Selective inhibition of heme oxygenase-2 activity by analogs of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole (clemizole): Exploration of the effects of substituents at the N-1 position

Article abstract of DOI:10.1016/j.bmc.2013.07.050

Several analogs based on the lead structure of 1-(4-chlorobenzyl)-2- (pyrrolidin-1-ylmethyl)-1H-benzimidazole (clemizole) were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). Many of the compounds were found to be potent and highly s

Full text of DOI:10.1016/j.bmc.2013.07.050

Bioorganic & Medicinal Chemistry 21 (2013) 6788–6795
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Selective inhibition of heme oxygenase-2 activity by analogs of
1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole
(clemizole): Exploration of the effects of substituents at the N-1
position
Jason Z. Vlahakis a, Dragic Vukomanovic b, Kanji Nakatsu b, Walter A. Szarek a,
⇑
a Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada
b Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario K7L 3N6, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Several analogs based on the lead structure of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benz-
imidazole (clemizole) were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). Many
of the compounds were found to be potent and highly selective for the HO-2 isozyme (constitutive), and
had substantially less inhibitory activity on the HO-1 isozyme (inducible). The compounds represent the
first report of highly potent and selective inhibitors of HO-2 activity, and complement our suite of selec-
tive HO-1 inhibitors. The study has revealed many candidates based on the inhibition of heme oxygen-
ases for potentially useful pharmacological and therapeutic applications.
Received 29 May 2013
Revised 22 July 2013
Accepted 30 July 2013
Available online 8 August 2013
Keywords:
Heme oxygenase
Selective inhibitors
Clemizole analogs
Carbon monoxide
Heme
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
CO, as a regulator of cellular processes in the brain, and in circula-
tory, respiratory, and immune systems has been recognized
Currently, there is great interest in gasotransmitters,1 sub-
stances that exist as gaseous compounds dissolved in biological
fluids at usual mammalian temperatures and pressures, and that
have the ability to regulate a variety of biological functions. Three
substances generally have been recognized as gasotransmitters,
namely, nitric oxide (NO), carbon monoxide (CO), and hydrogen
sulfide (H2S). Our laboratories have focussed on CO and the en-
zymes that are responsible for its biosynthesis, namely, the heme
oxygenases (HOs). CO is formed in mammals primarily through
the action of HO on the substrate heme.2,3 While most of the heme
oxidation occurs in the liver and spleen during the ‘housekeeping’
degradation of heme, the formation of CO from heme occurs in sig-
nificant quantities in sites such as blood vessels and the brain. In-
deed, the brain, testes, and spleen have the greatest specific
activity of HO.4 HO activity is attributable to two functional iso-
zymes. HO-1 (known also as HSP-32, molecular weight ꢀ32 kD)
is induced by various stimuli including heat shock, heavy metals,
heme, and reactive oxygen species. HO-2 has a molecular weight
of 36.5 kD and is constitutive. The role of the gasotransmitter,
widely,3,5 and its anti-inflammatory, anti-proliferative, and anti-
apoptotic effects have been demonstrated.6 The use of HO inhibi-
tors is critical in the elucidation of the physiological functions of
the CO/HO system and related physiological pathways.
The most intensively studied and best understood aspect of the
CO/HO system is the regulation of HO-1 synthesis, whereas the lit-
erature describing a role for HO-2 is relatively sparse. Initially,
information on the CO/HO pathway was obtained using metallo-
porphyrins (such as tin protoporphyrin, SnPP) as HO inhibitors.
However, there is a problem with the use of the metalloporphyrin
HO inhibitors owing to the lack of selectivity because of the close
structural similarity between heme and these inhibitors. We have
designed and synthesized several azole-based HO inhibitors that
are substantially more-selective than the metalloporphyrins. Many
of our compounds were found to be selective for the HO-1 isozyme,
and had substantially less inhibitory activity on HO-2.7–17 In this
publication we describe the identification of a highly-selective
HO-2 inhibitor and initial exploratory structure–activity relation-
ship (SAR) studies. The availability of selective HO-2 inhibitors,
in addition to earlier selective HO-1 inhibitors, will facilitate inves-
tigations of the involvement of HO-1 and HO-2 in a variety of phys-
iological roles. To our knowledge the compounds represent the
first reported highly selective HO-2 inhibitors.
⇑
Corresponding author. Tel.: +1 613 533 2643.
E-mail address: szarekw@chem.queensu.ca (W.A. Szarek).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.050

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 21 (2013) 6788–6795
6789
whereas compound 36, devoid of a substituent at N-1, was found
2. Results and discussion
to be inactive.
From the screening of a compound library, an initial hit for a
selective inhibitor of HO-2 was discovered, namely, the benzimid-
azole, clemizole, whose in vitro IC50 against HO-2 (rat brain micro-
3. Conclusions
somes) was 3.4 0.3
lM and against HO-1 (rat spleen microsomes)
We have synthesized several compounds that express high
selectivity as inhibitors of HO-2 relative to HO-1. Specifically, com-
pounds 1–3, 5, 13–15, 17–22, 25, 28–30, 32, and 33 are noteworthy
for their potency and selectivity towards HO-2. This initial SAR
study explored the effects of substituents at the N-1 position
of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimid-
azole (clemizole); the introduction of a variety of substituents on
the N-1 benzyl group was well-tolerated as regards HO-2 inhibi-
tory potency and selectivity. The synthetic protocol for the gener-
ation of analogs involved a straightforward two-step process. To
our knowledge, the work described represents the first report of
highly potent and selective inhibitors of HO-2. This study has pro-
vided new potentially useful pharmacological tools for the study of
the CO/HO system, and complement our suite of selective HO-1
inhibitors. The compounds could be of use in exploring the role
of enzymic products in those cells that are rich in HO-2 activity. These
novel compounds might also have useful therapeutic applications.
was >100 M. Here we present the chemical synthesis of candidate
l
compounds and an initial SAR study of analogs of clemizole
bearing a selected variety of substituents at the N-1 position
(see Fig. 1).
2.1. Chemical synthesis
The synthesis of the benzimidazole compounds is shown
in Scheme 1 and involved the alkylation of pyrrolidine with
2-(chloromethyl)benzimidazole in ethanol. The alkylation of the
resulting benzimidazole derivative was accomplished using two
different procedures. Procedure A involved the treatment of the
benzimidazole derivative with sodium hydride in tetrahydrofuran,
followed by the addition of an alkyl halide and tetra-n-butylammo-
nium bromide as phase-transfer catalyst (see Ref. 18). Alterna-
tively, Procedure B involved the treatment of the benzimidazole
derivative with sodium hydroxide in dimethyl sulfoxide, followed
by the addition of an alkyl halide. The final alkylated benzimidaz-
oles were isolated mainly in the dihydrochloride salt form. Many
novel compounds were synthesized using this simple two-step
procedure; an attractive feature for large-scale development.
