Antiparasitic activity of highly conjugated pyrimidine-2,4-dione derivatives

Article abstract of DOI:10.1016/j.farmac.2003.07.009

4-[2-(1,3-Dimethyl-5-nitro-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl) vinyl]benzaldehyde was synthesized in four steps from 6-methyl-1H,3H-pyrimidine- 2,4-dione. This aldehyde was functionalized by various substituted anilines or substituted benzylamines. Antiparasitic activities of the corresponding azomethines were assessed against Plasmodium falciparum, Trichomonas vaginalis and Leishmania infantum compared to their toxicity versus human cells.

Full text of DOI:10.1016/j.farmac.2003.07.009

Il Farmaco 58 (2003) 1263Á1270
/
www.elsevier.com/locate/farmac
Antiparasitic activity of highly conjugated pyrimidine-2,4-dione
derivatives
Nadine Azas a,*, Pascal Rathelot b, Serge Djekou b, Florence Delmas a, A. Gellis b,
Carole Di Giorgio a, Patrice Vanelle b, Pierre Timon-David a
a Laboratoire de Parasitologie, EA 864, Faculte´ de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France
b Laboratoire de Chimie Organique, UMR CNRS 6517, Faculte´ de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France
Received 26 February 2003; accepted 25 July 2003
Abstract
4-[2-(1,3-Dimethyl-5-nitro-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)vinyl]benzaldehyde was synthesized in four steps from 6-
methyl-1H,3H-pyrimidine-2,4-dione. This aldehyde was functionalized by various substituted anilines or substituted benzylamines.
Antiparasitic activities of the corresponding azomethines were assessed against Plasmodium falciparum, Trichomonas vaginalis and
Leishmania infantum compared to their toxicity versus human cells.
´
# 2003 Published by Editions scientifiques et me´dicales Elsevier SAS.
Keywords: Antiparasitic activity; Pyrimidine-2,4-dione derivatives; Toxicity; THP1 cells; Azomethines
1. Introduction
determined on a Bruker ARX 200 spectrometer. The 1H
chemical shifts were reported as parts per million
downfield from tetramethylsilane (Me4Si). Absorptions
were reported with the following notations: s, singlet; d,
doublet; t, triplet; q, quartet; m, a more complex
multiplet or overlapping multiplets. The following
adsorbent was used for column chromatography: silica
The pyrimidine-2,4-dione ring is frequently encoun-
tered in many drugs used for the treatment of hypothy-
roidy, hypertension, cancer chemotherapy or HIV
infection [1]. According to our interest toward nitrated
heterocycles and their significant antiparasitic activity
[2Á
/
4], we report here the synthesis of a new azomethine
gel 60 (Merck, particle size 0.063Á
/
0.200 mm, 70Á230
/
series after subjecting the 4-[2-(1,3-dimethyl-5-nitro-
2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)vinyl]benzal-
dehyde to classical reactions. Their antiparasitic activity
versus Plasmodium falciparum, Trichomonas vaginalis
and Leishmania infantum was assessed and compared to
their toxicity against THP1 cells.
mesh ASTM). Microanalyses for C, H, N were per-
formed by the Service de Microanalyse de l’Universite´
de Pharmacie de Chaˆtenay-Malabry and the results for
C, H, N elements were within 9
/
0.4% of the calculated
values, unless otherwise indicated.
2.1.1. 1,3,6-Trimethyl-1H,3H-pyrimidine-2,4-dione (1)
A solution of 2,4-dihydroxy-6-methylpyrimidine-2,4-
dione (15 g, 119 mmol) in 140 ml of 4 N NaOH was
heated at 70 8C. Then, dimethylsulfate (40 ml, 422
mmol) was added dropwise. The reaction mixture was
then heated for 1 h at 80 8C. After cooling at room
temperature (r.t.), the solution was neutralized with
concentrated acetic acid and extracted with chloroform.
The chloroformic layer was dried over anhydrous
MgSO4 and the solvent was removed in vacuo leading
to a white solid crystallized from ethanol in 70% yield
2. Experimental
2.1. Chemistry
Melting points were determined on a Buchi B-540
¨
1
apparatus and are uncorrected. H NMR spectra were
* Corresponding author.
E-mail address: nadine.azas@pharmacie.univ-mrs.fr (N. Azas).
´
0014-827X/03/$ - see front matter # 2003 Published by Editions scientifiques et me´dicales Elsevier SAS.
doi:10.1016/j.farmac.2003.07.009

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N. Azas et al. / Il Farmaco 58 (2003) 1263Á
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1270
1
(12.8 g), m.p. 112 8C (lit. m.p. 112 8C [6]). H NMR
(CDCl3) d 2.24 (s, 3H, C-CH3), 3.34 (s, 3H, NÁCH3),
3.41 (s, 3H, NÁCH3), 5.63 (s, 1H, H5).
mmol) in ethanol (5 ml). The reaction mixture was
refluxed for 2 h.
