Nematocidal and trichomonacidal activities of 2-substituted quinolines

Article abstract of DOI:10.1016/j.farmac.2004.11.010

Several quinolines were synthesized and evaluated in vitro and in vivo against the nematodes Caenorhabditis elegans, Heligmosomoides polygyrus and the protozoa Trichomonas vaginalis. If some of them have shown in vitro nematocide activity (at 10 μM), however, their trichomonacidal activity reached 50% reduction at only 100 μM. The in vivo activity on Trichinella spiralis model was evaluated for some of the most in vitro active quinolines.

Full text of DOI:10.1016/j.farmac.2004.11.010

Il Farmaco 60 (2005) 219–224
http://france.elsevier.com/direct/FARMAC/
Nematocidal and trichomonacidal activities of 2-substituted quinolines
a
b
a
Mercedes Martínez-Grueiro , Consuelo Giménez-Pardo , Alicia Gómez-Barrio ,
c
c,d
c
c,
Xavier Franck , Alain Fournet , Reynald Hocquemiller , Bruno Figadère *,
b,
Nieves Casado-Escribano *
a
Departamento de Parasitología, Universidad Complutense, 28040 Madrid, Spain
b
Departamento de Microbiología y Parasitología, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain
c
Laboratoire de Pharmacognosie (associé au CNRS-BioCIS) Faculté de Pharmacie, Université de Paris-Sud, rue J.B. Clément,
2296 Châtenay-Malabry, France
9
d
Institut de recherche pour le développement, 213, rue La Fayette, 75480 Paris cedex, France
Received 6 August 2004; revised 16 november 2004; accepted 23 November 2004
Abstract
Several quinolines were synthesized and evaluated in vitro and in vivo against the nematodes Caenorhabditis elegans, Heligmosomoides
polygyrus and the protozoa Trichomonas vaginalis. If some of them have shown in vitro nematocide activity (at 10 µM), however, their
trichomonacidal activity reached 50% reduction at only 100 µM. The in vivo activity on Trichinella spiralis model was evaluated for some of
the most in vitro active quinolines.
©
2005 Elsevier SAS. All rights reserved.
Keywords: Synthesis; Substituted quinolines; Nematocidal and Trichomonacidal activities
1
. Introduction
[3] or prepared by synthesis [4–6]. These compounds, with
low-molecular weight, exhibited a variety of biological prop-
erties such as leishmanicidal [7], trypanocidal [8], antima-
larial [9] and were found to be potent inhibitors of the human
immunodeficiency virus of type-1 (HIV-1) integrase [10,11],
as well as active against HTLV-1 transformed cell lines (HUT-
102) [12,13].A very few quinolines (imidazoquinolinamines)
such as imiquimod have also been demonstrated to be potent
inducers of IFN-a and cytokines in both in vitro and in vivo
experiments [14]. The interesting in vitro nematocidal and
trichomonacidal activity of the 2-substituted quinolines tested
(Scheme 1) as well as the promising in vivo nematocidal activ-
ity of some of them prompted us to disclose our results.
The chemotherapy of many parasitic infections is nowa-
days an unresolved problem. The widespread development
of resistance to drugs is increasing, not only among hemin-
thic [1] but also in protozoan infections. That is the case of
Trichomonas vaginalis, the most common parasite causing
sexually transmitted diseases [2]. Consequent failures of treat-
ment, in addition to side effects, are main reasons that main-
tain the interest of the pharmacological screening. In the field,
the search of new antiparasitic agents among natural com-
pounds with biological properties led us to investigate as new
drug candidates, quinolines, specially 2-alkylquinolines,
2-alkenylquinolines and 2-arylquinolines, isolated from plants
2
. Results
*
Corresponding authors. Dr. Bruno Figadère, Laboratoire de pharmaco-
gnosie, UMR 8076 CNRS (BioCIS), Faculté de pharmacie, rue Jean-
Baptiste Clément, 92296 Châtenay-Malabry cedex, France. Tel.: +33 1 46
2.1. Chemistry
8
3 55 92; fax: +33 1 46 3 53 99, and Dr. Nieves Casado, Departamento de
Compounds 2 and 3 were prepared using previously pub-
lished methods [4–6]. Quinolines 1 and 7 were prepared by
condensation of 2-quinaldehyde with the corresponding Wit-
tig reagents, according to Scheme 2. Compounds 5 and 6 were
microbiología y parasitología, Universidad deAlcalá, 28871Alcalá de Hena-
res, Madrid, Spain. Tel.: +34 91 88 54 638; fax: +34 91 88 54 663.
