Imidazole-dioxolane compounds as isozyme-selective heme oxygenase inhibitors

Article abstract of DOI:10.1021/jm0511435

Several imidazole-dioxolane compounds were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). These compounds, which include (2R,4R)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-methyl-1, 3-dioxolane (1) hydrochloride, are

Full text of DOI:10.1021/jm0511435

J. Med. Chem. 2006, 49, 4437-4441
4437
Brief Articles
Imidazole-Dioxolane Compounds as Isozyme-Selective Heme Oxygenase Inhibitors
Jason Z. Vlahakis,† Robert T. Kinobe,‡ Raymond J. Bowers,† James F. Brien,‡ Kanji Nakatsu,‡ and Walter A. Szarek†,*
Departments of Chemistry and Pharmacology & Toxicology, Queen’s UniVersity, Kingston, ON K7L 3N6, Canada
ReceiVed NoVember 14, 2005
Several imidazole-dioxolane compounds were synthesized and evaluated as novel inhibitors of heme
oxygenase (HO). These compounds, which include (2R,4R)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-
1-yl)methyl]-4-methyl-1,3-dioxolane (1) hydrochloride, are structurally distinct from metalloporphyrin HO
inhibitors and lack the aminothiophenol moiety of azalanstat. They were found to be highly selective for
the HO-1 isozyme (stress induced) and had substantially less inhibitory potency toward HO-2, the constitutive
isozyme. These imidazole-dioxolane compounds are the first of their type known to exhibit this isozyme-
selective HO inhibition.
Introduction
Heme oxygenase (HO) isozymes are involved in the biotrans-
formation of heme into biliverdin, releasing ferrous iron (Fe2+
)
and carbon monoxide (CO) in the process (Figure 1).1,2 This
reaction is the main source of CO in mammals. HO-1 (also
known as HSP-32) is the inducible form of the enzyme, and is
a stress protein induced by a number of stimuli including heat
shock, heme, heavy metals, and reactive oxygen species. HO-
2, the constitutive form of the enzyme, is the most prevalent
form in most tissues, except the spleen where HO-1 levels are
predominant.3 Although the role of HO in heme catabolism is
quite well understood, the physiological role of the CO produced
in this process is receiving much attention, as CO is increasingly
becoming recognized as a cellular regulator with actions in brain,
blood vessels, and immune systems. The availability of drugs
that can be used to investigate the CO/HO system has been
limited largely to metalloporphyrins such as tin protoporphyrin
(SnPP).4,5 Because of the close structural similarity between
heme and the metalloporphyrin HO inhibitors, specificity
becomes a problem. In several systems proteins bind heme and
employ it for functions such as regulation of enzyme activity
{for example, of soluble guanylyl cyclase (sGC)} or as the key
component of the active site of an enzyme {for example, nitric
oxide synthase (NOS), cytochromes P450 (CYP)}. Accordingly,
the use of metalloporphyrins to ascribe various physiological
roles to the CO/HO system has been the subject of some
criticism because they have been shown to affect the activity
of both NOS and sGC.6,7 Thus, a program in our laboratories is
concerned with the design of HO inhibitors that are not based
on the porphyrin nucleus and has the objective of obtaining more
selective HO inhibitors.
dependent enzymes (such as sGC and NOS), in addition to
isozyme-dependent HO selectivity. These aminothiophenol-
containing azalanstat analogues were observed to be excellent
inhibitors of HO, and most of them showed significantly more
potency as inhibitors of the inducible isoform of HO (HO-1)
than the constitutive isoform (HO-2). Here we describe the
extension of our synthetic program to include a methyl-
terminated series of imidazole-dioxolane compounds (1-4)
which were evaluated for their ability to inhibit HO-1 and HO-
2. Our results show that these imidazole-dioxolane compounds
are much more isozyme-selective than the previously studied
azalanstat analogues,8 as they are excellent inhibitors of HO-1
and virtually devoid of the ability to inhibit HO-2. These features
might result in useful therapeutic applications; for example, Fang
et al.10 have described the therapeutic potential of HO-1
inhibition, by targeting HO-1 in tumors, to induce tumor cell
apoptosis.