Azomethines which precipitated in boiling ethanol
were isolated by filtration and purified by washing with
boiling ethanol.
For azomethines which precipitated after cooling the
reaction mixture at r.t., the solvent was removed in
vacuo and the crude residue washed with water and
crystallized from ethanol.
For other azomethines the solvent was removed in
vacuo and was dissolved in chloroform. The organic
layer was washed with water, dried over anhydrous
MgSO4 and evaporated under reduced pressure. The
residue was crystallized from ethanol.
/
/
2.1.2. 1,3,6-Trimethyl-5-nitro-1H,3H-pyrimidine-2,4-
dione (2)
1,3,6-Trimethyl-1H,3H-pyrimidine-2,4-dione (1) (15
g, 97.3 mmol) was dissolved into concentrated sulfuric
acid (50 ml) previously cooled at 0 8C. Fuming nitric
acid (40 ml), cooled at 0 8C, was then added dropwise.
The solution was stirred at a such temperature for 1 h
and poured slowly over crushed ice. The yellow pre-
cipitate was filtered, dried in vacuo over P2O5 and
crystallized from ethanol to give the nitrated derivative 2
in 86% yield (16.7 g). Yellow solid, m.p. 153 8C (lit. m.p.
1
150 8C [7]). H NMR (CDCl3) d 2.42 (s, 3H, CÁ
/
CH3),
2.1.5.1. 1,3-Dimethyl-5-nitro-6-(2-{4-[(4-
3.37 (s, 3H, NÁ
/
CH3), 3.53 (s, 3H, NÁ
/
CH3).
trifluoromethylbenzylimino)-methyl]-phenyl}-vinyl)-
1H,3H-pyrimidine-2,4-dione (5a). Yellow crystals, m.p.
140 8C, yield 76% (230 mg). 1H NMR (CDCl3) d 3.37 (s,
2.1.3. 6-[2-(4-Diethoxymethylphenyl)-vinyl]-1,3-
dimethyl-5-nitro-1H,3H-pyrimidine-2,4-dione (3)
Compound 2 (5 g, 25.1 mmol) and 4-(diethoxy-
methyl)benzaldehyde (15.7 g, 75.3 mmol) were added
to a mixture of pyridine (2.5 ml) and ethanol (25 ml) and
3H, NÁ
6.65 (d, Jꢀ
Hz, 1H, ethylenic H), 7.41 (d, Jꢀ
benzyl), 7.45 (d, Jꢀ8.2 Hz, 2H, CHÁ
Jꢀ8.0 Hz, 2H, NÁbenzyl), 7.78 (d, Jꢀ
CHÁphenyl), 8.38 (s, 1H, CHÄN).
/
CH3), 3.42 (s, 3H, NÁ
16.4 Hz, 1H, ethylenic H), 7.02 (d, Jꢀ
8.0 Hz, 2H, NÁ
phenyl), 7.57 (d,
8.2 Hz, 2H,
/
CH3), 4.82 (s, 2H, CH2N),
/
/16.4
/
/
/
/
heated at 60Á
/
65 8C for 3 h. Then, the reaction mixture
/
/
/
was poured over water and extracted with chloroform.
The organic layer was dried over anhydrous MgSO4 and
the solvent was removed under reduced pressure. The
crude residue was purified by chromatography on a
silica gel column, eluting with petroleum etherÁethyle
acetate (1:1) to give 7.8 g (80%) of yellow crystals, m.p.
167 8C (ethanol). 1H NMR (CDCl3) d 1.00 (m, 6H,
/
/
2.1.5.2. 1,3-Dimethyl-5-nitro-6-(2-{4-[(3-
trifluoromethylbenzylimino)-methyl]-phenyl}-vinyl)-
1H,3H-pyrimidine-2,4-dione (5b). Yellow crystals, m.p.
156 8C, yield 68% (205 mg). 1H NMR (CDCl3) d 3.37 (s,
/
3H, NÁ
6.67 (d, Jꢀ
Hz, 1H, ethylenic H), 7.39Á
2H CHÁphenyl), 7.78 (d, Jꢀ
8.37 (s, 1H, CHÄN).
/
CH3), 3.45 (s, 3H, NÁ
16.4 Hz, 1H, ethylenic H), 7.01 (d, Jꢀ
7.58 (m, 6H, 4H NÁbenzyl,
8.2 Hz, 2H, CHÁphenyl),
/
CH3), 4.82 (s, 2H, CH2N),
2CH2CH3), 3.15 (s, 3H, NÁ
3.37 (m, 4H, 2CH2CH3), 5.27 [s, 1H, CH(OEt)2], 6.49
(d, Jꢀ16.4 Hz, 1H, ethylenic H), 6.77 (d, Jꢀ16.4 Hz,
1H, ethylenic H), 7.42 (d, Jꢀ8.3 Hz, 2H, CHÁphenyl),
7.63 (d, Jꢀ8.3 Hz, 2H, CHÁphenyl).
