
>238
35.6
74.65
109.7
132.97
74.53
116.02
155.02
>192
186.8
>341.9
>323.5
>323.5
96.9
5j
5k
6a
6b
6c
6d
6e
6f
6.3
3.8
6.66
4.08
7.52
7.19
5.63
>114.0
>107.8
28.75
5.97
>113.5
23.8
Tox
2.60
1.03
5.61
4.27
8.66
>19
>18
>18
ND
6g
6h
9a
9b
9c
9d
9e
9f
>114.0
>107.8
>107.8
ND
>113.5
>113.5
ND
>18.9
18.9
4.5
>340.6
>340.6
40.4
10
11
155.1
ND
Tox
18.0
20
spectra were recorded at a Bruker Avance DPX500 spectrometer
300 MHz and 500 MHz spectrometers using the applied solvent
simultaneously as internal standard. Chemical shifts (d) are given
in ppm together with the relative frequency, assignment, the
coupling constants (nJ(H,H)/Hz) and the multiplicity: singlet
(s), broad singlet (bs), doublet (d), triplet (t), quartet, quintuplet,
sextet, septuplet, multiplet (m), broad multiplet (bm). 13C-NMR
spectra were recorded at a Bruker 300 MHz NMR spectrometer
using the applied solvent simultaneously as internal standard.
Chemical shifts (d) are given in ppm. DMSO-d6/TFA was used in
some experiments as solvent for some insoluble compounds and
represents a mixture of DMSO-d6 with two drops of TFA. Extra
peaks associated to tautomeric effects observed in some groups
were indicated with an asterisk (*) after the formula. The 13C
signals for the triazine carbons are quaternary and hence very
weak and this is exacerbated by tautomerism, hence not all
triazine carbon signals are always visible. It is impossible with
the information that we have to assign those that are detected.
In some cases, the numbering and the nomenclature system used
for the structures are not referring to IUPAC nomenclature in
order to aid the NMR interpretation. Elemental analyses were
carried out by MEDAC Ltd. IR spectra were recorded at a
FT-IR spectrometer 1600 from Perkin Elmer using the Diffuse
Reflectance Accessory from Spectra Tech. (Refl.), a potassium
bromide pellet (KBr), or methylene chloride solution of the
compound between potassium bromide single crystals (film). The
band positions were characterised by their wave numbers (n/cm-1),
intensity (strong, medium, weak, broad), and assignment. Mass
spectra were recorded at a Platform II mass spectrometer
(Micromass) from Fisons. High resolution mass spectra were
performed respectively on a Waters ZQ4000 and a Finningan
MAT 95XP at EPSRC National Mass Spectrometry Service
centre in the Chemistry department, University of Wales Swansea,
Swansea, Wales, UK. Purification by column chromatography was
performed on Sorbosil C60A silica-gel-40–60 mm from Merck. In
some cases higher flow rates were maintained by using a slight
pressure. Qualitative thin-layer chromatography (TLC) was done
on pre-coated aluminium sheets Silica gel 60 F254 from Merck.
Compounds were detected either with iodine, ninhydrin or 254 nm
UV-light. Purification by reversed phased was performed on silica
gel 100 C18. In several experiments flash chromatography was
performed using Combiflash Companion and prepacked column
(silica gel and C18 reverse phase) purchased from Redisep e
Presearch. Purification by ion exchange chromatography was
performed on a Dowex 50WX8.200 resin (prewashed with MeOH,
HCl 1 M and water) using HCl gradient (1 M to 2 M) and eluting
first with water and then stepwise increasing amounts of HCl
and MeCN. Melting points were determined with a Gallenkamp
melting point apparatus and are not corrected. Solvents and
reagents were purchased from chemical companies and used
without further purification. Dry solvents were purchased in sure
sealed bottles stored over molecular sieves. Compound 5k was
purchased from Aldrich. Experimental details for the preparation
of compounds 2, 3 and 4 are in the supplementary data.
5-Nitro-2-furaldehyde (N4-propyl-4,6-diamino)-[1,3,5]-
triazin-2-yl-hydrazone (5b)
5-Nitrofuraldehyde (203.7 mg, 99%, 1.43 mmol) and N4-propyl-
4,6-diamino-1,3,5-triazin-2-yl-hydrazine (264 mg, 1.34 mmol)
were suspended in MeOH (5 mL) and left stirring overnight
at room temperature. The mixture was filtered and washed
several times with MeOH. The yellow product was dried over high
vacuum giving 5b (282 mg, 64%) as a pure yellow product; mp 236–
238 ◦C; (Found: C, 41.9; H, 4.5; N, 34.9; Cl, 0.6. C11H14N8O3·0.5
H2O·0.08 HCl requires C, 41.5; H, 4.8; N, 35.2; Cl, 0.9%);
1160 | Org. Biomol. Chem., 2009, 7, 1154–1166
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dH(500 MHz, DMSO-d6) 0.86 (3 H, t, J 7.32, -CH2CH3), 1.49
(2 H, m, -CH2CH3), 3.18 (2 H, m, CH2NH-), 6.41–7.13 (3 H, m,
NH-, NH2), 7.05 (1 H, d, J 3.45, furan-CH-), 7.76 (1 H, d, J 3.45,
hydrazine (170 mg, 0.40 mmol) were suspended in MeOH (5 mL)
and left stirring overnight at room temperature. The mixture was
filtered and washed with cold EtOH and with Et2O. The product
was dried over high vacuum giving 5e (0.020 g, 9%) as a solid;
mp 163–165 ◦C; dH(300 MHz, DMSO-d6) 0.99 (9 H, s, 3 CCH3),
3.48 (2 H, m, -CH2-NH-), 3.73 (2 H, m, -CH2-O-), 6.40–7.00 (3 H,
3 m, NH-, -NH2), 7.43 (6 H, m, Ar-C-H), 7.63 (4 H, m, Ar-C-H),
7.76 (1 H, d, J 3.24, furan-C-H), 7.84 (1 H, d, J 3.24, furan-C-H),
=
=
furan-CH-), 8.00–8.04 (1H, m, CH-), 11.15 (1 H, br s, -NH N-);
dC(125 MHz, DMSO-d6) 8.4 (CH2CH3), 22.3 (CH2CH2CH2), 22.5
(CH2C*H2CH2), 41.7 (CH2NH-), 113.6 (furan-CH), 114.0 (furan-
=
=
C*H), 115.2 (furan-CH), 128.9 ( CH-), 129.2 ( C*H-), 151.4
(furan-C), 152.9 (furan-C), 164.0 (triazine-C), 164.2 (triazine-C*),
166.0 (triazine-C), 166.9 (triazine-C), 167.1 (triazine-C*); m/z
=
=
8.02 (1 H, s, CH-), 11.10 (1 H, br s, -NH N-); dC(75 MHz,
DMSO-d6) 19.1 (CCH3), 26.9 (CCH3), 42.4 (CH2NH-), 62.8
(EI+) 306.2 (M+, 10%); 307.1261 (M + H+. C11H15N8O3 requires
+
=
307.1262).
