Synthesis and biological evaluation of genistein-O-alkylamine derivatives as potential multifunctional anti-Alzheimer agents

Article abstract of DOI:10.1111/cbdd.13414

A series of genistein derivatives were synthesized and evaluated as multifunctional anti-Alzheimer agents. The results showed that these derivatives had significant acetylcholinesterase (AChE) inhibitory activity; compound 5a exhibited the strongest inhib

Full text of DOI:10.1111/cbdd.13414

DR WEN LUO (Orcid ID : 0000-0002-0621-2886)
Article type
: Research Article
Synthesis and Biological Evaluation of Genistein-O-alkylamine
Derivatives as Potential Multifunctional Anti-Alzheimer Agents
Chen Hong, a Hui-yan Guo, b Shuai Chen, b Jian-wu Lv, b Xin Zhang, b Ya-cheng Yang, b Kang
Huang, c Yi-juan Zhang, c Zhi-yong Tian, c Wen Luo, b,* Yi-ping Chen, d,*
a Huaihe Hospital, Henan University, Kaifeng 475000, China
b Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University, Kaifeng
475004, China
c Pharmaceutical College, Henan University, Kaifeng 475004, China
d School of Pharmaceutical Sciences, Guangxi University of Chinese Medicine, Nanning
530200, China
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.13414
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* Wen Luo: Email luowen83@henu.edu.cn, Tel 086 0371-22864665, Fax 086 0371-22864665
* Yi-ping Chen: Email yipchen2009@foxmail.com, Tel 086 0771-4733943
KEYWORDS
Genistein
Alzheimer’s disease
Cholinesterase
Antioxidant
Multifunctional
ABSTRACT
A series of genistein derivatives were synthesized and evaluated as multifunctional
anti-Alzheimer agents. The results showed that these derivatives had significant
acetylcholinesterase (AChE) inhibitory activity; compound 5a exhibited the strongest
inhibition to AChE with an IC50 value (0.034 M) much lower than that of rivastigmine (6.53
M). A Lineweaver-Burk plot and molecular modeling study showed that 5a targeted both
the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. These
compounds also showed potent peroxy scavenging activity and metal chelating ability. The
compounds did not show obvious effect on HepG2 and PC12 cell viability at the
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concentration of 100 μM. Therefore, these genistein derivatives can be utilized as
multifunctional agents for the treatment of AD.
1. INTRODUCTION
Alzheimer’s disease (AD), a disastrous and highly prevalent neurodegenerative disorder, is
typically characterized by memory deficit and progressive impairment of cognitive
functions.1 Approximately 36 million people worldwide suffer from AD, and the number may
surpass 115 million by 2050.2 The pathogenesis of AD is extrmely complex which involves
several factors, such as low levels of the acetylcholine (ACh), aggregation of -amyloid (A),
oxidative damage and metal dysregulation.3
ACh, being a major neurotransmitter in the brain, plays a crucial role in learning, memory,
cognition and consciousness.4 The pathogenesis of AD may involve the destruction of the
cholinergic neurons in the brain of the AD patients. 5 Currently, the main therapeutic
approach in the AD treatment involves the enhancement of the cholinergic
neurotransmission by inhibiting cholinesterase (ChE). Four cholinesterase inhibitors such as,
tacrine, donepezil, rivastigmine and galantamine have been approved by FDA.6 However,
treatments with ChE inhibitors are palliative in nature, which can improve only the clinical
symptoms by modulating a single target, but cannot prevent the progression of the disease.
The multi-target-directed ligands (MTDLs) can simultaneously target multiple pathological
processes involved in the neurodegenerative cascade, and treatment with MTDLs can be
considered as a better therapeutic strategy than the treatment with ChE inhibitors. 7 MTDLs
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can be synthesized by conjugating or fusing two or more bioactive scaffolds for multiple
complementary biological activities.8, 9
High levels of biometals, like copper, zinc and iron bind to the A peptides and generate
toxic A oligomers.10 Redox-active metal ions, such as Cu2+ and Fe2+ are also involved in the
production of reactive oxygen species (ROS). Excessive ROS causes oxidative stress, which
eventually destroys the neuronal networks in AD patients.11 The progression of AD can be
retarded by modulating the biometals, inhibiting A aggregation and eliminating the ROS in
the brain of the patients.12
Genistein (4’,5,7-trihydroxyisoflavone, Fig. 1, A), a polyphenolic isoflavone was isolated
from Genista tinctoria (commonly known as dyer’s broom).13 The compound possesses a
broad range of biological activities, such as estrogenic, antioxidant, anti-inflammatory,
antimicrobial and metal chelting activity.14 Genistein also exhibited neuroprotective effect,
and ameliorated learning and memory deficits in AD rat model.15, 16 The curative potentials
of different genistein derivatives (Fig. 1, B, C) 17, 18 against AD have been reported in
different scientific studies, and genistein molecule can act as a lead compound to synthesize
different other derivatives with anti-AD activity.
As per the previous studies by our research group,19-21 genistein-polyamine conjugates
exhibited potent ChEs inhibition activity and metal chelating ability with low cytotoxicity in
vitro22 and thus, can be utilized as potent multifunctional agents for AD therapy. One of our
previous research also showed that flavonoid derivatives modified with a five-membered
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ring (such as pyrrolidine) possess potent ChE inhibitory activity.23, 24 Therefore, the present
study is aimed at synthesizing a series of genistein derivatives with enhanced
pharmacological potential by conjugating with pyrrolidine or imidazole using different
lengths of carbon spacers as the linker. Among these derivatives, 3h-i and 5e-i are new and
have not been reported previously. Although other compounds have been described in
several literature (Supporting Information, Table 1), we were unable to find their ChEs
inhibitory, antioxidative data (3a-g, 5a-d and 9a-b) and 1H NMR, 13C NMR data (3d, 5a-d and
9a). The strategy for synthesis of genistein derivatives is depicted in Fig. 2.
2. METHODS AND MATEREALS
4.1 Chemistry
Melting points (mp) were determined using an X-6 hot stage microscope and were not
corrected. 1H NMR and 13C NMR spectra were recorded with Bruker AV-300 or Bruker
AV-400 spectrometer in CDCl3 or DMSO-d6 at 300 or 400 MHz and 75 or 100 MHz,
respectively, using solvent peaks [CDCl3: 7.27 (D), 77.2 (C) ppm; and DMSO-d6: 2.49 (D), 40
(C) ppm] as internal reference. MS spectra were recorded on a Shimadzu LCMS-2010A
instrument with an ESI mass selective detector. Elemental analyses were performed on a
Gmbe VarioEL Elemental Instrument. Flash column chromatography was performed with
silica gel (200 - 300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd.