Synthesis. In an effort to evaluate the importance that certain
structural features of azalanstat have on the inhibition of HO,
we chose to focus initially on the modification of the amino-
thiophenol moiety. By simply replacing the aminothiophenol
functionality by a hydrogen atom, a methyl-terminated series
of compounds (1-4) were synthesized and evaluated for their
ability to inhibit HO-1 and HO-2. Further modifications involved
the synthesis of compound 5, which was devoid of the methyl
group in the 4-position of the dioxolane ring, and of its
dithiolane analogue 6; the new compounds were investigated
for inhibitory activity against HO. The diastereomeric tosylates
7-10 were prepared as previously reported.8 As shown in
In a previous publication,8 we described the synthesis and
HO inhibitory activity of a series of imidazole-dioxolanes that
arose from the lead compound azalanstat.9 Our initial approach
was to perform appropriate synthetic modifications to the
structure of azalanstat and develop imidazole-based analogues
having enhanced inhibitory potency for HO over other heme-
* To whom correspondence should be addressed. Telephone: (613) 533-
2643. Fax: (613) 533-6532. E-mail: walter.szarek@chem.queensu.ca.
† Department of Chemistry.
Figure 1. The oxidative degradation of heme in the CO/HO pathway.
‡ Department of Pharmacology & Toxicology.
10.1021/jm0511435 CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/21/2006

4438 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Brief Articles
Scheme 1a
a Reagents and conditions: (a) LiAlH4, THF, reflux; (b) p-TsOH‚H2O, ethylene glycol, toluene, reflux; (c) p-TsOH‚H2O, 1,2-ethanedithiol, toluene,
reflux.
Table 1. Inhibitory Potency and Selectivity of Imidazole-Dioxolane/Dithiolane Compounds against the Activity of Various Enzymesa
IC50 (µM)
HO-1
(rat spleen)
IC50 (µM)
HO-2
(rat brain)
selectivity index
rat IC50 (HO-2)/
IC50 (HO-1)
IC50 (µM)
nNOS
(rat brain)
IC50 (µM)
sGC
(rat lung)
IC50 (µM)
CYP3A1/3A2
(rat liver)
IC50 (µM)
CYP2E1
(rat liver)
compd
1
2
3
4
5
6
0.8 ( 0.2
2.6 ( 0.4
12 ( 4
305 ( 25
>100
381
>38
>8
362 ( 1
773 ( 426
>500
>1000
>500
>500
>500
>1000
>500
3.2 ( 0.8
8 ( 2
3.6 ( 0.5
6.0 ( 0.1
20 ( 2
30 ( 6
5 ( 1
>100
10 ( 5
7 ( 3
20 ( 4
>100
>100
16 ( 4
35 ( 6
28 ( 18
>5
>500
4 ( 2
>25
3.4
35
182 ( 119
34 ( 12
4 ( 1
5.9 ( 0.1
4.7 ( 0.6
1.0 ( 0.2
6 ( 1
14 ( 6
12
azalanstat
4.7
a Assays were performed as indicated in the Experimental Section. Data represent mean IC50 values ( standard deviation of replicate experiments. All
imidazole-dioxolane compounds were evaluated as HCl salts.
Scheme 1, the methyl-terminated compounds 1, 2, 3, and 4 were
obtained by the reduction of tosylates 7, 8, 9, and 10,
respectively, using lithium aluminum hydride in THF.11 Since
the reduction was performed on only one diastereomeric tosylate,
only one methyl-terminated diastereomer was produced, thus
avoiding the production of a mixture of all four diastereomers
which would have resulted by acid-catalyzed acetalation of 4-(4-
chlorophenyl)-1-(1H-imidazol-1-yl)-2-butanone (11) using ra-
cemic 1,2-propanediol. The 1,3-dioxolane compound 5 was
prepared from ketone 11 by an acid-catalyzed acetalation
reaction in toluene using ethylene glycol, according to a
procedure similar to that reported by Walker et al.12 The
corresponding 1,3-dithiolane derivative 6 was also prepared in
this manner from 11 using 1,2-ethanedithiol. All of the final
compounds were isolated/characterized and evaluated as the
hydrochloride salts of the structures shown.
membrane free; however, this changes the milieu for enzyme
activity. Each of the methyl-terminated compounds (1-4) were
potent inhibitors of HO-1, while showing very little activity for
HO-2 (Table 1). The results are in contrast to the results obtained
in our earlier study8 in that none of the present compounds were
potent inhibitors of HO-2. The selectivity index of these
compounds for HO-1 is given in the fourth column.
In contrast to metalloporphyrins such as SnPP which show
modest selectivity toward HO-2,16 the methyl-terminated com-
pounds show HO-1 selectivity. The selectivity indexes of
compounds 1 and 2 (and presumably 3-5) are also much higher
than with the previously studied8 azalanstat analogues containing
the aminothiophenol moiety. As reported in that study, the
highest index, namely ∼35, was observed for compound 12.
