Synthesis and potent antiprotozoal activity of mono/di amidino 2-anilinobenzimidazoles versus Plasmodium falciparum and Trypanosoma brucei rhodesiense

Article abstract of DOI:10.1016/j.bmc.2016.06.047

A series of mono and dicationic new 2-anilinobenzimidazole carboxamidines were prepared in a four step process starting from 4-amino-3-nitrobenzonitrile and corresponding o-phenylenediamines. Their antiparasitic activity against Plasmodium falciparum (P.

Full text of DOI:10.1016/j.bmc.2016.06.047

Accepted Manuscript
Synthesis and Potent Antiprotozoal Activity of Mono/Di amidino 2-Anilino-
benz-imidazoles versus Plasmodium falciparum and Trypanosoma brucei rho-
desiense
Cigdem Karaaslan, Marcel Kaiser, Reto Brun, Hakan Göker
PII:
S0968-0896(16)30470-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.047
BMC 13103
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 April 2016
21 June 2016
23 June 2016
Please cite this article as: Karaaslan, C., Kaiser, M., Brun, R., Göker, H., Synthesis and Potent Antiprotozoal Activity
of Mono/Di amidino 2-Anilinobenz-imidazoles versus Plasmodium falciparum and Trypanosoma brucei
rhodesiense, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.06.047
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Synthesis and Potent Antiprotozoal Activity of Mono/Di amidino 2-Anilinobenz-
imidazoles versus Plasmodium falciparum and Trypanosoma brucei rhodesiense
Cigdem Karaaslan a, Marcel Kaiser b, Reto Brun b, Hakan Göker a *
a. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100
Tandogan, Ankara, Turkey
b. Parasite Chemotherapy Unit, Swiss Tropical and Public Health Institute, Basel CH-4002,
Switzerland, and University of Basel, Basel CH-4001, Switzerland
I. Introduction
Protozoan parasites cause significant public health problems in many parts of the world,
particularly in tropical and subtropical regions, and rural developing countries [1]. At least 1
billion people - about 15% of the world's population - suffer from one or several neglected
tropical diseases (NTDs) [2]. Furthermore, these parasitic infections are frequently associated
with immunocompromised patients (e.g. leishmaniasis) or disseminated by immigrants and are
thus also seen in developed countries. As long as no vaccines are available for protozoan
diseases, control solely relies on vector control and drug therapies. Among the protozoal
infections malaria is the most important and sleeping sickness (Human Africa trypanosomiasis,
HAT) is one of the most fatal diseases for which control is still far from ideal. Malaria is caused
by Plasmodium falciparum and four other Plasmodium species which are transmitted via the bites
of certain species of infected Anopheles mosquitoes [3,4]. Human African trypanosomiasis is
caused by Trypanosoma brucei rhodesiense and T. b. gambiense. Both trypanosome subspecies
are transmitted by the bite of an infected tsetse fly [5,6]. The disease affects mostly poor
populations living in remote rural areas of Africa. It is usually fatal if untreated. Although
chemotherapeutic approaches are used effectively to treat and prevent parasitic infections,
satisfactory medications are still lacking. New safe and effective drugs against these parasitic
infections are desperately needed. Many of the currently used ones have major problems such as
significant toxicity, variable efficacy, lack of oral bioavailability, and therefore the need for
parenteral administration. Furthermore, drug resistant parasites are becoming an increasing
problem [3,7].

Aromatic amidine derivatives are known as a group of DNA interactive compounds. They bind to
the minor groove of DNA, especially A-T base pairs and show significant in vitro antiprotozoal
activity. These derivatives have been used in the treatment of protozoal diseases for many years.
Pentamidine (I) (Fig 1) is such a compound which is clinically used in the treatment of sleeping
sickness, pneumonia caused by Pneumocystis jirovecii and antimony-resistant leishmaniasis [8].
However, it is not effective when given orally and several toxic effects such as hypotension,
dysglycemia, renal and hepatic toxicity have been reported [9]. Based on Pentamidine, a large
number of different types of cationic derivatives were synthesized and screened for antiprotozoal
activity. Among them Furamidine (II) (Fig 1) has a significantly better activity against a number
of protozoan parasites with lower toxicity than Pentamidine. Furamidine and its methamidoxime
pro-drug Pafuramidine exhibited very potent inhibitory activity against protozoan parasites
[9,10].
O
O
H2N
HN
O
NH2
NH
H2N
NH2
NH
NH
I
II
Fig. 1 Pentamidine (I)
and Furamidine DB75 (II)
On the other hand, some recent studies have shown that 1H-benzimidazole carboxamidines such
as structures III [8] and IV [10] (Fig 2) strongly inhibited growth of protozoan parasites in vitro.
As a further contribution to this field, we have also reported [11,12] the synthesis of a series of 2'-
arylsubstituted-1H,1'H-[2,5']bisbenzimidazolyl-5-carboxamidines V, VI (Fig 2) and their very
potent antiparasitic activities against T.b. rhodesiense., P. falciparum, and Naegleria fowleri.