E-mail addresses: bruno.figadere@cep.u-psud.f (B. Figadère),
nieves.casado@uah.es (N. Casado-Escribano).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.11.010

220
M. Martínez-Grueiro et al. / Il Farmaco 60 (2005) 219–224
Scheme 1. Evaluated quinolines [1–7].
2
.2. Biology
The in vitro activity of the synthesized quinolines 1-7 was
tested against two nematodes Caenorhabditis elegans and
Heligmosomoides polygyrus. The effect of compounds on the
development and reproductive capacity of C. elegans was
determined by comparing the population levels attained in
the control and test wells after an incubation period of 7 days
at 20 ± 1 °C. Mebendazole was used in these tests as positive
control. Compound 5, and in a less extent compound 6,
showed remarkable inhibition activity at 10 and 1 µM, com-
parable to the reference drug, mebendazole (Table 1).
Ovicidal activity of the synthesized quinolines 1–7 was
then tested. Unembryonated eggs of H. polygyrus were recov-
ered from fresh faeces of experimental infected mice. Using
an inverted microscope, the following parameters were exam-
ined: embryonation (Table 2), after 24 h of incubation, and
Scheme 2. Synthesis of quinolines [1,4–7].
obtained by treating 2-quinaldine by acetic anhydride in the
presence of the desired aromatic aldehyde. Quinoline 4 was
obtained by coupling reaction of 2-chloroquinoline with pro-
pargyl alcohol in the presence of catalytic amounts of palla-
dium and copper iodide. All the compounds were character-
1
13
ized by nuclear magnetic resonance ( H- and C-NMR)
spectrometry, IR and mass spectrometry. They were dis-
solved in dimethyl sulfoxide (DMSO, Sigma) and were used
at concentrations of 100, 10 and 1 µM.
hatching and L motility, at the end of the incubation period
1
(40 h) (Table 2). In these assays, the sole compound 6 showed
some activity at 10 µM, comparable to the reference drug,
levamisole (86% hatching compared to 67% of reference
drug).
Then effects on acid phosphatase and acetylcholinest-
erase excretion/secretion by Heligmosomoides polygyrus
adults, in the presence of the quinolines 1–7, were evaluated
Table 1
In vitro nematocidal activity against Caenorhabditis elegans
Compound
Inhibition (%)
Inhibition (%)
(at 10 µM)
0
Inhibition (%)
(at 1 µM)
(
at 100 µM)
0
(Table 3). The level of activity of each tested compound was
1
0
expressed as the percentage decrease in absorbency in rela-
tion to the control wells. In these assays compound 1 showed
a similar activity at 100 µM compared to the reference drug,
albendazole (87.4 vs 82.3% for AChE, and 30.2% for both
products for AP). Compound 6 was a little bit less active
2
3
4
5
6
7
57.2
22.8
0
41.2
23.8
0
58.6
23.5
0
99.7
99.3
15.3
0
100
99.7
62.8
18.0
0
(67.7% for AChE) than albendazole, whereas other quino-
Mebendazole
-
96.7
18.6
lines showed much weaker activity.
Table 2
Percentage of embrionation and hatching of Heligmosomoides polygyrus eggs determined after 24 and 40 h of incubation, respectively
Compound
Embryonation (%)
at)
Hatching (%)
(100 µM)
Embryonation (%)
Hatching (%)
Embryonation (%)
(at)
Hatching (%)
(1 µM)
100
(
(at)
≥95
≥95
≥95
≥95
≥95
≥95
≥95
4
(10 µM)
100
100
100
100
100
86
1
2
3
4
5
6
7
0
0
≥95
≥95
≥95
≥95
≥95
≥95
≥95
(ND)
≥95
0
100
0
100
100
≥95
≥95
≥95
≥95
100
76
46
0
100
100
100
100
67
100
Levamisole (ND)
ND: not determined.