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2.2 General procedure for the synthesis of the intermediates 2a-e, 4a-e
A suspension of genistein (2.70 g, 10 mmol), anhydrous K2CO3 (1.38 g, 10.0 mmol) in
acetone (20 mL), α,ω-dibromoalkane (10.0 mmol) were mixed together and refluxed for 10
h. The solvent was evaporated under reduced pressure, the residue was poured into water,
extracted with EtOAc, dried and concentrated under vacuum. The intermediates 2a-e were
crystallized from EtOH, and they were used in the next step without further purification.
The intermediates 4a-e were synthesized following the previously mentioned procedure
for 2a-e, using 40 mmol of α,ω-dibromoalkane and keeping the quantities/concentrations of
other reactants same.
2.3 General procedure for the synthesis of compounds 3a-i, 5a-i
A solution of 2a-e (1.0 mmol), anhydrous K2CO3 (2.5 mmol) in CH3CN (15 mL) and
pyrrolidine or imidazole (1.2 mmol) were mixed together and stirred at 60 °C for 8 h. The
solvent was removed under vacuum, the mixture was diluted with CHCl3 and then washed
with water and brine. The organic layer was dried over Na2SO4, and then concentrated
under vacuum. The resulting crude product was purified on a silica gel chromatography
using a mixture of CH2Cl2/MeOH as eluent.
The compounds 5a-i were synthesized following the previously mentioned procedure for
3a-i, using 2.5 mmol of pyrrolidine or imidazole.
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2.3.1 5-hydroxy-3-(4-hydroxyphenyl)-7-(2-(pyrrolidin-1-yl)ethoxy)-4H-chromen-4-one (3a)
White solid (73% yield), 1H NMR (300 MHz, DMSO-d6) δ 12.94 (s, 1H), 9.66 (s, 1H), 8.37 (s,
1H), 7.39 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 6.61 (d, J = 2.1 Hz, 1H), 6.37 (d, J = 2.1
Hz, 1H), 4.16 (t, J = 5.7 Hz, 2H), 2.80 (t, J = 5.7 Hz, 2H), 2.53 (t, J = 5.4 Hz, 4H), 1.75 – 1.47 (m,
4H). 13C NMR (75 MHz, DMSO-d6) δ 180.80, 164.81, 162.18, 157.94, 157.88, 154.73, 130.58,
122.92, 121.50, 115.53, 105.82, 98.79, 93.23, 67.99, 54.40, 23.59. MS (ESI+): m/z:
368.1[M+H]+.
2.3.2 5-hydroxy-3-(4-hydroxyphenyl)-7-(3-(pyrrolidin-1-yl)propoxy)-4H-chromen-4-one (3b)
White solid (75% yield), 1H NMR (300 MHz, DMSO-d6) δ12.93 (s, 1H), 9.65 (s, 1H), 8.38 (s,
1H), 7.38 (d, J = 8.1 Hz, 2H), 6.82 (d, J = 8.2 Hz, 2H), 6.62 (d, J = 2.7 Hz, 1H), 6.37 (s, 1H), 4.10
(d, J = 6.6 Hz, 2H), 2.42 (s, 4H), 1.88 (t, J = 6.9 Hz, 2H), 1.67 (s, 4H). 13C NMR (75 MHz,
DMSO-d6) δ 180.82, 165.06, 162.18, 157.93, 154.81, 130.62, 122.92, 121.51, 115.53, 105.78,
98.79, 93.23, 67.33, 54.07, 52.48, 28.39, 23.56. MS (ESI+): m/z: 382.2[M+H]+.
2.3.3 5-hydroxy-3-(4-hydroxyphenyl)-7-(4-(pyrrolidin-1-yl)butoxy)-4H-chromen-4-one (3c)
White solid (67% yield), 1H NMR (300 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.39 (d, J = 9.0 Hz,
2H), 6.82 (d, J = 6.0 Hz, 2H), 6.64 (d, J = 6.0 Hz, 1H), 6.39 (d, J = 6.0 Hz, 1H), 4.10 (t, J = 6.0 Hz,
2H), 2.45 – 2.33 (m, 6H), 1.85 – 1.70 (m, 2H), 1.66 (t, J = 6.0 Hz, 4H), 1.62 – 1.51 (m, 2H). 13C
NMR (75 MHz, DMSO-d6) δ 180.84, 165.11, 162.21, 157.98, 157.93, 154.80, 130.61, 122.95,
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121.53, 115.55, 105.80, 98.81, 93.28, 68.84, 56.50, 55.57, 54.00, 26.87, 25.12, 23.55, 19.01.
MS (ESI+): m/z: 396.2[M+H]+.
2.3.4 5-hydroxy-3-(4-hydroxyphenyl)-7-((6-(pyrrolidin-1-yl)hexyl)oxy)-4H-chromen-4-one (3d)
White solid (60% yield), 1H NMR (300 MHz, DMSO-d6) δ 12.94 (s, 1H), 9.65 (s, 1H), 8.40 (s,
1H), 7.39 (d, J = 6.0 Hz, 2H), 6.82 (d, J = 7.5 Hz, 2H), 6.63 (s, 1H), 6.38 (s, 1H), 4.07 (s, 2H),
2.37 (s, 6H), 1.76 – 1.60 (m, 6H), 1.50 – 1.29 (m, 6H). 13C NMR (75 MHz, DMSO-d6) δ 180.84,
165.11, 162.20, 157.96, 157.93, 154.80, 130.61, 122.95, 121.52, 115.55, 105.79, 98.80,
93.26, 68.94, 56.13, 54.08, 28.82, 27.21, 25.82, 23.52. MS (ESI+): m/z: 424.2[M+H]+.
