Identification and characterization of diarylimidazoles as hybrid inhibitors of butyrylcholinesterase and amyloid beta fibril formation

Article abstract of DOI:10.1016/j.ejps.2011.11.004

In this contribution, a chemical collection of aromatic compounds was screened for inhibition on butyrylcholinesterase (BChE)'s hydrolase activity using Ellman's reaction. A set of diarylimidazoles was identified as highly selective inhibitors of BChE hyd

Full text of DOI:10.1016/j.ejps.2011.11.004

European Journal of Pharmaceutical Sciences 45 (2012) 169–183
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Identification and characterization of diarylimidazoles as hybrid inhibitors
of butyrylcholinesterase and amyloid beta fibril formation
Daniela Karlsson a, Adyary Fallarero a, Gerda Brunhofer a,b, Przemyslaw Guzik b, Michaela Prinz c,
Ulrike Holzgrabe c, Thomas Erker b, , Pia Vuorela a,
⇑
⇑
a Pharmaceutical Sciences, Department of Biosciences, Abo Akademi University, BioCity, Artillerigatan 6A, FI-20520 Turku, Finland
b Department of Medicinal Chemistry, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria
c Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In this contribution, a chemical collection of aromatic compounds was screened for inhibition on butyr-
ylcholinesterase (BChE)’s hydrolase activity using Ellman’s reaction. A set of diarylimidazoles was iden-
tified as highly selective inhibitors of BChE hydrolase activity and amyloid b (Ab) fibril formation. New
derivatives were synthesized resulting in several additional hits, from which the most active was 6c,
4-(3-ethylthiophenyl)-2-(3-thienyl)-1H-imidazole, an uncompetitive inhibitor of BChE hydrolase activity
Received 26 September 2011
Received in revised form 24 October 2011
Accepted 8 November 2011
Available online 16 November 2011
(IC50 BChE = 0.10 lM; Ki = 0.073 0.011 lM) acting also on Ab fibril formation (IC50 = 5.8 lM). With the
Keywords:
Butyrylcholinesterase
Inhibition
aid of structure–activity relationship (SAR) studies, chemical motifs influencing the BChE inhibitory activ-
ity of these imidazoles were proposed. These bifunctional inhibitors represent good tools in basic studies
of BChE and/or promising lead molecules for AD therapy.
Imidazole
Ó 2011 Elsevier B.V. All rights reserved.
ChemGPS-NP
Lead refinement
Acetylcholinesterase
1. Introduction
Through the use of inhibitors as the main line of AD therapy,
acetylcholinesterase (AChE, EC 3.1.1.7 acetylcholine acetylhydro-
Alzheimer’s disease (AD) is an age-related brain disorder that
affected 26.6 million people worldwide in 2006 and is expected
to affect as much as 106.8 million by 2050 (Brookmeyer et al.,
2007). This high incidence together with the severity of the disor-
der has made it a major research area, but no cure is still available.
The formation of amyloid b (Ab) plaques and neurofibrillary
tangles in nerve ends, the hallmarks of the disease, contribute to
the inhibition of the cholinergic neurotransmission in synapses
and thus, to decreased cognitive function (Ballatore et al., 2007;
LaFerla et al., 2007). Treatment options include the most widely
used cholinesterase inhibitors and recently also memantine, an
lase) has been the lead target in order to restore or increase the de-
pleted acetylcholine (ACh) levels in the AD brain, which in turn
should improve cognitive function. However, the second enzyme
of the cholinesterase family, butyrylcholinesterase (BChE, EC
3.1.1.8 acylcholine acylhydrolase), has lately gained much atten-
tion as an alternative target in AD therapy. This enzyme, BChE, is
known to hydrolyze several choline and non-choline esters and it
functions, with AChE, as a co-regulator of the levels of ACh in the
human brain (Mendel and Rudney, 1943; Mesulam et al., 2002).
In this co-regulation, the function of BChE is to hydrolyze the
excessive ACh, due to its higher KM for ACh compared to AChE
(Giacobini, 2004).
N-methyl D-aspartate receptor antagonist (Farlow et al., 2008).
In the AD scenario, BChE seems to have an attractive involve-
ment. Studies have shown that as the disease progresses, the activ-
ity of AChE decreases while the activity of BChE remains unaffected
or in some cases even increases (Giacobini, 2004; Perry et al.,
1978). Since BChE can compensate for AChE, when the activity of
AChE is inhibited (Xie et al., 2000), a more relevant role of BChE
in regulating central cholinergic transmission seems to take place
in the brain of advanced staged AD patients. Then, instead of
hydrolyzing the excessive ACh occurring in the healthy brain, BChE
hydrolyses the already depleted levels of ACh in the AD brain
Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s
disease; Ab, amyloid b; BChE, butyrylcholinesterase; CVA, coefficient of variation of
the assay; DTNB, 5,50-dithiobis(2-nitro-benzoic acid); GT1-7, mouse hypothalamic
immortalized cells; HL, human lung epithelial cells; SAR, structure–activity
relationship; S/B, signal-to-background ratio; S/N, signal-to-noise ratio; ThT,
thioflavin T; Z’ factor, signal window coefficient.
⇑
Corresponding authors. Tel.: +43 1 4277 55003; fax: +43 1 4277 9551 (T. Erker),
tel.: +358 2 215 4267; fax: +358 2 215 5018 (P. Vuorela).
E-mail addresses: thomas.erker@univie.ac.at (T. Erker), pia.vuorela@abo.fi
(P. Vuorela).
0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2011.11.004

170
D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
(Greig et al., 2005). Furthermore, it has been proposed that individ-
uals with low-activity BChE variants can sustain cognitive function
better when compared to individuals with the normal-activity
BChE (Holmes et al., 2005). Using in vivo models, selective BChE
inhibition has shown to be of potential clinical value in ameliorat-
ing the symptoms of AD. Selective AChE inhibition elevated the
levels of ACh in normal mice with functional AChE, but in the AChE
nullizygote animals only selective BChE inhibitors increased ACh
levels (Hartmann et al., 2007). In addition, restoration of ACh levels
by BChE inhibition seems to occur without apparent adverse ef-
fects (Greig et al., 2005; Hartmann et al., 2007). Selective inhibition
of BChE by one cymserine derivative has additionally been shown
to augment ACh levels, increase cognitive function and decrease
amyloid deposits in aged rats (Greig et al., 2005).
2. Material and methods
2.1. Chemistry
Unless otherwise stated, all chemicals were purchased from Sig-
ma–Aldrich Chemie GmbH (Schnelldorf, Germany), Merck (Darms-
tadt, Germany) or Apollo Scientific Ltd. (Bredbury, UK) at analytical
grade and used without further purification. Anhydrous THF was
freshly distilled over sodium metal and benzophenone. All reac-
tions were carried out under argon atmosphere. Purification was
performed using preparative separation column chromatography
(Merck silica gel 60, 230–400 mesh). Analytical TLC was carried
out on pre-coated aluminium plates (Merck silica gel 60, F254).
Melting points were determined with a Kofler melting point appa-
ratus and are uncorrected. 1H and 13C NMR spectra were recorded
on a Brucker Advance DPx200 (200 and 50 MHz). Chemical shifts
are reported in d values (ppm) relative to tetramethylsilane
(TMS) as internal standard for 1H NMR spectra and the solvent
peak for 13C NMR. Peak splitting patterns in the 1H NMR are re-
ported as follows: s, singlet; d, doublet; t, triplet; m, multiplet;
br, broad. J values are reported in Hertz. Mass spectra (MS) were
obtained with a Shimadzu (GC-17A; MS-QP5050A) spectrometer.
Purity of the compounds 1a–c, 3a–c, 4a–c, 5a–c, 6a–b, 8a–i, 9a
and 9d–i was established by combustion analysis with a Perkin–El-
mer 2400 CHN elemental analyzer, and of the compounds 6c, 9b
and 9c by high resolution mass spectroscopy (HRMS), conforming
a purity P95%.
The connection between BChE inhibition and Ab fibril forma-
tion is still not completely understood. The Ab peptide is pro-
duced by proteolytic cleavage of the amyloid precursor protein
mediated by b- and
c-secretase. Two forms of the Ab peptide,
the variants Ab(1–40) and Ab(1–42) are involved in the formation
of the Ab extracellular aggregates that are claimed responsible
for causing neuronal cell death (Lorenzo and Yankner, 1994). Sev-
eral studies have proven that BChE exists in the Ab neuritic pla-
ques and neurofibrillary tangles (Mesulam et al., 1987).
Moreover, in 2006, a peculiar claim of human BChE’s role in AD
was made, which states that it is responsible for significantly
slowing down the in vitro Ab fibrils formation. This putative role
is not related to the hydrolase activity of BChE but is connected
to the imperfect amphipathic characteristics (due to a Trp substi-
tution) of the human BChE’s C-terminus (Diamant et al., 2006).
Thus, inhibiting BChE with molecules binding far away from the
C-terminus could leave unaffected BChE’s putative ability to pre-
vent amyloid deposition. Moreover, compounds with dual ability
to inhibit BChE’s hydrolase activity and impede Ab fibril formation
could therefore potentiate BChE’s neuroprotective ability while
facilitating cholinergic restoration.
2.1.1. 4-(2-Methoxyphenyl)-2-phenyl-1H-imidazole (1a)
The synthesis of compound 1a is described elsewhere (Naruse
et al., 2010).
2.1.2. 4-(3-Methoxyphenyl)-2-phenyl-1H-imidazole (1b)
The synthesis of compound 1b is described elsewhere (Thurkauf
et al., 1999).
Recently discovered inhibitors acting on the hydrolase activity
of BChE include cymserine analogues (Greig et al., 2005), hetero-
bivalent tacrine derivatives (Elsinghorst et al., 2006), quinazolini-
mines (Decker et al., 2008), phenothiazines (Darvesh et al., 2007,
2010), benzofurans (Rizzo et al., 2008) and isosorbide-based com-
pounds (Carolan et al., 2008, 2010; Dillon et al., 2010). The most
potent and highly selective BChE inhibitor reported so far belongs
to the group of isosorbide-based compounds and displays an IC50
BChE value of 0.15 nM with a high selectivity of over 60,000 times
towards BChE (Carolan et al., 2010). The isosorbide-based mole-
cules as well as the potent and selective cymserine analogues (Gre-
ig et al., 2005) are carbamates, with scaffolds inspired by the
classical cholinesterase inhibitors physostigmine and rivastigmine.
However, from all reported BChE inhibitors only two (one cymser-
ine analog (Greig et al., 2005) and one benfozuran-based (Rizzo
et al., 2008)) have been proven to also act on Ab fibril formation
at concentrations on the micromolar range. Thus, the development
of novel hybrid inhibitors acting on both the hydrolase activity of
BChE and Ab fibrilogenesis is an underexplored strategy with high-
er chances of success in AD pharmacotherapy.
In this contribution, a unique collection of small aromatic com-
pounds was assayed for inhibition of BChE’s hydrolase activity. The
tested collection is not commercially available. It comprises mostly
new (not yet reported) synthetic compounds belonging to different
structural scaffolds, partly based on natural products. We found
that three diarylimidazoles could act as hybrid inhibitors of both
BChE hydrolase activity and Ab fibril formation. Lead optimization
generated a bifunctional molecule with a higher potency on both
activities. These diarylimidazoles-based hybrid inhibitors could
evolve into novel inspirational leads for use in AD treatment and
help assess the basic functions of BChE.
2.1.3. 4-(2,5-Dimethoxyphenyl)-2-phenyl-1H-imidazole (1c)
The general procedure for 1c is described elsewhere (Li et al.,
2005). To a solution of benzamidine hydrochloride monohydrate
(0.31 g, 0.002 mol) in tetrahydrofuran (4 ml) and water (1 ml),
potassium bicarbonate is added slowly portionwise (0.40 g,
0.004 mol). The reaction mixture is vigorously heated to reflux
and a solution of 2-bromo-20,50-dimethoxyacetophenone (0.52 g,
0.002 mol) in 3 ml of THF is then added dropwise via syringe and
refluxed for 16 h. The reaction mixture was cooled to RT, and the
solvent was removed under reduced pressure. 10 ml of water
was added, and the resulting suspension was stirred at 50 °C for
30 min. The mixture was cooled and the precipitate was filtered,
washed with water, and dried. The solid was purified by crystalli-
zation in EtOH 96%. The product was dried to give 0.32 g (57%) of
1c; mp 60–65 °C; 1H NMR (CDCl3): d 12.6–12.3 (s, 1H), 8.20–6.63
(m, 9H), 3.85 (s, 3H), 3.76 (s, 3H), 3.57–3.25 (m, 2H), 1.28–0.92
(m, 3H); 13C NMR (CDCl3): d 153.3, 150.1, 144.7, 136.6, 130.6,
128.7, 128.1, 125.1, 123.8, 117.9, 112.2, 111.7, 56.1, 55.7, 55.4,
18.6; MS: m/z 280 (82%, M+), 265 (17%), 162 (100%), 140 (14%),
69 (15%).
2.1.4. General procedure for compounds 3a–c
A mixture of 2 (0.005 mol), substituted phenylboronic acid
(0.0052 mol), tetrakis(triphenylphosphine)palladium(0) (0.0005
mol), toluene (90 ml), methanol (18 ml) and 2 M K2CO3 solution
(5.9 ml) was refluxed under argon for 16 h. The reaction mixture
was cooled to RT and washed with 30 ml saturated aq. ammonium
chloride solution. The organic layer was dried over anhydrous