Biological Evaluation. The compounds in Table 1 were
screened for evidence of their ability to inhibit HO-1 and HO-2
using an in vitro assay for HO in which heme was presented to
the enzyme complexed with albumin as described in the
Experimental Section. HO-1 was obtained from rat spleen and
HO-2 was obtained from rat brain as the microsomal fractions
prepared by differential centrifugation; the dominance of HO-1
and HO-2 proteins in the rat spleen and brain, respectively, has
been documented.3,13-15 These particular microsomal prepara-
tions were selected in order to use the most native (i.e., closest
to in vivo) forms of HO, and hence in order that they would be
most relevant to anticipated whole animal studies. Purer forms
of HO have been obtained by processes that render the enzymes
Thus, at least two members of the present methyl-terminated
series of compounds exhibit enhanced inhibitory selectivity for
HO-1 over HO-2 and are in fact the first compounds known to
exhibit this essentially isozyme-specific inhibition over a very
favorable concentration range. For instance, a representative
activity-inhibitor concentration curve showing the difference
in potency toward HO-1 (rat spleen) and HO-2 (rat brain)
activity exhibited by compound 1 is shown in Figure 2. The

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4439
Figure 2. Representative activity-inhibitor concentration curves for
the inhibition of HO-1 and HO-2 by 1. Enzyme activities were
determined as described in the Experimental Section. All of the values
of activity (ordinate) are expressed as a percentage of the control with
no inhibitor present. The values on the abscissa represent the log of
the inhibitor concentration in µM. Open circles O, HO-1 (rat spleen
microsomes) and filled squares 9, HO-2 (rat brain microsomes).
Inhibitor concentrations that inhibited activity by 50% (IC50) were
determined from such nonlinear regression plots (sigmoidal dose-
response curve) using GraphPad Prism version 3 and reported as an
average ( standard deviation of replicate experiments.
Figure 4. Representative activity-inhibitor concentration curves for
the inhibition of various enzymes by 1. Enzyme activities were
determined as described in the Experimental Section. Open squares 0,
CYP2E1 (rat liver microsomes), filled triangles 2, CYP3A1/3A2 (rat
liver microsomes) and filled circles b, nNOS (rat brain microsomes).
methyl group, namely 5, was synthesized and evaluated.
Interestingly, compound 5 also showed good potency and
selectivity toward inhibition of HO-1 (IC50 ) 4 ( 2 µM) over
HO-2 (IC50 > 100 µM). In the case of the dithiolane analogue
6, the HO-1 inhibitory potency (IC50 ) 4.7 ( 0.6 µM) was
slightly less than that of 5 and there was observed substantially
increased potency toward HO-2 (IC50 ) 16 ( 4 µM), resulting
in a drastic reduction in selectivity.
Previous work on azalanstat and its structural analogues
showed potent inhibition of both HO-1 and HO-2 which
appeared to be dependent on the position of the amino group
on the thiophenol moiety as well as the configurations of the
diastereomeric compounds.8 However, the aminothiophenol
moiety is not an absolute requirement for HO-inhibition since
from the present study it appears that its removal enhances the
selectivity for HO-1 inhibition (compounds 1-5). The amino-
thiophenol group (or other group in the analogous position) may
be an important factor for recognition in the HO-2 system, a
fact to keep in mind when designing inhibitors selective for
HO-2.
Figure 3. Representative activity-inhibitor concentration curves for
the inhibition of HO-1 (human spleen) and HO-2 (rat liver) by 1.
Enzyme activities were determined as described in the Experimental
Section. Filled diamonds [, HO-1 (human spleen microsomes) and
filled triangles 1, HO-2 (rat liver microsomes).
Inhibition of Other Enzymes. Compounds 1 (apparently as
a mixture of diastereomers) and 5 have been described in a
patent17 which purports that derivatives of substituted N-
alkylimidazoles could be useful as anticonvulsant agents. Also,
the closely related (2-aryl-4-phenylthioalkyl-1,3-dioxolan-2-
ylmethyl)azole derivatives are indicated18 to be useful as
antifungal and antineoplastic agents, presumably owing to the
inhibition of lanosterol 14R-demethylase. Thus, we also chose
to address the issue of selectivity with respect to other enzymes,
in particular, hemoproteins. Since a major drawback of metal-
loporphyrins is their ability to inhibit nNOS and sGC, selectivity
of the imidazole-dioxolane compounds for HO over other
heme-dependent enzymes such as nNOS, sGC and cytochromes
P45019 (CYP) was studied also.
With the exception of 6, the candidate compounds in Table
1 were found to be poor inhibitors of nNOS (with IC50 > 400
µM) and all of the compounds had virtually no effect on sGC
activity up to 1000 µM. These results are in contrast to those
of the metalloporphyrin HO inhibitors, which also interact with
the NOS and sGC systems. However, the candidate compounds
are potent inhibitors of CYP3A1/3A2 and CYP2E1 (Table 1)
obtained from rat liver. For instance, as exemplified for 1 in
Figure 4, potent inhibition of CYP3A1/3A2 (IC50 ) 3.2 ( 0.8
µM) and CYP2E1 (IC50 ) 3.6 ( 0.5 µM) was observed along
with only a weak inhibitory effect on nNOS (IC50 ) 362 ( 1
µM). The observed P450 inhibition is of significance since
cytochromes P450 are responsible for the metabolism of many
drugs used in therapy and as experimental tools. Such drugs
could be used in conjunction with our novel agents.
