Synthesis and biological evaluation of 2,3-dihydroimidazo[1,2-a] benzimidazole derivatives against Leishmania donovani and Trypanosoma cruzi

Article abstract of DOI:10.1016/j.ejmech.2014.07.038

A high-throughput (HTS) and high-content screening (HCS) campaign of a commercial library identified 2,3-dihydroimidazo[1,2-a]benzimidazole analogues as a novel class of anti-parasitic agents. A series of synthetic derivatives were evaluated for their in vitro anti-leishmanial and anti-trypanosomal activities against Leishmania donovani and Trypanosoma cruzi, which have been known as the causative parasites for visceral leishmaniasis and Chagas disease, respectively. In the case of Leishmania, the compounds were tested in both intracellular amastigote and extracellular promastigote assays. Compounds 4 and 24 showed promising anti-leishmanial activity against intracellular L. donovani (3.05 and 5.29 μM, respectively) and anti-trypanosomal activity against T. cruzi (1.10 and 2.10 μM, respectively) without serious cytotoxicity toward THP-1 and U2OS cell lines.

Full text of DOI:10.1016/j.ejmech.2014.07.038

Accepted Manuscript
Synthesis and biological evaluation of 2,3-dihydroimidazo[1,2-a]benzimidazole
derivatives against Leishmania donovani and Trypanosoma cruzi
Sangmi Oh, Sungbum Kim, Sunju Kong, Gyongseon Yang, Nakyung Lee, Dawoon
Han, Junghyun Goo, Jair L. Siqueira-Neto, Lucio H. Freitas-Junior, Rita Song
PII:
S0223-5234(14)00645-X
10.1016/j.ejmech.2014.07.038
EJMECH 7159
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 December 2013
Revised Date: 9 July 2014
Accepted Date: 10 July 2014
Please cite this article as: S. Oh, S. Kim, S. Kong, G. Yang, N. Lee, D. Han, J. Goo, J.L.
Siqueira-Neto, L.H. Freitas-Junior, R. Song, Synthesis and biological evaluation of 2,3-
dihydroimidazo[1,2-a]benzimidazole derivatives against Leishmania donovani and Trypanosoma cruzi,
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.07.038.
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ACCEPTED MANUSCRIPT
Graphical Abstract
2,3-Dihydroimidazo[1,2-a]benzimidazole derivatives were synthesized and evaluated against
Leishmania donovani and Trypanosoma cruzi.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of 2,3-dihydroimidazo[1,2-
a]benzimidazole derivatives against Leishmania donovani and
Trypanosoma cruzi
Sangmi Oh,a Sungbum Kim,a Sunju Kong,a Gyongseon Yang,b Nakyung Lee,b Dawoon Han,b
Junghyun Goo,b Jair L. Siqueira-Neto,b,† Lucio H. Freitas-Junior,b,‡ Rita Songa,*
a Medicinal Chemistry Group, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si,
Gyeonggi-do 463-400, Korea
b Center for Neglected Diseases Drug Discovery (CND3), Institut Pasteur Korea, 696 Sampyeong-dong,
Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Korea
† Present address: University of California, San Francisco, USA
‡ Present address: Laboratório Nacional de Biociências (LNBio), Brazil
Corresponding author. Tel.: +82-31-8018-8230; fax: +82-31-8018-8014; e-mail: rsong@ip-
korea.org
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Abstract
A high-throughput (HTS) and high-content screening (HCS) campaign of a commercial library
identified 2,3-dihydroimidazo[1,2-a]benzimidazole analogues as a novel class of anti-parasitic agents.
A series of synthetic derivatives were evaluated for their in vitro anti-leishmanial and anti-
trypanosomal activities against L. donovani and T. cruzi, which have been known as the causative
parasites for visceral leishmaniasis and Chagas disease, respectively. In the case of Leishmania, the
compounds were tested in both intracellular amastigote and extracellular promastigote assays.
Compounds 4 and 24 showed promising anti-leishmanial activity against intracellular L. donovani
(3.05 and 5.29 ꢀM, respectively) and anti-trypanosomal activity against T. cruzi (1.10 and 2.10 ꢀM,
respectively) without serious cytotoxicity toward THP-1 and U2OS cell lines.
Keywords:
Leishmaniasis
Chagas disease
Anti-parasitic activity
2,3-Dihydroimidazo[1,2-a]benzimidazole
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1. Introduction
The kinetoplastids are a group of flagella protozoan parasites which contain subcellular structure
known as the kinetoplast as the major distinguishing feature of parasites [1]. Three distinct
kinetoplastids are known to cause different human diseases: leishmaniasis caused by Leishmania,
Chagas disease caused by Trypanosoma cruzi (T. cruzi), and sleeping sickness caused by
Trypanosoma brucei. Among them, leishmaniasis is the most prevalent in the world found in about 88
countries affecting more than 12 million people worldwide [2], and visceral leishmaniasis caused by
Leishmania donovani (L. donovani) is the most severe form of three types of leishmaniasis being
mortal if left untreated [3,4]. In the case of Chagas disease, it has been found mainly in endemic areas
of Latin American countries, and over 8 million people are estimated to be infected with T. cruzi [4].
In spite of high unmet medical needs, the molecular basis and pathway for the diseases caused by the
kinetoplastids have been little elucidated and only very few target proteins have been reported [5].
Treatment options for visceral leishmaniasis are including the use of pentavalent antimonials,
amphotericin B, paromomycin, and miltefosine. Although several drugs are available in the clinic for
the treatment of leishmaniasis and Chagas disease, most of drugs have been faced with many
difficulties such as low efficacy, severe side effects, inconvenient administration, and emergence of
drug resistance [6]. For example, miltefosine is the only oral drug for the treatment of resistant
leishmaniasis but this drug is teratogenic and causes extremely severe gastrointestinal side effects [7].
Likewise, nifurtimox and benznidazole have been used for the treatment of Chagas disease, but both
drugs have also been reported to show limited effectiveness for the treatment of chronic patients and
undesirable side effects [8]. Therefore, identification and optimization of novel anti-parasitic
compounds with new mechanisms of action are urgently needed to overcome the current situation and
eventually eradicate these fatal diseases.
For the discovery of novel anti-parasitic compounds, drugable targets of the pathogenic parasite
have not been fully exploited so that traditional target-based drug discovery approaches become more
likely to be inefficient. Only a few targets such as topoisomerases [9], kinetoplast [10], trypanothione
reductase (TryR) [11], and fatty acid and sterol biosynthesis pathways [12] have been reported.
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Nalidixic acid, which is the first synthetic quinolone antibiotics, has been known to have inhibitory
effect on the proliferation of kinetoplastids by the inhibition of topoisomerase II [13,14]. On the other
hand, other quinolone antibacterial compounds have been reported to induce a defect on kinetoplast
segregation, which was not due to the topoisomerase II inhibition mechanism [15]. In addition, 2-
iminobenzimidazole derivatives have been reported as a new class of TryR inhibitors [16,17], and
numerous antifungal azole compounds have shown to effectively inhibit the ergosterol biosynthesis
which is a key process for the formation of protozoal membrane [18,19]. However, no success in the
drug discovery has been achieved so far based on the targets above mentioned, which have attracted
more attention for the validation of these targets and exploitation of novel targets. Target-free cell-
based assays have been widely used, as they bypass the need for a known target and have the potential
to lead to the discovery of unknown target. For instance, Genomic Institute of the Novartis Research
Foundation (GNF) and some laboratories supported by the Special Programme for Research and
Training in Tropical Disease (TDR/WHO) have performed phenotypic screening to identify novel
anti-parasitic compounds [20]. In this context, we previously reported a HTS and high-content
screening (HCS) of commercially available chemical libraries against L. donovani [21]. Here, we
report structure-activity relationship (SAR) studies of 2,3-dihydroimidazo[1,2-a]benzimidazole
derivatives identified through phenotypic screening of 200,000 chemical library against L. donovani
and T. cruzi.
