Synthesis and fasciolicidal activity of 5-chloro-2-methylthio-6-(1- naphthyloxy)-1H-benzimidazole

Article abstract of DOI:10.1248/cpb.50.649

The synthesis and fasciolicidal activity of 5-chloro-2-methylthio-6-(1- naphthyloxy)-1H-benzimidazole (6) is described. Compound 6 showed 100% activity in vitro at 146.70 and 29.34 μM concentrations. It also completely removed 3-d and 10-week-old Fasciola

Full text of DOI:10.1248/cpb.50.649

May 2002
Notes
Chem. Pharm. Bull. 50(5) 649—652 (2002)
649
Synthesis and Fasciolicidal Activity of 5-Chloro-2-methylthio-6-
(1-naphthyloxy)-1H-benzimidazole**
Alicia HERNÁNDEZ-CAMPOS,a Froylán IBARRA-VELARDE,b Yolanda VERA-MONTENEGRO,b
b
,a
*
Norma RIVERA-FERNÁNDEZ, and Rafael CASTILLO
a Departamento de Farmacia, Facultad de Química, UNAM, CU; México D.F. 04510, México: and b Departamento de
Parasitología, Facultad de Medicina Veterinaria y Zootecnia, UNAM, CU; México D.F. 04510, México.
Received October15, 2001; accepted February 5, 2002
The synthesis and fasciolicidal activity of 5-chloro-2-methylthio-6-(1-naphthyloxy)-1H-benzimidazole (6) is
described. Compound 6 showed 100% activity in vitro at 146.70 and 29.34 mM concentrations. It also completely
removed 3-d and 10-week-old Fasciola hepatica in sheep at a dose of 15 mg/kg.
Key words fasciolicidal activity; bioisostere; benzimidazole synthesis; Fasciola hepatica
Fasciolosis, caused by Fasciola hepatica and Fasciola gi- Chart 1. In the first step, 4,5-dichloro-2-nitroaniline (1) was
gantica, is a very serious parasitic disease, which is responsi- subjected to a nucleophilic substitution reaction with 1-naph-
ble for heavy economic losses in sheep and cattle production thol (2) under known conditions,14) and the ether obtained (3)
in many countries of the world.1)
was reduced with SnCl2·2H2O.15) The corresponding o-
Besides domestic animals, other animal species and man phenylenediamine (4) formed was immediately cyclocon-
may be infected as well.2) There are effective strategies for densed with CS2 in an EtOH–KOH solution to give the
the control of fasciolosis which are largely based on drug 2-mercaptobenzimidazole (5).16) In the last step, 5 was
(fasciolicide) use on the definitive host.3) So far the only ef- monoalkylated with CH3I in acetone and KOH solution17) to
fective drug against immature and adult flukes alike is tri- afford the title compound (6).
clabendazole (TCBZ, Fig. 1), a potent fasciolicide with a bet-
The synthesized compounds were purified by recrystalliza-
ter bioavailability than that of the benzimidazole carbamates, tion and their physical constants were determined. The struc-
such as albendazole. It differs structurally from the latter in ture of all new compounds was established by spectroscopic
having a 2-methylthio group instead of a 2-methylcarbamate and spectrometric data.
group in the benzimidazole ring.4)
TCBZ appears to be highly specific for flukes, possessing Pharmacology
97—100% activity against all stages of Fasciola spp. The
Compound 6 was evaluated in vitro against newly excysted
usual oral dose of TCBZ for the elimination of immature and F. hepatica metacercariae at 146.70, 29.34, 9.68, 3.22, and
adult forms of F. hepatica is 5—10 mg/kg for sheep and 1.08 mM concentrations.18,19) In this experiment we used the
goats, and 12 mg/kg for cattle.5)
deacetylated (amine) metabolite of diamphenetide (DAMD),
For safety and efficacy, TCBZ is the drug of choice for the as a reference standard. Diamphenetide is a phenoxyalkane
treatment of human fasciolosis.6—10) Unlike nematocides, no compound which is highly effective against early immature
new fasciolicides have been marketed recently and appar- flukes from 1 d up to 6 weeks, at concentrations of 1.08—
ently no compounds are yet under development.1) Consider- 3.81 mM.19) The activity was assessed by comparing the sur-
ing that fasciolosis is also a health problem in Mexico, a re- vival of flukes with the untreated control after a 4-d period of
search project was undertaken in our laboratory with the pur- exposure to 6; the results of these studies are shown in Table
pose of developing new compounds with potential fasciolici- 1.
dal activity.11,12)
For the in vivo evaluation two experiments were carried
For the design of one of the new compounds described in out. In the former, 27 crossbred sheep were infected each
this paper, we use as a model pronethalol, an adrenergic with 150 metacercariae; ten weeks after infection the sheep
blocker which was formed by replacing the 3,4-dichlorophenyl were randomly divided in three groups of 9 animals each.
group in dichloroisoproterenol (DCI, Fig. 1) by the 2-naphtyl
group.13) DCI is also an adrenergic blocker with some ago-
nist activity thus establishing the fact that pronethalol is a
bioisostere of DCI.
