Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bbagen.2014.05.005

Background Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that is characterized by dementia, cognitive impairment, and memory loss. Diverse factors are related to the development of AD, such as increased level of β-amyloid (Aβ), acetylcholine, metal ion deregulation, hyperphosphorylated tau protein, and oxidative stress. Methods The following methods were used: organic syntheses of 1H-phenanthro[9,10-d] imidazole derivatives, inhibition of self-mediated and metal-induced Aβ_1-42 aggregation, inhibition studies for acetylcholinesterase and butyrylcholinesterase, anti-oxidation activity studies, CD, MTT assay, transmission electron microscopy, dot plot assay, gel electrophoresis, Western blot, and molecular docking studies. Results We synthesized and characterized a new type of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for AD treatment. Our results showed that most of these derivatives exhibited strong Aβ aggregation inhibitory activity. Compound 9g had 74% Aβ_1-42 aggregation inhibitory effect at 10 μM concentration with its IC₅₀ value of 6.5 μM for self-induced Aβ_1-42 aggregation. This compound also showed good inhibition of metal-mediated (Cu^{2 +} and Fe^{2 +}) and acetylcholinesterase-induced Aβ_1-42 aggregation, as indicated by using thioflavin T assay, transmission electron microscopy, gel electrophoresis, and Western blot. Besides, compound 9g exhibited cholinesterase inhibitory activity, with its IC₅₀ values of 0.86 μM and 0.51 μM for acetylcholinesterase and butyrylcholinesterase, respectively. In addition, compound 9g showed good anti-oxidation effect with oxygen radical absorbance capacity (ORAC) value of 2.29. Conclusions Compound 9g was found to be a potent multi-target-directed agent for Alzheimer's disease. General significance Compound 9g could become a lead compound for further development as a multi-target-directed agent for AD treatment.

Full text of DOI:10.1016/j.bbagen.2014.05.005

BBAGEN-27942; No. of pages: 18; 4C: 14, 15, 16
Biochimica et Biophysica Acta xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
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Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole
derivatives as multifunctional agents for treatment of
Alzheimer's disease
4Q1 Jinggong Liu a, Jun Qiu a, Mingxue Wang a, Ling Wang a, Lijuan Su a, Jinbo Gao b, Qiong Gu a, Jun Xu a,
⁎
a,⁎⁎
5
Shi-Liang Huang a, Lian-Quan Gu a, Zhi-Shu Huang a, , Ding Li
a
6
7
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou University City, 132 Waihuan East Road, Guangzhou 510006, PR China
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, PR China
b
OOF
8
a r t i c l e i n f o
a b s t r a c t
9
Article history:
Background: Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that is characterized by 20
dementia, cognitive impairment, and memory loss. Diverse factors are related to the development of AD, such as 21
increased level of β-amyloid (Aβ), acetylcholine, metal ion deregulation, hyperphosphorylated tau protein, and 22
10
11
12
13
Received 27 February 2014
Received in revised form 25 April 2014
Accepted 5 May 2014
oxidative stress.
23
Available online xxxx
Methods: The following methods were used: organic syntheses of 1H-phenanthro[9,10-d]imidazole derivatives, 24
inhibition of self-mediated and metal-induced Aβ1–42 aggregation, inhibition studies for acetylcholinesterase 25
and butyrylcholinesterase, anti-oxidation activity studies, CD, MTT assay, transmission electron microscopy, 26
14
15
16
17
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Keywords:
Alzheimer's disease
Imidazole derivative
Aβ aggregation
Cholinesterase
Anti-oxidation
dot plot assay, gel electrophoresis, Western blot, and molecular docking studies.
27
Results: We synthesized and characterized a new type of 1H-phenanthro[9,10-d]imidazole derivatives as multi- 28
functional agents for AD treatment. Our results showed that most of these derivatives exhibited strong Aβ aggre- 29
gation inhibitory activity. Compound 9g had 74% Aβ1–42 aggregation inhibitory effect at 10 μM concentration 30
with its IC50 value of 6.5 μM for self-induced Aβ1–42 aggregation. This compound also showed good inhibition 31
of metal-mediated (Cu2+ and Fe2+) and acetylcholinesterase-induced Aβ1–42 aggregation, as indicated by 32
using thioflavin T assay, transmission electron microscopy, gel electrophoresis, and Western blot. Besides, com- 33
pound 9g exhibited cholinesterase inhibitory activity, with its IC50 values of 0.86 μM and 0.51 μM for acetylcho- 34
linesterase and butyrylcholinesterase, respectively. In addition, compound 9g showed good anti-oxidation effect 35
with oxygen radical absorbance capacity (ORAC) value of 2.29.
36
Conclusions: Compound 9g was found to be a potent multi-target-directed agent for Alzheimer's disease.
37
General significance: Compound 9g could become a lead compound for further development as a multi-target- 38
directed agent for AD treatment. 39
40
© 2014 Published by Elsevier B.V.
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1. Introduction
functions among elderly adults [1–3], with expected number of patients 48
increased to 25 million by 2025 [4]. Over one century since the first de- 49
scription of AD and its predicted increase in incidence in the coming 50
years as well as the lack of effective treatment strategies make this a 51
very important research area [2,5–8]. The molecular etiology of AD 52
remains not completely known, but diverse factors are suggested to be re- 53
lated to the development of AD, including increased level of β-amyloid 54
(Aβ), metal ion deregulation, hyperphosphorylated tau protein, oxidative 55
stress, inflammation, cell cycle regulatory failure, and low level of 56
46
47
Alzheimer's disease (AD) is the most prevalent form of neurodegener-
ation which is the most common cause of dementia and other cognitive
Abbreviations: AAPH, 2,2′-azobis(amidinopropane) dihydrochloride; Aβ, β-amyloid;
AChE, acetylcholinesterase; AD, Alzheimer's disease; ATC, acetylthiocholine chloride;
UNCORRECTED PR
BTC, butyrylthiocholine chloride; BuChE, butyrylcholinesterase; CAS, catalytic binding
site; ChE, cholinesterase; DCFH-DA, dichlorofluorescein diacetate; DMEM, Dulbecco's
modified Eagle's medium; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); MTDLs, multi-
target-directed ligands; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide; ORAC, oxygen radical absorbance capacity; PAS, peripheral anionic site; RMSD,
root mean square deviation; ROS, reactive oxygen species; TEM, transmission electron mi-
croscopy; ThT, thioflavin T; TLC, thin layer chromatography
acetylcholine.
57
It is well known that Aβ plaques play a central role in the neuropa- 58
thology of AD [8–12], and the accumulation of aggregated Aβ species 59
in brain tissues has been a key feature of the amyloid cascade hypothe- 60
sis [10,13–15], which cites that these aggregates are possible causative 61
agents in AD. Therefore, the prevention of Aβ aggregation attracts 62
much current attention. Besides, elevated concentrations of transition 63
⁎
Corresponding author. Tel.: +86 20 3994 3056.
⁎⁎ Corresponding author. Tel.: +86 20 3994 3058.
E-mail addresses: ceshzs@mail.sysu.edu.cn (Z.-S. Huang), liding@mail.sysu.edu.cn
(D. Li).
http://dx.doi.org/10.1016/j.bbagen.2014.05.005
0304-4165/© 2014 Published by Elsevier B.V.
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

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metals such as Fe, Cu, and Zn play important roles in Aβ aggregate depo-
sition [11,16–19] and neurotoxicity as well as inducing formation of re-
active oxygen species (ROS) [20–23]. Thus, lowering the concentration
of metals in brain by chelating metals represents another rational ther-
apeutic approach for halting AD pathogenesis. In addition, cholinergic
hypothesis play important role in the development of AD, acetylcholine
can be degraded by two types of cholinesterases, namely acetylcholin-
esterase (AChE) and butyrylcholinesterase (BuChE) [24]. AChE contains
two binding sites: the catalytic active site (CAS) at the bottom and the
peripheral anionic site (PAS) near the entrance of the gorge [25]. It
has been indicated that AChE promotes amyloid fibril formation by
interacting with the PAS of the enzyme, giving stable AChE–Aβ com-
plexes, which are more toxic than single Aβ peptides [26]. Thus, small
molecules that could bind the CAS site and the PAS site appear to be
very promising therapeutic lead compounds. Due to the multiple factors
and the lack of effective drugs, scientists pay more and more attention
to the multi-target-directed ligands (MTDLs) designing strategies for
AD treatment [27–30].
The benzazole scanffold [31] and benzimidazole derivatives [32,
33] have been used in bioactive molecular design for the treatment
of AD. Based on these structures, we tried to expand their aromatic
plane to improve their π–π interactions and hydrophobic interac-
tions with the targets. The “click” chemistry has been used in the de-
sign of AD modulating agents [34]. Here, we report our design,
synthesis, and evaluation of 1H-phenanthro[9,10-d]imidazole deriv-
atives as multifunctional inhibitors for the treatment of AD. We
found that our compound 9g showed 74% Aβ1–42 aggregation inhib-
itory effect when used at 10 μM concentration, which is better than
resveratrol with only 53% Aβ1–42 aggregation inhibitory effect. Com-
pound 9g could also inhibit and disaggregate metal-induced aggre-
gation of Aβ1–42, which was supported by using thioflavin T (ThT)
assay, transmission electron microscopy (TEM), and gel electropho-
resis. Besides, compound 9g was found to be potent inhibitor for
both AChE and BuChE, with its IC50 values of 0.86 μM and 0.51 μM
for AChE and BuChE, respectively. In addition, compound 9g was
also found to be an anti-oxidation agent with its oxygen radical ab-
2.2.1. General procedure for the preparation of 4-(2-bromoethoxy) 123
benzaldehyde (1), 4-(3-bromopropoxy)benzaldehyde (2), and
4-(4-bromobutoxy)benzaldehyde (3)
124
125
To a solution of 4-hydroxybenzaldehyde (20 mmol, 2.44 g) and an- 126
hydrous K2CO3 (30 mmol, 5.14 g) in 100 mL dry acetone was added 1,2- 127
dibromoethane (40 mmol, 7.51 g) or 1,3-dibromopropane (40 mmol, 128
8.08 g) or 1,4-dibromobutane (40 mmol, 8.64 g). The resulting mixture 129
was heated under reflux for 6 h until the starting material disappeared, 130
and then the remaining solution was filtered and washed with acetone 131
for three times. After concentration, the crude product was purified 132
with gel chromatography to give desired product 1 or 2 or 3 as a 133
white liquid.
134
2.2.2. General procedure for the preparation of 2-(4-(bromomethoxy) 135
phenyl)-1H-phenanthro[9,10-d]imidazole (4), 2-(4-(3-bromopropoxy) 136
phenyl)-1H-phenanthro[9,10-d]imidazole (5), and 2-(4-(4-bromobutoxy) 137
phenyl)-1H-phenanthro[9,10-d]imidazole (6)
138
A mixture of phenanthrene-9,10-dione (10 mmol, 2.08 g) and 1 139
(11 mmol, 2.51 g) or 2 (11 mmol, 2.66 g) or 3 (11 mmol, 2.82 g) in 140
50 mL acetic acid was added ammonium acetate (100 mmol, 7.8 g), 141
and the solution was heated under reflux at 120 °C for 2 h, resulting 142
in a gray precipitate. After cooling, the mixture was poured into 143
100 mL of water and neutralized with concentrated aqueous ammonia. 144
The gray product was filtered and washed with water to give a crude 145
product, which was purified with flash gel chromatography to give 146
compound 4 or 5 or 6 as an off white solid.
147
2.2.2.1. 2-(4-(bromomethoxy)phenyl)-1H-phenanthro[9,10-d]imidazole 148
(4). The compound was obtained with a yield of 56%. 1H NMR 149
(400 MHz, DMSO) δ 13.32 (s, 1H), 8.84 (d, J = 8.2 Hz, 2H), 8.55 (d, 150
J = 7.6 Hz, 2H), 8.26 (d, J = 8.4 Hz, 2H), 7.72 (t, J = 7.2 Hz, 2H), 151
7.62 (t, J = 7.4 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 4.43 (t, J = 5.8 Hz, 152
2H), 3.86 (t, J = 6.0 Hz, 2H);13C NMR (100 MHz, DMSO) δ 171.9, 153
158.8, 149.1, 127.7, 127.5, 127.0, 125.0, 123.8, 123.4, 121.8, 115.0, 154
67.9, 31.3.
155
100 sorbance capacity (ORAC) value of 2.29. Thus, compound 9g could
101 become a lead compound for further development as a multi-
102 target-directed agent for the treatment of AD.
2.2.2.2. 2-(4-(3-bromopropoxy)phenyl)-1H-phenanthro[9,10-d]imidazole 156
(5). The compound was obtained with a yield of 66%. 1H NMR 157
(400 MHz, CDCl3) δ 8.72 (d, J = 8.4 Hz, 2H), 8.43 (d, J = 8.0 Hz, 2H), 158
8.01 (d, J = 8.8 Hz,2H), 7.65–7.58 (m, 2H), 6.89 (d, J = 8.4 Hz, 2H), 159
4.06 (t, J = 5.6 Hz, 2H), 3.72 (t, J = 6.2 Hz, 2H), 2.23–2.17 (m, 2H); 160
13C NMR (100 MHz, CDCl3) δ 159.2, 149.0, 127.7, 127.3, 127.0, 125.0, 161
103 2. Materials and methods
104 2.1. Materials
123.7, 123.2, 121.8, 114.8, 67.2, 32.6, 29.8.
162
2.2.2.3. 2-(4-(4-bromobutoxy)phenyl)-1H-phenanthro[9,10-d]imidazole 163
(6). The compound was obtained with a yield of 58%. 1H NMR 164
(400 MHz, DMSO) δ 13.31 (s, 1H), 8.85 (d, J = 8.4 Hz, 2H), 8.54 (d, 165
J = 8.0 Hz, 2H), 8.24 (d, J = 8.8 Hz, 2H), 7.72 (t, J = 7.4 Hz, 2H), 166
7.63 (t, J = 7.4 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 4.12 (t, J = 6.2 Hz, 167
2H), 3.64 (t, J = 6.6 Hz, 2H), 2.05–1.95 (m, 2H), 1.89–1.82 (m, 2H); 168
13C NMR (100 MHz, DMSO) δ 159.5, 149.2, 127.7, 127.4, 127.0, 169
105
All the reagents used in the biological assay were analytically pure.
106 Chemical reagents used in the synthesis were of research grade or better
107 and were obtained from commercial sources.
108 2.2. Organic syntheses of imidazole derivatives
125.0, 123.8, 122.9, 121.8, 114.8, 66.8, 34.7, 29.1, 27.4.
