Discovery of 2-iminobenzimidazoles as a new class of trypanothione reductase inhibitor by high-throughput screening

Article abstract of DOI:10.1016/j.bmcl.2006.11.090

A high-throughput screening campaign of a library of 100,000 lead-like compounds identified 2-iminobenzimidazoles as a novel class of trypanothione reductase inhibitors. These 2-iminobenzimidazoles display potent trypanocidal activity against Trypanosoma

Full text of DOI:10.1016/j.bmcl.2006.11.090

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1422–1427
Discovery of 2-iminobenzimidazoles as a new class of
trypanothione reductase inhibitor by high-throughput screening
Georgina A. Holloway,a Jonathan B. Baell,a Alan H. Fairlamb,b Patrizia M. Novello,a
John P. Parisot,a John Richardson,b Keith G. Watsona and Ian P. Streeta,*
aThe Walter and Eliza Hall Institute of Medical Research, Biotechnology Centre, 4 Research Avenue,
La Trobe Research and Development Park, Bundoora, Vic. 3086, Australia
bDivision of Biological Chemistry and Molecular Microbiology, School of Life Sciences, Wellcome Trust Biocentre,
University of Dundee, Dundee, Scotland, UK
Received 8 September 2006; revised 21 November 2006; accepted 30 November 2006
Available online 3 December 2006
Abstract—A high-throughput screening campaign of a library of 100,000 lead-like compounds identified 2-iminobenzimidazoles as a
novel class of trypanothione reductase inhibitors. These 2-iminobenzimidazoles display potent trypanocidal activity against Trypan-
osoma brucei rhodesiense, do not inhibit closely related human glutathione reductase and have low cytotoxicity against mammalian
cells.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Parasitic protozoa of the family Trypanosomatidae are
the causative agent of many significant tropical diseases
including African trypanosomiasis, Chagas disease and
Leishmaniasis. In the 2004 world health report1 African
trypanosomiasis was reported to cause 48 thousand
deaths and a disease burden of 1525 thousand DALYs
(disability adjusted life years) annually, Chagas disease
14 thousand deaths and a disease burden of 667 thou-
sand DALYs and Leishmaniasis 51 thousand deaths
and a disease burden of 2090 DALYs. There are cur-
rently nine key drugs in use for the treatment of these
disease states (Fig. 1): suramin and pentamidine against
early stage African trypanosomiasis, and eflornithine
and melarsoprol against late stage disease; nifurtimox
and benznidazole against early stage Chagas disease;
meglumine antimoniate and sodium stibogluconate
against Leishmaniasis, and amphotericin B against anti-
mony-resistant strains. All of these drugs have severe
limitations including administration difficulties, long
treatment regimes, life-threatening side effects, varied
parasitological cure rates for different strains, lack of
efficacy against late stage diseases, and increasing inci-
dence of drug resistance.2–5
The intracellular reducing environment of trypanosoma-
tids is maintained by a unique thiol redox system where
the glutathione/glutathione reductase (GR) couple
found in mammalian cells is replaced by the (bis-gluta-
thionyl)spermidine trypanothione/trypanothione reduc-
tase (TR) couple.6 TR is a key enzyme of the parasite
antioxidant defence,7,8 does not occur in the mammalian
host and has been found to be essential for all trypano-
somatids currently studied.9,10
TR and human GR have similar catalytic mechanisms
with 14 of the 19 amino acid residues close to the sub-
strate binding site being conserved. However, they are
specific to their respective disulfide substrates
(Fig. 2).11 GR has a hydrophilic, positively charged
region in its active site that interacts with the glycine
carboxylates of glutathione disulfide, while TR has a
larger binding site with a negatively charged region with
which the spermidine moiety of trypanothione disulfide
binds.12 The absence of TR from the mammalian host
and the sensitivity of trypanosomatids to oxidative
stress makes TR an attractive target for trypanosomiasis
therapeutics.13 The objective of this work, therefore,
Keywords: Tropical diseases; Trypanosomiasis therapeutics; Trypano-
thione reductase inhibitors; High-throughput screening; Medicinal
chemistry; Iminobenzimidazoles.
