Bis(oxyphenylene)benzimidazoles: A novel class of anti-Plasmodium falciparum agents

Article abstract of DOI:10.1016/j.bmc.2011.10.039

A small library of 26 2,2′-[alkane-α,ω- diylbis(oxyphenylene)]bis-1H-benzimidazoles has been prepared and evaluated against Giardia intestinalis, Entamoeba histolytica, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium

Full text of DOI:10.1016/j.bmc.2011.10.039

Bioorganic & Medicinal Chemistry 19 (2011) 7493–7500
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bis(oxyphenylene)benzimidazoles: A novel class of anti-Plasmodium
falciparum agents
Annie Mayence a, Jean Jacques Vanden Eynde a,b, , Marcel Kaiser c,d, Reto Brun c,d, Nigel Yarlett e,
⇑
Tien L. Huang a,
⇑
a Xavier University of Louisiana, College of Pharmacy, Division of Basic Pharmaceutical Sciences, 1 Drexel drive, New Orleans, LA 70125, USA
b University of Mons-UMONS, laboratory of Organic Chemistry, 20 Place du Parc, B-7000 Mons, Belgium
c Parasite Chemotherapy, Swiss Tropical Institute, Basel CH-4002, Switzerland
d University of Basel, Petersplatz 1, CH-4051 Basel, Switzerland
e Pace University, Department of Chemistry and Physical Sciences, and Haskins Laboratories, 1 Pace Plaza, New York, NY 10038, USA
a r t i c l e i n f o
a b s t r a c t
A small library of 26 2,20-[alkane-
a,x-diylbis(oxyphenylene)]bis-1H-benzimidazoles has been prepared
and evaluated against Giardia intestinalis, Entamoeba histolytica, Trypanosoma brucei rhodesiense, Trypan-
osoma cruzi, Leishmania donovani, and Plasmodium falciparum. Among the tested compounds, eight deriv-
atives 1(7, 19, 20, 24, 27, 30, 32 and 35) exhibited an anti-Plasmodium falciparum activity characterized by
Article history:
Received 2 June 2011
Revised 6 October 2011
Accepted 14 October 2011
Available online 20 October 2011
IC50 values in the range of 180–410 nM (0.11–0.21 lg/mL) and selectivity indexes (IC50 rat skeletal myo-
blasts L6 cells vs IC50 P. falciparum K1 strain) varying between 92 and more than 450. Two of the eight
novel drug leads, namely compounds 19 and 32, were also active against G. intestinalis and L. donovani
with selectivity indexes of 122 and >164 respectively.
Keywords:
Benzimidazole
Malaria
Plasmodium
Protozoan parasites
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
against L. donovani. Later Navarrete and co-workers19 demon-
strated that the 5,50-dimethoxy analog exhibited significant
The benzimidazole skeleton has attracted and still attracts
much attention from medicinal chemists because of its structural
resemblance to various moieties present in fundamental constitu-
ents of proteins and nucleic acids. Benzimidazoles are also well-
known for their broad spectrum of anti-parasitic properties. For
example, thiabendazole (1, Fig. 1) is used primarily to control mold,
blight, and other fungi in fruits and vegetables.1 Thiabendazole (1),
ciclobendazole (2), mebendazole (3), flubendazole (4), fenbendaz-
ole (5), and albendazole (6) are prescribed to eliminate gastrointes-
tinal parasites and worms in wild and domestic animals, livestock,
as well as humans.2–7 Albendazole (6) is also used for the treat-
ment of lymphatic filariasis and giardiasis in some parts of the
world.8,9 During the last decade some bisbenzimidazoles have
been screened for inhibition of hepatitis C virus serine prote-
ases10,11 (e.g., 7), and anti-cancer drug leads12–15 (e.g., 8). Mean-
while, structurally related congeners of 8 (e.g., 9) and other
bisbenzimidazoles (e.g., 10) have been evaluated as anti-microbial,
anti-fungal, and anti-parasitic agents.16–20 For example we18 have
established that 2,20-[1,5-pentanediylbis(oxy-1,4-phenylene)]bis-
1H-benzimidazole 15 could be considered as a promising hit
in vitro effect against Trichomonas vaginalis, Giardia lamblia, Ent-
amoeba histolytica, and Leishmania mexicana also.
Because of our combined interest in the chemistry of benzimi-
dazoles21,22 and in the study of their structure–activity relation-
ships18,23–25 we now wish to report on the preparation of a
library of bisbenzimidazoles whose structures are depicted in
Figure 2 and on their ability to inhibit (or not) the growth of vari-
ous strains of parasites.
2. Results and discussion
2.1. Chemistry
The targeted bisbenzimidazoles were obtained by a two-step
sequence following experimental protocols already de-
scribed.18,21–27 All compounds have been fully characterized by
their spectroscopic data (1H NMR, 13C NMR, IR, HRMS) and purity
assessed by HPLC. In the 13C NMR spectra some signals due to
the benzimidazole moieties were sometimes broadened or even
not observed. As reported in the literature28,29 this is linked to
the well-known prototropic tautomerism in the (benz)azoles ser-
ies. From the HPLC analyses, the purity of the compounds ranged
from 95.6% to 100%.
⇑
Corresponding author. Tel.: +1 504 520 7603; fax: +1 504 520 7954.
E-mail address: thuang@xula.edu (T.L. Huang).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.039

7494
A. Mayence et al. / Bioorg. Med. Chem. 19 (2011) 7493–7500
R
O
C
O
cyclo-C3H5
2
3
N
H2N
O
CH3
O
N
H
C6H5
C
N
R
O
CH3
N
N
N
S
O
C
NH
O
H
N
N
H
N
N
H
4-F-C6H4
4
7
OH
OH
O
O
1
C6H5
C3H7
S
S
5
6
P
HO
2 - 6
OH
F
HO
N
N
N
N
N
N
N
H
NH
NH
HN
N
H
OH
N
H
N
H
N
N
NH2
10
H3C
9
8 (Hoechst 33258)
Figure 1. Some benzimidazole-containing derivatives of pharmacological interest.
O
CH2
O
N
N
n
O
CH2
O
n
N
H
N
H
N
NH
NH
N
11 20
-
(n = 1 - 10)
21 - 30 (n = 1 - 10)
CH2
O
CH2
O
H2C
O
N
H2C
O
N
H
NH
N
N
34 36
-
(o, m, p)
NH
NH
N
31 33
-
(o, m, p)
Figure 2. Structure of the bisbenzimidazoles studied in this work.
