Synthesis and activity of 1H-benzimidazole and 1H-benzotriazole derivatives as inhibitors of Acanthamoeba castellanii

Article abstract of DOI:10.1016/j.bmc.2004.03.022

Chloro-, bromo- and methyl- analogues of 1H-benzimidazole and 1H-benzotriazole and their N-alkyl derivatives have been synthesized and tested in vitro against the protozoa Acanthamoeba castellanii. The results indicate that 5,6-dimethyl-1H-benzotriazole (11) and 5,6-dibromo-1H-benzotriazole (14) have higher efficacy than the antiprotozoal agent chlorohexidine.

Full text of DOI:10.1016/j.bmc.2004.03.022

Bioorganic & Medicinal Chemistry 12 (2004) 2617–2624
Synthesis and activity of 1H-benzimidazole and 1H-benzotriazole
derivatives as inhibitors of Acanthamoeba castellanii
a
Katarzyna Kopanska (nee Zastapiło), Andzelika Najda, Justyna Zebrowska,
a
b
_
ꢀ
ꢀ
_
z
Lidia Chomicz,b Janusz Piekarczyk,c Przemysław Myjakd and Maria Bretnera,*
aInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, 5a Pawiꢀnskiego, 02-106 Warsaw, Poland
bDepartment of Medical Biology, Medical University of Warsaw, 73 Nowogrodzka, 02-018 Warsaw, Poland
c2nd Department of Maxillofacial Surgery, Medical University of Warsaw, 4 Lindleya, 02-005 Warsaw, Poland
dDepartment of Tropical Parasitology, Medical University of Gdaꢀnsk, 9b Powstania Styczniowego, 081-516 Gdynia, Poland
Received 22 December 2003; accepted 10 March 2004
Available online 12 April 2004
Abstract—Chloro-, bromo- and methyl- analogues of 1H-benzimidazole and 1H-benzotriazole and their N-alkyl derivatives have
been synthesized and tested in vitro against the protozoa Acanthamoeba castellanii. The results indicate that 5,6-dimethyl-1H-
benzotriazole (11) and 5,6-dibromo-1H-benzotriazole (14) have higher efficacy than the antiprotozoal agent chlorohexidine.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
affecting contact lens wearers.2;9;10 In our comparative
studies11–13 on microorganisms colonizing the oral cavity
of 77 patients with or without systemic diseases, we
found mixed amoebic infection with Entamoeba gingi-
valis trophozoites and Acanthamoeba sp. trophozoites
and cysts in the mouths of four patients with deterio-
ration of periodontium and gingiva.
During the last 10 years it has become apparent that
several species of free-living amoebae belonging to the
genus Acanthamoeba present a serious risk to human
health. They act as causative agents of serious infections
involving the human brain, lung, skin and eyes.1–3 When
the amoebae invade the human brain through the nasal
cavity by penetrating the ethmoid bone (e.g., during
swimming), primary granulomatous encephalitis (GAE)
develops, unlike during infection with Entamoeba his-
tolytica, where brain involvement is usually secondary.
GAE as well as the pneumonitis and dermatitis caused
by Acanthamoeba sp. are serious progressive diseases
occurring predominantly in patients undergoing immu-
nosuppressive therapy for organ transplants and in
immunocompromized patients with AIDS.4–6 For this
reason, the amphizoic amoebae are now considered
opportunistic pathogens.2–5;7–9
The above data, together with the fact that Acanth-
amoeba may serve as a protective host for pathogenic
microorganisms, such as Legionella, Escherichia or
Pseudomonas,14 indicate the importance of the Acanth-
amoeba for human health. The amoebae are ubiquitous in
nature: they are found in water (fresh, sea, chlorinated,
bottled mineral and domestic tap water), soil and air in
many parts of the world.1;15;16 Acanthamoeba castellanii
occurs in two developmental stages: as amoeboid
trophozoites––with filamentous pseudopods (acantho-
podia) and as cysts. Cysts are highly resistant to changes
of temperature, pH and to extreme dryness; they are
found in dust and may be dispersed by wind. Generally,
recent studies9 indicate that these protozoans are more
resistant to environmental factors as well as to anti-
microbial and antiparasitic drugs than other amphizoic
amoebae.
Infection with Acanthamoeba sp. can result in eye dis-
ease––acanthamoeba keratitis, with 85% of cases
Keywords: Analogues of 1H-benzimidazole; Analogues of 1H-benzo-
triazole; Acanthamoeba castellanii; Antiprotozoal activity.
