Design, Synthesis, and Biological Evaluation of Scutellarein Carbamate Derivatives as Potential Multifunctional Agents for the Treatment of Alzheimer's Disease

Article abstract of DOI:10.1111/cbdd.12580

A series of scutellarein carbamate derivatives were designed and synthesized based on the multitarget-directed drug design strategy for treatment of Alzheimer's disease. Their acetylcholinesterase and butyrylcholinesterase inhibitory activities, antioxida

Full text of DOI:10.1111/cbdd.12580

Received Date : 08-Dec-2014
Revised Date : 15-Mar-2015
Accepted Date : 28-Apr-2015
Article type
: Research Article
Design, synthesis and biological evaluation of scutellarein carbamate derivatives
as potential multifunctional agents for the treatment of Alzheimer's disease
Zhi-pei Sanga, Xiao-ming Qianga, Yan Lia, Bei Wua, Hui Zhanga, Ming-gao Zhaob, Yong Denga,*
a Department of Medicinal Chemistry, Key Laboratory of Drug Targeting and Drug Delivery System
of the Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, 610041,
China
b Department of Pharmacology, Fourth Military Medical University, Xi'an, 710032, China
* Corresponding Author. Phone, +86-28-85503790; E-mail: dengyong@scu.edu.cn (Yong Deng)
Abstract
A series of scutellarein carbamate derivatives were designed and synthesized based on the
multi-target-directed drug design strategy for treatment of Alzheimer’s disease. Their
acetylcholinesterase and butyrylcholinesterase inhibitory activities, antioxidant activities, metals
chelation and neuroprotective effects against hydrogen peroxide induced PC12 cell injury were
evaluated in vitro. The preliminary results indicated that compound 7b exhibited good inhibitory
potency toward AChE and BuChE with IC50 values of 1.2 ± 0.03 μM and 22.1 0.15 μM,
respectively, possessed the strong antioxidant potency (10.3 trolox equivalents), as well as acted
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as a selective metal chelator and neuroprotective agent. Furthermore, 7b could improve memory
impairment induced by scopolamine, ethanol and sodium nitrite using the step-down passive
avoidance task in vivo, and could remarkably decrease the activity of acetylcholinesterase in mice
brain. The present study indicated that 7b could be considered as a potential multi-target agent
against AD.
Keywords: Alzheimer’s disease; Scutellarein carbamates; Synthesis; AChE inhibitors; Antioxidant;
Metal chelator; Neuroprotective effects.
1. Introduction
Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder with
noticeable impairment of cognitive function (1). By 2050, one new case of AD is expected to
develop every 33 seconds, or nearly a million new cases per year, and the total estimated
prevalence is expected to be 13.8 million (2). Although the etiology of AD is not completely known
until now, several factors, including low levels of acetylcholine (ACh), oxidative stress,
dyshomeostasis of biometals and amyloid-β (Aβ) deposits, have been considered to play key roles
in the pathogenesis of AD (3). Oxidative stress plays an important role in neuronal degeneration.
Oxidative damage marked by lipid peroxidation, nitration, reactive carbonyls, and nucleic acid
oxidation is increased in vulnerable neurons in AD (4). Therefore, the successful protection of
neuronal cells from oxidative damage could potentially prevent AD. Recent studies have indicated
that metal dyshomeostasis is frequently observed in AD due to anomalous binding of metals such
as iron (Fe), copper (Cu), and zinc (Zn), or impaired regulation of redox-active metals which can
induce the formation of cytotoxic reactive oxygen species and neuronal damage (5). Thus, metal
chelators have been administered therapeutically in transgenic mice models for AD and in clinical
human AD studies, with positive outcomes (6, 7).
Nevertheless, the primary therapeutic treatment for the sympt
omatic cognitive loss associated with AD is a cholinergic replacement strategy, represented by
FDA approved acetylcholinesterase inhibitors (AChEIs) including tacrine (now withdrawn due to
hepatotoxicity), donepezil, rivastigmine and galantamine (8). Of which, donepezil and galantamine
are reversible, selective inhibitors of acetylcholinesterase (AChE), while rivastigmine inhibits both
AChE and butyrylcholinesterase (BuChE) (9). BuChE is an enzyme closely related to AChE and
serves as a co-regulator of cholinergic neurotransmission. However, BuChE is mainly localized in
the peripheral tissues including plasma and a few regions of the brain (10). Therefore, the
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potential advantages of selective inhibition of AChE over BuChE may include lesser degree of
associated side effects caused by peripheral inhibition of cholinesterase.
Scutellarein (2) possesses an impressive array of biological properties including free radical
scavenging effect, metal chelation, anti-inflammatory effect, neuroprotective action (11, 12).
However, the poor solubility, low oral absorption and poor ability of penetrating the blood–brain
barrier restrict its clinical using as an anti-AD drug (13). Rivastigmine (1) is approved by the FDA as
an AChE inhibitor for the treatment of AD, and the carbamate moiety has been proved to be the
cholinesterase-inhibitory pharmacophore. Moreover, our group has reported the pterostilbene
and resveratrol carbamate derivatives, and genistein carbamate derivatives as potential dual
cholinesterase inhibitors recently (14, 15). However, the pterostilbene and resveratrol carbamate
derivatives have no metal chelation and the genistein carbamate derivatives have low antioxidant
activity. Herein, scutellarein was selected to combine with different carbamate fragments to
design a series of novel scutellarein carbamate derivatives, to test whether these novel molecules
might possess more potency in various multifunctional activities and improve the shortcomings.
In this article, a series of scutellarein carbamate derivatives (7a~l) were designed and
synthesized based on the multitarget-directed ligands strategy (MTDLs). This may be regarded as
the buildup of hybrid molecules composed of distinct pharmacophores of different drugs. Thus,
each pharmacophore of the new hybrid drug would preserve the capacity of interacting with their
specific sites on the targets and, therefore, generate multiple specific pharmacological responses
which would enable the treatment of the multi-factorial Alzheimer’s disease (16). The design
strategy of the target compounds are depicted in Figure 1. Furthermore, their inhibitory activity
for acetylcholinesterase and butyrylcholinesterase, antioxidant activity, metals chelation and
neuroprotective effects against hydrogen peroxide induced PC12 cell injury were investigated in
vitro. Further, in vivo memory impairment induced by scopolamine, ethanol and sodium nitrite
was carried out using the step-down passive avoidance task, and the activity of
acetylcholinesterase in mice brain was also studied.
