Synthesis and evaluation of imidazole-dioxolane compounds as selective heme oxygenase inhibitors: Effect of substituents at the 4-position of the dioxolane ring

Article abstract of DOI:10.1016/j.bmc.2009.01.078

Several imidazole-dioxolane compounds were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). These compounds, which include a series of substituted thiophenol and substituted phenol derivatives of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-

Full text of DOI:10.1016/j.bmc.2009.01.078

Bioorganic & Medicinal Chemistry 17 (2009) 2461–2475
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Bioorganic & Medicinal Chemistry
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Synthesis and evaluation of imidazole–dioxolane compounds as selective
heme oxygenase inhibitors: Effect of substituents at the 4-position
of the dioxolane ring
Jason Z. Vlahakis a, Maaike Hum b, Mona N. Rahman c, Zongchao Jia c, Kanji Nakatsu b, Walter A. Szarek a,
*
a Department of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6
b Department of Pharmacology & Toxicology, Queen’s University, Kingston, Ontario, Canada K7L 3N6
c Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6
a r t i c l e i n f o
a b s t r a c t
Article history:
Several imidazole–dioxolane compounds were synthesized and evaluated as novel inhibitors of heme
oxygenase (HO). These compounds, which include a series of substituted thiophenol and substituted phe-
nol derivatives of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[(phenylsulfa-
nyl)methyl]-1,3-dioxolane hydrochloride (3), in addition to smaller functionalized derivatives,
continue our structure–activity studies by exploration of the aminothiophenol region (‘northeastern
region’) in our original target structure azalanstat (1). In vitro, most of the compounds in this series were
found to be highly potent inhibitors of the stress-induced isozyme HO-1 and the constitutive isozyme
HO-2, showing only moderate selectivity for HO-1. Nevertheless, a few of the compounds displayed
higher selectivity toward HO-1. None of the compounds having a larger appendage in the northeastern
region were inhibitors of CYP2E1, whereas a compound having a relatively small fluorine substituent
in this region did inhibit CYP2E1; all of the compounds tested exhibited high inhibitory potency against
CYP3A1/3A2.
Received 23 October 2008
Revised 30 January 2009
Accepted 31 January 2009
Available online 12 February 2009
Keywords:
Heme oxygenase
Imidazole–dioxolane inhibitors
Carbon monoxide
Heme
Molecular modeling
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
The heme oxygenase (HO) system comprises two active iso-
zymes, namely, HO-1 (inducible) and HO-2 (constitutive), and is
involved in the biotransformation of heme into biliverdin, releas-
ing carbon monoxide (CO) and ferrous iron (Fe2+) in the process
(Fig. 1).1,2 The cellular regulatory actions of CO have recently been
acknowledged widely,3–5 and the use of HO inhibitors as pharma-
cological tools is critical in the elucidation of the physiological
functions of the CO/HO system and related physiological pathways.
Such inhibitors may be useful when investigating the role of HO-1
in various clinical situations such as: a protective effect in acute re-
nal failure,6 its role in the response to cardiovascular-system inju-
ries,7 a role in neurodegenerative diseases,8 and its promotion of
the growth of solid tumors.9 Owing to the lack of selectivity of
first-generation HO inhibitors, which are metalloporphyrin-based,
our laboratory has been concerned with the design of new HO
inhibitors that are not based on the porphyrin nucleus, having
the objective of obtaining more-selective HO inhibitors. The dis-
covery2 of imidazole-based azalanstat (1) as a lead compound al-
lowed the design of novel, more-selective inhibitors of these
Figure 1. The oxidative degradation of heme in the CO/HO pathway.
enzymes; azalanstat itself is a potent inhibitor of HO-1 and HO-2
and has minimal effects on other enzymes usually inhibited by
metalloporphyrins such as nitric oxide synthase (NOS) or soluble
guanylyl cyclase (sGC).10 Our initial structure–activity study of a
series of twelve azalanstat analogs11 showed that different degrees
of selectivity in the inhibition of HO-1 relative to HO-2 could be
achieved by modifying the position of the amino functionality on
the aminothiophenol moiety in the northeastern region of azalan-
stat (1) or by modifying the diastereomeric configuration of the
substituents attached to the dioxolane ring in the central region
(Fig. 2). Thus, we embarked on a mission to systematically explore
the various regions of the azalanstat structure with a view of
designing highly isozyme-selective HO inhibitors.
The present work focuses initially on the investigation of the
structure–activity relationship brought about by subtle modifica-
* Corresponding author. Tel.: +1 613 533 2643; fax: +1 613 533 6532.
E-mail address: szarekw@chem.queensu.ca (W.A. Szarek).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.078

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
mized (2R,4S) dioxolane ring configuration, and the rest of the aza-
lanstat structure including the 4-chlorophenethyl motif in the
western region and the 1-substituted imidazole moiety in the east-
ern region. Each of these regions is being studied systematically,
and is the subject of a separate publication; for example, the re-
sults of our efforts in optimizing the western region have been
published,13 and work on the eastern region is in progress.
Following an approach similar to that previously reported,11 our
synthetic strategy involved utilizing the diastereomeric tosylate
intermediate 4, and generating analogs by modification in the last
synthetic step. As shown in Scheme 1, the arylthio and alkylthio
analogs of 3 (namely, 5–19) were synthesized by a nucleophilic
displacement reaction on tosylate 4 using the appropriately substi-
tuted thiophenol or alkanethiol in acetone/Cs2CO3 in a manner
similar to that previously performed using acetone/K2CO3.11 Aryl-
oxy analogs of 3 (namely, 20–29) were also synthesized by nucle-
Figure 2. Topological representation of the azalanstat molecule 1.
ophilic displacement reactions on tosylate
4
using the
appropriately substituted phenol in N,N-dimethylformamide
(DMF)/Cs2CO3; higher reaction temperatures and longer reaction
times were required. In a similar approach, other northeastern-re-
gion analogs (namely, 30–37) were synthesized by nucleophilic
displacement reactions on tosylate 4 using various nucleophiles,
as outlined in Scheme 2. The hydroxy-terminated derivative 30
was obtained from 4 using potassium hydroxide/lithium hydroxide
in DMF. Similarly, treatment of 4 with sodium methoxide in DMF
gave the methoxy-containing derivative 37. The thiomethoxide
derivative 31 was prepared using sodium thiomethoxide in ace-
tone/Cs2CO3 in a manner similar to that previously performed
using acetone/K2CO3.11 Displacement by chloride to afford com-
pound 34 was achieved using lithium chloride in DMF.14 Displace-
ment by fluoride to afford compound 32 was achieved using tetra-
n-butylammonium fluoride in THF. The displacement reaction on
tosylate 4 using sodium azide in DMF15 afforded compound 35.
The azide 35 was fully reduced to the amine 36 (the chloro group
was also reduced) using ammonium formate and 10% Pd/C catalyst
in methanol (catalytic hydrogen transfer conditions) by the pub-
lished procedure.16 The diimidazole compound 33 was obtained
by treatment of 4 with an excess of imidazole in DMF.
tions to the northeastern (NE) region of azalanstat, namely, replac-
ing the aminothiophenol moiety by other substituted thiophenol
or substituted phenol moieties (see Fig. 3). For proper comparison
we chose to adopt only the (2R,4S) configuration of the dioxolane
ring system as a template, as this configuration was shown11 to
produce both potent and selective HO-1 inhibitors. Further inves-
tigations12 showed that the complete removal of the aminothio-
phenol moiety (replacement by hydrogen) in the NE region
produced a methyl-terminated series of compounds (including 2)
having enhanced selectivity for the inhibition of HO-1 relative to
HO-2. Thus, we also studied structure–activity relationships that
result from replacing the aminothiophenol moiety by smaller func-
tional groups such as halo, hydroxy, methoxy, thiomethoxy, cyano,
thiocyanato, azido, amino, and imidazolyl.
2. Results and discussion
2.1. Synthesis
In an effort to fully evaluate the importance that structural fea-
tures in the northeastern region have on HO-1 and HO-2 activities,
a simple synthetic strategy was devised to produce many substi-
tuted analogs of the parent compound 3, a compound that simply
contains a benzenethiol moiety in the northeastern region, an opti-
Since all of these reactions were performed on only one purified
diastereomeric tosylate, only one diastereomeric product was pro-
duced in each case; these imidazole-containing compounds were
isolated/characterized/evaluated in their hydrochloride or dihy-
Figure 3. General structure of northeastern region analogs and one such compound (3).
Scheme 1. Reagents and conditions: (a) ArSH (or RSH), Cs2CO3, acetone, reflux, 6 h; (b) ArOH, Cs2CO3, DMF, 90 °C, 8 h.

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
2463
Scheme 2. Reagents and conditions: (a) KOH, LiOH, DMF, 120 °C, 9 h; (b) NaSCH3, Cs2CO3, acetone, reflux, 6 h; (c) Bu4NF, THF, reflux, 18.5 h; (d) imidazole, DMF, 110 °C, 30 h;
(e) LiCl, DMF, 110 °C, 1 h; (f) NaN3, DMF, 110 °C, 2 h; (g) NH4OCOH, 10% Pd/C, CH3OH, reflux, 3 h; (h) NaOCH3, DMF, 120 °C, 7 h.
drochloride salt forms of the structures shown. Following these
strategies, 31 new, northeastern-region analogs were synthesized
and evaluated for their ability to inhibit HO-1 and HO-2.
thesizing many NE-region analogs because this approach had the
potential to identify inhibitors selective for HO-2. However, HO-2
selectivity was not achieved, as most of the compounds tested
were potent inhibitors of both HO-1 and HO-2, always showing
less activity toward HO-2 (Table 1). In contrast to the metallopor-
phyrins which show modest selectivity toward HO-2,20 all of the
imidazole–dioxolane compounds studied herein appear to be
selective for HO-1.
Our systematic investigation of analogs of 3 originally focused
on the modification of the benzenethiol moiety; incorporating
other substituted thiophenol appendages. As shown in Table 1,
nearly all of the compounds in the thiophenol-containing series ex-
hibit high inhibitory potency against HO-1 (13 out of the 16 com-
2.2. Biological evaluation
As described in Section 4, 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. 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 protein in the rat spleen and HO-2 protein in the rat brain,
has been documented.17–19,10 These particular microsomal prepa-
rations were selected in order to use the most native (i.e., closest
to in vivo) forms of HO, that is, those being most relevant to antic-
ipated whole-animal studies. We appreciate that pure forms of HO
can be obtained but these enzymes are membrane-free and trun-
cated. The data from biological screening assays are presented in
Table 1; the ratio of IC50 values measured for HO-2 and HO-1 for
each compound is shown in the fifth column, and is used as the in-
dex of selectivity for HO-1 compared to HO-2.
pounds exhibit IC50 values of less than 5 lM for HO-1). Although
the HO-2 inhibitory activity of these compounds is consistently
lower, only 5 of the 16 compounds (namely, 3, 5, 6, 16, and 17)
show selectivity indexes greater than 10. Although not as striking
as with our most-selective inhibitor to date (compound 2, selectiv-
ity index ꢀ381), a few of the new compounds show modest selec-
tivity for HO-1. For instance, representative activity–inhibitor
concentration curves showing the difference in potency toward
HO-1 (rat spleen) and HO-2 (rat brain) activity exhibited by com-
Previous work11 on azalanstat and its structural analogs
(including 5–7) 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. Further exploration12 revealed
that the aminothiophenol moiety is not an absolute requirement
for HO-inhibition because its removal enhanced the selectivity
for HO-1 inhibition (compound 2). We concluded that the amino-
thiophenol group (or other group in the analogous northeastern
position) may be an important factor for recognition in the HO-2
system. Thus, we chose to investigate this region further by syn-
pound 17 are shown in Figure 4. The IC50 was 0.9 0.1
lM for
HO-1 (rat spleen) and 30 M for HO-2 (rat brain), with a mod-
4
l
est selectivity index of 30. For comparison, representative activity–
inhibitor concentration curves showing the difference in potency
toward HO-1 (rat spleen) and HO-2 (rat brain) activity exhibited
by the essentially non-selective HO inhibitor 19 are shown in
Figure 5.
With respect to potency and selectivity, similar results (Table 1)
were noted with the analogous phenol-containing derivatives; 8 of
the 10 compounds exhibit IC50 values of less than 5 lM for HO-1,