D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
171
Na2SO4 and evaporated. The residue was purified using column
chromatography.
7.09 (s, 1H), 7.02–6.94 (m, 1H), 5.24 (s, 2H), 3.84 (s, 3H), 3.57 (t,
J = 8.2 Hz, 2H), 0.93 (t, J = 8.2 Hz, 2H), ꢀ0.0003 (s, 9H); 13C NMR
(CDCl3): d 159.7, 148.5, 130.2, 129.6, 121.2, 120.3, 115.9, 115.5,
113.9, 75.6, 66.7, 55.4, 17.8, ꢀ1.4; MS: m/z 384 (M+, 16%), 326 (49%),
245 (100%), 103 (74%).
2.1.4.1. 4,5-Dibromo-2-(2,5-dimethoxyphenyl)-1-{[2-(1,1,1-trimeth-
ylsilyl)ethoxy]methyl}-1H-imidazole (3a). The title compound was
synthesized from
2
(2.18 g, 0.005 mol) and 2,5-dim-
ethoxyphenylboronic acid (0.95 g, 0.0052 mol) and was purified
by column chromatography (ethyl acetate/n-hexane 10:90) to af-
ford 3a as an oil (1.80 g, 74%); 1H NMR (CDCl3): d 7.05–6.85 (m,
3H), 5.23 (s, 2H), 3.77 (s, 3H), 3.75 (s, 3H), 3.23 (t, J = 8.3 Hz, 2H),
0.72 (t, J = 8.3 Hz, 2H), ꢀ0.10 (s, 9H); 13C NMR (CDCl3): d 153.8,
151.0, 119.3, 117.6, 116.9, 112.7, 104.3, 75.2, 66.5, 56.3, 55.8,
17.5, ꢀ1.58; MS: m/z 492 (M+, 2%), 354 (9%), 174 (13%), 103
(12%), 73 (100%).
2.1.5.3.
4-Bromo-2-(3-thienyl)-1-{[2-(1,1,1-trimethylsilyl)ethoxy]
methyl}-1H-imidazole (4c). The title compound was synthesized
from 3c (1.75 g, 0.004 mol) and was purified by column chroma-
tography (ethyl acetate/toluene 10:90) and crystallization in EtOH
90%. The product was dried to afford 4c as crystalline solid (1.16 g,
81%); mp 65–71 °C; 1H NMR (CDCl3): d 7.88–7.81 (m, 1H), 7.62–
7.54 (m, 1H), 7.43–7.35 (m, 1H), 7.02 (s, 1H), 5.27 (s, 2H), 3.61 (t,
J = 8.2 Hz, 2H), 0.95 (t, J = 8.3 Hz, 2H), 0.007 (s, 9H); 13C NMR
(CDCl3): d 144.6, 129.9, 128.1, 126.2, 125.6, 120.1, 115.0, 75.6,
66.8, 17.9, ꢀ1.3; MS: m/z 360 (M+, 0.7%), 302 (2%), 221 (4%), 103
(4%), 73 (100%), 69 (6%).
2.1.4.2. 4,5-Dibromo-2-(3-methoxyphenyl)-1-{[2-(1,1,1-trimethylsilyl)
ethoxy]methyl}-1H-imidazole (3b). The title compound was synthe-
sized from 2 (2.18 g, 0.005 mol) and 3-methoxyphenylboronic acid
(0.79 g, 0.0052 mol) and was purified by column chromatography
(ethyl acetate/n-hexane 10:90) to afford 3b as crystalline solid
(1.90 g, 83%); mp 61–63 °C; 1H NMR (CDCl3): d 7.40–7.30 (m, 3H),
7.05–6.94 (m, 1H), 5.31 (s, 2H), 3.84 (s, 3H), 3.65 (t, J = 8.3 Hz, 2H),
0.95 (t, J = 8.3 Hz, 2H), 0.004 (s, 9H); 13C NMR (CDCl3): d 159.7,
149.6, 130.3, 129.7, 121.1, 117.7, 116.2, 113.8, 105.2, 74.5, 66.8,
55.3, 17.9, ꢀ1.5; MS: m/z 462 (M+, 0.6%), 404 (9%), 352 (12%), 103
(25%), 73 (100%).
2.1.6. General procedure for compounds 5a–c
Compounds 5a–c were synthesized using the same general pro-
cedure as described in Section 2.1.4 using 4a, 4b and 4c,
respectively.
2.1.6.1. 2,4-Bis(2,5-dimethoxyphenyl)-1-{[2-(1,1,1-trimethylsilyl)eth-
oxy]methyl}-1H-imidazole (5a). The title compound was synthe-
sized from 4a (1.24 g, 0.003 mol) and 2,5-dimethoxyphenylbo
ronic acid (0.58 g, 0.0032 mol) and was purified by column chro-
matography (ethyl acetate/n-hexane 30:70) to afford 5a as an oil
(0.84 g, 60%); 1H NMR (CDCl3): d 7.92–7.88 (m, 1H), 7.82 (s, 1H),
7.17–7.12 (m, 1H), 7.02–6.85 (m, 3H), 6.81–6.72 (m, 1H), 5.21 (s,
2H), 3.92 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H), 3.73 (s, 3H), 3.29 (t,
J = 8.3 Hz, 2H), 0.77 (t, J = 8.3 Hz, 2H), ꢀ0.08 (s, 9H); 13C NMR
(CDCl3): d 153.8, 153.7, 151.2, 150.4, 144.2, 136.9, 123.5, 120.6,
120.0, 117.1, 116.5, 113.1, 112.7, 111.9, 111.8, 75.7, 65.8, 56.2,
55.8, 55.7, 55.7, 17.4, ꢀ1.64; MS: m/z 470 (M+, 1%), 370 (1%), 277
(2%), 161 (5%), 117 (19%), 59 (100%).
2.1.4.3. 4,5-Dibromo-2-(3-thienyl)-1-{[2-(1,1,1-trimethylsilyl)ethoxy]
methyl}-1H-imidazole (3c). The title compound was synthesized
from 2 (2.18 g, 0.005 mol) and 3-thienylboronic acid (0.67 g,
0.0052 mol) and was purified by column chromatography (ethyl
acetate/n-hexane 10:90) to afford 3c as an oil (1.83 g, 84%); 1H
NMR (CDCl3): d 7.96–7.89 (m, 1H), 7.62–7.54 (m, 1H), 7.45–7.35
(m, 1H), 5.36 (s, 2H), 3,71 (t, J = 8.3 Hz, 2H), 0.97 (t, J = 8.3 Hz,
2H), 0.02 (s, 9H); 13C NMR (CDCl3): d 145.8, 130.1, 128.0, 126.2,
125.9, 117.4, 104.7, 74.4, 67.0, 18.0, ꢀ1.4; MS: m/z 438 (M+,
0.5%), 376 (1%), 103 (4%), 73 (100%), 57 (7%).
2.1.6.2. 2,4-Bis(3-methoxyphenyl)-1-{[2-(1,1,1-trimethylsilyl)ethoxy]
methyl}-1H-imidazole (5b). The title compound was synthesized
from 4b (0.77 g, 0.002 mol) and 3-methoxyphenylboronic acid
(0.33 g, 0.0032 mol) and was purified by column chromatography
(ethyl acetate/n-hexane 20:80) to afford 5b as an oil (0.69 g,
84%); 1H NMR (CDCl3): d 7.51–7.28 (m, 7H), 7.05–6.94 (m, 1H),
6.87–6.78 (m, 1H), 5.31 (s, 2H), 3.88 (s, 3H), 3.87 (s, 3H), 3.60 (t,
J = 8.2 Hz, 2H), 0.94 (t, J = 8.2 Hz, 2H), 0.004 (s, 9H); 13C NMR
(CDCl3): d 160.0, 159.8, 148.6, 141.2, 135.4, 131.4, 129.6, 129.5,
121.4, 117.6, 117.2, 115.4, 114.2, 113.1, 110.2, 75.6, 66.5, 55.4,
55.3, 17.8, ꢀ1.4; MS: m/z 410 (M+, 2%), 352 (8%), 293 (5%), 117
(15%), 102 (18%), 73 (100%).
2.1.5. General procedure for dehalogenization via lithiation for
compounds 4a–c
A solution of n-BuLi in n-hexane (1.6 M, 1 eq.) was added drop-
wise under argon at ꢀ78 °C to a solution of 3a–c (1 eq.), respec-
tively, in anhydrous THF (5 ml). After stirring the reaction
mixture for 1 min at ꢀ78 °C, water (1 ml) was added. The reaction
mixture was allowed to warm to RT, and after that was washed
with 30 ml of a saturated aqueous solution of ammonium chloride.
The organic layer was dried over anhydrous sodium sulfate and
evaporated. The residue was subjected to column chromatography
(Langhammer and Erker, 2005).
2.1.5.1. 4-Bromo-2-(2,5-dimethoxyphenyl)-1-{[2-(1,1,1-trimethylsilyl)
ethoxy]methyl}-1H-imidazole (4a). The title compound was synthe-
sized from 3a (1.480 g, 0.003 mol) and was purified by column chro-
matography (ethyl acetate/n-hexane20:80) to afford 4a as crystalline
solid(1.20 g, 97%);mp71–76 °C;1HNMR(CDCl3):d 7.13 (s, 1H), 7.05–
6.85 (m, 3H), 5.12 (s, 2H), 3.78 (s, 3H), 3.73 (s, 3H), 3.26 (t, J = 8.3 Hz,
2H), 0.76 (t, J = 8.3 Hz, 2H), ꢀ0.08 (s, 9H); 13C NMR (CDCl3): d 153.8,
151.1, 145.1, 119.2, 118.6, 117.4, 116.8, 115.6, 112.6, 76.1, 66.4,
56.3, 55.8, 17.6, ꢀ1.46; MS: m/z 414 (M+, 9%), 296 (10%), 275 (27%),
176 (18%), 73 (100%).
2.1.6.3. 4-(3-Ethylthiophenyl)-2-(3-thienyl)-1-{[2-(1,1,1-trimethylsilyl)
ethoxy]methyl}-1H-imidazole (5c). The title compound was synthe-
sized from 4c (0.72 g, 0.002 mol) and 3-ethylthiophenylboronic acid
(0.40 g, 0.0022 mol) and was purified by column chromatography
(toluene/ethyl acetate 95:5) to afford 5c as an oil (0.63 g, 76%); 1H
NMR (CDCl3): d 7.89–7.77 (m, 2H), 7.66–7.57 (m, 2H), 7.41–7.14 (m,
4H), 5.29 (s, 2H), 3.60 (t, J = 8,2 Hz, 2H), 2.95 (q, J = 7.3 Hz, 2H), 1.28
(t, J = 7.3 Hz, 2H), 0.92 (t, J = 8.2 Hz, 2H), ꢀ0.04 (s, 9H); 13C NMR
(CDCl3): d 144.8, 140.2, 136.9, 134.4, 129.0, 128.3, 127.7, 126.0,
125.8, 125.3, 122.7, 117.0, 75.6, 66.6, 27.8, 17.9, 14.4, ꢀ1.4; MS: m/z
416 (M+, 1.4%), 154 (29%), 91 (19%), 73 (100%), 69 (58%).
2.1.5.2. 4-Bromo-2-(3-methoxyphenyl)-1-{[2-(1,1,1-trimethylsilyl)eth-
oxy]methyl}-1H-imidazole (4b). The title compound was synthesized
from3b(1.39 g,0.003 mol)andwaspurifiedbycolumnchromatogra-
phy (ethyl acetate/n-hexane 10:90) to afford 4b as crystalline solid
(0.99 g, 86%); mp 55–57 °C; 1H NMR (CDCl3): d 7.40–7.28 (m, 3H),
2.1.7. General procedure for cleavage of the protecting group for
compounds 6a–c
A solution of 5a–c (0.001 mol) in 25 ml ethanol and 7 ml
concentrated HCl was refluxed for 16 h. After cooling to RT, the