170
109
110
111
112
113
114
115
All the chemical reagents used in the synthesis were of research grade
or better and were obtained from commercial sources. 1H and 13C NMR
spectra were recorded using TMS as the internal standard in DMSO-d6
or CDCl3 with a Bruker BioSpin GmbH spectrometer at 400 MHz and
100 MHz, respectively. High resolution mass spectra (HRMS) were re-
corded on Shimadzu LCMS-IT-TOF spectrometer. Flash column chroma-
tography was performed with silica gel (200–300 mesh) purchased
2.2.3. General procedure for the preparation of 7a–9f
171
To a stirred suspension of compound 4 or 5 or 6 (1 mmol) and an- 172
hydrous K2CO3 (2 mmol) in dry acetonitrile (50 mL) was added ex- 173
cess alkylamine (3 mmol), and the resulting mixture was heated 174
under reflux for 6 h until the starting material disappeared. The 175
K2CO3 was removed through filtration, and the remaining solution 176
was concentrated under reduced pressure. The crude product was 177
purified by using gel chromatography with CH2Cl2/MeOH (30:1 to 178
116 from Qingdao Haiyang Chemical Co. Ltd. The purities of synthesized
117 compounds were confirmed to be higher than 95% by using analyti-
118 cal HPLC with a dual pump Shimadzu LC-20AB system equipped
119 with an Ultimate-QB-C18 column (4.6 × 250 mm, 5 μm) and eluted
120 with methanol/water (60:40 to 70:30) containing 0.1% trifluoroacetic
121 acid at a flow rate of 0.6 mL/min. Organic syntheses of imidazole deriv-
122 atives were shown in Schemes 1–4.
10:1) as elution solvents to give the desired products.
179
2.2.3.1. 2-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)-N,N- 180
diethylethanamine (7a). Compound 4 was treated with diethylamine 181
following the general procedure to give the desired product 7a as a 182
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
3
OOF
Scheme 1. The organic synthesis of target compounds 7a–9f. Reagents and conditions: (a) 1,2-dibromoethane or 1,3-dibromopropane, or 1,4-dibromobutane, K2CO3, acetone, reflux 6 h;
(b) phenanthrene-9,10-dione, NH4OAc, AcOH, 120 °C, 3 h; (c) RNH, K2CO3, acetonitrile, reflux, 5–6 h.
183 white solid with a yield of 62%, with purity of 98% determined by using
184 HPLC. 1H NMR (400 MHz, DMSO) δ 13.30 (s, 1H), 8.87 (d, J = 8.4 Hz,
185 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.58 (d, J = 7.8 Hz, 1H), 8.58 (d, J =
186 8.0 Hz, 1H), 7.77–7.70 (m, 2H), 7.65–7.58 (m, 2H), 4.17 (t, J = 5.6 Hz,
187 2H), 2.92 (t, J = 5.6 Hz, 2H), 2.76–2.57 (m, 4H), 1.04 (t, J = 7.0 Hz,
188 6H); 13C NMR (100 MHz, DMSO) δ 159.3, 149.3, 136.9, 127.7, 127.4,
189 127.0, 125.0, 124.9 124.0, 123.6, 123.1, 122.4, 121.9, 114.8, 66.0, 51.1,
190 47.0, 11.4. ESI-HRMS m/z: calcd for C27H27N3O [M + H]+ 410.2227,
191 found 410.2245.
54.0, 23.0. ESI-HRMS m/z: calcd for C27H25N3O [M + H]+ 408.2070, 202
found 408.2072. 203
2.2.3.3. 2-(4-(2-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)ethyl) 204
piperazin-1-yl)ethanol (7c). Compound 4 was treated with N-(2- 205
hydroxyethyl)piperazine following the general procedure to give the 206
desired product 7c as a white solid with a yield of 28%, with purity of 207
95% determined by using HPLC. 1H NMR (400 MHz, MeOD) δ 8.66 208
(d, J = 8.4 Hz, 2H), 8.41 (d, J = 7.6 Hz, 2H), 8.03 (d, J = 8.8 Hz, 209
2H), 7.57 (t, J = 7.2 Hz, 2H), 7.50 (t, J = 7.0 Hz, 2H), 6.98 (d, J = 210
8.8 Hz, 2H), 4.07 (t, J = 5.2 Hz, 2H), 3.69 (t, J = 5.4 Hz, 2H), 3.02–2.90 211
(m, 4H), 2.89–2.80 (m, 4H), 2.80–2.68 (m, 4H); 13C NMR (100 MHz, 212
MeOD) δ 161.1, 151.4, 129.6, 129.3, 128.1 126.4, 125.9 124.8, 124.2, 213
123.0, 116.0, 66.4, 60.1, 57.8, 57.4 53.3, 52.3. ESI-HRMS m/z: calcd for 214
192 2.2.3.2. 2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-1H-phenanthro[9,10-d]
193 imidazole (7b). Compound 4 was treated with pyrrolidine following
194 the general procedure to give the desired product 7b as a light grey
195 solid with a yield of 47%, with purity of 98% determined by using
196 HPLC. 1H NMR (400 MHz, DMSO) δ 13.37 (s, 1H), 8.88–8.83 (m, 2H),
197 8.59 (d, J = 7.2 Hz, 2H), 8.30–8.28 (m, 2H), 7.76 (d, J = 7.2 Hz, 1H),
198 7.72 (d, J = 9.0 Hz, 1H), 7.63 (t, J = 7.4 Hz, 2H), 7.20–7.18 (m, 2H),
199 4.23 (t, J = 5.2 Hz, 2H), 3.01 (t, J = 5.2 Hz, 2H), 3.81–2.68 (m, 4H),
200 1.82–1.73 (m, 4H); 13C NMR (100 MHz, DMSO) δ 159.2, 149.2, 136.8,
201 127.7, 127.4, 127.0, 125.0, 124.0 123.7, 123.2, 122.4, 121.9, 114.8, 66.2,
C29H30N4O2 [M + H]+ 467.2442, found 467.2451.
215
2.2.3.4. 2-(4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-1H-phenanthro 216
[9,10-d]imidazole (7d). Compound 4 was treated with N-methyl pipera- 217
zine following the general procedure to give the desired product 7d as a 218
white solid with a yield of 45%, with purity of 95% determined by using 219
UNCORRECTED PR
Scheme 2. The organic synthesis of target compounds 7g–9g. Reagents and conditions: (a) pyridine, 80 °C, 8 h.
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

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J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
Scheme 3. The organic synthesis of target compounds 19a–20e. Reagents and conditions: (a) propargyl bromide, K2CO3, acetone, reflux 6 h; (b) phenanthrene-9,10-dione, NH4OAc, AcOH,
120 °C, 3 h; (c) chloroacetyl chloride or 3-chloropropionyl chloride, THF, K2CO3, 0 °C to 50 °C, 16 h; (d) CH3OH, THF, 0 °C to rt, 16 h; (e) NH4CO2, Pb/C, DMF, rt, 4 h; (f) conc. HCl, t-BuONO,
NaN3, H2O; (g) sodium ascorbate, CuSO4∙5H2O, t-BuOH, H2O.
220 HPLC. 1H NMR (400 MHz, DMSO) δ 13.28 (s, 1H), 8.88 (d, J = 8.4 Hz,
221 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.54 (d, J =
222 8.0 Hz, 1H), 8.25 (d, J = 8.8 Hz, 2H), 7.76–7.70 (m, 2H), 7.63 (t, J =
223 6.4 Hz, 2H), 7.17 (d, J = 8.8 Hz, 2H), 4.18 (t, J = 5.8 Hz, 2H), 2.74 (t,
224 J = 5.8 Hz, 2H), 2.52–2.49 (m, 4H), 2.43–2.38 (m, 4H), 2.19 (s, 3H);
ESI-HRMS m/z: calcd for C28H28N4O [M + H]+ 437.2336, found 227
437.2352. 228
2.2.3.5. 2-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-1H-phenanthro[9,10-d] 229
imidazole (7e). Compound 4 was treated with piperidine following the 230
general procedure to give the desired product 7e as a white solid with 231
a yield of 73%, with purity of 99% determined by using HPLC. 1H NMR 232
225
13C NMR (100 MHz, DMSO) δ 159.4, 149.3, 127.6, 127.4, 127.0,
226 125.0, 124.0, 123.6, 123.0, 121.8, 114.8, 65.7, 56.5, 54.6, 52.8, 45.5.
Scheme 4. The organic synthesis of target compounds 22a–22f. Reagents and conditions: (a) NaN3, H2O; (b) sodium ascorbate, CuSO4∙5H2O, t-BuOH, H2O.
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
5
233 (400 MHz, DMSO) δ 13.34 (s, 1H), 8.84 (t, J = 9.4, 2H), 8.63 (d, J =
234 7.2 Hz, 1H), 8.58 (d, J = 7.6 Hz, 1H), 8.29 (d, J = 8.0 Hz, 2H), 7.75
235 (d, J = 6.8 Hz, 2H), 7.63 (t, J = 6.8 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H),
236 4.16 (t, J = 5.8 Hz, 2H), 2.73 (t, J = 5.4 Hz, 2H), 2.53–2.42 (m, 4H),
237 1.57–1.46 (m, 4H), 1.42–1.33 (m, 2H); 13C NMR (100 MHz, DMSO)
238 δ 159.4, 149.3, 136.9, 127.7, 127.4, 127.0, 125.0, 124.9, 124.0, 123.6,
239 123., 122.4, 121.8, 114.8, 65.5, 57.2, 54.3, 25.4, 23.7. ESI-HRMS m/z:
240 calcd for C28H28N3O [M + H]+ 422.2227, found 422.2228.
99% determined by using HPLC. 1H NMR (400 MHz, DMSO) δ 13.30 294
(s, 1H), 8.85 (s, 2H), 8.59–8.54 (m, 2H), 8.25 (d, J = 6.8 Hz, 2H), 295
7.73 (d, J = 6.4 Hz, 2H), 7.63 (t, J = 7.2 Hz, 2H), 7.15 (d, J = 6.8 Hz, 296
2H), 4.10 (t, J = 5.4 Hz, 2H), 2.45 (t, J = 6.2 Hz, 2H), 2.39–2.34 (m, 297
8H), 2.16 (s, 3H), 1.94–1.89 (m, 2H); 13C NMR (100 MHz, DMSO) δ 298
159.5, 149.3, 127.7, 127.4, 127.0, 125.0, 123.9, 122.9, 121.8, 114.8, 299
66.0, 54.7, 54.3, 52.7, 45.7, 26.2. ESI-HRMS m/z: calcd for 300
C29H30N4O [M + H]+ 451.2492, found 451.2503.
301
241 2.2.3.6. 4-(2-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)ethyl)
242 morpholine (7f). Compound 4 was treated with morpholine following
243 the general procedure to give the desired product 7f as an off white
244 solid with a yield of 65%, with purity of 99% determined by using
245 HPLC. 1H NMR (400 MHz, DMSO) δ 13.29 (s, 1H), 8.88–8.83 (m, 2H),
246 8.58 (d, J = 8.0 Hz, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 2.8 Hz,
247 1H), 8.28 (d, J = 2.4 Hz, 1H), 7.77–7.70 (m, 2H), 7.63 (t, J = 6.8 Hz,
248 2H), 7.19 (d, J = 2.4 Hz, 2H), 7.17 (d, J = 2.0 Hz, 2H), 4.20 (t, J =
249 6.0 Hz, 2H), 3.66–3.56 (m, 4H), 2.75 (t, J = 5.8 Hz, 2H), 2.53–2.49 (m,
250 4H); 13C NMR (100 MHz, DMSO) δ 159.4, 149.2, 127.7, 127.4, 127.0,
251 125.0, 124.0, 123.7, 123.0, 121.8, 114.9, 66.1, 65.5, 57.0, 53.6. ESI-
252 HRMS m/z: calcd for C27H25N3O2 [M + H]+ 424.2020, found 424.2035.
2.2.3.11. 2-(4-(3-(piperidin-1-yl)propoxy)phenyl)-1H-phenanthro[9,10- 302
d]imidazole (8e). Compound 5 was treated with piperidine following 303
the general procedure to give the desired product 8e as a white 304
solid with a yield of 33%, with purity of 99% determined by using 305
HPLC. 1H NMR (400 MHz, DMSO) δ 13.31 (s, 1H), 8.85 (d, J = 306
8.0 Hz, 2H), 8.56 (d, J = 7.6 Hz, 2H), 8.25 (d, J = 8.8 Hz, 2H), 7.73 307
(t, J = 7.4 Hz, 2H), 7.65–7.61 (m, 2H), 7.15 (d, J = 8.8 Hz, 2H), 4.10 308
(t, J = 6.4 Hz, 2H), 2.46 (t, J = 7.2 Hz, 2H), 2.42–2.35 (m, 4H), 1.91 309
(s, 2H), 1.56–1.48 (m, 4H), 1.43–1.36 (m, 2H); 13C NMR (100 MHz, 310
DMSO) δ 172.3, 159.5, 149.3, 127.7, 127.4, 126.9, 124.9, 123.8, 311
123.0, 121.9, 114.7, 66.0, 54.9, 53.8, 25.9, 25.2, 23.8, 21.3. ESI-HRMS 312
m/z: calcd for C29H30N3O [M + H]+ 436.2383, found 436.2384.
313
OOF
253 2.2.3.7.