(-CH2O-), 114.4 (furan-CH), 115.6 (furan-CH), 120.1 ( CH-),
128.2 (Ar-CH), 130.1 (Ar-CH), 133.5 (Ar-CH), 135.4 (Ar-CH),
151.8 (furan-C), 166.6 (triazine-C); m/z (ES+) 547.5 ((M + H)+,
100%); 569.2069 (M + Na+. C26H30N8O4SiNa+ requires 569.2057).
5-Nitro-2-furaldehyde (N4-isopropyl-4,6-diamino)-[1,3,5]-
triazin-2-yl-hydrazone (5c)
5-Nitrofuraldehyde (92.6 mg, 99%, 0.65 mmol) and N4-isopropyl-
4,6-diamino-[1,3,5]-triazin-2-yl-hydrazine (120 mg, 0.65 mmol)
were suspended in MeOH (3 mL) and the suspension left stirring
overnight at room temperature. The mixture was filtered and
recrystallised by EtOH. The yellow product was dried over high
vacuum giving 5c (0.050 g, 25%) as a yellow solid; mp 178–
180 ◦C; (Found: C, 38.6; H, 4.2; N, 29.6; Cl, 4.0. C11H14N8O3·2
H2O·0.2 CH2Cl2·EtOH requires C, 38.5, H, 5.7, N, 29.0, Cl, 3.7%);
dH(500 MHz, DMSO-d6) 1.17 (6 H, d, J 6.22, CH(CH3)2), 4.10
(1 H, m, CH(CH3)2), 7.15–9.18 (3 H, m, NH-, NH2), 7.85 (1 H,
5-Nitro-2-furaldehyde {N4-[3-(t-butyl-diphenylsilyl)-oxy]-propyl}-
(4,6-diamino)-[1,3,5]-triazin-2-yl-hydrazone (5f)
5-Nitrofuraldehyde (81.2 mg, 99%, 0.57 mmol) and N4-{[3-(t-butyl
-diphenylsilyl)-oxy]-propyl}-4,6-diamino-[1,3,5]-triazin-2-yl-
hydrazine (250 mg, 0.57 mmol) were suspended in EtOH (5 mL)
and left stirring at room temperature. The yellow colour turned
into a dark orange colour after 30 min. The mixture was left
stirring overnight at room temperature and then was filtered and
washed with cold EtOH and with Et2O. The product was dried
over high vacuum giving 5f (264 mg, 84%) as a solid; mp >320 ◦C;
dH(500 MHz, DMSO-d6) 0.99 (9 H, s, CCH3), 1.81 (2 H, t, J 6.23,
-CH2NH-), 3.40 (2 H, m, CH2CH2CH2), 3.72 (2 H, t, J 6.23, -
CH2O-), 5.95–6.85 (3 H, br m, 3H, NH-, -NH2), 7.05 (1 H, d, J
3.59, furan-CH), 7.43 (6 H, m, Ar-CH), 7.62 (4 H, m, Ar-CH),
=
m, furan-CH), 8.20 (1 H, s, furan-CH), 8.25–8.30 (1 H, s, CH-),
=
13.0 (1 H, br s, -NH N-); dC(125 MHz, DMSO-d6) 22.0 (C(CH3)2),
=
42.7 (C(CH3)2), 114.4 (furan-CH), 115.5 (furan-CH), 134.4 ( CH-
), 151.7 (furan-C-), 151.8 (furan-C*-), 152.0 (furan-C-); m/z (ES+)
307.11 ((M + H)+, 100%), 329.2 ((M + Na)+, 20%); 307.1258 (M +
H+. C11H15N8O3 requires 307.1262).
+
=
7.77 (1 H, d, J 3.59, furan-CH), 8.05–8.10 (1 H, m, CH-), 11.10
=
(1 H, br s, -NH N-); dC(125 MHz, DMSO-d6) 18.7 (CCH3), 19.6
5-Nitro-2-furaldehyde (N4-butyl-4,6-diamino)-[1,3,5]-
triazin-2-yl-hydrazone (5d)
(CH2NH-), 26.6 (CCH3), 36.9 (CH2CH2CH2), 56.0 (CH2-NH-),
61.5 (-CH2-O-), 115.1 (furan-CH), 115.2 (furan-CH), 127.8 (Ar-
=
C-H), 129.7 ( CH-), 133.2 (Ar-C-H), 134.9 (Ar-C), 151.4 (furan-
5-Nitrofuraldehyde (191 mg, 99%, 1.34 mmol) and N4-butyl-4,6-
diamino-1,3,5-triazin-2-yl-hydrazine (264 mg, 1.34 mmol) were
suspended in MeOH (5 mL) and left stirring overnight at room
temperature. The mixture was filtered and washed several times
with MeOH. The yellow product was dried over high vacuum
giving 5d (317 mg, 73%) as a pure yellow product; mp 226–228 ◦C;
(Found: C, 43.9; H, 4.9; N, 33.5; Cl, 0.7. C12H16N8O3·0.5 H2O·0.05
HCl requires C, 43.5; H, 5.2; N, 33.8; Cl, 0.5%); dH(500 MHz,
DMSO-d6) 0.88 (3 H, t, J 7.28, -CH2CH3), 1.30 (2 H, m,
-CH2CH3), 1.47 (2 H, m, CH2CH2CH2), 3.22 (2 H, m, CH2NH-),
6.58–7.13 (3 H, m, NH-, NH2), 7.04 (1 H, d, J 3.20, furan-CH-),
C), 152.9 (furan-C), 166.1 (triazine-C); m/z (ES+) 561.3 (M + H+,
100%); 561.239 (M + H+. C27H33N8O4Si+ requires 561.2389).