* Corresponding author. Tel.: +48-22-658-44-99; fax: +48-22-658-46-
36; e-mail: majka@ibb.waw.pl
Treatment of diseases caused by Acanthamoeba sp. is
difficult and the results are disappointing. Various
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.022

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K. Kopaꢀnska et al. / Bioorg. Med. Chem. 12 (2004) 2617–2624
agents such as clotrimazole, diminazine, paromomycin
and ketoconazole were cysticidal in vitro, but only at
high concentrations. Neomycin was ineffective against
Acanthamoeba cysts in vivo. Introduction of chloro-
hexidine has improved the treatment of acanthamoeba
keratitis, but relapses occur with continued culture-
positive isolation of Acanthamoeba.17 The long duration
of therapy, its toxicity and development of resistance
make the search for more effective treatment necessary.
pared by different methods. Analogues, which have not
been synthesized previously (6–10, 14) were prepared by
adapting the known methods.
Methyl derivatives 1, 2 and 12 were synthesized from
pentamethylbenzene (Scheme 1), which was nitrated22 to
obtain 1,2-dinitro-3,4,5,6-tetramethylbenzene. After
reduction with SnCl2 in solution of concentrated HCl,
1,2-diamino-3,4,5,6-tetramethylbenzene was prepared as
starting material. The reaction with formic acid23 gave
4,5,6,7-tetramethyl-1H-benzimidazole. The method of
Smith and Harris22 was followed for synthesis of
2,4,5,6,7-pentamethyl-1H-benzimidazole (PMBI, 2).
Diazotization with NaNO2 yielded 4,5,6,7-tetramethyl-
1H-benzotriazole (TMBT, 12), as was previously
reported by us.24
Benzimidazole derivatives are of wide interest because of
their diverse biological activity and clinical applications.
Some of them, such as mebendazol, chlorimidazol and
albendazol, are clinically and agriculturally useful drugs.
Most recently, antiprotozoal activity of substituted
nitro-, halogenobenzimidazoles and 2-trifluorobenzimi-
dazoles has been reported18;19 while earlier studies
showed antigiardial activity of various benzimidazole
derivatives.20;21
Chlorination of 1H-benzimidazole, with the use of the
procedure reported for chlorination of 1H-benzotri-
azole25 and published recently,26 gave the 4,5,6,7-tetra-
chloro-1H-benzimidazole (TCBI, 3) (Scheme 2). The
4,5,6,7-tetrachloro derivative was N-alkylated using
appropriate alkyl iodides in acetonitrile with 1,8-diaza-
bicyclo[5.4.0]undec-7-ene (DBU) as a base, to give
N-methyl- (MTCBI, 5), ethyl- (ETCBI, 7) and propyl-
(PTCBI, 9) derivatives (Scheme 2). Of the N-alkylated
4,5,6,7-tetrachloro-1H-benzimidazoles only 4,5,6,7-tetra-
chloro-1-methyl-1H-benzimidazole had previously been
synthesized27;28 using a different method. The 4,5,6,7-
tetrabromo derivatives (TBBI, 4; TBBT, 15) were
synthesized by bromination of 1H-benzimidazole or 1H-
benzotriazole according to published procedures.24;29
The 4,5,6,7-tetrabromo derivatives 6, 8, 10 (Scheme 2)
and 16–17 (Scheme 3) were obtained by alkylation as
mentioned above. N1 and N2-alkyltetrabromo-1H-ben-
zotriazoles 16, 16a, 17 and 17a were synthesized,25 but
reaction in acetonitrile and with DBU improved the
yield and simplified the purification. The 5,6-dimethyl-
1H-benzotriazole (DMBT, 11) was synthesized from
4,5-dimethylphenylenediamine, and the 5,6-dichloro-
1H-benzotriazole (DCBT, 13) was synthesized from 4,5-
dichlorophenylenediamine, by closing the triazole ring
with the use of NaNO2. Bromination of 1H-benzo-
triazole (Scheme 3) with the use of a procedure
We synthesized chloro-, bromo- and methyl- derivatives
of 1H-benzimidazole and 1H-benzotriazole, and their
N-alkylated analogues and tested them in vitro against
the protozoa A. castellanii.
2. Materials and methods
2.1. Chemistry
Compounds (1–5, 11–13, 15–17) shown in Figure 1 have
been reported previously but some of them were pre-
Figure 1. Benzimidazole and benzotriazole analogues synthesized in
this work.
CH3
H3C
H3C
N
CH3
CH3
CH3
CH3
CH3
H3C
H3C
CH3
H
H3C
H3C
NO2
NO2
H3C
H3C
NH2
NH2
N
H
iii
i
ii
CH3
CH3
1
iv
CH3
v
H3C
H3C
N
CH3
N
H
CH3
CH3
H3C
H3C
N
2
N
N
H
CH3
12
Scheme 1. Reagents and conditions: (i) fuming HNO3, concentrated H2SO4, <10 ꢁC; (ii) SnCl2, HCl, reflux; (iii) HCOOH; (iv) CH3COOH;
(v) NaNO2, HCl.

