Synthesis and antiparasitic and antifungal evaluation of 2′-arylsubstituted-1H,1′H-[2,5′]bisbenzimidazolyl-5-carboxamidines

Article abstract of DOI:10.1016/j.ejmech.2008.10.003

A series of 2′-arylsubstituted-1H,1′H-[2,5′]-bisbenzimidazolyl-5-carboxamidines were prepared in a six-step process starting from 4-amino-3-nitrobenzonitrile. The antiparasitic activity against Trypanosoma brucei rhodesiense (T.b.r.), Plasmodium falciparu

Full text of DOI:10.1016/j.ejmech.2008.10.003

European Journal of Medicinal Chemistry 44 (2009) 2002–2008
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiparasitic and antifungal evaluation of 20-arylsubstituted-1H,
10H-[2,50]bisbenzimidazolyl-5-carboxamidines
,
b
c
Mehmet Alp a, Hakan Go¨ker a , Reto Brun , Sulhiye Yıldız
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
b Parasite Chemotherapy, Swiss Tropical Institute, Basel CH-4002, Switzerland
c Department of Microbiology, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
A series of 20-arylsubstituted-1H,10H-[2,50]-bisbenzimidazolyl-5-carboxamidines were prepared in a six-
step process starting from 4-amino-3-nitrobenzonitrile. The antiparasitic activity against Trypanosoma
brucei rhodesiense (T.b.r.), Plasmodium falciparum (P.f.), Leishmania donovani (L.d.) and Trypanosoma cruzi
(T.c.) and antifungal activity against Candida albicans and Candida krusei were evaluated in vitro. Several
compounds showed promising in vitro activity against T.b.r., P.f. and C. albicans and had superior activity
against P.f. as compared to chloroquine.
Article history:
Received 24 May 2008
Received in revised form
22 September 2008
Accepted 2 October 2008
Available online 11 October 2008
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords:
Bisbenzimidazoles
Carboxamidine
Antiparasitic agents
Antifungal agents
1. Introduction
countries of sub-Saharan Africa are at risk and approximately
40,000 die each year [4]. The disease is fatal if untreated.
At least 1 billion people – one sixth of the world’s population
suffer from one or several neglected tropical diseases (NTDs)
according to the WHO reports. Among them malaria, African
(sleeping sickness) and American (Chagas disease) trypanosomiasis
and leishmaniasis are widespread in tropical and subtropical areas
of the world. Furthermore, these parasitic infections are frequently
associated with immunocompromised patients and are thus seen
also in developed countries [1].
Malaria, the most prevalent and most pernicious protozoan
parasitic disease of humans is estimated to kill between one and
two million people, mainly children, each year [2,3]. It is widely
distributed throughout the tropic and transmitted by certain
species of anopheline mosquitoes.
Four species of Plasmodium commonly infect humans, but
particularly two of them, Plasmodium falciparum and Plasmodium
vivax account for the majority of instances of morbidity and
mortality [2,3]. Sleeping sickness is caused by the parasites Trypa-
nosoma brucei rhodesiense (T.b.r.) and T. brucei gambiense (T.b.g.).
More than 50 million women, men and children in over 25
In South America, a different trypanosome, Trypanosoma cruzi,
causes Chagas disease [5,6]. This parasite is transmitted by blood-
sucking reduviid bugs. T. cruzi infection is an important cause of
mortality and morbidity, no vaccines or safe and effective chemo-
therapeutic agents are avaliable. Leishmaniasis is an another
parasitic disease spread by the bite of infected sand flies [7,8]. There
are several different forms of leishmaniasis. The most common
forms are cutaneous leishmaniasis, which causes skin sores, and
visceral leishmaniasis, which affects internal organs of the body
such as spleen, liver, bone marrow. Leishmaniasis is found in 88
countries, most of them in tropical and subtropical areas [7,8].
Newsafe and effectivedrugs against these parasitic infections are
desperately needed. Many of them currently in use for the treatment
have major problems such as significant toxicity, variable efficacy,
lack of oral bioavailability, and the need for parenteral administra-
tion [9,10]. Furthermore, drug resistance is becoming an increasing
problem [9–12]. The ideal drug should target a biochemical pathway
in the parasite which is not present in the human host.
Aromatic diamidines have shown promise as effective agents for
the treatment of parasitic infections [9,10]. Among them pentamidine
is the only compound from this class which has been significantly
used in humans for the treatment of early stage human African
trypanosomiasis (HAT), antimony-resistant leishmaniasis and AIDS-
related Pneumocystis jiroveci pneumonia. Another bis-amidine
* Corresponding author. Tel.: þ91 9411 80 3461; fax: þ90 312 2131081.