5-Nitro-2-furaldehyde {[N4-(3-hydroxypropyl-amino)-4,6-diamino-
[1,3,5]-triazin]-2-yl}-hydrazone (5g)
Crude
N4-(3-hydroxy-propyl)-2,4-diamino-1,3,5-triazin-2-yl-
hydrazine (1.466 g, 14.72 mmol) was dissolved in MeOH (10 mL)
and 5-nitrofuraldehyde (2.096 g, 99%, 14.72 mmol), previously
dissolved in MeOH (10 mL), was added to the solution. The
solution turned into a suspension after 30 min. The mixture
was left stirring at room temperature for 2 h. The solvent was
then removed under vacuum and the solid purified by flash
chromatography (2 ¥ 200 mL DCM 100%, 2 ¥ 200 mL MeOH
0.5% in DCM, 2 ¥ 200 mL MeOH 1% in DCM) giving 5g
(417 mg, 17%) as an orange solid; (Found: C, 39.7; H, 4.5; N,
33.6; Cl, 1.6. C11H14N8O4·0.2 MeOH·0.2 HCl requires C, 40.0; H,
4.5; N, 33.3; Cl, 2.1%); mp 143–145 ◦C; dH(500 MHz, DMSO-d6)
1.64 (2 H, m, CH2CH2CH2), 3.34 (2 H, m, CH2-NH-), 3.44 (2 H,
m, CH2-OH), 4.46–4.65 (1 H, br m, -OH), 6.32–7.11 (3 H, br m,
NH2, -NH-), 7.05 (1 H, d, J 2.84, furan-CH), 7.77 (1 H, d, J 2.84,
=
7.77 (1 H, d, J 3.20, furan-CH-), 8.00–8.04 (1 H, m, CH-), 11.06
=
(1 H, br s, NH C-); dC(125 MHz, DMSO-d6) 13.7 (CH2CH3),
19.6 (-CH2CH3), 30.6 (CH2CH2CH2), 31.2 (CH2C*H2CH2), 40.0
(CH2-NH-), 113.6 (furan-CH), 114.0 (furan-C*H), 115.2 (furan-
=
=
CH), 128.9 ( CH-), 129.1 ( C*H-), 151.4 (furan-C), 152.9 (furan-
C), 164.0 (triazine-C), 164.2 (triazine-C*), 166.0 (triazine-C),
166.9 (triazine-C), 167.1 (triazine-C*); m/z (EI+) 320.2 (M+, 10%);
321.1415 (M + H+. C12H17N8O3 requires 321.1418).
+
5-Nitro-2-furaldehyde {N4-[3-(t-butyl-diphenylsilyl)-oxy]-ethyl}-
(4,6-diamino)-[1,3,5]-triazin-2-yl-hydrazone (5e)
=
furan-CH), 8.02–8.04 (1 H, m, CH-), 11.08–11.23 (1 H, br s,
1H, -NH N-); dC(125 MHz, DMSO-d6) 32.4 (CH2CH2CH2),
32.7 (CH2C*H2CH2), 36.9 (CH2-NH-), 37.2 (C*H2-NH-),
5-Nitrofuraldehyde (57.0 mg, 99%, 0.40 mmol) and N4-{[3-(t-butyl
-diphenylsilyl)-oxy]-ethyl}-4,6-diamino-[1,3,5]-triazin-2-yl-
=
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58.5 (CH2-OH), 113.7 (furan-CH), 115.2 (furan-CH), 128.9
(C-NO2); dH(500 MHz, DMSO-d6) 1.72 (2 H, m, CH2), 2.24 (2 H,
m, CH2), 3.28 (2 H, m, CH2), 7.81 (1 H, m, furan-CH), 7.83 (s,
=
=
( CH-), 129.2 ( C*H-), 151.5 (furan-C), 152.9 (furan-C), 164.0
(triazine-C), 166.1 (triazine-C), 167.1 (triazine-C); dC(100 ◦C,
125 MHz, DMSO-d6) 34.6 (CH2CH2CH2), 60.7 (CH2-OH), 114.4
=
1 H, N CH), 8.15 (1H, d, J 16.4, furan-CH), 8.20–9.20 (4H, 3 br s,
NH2 + 2 NH), 12.90 (1H, m, CO2H); dC(125 MHz, DMSO-d6)
24.0 (CH2), 30.9 (CH2), 39.6 (CH2), 115.0 and 115.1 (2 furan-CH),
=
(furan-CH), 116.6 (furan-CH), 131.6 ( CH-), 153.5 (furan-C),
=
155.3 (furan-C), 166.3 (triazine-C), 168.4 (triazine-C), 169.2
134.9 (N CH), 151.7 (furan-C), 151.8 (furan-C), 156.2, 156.6 and
(triazine-C); m/z (ES+) 323.12 ((M + H)+, 100%); 323.1211 (M +
158.2 (triazine-C), 174.0 (C O); m/z (ES ) 351(M + H) ; 351.1163
+
+
=
H+. C11H15N8O4 requires 323.1211).
(M + H+. C12H15N8O5 requires 351.1160).
+
+
3-(4-(2-((5-Nitrofuran-2-yl)methylene)hydrazinyl)-6-amino-1,3,5-
triazin-2-ylamino)propanoic acid (5h)
6-(4-(2-((5-Nitrofuran-2-yl)methylene)hydrazinyl)-6-amino-1,3,5-
triazin-2-ylamino)hexanoic acid (5j)
5-Nitro-furaldehyde (721 mg, 5.1 mmol) added to starting material
4h (700 mg, 3.27 mmol) dissolved in MeOH (20 mL) and resultant
yellow suspension was stirred overnight at room temperature. Tlc
analysis (RP C18 plates, 1:1 MeCN:H2O) showed the triazine
starting material (Rf = 0.7) still present. A further aliquot of
5-nitro-furaldehyde (400 mg, 2.8 mmol) was added and the
reaction was stirred at 35 ◦C overnight whereupon tlc analysis
showed complete loss of triazine starting material. The reaction
was cooled to room temperature, filtered, washed with MeOH
(10 ml) and air dried 10 min to yield the crude compound as a
dark brown solid (1.284 g). The crude material was purified by ion
exchange chromatography on DOWEX WX8/200 sulfonic acid
resin eluting with a step wise gradient of water, 0.5 M HCl, 1.0 M
HCl, 1.5 M HCl, (200 mL per step), 2.0 M HCl (the compound
now begins to elute 1000 mL), 5% MeCN in 2.0 M HCl, (400 mL),
10% MeCN in 2.0 M HCl, (400 mL). UV absorbance of each
fraction was measured at 240 nm and the fractions containing the
compound were reduced in batches of 5 and checked by mass spec
analysis before combining to yield the title compound 5h (32%)
as a pale yellow solid; nmax/cm-1 (KBr) 3500–2700 {OH (water),
5-Nitrofuraldehyde (77 mg, 0.5 mmol) was added to starting
material 4j (76 mg, 0.3 mmol) dissolved in MeOH (2.8 mL) with
˚
molecular sieves (4 A) and the resulting yellow suspension stirred
for 24 h at room temperature. Mass spec analysis of the solution
showed the presence of starting material 4j, therefore, more
5-nitrofuraldehyde (25 mg, 0.2 mmol) was added and the resulting
solution stirred a further 4 h at 40 ◦C. Mass spec of the solution
showed the only presence of compound 5j. The solution was left
to cool down to room temperature (1 h), filtered and washed with
MeOH (10 mL) to afford a pure solid as 5j (63 mg, 56%). mp 174–
◦
176 C; nmax/cm-1 (KBr) 3500–2700 {OH (water), CO2H, NH2},
=
=
~2900 (CH2), ~1700 (C O acid), 1641 (C N), 1522, 1354 (C-NO2);
dH(500 MHz, DMSO-d6) 1.29 (2 H, m, CH2), 1.50 (4 H, m, 2 x
CH2), 2.21 (2 H, t, J 7.0, CH2), 3.21 (2 H, m, CH2), 6.2–7.3 (3 H, 3
br s, NH2 and NH), 7.06 (1 H, d, J 3.7, furan-CH), 7.78 (1H, d, J
=
3.7, furan-CH), 8.02 (1H, d, J 17.9, N CH), 11.12 (1 H, m, NH);
dC(125 MHz, DMSO-d6) 24.3 and 29.0 (2 CH2), 26.0 (CH2), 33.6
=
(CH2), 39.7 (CH2), 113.8 and 115.3 (2 furan-CH), 128.8 (N CH),
151.5 (furan-C), 153.0 (furan-C), 164.1, 166.1 and 167.2 (triazine-
+
+
+
=
C), 174.5 (C O); m/z (ES ) 379 (M + H) ; 379.1473 (M + H .