IC50 was 0.8 ( 0.2 µM for HO-1 (rat spleen) and approximately
305 ( 25 µM for HO-2 (rat brain), with a selectivity index
∼381. Similar selectivity for HO-1 was observed also for 1 when
comparing inhibition of HO isolated from other sources. For
example, 1 also showed strong potency toward the activity of
HO-1 isolated from human spleen (IC50 ) 1.5 ( 0.4 µM), while
inhibition of HO-2 (predominantly) isolated from rat liver (IC50
532 ( 135 µM) required much higher inhibitor concentrations
(Figure 3).
The selectivity between HO-1/HO-2 noticed for 1 was
observed also in diastereomers 2-4. As with the aminothiophe-
nol-containing azalanstat series of HO inhibitors studied earlier,8
the potency and selectivity of these methyl-terminated com-
pounds (1-4) do appear to be dependent on the diastereomeric
configuration of the dioxolane core (which determines 3-D
molecular shape). All of the other three diastereomers of 1 were
synthesized and evaluated as inhibitors of HO. It is the (2R,
4R) isomer 1 in the methyl-terminated series that is the most
potent toward HO-1 (IC50 ) 0.8 ( 0.2 µM); this compound is
directly comparable to the (2R, 4S) azalanstat analogues with
respect to 3-dimensional shape, compounds which also are the
most potent against HO-1 (IC50 in the 0.5-2.5 µM range).8
These methyl-terminated compounds have low potency toward
HO-2 (>100 µM); selectivity of these compounds approximately
parallels HO-1 inhibitory potency, the order of which is
presumably 1 > 2 > 3 > 4.
Since HO inhibition potency appeared to be related to the
orientation of the methyl group, the compound lacking the

4440 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Brief Articles
OD): δ 1.04 (d, J ) 6.0 Hz, 3 H), 1.95-2.05 (m, 2 H), 2.70-2.85
(m, 3 H), 4.08 (dd, J ) 8.0, 6.0 Hz, 1 H), 4.26-4.35 (m, 1 H),
4.49 (s, 2 H), 7.22 (d, J ) 8.4 Hz, 2 H), 7.28 (d, J ) 8.4 Hz, 2 H),
7.58 (s, 1 H), 7.62 (s, 1 H), 8.96 (s, 1 H); 13C NMR (100 MHz,
CD3OD): δ 18.0, 30.1, 39.2, 54.7, 73.0, 74.7, 109.2, 120.2, 125.4,
129.6, 130.9, 132.8, 137.9, 141.5; HRMS (ES) [M + H]+ Calcd.
for C16H20ClN2O2: 307.1207. Found: 307.1203.
Conclusion
We have synthesized several compounds that express excel-
lent selectivity as inhibitors of HO-1 relative to HO-2. In
particular, compound 1 is noteworthy for its high potency and
selectivity toward HO-1; compound 5, although not as potent,
is prepared relatively easily. As HO-1 is the isozyme that has
attracted the most research interest, these compounds are
anticipated to become very useful tools in elucidating the
physiological roles of HO-1 and carbon monoxide in mammalian
and other biological systems. These novel compounds might
also have useful therapeutic applications, as indicated earlier,
for example, by the studies of Fang et al.10
(2S,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)m-
ethyl]-4-methyl-1,3-dioxolane Hydrochloride (3‚HCl). White
solid in 38% yield from 98: mp 172-173 °C; Rf ) 0.18 (EtOAc);
[R]2D2 ) +15.7° (c ) 1.65, D2O); H NMR (400 MHz, D2O): δ
1
1.22 (d, J ) 6.0 Hz, 3 H), 1.92-2.08 (m, 2 H), 2.72 (t, J ) 8.4
Hz, 2 H), 3.50 (t, J ) 8.6 Hz, 1 H), 3.67-3.75 (m, 1 H), 4.08 (dd,
J ) 8.0, 6.0 Hz, 1 H), 4.41 (d, J ) 2.0 Hz, 2 H), 7.22 (d, J ) 8.4
Hz, 2 H), 7.33 (d, J ) 8.4 Hz, 2 H), 7.48 (s, 1 H), 7.50 (s, 1 H),
8.74 (s, 1 H); 13C NMR (100 MHz, D2O): δ 16.9, 28.2, 37.5, 54.0,
71.9, 74.6, 108.4, 119.7, 123.7, 128.8, 130.2, 131.5, 136.0, 140.2;
HRMS (ES) [M + H]+ Calcd. for C16H20ClN2O2: 307.1207.
