Heme Oxygenase Inhibition by α-(1H-Imidazol-1-yl)-ω-phenylalkanes: Effect of Introduction of Heteroatoms in the Alkyl Linker

Article abstract of DOI:10.1002/cmdc.201100602

Several α-(1H-imidazol-1-yl)-ω-phenylalkanes were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). These compounds were found to be potent and selective for the stress-induced isozyme HO-1, showing mostly weak activity toward the cons

Full text of DOI:10.1002/cmdc.201100602

MED
DOI: 10.1002/cmdc.201100602
Heme Oxygenase Inhibition by a-(1H-Imidazol-1-yl)-w-
phenylalkanes: Effect of Introduction of Heteroatoms in
the Alkyl Linker
Jason Z. Vlahakis,[a] Carmen Lazar,[a] Gheorghe Roman,[a] Dragic Vukomanovic,[b]
Kanji Nakatsu,[b] and Walter A. Szarek*[a]
Several a-(1H-imidazol-1-yl)-w-phenylalkanes were synthesized
and evaluated as novel inhibitors of heme oxygenase (HO).
These compounds were found to be potent and selective for
the stress-induced isozyme HO-1, showing mostly weak activity
toward the constitutive isozyme HO-2. The introduction of an
oxygen atom in the alkyl linker produced analogues with de-
creased potency toward HO-1, whereas the presence of
a sulfur atom in the linker gave rise to analogues with greater
potency toward HO-1 than the carbon-containing analogues.
The most potent compounds studied contained a five-atom
linker between the imidazolyl and phenyl moieties, whereas
the most HO-1-selective compounds contained a four-atom
linker between these groups. The compounds with a five-atom
linker containing a heteroatom (O or S) were found to be the
most potent inhibitors of HO-2; 1-(N-benzylamino)-3-(1H-imida-
zol-1-yl)propane dihydrochloride, with a nitrogen atom in the
linker, was found to be inactive.
Introduction
A program in our research group is focused on the design and
synthesis of selective heme oxygenase (HO) inhibitors. In par-
ticular, we are interested in compounds that inhibit the two
active isozymes of HO, namely, HO-1 (inducible) and HO-2
(constitutive), especially regarding the role of these HO inhibi-
tors in biology and medicine.[1–4] Notably, inhibitors of HO have
been studied for the treatment of hyperbilirubinemia,[5–7] neu-
rodegenerative disorders,[8] certain types of cancer,[9,10] and
bacterial and fungal infections;[11] HO inhibitors also provide
useful tools for the elucidation of the physiological roles of
heme oxygenases. We have identified a number of potent and
selective compounds for the inhibition of HO-1.[12–16] The key
functional groups of these compounds include an azolyl
moiety, a phenyl group, and an alkyl linkage between these.
Successful compounds identified to date incorporate a dioxo-
lanyl, a hydroxy, or a carbonyl group in the linker. Herein we
describe the effect of introducing heteroatoms in the alkyl
linker of a-(1H-imidazol-1-yl)-w-phenylalkanes on heme oxy-
genase inhibition.
with imidazole and potassium carbonate in THF. Compound 4
was obtained by treatment of 1-bromo-3-phenylpropane with
imidazole and sodium hydroxide in DMSO. Analogously, com-
pounds 5 and 6 were obtained by treatment of 4-phenylbutyl
toluene-4-sulfonate and 5-phenylpentyl toluene-4-sulfonate,
respectively, with imidazole and sodium hydroxide in DMSO.
The a-(1H-imidazol-1-yl)-w-phenoxyalkanes 7, 8, and 10 were
obtained by treatment of the corresponding a-bromo-w-phe-
noxyalkane with imidazole (in either NaH–THF or NaOH–
DMSO), followed by hydrochloride salt formation. The a-(1H-
imidazol-1-yl)-w-(phenylsulfanyl)alkanes 11, 12, and 14 were
obtained by treatment of the corresponding a-bromo-w-(phe-
nylsulfanyl)alkane with imidazole–NaH–THF, followed by hydro-
chloride salt formation. 2-(Benzyloxy)-1-(1H-imidazol-1-yl)-
ethane (9) was commercially available. Compound 13 was ob-
tained by treatment of 1-(2-chloroethyl)imidazole with pre-
formed sodium benzylthiolate in ethanol. The secondary amine
15 was prepared by treatment of 1-(3-aminopropyl)imidazole
with benzaldehyde in methanol, followed by reduction of the
resultant imine with sodium borohydride, and finally dihydro-
chloride salt formation.
Results and Discussion
Synthesis
The structures of the compounds studied in this work are
shown in Table 1. Compounds 1 and 2 were obtained simply
by treatment of 1-phenyl-1H-imidazole and 1-benzyl-1H-imida-
zole, respectively, with hydrochloric acid in ethanol. The gener-
al approach for the synthesis of (phenylalkyl)imidazoles 3–6,
(phenoxyalkyl)imidazoles 7, 8, and 10, and {(phenylsulfanyl)al-
kyl}imidazoles 11, 12, and 14 is summarized in Table 2. Com-
pound 3 was obtained by treatment of (2-bromoethyl)benzene
[a] Dr. J. Z. Vlahakis, Dr. C. Lazar, Dr. G. Roman, Prof. W. A. Szarek
Department of Chemistry, Queen’s University
Chernoff Hall, 90 Bader Lane, Kingston, ON K7L 3N6 (Canada)
E-mail: walter.szarek@chem.queensu.ca
[b] Dr. D. Vukomanovic, Prof. K. Nakatsu
Department of Biomedical and Molecular Sciences, Queen’s University
Botterell Hall, 18 Stuart Street, Kingston, ON K7L 3N6 (Canada)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100602.