3-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)-N,N-
2.2.3.12. 4-(3-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)propyl) 314
morpholine (8f). Compound 5 was treated with morpholine following 315
the general procedure to give the desired product 8f as an off white 316
solid with a yield of 52%, with purity of 99% determined by using 317
HPLC. 1H NMR (400 MHz, DMSO) δ 13.28 (s, 1H), 8.87 (d, J = 8.4 Hz, 318
1H), 8.84 (d, J = 8.4 Hz, 1H), 8.58 (d, J = 7.6 Hz, 1H), 8.54 (d, J = 319
7.6 Hz, 2H), 8.25 (d, J = 8.4 Hz, 2H), 7.77–7.70 (m, 2H), 7.63 (m, 2H), 320
7.16 (d, J = 8.4 Hz, 2H), 4.12 (t, J = 6.4 Hz, 2H), 3.59 (t, J = 4.4 Hz, 321
2H), 2.46 (t, J = 7.0 Hz, 2H), 2.42–2.33 (m, 2H), 1.96–1.89 (m, 2H); 322
13C NMR (100 MHz, DMSO) δ 159.5, 149.3, 136.9, 127.7, 127.4, 127.3, 323
127.0, 125.0, 124.9, 124.0, 123.7, 122.9, 122.4, 121.8, 114.8, 66.1, 66.0, 324
54.8, 53.3, 25.8. ESI-HRMS m/z: calcd for C28H27N3O2 [M + H]+ 325
254 diethylpropan-1-amine (8a). Compound 5 was treated with di-
255 ethylamine following the general procedure to give the desired
256 product 8a as a white solid with a yield of 38%, with purity of 99%
257 determined by using HPLC. 1H NMR (400 MHz, DMSO) δ 9.07 (s, 1H),
258 8.87 (d, J = 7.6 Hz, 2H), 8.57 (d, J = 7.6 Hz, 2H), 8.29 (d, J = 6.4 Hz,
259 2H), 7.75 (t, J = 7.0 Hz, 2H), 7.65 (t, J = 7.0 Hz, 2H), 7.21 (d, J =
260 6.4 Hz, 2H), 4.21 (t, J = 5.8 Hz, 2H), 3.35 (t, J = 5.6 Hz, 2H), 3.23–3.18
261 (m, 4H), 2.22–2.09 (m, 2H), 1.24 (t, J = 7.0 Hz, 6H); 13C NMR
262 (100 MHz, DMSO) δ 159.2, 149.0, 127.9, 127.5, 127.1, 125.2, 123.9,
263 122.8, 121.8, 114.9, 64.9, 48.1, 46.6, 23.3, 8.6. ESI-HRMS m/z: calcd for
264 C28H29N3O [M + H]+ 424.2383, found 424.2396.
438.2176, found 438.2192.
326
265 2.2.3.8. 2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)-1H-phenanthro[9,10-
266 d]imidazole (8b). Compound 5 was treated with pyrrolidine following
267 the general procedure to give the desired product 8b as a light grey
268 solid with a yield of 32%, with purity of 99% determined by using
269 HPLC. 1H NMR (400 MHz, DMSO) δ 13.35 (s, 1H), 8.87 (d, J =
270 8.4 Hz, 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.58 (d, J = 7.6 Hz, 2H), 8.28
271 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 7.6 Hz,
272 1H), 7.63 (t, J = 6.0 Hz, 2H), 7.17 (d, J = 8.8 Hz, 2H), 4.16 (t, J =
273 5.8 Hz, 2H), 3.01–2.91 (m, 4H), 2.11–2.03 (m, 2H), 1.90–1.78 (m, 4H);
2.2.3.13. 4-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)-N,N- 327
diethylbutan-1-amine (9a). Compound 6 was treated with diethylamine 328
following the general procedure to give the desired product 9a as a 329
white solid with a yield of 45%, with purity of 99% determined by 330
using HPLC. 1H NMR (400 MHz, DMSO) δ 13.38 (s, 1H), 8.92–8.87 (m, 331
2H), 8.62 (d, J = 6.8 Hz, 2H), 8.31 (d, J = 7.6 Hz, 2H), 7.81–7.75 (m, 332
2H), 7.69–7.66 (m, 2H), 7.20 (d, J = 8.0 Hz, 2H), 4.15 (t, J = 6.0 Hz, 333
2H), 2.67 (m, 6H), 1.86–1.80 (m, 2H), 1.71–1.64 (m, 2H), 1.07 (t, J = 334
6.8 Hz, 6H); 13C NMR (100 MHz, DMSO) δ 159.4, 149.3, 136.8, 129.3, 335
127.8, 127.4, 127.0, 125.0, 123.9, 123.7, 123.1, 122.5, 122.1, 121.9, 336
118.7, 115.2, 114.8, 67.0, 53.8, 53.1, 25.9, 22.6, 22.5. ESI-HRMS m/z: 337
274
13C NMR (100 MHz, DMSO) δ 159.3, 149.2, 136.8, 127.7, 127.4, 127.0,
275 125.0, 124.9, 124.0, 1237, 123.1, 122.4, 122.0, 121.8, 99.5, 65.4, 53.3,
276 51.7, 26.5, 22.8. ESI-HRMS m/z: calcd for C28H27N3O [M + H]+
277 422.2227, found 422.2227.
calcd for C29H31ON3 [M + H]+ 438.2540, found 438.2543.
338
278 2.2.3.9. 2-(4-(3-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)
279 propyl)piperazin-1-yl)ethanol (8c). Compound 5 was treated with
280 N-(2-hydroxyethyl)piperazine following the general procedure to
281 give the desired product 8c as a white solid with a yield of 59%,
282 with purity of 99% determined by using HPLC. 1H NMR (400 MHz,
283 MeOD) δ 8.64 (d, J = 8.4 Hz, 2H), 8.40 (d, J = 7.6 Hz, 2H), 7.99 (d,
284 J = 8.4 Hz, 2H), 7.56 (t, J = 7.4 Hz, 2H), 7.49 (t, J = 7.2 Hz, 2H),
285 6.90 (d, J = 8.8 Hz, 2H), 3.87 (t, J = 5.8 Hz, 2H), 3.59 (t, J = 5.8 Hz,
286 2H), 2.59–2.46 (m, 12H), 1.85–1.79 (m, 2H); 13C NMR (100 MHz,
287 MeOD) δ 161.6, 151.6, 129.6, 129.3, 128.1, 126.4, 124.8, 124.0, 122.9,
288 115.9, 67.1, 60.8, 59.2, 55.9, 53.7, 53.2, 27.2. ESI-HRMS m/z: calcd for
289 C30H32N4O2 [M + H]+ 481.2598, found 481.2597.
2.2.3.14. 2-(4-(4-(pyrrolidin-1-yl)butoxy)phenyl)-1H-phenanthro[9,10- 339
d]imidazole (9b). Compound 6 was treated with pyrrolidine following 340
the general procedure to give the desired product 9b as a light grey 341
solid with a yield of 38%, with purity of 99% determined by using 342
HPLC. 1H NMR (400 MHz, DMSO) δ 13.38 (s, 1H), 8.92–8.87 (m, 2H), 343
8.62 (d, J = 6.4 Hz, 2H), 8.31 (d, J = 7.6 Hz, 2H), 7.81–7.74 (m, 2H), 344
7.71–7.63 (m, 2H), 7.20 (d, J = 8.0 Hz, 2H), 4.15 (t, J = 6.0 Hz, 2H), 345
2.72–2.60 (m, 6H), 1.85–1.80 (m, 2H), 1.71–1.64 (m, 2H), 1.07 (t, J = 346
6.8 Hz, 6H); 13C NMR (100 MHz, DMSO) δ 159.5, 149.3, 136.8, 127.7, 347
127.4, 126.9, 124.9, 123.9, 123.7, 122.9, 121.9, 114.8, 67.5, 51.5, 46.2, 348
26.4, 22.4, 10.9. ESI-HRMS m/z: calcd for C29H30ON3 [M + H]+ 349
UNCORRECTED PR
436.2383, found 436.2385.
350
290 2.2.3.10.
2-(4-(3-(4-methylpiperazin-1-yl)propoxy)phenyl)-1H-
2.2.3.15. 2-(4-(4-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy) 351
butyl)piperazin-1-yl)ethanol (9c). Compound 6 was treated with N- 352
(2-hydroxyethyl)piperazine following the general procedure to 353
give the desired product 9c as a white solid with a yield of 75%, 354
291 phenanthro[9,10-d]imidazole (8d). Compound 5 was treated with N-
292 methyl piperazine following the general procedure to give the de-
293 sired product 8d as a white solid with a yield of 41%, with purity of
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

6
J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
355 with purity of 99% determined by using HPLC. 1H NMR (400 MHz,
356 DMSO) δ 13.34 (s, 1H), 8.87–8.82 (m, 2H), 8.59 (d, J = 6.8 Hz, 2H),
357 8.27 (d, J = 8.0 Hz, 2H), 7.74–7.70 (m, 2H), 7.63 (d, J = 6.8 Hz, 2H),
358 7.15 (d, J = 8.0 Hz, 2H), 4.59 (s, 1H), 4.10 (t, J = 5.4 Hz, 2H), 3.55
359 (t, J = 5.8 Hz, 2H), 3.36 (t, J = 5.8 Hz, 2H), 2.78–2.54 (m, 10H),
360 1.86–1.73 (m, 2H), 1.72–1.55 (m, 2H); 13C NMR (100 MHz, DMSO)
361 δ 159.5, 149.3, 136.8, 127.8, 127.4, 126.9, 125.0, 124.0, 123.6, 122.9,
362 122.5, 122.1, 121.8, 114.8, 67.4, 59.5, 57.7, 56.8, 52.2, 51.6, 26.4,
363 22.2. ESI-HRMS m/z: calcd for C31H35O2N4[M + H]+ 495.2755,
364 found 495.2759.
1H), 7.15 (d, J = 2.0 Hz, 1H), 5.13 (t, J = 6.4 Hz, 2H), 4.66 (t, J = 416
6.4 Hz, 2H); 13C NMR (100 MHz, DMSO) δ 158.3, 149.0, 146.1, 145.5, 417
127.8, 127.5, 127.0, 1251, 123.9, 121.9, 115.0, 66.2, 59.9. ESI-HRMS 418
m/z: calcd for C28H21N3O [M–Br + H]+ 416.1757, found 416.1772. 419
2.2.4.2. 1-(3-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)propyl) 420
pyridin-1-ium bromide (8g). Compound 5 was treated with pyridine fol- 421
lowing the general procedure to give the desired product 8g as a yellow 422
solid with a yield of 76%, with purity of 98% determined by using HPLC. 423
1H NMR (400 MHz, DMSO) δ 13.37 (s, 1H), 9.21 (s, 2H), 8.86 (d, J = 424
6.4 Hz, 2H), 8.68–8.64 (m, 1H), 8.60 (d, J = 6.4 Hz, 2H), 8.28 (d, J = 425
7.2 Hz, 2H), 8.23–8.14 (m, 2H), 7.79–7.69 (m, 2H), 7.68–7.57 (m, 2H), 426
7.02 (d, J = 7.6 Hz, 2H), 4.88 (t, J = 6.4 Hz, 2H), 4.23 (t, J = 6.4 Hz, 427
2H); 13C NMR (100 MHz, DMSO) δ 158.8, 149.2, 145.6, 145.0, 127.9, 428
127.4, 127.0, 125.0, 123.8, 123.2, 122.0, 114.7, 65.0, 58.8, 29.9. ESI- 429
HRMS m/z: calcd for C29H23N3O [M–Br + H]+ 430.1914, found 430
365 2.2.3.16.
2-(4-(4-(4-methylpiperazin-1-yl)butoxy)phenyl)-1H-
366 phenanthro[9,10-d]imidazole (9d). Compound 6 was treated with N-
367 methyl piperazine following the general procedure to give the de-
368 sired product 9d as a white solid with a yield of 37%, with purity of
369 99% determined by using HPLC. 1H NMR (400 MHz, DMSO) δ 13.30
370 (s, 1H), 8.45 (t, J = 10.0, 2H), 8.58 (t, J = 8.2 Hz, 2H), 8.26 (d, J =
371 8.4 Hz, 2H), 7.75 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.62
372 (t, J = 7.0 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H), 4.08 (t, J = 6.2 Hz, 2H),
373 2.50 (t, J = 5.8 Hz, 2H), 2.48–2.36 (m, 8H), 2.25 (s, 3H), 1.81–1.73
374 (m, 2H), 1.64–1.56 (m, 2H); 13C NMR (100 MHz, DMSO) δ 159.5,
375 149.3, 136.9, 127.7, 127.4, 126.9, 125.0, 124.0, 123.6, 122.9, 122.4,
376 121.9, 114.7, 67.4, 57.0, 54.2, 52.0, 45.1, 26.5, 22.5. ESI-HRMS m/z:
377 calcd for C30H33ON4[M + H]+ 465.2649, found 465.2653.
430.1917.
431
2.2.4.3. 1-(2-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)ethyl) 432
pyridin-1-ium bromide (9g). Compound 6 was treated with pyridine fol- 433
lowing the general procedure to give the desired product 9g as a yellow 434
solid with a yield of 82%, with purity of 99% determined by using HPLC. 435
1H NMR (400 MHz, DMSO) δ 13.31 (s, 1H), 9.18 (d, J = 5.6 Hz, 2H), 8.85 436
(s, 2H), 8.66–8.57 (m, 4H), 8.28 (d, J = 8.8 Hz, 2H), 8.20 (t, J = 6.6 Hz, 437
2H), 7.73 (s, 2H), 7.63 (t, J = 7.2 Hz, 2H),7.38 (t, J = 6.4 Hz, 1H), 7.16 438
(d, J = 8.4 Hz, 2H), 4.75 (t, J = 7.2 Hz, 2H), 4.14 (t, J = 5.8 Hz, 2H), 439
2.20–2.10 (m, 2H), 1.88–1.78 (m, 2H); 13C NMR (100 MHz, DMSO) δ 440
159.8, 150.0, 149.7, 146.0, 145.2, 136.6, 128.6, 128.2, 127.9, 127.5, 441
125.5, 124.3, 123.6, 122.4, 115.3, 67.4, 60.9, 28.2, 25.7. ESI-HRMS m/z: 442
378 2.2.3.17. 2-(4-(4-(piperidin-1-yl)butoxy)phenyl)-1H-phenanthro[9,10-d]
379 imidazole (9e). Compound 6 was treated with piperidine following the
380 general procedure to give the desired product 9e as a white solid with
381 a yield of 23%, with purity of 99% determined by using HPLC. 1H NMR
382 (400 MHz, DMSO) δ 13.36 (s, 1H), 8.87 (d, J = 8.8 Hz, 1H), 8.84 (d,
383 J = 8.8 Hz, 1H), 8.58 (d, J = 7.6 Hz, 2H), 8.27 (d, J = 8.8 Hz, 2H), 7.75
384 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 6.8 Hz, 2H),
385 7.15 (d, J = 8.4 Hz, 2H), 4.09 (t, J = 6.2 Hz, 2H), 3.35 (m, 2H), 2.48–
386 2.33 (m, 4H), 1.80–1.73 (m, 2H), 1.68–1.61 (m, 2H), 1.59–1.50 (m,
387 4H), 1.44–1.37 (m, 2H); 13C NMR (100 MHz, DMSO) δ 159.5, 149.3,
388 136.8, 127.7, 127.4, 126.9, 124.9, 123.9, 123.6, 122.9, 122.5, 122.0,
389 121.8, 114.7, 99.5, 67.4, 57.6, 53.5, 48.6, 26.5, 24.9, 23.6, 22.2. ESI-
390 HRMS m/z: calcd for C30H32ON3 [M + H]+ 450.2540, found 450.2540.
calcd for C30H26N3O [M–Br + H]+ 444.2070, found 444.2071.