K. Kopaꢀnska et al. / Bioorg. Med. Chem. 12 (2004) 2617–2624
2619
Cl
Cl
Cl
Cl
Cl
Cl
N
N
N
iii
N
H
R
i
Cl
Cl
H
H
3
H
H
N
5 R=CH3, 7 R=C2H5, 9 R=C3H7
N
H
ii
Br
Br
Br
Br
Br
Br
Br
N
N
N
iii
N
H
R
Br
4
6 R=CH3, 8 R=C2H5, 10 R=C3H7
Scheme 2. Reagents and conditions: (i) HCl, HNO3, reflux; (ii) HNO3, Br2, reflux; (iii) CH3I (or C2H5I or C3H7I), CH3CN, DBU.
H
Br
Br
N
N
N
H
i
Br
Br
H
H
H
Br
Br
H
H
N
N
N
14
N
R
N
N
H
ii
Br
Br
16a R=CH3, 17a R=C2H5
Br
Br
N
iii
N
N
H
Br
Br
Br
N
N
15
N
R
Br
16 R=CH3, 17 R=C2H5
Scheme 3. Reagents and conditions: (i) H2SO4, Ag2SO4, Br2, room temperature; (ii) HNO3, Br2, reflux; (iii) CH3I (or C2H5I), CH3CN, DBU.
published by Cheeseman30 for bromination of 2,3-di-
hydroxyquinoxaline yielded the 5,6-dibromo-1H-ben-
zotriazole (DBBT, 14).
status of the protozoans. A similar dilution of DMSO
was used for the drug susceptibility assays. 7 benzo-
triazole and 10 benzimidazole derivatives at two differ-
ent concentrations were examined (see Table 1) for their
antiprotozoal activity. For each compound and control
assay, 1 mL of vortexed culture containing A. castellanii
was transferred to individual 1.5 mL Eppendorf tubes to
which 20 lL dilution of the proper compound in DMSO
or DMSO without the analogue were added. Each
compound concentration was tested in three replicates
including control assays without drugs and DMSO.
After 24 h, the tube cultures of amoebae with tested
compound as well as the controls were intensively vor-
texed, and subsequently 20 lL samples were withdrawn
from each of them for preparation of wet slides covered
with 24 · 24 mm glass slips. The number of amoebae as
well as the trophozoites and cysts were counted under
the microscope at 100-fold magnification. Percentage
content of particular stage of the Acanthamoeba was
assessed and compared in the tested compound versus
the control.
2.2. Biological assays
The A. castellanii Neff strain from the reference labo-
ratory of the Department of Tropical Parasitology,
Institute of Maritime and Tropical Medicine (Gdynia,
Poland) was used in these studies. All protozoans were
extensively grown in bacteria-free (axenic) condition in
sterile 15 mL tubes containing BSC culture medium31
enriched with calf serum, at 24–26 ꢁC. The amoebae
were subcultured twice a month. All amoebae used both
in the control and in the drug susceptibility assays were
grown for 4 days following regular subculturing. For the
control dimethyl sulfoxide (CDMSO) assays, a concen-
tration of 20 lL dimethyl sulfoxide (DMSO) added to
1 mL of culture medium containing Acanthamoeba has
been determined as having no effect on the number and

2620
K. Kopaꢀnska et al. / Bioorg. Med. Chem. 12 (2004) 2617–2624