N
NH2
NH
N
HN
H2N
S
C
H2N
HN
N
H
C
NH
NH
H2N
III
IV
F
O
OCH3
H
N
H
N
H
N
H
N
OCH3
N
N
HN
N
HN
N
NH2
NH2
V
VI
Fig. 2
The structures of previously reported amidinobenzimidazoles possessing potent
antiprotozoal activity.
We now report the in vitro antiprotozoal evaluation of new mono and dicationic
2-anilinobenzimidazoles against P. falciparum and T.b. rhodesiense.
2. Materials and Methods
2.1. Chemistry
Compounds 1 – 21 (Table 1) were prepared using the methods outlined in Scheme 1. Reduction
of 4-amino-3-nitrobenzonitrile with H2/Pd-C afforded 3,4-diaminobenzonitrile 1. Cyclization of
1 and other commercially available corresponding o-phenylenediamines with substituted
phenylisothiocyanates using N-(3-dimetilaminopropyl)-N’-ethyl carbodiimide hydrochloride
(EDCI. HCl) gave the 2-anilinobenzimidazolecarbonitriles 2 - 9. Both nitrile groups of 2 were
converted to imidate esters (not characterized, because of the instability), with dry HCl gas in
absolute ethanol, by Pinner reaction [13], and then treatment of imidate ester with NH3 gas /
several amines and in ethanol gave the dicationic amidines 10 - 14. Similarly, other
anilinobenzimidazole mono cationic amidines 15 - 18 and 19 - 21 were obtained from 3 - 6 and
7 - 9, respectively.
2.1.1. Experimental
Uncorrected melting points were measured on a Büchi B-540 capillary melting point apparatus.
1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded employing a Varian Mercury 400
MHz FT spectrometer, chemical shifts (δ) are in ppm relative to TMS, and coupling constants (J)

are reported in Hertz. Mass spectra were taken on a Waters Micromass ZQ connected with
Waters Alliance HPLC, using ESI (+) or ESI (-) method, with C-18 column. Elemental analyses
were performed by Leco CHNS-932. The compounds reported as salts frequently analyzed
correctly for fractional moles of water and/or organic solvent of solvation.
Because of the tautomeric effect of the imidazole ring, the 1H-NMR spectra of some compounds
was not clear enough, so in order to prevent the tautomerism, compounds were dissolved in
DMSO-d6, followed by a tiny amount of dry NaH and 2-3 drops of D2O were added to NMR
tubes and stirred well. In case of any turbidity, the tubes were centrifuged. Clear NMR spectra
were observed as reported in the experimental part.
3,4-Diaminobenzonitrile (1)
1 g (6.1 mmol ) 4-Amino-3-nitrobenzonitrile was dissolved in 50 ml ethanol and was subjected to
hydrogenation using 35 psi of H2 and 10 % Pd.C until the end of H2 uptake. The catalyst was
filtered on a bed of Celite, washed with ethanol and concentrated in vacuo. Powder residue was
used for the subsequent steps without crystallization. Yield: 88 % (0.72 g). Mp: 144-146°C [14].
2.1.1.2. General Procedure for Synthesis of Anilinobenzimidazole derivatives (2 - 9)
A mixture of corresponding o-phenylenediamines (2 mmol), substituted phenylisothiocyanates
(2 mmol) and 0.764 g (4 mmol) of N-(3-dimetilaminopropyl)-N’-ethyl carbodiimide
hydrochloride (EDCI.HCl) in THF (40 ml) was heated in sealed tubes at 70oC, for 3h. The
reaction mixture was then cooled, water was added, and the precipitate was filtered. The crude
product was recrystallised from the ethanol/methanol-water mixture [15].
2-[(4-Cyanophenyl)amino]-1H-benzimidazol-5(6)-carbonitrile (2)
0.266 g (2 mmol) 3,4-Diaminobenzonitrile (1), 0.320 g (2 mmol) 4-cyanophenylisothiocyanate
and 0.764 g (4 mmol) EDCI.HCl were reacted according to the general method. Yield 47 %
1
(0.245 g). Mp: 303-307°C. H-NMR δ ppm (DMSO-d6 +NaH+D2O): 7.04 (dd, 1H, Jo=8 Hz,
Jm=2 Hz, H-6), 7.24 (d, 1H, Jo=8 Hz, H-7), 7.48 (d, 1H, Jm=1.6 Hz, H-4), 7.54 (d, 2H, Jo=8 Hz,
H-2',6'), 7.87 (d, 2H, Jo=8 Hz, H-3',5'). 13C-NMR & HSQC δ ppm (DMSO-d6 +NaH +D2O) :
95.9 (Cq), 97.4 (Cq), 113.7 (CH-7), 116.5 (CH-4), 116.8 (CH-3',5'), 120.8 (CH-6), 121.4 (Cq),