0
(ND)

M. Martínez-Grueiro et al. / Il Farmaco 60 (2005) 219–224
221
Table 3
were not performed. A clear structure–activity is difficult to
ascertain, and further screenings are necessary in order to get
a full rationalization of the observed activities. Nevertheless,
these interesting results open a new area of investigation of
the biological activity of 2-substituted quinolines.
Effect of compounds (100 µM) on acetylcholinesterase and acid phospha-
tase excretion–secretion by Heligmosomoides polygyrus
Compound
Inhibition (%)
of AChE
87.4
Inhibition (%)
of AP
30.2
20.1
29.9
0
1
2
3
4
26.8
33.8
44.5
3. Experimental section
5
6
7
10.0
11.4
42.0
0
67.7
3
.1. Chemistry
55.9
Albendazole
82.3
30.2
Solvents were purified according to the procedures de-
scribed. Infra red spectra (IR) were recorded on a Perkin-
In view of these encouraging preliminary in vitro nemato-
cidal activities displayed by the synthesized quinolines, we
decided to evaluate the in vivo activity of quinolines 1, 5, 6,
–1
1
13
Elmer-257 apparatus (m expressed in cm ). H- and C-
NMR spectra were recorded either at 200 or 400 MHz.
Chemical shifts (d) were expressed in ppm with the proto-
nated solvent as reference. Patterns were described accord-
ing to Hoye et al. [15] and coupling constants (J) were given
in hertz (Hz). Mass spectra (MS) have been recorded on:
Nermag-Sidar R10-10C (CIMS) with either CH or NH and
7
. The quinolines were tested on Trichinella spiralis model.
Oral treatment of infected mice was applied as single dose at
4 h postinfection (p.i.). Among the four compounds tested,
2
sole the bis-quinoline 6 showed significant (P < 0.01) in vivo
nematocidal activity against T. spiralis (Table 4).
4
3
for the electronic impacts (EIMS) at 70 ev. Electrospray mass
spectra were recorded on a Bruker-Esquire equipment. Flash
chromatography was performed with silica gel 60
Then quinolines 1–7 were tested in vitro against Trichomo-
nas vaginalis. In these assays compounds 2, 3, 4, 6 and 7
reached reductions >50% at 100 µM (Table 5) when com-
pared to controls, but unfortunately the activity drastically
decreased when concentrations are lower.
(9385 Merck), silica gel S (31607 Riedel-de-Haën), and silica
gel 60H (7736 Merck). TLC was performed on plates coated
with silica gel 60F254 (554 Merck).
In conclusion, quinolines 1, 5, 6 and 7 showed interesting
nematocidal and trichomonacidal activity, although only one
of them, the bis-quinoline 6 showed in vivo activity. Relative
to trichomonacidal activity, the quinolines 2, 3, 4, 6 and 7
exhibited some activity at the higher concentration assayed.
However, as their activity disappears at 10 µM, in vivo assays
2
-(2-Methoxy-vinyl)-quinoline 1: 2-quinaldehyde (1.03 g,
6
.6 mmol) was dissolved in 50 ml of THF then methoxym-
ethyltriphenylphosphonium chloride (2.71 g, 7.9 mmol) was
added. The mixture was cooled to 0 °C before slowly adding
tBuOK (0.89 g, 7.9 mmol). The mixture was stirred one hour
before hydrolysis with satured aq. NH Cl. The mixture was
4
extracted twice with ethyl acetate and the combined organic
layers were washed with water then brine before drying with
Table 4
In vivo nematocidal activity against Trichinella spiralis preadults. Com-
pounds 1, 5, 6 and 7 were administered at 200 mg/kg and mebendazole at
MgSO , filtered and concentrated. The crude mixture was
4
25 mg/kg
purified by flash chromatography (cyclohexane:Et O 5:5) to
2
Number of worms ± S. D. and % reduction
give 0.34 g (36%) of the mixture of (E) and (Z)-isomers 1.