2.2. Biological assays
2.2.1. Cytotoxicity towards rat skeletal myoblasts, L-6 cells30
The L-6 cell line consists of rat muscle precursor cells. It is a
commercially available cell line with low culture medium require-
ments. It can readily be handled and stops proliferation when
reaching confluency.
Most compounds in this study were characterized by an IC50 va-
lue in the range of 1–12 lg/mL (3–25 lM). Derivatives 17, 19, 20,
27, 30, 32, and 35 emerged as the less toxic members of the library.
The so-obtained 26 bisbenzimidazoles were evaluated in vitro
for their cytotoxicity towards the non-carcinoma L-6 rat skeletal
myoblasts cell line and for their activity against various protozoan
parasites infecting people in developing countries essentially (but
not exclusively), namely Giardia intestinalis, E. histolytica, Trypano-
soma brucei rhodesiense, Trypanosoma cruzi, L. donovani, and Plas-
modium falciparum.
The structures of the reference drugs are represented in Figure
3. Experimental data and some relevant calculated data (IC50 val-
ues, selectivity index, molecular weights and LogP) are compiled
in Tables 1 and 2. The calculated partition coefficients of these
compounds ranged from 6.83 to 9.28 indicating high lipid and
low aqueous solubility. The high lipophilicity of the tested com-
pounds could present some solubility problems and may enhance
binding to plasma proteins in the assay mixture, thereby decreas-
ing the real potency of the compounds. With this limitation in
mind, the aim of this work is limited to identification of some po-
tential active leads and not to determine why some compounds
were inactive.
2.2.2. Anti-Giardia intestinalis activity31
Giardiasis is a form of contagious dysentery that is often related
to poor sanitary conditions. It is caused by swallowing resting cysts
of a protozoan parasite, Giardia intestinalis, which can be found in
water, food, and soil. The illness is very common in developing
countries but also affects all other parts of the world.
Metronidazole (a 2-nitroimidazole derivative, see Fig. 3), the
first-line drug used in the treatment of giardiasis, was chosen
as the reference drug for that study. It exhibited an IC50 value
of 0.267
compounds (13, 15, 17, and 19) were efficient against that pro-
tozoan at a dose lower than 1.50 g/mL. Amazingly those four
lg/mL (1.56 lM) against Giardia intestinalis G1. Four
l

A. Mayence et al. / Bioorg. Med. Chem. 19 (2011) 7493–7500
7495
OH
H
H
NH2
O
O
N
HO
O
N
N
NO2
OH
S
S
N
As
O
H2N
N
NH
metronidazole
O
O
melarsoprol
O
podophyllotoxin
N
HN
O (-)
N
(+)
N
O
P
O2N
H3C CH2
CH2O
N
O
14
NH
Cl
N
miltefosine
O
chloroquine
benznidazole
Figure 3. Reference drugs used in this study.
Table 1
IC50 values determined towards rat skeletal myoblasts L-6 cells, Gardia intestinalis, Entemoeba hystolytica, T. brucei rhodesiense, T. cruzi, and L. donovani
b
Compounda
IC50
(
lM)
L-6 cells
0.012
G. intestinalis
E. hystolytica Hk-9
T. brucei rhodesiense
T. cruzi
L. donovani
Podophyllotoxin
Metronidazole
Melarsoprol
Benznidazol
Miltefosine
11 (n = 1)
12 (n = 2)
13 (n = 3)
14 (n = 4)
15 (n = 5)
16 (n = 6)
17 (n = 7)
18 (n = 8)
19 (n = 9)
20 (n = 10)
21 (n = 1)
22 (n = 2)
23 (n = 3)
24 (n = 4)
25 (n = 5)
26 (n = 6)
27 (n = 7)
28 (n = 8)
29 (n = 9)
30 (n = 10)
31 (ortho)
32 (meta)
33 (para)
1.560
5.820
0.010
1.134
0.445
16.49
> 202
2.67
3.25
0.80
6.59
1.53
98.22
5.93
123.80
6.19
24.10
3.91
9.99
1.19
2.57
2.69
5.90
6.59
13.30
2.33
1.05
0.80
3.00
4.63
8.44
14.40
25.38
3.56
7.84
6.82
2.13
154.13
1.50
2.76
1.02
7.22
1.08
39.12
0.81
9.34
14.20
16.57
8.01
18.48
1.33
2.37
1.37
4.50
2.37
2.08
1.32
3.56
2.12
2.66
3.60
24.01
11.26
133.15
3.91
5.75
2.42
124.08
0.74
17.85
0.82
3.58
1.34
10.44
0.83
22.08
190.82
3.37
8.13
22.72
>199
21.74
65.75
30.72
19.29
9.61
6.05
48.39
19.80
101.25
45.98
18.94
5.04
9.82
21.23
3.56
14.75
56.97
> 165
88.45
11.63
4.68
8.92
23.94
1.70
3.88
1.82
4.28
4.98
11.65
56.07
36.03
18.46
17.71
25.76
52.44
45.53
171.95
83.99
78.26
211
27.84
73.70
56.73
23.54
46.21
22.19
7.98
118.34
13.08
21.92
72.20
5.89
>172
16.01
7.02
83.23
6.07
113.20
26.31
2.53
34 (ortho)
35 (meta)
36 (para)
79.58
10.39
5.99
10.89
Compounds with low cytotoxicity toward L-6 cells or significant activity against the protozoan parasites are shown in bold.
a
The number of methylene groups or the relative positions of the substituents in the central ring are given in the brackets.
The IC50 values are the average of duplicate determinations, the individual values vary less than a factor 2. Standard deviations or confidence intervals could not be
b
calculated.
bisbenzimidazoles contain a central chain constituted by 3, 5, 7,
calculated. Compound 19 also showed a high selectivity index
of 344 against P. falciparum, and was relatively non-toxic to
the L-6 cells. Therefore this compound is an interesting lead that
deserves further attention.
and 9 methylene groups respectively and all of them are charac-
terized by the presence of the benzimidazole moieties in the
para position relative to the ether bonds linking the central
chain and the phenylene rings. In that series of para-substituted
congeners, cytotoxicity against L-6 cells roughly decreased when
the length of the central alkanediyl chain increases, therefore the
most interesting hit for further studies is derivative 19 for which
a selectivity index (IC50 L-6 cells/IC50 parasite) of 123 could be
It is interesting to note that Navarrete and co-workers19 re-
ported that compound 15 was less active against G. lamblia and
more active against E. histolytica when compared to metronidazole,
but the strains of the parasites and the protocols used were
different.