Found: 307.1204.
Experimental Section
The 1H and 13C NMR spectra were recorded on a Bruker Avance
400 MHz spectrometer in CD3OD or D2O. The signals owing to
residual protons in the deuterated solvents were used as internal
standards. Chemical shifts (δ) are reported in ppm downfield from
tetramethylsilane.20 Carbon chemical shifts are given relative to
CD3OD: δ ) 49.00. High-resolution electrospray mass spectra were
recorded on an Applied Biosystems/MDS Sciex QSTAR XL
spectrometer with an Agilent HP1100 Cap-LC system. Samples
were run in 50% aqueous MeOH at a flow rate of 6 µL/min.
Elemental analyses were performed by MHW Laboratories (Phoe-
nix, AZ). Melting points were determined on a Mel-Temp II melting
point apparatus and are uncorrected. Optical rotations were
measured using an Autopol II automatic polarimeter for solutions
in a 1-dm cell at room temperature. Thin-layer chromatography
was performed using glass- or aluminum-backed Silica Gel 60 F254
plates (Silicycle, Quebec City, Quebec, Canada). Plates were viewed
under UV light or by charring after spraying with phosphomolybdic
acid (PMA) in EtOH.
(2S,4R)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)-
methyl]-4-methyl-1,3-dioxolane Hydrochloride (4‚HCl). White
solid in 51% yield from 108: mp 152-153 °C; Rf ) 0.21 (EtOAc);
[R]2D2 ) -21.0° (c ) 1.33, D2O); H NMR (400 MHz, D2O): δ
1
0.97 (d, J ) 6.0 Hz, 3 H), 1.88-1.98 (m, 2 H), 2.64 (t, J ) 8.2
Hz, 2 H), 2.73 (t, J ) 8.4 Hz, 1 H), 4.02 (t, J ) 7.2 Hz, 1 H),
4.19-4.27 (m, 1 H), 4.37 (s, 2 H), 7.15 (d, J ) 8.4 Hz, 2 H), 7.25
(d, J ) 8.0 Hz, 2 H), 7.43 (s, 1 H), 7.44 (s, 1 H), 8.70 (s, 1 H); 13
C
NMR (100 MHz, D2O): δ 16.8, 28.5, 37.6, 53.7, 71.8, 74.1, 108.3,
119.5, 123.9, 128.7, 130.1, 131.4, 136.1, 140.2; HRMS (ES)
[M + H]+ Calcd. for C16H20ClN2O2: 307.1207. Found: 307.1207.
2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-
dioxolane Hydrochloride (5‚HCl). Under a N2 atmosphere, a
mixture of the ketone 118 (400 mg, 1.61 mmol) and p-toluene-
sulfonic acid monohydrate (536 mg, 2.82 mmol, 1.75 eq) in toluene
(12 mL) was heated at reflux temperature (Dean-Stark apparatus)
for 1.5 h; a solution of ethylene glycol (156 mg, 2.51 mmol, 1.56
eq) in toluene (4 mL) was added and the mixture was heated at
reflux temperature for an additional 8.5 h. The mixture was cooled
to room temperature and diluted with H2O, and the mixture was
extracted twice with EtOAc. The combined extracts were washed
sequentially with aqueous Na2CO3 solution and water, dried
(MgSO4), and concentrated to a dark brown oil. Purification by
flash chromatography on silica gel (EtOAc and then acetone) gave
242 mg (0.83 mmol, 52%) of the free base which was dissolved in
hot EtOH (2 mL) and treated with a solution of 37% aqueous HCl
(100 mg, 1.02 mmol) in EtOH (1 mL). The mixture was
concentrated and dried under high vacuum. Hot EtOAc (10 mL)
was added, followed by a minimum amount of hot EtOH to produce
a solution. The solution was cooled in the freezer, hexanes (5 mL)
added, and the product allowed to crystallize overnight. The solid
was removed by filtration and washed with hexanes. High-vacuum-
drying afforded 125 mg (0.38 mmol, 24%) of 5 as a white solid:
Representative procedure for the reduction of tosylates using
lithium aluminum hydride to afford 1-4:
(2R,4R)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)-
methyl]-4-methyl-1,3-dioxolane Hydrochloride (1‚HCl). Under
a N2 atmosphere, a solution of the tosylate 78 (200 mg, 0.42 mmol)
in THF (2 mL) was cooled to 0 °C, and a suspension of LiAlH4
(31 mg, 0.83 mmol) in THF (2 mL) was added; the mixture was
heated at reflux temperature for 4 h. The mixture was cooled to 0
°C, diluted with Et2O, and then carefully quenched with wet Et2O.