4. Experimental
4.1. General
Flash column chromatography was performed on Silicycle silica
gel (230–400 mesh, 60 Å). Analytical thin-layer chromatography
was performed on glass-backed pre-coated silica gel 60 F254 plates
(Silicycle), and the compounds were visualized either by UV illumi-
nation (254 nm), or by heating after spraying with phosphomolyb-
dic acid in ethanol. Melting points were measured on a Mel-Temp
II apparatus and are uncorrected. 1H and 13C NMR spectra were re-
corded on a Bruker Avance 400 spectrometer in CDCl3, CD3OD, or
DMSO-d6. The chemical shifts are reported in d (ppm) relative to
tetramethylsilane.19 The compounds synthesized were deemed
>95% pure by 1H NMR analysis. High-resolution ESI mass spectra
were recorded on an Applied Biosystems/MDS Sciex QSTAR XL
mass spectrometer with an Agilent HP1100 Cap-LC system. Sam-
2.2. Biological evaluation
Results for inhibition of HO activity, in vitro, through determin-
ing the concentration necessary to decrease enzyme activity by
50% (inhibitory concentration 50%, IC50) are shown in Table 1.
Compounds 1–12 represent a series of 4-substituted benzyl com-
pounds. The 4-fluoro, 4-bromo, and 4-iodo analogs (2–4) were
found to have essentially the same HO-2 inhibitory activity as
the hit clemizole (1). In the case of compounds 5–12, having a vari-
ety of electron-donating and electron-withdrawing substituents,
the least potent compound was found to be the 4-benzyloxy analog
9. Moreover, compounds 1–3 had high selectivity indices for HO-2.
In the case of the 3-substituted compounds 13–17, the 3-chloro,
3-bromo, and 3-nitro derivatives were all found to be highly potent
inhibitors of HO-2; the least potent was the 3-cyano analog 16. The
3-nitro compound 17 was found to be the most potent, having a
correspondingly high selectivity index. Interestingly, the 2-substi-
tuted analogs 18–22 were found all to be highly potent inhibitors
of HO-2. The dichlorinated analogs 23–28 were found to be either
highly potent or at least moderately potent towards HO-2 inhibi-
tion. Compounds 29–31 have different linkers between N-1 and
the phenyl group. The methylene (29) and ethylene (30) analogs
were found to exhibit essentially the same inhibitory activity;
however, the introduction of an oxygen atom in 30 to give 31
resulted in a loss of activity. Compounds 32–36 represent a depar-
ture from the monophenyl analogs described thus far; compounds
32–34 were found to exhibit essentially the same high potency,
ples were run in 50% aqueous MeOH at a flow rate of 6 lL/min.
High-resolution EI mass spectra were recorded on a Waters/Micro-
mass GC-TOF instrument. 2-(Chloromethyl)benzimidazole and all
other chemicals were obtained from Sigma–Aldrich.
4.2. Representative Procedure A for the formation of alkylated
benzimidazole derivatives, as outlined in Scheme 1
1-(4-Chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole
dihydrochloride (1). Under an atmosphere of nitrogen, compound
36 (250 mg, 1.24 mmol, 1 equiv) was dissolved in freshly distilled
warm tetrahydrofuran (THF) (2.5 mL), then cooled to 0 °C. To this
solution was added 60% NaH in oil (100 mg, 60 mg pure,
2.48 mmol, 2 equiv) and the mixture stirred at 0 °C for 5 min.
4-Chlorobenzyl bromide (255 mg, 1.24 mmol, 1 equiv) was added
followed by the addition of tetra-n-butylammonium bromide
(23 mg, 0.07 mmol, 6 mol %). The mixture was stirred at rt over-
night, then diluted with a solution of water (2 drops) in THF
(5 mL). The solution was filtered through Celite, and the Celite
was washed with THF (50 mL) and then EtOAc (50 mL). The filtrate
was concentrated and the remaining residue purified by flash col-
umn chromatography on silica gel (EtOAc) to give the free base
(195 mg) as an oil (Rf = 0.46, EtOAc). To a solution of the free base
in MeOH (2 mL) was added a solution of 37% aqueous HCl
(158 mg, 1.60 mmol, 2.7 equiv) in MeOH (2 mL). The mixture was
Figure 1. The structure of clemizole (1).

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. 21 (2013) 6788–6795
Scheme 1. Generation of benzimidazole derivatives.
Table 1
Inhibitory potency and selectivity of candidate compounds against HO-1 and HO-2 activity
N
N
2 HCl
N
R
Compound
Substituent
R
IC50
rat spleen
(
l
M)
IC50
rat brain
(
l
M)
Selectivity index
IC50 (HO-1)/IC50 (HO-2)
(HO-1)
(HO-2)
1
2
3
4
5
6
7
8
4-Cl-PhCH2
4-F-PhCH2
4-Br-PhCH2
4-I-PhCH2
4-Me-PhCH2
4-i-Pr-PhCH2
4-OMe-PhCH2
4-SMe-PhCH2
4-OBn-PhCH2
4-CN-PhCH2
4-CF3-PhCH2
4-NO2-PhCH2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.4 0.3
2.0 0.2
2.6 0.4
7.6 0.6
>29
>50
>38
>13
>20
>1.8
>5
>6
>1.5
>9
5
1
55 18
18
15
5
3
9a
10
11
12
63 23
11
15.6 0.4
27
1
>6.4
>3
5
13
14
15
16
17
3-Cl-PhCH2
3-Br-PhCH2
3-Me-PhCH2
3-CN-PhCH2
3-NO2-PhCH2
>100
>100
>100
>100
>100
1.65 0.08
1.5 0.3
>60
>66
>20
>6
5
1
15.8 0.3
1.3 0.2
>76
18
19
20
21
22
2-Cl-PhCH2
2-Br-PhCH2
2-Me-PhCH2
2-CN-PhCH2
2-NO2-PhCH2
>100
4.8 0.4
1.6 0.3
1.8 0.2
>20
26
42
>25
>28
42
1
76 17
>100
>100
4
1
3.5 0.6
23
24
25
26
27
28
2,3-diCl-PhCH2
2,4-diCl-PhCH2
2,5-diCl-PhCH2
2,6-diCl-PhCH2
3,4-diCl-PhCH2
3,5-diCl-PhCH2
>100
62 10
14.7 0.5
6.1 0.3
>6
10
24
>10
>5
96
5
4
3
>100
>100
>100
9.2 0.2
17
3
2.1 0.3
>47
29
30
31
PhCH2
PhCH2CH2
PhOCH2CH2
>100
>100
>100
3.1 0.7
2.7 0.2
>100
>32
>37
—
32b
33
34
(Ph)2CH
(Naphthalene-2-yl)-CH2
Cyclohexyl-CH2
>100
>100
>100
4.1 0.1
>24
>20
>16
5
6
2
1
35
36
CH3CH2CH2
H
>100
>100
27
>100
5
>3
—
Data represent mean IC50 values SD of replicate experiments (n = 4).
a
Isolated in the free base form.
Isolated in the monohydrochloride form.
b

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 21 (2013) 6788–6795
6791
concentrated, Et2O (5 mL) was added, and the mixture concen-
trated again. High-vacuum drying gave the product (242 mg,
0.61 mmol, 49%) as a white solid; mp 232–234 °C; Rf = 0 (EtOAc);
2H), 7.30–7.37 (m, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.55–7.61
(m, 1H), 7.73–7.81 (m, 1H); 13C NMR (100 MHz, DMSO-d6): d
22.7, 46.8, 47.8, 53.9, 111.7, 118.4, 121.0, 123.4, 124.0, 129.3,
131.7, 134.3, 135.4, 139.7, 145.8; HRMS (ESI) [MꢁHCl2]+ Calcd
for C19H21N3Br: 370.0918. Found: 370.0921.