In this paper we report the synthesis and preliminary fasci-
olicidal activity, in vitro and in vivo, against F. hepatica, of 5-
chloro-2-methylthio-6-(1-naphthyloxy)-1H-benzimidazole
(6), a TCBZ analog. In this compound, the 1-naphthyl group
replaces the 3,4-dichlorophenyl group in TCBZ, a moiety
change, which is analogous to that done with pronethalol–
DCI.
Chemistry
Fig. 1. Isosteric Substitution in TCBZ and Dichoroisoproterenol (DCI)
That Led to 6 and Pronethalol, Respectively
The synthetic sequence in the preparation of 6 is shown in
To whom correspondence should be addressed. e-mail: rafaelc@servidor.unam.mx
Taken in part from the PhD thesis of Alicia Hernández-Campos.
© 2002 Pharmaceutical Society of Japan

650
Vol. 50, No. 5
Chart 1. Synthesis of 5-Chloro-2-methylthio-6-(1-naphthyloxy)-1H-benzimidazole (6)
Table 1. Biological Activity in Vitro of 6 against F. hepatica Metacer-
Groups 1 and 2 were treated orally with a dose of 10 and 15
mg/kg of compound 6, respectively, in gelatin capsules.
Group 3 was the untreated control. Two weeks after treat-
ment, all sheep were killed; the flukes were collected from
the liver and counted. Efficacy was determined as a percent-
age of fluke reduction by the treatment in comparison with
the control (Table 2).12)
In a second experiment, sixty 10 to 12 month-old cross-
bred sheep were orally infected, each with 150 metacercariae
of F. hepatica. The animals were divided into 12 groups of 5
each. Groups 1, 3, 5, 7, 9, and 11 with 3-d, 2-week, 4-week,
6-week, 8-week and 10-week-old flukes, respectively, were
orally treated with 15 mg of 6 in a freshly prepared suspen-
sion;20,21) while groups 2, 4, 6, 8, 10, and 12 remained as un-
treated controls. Four weeks after treatment, the animals were
killed in order to collect and count the flukes in the liver. Ef-
ficacy (Table 3) was assessed as a percentage of fluke reduc-
tion in the treated groups in comparison with the control ac-
cording to the formula of Forey.22)
cariaea)
(%) Efficacy
Concentration (mM)
Compound 6
Diamphenetide deacetylated
146.7
34.68c)
29.34c)
9.68
3.81
3.22
100b)
nd
nd
100
nd
100b)
77.5
nd
nd
100d)
nd
0.0
0.0
1.08
100d)
a) Experiment was carried out in triplicate. b) Caused lysis of the metacercariae.
c) Dose equivalent to 10 mg/ml. d) Ref. 19. ndϭnot determined.
Table 2. Efficacy of 6 against 10 Week-Old F. hepatica in Experimentally
Infected Sheep at Two Dose Levels
Dose
Group
(nϭ9)
Efficacy
(%)
(mg/kg/bw)a)
AϪB
per os
% efficacyϭ
ϫ100
A
1
2
3
10
15
80.6
86.9
0.0
Where A is the mean number of flukes in the control group;
B is the mean number of flukes in the treated group.
Non treated control
a) Body weight.