*
Corresponding author. Tel.: +61 3 9345 2200; fax: +61 3 9345
2211; e-mail: istreet@wehi.edu.au
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.090

G. A. Holloway et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1422–1427
1423
Figure 1. Current trypanosomiasis drugs on the market.
25 lM. The potencies of the hits were confirmed by
assaying compounds as 11 point titrations. In summary,
the hit set contained compounds from 13 distinct struc-
tural classes, and the IC50 values of the hits ranged from
1 to 67 lM. A focus set of 43 compounds was selected
from the population of primary screen hits based on
inhibitory potency, synthetic accessibility and com-
pound novelty. These compounds were re-ordered from
the original chemical vendors and the TR inhibitory
potency, structural identity and purity of the re-supplied
material confirmed. The focus set contained compounds
from nine distinct structural classes and the 2-imino-
benzimidazoles were prominent having four close ana-
logues in the focus set. The development of this 2-
iminobenzimidazole structural class will be discussed
in this communication.
Figure 2. Trypanothione disulfide and glutathione disulfide.
was to identify novel classes of TR inhibitors by
high-throughput screening (HTS) of a diverse chemical
library.
The 2-iminobenzimidazoles are a novel class TR inhibi-
tors that are chemically suitable for optimization and
scored well in a drug-likeness analysis. A search of the
patent literature revealed few 2-iminobenzimidazoles,
none of which were reported to have anti-trypanosomal
activity. A number of other 2-iminobenzimidazoles were
contained within the lead discovery library; eight were
selected and their potency determined to investigate
structure–activity relationships (SAR) (Table 1).
Our chemical compound library is a collection of
approximately 100,000 compounds purchased from
commercial vendors. The compounds in this chemical
library were selected to provide ‘lead-like’ chemical
structures with a guiding philosophy that most success-
ful drug development projects have started from leads
that are smaller and more polar than the drug itself.
‘Lead-like’ chemical structures were defined as having
simple molecular structures that are chemically unreac-
tive, synthetically accessible and have ‘drug-like’ proper-
ties. The 100,000 compounds in our ‘lead discovery
library’ represent a diverse set of molecules as judged
by Tanimoto dissimilarity analysis (T 6 0.85), and
although simple filters based on the Lipinski criteria
were not used in the selection process, 89% of the com-
pounds in the library are Lipinski compliant14 and 81%
conform with Oprea’s criteria for ‘lead-likeness’.15
Defined SAR were observed with the highest inhibitory
activity obtained when one side chain contained a basic
moiety (R2) and the other (R1) an aromatic ring (3, 5, 7).
Replacement of the basic group in the R2 side chain with
a hydrophobic group (1, 2, 4), or removal of the phenyl
ring in the R1 side chain (6, 8) resulted in a significant
loss of inhibitory activity. None of the 2-iminobenzimi-
dazoles in the patent literature possessed the general
structure I (Table 2). A search of chemical vendors re-
vealed 26 analogues with the general structure I that
were purchased and tested, revealing further SAR
(selected compounds: Table 2). The most potent com-
pounds (IC50 6 10 lM) possessed a piperidine (9, 11,
14, 16, 20) or diethylamine (13, 15, 7, 22) basic amine
moiety. The inhibitory activity was significantly reduced
by extension of the R2 side chain (18) and completely
lost when the basic amine group was a morpholine moi-
ety (10, 12). A variety of alcohol substituents (9, 11, 15,
An automated screening protocol for TR was developed
using the photometric assay described by Hamilton
et al.16,17 The assay was adapted for operation in 384-
well microtitre plates and was found to be exceptionally
robust and reproducible. The average Z0 and Z values18
achieved over the entire primary screen were 0.72 and
0.54, respectively. The primary screen of 100,000 com-
pounds identified 120 compounds that inhibited TR
activity by more than 50% at a concentration of

1424
G. A. Holloway et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1422–1427
Table 1. Inhibition of TR by selected 2-iminobenzimidazoles in the lead discovery library
R1
R2
% inhibitiona
IC50 (lM)
b
Compound
1
2
3
4
5
6
7
8
4-BrPhCH(OH)CH2
4-ClPhCH(OH)CH2
3,4-DiClPhCH(OH)CH2
4-(EtO)PhCH(OH)CH2
4-(MeO)PhCH(OH)CH2
CH2@CHCH2
CH2CH2CH2CH3
CH2Ph
42
32
92
6
>100
>100
10
CH2CH2NMe2
CH2CH@CH2
CH2CH2Npiperidine
CH2CH2NEt2
>100
24
85
31
91
66
>100
7
>100
4-MePhC(O)CH2
Me3CC(OH)CH2
CH2CH2NEt2
CH2CH2Npiperidine
a At 25lM.