=
=
CO2H, NH2}, ~2900 (CH2), ~1700 (C O acid), 1642 (C N), 1526,
(C14H19N8O5)+ requires 379.1478).
1352 (C-NO2); dH(500 MHz, D2O) 2.5 (2 H, m, CH2), 3.5 (2 H, m,
=
CH2), 7.0 (1 H, m, furan-CH), 7.5 (1 H, s, N CH), 8.1 (1 H, m,
5-Nitro-2-furaldehyde [N4-(3-acetyl-oxy)-propyl]-4,6-diamino-
[1,3,5]-triazin-2-yl-hydrazone (6a)
furan CH); dC(125 MHz, DMSO-d6) 33.3 (CH2), 36.8 (CH2), 114.9
=
(2 furan-CH), 134.9 (N CH), 151.7 (2 furan-C), 156.2 (triazine-
C-NH-alkyl), 172.5 (C O), 187.3 (triazine-C-NHN ); m/z 337
5-Nitro-2-furaldehyde
[N4-(3-hydroxypropyl)-4,6-diamino-
=
=
(M +H)+; 337.1003 (M + H+. C11H13N8O5 requires 337.1014).
[1,3,5]-triazin]-2-yl-hydrazone (117 mg, 0.36 mmol) was
suspended in dry pyridine (5 mL) and acetic anhydride (39.8 mg,
d = 1.08, 0.04 mL, 0.39 mmol) was added to the suspension
at 0 ◦C. The mixture was left stirring for 30 min and DMAP
(catalytic, 10 mg) was then added. The temperature was left
stirring at room temperature for 20 min. The reaction was
quenched with MeOH and the solvent was removed under
vacuum. The crude was purified by flash chromatography
(200 mL DCM, 2 ¥ 100 mL 2% MeOH in DCM, 2 ¥ 100 mL
5% MeOH in DCM) giving 6a (85 mg, 65%) as an orange
product; (Found: C, 42.8; H, 4.5; N, 29.2; Cl, 0.4. C13H16N8O5·0.5
MeOH ·0.05 HCl requires C, 42.4; H, 4.7; N, 29.3; Cl, 0.5%);
mp 142–144 ◦C; dH(500 MHz, DMSO-d6) 1.81 (2 H, t, J 6.33,
+
4-(4-(2-((5-Nitrofuran-2-yl)methylene)hydrazinyl)-6-amino-1,3,5-
triazin-2-ylamino)butanoic acid (5i)
5-Nitrofuraldehyde (300 mg, 2.1 mmol) was added to starting
material 4i (263 mg, 1.2 mmol) dissolved in MeOH (10 mL)
and the resulting yellow suspension stirred overnight at room
temperature. The orange mixture was filtered, washed with MeOH
(2 ¥ 20 mL) and air dried 20 min to afford 551 mg of crude. The
crude material was purified by ´ıon exchange chromatography on
DOWEX WX8/200 sulfonic acid resin eluting with a step wise
gradient of water, 200 mL HCl (0.5M), 200 mL HCl (1.0M),
200 mL HCl (1.5M), 1600 mL HCl (2.0M) (the compound now
begins to elute), 400 mL 5% MeCN in HCl (2.0M), 400 mL 10%
MeCN in HCl (2.0M), 400 mL 15% MeCN in HCl (2.0M), 400 mL
20% MeCN in HCl (2.0M). UV absorbance of each fraction was
measured at 240 nm and the fractions containing the compound
reduced under vacuum to yield a pale yellow solid 5i (321 mg,
79%). mp 185–188 ◦C; nmax/cm-1 (KBr) 3500–2700 {OH (water),
=
CH2CH2CH2), 2.01 (3 H, s, CH3-C O), 3.25 (2 H, m, CH2-NH,
4.04 (2 H, t, J 6.33, CH2-O-), 6.40–7.58 (3 H, br m, NH-,
NH2-), 7.05 (1 H, s, furan-CH), 7.77 (1 H, d, J 3.93, furan-CH),
=
=
8.01, 8.04 (1 H, m, N CH), 11.08, 11.16 (1 H, br m, N CH);
=
=
dC(125 MHz, DMSO-d6) 20.6 (CH3-C O), 20.7 (C*H3-C O),
28.2 (-CH2CH2CH2-), 28.4 (-CH2C*H2CH2), 36.9 (-CH2-NH-),
37.1 (-C*H2-NH-), 61.7 (-CH2-O-), 61.8 (-C*H2-O-), 113.7
(furan-CH), 114.4 (furan-C*H), 115.2 (furan-CH), 116.0
=
=
NH2}, ~2900 (CH2), ~1700 (C O acid), 1642 (C N), 1526, 1352
1162 | Org. Biomol. Chem., 2009, 7, 1154–1166
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=
(furan-C*H), 129.0 (N CH), 151.5 (furan-C), 152.9 (furan-C),
and then dried under high vacuum for 4 h to yield the t◦itle
compound (24 mg, 88%) as a pale yellow solid. mp 172–174 C;
164.0 (triazine-C-NH-alkyl), 166.1 (triazine-C-NHN=), 167.1
+
+
(triazine-C-NH2), 170.4 (C O); m/z (ES ) 365 (M + H , 100%);
n
max(KBr)/cm-1 3500–2700 OH (water), NH2, ~2900 CH2, CH3,
=
∑
365.1331 (M + H+. C13H17N8O5 requires 365.1316).