Results and Discussion
The synthesis of compound 3 implies a nucleophilic sub-
stitution which can be carried out either by melting 1 and
2 in the presence of KOH,23) or by heating the mixture at
115 °C in a polar solvent (N,N-dimethylformamide (DMF)),
and a base (K2CO3).14) We used the second method; which
gave higher yield % of the desired product, however, it was
severely decreased by recrystallization. Washing the crude
product with cold solvent gave a product of high quality and
a good yield %. Compound 4 was obtained in good yield %,
but the crude amine had to be stabilized with acid to avoid a
fast decomposition. Although it was possible to isolate 4, we
preferred to use it immediately in the next reaction. The
preparation of 2-mercaptobenzimidazoles is also a known
process;16,17) it was adapted here to obtain 5. Although this
compound had a poor solubility in most organic solvents, it
was possible to purify it by recrystallization from AcOEt–
EtOH. The reaction to obtain 6 was very fast. In this final
Table 3. Efficacy of 6 against F. hepatica of Different Ages in Experimen-
tally Infected Sheep
Group
(nϭ5)
Age of F. hepatica at the
moment of treatment
Efficacy (%)
at 15 mg/kga)
1
2
3
4
5
6
7
8
9
3 d
100
0
100
0
100
0
100
0
100
0
100
0
Control
2 weeks
Control
4 weeks
Control
6 weeks
Control
8 weeks
Control
10 weeks
Control
10
11
12
a) Previously formulated.21)

May 2002
651
step, the amount of reagent and the temperature were care- was basified (pHϭ9) with a 50% NaOH solution. The solvent was removed
carefully in vacuo, and the solid residue was extracted with AcOEt (200
mlϫ3). The combined organic extracts were washed with brine, dried with
anhydrous Na2SO4 and concentrated in vacuo to give 89.6 g (99.08%) of 4 as
an oil, which was immediately cyclocondesed in the next step. An analytical
fully controlled; otherwise dimethylation products were
formed.
The in vitro assay (Table 1) showed that compound 6
killed early immature flukes (1—4 d) at concentrations of
29.34—9.68 mM (77.5%). These results motivated us to carry
out in vivo tests.
The preliminary in vivo assay (Table 2) showed that com-
pound 6 had an efficacy of 86.90% at a dose of 15 mg/kg
against 10-week-old flukes. However, compound 6 was not
formulated in our assay; this fact might as well have reduced
significantly the efficacy.
sample, which was dissolved in anhydrous ethanol, was converted into the
monohydrochloride by treatment with HCl gas; the solid formed was sepa-
rated in vacuo, washed with ethanol and recrystallized from ethanol to give
yellow crystals. mp 195—197 °C. IR (KBr) cmϪ1: 3461, 3345, 1629, 1493,
1
1227, 766. H-NMR (DMSO-d6) d: 3.80 (4H, br, 2NH2), 6.50 (1H, s, H-6),
6.89 (1H, dd, J1ϭ7.6, J2ϭ0.9, H-2Ј), 7.53 (1H, s, H-3), 7.47 (1H, t, J1ϭ8.1,
Hz, J2ϭ7.8 Hz, H-3Ј), 7.50—7.61 (2H, m, H-6Ј, H-7Ј), 7.73 (1H, d, Jϭ8.4
Hz, H-4Ј), 7.95—8.02 (2H, m, H-5Ј, H-8Ј). 13C-NMR (DMSO-d6) d: 108.51
(C-6), 112.38 (C-4), 112.90 (C-2Ј), 119.10 (C-8Јa), 121.12 (C-8Ј), 123.67
(C-3), 123.82 (C-4Ј), 125.39 (C-4Јa), 126.13 (C-3Ј), 126.52 (C-6Ј), 126.93
(C-7Ј), 127.98 (C-5Ј), 134.59 (C-2), 137.96 (C-1), 150.16 (C-1Ј), 151.58 (C-
5). MS (EI) m/z: 284 (Mϩ), 232. HR-MS (EI) Calcd for C16H13ClN2O (Mϩ)
m/z: 284.0716. Found: 284.0716. Anal. Calcd for C16H13ClN2O·HCl: C,
59.83; H, 4.39; N, 8.72. Found: C, 59.80; H, 4.38; N, 8.67.
The third trial showed that compound 6 given in an oral
suspension was 100% effective (Table 3) against all stages of
F. hepatica (3 d—12 weeks) at a dose of 15 mg/kg.
5-Chloro-2-mercapto-6-(1-naphtyloxy)-1H-benzimidazole (5) A so-
lution of 4 (50 g, 0.175 mol) in 250 ml of EtOH : H2O (80 : 20), KOH (14.77
g, 0.263 mol), and CS2 (15.84 ml, 20.05 g) was stirred at 70 °C for 3 h under
a N2 atmosphere. During this time a yellow precipitate was formed. The cold
Conclusions
The substitution of the 1-naphthyl group for the 2,3-
dichlorophenyl group in TCBZ gives a bioisostere of this po-
tent fasciolicide. The in vitro assay showed that compound 6 mixture was poured into 600 ml of H2O and treated with 20% AcOH solu-
tion to a pH 5—6. The precipitate was separated by suction filtration,
washed with water and dried in an oven at 90 °C. The crude product was pu-
rified by recrystallization from AcOEt–EtOH and activated carbon twice to
is very active and capable of disrupting the metacercariae.