172
D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
reaction mixture was neutralized with 6 M aq NaOH. Ethanol was
evaporated and the residue extracted with dichloromethane. The
combined organic extracts were dried over anhydrous sodium sul-
fate and evaporated. The product was purified by column chroma-
tography or recrystallized from ethanol (Langhammer and Erker,
2005).
7.40–7.30 (m, 1H), 7.22 (s, 1H), 7.20–7.13 (m, 2H), 6.96–6.88 (m,
1H), 5.14 (s, 2H), 4.1 (q, J = 7.0 Hz, 2H), 3.45 (t, J = 8.4 Hz, 2H),
1.44 (t, J = 7.0 Hz, 3H), 0.97 (t, J = 8.3 Hz, 2H), 0.004 (s, 9H); 13C
NMR (CDCl3): d 159.2, 150.1, 135.4, 131.2, 130.6, 129.8, 129.0,
128.9, 128.6, 127.5, 121.0, 114.8, 114.3, 73.3, 65.5, 63.5, 18.0,
14.8, ꢀ1.5; MS: m/z 394 (M+, 14%), 336 (62%), 277 (9%), 73
(100%), 45 (13%).
2.1.7.1. 2,4-Bis(2,5-dimethoxyphenyl)-1H-imidazole (6a). The title
compound was synthesized from 5a and was purified by recrystal-
lization from ethanol 96% to afford 6a as crystalline solid (0.31 g,
92%); mp 166–168 °C; 1H NMR (CDCl3): d 11.78 (br s, 1H), 7.94
(s, 1H), 7.57 (s, 1H), 7.26 (s, 1H), 6.99–6.73 (m, 4H), 4.00 (s, 3H),
3.95 (s, 3H), 3.88 (s, 3H), 3.84 (s, 3H); 13C NMR (CDCl3): d 154.2,
154.1, 150.0, 143.9, 118.9, 116.0, 112.8, 112.7, 111.9, 111.5, 56.3,
56.2, 55.9, 55.8; MS: m/z 340 (M+, 91%), 307 (24%), 205 (26%),
162 (100%), 155 (38%).
2.1.8.3. 5-(3-Propoxyphenyl)-2-phenyl-1-{[2-(1,1,1-trimethylsilyl)eth-
oxy]methyl}-1H-imidazole (8c). The title compound was synthesized
from
7 (0.80 g, 0.002 mol) and 3-propoxyphenylboronic acid
(0.40 g, 0.0022 mol) and was purified by column chromatography
(ethyl acetate/n-hexane 20:80) to afford 8c as an oil (0.64 g, 79%);
1H NMR (CDCl3): d 7.95–7.83 (m, 2H), 7.55–7.42 (m, 3H), 7.41–7.30
(m, 1H), 7.25–7.12 (m, 3H), 7.01–6.89 (m, 1H), 5.14 (s, 2H), 3.96 (t,
J = 6.6 Hz, 2H), 3.46 (t, J = 8.3 Hz, 2H), 1.94–1.74 (m, 2H), 1.06 (t,
J = 7.3 Hz, 2H), 0.98 (t, J = 8.4 Hz), 0.004 (s, 9H); 13C NMR (CDCl3): d
159.5, 150.1, 135.5, 131.2, 130.5, 129.8, 129.2, 129.0, 128.6, 127.3,
121.0, 114.9, 114.4, 73.3, 69.6, 65.5, 22.7, 18.1, 10.6, ꢀ1.5; MS: m/z
408 (M+, 25%), 350 (52%), 308 (12%), 103 (10%), 73 (100%).
2.1.7.2. 2,4-Bis(3-methoxyphenyl)-1H-imidazole (6b). The title com-
pound was synthesized from 5b and was purified by recrystalliza-
tion from ethanol 96% to afford 6b as crystalline solid (0.24 g, 86%);
mp 148–154 °C; 1H NMR (DMSO-d6): d 8.27 (s, 1H), 7.93 (s, 1H),
7.86–7.77 (m, 1H), 7.69–7.63 (m, 1H), 7.61–7.42 (m, 3H), 7.25–
7.15 (m, 1H), 7.05–6.96 (m, 1H), 3.90 (s, 3H), 3.86 (s, 3H); 13C
NMR (DMSO-d6): d 159.7, 159.6, 144.4, 134.5, 130.4, 130.1, 129.2,
125.3, 119.3, 118.0, 117.6, 116.9, 114.6, 112.3, 111.3, 55.7, 55.5;
MS: m/z 280 (M+, 100%), 279 (45%), 250 (18%), 140 (16%), 89
(17%), 77 (31%).
2.1.8.4. 5-(3-Butoxyphenyl)-2-phenyl-1-{[2-(1,1,1-trimethylsilyl)eth-
oxy]methyl}-1H-imidazole (8d). The title compound was synthe-
sized from 7 (0.80 g, 0.002 mol) and 3-butoxyphenylboronic acid
(0.43 g, 0.0022 mol) and was purified by column chromatography
(ethyl acetate/n-hexane 30:70) to afford 8d as an oil (0.68 g,
80%); 1H NMR (CDCl3): d 7.94–7.82 (m, 2H), 7.55–7.42 (m, 3H),
7.40–7.29 (m, 1H), 7.27–7.12 (m, 3H), 6.98–6.88 (m, 1H), 5.13
(s, 2H), 4.00 (t, J = 6.4 Hz, 2H), 3.46 (t, J = 8.3 Hz, 2H), 1.89–1.71
(m, 2H), 1.62–1.40 (m, 2H), 1.06–0.92 (m, 5H), 0.01 (s, 9H); 13C
NMR (CDCl3): d 159.4, 150.1, 135.4, 131.2, 130.6, 129.7, 129.0,
128.9, 128.5, 127.5, 120.9, 114.8, 114.3, 73.2, 67.7, 65.4, 31.3,
19.2, 18.0, 13.8, ꢀ1,5; MS: m/z 422 (M+, 50%), 392 (17%), 364
(96%), 308 (34%), 73 (100%).
2.1.7.3. 4-(3-Ethylthiophenyl)-2-(3-thienyl)-1H-imidazole (6c). The
title compound was synthesized from 5c and was purified by col-
umn chromatography (toluene/ethyl acetate 80:20) to afford 6c
as an oil (0.22 g, 77%). For converting 6c into the appropriate
hydrochloride, a solution of hydrochloric acid in ether (1.0 M,
1 eq.) was added drop wise under argon to a solution of 6c in dry
THF. After stirring the reaction mixture for 1 h, the precipitate
was filtered and dried; mp (HCl–salt) 153–160 °C; 1H NMR (CDCl3):
d 9.18 (br s, 1H), 7.79–7.65 (m, 2H), 7.57–7.46 (m, 2H), 7.34–7.14
(m, 4H), 2.90 (q, J = 7.3 Hz, 2H), 1.26 (t, J = 7.3 Hz, 2H); 13C NMR
(CDCl3): d 144.1, 138.0, 137.3, 132.8, 131.4, 129.2, 127.6, 126.8,
125.6, 125.5, 122.7, 122.6, 117.0, 27.5, 14.3; MS: m/z 286 (M+,
100%), 258 (37%), 201 (33%), 69 (46%), 57 (61%).
2.1.8.5. 5-(3-Isobutoxyphenyl)-2-phenyl-1-{[2-(1,1,1-trimethylsilyl)
ethoxy]methyl}-1H-imidazole (8e). The title compound was synthe-
sized from 7 (0.80 g, 0.002 mol) and 3-butoxyphenylboronic acid
(0.43 g, 0.0022 mol) and was purified by column chromatography
(ethyl acetate/n-hexane 30:70) to afford 8e as an oil (0.72 g,
85%); 1H NMR (CDCl3): d 7.95–7.82 (m, 2H), 7.55–7.41 (m, 3H),
7.40–7.29 (m, 1H), 7.27–7.11 (m, 3H), 6.99–6.88 (m, 1H), 5.10 (s,
2H), 3.83–3.72 (m, 2H), 3.47 (t, J = 8.3 Hz, 2H), 2.22–2.00 (m, 1H)
1.14–0.91 (m, 8H), 0.004 (s, 9H); 13C NMR (CDCl3): d 159.6,
150.1, 135.5, 131.2, 130.6, 129.7, 129.0, 128.9, 128.6, 127.5,
120.9, 114.8, 114.3, 74.5, 73.3, 65.5, 28.3, 19.3, 18.1, ꢀ1.5; MS:
m/z 423 (M+, 1.2%), 364 (8%), 307 (7%), 103 (5%), 73 (100%), 41
(12%).
2.1.8. General procedure for compounds 8a–i
Compounds 8a–i were synthesized using the same general pro-
cedure as in Section 2.1.4 but using compound 7 as starting
material.
2.1.8.1. 5-(2,3-Dimethoxyphenyl)-2-phenyl-1-{[2-(1,1,1-trimethylsilyl)
ethoxy]methyl}-1H-imidazole (8a). The title compound was synthe-
sized from 7 (0.80 g, 0.002 mol) and 2,3-dimethoxyphenylboronic
acid (0.40 g, 0.0022 mol) and was purified by column chromatogra-
phy (ethyl acetate/n-hexane 20:80) to afford 8a as an oil (0.56 g,
68%); 1H NMR (CDCl3): d 7.93–7.82 (m, 3H), 7.77 (s, 1H), 7.53–7.41
(m, 3H), 7.18–7.08 (m, 1H), 6.88–6.80 (m, 1H), 5.32 (s, 2H), 3.91 (s,
3H), 3.87 (s, 3H), 3.64 (t, J = 8.2 Hz, 2H), 0.96 (t, J = 8.2 Hz, 2H),
ꢀ0.006 (s, 9H); 13C NMR (CDCl3): d 152.9, 147.7, 145.9, 136.3, 130.2,
129.1, 129.0, 128.6, 127.8, 124.3, 123.3, 123.2, 121.4, 119.8, 111.6,
110.7, 75.6, 66.4, 59.5, 55.8, 17.8, ꢀ1.4; MS: m/z 410 (M+, 36%), 293
(43%), 274 (100%), 228 (51%), 176 (29%), 73 (86%).
2.1.8.6. 5-(2-Ethoxyphenyl)-2-phenyl-1-{[2-(1,1,1-trimethylsilyl)eth-
oxy]methyl}-1H-imidazole (8f). The title compound was synthe-
sized from 7 (1.20 g, 0.003 mol) and 2-ethoxyphenylboronic acid
(0.53 g, 0.0032 mol) and was purified by column chromatography
(ethyl acetate/n-hexane 30:70) to afford 8f as crystalline solid
(0.91 g, 77%); mp 42–49 °C; 1H NMR (CDCl3): d 7.97–7.86 (m,
2H), 7.50–7.33 (m, 5H), 7.17 (s, 1H), 7.08–6.94 (m, 2H), 5.23
(s, 2H), 4.06 (q, J = 6.9 Hz, 2H), 3.19 (t, J = 8.4 Hz, 2H), 1.33
(t, J = 7.0 Hz, 3H), 0.73 (t, J = 8.3 Hz, 2H), ꢀ0.11 (s, 9H); 13C NMR
(CDCl3): d 156.4, 149.3, 132.1, 131.4, 130.8, 130.1, 129.0, 128.7,
128.4, 128.3, 120.8, 119.4, 112.2, 73.6, 65.8, 63.9, 17.7, 14.7,
ꢀ1.60; MS: m/z 394 (M+, 21%), 321 (53%), 277 (18%), 176 (15%),
73 (100%).
2.1.8.2. 5-(3-Ethoxyphenyl)-2-phenyl-1-{[2-(1,1,1-trimethylsilyl)eth-
oxy]methyl}-1H-imidazole (8b). The title compound was synthe-
sized from 7 (1.20 g, 0.003 mol) and 3-ethoxyphenylboronic acid
(0.53 g, 0.0032 mol) and was purified by column chromatography
(ethyl acetate/n-hexane 30:70) to afford 8b as an oil (0.75 g,
64%); 1H NMR (CDCl3): d 7.91–7.82 (m, 2H), 7.53–7.42 (m, 3H),
2.1.8.7. 5-(3-Methylthiophenyl)-2-phenyl-1-{[2-(1,1,1-trimethylsilyl)
ethoxy]methyl}-1H-imidazole (8g). The title compound was synthe-
sized from 7 (1.20 g, 0.003 mol) and 3-methylthiophenylboronic

