Benzimidazole derivatives endowed with potent antileishmanial activity

Article abstract of DOI:10.1080/14756366.2017.1410480

Two sets of benzimidazole derivatives were synthesised and tested in vitro for activity against promastigotes of Leishmania tropica and L. infantum. Most of the tested compounds resulted active against both Leishmania species, with IC₅₀ values in the low micromolar/sub-micromolar range. Among the set of 2-(long chain)alkyl benzimidazoles, whose heterocyclic head was quaternised, compound 8 resulted about 100-/200-fold more potent than miltefosine, even if the selectivity index (SI) versus HMEC-1 cells was only moderately improved. In the set of 2-benzyl and 2-phenyl benzimidazoles, bearing a basic side chain in position 1, compound 28 (2-(4-chlorobenzyl)-1-lupinyl-5-trifluoromethylbenzimidazole) was 12-/7-fold more potent than miltefosine, but exhibited a further improved SI. Therefore, compounds 8 and 28 represent interesting hit compounds, susceptible of structural modification to improve their safety profiles.

Full text of DOI:10.1080/14756366.2017.1410480

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Benzimidazole derivatives endowed with potent
antileishmanial activity
Michele Tonelli, Elena Gabriele, Francesca Piazza, Nicoletta Basilico, Silvia
Parapini, Bruno Tasso, Roberta Loddo, Fabio Sparatore & Anna Sparatore
To cite this article: Michele Tonelli, Elena Gabriele, Francesca Piazza, Nicoletta Basilico, Silvia
Parapini, Bruno Tasso, Roberta Loddo, Fabio Sparatore & Anna Sparatore (2018) Benzimidazole
derivatives endowed with potent antileishmanial activity, Journal of Enzyme Inhibition and Medicinal
Chemistry, 33:1, 210-226, DOI: 10.1080/14756366.2017.1410480
To link to this article: https://doi.org/10.1080/14756366.2017.1410480
© 2017 The Author(s). Published by Informa
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Date: 16 December 2017, At: 14:04

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 33, NO. 1, 210–226
https://doi.org/10.1080/14756366.2017.1410480
RESEARCH PAPER
Benzimidazole derivatives endowed with potent antileishmanial activity
Michele Tonellia , Elena Gabrieleb , Francesca Piazzab, Nicoletta Basilicoc, Silvia Parapinid, Bruno Tassoa,
Roberta Loddoe, Fabio Sparatorea and Anna Sparatoreb
a
b
ꢀ
ꢀ
Dipartimento di Farmacia, Universita di Genova, Genova, Italy; Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Milano,
c
d
ꢀ
Milano, Italy; Dipartimento di Scienze Biomediche Chirurgiche e Odontoiatriche, Universita degli Studi di Milano, Milano, Italy; Dipartimento di
Scienze Farmacologiche e Biomolecolari, Universita degli Studi di Milano, Milano, Italy; Dipartimento di Scienze e Tecnologie Biomediche,
Universita di Cagliari, Cittadella Universitaria, Monserrato, Italy
e
ꢀ
ꢀ
ABSTRACT
ARTICLE HISTORY
Received 27 October 2017
Revised 22 November 2017
Accepted 22 November 2017
Two sets of benzimidazole derivatives were synthesised and tested in vitro for activity against promasti-
gotes of Leishmania tropica and L. infantum. Most of the tested compounds resulted active against both
Leishmania species, with IC50 values in the low micromolar/sub-micromolar range. Among the set of
2-(long chain)alkyl benzimidazoles, whose heterocyclic head was quaternised, compound 8 resulted about
100-/200-fold more potent than miltefosine, even if the selectivity index (SI) versus HMEC-1 cells was only
moderately improved. In the set of 2-benzyl and 2-phenyl benzimidazoles, bearing a basic side chain in
position 1, compound 28 (2-(4-chlorobenzyl)-1-lupinyl-5-trifluoromethylbenzimidazole) was 12-/7-fold more
potent than miltefosine, but exhibited a further improved SI. Therefore, compounds 8 and 28 represent
interesting hit compounds, susceptible of structural modification to improve their safety profiles.
KEYWORDS
Leishmania tropica and
infantum; promastigotes;
anti-leishmania agents; alkyl
benzimidazolium salts;
2-benzyl-1-lupinylbenzimi-
dazole derivatives
SI
IC50
Compd.
(µM)
HMEC Vero76
L. tropicaa
5.05
>27.1
-
L. infantuma
10.09
>13.6
-
L. tropicaa
L. infantuma
L. infantumb
0.19
0.34
0.31
4.10
2.29
-
30.5
17.1
-
L. tropicaa
3.70
4.76
4.58
3.61
>27
>21
L. infantuma
L. tropicaa
L. infantuma
L. infantumb
43.26
31.26
1.05
2.3
3.2
-
-
-
-
Miltefosine
apromastigotes; bamastigotes
ꢀ
CONTACT Michele Tonelli
basilico@unimi.it
Sparatore
tonelli@difar.unige.it
Dipartimento di Farmacia, Universita degli Studi di Genova, 16132 Genova, Italy; Nicoletta Basilico
nicoletta.
ꢀ
Dipartimento di Scienze Biomediche Chirurgiche e Odontoiatriche, Universita degli Studi di Milano, Via Pascal 36, Milano, 20133 Italy; Anna
anna.sparatore@unimi.it Dipartimento di Scienze e Tecnologie Biomediche, Universita degli Studi di Milano, Via L. Mangiagalli, 25, 20133 Milano,
Italy
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
211
paromomycin and pentamidine are considered second-line
drugs3a–c. Some other drugs as edelfosine, sitamaquine, fexinida-
zole, tamoxifene, imiquimod and pentoxyphylline are reported to
give variable cure rates when used either alone or, better, in asso-
ciation with antimonials to overcome resistance4 (Figure 1).
Introduction
After malaria, leishmaniasis is the second most prevalent parasite
infection worldwide for mortality in humans1. It is transmitted by
the bite of a sand-fly infected by a flagellate protozoan of the
genus Leishmania. Three different forms of the disease are
described: visceral, cutaneous and muco-cutaneous leishmaniasis.
The disease is endemic in many tropical and subtropical Countries,
leading annually to an estimated 700,000–1 million new cases and
20,000–30,000 deaths1, mostly due to the visceral form caused by
Leishmania donovani. The parasite exists in the ovoid non-flagel-
late form (amastigote) and in the flagellate promastigote, found in
the sand-fly.
All these drugs may cause several side effects and most of
them are also expensive, and thus out of reach for the poor peo-
ple living in tropical and sub-tropical countries, where the disease
is endemic. The cited drugs exhibit very different chemical struc-
tures and hit a variety of biological targets, but in several cases
the mechanism of action is still undefined or only partially known.
To meet the need of novel more efficacious, safe and unexpen-
sive drugs to treat leishmaniasis, a number of studies are on-
going, exploring a wide chemical space from several classes of
natural products5 and or their semi-synthetic derivatives (sterols5,
The therapy of leishmaniasis is still based on pentavalent anti-
monials (sodium stibogluconate and meglumine antimoniate) as
first choice drug2a,2b
,
whereas amphotericin B, miltefosine, mono-, sequi-, di- and tri-terpens6, alkaloids7, flavonoids8, etc.) to
Figure 1. First and second line or synergistic agents to treat leishmaniasis: (a) meglumine antimoniate (predominant species in aqueous solution); (b) sodium stibogluc-
onate (predominant species in aqueous solution); (c) amphotericin B; (d) miltefosine; (e) edelfosine; (f) paromomycin; (g) pentamidine; (h) sitamaquine; (i) imiquimod; (j)
tamoxifene; (k) fexinidazole; (l) pentoxyphylline.

212
M. TONELLI ET AL.
the most diversified synthetic compounds, from the simple chlor-
Among the benzazolic derivatives, an important position is
oacetoanilides9, to organometallics10a (as auranofin10b), aryldisele- held by the 2-trifluoromethyl-17 and 2-arylbenzimidazole18 deriva-
nides11, adamantylidene alkyl phosphocoline12 and a variety of tives that, besides activity versus several other protozoa, display
heterocycles13, particularly indole14, indazole15, benzotriazole16 antileishmanial action with potency in the low micromolar range.
and benzimidazole17–20 derivatives. Examples of these compounds Interestingly, some bis-benzimidazoles19,20 exhibit sub-micromolar
are depicted in Figures 2 and 3.
IC50, resulting 7- to 26-fold more potent than pentamidine.
Figure 2. Examples of investigational anti-leishmanial agents5–16
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
213
Figure 3. Benzimidazole derivatives previously tested as anti-leishmanial agents17–20
.
Since many years we are interested in the chemistry and bio- responsible for cutaneous leishmaniasis (CL), and 33 of them
logical properties of benzimidazole derivatives, pursuing varied
pharmacological aims, from analgesic-anti-inflammatory action21
(depending on availability) were also tested against L. infantum,
the causative agent of visceral leishmaniasis (VL). The two best
compounds were also assayed against L. infantum amastigotes.
,
conditioned avoidance response (CAR) inhibition22, choleretic
activity and gastric protection23, antiviral24 and antitumoral25 activ-
ities. In order to further explore the biocidal potential of benzimi-
dazole derivatives, we deemed interesting to evaluate the
antileishmanial activity of a set of 2-alkyl/2-benzyl benzimidazoles
whose heterocyclic head was quaternised to mimic the ammo-
nium head of miltefosine and edelfosine. Additionally, we selected,
among our in house library of benzimidazoles, a second set of
2-arylbenzimidazoles 1-substituted with basic side chains that
might be loosely related to sitamaquine. As the anti-leishmanial
activity of sitamaquine analogues is mainly related to the length
and structure of their basic side chains4a, in this subset of benzimi-
dazoles a variety of basic chains, featured by different sizes, steric
hindrance and lipophilicity, have been included. The bicyclic qui-
nolizidine (lupinyl) moiety is of particular relevance, having been
shown to produce analogous or superior activity against
Leishmania promastigotes in comparison to sitamaquine4c when
replacing the diethylaminohexyl side chain of the latter (our
unpublished results). On the whole 38 compounds (Figures 4 and
Materials and methods
General
Chemicals, solvents and reagents used for the syntheses were pur-
chased from Sigma-Aldrich, Fluka or Alfa Aesar, and were used
without any further purification unless otherwise stated. CC ¼ flash
column chromatography. Melting points (uncorrected) were deter-
1
mined with a Buchi apparatus. H NMR and 13C NMR spectra were
€
recorded with a Varian Mercury 300VX or Varian Gemini-200 spec-
trometers in CDCl3 or acetone-d6; the chemical shifts were
expressed in ppm (d), coupling constants (J) in Hertz (Hz). High-
resolution mass spectra (HRMS) were performed on a FT-Orbitrap
mass spectrometer in positive electrospray ionisation (ESI).
Elemental analyses were performed on a Carlo Erba EA-1110 CHNS
instrument in the Microanalysis Laboratory of the Department of
Pharmacy of Genoa University. Compounds were generally charac-
5) were tested against the promastigotes of Leishmania tropica, terised by 1H and 13C NMR spectra and elemental analysis or