1H NMR (400 MHz, CD3OD):
d 2.10–2.26 (m, 4H), 3.55–3.78
(m, 4H), 5.21 (s, 2H), 5.95 (s, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.40
(d, J = 8.4 Hz, 2H), 7.53–7.68 (m, 3H), 7.92 (d, J = 8.0 Hz, 1H); 13C
NMR (100 MHz, CD3OD): d 24.0, 48.0, 50.0, 56.5, 114.4, 117.3,
128.0, 128.1, 129.9, 130.4, 134.0, 134.3, 135.1, 135.7, 144.5; HRMS
(ESI) [MꢁCl]+ Calcd for C19H21N3Cl: 326.1424. Found: 326.1433.
4.4.3. 1-(4-Iodobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimid-
azole dihydrochloride (4)
Compound 4 was prepared using Procedure A with 36 and 4-iod-
obenzyl bromide as starting materials to give the product (387 mg,
60%) as a beige solid; mp 141–144 °C; 1H NMR (400 MHz, DMSO-
d6): d 1.80–2.20 (m, 4H), 3.20–3.40 (m, 2H), 3.60–3.80 (m, 2H),
4.88 (s, 2H), 5.67 (s, 2H), 7.01 (d, J = 8.4 Hz, 2H), 7.28–7.35 (m,
2H), 7.52–7.58 (m, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.72–7.77 (m,
1H); 13C NMR (100 MHz, DMSO-d6): d 22.7, 46.7, 48.0, 54.0,
111.6, 118.6, 123.2, 123.8, 129.3, 132.8, 134.5, 135.9, 137.5,
140.2, 145.9; HRMS (ESI) [MꢁHCl2]+ Calcd for C19H21N3I:
418.0780. Found: 418.0780.
4.3. Representative Procedure B for the formation of alkylated
benzimidazole derivatives, as outlined in Scheme 1
1-Benzyl-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole
dihydro-
chloride (29). Under an atmosphere of nitrogen, compound 36
(511 mg, 2.54 mmol, 1 equiv) was combined with sodium hydrox-
ide (102 mg, 2.54 mmol, 1 equiv) and DMSO (4 mL). The mixture
was heated at 75–80 °C for 15 min, cooled to rt, then benzyl bro-
mide (0.30 mL, 434 mg, 2.54 mmol, 1 equiv) was added. The mix-
ture was stirred at rt for 2 h, then at ꢀ60 °C for 21 h. After
cooling to rt, a saturated aqueous solution of NaHCO3 was added
and the mixture extracted with EtOAc (2ꢂ). The combined organic
extracts were washed with a saturated aqueous solution of
NaHCO3 (2ꢂ), then with brine, and dried (Na2SO4). The solution
was concentrated and the remaining brown oil residue purified
by flash column chromatography on silica gel (EtOAc) to give the
free base (97 mg, 0.33 mmol) as an oil (Rf = 0.53, EtOAc). To a solu-
tion of the free base in MeOH (3 mL) was added a solution of 37%
aqueous HCl (98 mg, 0.99 mmol, 3 equiv) in MeOH (3 mL). The
mixture was concentrated, Et2O (5 mL) was added, and the mixture
concentrated again. High-vacuum drying gave the product
(130 mg, 0.36 mmol, 14%) as a white solid; mp 126–128 °C (dec);
1H NMR (400 MHz, DMSO-d6): d 1.80–2.16 (m, 4H), 3.22–3.50
(m, 2H), 3.50–3.80 (m, 2H), 4.97 (s, 2H), 5.78 (s, 2H), 7.22–7.43
(m, 7H), 7.56–7.62 (m, 1H), 7.76–7.81 (m, 1H); 13C NMR
(100 MHz, DMSO-d6): d 22.7, 47.4, 47.7, 53.9, 112.1, 118.0, 123.8,
124.3, 127.1, 127.9, 128.8, 134.1, 135.8, 138.7, 145.4; HRMS (ESI)
[MꢁHCl2]+ Calcd for C19H22N3: 292.1813. Found: 292.1810.
4.4.4. 1-(4-Methylbenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (5)
Compound 5 was prepared using Procedure A with 36 and 4-
methylbenzyl bromide as starting materials to give the product
(648 mg, 87%) as
a
beige solid; mp 120–125 °C; 1H NMR
(400 MHz, CD3OD): d 2.14–2.23 (m, 4H), 2.32 (s, 3H), 3.58–3.72
(m, 4H), 5.23 (s, 2H), 5.92 (s, 2H), 7.18–7.24 (m, 4H), 7.55–7.60
(m, 1H), 7.64 (td, J = 7.7, 0.9 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.92
(d, J = 8.4 Hz, 1H); 13C NMR (100 MHz, CD3OD): d 21.1, 24.0, 47.8,
50.6, 56.4, 114.7, 116.9, 128.1, 128.3 (2C), 130.9, 132.0, 134.3,
134.4, 140.1, 144.2; HRMS (MALDI) [MꢁHCl2]+ Calcd for
C20H24N3: 306.1970. Found: 306.1973.
4.4.5. 1-(4-Isopropylbenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (6)
Compound 6 was prepared using Procedure A with 36 and
4-isopropylbenzyl chloride as starting materials to give the
product (155 mg, 31%) as a beige solid; mp 110–112 °C; 1H NMR
(400 MHz, DMSO-d6):
d 1.15 (d, J = 6.8 Hz, 6H), 1.86–2.10
(m, 4H), 2.84 (7-tet, 1H), 3.22–3.44 (m, 2H), 3.57–3.77 (m, 2H),
4.94 (s, 2H), 5.70 (s, 2H), 7.15 (d, J = 8.0 Hz, 2H), 7.21 (d,
J = 8.0 Hz, 2H), 7.34 (d, J = 3.2 Hz, 1H), 7.36 (d, J = 3.2 Hz, 1H),
7.62 (dd, J = 6.4, 2.8 Hz, 1H), 7.77 (dd, J = 6.4, 2.8 Hz, 1H);
13C NMR (100 MHz, DMSO-d6): d 22.7, 23.8, 33.1, 47.2, 47.7, 53.9,
111.9, 118.2, 123.5, 124.1, 126.7, 127.0, 133.3, 134.7, 139.3,
145.6, 148.1; HRMS (ESI) [MꢁHCl2]+ Calcd for C22H28N3:
334.2283. Found: 334.2279.
4.4. Characterization of the compounds synthesized following
the representative procedure for the formation of alkylated
benzimidazole derivatives
4.4.1. 1-(4-Fluorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzim-
idazole dihydrochloride (2)
Compound 2 was prepared using Procedure B with 36 and 4-flu-
orobenzyl bromide as starting materials to give the product
(35 mg, 3%) as a beige solid (after recrystallization from 2-propa-
nol); mp 166–168 °C; Rf = 0 (EtOAc); 1H NMR (400 MHz, CD3OD):
d 2.08–2.22 (m, 4H), 3.50–3.73 (m, 4H), 4.90 (s, 2H), 5.66 (s, 2H),
7.08 (app t, J = 8.4 Hz, 2H), 7.20–7.28 (m, 2H), 7.34–7.42 (m, 2H),
7.48–7.55 (m, 2H), 7.74–7.81 (m, 2H); 13C NMR (100 MHz, CD3OD):
4.4.6. 1-(4-Methoxybenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (7)
Compound 7 was prepared using Procedure A with 36 and
4-methoxybenzyl chloride as starting materials to give the product
(146 mg, 30%) as a beige solid; mp 116–118 °C; 1H NMR (400 MHz,
DMSO-d6): d 1.80–2.15 (m, 4H), 3.28–3.44 (m, 2H), 3.60–3.70
(m, 2H), 3.71 (s, 3H), 4.96 (s, 2H), 5.67 (s, 2H), 6.89 (d, J = 8.4 Hz,
2H), 7.24 (d, J = 8.4 Hz, 2H), 7.31–7.39 (m, 2H), 7.58–7.65 (m,
1H), 7.73–7.79 (m, 1H), 11.68 (br s, 1H); 13C NMR (100 MHz,
DMSO-d6): d 22.7, 47.2, 47.6, 53.9, 55.1, 112.1, 114.2, 118.1,
123.6, 124.1, 127.6, 128.7, 134.1, 138.9, 145.4, 158.9; HRMS (ESI)
[MꢁHCl2]+ Calcd for C20H24N3O: 322.1919. Found: 322.1915.
