Synthesis and antiparasitic activity of Albendazole and Mebendazole analogues

Article abstract of DOI:10.1016/S0968-0896(03)00497-8

Albendazole (Abz) and Mebendazole (Mbz) analogues have been synthesized and in vitro tested against the protozoa Giardia lamblia, Trichomonas vaginalis and the helminths Trichinella spiralis and Caenorhabditis elegans. Results indicate that compounds 4a, 4b (Abz analogues), 12b and 20 (Mbz analogues) are as active as antiprotozoal agents as Metronidazole against G. lamblia. Compound 9 was 58 times more active than Abz against T. vaginalis. Compounds 8 and 4a also shown high activity against this protozoan. Compounds 4b and 5a were as active as Abz. None of the Mbz analogues showed activity against T. vaginalis. The anthelmintic activity presented by these compounds was poor.

Full text of DOI:10.1016/S0968-0896(03)00497-8

Bioorganic & Medicinal Chemistry 11 (2003) 4615–4622
Synthesis and Antiparasitic Activity of Albendazole
and Mebendazole Analogues§
Gabriel Navarrete-Vazquez,a,* Lilian Yepez,b Alicia Hernandez-Campos,a
Amparo Tapia,b Francisco Hernandez-Luis,a Roberto Cedillo,c Jose Gonzalez,a
Antonio Martınez-Fernandez,d Mercedes Martınez-Grueirod and Rafael Castilloa,*
a
´
Departamento de Farmacia, Facultad de Quımica, UNAM, CU, DF 04510, Mexico
b
´
´
Unidad de Investigacion Medica en Enfermedades Infecciosas y Parasitarias, IMSS, DF 06720, Mexico
c
´
´
´
´
Unidad Interinstitucional de Investigacion Medica, IMSS-UADY, Merida, Yucatan, Mexico
d
´
Departamento de Parasitologıa, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040, Spain
Received 6 May 2003; revised 22 July 2003; accepted 25 July 2003
Abstract—Albendazole (Abz) and Mebendazole (Mbz) analogues have been synthesized and in vitro tested against the protozoa
Giardia lamblia, Trichomonasvaginalis and the helminths Trichinella spiralis and Caenorhabditiselegans . Results indicate that
compounds 4a, 4b (Abz analogues), 12b and 20 (Mbz analogues) are as active as antiprotozoal agents as Metronidazole against G.
lamblia. Compound 9 was 58 times more active than Abz against T. vaginalis. Compounds 8 and 4a also shown high activity against
this protozoan. Compounds 4b and 5a were as active as Abz. None of the Mbz analogues showed activity against T. vaginalis. The
anthelmintic activity presented by these compounds was poor.
# 2003 Published by Elsevier Ltd.
Introduction
Since the site of action of the different BZC is the same,
these compounds have additive action and show crossed
resistance, which may undermine their future thera-
peutic value.11 Due to their poor solubility and absorp-
tion in vivo, BZC have successfully been used to treat
gastrointestinal helmintic diseases.
Parasitic infections are still a major health problem in
developing countries, affecting mainly the infantile
population. It has been reported that benzimidazole 2-
carbamates (BZC), such as Albendazole (Abz) and
Mebendazole (Mbz), used mainly as anthelmintic agents
(Fig. 1), inhibit the in vitro growth of protozoa Giardia
1ꢀ3
Although systemic infections have also been treated
with these agents, high doses and long treatments are
required.12
lamblia and Trichomonasvaginali.s
Clinical assays
have shown that Abz is as effective as Metronidazole,
the choice drug for the treatment of giardiasis.4ꢀ6
As part of our search for basic information about the
structural requirements for antiprotozoal and anthel-
mintic activity,10,13,14 we have synthesized a series of
novel 2-(trifluoromethyl)benzimidazole derivatives, ana-
logues of Abz (4a–b, 5a–b, 8, and 9) and Mbz (12a–b,
and 20), reported in Table 1. The in vitro antiparasitic
activity of these compounds on an intestinal protozoan
(G. lamblia) and urogenital tract parasite (T. vaginalis);
and helminths Trichinella spiralis and Caenorhabditis
elegans, is also reported.
The anthelmintic activity of benzimidazole 2-carba-
mates has been related to their selective antimitotic
activity due to the preferential binding of these agents to
helmintic tubulin over mammalian tubulin.7 Similarly,
the action of Abz against Giardia lamblia also involves
the interaction with tubulin of the Giardia cytoskele-
ton.2,8 It is suggested that one of the requirements for
this action is that the substituted benzimidazole bear a
hydrogen atom at the 1-position and a methylcarbamate
group at the 2-position.9,10
Chemistry
§Taken in part from the PhD thesis of Gabriel Navarrete-Vazquez
*Corresponding authors. Tel./fax:+52-5622-5287; e-mail: gabriel_na-
varrete@correo.unam.mx; rafaelc@servidor.unam.mx
Compounds 4a–b, 5a–b, 8 and 9 were prepared from the
adequate substituted 2-nitroaniline 1a, 1b, or 1c,
0968-0896/$ - see front matter # 2003 Published by Elsevier Ltd.
doi:10.1016/S0968-0896(03)00497-8

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 11 (2003) 4615–4622
4616
respectively. The sequence shown in Scheme 1 was fol-
lowed. Aromatic nucleophilic substitution in 1a–b with
1-propanothiol afforded 2a–b,15,17 which upon reduc-
tion with H2, Pd/C gave 1,2-phenylenediamine 3a–b.