2. Result and discussion
2.1. Synthesis of 2,3-dihydroimidazo[1,2-a]benzimidazole derivatives
General synthetic route for 2,3-dihydroimidazo[1,2-a]benzimidazole derivatives was depicted in
Scheme 1. For the search of compounds with improved anti-leishmanial and anti-trypanosomal
activities, SAR studies of 2,3-dihydroimidazo[1,2-a]benzimidazole derivatives were conducted with
the modification of three different parts: tricyclic ring, substitution in benzene ring (R1 and R2), and
substitution in imidazole nitrogen (R3). Intermediates 1c having benzimidazolone bicyclic ring were
obtained by N-alkylation of aniline moiety of commercially available 2-nitroaniline derivatives (1a)
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and then nitro reduction (1b), followed by intramolecular ring formation in the presence of urea [22].
These benzimidazolone compounds were chlorinated with POCl3 and catalytic amount of HCl to give
N-alkylated 2-chlorobenzimidazole intermediates 1d [23]. Alternatively, in the case of compounds
without substituent in the benzimidazole ring (R1 = R2 = H), 1d was easily synthesized by reacting
commercially available 2-chlorobenzimidazole with various alkyl bromide in the presence of organic
base, DIPEA. Compounds 1e, key intermediates for the tricyclic ring formation, were obtained by
nucleophilic substitution with ethanolamine under microwave irradiation. After alcohol group was
converted to chloride under POCl3 condition, intramolecular cyclization in the presence of TEA was
performed to yield final compounds 4 and 7−33 [24,25].
Synthesis of compound 6 containing a phenacyl substituent in benzimidazole nitrogen was started
from the reaction of 2-chlorobenzimidazole with 2-bromo-4’-tertbutylacetophenone in the presence of
base to yield 2a (see Route I in Scheme 2). However, the reaction of N-phenacylated 2-
chlorobenzimidazole 2a with ethanolamine was failed to produce 2c, a key intermediate for the final
tricyclic compound. Due to the harsh reaction condition using microwave irradiation at 200 °C,
tricyclic byproduct 2 was formed via intramolecular enamine formation. Therefore, 2c was obtained
by substitution reaction of 2-chlorobenzimidazole with ethanolamine and then subsequent N-
alkylation with phenacyl bromide (see Route II in Scheme 2). Chlorination of alcohol moiety (2d)
and intramolecular substitution reaction gave the final tricyclic compound 6. All final compounds
newly synthesized (6−12, 14−18, 21−33) were characterized by melting point, low resolution ESI
mass and 1H NMR.
2.2. Biological evaluation against L. donovani
For assessing anti-leishmanial activity of the compounds, L. donovani in two different stages of the
life cycle was used. Intracellular amastigote form is specifically found in the host cell, and the other
extracellular promastigote form is predominately found in the insect vector. Promastigotes or axenic
amastigotes have been generally used in the screening for the discovery of anti-leishmanial drug
candidates because of easier adaptation to the high-throughput assay format. In this study, newly
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synthesized 2,3-dihydroimidazo[1,2-a]benzimidazole derivatives were tested in in vitro intracellular
amastigote assays as well as extracellular promastigote, because amastigotes are substantially
different from promastigotes in a molecular perspective and known to be more physiologically
relevant parasite stage to evaluate anti-leishmanial activity of the compounds.
The amastigote assay was established by using metacyclic promastigote of L. donovani to infect
differentiated THP-1 macrophages. Amastigotes transformed from metacyclic promastigotes were
allowed to multiply inside host macrophages for 24 h and then the compounds were added. The
quantification of the infection ratio was done 3 days after compound treatment. Fluorescent images of
DNA-stained cells were acquired using automated confocal microscopy and customized software
developed for image analysis allowed to assess diverse information such as number of cells per well,
number of parasites per cell, infection ratio, and cell cytoplasmic area. Cytotoxicity against host cells
was also easily determined by counting cell number in the same assay. Viability of promastigotes were
independently accessed by fluorometric measurement of resazurin reduction after the treatment of
extracellular promastigotes with each compound. In vitro anti-leishmanial activity of the synthesized
compounds was summarized in Table 1 and 2.
As shown in Table 1, compound 4 with para-tertbutylphenyl moiety at R3 position was found to be
the most active among all the synthesized compounds with EC50 value of 3.05 µM which is
comparable to that of the reference drug, miltefosine (4.83 µM). In addition, compound 4 didn’t show
any cytotoxicity against host macrophages (CC50 > 50 µM) resulting in high selectivity index (SI >
16.4) while that of miltefosine was 3.91. 2,3-Dihydroimidazo[1,2-a]benzimidazole tricyclic ring is
essential for anti-leishmanial activity because when this tricyclic ring was changed to 6-membered
tetrahydropyrimidine conjugated tricyclic ring, 2,3,4,10-tetrahydropyrimido[1,2-a]benzimidazole (5)
or 2,3-hydroimidazo[1,2-a]benzimidazol-2-one (3), the anti-amastigote activity was found to be less
(12.5 µM) or disappeared (> 50 µM), respectively. Phenyl group at R3 position is also critical for anti-
leishmanial activity because phenacyl-substituted compound 6 and benzyl-substituted compound 8
showed decreased activities (39.4 and 25.3 µM, respectively) compared to compound 4. In the case of
compound 7 bearing phenoxymethyl substituent in R3 position showed not only low anti-amastigote
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activity (27.4 µM) but also cytotoxicity toward host macrophages in the similar concentration (33.1
µM).
A series of compounds described in Table 2 were synthesized to determine substituent effects of
benzene included in the tricyclic ring (R1 and R2) and phenyl in R3 position. Different substituents at
R1 and R2 such as fluoro (9), chloro (10), or methyl (11 and 12) instead of hydrogen (4) were not
effective to improve anti-leishmanial activity. In addition, some of the compounds (10 and 11) were
toxic toward host macrophages even though they maintained good anti-promastigote activities (2.22
and 1.80 µM, respectively). Only fluoro-substituted compound 9 exhibited moderate activity (14.0
µM) without cytotoxicity.
Concerning substituent effect at R3 position, potency and selectivity index were influenced by the
position of the substituents and physicochemical properties such as size, electronic effect and
lipophilicity. A significant decrease of anti-leishmanial activity was observed when tertbutyl group was
changed from para to meta position (18) of the phenyl ring in R3. The presence of hydrophobic and
sterically hindered substituent in para position such as tertbutyl (4), cyclohexyl (16), or phenyl (24)
was also necessary to retain anti-parasitic activities, while the compounds with hydrogen (13), ethyl
(14), and isopropyl (15) as well as other compounds containing electron withdrawing group at para
position (19−23) showed significant loss of activity. In the case of compound 24 bearing
biphenylmethyl substituent, the anti-amastigote activity (5.29 µM) was comparable to that of
miltefosine and selectivity index (7.5) was superior to miltefosine. To study substituent effects on
biphenyl ring in R3, compounds 25−31 were prepared and evaluated their biological activities.
However, most of compounds didn’t show any improvement in terms of activity and cytotoxicity.
New compounds showing promising anti-amastigote activities in cell-based assay also revealed
good anti-promastigote activities in extracellular promastigote assay. In particular, compounds 4 and
24 exhibiting anti-amastigote activities comparable to that of miltefosine in cell-based assay showed
better anti-promastigote activities (1.25 and 1.48 µM, respectively) than that of miltefosine (11.1 µM).
