Novel bisbenzimidazoles with antileishmanial effectiveness

Article abstract of DOI:10.1016/j.bmcl.2008.03.020

A small library of 2,2′-[(α,ω-alkanediylbis(oxyphenylene)]bis-1H-benzimidazoles has been prepared and screened in vitro against Pneumocystis carinii, Trypanosoma brucei rhodesiense, and Leishmania donovani. Among the six tested compounds two derivatives e

Full text of DOI:10.1016/j.bmcl.2008.03.020

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 2658–2661
Novel bisbenzimidazoles with antileishmanial effectiveness
a
b
c,d
c,d
Annie Mayence, Aur e´ lie Pietka, Margaret S. Collins, Melanie T. Cushion,
e
a
a,b,
*
Babu L. Tekwani, Tien L. Huang and Jean Jacques Vanden Eynde
a
Xavier University of Louisiana, College of Pharmacy, Division of Basic Pharmaceutical Sciences,
Drexel Drive, New Orleans, LA 70125, USA
University of Mons-Hainaut, Laboratory of Organic Chemistry, Faculty of Sciences, 20 place du Parc, B-7000 Mons, Belgium
1
b
c
Research Service, Veterans Affairs Medical Center, Cincinnati, OH 45220, USA
Division of Infectious Diseases, Department of Internal Medicine, University of Cincinnati, Cincinnati, OH 45267-0560, USA
d
e
National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA
Received 11 February 2008; revised 4 March 2008; accepted 6 March 2008
Available online 10 March 2008
0
Abstract—A small library of 2,2 -[(a,x-alkanediylbis(oxyphenylene)]bis-1H-benzimidazoles has been prepared and screened in vitro
against Pneumocystis carinii, Trypanosoma brucei rhodesiense, and Leishmania donovani. Among the six tested compounds two deriv-
atives emerged as promising hits characterized by IC50 values lower than that determined for pentamidine against L. donovani.
ꢀ
2008 Elsevier Ltd. All rights reserved.
Whereas the amidine group is seldom encountered in
biomolecules, it is present in a number of anticoagulant
emerged as promising drug candidates for the treatment
of trypanosomiasis and P. carinii pneumonia,
respectively
1
drug candidates and some pharmaceutical specialties
prescribed to cure antifungal and antimicrobial infec-
2
tions. Among those specialties, let us mention (Fig. 1)
propamidine (1, Brolene ) used in the treatment of eyes
ꢁ
infections caused by Acanthamoeba keratitis, hexami-
dine (2, Hexomedine ) used as a topical antiseptic found
ꢁ
in many skin care compositions, and pentamidine (3,
Pentacarinat , Pentam , NebuPent ), the most repre-
sentative example of the series, clinically used for the
ꢁ
ꢁ
ꢁ
3
treatment of Pneumocystis carinii pneumonia, Human
4
African trypanosomiasis (sleeping sickness), and leish-
5
maniasis. However despite their efficiency those com-
pounds, and pentamidine more particularly, are
plagued by important drawbacks including poor bio-
6
availability and some unpleasant side effects. In order
to circumvent those drawbacks, many structural varia-
tions have already been considered and they essentially
deal with modifications of the moiety linking both ben-
7
0
zamidine groups. In that sense furamidine (4) and 4,4 -
8
(
piperazine-1,4-diyl)bisbenzamidine (5) have recently
Keywords: Bisbenzamidine; Bisbenzimidazole; Pentamidine; Pneumo-
cystis carinii pneumonia; Human African trypanosomiasis;
Leishmaniasis.
*
Corresponding author. Tel.: +1 32 65 373337; e-mail: jjvde@
umh.ac.be
Figure 1. Some pharmacologically important bisbenzamidines.