Figure 1. The multi-target-directed ligand (MTDL) strategy for the design of target compounds.
2. Materials and Methods
2.1. Reagents and analysis
Melting points were recorded on the YRT-3 melting-point apparatus (China) and were
uncorrected. 1H and 13C NMR spectra were recorded in CDCl3 or DMSO-d6 with a Varian INOVA
spectrometer at 400 and 100 MHz, respectively. Mass spectra were recorded on an Agilent-6210 TOF
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LC/MS spectrometer. All commercially available reagents and anhydrous solvents were purchased at
the highest commercial quality and were used without further purification. Column chromatography
using silica gel (230–400 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. (China).
2.2. Chemistry
The synthetic process for the preparation of the target compounds was outlined in Scheme 1
and Scheme 2. The structures of the synthesized compounds were supported by the spectral 1H
NMR and 13C NMR data, the results of HR-ESI-MS which were in agreement with the proposed
structures.
9-Hydroxy-6-(4-hydroxyphenyl)-2,2-diphenyl-8H-1,3-dioxolo[4,5-g][1]benzopyran-8-one (4)
To a mixture of scutellarein 2 (5.0 g, 17.5 mmol) and diphenyl dichloromethane 3 (3.2 mL, 26.2
mmol) in 1,2-dichlorobenzene (30 mL), the mixture was heated at 170 °C for 1.5 h. Cooled to room
temperature, acetone (10 mL) was added to the solution, filtered and dried, gave the compound 4
as light brown solid (6.74 g, 85.6% yield). 1H NMR (400 MHz, DMSO-d6) δ 13.18 (s, 1H), 10.41 (s,
1H), 7.95 (d, J = 8.8 Hz, 2H), 7.61 (s, 1H), 7.58-7.56 (m, 4H), 7.51-7.47 (m, 6H), 6.94 (d, J = 8.8 Hz,
2H), 6.88 (s, 1H).
General procedure for the synthesis of compounds 6a~h
To a solution of compound 4 (100 mg, 0.22 mmol), anhydrous K2CO3 (0.27 mmol), and 4-DMAP
(0.02 mmol) in anhydrous MeCN (5 mL), N,N-disubstituted carbamoyl chlorides 5a–h (0.24
mmol)(14) was added dropwise at 0 °C. The mixture was then heated at 60–65 °C for 6–10 h under
an argon atmosphere. After complete reaction, the solvent was evaporated under reduced
pressure. Water (20 mL) was added to the residue and the mixture was extracted with
dichloromethane (2 × 20 mL). The combined organic phases were washed with saturated aqueous
sodium chloride (30 mL), dried over anhydrous Na2SO4 and filtered. The solvent was evaporated to
dryness under reduced pressure to give the crude product which was purified on a silica gel
chromatography using petroleum/acetone (10:1) as eluent to give the desired products 6a~h.
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N,N-dimethylcarbamic acid-4-(9-hydroxy-8-oxo-2,2-diphenyl-8H-1,3-dioxolo[4,5-g][1]
benzopyran-6-yl)phenyl ester (6a)
Light yellow solid, yield: 71.2 %, mp: 230.2-232.1 °C. 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.8
Hz, 2H), 7.63-7.61 (m, 4H), 7.41-7.38 (m, 6H), 7.28 (d, J = 8.8 Hz, 2H), 6.65 (s, 1H), 6.62 (s, 1H), 3.13
(s, 3H), 3.04 (s, 3H).
N,N-diethylcarbamic acid-4-(9-hydroxy-8-oxo-2,2-diphenyl-8H-1,3-dioxolo[4,5-g][1]
benzopyran-6-yl)phenyl ester (6b)
Light yellow solid, yield: 67.9 %, mp: 216.3-218.1 °C. 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.8
Hz, 2H), 7.64-7.60 (m, 4H), 7.43-7.37 (m, 6H), 7.29 (d, J = 8.8 Hz, 2H), 6.66 (s, 1H), 6.63 (s, 1H),
3.47-3.45 (m, 2H), 3.42-3.40 (m, 2H), 1.27 (t, J = 6.8 Hz, 3H), 1.22 (t, J = 6.8 Hz, 3H).
N,N-bis(1-methylethyl) carbamic acid-4-(9-hydroxy-8-oxo-2,2-diphenyl-8H-1,3-dioxolo[4,5-g]
[1]benzopyran-6-yl)phenyl ester (6c)
Light yellow solid, yield: 50.4 %, mp: 239.4-241.1 °C. 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.8
Hz, 2H), 7.64-7.61 (m, 4H), 7.41-7.38 (m, 6H), 7.29 (d, J = 9.2 Hz, 2H), 6.66 (s, 1H), 6.62 (s, 1H), 4.10
(s, 1H), 3.99 (s, 1H), 1.35 (s, 6H), 1.32 (s, 6H).
N-ethyl-N-methylcarbamic acid-4-(9-hydroxy-8-oxo-2,2-diphenyl-8H-1,3-dioxolo[4,5-g][1]
benzopyran-6-yl)phenyl ester (6d)
Light yellow solid, yield: 80.8 %, mp: 192.3-194.1 °C. 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.8
Hz, 2H), 7.65-7.60 (m, 4H), 7.42-7.36 (m, 6H), 7.29 (d, J = 8.8 Hz, 2H), 6.65 (s, 1H), 6.62 (s, 1H),
3.50-3.49 (m, 1.5H), 3.44-3.41 (m, 1.5H), 3.09 (s, 1H), 3.01 (s, 1H), 1.26 (t, J = 7.2 Hz, 1.5H), 1.21 (t,
J = 7.2 Hz, 1.5H).
1-Pyrrolidinecarboxylic acid-4-(9-hydroxy-8-oxo-2,2-diphenyl-8H-1,3-dioxolo[4,5-g][1]
benzopyran-6-yl)phenyl ester (6e)
Light yellow solid, yield: 49.3 %, mp: 226.3-228.1 °C. 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.8
Hz, 2H), 7.63-7.61 (m, 4H), 7.41-7.38 (m, 6H), 7.29 (d, J = 8.8 Hz, 2H), 6.65 (s, 1H), 6.62 (s, 1H), 3.56
(d, J = 6.8 Hz, 2H), 3.53 (d, J = 6.8 Hz, 2H), 1.92 (t, J = 6.8 Hz, 2H), 1.87 (t, J = 6.8 Hz, 2H).
