Design, synthesis and evaluation of scutellarein- O -alkylamines as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2015.02.063

A series of scutellarein-O-alkylamine derivatives were designed, synthesized and tested as multifunctional agents for the treatment of Alzheimer's disease (AD). The results showed that most of these compounds exhibited good multifunctional activities. Among them, compound 16d demonstrated significant metal chelating properties, moderate acetylcholinesterase (AChE) inhibitory and anti-oxidative activity, and excellent inhibitory effects on self-induced Aβ2_1-42 aggregation, Cu²⁺-induced Aβ2_1-42 aggregation, human AChE-induced Aβ2_1-40 aggregation and disassembled Cu²⁺-induced aggregation of the well-structured Aβ2_1-42 fibrils. Both kinetic analysis of AChE inhibition and molecular modeling study suggested that 16d binds simultaneously to the catalytic active site and peripheral anionic site of AChE. Moreover, compound 16d showed a good protective effect against H₂O₂-induced PC12 cell injury, with low toxicity in SH-SY5Y cells. Furthermore, the step-down passive avoidance test showed this compound significantly reversed scopolamine-induced memory deficit in mice. Thus, 16d was shown to be an interesting multifunctional lead compound worthy of further study.

Full text of DOI:10.1016/j.ejmech.2015.02.063

Accepted Manuscript
Design, synthesis and evaluation of scutellarein-O-alkylamines as multifunctional
agents for the treatment of Alzheimer's disease
Zhipei Sang, Xiaoming Qiang, Yan Li, Wen Yuan, Qiang Liu, Yikun Shi, Wei Ang,
Youfu Luo, Zhenghuai Tan, Yong Deng
PII:
S0223-5234(15)00161-0
10.1016/j.ejmech.2015.02.063
EJMECH 7747
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 November 2014
Revised Date: 27 February 2015
Accepted Date: 28 February 2015
Please cite this article as: Z. Sang, X. Qiang, Y. Li, W. Yuan, Q. Liu, Y. Shi, W. Ang, Y. Luo, Z. Tan,
Y. Deng, Design, synthesis and evaluation of scutellarein-O-alkylamines as multifunctional agents for
the treatment of Alzheimer's disease, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.02.063.
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ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of scutellarein-O-alkylamines as multifunctional agents for
the treatment of Alzheimer's disease
1
,#
1,#
1
1
1
3
1
2
Zhipei Sang , Xiaoming Qiang , Yan Li , Wen Yuan , Qiang Liu , Yikun Shi , Wei Ang , Youfu Luo ,
3,*
1,*
Zhenghuai Tan and Yong Deng
Graphical Abstract
antioxidant
metal chelation
OH
O
O
H CO
3
CH3
H CO
3
N
O
OCH3
1
6d
Rat AChE IC50: 0.62
M
M
M
Self-induced A
Cu2+-induced A
aggregation: 69.1% inhibition at 25
aggregation: 85.7% inhibition at 25
M
M
1-42
EeAChE IC50: 0.85
HuAChE IC50: 0.25
Rat BuChE IC50: 62.0
1-42
AChE-induced A 1-40 aggregation: 62.2% inhibition at 100
M
M
Disaggregation Cu2+-induced A
aggregation:
1-42
Blockade of CAS and PAS of AChE
ORAC-FL value: 0.84 (Trolox equiv)
Cytotoxicity (SH-SY5Y cells): IC50 >100
81.3% disaggregation at 25
Good protective effect against H O -induced PC12 cell injury
M
2
2
M
A series of scutellarein-O-alkylamine derivatives were designed, synthesized and tested as
multifunctional agents for the treatment of Alzheimer’s disease (AD). Compound 16d was shown to
be an interesting multifunctional lead compound for AD treatment.
1

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Design, synthesis and evaluation of scutellarein-O-alkylamines as multifunctional agents for
the treatment of Alzheimer's disease
1
,#
1,#
1
1
1
3
1
2
Zhipei Sang , Xiaoming Qiang , Yan Li , Wen Yuan , Qiang Liu , Yikun Shi , Wei Ang , Youfu Luo ,
3,*
1,*
Zhenghuai Tan and Yong Deng
1
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
2
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan
University, Chengdu, 610041, China
3
Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan Academy of
Chinese Medicine Sciences, Chengdu, 610041, China
Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterase; BuChE, butyrylcholinesterase;
HuAChE, human AChE; EeAChE, Electrophorus electricus AChE; MTDL, multitarget-directed
ligand; Aβ, β-amyloid peptide; CAS, catalytic active site; PAS, peripheral anionic site; MOM,
methoxymethyl ether; PBS, phosphate-buffered saline; DTNB, 5,5’-dithiobis-2-nitrobenzoic acid;
PDB, Protein Data Bank; TcAChE, Torpedo californica AChE; ROS, reactive oxygen species;
ORAC-FL, Oxygen Radicals Absorbance Capacity by Fluorescence; AAPH, 2,2’-Azobis
(amidinopropane) dihydrochloride; ThT, thioflavin T; HEPES, 4-(2-Hydroxyethyl)-1-
piperazineethanesulfonic acid; TEM, transmission electron microscopy.
#
These authors contributed equally to this work.
*
Corresponding Author.
Phone, +86-28-85258982; E-mail: tanzhh616@163.com (Zhenghuai Tan)
Phone, +86-28-85503790; E-mail: dengyong@scu.edu.cn (Yong Deng)
1

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Abstract
A series of scutellarein-O-alkylamine derivatives were designed, synthesized and tested as
multifunctional agents for the treatment of Alzheimer’s disease (AD). The results showed that most
of these compounds exhibited good multifunctional activities. Among them, compound 16d
demonstrated significant metal chelating properties, moderate acetylcholinesterase (AChE)
inhibitory and anti-oxidative activity, and excellent inhibitory effects on self-induced Aβ1-42
2
+
aggregation, Cu -induced Aβ
aggregation, human AChE-induced Aβ1-40 aggregation and
-42
1
2
+
disassembled Cu -induced aggregation of the well-structured Aβ
fibrils. Both kinetic analysis of
-42
1
AChE inhibition and molecular modeling study suggested that 16d binds simultaneously to the
catalytic active site and peripheral anionic site of AChE. Moreover, compound 16d showed a good
protective effect against H O -induced PC12 cell injury, with low toxicity in SH-SY5Y cells.
2
2
Furthermore, the step-down passive avoidance test showed this compound significantly reversed
scopolamine-induced memory deficit in mice. Thus, 16d was shown to be an interesting
multifunctional lead compound worthy of further study.
Keywords: Alzheimer’s disease; Scutellarein-O-alkylamines; Multifunctional agents;
Acetylcholinesterase inhibitors; Metal-chelating agents; Aβ aggregation inhibitors.
2