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A. Mayence et al. / Bioorg. Med. Chem. 19 (2011) 7493–7500
2.2.5. Anti-Leishmania donovani activity34,36–39
Table 2
IC50 values determined towards P. falciparum and some calculated values (selectivity
indexes, molecular weights, and logP)
Leishmaniases are visceral, cutaneous, and mucocutaneous dis-
eases caused by intracellular protozoan parasites of the genus
Leishmania. The World Health Organization reports that several
million people are infected each year with visceral leishmaniasis.
Depending on the geographic area, either L. donovani or Leishmania
infantum is the causative agent.
Compounda
IC50
SIb
MW
cLogPc
l
g/mL
lM
P. falciparum
Chloroquine
11 (n = 1)
12 (n = 2)
13 (n = 3)
14 (n = 4)
15 (n = 5)
16 (n = 6)
17 (n = 7)
18 (n = 8)
19 (n = 9)
20 (n = 10)
21 (n = 1)
22 (n = 2)
23 (n = 3)
24 (n = 4)
25 (n = 5)
26 (n = 6)
27 (n = 7)
28 (n = 8)
29 (n = 9)
30 (n = 10)
31 (ortho)
32 (meta)
33 (para)
34 (ortho)
35 (meta)
36 (para)
0.059
0.30
0.90
0.13
0.21
0.13
0.12
0.12
0.16
0.16
0.10
0.73
1.15
1.11
0.11
0.17
0.14
0.21
0.24
0.31
0.20
0.18
0.20
0.12
0.18
0.21
0.10
0.184
0.69
2.01
0.28
0.44
0.27
0.24
0.23
0.30
0.29
0.18
1.69
2.58
2.41
0.23
0.34
0.28
0.41
0.45
0.57
0.36
0.34
0.38
0.23
0.34
0.40
0.19
319.87
432.47
446.50
460.53
474.55
488.58
502.61
516.63
530.66
544.69
558.71
432.47
446.50
460.53
474.55
488.58
502.61
516.63
530.66
544.69
558.71
522.60
522.60
522.60
522.60
522.60
522.60
5.01
6.88
6.89
7.16
7.43
7.94
8.37
8.67
8.90
9.09
9.24
6.83
6.84
7.11
7.38
7.89
8.33
8.64
8.88
9.07
9.28
8.34
8.36
8.38
8.33
8.34
8.34
The activities of bisbenzimidazoles 11–36 were compared to
21
13
13
18
26
40
208
67
344
257
11
2
4
92
10
29
291
29
39
202
17
that of miltefosine in this assay. An IC50 value of 0.45
determined for the reference drug whereas the lowest IC50 values
measured in the bisbenzimidazoles library were 0.80 M for 15
and 0.80 M for 33. Unfortunately, those two derivatives were also
lM was
l
l
toxic in the presence of the rat skeletal myoblasts and were there-
fore excluded from any further development. However compound
32 in which the central phenylene ring is substituted at the meta
positions is a good lead based on its selectivity index of >164.
2.2.6. Anti-Plasmodium falciparum activity36,40
It is estimated that over one billion people (one sixth of the
world population) suffers from one or more tropical protozoal
infections. Among them malaria, caused by P. falciparum, is the
most dangerous disease with the highest rates of complications
and mortality. The World Health Organization reports that in
2008 there were 247 million cases of the sickness causing almost
one million deaths. Fight against female Anopheles mosquitoes,
the vector of Plasmodium, can be affected by indoor spraying with
insecticides or distribution of insecticide treated nets to cover
beds. Chemoprophylaxis and chemotherapy, including the arte-
misin-based combination therapies (ACT) constitute other ways
to eliminate the parasite. However excessive use of those chemical
weapons led to numerous mutations and resistance in Plasmodium,
thus justifying a continuous search for novel anti-malarial agents.
The WHO Special Program for Research and Training in Tropical
Diseases defines an activity criterion to have an IC50 value lower
>450
70
20
198
54
Compounds with high selectivity indexes are shown in bold.
The number of methylene groups or the relative positions of the substituents in
the central ring are given in the brackets.
a
b
Selectivity index = IC50 L-6 cells/IC50 parasite.
From www.molinspiration.com.
c
2.2.3. Anti-Entamoeba histolytica Hk-9 activity31
than 0.20 lg/mL. Interestingly only six out of the 26 members of
the library exhibited IC50 values higher than 0.30
pounds exhibited values ranging from 0.20 to 0.24
teen derivatives were active at doses lower than 0.20
l
l
g/mL, six com-
g/mL and four-
g/mL.
E. histolytica is another parasitic protozoal organism that is
responsible for amebiasis that manifests under the forms essen-
tially of amebic dysentery or amebic liver abscess. These diseases
are prevalent in tropical countries with an estimated 40 million
cases and 100,000 deaths annually. They come in second place,
after malaria, in terms of mortality.
Metronidazole is prescribed also to cure amebiasis and was
again the reference compound for that study with an IC50 value
of 5.82 lM against E. histolytica HK-9. Only two compounds,
namely 13 and 14, were found to be active enough against the par-
asite to deserve attention. However they were characterized with
poor selectivity indexes (IC50 L-6 cells/IC50 parasite) neighboring
one unit and therefore they had to be classified as devoid of prac-
tical interest.
l
It must be pointed out that the majority of the less active sub-
stances are those in which the linker is constituted by a short alka-
nediyl chain of one, two, or eventually three carbon atoms, namely
11, 12, 21, 22, 23 (derivative 29 is the sixth substance of the group
of the poorly active substances). All 20 other compounds were
characterized by IC50 values ranging from 0.10 to 0.24 lg/mL,
which corresponds to molar concentrations comprised between
180 and 450 nM. This means that those compounds are as active,
in vitro, as chloroquine, the reference drug, or at the worst 2.5-fold
less active than chloroquine which has an IC50 value of 0.059
mL (184 nM).
lg/
As far as cytotoxicity is concerned, inspection of the selectivity
indexes (IC50 L-6 cells/IC50 parasite) reported in Table 2 indicated
that eight bisbenzimidazoles could emerge as promising drug
leads. Those are the bisbenzimidazoles 17, 19, 20, 24, 27, 30, 32,
35, for which selectivity indexes of 208, 344, 257, 92, 291, 202,
>450, and 198 respectively have been calculated. The main draw-
back of those novel anti-Plasmodium falciparum agents is their
low water solubility which could affect their bioavailability when
administered by the oral route. Indeed calculated partition coeffi-
cients between n-octanol and water (clogP, see Table 2) are higher
than 7 units and therefore do not obey the Lipinski’s rules.41 That
drawback could however be circumvented by preparing proton-
ated forms of the bisbenzimidazoles. Indeed, softwares used to cal-
culate logP values predicted a decrease of several units when going
from the neutral bisbenzimidazole species to the diprotonated
ones. In our opinion, molecular weights, which are at the limit of
2.2.4. Anti-Trypanosoma activity32–35
T. brucei rhodesiense is responsible for sleeping sickness, which
is an endemic disease affecting several dozens of thousands people
across sub-Saharan Africa, most of them being undiagnosed and
therefore untreated. T. cruzi is a different parasite found in Central
and South America. It causes Chagas disease. Approximately
20,000 persons die from Chagas disease every year. Depending
on the strain considered, two different drugs of reference were
used: melarsoprol in the case of T. brucei rhodesiense and benzni-
dazole (a monobenzimidazole derivative) in the case of T. cruzi.