214
M. TONELLI ET AL.
Figure 4. Investigated benzimidazole derivatives without basic side chain.
HRMS; a few intermediates were characterised by elemental ana- (50 MHz, CDCl3): 154.1, 136.5, 121.1, 113.6, 30.9, 28.6, 28.4, 28.3,
1
lysis and H NMR.
27.2, 21.6, 13.0. Anal. Calcd for C22H36N2: C, 80.43; H, 11.04; N,
8.53. Found: C, 80.50; H, 11.39; N, 8.42.
General procedure for the synthesis of 1 H-benzimidazoles 1, 6
General procedure for the synthesis of 2-alkyl-1-methyl-1 H-
benzimidazoles (2, 7) and 2-alkyl-1,3-dimethyl-1 H-benzimidazol-
3-ium iodides (3, 8)
Benzene-1,2-diamine (500 mg, 4.62 mmol) and the appropriate acid
(5.55 mmol) were stirred at 145 ꢀC for 24 h under inert atmosphere.
The resulting residue was purified by CC (silica gel; eluent as indi-
cated for each compound). These compounds were already To a solution of the appropriate 1H-benzimidazole (1 or 6,
obtained through different procedure26,27
.
0.37 mmol) in anhydrous THF (2 ml), K2CO3 (50.7 mg, 0.37 mmol)
2-Undecyl-1 H-benzimidazole (1): CC (silica gel; cyclohexane/ and methyl iodide (102 lL, 1.65 mmol) were added. The mixture
EtOAc; in gradient up to 92:8). The solid residue was rinsed with was stirred at 40 ꢀC for 76 h under inert atmosphere. After cool-
petroleum ether and the title compound was obtained as a white ing at room temperature, inorganic salts were filtered and the
solid. Yield: 32%. m.p. 108.1–109.3 ꢀC (lit.26, 107.5 ꢀC). 1H NMR solution was evaporated under reduced pressure. The resulting
(300 MHz, CDCl3): 9.41 (s, 1H, NH). 7.56 (dd, 2H, J ¼ 5.9 and 3.1 Hz), residue was treated with ethyl ether and rinsed with the same
7.22 (dd, 2H, J ¼ 5.9 and 3.1 Hz), 2.94 (t, 2H, J ¼ 7.7 Hz), 1.91–1.81 solvent giving compound 3 or 8 as a white-cream solid. The
(m, 2H), 1.39–1.23 (m, 16H), 0.88 (t, 3H, J ¼ 6.6 Hz). 13C NMR ethereal solution was then purified by CC (silica gel; eluent as
(50 MHz, CDCl3): 154.5, 137.3, 121.1, 113.5, 30.9, 28.6, 28.4, 28.35, indicated for each compound). Compounds 2, 3 and 8 were
28.3, 27.4, 21.6, 13.1. Anal. Calcd for C18H28N2: C, 79.36; H, 10.36; already described in the literature27–29, obtained by different
N, 10.28. Found: C, 79.29; H, 10.53; N, 10.06.
methods.
1-Methyl-2-undecyl-1H-benzimidazole (2): CC (CH2Cl2; isocratic).
2-Pentadecyl-1 H-benzimidazole (6): CC (silica gel; CH2Cl2/MeOH;
in gradient up to 99.4:0.6). The solid residue was rinsed with ethyl The solid residue was rinsed with ethyl ether and the final product
ether and the title compound was obtained as a white solid. Yield: was obtained as a white-cream solid. Yield: 45%. m.p. 40.8–43.4 ꢀC
63%. m.p. 88.8–94.2 ꢀC (lit.26, 96.5–97; lit27, 98–100 ꢀC). 1H NMR (lit.28, yellow oil). 1H NMR (300 MHz, CDCl3): 7.75–7.72 (m, 1 H),
(300 MHz, CDCl3): 9.36 (s, 1H, NH), 7.56 (dd, 2H, J ¼ 5.8 and 3.1 Hz), 7.41–7.23 (m, 3H), 3.74 (s, 3H), 2.90 (t, 2H, J ¼ 7.7 Hz), 1.87–1.84 (m,
7.22 (dd, 2H, J ¼ 5.8 and 3.1 Hz), 2.29 (t, 2H, J ¼ 7.7 Hz), 1.90–1.80 2H), 1.45–0.85 (m, 19H); conforming to the previously described
(m, 2H), 1.39–1.24 (m, 24H), 0.88 (t, 3H, J ¼ 6.5 Hz). 13C NMR spectrum28
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
215
Figure 5. Investigated benzimidazole derivatives with basic side chain.
1,3-Dimethyl-2-undecyl-1 H-benzimidazol-3-ium iodide (3): Yield: 14.1. HRMS (ESI) m/z Calcd for C24H41N2 [M]þ: 357.3264; found:
þ
22%. m.p. 157.3–161.3 ꢀC (lit.29 167–168 ꢀC). 1H NMR (300 MHz,
357.3263.
CDCl3): 7.68–7.62 (m, 4H), 4.11 (s, 6H), 3.55 (t, 2H, J ¼ 7.2 Hz),
1.74–1.73 (m, 2H), 1.60–1.59 (m, 2H), 1.48–1.47 (m, 2H), 1.25–1.24
(m, 12H), 0.87–0.86 (m, 3H).13C NMR (75 MHz, CDCl3): 154.0, 131.4,
General procedure for the synthesis of 1-methyl-3-propyl-1 H-
benzimidazol-3-ium iodides 4 and 12
126.7, 112.5, 33.2, 31.6, 29.3, 29.1, 29.0,þ28.9, 27.1, 26.1, 22.4, 13.8.
HRMS (ESI) m/z Calcd for C20H33N2 [M]þ: 301.2638; found:
To a solution of the appropriate 1-methyl-1H-benzimidazole (2 or
301.2637.
1-Methyl-2-pentadecyl-1H-benzimidazole (7): CC (CH2Cl2; iso-
7, 0.16 mmol) in anhydrous THF (1 mL), 1-iodopropane (160 lL,
1.640 mmol) was added. The mixture was stirred at reflux for
24–60 h under nitrogen. After cooling at room temperature, ethyl
ether was added to the reaction and the formed solid was then fil-
tered and rinsed with the same solvent giving compound 4 or 12
as a white solid.
1-Methyl-3-propyl-2-undecyl-1 H-benzimidazol-3-ium iodide (4):
Yield: 57%. m.p. 140.2–145.0 ꢀC. 1H NMR (300 MHz, CDCl3):
7.71–7.61 (m, 4H), 4.40 (t, 2H, J ¼ 7.2 Hz), 4.17 (s, 3H), 3.54 (t, 2H,
J ¼ 6.9 Hz), 2.05–2.03 (m, 2H), 1.74–1.73 (m, 2H), 1.55–1.52 (m, 3H),
1.25–1.24 (m, 13H), 1.10 (t, 3H, J ¼ 7.4 Hz), 0.87–0.86 (m, 3H).13C
cratic). The solid residue was rinsed with petroleum ether and the
final product was obtained as a white-cream solid. Yield: 17%.
m.p. 64.2–65.6 ꢀC. 1H NMR (300 MHz, CDCl3): 7.74–7.71 (m, 1H),
7.32–7.23 (m, 3H), 3.74 (s, 3H), 2.88 (t, 2H, J ¼ 7.7 Hz), 1.92–1.82 (m,
2H), 1.45–1.25 (m, 24H), 0.88 (t, 3H, J ¼ 6.6 Hz). Anal. Calcd for
C23H38N2: C, 80.64; H, 11.18; N, 8.18. Found: C, 80.44; H, 11.20; N,
7.90.
1,3-Dimethyl-2-pentadecyl-1 H-benzimidazol-3-ium iodide (8):
Yield: 43%. m.p. 169.0–172.0 ꢀC (lit.27 187–188 ꢀC). 1H NMR
(300 MHz, CDCl3): 7.72–7.70 (m, 2H), 7.69–7.59 (m, 2H), 4.11 (s, NMR (75 MHz, CDCl3): 153.7, 131.8, 130.9, 126.8, 112.9, 112.7, 48.1,
33.7, 31.8, 29.5, 29.4, 29.3, 29.2, 29.1, 29.0, 27.1, 26.1, 23.1, 22.6,
6H), 3.55 (t, 2H, J ¼ 7.7 Hz), 1.77–1.60 (m, 2H), 1.50–1.18 (m, 24H),
14.1, 11.5. HRMS (ESI) m/z Calcd for C22H37N2 [M]þ: 329.2951;
þ
0.87 (t, 3H, J ¼ 6.6 Hz).13C NMR (75 MHz, CDCl3): 154.2, 131.6,
found: 329.2949.
126.9, 112.7, 33.3, 31.8, 29.6, 29.5, 29.3, 29.2, 27.3, 26.3, 22.6,

