Synthesis and antitrichinellosis activity of some bis(benzimidazol-2-yl)amines

Article abstract of DOI:10.1016/j.bmc.2007.06.017

Novel bis(benzimidazol-2-yl)amines were synthesized using two methods and studied for antitrichinellosis activity. DFT calculations were performed in order to determine the geometry of molecules. All derivatives of 2-aminobenzimidazole exhibited higher activity in vitro against Trichinella spiralis larvae in regard to the activity of albendazole, moreover compounds 4f-i manifested antitrichinellosis effect, which surpassed five times the activity of albendazole. The in vivo screening of intestinal phase of the T. spiralis revealed 100% effectiveness of compounds 4g-i at oral dosages of 50 and 100 mg/kg mw, while albendazole possesses 100% efficacy only at a dose of 100 mg/kg mw.

Full text of DOI:10.1016/j.bmc.2007.06.017

Bioorganic & Medicinal Chemistry 15 (2007) 6291–6297
Synthesis and antitrichinellosis activity of some
bis(benzimidazol-2-yl)amines
Anelia Ts. Mavrova,a,* Pavletta Denkova,b Yordan A. Tsenov,b
Kameliya K. Anichinaa and Dimitar I. Vutchevc
aDepartment of Organic Synthesis, University of Chemical Technology and Metallurgy, 8 Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
bNational Center of Infectious and Parasitic Diseases, 26 Yanko Sakazov Blvd., 1504 Sofia, Bulgaria
cInstitute of Organic Chemistry, Bulgarian Academy of Science, 1113 Sofia, Bulgaria
Received 14 December 2006; revised 29 May 2007; accepted 8 June 2007
Available online 13 June 2007
Abstract—Novel bis(benzimidazol-2-yl)amines were synthesized using two methods and studied for antitrichinellosis activity. DFT
calculations were performed in order to determine the geometry of molecules.
All derivatives of 2-aminobenzimidazole exhibited higher activity in vitro against Trichinella spiralis larvae in regard to the activity
of albendazole, moreover compounds 4f–i manifested antitrichinellosis effect, which surpassed five times the activity of albendazole.
The in vivo screening of intestinal phase of the T. spiralis revealed 100% effectiveness of compounds 4g–i at oral dosages of 50 and
100 mg/kg mw, while albendazole possesses 100% efficacy only at a dose of 100 mg/kg mw.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
the development of various drugs as mebendazole, fen-
bendazole, oxbendazole and albendazole support fur-
ther the importance of this heterocycle in generating
better chemotherapeutical agents against parasitic
diseases.13–15 The presence of the 2-aminobenzimidazole
moiety presumes that probably that fragment is respon-
sible for the anthelmintic properties of these
compounds. Unless the high efficacy of the above-men-
tioned drugs the definitive treatment of trichinellosis,
one of the most disseminated tissue helminthosis,
remains pending. Therefore, it is of pharmacological
interest to synthesize some new derivatives of 2-amino-
benzimidazole and to study their anthelmintic activity.
The 2-aminobenzimidazoles are promising class of
chemical compounds with different biological effects as
immunotropic, diuretic, antihistaminic as well as highly
selective p38a MAP inhibition properties.1–5 The poly-
functionality of the 2-aminobenzimidazole molecule
resulting from the cyclic guanidine moiety has made it
a building block for the synthesis of a large number of
benzimidazole derivatives of pharmacological interest.
Following enviradene and enviroxime, many other 2-
aminobenzimidazole derivatives have been synthesized
and evaluated for antiviral activity.6,7 Moreover, a
number of 2-aminobenzimidazoles have exhibited
antiproliferative properties.8,9 Different substituted
2-aminobenzimidazoles have been found to possess
in vivo and in vitro growth inhibition activity against
various strains of bacteria, fungi and yeasts.10–12
Continuing our studies aimed at developing new potent
antitrichinellosis agents, 2-aminobenzimidazole deriva-
tives were prepared. In this paper, we report the synthe-
sis of some novel bis(benzimidazol-2-yl)amines as well
as their pharmacological evaluation for antitrichinellosis
activity. That set of compounds is structurally different
from the already known antitrichinellosis drug albenda-
zole because other benzimidazole cycle is bonded to the
amino group at second position instead of a methoxy-
carbonyl group and an alkyl group is introduced to
the imino group of the benzimidazole ring in some of
the compounds. As 2-aminobenzimidazole compounds
are usually associated with antiparasitic activity it may
be expected the bis(benzimidazol-2-yl)amines to display
The demonstration of potent anthelmintic activity of the
different substituted 2-aminobenzimidazoles as well as
Keywords: 2-Aminobenzimidazole; Bis(benzimidazol-2-yl)amines; An-
titrichinellosis activity; DFT.