0
960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.020

A. Mayence et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2658–2661
2659
Based on a disconnection strategy, the targeted deriva-
tives can be obtained either (i) by forming 2-(hydroxy-
phenyl)-1H-benzimidazoles and subsequently the ether
bonds by reaction with a dihaloalkane or (ii) by reacting
a dihaloalkane with an hydroxybenzaldehyde and then
only forming the heterocyclic entities. In our hands,
the first route led to mixtures of compounds (including
N-alkylated benzimidazoles) whereas the second syn-
thetic scheme afforded the expected derivatives in good
yields (75–98%) and purity. Intermediate dialdehydes
Figure 2. Structure of the bisbenzimidazole 6.
Our approach is quite different as it consists in masking
the amidine functions by incorporation into conjugated
cyclic systems and more particularly into benzimidazole
systems. In that way, we have already observed that bis-
benzimidazole 6 (Fig. 2), structurally related to 5, was
1
3
have been described in the literature and reaction con-
ditions have been optimized for the cyclization step
only. Indeed activation of the dialdehydes by reaction
with sodium bisulfite and coupling with the diamines
were performed by a one-pot procedure and were
advantageously conducted under microwave irradiation
9
inactive against Trypanosoma brucei rhodesiense and
Trypanosoma brucei brucei but active, and far more ac-
tive than 5 or even pentamidine 3, against Leishmania
1
0
14
donovani. In this report, we extend that preliminary re-
sult and we disclose our recent findings on the prepara-
within a few minutes .
tion
and
bisbenzimidazoles.
the
evaluation
of
other
novel
In order to establish the pharmacological profile of com-
pounds 7–12 they have been tested in vitro against P.
carinii, Trypanosoma brucei rhodesiense, and L. donovan-
i, the three species that can be efficiently killed by the ac-
tion of pentamidine 3. Inspection of the data gathered in
Table 1 clearly indicates that inclusion of the amidine
functions in benzimidazole systems reduced the
in vitro activity against P. carinii and Trypanosoma bru-
cei rhodesiense by a tenfold factor and even more when
compared to 3. For both strains the most active deriva-
tive was compound 12 bearing the benzimidazole groups
in the meta position relative to the ether bonds. Except 7
the other derivatives were inactive against both para-
sites. The situation was quite different in the screening
against L. donovani. Indeed the IC50 determined for
pentamidine 3 was 2.2 lM and the bisbenzimidazoles
1
1
Benzimidazoles are generally prepared (cfr. Scheme 1)
by the reaction of 1,2-phenylenediamine with carboxylic
acid under drastic conditions. Another popular route
1
1
involves 1,2-phenylenediamines and aldehydes to form
transient imines that must be oxidized in a second step.
A few papers indicate that starting from the bisulfite ad-
duct of the aldehydes enables to avoid the oxidation
step and we decided to test the possibility of exploiting
that protocol to prepare bisbenzimidazoles 7–12 (cfr.
Scheme 2). In those substances the heterocyclic systems
are linked by oxyphenylene groups separated by a num-
ber of methylene units ranging from 3 to 7. Therefore,
they are structurally related to the bisbenzamidines 1–3.
1
2
Scheme 1. Disconnection approach to the targeted bisbenzimidazoles.

2660
A. Mayence et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2658–2661
the tested compounds, 7 and 9 emerged as promising
hits exhibiting a more efficient antiparasitic behavior
than 3, when either IC50 or IC90 values are considered.
Interestingly, 7 and 9 possess structural features closely
related to propamidine 1 and pentamidine 3 itself.
All compounds were subsequently tested for cytotoxic-
ity. Only compound 8 displayed no cytotoxicity in the
assay considered in this study. Hits 7 and 9, effectively
active against L. donovani, gave rise to modest selectivity
indexes expressed as ratio IC50 Vero cells/IC50 L. dono-
vani in the range of 20.
Although it is out of the scope of this preliminary com-
munication to suggest any mechanism of action of the
bisbenzimidazoles under study, it is noteworthy that,
1
5
contrary to bisbenzamidines, they did not exhibit sig-
nificant binding, if any, to DNA (data not shown).