216
M. TONELLI ET AL.
1-Methyl-2-pentadecyl-3-propyl-1H-benzimidazol-3-ium iodide (12): 1-(Ferrocenylmethyl)-2-pentadecyl-1 H-benzimidazole (16)
Yield: 28%. m.p. 144.7–147.4 ꢀC. 1H NMR (300 MHz, CDCl3):
7.73–7.59 (m, 4H), 4.40 (t, 2H, J ¼ 7.5 Hz), 4.18 (s, 3H), 3.56 (t, 2H,
J ¼ 7.7 Hz), 2.08–2.01 (m, 2H), 1.77–1.70 (m, 2H), 1.59–1.53 (m, 3H),
1.40–1.25 (m, 21H), 1.12 (t, 3H, J ¼ 7.4 Hz), 0.87 (t, 3H, J ¼ 6.0 Hz).
13C NMR (75 MHz, CDCl3): 153.7, 131.8, 130.9, 126.8, 112.9, 112.7,
48.1, 33.7, 31.8, 29.6, 29.5, 29.4, 29.3, 29.1, 27.8, 26.1, 23.1, 22.6,
To a mixture of K2CO3 (63 mg, 0.45mmol) and 2-pentadecyl-1H-ben-
zimidazole (compound 6, 0.30 mmol) in anhydrous THF/CH3CN
(1:3.5mL), ferrocenylmethyl trimethylammonium iodide (117 mg,
0.46 mmol) was added. The reaction was stirred at room tempera-
ture for 18 h under inert atmosphere. After the completion of reac-
tion, the solution was evaporated under reduced pressure. The
resulting residue was taken up with CH2Cl2 and washed several
times with H2O. The organic layer was dried with anhydrous
Na2SO4, filtered and evaporated to dryness to obtain a pale orange
oil, which was purified by CC (silica gel; CH2Cl2; isocratic). The title
compound was obtained as a pale yellow solid. Yield: 75%. m.p.
þ
14.1, 11.5. HRMS (ESI) m/z Calcd for C26H45N2 [M]þ: 385.3577;
found: 385.3580.
General procedure for the synthesis of 2-alkyl-1,3-dibenzyl-1 H-
benzimidazol-3-ium chlorides 5, 15 and 1-benzyl-2-pentadecyl-
1 H-benzimidazole 13
1
77.2–78.7 ꢀC. H NMR (300 MHz, CDCl3): 7.79 (d, 1H, J ¼ 6.3 Hz), 7.38
(s, 1H), 7.23–7.22 (m, 2H), 5.05 (s, 2H), 4.42–4.11 (m, 9H), 2.90 (t, 2H,
J ¼ 7.1 Hz), 1.89–1.88 (m, 2H), 1.64–1.25 (m, 24H), 0.88–0.85 (m, 3H).
Hydrochloride: m.p. 149.8–150.2 ꢀC. Anal. calcd for C33H47ClFeN2: C,
70.40; H, 8.41; N, 4.98. Found: C, 70.42; H, 8.91; N, 5.02.
To a mixture of K2CO3 (70 mg, 0.50 mmol) and the appropriate
1 H-benzo[d]imidazole (1 or 6, 0.30 mmol) in anhydrous THF
(2.5 ml), benzyl chloride (183 lL, 1.52 mmol) was added, then
stirred at reflux for 60 h under inert atmosphere. After cooling at
room temperature, inorganic salts were filtered and the solution
was evaporated under reduced pressure. The resulting residue was
treated with THF and rinsed with the same solvent giving com-
pound 5 or 15 as a white-cream solid. The solution was then puri-
fied by CC (silica gel; eluent as indicated for each compound).
1,3-Dibenzyl-2-undecyl-1H-benzimidazol-3-ium chloride (5): Yield:
77%. m.p. 223.3–225.3 ꢀC. 1H NMR (300 MHz, CDCl3): 7.60–7.51 (m,
4H), 7.37–7.26 (m, 10H), 5.90 (s, 4H), 3.64 (t, 2H, J ¼ 7.7 Hz),
1.20–0.99 (m, 18H), 0.86 (t, 3H, J ¼ 6.7 Hz). 13C NMR (75 MHz,
CDCl3): 155.9, 133.4, 131.7, 129.4, 128.8, 126.9, 126.7, 113.3, 49.8,
31.8, 29.6, 29.4, 29.3, 29.2, 29.1, 28.8, 27.3, 26.1, 22.6, 14.1. HRMS
1-(Ferrocenylmethyl)-3-methyl-2-pentadecyl-1 H-benzimidazol-3-
ium iodide (17)
Methyl iodide (200 lL, 3.24 mmol) was added to a solution of
1-ferrocenyl-3-methyl-2-pentadecyl-1 H-benzo[d]imidazole
(com-
pound 16, 0.09 mmol) in anhydrous ethyl ether (1.5 mL). The reac-
tion was stirred at 40 ꢀC for 80 h under inert atmosphere. After
cooling at room temperature, the formed solid was filtered and
rinsed with ethyl ether giving compound 17 as a white solid.
Yield: 52%. m.p. 162.2–165.7 ꢀC. 1H NMR (300 MHz, CDCl3):
7.74–7.73 (m, 1H), 7.60–7.59 (m, 3H), 5.55 (s, 2H), 4.36–4.21 (m,
9H), 4.05 (s, 3H), 3.53–3.52 (m, 2H), 1.52–1.51 (m, 4H), 1.26–1.24
(m, 22H), 0.88–0.87 (m, 3H). 13C NMR (75 MHz, CDCl3): 153.8, 131.6,
130.9, 126.7, 113.0, 112.6, 79.3, 69.4, 69.3, 47.1, 33.1, 31.9, 29.7,
29.6, 29.5, 29.3, 29.2, 27.5, 26.6, 22.6, 14.1. HRMS (ESI) m/z Calcd
for C34H49N2Feþ [M]þ: 541.3240; found: 541.3234.
(ESI) m/z Calcd for C32H41N2 [M]þ: 453.3264; found: 453.3257.
þ
1-Benzyl-2-pentadecyl-1H-benzimidazole (13): CC (CH2Cl2; iso-
cratic). The solid residue was rinsed with cold MeOH and the final
product was obtained as a white-cream solid. Yield: 17%. m.p.
1
60.7–61.8 ꢀC. HNMR (300 MHz, CDCl3): 7.78–7.75 (d, 1H, J ¼ 7.5 Hz),
7.30–7.18 (m, 6H), 7.05–7.03 (m, 2H), 5.34 (s, 2H), 2.82 (t, 2H,
J ¼ 7.6 Hz), 1.87–1.77 (m, 2H), 1.34–1.25 (m, 24H), 0.88 (t, 3H,
J ¼ 6.5 Hz). Anal. calcd for C29H42N2: C, 83.20; H, 10.11; N, 6.69.
Found: C, 83.16; H, 10.41; N, 6.86.
2-(Heptan-4-yl)-1 H-benzimidazole (18)
1,3-Dibenzyl-2-pentadecyl-1H-benzimidazol-3-ium chloride (15): CC 2-Propylpentanoyl chloride (338 mg, 2.08 mmol) was added at 0 ꢀC
(silica gel; CH2Cl2/MeOH; in gradient up to 99.5:0.5). The solid resi- to a solution of benzene-1,2-diamine (225 mg, 2.08 mmol) in
due was rinsed with THF and the final product was obtained as a
white-cream solid. Yield: 40%. m.p. 214.9–216.3 ꢀC. 1H NMR
(300 MHz, CDCl3): 7.56–7.54 (m, 4H), 7.36–7.29 (m, 10H), 5.90 (s, 4H),
3.67–3.65 (m, 2H), 1.25–0.87 (m, 29H). 13C NMR (75 MHz, CDCl3):
156.0, 133.3, 131.6, 129.4, 128.8, 126.9, 126.7, 113.2, 49.8, 31.8, 29.7,
29.6, 29.5, 29.4, 29.þ3, 29.0, 28.8, 27.3, 26.1, 22.6, 14.1. HRMS (ESI) m/z
Calcd for C36H49N2 [M]þ: 509.3890; found: 509.3878.
anhydrous 1,4-dioxane (1 mL) and the reaction mixture was stirred
at room temperature for 15 h under nitrogen. After that time,
BF3 Et2O (263 lL) was added and the mixture was stirred at reflux
.
for other 12 h. The solvent was then stripped off and the obtained
residue was diluted with EtOAc, washed with a cold solution of
5% HCl and with 2 M NaOH. The organic layer was dried with
anhydrous Na2SO4, filtered and evaporated to dryness to obtain a
residue that was purified by CC (silica gel; CH2Cl2/cyclohexane; in
gradient up to 80:20). The fractions containing the purified prod-
uct were gathered up and rinsed with diethyl ether to provide a
white solid. Yield: 18%. m.p. 224.7–225.8 ꢀC. 1H NMR (300 MHz,
acetone-d6): 7.49–7.46 (m, 2H), 7.39 (s, 1H), 7.13–7.11 (m, 2H),
3.03–2.95 (m, 1H), 1.91–1.81 (m, 2H), 1.79–1.64 (m, 2H), 1.33–1.20
(m, 4H), 0.89–0.84 (m, 6H). 13C NMR (50 MHz, CDCl3): 157.8, 136.8,
121.2, 113.6, 39.4, 36.1, 19.2, 12.9. Anal. calcd for C14H20N2: C,
77.73; H, 9.32; N, 12.95. Found: C, 77.71; H, 9.66; N, 12.83.
3-Benzyl-1-methyl-2-pentadecyl-1 H-benzimidazol-3-ium chloride
(14)
Benzyl chloride (168 lL, 1.43 mmol) was added to a solution of
1-methyl-2-pentadecyl-1H-benzimidazole (compound 7, 0.15 mmol)
in anhydrous THF (1 ml). The reaction was stirred at reflux for 80 h
under inert atmosphere. After cooling at room temperature, the
formed solid was filtered and rinsed first with THF and then with
ethyl ether, providing compound 14 as a white solid. Yield: 18%.
m.p. 215.7–219.3 ꢀC. 1H NMR (300 MHz, CDCl3): 7.77–7.26 (m, 9H),
5.84 (s, 2H), 6.87 (s, 3H), 3.67 (s, 2H), 1.34–1.14 (m, 26H), 0.88 (t,
3H, J ¼ 6.1 Hz). 13C NMR (75 MHz, CDCl3): 155.5, 133.5, 131.8, 131.5,
129.4, 128.9, 126.9, 126.8, 112.9, 112.8, 77.0, 49.7, 32.9, 31.9, 29.7,
General procedure for the synthesis of 2-(heptan-4-yl)-1-methyl-
1 H-benzimidazole 19 and 1,3-dimethyl-2-(heptan-4-yl)-1 H-
benzimidazol-3-ium iodide 20
29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 27.2, 25.8, 22.7, 14.1. HRMS (ESI) To a mixture of K2CO3 (33.0 mg, 0.24 mmol) and 2-(heptan-4-yl)-
m/z Calcd for C30H45N2 [M]þ: 433.3577; found: 433.3576.
1H-benzimidazole (compound 18, 0.24 mmol) in anhydrous THF
þ