These results led us to speculate that the major target of these compounds mmight be in common
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pathway of both stages of parasites, which allows of further target identification approaches using
extracellular promastigotes more prone to handle.
Automated confocal microscopic images for the representative compounds 4 and 15 are shown in
Fig. 1. In the case of compound 4, no parasite was observed inside of host THP-1 cells as seen in
reference drugs, miltefosine and amphotericin B, at the concentration of 12.5 µM. However, most of
parasites still remained at the same concentration when they were treated with inactive compound 15.
Confocal images clearly show that no serious decrease of cell number was detected when THP-1 cells
were treated with both compounds 4 and 15. The calculated percentage of infection based on the
EC100 of amphotericin B, which is the first effective concentration to reach 100 % activity determined
as 4 µM and 1 % DMSO (0 % activity as maximum infection ratio) was normalized for the activity.
Cytotoxicity was determined by percentage of cell number normalized against DMSO control.
2.3. Biological evaluation against T. cruzi
All compounds tested against L. donovani were also phenotypically screened for their in vitro anti-
parasitic activity against T. cruzi. For the image-based assay, U2OS cells were used as host cells and
infected with tissue culture trypomastigotes (TCT) of T. cruzi. In the field of drug discovery for
Chagas disease, colorimetric assay using recombinant T. cruzi modified to express fluorescent β-
galactosidase is one of the popular HTS methods to find active compounds [26]. However, wild-type
T. cruzi was used in this study to improve the relevance between in vitro assay and real disease state,
avoiding using genetically modified parasites [27].
In vitro anti-T. cruzi activity of the synthesized compounds was described in Table 3. Structure-
activity relationship of 2,3-dihydroimidazo[1,2-a]benzimidazole derivatives against T. cruzi was very
similar to that against L. donovani. The most active compound 4 in L. donovani assay also showed the
best activity toward T. cruzi with an EC50 value of 1.10 µM and selectivity index of 33.2. Other
compounds such as 6-membered tetrahydropyrimidine-conjugated tricyclic compound (5), fluoro-
substituted compound in R1 position (9), and biphenyl-substituted compound in R3 position (24),
which showed moderate anti-leishmanial activities (12.5, 14.0, and 5.29 µM , respectively), also
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exhibited good anti-trypanosomal activities against T. cruzi (3.09, 8.24, and 2.10 µM, respectively).
Moreover, their cytotoxicity determined in the same assay using U2OS cell lines were very low
leading to high selectivity indexes.
The representative confocal images of T. cruzi infected cells during the assay were shown in Fig. 2.
DNA of host cells and parasites stained by fluorescent dye was very clearly shown to be able to
differentiate each other so that the anti-parasitic activity was determined by quantifying parasitemia in
the presence of each compound. Benznidazole was used as a reference drug for positive control at the
concentration of 400 µM, while 1 % of DMSO was used as a negative control. Even though the
parasites were completely disappeared from the host cells at the concentration of 400 µM, some
parasites still remained when 12.5 µM of benznidazole was treated (EC50 = 20.7 µM). However, in the
case of compound 4 which showed the best anti-parasitic activity in this assay (EC50 = 1.10 µM), no
parasite was observed in the host cells even at the concentration of 6.25 µM. Compound 4 showed
dose-dependent clearance of the parasites from the host cells.
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3. Conclusions
A series of 2,3-dihydroimidazo[1,2-a]benzimidazole derivatives were identified as a novel class of
anti-parasitic agents. The synthetic analogues were evaluated for their in vitro anti-leishmanial and
anti-trypanosomal activities against L. donovani and T. cruzi, respectively, which have been known as
the causative protozoa for visceral leishmaniasis and Chagas disease. In the case of Leishmania, the
compounds were tested in both intracellular amastigote and extracellular promastigote assays.
Analysis of the structure-activity relationship revealed that 2,3-dihydroimidazo[1,2-a]benzimidazole
tricyclic ring and sterically hindered hydrophobic substituent in para-position of benzyl group played
a critical role to show anti-parasitic activity and selectivity. Compounds 4 and 24 showed promising
anti-parasitic activities against L. donovani and T. cruzi without serious cytotoxicity toward human
THP-1 and U2OS cell lines resulting in high selectivity indexes. A follow-up studies including
optimization of anti-parasitic activity, evaluation of in vivo efficacy, and elucidation of mechanism of
action are currently underway.
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4. Experimental section
4.1. Chemistry
1H and 13C NMR spectra were recorded on a Varian High Resolution FT-NMR Spectrometer-400, and
chemical shifts were measured in ppm relative to internal tetramethylsilane (TMS) standard or
specific solvent signal. Routine mass analyses were performed on Waters LC/MS ZQ2000 system
equipped with a reverse phase column (XBridgeTM C18 × 3.5 ꢀm, 50 × 2.1 mm) and photodiode array
detector using electron spray ionization (ESI). Melting point analysis was performed on BUCHI
Melting point M-565. Most of reagents used in the synthetic procedure were purchased from Sigma-
Aldrich, Alfa Aesar, and TCI. The progress of reaction was monitored using thin-layer
chromatography (TLC) (silica gel 60 F254, 0.25 mm), and the products were visualized by UV light
(254 and 365 nm) or by ninhydrin staining followed by heating. Silica gel 60 (0.040–0.063 mm) used
in flash column chromatography was purchased from Merck. Other solvents and organic reagents
were purchased from commercial venders and used without further purification unless otherwise
mentioned.
4.1.1. Synthetic procedure for N-alkylation (1a)
To a stirred mixture of 2-nitroanilne compound (1.0 eq.) and K2CO3 (1.3 eq.) in dimethylformamide
was added dropwise R3CH2Br (1.3 eq.). The reaction mixture was heated to 120 ˚C and stirred for 4 h.
After the reaction was completed, the reaction mixture was diluted with ethyl acetate and washed with
water. The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude product was
purified by flash column chromatography (ethyl acetate / hexane) to give desired product 1a.
4.1.2. Synthetic procedure for nitro reduction (1b)
To a stirred solution of 1a (1.0 eq.) in MeOH was added 10 % Pd/C (10 % w/w). The reaction mixture
was stirred for 3 h at room temperature under H2 environment. After the reaction was completed, the
reaction mixture was filtered and then concentrated in vacuo. The crude product was purified by flash
column chromatography (ethyl acetate / hexane) to give desired product 1b.
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4.1.3. Synthetic procedure for bicyclic ring formation (1c)
To a stirred solution of 1b (1.0 eq.) in dimethylformamide was added urea (2.0 eq.). The reaction
mixture was heated to 200 ˚C and stirred for 30 min. After the reaction was completed, the reaction
mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with
brine, dried over Na2SO4 and concentrated in vacuo. The crude product was suspended in hexane and
filtered to give desired product 1c.
4.1.4. Synthetic procedure for chlorination (1d)
To a stirred solution of 1c (1.0 eq.) and POCl3 (10 eq.) was added a few drops of conc. HCl. The
reaction mixture was heated to 150 ˚C and stirred for 3 h. After the reaction was completed, the
reaction mixture was poured into ice water, neutralized with 1N aq. NaOH and extracted with ethyl
acetate. The organic layer was washed with water, dried over Na2SO4 and concentrated in vacuo to
give crude product 1d.
Alternatively, to a stirred mixture of 2-chlorobenzimidazole (1.0 eq.) and R3CH2Br (1.0 eq.) in
dimethylformamide was added N,N-diisopropylethylamine (1.5 eq.). The reaction mixture was heated
to 110 ˚C and stirred for 12 h. After the reaction was completed, the reaction mixture was cooled
down to room temperature, diluted with ethyl acetate and washed with water. The organic layer was
dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by flash
column chromatography (ethyl acetate / hexane) to give desired product 1d.