123.5 (Cq), 133.2 (CH-2',6'), 145.9 (Cq), 148.9 (Cq), 150.4 (Cq), 163.7 (Cq). MS (ESI+) m/z:
260 (M+H, %23), 301 (M + H + CH3CN, %100).
4-[(1H-benzimidazol-2-yl)amino]benzonitrile (3)
0.216 g (2 mmol) 1,2-Diaminobenzene, 0.320 g (2 mmol) 4-cyanophenylisothiocyanate and
0.764 g (4 mmol) EDCI.HCl were reacted according to the general method. Yield 40.5 % (0.19
1
g). Mp: 214 -216°C. H-NMR δ ppm (DMSO-d6 +NaH+D2O): 6.71-6.74 (m, 2H, H-5,6), 7.19-
7.22 (m, 2H, H-4,7), 7.57 (d, 2H, Jo= 8.8 Hz, H-2',6'), 7.86 (d, 2H, Jo= 8.8 Hz, H-3',5'). MS
(ESI+) m/z: 235.3 (M+H, %100), 276.4 (M + H + CH3CN, %68).
4-[(5(6)-Chloro-1H-benzimidazol-2-yl)amino]benzonitrile (4)
0.284 g (2 mmol) 4-Chlorobenzen-1,2-diamine, 0.320 g (2 mmol) 4-cyanophenylisothiocyanate
and 0.764 g (4 mmol) EDCI.HCl were reacted according to the general method. Yield 50.0 %
(0.27 g). Mp: 309-313°C. 1H-NMR δ ppm (DMSO-d6 +NaH+D2O): 6.56 (dd, 1H, Jo=8.4 Hz, H-
6), 7.03-7.05 (m, 2H, H-4, 7), 7.45 (d, 2H, Jo=9.2 Hz, H-2',6'), 7.86 (d, 2H, Jo=8.8 Hz, H-3',5').
MS (ESI+) m/z: 269 (M+H, %100), 271 (M+H+2, % 33), 310.3 (M + H + CH3CN, %81), 312.3
(M+H+2+CH3CN, %25).
4-[(5(6)-Trifluoromethyl-1H-benzimidazol-2-yl)amino]benzonitrile (5)
0.352 g (2 mmol) 4-(Trifluoromethyl)benzen-1,2-diamine, 0.320 g (2 mmol) 4-cyanophenyl-
isothiocyanate and 0.764 g (4 mmol) EDCI.HCl were reacted according to the general method.
1
Yield 29.8% (0.18 g). Mp: 275-279°C. H-NMR δ ppm (DMSO-d6 +NaH+D2O): 6.91 (dd, 1H,
Jo=8.4 Hz, Jm=1.6 Hz, H-6), 7.18 (d, 1H, Jo=8.4 Hz, H-7), 7.36 (d, 1H, Jm=1.6 Hz, H-4), 7.45 (d,
1H, Jo=8.8 Hz, H-2',6'), 7.87 (d, 2H, Jo=9.2 Hz, H-3',5'). MS (ESI+) m/z: 303 (M+H, %60), 344
(M+H+CH3CN, %100).
4-[( 5(6)-Chloro-6(5)-methyl-1H-benzimidazol-2-yl)amino]benzonitrile (6)
0.312 g (2 mmol) 4-Chloro-5-methylbenzene-1,2-diamine, 0.320 g (2 mmol) 4-cyanophenyl-
isothiocyanate and 0.764 g (4 mmol) EDCI.HCl were reacted according to the general method.
1
Yield 53,2 % (0.300 g). Mp: 282-285°C. H-NMR δ ppm (DMSO-d6 +NaH+D2O): 2.29 (s, 3H,
CH3), 7.05 (s, 1H, H-7), 7.12 (s, 1H, H-4), 7.48 (d, 2H, Jo=9.2 Hz, H-2',6'), 7.86 (d, 2H, Jo=9.2

Hz, H-3',5'). 13C-NMR δ ppm (DMSO-d6 +NaH+D2O) : 20.5 (CH3), 97.3, 112.9, 114.9, 116.0,
121.0, 121.2, 121.4, 132.9, 145.4, 145.5, 148.3, 160.8. MS (ESI+) m/z: 283 (M+H, %98), 285
(M+H+2, %32), 324 (M+H+CH3CN, %100), 326 (M+H+2+CH3CN, %34).
2-(Phenylamino)-1H-benzimidazole-5(6)-carbonitrile (7)
0.266 g (2 mmol) 3,4-Diaminobenzonitrile (1), 0.270 g (2 mmol) phenylisothiocyanate and
0.764 g (4 mmol) EDCI.HCl were reacted according to the general method. Yield 25.6% (0.124
1
g). Mp: 290 - 294°C. H-NMR δ ppm (DMSO-d6 +NaH+D2O) : 6.75 (t, 1H, Jo= 7.2 Hz, H-4’),
6.98 (dd, 1H, Jo= 8.4 Hz, H-6), 7.16-7.22 (m, 3H, H-4,3',5'), 7.40 (d, 1H, Jm= 1.6 Hz, H-7), 7.78
13
(d, 2H, Jo= 7.6 Hz, H-2',6'). C-NMR, HSQC & HMBC δ ppm (DMSO-d6 +NaH+D2O): 95.42
(C-1’), 113.12 (CH-4), 115.93 (C-5), 116.49 (CH-3’,5’), 118.79 (CH-4’), 120.48 (CH-7), 123.70
(-CN), 128.82 (CH-2’,6’), 143.28 (C-7a), 146.38 (CH-6), 150.84 (C-3a), 163.99 (C-2). MS
(ESI+) m/z: 235 (M+H, %100), 276 (M+H+ CH3CN, %62).
2-[(4-Chlorophenyl)amino]-1H-benzimidazole-5(6)-carbonitrile (8)
0.266 g (2 mmol) 3,4-Diaminobenzonitrile (1), 0.338 g (2 mmol) 4-chloroisothiocyanate and
0.764 g (4 mmol) EDCI.HCl were reacted according to the general method. Yield 42.9% (0.230
1
g). Mp: 280-284°C. H-NMR δ ppm (DMSO-d6 +NaH+D2O): 7.02 (dd, 1H, Jo= 8.4 Hz, Jm= 2
Hz, H-6), 7.19-7.24 (m, 3H, H-7,2',6'), 7.44 (d, 1H, Jm= 1.6 Hz, H-4), 7.81 (d, 2H, Jo= 8.8 Hz, H-
3',5'). MS (ESI+) m/z: 269 (M+H, % 87), 271 (M+H+2, %33), 310 (M+H+CH3CN, %100), 312
(M+H+CH3CN+2, %33).
2-[(3,4-Dichlorophenyl)amino]-1H-benzimidazol-5(6)-carbonitrile (9)
0.266 g (2 mmol) 3,4-Diaminobenzonitrile (1), 0.404 g (2 mmol) 3,4-dichlorophenyl
isothiocyanate and 0.764 g (4 mmol) EDCI.HCl were reacted according to the general method.
Yield 36.0 % (0.185 g). Mp: 357-358°C [16].
2.1.1.2. General Procedure for Synthesis of Mono/Dicationic Anilinobenzimidazole
Carboxamidine Derivatives (10-21)
2-((4-Cyanophenyl)amino)-1H-benzimidazole-5(6)-carbonitrile (2), 4-((1H-benzimidazol-2-
yl)amino)benzonitrile (3, 4, 5, 6), 2-(phenylamino)-1H-benzimidazole-5(6)-carbonitrile (7, 8, 9)