443
2.2.5. General procedure for the preparation of 4-(prop-2-yn-1-yloxy) 444
benzaldehyde (10) 445
To a solution of 4-hydroxybenzaldehyde (10 mmol, 1.22 g) and an- 446
hydrous K2CO3 (15 mmol, 2.57 g) in 100 mL dry acetone was added 447
propargyl bromide (30 mmol, 3.57 g). The resulting mixture was heated 448
under reflux for 6 h until the starting material disappeared, and then the 449
remaining solution was filtered and washed with acetone for three 450
times. After concentration, the desired product 10 was obtained as a yel- 451
391 2.2.3.18. 4-(4-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)butyl)
392 morpholine (9f). Compound 6 was treated with morpholine following
393 the general procedure to give the desired product 9f as an off white
394 solid with a yield of 74%, with purity of 99% determined by using
395 HPLC. 1H NMR (400 MHz, DMSO) δ 13.27 (s, 1H), 8.85 (d, J =
396 8.4 Hz, 2H), 8.56 (d, J = 7.6 Hz, 2H), 8.25 (d, J = 8.8 Hz, 2H), 7.73
397 (t, J = 7.4 Hz, 2H), 7.62 (t, J = 7.0 Hz, 2H), 7.15 (d, J = 8.8 Hz, 2H),
398 4.09 (t, J = 6.4 Hz, 2H), 3.57 (t, J = 4.6 Hz, 4H), 2.37–2.31 (m, 8H),
399 1.81–1.74 (m, 2H), 1.63–1.56 (m, 2H); 13C NMR (100 MHz, DMSO)
400 δ 159.6, 149.3, 127.7, 127.4, 126.9, 124.9, 123.8, 122.9, 121.8, 114.8,
401 67.5, 66.2, 57.7, 53.3, 26.5, 22.3. ESI-HRMS m/z: calcd for C29H30O2N3
402 [M + H]+ 452.2333, found 452.2336.
low solid with nearly 100% yield.
452
2.2.6. General procedure for the preparation of 2-(4-(prop-2-yn-1-yloxy) 453
phenyl)-1H-phenanthro[9,10-d]imidazole (11) 454
The synthesis of 11 was carried out following the procedure for 455
the synthesis of 4, 5, and 6. The desired product 11 was obtained as 456
a gray solid with 53% yield. 1H NMR (400 MHz, DMSO) δ 13.32 (s, 457
1H), 8.86 (s, 2H), 8.57 (s, 2H), 8.28 (s, 2H), 7.74 (s, 2H), 7.64 (s, 1H), 458
7.24 (s, 2H), 4.93 (s, 2H), 3.64 (s, 1H); 13C NMR (100 MHz, DMSO) δ 459
158.0, 149.1, 136.9, 135.4, 129.2, 129.1, 127.6, 127.4, 127.0, 125.0, 460
123.7, 121.8, 115.2, 79.0, 78.4, 55.6.
461
2.2.7. General procedures for the preparation of 12a–12b, 13a–13d, 14a– 462
14e, 15a–15d, 16a–16e, 17a–17d, and 18a–18e 463
403 2.2.4. General procedure for the preparation of 7g, 8g, and 9g
404
A solution of 4 or 5 or 6 (0.5 mmol) in pyridine (5 mL) was heated
The syntheses of 12a–12b, 13a–13d, 14a–14e, 15a–15d, 16a–16e, 464
17a–17d, and 18a–18e were carried out following procedures reported 465
previously. All these compounds were prepared and identified by using 466
405 for 8 h at 80 °C. 5 mL of ether was added to cooled reaction mixture
406 and the pale yellow precipitate was collected by filtration. The precipi-
407 tate was washed with ether three times and dried under vacuum to
408 give 7g, 8g, or 9g.
LC-MS.
467
2.2.8. General procedures for the preparation of 19a–19d, 20a–20e
468
409 2.2.4.1. 1-(2-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)ethyl)
410 pyridin-1-ium bromide (7g). Compound 4 was treated with pyridine fol-
411 lowing the general procedure to give the desired product 7g as a yellow
412 solid with a yield of 85%, with purity of 96% determined by using HPLC.
To a solution of azide (17a–18e, 0.4 mmol, 2 equiv.) dissolved in 469
t-BuOH (2.5 mL) and distilled water (2.5 mL) was added the intermedi- 470
ate 11 (0.2 mmol, 1 equiv.), sodium ascorbate (19 mg, 0.10 mmol, 0.5 471
equiv.) and CuSO4∙5H2O (2 mg, 10 μmol, 5 mol%), and the mixture 472
was heated at 140 °C for 5 h. The reaction was cooled and diluted 473
with distilled water. The crude product was isolated by filtration, 474
washed with ice cold distilled water (2 mL), ether (2 × 2 mL), and 475
413
1H NMR (400 MHz, DMSO) δ 13.36 (s, 1H), 9.22 (d, J = 6.4 Hz, 2H), 8.86
414 (s, 2H), 8.69 (t, J = 7.2 Hz, 2H), 8.57 (d, J = 7.6 Hz, 2H), 8.31–8.21 (m,
415 4H), 7.77–7.70 (m, 2H), 7.63 (t, J = 7.2 Hz, 2H), 7.18 (d, J = 2.0 Hz,
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
7
476 was purified with gel chromatography to give desired product 19a–19d,
477 20a–20e.
(100 MHz, DMSO) δ 168.5, 158.9, 149.2, 143.5, 139.0, 136.9, 131.9, 537
127.8, 127.4, 127.0, 125.0, 124.0, 123.7, 123.6, 122.7, 122.4, 121.9, 538
120.7, 120.3, 115.1, 61.2, 59.1, 53.7, 23.3. ESI-HRMS m/z: calcd for 539
478 2.2.8.1. N-(4-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)
479 methyl)-1H-1,2,3-triazol-1-yl)phenyl)-3-(pyrrolidin-1-yl)propanamide
480 (19a). It was obtained with purity of 99% determined by using HPLC. 1H
481 NMR (400 MHz, DMSO) δ 13.33 (s, 1H), 10.36 (s, 1H), 8.92 (s, 1H), 8.87
482 (d, J = 8.4 Hz, 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.59 (d, J = 7.6 Hz, 1H), 8.59
483 (d, J = 8.0 Hz, 1H), 8.29 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 9.2 Hz, 2H), 7.81
484 (d, J = 9.2 Hz, 2H), 7.77–7.70 (m, 2H), 7.66–7.61 (m, 2H), 7.33 (d, J =
485 8.8 Hz, 2H), 5.36 (s, 2H), 3.39–3.26 (m, 2H), 2.78 (t, J = 6.8 Hz, 2H),
486 2.57–2.54 (m, 2H), 2.52–2.50 (m, 2H), 1.73–1.68 (m, 4H); 13C NMR
487 (100 MHz, DMSO) δ 170.3, 158.9, 149.2, 143.5, 139.6, 136.9, 131.5,
488 127.7, 127.4, 127.0, 125.1, 125.0, 124.0, 123.7, 123.5, 122.7, 122.4,
489 121.8, 120.8, 119.7, 115.1, 61.2, 53.4, 51.3, 35.9, 23.1. ESI-HRMS m/z:
490 calcd for C37H33N7O2 [M + H]+ 608.2768, found 608.2773.
C36H31N7O2 [M + H]+ 594.2612, found 594.2625.
540
2.2.8.6. N-(4-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy) 541
methyl)-1H-1,2,3-triazol-1-yl)phenyl)-2-(diethylamino)acetamide (20b). 542
It was obtained with purity of 99% determined by using HPLC. 1H 543
NMR (400 MHz, DMSO) δ 13.31 (s, 1H), 9.90 (s, 2H), 8.94 (s, 1H), 544
8.87 (d, J = 8.4 Hz, 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.58 (d, J = 545
7.6 Hz, 1H), 8.53 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.4 Hz, 2H), 7.91– 546
7.85 (m, 4H), 7.78–7.70 (m, 2H), 7.66–7.60 (m, 2H), 7.32 (d, J = 547
8.4 Hz, 2H), 5.37 (s, 2H), 3.20 (s, 2H), 2.65–2.60 (m, 4H), 1.03 (t, J = 548
7.0 Hz, 4H); 13C NMR (100 MHz, DMSO) δ 171.2, 159.8, 150.1, 144.5, 549
139.7, 137.8, 132.8, 128.7, 128.4, 128.3, 127.9, 127.9, 126.0, 125.9, 550
124.9 124.6, 124.4, 123.7, 123.3, 122.8, 121.6, 121.1, 116.1, 62.1, 58.3, 551
48.7, 12.8. ESI-HRMS m/z: calcd for C36H34N7O2 [M + H]+ 596.2769, 552
491 2.2.8.2. N-(4-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)
492 methyl)-1H-1,2,3-triazol-1-yl)phenyl)-3-(diethylamino)propanamide
493 (19b). It was obtained with purity of 99% determined by using HPLC. 1H
494 NMR (400 MHz, DMSO) δ 13.37 (s, 1H), 10.45 (s, 1H), 8.93 (s, 1H), 8.87
495 (d, J = 8.4 Hz, 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.57 (t, J = 8.4 Hz, 2H), 8.30
496 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H),
497 7.77–7.70 (m, 2H), 7.66–7.61 (m, 2H), 7.32 (d, J = 8.4 Hz, 2H), 5.36
498 (s, 2H), 2.92 (t, J = 8.4 Hz, 2H), 2.73–2.64 (m, 4H), 2.58 (t, J =
499 8.4 Hz, 2H), 1.05 (t, J = 5.8 Hz, 6H); 13C NMR (100 MHz, DMSO) δ
500 170.2, 158.8, 149.2, 143.5, 139.5, 136.8, 131.6, 127.8, 127.4, 127.0,
501 126.9, 125.1, 124.9, 124.0, 123.7, 123.5, 122.8, 122.4, 121.9, 121.8,
502 120.8, 119.7, 115.1, 61.2, 47.9, 46.2, 33.3, 11.0. ESI-HRMS m/z: calcd
503 for C37H35N7O2 [M + H]+ 610.2925, found 610.2942.
found 596.2773.
553
2.2.8.7. N-(4-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy) 554
methyl)-1H-1,2,3-triazol-1-yl)phenyl)-2-(piperidin-1-yl)acetamide 555
OOF
(20c). It was obtained with purity of 99% determined by using HPLC. 1H 556
NMR (400 MHz, DMSO) δ 13.31 (s, 1H), 9.93 (s, 1H), 8.93 (s, 1H), 8.58 557
(d, J = 7.6 Hz, 1H), 8.53 (d, J = 7.6 Hz, 1H), 8.28 (d, J = 8.4 Hz, 2H), 558
7.88 (s, 4H), 7.77–7.70 (m, 2H), 7.67–7.58 (m, 2H), 7.32 (d, J = 8.4 Hz, 559
2H), 5.36 (s, 2H), 3.11 (s, 2H), 2.58 (s, 2H), 2.49–2.43 (m, 2H), 1.61– 560
1.54 (m, 4H), 1.47–1.38 (m, 2H);13C NMR (100 MHz, DMSO) δ 168.9, 561
158.9, 149.2, 143.6, 138.9, 136.9, 131.8, 127.7, 127.5, 127.4, 127.0, 562
126.9 125.1, 125.0, 124.0, 123.6, 123.5, 122.7, 122.4, 121.8, 120.7, 563
120.2, 115.1, 62.6, 61.2, 54.0, 25.3, 23.5. ESI-HRMS m/z: calcd for 564
C37H34N7O2 [M + H]+ 608.2769, found 608.2773.
565
504 2.2.8.3. N-(4-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)
505 methyl)-1H-1,2,3-triazol-1-yl)phenyl)-3-(piperidin-1-yl)propanamide
506 (19c). It was obtained with purity of 99% determined by using HPLC. 1H
507 NMR (400 MHz, CDCl3) δ 11.65 (s, 1H), 8.74 (t, J = 7.4 Hz, 2H), 8.63 (d, J
508 = 7.6 Hz, 1H), 8.34 (d, J = 6.8 Hz, 3H), 8.05 (s, 1H), 7.74–7.68 (m, 8H),
509 7.21 (d, J = 7.4 Hz, 2H), 5.40 (s, 2H), 2.71 (t, J = 8.4 Hz, 2H), 2.57–2.50
510 (m, 6H), 1.76–1.69 (m, 4H), 1.65–1.57 (m, 2H); 13C NMR (100 MHz,
511 CDCl3) δ 171.0, 160.5, 144.7, 144.3, 139.7, 135.6, 132.3, 129.2, 129.0
512 128.9, 127.4, 127.3, 126.3, 126.2, 126.1, 125.3, 123.9, 123.8, 123.4,
513 123.0, 121.4, 121.2, 121.0, 120.8, 120.2, 115.2, 62.2, 54.2, 53.6, 32.5,
514 26.3, 24.1. ESI-HRMS m/z: calcd for C38H35N6O3 [M + H]+ 623.2763,
515 found 623.2768.
2.2.8.8. N-(4-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy) 566
methyl)-1H-1,2,3-triazol-1-yl)phenyl)-2-morpholinoacetamide (20d). It 567
was obtained with purity of 98% determined by using HPLC. 1H NMR 568
(400 MHz, DMSO) δ 13.36 (s, 1H), 10.06 (s, 1H), 8.98 (s, 1H), 8.92 569
(d, J = 8.4 Hz, 1H), 8.88 (d, J = 8.0 Hz, 1H), 8.63 (d, J = 7.6 Hz, 570
1H), 8.58 (d, J = 7.6 Hz, 1H), 8.32 (d, J = 8.4 Hz, 2H), 7.92 (s, 4H), 571
7.82–7.75 (m, 2H), 7.68 (d, J = 3.2 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 572
5.41 (s, 2H), 3.73–3.67 (m, 4H), 3.22 (s, 2H), 2.62–2.56 (m, 4H); 13C 573
NMR (100 MHz, DMSO) δ 168.4, 158.9, 149.2, 143.6, 138.9, 137.5, 574
131.9, 127.7, 127.4, 127.0, 125.1, 125.0, 121, 123.6, 123.5, 122.7, 122.4, 575
121.8, 120.7, 120.3, 115.1, 66.0, 62.0, 61.2, 53.1. ESI-HRMS m/z: calcd 576
for C37H32N7O3 [M + H]+ 610.2561, found 610.2563.