1
Control
9.0 ± 17.0
Compound 1
71.9 ± 17.7 NS
Compound 5
59.0 ± 17.0 NS
Mebendazole
9.3 ± 5.0, 84.2
Spectroscopic data for Z-isomer H-NMR (d, ppm
a
a
5
2
7
00 MHz): 8.11–7.97 (m, 3H), 7.71 (dd, 1H, J = 8.0 and
.3 Hz), 7.63 (dd, 1H, J = 8.2 and 7.2 Hz), 7.43 (dd, 1H,
(
P < 0.01)
Control
Compound 6
Compound 7
100.00 ± 16.7 NS
a
J = 7.7 and 7.0 Hz), 7.46 (d, 1H, J = 7.2 Hz), 5.71 (d, 1H,
84.1 ± 16.4 57.9 ± 9.4, 31.2
1
3
a
J = 7.2 Hz), 3.86 (s, 3H). C-NMR (d, ppm 50 MHz): 155.5,
(
P < 0.01)
a
151.9, 147.7, 135.3, 128.9, 128.6, 127.0, 126.2, 125.2, 121.9,
Versus control.
1
07.2, 60.9.
1
Table 5
Spectroscopic data for E-isomer H-NMR (d, ppm
00 MHz): 7.96 (d, 1H, J = 8.2 Hz), 7.94 (d, 1H, J = 8.4 Hz),
.68 (d, 1H, J = 12.6 Hz), 7.69–7.58 (m, 2H), 7.38 (dd, 1H,
Growth inhibition (%) of Trichomonas vaginalis after 24 h of incubation.
Metronidazole was assayed at 20, 10 and 5 µM
2
7
Compound
Inhibition (%)
at 100 µM
22.4
Inhibition (%)
at 10 µM
8.5
Inhibition (%)
J = 7.7 and 7.2 Hz), 7.36 (d, 1H, J = 8.7 Hz), 6.07 (d, 1H,
J = 12.9 Hz), 3.75 (s, 3H). C-NMR (d, ppm 50 MHz): 155.9,
at 1 µM
11.9
0
1
3
1
2
3
4
5
6
7
1
1
54.3, 148.0, 135.9, 129.3, 128.4, 127.3, 125.6, 125.0, 118.5,
05.8, 56.7. MS (ESI): 186 (MH , 100).
99.7
17.3
2.2
+
61.3
11.6
10.9
0
54.8
7.3
3-Quinolin-2-yl-prop-2-yn-1-ol4:Toasolutionof2-chloro-
3.5
0
quinoline (0.33 g, 2 mmol) in triethylamine (5 ml) were suc-
cessively added CuI (19 mg, 0.1 mmol), propargyl alcohol
69.4
3.3
0
51.6
0
0
(0.3 ml, 5 mmol) and PdCl (PPh ) (35 mg, 0.05 mmol). The
2 3 2
Metronidazole
100
100
98.7
mixture was stirred overnight at 70 °C, then, after cooling to

222
M. Martínez-Grueiro et al. / Il Farmaco 60 (2005) 219–224
room temperature, quenched with water and extracted twice
with Et O, dried over MgSO filtered and concentrated. The
ppm 50 MHz): 154.6, 149.8, 148.2, 148.1, 142.3, 136.6, 134.9,
129.9, 129.6, 129.5, 129.3, 128.2, 127.6, 127.5, 126.7, 126.6,
2
4
+
crude mixture was purified by flash chromatography using
:8 cyclohexane:EtOAc mixture as solvent to give 0.185 g
126.3, 123.5, 119.8, 117.4. MS (ESI): 283 (MH , 100). IR (m,
–
1
2
cm ): 3060; 1590; 1580; 1560; 1510; 1430; 1390; 960; 840;
760.
1
(
50%) of compound 4. H-NMR (d, ppm 400 MHz): 8.11 (d,
H, J = 8.5 Hz); 8.08 (d, 1H, J = 8.6 Hz); 7.79 (d, 1H,
J = 8.2 Hz); 7.72 (dd, 1H, J = 7.3, 7.4 Hz), 7.54 (dd, 1H,
1
(E)-3-Quinolin-2-yl-acrylonitrile 7: To a solution of
2-quinaldehyde (1.57 g, 10 mmol) in THF (25 ml) were suc-
cessively added at 0 °C diethylphosphonoacetonitrile (2.12 g,
1
3
J = 7.2, 7.8 Hz); 7.49 (d, 1H, J = 8.4 Hz); 4.62 (s, 2H). C-
NMR (d, ppm 50 MHz): 147.8, 142.9, 136.4, 130.2, 129.1,
1
2 mmol) and tBuOK (1.34 g, 12 mmol). After 1 h at 0 °C,
1
2
2
27.5, 127.3 (2C), 124.0, 88.7, 85.9, 51.3. MS (ESI):
06(M + Na, 100), 184 (MH , 89). IR (m, cm ): 3230; 3060;
910; 2850; 1590; 1500; 1430; 1240; 1040; 830; 760.