D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
173
acid (0.54 g, 0.0032 mol) and was purified by column chromatogra-
phy (ethyl acetate/n-hexane 20:80) to afford 8g as an oil (0.65 g,
54%): 1H NMR (CDCl3): d 7.91–7.82 (m, 2H), 7.55–7.44 (m, 4H),
7.39–7.34 (m, 2H), 7.30–7.22 (m, 2H), 5.11 (s, 2H), 3.47 (t,
J = 8.3 Hz, 2H), 2.53 (s, 3H), 1.00 (t, J = 8.4 Hz, 2H), 0.017 (s, 9H);
13C NMR (CDCl3): d 150.2, 139.5, 135.1, 130.6, 130.4, 129.2,
129.2, 129.0, 128.7, 127.5, 126.5, 126.1, 125.4, 73.3, 65.6, 18.1,
15.7, ꢀ1.5; MS: m/z 396 (M+, 12%), 338 (77%), 73 (100%), 57
(36%), 43 (55%).
2.1.9.3. 4-(3-Propoxyphenyl)-2-phenyl-1H-imidazole (9c). The title
compound was synthesized from 8c and was purified by column
chromatography (ethyl acetate/n-hexane 40:60) to afford 9c as
oil (0.25 g, 89%). For converting 9c into the appropriate hydrochlo-
ride, a solution of hydrochloric acid in ether (1.0 M, 1 eq.) was
added drop wise under argon to a solution of 9c in dry THF. After
stirring the reaction mixture for 1 h, the precipitate was filtered
and dried; mp (HCl–salt) 147–151 °C; 1H NMR (CDCl3): d 8.80–
8.00 (br s, 1H), 7.98–7.78 (m, 2H), 7.50–7.23 (m, 7H), 6.91–6.72
(m, 1H), 3.93 (t, J = 6.6 Hz, 2H), 1.90–1.69 (m, 2H), 1.02
(t, J = 7.3 Hz, 2H); 13C NMR (CDCl3): d 159.5, 146.9, 133.6, 129.7,
129.6, 128.7, 125.3, 117.2, 113.4, 110.9, 69.4, 22.5, 10.4; MS: m/z
278 (M+, 100%), 236 (81%), 208 (26%), 77 (42%), 43 (32%), 41 (30%).
2.1.8.8. 5-(2-Methylthiophenyl)-2-phenyl-1-{[2-(1,1,1-trimethylsilyl)
ethoxy]methyl}-1H-imidazole (8h). The title compound was synthe-
sized from 7 (1.20 g, 0.003 mol) and 2-methylthiophenylboronic
acid (0.54 g, 0.0032 mol) and was purified by column chromatogra-
phy (ethyl acetate/n-hexane 20:80) to afford 8h as an oil (0.98 g,
82%); 1H NMR (CDCl3): d 7.86–7.74 (m, 2H), 7.52–7.39 (m, 3H),
7.20–7.09 (m, 2H), 5.29 (s, 2H), 3.57 (t, J = 8.2 Hz, 2H), 2.40 (s,
3H), 0.92 (t, J = 8.3 Hz, 2H), ꢀ0.009 (s, 9H); 13C NMR (CDCl3): d
149.4, 140.7, 132.2, 131.8, 130.3, 129.7, 129.1, 129.0, 128.6,
128.3, 127.9, 124.6, 124.4, 73.4, 66.1, 17.8, 15.2, ꢀ1.5; MS: m/z
396 (M+, 61%), 323 (18%), 279 (35%), 265 (29%), 176 (50%), 73
(100%).
2.1.9.4. 4-(3-Butoxyphenyl)-2-phenyl-1H-imidazole (9d). The title
compound was synthesized from 8d and was purified by column
chromatography (ethyl acetate/n-hexane 30:70) to afford 9d as
oil (0.26 g, 86%). For converting 9d into the appropriate hydrochlo-
ride, a solution of hydrochloric acid in ether (1.0 M, 1 eq.) was
added drop wise under argon to a solution of 9d in dry THF. After
stirring the reaction mixture for 1 h, the precipitate was filtered
and dried; mp (HCl–salt) 153–158 °C; 1H NMR (CDCl3): d 10.6–
10.1 (br s, 1H), 7.93–7.75 (m, 2H), 7.46–7.14 (m, 7H), 6.88–6.71
(m, 1H), 4.00–3.83 (m, 2H), 1.83–1.63 (m, 2H), 1.56–1.36 (m,
2H), 1.05–0.83 (m, 3H); 13C NMR (CDCl3): d 159.6, 147.4, 139.0,
133.8, 130.0, 129.7, 128.8, 128.7, 125.6, 118.1, 117.4, 113.6,
110.9, 67.7, 31.4, 19.2, 13.9; MS: m/z 292 (M+, 1.7%), 81 (22%), 69
(88%), 55 (46%), 43 (100%), 41 (65%).
2.1.8.9.
5-(3-Ethylthiophenyl)-2-phenyl-1-{[2-(1,1,1-trimethylsilyl)
ethoxy]methyl}-1H-imidazole (8i). The title compound was synthe-
sized from 7 (1.20 g, 0.003 mol) and 3-ethylthiophenylboronic acid
(0.58 g, 0.0032 mol) and was purified by column chromatography
(ethyl acetate/n-hexane 20:80) to afford 8i as an oil (0.93 g, 76%);
1H NMR (CDCl3): d 7.90–7.79 (m, 2H), 7.60–7.54 (m, 1H), 7.51–
7.41 (m, 3H), 7.40–7.29 (m, 3H), 7.25–7.20 (m, 1H), 5.08 (s, 2H),
3.44 (t, J = 8.3 Hz, 2H), 2.97 (q, J = 7.3 Hz, 2H), 1.32 (t, J = 7.3 Hz,
3H), 0.98 (t, J = 8.3 Hz, 2H), ꢀ0.01 (s, 9H); 13C NMR (CDCl3): d
150.0, 137.8, 135.0, 130.3, 129.8, 129.4, 129.2, 129.1, 129.0,
128.7, 128.6, 128.2, 126.9, 126.0, 73.3, 65.6, 27.4, 18.0, 14.3,
ꢀ1.5; MS: m/z 410 (M+, 4%), 352 (12%), 75 (10%), 73 (100%), 57
(37%).
2.1.9.5. 4-(3-Isobutoxyphenyl)-2-phenyl-1H-imidazole (9e). The title
compound was synthesized from 8e and was purified by column
chromatography (ethyl acetate/n-hexane 30:70) to afford 9e as
oil (0.27 g, 92%). For converting 9e into the appropriate hydrochlo-
ride, a solution of hydrochloric acid in ether (1.0 M, 1 eq.) was
added drop wise under argon to a solution of 9e in dry THF. After
stirring the reaction mixture for 1 h, the precipitate was filtered
and dried; mp (HCl–salt) 162–168 °C; 1H NMR (CDCl3): d 9.99 (br
s, 1H), 7.99–7.75 (m, 2H), 7.49–7.16 (m, 7H), 6.94–6.70 (m, 1H),
3.77–3.64 (m, 2H), 2.17–1.93 (m, 1H), 1.14–0.84 (m, 6H); 13C
NMR (CDCl3): d 159.5, 147.1, 138.8, 133.6, 129.8, 129.5, 128.6,
128.5, 125.4, 117.8, 117.1, 113.4, 110.8, 74.2, 28.2, 19.1; MS: m/z
292 (M+, 2.6%), 236 (5%), 69 (31%), 57 (17%), 43 (100%), 41 (26%).
2.1.9. General procedure for compounds 9a–i
Compounds 9a–i were synthesized using the same general pro-
cedure as described in Section 2.1.7 but using compounds 8a–i.
2.1.9.1. 4-(2,3-Dimethoxyphenyl)-2-phenyl-1H-imidazole (9a). The
title compound was synthesized from 8a and was purified by col-
umn chromatography (ethyl acetate/n-hexane 30:70) to afford 9a
as an oil (0.18 g, 64%). For converting 9a into the appropriate
hydrochloride, a solution of hydrochloric acid in ether (1.0 M,
1 eq.) was added drop wise under argon to a solution of 9a in
dry THF. After stirring the reaction mixture for 1 h, the precipitate
was filtered and dried; mp (HCl–salt) 94–97 °C; 1H NMR (CDCl3): d
7.95–7.80 (m, 2H), 7.57 (s, 1H), 7.50–7.30 (m, 4H), 7.16–7.04 (m,
1H), 6.90–6.80 (m, 1H), 3.91 (s, 3H), 3.88 (s, 3H); 13C NMR (CDCl3):
d 153.1, 146.3, 144.7, 131.1, 129.8, 129.0, 128.8, 125.1, 124.8,
124.0, 118.8, 111.0, 60.5, 55.9; MS: m/z 280 (M+, 84%), 264 (17%),
162 (100%), 132 (29%), 104 (26%), 77 (26%).
2.1.9.6. 4-(2-Ethoxyphenyl)-2-phenyl-1H-imidazole (9f). The title
compound was synthesized from 8f and was purified by recrystal-
lization from ethanol 96% to afford 9f as crystalline solid (0.12 g,
45%); mp 54–59 °C; 1H NMR (DMSO-d6/CDCl3): d 8.23–8.04 (m,
3H), 7.73 (s, 1H), 7.60–7.45 (m, 3H), 7.38–7.23 (m, 1H), 7.10–6.98
(s, 2H), 4.20 (q, J = 6.9 Hz, 2H), 1.53 (t, J = 6.9 Hz, 3H); 13C NMR
(DMSO-d6): d 154.9, 144.1, 132.2, 129.5, 128.4, 127.1, 126.3,
125.9, 120.0, 118.9, 118.1, 111.4, 63.3, 14.3; MS: m/z 264 (M+,
81%), 249 (14%), 132 (100%), 104 (30%), 77 (30%). HRMS (ESI/MS)
calcd for C17H16N2OH: 265.1341, found: 265.1323.
2.1.9.7. 4-(3-Methylthiophenyl)-2-phenyl-1H-imidazole (9g). The ti-
tle compound was synthesized from 8g and was purified by recrys-
tallization from ethanol 96% to afford 9g as crystalline solid (0.25 g,
92%); mp 153–156 °C; 1H NMR (CDCl3): d 8.10–8.01 (m, 2H), 7.77–
7.72 (m, 1H), 7.60–7.52 (m, 1H), 7.48–7.29 (m, 5H), 7.16–7.07 (m,
1H), 2.54 (s, 3H); 13C NMR (CDCl3): d 146.3, 138.0, 137.4, 132.8,
129.5, 128.3, 127.8, 127.7, 124.8, 123.8, 122.0, 120.9, 14.9; MS:
m/z 266 (M+, 100%), 265 (40%), 220 (19%), 89 (18%), 43 (19%).
2.1.9.2. 4-(3-Ethoxyphenyl)-2-phenyl-1H-imidazole (9b). The title
compound was synthesized from 8b and was purified by recrystal-
lization from ethanol 96% to afford 9b as crystalline solid (0.25 g,
95%); mp 132–137 °C; 1H NMR (CDCl3): d 7.91–7.78 (m, 2H),
7.38–7.22 (m, 7H), 6.83–6.73 (m, 1H), 4.00 (q, J = 6.9 Hz, 2H),
1.37 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3): d 159.3, 147.3, 138.9,
133.8, 129.9, 129.7, 128.8, 128.7, 125.5, 117.4, 113.5, 110.9, 63.4,
14.9; MS: m/z 264 (M+, 100%), 236 (42%), 208 (20%), 104 (23%),
77 (42%); HRMS (ESI/MS) calcd for C17H16N2OH: 265.1341, found:
265.1338.
2.1.9.8. 4-(2-Methylthiophenyl)-2-phenyl-1H-imidazole (9h). The ti-
tle compound was synthesized from 8h and was purified by recrys-
tallization from ethanol 70% to afford 9h as crystalline solid (0.20 g,

