Heme oxygenase inhibition by 2-oxy-substituted 1-(1H-imidazol-1-yl)-4-phenylbutanes: Effect of halogen substitution in the phenyl ring

Article abstract of DOI:10.1016/j.bmc.2007.02.034

A series of 2-oxy-substituted 1-(1H-imidazol-1-yl)-4-phenylbutanes comprising imidazole-ketones, imidazole-dioxolanes, and imidazole-alcohols substituted with halogens in the phenyl ring were synthesized and evaluated as novel inhibitors of heme oxygenase which are structurally distinct from metalloporphyrins. The entire library of compounds was found to be highly active, with the bromine- and iodine-substituted derivatives being the most potent. The imidazole-dioxolanes were all selective for the HO-1 isozyme (inducible) and exhibited substantially lower activity toward the HO-2 isozyme (constitutive). The corresponding imidazole-ketones and imidazole-alcohols showed selectivity toward HO-1 to a lesser degree than the similarly substituted imidazole-dioxolanes.

Full text of DOI:10.1016/j.bmc.2007.02.034

Bioorganic & Medicinal Chemistry 15 (2007) 3225–3234
Heme oxygenase inhibition by 2-oxy-substituted 1-(1H-imidazol-1-
yl)-4-phenylbutanes: Effect of halogen substitution in the phenyl ring
Gheorghe Roman,a John G. Riley,a Jason Z. Vlahakis,a Robert T. Kinobe,b
James F. Brien,b Kanji Nakatsub and Walter A. Szareka,*
aDepartment of Chemistry, Queen’s University, Kingston, Ont., Canada K7L 3N6
bDepartment of Pharmacology and Toxicology, Queen’s University, Kingston, Ont., Canada K7L 3N6
Received 23 November 2006; revised 13 February 2007; accepted 19 February 2007
Available online 22 February 2007
Abstract—A series of 2-oxy-substituted 1-(1H-imidazol-1-yl)-4-phenylbutanes comprising imidazole–ketones, imidazole–dioxol-
anes, and imidazole–alcohols substituted with halogens in the phenyl ring were synthesized and evaluated as novel inhibitors of
heme oxygenase which are structurally distinct from metalloporphyrins. The entire library of compounds was found to be highly
active, with the bromine- and iodine-substituted derivatives being the most potent. The imidazole–dioxolanes were all selective
for the HO-1 isozyme (inducible) and exhibited substantially lower activity toward the HO-2 isozyme (constitutive). The corre-
sponding imidazole–ketones and imidazole–alcohols showed selectivity toward HO-1 to a lesser degree than the similarly substituted
imidazole–dioxolanes.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ally inhibited by metalloporphyrins such as nitric oxide
synthase (NOS) or soluble guanylyl cyclase (sGC).8 The
initial structure–activity study of a series of azalanstat
analogs9 showed that different degrees of selectivity in
the inhibition of HO-1 over HO-2 could be achieved
by modifying the aminophenylmercapto moiety in the
northeastern region of azalanstat or the diastereomeric
configuration of the substituents attached to the
dioxolane ring (Fig. 1). Further investigations10 showed
that the removal of the aminophenylmercapto moiety
Heme oxygenases (HOs), comprising two active iso-
zymes HO-1 (inducible) and HO-2 (constitutive), are en-
zymes responsible for heme degradation in vivo, a
process which generates carbon monoxide (CO) whose
cellular regulatory actions have recently been acknowl-
edged.1–3 Inhibitors of these enzymes may prove to be
indispensable pharmacological tools for the investiga-
tion of the CO/HO system and related physiological
pathways. Furthermore, investigation of the mechanism
of HO-1’s protective effect in acute renal failure,4 its
beneficial involvement in cardiovascular system inju-
ries,5 or its central role in neurodegenerative diseases6
significantly depends on the availability of efficient
inhibitors of this enzyme. Because the previously used
metalloporphyrin-based HO inhibitors lack specificity,
the discovery7 of azalanstat 1 as a compound active to-
ward HO-1 and HO-2 has opened the way for the design
of novel, more-selective non-metalloporphyrin inhibi-
tors of these enzymes. Azalanstat itself is a good HO
inhibitor and has minimal effects on other enzymes usu-
Keywords: Heme oxygenase; Imidazole-based inhibitors; Carbon mon-
oxide; Heme.
*
Corresponding author. Tel.: +1 613 533 2643; fax: +1 613 533
6532; e-mail: szarekw@chem.queensu.ca
Figure 1. Topological analysis of azalanstat 1.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.034

3226
G. Roman et al. / Bioorg. Med. Chem. 15 (2007) 3225–3234
selectively enhanced the inhibition of HO-1 relative to
HO-2. As our previous reports have dealt only with
compounds featuring the typical chloro substituent in
the benzene ring in the southwestern region of azalan-
stat (Fig. 1), the present work focuses on the investiga-
tion of the structure–activity relationship brought
about by the modification of the substitution pattern
in the benzene ring in imidazole–dioxolanes and struc-
turally related compounds.
Two different synthetic approaches to the targeted com-
pounds have been considered. The first multi-step reac-
tion sequence, illustrated in Scheme 1, was devised as a
general strategy granting access to imidazole–dioxolanes
variously substituted in the benzene ring, and debuts
with the preparation of 4-aryl-2-butanones 3 by way
of a base-catalyzed condensation of the appropriate
benzyl halides with 2,4-pentanedione,11 followed by
the bromination of these materials to afford the interme-
diate 1-bromoketones 4. The formation of the undesired
isomeric 3-bromoketones has been abated by the use of
methanol as a solvent instead of the usual halogenated
solvents.12 Alkylation of imidazole with the bromoke-
tones 4 provided easy access to the key intermediate
imidazole–ketones 5, which were subsequently con-
verted into either the corresponding imidazole–dioxol-
anes upon heating at refluxing temperature in ethylene
glycol–toluene in the presence of p-toluenesulfonic acid
and with continuous azeotropic removal of water, or
into the corresponding imidazole–alcohols by reduction
with sodium borohydride in methanol. Imidazole–
ketones 5 have been characterized as hydrochlorides 6,
2. Results and discussion
2.1. Chemistry
Prompted by the discovery of the selective inhibitory
activity of the imidazole–dioxolane 2 (R = H) toward
HO-1,10 a series of analogs bearing various halogen sub-
stituents on the benzene ring has been designed for the
present study. The imidazole–dioxolane having an
unsubstituted benzene ring has also been incorporated
in this series. Including two other series of compounds
(the related imidazole–ketones and the corresponding
imidazole–alcohols) has further broadened the scope
of the investigation.
whereas the imidazole–dioxolane hydrochlorides
7
and the imidazole–alcohol hydrochlorides 8 have been
made available for the biological evaluation from the
respective free bases.
The second synthetic pathway (Scheme 2) is based on
the methodology of Walker et al.,13 and was used for
the synthesis of imidazole–alcohols 8 only in the case
of the commercially available halogen-substituted ben-
zyl halides whose benzylic halogen atom is more reactive
than the halogen substituent in the aromatic ring. Treat-
ment of the Grignard reagents derived from either p-flu-
orobenzyl chloride or p-chlorobenzyl chloride with
Scheme 1. General approach for the synthesis of imidazole–ketones, imidazole–dioxolanes, and imidazole–alcohols related to azalanstat. Reagents
and conditions: (a) anhyd K2CO3, MeOH, reflux, 16 h; (b) Br2, MeOH, rt, 2 h; (c) imidazole, DMF, rt, 1 h; (d) ethylene glycol, p-TsOHÆH2O, toluene,
reflux, 2 h; (e) NaBH4, MeOH, rt, 3 h; (f) 37% aq HCl, 2-propanol, rt.