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
897

W. A. Szarek et al.
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Table 1. Inhibitory potency and selectivity of candidate compounds
against HO-1 and HO-2 activity.
Table 2. General approach for the synthesis of (phenylalkyl)imidazoles 3–
6, (phenoxyalkyl)imidazoles 7, 8, and 10, and {(phenylsulfanyl)alkyl}imida-
zoles 11, 12, and 14.
Compd
Structure
IC50 [mm][a]
SI[b]
HO-1 (rat
HO-2 (rat
brain)
spleen)
inactive
44ꢀ6
X
n
Z
Reagents/Solvent
Compd
1
2
3
4
5
inactive
–
CH2
CH2
CH2
CH2
O
O
O
S
S
1
2
3
4
2
3
4
2
3
4
Br
Br
OTs
OTs
Br
Br
Br
Br
Br
imidazole, K2CO3, THF
imidazole, NaOH, DMSO
imidazole, NaOH, DMSO
imidazole, NaOH, DMSO
imidazole, NaH, THF
imidazole, NaOH, DMSO
imidazole, NaOH, DMSO
imidazole, NaH, THF
3[a]
4
5
6
7
8
10
11
12
14
>100
>2.3
>1.4
>7.1
>28.6
72ꢀ8
>100
>100
14ꢀ3
imidazole, NaH, THF
imidazole, NaH, THF
S
Br
3.5ꢀ0.7 >100
[a] Compound 3 was evaluated as the free base.
6
7
2.8ꢀ0.3
61ꢀ20
20ꢀ11
7.1
methylene and ethylene analogues (compounds 2 and 3) were
found to be weak inhibitors of HO-1, whereas the propylene,
butylene, and pentylene analogues (compounds 4, 5, and 6)
progressively showed increased inhibitory potency, with penty-
lene analogue 6 being the most potent (IC50 =2.8ꢀ0.3 mm).
With respect to HO-2, notable inhibition was not observed in
this series until compound 6, with an alkyl chain length of five
carbon units, was examined (IC50 =20ꢀ11 mm). The butylene
analogue 5 was observed to be the most selective for HO-
1 over HO-2. Compound 1, in which the phenyl and imidazolyl
groups are joined directly together, is essentially inactive
toward both HO-1 and HO-2.
>100
>1.6
8
9
42ꢀ9
>100
>2.4
>3.1
32ꢀ1
4ꢀ1
>100
10
11
4.6ꢀ0.4
1.2
6.0ꢀ0.1 >100
>16.7
12
13
2.4ꢀ0.1
16ꢀ2
6.7
The effect of introducing oxygen in the alkyl chain on HO
activity was determined by the study of compounds 7–10. In
general, replacement of a methylene unit with an oxygen
atom led to a decrease in HO-1 inhibitory potency. Specifically,
analogue 7 (IC50 =61ꢀ20 mm) was fourfold less potent than
compound 4 (IC50 =14ꢀ3 mm). Similarly, the two oxygen-con-
4.4ꢀ0.7 >100
>22.7
14
15
1.2ꢀ0.1
5ꢀ2
inactive
4.2
–
taining structural analogues of compound
5
(IC50 =3.5ꢀ
inactive
0.7 mm), namely compounds 8 (IC50 =42ꢀ9 mm) and 9 (IC50 =
32ꢀ1 mm), were less potent by factors of 12 and 9, respective-
ly. The direct oxygen-containing analogue of 6 (IC50 =2.8ꢀ
0.3 mm), namely compound 10 (IC50 =4ꢀ1 mm), was also less
potent with respect to HO-1 inhibition. In accordance with the
results obtained in the alkyl series 2–6, significant HO-2 inhibi-
tion in the ether series of compounds 7–10 was observed only
for the compound with a five-atom spacer, namely compound
10, although this compound is essentially unselective for HO-
1 (IC50 =4ꢀ1 mm) over HO-2 (IC50 =4.6ꢀ0.4 mm).
[a] Data represent mean values ꢀSEM of replicate (n=4) experiments.
[b] Selectivity index: IC50(HO-2)/IC50(HO-1)
.
Biological evaluation
The compounds listed in Table 1 were tested as inhibitors of
HO-1 (rat spleen microsomal fraction) and HO-2 (rat brain mi-
crosomal fraction) using an in vitro assay for heme oxygenase.
Compounds 2–6 contain an a-(imidazol-1-yl) moiety, an w-
phenyl moiety, and an alkyl linkage between these. The intro-
duction of oxygen, sulfur, or nitrogen into the alkyl linkage af-
forded compounds 7–10, 11–14, and 15, respectively.