E-mail address: goker@pharmacy.ankara.edu.tr (H. Go¨ker).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.003

M. Alp et al. / European Journal of Medicinal Chemistry 44 (2009) 2002–2008
2003
R
OH
H
H
N
N
H
N
H
N
N
N
H2N
N
N
N
NH
N
DB 183 R=OH
DB 210 R=OMe
H3C
Hoechst 33258
Fig. 1. DB183, DB210 and Hoechst 33258.
derivative, Furamidine, also exhibits very potent activities against
some parasites and its methamidoxime prodrug, Pafuramidine, is
currently undergoing Phase III clinical trials against HAT [13]. This
class of compounds binds to the minor-groove of DNA at AT-rich sites.
Binding to DNA does not directly kill the parasite, but causes inhibi-
tion of DNA dependant enzymes or direct inhibition of transcription
[14]. Recently, aromatic diguanidine types of molecules [15–19] as
bioisosters of amidines were reported to exhibit significant anti-
protozoal activity as well. In addition, it has already been reported
that bisbenzimidazole-amidine systems, such as in compounds
DB183, DB210 [20,21] (Fig. 1) with potent antifungal activity against
Candidaalbicansand Crytococcusneoformans, provideaveryfavorable
and flexible DNA recognition module. Benzimidazoles, for example,
form the core of Hoechst 33258 (H258) [20,21] one of the most
Table 1
Formulas and in vitro antiparasitic and antifungal activities of 9–24.
R2
R3
R4
R1
N
H
N
H
N
H2N
N
NH
Antiparasitic activity
IC50 g/ml)
Antifungal activity
Cytotoxicity
(
m
MIC (
m
g/ml)
IC50
L-6a
(mg/ml)
a
No
9
R1
F
R2
R3
R4
T.b.r.a
0.08
0.04
0.33
1.06
ND
P.f.
C.a.
12.5
25
C.k.
12.5
12.5
25
0.007
0.010
0.015
0.031
0.211
0.064
0.630
0.017
0.008
0.017
0.023
0.061
26.8
2.48
29.8
13
13.4
31.8
76.8
25.5
10
11
12
13
14
15
16
17
18
19
20
F
F
F
25
C(CH3)3
1.56
3.12
12.5
6.25
OH
Cl
C(CH3)3
CF3
CF3
OCH3
ND
ND
NT
NT
25
50
25
25
50
50
CF3
OCH3
OCH3
50
50
25
12.5
25
0.59
0.06
0.81
0.57
1.06
OCH3
>90
17.4
19.8
OCH2Ph
OCH2Ph
OCH2Ph
OCH3
OCH2Ph
25
>90
O
O
Cl
Cl
21
22
23
ND
ND
0.193
0.634
0.028
50
50
25
50
50
25
>90
>90
46
F
O
O
Cl
0.708
0.036
OCH3
OCH3
24
0.015
3.12
6.25
29.4
25
26
DB183
DB210
Mel.
Chl.
0.48
0.016
0.016
0.013
0.015
0.78
6.25
0.78b
12.5
25
NT
8.72
34.7
4.2
0.389
0.017
0.009
0.003
OH
OCH3
>100b
NT
10.0
0.086
Flu.
0.78
25
Pod.
0.007
a
Activities represent the mean of at least two independent experiments; IC50 values used to calculate the average for a given compound were within a factor of two. T.b.r.:
Trypanosoma brucei rhodesiense STIB 900, P.f.: Plasmodium falciparum K1, C.a.: Candida albicans (ATCC 10231), C.k.: Candida krusei (ATTC 6258), Mel: Melarsoprol, Chl: Chlo-
roquine, Flu: Fluconazole, Pod: Podophyllotoxin.
b
These data were taken from lit. [20], NT: not tested, ND: not determined since activity criteria were not fulfilled in the first screening.

Table 2
Formulas, spectroscopic data, m.p. and yields of 9–26.
Compound
m.p. (ꢀC)
Yield (%)
Formula
Calculated
Found
1H NMR,
(DMSO-d6, Me4Si)
d
ppm
13C NMR,
(DMSO-d6)
d
ppm
MS (ESIþ) m/z
(if not stated otherwise)
(if not stated otherwise)
9
>300
24
C21H15FN6$3HCl$2.66H2O
C: 47.80, H: 4.45, N: 15.93
C: 48.06, H: 4.82, N: 15.40
(Base) 7.37–7.47 (m, 2H), 7.54–7.62 (m, 2H),
(Base) 115.01, 115.28, 116.4, 116.55,
117.14, 117.35, 118.59, 118.7, 121.61,
121.90, 122.44, 125.1, 125.78, 131.09,
132.80, 132.88, 140.26, 141.2, 141.44,
144.32, 149.05, 156.9, 158.98, 161.47, 166.80.