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1. Introduction
Alzheimer’s disease (AD), the most common form of adult onset dementia affecting more than 44
million people worldwide, is a complex neurodegenerative process occurring in the central nervous
system (CNS), characterized by progressive and irreversible cognitive impairment, severe
behavioral abnormalities and ultimately causing death [1]. Current clinical treatment of AD is
mainly focused on the symptomatic aspects. The drugs that are currently approved by the FDA
include four acetylcholinesterase inhibitors (AChEs), tacrine (now withdrawn due hepatotoxicity),
donepezil, rivastigmine and galantamine, and one N-methyl-D-aspartate (NMDA) receptor
antagonist, memantine [2]. These agents can increase the levels of the neurotransmitter
acetylcholine, which is depleted in AD brains, or prevent aberrant neuronal stimulation. Although
these drugs are beneficial in improving cognitive, behavioral, and functional impairments they
cannot prevent, halt, or reverse the progression of the disease. These are just palliative treatment
and do not address the molecular mechanisms that underlie the pathogenic processes because of the
multifactorial nature of AD [3]. However, treatment with a combination of an AChE inhibitor and
memantine appears to produce an additional effect resulting in a well-tolerated, effective clinical
strategy that increases the time before patients require nursing home care [4]. Thus, the
development of agents that affect two or more AD-relevant targets has drawn considerable attention
for their advancement in the treatment of AD [5,6]. In this regard, the multitarget-directed ligand
strategy (MTDLs), i.e., single chemical entities able to hit different targets involved in the cascade
of AD pathological events, has been applied by many research groups [7,8], and the results obtained
have been encouraging and convince researchers that MTDLs might present the best
pharmacological option for tackling the multifactorial nature of AD and for halting the progression
of the disease. Several MTDLs candidate drugs with disease modifying potential are now in the
pipeline and have reached testing stage in clinical trials [9].
Although the etiology of AD is still not completely known, several factors are considered to play
definitive roles in AD pathogenesis. These include low levels of acetylcholine (ACh), oxidative
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stress and free radical formation, the dyshomeostasis of biometals and β-amyloid (Aβ) deposits
[
10,11]. These insights have provided the rationale for therapies directly targeting the molecular
causes of AD. Therefore the discovery of a lead compound that can modulate these four factors
simultaneously is a crucial step in the search for a candidate for the clinical treatment of AD.
Scutellarin (4′,5,6-trihydroxyflavone-7-glucuronide), the major active component in breviscapine
extracted from the Chinese herb Erigeron breviscapus (vant.) Hand.-Mazz., possesses a broad range
of pharmacological properties related to neurological disorders, such as anti-inflammatory effect,
metal chelating ability, neuroprotective action, Aβ fibril formation inhibition and free radical
scavenging effect [12-14]. However, scutellarin’s poor solubility, low oral absorption and difficultly
of passing through the blood–brain barrier restricts its clinical uses as an anti-AD drug [15,16].
Recent studies have indicated that scutellarin is mainly hydrolyzed in the intestinal tract and is
absorbed as aglycone scutellarein (I). This is the real bioactive component and much more easily
absorbed [17]. Recently, our group has reported the synthesis of genistein-O-alkylbenzylamines and
discovered a 7,4′-O-modified genistein derivative (II) as a potential multifunctional agent for the
treatment of AD [18]. However, this compound has low inhibitory effects on self-induced Aβ1-42
aggregation and human AChE-induced Aβ1-40 aggregation. In this paper, 5,6,7-trimethoxy and
5-hydroxy-6,7-dimethoxy flavonoids were selected to combine with different length
alkylbenzylamine or alkylpiperazine fragments to design series of novel
a
scutellarein-O-alkylamine derivatives, to test whether these novel molecules might possess more
potency in various multifunctional activities.
In this study, a series of scutellarein-O-alkylamine derivatives were designed based on MTDLs
(
Figure 1). They were also synthesized and evaluated for their multifunctional biological activities.
The biological evaluation included anti-oxidative effects, AChE and butyrylcholine esterase
BuChE) inhibition, the kinetics of enzyme inhibition, metal-chelating properties, effects on Aβ
aggregation and disaggregation, protective effect against H O -induced PC12 cell injury, cytotoxic
(
2
2
effects on SH-SY5Y cells, and neuroprotective effects in the mouse scopolamine model of memory
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impairment. Finally, molecular modeling studies were also performed to afford insight into the
binding mode and the structure-activity relationships of the novel scutellarein-O-alkylamine
derivatives.
<
Insert Figure 1 >
2. Results and discussion
2.1. Chemistry
The synthesis of scutellarein-O-alkylamine derivatives (15–18) occurred via the general
pathway, in which 6-hydroxy-2,3,4-trimethoxyacetophenone (1) was used as the starting material
Scheme 1). Compounds 7a–b and 8a–b were the key intermediates, usually prepared from
(
condensation of compound 1 [19] with para-methoxymethoxybenzaldehyde (2) [20] or
m-hydroxybenzaldehyde (3) in the presence of KOH at room temperature for 3 days to afford the
chalcone intermediates 4 or 6, respectively. Removal of the methoxymethyl ether (MOM)
protection group was then carried out in a 10% aqueous hydrochloric acid solution at 50°C to obtain
the corresponding 4′-OH chalcone compound (5). Subsequently, treatment of compound 5 or 6 with
conc. H SO and a catalytic amount of KI in DMSO at 100°C for 3–5 hours gave the flavonoids 7a
2
4
and 7b respectively. Finally, removal of 5-methyl at flavonoid nucleus by anhydrous AlCl in
3
CH CN at 55–60°C for 1 h gave the corresponding 5-OH scutellarein derivatives 8a–b [21].
3
Alkylation of 7a–b or 8a–b with excessive amounts of 1,3-dibromopropane, 1,4-dibromobutane or
1
1
1
,6-dibromohexane in the presence of K CO in CH CN at 60–65°C resulted in the intermediates,
2 3 3
0–13 [18]. In turn, these intermediates were reacted with the corresponding secondary amines
4A–F in the presence of anhydrous K CO at 60–65°C to provide the final
2
3
scutellarein-O-alkylamine compounds, 15–18 [18]. All new compounds were purified by
recrystallization or chromatography, and the analytical and spectroscopic data confirmed their
structures, as detailed in the experimental section.
<
Insert Scheme 1 >
2.2. Pharmacology
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2.2.1. Antioxidant activity
Reactive oxygen species (ROS) have been identified as important mediators of cell structure
damage to lipids, proteins and nucleic acids. ROS have been associated with aging, AD and other
neurodegenerative disorders [22]. In normal physiological conditions the host antioxidant defenses
would control the level of reactive free radicals, but when free radicals have overwhelmed these
defenses, cellular damage occurs [23]. Thus, an antioxidant might contribute to a therapeutic
strategy that prevents the development of AD and other neurodegenerative disorders.
The antioxidant activities of all synthesized scutellarein-O-alkylamine derivatives were evaluated
by following the well-established ORAC-FL method (oxygen radical absorbance capacity by
fluorescein) [24] and the results were shown in Table 1. Trolox, a vitamin E analogue, was used as
the standard, and the results were expressed as Trolox equivalents. Scutellarein was also tested,
which has an ORAC-FL value of 10.0 Trolox equivalents. All the tested compounds exhibited
potent peroxyl radical absorbance capacities, ranging from 0.12 to 0.84-fold of Trolox. As expected,
the introduction of alkylamine group side chain in the flavonoid nucleus decreased the radical
capture capacity but the different tertiary amine unit on the side chain has little influence on the
radical capture capacity. Generally, the compounds with 5-hydroxyl at the flavonoid nucleus (16
and 18) exhibited higher radical capture capacity than that of 5-methoxyl (15 and 17). Compound
16d showed the highest antioxidant activity, with an activity of 0.84 Trolox equivalents.
2.2.2. AChE and BuChE Inhibition
To evaluate the potential of the target compounds 15–18 for the treatment of AD, their
cholinesterase inhibitory activities were determined by the modified Ellman method [25] using
AChE from rat cortex homogenate (RatAChE) and electrophorus electricus (EeAChE) and BuChE
from rat serum (RatBuChE), with commercially available rivastigmine and donepezil as the
reference standards. Furthermore, the most potent and representative compounds were reevaluated
using human erythrocyte AChE (HuAChE). The AChE and BuChE inhibitory activities of the
synthesized scutellarein-O-alkylamine derivatives are listed in Table 1 and Table 2, expressed as
6

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IC values.
5
0
<
Insert Table 1 >
The tested target compounds displayed significant inhibitory activity against AChE. Almost all the
compounds exhibited very weak activity against BuChE and showed higher selectivity for ratAChE
over ratBuChE. The results showed that introduction of the O-alkylamines increased the ChEs
inhibitory capacity and improved the selectivity for ratAChE over ratBuChE. This selectivity
profile may be a limitation, but this also may be beneficial to diminish peripheral cholinergic side
effects and provide lower toxicity. Because severe side effects of AChE inhibitors, such as tacrine,
have been suggested to be attributed to their poor selectivity [26]. As shown in Table 1 and Figure
2
, the properties of the substitutions at flavonoid nucleus 5 position had significant effects on AChE
inhibitory activities, 5-methoxy derivatives (15c, 15d, 15i, 15j, 15o, 15p, 17h, 17i, 17n, 17o, 17t,
7u) showed higher activity than the 5-hydroxyl analogues (16a–f, 18a–f). The 5-methoxy at the
flavonoid nucleus may play a significant role in the AChE inhibitory activity.
Insert Figure 2 >
1
<
As shown in Table 1, compounds with different length alkyamino side chains at 4′ positon showed
better AChE inhibitory activities than that at 3′ positon, but the compounds (15e, 15f, 15k, 15l, 15q,
17d, 17e, 17j, 17k, 17p, 17q, 17v) containing two basic centers (methyl piperazine and
benzylpiperazine group) demonstrated the opposite result. This phenomenon revealed that the
alkyamino side chain can interact with catalytic active site (CAS) of AChE in different ways. The
screening data also showed that the structure of terminal groups NR R of side chain affected the
1
2
inhibitory activities. The potencies to inhibit AChE were in the order N-(2-methoxybenzyl)
ethanamine > N-(2-methoxybenzy)methanamine > N-benzylpiperazine > N-benzylethanamine >
N-benzyl methanamine > 1-methylpiperazine. In most conditionsꢀcompounds (15b, 15d, 15h, 15j,
15n, 15p, 16b, 16d, 16f, 17b, 17g, 17i, 17m, 17o, 17s, 17u, 18b, 18d, 18f) possessing a N-ethyl
group showed better AChE inhibitory activity than compounds (15a, 15c, 15g, 15i, 15m, 15o, 16a,
16c, 16e, 17a, 17c, 17f, 17h, 17l, 17n, 17r, 17t, 18a, 18c, 18e) with a N-methyl group. The terminal
7

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groups NR R containing a methoxyl group showed more potent AChE inhibitory activity than that
1
2
without a methoxyl. In particular, the N-(2-methoxybenzyl)ethanamine (15j, IC = 0.12±0.01ꢀM)
5
0
at the end of side chain expressed high inhibitory activity. As shown in Table 1, with the same
scaffold and terminal groups NR R , AChE inhibitory activity increased with an increase in
1
2
methylene chain length, except compounds 15j, 16c and 16d, where the optimal chain length was
four. However, inhibitory activity decreased with an increase of methylene chain length when the
terminal groups NR R was 1-benzylpiperazine, for example compounds (15f, 15l, 15q) and
1
2
compounds (17e, 17k, 17q, 17v). Finally, compound 15j showed the most potent inhibition for
RatAChE with an IC50 value of 0.12ꢀM and a 568.3-fold selectivity for AChE over BuChE.
Furthermore, compound 16d also exhibited a good AChE inhibition activity (IC = 0.62±0.01ꢀM)
5
0
and a high selectivity (100.0-fold of inhibition selectivity for AChE over BuChE). From the
re-evaluated results by additional testing using EeAChE and HuAChE, it is notable that most of the
tested compounds exhibited a 3–6-fold higher inhibitory activity towards RatAChE than towards
EeAChE (Table 1). However, the inhibitory activity of compounds (15j, 16d) against RatAChE is
about 2–4-fold lower than that against HuAchE (Table 2).
2.2.3. Kinetic studies of AChE inhibition
To investigate the AChE inhibitory mechanism for this class of scutellarein-O-alkylamine
derivatives, a kinetic study was carried out with a representative compound 16d using EeAChE
(Figure 3). The Lineweaver–Burk plots exhibited that both inhibitions had rising slopes and
increasing intercepts at higher concentration, which indicated a mixed-type inhibition. The result
demonstrated that compound 16d could bind to both CAS and PAS of AChE, which is similar to
that of donepezil and consistent with the results of molecular modeling studies.
<
Insert Figure 3 >
2.2.4. Molecular modeling study
To explore the possible interacting mode of the scutellarein-O-alkylamine derivatives with Torpedo
californica (Tc)AChE (PDB code: 1EVE), a molecular modeling study was performed using the
8