Table 1. The reduction in viability of A. castellanii trophozoites and cysts after 24 h of incubation with synthesized compounds or with chloro-
hexidine
Compounds
Concentrations [lmol/L]
Percentage of survivors
Cysts
Percentage content of particular stages
Trophozoites
Total
Trophozoites
Cysts
1 TMBI
4.5
9.0
5.4
10.8
4.5
9.0
5.0
7.6
3.8
7.6
4.0
8.0
5.2
7.9
3.5
7.0
5.5
11.1
3.6
10.9
3.7
7.5
3.9
7.8
4.1
8.3
4.0
8.2
5.2
7.8
5.4
10.8
3.7
7.5
4.4
11.0
32.3 1.8
16.3 1.3
30.0 3.5
35.5 2.6
31.2 3.1
18.0 1.5
40.0 4.4
47.9 5.3
58.0 5.1
36.0 3.8
52.4 4.1
41.5 1.5
26.5 2.3
22.0 1.8
36.4 1.7
53.8 6.5
23.3 2.0
41.2 2.8
53.0 5.4
5.7 1.0
16.4 3.2
93.0 11.4
130.0 10.2
51.0 7.6
332.0 23.2
25.0 3.8
79.0 13.5
165.0 27.3
173.0 27.5
60.0 9.0
30.0 5.1
52.0 11.3
19.0 3.4
121.0 12.6
12.3 2.1
110.0 19.9
15.0 2.3
76.0 9.7
61.6 11.0
66.6 5.0
8.0 1.5
30.1 1.9
23.8 2.3
39.2 4.2
37.0 3.1
59.8 5.1
18.7 1.7
44.0 5.3
59.4 7.4
69.0 7.3
38.4 4.3
50.0 4.2
42.5 2.4
25.8 2.4
31.6 2.9
34.0 1.7
59.4 7.8
22.5 2.0
44.5 3.5
53.9 5.9
11.7 1.4
33.2 3.3
36.5 3.6
71.8 7.0
37.5 5.3
42.0 4.3
48.5 2.5
18.0 0.8
24.8 1.3
35.0 4.2
23.0 1.7
32.0 2.0
13.5 1.8
52.0 4.9
34.7 1.8
22.3 0.8
24.8 2.6
94.8 5.2
61.7 4.8
69.3 8.2
86.6 6.3
48.6 4.9
87.7 7.1
82.5 9.1
72.8 8.0
75.5 6.7
84.6 9.0
94.1 7.4
88.0 3.1
92.7 7.9
62.5 5.2
96.5 4.4
81.8 9.9
93.4 8.0
83.6 5.7
88.8 9.0
44.2 7.7
97.8 9.5
96.9 9.3
79.9 6.4
60.0 7.8
96.0 9.5
89.6 3.2
93.6 3.7
94.3 4.1
82.6 8.9
93.0 6.0
98.1 5.9
72.1 8.1
89.8 7.4
95.2 4.3
95.3 2.9
88.4 3.9
5.2 1.0
38.3 4.7
30.7 2.4
13.4 2.0
51.5 3.6
12.4 1.9
17.5 3.0
27.2 4.5
24.5 3.9
15.4 2.3
5.9 1.0
12.0 2.6
7.3 1.3
37.5 3.9
3.6 0.6
18.2 3.3
6.6 1.0
16.4 2.1
11.2 2.0
55.8 4.2
2.2 0.4
3.1 0.5
20.1 3.3
40.0 6.2
4.0 0.7
10.4 2.0
6.4 0.8
5.7 1.1
17.4 3.0
7.0 1.2
1.9 0.3
27.9 5.0
10.2 2.0
4.8 0.8
4.7 0.7
11.6 1.8
2 PMBI
3 TCBI
4 TBBI
5 MTCBI
6 MTBBI
7 ETCBI
8 ETBBI
9 PTCBI
10 PTBBI
11 DMBT
12 TMBT
13 DCBT
14 DBBT
15 TBBT
16 N1-MTBBT
17 N1-ETBBT
Chlorohexidine
35.8 3.5
39.0 3.7
63.2 5.1
24.0 3.1
44.8 4.4
48.2 1.7
18.7 0.7
26.0 1.1
32.0 3.4
23.0 1.5
35.0 2.1
10.7 1.2
51.6 4.3
36.6 1.7
23.4 0.7
24.2 1.1
11.8 1.9
162.0 26.6
162.0 25.1
17.3 2.8
51.5 9.9
12.5 1.6
14.5 2.8
62.5 10.8
16.4 2.8
6.3 1.0
38.4 6.9
54.2 10.6
17.3 2.8
11.0 1.6
31.0 4.8
All assays were performed in triplicate and repeated on at least twice to check the results. Values are means of two experiments SD.
3. Results and discussion
triazole (14), whereas in reduction of cysts it was 5,6-
dimethyl-1H-benzotriazole (11) and 4,5,6,7-tetrabromo-
1-methyl-1H-benzotriazole (16). The reduction of the
number of surviving cysts was more effective than with
the use of the known drug, chlorohexidine. When a
higher concentration of compounds was used, com-
pounds 3 and 14–16 showed higher efficacy than chloro-
hexidine.