derivatives, were suspended in 2 ml of absolute ethanol which was saturated by dry HCl gas , the
flask was stoppered and the contents were stirred at room temperature until the nitrile group
disappeared (4 days, by TLC). After complete consumption of the starting material, dry ether was
added, then the precipitate was collected by filtration, washed with anhydrous Et2O, dried in
vacuo at room temperature to give imidate ester HCl. They were used without purification as HCl
salts for the next steps since they were prepared completely pure. To a solution of imidate ester
HCl in absolute EtOH, corresponding amine derivatives (1.5-2 fold excess) were added to obtain
mono/di cationic benzimidazole carboxamidine derivatives.
2-[(4-Amidinophenyl)amino]-1H-benzimidazole-5(6)-carboxamidine HCl (10) [17,18]
0.259
g
2-[(4-Cyanophenyl)amino]-1H-benzimidazole-5(6)-carbonitrile (2) was reacted
according to the general method 2. Obtained imidate ester was stirred in 2 ml of absolute ethanol
saturated with ammonia gas, at room temperature, for 2 days. The crude product was precipitated
by ether, filtrated and crystallised from ethanol. Salt was prepared by ethanolic HCl. Yield 17 %
(0.070 g). Mp: 302-306 °C. 1H-NMR δ ppm (CD3OD): 7.70 (d, 1H, Jo= 8.4 Hz, H-7), 7.77-7.78
13
(m, 2H, H-2', 6'), 7.80 (d, 1H, Jm= 2 Hz, H-4), 7.97-8.00 (m, 3H, H-6, 3', 5'). C-NMR δ ppm
(CD3OD): 112.14, 112.61, 121.81, 123.59, 123.79, 125.09, 129.78, 135.07, 141.52, 149.44,
.
.
165.96, 166.67 . MS (ESI+) m/z : 294.3 (M+H, %100). Anal. for C15H15N7 2HCl 3H2O
.
0.25 C2H6O calculated, %: C 43.11; H 5.71; N 22.70; found, %: C 43.27; H 5.30; N 22.85.
N-Isopropyl-2-[(4-(N-isopropylamidino)phenyl)amino]-1H-benzimidazole-5(6)-carboxamidine
HCl (11)
0.259
g
2-[(4-Cyanophenyl)amino]-1H-benzimidazole-5(6)-carbonitrile (2) was reacted
according to the general method 2. 0.25 ml Isopropylamine was added to the obtained imidate
ester and was stirred in 2 ml of absolute ethanol at room temperature, for 2 days. The crude
product was purified by chromatography on silica gel (Chloroform/isopropanol/ammonia 10 : 4 :
0.7, saturated with ammonia gas) and recrystallised from ethanol. Salt was prepared by ethanolic
1
HCl. Yield 28.9 % (0.150 g). Mp: 270-274 °C. H-NMR δ ppm (CD3OD): 1.34 (d, 6H, J= 6,
Hz, CH3), 1.38 (d, 6H, J= 6 Hz, CH3), 3.96-3.99 (m, 2H, CH), 7.26 (dd, 1H, Jo= 8 Hz, Jm= 1.6
Hz, H-6), 7.39 (d, 1H, Jo= 8 Hz, H-7), 7.60-7.62 (m, 3H, H-4,2',6'), 7.77 (d, 2H, Jo= 8.4 Hz, H-
3',5'). 13C-NMR δ ppm (CD3OD): 20.74 (CH3), 20.81 (CH3), 44.87 (CH), 45.17 (CH), 112.61,

113.22, 116.12, 117.87, 118.01, 121.22, 128.46, 144.60, 147.01, 149.59, 161.28, 163.15, 164.99.
.
.
2HCl 2H2O
.
MS (ESI+) m/z: 378 (M+H, %20), 189.8 (%100). Anal. for C21H27N7
0.75C2H6O; calculated, %: C 51.87; H 7.25; N 18.81; found, %: C 52.20; H 7.43; N 18.56.
N-Isobutyl-2-[(4-(N-isobutylamidino)phenyl)amino]-1H-benzimidazole-5(6)-carboxamidine HCl
(12)
0.259
g
2-[(4-Cyanophenyl)amino]-1H-benzimidazole-5(6)-carbonitrile (2) was reacted
according to the general method 2. 0.25 ml Isobutylamine was added to the obtained imidate
ester and was stirred in 2 ml of absolute ethanol at room temperature, for 2 days. The crude
product was purified by chromatography on silica gel (Chloroform/isopropanol/ammonia
10:4:0.7, saturated with ammonia gas) and recrystallised from ethanol. Salt was prepared by
1
ethanolic HCl. Yield 26,9 % (0,142 g). Mp: 276-280 °C . H-NMR δ ppm (CD3OD): 1.02-
1.07 (m, 12H, CH3), 2.02-2.10 (m, 2H, CH), 3.18 (d, 2H, J= 7.2 Hz, CH2), 3.25 (d, 2H, J= 7.2
Hz, CH2), 7.27 (dd, 1H, Jm= 2 Hz, Jo= 8 Hz, H-6), 7.38 (d, 1H, Jo= 8 Hz, H-7), 7.60-7.65 (m,
13
3H, H-4,2',6'), 7.75 (d, 2H, Jo= 8.4 Hz, H-3',5'). C-NMR δ ppm (CD3OD): 19.27 (CH3), 19.40
(CH3), 27.72 (CH), 27.77 (CH), 49.94 (CH2), 50.07 (CH2), 112.41, 113.22, 116.20, 117.69,
118.04, 122.34, 128.16, 144.68, 146.62, 149.64, 161.41, 164.29, 166.19 . MS (ESI+) m/z:
.
406.47 (M+H, %100). Anal. for C23H31N7 2HCl . 1.5H2O . 0.5C2H6O; calculated, %: C 54.54; H
7.43; N 18.55 ; found, %: C 54.70; H 7.56; N 18.53.
N-secbutyl-2-[(4-(N-secbutylamidino)phenyl)amino]-1H-benzimidazole-5(6)-carboxamidine
HCl (13)
0.259
g
2-[(4-Cyanophenyl)amino]-1H-benzimidazole-5(6)-carbonitrile (2) was reacted
according to the general method 2. 0.25 ml sec-Butylamine was added to the obtained imidate
ester and was stirred in 2 ml of absolute ethanol at room temperature, for 2 days. The crude
product was purified by chromatography on silica gel (Chloroform/isopropanol/ammonia 10 : 4 :
0.7, saturated with ammonia gas) and recrystallised from ethanol. Salt was prepared by ethanolic
1
HCl. Yield 14 % (0.082 g). Mp: 278-284 °C. H-NMR δ ppm (CD3OD) : 1.02-1.16 (m, 6H,
CH3), 1.35(t, 6H, J= 6.8 Hz, CH3), 1.68-1.79 (m, 4H, CH2), 3.75-3.82 (m, 2H, CH), 7.29 (dd, 1H,
Jo= 8.4 Hz, Jm= 2 Hz, H-6), 7.42 (d, 1H, Jo= 8.4 Hz, H-7), 7.63-7.66 (m, 3H, H-4, 2',6'), 7.82 (d,
2H, Jo= 9.2 Hz, H-3',5'). 13C-NMR δ ppm (CD3OD): 9.59 (CH3), 18.27 (CH3), 28.44 (CH2),

