Benzimidazole-4,7-diones as inhibitors of protozoal (Toxoplasma gondii) purine nucleoside phosphorylase

Article abstract of DOI:10.1016/S0960-894X(02)00064-1

Benzimidazole-4,7-diones derivatives substituted at 1- and/or 2-position have been synthetized and tested as inhibitors of purine nucleoside phosphorylase (PNP), isolated from two strains of Toxoplasma gondii (RH and ME 49). They were identified as inhibi

Full text of DOI:10.1016/S0960-894X(02)00064-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 977–979
Benzimidazole-4,7-diones as Inhibitors of Protozoal
(Toxoplasma gondii) Purine Nucleoside Phosphorylase
Frederic Alvarez,a Arnaud Gherardi,b Pascal Nebois,a Marie-Elizabeth Sarciron,b
Anne-Francoise Petavyb and Nadia Walchshofera,*
a
´ ´
Laboratoire de Chimie Organique (EA 635), Faculte de Pharmacie, Universite Lyon I, 8, Avenue Rockefeller,
69373 Lyon Cedex 08, France
´
69373 Lyon Cedex 08, France
b
´
Laboratoire de Parasitologie (EA 1887), Faculte de Pharmacie, Universite Lyon I, 8, Avenue Rockefeller,
Received 8 October 2001; revised 21 December 2001; accepted 22 January 2002
Abstract—Benzimidazole-4,7-diones derivatives substituted at 1- and/or 2-position have been synthetized and tested as inhibitors of
purine nucleoside phosphorylase (PNP), isolated from two strains of Toxoplasma gondii (RH and ME 49). They were identified as
inhibitors of both enzymes. # 2002 Elsevier Science Ltd. All rights reserved.
Toxoplasma gondii is a unicellular protozoan which
causes severe disease in immunocompromized patients.
Toxoplasmic encephalitis is a common cause of mortal-
ity in AIDS patients, either by primary infection, or by
reactivation of CNS tissue cysts.1 Only a few drugs are
active against this parasite, and they lead to severe side
effects.2 Progress in treatment of toxoplasmosis with
more potent or less toxic drugs is required and implies a
greater knowledge of the parasite biology to determine
novel chemotherapeutic targets.
bed.4,6 They are closely related to purine structure, with
a pyrimidinone ring fused to an imidazole, a pyrole or a
triazole nucleus.
On the other hand, heterocyclic quinones, like
naphto[2,3-d]imidazole-4,9-dione, have been described
as inhibitors of hypoxanthine phosphoribosyltransfer-
ase HGPRT (E.C. 2.4.2.8), another enzyme of the
purine salvage pathway.7
Thus, we decided to synthetize benzimidazole-4,7-dione
derivatives 8a–j (Table 1), in order to evaluate their
ability to inhibit in vitro T. gondii purine nucleoside
phosphorylase from two strains, a virulent strain RH
and a cystic strain ME 49.
All classified protozoa lack the de novo purine bio-
synthesis and require host-supplied preformed purines.3
Purine salvage pathway is therefore crucial for the
parasite survival. Purine nucleoside phosphorylase PNP
(E.C. 2.4.2.1) is a ubiquitous key enzyme in this
metabolic way.4 It reversibly catalyses the phosphoro-
lysis of purine nucleosides to corresponding bases and
sugar 1-phosphate.
PNP inhibition activity of 8a–j was compared to that of
three reference compounds: 8-aminoguanosine (AG), a
Plasmodium falciparum and human PNP inhibitor,9 for-
mycine A (FoA), an Escherichia coli PNP inhibitor,6b
and allopurinol riboside (alloR), an inhibitor of human
PNP, but not of Plasmodium lophurae PNP6a (Fig. 1).
We have recently characterized the PNP of T. gondii.5 It
possesses properties different from those of other species
and may be therefore a potential target for antiparasitic
compounds.
Chemistry
Many inhibitors of PNP, mainly of the human ery-
throcyte and calf spleen enzymes, have been descri-
The precursors, compounds 1–2,10 3–4,8 5,11 6a–7a and
6g–7g,8 are reported in the literature.
Benzimidazolediones 8a–j were synthetized according to
procedures modified with regard to those published.
*Corresponding author. Fax +33-4-7877-7158; e-mail: nadia.
walchshofer@univ-lyon1.fr
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00064-1