The effect of alkyl chain length on HO activity was addressed
by the study of compounds 2–6. Increasing the alkyl chain
length increased potency in HO-1 inhibition. For example, the
The effect of introducing sulfur into the alkyl chain on HO
activity was determined by the study of compounds 11–14. In
contrast to the oxygen-containing compounds, the sulfur-con-
taining compounds (apart from 13) are more potent HO-1 in-
hibitors than their carbon analogues. For instance, the thioeth-
er 11 (IC50 =6.0ꢀ0.1 mm) was twofold more potent than the
corresponding propylene compound 4 (IC50 =14ꢀ3 mm). The
sulfur-containing analogue of 5 (IC50 =3.5ꢀ0.7 mm), namely
898
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Heme Oxygenase Inhibitors
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zole, 1-benzyl-1H-imidazole, benzylthiol, 1-(3-aminopropyl)imida-
zole, 2-(benzyloxy)-1-(1H-imidazol-1-yl)ethane (9) and all other re-
agents were obtained from Sigma–Aldrich (Oakville, ON, Canada).
Flash column chromatography was performed on Silicycle silica gel
(230–400 mesh, 60 ꢁ). Analytical thin-layer chromatography was
performed on glass-backed pre-coated Silica Gel 60 F254 plates (Sili-
cycle), and the compounds were visualized either by UV illumina-
tion (l 254 nm), or by heating after spraying with phosphomolyb-
dic acid in ethanol. Melting points were determined on a Mel-
Temp II apparatus and are uncorrected. 1H and 13C NMR spectra
were recorded on a Bruker Avance 400 MHz spectrometer in CDCl3
or CD3OD. The chemical shifts (d) are reported in ppm relative to
tetramethylsilane using the residual solvent peak as an internal ref-
erence.[19] High-resolution ESI mass spectra were recorded on an
Applied Biosystems/MDS Sciex QSTAR XL mass spectrometer with
an Agilent HP1100 Cap-LC system. Samples were run in 50% aque-
ous methanol at a flow rate of 6 mLminꢁ1. High-resolution EI mass
spectra were recorded on a Waters/Micromass GC-ToF instrument.
compound 12 (IC50 =2.4ꢀ0.1 mm), was also slightly more
active against HO-1, whereas the structural analogue 13 (IC50 =
4.4ꢀ0.7 mm) was only slightly less active against HO-1 than 5.
The sulfur-containing analogue 14 (IC50 =1.2ꢀ0.1 mm) was ap-
proximately twice as potent toward HO-1 than the directly
comparable alkyl compound 6 (IC50 =2.8ꢀ0.3 mm). Regarding
HO-2 inhibition, the series of sulfur-containing compounds did
show significant activity in the cases of the four- and five-atom
linker lengths, and this attribute could be a result of the larger
size of the sulfur atom; compounds 12 (IC50 =16ꢀ2 mm) and
14 (IC50 =5ꢀ2 mm) are both potent HO-2 inhibitors. In general,
the compounds with a five-atom linker length are the most
potent HO-2 inhibitors (compounds 6, 14, and 10); potency in-
creased in the order CH2 <S<O. Thus, the introduction of het-
eroatoms in the alkyl linkage may play an important role in
the design of HO-2 inhibitors.
Only one nitrogen-containing analogue was studied (com-
pound 15) owing largely to the fact that it was inactive against
both HO-1 and HO-2, even though it contains a spacer length
deemed to be desirable as observed in the CH2, O, and S series
of compounds; no other nitrogen-containing analogues were
pursued.
1-Phenyl-1H-imidazole hydrochloride (1): To a solution of 1-
phenyl-1H-imidazole (310 mg, 2.15 mmol, 1 equiv) in EtOH (2 mL)
was added 37% aqueous HCl (262 mg, 2.66 mmol, 1.2 equiv) in
EtOH (2 mL) and the mixture was concentrated. High-vacuum
drying gave compound 1 as a white solid (380 mg, 98%); mp:
127–1288C; 1H NMR (400 MHz, CD3OD): d=7.56–7.69 (m, 3H),
7.71–7.81 (m, 2H), 7.80 (t, J=1.6 Hz, 1H), 8.11 (t, J=1.6 Hz, 1H),
9.52 ppm (s, 1H); 13C NMR (100 MHz, CD3OD): d=121.9, 122.7,
123.6, 131.3, 131.5, 135.7, 136.5 ppm; HRMS-ESI: m/z [MꢁCl]+
calcd for C9H8N2: 144.0687, found: 144.0682.
Conclusions
The incorporation of a heteroatom into the alkyl chain be-
tween the a-(imidazol-1-yl) moiety and the w-phenyl group
imparts dramatic effects on HO activity. Potency in HO-1 inhibi-
tion increased in the order O<CH2 <S, and this trend was ob-
served in each of the following three direct comparisons of
HO-1 inhibitor potency: 7<4<11, 8<5<12, and 10<6<14
(see Table 1). In each of the three series of compounds studied
(the CH2, O, and S series), the compound with the longest
spacer in each series, namely compounds 6, 10, and 14, were
observed to be the most potent HO-1 and HO-2 inhibitor in
that series; however, they were not the most selective HO-1 in-
hibitors. The data suggest that the most selective HO-1 inhibi-
tor in each respective series contains a four-atom linker be-
tween the a-(imidazol-1-yl) moiety and the w-phenyl group, as
observed for compounds 5, 9, and 13. Thus, further modifica-
tions to increase the length of the linkage (beyond five atoms)
may generate more potent HO-1 and HO-2 inhibitors, although
it is doubtful that pronounced HO-1 selectivity will be attained
through this approach.