/
CH3), 3.24 (s, 3H, NÁ
/
CH3),
/
/16.4
/
/
/
/
/
/
/
/
/
/
/
/
2.1.5.3. 1,3-Dimethyl-5-nitro-6-(2-{4-[(2-
2.1.4. 4-[2-(1,3-Dimethyl-5-nitro-2,6-dioxo-1,2,3,6-
tetrahydropyrimidin-4-yl)-vinyl]-benzaldehyde (4)
Water (10 ml) and 37% hydrochlorid acid (5 ml) were
added to a solution of 3 (3 g, 7.7 mmol) in 30 ml of
THF. The reaction mixture was stirred at r.t. for 24 h.
The residue was filtered and dried in vacuo over P2O5.
The yellow precipitate was, then, purified by chromato-
graphy on a silica gel column, eluting with petroleum
trifluoromethylbenzylimino)-methyl]-phenyl}-vinyl)-
1H,3H-pyrimidine-2,4-dione (5c). Yellow crystals, m.p.
195 8C, yield 60% (180 mg). 1H NMR (CDCl3) d 3.43 (s,
3H, NÁ
6.70 (d, Jꢀ
Hz, 1H, ethylenic H), 7.37 (dd, Jꢀ
NÁbenzyl), 7.51Á7.69 (m, 5H, 3H NÁ
phenyl), 8.40 (s, 1H, CHÄN).
/
CH3), 3.50 (s, 3H, NÁ
16.4 Hz, 1H, ethylenic H), 7.08 (d, Jꢀ
7.7 and 7.2 Hz, 1H,
benzyl, 2H CHÁ
/
CH3), 5.01 (s, 2H, CH2N),
/
/16.4
/
/
/
/
/
/
etherÁ
yielding 1.7 g (70%) of a yellow solid, m.p. 177 8C. H
NMR (CDCl3) d 3.34 (s, 3H, NÁCH3), 3.42 (s, 3H, NÁ
CH3), 6.75 (d, Jꢀ16.4 Hz, 1H, ethylenic H), 7.05 (d,
Jꢀ16.4 Hz, 1H, ethylenic H), 7.60 (d, Jꢀ8.3 Hz, 2H,
CHÁphenyl), 7.90 (d, Jꢀ8.3 Hz, 2H, CHÁphenyl),
10.01 (s, 1H, CHO).
/
ethyle acetate (1:1) and crystallized from ethanol
1
2.1.5.4. 1,3-Dimethyl-5-nitro-6-(2-{4-[(1-
/
/
phenylethylimino)-methyl]-phenyl}-vinyl)-1H,3H-
/
pyrimidine-2,4-dione (5d). Yellow crystals, m.p. 148 8C,
yield 71% (190 mg). 1H NMR (CDCl3) d 1.54 (d, Jꢀ
6.6
Hz, 3H, CH-CH3), 3.37 (s, 3H, NÁCH3), 3.45 (s, 3H,
NÁCH3), 4.50 (q, Jꢀ6.6 Hz, 1H, CH ÁCH3), 6.60 (d,
Jꢀ16.4 Hz, 1H, ethylenic H), 7.00 (d, Jꢀ16.4 Hz, 1H,
ethylenic H), 7.12Á7.39 (m, 5H, NÁbenzyl), 7.43 (d, Jꢀ
8.2 Hz, 2H, CHÁphenyl), 7.75 (d, Jꢀ8.2 Hz, 2H, CHÁ
phenyl), 8.29 (s, 1H, CHÄN).
/
/
/
/
/
/
/
/
/
/
/
/
2.1.5. General procedure for the preparation of aldimines
A solution of amine (0.96 mmol, 1.5 equiv.) in ethanol
(5 ml) was added to a solution of aldehyde 4 (0.2 g, 0.64
/
/
/
/
/
/
/

N. Azas et al. / Il Farmaco 58 (2003) 1263Á
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1270
1265
2.1.5.5. 6-(2-{4-[(2-Chlorobenzylimino)-methyl]-
phenyl}-vinyl)-1,3-dimethyl-5-nitro-1H,3H-pyrimidine-
3H, CH3), 3.43 (s, 3H, NÁ
6.76 (d, Jꢀ16.4 Hz, 1H, ethylenic H), 7.08Á
2H, ethylenic H and NÁphenyl), 7.33 (dd, Jꢀ
8.0 Hz, 1H, NÁphenyl), 7.58 (d, Jꢀ8.3 Hz, 2H, CHÁ
phenyl), 7.73 (d, Jꢀ7.9 Hz, 1H, NÁphenyl), 7.96 (d,
Jꢀ8.3 Hz, 2H, CHÁphenyl), 8.36 (s, 1H, CHÄN).
