M. Andrzejewska et al. / European Journal of Medicinal Chemistry 37 (2002) 973Á
/978
977
Merck silica gel 60 (200Á
carried out on precoated silica gel F254 (Merck) plates
(0.25 mm thickness). Analyses of the new compounds,
indicated by the symbols of the elements, were within 9
0.4% of the theoretical values.
/
400 mesh). Analytical TLC was
H2O (70 mL, 9:1), carbon disulfide (3.8 g, 49.5 mmol)
and KOH (2.4 g, 43 mmol) were added. The reaction
mixture was stirred and refluxed for 3 h. The mixture
was cooled and acidified to pH 2 with HCl. The
precipitate formed was filtered off and purified by twice
/
crystallization from EtOHÁ
powder (2.5 g, 60%) of m.p.ꢀ
DMSO): 7.79 and 7.91 (2ꢁd, 2H, H-5 and H-7); UV:
(pH 2) 251 (18 900), 314 (21 300); (pH 12) 242 (11 100),
314 (10 500); TLC (CHCl3ÁMeOH, 9:1, v/v): Rf 0.58.
/
H2O (1:1) to give white
1
4.1.1. General procedure for the synthesis 1b,i and j
To a solution of respective o-phenylene diamine (5
mmol) in 4 N HCl (20 mL), trifluoroactic or penta-
fluoropropionic acids, respectively (10 mmol), was
added. The stirred reaction mixture was stirred under
reflux for 5 or 24 h, respectively, diluted with water (30
/
350 8C. H-NMR (D6-
/
/
Anal. Calc. for C7H4Br2N2S: C, 27.30; H, 1.30; N, 9.10.
Found: C, 27.14; H, 1.42; N, 8.97%.
mL), treated with charcoal and brought to pH 4Á
aq. ammonia. The precipitate formed was filtered off
and crystallized from EtOHÁH2O.
/
5 with
4.1.4. 4,6-Dibromo-2-
dimethylaminoethylthiobenzimidazole (8)
/
To a solution of 7 (0.92 g, 3 mmol) in dry MeCN (15
mL) were added DBU (1.02 g, 6.7 mmol) and dimethy-
lamineethylchloride hydrochloride (0.57 g, 4 mmol). The
reaction mixture was stirred at 70 8C (bath tempera-
ture) for 30 min. The mixture was adsorbed on silica gel
4.1.1.1. 4,5,6-Trifluoro-2-trifluoromethylbenzimidazole
(1b). Yield: 45%; m.p. 169Á
171 8C; 1H-NMR (D6-
DMSO): 7.67 (s, 1H, H-7); UV: (pH 6) 250 (4600), 271
(4800); (pH 12) 273 (5800); TLC (CHCl3ÁMeOH, 9:1, v/
/
/
v): Rf 0.51. Anal. Calc. for C8H2F6N2: C, 40.02; H, 0.84;
N, 11.67. Found: C, 39.89; H, 1.01; N, 11.55%.
and chromatographed on silica gel column (3ꢁ
/
15 cm)
with CHCl3ÁMeOH (9:1). The oily product was trans-
/
formed in colourless crystalline hydrochloride by treat-
ing it with HCl-saturated MeOH (0.24 g, 19%). M.p.
4.1.1.2. 5,6-Dichloro-2-pentafluoroethylbenzimidazole
1
1
(1i). Yield: 38%; m.p. 207 8C; H-NMR (D6-DMSO):
8.05 (s, 2H, H-4 and H-7). UV: (pH 6) 260 (3500), 291
230Á
/
232 8C. H-NMR (D6-DMSO): 2.84 (s, 6H, 2ꢁ
/
CH3), 3.46 and 3.68 (2t, 4H, 2ꢁ
(2d, H-5 and H-7); UV: (pH 6) 301 (13 500), (pH 12) 307
(1500); TLC (CHCl3ÁMeOH, 9:1, v/v): Rf 0.21. Anal.
Calc. for C11H13Br2N3SꢁHCl: C, 31.79; H, 3.40; N,
/
CH2), 7.54 and 7.67
(5600), 301 (4900); TLC (CHCl3ÁMeOH, 9:1, v/v): Rf
/
0.60. Anal. Calc. for C9H3Cl2F5N2: C, 35.44; H, 0.99; N,
9.18. Found: C, 35.31; H, 1.11; N, 9.29%.
/
/
10.11. Found: C, 31.91, H, 3.53; N, 10.00%.
4.1.1.3. 5,6-Dimethyl-2-pentafluoroethylbenzimidazole
206 8C; 1H-NMR (D6-
DMSO): 2.32 (s, 6H, 2CH3), 7.46 (s, 2H, H-4 and H-
(1j). Yield: 63%; m.p. 204Á
/
4.2. Biology
7); UV: (pH 6) 262 (3600), 284 (5000), 291 (4800); (pH
MeOH, 9:1, v/v): Rf 0.71.
Anal. Calc. for C11H9F5N2: C, 50.01; H, 3.43; N, 10.60.
Found: C, 50.17; H, 3.45; N, 10.47%.
4.2.1. Parasites
12) 282 (7200). TLC (CHCl3Á
/
E. histolytica strain HM1-IMSS, T. vaginalis strain
GT3 and G. intestinalis isolate IMSS:1090:1 were used
in all experiments. Trophozoites of E. histolytica and T.
vaginalis were maintained in TYI-S-33 medium supple-
mented with 10% bovine serum. Giardia trophozoites
were cultured in TYI-S-33 modified medium supple-
mented with 10% calf serum and bovine bile [16]17.
4.1.2. 4,5,6,7-Tetrabromo-2-
pentafluoroethylbenzimidazole (5)
To a stirred and refluxed suspension of 2-pentafluor-
oethylbenzimidazole (1.4 g, 5.9 mmol) in water (70 mL),
bromine (7.5 g, 47 mmol) was added portionwise within
8 h. The reflux was continued for 3 days. The reaction
mixture was cooled and the pale yellow precipitate
formed was filtered off, washed with cold water and
4.2.2. Protozoa susceptibility assays
In vitro susceptibility assays were performed using a
method previously described [16,17]. Briefly: 4ꢁ
/
104 G.
intestinalis, 6ꢁ
/
103 E. histolytica or 4ꢁ104 T. vaginalis
/
crystallized from EtOHÁ
of colourless crystals of m.p. 235Á
(D6-DMSO): 14.54 (bs, 1H, NÃH); UV (MeOHÁ
1:1): 233 (4300), 304.5 (2700); TLC (CHCl3ÁMeOH, 9:1,
v/v): Rf 0.77. Anal. Calc. for C9HBr4F5N2: C, 19.59; H,
0.18; N, 5.08. Found: C, 19. 43; H, 0.28; N, 4.95%.
/
H2O (1:1) to give (1.59 g, 49%)
237 8C. 1H-NMR
H2O,
trophozoites were incubated for 48 h at 37 8C with
different concentrations of compounds 1c, 1d, 1e, 1f, 1g,
1i, 1k, 5, 8, albendazole, or metronidazole, each added
as a solution in dimethyl sulfoxide (DMSO). As the
negative control, trophozoites were incubated with
DMSO. At the end of the treatment period, the cells
were washed and subcultured for another 48 h in fresh
medium to which no drug was added. The trophozoites
were then counted with a haemocytometer and the 50%
(IC50) inhibitory concentrations, together with the
/
/
/
/
4.1.3. 4,6-Dibromo-2-thiobenzimidazole (7)
To a stirred suspension of 3,5-dibromo-1,2-diamino-
benzene dihydrochloride (4.6 g, 1.5 mmol) in EtOHÁ
/