1,3-Diphenylpyrazoles: Synthesis and antiparasitic activities of azomethine derivatives

Article abstract of DOI:10.1016/S0223-5234(02)01388-0

1,3-Diphenylpyrazole-4-carboxaldehyde and 1-(4-nitrophenyl)-3-phenylpyrazole-4-carboxaldehyde were obtained from the appropriated phenylhydrazones via the Vilsmeier-Haack reaction. These two aldehydes were functionalized by various substituted anilines or substituted benzylamines. Antiparasitic activities of the corresponding azomethines were assessed. In the most cases, nitrated compounds were found to be more efficient than non-nitrated ones against Plasmodium falciparum, Trichomonas vaginalis and Leishmania infantum.

Full text of DOI:10.1016/S0223-5234(02)01388-0

Eur. J. Med. Chem. 37 (2002) 671–679
www.elsevier.com/locate/ejmech
Original article
1,3-Diphenylpyrazoles: synthesis and antiparasitic activities of
azomethine derivatives
Pascal Rathelot a, Nadine Azas b, Hussein El-Kashef c, Florence Delmas b,
Carole Di Giorgio b, Pierre Timon-David b, Jose´ Maldonado a, Patrice Vanelle a,*
a Laboratoire de Chimie Organique, UMR CNRS 6517, Faculte´ de Pharmacie, 27 Boule6ard Jean Moulin, 13385 Marseille Cedex 5, France
b Laboratoire de Parasitologie, EA 864, Faculte´ de Pharmacie, 27 Boule6ard Jean Moulin, 13385 Marseille Cedex 5, France
c Chemistry Department, Faculty of Science, Assiut Uni6ersity, 71516 Assiut, Egypt
Received 13 March 2002; received in revised form 6 May 2002; accepted 13 May 2002
Abstract
1,3-Diphenylpyrazole-4-carboxaldehyde and 1-(4-nitrophenyl)-3-phenylpyrazole-4-carboxaldehyde were obtained from the ap-
propriated phenylhydrazones via the Vilsmeier–Haack reaction. These two aldehydes were functionalized by various substituted
anilines or substituted benzylamines. Antiparasitic activities of the corresponding azomethines were assessed. In the most cases,
nitrated compounds were found to be more efficient than non-nitrated ones against Plasmodium falciparum, Trichomonas 6aginalis
´
and Leishmania infantum. © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
Keywords: Diphenylpyrazole; Nitrophenylpyrazole; Azomethine; Antiplasmodial activity; Trichmonocidal activity; Leishmanicidal activity
1. Introduction
between acetophenone and phenylhydrazine. The hy-
drazone derivative was treated with the Vilsmeier–
Haack reagent (DMF–POCl3) leading to the
corresponding 4-carboxaldehyde functionalized pyra-
zole heterocyclic ring in mild operating conditions.
Three equivalents of this reagent, instead of two as
described by Kira [15], were necessary to obtain the
aldehyde 3a in good yields (see Fig. 1).
Pyrazole nucleus [1] has long shown its pharmacolo-
gical interest as antianxiety [2,3], antipyretic, analgesic
and anti-inflammatory drugs [4–6], as well as its good
antimicrobial activities [7–11]. The results we had ob-
tained in previous studies about antiparasitic activity in
nitroheterocyclic series [12–14] and our will to confirm
some structure–activity relationships led us to synthe-
size the 1,3-diphenylpyrazole-4-carboxaldehyde and the
1-(4-nitrophenyl)-3-phenylpyrazole-4-carboxaldehyde.
These two aldehydes were functionalized by condensa-
tion with various amines. The antiparasitic activity of
the corresponding azomethines was assessed against
Plasmodium falciparum, Trichomonas 6aginalis and
Leishmania infantum.
In order to prepare the 1-(4-nitrophenyl)-3-
phenylpyrazole-4-carboxaldehyde 3b, we tried, first, to
nitrate 3a. Hurst [16] had shown that the nitration of
3-methyl-1-phenylpyrazole only occurred on the para
position of the phenyl ring when 1 equiv. of concen-
trated nitric acid was used. With an excess of nitric
acid, the 4-position of the pyrazole ring was also able to
be nitrated in a lower yield. We performed such a
reaction with 3a, using 1 equiv. of concentrated nitric
1
acid in concentrated sulfuric acid. The H-NMR data
2. Chemistry
of the crude residue has been shown four different
CHO proton signals (singlets l=9.90, 10.06, 10.09 and
10.13 ppm) which indicated the multiplicity of the
nitrated positions. Owing to the number of nitrated
compounds obtained, and because of its low solubility,
the crude residue was not purified. Thus, the nitrated
carboxaldehyde 3b has been prepared by the pathway
The 1,3-diphenylpyrazole-4-carboxaldehyde 3a was
prepared in two steps. The first one was the reaction
* Correspondence and reprints
E-mail address: patrice.vanelle@pharmacie.univ-mrs.fr (P. Vanelle).