G. Roman et al. / Bioorg. Med. Chem. 15 (2007) 3225–3234
3227
Scheme 2. Alternative synthesis of imidazole–alcohols related to azalanstat. Reagents and conditions: (a) Mg, anhyd diethyl ether, reflux, 15 min; (b)
( )-epichlorohydrin, anhyd diethyl ether, reflux, 2 h; (c) imidazole, NaH, anhyd DMF, 70–80 ꢁC, 4.5 h; (d) 37% aq HCl, 2-propanol, rt.
racemic epichlorohydrin led to the intermediate opti-
cally inactive 1-chloro-2-butanols 9, which yielded the
imidazole–alcohols 10 as free bases through the N-alkyl-
ation of imidazole. The free bases led to the correspond-
ing hydrochlorides 8 upon treatment with hydrochloric
acid.
of each compound, have been used to express quantita-
tively the selectivity of these inhibitors toward the HO
isozymes.
Imidazole–ketones 6 have been evaluated for the first
time for HO inhibitory activity, and all of them proved
to be excellent inhibitors of both HO-1 and HO-2. When
compared to azalanstat 1, the 4-chlorophenyl compound
6e had similar inhibitory potency for HO-1, but it was
the least potent HO-1 inhibitor in this series. The
dose–response curves for the iodo-substituted imid-
azole–ketone 6d, which exhibited the highest values for
the inhibition of both HO-1 and HO-2 while having
the best selectivity index in this series, are shown in Fig-
ure 2a. However, none of the imidazole–ketones 6
showed any marked selectivity toward any of the two
HO isozymes. At the lowest concentration used in the
study (0.1 lM), compounds 6a, 6b, and 6c showed al-
most no inhibition of HO-1, whereas compounds 6d
and 6e inhibited 44% and 27%, respectively, of the con-
trol activity of HO-1. However, a very slight inhibition
of HO-1 by compound 6d was noted upon a 10-fold de-
crease of its concentration. Even at the highest concen-
tration of the evaluated imidazole–ketones 6, the
greatest inhibition observed was approximately 80%.
2.2. Biological evaluation
Three different series of compounds, namely imidazole–
ketones 6, imidazole–dioxolanes 7, and imidazole–alco-
hols 8, all as hydrochlorides, were evaluated for their
ability to inhibit HO using an in vitro assay for HO in
which heme was presented to the enzyme complexed
with albumin as described in Section 4. HO-1 was ob-
tained from rat spleen, and HO-2 was obtained from
rat brain in the microsomal fractions prepared by differ-
ential centrifugation. The dominance of HO-1 and HO-2
proteins in the rat spleen and brain, respectively, has
been documented.8,14–16 As shown in Table 1, all of
the compounds exhibited good potency as inhibitors of
HO-1 activity, but only imidazole–ketones 6 and imidaz-
ole–alcohols 8 inhibited HO-2 significantly. The selectiv-
ity indices given in Table 1, calculated as the ratio of
IC50 values determined for HO-2 and HO-1 in the case
Table 1. Inhibitory potency and selectivity of imidazole–ketones 6, imidazole–dioxolanes 7, and imidazole–alcohols 8
Compound
IC50 (lM)
Selectivity index IC50 (HO-2)/IC50 (HO-1)
Rat spleen (HO-1)
Rat brain (HO-2)
6a (X = H)
6b (X = F)
6c (X = Br)
6d (X = I)
4.0 1.8
2.7 0.9
1.7 0.7
0.11 0.06
4.7 0.5
0.7 0.4
3.8 1.1
1.9 0.2
3.7 0.9
4.3 2.1
6.2 0.8
1.4 1.1
0.14 0.06
0.06 0.03
0.5 0.1
11.3 4.7
1.9 0.2
9.5 4.6
1.8 0.7
43.1 5.4
>100
2.8
1.4
9.2
5.6
6e (X = Cl)
7a (X = H)
7b (X = F)
7c (X = Br)
7d (X = I)
7e (X = Cl)10
8a (X = H)
8b (X = F)
8c (X = Br)
8d (X = I)
16.4
>143
>26.3
>52.6
>27
>23.2
2.6
>100
>100
>100
>100
16.0 8.2
17.9 11.8
2.6 0.5
1.8 1.5
4.0 0.6
24.5 2.1
12.8
18.6
30
8e (X = Cl)
1 (azalanstat)
8
4.6
5.3
2
Data represent mean IC50 values standard deviation of replicate experiments.

3228
G. Roman et al. / Bioorg. Med. Chem. 15 (2007) 3225–3234
toward HO-1. The selectivity indices ranged from
more than 143 to at least 23 for compounds 7, and are
much higher than those determined for any azalanstat
analogs,9 but comparable with the indices recorded for
the methyl-terminated imidazole–dioxolanes 2 (R =
CH3).10 The dose–response curves (Fig. 2b) for the
iodo-substituted imidazole–dioxolane 7d illustrate the
difference in potency toward HO-1 and HO-2. All of
the imidazole–dioxolanes did not inhibit significantly
HO-1 or HO-2 at the lowest concentration (0.1 lM),
whereas the extent of HO-1 inhibition reached 80–85%
at the highest concentration of the inhibitor. On the
other hand, the inhibition of HO-2 by imidazole–dioxol-
anes 7 was less than 50% even at 100 lM. Surprisingly,
the iodo-substituted imidazole–dioxolane 7d was not the
best HO-1 inhibitor in this series.
Also under scrutiny for the first time as inhibitors of
HO, imidazole–alcohols 8 behaved similarly to the
structurally related imidazole–ketones 6 in this respect.
Compounds 8 inhibited both HO-1 and HO-2 almost
to the same extent, their selectivity indexes varying from
2.6 to 30. Again, the iodo-substituted imidazole–alcohol
8d was the most selective analog in this series, but the
bromo-substituted analog 8c, having an average HO-1
inhibition of 55% and 8% from control at 0.1 and
0.01 lM, respectively, was one of the most potent in this
group. HO-2 inhibition in this series ranges from 55%
for 8c to 90% for 8e at the highest concentration
(100 lM).
As far as the substitution pattern in the benzene ring is
concerned, no striking differences in potency were noted
in the three series of structurally related compounds. It
seems that the less electronegative and the bulkier the
para substituent, the greater the potency toward both
HO-1 and HO-2; compounds having a bulky bromo or
iodo substituent in the para position of the phenyl ring
had an improved inhibitory activity than their analogs
containing a less bulky atom in that position, and this
pattern is reflective also of the variation of the selectivity
index of these compounds. The observation18 that there
is a strong correlation between the hydrophilicity of the
substituent at the far end in a series of 4-(substituted
biphenyl)methylimidazoles and some unidentified resi-
dues at the end of the substrate binding loops of
P45017a allowed the hypothesis that the ability of the ter-
minal substituent to undergo polar–polar interaction
with a hydrogen-bonding group at the active site is
responsible for the enhanced inhibition of this enzyme
by the bromine-substituted inhibitors compared, for
example, with the one exhibited by the fluorine-substi-
tuted compounds.19 The same effect could be responsible
for the greater inhibitory action of the iodo- and bromo-
substituted compounds reported in this study. Even
though the current findings lead to the conclusion that
the replacement of chlorine with other halogens or with
hydrogen does not result in substantial changes in the
HO inhibitory activity of such imidazole-containing
compounds, one cannot preclude the possibility of a sig-
nificant increase or decrease in activity upon dramatic
changes in this part of the molecule. The results of the
biological evaluation of these compounds seem to
Figure 2. Inhibition of HO-1 and HO-2 by imidazole–ketone 6d
(graph a) and imidazole–dioxolane 7d (graph b). All 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. Solid triangles (m), HO-1 (rat spleen micro-
somes) and open squares (h), HO-2 (rat brain microsomes).