2
d 24.1, 48.1, 50.7, 56.5, 112.5, 116.8, 117.0, 119.7, 125.4 (d, JC–
3
4
F = 67.6 Hz), 130.0 (d, JC–F = 8.3 Hz), 132.7 (d, JC–F = 3.4 Hz),
136.1, 141.2, 146.3, 163.9 (d, JC–F = 246.0 Hz); 19F NMR
1
3
(376 MHz, CD3OD):
d
ꢁ116.9 (t, JF–H = 4.3 Hz); HRMS (EI)
[MꢁHCl2]+ Calcd for C19H21N3F: 310.1720. Found: 310.1711.
4.4.2. 1-(4-Bromobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (3)
Compound 3 was prepared using Procedure A with 36 and 4-
bromobenzyl bromide as starting materials to give the product
4.4.7. 1-(4-Thiomethylbenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (8)
Compound 8 was prepared using Procedure A with 36 and 4-thi-
omethylbenzyl bromide as starting materials to give the product
(117 mg, 66%) as a yellow-white solid; mp 126–129 °C; 1H NMR
(133 mg, 44%) as
a
beige solid; mp 122–124 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.20 (m, 4H), 3.22–3.50 (m, 2H),
3.50–3.80 (m, 2H), 4.92 (s, 2H), 5.73 (s, 2H), 7.19 (d, J = 8.4 Hz,

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. 21 (2013) 6788–6795
(400 MHz, DMSO-d6): d 1.83–1.99 (m, 2H), 1.99–2.14 (m, 2H), 2.43
(s, 3H), 3.26–3.43 (m, 2H), 3.61–3.76 (m, 2H), 4.95 (s, 2H), 5.70
(s, 2H), 7.17–7.24 (m, 4H), 7.32–7.38 (m, 2H), 7.57–7.62 (m, 1H),
7.74–7.80 (m, 1H); 13C NMR (100 MHz, DMSO-d6): d 14.6, 22.7,
47.2, 47.7, 54.0, 112.0, 118.2, 123.6, 124.1, 126.1, 127.8, 132.3,
134.2, 138.0, 139.2, 145.6; HRMS (ESI) [MꢁHCl2]+ Calcd for
4.4.12. 1-(3-Chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (13)
Compound 13 was prepared using Procedure A with 36 and
3-chlorobenzyl bromide as starting materials to give the product
(285 mg, 57%) as an off-white solid; mp 115–120 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.20 (m, 4H), 3.20–3.50 (m, 2H),
3.50–3.80 (m, 2H), 3.50–3.80 (m, 2H), 4.95 (s, 2H), 5.77 (s, 2H),
7.14–7.21 (m, 2H), 7.30–7.41 (m, 5H), 7.53–7.61 (m, 1H), 7.74–
7.81 (m, 1H); 13C NMR (100 MHz, DMSO-d6): d 22.7, 46.9, 47.7,
53.9, 111.8, 118.4, 123.6, 124.1, 125.7, 126.9, 127.9, 130.7, 133.4,
134.2, 138.4, 139.5, 145.8; HRMS (ESI) [MꢁHCl2]+ Calcd for
C20H24N3S: 338.1690. Found: 338.1694.
4.4.8. 1-(4-Benzyloxybenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole (9)
Compound 9 was prepared using Procedure A with 36 and
4-benzyloxybenzyl chloride as starting materials to give the prod-
uct (144 mg, 29%) as a golden oil isolated in the free base form; 1H
NMR (400 MHz, DMSO-d6): d 1.61–1.73 (m, 4H), 2.45–2.55 (m, 4H),
3.85 (s, 2H), 5.04 (s, 2H), 5.49 (s, 2H), 6.96 (d, J = 8.8 Hz, 2H), 7.12–
7.19 (m, 4H), 7.31 (d, J = 6.8 Hz, 1H), 7.36 (app t, J = 7.4 Hz, 2H),
7.39–7.45 (m, 3H), 7.56–7.62 (m, 1H); 13C NMR (100 MHz,
DMSO-d6): d 23.2, 46.1, 52.1, 53.5, 69.2, 110.5, 114.8, 118.9,
121.3, 122.1, 127.6, 127.8, 128.3, 128.4, 129.3, 135.5, 137.0,
142.1, 152.1, 157.6; HRMS (ESI) [M+H]+ Calcd for C26H28N3O:
398.2232. Found: 398.2315.
C19H21ClN3: 326.1424. Found: 326.1428.
4.4.13. 1-(3-Bromobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (14)
Compound 14 was prepared using Procedure A with 36 and
3-bromobenzyl bromide as starting materials to give the product
(259 mg, 47%) as
a
white solid; mp 162–165 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.20 (m, 4H), 3.20–3.52 (m, 2H),
3.52–3.83 (m, 2H), 4.96 (s, 2H), 5.77 (s, 2H), 6.82 (br s, ꢀ1H),
7.21 (d, J = 7.6 Hz, 1H), 7.30 (t, J = 8.2 Hz, 1H), 7.33–7.40 (m, 2H),
7.47–7.53 (m, 2H), 7.54–7.61 (m, 1H), 7.75–7.81 (m, 1H), 11.68
(br s, 1H); 13C NMR (100 MHz, DMSO-d6): d 22.7, 47.0, 47.6, 53.9,
111.8, 118.3, 122.1, 123.8, 124.3, 126.1, 129.8, 130.9, 131.0,
134.1, 138.6, 139.1, 145.7; HRMS (ESI) [MꢁHCl2]+ Calcd for C19H21-
N3Br: 370.0918. Found: 370.0903.
4.4.9. 1-(4-Cyanobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (10)
Compound 10 was prepared using Procedure A with 36 and
4-(chloromethyl)benzonitrile as starting materials to give the
product (129 mg, 27%) as a white solid; mp 126–130 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.18 (m, 4H), 3.20–3.45 (m, 2H),
3.52–3.80 (m, 2H), 4.90 (s, 2H), 5.86 (s, 2H), 7.33 (app t,
J = 3.8 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.53 (dd, J = 5.8, 2.6 Hz,
1H), 7.77 (dd, J = 6.2, 2.6 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H); 13C
NMR (100 MHz, DMSO-d6): d 22.7, 47.0, 47.8, 53.9, 110.6, 111.6,
118.5, 118.6, 123.4, 124.0, 127.9, 132.7, 134.3, 140.0, 141.7,
146.0; HRMS (ESI) [MꢁHCl2]+ Calcd for C20H21N4: 317.1766.