(1H-Imidazol-1-yl)phenylmethane hydrochloride (2): To a solution
of 1-benzyl-1H-imidazole (396 mg, 2.50 mmol, 1 equiv) in EtOH
(2 mL) was added 37% aqueous HCl (494 mg, 5.01 mmol, 2 equiv)
in EtOH (2 mL) and the mixture was concentrated. High-vacuum
drying gave compound 2 as a hygroscopic white solid (423 mg,
1
2.17 mmol, 87%); mp: 134–1358C; H NMR (400 MHz, CD3OD): d=
5.48 (s, 2H), 7.39–7.46 (m, 5H), 7.59 (t, J=1.6 Hz, 1H), 7.65 (t, J=
1.6 Hz, 1H), 9.10 ppm (s, 1H); 13C NMR (100 MHz, CD3OD): d=53.9,
121.4, 123.3, 129.6, 130.3, 130.4, 135.5, 136.5 ppm; HRMS-EI: m/z
[MꢁHCl]+ calcd for C10H10N2: 158.0844, found: 158.0846.
1-(1H-Imidazol-1-yl)-2-phenylethane (3): Under an atmosphere of
N2, a mixture of imidazole (788 mg, 11.57 mmol, 2.1 equiv) and
K2CO3 (370 mg, 2.68 mmol, 1 equiv) in dry THF (18 mL) was stirred
at RT for 10 min. To this mixture was added a solution of (2-bro-
moethyl)benzene (1.00 g, 5.40 mmol, 1 equiv) in THF (1 mL), and
the mixture was heated at reflux for 14 h. The mixture was filtered,
and the filtrate was concentrated to afford a clear oil. The oil was
dissolved in CH2Cl2, and the solution was washed with H2O (2ꢂ).
The CH2Cl2 layer was then extracted with dilute aqueous HCl (3ꢂ).
The aqueous extract was then neutralized with solid NaHCO3, and
the free base extracted using CH2Cl2 (3ꢂ). The combined organic
extracts were dried (Na2SO4) and concentrated. High-vacuum
drying gave compound 3 as a clear oil (420 mg, 2.44 mmol, 45%);
1H NMR (400 MHz, CDCl3): d=3.05 (t, J=7.0 Hz, 2H), 4.17 (t, J=
7.0 Hz, 2H), 6.83 (s, 1H), 7.01–7.09 (m, 3H), 7.22–7.34 ppm (m, 4H);
13C NMR (100 MHz, CDCl3): d=38.0, 48.6, 118.9, 127.1, 128.7, 128.9,
129.6, 137.2, 137.6 ppm; HRMS-EI: m/z [M]+ calcd for C11H12N2:
172.1000, found: 172.0999.
Experimental Section
Chemistry
Materials and methods: 1-Bromo-2-phenoxyethane, 1-bromo-3-
phenoxypropane, 1-bromo-4-phenoxybutane, and 1-bromo-2-(phe-
nylsulfanyl)ethane were obtained from Alfa Aesar (Ward Hill, MA,
USA). 1-Bromo-3-(phenylsulfanyl)propane and 1-bromo-4-(phenyl-
sulfanyl)butane were prepared as previously reported.[17] 5-Phenyl-
pentyl toluene-4-sulfonate and 4-phenylbutyl toluene-4-sulfonate
were prepared by following previously reported procedures.[18] N-
(2-Chloroethyl)imidazole hydrochloride was obtained from Oak-
wood Products Inc. (West Columbia, SC, USA). 1-Phenyl-1H-imida-
1-(1H-Imidazol-1-yl)-3-phenylpropane hydrochloride (4): Under
an atmosphere of N2, a mixture of imidazole (376 mg, 5.52 mmol,
1.1 equiv) and NaOH (221 mg, 5.52 mmol, 1.1 equiv) in DMSO
(2 mL) was heated at 70–808C with stirring for 1.5 h. To this mix-
ture was added a solution of 1-bromo-3-phenylpropane (1.00 g,
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W. A. Szarek et al.
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5.02 mmol, 1 equiv) in DMSO (2 mL), and the mixture heated at
70–808C with stirring for 13 h. The temperature was slightly elevat-
ed, and the DMSO was removed by blowing a stream of air over
the reaction mixture. High-vacuum drying left a yellow residue.