The results for the inhibition of HO-2 parallel those
recorded for HO-1, with compounds 6a and 6c being
almost devoid of any inhibition toward HO-2 at the low-
est concentration. Under the same conditions, the rest of
the imidazole–ketones 6 inhibited about 10% of the con-
trol activity of HO-2, and at the highest concentration
(100 lM) all of the compounds in this series inhibited
only 60–70% of the control activity of HO-2.
The development of an optimally selective HO inhibitor
still remains a problem. Metalloporphyrins show a mod-
est degree of selectivity toward HO-2,17 but there are
certain drawbacks that prevent their use in the charac-
terization of the physiological role of these enzymes.
Although all of the imidazole–dioxolanes 7 were potent
inhibitors of HO-1, none of these compounds inhibited
HO-2 activity significantly in the range of concentra-
tions used in the assay. These results confirm the initial
observation10 of isozyme-selective HO inhibition within
this class of compounds, and are consistent with the
working hypothesis that the presence of an imidazole
ring and a minimally substituted dioxolane moiety in
the structure of HO inhibitors enhanced selectivity

G. Roman et al. / Bioorg. Med. Chem. 15 (2007) 3225–3234
3229
indicate that the southwestern region of compounds 6,
4. Experimental
7, and 8 is not actively involved in the binding of these
inhibitors to HO. Nevertheless, the modification of the
southwestern region may become important in the opti-
mization process of a selective inhibitor of HO.
All chemical reagents were obtained from Sigma–
Aldrich and were used without prior purification.
Column chromatography was performed on Silicycle
˚
silica gel (230–400 mesh, 60 A). Analytical thin-layer
It can be inferred, from the reports20 of antifungal drugs
exerting their action through the coordination of their
azole moiety with the protoporphyrin iron atom, that
the imidazole ring is essential for the expression of HO
inhibitory activity in compounds 6, 7, and 8. Further-
more, a comparative molecular-field analysis study21
has emphasized the importance of the hydrophobic
interactions of the N-1 moiety in these antifungal agents
with the active site of an enzyme. The limited up-to-date
knowledge on the inhibition of HO by azalanstat and its
structurally simpler analogs suggests that the presence of
at least one oxygen-containing function strategically
located on the hydrophobic backbone of the N-1 substi-
tuent is required for the materialization of the inhibitory
activity. The replacement of the oxygen atoms of com-
pound 7e with sulfur yielded an imidazole–dithiolane
having a slightly less effect on HO-1, but a significantly
less effect on HO-2;10 this observation is interpreted to
mean that the nature of the heteroatom in this five-mem-
bered ring on the hydrophobic backbone is relevant for
the enhancement of selectivity toward HO-1. The
presence of two oxygen atoms in the dioxolane moiety
and the bulkiness of the dioxolane ring compared to
the size of the carbonyl or hydroxyl functions in
compounds 6 or 8, respectively, may concurrently
contribute to the particularly selective inhibition of the
two HO isoforms by compounds 7.
chromatography was performed on glass- or alumi-
num-backed Silicycle precoated silica gel 60 F254
plates, and the compounds were visualized either by
UV illumination (254 nm), or by heating after spraying
with phosphomolybdic acid in ethanol. Melting points
were recorded on a Mel-Temp II apparatus and are
uncorrected. 1H NMR spectra were recorded on
Bruker Avance 300- and 400-MHz spectrometers.
The signals owing to residual protons in the deuterated
solvents were used as internal standards for the 1H
NMR spectra. The chemical shifts for the carbon
atoms are given relative to CDCl3 (d = 77.16 ppm) or
CD3OD (49.00 ppm). High-resolution mass spectra
were obtained on an Applied Biosystems/MDS Sciex
QSTAR XL spectrometer equipped with an Agilent
HP1100 Cap-LC system. The data for elemental anal-
ysis, determined at MHW Laboratories (Pheonix, AZ),
were within 0.4% of theoretical values. 4-(4-Chlor-
ophenyl)-1-(1H-imidazol-1-yl)-2-butanone hydrochlo-
ride (6e)13 and 1-((2-(2-(4-chlorophenyl)ethyl)-1,
3-dioxolan-2-yl)methyl)-1H-imidazole hydrochloride
(7e)10 were prepared according to the reported
procedures.
4.1. General procedure for the synthesis of 4-aryl-2-butanones
3a–d
A
mixture of 2,4-pentanedione (200 mg, 206 lL,
2 mmol), the 4-substituted benzyl halide (chloride in
the case of 3b, bromide in all other cases, 2 mmol),
and anhydrous potassium carbonate (276 mg, 2 mmol)
in methanol (10 mL) was heated at reflux temperature
for 16 h. The mixture was then cooled to room temper-
ature, methanol was removed under reduced pressure,
and the resulting residue was partitioned between ethyl
acetate (10 mL) and water (10 mL). The organic layer
was separated, and the aqueous layer was extracted fur-
ther with ethyl acetate (3· 10 mL). The combined organ-
ic phase was washed with water (10 mL), dried over
anhydrous Na2SO4, and then the solvent was removed
under pressure. The resulting oil was chromatographed
on a silica gel column using hexanes–ethyl acetate as
the mobile phase to give the title compounds.
3. Conclusions
Instigated by the discovery of imidazole–dioxolane ana-
logs of azalanstat as selective inhibitors of HO isoforms,
the design and synthesis of a series of 2-oxy-substituted
1-(1H-imidazol-1-yl)-4-phenylbutanes has been under-
taken with the view to explore simultaneously the effect
of the substitution pattern in the phenyl ring and the
outcome of the replacement of the dioxolane moiety
by a secondary alcohol or a carbonyl function on the
inhibition of HO. All of the compounds were effective
against HO-1, but only the imidazole–ketones and the
imidazole–alcohols were active toward HO-2. Thus,
the imidazole–dioxolanes were selective toward HO-1,
whereas the imidazole–ketones and the corresponding
imidazole–alcohols showed selectivity for HO-1 to a les-
ser degree. The substitution in the phenyl ring did not
seem to have a remarkable contribution to the HO inhi-
bition by these compounds; however, the electronegativ-
ity of the para substituent in the case of the bromo- and
iodo-substituted compounds may have improved their
effect against both isoforms in terms of potency com-
pared to the typically chloro-substituted analogs. Fur-
thermore, the significance of an oxygen-containing
backbone of the N-1 substituent of imidazole for the
hydrophobic interaction of an inhibitor with the binding
site of the enzyme is supported by the good activity of
the entire collection of compounds.
4.1.1. 4-Phenyl-2-butanone (3a).22,23 Clear liquid
(169 mg, 57%), Rf = 0.63 (hexanes–ethyl acetate 3:1 v/
1
v); H NMR (300 MHz, CDCl3): d 2.14 (s, 3H), 2.76
(t, J = 7.2 Hz, 2H), 2.90 (t, J = 7.2 Hz, 2H), 7.15–7.23
(m, 3H), 7.26–7.32 (m, 2H); 13C NMR (75 MHz,
CDCl3): d 29.8, 30.1, 45.2, 126.2, 128.4, 128.6, 141.1,
208.0; HRMS (EI) Calcd for C10H12O: 148.0888 (M+).
Found: 148.0885.
4.1.2. 4-(4-Fluorophenyl)-2-butanone (3b).24 Clear liquid
(177 mg, 54%), Rf = 0.62 (hexanes–ethyl acetate 3:1
1
v/v); H NMR (400 MHz, CDCl3): d 2.14 (s, 3H), 2.74
(t, J = 7.2 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H), 6.92–7.00

3230
G. Roman et al. / Bioorg. Med. Chem. 15 (2007) 3225–3234
(m, 2H), 7.10–7.17 (m, 2H); 13C NMR (100 MHz,
CDCl3): d 29.0, 30.2, 45.3, 115.2 (d, J2C;F ¼ 21 Hz),
129.8 (d, J3C;F ¼ 8 Hz), 136.6 (d, JC4 ;F ¼ 3 Hz), 161.5 (d,
J1C;F ¼ 242 Hz), 208; 19F NMR (376 MHz, CDCl3): d
ꢀ118.3.