Reaction of these with CF3COOH yielded 4a–b. The
sulfoxides 5a–b were obtained by treatment of 4a–b with
m-CPBA. Reaction of 1c with NH4SCN and Br2 gave
6,16,17 which was converted to 7 with a mixture of 1-
PrOH, 1-PrBr, KCN, H2O and then reduced and cyclo-
condensed with CF3COOH to give 8.13 The same proce-
dure employed above was used to prepare sulfoxide 9.
and oxidation of the nitration products 14 with
K2Cr2O7 and H2SO4, preferably gave 3-chloro-4-nitro-
benzoic acid 15. The activation of 15 with SOCl2, and
Friedel–Crafts acylation of benzene with chloride 16 led
to benzophenone 17, which upon nucleophilic substitu-
tion with CH3NH2 followed by reduction of 18 with
.
SnCl2 2H2O afforded the required compound 19.
Cyclocondensation of 19 with CF3COOH, as shown
before, gave 20. Solid compounds were purified by
recrystallization. The structure of the purified com-
pounds was established by spectroscopic and spectro-
metric data.
Starting with 4-benzoyl-2-nitroanilines 10a–b, the
.
reduction with SnCl2 2H2O and cyclocondensation of
the o-phenylenediamine intermediate with CF3COOH
afforded the corresponding benzimidazole derivatives
12a–b, respectively (Scheme 2). For the synthesis of the
regioisomeric derivative 20, o-phenylenediamine 19 was
first prepared through the series of reactions shown in
Scheme 3. Thus, 3-chlorotoluene 13, through nitration
Results and Discussion
In this study, 9 new 2-(trifluoromethyl)benzimidazole deri-
vatives, analogues of Abz and Mbz, have been synthesized
Table 1. Synthesized 2-(trifluoromethyl)benzimidazoles
Compd
R1
R2
R3
4a
4b
5a
5b
8
H
CH3
H
CH3
CH3
CH3
H
CH3
CH3
Propylthio
Propylthio
Propylsulfinyl
Propylsulfinyl
H
H
H
H
H
H
H
Propylthio
Propylsulfinyl
Benzoyl
Benzoyl
H
9
12a
12b
20
H
Benzoyl
Figure 1. Benzimidazole anthelmintics albendazole and mebendazole
and their analogues prepared in this work.
Scheme 1. Reagents: (i) 1-PrSH, ethylene glycol, 120 ꢁC; (ii) NH4SCN, Br2, MeOH; (iii) 1-PrBr, KCN, 1-PrOH, (Bu)4NCl, CH2Cl2, reflux; (iv) H2,
Pd/C 10%, MeOH; (v) CF3COOH, HCl, reflux; (vi) m-CPBA, CHCl3, 5 ꢁC.

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 11 (2003) 4615–4622
4617
.
Scheme 2. Reagents: (i) SnCl2 2H2O, EtOH, reflux; (ii) CF3COOH, HCl, reflux.
ꢁ
.
Scheme 3. Reagents:ꢁ(i) H2SO4–HNO3, 20 C; (ii) K2Cr2O7, H2SO4; (iii) SOCl2, benzene; (iv) benzene, AlCl3; (v) CH3NH2 HCl, 1,2-dimethox-
.
yethane, K2CO3, 130 C; (vi) SnCl2 2H2O, EtOH, reflux; (vii) CF3COOH, HCl, reflux.
Table 2. In vitro susceptibility of G. lamblia and T. vaginalis to syn-
thesized compounds, Metronidazole and Abz
Table 3. Percentage of viability reduction of T. spiralis muscle lar-
vae after 3 days of incubation with synthesized compounds and Abza
Compd
G. lamblia
IC50, mM
T. vaginalis
IC50, mM
Compd
0.037 mM
0.188 mM
0.37 mM
1.88 mM
Abz
4a
4b
5a
5b
8
57
nr
11
10
16
28
8
27
26
21
58
18
30
18
24
41
21
33
32
24
61
27
48
30
29
43
29
36
33
28
67
38
56
45
32
48
42
45
37
31
4a
4b
5a
5b
8
9
12a
1.515
1.403
6.37
4.312
10.45
20.89
2.31
1.098
1.285
1.22
0.345
2.58
2.78
4.42
0.2
0.058
28.44
29.59
10.64
0.2161
3.39
9
12a
12b
20
12b
20
Metronidazole
Abz
nr=no reduction observed.
aValues are means of three experiments.
0.037
and tested as antiprotozoal and anthelmintic agents.
The main features of these compounds are the substitu-
tion of the 2-methylcarbamate group by a 2-tri-
fluoromethyl group in order to enhance solubility and
absorption properties and hopefully, antiparasitic
activity; and the synthesis of regioisomeric 1-methyl-
benzimidazole derivatives in order to determine the
importance of hydrogen at position 1 on the anti-
parasitic activity.
pattern 1,2,5-trisubstituted; also, that those compounds
with a propylthio group (4a, 4b, 8) were more active
than their propylsulfinyl analogues (5a, 5b, 9).
In the other assay, against T. vaginalis, Abz analogues
(4a, 5a) with a hydrogen at position 1 were more active
than Abz, where as Mbz analogue 12a did not show
significant activity. Although these compounds showed
good activity, however, none of them were as active as
Metronidazole. Another observation is that Abz analo-
gues 1,2,5-trisubstituted (8, 9) were more active than
regioisomers 1,2,6-trisubstituted (4b, 5b). Although Abz
analogues with a propylthio group (4a, 4b) were more
active than those with a propylsulfinyl group (5a, 5b),
this fact was not observed with 8 and 9. Interesting to
note is that compound 9 was 4 and 58 times more active
than Metronidazole and Abz, respectively.