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
Table 1
Inhibitory potency and selectivity of thiophenol- and phenol-containing imidazole–dioxolanes against the activity of HO-1 and HO-2a
X
Ar
N
o
o
N
Cl
Compound
X–Ar
IC50
(l
M) rat spleen (HO-1)
IC50
(
lM) rat brain (HO-2)
Selectivity index IC50 (HO-2)/IC50 (HO-1)
3
1.03 0.07
0.33 0.07b
34 12
33
S
5b
8
1b
20
10
2
NH2
S
H2N
6b
4
2b
42 28b
S
NH2
7b
4
2b
6
1b
S
S
8
25
5
69
7
8
2.8
4
N
9c
1.59 0.03
2.1 0.9
0.7 0.3
2.8 0.4
2.2 0.2
2
S
S
S
S
S
OH
Br
10
11
12
13
14
15
2.4 0.1
2.5 0.4
1.1
4
OCH3
Cl
12
5
5
4.3
2
4
F
6
2
19
16
2
8
3
S
S
NO2
CF3
2.1 0.6
7.6
N

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
2465
Table 1 (continued)
Compound
X–Ar
IC50
(
l
M) rat spleen (HO-1)
IC50
13
(
l
M) rat brain (HO-2)
Selectivity index IC50 (HO-2)/IC50 (HO-1)
16d
0.94 0.09
2
14
S
17
18
19
0.9 0.1
30
22
4
9
30
4
S
S
Br
5
6
2
1
Br
12.3 0.5
2
S
20
21
1.4 0.3
1.8 0.5
13
4
9.3
3.9
O
NH2
OH
7.1 0.7
O
22
23
24
25
> 100
> 100
—
O
O
0.59 0.04
3.5 0.2
0.28 0.01
1.6 0.3
2.7
6.3
2
22
8
O
O
Br
F
0.5 0.2
26
27
28
2
1
43
19
15
3
7
4
20
14
O
O
1.33 0.03
OCH3
9
3
2
O
O
I
29
0.67 0.02
1.7 0.2
2.5
CN
a
Assays were performed as indicated in Section 4. Data represent mean IC50 values standard deviation of replicate experiments. All imidazole–dioxolane compounds
were evaluated as the hydrochloride or dihydrochloride salt of the structure shown.
b
Biological data and compound synthesis originally published in Ref. 11; biological data included in this table for comparison.
Compound synthesis originally published in Ref. 29.
The cyclohexylsulfanyl-containing derivative 16 is included in this table for comparison.
c
d

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
Table 2
Inhibitory potency and selectivity of various imidazole–dioxolanes, having a small
substituent at the 4-position of the dioxolane ring, against the activity of HO-1 and
HO-2a
Z
N
o
o
N
Cl
Figure 4. Inhibition of HO-1 and HO-2 by a thionaphthyl-substituted imidazole–
dioxolane (compound 17). Enzyme activities were determined as described in
Section 4. Activity (ordinate) is expressed as a percentage of the control with no
inhibitor present, mean SD. The values on the abscissa represent the log of the
Compound
Z
IC50
rat spleen
(HO-1)
(
l
M)
IC50
brain (HO-2)
(
l
M) rat
Selectivity index IC50
(HO-2)/IC50 (HO-1)
2b
30
31
32
H
0.8 0.2b
305 25b
>100
ꢀ381b
>8
2
inhibitor concentration in
microsomes).
lM. s, HO-1 (rat spleen microsomes). j, HO-2 (rat brain
OH
SCH3
F
12
9
2
2
19
7
1.20 0.01
4.4 0.4
3.7
N
33
10
6
26
3
2.6
N
34
35
36c
37
Cl
N3
NH2
OCH3
3.5 0.1
3.6 0.2
122 30
38
>100
35
11
>5
1.9
5
21
3
1.73 0.01
3.3 0.9
a
Assays were performed as indicated in Section 4. Data represent mean IC50
values standard deviation of replicate experiments. All imidazole–dioxolane
compounds were evaluated as the hydrochloride or dihydrochloride salt of the
structure shown.
b
Biological data and compound synthesis originally published in Ref. 12; bio-
logical data included in this table for comparison.
c
The chlorine atom in the 4-position of the phenyl ring in the general structure is
replaced by a hydrogen atom.
Figure 5. Inhibition of HO by compound 19—lack of isozyme selectivity. Enzyme
activities were determined as described in Section 4. s, HO-1. j, HO-2.
but only 2 of the 10 compounds (namely, 26 and 27) show selectiv-
ity indices greater than 10. From this study, the effect of the
replacement of sulfur by oxygen on biological activity is not imme-
diately apparent. No trend in potency and/or selectivity is noticed
when comparing appropriate compound pairs such as 3/23, 9/21,
10/24, 11/27, and 13/25.
It appears that the addition of an extra aromatic ring system
(compared to that of 3) to the northeastern region (as in com-
pounds 17 and 26) is well tolerated, and even beneficial to attain
higher selectivity for HO-1. However, the system can be extended
too far, as noticed with the incorporation of the 4-adamantyl moi-
ety to produce compound 22, a compound inactive toward both
HO-1 and HO-2. It is interesting to note that, of the compounds
in the thiophenol/phenol series, 4 of the 6 most selective com-
pounds (namely, 3, 16, 17, and 26) all contain hydrocarbon moie-
ties in the northeastern region (as in the case of our most-
selective inhibitor 2). Thus, further modification to include more
non-polar functionality in the NE-region may be worth exploring.
The compounds in Table 2 possess more-dramatic changes to
the azalanstat skeleton in the NE region, and represent our efforts
at the incorporation of relatively small, polar functionality to this
area. Although the drastic structural changes did have significant
impact on activity/selectivity, we observed similar results as with
the thiophenol/phenol-containing derivatives; the compounds
are HO-1 selective, with a few derivatives showing modest selec-
tivity. For instance, representative activity–inhibitor concentration
curves showing a difference in potency toward HO-1 (rat spleen)
and HO-2 (rat brain) activity exhibited by compound 34 are shown
Figure 6. HO isozyme selectivity exhibited by
a compound having a small
substituent at the 4-position of the dioxolane ring. Representative activity–
inhibitor concentration curves for the inhibition of HO-1 and HO-2 by compound
34. Enzyme activities were determined as described in Section 4. s, HO-1. j, HO-2.
of 35. Because of the wide variety of functionality incorporated
into these series of compounds, structure–activity relationships
are difficult to formulate. Nonetheless, most of the compounds in
Table 2 have lower potency against both HO-1 and HO-2 compared
to the compounds in Table 1 which have larger groups in the
northeastern region. As with all of the imidazole–dioxolane com-
pounds studied thus far, HO-1 selectivity is apparent; the inhibi-
tors having smaller groups in the northeastern region show mild
to modest selectivity, although never achieving the isozyme-spe-
cific characteristic of compound 2. The thiomethyl-containing ana-
log 31 and the diimidazole compound 33 show little preference for
HO-1 inhibition, while the halogen-containing derivatives 32 and
34 are quite selective.
in Figure 6. The IC50 was 3.5 0.1
lM for HO-1 (rat spleen) and
122 30 M for HO-2 (rat brain), with a modest selectivity index
l

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
2467
We have addressed also the issue of selectivity with respect to
other hemoproteins such as cytochromes P45021 (CYP). The in vitro
screening of our compounds for the inhibition of CYP is necessary
because cytochromes P450 are responsible for the metabolism of
many drugs used in therapy and as experimental tools. As shown
in Table 3, all of the 11 compounds tested exhibit high inhibitory
Table 3
Inhibition of CYP2E1 and CYP3A1/3A2 by various imidazole–dioxolanesa
Z
Figure 7. Representative activity–inhibitor concentration curves for the inhibition
of CYP2E1 and CYP3A1/3A2 by compound 8—compound 8 inhibited CYP3A1/3A2
but not CYP2E1. Enzyme activities were determined as described in Section 4. s,
CYP2E1. j, CYP3A1/3A2.
N
o
o
N
Cl
potency against CYP3A1/3A2. Interestingly, with the exception of
compound 32, none of the compounds were inhibitors of CYP2E1.
It is noteworthy that the majority of compounds in Table 3
have large appendages in the NE region and did not inhibit CYP2E1,
whereas compound 32 contains only a relatively small F substitu-
ent in this region, and did inhibit CYP2E1. These results are consis-
tent with our previous investigations,10,12 in that analogs devoid of
large NE-region substituents were potent inhibitors of CYP2E1.
Thus, for compounds having large NE-region substituents,
although there was a low selectivity for HO-1 over HO-2, there
was a noticeable selectivity for CYP3A1/3A2 over CYP2E1. For in-
stance, representative activity–inhibitor concentration curves in
Compound
Z
IC50
(CYP2E1)
(
l
M)
IC50
3A2)
(lM) (CYP3A1/
3
>100
2
7
1
4
S
8
9
>100
>100
>100
>100
>100
>100
>100
>100
S
S
S
S
N
Figure
(IC50 > 100
7
showed
a
difference in potency toward CYP2E1
M) activity by
lM) and CYP3A1/3A2 (IC50
7
4 l
compound 8. We plan to continue our study of selectivity to in-
clude other hemoenzymes, namely, soluble guanylyl cyclase
(sGC) and nitric oxide synthase (NOS); we have reported
previously12 that similar imidazole–dioxolane compounds
(analogs of 2) are extremely weak inhibitors of sGC and neuronal
NOS.
Most of our synthetic modifications to the NE region led to com-
pounds active against both HO-1 and HO-2 but lacking strikingly
high selectivity. Inhibitors with high potency against HO-2 were al-
ways even more potent against HO-1. Thus, at least for this diaste-
reomeric series, it seems unlikely that HO-2 selectivity will be
achieved by such modifications in this region. Conversely, coupled
with our knowledge12 that removal of the northeastern portion
leads to highly selective HO-1 inhibitors, it seems unlikely that ex-
tremely selective inhibitors of HO-1 will be developed from simple
modifications to appendages in the NE region (the hydrocarbon
substituents appear to be an exception, and may be worthwhile
exploring).
1.4 0.2
2.1 0.4
2.8 0.8
1.8 0.4
2.0 0.8
3.4 0.6
OH
Br
10
16
17
21
24
29
S
O
OH
Br
2.3. Modeling of HO–inhibitor complexes
In an effort to rationalize some of our experimental observations,
amodelingstudywasperformedbasedon theX-raycrystallographic
structural data of hHO-1 in complex with analogous inhibitors. We
have previously published22 the structure of hHO-1 in which azalan-
stat had been docked (AUTODOCK version 3.0) to show a secondary
hydrophobic pocket into which the northeastern region of the mol-
ecule fit. Using this model as a guide, inhibitor 3 was placed (CCP4:
O
O
6.9 0.5
2.2 0.6
CN
XFit)
in
the
inhibitor
binding
site
of
hHO-1
31
32
SCH3
F
>100
(Fig. 8A). The structure of hHO-1 utilized was based on the X-ray
crystallographic structure of hHO-1 in complex with 1-(adaman-
tan-1-yl)-2-(1H-imidazol-1-yl)ethanone (PDB 3CZY) from which
the inhibitor had been omitted. This template was the same as that
utilized for the original automated docking of azalanstat to hHO-1.
3.6 0.2
4
1
a
Assays were performed as indicated in Section 4. Data represent mean IC50
values standard deviation of replicate experiments. All of the imidazole–dioxo-
lane compounds were evaluated as the hydrochloride or dihydrochloride salts of
the structure shown.