Deceptively the lowest IC50 value recorded against T. brucei rho-
desiense for a bisbenzimidazole considered in this study remained
100-fold higher than the value measured for melarsoprol. None of
the tested compounds were more active than benznidazole or
showed good selectivity against T. cruzi.

A. Mayence et al. / Bioorg. Med. Chem. 19 (2011) 7493–7500
7497
the violation of the Lipinski’s rules, would not hamper further
development of the hits identified in this work.
commercially available (Aldrich, Alfa Aesar, Acros Organics, Fisher
Scientific) and were used without further purification.
Chloroquine and some other anti-malarial drugs are known to
exert their effect by inhibiting a detoxication process by which Plas-
modium, in its food vacuole, converts heme, a fatal poison for the
parasite, into hemozoin, the non-toxic malaria pigment.42–44 Such
a chemical transformation can readily be mimicked in a cell-free as-
say.45–50 In particular, Egan45 reported that, under acidic condition
(pH 5.0) similar to that found in the lysosomal vacuole of P. falcipa-
rum, ferriprotoporphyrin IX chloride (hemin) evolved to yield a pre-
cipitate of b-hematin, the synthetic form of hemozoin. However,
that reaction can be inhibited in the presence of some chemicals,
which include quinine, chloroquine, and amodiaquin. Therefore
observation of that inhibition constitutes a simple test enabling to
identify potential anti-Plasmodium falciparum agents. In our hands,
a randomized series of bisbenzimidazoles described in this work
(13, 14, 15, 16, 17, 22, 23, 24, and 27) have been evaluated in that
cell-free assay and have been shown to effectively inhibit precipita-
tion of b-hematin from hemin in an aqueous acetate buffer. These
results suggest that the tested derivatives could contribute to the
accumulation of heme in the food vacuole of Plasmodium and in this
way lead to the death of the parasite. Similar conclusions had
already been made to explain the anti-Plasmodium activity of
pentamidine49 and structurally related bisbenzamidines,50 but
obviously other modes of action cannot be excluded.51,52
4.1. Chemistry
4.1.1. Preparation of the bisbenzaldehydes
A mixture of a hydroxybenzaldehyde (6.72 g, 55 mmol), a dibro-
moalkane (25 mmol), and tetrabutylammonium sulfate (0.34 g;
1 mmol) in an aqueous 4 M solution of sodium hydroxide was
heated under reflux for 7 h. After cooling the precipitate was fil-
tered and successively washed with water, and ether. Yields (not
optimized) ranged from 20% (for the precursor of 11) to 95% for
the precursors of 25, 32, 34, and 35). The bisbenzaldehydes were
pure enough to be engaged in the second step.
The bisbenzaldehydes precursors of 11–20,53 21,54 22,55 23,56
24,57 25,58 26,58 27,59 28–30,58 31,60 32,61 33,62 34,63 35,64 and
36.65 have been described in the literature.
4.1.2. Preparation of the bisbenzimidazoles
A mixture of pyridine (1.62 mL; 1.58 g; 20 mmol) and thionyl
chloride (1.46 mL; 2.38 g; 20 mmol) in dichloromethane (50 mL)
was slowly added to a solution of bisbenzaldehyde (10 mmol) in
dichloromethane (25 mL) maintained at 0 °C. After addition, the
reaction medium was allowed to warm up to room temperature
for 10 min and
a solution of 1,2-phenylenediamine (4.32 g;
40 mmol) in dichloromethane (25 mL) was slowly added. The mix-
ture was stirred overnight. The solid was filtered and successively
washed with N,N-dimethylformamide (or acetonitrile when the
bisbenzimidazole was soluble in N,N-dimethylformamide), hot
water, and hot ethanol. The so-obtained dihydrochloride salt
(100 mg) was neutralized in a hot mixture of NaOH 0.4 M (4 mL)
and ethanol (1 mL). Yields have not been optimized.
3. Conclusion
A small library of 26 bisbenzimidazoles have been designed,
prepared, and evaluated for their in vitro effects toward rat skeletal
myoblasts and several protozoan parasites responsible for diseases
essentially infecting large number of people living in developing
countries. Those parasites are Giardia intestinalis, E. histolytica, T.
brucei rhodesiense, T. cruzi, L. donovani, and P. falciparum. From
the collected data, it is clear that the library of benzimidazoles con-
stitutes an interesting source for the identification of a novel drug
lead for the fight against P. falciparum. Indeed more than two-third
of the derivatives was significantly active against the parasite and
almost one-third is characterized by selectivity indexes (IC50 L-6
cells/IC50 parasite) higher than 90 and even exceeding 450. In addi-
tion let us emphasize that the bisbenzimidazoles described in this
work are readily accessible from cheap precursors and are charac-
terized by simple chemical structures devoid of stereogenic cen-
ters. These promising in vitro results however need to be
confirmed by in vivo screenings, which will be a challenge because
the physicochemical properties of these derivatives must be im-
proved prior to the in vivo studies. In particular their solubility
in water has to be increased and appropriate formulations should
be found in order to reach acceptable oral absorption. Based on
the leads identified in this work, future studies are directed to
the preparation of novel compounds bearing substituents that
could modulate not only the physicochemical properties, but also
increase their selectivity indexes.
The bisbenzimidazoles 13–17 and 25 have been described in
the literature.18
4.1.2.1.
imidazole (11).
2,20-[Methylenebis(oxy-1,4-phenylene)]bis-1H-benz-
Yield: 70%. Mp: >300 °C. 1H NMR (DMSO-
d6): 8.1 (d, 4H, J = 8 Hz); 7.6 (d, 2 H, J = 7 Hz); 7.5 (d, 2 H, J = 7 Hz);
7.2 (m, 8 H); 6.0 (s, 2 H) ppm. 13C NMR (DMSO-d6): 157.5; 151.0;
144.8; 135.0; 128.8; 124.5; 122.2; 121.5; 118.6; 116.5; 111.1;
89.9 ppm. IR: 3300–2200; 3058; 1616; 1500; 1439; 1233 cmꢀ1
HRMS: found 455.1484, calcd 455.1470 for C27H20N4O2 + Na.