After dilution with H2O, the mixture was extracted with benzene
(3ꢂ50 mL) and the combined organic extracts were washed with
brine (2ꢂ), dried (MgSO4), and concentrated to give the free base
(914 mg, 4.91 mmol, 98%). To a solution of this free base in hot
EtOH (3 mL) was added a solution of 37% aqueous HCl (500 mg,
5.08 mmol, 1 equiv) in EtOH (2 mL). The warm mixture was filtered
through a syringe filter (0.45 mm), and the filtrate was concentrated
and dried under high vacuum. The residue was recrystallized from
2-propanol/Et2O to give compound 4 as a white solid (1.05 g,
4.71 mmol, 94%); mp: 95–968C; 1H NMR (400 MHz, CD3OD): d=
2.25 (p, 2H), 2.70 (t, J=7.6 Hz, 2H), 4.29 (t, J=7.4 Hz, 2H), 7.16–
7.22 (m, 3H), 7.22–7.30 (m, 2H), 7.55 (s, 1H), 7.67 (s, 1H), 8.95 ppm
(s, 1H); 13C NMR (100 MHz, CD3OD): d=32.6, 33.4, 50.2, 121.1,
123.3, 127.4, 129.4, 129.6, 136.4, 141.5 ppm; HRMS-ESI: m/z
[MꢁCl]+ calcd for C12H15N2: 187.1235, found: 187.1242.
3.71 mmol, 37%). The free base was treated with 37% aqueous
HCl (1 equiv) in 2-propanol, and the mixture was concentrated to
afford compound 7 as a white solid (488 mg, 2.17 mmol, 22%);
Rf =0.18 (EtOAc/MeOH, 4:1); mp: 85–888C; 1H NMR (400 MHz,
CD3OD): d=4.38 (t, J=4.8 Hz, 2H), 4.69 (t, J=4.8 Hz, 2H), 6.95–
7.01 (m, 3H), 7.21–7.26 (m, 2H), 7.59 (t, J=1.6 Hz, 1H), 7.76 (t, J=
1.6 Hz, 1H), 9.08 ppm (s, 1H); 13C NMR (100 MHz, CD3OD): d=50.2,
67.1, 115.6, 121.0, 122.7, 123.8, 130.6, 137.0, 159.3 ppm; HRMS-EI:
m/z [MꢁHCl]+ calcd for C11H12N2O: 188.0950, found: 188.0958.
General procedure for the formation of (phenoxyalkyl)imida-
zoles: Under an atmosphere of N2, a mixture of imidazole (1.00 g,
14.70 mmol, 3 equiv) and NaOH (588 mg, 14.70 mmol, 3 equiv) in
DMSO (5 mL) was stirred at 758C for 1 h. A solution of the a-
bromo-w-phenoxyalkane (4.90 mmol, 1 equiv) in DMSO (3 mL) was
added, and the mixture was stirred at 75–1008C for 24 h. The sol-
vent was removed, 5% aqueous NaOH (50 mL) was added to the
residue, and the mixture was extracted with EtOAc (2ꢂ25 mL). The
combined organic phase was dried (Na2SO4) and concentrated to
give a yellow oil. Purification by flash column chromatography on
silica gel (EtOAc/hexanes 3:1 v/v as eluent) afforded the corre-
sponding a-(1H-imidazol-1-yl)-w-phenoxyalkane. The free base was
treated with 37% aqueous HCl (1 equiv) in 2-propanol, and the
mixture was concentrated to afford the product.
1-(1H-Imidazol-1-yl)-4-phenylbutane hydrochloride (5): Com-
pound 5 was prepared by a procedure analogous to that used to
form 6 below, except that 4-phenylbutyl toluene-4-sulfonate was
used instead of 5-phenylpentyl toluene-4-sulfonate, to give the
product as a hygroscopic white solid (632 mg, 2.67 mmol, 89%);
mp: 88–908C; 1H NMR (400 MHz, CD3OD): d=1.60–1.71 (m, 2H),
1.87–1.98 (m, 2H), 2.68 (t, J=7.6 Hz, 2H), 4.28 (t, J=7.6 Hz, 2H),
7.13–7.21 (m, 3H), 7.22–7.29 (m, 2H), 7.56 (t, J=1.6 Hz, 1H), 7.65 (t,
J=1.6 Hz, 1H), 8.97 ppm (s, 1H); 13C NMR (100 MHz, CD3OD): d=
29.1, 30.7, 36.0, 50.4, 121.1, 123.3, 127.0, 129.4, 129.5, 136.3,
142.9 ppm; HRMS-ESI: m/z [MꢁCl]+ calcd for C13H17N2: 201.1391,
found: 201.1390.
1-(1H-Imidazol-1-yl)-3-phenoxypropane hydrochloride (8): Com-
pound
8
was prepared from 1-bromo-3-phenoxypropane
(4.90 mmol, 1 equiv) following the general procedure for the for-
mation of (phenoxyalkyl)imidazoles to afford compound 8 as
a white solid (571 mg, 2.39 mmol, 49%); Rf =0.20 (EtOAc/MeOH,
1
4:1); mp: 123–1248C; H NMR (400 MHz, CD3OD): d=2.34–2.43 (m,
2H), 4.05 (t, J=5.6 Hz, 2H), 4.51 (t, J=7.2 Hz, 2H), 6.84–7.96 (m,
3H), 7.21–7.30 (m, 2H), 7.58 (s, 1H), 7.70 (s, 1H), 9.02 ppm (s, 1H);
13C NMR (100 MHz, CD3OD): d=30.7, 48.2, 65.5, 115.4, 121.1, 122.1,
123.5, 130.5, 136.6, 159.8 ppm; HRMS-EI: m/z [MꢁHCl]+ calcd for
C12H14N2O: 202.1106, found: 202.1109.