2.3.5 7-(2-(1H-imidazol-1-yl)ethoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (3e)
White solid (53% yield), 1H NMR (300 MHz, DMSO-d6) δ 12.94 (s, 1H), 9.60 (s, 1H), 8.40 (s,
1H), 7.69 (s, 1H), 7.39 (d, J = 9 Hz, 2H), 7.25 (s, 1H), 6.90 (s, 1H), 6.82 (d, J = 6 Hz, 2H), 6.66
13
(d, J =3 Hz, 1H), 6.42 (d, J = 3 Hz, 1H), 4.39 (s, 4H). C NMR (75 MHz, DMSO-d6) δ 180.90,
164.31, 162.26, 157.96, 154.93, 138.13, 130.63, 128.83, 123.04, 121.47, 120.21, 115.56,
106.13, 98.90, 93.48, 68.42, 45.72. MS (ESI+): m/z: 365.1[M+H]+.
2.3.6 7-(3-(1H-imidazol-1-yl)propoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (3f)
White solid (55% yield), 1H NMR (300 MHz, DMSO-d6) δ 12.96 (s, 1H), 9.63 (s, 1H), 8.41 (s,
1H), 7.64 (s, 1H), 7.39 (d, J = 8.7 Hz, 2H), 7.21 (d, J = 1.2 Hz, 1H), 6.90 (t, J = 1.2 Hz, 1H), 6.83
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(d, J = 8.7 Hz, 2H), 6.65 (d, J = 2.4 Hz, 1H), 6.41 (d, J = 2.1 Hz, 1H), 4.15 (t, J = 6.9 Hz, 2H), 4.04
(t, J = 6.0 Hz, 2H), 2.19 (t, J = 6.6 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 180.88, 164.76,
162.20, 157.94, 154.90, 137.84, 130.63, 128.93, 122.99, 121.50, 119.85, 115.55, 105.98,
98.88, 93.34, 66.02, 43.27, 30.35. MS (ESI+): m/z: 379.1[M+H]+.
2.3.7 7-(4-(1H-imidazol-1-yl)butoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (3g)
White solid (56% yield), 1H NMR (300 MHz, DMSO-d6) δ 12.95 (s, 1H), 9.67 (s, 1H), 8.41 (s,
1H), 7.65 (s, 1H), 7.38 (d, J = 8.1 Hz, 2H), 7.20 (s, 1H), 6.89 (s, 1H), 6.82 (d, J = 8.1 Hz, 2H),
6.64 (s, 1H), 6.40 (s, 1H), 4.13 – 3.98 (m, 4H), 1.92 – 1.79 (m, 2H), 1.70 – 1.63 (m, 2H). 13C
NMR (75 MHz, DMSO-d6) δ 180.84, 164.94, 162.19, 157.92, 154.86, 137.72, 130.63, 128.82,
122.93, 121.50, 119.74, 115.54, 105.82, 98.80, 93.30, 68.36, 46.00, 27.65, 25.90. MS (ESI+):
m/z: 393.1[M+H]+.
2.3.8 7-((6-(1H-imidazol-1-yl)hexyl)oxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one
(3h)
1
White solid (48% yield), mp 200202 C; H NMR (300 MHz, DMSO-d6) δ 12.95 (s, 1H),
9.64 (s, 1H), 8.40 (s, 1H), 7.61 (s, 1H), 7.39 (d, J = 9.0 Hz, 2H), 7.16 (s, 1H), 6.87 (s, 1H), 6.82
(d, J = 6.0 Hz, 2H), 6.64 (s, 1H), 6.38 (s, 1H), 4.05 (t, J = 6.0 Hz, 2H), 3.94 (t, J =6.0 Hz, 2H),
1.71 (t, J = 6.0 Hz, 4H), 147 – 1.39 (m, 2H), 1.30 – 1.23 (m, 2H). 13C NMR (75 MHz, DMSO-d6)
δ 180.84, 165.07, 162.19, 157.96, 157.92, 154.87, 137.67, 130.64, 128.73, 122.93, 121.51,
119.72, 115.54, 105.78, 98.80, 93.26, 68.83, 46.31, 30.95, 28.69, 26.09, 25.34. MS (ESI+):
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m/z: 421.2[M+H]+. Elemental Anal. Calcd for C24H24N2O5: C, 68.56; H, 5.75; N, 6.66. Found: C,
68.77; H, 5.90; N, 6.45.
2.3.9 7-((8-(1H-imidazol-1-yl)octyl)oxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one
(3i)
White solid (46% yield), mp 173175 C; 1H NMR (300 MHz, DMSO-d6) δ 12.95 (s, 1H),
9.67 (s, 1H), 8.40 (s, 1H), 7.61 (s, 1H), 7.38 (d, J = 9.0 Hz, 2H), 7.15 (s, 1H), 6.88 – 6.79 (m,
3H), 6.63 (s, 1H), 6.38 (s, 1H), 4.05 (t, J = 6.0 Hz, 2H), 3.93 (t, J = 6.0 Hz, 2H), 1.73 – 1.66 (q, J
= 6.9 Hz, 4H), 1.37 – 1.18 (m, 8H). 13C NMR (75 MHz, DMSO-d6) δ 180.83, 165.09, 162.17,
157.95, 157.92, 154.86, 137.66, 130.63, 128.71, 122.91, 121.50, 119.70, 115.53, 105.76,
98.78, 93.24, 68.91, 46.35, 31.01, 29.06, 28.86, 28.80, 26.32, 25.76. MS (ESI+): m/z: 449.2
[M+H]+. Elemental Anal. Calcd for C26H28N2O5: C, 69.63; H, 6.29; N, 6.25. Found: C, 69.80; H,
6.12; N, .6.04.
2.3.10
5-hydroxy-7-(2-(pyrrolidin-1-yl)ethoxy)-3-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4H-chromen-4
-one (5a)
White solid (52% yield), 1H NMR (400 MHz, CDCl3) δ 12.31 (s, 1H), 7.81 (s, 1H), 7.38 (d, J =
8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.35 (d, J = 2.0 Hz, 1H), 6.27 (d, J = 2.0 Hz, 1H), 4.29 (q, J
= 4.8 Hz, 4H), 3.39 – 3.33 (m, 2H), 3.33 – 3.28 (m, 2H), 3.20 (s, 4H), 3.13 (s, 4H), 1.98 – 1.93
(s, 8H). 13C NMR (100 MHz, CDCl3) δ 180.78, 176.39, 163.68, 162.69, 157.96, 152.96, 130.29,
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123.73, 123.57, 114.72, 106.65, 98.87, 92.75, 65.11, 64.31, 53.94, 53.88, 53.62, 53.49,
23.18, 22.13. MS (ESI+): m/z: 465.2 [M+H]+.