Compound 6 showed good activity when it was administered
in a suspension. At a dose of 15 mg/kg it was 100% effective
against 3-d and 10-week-old flukes. The results so far ob-
tained are promising, since most fasciolicidal agents such as
rafoxanide, nitroxynil, closantel or clorsulon act only against
6-week-old flukes or older; or at the immature stage, as di-
amphenetide.1) Further studies are in progress in order to get
a better formulation and to optimize the dose. The determi-
nation of the efficacy of 6 in cattle is in progress as well.
give 41.82 g (73%) of 5 as a white powder. mp 273—275 °C. IR (KBr)
1
cmϪ1: 3380, 3053, 1460, 1391, 1328, 1229, 1157. H-NMR (DMSO-d6) d:
6.75 (1H, dd, J1ϭ6.6, J2ϭ2.0, H-2Ј), 6.84 (1H, s, H-7), 7.34 (1H, s, H-4),
7.40 (1H, t, J1ϭ8.1 Hz, J2ϭ8.1 Hz, H-3Ј), 7.54—7.62 (2H, m, H-6Ј, H-7Ј),
7.68 (1H, d, Jϭ8.4 Hz, H-4Ј), 7.95—8.00 (1H, m, H-5Ј), 8.13—8.17 (1H, m,
H-8Ј), 12.60 (1H, s, NH or SH), 12.72 (1H, s, NH or SH). 13C-NMR
(DMSO-d6) d: 102.27 (C-7), 110.60 (C-2Ј), 110.95 (C-4Ј), 118.96 (C-5),
121.19 (C-6Ј), 123.04 (C-4), 124.97 (C-8Јa), 126.01 (C-8Ј), 126.31 (C-7Ј),
126.89 (C-5Ј), 127.82 (C-3Ј), 129.57 (C-4Јa), 132.04 (C-3a), 134.46 (C-7a),
147.10 (C-1Ј), 152.66 (C-6), 169.66 (C-2). MS (EI) m/z 326 (Mϩ), 291, 290.
HR-MS (EI) Calcd for C17H11ClN2OS (Mϩ) m/z: 326.0280. Found:
326.0281. Anal. Calcd for: C17H11ClN2OS: C, 62.48; H, 3.39; N, 8.57; S,
9.81. Found: C, 62.49; H, 3.29; N, 8.44; S, 9.93.
Experimental
Melting points were determined on a Büchi B-540 melting point appara-
tus and are uncorrected. Reactions were monitored by TLC on 0.2 mm pre-
coated silica gel 60 F254 plates (E. Merck). IR spectra were recorded on a
Perkin Elmer FT-IR-1600 spectrometer as KBr disks. 1H-NMR spectra were
measured with a Varian EM-390 (300 MHz) spectrometer. Chemical shifts
are given in ppm relative to Me4Si (dϭ0) in DMSO-d6, J values are given in
Hz. The following abbreviations are used: s, singlet; d, doublet; dd, doublet
of doublet; t, triplet; m, multiplet; br, broad. MS were recorded on a JEOL
JMS-SX102A spectrometer by electron impact (EI) low and high resolution
(HR). Elemental analyses were carried out on a FISONS EA11108 CHNSO
analyzer. Starting material 1 was synthesized in our laboratory from the
commercially available 3,4-dichloroaniline via acetylation, nitration and hy-
drolysis of the nitroacetanilide.
4-Chloro-5-(1-naphtyloxy)-2-nitroaniline (3) A suspension of 1 (100
g, 0.483 mol), 2 (70.33 g, 0.487 mol), K2CO3 (100 g, 0.724 mol), and DMF
(360 ml) was stirred at 115 °C for 3.5 h. Then the mixture was cooled at
room temperature and filtered by suction. The residue was washed with hot
DMF (300 ml) and the solvent removed in vacuo. The crude product was
washed with cold methanol (300 ml) twice, and the orange solid was air-
dried to give 108.0 g (71%) of essentially pure 3. An analytical sample was
recrystallized from toluene–ethanol affording 98% of yellow crystals. mp
145—146 °C. IR (KBr) cmϪ1: 3462, 3347, 1628, 1560, 1387, 1320, 1225.