1-(1H-Imidazol-1-yl)-5-phenylpentane hydrochloride (6): Under
an atmosphere of N2, a mixture of imidazole (612 mg, 9 mmol,
3 equiv) and NaOH (360 mg, 9 mmol, 3 equiv) in DMSO (4 mL) was
heated at 70–808C with stirring for 1 h. A solution of 5-phenylpen-
tyl toluene-4-sulfonate (954 mg, 3 mmol, 1 equiv) in DMSO (3 mL)
was added, and the reaction mixture was stirred at 70–808C over-
night. The reaction mixture was partitioned between H2O (100 mL)
and EtOAc (30 mL), the aqueous phase was further extracted with
EtOAc (20 mL), and then the combined organic phase was washed
with H2O (2ꢂ50 mL), dried (Na2SO4) and concentrated. The result-
ing clear oil (626 mg, 2.92 mmol) was dissolved in Et2O and treated
with an excess of ethereal HCl. The solid that separated was col-
lected by filtration and recrystallized from 2-propanol/Et2O to give
compound 6 as a white solid (557 mg, 2.22 mmol, 74%); mp: 86–
1-(1H-Imidazol-1-yl)-4-phenoxybutane hydrochloride (10): Com-
pound 10 was prepared from 1-bromo-4-phenoxybutane
(4.90 mmol, 1 equiv) following the general procedure for the for-
mation of (phenoxyalkyl)imidazoles to afford the product as
a white solid (517 mg, 2.05 mmol, 42%); Rf =0.18 (EtOAc/MeOH,
1
4:1); mp: 99–1008C; H NMR (400 MHz, CD3OD): d=1.77–1.87 (m,
2H), 2.04–2.16 (m, 2H), 4.02 (t, J=6.0 Hz, 2H), 4.36 (t, J=7.2 Hz,
2H), 6.87–6.94 (m, 3H), 7.21–7.29 (m, 2H), 7.58 (t, J=1.6 Hz, 1H),
7.71 (t, J=1.6 Hz, 1H), 9.04 ppm (s, 1H); 13C NMR (100 MHz,
CD3OD): d=27.0, 28.2, 50.4, 68.0, 115.5, 121.1, 121.8, 123.3, 130.5,
136.3, 160.2 ppm; HRMS-EI: m/z [MꢁHCl]+ calcd for C13H16N2O:
216.1263, found: 216.1263.
1
878C; H NMR (400 MHz, CD3OD): d=1.29–1.41 (m, 2H), 1.63–1.74
(m, 2H), 1.87–1.98 (m, 2H), 2.63 (t, J=7.4 Hz, 2H), 4.25 (t, J=
7.4 Hz, 2H), 7.10–7.19 (m, 3H), 7.20–7.27 (m, 2H), 7.56 (t, J=1.6 Hz,
1H), 7.64 (t, J=1.6 Hz, 1H), 8.95 ppm (s, 1H); 13C NMR (100 MHz,
CD3OD): d=26.6, 31.0, 31.8, 36.5, 50.5, 121.1, 123.3, 126.8, 129.3,
129.4, 136.2, 143.4 ppm; HRMS-ESI: m/z [MꢁCl]+ calcd for
C14H19N2: 215.1548, found: 215.1545.
General procedure for the formation of {(phenylsulfanyl)alkyl}i-
midazoles: To
a mixture of imidazole (3 equiv) in dry THF
(10 mLgꢁ1 imidazole) under an atmosphere of N2 was added NaH
(2.3 equiv), and the mixture was stirred at RT for 1 h. The corre-
sponding a-bromo-w-(phenylsulfanyl)alkane (1 equiv) in dry THF
(10 mLgꢁ1 imidazole) was added, and the mixture was stirred at RT
overnight. The solvent was removed, 5% aqueous NaOH (50 mL)
was added to the residue, and the mixture was extracted with
CH2Cl2 (2ꢂ50 mL), dried (Na2SO4), and then concentrated to give
a clear oil. Purification by flash column chromatography on silica
gel (EtOAc/MeOH 9:1 v/v as eluent) afforded the a-(1H-imidazol-1-
yl)-w-(phenylsulfanyl)alkane. The free base was treated with 37%
aqueous HCl (1 equiv) in 2-propanol, and the mixture was concen-
trated to afford the product.