Biological assays were performed as described in
Materials and methods. Comparison was made among
new compounds and the previously used drug, the
chlorohexidine. The number of trophozoites and cysts
surviving the assay was calculated, compared with the
controls and the results are presented in Table 1. In
general all the tested compounds showed activity against
A. castellanii. Compounds with a methyl group at
position 4,5,6,7- of the benzene ring did not reduce the
viability of the cysts, but reduced the viability of the
trophozoites. Among the 4,5,6,7-tetrachloro-1H-benz-
imidazole derivatives at low concentration (Table 1), the
most effective in reducing the number of trophozoites
and cysts were 4,5,6,7-tetrachloro-1-propyl-1H-benz-
imidazole (9) (23.3% T and 15% C), and 4,5,6,7-tetra-
chloro-1-ethyl-1H-benzimidazole (7) (26.5% T and 19%
C). The 4,5,6,7-tetrabromo-1H-benzimidazole deriva-
tives appeared to be less effective. Among the 1H-benzo-
triazole derivatives the most effective in reducing the
number of trophozoites was 5,6-dibromo-1H-benzo-
The mode of action of therapeutic agents against
Acanthamoeba is not known. In bacteria, chlorohexidine
causes cytoplasmic membrane damage, resulting in an
irreversible loss of essential cellular components.32 The
different chemical structure and low molecular weight of
benzimidazole and benzotriazole analogues (molecular
weight 200–500, compared to 898 for chlorohexidine
digluconate) might permit better penetration through
the membrane to achieve the therapeutic effect. It was
previously reported that 4,5,6,7-tetrabromo-1H-benzo-
triazole is a highly selective inhibitor of protein kinase 2
(CK2),33;34 and of the NTPase/helicase from hepatitis C
virus and West Nile virus.26 A subsequent study with

K. Kopaꢀnska et al. / Bioorg. Med. Chem. 12 (2004) 2617–2624
2621
Table 2. Calculated ClogP for 1H-benzimidazole and 1H-benzotri-
azole derivatives
million (ppm) relative to tetramethylsilane (Me4Si,
d ¼ 0). Starting materials: 1H-benzimidazole, 1H-ben-
zotriazole, 4,5-dichloro-1,2-phenylenediamine, penta-
methylbenzene and 4,5-dimethyl-1,2-phenylenediamine
were purchased from Aldrich Co. 4,5,6,7-Tetrachloro-
1H-benzimidazole (TCBI, 3), 4,5,6,7-tetrabromo-1H-
benzimidazole (TBBI, 4) and 4,5,6,7-tetramethyl-
1H-benzotriazole (TMBT, 12) were synthesized by
adapting the published methods,24 whereas 4,5,6,7-tetra-
bromo-1H-benzotriazole (TBBT, 15) as published.26
The ClogP values were obtained using ChemDraw 8 Pro
software.
Compound
ClogP
1 TMBI
2 PMBI
3.41
3.68
4.17
4.53
4.11
4.47
4.64
5.00
5.17
5.53
2.35
3.23
2.71
2.93
4.26
4.07
4.60
4.81
3 TCBI
4 TBBI
5 MTCBI
6 MTBBI
7 ETCBI
8 ETBBI
9 PTCBI
10 PTBBI
11 DMBT
12 TMBT
13 DCBT
14 DBBT
15 TBBT
16 N1-MTBBT
17 N1-ETBBT
Chlorohexidine
5.1. 4,5,6,7-Tetramethyl-1H-benzimidazol (TMBI, 1)
To a stirred, cooled (2–5 ꢁC) nitrating mixture consisting
of 5 mL of fuming nitric acid, 18 mL of concentrated
H2SO4 and 18 mL of chloroform, the solution of penta-
methylbenzene (2.5 g, 16.9 mmol) in 5 mL of CHCl3 was
added dropwise. The whole mixture was stirred vigor-
ously with temperature never exceeding 10 ꢁC. The
chloroform layer was separated, washed with water and
sodium bicarbonate and dried with anhydrous sodium
sulfate. After evaporation the residue was crystallized
from ethanol to give 3,4,5,6-tetramethyl-1,2-dinitro-
benzene (2.5 g, 11.16 mmol, 66% yield) as light yellow
needles, mp 178 ꢁC, (lit.22 176–177 ꢁC).
other analogues of benzotriazole and benzimidazole
demonstrated that 4,5,6,7-tetrabromo-1H-benzimid-
azole is more effective versus CK2 than TBBT,24 while
TMBT is inactive. Appropriate physicochemical prop-
erties (e.g., molecular weight, ClogP and solubility)
together with pharmacokinetic properties and toxicity
are the major indicators for progressing from a good
lead to a good drug. The compounds synthesized in this
work are hydrophobic (their ClogP are shown in Table
2), and can reach hydrophobically protected regions.
To the solution of the above nitro compound (268 mg,
1.2 mmol) in 1.6 mL of concentrated HCOOH, SnCl2
(2.68 g, 14 mmol) dissolved in 2.4 mL of concentrated
HCl was added dropwise during 5 h with heating and
stirring. The mixture was cooled, and the solid filtered
off and washed with water and ether. The solid was
dissolved in MeOH and 1 N NaOH was added to obtain
pH 8. The solution was evaporated and the residue was
crystallized from ethanol and water (1:1) to give 120 mg
of product (0.68 mmol, 57% yield), mp 243–244 ꢁC (lit.22
187–188 ꢁC); Rf 0.32 (CHCl3/CH3OH, 9:1). UV kmax ðeÞ
pH 2: 275 (6500), 301 (3930); pH 7: 253 (6900), 276
(4200), 285 (3500); pH 12: 253 (7000), 276 (4000), 285
(3300); MeOH: 253 (7200), 278 (3900), 288 (3500); 13C
NMR (DMSO-d6) d 139.970; 128.102; 15.632; 14.013;
MS [MþH]þ: found 175.3882, calcd 175.2556.