b Number of assay replicates performed = 3.
Table 2. Inhibition of TR by selected commercially available 2-iminobenzimidazoles
R1
R2
IC50 (lM)
a
Compound
9
PhOCH2CH(OH)
PhOCH2CH(OH)
4-MePhOCH2CH(OH)
4-MePhOCH2CH(OH)
PhOCH2
Npiperidine
5
>100
9
10
11
12
13
14
15
16
17
18
7
Nmorpholine
Npiperidine
Nmorpholine
NEt2
>100
8
PhOCH2
Npiperidine
NEt2
4
3,4-DiClPhCH(OH)
3,4-DiClPhCH(OH)
4-MePhCH(OH)
4-MePhCH(OH)
4-MePhC(O)
9
Npiperidine
NMe2
4
29
35
7
CH2NMe2
NEt2
NEt2
19
20
21
5
4-PhPhC(O)
25
5
2,4-diClPhOCH2CH(OH)
4-(MeO)PhOCH2CH(OH)
4-(MeO)PhCH(OH)
3,4-DiClPhC(O)
4-MePhCH(OH)
Npiperidine
Npiperidine
Npiperidine
NEt2
16
24
10
19
22
23
NEt2
a Number of assay replicates performed = 3.
16, 20), carbonyl groups (7, 22) or ether linkages (9, 11,
13, 14, 20) were accommodated in the R1 side chain
without significant loss of inhibitory activity. Further-
more, variation of the number of methylene groups
(n = 2–4) linking the endocyclic benzimidazole nitrogen
atom and the R1 aryl group also did not result in a sig-
nificant loss of inhibitory activity (e.g., 7 15, 16 and 22
(n = 2) cf 13 and 14 (n = 3) cf. 9, 11 and 20 (n = 4)). With
n = 2, the 3,4-dichloro aryl group was tolerated regard-
less of linker composition (15, 16, 22). The 4-methoxy-
phenyl substituent was distinctly less favoured (5). A
4-methyl group was well tolerated in 7 where the linker
contained a carbonyl but less so in 23 where the linker
contained an alcohol. Replacement of the 4-methyl
group (7) with a 4-phenyl group (19) resulted in a signif-
icant drop in inhibitory activity. The results obtained on
this limited number of analogues indicate that the TR
binding site will tolerate a relatively wide range of differ-
ent linker structures and substitution patterns on the R1
aromatic group. This is consistent with previous studies
in which inhibitors have been reported to have multiple
binding modes.19–21
The 2-iminobenzimidazole dimer, 25, is one of the
most potent compounds identified from the screen
(Table 3). It does not fit the general pharmacophore
pattern of one basic amine and one bulky hydrophobic
side chain, but it is possible that one of the 2-imino-
benzimidazoles is acting as the basic amine moiety.
This hypothesis is supported by the observation that
when the benzyl side chain was replaced with an ethyl
group (24), there is a significant loss of inhibitory
activity.