/
CH3), 3.51 (s, 3H, NÁ
/
CH3),
/
/7.15 (m,
2,4-dione (5e). Yellow crystals, m.p. 184 8C, yield 66%
/
/8.2 and
1
(185 mg). H NMR (CDCl3) d 3.36 (s, 3H, NÁ
/
CH3),
CH3), 4.86 (s, 2H, CH2N), 6.63 (d, Jꢀ
16.4 Hz, 1H, ethylenic H), 7.00 (d, Jꢀ16.4 Hz, 1H,
ethylenic H), 7.12Á7.22 (m, 2H, NÁbenzyl), 7.28Á7.36
(m, 2H, NÁbenzyl), 7.46 (d, Jꢀ8.2 Hz, 2H, CHÁ
phenyl), 7.77 (d, Jꢀ8.2 Hz, 2H, CHÁphenyl), 8.35 (s,
1H, CHÄN).
/
/
/
3.44 (s, 3H, NÁ
/
/
/
/
/
/
/
/
/
/
/
/
/
/
2.1.5.11. 1,3-Dimethyl-6-(2-{4-[(4-methyl-3-
nitrophenylimino)-methyl]-phenyl}-vinyl)-5-nitro-
/
/
/
1H,3H-pyrimidine-2,4-dione (5k). Yellow crystals, m.p.
116 8C, yield 49% (140 mg). 1H NMR (CDCl3) d 2.62 (s,
2.1.5.6. 6-(2-{4-[(4-Chlorophenylimino)-methyl]-
phenyl}-vinyl)-1,3-dimethyl-5-nitro-1H,3H-pyrimidine-
3H, CH3), 3.44 (s, 3H, NÁ
6.78 (d, Jꢀ16.4 Hz, 1H, ethylenic H), 7.13 (d, Jꢀ
Hz, 1H, ethylenic H), 7.36Á7.41 (m, 2H, NÁphenyl),
7.58 (d, Jꢀ8.3 Hz, 2H, CHÁphenyl), 7.85 (d, Jꢀ1.9
Hz, 1H, NÁphenyl), 7.95 (d, Jꢀ8.3 Hz, 2H, CHÁ
phenyl), 8.36 (s, 1H, CHÄN).
/
CH3), 3.51 (s, 3H, NÁ
/
CH3),
/
/16.4
2,4-dione (5f). Yellow crystals, m.p. 250 8C, yield 70%
/
/
1
(190 mg). H NMR (CDCl3) d 3.43 (s, 3H, NÁ
3.51 (s, 3H, NÁCH3), 6.71 (d, Jꢀ16.4 Hz, 1H, ethylenic
H), 7.06 (d, Jꢀ16.4 Hz, 1H, ethylenic H), 7.16 (d, Jꢀ
8.7 Hz, 2H, CHÁ
phenyl), 7.55 (d, Jꢀ
Jꢀ8.2 Hz, 2H, NÁ
/
CH3),
/
/
/
/
/
/
/
/
/
/
/
/
phenyl), 7.38 (d, Jꢀ
/
8.7 Hz, 2H, CHÁ
phenyl), 7.94 (d,
/
/
8.0 Hz, 2H, NÁ
phenyl), 8.44 (s, 1H, CHÄ
/
2.2. Pharmacology
2.2.1. Antiplasmodial activity
/
/
/
N).
2.1.5.7. 6-(2-{4-[(2,6-Dichlorophenylimino)-methyl]-
phenyl}-vinyl)-1,3-dimethyl-5-nitro-1H,3H-pyrimidine-
Assays were performed against a chloroquine-resis-
tant strain (W2) of P. falciparum maintained in con-
tinuous culture according to the methodology described
by Trager and Jensen [9]. Parasites were cultivated in
2,4-dione (5g). Yellow crystals, m.p. 252 8C, yield 61%
1
(180 mg). H NMR (CDCl3) d 3.43 (s, 3H, NÁ
/
CH3),
16.4 Hz, 1H, ethylenic
7.20 (m, 2H, ethylenic H and NÁphenyl), 7.36
8.5 and 1.5 Hz, 2H, NÁphenyl), 7.55 (d, Jꢀ8.2
Hz, 2H, CHÁphenyl), 7.93 (d, Jꢀ8.2 Hz, 2H, CHÁ
phenyl), 8.44 (s, 1H, CHÄN).
3.50 (s, 3H, NÁ
H), 7.03Á
(dd, Jꢀ
/
CH3), 6.73 (d, Jꢀ
/
group Aꢁ
hematocrit in RPMI 1640 medium supplemented with
Hepes, NaHCO3, 10% Aꢁ human serum and Neomycin
(Sigma, St Louis, MO) at 37 8C in a gas mixture of 5%
O2Á6% CO2Á90% N2; RPMI 1640, Hepes, NaHCO3
were obtained from GibcoÁBRL (Paisley, Scotland).