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
tutions in hHO-2 would result in a larger hydrophobic pocket (see
Fig. 8). Thus, the shorter residues in hHO-2 would be farther away
from the phenyl group in the northeastern region of inhibitor 3. For
example, replacement of the Leu213 in hHO-1 by the correspond-
ing Ile of hHO-2 increases the distance between the phenyl ring of
inhibitor 3 and the residue from 3.6 Å to 4.4 Å. Replacement of
Val50 by Ala would also increase the distance between residue
and inhibitor by ꢀ0.8 Å (3.1–3.9 Å). Although the distance from
the Ser53 of hHO-1 to inhibitor 3 would be similar if substituted
with Ala (4.5 Å), the hydroxyl group of Ser53 in the hHO-1 model
would be close enough to be within van der Waals contact of the
inhibitor if it were rotated toward the hydrophobic pocket. More-
over, this may also provide a site for hydrogen-bond interactions
with a suitable inhibitor, a situation which would not be present
in hHO-2. The replacement of Met34 by Val would increase the dis-
tance to the phenyl group in the northeastern region of compound
3 by ꢀ2 Å; the distance between this residue and the phenyl moi-
ety increased from 4.4 to 6.7 Å. In general, the slightly larger, sec-
ondary hydrophobic pocket of hHO-2 may result in decreased
contacts with the northeastern portion of our inhibitors; this
may explain the general selectivity observed for HO-1 over HO-2
with these compounds.
As previously noted, some of the most selective compounds
for HO-1 in this series contain hydrocarbon moieties in the
northeastern
region.
Conversely,
compounds
having
electronegative substituents in this region would impart a nega-
tive-charge distribution in an area (the secondary hydrophobic
pocket) which is generally hydrophobic; accordingly, a general
decrease in potency was observed with each of HO-1 and HO-
2 for most compounds not containing non-polar moieties in the
northeastern region. Although one can infer, based on the previ-
ous X-ray crystallographic structures of the native24,25 and
inhibitor-bound HO-1 (both rat26 and human22), that there
may be some flexibility in the secondary hydrophobic pocket,
there are length limitations. For example, compound 22 pos-
sesses an extended northeastern region that contains, in addition
to the phenyl group, a bulky adamantyl terminus. This group is
clearly too large and bulky to fit into the pocket owing to steric
hindrance, an observation reflected by the lack of inhibitory
activity toward HO we observed using this compound.
It should be noted, that the hHO crystal structures solved to
date do not represent the full-length enzymes found in vivo.23–25
Both proteins contain a membrane-binding tail, while hHO-2 also
contains three heme-regulatory motifs which have been removed
for crystallization.23,27 One should not overlook the possibility that
these absent regions may have some influence on the binding/po-
tency of inhibitors.
Figure 8. (A) Model of inhibitor 3 (magenta) in complex with hHO-1. (B) Model of
inhibitor 3 (magenta) in complex with hHO-1 in which the residues involved in
inhibitor binding have been changed to those of hHO-2. The modified residues are
indicated and labeled accordingly. Heme is colored orange.
For comparison with the hHO-2 system, residues within the inhibi-
tor binding site of hHO-1 were mutated to those of hHO-223 (i.e.,
Met34?Val, Leu213?Ile, Ser53?Ala, Val50?Ala, Met111?Ala,
Ile211?Met, Ala31?Thr, Gln38?Leu, Phe167?Tyr, and Met51?
Thr, Leu138?Met) ( Fig. 8B). Since the structure of the inhibitor
compounds in this study differ only in the northeastern region, only
residues in the putative secondary hydrophobic pocket were
analyzed.
Previous crystallographic studies22 have demonstrated that
when hHO-1 binds an inhibitor, there is substantial movement in
the distal helix as well as, to a lesser extent, in the proximal pocket
to open up the heme-binding pocket in order to accommodate the
inhibitor. Indeed, the presence of the putative secondary hydro-
phobic pocket was apparent only in the crystal structure of hHO-
1 in complex with the bulky 1-(adamantan-1-yl)-2-(1H-imidazol-
1-yl)ethanone22 and not in the native structure,24,25 or in the struc-
ture of rat HO-1 in complex with 2-[2-(4-chlorophenyl)ethyl]-2-
[(1H-imidazol-1-yl)methyl]-1,3-dioxolane.26 Thus, the heme-bind-
ing pocket of the native (truncated) hHO-1 structure could not be
used in our modeling studies as the pocket was not large enough.
Given that the structures of hHO-1 and hHO-2 are so similar, we
anticipate that hHO-2 would have to undergo similar changes in
structure (i.e., expansion) to accommodate the inhibitor. Since an
X-ray crystallographic structure of hHO-2 in complex with an
inhibitor is unavailable, we changed residues in the inhibitor-
bound hHO-1 binding pocket with residues of hHO-2 to effectively
approximate the inhibitor-bound binding pocket of hHO-2. With
respect to the residues that differ between hHO-1 and hHO-2 in
the secondary hydrophobic pocket, it would appear that the substi-
3. Conclusions
We have synthesized several compounds that express moder-
ate selectivity as inhibitors of HO-1 relative to HO-2. In particular,
compounds 2, 3, 5, 6, 16, 17, 26, 27, 34, and 35 are noteworthy
for moderately high potency and selectivity toward HO-1. In addi-
tion, we have synthesized potent but non-selective inhibitors of
HO-2 such as 10, 11, 23, 25, 29, 32, and 37. Our systematic inves-
tigation of the northeastern section of the lead compound azalan-
stat has shed light on our overall regional study, suggesting that
we focus our modifications elsewhere. Although synthetically
demanding, these compounds are anticipated to become useful
tools in elucidating the physiological roles of HO and carbon
monoxide in mammalian and other biological systems, as regards
both HO-1 and HO-2. These novel compounds might also have
useful therapeutic applications, either alone or in conjunction
with other novel agents.

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
2469
night. The solid was removed by filtration and washed with Et2O.
4. Experimental
4.1. General
High-vacuum drying left 3 (150 mg, 0.33 mmol, 89%) as a white
solid: mp 134–135 °C; Rf = 0.24 (EtOAc); ½a D23
ꢃ8.2 (c 1.7 in
ꢂ
CD3OD); 1H NMR (400 MHz, CD3OD): d 1.97 (t, J = 8.4 Hz, 2H),
2.66–2.84 (m, 2H), 3.09 (dd, J = 13.8, 5.8 Hz, 1H), 3.19 (dd,
J = 14.0, 4.8 Hz, 1H), 3.66–3.75 (m, 2H), 4.00–4.90 (m, 1H), 4.45
(s, 2H), 7.16 (d, J = 8.4 Hz, 2H), 7.20–7.42 (m, 7H), 7.50 (br s,
1H), 7.58 (br s, 1H), 8.92 (br s, 1H); 13C NMR (100 MHz, CD3OD):
d 29.8, 36.7, 39.2, 54.7, 70.9, 78.0, 109.9, 120.5, 125.0, 127.7,
129.5, 130.2, 130.9, 131.0, 132.8, 136.8, 137.7, 141.4; HRMS
(ESI) [MꢃCl]+ Calcd for C22H24ClN2O2S: 415.1247. Found:
415.1233. Anal. Calcd for C22H24Cl2N2O2S: C, 58.54; H, 5.36; N,
6.21. Found: C, 58.44; H, 5.28; N, 6.06.
The 1H and 13C NMR spectra were recorded on a Bruker
Avance 400 MHz spectrometer in CD3OD or D2O. The signals ow-
ing to residual protons in the deuterated solvents were used as
internal standards in the measurement of 1H NMR spectra.
Chemical shifts (d) are reported in ppm downfield from tetra-
methylsilane.28 Carbon chemical shifts are given relative to
CD3OD: d = 49.00 or CDCl3: d = 77.16. High-resolution electro-
spray 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
4.4. Characterization of the new compounds synthesized
following the representative procedure described above for
compound 3, as outlined in Scheme 1
flow rate of 6 lL/min. Elemental analyses were performed by
MHW Laboratories (Phoenix, AZ, USA). Melting points were
determined on a Mel-Temp II melting point apparatus and are
uncorrected. Optical rotations were measured using an AutopolÒ
II automatic polarimeter for solutions in a 1-dm cell at rt. Col-
umn chromatography was performed on Silicycle silica gel
(230–400 mesh, 60 Å). Analytical thin-layer chromatography
was performed using glass- or aluminum-backed Silica Gel 60
F254 plates (Silicycle, Quebec City, QC, Canada). Plates were
viewed under UV light or by charring after spraying with phos-
phomolybdic acid (PMA) in EtOH.
4.4.1. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-[(pyridin-4-ylsulfanyl)methyl]-1,3-dioxolane
dihydrochloride (8)
Hygroscopic white solid in 76% yield from 411: ½a D24
ꢂ
+22.3 (c 2.0
in CD3OD); 1H NMR (400 MHz, CD3OD): d 1.93–2.05 (m, 2H), 2.65–
2.82 (m, 2H), 3.45 (dd, J = 14.2, 5.8 Hz, 1H), 3.64 (dd, J = 14.8, 4.8
Hz, 1H), 3.87 (t, J = 8.2 Hz, 1H), 4.00–4.10 (m, 1H), 4.19 (dd,
J = 8.6, 6.2 Hz, 1H), 4.47 (s, 2H), 7.14 (d, J = 8.4 Hz, 2H), 7.26 (d,
J = 8.4 Hz, 2H), 7.59 (t, J = 1.6 Hz, 1H), 7.65 (t, J = 1.6 Hz, 1H), 7.90
(d, J = 7.2 Hz, 2H), 8.50 (d, J = 6.4 Hz, 2H), 8.99 (s, 1H); 13C NMR
(100 MHz, CD3OD): d 29.8, 33.8, 38.7, 54.3, 70.2, 76.7, 110.3,
120.7, 123.9, 125.1, 129.6, 130.9, 132.9, 137.8, 140.7, 141.1,
165.9; HRMS (ESI) [MꢃCl]+ Calcd for C21H23ClN3O2S: 416.1199.
Found: 416.1183.
4.2. Materials
Dexamethasone-5 was obtained from Vetoquinol Canada Inc,
Lavaltrie, QC, Canada. Erythromycin, p-nitrophenol, b-NADPH,
ethylenediamine tetraacetic acid disodium salt (EDTA), and 70%
perchloric acid were obtained from Sigma Chemical Company, St.
Louis, MO, USA. Acetone, glycerol, ammonium acetate, glacial ace-
tic acid, KCl, KH2PO4, and KOH were obtained from Fisher Scientific
Ltd, Ottawa, ON, Canada. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-
[(1H-imidazol-1-yl)methyl]-4-[(p-toluenesulfonyloxy)methyl]-1,
3-dioxolane (4) and compounds 5–7 were prepared according to
the published procedures.11 The synthesis of compound 9 has been
published in a previous communication.29 All other chemical re-
agents were obtained from Sigma–Aldrich and used without prior
purification.
4.4.2. (2R,4S)-4-[(4-Bromophenylsulfanyl)methyl]-2-[2-(4-
chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-
dioxolane hydrochloride (10)
White solid in 96% yield from 411: mp 141–142 °C; Rf = 0.23
(EtOAc); ½a 2D2
ꢂ
ꢃ4.5 (c 0.9 in CD3OD); 1H NMR (400 MHz, CD3OD):
d 1.95 (t, J = 8.4 Hz, 2H), 2.64–2.80 (m, 2H), 3.13 (dd, J = 14.0, 5.6
Hz, 1H), 3.19 (dd, J = 14.0, 5.2 Hz, 1H), 3.69–3.79 (m, 2H), 4.01–
4.09 (m, 1H), 4.45 (s, 2H), 7.14 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4
Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.52 (br s,
1H), 7.59 (br s, 1H), 8.92 (br s, 1H); 13C NMR (100 MHz, CD3OD):
d 29.8, 36.6, 39.1, 54.6, 70.8, 78.0, 110.0, 120.6, 121.2, 125.1,
129.6, 131.0, 132.3, 132.9, 133.2, 136.6, 137.8, 141.3; HRMS (ESI)
[MꢃCl]+ Calcd for C22H23BrClN2O2S: 493.0352. Found: 493.0336.
Anal. Calcd for C22H23BrCl2N2O2S: C, 49.83; H, 4.37; N, 5.28. Found:
C, 50.52; H, 4.50; N, 4.66.
4.3. Representative procedure for the displacement of the p-
toluenesulfonyloxy group in compound 4 using thiophenol-
containing nucleophiles
4.3.1. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-[(phenylsulfanyl)methyl]-1,3-dioxolane
hydrochloride (3)
Under a N2 atmosphere, a mixture of (2R,4S)-2-[2-(4-chloro-
phenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[(p-toluenesulfony-
loxy)methyl]-1,3-dioxolane (4)11 (178 mg, 0.37 mmol), benzene-
thiol (82 mg, 0.74 mmol, 2 equiv), and cesium carbonate (241
mg, 0.74 mmol, 4 equiv) in acetone (7 mL) was heated at reflux
temperature with stirring for 6 h. The solids were removed by
filtration, and washed with hot acetone and then with hot ethyl
acetate. The filtrate was concentrated, and the residue (Rf ꢁ 0.2
in EtOAc) purified by flash chromatography on silica gel (EtOAc)
to give the free base (150 mg, 0.36 mmol, 98%) as an oil. To a
solution of the oil in warm 2-propanol (2 mL) was added a solu-
tion of 37% aqueous HCl (50 mg, 0.51 mmol, 1.4 equiv) in 2-pro-
panol (2 mL). The mixture was concentrated and dried under
high vacuum. The residue was dissolved in 2-propanol (0.5
mL), the solution cooled in the freezer, and then a few drops
of Et2O were added and the product allowed to crystallize over-
4.4.3. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-[(4-methoxyphenylsulfanyl)methyl]-1,3-
dioxolane hydrochloride (11)
Beige solid in 73% yield from 411: mp 139–140 °C; Rf = 0.16
(EtOAc);
½
a 2D2
ꢂ
ꢃ9.2 (c 0.9 in CD3OD); 1H NMR (400 MHz,
CD3OD): d 1.95 (t, J = 8.4 Hz, 2H), 2.64–2.78 (m, 2H), 2.93 (dd,
J = 13.8, 5.8 Hz, 1H), 3.07 (dd, J = 13.8, 5.0 Hz, 1H), 3.59–3.68
(m, 2H), 3.79 (s, 3H), 3.98–4.05 (m, 1H), 4.43 (s, 2H), 6.89 (d,
J = 8.8 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H),
7.36 (d, J = 8.4 Hz, 2H), 7.51 (br s, 1H), 7.58 (br s, 1H), 8.89
(br s, 1H); 13C NMR (100 MHz, CD3OD): d 29.8, 38.9, 39.2,
54.7, 55.8, 71.0, 78.4, 109.8, 115.8, 120.7, 125.0, 126.6, 129.5,
131.0, 132.8, 134.8, 137.8, 141.4, 161.0; HRMS (ESI) [MꢃCl]+
Calcd for C23H26ClN2O3S: 445.1353. Found: 445.1362. Anal. Calcd
for C23H26Cl2N2O3S: C, 57.38; H, 5.44; N, 5.82. Found: C, 57.68;
H, 5.73; N, 6.06.