51.59 (CH), 51.64 (CH), 112.25, 112.73, 122.43, 124.19, 125.41, 127.12, 130.06, 130.7, 134.21,
.
140.80, 149.39, 163.13, 163.63. MS (ESI+) m/z : 406.3 (M+H, %100). Anal. for C23H31N7 HCl
.
.
H2O 1.5C2H6O ; calculated, % : C 59.01; H 8.19; N 18.53 ; found, %: C 59.34; H 8.21; N
18.84.
N-Cyclohexyl-2-[(4-(N-cyclohexylamidino)phenyl)amino]-1H-benzimidazol-5(6)-carboxamidine
HCl (14)
0.259
g
2-[(4-Cyanophenyl)amino]-1H-benzimidazole-5(6)-carbonitrile (2) was reacted
according to the general method 2. 0.26 ml of Cyclohexylamine was added to the obtained
imidate ester and was stirred in 2 ml of absolute ethanol at room temperature, for 2 days. The
crude product was purified by chromatography on silica gel (Chloroform/isopropanol/ammonia
10 : 4 : 0.7, saturated with ammonia gas) and recrystallised from ethanol. Salt was prepared by
1
ethanolic HCl. Yield 25.6 % (0,146 g). Mp : 296-298 °C. H-NMR δ ppm (DMSO-d6
+NaH+D2O) : 1.06-1.32 (m, 10H, cyclohexyl CH2), 1.49-1.52 (m, 2H, cyclohexyl CH2), 1.61-
1.64 (m, 4H, cyclohexyl CH2), 1.79-1.86 (m, 4H, cyclohexyl CH2), 3.40-3.55 (m, 2H, CH), 6.95
(dd, 1H, Jo= 8 Hz, Jm = 2 Hz, H-6), 7.09 (d, 1H, Jo = 8.4 Hz, H-7), 7.36 (d, 1H, Jm = 1.6 Hz, H-4),
13
7.42 (d, 2H, Jo = 8.8 Hz, H-2',6'), 7.59 (d, 2H, Jo = 8.8 Hz, H-3',5'). C-NMR & HSQC δ ppm
(DMSO-d6 +NaH +D2O) : 25.72 (CH2), 25.76 (CH2), 26.32 (CH2), 33.38 (CH2), 33.44 (CH2),
50.72 (CH), 51.15 (CH), 111.73 (CH-4), 113.22 (CH-7), 116.14 (CH-3’,5’), 116.35 (CH-6) ,
126.48 (Cq), 127.61 (Cq), 128.00 (CH-2’,6’), 145.23 (Cq), 145.84 (Cq), 148.07 (Cq), 161.71
.
(Cq), 165.70 (Cq). MS (ESI+) m/z: 458.9 (M+H, %28). Anal. for C27H35N7 2HCl 0.75H2O
.
.
C2H6O; calculated, %: C 59.02; H 7.60; N 16.61; found, %: C 59.34; H 8.04; N 16.84.
4-[(1H-Benzimidazole-2-yl)amino]benzene-1-carboxamidine HCl (15) [19]
0.234 g 4-[(1H-benzimidazole-2-yl)amino]benzonitrile (3) was reacted according to the general
method 2. Obtained imidate ester was stirred in 2 ml of absolute ethanol saturated with ammonia
gas, at room temperature, for 2 days. The crude product was precipitated by ether, filtrated and
crystallised from ethanol. Salt was prepared by ethanolic HCl. Yield 56.9 % (0.184 g). Mp: 167-
1
171 °C (bubbling). H-NMR δ ppm (CD3OD): 6.86-6.89 (m, 2H, H-4,7), 7.30-7.32 (m, 2H, H-
5,6), 7.61-7.66 (m, 4H, H-2',3',5',6'). 13C-NMR δ ppm (CD3OD): 113.20, 115.75, 118.19, 125.46,
127.40, 143.83, 145.43, 158.40, 166.82 . MS (ESI+) m/z: 252.3 (M+H, %85), 167.7 (%100).

