Synthesis and uptake of nitric oxide-releasing drugs by the P2 nucleoside transporter in trypanosoma equiperdum

Article abstract of DOI:10.1016/S0960-894X(00)00233-X

A series of S-nitrosothiols, structurally related to the NO-donor S-nitroso-N-acetylpenicillamine, and of organic nitrate esters that contain amidine groups which specify a recognition via the trypanosomal purine transporter P2, were synthesized and tested for their ability to inhibit the uptake of [2-³H]adenosine on Trypanosoma equiperdum. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00233-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1347±1350
Synthesis and Uptake of Nitric Oxide-Releasing Drugs by the P2
Nucleoside Transporter in Trypanosoma equiperdum
Laurent Soulere,a Pascal Ho€mann,a,* Frederic Bringaudb
and Jacques Periea
a
Á Â Â Ã
Laboratoire de Synthese et Physico-Chimie de Molecules d'Interet Biologique- ESA-CNRS 5068. Universite de Toulouse III,
118 route de Narbonne, 31062 Toulouse Cedex 4, France
Â
b
  Â
Laboratoire de Parasitologie Moleculaire- ESA-CNRS 5016. Universite de Bordeaux II, 146 rue Leo Saignat,
33076 Bordeaux Cedex, France
Received 25 January 2000; accepted 17 April 2000
AbstractÐA series of S-nitrosothiols, structurally related to the NOꢀ-donor S-nitroso-N-acetylpenicillamine, and of organic nitrate
esters that contain amidine groups which specify a recognition via the trypanosomal purine transporter P2, were synthesized and
tested for their ability to inhibit the uptake of [2-3H]adenosine on Trypanosoma equiperdum. # 2000 Elsevier Science Ltd. All rights
reserved.
Introduction
superoxide dismutase and a spermidine-glutathion con-
jugate, called trypanothione, associated with the enzyme
trypanothione reductase.6 A strategy of development
for the treatment of typanosomiasis could be considered
by combining both speci®c targeted nitric oxide genera-
tion and parasite iron superoxide dismutase inhibition
for an accumulation of superoxide anion whose dis-
mutation into hydrogen peroxide and oxygen represents
a mechanism for evasion of the host immune attack. As
a consequence of the combination of these two radicals,
peroxynitrite should be produced that may cause the
killing of trypanosomes. This present work is focused
on the ®rst component of this strategy, outlining how
nitric oxide can be selectively delivered to the parasite
using speci®c transporters. Most of the parasitic proto-
zoa are unable to biosynthesize purines de novo, and
consequently must use active transporters to salvage
them from the hosts. The transport of adenosine in the
African trypanosomes T. brucei and T. equiperdum is
ensured by two transporter systems: a P1 type, which
also transports inosine, and a P2 type, which also
enables adenine uptake. The P2 transporter has been
shown to be implicated in the selective uptake of some
melaminophenyl arsenicals7 1, or pentamidine8 2, and
probably of the nitroheterocyclic compound megazol9 3
(Fig. 1) that has been shown to be active against many
micro-organisms, including T. cruzi.
The radical nitric oxide (NOꢀ) is involved in numerous
biological processes1 including vasodilatation or neuro-
transmission, and also plays a critical role in protection
against parasitic infections as a regulatory molecule and
cytotoxic mediator of the immune system.2 Although
few physiological-target molecules of nitric oxide have
been clearly identi®ed, its role in protective mechanisms
is believed to occur through inactivation of critical
enzymes and nitrosation of nucleophilic residues.3 NOꢀ
can also react rapidly with a variety of radical species,
such as superoxide radical anion Oꢀ2 to produce the
potent toxic agent peroxynitrite anion ONOO which is
able to oxidize a great variety of biomolecules, and that
could act as a source of toxic hydroxyl radicals.4 Per-
oxynitrite anion, like NOꢀ, seems to play a major role in
the protective mechanisms of the host against parasitic
infections. For instance, it has been shown to be highly
cytotoxic against T. cruzi epimastigotes, the causative
agents of Chagas' disease, inactivating two key
enzymes, succinate dehydrogenase and NADH-fuma-
rate reductase.5 This high vulnerability of trypanosomes
towards active oxygen and nitrogen species is due to a
weak enzymatic antioxidant system which is, in the
absence of catalase, essentially assured by an iron-
Recently, the gene that encodes the T. brucei P2-trans-
porter activity has been identi®ed, cloned and expressed
*Corresponding author. Tel.: +33-5-6155-6486; fax: +33-5-6155-
6011; e-mail: ho€mann@cict.fr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00233-X

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L. Soulere et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1347±1350
1348
(4-aminophenylamino)-4,6-diamino-1,3,5-triazine,13 and
with 9-(2-amino-ethyl)-9H-purin-6-ylamine14 gave the
thiols 6a, 6b and 6c, respectively. Compound 6d was
obtained by reaction of 5 with 50-amino-20,30-(O-iso-
propylidene)-50-deoxyadenosine15 and subsequent deace-
talization. Thiols 6a±d were fully characterized by mass
spectrometry, NMR and IR spectroscopies, and gave
satisfactory elemental analysis.16 For all these coupling
reactions, no protection was required for amidine func-
tions. The nitrosation reactions to obtain the thioni-
trites necessitated an investigation of various methods
so that they precipitate once formed: only sodium nitrite
as nitrosating agent in acidic methanolic conditions
gave a satisfactory result in obtaining the thionitrite 7b,
and no diazotization/deamination reaction was observed.