.
4.1.2.2.
benzimidazole (12).
2,20-[1,2-Ethanediylbis(oxy-1,4-phenylene)]bis-1H-
Yield: 50%. Mp: >300 °C. 1H NMR
(DMSO-d6): 8.1 (d, 4 H, J = 8 Hz); 7.6 (d, 2H, J = 7 Hz); 7.5 (d, 2 H,
J = 7 Hz); 7.2 (m, 8 H); 4.5 (s, 4 H) ppm. 13C NMR (DMSO-d6):
159.7; 151.3; 143.8; 134.9; 128.1; 122.9; 122.1; 121.5; 118.5;
114.9; 111.0; 66.5 ppm. IR: 3300–2200; 3057; 1609; 1496; 1437;
1246 cmꢀ1. HRMS: found 447.1823, calc. 447.1821 for C28H23N4O2.
4.1.2.3.
benzimidazole (18).
2,20-[1,8-Octanediylbis(oxy-1,4-phenylene)]bis-1H-
Yield: 35%. Mp: 292–294 °C (decompo-
sition). 1H NMR (DMSO-d6): 8.1 (d, 4 H, J = 8 Hz); 7.6 (d, 2 H,
J = 7 Hz); 7.5 (d, 2 H, J = 7 Hz); 7.2 (m, 8 H); 4.1 (t, 4 H, J = 6 Hz);
1.8 (m, 4 H, J = 6 Hz); 1.5 (br, 4 H); 1.4 (br, 4 H) ppm. 13C NMR
(DMSO-d6): 160.1; 151.4; 143.8; 135.0; 128.0; 122.5; 122.0;
121.4; 118.4; 114.8; 111.0; 67.6; 28.7; 28.6; 25.4 ppm. IR: 3300-
2200; 2938; 1614; 1551; 1503; 1445 1258 cmꢀ1. HRMS: found
531.2762, calcd 531.2760 for C34H35N4O2.
4. Experimental
1H NMR spectra were obtained using a Varian Inova instrument
(500 MHz), 13C NMR spectra using
a Bruker Advance 500
(125 MHz), chemical shifts (d) are given in ppm using TMS as inter-
nal reference and coupling constants (J) in Hz. IR spectra were re-
corded on a Perkin-Elmer Spectrum One spectrometer operating in
the diffuse reflectance mode. High resolution mass spectra (HRMS)
were recorded on a Waters Q-ToF 2 instrument. A Merck Hitachi
LaChrom HPLC equipped with a HibarÒ 125-4 PurospherÒ STAR
RP-18e column has been used (solvents: methanol/water) to assess
purity of the final compounds. Solvents and reagents were
4.1.2.4.
benzimidazole (19).
2,20-[1,9-Nonanediylbis(oxy-1,4-phenylene)]bis-1H-
Yield: 50%. Mp: 271–273 °C (decomposi-
tion). 1H NMR (DMSO-d6): 8.1 (d, 4 H, J = 8 Hz); 7.6 (d, 2 H,
J = 7 Hz); 7.5 (d, 2 H, J = 7 Hz); 7.2 (m, 8 H); 4.1 (t, 4 H, J = 6 Hz);
1.8 (m, 4 H, J = 6 Hz); 1.5 (br, 4 H); 1.4 (br, 6 H) ppm. 13C NMR

7498
A. Mayence et al. / Bioorg. Med. Chem. 19 (2011) 7493–7500
(DMSO-d6): 160.0; 151.4; 143.8; 134.9; 128.0; 122.5; 122.0; 121.4;
118.4; 114.8; 111.0; 67.64; 28.9; 28.7; 28.6; 25.5 ppm. IR: 3300–
2200; 2933; 1613; 1435; 1256 cmꢀ1. HRMS: found 567.2745, calcd
567.2736 for C35H36N4O2 + Na.
151.1; 143.7; 134.9; 131.4; 130.1; 122.6; 121.7; 118.8; 118.6;
116.3; 112.0; 111.3; 67.6; 28.6; 28.5; 25.5 ppm. IR: 3300–2200;
2937; 1603; 1492; 1463; 1275; 1242 cmꢀ1
539.2441, calcd 539.2423 for C33H32N4O2 + Na.
. HRMS: found
4.1.2.5.
benzimidazole (20).
2,20-[1,10-Decanediylbis(oxy-1,4-phenylene)]bis-1H-
4.1.2.12.
benzimidazole (28).
2,20-[1,8-Octanediylbis(oxy-1,3-phenylene)]bis-1H-
Yield: 30%. Mp: 270–271 °C (decomposi-
Yield: 50%. Mp: 246–248 °C (decomposi-
tion). 1H NMR (DMSO-d6): 8.1 (d, 4 H, J = 8 Hz); 7.6 (d, 2 H,
J = 7 Hz); 7.5 (d, 2 H, J = 7 Hz); 7.2 (m, 8 H); 4.1 (t, 4 H, J = 7 Hz);
1.8 (m, 4 H, J = 7 Hz); 1.4 (br, 4 H); 1.3 (br, 4 H) ppm. 13C NMR
(DMSO-d6): 159.3; 150.6; 142.5; 134.0; 127.2; 122.5; 121.7;
117.7; 114.0; 110.0; 66.9; 28.1; 28.0; 27.8; 24.7 ppm. IR: 3300–
tion). 1H NMR (DMSO-d6): 8.0 (t, 2 H, J = 2 Hz); 7.9 (d, 2 H, J = 8 Hz);
7.7 (d, 2 H, J = 6 Hz); 7.5 (m, 4 H); 7.2 (m, 6 H); 4.1 (t, 4 H, J = 6 Hz);
1.8 (m, 4 H, J = 6 Hz); 1.5 (br, 4 H); 1.4 (br, 4 H) ppm. 13C NMR
(DMSO-d6): 159.0; 151.1; 143.7; 134.9; 131.4; 130.1; 122.6;
120.7; 118.8; 118.6; 116.3; 111.9; 111.3; 67.6; 28.7; 28.6;
2200; 2924; 1614; 1498; 1436; 1252 cmꢀ1
559.3069, calcd 559.3073 for C36H39N4O2.
.
HRMS: found
25.5 ppm. IR: 3300–2200; 2930; 1602; 1491; 1458; 1235 cmꢀ1
HRMS: found 531.2768, calcd 531.2760 for C34H35N4O2.
.
4.1.2.6.