4. Conclusions
We have synthesized bromo-, chloro- and methyl- ana-
logues of benzimidazole and benzotriazole and their
alkyl derivatives, and screened them for their in vitro
antiprotozoal activity. The obtained results are very
promising since many of the compounds showed activity
comparable with the currently used drug chlorohex-
idine, whereas compounds 11 and 14 exhibited even
higher activity, especially towards Acanthamoeba cysts.
It is known35 that Acanthamoeba cysts may maintain
their viability and virulence for as long as 25 years.
Further optimization and pharmacokinetic character-
ization of this series of compounds are in progress.
5.2. 2,4,5,6,7-Pentamethyl-1H-benzimidazole (PMBI, 2)
5. Experimental
To the solution of 3,4,5,6-tetramethyl-1,2-dinitrobenz-
ene (1.1 g, 5 mmol) in 5 mL concentrated HCl, the
solution of SnCl2 (5.7 g, 30 mmol) in 20 mL of concen-
trated HCl was added dropwise, followed by addition of
2.5 mL CH3COOH. The reaction mixture was stirred
and heated for 5 h. The resulting solid was filtered off
and crystallized from CH3OH. The white solid of
3,4,5,6-tetramethyl-1,2-phenylenediamine was filtered
off, and the brown filtrate was evaporated and crystal-
lized from EtOH to obtain 1.2 g of hydrochloride of 2.
The solid was dissolved in MeOH and 1 N NaOH was
added to obtain pH 8. The solution was evaporated
and the residue was crystallized from ethanol and water
(1:1) to give 2,4,5,6,7-pentamethyl-1H-benzimidazole
Melting points were determined in open capillary tubes
€
using Buchi apparatus B504 and are uncorrected. UV
absorption spectra were recorded using Cary 300
instrument. All compounds were checked by thin-layer
chromatography (TLC) on 0.2 mm Merck silica gel 60
F254 plates. Preparative separations were carried out by
column chromatography using Merck silica gel (230–400
mesh), or on preparative glass plates (2 mm, Merck silica
gel 60 F254). Mass spectrometry was recorded on Micro-
mass ESI Q-TOF spectrometer at IBB PAS. 13C NMR
spectra were obtained with a Varian 500 or Varian 200
spectrometer. Chemical shifts are given in parts per

2622
K. Kopaꢀnska et al. / Bioorg. Med. Chem. 12 (2004) 2617–2624
(715 mg, 3.8 mmol, 76% yield), mp 265 ꢁC (lit.22 264 ꢁC),
Rf 0.24 (CHCl3/CH3OH, 9:1). UV kmax ðeÞ pH 2: 266
(5400), 275 (6700), 284 (6500); pH 7: 251 (6900), 276
(5100), 285 (4800); pH 12: 251 (7600), 276 (4300), 286
(4100); MeOH: 251 (7900), 278 (4400), 287 (4300); 13C
NMR (DMSO-d6) d 149.546; 135.830; 127.363; 118.416;
15.583; 14.147; 13.989; MS [MþH]þ: found 189.1083;
calcd 189.274.
ethyl-1H-benzimidazole (7) was obtained (yield 65%),
mp 140 ꢁC, Rf 0.54 (CHCl3/MeOH, 100:1), 0.19 (CHCl3/
hexane, 75:25). UV kmax ðeÞ pH 2: 264 (8080), 272 (8800),
287 (5100), 298 (3600), 305 (2760); pH 12: 264 (9180),
272 (10,400), 289 (5780), 300 (4300); MeOH: 265 (7710),
272 (9000), 289 (4200), 300 (3300). 13C NMR (DMSO-
d6) d 148.288; 141.202; 129.478; 125.433; 124.247;
122.443; 114.797; 41.577; 17.136. MS for C9H7Cl4N2
[MþH]þ obtained 284.9821; calcd 284.976.
5.3. General procedure for alkylation of halogeno 1H-
benzimidazoles 5–10 and halogeno-1H-benzotriazoles
16–17
5.7. 4,5,6,7-Tetrabromo-1-ethyl-1H-benzimidazole
(ETBBI, 8)
To the solution of 1 mmol of appropriate halogeno 1H-
benzimidazole (3 or 4) or 4,5,6,7-tetrabromo-1H-ben-
zotriazole (15) in 10 mL of acetonitrile with 0.5 mL of
DBU, the alkyl iodide was added (5 mM) and the
solution was heated and mixed for 12–20 h. The reaction
was checked by TLC, and DBU and alkyl iodide was
added to complete the reaction. The solution was con-
centrated and products were purified with column
chromatography or preparative silica gel plates chro-
matography.