After dilution with H2O, the mixture was extracted twice with Et2O.
The combined extracts were washed sequentially with a saturated
aqueous solution of Na2CO3 and water, dried (MgSO4), and
concentrated to a yellow oil. Purification by preparative scale (1-
cm thick) thin-layer chromatography (EtOAc) gave 73 mg (0.24
mmol, 57%) of the free base which was dissolved in hot 2-propanol
(2 mL), and the solution was treated with a solution of 37% aqueous
HCl (40 mg, 0.41 mmol) in 2-propanol (1 mL). The mixture was
concentrated and dried under high vacuum. The residue was
recrystallized (2-propanol-Et2O), and the solid was removed by
filtration and washed with Et2O. High-vacuum-drying afforded 85
mg (0.25 mmol, 59%) of 1 as a white solid: mp 172-173 °C;
1
mp 168-169 °C; Rf ) 0.17 (EtOAc); H NMR (400 MHz, D2O):
Rf ) 0.24 (EtOAc); [R]22 ) -10.2° (c ) 2.17, D2O); H NMR
1
δ 1.94-2.02 (m, 2 H), 2.64-2.72 (m, 2 H), 3.58-3.68 (m, 2 H),
3.92-4.02 (m, 2 H), 4.41 (s, 2 H), 7.18 (d, J ) 8.0 Hz, 2 H), 7.29
(d, J ) 8.0 Hz, 2 H), 7.47 (s, 2 H), 8.72 (s, 1 H); 13C NMR (100
MHz, D2O): δ 28.3, 37.1, 53.7, 66.1, 108.2, 119.7, 123.7, 128.8,
130.1, 131.4, 136.1, 140.3; HRMS (ES) [M + H]+ Calcd. for
C15H18ClN2O2: 293.1051. Found: 293.1040.
(400 MHz, D2O): δ 1.2D4 (d, J ) 6.0 Hz, 3 H), 1.93-2.08 (m, 2
H), 2.73 (t, J ) 8.2 Hz, 2 H), 3.51 (t, J ) 8.6 Hz, 1 H), 3.69-3.78
(m, 1 H), 4.10 (dd, J ) 8.0, 6.0 Hz, 1 H), 4.43 (d, J ) 2.4 Hz, 2
H), 7.23 (d, J ) 8.4 Hz, 2 H), 7.34 (d, J ) 8.4 Hz, 2 H), 7.49-
7.52 (m, 2 H), 8.76 (s, 1 H); 13C NMR (100 MHz, D2O): δ 17.0,
28.3, 37.5, 54.0, 71.9, 74.7, 108.4, 119.7, 123.7, 128.8, 130.2, 131.5,
136.0, 140.2; HRMS (ES) [M + H]+ Calcd. for C16H20ClN2O2:
307.1207. Found: 307.1193.
2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-
dithiolane Hydrochloride (6‚HCl). The title compound was
synthesized from ketone 118 by the procedure employed for the
synthesis of 5, except using 1,2-ethanedithiol instead of ethylene
glycol, to afford a beige solid in 32% yield after recrystallization
(2-propanol): mp 204-205 °C; Rf ) 0.21 (EtOAc); 1H NMR (400
MHz, CD3OD): δ 2.17-2.25 (m, 2 H), 2.94-3.06 (m, 4 H), 3.28-
3.38 (m, 2 H), 4.65 (s, 2 H), 7.21 (d, J ) 8.4 Hz, 2 H), 7.28 (d,
J ) 8.4 Hz, 2 H), 7.56 (s, 1 H), 7.81 (s, 1 H), 9.11 (s, 1 H); 13C
NMR (100 MHz, D2O): δ 32.6, 41.6, 43.4, 59.8, 71.2, 119.9, 125.5,
Characterization of the new compounds (2-4) synthesized
following the representative procedure for the reduction of tosylates
(shown above for 1) as outlined in Scheme 1:
(2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)-
methyl]-4-methyl-1,3-dioxolane Hydrochloride (2‚HCl). Beige
solid in 64% yield from 88: mp 148-149 °C; Rf ) 0.21 (EtOAc);
[R]2D2 ) +15.1° (c ) 1.19, CD3OD); H NMR (400 MHz, CD3-
1

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4441
129.6, 131.1, 133.0, 138.2, 141.3; HRMS (ES) [M + H]+ Calcd.
for C15H18ClN2S2: 325.0600. Found: 325.0587.
(4) Johnson, R. A.; Colombari, E.; Colombari, D. S.; Lavesa, M.; Talman,
W. T.; Nasjletti, A. Role of endogenous carbon monoxide in central
regulation of arterial pressure. Hypertension 1997, 30, 962-967.