2,20-[Methylenebis(oxy-1,3-phenylene)]bis-1H-benz-
4.1.2.13.
2,20-[1,9-Nonanediylbis(oxy-1,3-phenylene)]bis-1H-
imidazole (21).
Yield: 35%. Mp: 289–291 °C (decomposition).
benzimidazole (29).
Yield: 30%. Mp: 226–228 °C (decomposi-
1H NMR (DMSO-d6): 8.0 (t, 2 H, J = 2 Hz); 7.9 (d, 2 H, J = 8 Hz); 7.7
(d, 2 H, J = 6 Hz); 7.5 (m, 4 H); 7.2 (m, 6 H); 6.1 (s, 2 H) ppm. 13C
NMR (DMSO-d6): 156.8; 150.8; 144.8; 135.0; 131.7; 130.3; 122.6;
121.7; 120.5; 118.9; 117.7; 114.1; 111.3; 90.1 ppm. IR: 3300–
tion). 1H NMR (DMSO-d6): 8.0 (t, 2 H, J = 2 Hz); 7.9 (d, 2 H, J = 8 Hz);
7.7 (d, 2 H, J = 6 Hz); 7.5 (m, 4 H); 7.2 (m, 6 H); 4.1 (t, 4 H, J = 6 Hz);
1.8 (m, 4 H, J = 6 Hz); 1.5 (br, 4 H); 1.4 (br, 6 H) ppm. 13C NMR
(DMSO-d6): 159.0; 151.1; 131.3; 130.6; 122.2; 118.6; 116.3;
112.0; 67.6; 29.0; 28.7; 28.6; 25.5 ppm. IR: 3300–2200; 2920;
1604; 1539; 1492; 1456; 1236 cmꢀ1. HRMS: found 545.2928, calcd
545.2917 for C35H37N4O2.
2200; 2798; 1590; 1490; 1445; 1205 cmꢀ1
455.1483, calcd 455.1484 for C27H20N4O2 + Na.
. HRMS: found
4.1.2.7.
benzimidazole (22).
2,20-[1,2-Ethanediylbis(oxy-1,3-phenylene)]bis-1H-
Yield: 20%. Mp: >300 °C. 1H NMR
4.1.2.14.
benzimidazole (30).
2,20-[1,10-Decanediylbis(oxy-1,3-phenylene)]bis-1H-
Yield: 20%. Mp: 251–253 °C (decomposi-
(DMSO-d6): 8.0 (t, 2 H, J = 2 Hz); 7.9 (d, 2 H, J = 8 Hz); 7.7 (d, 2 H,
J = 6 Hz); 7.5 (m, 4 H); 7.2 (m, 6 H); 4.5 (s, 4 H) ppm. 13C NMR
(DMSO-d6): 158.7; 151.0; 143.7; 134.9; 131.5; 130.2; 122.6;
121.7; 119.1; 118.9; 116.4; 112.0; 111.3; 66.5 ppm. IR: 3200–
2500; 3049; 1589; 1490; 1446; 1400; 1233 cmꢀ1. HRMS: found
447.1812, calcd 447.1821 for C28H23N4O2.
tion). 1H NMR (DMSO-d6): 8.0 (t, 2 H, J = 2 Hz); 7.9 (d, 2 H, J = 8 Hz);
7.7 (d, 2 H, J = 6 Hz); 7.5 (m, 4 H); 7.2 (m, 6 H); 4.1 (t, 4 H, J = 6 Hz);
1.8 (m, 4 H, J = 6 Hz); 1.4 (br, 4 H); 1.3 (br, 8 H) ppm. 13C NMR
(DMSO-d6): 159.0; 151.1; 131.3; 130.1; 122.2; 118.6; 116.3;
114.6; 111.9; 67.58; 28.9; 28.7; 28.6; 25.5 ppm. IR: 3300–2200;
2928; 1601; 1460; 1236 cmꢀ1. HRMS: found 559.3085, calcd
559.3073 for C36H39N4O2.
4.1.2.8.
benzimidazole (23).
2,20-[1,3-Propanediylbis(oxy-1,3-phenylene)]bis-1H-
Yield: 20%. Mp: 285–286 °C (decomposi-
tion). 1H NMR (DMSO-d6): 8.0 (t, 2 H, J = 2 Hz); 7.9 (d, 2 H, J = 8 Hz);
7.7 (d, 2 H, J = 6 Hz); 7.5 (m, 4 H); 7.2 (m, 6 H); 4.3 (t, 4 H, J = 6 Hz);
2.3 (m, 2 H, J = 6 Hz) ppm. 13C NMR (DMSO-d6): 158.9; 151.0;
131.4; 130.2; 122.4; 118.8; 116.4; 111.9; 64.5; 28.7 ppm. IR:
3300-2200; 2960; 1590; 1489; 1455; 1235 cmꢀ1. HRMS: found
461.1989, calcd 461.1978 for C29H25N4O2.
4.1.2.15.
lene)]bis-1H-benzimidazole (31).
2,20-[1,2-Phenylenebis(methyleneoxy-1,4-pheny-
Yield: 60%. Mp: >300 °C.
1H NMR (DMSO-d6): 8.1 (d, 4 H, J = 8 Hz); 7.6–7.4 (m, 8 H); 7.2
(d, 4 H, J = 8 Hz); 7.2 (dd, 4 H, J = 6 Hz and 3 Hz); 5.4 (s, 4 H)
ppm. 13C NMR (DMSO-d6): 159.6; 151.2; 135.0; 128.6; 128.2;
128.0; 123.0; 121.8; 67.2 ppm. IR: 3300–2200; 2881; 1611;
1499; 1435; 1246; 1176 cmꢀ1. HRMS: found 545.1945, calc.
545.1953 for C34H26N4O2 + Na.
4.1.2.9.
benzimidazole (24).
2,20-[1,4-Butanediylbis(oxy-1,3-phenylene)]bis-1H-
Yield: 35%. Mp: 287–287 °C (decompo-
sition). 1H NMR (DMSO-d6): 8.0 (t, 2 H, J = 2 Hz); 7.9 (d, 2 H,
J = 8 Hz); 7.7 (d, 2 H, J = 6 Hz); 7.5 (m, 4 H); 7.2 (m, 6 H); 4.2 (t, 4
H, J = 6 Hz); 2.0 (t, 4 H, J = 6 Hz) ppm. 13C NMR (DMSO-d6):
159.0; 151.1; 131.3; 130.1; 122.2; 118.7; 116.3; 112.1; 67.4;
25.4 ppm. IR: 3300-2200; 2922; 1589; 1491; 1455; 1401;
1224 cmꢀ1. HRMS: found 475.2125, calc. 475.2134 for C30H27N4O2.