After silica gel column chromatography with chloro-
form and crystallization 305 mg of 4,5,6,7-tetrabromo-1-
ethyl-1H-benzimidazole was obtained (yield 66%), mp
151–152 ꢁC, Rf 0.63 (CHCl3/MeOH, 100:1); 0.21
(CHCl3/hexane, 75:25). UV kmax ðeÞ pH 2: 269 (9240),
274 (9500), 302 (5300), pH 12: 268 (12,300), 274
(12,000), 304 (6400); MeOH: 269 (10700), 274 (10800),
302 (4100). 13C NMR (DMSO-d6) d 148.372; 143.588;
131.166; 122.159; 120.239; 116.423; 106.343; 41.646;
17.326. MS for C9H7Br4N2 [MþH]þ obtained 462.8498;
calcd 462.7812.
5.4. 4,5,6,7-Tetrachloro-1-methyl 1H-benzimidazole
(MTCBI, 5)
5.8. 4,5,6,7-Tetrachloro-1-propyl-1H-benzimidazole
(PTCBI, 9)
After column chromatography with chloroform, 195 mg
of
4,5,6,7-tetrachloro-1-methyl-1H-benzimidazole
After chromatography on silica gel plates (CHCl3/
MeOH, 100:2) 155 mg of 4,5,6,7-tetrachloro-1-propyl-
1H-benzimidazole was obtained (yield 67%), mp 103–
104 ꢁC, Rf 0.62 (CHCl3/MeOH, 100:1); 0.24 (CHCl3/
hexane, 75:25). UV kmax ðeÞ pH 2: 265 (10,900), 272
(11,700), 287 (8070), 298 (5960); pH 12: 265 (12,980),
273 (14,000), 289 (10,300), 300 (7700); MeOH: 264
(8080), 272 (9500), 289 (4500), 300 (3500). 13C NMR
(DMSO-d6) d 148.779; 141.180; 129.511; 125.495;
124.258; 122.450; 114.852; 47.699; 24.550; 10.392. MS
for C10H9Cl4N2 [MþH]þ obtained 298.9992; calcd
299.0032.
(MTCBI) was obtained (yield 72%), mp 258 ꢁC (lit.27
258–261 ꢁC), Rf 0.40 (CHCl3/MeOH, 100:1) 0.13
(CHCl3/hexane, 75:25). UV kmax ðeÞ pH 2: 272 (5600),
278 (5660), 289 (4520), 305 (2760); pH 12: 271 (5500),
278 (5600), 290 (4080), 304 (2400); MeOH: 264 (7900),
272 (9250), 289 (4400), 300 (3370); 13C NMR (DMSO-
d6) d 149.052; 140.958; 130.358; 125.208; 124.131;
122.338; 115.284; 34.138–34.120. MS for C8H5Cl4N2
[MþH]þ: obtained 270.9234; calcd 270.942.
5.5. 4,5,6,7-Tetrabromo-1-methyl-1H-benzimidazole
(MTBBI, 6)
5.9. 4,5,6,7-Tetrabromo-1-propyl-1H-benzimidazole
(PTBBI, 10)
After column chromatography with chloroform 277 mg
of 4,5,6,7-tetrabromo-1-methyl-1H-benzimidazole was
obtained (yield 62%), mp 242–243 ꢁC, Rf 0.44 (CHCl3/
MeOH, 100:1), 0.14 (CHCl3/hexane, 75:25). UV kmax ðeÞ
pH 2: 275 (7750), 283 (7700), 307 (4100); pH 7: 275
(7400), 282 (7300), 307 (3800); pH 12: 274 (7400), 281
(7200), 307 (4000); MeOH: 268 (11,000), 274 (11,000),
302 (4260); 13C NMR (DMSO-d6) d 149.084; 143.257;
132.039; 121.865; 120.115; 116.299; 106.725; 34.880. MS
for C8H5Br4N2 [MþH]þ obtained 448.7366; calcd
448.747.
After column chromatography with chloroform 304 mg
of 4,5,6,7-tetrabromo-1-propyl-1H-benzimidazole was
obtained (yield 64%), mp 128–129 ꢁC, Rf 0.70 (CHCl3/
MeOH, 100:1); 0.28 (CHCl3/hexane, 75:25). UV kmax ðeÞ
pH 2: 277 (10,000), 308 (6200); pH 12: 278 (10,500), 308
(6000); MeOH: 269 (10,500), 274 (10,600), 302 (4000).
13C NMR (DMSO-d6) d 148.837; 143.585; 131.210;
122.283; 120.293; 116.445; 106.496; 47.724; 24.739;
10.305. MS for C10H9Br4N2 [MþH]þ obtained
476.7985; calcd 476.808.