2.3.11 4.4.2
5-hydroxy-7-(3-(pyrrolidin-1-yl)propoxy)-3-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)-4H-chrome
n-4-one (5b)
White solid (68% yield), 1H NMR (400 MHz, CDCl3) δ 12.84 (s, 1H), 7.85 (s, 1H), 7.44 (d, J =
8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.40 (d, J = 2.0 Hz, 1H), 6.36 (d, J = 2.0 Hz, 1H), 4.12 –
4.05 (m, 4H), 2.85 – 2.76 (m, 2H), 2.72 (t, J = 6.8 Hz, 6H), 2.64 (s, 4H), 2.14 – 2.07 (m, 4H),
1.89 – 1.83 (m, 8H). 13C NMR (100 MHz, CDCl3) δ 180.81, 164.93, 162.62, 159.12, 157.94,
152.70, 130.09, 123.58, 122.97, 114.65, 106.20, 98.70, 92.80, 66.90, 66.22, 54.23, 53.14,
52.88, 28.31, 28.26, 23.48. MS (ESI+): m/z: 493.3 [M+H]+.
2.3.12
5-hydroxy-7-(4-(pyrrolidin-1-yl)butoxy)-3-(4-(4-(pyrrolidin-1-yl)butoxy)phenyl)-4H-chromen-4
-one (5c)
White solid (60% yield), 1H NMR (400 MHz, CDCl3) δ 12.86 (s, 1H), 7.86 (s, 1H), 7.45 (d, J =
8.4 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 6.39 (d, J = 2.0 Hz, 1H), 6.36 (d, J = 2.0 Hz, 1H), 4.15 –
3.94 (m, 4H), 3.16 – 2.38 (m, 12H), 1.87 – 1.79 (m, 16H). 13C NMR (100 MHz, CDCl3) δ 180.84,
164.93, 162.64, 159.15, 157.94, 152.72, 130.11, 123.61, 122.86, 114.63, 106.18, 98.55,
92.84, 68.28, 67.61, 55.97, 54.13, 27.15, 26.95, 25.06, 23.42. MS (ESI+): m/z: 521.3 [M+H]+.
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2.3.13
5-hydroxy-7-((6-(pyrrolidin-1-yl)hexyl)oxy)-3-(4-((6-(pyrrolidin-1-yl)hexyl)oxy)phenyl)-4H-chro
men-4-one (5d)
White solid (57% yield), 1H NMR (400 MHz, CDCl3) δ 12.85 (s, 1H), 7.86 (s, 1H), 7.44 (d, J =
8.8 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 6.38 (d, J = 2.2 Hz, 1H), 6.35 (d, J = 2.2 Hz, 1H), 4.01 (t, J =
5.2 Hz, 2H), 3.98 (t, J = 5.2 Hz, 2H), 2.71 (d, J = 6.4 Hz, 8H), 2.65 – 2.57 (m, 4H), 1.92 – 1.85
(m, 8H), 1.85 – 1.78 (m, 4H), 1.67 –1.64 (m, 4H), 1.53 – 1.46 (m, 4H), 1.46 – 1.39 (m, 4H). 13C
NMR (100 MHz, CDCl3) δ 180.84, 165.06, 162.65, 159.30, 157.96, 152.67, 130.07, 123.62,
122.80, 114.66, 106.15, 98.61, 92.82, 68.51, 67.89, 56.27, 54.07, 54.04, 29.09, 28.83, 28.12,
28.05, 27.22, 27.19, 25.90, 25.83, 23.41. MS (ESI+): m/z: 577.4 [M+H]+.
2.3.14
7-(2-(1H-imidazol-1-yl)ethoxy)-3-(4-(2-(1H-imidazol-1-yl)ethoxy)phenyl)-5-hydroxy-4H-chrom
en-4-one (5e)
White solid (59% yield), mp 8587 C; 1H NMR (300 MHz, CDCl3) δ 12.83 (s, 1H), 7.85 (s,
1H), 7.59 (s, 2H), 7.43 (d, J = 9 Hz, 2H), 7.05 (d, J = 9 Hz, 4H), 6.93 (d, J = 6 Hz, 2H), 6.34 (d, J =
6 Hz, 2H), 4.35 (t, J = 6 Hz, 4H), 4.26 (t, J = 6 Hz, 4H). 13C NMR (75 MHz, CDCl3) δ 180.76,
163.63, 162.87, 158.19, 157.80, 152.93, 137.57, 137.52, 130.26, 129.82, 129.62, 123.70,
123.53, 119.37, 119.33, 114.72, 106.66, 98.34, 93.05, 67.64, 67.34, 46.43, 46.11. MS (ESI+):
m/z: 459.2 [M+H]+. Elemental Anal. Calcd for C25H22N4O5: C, 68.49; H, 4.84; N, 12.22. Found:
C, 68.72; H, 4.91; N, 12.32.
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2.3.15
7-(3-(1H-imidazol-1-yl)propoxy)-3-(4-(3-(1H-imidazol-1-yl)propoxy)phenyl)-5-hydroxy-4H-chr
omen-4-one (5f)
White solid (52% yield), mp 9597 C; 1H NMR (300 MHz, CDCl3) δ 12.84 (s, 1H), 7.87 (s,
1H), 7.47 (s, 3H), 7.43 (s, 1H), 7.06 (d, J = 3.6 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 1.5
Hz, 2H), 6.35 (s, 2H), 4.19 (t, J = 6.3 Hz, 4H), 3.94 (q, J = 6.0 Hz, 4H), 2.30 – 2.19 (m, 4H). 13C
NMR (75 MHz, CDCl3) δ 180.80, 164.26, 162.80, 158.67, 157.95, 152.80, 137.32, 137.27,
130.23, 129.85, 129.68, 123.64, 123.37, 119.01, 118.90, 114.62, 106.55, 98.60, 92.73, 77.26,
64.39, 63.80, 43.41, 43.26, 30.74, 30.47. MS (ESI+): m/z: 487.2 [M+H]+. Elemental Anal. Calcd
for C27H26N4O5: C, 66.66; H, 5.39; N, 11.52. Found: C, 66.43; H, 5.57; N, 5.09.