4-Morpholinecarboxylic acid-4-(9-hydroxy-8-oxo-2,2-diphenyl-8H-1,3-dioxolo[4,5-g][1]
benzopyran-6-yl)phenyl ester (6f)
Light yellow solid, yield: 52.5 %, mp: > 242 °C. 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.8 Hz,
2H), 7.63-7.61 (m, 4H), 7.41-7.38 (m, 6H), 7.28 (d, J = 8.8 Hz, 2H), 6.66 (s, 1H), 6.63 (s, 1H), 3.77 (t, J
= 4.8 Hz, 4H), 3.70 (d, J = 4.8 Hz, 2H), 3.60 (d, J = 4.0 Hz, 2H).
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1-Piperidinecarboxylic acid-4-(9-hydroxy-8-oxo-2,2-diphenyl-8H-1,3-dioxolo[4,5-g][1]
benzopyran-6-yl)phenyl ester (6g)
Light yellow solid, yield: 86.7 %, mp: 220.3-222.2 °C. 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.8
Hz, 2H), 7.66-7.63 (m, 4H), 7.43-7.41 (m, 6H), 7.29 (d, J = 8.8 Hz, 2H), 6.67 (s, 1H), 6.65 (s, 1H),
3.65-3.63 (m, 2H), 3.57-3.55 (m, 2H), 1.69-1.66 (m, 6H).
4-(Phenylmethyl)-1-piperidinecarboxylic acid-4-(9-hydroxy-8-oxo-2,2-diphenyl-8H-1,3-
dioxolo[4,5-g][1]benzopyran-6-yl)phenyl ester (6h)
Light yellow solid, yield: 71.5 %, mp: 98.6-100.2 °C. 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 9.2
Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.63-7.59 (m, 4H), 7.43-7.38 (m, 6H), 7.33-7.19 (m, 5H), 7.17 (d, J =
8.0 Hz, 1H), 7.14 (d, J = 8.8 Hz, 1H), 6.65 (s, 0.5H), 6.62 (s, 0.5H), 6.58 (s, 0.5H), 6.50 (s, 0.5H), 4.27
(t, J = 15.2 Hz, 2H), 2.95 (t, J = 12.8 Hz, 1H), 2.74 (t, J = 12.8 Hz, 1H), 2.61 (s, 1H), 2.59 (s, 1H).
General procedure for the synthesis of compounds 7a~h
To a solution of compounds 6a~h (0.25 mmol) in 80% AcOH (5 mL). The mixture was then
heated at reflux for 2-3 h under an argon atmosphere. After complete reaction, cooled, filtered,
the crude product was recrystallized with ethanol to afford the desired products 7a~h.
N,N-dimethylcarbamic acid-4-(5,6,7-trihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenyl ester (7a)
Light yellow solid, yield: 85.5 %, mp: 254-256 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.68 (brs,
1H), 10.61 (brs, 1H), 8.85 (brs, 1H), 8.10 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 6.94 (s, 1H), 6.63
(s, 1H), 3.07 (s, 3H), 2.94 (s, 3H). HR-ESI-MS: Calcd. for C18H16NO7 [M+H]+: 358.0927, found:
358.0920.
N,N-diethylcarbamic acid-4-(5,6,7-trihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenyl ester (7b)
Light yellow solid, yield: 93.0%, mp: 205-206 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.68 (brs,
1H), 10.60 (brs, 1H), 8.85 (brs, 1H), 8.10 (d, J = 8.8Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 6.94 (s, 1H), 6.63
(s, 1H), 3.43 (q, J = 6.8 Hz, 2H), 3.13 (q, J = 6.8 Hz, 2H), 1.22 (t, J = 6.8 Hz, 3H), 1.13 (t, J = 6.8 Hz,
3H). 13C NMR (100 MHz, DMSO-d6) δ: 182.4, 162.6, 154.2, 153.9, 153.0, 150.1, 147.2, 129.6, 127.9,
127.8, 122.7, 104.5, 104.4, 94.3, 42.1, 41.9, 14.4, 13.5. HR-ESI-MS: Calcd. for C20H20NO7 [M+H]+:
386.1240, found: 386.1231.
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N,N-bis(1-methylethyl)carbamic acid-4-(5,6,7-trihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenyl
ester (7c)
Light yellow solid, yield: 90.3%, mp: 206-208 °C; 1H NMR (400 MHz, DMSO-d6) δ: 12.67 (brs,
1H), 10.56 (brs, 1H), 8.81 (brs, 1H), 8.09 (d, J = 8.8 Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H), 6.92 (s, 1H), 6.63
(s, 1H), 4.01 (q, J = 6.8 Hz, 2H), 1.27 (d, J = 6.8 Hz, 12H). HR-ESI-MS: Calcd. for C22H24NO7 [M+H]+:
414.1553, found: 414.1537.
N-ethyl-N-methylcarbamic acid-4-(5,6,7-trihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenyl ester
(7d)
Light yellow solid, yield: 92.6%, mp: 200-202 °C; 1H NMR (400 MHz, DMSO-d6) δ: 12.67 (brs,
1H), 10.56 (brs, 1H), 8.81 (brs, 1H), 8.09 (d, J = 8.8 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 6.92 (s, 1H), 6.63
(s, 1H), 3.45 (q, J = 7.2Hz, 1H), 3.38 (q, J = 7.2Hz, 1H), 3.05 (s, 1.5H), 2.92 (s, 1.5H), 1.20 (t, J = 7.2
Hz, 1.5H), 1.12 (t, J = 7.2 Hz, 1.5H). HR-ESI-MS: Calcd. for C19H18NO7 [M+H]+: 372.1083, found:
372.1078.
1-Pyrrolidinecarboxylic acid-4-(5,6,7-trihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenyl ester (7e)
Light yellow solid, yield: 85.0%, mp: 212-214 °C; 1H NMR (400 MHz, DMSO-d6) δ: 12.68 (brs,
1H), 10.59 (brs, 1H), 8.63 (brs, 1H), 8.09 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 6.93 (s, 1H), 6.63
(s, 1H), 3.53 (t, J = 6.8 Hz, 2H), 3.36 (t, J = 6.8 Hz, 2H), 1.92 (t, J = 6.8 Hz, 2H), 1.87 (t, J = 6.8 Hz, 2H).
HR-ESI-MS: Calcd. for C20H18NO7 [M+H]+: 384.1083, found: 384.1080.