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docking program named AUTODOCK 4.2 package with Discovery Studio 2.1 [27]. As shown in
Figure 4, the results indicated that 16d occupied the entire enzymatic CAS, the mid-gorge sites and
the PAS, and could simultaneously bind to both the catalytic site and the peripheral site thus
providing an explanation for its highly potent inhibitory activity against AChE. In the 16d-TcAChE
complex, the N-(2-methoxybenzyl)ethanamine moiety of 16d was observed to bind to the CAS via
a π-π interaction with Trp84 and potential hydrophobic interactions with residues Tyr130, Asn85,
Ser81, Asp72, Gly123, Gly117, Gly118 and Ser122. The flavonoid moiety occupied the PAS of
AChE, the benzene ring of flavonoid moiety in 16d binding to the PAS via a π-π interaction with
Trp279 and the benzene ring at 2-position of flavonoid nucleus could bind to the mid-gorge sites via
a π-π interaction with Tyr334. The 5-hydroxy and 4-carbonyl group at the flavonoid nucleus could
simultaneously bind to the Arg289 via two intermolecular hydrogen bonds, and the 4-chromanones
moiety possessed potential hydrophobic interaction with residues Ser286, Leu282, Ile287 and
Phe290. In addition, the long chain of methylene and the benzene ring at the 2-position of flavonoid
nucleus could fold into a conformation in the gorge that allowed them to interact with Tyr70,
Tyr121, Phe330 and Phe331 through hydrophobic interaction. The results of the docking also
explained why 5-methoxy substituted derivatives showed higher AChE inhibition activity than that
5-hydroxyl substituted ones. It might be because the amino acid residues taking part in hydrogen
bond interaction were different. As shown in Fig 4, 16d took part in hydrogen bond interaction with
Arg289, while 15j formed hydrogen bond interaction with Phe288, a very important amino acid of
the acyl pocket of AChE [28]. Another explanation for this phenomenon, that as with donepezil
which has no direct hydrogen bond interaction, a water bridged hydrogen bonding may have
occurred under biological circumstances between the 5-methoxy of compound 15j and TcAChE
[
29].
<
Insert Figure 4 >
2.2.5. Metal-chelating properties of compounds 15j and 16d
2
+
2+
2+
The complexing ability of compounds 15j and 16d toward biometals such as Cu , Fe , Zn and
9

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3
+
Al was studied by UV-visual spectrometry, and the results are shown in Figure 5. The electronic
spectra of 16d demonstrated a red shift (the peak at 330 nm shifted to 337 nm) after the addition of
2
+
CuCl . The result indicated that compound 16d could interact effectively with the Cu . Similar
2
results were also obtained when AlCl was added, the peak in the electronic spectra of 16d at 330
3
nm shift to 340 nm. When FeSO and ZnCl were added, the electronic spectra of 16d showed no
4
2
obvious change. Therefore, 16d has the selectivity of metal chelation. However, when the same
experiment was performed using compound 15j and biometal ions, no significant differences were
observed in the UV spectrum (see Supporting Information), suggesting little or no complex
2+
3+
2+
2+
formation between 15j with Cu , Al , Fe or Zn . The results revealed that the 5-hydroxyl and
4-carbonyl group of the flavonoid nucleus may contribute to the chelation.
2+
3+
The observed selectivity of the compound 16d toward Cu compared with Fe may have
2+
interesting therapeutic applications. Several studies have shown that Cu contributes more to the
formation of senile plaques than iron does because of its superior affinity for Aβ1-42 [30]. In addition,
2+
the complex Aβ1-42-Cu produces more reactive oxygen species due to its higher reduction
3
+
potential (500 mV) than the corresponding complex with Fe [31]. Therefore, the selective
2
+
complexation of Cu by the compound 16d could simultaneously stop the formation of amyloid
plaques and relieve oxidative stress.
<
Insert Figure 5 >
2
+
The stoichiometry of the 16d-Cu complex was determined using the molar ratio method, by
preparing the solution of compound 16d with ascending amounts of CuCl . The UV spectra were
2
recorded and treated by numerical subtraction of CuCl and 16d at corresponding concentrations at
2
337nm. As shown in Figure 6, the absorbance linearly increased initially and then plateaued. The
two straight lines intersected at a mole fraction of 0.95, indicating a 1:1 stoichiometry for the
2
+
complex 16d-Cu .
<
Insert Figure 6 >
2.2.6. Effects on the self-induced Aβ1-42 aggregation and HuAChE-induced Aβ1-40 aggregation
1
0

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A number of dual binding site AChE inhibitors have been found to exhibit a significant inhibitory
activity on Aβ self-aggregation [32,33]. Therefore, some compounds (15d, 15g, 15h, 15i, 15j, 15p,
16b, 16d and 16f) that showed good potency for AChE inhibition were selected to assess their
ability to inhibit self-induced Aβ1-42 aggregation via the thioflavin T fluorescence method [18],
using curcumin, a known active natural product that inhibits self-induced Aβ aggregation, as a
reference compound. As summarized in Table 2, the tested compounds showed moderate
(31.3–37.1%) to good (66.5–71.4%) inhibitory effect, compared with curcumin (43.1%). The
marketed AD drug, donepezil, did not show any significant inhibitory activity under the same
experimental conditions. The results also indicated that the 5-hydroxy at flavonoid nucleus (16b,
1
6d, 16f) showed remarkably higher inhibitory activity than that of 5-methoxyl (15d, 15g, 15h, 15i,
5j and 15p), the inhibitory activity increased slightly as the length of the side chain carbon spacer
1
increased, for example, 15d (32.1%) < 15j (34.3%) < 15p (35.6%); 16b (66.5%) < 16d (69.1%) <
6f (71.4%), and different tertiary amine units on the side chain had little influence on the inhibitory
1
activity. The results revealed that the hydrogen bonds are crucial for the interactions between ligand
and proteins. Compound 16d also showed a dose-dependent inhibitory effect on self-induced Aβ1-42
aggregation (43.5±0.8% at 12.5ꢀM, 69.1±1.8% at 25ꢀM, 87.2±2.6% at 50ꢀM).
Numerous studies have revealed that AChE played an important role not only in the hydrolysis of
ACh, but also in promoting the aggregation of Aβ into amyloid fibrils [33]. Furthermore, the PAS of
AChE can bind to Aβ, accelerating the formation of amyloid fibrils [34]. Therefore, inhibition of
AChE, particularly the inhibition of PAS of AChE, may affect Aβ aggregation. The kinetic study
showed that compound 16d exhibited a mix-type inhibition and was able to bind to both the
catalytic and peripheral site of AChE. Therefore, to further explore the dual action, compound 15j
and 16d were selected to assess their ability to inhibit the HuAChE-induced Aβ1-40 aggregation via
the thioflavin T fluorescence method [18], using donepezil as the reference compound. As
summarized in Table 2, compounds 15j and 16d (all at 100 ꢀM) prevented HuAChE-induced Aβ1-40
aggregation by 37.0% and 62.2%, respectively, which was higher than that of donepezil (18.8%). It
11

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revealed that 15j and 16d may had higher affinity for PAS than donepezil. The results also showed
that compound 16d has about two-fold more effective than that of 15j, indicating that 5-hydroxy at
flavonoid nucleus played an important role in binding to PAS of AChE.
<
Insert Table 2 >
2
+
2
.2.7. Effects on Cu -induced Aβ aggregation and disaggregation
1
-42
2
+
To investigate the effects of the derivatives on Cu -induced Aβ
aggregation, we carried out two
-42
1
2
+
individual studies (Table 2): The inhibitory activity of Cu -induced Aβ
aggregation by the
-42
1
compounds (15d, 15g, 15h, 15i, 15j, 15p, 16b, 16d and 16f) and the disaggregation effects of two
2
+
representative compounds (15j and 16d) on Cu -induced Aβ
aggregation, using Thioflavin T
-42
1
(ThT) binding assay with curcumin as the reference compound [35]. The degree of Aβ1-42
aggregation was probed mainly by transmission electron microscopy (TEM) [35]. The Aβ1-42
2
+
peptide (25ꢀM) was first treated with one equivalent of Cu for 2 min at room temperature and
2
+
then incubated with or without the testing compounds for 24 h at 37°C. Cu could accelerate the
aggregation of Aβ. In contrast, the fluorescence was markedly reduced by 84.8± 1.1%, 85.7±2.5%,
2
+
or 87.2± 1.7% when Aβ1-42 was treated with Cu and compounds 16b, 16d, or 16f, respectively,
which exhibited more potency than that of curcumin (58.0±2.3%). However, the other tested
compounds showed lower inhibition potent than that of curcumin (Table 2). Donepezil did not
show any inhibitory activity under the same experimental conditions. Structure-activity relationship
analysis indicated that the long carbon chain side-chain at flavonoid 4′-position might increase the
inhibition activity, for example 15d < 15j < 15p or 16b < 16d < 16f. Moreover, compound 16d
2
+
significantly inhibited Cu -induced Aβ
aggregation in a concentration-dependent manner
-42
1
(46.5±3.5% at 5ꢀM, 64.6±1.2% at 10ꢀM and 85.7±2.5% at 25ꢀM). Interestingly, the rate of
inhibition of 16d showed nearly two-fold greater potency than that of 15j (46.8±1.5%). It revealed
that the 5-hydroxyl and 4-carbonyl at the flavonoid nucleus were key structural units to chelate with
2
+
Cu . Further, the results of TEM are consistent with ThT binding assay results. More well-defined
2
+
Aβ aggregates were observed in the presence of Cu than Aβ alone, and fewer Aβ fibrils were
1
2