174
D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
75%); mp 167–169 °C; 1H NMR (CDCl3): d 8.33–8.01 (br s, 1H),
7.96–7.83 (m, 2H), 7.74–7.63 (m, 1H), 7.48 (s, 1H), 7.44–7.17 (m,
6H), 2.42 (s, 3H); 13C NMR (CDCl3): d 146.4, 135.0, 134.7, 130.8,
129.7, 129.1, 128.9, 128.1, 127.9, 125.9, 125.4, 123.5, 16.9; MS:
m/z 266 (M+, 100%), 220 (11%), 162 (14%), 148 (41%), 121 (32%),
117 (34%).
iodide (ATCI) was used as substrate (diluted in H2O, final concentra-
tion of 1.5 mM) and 0.224 U/ml AChE (EC 3.1.1.7, from Electric eel,
Sigma–Aldrich, USA) was used as enzyme (diluted in Tris–HCl
50 mM pH 8, containing 0.1% BSA).
2.3.1. Potency determinations
Depending on the results from the primary screening, concen-
2.1.9.9. 4-(3-Ethylthiophenyl)-2-phenyl-1H-imidazole (9i). The title
compound was synthesized from 8i and was purified by column
chromatography (ethyl acetate/n-hexane 50:50) to afford 9i as an
oil (0.22 g, 69%). For converting 9i into the appropriate hydrochlo-
ride, a solution of hydrochloric acid in ether (1.0 M, 1 eq.) was
added dropwise under argon to a solution of 9i in dry THF. After
stirring the reaction mixture for 1 h, the precipitate was filtered
and dried; mp (HCl–salt) 173–177 °C; 1H NMR (DMSO-d6): d
15.12 (br s, 1H), 8.45–8.26 (m, 3H), 8.01 (s, 1H), 7.92–7.80 (m,
1H), 7.79–7.63 (m, 3H), 7.58–7.35 (m, 2H), 3.20–3.85 (m, 2H),
1.39–1.20 (m, 3H); 13C NMR (DMSO-d6): d 144.6, 137.7, 133.5,
131.8, 129.6, 129.2, 128.1, 128.0, 127.4, 124.6, 123.5, 122.9,
117.0, 25.9, 14.1; MS: m/z 280 (M+, 100%), 252 (33%), 121 (30%),
89 (27%), 77 (37%).
tration series of the active compounds between 0.02 and 60 lM
were selected. At least seven different concentrations were used
in four replicates. In order to quantify the inhibitor’s selectivity,
concentration-dependency effects on AChE were also measured
using the same concentrations and further tested up to 250 lM.
2.3.2. Kinetic studies
These experiments were carried out according to the previously
described BChE assay (Section 2.3). Inhibitors were tested at differ-
ent concentrations (0.03–5 lM) with substrate (BTCCl) concentra-
tion ranging from 0.065 to 1.5 mM. Rates of the enzymatic
reactions were calculated for each experimental condition tested
and compared to the rates obtained in absence of inhibitors [I].
The results were fitted into the conventional Lineweaver–Burk
double-reciprocal (1/V vs. 1/[S]) and Dixon (1/V vs. [I]) kinetic plots
to identify the inhibition types and calculate Ki values. The appar-
ent Ki constants were calculated using following equation:
2.2. Chemical library
Stock solutions of all tested compounds were made by scaling
the compounds in cryogenic tubes (external threaded polypropyl-
ene vials with plug seal caps) and dissolving them in dry DMSO to a
final concentration of 20 mM. They were maintained at ꢀ20 °C. In
order to facilitate the screening process, the chemical library was
transferred to 96-well microplates using Biomek 3000 liquid-han-
dling workstation (Beckman Coulter, USA). In this step, the com-
pounds were diluted in Tris–HCl 50 mM pH 8 (the reaction
buffer) to a concentration of 0.5 mM and kept at +4 °C before using
them for bioassays.
Kiuc ¼ Vmax app½Iꢂ=Vmax ꢀ Vmax app
where Vmax corresponded to the kinetic constants determined in ab-
sence of inhibitors and Vmax app corresponded to the constants mea-
sured in the presence of different concentrations of the inhibitors.
2.4. Assay for measuring inhibition of fibril formation
Fibril formationwas measured by the thioflavinT (ThT) assay (Le-
Vine, 1993) using two different peptides; a shorter 11-aminoacids
fragment containing the sequence KLVFF and the Ab(1–40) peptide.
The fluorescent stain used to identify amyloid deposits, ThT, is based
on a shift in the emission and excitation spectra (kexcitation = 385 nm
and kemission = 445 nm to kexcitation = 450 nm and kemission = 482 nm)
that occurs upon interaction with amyloid fibrils (LeVine, 1993).
The first study with the lead compounds was carried out as
described in Alptüzün et al. (2010). The inhibition of fibril forma-
tion was tested using the 11-aminoacids peptide with three differ-
2.3. Assays for measuring BChE and AChE activity
The kinetic assay applied to screen for BChE inhibitors is based
on the Ellman’s reaction (Ellman et al., 1961). It uses 5,50-dithiobis
(2-nitro-benzoic acid) (DTNB, Ellman’s reagent) which reacts with
the thiocholine produced by enzyme hydrolysis of the substrate. As
a result, a colored anion (5-thio-2-nitro-benzoic acid) is formed
which can be spectrophotometrically detected at 412 nm. The final
ent concentrations of the inhibitors (2.5, 25 and 250 lM). The
reaction components in the 96-well microplate were 10
l
M com-
compounds were further assayed using the full Ab(1–40) peptide
according to the protocol described by Liu et al. (2004), with sev-
eral modifications, as follows. Ab-protein (1–40) was dissolved in
100% 1,1,1,3,3,3-hexafluoro-2-propanol and sonicated in a water
bath for 20 min to eliminate pre-existing aggregates. Then, the
solution was aliquoted, lyophilized and stored at ꢀ20 °C. Immedi-
pound in Tris–HCl 50 mM pH 8 (or 10 M controls or correspond-
l
ing solvent), 1.5 mM S-bytyrylthiocholine chloride (BTCCl) in H2O,
3 mM DTNB in Tris–HCl 50 mM pH 8 containing 0.1 M NaCl and
0.02 M MgCl ꢁ 6 H2O and 0.1% (w/v) BSA in Tris–HCl 50 mM pH
8. The enzymatic reaction was started by adding 0.350 U/ml BChE
(EC 3.1.1.8, from horse serum, Sigma–Aldrich, USA) or 0.192 U/ml
recombinant human BChE (EC 3.1.1.8, expressed in transgenic goat,
Sigma–Aldrich, USA) in Tris–HCl 50 mM pH 8 containing 0.1% BSA.
Spontaneous and enzymatic hydrolysis was measured 10 times
during 10 min at k = 405 nm using a Victor2 1420 multilabel coun-
ter (PerkinElmer, Finland) before and after enzyme addition. Phy-
ately prior to use, an Ab aliquot (50 lg) was dissolved in MQ-H2O
containing 0.05% NH4OH. Stock solutions of the compounds were
prepared in methanol/buffer (0.01 M PBS, 0.1 M NaCl pH 7.4, 10%
DMSO, 2% HFIP) 2:1. The ThT solution was prepared in 0.025 M
phosphate buffer pH 6.0, filtered through 0.22 lm PTFE filters
and kept in aluminum foil prior to use. Inhibitors, ThT and protein
sostigmine 10 lM (diluted in methanol) was used as control
were added into 96-well plates, and concentrations of ThT and pro-
compound while DMSO: buffer Tris–HCl 50 mM pH 8 was used
as solvent control in all experiments. The effect of methanol and
water was also tested and found not to influence the activity of
BChE. When indicated, galanthamine and N1,N8-bisnorcymserine
were also added as reference inhibitors. Galanthamine was dis-
solved in water while N1,N8-bisnorcymserine was diluted in
DMSO:buffer, as detailed earlier. Reaction took place at room tem-
perature (25 °C).
tein were 23
concentrations between 5 and 250
N1,N8-bisnorcymserine, that was soluble only up to 100
fluorescence was measured immediately after addition of the pro-
tein and after 45 min of incubation at 37 °C and vigorous shaking
(1000 rpm). Inhibitors as well as the reference compounds, 4-ami-
nophenol (De Felice et al., 2004) and nordihydroguaretic acid (Ono
et al., 2004) were dissolved in DMSO and stock solutions were
prepared, as for the compounds, in methanol/buffer 2:1. The typi-
cal final DMSO concentration was 1%. The IC50 determination was
l
M and 0.7
l
M, respectively. Inhibitors were tested in
M, with the exception of
M. The
l
l
The AChE assay was conducted following the same procedure de-
scribed for the BChE assay with the exception that acetylthiocholine