4.1.5. Synthetic procedure for substitution reaction (1e)
A mixture of 1d (1.0 eq.) and ethanolamine (10 eq.) was heated to 150 ˚C for 30 min by use of
microwave irradiation. After the reaction was completed, the reaction mixture was cooled down to
room temperature. The precipitated solid was filtered and washed with diethyl ether. The crude
product was purified by flash column chromatography (MeOH / methylene chloride) to give desired
product 1e.
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4.1.6. Synthetic procedure for chlorination (1f)
A mixed solution of 1e (1.0 eq.) and POCl3 (10 eq.) was heated to 110 ˚C and stirred for 2 h. After the
reaction was completed, the reaction mixture was cooled down to 0 ˚C, quenched with ice water and
extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO4 and concentrated in
vacuo. The crude product was purified by flash column chromatography (MeOH / methylene chloride)
to give desired product 1f.
4.1.7. Synthetic procedure for intramolecular cyclization for 2,3-dihydroimidazo[1,2-a]benzimidazole
derivatives (7−12, 14−19, 22−32)
To a stirred solution of 1f (1.0 eq.) in toluene was added triethylamine (1.0 eq.). The reaction mixture
was heated to 110 ˚C and stirred for 8 h. After the reaction was completed, the reaction mixture was
cooled down to room temperature and diluted with ethyl acetate. This organic layer was washed with
water, dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by
flash column chromatography (MeOH / methylene chloride) to give desired product.
4.1.7.1. 9-(4-tert-Butylphenoxyethyl)-2,3-dihydroimidazo[1,2-a]benzimidazole (7)
Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.26 (s, 1H), 7.24 (s, 1H), 7.07–7.05 (m, 1H), 6.98–6.96 (m,
2H), 6.79 (d, J = 8.8 Hz, 2H), 6.74–6.72 (m, 1H), 4.31−4.25 (m, 4H), 4.20 (t, J = 5.2 Hz, 2H), 3.89 (t,
J = 8.4 Hz, 2H), 1.26 (s, 9H); LRMS (electrospray) m/z (M+H)+ 336.
4.1.7.2. 9-(4-(tert-Butyl)phenethyl)-2,9-dihydro-3H-benzo[d]imidazo[1,2-a]imidazole (8)
1
Yellow oil; H NMR (400 MHz, CD3OD) δ 7.26 (d, J = 6.4 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H),
6.95−6.91 (m, 1H), 6.87 (t, J = 7.8 Hz, 1H), 6.82 (d, J = 6.4 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 4.16 (t,
J = 8.0 Hz, 2H), 3.97 (t, J = 7.2 Hz, 2H), 3.89 (t, J = 8.4 Hz, 2H), 2.97 (t, J = 7.4 Hz, 2H), 1.26 (s, 9H);
LRMS (electrospray) m/z (M+H)+ 320.
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4.1.7.3. 9-(4-(tert-Butyl)benzyl)-6-fluoro-2,9-dihydro-3H-benzo[d]imidazo[1,2-a]imidazole (9)
Brown solid; mp = 263.8 ˚C; 1H NMR (400 MHz, CDCl3) δ 7.40–7.38 (m, 6H), 7.12 (s, 1H), 5.34 (s,
2H), 4.33 (s, 4H), 1.25 (s, 9H); LRMS (electrospray) m/z (M+H)+ 324.
4.1.7.4. 9-(4-(tert-Butyl)benzyl)-6-chloro-3,9-dihydro-2H-benzo[d]imidazo[1,2-a]imidazole (10)
1
Pale brown solid; H NMR (400 MHz, CD3OD) δ 7.62–7.59 (m, 1H), 7.40–7.32 (m, 3H), 7.24–7.19
(m, 1H), 7.01–6.99 (m, 2H), 5.44 (s, 2H), 4.28 (t, J = 8.5 Hz, 2H), 4.05 (t, J = 8.5 Hz, 2H), 1.27 (s,
9H); LRMS (electrospray) m/z (M+H)+ 341.
4.1.7.5. 9-(4-(tert-Butyl)benzyl)-6-methyl-2,9-dihydro-3H-benzo[d]imidazo[1,2-a]imidazole (11)
1
Brown solid; H NMR (400 MHz, CDCl3) δ 7.34 (m, 5H), 7.25 (d, 1H), 7.06 (d, 1H), 5.27 (s, 2H),
4.32 (m, 4H), 2.37 (s, 3H), 1.24 (s, 9H); LRMS (electrospray) m/z (M+H)+ 320.
4.1.7.6. 9-(4-(tert-Butyl)benzyl)-6,7-dimethyl-3,9-dihydro-2H-benzo[d]imidazo[1,2-a]imidazole (12)
1
Pale yellow solid; H NMR (400 MHz, CD3OD) δ 7.28–7.22 (m, 4H), 7.12–7.09 (m, 1H), 6.82–6.80
(m, 1H), 5.31 (s, 2H), 4.07 (t, J = 8.5 Hz, 2H), 3.69 (t, J = 8.5 Hz, 2H), 2.31 (s, 3H), 2.26 (s, 3H), 1.35
(s, 9H); LRMS (electrospray) m/z (M+H)+ 334.
4.1.7.7. 9-(4-Ethylbenzyl)-2,3-dihydroimidazo[1,2-a]benzimidazole (14)
Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.31 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 7.02 (t, J
= 7.6 Hz, 1H), 6.95 (t, J = 7.8 Hz, 1H), 6.85 (d, J = 4.8 Hz, 1H), 6.83 (d, J = 4.4 Hz, 1H), 5.13 (s, 2H),
4.35 (t, J = 8.4 Hz, 2H), 4.01 (t, J = 8.4 Hz, 2H), 2.60 (q, J = 7.5 Hz, 2H), 1.19 (t, J = 5.6 Hz, 3H);
LRMS (electrospray) m/z (M+H)+ 278.
4.1.7.8. 9-(4-Isopropylbenzyl)-2,9-dihydro-3H-benzo[d]imidazo[1,2-a]imidazole (15)
1
White solid; mp = 133.2 ˚C; H NMR (400 MHz, CD3OD) δ 7.44 (d, J = 8.4 Hz, 1H), 7.39−7.31 (m,
3H), 7.28−7.22 (m, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.01 (s, 2H), 4.79−4.75 (m, 2H), 4.43 (t, J = 8.6 Hz,
14

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2H), 2.85−2.82 (m, 1H), 1.18 (d, J = 6.8 Hz, 6H); LRMS (electrospray) m/z (M+H)+ 292.
4.1.7.9. 9-(4-Cyclohexylbenzyl)-2,9-dihydro-3H-benzo[d]imidazo[1,2-a]imidazole (16)
1
Yellow oil; H NMR (400 MHz, CD3OD) δ 7.23 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 8.0 Hz, 2H),
6.97−6.93 (m, 1H), 6.89−6.80 (m, 3H), 4.94 (s, 2H), 4.20 (t, J = 7.8 Hz, 2H), 3.92 (t, J = 8.0 Hz, 2H),
2.45 (brs, 1H), 1.80−1.71 (m, 5H), 1.45−1.24 (m, 5H); LRMS (electrospray) m/z (M+H)+ 332.