/
human erythrocytes and suspended at a 4%
/
/
/
/
/
/
/
/
/
/
/
/
/
2.1.5.8. 6-(2-{4-[(4-Bromophenylimino)-methyl]-
phenyl}-vinyl)-1,3-dimethyl-5-nitro-1H,3H-pyrimidine-
2,4-dione (5h). Yellow crystals, m.p. 244 8C, yield 62%
Assays were performed in triplicate in 96 well tissue
culture plates (Nunc Brand products, Fisher, Paris,
France) containing 200 ml of W2 asynchronous parasite
cultures at 2% of parasitemia and 2% hematocrit, and 5
ml of the appropriate drug or drug combination
dissolved in DMSO (dimethylsulfoxide, Sigma, St
Louis, MO). Negative controls treated by solvent
(DMSO).
1
(185 mg). H NMR (CDCl3) d 3.43 (s, 3H, NÁ
3.51 (s, 3H, NÁCH3), 6.74 (d, Jꢀ16.4 Hz, 1H, ethylenic
H), 7.06Á7.12 (m, 3H, ethylenic H and NÁphenyl), 7.51
(d, Jꢀ8.7 Hz, 2H, NÁphenyl), 7.56 (d, Jꢀ8.2 Hz, 2H,
CHÁphenyl), 7.74 (d, Jꢀ8.2 Hz, 2H, CHÁphenyl), 8.44
(s, 1H, CHÄN).
/
CH3),
/
/
/
/
/
/
/
/
/
/
/
2.2.1.1. Flow cytometric assessment of parasitemia
[10,11]. Parasitemia was evaluated after 48 h by a
flow cytometric method derived from the protocol
published by Wyatt et al. [10] and adapted to Plasmo-
dium strain by Van der Heyde et al. [11]. Parasite growth
was assessed by a flow cytometric method in order to
determine the number and viability of the intraerythro-
cytic P. falciparum on the basis of the ability of the
parasite to take up and metabolize hydroethidine (HE)
into ethidium, a DNA-binding fluorochrome. After
incubation with hydroethidine, parasitized and unin-
fected erythrocytes were all identified on the basis of
fluorescence intensity and size. For HE staining, a stock
2.1.5.9. 6-(2-{4-[(3-Bromophenylimino)-methyl]-
phenyl}-vinyl)-1,3-dimethyl-5-nitro-1H,3H-pyrimidine-
2,4-dione (5i). Yellow crystals, m.p. 248 8C, yield 38%
1
(115 mg). H NMR (CDCl3) d 3.37 (s, 3H, NÁ
/
CH3),
16.4 Hz, 1H, ethylenic
7.06 (m, 2H, ethylenic H and NÁphenyl),
7.24 (m, 1H, NÁphenyl), 7.43Á7.56 (m, 3H, CHÁ
phenyl and NÁphenyl), 7.61 (d, Jꢀ7.6 Hz, 1H, NÁ
phenyl), 7.90 (d, Jꢀ8.2 Hz, 2H, CHÁphenyl), 8.31 (s,
1H, CHÄN).
3.46 (s, 3H, NÁ
H), 6.94Á
7.18Á
/
CH3), 6.67 (d, Jꢀ
/
/
/
/
/
/
/
/
/
/
/
/
/
2.1.5.10. 1,3-Dimethyl-6-(2-{4-[(2-methyl-3-
nitrophenylimino)-methyl]-phenyl}-vinyl)-5-nitro-
solution of HE (10 mg/ml) (Interchim, Montlucon,
¸
France) in DMSO was prepared and stored at
1H,3H-pyrimidine-2,4-dione (5j). Yellow crystals, m.p.
103 8C, yield 52% (150 mg). 1H NMR (CDCl3) d 2.51 (s,
ꢂ20 8C. The culture medium was removed from each
/

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N. Azas et al. / Il Farmaco 58 (2003) 1263Á
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1270
Scheme 1.
well of parasite culture plates. Two hundred microliters
of HE diluted at 1/200 in phosphate buffered saline
(PBS; Sigma, St Louis, MO) was added to each well and
incubated for 20 min at 37 8C in the dark. Parasites were
porated into duplicate cultures. After a 48-h incubation
period at 37 8C, viable Trichomonas were identified and
counted microscopically on the basis of their aspect and
motility.
then washed twice in PBS by centrifugation at 400ꢃg
/
for 5 min and were resuspended in a final volume of 1 ml
of PBS in the tubes for fluorescence-activated cell sorter
(FACS) analysis. Flow cytometry data acquisition and
analysis were performed on a FACSort instrument
(Becton-Dickinson, San Jose, CA). The detectors for
forward and side scatter of the FACSort were set to
E-01 and 250, respectively, and both detectors were set
to the logarithmic scale. The FL2 detectors were also
adjusted to bring events within the detection range of
the instrument (generally 459 for FL2). Both infected
and uninfected erythrocytes were gated in the analysis
and the percentage of parasitemia (number of infected
2.2.3. Antileishmanial activity versus promastigotes
[13,14]
Experiments were performed on the referenced strain
L. infantum MHOM/FR/78/LEM75. Promastigotes in
the late log phase were incubated in RPMI medium
supplemented with 12% foetal calf serum (Eurobio,
Paris, France), at an average of 105 cells/ml. Various
concentrations of active compounds were aseptically
dissolved in DMSO (final concentration less than 1.1%
volume/volume) and incorporate in duplicate cultures.