1H-NMR (DMSO-d6) d: 6.22 (1H, s, H-6), 7.37 (1H, dd, J1ϭ7.5 Hz, J2ϭ1.0
Hz, H-2Ј), 7.44 (2H, br, NH2), 7.55—7.64 (3H, m, H-3, H-6Ј, H-7Ј), 7.79
(1H, dd, J1ϭ8.0 Hz, J2ϭ2.4 Hz, H-5Ј), 7.93 (1H, d, Jϭ8.4 Hz, H-4Ј), 8.05
(1H, dd, J1ϭ7.2 Hz, J2ϭ1.8 Hz, H-8Ј), 8.18 (1H, s, H-3). 13C-NMR (DMSO-
d6) d: 103.59 (C-6), 109.54 (C-4), 117.14 (C-2Ј), 120.73 (C-5Ј), 125.69 (C-
8Јa), 125.79 (C-4Јa), 129.99 (C-4Ј), 126.26 (C-6Ј), 127.06 (C-3Ј), 127.12
(C-3), 127.15 (C-7Ј), 128.26 (C-8Ј), 134.77 (C-2), 146.91 (C-1), 149.14 (C-
1Ј), 159.04 (C-5). MS EI m/z 314 (Mϩ), 279, 233. HR-MS (EI) Calcd for
C16H11ClN2O3 (Mϩ) m/z: 314.0458. Found: 314.0458. Anal. Calcd for
C16H11ClN2O3: C, 61.06; H, 3.52; N, 8.90. Found: C, 61.18; H, 3.58; N,
8.62.
5-Chloro-2-methylthio-6-(1-naphthyloxy)-1H-benzimidazole (6) A sus-
pension of 100 g (0.3059 mol) of 5 in acetone (300 ml) was treated with a so-
lution of KOH (18.8 g, 0.336 mol) in water (50 ml), at room temperature,
under a N2 atmosphere. The dark solution formed was cooled to 0—5 °C and
treated slowly (1 h) with a solution of CH3I (43.41 g, 0.3059 mol) in acetone
(43 ml). The mixture was stirred at 10 °C for 30 min, neutralized with 20%
HCl solution, and the solvent was eliminated in vacuo. The solid residue
was taken up with 400 ml of AcOEt, washed with brine, dried with anhy-
drous Na2SO4, and the solvent was evaporated up to 200 ml. To the concen-
trated solution was added an equal volume of methanol and it was stirred in
a cold bath. The resulting precipitate was collected, washed with methanol
and dried. The crude product was recrystallized from CHCl3–MeOH to give
86.22 g (82.70%) of 6 as white powder. mp 192—195 °C. IR (KBr) cmϪ1
:
3402, 2720, 1453, 1316, 1157. 1H-NMR (DMSO-d6) d: 2.84 (3H, s,
SϪCH3), 6.48 (1H, br, NH), 6.77 (1H, d, Jϭ7.8 Hz, H-2Ј), 7.29 (1H, s, H-4),
7.40 (1H, t, J1ϭ7.8 Hz, J2ϭ8.1 Hz, H-3Ј), 7.53—7.66 (2H, m, H-6Ј, H-7Ј),
7.70 (1H, d, Jϭ8.1 Hz, H-4Ј), 7.89 (1H, s, H-7), 7.94—8.20 (1H, m, H-5Ј),
8.12—8.22 (1H, m, H-8Ј). 13C-NMR (DMSO-d6) d: 13.46 (S-CH3), 102.95
(C-7), 110.20 (C-2Ј), 113.16 (C-4), 119.70 (C-8Ј), 120.83 (C-5), 122.04 (C-
4Ј), 123.80 (C-8Јa), 124.12 (C-3Ј), 124.66 (C-6Ј), 125.25 (C-7Ј), 126.21 (C-
5Ј), 128.72 (C-4Јa), 131.34 (C-3a), 133.08 (C-7a), 148.05 (C-1Ј), 150.75 (C-
2), 152.03 (C-6). MS (EI) m/z 340 (Mϩ), 305, 290. HR-MS (EI) Calcd for
C18H13ClN2OS (Mϩ) m/z: 340.0437. Found: 340.0437. Anal. Calcd for
C18H13ClN2OS: C, 63.43; H, 3.84; N, 8.22; S, 9.41. Found: C, 62.82; H,
3.69; N, 7.97; S, 9.86.