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
217
(1 mL), methyl iodide (814 lL, 13.14 mmol) was added. The reac- was basified with 2 N NaOH and shaken with CH2Cl2. The organic
tion was stirred at 40 ꢀC for 26 h under inert atmosphere. After layer was dried (Na2SO4) and evaporated to afford the benzimida-
cooling at room temperature, inorganic salts were filtered and the zole that was thoroughly washed with dry Et2O/hexane (1:1).
solution was evaporated under reduced pressure. The resulting 2-Pentadecyl-5-trifluoromethyl-1H-benzimidazole was yield as an
residue was treated with ethyl ether and rinsed with the same oil and was used as such for the preparation of the corresponding
solvent giving compound 20 as a white solid. The ethereal solu- 1-methylbenzimidazole derivative.
tion was then purified by CC (silica gel; eluent as indicated for
each compound).
2-Pentadecyl-5-trifluoromethyl-1 H-benzimidazole (43): Yield: 76%.
Oil. H NMR (200 MHz, CDCl3): 9.24 (s, 1H, NH, collapses with D2O),
1
2-(Heptan-4-yl)-1-methyl-1 H-benzimidazole (19): CC (CH2Cl2; iso- 8.21 (s, 1H), 8.08 (d, 1H, J¼ 8.6 Hz), 7.80 (d, 1H, J ¼ 8.6 Hz), 2.45 (t,
cratic). The title compound was obtained as a pale grey oil. Yield: 2H, J ¼ 7.0 Hz), 1.84–1.1.59 (m, 2H), 1.29 (pseudo s, 24H), 0.91 (t,
47%. 1H NMR (300 MHz, CDCl3): 7.80–7.77 (m, 1H), 7.33–7.24 (m, 3H, J ¼ 6.8 Hz). Anal. calcd for C23H35F3N2: C, 69.67; H, 8.90; N, 7.06.
3H), 3.76 (s, 3H), 3.03–2.97 (m, 1H), 2.01–1.89 (m, 2H), 1.81–1.70 Found: C, 69.45; H, 9.00; N, 8.75.
(m, 2H), 1.34–1.17 (m, 4H), 0.87 (t, 6H, J ¼ 7.4 Hz). 13C NMR
5-Nitro-2-pentadecyl-1 H-benzimidazole (44): Yield: 45%. m.p.
(50 MHz, CDCl3): 155.6, 130.7, 129.9, 125.4, 124.9, 114.9, 110.0, 36.6, 86–88 ꢀC (hexane/Et2O an.). 1H NMR (200 MHz, CDCl3): 9.54 (s, 1H,
34.9, 30.5, 19.9, 12.7. Hydrochloride, m.p. 169.2–173.2 (EtOH/Et2O). NH, collapses with D2O), 8.52 (s, 1H), 8.21 (d, 1H, J ¼ 9.0 Hz), 7.64
Anal. calcd for C15H23ClN2: C 67.51, H 8.69, N 10.50, found: C (d, 1H, J ¼ 8.8 Hz), 3.04 (t, 2H, J ¼ 8.0 Hz), 2.03–1.80 (m, 2H), 1.26
67.71, H 8.88, N 10.40.
(pseudo s, 24H), 0.90 (t, 3H, J ¼ 6.8 Hz). Anal. calcd for C22H35N3O2:
1,3-Dimethyl-2-(heptan-4-yl)-1 H-benzimidazol-3-ium iodide (20): C, 70.74; H, 9.44; N, 11.25. Found: C, 70.60; H, 9.59; N, 11.55.
White powder. Yield: 25%. m.p. 192.2–192.9 ꢀC. 1H NMR (300 MHz,
5,6-Dichloro-2-pentadecyl-1 H-benzimidazole (45): Yield: 44%.
1
CDCl3): 7.88–7.86 (m, 2H), 7.68–7.65 (m, 2H), 4.25 (m, 6H), m.p. 74–76 ꢀC (hexane/Et2O an.). H NMR (200 MHz, CDCl3): 9.38 (s,
3.78–3.73 (m, 1H), 2.06–1.98 (m, 2H), 1.60–1.43 (m, 4H), 1.22–1.18 1H, NH, collapses with D2O), 7.60 (s, 1H), 7.26 (s, 1H), 3.01 (t, 2H,
(m, 2H), 0.98–0.93 (t, 6H, J ¼ 7.1 Hz). 13C NMR (75 MHz, CDCl3): J ¼ 8.0 Hz), 2.01–1.80 (m, 2H), 1.23 (pseudo s, 24H), 0.90 (t, 3H,
154.9, 133.1, 131.6, 127.4, 37.3, 34.5, 21.4, 13.9. HRMS (ESI) m/z J ¼ 6.6 Hz). Anal. calcd for C22H34Cl2N2: C, 66.49; H, 8.62; N, 7.05.
þ
Calcd for C16H25N2 [M]þ: 245.2012; found: 245.2011.
Found: C, 66.70; H, 8.95; N, 7.35.
General procedure for the synthesis of N-(2-aminophenyl)
palmitamide derivatives 40–42
General procedure for the synthesis of N-methyl-1 H-
benzimidazole derivatives 46–48, 50 and 51
To a solution of the proper 4- or 4,5-substituted 1,2-phenylendi-
amine (2.5 mmol) in THF (8 mL) in presence of Hunig base In a sealed tube, to a solution of the proper benzimidazole
(5 mmol), a solution of palmitoyl chloride (2.5 mmol) in 5 ml of THF (0.10 mmol) in 5 mL of THF were added, in the order, Cs2CO3
was added dropwise. The mixture was reacted at r.t. for 24 h with (0.30 mmol) and iodomethane (0.15 mmol). The mixture was
stirring. After removing the solvent, the residue was taken up heated at 60 ꢀC for 6–8 h with stirring. The solvent was evaporated
with water, alkalinised with 2 N NaOH and exhaustively extracted and the residue was taken up with water, alkalinised with 2 N
with CHCl3. The dried organic layer (Na2SO4) was concentrated to NaOH and extracted with CH2Cl2. After drying, the solvent was
dryness leaving a residue that was thoroughly washed with dry removed obtaining an oily residue that was washed with hexane.
Et2O/hexane (1:1).
N-Methyl-2-pentadecyl-5(6)-trifluoromethyl-1 H-benzimidazole (46):
N-[2-Amino-4(5)-trifluoromethylphenyl]palmitamide (40): White White powder. Yield: 90%. m.p. 65.8–67.9 ꢀC (hexane). 1H NMR
powder. Yield: 42%. m.p. 71–73.5ꢀC (hexane/Et2O an.). 1H NMR (200 MHz, CDCl3): 8.03 (s, 1H), 7.81 (d, 1H, J¼ 9.6 Hz), 7.51 (d, 1H,
(200 MHz, CDCl3): 7.63 (s, 1H, NHCO, collapses with D2O), 7.39 (s, J ¼ 9.6 Hz), 3.79 (s, 3H, NCH3), 2.90 (t, 2H, J ¼ 8.0 Hz), 2.01–1.80 (m,
1H), 7.26 (d, 1H, J ¼ 8.8 Hz), 6.75 (d, 1H, J ¼ 8.8 Hz), 3.92 (s, 2H, NH2, 2H), 1.28 (pseudo s, 24H), 0.89 (t, 3H, J ¼ 6.4 Hz). Anal. calcd for
collapse with D2O), 2.41 (t, 2H, J ¼ 7.8Hz), 1.87–1.56 (m, 2H), 1.29 C24H37F3N2: C, 70.21; H, 9.08; N, 6.82. Found: C, 69.72; H, 9.23; N,
(pseudo s, 24H), 0.91 (t, 3H, J ¼ 6.8Hz). Anal. calcd for C23H37F3N2O: 6.00.
C, 66.64; H, 9.00; N, 6.76. Found: C, 66.72; H, 9.09; N, 6.85.
N-Methyl-5(6)-nitro-2-pentadecyl-1H-benzimidazole (47): Yellowish
N-[2-Amino-4(5)-nitrophenyl]palmitamide (41): Yellowish powder. powder. Yield: 45%. m.p. 69.7–71.4 ꢀC (hexane). 1H NMR
Yield: 29%. m.p. 142–143 ꢀC (hexane/Et2O an.). 1H NMR (200 MHz, (200 MHz, CDCl3): 8.55 (s, 1H), 8.38 (d, 1H, J¼ 9.8 Hz), 7.68 (d,
CDCl3): 8.06 (br. s, 1H and 1H, NHCO, collapses with D2O, superim- 1H, J ¼ 9.8 Hz), 4.09 (s, 3H, NCH3), 3.28 (t, 2H, J ¼ 7.8 Hz),
posed), 7.42 (m, 1H), 6.82 (d, 1H, J ¼ 8.8 Hz), 3.40 (s, 2H, NH2, col- 2.09–1.85 (m, 2H), 1.28 (pseudo s, 24H), 0.90 (t, 3H, J ¼ 6.2 Hz).
lapse with D2O), 2.49 (pseudo s, 2H), 1.96–1.70 (m, 2H), 1.30 Anal. calcd for C23H37N3O2: C, 71.28; H, 9.62; N, 10.84. Found: C,
(pseudo s, 24 H), 0.92 (pseudo s, 3H). Anal. calcd for C22H37N3O3: C, 71.28; H, 9.67; N, 11.18.
67.49; H, 9.53; N, 10.73. Found: C, 67.74; H, 9.57; N, 10.93.
N-Methyl-5,6-dichloro-2-pentadecyl-1H-benzimidazole (48): White
1
N-(2-Amino-4,5-dichlorophenyl)palmitamide (42): White powder. powder. Yield: 42%. m.p. 64.8–67.3 ꢀC (hexane). H NMR (200 MHz,
Yield: 35%. m.p. 96–98 ꢀC (hexane/Et2O an.). 1H NMR (200 MHz, CDCl3): 8.15 (s, 1H), 7.69 (s, 1H), 3.97 (s, 3H, NCH3), 3.28 (t, 2H,
CDCl3): 7.40 (s, 1H, NHCO, collapses with D2O), 7.36 (s, 1H), 6.95 (s, J ¼ 8.4 Hz), 2.06–1.87 (m, 2H), 1.28 (pseudo s, 24H), 0.91 (t, 3H,
1H), 3.38 (s, 2H, NH2, collapse with D2O), 2.42 (t, 2H, J ¼ 7.8 Hz), J ¼ 6.4 Hz). Anal. calcd for C23H36Cl2N2: C, 67.14; H, 8.82; N, 6.81.
1.82–1.54 (m, 2H), 1.26 (pseudo s, 24H), 0.91 (t, 3H, J ¼ 7.0 Hz). Found: C, 67.17; H, 8.80; N, 7.15.
Anal. calcd for C22H36Cl2N2O: C, 63.60; H, 8.73; N, 6.74. Found: C,
63.51; H, 8.73; N, 7.00.
N-Methyl-2–(4-chlorobenzyl)-1 H-benzimidazole (50): White pow-
der. Yield: 23%. m.p. 117–119 ꢀC (hexane) conforming to the
literature30.
N-Methyl-2-(4-chlorobenzyl)-5-trifluoromethyl-1 H-benzimidazole
(51): White powder. Yield: 100%. Oil. 1H NMR (200 MHz, CDCl3):
7.80–7.04 (m, 7H), 4.36 (s, 2H), 3.95 (s, 3H, NCH3). Anal. calcd
General procedure for the synthesis of 2-pentadecyl-5/6-1H-
benzimidazole derivatives 43–45
The N-(2-aminophenyl)palmitamides (0.50 mmol) in 4 N HCl (10 mL) for C16H12ClF3N2: C, 59.18; H, 3.72; N, 8.63. Found: C, 59.30; H, 3.65;
were refluxed at 120 ꢀC for 4 h. After cooling, the acidic solution N, 8.49.

218
M. TONELLI ET AL.
General procedure for the synthesis of benzimidazole
quaternary ammonium salts 9–11, 21, 22 and 39
diluted with medium to achieve the required concentrations.
Drugs were placed in 96 wells round-bottom microplates and
seven serial dilutions made. Amphotericin B or miltefosine were
used as reference anti-Leishmania drugs. Parasites were diluted in
complete medium to 5 ꢁ 106 parasites/mL and 100 lL of the sus-
pension was seeded into the plates, incubated at 24 ꢀC for 72 h
and then 20 mL of MTT solution (5 mg/mL) was added into each
well for 3 h. The plates were then centrifuged at 1000 ꢁ g for
8 min at r.t., the supernatants discarded and the resulting pellets
dissolved in 100 mL of lysing buffer consisting of 20% (w/v) of a
solution of SDS (Sigma), 40% of DMF (Merck) in H2O. The absorb-
ance was measured spectrophotometrically at a test wavelength
of 550 nm and a reference wavelength of 650 nm. The results are
expressed as IC50 which is the dose of compound necessary to
inhibit parasite growth by 50%; each IC50 value is the mean of
separate experiments performed in duplicate.
(b) In vitro intracellular amastigote susceptibility assays. THP-1
cells (human acute monocytic leukaemia cell line) were maintained
in RPMI supplemented with 10% FBS, 50 lM 2-mercaptoethanol,
20 mM Hepes, 2 mM glutamine, at 37 ꢀC in 5% CO2. For Leishmania
infections, THP-1 cells were plated at 5 ꢁ 105 cells/mL in 16-cham-
ber Lab-Tek culture slides (Nunc) and treated with 0.1 lM phorbol
myristate acetate (PMA, Sigma) for 48 h to achieve differentiation
into macrophages. Cells were washed and infected with metacyclic
L. infantum promastigotes at a macrophage/promastigote ratio of
1/10 for 24 h. Cell monolayers were then washed and incubated
with compounds for 72 h. Slides were fixed with methanol and
stained with Giemsa. The percentage of infected macrophages in
treated and non-treated cells was determined by light microscopy.
The suitable N-methylbenzimidazole derivative or N-lupinyl-5-tri-
fluoromethyl-2-(4-chlorophenyl)benzimidazole (0.20 mmol) was
reacted with iodomethane (0.5 mL, 8 mmol) at r.t. for 24 h with stir-
ring. The reaction mixture was washed with dry Et2O affording the
title quaternary ammonium salt.
1,3-Dimethyl-2-pentadecyl-5-trifluoromethyl-1H-benzimidazol-3-ium
1
iodide (9): Yield: 83%. m.p. 116–118 ꢀC (Et2O an.). H NMR (200 MHz,
CDCl3): 8.10–7.70 (m, 3H), 4.15 (s, 6H), 3.52 (t, 2H, J ¼ 7.05 Hz),
1.93–1.64 (m, 2H), 1.57–0.80 (m, 27H). 13C NMR (50 MHz, CDCl3):
156.2, 132.7, 130.3, 122.7, 113.2, 109.6, 33.0, 32.6, 30.9, 28.6, 28.5,
28.4, 28.3, 28.1, 26.2, 26.0, 21.6, 13.1. Anal. calcd for C25H40F3IN2: C,
54.35; H, 7.30; N, 5.07. Found: C, 54.33; H, 6.92; N, 5.21.
1,3-Dimethyl-5-nitro-2-pentadecyl-1 H-benzimidazol-3-ium iodide
(10): Yield: 51%. m.p. 154–156 ꢀC (Et2O an.). 1H NMR (200 MHz,
CDCl3): 8.63 (s, 1H), 8.29–8.17 (m, 1H), 7.88 (d, 1H, J ¼ 8.10 Hz), 4.21
(s, 6H), 2.94 (t, 2H, J ¼ 7.15 Hz), 2.00–0.80 (m, 29H). 13C NMR
(50 MHz, CDCl3): 157.0, 133.0, 129.7, 117.0, 114.8, 107.7, 30.9, 29.2,
28.6, 28.4, 28.3, 26.7, 26.3, 21.7, 13.1; Anal. calcd for C24H40IN3O2:
C, 54.44; H, 7.61; N, 7.94. Found: C, 54.37; H, 7.63; N, 7.98.
5,6-Dichloro-1,3-dimethyl-2-pentadecyl-1 H-benzimidazol-3-ium
iodide (11): Yield: 100%. m.p. 200–203 ꢀC (Et2O an.). 1H NMR
(200 MHz, CDCl3): 7.96 (s, 2H), 4.14 (s, 6H), 3.51 (t, 2H, J ¼ 7.15 Hz),
1.84–0.75 (m, 29H). 13C NMR (50 MHz, CDCl3): 155.5, 130.8, 129.7,
113.4, 32.4, 30.9, 28.6, 28.3, 26.2, 21.7, 13.1. Anal. calcd for
C24H39Cl2IN2: C, 52.09; H, 7.10; N, 5.06. Found: C, 52.18; H, 7.17; N,
5.41.
2-(4-Chlorobenzyl)-1,3-dimethyl-1 H-benzimidazol-3-ium
iodide
(21): Yield: 35%. m.p. 94–98 ꢀC (Et2O an.). 1H NMR (200 MHz,
CDCl3): 8.10–7.00 (m, 8H), 4.32 (s, 3H), 4.17 (s, 3H), 3.45 (s, 2H). 13C
NMR (50 MHz, CDCl3): 128.6, 126.6, 126.0, 120.2, 111.9, 106.3, 64.0,
32.5, 26.1. Anal. calcd for C16H16ClIN2: C, 48.20; H, 4.05; N, 7.03.
Found: C, 42.45; H, 4.90; N, 6.16.
Cell cytotoxicity assays
(a) The long-term human microvascular endothelial cell line
(HMEC-1) was maintained in MCDB 131 medium (Invitrogen, Milan,
Italy) supplemented with 10% fetal calf serum (HyClone, Celbio,
Milan, Italy), 10 ng/mL of epidermal growth factor (Chemicon),
1 mg/mL of hydrocortisone, 2 mM glutamine, 100 U/mL of penicillin,
100 l g/mL of streptomycin and 20 mM Hepes buffer (EuroClone).
Unless stated otherwise, all reagents were from Sigma Italia, Milan,
Italy. For the cytotoxicity assays, cells were treated with serial dilu-
tions of test compounds and cell proliferation evaluated using the
MTT assay already described33. The results are expressed as IC50,
which is the dose of compound necessary to inhibit cell growth
by 50%.
2-(4-Chlorobenzyl)-1,3-dimethyl-5-trifluoromethyl-1 H-benzimida-
zol-3-ium iodide (22): Yield: 41%. m.p. 60 ꢀC (Et2O an.). 1H NMR
(200 MHz, CDCl3): 8.16–7.08 (m, 7H), 4.25 (s, 3H), 4.18 (s, 3H), 3.58
(s, 2H). 13C NMR (50 MHz, CDCl3): 153.4, 132.7, 130.4, 128.7, 126.1,
123.2, 113.4, 110.0, 64.1, 33.4, 33.1, 30.9, 28.9. Anal. calcd for
C17H15ClF3IN2: C, 43.75; H, 3.24; N, 6.00. Found: C, 43.92; H, 3.49; N,
6.00.
2-(4-Chlorophenyl)-3-methyl-1-{[5-methylammonio-(1 S,9aR)-octa-
hydroquinolizin-1-yl]- methyl}-5-trifluoromethyl-1 H-benzimidazol-3-
ium diiodide (39): Yield: 35%. m.p. 165–169 ꢀC (Et2O an.). 1H NMR
(200 MHz, DMSO): 8.75 (s, 1 arom. H), 8.56 (d, J ¼ 9.0, 1H), 8.07 (d,
J ¼ 8.5, 2H), 7.91 (d, J ¼ 8.5, 2H), 7.26 (s, 1H), 3.93 (s, 3H), 3.16 (s,
3H), 3.11–2.88 (m, 2H), 2.78–2.63 (m, 2H), 2.17–1.05 (m, 14H). 13C
NMR (DMSO): 151.5, 138.2, 133.1, 132.3, 131.5, 130.0, 127.2, 123.0,
119.0, 115.1, 66.0, 64.5, 50.3, 49.1, 47.2, 33.4, 33.0, 20.6, 19.1, 18.7,
18.1. Anal. calcd for C26H31ClF3I2N3: C, 40.52; H, 4.97; N 5.47.
Found: C, 40.67; H, 4.59; N 5.47.
(b) Vero-76 cells were seeded at an initial density of 4 ꢁ 105
cells/mL in 24-well plates, in culture medium (Dulbecco’s Modified
Eagle Medium (D-MEM) with L-glutamine, supplemented with foe-
tal bovine serum (FBS), 0.025 g/L kanamycin). Cell cultures were
then incubated at 37 ꢀC in a humidified, 5% CO2 atmosphere in
the absence or presence of serial dilutions of test compounds. Cell
viability was determined after 48–96 h at 37 ꢀC by the Crystal vio-
let staining method.
The results are expressed as CC50, which is the concentration of
compound necessary to inhibit cell growth by 50%. Each CC50
value is the mean and standard deviation of at least three separ-
ate experiments performed in duplicate.
Evaluation of anti-leishmanial activity
(a) Promastigote stage of L. infantum strain MHOM/TN/80/IPT1
(kindly provided by Dr M. Gramiccia, ISS, Roma) and L. tropica
(MHOM/IT/2012/ISS3130) were cultured in RPMI 1640 medium
(EuroClone) supplemented with 10% heat-inactivated fetal
calf serum (EuroClone), 20 mM Hepes, and 2 mM L-glutamine Results and discussion
at 24 ꢀC.
Synthesis
To estimate the 50% inhibitory concentration (IC50), the MTT
The 1-unsubstituted 2-alkylbenzimidazoles were prepared either
(3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) method
was used31,32. Compounds were dissolved in DMSO and then by dry heating at 145 ꢀC of a mixture of 1,2-phenylenediamine