On the other hand, thionitrites 7a, 7c and 7d were
obtained by electrophilic nitrosation of the correspond-
ing parent thiols with tert-butyl nitrite in chloroform
(7a) or in acetone (7c and 7d) with yields ranging from
60 to 86%. All thionitrites were characterized by 1H/13C
NMR and IR spectroscopies.17 They were stable at the
solid stage and gave a green colour in solution in organic
or aqueous media which indicates the presence of an S-
nitroso group. Their decomposition studies were carried
out by UV±visible spectroscopy noting the disappear-
ance of the characteristic-S NˆO absorbance at 340
nm. Compound 7a±d decomposed slowly in phosphate
bu€ers (pH 7.5) with half-life times comprised between
2 and 3 h, comparable to the half-life of S-nitroso-N-
acetylpenicillamine (SNAP; 7 where R=OH, Scheme 1)
in the same conditions.18 In the presence of transition
metal ion chelators such as EDTA, release of nitric
oxide from thionitrites 7a±d decreased dramatically.19
Figure 1.
in yeast, thus opening up prospects for the therapy of
sleeping sickness.10 In view of the analogies between
adenine (or adenosine), benzamidine and melamine, it
has been suggested that a speci®c P2-recognition may be
related to the presence of the amidine motif (NˆC-
NH2).11
In this paper we report the synthesis and the uptake
studies on adenosine transport in T. equiperdum of a
number of drugs that contain both a group which spe-
ci®cally could target these drugs into the parasite via the
P2-transporter, and a thionitrite-based or organic
nitrate-based NOꢀ-donor group that could exert the
speci®c pharmacological e€ect by increasing the nitric
oxide level. Furoxan 9 (Fig. 1), structurally-related to
adenine, was also investigated.
Results
Synthesis and stability studies of thionitrites 7a±d
(Scheme 1)
Synthesis of organic nitrates 8a±d (Scheme 2) and fur-
oxan 9. Organic nitrates 8b and 8c were readily pre-
pared from the corresponding alcohol derivatives20 by
nitration using 60% nitric acid in the presence of acetic
anhydride. They were fully characterized by mass spectro-
metry, NMR and IR spectroscopies, and gave satisfactory
elemental analysis.21 Synthesis of 8a was achieved from
benzyl bromide,22 and 8d was synthesized by nitration
of 20,30-isopropylidene adenosine followed by an acidic
deacetalization as previously published.23 Nitration of
2,4,6-triaminopyrimidine followed by an oxidative intra-
molecular cyclization with iodosylbenzene diacetate
gave furoxan 9.24
d,l-Penicillamine 4 was activated for coupling with
amines by conversion into 3-acetamido-4,4-dimethyl-
thietan-2-one 5, which was prepared from the racemate
of penicillamine by reaction with acetic anhydride in
pyridine (40% yield).12 The conversion of penicillamine
into its corresponding thietanone for coupling with
amino compounds was found to be a convenient and
general method that can be run either in organic solvents
or in biphasic conditions. Reaction of thietanone 5 with
2,4-diamino pyridine (commercially available), with 2-
Scheme 1. Synthesis of thionitrites.

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L. Soulere et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1347±1350
1349
use, and are capable of slowly generating NOꢀ in physio-
logical conditions with half-life times of several hours,
i.e., a rate of decomposition comparable to those of
SNAP. Beside S-nitrosothiols, organic nitrates con-
stitute a class of drugs used as therapy for a large vari-
ety of cardiovascular diseases, but their use is often
limited by the development of nitrate tolerance.