371 (100)
7.70 (d, 1H, Jo ¼ 8.4), 7.76 (d, 1H, Jo ¼ 8.4),
8.11 (s,1H), 8.17 (d, 1H, Jo ¼ 8.4), 8.25 (t, 1H, J ¼ 8), 8.52 (s, 1H)
10
11
12
>300
>300
>300
23
27
41
C21H15FN6$3HCl$3H2O
(Base) 7.39 (t, 2H, J ¼ 8.4), 7.52 (d, 1H, Jo ¼ 8.4),
7.60 (d, 1H, Jo ¼ 8), 7.66 (d, 1H, Jo ¼ 8.4), 8.04 (s, 1H),
8.12 (d, 1H, Jo ¼ 8.4 Hz), 8.26 (t, 2H, J ¼ 8), 8.42 (s, 1H)
(CD3OD) 7.66 (q, 1H, J ¼ 8.4), 7.96 (dd, 1H, Jo ¼ 8.8, Jm ¼ 1.6),
8.02–8.09 (m, 3H), 8.16–8.21 (m, 1H), 8.30–8.33 (m, 2H),
8.67 (d, 1H, Jm ¼ 0.8)
371 (100)
389 (100)
409 (100)
C: 47.25, H: 4.53, N: 15.74
C: 47.57, H: 4.73, N: 15.11
C21H14F2N6$3HCl$3.33H2O
C: 45.22, H: 4.28, N: 15.07
C: 45.12, H: 4.43, N: 14.81
C25H24N6$3HCl$3.5H2O
C: 51.69, H: 5.90, N: 14.47
C: 51.52, H: 5.67, N: 14.34
(Base) 1.390 (s, 9H), 7.52–7.60 (m, 4H),
(Base) 31.67, 35.25, 113.82, 115.17, 115.81,
115.98, 120.39, 122.00, 122.12, 126.35,
126.89, 127.10, 128.46, 140.55, 142.21,
143.25, 146.51, 153.22, 153.90, 159.43, 166.28
(CD3OD): 28.75, 34.83, 111.83, 114.44, 114.65,
115.32, 115.89, 116.42, 118.10, 123.04, 124.54,
125.61, 126.06, 130.09, 132.11, 135.58, 138.37,
153.25, 156.13, 158.05, 166.51
7.66 (d, 1H, Jo ¼ 8.8), 8.07 (s, 1H), 8.17 (d, 3H, J ¼ 8.8), 8.48 (s, 1H)
13
>300
50
C25H24N6O$2HCl$2.5H2O
C: 55.35, H: 5.76, N: 15.49
C: 55.50, H: 5.75, N: 15.38
(CD3OD) 1.40 (s, 9H), 6.84 (t, 1H, Jo ¼ 8),
425 (100)
7.32 (dd, 1H, Jo ¼ 7.6, Jm ¼ 1.2), 7.65 (dd, 1H, Jo ¼ 7.6,
Jm ¼ 1.2), 7.8–7.84 (m, 2H), 7.89(d, 1H, Jo ¼ 8.4),
7.98 (dd, 1H, Jo ¼ 8, Jm ¼ 2), 8.2 (d, 1H, Jm ¼ 0.8),
8.38 (d, 1H, Jm ¼ 1.2)
14
15
16
17
>300
>300
21
17
22
24
C
22H14ClF3N6$2.5HCl$3.3H2O
(Base) 7.56 (d, 1H, Jo ¼ 8.4), 7.65 (d, 1H, Jo ¼ 8.8),
7.70 (d, 1H, Jo ¼ 8.8), 7.79–7.85 (m, 2H),
455 (100),
457 (33)
C: 43.64, H: 3.85, N: 13.88
C: 43.69, H: 4.20, N: 13.60
C23H14F6N6$3HCl$1.66H2O
C: 44.01, H: 3.26, N: 13.38
C: 44.10, H: 3.62, N: 12.98
C23H20N6O2$3HCl$2.5H2O
C: 48.73, H: 4.98, N: 14.83
C: 48.92, H: 4.94, N: 14.58
C23H20N6O2$3HCl$2H2O
C: 49.52, H: 4.88, N: 15.07
C: 49.56, H: 4.98, N: 15.05
8.06–8.08 (2H), 8.52 (d, 1H, Jm ¼ 1.2), 8.48 (s, 1H)
(Base) 7.61 (dd, 1H, Jo ¼ 8.2, Jm ¼ 1.8), 7.66 (d, 1H, Jo ¼ 8.4),
7.71 (d, 1H, Jo ¼ 8.8), 7.96 (d, 1H, Jo ¼ 8.8),
489 (100)
413 (100)
413 (100)
8.07–8.08 (2H), 8.41 (s, 1H), 8.83 (s, 2H)
286–290
295–300
3.91 (s, 6H), 6.82 (s, 1H), 7.72 (s, 2H), 7.80 (d, 1H, Jo ¼ 8.4),
7.92 (d, 1H, Jo ¼ 8.4), 8.02 (d, 1H, Jo ¼ 8.4), 8.25 (s, 1H),
8.47 (d, 1H, Jo ¼ 8.8), 8.81 (s, 1H), 9.17 (s, 2H), 9.51 (s, 2H)