.
Anal. for C14H13N5 HCl 0.75 H2O 0.5 C2H6O. calculated, %: C 55.55; H 5.75; N 21.59;
.
.
found, %: C 55.39; H 5.31; N 21.87.
4-[(5(6)-Chloro-1H-benzimidazole-2-yl)amino]benzene-1-carboxamidine HCl (16)
0.268 g 4-[(5(6)-Chloro-1H-benzimidazole-2-yl)amino]benzonitrile (4), was reacted according to
the general method 2. Obtained imidate ester was stirred in 2 ml of absolute ethanol saturated
with ammonia gas, at room temperature, for 2 days. At the end of the time, crude product was
precipitated by ether, filtered and crystallised from ethanol. Salt was prepared by ethanolic HCl.
1
Yield 33 % (0.128 g). Mp: 170-174 °C (bubbling). H-NMR δ ppm (CD3OD): 6.80 (dd, 1H,
Jo= 8 Hz, Jm=1.6 Hz, H-6), 7.20 (d, 1H, Jo= 8.4 Hz, H-7), 7.23 (d, 1H, Jm= 2 Hz, H-4), 7.64 (m,
13
4H, H-2', 3', 5', 6'). C-NMR δ ppm (CD3OD): 112.80, 113.59, 115.81, 117.70, 123.08, 124.55,
127.56, 143.04, 145.41, 145.72, 159.84, 166.72 . MS (ESI+) m/z: 286.3 (M+H, %21), 288.3
.
(M+H+2, %7), 184.9 (%100). Anal. for C14H12ClN5 2HCl . 0.5 H2O . 0,5 C2H6O calculated, %:
C 46.11; H 4.64; N 17.92 ; found, %: C 46.57; H 4.57; N 17.73.
4-[(5(6)-(Trifluoromethyl)-1H-benzimidazole-2-yl)amino]benzene-1-carboxamidine HCl (17)
0.302 g 4-[(5-(Trifluoromethyl)-1H-benzimidazole-2-yl)amino]benzonitrile (5), was reacted
according to the general method 2. Obtained imidate ester was stirred in 2 ml of absolute ethanol
saturated with ammonia gas, at room temperature, for 2 days. Crude product was precipitated by
ether, filtered and crystallised from ethanol. Salt was prepared by ethanolic HCl. Yield 22.9 %
1
(0.096 g). Mp: 304-306°C. H-NMR δ ppm (CD3OD): 7.40 (d, 1H, Jo= 8.4 Hz, H-6), 7.51 (d,
1H, Jo= 8 Hz, H-7), 7.67 (s, 1H, H-4), 7.82-7.89 (m, 4H, H-2', 3', 5',6'). MS (ESI+) m/z: 320.3
.
(M+H, %100). Anal. for C15H12 F3N5 HCl . 3,5 H2O calculated, %: C 43.01; H 4.81; N 16.72;
found, %: C 42.75; H 4.90; N 17.08.
4-[(5(6)-Chloro-6(5)-methyl-1H-benzimidazole-2-yl)amino]benzene-1-carboxamidine HCl (18)
0.282 g 4-[(5(6)-Chloro-6(5)-methyl-1H-benzimidazole-2-yl)amino]benzonitrile (6), was reacted
according to the general method 2. Obtained imidate ester was stirred in 2 ml of absolute ethanol
saturated with ammonia gas, at room temperature, for 2 days. The crude product was precipitated
by ether, filtered and crystallised from ethanol. Salt was prepared by ethanolic HCl. Yield 17.6 %
(0.076 g). Mp: 315-317 °C . 1H-NMR δ ppm (DMSO-d6): 2.32 (s, 3H, -CH3), 7.08 (s, 1H, H-7),

7.14 (s, 1H, H-4), 7.62 (dd, 2H, Jo= 8.8 Hz, Jm= 2 Hz, H-2', 6'), 7.73(dd, 2H, Jo= 8.8 Hz, Jm= 2
Hz, H-3', 5'). 13C-NMR δ ppm (DMSO-d6): 20.86 (CH3), 112.97, 115.06, 115.44, 121.31, 121.85,
124.82, 127.76, 145.56, 145.62, 145.66, 161.08, 164.49. MS (ESI+) m/z: 300.3 (M+H, %21),
.
302.3 (M+H+2, %7), 192.0 (%100). Anal. for C15H14 ClN5 2HCl 2,5 H2O calculated, %: C
.
43.13; H 5.06; N 16.76; found, %: C 42.89; H 5.63; N 16.95 .
2-(Phenylamino)-1H-benzimidazole-5(6)-carboxamidine HCl (19)
0.234 g 2-(phenylamino)-1H-benzimidazole-5(6)-carbonitrile (7), was reacted according to the
general method 2. Obtained imidate ester was stirred in 2 ml of absolute ethanol saturated with
ammonia gas, at room temperature, for 2 days. The crude product was precipitated by ether,
filtered and crystallised from ethanol. Salt was prepared by ethanolic HCl. Yield 21 % (0.070 g).
1
Mp: 291-295 °C. H-NMR δ ppm (DMSO-d6): 6.72 (t, 1H, J= 7.2 Hz, H-4’), 7.08 (s, 2H, H-2’,
6’), 7.19 (t, 2H, J= 7.2 Hz H-3’, 5’), 7.51 (s, 1H, H-7), 7.79 (d, 2H, Jo=7.6 Hz, H-4, 5). 13C-NMR
δ ppm (DMSO-d6): 111.35, 112.11, 115.32, 116.29, 118.36, 123.19, 128.9, 143.97, 146.26,
.
148.83, 162.65, 167.36. MS (ESI+) m/z: 252.3 (M+H, %56), 167.6 (%100). Anal. for C14H13N5
1.5HCl . 0.75H2O . 0.25C2H6O calculated, %: C 52.61; H 5.33; N 21.16; found, %: C 52.56; H
5.27; N 20.99 .
2-[(4-Chlorophenyl)amino]-1H-benzimidazole-5(6)-carboxamidine HCl (20)
0.268 g 2-[(4-Chlorophenyl)amino]-1H-benzimidazole-5(6)-carbonitrile (8), was reacted
according to the general method 2. Obtained imidate ester was stirred in 2 ml of absolute ethanol
saturated with ammonia gas, at room temperature, for 2 days. The crude product was precipitated
by ether, filtered and crystallised from ethanol. Salt was prepared by ethanolic HCl. Yield 33 %
(0.132 g). Mp : 182-186°C (bubbling). 1H-NMR δ ppm (DMSO-d6): 7.04–7.09 (m, 2H, H-6,7),
13
7.15 (d, 2H, Jo= 8.8 Hz H-2',6'), 7.49 (s, 1H, H-4), 7.77 (d, 2H, Jo= 8.8 Hz, H-3',5'). C-NMR δ
ppm (DMSO-d6): 111.19, 112.09, 115.15, 117.52, 121.28, 123.15, 128.32, 142.61, 145.79,
148.36, 161.88, 167.15. MS (ESI+) m/z: 286.3 (M+H, %48), 288.5 (M+H+2, %16), 205.5
.
(%100). Anal. for C14H12ClN5 2HCl H2O 0,5 C2H6O calculated, %: C 45.07; H 4.79; N
.
.
17.52; found, %: C 45.09; H 4.56; N 17.24 .