~1750 C O ester, 1641 C N, 1530, 1354 C-NO2; dH(500 MHz,
DMSO-d6) d 1.12 (3 H, m, CH3) 1.73 (2 H, t, J 6.5, CH2), 2.29
(2 H, t, J 7.2, CH2), 3.27 (2 H, m, CH2), 3.99 (2 H, q, J 7.0, OCH2),
7.80 (1 H, m, furan-CH), 8.13 (1 H, d, J 9.1, furan-CH), 8.30 (1 H,
+
=
=
Ethyl 3-(4-(2-((5-nitrofuran-2-yl)methylene)-hydrazinyl)-6-amino-
1,3,5-triazin-2-ylamino)propanoate (6b)
=
m, N CH), 8.5–9.2 (3H, m, NH2 and N-NH), 12.80 (1H, bs, NH);
HCl (g) generated by the addition of H2SO4 to NaCl was bubbled
for 30 min through a suspension of the starting material 5h (40 mg,
1.1mmol) in EtOH (20 mL) at room temperature. The flask was
sealed and the reaction mixture was stirred overnight at room
temperature. The resultant solution was reduced in vacuum to
yield the title compound (36 mg, 84%) as a pale yellow solid.
dC(125 MHz, DMSO-d6) 14.1 (CH3), 24.1 (CH2), 30.7 (CH2), 39.8
(CH2), 59.9 (OCH2), 115.0 (2 furan-CH), 135.0 (N CH), 151.4
=
=
(furan-C), 151.8 (furan-C), 151.9 (furan-C*), 172.5 (C O). m/z
∑
+
(ES+) 379.1 (M + H+, 100%); 379.1473 (M + H+. C14H19N8O5
requires 379.1473).
n
max(KBr)/cm-1 3500–2700 OH (water), NH2, ~2900 CH2, CH3,
=
=
1760 C O ester, 1639 C N, 1529, 1354 C-NO2; dH(500 MHz,
acetone-d6) 1.3 (3 H, m, CH3), 2.7 (2 H, bm, CH2), 3.7 (2 H,
bm, CH2), 4.2 (2 H, m, -OCH2-), 7.5 (1 H, m, furan-CH), 8.0
Benzyl 4-(4-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)-6-amino-
1,3,5-triazin-2-ylamino)butanoate (6e)
Thionyl chloride (30 ml, 0.39 mmol) was added to a solution
of the starting material 5i (40 mg) in BnOH (0.5 mL) at room
temperature. The resultant viscous solution was heated at 85 C
for 30 min. The viscous reaction mixture was allowed to cool to
room temperature, washed with hexane (2 ¥ 5 mL), diethyl ether
(2 ¥ 5 mL) and dried under high vacuum for 2 ¥ 8 h to yield the title
compound (27 mg, 54%) as a pale yellow solid. nmax(KBr)/cm-1
=
(1 H, bs, N CH), 8.4 (1 H, m, furan-CH); dC(125 MHz, DMSO-
d6) 14.2 (CH3), 33.2 (CH2), 36.6 (CH2), 60.2 (-OCH2), 115.2 (2
◦
=
=
furan-CH), 135 (N CH), 151.7 (furan-C), 151.8, 170.9 (C O).
∑
m/z (ES+) 365 (M + H+, 100%); 365.1310 (M + H+. C13H17N8O5
+
requires 365.1316).
Benzyl 3-(4-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)-6-amino-
1,3,5-triazin-2-ylamino)propanoate (6c)
=
3500–2700 OH (water), NH2, ~2900 CH2, ~1710 C O ester, 1636
C N, 1522, 1397 C-NO2, 737, 698 5 adjacent Ph; dH(500 MHz,
=
Thionyl chloride (10 ml, 0.13 mmol) was added to a solution of the
starting material 5h (45 mg, 0.13 mmol) in BnOH (0.5 mL) cooled
to 0 ◦C. The resultant solution was stirred at 0 ◦C for 10 min and
then at room temperature for 48 h. Mass spec analysis showed
the starting material triazine was still present therefore a further
aliquot of thionyl chloride (10 mL, 0.13 mmol) was added and
the mixture was stirred at 65 ◦C for 90 min whereupon mass spec
analysis showed complete loss of the triazine starting material.
The viscous reaction mixture was washed with hexane (3 ¥ 5 ml),
diethyl ether (2 ¥ 5 mL) and dried under high vacuum for 4 h
to yield the title compound (36 mg, 57%) as a pale yellow solid.
DMSO-d6) d 1.8 (2 H, bm, CH2), 2.4 (2 H, bm, CH2), 3.4 (2 H,
bm, CH2), 5.1 (2 H, s, Bn-CH2), 7.4-7.3 (6 H, m, furan-CH, 5 Ar-
=
CH), 7.9 (1 H, bs, -N CH), 8.3 (1 H, m, furan-CH), 8.6 (1 H, bm,
NH); dC(125 MHz, DMSO-d6) 24.0 (CH2) 30.7 (CH2), 65.4 (Bn-
CH2), 114.9 (furan-CH), 115 (furan-CH), 126.4 (Ar-CH), 126.6
(Ar-CH), 127.9 (Ar-CH), 128.0 (Ar-CH), 128.4 (Ar-CH), 134.5
=
(N CH), 136.1 (Ar-C), 151.5 (furan-C), 151.7 (furan-C), 151.8
+
=
(furan-C*), 172.4 (triazine-C), 187.4 (C O). m/z (ES ) 441 (M +
∑
H+, 100%); 441.1629 (M + H+. C13H17N8O5 requires 441.1630).