*
Corresponding author. Tel.: +359 28163207; fax: +359 2685488;
e-mail: anmav@abv.bg
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.017

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A. Ts. Mavrova et al. / Bioorg. Med. Chem. 15 (2007) 6291–6297
potential biological activity against different parasites
and physiological disorders.
with physiological solution and the samples only in
DMSO all T. spiralis larvae had spiral form, i.e., vital.
The in vivo screening of intestinal phase of the T. spiralis
done by us revealed 100% effectiveness of compounds
4g–i after a 3-day-treatment course with oral dosages
of 50 and 100 mg/kg mw, beginning on the third day
of infection. We obtained the same results using
2. Results
The synthesis of the compounds 4a–i was accomplished
using two reaction methods: substitution of the sulfonic
group by fusion of 1-(un)substituted benzimidazol-2-yl-
sulfonic acid 2a–d with 1-alkyl-2-amino-benzimidazole
3a–d (method A) and reaction of 1-alkyl-2-(methyl-
thio)benzimidazoles 5a–c with 1-alkyl-2-aminobenzimi-
dazole 5a–c (method B), as outlined in Figures 1 and 2.
albendazole as standard at
a
concentration of
100 mg/kg mw.22,23 White immature mice were used as
experimental animals.
3. Discussion
The reaction of 1,2-diaminobenzene, carbon disulfide
and sodium hydroxide in ethanol medium yielded com-
pound 1a.16,17
From the large diversity of methods, described in the lit-
erature for the synthesis of the intermediate compounds
1–3, we chose the methods marked by their perfor-
mance, simplicity and the possibility of every one of
the synthesized compounds being used as starting com-
ponent in the next reaction.
The starting compounds 1b–d were obtained by fusion
of the corresponding 1-alkylbenzimidazoles with sulfur
at 180 °C for 30 min according to the cited procedure.18
The oxidation of the 1-(un)substituted-1H-ben-
zimidazol-2-yl-thiols 1a–d with KMnO4 in 25% water
solution of sodium hydroxide led to the formation of
1-(un)substituted-1H-benzimidazol-2-yl-sulfonic acids
2a–d.19 The reaction of benzimidazol-2-yl-sulfonic acids
2a–d, carried out with 25% ammonium hydroxide in
welded ampoule at 145–150 °C for 5 h, resulted in 2-
aminobenzimidazoles 3a–d.20
It is well known that sulfonic group at the second posi-
tion in the benzimidazole can be easily replaced with
alkoxy, hydrazine, amino, alkyl, aryl and heteroamino-
group.18–20 On the base of these facts we used
1-(un)substituted benzimidazol-2-yl-sulfonic acids to
synthesize bis(benzimidazol-2-yl)amines. The reaction
between benzimidazol-2-yl-sulfonic acids 2 and 2-
aminobenzimidazoles 3 (method A), as it is shown in
Figure 1, was carried out at molar ratio of reagents
1:2 and temperature 180 °C for 30 min. The yields are
in the range of 48–70%. It was ascertained that the yields
of bis(benzimidazol-2yl)amines increased as the alkyl
chain in the (H)-benzimidazol-2-yl-sulfonic acids was
longer compared to the alkyl chain in the 2-aminobenz-
imidazoles. For compound 4h the yield was 42% when
compounds 2c and 3d were used, but the reaction
between 2d and 3c led to an increase of the yield up to
70%. The same relations were observed for the yields
of compounds 4a–c, 4e and 4f.
The bis(benzimidazol-2-yl)amines 4a–d were synthesized
by heating of benzimidazol-2-yl-sulfonic acids 2a–d and
2-aminobenzimidazoles 3a–d.
The chemical structures of all new compounds were
established by IR, 1H NMR and 13C NMR spectra
as well as elemental analysis. The IR-spectral charac-
teristics (all spectra taken in KBr) are quite similar
and could be summarized in the range as follows:
m(N–H): 3159.67–3175.76 cmꢀ1; m(CH3): 2962.88 cmꢀ1
;
m(AC@NA): 1627.35–1808.19 cmꢀ1; d (Ar) 732.69–
800.26 cmꢀ1. Detail assignment of the 1H NMR and
13C NMR spectra of the studied compounds is given
in the Experimental part. The elemental analyses indi-
cated by the symbols of the element were within
0.4% of theoretical values (Table 1).