Selected analogues were synthesized to further explore
structure–inhibitory activity relationships in the

G. A. Holloway et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1422–1427
1425
Table 3. Inhibition of TR by commercially available dimer 2-imino-
benzimidazoles
(Scheme 1, Method A). Despite the good stability of
the compounds once isolated, a number of challenges
were encountered during the purification of the 2-imino-
benzimidazoles and their intermediates. In addition, the
second substitution in Method A was slow and accom-
panied by significant decomposition. Therefore, an
alternative method was explored to synthesize further
2-iminobenzimidazole analogues (Scheme 1, Method
B). 1,2-Phenylenediamine was sequentially substitut-
ed25–27 and then the ring closed upon treatment with
cyanogen bromide.28 Finally, a 2-acyliminobenzimidaz-
ole analogue was synthesized by treatment of the 2-imi-
nobenzimidazole with acetyl chloride in the presence of
four equivalents of N,N-diisopropylethylamine and
catalytic 4-dimethylaminopyridine in acetonitrile at
60 ꢁC for 6 h (Scheme 1, Method A).
a
Compound
R
IC50 (lM)
24
25
Me
Ph
>100
4
a Number of assay replicates performed = 3.
The compounds in Table 4 both confirm earlier observa-
tions and reveal more information about the SAR with-
in this chemical series. First, the general structure I was
confirmed as the essential pharmacophore since com-
pound 30, which contains two basic amine side chains,
also has poor inhibitory activity (Table 4). Second, in
agreement with earlier observations, the results obtained
with 14, 28 and 29 indicate that the enzyme binding site
will tolerate a range of R1 linker lengths. Third, the loss
of inhibitory activity of the acyclic compound 26 sug-
gests that the 2-iminobenzimidazole moiety is an impor-
tant part of the pharmacophore and does not merely
serve as a scaffold to appropriately present the R1 and
R2 side chains for binding. Finally, we postulate that
the loss of inhibitory activity of the acylimino analogue,
27, is likely to be due to the reduced basicity of its 2-im-
ino group, and that the guanidyl moiety of inhibitory
2-iminobenzimidazoles must be protonated for high
affinity binding.29 This hypothesis is supported by the
observation that 3, 16, and 25 are competitive inhibitors
of trypanothione binding30 (Table 5) and that the trypa-
nothione binding site has a negatively charged region
which binds the spermidine moiety of trypanothione
disulfide.12
Scheme 1. Synthesis of 2-iminobenzimidazoles. Yields displayed are
for the synthesis of 27, 29 via Method A and 30 via Method B. (i)
R2ClÆHCl, KOH, EtOH, D, 16 h (42%); (ii) R1Br, 2-butanone, D, 48 h
(64%); (iii) AcCl, N,N-diisopropylamine, DMAP, CH3CN, 60 ꢁC, 6 h
(59%); (iv) R2Br, MeOH, D, 24 h (77%); (v) R1Br, NaI, DMF, D, 2 h or
R1OTs, Na2CO3, 120 ꢁC, 24 h; (vi) CNBr, EtOH, rt, 48 h (74%).
2-iminobenzimidazole series that were not accessible
through the commercially available analogues.22
Two methods were utilized to synthesize the desired
2-iminobenzimidazole analogues. In Method
A
2-aminobenzimidazole was substituted with a alkyla-
mine hydrochloride23 and then the monosubstituted
product was reacted with a series of phenoxyalkylbro-
mides24 to give the desired 2-iminobenzimidazoles
Table 4. Inhibition of TR by 2-iminobenzimidazole analogues
R1
R2
R3
IC50 (lM)
c
Compound
Gen Struct
14a
26b
27a
28a
29a
30b
31b
III
II
PhO
PhO
Npiperidine
Npiperidine
Npiperidine
Npiperidine
Npiperidine
NEt2
H
4d
>100
>100
7
—
Ac
H
III
III
III
III
IV
PhO
PhOCH2
PhOCH2CH2CH2
NEt2
H
8
59
H
—
PhO
—
>100
a Synthesized using Method A.
b Synthesized using Method B.
c Number of assay replicates performed = 2.
d IC50 of batch synthesized in-house.

1426
G. A. Holloway et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1422–1427
Table 5. Biological activity of selected 2-iminobenzimidazoles
Compound
TR enzyme Ki for
competitive binding (lM)
T. brucei rhodesiense
Cytotoxicity
IC50 (lM)
Human GR enzyme
IC50 (lM)
IC50 (lM)
3
5.4
3.6
1.9
0.6
0.2
1.3
46
9
>100
>100
>100
16
25
144
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The biological inhibitory activity of a small selection of
2-iminobenzimidazoles (3, 16, 25) was further explored
in whole parasite and cytotoxicity assays (Table 5).31
The 2-iminobenzimidazoles tested displayed potent try-
panocidal activity against Trypanosoma brucei rhodes-
iense (STB 900) and relatively low cytotoxicity
against a human bladder carcinoma cell line (HT-29).