Found: 317.1759.
4.4.14. 1-(3-Methylbenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (15)
Compound 15 was prepared using Procedure A with 36 and 3-
methylbenzyl bromide as starting materials to give the product
(207 mg, 44%) as
a
white solid; mp 121–125 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.85–2.15 (m, 4H), 3.25–3.50 (m, 2H),
3.50–3.80 (m, 2H), 4.95 (s, 2H), 5.72 (s, 2H), 7.01 (d, J = 7.6 Hz,
1H), 7.06–7.14 (m, 2H), 7.22 (t, J = 7.6 Hz, 1H), 7.32–7.40 (m, 2H),
7.55–7.63 (m, 1H), 7.74–7.82 (m, 1H), 7.84 (br s, 1H), 11.73 (br s,
1H); 13C NMR (100 MHz, DMSO-d6): d 20.9, 22.7, 47.5, 47.6, 53.9,
112.0, 118.1, 123.7, 124.1, 124.2, 127.5, 128.6, 128.7, 134.2,
135.7, 138.1, 138.9, 145.5; HRMS (ESI) [MꢁHCl2]+ Calcd for
4.4.10. 1-(4-Trifluoromethylbenzyl)-2-(pyrrolidin-1-ylmethyl)-
1H-benzimidazole dihydrochloride (11)
C20H24N3: 306.1970. Found: 306.1978.
Compound 11 was prepared using Procedure A with 36 and 4-
trifluoromethylbenzyl bromide as starting materials to give the
product (225 mg, 42%) as a white solid; mp 236–237 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.20 (m, 4H), 3.20–3.40 (m, 2H),
3.60–3.80 (m, 2H), 4.90 (s, 2H), 5.85 (s, 2H), 7.28–7.36 (m, 2H),
7.39 (d, J = 8.0 Hz, 2H), 7.53–7.58 (m, 1H), 7.71 (d, J = 8.0 Hz, 2H),
7.74–7.80 (m, 1H); 13C NMR (100 MHz, DMSO-d6): d 22.7, 46.7,
4.4.15. 1-(3-Cyanobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (16)
Compound 16 was prepared using Procedure A with 36 and
3-cyanobenzyl chloride as starting materials to give the product
(202 mg, 42%) as a yellow-white solid; mp 133–136 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.20 (m, 4H), 3.22–3.52 (m, 2H),
3.52–3.84 (m, 2H), 4.98 (s, 2H), 5.82 (s, 2H), ꢀ6.6 (br s, ꢀ1H),
7.30–7.41 (m, 2H), 7.50–7.65 (m, 3H), 7.73–7.84 (m, 3H), 11.68
(br s, 1H); 13C NMR (75 MHz, DMSO-d6): d 22.7, 46.9, 47.7, 54.0,
111.7, 111.8, 118.3, 118.6, 123.7, 124.2, 130.0, 130.8, 131.8,
132.2, 134.0, 137.5, 139.3, 145.9; HRMS (ESI) [MꢁHCl2]+ Calcd for
1
48.0, 53.9, 111.5, 118.8, 123.2, 123.8, 123.9 (quartet, JC–
3
2
F = 263.3 Hz), 125.7 (d, JC–F = 3.6 Hz), 127.6, 128.3 (d, JC–F
=
31.7 Hz), 134.5, 140.4, 140.9, 146.1; 19F NMR (376 MHz,
DMSO-d6): d ꢁ62.0; HRMS (ESI) [MꢁHCl2]+ Calcd for C20H21N3F3:
360.1687. Found: 360.1686.
C20H21N4: 317.1766. Found: 317.1762.
4.4.11. 1-(4-Nitrobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (12)
4.4.16. 1-(3-Nitrobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (17)
Compound 12 was prepared using Procedure A with 36 and
4-nitrobenzyl bromide as starting materials to give the product
(137 mg, 27%) as an orange solid; mp 132–135 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.00 (m, 2H), 2.00–2.20 (m, 2H),
3.20–3.44 (m, 2H), 3.53–3.80 (m, 2H), 4.90 (s, 2H), 5.91 (s, 2H),
7.28–7.38 (5-tet, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.51–7.57 (m, 1H),
7.73–7.81 (m, 1H), 8.19 (d, J = 8.8 Hz, 2H), 11.61 (br s, 1H);
13C NMR (100 MHz, DMSO-d6): d 22.7, 46.7, 48.0, 53.9, 111.5,
118.8, 123.3, 123.9, 124.0, 128.1, 134.5, 140.4, 143.8, 146.1,
147.0; HRMS (ESI) [MꢁHCl2]+ Calcd for C19H21N4O2: 337.1664.
Found: 337.1663.
Compound 17 was prepared using Procedure A with 36 and
3-nitrobenzyl bromide as starting materials to give the product
(368 mg, 73%) as an orange solid; mp 130–134 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.20 (m, 4H), 3.20–3.50 (m, 2H),
3.50–3.82 (m, 2H), 4.94 (s, 2H), 5.91 (s, 2H), 7.30–7.39 (m, 2H),
7.55–7.67 (m, 4H), 7.75–7.81 (m, 1H), 8.08–8.13 (m, 1H), 8.13–
8.19 (m, 1H), 11.60 (br s, 1H); 13C NMR (100 MHz, DMSO-d6): d
22.7, 46.6, 47.9, 53.9, 111.6, 118.6, 121.8, 122.8, 123.5, 124.1,
130.4, 133.6, 134.3, 138.3, 140.0, 146.0, 148.0; HRMS (ESI)
[MꢁHCl2]+ Calcd for C19H21N4O2: 337.1664. Found: 337.1660.

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 21 (2013) 6788–6795
6793
4.4.17. 1-(2-Chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (18)
7.70 (m, 3H), 7.96 (d, J = 8.0 Hz, 1H), 8.28–8.37 (m, 1H); 13C NMR
(100 MHz, CD3OD): d 24.0, 48.1, 48.6, 56.5, 114.0, 117.8, 127.1,
127.90, 127.97, 129.0, 130.9, 131.0, 134.6, 135.7, 135.8, 145.4,
148.8; HRMS (ESI) [MꢁHCl2]+ Calcd for C19H21N4O2: 337.1659.
Found: 337.1648.
Compound 18 was prepared using Procedure A with 36 and
2-chlorobenzyl bromide as starting materials to give the product
(310 mg, 63%) as
a
white solid; mp 203–204 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.84–2.15 (m, 4H), 3.21–3.45 (m, 2H),
3.55–3.80 (m, 2H), 4.88 (s, 2H), 5.78 (s, 2H), 6.72 (d, J = 6.8 Hz,
1H), 7.24 (td, J = 7.5, 0.9 Hz, 1H), 7.28–7.42 (m, 4H), 7.56 (dd,
J = 8.0, 0.8 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 9.57 (br s, 1H), 11.66
(br s, 1H); 13C NMR (100 MHz, DMSO-d6): d 22.7, 45.6, 47.9, 53.9,
111.4, 118.7, 123.4, 124.0, 127.7, 127.9, 129.6, 129.8, 131.8,
133.1, 134.4, 140.0, 146.2; HRMS (ESI) [MꢁHCl2]+ Calcd for C19H21-
ClN3: 326.1424. Found: 326.1419.