3.76 (s, 3H), 3.81 (s, 3H), 7.16 (d, 1H, Jo ¼ 8.8), 7.64 (dd, 1H,
Jo ¼ 8.6, Jm ¼ 1.2), 7.75 (d,1H, Jo ¼ 8.8), 7.86 (d, 1H, Jo ¼ 8.4),
7.99 (dd, 1H, Jo ¼ 8.8, Jm ¼ 2), 8.08 (d, 1H, Jm ¼ 1.6), 8.10 (s, 1H),
8.33 (dd, 1H, Jo ¼ 8.6, Jm ¼ 1.2), 8.61 (s, 1H),
9.03 (s, 2H), 9.35 (s, 2H)
18
19
270–275 bubb
265–270
20
31
C28H22N6O$3HCl$4H2O$0.5C2H6O
C: 52.54, H: 5.47, N: 12.68
C: 52.56, H: 5.37, N: 12.54
5.27 (s, 2H), 7.35–7.5 (m, 7H), 7.77 (dd, 1H, Jo ¼ 8.4 Hz,
Jm ¼ 1.6 Hz), 7.89 (d, 1H, Jo ¼ 8.4), 7.99 (d, 1H, Jo ¼ 8.8 Hz),
8.23 (s, 1H), 8.45–8.49 (m, 3H), 8.77 (s, 1H),
9.18 (s, 2H), 9.50 (s, 2H)
3.82 (s, 3H), 5.09 (s, 2H), 7.20–7.34 (m, 6H), 7.62 (d, 1H, Jo ¼ 8.8),
7.71 (d, 1H, Jo ¼ 8.8), 7.82 (d, 1H, Jo ¼ 8.4), 7.96 (d, 1H, Jo ¼ 8.8),
8.08–8.09 (2H), 8.31 (d, 1H, Jo ¼ 8.8), 8.57 (s, 1H),
9.07 (s, 2H), 9.37(s, 2H)
459 (100)
489 (100)
C29H24N6O2$2HCl$3.3H2O
C: 56.10, H: 5.29, N: 13.54
C: 56.09, H: 5.22, N: 13.27
57.09, 70.79, 112.32, 113.68, 114.18,
115.19, 115.42, 116.18, 116.54, 122.95,
123.40, 124.02, 124.49, 125.46, 128.68,
128.81, 129.21, 133.55, 135.30, 136.98,
137.55, 140.66, 150.11, 151.66, 152.78,
153.45, 166.53
20
>300
33
C35H28N6O2$4HCl$3.3H2O
C: 54.60, H: 5.05, N: 10.92
C: 54.69, H: 5.31, N: 11.19
(Base) 5.21 (s, 2H), 5.23 (s, 2H), 7.25 (d, 1H, Jo ¼ 8.8),
7.33–7.35 (m, 2H),
7.38–7.42 (m, 4H), 7.47–7.54 (m, 5H), 7.6 (d, 1H, Jo ¼ 8.4),
7.65 (d, 1H, Jo ¼ 8), 7.8 (dd, 1H, Jo ¼ 8.4, Jm ¼ 1.6), 7.97 (d, 1H,
Jm ¼ 1.2), 8.06 (s, 1H), 8.14 (dd, 1H, Jo ¼ 7.8, Jm ¼ 1.4), 8.43 (s, 1H)
565 (100)

21
22
290–295 bubb
288–296 bubb
28
42
C27H19ClN6O$3HCl$3H2O
C: 50.48, H: 4.39, N: 13.08
C: 50.78, H: 4.59, N: 13.11
(Base) 7.01 (d, 1H, Jo ¼ 8 Hz), 7.18–7.20 (m, 2H), 7.35(td, 1H,
Jo ¼ 7.6, Jm ¼ 1.2),7.47–7.56 (m, 4H), 7.62 (d, 1H, Jo ¼ 8.8),
7.71 (d, 1H, Jo ¼ 8.8), 8.06 (d, 1H, Jm ¼ 1.2), 8.15 (dd, 1H,
Jo ¼ 8, Jm ¼ 1.6), 8.36 (dd, 1H, Jo ¼ 7.6, Jm ¼ 2), 8.51 (d, 1H, Jm ¼ 1.2)
(Base) 115.24, 116.12, 119.34, 120.10,
121.74, 121.92, 122.09, 122.25, 124.77,
126.28, 128.69, 130.57, 131.56, 132.32,
142.43, 144.25, 145.53, 145.60, 150.29,
155.00, 155.52, 158.31, 166.42
(Base) 114.87, 115.26, 116.12, 116.33, 117,
117.25, 118.71, 118.93, 120.87, 121.53, 121.77,
122.03, 122.10, 122.36, 123.91, 124.00, 126.48,
128.48, 130.51, 140.26, 141.29, 142.57, 145.7,
149.45, 150.93, 156.01, 157.60, 158.46, 159.9, 166.60
(Base) 114.03, 115.17, 116.02, 119.27, 120.81,
121.66, 121.84, 121.95, 126.12, 126.73, 128.56,
129.32, 130.73, 140.84, 142.49, 142.71, 145.90,
153.71, 155.60, 158.55, 158.83, 160.88, 166.52