577
516 2.2.8.4. N-(4-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)
517 methyl)-1H-1,2,3-triazol-1-yl)phenyl)-3-(4-(2-hydroxyethyl)piperazin-
518 1-yl)propanamide (19d). It was obtained with purity of 99% determined
519 by using HPLC. 1H NMR (400 MHz, DMSO) δ 13.33 (s, 1H), 10.36 (s, 1H),
520 8.91 (s, 2H), 8.87 (d, J = 8.4 Hz, 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.58 (d, J =
521 7.6 Hz, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.4 Hz, 2H), 7.86 (d, J =
522 8.8 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 7.77–7.70 (m, 2H), 7.66–7.60 (m,
523 2H), 7.32 (d, J = 8.4 Hz, 2H), 5.36 (s, 2H), 3.51 (s, 1H), 3.49 (t, J =
524 8.4 Hz, 2H), 2.65 (t, J = 8.4 Hz, 2H), 2.48–2.37 (m, 10H); 13C NMR
525 (100 MHz, DMSO) δ 170.5, 158.9, 149.2, 143.5, 139.6, 136.8, 131.5,
2.2.8.9. N-(4-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy) 578
methyl)-1H-1,2,3-triazol-1-yl)phenyl)-2-(dimethylamino)acetamide 579
(20e). It was obtained with purity of 99% determined by using HPLC. 1H 580
NMR (400 MHz, DMSO) δ 13.31 (s, 1H), 10.01 (s, 1H), 8.93 (s, 1H), 8.87 581
(d, J = 8.4 Hz, 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.56 (d, J = 7.2 Hz, 1H), 8.56 582
(d, J = 7.6 Hz, 1H), 8.28 (d, J = 8.8 Hz, 2H), 7.92–7.85 (m, 4H), 7.75–7.70 583
(m, 2H), 7.66–7.61(m, 2H), 7.32 (d, J = 8.8 Hz, 2H), 5.36 (s, 2H), 3.12 584
(s, 2H), 2.30 (s, 6H); 13C NMR (100 MHz, DMSO) δ 169.0, 158.9, 585
149.2, 143.5, 139.1, 136.9, 131.8, 127.7, 127.4, 127.0, 125.1, 124.9, 586
124.0, 123.6, 123.5, 122.7, 122.4, 121.8, 120.6, 120.3, 115.1, 63.2, 61.2, 587
45.3. ESI-HRMS m/z: calcd for C34H29N7O2 [M + H]+ 568.2455, found 588
UNCORRECTED PR
526 129.6, 129.3, 127.7, 127.5, 127.4, 127.0, 126.9, 125.1, 125.0, 124.0,
527 123.6, 123.4, 122.7, 122.4, 121.8, 120.8, 119.7, 115.1, 69.7, 61.2, 60.0,
528 58.3, 53.6, 53.0, 52.2, 34.1. ESI-HRMS m/z: calcd for C40H42N8O3 [M
529 + H]+ 667.3140, found 667.3140.
568.2469.
589
2.2.9. General procedure for the preparation of 21a–21f
590
530 2.2.8.5. N-(4-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)
531 methyl)-1H-1,2,3-triazol-1-yl)phenyl)-2-(pyrrolidin-1-yl)acetamide
532 (20a). It was obtained with purity of 96% determined by using HPLC. 1H
533 NMR (400 MHz, DMSO) δ 13.36 (s, 1H), 10.11 (s, 1H), 8.94 (s, 1H), 8.88–
534 8.83 (m, 2H), 8.60–8.55 (m, 2H), 8.30 (d, J = 8.4 Hz, 2H), 7.88 (m, 4H),
535 7.77–7.70 (m, 2H), 7.67–7.60 (m, 2H), 7.33 (d, J = 8.4 Hz, 2H), 5.37 (s,
536 2H), 3.41 (s, 2H), 2.78–2.62 (m, 4H), 1.86–1.70 (m, 4H); 13C NMR
The syntheses of 21a–21f were carried out following procedures re- 591
ported previously with minor modification [35]. To a solution of amine 592
salt (30.8 mmol) in 30 mL water was added sodium azide (6.0 g, 593
92.3 mmol, 3 equiv.), and the mixture was stirred at 80 °C for 16 h. 594
After removing most of the water by rotary evaporation, the reaction 595
mixture was cooled in an ice bath. Diethyl ether (50 mL) and then 596
KOH pellets (4.0 g) were added keeping the temperature below 10 °C. 597
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

8
J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
598 After separation of the organic phase, the aqueous layer was further ex-
599 tracted with diethyl ether (2 × 20 mL). The combined organic layers
600 were dried over Na2SO4 and concentrated to give oil products 21a–
601 21f, which were used directly for the next step reaction without further
602 purification.
121.1, 120.8, 120.7, 115.2, 62.2, 58.2, 54.5, 47.9, 26.0, 24.1. ESI- 658
HRMS m/z: calcd for C31H30N5O2 [M + H]+ 504.2394, found 659
504.2392.
660
2.2.10.6. 3-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy) 661
methyl)-1H-1,2,3-triazol-1-yl)-N,N-dimethylpropan-1-amine (22f). It 662
was obtained with purity of 99% determined by using HPLC. 1H 663
NMR (400 MHz, CDCl3) δ 8.72 (t, J = 7.4 Hz, 2H), 8.60 (d, J = 664
7.4 Hz, 1H), 8.30 (d, J = 7.8 Hz, 3H), 7.77–7.63 (m, 5H), 7.15 (d, J = 665
8.0 Hz, 2H), 5.32 (s, 2H), 4.44 (t, J = 6.0 Hz, 2H), 2.26 (t, J = 6.0 Hz, 666
2H), 2.21 (s, 6H), 2.11–2.05 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 667
162.1, 160.5, 144.6, 143.5, 135.6, 129.1, 128.9, 128.8, 127.3, 127.2, 668
126.2, 126.1, 126.0, 123.7, 123.4, 123.2, 122.9, 121.1, 120.8, 120.7, 669
115.2, 62.2, 55.7, 48.1, 45.2, 28.0. ESI-HRMS m/z: calcd for C29H28N5O2 670
603 2.2.10. The syntheses of 22a–22f were carried out following the procedures
604 for the syntheses of 19a–19d
605 2.2.10.1. 3-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)
606 methyl)-1H-1,2,3-triazol-1-yl)propan-1-amine (22a). It was obtained
607 with purity of 99% determined by using HPLC. 1H NMR (400 MHz,
608 DMSO) δ 8.95 (s, 2H), 8.49 (d, J = 5.2 Hz 1H), 8.37 (d, J = 4.8 Hz
609 1H), 8.30 (d, J = 8.0 Hz, 2H), 7.82 (d, J = 2.8 Hz, 2H), 7.82 (d, J =
610 2.4 Hz, 2H), 7.32 (d, J = 3.2 Hz, 2H), 5.31 (s, 2H), 4.48 (s, 2H), 2.62
611 (s, 2H), 2.03–1.89 (m, 2H); 13C NMR (100 MHz, DMSO) δ 161.7,
612 160.5, 143.9, 142.2, 134.7, 128.7, 128.4, 128.4, 127.7, 127.7, 126.7,
613 126.4, 125.3, 124.7, 124.2, 124.0, 122.2, 120.4, 120.2, 119.4, 115.5,
614 61.4, 47.1. ESI-HRMS m/z: calcd for C34H29N7O2 [M + H]+ 568.2455,
615 found 568.2469.
[M + H]+ 478.2238 found 478.2238.
671
2.3. Thioflavin T (ThT) assay
672
Aβ1–42 (Sigma-Aldrich, USA) was dissolved in ammonium hy- 673
droxide (1% v/v) to give a stock solution (1 mM), which was 674
aliquoted into small samples and stored at −80 °C. Thioflavin T 675
(ThT) assay was performed to determine the activities of our com- 676
pounds on inhibiting Aβ1–42 self-aggregation. Experiments were 677
performed by incubating the peptides (Aβ1–42, 20 μM, final concen- 678
tration) in 20 mM phosphate buffer (pH 7.4) at 37 °C for 48 h with 679
the tested compounds (10 μM, final concentration). After incubation, 680
the samples were diluted to a final volume of 40 μL with 50 mM gly- 681
cine–NaOH buffer (pH 8.5) containing 5 μM thioflavin-T. Fluores- 682
cence signal was measured (excitation wavelength 450 nm, 683
emission wavelength 485 nm, and slit widths set to 5 nm) on a 684
monochromators based multimode microplate reader (INFINITE 685
M1000), adapted for 384 well microtiter plates. Each inhibitor was 686
examined in triplicate. The fluorescence intensities were recorded, 687
and the percentage of inhibition on aggregation was calculated by 688
using the following equation: (1 − IFi/IFc) × 100% in which IFi and 689
IFc were the fluorescence intensities obtained for absorbance in the 690
presence and absence of inhibitors, respectively, after subtracting 691
616 2.2.10.2.
2-(4-((1-(2-(pyrrolidin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)
617 methoxy)phenyl)-1H-phenanthro[9,10-d]imidazole (22b). It was obtain-
618 ed with purity of 99% determined by using HPLC. 1H NMR (400 MHz,
619 CDCl3) δ 8.72 (t, J = 8.0 Hz, 2H), 8.60 (d, J = 8.0 Hz, 1H), 8.30 (d, J =
620 8.8 Hz, 3H), 7.79 (s, 1H), 7.75–7.64 (m, 4H), 7.16 (d, J = 8.8 Hz, 2H),
621 5.31 (s, 2H), 4.50 (t, J = 6.4 Hz, 2H), 2.97 (t, J = 6.4 Hz, 2H), 2.60–2.49
622 (m, 4H), 1.80–1.74 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 162.1,
623 160.5, 144.5, 143.4, 135.5, 129.0, 128.9, 128.8, 127.3, 127.2, 126.2,
624 126.1 126.0, 123.7 123.4, 123.4, 122.9, 121.1, 120.7, 120.6, 115.2, 62.2,
625 55.4, 54.0, 49.5, 23.6. ESI-HRMS m/z: calcd for C30H28N5O2 [M + H]+
626 490.2238, found 490.2242.
627 2.2.10.3. 4-(2-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)
628 methyl)-1H-1,2,3-triazol-1-yl)ethyl)morpholine (22c). It was obtained
629 with purity of 96% determined by using HPLC. 1H NMR (400 MHz,
630 CDCl3) δ 8.74 (t, J = 8.2 Hz, 2H), 8.61 (d, J = 7.6 Hz, 1H), 8.32 (d, J =
631 8.4 Hz, 3H), 7.79 (s, 1H), 7.76–7.65 (m, 4H), 7.17 (d, J = 8.4 Hz, 2H),
632 5.34 (s, 2H), 4.49 (t, J = 6.0 Hz, 2H), 3.68 (t, J = 7.8, 4H), 2.84 (t, J =
633 5.6 Hz, 2H), 2.49 (t, J = 4.4 Hz, 4H); 13C NMR (100 MHz, CDCl3) δ
634 162., 160.4, 144.6, 143.6, 135.6, 129.1, 128.9, 127.3, 127.2, 126.2, 126.0,
635 123.7, 123., 122.9, 121.1, 120.8, 120.7, 115.2, 66.8, 62.2, 57.8, 53.5,
636 47.5. ESI-HRMS m/z: calcd for C30H28N5O3 [M + H]+ 506.2187, found
637 506.2190.
the background fluorescence of 5 μM thioflavin-T solution.
692
For the disaggregation of self-induced Aβ fibrils, the Aβ1–42 stock so- 693
lution was diluted with 20 mM phosphate buffer (pH 7.4). 20 μM Aβ1–42 694
was incubated at 37 °C for 24 h. Then, the 20 μM tested compound was 695
added and incubated at 37 °C for another 24 h. The sample was diluted 696
to a final volume of 40 μL with 50 mM glycine–NaOH buffer (pH 8.0) 697
containing thioflavin T (5 μM). The detection method was the same as 698
above.
699
638 2.2.10.4. 2-(4-((4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)
639 methyl)-1H-1,2,3-triazol-1-yl)-N,N-diethylethanamine (22d). It was
640 obtained with purity of 98% determined by using HPLC. 1H NMR
641 (400 MHz, CDCl3) δ 8.73 (t, J = 8.2 Hz, 2H), 8.61 (d, J = 7.6 Hz, 1H),
642 8.30 (d, J = 8.8 Hz, 3H), 7.79 (s, 1H), 7.80–7.64 (m, 4H), 7.16 (d, J =
643 8.4 Hz, 2H), 5.32 (s, 2H), 4.42 (t, J = 6.0 Hz, 2H), 2.89 (t, J = 6.0 Hz,
644 2H), 2.54 (m, 4H), 0.96 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3)
645 δ 162.2, 160.5, 144.6, 143.3, 135.6, 129.1, 128.9, 128.9, 127.3, 127.2,
646 126.2, 126.1, 126.0, 123.7, 123.7, 123.4 122.9, 121.1, 120.8, 120.7,
647 115.2, 62.2, 53.0, 49.2, 47.4 11.9. ESI-HRMS m/z: calcd for C30H30N5O2
648 [M + H]+ 492.2394, found 492.2393.
The effects of compounds on metal-induced Aβ1–42 aggregation 700
were also determined by using thioflavin T. The Aβ1–42 stock solution 701
was diluted with 20 μM HEPES (pH 6.6) containing 150 μM NaCl. 20 702
μM Aβ1–42 was incubated with or without 20 μM copper or 20 μM 703
iron and 20 μM tested compounds at 37 °C for 24 h. The sample 704
was diluted to a final volume of 40 μL with 50 mM glycine–NaOH 705
buffer (pH 8.0) containing thioflavin T (5 μM) and assayed as de- 706
scribed above.
707
The inhibitory potency of compounds on AChE-induced Aβ1–42 ag- 708
gregation was determined by using thioflavin T. The mixtures contain- 709
ing 2 μL of Aβ1–42 and 16 μL of AChE in the presence or absence of the 710
tested compounds (2 μL) were incubated for 6 h at 37 °C. The final vol- 711
ume of each vial was 20 μL, and the final concentrations of Aβ1–42 (dilut- 712
ed in 0.215 M sodium phosphate buffer, pH 8.0) and AChE (dissolved in 713
0.1 M sodium phosphate buffer, pH 8.0) were 25 μM and 0.25 μM. Blanks 714
containing Aβ1–42, AChE, Aβ1–42 plus the tested compounds in 0.215 M 715
sodium phosphate buffer were prepared. The percent inhibition on 716
AChE-induced aggregation was calculated with the following equation: 717
(1 − IFi/IFc) × 100% where IFi and IFc were the fluorescence intensities 718
obtained for Aβ1–42 plus AChE in the presence and absence of inhibitors, 719
649 2.2.10.5.