4-Morpholinecarboxylic acid-4-(5,6,7-trihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenyl ester (7f)
Light yellow solid, yield: 80.0%, mp: > 255 °C; 1H NMR (400 MHz, DMSO-d6) δ: 12.66 (brs, 1H),
10.57 (brs, 1H), 8.81 (brs, 1H), 8.10 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H), 6.93 (s, 1H), 6.63 (s,
1H), 3.67 (t, J = 4.8 Hz, 4H), 3.63-3.59 (m, 2H), 3.46-3.42 (m, 2H). HR-ESI-MS: Calcd. for C20H18NO8
[M+H]+: 400.1032, found: 400.1040.
1-Piperidinecarboxylic acid-4-(5,6,7-trihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenyl ester (7g)
Light yellow solid, yield: 86.8%, mp: 196-198 °C; 1H NMR (400 MHz, DMSO-d6) δ: 12.69 (brs,
1H), 10.60 (brs, 1H), 8.86 (brs, 1H), 8.09 (d, J = 8.8Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 6.94 (s, 1H), 6.63
(s, 1H), 3.58 (m, 2H), 3.42 (m, 2H), 1.60 (m, 6H). HR-ESI-MS: Calcd. for C21H20NO7 [M+H]+:
398.1240, found: 398.1232.
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4-(Phenylmethyl)-1-piperidinecarboxylic acid-4-(5,6,7-trihydroxy-4-oxo-4H-1-benzopyran-2-
yl)phenyl ester (7h)
Light yellow solid, yield: 80.9%, mp: 176-178 °C; 1H NMR (400 MHz, CDCl3) δ: 7.72 (d, J = 8.8 Hz,
1H), 7.68 (d, J = 8.8 Hz, 1H), 7.34~7.17 (m, 5H), 7.19 (d, J = 8.8 Hz, 1H), 7.13 (d, J = 8.8 Hz, 1H), 6.51
(s, 0.5H), 6.50 (s, 0.5H), 6.42 (s, 0.5H), 6.13 (s, 0.5H), 4.29 (t, J = 15.2 Hz, 2H), 2.99-2.95 (m, 1H),
2.87-2.83 (m, 1H), 2.64-2.62 (m, 1H), 2.62-2.60 (m, 1H), 1.79-1.77 (m, 1.5H), 1.77-1.74 (m, 1.5H),
1.34-1.30 (m, 1H), 1.31-1.27 (m, 1H). HR-ESI-MS: Calcd. for C28H26NO7 [M+H]+: 488.1709, found:
488.1697.
General procedure for the synthesis of compounds 7i and 7j
To a solution of compound 7b (1.0 mmol) and anhydrous K2CO3 (12.0 mmol) in anhydrous
acetone (30 mL), Me2SO4 (3.0 mmol) was added dropwise. The mixture was then heated at reflux
for 12 h under an argon atmosphere. After complete reaction, filtered and washed with acetone,
the filtrate was evaporated under reduced pressure. Ice-water (20 mL) was added to the residue,
and filtered, the crude product was purified on a silica gel chromatography using CHCl3/CH3OH
(50:1) as eluent to give the desired product 7i and 7j, respectively.
N,N-diethylcarbamic acid-4-(5,6,7-trimethoxy-4-oxo-4H-1-benzopyran-2-yl)phenyl ester (7i)
Yellow solid, yield: 58.0%, mp: 138~139 °C. 1H NMR (CDCl3 400 MHz) δ: 7.88 (d, 2H, J = 8.8 Hz),
7.28 (d, 2H, J = 8.8 Hz), 6.82 (s, 1H), 6.65 (s, 1H), 3.99 (s, 6H), 3.93 (s, 3H), 3.45 (q, 2H, J = 7.2 Hz),
3.42 (q, 2H, J = 7.2 Hz),1.28 (t, 3H, J = 7.2 Hz), 1.23 (t, 3H, J = 7.2 Hz). 13C NMR (CDCl3 100 MHz) δ:
177.0, 160.4, 157.6, 154.3, 153.8, 153.4, 152.3, 140.2, 128.0, 126.9, 122.2, 112.7, 107.9, 96.2, 62.0,
61.4, 56.2, 42.2, 41.8, 14.1, 13.2. HR-ESI-MS: Calcd. for C23H26NO7 [M+H]+: 428.1709, found:
428.1711.
N,N-diethylcarbamic acid-4-(5-hydroxy-6,7-dimethoxy-4-oxo-4H-1-benzopyran-2-yl)phenyl ester
(7j)
Yellow solid, yield: 32.5%, mp: 167~168 °C. 1H NMR (CDCl3 400 MHz) δ: 12.70 (s, 1H), 7.89 (d,
2H, J = 8.8 Hz), 7.30 (d, 2H, J = 8.8 Hz), 6.65 (s, 1H), 6.57 (s, 1H), 3.98 (s, 3H), 3.93 (s, 3H), 3.46 (q,
2H, J = 6.8 Hz), 3.42 (q, 2H, J = 6.8 Hz),1.28 (t, 3H, J = 7.2 Hz), 1.22 (t, 3H, J = 7.2Hz), 13C NMR (CDCl3
100 MHz) δ: 182.4, 163.2, 158.7, 154.2, 153.2, 153.0, 152.7, 132.4, 127.6, 127.2, 122.2, 105.9,
105.0, 90.5, 60.6, 56.1, 42.2, 41.8, 14.1, 13.2. HR-ESI-MS: Calcd. for C22H24NO7 [M+H]+: 414.1553,
found: 414.1548.
General procedure for the synthesis of compounds 7k and 7l
To a solution of compound 7b (1.0 mmol) and pyridine (4.0 mmol) in anhydrous acetone (30
mL), acetic anhydride (4.0 mmol) was added dropwise. The mixture was stirred at room
temperature for 15 h under an argon atmosphere. After complete reaction, filtered and washed
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with acetone, the filtrate was evaporated under reduced pressure. Ice-water (30 mL) was added to
the residue, and filtered, the crude product was purified on a silica gel chromatography using
CHCl3/CH3OH (100:1) as eluent to give the desired product 7k and 7l, respectively.