4.4.22. 1-(2,3-Dichlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (23)
Compound 23 was prepared using Procedure A with 36 and 2,3-
dichlorobenzyl chloride as starting materials to give the product
(306 mg, 57%) as
a
white solid; mp 163–167 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.70–2.20 (m, 4H), 3.20–3.50 (m, 2H),
3.50–3.80 (m, 2H), 4.84 (s, 2H), 5.81 (s, 2H), 6.51 (d, J = 7.6 Hz,
1H), 7.25 (t, J = 7.8 Hz, 1H), 7.28–7.40 (m, 2H), 7.46 (d, J = 8.0 Hz,
1H), 7.61 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 8.69 (br s,
1H), 11.63 (br s, 1H); 13C NMR (100 MHz, DMSO-d6): d 22.7, 46.1,
47.9, 53.9, 111.4, 118.8, 123.3, 124.0, 125.9, 128.6, 129.8, 132.3,
134.5, 136.0, 146.3; HRMS (ESI) [MꢁHCl2]+ Calcd for C19H20Cl2N3:
360.1034. Found: 360.1044.
4.4.18. 1-(2-Bromobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (19)
Compound 19 was prepared using Procedure A with 36 and
2-bromobenzyl bromide as starting materials to give the product
(431 mg, 78%) as
a
white solid; mp 238–240 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.20 (m, 4H), 3.18–3.50 (m, 2H),
3.50–3.83 (m, 2H), 4.89 (s, 2H), 5.74 (s, 2H), 6.64 (dd, J = 5.4,
3.8 Hz, 1H), ꢀ7.07 (br s, ꢀ1H), 7.23–7.30 (m, 2H), 7.30–7.40 (m,
3H), 7.69–7.76 (m, 1H), 7.79 (d, J = 7.6 Hz, 1H), 11.67 (br s, 1H);
13C NMR (100 MHz, DMSO-d6): d 22.8, 47.9, 48.0, 54.0, 111.5,
118.7, 121.9, 123.5, 124.1, 127.9, 128.3, 129.9, 133.1, 134.3,
134.6, 139.9, 146.2; HRMS (ESI) [MꢁHCl2]+ Calcd for C19H21N3Br:
370.0918. Found: 370.0906.
4.4.23. 1-(2,4-Dichlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (24)
Compound 24 was prepared using Procedure A with 36 and
2,4-dichlorobenzyl chloride as starting materials to give the
product (236 mg, 44%) as a white solid; mp 252–253 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.15 (m, 4H), 3.18–3.46 (m, 2H),
3.52–3.80 (m, 2H), 4.85 (s, 2H), 5.75 (s, 2H), 6.70 (d, J = 8.4 Hz,
1H), ꢀ7.25 (br s, 1H), 7.27–7.37 (m, 3H), 7.41 (d, J = 8.0 Hz, 1H),
7.74 (d, J = 2.0 Hz, 1H), 7.78 (d, J = 7.2 Hz, 1H), 11.61 (br s, 1H);
13C NMR (100 MHz, DMSO-d6): d 22.7, 45.2, 48.0, 53.9, 111.3,
118.9, 123.3, 124.0, 127.9, 129.2, 132.5, 132.8, 133.2, 134.4,
140.4, 146.3; HRMS (ESI) [MꢁHCl2]+ Calcd for C19H20Cl2N3:
360.1034. Found: 360.1020.
4.4.19. 1-(2-Methylbenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (20)
Compound 20 was prepared using Procedure A with 36 and
2-methylbenzyl chloride as starting materials to give the product
(345 mg, 73%) as a pale yellow solid; mp 138–140 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.85–2.10 (m, 4H), 2.42 (s, 3H), 3.20–3.45
(m, 2H), 3.55–3.88 (m, 2H), 4.79 (s, 2H), 5.74 (s, 2H), 6.25 (d,
J = 7.6 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H),
7.25–7.43 (m, 3H), 7.47 (d, J = 7.6 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H),
11.58 (br s, 1H); 13C NMR (100 MHz, DMSO-d6): d 18.9, 22.7,
45.5, 47.8, 53.9, 111.7, 118.5, 123.3, 123.9, 124.3, 126.2, 127.4,
130.4, 134.1, 134.7, 135.4, 139.9, 146.1; HRMS (ESI) [MꢁHCl2]+
Calcd for C20H24N3: 306.1965. Found: 306.1960.
4.4.24. 1-(2,5-Dichlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (25)
Compound 25 was prepared using Procedure A with 36 and 2,5-
dichlorobenzyl bromide as starting materials to give the product
(441 mg, 82%) as
a
white solid; mp 192–194 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.20 (m, 4H), 3.20–3.50 (m, 2H),
3.50–3.82 (m, 2H), 4.92 (s, 2H), 5.79 (s, 2H), 6.78 (d, J = 2.4 Hz,
1H), 7.29–7.42 (m, 3H), 7.44 (dd, J = 8.4, 2.4 Hz, 1H), 7.61 (d,
J = 8.4 Hz, 1H), 7.80 (d, J = 7.2 Hz, 1H), 8.44 (br s, 1H), 11.67 (br s,
1H); 13C NMR (100 MHz, DMSO-d6): d 22.7, 45.6, 47.8, 53.9,
111.4, 118.7, 123.6, 124.3, 127.6, 129.6, 130.7, 131.5, 132.3,
134.1, 135.3, 139.8, 146.4; HRMS (ESI) [MꢁHCl2]+ Calcd for C19H20-
Cl2N3: 360.1034. Found: 360.1050.
4.4.20. 1-(2-Cyanobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (21)
Compound 21 was prepared using Procedure A with 36 and
2-cyanobenzyl chloride as starting materials to give the product
(268 mg, 56%) as
a
beige solid; mp 138–140 °C; 1H NMR
4.4.25. 1-(2,6-Dichlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (26)
(400 MHz, DMSO-d6): d 1.80–2.17 (m, 4H), 3.22–3.50 (m, 2H),
3.50–3.84 (m, 2H), 4.93 (s, 2H), 5.95 (s, 2H), 6.95 (d, J = 7.6 Hz,
1H), ꢀ7.02 (br s, ꢀ1H), 7.25–7.35 (m, 2H), 7.38 (t, J = 7.4 Hz, 1H),
7.52 (t, J = 7.6 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.79 (t, J = 7.6 Hz,
1H), 7.93 (t, J = 7.6 Hz, 1H), 11.69 (br s, 1H); 13C NMR (100 MHz,
DMSO-d6): d 22.7, 46.4, 47.8, 53.9, 110.2, 111.5, 117.2, 118.7,
123.5, 124.1, 127.2, 128.7, 133.7, 133.9, 134.3, 139.3, 140.1,
146.4; HRMS (ESI) [MꢁHCl2]+ Calcd for C20H21N4: 317.1766.
Found: 317.1761.