2-[(3,4-Dichlorophenyl)amino]-1H-benzimidazole-5(6)-carboxamidine HCl (21)
0.302 g 2-[(3,4-Dichlorophenyl)amino]-1H-benzimidazole-5(6)-carbonitrile (9), was reacted
according to the general method 2. Obtained imidate ester was stirred in 2 ml of absolute ethanol
saturated with ammonia gas, at room temperature, for 2 days. The crude product was precipitated
by ether, filtered and crystallised from ethanol. Salt was prepared by ethanolic HCl. Yield 18 %
(0.068 g). Mp: 187-191 °C (bubbling). 1H-NMR δ ppm (DMSO-d6): 7.06 – 7.11 (m, 2H, H-
6,5’), 7.29 (d, 1H, Jo=9.2 Hz, H-7), 7.50 (s, 1H, H-2'), 7.54 – 7.57 (dd, 1H, Jo=8.8 Hz, Jm=2.8 Hz,
H-6'), 8.13 (d, 1H, Jm=2.4 Hz, H-4). 13C-NMR δ ppm (DMSO-d6): 111.84, 112.82, 115.98,
116.62, 117.20, 119.24, 123.72, 130.61, 131.41, 143.89, 145.66, 148.26, 161.43, 167.46 . MS
(ESI+) m/z: 320.2 (M+H, %85), 322.2 (M+H+2, %56), 324.2 (M+H+4, %8), 222.3 (%100).
.
Anal. for C14H11Cl2N5 HCl 1.5H2O 0.5C2H6O calculated, %: C 44.29; H 4.46; N 17.22;
.
.
found, %: C 44.15; H 4.17; N 17.30.
2.2 Biology
2.2.1. Antiparasitic activity
In vitro assays with trypomastigote bloodstream forms of T.b. rhodesiense STIB 900 and
erythrocytic forms of P. falciparum K1 (chloroquine and pyrimethamine resistant) were carried
out as previously reported [20].
2.2.2. Cytotoxicity studies
Cytotoxicity was evaluated using cultured L6 rat myoblast cells and an Alamar Blue assay as
previously reported [20]. A selectivity index (SI) could be calculated based on the following
formula: IC50 for L6 cells/IC50 for parasite.
3. Results and Discussion
All described anilinobenzimidazole carboxamidines 10 - 21 were tested in vitro for antiparasitic
activity against P. falciparum erythrocytic stages and T. b. rhodesiense trypomastigote
bloodstream forms. As a first step to clarify the antiparasitic effect of the synthesized compounds,

% inhibition values were determined at two different concentrations (4.85 µg/mL and 0.81
µg/mL). Activity was defined as ≥50 % inhibition at the lower concentration for T. b. rhodesiense
and ≥70 % inhibition at the higher concentration for P. falciparum. Subsequently, the IC50 values
were determined for the compounds which achieved these criteria. The IC50 values of the selected
compounds against T. b. rhodesiense ranged from 0.00466 to 54.66 µM (Table 1). Dicationic
compounds 10 - 14 revealed higher inhibitory activity against T. b. rhodesiense when compared
to mono cationic derivatives 15 – 21. Among the dicationic compounds, the best activity was
determined for 10 (IC50 = 0.00466 0.0009 µM), 14 (IC50 = 0.0051 0.0017 µM) and 12 (IC50 =
0.0095
0.0057 µM) which are having N-unsubstituted, N-cyclohexyl and N-isobutyl
substitution on the carboxamidine group of the dicationic anilinobenzimidazole structure. The
antitrypanosomal activity of these three compounds is comparable to the standard drug
melarsoprol. The inhibitory activity is dramatically decreased in monocationic compounds 15 -
21 with IC50 values around 20 µM. Another striking difference between dicationic and
monocationic compounds can be seen in the selectivity index (SI: IC50 for L6/IC50 for parasite)
which is <10 for monocationic compounds and between 2060 and 22600 for dicationic
compounds (Table 1).
Against P. falciparum, none of these series of compounds have superior activity to that of the
reference compound chloroquine. However, again dicationic compound 14 (IC50 = 0.0459
0.017 µM), 10 (IC50 = 0.0675 0.025 µM) and 12 (IC50 = 0.0913 0.048 µM) exhibit similar
activity to the reference compound chloroquine (IC50 = 0.0125 0.002 µM) which still has a 4-7
times higher activity. Among the monocationic compounds 15 - 21, only compound 21 revealed
the good inhibitory activity with an IC50 value of 0.138 0.017 µM which is almost equal to that
of compound 12. These data show that the introduction of a mono carboxamidine on the
condensed benzene moiety of the benzimidazole ring results in a better inhibitory activity against
P. falciparum. The dicationic compounds showed a better selectivity (SI values between 320 and
1560) than the monocationic compounds which had SI values between 30 and 60 except for 21
with an SI of 280.
Cytotoxicity of the synthesized compounds was determined using the Alamar Blue assay with rat
skeletal myoblasts (L6 cells). Based on the results of this in vitro assay these compounds seem to
have no cytotoxicity liability. Nevertheless, the absence of cytotoxicity should be confirmed