479 (100),
481 (35)
C27H18ClFN6O$2HCl$3.3H2O
C: 51.53, H: 4.26, N: 13.35
C: 51.56, H: 4.18, N: 13.20
(Base) 7.10 (dd, 1H, Jo ¼ 8.8, J ¼ 4.8), 7.15–7.18 (m, 2H),
7.33–7.38 (m, 1H), 7.44–7.46 (m, 2H), 7.56 (dd, 1H, Jo ¼ 8,
Jm ¼ 1.6), 7.63 (d, 1H, Jo ¼ 8), 7.71 (d, 1H, Jo ¼ 8.8), 8.08–8.11
(m, 2H), 8.15 (dd, 1H, Jo ¼ 8.6, Jm ¼ 1.6), 8.51 (s, 1H)
497 (100),
499 (33)
23
24
>300
38
28
C27H19ClN6O$3HCl$2H2O
C: 51.94, H: 4.20, N: 13.46
C: 51.73, H: 4.50, N: 13.14
(Base) 7.09–7.14 (m, 4H), 7.44 (d, 2H, Jo ¼ 8), 7.52 (d, 1H,
Jo ¼ 8.4), 7.59 (d, 1H, Jo ¼ 8.4), 7.64 (d, 1H, Jo ¼ 8),
8.06 (s, 1H), 8.12 (d, 1H, Jo ¼ 8.4), 8.23
(d, 2H, Jo ¼ 8.8), 8.44 (s, 1H)
479 (100),
481 (34)
295–300 bubb
C
29H24N6O3$3HCl$2H2O
3.78 (s, 3H), 3.80 (s, 3H), 6.74 (dd,
1H, Jo ¼ 8.4, Jm ¼ 2.8),
505 (100)
C: 53.59, H: 4.81, N: 12.93
C: 53.47, H: 4.99, N: 12.68
6.88 (d, 1H, Jm ¼ 2.8), 7.05 (d, 1H,
Jo ¼ 8.8), 7.23 (d, 2H,
Jo ¼ 8.4), 7.80 (dd, 1H, Jo ¼ 8.8,
Jm ¼ 1.6), 7.91 (d, 1H, Jo ¼ 8.8),
8.06 (d, 1H, Jo ¼ 8.8), 8.25 (d, 1H,
Jm ¼ 0.8), 8.45–8.49
(m, 3H), 8.79 (d, 1H, Jm ¼ 0.8),
9.19 (s, 2H), 9.5 (s, 2H)
25
26
>300
>300
22
31
C25H18N6$3HCl$3.5H2O
C: 52.23, H: 4.91, N: 14.62
C: 52.38, H: 4.99, N: 14.55
(CD3OD): 7.65–7.76 (m, 3H), 7.95 (dd, 1H,
Jo ¼ 8.4, Jm ¼ 1.2), 8.03–8.10 (m, 3H),
8.19 (d, 2H, Jo ¼ 8.4), 8.27 (d, 1H, Jo ¼ 8.4),
8.33 (d, 1H, Jm ¼ 0.8), 8.43 (dd, 1H, Jo ¼ 8.4, Jm ¼ 1.2),
8.81 (s, 1H), 9.01 (s), 9.50 (s)
(Base) 7.51–7.61 (m,4H), 7.70 (d, 1H, Jo ¼ 8.4),
7.95–8.06 (m, 4H), 8.16 (dd, 1H, Jo ¼ 8.8, Jm ¼ 1.2),
8.36 (dd, 1H, Jo ¼ 8.8, Jm ¼ 1.6), 8.48 (s, 1H), 8.77 (s, 1H)
(CD3OD): 115.14, 115.21, 115.37, 116.04, 120.37,
121.26, 123.77, 125.29, 125.75, 126.16, 126.31,
127.60, 129.03, 129.20, 130.32, 130.89, 132.98,
133.04, 134.13, 134.34, 135.70, 136.34, 152.18,
152.95, 166.72
403 (100)
403 (100)
C25H18N6$3HCl$2.5H2O
C: 53.92, H: 4.71, N: 15.09
C: 53.96, H: 4.86, N: 14.74

2006
M. Alp et al. / European Journal of Medicinal Chemistry 44 (2009) 2002–2008
NH2
NH2
NH2
NO2
NH2
NO2
NH2
NO2
b
c
a
H2N
EtO
H2N
NC
NH HCl
2
NH
HCl
1
NH
HCl
NH2
NH2
NO2
NO2
H
N
H
N
c
d
H2N
H2N
N
N
NH
NH
3
4
OH
X
O
H
R
O
e
+
X1
H
X
X1
R
2-F
2,5-di-F
4-F
4-Cl
4-Cl
4-Cl
5
6
7
8
2-(4-chlorophenoxy)
2-(4-chlorophenoxy)-5-fluoro
4-(4-chlorophenoxy)
4-(3,4-dimethoxyphenoxy)
4-F
3,4-di-OMe
4
f
R2
R1
H
R3
R4
N
H
N
H
N
N
H
N
H2N
N
N
H2N
NH
N
25 : 1-naphtyl
26 : 2-naphtyl
NH
9 - 24
Scheme 1. Reagents, a: HCl(g)/EtOH, b: NH3(g)/EtOH, c: H2/PdC, d: 3,4-dinitrobenzoic acid/PPA, e: K2CO3/DMAC, f: Na2S2O5 adduct of compounds 5–8 and other aldehydes.