2.3.16
7-(4-(1H-imidazol-1-yl)butoxy)-3-(4-(4-(1H-imidazol-1-yl)butoxy)phenyl)-5-hydroxy-4H-chrom
en-4-one (5g)
White solid (48% yield), mp 104106 C; 1H NMR (300 MHz, CDCl3) δ 12.85 (s, 1H), 7.86 (s,
1H), 7.50 (s, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.07 (s, 2H), 6.94 (d, J = 4.5 Hz, 4H), 6.35 (d, J = 6.3
Hz, 2H), 4.06 – 3.98 (m, 8H), 2.04 –1.95 (m, 4H), 1.86 – 1.74 (m, 4H). 13C NMR (75 MHz,
CDCl3) δ 180.82, 164.61, 162.76, 158.96, 157.93, 152.72, 137.11, 130.17, 129.72, 129.61,
123.63, 123.08, 118.77, 118.72, 114.60, 106.33, 98.48, 92.81, 67.73, 67.18, 46.75, 46.68,
28.08, 27.93, 26.26, 26.08. MS (ESI+): m/z: 515.3 [M+H]+. Elemental Anal. Calcd for
C29H30N4O5: C, 67.69; H, 5.88; N, 10.89. Found: C, 67.53; H, 5.61; N, 11.00.
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2.3.17
7-((6-(1H-imidazol-1-yl)hexyl)oxy)-3-(4-((6-(1H-imidazol-1-yl)hexyl)oxy)phenyl)-5-hydroxy-4H
-chromen-4-one (5h)
White solid (50% yield), mp 8789 C; 1H NMR (300 MHz, CDCl3) δ 12.85 (s, 1H), 7.85 (s,
1H), 7.45 (d, J = 3.6 Hz, 3H), 7.42 (s, 1H), 7.05 (s, 2H), 6.95 (s, 1H), 6.91 (d, J = 6.9 Hz, 3H),
6.35 (d, J = 7.2 Hz, 2H), 4.01 – 3.92 (m, 8H), 1.84 – 1.76 (m, 8H), 1.51 – 1.45 (m, 4H), 1.41 –
1.33 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 180.82, 164.94, 162.74, 159.22, 157.95, 152.62,
137.05, 130.10, 129.48, 129.43, 123.67, 122.89, 118.74, 114.66, 106.22, 98.54, 92.84, 68.28,
67.70, 46.94, 31.03, 31.00, 29.01, 28.77, 26.31, 26.30, 25.62, 25.56. MS (ESI+): m/z: 571.3
[M+H]+. Elemental Anal. Calcd for C33H38N4O5: C, 69.45; H, 6.71; N, 9.82. Found: C, 69.59; H,
6.39; N, 9.73.
2.3.18
7-((8-(1H-imidazol-1-yl)octyl)oxy)-3-(4-((8-(1H-imidazol-1-yl)octyl)oxy)phenyl)-5-hydroxy-4H-
chromen-4-one (5i)
White solid (43% yield), mp 7577 C; 1H NMR (300 MHz, CDCl3) δ 12.85 (s, 1H), 7.85 (s,
1H), 7.43 (d, J = 9.6 Hz, 4H), 7.05 (s, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.90 (s, 2H), 6.36 (d, J = 7.5
Hz, 2H), 4.02 – 3.90 (m, 8H), 1.77 (t, J = 7.2 Hz, 8H), 1.46 – 1.24 (m, 16H). 13C NMR (75 MHz,
CDCl3) δ 180.84, 165.04, 162.66, 159.29, 157.94, 152.64, 137.09, 130.07, 129.40, 123.64,
122.76, 118.78, 114.64, 106.14, 98.56, 92.83, 68.54, 67.93, 47.03, 31.08, 29.17, 29.14,
29.02, 28.88, 26.50, 25.93, 25.85. MS (ESI+): m/z: 626.4 [M+H]+. Elemental Anal. Calcd for
C37H46N4O5: C, 70.90; H, 7.40; N, 8.94. Found: C, 70.82; H, 7.68; N, 9.08.
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2.4 Synthesis of compounds 9a-b
The intended compounds 9a-b were obtained following the literature reported
procedure,25 as shown in the Scheme 2.
2.4.1 3-(4-(4-(diethylamino)butoxy)phenyl)-5,7-dihydroxy-4H-chromen-4-one (9a)
1H NMR (300 MHz, DMSO-d6) δ 12.91 (s, 1H), 11.06 (s, 1H), 10.39 (s, 1H), 8.36 (s, 1H), 7.50
(d, J = 8.1 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.45 (d, J = 2.1 Hz, 1H), 6.28 (d, J = 2.1 Hz, 1H),
4.06 (t, J = 5.7 Hz, 2H), 3.12 –3.07 (m, 6H), 1.89 – 1.77 (m, 4H), 1.23 (t, J = 7.2 Hz, 6H).13C
NMR (75 MHz, DMSO-d6) δ 180.55, 164.95, 162.38, 158.82, 158.00, 154.76, 130.64, 123.47,
122.34, 114.71, 104.85, 99.54, 94.22, 67.36, 50.61, 46.47, 26.29, 20.43, 8.88. MS (ESI+): m/z:
398.2 [M+H]+.
2.4.2 5,7-dihydroxy-3-(4-(4-(pyrrolidin-1-yl)butoxy)phenyl)-4H-chromen-4-one (9b)
1H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H), 11.05 (s, 1H), 10.73 (s, 1H), 8.37 (s, 1H), 7.49
(d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.43 (d, J = 2.0 Hz, 1H), 6.27 (d, J = 2.0 Hz, 1H),
4.03 (t, J = 5.6 Hz, 2H), 3.56 – 3.47 (m, 2H), 3.18 – 3.13 (m, 2H), 2.98 – 2.93 (m, 2H), 1.98 (s,
2H), 1.86 – 1.78 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 180.56, 164.94, 162.40, 158.83,
158.02, 154.77, 130.64, 123.47, 122.36, 114.73, 104.87, 99.54, 94.22, 67.33, 53.99, 53.26,
26.32, 23.15, 22.57. MS (ESI+): m/z: 396.2 [M+H]+.