We have investigated also the possibility to replace the
starting benzimidazol-2-yl-sulfonic acids 2 with 2-(meth-
ylthio)benzimidazoles 5 (method B, Fig. 2). We have
experimentally proved that the necessary temperature
for the proceeding of the reaction is about 250 °C and
that the duration of the process is 15 min, but the yields
of bis(benzimidazol-2-yl)amines were lower in compari-
son to the yields obtained by means of method A.
2.1. Computations
In order to determine the electronic properties and the
geometry of the molecule of the synthesized compounds
DFT computations were performed with standard
Gaussian 98 program package. We employed the
B3LYP hybrid functional with 6-31G basis set.21
Therefore, method A is the preferable reaction way. This
method permits pure bis(benzimidazol-2-yl)amines to be
obtained with reasonable yields. The basic advantages
of the proposed method are its simplicity, lower reaction
temperature, and the use of easily accessible and non-
toxic raw materials.
2.2. Anthelmintic activity
The parasitological study in vitro showed that most of
the tested compounds possess higher activity than both
the activity of albendazole and the efficacy of ivermectin
against T. spiralis. Moreover, the efficacy of compound
4g was 98% and that of compounds 4h and 4i was 100%.
The results are given in Table 2. In the control samples
In the 13C NMR spectra of compounds substituted in
one of the two benzimidazole rings (compounds 4a–c,
R = H, see Fig. 3) we observed broadening of the sig-
nals for some carbon atoms from the unsubstituted
benzimidazole ring. The respective carbons are

A. Ts. Mavrova et al. / Bioorg. Med. Chem. 15 (2007) 6291–6297
6293
O
N
N
S O
OH
R1
O
(2 a-d)
NH4OH
145 0C
N
N
N
N
KMnO4
NaOH
N
N
NH2
S O
OH
SH
R
R
R
1 a - d
1 a: R = H;
2 a - d
2 a: R = H;
3 a - d
3 a: R = H;
1 b: R = CH3
1 c: R = C2H5
1 d: R = C3H7
2 b: R = CH3
2 c: R = C2H5
2 d: R = C3H7
3 b: R = CH3
3 c: R = C2H5
3 d: R = C3H7
19
14
N
N
18
17
11
R1
N
5
NH
2
13
16
R
N
4
6
9
7
8
4 a - i
4 a: R = H, R1 = CH3; 4 b: R = H, R1 = C2H5; 4 c: R = H, R1 = C3H7;
4 d: R = CH3; R1 = CH3; 4 e: R = CH3, R1 = C2H5; 4 f: R = CH3, R1 = C3H7;
4 g: R = C2H5, R1 = C2H5; 4 h: R = C2H5, R1 = C3H7; 4 i: R = C3H7, R1 = C3H7;
Figure 1. Synthesis of bis(benzimidazol-2-yl)amines (method A).
3 b: R =CH3
3 c: R = C2H5
3 d: R = C3H7
N
N
NH2
R
3 b - d
N
N
N
CH3I
NH
N
SH
SCH3
R1
250 oC
N
N
R
N
R1
N
R1
NaOH
5 a - c
1 a - b
4 e-h
5 a: R1 =CH3
5 b: R1 = C2H5
5 c: R1= C3H7
4 e: R = CH3; R1 = C2H5
4 f: R = C3H7; R1 = CH3
4 g: R = C2H5; R1 = C2H5
4 h: R = C3H7; R1 = C2H5
1 a: R1 =CH3
1 b: R1 = C2H5
Figure 2. Synthesis of bis(benzimidazol-2-yl)amines (method B).
indicated as broad singlet (br s) in the description of
the NMR spectra. These observations imply the pres-
ence of dynamic equilibrium between at least two pos-
sible tautomeric forms for the monosubstituted
compounds. Each of the compounds 4a–c has seven
potential tautomeric forms. To determine the most
favourable tautomeric structures of compounds 4a–c
we used ab initio calculations with complete optimiza-
tion of the molecular geometry. We carried out the cal-
culations with the standard Gaussian 98 package at
RHF level using 6-31G basis set. The calculations per-
formed in a gas phase show that the most favorable
forms are those presented in Figure 3 with tautomer
B being the more stable one. These results however
are not consistent with the observed small chemical dif-
ference of carbon atoms C-11 and C-2 which is
0.2 ppm only. If the tautomeric form B is predominant
larger chemical shift difference of the two atoms is ex-
pected because one of the C@N bonds is exocyclic,
while the other one is in the benzimidazole ring. The
small chemical shift difference of the two atoms sug-
gests that both C@N bonds are included in the cyclic
structure. It is well known that the tautomeric equilib-
rium is very sensitive to the type of the solvent. To ac-
count for the effect of the polar solvent we performed
the theoretical calculations including two molecules of
DMSO in the vicinity of the NH molecular fragments.