extensively studied minor-groove binding agents, as well as of
a variety of other related DNA binding compounds.
These findings prompted us to synthesize new analogues of
a series of novel 20-arylsubstituted-1H,10H-[2,50]bis-benzimida-
zolyl-5-carboxamidine derivatives in order to develop new anti-
parasitic and/or antifungal agents.
antiparasitic effect of the synthesized compounds, % inhibition
values were determined at two different concentrations (4.85 g/
mL and 0.81
g/mL). Activity was defined as ꢁ50% inhibition at the
m
m
lower concentration and/or ꢁ70% inhibition at the higher concen-
tration for T.b.r., L.d., P.f. and for T.c., respectively. Subsequently, the
IC50 values were determined for the compounds which achieved
these criteria. None of the compounds fulfilled the activity criteria
in the first screening for L.d. and T.c., and hence their IC50 values
were not determined. The IC50 values of the selected compounds
2. Chemistry
Compounds 9–26 (Tables 1 and 2) were prepared using the
methods outlined in Scheme 1. The nitrile group of 4-amino-3-nitro-
benzonitrile was converted to imidate esters (the reason of the
instability they were not characterized), with dry HCl gas in absolute
ethanol, by Pinner reaction, and then treatment of imidate ester with
NH3 gas in ethanol gave the amidine 1. Reduction of nitro group yiel-
ded 2. Compound 3 was obtained by condensation of 2 with 3,4-
dinitrobenzoic acide in PPA. Reduction of both nitro groups of 3
afforded 4. Condensation of 4-chlorophenol with 2-fluorobenz-alde-
hyde, 4-fluorobenzaldehyde, 2,5-difluorobenzaldehyde and 3,4-
dimethoxyphenol with 4-fluorobenzaldehyde yielded the corres-
ponding substituted-phenyloxybenzaldehydes, 5–8 in dimethylace-
tamide. The final compounds 9–26 were prepared by condensation of
4 with Na2S2O5 adduct of 5–8 and other aldehydes [22] in
dimethylformamide.
against T.b.r. ranged from 0.036 to 1.06
m
g/mL (Table 1). Among
g/mL)
mg/mL) which are having 4-fluorophenyl and
them the best activity were obtained with 10 (IC50 ¼ 0.041
m
and 24 (IC50 ¼ 0.036
4-(3,4-dimethoxyphenoxy)phenyl groups at the C-20 position of the
amidinobisbenzimidazole moiety. While compound 17 (having
a 3,4-dimethoxyphenyl group at the same position) exhibited good
result with an IC50 value of 0.06
mg/mL, the inhibitory activity is
dramatically decreased in 16 (IC50 ¼ 0.594
mg/mL) which is having
a 3,5-dimethoxyphenyl group at the C-20 position.