2470
J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
4.4.4. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-4-[(4-chlorophenyls-
ulfanyl)methyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
hydrochloride (12)
484.1073. Found: 484.1056. Anal. Calcd for C22H22Cl2F3N3O2S: C,
50.78; H, 4.26; N, 8.07. Found: C, 50.59; H, 4.26; N, 7.97.
White solid in 80% yield from 411: mp 128–129 °C; Rf = 0.20
4.4.8. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-4-[(cyclohexylsulfanyl)
methyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
hydrochloride (16)
(EtOAc); ½a 2D2
ꢂ
ꢃ5.2 (c 0.8 in CD3OD); 1H NMR (400 MHz, CD3OD):
d 1.96 (t, J = 8.4 Hz, 2H), 2.64–2.81 (m, 2H), 3.12 (dd, J = 14.2, 5.4
Hz, 1H), 3.19 (dd, J = 14.0, 5.2 Hz, 1H), 3.69–3.78 (m, 2H), 4.01–
4.09 (m, 1H), 4.45 (s, 2H), 7.15 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4
Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.52 (br s,
1H), 7.59 (br s, 1H), 8.92 (br s, 1H); 13C NMR (100 MHz, CD3OD):
d 29.8, 36.8, 39.1, 54.6, 70.8, 78.0, 109.9, 120.6, 125.1, 129.6,
130.2, 131.0, 132.2, 132.9, 133.5, 135.9, 137.8, 141.3; HRMS (ESI)
[MꢃCl]+ Calcd for C22H23Cl2N2O2S: 449.0857. Found: 449.0851.
Anal. Calcd for C22H23Cl3N2O2S: C, 54.39; H, 4.77; N, 5.77. Found:
C, 54.53; H, 4.71; N, 5.64.
White solid in 81% yield from 411: mp 172–173 °C; Rf = 0.36
(free base, EtOAc); ½a D23
ꢂ
ꢃ18.1 (c 0.4 in CD3OD); 1H NMR (400
MHz, CD3OD): d 1.20–1.40 (m, 5H), 1.58–1.68 (m, 1H), 1.71–1.82
(m, 2H), 1.90–2.00 (m, 2H), 2.00 (dd, J = 9.2, 7.6 Hz, 2H), 2.62–
2.73 (m, 2H), 2.73–2.86 (m, 3H), 3.68–3.76 (m, 2H), 4.04–4.12
(m, 1H), 4.47 (s, 2H), 7.21 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz,
2H), 7.59 (ꢀt, J = 1.6 Hz, 1H), 7.64 (ꢀt, J = 1.6 Hz, 1H), 8.96 (br s,
1H); 13C NMR (100 MHz, CD3OD): d 26.9, 27.0, 29.8, 33.0, 34.8,
34.9, 39.2, 45.3, peak under solvent, 54.7, 71.1, 79.1, 109.8, 120.6,
125.1, 129.6, 131.0, 132.9, 137.8, 141.4; HRMS (ESI) [MꢃCl]+ Calcd
for C22H30ClN2O2S: 421.1716. Found: 421.1698. Anal. Calcd for
C22H30Cl2N2O2S: C, 57.76; H, 6.61; N, 6.12; S, 7.01. Found: C,
58.11; H, 6.70; N, 6.13; S, 6.79.
4.4.5. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-4-[(4-fluorophenyl-
sulfanyl)methyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
hydrochloride (13)
Hygroscopic beige solid in 70% yield from 411: mp 112–113 °C;
Rf = 0.25 (EtOAc); ½a D24
ꢂ
ꢃ9.4 (c 1.9 in CD3OD); 1H NMR (400 MHz,
4.4.9. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-[(naphthalen-2-ylsulfanyl)methyl]-1,3-dioxolane
hydrochloride (17)
CD3OD): d 1.96 (t, J = 8.4 Hz, 2H), 2.65–2.80 (m, 2H), 3.05 (dd,
J = 14.0, 5.6 Hz, 1H), 3.15 (dd, J = 13.8, 5.0 Hz, 1H), 3.64–3.74 (m,
2H), 4.01–4.09 (m, 1H), 4.46 (s, 2H), 7.07 (ꢀt, J = 8.8 Hz, 2H), 7.17
(d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 7.44 (ꢀdd, J = 8.8, 5.2
Hz, 2H), 7.53 (br s, 1H), 7.60 (br s, 1H), 8.93 (br s, 1H); 13C NMR
White solid in 90% yield from 411: mp 156–157 °C; Rf = 0.26
(free base, EtOAc); ½a D24
ꢂ
ꢃ7.4 (c 0.5 in CD3OD); 1H NMR (400
MHz, CD3OD): d 1.94 (t, J = 8.4 Hz, 2H), 2.62–2.78 (m, 2H), 3.19–
3.34 (m, 2H), 3.73–3.83 (m, 2H), 4.07 (dd, J = 7.2, 4.8 Hz, 1H),
4.43 (s, 2H), 7.08 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 7.38
(ꢀt, J = 1.6 Hz, 1H), 7.43–7.53 (m, 3H), 7.56 (ꢀt, J = 1.6 Hz, 1H),
7.75–7.88 (m, 4H), 8.90 (br s, 1H); 13C NMR (100 MHz, CD3OD): d
29.8, 36.5, 39.2, 54.6, 70.9, 78.2, 109.9, 120.4, 125.0, 127.1, 127.9,
128.2, 128.6, 128.7, 128.8, 129.5, 129.7, 130.9, 132.8, 133.5,
134.4, 135.2, 137.7, 141.3; HRMS (EI) [MꢃHCl]+ Calcd for
C26H25ClN2O2S: 464.1325. Found: 464.1347. Anal. Calcd for
C26H26Cl2N2O2S: C, 62.27; H, 5.23; N, 5.59. Found: C, 62.16; H,
5.05; N, 5.53.
(100 MHz, CD3OD): d 29.8, 38.0, 39.1, 54.6, 70.9, 78.1, 109.9,
4
117.1 (d, 2JC–F = 22.2 Hz), 120.6, 125.1, 129.5, 131.0, 132.0 (d, JC–F
=
3
3.6 Hz), 132.8, 134.0 (d, JC–F = 8.0 Hz), 137.8, 141.3, 163.5 (d,
1
1JC–F = 44.5 Hz); 19F NMR (376 MHz, CD3OD): d ꢃ118.1 (t, JF–C
=
6.6 Hz); HRMS (ESI) [MꢃCl]+ Calcd for C22H23ClFN2O2S: 433.1153.
Found: 433.1154. Anal. Calcd for C22H23Cl2FN2O2S: C, 56.29; H,
4.94; N, 5.97. Found: C, 56.12; H, 5.04; N, 6.02.
4.4.6. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-[(4-nitrophenylsulfanyl)methyl]-1,3-dioxolane
hydrochloride (14)
Hygroscopic yellow solid in 55% yield from 411: mp moistens at
4.4.10. (2R,4S)-4-[(3-Bromophenylsulfanyl)methyl]-2-[2-(4-
chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
hydrochloride (18)
70 °C; Rf = 0.14 (EtOAc); ½a D23
ꢂ
+8.9 (c 0.7 in CD3OD); 1H NMR (400
MHz, CD3OD): d 1.97 (t, J = 8.4 Hz, 2H), 2.64–2.81 (m, 2H), 3.30–
3.41 (m, 2H), 3.80 (t, J = 8.2 Hz, 1H), 3.84–3.92 (m, 1H), 4.12 (dd,
J = 8.0, 5.6 Hz, 1H), 4.45 (s, 2H), 7.12 (d, J = 8.4 Hz, 2H), 7.24 (d,
J = 8.4 Hz, 2H), 7.52 (d, J = 9.2 Hz, 2H), 7.54 (br s, 1H), 7.62 (br s,
1H), 8.14 (d, J = 8.8 Hz, 2H), 8.94 (br s, 1H); 13C NMR (100 MHz,
CD3OD): d 29.8, 34.6, 39.0, 54.5, 70.6, 77.5, 110.1, 120.7, 124.9,
125.1, 128.1, 129.5, 130.9, 132.9, 137.8, 141.2, 146.8, 147.9; HRMS
(ESI) [MꢃCl]+ Calcd for C22H23ClN3O4S: 460.1098. Found:
460.1075. Anal. Calcd for C22H23Cl2N3O4S: C, 53.23; H, 4.67; N,
8.46. Found: C, 53.09; H, 4.70; N, 8.23.
White solid in 90% yield from 411: mp 128–129 °C; Rf = 0.32
(free base, EtOAc); ½a D23
ꢂ
ꢃ6.7 (c 0.7 in CD3OD); 1H NMR (400
MHz, CD3OD): d 1.96 (t, J = 8.6 Hz, 2H), 2.65–2.79 (m, 2H), 3.14–
3.25 (m, 2H), 3.70–3.82 (m, 2H), 4.07 (dd, J = 7.2, 4.8 Hz, 1H),
4.46 (s, 2H), 7.14 (d, J = 8.0 Hz, 2H), 7.19–7.25 (m, 1H), 7.26 (d,
J = 8.4 Hz, 2H), 7.33–7.39 (m, 2H), 7.54 (br s, 1H), 7.56 (t, J = 1.8
Hz, 1H), 7.60 (br s, 1H), 8.93 (br s, 1H); 13C NMR (100 MHz,
CD3OD): d 29.8, 36.2, 39.1, 54.6, 70.7, 78.0, 110.0, 120.6, 123.8,
125.1, 129.0, 129.5, 130.4, 131.0, 131.7, 132.6, 132.9, 137.8,
139.9, 141.3; HRMS (ESI) [MꢃCl]+ Calcd for C22H23BrClN2O2S:
493.0352. Found: 493.0333. Anal. Calcd for C22H23BrCl2N2O2S: C,
49.83; H, 4.37; N, 5.28. Found: C, 49.95; H, 4.58; N, 5.19.
4.4.7. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-[{(5-trifluoromethyl)pyridin-2-
ylsulfanyl}methyl]-1,3-dioxolane hydrochloride (15)
4.4.11. (2R,4S)-4-[(2-Bromophenylsulfanyl)methyl]-2-[2-(4-
chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-
dioxolane hydrochloride (19)
White solid in 90% yield from 411: mp 156–157 °C; ½a 2D0
ꢃ24.2 (c
ꢂ
0.8 in CD3OD); 1H NMR (400 MHz, CD3OD): d 2.00 (t, J = 8.4 Hz, 2H),
2.69–2.88 (m, 2H), 3.48 (dd, J = 14.0, 6.0 Hz, 1H), 3.57 (dd, J = 14.0,
5.2 Hz, 1H), 3.77 (t, J = 8.4 Hz, 1H), 3.88–3.97 (m, 1H), 4.08 (dd,
J = 8.4, 6.0 Hz, 1H), 4.47 (ꢀs, 2H), 7.19 (d, J = 8.4 Hz, 2H), 7.27 (d,
J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 1H), 7.54 (br s, 1H), 7.62 (br s,
1H), 7.85 (dd, J = 8.6, 2.2 Hz, 1H), 8.68 (br s, 1H), 8.94 (br s, 1H);
13C NMR (100 MHz, CD3OD): d 29.8, 32.3, 39.1, 54.6, 70.6, 77.7,
Hygroscopic white solid in 100% yield from 411: Rf = 0.28 (free
base, EtOAc); ½a D22
ꢂ
ꢃ14.4 (c 0.6 in CD3OD); 1H NMR (400 MHz,
CD3OD): d 1.97 (t, J = 8.6 Hz, 2H), 2.67–2.80 (m, 2H), 3.14–3.25
(m, 2H), 3.73–3.80 (m, 2H), 4.04–4.11 (m, 1H), 4.46 (s, 2H), 7.11
(ꢀtd, J = 7.8, 1.6 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4
Hz, 2H), 7.34 (ꢀtd, J = 7.6, 1.2 Hz, 1H), 7.44 (dd, J = 8.0, 1.6 Hz,
1H), 7.52 (br s, 1H), 7.57 (dd, J = 8.0, 1.2 Hz, 1H), 7.60 (br s, 1H),
8.92 (br s, 1H); 13C NMR (100 MHz, CD3OD): d 29.8, 35.8, 39.2,
54.6, 70.8, 77.7, 110.0, 120.7, 124.9, 125.1, 128.5, 129.2, 129.5,
130.4, 131.0, 132.8, 134.2, 137.8, 138.3, 141.3; HRMS (ESI) [MꢃCl]+
2
109.9, 120.6, 123.2, 123.9 (d, JC–F = 33.2 Hz), 125.1, 125.3 (q,
3
1JC–F = 271.0 Hz), 129.6, 130.9, 132.9, 134.4 (d, JC–F = 3.5 Hz),
3
137.8, 141.3, 147.2 (d, JC–F = 3.9 Hz), 164.6; 19F NMR (376 MHz,
CD3OD): d ꢃ64.6; HRMS (ESI) [MꢃCl]+ Calcd for C22H22ClF3N3O2S:

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
2471
Calcd for C22H23BrClN2O2S: 493.0352. Found: 493.0343. Anal. Calcd
for C22H23BrCl2N2O2S: C, 49.83; H, 4.37; N, 5.28. Found: C, 49.66; H,
4.47; N, 5.08.
d 2.03 (t, J = 8.6 Hz, 2H), 2.70–2.87 (m, 2H), 3.88–4.12 (m, 5H), 4.51
(s, 2H), 6.70 (d, J = 8.8 Hz, 2H), 6.75 (d, J = 9.2 Hz, 2H), 7.15 (d,
J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.58 (ꢀt, J = 1.6 Hz, 1H),
7.65 (ꢀt, J = 1.6 Hz, 1H), 8.97 (br s, 1H); 13C NMR (100 MHz,
CD3OD): d 29.8, 39.1, 54.7, 68.3, 69.4, 77.7, 110.0, 116.8, 116.9,
120.6, 125.2, 129.5, 131.0, 132.8, 137.8, 141.5, 152.9, 153.3; HRMS
(ESI) [MꢃCl]+ Calcd for C22H24ClN2O4: 415.1425. Found: 415.1407.
Anal. Calcd for C22H24Cl2N2O4: C, 58.54; H, 5.36; N, 6.21. Found: C,
58.50; H, 5.47; N, 6.13.
4.5. Representative procedure for the displacement of the p-
toluenesulfonyloxy group in compound 4 using phenol-
containing nucleophiles
4.5.1. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-(phenoxymethyl)-1,3-dioxolane hydrochloride
(23)
4.6.3. (2R,4S)-4-[{(4-Adamantan-1-yl)phenoxy}methyl]-2-[2-
(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-
dioxolane hydrochloride (22)
Under a N2 atmosphere, a mixture of (2R,4S)-2-[2-(4-chloro-
phenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[(p-toluenesulfony-
loxy)methyl]-1,3-dioxolane (4)11 (100 mg, 0.21 mmol), phenol (79
mg, 0.84 mmol, 4 equiv), and cesium carbonate (205 mg, 0.63 mmol,
6 equiv) in N,N-dimethylformamide (3 mL) was heated at 90 °C with
stirring for 8 h. The mixture was diluted with H2O, extracted with
EtOAc (3ꢄ), and the combined organic extracts were washed
sequentially with a saturated aqueous solution of Na2CO3, and brine,
and then dried (MgSO4). The solution was concentrated, and the res-
idue was purified by flash chromatography on silica gel (EtOAc) to
give the free base (60 mg, 0.15 mmol) as an oil (Rf = 0.38, EtOAc).
To a solution of this oil in warm 2-propanol (2 mL) was added a solu-
tion of 37% aqueous HCl (34 mg, 0.35 mmol, 2.3 equiv) in 2-propanol
(2 mL). The mixture was concentrated and dried under high vacuum.
The residue was dissolved in 2-propanol (0.5 mL), the solution
cooled in the freezer, and then a few drops of Et2O were added
and the product allowed to crystallize overnight. The solid was re-
moved by filtration and washed with Et2O. High-vacuum drying
gave 23 (79 mg, 0.18 mmol, 86%) as a white solid: mp 139–140 °C;
White solid in 72% yield from 411: mp 132–134 °C; Rf = 0.22
(free base, EtOAc); ½a D22
ꢂ
ꢃ11.8 (c 0.6 in CD3OD); 1H NMR (400
MHz, CD3OD): d 1.74–1.91 (m, 13H), 2.02 (t, J = 8.6 Hz, 2H), 2.04–
2.10 (m, 2H), 2.70–2.84 (m, 2H), 3.94–4.05 (m, 3H), 4.06–4.15
(m, 2H), 4.51 (s, 2H), 6.84 (d, J = 9.2 Hz, 2H), 7.13 (d, J = 8.4 Hz,
2H), 7.23 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H), 7.59 (br s,
1H), 7.66 (br s, 1H), 8.98 (br s, 1H); 13C NMR (100 MHz, CD3OD):
d 29.8, 30.5, 36.7, 37.9, 39.0, 44.6, 54.6, 68.2, 68.5, 77.7, 110.0,
115.2, 120.6, 125.1, 126.9, 129.5, 131.0, 132.8, 137.8, 141.5,
145.6, 157.8; HRMS (ESI) [MꢃCl]+ Calcd for C32H38ClN2O3:
533.2571. Found: 533.2581. Anal. Calcd for C32H38Cl2N2O3: C,
67.48; H, 6.72; N, 4.92. Found: C, 66.85; H, 7.33; N, 4.37.
4.6.4. (2R,4S)-4-[(4-Bromophenoxy)methyl]-2-[2-(4-chlorophe-
nyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
hydrochloride (24)
Hygroscopic white solid in 76% yield from 411: mp 55–57 °C in
½
a 2D3
ꢂ
ꢃ18.3 (c 0.5 in CD3OD); 1H NMR (400 MHz, CD3OD): d 2.04 (t,
air; Rf = 0.22 (free base, EtOAc); ½a D25
ꢂ
ꢃ16.3 (c 0.6 in CD3OD); 1H
J = 8.4 Hz, 2H), 2.70–2.88 (m, 2H), 3.96 (t, J = 7.6 Hz, 1H), 4.00–4.08
(m, 2H), 4.09–4.17 (m, 2H), 4.52 (s, 2H), 6.88–6.91 (m, 2H), 6.94 (t,
J = 7.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 7.20–7.31 (m, 4H), 7.59 (br
s, 1H), 7.67 (br s, 1H), 9.00 (br s, 1H); 13C NMR (100 MHz, CD3OD):
d 29.8, 39.0, 54.6, 68.2, 68.3, 77.5, 110.0, 115.6, 120.6, 122.3, 125.1,
129.5, 130.6, 131.0, 132.8, 137.8, 141.5, 160.0; HRMS (ESI) [MꢃCl]+
Calcd for C22H24ClN2O3: 399.1475. Found: 399.1466. Anal. Calcd for
C22H24Cl2N2O3: C, 60.70; H, 5.56; N, 6.43. Found: C, 60.90; H, 5.51; N,
6.39.
NMR (400 MHz, CD3OD): d 2.02 (t, J = 8.4 Hz, 2H), 2.69–2.84 (m,
2H), 3.88–4.06 (m, 3H), 4.11 (t, J = 6.8 Hz, 2H), 4.51 (s, 2H), 6.85
(d, J = 9.2 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H),
7.39 (d, J = 8.8 Hz, 2H), 7.59 (br s, 1H), 7.66 (br s, 1H), 8.98 (br s,
1H); 13C NMR (100 MHz, CD3OD): d 29.8, 38.9, 54.6, 68.1, 68.7,
77.4, 110.1, 114.3, 117.6, 120.7, 125.1, 129.6, 131.0, 132.9, 133.5,
137.9, 141.4, 159.2; HRMS (ESI) [MꢃCl]+ Calcd for C22H23BrClN2O3:
477.0581. Found: 477.0557. Anal. Calcd for C22H23BrCl2N2O3: C,
51.38; H, 4.51; N, 5.45. Found: C, 51.44; H, 4.37; N, 5.25.
4.6. Characterization of the new compounds synthesized
following the representative procedure described above for
compound 23, as outlined in Scheme 1
4.6.5. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-4-[(4-fluorophenoxy)
methyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
hydrochloride (25)
Hygroscopic white solid in 86% yield from 411: mp 50–52 °C in
4.6.1. (2R,4S)-4-[(4-Aminophenoxy)methyl]-2-[2-(4-chlorophe-
nyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
dihydrochloride (20)
air; Rf = 0.23 (free base, EtOAc); ½a D24
ꢂ
ꢃ20.8 (c 0.5 in CD3OD); 1H
NMR (400 MHz, CD3OD): d 2.04 (t, J = 8.4 Hz, 2H), 2.71–2.87 (m,
2H), 3.88–4.15 (m, 5H), 4.52 (s, 2H), 6.86–6.93 (m, 2H), 7.00 (ꢀt,
J = 8.8 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H),
7.59 (br s, 1H), 7.66 (br s, 1H), 8.98 (br s, 1H); 13C NMR (100
MHz, CD3OD): d 29.8, 39.0, 54.6, 68.2, 69.2, 77.5, 110.1, 116.8 (d,
Hygroscopic white solid in 54% yield from 411: Rf = 0.17 (free
base, EtOAc); ½a D22
ꢂ
ꢃ12.9 (c 0.9 in CD3OD); 1H NMR (400 MHz,
CD3OD): d 2.04 (t, J = 8.4 Hz, 2H), 2.70–2.88 (m, 2H), 3.97 (t,
J = 6.8 Hz, 1H), 4.03–4.20 (m, 4H), 4.53 (s, 2H), 7.06 (d, J = 8.8 Hz,
2H), 7.16 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.33 (d,
J = 8.8 Hz, 2H), 7.60 (br s, 1H), 7.67 (br s, 1H), 9.00 (br s, 1H); 13C
NMR (100 MHz, CD3OD): d 29.8, 38.9, 54.5, 68.1, 68.9, 77.3,
110.1, 117.0, 120.6, 125.1, 125.2, 125.3, 129.5, 131.0, 132.8,
137.9, 141.5, 160.2; HRMS (ESI) [MꢃHꢃ2Cl]+ Calcd for
C22H25ClN3O3: 414.1584. Found: 414.1565. Anal. Calcd for
C22H26Cl3N3O3: C, 54.28; H, 5.38; N, 8.63. Found: C, 54.48; H,
5.60; N, 8.59.
3
2JC–F = 11.9 Hz), 116.9 (d, JC–F = 3.5 Hz), 120.6, 125.1, 129.5,
4
1
131.0, 132.8, 137.8, 141.5, 156.2 (d, JC–F = 1.8 Hz), 158.9 (d, JC–
F = 237.5 Hz); 19F NMR (376 MHz, CD3OD): d ꢃ126.6; HRMS (ESI)
[MꢃCl]+ Calcd for C22H23ClFN2O3: 417.1381. Found: 417.1366.
Anal. Calcd for C22H23Cl2FN2O3: C, 58.29; H, 5.11; N, 6.18. Found:
C, 58.27; H, 5.16; N, 6.00.
4.6.6. (2R,4S)-4-[(Biphenyl-4-yloxy)methyl]-2-[2-(4-chlorophe-
nyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
hydrochloride (26)
4.6.2. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-4-[(4-hydroxyphe-
noxy)methyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
hydrochloride (21)
White solid in 43% yield from 411: mp 162–163 °C; Rf = 0.12
(EtOAc); ½a 2D3
ꢂ
ꢃ23.5 (c 0.6 in CD3OD); 1H NMR (400 MHz, CD3OD):
d 2.04 (t, J = 8.4 Hz, 2H), 2.70–2.90 (m, 2H), 3.98 (t, J = 7.4 Hz, 1H),
4.02–4.10 (m, 2H), 4.10–4.15 (m, 1H), 4.16–4.22 (m, 1H), 4.52 (s,
2H), 6.99 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 7.23 (d,
White solid in 33% yield from 411: mp 128–130 °C; Rf = 0.17
(EtOAc); ½a 2D2
ꢂ
ꢃ14.4 (c 0.8 in CD3OD); 1H NMR (400 MHz, CD3OD):