D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
175
carried out accordingly using six different concentrations between
5 and 50 M in three replicates. Due to solubility problems of 6c at
higher concentrations, protein was added after incubation of the
compound in the buffer/ThT mixture at 37 °C and 1000 rpm for
90 min.
repeated twice. Values of IC50 were calculated using non-linear
regression analysis (sigmoidal fitting with variable slope) with
the aid of GraphPad Prism v. 4.0 (GraphPad software Inc., US). Typ-
ical statistical parameters (S/N ratio, S/B ratio, Z’ factor and CVA)
characterizing the performance of screening assays were rigor-
ously used to monitor the overall assay performance and specific
quality of the screening runs. Calculations were made based on
the original equations described by Zhang et al. (1999), Bollini
et al. (2002) and Iversen et al. (2006):
l
2.5. Cytotoxicity studies
Mouse hypothalamic immortalized (GT1-7) cells (Mellon et al.,
1990) where grown in Dulbecco’s Modified Eagle Medium (DMEM)
containing inactivated fetal bovine serum (FBS) 10%, penicillin
50 IU/ml and streptomycin 50 lg/ml. Human lung (HL) epithelial
cells where grown in RN-media consisting of RPMI 1640 (BioWhit-
Z0 ¼ 1 ꢀ ½ð3SDS þ 3SDBÞ=jXS ꢀ XBjꢂ
S=B ¼ XS=XB
ꢀ
ꢁ
1=2
S=N ¼ ðXS ꢀ XBÞ= SD2 þ SD2
CVA ¼ SDS=ðXS ꢀ XBÞ
S
B
taker, US) supplemented with inactivated FBS 7% (BioWhittaker,
US), L-glutamine 2 mM and gentamicin 20 lg/ml. Both cell lines
were cultured in 75 cm2 cell culture flasks at 37 °C in 5% CO2 in
an air-ventilated humidified incubator to confluence. Cells were
harvested using 0.05% (v/v) trypsin and 0.02% (w/v) EDTA in PBS
(w/o Ca and Mg) for GT1-7 cells and 0.25% trypsin in PBS (w/o Ca
and Mg) for HL cells.
where XS and SDS correspond to the mean value and the standard
deviation of the maximal signal (represented by the spectrophoto-
metric absorbance registered 10 min after adding the enzyme),
and XB and SDB are the mean value and standard deviation of the
minimal signal, the background signal (represented by the spectro-
photometric absorbance registered 1 min after adding the enzyme),
in solvent (negative) control wells. In the absence of inhibition, val-
ues resembling maximal signal absorbance data points were ob-
tained whereas in the presence of inhibitory compounds, values
closer to the background signal were achieved.
Cell viability was measured using In vitro Toxicology Assay Kit
(Resazurin based, Sigma–Aldrich, US). Resazurin is a chemical
non-fluorescent probe that is reduced to the fluorescent resorufin
(kexcitation = 570 nm and kemission = 590 nm) inside the viable cells
due to cellular metabolic activity (O’Brien et al., 2000). Cell suspen-
sions of 200
l
l (4 ꢁ 105 cells/ml) were added into 96-well micro-
plates and incubated at 37 °C for 24 h. After that, 20 ll of culture
media was replaced with a similar volume of compound solutions
3. Results and discussion
at different concentrations (between 1 and 100 M) and incubated
l
for another 24 h at 37 °C. Then, culture media was replaced with a
solution of resazurin 10% (diluted in PBS) and incubated for 2 h at
37 °C in the incubator conditions. Cell viability was measured using
a Varioskan Flash multimode plate reader (Thermo Fisher Scientific,
Finland). Results were expressed as relative fluorescence units
(RFU). Cells without test compounds were used as positive control,
cell free wells (containing only media) as negative control and 0.5%
DMSO as solvent control.
3.1. Screening of the chemical library
The tested chemical collection contains 697 natural and syn-
thetic molecules that are based on naturally-present scaffolds,
and includes flavonoids, coumarins, alkaloids, imidazoles, chal-
cones, curcumins, benzanilides and thiophenes, among others
(Supplementary data Table S1). Prior to bioactivity testing, and to
get a first overview of the chemical space occupied by this library,
compounds were mapped using ChemGPS-NP (Larsson et al., 2007;
Rosén et al., 2009b) (Fig. 1). ChemGPS-NP is based on the analysis
of 8 principal components (PC) (dimensions) that describe relevant
physico-chemical properties such as size, shape, aromaticity, lipo-
philicity, polarity, flexibility, and hydrogen bond capacity for a ref-
erence set of compounds. With the program, new compounds are
mapped into the chemical space using interpolation based on PC
analysis score prediction (Oprea and Gottfries, 2001). From Fig. 1
(white spheres) it can be seen that the collection covers mostly
the range of low size molecules (described by PC1), and contains
a major proportion of aromatic compounds (described by PC2)
while displaying a wider range of polarities, with a fairly similar
amount of lipophilic (located in the positive direction of PC3)
and polar molecules (located in the negative direction of PC3).
The collection is also preferentially situated on the positive direc-
tion of PC4, which indicates a high degree of structural flexibility.
It has been previously shown that natural and synthetic com-
pounds exhibit no radical differences between positioning in PC1
and PC3, but natural compounds tend to be located on the negative
direction of PC2 and they are more rigid (Rosén et al., 2009a). Here,
coverage of positive directions of PC2 and PC4 seems to clearly re-
flect the fact that this collection contains mostly synthetic com-
pounds based on naturally existing scaffolds.
2.6. LogP predictions
The molecular properties (logP) of the compounds were calcu-
lated using MOE 2008.10 (Molecular Operating Environment,
Chemical Computing Group) and evaluated according to Lipinski
et al. (1997) and Norinder and Haeberlein (2002).
2.7. Exploration of chemical diversity of the library
The chemical space occupied by the screened library was stud-
ied using the Principal Component Analysis (PCA)-based chemical
space navigation tool ChemGPS-NP (Larsson et al., 2007; Rosén
et al., 2009b). This analysis is made from 2D descriptors (total of
35) that describe physical–chemical properties of the compounds
and are calculated from SMILES. SMILES for all compounds were
obtained using web based programs ChemSpider or Molinspiration
Chemoinformatics v2009.01. All salts, hydration information, and
counter-ions were excluded from the SMILES annotation, and dif-
ferences in stereochemistry ignored, since ChemGPS-NP only uses
2D descriptors. Analysis was focused on the first four dimensions
of ChemGPS-NP (PC1–PC4).
2.8. Statistical analysis and data processing
This chemical library was screened for inhibition of horse
BChE’s hydrolase activity using a kinetic assay based on Ellman’s
reaction (Ellman et al., 1961). To ensure high quality from the
screening viewpoint, the screening assay was evaluated using
statistical parameters; signal-to-noise (S/N) ratio (Bollini et al.,
2002), signal-to-background (S/B) ratio, signal window coefficient
In the first primary screening against both targets (BChE and
AChE), samples were tested in a single-well composition. In the
rest of the analysis at least 2 replicates were used for each exper-
imental treatment. Potency determination experiments were

176
D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
Fig. 1. Three dimensional representations of the chemical space occupied by the chemical library using PCA-based navigation tool ChemGPS-NP (Larsson et al., 2007; Rosén
et al., 2009b). White spheres refer to the 697 compounds screened first and red spheres refer to the 12 imidazole derivatives that were synthesized and tested during the lead
optimization stage. The results were obtained using the first four dimensions of ChemGPS-NP. The data can be interpreted as follows: size increases in the positive direction of
PC1, aromaticity and conjugation-related properties increase in the positive direction of PC2, compounds are increasingly lipophilic in the positive direction of PC3 and they
are more flexible in the positive direction of PC4.
(Z’ factor) (Zhang et al., 1999) and coefficient of variation of the as-
say (CVA) (Iversen et al., 2006), which altogether provide an indica-
tion of the assay performance. For instance, an assay with Z0 P 0.5
is regarded as a good-to-excellent assay, depending on the proxim-
ity to Z0 = 1 (the ideal assay) (Zhang et al., 1999). The following val-
ues obtained during the screening runs were set as acceptance
parameters: Z0 > 0.5, S/N > 7 and S/B > 4. In addition, the assay
was proven to be both reliable and accurate (data not shown).
The theoretical hit limit was determined using three times the
standard deviation (3xSD) of the maximal signal (negative control)
(Zhang et al., 1999) and was situated at 36% of inhibition. However,
since the screening was planned at a compound concentration in
the micromolar range (10 lM) and the aim was to identify potent
inhibitors, the threshold was empirically set at a higher value, 50%
inhibition. At the same time, exclusion of off-target effects on
AChE’s hydrolase activity was conducted by screening the entire
collection against AChE, which confirmed the overall selectivity
of the compound collection for inhibiting BChE (Fig. 2). Perfor-
mance of this screening assay was also strictly monitored using
the statistical parameters previously described.
Five compounds were shown to inhibit BChE’s hydrolase activity
over the established hit threshold, representing a hit rate of 0.7%,
which can be considered as a typical rate in screening campaigns
(Coma et al., 2009). Due to structural and functional similarities
of the two cholinesterases (Nicolet et al., 2003), inhibitors are com-
monly found to act on both AChE and BChE (Musiał et al., 2007).
However, none of the active compounds were found to display
off-target inhibitory effects on AChE, thus giving a first indication
of high selectivity. During the reconfirmation trial to assess the
inhibitors’ potencies, 5-chloro-2-thienyl[4-(2-methoxyphenyl)pip-
erazinyl]methanone, situated just above the hit limit (50% inhibi-
Fig. 2. Correlation plotting of the BChE and AChE inhibitory activities of the
chemical collection. Compounds (697) were tested at 10 M. Selective BChE
inhibitors locate close to the x-axis while selective AChE inhibitors locate close to
the y-axis. Active compounds are situated inside the marked area and the active
compounds are indicated with filled circles.
l
AChE but did not display inhibitory activity higher than 50%. This
indicated an IC50 AChE over 250 lM, thus confirming the high
selectivity of the hits. The imidazole core structure is frequently
present in drug molecules (Bemis and Murcko, 1996) and a few
imidazole compounds have previously been reported to display
BChE and/or AChE inhibitory activity (Andreani et al., 2008; Ková-
rová et al., 2010). However, the imidazoles found in our study are
highly selective towards BChE and the most active is at least 37
times more potent as inhibitor of BChE’a hydrolase activity.
tion at 10
BChE > 10
lide chemical class, 3,5-diamino-N-(2,4,6-trifluorophenyl)benzam-
ide, and displayed an IC50 BChE = 8.6 2.9 M. To our knowledge,
lM) (Fig. 2), was demonstrated to have an IC50
lM. The second least active hit belongs to the benzani-
l
no earlier studies on the inhibition of cholinesterases by benzani-
lides have been published to date. However, because of the low
potency of these two initially identified hits, they were not studied
further.
3.2. Biological profile of 1a–c
Thus, we focused on the three most active hits, which were
from the imidazole chemical class (1a, 1b and 1c). Their activities
were characterized (Table 1) and the compounds were proven to
Classical enzyme-inhibitor kinetic analysis (Lineweaver–Burk
and Dixon plots) was used to establish the inhibition mechanisms
of these three active compounds. In all cases, the lines cross the x-
axis at different positions with unchanged slope values indicating a
pattern of uncompetitive inhibition (Supplementary data Fig. S1).
display IC50 values for BChE inhibition within 0.2–4
three active compounds were also tested up to 250
l
M. These
l
M against