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
219
with the suitable acid (1 and 6), or by treating the diamine with 1,3-dibenzyl benzimidazolium chlorides 5 and 15 were obtained,
but the mono-benzylated compound (13) was isolated only in the
case of 6 (Scheme 1). Compounds 2, 3 and 8 were already
described (see Materials and methods).
valproyl chloride, in dioxane solution, followed by the action of
ethereal boron trifluoride (18) (Schemes 1 and 4). The last
method, in contrast with the indication of Tandon and Kumar34
,
An attempt to improve the yield of 1-methyl-2-pentadecyl ben-
zimidazole (7) by reacting directly the palmitic acid with N-methyl-
1,2-phenylendiamine gave disappointing result (yield 17%).
To obtain the 5-substituted compounds 9–11, the 4-substituted
or 4,5-disubstituted-1,2-phenylenediamines were mono-acylated
with palmitoyl chloride and the monoamides 40–42 were cyclised
by the action of 4 N HCl. The benzimidazoles 43–45 were methy-
lated with methyl iodide in the presence of Cs2CO3 (46–48) and,
finally, quaternised at r.t. with excess of methyl iodide (Scheme 2).
The intermediates 40, 41, 46 and 47 (Scheme 2) could be a mix-
ture of two regioisomers, however we did not succeed in separat-
ing them, but it is not important for the structures of the final
compounds 9–11.
gave only a modest yield of benzimidazole, being prevailing the
formation of the N,N’-divalproyl-1,2-phenylendiamine (49).
Compounds 1 and 6 were already described (see Materials and
methods).
The treatment of the 1-unsubstituted benzimidazoles with
excess of methyl iodide, in the presence of anhydrous K2CO3, gave
place to mixtures of 1-methyl-2-substituted benzimidazoles (2, 7
and 19) and 1,3-dimethyl-2-substituted benzimidazolium iodides
(3, 8 and 20) (Schemes 1 and 4). The dimethylated compounds
were easily isolated being insoluble in dry ether, while the mono-
methylated compounds were separated from the N-unsubstituted
benzimidazoles by CC on silica, eluting with CH2Cl2. Similarly, by
treating compounds 1 and 6 with an excess of benzyl chloride the
Scheme 1. Reagents and conditions: (a) 145 ꢀC, N2, 24 h; (b) CH3I, THF, K2CO3, 40 ꢀC, 76 h; (c) C3H7I, THF, 24–60 h; (d) C6H5–CH2–Cl, THF, K2CO3, N2, reflux, 60 h; (e)
C6H5–CH2–Cl, THF, N2, reflux, 80 h.
Scheme 2. Reagents and conditions: (a) THF, N2, Hunig base (2 equiv), r.t., 24 h; (b) HCl 4 N, reflux, 4 h; (c) CH3I, THF, Cs2CO3, 60 ꢀC, 6–8 h; (d) CH3I excess, r.t., 24 h.
€