ACCEPTED MANUSCRIPT
observed when compound 16d was added to the samples (Figure 7). These observations suggest
that the chelating properties of the tested compounds might be crucial to changing the structural
organization of Aβ aggregation fibrils.
<
Insert Figure 7 >
For the disaggregation studies, compound 15j or 16d (25ꢀM) were added to Aβ fibrils, which were
2
+
generated by reacting Aβ1-42 with one equivalent of Cu for 24 h at 37°C. The ThT binding assay
showed that 15j exhibited lower disaggregation potency (41.3±1.7%) than that of curcumin
(56.5±2.1%), but 16d manifested good disaggregation potency (81.3±3.4%) (Table 2). The TEM
figures illustrated that the sample of Aβ1-42 alone had aggregated into amyloid fibrils after 24 h of
incubation, while only small bulk aggregates were visible and no characteristic fibrils were
observed after adding 16d (Figure 8). This suggests that 16d could decompose the structure of
2
+
Cu -mediated Aβ aggregation fibrils. In summary, we can conclude that compound 16d can inhibit
2
+
Cu -induced Aβ aggregation and disassemble the well-structured Aβ fibrils.
Insert Figure 8 >
<
2.2.8. Cytotoxic effects on SH-SY5Y cells
To examine the cytotoxicity of the representative compounds 15j and 16d, human neuronal cell line
SH-SY5Y cells were exposed to the test compounds at three different concentrations (1, 10 and
100ꢀM) for 24 h and the cell viability was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium (MTT) assays [36]. As shown in Figure 9, 15j and 16d did not show modified
cell viability up to the concentration of 10ꢀM. With increased concentration to 100ꢀM, 15j or 16d
induced a decrease of cell viability (73% and 80.8%, respectively), it may be that the compound
with a 5-methoxy at flavonoid nucleus exhibited more cytotoxicity than that of a 5-hydroxy. These
results showed that the target compounds had a wide therapeutic safety range. Moreover, these
preliminary results should encourage further studies to elucidate the neuroprotective mechanisms of
15j and 16d.
<
Insert Figure 9 >
1
3

ACCEPTED MANUSCRIPT
2
.2.9. Cell protective effects on H O induced PC12 cell injury
2 2
AD brains exhibit constant evidence of reactive oxygen species (ROS) and reactive nitrogen species
2
+
(
RNS) mediated injury. These, like H O in the presence of transition metals, such as Fe , generate
2 2
.
hydroxyl radicals (OH ) which can feed into a self-propagating cascade of neurodegenerative events
22]. The protective effects of 15j and 16d against free radicals damage were assessed by measuring
the ability of the compounds to protect against H O injury according to the reported protocol [37].
[
2
2
After 100 ꢀM H O exposure, cell viability as determined by MTT reduction was markedly
2
2
decreased to 56.1 % (P < 0.01 vs control), suggesting high sensitivity to H O -induced injury.
2
2
However, compounds 15j and 16d showed protective effects in a dose-dependent manner against
hydrogen peroxide-induced PC12 cell injury (Figure 10). At concentrations of 10.0 ꢀM,
compounds 15j and 16d exhibited significantly neuroprotective effects and the cell viabilities were
60.3% and 75.1%, respectively. When the concentration is reduced to 1.0 ꢀM, these cell viabilities
decreased to 58.7% and 65.9%, respectively. It suggested the compound with a 5-hydroxy on the
flavonoid nucleus had higher cell survival ratio than that with a 5-methoxy group. This may be
associated with the ease which the 5-hydroxy can capture the hydroxyl radical, generated by H O .
2
2
<
Insert Figure 10 >
2.2.10. In vivo assay
Scopolamine is a muscarinic receptor antagonist that inhibits central cholinergic neuronal activity
and influences the expression of a broad spectrum of genes associated with muscarinic receptor
signaling pathways, apoptosis, and cell differentiation in rat brain [38]. Scopolamine-induced
amnesia has been used extensively to evaluate potential therapeutic agents for treating AD. To
determine whether the treatment of 16d improve the contextual memory in scopolamine-induced
mice, we performed the step-down passive avoidance tests [39]. As shown in Figure 11, the latency
of mice treated with scopolamine alone (1 mg/kg, control group) showed no significant differences
compared with vehicle-treated mice (normal group), when the dose of scopolamine up to 3 mg/kg,
the step-down latency of mice treated with scopolamine alone (control group) was significantly
1
4

ACCEPTED MANUSCRIPT
shorter than that of vehicle-treated mice (normal group) (p < 0.01). Moreover, we found that the
latency of mice showed no significant differences between the treatment with donepezil group (0.5
mg/kg, positive group) and scopolamine alone (3 mg/kg, control group), while treatment with
donepezil group (5 mg/kg) showed longer latency time than the control group with scopolamine (3
mg/kg) (p < 0.01) and clearly reversed the cognitive deficit induced by scopolamine. This was in
agreement with our previous work and other groups in which similar doses also increased the
latency time [18, 40]. According to the screening data, treatment with compound 16d (0.66, 2.2, 6.6
and 19.8 mg/kg) increased the latency time in a dose-dependent manner, but the medium dose group
(6.6 mg/kg) presented the longest latency time (136.2 sec) (p < 0.01) of the four dose groups. The
medium dose (6.6 mg/kg) of compound 16d showed slightly longer latency time than the mole
equivalent of donepezil (5 mg/kg) (132.8 sec), which was a representative AChE inhibitor and had
been approved by FDA for treating AD. Meanwhile, the latency time of the high dose of 16d (19.8
mg/kg) demonstrated slightly shorter (118.2 sec) than that of the medium dose and donepezil (5
mg/kg). The reason maybe that the high dose has some neurotoxicity, the medium dose may close to
full saturate AChE and would be the optimal dose. The latency time of the lowest dose of 16d (0.66
mg/kg) demonstrated no significant differences compared with scopolamine alone (3 mg/kg, control
group). Therefore, the optimal dose of 16d might be 6.6 mg/kg. These results indicated that
compound 16d might not only have reversed cognitive deficit induced by scopolamine through
increasing brain cholinergic activity due to the inhibition of AChE but also revealed some other
properties influencing memory processes.
<
Insert Figure 11 >
3. Conclusion
In summary, to explore for effective drugs for the treatment of Alzheimer’s disease (AD), a
novel series of scutellarein-O-alkylamine derivatives 15–18 were designed, synthesized and
evaluated as multi-target anti-Alzheimer agents. Most of the synthesized compounds were potent in
1
5

ACCEPTED MANUSCRIPT
inhibiting AChE activity in vitro and displayed high selectivity for AChE over BuChE. Compound
1
6d demonstrated a useful inhibitory activity toward AChE with IC value of 0.62±0.01 ꢀM. The
5
0
kinetic analysis suggested that 16d showed mixed-type inhibition, and could bind to both CAS and
PAS of AChE, which was consistent with the molecular modeling study. Compound 16d also
possessed the modest antioxidant activity (0.84 eq of Trolox), excellent inhibitory effects on
2
+
self-induced Aβ1-42 aggregation (69.1±1.8%), Cu -induced Aβ
aggregation (85.7±2.5%) and
1-42
HuAChE-induced Aβ1-40 aggregation (62.2±1.8%). Moreover, 16d was found to be able to bind
2
+
3+
2+
Cu and Al and assemble into metal complexes, which disaggregated Cu -induced Aβ1
-42
aggregation (81.3±3.4%). Furthermore, 16d had a good neuroprotective effect against
H O -induced PC12 cell injury and exhibited a low toxicity in SH-SY5Y cells. More interestingly,
2
2
our in vivo study proved that mice treated with 16d (6.6 mg/kg, p.o.) displayed slightly longer
step-down latency time than the drug control group donepezil (5.0 mg/kg, p.o.) in the step-down
passive avoidance test. The present study indicated that 16d could be considered as an interesting
multi-targeted lead compound for the further study in the treatment of AD. Further studies to
evaluate compound 16d in vivo and to develop structural refinements are underway and will be
reported in due course.
4. Experimental section
4.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and used without
further purification. The purity of all final compounds was determined by high-performance liquid
chromatography (HPLC) analysis to be over 96%. HPLC analysis was carried out on a Shimadzu
1
LC-10Avp plus system with the use of a Kromasil C column (4.6 mm × 250 mm, 5um). H and
1
8
1
3
C NMR spectra were recorded using TMS as the internal standard in CDCl or DMSO-d with a
3
6
Varian INOVA spectrometer at 400 and 100 MHz, respectively. Coupling constants were given in
Hz. Multiplicities are given as s (singlet), d (doublet), dd (double-doublet), t (triplet), q (quadruplet),
1
6