D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
177
Table 1
Effects of imidazoles on BChE and AChE’s hydrolase activities.
Compound
Horse BChE
Electric eel AChE
Selectivitya
Ki valueb
(l
M)
logP-(N + O) >0f
% Inh (10
l
M)
IC50
(
l
M)
% Inh (10
lM)
% Inh (250
15
7.7 1.3
lM)
1a
1b
1c
6a
6b
6c
9a
9b
9c
9d
9e
9f
61
82
93
3.6 0.7
1.5 0.4
0.20 0.03
>10
1.2
4
70
4.9 1.2
1.0 0.1
0.17 0.02
0.5
0.5
ꢀ0.5
ꢀ2.2
ꢀ0.2
2.3
ꢀ1.7
ꢀ0.31
0.91
2.1
170
1300
n.d.
n.d.
2500
20
140
370
290
260
74
14
39
58
3
5
3
ꢀ0.72
30
>10
100
54
83
97
99
0.10 0.01
3.7
ꢀ0.34 2.32
0.073 0.011
12
2
0.11
ꢀ2.9
ꢀ1.4
0.38
0.82
2.1
54
3
ꢀ0.5
1.8 0.1
9.9 2.9
5.2 0.0
ꢀ0.59 2.18
ꢀ7.5 0.6
8.7 1.6
1.1
0.68 0.05
0.86 0.04
0.98 0.04
3.4 0.1
1.5
1.9
1.9
1.1
98
76
9g
9h
9i
98
85
99
0.18 0.06
3.2 0.5
0.16 0.07
2.1 0.5
ꢀ2.9
ꢀ0.21
10
14
1
1400
78
1600
0.86
0.07
620
2.1
2.1
2.5
9.4 3.6
9.7 3.3
1.8 0.2c
1.4 0.2d
24 1e
0.11 0.05
Physostigmine
Galanthamine
N1,N8-bisnorcymserine
20
3
0.038 0.004
a
IC50AChE/IC50 BChE. IC50 AChE was estimated to be above 250
was not determined (n.d.).
lM since 50% inhibition was not yet reached at this concentration. For compounds 6a and 6b the selectivity
b
Calculation of Ki based on an uncompetitive inhibition mechanism.
c
d
e
f
Potency value reported previously by our group, Järvinen et al. (2011), under similar assay conditions.
Potency value reported by Adsersen et al. (2007), under similar assay conditions.
Potency value obtained in current study.
LogP calculated using MOE 2008.10 (Molecular Operating Environment, Chemical Computing Group).
Table 2
Effects of imidazoles on Ab fibril formation in vitro.
Compound
Inhibition of Ab fibril formation (%)
HHQKLVFFAED
Inhibition of Ab fibril formation (%)
Ab(1–40)
2.5
l
M
25
35 14
41
48 10
n.t.
l
M
250
l
M
2.5
l
M
25
18
l
M
5
250
lM
1a
1b
ꢀ3.8 3.1
45 11
58
65
n.t.
n.t.
n.t.
8.0 7.0
ꢀ1.9 3.5
28 12
n.t.
n.t.
0.50 1.55
57 40
24
3
3
4
4
2
8.7 11.3
21
0.25 6.60
69
0.11 2.47
38
62
64
n.t.
2
3
1
1c
30
6
a
4-Aminophenol
n.t.
n.t.
n.t.
b
Nordihydroguaretic acid
n.t.
n.t.
2
N1,N8-Bisnorcymserine
0.05 1.10c
c
n.t.- not tested.
a
The potency value for 4-aminophenol on the short peptide-based assay was 83
8 lM.
b
c
The potency value for nordihydroguaretic acid on the Ab(1–40) assay was 18
2 lM.
The highest concentration tested was 100 M (instead of 250 M) due to the poor solubility of the compound at concentrations higher than 100 lM.
l
l
The apparent Ki values were calculated for all inhibitors consider-
ing the proposed kinetic mechanisms (Table 1). The most potent
sequence KLVFF, present in the short peptide, has been described
as responsible for fibril formation (Gazit, 2005; Tjernberg et al.,
1996). The most active hit on preventing amyloid fibril formation
compound, 1c, exhibited an IC50 BChE = 0.20 0.03
uncompetitive mechanism of action and Ki = 0.17 0.02
l
M, with an
M. Even
l
was again 1c, with IC50 values below 250
lM and on the vicinity
though it was fairly active, the potency of 1c was found to be still
lower than other reported inhibitors, for instance N1,N8-bisnor-
cymserine (IC50 of 38 nM, Table 1).
to 25 M for the peptide-based assay (exact potency values were
l
not quantified, since a good non-linear fitting could not be attained
with only 3 concentrations points). Albeit in the micromolar range,
the activity of 1c can be regarded as acceptable. The reference com-
pounds included here, 4-aminophenol (De Felice et al., 2004) (for
the short peptide assay) and nordihydroguaretic acid (Ono et al.,
However, unlike N1,N8-bisnorcymserine, these three com-
pounds were found to inhibit Ab fibril formation in a concentra-
tion-dependent manner (Table 2). The assays used here are based
on ThT fluorescence changes. It is known that some variations be-
tween runs and Ab protein batches can occur with the ThT assay.
Measuring amyloid formation has previously been described as
difficult due to the possibility of protein precipitation, aggregate
transformation and fibril morphology alterations (Sabaté and Sau-
pe, 2007). Nevertheless, the ThT assay is among the most exten-
sively used methods for quantification of Ab fibril formation
(Reinke and Gestwicki, 2011; Sabaté and Saupe, 2007). In the first
case, the aggregation of the short peptide HHQKLVFFAED
(Alptüzün et al., 2010; LeVine, 1993) was followed, while in the
second, the Ab(1–40) peptide (Liu et al., 2004) was monitored. The
2004) (for the Ab(1–40) peptide) displayed potencies of 83
18 M, respectively (Table 2). One of the most active compounds
that inhibit Ab fibril formation, propidium (a non competitive AChE
inhibitor) has a potency of 12 M (82% of inhibitory effect at
100 M) (Bartolini et al., 2003). In addition, other potency values
lM and
l
l
l
of compounds specifically developed for inhibiting Ab fibril forma-
tion are also in the micromolar range (e.g. 4-(2-(2,6-dichloroben-
zylidene)hydrazinyl)-1-(3-phenylpropyl)pyridin-1-ium bromide,
IC50 = 60 lM or 4-hydroxyindole, IC50 = 85 lM) (Alptüzün et al.,
2010; Cohen et al., 2006). More importantly, the fact that 1c was
identified as an inhibitor with mixed pharmacological activities

178
D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
NH2
N
NH
HCl*H2O
+
N
H
R
R
Br
O
1a-c
Compound
R
1a
1b
1c
2-(OCH3)
3-(OCH3)
2,5-(OCH3)
a Reagents and conditions: KHCO3, THF/H2O, reflux.
Scheme 1. Synthesis of the lead compounds 1a–ca.
proved the feasibility of utilizing diarylimidazoles as starter scaf-
fold in developing bifunctional hits. By comparison, the hybrid
inhibitor (benzofuran-based) has been shown to combine an IC50
with either phenyl or thienyl building blocks (compounds 3a–c)
on the SEM-protected imidazole, as well as the follow-up substitu-
tion of its position 4 to give compounds 5a–c was achieved by a Su-
zuki coupling reaction. The selective dehalogenization on position
5 of the dibromoderivatives was achieved by bromine–lithium ex-
change of compounds 3a–c at ꢀ78 °C followed by quenching with
water. The substituted 2,4-diarylimidazoles 6a–c were deprotected
with hydrochloric acid in ethanol by refluxing overnight. Com-
pounds 8a–i were synthesized from 5-iodo-2-phenyl-1-{[2-
(1,1,1-trimethylsilyl)ethoxy]methyl}-1H-1-imidazole (7) by Suzuki
coupling reaction, as shown in Scheme 3 (Knapp et al., 1993). The
deprotection of 8a–i gave compounds 9a–i. Characterization as
well as purification of the compounds was confirmed by elemental
analysis (Supplementary data Table S2). For compounds 6c, 9b and
9c the elemental analysis provided inconclusive results and there-
fore these compounds were further characterized by HRMS. Since
compound 3c and its starting material compound 2 could not be
completely separated from each other (10% remaining in the prod-
uct fraction), characterization of this compound by elemental anal-
ysis was not feasible. Hence, the reaction mixture was used
without further purification.
of 0.28
of 7.0
l
M for inhibition of BChE hydrolase activity with an IC50
lM for inhibition of Ab aggregation (Rizzo et al., 2008).
The lead compounds were also subjected to preliminary in silico
ADME predictions to assess if they could comply with Lipinski’s
rule of five (Lipinski et al., 1997) to predict their likelihood of oral
bioavailability as well as to estimate their chances for blood-brain
barrier (BBB) penetration, according to Norinder and Haeberlein
(2002). All of the inhibitors fulfill the Lipinski’s rule of five, contain-
ing one H-bond donor, no more than three H-bond acceptors,
molecular weights <500 g/mol and logP <5. In accordance with
the computational work published by Norinder and Haeberlein
(2002), an increased likelihood for BBB penetration is associated
with a compound not containing more than five nitrogen and oxy-
gen molecules (N + O) and with values of logP-(N + O) above zero.
These rules were established based on experimental and computa-
tional data provided by several groups and reviewed by Norinder
and Haeberlein (2002). Given its high content of nitrogen and oxy-
gen atoms when compared to its low logP value, the most active
inhibitor (1c), however, revealed small likelihood to penetrate
the BBB with a logP-(N + O) value of ꢀ0.5 (Table 1). Upon in vitro
testing, none of these lead compounds produced any cytotoxicity
3.4. SAR studies based on inhibition of BChE’s hydrolase activity
at concentrations up to 100 lM using two distinct cell lines of dif-
Lead optimization of the imidazoles was carried out with two
main objectives. First, we focused on trying to improve the potency
and/or the likelihood of bioavailability. Likelihood of oral bioavail-
ability was estimated using Lipinski’s rule of five (Lipinski et al.,
1997) (logP calculated using MOE 2008.10), all derivatives com-
plied with this rule, and chances of BBB penetration were assessed
using the guidelines of Norinder and Haeberlein (2002), as detailed
previously in Section 3.2. According to this the most active com-
pounds have a good likelihood for BBB penetration with logP-
(N + O) of 2.3 (6c) and 2.5 (9i) (Table 1).
Second, we attempted to gather data concerning the most rele-
vant chemical motifs influencing the selective inhibition of the
BChE’s hydrolase activity by means of SAR studies. Andreani
et al. (2008) had already reported that imidazo[2,1-b]thiazole
derivatives showed weaker inhibition of BChE, as compared to
AChE. Therefore, the lead optimization conducted here paid atten-
tion to the influence of the different substitution patterns at the
central imidazole ring. A total of 12 derivatives were synthesized
and tested for their BChE inhibitory activity. The chemical space
ferent origins, namely hypothalamic mice neurons (GT1-7 cells)
and human lung epithelial cells (HL-cells) (data not shown). Lead
optimization was then conducted and targeted to increase potency
of the leads and solve these predicted ADMET limitations.
3.3. Synthesis of lead compounds 1a–c and structural optimization
The lead compounds 1a–c were synthesized by a condensation
reaction in presence of KHCO3 from benzamidine and the appropri-
ate 2-bromoacetophenone derivative (Scheme 1) (Li et al., 2005).
For structure optimization, the 2,4-diarylimidazole derivatives
were synthesized using procedures adapted from the literature
(Kawasaki et al., 1996; Knapp et al., 1993; Langhammer and Erker,
2005). N-1 Alkylated derivatives of the 2,4,5-tribromoimidazole
were synthesized by deprotonation with sodium hydride and
alkylation with 2-(trimethylsilyl)-ethoxymethylchloride (SEM
chloride) as protection group to give compound 2 (Kawasaki
et al., 1996). As stated in Scheme 2 the substitution of position 2

D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
179
Br
Br
Br
Br
Br
N
N
N
i
ii
Br
Si
Ar
Ar
N
N
N
Ar-B(OH)2
O
O
O
Si
Si
4a-c
2
3a-c
R
N
R
N
iii
iv
Ar
N
Ar
N
H
B(OH)
2
O
R
Si
5a-c
N
Ar
N
H
R
6a-c
Compound
Ar
R
-
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
2,5-(OCH3)2C6H3
3-(OCH3)C6H4
3-thienyl
-
-
2,5-(OCH3)2C6H3
3-(OCH3)C6H4
3-thienyl
-
-
-
2,5-(OCH3)2C6H3
3-(OCH3)C6H4
3-thienyl
2,5-(OCH3)2
3-(OCH3)
3-(SCH2CH3)
2,5-(OCH3)2
3-(OCH3)
2,5-(OCH3)2C6H3
3-(OCH3)C6H4
3-thienyl
3-(SCH2CH3)
a Reagents and conditions: (i/iii) Pd[PPh3]4, K2CO3, toluene, MeOH, 90°C 16h; (ii) n-BuLi, THF, -78°C; (iv) HCl,
EtOH, reflux.
Scheme 2. Structural optimization. Synthesis of the derivatives 3–6a–ca.
occupied by these compounds was also assessed using ChemGPS-
NP (Fig. 1,1 red spheres) and compared to the entire chemical col-
lection (Fig. 1, white spheres). This smaller subset of imidazoles
overlaps with the previously screened collection, but the molecules
occupy a more narrowly defined space. For example, all the deriv-
atives are aromatic compounds (PC2) and they are exclusively of
high lipophilicity (PC3).
1
This biological investigation clearly demonstrated that com-
pounds with a 2,4(5)-disubstituted imidazole scaffold are favored
For interpretation of color in Fig. 1, the reader is referred to the web version of
this article.