The results show that in that case the tautomeric form
A is more stable, which is in agreement with the chemi-
cal shifts of carbon atoms C-11 and C-2.

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A. Ts. Mavrova et al. / Bioorg. Med. Chem. 15 (2007) 6291–6297
Table 1. Bis(benzimidazol-2-yl)amines 4a–i
Compound
R
R0
Mol. Form. (MW)
Calculated (%)
Found (%)
Rf
Yield (%)
C
H
N
C
H
N
4a
4b
4c
4d
4e
4f
H
CH3
H
C15H13N5
(263.30)
68.42
4.98
26.60
25.25
24.02
25.25
24.04
22.93
68.21
5.14
26.48
0.58
0.56
0.34
0.66
14
40
20
49
28
55
38
CH3
H
C2H5
H
C16H15N5
(277.13)
69.29
70.08
69.29
70.08
70.08
5.45
5.88
5.45
5.88
6.27
69.45
70.31
69.19
70.23
70.21
5.34
5.67
5.61
5.68
6.38
25.41
23.98
25.30
24.33
22.74
C2H5
H
C3H7
H
CH3
C17H17N5
(291.35)
C3H7
CH3
C16H15N5
(277.13)
CH3
C2H5
CH3
C2H5
CH3
C17H17N5
(291.35)
0.47
0.47
0.70
0.70
0.71
0.76
0.76
0.78
34
47
37
64
35
42
70
50
C3H7
CH3
C18H19N5
(305.38)
C3H7
C2H5
C2H5
C3H7
C3H7
4g
4h
C2H5
C3H7
C2H5
C3H7
C18H19N5
C19H21N5
(319.40)
70.08
71.45
6.27
6.63
22.93
21.93
70.21
71.60
6.38
7.06
22.74
22.09
4i
C20H23N5
72.04
6.95
21.00
72.23
7.09
20.89
Table 2. In vitro efficacy of compounds 4a–i against Trichinella larvae
Compound
Efficacy (%) after 24 h
Compound
Efficacy (%) after 48 h
50 lg/ml
100 lg/ml
200 lg/ml
50 lg/ml
100 lg/ml
200 lg/ml
4a
4b
9.1
15.7
12.0
8.8
13.5
20.0
17.6
17.3
65.7
73.8
92.0
84.0
81.2
10.7
48.6
30.2
38.2
67.7
45.7
76.3
80.0
94.0
96.0
100
4a
4b
17.2
25.3
20.0
14.0
65.3
43.4
66.3
46.0
58.6
14.8
62.4
28.3
38.6
34.5
18.3
73.0
80.0
97.5
92.1
95.3
15.1
78.6
58.3
65.0
78.0
67.9
84.0
88.4
98.0
100
4c
4d
4c
4d
4e
35.3
33.3
45.8
42.0
45.9
10.6
45.5
4e
4f
4g
4f
4g
4h
4i
4h
4i
100
Albendazole
Ivermectin
13.3
54.3
Albendazole
Ivermectin
17.4
88.2
a
b
pounds 4a–i exert different anthelmintic effect, expressed
in the suppressing of larvae motor activity and the open-
ing of their spiral form, which is a mark of nonviability.
In the first control group with physiological solution
and in the second one with DMSO practically all larvae
were in spiral form.
H
H
N
N
NH
N
N
N
N
NH
N
R1
R1
Figure 3. The most favourable tautomeric forms of compounds 4a–c
as determined by ab initio calculations.
All derivatives of 2-aminobenzimidazole revealed high-
er activity against T. spiralis larvae in regard to the
activity of albendazole, moreover compounds 4f–i
manifested antitrichinellosis effect, which surpassed
five times the activity of albendazole. The compounds
4e–i demonstrated higher effect against T. spiralis larvae
than ivermectin after 24 h. The efficacy of compounds
4g–i was also higher than the efficacy of ivermectin
after 48 h.
The ab initio computations performed for compounds
4d–i indicated that they possess nonplanar geometry of
molecule in contrast to the other broad spectrum benz-
imidazole anthelmintics. The dihedral angle between
both cycles increases and is 45° for compound 4i. It
may be supposed that the nonplanar structure facilitates
the interaction with the biological targets.