2472
J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
J = 8.4 Hz, 2H), 7.28 (t, J = 7.4 Hz, 1H), 7.39 (t, J = 7.8 Hz, 2H), 7.50–
7.56 (m, 4H), 7.60 (br s, 1H), 7.67 (br s, 1H), 9.00 (br s, 1H); 13C
NMR (100 MHz, CD3OD): d 29.8, 39.0, 54.6, 68.2, 68.5, 77.6,
110.1, 116.0, 120.6, 125.1, 127.6, 127.8, 129.1, 129.5, 129.8,
131.0, 132.8, 135.6, 137.8, 141.5, 141.9, 159.6; HRMS (ESI) [MꢃCl]+
Calcd for C28H28ClN2O3: 475.1788. Found: 475.1779. Anal. Calcd for
C28H28Cl2N2O3: C, 65.76; H, 5.52; N, 5.48. Found: C, 65.58; H, 5.42;
N, 5.37.
tion mixture was cooled to room temperature, diluted with H2O,
and extracted with EtOAc (2ꢄ), and the combined organic extracts
were washed sequentially with a saturated aqueous solution of
Na2CO3, and water, and then dried (MgSO4). The solution was con-
centrated, and the residue was purified by preparative thin-layer
chromatography on silica gel (load with MeOH, elute with EtOAc)
to give the free base (40 mg, 0.12 mmol) as an oil (Rf ꢀ 0.2–0.3,
EtOAc). To a solution of the free base in warm 2-propanol (2 mL)
was added a solution of 37% aqueous HCl (20 mg, 0.20 mmol,
1.7 equiv) in 2-propanol (2 mL). The mixture was concentrated,
dried under high vacuum, and washed with Et2O. High-vacuum
drying gave 30 (30 mg, 0.08 mmol, 42%) as a white solid: mp
4.6.7. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-[(4-methoxyphenoxy)methyl]-1,3-dioxolane
hydrochloride (27)
White solid in 81% yield from 411: mp 128–129 °C; Rf = 0.29
159–160 °C; Rf = 0.07 (EtOAc); ½a D22
ꢂ
ꢃ6.3 (c 0.6 in CD3OD); 1H
(free base, EtOAc); ½a D23
ꢂ
ꢃ16.8 (c 0.5 in CD3OD); 1H NMR (400
NMR (400 MHz, CD3OD): d 1.98 (t, J = 8.6 Hz, 2H), 2.70–2.88 (m,
2H), 3.53–3.67 (m, 2H), 3.68–3.79 (m, 2H), 4.01 (t, J = 6.6 Hz, 1H),
4.47 (s, 2H), 7.20 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 7.57
(br s, 1H), 7.63 (br s, 1H), 8.94 (br s, 1H); 13C NMR (100 MHz,
CD3OD): d 29.7, 39.2, 54.7, 62.8, 68.4, 79.6, 109.7, 120.7, 125.1,
129.5, 131.0, 132.8, 137.9, 141.5; HRMS (ESI) [MꢃCl]+ Calcd for
C16H20ClN2O3: 323.1162. Found: 323.1170.
MHz, CD3OD): d 2.03 (t, J = 8.4 Hz, 2H), 2.71–2.86 (m, 2H), 3.73
(s, 3H), 3.90–4.04 (m, 3H), 4.04–4.13 (m, 2H), 4.51 (s, 2H), 6.83
(br s, 4H), 7.15 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.59 (br
s, 1H), 7.66 (br s, 1H), 8.98 (br s, 1H); 13C NMR (100 MHz, CD3OD):
d 29.8, 39.0, 54.6, 56.1, 68.3, 69.2, 77.6, 110.0, 115.7, 116.6, 120.6,
125.1, 129.5, 131.0, 132.8, 137.8, 141.5, 154.1, 155.8; HRMS (ESI)
[MꢃCl]+ Calcd for C23H26ClN2O4: 429.1581. Found: 429.1567. Anal.
Calcd for C23H26Cl2N2O4: C, 59.36; H, 5.63; N, 6.02. Found: C, 59.50;
H, 5.56; N, 6.03.
4.7.2. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-[(methylthio)methyl]-1,3-dioxolane
hydrochloride (31)
4.6.8. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-[(4-iodophenoxy)methyl]-1,3-dioxolane
hydrochloride (28)
A mixture of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imi-
dazol-1-yl)methyl]-4-[(p-toluenesulfonyloxy)methyl]-1,3-dioxo-
lane (4)11 (135 mg, 0.28 mmol), sodium thiomethoxide (51 mg,
0.73 mmol, 2.6 equiv), and cesium carbonate (91 mg, 0.28 mmol,
2 equiv) in acetone (6 mL) was heated at reflux temperature with
stirring for 6 h. The reaction mixture was concentrated, and hot
EtOAc was added. The solids were removed by filtration and
washed with hot EtOAc and then with acetone. The organic fil-
trate was concentrated, and the residue was purified by flash
chromatography on silica gel (EtOAc) to give the free base
(ꢀ110 mg) as a golden oil (Rf = 0.28, EtOAc). To a solution of the
free base in warm 2-propanol (2 mL) was added a solution of
37% aqueous HCl (35 mg, 0.36 mmol, 1.3 equiv) in 2-propanol
(2 mL). The mixture was concentrated and dried under high vac-
uum. The residue was dissolved in 2-propanol (1 mL), the solu-
tion cooled in the freezer, and then a few drops of Et2O were
added and the product allowed to crystallize overnight. The solid
was removed by filtration and washed with Et2O. High-vacuum
drying gave 31 (98 mg, 0.25 mmol, 89%) as a white solid: mp
White solid in 67% yield from 411: mp 113–114 °C; Rf = 0.29
(free base, EtOAc); ½a D24
ꢂ
ꢃ12.7 (c 0.7 in CD3OD); 1H NMR (400
MHz, CD3OD): d 2.01 (t, J = 8.4 Hz, 2H), 2.69–2.84 (m, 2H), 3.90–
3.97 (m, 1H), 3.98–4.07 (m, 2H), 4.08–4.16 (m, 2H), 4.51 (s, 2H),
6.73 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz,
2H), 7.56 (d, J = 8.8 Hz, 2H), 7.58 (br s, 1H), 7.65 (br s, 1H), 8.97
(br s, 1H); 13C NMR (100 MHz, CD3OD): d 29.8, 38.9, 54.6, 68.1,
68.5, 77.4, 110.1, 118.1, 120.7, 125.1, 129.5, 131.0, 132.8, 137.9,
139.5 (2C), 141.4, 160.0; HRMS (ESI) [MꢃCl]+ Calcd for C22H23ClI-
N2O3: 525.0442. Found: 525.0440. Anal. Calcd for C22H23Cl2IN2O3:
C, 47.08; H, 4.13; N, 4.99. Found: C, 47.21; H, 4.33; N, 4.95.
4.6.9. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-4-[(4-cyanophenoxy)
methyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
hydrochloride (29)
Hygroscopic white solid in 100% yield from 411: Rf = 0.26 (free
base, EtOAc); ½a D21
ꢂ
ꢃ15.3 (c 0.5 in CD3OD); 1H NMR (400 MHz,
142–143 °C; Rf = 0.20 (EtOAc); ½a D22
ꢂ
ꢃ11.9 (c 1.0 in CD3OD); 1H
CD3OD): d 2.03 (dd, J = 9.2, 7.6 Hz, 2H), 2.70–2.85 (m, 2H), 3.96
(t, J = 7.8 Hz, 1H), 4.04–4.17 (m, 3H), 4.23 (dd, J = 10.2, 3.0 Hz,
1H), 4.52 (s, 2H), 7.06 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H),
7.24 (d, J = 8.4 Hz, 2H), 7.60 (br s, 1H), 7.63–7.68 (m, 3H), 8.99
(br s, 1H); 13C NMR (100 MHz, CD3OD): d 29.7, 38.8, 54.5, 68.0,
68.7, 77.2, 105.4, 110.2, 116.6, 119.9, 120.7, 125.1, 129.6, 131.0,
132.9, 135.3, 137.9, 141.4, 163.4; HRMS (ESI) [MꢃCl]+ Calcd for
C23H23ClN3O3: 424.1428. Found: 424.1409. Anal. Calcd for
C23H23Cl2N3O3: C, 60.01; H, 5.04; N, 9.13. Found: C, 59.84; H,
5.09; N, 8.96.
NMR (400 MHz, CD3OD): d 2.00 (dd, J = 9.4, 7.8 Hz, 2H), 2.65
(dd, J = 13.6, 6.0 Hz, 1H), 2.70–2.85 (m, 3H), 3.71 (t, J = 8.0 Hz,
1H), 3.75–3.82 (m, 1H), 4.09 (dd, J = 8.0, 5.6 Hz, 1H), 4.48 (s,
2H), 4.86 (s, 3H), 7.21 (d, J = 8.8 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H),
7.59 (br s, 1H), 7.64 (br s, 1H), 8.96 (br s, 1H); 13C NMR (100
MHz, CD3OD): d 16.4, 29.9, 37.0, 39.2, 54.7, 71.1, 78.8, 109.8,
120.6, 125.1, 129.6, 131.0, 132.9, 137.8, 141.4; HRMS (ESI)
[MꢃCl]+ Calcd for C17H22ClN2O2S: 353.1090. Found: 353.1086.
Anal. Calcd for C17H22Cl2N2O2S: C, 52.44; H, 5.70; N, 7.20. Found:
C, 52.51; H, 5.51; N, 7.12.
4.7. Procedures for the displacement of the p-toluenesulfonylo-
xy group in compound 4 using various nucleophiles as outlined
in Scheme 2
4.7.3. (2R,4S)-2-[2-(4-Chlorophenyl)ethyl]-4-(fluoromethyl)-2-
[(1H-imidazol-1-yl)methyl]-1,3-dioxolane hydrochloride (32)
To a sample of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imi-
dazol-1-yl)methyl]-4-[(p-toluenesulfonyloxy)methyl]-1,3-dioxo-
lane (4)11 (120 mg, 0.25 mmol) was added a 1 M solution of tetra-
n-butylammonium fluoride in THF (5 mL, 5.0 mmol, 20 equiv) and
the mixture was heated at reflux temperature with stirring for
18.5 h. The reaction mixture was cooled to room temperature, di-
luted with H2O, and extracted with EtOAc (3ꢄ), and the combined
organic extracts were washed sequentially with a saturated aque-
ous solution of Na2CO3, and water, and then dried (MgSO4). The
4.7.1. (2R,4R)-2-[2-(4-Chlorophenyl)ethyl]-4-(hydroxymethyl)-
2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane hydrochloride (30)
To a mixture of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imi-
dazol-1-yl)methyl]-4-[(p-toluenesulfonyloxy)methyl]-1,3-dioxo-
lane (4)11 (91 mg, 0.19 mmol) in N,N-dimethylformamide (6 mL)
was added KOH (210 mg, 3.74 mmol, 20 equiv) and a small amount
of LiOH. The mixture heated at 120 °C with stirring for 9 h. The reac-