(5) Tulis, D. A.; Durante, W.; Peyton, K. J.; Evans, A. J.; Schafer, A. I.
Heme oxygenase-1 attenuates vascular remodelling following balloon
injury in rat carotid arteries. Atherosclerosis 2001, 155, 113-122.
(6) Luo, D.; Vincent, S. R. Metalloporphyrins inhibit nitric oxide-
dependent cGMP formation in vivo. Eur. J. Pharmacol. 1994, 267,
263-267.
(7) Meffert, M. K.; Haley, J. E.; Schuman, E. M.; Schulman, H.;
Madison, D. V. Inhibition of hippocampal heme oxygenase, nitric
oxide synthase, and long-term potentiation by metalloporphyrins.
Neuron 1994, 13, 1225-1233.
(8) Vlahakis, J. Z.; Kinobe, R. T.; Bowers, R. J.; Brien, J. F.; Nakatsu,
K.; Szarek, W. A. Synthesis and evaluation of azalanstat analogues
as heme oxygenase inhibitors. Bioorg. Med. Chem. Lett. 2005, 15,
1457-1461.
(9) Walker, K. A. M.; Kertesz, D. J.; Rotstein, D. M.; Swinney, D. C.;
Berry, P. W.; So, O.-Y.; Webb, A. S.; Watson, D. M.; Mak, A. Y.;
Burton, P. M.; Mills-Dunlap, B.; Chiou, M. Y.; Tokes, L. G.; Kurz,
L. J.; Kern, J. R.; Chan, K. W.; Salari, A.; Mendiza´bal, G. R. J.
Med. Chem. 1993, 36, 2235-2237.
In Vitro HO Activity Assay. HO activity in rat spleen and brain
microsomal fractions was determined by the quantitation of CO
formed from the degradation of methemalbumin (heme complexed
with albumin).21,22 Using spleen and brain tissue from Sprague-
Dawley rats, microsomal fractions were prepared according to the
procedure outlined by Appleton et al.23 Protein concentration of
microsomal fractions was determined by a modification of the
Biuret method.22 Incubations for HO activity analysis were done
under conditions for which the rate of CO formation (pmol CO
min-1 mg protein-1) was linear with respect to time and microsomal
protein concentration. Briefly, reaction mixtures (150 µL) consisting
of 100 mM phosphate buffer (pH 7.4), 50 µM methemalbumin,
and 1 mg/mL protein were preincubated with the imidazole-
dioxolane compounds (as HCl salts) at final concentrations ranging
from 0.1 to 100 µM 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.
Blanks for which NADPH was omitted were subtracted to correct
for CO that might have formed by a route other than HO. Reactions
were stopped by instantly freezing the reaction mixture on dry ice,
and CO formation was monitored by gas chromatography according
to the method described by Vreman and Stevenson.21
In Vitro nNOS Assay. The activity of nNOS was measured by
monitoring the conversion of 14C-L-arginine into 14C-L-citrulline
in rat brain cytosolic fractions according to a modification of
previously outlined procedures,24,25 as described by Kinobe et al.15
In Vitro sGC Assay. The activity of sGC in rat lung cytosolic
fractions was measured by monitoring the formation of cGMP using
a competitive enzyme-linked immunosorbent assay (ELISA), as
described by Kinobe et al.15
In Vitro CYP3A1/3A2 and CYP2E1 Assay. CYP3A1/3A2-
catalyzed erythromycin N-demethylase activity in rat liver mi-
crosomal fractions was determined by the spectrophotometric
measurement of formaldehyde, whereas the hydroxylation of
p-nitrophenol by CYP2E1 was determined by the spectrophoto-
metric measurement of 4-nitrocatechol, as described by Kinobe et
al.15
(10) Fang, J.; Akaike, T.; Maeda, H. Antiapoptotic role of heme oxygenase
(HO) and the potential of HO as a target in anticancer treatment.
Apoptosis 2004, 9, 27-35.
(11) Rapoport, H.; Bonner, R. M. ∆7- and ∆8-Desoxycodeine. J. Am.
Chem. Soc. 1951, 73, 2872-2876.
(12) Walker, K. A. M.; Burton, P. M.; Swinney, D. C. 1,3-Dioxolane
Derivatives as Cholesterol-lowering Agents. Eur. Patent 0 492 474
B1, March 5, 1997.
(13) Xia, Z. W.; Cui, W. J.; Zhang, X. H.; Shen, Q. X.; Wang, J.; Li, Y.
Z.; Chen, S. N.; Yu, S. C. Analysis of heme oxygenase isomers in
rat. World J. Gastroenterol. 2002, 8, 1123-1128.