using human cell cultures and ultimately be tested for acute toxicity in a mouse model.
4. Conclusions
This work demonstrated that 2-anilinobenzimidazole carboxamidines show a good inhibitory
activity profile against T. b. rhodesiense and P. falciparum. In particular, some of the dicationic
compounds (10, 12, 14) showed equal or very close activity against T.b. rhodesiense with
reference drug Melarsoprol. Dicationic compounds also showed promising activity against P.
falciparum as compared to the reference drug chloroquine, furthermore among the monocationic
compounds 21 exhibited good inhibitory activity against this parasite. These compounds,
especially the dicationic ones, have a good selectivity for the parasites. In vivo studies of
compounds 10 and 14 are in progress, to test efficacy in an animal model of infection and to
establish pharmacokinetic profiles.
Acknowledgement: Central Laboratory of Pharmacy Faculty of Ankara University provided
support for acquisition of the NMR, Mass spectrometer and elemental analyzer used in this work.

Scheme 1. Synthesis of targeted 1H-anilinobenzimidazolecarboxamidines
NH2
NO2
NH2
NH2
a
R6
R5
R3' R4'
NC
NC
CN
H
H
H
H
H
H
H
CN
CN
CN
1
2
3
4
5
6
7
8
9
R3'
Cl
R3'
H
N
SCN
R4'
R6
R5
NH2
NH2
2'
6'
R6
CF3
H
H
CN
CN
H
H
N
R4'
Cl CH3
H
N
CN
CN
CN
H
H
H
H
b
R5
5'
Cl
Cl
H
2 - 9
Cl
H
N
H
N
NH
d
c
H
N
H
N
CN
C
OEt
HN
N
N
C
NC
EtO
2
H
CH3
H3C
NH
N
H
N
CH3
CH
CH3
C
CH
CH CH3
HN
,
,
R=
H
,
N
,
NHR
C
H2C
CH3
CH2
12
NHR
10 - 14
10
11
13
14
H
N
H
R6
R6
R5
NH
c
N
d
H
N
H
N
CN
C
OEt
N
N
R5
3 - 6
R6
R5
H
R6
R5
NH
C
N
H
N
15
16
17
18
H
H
H
H
NH2
N
Cl
CF3
15 - 18
Cl CH3
R3'
R3'
H
N
H
N
H
N
d
H
N
c
R4'
R4'
EtO
N
N
C
NC
NH
7 - 9
R4'
R3'
R3'
H
N
19
20
21
H
H
H
H
N
R4'
Cl
H2N
N
C
Cl
Cl
NH
19 - 21
Reagents : a: Pd-C / H2 ; b: EDCI, THF, 700C; c: EtOH-HCl gas; d: NH3 or corresponding amines

Table 1. Formulas and in vitro antiparasitic activities of 10 – 21
.
H
N
R6
NH
N
R5
R3'
R4 '
T. b.
rhodesiense
Cytotoxicity
L6 cells
P. falciparum
R5
R6
R3’
R4’
Compound
IC50 µM
IC50 µM
IC50 µM
NH
NH
0.00466 0.0009
SI: 22600
0.0675 0.025
SI: 1560
105.23 29.34
107.62 30.67
84.41 4.30
93.64 19.73
-H
-H
-H
-H
-H
-H
-H
-H
10
11
12
13
NH2
NH
NH2
NH
0.027 0.0135
SI: 4000
0.164 0.058
SI: 660
HN
HN
NH
NH
0.0095 0.0057
SI: 8880
0.0913 0.048
SI: 925
HN
NH
HN
NH
0.0455 0.0341
SI: 2060
0.294 0.082
SI: 320
HN
HN
NH
NH
0.0051 0.0017
SI: 12470
0.0459 0.017
SI: 1390
63.66 3.73
-H
-H
14
HN
HN
NH
15.85 6.07
SI: 7.7
3.29 0.371
SI: 37
122.60 21.05
46.41 9.23
41.44 5.84
23.25 6.48
236.58 43.93
-H
-H
-H
-H
-H
-H
-H
15
16
17
18
19
NH2
NH
19.62 9.62
SI: 2.4
0.877 0.033
SI: 53
-Cl
NH2
NH
20.86 10.68
SI: 2.0
0.853 0.072
SI: 49
-CF3
-Cl
NH2
NH
20.67 10.43
SI: 1.1
0.795 0.072
SI: 29
-CH3 -H
NH2
NH
54.66 11.92
SI: 4.3
5.46 1.024
SI: 43
-H
-H
-H
NH2

NH
17.00 6.90
SI: 2.4
0.688 0.171
SI: 60
41.56 2.57
39.06 0.47
-H
-H
-H
-Cl
-Cl
20
21
NH2
NH
21.80 8.01
SI: 1.8
0.138 0.017
SI: 280
-Cl
NH2
Melarsoprol
-
0.005 0.0025
-
Chloroquine
Podophyllotoxin
-
-
0.0125 0.002
-
-
0.0169 0.001
Values represent means and standard deviations of 3 or 4 independent assays. SI = Selectivity Index (IC50 for L6/IC50 for parasite).

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R= H, IC50= 0.00466 0.0009
SI: 22600
µM