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
2473
solution was concentrated and the residue purified by flash col-
umn chromatography on silica gel (EtOAc) to give the free base
(70 mg, 0.22 mmol) as a golden oil (Rf = 0.21, EtOAc). To a solution
of the free base in warm 2-propanol (2 mL) was added a solution
of 37% aqueous HCl (25 mg, 0.25 mmol, 1.1 equiv) in 2-propanol
(2 mL). The mixture was concentrated and dried under high vac-
uum. The residue was dissolved in 2-propanol (1 mL), the solu-
tion cooled in the freezer, and then a few drops of Et2O were
added and the product allowed to crystallize overnight. The solid
was removed by filtration and washed with Et2O. High-vacuum
drying gave 32 (72 mg, 0.20 mmol, 80%) as a white solid: mp
and extracted with EtOAc (3ꢄ), and the combined organic extracts
were washed sequentially with a saturated aqueous solution of
Na2CO3, and H2O, and then dried (Na2SO4). The solution was con-
centrated and dried under high vacuum to give the clean free base
(90 mg) as an oil (Rf = 0.24, EtOAc). To a solution of the free base in
warm 2-propanol (2 mL) was added a solution of 37% aqueous HCl
(40 mg, 0.41 mmol, 2.7 equiv) in 2-propanol (2 mL). The mixture
was concentrated and dried under high vacuum. The residue was
dissolved in the least amount of hot 2-propanol, the solution
cooled at room temperature, and then at ꢃ25 °C in a freezer prior
to the gradual addition of diethyl ether to complete the precipita-
tion of the hydrochloride. The product was collected by filtration
and washed with diethyl ether. High-vacuum drying gave 34 (74
mg, 0.19 mmol, 83%) as a white solid in the hydrochloride mono-
128–129 °C; ½a 2D2
ꢂ
ꢃ6.0 (c 1.0 in CD3OD); 1H NMR (400 MHz,
CD3OD): d 1.99 (t, J = 8.6 Hz, 2H), 2.69–2.84 (m, 2H), 3.86 (t,
J = 7.8 Hz, 1H), 3.90–4.00 (m, 1H), 4.06 (t, J = 6.6 Hz, 1H), 4.35
(ꢀdd, J = 10.8, 4.0 Hz, 0.5H), 4.44–4.49 (m, 1H), 4.51 (s, 2H),
4.61 (ꢀdd, J = 10.6, 2.6 Hz, 0.5H), 7.20 (d, J = 8.4 Hz, 2H), 7.27 (d,
J = 8.4 Hz, 2H), 7.59 (br s, 1H), 7.64 (br s, 1H), 8.98 (br s, 1H);
hydrate form: mp 118–119 °C; ½a D24
ꢂ
ꢃ16.2 (c 0.4 in CD3OD); 1H
NMR (400 MHz, CD3OD): d 1.96–2.05 (m, 2H), 2.72–2.86 (m, 2H),
3.63–3.73 (m, 2H), 3.86 (t, J = 8.0 Hz, 1H), 3.91–3.98 (m, 1H),
4.09 (dd, J = 8.4, 6.0 Hz, 1H), 4.44–4.55 (m, 2H), 7.21 (d, J = 8.8
Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 7.59 (s, 1H), 7.65 (s, 1H), 8.97 (s,
1H); 13C NMR (100 MHz, CD3OD): d 29.8, 39.0, 44.7, 54.4, 69.3,
78.2, 110.2, 120.7, 125.1, 129.6, 131.0, 132.9, 137.8, 141.3; HRMS
(ESI) [Mꢃ2HꢃClꢃO]+ Calcd for C16H19Cl2N2O2: 341.0824. Found:
341.0817. Anal. Calcd for C16H21Cl3N2O3: C, 48.56; H, 5.35; N,
7.08. Found: C, 49.88; H, 5.16; N, 6.30.
13C NMR (100 MHz, CD3OD):
d 29.7, 38.9, 54.4, 66.7 (d,
3JC–F = 7.6 Hz), 77.7 (d, JC–F = 19.5 Hz), 82.8 (d, JC–F = 172.7 Hz),
110.1, 120.6, 125.1, 129.6, 131.0, 132.8, 137.8, 141.4; 19F–1Hdec
(376 MHz, CD3OD): d -234.1; HRMS (ESI) [MꢃCl]+ Calcd for
C16H19ClFN2O2: 325.1119. Found: 325.1124. Anal. Calcd for
C16H19Cl2FN2O2: C, 53.20; H, 5.30; N, 7.75. Found: C, 53.21; H,
5.23; N, 7.59.
2
1
4.7.4. (2R,4R)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane
4.7.6. (2R,4R)-4-(Azidomethyl)-2-[2-(4-chlorophenyl)ethyl]-2-
[(1H-imidazol-1-yl)methyl]-1,3-dioxolane (35)
dihydrochloride dihydrate (33)
To a mixture of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-
imidazol-1-yl)methyl]-4-[(p-toluenesulfonyloxy)methyl]-1,3-dioxo-
lane (4)11 (201 mg, 0.42 mmol) in N,N-dimethylformamide (3 mL)
was added sodium azide (546 mg, 8.40 mmol, 20 equiv). The mix-
ture was heated at 110 °C with stirring for 2 h. The reaction mixture
was cooled to room temperature, diluted with H2O, and extracted
with EtOAc (3ꢄ), and the combined organic extracts were washed
sequentially with a saturated aqueous solution of Na2CO3, and
H2O, and then dried (Na2SO4). The solution was concentrated and
the golden oily residue purified by flash column chromatography
on silica gel (EtOAc) to give the free base as an oil (Rf = 0.29, EtOAc).
High-vacuum drying gave 35 (121 mg, 0.35 mmol, 83%) as a color-
To a mixture of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-
imidazol-1-yl)methyl]-4-[(p-toluenesulfonyloxy)methyl]-1,3-dioxo-
lane (4)11 (105 mg, 0.22 mmol) in N,N-dimethylformamide (2.5
mL) was added imidazole (120 mg, 1.76 mmol, 8 equiv). The mix-
ture heated at 110 °C with stirring for 30 h. The reaction mixture
was cooled to room temperature, diluted with H2O, and extracted
with CHCl3 (3ꢄ), and the combined organic extracts were washed
with water, and then dried (Na2SO4). The solution was concen-
trated, and the residue was purified by flash column chromatogra-
phy on silica gel (load with hot EtOAc, elute with acetone) to give
the free base (80 mg, 0.21 mmol) as an oil (Rf ꢀ 0.16, EtOAc). To a
solution of the free base in warm 2-propanol (1 mL) was added a
solution of 37% aqueous HCl (56 mg, 0.57 mmol, 2.7 equiv) in 2-
propanol (1 mL). The mixture was concentrated, CH2Cl2 (5 mL)
added, and the mixture concentrated again. High-vacuum drying
gave 33 (90 mg, 0.19 mmol, 86%) as a hygroscopic white solid in
the dihydrochloride dihydrate form: mp 145 °C (fully melted);
less oil: ½a 2D4
ꢂ
+6.6 (c 0.7 in CDCl3); 1H NMR (400 MHz, CDCl3): d
1.90–2.05 (m, 2H), 2.66–2.81 (m, 2H), 3.20 (dd, J = 13.2, 5.2 Hz,
1H), 3.39 (dd, J = 13.2, 4.0 Hz, 1H), 3.44–3.51 (m, 1H), 3.64 (t,
J = 8.0 Hz, 1H), 3.78 (dd, J = 8.0, 6.4 Hz, 1H), 4.02 (s, 2H), 6.98 (br s,
1H), 7.05 (br s, 1H), 7.12 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H),
7.49 (br s, 1H); 13C NMR (100 MHz, CDCl3): d 29.0, 38.7, 51.8, 53.0,
67.9, 76.1, 109.9, 120.9, 128.8, 129.3, 129.8, 132.1, 138.7, 139.6;
HRMS (EI) [M+H]+ Calcd for C16H19ClN5O2: 348.1227. Found:
348.1234. Anal. Calcd for C16H18ClN5O2: C, 55.25; H, 5.22; N,
20.14. Found: C, 55.10; H, 5.18; N, 19.88.
½
a 2D2 +6.8 (c 0.9 in CD3OD); 1H NMR (400 MHz, CD3OD): d 1.90–
ꢂ
2.06 (m, 2H), 2.63–2.82 (m, 2H), 3.79 (t, J = 8.6 Hz, 1H), 4.07–4.15
(m, 1H), 4.25 (dd, J = 8.6, 6.6 Hz, 1H), 4.39 (dd, J = 14.4, 7.2 Hz,
1H), 4.54 (s, 2H), 4.60 (dd, J = 14.4, 2.8 Hz, 1H), 7.22 (d, J = 8.4 Hz,
2H), 7.28 (d, J = 8.4 Hz, 2H), 7.56–7.72 (m, 4H), 8.98 (s, 1H), 9.02
(s, 1H); 13C NMR (100 MHz, CD3OD): d 29.6, 38.6, 51.6, 54.2,
68.5, 76.9, 110.8, 120.8, 121.2, 124.2, 125.0, 129.6, 131.0, 133.0,
137.3, 137.8, 141.1; HRMS (ESI) [Mꢃ5Hꢃ2Clꢃ2O]+ Calcd for
C19H22ClN4O2: 373.1431. Found: 373.1429. Anal. Calcd for
C19H27Cl3N4O4: C, 47.36; H, 5.65; N, 11.63. Found: C, 47.83; H,
5.51; N, 11.41.
4.7.7. (2R,4R)-4-(Aminomethyl)-2-[(1H-imidazol-1-yl)methyl]-
2-[(2-phenyl)ethyl]-1,3-dioxolane dihydrochloride (36)
To a sample of 10% Pd/C catalyst (25 mg) under an atmosphere
of N2, was carefully added MeOH (5 mL). To this suspension was
then added the azide 35 (87 mg, 0.25 mmol) along with ammo-
nium formate (100 mg, 1.59 mmol, 6.4 equiv). The mixture was
heated to reflux temperature for 3 h, and then filtered through Cel-
ite. The filter cake was washed with MeOH, and the filtrate and
washings were combined and concentrated. The residue was di-
luted with a saturated aqueous solution of Na2CO3, and extracted
with EtOAc (2ꢄ), and the combined organic extracts were washed
sequentially with a saturated aqueous solution of Na2CO3, and
brine, and then dried (MgSO4). The solution was concentrated
and dried under high vacuum. To a solution of the free base (ꢀ30
mg, 0.10 mmol) in warm EtOH (2 mL) was added a solution of
37% aqueous HCl (40 mg, 0.41 mmol, 4.6 equiv) in EtOH (2 mL);
4.7.5. (2R,4S)-4-(Chloromethyl)-2-[2-(4-chlorophenyl)ethyl]-2-
[(1H-imidazol-1-yl)methyl]-1,3-dioxolane hydrochloride
monohydrate (34)
To a mixture of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-
imidazol-1-yl)methyl]-4-[(p-toluenesulfonyloxy)methyl]-1,3-dio-
xolane (4)11 (108 mg, 0.23 mmol) in N,N-dimethylformamide (3
mL) was added lithium chloride (192 mg, 4.53 mmol, 19.7 equiv).
The mixture was heated at 110 °C with stirring for 1 h. The reac-
tion mixture was cooled to room temperature, diluted with H2O,