the mixture was quenched with satured aq. NH Cl solution
4
+
–1
and extracted with cyclohexane (three times). The combined
organic layers were dried over MgSO then filtered and con-
4
(E,E)-2-(4-Phenyl-buta-1,3-dienyl)-quinoline 5: To a solu-
centrated. The crude mixture was purified by flash chroma-
tography (cyclohexane:EtOAc 8:2) to give 1.31 g (73%) of
tion of quinaldine (4 ml, 30 mmol) in 50 ml Ac O was added
2
cinnamaldehyde (5 ml, 40 mmol) and the solution was heated
under reflux two hours. After cooling to room temperature,
the mixture was carefully and portionwise added to 200 ml
1
the E-isomer 7. H-NMR (d, ppm 400 MHz): 8.22 (d, 1H,
J = 8.4 Hz), 8.10 (d, 1H, J = 8.5 Hz), 7.84 (d, 1H, J = 8.2 Hz),
7
.77 (dd, 1H, J = 8.1 and 7.3 Hz), 7.61 (d, 1H, J = 16.3 Hz),
of saturated aq. NaHCO with vigorous stirring. After the
3
7.60 (dd, 1H, J = 8.0 and 7.1 Hz), 7.48 (d, 1H, J = 8.4 Hz),
hydrolysis being complete, the solution was extracted twice
with 5:5 hexane:EtOAc mixture. After drying over MgSO4
filtration and evaporation, the crude mixture was dissolved in
EtOH and sodium borohydride was added until no cinnama-
ldehyde remained (as judged by TLC). The solution was
hydrolyzed by addition of water, extracted with 5:5 hexane:E-
tOAc mixture, then dried, filtered and concentrated. The crude
mixture was purified by flash chromatography using
13
6
1
1
2
.72 (d, 1H, J = 16.3 Hz). C-NMR (d, ppm 50 MHz): 151.1,
49.2, 148.1, 137.2, 130.5, 129.9, 128.5, 127.8, 127.6, 120.5,
17.8, 102.1. MS (ESI): 180(MH , 100). IR (m, cm ): 3060;
220; 1590; 1500; 970; 910; 820; 730.
+
–1
3.2. Biology
8
:2 pentane:Et O mixture to give 1.5 g (19%) of the com-
2
1
pound 5. H-NMR (d, ppm 400 MHz): 8.09 (d, 1H,
J = 8.5 Hz), 8.05 (d, 1H, J = 8.6 Hz), 7.74 (d, 1H, J = 8.2 Hz),
3.2.1. In vitro activity against Caenorhabditis elegans
Assays were performed following the method of Simpkin
and Coles [16] with slight modifications. Tests were carried
out in 24-well plates (Costar®) and 4 wells were used for
each experiment group. To each well, 1.0 ml of culture
medium (with 9 mg/ml chloramphenicol instead of ampicil-
lin) was added followed by 7.5 µl of the appropriate com-
pound solution or solvent. Finally, 0.5 ml of culture medium
containing 10–15 C. elegans (L or L obtained from syn-
chronous cultures) was added to each well. The effect of com-
pound on the development and reproductive capacity of C.
elegans was determined by comparing the population levels
attained in the control and test wells after an incubation period
of 7 days at 20 ± 1 °C. Mebendazole was used in this test as
positive control.