N,N-diethylcarbamic acid-4-[5,6,7-tris(acetyloxy)-4-oxo-4H-1-benzopyran-2-yl]phenyl ester (7k)
Yellow solid, yield: 42.6%, mp: 171~172 °C. 1H NMR (CDCl3 400 MHz) δ: 7.85 (d, 2H, J = 8.4 Hz),
7.50 (s, 1H), 7.29 (d, 2H, J = 8.4 Hz), 6.63 (s, 1H), 3.47-3.40 (m, 4H), 2.45 (s, 3H), 2.35 (s, 3H), 2.34
(s, 3H), 1.29-1.21 (m, 6H). HR-ESI-MS: Calcd. for C26H26NO10 [M+H]+: 512.1557, found: 512.1550.
N,N-diethylcarbamic acid-4-[6,7-bis(acetyloxy)-5-hydroxy-4-oxo-4H-1-benzopyran-2-yl] phenyl
ester (7l)
Yellow solid, yield: 25.0%, mp: 188~189 °C, 1H NMR (CDCl3 400 MHz) δ: 12.35 (brs, 1H), 7.88 (d,
2H, J = 8.8 Hz), 7.31 (d, 2H, J = 8.8 Hz), 6.97 (s, 1H), 6.70 (s, 1H), 3.49-3.39 (m, 4H), 2.37 (s, 3H),
2.35 (s, 3H), 1.30-1.21 (m, 6H). HR-ESI-MS: Calcd. for C24H24NO9 [M+H]+: 470.1451, found:
470.1442.
2.3. Biological assays
2.3.1. Inhibition experiments of AChE and BuChE
The determination of AChE and BuChE inhibitory capacity of the compounds were tested by
Ellman assay with slight modification (17), using AChE from 5% rat cortex homogenate and BuChE
from rat serum. The brain homogenate was pre-incubated for 5 min with tetraisopropyl
pyrophosphoramide (iso-OMPA, selective inhibitor of BuChE, 4.0 mmol/L) (Sigma-Aldrich Co.)
before use. For rat AChE or BuChE inhibition assays, a reaction mixture (100 µL) containing
acetylthiocholine iodide (1 mmol/L, 30 µL) (J&K Scientific) or butyrylthiocholine iodide (1 mmol/L,
30 µL) (TCI Shanghai Development), phosphate-buffered solution (0.1 mmol/L, pH = 7.4, 40 µL), 5%
homogenate or 25% serum (10 µL) and different concentrations (DMSO < 1%) of test compounds
(20 µL) was incubated at 37 °C for 15 min. Then 5,5’-dithiobis-2-nitrobenzoic acid (DTNB, 0.2%, 30
µL) (J&K Scientific) was added. The activities were determined by using a Varioskan Flash
Multimode Reader (Thermo Scientific) at 405 nm. Data presented were the average of three
independent experiments ± SD.
2.3.2. Metal binding studies (17)
The metal binding studies were carried out in a Varioskan Flash Multimode Reader (Thermo
Scientific). The UV absorption of the tested compounds 7b, 7i and 7j, in the absence or presence
of CuCl2, ZnCl2, AlCl3 and FeSO4, was recorded with wavelength ranging from 200 to 600 nm after
incubating for 2 h in methanol at room temperature. The final volume of reaction mixture was 200
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μL, and the final concentrations of tested compound and metals were 37.5 μM. The stoichiometry
of the compound-Cu2+ complex was determined by titrating the methanol solution of tested
compound with ascending of CuCl2. The final concentration of tested compound was 37.5 μM, and
the final concentration of Cu2+ ranged from 3.75 to 112.5 μM. The UV spectra was recorded and
treated by numerical subtraction of CuCl2 and tested compound at corresponding concentrations,
plotted versus the mole fraction of tested compound.
2.3.3. Antioxidant activity assay.
The antioxidant activity was determined by the oxygen radical absorbance capacity fluorescein
(ORAC-FL) assay (17). 2,2’-Azobis(amidinopropane) dihydrochloride (AAPH) was purchased from
Accela ChemBio Co., Ltd. 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) and
fluorescein (FL) were purchased from TCI (Shanghai) Development. All the assays were conducted
with 75 mM phosphate buffer (pH 7.4), and the final reaction mixture was 200 μL. Antioxidant (20
μL) and fluorescein (120 μL, 150 nM final concentration) were placed in the wells of a black
96-well plate. The mixture was pre-incubated for 15 min at 37 °C, and then AAPH solution (60 μL,
12 mM final concentration) was added rapidly using an autosampler. The plate was immediately
placed in a Varioskan Flash Multimode Reader (Thermo Scientific) and the fluorescence recorded
every minute for 90 min with excitation at 485 nm and emission at 535 nm. The plate was
automatically shaken prior to each reading. Trolox was used as standard (1-8 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 concentration
(1-10 μM). All reaction mixtures were prepared in duplicate, and at least three independent runs
were performed for each sample. Fluorescence measurements were normalized to the curve of
the blank (without antioxidant). The ORAC-FL values were calculated as described in the reference,
and the final results were in (μM Trolox equivalent)/(μM pure compound).
2.3.4. Hydrogen peroxide induced PC12 cell injury (14)
PC12 cells were seeded into 96-well plates at a density of 1×105 cells per mL in DMEM medium
(GIBCO.) supplemented with 10% heat-inactivated bovine calf serum (Hyclone). After overnight
incubation, tested compounds at 10.0 μmol/L and 1.0 μmol/L were added and incubated with the
cells. After pretreatment with the compounds for 2 h, H2O2 (100 μM) was added to the PC12 cell
cultures for 30 min, then replaced with fresh DMEM medium without calf serum and phenol red.
Cell-viability assays were performed 24 h after culture in fresh medium. At the end of the
experiments, the cells were incubated with 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) for 2 h at 37 °C. The supernatant layer was then carefully
removed, 100 μL DMSO was added into each well, and the absorbance at 490 nm of the MTT
formazan was determined by use of an Elx-800 microplate recorder (Bio-Tek Instruments). Data
are expressed as mean ± SD.
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2.3.5. Step-down passive avoidance test
Materials and animals
Donepezil hydrochloride was purchased from Eisai China Inc. Scopolamine was purchased from
J&K Scientific. Ethanol and sodium nitrite were from Sigma. Kunming mice at body weight of
18–22 g (six weeks old, either gender) were supplied by the Center of Experimental Animals of
Sichuan Academy of Chinese Medicine Sciences (eligibility certification no. SCXK-Sichuan 2008-19).
Mice were maintained under standard conditions with a 12 h:12 h light–dark cycle, a temperature
and humidity controlled environment with access to food and water ad libitum. The mice were
submitted to behavioral tests one day after 7 days of treatment with compounds.