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2.5 Biological activity
2.5.1 Inhibition assays on AChE and BuChE in vitro
AChE (E.C. 3.1.1.7, from electric eel), BuChE (E.C. 3.1.1.8, from equine serum),
5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine chloride (ATC),
butylthiocholine chloride (BTC) were purchased from Sigma-Aldrich. The compounds were
dissolved in a minimum volume of DMSO (1%) and diluted in phosphate-buffered solution
(0.1 M, pH 8.0) to provide a final concentration range. All the assays were under the
phosphate-buffered solution, using a Shimadzu UV-2450 Spectrophotometer. Enzyme
solutions were prepared to give 2.0 units/mL in 2 mL aliquots. The assay medium contained
10 L of enzyme, 50 L of DTNB (0.01 M) and 50 L of substrate (ATC, 0.01 M). The
substrate was added to the assay medium containing enzyme, buffer, and DTNB with
inhibitor after 15 min of incubation time at 37 C. The activity was determined by measuring
the increase in absorbance at 412 nm at 1 min intervals at 37 C. Calculations were
performed according to the method of the equation in Ellman et al.26 In vitro BuChE assay
use the similar method described above. Each concentration was assayed in triplicate.
2.5.2 Kinetic characterization of AChE inhibition
Three different concentrations of substrate were mixed in the 1.0 mL phosphate buffer
(0.1 M, pH 8.0), containing 50 L of DTNB, 10 L AChE, and 50 L substrate. Test compound
was added into the assay solution and pre-incubated with the enzyme at 37C for 15 min,
followed by the addition of substrate. Kinetic characterization of the hydrolysis of ATC
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catalyzed by AChE was done spectrometrically at 412 nm. A parallel control with no inhibitor
in the mixture, allowed adjusting activities to be measured at various times.
2.5.3 Molecular modeling
The crystal structure of the torpedo AChE (code ID: 1ACJ) were obtained in the Protein
Data Bank after eliminating the inhibitor and water molecules. The 3D structure of 5a was
built and performed geometry optimization by molecular mechanics. Further preparation of
substrates included addition of Gasteiger charges, removal of hydrogen atoms and addition
of their atomic charges to skeleton atoms, and finally, assignment of proper atomic types.
Autotors was then used to define the rotatable bonds in the ligand.
Docking studies were carried out using the AutoDock 4.2 program (The Scripps Research
Institute, San Diego, CA, USA). The resulting enzyme structure was used as an input for the
AutoGrid program. AutoGrid performed a pre-calculated atomic affinity grid maps for each
atom type in the ligand plus an electrostatics map and a separate desolvation map present
in the substrate molecule. All maps were calculated with 0.375 Å spacing between grid
points. The center of the grid box was placed at the bottom of the active site gorge (AChE
[2.781 64.383 67.971]. The dimensions of the active site box were set at 50  50  50 Å.
Flexible ligand docking was performed for the compounds. Each docked system was
performed by 100 runs of the AutoDock search by the Lamarckian genetic algorithm (LGA).
Other than the referred parameters above, the other parameters were accepted as default.
Flexible ligand docking was performed for the compounds. Docking calculations were
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carried out using the Lamarckian genetic algorithm (LGA) and all parameters were the same
for each docking, the best 10 poses of molecules were retained. After docking, the geometry
of resulting complex was studied using the PyMOL program.
2.5.4 Measurement of the anti-oxidation activity
The antioxidation activity was determined by using the oxygen radical absorbance
capacity-fluorescein (ORAC-FL) assay. The ORAC-assay measures antioxidant scavenging
activity against peroxyl radical induced by 2,2’-azobis(2-amidinopropane) dihydrochloride
(AAPH) at 37 °C. The reaction was carried out in 75 mM phosphate buffer (pH 7.4), and the
final reaction mixture was 200 µL. Antioxidant (20 µL) and fluorescein (120 µL, 300 nM final
concentration) were placed in the wells of a black 96 well plate, and the mixture was incubated
for 15 min at 37 °C. Then AAPH solution (60 µL, 12 mM final concentration) was added
rapidly. The plate was immediately placed into a Spectrafluor Plus plate reader (BMG,
German), and the fluorescence was measured every 60 seconds for 180 minutes with exitation
at 485 nm and emission at 535 nm. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox) was used as a standard (1-5 µM, final concentration). A blank (FL+AAPH) using
phosphate buffer instead of antioxidant and Trolox calibration were carried out in each assay.
The samples were measured at different concentrations (1-5 µM), and at least three
independent runs were performed for each sample. The ORAC values calculated as previously
described.21
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2.5.5 MTT assay of HepG2 and PC12 cell viability
MTT assay of PC12 and HepG2 was conducted according to the previous report.24
2.5.6 Metal-chelating study
The chelating studies were made in methanol using a Shimadzu UV-2450
Spectrophotometer. The absorption spectrum of test compounds (20 M), alone or in the
presence of FeCl3, CuSO4 or ZnCl2 (20 M) was recorded with wavelength ranging from 200
to 500 nm after incubating for 30 min at room temperature. The final volume of reaction
mixture was 1.0 mL, and the final concentrations of tested compounds and metals were 20
µM. The molar ratio method was performed to determine the stoichiometry of the complex
compound-metal by titrating the methanol solution of the compounds with ascending of
CuCl2. The final concentration of the compounds was 20 µM, and the final concentration of
Cu2+ ranged from 2.0 to 200 µM. The spectra was recorded and treated by numerical
subtraction of CuCl2 and the compounds at corresponding concentrations, plotted versus
the mole fraction of the test compounds.
The complex formation constant (1) was calculated as: 1 = [ML]/[M][L] = (A/A0)/{[1-(
A/A0)]2·c}, where A is the absorption in the curve at the molar ratio of 0.98, A0 is the
absorption at intersection of the two lines (Fig. 5), c is the concentration of the test
compound (20 M).
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3. RESULTS AND DISCUSSION
3.1 Chemistry
The 4’-OH group in genistein molecule is more nucleophilic than 7-OH and 5-OH group,
but less acidic than 7-OH group, and thus 4’-OH or/and 7-OH can be easily substituted by
desired substituents.27, 28 The intended compounds (3a–i, 5a-i) were synthesized following
the synthetic route as depicted in the Scheme 1.