Statistically significant differences in the level of para-
sites in both control and experimental groups were
determined (p 6 0.05).
3.1. Antitrichinellosis activity
Most of the tested bis(benzimidazol-2-yl)amines exhib-
ited significant activity against T. spiralis larvae. In par-
asitological experiment in vitro for antitrichinellosis
activity it was ascertained that the investigated com-
The obtained results prompted us to study further three
of the compounds—4g–i for parasitocide effect in vivo
using white mice, infected with T. spiralis.

A. Ts. Mavrova et al. / Bioorg. Med. Chem. 15 (2007) 6291–6297
6295
As a result of the in vivo screening of intestinal phase of
the T. spiralis in white mice, 100% effectiveness of com-
pounds 4g–i was established after a 3-day-treatment
course with oral dosages of 50 and 100 mg/kg mw,
beginning on the third day of infection. Diversions in
the behaviour of the experimental mice during the treat-
ment were not observed. By means of microscopic con-
trol study of the intestinal content after autopsy of all
tested animals no larvae were found. That fact indicated
that all three compounds possess remarkable antitrich-
inellosis efficacy while albendazole exhibits 100% activ-
ity at a dose of 100 mg/kg mw.
description of the spectra is shown in Figure 1. The
microanalyses for C, H and N were performed on Per-
kin-Elmer elemental analyzer.
5.1. Chemistry
5.1.1. General procedure for compound 1a. 1,2-Diamino-
benzene (0.019 mol) and water (3 ml) were added to a
solution of sodium hydroxide (0.022 mol) in ethanol
(20 ml) and carbon disulfide (0.022 mol). The mixture
was heated under reflux for 3 h. Charcoal was added
cautiously and removed by filtration after the mixture
has been refluxed for 10 min more. The filtrate was
heated to 60–70 °C and quenched with warm water
(70°, 20 ml), and then 50% acetic acid (9 ml) was added
by good stirring. The product was separated and after
cooling in refrigerator for 3 h the crystallization was
completed.
4. Conclusion
New derivatives of 2-amino-benzimidazole were synthe-
sized by use of two methods and studied for their
anthelmintic activity. The ab initio computations
indicated that the investigated compounds 4a–c possess
planar geometry of molecule, while for the disubstituted
compounds 4d–i nonplanar structure was determined in
contrast to the other broad spectrum benzimidazole
anthelmintics.
5.1.2. General procedure for compounds 1b–d. 1-Alkyl-
benzimidazole (0.01 mol) and sulfur (0.01 mol) were
heated at 260 °C for 30 min. About 10% water solution
of sodium hydroxide was added after cooling of the
fusion. The 1-alkyl-benzimidazole-2-thiol crystallized
by neutralization of the obtained solution with acetic
acid. After filtration the compound was washed with
water and re-crystallized with ethanol.
The in vitro screening revealed significant antiparasitic
activity of the tested compounds. All benzimidazole
derivatives of 2-aminobenzimidazole showed higher
activity against T. spiralis larvae in comparison to alben-
dazole, furthermore, some of the compounds (4g–i)
demonstrated antitrichinellosis activity, which surpassed
five times the activity of albendazole. In regard to iver-
mectin compounds 4c–d and 4g–i demonstrated higher ef-
fect against T. spiralis larvae at concentration 200 lg/ml
after 24 h. The results obtained from the in vivo screen-
ing indicated that compounds 4g–i possess 100% effec-
tiveness at a concentration of 50 mg/kg mw.
5.1.3. General procedure for compounds 2a–d. To a boil-
ing solution of 1-(un)substituted-1H-benzimidazol-2-yl-
thiol (0.04 mol) in water (25 ml) and 50% sodium
hydroxide (15 ml), a solution of potassium permanga-
nate (0.08 mol) in 200 ml was added in small portions
by stirring. After the complete addition of potassium
permanganate the reaction solution was refluxed for
30 min more. The formed manganese dioxide was fil-
tered. The filtrate was treated with hydrochloric acid
to pH 1 by cooling. The obtained precipitate of
These above results confirmed also the hypothesis that
the introduction of a second benzimidazole ring in the
structure of 2-aminobenzimidazoles and the nonplanar
geometry are auspicious to the interaction of these mol-
ecules with the biological targets.