It is possible that the cytotoxicity observed, particular-
ly with compound 16, may be due to inhibition of hu-
man GR. However this is unlikely due to their lack of
activity in an in vitro GR assay (Table 5).32 Consider-
ing the low micromolar Ki values obtained for these
compounds against the TR enzyme, the trypanocidal
activity particularly for 3 and 16 is unexpectedly po-
tent. This could be due to the active uptake of these
compounds by the parasite which is known to occur
via a variety of mechanisms for other basic trypanoci-
dal compounds.33,34
11. Marsh, I. R.; Bradley, M. Eur. J. Biochem. 1997, 243, 690.
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17. Briefly the assay determined the rate of TNB formation
which was measured continuously for 15 min at 412 nm in
a 40 lL assay volume at room temperature. Each reaction
mixture contained the following constituents: TR
(0.2 mU), T[S]2 (6 lM), DTNB (100 lM), compound
(25 lM) and NADPH (150 lM).
In summary, the application of high-throughput screen-
ing of a lead discovery library of 100,000 compounds
identified nine novel chemical classes of TR inhibitors.
In particular the 2-iminobenzimidazole class was found
to have good development potential. The essential phar-
macophore for TR inhibitory activity was identified by
investigation of a series of analogues and further biolog-
ical testing revealed that members of this new class of
TR inhibitor have potent trypanocidal activity against
T. brucei rhodesiense, and low cytotoxicity against hu-
man cells. This chemical series has significant potential
for further development as a new class of therapeutics
for trypanosome-mediated diseases.
18. Zhang, J.-H.; Chung, T. D. Y.; Oldenburg, K. R.
J. Biomol. Screen. 1999, 4, 67.
Acknowledgments
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M. R.; Hunter, W. N.; Fairlamb, A. H. J. Biol. Chem.
2004, 279, 29493.
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R.; Speers, P.; Douglas, K. T.; Rock, P. J.; Yardley, V.;
Croft, S. L.; Fairlamb, A. H. J. Med. Chem. 1998, 41,
148.
21. Garforth, J.; Yin, H.; McKie, J. H.; Douglas, K. T.;
Fairlamb, A. H. J. Enzym. Inhib. 1997, 12, 161.
22. The identity of synthesized compounds was confirmed by
NMR and MS and their purity was determined to be
>95%.
23. Da Settimo, A.; Da Settimo, F.; Marini, A. M.; Primofi-
ore, G.; Salerno, S.; Viola, G.; Da Via, L.; Magno, S. M.
Eur. J. Med. Chem. 1998, 33, 685.
This investigation received financial support from the
UNICEF/UNDP/World Bank/WHO special program
for research and training in tropical diseases (TDR).
We gratefully acknowledge Bill Charman, the center
for drug candidate optimization (CDCO), for advice,
valuable discussion and encouragement. We also express
our gratitude for the support of the TDR screening net-
work, in particular Reto Brun and his group at the Swiss
tropical institute for conducting the anti-trypanosomal
and cytoxicity assays. We also acknowledge Ahilan
Saravanamuthu for conducting the early TR assay
development.
24. Morishita, S.; Saito, T.; Hirai, Y.; Shoji, M.; Mishima, Y.;
Kawakami, M. J. Med. Chem. 1988, 31, 1205.
25. Monosubstitution: Shinkai, S.; Harada, A.; Ishikawa, Y.;
Manabe, O.; Yoneda, F. J. Chem. Soc., Perkin. Trans.
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32. An automated screening protocol for GR was developed
based on our TR assay. Higher concentrations of GR and
G[S]2 were required because of their higher Km. The assay
determined the rate of TNB formation which was measured
continuously for 15 min at 412 nm in a 40 lL assay volume
at room temperature. Each reaction mixture contained the
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conditions.
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trations (0.1–10 · Km) were performed in the absence/
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