´
0223-5234/02/$ - see front matter © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
PII: S0223-5234(02)01388-0

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P. Rathelot et al. / European Journal of Medicinal Chemistry 37 (2002) 671–679
against THP1 cells: IC50-THP1 and LC50-THP1 expressed
the drug concentrations required to induce, respec-
tively, a 50% reduction of cell growth and a 50% cell
viability drop. IC50 were calculated by non-linear re-
gression analysis of dose–response curves representing
the number of viable parasites, infected cells or non-in-
fected THP1 cells according to drug concentrations and
were expressed as the mean values of three independent
experiments. A therapeutic index (TI) was calculated
for each parasite according to the following formula:
used to prepare 3a, starting with para-acetophenone
instead of acetophenone (see Fig. 1).
The two aldehydes 3a and 3b were functionalized by
a classical condensation reaction [17] with various sub-
stituted anilines and substituted benzylamines in
refluxed methanol or ethanol, with traces of concen-
trated acetic acid, to give the corresponding azomethine
derivatives (see Fig. 2 and Table 1).
3. Pharmacology
TItrichomonocidal=IC50-THP1/IC50-trichomonocidal
TIantileishmanial=IC50-THP1/IC50-antileishmanial
TIantimalarial=IC50-THP1/IC50-antimalarial
and
Antimalarial activity was assessed against
a
.
chloroquine resistant strain of P. falciparum (W2)
maintained in continuous culture in human erythro-
cytes. IC50-antimalarial represented the drug concentration
able to induce a 50% decrease of infected erythrocytes.
Antileishmanial activity was measured on amastigote
forms of L. infantum infecting human monocyte-
derived macrophages (THP1 cells). IC50-antileishmanial
evaluated the drug concentration that produces a 50%
reduction of infected macrophages. Activity against T.
6aginalis was evaluated versus in vitro parasite culture.
IC50-trichomonocidal was expressed as the drug concentra-
tion necessary to induce a 50% decrease of the parasite
growth. Toxicity versus human cells was assessed
The highest concentration assessed against the para-
sites was 100 mg/mL. In Tables 2–4, the sign ‘\’
indicated that the corresponding product was ineffec-
tive against the parasite at such a concentration. Con-
cerning the toxicity against THP1 cells, the sign ‘\’
indicated that higher concentrations of the correspon-
ding compound were not able to be assessed owing to
it’s low solubility.
The THP1 cells were employed in the toxicity assays,
because of their use in the culture of the amastigote
forms of Leishmania so the products could be tested
against the parasites only at a concentration lower than
the IC50 against THP1 cells.
Fig. 1. Synthesis of 1,3-diphenylpyrazole-4-carboxaldehyde (3a) and 1-(4-nitrophenyl)-3-phenylpyrazole-4-carboxaldehyde (3b).
Fig. 2. Reactions of 3a–b with various substituted anilines or substituted benzylamines.

P. Rathelot et al. / European Journal of Medicinal Chemistry 37 (2002) 671–679
673
Table 1
Azomethine derivatives of the aldehydes 3a and 3b
4. Results and discussion
activity compared with aldehyde 3a, excepted for com-
pound 4a10, substituted by a 2-trifluoromethylbenzyl
group, which exhibits a TI of 4.9. Among the deriva-
tives from the nitrated aldehyde 3b (4b1–7), com-
pounds 4b2, 4b5 and 4b7, respectively, substituted by a
The Table 2 displayed the antiplasmodial activity of
aldehyde derivatives. The azomethines synthesized from
the non-nitrated aldehyde 3a have been shown a lower

674
P. Rathelot et al. / European Journal of Medicinal Chemistry 37 (2002) 671–679
p-tolyl, a 2-methyl-3-nitrophenyl or a 3-trifluoromethyl-
benzyl group, have been shown a better antiplasmodial
activity compared with 3b. The TI of the most active
compound 4b7 was 20.2. The IC50 observed for 4a10
dal activity. Similarly to antiplasmodial activity, func-
tionalization of aldehyde 3a had not enhanced the
efficiency against the T. 6aginalis strain. Nitrated deriva-
tives are more interesting than 4a1–10 derivatives, but
their low solubility in dimethylsulfoxyde as solvent did
not allow to assess higher concentrations on THP1 cells,
which explain the limit values reported for their TI.
These azomethines (4b1–7) have been shown a similar
activity compared with aldehyde 3b.
and 4b7 have confirmed the interest of
a tri-
fluoromethylbenzyl substituent for antiplasmodial acti-
vity as observed previously in nitroisoquinolines series
[12].