+
n
max(KBr)/cm-1 3500–2700 OH (water), NH2, ~2900 CH2, ~1710
Ethyl 6-(4-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)-6-amino-
1,3,5-triazin-2-ylamino)hexanoate (6f)
=
=
C O ester, 1636 C N, 1530, 1402 C-NO2, 740, 698 5 adjacent
Ph; dH(500 MHz, DMSO-d6) 2.7 (2 H, bm, CH2), 3.6 (2 H, bm,
CH2), 5.1 (2 H, s, Bn-CH2), 7.4–7.3 (5 H, m, Ar-CH), 7.6–7.4
Thionyl chloride (30 ml, 0.39 mmol) was added to a solution of
the starting material 5j (45 mg, 0.11 mmol) in EtOH (5 mL) at
room temperature. The resultant solution was stirred at room
temperature for 15 h whereupon a faint precipitate formed. The
reaction mixture was reduced in vacuum to give a pale orange solid
which was dissolved in MeOH (0.5 mL), triturated with hexane
(5 mL), washed with diethyl ether (5 mL), reduced in vacuum and
then dried under high vacuum for 4 h to yield the title compound
(32 mg, 67%) as a pale yellow solid. nmax(KBr)/cm-1 3500–2700
=
(5 H, m, furan-CH, -NH2, 2 NH), 7.9 (1 H, bs, N CH), 8.4 (1 H,
m, furan CH); dC(125 MHz, DMSO-d6) 33.1 (CH2), 38.9 (CH2),
65.7 (Bn-CH2), 115 (2 furan-CH), 126.4 (Ar-CH), 126.6 (Ar-CH),
=
127.9 (Ar-CH), 128.0 (Ar-CH), 128.4 (Ar-CH), 135.9 (N CH),
142.4 (Ar-C), 151.6 (furan-C), 151.8 (furan-C), 170.8 (triazine-
+
+
=
C), 187.3 (C O). m/z (ES ) 427.1 (M + H , 100%); 427.1473
∑
(M + H+. C13H17N8O5 requires 427.1476).
+
=
=
OH (water), NH2, ~2900 CH2, CH3, ~1750 C O ester, 1641 C N,
1530, 1354 C-NO2; dH(500 MHz, DMSO-d6) 1.2 (3 H, m, CH3)
1.3 (2 H, m, CH2), 1.6 (4 H, m, 2 CH2), 2.3 (2 H, m, CH2), 3.4
(2 H, m, CH2), 4.1 (2 H, quartet, OCH2), 7.7 (1 H, m, furan-
Ethyl 4-(4-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)-6-amino-
1,3,5-triazin-2-yl amino)butanoate (6d)
Thionyl chloride (30 ml, 0.39 mmol) was added to a solution of
the starting material 5i (25 mg, 0.07 mmol) in EtOH (5 mL) at
room temperature. The resultant solution was stirred at room
temperature for 15 h whereupon a faint precipitate formed. The
reaction mixture was reduced in vacuum to give a pale orange solid
which was dissolved in MeOH (0.5 mL), triturated with hexane
(5 mL), washed with diethyl ether (5 mL), reduced in vacuum
=
CH), 7.9 (1 H, bs, -N CH), 8.3 (1 H, m, furan-CH); dC(125 MHz,
DMSO-d6) 18.4 (CH3) 21.9, 24.0 (CH2), 25.7 (CH2), 28.3 (CH2),
33.5 (CH2), 55.8 (OCH2), 114.8 (furan-CH), 114.9 (furan-CH),
=
134.9 (N CH), 151.6 (furan-C), 151.7 (furan-C), 151.8 (furan-
+
+
=
C*), 172.8 (C O), 174.3 (triazine-C). m/z (ES ) 407 (M + H ,
∑
+
100%); 407.1785 (M + H+. C13H17N8O5 requires 407.1787).
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Org. Biomol. Chem., 2009, 7, 1154–1166 | 1163
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Benzyl 6-(4-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)-6-amino-
1,3,5-triazin-2-ylamino)hexanoate (6g)
second flask, nitric acid 70% (0.14 mL, 2.2 mmol) was slowly
added to acetic anhydride (0.34 mL, 3.6 mmol) at 0 ◦C. The
nitric acid solution was then dropwise added to the starting
material at 0 ◦C. The resulting mixture was left stirring 3 h at
room temperature. TEA (0.13 mL, 0.9 mmol) was then added
and the solution stirred a further 1 h at room temperature,
extracted with CHCl3 (2 x 15 mL), dried, concentrated and purified
by column chromatography (CHCl3/MeOH: 100–96%/0–4%) to
afford product 8c (69 mg, 53%); dH(500 MHz, MeOD) 1.04 (3 H,
t, J 7.4, CH3), 1.80 (2 H, qt, J 7.4, CH2-CH3), 2.95 (2 H, t,
J 7.3, -CH2-CO-), 7.28 (1 H, d, J 3.8, furan-CH), 7.39 (1 H,
d, J 3.8, furan-CH,); dC(125 MHz, MeOD): 13.7 (CH3-), 17.0
(-CH2-CH3,), 40.6 (-CH2-CO-), 111.9 (furan-CH), 116.5 (furan-
Thionyl chloride (30 ml, 0.39 mmol) was added to a solution of the
starting material 5j (35 mg, 0.1 mmol) in BnOH (0.5 mL) at room
temperature overnight. The resultant viscous solution was stirred
at room temperature. The viscous reaction mixture was washed
with hexane (2 ¥ 5 ml), diethyl ether (2 ¥ 5 mL) and dried under
high vacuum for 2 ¥ 8 h to yield the title compound (42 mg, 97%)
as a pale yellow solid. nmax(KBr)cm-1 3500–2700 OH (water), NH2,
=
=
~2900 CH2, ~1710 C O ester, 1636 C N, 1522, 1397 C-NO2, 737,
698 5 adjacent Ph; dH(500 MHz, DMSO-d6) 1.3 (2 H, m, CH2), 1.5
(4 H, bm, 2 CH2), 2.3 (2 H, t, J 6.9, CH2), 3.4 (2 H, t, J 7.0, CH2),
5.1 (2 H, s, Bn-CH2), 7.4–7.3 (6 H, m, furan-CH, 5 Ar-CH), 7.9
=
CH), 151.6 (furan-C), 189.6 (C O).
=
(1 H, s, N CH), 8.3 (1 H, m, furan-CH); dC(125 MHz, DMSO-d6)
22.4 (CH2) 24.1 (CH2), 25.7 (CH2), 28.3 (CH2), 33.4 (CH2), 65.4
(Bn-CH2), 114.9 (furan-CH), 115 (furan-CH), 126.4 (Ar-CH),
126.6 (Ar-CH), 127.9 (Ar-CH), 128.0 (Ar-CH), 128.4 (Ar-CH),
5-Nitro-2-imidazolcarboxaldehyde (8d)
Nitric acid 70% (3.0 mL, 46.5 mmol) was added to 2-imidazolecar-
boxaldehyde 7c (127 mg, 1.3 mmol) at room temperature and
stirred 30 min at room temperature. Then the solution was
concentrated under vacuum to afford compound 8d (185 mg,
100%); mp = 148–149 ◦C; (Found: C, 29.6; H, 3.1; N,
25.8 C4H3N3O3·1.2H2O Requires C, 29.5 H, 3.3; N, 25.8%)
dH(500 MHz, DMSO) 6.06 (1H, s, CH*(OH)2), 7.61 (2 H, s,
imidaz-CH* and NH*), 7.81 (2 H, s, imidaz-CH and NH),
9.69 (1 H, s, -CHO), 14.30 (2 H, br s, 2-OH*); dC(125 MHz,
DMSO) 83.2 (-CH*(OH)2), 119.2 (imidaz-CH* and -C*-NO2),
125.0 (imidaz-CH and -C-NO2), 142.6 (-C-CHO), 148.2 (-C*-
=
134.0 (N CH), 136.2 (Ar-C), 151.7 (furan-C), 152.2 (furan-C),
+
+
=
172.7 (C O). m/z (ES ) 469.2 (M + H , 100%); 469.1942 (M +
∑
H+. C13H17N8O5 requires 469.1942).