4.1.2.16.
lene)]bis-1H-benzimidazole (32).
244 °C (decomposition). 1H NMR (DMSO-d6): 8.1 (d,
2,20-[1,3-Phenylenebis(methyleneoxy-1,4-pheny-
Yield: 40%. Mp: 242–
H,
4
J = 8 Hz); 7.6 (br, 2 H); 7.5–7.4 (br, 8 H); 7.2–71 (m, 8 H); 5.2 (s,
4 H) ppm. 13C NMR (DMSO-d6): 159.7; 151.3; 137.1; 128.7;
128.0; 127.4; 127.0; 122.9; 121.8; 115.2; 69.3 ppm. IR: 3300–
2200; 3053; 1613; 1435; 1251 cmꢀ1. HRMS: found 545.1939, calc.
545.1953 for C34H26N4O2 + Na.
4.1.2.10.
benzimidazole (26).
2,20-[1,6-Hexanediylbis(oxy-1,3-phenylene)]bis-1H-
Yield: 40%. Mp: 294–296 °C (decomposi-
tion). 1H NMR (DMSO-d6): 8.0 (t, 2 H, J = 2 Hz); 7.9 (d, 2 H, J = 8 Hz);
7.7 (d, 2 H, J = 6 Hz); 7.5 (m, 4 H); 7.2 (m, 6 H); 4.1 (t, 4 H, J = 6 Hz);
1.8 (m, 4 H); 1.6 (m, 4 H) ppm. 13C NMR (DMSO-d6): 157.5; 149.6;
142.1; 133.4; 129.6; 128.5; 121.0; 120.1; 117.3; 117.1; 114.8;
110.4; 109.8; 66.0; 27.1; 23.8 ppm. IR: 3300–2200; 2929; 1603;
1541; 1492; 1457; 1364; 1237 cmꢀ1. HRMS: found 525.2272, calcd
525.2266 for C32H30N4O2 + Na.
4.1.2.17.
lene)]bis-1H-benzimidazole (33).
291 °C (decomposition). 1H NMR (DMSO-d6): 8.1 (d,
2,20-[1,4-Phenylenebis(methyleneoxy-1,4-pheny-
Yield: 70%. Mp: 289–
H,
4
J = 8 Hz); 7.6 (br, 2 H); 7.5 (s, 4 H); 7.5 (br, 2 H); 7.3 (d, 4 H,
J = 8 Hz); 7.2 (br, 4 H); 5.2 (s, 4H) ppm. 13C NMR (DMSO-d6):
159.7; 151.3; 143.9; 136.6; 135.0; 128.0; 127.8; 123.0; 122.1;
121.4; 118.5; 114.6; 111.0; 69.2 ppm. IR: 3300–2200; 3060;
1614; 1499; 1248 cmꢀ1. HRMS: found 545.1960, calcd 545.1953
for C34H26N4O2 + Na.
4.1.2.11.
benzimidazole (27).
2,20-[1,7-Heptanediylbis(oxy-1,3-phenylene)]bis-1H-
Yield: 35%. Mp: 258–259 °C (decomposi-
tion). 1H NMR (DMSO-d6): 8.0 (t, 2 H, J = 2 Hz); 7.9 (d, 2 H, J = 8 Hz);
7.7 (d, 2 H, J = 6 Hz); 7.5 (m, 4 H); 7.2 (m, 6 H); 4.1 (t, 4 H, J = 6 Hz);
1.8 (m, 4 H, J = 6 Hz); 1.5 (br, 6 H) ppm. 13C NMR (DMSO-d6): 159.1;
4.1.2.18.
lene)]bis-1H-benzimidazole (34).
1H NMR (DMSO-d6): 7.8 (s, 2 H); 7.8 (d, 2 H, J = 8 Hz); 7.7–7.4
2,20-[1,2-Phenylenebis(methyleneoxy-1,3-pheny-
Yield: 30%. Mp: >300 °C.

A. Mayence et al. / Bioorg. Med. Chem. 19 (2011) 7493–7500
7499
(m, 10 H); 7.2 (m, 6 H) 5.4 (s, 4 H) ppm. 13C NMR (DMSO-d6):
158.7; 151.0; 143.7; 135.1; 134.9; 131.5; 130.1; 128.7; 128.2;
122.6; 121.7; 119.1; 118.9; 116.5; 112.6; 111.3; 67.3 ppm. IR:
3300–2200; 2880; 1589; 1533; 1455; 1226 cmꢀ1. HRMS: found
545.1955, calcd 545.1953 for C34H26N4O2 + Na.
against the drug concentrations. IC50 values were calculated from
the sigmoidal inhibition curves. Metronidazole was used as refer-
ence drug.
4.2.3. Anti-Entamoeba histolytica activity69
Entamoeba histolytica Hk-9 trophozoites were cultivated in a
TYI-S-33 culture medium supplemented with 10% heat-inactivated
bovine serum and enriched with 3% vitamin-mix NCTC. A proce-
dure similar to that described for the determination of the anti-
Giardia intestinalis activity was developed. Metronidazole was also
used as reference drug.
4.1.2.19.
lene)]bis-1H-benzimidazole (35).
2,20-[1,3-Phenylenebis(methyleneoxy-1,3-pheny-
Yield: 20%. Mp: 253–
255 °C (decomposition). 1H NMR (DMSO-d6): 7.9 (s, 2 H); 7.8 (d,
2 H, J = 8 Hz); 7.7 (s, 1 H); 7.6-7.4 (m, 9 H); 7.2 (br, 4 H); 7.1 (dd,
2 H, J = 8 Hz and 2 Hz); 5.3 (s, 4 H) ppm. 13C NMR (DMSO-d6):
158.7; 151.0; 143.7; 137.2; 134.9; 131.5; 130.2; 128.7; 127.3;
126.9; 122.6; 121.7; 119.0; 118.9; 116.5; 112.5; 111.3; 69.3 ppm.
IR: 3300–2200; 2874; 1590; 1540; 1455; 1229 cmꢀ1. HRMS: found
545.1967, calcd 545.1953 for C34H26N4O2 + Na.
4.2.4. Anti-Trypanosoma activity
4.2.4.1.
Anti-Trypanosoma brucei rhodesiense activ-
Minimum Essential Medium–MEM—(50 L) supple-
ity70
.
l
mented with 25 mM HEPES, 1 g/L additional glucose, 1% MEM
non-essential amino acids (100ꢁ), 0.2 mM 2-mercaptoethanol,
1 mM Na-pyruvate, and 15% heat inactivated horse serum were
added to each well of a 96-well microtiter plate. Serial drug dilu-
tions of seven threefold dilution steps covering a range from 90
4.1.2.20.
lene)]bis-1H-benzimidazole (36).