Biological assay results shown in Table 2, against G.
lamblia, indicate that compounds with a hydrogen at
position 1 (4a, 5a, 12a) were less active than Abz and
Metronidazole. Compounds with a methyl group at
position 1 (4b, 5b, 8, 9, 12b, 20) were less active than
Abz; however, regioisomeric 1-methyl Mbz analogues
(12b, 20) were as active as Metronidazole. Taking in
consideration the pattern of substitution in Abz analo-
gues, compounds 4b and 5b, with the pattern 1,2,6-tri-
substituted were more active than 8 and 9, with the
The in vitro anthelmintic activity results against T.
spiralis and C. elegans are shown in Tables 3 and 4,

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 11 (2003) 4615–4622
4618
Table 4. Percentage of viability reduction of C. elegans after 7 days
of incubation with synthesized compounds and Mbza
Conclusion
The results obtained with the synthesized analogues as
antiprotozoal agents are very promising indeed since
they broaden the knowledge of the activity of these
versatile derivatives of benzimidazole. The fact that
some of the 1-methyl analogues were active against G.
lamblia and T. vaginalis confirm what we found in our
previous studies, viz. that the H at position 1 and the
methylcarbamate group at position 2, of the benzimi-
dazole ring, are not required for antiprotozoal activity;
however, they are required for a good anthelmintic activ-
ity.10,13 These findings also imply that the mechanism of
action of these novel 2-(trifluoromethyl)benzimidazole
derivatives is different from that of the benzimidazole 2-
carbamates through inhibition of the polymerization of
tubulin.
Compd
100 mM
10 mM
1 mM
Mbz
4a
4b
5a
5b
8
98
28
62.4
nr
nr
62.2
nr
nr
nr
nr
99
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
9
12a
12b
20
anr, no reduction observed.
Table 5. The antiprotozoal benzimidazoles have physical properties
compatible with reasonable pharmacokinetics and drug availability
Compd
Mol
wt
C log P
No. of H
bond
donors
No. of H
bond
acceptors
No. of
criteria met
Experimental
Melting points were determined on a Buchi B-450 melt-
ing point apparatus and are uncorrected. Reactions
were monitored by TLC on 0.2 mm precoated silica gel
60 F254 plates (E. Merck). 1H NMR and 13C NMR
spectra were measured with a Varian EM-390 (300 and
75.5 MHz) spectrometer. Chemical shifts are given in
ppm relative to tetramethylsilane (Me4Si, d=0) in
CDCl3; J values are given in Hz. The following abbre-
viations are used: s, singlet; d, doublet; q, quartet; dd,
doublet of doublet; t, triplet; m, multiplet; bs, broad
signal. MS were recorded on a JEOL JMS-SX102A
spectrometer by electron impact (EI). Catalytic hydro-
genations were carried out in a Parr hydrogenation
apparatus. Starting materials 1a–c and 10b were syn-
thesized in our laboratory from the commercially avail-
able 3-chloroaniline, and 4-aminobenzophenone
(Aldrich), respectively, via acetylation, nitration,
methylation and hydrolysis of the corresponding N-
Methyl-2-nitroacetanilide. m-Chloroperbenzoic acid (m-
CPBA) 57–86% was from Aldrich. The C log P values
were obtained using ACD/labs software v.4.5.
Rule
4a
4b
5a
5b
8
9
12a
12b
20
<500
260
274
276
290
274
290
290
304
304
<5
<5
1
0
1
0
0
0
1
0
<10
1
At least 3
All
All
3.95
4.26
1.79
2.1
4.26
2.1
3.36
3.67
3.67
1
2
2
1
2
2
2
2
All
All
All
All
All
All
All
0
respectively. In general, Abz and Mbz analogues
showed modest activity, less than that of Abz. However,
the pattern of substitution and the electronic properties
of the substituent at position 5(6) play an important
role. Thus, 1-methyl Abz analogue (4b) with an electron
donating group at position 6 was more active than 8,
with the same group at position 5. The 1-H Abz analo-
gue (4a) with an electron donating group at position
5(6) was the least active of all. On the other hand,
compounds 5a, 12a with an electron withdrawing group
at position 5(6) were more active than their corre-
sponding 1-methyl analogues (5b, 9, 12b, 20). None of
the compounds showed appreciable activity against
C. elegans under 100 mM concentrations.
General method of synthesis of 2-(trifluoromethyl)-1H-
benzimidazoles 4a, 4b, 8, 12a, 12b, 20
The appropriate 1,2-phenylenediamine (0.0313 mol), 1.6
equivalents of CF3COOH and one drop of concentrated
HCl were heated under reflux in a N2 atmosphere for 3–
4 h. TLC was used to monitor the reaction. The cooled
mixture was neutralized with saturated NaHCO3 solu-
tion, and the crude benzimidazole was extracted with
AcOEt. The solvent was removed under high vacuum (1
mm Hg), and the resulting solid was isolated by fil-
tration through a fritted 60-mL glass funnel packed
with Al2O3, acid type.
These compounds are fully compatible with Lipiski’s
rule18 (Table 5), which should allow for the develop-
ment of additional antiprotozoal analogues. Their
advantages include: (i) physical properties known to be
compatible with desirable pharmacokinetic (low mole-
cular weight, favorable C log P, favorable hydrogen
bond donating and accepting capabilities), (ii) potency
and efficacy, with IC50 values at the low micromolar
level, (iii) simple synthetic access and thus low produc-
tion costs, and (iv) non carbamated groups improving
the likelihood of reasonable solubility. Further optimi-
zation and pharmacokinetic characterization of this
series are ongoing.