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Frederic Alvarez et al. / Bioorg. Med. Chem. Lett. 12 (2002) 977–979
978
Nitration of 1,4-dimethoxybenzene (Scheme 1) afforded
a mixture of 2,3-dinitro-1,4-dimethoxybenzene 1 and its
2,5-dinitro isomer 1 in a ratio of 40/60, respectively
(quantitative overall yield).
respectively, to 4,7-dimethoxybenzimidazoles 3, 4 and 5.
N-substituted compounds 6a–j were obtained from 3–5
by treatment with NaH in DMF and subsequent addi-
tion of the suitable alkylating agent, except for 6g. The
latter was prepared by treatment of 3 with KOH in
acetone following by addition of iodomethane.
For the reduction of 1, we obtained a better yield (98%)
with Sn/HCl than with catalytic hydrogenation (Pd/C,
MeOH/HCl). As starting from purified 2,3-dinitro com-
pound 1 did not improve final yields, our sequence was
carried out with the mixture of the two dinitro isomers.
All our attempts to submit compound 6b to an oxida-
tive demethylation with ceric ammonium nitrate (CAN)
or AgO led mainly to dimerization products and the
final quinones were successfully obtained through a
two-step process. Demethylation of 6a–j with HBr pro-
vided 7a–j. Oxidation of 7a with aqueous FeCl3 led to
8a, whereas K2Cr2O7 gave better results for N-sub-
stituted compounds 8b–j.12
Treatment of the mixture 2+20 with formic acid, acetic
acid or phenylacetic acid in hydrochloric acid 6N led,
Table 1. Synthetized benzimidazole-4,7-diones 8a–j
Biology
Purine nucleoside phosphorylase was isolated from the
RH and ME 49 strains of T. gondii. Both strains are
maintained in culture with the human myelomonocytic
cell line THP-1.13 PNP was purified by the followings
steps: precipitation with (NH4)2SO4, DEAE-Sephacel
and hydroxyapatite columns. The amount of the protein
was determined by the Bensadoun’s method.14
Compd
R1
R2
8a8
8b
8c
8d
8e
H
H
H
H
H
H
CH2C6H5
CH2-pF-C6H4
CH2-mF-C6H4
CH2-oF-C6H4
The drugs were tested on both PNP at the doses of 1
and 4 mM. The inhibition percentage of PNP specific
activity5 was defined as the ratio of PNP specific activity
with drug on PNP specific activity without drug. Ki
values were determined by using the Lineweaver–Burk
method, with inosine as the variable substrate ranging
from 0.5 to 3 mM and fixed inorganic phosphate con-
centration (50 mM).
8f
H
8g8
8h
8i
H
H
CH3
n-C4H9
CH2C6H5
CH2C6H5
CH3
CH2C6H5
8j
Results and Discussion
8-Aminoguanosine (AG) was the first reported sig-
nificant inhibitor of human erythrocyte PNP (Ki=7
mM). We found (Table 2) that AG, albeit the most
potent of the reference compounds, inhibits T. gondii
PNP with a Ki of 200 mM, suggesting a marked differ-
ence in the active sites between T. gondii PNP and
human PNP.
Figure 1. Structure of the reference compounds.
Furthermore, compounds 8 seem to have a slightly dif-
ferent behavior towards the enzymes of ME 49 strain
and RH strain, indicating some differences between
them.
All compounds 8a–j possess some potency as PNP
inhibitor but inhibit T. gondii PNP by different
mechanisms. 8f and 8i–j are competitive inhibitors,
while the others are noncompetitive.
It is noteworthy that 8i–j, which are substituted at
position 2, are among the most potent inhibitors of
PNP from ME 49 strain, while the substitution of
purine analogues at similar position, except with an
amino group, leads generally to a marked decrease of
the human PNP inhibition.15,16
Scheme 1. (a) HNO3 70% 2 equiv/(CH3CO)2O, 0 ꢀC, 3 h; (b) Sn/HCl
12 N, reflux, 2 h, (a) and (b) 98% overall yield; (c) R1COOH, reflux, 5
h, 57–80% yield from pure 1; (d) (1) NaH 1.05 equiv/DMF or KOH 5
equiv/CH3COCH3, rt; (2) R2X. 0.95 equiv, rt, 12 h, 65–83% yield; (e)
48% aqueous HBr, 125ꢀC, 5 h, 60–94% yield; (f) FeCl3 0.7 M, 30 min
rt or K2Cr2O7 0.3 M/HCl 7 N, HCCl3, 30 min, rt 68–95% yield.