2-(4-((1-(2-(piperidin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)
650 methoxy)phenyl)-1H-phenanthro[9,10-d]imidazole (22e). It was obtain-
651 ed with purity of 98% determined by using HPLC. 1H NMR (400 MHz,
652 CDCl3) δ 8.73 (t, J = 8.0 Hz, 2H), 8.61 (d, J = 7.6 Hz, 1H), 8.31 (d, J =
653 8.4 Hz, 3H), 7.82 (s, 1H), 7.75–7.64 (m, 4H), 7.16 (d, J = 8.4 Hz, 2H),
654 5.32 (s, 2H), 4.47 (t, J = 6.2 Hz, 2H), 2.77 (t, J = 6.2 Hz, 2H), 2.42
655 (t, J = 6.2 Hz, 4H), 1.58–1.53 (m, 4H), 1.45–1.39 (m, 2H); 13C NMR
656 (100 MHz, CDCl3) δ 162.2, 160.5, 144.6, 143.4, 135.5, 129.1, 128.,
657 128.8, 127.3, 127.2, 126.2, 126.1, 126.0, 123.7, 123.5, 123.4, 122.9,
respectively, minus the fluorescence of respective blanks.
720
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
9
721 2.4. Transmission electron microscope (TEM)
722
The Aβ1–42 stock solution was diluted with 20 mM phosphate buffer
inhibitors were added to the assay solution and pre-incubated for 10 782
min at 37 °C with the enzyme followed by the addition of substrate in 783
different concentrations. Kinetic characterization for the hydrolysis of 784
ATC catalyzed by AChE was carried out using spectrometric method at 785
412 nm. The parallel control experiments were performed without in- 786
723 (pH 7.4) for the metal free experiment and 20 μM HEPES (pH 6.6) in-
724 cluding 150 μM NaCl for the copper and iron containing experiment.
725 Then concentrations of Aβ1–42, metal ions and tested compounds were
726 20 μM, 20 μM, and 40 μM, respectively. After incubation at 37 °C for
727 24 h, aliquots of 10 μL samples were placed on carbon-coated copper/
728 rhodium grid. After 1 min, the grid was washed with water and nega-
729 tively stained with 2% uranyl acetate solution for 1 min. After draining
730 off the excess staining solution by filter paper, the specimen was trans-
731 ferred for examination in a transmission electron microscope (JEOL
732 JEM-1400).
hibitor in the assay.
787
2.7. Anti-oxidation activity with in vitro-ORAC-FL assay
788
The anti-oxidation activity was determined based on the oxygen 789
radical absorbance capacity-fluorescein (ORAC-FL) assay. The reaction 790
was carried out in 75 mM potassium phosphate buffer (pH 7.4), and 791
the final volume of reaction mixture was 200 μL. The tested compound 792
or Trolox standard substance was dissolved in DMSO to 10 mM and 793
diluted in 75 mM potassium phosphate buffer (pH 7.4). Antioxidant 794
(20 μL) and fluorescein (FL, 120 μL, final concentration of 140 nM) 795
were incubated for 15 min at 37 °C placing in the wells of a black 96 796
well plate. Then 60 μL of 2,2′-azobis(amidinopropane) dihydrochloride 797
(AAPH, final concentration of 40 mM) solution was added rapidly. 798
The fluorescence was recorded every minute for 240 min at 485 nm 799
733 2.5. Inhibition studies on AChE and BuChE
734
Acetylcholinesterase (from electric eel), butyrylcholinesterase (from
735 equine serum), 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent,
736 DTNB), acetylthiocholine chloride (ATC), butylthiocholine chloride
737 (BTC), and Tacrine hydrochloride were purchased from Sigma Aldrich.
738
The metal free assays were carried out in 0.1 M KH2PO4/K2HPO4
(excitation wavelength) and 535 nm (emission wavelength). The 800
final concentration of tested compound or Trolox standard substance 801
OOF
739 buffer (pH 8.0). Enzyme solutions were prepared by dissolving lyophi-
740 lized powder in double-distilled water. Stock solutions of tested com-
741 pounds (10 mM) were prepared in DMSO and diluted in phosphate
742 buffer (pH 8.0). The assay solution (200 μL) consists of phosphate buffer
743 (pH 8.0), with the addition of 10 μL of 0.01 M DTNB, 10 μL of enzyme,
744 and 10 μL of 0.01 M substrate (ATC or BTC). Five increasing concentra-
745 tions of inhibitors with their inhibitory activity ranged from 20% to
746 80% were added to the assay solution and pre-incubated for 10 min at
747 37 °C with the enzyme followed by the addition of corresponding sub-
748 strate. Initial rate measurement assays were performed at 37 °C with a
749 PowerWave XS2 microplate spectrophotometer. Absorbance value at
750 412 nm was recorded for 2 min, and the calculations were performed
751 based on the method of Ellman et al. Each concentration was tested in
752 triplicate, and IC50 values were calculated graphically from log concen-
753 tration inhibition curve (Origin 7.5 software).
was 1–5 μM. A blank (FL + AAPH in 75 mM potassium phosphate 802
buffer) instead of the sample and Trolox calibration solution was 803
used in each assay. All the reactions were carried out in triplicate, 804
and each reaction was repeated for at least three times. Anti- 805
oxidation curves (fluorescence versus time) were normalized to 806
the curve of the blank in the same assay, and then the area under 807
the fluorescence decay curve (AUC) was calculated. The net AUC of 808
a sample was obtained by subtracting the AUC of the blank. ORAC- 809
FL values were expressed as Trolox equivalents by using the standard 810
curve calculated for each sample, where the ORAC-FL value of Trolox 811
was taken as 1, indicating the anti-oxidative potency of the tested 812
compounds.
813
2.8. Anti-oxidation activity in SH-SY5Y cells
814
754
The determination of the inhibition effects on ChE in the presence of
755 metal ions and Aβ1–42 was carried out following the previously reported
756 method. Compound 9g was dissolved in HEPES buffer (pH 6.6) and one
757 of the four conditions was followed. The assay solution was 200 μL. Con-
758 dition (a): AChE or BuChE (0.08 U/mL final concentration) was added to
759 the inhibitor solutions (100 μL) and incubated for 10 min. Then, the re-
760 actions were initialized with the addition of 10 μL of 0.01 M DTNB, and
761 10 μL of 0.01 M substrate (ATC or BTC). Condition (b): AChE or BuChE
762 (0.08 U/mL final concentration) was added to the inhibitor solutions
763 (100 μL) and incubated for 10 min. Then CuSO4 or FeSO4 or Aβ1–42 pep-
764 tide (10 μM) was added, and the mixture was incubated for another
765 10 min. The reactions were initialized with the addition of 10 μL of
766 0.01 M DTNB, and 10 μL of 0.01 M substrate (ATC or BTC). Condition
767 (c): CuSO4 or FeSO4 or Aβ1–42 peptide (10 μM) was added to the inhib-
768 itor solutions (100 μL) and incubated for 10 min. Then AChE or BuChE
769 (0.08 U/mL final concentration) was added, and the mixture was incu-
770 bated for another 10 min. The reactions were initialized with the addi-
771 tion of 10 μL of 0.01 M DTNB, and 10 μL of 0.01 M substrate (ATC or
Intracellular ROS were measured with the a fluorescent probe (2′,7′- 815
dichlorofluorescein diacetate, DCFH-DA) as reported with some varia- 816
tion. Human neuroblastoma cells, SH-SY5Y, were routinely grown at 817
37 °C in a humidified incubator with 5% CO2 in Dulbecco's modified 818
Eagle's medium (DMEM, GIBCO) containing 15 nonessential amino 819
acid and supplemented with 10% fetal calf serum (FCS, GIBCO), 1 mM 820
glutamine, 50 mg/μL penicillin, and 50 mg/μL streptomycin. For assays, 821
SH-SY5Y cells were sub-cultured in 96-well plates at a seeding density 822
of 3 × 104 cells per well. After 24 h, the cells were treated with the syn- 823
thesized compounds at concentrations of 3.125 μM, 6.25 μM, 12.5 μM, 824
and 25 μM. Trolox was used as a reference compound. After 24 h of 825
treatment with the compounds, the cells were washed with phosphate 826
buffer, and then incubated with 5 μM DCFH-DA in phosphate buffer at 827
37 °C in 5% CO2 for 30 min. After DCFH-DA was removed, the cells 828
were washed and incubated with 0.1 mM t-BuOOH in phosphate buffer 829
for 30 min. At the end of the incubation, the fluorescence of the cells 830
from each well was measured at 485 nm excitation and 535 nm emis- 831
sion with a monochromators based multimode microplate reader (INFI- 832
NITE M1000). Results are expressed as a percentage of the sample 833
average divided by the control group data, calculated as follows: 834
UNCORRECTED PR
772 BTC). Condition (d): A mixture of AChE or BuChE (0.08 U/mL final con-
773 centration) and CuSO4 or FeSO4 or Aβ1–42 peptide (10 μM) was added to
774 the inhibitor solutions (100 μL) and incubated for 10 min. The reactions
775 were initialized with the addition of 10 μL of 0.01 M DTNB, and 10 μL of
776 0.01 M substrate (ATC or BTC).
(ODsample − ODblank)/(ODcontrol − ODblank) × 100%.
835
2.9. Metal-chelating study
836
777 2.6. Kinetic characterization of AChE inhibition
The chelating studies were made in buffer (20 mM HEPES, 150 mM 837
NaCl, pH 7.4) using a UV–vis spectrophotometer (SHIMADZC UV- 838
2450PC). The absorption spectra of compound 9g (20 μM), alone or in 839
the presence of CuSO4, FeSO4, and ZnCl2 (40 μM), were recorded at 840
778
Kinetic characterization of AChE inhibition was performed based on
779 a reported method. The assay solution (200 μL) consists of 0.1 M phos-
780 phate buffer (pH 8.0), with the addition of 10 μL of 0.01 M DTNB, 10 μL
781 of enzyme, and 10 μL of substrate (ATC). Four different concentrations of
room temperature in a 1 cm quartz cell.
841
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

10
J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
842 2.10. Cell culture and MTT assay
secondary antibody (1:5000 dilution). Invitrogen's Western Breeze 901
Chemiluminescent kit was used to visualize the protein bands, and 902
these bands were imaged using a FUJIFILM Luminescent Image Analyzer 903
843
Cytotoxicity was evaluated with the colorimetric MTT [3-(4,5-di-
844 methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay. SH-
845 SY5Y cells were sub-cultured in 96-well plates at a seeding density of
846 10,000 cells per well. After 24 h, the cells were treated with different
847 concentrations of compounds (0–50 μM). After 48 h, the survival of
848 cells was determined with MTT assay. Briefly, 20 μL of MTT (5 mg/mL)
849 was added to each well and incubated for 4 h. The MTT medium in
850 each well was carefully removed and 100 μL DMSO was added into
851 each well, followed by incubation at 37 °C for 10 min with horizontal
852 shaking. The absorbance of each well was measured with a micro cul-
853 ture plate reader at the wavelength of 570 nm. The IC50 values were cal-
854 culated graphically from log concentration–inhibition curve (Origin 7.5
855 software).
LAS-1000CH.
904
2.14. CD spectroscopy
905
The secondary structure of Aβ1–42 aggregates was evaluated 906
using a Jasco-810-150S spectropolarimeter (Jasco, Japan) at room 907
temperature as described previously [38]. Aβ1–42 (20 μM) was 908
mixed with and without 10, 20 μM 9g in 20 mM sodium phosphate 909
buffer (pH 7.4). All solutions were incubated at 37 °C for 48 h. Spec- 910
tra were recorded at 25 °C between 190 and 260 nm with a band 911
width of 0.5 nm, a 3 s response time, and scan speed of 10 nm/min. 912
Background spectra and when applicable, spectra of 9g were 913
subtracted.
914
856 2.11. Determination of neuroprotective activity
2.15. Molecular docking study
915
857
Aβ1–42 was dissolved to 10 mM with DMSO and further diluted into
858 100 μL of 40, 20, and 10 μM, with Dulbecco's modified Eagle's medium,
859 respectively. DMSO was added to 100 μL Dulbecco's modified Eagle's
860 medium to give the blank control. Then the Aβ1–42 of 40, 20, 10 μM
861 and the blank control were pre-incubated for 48 h for aging fibrils at
862 37 °C (as prepared in aggregation studies). Aggregated Aβ1–42 (40, 20,
863 10 μM) were added to the SH-SY5Y cells, which had been incubated
864 for 24 h. Absorbance value was measured at 570 nm through MTT
865 methods. To study the protective effect of 9g on the neurotoxicity of
866 Aβ1–42, 20 μM Aβ1–42 seed samples, with or without 9g (3.125–12.5
867 μM), were pre-incubated at 37 °C for 48 h. Then the prepared samples
868 were added to SH-SY5Y cells, and incubated at 37 °C for 48 h. DMSO di-
869 luted with phosphate buffer solution was also added to the blank con-
870 trol wells. The final concentration of DMSO in each well was less than
871 0.5%. At the end of the experiment, samples were tested via MTT
872 methods. Absorbance value was measured to determine cell viability.
The crystal structure of human AChE (PDB ID: 1B41, resolution = 916
2.76 Å) was retrieved from the Protein Data Bank. For docking study, 917
short peptide and water molecules were removed. The apo structure 918
was superposed with the co-crystal structure of Torpedo californica 919
AChE and F11 (PDB ID: 2CMF [39], resolution = 2.5 Å), and then the 920
complex of human AChE and F11 was kept. The complex was prepared 921
in the“Protein Preparation Wizard” workflow in Maestro, version 9.4 922
[40], and bond orders were assigned. All the heavy atoms were mini- 923
mized to reach the converge root mean square deviation (RMSD) of 924
0.30 Å with the OPLS_2005 force field. After preparation, the docking 925
grid was generated using “Receptor Grid Generation”, the grid enclosing 926
box was centered on the original ligand (F11) a size of 10 × 8 × 10 (x × y 927
× z, Å), and a scaling factor of 0.80 was set to van der Waals radii of those 928
receptor atoms with a partial atomic charge less than 0.15. Compound 929
9g was optimized using MMFF force field [41] and the Powell method 930
was used for energy minimization by default parameters in Discovery 931
studio 2.5 (Accelrys Inc.). Extra precision mode (Glide XP) was 932
employed for identifying the potential binding of compound 9g to the 933
873 2.12. Dot blot assays
874
Dot blot assays to detect Aβ1–42 fibril aggregation with B10 were
human AChE.
934
875 performed as described previously with minor modification [36]. Brief-
876 ly, Aβ1–42 was diluted into 20 μM with buffer (20 mM HEPES, 150 mM
877 NaCl, pH 7.4) and then incubated with or without CuSO4 or FeSO4
878 (20 μM), and the concentrations of 9g were 20 μM and 40 μM. After in-
879 cubation at 37 °C for 24 h, 10 μL aliquots of 20 μM Aβ1–42 reactions were
880 spotted onto nitrocellulose membranes. Membranes were blocked for
881 2 h with 10% non-fat milk in TBS. After washing, membranes were incu-
882 bated with the anti-Aβ fibril antibody (1:2000 dilution) dissolved in TBS
883 containing 3% BSA and 0.01% Tween-20 and developed using an
884 alkaline-phosphatase antirabbit secondary antibody (1:5000 dilution).