5(6)-(Propylthio)-2-(trifluoromethyl)-1H-benzimidazole
(4a). Eluted with CHCl3 and recrystallized from cyclo-
hexane-CHCl3. Yield 6.92 g (85%) of white solid. Mp
102–105 ꢁC. H NMR (300 MHz, CDCl3) d 1.0 (t, 3H,
1
CH3CH2), 1.68 (m, 2H, CH3CH2CH2), 2.90 (t, 2H,
CH2CH2S), 7.42 (dd, 1H, H-6, J=8.55, J=1.50 Hz),

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 11 (2003) 4615–4622
4619
7.65 (d, 1H, H-7, J=8.85 Hz), 7.69 (d, 1H, H-4, J=1.65
Hz), 10.86 (bs, 1H, N-H) ppm; 13C NMR (75.5 MHz,
CDCl3) d 13.27 (CH3CH2), 22.38 (CH3CH2CH2), 36.68
(CH2CH2S), 116.53 (C-4), 116.92 (C-7), 118.72 (q, CF3,
J=270.96 Hz), 127.34 (C-6), 133.91 (C-5), 136.29 (C-
7a), 137.58 (C-3a), 141.16 (q, C-2, J=40.77 Hz) ppm;
MS: m/z (% rel. int.) 260 (M+, 100), 231 (78), 218 (95),
187 (50); HRMS: calcd for C11H11F3N2S: 260.0595,
found: 260.0595.
H-6, J=8.40, J=1.50 Hz), 7.94 (d, 1H, H-7, J=8.40
Hz), 8.01 (d, 1H, H-4, J=0.90 Hz) ppm; 13C NMR
(75.5 MHz, CDCl3) d 31.14 (N-CH3), 112.82 (C-7),
118.74 (q, CF3, J=272 Hz), 121.10 (C-6), 125.79 (C-6),
128.36 (C-30, C-50), 130.01 (C-20, C-60), 132.53 (C-4),
134.77 (C-40), 135.80 (C-5), 137.66 (C-10, C-3a), 143.29
(q, C-2, J=39.48 Hz), 143.63 (C-7a), 196.14 (C¼O)
ppm; MS: m/z (% rel. int.) 304 (M+, 90), 227 (100), 199
(20); HRMS: calcd for C16H11F3N2O: 304.0823, found:
304.0832.
1-Methyl-6-(propylthio)-2-(trifluoromethyl)-1H-benzimi-
dazole (4b). Eluted with CHCl3 and recrystallized from
cyclohexane. Yield 7.46 g (87%) of white solid. Mp 55–
56.9 ꢁC. 1H NMR (300 MHz, CDCl3) d 1.05 (t, 3H,
CH3CH2), 1.71 (m, 2H, CH3CH2CH2), 2.97 (t, 2H,
CH2CH2S), 3.93 (s, 3H, N-CH3), 7.39 (dd, 1H, H-5,
J=8.40, J=1.80 Hz), 7.43 (d, 1H, H-7, J=1.50 Hz),
7.78 (d, 1H, H-4, J=8.10 Hz,) ppm; 13C NMR
6-Benzoyl-1-methyl-2-(trifluoromethyl)-1H-benzimidazole
(20). Eluted with CHCl3 and recrystallized from hexane.
Yield 7.71 g (81%) of white solid. Mp 156.8–158.1 ꢁC.
1H NMR (300 MHz, CDCl3) d 4.0 (s, 3H, N-CH3),
7.46–7.63 (m, 4H, H-20,H-30, H-50, H-60), 7.79–7.82 (m,
2H, H-4, H-40, J=8.40 Hz), 8.06 (dd, 1H, H-5, J=8.70,
J=1.80 Hz), 8.29 (d, 1H, H-7, J=0.90 Hz) ppm; 13C
NMR (75.5 MHz, CDCl3) d 31.15 (N-CH3), 110.36 (C-
7), 118.74 (q, CF3, J=271.80 Hz), 125.15 (C-5), 127.02
(C-4), 128.28 (C-30, C-50), 129.98 (C-20, C-60), 132.37 (C-
40), 133.38 (C-10), 137.680 (C-6), 138.66 (C-7a), 140.10
(C-3a), 142.60 (q, C-2, J=39.10 Hz), 196.01 (C¼O)
ppm; MS: m/z (% rel. int.) 304 (M+, 70), 227 (100), 199
(15); HRMS: calcd for C16H11F3N2O: 304.0823, found:
304.0844.
(75.5 MHz, CDCl3)
d
13.33 (CH3CH2), 22.46
(CH3CH2CH2), 30.76 (N-CH3), 36.78 (CH2CH2S),
110.91 (C-7), 118.97 (q, CF3, J=271.49 Hz), 121.63 (C-
5), 125.88 (C-6), 134.34 (C-4), 136.47 (C-7a), 139.54 (C-
3a), 140.75 (q, C-2, J=38.70 Hz) ppm; MS: m/z (% rel.
int.) 274 (M+, 100), 245 (20), 232 (75); HRMS: calcd for
C12H13F3N2S: 274.0751, found: 274.0767.
1-Methyl-5-(propylthio)-2-(trifluoromethyl)-1H- benzimi-
dazole (8). Eluted with hexane and recrystallized from
cyclohexane-CH2Cl2. Yield 7.98 g (93%) of white solid.
Mp 52.4–54.5 ꢁC. 1H NMR (300 MHz, CDCl3) d 1.01 (t,
3H, CH3CH2), 1.65 (m, 2H, CH3CH2CH2), 2.92 (t, 2H,
CH2CH2S), 3.94 (s, H, N-CH3), 7.36 (d, 1H, H-7,
J=8.7 Hz), 7.47 (dd, 1H, H-6, J=8.55, J=1.50 Hz),
7.87 (d, 1H, H-4, J=1.50 Hz) ppm; 13C NMR
General method of synthesis of propylsulfinyl derivatives
5a, 5b, 9
A stirred suspension of 4a, 4b, or 8 in CHCl3 was trea-
ted, dropwise, with a solution of m-CPBA in CHCl3 at
0–5 ꢁC. The mixture was stirred at 5 ꢁC for 30 min,
neutralized with 50% NaHCO3 solution, and the
organic layer was eliminated in vacuo. The crude pro-
duct was then purified.