ACCEPTED MANUSCRIPT
m (multiplet), and br (broad signal). MS spectra data were obtained on an Agilent-6210 TOF
LC-MS spectrometer. Melting points were recorded on YRT-3 melting-point apparatus (China) and
are uncorrected. All the reactions were monitored by thin-layer chromatography (TLC) using silica
gel GF254 plates from Qingdao Haiyang Chemical Co. Ltd. (China) with an UV lamp (254nm).
Column chromatography using silica gel (230–400 mesh) purchased from Qingdao Haiyang
Chemical Co. Ltd. (China).
4
.1.1. (E)-1-(6-Hydroxy-2,3,4-trimethoxyphenyl)-3-(4-methoxymethoxyphenyl)-2-propen-1-one (4).
To a stirred solution of 6-hydroxy-2,3,4-trimethoxyacetophenone (10.0 g, 44.0 mmol) and
-methoxymethoxybenzaldehyde (8.10 g, 49.0 mmol) in ethanol (120 mL) was added a solution of
4
KOH (7.4 g, 0.14 mol) in water (7.4 mL). The reaction mixture was stirred for 72 h at room
temperature and quenched in ice-cold water, acidified with 10% HCl, filtered and washed with
water. The crude product was recrystallized with alcohol to afford the title product as yellow solid
1
(
14.9 g, 90% yield), mp 94.2-94.5 °C. H NMR (400 MHz, CDCl ) δ 13.78 (s,1H, OH), 7.87 (d, J =
3
16.0 Hz, 1H, =CH), 7.81 (d, J = 16.0 Hz, 1H, =CH), 7.60 (d, J = 8.8Hz, 2H, Ar-H), 7.08 (d, J =
8
.8Hz, 2H, Ar-H), 6.30 (s, 1H, Ar-H), 5.23 (s, 2H, OCH O), 3.93 (s, 3H, OCH ), 3.91 (s, 3H,
2
3
OCH ), 3.84 (s, 3H, OCH ), 3.50 (s, 3H, OCH ).
3
3
3
4.1.2. (E)-1-(6-Hydroxy-2,3,4-trimethoxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one (5). To a
solution of compound 4 (3.74 g, 10.0 mmol) and 10%HCl (6.0 mL) in ethanol (8.0 mL), the mixture
was heated for 4 h at 50 °C, cooled to room temperature. The reaction mixture was poured into
crushed ice. The resulting precipitate was filtered and washed with water. The crude product was
1
recrystallized with alcohol to give yellow solid (3.0 g, 91% yield), mp 146.2-147.2 °C. H NMR
(
400 MHz, CDCl ) δ 13.79 (s,1H, OH), 7.86 (d, J = 16.0 Hz, 1H, Ar-H), 7.81 (d, J = 16.0 Hz, 1H,
3
Ar-H), 7.54 (d, J = 8.4Hz, 2H, Ar-H), 6.88 (d, J = 8.4Hz, 2H, Ar-H), 6.30 (s, 1H, Ar-H), 5.41 (brs,
1
H, OH), 3.93 (s, 3H, OCH ), 3.90 (s, 3H, OCH ), 3.84 (s, 3H, OCH ).
3 3 3
4.1.3. (E)-1-(6-Hydroxy-2,3,4-trimethoxyphenyl)-3-(3-hydroxyphenyl)-2-propen-1-one (6). To a
stirred solution of 6-hydroxy-2,3,4-trimethoxyacetophenone (5.0 g, 22.0 mmol) and
1
7

ACCEPTED MANUSCRIPT
m-hydroxybenzaldehyde (3.3 g, 26.4 mmol) in ethanol (40 mL) was added a solution of KOH (3.7 g,
66.0 mmol) in water (6.0 mL). The reaction mixture was stirred for 72 h at room temperature and
quenched in ice-cold water, acidified with 10% HCl, filtered and washed with water. The crude
product was recrystallized with alcohol to afford the title product as yellow solid (6.1 g, 83.0%
1
yield), mp 87.8-88.9 °C. H NMR (400 MHz, CDCl ) δ 13.67 (brs,1H, OH), 7.91 (d, J = 15.6 Hz,
3
1H, =CH), 7.75 (d, J = 15.6 Hz, 1H, =CH), 7.29 (t, J = 8.0 Hz, 1H, Ar-H), 7.21 (d, J = 7.6 Hz, 1H,
Ar-H), 7.11 (s, 1H, Ar-H), 6.88 (dd, J = 1.6 Hz, 7.6Hz, 1H, Ar-H), 6.29 (d, J = 5.6 Hz, 1H, Ar-H),
5
4
1
.03 (brs, 1H, OH), 3.92 (s, 3H, OCH ), 3.90 (s, 3H, OCH ), 3.83 (s, 3H, OCH ).
3 3 3
.1.4. General procedure for the synthesis of intermediates 7a-b. Chalcones compound 5 or 6 (3.6 g
0.8 mmol), potassium iodide (70 mg, 0.42 mmol) were added to a solution of conc. H SO (0.2 mL)
2
4
in DMSO (16 mL). The solution was heated to 100 °C for 3-5 h, and then the reaction mixture was
poured into crushed ice. The resulting precipitate was collected and washed with 5% sodium
thiosulfate solution (50 mL) and water. Recrystallization from 80% ethanol afforded the flavones
compound 7a-b, respectively.
4
2
7
3
4
2
7
.1.4.1. 4′-Hydroxy-5,6,7-trimethoxyflavone (7a). Light yellow solid; 84.0 % yield; mp
1
24-225.4 °C. H NMR (400 MHz, DMSO-d ) δ 10.25 (s, 1H, OH), 7.91 (d, J = 8.4 Hz, 2H, Ar-H),
6
.19 (s, 1H, Ar-H), 6.92 (d, J = 8.4 Hz, 2H, Ar-H), 6.64 (s, 1H, CH), 3.95 (s, 3H, OCH ), 3.80 (s,
3
H, OCH ), 3.76 (s, 3H, OCH ).
3
3
.1.4.2. 3′-Hydroxy-5,6,7-trimethoxyflavone (7b). Light yellow solid; 90.0 % yield; mp
1
27-228.8 °C. H NMR (400 MHz, DMSO-d ) δ 9.85 (s,1H, OH), 7.48 (d, J = 7.6 Hz, 1H, Ar-H),
6
.40 (s, 1H, Ar-H), 7.36 (t, J = 8.0 Hz, 1H, Ar-H), 7.20 (s, 1H, CH), 6.98 (dd, J = 2.0, 8.0 Hz, 1H,
Ar-H), 6.69 (s, 1H, Ar-H), 3.96 (s, 3H, OCH ), 3.81 (s, 3H, OCH ), 3.77 (s, 3H, OCH ).
3
3
3
4
.1.5. General procedure for the synthesis of intermediates 8a-b. Anhydrous AlCl (1.62 g, 12.1
3
mmol) was added to a solution of compound 7a-b (2.0 g, 6.1 mmol) in anhydrous acetonitrile (20
mL) at room temperature. The mixture was heated to 55-60 °C for 1 h, then added 10% HCl (20 mL)
and refluxed for 40 min. Then the mixture was poured into ice water (50 mL). The resulting
1
8