The intermediates 2a-e and bis-substituted intermediates 4a-e were synthesized in the
presence of excess anhydrous K2CO3 by alkylating genistein 1 with equivalent amount
dibromoalkanes and 4 equivalent of dibromoalkanes in acetone, respectively for 10 h. The
reaction did not produce the tri-substituted compounds as the 5-OH group was stabilized by
hydrogen bonds. Finally, 2a-e or 4a-e were conjugated with pyrrolidine or imidazole to
obtain the intended compounds 3a-i and 5a-i with satisfactory yields. We also synthesized
GS-14 (Fig. 1, B) and two of 4’-O-modified genistein derivatives 9a-b to calculate the
complete structure-activity relationships (SAR) of these series of compounds. While
synthesizing the compounds 9a-b, the 7-OH group of genistein was protected with
chloromethyl methyl ether (MOM-Cl) (Scheme 2),29 and the protected intermediate 6 was
alkylated with the equivalent amount of 1,4-dibromobutane in the presence of excess K2CO3
in acetone to obtain the intermediate 7, which in turn was conjugated with diethylamine or
pyrrolidine to yield the corresponding intermediates 8a and 8b, respectively. Finally, the
MOM protecting groups were detached by hydrolyzing with 4 M HCl at room temperature
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to obtain the corresponding compounds 9a and 9b as hydrochloride salts. The structure and
purity of these synthesized new compounds were confirmed by 1H NMR, 13H NMR, ESI-MS
and elemental analysis.
3.2 In vitro AChE and BuChE inhibition studies
The synthesized compounds (3a-i, 5a-i, 9a-b and GS-14) were screened for AChE and
BuChE inhibitory activities in vitro according to the Ellman method. Donepezil and
rivastigmine were used as positive controls. The IC50 values and selectivity ratio for AChE
and BuChE inhibition are summarized in Table 1. The synthesized compounds showed
different inhibitory activities towards AChE with their IC50 values ranging from the
nanomolar to micromolar range (IC50 0.034  7.25 µM), and exhibited better inhibition
selectivity towards AChE than BuChE. Compound 5a was obtained as the most potent AChE
inhibitor (IC50 = 0.034 µM) with a potency of about 200-fold higher than rivastigmine (IC50 =
6.53 µM) and 10-fold higher than a reported compound GS-14 (IC50 = 0.29 µM).
The pyrrolidine conjugated compounds (3a-d, 5a-d) exhibited higher potencies (IC50 =
0.034  1.11 µM) than the positive control, rivastigmine (IC50 = 6.53 µM). While most of the
imidazole conjugated derivatives (3e-i and 5g-i) did not exhibit effective AChE inhibitory
activity with IC50 values more than 10 µM. The results indicated that O-alkyl-pyrrolidino
genistein (but not the imidazole substituted genistein) exhibited significant AChE inhibitory
activity, which signifies that the pyrrolidine moiety is crucial for AChE inhibition. On the
other hand, imidazole conjugated derivatives 5e and 5f (n=2, 3) displayed stronger
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inhibitory activity than 5g-i, the results indicated that the potency of the derivatives to
inhibit AChE was related to the length of alkylene. In general, two methylene groups (3a, 5a,
5e) between genistein and pyrrolidine/imidazole constituted the optimal chain length for
this series.
The 4’-O-substituted genistein derivatives 9a-b showed moderate inhibitory activity (IC50=
4.98, 7.25 µM) compared with GS-14 (7-O-substituted, IC50= 0.29 µM), 3c (7-O-substituted,
IC50= 0.44 µM) and 5c (7,4’-O-substituted, IC50= 0.08 µM). The derivatives exhibited AChE
inhibition potencies in the following order: 7,4’-O-substituted > 7-O-substituted >
4’-O-substituted genistein derivatives.
BuChE is considered as a potential target for AD therapy owing to its crucial role in
regulating acetylcholine levels.30 The pyrrolidine substituted derivatives (except 3b, 3c)
exhibited higher BuChE inhibitory activities than rivastigmine (IC50 = 1.41 µM). The 7,4’-O-
pyrrolidino compounds 5a and 5c exhibited the strongest BuChE inhibition with IC50 values
of 0.33 and 0.35 M, respectively. The concurrent inhibition of both AChE and BuChE can
provide additional benefits in AD therapy (eg. rivastigmine). 31 The present study showed
that the compounds 3a-d, 5a-d can significantly inhibit both AChE and BuChE enzymes.
3.3 Kinetic characterization of AChE inhibition
The most potent AChE inhibitor (5a) was selected for kinetic analysis to characterize the
inhibition mechanism (Fig. 3, left). Lineweaver-Burk plots showed both increasing slopes and
increasing intercepts with higher inhibitor concentration, indicating a mixed-type inhibition.
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These results indicate the ability of the compound 5a to interact with both catalytic active
site (CAS) and peripheral anionic site (PAS) of AChE.
3.4 Molecular modeling study
Molecular modeling was carried out by Autodock 4.2 package and PyMOL program32, 33 to
obtain the functional and structural insight into the bond/interaction between the
compound 5a and TcAChE (PDB code: 1ACJ). After docking, the best pose of molecule was
chosen and the geometry of complex was studied. (The affinity was shown in Supporting
Information, Table 2) The docking result (Fig. 3, right) demonstrated multiple binding modes
between 5a and TcAChE. In the 5a-TcAChE complex, 5a occupied the entire enzymatic CAS,
mid-gorge and PAS. The pyrrolidine moiety was bound to CAS with cation- interaction
between Trp84 and Phe330; the distance was 3.5 and 3.7 Å, respectively. At the midgorge
recognition site, the chromone moiety displayed H-bond with Tyr70, and the distance was
3.2 Å. Another pyrrolidine moiety was bound to PAS with hydrophobic interaction. The
docking result showed that the compound 5a could bind to both CAS and PAS of AChE, and
this finding was consistent with the results of the kinetic study.
3.5 Studies of antioxidation activity
The antioxidant activities of the synthesized compounds were evaluated by oxygen
radical absorbance capacity (ORAC) assay. Trolox (a vitamin E analogue) was used as the
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standard and the antioxidative capacity of the compounds were represented as a Trolox
equivalent. Genistein was also tested, with an ORAC value of 4.20 trolox equivalents. All of
the target compounds demonstrated weak to moderate antioxidant activity with ORAC
values ranging from 0.56 to 2.31 Trolox equivalents (Table 1). Compounds 3e showed the
most potent antioxidant activity (ORAC value = 2.31), whereas compounds 3a, 3b, 3f, 3h and
GS-14 exhibited slightly lower activities (ORAC value = 1.85, 1.94, 2.13, 2.04 and 2.15,
respectively). The 7,4’-O-substituted derivatives 5a-5i showed weak antioxidant activities
with ORAC value ranging from 0.56 to 1.23. Donepezil and rivastigmine exhibited minimum
free radical scavenging activities with ORAC values < 0.2. These results indicated that the
free 4’-OH of genistein was crucial for the free radical scavenging activity.