The Table 3 reported the assessment of trichomonaci-
Table 2
Antimalarial activity against W2 P. falciparum strain
Compound IC50 (mM) P. falciparum
Toxicity versus human cells (THP1 cells)
Therapeutic index
Toxicity LC50 (mM)
Antiproliferative activity IC50 (mM)
3a
32
\60
56
\60
48
\50
\50
\52
\52
\54
50
68
\262
35
\201
50
137
56
140
27
28
65
105
\201
47
74
56
70
3
13
13
4
\6.3
1.3
4a1
4a2
4a3
4a4
4a5
4a6
4a7
4a8
4a9
4a10
3b
4b1
4b2
4b3
4b4
4b5
4b6
4b7
1.5
13
246
13
246
\340
262
262
\248
\224
\234
\234
\222
4.9
5
\340
\262
\262
\248
\224
\234
\234
\222
7.5
\2
122
\224
35
61
11
\6.7
\3.8
\20.2
Table 3
Trichomonocidal activity against TVR87 T. 6aginalis strain
Compound
IC50 (mM) T. 6aginalis
Toxicity versus human cells (THP1 cells)
Therapeutic index
Toxicity LC50 (mM)
Antiproliferative activity IC50 (mM)
3a
50
\201
50
137
56
140
27
28
65
105
\201
47
74
56
70
3
13
13
4
\4
4a1
4a2
4a3
4a4
4a5
4a6
4a7
4a8
4a9
4a10
3b
4b1
4b2
4b3
4b4
4b5
4b6
4b7
\148
\142
\148
\140
\124
\76
\130
\130
\134
\246
51
130
65
122
33
81
61
55
13
246
13
246
\340
262
262
\248
\224
\234
\234
\222
\340
\262
\262
\248
\224
\234
\234
\222
\6.6
2
4
\2
\6
\3
\3.8
\4

P. Rathelot et al. / European Journal of Medicinal Chemistry 37 (2002) 671–679
675
Table 4
Antileishmanial activity against MHOM/FR/78/LEM75 L. infantum strain (amastigote forms)
Compound
IC50 (mM) L. infantum
Toxicity versus human cells (THP1 cells)
Therapeutic index
Toxicity LC50 (mM)
Antiproliferative activity IC50 (mM)
3a
\201
\47
\74
14
\70
\3
13
\13
\4
\13
\246
\340
\262
\262
12.4
\224
\233
23
\201
50
137
56
140
27
28
65
105
\201
47
74
56
70
3
13
13
4
4a1
4a2
4a3
4a4
4a5
4a6
4a7
4a8
4a9
4a10
3b
4b1
4b2
4b3
4b4
4b5
4b6
4b7
4
1
13
246
13
246
\340
262
262
\248
\224
\234
\234
\222
\340
\262
\262
\248
\224
\234
\234
\222
\20
\10.2
\222
Results of antileishmanial activity were presented in
Table 4. Most of the compounds assessed, including
aldehydes 3a and 3b, were ineffective against L. infan-
tum at the highest concentration assessed. Among the
non-nitrated derivatives, 4a3 showed a TI of 4. Nitrated
azomethine 4b6 substituted by a 4-methyl-3-nitrophenyl
moiety presented a TI upper than 10. The most effec-
tive compound is the 4-chlorophenyl substituted azome-
thine 4b3, which exhibited a TI upper than 20.
singlet; d, doublet; t, triplet; q, quartet; m, a more
complex multiplet or overlapping multiplets. The fol-
lowing adsorbent was used for column chromatogra-
phy: silica gel 60 (Merck, particle size 0.063–0.200 mm,
70–230 mesh ASTM). Microanalyses for C, H, N were
performed by the Ser6ice de Microanalyse de l’Uni6er-
site´ de Pharmacie de Chaˆtenay-Malabry.
6.1.1. Synthesis of the hydrazones 2a and 2b
Concentrated acetic acid (1 mL) and phenylhydrazine
hydrochloride (1a) (3 g, 20 mmol) or 4-nitrophenylhy-
drazine (1b) (3.68 g, 24 mmol) were added to a solution
of acetophenone (2.88 g, 24 mmol) in 90 mL of ethanol.
Then, the reaction mixture was refluxed for 1 h at room
temperature (r.t.). The precipitate was filtered and
washed with ethanol. After drying in vacuum over
P2O5, 8.05 g (92%) of 2a or 4.24 g (83%) of 2b were
obtained.
5. Conclusions
The derivatives of this 1,3-diphenylpyrazole series
have shown modest antiparasitic activities. Their low
solubility did not allow to assess higher concentrations
on THP1 cells, which limit the in vitro TI values.
According to the mutagenicity of nitro derivatives,
further studies related to the assessment of mutagenic
potentialities of this series will be necessary to complete
this pharmacological evaluation.
Acetophenone phenylhydrazone (2a): yellow solid,
1
m.p.=104 °C (litt. 105 °C [19]). H-NMR (CDCl3) l
2.24 (s, 3H, CH3), 7.20–7.46 (m, 9H, H3,4,5 1-phenyl,
H2,3,4,5,6 3-phenyl, NH), 7.81–7.85 (m, 2H, H2,6 1-
phenyl).