4.2.3.
1-Bromo-4-(4-bromophenyl)-2-butanone
(4c).
White solid (193 mg, 63%), mp 63–64 ꢁC, Rf = 0.42 (hex-
anes–ethyl acetate 4:1 v/v); 1H NMR (400 MHz,
CDCl3): d 2.89 (t, J = 6.8 Hz, 2H), 2.96 (t, J = 6.8 Hz,
2H), 3.84 (s, 2H), 7.07 (d, J = 8 Hz, 2H), 7.41 (d,
J = 8 Hz, 2H); 13C NMR (100 MHz, CDCl3): d 29.3,
34.3, 41.2, 120.2, 130.2, 131.7, 139.4, 201.0; HRMS
(EI) Calcd for C10H10Br2O: 303.9098 (M+). Found:
303.9090.
4.1.3. 4-(4-Bromophenyl)-2-butanone (3c).25 Clear liquid
(302 mg, 67%), Rf = 0.38 (hexanes–ethyl acetate 3:1
1
v/v); H NMR (400 MHz, CDCl3): d 2.15 (s, 3H), 2.75
(t, J = 7.2 Hz, 2H), 2.86 (t, J = 7.2 Hz, 2H), 7.07 (d,
J = 8 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d 29.2, 30.2, 44.9, 120.0, 130.2,
131.7, 140.2, 207.4; HRMS (ESI) Calcd for C10H11BrO-
Na: 248.9891 [M+Na+]. Found: 248.9880.
4.2.4. 1-Bromo-4-(4-iodophenyl)-2-butanone (4d). White
solid (208 mg, 59%), mp 76–77 ꢁC, Rf = 0.50 (hexanes–
1
ethyl acetate 4:1 v/v); H NMR (400 MHz, CDCl3): d
2.88 (t, J = 7.2 Hz, 2H), 2.96 (t, J = 7.2 Hz, 2H), 3.84
(s, 2H), 6.94 (d, J = 8 Hz, 2H), 7.60 (d, J = 8 Hz, 2H);
13C NMR (100 MHz, CDCl3): d 29.4, 34.2, 41.1, 91.6,
130.6, 137.8, 140.1, 201.0; HRMS (EI) Calcd for
C10H10BrIO: 351.8960 (M+). Found: 351.8963.
4.1.4. 4-(4-Iodophenyl)-2-butanone (3d). White solid
(318 mg, 58%), mp 75–76 ꢁC, Rf = 0.60 (hexanes–ethyl
1
acetate 3:1 v/v); H NMR (300 MHz, CDCl3): d 2.13
(s, 3H), 2.73 (t, J = 7.2 Hz, 2H), 2.83 (t, J = 7.2 Hz,
2H), 6.94 (d, J = 7.8 Hz, 2H), 7.59 (d, J = 7.8 Hz, 2H);
13C NMR (75 MHz, CDCl3): d 29.2, 30.2, 44.9, 91.3,
130.6, 137.6, 140.8, 207.6; HRMS (EI) Calcd for
C10H11IO: 273.9855 (M+). Found: 273.9853.
4.3. General procedure for the preparation of imidazole–
ketones 5 and their hydrochlorides 6
A mixture of bromoketone 4 (0.5 mmol) and imidazole
(102 mg, 1.5 mmol) in dry N,N-dimethylformamide
(2 mL) was stirred at room temperature under a nitro-
gen atmosphere for 1 h. The mixture was then diluted
with ethyl acetate (15 mL), and the solution was washed
with water (4· 15 mL). The separated organic phase was
dried over anhydrous Na2SO4, and then the solvent was
removed under reduced pressure to afford a residue that
was chromatographed on a silica gel column using ethyl
acetate–methanol as the mobile phase to give the imid-
azole–ketones 5 as free bases. The free bases 5
(0.2 mmol) were turned into the corresponding hydro-
chlorides upon treatment with 37% aqueous HCl
(26 mg, 22 lL, 0.26 mmol) in 2-propanol (1 mL). The
mixture was then concentrated and dried under high
vacuum to afford a residue that was dissolved in the least
amount of hot 2-propanol. The solution was cooled at
room temperature, and then to ꢀ25 ꢁC in a freezer prior
to gradual addition of diethyl ether to complete the pre-
cipitation of the hydrochlorides 6, which were collected
by filtration and washed with diethyl ether.
4.2. General procedure for the bromination of ketones 3
To a solution of ketone 3 (1 mmol) in methanol (8 mL)
stirred at room temperature, a solution of bromine
(160 mg, 51.6 lL, 1 mmol) in methanol (1 mL) was
added in one portion. The orange reaction mixture
was then stirred at room temperature for 2 h, and, after
the ketone 3 had been consumed (TLC monitoring, hex-
anes–ethyl acetate 4:1 v/v), the reaction was quenched
by adding
a 0.3 M sodium thiosulfate solution
(618 lL), and diluted with ethyl acetate (15 mL). The
resulting mixture was washed with water (15 mL), the
organic layer was separated, and the aqueous layer
was extracted further with ethyl acetate (3· 15 mL).
The combined organic phase was dried over anhydrous
Na2SO4 and concentrated under reduced pressure to
give a residue that was chromatographed on a silica
gel column using hexanes–ethyl acetate (15:1 v/v) as
the mobile phase to give the desired 1-bromo ketones 2.
4.2.1. 1-Bromo-4-phenyl-2-butanone (4a).26,27 White so-
lid (131 mg, 58%), mp 37–38 ꢁC, Rf = 0.53 (hexanes–
4.3.1. 1-(1H-Imidazol-1-yl)-4-phenyl-2-butanone (5a).28
White solid (64 mg, 60%), mp 71–72 ꢁC, Rf = 0.44 (ethyl
acetate–methanol 8:1 v/v); 1H NMR (400 MHz, CDCl3):
d 2.74 (t, J = 7.2 Hz, 2H), 2.93 (t, J = 7.2 Hz, 2H), 4.61
(s, 2H), 6.78 (s, 1H), 7.06 (s, 1H), 7.15 (d, J = 7.6 Hz,
2H), 7.23 (d, J = 7.2 Hz, 1H), 7.28–7.33 (m, 3H); 13C
NMR (100 MHz, CDCl3): d 29.6, 41.1, 55.6, 120.0,
126.5, 128.4, 128.7, 129.6, 137.9, 140.1, 202.7; HRMS
(ESI) Calcd for C13H15N2O: 215.1184 [M+H]+. Found:
215.1195.
1
ethyl acetate 4:1 v/v); H NMR (400 MHz, CDCl3): d
2.98 (t, J = 6.8 Hz, 2H), 3.02 (t, J = 6.8 Hz, 2H), 3.88
(s, 2H), 7.19–7.24 (m, 3H), 7.28–7.33 (m, 2H); 13C
NMR (100 MHz, CDCl3): d 30.0, 34.4, 41.6, 126.6,
128.6, 128.7, 140.5, 201.4; HRMS (EI) Calcd for
C10H11BrO: 225.9993 (M+). Found: 225.9997.