ACCEPTED MANUSCRIPT
precipitate was collected and washed with water. The residue was recrystallized with
methoxyethanol to afford compounds 8a-b, respectively.
4.1.5.1. 5,4′-Dihydroxy-6,7-dimethoxyflavone (8a). Light yellow solid; 88.0 % yield; mp
1
2
61-262.3 °C. H NMR (400 MHz, DMSO-d ) δ 12.94 (s, 1H, OH), 10.42 (s, 1H, OH), 7.98 (d, J
6
=8.8 Hz, 2H, Ar-H), 6.94 (d, J = 8.8Hz, 2H, Ar-H), 6.93 (s, 1H, Ar-H), 6.87 (s, 1H, CH), 3.93 (s, 3H,
OCH ), 3.74 (s, 3H, OCH ).
3
3
4
2
8
.1.5.2. 5,3′-Dihydroxy-6,7-dimethoxyflavone (8b). Light yellow solid; 85.0 % yield; mp
1
10-211.4 °C. H NMR (400 MHz, DMSO-d ) δ 12.77 (s, 1H, OH), 9.85 (s, 1H, OH), 7.53 (d, J =
6
.0 Hz, 1H, Ar-H), 7.45-7.42 (m, 1H, Ar-H), 7.38 (t, J = 8.0 Hz, 1H, Ar-H), 7.03 (d, J = 8.0 Hz, 1H,
Ar-H), 6.94 (s, 1H, CH), 6.90 (s, 1H, Ar-H), 3.95 (s, 3H, OCH ), 3.75 (s, 3H, OCH ).
3
3
4.1.6. General procedure for the synthesis of intermediates 10-13. The appropriate dibromoalkane
derivative 9 (6.6 mmol) was added to a mixture of the intermediate 7a-b or 8a-b (3.0 mmol),
anhydrous K CO (430 mg, 3.1 mmol) and KI (53 mg, 0.32mmol) in CH CN (30 ml). The reaction
2
3
3
mixture was warmed to 60-65 °C and stirred for 8-10 h under an argon atmosphere. After complete
reaction, the solvent was evaporated under reduced pressure. Water (30 mL) was added to the
residue and the mixture was extracted with dichloromethane (30 mL×3). The combined organic
phases were washed with saturated aqueous sodium chloride, dried over sodium sulfate, and filtered.
The solvent was evaporated to dryness under reduced pressure. The residue was purified on a silica
gel chromatography using petroleum ether/acetone (15:1) as eluent to give the intermediates 10-13.
4
1
2
.1.6.1. 4′-(3-Bromopropoxy)-5,6,7-trimethoxyflavone (10a). Light yellow solid; 68.7 % yield; mp
1
42.1-143.2 °C. H NMR (400 MHz, CDCl ) δ 7.83 (d, J = 8.8 Hz, 2H, Ar-H), 7.02 (d, J = 8.8 Hz,
3
H, Ar-H), 6.80 (s, 1H, =CH), 6.59 (s, 1H, Ar-H), 4.19 (t, J = 6.0 Hz, 2H, OCH ), 3.99 (s, 3H,
2
OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.63 (t, J = 6.4 Hz, 2H, CH Br), 2.39-2.34 (m, 2H,
3
3
3
2
CH2).
4.1.6.2. 4′-(4-Bromobutoxy)-5,6,7-trimethoxyflavone (10b). Light yellow solid; 74.3 % yield; mp
1
1
57.3-158.4 °C. H NMR (400 MHz, CDCl ) δ 7.82 (d, J = 8.8 Hz, 2H, Ar-H), 6.99 (d, J = 8.8 Hz,
3
1
9

ACCEPTED MANUSCRIPT
2
H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H, Ar-H), 4.08 (t, J = 6.0 Hz, 2H, OCH ), 3.99 (s, 3H,
2
OCH ), 3.98(s, 3H, OCH ), 3.92(s, 3H, OCH ), 3.51(t, J = 6.4 Hz, 2H, CH Br), 2.11-2.07(m, 2H,
3
3
3
2
CH ), 2.01-1.97(m, 2H, CH ).
2
2
4.1.6.3. 4′-(6-Bromohexyloxy)-5,6,7-trimethoxyflavone (10c). Light yellow solid; 72.8 % yield, mp
1
1
17.3-118.2 °C. H NMR (400 MHz, CDCl ) δ 7.83 (d, J = 8.8 Hz, 2H, Ar-H), 6.99 (d, J = 8.8 Hz,
3
2
H, Ar-H), 6.80 (s, 1H, =CH), 6.64 (s, 1H, Ar-H), 4.04 (t, J = 6.4 Hz, 2H, OCH ), 3.99 (s, 3H,
2
OCH ), 3.99 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.44 (t, J = 6.8 Hz, 2H, CH Br), 1.92-1.88 (m, 2H,
3
3
3
2
CH ), 1.87-1.83 (m, 2H, CH ), 1.56-1.52 (m, 4H, CH ).
2
2
2
4.1.6.4. 4′-(3-Bromopropoxy)-5-hydroxy-6,7-dimethoxyflavone (11a). Yellow solid; 63.7 % yield;
1
mp 143.1-144.5 °C. H NMR (400 MHz, CDCl ) δ 12.79 (brs, 1H, OH), 7.85 (d, J = 8.8 Hz, 2H,
3
Ar-H), 7.03 (d, J = 9.2 Hz, 2H, Ar-H), 6.59 (s, 1H, =CH), 6.55 (s, 1H, Ar-H), 4.20 (t, J = 6.0 Hz, 2H,
OCH ), 3.97 (s, 3H, OCH ), 3.93 (s, 3H, OCH ), 3.63 (t, J = 6.4 Hz, 2H, CH Br), 2.39-2.34 (m, 2H,
2
3
3
2
CH2).
4
1
7
.1.6.5. 4′-(4-Bromobutoxy)-5-hydroxy-6,7-dimethoxyflavone (11b). Yellow solid; 68.4 % yield; mp
1
49.1-150.5 °C. H NMR (400 MHz, CDCl ) δ 12.80 (brs, 1H, OH), 7.84 (d, J = 9.2 Hz, 2H, Ar-H),
3
.00 (d, J = 9.2 Hz, 2H, Ar-H), 6.59 (s, 1H, =CH), 6.55 (s, 1H, Ar-H), 4.09 (t, J = 6.0 Hz, 2H,
OCH ), 3.97 (s, 3H, OCH ), 3.93 (s, 3H, OCH ), 3.51 (t, J = 6.4 Hz, 2H, CH Br), 2.12-2.08 (m, 2H,
2
3
3
2
CH ), 2.02-1.98 (m, 2H, CH ).
2
2
4.1.6.6. 4′-(6-Bromohexyloxy)-5-hydroxy-6,7-dimethoxyflavone (11c). Yellow solid; 67.4 % yield;
1
mp 133.3-134.5 °C. H NMR (400 MHz, CDCl ) δ 12.80 (brs, 1H, OH), 7.84 (d, J = 8.8 Hz, 2H,
3
Ar-H), 7.00 (d, J = 8.8Hz, 2H, Ar-H), 6.59 (s, 1H, =CH), 6.55 (s, 1H, Ar-H), 4.05 (t, J = 6.4 Hz, 2H,
OCH ), 3.97 (s, 3H, OCH ), 3.93 (s, 3H, OCH ), 3.44 (t, J = 6.8 Hz, 2H, CH Br), 1.94-1.90 (m, 2H,
2
3
3
2
CH ), 1.87-1.83 (m, 2H, CH ), 1.56-1.52 (m, 2H, CH ).
2
2
2
4.1.6.7. 3′-(2-Bromoethoxy)-5,6,7-trimethoxyflavone (12a). Light yellow solid; 67.3 % yield; mp
1
9
4.5-95.9 °C. H NMR (400 MHz, CDCl ) δ 7.49 (d, J = 8.0 Hz, 1H, Ar-H), 7.43 (t, J = 8.0 Hz, 2H,
3
Ar-H), 7.07 (d, J = 8.0 Hz 1H, Ar-H), 6.83 (s, 1H, =CH), 6.66 (s, 1H, Ar-H), 4.38 (t, J = 6.0 Hz, 2H,
2
0

ACCEPTED MANUSCRIPT
OCH ), 4.00 (s, 6H, 2 × OCH ), 3.93 (s, 3H, OCH ), 3.70 (t, J = 6.0 HZ, 2H, CH Br).
2
3
3
2
4.1.6.8. 3′-(3-Bromopropoxy)-5,6,7-trimethoxyflavone (12b). Light yellow solid; 70.6 % yield; mp
1
8
9.7-90.4 °C. H NMR (400 MHz, CDCl ) δ 7.47 (d, J = 8.0 Hz, 1H, Ar-H), 7.43-7.39 (m, 2H,
3
Ar-H), 7.06 (dd, J = 1.6, 8.0 Hz, 1H, Ar-H), 6.83 (s, 1H, =CH), 6.74 (s, 1H, Ar-H), 4.19 (t, J = 6.4
Hz, 2H, OCH ), 3.99 (s, 6H, 2 × OCH ), 3.92 (s, 3H, OCH ), 3.64 (t, J = 6.4 Hz, 2H, CH Br),
2
3
3
2
2
4
1
1
.39-2.34 (m, 2H, CH2).
.1.6.9. 3′-(4-Bromobutoxy)-5,6,7-trimethoxyflavone (12c). Light yellow solid; 67.6 % yield; mp
1
08.2-109.1 °C. H NMR (400 MHz, CDCl ) δ 7.45 (d, J = 8.0 Hz, 1H, Ar-H), 7.42 (d, J = 8.0 Hz,
3
H, Ar-H), 7.40-7.36 (m, 1H, Ar-H), 7.04 (dd, J = 1.6, 8.0 Hz, 2H, Ar-H), 6.82 (s, 1H, =CH), 6.67
(
s, 1H, Ar-H), 4.08 (t, J = 6.4 Hz, 2H, OCH ), 3.99 (s, 6H, 2 × OCH ), 3.93 (s, 3H, OCH ), 3.52 (t, J
2 3 3
=
6.4 Hz, 2H, CH Br), 2.13-2.09 (m, 2H, CH ), 2.02-1.98 (m, 2H, CH ).
2 2 2
4
1
2
6
1
4
.1.6.10. 3′-(6-Bromohexyloxy)-5,6,7-trimethoxyflavone (12d). Light yellow solid; 71.4 % yield; mp
1
28.9-130.2 °C. H NMR (400 MHz, CDCl ) δ 7.45 (d, J = 8.0 Hz, 1H, Ar-H), 7.41 (d, J = 8.0 Hz,
3
H, Ar-H), 7.04 (dd, J = 1.6, 8.0 Hz, 1H, Ar-H), 6.82 (s, 1H, =CH), 6.69 (s, 1H, Ar-H), 4.04 (t, J =
.4 Hz, 2H, OCH ), 3.99 (s, 6H, 2 × OCH ), 3.93 (s, 3H, OCH ), 3.45 (t, J = 6.8 Hz, 2H, CH Br),
2
3
3
2
.93-1.89 (m, 2H, CH ), 1.87-1.82 (m, 2H, CH ), 1.57-1.53 (m, 4H, CH ).
2
2
2
.1.6.11. 3′-(3-Bromopropoxy)-5-hydroxy-6,7-dimethoxyflavone (13a). Yellow solid; 68.7 % yield;
mp 138.8-139.9 °C. 1H NMR (400 MHz, CDCl ) δ 12.67 (brs, 1H, OH), 7.49-7.42 (m, 3H, Ar-H),
3
7.09 (dd, J = 1.2, 7.6 Hz, 1H, Ar-H), 6.67 (s, 1H, =CH), 6.58 (s, 1H, Ar-H), 4.21 (t, J = 5.6 Hz, 2H,
OCH ), 3.98 (s, 3H, OCH ), 3.93 (s, 3H, OCH ), 3.65 (t, J = 6.4 Hz, 2H, CH Br), 2.40-2.36 (m, 2H,
2
3
3
2
CH2).
4.1.6.12. 3′-(3-Bromobutoxy)-5-hydroxy-6,7-dimethoxyflavone (13b). Yellow solid; 63.4 % yield;
1
mp 115.5-116.9 °C. H NMR (400 MHz, CDCl ) δ 12.67 (brs, 1H, OH), 7.47 (dt, J = 1.6, 8.0 Hz,
3
1H, Ar-H), 7.43 (d, J = 8.0 Hz, 1H, Ar-H), 7.41-7.36 (m, 1H, Ar-H), 7.07 (dd, J = 1.6, 8.0 Hz, 1H,
Ar-H), 6.66 (s, 1H, =CH), 6.58 (s, 1H, Ar-H), 4.09 (t, J = 6.0 Hz, 2H, OCH ), 3.98 (s, 3H, OCH ),
2
3
3
.93 (s, 3H, OCH ), 3.52 (t, J = 6.4 Hz, 2H, CH Br), 2.14-2.10 (m, 2H, CH ), 2.03-1.99 (m, 2H,
3
2
2
2
1