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Frederic Alvarez et al. / Bioorg. Med. Chem. Lett. 12 (2002) 977–979
979
Table 2. PNP inhibition of compounds 8a–j
References and Notes
Compd
Inh (%) RH
1 mM 4 mM
Ki (mM)
Inh (%) ME49
1 mM 4 mM
Ki (mM)
1. Tenter, A. M.; Heekeroth, A. R.; Weiss, L. M. Int. J.
Parasitol. 2000, 30, 1217.
RH
ME 49
2. Scholer, N.; Krause, K.; Kayser, O.; Muller, R. H.; Borner,
K.; Hahn, H.; Liesenfeld, O. Antimicrob. Agents Chemother.
2001, 45, 1771.
3. Hwang, H.-Y; Ullman, B. J. Biol. Chem. 1997, 31, 19488.
4. For a review on PNP, see: Bzowska, A.; Kulikowska, E.;
Shugar, D. Pharmacol. Ther. 2000, 88, 349.
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
AG
FoA
AlloR
47ꢁ1.4 68ꢁ1.4
55ꢁ1.7 77ꢁ1.7
80ꢁ2.4 88ꢁ1.8
65ꢁ2.0 100ꢁ2
70ꢁ2.1 87ꢁ1.7
50ꢁ1.5 75ꢁ1.5
55ꢁ1.7 85ꢁ1.7
45ꢁ1.4 68ꢁ1.4
54ꢁ1.6 82ꢁ1.6
65ꢁ2.0 80ꢁ1.6
85ꢁ2.6 91ꢁ2.7
12ꢁ0.4 37ꢁ1.1
8ꢁ0.2 28ꢁ0.8
1.65a
0.58a
0.78a
0.99a
1.48a
0.10b
2.21a
2.96a
1.05b
0.67b
0.20b
1.68b
2.00b
52ꢁ2.3 71ꢁ2.5
57ꢁ3.0 89ꢁ3.0
56ꢁ2.8 100ꢁ2.8
60ꢁ2.0 100ꢁ2.0
62ꢁ2.2 89ꢁ1.5
44ꢁ3.0 77ꢁ2.1
31ꢁ1.2 82ꢁ2.0
53ꢁ2.2 65ꢁ2.0
66ꢁ1.8 92ꢁ2.8
61ꢁ3.2 78ꢁ2.3
92ꢁ1.5 95ꢁ1.0
12ꢁ0.3 30ꢁ0.5
11ꢁ0.2 22ꢁ0.5
0.55a
0.67a
0.99a
0.52a
1.50a
0.11b
3.41a
0.56a
0.30b
0.35b
0.20b
1.70b
1.80b
5. Gherardi, A.; Sarciron, M.-E.; Petavy, A.-F.; Peyron, F.
Life Sci. 1999, 65, 1733.
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Chem. Pharm. Bull. 2001, 49, 10.
The values are the meansꢁSD of triplicate experiments.
aNoncompetitive inhibition.
bCompetitive inhibition.
7. Pickering, M. V.; Dea, P.; Strater, D. G.; Witkowski, J. T.
J. Med. Chem. 1977, 20, 818.
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12. All new synthetized compounds gave satisfactory ele-
mental analysis and were characterized by NMR spectro-
scopy.
In conclusion, T. gondii PNP represents a reasonable
target for chemotherapy. Benzimidazole-4,7-diones are
weak inhibitors of this enzyme, but some of them have
shown a similar potency than 8-aminoguanosine, our
best reference compound.
Further studies on new derivatives are at the present
time in progress, as well as the evaluation of compounds
8 activity against T. gondii in culture.
13. Nebois, P.; Sarciron, M.-E.; Bibal, B.; Bouammali, B.;
Cherkaoui, O.; Pautet, F.; Petavy, A.-F.; Walchshofer, N.;
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Acknowledgements
The authors wish to thank Lyon I University (France)
for financial support (BQR 2000). They are also grate-
ful to Prof. H. Fillion (Lyon I University) for helpful
discussions.