Although a large body of evidence indicates that nitric
oxide is formed from this class of NOꢀ-donors, the
mechanisms by which this occurs that, it is thought,
involve both ¯avins and thiols, are not fully under-
stood.27 In the case of furoxans, an analogous mechan-
ism involving thiols as cofactors has been assumed to
occur resulting in the NOꢀ generation. Stability studies of
compounds 8a±d revealed that they are stable in physi-
ological conditions. All compounds which possess a
melaminyl-, adenine- or adenosine-based P2 recognition
motif strongly inhibit the uptake of adenosine by the
transporter P2, that is, these compounds, like the mela-
minophenyl arsenical cymelarsen, have a speci®c inter-
action with this transporter. In the same experimental
conditions, 7a (benzamidine) and 9 (furoxan) are poor
substrates of P2, whereas 8a and SNAP do not compete
with adenosine for transport, suggesting that recogni-
tion is strongly dependent on the nature of the struc-
tural features carried by the NOꢀ-releasing moiety.
Production of nitric oxide from NOꢀ donors, including
SNAP, has been shown to inhibit the growth of Trypa-
nosoma cruzi and Leishmania major.28 The mechanism
of this toxicity has been attributed, at least in part, to
the inhibition of the glycolytic enzyme glyceraldehyde-
3-phosphate dehydrogenase, or to its conversion into
more reactive species like peroxynitrite. Conversely, all
NOꢀ donors of this study show a weak in vitro anti-
parasitic activity against T. equiperdum at relevant con-
centrations, except for compound 8c which shows a
cytotoxic (LD100=60 mM) and a cytostatic activity (8
mM). A better enzymatic defence system in the case of
T. equiperdum could account for this discrepancy. In the
case of thionitrites, these poor activities may be also
related to their relatively rapid homolytic clivage in
physiological conditions, and thus to a reduced NOꢀ
release when the molecules reach the parasite.
Scheme 2. Synthesis of organic nitrates. For 8a, 8b and 8c, R=R0.
For 8d, R0 is the O-isopropylidene derivative of R.
Biological evaluation25
All compounds were tested on T. equiperdum E1 for
their ability to inhibit the uptake of [2-3H]adenosine via
transporter P2 in the presence of a saturating concentra-
tion of inosine to inhibit the P1 transporter. The values
of Ki (Table 1) show that all compounds structurally-
related to the melaminyl, adenine and adenosine deri-
vatives eciently inhibit adenosine transport in T. equi-
perdum in the presence of inosine, with anity constant
values (Ki) ranging from 0.2 to 12.3 mM, i.e., of the same
order of magnitude of the Km values of the natural
substrate adenosine which enters through both adeno-
sine transporters P1 (Km=0.6 mM) and P2 (Km=0.7
mM). In comparison, the melaminyl drug cymelarsen
(compound 1 where R=R0=-CH2-CH2-NH2) uptake
via P2 is less ecient (Ki=22.9 mM). In the same con-
ditions, thionitrite 7a, which possesses a benzamidine
function, and furoxan 9 exhibit a weak anity for the
transporter, whereas SNAP and 8c which lack a P2-
recognition moiety do not have any anity for it with
Ki values higher than 1 mM.
Discussion
The uncommon stability of SNAP26 prompted us to
choose penicillamine derivatives for the synthesis of
thionitrites 7a±d. In spite of the presence of amino
groups, they exhibit signi®cant stability to allow a
structural characterization and a potential therapeutic
Conclusion
The presence of purine transporters constitutes a
weighty di€erence between parasites and their hosts that
could be exploited for the development of drugs with
trypanocidal activities. The molecules that bear a mela-
minyl, adenine or adenosine moiety were found to have
a high anity for the P2-transporter in T. equiperdum
suggesting that these recognition moieties could be
considered as a mode of transport for such drugs. By
analogy with the detrimental e€ect of nitric oxide in the
growth of T. cruzi or Leishmania, we expected that these
NOꢀ-carriers could by themselves provoke a nitrosative
stress on T. equiperdum, leading to the death of the
parasite. The poor in vitro trypanotoxic activity of these
compounds could be attributed either to their rapid
metabolization or to an ecient antioxidant defence
system. In a complementary approach to the present
Table 1. Inhibition of P2 adenosine transporter by cymelarsen and
NOꢀ-donors in T. equiperdum
Compounds
Ki (mM)
Compounds
Ki (mM)
Adenosine
Cymelarsen
7a
7b
7c
7d
0.70aÆ0.01
22.9Æ0.2
404Æ54
8a
8b
8c
8d
9
SNAP
>1000
0.20Æ0.02
1.9Æ0.3
12.3Æ0.2
117Æ20
>1000
0.58Æ0.05
0.62Æ0.05
3.47Æ0.35
aKm=value of adenosine.