ACCEPTED MANUSCRIPT
CH2).
4.1.6.13. 3′-(3-Bromohexyloxy)-5-hydroxy-6,7-dimethoxyflavone (13c). Yellow solid; 69.3 % yield;
1
mp 98.4-99.2 °C. H NMR (400 MHz, CDCl ) δ 12.68 (brs, 1H, OH), 7.46 (dt, J = 1.2, 8.0 Hz, 1H,
3
Ar-H), 7.43 (d, J = 8.0 Hz, 1H, Ar-H), 7.42-7.38 (m, 1H, Ar-H), 7.07 (dd, J = 1.2, 8.0 Hz, 1H, Ar-H),
6
3
1
4
.67 (s, 1H, =CH), 6.57 (s, 1H, Ar-H), 4.05 (t, J = 6.4 Hz, 2H, OCH ), 3.98 (s, 3H, OCH ), 3.93 (s,
2 3
H, OCH ), 3.47 (t, J = 6.4 Hz, 2H, CH Br), 1.94-1.90 (m, 2H, CH ), 1.88-1.84 (m, 2H, CH ),
3
2
2
2
.56-1.52 (m, 2H, CH2).
.1.7. General procedure for the synthesis of scutellarein-O-alkylamine derivatives 15–18 [18]. To a
mixture of the corresponding secondary amines 14A–F (0.6 mmol), anhydrous K CO (89.7 mg,
2
3
0
.65 mmol) and KI (8.6 mg, 0.052 mmol) in anhydrous CH CN (12 ml) were added the appropriate
3
intermediates 10–13 (0.5 mmol). The reaction mixture was warmed to 60-65 °C and stirred for 6–10
h under an argon atmosphere. After complete reaction, the solvent was evaporated under reduced
pressure. The residue was dissolved in water (40 mL) and the mixture was extracted with
dichloromethane (25 mL×3). The combined organic phases were washed with saturated aqueous
sodium chloride (20 mL), dried over sodium sulfate, and filtered. The solvent was evaporated to
dryness under reduced pressure. The residue was purified on a silica gel chromatography using
mixtures of CH Cl /CH OH (100:1) as eluent to afford the corresponding scutellarein-O-alkylamine
2
2
3
derivatives 15-18.
4
7
7
4
.1.7.1. 4′-(3-(Benzyl(methyl)amino)propoxy)-5,6,7-trimethoxyflavone (15a). Light yellow oil,
1
2.7 % yield, 97.6 % HPLC purity. H NMR (400 MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz, 2H, Ar-H),
3
.29-7.21 (m, 5H, Ar-H), 6.97 (d, J = 8.8 Hz, 2H, Ar-H), 6.80 (s, 1H, =CH), 6.59 (s, 1H, Ar-H),
.10 (t, J = 6.4 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.54
2
3
3
3
(
s, 2H, CH Ph), 2.58 (t, J = 6.8 Hz, 2H, CH N), 2.56 (s, 3H, CH N), 2.06-2.00 (m, 2H, CH );
2 2 3 2
+
HR-ESI-MS: Calcd. for C H NO [M+H] : 490.2230, found: 490.2225.
29
32
6
4.1.7.2. 4′-(3-(Benzyl(ethyl)amino)propoxy)-5,6,7-trimethoxyflavone (15b). Light yellow oil, 77.5 %
1
yield, 98.0 % HPLC purity. H NMR (400 MHz, CDCl ) δ 7.80 (d, J = 8.8 Hz, 2H, Ar-H), 7.34-7.26
3
2
2

ACCEPTED MANUSCRIPT
(
m, 5H, Ar-H), 6.95 (d, J = 8.8 Hz, 2H, Ar-H), 6.80 (s, 1H, =CH), 6.59 (s, 1H, Ar-H), 4.07 (t, J =
6
.4 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.62 (s, 2H,
2
3
3
3
CH Ph), 2.65 (t, J = 6.4 Hz, 2H, CH N), 2.56 (J = 6.8 Hz, 2H, CH N), 2.00-1.96 (m, 2H, CH ), 1.08
2
2
2
2
+
(
t, J = 6.4 Hz, 3H, CH ); HR-ESI-MS: Calcd. for C H NO [M+H] : 504.2386, found: 504.2391.
3 30 34 6
4.1.7.3. 4′-(3-((2-Methoxybenzyl)(methyl)amino)propoxy)-5,6,7-trimethoxyflavone (15c). Light
1
yellow oil, 87.1 % yield, 98.9% HPLC purity. H NMR (400 MHz, CDCl ) δ 7.80 (d, J = 8.8 Hz,
3
2
6
3
H, Ar-H), 7.41-7.35 (m, 1H, Ar-H), 7.29 (t, J = 7.2 Hz, 1H, Ar-H), 6.97 (d, J = 8.8 Hz, 2H, Ar-H),
.92-6.88 (m, 2H, Ar-H), 6.79 (s, 1H, =CH), 6.57 (s, 1H, Ar-H), 4.14 (t, J = 6.4 Hz, 2H, OCH2),
.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.91 (s, 3H, OCH ), 3.84 (s, 3H, OCH ), 3.81 (s, 2H,
3
3
3
3
CH Ph), 2.60-2.54 (m, 2H, CH N), 2.43 (s, 3H, CH N), 2.24-2.20 (m, 2H, CH ); HR-ESI-MS:
2
2
3
2
+
Calcd. for C H NO [M+H] : 520.2335, found: 520.2342.
3
0
34
7
4.1.7.4. 4′-(3-((Ethyl)(2-methoxybenzyl)amino)propoxy)-5,6,7-trimethoxyflavone (15d). Light
1
yellow oil, 72.6 % yield, 98.6% HPLC purity. H NMR (400 MHz, CDCl ) δ 7.80 (d, J = 8.8 Hz,
3
2
H, Ar-H), 7.43 (brs, 1H, Ar-H), 7.23 (t, J = 6.4 Hz, 1H, Ar-H), 6.96 (d, J = 8.8 Hz, 2H, Ar-H), 6.92
t, J = 7.2 Hz, 1H, Ar-H), 6.86 (d, J = 8.0 Hz, 1H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H, Ar-H),
.10 (t, J = 6.0 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.82
(
4
2
3
3
3
(s, 3H, OCH ), 3.69 (s, 2H, CH Ph), 2.74-2.70 (m, 2H, CH N), 2.65-2.61 (m, 2H, CH N), 2.06-2.02
3 2 2 2
+
(
m, 2H, CH ), 1.14-1.10 (m, 3H, CH ); HR-ESI-MS: Calcd. for C H NO [M+H] : 534.2492,
2
3
31 36
7
found: 534.2484.
4
7
7
4
.1.7.5. 4′-(3-(4-Benzylpiperazin-1-yl)propoxy)-5,6,7-trimethoxyflavone (15e). Light yellow oil,
1
6.1 % yield, 97.8 % HPLC purity. H NMR (400 MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz, 2H, Ar-H),
3
.35-7.31 (m, 5H, Ar-H), 6.99 (d, J = 8.8 Hz, 2H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H, Ar-H),
.10 (t, J = 6.0 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.55
2
3
3
3
(
s, 2H, CH Ph), 2.60-2.56 (m, 10H, CH N), 2.07-2.03 (m, 2H, CH ); HR-ESI-MS: Calcd. for
2 2 2
+
C H N O [M+H] : 545.2652, found: 545.2645.
32
37
2
6
4.1.7.6. 4′-(3-(4-Methylpiperazin-1-yl)propoxy)-5,6,7-trimethoxyflavone (15f). Light yellow oil,
2
3