After a 48-h incubation period at 25 8C, parasite growth
was estimated by counting promastigotes with haema-
cytometer. The IC50 represented the concentration of
drug that induced a decrease of 50% promastigotes
compared to a control culture.
erythrocytes/total erythrocytes ꢃ100) was determined
/
using the LYSIS II program (Becton-Dickinson). Ten
thousand cells were used for data acquisition.
The antimalarial activity of derivatives was expressed
by the inhibitory concentrations 50% (IC50), represen-
ting the drug concentration that induced a 50% para-
sitemia decrease compared to control culture. IC50 were
calculated by non-linear regression analysis processed
2.2.4. Toxicity on THP1 cells
In vitro toxicity and antiproliferative activity of
azomethine derivatives were assessed on human mono-
cytes (THP1 cells) maintained in RPMI medium (Euro-
bio, Paris, France) supplemented with 10% foetal calf
serum (Eurobio, Paris, France) at 37 8C in 5% CO2 and
replicated every 7 days. Human monocytes in late log
phase culture (105 cells/ml) were treated in duplicate
assays with various concentrations of drugs and incu-
bated in RPMI medium during 72 h at 37 8C with 5%
CO2 [14]. At the end of the incubation period, cell
growth and viability were estimated by flow cytometry
after staining by propidium iodide. The antiproliferative
activity of chemical compounds was determined by the
inhibitory concentration IC50 that represented the con-
centration of chemical products that induced a 50%
on doseÁresponse curves by the Table Curve software
/
(Jandel Scientific, Paris, France).
2.2.2. Activity versus T. vaginalis
The antiproliferative activity of derivatives versus
parasites of the genus Trichomonas was assessed on
the referenced strain of T. vaginalis (TVR87) maintained
in continuous culture in Trichomonas medium TM 161
(Oxoid) supplemented with 8% heat-inactivated horse
serum (Eurobio, Paris, France) [12]. Parasites in late log-
phase were incubated at an average of 104 cells/ml and a
range of product concentrations were aseptically incor-

N. Azas et al. / Il Farmaco 58 (2003) 1263Á
/
1270
1267
Scheme 2.
Table 1
Azomethine derivatives of the aldehyde 4

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N. Azas et al. / Il Farmaco 58 (2003) 1263Á1270
/
Table 2
Antiplasmodial activity against W2 P. falciparum strain
Comp.
P. falciparum IC50 (mM)
Toxicity versus human cells (THP1 cells)
Therapeutic index (TI)
IC50 (mM)
LC50 (mM)
4
15
32
105
10.5
8
320
2
10
5a
5b
5c
5d
5e
5f
5g
5h
5i
10.5
10.5
2.1
12
ꢀ
/
105
105
40
4
12
24
11.3
5.6
12
11.3
59
2
5
5
59
108
106
53
21
43
108
ꢀ
ꢀ
/
ꢀ/
106
53
/
ꢀ/2.4
5
21
5j
5k
44
15
ꢀ
ꢀ
/
111
ꢀ
ꢀ
/
111
56
ꢀ
ꢀ/
/
2.5
/56
/
4
Table 3
Antileishmanial activity against MHOM/FR/78/LEM75 L. infantum strain
Comp.
L. infantum IC50 (mM)
Toxicity versus human cells (THP1 cells)
Therapeutic index (TI)
IC50 (mM)
LC (mM)
50
3
3.2
105
10.5
2.1
32
105
10.5
8
320
10
5a
5b
5c
5d
5e
5f
5g
5h
5i
ꢀ
/
105
105
40
3.8
12
12
24
11.3
2.2
11.3
59
5
5
12
59
108
106
53
ꢀ
ꢀ/
/
108
106
108
106
ꢀ
/
ꢀ
/
ꢀ/
21.3
53
2.5
5j
5k
55.6
11.1
ꢀ
ꢀ
/
111
ꢀ
ꢀ
/
111
56
ꢀ
ꢀ/
/
2
5
/56
/
decrease of cell growth compared to the control culture.
Toxicity versus human cells was measured by the lethal
concentration LC50 representing the concentrations of
chemical compounds that produced a 50% cell death.
to lead to 1,3,6-trimethyl-5-nitropyrimidine-2,4-dione
(2) in 72% yield [5]. Senda isolated 2 in 93% yield,
stirring 1 in a mixture of concentrated nitric and sulfuric
acids for 1 h at 0 8C [7]. Thus, we used this second
procedure owing to its better yield in 2 and its easier
workup.