(14) Trakshel, G. M.; Kutty, R. K.; Maines, M. D. Resolution of the rat
brain heme oxygenase activity: absence of a detectable amount of
the inducible form (HO-1). Arch. Biochem. Biophys. 1988, 260, 732-
739.
(15) Kinobe, R. T.; Vlahakis, J. Z.; Vreman, H. J.; Stevenson, D. K.;
Brien, J. F.; Szarek, W. A.; Nakatsu, K. Selectivity of imidazole-
dioxolane compounds for in Vitro inhibition of microsomal haem
oxygenase isoforms. Br. J. Pharmacol. 2006, 147, 307-315.
(16) Vreman, H. J.; Cipkala, D. A.; Stevenson, D. K. Characterization of
porphyrin heme oxygenase inhibitors.Can. J. Physiol. Pharmacol.
1996, 74, 278-285.
(17) Walker, K. A. M. Derivatives of Substituted N-Alkylimidazoles. U.
S. Patent 4,321,272, March 23, 1982.
(18) Heeres, J.; Van der Veken, L. J. E. (2-Aryl-4-phenylthioalkyl-1,3-
dioxolan-2-ylmethyl)azole Derivatives. U.S. Patent 4,490,540, Dec
25, 1984.
(19) Ortiz de Montellano, P. R. In Cytochrome P-450: Structure,
Mechanism and Biochemistry; Ortiz de Montellano, P. R., Ed.;
Plenum Press: New York and London, 1986.
(20) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62,
7512-7515.
(21) Vreman, H. J.; Stevenson, D. K. Heme oxygenase activity as
measured by carbon monoxide production. Anal. Biochem. 1988, 168,
31-38.
(22) Cook, M. N.; Nakatsu, K.; Marks, G. S.; McLaughlin, B. E.; Vreman,
H. J.; Stevenson, D. K.; Brien, J. F. Heme oxygenase activity in the
adult rat aorta and liver as measured by carbon monoxide formation.
Can. J. Physiol. Pharmacol. 1995, 73, 515-518.
(23) Appleton, S. D.; Chretien, M. L.; McLaughlin, B. E.; Vreman, H.
J.; Stevenson, D. K.; Brien, J. F.; Nakatsu, K.; Maurice, D. H.; Marks,
G. S. Selective inhibition of heme oxygenase, without inhibiton of
nitric oxide synthase or soluble guanylyl cyclase, by metalloporphy-
rins at low concentrations. Drug Metab. Dispos. 1999, 27, 1214-
1219.
Analysis of Enzyme Inhibition. Inhibitions of the catalytic
activities of HO, nNOS, sGC, CYP2E1, and CYP3A1/3A2 by the
imidazole-dioxolane compounds (as HCl salts) studied herein were
evaluated by the percentage of control activity of each enzyme
remaining in the presence of different concentrations of inhibitors
with reference to control reactions. Replicate experiments were
performed to generate multiple (two or three) activity-inhibitor
concentration curves for each imidazole-dioxolane compound
tested. For each curve, an IC50 value (inhibitor concentration that
decreased enzyme activity by 50%) was calculated from the
equation generated from a nonlinear regression plot of the data using
a sigmoidal dose-response curve (GraphPad Prism version 3). The
IC50 values in Table 1 are presented as the mean ( standard
deviation from replicate experiments.
Acknowledgment. This work was supported by a grant-in-
aid from the Canadian Institutes of Health Research, MOP
64305.
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(24) Brien, J. F.; Reynolds, J. D.; Cunningham, M. A.; Parr, A. M.;
Waddock, S.; Kalisch, B. E. Nitric oxide synthase activity in the
hippocampus, frontal cerebral cortex, and cerebellum of the guinea
pig: ontogeny and in vitro ethanol exposure. Alcohol 1995, 12, 329-
333.
References
(1) Maines, M. D. The heme oxygenase system: a regulator of second
messenger gases. Annu. ReV. Pharmacol. Toxicol. 1997, 37, 517-
554.
(25) Kimura, K. A.; Parr, A. M.; Brien, J. F. Effect of chronic maternal
ethanol administration on nitric oxide synthase activity in the
hippocampus of the mature fetal guinea pig. Alcohol Clin. Exp. Res.
1996, 20, 948-953.
(2) Vreman, H. J.; Wong, R. J.; Stevenson, D. K. In Carbon Monoxide
and CardioVascular Function; Wang, R., Ed.; CRC Press: Boca
Raton, London, New York, and Washington, 2002; p 273.
(3) Maines, M. D. Heme oxygenase: function, multiplicity, regulatory
mechanisms, and clinical applications. FASEB J. 1988, 2, 2557-
2568.
JM0511435