1-(un)substituted-1H-benzimidazol-2-yl-sulfonic
was filtered and washed with water.
acid
5.1.4. General procedure for compounds 3a–d. 1-(Un)
substituted-1H-benzimidazol-2-sulfonic acid (0.002 mol)
and 25% ammonium hydroxide (1 ml) were heated in
welded ampoule for 5 h at 145–150 °C. After cooling
the formed crystals of 2-aminobenzimidazole were fil-
tered and washed with ammonium hydroxide (0.3 ml)
and water (4 ml) and re-crystallized with ethanol.
5. Experimental
Melting points (mp) were determined on an Electrother-
mal AZ 9000 3MK4 apparatus and are uncorrected. The
thin layer chromatography (TLC, Rf values) was per-
formed on silica gel 60 plates F254 (Merck, 0.2 mm thick-
ness) using mobile phase n-heptane/ethyl acetate ꢀ2:1
and visualization was effected with ultraviolet light. IR
spectra were recorded on a HP-ST-IR spectrophotome-
ter as potassium bromide discs. NMR spectra were
recorded on a Bruker Avance DRX 250 spectrometer
(Bruker, Faelanden, Switzerland), with working fre-
quency for 1H 250 MHz, using a dual 1H/13C 5 mm pro-
behead. CDCl3 and DMSO-d6 were used as solvents.
Chemical shifts were expressed relative to tetramethylsi-
lane (TMS) as internal standard and were reported as d
(ppm). The measurements were carried out at ambient
temperature (300 K). The atom numbering used for
5.1.5. General procedure for compounds 4a–d. Method
A: 1-(un)substituted-1H-benzimidazol-2-sulfonic acid
(1.55 mmol) and the corresponding 2-aminobenzimi-
dazole (3.1 mmol) were heated at 180 °C for 30 min.
After cooling the fusion was diluted with ethanol.
The obtained sediment was filtered and re-crystallized
with ethanol.
Method B: 1-(un)substituted-2-(methylthio)benzimid-
azole (1.4 mmol) and 1-(un)substituted-2-amino-benz-
imidazoles 3a–c were heated at 250 °C for 15 min. The
fusion was cooled and minimum quantity of ethanol
was added. The obtained precipitate was re-crystallized
with ethanol.

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A. Ts. Mavrova et al. / Bioorg. Med. Chem. 15 (2007) 6291–6297
5.1.5.1. N-1H-Benzimidazol-2-yl-1-methyl-1H-ben-
zimidazol-2-amine (4a). Method A: yield ꢀ40% (start-
(starting reagents 2d and 3b) resp. 37% (3d and 2b);
1
Method B: 42%; mp 136–138 °C; Rf = 0.69; H NMR
1
ing reagents 2b and 3a); 14% (starting reagent 2a
(CDCl3, d ppm): H NMR: 0.99 (t, 3H, N–CH2–CH2–
1
and3b); mp 240–242 °C; Rf = 0.58; H NMR (DMSO,
CH3, J = 7.34 Hz), 1.89 (m, 2H, N–CH2–CH2–CH3,
J = 7.34 Hz, J = 7.09 Hz), 3.67 (s, 3H, N–CH3), 4.15
(t, 2H, N–CH2–CH2–CH3, J = 7.09 Hz), 7.17–7.09 (m,
6H, Ar–Bz), 7.45–7.40 (m, 2H, Ar–Bz), 8.63 (br s, 1H,
NH); 13C NMR: 11.6 (N–CH2–CH2–CH3), 22.0 (N–
CH2–CH2–CH3), 28.0 (N–CH3), 43.5 (N–CH2–CH2–
CH3), 107.6 (CH-16), 108.0 (CH-6), 112.9 (CH-9),
113.0 (CH-19), 120.8 (CH-8, CH-18), 121.2 (CH-7),
121.3 (CH-17), 132.2 (C-5), 132.9 (C-13), 135.0 (C-14),
135.4 (C-4), 154.2 (C-2), 154.6 (C-11).
d ppm): 3.61 (s, 3H, N–CH3), 7.34–7.04 (m, 8H, Ar–
Bz); 13C NMR (DMSO, d ppm): 18.9 (N–CH3), 108.4
(CH-6), 111.5 (CH-16, br s), 111.8 (CH-19, br s), 113.8
(CH-9), 120.7 (CH-8), 120.9 (CH-18), 121.2 (CH-17),
121.3 (CH-7), 133.2 (C-5), 133.7 (C-14, br s), 134.8 (C-
13, br s), 136.6 (C-4), 154.6 (C-2), 154.8 (C-11).