4.2.2. 1-Bromo-4-(4-fluorophenyl)-2-butanone (4b). White
solid (159 mg, 65%), Rf = 0.45 (hexanes–ethyl acetate
4:1 v/v); 1H NMR (400 MHz, CDCl3): d 2.95 (t,
J = 6.8 Hz, 2H), 2.99 (m, J = 6.8 Hz, 2H), 3.88 (s, 2H),
6.93–7.01 (m, 2H), 7.11–7.18 (m, 2H); 13C NMR
(100 MHz, CDCl3): d 29.1, 34.4, 41.5, 115.4 (d,
4.3.2. 1-(1H-Imidazol-1-yl)-4-phenyl-2-butanone hydro-
chloride (6a). White solid (43 mg, 86%), mp 170–
171 ꢁC (lit.29 mp 171–173 ꢁC), Rf = 0.0 (ethyl acetate);
1H NMR (400 MHz, D2O): d 2.96 (t, J = 6.8 Hz, 2H),
3.04 (t, J = 6.8 Hz, 2H), 5.27 (s, 2H), 7.27–7.33 (m,
4H), 7.34–7.41 (m, 2H), 7.48 (s, 1H), 8.59 (s, 1H); 13C
NMR (100 MHz, D2O): d 28.7, 40.6, 57.0, 119.5,
122.9, 126.6, 128.4, 128.8, 135.7, 140.5, 204.7; HRMS
J2C;F ¼ 21 Hz),
129.9
(d,
J3C;F ¼ 8 Hz),
136.1
(d, J4C;F ¼ 3 Hz), 161.6 (d, JC1 ;F ¼ 243 Hz), 201.1; 19F
NMR (376 MHz, CDCl3): d ꢀ117.8; HRMS (EI) Calcd
for C10H10BrFO: 243.9902 (M+). Found: 243.9899.

G. Roman et al. / Bioorg. Med. Chem. 15 (2007) 3225–3234
3231
(ESI) Calcd for C13H15N2O: 215.1184 [M+H]+. Found:
215.1195. Anal. Calcd for C13H15ClN2O: C, 62.28; H,
6.03; N, 11.17. Found: C, 62.33; H, 5.85; N, 10.99.
130.6, 137.9, 138.0, 139.8, 202.3; HRMS (EI) Calcd for
C13H13IN2O: 340.0073 (M+). Found: 340.0074.
4.3.8. 1-(1H-Imidazol-1-yl)-4-(4-iodophenyl)-2-butanone
hydrochloride (6d). Off-white solid (46 mg, 61%), mp
202–203 ꢁC, Rf = 0.0 (ethyl acetate); 1H NMR
(400 MHz, CD3OD): d 2.90 (t, J = 6.8 Hz, 2H), 2.96 (t,
J = 6.8 Hz, 2H), 5.31 (s, 2H), 7.04 (d, J = 8 Hz, 2H),
7.49 (s, 2H), 7.56–7.64 (m, 3H), 8.84 (s, 1H); 13C
NMR (100 MHz, CD3OD): d 29.4, 41.7, 57.9, 91.6,
120.5, 124.7, 131.7, 137.7, 138.7, 141.8, 201.7; HRMS
(ESI) Calcd for C13H14IN2O: 341.0151 [M+H]+. Found
341.0147. Anal. Calcd for C13H14ClIN2O: C, 41.46; H,
3.75; N, 7.44. Found: C, 41.59; H, 4.00; N, 7.37.
4.3.3. 4-(4-Fluorophenyl)-1-(1H-imidazol-1-yl)-2-buta-
none (5b). White solid (76 mg, 66%), mp 69–70 ꢁC,
Rf = 0.52 (ethyl acetate–methanol 8:1 v/v); H NMR
1
(400 MHz, CDCl3): d 2.71 (t, J = 7.2 Hz, 2H), 2.90
(t, J = 7.2 Hz, 2H), 4.66 (s, 2H), 6.83 (br s, 1H),
6.93–6.99 (m, 2H), 7.08–7.14 (m, 3H), 7.44 (br s,
1H); 13C NMR (100 MHz, CDCl3): d 28.7, 41.3,
55.8, 115.6 (d, J2C;F ¼ 21 Hz), 120.0, 129.7, 129.9 (d,
J3C;F ¼ 8 Hz), 135.8 (d, JC4 ;F ¼ 3 Hz), 137.9, 161.7
(d, J1C;F ¼ 243 Hz), 202.5; 19F NMR (376 MHz,
CDCl3): d ꢀ117.5; HRMS (EI) Calcd for C13H13
FN2O: 232.1012 (M+). Found: 232.1006.
4.4. General procedure for the preparation of imidazole–
dioxolane hydrochlorides 7
4.3.4. 4-(4-Fluorophenyl)-1-(1H-imidazol-1-yl)-2-buta-
none hydrochloride (6b). White solid (43 mg, 80%),
mp 160–162 ꢁC, Rf = 0.0 (ethyl acetate); 1H NMR
(400 MHz, D2O): d 2.90 (t, J = 7.2 Hz, 2H), 3.00 (t,
J = 7.2 Hz, 2H), 5.26 (s, 2H), 7.01–7.08 (m, 2H),
7.20–7.27 (m, 2H), 7.29 (s, 1H), 7.46 (s, 1H), 8.60 (s,
1H); 13C NMR (100 MHz, D2O): d 27.8, 40.6, 57.0,
115.2 (d, J2C;F ¼ 21 Hz), 119.4, 122.9, 129.9 (d,
J3C;F ¼ 8 Hz), 135.7, 136.2 (d, JC4 ;F ¼ 3 Hz), 161.2
(d, J1C;F ¼ 240 Hz), 204.5; 19F NMR (376 MHz,
D2O): d ꢀ118.5; HRMS (ESI) Calcd for C13H14FN2O:
233.1090 [M+H]+. Found: 233.1089. Anal. Calcd for
C13H14ClFN2O: C, 58.11; H, 5.25; N, 10.43. Found:
C, 58.25; H, 5.17; N, 10.61.
A mixture of an imidazole–ketone 5 (0.5 mmol), ethyl-
ene glycol (62 mg, 58 lL, 1 mmol), and p-toluenesulfon-
ic acid monohydrate (190 mg, 1 mmol) in toluene
(20 mL) was heated at reflux temperature under nitrogen
until the Dean–Stark trap had filled (1 h). The trap was
then emptied, fresh toluene (10 mL) was added to the
reaction mixture, and heating at reflux temperature con-
tinued for another hour until the trap had refilled. The
reaction mixture was then cooled to room temperature,
diluted with ethyl acetate (15 mL), and washed sequen-
tially with saturated NaHCO3 solution (15 mL), water
(15 mL), and brine (15 mL). The organic layer was dried
over anhydrous Na2SO4 and then concentrated under
reduced pressure to give a residue that was chromato-
graphed on silica gel to afford the dioxolanes as free
bases. The hydrochlorides 7 were prepared starting from
the corresponding free bases and 37% aqueous HCl
(molar ratio 1:1.3) in 2-propanol (1–2 mL) in a manner
identical to the one described for the hydrochlorides of
the imidazole–ketones 5.
4.3.5. 4-(4-Bromophenyl)-1-(1H-imidazol-1-yl)-2-buta-
none (5c). White solid (111 mg, 76%), mp 79–80 ꢁC,
Rf = 0.50 (ethyl acetate–methanol 4:1 v/v); H NMR
1
(400 MHz, CDCl3): d 2.70 (t, J = 7.2 Hz, 2H), 2.83
(t, J = 7.2 Hz, 2H), 4.65 (s, 2H), 6.81 (br s, 1H), 7.02
(d, J = 7.6 Hz, 2H), 7.09 (s, 1H), 7.37–7.40 (m, 3H);
13C NMR (100 MHz, CDCl3): d 28.6, 40.9, 55.7,
120.1, 120.4, 130.0, 131.8, 138.0, 139.1, 202.3; HRMS
(EI) Calcd for C13H13BrN2O: 292.0211 (M+). Found:
292.0219.