3.6 Metal-chelating study
High levels of redox-active biometals in the brain catalyze the formation of ROS, which
further aggravates oxidative stress and thus, contributing to the AD pathogenesis.34 Metal
chelators can form complexes with these redox-active biometals and prevent the formation
of ROS. The chelating abilities of the compound 5a for the biologically relevant metal ions:
Cu2+, Fe3+ and Zn2+ were studied by UV-vis spectrometry. A series of UV-vis spectra of
compound 5a are demonstrated in the Figure 4. The absorbance spectra of 5a showed a
significant decrease after the addition of Cu2+. An apparent red shift in the maximum
absorption from 260 nm to 268 nm indicated an interaction between compound 5a and Cu2+
due to complex formation.35 Addition of Fe3+ or Zu2+ resulted minor changes in the UV
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spectrum of 5a indicating poor chelating capacity of the compound 5a for Fe3+ or Zu2+ ion.
Similar results were observed for the compounds 5b-d (Fig. 4, B-D).
The stoichiometry of the 5a-Cu2+ complex was determined using the molar ratio method,
by preparing solutions of compound 5a with increasing amounts of CuCl2. The UV spectra
were used to obtain the absorbance of the 5a complex and differing concentrations of CuCl2
at 373 nm. Two lines with different slopes were plotted to distal parts of the curve
(approximations to tangents), with the interception at a molar ratio of 0.98 (Fig. 5), it
revealed that a 1:1 stoichiometry for complex 5a-Cu2+. The complex formation constant (1)
obtained after calculation is 1.10106 L·mol-1, log 1=6.04.
3.7 MTT assay of cell viability
Tacrine, a well-known AChE inhibitor, was withdrawn from the market for its severe
reversible hepatotoxicity. Therefore, tacrine was used as the positive control. To determine
whether our compounds had hepatotoxicity in comparison to tacrine, the
representative compounds (3a-d, 5a-d) were selected for the MTT assay in PC12 and HepG2
cell lines at the concentrations of 1, 10 and 100 M, respectively. The results showed that
the inhibition rates of all the tested compounds were less than 5% at 1 μM, and most of the
compounds showed no obvious effect on cell viability at concentrations of 10 and 100 μM,
as shown in the Table 2. The compounds exhibited lower toxicity towards the cell lines when
compared with tacrine.
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4. CONCLUSION
A series of genistein-O-alkylamine derivatives were synthesized and evaluated for
multifunctional anti-AD agents. Among these compounds 3h-i and 5e-i were novel. The
results showed that pyrrolidine modified derivatives (3a-3d, 5a-5d) exhibited strong AChE
inhibitory activity, and 7,4’-O-bis-substituted derivative 5a exhibited the most potent
inhibition with IC50 value of 0.034 M. For BuChE inhibition, 5a and 5c were the most potent
compounds with IC50 values of 0.33 and 0.35 M, respectively. A Lineweaver-Burk plot and
molecular modeling study suggested that the compound 5a interacted with both the CAS
and PAS of AChE, and induced a strong inhibition effect. Compound 5a also displayed
antioxidant activity with ORAC value of 1.21. The compounds 5a-5d showed potent Cu2+
chelating ability. In addition, these compounds showed low toxicity towards PC12 and
HepG2 cell lines by MTT assay in vitro. The results of the study showed that the compound
5a is a potent, multifunctional, nontoxic drug and a suitable candidate for the treatment of
AD.
ACKNOWLEDGEMENTS
We thank the Foundation of Henan Educational Committee (No. 182102310504,
14A350008), the Natural Science Foundation of China (No. U1704176, 81460528,
21762020), the Foundation for University Key Teacher by Educational Commission of Henan
Province (2016GGJS-030), the Postdoctoral Science Foundation of China (No.
2012M521391), and the Postdoctoral Science Foundation of Henan Province (No. 2011015)
for financial support of this study.
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CONFLICT OF INTEREST
We declare that we have no conflict of interest.
List of the figure legends
Figure 1. Chemical structures of genistein and genistein derivatives.
Figure 2. Design strategy for the genistein derivatives.
Scheme 1. Synthesis of genistein derivatives. Reagents and conditions: (a) 1 equiv.
Br(CH2)nBr, K2CO3, acetone, reflux; (b) 4 equiv. Br(CH2)nBr, K2CO3, acetone, reflux; (c)
HNR1R2, K2CO3, CH3CN, 60 C.
Scheme 2. Synthesis of 4’-O-modified genistein derivatives 9a-b. Reagents and conditions:
(a) KOH, DMF, MOM-Cl, rt; (b) 1 equiv. Br(CH2)4Br, K2CO3, acetone, reflux; (c) HNR1R2, K2CO3,
CH3CN, 60 C; (d) 10% HCl/EtOH, reflux.
Figure 3. Lineweaver-Burk plots (left) and docking model (right) for the compound 5a with
TcAChE.
Figure 4. UV spectrum of compounds 5a-d (20 M) alone or at the presence of 20 M Fe3+,
Cu2+ and Zn2+.
Figure 5. Determination of the stoichiometry of complex-Cu2+ using the molar ratio method
of titrating the methanol solution of 5a with ascending amounts of CuCl2. The final
concentration of tested compound was 20 µM, and the final concentration of Cu2+ ranged
from 2.0 to 200 µM.
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Table 1. Inhibition of AChE, BuChE and oxygen radical absorbance capacity (ORAC, Trolox
equivalents) of the compounds.
IC50a for
IC50b for
Selectivity ORAC d
for AChEc
Compd.
R
n
AChE (M)
BuChE (M)
2.4
5.8
8.9
1.4
-
1.85  0.14
1.94  0.10
1.42  0.06
1.15  0.08
2.31  0.06
2.13  0.04
1.58  0.11
2.04  0.09
1.39 0.17
1.21  0.07
3a
3b
3c
3d
3e
3f
2
3
4
6
2
3
4
6
8
2
3
4
6
0.56±0.03
1.11±0.17
0.44±0.01
0.88±0.12
>10
1.37±0.08
6.48±0.42
3.90±0.26
1.21±0.11
>10
-
>10
>10
-
3g
3h
3i
>10
>10
-
>10
>10
-
>10
>10
9.7
0.034±
5a
5b
5c
5d
0.33±0.05
0.61±0.10
0.35±0.08
0.58±0.02
0.003
2.1
4.4
1.9
1.02  0.03
0.65  0.12
0.78  0.05
0.29±0.02
0.08±0.01
0.31±0.04
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