5.6. 4,5,6,7-Tetrachloro-1-ethyl-1H-benzimidazole
(ETCBI, 7)
5.10. 5,6-Dimethyl-1H-benzotriazole (DMBT, 11)
After silica gel column chromatography with chloro-
form and crystallization 184 mg of 4,5,6,7-tetrachloro-1-
This compound was synthesized from 4,5-dimethyl-1,2-
phenylenediamine according to the method of Plaut.36

K. Kopaꢀnska et al. / Bioorg. Med. Chem. 12 (2004) 2617–2624
2623
Yield 78%; mp 156.6–156.8 ꢁC after crystallization from
MeOH (lit.36 156–157 ꢁC), Rf 0.59 (CHCl3/MeOH, 9:1).
UV kmax ðeÞ pH 2: 268 (6300), 284 (6000); pH 7: 267
(6400), 282 (5900); pH 12: 281 (9100), 289 (7500);
MeOH: 263 (5400), 283 (4800); (lit.37 H2O: 266 (5623),
280 (5248)). 13C NMR (DMSO-d6) d 143.308; 137.066;
133.120; 131.897; 117.511; 109.894; 19.999. MS for
C8H10N3 [MþH]þ obtained 148.2683; calcd 148.281.
NMR (CDCl3) d 143.352; 126.460; 113.615; 44.076. UV
kmax ðeÞ MeOH: 296 (12,700), 306 (13,100); (lit.29 MeOH:
294 (12,590), 299 (12,590), 305 (13,180)). MS for
C7H4Br4N3 [MþH]þ found 449.7848; calcd 449.752.
5.14. 4,5,6,7-Tetrabromo-1-ethyl-1H-benzotriazole
(N1-ETBBT, 17)
To a solution of TBBT (215 mg, 0.5 mmol) in 4 mL of
acetonitrile with 0.24 mL of DBU, 150 lL of ethyl iodide
was added. The mixture was heated to 50–60 ꢁC. After
24 h the reaction mixture was evaporated, residue was
dissolved in the mixture of methanol/chloroform and the
products were separated with the use of preparative
plate chromatography (CHCl3/hexane, 1:1). After elu-
tion and crystallization 81 mg of N1-ethyl TBBT (yield
35%) was obtained, mp 151 ꢁC (lit.29 150–152 ꢁC), Rf
0.81 (MeOH/CHCl3, 100:1); 0.48 (CHCl3/hexane, 1:1).
UV kmax ðeÞ MeOH: 277 (8800), 289 (8500), 308 (6000);
(lit.29 MeOH: 278 (8710), 290 (8710), 308 (6166)). 13C
NMR (CDCl3) d 145.704; 131.515; 129.067; 124.266;
116.702; 105.646; 45.744; 16.832. MS for C8H6Br4N3
[MþH]þ found 463.7757; calcd 463.7652.
5.11. 5,6-Dichloro-1H-benzotriazole (DCBT, 13)
This compound was synthesized from 4,5-dichloro-1,2-
phenylenediamine according to Wiley and Hussung.29
Yield 60%, mp 264 ꢁC, (lit.29 264–266 ꢁC); Rf 0.58
(CHCl3/MeOH, 9:1). UV kmax ðeÞ pH 2: 264 (5400), 270
(5440), 292 (5750); pH 7: 290 (7280), pH 12: 290 (9100),
298 (7350); CH3OH: 262 (4850), 269 (5000), 292 (5800).
13C NMR (DMSO-d6) d 137.467; 128.472; 116.525. MS
for C6H4Cl2N3 [MþH]þ obtained 189.0765; calcd
189.0269.
5.12. 5,6-Dibromo-1H-benzotriazole (DBBT, 14)
To the solution of 1H-benzotriazole (600 mg, 5 mmol)
and 1.56 g (5 mmol) of Ag2SO4 in 20 mL of concentrated
H2SO4, 2 mL of Br2 was added dropwise during 4 h. The
reaction was stirred at room temperature for 12 h. The
precipitate was filtered off, and ethanol with ice was
added to the filtrate. The new precipitate was crystal-
lized from EtOAc and water, to give 858 mg (62% yield)
of product, mp 248–250 ꢁC, Rf 0.56 (CHCl3/MeOH,
9:1). UV kmax ðeÞ pH 2: 266 (11,600), 273 (12,500), 291
(11,260); pH 6: 285 (12,600), 294 (11,800), 305 (6560);
pH 12: 284 (16,890), 294 (15,100), 305 (7790); MeOH:
265 (7300), 272 (8600), 289 (8700), 305 (4700). 13C NMR
(DMSO-d6) d 139.138; 129.465; 106.725. MS for
C6H4Br2N3 [MþH]þ obtained 277.9385; calcd 277.924.
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