Therefore, their antiparasitic properties cannot be
understood in terms of inhibition of DNA dependent
1
6
17
enzymes (e.g., topoisomerases and nucleases ). The
lack of efficacy of the bisbenzimidazoles against the Try-
panosoma parasite can tentatively be linked to poor
interactions with the P2 aminopurine transporter be-
cause the substrate structural recognition motif (i.e.,
1
8
the amidine group) is masked in those compounds.
In conclusion we have successfully prepared a series of
novel bisbenzimidazoles structurally related to clinically
used bisbenzamidines. Although those compounds must
be carefully considered due to their potential cytotoxic-
ity, our approach led to the identification of some prom-
ising hits that were selectively active against L. donovani
at doses deserving further investigations. Other screen-
ings involving a larger library of bisbenzimidazoles are
currently in progress.
Acknowledgments
Scheme 2. Preparation of compounds 7–12. Reaction conditions: (a)
EtOH, K CO (1 equiv), reflux 8 h; (b) EtOH/H O (3/1), Na (1
equiv), 1,2-phenylenediamine (2 equiv), MW 140 ꢂC, 15 min.
2
3
2
2 2 5
S O
J.J.V.E. and A.P. are grateful to the Belgian Fonds
National de la Recherche Scientifique for a financial
support. T.L.H. is supported by NIH Grant
2S06GM08008. The authors thank WHO for the anti-
Trypanosomal data.
1
9
Table 1. Pharmacological activities for compounds 7–12 and pent-
amidine 3
Compound Pneumocystis Trypanosoma Leishmania Vero
2
0
22
23
carinii
brucei
donovani
cells
References and notes
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21
rhodesiense
IC50 (lM)
IC50 (lM)
IC50 IC90 IC50
lM) (lM) (lM)
1
(
M. L.; Amparo, E.; Cacciola, J.; Rossi, K. A.; Alexander,
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7
8
9
1
1
1
3
10.2
169.4
54.1
>100
>100
5.2
>60
>60
>60
>60
>60
0.85
0.05
1.5 3.3 27.2
4.9 12.9 >100
1.4 3.1 28.7
0
1
2
11.8 53.8 27.9
13.6 56.2 46.1
10.3 45.1 12.3
2. Soeiro, M. N. C.; De Souza, E. M.; Stephens, C. E.;
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2.2
9.8 >100
4
5
6
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that we prepared were characterized by IC50 values
ranging from 1.4 lM, more active than pentamidine,
to 13.6 lM, sixfold less active than pentamidine. Among
7. Boykin, D. W.; Kumar, A.; Spychala, J.; Zhou, M.;
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A. Mayence et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2658–2661
2661
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J = 6 Hz, O–CH
CH –CH ); 1.6 (m.large, 2H, O–CH
(ESI-ToF) MH C H N O : MW experimental: m/z
2
–CH
2
–CH
2
); 1.8 (m.large, 4H, O–CH
2
–
2
2
2
–CH –CH ). HRMS
2
2
+
8
3
1
29
4
2
489.2275; MW calculated: m/z 489.2291. Mp (ꢂC):
>300 ꢂC. Yield: 94%.
2
337; (b) Walzer, P.; Cushion, M. T.; Mayence, A.;
Huang, T. L.; Vanden Eynde, J. J. WO Patent
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2
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9
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1
0. Mayence, A.; Vanden Eynde, J. J.; LeCour, L., Jr.;
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19. Reported values are means of 2–4 determinations.
20. Pneumocystis carinii were obtained from chronically
immunocompressed Long Evans and Brown Norway rats.
1
1
1. Preston, P. N. Chem. Rev. 1974, 74, 279.
1
0
2. (a) Ridley, H. F.; Spickett, R. G.; Timmis, G. M.