885 Invitrogen's Western Breeze Chemiluminescent kit was used to visual-
886 ize the protein dots, and these dots were imaged using a FUJIFILM Lumi-
887 nescent Image Analyzer LAS-1000CH.
The initial coordinates of human BuChE used in our computational 935
studies came from the X-ray crystal structure (PDB ID: 1POP) [42]. The 936
missing residues (D2, D3, E255, D378, D379, N455, L530, E531, and 937
M532) were built using the automated homology modeling tools in Dis- 938
covery Studio 2.5. The binding site was defined as a box with the center 939
of the native ligand. Extra precision mode (Glide XP) was applied for 940
identifying the potential binding of compound 9g to the BuChE with 941
the default parameters.
942
For Aβ1–42 docking study, the initial structure of Aβ1–42 was taken 943
from the NMR structure (PDB ID: 1IYT) [43]. Autodock 4.0 was 944
employed to identify the binding poses of 9g for Aβ1–42 with a La- 945
marckian genetic algorithm [44]. The grid map, with 80 × 80 × 80 946
points spaced equally at 0.375 Å, was generated using the Auto 947
Grid program to evaluate the binding energies between ligand and 948
receptor. All docked poses of compound 9g were clustered using a 949
tolerance of 2 Å for the RMSD and ranked on the basis of the binding 950
docking energies, and the lowest energy conformation in the most 951
888 2.13. Native gel electrophoresis and Western blot
889
Gel electrophoresis and Western blot [37] were performed to deter-
890 mine the inhibition of Aβ1–42 aggregation by 9g in the presence or ab-
891 sence of metal ions with antibody 6E10. The preparation of samples
892 were the same as that for ThT assay, and 20 μM Aβ1–42 was incubated
893 with various concentrations of 9g (20 μM, 40 μM) in the presence or ab-
894 sence of 20 μM CuSO4 or FeSO4 at 37 °C for 24 h. Samples were separat-
895 ed on 10–20% gradient Tris–tricine mini gels. The gel was transferred to
896 a nitrocellulose membrane in an ice bath, and the protocol was followed
897 as suggested except that the membrane was blocked for 2 h with 10%
898 non-fat milk in TBS. After blocking, the membrane was incubated in a
899 solution (1:2000 dilution) of 6E10 anti-Aβ primary antibody (Covance)
900 for 2 h, and developed using an alkaline-phosphatase antimouse
populated cluster was chosen for further study.
952
953
954
3. Results and discussion
3.1. Organic syntheses of imidazole derivatives
Compounds 7a–9f, 7g–9g, 19a–20e, 22a–22f were synthesized as de- 955
scribed in Schemes 1–4. As shown in Scheme 1, commercially available 956
4-hydroxybenzaldehyde was first reacted with 1,2-dibromoethane, 957
1,3-dibromopropane, or 1,4-dibromobutane to give product 1, 2, or 3, re- 958
spectively. The product 1, 2, or 3 was reacted with phenanthrene-9,10- 959
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
11
960
961
962
963
964
965
966
967
968
969
970
971
dione to produce intermediate 4, 5, or 6, which was then reacted with
various amines to give final products 7a–9f. In Scheme 2, the intermedi-
ate 4, 5, or 6 was used to react with pyridine to produce 7g, 8g, and 9g.
Similarly, as shown in Scheme 3, 4-hydroxybenzaldehyde was first
reacted with propargyl bromide, followed with phenanthrene-9,10-
dione to give intermediate 11. The syntheses of 12a–12b, 13a–13d,
14a–14e, 15a–15d, 16a–16e, 17a–17d, and 18a–18e were carried out
by following the procedures reported previously [45–47]. The intermedi-
ate 11 was then reacted with 17a–18e through “click” chemistry with in-
troduction of triazole rings to afford products 19a–20e [45–48]. The
intermediate 11 was also reacted with azide 21a–21f to give products
22a–22f as shown in Scheme 4.
972 3.2. Inhibition of self-mediated Aβ1–42 aggregation
973
The inhibition of self-mediated Aβ1–42 aggregation by our synthe-
974 sized imidazole derivatives were studied by using thioflavin T (ThT)
975 assay [49] with resveratrol as a reference compound. The effects of
976 these compounds on Aβ1–42 peptide aggregation at concentration of
977 10 μM and the IC50 of these compounds against self-mediated Aβ1–42
978 aggregation were summarized as shown in Table 1. The IC50 value of
979 resveratrol against Aβ1–42 aggregation was 10 μM, in comparison,
980 more than half of our compounds displayed better inhibition effects
981 than resveratrol. The first series of our compounds 7a–9g almost all
982 showed more than 50% inhibition effect except 7f with 28% inhibitory
983 activity when used at 10 μM concentration. Compound 9g was found
984 to have 74% Aβ1–42 aggregation inhibitory effect when used at 10 μM
985 concentration with its IC50 value of 6.5 μM. The compounds with incor-
986 poration of triazole rings also displayed some inhibitory activity from
987 13% to 71% for Aβ1–42 aggregation when used at 10 μM concentration,
988 and the IC50 value of 19a against Aβ1–42 aggregation was 6.7 μM. The
989 reason of the similarities of anti-Aβ1–42 aggregation activity of our com-
990 pounds were explored by using molecular docking experiment and the
991 scores of the representative compounds 9d, 19a, 22e, and 9g were
992 −3.9514, −4.8855, −4.2690, −4.6432, respectively, and the binding
993 modes of these compounds with Aβ1–42 were similar (Fig. 3S).
OOF
Fig. 1. TEM image analysis of Aβ1–42 aggregation in the presence of 9g. (A) Aβ1–42 (20 μM),
0 h. (B) Aβ1–42 (20 μM) alone was incubated at 37 °C for 24 h. (C) Aβ1–42 (20 μM) and res-
veratrol (20 μM) were incubated at 37 °C for 24 h. (D) Aβ1–42 (20 μM) and 9g (20 μM)
were incubated at 37 °C for 24 h.
serum were used in this study because of their high sequence homology 998
to the human enzymes. As shown in Table S1, our most synthesized im- 999
idazole derivatives displayed strong inhibitory activity to ChE at micro- 1000
mole level, which were weaker than Tacrine. Compound 9g had potent 1001
inhibitory activity for both AChE and BuChE with its IC50 values of 1002
860 nM and 510 nM for AChE and BuChE, respectively, which were 1003
much better than those reported previously for other imidazole deriva- 1004
tives [32,33]. The Lineweaver Burk plots showed a mixed-type inhibi- 1005
tion of 9g to AChE (Fig. S1), indicating that 9g could bind to both 1006
catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. 1007
The expanded aromatic plane and the pyridine moiety at the end of 1008
the chain facilitated the binding of 9g to the ChE, which is consistent 1009
994 3.3. Inhibition studies for AChE and BuChE
995
The inhibitory activity of our synthetic derivatives was evaluated
996 against AChE and BuChE using the method of Ellman et al. [50] with Tac-
997 rine as a positive control. AChE from electric eel and BuChE from equine
with our following molecular modeling studies.
1010
t1:1
t1:2
Table 1
Effects of our compounds on Aβ1\42 aggregation.
t1:3
Compound
Aβ1\42 aggregation inhibition (%)a
Aβ1\42 IC50 (μM)b
Compound
Aβ1\42 aggregation inhibition (%)
Aβ1\42 IC50 (μM)
t1:4
7a
7b
7c
7d
7e
7f
7 g
8a
8b
8c
8d
8e
8f
8 g
9a
9b
9c
9d
9e
51
65
54
77
62
28
55
55
56
59
61
72
60
64
58
68
72
73
52
3
1
1
1
1
3
1
1
5
3
3
7
2
4
8
2
5
4
2
8.9
7.9
9.2
7.0
8.0
n.d.c
9.0
9.7
9.2
9.0
7.9
7.0
8.5
7.4
8.5
8.3
7.3
7.0
9.8
0.2
0.4
0.3
0.6
0.1
9f
g
70
74
71
63
13
58
60
51
50
55
65
41
23
35
57
24
54
53
2
2
1
4
1
4
3
1
1
2
3
6
3
2
3
4
2
2
7.0
6.5
6.7
8.0
n.d.
7.5
8.6
10.0
n.d.
8.4
0.8
0.4
0.4
1.2
t1:5
t1:6
19a
19b
19c
19d
20a
20b
20c
20d
20e
22a
22b
t1:7
t1:8
t1:9
1.2
2.2
1.1
t1:10
t1:11
t1:12
t1:13
t1:14
t1:15
t1:16
t1:17
t1:18
t1:19
t1:20
t1:21
t1:22
0.4
0.7
1.2
UNCORRECTED PR
1.0
1.5
0.8
2.2
1.4
2.1
0.3
0.6
1.0
1.1
0.3
0.8
7.4
n.d.
n.d.
n.d.
9.0
n.d.
8.4
22c
22d
22e
22f
1.0
0.6
Resveratrol
10.0
0.6
a
t1:23
The thioflavin-T fluorescence method was used. The values are expressed as the mean
SD of at least three independent measurements. All values were obtained at a compound
concentration of 10 μM.
b
t1:24
t1:25
The IC50 (μM) values shown are the mean
n.d. means not determined.
SD of three experiments.
c
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

12
J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
Fig. 2. Disaggregation experimental result. (A) ThT binding assay for Aβ1–42 without and with test compound. (B) TEM images for Aβ1–42 disaggregation. (a) Aβ1–42 (20 μM) was incubated
at 37 °C for 24 h in phosphate buffer. (b) Aβ1–42 (20 μM) was incubated with 9g (20 μM) at 37 °C for 24 h. (c) Aβ1–42 (20 μM) was incubated with resveratrol (20 μM) at 37 °C for 24 h.
1011 3.4. Anti-oxidation activity studies
imidazole derivatives have been studied by using the oxygen radical ab- 1014
sorbance capacity assay method with fluorescein (ORAC-FL) [51,52], 1015
and vitamin E analogue Trolox has been used as a standard. We studied 1016
our compounds for anti-oxidation activity with this assay method, and 1017
1012
The reduction of the oxidative stress is another crucial aspect in de-
1013 signing agents for AD treatment. The anti-oxidation activities of the
Fig. 3. Metal induced Aβ aggregation was carried out with incubation for 2 min, followed with test compound induced Aβ disaggregation upon incubation at 37 °C for 24 h. (A) ThT binding
assay for metal induced Aβ aggregation, and test compound induced Aβ disaggregation. (B) TEM images for metal induced Aβ aggregation, and test compound induced Aβ disaggregation.
The incubations were carried out with following reagents: (a) 20 μM Aβ1–42 alone. (b) 20 μM Aβ1–42 and 20 μM Cu2+. (c) 20 μM Aβ1–42 and 20 μM Fe2+. (d) 20 μM Aβ1–42, 20 μM Cu2+, and
40 μM 9g. (e) 20 μM Aβ1–42, 20 μM Fe2+, and 40 μM 9g. (f) 20 μM Aβ1–42, 20 μM Cu2+, and 40 μM resveratrol. (g) 20 μM Aβ1–42, 20 μM Fe2+, and 40 μM resveratrol.
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
13
Fig. 4. Percentage increase in intracellular ROS induced by exposure to t-BuOOH, as deter-
mined by DCFH-DA. The concentrations of compounds were 3.125 μM, 6.25 μM, 12.5 μM,
and 25 μM.
Fig. 6. The inhibitory activity of our imidazole derivatives to AChE-induced Aβ1–42 aggre-
gation. The concentration of Aβ1–42 was 25 μM, and the concentration ratio of Aβ1–42
,
AChE, compound was 100:1:100.
OOF
1018 found that most of our compounds showed relatively low ORAC-FL
1019 values compared to Trolox, as shown in Table S1. In comparison, com-
1020 pound 9g displayed relatively high anti-oxidation activity, with approx-
1021 imately 2.29 times higher activity than Trolox when used at 5 μM
1022 concentration. The anti-oxidation mechanism of 9g is not clear at this
1023 time. We think that 9g could have several possible mechanisms for its
1024 anti-oxidation activity in cells. The core structure of phenanthro-
1025 imidazole is somehow similar to that of melatonin, a well-known anti-
1026 oxidation agent, which could prevent the generation of ROS through
1027 its interaction with intracellular superoxide anion. Besides, 9g may
1028 play an indirect anti-oxidation role through activation of some anti-
1029 oxidation enzymes in cells. In addition, higher concentration of metal
1030 ions could cause increased ROS, and 9g could reduce the generation of
1031 ROS through metal chelation.
aggregation, and had its IC50 values of 860 nM and 510 nM for AChE 1041
and BuChE respectively, with 2.29 times higher anti-oxidation activity 1042
than Trolox and very low cytotoxic effect to neural cells, this compound 1043
could become a promising lead compound for further development for 1044
AD treatment.
1045
3.6. Inhibition of Aβ1–42 fibril formation monitored by using transmission 1046
electron microscopy (TEM) 1047
To further study the activity of compound 9g for its inhibition of 1048
Aβ1–42 aggregation, its inhibitory activity was monitored by using trans- 1049
mission electron microscopy (TEM) [53], with resveratrol as a reference 1050
compound. After 24 h of incubation at 37 °C, Aβ1-42 alone aggregated 1051
into well-defined Aβ fibrils (Fig. 1B). In contrast, few and slender Aβ fi- 1052
brils were observed in the presence of compound 9g (Fig. 1D) under 1053
identical conditions. Therefore, based on the TEM and ThT measure- 1054
ment results, we can conclude that compound 9g could effectively in- 1055
1032 3.5. Cytotoxic studies on SH-SY5Y neuroblastoma cells
1033
The cytotoxicity of our imidazole derivatives to the human neuro-
1034 blastoma SH-SY5Y cells was evaluated by using colorimetric MTT
1035 assay. The cells were treated with various concentrations of compounds
1036 for 48 h with maximum concentration of 50 μM. As shown in Table 1,
1037 most compounds showed their IC50 values of more than 10 μM, indicat-
1038 ing their low neural cytotoxic effects. Compound 9g had its IC50 value of
1039 more than 50 μM, indicating its very low neural cytotoxicity. As men-
1040 tioned above, considering that compound 9g inhibited 74% Aβ1–42
hibit Aβ1–42 fibril formation.