(75.5 MHz, CDCl3)
d
13.30 (CH3CH2), 22.41
(CH3CH2CH2), 30.94 (N-CH3), 36.93 (CH2CH2S),
110.35 (C-7), 118.87 (q, CF3, J=271.42 Hz), 122.26 (C-
4), 128.31 (C-6), 132.09 (C-5), 134.60 (C-7a), 140.96 (q,
C-2, J=39.10 Hz), 141.21 (C-3a) ppm; MS: m/z (% rel.
int.) 274 (M+, 98), 245 (30), 232 (100); HRMS: calcd for
C12H13F3N2S: 274.0751, found: 274.0752.
5(6)-(Propylsulfinyl)-2-(trifluoromethyl)-1H-benzimida-
zole (5a). Following the general procedure described
above, 4a (3.5 g, 0.0134 mol) in 30 mL of CHCl3 and m-
CPBA (3.24 g, 0.0188 mol) in 20 mL of CHCl3 gave 5a
(3.2 g, 0.0116 mol, 86%) as a white solid, after recrys-
tallization from cyclohexane-toluene. Mp 123.2–
5(6)-Benzoyl-2-(trifluoromethyl)-1H-benzimidazole (12a).
Eluted with CHCl3 and recrystallized from EtOH. Yield
125.2 ꢁC. H NMR (300 MHz, CDCl3) d 0.93 (t, 3H,
1
6.53 g (72%) of white solid. Mp 55–58 ꢁC. H NMR
CH3CH2), 1.34–1.72 (m, 2H, CH3CH2CH2), 2.73–2.97
(m, 2H, CH2CH2SO), 7.62 (dd, 1H, H-6, J=8.40,
J=1.50 Hz), 7.89 (d, 1H, H-7, J=8.40 Hz), 8.01 (d, 1H,
H-4, J=0.90 Hz), 13.84 (bs, 1H, N-H) ppm; 13C NMR
1
(300 MHz, DMSO-d6) d 7.53–7.78 (m, 5H, H-20, H-30,
H-40, H-50, H-60), 7.83 (dd, 1H, H-6, J=8.40, J=1.20
Hz), 7.85 (d, 1H, H-7, J=8.40 Hz), 8.05 (d, 1H, H-4,
J=1.20 Hz), 10.40 (bs, 1H, N-H) ppm; 13C NMR
(75.5 MHz, DMSO-d6) d 113.52 (C-7), 114.66 (q, CF3,
J=270.96 Hz), 120.08 (C-4), 121.57 (C-6), 124.46 (C-20,
C-60), 125.55 (C-30, C-50), 128.43 (C-40), 128.85 (C-5, C-10),
133.42 (C-3a, C-7a), 138.34 (q, C-2, J=39.80 Hz),
196.91 (C¼O) ppm; MS: m/z (% rel. int.) 290 (M+,
100), 213 (98), 185 (50), 105 (70); HRMS: calcd for
C15H9F3N2O: 290.0666, found: 290.0690.
(75.5 MHz, CDCl3)
d
12.88 (CH3CH2), 15.18
(CH3CH2CH2), 57.71 (CH2CH2SO), 113.43 (C-4),
117.38 (C-7), 118.81 (q, CF3, J=271.42 Hz), 119.53 (C-
6), 138.20 (C-3a), 139.12 (C-7a), 140.04 (C-5), 141.76 (q,
C-2, J=39.40 Hz) ppm; MS: m/z (% rel. int.) 276 (M+,
100), 260 (30); HRMS: calcd for C11H11F3N2OS:
276.0544, found: 276.0563.
1-Methyl-6-(propylsulfinyl)-2-(trifluoromethyl)-1H-benzi-
midazole (5b). Following the general procedure descri-
bed above, 4b (5 g, 0.0182 mol) in 42 mL of CHCl3 and
m-CPBA (4.1 g, 0.0241 mol) in 30 mL of CHCl3 gave 5b
(4.8 g, 0.0165 mol, 90%) as a white solid, after recrys-
tallization from cyclohexane–petroleum ether. Mp 90.7–
91.8 ꢁC. 1H NMR (300 MHz, CDCl3) d 1.0 (t, 3H,
5-Benzoyl-1-methyl-2-(trifluoromethyl)-1H-benzimidazole
(12b). Eluted with CHCl3 and recrystallized from
cyclohexane. Yield 7.61 g (80%) of white solid. Mp
103.1–104.2 ꢁC. Mp 55–56.9 ꢁC. 1H NMR (300 MHz,
CDCl3) d 4.01 (s, 3H, N-CH3), 7.49–7.54 (m, 2H, H-30,
H-50), 7.60–7.66 (m, 3H, H-20, H-40, H-60), 7.81 (dd, 1H,

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 11 (2003) 4615–4622
4620
CH3CH2), 1.56–1.77 (m, 2H, CH3CH2CH2), 2.72–2.97
(m, 2H, CH2CH2SO), 4.14 (s, 3H, N-CH3), 7.59 (dd,
1H, H-5, J=8.70, J=1.50 Hz), 7.93 (d, 1H, H-4,
J=8.70 Hz), 8.05 (d, 1H, H-7, J=1.80 Hz) ppm; 13C
NMR (75.5 MHz, CDCl3) d 13.34 (CH3CH2), 16.33
(CH3CH2CH2), 31.74 (N-CH3), 59.64 (CH2CH2SO),
108.64 (C-7), 119.44 (C-4), 120.0 (q, CF3, J=272.0 Hz),
122.56 (C-5), 138.20 (C-7a), 141.12 (q, C-2, J=39.20
Hz), 143.35 (C-3a) ppm; MS: m/z (% rel. int.) 290 (M+,
20), 273 (15), 248 (90), 200 (100); HRMS: calcd for
C12H13F3N2OS: 290.0700, found: 290.0706.
H2 uptake. The reaction mixture was filtered off on a
Whatman paper No. 2, washed with EtOH, and the fil-
trate concentrated to provide a dark violet-colored
liquid, which was used immediately in a subsequent step
without purification.