+
Methyl 6-(4-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)-6-
amino-1,3,5-triazin-2-ylamino)hexanoate (6h)
Thionyl chloride (30 ml, 0.39 mmol) was added to a solution of the
starting material 5j (45 mg, 0.11 mmol) in BnOH (0.5 mL) at room
temperature. The resultant viscous solution was stirred at room
temperature overnight. The viscous reaction mixture was washed
with hexane (2 ¥ 5 ml), diethyl ether (2 ¥ 5 mL) and dried under
high vacuum for 2 ¥ 8 h to yield the title compound (30 mg, 65%) as
a pale yellow solid. dH (500 MHz, DMSO-d6) 1.3 (2 H, bm, CH2),
1.5 (4 H, m, 2 CH2), 2.3 (2 H, bm, CH2), 3.3 (2 H, bm, CH2), 7.3
=
CH(OH)2), 178.6 (C O).
2-Nitro-5-imidazolcarboxaldehyde (8e)
=
Nitric acid 70% (3.0 mL, 46.5 mmol) was added to 4(5)-
imidazolecarboxaldehyde 7e (143 mg, 1.5 mmol) at room tempera-
ture and stirred 30 min at room temperature. Then the solution was
concentrated under vacuum to afford compound 8e (197 mg, 94%);
* indicates the hemiacetal form. mp = 160–161 ◦C; dH(500 MHz,
DMSO-d6) 5.90 (1 H, s, CH*(OH)2), 7.44 (1 H, s, imidaz-CH*),
8.57 (1 H, s, imidaz-CH), 8.80 (1 H, s, imidaz-NH*) 9.28 (1 H, s,
imidaz-NH), 9.86 (1 H, s, -CHO); dC(125 MHz, DMSO-d6)
83.0 (CH*(OH)2), 115.5 (imidaz-CH*), 128.4 (imidaz-CH), 132.9
(-C-CHO), 133.5 (-C*-NO2), 135.9 (-C*-C(OH)2), 138.2 (-C-
(1 H, m, furan CH), 7.9 (1 H, bs, N CH), 8.3 (1 H, m, furan-CH);
dC (125 MHz, DMSO-d6) 24.0 (CH2), 25.6 (CH2), 28.3 (CH2), 33.1
(CH2), 33.5 (CH2), 51.2 (CH3-O), 114.3 (furan-CH), 114.7 (furan-
CH), 134.5 (N CH), 151.5 (furan-C), 151.7 (furan-C), 151.8
=
+
=
(furan-C*), 173.4 (C O), 174.3 (triazine C). m/z (ES ) 393.2 (M +
∑
H+, 100%); 393.1629 (M + H+. C13H17N8O5 requires 393.1633).
+
2-Acetyl-5-nitro-furan (8b)
In a first flask, 2-acetylfuran 7b (252 mg, 2.27 mmol) was dissolved
in acetic anhydride (0.4 mL, 4.20 mmol) and stirred at 0 C. In
◦
=
NO2), 181.1 (C O).
a second flask, nitric acid 70% (0.2 mL, 3.10 mmol) was slowly
added to acetic anhydride (0.6 mL, 6.30 mmol) at 0 ◦C. The nitric
acid solution was then added dropwise to the starting material at
0 ◦C. The resulting mixture was left stirring 3 h at rt. TEA was then
added and the solution stirred a further 1 h at rt, extracted with
CHCl3 (2 ¥ 25 mL), dried, concentrated and purified by column
chromatography (CHCl3/MeOH: 100–96%/0–4%) to afford the
pure product (90 mg, 26%) 8b; dH(500 MHz, MeOD) 2.45 (3 H, s,
CH3), 7.35 (1 H, d, J 3.9, furan-CH), 7.44 (1 H, d, J 3.9, furan-
CH); dC(125 MHz, MeOD) 26.2 (CH3), 113.0 (furan-CH), 119.0
5-Carboxylic acid-2-furaldehyde-(4,6-diamino)-[1,3,5]-
triazin-2-yl-hydrazone (9a)
A mixture of 2-hydrazino-4,6-diamino-1,3,5-triazine (51 mg,
0.36 mmol) and 5-formyl-2-furancarboxylic acid (50 mg,
0.36 mmol) were stirred in methanol (3 mL) at room temperature
for 6 h. The mixture was filtered and the solid washed with
methanol (20 mL) to afford a pure product (50 mg, 53%)
(C, 37.3 H, 3.8; N, 32.9. C9H9N7O7·0.5HCl·0.5H2O·0.2MeOH
requires C, 37.2; H, 3.8; N, 33.0); dH(500 MHz, DMSO-d6) 6.40
(4H, br s, 2 NH2), 6.83 (1 H, d, J 3.6, furan-CH), 7.26 (1 H, d,
+
+
=
(furan-CH), 152.7 (2 furan-C), 188.3 (C O); m/z (ES ) 155 (M ,
∑
100%); 155.0211 (M+. C6H5NO4 requires 155.0213).
+
=
=
J 3.6, furan-CH), 7.97 (1 H, s, N CH), 10.85 (1 H, bs, N NH);
dC(125 MHz, DMSO-d6) 112.9 (furan-CH), 119.3 (furan-CH),
2-Butyryl-5-nitro-furan (8c)
=
130.4 (N CH), 144.8 (furan-C), 153.1 (furan-C), 159.3 (COOH),
164.6 (2 triazine-C-NH2), 167.2 (triazine-C-NH-N ); m/z (ES )
In a first flask, 2-butyrylfuran (97 mg, 0.7 mmol) was dissolved
in acetic anhydride (0.24 mL, 2.5 mmol) and stirred at 0 ◦C. In a
+
=
1164 | Org. Biomol. Chem., 2009, 7, 1154–1166
This journal is The Royal Society of Chemistry 2009
©

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∑
m/z 264.1 (M + H+, 100%), 264.0836 (M + H+. C9H10N7O3
requires 264.0840).
at room temperature. The precipitate was filtered off and washed
with methanol (10 mL). The organic phase was concentrated
and the product purified by reverse phase column chromatog-
raphy (H2O/CH3CN: 100–85%/0–15%) to afford product 9e
(14 mg, 19%); mp 247–250 ◦C; (C, 26.7; H, 3.6; N, 43.8.