220
M. TONELLI ET AL.
The mono-methylated benzimidazoles 2 and 7 were converted L. tropica, while 33 of them were also tested against L. infantum,
using the MTT assay31,32. Results are expressed as IC50 SD (mM)
into the quaternary salts 4, 12 and 14 (Scheme 1), by heating
them with propyl iodide or with benzyl chloride for the latter. As
suggested by Howarth and Hanlon35 for analogous compounds,
by treating the 2-pentadecyl benzimidazole with (ferrocenylme-
thyl)trimethyl ammonium iodide at r.t., the 1-ferrocenylmethylben-
zimidazole 16 was obtained in high yield, the latter was then
quaternised with methyl iodide to 17 (Scheme 3).
and reported in Table 1, together with the corresponding selectiv-
ity indexes (ratio of IC50 versus human microvascular endothelial
cell line (HMEC-1), or monkey kidney cells (Vero76), and IC50 of
compounds versus the two Leishmania species.
The results collected in Table 1 show that most of the tested
compounds were active against L. tropica (30 over 38) and
L. infantum (25 over 33). Among the compounds considered
inactive (1, 7, 13, 16, 20, 21, 25 and 39), two (1 and 13) were
tested only at concentrations up to 16 and 12 lM, respectively, and
it is not excluded that they could exhibit some activity at higher
concentrations. The active compounds resulted less potent than the
reference drug amphotericin B, reaching, at the best, the 43% of its
potency versus L. tropica (cpd 8) and the 58% versus L. infantum
Finally, by treating the 2-(4-chlorobenzyl)benzimidazole36 and
2-(4-chlorobenzyl)-5-trifluoromethylbenzimidazole37 with methyl
iodide in the presence of Cs2CO3, the corresponding 1-methylben-
zimidazoles were obtained, that with excess of methyl iodide gave
the quaternary salts 21 and 22 (Scheme 5).
All but one (39) of the benzimidazole derivatives bearing a
basic side chain were already described by some of us: 24, 28 and
32–3421a; 23, 25 and 29–3121b; 26 and 2724c; 35, 36 and 3821c
;
3725b. The novel bisquaternary salt 39 was obtained by treating
with methyl iodide the previously described benzimidazole deriva-
tive 3621c (Scheme 6). Attempts of selective quaternisation of qui-
nolizidine nitrogen were unsuccessful.
Antileishmanial activity
With the exception of compound 2, all the (38) compounds of
Figures 4 and 5 were tested in vitro against promastigotes of
Scheme 6. Reagents and conditions: (a) CH3I excess, r.t., 24 h.
Scheme 3. Reagents and conditions: (a) CH3CN/THF, K2CO3, N2, r.t., 18 h; (b) CH3I, dry Et2O, 40 ꢀC, 80 h.
ꢀ
ꢂ
Scheme 4. Reagents and conditions: (a) dioxane, N2, r.t., 15 h; (b) BF3 Et2O, reflux, 12 h; (c) CH3I, THF, K2CO3, 50 C, 26 h.
Scheme 5. Reagents and conditions: (a) CH3I, THF, Cs2CO3, 60 ꢀC, 6 h; (b) CH3I excess, r.t., 24 h.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
221
Table 1. In vitro data on antileishmanial activity against L. tropica and L. infantum promastigotes and cytotoxicity on the human endothelial cell line (HMEC-1) and/
or monkey kidney cell (Vero-76) of benzimidazole derivatives 1 and 3–39.
IC50 amph. B
ꢁ100/IC50
compd.b
IC50 amph. B
ꢁ100/IC50
compd.b
IC50 (mM)
IC50 (mM)a L.
tropica
Ratioc IC50 mil-
tef./IC50 compd.
IC50 (mM)a L.
infantum
Ratioc IC50 mil- HMEC-1 and or
tef./IC50 compd.
Compd.
Vero76d
SIe L. tropica SIe L. infantum
ꢂ
1
3
4
>16.20
1.68
0.46
/
4.9
17.9
/
nt
/
/
51.00 7.5
2.64 0.49
2.01 0.24/
2.6 0.3
1.38 0.17
>37
/
/
ꢂ
25.8
94.0
0.28 0.07
0.27 0.01
55.7
57.8
111.6
115.8
1.57
4.37/5.65
9.43
7.44/9.63
ꢂ
ꢂ
5
6
7
8
0.78
10.5
1.6
/
55.5
8.6
/
0.61 0.05
10.09 4.9
>58.0
25.6
0.93
/
51.3
3.1
/
1.77
>27.1
/
2.26
>13.6
/
5.05 0.01
>58.0
nt
0.19 0.06
43.5
227.7
0.34 0.12
27.8
91.9
0.78 0.06/
5.8 0.3
1.86 0.34
2.11 0.45
1.68 0.75
0.73 0.37
>47.0
0.91 0.19
0.94 0.17
>35.0
4.10/30.5
2.29/17.1
9
10
11
12
13
14
15
16
0.87 0.16
1.40 0.58
1.06 0.54
11.9
7.35
9.1
16.1
/
16.7
5.1
/
49.7
30.9
40.8
84.8
/
88.3
26.9
/
1.32 0.30
0.96 0.17
0.84 0.13
0.42 0.14
>11.90
0.64 0.01
3.26 0.80
nt
8.9
12.3
14.1
37.1
/
24.4
2.9
/
23.7
32.6
37.2
74.4
/
48.8
9.6
/
2.14
1.51
1.58
1.43
/
1.86
0.60
/
1.41
2.20
2.00
1.74
ꢂ
0.51
>11.90
/
ꢂ
0.49
1.42
0.29
/
1.61 0.15
>35.0
17
18
19
20
3.56 0.84
11.08 2.73
30.02 10.27
>73.0
3.34
1.07
0.40
/
12.2
3.9
1.4
/
nt
>39.6
nt
nt
/
/
/
/
/
/
/
/
1.26 0.24
65.10 12.21
>74.0
0.35
5.87
>2.43
/
/
/
/
/
>73.0
21
22
23
24
>50.0
/
/
>50.0
16.74 7.07
>56.0
20.89 7.57
>51.0
/
/
1.9
/
1.5
/
nt
nt
nt
>100
/
/
/
/
/
/
33.93 12.62
47.26 13.25
15.04 1.03
>51.0
0.30
0.36
1.18
/
1.3
0.9
2.9
/
0.70
/
1.0
/
>6.65
/
>4.79
/
25
nt
26
27
28
29
30
31
32
33
34
35
36
37
38
39
29.64 0.30
21.09 5.84
3.70 1.19
23.71 8.54
12.13 4.06
23.89 7.78
7.22 3.13
3.92 1.43
3.44 1.43
7.31 2.75
12.61 4.54
13.53 2.21
6.54 0.18
>27.0
0.60
0.84
4.8
1.5
2.1
11.7
1.8
3.6
1.8
6.0
11.0
12.6
5.9
3.4
3.2
6.6
/
>55.0
/
/
>100
>100
16.95/>100
nt
>100
>3.37
>4.74
4.58/>27
/
>8.24
/
6.93
17.35
14.24
12.3
1.90
5.76
11.47
/
/
>3
3.56/>21
/
>6.30
/
5.23
9.97
7.33
7.44
1.92
4.49
6.68
/
32.68 3.92
4.76 1.60
28.51 8.66
15.87 3.21
28.52 4.47
9.55 3.11
6.82 1.25
6.68 1.74
12.09 1.82
12.47 3.51
17.36 1.49
11.23 1.33
>27.0
0.64
4.4
0.73
1.32
0.73
1.24
1.73
1.77
1.73
0.95
1.20
1.86
/
1.0
6.6
1.1
2.0
1.1
3.3
4.6
4.7
2.6
2.5
1.8
2.8
/
0.75
1.46
0.74
1.43
2.63
2.99
2.42
0.69
1.31
2.71
/
nt
50
ꢂ
68
49
90
24
78
75
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
nt
Amph. B
Miltefosine
0.113 0.03f
43.26 11.36
100
0.26
0.135 0.03f
31.26 10.43
100
0.27
25.7 1.90g
227.4
2.3
190.4
3.2
h
ꢂ
1.0
1.0
99.8
aThe results are expressed as IC50 SD of at least three different experiments performed in duplicate or triplicate, with the exception of the starred values that are
the means of two experiments performed in duplicate.
ꢂ
bRatios between the IC50 of amphotericin B ꢁ 100 and IC50 of each compound against L. tropica or L. infantum, calculated for each experiment. The IC50 values of
amphotericin B ranged from 0.082 to 0.177 mM for L. tropica, and from 0.094 to 0.209 mM for L. infantum.
cRatios between the IC50 of miltefosine and that of each compound against L. tropica or L. infantum.
dThe cytotoxicity was assayed in vitro on the human microvascular endothelial cell line (HMEC-1) for compounds 1–20 and 28, and on monkey kidney (Vero76) cells
for compounds 4, 8, 23, 26–28, 30 and 32–39.
eSelectivity index: IC50 HMEC-1 or Vero76/IC50 for the two species of Leishmania.
fMean values from many different experiments; range 0.082–0.177 mM for L. tropica, and 0.094–0.209 mM for L. infantum.
gCytotoxicity of amphotericin B on HMEC-1 cells.
hCytotoxicity of miltefosine on HMEC-1 cells.
(cpd 4), respectively. However, comparing the tested compounds
L. infantum was commonly (with the exception of compounds
with miltefosine, another commonly used drug, they frequently 4, 5, 10–12 and 22) less sensitive than L. tropica, which in two
resulted many fold (up to 228-fold) more potent. It is worth noting cases (compounds 23 and 26) was the only affected species.
that in our experimental conditions miltefosine displayed an IC50
Activity was largely present in both subsets of compounds, but
value versus the promastigote stage of L. infantum (31.26 lM) quite the higher potencies (IC50ꢃ 5 mM) were mainly found among the
higher than the corresponding values found in the literature 2-undecyl- and 2-pentadecylbenzimidazole derivatives, in which
(15.0 lM11a; 16.7lM6c; 19.6lM13b), while no data are available in the subset the activity was particularly high (IC50 minor or around
literature for miltefosine activity versus L. tropica to compare with 1 mM) when the benzimidazole ring was quaternised (compounds
our results (43lM). Indeed, substantial variability has been observed 3–5, 8–12 and 14).
for miltefosine susceptibility of several other Leishmania species38.
Considering the quite different structural features that charac-
Anyhow, even taking into account the lowest IC50 value (15 lM) terise the two subsets of compounds, the structure–activity rela-
aforementioned, most of the tested compounds remain many-fold tionships will be discussed separately for each subset. The
(up to 55- and 43-fold for compounds 4 and 8, respectively) more distribution of activity among the two subsets is illustrated in
potent than miltefosine against L. infantum.
Figure 6.

222
M. TONELLI ET AL.
Figure 6. Number of compounds inhibiting the growth of L. tropica and L. infantum promastigotes and range of their IC50 (mM).
Regarding the subset of benzimidazole derivatives bearing in developing better anti-leishmania agents by increasing activity or
position 2 an aliphatic chain, it is observed that the 1-unsubsti- reducing toxicity through further chemical manipulation (chain
tuted-2-alkylbenzimidazoles (1, 6 and 18) were not only either length, chain branching and unsaturation, number and nature of
inactive or only moderately active, but also the least toxic versus substituents on the benzimidazole and eventual benzyl group).
Compound 8 and its analogues (3–5, 9–12, 14, 15 and 17)
HMEC-1 cells. The introduction in position 1 of a methyl, benzyl
and ferrocenylmethyl residue abolished (7, 13 and 16) or reduced may display their activity (as well as their toxicity) acting as cat-
(19) the activity. However generating a fixed positive charge on ionic surfactants able to modify, like miltefosine38, the cell mem-
the benzimidazole ring of the aforementioned compounds, by brane permeability; moreover, once inside the cell, they may
treating them with methyl or propyl iodide or benzyl chloride, a activate several stress pathways, inhibit fatty acids and sterol bio-
striking increase of activity was observed, obtaining compounds synthesis, and/or cytochrome-C oxidase and other targets.
with IC50 in submicromolar (4, 5, 8, 12 and 14) or low micromolar Moreover, it is known that quaternary ammonium compounds are
range (3, 9–11, 15 and 17). Somewhat unexpected was the lack of able to impair the uptake of choline39, required for the synthesis
activity observed for the quaternised compound 20 (1,3-dimethyl- of parasite membrane phospholipids, but also to inhibit the 3-fold
2-(4-heptyl)benzimidazolium iodide), which was inactive even at a methylation of phosphatidyl ethanolamine that represents the pri-
concentration up to 73 mM. Commonly, the quaternisation mary route to the Leishmania phosphatidyl choline40. It is worth
increased both the activity and the cytotoxicity, while quaternising noting that sodium 2-pentadecylbenzimidazole-5-carboxylate
compound 19 to 20, its activity was abolished leaving unchanged
the low cytotoxicity.
(M&B35347B) besides acting as anionic surfactant, is an inhibitor of
acetyl-CoA carboxylase able to derange fatty acid and cholesterol
biosynthetic pathways41.
In this subset of benzimidazole derivatives, compounds 4 and
8 were the most potent versus L. infantum and L. tropica, respect-
ively, with IC50¼ 0.27 and 0.19 mM corresponding to the 58 and
Concerning the subset of 2-phenyl- and 2-benzylbenzimida-
zoles, the compounds bearing an open-chain basic head were
only moderately active (24, 26 and 27) or inactive (23 and 25),
while those bearing a lupinyl residue were all, but one (39),
endowed with valuable activity. Among the 1-lupinylbenzimida-
zole the activity was influenced by the substituents in 2 and 5
positions. The 5-acetyl derivatives were less potent than the corre-
sponding 5-trifluoromethyl- and 5-nitro derivatives (compare
28–34, particularly 28, 30 and 33). The negative effect of the
5-acetyl group was also evident among the 1-dialkylaminoalkyl
derivatives 23–27.
The higher potency of compound 28 in comparison to 36 sug-
gests that the 2-benzylbenzimidazoles may be more potent than
the corresponding 2-phenyl analogues, and indeed, excluding
from comparison compounds 29–31 for the presence of the acetyl
group (negatively affecting the activity), the 2-benzyl-1-lupinylben-
zimidazole were, on average, more potent than the 2-phenyl-1-
lupinyl derivatives.
28% of amphotericin
B potency with respect to the two
Leishmania species. In comparison to miltefosine, compound 4
was 116-fold more effective versus L. infantum, whilst compound
8 was 228 more potent versus L. tropica. The introduction of elec-
tron-withdrawing substituents on the benzimidazole ring reduced
the activity (compare 8 with 9–11), but the activity-lowering effect
was stronger versus L. tropica than versus L. infantum. However,
comparing the couple of compounds 8–9 to 21–22, where the
pentadecyl chain is replaced by a 4-chlorobenzyl moiety, it is
observed that the introduction of a 5-trifluoromethyl group had a
positive effect on the activity. Also in this case the activity on
L. infantum was higher than on L. tropica. The toxicity of 4 and 8
(and similar compounds) versus the HMEC cells was not negligible,
with selectivity index (SI) in the range 2.3–7.4, that, however, were
better than the corresponding SI of miltefosine (2.0 and 3.2).
Indeed, the HMEC cells are particularly sensitive to most kinds of
chemicals, thus the best compounds 4 and 8 were also tested for
toxicity against Monkey kidney Vero76 cells, sharing a quite more
valuable SI value. Interestingly, the 1-unsubstituted benzimidazole
6, even displaying a moderate activity, exhibited a very valuable SI
versus the sensitive HMEC cells (SI> 37 and >13). Thus, com-
With this kind of compounds we did not succeed to quaternise
the lupinyl moiety without affecting also the benzimidazole ring
and it was observed that the double quaternisation produced the
loss of activity (compare compounds 36 and 39).
In this subset of 2-arylbenzimidazoles, compound 28 appears
pounds 4, 6 and 8 represent interesting hit compounds for as the most interesting because resulted 12-/7-fold more potent