180
D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
N
N
N
i
ii
I
N
N
N
H
R1
R1
B(OH)2
O
O
R1
Si
Si
8a-i
9a-i
7
Compound R1
Compound R1
8a
8b
8c
8d
8e
8f
2,3-(OCH3)2
9a
9b
9c
9d
9e
9f
2,3-(OCH3)2
3-(OCH2CH3)
3-(OC3H7)
3-(OCH2CH3)
3-(OC3H7)
3-(OC4H9)
3-(OC4H9)
3-(OCH2CH(CH3)2)
2-(OCH2CH3)
3-(SCH3)
3-(OCH2CH(CH3)2)
2-(OCH2CH3)
3-(SCH3)
8g
8h
8i
9g
9h
9i
2-(SCH3)
2-(SCH3)
3-(SCH2CH3)
3-(SCH2CH3)
a Reagents and conditions: (i) Pd[PPh3]4, K2CO3, toluene, MeOH, 90°C 16h; (ii) HCl, EtOH, reflux.
Scheme 3. Structural optimization. Synthesis of the derivatives 8–9a–ia.
in regard to inhibition of BChE’s hydrolase activity. In total, 15
compounds (1a–c, 6a–c, 9a–i) showing this pattern were chosen
for SAR studies (Schemes 1–3). As can be seen in the case of cym-
serine and its derivatives, the selective activity towards BChE de-
pends on only minor modifications of the active scaffold.
Changing of the N1 and N8 methyl groups of cymserine to a H
obtaining N1,N8-bisnorcymserine led to a 50-fold enhancement of
the inhibitory activity towards BChE (Yu et al., 1999). Thus, to re-
tain the feature of the compounds 1a–c as Ab fibril formation
inhibitors, ensuring the bifunctionality of the compound class to
a certain extent, the focus was paid on compounds covering a nar-
row chemical space with limited structural diversity (different alk-
oxy and thioether groups, phenyl vs. thienyl systems). The 2,4-
disubstituted rings at the imidazole core unit are either (substi-
tuted) phenyl or thienyl moieties. According to the BChE inhibitory
activity obtained for 1a, 9f, and 9h, it is obvious that methoxy, eth-
oxy and even thiomethyl substitution of the phenyl ring in position
two (ortho position) is unfavorable for the inhibitory potency.
Compound 9a with 2,3-dimethoxy substituents at ring A was also
less favorable and resulted in a reduced activity. It could be sug-
gested that residues in position two as well as vicinal substituents
are able to decrease the ring flexibility making it more difficult for
the molecule to get into the appropriate conformation for enzyme
inhibition. An exception is 1c, which shows a 2,5-dimethoxy sub-
stitution and good inhibitory activity. Evaluation of alkoxy groups
in the meta position revealed to be beneficial for the inhibitory
potency of the compounds. Alkoxy residues showed an increase
in activity in the order ethoxy < methoxy < isobutoxy < but-
oxy < propoxy. Moreover, replacing the oxygen of the alkoxy group
against a sulfur atom resulted in a 4.7-fold increase of the inhibi-
tory activity whereas thioethyl (9i) seems to be more beneficial
when compared to thiomethyl (9g). Since sulfur is less electroneg-
ative than oxygen these data suggest that electrondonating, lipo-
philic substituents (methylthio, ethylthio) in position three of
ring A are required for a good biological response. BChE inhibitory
activity was limited to derivatives with an unsubstituted ring B.
Compounds with mono- or disubstituents at this part of the mole-
cule were inactive confirming a relationship between the potency
for BChE inhibition and the nature of ring B. This can be seen from
compound 1c and 6a as well as 1b and 6b. Furthermore, these
observations indicate that those derivatives with substituents at
ring B may be electronically unfavored and/or act as a steric
obstruction and thus prevents binding to BChE. Replacement of
the phenyl ring by a thiophene structure resulted in the most ac-
tive compound 6c indicating that a high electron density at this
area of the molecule is beneficial for the inhibitory BChE activity.
Based on our data, we hypothesize that the following structural
requirements are necessary to favor BChE inhibitory activity: (i)
ring A is supposed to present electron-donating, lipophilic substit-
uents in position three and (ii) ring B has to be electron rich,
unsubstituted and thiophene is more favorable than phenyl. In to-
tal, nine of the tested compounds showed good potencies with IC50
BChE values below 5
lM and six of them even below 1 lM.
3.5. Biological profile of the most active hit
From the activity viewpoint, the most promising identified
inhibitor is 6c (4-(3-ethylthiophenyl)-2-(3-thienyl)-1H-imidazole)

D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
181
Fig. 3. Concentration-response curve of the inhibitory effect of 6c on the esterase
activity of human BChE.
Fig. 4. In vitro inhibitory effect of 6c on Ab fibril formation, as measured with the
Ab(1–40) peptide.
which displayed the highest potency of the imidazoles series (IC50
BChE = 0.10 0.01
lished to be uncompetitive, as presented in Supplementary data
Fig. S1, with a Ki value of 0.073 0.011 M. This type of inhibition
mechanism (uncompetitive) may serve as an advantage since an
increase in substrate concentration, which in AD therapy translates
into achieving higher ACh levels, will enhance the potency of an
uncompetitive inhibitor in contrast to the potency of a competitive
inhibitor which would decrease (Copeland, 2005).
lM). The inhibition mechanism of 6c was estab-
the extent of our knowledge, no other studies have been published
on the interaction of imidazoles with the Ab peptide.
l
A third improvement achieved with 6c is related to the pre-
dicted ADMET properties, since its BBB permeability (Norinder
and Haeberlein, 2002) was better than for 1c with a logP-(N + O)
value of 2.3 compared to ꢀ0.5. The cytotoxicity of 6c was also mea-
sured and was found not to affect cell viability of GT1-7 and HL-
cells at concentrations up to 50 lM. Thus, it seems that, at least
A first improvement obtained with 6c is the registered increase
in the inhibitory activity towards BChE’s hydrolase activity (IC50
BChE value of 0.10 lM as opposed to 0.20 lM for 1c). Compound
based on this preliminary ADMET data, compound 6c could behave
as a bifunctional inhibitor with good bioavailability and tolerability
profiles. Restoring the balance of AChE:BChE activity ratio in the
brain with an acceptable potency, together with counteracting
the Ab fibril formation process, makes this molecule a very attrac-
tive and novel alternative for combating the progression of AD.
6c was further tested against the human BChE and the potency
was compared to N1,N8-bisnorcymserine. The IC50 values against
the human enzyme were in the micromolar range for both com-
pounds, 1.11 lM for 6c (Fig. 3) and 0.67 l
M for N1,N8-bisnorcym-
serine. The second element, which makes this compound
particularly attractive, is the fact that it also displays a good po-
4. Conclusions
tency for inhibition of Ab fibril formation with an IC50 of 5.8 lM
Given the complexity of AD pathogenesis, targeting only one
protein may be insufficient to achieve a relevant pharmacological
response. Thus, developing multifunctional molecules (i.e. hybrid
or bifunctional inhibitors) is a rational approach with higher likeli-
hood of success. In one direction, efforts have focused on synthe-
sizing bifunctional molecules starting from two known inhibitors
that display a high potency towards their respective targets (e.g.
Decker et al., 2008). In other cases, biomolecular screening has
served instead as the starting point to identify novel inhibitors that
can be later refined by medicinal chemists. In this contribution we
succeed with the second strategy in identifying diarylimidazoles as
a new type of hybrid inhibitors acting selectively on BChE’s hydro-
lase activity and Ab fibril formation. They represent an easily
accessible chemical class (Greig et al., 2005; Greig et al., 2008)
and the most active hit (6c) was obtained in good yield in less than
five reaction steps. Additionally, the 2,4(5)-disubstituted imidazole
scaffold provides the possibility of further structural modification
to improve the mixed pharmacological effects. Hence, the here pre-
sented chemical compound class possesses the capability to open a
new area of BChE inhibitors from a biological as well as a synthetic
point of view.
(Fig. 4), a value that is lower than the ones registered for most of
the compounds designed to target Ab aggregation and BChE activ-
ity (Greig et al., 2005; Rizzo et al., 2008). The structural factors
influencing the ability to inhibit Ab fibril formation (i.e. via SAR
studies) have not been addressed in this project, mainly because
of the high cost associated to the assay for measuring inhibition
of Ab aggregation. Therefore, at the moment, it is not possible to
postulate the specific mechanism by which this hybrid inhibitor af-
fects Ab fibril formation. However, some theoretical insights can be
speculated judging from the various studies that have been con-
ducted regarding the interactions between ThT and Ab amyloid
aggregates (recently reviewed by Reinke and Gestwicki (2011)).
The structure of the ThT can be roughly described as a benzothia-
zole ring system attached to substituted aniline, and this molecule
is known to bind to amyloid fibrils even during the early stages of
the fibril formation process via multiple sites. Studies have shown
that replacing the benzothiazole ring of the ThT with an imidazole
maintains the affinity towards the protein (Reinke and Gestwicki,
2011) and replacing the aniline with a bithiophene causes a posi-
tive modulation of the interaction towards the Ab (Cui et al.,
2010). Since 6c is an imidazole with a thiophene substitution in po-
sition 2, it thus seems reasonable to speculate that it can compete
with the ThT for the binding to prefibrils, this way preventing or at
least delaying the formation of Ab fibrils. It is plausible to hypoth-
esize that a physical interaction of 6c with the protein takes place,
which could slow down the aggregation process, resulting in a low-
er proportion of bound ThT and reduced fluorescence. At least to
Acknowledgments
The financial support of the National Doctoral Programme in
Informational and Structural Biology (ISB) (to D.K.) is greatly appre-
ciated. A.F. acknowledges the financial support of Tor, Joe och Pentti

182
D. Karlsson et al. / European Journal of Pharmaceutical Sciences 45 (2012) 169–183
Elsinghorst, P.W., Tanarro, C.M., Gütschow, M., 2006. Novel heterobivalent tacrine
derivatives as cholinesterase inhibitors with notable selectivity toward
butyrylcholinesterase. J. Med. Chem. 49, 7540–7544.
Farlow, M., Miller, M., Pejovic, V., 2008. Treatment options in Alzheimer’s disease:
maximizing benefit, managing expectations. Dement. Geriatr. Cogn. Disord. 25,
408–422.
Borgs minnesfond to the research project. In addition, Academy of
Finland (BIOARMI project, decision 128870) is sincerely acknowl-
edged as well as the support received from the Drug Discovery
and Chemical Biology (DDCB) network of Biocenter Finland (P.V.).
Gazit, E., 2005. Mechanisms of amyloid fibril self-assembly and inhibition. Model
short peptides as a key research tool. FEBS J. 272, 5971–5978.
Giacobini, E., 2004. Cholinesterase inhibitors: new roles and therapeutic
alternatives. Pharmacol. Res. 50, 433–440.
Appendix A. Supplementary data
Greig, N., Utsuki, T., Ingram, D., Wang, Y., Pepeu, G., Scali, C., Yu, Q., Mamczarz, J.,
Holloway, H., Giordano, T., Chen, D., Furukawa, K., Sambamurti, K., Brossi, A.,
Lahiri, D., 2005. Selective butyrylcholinesterase inhibition elevates brain
acetylcholine, augments learning and lowers Alzheimer beta-amyloid peptide
in rodent. Proc. Natl. Acad. Sci. USA 102, 17213–17218.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejps.2011.11.004.
Greig, N., Yu, Q., Brossi, A., Martin, E., Lahiri, D., Darvesh, S., 2008.
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