Acknowledgements We thank CONACyT for the financial support of
project 34942B. We also thank DGAPA for the financial help of projects
IN227998 and IN204998. We are grateful to Rosa Isela del Villar, Georgina
Duarte, Margarita Guzmán and Marisela Gutiérrez, from the School of
Chemistry, UNAM, for the determination of all spectra, and Socorro
Alpizar, from the Laboratorio de Tecnología Farmacéutica, for the formula-
tions. We are very grateful to Dr. Eugene Bratoeff for critical review of the
manuscript.
4-Chloro-5-(1-naphtyloxy)-1,2-phenylenediamine (4) A mixture of 3
(100 g, 0.317 mol), SnCl2·2H2O (430 g, 1.9 mol), and EtOH (640 ml) was
stirred at 70 °C for 3 h in a nitrogen atmosphere. After cooling, the mixture

652
Vol. 50, No. 5
13) Black J. W., Stephenson J. S. Lancet, ii, 311—314 (1962).
References and Notes
1) Fairweather I., Boray J. C., Vet. J., 158, 81—112 (1999).
2) Chen M. G., Mott K. E., Trop. Dis. Bull., 1990, R1—R37 (1990).
3) Boray J. C. “Immunology, Pathobiology and Control of Fasciolosis,”
14) Averkin E. A., Beard C. C., Dvorak C. A., Edwards J. A., Fried J. H.,
Kilian J. G., Schiltz R. A., Kistner T. P., Drudge J. H., Lyons E. T.,
Sharp M. L., Corwin R. M. J., J. Med. Chem., 18, 1164—1166 (1975).
ed. by Boray J. C., MSD AGVET Rahway, New Jersey, 1997, pp. 83— 15) Manjarrez N., Luna H. M., Pérez H. I., Soria O., Rev. Soc. Quím. Méx.,
89.
30, 52—54 (1986).
4) Thorpe P. J., Drugs Future, 9, 593—594 (1984).
5) Boray J. C., Crowfoot P. D., Strong M. B., Allison J. R., Schellenbaum
M., Von Orelli M., Sarisin G., Vet. Rec., 113, 315—317 (1983).
6) Wolf K., Eckert J., Schneiter G., Lutz H., Vet. Parasitol., 13, 145—150
(1983).
16) Van Allan J. A., Deacon B. D., “Organic Syntheses Collective Vol-
ume,” Vol. IV, ed. by Rabjhon N., John Wiley and Sons, N.Y., 1976,
pp. 569—570.
17) Ciba-Geigy A. G., Belg. Pat. 865870 (1978) [Chem. Abstr., 90,
137817 z (1979)].
7) Craig T. M., Huey R. L., Am. J. Vet. Res., 45, 1644—1645 (1984).
18) Ibarra O. F., Jenkins D. C., Z. Parasitenk., 70, 655—671 (1984).
8) Arjona R., Riancho J. A., Aguado J. M., Salesa R., Gonzalez-Macias 19) Sánchez E. B., Ibarra V. F., Tec. Pec. Méx., 25, 397—403 (1988).
J., Medicine (Baltimore), 74, 13—23 (1995).
20) Ibarra-Velarde F., Montenegro-Cristino N., Flores-Crespo J.,
Hernéndez-Campos A., Castillo R., Vet. Méx., 31, 47—51 (2000).
21) The freshly prepared suspension contained: compound 6 (10 g), pectin
(1.5 g), low viscosity carboxymethylcellulose (0.5 g), medium viscos-
ity carboxymethylcellulose (0.5 g), USP saccharine (0.2 g), methyl-
paraben (0.5 g), propylparaben (0.03 g), and water to make 100 ml.
9) Apt W., Aguilera X., Vega F., Miranda C., Zulantay I., Perez C., Gabor
M., Apt P., Am. J. Trop. Med. Hyg. 52, 532—535 (1995).
10) el-Karaksy H., Hassanein B., Okasha S., Behairy B., Gadallah I., J.
Trop. Pediatr., 45, 135—138 (1999).
11) Ibarra-Velarde F., García-Sánchez E., Fernández-Ruvalcaba M., Vera-
Montenegro Y., Castillo R., Hernández-Campos A., Vet. Méx. 28, 22) Forey W. S., Vet. Parasitol., 26, 265 271 (1988).
—
291—296 (1997).
23) Frick W. E., Weiss A. G., Wenger T., Traber W., U.S. Pat. 3506767
12) Ibarra-Velarde F., Vera-Montenegro Y., Hernández-Campos A.,
Castillo R., R. Vet. Méx. 28, 297—301 (1997).
(1968) [Chem. Abstr., 69, 37061w (1968)].
24) Bennett J. L., Köhler P., Exp. Parasitol., 63, 49—57 (1987).