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L. Soulere et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1347±1350
1350
work, further studies are being carried out to investigate
the inhibition of parasitic iron superoxide dismutase for a
synergistically production of nitric oxide and superoxide.
1H), 8.41 (s, 1H), 8.69 (d, J=9 Hz, 1H), 10.94 (s, 1H). 13C
NMR (DMSO-d6, 60 MHz): 22.2, 24.7, 26.4, 59.1, 59.5, 114.1,
124.9, 125.0, 137.6, 151.3, 167.6, 169.5. 7b: IR (KBr) 1654,
1
1510, 668 cm 1. H NMR (C5D5N, 300 MHz): d 2.12 (s, 3H),
2.17 (s, 3H), 2.24 (s, 3H), 6.16 (d, J=9.5 Hz, 1H), 6.57 (br. s,
6H), 7.89 (d, J=9 Hz, 2H), 7.97 (d, J=9 Hz, 2H), 9.53 (d,
J=9.5 Hz, 1H), 10.74 (s, 1H); 11.73 (s, 1H). 13C NMR
(C5D5N, 75 MHz): d 22.9, 25.6, 27.0, 60.0, 61.1, 121.2, 122.2,
Acknowledgements
The authors would like to thank the CNRS-DRET
(GDR 1077) for their ®nancial support.
134.6, 136.7, 164.4, 168.5, 170.4. 7c: IR (KBr) 1700, 1655,
1
1512, 668 cm
.
1H NMR (DMSO-d6, 200 MHz): d 1.82 (s,
6H), 1.85 (s, 3H), 3.59.62 (m, 2H), 4.33 (m, 2H), 5.05 (d,
J=9.5 Hz, 1H), 8.35 (d, J=9.5 Hz, 1H), 8.43 (s, 1H), 8.45 (s,
1H), 8.59 (m, 1H), 8.77 (br. s, 1H), 9.55 (br. s, 1H). 13C NMR
(DMSO-d6, 50 MHz): d 22.2, 24.6, 26.7, 38.2, 43.3, 58.8, 59.1,
117.9, 144.1, 144.6, 148.7, 148.8, 168.6, 169.2. 7d: IR (KBr)
1686, 1654, 1510, 669 cm. 1 1H NMR (DMSO-d6, 250 MHz): d
1.84, 1.90 (2 s, 3H), 1.92, 1.96 (2 s, 3H), 1.95 (s, 3H), 3.37.50
(m, 2H), 3.99.10 (m, 2H), 4.60.65 (m, 1H), 5.22, 5.24 (d, J=9.5
Hz, 1H), 5.92.94 (m, 1H), 8.40.53 (m, 2H), 8.67.73 (m, 2H).
13C NMR (DMSO-d6, 60 MHz): d 22.4, 24.5, 24.7, 26.6, 40.0,
40.4, 58.8, 58.9, 59.6, 71.2, 73.2, 83.2, 83.5, 87.5, 87.6, 118.8,
119.0, 142.3, 146.6, 148.4, 148.5, 151.0, 151.2, 168.5, 169.3.
18. Singh, R. J.; Hogg, N.; Joseph, J.; Kalyanaraman, B. J.
Biol. Chem. 1996, 271, 18596.
19. Askew, S. C.; Barnett, D. J.; McAninly, J.; Williams, D. L.
H. J. Chem. Soc. Perkin Trans. 2 1995, 741.
20. Precursor of 8b was synthesized from 2-chloro-4,6-dia-
mino-1,3,5-triazine and 4-aminophenethyl alcohol in re¯uxing
aq NaOH (yield: 96%). Precursor of 8c was synthesized from
adenine and ethylene carbonate in re¯uxing DMF with a cat-
References and Notes
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9. Barrett, M. P.; Fairlamb, A. H.; Rousseau, B.; Perie, J.;
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10. Maser, P.; Sutterlin, C.; Kralli, A.; Kaminsky, R. Science
1999, 285, 242.
alytic amount of NaOH (yield: 46%).
21. 8b: IR (KBr) 3411, 3323, 1632, 1277 cm
1
.
1H NMR
11. Tye, C.-K.; Kasinathan, G.; Barrett, M. P.; Brun, R.;
Doyle, V. E.; Fairlamb, A. H.; Weaver, R.; Gilbert, I. H.