2,20-[1,4-Phenylenebis(methyleneoxy-1,3-pheny-
Yield: 20%. Mp: >300 °C.
1H NMR (DMSO-d6): 7.9 (s, 2 H); 7.8 (d, 2 H, J = 8 Hz); 7.6 (br, 4
H); 7.6 (s, 4 H); 7.5 (t, 2 H, J = 8 Hz); 7.2 (dd, 4 H, J = 8 Hz and
3 Hz); 7.2 (dd, 2 H, J = 8 Hz and 3 Hz); 5.2 (s, 4 H) ppm. 13C NMR
(DMSO-d6): 158.7; 151.0; 136.6; 131.5; 130.1; 127.8; 122.2;
119.1; 116.6; 112.6; 69.2 ppm. IR: 3300–2200; 2870; 1601;
1491; 1455; 1219 cmꢀ1. HRMS: found 545.1960, calc. 545.1953
for C34H26N4O2 + Na.
to 0.123
l
g/mL were prepared. Then 2 ꢁ 103 bloodstream forms
of T. brucei rhodesiense STIB 900 in 50 lL were added to each well
and the plate was incubated at 37 °C under a 5% CO2 atmosphere
for 70 h. Ten microliters resazurin solution (12.5 mg resazurin dis-
solved in 100 mL phosphate-buffered saline) were then added to
each well and incubation continued for a further 2–4 h. Then the
plate was read with a Spectramax Gemini XS microplate fluorom-
eter (Molecular Devices Corporation, Sunnyvale, CA, USA) using an
excitation wavelength of 536 nm and an emission wavelength of
588 nm. Data were analyzed using the microplate reader software
Softmax Pro (Molecular Devices Corporation, Sunnyvale, CA, USA).
Melarsoprol was used as reference drug.
4.2. Biological assays
4.2.1. Cytotoxicity towards rat skeletal myoblasts, L-6 cells66,67
Assays were performed in 96-well microtiter plates, each well
containing 100 lL of RPMI 1640 medium supplemented with 1%
L
-glutamine (200 mM) and 10% fetal bovine serum, and 4 ꢁ 104
L6 cells. Serial drug dilutions of seven threefold dilution steps cov-
ering a range from 90 to 0.123
l
g/mL were prepared. After 72 h of
4.2.4.2. Anti-Trypanosoma cruzi activity71
blasts (L6 cells) were seeded in 96-well microtiter plates at 2000
cells/well in 100 L RPMI 1640 medium with 10% FBS and 2 mM
-glutamine. After 24 h the medium was replaced by 100 L per
.
Rat skeletal myo-
incubation, the plates were inspected under an inverted micro-
scope to assure growth of the controls and sterile conditions. Ten
microliters of the viability marker resazurin (12.5 mg resazurin
dissolved in 100 mL phosphate-buffered saline) were then added
to each well and the plates incubated for another 2 h. Then the
plates were read with a Spectramax Gemini XS microplate fluo-
rometer (Molecular Devices Corporation, Sunnyvale, CA, USA)
using an excitation wavelength of 536 nm and an emission wave-
length of 588 nm. Data were analyzed using the microplate reader
software Softmax Pro. Podophyllotoxin was the reference drug
used.
l
L
l
well containing 5000 trypomastigote forms of T. cruzi Tulahuen
strain C2C4 containing the b-galactosidase (Lac Z) gene. After
48 h, the medium was removed from the wells and replaced by
100
lL fresh medium with or without a serial drug dilution of se-
ven threefold dilution steps covering a range from 90 to 0.123
lg/
mL. After 96 h of incubation, the plates were inspected under an in-
verted microscope to assure growth of the controls and sterility.
Then, the substrate CPRG/Nonidet (50 lL) was added to all wells.
A color reaction developed within 2–6 h and could be read photo-
metrically at 540 nm. Data were transferred into the graphic pro-
gramme Softmax Pro, which calculated IC50 values. Benznidazole
was used as reference drug.
4.2.2. Anti-Giardia intestinalis activity68
One hundred microliters of a slightly modified TPS-1 culture
medium were added to wells of a 96-well microtiter plate. Serial
drug dilutions covering a range from 100 to 0.137
lg/mL were pre-
pared. Then a suspension (100 L) of the Giardia intestinalis G1
l
4.2.5. Anti-Leishmania donovani activity72
strain was added to each well leading to an initial Giardia density
of 5 ꢁ 105 microorganisms per mL. After 70 h of incubation at
37 °C in a nitrogen atmosphere, the plate was inspected under an
inverted microscope to assure growth of the controls and sterile
conditions. The medium was removed from all wells and replace
Amastigotes ofL. donovani strain MHOM-ET-67/L82 were grown
in axenic culture at 37 °C in SM medium at pH 5.4 supplemented
with l0% heat-inactivated fetal bovine serum under an atmosphere
of 5% CO2 in air. One hundred microliters of culture medium with
105 amastigotes from axenic culture with or without a serial drug
dilution were seeded in 96-well microtitre plates. Serial drug dilu-
by 100 lL of the viability marker resazurin (12.5 mg resazurin dis-
solved in 100 mL phosphate-buffered saline–PBS—further diluted
10ꢁ in PBS to afford a final concentration of 12.5 mg/L). Then the
plate was sealed again and incubated for a further 2 h at 37 °C.
The plate was finally read with a Spectramax Gemini XS microplate
fluorometer using an excitation wavelength of 536 nm and an
emission wavelength of 588 nm. Data were analyzed using the
software Softmax Pro (Molecular Devices Corporation, Sunnyvale,
CA, USA). Decrease of fluorescence (inhibition) was expressed as
percentage of the fluorescence of control cultures and plotted
tions covering a range from 90 to 0.123 lg/mL were prepared.
After 72 h of incubation the plates were inspected under an in-
verted microscope to assure growth of the controls and sterile con-
ditions. 10 lL of resazurin (12.5 mg resazurin dissolved in 100 mL
phosphate-buffered saline) were then added to each well and the
plates incubated for another 2 h. Then, the plates were read with
a Spectramax Gemini XS microplate fluorometer (Molecular De-
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length of 536 nm and an emission wavelength of 588 nm. Data

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Ci 3H-hypoxanthine was added to each
well. Cultures were incubated for a further 24 h before they were
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2
l
mol of that compound in a mixture of DMSO (1 mL) and HEPES
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l
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by mixing solution A (1 mL), solution B (1 mL), acetate buffer pH
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