1-(1H-Imidazol-1-yl)-2-phenoxyethane hydrochloride (7): To a so-
lution of imidazole (1.00 g, 14.70 mmol) in dry THF (20 mL) under
an atmosphere of N2 was added NaH (360 mg, 15.65 mmol), and
the mixture was stirred at RT for 1 h. 1-Bromo-2-phenoxyethane
(2.00 g, 9.95 mmol) in dry THF (20 mL) was added, and the mixture
was stirred at RT overnight. The solvent was removed, 5% aqueous
NaOH (50 mL) was added to the residue, and the mixture was ex-
tracted with CH2Cl2 (2ꢂ50 mL) to give a yellow oil. Purification by
flash column chromatography on silica gel (EtOAc/hexanes 1:3 v/v
as eluent) afforded 1-(1H-imidazol-1-yl)-2-phenoxyethane (699 mg,
900
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ChemMedChem 2012, 7, 897 – 902

Heme Oxygenase Inhibitors
MED
1-(1H-Imidazol-1-yl)-2-(phenylsulfanyl)ethane hydrochloride (11):
Compound 11 was prepared from 1-bromo-2-(phenylsulfanyl)-
ethane (1 g, 4.61 mmol, 1 equiv) following the general procedure
for the formation of {(phenylsulfanyl)alkyl}imidazoles to afford the
product as a yellow oil (112 mg, 0.47 mmol, 10%); Rf =0.10 (EtOAc/
resulting residue was partitioned between EtOAc (30 mL) and H2O
(30 mL). The aqueous phase was extracted further with EtOAc
(20 mL). The combined organic phase was washed with H2O (2ꢂ
20 mL), dried (Na2SO4), and concentrated. The residue was purified
using flash column chromatography on silica gel (EtOAc/MeOH 1:1
v/v as eluent) to give the free base (1.46 g, 6.78 mmol, 68%),
which was dissolved in 2-propanol (4 mL), and the solution was
treated with a solution of 37% aqueous HCl (1.34 g, 13.60 mmol,
2 equiv) in 2-propanol (2 mL). The mixture was concentrated and
dried under high vacuum. Recrystallization from 2-propanol
(10 mL) gave compound 15 as a hygroscopic white solid (1.63 g,
1
MeOH, 4:1); H NMR (400 MHz, CD3OD): d=3.50 (t, J=6.0 Hz, 2H),
4.48 (t, J=6.4 Hz, 2H), 7.20–7.26 (m, 1H), 7.27–7.34 (m, 2H), 7.35–
7.40 (m, 2H), 7.47 (s, 1H), 7.64 (s, 1H), 8.95 ppm (s, 1H); 13C NMR
(100 MHz, CD3OD): d=34.7, 50.1, 121.0, 123.5, 128.1, 130.4, 131.2,
135.2, 136.8 ppm; HRMS-ESI: m/z [MꢁCl]+ calcd for C11H13N2S:
205.0799, found: 205.0796.
1
5.66 mmol, 57%); mp: 144–1458C; H NMR (400 MHz, CD3OD): d=
1-(1H-Imidazol-1-yl)-3-(phenylsulfanyl)propane
hydrochloride
2.38 (p, 2H), 3.16 (t, J=7.8 Hz, 2H), 4.25 (s, 2H), 4.44 (t, J=7.2 Hz,
2H), 7.42–7.50 (m, 3H), 7.53–7.58 (m, 2H), 7.62 (t, J=1.6 Hz, 1H),
7.75 (t, J=1.6 Hz, 1H), 9.09 ppm (s, 1H); 13C NMR (100 MHz,
CD3OD): d=27.9, 45.3, 47.5, 52.5, 121.4, 123.3, 130.3, 130.7, 131.1,
132.4, 136.7 ppm; HRMS-ESI: m/z [MꢁHꢁ2Cl]+ calcd for C13H18N3:
216.1500, found: 216.1509.
(12): Compound 12 was prepared from 1-bromo-3-(phenylsulfa-
nyl)propane (1.20 g, 4.33 mmol, 1 equiv) following the general pro-
cedure for the formation of {(phenylsulfanyl)alkyl}imidazoles to
afford the product as a yellow solid (740 mg, 2.90 mmol, 67%);
Rf =0.30 (EtOAc/MeOH, 4:1); mp: 70–728C; 1H NMR (400 MHz,
CD3OD): d=2.15–2.23 (m, 2H), 2.98 (t, J=6.8 Hz, 2H), 4.42 (t, J=
6.8 Hz, 2H), 7.18–7.25 (m, 1H), 7.28–7.38 (m, 4H), 7.58 (s, 1H), 7.66
(s, 1H), 9.01 ppm (s, 1H); 13C NMR (100 MHz, CD3OD): d=30.5, 30.9,
49.2, 121.2, 123.3, 127.5, 130.2, 130.7, 136.4, 136.6 ppm; HRMS-ESI:
m/z [MꢁCl]+ calcd for C12H15N2S: 219.0955, found: 219.0953.