Compound 26 was prepared using Procedure A with 36 and
2,6-dichlorobenzyl chloride as starting materials to give the prod-
uct (182 mg, 34%) as a white solid; mp 253–254 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.22 (m, 4H), 3.22–3.50 (m, 2H),
3.50–3.84 (m, 2H), 5.04 (s, 2H), 5.94 (s, 2H), 6.96 (d, J = 8.0 Hz,
1H), 7.19 (t, J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.47 (t,
J = 8.2 Hz, 1H), 7.55–7.61 (m, 2H), 7.72 (d, J = 8.0 Hz, 1H), 11.62
(br s, 1H); 13C NMR (100 MHz, DMSO-d6): d 22.7, 45.0, 48.6, 53.9,
110.7, 119.1, 122.9, 123.8, 129.5, 129.8, 131.5, 133.9, 135.5,
140.6, 146.9; HRMS (ESI) [MꢁHCl2]+ Calcd for C19H20Cl2N3:
360.1034. Found: 360.1042.
4.4.21. 1-(2-Nitrobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (22)
Compound 22 was prepared using Procedure A with 36 and
2-nitrobenzyl chloride as starting materials to give the product
(25 mg, 5%) as an beige solid; mp 114–116 °C; 1H NMR
(400 MHz, CD3OD): d 2.10–2.33 (m, 4H), 3.55–3.85 (m, 4H), 5.19
(s, 2H), 6.34 (s, 2H), 6.82–6.92 (m, 1H), 7.50–7.60 (m, 2H), 7.60–
4.4.26. 1-(3,4-Dichlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (27)
Compound 27 was prepared using Procedure A with 36 and
3,4-dichlorobenzyl chloride as starting materials to give the

6794
J. Z. Vlahakis et al. / Bioorg. Med. Chem. 21 (2013) 6788–6795
product (306 mg, 56%) as a white solid; mp 218–220 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.18 (m, 4H), 3.22–3.50 (m, 2H),
3.50–3.82 (m, 2H), 4.94 (s, 2H), 5.76 (s, 2H), 7.19 (dd, J = 8.4,
2.0 Hz, 1H), 7.30–7.40 (m, 2H), 7.54–7.63 (m, 2H), 7.74–7.81
(m, 2H), 7.79 (br s, 1H), 11.63 (br s, 1H); 13C NMR (100 MHz,
DMSO-d6): d 22.7, 46.3, 47.8, 53.9, 111.6, 118.5, 123.4, 124.0,
127.4, 129.1, 130.5, 130.9, 131.4, 134.2, 137.1, 139.7, 145.9; HRMS
(ESI) [MꢁHCl2]+ Calcd for C19H20Cl2N3: 360.1034. Found: 360.1030.
product (123 mg, 24%) as a white solid; mp 134–138 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.24 (m, 4H), 3.20–3.54 (m, 2H),
3.54–3.90 (m, 2H), 4.97 (s, 2H), 5.89 (s, 2H), 7.27–7.36 (m, 2H),
7.38 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 3.2 Hz, 1H), 7.51 (d, J = 3.2 Hz,
1H), 7.61 (d, J = 7.2 Hz, 1H), 7.73–7.82 (m, 2H), 7.85 (dd, J = 6.0,
3.2 Hz, 1H), 7.87–7.95 (m, 2H), 8.39 (br s, 1H), 11.61 (br s, 1H);
13C NMR (100 MHz, DMSO-d6): d 22.7, 47.6, 48.0, 54.0, 111.7,
118.5, 123.3, 123.8, 125.0, 125.5, 126.3, 126.5, 127.6, 127.7,
128.5, 132.4, 132.8, 133.5, 134.5, 140.0, 145.9; HRMS (ESI)
[MꢁHCl2]+ Calcd for C23H24N3: 342.1964. Found: 342.1963.
4.4.27. 1-(3,5-Dichlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (28)
Compound 28 was prepared using Procedure A with 36 and
3,5-dichlorobenzyl chloride as starting materials to give the
product (288 mg, 65%) as a pale yellow solid; mp 144–147 °C; 1H
4.4.32. 1-(Cyclohexylmethyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (34)
Compound 34 was prepared using Procedure A with 36 and
(bromomethyl)cyclohexane as starting materials to give the prod-
uct (284 mg, 62%) as a white solid; mp 127–130 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.01–1.21 (m, 5H), 1.40–1.54 (m, 2H),
1.54–1.71 (m, 3H), 1.76–1.89 (m, 1H), 1.89–2.12 (m, 4H),
3.25–3.80 (m, 4H), 4.33 (d, J = 7.2 Hz, 2H), 4.94 (s, 2H), 7.37–
7.48 (m, 2H), 7.74–7.81 (m, 1H), 7.81–7.89 (m, 1H), 11.70 (br s,
1H); 13C NMR (100 MHz, DMSO-d6): d 22.7, 25.1, 25.7, 29.7,
37.8, 47.1, 50.1, 54.0, 112.4, 117.4, 124.0, 124.3, 134.3, 137.3,
145.0; HRMS (EI) [MꢁHCl2]+ Calcd for C19H28N3: 298.2283.
Found: 298.2276.
NMR (400 MHz, DMSO-d6):
d 1.80–2.20 (m, 4H), 3.21–3.50
(m, 2H), 3.50–3.80 (m, 2H), 4.95 (s, 2H), 5.77 (s, 2H), 7.28–7.33
(m, 2H), 7.33–7.40 (m, 2H), 7.52–7.59 (m, 2H), 7.75–7.81 (m,
1H), 9.79 (br s, 1H), 11.63 (br s, 1H); 13C NMR (100 MHz, DMSO-
d6): d 22.7, 46.5, 47.7, 53.9, 111.6, 118.5, 123.6, 124.2, 125.9,
127.6, 134.1, 134.4, 139.6, 140.2, 146.0; HRMS (ESI) [MꢁHCl2]+
Calcd for C19H20Cl2N3: 360.1034. Found: 360.1030.
4.4.28. 1-(2-Phenylethyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (30)
Compound 30 was prepared using Procedure A with 36 and (2-
bromoethyl)benzene as starting materials to give the product
(119 mg, 25%) as a hygroscopic white solid; mp ꢀ70–80 °C; 1H
4.4.33. 1-n-Propyl-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole
dihydrochloride (35)
NMR (400 MHz, DMSO-d6):
d 1.80–2.10 (m, 4H), 3.09 (t,
Compound 35 was prepared using Procedure A with 36 and 1-
bromopropane as starting materials to give the product (227 mg,
58%) as a white solid; mp 190–195 °C; 1H NMR (400 MHz,
DMSO-d6): d 0.93 (t, J = 7.4 Hz, 3H), 1.74–1.87 (m, 2H), 1.87–
2.15 (m, 4H), 3.32–3.54 (m, 2H), 3.54–3.80 (m, 2H), 4.49 (t,
J = 7.4 Hz, 2H), 4.99 (s, 2H), 7.42–7.54 (m, 2H), 7.81 (d,
J = 7.6 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 10.40 (br s, 1H), 11.94
(br s, 1H); 13C NMR (100 MHz, DMSO-d6): d 10.9, 22.6, 22.7,
46.3, 46.4, 53.9, 112.5, 116.9, 124.7, 124.8, 133.4, 135.9, 144.3;
HRMS (ESI) [MꢁHCl2]+ Calcd for C15H22N3: 244.1813. Found:
244.1811.
J = 7.2 Hz, 2H), 3.18–3.42 (m, 2H), 3.42–3.78 (m, 2H), 4.58–4.72
(m, 4H), 7.15–7.30 (m, 5H), 7.30–7.42 (m, 2H), 7.68–7.77 (m,
2H), 11.53 (br s, 1H); 13C NMR (100 MHz, DMSO-d6): d 22.7, 34.9,
45.6, 47.4, 53.9, 111.7, 117.8, 123.5, 123.9, 126.7, 128.4, 129.1,
133.9, 137.7, 138.5, 145.1; HRMS (ESI) [MꢁHCl2]+ Calcd for
C20H24N3: 306.1970. Found: 306.1974.