4.4.1. 1-((2-(2-Phenylethyl)-1,3-dioxolan-2-yl)methyl)-
1H-imidazole hydrochloride (7a). White solid (90 mg,
61%), mp 164–165 ꢁC, Rf = 0.0 (ethyl acetate); 1H
NMR (400 MHz, D2O): d 2.00–2.06 (m, 2H), 2.72–
2.78 (m, 2H), 3.66 (t, J = 8 Hz, 2H), 4.02 (t,
J = 7.2 Hz, 2H), 4.43 (s, 2H), 7.26–7.31 (m, 3H),
7.34–7.40 (m, 2H), 7.48 (s, 1H), 7.49 (s, 1H), 8.72 (s,
1H); 13C NMR (100 MHz, D2O): d 28.6, 37.0, 53.4,
65.8, 108.0, 119.3, 123.4, 126.3, 128.4, 128.8, 135.7,
141.4; HRMS (ESI) Calcd for C15H19N2O2: 259.1446
[M+H]+. Found: 259.1441. Anal. Calcd for
C15H19ClN2O2ÆH2O: C, 57.60; H, 6.77; N, 8.96.
Found: C, 57.79; H, 6.53; N, 8.99.
4.3.6. 4-(4-Bromophenyl)-1-(1H-imidazol-1-yl)-2-buta-
none hydrochloride (6c). White solid (51 mg, 77%), mp
174–175 ꢁC, Rf = 0.0 (ethyl acetate); 1H NMR
(400 MHz, D2O): d 2.91 (t, J = 7.2 Hz, 2H), 3.01 (t,
J = 7.2 Hz, 2H), 5.27 (s, 2H), 7.18 (d, J = 8 Hz, 2H),
7.30 (s, 1H), 7.46–7.50 (m, 3H), 8.61 (s, 1H); 13C
NMR (100 MHz, CD3OD): d 29.4, 41.8, 57.9, 120.8,
121.0, 124.6, 131.5, 132.6, 137.7, 141.2, 201.7; HRMS
(ESI) Calcd for C13H14BrN2O: 293.0290 [M+H]+.
Found: 293.0279. Anal. Calcd for C13H14BrClN2O:
C, 47.37; H, 4.28; N, 8.50. Found: C, 47.60; H, 4.13;
N, 8.34.
4.4.2. 1-((2-(2-(4-Fluorophenyl)ethyl)-1,3-dioxolan-2-
yl)methyl)- 1H-imidazole hydrochloride (7b). White
solid (91 mg, 58%), mp 153–154 ꢁC, Rf = 0.0 (ethyl
1
4.3.7.
1-(1H-Imidazol-1-yl)-4-(4-iodophenyl)-2-butanone
acetate); H NMR (400 MHz, D2O): d 1.99–2.04 (m,
(5d). Off-white solid (121 mg, 71%), mp 124–125 ꢁC,
2H), 2.69–2.75 (m, 2H), 3.64 (t, J = 6.4 Hz, 2H), 4.00
(t, J = 7.2 Hz, 2H), 4.43 (s, 2H), 7.02–7.09 (m, 2H),
7.22–7.27 (m, 2H), 7.46 (s, 1H), 7.48 (s, 1H), 8.71 (s,
1H); 13C NMR (100 MHz, D2O): d 27.8, 37.1, 53.4,
65.8, 108.0, 115.2 (d, J2C;F ¼ 21 Hz), 119.3, 123.4,
129.8 (d, JC3 ;F ¼ 8 Hz), 135.8, 137.1 (d, J4C;F ¼ 3 Hz),
1
Rf = 0.19 (ethyl acetate); H NMR (400 MHz, CDCl3): d
2.71 (t, J = 7.6 Hz, 2H), 2.86 (t, J = 7.6 Hz, 2H), 4.65 (s,
2H), 6.81 (s, 1H), 6.90 (d, J = 8 Hz, 2H), 7.09 (s, 1H),
7.39 (s, 1H), 7.60 (d, J = 8 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d 29.0, 40.9, 55.7, 91.8, 120.0, 130.1,

3232
G. Roman et al. / Bioorg. Med. Chem. 15 (2007) 3225–3234
161.1 (d, J1C;F ¼ 240 Hz); 19F NMR (376 MHz, D2O): d
ꢀ118.9; HRMS (ESI) Calcd for C15H18FN2O2:
277.1352 [M+H]+. Found: 277.1340. Anal. Calcd for
C15H18ClFN2O2: C, 57.60; H, 5.80; N, 8.96. Found:
C, 57.86; H, 5.82; N, 8.98.
C13H17ClN2O: C, 61.78; H, 6.78; N, 11.08. Found: C,
61.68; H, 6.87; N, 10.95.
4.5.2.
( )-4-(4-Bromophenyl)-1-(1H-imidazol-1-yl)-2-
butanol hydrochloride (8c). White solid (111 mg, 67%),
mp 174–175 ꢁC, Rf = 0.0 (ethyl acetate); 1H NMR
(400 MHz, CD3OD): d 1.65–1.76 (m, 1H), 1.78–189
(m, 1H), 2.64–2.74 (m, 1H), 2.77–2.88 (m, 1H), 3.81–
3.89 (m, 1H), 4.17 (dd, J = 8.4 and 13.6 Hz, 1H), 4.36
(dd, J = 3.0 and 13.8 Hz, 1H), 7.16 (d, J = 8.0 Hz,
1H), 7.42 (d, J = 8.4 Hz, 1H), 7.56 (s, 1H), 7.63 (s,
1H), 8.90 (s, 1H); 13C NMR (100 MHz, CD3OD): d
31.9, 37.07, 56.2, 70.0, 120.6, 124.1, 131.5, 132.5,
136.9, 142.1; HRMS (ESI) Calcd for C13H16BrN2O:
295.0446 [M+H]+. Found: 295.0432. Anal. Calcd for
C13H16BrClN2O: C, 47.08; H, 4.86; N, 8.45. Found: C,
47.19; H, 5.00; N, 8.56.
4.4.3.
1-((2-(2-(4-Bromophenyl)ethyl)-1,3-dioxolan-2-yl)
methyl)- 1H-imidazole hydrochloride (7c). White solid
(110 mg, 59%), mp 205–207 ꢁC, Rf = 0.0 (ethyl acetate);
1H NMR (400 MHz, D2O): d 1.97–2.03 (m, 2H), 2.66–
2.72 (m, 2H), 3.60–3.65 (m, 2H), 3.96–4.01 (m, 2H), 4.42
(s, 2H), 7.16 (d, J = 8 Hz, 2H), 7.45–7.49 (m, 4H), 8.72
(s, 1H); 13C NMR (100 MHz, D2O): d 28.0, 36.7, 53.3,
65.8, 107.9, 119.2, 119.3, 123.4, 130.2, 131.4, 135.7,
140.5; HRMS (ESI) Calcd for C15H1881BrN2O2:
339.0525 [M+H]+. Found: 339.0510. Anal. Calcd for
C15H18BrClN2O2: C, 48.21; H, 4.86; N, 7.50. Found: C,
48.40; H, 4.73; N, 7.43.
4.5.3. ( )-1-(1H-Imidazol-1-yl)-4-(4-iodophenyl)-2-buta-
nol hydrochloride (8d). White solid (136 mg, 72%), mp
196–197 ꢁC, Rf = 0.0 (ethyl acetate); 1H NMR
(400 MHz, D2O): d 1.64–1.75 (m, 1H), 1.78–1.88 (m,
1H), 2.63–2.73 (m, 1H), 2.75–2.85 (m, 1H), 3.81–3.89
(m, 1H), 4.13 (dd, J = 8.4 and 14.6 Hz, 1H), 4.36 (d,
J = 14 Hz, 1H), 7.03 (d, J = 8 Hz, 2H), 7.56 (s, 1H),
7.58–7.65 (m, 3H), 8.92 (s, 1H); 13C NMR (100 MHz,
D2O): 32.0, 37.0, 56.3, 70.0, 91.6, 120.7, 124.1, 131.8,
136.9, 138.7, 142.7; HRMS (ESI) Calcd for
C13H16IN2O: 343.0307 [M+H]+. Found: 343.0319. Anal.