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
223
than miltefosine and did not manifest any discernible cytotoxicity
on Vero cells (CC50> 100 mM and SI >27 and >21 versus L. tropica
and L. infantum, respectively), while the toxicity on HMEC-1 cells
was only moderate (SI¼ 4.58 and 3.56 versus the two Leishmania
species). It is worth noting that compound 28 was already shown
to possess antiproliferative activity, with GI50< 5 mM, against 24
human cancer cell lines, among which the renal cancer cell line
UO31 was particularly sensitive (GI50¼ 0.019 mM25b). Moreover, the
same compound displayed moderate antiviral activity against
Coxsackie virus B5 (CVB-5) and respiratory syncytial virus (RSV)
with EC50 13 and 15 mM, respectively24c. Also compounds 33 and
34 displayed good level of antileishmanial activity associate with
modest toxicity on Vero76 cells and represent, together with 28,
interesting hit compounds.
Possessing a basic side chain, compounds 23–39 might, like
sitamaquine3b,4d, anchor to the anionic phospholipidic compo-
nents of Leishmania cell membrane, disrupting its function.
Eventually, they could permeate the cell and accumulate into
cytosolic acidic compartments. Once inside the cells, the benzimi-
dazole derivatives might inhibit some of the enzymes that are
Conclusions
Two sets of benzimidazole derivatives (38 compounds) were
tested in vitro for activity against promastigotes of L. tropica and
L. infantum. A first set was formed by 2-(long chain)-alkyl/benzyl
benzimidazoles (1–22), whose heterocyclic head was, in most
cases, quaternised to mimic the ammonium head of miltefosine
and related analogous anti-leishmanial drugs. The second set was
composed of 2-benzyl and 2-phenyl benzimidazoles (23–39) bear-
ing in position 1 a basic side chain (dialkylaminoalkyl- or lupinyl-).
Most of the tested compounds of both sets resulted active
against L. tropica (30 over 38) and L. infantum (25 over 33) (Figure
6). The IC50 values for the quaternised 2-alkylbenzimidazoles were
in the low micromolar/submicromolar range. Compound
8
(IC50¼ 0.19 mM and 0.34 mM versus L. tropica and L. infantum,
respectively) resulted 228- and 93-fold more potent than miltefo-
sine, with SI in the range 4.1–2.3 versus HMEC cells, but displaying
SI¼ 30 and 17 versus Vero76 cells. Among the compounds bearing
a basic side chain, the 1-lupinyl derivatives were commonly more
active than dialkylaminoalkyl ones, and compound 28 [2-(4-chloro-
essential for Leishmania survival and proliferation and are absent benzyl)-1-lupinyl-5-trifluoromethylbenzimidazole] displayed the
from their mammalian host42, like those involved in the biosyn-
highest potency (IC50¼ 3.70 mM and 4.76 mM for the two
thesis of membrane ergosterol and the 24-alkylsterols3b,43 or the
Leishmania species). This compound was just a little less toxic
zinc metalloprotease (leishmanolysin)18,44, playing crucial roles in
the Leishmania parasite physiology and in host-parasite
interaction.
than 8 on HMEC cells (SI¼ 4.6 and 3.6 versus L. tropica and
L. infantum, respectively), but did not manifest any discernible
cytotoxicity against Vero76 cells (CC50> 100 mM and SI¼ 27 and 21
versus the two Leishmania species). Therefore, several compounds
and particularly the benzimidazoles 8 and 28, whose activity was
confirmed on intramacrophagic amastigote stage of L. infantum,
represent interesting hit compounds, whose structure can be fur-
ther variate in order to improve their safety profiles (toxicity/activ-
ity ratios).
Some benzimidazole derivatives bearing a basic side chain
have, already, been shown to somewhat affect sterol biosynthesis,
like 2-[(4-diethylaminoethoxy)phenyl]benzimidazole that blocks the
reduction of 7-dehydrocholesterol to cholesterol45 and 2-(4-chloro-
benzyl)-1-(3-diethylaminopropyl)-5-trifluoromethylbenzimidazole21
(structurally close to compounds 23–25) that, at 50 mg/kg p.os,
reduced significantly (>15%) the serum cholesterol concentration
in hypercholesterolemic mice. The mechanism of action of these
two kinds of benzimidazole derivatives was not further investi-
gated, and the possibility of their interference in parasite ergos-
terol biosynthesis may be only conjectural.
On the other hand, some 2-aryl-5-substituted benzimidazoles,
devoid of basic chain, have been shown to inhibit the stearoyl
coenzyme A desaturase (SCD1), blocking the formation of oleic
and palmitoleic triglycerides, cholesterol esters and phospholi-
pids46. The SCD1, besides being investigated for the treatment of
dislipidemic diseases and body weight control, has been found to
participate, together with other desaturase enzymes, in the de
novo synthesis of mono- and poly-unsaturated fatty acids
(C18–C22 PUFA) of parasitic membrane. These biosynthetic path-
ways play a crucial role for parasitic viability at different life cycle
stages47. Some other 2-arylbenzimidazoles, still lacking basic side
chain (Figure 3, central row), have been shown to exhibit leishma-
nicidal effect and to dock successfully in the binding pocket of the
promastigote surface protease (leishmanolysin, GP63 protein),
which contributes to parasite virulence18. Of course, for the dis-
cussed compounds, other, even multiple, mechanisms of action,
not yet identified, may take place.
Based on the chemical features of the relevant compounds,
their interaction with the acidic components (mainly the phospho-
lipids) of cell membrane, with consequent disruption of its func-
tion, may explain the observed anti-leishmanial activity. The
internalisation of compounds and their interaction with different
targets inside the cell might also have an important role, but its
investigation is beyond the aim of the present preliminary study.
Acknowledgments
This work was in part supported by Ministero dell’Istruzione,
ꢀ
dell’Universita e della Ricerca (PRIN) Projects 2010C2LKKJ_006;
20154JRJPP_004.
Disclosure statement
All authors declare no conflicts of interest.
ORCID
Michele Tonelli
Elena Gabriele
Anna Sparatore
http://orcid.org/0000-0003-1518-2890
http://orcid.org/0000-0002-0643-7372
http://orcid.org/0000-0003-2135-2649
Finally, for a better insight of the real value of the studied com-
pounds as antileishmanial agents, compounds 8 and 28, represen-
tative of the two subsets of benzimidazole derivatives that display
the highest activity against the promastigote stage, were tested
against the intramacrophagic amastigote stage of L. infantum.
Compound
8
exhibited an IC50¼ 0.313 lM, with
a 3.35-fold
References
increased potency with respect to miltefosine, while compound
28, at 2 lM concentration (42% of its IC50 versus promastigotes)
reduced the amastigote infection of THP-1 cells by 33.2% (human
acute monocytic leukaemia cell line; IC50> 2 lM).
1. World Health Organization (WHO). Available from: http://
www.who.int/neglected_diseases/diseases/en [last accessed
11 Jul 2017].