1,3,6-Trimethyl-5-nitro-1H,3H-pyrimidine-2,4-dione
(2) reacted by base-catalyzed condensation of 1 with 4-
diethoxymethylbenzaldehyde to give 6-[2-(4-diethoxy-
methylphenyl)vinyl]-1,3-dimethyl-5-nitro-1H, 3H-pyri-
midine-2,4-dione (3) in high yield. A such reaction has
previously reported in our laboratory on a 5-nitroimi-
dazole substrate [8]. After deprotection, the styryl
compound 3 led to the 4-[2-(1,3-dimethyl-5-nitro-2,6-
dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)vinyl]benzalde-
hyde 4 (Scheme 1).
3. Results and discussion
3.1. Chemistry
The required aldehyde 4 was obtained in a four steps
process (Scheme 1), starting from 6-methyl-1H,3H-
pyrimidine-2,4-dione. The first step performed the N-
alkylation of 1H,3H-pyrimidine-2,4-dione ring by di-
methylsulfate (DMS) while the reaction mixture was
maintained alkaline [5,6]. KromovÁ
the nitration stirring 1 in a mixture of concentrated
nitric acid and phosphorus pentoxide for 18 h at ꢂ6 8C
/
Borisov performed
Then, the benzaldehyde derivative 4 was functiona-
lized by a classical condensation reaction with various
/

N. Azas et al. / Il Farmaco 58 (2003) 1263Á
/
1270
1269
Table 4
Trichomonocidal activity against TVR87 T. vaginalis strain
Comp.
T. vaginalis IC50 (mM)
Toxicity versus human cells (THP1 cells)
Therapeutic index (TI)
IC50 (mM)
LC50 (mM)
4
22.2
78
32
105
10.5
8
320
1.4
1.3
5a
5b
5c
5d
5e
5f
5g
5h
5i
ꢀ
/
105
105
40
38
38
16.7
7
12
24
11.3
11.3
59
1.6
1.4
42.3
59
108
106
53
ꢀ
ꢀ/
/
106
106
2
108
106
ꢀ
ꢀ
/
ꢀ
/
/
53
26.5
5j
5k
ꢀ/111
40
ꢀ
ꢀ
/
111
ꢀ
ꢀ
/
111
/56
/56
ꢀ/1.4
substituted anilines and substituted benzylamines in
refluxed ethanol, to give the corresponding azomethine
compounds in good yields (Scheme 2, Table 1).
trations of the corresponding compound were not able
to be assessed owing to it’s low solubility.
Most of the compounds of these series have shown a
more or less interesting antiparasitic activity compared
to their cytotoxicity on THP1 cells. The lower solubility
in DMSO of some derivatives explain the limit values
reported for the IC50 on THP1 cells. Higher concentra-
tions could not be assessed.
Concerning the activity against the Plasmodium
falciparum W2 strain, the functionalization increases
the therapeutic index values for seven azomethines
among these series (Table 2). The best therapeutic index
was obtained for compound 5a which confirmed the
interest of a trifluoromethylbenzyl substituant for anti-
plasmodial activity as shown in previous studies [2].
The functionalization of aldehyde 4 decreases the
leishmanicidal activity: the best therapeutic index (10)
was observed with 4 (Table 3).
The results observed on T. vaginalis show only one
interesting product: compound 5i which presents 26.5 as
therapeutic index value (Table 4).
The derivatives of this new azomethines series dis-
played antiparasitic activities modulated by their amine
moiety. The low solubility in dimethylsulfoxyde of
several compounds did not allow to assess higher
concentrations on human cells which limited their in
vitro therapeutic index values.
3.2. Antiparasitic activity
Antimalarial activity was assessed against a chloro-
quine resistant strain of P. falciparum (W2) maintained
in continuous culture in human erythrocytes. IC50-
represented the drug concentration able to
antimalarial
induce a 50% decrease of infected erythrocytes (Table 2).
Antileishmanial activity was measured on the referenced
strain L. infantum (MHOM/FR/78/LEM75). IC50-antil-
was determined as the concentration of drug
eishmanial
necessary to inhibit 50% of parasite growth (Table 3).
Antitrichomonal activities were assessed on the refe-
renced strain T. vaginalis (TVR87). IC50-trichomonocidal
was expressed as the drug concentration necessary to
induce a 50% decrease of the parasite growth (Table 4).
Toxicity versus human cells was assessed against THP1
cells: IC50-THP1 and LC50-THP1 expressed the drug
concentrations required to induce respectively a 50%
reduction of cell growth and a 50% cell viability drop.