4.1.7.10. 9-(3,5-Dimethylbenzyl)-2,9-dihydro-3H-benzo[d]imidazo[1,2-a]imidazole (17)
Yellow solid; mp = 274.6 ˚C; 1H NMR (400 MHz, CD3OD) δ 7.02−6.85 (m, 7H), 4.94 (s, 2H), 4.25 (t,
J = 8.6 Hz, 2H), 4.00 (t, J = 8.4 Hz, 2H), 2.25 (s, 6H); LRMS (electrospray) m/z (M+H)+ 278.
4.1.7.11. 9-(3-(tert-Butyl)benzyl)-2,9-dihydro-3H-benzo[d]imidazo[1,2-a]imidazole (18)
White solid; mp = 248.6 ꢀC; 1H NMR (400 MHz, CDCl3) δ 7.34 (m, 5H), 7.25 (d, 1H), 7.06 (d, 1H),
7.17 (m, 3H), 5.27 (s, 2H), 4.32 (m, 4H), 1.24 (s, 9H); LRMS (electrospray) m/z (M+H)+ 306.
4.1.7.12. 9-(4-(Methylsulfonyl)benzyl)-2,9-dihydro-3H-benzo[d]imidazo[1,2-a]imidazole (19)
Yellow solid; mp = 149.5 ˚C; 1H NMR (400 MHz, CD3OD) δ 7.93 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.0
Hz, 2H), 7.04−6.87 (m, 4H), 5.15 (s, 2H), 4.24 (t, J = 8.2 Hz, 2H), 4.01 (t, J = 8.6 Hz, 2H), 3.09 (s,
3H); LRMS (electrospray) m/z (M+H)+ 328.
4.1.7.13. 9-(4-(Trifluoromethoxy)benzyl)-3,9-dihydro-2H-benzo[d]imidazo[1,2-a]imidazole (22)
White solid; mp = 183.9 ˚C;1H NMR (400 MHz, CD3OD) δ 7.93–7.91 (m, 2H), 7.48–7.46 (m, 2H),
7.43–7.41 (m, 1H), 7.38–7.34 (m, 2H), 7.29–7.27 (m, 2H), 5.43 (s, 2H), 4.45 (t, J = 8.5 Hz, 2H), 4.10
(t, J = 8.5 Hz, 2H). LRMS (electrospray) m/z (M+H)+ 334.
4.1.7.14. 9-(4-Cyanobenzyl)-3,9-dihydro-2H-benzo[d]imidazo[1,2-a]imidazole (23)
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1
White solid; mp = 190.0 ˚C; H NMR (400 MHz, CDCl3) δ 7.45–7.42 (m, 2H), 7.26–7.23 (m, 2H),
6.97–6.84 (m, 2H), 6.75–6.65 (m, 2H), 4.95 (s, 2H), 4.29 (t, J = 8.5 Hz, 2H), 3.91 (t, J = 8.5 Hz, 2H).
LRMS (electrospray) m/z (M+H)+ 275.
4.1.7.15. 9-(4-Phenylbenzyl)-2,3-dihydroimidazo[1,2-a]benzimidazole (24)
1
Yellow solid; mp = 139.5 ˚C; H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 8.4 Hz, 4H), 7.44–7.40 (m,
4H), 7.33 (t, J = 7.4 Hz, 1H), 6.96 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 7.8 Hz, 1H), 6.76 (d, J = 7.6 Hz,
2H), 5.30 (s, 2H), 4.29 (t, J = 8.6 Hz, 2H), 3.94 (t, J = 8.4 Hz, 2H); LRMS (electrospray) m/z (M+H)+
326.
4.1.7.16. 9-((2',6'-Difluoro-[1,1'-biphenyl]-4-yl)methyl)-2,9-dihydro-3H-benzo[d]imidazo[1,2-
a]imidazole (25)
Brown solid; mp = 210.4 ˚C; 1H NMR (400 MHz, CD3OD) δ 7.43−7.36 (m, 5H), 7.24 (d, J = 7.6 Hz,
1H), 7.13−7.08 (m, 3H), 7.03 (t, J = 7.6 Hz, 2H), 5.18 (s, 2H), 4.25 (t, J = 8.6 Hz, 2H), 3.04 (t, J = 6.4
Hz, 2H); LRMS (electrospray) m/z (M+H)+ 362.
4.1.7.17.
9-((4'-Fluoro-[1,1'-biphenyl]-4-yl)methyl)-3,9-dihydro-2H-benzo[d]imidazo[1,2-
a]imidazole (26)
Brown solid; mp = 199.5 ˚C; 1H NMR (400 MHz, CD3OD) δ 7.64–7.48 (m, 4H), 7.40 (d, J = 8.1 Hz,
2H), 7.12 (t, J = 8.7 Hz, 2H), 7.02 (dd, J = 6.9 and 2.2 Hz, 1H), 6.94 (d, J = 6.2 Hz, 3H), 5.08 (s, 2H),
4.26 (t, J = 8.5 Hz, 2H), 4.02 (t, J = 8.5 Hz, 2H); LRMS (electrospray) m/z (M+H)+ 344.
4.1.7.18.
9-((4'-Methyl-[1,1'-biphenyl]-4-yl)methyl)-3,9-dihydro-2H-benzo[d]imidazo[1,2-
a]imidazole (27)
1
Pale yellow solid; mp = 202.1 ˚C; H NMR (400 MHz, CD3OD) δ 7.56–7.54 (m, 2H), 7.47–7.42 (m,
2H), 7.40–7.38 (m, 2H), 7.28–7.23 (m, 2H), 7.12–7.01 (m, 1H), 6.99–6.95 (m, 3H), 5.09 (s, 2H), 4.26
(t, J = 8.5 Hz, 2H), 4.13 (t, J = 8.5 Hz, 2H), 2.34 (s, 3H); LRMS (electrospray) m/z (M+H)+ 340.
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4.1.7.19.
9-((4'-Methyl-[1,1'-biphenyl]-4-yl)methyl)-3,9-dihydro-2H-benzo[d]imidazo[1,2-
a]imidazole (28)
Pale brown solid; mp = 202.1 ˚C; 1H NMR (400 MHz, CD3OD) δ 7.77 (d, J = 8.3 Hz, 2H), 7.71 (d, J
= 8.3 Hz, 2H), 7.65 (s, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.10–7.00 (m, 1H), 7.00–6.92 (m, 3H), 5.14 (s,
2H), 4.28 (t, J = 8.5 Hz, 2H), 4.07 (t, J = 8.5 Hz, 2H); LRMS (electrospray) m/z (M+H)+ 394.
4.1.7.20. 9-((4'-Chloro-[1,1'-biphenyl]-4-yl)methyl)-3,9-dihydro-2H-benzo[d]imidazo[1,2-
a]imidazole (29)
1
Pale brown solid; mp = 198.6 ˚C; H NMR (400 MHz, CD3OD) δ 7.72–7.65 (m, 2H), 7.59–7.49 (m,
3H), 7.47–7.30 (m, 4H), 7.02 (s, 1H), 6.72 (d, J = 7.0 Hz, 2H), 5.24 (s, 2H), 4.13–4.09 (m, 2H), 3.73–
3.69 (m, 2H); LRMS (electrospray) m/z (M+H)+ 361.
4.1.7.21. 9-((4'-Methoxy-[1,1'-biphenyl]-4-yl)methyl)-3,9-dihydro-2H-benzo[d]imidazo[1,2-
a]imidazole (30)
1
Pale brown solid; H NMR (400 MHz, CD3OD) δ 7.67–7.58 (m, 2H), 7.58–7.54 (m, 2H), 7.53–7.44
(m, 2H), 7.42–7.39 (m, 1H), 7.07–7.00 (m, 3H), 6.83 (d, J = 6.1 Hz, 2H), 5.31 (s, 2H), 4.32 (t, J = 8.5
Hz, 2H), 3.83 (t, J = 8.5 Hz, 2H), 3.40 (s, 3H); LRMS (electrospray) m/z (M+H)+ 356.