Bioorg. Med. Chem. Lett. 1998, 8, 811.
12. Al-Zaidi, S. M. R.; Crilley, M. M. L.; Stoodley, R. J. J.
Chem. Soc. Perkin Trans. 1 1983, 2259.
13. Synthesized from 2-chloro-4,6-diamino-1,3,5-triazine and
4-aminoacetanilide in re¯uxing aq NaOH (1 eq.) and sub-
sequent deacetalization with aq HCl (yield: 95%).
14. Synthesized from adenine and 2-bromo-1-[(tert-butylox-
ycarbonyl)amino]ethane in dry N,N0-dimethylformamide in
presence of tetrabutylammonium iodide and potassium car-
bonate, and subsequent Boc-deprotection with tri¯uoroacetic
acid (yield: 20%).
(DMSO-d6, 250 MHz): d 2.92 (t, J=5 Hz, 2H), 4.70 (t, J=5
Hz, 2H), 6.30 (s, 4H), 7.12 (d, J=8.5 Hz, 2H), 7.71 (d, J=8.5
Hz, 2H), 8.81 (s, 1H). 13C NMR (DMSO-d6, 60 MHz): d 31.6,
73.0, 119.7, 128.57, 129.1, 139.19, 164.7, 166.0. Mass (DCI/
NH3) m/z 292 [M+H]+. Anal. calcd for C11H13N7O3: C,
45.36; H, 4.50; N, 33.66. Found C, 45.14; H, 4.50; N, 33.77.
1
8c: IR (KBr) 3301, 3157, 1689, 1632, 1281 cm. 1H NMR
(DMSO-d6, 250 MHz): d 4.55 (t, J=5 Hz, 2H), 4.93 (t, J=5
Hz, 2H), 7.27 (s, 2H), 8.15 (s, 1H), 8.18 (s, 1H). 13C NMR
(DMSO-d6, 60 MHz): d 40.4, 71.4, 118.4, 140.7, 149.6, 152.5,
155.9. Mass (DCI/NH3) m/z 225 [M+H]+, 242 [M+NH4]+.
Anal. calcd for C7H8N6O3: C, 37.50; H, 3.60; N, 37.49. Found
C, 37.68; H, 3.60; N, 37.12.
15. Kolb, M.; Danzin, C.; Barth, J.; Claverie, N. J. Med.
Chem. 1982, 25, 550.
22. 8a was prepared from benzyl bromide and silver nitrate in
acetonitrile at room temperature (yield: 97%).
16. 6a: Anal. calcd for C12H18N4O2S: C, 51.04; H, 6.43; N,
19.84. Found C, 50.99; H, 6.41; N, 19.81. 6b: Anal. calcd for
C16H22N8O2S, 1.5 H2CO3: C, 43.47; H, 5.21; N, 23.18. Found
C, 42.71; H, 5.67; N 22.98. 6c: Anal. calcd for C14H21N7O2S,
1.5 H2CO3: C, 41.89; H, 5.44; N, 22.06. Found C, 41.01; H,
5.40; N, 22.06. O-isopropylidene derivative of 6d: Anal. calcd
for C20H29N7O5S, 1.5 H2CO3: C, 46.57; H, 5.77; N, 18.10.
Found C, 46.06; H, 5.84; N, 18.08.
23. Lichtenthaler, F. W.; Muller, H. J. Synthesis 1974, 199.
24. Sako, M.; Oda, S.; Ohara, S.; Hirota, K.; Maki, Y. J. Org.
Chem. 1998, 63, 6947.
25. For the experimental determination of Ki, see: Barrett, M.
P.; Zhang, Z. Q.; Denise, H.; Giroud, C.; Baltz, T. Mol. Bio-
chem. Parasitol. 1995, 73, 223.
26. Field, L.; Dilts, R. V.; Ravichandran, R.; Lenhert, P. G.;
Carnahan, G. E. J. Chem. Soc. Chem. Commun. 1978, 249
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28. Bourguignon, S. C.; Alves, C. R.; Giovanni De Simone, S.
Acta Trop. 1997, 66, 109.
1
17. 7a: IR (KBr) 1657, 1531, 667 cm 1. H NMR (DMSO-d6,
250 MHz): d 1.89 (s, 3H), 1.98 (s, 3H), 2.03 (s, 3H), 5.40 (d,
J=9.5 Hz, 1H), 7.06 (d, J=9.5 Hz, 1H), 7.96 (d, J=9 Hz,