Calcd for C13H16ClIN2O: C, 41.24; H, 4.26; N, 7.40.
Found: C, 41.20; H, 4.44; N, 7.32.
4.4.4.
1-((2-(2-(4-Iodophenyl)ethyl)-1,3-dioxolan-2-yl)
methyl)- 1H-imidazole hydrochloride (7d). White solid
(105 mg, 50%), mp 241–243 ꢁC (dec), Rf = 0.0 (ethyl ace-
tate); 1H NMR (400 MHz, CD3OD): d 1.94–2.01 (m,
2H), 2.68–2.77 (m, 2H), 3.58–3.67 (m, 2H), 3.93–4.03
(m, 2H), 4.47 (s, 2H), 7.02 (d, J = 8.4 Hz, 2H), 7.56 (s,
1H), 7.59–7.64 (m, 3H), 8.93 (s, 1H); 13C NMR
(100 MHz, CD3OD): d 30.0, 38.7, 54.6, 66.9, 91.6,
109.0, 120.4, 125.2, 131.6, 137.8, 138.7, 142.5; HRMS
(ESI) Calcd for C15H18IN2O2: 385.0413 [M+H]+. Found:
385.0406. Anal. Calcd for C15H18ClIN2O2: C, 42.83; H,
4.31; N, 6.66. Found: C, 42.87; H, 4.38; N, 6.54.
4.5. General procedure for the preparation of imidazole–
alcohol hydrochlorides 8 through the reduction of ketones 5
4.6. General procedure for the preparation of alcohols 9
To a suspension of magnesium turnings (168 mg,
6.92 mmol) in diethyl ether (3 mL), stirred under a nitro-
gen atmosphere, was added a small portion (0.15 mL) of
a solution of 4-halobenzyl chloride (6.92 mmol) in
diethyl ether (2 mL), followed by a crystal of iodine.
The remaining solution of 4-halobenzyl chloride was
then added over a period of 15 min, and then the mix-
ture was heated at reflux temperature for 15 min. The
resulting Grignard reagent was then cooled to room
temperature, and added dropwise, using a syringe, to a
solution of ( )-epichlorohydrin (640 mg, 541 lL,
6.92 mmol) in diethyl ether (3 mL) over a period of
10 min. The reaction mixture was then stirred at room
temperature for 30 min, then heated at reflux tempera-
ture for 2 h, and diluted with water (10 mL) and ethyl
acetate (10 mL). Hydrochloric acid (10 mL, 1.0 M) was
then added dropwise until all of the solids dissolved.
The organic layer was then separated, and the aqueous
layer extracted with ethyl acetate (3· 10 mL). The com-
bined organic phase was then washed with water
(10 mL), dried over anhydrous Na2SO4, and concen-
trated. The resulting oil was chromatographed on a col-
umn of silica gel using hexanes–ethyl acetate as the
mobile phase to give the title compounds.
A solution of an imidazole–ketone 5 (0.5 mmol) in
methanol (10 mL) was gradually treated with sodium
borohydride (57 mg, 1.5 mmol). After the reducing
agent had been added, the reaction mixture was further
stirred for 3 h, and then the solvent was removed in va-
cuo to give a solid residue that was partitioned between
ethyl acetate (10 mL) and water (10 mL). The aqueous
phase was extracted further with ethyl acetate (2·
10 mL), the combined organic phase was dried over
anhydrous Na2SO4, and then the solvent was removed
to give the desired alcohols as free bases. These com-
pounds were turned into the corresponding hydrochlo-
rides 8 upon treatment with 37% aqueous HCl (molar
ratio 1:1.3) in 2-propanol (1–2 mL) in a manner similar
to the one described for the preparation of the hydro-
chlorides of the imidazole–ketones 5.
4.5.1. ( )-1-(1H-Imidazol-1-yl)-4-phenyl-2-butanol hydro-
chloride (8a). White solid (102 mg, 81%), mp 56–57 ꢁC,
Rf = 0.16 (ethyl acetate); 1H NMR (400 MHz, CD3OD):
d 1.66–1.80 (m, 1H), 1.82–1.93 (m, 1H), 2.71–2.81 (m,
1H), 2.84–2.94 (m, 1H), 3.86–3.95 (m, 1H), 4.19 (dd,
J = 8.0 and 14.0 Hz, 1H), 4.41 (dd, J = 3.0 and
13.8 Hz, 1H), 7.21–7.38 (m, 5H), 7.64 (s, 1H), 7.70 (s,
1H), 8.99 (s, 1H); 13C NMR (100 MHz, CD3OD): d
32.5, 37.4, 56.0, 69.9, 120.8, 124.0, 127.1, 129.6, 129.7,
137.0, 143.1; HRMS (ESI) Calcd for C13H17N2O:
217.1341 [M+H]+. Found: 217.1344. Anal. Calcd for
4.6.1. ( )-1-Chloro-4-(4-fluorophenyl)-2-butanol (9b).
Clear oil (964 mg, 69%), Rf = 0.68 (hexanes–ethyl ace-
1
tate 1:1 v/v); H NMR (400 MHz, CDCl3): d 1.83 (m,
2H), 2.70–2.75 (m, 1H), 2.80–2.83 (m, 1H), 3.51 (dd,

G. Roman et al. / Bioorg. Med. Chem. 15 (2007) 3225–3234
3233
J = 7.1 and 11.1 Hz, 1H), 3.65 (dd, J = 3.2 and 7.8 Hz,
1H), 3.81 (m, 2H), 7.00 (t, J = 8.6 Hz, 2H), 7.18 (t,
J = 5.6 Hz, 2H); 13C NMR (100 MHz, CDCl3): d 30.9,
35.9, 50.5, 70.4, 115.2 (d, J2C;F ¼ 21:2 Hz), 129.8 (d,
J3C;F ¼ 7:8 Hz), 161.4 (d, JC1 ;F ¼ 243:8); HRMS (EI)
Calcd for C10H12ClFO: 202.0561 (M+). Found:
202.0566.
3.86–3.96 (m, 1H), 4.13 (dd, J = 8.1 and 14.2 Hz,
1H), 4.32 (dd, J = 2.9 and 14.2 Hz, 1H), 7.06 (t,
J = 8.9 Hz, 2H), 7.24–7.32 (m, 2H), 7.43 (s, 2H), 8.66
(s, 1H); 13C NMR (100 MHz, D2O): d 30.0, 34.9,
54.6, 68.6, 115.1 (d, J2C;F ¼ 21:2 Hz), 119.6, 122.3,
130.0 (d, J3C;F ¼ 8:0 Hz), 137.2 (d, JC4 ;F ¼ 3:0 Hz),
161.1 (d, J1C;F ¼ 241:2); 19F NMR (376 MHz, D2O): d
-119.0; HRMS (ESI) Calcd for C13H16FN2O:
235.1247 [M+H]+. Found: 235.1247. Anal. Calcd for
C13H16ClFN2O: C, 57.67; H, 5.96; N, 10.35. Found:
C, 57.75; H, 5.94; N, 10.49.
4.6.2. ( )-1-Chloro-4-(4-chlorophenyl)-2-butanol (9e).13
Golden oil (860 mg, 57%), Rf = 0.18 (hexanes–ethyl ace-
1
tate 9:1 v/v); H NMR (400 MHz, CDCl3): d 1.73–1.90
(m, 2H), 2.20 (br s, 1 H), 2.63–2.73 (m, 1H), 2.76–2.87
(m, 1H), 3.49 (dd, J = 7.0 and 11.0 Hz, 1H), 3.62 (dd,
J = 3.2 and 11.2 Hz, 1H), 3.74–3.83 (m, 1H), 7.13 (d,
J = 8.0 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d 31.2, 35.8, 50.6, 70.6, 128.8,
129.9, 132.0, 139.9; HRMS (EI) Calcd for C10H12Cl2O:
218.0265 (M+). Found 218.0260.