Biology
In vitro HO activity assays: HO activity in rat spleen (HO-1) and rat
brain (HO-2) microsomal fractions was determined by the quantita-
tion of CO formed from the degradation of methemalbumin (heme
complexed with albumin).[20,21] Spleen and brain (Sprague–Dawley
rats) microsomal fractions were prepared according to the proce-
dure outlined by Appleton et al.[22] Protein concentration of micro-
somal fractions was determined by a modification of the biuret
method.[21] Incubations for HO activity analysis were done under
conditions for which the rate of CO formation [(pmol CO)
minꢁ1 (mgprotein)ꢁ1] was linear with respect to time and microso-
mal protein concentration. Briefly, reaction mixtures (150 mL) con-
sisting of 100 mm phosphate buffer (pH 7.4), 50 mm methemalbu-
min, and 1 mgmLꢁ1 protein were pre-incubated with the inhibitors
at final concentrations ranging from 0.1 to 100 mm for 10 min at
378C. Reactions were initiated by adding NADPH at a final concen-
tration of 1 mm, and incubations were performed for an additional
15 min at 378C. Reactions were stopped by instantly freezing the
reaction mixture on dry ice, and CO formation was monitored by
GC according to the method described by Vreman and Steven-
son.[20]
1-(Benzylsulfanyl)-2-(1H-imidazol-1-yl)ethane (13): Under an at-
mosphere of N2, sodium (230 mg, 10.00 mmol, 1 equiv) was added
to EtOH (20 mL) to form a NaOEt/EtOH solution. To this solution
was added benzylthiol (1.24 g, 10.00 mmol, 1 equiv), and the mix-
ture was heated at reflux with stirring for 0.5 h. The mixture was
cooled to RT, and a mixture of N-(2-chloroethyl)imidazole hydro-
chloride (835 mg, 5.00 mmol, 0.5 equiv) and EtOH (10 mL) was
added. The reaction mixture was heated at reflux with stirring for
20 h, then cooled to RT. The mixture was filtered and the filtrate
concentrated. The resulting residue was dissolved in 10% aqueous
HCl, and the solution was washed with EtOAc (3ꢂ15 mL). The
aqueous phase was basified using NaOH and then extracted with
EtOAc. The organic extract was washed sequentially with H2O and
brine, dried (Na2SO4), and concentrated. High-vacuum drying gave
compound 13 as a clear oil (1.03 g, 4.72 mmol, 94%); Rf =0.17
1
(EtOAc/MeOH, 9:1); H NMR (400 MHz, CDCl3): d=2.70 (t, J=6.8 Hz,
2H), 3.58 (s, 2H), 3.94 (t, J=6.8 Hz, 2H), 6.86 (s, 1H), 7.04 (s, 1H),
7.23–7.29 (m, 3H), 7.30–7.36 (m, 2H), 7.42 ppm (s, 1H); 13C NMR
(100 MHz, CDCl3): d=32.2, 36.6, 47.0, 118.9, 127.5, 128.8, 129.0,
129.7, 137.3, 137.8 ppm; HRMS-EI: m/z [M]+ calcd for C12H14N2S:
218.0878, found: 218.0871.
Analysis of enzyme inhibition: The data resulting from the above ex-
periments were plotted as nonlinear regression (sigmoidal dose–re-
sponse) curves using GraphPad Prism version 3. The values on the
abscissa represent the logarithm of inhibitor concentration (in mm),
whereas the values of the activity on the ordinate are expressed as
a percentage of the control experiments without inhibitor. From
these curves, the inhibitor concentration (EC50) at which enzyme
activity is halfway between the bottom and top plateau of the
curve, as well as the top and the bottom plateau values of the
curves, were retrieved by using the same program, and input into
the following formula to give the calculated values of the concen-
tration (IC50) of the compound under evaluation, for which the ac-
tivity of the enzyme is inhibited by 50% relative to control
[Eq. (1)].
1-(1H-Imidazol-1-yl)-4-(phenylsulfanyl)butane
hydrochloride
(14): Compound 14 was prepared from 1-bromo-4-(phenylsulfa-
nyl)butane (1.16 g, 4.73 mmol, 1 equiv) following the general pro-
cedure for the formation of {(phenylsulfanyl)alkyl}imidazoles to
afford the product as a white solid (380 mg, 1.41 mmol, 30%); Rf =
1
0.21 (EtOAc/MeOH, 4:1); mp: 90–928C; H NMR (400 MHz, CD3OD):
d=1.58–1.68 (m, 2H), 2.00–2.10 (m, 2H), 3.00 (t, J=7.0 Hz, 2H),
4.27 (t, J=7.2 Hz, 2H), 7.15–7.21 (m, 2H), 7.24–7.35 (m, 3H), 7.55 (t,
J=1.6 Hz, 1H), 7.64 (t, J=1.6 Hz, 1H), 8.95 ppm (s, 1H); 13C NMR
(100 MHz, CD3OD): d=26.7, 30.0, 33.5, 50.1, 121.2, 123.3, 127.2,
130.0, 130.5, 136.3, 137.3 ppm; HRMS-ESI: m/z [MꢁCl]+ calcd for
C13H17N2S: 233.1112, found: 233.1108.
EC50
IC50
¼
ð1Þ
1-(N-Benzylamino)-3-(1H-imidazol-1-yl)propane dihydrochloride
(15): Under an atmosphere of N2, a mixture of 1-(3-aminopropyl)i-
midazole (1.25 g, 9.99 mmol, 1 equiv) and benzaldehyde (1.27 g,
11.97 mmol, 1.2 equiv) in MeOH (20 mL) was stirred at RT for 6 h.
NaBH4 (1.11 g, 29.34 mmol, 11.7 equiv) was added, and the mixture
was stirred at RT overnight. The mixture was concentrated, and the
bottom
50
ꢁ
top
ꢁ 1
ꢁ
top
The IC50 value reported for each compound is the mean of the
values recorded in replicate experiments, and for each of these
replicate experiments (consisting of two separate assays) an indi-
ChemMedChem 2012, 7, 897 – 902
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
901

W. A. Szarek et al.
MED
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Acknowledgements
The authors thank the Canadian Institutes of Health Research for
a grant-in-aid (MOP 64305).
Keywords: carbon monoxide · heme · heme oxygenases ·
imidazoles · inhibitors
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Received: December 21, 2011
Revised: January 25, 2012
Published online on March 16, 2012
902
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 897 – 902