The inhibitory activity of these compounds against P.f. is very
important, since most of them have superior activity to that of the
reference compound chloroquine. The IC50 values range from 0.007
to 0.634
mg/mL against P.f. with the most active antimalarials being
9, 10, 17 with IC50 values of 0.007, 0.010 and 0.008
mg/mL, respec-
tively. The antifungal activity of the compounds were tested in vitro
against C. albicans and Candida krusei by the well known tube
dilution method. The results are given as MIC values (Minimum
Inhibitory Concentration) in Table 1. The data show that, intro-
duction of 1-naphtyl group at C-20 gives a good profile of antifungal
3. Results and discussion
All described amidinobisbenzimidazoles 9–26, were tested in
vitro for antiparasitic activity against T.b. rhodesiense, Leishmania
donovani, P. falciparum and T. cruzi. As a first step, to clarify the
activity (25, MIC value of 0.78
mg/mL against C. albicans). It is
surprising that the introduction of a 2-naphtyl group at the same

M. Alp et al. / European Journal of Medicinal Chemistry 44 (2009) 2002–2008
2007
position (26), lead to a 8-fold reduction in antifungal activity (MIC
6.25 g/mL). However, similiar structure–activity relationships are
5.1.2. 2-(3,4-Diaminophenyl)-1H-benzimidazole-5-carboxamidine
(4)
m
also observed for their cytotoxicity. With the exception of 10 and
25, the compounds do not show cytotoxicity and thus are reason-
ably selective for P. falciparum.
A solution of 2-(3,4-dinitrophenyl)-1H-benzimidazole-5-car-
boxamidine (3) (400 mg, 15 mmol) in EtOH (30 ml) was subjected
to hydrogenation using 40 psi of H2 and 10% Pd-C (0.100 g) until of
H2 uptake ceased. The catalyst was filtered on a bed of Celite,
washed with EtOH, and the filtrate was concentrated in vacuo;
4. Conclusions
yield 73%; m.p.: 98–105 ꢀC bubb.; 1H NMR
d ppm (DMSO-d6): 4.67
(s, 2H, NH2), 4.99 (s, 2H, NH2), 6.59(d, 1H, Jo ¼ 8), 7.25 (dd, 1H,
Jo ¼ 8.4, Jm ¼ 2), 7.41 (d, 1H, Jm ¼ 2), 7.47 (d, 1H, Jo ¼ 8.4), 7.54 (dd,
1H, Jo ¼ 8.4, Jm ¼ 1.6), 7.9 (s, 1H). MS (ESIþ) m/z: 267 (M þ H, 100%).
This work demonstrates that 20-arylsubstituted-1H,10H-
[2,50]bis-benzimidazolyl-5-carboxamidine derivatives show a good
activity profile especially against P f. and C. albicans. Most of them
showed higher activity against P.f. that chloroquine and compound
25 exhibited the greatest activity against C. albicans comparable to
fluconazole. In vivo studies of compounds 9, 10, 17, 25 for efficacy in
an animal model of infection and the establishment of pharmaco-
kinetic profiles are in progress.
5.1.3. General procedure for synthesis of aryloxybenzaldehydes
(5–8)
A
mixture of substituted phenols (10 mmol), fluo-
robenzaldehyde (10 mmol) and anhydrous K2CO3 (10 mmol) in
dimethylacetamide (5 ml) were heated for 4 h at 140 ꢀC. The cooled
reaction mixture was treated with water (30 mL) and extracted
with CHCl3 (60 mL). The organic layer was dried (MgSO4), and
evaporated in vacuo. Further purification methods are given in
Table 3.
5. Experimental
5.1. General methods
Uncorrected melting points were measured on an Bu¨chi B-540
capillary melting point apparatus. 1H (400 MHz) and 13C (100 MHz)
NMR spectra were recorded employing a Varian Mercury 400 MHz
5.1.4. General procedure for synthesis of amidinobisbenzimidazoles
(9–26)
The corresponding benzaldehydes (15 mmol) were dissolved in
EtOH (50 ml) and sodium metabisulfite (1.6 g) in H2O (10 mL) was
added in portions. The reaction mixture was stirred vigorously and
more EtOH was added. The mixture was kept in a refrigarator for
several hours. The precipitate was filtered and dried. The mixture of
these salts (1 mmol) and 4 (1 mmol) in DMF (1–2 mL) was heated at
120 ꢀC for 4 h. The reaction mixture was cooled, poured into water and
the solid was filtered. Crude product was purified by column chro-
matography, using the mixture of Chloroform:NH3(g) saturated
Methanol (12:3) solvent system. For the HCl salts of the synthesized
compounds, the free bases were dissolved in ethanol and dry HCl gas
was passed through the solution.