Assay method
A modification of step-down passive avoidance test was used to assess learning and memory
in mice (18). The apparatus consisted of a grid floor with a wooden block placed in the center. The
block served as a shock free zone. The mice underwent two separate trials: a training trial and a
test trial 24 h later. For the training trial, mice were initially placed on the block and were given an
electrical foot shock (0.5 mA, 2 s) through the grid floor on stepping down. We used a total of 180
mice in the passive avoidance test with 10 mice were used per treatment. Compounds 7b (2, 4 and
8 mg/kg, p.o.) or donepezil (as a positive control, 1.6 mg/kg, p.o.) were administered 1 h before
each training trial. After 30 min, memory impairment was induced by administering scopolamine
(3 mg/kg, i.p.), ehanol (40% v/v, 10 mL/kg, i.g.) and sodium nitrite (200 mg/kg, i.p.). Twenty-four
hours after the training trial, mice were placed on the block and the time for the animal to step
down was measured as latency time for test trial. An upper cut-off time was set at 300 s.
Statistical analysis
All data are expressed as mean ± SEM. Differences between groups were examined for
statistical significance using one-way ANOVA with Student’s t test. A P value less than 0.05
denoted the presence of a statistically significant difference.
2.3.6. Estimation of brain acetylcholinesterase (AChE)
Materials and animals
Donepezil hydrochloride was purchased from Eisai China Inc. Kunming mice at body weight of
18–22 g (six weeks old, either gender) were supplied by the Center of Experimental Animals of
Sichuan Academy of Chinese Medicine Sciences (eligibility certification no. SCXK-Sichuan 2008-19).
The control group mice were treated with saline. For positive control group donepezil was
administered 1.6 mg/kg and the test groups were intragastric administration with compound 7b
(2, 4, 8 mg/kg) for 3 weeks.
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Assay method
The estimation of brain acetyl cholinesterase activity was performed based on Ellman’s
method with slight modifications (19). The animals were decapitated and brains were dissected
out immediately and placed in ice-cold saline. The tissue was weighed and homogenized in 0.1 M
phosphate buffer pH 8, the homogenized tissue was centrifuged to 10,000 rpm for 10 min, 20 µL
aliquot of the supernatant is added into a cuvette containing 30 µL phosphate buffer (0.1 M, pH 8)
and 30 µL of DTNB. The contents of the cuvette are mixed thoroughly by bubbling air and
absorbance is measured at 405 nm in Varioskan Flash Multimode Reader (Thermo Scientific).
When absorbance reaches a stable value, it is recorded as the basal reading. A 20 µL of substrate
acetylthiocholine is added and change in absorbance is recorded for a period of 10 min at intervals
of 2 min. Change in the absorbance per minute is thus determined. AChE activity is calculated
using the formula reported previously (19).
3. Results and discussion
3.1. Chemistry
The target compounds 7a~l were synthesized according to procedures as outlined in Scheme 1
and Scheme 2. The starting material scutellarein (2) with 1.2 equivalents diphenyl
dichloromethane (3) in 1,2-dichlorobenzene at 170 °C gave 86.5% yield after purification, was
subsequently reacted with 1.2 equivalents N,N-disubstituted carbamoyl chlorides (5a~h) (14) in
the presence of anhydrous K2CO3 in CH3CN at 60~65 °C to afford the desired products 6a-h.
Subsequently, deprotection of the catecholic hydroxyls with a HOAc-H2O (2:1) solution at reflux
and then purification by recrystallisation with ethanol gave the desired target compounds 7a-h in
good yield. Furthermore, 7b was reacted with 3.0 equivalents Me2SO4 in the presence of
anhydrous K2CO3 in acetone at reflux for 12 h to furnish the products 7i and 7j. Meanwhile, 7b was
reacted with 4.0 equivalents Ac2O in the presence of anhydrous pyridine in acetone at room
temperature for 15 h to afford the desired products 7k and 7l.
Scheme 1. Synthesis of scutellarein carbamate derivatives 7a-h. Reagents and conditions: a)
diphenyl dichloromethane (3), 1,2-dichlorobenzene, 170 °C, 40 min, 85.6%; b) RCOCl (5a~h),
K2CO3, CH3CN, reflux, 5 h, 49.3%-86.7%; c) HOAc/H2O (2:1), reflux, 2-3 h, 80.0%-93.0%.
Scheme 2. Synthesis of scutellarein carbamate derivatives 7i-l. Reagents and conditions: a) 3.0 eq.
Me2SO4, K2CO3, acetone, reflux, 12 h; b) 4.0 eq. Ac2O, 4.0 eq pyridine, acetone, r.t., 15 h.
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3.2. AChE and BuChE inhibition
The AChE and BuChE inhibitory activities of scutellarein carbamate derivatives 7a~l were
determined by the modified Ellman method using AChE from rat cortex homogenate (RatAChE)
and BuChE from rat serum (RatBuChE) (17). Rivastigmine, the well-known AChE and BuChE dual
inhibitor, was applied as positive drug. And previously reported compound scutellarein (1) was
also evaluated for comparative purpose. Their IC50 values and selectivity index for the inhibition of
AChE and BuChE were summarized in Table S1. In the preliminary experiments, target compounds
7a~h were synthesized first, and indicated better inhibitory activity than scutellarein with IC50
>
500μM, in particular, compounds 7b, 7c and 7d, with IC50 values of 1.2 μM, 4.0 μM and 2.7 μM,
respectively, exhibited higher inhibitory capacities than rivastigmine (IC50 = 5.6 μM). It showed
that the introduction of carbamate group improved AChE inhibitory activity, which was consistent
with our design strategy. Among these compounds, compound 7b, with a diethylamine carbamate
group, showed the most potent inhibition for AChE with IC50 value of 1.2 μM. According to the
screening data, the properties of the carbamate groups at scutellarein 4′ position had significant
effects on AChE inhibitory activities, generally, the potencies to inhibit AChE were in the order,
diethylamine > N-methylethylamine > diisopropylamine > dimethylamine > piperidine >
morpholine > pyrrole > benzylpiperidine. The results suggested that diethylamine may be the
optimal substituent pattern for AChE inhibition. To explore the inhibitory effects of 5, 6 and
7-substituted groups on AChE, compounds 7i~l were synthesized and evaluated their activities. As
shown in Table S1, compounds 7i and 7j with methyl substituted showed better inhibitory
activities than compounds 7k and 7l, which were substituted with acetyl. Compounds 7i and 7j
also exhibited higher inhibitory activity than precursor compound 7b.