5.1.5.2. N-1H-Benzimidazol-2-yl-1-ethyl-1H-ben-
zimidazol-2-amine (4b). Method A: yield 49% (starting
reagents 2c and 3a) resp. 20% (starting reagent 2a and
1
3c); mp 269–272 °C; Rf = 0.56; H NMR (DMSO3, d
5.1.5.7. 1-Ethyl-N-(1-ethyl-1H-benzimidazol-2-yl)-1H-
benzimidazol-2-amine (4g). Yield: Method A: 64%;
ppm): 1.30 (t, 3H, –N–CH2–CH3, J = 6.95 Hz), 4.18
(q, 2H, –N–CH2–CH3, J = 6.95 Hz), 7.51–7.02 (m, 8H,
Ar–Bz); 13C NMR: 13.8 (–N–CH2–CH3), 36.0 (–N–
CH2–CH3), 108.1 (CH-6), 111.0 (CH-19, CH-16, br s),
113.4 (CH-9), 120.4 (CH-8), 120.9 (CH-7, CH-17, CH-
18), 131.7 (C-5), 133.1 (C-13, C-14, br s), 136.1 (C-4),
153.6 (C-2), 154.2 (C-11).
1
Method B: 48%; mp 156–158 °C; Rf = 0.71; H NMR
(CDCl3, d ppm): 1H NMR: 1.41 (t, 6H, 2 N–CH2–
CH3, J = 7.10 Hz), 4.22 (q, 4H,
2 N–CH2–CH3,
J = 7.10 Hz), 7.16–7.11 (m, 6H, Ar), 7.43–7.40 (m, 2H,
Ar), 9.40 (br s, 1H, NH); 13C NMR: 13.8 (2 N–CH2–
CH3), 36.6 (2 N–CH2–CH3), 107.7 (CH-6, CH-16),
113.1 (CH-9, CH-19), 120.7 (CH-8, CH-18), 121.1
(CH-7, CH-17), 131.8 (C-5, C-13), 135.4 (C-4, C-14),
154.0 (C-2, C-11).
5.1.5.3. N-1H-Benzimidazol-2-yl-1-propyl-1H-ben-
zimidazol-2-amine (4c). Method A: yield 55% (starting
reagents 2d and 3a) resp. 28% (2a and 3d); mp 209–
1
211 °C; Rf = 0.34; H NMR (DMSO, d ppm): 0.91 (t,
5.1.5.8. 1-Ethyl-N-(1-propyl-1H-benzimidazol-2-yl)-1H-
benzimidazol-2-amine (4h). Method A: yield: 70% (start-
ing reagents 2d and 3c) resp. 42% (2c and 3d); Meth-
od B: 52%; mp 145–146 °C; Rf = 0.76; 1H NMR
(CDCl3, d ppm): 1H NMR: 0.99 (t, 3H, N–CH2–
CH2–CH3, J = 7.34 Hz), 1.41 (t, 3H, N–CH2–CH3,
J = 7.36 Hz), 1.89 (m, 2H, N–CH2–CH2–CH3,
J = 7.34 Hz, J = 6.85 Hz), 4.12 (t, 2H, N–CH2–CH3,
J = 6.85 Hz), 4.23 (q, 2H, N–CH2–CH2–CH3,
J = 7.36 Hz), 7.19–7.11 (m, 6H, Ar), 7.46–7.41 (m,
2H, Ar), 8.25 (br s, 1H, NH); 13C NMR: 11.6
3H, N–CH2–CH2–CH3, J = 7.35 Hz), 1.78 (m, 2H, N–
CH2–CH2–CH3), 4.09 (t, 2H, N–CH2–CH2–CH3,
J = 7.05 Hz), 7.49–7.04 (m, 8H, Ar–Bz); 13C NMR:
11.2 (–N–CH2–CH2–CH3), 21.7 (N–CH2–CH2–CH3),
42.7 (N–CH2–CH2–CH3), 108.2 (CH-6), 110.9 (CH-16,
CH-19, br s), 113.5 (CH-9), 120.2 (CH-8), 120.7 (CH-
7), 120.8 (CH-17, CH-18), 132.3 (C-5), 132.9 (C-13, br
s), 134.2 (C-14, br s), 136.3 (C-4), 154.1 (C-2), 154.2
(C-11).