4.1.7.22. 9-((4'-(Trifluoromethoxy)-[1,1'-biphenyl]-4-yl)methyl)-3,9-dihydro-2H-
benzo[d]imidazo[1,2-a]imidazole (31)
1
Pale yellow solid; H NMR (400 MHz, CD3OD) δ 7.65–7.50 (m, 4H), 7.48–7.43 (m, 2H), 7.40–7.38
(m, 1H), 7.14–6.99 (m, 3H), 6.84 (d, J = 4.6 Hz, 2H), 5.37 (s, 2H), 4.25–3.93 (m, 2H), 3.76–3.72 (m,
2H); LRMS (electrospray) m/z (M+H)+ 410.
4.1.7.23. 9-(4-Phenoxybenzyl)-2,9-dihydro-3H-benzo[d]imidazo[1,2-a]imidazole (32)
17

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1
Pale yellow solid; H NMR (400 MHz, CD3OD) δ 7.37–7.31 (m, 4H), 7.21–7.06 (m, 6H), 6.97–6.94
(m, 3H), 5.11 (s, 2H), 4.33–4.29 (m, 2H), 4.18–4.13 (m, 2H); LRMS (electrospray) m/z (M+H)+ 342.
4.1.8. Synthetic procedure for substitution reaction (2-(1H-Benzo[d]imidazol-2-ylamino)ethanol, 2b)
A mixture of 2-chloro-1H-benzo[d]imidazole (1.0 eq.) and ethanolamine (10 eq.) was heated to 200
˚C for 30 min by use of microwave irradiation. After the reaction was completed, the reaction mixture
was cooled down to room temperature. The precipitated solid was filtered and washed with water to
1
give desired product 2b. White solid (96 % yield); mp = 177.4 ˚C; H NMR (400 MHz, CD3OD) δ
7.20–7.18 (m, 2H), 6.97–6.95 (m, 2H), 3.75 (t, J = 5.6 Hz, 2H), 3.49 (t, J = 5.4 Hz, 2H); LRMS
(electrospray) m/z (M+H)+ 178.
4.1.9.
Synthetic
procedure
for
substitution
reaction
(1-(4-tert-Butylphenyl)-2-(2-(2-
hydroxyethylamino)-1H-benzo[d]imidazol-1-yl)ethanone, 2c)
To a stirred mixture of 2b (1.0 eq.) in dimethylformamide was added 2-bromo-4’-
tertbutylacetophenone (1.0 eq.). The reaction mixture was stirred for 15 h. After the reaction was
completed, the reaction mixture was diluted with distilled water. The precipitated solid was filtered,
washed with distilled water and dried to give desired product 2c. White solid (55 % yield); mp =
1
230.7 ˚C; H NMR (400 MHz, CD3OD) δ 8.09 (d, J = 6.8 Hz, 2H), 7.67 (d, J = 6.8 Hz, 2H), 7.47 (d, J
= 7.6 Hz, 1H), 7.34−7.29 (m, 3H), 5.84 (s, 2H), 3.79 (t, J = 5.2 Hz, 2H), 3.59 (t, J = 5.4 Hz, 2H), 1.38
(s, 9H); LRMS (electrospray) m/z (M+H)+ 352.
4.1.10. Synthetic procedure for substitution reaction (2d)
A mixed solution of 2c (1.0 eq.) and POCl3 (10 eq.) was heated to 110 ˚C and stirred for 2 h. After the
reaction was completed, the reaction mixture was cooled down to 0 ˚C, quenched with ice water and
extracted with methylene chloride. The organic layer was dried over anhydrous MgSO4 and
concentrated in vacuo. The crude product 2d was used in the next step without further purification.
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4.1.11. Synthetic procedure for intramolecular ring formation (9-(4-tert-Butylphenacyl)-2,3-
dihydroimidazo[1,2-a]benzimidazole, 6)
To a stirred solution of 2d (1.0 eq.) in toluene was added triethylamine (1.0 eq.). The reaction mixture
was heated to 110 ˚C and stirred for 8 h. After the reaction was completed, the reaction mixture was
cooled down to room temperature and diluted with ethyl acetate. This organic layer was washed with
water, dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by
flash column chromatography (MeOH / methylene chloride) to give desired product 6. Yellow oil (19 %
1
overall 2 steps yield); H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H),
7.12 (t, J = 7.6 Hz, 1H), 7.05 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 7.6 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H),
5.70 (s, 2H), 4.38 (t, J = 8.6 Hz, 2H), 4.15 (t, J = 8.4 Hz, 2H), 1.34 (s, 9H); LRMS (electrospray) m/z
(M+H)+ 334.
4.2. Biology
4.2.1. Parasite and cell cultures
L. donovani parasites (MHOM/ET/67/HU3, ATCC 50127) were cultivated as promastigotes at 28 ºC
in M199 with 40 mM HEPES, 0.1 mM adenine, 0.0001 % biotin and 4.62 mM NaHCO3,
supplemented with 10 % heat-inactivated FBS. Parasites were sub-cultured every 2 or 3 days. THP-1
cells (ATCC TIB-202) were cultivated in RPMI supplemented with 10 % FBS at 37 ºC with 5 % CO2.
Tissue culture trypomastigotes (TCT) of T. cruzi were maintained in LLC-MK2 cell line. Every 6−8
days after infection, TCT was released in the supernatant and re-infect LLC-MK2 cell line. LLC-MK2
and U2OS cell line were cultivated in DMEM with 10% FBS at 37 ºC with 5 % CO2.
4.2.2. Intracellular Leishmania (amastigote) screening assay
The screening assay for Leishmania was performed using previously reported protocol with slight
modification in incubation period [21]. Briefly, L. donovani culture was incubated 6 days before
infection to generate metacylic promastigotes which is macrophage-infective form. THP-1 cells were
differentiated with 50 ng/mL of phorbol 12-myristate 13-acetate (PMA, Sigma P1585) before
19

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infection. Cells and parasites were seeded with 1:20 infection ratio in 384 well plates, using a
WellMateTM liquid handler. After 24 h incubation at 37 ºC with 5 % CO2, 4 µM of reference drug
amphotericin B (Sigma A2942) as EC100 (positive control), 1 % DMSO (negative control), and the
compounds were added to the wells followed by incubation at 37 ºC with 5 % CO2 for 3 days. Wells
were then washed with PBS, fixed with 4 % paraformaldehyde, and then DNA was stained with
DRAQ5TM. Automated confocal microscope (Perkin Elmer) was used to take pictures of the plates and
images were analyzed by software developed in house.
4.2.3. Extracellular Leishmania (promastigote) assay
L. donovani (1 × 106 parasites/mL) were seeded in 384 plate (EvotecTM) containing each compound
dissolved in DMSO. Leishmania was incubated with the compounds for 28 h and 400 µM of resazurin
sodium salt (Sigma R7017) was added in the plate. After additional incubation with the compounds
for 20 h, the parasites were fixed with 4 % paraformaldehyde and the plates were analyzed in
Victor3TM (Perkin Elmer) at 590 nm (emission) with the excitation at 530 nm [28].
4.2.4 Intracellular Trypanosoma cruzi assay
Tissue culture trypomastigotes (TCT) of T. cruzi were obtained from metacyclic trypomastigotes after
infection to LLC-MK2 cell line. U2OS cell line was used as host cells for the phenotypic assay. In the
plate containing each compound, homogeneous mixture of U2OS cells and parasites was dispensed
and incubated for 48 h at 37 ºC with 5 % CO2. After incubation was completed, the parasites were
fixed with 4 % paraformaldehyde, and then DNA was stained with DRAQ5TM. Automated confocal
microscope (Perkin Elmer) was used to take pictures of the plates and then images were analyzed by
software developed in house [27].