1056
3.7. Disaggregation of self-induced Aβ1–42 aggregation fibrils by 9g
1057
The ability of 9g to disaggregate self-induced Aβ1–42 aggregation fi- 1058
brils was investigated. Aβ1–42 fibrils were generated by incubating 1059
fresh Aβ1–42 for 24 h at 37 °C. The test compound was then added to 1060
UNCORRECTED PR
Fig. 5. Protective effect of 9g on Aβ1–42-induced toxicity in SH-SY5Y cell lines. (A) Aβ1–42 (10, 20 and 40 μM) were incubated with SH-SY5Y cells for 48 h. (B) Aβ1–42 (20 μM) was incubated
with SH-SY5Y cells in the absence or presence of various concentrations of 9g for 48 h. Cell viability was determined by using MTT methods.
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

14
J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
t2:1
t2:2
Table 2
3.9. Anti-oxidation activity of 9g in SH-SY5Y cells
1088
IC50 values for inhibition of ChE by compound 9g in HEPES buffer (nM).
The ability of 9g to counteract the formation of ROS was assayed in 1089
human neuroblastoma cells (SH-SY5Y) based on dichlorofluorescein 1090
diacetate (DCFH-DA), after the treatment with tert-butyl hydroperoxide 1091
(t-BuOOH), a compound used to induce oxidative stress [56,57]. Trolox 1092
was used as a reference control compound. The concentrations of tested 1093
compounds were 3.125 μM, 6.25 μM, 12.5 μM, and 25 μM. As shown in 1094
Fig. 4, both Trolox and compound 9g exhibited dose-dependent anti- 1095
oxidant activity, and the activity of 9g was higher than that of Trolox. 1096
t2:3
t2:4
t2:5
IC50 (nM)
AChE
BuChE
CuSO4
Experiment
CuSO4
FeSO4
Aβ1–42
FeSO4
Aβ1–42
t2:6
t2:7
t2:8
t2:9
a
b
c
796
820
766
753
796
845
642
822
796
903
800
829
495
527
445
400
495
478
483
367
495
493
511
515
d
t2:10 Experiments: (a) 9g was incubated with AChE (or BuchE) for 10 min. (b) 9g was
t2:11 incubated with AChE (or BuchE) for 10 min, followed with addition of Cu2+ or Fe2+ or
t2:12 Aβ1–42 and incubation of the mixture for additional 10 min. (c) 9g was incubated with
t2:13 Cu2+ or Fe2+ or Aβ1–42 for 10 min, followed with addition of AChE (or BuChE) and
t2:14 incubation of the mixture for additional 10 min. (d) AChE (or BuChE) was incubated
t2:15 with Cu2+ or Fe2+ or Aβ1–42 for 10 min, followed with addition of 9g and incubation of
t2:16 the mixture for additional 10 min.
3.10. Compound 9g protects cells against Aβ1–42-induced toxicity
1097
As mentioned before, compound 9g had low cell toxicity, with its 1098
IC50 value of more than 50 μM for SH-SY5Y cells. The neuroprotective 1099
activity of 9g was further studied following a procedure reported 1100
previously with minor modification [58]. SH-SY5Y cells were treated 1101
with Aβ1–42 solutions at 10 μM, 20 μM, and 40 μM concentrations. 1102
Aβ1–42-induced cytotoxicity was determined as shown in Fig. 5a, 1103
which indicated that Aβ1–42 significantly reduced cell viability in a 1104
dose-dependent manner. It was found that the cells were well 1105
protected when 20 μM Aβ1–42 was mixed with compound 9g at 1106
3.125 μM, 6.25 μM, and 12.5 μM concentrations, as shown in Fig. 5b. 1107
This result showed that 9g was a neuroprotective agent against 1108
1061 the sample and incubated for another 24 h at 37 °C. Our ThT binding
1062 assay result showed that compound 9g, resveratrol, and compound
1063 19a could all disaggregate Aβ1–42 fibrils at 20 μM concentration with
1064 ratio of 73.3%, 68.7%, and 52%, respectively, as shown in Fig. 2(A). Our
1065 TEM result further demonstrated the disaggregation effect of compound
1066 9g in comparison with resveratrol, as shown in Fig. 2(B).
Aβ1–42-induced toxicity at low concentration.
1109
1067 3.8. Effect of 9g on metal-induced Aβ1–42 aggregation
3.11. Inhibition of 9g on AChE-induced Aβ1–42 aggregation
1110
1068
Transition metal ions, especially Cu, Fe, Zn, play important role in
1069 the development of AD. The interaction of 9g with Cu(II), Fe(II), and
1070 Zn(II) was studied by using UV–vis spectroscopy [37,54,55]. As
1071 shown in Fig. S2, the addition of 2 equivalents of metal ions to
1072 20 μM 9g caused decreased UV absorbance for 9g, indicating the in-
1073 teraction of metal ions with 9g.
It has been reported that Aβ deposition in AD brain is linked to AChE 1111
expression, and the PAS of AChE can bind to the Aβ, accelerating the for- 1112
mation of amyloid fibrils [59,60]. The inhibitory activity of our imidazole 1113
derivatives to AChE-induced Aβ1–42 aggregation was also determined 1114
by using thioflavin T (ThT) assay, with Tacrine, propidium iodide, and 1115
Congo-red as reference compounds. As shown in Fig. 6, Tacrine, with 1116
its high affinity for CAS rather than PAS of AChE, showed 33.4% inhibito- 1117
ry effect against AChE-induced Aβ1–42 aggregation. In comparison, 1118
propidium iodide significantly reduced about 85.9% Aβ1–42 aggregation 1119
as a result of its noncompetitive inhibition type. Our imidazole deriva- 1120
tives 9g and 19a displayed 65.2% and 36.0% inhibition of ChE-induced 1121
Aβ1–42 aggregation, respectively. Compound 9g was better than the sin- 1122
gle site inhibitor Tacrine, indicating its possible dual binding effect to 1123
1074
To investigate the ability of our imidazole derivatives to inhibit
1075 metal-induced Aβ aggregation, we studied the effect of 9g on
1076 metal-induced Aβ1–42 aggregation by ThT fluorescence and TEM.
1077 After incubation at 37 °C for 24 h, Cu2+ and Fe2+ could induce the
1078 fibrillization of Aβ1–42 at the levels of 151.6% and 176.9%, respective-
1079 ly, in comparison with the fibrillization of Aβ1–42 alone as 100%. More
1080 fibrils could be observed with the TEM (Fig. 3b and c). The addition of
1081 9g could significantly reverse these effects, rescuing 129.5% and
1082 151.4% Aβ1–42 aggregation induced by Cu2+ and Fe2+ respectively,
1083 and its disaggregation effect was better than resveratrol (rescuing
1084 104.0% aggregation induced by Cu2+, and 120.0% aggregation in-
1085 duced by Fe2+). These disaggregation effects were consistent with
1086 our TEM image results, and we could barely see the fibrils for 9g-
1087 treated Aβ1–42 (Fig. 3d and e).
AChE with mixed type inhibitory activity.
1124
3.12. Effects of metal ions and Aβ1–42 on inhibition of ChE by compound 9g 1125
It is possible that Aβ and metal ions could interact with compound 1126
9g, which could prevent the binding of 9g with ChE. Therefore, the 1127
Fig. 7. Dot plot analysis for the inhibition of self-aggregation and metal-induced formation of Aβ fibrils by 9g with Aβ fibrils antibody B10. The concentrations of Aβ1–42 and metal ions were
20 μM, and the concentrations of 9g were 20 μM and 40 μM. (A) Dot plot image of Aβ1–42 in the presence or absence of metal ions and/or 9g after incubation at 37 °C for 24 h. (B) Bar
graphical analysis for the relative intensity of the dot plot result.
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
15
accelerate the formation of Aβ1–42, which is consistent with our ThT re- 1146
sult. The incubation of Aβ1–42 with 9g reduced Aβ fibril formation, as in- 1147
dicated by B10 immunoreactivity. Our results showed that 9g could 1148
reduce both Aβ self-aggregation and metal-induced Aβ-aggregation. 1149
Besides, as shown in Fig. 8b and c, more lower molecular weight 1150
(≤25 kDa) Aβ species were visualized by using native gel electrophore- 1151
sis followed by Western blot with 6E10. Compound 9g had dose- 1152
dependent inhibition of Aβ fibril formation, and stabilized lower molec- 1153
ular weight Aβ species. For Cu2+-induced (Fig. 8f) and Fe2+-induced 1154
(Fig. 8g) Aβ-aggregation, more lower molecular weight Aβ species 1155
could be observed upon incubation with 9g (Fig. 8d and e), indicating 1156
that 9g could reduce metal-induced Aβ-aggregation.
1157
3.14. Effect of 9g on Aβ β-sheet formation
1158
Fig. 8. Gel electrophoresis and Western blot analysis for the inhibition of Aβ1–42 aggrega-
tion by 9g in the presence or absence of metal ions with antibody 6E10. Aβ1–42 used was
20 μM, the metal ions used was 20 μM, and the concentrations of 9g were 20 μM and 40 μM
for the inhibition of Aβ1–42 aggregation and 40 μM for the inhibition of metal-induced Aβ1–
42 aggregation. (a) Incubation of Aβ1–42 alone for 24 h at 37 °C. (b) Incubation of Aβ1–42
with 20 μM 9g for 24 h at 37 °C. (c) Incubation of Aβ1–42 with 40 μM 9g for 24 h at
37 °C. (d) Incubation of Aβ1–42 with 20 μM Cu2+ and 40 μM 9g for 24 h at 37 °C. (e) Incu-
bation of Aβ1–42 with 20 μM Fe2+ and 40 μM 9g for 24 h at 37 °C. (f) Incubation of Aβ1–42
with 20 μM Cu2+ for 24 h at 37 °C. (g) Incubation of Aβ1–42 with 20 μM Fe2+ for 24 h at
37 °C.
It has been known that Aβ adopt a conformational mixture of α- 1159
helix, β-sheet, and random coil in aqueous solution, and undergo a con- 1160
formational change to form intramolecular β-sheet structure in the 1161
fibrillization [63]. These β-sheets have been suggested of contributing 1162
to the toxicity of Aβ [64]. In order to further investigate the mechanism 1163
of 9g-induced Aβ conformational transformation, CD spectroscopy was 1164
OOF
used to monitor the change of Aβ1–42 secondary structure during the as- 1165
sembly stage without or with different concentrations of 9g. As shown 1166
in Fig. 9, freshly prepared Aβ1–42 in phosphate buffer solution had no 1167
obvious α-helix and β-sheet (Fig. 9A), which indicates unfolded peptide 1168
structure. After 48 h of incubation at 37 °C, a maximal positive absor- 1169
bance around 195 nm (the general characteristic of α-helix) and a neg- 1170
ative absorbance around 218 nm (the general characteristic of β-sheets) 1171
were observed (Fig. 9b). The addition of 9g decreased the absorbance at 1172
both 195 nm and 218 nm. Thus, 9g could stabilize random Aβ1–42 and 1173
reduce the formation of α-helix and β-sheet structure of the peptide, 1174
1128 ability of 9g to inhibit ChE in the presence of metal ions and Aβ1–42 was
1129 examined by following a procedure reported previously [61]. As shown
1130 in Table 2, 9g showed good inhibition to both AChE and BuChE, with IC50
1131 values of 796 nM and 495 nM for AChE and BuChE respectively in HEPES
1132 buffer (pH 6.6, Condition a). The presence of metal ions or Aβ1–42 (Con-
1133 ditions b–d) had little effect to the inhibitory activity of 9g to ChE. Com-
1134 pound 9g retained good inhibition to ChE in the presence of metal ions
1135 or Aβ1–42, indicating that inhibition of ChE by compound 9g is not influ-
1136 enced by its Aβ disaggregation and anti-oxidation activity for
1137 Alzheimer's disease and the multifunctional effects of 9g.
which could partially explain its inhibition of Aβ aggregation.
1175
3.15. Molecular modeling study for the binding mode of 9g with AChE and 1176
BuChE as well as Aβ1–42 1177
1138 3.13. Dot plot, gel electrophoresis and Western blot analysis for the inhibi-
1139 tion of Aβ1–42 aggregation by 9g
To investigate the binding modes between compound 9g and AChE, 1178
BuChE, as well as Aβ, molecular docking studies were carried out. As 1179
shown in Fig. 10A, compound 9g could well occupy the PAS of AChE 1180
and forms hydrophobic interactions with residues Phe297, Phe295, 1181
Leu289, Val294, Trp286, Tyr337, Phe338, Tyr341, and Trp86. The intro- 1182
duction of pyridine moiety at the end of chain can enhance AChE inhibi- 1183
tion activity because it can form π–π stacking interactions with Tyr336 1184
and Trp86 (Fig. 10B). Compared the binding modes of compound 9g 1185
with ligand F11 (a known inhibitor occupies two binding sites of 1186
1140
The inhibition of Aβ1–42 aggregation by 9g was further analyzed by
1141 using dot plot, gel electrophoresis, and Western Blotting. Dot-blot as-
1142 says of the Aβ samples were performed using Aβ fibril-specific antibody
1143 B10 [62], which preferentially recognizes amyloid fibrils but does not
1144 bind to monomers or small oligomers. The antibody used in the gel
1145 electrophoresis was 6E10. As shown in Fig. 7, Cu2+ and Fe2+ could
UNCORRECTED PR
Fig. 9. (A) CD spectroscopy for incubation of Aβ1–42 alone (20 μM) and Aβ1–42 with compound 9g (20 μM, 40 μM) at 37 °C for 0 h. (B) CD spectroscopy for incubation of Aβ1–42 alone
(20 μM) and Aβ1–42 with compound 9g (20 μM, 40 μM) at 37 °C for 48 h.
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

16
J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
Fig. 10. Binding mode for 9g–AChE complex. (A) 3D ligand-interaction diagram. (B) 2D ligand-interaction diagram. (C) The polar and hydrophobic surface profile of human AChE with
compound 9g. (D) Superposition of 9g with F11 in ligand-binding pocket. Blue carbon represents compound 9g and cyan carbon represents compound F11.
Fig. 11. Binding modes for 9g–BuChE and 9g–Aβ1–42 complexes. (A) 3D ligand-interaction diagram of 9g–BuChE. (B) 2D ligand-interaction diagram of 9g–BuChE. (C) 3D ligand-interaction
diagram of 9g–Aβ1–42. (D) 2D ligand-interaction diagram of 9g–Aβ1–42
.
Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
treatment of Alzheimer's disease, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagen.2014.05.005

J. Liu et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
17
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1188 ligand F11 (Fig. 10D), suggesting its dual binding mode and mixed
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1212 treatment of AD.
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Please cite this article as: J. Liu, et al., Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for
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