C7H8N10O2·1.4H2O·0.8HCl requires C, 26.4 H, 3.7; N, 44.0%),
dH(500 MHz, DMSO-d6) 6.37 (4 H, bs, 2 NH2), 7.01 (1 H, s,
+
Methyl-5-nitro-furyl ketone-(4,6-diamino)-[1,3,5]-
triazin-2-yl-hydrazone (9b and 9c)
2-Hydrazino-4,6-diamino-1,3,5-triazine (4a, 245 mg, 1.74 mmol.)
and 5-nitro-2-acetylfuran (8b, 235 mg, 1.52 mmol) were dissolved
in methanol (30 mL) and stirred 24 h at room temperature with
molecular sieves. Starting material was filtered off and washed
with methanol (50 mL). The organic phase was concentrated
and purified by column chromatography (CHCl3/MeOH: 100–
70/0–30) to afford a mixture of diastereoisomers. Preparative
chromatography (CHCl3/MeOH: 90/10) allowed the purification
of compounds 9c (80 mg, 32%) and 9b (46 mg, 18%).
=
NH-N CH), 7.17 (1 H, s, imidaz-CH), 8.00 (1 H, s, triazine-NH-
N), 12.50 (1 H, s, imidaz-NH); dC(125 MHz, DMSO-d6) 118.3
=
(imidaz-CH), 129.3 (N CH) and 133.4 (imidaz-C-NO2), 143.3
=
(imidaz-C-C ), 164.6 (triazine-C), 167.2 (2 triazine-C-NH2); m/z
220.1 (M + H - NO2 , 100%).
+
2-Nitro-5-imidazolcarboxaldehyde-(4,6-diamino)-[1,3,5]-
triazin-2-yl-hydrazone (9f)
Compound 9c. dH(500 MHz, MeOD) 1.30 (3 H, s, CH3-), 3.61
(4 H, bs, 2-NH2), 7.14 (1 H, bs, N NH), 7.34 (1 H, d, J 8.6, furan-
CH), 8.05 (1 H, d, J 8.7, furan C-H). dC(125 MHz, MeOD) 23.8
(CH3), 119.9 (furan-CH) and 130.1 (furan-CH), 136.6 and 156.5
2-Nitro-5-imidazolecarboxaldehyde 8e (115 mg, 0.82 mmol) and
compound 4a (140 mg, 0.99 mmol) were dissolved in methanol
(7 mL) in the presence of molecular sieves, and stirred overnight
at room temperature. The precipitate was filtered off and washed
with methanol (40 mL). The organic phase was concentrated and
the product purified by reverse phase column chromatography
(H2O/CH3CN: 100–75%/0–25%) to afford product 9f (32 mg,
=
=
(N CH) and (furan-C), 159.3 (furan-C-NO2), 165.7 (triazine-C-
+
+
=
NH-N ), 168.6 (triazine-C-NH2). m/z (ES ): m/z 278.9 (M + H ,
∑
100%); 279.0957 (M + H+. C9H11N8O3 requires 279.0949).
+
◦
12%); mp 270–272 C; dH (500 MHz, DMSO-d6) 6.40 (4 H bs, 2
NH2), 7.16 and 7.33 (1 H, 2 s, imidaz-CH), 7.59 and 7.70 (1 H,
Compound 9b. dH(500 MHz, MeOD) 1.34 (3 H, s, CH3), 3.52
=
(4 H, bs, 2-NH2), 7.17 (1 H, bs, N NH), 7.53 (1 H, d, J 8.5, furan-
=
=
2 s, N C-H), 7.99 and 8.01 (1 H, 2 s, N NH), 12.57 and 12.72
(1 H, 2bs, imidaz-NH); dC(125 MHz, DMSO-d6) 119.7 (imidaz-
C-H), 7.65 (1 H, d, J 8.5, furan-C-H); dC(125 MHz, MeOD) 23.6
(CH3), 119.2 (furan-CH) and 128.8 (furan-CH), 136.4 and 153.0
=
=
=
C-C N), 130.1 (imidaz-C-H), 135.8 (N C-H) and 136.1 (imidaz-
(N C and furan-C), 159.1 (furan-C-NO2), 165.9 (triazine-C-NH-
=
C-NO2), 164.3 (triazine-C) 167.1 (2 triazine-C).
N ), 168.3 (triazine-C-NH2).
Propyl-5-nitro-furyl ketone-(4,6-diamino)-[1,3,5]-
triazin-2-yl-hydrazone (9d)
Acknowledgements
We would like to thank DNDi and Cardiff University for funding.
The EPSRC National Mass Spectrometry Service Centre at
Swansea is acknowledged for accurate mass spectroscopy.
2-Hydrazino-4,6-diamino-1,3,5-triazine (4a, 138 mg, 1.0 mmol)
and 5-nitro-2-butyrylfuran (163 mg, 0.9 mmol) were dissolved in
methanol (21 mL) and stirred 48 h at room temperature with
molecular sieves. Molecular sieves were filtered, washed with
methanol (20 mL) and the solute concentrated. The organic phase
was purified by column chromatography (CHCl3/MeOH:100–
0/0–10) to afford a non separable mixture of diastereoisomers 9d
(49 mg, 18%). mp: 249–251 ◦C; dH(500 MHz, MeOD) 0.91 (3 H, t,
J 7.4, CH3), 0.96 (3 H, t, J 7.4, CH¢3), 1.58 (4 H, m, CH3-CH2- and
CH3-C¢H2-), 2.60 (2 H, m, -CH2-CH2-), 2.69 (2 H,m, -CH2-C¢H2-),
7.14 (1 H, d, J 4.0, furan-C-H), 7.21 (1 H, d, J 3.9, furan-C¢-H),
7.44 (1 H, d, J 3.9, furan-C-H,), 7.50 (1 H, d, J 4.0, furan-C¢-
H,); dC(125 MHz, MeOD) 14.1 (CH3), 14.2 (C¢H3), 20.5 (CH3-
C¢H2-), 22.8 (CH3-CH2-), 28.3 (-CH2-C¢H2-), 30.8 (-CH2-CH2-),
112.3 (furan-C¢H), 113.4 (furan-CH), 114.9 (furan-C¢H), 116.9
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=
=
(furan-CH), 143.5 (N CH) + 150.3 (furan-C) + 153.0 (N C¢H) +
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