For BuChE inhibition assay, almost all compounds showed very weak activity against BuChE.
These results demonstrated that the target compounds were potent AChE inhibitors with
selectivity toward AChE over BuChE, which might be beneficial to the treatment of AD according
to the previously reported (17). Therefore, compounds 7b, 7i and 7j were selected for further
study to evaluate the metal-chelating ability.
3.3. Metal-chelating properties of compounds 7b, 7i and 7j
The ability of compounds 7b, 7i and 7j to chelate biometals such as Cu2+, Zn2+, Al3+ and Fe2+ was
studied by UV-vis spectrometry (17). The results are shown in Figure S1, after CuCl2 was added to
a solution of 7b, the maximum absorption wavelength shifted from 322 nm to 358 nm, indicating
the formation of 7b-Cu2+ complex. It was reasonable that the chelation attributed to the
5-hydroxyl and 4-carbonyl groups at the flavonoid nucleus. Similar result was also obtained when
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AlCl3 and FeSO4 were added, the peak in the electronic spectra of 7b at 322 nm shifted to 366 nm
and 360nm, respectively. However, when ZnCl2 was added, the electronic spectra of 7b exhibited
no significant change. Therefore, 7b had the selectivity for metal chelation. Moreover, the same
experiment was performed using compound 7j and biometal ions (Figure S2). When CuCl2 and
AlCl3 were added to a solution of 7j, the maximum absorption wavelength shifted from 322 nm to
388 nm and 341 nm, respectively, indicating the formation of 7j -Cu2+ and 7j - Al3+ complexs.
However, when FeSO4 and ZnCl2 were added, the electronic spectra of 7j exhibited no significant
change. On the other hand, the same experiment was carried out on compound 7i (Figure S3), no
significant differences were observed in the UV spectrum, suggesting little or no complex
formation between 7i with metal ions, such as Cu2+, Zn2+, Al3+ or Fe2+.
The stoichiometry of the 7b-Cu2+ complex was determined using molar ratio method by
preparing solutions of compound 7b with increasing amounts of CuCl2. The UV spectra was used to
obtain the absorbance of the complex of 7b and CuCl2 at different concentrations at 358 nm.
According to the Figure S4, the absorbance linearly increased at first and then tended to be stable.
The two straight lines intersected at a mole fraction of 1.98, revealing a 1:2 stoichiometry for
complex 7b-Cu2+. Meanwhile, the same experiment was performed on compound 7j at 388 nm,
Figure S5 showed the two straight lines intersected at a mole fraction of 0.98, revealing a 1:1
stoichiometry for complex 7j-Cu2+.
3.4. Antioxidant activity
The antioxidant activities of the selected target compounds were evaluated by following the
ORAC-FL method (Oxygen Radicals Absorbance Capacity by Fluorescence) (17). The vitamin E
analogue Trolox was used as a standard, and the results were expressed as Trolox equivalents. As
shown in Table S1, the chosen compounds 7a, 7b, 7d, 7i, 7j, 7k and 7l exhibited significant
ORAC-FL with values of 10.0, 10.3, 9.5, 0.4, 1.3, 0.7 and 2.0 Trolox equivalents respectively, but
lower than the parent scutellarein 2 (12.5 Trolox equivalents). As expected, the introduction of
carbamate groups in the scutellarein 4′ position decreased the antioxidant capacity but the
different carbamate moieties had little influence on the radical capture capacity. According to the
assay data, compound 7b (10.3 Trolox equivalents) with 5,6,7-trihydroxy at the flavonoid nucleus
exhibited better antioxidant capacity than 7i (0.4 Trolox equivalents) with 5,6,7-trimethoxy and 7j
with 5-hydroxyl-6,7-dimethoxy (1.3 Trolox equivalents) respectively. The compound 7b, possessing
the better AChE inhibitory activity, showed the remarkable antioxidant activity with ORAC-FL value
of 10.3 Trolox equivalents.
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3.5. Neuroprotective effect on cell injury induced by hydrogen peroxide
The neuroprotective capacities of the selected carbamate derivatives against oxidative stress
were assayed by measuring the ability of the tested compounds to protect against hydrogen
peroxide-induced PC12 cell injury, and using the MTT assay evaluated the cell viability (20). In the
test, exposure of PC12 cells to 100 μM H2O2 markedly reduced viability to 59.3%, suggesting high
sensitivity to H2O2-induced injury. The cell viabilities attributable to the protective efficiency of
tested compounds (10 μM) against H2O2 were listed in Table S1. However, the parent scutellarein
2 only increased the cell survival to 65.54%. We speculate that the weak neuroprotective effect of
compound 2 is due to its high polarity, poor solubility and poor ability to penetrate the cell
membrane. The tested compounds exhibited higher cell viability than scutellarein (2). It was
reasonable that the introduction of carbamate moiety improved neuroprotective effect. On the
other hand, for compounds 7b, 7i and 7j, with different substituted groups at the 5, 6 and 7
position of the flavonoid nucleus, showed significantly neuroprotective capacities with the cell
viabilities in 74.70%, 70.31% and 75.52%, respectively. Therefore, according to the above results,
compound 7b was chosen for further study.
3.6. Effects of 7b on learning and memory impairment induced by scopolamine, ethanol and
sodium nitrite (passive avoidance test)
We examined whether 7b could improve learning and memory impairment induced by
scopolamine, ethanol and sodium nitrite using the step-down passive avoidance test, which had
been used extensively to evaluate potential therapeutic agents for treating AD (18). For
scopolamine-induced memory impairment (Figure S6), in the retention trial, the vehicle-treated
mice (sham group given the same dose of normal saline) indicated the step-down latency time up
to 207.5 sec. The latency time (47.5 sec) (p < 0.01) of mice treated with scopolamine alone
(control group) showed a dramatic decrease compared to the sham group (p < 0.01). Treatment
with compound 7b (2, 4 and 8 mg/kg) increased the latency time in a dose-dependent manner,
and the high-dose group (8 mg/kg) presented the longest latency time (170.6 sec) (p < 0.01) in the
three groups. The high-dose (8 mg/kg) of compound 7b also showed slightly longer latency time
than the control drug group with donepezil (1.6 mg/kg) (164.4 sec), which is a representative AChE
inhibitor and had been approved by FDA for the treatment of AD.