FT spectrometer, chemical shifts (d) are in ppm relative to TMS, and
coupling constats (J) are reported in Hertz. Mass spectra were taken
on a Waters Micromass ZQ connected with Waters Alliance HPLC,
using ESI(þ) 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
ethanol of solvation; for compound 18, proton NMR confirmed the
presence of solvent. All chemical and solvents were purchased from
Aldrich Chemical Co. or Fischer Scientific. Compounds 1 and 2 were
synthesized as described in our previous study [23].
5.1.1. 2-(3,4-Dinitrophenyl)-1H-benzimidazole-5-carboxamidine
(3)
5.2. Biology
3,4-Diaminobenzamidine HCl 2, 0.575 g (30 mmol) and 3,4-
dinitrobenzoic acid 0.635 g (30 mmol) in polyphosphoric acid
(25 g) was heated to 160 ꢀC for 4 h. After cooling to room temper-
ature, the resultant reaction solution was poured into a mixture of
ice and water, and the solution was basified with 40% NaOH solu-
tion. The resultant red precipitate was filtered, washed with water;
5.2.1. Antifungal activity
Antifungal activity against C. albicans and C. krusei was deter-
mined by the macro-broth dilution [26] assay. The synthesized
compounds and reference drugs were dissolved in water or DMSO–
water (40%) at a concentration of 400
mg/mL. The concentration
yield 46%; m.p.: >300 ꢀC; 1H NMR
d
ppm (DMSO-d6 þ D2O): 7.36
was adjusted to 100 g/mL by 4-fold dilution with culture medium
m
(dd, 1H, H-60, Jo ¼ 8.4, Jm ¼ 1.6), 7.59 (d, 1H, H-50, Jo ¼ 8.4), 8.05 (d,
and fungi solution at the first tube. Data was not taken for the initial
solution because of the high DMSO concentration (10%).
1H, H-20, Jm ¼ 1.2), 8.21 (d, 1H, H-7, Jo ¼ 8.4), 8.62 (dd, 1H, H-6,
Jo ¼ 8.4, Jm ¼ 2), 8.76 (d, 1H, H-4, Jm ¼ 2), 13C NMR
d ppm (DMSO-
d6 þ D2O): 117.05 (C-50), 117.83 (C-20), 118.85 (C-60), 118.96, 122.82
(C-4), 127.01 (C-7), 130.69 (C-6), 140.22, 143.98, 144.25, 148.40,
153.34,160.96(C-2),167.33 (C-amidine), MS (ESIþ) m/z: 327 (M þ H,
100%).
5.2.2. Antiparasitic activity
In vitro assays with T.b. rhodesiense STIB 900, P. falciparum K1, T. cruzi
Tulahen Lac Z C4 and L. donovani MHOM-ET/67/L82 were carried out as
previously reported [27]. For T. cruzi and L. donovani a medium
Table 3
Formulas, spectroscopic data, m.p. and yields of aryloxybenzaldehydes 5–8.
Compound
Formula
13H9ClO2
1H NMR,
ppm (CDCl3)
m.p. (ꢀC)
Yield (%)
44
Purification,
column chromatography
d
5
6
C
55
Lit [24] 56.5–57
87–88
C13H8ClFO2
C13H9ClO2
6.91 (dd, 1H, J ¼ 9.2, J ¼ 4), 6.97 (d, 2H, Jo ¼ 8.8),
7.22–7.27 (m, 1H), 7.31 (d, 2H, Jo ¼ 9.2),
n-Hexane:EtOAc (1:1)
n-Hexane:EtOAc (2:1)
7.58 (dd, 1H, J ¼ 8.4, J ¼ 3.6), 10.37 (d, 1H, J ¼ 2.8)
7
8
47
Lit [25] 47–48.5
115
C
15H14O4
3.84 (s, 3H), 3.89 (s, 3H), 6.65 (m, 2H),
6.85 (d, 1H, Jo ¼ 8.8), 7.01 (d, 2H, Jo ¼ 8.4),
7.8 (d, 2H, Jo ¼ 8.4), 9.90 (s, 1H)
41

2008
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This work was supported by Ankara University Research Fund
(Project No: 20060803054). Central Lab. of Pharmacy, Faculty of
Ankara University provided support for acquisition of the NMR,
Mass spectrometer and elemental analyser used in this work. We
thank Marcel Kaiser, Monica Cal and Sacha Schneeberger (all of the
Swiss Tropical Institute) for screening the compounds for antipar-
asitic activities. We thank Dr. D. Boykin (Georgia State University,
Chemistry Department, Atlanta, USA) for having donated the
compounds DB183 and DB210.
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