7
(
7
.69 (dd, 1H, J = 8.2 and 7.1 Hz), 7.55–7.46 (m, 5H), 7.36
dd app., 2H, J = 7.3 and 7.7 Hz), 7.28 (dd, 1H, J = 7.2 and
.3 Hz), 7.61 (dd, 1H, J = 15.5 and 10.7 Hz), 6.96 (d, 1H,
13
J = 15.5 Hz), 6.85 (d, 1H, J = 15.6 Hz). C-NMR (d, ppm
5
1
1
3
1
0 MHz): 155.9, 148.3, 136.9, 136.1, 135.9, 134.8, 132.8,
29.6, 129.1, 128.6, 128.5, 128.0, 127.4, 127.2, 126.7, 126.0,
19.4. MS (ESI): 258 (MH , 100), 259 (40). IR (m, cm ):
060; 3030; 1610; 1590; 1550; 1500; 1430; 1310; 1280; 1140;
120; 990; 840; 820; 750; 690. C H N: calcd. C (88.68); H
+
–1
2
3
19 15
(5.87); N(5.44); Found C (88.65); H (5.91); N (5.33).
(E)-2-[(2-quinol-4-yl)-ethylene)]-quinoline 6: To a solu-
tion of quinaldine (2.7 ml, 20 mmol) in 20 ml of acetic anhy-
dride was added 4-quinaldehyde (3.1 g, 20 mmol) and the
solution heated under reflux for 2 h. After this time, the solu-
tion was cooled to room temperature and added portionwise
to a vigorously stirred saturated aq. NaHCO (200 ml) solu-
tion. After complete hydrolysis, the solution was extracted
twice with ethyl acetate and the combined organic layers were
3
.2.2. In vitro ovicidal activity on Heligmosomoides
polygyrus
Unembryonated eggs of H. polygyrus were recovered from
3
fresh feaces of experimental infected mice. [17] For the assay,
50 eggs/well (0.1 ml) were placed in culture plates of 24 wells,
which contained distilled water (0.9 ml/well) and appropri-
ate diluting of the drug solution or solvent (5 µl). There were
four replicates for each concentration and four replicates for
controls. The plates were then incubated at 20 ± 1 °C for 40 h.
Using an inverted microscope, the following parameters were
examined: embryonation, after 24 h of incubation, and hatch-
dried over MgSO , filtered and concentrated. The residue was
4
crystallized in cyclohexane and further purified by filtration
over a pad of silica eluting with EtOAc yielding 3 g of com-
1
pound 6 (53%). H-NMR (d, ppm 400 MHz): 8.95 (d, 1H,
J = 4.6 Hz); 8.48 (d, 1H, J = 15.5 Hz); 8.34 (d, 1H,
J = 8.3 Hz); 8.21 (d, 1H, J = 8.6 Hz), 8.18 (d, 1H, J = 9.1 Hz),
8.15 (d, 1H, J = 8.6 Hz), 7.83 (d, 1H, J = 8.1 Hz), 7.79–7.71
(
m, 4H), 7.63 (dd, 1H, J = 8.2 and 7.0 Hz), 7.61 (d, 1H,
13
J = 16.1 Hz), 7.55 (dd, 1H, J = 7.1 and 7.3 Hz). C-NMR (d,
ing and L motility, at the end of the incubation period.
1

M. Martínez-Grueiro et al. / Il Farmaco 60 (2005) 219–224
223
3.2.3. In vitro effect on acid phosphatase
dent’s t-test. A P value of less than 0.05 was considered as
and acetylcholinesterase excretion/secretion by
Heligmosomoides polygyrus adults
significant.
H. polygyrus adults were recovered from NMRI mice 12,
13 or 14 days after oral infection as previously described [18].
Acknowledgements
The in vitro effects of extracts on nematodes were investi-
gated by placing groups of eight worms (four males and four
females) into wells of the 48-well plates, containing 0.5 ml
of phenol-red free RPMI-1640 medium supplemented with
We wish to thank Drs. Ph. Loiseau and Ch. Bories for fruit-
ful discussions, and B. Seon-Méniel for her technical assis-
tance.
25 mM HEPES, 500 U/ml penicillin, 500 µg/ml streptomy-
cin and 0.6 µg/ml amphotericin B (pH 7.2), together with the
desired concentration of the product or solvent. Four wells
were observed for each concentration. Dimethylsulfoxide was
used to prepare the drug solutions and an equivalent quantity
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3.2.4. In vitro Trichomonas vaginalis assays
T. vaginalis strain JH31A n° 4 was cultured at 37 °C with
5
% CO in TYM (Trypticase—Yeast extract-Maltose) me-
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