5.1.5.4. 1-Methyl-N-(1-methyl-1H-benzimidazol-2-yl)-
1H-benzimidazol-2-amine (4d). Yield 38%: Method A;
mp 200–202 °C; Rf = 0.66; H NMR (CDCl3, d ppm):
3.66 (s, 6H, 2 N–CH3), 7.19–7.08 (m, 6H, Ar–Bz),
7.44–7.37 (m, 2H, Ar–Bz), 9.78 (br s, 1H, NH); 13C
NMR: 27.9 (2 N–CH3), 107.7 (CH-6, CH-16), 113.0
(CH-9, CH-19), 120.8 (CH-8, CH-18), 121.4 (CH-7,
CH-17), 132.8 (C-5, C-13), 135.2 (C-14, C-4), 154.5
(C-11, C-2).
(N–CH2–CH2–CH3),
13.8
(N–CH2–CH3),
22.0
(N–CH2–CH2–CH3), 36.7 (N–CH2–CH3), 43.4 (N–
CH2–CH2–CH3), 107.7 (CH-16), 107.9 (CH-6), 113.0
(CH-9, CH-19), 120.8 (CH-8, CH-19), 121.2 (CH-7,
CH-17), 131.8 (C-13), 132.3 (C-5), 135.2 (C-4), 135.3
(C-14), 153.8 (C-2) 154.3 (C-11).
1
5.1.5.9. 1-Propyl-N-(1-propyl-1H-benzimidazol-2-yl)-
1H-benzimidazol-2-amine (4i). Method A: yield: 50%;
1
mp 132–134 °C; Rf = 0.78; H NMR (CDCl3, d ppm):
5.1.5.5. 1-Ethyl-N-(1-methyl-1H-benzimidazol-2-yl)-1H-
benzimidazol-2-amine (4e). Method A: yield 47% (starting
reagents 2c and 3b) resp. 34% (2b and 3c); Method B:
1H NMR: 0.99 (t, 6H,
J = 7.38 Hz), 1.89 (m, 4H,
2 N–CH2–CH2–CH3,
2 N–CH2–CH2–CH3,
J = 7.38 Hz, J = 7.02 Hz), 4.13 (t, 4H, 2 N–CH2–CH2–
CH3, J = 7.02 Hz), 7.18–7.09 (m, 6H, Ar), 7.45–7.40
(m, 2H, Ar), 9.22 (br s, 1H, NH); 13C NMR: 11.6 (2
N–CH2–CH2–CH3), 22.0 (2 N–CH2–CH2–CH3), 43.5
(2 N–CH2–CH2–CH3), 107.9 (CH-6, CH-16), 113.0
(CH-9, CH-19), 120.7 (CH-8, CH-18), 121.1 (CH-7,
CH-17), 132.3 (C-5, C-13), 135.3 (C-4, C-14), 154.4
(C-2, C-11).
1
31%; mp 147–148 °C; Rf = 0.47; H NMR (CDCl3, d
ppm): 1.42 (t, 3H, N–CH2–CH3, J = 7.21 Hz), 3.68 (s,
3H, N–CH3), 4.22 (q, 2H, N–CH2–CH3, J = 7.21 Hz),
7.20–7.13 (m, 6H, Ar–Bz), 7.43–7.40 (m, 2H, Ar–Bz),
8.92 (br s, 1H, NH); 13C NMR: 13.8 (N–CH2–CH3),
28.0 (N–CH3), 36.6 (N–CH2–CH3), 107.7 (CH-16),
107.8 (CH-6), 113.1 (CH-9, CH-19), 120.8 (CH-8, CH-
18), 121.2 (CH-7), 121.3 (CH-17), 131.8 (C-5), 132.9
(C-13), 135.2 (C-14), 135.4 (C-4), 153.9 (C-2), 154.7
(C-11).
5.2. Biological screening
5.2.1. Antitrichinellosis activity in vitro. The parasitolog-
ical pharmaco-therapeutic experiments in vitro and
in vivo for antitrichinellosis activity of the tested
5.1.5.6. 1-Methyl-N-(1-propyl-1H-benzimidazol-2-yl)-
1H-benzimidazol-2-amine (4f). Method A: yield 64%

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5.2.2. Antitrichinellosis activity in vivo. The biological
test for antitrichinellosis activity in intestinal phase
in vivo was accomplished using 45 white immature mice,
infected under same conditions with 150 Trichinella lar-
vae. The tested animals were divided in six groups of five
mice for treatment with each one of the compounds as
well as in two control groups of five mice one of them
without treatment and one control group treated with
albendazole, applied at dose of 100 mg/mw. The treat-
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50 and 100 mg/kg mice-weight per os, beginning on
the third day after infection. The compounds were used
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