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organism nuclei with the vast majority (about 95%) of
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remainder (about 5%) being composed of cysts. P. carinii
preparations were evaluated for microbial contamination,
ATP content, karyotype, and host cell content prior to use
in the ATP assay. See, Cushion, M. T.; Walzer, P. D.;
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1
1
4. General procedure. A mixture of an hydroxybenzaldehyde
(
5.12 g; 42 mmol), a dibromoalkane (20 mmol), and
21. Assays were performed in 96-well microtiter plates, each
3
potassium carbonate (2.76 g; 20 mmol) in ethanol (10 ml)
was heated under reflux for 8 h. After cooling, the
precipitate was filtered and successively washed with
water, ethanol, and ether. The bisbenzaldehyde was pure
enough to be engaged in the second step. A mixture of the
bisbenzaldehyde (3 mmol), sodium pyrosulfite (0.57 g;
well containing 100 ll of culture medium with 8 · 10
bloodstream-form trypomastigotes of the strain Trypano-
soma brucei rhodesiense STIB 900, which is known to be
susceptible to all currently drugs. After 3 days of incuba-
tion, parasite growth is assessed fluorimetrically after the
addition of resazurin. Fluorescence is measured after 24 h
at 37 ꢂC. See, http://www.who.int/tdr/grants/workplans/
pdf/.
3
(
mmol), ortho-phenylenediamine (6 mmol), and water
3 ml) in ethanol (9 ml) was irradiated in a microwave
oven (Biotage) for 15 min at 140 ꢂC. After cooling the
precipitate was filtered and thoroughly washed with water,
ethanol, and ether. 4,4 -[1,5-Pentanediyl(oxy)]bisbenzald e´ -
22. The compounds were dissolved in DMSO at the concen-
ꢀ
1
tration of 2 mg ml and diluted with the culture medium
to obtain the desired final concentrations. The solutions
were added, in a 96-well microplate assay, to the L.
0
ꢀ
1
1
hyde: IR (KBr, cm ): 2945, 1692, 1604, 1576, 1510. H
NMR (DMSO-d , ppm): 9.9 (s, 2H, CHO); 7.9 (d, 4H,
J = 8 Hz,
6
6
donovani promastigotes culture (2 · 10 cells/ml). Controls
2
6
C
6
H
4
:H ,H ); 7.1 (d, 4H, J = 8 Hz,
with corresponding dilutions of DMSO indicated that
growth of the parasite was not altered under those
experimental conditions. The plates were incubated at
26 ꢂC for 72 h and growth of Leishmania promastigotes
was determined by Alamar blue assay. See, Mikus, J.;
Steverding, D. Parasitol. Int. 2000, 48, 265.
3
5
C H :H ,H ); 4.2 (t, 4H, J = 6 Hz, O–CH –CH CH2);
2ꢀ
.8 (m, 4H, J = 6 Hz, O–CH
6
4
2
1
2
–CH
2 2
–CH ); 1.5 (m, 2H,
J = 6 Hz, O–CH
Yield: 90%.
yl e` ne)]bis-1H-benzimidazole (8): IR (KBr, cm ): 3623–
2
–CH
2
0
–CH
2,2 -[1,5-Pentanediylbis(oxy-1,4-phen-
2
). Mp (ꢂC): 90.7–94.5 ꢂC.
ꢀ
1
1
2
d
(
400 (N–H bz), 1762, 1611,1500, 1254. H NMR (DMSO-
23. The compounds were tested on Vero cells (monkey
kidney fibroblast) by a Neutral Red assay. See,
0
0
6
2
6
, ppm): 8.1 (d, 4H, J = 9 Hz, C
0
6
H
0
4
:H ,H ); 7.6
0
4
7
0
AA BB , 4H, benz: H ,H ); 7.2 (AA BB , 4H, benz:
Babich, H.; Borenfreund, E. Appl. Environ. Microbiol.
1991, 57, 2101.
0
0
5
5
6
3
6 4
H ,H ); 7.1 (d, 4H, J = 9 Hz, C H :H ,H ); 4.1 (t, 4H,