Acetophenone 4-nitrophenylhydrazone (2b): orange
solid, m.p.=183 °C (litt. 185 °C [19]). 1H-NMR
(CDCl3) l 2.32 (s, 3H, CH3), 7.21 (d, J=9.2 Hz, 2H,
H2,6 nitrophenyl), 7.37–7.46 (m, 3H, H3,4,5 3-phenyl),
7.79–7.84 (m, 3H, H2,6 3-phenyl, NH), 8.21 (d, J=9.2
Hz, 2H, H3,5 nitrophenyl).
6. Experimental
6.1. Chemistry
Melting points (m.p.) were determined on a Bu¨chi
1
B-540 apparatus and are uncorrected. H-NMR spectra
were determined on a Bruker ARX 200 spectrometer.
The 1H chemical shifts were reported as parts per
million downfield from tetramethylsilane (Me4Si). Ab-
sorptions were reported with the following notations: s,
6.1.2. Vilsmeier–Haack reaction
Dimethylformamide (2.58 g, 35.3 mmol) and POCl3
(5.4 g, 35.3 mmol) were previous separately cooled at

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P. Rathelot et al. / European Journal of Medicinal Chemistry 37 (2002) 671–679
0 °C before being stirred at such temperature. A
solution of 2a (3 g, 11.76 mmol) or 2b (3 g, 11.76
mmol) in DMF (3 mL) was added dropwise to the
reaction mixture which was, then, warmed at r.t. and
heated at 70–80 °C for 5 h. After cooling at r.t., the
mixture was basified with a cool saturated K2CO3
solution. The precipitate was filtered, strongly washed
with water and crystallized from ethanol, yielding 95%
(2.78 g) of 3a or 94% (3.25 g) of 3b.
1,3-Diphenylpyrazole-4-carboxaldehyde (3a): beige
needles, m.p.=145–147 °C (litt. 140 °C [15]), 1H-
NMR (CDCl3) l 7.29–7.56 (m, 6H, H3,4,5 1-phenyl,
H3,4,5 3-phenyl), 7.77–7.85 (m, 4H, H2,6 1-phenyl, H2,6
3-phenyl), 8.59 (s, 1H, H5), 10.11 (s, 1H, CHO). Anal.
Calc. for C16H12N2O: C, 77.40; H, 4.87; N, 11.28.
Found: C, 77.28; H, 4.94; N, 11.19%.
(s, 1H, CHꢀN). Anal. Calc. for C24H21N3: C, 82.02; H,
6.02; N, 11.96. Found: C, 81.90; H, 6.09; N, 11.93%.
6.1.3.3.
(1,3-Diphenylpyrazol-4-ylmethylene)-p-tolyl-
amine (4a3). Beige crystals, m.p.=131 °C (litt. 128–
1
130 °C [18]). H-NMR (CDCl3) l 2.37 (s, 3H, CH3),
7.10 (d, J=8.4 Hz, 2H, H3,5 N-phenyl), 7.20 (d, J=8.4
Hz, 2H, H2,6 N-phenyl), 7.41–7.56 (m, 6H, H3,4,5 1-
phenyl, H3,4,5 3-phenyl), 7.70–7.76 (m, 2H, H2,6 3-
phenyl), 7.81–7.86 (m, 2H, H2,6 1-phenyl), 8.54 (s, 1H,
H5), 8.67 (s, 1H, CHꢀN). Anal. Calc. for C23H19N3: C,
81.87; H, 5.68; N, 12.45. Found C: 81.92; H, 5.70; N,
12.41%.
6.1.3.4.
(4-Chlorophenyl)-(1,3-diphenylpyrazol-4-yl-
1-(4-Nitrophenyl)-3-phenylpyrazole-4-carboxaldehy-
de (3b): brown solid, m.p.=210–212 °C (litt. 210 °C
[15]), 1H-NMR (CDCl3) l 7.52–7.57 (m, 3H, H3,4,5
3-phenyl), 7.80–7.85 (m, 2H, H2,6 3-phenyl), 8.03 (d,
J=9.1 Hz, 2H, H2,6 nitrophenyl), 8.41 (d, J=9.1 Hz,
2H, H3,5 nitrophenyl), 8.67 (s, 1H, H5), 10.09 (s, 1H,
CHO). Anal. Calc. for C16H11N3O3: C, 65.53; H, 3.78;
N, 14.33. Found: C, 65.61; H, 3.96; N, 13.99%.
methylene)-amine (4a4). Yellow solid, m.p.=161 °C
(litt. 163–165 °C [18]). H-NMR (CDCl3) l 7.11 (d,
1
J=8.7 Hz, 2H, H3,5 N-phenyl), 7.34 (d, J=8.7 Hz, 2H,
H2,6 N-phenyl), 7.39–7.55 (m, 6H, H3,4,5 1-phenyl,
H3,4,5 3-phenyl), 7.69-7.78 (m, 2H, H2,6 3-phenyl), 7.80-
7.87 (m, 2H, H2,6 1-phenyl), 8.49 (s, 1H, H5), 8.66 (s,
1H, CHꢀN). Anal. Calc. for C22H16ClN3: C, 73.84; H,
4.51; N, 11.74. Found: C, 73.76; H, 4.47; N, 11.80%.