4.2.5. Data analysis
The acquired images were analyzed with in-house software to quantify cell numbers, parasites
numbers, and infection ratio. 4 µM of amphotericin B for L. donovani, 400 µM of benznidazole for T.
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cruzi, and 1 % of DMSO were used for positive and negative controls, respectively. 2-Fold dilution
with 10 points of reference drugs were tested for assay-to-assay quality control by comparing EC50
and DRC graph. Collected data were used to determine the activities of synthesized compounds in
terms of EC50, CC50 and SI value. EC50 was calculated by infection ratio normalized by positive and
negative controls. CC50 value was obtained from counting THP-1 and U2OS cell numbers, and SI
value was determined by EC50 / CC50 value. Quality of all performed assay was controlled by Z’ value,
windows and CV [29].
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Acknowledgements
This work was supported by the National Research foundation of Korea (NRF) grant funded by the
Korea government (MSIP, No. 2007-00559), Gyeonggi-do (No. K204EA000001-09E0100-00110),
and KISTI.
References
[1] K. Stuart, R. Brun, S. Croft, A. Fairlamb, R.E. Gürtler, J. McKerrow, S. Reed, R. Tarleton,
Kinetoplastids: related protozoan pathogens, different diseases, J. Clin. Invest. 118 (2008),
1301−1310.
[2] R. Reithinger, J.C. Dujardin, Molecular diagnosis of leishmaniasis: current status and future
applications, J. Clin. Microbiol. 45 (2007), 21 25.
−
[3] G.G. Baily, A. Nandy, Visceral leishmaniasis: more prevalent and more problematic. J. Infect. 29
(1994), 241 247.
−
[4] F. Chappuis, S. Sundar, A. Hailu, H. Ghalib, S. Rijal, R.W. Peeling, J. Alvar, M. Boelaert, Visceral
leishmaniasis: what are the needs for diagnosis, treatment and control? Nat. Rev. Microbiol. 5 (2007),
873−882.
[5] S.L. Croft, G.H. Coombs, Leishmaniasis – current chemotherapy and recent advances in the
search for novel drugs, Trends Parasitol. 19 (2003), 502–508.
[6] B. Purkait, A. Kumar, N. Nandi, A.H. Sardar, S. Das, S. Kumar, K. Pandey, V. Ravidas, M.
Kumar, T. De, D. Singh, P. Das, Mechanism of amphotericin B resistance in clinical isolates of
Leishmania donovani, Antimicrob. Agents Chemother. 56 (2012), 1031–1041.
[7] F.J. Pérez-Victoria, S. Castanys, F. Gamarro, Leishmania donovani resistance to miltefosine
involves a defective inward translocation of the drug, Antimicrob. Agents Chemother. 47 (2003),
2397–2403.
[8] J.A. Pérez-Molina, A. Pérez-Ayala, S. Moreno, M.C. Fernández-González, J. Zamora, R. López-
Velez, Use of benznidazole to treat chronic Chagas' disease: a systematic review with a meta-analysis,
J. Antimicrob. Chemother. 64 (2009), 1139−1147.
22

ACCEPTED MANUSCRIPT
[9] B.B. Das, A. Ganguly, H.K. Majumder, DNA topoisomerases of Leishmania: the potential targets
for anti-leishmanial therapy, Adv. Exp. Med. Biol. 625 (2008), 103−115.
[10] M.C. Motta, Kinetoplast as a potential chemotherapeutic target of trypanosomatids, Curr. Pharm.
Des. 14 (2008), 847−854.
[11] R.L. Krauth-Siegel, S.K. Meiering, H. Schmidt, The parasite-specific trypanothione metabolism
of Trypanosoma and Leishmania, Biol. Chem. 384 (2003), 539−549.
[12] C.W. Roberts, R. McLeord, D.W. Rice, M. Ginger, M.L. Chance, L.J Goad, Fatty acid and sterol
metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa,
Mol. Biochem. Parasitol. 126 (2003), 129−142.
[13] A. Cavalli, M.L. Bolognesi, Neglected tropical diseases: Multi-target-directed ligands in the
search for novel lead candidates against Trypanosoma and Leishmania, J. Med. Chem. 52 (2009),
7339−7359.
[14] D.P. Cavalcanti, S.P. Fragoso, S. Goldenberg, W. de Souza, M.C. Motta, The effect of
topoisomerase II inhibitors on the kinetoplast ultrastructure, Parasitol. Res. 94 (2004), 439 448.
−
[15] G. Hiltensperger, N.G. Jones, S. Niedermeier, A. Stich, M. Kaiser, J. Jung, S. Puhl, A. Damme, H.
Braunschweig, L. Meinel, M. Engstler, U. Holzgrabe, Synthesis and structure-activity relationships of
new quinolone-type molecules against Trypanosoma brucei, J. Med. Chem. 55 (2012), 2538−2548.
[16] G.A. Holloway, J.B. Baell, A.H. Fairlamb, P.M. Novello, J.P. Parisot, J. Richardson, K.G. Watson,
I.P. Street, Discovery of 2-iminobenzimidazoles as a new class of trypanothione reductase inhibitor by
high-throughput screening, Bioorg. Med. Chem. Lett. 17 (2007), 1422−1427.
[17] G.A. Holloway, W.N. Charman, A.H. Fairlamb, R. Brun, M. Kaiser, E. Kostewicz, P. M. Novello,
J.P. Parisot, J. Richardson, I.P. Street, K.G. Watson, J.B. Baell, Trypanothione reductase high-
throughput screening campaign identifies novel classes of inhibitors with antiparasitic activity,
Antimicrob. Agents Chemother. 53 (2009), 2824−2833.
[18] A.R. Renslo, J.H. McKerrow, Drug discovery and development for neglected parasitic diseases,
Nat. Chem. Biol. 2 (2006), 701−710.
[19] S.L. Croft, M.P. Barrett, J.A. Urbina, Chemotherapy of trypanosomiases and leishmaniasis,
Trends Parasitol. 21 (2005), 508−512.
23

ACCEPTED MANUSCRIPT
[20] L.H. Freitas-Junior, E. Chatelain, H.A. Kim, J.L. Siqueira-Neto, Visceral leishmaniasis treatment:
What do we have, what do we need and how to deliver it? Int. J. Parasitol. Drugs Drug Resist. 2
(2012), 11−19.
[21] J.L. Siqueira-Neto, S. Moon, J. Jang, G. Yang, C. Lee, H.K. Moon, E. Chatelain, A. Genovesio, J.
Cechetto, L.H. Freitas-Junior, An image-based high-content screening assay for compounds targeting
intracellular Leishmania donovani Amastigotes in human, PLoS Negl. Trop. Dis. 6 (2012), e1671.
[22] R. Iemura, M. Hori, H. Ohtaka, Syntheses of the metabolites of 1-(2-ethoxyehtyl)-2-(hexahydro-
4-methyl-1H-1,4-diazepin-1-yl)-1H-benzimidazole difumarate (KG-2413) and related compounds,
Chem. Pharm. Bull. 37 (1989), 962−966.
[23] B. Buckman, J.B. Nicholas, L. Beigelman, V. Serebryany, A.D. Stoycheva, T. Thrailkill, S.D.
Seiwert, Cyclic peptide inhibitors of hepatitis C virus replication, PCT Int. Appl. WO2011038293,
2011.
[24] V.A. Anisimova, I.E. Tolpygin, A.A. Spasov, V.A. Kosolapov, A.V. Stepanov, A.F.