2474
J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
the mixture was concentrated. High-vacuum drying gave 36 (40
at 100,000 g for 60 min at 4 °C. The resulting pellet was resus-
pended in buffer (100 mM KH2PO4, 20% v/v glycerol and 1 mM
EDTA adjusted to pH 7.4) and stored at ꢃ80 °C until use. Protein
concentrations were determined by a modification of the Biuret
method as described by Marks et al.31
mg, 0.10 mmol, 40%) as a white hygroscopic solid: ½a D19
ꢂ
+3.7 (c
1.7 in CD3OD); 1H NMR (400 MHz, CD3OD): d 2.00–2.10 (m, 2H),
2.76–2.86 (m, 2H), 3.14 (dd, J = 13.4, 9.8 Hz, 1H), 3.26 (dd,
J = 13.2, 2.0 Hz, 1H), 3.78 (t, J = 8.0 Hz, 1H), 4.14–4.24 (m, 1H),
4.25 (dd, J = 8.4, 6.4 Hz, 1H), 4.56 (s, 2H), 7.15–7.34 (m, 5H), 7.61
(s, 1H), 7.69 (s, 1H), 9.06 (s, 1H); 13C NMR (100 MHz, CD3OD): d
30.3, 38.8, 48.0, 54.3, 69.5, 74.7, 111.3, 120.8, 125.0, 127.2, 129.4,
129.6, 137.8, 142.3; HRMS (ESI) [MꢃHꢃ2Cl]+ Calcd for
C16H22N3O2: 288.1712. Found: 288.1705.
4.10. Measurement of CYP2E1 and CYP3A1/3A2 enzymatic
activity
In order to determine the in vitro CYP2E1 and CYP3A1/3A2
enzymatic activities, acetone-treated and dexamethasone-treated
Sprague–Dawley rats were used, respectively. An acetone solution
(5% v/v) was provided in place of water for 3 days. Dexametha-
sone-5 (Vetoquinol Canada Inc, Lavaltrie, QC, Canada) was injected
i.p. (15 mg/kg/day for 3 days). The animals were sacrificed by
decapitation and the livers perfused. Microsomes were prepared
according to the procedures outlined above. CYP2E1 hydroxylation
of p-nitrophenol was determined by the spectrophotometric
measurement of 4-nitrocatechol.32 CYP3A1/3A2-catalyzed eryth-
romycin N-demethylase activity was determined by the spectro-
photometric measurement of formaldehyde.33,34 Briefly, reaction
4.7.8. (2R,4R)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-
yl)methyl]-4-(methoxymethyl)-1,3-dioxolane hydrochloride
monohydrate (37)
A solution of sodium methoxide in methanol was prepared by
carefully adding sodium (195 mg, 8.48 mmol) to dry methanol (3
mL) under a nitrogen atmosphere, and allowing this solution to
cool back to room temperature. This solution was then added to
a
mixture of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imida-
zol-1-yl)methyl]-4-[(p-toluenesulfonyloxy)methyl]-1,3-dioxolane
(4)11 (133 mg, 0.28 mmol) in N,N-dimethylformamide (2 mL). The
mixture was heated at 120 °C with stirring for 7 h. The solution
was then concentrated to remove methanol, diluted with a satu-
rated aqueous solution of Na2CO3, and extracted with EtOAc
(3ꢄ). The combined organic extracts were washed sequentially
with a saturated aqueous solution of Na2CO3, and brine, and then
dried (Mg2SO4). The solution was concentrated and the golden oily
residue purified by flash column chromatography on silica gel
(EtOAc) to give the free base as an oil (Rf = 0.13, EtOAc). To a solu-
tion of the free base (61 mg, 0.18 mmol, 64%) in warm 2-propanol
(2 mL) was added a solution of 37% aqueous HCl (31 mg, 0.31
mmol, 1.7 equiv) in 2-propanol (2 mL); the mixture was concen-
trated. High-vacuum drying gave 37 (63 mg, 0.17 mmol, 61%) as
mixtures (500
lL) consisting of 100 mM phosphate buffer (pH
7.4), 100 M p-nitrophenol (CYP2E1) or 400
l
lM erythromycin
(CYP3A1/3A2), 2 mg/mL protein, and inhibitors were pre-incu-
bated for 10 min at 37 °C. Reactions were initiated by adding
NADPH at a final concentration of 1 mM and were incubated for
an additional 30 min (CYP2E1) or 60 min (CYP3A1/3A2) at 37 °C.
Reactions were stopped upon the addition of perchloric acid
(0.6 N or 22% for CYP2E1 or CYP3A1/3A2, respectively). The effects
of the HO inhibitors on enzyme activity were tested at concentra-
tions in the range of 0.01–100
lM.
4.11. In vitro HO activity assay
a white hygroscopic solid: mp 92–93 °C; ½a D24
ꢃ11.6 (c 0.8 in
ꢂ
CD3OD); 1H NMR (400 MHz, CD3OD): d 1.98 (t, J = 8.4 Hz, 2H),
2.69–2.84 (m, 2H), 3.36 (s, 3H), 3.42–3.50 (m, 2H), 3.75 (dd,
J = 16.0, 8.4 Hz, 1H), 3.76–3.84 (m, 1H), 4.02 (dd, J = 7.4, 5.8 Hz,
1H), 4.48 (s, 2H), 7.20 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H),
7.58 (br s, 1H), 7.63 (br s, 1H), 8.96 (br s, 1H); 13C NMR (100
MHz, CD3OD): d 29.8, 39.0, 54.7, 59.6, 68.5, 73.5, 77.9, 109.9,
120.5, 125.1, 129.6, 131.0, 132.8, 137.8, 141.5; HRMS (EI)
[Mꢃ2HꢃClꢃO]+ Calcd for C17H22ClN2O3: 337.1319. Found:
337.1306. Anal. Calcd for C17H24Cl2N2O4: C, 52.18; H, 6.18; N,
7.16. Found: C, 52.14; H, 5.64; N, 6.96.
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).35,36 Spleen
and brain (Sprague–Dawley rats) microsomal fractions were pre-
pared according to the procedure outlined by Appleton et al.30 Pro-
tein concentration of microsomal fractions was determined by a
modification of the biuret method.36 Incubations for HO activity
analysis were done under conditions for which the rate of CO for-
mation (pmol CO ꢄ minꢃ1 ꢄ mg proteinꢃ1) was linear with respect
to time and microsomal protein concentration. Briefly, reaction
mixtures (150
7.4), 50 M methemalbumin, and 1 mg/mL protein were pre-incu-
bated with the inhibitors at final concentrations 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 per-
formed for an additional 15 min at 37 °C. Reactions were stopped
by instantly freezing the reaction mixture on dry ice, and CO for-
mation was monitored by gas chromatography according to the
method described by Vreman and Stevenson.35
lL) consisting of 100 mM phosphate buffer (pH
4.8. Animals
l
Liver tissue was obtained from adult male Sprague–Dawley rats
(250–300 g) purchased from Charles River Inc. (Montreal, QC, Can-
ada). Rats were cared for in accordance with the principles and
guidelines of the Canadian Council on Animal Care and the experi-
mental protocols were approved by Queen’s University Animal Care
Committee. The animals were maintained on 12-h light cycles and
were provided with ad libitum access to standard RalstonPurina Lab-
oratory Chow (Ren’s Feed Supplies Ltd, Oakville, ON, Canada) and
water.
l
4.12. Analysis of enzyme inhibition
The data resulting from the above experiments were plotted as
non-linear regression (sigmoidal dose–response) curves using
GRAPHPAD Prism (Version 3). The values on the abscissa represent
4.9. Preparation of liver microsomal fractions
Microsomal fractions were prepared by differential centrifuga-
tion according to previously published methods.10,30 Briefly, tissue
homogenate (15% w/v) was prepared in buffer (20 mM KH2PO4,
135 mM KCl, and 0.10 mM EDTA; adjusted to pH 7.4 at 4 °C with
1 M KOH) using a 60S Sonic Dismembrator (Fisher Scientific Ltd,
Ottawa, ON, Canada). The homogenate was centrifuged at 10,000
g for 20 min at 4 °C, followed by centrifugation of the supernatant
the logarithm of inhibitor concentration (in lM), whereas the values
of the activity on the ordinate are expressed as a percentage of the
control experiments without inhibitor. From these curves, the value
of the concentration (EC50) of the inhibitor at which the enzyme’s
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, have
been retrieved using the same program, and input into the following

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 17 (2009) 2461–2475
2475
7. Abraham, N. G.; Kappas, A. Free Radical Biol. Med. 2005, 39, 1.
8. Takeda, A.; Itoyama, Y.; Kimpara, T.; Zhu, X.; Avila, J.; Dwyer, B. E.; Perry, G.;
Smith, M. A. Antioxid. Redox Signal. 2004, 6, 888.
9. Fang, J.; Akaike, T.; Maeda, H. Apoptosis 2004, 9, 27.
10. Kinobe, R. T.; Vlahakis, J. Z.; Vreman, H. J.; Stevenson, D. K.; Brien, J. F.; Szarek,
W. A.; Nakatsu, K. Br. J. Pharmacol. 2006, 147, 307.
11. Vlahakis, J. Z.; Kinobe, R. T.; Bowers, R. J.; Brien, J. F.; Nakatsu, K.; Szarek, W. A.
Bioorg. Med. Chem. Lett. 2005, 15, 1457.
formula to give the calculated values of the concentration (IC50) of
the compound under evaluation for which the activity of the enzyme
was inhibited by 50% compared to the control.
EC50
IC50
¼
:
bottomꢃtop
ꢃ 1
50ꢃtop
12. Vlahakis, J. Z.; Kinobe, R. T.; Bowers, R. J.; Brien, J. F.; Nakatsu, K.; Szarek, W. A. J.
Med. Chem. 2006, 49, 4437.
13. Roman, G.; Riley, J. G.; Vlahakis, J. Z.; Kinobe, R. T.; Brien, J. F.; Nakatsu, K.;
Szarek, W. A. Bioorg. Med. Chem. Lett. 2007, 15, 3225.
14. Tani, K.; Suwa, K.; Tanigawa, E.; Ise, T.; Yamagata, T.; Tatsuno, Y.; Otsuka, S. J.
Organomet. Chem. 1989, 370, 203.
15. Grotenbreg, G. M.; Kronemeijer, M.; Timmer, M. S. M.; El Oualid, F.; van Well, R.
M.; Verdoes, M.; Spalburg, E.; van Hooft, P. A. V.; de Neeling, A. J.; Noort, D.; van
Boom, J. H.; van der Marel, G. A.; Overkleeft, H. S.; Overhand, M. J. Org. Chem.
2004, 69, 7851.
The IC50 value reported for each compound is the mean of the
values recorded in replicate experiments, and for each of these rep-
licate experiments (consisting of two separate assays) an individ-
ual IC50 value was calculated in the manner described. The IC50
values for the replicate experiments were employed to generate
the reported standard deviation value.
4.13. Molecular modeling of compound 3 in the binding site of
hHO-1
16. Gartiser, T.; Selve, C.; Delpuech, J.-J. Tetrahedron Lett. 1983, 24, 1609.
17. Maines, M. D. FASEB J. 1988, 2, 2557.
18. Xia, Z. W.; Cui, W. J.; Zhang, X. H.; Shen, Q. X.; Wang, J.; Li, Y. Z.; Chen, S. N.; Yu,
S. C. World J. Gastroenterol. 2002, 8, 1123.
An initial template of hHO-1 was based on the x-ray crystallo-
graphic structure of hHO-1 in complex with 1-(adamantan-1-yl)-
2-(1H-imidazol-1-yl)ethanone (PDB 3CZY) from which the inhibi-
tor had been omitted.22 An initial template of inhibitor 3 was gen-
erated using CS CHEM3D ULTRA (version 6) and the Dundee PRODRG2
Server.37 Inhibitor 3 was then manually inserted into the inhibitor
binding site of hHO-1 using XFit in the CCP4 suite.38 The previously
published structure of hHO-1 into which azalanstat had been
docked was utilized as a guide.22 To compare binding of the inhib-
itor to hHO-2, residues within the inhibitor binding site of hHO-1
19. Trakshel, G. M.; Kutty, R. K.; Maines, M. D. Arch. Biochem. Biophys. 1988, 260,
732.
20. Vreman, H. J.; Cipkala, D. A.; Stevenson, D. K. Can. J. Physiol. Pharmacol. 1996, 74,
278.
21. Ortiz de Montellano, P. R. In Cytochrome P-450: Structure, Mechanism and
Biochemistry; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York,
1986.
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were changed to the corresponding residues of hHO-223 using
39
either XFit or PYMOL
.
Analyses of contact distances between the
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Acknowledgments
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The authors thank the Canadian Institutes of Health Research
for a grant-in-aid from the Canadian Institutes of Health Research
(MOP 64305), and Tracy Gifford, Brian McLaughlin, and Dragic
Vukomanovic for assistance with the biological evaluations.
33. Nash, T. Biochem. J. 1953, 55, 416.
34. Kim, H.; Putt, D. A.; Zangar, R. C.; Wolf, R.; Guengerich, F. P.; Edwards, R. J.;
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