6.1.3. General procedure for preparation of aldimines
Compound 3a (0.5 g, 2.02 mmol) was dissolved in
boiling methanol (15 mL). Then, 0.2 mL of concen-
trated acetic acid and 2.22 mmol of amine derivative
were added and the reaction mixture was refluxed for 7
h. After cooling at r.t., the precipitate was isolated by
filtration, washed with water and crystallized.
Compound 3b (0.5 g, 1.70 mmol) was dissolved in
boiling ethanol (40 mL). Then, 0.2 mL of concentrated
acetic acid and 1.87 mmol of amine derivative were
added and the reaction mixture was refluxed for 7 h.
After cooling at r.t., the precipitate was isolated by
filtration, and washed with water and crystallized.
6.1.3.5.
(4-Bromophenyl)-(1,3-diphenylpyrazol-4-yl-
methylene)-amine (4a5). Yellow powder, m.p.=157 °C.
1H-NMR (CDCl3) l 7.05 (d, J=8.7 Hz, 2H, H3,5
N-phenyl), 7.38 (d, J=8.7 Hz, 2H, H2,6 N-phenyl),
7.42–7.54 (m, 6H, H3,4,5 1-phenyl, H3,4,5 3-phenyl),
7.71–7.75 (m, 2H, H2,6 3-phenyl), 7.82–7.86 (m, 2H,
H2,6 1-phenyl), 8.49 (s, 1H, H5), 8.66 (s, 1H, CHꢀN).
Anal. Calc. for C22H16BrN3: C, 65.68; H, 4.01; N,
10.45. Found: C, 65.69; H, 4.06; N, 10.34%.
6.1.3.6.
(1,3-Diphenylpyrazol-4-ylmethylene)-(3-tri-
fluoromethylphenyl)-amine (4a6). White crystals, m.p.=
133 °C. 1H-NMR (CDCl3) l 7.30–7.38 (m, 2H, N-
phenyl), 7.40–7.57 (m, 8H, H3,4,5 1-phenyl, H3,4,5
3-phenyl, 2H, N-phenyl), 7.72–7.78 (m, 2H, H2,6 3-
phenyl), 7.83–7.88 (m, 2H, H2,6 1-phenyl), 8.53 (s, 1H,
H5), 8.68 (s, 1H, CHꢀN). Anal. Calc. for C23H16F3N3:
C, 70.58; H, 4.12; N, 10.74. Found: C, 70.57; H, 4.11;
N, 10.77%.
6.1.3.1.
(1,3-Diphenylpyrazol-4-ylmethylene)-o-tolyl-
amine (4a1). White crystals, m.p.=130 °C (litt. 125–
1
127 °C [18]). H-NMR (CDCl3) l 2.38 (s, 3H, CH3),
6.88 (dd, J=6.8 Hz, J=1.7 Hz, 1H, H3 N-phenyl),
7.11–7.24 (m, 2H, H4,5 N-phenyl), 7.32–7.55 (m, 6H,
H3,4,5 1-phenyl, H3,4,5 3-phenyl), 7.73–7.79 (m, 2H, H2,6
3-phenyl), 7.82–7.88 (m, 2H, H2,6 1-phenyl), 8.44 (s,
1H, H5), 8.66 (s, 1H, CHꢀN). Anal. Calc. for C23H19N3:
C, 81.87; H, 5.68; N, 12.45. Found: C, 81.75; H, 5.59;
N, 12.53%.
6.1.3.7. (2-Methyl-3-nitrophenyl)-(1,3-diphenylpyrazol-
4-ylmethylene)-amine (4a7). Beige solid, m.p.=188 °C.
1H-NMR (CDCl3) l 2.52 (s, 3H, CH3), 7.08 (d, J=7.7
Hz, 1H, H6 N-phenyl), 7.30–7.56 (m, 7H, H3,4,5 1-
phenyl, H3,4,5 3-phenyl, H6 N-phenyl), 7.67 (d, J=7.7
Hz, 1H, H4 N-phenyl), 7.72–7.77 (m, 2H, H2,6 3-
phenyl), 7.83–7.87 (m, 2H, H2,6 1-phenyl), 8.41 (s, 1H,
H5), 8.67 (s, 1H, CHꢀN). Anal. Calc. for C23H18N4O2:
C, 72.24; H, 4.74; N, 14.65. Found: C, 72.25; H, 4.69;
N, 14.52%.
6.1.3.2. (2,6-Dimethylphenyl)-(1,3-diphenylpyrazol-4-yl-
methylene)-amine (4a2). Brown crystals, m.p.=146 °C.
1H-NMR (CDCl3) l 2.21 (s, 6H, 2CH3), 6.92–7.10 (m,
3H, H3,4,5 N-phenyl), 7.37–7.56 (m, 6H, H3,4,5 1-phenyl,
H3,4,5 3-phenyl), 7.69–7.76 (m, 2H, H2,6 3-phenyl),
7.83–7.90 (m, 2H, H2,6 1-phenyl), 8.30 (s, 1H, H5), 8.74

P. Rathelot et al. / European Journal of Medicinal Chemistry 37 (2002) 671–679
677
6.1.3.8. (4-Methyl-3-nitrophenyl)-(1,3-diphenylpyrazol-
4-ylmethylene)-amine (4a8). Beige solid, m.p.=204 °C.