2
N -Methyl-4-(propylthio)-1,2-phenylenediamine (3b). A
mixture of 2b (14.2 g, 0.0627 mol), MeOH (120 mL) and
10% Pd/C (700 mg) was hydrogenated at 40 ꢁC until
cessation of H2 uptake. The reaction mixture was fil-
tered off on a Whatman paper No. 2, washed with
EtOH, and the filtrate concentrated to provide a dark
violet-colored liquid, which was used immediately in a
subsequent step without purification.
1-Methyl-5-(propylsulfinyl)-2-(trifluoromethyl)-1H- ben-
zimidazole (9). Following the general procedure descri-
bed above, 8 (1.58 g, 0.0057 mol) in 15 mL of CHCl3
and m-CPBA (1.37 g, 0.0079 mol) in 15 mL of CHCl3
gave 9 (1.34 g, 0.0045 mol, 80%) as a white solid, after
crystallization from cyclohexane–petroleum ether. Mp
N-Methyl-2-nitro-4-thiocyanoaniline (6). To a stirred,
cooled (3–5 ꢁC) solution of N-methyl-2-nitroaniline (12
g, 0.0788 mol) and NH4SCN (14.9 g, 0.1891 mol) in 80
mL of MeOH was added, droppingly, a solution of Br2
(13.88 g, 0.0866 mol) in MeOH (16 mL) previously
saturated with NaBr. The reaction mixture was stirred
for one more h at 5 ꢁC, and then, poured in 200 g of ice-
water. Stirring was continued in the hood until the for-
mation of a precipitate. After work up by filtration, the
crude product was recrystallized from toluene. Yield
ꢁ
1
130–132 C. H NMR (300 MHz, CDCl3) d 1.0 (t, 3H,
CH3CH2), 1.59–1.84 (m, 2H, CH3CH2CH2), 2.72–2.98
(m, 2H, CH2CH2SO), 4.05 (s, 3H, N-CH3), 7.65 (d, 1H,
H-7, J=8.70 Hz), 7.82 (dd, 1H, H-6, J=8.70, J=1.80
Hz), 8.12 (d, 1H, H-4, J=1.50 Hz) ppm; 13C NMR
(75.5 MHz, CDCl3)
d
13.21 (CH3CH2), 15.89
(CH3CH2CH2), 31.18 (N-CH3), 59.64 (CH2CH2SO),
111.49 (C-7), 118.47 (C-4), 119.03 (q, CF3, J=272.0
Hz), 120.65 (C-6), 127.90 (C-3a), 139.40 (C-7a), 140.87
(C-5), 142.01 (q, C-2, J=39.20 Hz) ppm; MS: m/z (%
rel. int.) 290 (M+, 100), 273 (15), 248 (20), 200 (10);
HRMS: calcd for C12H13F3N2OS: 290.0700, found:
290.0701.
ꢁ
1
14.81 g (90%) of orange crystals. Mp 109–111 C. H
NMR (300 MHz, DMSO-d6) d 3.03 (s, 3H, N-CH3),
6.77 (d, 1H, H-6, J=6.0 Hz), 7.48 (dd, 1H, H-5, J=6.0
Hz, J=2.0 Hz), 8.24 (d, 1H, H-3, J=2.0 Hz) ppm; MS:
m/z (% rel. int.) 209 (M+, 100), 192 (4), 183 (4).
N-Methyl-2-nitro-4-(propylthio)aniline (7). A mixture of
6 (6 g, 0.0287 mol), 1-PrBr (23 g, 0.187 mol), CH2Cl2
(60 mL), 1-PrOH (11.16 g), KCN (9 g, 0.1836 mol),
(Bu)4NCl (1 g) and H2O (27 mL) was heated under
reflux for 4 h in a N2 atmosphere. Workup by extraction
with EtOAc and concentration under vacuum left a red
oil, which was purified by column chromatography
(4ꢂ60 cm, 60 g of silica gel, petroleum ether). Yield
6.23g (93%) of red oil. This oil was reduced exactly as
2-Nitro-5-(propylthio)aniline (2a). A stirred mixture of
1a (42.1 g, 0.244 mol), NaOH (12 g, 0.3000 mol), ethyl-
ene glycol (11.5 mL), H2O (587 mL) and 1-PrSH (27.88
g, 0.2527 mol) was heated under reflux for 5 h. The
mixture was cooled, filtered by suction and the crude
product recrystallized from 2-PrOH. Yield 51 g (98.5%)
of a yellow solid. Mp 69–71 C. H NMR (300 MHz,
CDCl3) d 1.10 (t, 3H, CH3CH2), 1.80 (m, 2H,
CH3CH2CH2), 2.90 (t, 2H, CH2CH2S), 6.10 (bs, 2H,
NH2), 6.50 (m, 2H, H-4, H-6, J=9.0, J=1.50, J=1.70
Hz), 7.90 (dd, 1H, H-3, J=9.0 Hz) ppm; MS: m/z (%
rel. int.) 212 (M+, 100).
ꢁ
1
1
2a, 2b. H NMR (300 MHz, DMSO-d6) d 0.95 (t, 3H,
CH3CH2), 1.53 (m, 2H, CH3CH2CH2), 2.72 (t, 2H,
CH2CH2S), 3.03 (s, 3H, N-CH3), 6.45 (d, 1H, H-6,
J=6.0 Hz), 7.30 (dd, 1H, H-5, J=6.0, J=2.0 Hz), 7.95
(d, 1H, H-3, J=2.0 Hz) ppm; MS: m/z (% rel. int.) 226
(M+, 100), 197 (10), 183 (33), 151 (12).