224
M. TONELLI ET AL.
ꢁ
2. (a) Frezard F, Demicheli C, Ribeiro RR. Pentavalent antimoni-
investigations of leishmanicidal N-benzylcytisine derivatives.
Chem Biol Drug Des 2011;78:183–9.
als: new perspectives for old drugs. Molecules
ꢁ
8. Kirmizibekmez H, Calis I, Perozzo R, et al. Inhibiting activities
of the secondary metabolites of Phlomis brunneogaleata
against parasitic protozoa and plasmodial enoyl-ACP
Reductase, a crucial enzyme in fatty acid biosynthesis. Planta
Med 2004;70:711–17.
2009;1:2317–36. (b) Frezard F, Martins PS, Barbosa MC, et al.
New insights into the chemical structure and composition of
the pentavalent antimonial drugs, meglumine antimonate
and sodium stibogluconate.
J
Inorg Biochem 2008;
102:656–65.
9. Hiam A, Sebastien D, George B, et al. Microtubule target for
new antileishmanial drugs based on ethyl 3-haloacetamido-
benzoates. J Enzyme Inhib Med Chem 2006;21:305–12.
3. (a) Murray HW, Berman JD, Davies CR, et al. Advances in
leishmaniasis. Lancet 2005;3:1561–77. (b) Singh N, Kumar M,
Singh RK, Leishmaniasis: current status of available drugs
and new potential drug targets. J Trop Med 2012;5:485–97.
(c) Ameen M. Cutaneous leishmaniasis: advances in disease
pathogenesis, diagnostics and therapeutics. Clin Exp
Dermatol 2010;35:699–705.
4. (a) Berman JD, Lee LS. Activity of 8-aminoquinolines against
Leishmania tropica within human macrophages in vitro. Am
J Trop Med Hyg 1983;32:753–9. (b) Singh S, Sivakumar R.
Challenges and new discoveries in the treatment of leish-
maniasis. J Infect Chemother 2004;10:307–15. (c) Garnier T,
Brown MB, Lawrence MJ, Croft SL. In-vitro and in-vivo stud-
ies on a topical formulation of sitamaquine dihydrochloride
ꢁ
10. (a) Sanchez-Delgado RA, Anzellotti A. Metal complexes as
chemotherapeutic agents against tropical diseases: trypano-
somiasis, malaria and leishmaniasis. Mini Rev Med Chem
2004;4:23–30. (b) Ilari A, Baiocco P, Messori L, et al. A gold-
containing drug against parasitic polyamine metabolism: the
X-ray structure of trypanothione reductase from Leishmania
infantum in complex with auranofin reveals a dual mechan-
ism of enzyme inhibition. Amino Acids 2012;42:803–11.
11. (a) Plano D, Baquedano Y, Moreno-Mateos D, et al.
Selenocyanates and diselenides: a new class of potent anti-
leishmanial agents. Eur J Med Chem 2011;4:3315–23. (b)
Baquedano Y, Moreno E, Espuelas S, et al. Novel hybrid sele-
nosulfonamides as potent antileishmanial agents. Eur J Med
Chem 2014;74:116–23.
for cutaneous leishmaniasis.
J
Pharm Pharmacol
ꢁ
2006;58:1043–54. (d) Loiseau PM, Cojean S, Schrevel J.
Sitamaquine as a putative antileishmanial drug candidate:
from the mechanism of action to the risk of drug resistance.
Parasite 2011;18:115–19. (e) Almeida OL, Santos JB.
Advances in the treatment of cutaneous leishmaniasis in the
new world in the last ten years: a systematic literature
review. An Bras Dermatol 2011;86:497–506.
12. Papanastasiou I, Prousis KC, Georgikopoulou K, et al. Design
and synthesis of new adamantyl-substituted antileishmanial
ether phospholipids. Bioorg Med Chem Lett 2010;20:5484–7.
13. (a) Pathak D, Yadav M, Siddiqui N, et al. Antileishmanial
agents: an updated review. Pharm Chem 2011;3:239–49. (b)
ꢁ
Vale-Costa S, Costa-Gouveia J, Perez B, et al. N-cinnamoy-
5. (a) Singh N, Mishra BB, Bajpai S. Natural product based leads
to fight against leishmaniasis. Bioorg Med Chem
2014;22:18–45. (b) Cheuka PM, Mayoka G, Mutai P, et al. The
role of natural products in drug discovery and development
against neglected tropical diseases. Molecules 2017;22:E58.
6. (a) do Socorro S, Rosa M, Mendonc¸a-Filho RR, Bizzo HR,
et al. Antileishmanial activity of a linalool-rich essential oil
from Croton cajucara. Antimicrob Agents Chemother
2003;47:1895–901. (b) De Monte C, Bizzarri B, Gidaro MC,
et al. Bioactive compounds of Crocus sativus L. and their
semi-synthetic derivatives as promising anti-Helicobacter
pylori, anti-malarial and anti-leishmanial agents. J Enzyme
Inhib Med Chem 2015;30:1027–33. (c) Wulsten IF, Costa-Silva
TA, Mesquita JT, et al. Investigation of the anti-Leishmania
(Leishmania) infantum activity of some natural sesquiterpene
lactones. Molecules 2017;22:e685. (d) Barrera PA, Jimenez-
Ortiz V, Tonn C, et al. Natural sesquiterpene lactones are
lated aminoquinolines as promising antileishmanial agents.
Antimicrob Agents Chemother 2013;5:5112–15. (c) Brindisi
M, Brogi S, Relitti N, et al. Structure-based discovery of the
first non-covalent inhibitors of Leishmania major trypare-
doxin peroxidase by high throughput docking. Sci Rep
2015;5:9705. (d) Barteselli A, Casagrande M, Basilico N, et al.
Clofazimine analogs with antileishmanial and antiplasmodial
activity. Bioorg Med Chem 2015;23:55–65.
14. (a) Pagniez F, Abdala-Valencia H, Marchand P, et al.
Antileishmanial activities and mechanisms of action of
indole-based azoles.
J
Enzyme Inhib Med Chem
2006;21:277–83. (b) Gupta L, Talwar A, Nishi, et al. Synthesis
of marine alkaloid: 8,9-dihydrocoscinamide B and its ana-
logues as novel class of antileishmanial agents. Bioorg Med
Chem Lett 2007;17:4075–9. (c) Bharate SB, Bharate JB, Khan
SI, et al. Discovery of 3,30-diindolylmethanes as potent anti-
leishmanial agents. Eur J Med Chem 2013;63:435–43. (d) Roy
A, Chowdhury S, Sengupta S, et al. Development of deriva-
tives of 3, 3'-diindolylmethane as potent Leishmania dono-
vani bi-subunit topoisomerase IB poisons. PLoS One
2011;6:e28493.
active
against
Leishmania
mexicana.
J
Parasitol
2008;5:1143–9. (e) Sairafianpour M, Christensen J, Staerk D,
et al. Leishmanicidal, antiplasmodial, and cytotoxic activity
of novel diterpenoid 1,2-quinones from Perovskia abrota-
noides: new source of tanshinones.
J
Nat Prod
15. Danan A, Charon D, Kirkiacharian S, et al. Synthesis and anti-
parasitic activities of amidinic azolated derivatives. Farmaco
1997;52:227–9.
16. Jagu E, Pomel S, Diez-Martinez A, et al. Synthesis and in
vitro antikinetoplastid activity of polyamine-hydroxybenzo-
triazole conjugates. Bioorg Med Chem 2017;25:84–90.
2001;64:1398–403. (f) Kayser O, Kiderlen AF, Bertels S, et al.
Antileishmanial activities of aphidicolin and its semisynthetic
derivatives. Antimicrob Agents Chemother 2001;45:288–92.
(g) Sousa MC, Varandas R, Santos RC, et al. Antileishmanial
activity of semisynthetic lupane triterpenoids betulin and
betulinic acid derivatives: synergistic effects with miltefosine.
PLoS One 2014;9:e89939.
ꢁ
ꢁ
17. Hernandez-Luis F, Hernandez-Campos A, Castillo R, et al.
Synthesis and biological activity of 2-(trifluoromethyl)-1H-
benzimidazole derivatives against some protozoa and
Trichinella spiralis. Eur J Med Chem 2010;45:3135–41.
7. (a) Di Giorgio C, Delmas F, Ollivier E, et al. In vitro activity of
the beta-carboline alkaloids harmane, harmine, and harma-
line toward parasites of the species Leishmania infantum.
Exp Parasitol 2004;1:67–74. (b) Turabekova MA, Vinogradova
VI, Werbovetz KA, et al. Structure-activity relationship
18. Shaukat A, Mirza HM, Ansari AH, et al. Benzimidazole deriva-
tives: synthesis, leishmanicidal effectiveness, and molecular
docking studies. Med Chem Res 2013;22:3606–20.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
225
19. (a) Mayence A, Vanden Eynde JJ, LeCour L, Jr, et al. 27. Shi Z, Ta J-T. Synthesis of the b-keto acids from benzimida-
zolium iodides and ethyl malonate. Chin
2000;18:940–1.
J
Chem
Piperazine-linked bisbenzamidines: a novel class of antileish-
manial agents. Eur J Med Chem 2004;3:547–53. (b) Mayence
A, Pietka A, Collins MS, et al. Novel bisbenzimidazoles with
antileishmanial effectiveness. Bioorg Med Chem Lett
2008;18:2658–61.
28. Babu KR, Zhu N, Bao H. Iron-catalyzed C-H alkylation of het-
erocyclic C-H bonds. Org Lett 2017;19:46–9.
29. Guo Y, Lu Z, Yao L, et al. A novel synthetic method for the
preparation of aliphatic aldehydes from the corresponding
carboxylic acids. Chin J Chem 2011;29:489–92.
30. She J, Jiang Z, Wang Y. One-pot synthesis of functionalized
benzimidazoles and 1H-pyrimidines via cascade reactions of
o-aminoanilines or naphthalene-1,8-diamine with alkynes
and p-tolylsulfonyl azide. Synlett 2009;12:2023–7.
31. Mosmann T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 1983;65:55–63.
ꢁ
ꢁ
ꢁ~
ꢁ
20. (a) Torres-Gomez H, Hernandez-Nunez E, Leon-Rivera I, et al.
Design, synthesis and in vitro antiprotozoal activity of benzi-
midazole-pentamidine hybrids. Bioorg Med Chem Lett
2008;1:3147–51. (b) Mendez-Cuesta CA, Herrera-Rueda MA,
Hidalgo-Figueroa S, et al. Synthesis, screening and in silico
simulations of anti-parasitic propamidine/benzimidazole
derivatives. Med Chem 2017;13:137–48.
21. (a) Sparatore F, Boido V, Fanelli F. Dialkylaminoalkylbenzimi-
dazoles of pharmacological interest. Farmaco Sci
32. Baiocco P, Ilari A, Ceci P, et al. Inhibitory effect of silver
nanoparticles on trypanothione reductase activity and leish-
mania infantum proliferation. ACS Med Chem Lett
2010;2:203–33.
33. D'Alessandro S, Gelati M, Basilico N, et al. Differential effects
on angiogenesis of two antimalarial compounds, dihydroar-
temisinin and artemisone: implications for embryotoxicity.
Toxicology 2007;241:66–74.
34. Tandon VK, Kumar M. BF3ꢄEt2O promoted one-pot expeditious
and convenient synthesis of 2-substituted benzimidazoles
and 3,1,5-benzoxadiazepines. Tetrahedr Lett 2004;45:4185–7.
35. Howarth J, Hanlon K. N-ferrocenylmethyl, N'-methyl-2-substi-
tuted benzimidazolium iodide salts with in vitro activity
against the Leishmania infantum parasite strain L1. Bioorg
Med Chem Lett 2003;13:2017–20.
1968;23:344–59.
(b)
Paglietti
G,
Sparatore
F.
Dialkylaminoalkyl-benzimidazoles of pharmacological inter-
est. 3. Farmaco Sci 1972;27:333–42. (c) Boido A, Vazzana I,
Sparatore F, et al. Preparation and pharmacological activity
of some 1-lupinylbenzimidazoles and 1-lupinylbenzotriazoles.
Farmaco 1991;46:775–88.
22. (a) Paglietti G, Pirisi MA, Loriga M, et al. Preparation and
pharmacologic activity of 2-(4'R')benzyl-5R-benzimidazole.
Analgesic activity and effect on conditioned avoidance
response. Farmaco Sci 1988;43:203–14. (b) Paglietti G, Pirisi
MA, Loriga M, et al. Preparation and pharmacologic activity
of 2-(4'R')benzyl-5R-benzimidazole and 2-(4'-pyridinyl)-5R-
benzimidazoles. Analgesic activity and effect on conditioned
avoidance response. Farmaco Sci 1988;43:215–26.
23. (a) Paglietti G, Sparatore F. Preparation of beta-benzimida-
zolyl- and indazolylbutyric acids as potential choleretic
agents. Farmaco Sci 1972;27:471–9. (b) Grella G, Paglietti G,
Sparatore F, et al. Synthesis and choleretic activity of 3-(2-
aryl-5R-benzimidazol-1-yl)butanoic acids. Farmaco Sci
1987;42:475–90. (c) Grella G, Paglietti G, Sparatore F, et al.
Synthesis and choleretic activity of 3-[2-(3-R', 4-R'', 5-R'''-ben-
zyl)-5-R-benzimidazol-1-yl]-butanoic acids. Farmaco Sci
1992;47:21–35. (d) Loriga M, Paglietti G, Piras S, et al.
Synthesis and evaluation of gastroprotective and antiulcer
activity of some 2-substituted-1H-imidazo[4,5-b] pyridines
and -1H-benzimidazoles. Farmaco 1992;47:287–303.
36. Hunger A, Kebrle J, Rossi A, et al. Benzimidazol-derivate und
verwandte Heterocyclen. II. Synthese von 1-aminoalkyl-2-
benzyl-benzimidazolen. Helv Chim Acta 1960;43:800–9.
37. Boido V, Sparatore F. Simple molecular analogs of anti-
inflammatory
1-lupinyl-2-(p-methoxy)benzyl-5-trifluorome-
thylbenzimidazole. Farmaco Sci 1974;29:517–25.
38. Dorlo TP, Balasegaram M, Beijnen JH, et al. Miltefosine: a review
of its pharmacology and therapeutic efficacy in the treatment
of leishmaniasis. J Antimicrob Chemother 2012;67:2576–97.
39. Ancelin ML, Vial HJ. Quaternary ammonium compounds effi-
ciently inhibit Plasmodium falciparum growth in vitro by
impairment of choline transport. Antimicrob Agents
Chemother 1986;29:814–20.
40. Bibis SS, Dahlstrom K, Zhu T, et al. Characterization of
Leishmania major phosphatidylethanolamine methyltransfer-
ases LmjPEM1 and LmjPEM2 and their inhibition by choline
analogs. Mol Biochem Parassitol 2014;196:90–9.
41. Whittington FM, Enser M, Pratt J, et al. Effect of sodium 2-n-
pentadecyl-benzimidazole-5-carboxylate (M & B 35347B), an
inhibitor of acetyl-CoA carboxylase, on lipogenesis and fat
deposition in obese hyperglycaemic (ob/ob) and lean mice.
Int J Obes 1987;11:619–29.
24. (a) Tonelli M, Paglietti G, Boido V, et al. Antiviral activity of
benzimidazole derivatives. I. Antiviral activity of 1-substi-
tuted-2-[(benzotriazol-1/2-yl)methyl]benzimidazoles.
Chem
Biodivers 2008;5:2386–401. (b) Tonelli M, Simone M, Tasso B,
et al. Antiviral activity of benzimidazole derivatives. II.
Antiviral activity of 2-phenylbenzimidazole derivatives.
Bioorg Med Chem 2010;1;2937–53. (c) Tonelli M, Novelli F,
Tasso B, et al. Antiviral activity of benzimidazole derivatives.
III. Novel anti-CVB-5, anti-RSV and anti-Sb-1 agents. Bioorg
Med Chem 2014;22:4893–909.
25. (a) Novelli F, Tasso B, Sparatore F. Synthesis and biological
investigations of 2-(tetrahydropyran-2'-yl) and 2-(tetrahydro-
furan-2'-yl)benzimidazoles. Farmaco 1997;52:499–507. (b)
Tonelli M, Tasso B, Mina L, et al. Primary anti-proliferative
activity evaluation of 1-(quinolizidin-1'-yl)methyl- and
1-(x-tert-amino)alkyl-substituted 2-phenyl-, 2-benzyl- and
2-[(benzotriazol-1/2-yl)methyl]benzimidazoles on human
cancer cell lines. Mol. Divers 2013;17:409–19.
42. Chawla B, Madhubala R. Drug targets in Leishmania. J
Parasit Dis 2010;34:1–13.
43. (a) Fernandes Rodrigues JC, Concepcion JL, Rodrigues C,
et al. In vitro activities of ER-119884 and E5700, two potent
squalene synthase inhibitors, against Leishmania amazonen-
sis: antiproliferative, biochemical, and ultrastructural effects.
Antimicrob Agents Chemother 2008;5:4098–114. (b) de
Macedo-Silva ST, Visbal G, Urbina JA, et al. Potent in vitro
antiproliferative synergism of combinations of ergosterol
biosynthesis inhibitors against Leishmania amazonensis.
Antimicrob Agents Chemother 2015;59:6402–18.
26. Pool WO, Harwood HJ, Ralston AW. 2-Alkylbenzimidazoles as
derivatives for the identification of aliphatic acids. J Am
Chem Soc 1937;59:178–9.

226
M. TONELLI ET AL.
44. Das P, Alam MN, Paik D, et al. Protease inhibitors in poten- 46. Powell DA, Ramtohul Y, Lebrun ME, et al. 2-Aryl benzimida-
tial drug development for Leishmaniasis. Indian J Biochem
zoles: human SCD1-specific stearoyl coenzyme-A desaturase
Biophys 2013;50:363–76.
inhibitors. Bioorg Med Chem Lett 2010;20:6366–9.
45. (a) Rodney G, Black ML, Bird OD, The common mode of 47. (a) Maldonado RA, Kuniyoshi RK, Linss JG, et al. Trypanosoma
action of three new classes of inhibitors of cholesterol bio-
synthesis. Biochem Pharmacol 1965;1:445–56. (b) Black ML,
Rodney G, Capps DB. Simultaneous inhibition of alternative
pathways of cholesterol biosynthesis by two related
hypocholesteremic agents. Biochem Pharmacol 1968;17:
1803–14.
cruzi oleate desaturase: molecular characterization and com-
parative analysis in other trypanosomatids. Parassitol
2006;92:1064–74. (b) Ramakrishnan S, Serricchio M, Striepen
B, et al. Lipid synthesis in protozoan parasites: a comparison
between kinetoplastids and apicomplexans. Prog Lipid Res
2013;52:488–512.
J