IC50 were calculated by non linear regression analysis of
doseÁresponse curves representing the number of viable
/
parasites and THP1 cells according to drug concentra-
tions and were expressed as the mean values of three
independent experiments. A therapeutic index (TI) was
calculated for each parasite according to the following
Acknowledgements
formula: TItrichomonocidal
TIantileishmanial IC50-THP1/IC50-antileishmanial
TIantimalarial IC50-THP1/IC50-antimalarial. In Tables 2Á
the sign ‘ꢀ’ indicated that the corresponding product
was ineffective against the parasite at the highest
concentration assessed. Concerning the toxicity against
ꢀ
/
IC50-THP1/IC50-trichomonocidal
and
4,
,
ꢀ
/
We are grateful to the Centre National de la Recherche
Scientifique for its financial support. We express our
thanks to Gilles Lanzada for his technical collaboration
and to the Service de Microanalyse de l’Universite´ de
Pharmacie de Chatenay-Malabry which performed the
elemental analyses.
ꢀ
/
/
/
THP1 cells, the sign ‘ꢀ’ indicated that higher concen-
/

1270
N. Azas et al. / Il Farmaco 58 (2003) 1263Á1270
/
series: influence of the nucleophile, Heterocycles 51 (1999) 557Á
565.
[9] W. Trager, J. Jensen, Human malaria parasites in continuous
/
References
[1] Kathleen Parfitt (Ed.), Martindale. The Complete Drug Refer-
ence, 32nd ed., Pharmaceutical Press, London, 1999.
culture, Science 193 (1976) 673Á675.
/
[10] C.R. Wyatt, W. Goff, W.C. Davis, A flow cytometric method for
[2] P. Rathelot, P. Vanelle, M. Gasquet, F. Delmas, M.P. Crozet, P.
Timon-David, J. Maldonado, Synthesis of novel functionalized 5-
nitroisoquinolines and evaluation of in vitro antimalarial activity,
assessing viability of intraerythrocytic hemoparasites, J. Immu-
nol. Methods 140 (1991) 23Á30.
/
[11] H.C. Van der Heyde, M.M. Elloso, J. Vande Waa, K. Schell,
W.P. Weidanz, Use of hydroethidine and flow cytometry to
assess the effects of leukocytes on the malarial parasite
Plasmodium falciparum, Clin. Diagn. Lab. Immunol. 4 (1995)
Eur. J. Med. Chem. 30 (1995) 503Á506.
/
[3] F. Tedlaouti, M. Gasquet, F. Delmas, P. Timon-David, E.
Madadi, P. Vanelle, J. Maldonado, Evaluation of the antimalarial
activity of new compounds against Plasmodium falciparum in
vitro, and Plasmodium berghei in vivo, J. Pharm. Belg. 45 (1990)
417Á425.
/
[12] F. Delmas, C. Di Giorgio, M. Robin, N. Azas, M. Gasquet, C.
Detang, M. Costa, P. Timon-David, In vitro activities of position
2 substitution-bearing 6-nitro- and 6-amino-benzothiazoles and
their corresponding anthranilic acid derivatives against Leishma-
nia infantum and Trichomonas vaginalis, Antimicrob. Agents
306Á310.
/
[4] F. Tedlaouti, M. Gasquet, F. Delmas, B. Majester, P. Timon-
David, E. Madadi, P. Vanelle, J. Maldonado, Synthesis and study
of antiprotozoal activity of some 5-nitrothiophene aldimines,
Farmaco 46 (1991) 1195Á1201.
[5] M.V. Khromov-Borisov, I.M. Yurist, Syntheses and transforma-
tions of pyrimidine derivatives. VI. Preparation of 1-methyl and
/
Chemother. 46 (2002) 2588Á2594.
/
[13] B.W. Ogunkolade, I. Colomb-Valet, L. Monjour, A. Rhodes-
Feuillette, J.P. Abita, D. Frommel, Interactions between the
human monocytic leukaemia THP-1 cell line and Old
and New World species of Leishmania, Acta Trop. 47 (1990)
1,3,7-trimethylisoxanthines, Akad. Nauk. SSSR 1 (1953) 657Á
/
662.
[6] W. Pfleiderer, H. Mosthaf, Investigations in the pyrimidine series.
II. The preparation of pyrido[3,2-d]- and pyrrolo[3,2-d]pyrimi-
171Á176.
/
[14] F. Delmas, C. Di Giorgio, R. Elias, M. Gasquet, N. Azas, V.
Mshvildadze, G. Dekanosidze, E. Kemertelidze, P. Timon-David,
Antileishmanial activity of three saponins isolated from ivy,
alpha-hederin, beta-hederin and hederacolchiside A1, as com-
pared to their action on mammalian cells cultured in vitro, Planta.
dines, Chem. Ber. 90 (1957) 728Á745.
[7] S. Senda, A. Suzui, M. Honda, H. Fujimura, Uracil derivatives
and related compounds. III. 5-amino-1,3,6-trialkyluracil deriva-
/
tives. 1, Chem. Pharm. Bull 6 (1958) 482Á487.
[8] K. Benakli, M. Kaafarani, M.P. Crozet, P. Vanelle, Competition
/
between C- and O-alkylation reactions in the 5-nitroimidazole
Med. 66 (2000) 343Á347.
/