N-Methyl-2-nitro-5-(propylthio)aniline (2b). A stirred
mixture of 5-chloro-N-methyl-2-nitroaniline (12.1 g
(0.0648 mol), KOH (4.55 g, 0.0810 mol), ethylene glycol
(33 mL), H2O (150 mL) and 1-PrSH (5.68 g, 0.0745
mol) was heated at 115 ꢁC for 4 h. The mixture was
cooled, filtered by suction and the crude product
recrystallized from hexane. Yield 14.2 g (96.8%) of
orange crystals. Mp 61–62 ꢁC. 1H NMR (300 MHz,
DMSO-d6). d 0.99 (t, 3H, CH3CH2), 1.65 (m, 2H,
CH3CH2CH2), 2.91 (s, 3H, N-CH3), 3.10 (t, 2H,
CH2CH2S), 6.50 (dd, 1H, H-3, J=9.0, J=2.0 Hz), 6.69
(dd, 1H, H-6, J=2.0 Hz), 7.90 (d, 1H, H-4, J=9.0 Hz),
8.20 (bs, 1H, N-H) ppm; MS: m/z (% rel. int.) 226 (M+,
100).
3-Amino-4-(methylamino)benzophenone (11b). A mixture
.
of 10b (9.6 g, 0.0395 mol), SnCl2 2H2O (53.47 g, 0.2370
mol) and 150 mL of ethanol was stirred at 75ꢁ C for 3.5
h under a N2 atmosphere. After cooling, the mixture
was basified (pH=9–10) with a 50% NaOH solution
and then filtered by suction. The solvent was carefully
removed in vacuo and the solid residue was extracted
with AcOEt (3ꢂ50 mL). The combined organic extracts
were washed with brine, dried with anhydrous Na2SO4
and concentrated in vacuo to give 7.62 g (86%) of 11b
as an orange solid, which was immediately ciclo-
condensed in the next step. An analytical sample was
obtained by recrystallization from EtOH to give orange
1
4-(Propylthio)-1,2-phenylenediamine (3a). A mixture of
2a (6.37 g, 0.0282 mol), EtOH (100 mL) and 10% Pd/C
(300 mg) was hydrogenated at 25 ꢁC until cessation of
crystals. Mp 129–131 ꢁC. H NMR (300 MHz, DMSO-
d6) d 2.85 (s, 3H, N-CH3), 3.40 (s, 3H, N–H, NH2), 6.50
(m, 1H, H-2), 6.91-7.56 (m, 6H, H-5, H-20, H-30, H-40,

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 11 (2003) 4615–4622
4621
H50, H-60), 7.72 (m, 1H, H-6) ppm; MS: m/z (% rel. int.)
by suction. The solvent was removed carefully in vacuo,
and the solid residue was extracted with AcOEt (3ꢂ50
mL). The combined organic extracts were washed with
brine, dried with anhydrous Na2SO4 and concentrated
in vacuo. The yellow residue was recrystallized from
benzene. Yield 4.3 g (81.3%) of 19 as yellow crystals,
which were immediately ciclocondensed in the next step.
Mp 118.7–120.4 ꢁC. 1H NMR (300 MHz, CDCl3) d 2.71
(s, 3H, N-CH3), 4.92 (s, 1H, N-H), 5.52 (s, 2H, NH2),
6.55 (d, 1H, H-5, J=8.70 Hz), 6.88 (m, 2H, H-2, H-6,
J=8.70, J=1.50, J=1.80 Hz), 7.43–7.62 (m, 5H, H-20,
H-30, H-40, H-50, H-60) ppm; MS: m/z (% rel. int.) 226
(M+, 100), 211 (27), 149 (81).
226 (M+, 100), 211 (27), 149 (58).
3-Chloro-4-nitrobenzoic acid (15). To 3-chlorotoluene
(100 g, 0.7900 mol) was added a cold (0–10 ꢁC) mixture
of H2SO4–HNO3 (95-79) at 15–22 ꢁC. After the addi-
tion, the organic layer was separated, washed with brine
and dried with CaCl2 to give a yellow oil (126.48 g) as a
mixture of three compounds. To a stirred suspension of
this mixture (120 g, 0.699 mol) and K2Cr2O7 (236.2 g,
0.9480 mol) was carefully added H2SO4 (520 mL). The
hot reaction mixture was heated under reflux for 2 h and
then cooled with 1000 mL of water. The mixture was
filtered and the residue washed with 5% solution of
H2SO4, water, and filtered to afford 58 g (40%) of yel-
low-green solid. Mp 181–183.4 ꢁC. An analytical sample
was obtained by recrystallization from CHCl3 to give
white crystals, Mp 183–184 ꢁC. 1H NMR (300 MHz,
CDCl3) d 8.08 (d, 1H, H-2, J=1.50 Hz), 8.15 (d, 1H, H-
5, J=8.50 Hz), 8.18 (dd, 1H, H-6, J=9.0, J=1.70 Hz),
14.0 (s, 1H, O–H) ppm; MS: m/z (% rel. int.) 201 (M+,
100), 171 (38), 143 (24).
Biological assays
Culture. G. lamblia strain IMSS:0989:1 and Trichomo-
nasvaginalis strain GT3 were cultured in TYI-S-33
modified medium, supplemented with 10% calf serum
and bovine bile.1 In vitro susceptibility assays were per-
formed using a method previously described.1 Briefly:
4ꢂ104 trophozoites of G. lamblia or T. vaginalis were
incubated for 48 h at 37 ꢁC with increasing concentra-
tions of synthesized compounds, Abz, and Metronida-
zole. As the negative control, trophozoites were
incubated with dimethylsulphoxide (DMSO) used in the
experiments. After the incubation, the trophozoites
were washed and subcultured for another 48 h in fresh
medium alone. At the end of this period, trophozoites
were counted and the 50% inhibitory concentration
(IC50) was calculated by Probit analysis. Experiments
were carried out in triplicate and repeated at least
twice.