1H-NMR (CDCl3) l 2.60 (s, 3H, CH3), 7.33–7.54 (m,
8H, H3,4,5 1-phenyl, H3,4,5 3-phenyl, H5,6 N-phenyl),
7.70–7.77 (m, 3H, H2,6 3-phenyl, H2 N-phenyl), 7.83–
7.87 (m, 2H, H2,6 1-phenyl), 8.53 (s, 1H, H5), 8.67 (s,
1H, CHꢀN). Anal. Calc. for C23H18N4O2: C, 72.24; H,
4.74; N, 14.65. Found: C, 72.30; H, 4.71; N, 14.71%.
phenyl), 8.50 (s, 1H, H5), 8.76 (s, 1H, CHꢀN). Anal.
Calc. for C22H15ClN4O: C, 65.59; H, 3.75; N, 13.91.
Found: C, 65.65; H, 3.78; N, 13.76%.
6.1.3.14. (4-Bromophenyl)-[1-(4-nitrophenyl)-3-phenyl-
pyrazol-4-ylmethylene]-amine (4b4). Yellow needles,
m.p.=191 °C. 1H-NMR (CDCl3) l 7.06 (d, J=8.7
Hz, 2H, H3,5 N-phenyl), 7.48-7.56 (m, 5H, H3,4,5 3-
phenyl, H2,6 N-phenyl), 7.70–7.75 (m, 2H, H2,6 3-
phenyl), 8.04 (d, J=9.2 Hz, 2H, H2,6 nitrophenyl), 8.40
(d, J=9.2 Hz, 2H, H3,5 nitrophenyl), 8.50 (s, 1H, H5);
8.76 (s, 1H, CHꢀN). Anal. Calc. for C22H15BrN4O: C,
59.08; H, 3.38; N, 12.53. Found: C, 59.18; H, 3.47; N,
12.39%.
6.1.3.9.
(2-Chlorobenzyl)-(1,3-diphenylpyrazol-4-yl-
methylene)-amine (4a9). White crystals, m.p.=131 °C.
1H-NMR (CDCl3) l 4.86 (s, 2H, CH2), 7.18–7.54 (m,
10H, H3,4,5 1-phenyl, H3,4,5 3-phenyl, H3,4,5,6 N-benzyl),
7.67–7.74 (m, 2H, H2,6 3-phenyl), 7.77–7.83 (m, 2H,
H2,6 1-phenyl), 8.50 (s, 1H, H5), 8.57 (s, 1H, CHꢀN).
Anal. Calc. for C23H18ClN3: C, 74.29; H, 4.88; N,
11.30. Found: C, 74.27; H, 4.91; N, 11.19%.
6.1.3.15. (2-Methyl-3-nitrophenyl)-[1-(4-nitrophenyl)-3-
phenylpyrazol-4-ylmethylene]-amine (4b5). Yellow solid,
1
m.p.=219 °C. H-NMR (CDCl3) l 2.52 (s, 3H, CH3),
6.1.3.10.
(1,3-Diphenylpyrazol-4-ylmethylene)-(2-tri-
7.08 (d, J=7.5 Hz, 1H, H6 N-phenyl), 7.33 (dd, J=8.0
Hz, J=7.5 Hz, 1H, H5 N-phenyl), 7.51–7.53 (m, 3H,
H3,4,5 3-phenyl), 7.67–7.76 (m, 3H, H2,6 3-phenyl, H4
N-phenyl), 8.06 (d, J=9.2 Hz, 2H, H2,6 nitrophenyl),
8.40 (d, J=9.2 Hz, 2H, H3,5 nitrophenyl), 8.42 (s, 1H,
H5), 8.78 (s, 1H, CHꢀN). Anal. Calc. for C23H17N5O4:
C, 64.63; H, 4.01; N, 16.39. Found C, 64.48; H, 4.02;
N, 16.21%.
fluoromethylbenzyl)-amine (4a10). White crystals,
m.p.=99 °C. H-NMR (CDCl3) l 4.95 (s, 2H, CH2),
1
7.30–7.58 (m, 10H, H3,4,5 1-phenyl, H3,4,5 3-phenyl,
H3,4,5,6 N-benzyl), 7.62–7.73 (m, 2H, H2,6 3-phenyl),
7.79–7.84 (m, 2H, H2,6 1-phenyl), 8.49 (s, 1H, H5), 8.57
(s, 1H, CHꢀN). Anal. Calc. for C24H18F3N3: C, 71.10;
H, 4.48; N, 10.36. Found: C, 71.17; H, 4.54; N, 10.30%.
6.1.3.11.