4.7.2. ( )-4-(4-Chlorophenyl)-1-(1H-imidazol-1-yl)-2-
butanol hydrochloride (8e). Colorless solid (775 mg,
54%), mp 138–140 ꢁC, Rf = 0.0 (ethyl acetate); 1H
NMR (400 MHz, D2O): d 1.70–1.78 (m, 1H), 1.82–
1.91 (m, 1H), 2.67–2.74 (m, 1H), 2.78–2.86 (m, 1H),
3.88–3.94 (m, 1H), 4.15 (dd, J = 8.0 and 14.0 Hz,
1H), 4.34 (dd, J = 3.0 and 14.2 Hz, 1H), 7.25 (d,
J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.45 (s,
1H), 7.46 (s, 1H), 8.68 (s, 1H); 13C NMR (100 MHz,
D2O): d 30.5, 35.0, 54.9, 69.0, 120.0, 122.6, 128.8,
130.4, 131.4, 135.3, 140.5; HRMS (ESI) Calcd for
C13H16ClN2O: 251.0945 [M+H]+. Found: 251.0949.
Anal. Calcd for C13H16Cl2N2O: C, 54.37; H, 5.62; N,
9.75. Found: C, 54.56; H, 5.72; N, 9.18.
4.7. General procedure for the preparation of imidazole–
alcohol hydrochlorides 8 through the N-alkylation of imidaz-
ole with alcohols 9
A dispersion of 60% sodium hydride in mineral oil
(960 mg, 24 mmol) was washed twice with hexanes un-
der a nitrogen atmosphere, the solid suspended in dry
DMF (5 mL), and added portionwise to a cooled stirred
solution of imidazole (1.7 g, 25 mmol) in dry DMF
(5 mL). The mixture was brought to room temperature
and stirred until the evolution of hydrogen ceased, then
warmed at 70–80 ꢁC. A solution of compound 9
(5 mmol) in DMF (5 mL) was then added dropwise,
using a syringe, and the reaction mixture was further
stirred at 70–80 ꢁC for 4.5 h, then cooled to room tem-
perature. ( )-4-(4-Fluorophenyl)-1-(1H-imidazol-1-yl)-
2-butanol was isolated by pouring the mixture onto ice
(50 g), followed by extraction with ethyl acetate
(50 mL). The organic phase was washed with brine (3·
50 mL), dried over anhydrous Na2SO4, then the solvent
was removed to give a residue from which the desired
compound was separated by chromatography on silica
gel using ethyl acetate–methanol (4:1 v/v) as the mobile
phase. Alternatively, ( )-4-(4-chlorophenyl)-1-(1H-imi-
dazol-1-yl)-2-butanol was isolated from the reaction
mixture by addition of hexanes (10 mL), followed by
addition of ice-cold water until a precipitate started to
form. This mixture was then poured in small portions
onto ice–water, the separated solid was removed by fil-
tration, washed thoroughly, sequentially with cold
water, cold hexanes, and finally with cold water again
to give the desired imidazole–alcohol as a free base. In
order to prepare the corresponding hydrochlorides 9a
and 9e, the free bases of imidazole–alcohols were treated
with 37% aqueous HCl (molar ratio 1:1.3) in 2-propanol
(5–7 mL) in a manner similar to the one described for
the preparation of the hydrochlorides of the imidaz-
ole–ketones 5.
4.8. Biological evaluation
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).30,31 Spleen and brain (Sprague–Dawley
rats) microsomal fractions were prepared according to
the procedure outlined by Appleton et al.32 Protein con-
centration of microsomal fractions was determined by a
modification of the biuret method.31 Incubations for
HO activity analysis were done under conditions for
which the rate of CO formation (pmol CO · minꢀ1 · mg
proteinꢀ1) was linear with respect to time and micro-
somal protein concentration. Briefly, reaction mixtures
(150 lL) consisting of 100 mM phosphate buffer (pH
7.4), 50 lM methemalbumin, and 1 mg/mL protein were
pre-incubated with the inhibitors at final concentrations
ranging from 0.1 to 100 lM for 10 min at 37 ꢁC. Reac-
tions were initiated by adding NADPH at a final con-
centration of 1 mM and incubations were performed
for an additional 15 min at 37 ꢁC. Reactions were
stopped by instantly freezing the reaction mixture on
dry ice, and CO formation was monitored by gas chro-
matography according to the method described by Vre-
man and Stevenson.30
The data resulting from the above experiments were
plotted as non-linear regression (sigmoidal dose–re-
sponse) curves using the GraphPad Prism (version 3)
computer program. The values on the abscissa represent
the logarithm of the inhibitor’s concentration (in lM),
whereas the values of the activity on the ordinate are ex-
pressed as a percentage of the control experiments with-
out 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
4.7.1. ( )-4-(4-Fluorophenyl)-1-(1H-imidazol-1-yl)-2-
butanol hydrochloride (8b). Colorless solid (839 mg,
62%), mp 86–87 ꢁC, Rf = 0.0 (ethyl acetate); 1H
NMR (400 MHz, D2O): d 1.65–1.78 (m, 1H), 1.82–
1.92 (m, 1H), 2.64–2.75 (m, 1H), 2.77–2.87 (m, 1H),

3234
G. Roman et al. / Bioorg. Med. Chem. 15 (2007) 3225–3234
9. Vlahakis, J. Z.; Kinobe, R. T.; Bowers, R. J.; Brien, J. F.;
Nakatsu, K.; Szarek, W. A. Bioorg. Med. Chem. Lett.
2005, 15, 1457.
10. Vlahakis, J. Z.; Kinobe, R. T.; Bowers, R. J.; Brien, J. F.;
Nakatsu, K.; Szarek, W. A. J. Med. Chem. 2006, 49, 4437.
11. Boatman, S.; Harris, T. M.; Hauser, C. R. J. Org. Chem.
1965, 60, 3321.
12. Gaudry, M.; Marquet, A. Tetrahedron 1970, 26, 5611.
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Matthews, T. R. J. Med. Chem. 1978, 21, 840.
14. 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, 6, 1123.
top plateau of the curve, as well as the top and the bot-
tom plateau values of the curves, have been retrieved
using the same program, and input in the following for-
mula 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% com-
pared to the control.
EC50
bottomꢀtop
IC50
¼
ꢀ 1
50ꢀtop
The IC50 value reported for each compound is the
average of the values recorded in replicate experiments,
and for each of these replicate experiments (consisting
of two separate assays) an individual IC50 value was
calculated in the manner described. The IC50 values
for the replicate experiments were employed to generate
the reported standard deviation value.
15. Trakshel, G. M.; Kutty, R. K.; Maines, M. D. Arch.
Biochem. Biophys. 1988, 260, 732.
16. Maines, M. D. FASEB J. 1988, 10, 2557.
17. Vreman, H. J.; Cipkala, D. A.; Stevenson, D. K. Can. J.
Physiol. Pharmacol. 1996, 74, 278.
18. Wachall, B. G.; Hector, M.; Zhuang, Y.; Hartmann, R.
W. Bioorg. Med. Chem. 1999, 7, 1913.
19. Patel, C. H.; Dhanani, S.; Owen, C. P.; Ahmed, S. Bioorg.
Med. Chem. Lett. 2006, 16, 4752.
Acknowledgments
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The authors thank the Canadian Institutes of Health
Research for a Grant-in-Aid from the Canadian Insti-
tutes of Health Research (MOP 64305), and Ms. Tracy
Gifford and Mr. Brian McLaughlin for assistance with
the biological evaluations.
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