Kucheryavenko, Synthesis and biological activity of N-acylmethyl derivatives of 9H-2,3-
dihydroimidazo- and 10H-2,3,4,10-tetrahydropyrimido[1,2-a]benzimidazoles and their reduction
products, Pharmaceutical Chem. J. 40 (2006), 261−267.
[25] V.A. Anisimova, I.E. Tolpygin, G.S Borodkin, Research in the field of imidazo[1,2-
a]benzimidazole derivatives: XXVII. 1-Acylmethyl-2-( -hydroxyalkylamino)benzimidazoles and
their transformation into derivatives of tricyclic systems, Russian J. Org. Chem. 46 (2010), 275 285.
ω
−
[26] F.S. Buckner, C.L. Verlinde, A.C. La Flamme, W.C. Van Voorhis, Efficient technique for
screening drug for activity against Trypanosoma cruzi using parasites expressing β-galactosidase,
Antimicrob. Agents Chemother. 40 (1996), 2592−2597.
[27] Moon et al., PLoS one, submitted.
[28] J.L. Siqueira-Neto, O.-R. Song, H. Oh, J.-H. Sohn, G. Yang, J. Jang, J. Cechetto, C.B. Lee, S.
Moon, A. Genovesio, E. Chatelain, T. Christophe, L.H. Freitas-Junior, Antileishmanial high-
throughput drug screening reveals drug candidates with new scaffolds, PLoS Negl. Trop. Dis. 4
(2010), e675.
[29] J.H. Zhang, T.D. Chung, K.R. Oldenburg, A simple statistical parameter for use in evaluation and
validation of high throughput screening assays, J. Biomol. Screen. 4 (1999), 67−73.
24

ACCEPTED MANUSCRIPT
Scheme 1. Reagents and conditions: (A) R3CH2Br 1.3 eq, K2CO3 1.3 eq, DMF, 120 °C, 4 h; (B) H2, 10 % Pd/C,
MeOH, RT, 3 h; (C) urea 2.0 eq, DMF, 200 °C, 30 min; (D) POCl3 10.0 eq, conc. HCl (cat.), 150 °C, 3 h; (E)
ethanolamine 10.0 eq, microwave irradiation, 200 °C, 30 min; (F) POCl3 10.0 eq, 110 °C, 2 h; (G) TEA 1.0 eq,
toluene, 110 °C, 8 h; (H) R3CH2Br 1.0 eq, DIPEA 1.5 eq, DMF, 100 °C, 12 h.
26

ACCEPTED MANUSCRIPT
Scheme 2. Reagents and conditions: (A) Ethanolamine 10.0 eq, microwave irradiation, 200 °C, 30 min; (B) 2-
bromo-4’-tertbutylacetophenone 1.0 eq, DMF, 15 h; (C) POCl3 10.0 eq, 110 °C, 2 h; (D) TEA 1.0 eq, toluene,
110 °C, 8 h; (E) 2-bromo-4’-tertbutylacetophenone 1.0 eq, DIPEA 1.5 eq, DMF, 100 °C, 8 h.
27

ACCEPTED MANUSCRIPT
Table 1. In vitro activity of 2,3-dihydroimidazo[1,2-a]benzimidazole derivatives against amastigotes and
promastigotes of L. donovani
Activity against L. donovani
Amastigote form
Promastigote form
Compounda
Chemical Structure
EC50
SId
CC50 (µM)c
EC50 (µM)e
(µM)b
3
> 50
> 50
N/Af
N/Tg
4
5
3.05
12.5
> 50
> 50
> 16.4
> 3.98
1.25
2.73
6
7
39.4
> 50
> 1.27
N/T
27.4
25.3
33.1
> 50
1.21
N/T
20.8
8
> 1.98
Miltefosine
4.83
0.25
18.9
7.57
3.91
30.2
11.1
0.22
Amphotericin B
a
b
Compound 3−5 are already known molecules. EC50 indicates half maximal effective concentration on intracellular L. donovani (2-fold
c
dilution with 10 points dose response). Average of duplicate determinations. CC50 indicates cytotoxicity against THP-1 cells, which was
determined by counting of THP-1 cell numbers in the taken image from intracellular assay. Average of duplicate determinations. d SI is
e
selective index (EC50 / CC50). EC50 indicates half maximal effective concentration on extracellular L. donovani. Average of duplicate
determinations. f N/A, not available. g N/T, not tested.
28

ACCEPTED MANUSCRIPT
Table 2. In vitro activity of 2,3-dihydroimidazo[1,2-a]benzimidazole derivatives against amastigotes and
promastigotes of L. donovani
Chemical Structure
Activity against L. donovani
Compounda
Amastigote form
Promastigote form
R1
R2
R3
SIc
EC50 (µM)a
CC50 (µM)b
EC50 (µM)d
4
H
F
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-tertbutylphenyl
3.05
14.0
7.33
11.0
> 50
> 50
42
> 50
> 50
12.5
28.1
> 50
> 50
> 50
> 50
22.8
> 50
> 50
> 50
> 50
> 50
> 50
> 50
39.7
> 50
35.0
27.1
30.0
20.0
23.0
11.0
48.0
> 50
18.9
7.57
> 16.4
> 3.57
1.71
2.55
N/A
N/A
> 1.19
N/A
4.19
N/A
> 2.21
N/A
N/A
N/A
N/A
N/A
7.5
1.25
1.26
2.22
1.80
N/T
N/T
N/T
N/T
1.71
N/T
4.98
N/T
N/T
N/T
N/T
N/T
1.48
9.22
1.49
1.75
3.11
N/T
2.34
1.53
2.01
N/T
11.1
0.22
9
4-tertbutylphenyl
10
Cl
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-tertbutylphenyl
11
4-tertbutylphenyl
12
4-tertbutylphenyl
13
phenyl
14
4-ethylphenyl
15
4-isopropylphenyl
> 50
5.4
16
4-cyclohexylphenyl
3,5-dimethylphenyl
3-tertbutylphenyl
17
> 50
22.6
> 50
> 50
> 50
> 50
> 50
5.29
32.5
8.88
17.8
10.3
13.7
10.2
5.20
12.5
> 50
4.83
0.25
18
19
4-methylsulfonylphenyl
4-fluorophenyl
20
21
4-trifluoromethylphenyl
4-trifluoromethoxyphenyl
4-cyanophenyl
22
23
24
4-(1,1’-biphenyl)
25
4-(2’,5’-difluoro-1,1’-biphenyl)
4-(4’-fluoro-1,1’-biphenyl)
4-(4’-methyl-1,1’-biphenyl)
4-(4’-trifluoromethyl-1,1’-biphenyl)
4-(4’-chloro-1,1’-biphenyl)
4-(4’-methoxy-1,1’-biphenyl)
4-(4’-trifluoromethoxy-1,1’-biphenyl)
4-phenoxyphenyl
> 1.54
3.94
1.52
2.91
1.46
2.25
2.11
3.82
N/A
3.91
30.2
26
27
28
29
30
31
32
33
2-pyridyl
Miltefosine
Amphotericin B
a Compound 13, 20, and 21 are already known molecules. b EC50 indicate half maximal effective concentration on intracellular L. donovani
(2-fold dilution with 10 points dose response). Average of duplicate determinations. c CC50 indicate cytotoxicity against THP-1 cells, which
was determined by counting of THP-1 cell numbers in the taken image from intracellular assay. Average of duplicate determinations. d SI is
e
selective index (EC50 / CC50). EC50 indicate half maximal effective concentration on extracellular L. donovani. Average of duplicate
determinations. f N/A, not available. g N/T, not tested.
29