[1-(4-Nitrophenyl)-3-phenylpyrazol-4-yl-
6.1.3.16. (4-Methyl-3-nitrophenyl)-[1-(4-nitrophenyl)-3-
phenylpyrazol-4-ylmethylene]-amine (4b6). Brown solid,
m.p.=190 °C. H-NMR (CDCl3) l 2.61 (s, 3H, CH3),
methylene]-o-tolylamine (4b1). Yellow crystals, m.p.=
194 °C. H-NMR (CDCl3) l 2.38 (s, 3H, CH3), 6.88
1
1
(dd, J=7.5 Hz, J=1.8 Hz, 1H, H3 N-phenyl), 7.13–
7.22 (m, 3H, H4,5,6 N-phenyl), 7.47-7.54 (m, 3H, H3,4,5
3-phenyl), 7.74–7.78 (m, 2H, H2,6 3-phenyl), 8.05 (d,
J=9.2 Hz, 2H, H2,6 nitrophenyl), 8.40 (d, J=9.2 Hz,
2H, H3,5 nitrophenyl), 8.44 (s, 1H, H5), 8.76 (s, 1H,
CHꢀN). Anal. Calc. for C23H18N4O2: C, 72.24; H, 4.74;
N, 14.65. Found: C, 72.25; H, 4.75; N, 14.67%.
7.34–7.35 (m, 2H, H5,6 N-phenyl), 7.51-7.59 (m, 3H,
H3,4,5 3-phenyl), 7.70–7.78 (m, 2H, H2,6 3-phenyl), 7.95
(s, 1H, H2 N-phenyl), 8.05 (d, J=9.2 Hz, 2H, H2,6
nitrophenyl), 8.41 (d, J=9.2 Hz, 2H, H3,5 nitrophenyl),
8.53 (s, 1H, H5), 8.78 (s, 1H, CHꢀN). Anal. Calc. for
C23H17N5O4: C, 64.63; H, 4.01; N, 16.39. Found: C,
64.54; H, 3.92; N, 16.46%.
6.1.3.12.
[1-(4-Nitrophenyl)-3-phenylpyrazol-4-yl-
6.1.3.17.
[1-(4-Nitrophenyl)-3-phenylpyrazol-4-yl-
methylene]-p-tolylamine (4b2). Brown needles, m.p.=
192 °C. 1H-NMR (CDCl3) l 2.39 (s, 3H, CH3), 7.10 (d,
J=8.4 Hz, 2H, H3,5 N-phenyl), 7.21 (d, J=8.4 Hz, 2H,
H2,6 N-phenyl), 7.47–7.57 (m, 3H, H3,4,5 3-phenyl),
7.70–7.76 (m, 2H, H2,6 3-phenyl), 8.03 (d, J=9.2 Hz,
2H, H2,6 nitrophenyl), 8.39 (d, J=9.2 Hz, 2H, H3,5
nitrophenyl), 8.53 (s, 1H, H5), 8.76 (s, 1H, CHꢀN).
Anal. Calc. for C23H18N4O2: C, 72.24; H, 4.74; N,
14.65. Found: C, 72.26; H, 4.73; N, 14.69%.
methylene]-(3-trifluoromethylbenzyl)-amine (4b7). Yel-
low solid, m.p.=105 °C. H-NMR (CDCl3) l 4.83 (s,
1
2H, CH2), 7.51–7.60 (m, 7H, H3,4,5 3-phenyl, H2,4,5,6
N-phenyl), 7.69–7.73 (m, 2H, H2,6 3-phenyl), 8.00 (d,
J=9.2 Hz, 2H, H2,6 nitrophenyl), 8.41 (d, J=9.2 Hz,
2H, H3,5 nitrophenyl), 8.50 (s, 1H, H5), 8.66 (s, 1H,
CHꢀN). Anal. Calc. for C24H17F3N4O2: C, 64.00; H,
3.80; N, 12.44. Found: C, 63.83; H, 3.87; N, 12.30%.
6.2. Pharmacology
6.1.3.13. (4-Chlorophenyl)-[1-(4-nitrophenyl)-3-phenyl-
pyrazol-4-ylmethylene]-amine (4b3). Yellow crystals,
m.p.=194 °C. 1H-NMR (CDCl3) l 7.12 (d, J=8.7
Hz, 2H, H3,5 N-phenyl), 7.35 (d, J=8.7 Hz, 2H, H2,6
N-phenyl), 7.50–7.58 (m, 3H, H3,4,5 3-phenyl), 7.71–
7.75 (m, 2H, H2,6 3-phenyl), 8.04 (d, J=9.2 Hz, 2H,
H2,6 nitrophenyl), 8.39 (d, J=9.2 Hz, 2H, H3,5 nitro-
6.2.1. Antiplasmodial acti6ity
Assays were performed against a chloroquino-resis-
tant strain (W2) of P. falciparum maintained in conti-
nuous culture according to the methodology described
by Trager [20]. Parasites were cultivated in group A+
human erythrocytes and suspended at a 4% hematocrit

678
P. Rathelot et al. / European Journal of Medicinal Chemistry 37 (2002) 671–679
in RPMI 1640 medium supplemented with Hepes,
NaHCO3, 10% A+human serum and Neomycin
(Sigma, St Louis, MO, USA) at 37 °C in a gas mixture
of 5% O2–6% CO2–90% N2; RPMI 1640, Hepes,
NaHCO3 were obtained from Gibco-BRL (Paisley,
Scotland).