3-Chloro-4-nitrobenzoyl chloride (16). A mixture of 15
(50 g, 0.248 mol) and thionyl chloride (87.8 g, 0.744
mol) was heated in a dry atmosphere until gas evolution
was ceased. The excess of thionyl chloride was distilled
from the reaction and the residue was used immediately
for the next step.
3-Chloro-4-nitrobenzophenone (17). A stirred mixture of
16 (64 g, 0.248 mol), benzene (130 mL) and AlCl3 (45 g,
0.3383 mol) was heated at 80 ꢁC, under a N2 atmo-
sphere, for 3 h. The cooled mixture was poured in ice-
water (500 g). After work up by filtration and extraction
with EtOAc, the crude product was recrystallized from
ethanol. Yield 52.3 g (80.7%) of a light brown solid. Mp
Trichinella spiralis muscle larvae (parenteral phase) were
obtained according to the procedure of Dennis et al.19
For the assay, 1000 larvae were placed in culture plates
of 24 wells (Nunclon), which contained RPMI 1640
medium, with 0.037, 0.188, 0.37, 1.88 mM of the com-
pounds tested. The parasites were then incubated in a
humid 5% CO2 atmosphere at 37 ꢁC for 3 days, chan-
ging the medium and the compounds each day. Abz was
used in this test as a positive control, and the solvent
employed, as a negative control. After the incubation,
the viability of the parasites was determined by the col-
orimetric method described by Townson et al.,20 with
some modifications.21
97.5–98.2 ꢁC. H NMR (300 MHz, CDCl3) d 7.51–7.57
1
(m, 2H, H-30, H-50), 7.64–7.70 (m, 1H, H-40), 7.77–7.81
(m, 3H, H-20, H-60, H-6), 7.94 (d, 1H, H-2, J=2.10 Hz),
7.95 (d, 1H, H-5, J=6.30 Hz) ppm; MS: m/z (% rel.
int.) 261 (M+, 78), 105 (100), 77 (44).
3-(Methylamino)-4-nitrobenzophenone (18). A stirred
mixture of 17 (10 g, 0.0388 mol), 1,2-dimethoxyethane
.
(150 mL), CH3NH2 HCl (7.74 g, 0.1147 mol) and
K2CO3 (15.82 g, 0.1147 mol) in H2O (8 mL) was heated
for 6 h at 130ꢁ C in a Parr reactor. Then, the cooled
reaction mixture was poured over H2O (500 mL). The
orange precipitate was filtered off and recrystallized
from ethanol. Yꢁield 8.6 g (88.6%) of an orange solid.
C. elegans assay was performed following the method of
Simpkin and Coles22 with slight modifications. Tests
were carried out in 24-well plates and four wells were
used for each experimental group. To each well, 1.0 mL
of culture medium (with 9 mg/mL chloramphenicol
instead of ampicillin) was added followed by 7.5 mL of
the appropriate compound solution or solvent (DMSO).
Finally, 0.5 mL of culture medium containing 10–15 C.
elegans larvae (L2 or L3 obtained of synchronous cul-
tures) was added to each well. 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 20ꢃ1 ꢁC. Mebendazole
was used in this test as positive control.
1
Mp 121.2–122.5 C. H NMR (300 MHz, CDCl3) d 3.0
(s, 3H, N-CH3), 6.92 (dd, 1H, H-6, J=8.70, J=1.80
Hz), 7.23 (d, 1H, H-2, J=1.50 Hz), 7.48–7.53 (m, 3H,
H-20, H-40, H-60), 7.82–7.85 (m, 2H, H-30, H-50), 8.07 (s,
1H, N-H), 8.25 (d, 1H, H-5, J=8.70 Hz) ppm; MS: m/z
(% rel. int.) 256 (M+, 100), 161 (35), 105 (70).
4-Amino-3-(methylamino)benzophenone (19). A mixture
.
of 18 (6 g, 0.0234 mol), SnCl2 2H2O (31.7 g, 0.1406 mol)
and EtOH (120 mL) was stirred at 75ꢁ C for 4 h under a
N2 atmosphere. After cooling, the mixture was basified
(pH=9–10) with a 50% NaOH solution and then filtered

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 11 (2003) 4615–4622
4622
Acknowledgements
9. Lacey, E. Int. J. Parasitol. 1998, 18, 885.
10. Valdez, J.; Cedillo, R.; Hernandez-Campos, A.; Yepez-
Mulia, L.; Hernandez-Luis, F.; Navarrete-Vazquez, G.; Mor-
ales, R.; Cortes, R.; Hernandez, M.; Castillo, R. Bioorg. Med.
Chem. Lett. 2002, 12, 2221.
11. Singh, S.; Sharma, S. Med. Res. Rev. 1991, 11, 581.
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13. Navarrete-Vazquez, G.; Cedillo, R.; Hernandez-Campos,
A.; Yepez-Mulia, L.; Hernandez-Luis, F.; Valdez, J.; Morales,
R.; Cortes, R.; Hernandez, M.; Castillo, R. Bioorg. Med.
Chem. Lett. 2001, 11, 187.
This work was supported by grants from CONACyT
G34851-M and DGAPA IN202101. Gabriel Navarrete-
Vazquez acknowledges the fellowship awarded by CON-
ACyT and DGEP-UNAM to carry out graduate studies.
We are grateful to Rosa Isela del Villar, Georgina Duarte,
Margarita Guzman, and Marisela Gutierrez from the
School of Chemistry, UNAM, for the determination of all
spectra. Also, we thank Jessica Gonzalez and Marıa de
Lourdes Enrıquez for carrying out the biological assays.
14. Andrzejewska, M.; Yepez-Mulia, L.; Cedillo-Rivera, R.;
Tapia, A.; Vilpo, L.; Vilpo, J.; Kazimierczuk, Z. Eur. J. Med.
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