For ethanol-induced learning and memory impairment, ethanol is one of the most commonly
used and frequently abused psychoactive substances. Its consumption is associated with cognitive
deficit, including memory and learning impairment (21). Figure S7 showed the step-down latency
time of mice treated with ethanol alone (control group) (78.0 sec) was significantly shorter than
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that of vehicle-treated mice (sham group) (179.2 sec) (p < 0.01). Treatment with compound 7b (2,
4 and 8 mg/kg) increased the latency time in a dose-dependent manner, and the high-dose group
(8 mg/kg) presented the longest latency time (169.9 sec) (p < 0.01) in the three groups. The
high-dose (8 mg/kg) of compound 7b also showed slightly shorter latency time than the control
drug group with donepezil (1.6 mg/kg) (179.1 sec).
For sodium nitrite-induced memory impairment, Figure S8 indicated the step-down latency
time of mice treated with sodium nitrite alone (control group) (120.3 sec) was significantly shorter
than that of vehicle-treated mice (sham group) (225.7 sec) (p < 0.01). Treatment with compound
7b (2, 4 and 8 mg/kg) increased the latency time in a dose-dependent manner, and the high-dose
group (8 mg/kg) presented the longest latency time (223.4 sec) (p < 0.01) in the three groups. The
high-dose (8 mg/kg) of compound 7b also showed similar latency time compared with donepezil
(drug control group) (1.6 mg/kg) (224.0 sec).
Altogether, the above results exhibited that compound 7b could improve learning and memory
induced by scopolamine, ethanol and sodium nitrite.
3.7. Effects of 7b on the activity of acetylcholinesterase (18)
As shown in Figure S9, the vehicle-treated (sham group) indicated the activity of
acetylcholinesterase up to 0.772 U/mgprot, the activity of acetylcholinesterase of mice treated
with donepezil (0.286 U/mgprot) showed a dramatic decrease as compared to the sham group (p <
0.01). Treatment with compound 7b (2, 4 and 8 mg/kg) reduced the activity of
acetylcholinesterase in a dose-dependent manner, and the high-dose group (8 mg/kg) presented
the lowest activity of acetylcholinesterase (0.354 U/mgprot) in the three groups (p < 0.01). But the
inhibitory potency of compound 7b with acetylcholinesterase at the high-dose (8 mg/kg) was
slightly lower than the control group with donepezil (1.6 mg/kg). These results showed that
compound 7b could significantly decrease the activity of acetylcholinesterase in mice brain.
4. Conclusion
In summary, a series of scutellarein carbamate derivatives were designed, synthesized and
evaluated as multifunctional agents for the treatment of AD. Most of the synthesized compounds
showed significant AChE inhibitory activity in vitro and displayed selectivity toward AChE over
BuChE. Among these compounds, compound 7b demonstrated good inhibitory potency toward
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AChE and BuChE with IC50 values of 1.2 ± 0.03 μM and 22.1 0.15 μM, respectively. Compound 7b
also possessed the strongest antioxidant potency (10.3 trolox equivalents), and acted as a
selective metal chelator. Moreover, 7b showed a neuroprotective effect against H2O2-induced
PC12 cell injury. Furthermore, 7b could improve memory impairment induced by scopolamine,
ethanol and sodium nitrite using the step-down passive avoidance task, and could remarkably
decrease the activity of acetylcholinesterase in mice brain. The present study indicated that 7b
could be considered as an interesting multi-targeted lead compound for the further study in the
treatment of AD. Further studies to evaluate compound 7b in vivo and to develop structural
refinements are underway and will be reported in due course.
Acknowledgments
This work was supported in part by the Chinese National Natural Science Foundation (20872099),
the Research Fund for the Doctoral Program of Higher Education (20110181110079), the Sichuan
Academic and Technical Leader Training Foundation (2012DTPY003) and the National Science and
Technology Major Project on “Key New Drug Creation and Manufacturing
Program”(2009ZX09103111, 2013ZX09301304-002).
Conflict of interest
The authors declare no competing financial interest.
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Supporting Information
Figure S1. The UV spectrum of compounds 7b (37.5 μM, in methanol) alone or in the presence of
CuCl2, ZnCl2, AlCl3 and FeSO4 (37.5 μM, in methanol).
Figure S2. The UV spectrum of compounds 7j (37.5 μM, in methanol) alone or in the presence of
CuCl2, ZnCl2, AlCl3 and FeSO4 (37.5 μM, in methanol).
Figure S3. The UV spectrum of compounds 7i (37.5 μM, in methanol) alone or in the presence of
CuCl2, ZnCl2, AlCl3 and FeSO4 (37.5 μM, in methanol).
Figure S4. Determination of the stoichiometry of complex by molar ratio method. The final
concentration of tested compound 7b was 37.5 μM, and the final concentration of Cu2+ ranged
from 7.5 to 112.5 μM.
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Figure S5. Determination of the stoichiometry of complex by molar ratio method. The final
concentration of tested compound 7j was 37.5 μM, and the final concentration of Cu2+ ranged
from 7.5 to 93.75 μM.
Figure S6. Effects of compound 7b on memory deficit induced by scopolamine in the step-down
passive avoidance test. Values are expressed as the mean ± SEM (n=10). ## p < 0.01 vs normal
group. ** p < 0.01 vs scopolamine-treated control group.
Figure S7. Effects of compound 7b on memory deficit induced by ethanol in the step-down passive
avoidance test. Values are expressed as the mean ± SEM (n=10). ## p < 0.01 vs normal group. * p <
0.05 and ** p < 0.01 vs ethanol-treated control group.
Figure S8. Effects of compound 7b on memory deficit induced by sodium nitrite in the step-down
passive avoidance test. Values are expressed as the mean ± SEM (n=10). ## p < 0.01 vs normal
group. * p < 0.05 and ** p < 0.01 vs sodium nitrite -treated control group.
Figure S9. Effects of compound 7b on the activity of acetylcholinesterase in mice. Each group
consists of 10 animals (n = 10). Values are expressed as the mean ± SEM (n=10). * p < 0.05 and **
p < 0.01 vs sham group.
Table S1. Rat.AChE and Rat.BuChE inhibitory activities, neuroprotective effect, and oxygen radical
absorbance capacity (ORAC, Trolox equivalent) of scutellarein carbamate derivatives 7,
rivastigmine and scutellarein.
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.