4.4.29. 1-(2-Phenoxyethyl)-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole dihydrochloride (31)
Compound 31 was prepared using Procedure A with 36 and
2-phenoxyethyl bromide as starting materials to give the product
(273 mg, 56%) as
a
white solid; mp 98–100 °C; 1H NMR
4.5. Other synthetic procedures
(400 MHz, DMSO-d6): d 1.80–2.20 (m, 4H), 3.30–3.50 (m, 2H),
3.60–3.80 (m, 2H), 4.32 (t, J = 4.8 Hz, 2H), 4.93 (t, J = 4.6 Hz, 2H),
5.02 (s, 2H), 6.86 (d, J = 8.0 Hz, 2H), 6.90 (t, J = 7.4 Hz, 1H), 7.24
(t, J = 8.0 Hz, 2H), 7.38 (t, J = 7.6 Hz, 1H), 7.44 (t, J = 7.4 Hz, 1H),
7.75 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 8.35 (br s, 1H),
11.69 (br s, 1H); 13C NMR (100 MHz, DMSO-d6): d 22.8, 44.0,
47.6, 54.0, 66.0, 112.1, 114.3, 117.8, 121.0, 123.7, 124.1, 129.5,
134.2, 138.5, 145.8, 157.7; HRMS (ESI) [MꢁHCl2]+ Calcd for
2-(Pyrrolidin-1-ylmethyl)-1H-benzimidazole (36). Under an
atmosphere of nitrogen, 2-(chloromethyl)benzimidazole (1.00 g,
6.00 mmol, 1 equiv) was dissolved in ethanol (20 mL). To this
was added pyrrolidine (10 mL, 8.53 g, 119.94 mmol, 20 equiv).
The mixture was stirred at rt for 0.5 h, heated at reflux tempera-
ture for 2 h, then stirred at rt for an additional 48 h. The mixture
was concentrated, diluted with a saturated aqueous solution of
NaHCO3 (100 mL), and extracted with CH2Cl2 (2 ꢂ 100 mL). The
combined organic extracts were washed with brine (75 mL), dried
(Na2SO4), and concentrated. High-vacuum drying gave the
C20H24N3O: 322.1919. Found: 322.1908.
4.4.30. 1-Benzhydryl-2-(pyrrolidin-1-ylmethyl)-1H-
benzimidazole hydrochloride (32)
product (1.13 g, 5.61 mmol, 94%) as
a
beige solid; mp
Compound 32 was prepared using Procedure A with 36 and bro-
modiphenylmethane as starting materials to give the product
142–143 °C; Rf = 0.13 (EtOAc); 1H NMR (400 MHz, DMSO-d6):
d 1.65–1.75 (m, 4H), 2.46–2.56 (m, 4H), 3.81 (s, 2H), 7.06–7.16
(m, 2H), 7.35–7.59 (m, 2H), 12.31 (br s, 1H); 13C NMR
(100 MHz, DMSO-d6): d 23.3, 53.2, 53.7, 111.1, 118.4, 120.9,
121.6, 134.4, 143.0, 152.7; HRMS (ESI) [M+H]+ Calcd for
(147 mg, 29%) as
a
white solid; mp 120–124 °C; 1H NMR
(400 MHz, DMSO-d6): d 1.80–2.15 (m, 4H), 3.10–3.40 (m, 2H),
3.60–3.90 (m, 2H), 4.86 (s, 2H), 6.55 (d, J = 8.4 Hz, 1H), 6.90 (br s,
ꢀ1H), 7.03 (t, J = 7.8 Hz, 1H), 7.17–7.29 (m, 5H), 7.35–7.47 (m,
6H), 7.51 (s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 11.36 (br s, ꢀ1H); 13C
NMR (100 MHz, DMSO-d6): d 22.8, 49.2, 54.2, 61.8, 112.8, 119.3,
122.3, 123.2, 128.2, 128.4, 128.8, 134.5, 137.6, 141.7, 147.0; HRMS
(ESI) [MꢁCl]+ Calcd for C25H26N3: 368.2126. Found: 368.2120.
C12H16N3: 202.1344. Found: 202.1349.
4.6. In vitro HO activity assay
HO activity in rat spleen and rat brain microsomal fractions was
determined by the quantitation of CO formed from the degradation
of methemalbumin (heme complexed with albumin).20,21 Micro-
somal fractions of spleen and brain (Sprague–Dawley rats) were
prepared according to the procedure outlined by Appleton et al.;22
4.4.31. 1-(Naphthalen-2-ylmethyl)-2-(pyrrolidin-1-ylmethyl)-
1H-benzimidazole dihydrochloride (33)
Compound 33 was prepared using Procedure A with 36 and
2-(bromomethyl)naphthalene as starting materials to give the

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 21 (2013) 6788–6795
6795
these fractions have been characterized for HO-1 and HO-2 content
Acknowledgment
(see Ref. 9). Protein concentration of microsomal fractions was
determined by a modification of the biuret method.21 Incubations
for HO activity analysis were performed under conditions for
The authors thank the Canadian Institutes of Health Research
for a Grant-in-aid (MOP 119467).
which the rate of CO formation (pmol CO ꢂ minꢁ1 ꢂ mg proteinꢁ1
)
was linear with respect to time and microsomal protein concentra-
References and notes
tion. Briefly, reaction mixtures (150
phosphate buffer (pH 7.4), 50 M methemalbumin, and 1 mg/mL
protein were pre-incubated with the inhibitors at final concentra-
tions ranging from 0.1 to 100 M for 10 min at 37 °C. Reactions
were initiated by adding NADPH at a final concentration of 1 mM
and incubations were performed for an additional 15 min at
37 °C. Reactions were stopped by freezing the reaction mixture
on dry ice, and CO formation was monitored by gas chromatogra-
phy according to the method described by Vreman and
Stevenson.20
lL) consisting of 100 mM
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6. Ryter, S. W.; Alam, J.; Choi, A. M. Physiol. Rev. 2006, 86, 583.
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4.7. Analysis of enzyme inhibition
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Potencies of the compounds as inhibitors of HO are reported as
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Dercho, R. A.; Kinobe, R. T.; Hum, M.; Brien, J. F.; Jia, Z.; Szarek, W. A.; Nakatsu,
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their IC50 values, which were calculated using the formula
EC50
IC50
¼
bottomꢁtopꢁ1. The data resulting from the above experiments
50ꢁtop
were plotted as non-linear regression (sigmoidal dose–response)
curves using GraphPad Prism (version 3) software. The values
on the abscissa represent the logarithm of the inhibitor concen-
tration (in lM), whereas the values of the activity on the ordinate
are expressed as a percentage of the control experiments without
inhibitor. Bottom and top refer to the lower and upper plateaus;
EC50 is the value halfway between the top and bottom plateaus.
The IC50 value reported for each compound is reported as the
mean of the values recorded in replicate experiments, and for
each of these replicate experiments an individual IC50 value was
calculated in the manner described. The IC50 values for the repli-
cate experiments were employed to generate the reported stan-
dard deviation value.
17. Rahman, M. N.; Vukomanovic, D.; Vlahakis, J. Z.; Szarek, W. A.; Nakatsu, K.; Jia,
Z. J. R. Soc. Interface 2013, 10, 20120697.
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Gaikwad, A. N.; Tripathi, R. P. Eur. J. Med. Chem. 2009, 44, 3350.
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