Assays were performed in triplicate in 96 well tissue
culture plates (Nunc Brand products, Fisher, Paris,
France) containing 200 mL of W2 asynchronous para-
site cultures at 2% of parasitemia and 2% hematocrit,
and 5 mL of the appropriate drug or drug combination
dissolved in dimethylsulfoxide (DMSO, Sigma). Nega-
tive controls treated by solvent (DMSO) and positive
controls containing a range of chloroquine (Sigma)
concentrations were added to each set of experiments.
were calculated by non-linear regression analysis pro-
cessed on dose–response curves by the Table Curve
software (Jandel Scientific, Paris, France).
6.2.2. Acti6ity 6ersus Trichomonas 6aginalis
The antiproliferative activity of derivatives versus
parasites of the genus Trichomonas was assessed on the
referenced strain of T. 6aginalis (TVR87) maintained in
continuous culture in Trichomonas medium TM 161
(Oxoid) supplemented with 8% heat-inactivated horse
serum (Eurobio, Paris, France) [23]. Parasites in late
log-phase were incubated at an average of 104 cells per
mL and a range of product concentrations was asepti-
cally incorporated into duplicate cultures. After a 48 h
incubation period at 37 °C, viable Trichomonas were
identified and counted microscopically on the basis of
their aspect and motility.
6.2.1.1. Flow cytometric assessment of parasitemia. Para-
sitemia was evaluated after 48 h by a flow cytometric
method derived from the protocol published by Wyatt
et al. [21] and adapted to Plasmodium strain by Van der
Heyde et al. [22]. Parasite growth was assessed by a
flow cytometric method in order to determine the num-
ber and viability of the intraerythrocytic 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 HE, para-
sitized and uninfected erythrocytes were all identified
on the basis of fluorescence intensity and size. For HE
staining, a stock solution of HE (10 mg/mL) (Inter-
chim, Montluc¸on, France) in DMSO was prepared and
stored at −20 °C. The culture medium was removed
from each well of parasite culture plates. Two hundred
microliters of HE diluted at 1/200 in phosphate
buffered saline (PBS; Sigma) was added to each well
and incubated for 20 min at 37 °C in the dark. Para-
sites were then washed twice in PBS by centrifugation
at 400×g for 5 min and were resuspended in a final
volume of 1 mL of PBS in the tubes for fluorescence-ac-
tivated 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 erythrocytes/total erythrocytes×
100) was determined using the LYSIS II program (Bec-
ton-Dickinson). 10 000 cells were used for data
acquisition.
6.2.3. Antileishmanial acti6ity
Experiments were performed on the referenced strain
L. infantum MHOM/FR/78/LEM75. Intracellular
amastigote cultures were performed in human-derived
macrophages (THP1) cells according to the methodo-
logy described by Ogunkolade et al. [24]. Maturation of
adherent macrophages was performed by treating expo-
nentially-growing monocytes (105 cells per mL) with 1
mM phorbol myristate acetate (Sigma). After a 48 h
incubation period at 37 °C in chamber-slides (Fisher),
cells were rinsed with fresh medium and suspended in
RPMI medium containing stationary-phase Leishmania
promastigotes (cells/promastigotes ratio=1/10). After
a 24 h incubation period at 37 °C, promastigotes were
removed by four successive watches with fresh medium.
Adapted dilutions of drugs were added in duplicate
chambers and cultures were incubated for 96 h at
37 °C. Cells were stained with 10% Giemsa stain (Eu-
robio). The percentage of infected macrophages in each
assay was determined microscopically at 1000 times
magnification. The IC50 represented the concentration
of drug that induced a decrease of 50% of infected
human monocytes compared with a control culture.
6.2.4. 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) supplemented with 10% foetal calf serum
(Eurobio) at 37 °C in 5% CO2 and replicated every 7
days. Human monocytes in late log phase culture (105
cells per mL) were treated in duplicate assays with
various concentrations of drugs and incubated in
RPMI medium during 72 h at 37 °C with 5% CO2 [25].
At the end of the incubation period, cell growth and
viability were estimated by flow cytometry after stain-
ing by propidium iodide. The antiproliferative activity
of chemical compounds was determined by the in-
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 with control culture. IC50

P. Rathelot et al. / European Journal of Medicinal Chemistry 37 (2002) 671–679
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Acknowledgements
We are grateful to the Centre National de la
Recherche Scientifique for its financial support. We
express our thanks to Muriel Costa and Gilles Lanzada
for their technical collaboration and to the Ser6ice de
Microanalyse de l’Uni6ersite´ de Pharmacie de Chatenay-
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