ACCEPTED MANUSCRIPT
1
7
6
0.9 % yield, 99.2 % HPLC purity. H NMR (400 MHz, CDCl ) δ 7.80 (d, J = 8.8 Hz, 2H, Ar-H),
3
.99 (d, J = 8.8 Hz, 2H, Ar-H), 6.79 (s, 1H, =CH), 6.57 (s, 1H, Ar-H), 4.08 (t, J = 6.4 Hz, 2H,
OCH ), 3.98 (s, 3H, OCH ), 3.97 (s, 3H, OCH ), 3.91 (s, 3H, OCH ), 2.58-2.54 (m, 10H, CH N),
2
3
3
3
2
+
2
4
4
7
7
4
.33 (s, 3H, NCH ), 2.03-1.99 (m, 2H, CH ); HR-ESI-MS: Calcd. for C H N O [M+H] :
3
2
26 33
2
6
69.2339, found: 469.2336.
.1.7.7. 4′-(4-(Benzyl(methyl)amino)butoxy)-5,6,7-trimethoxyflavone (15g). Light yellow oil,
1
5.1 % yield, 97.5 % HPLC purity. H NMR (400MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz, 2H, Ar-H),
3
.33-7.26 (m, 5H, Ar-H), 6.97 (d, J = 8.8 Hz, 2H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H, Ar-H),
.03 (t, J = 6.0 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.56
2
3
3
3
(
s, 2H, CH Ph), 2.49 (t, J = 6.8 Hz, 2H, CH N), 2.25 (s, 3H, NCH ), 1.89-1.85 (m, 2H, CH ),
2 2 3 2
+
1
4
.77-1.73 (m, 2H, CH ); HR-ESI-MS: Calcd. for C H NO [M+H] : 504.2386, found: 504.2380.
2 30 34 6
.1.7.8. 4′-(4-(Benzyl(ethyl)amino)butoxy)-5,6,7-trimethoxyflavone (15h). Light yellow oil, 70.7 %
1
yield, 99.0% HPLC purity. H NMR (400MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz, 2H, Ar-H), 7.46-7.42
3
(m, 2H, Ar-H), 7.36-7.30 (m, 3H, Ar-H), 6.96 (d, J = 8.8 Hz, 2H, Ar-H), 6.80 (s, 1H, =CH), 6.59 (s,
1
H, Ar-H), 4.01 (t, J = 6.4 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H,
2
3
3
OCH ), 3.79 (s, 2H, CH Ph), 2.72-2.68 (m, 4H, CH N), 1.87-1.83 (m, 4H, CH ), 1.21 (t, J = 6.4 Hz,
3
2
2
2
+
3
H, CH ); HR-ESI-MS: Calcd. for C H NO [M+H] : 518.2543, found: 518.2552.
3 31 36 6
4.1.7.9. 4′-(4-((2-Methoxybenzyl)(methyl)amino)butoxy)-5,6,7-trimethoxyflavone (15i). Light
1
yellow oil, 77.5 % yield, 99.2 % HPLC purity. H NMR (400MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz,
3
2H, Ar-H), 7.36 (d, J = 7.6 Hz, 1H, Ar-H), 7.26 (t, J = 7.6 Hz, 1H, Ar-H), 6.98 (d, J = 8.8 Hz, 2H,
Ar-H), 6.94 (t, J = 7.6 Hz, 2H, Ar-H), 6.88 (d, J = 8.0 Hz, 1H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H,
Ar-H), 4.05 (t, J = 6.0 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ),
2
3
3
3
3
.83 (s, 3H, OCH ), 3.64 (s, 2H, CH Ph), 2.59-2.55 (m, 2H, CH N), 2.31 (s, 3H, NCH ), 1.90-1.86
3
2
2
3
+
(
m, 2H, CH ), 1.82-1.78 (m, 2H, CH ); HR-ESI-MS: Calcd. for C H NO [M+H] : 534.2492,
2
2
31 36
7
found: 534.2480.
.1.7.10. 4′-(4-((Ethyl)(2-methoxybenzyl)amino)butoxy)-5,6,7-trimethoxyflavone (15j). Light yellow
4
2
4

ACCEPTED MANUSCRIPT
1
oil, 72.7 % yield, 99.5% HPLC purity. H NMR (400MHz, CDCl ) δ 7.80 (d, J = 8.8 Hz, 2H, Ar-H),
3
7
.42 (d, J = 7.2 Hz, 1H, Ar-H), 7.20 (t, J = 6.8 Hz, 1H, Ar-H), 6.97 (d, J = 8.8 Hz, 2H, Ar-H), 6.92
t, J = 7.2 Hz, 1H, Ar-H), 6.86 (d, J = 8.0 Hz, 1H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H, Ar-H),
.01 (t, J = 6.4 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.82
(
4
2
3
3
3
(
s, 3H, OCH ), 3.62 (s, 2H, CH Ph), 2.57-2.53 (m, 4H, CH N), 1.86-1.84 (m, 2H, CH ), 1.71-1.67
3 2 2 2
1
3
(
m, 2H, CH ), 1.08 (t, J = 6.8 Hz, 3H, CH ); C NMR (100MHz, CDCl ) δ 177.2, 161.4, 161.1,
2 3 3
1
1
57.8, 157.5, 154.4, 152.4, 140.2, 131.3, 129.1, 127.5, 123.6, 120.6, 114.9, 114.8, 112.7, 110.4,
06.9, 96.2, 67.7, 62.1, 61.5, 56.2, 55.4, 52.2, 50.7, 47.4, 26.8, 22.4, 10.7; HR-ESI-MS: Calcd. for
+
C H NO [M+H] : 548.2648, found: 548.2657.
32
38
7
4
7
7
4
.1.7.11. 4′-(4-(4-Benzylpiperazin-1-yl)butoxy)-5,6,7-trimethoxyflavone (15k). Light yellow oil,
1
2.8% yield, 98.3% HPLC purity. H NMR (400MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz, 2H, Ar-H),
3
.33-7.27 (m, 5H, Ar-H), 6.98 (d, J = 8.8 Hz, 2H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H, Ar-H),
.06 (t, J = 6.0 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.54
2
3
3
3
(
s, 2H, CH Ph), 2.52-2.48 (m, 10H, CH N), 1.85-1.81 (m, 2H, CH ), 1.76-1.72 (m, 2H, CH );
2 2 2 2
+
HR-ESI-MS: Calcd. for C H N O [M+H] : 559.2808, found: 559.2820.
33
39
2
6
4
7
6
.1.7.12. 4′-(4-(4-Methylpiperazin-1-yl)butoxy)-5,6,7-trimethoxyflavone (15l). Light yellow oil,
1
8.8% yield, 98.9% HPLC purity. H NMR (400MHz, CDCl ) δ 7.82 (d, J = 8.8 Hz, 2H, Ar-H),
3
.99 (d, J = 8.8 Hz, 2H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H, Ar-H), 4.06 (t, J = 6.0 Hz, 2H,
OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 2.70-2.63 (m, 6H, CH N),
2
3
3
3
2
2
.52 (t, J = 7.2 Hz, 4H, CH N), 2.41 (s, 3H, NCH ), 1.88-1.84 (m, 2H, CH ), 1.77-1.73 (m, 2H,
2 3 2
+
CH ); HR-ESI-MS: Calcd. for C H N O [M+H] : 483.2495, found: 483.2490.
2
27 35
2
6
4
8
7
4
.1.7.13. 4′-(6-(Benzyl(methyl)amino)hexyloxy)-5,6,7-trimethoxyflavone (15m). Light yellow oil,
1
2.5 % yield, 98.7% HPLC purity. H NMR (400MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz, 2H, Ar-H),
3
.35-7.26 (m, 5H, Ar-H), 6.99 (d, J = 8.8 Hz, 2H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H, Ar-H),
.02 (t, J = 6.4 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.59
2
3
3
3
(
s, 2H, CH Ph), 2.49-2.45 (m, 2H, CH N), 2.27 (s, 3H, NCH ), 1.82-1.78 (m, 2H, CH ), 1.65-1.61
2 2 3 2
2
5

ACCEPTED MANUSCRIPT
(
m, 2H, CH ), 1.52-1.46 (m, 2H, CH ), 1.44-1.40 (m, 2H, CH ); HR-ESI-MS: Calcd. for C H NO
2 2 2 32 38 6
+
[
M+H] : 532.2699, found: 532.2706.
4
7
7
4
2
.1.7.14. 4′-(6-(Benzyl(ethyl)amino)hexyloxy)-5,6,7-trimethoxyflavone (15n). Light yellow oil,
1
6.5 % yield, 98.6 % HPLC purity. H NMR (400MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz, 2H, Ar-H),
3
.35-7.26 (m, 5H, Ar-H), 6.98 (d, J = 8.8 Hz, 2H, Ar-H), 6.79 (s, 1H, =CH), 6.58 (s, 1H, Ar-H),
.00 (t, J = 6.4 Hz, 2H, OCH ), 3.98 (s, 6H, 2 × OCH ), 3.91 (s, 3H, OCH ), 3.64 (s, 2H, CH Ph),
2
3
3
2
.61-2.57 (m, 2H, CH N), 2.53-2.49 (m, 2H, CH N), 1.82-1.78 (m, 2H, CH ), 1.58-1.54 (m, 2H,
2
2
2
CH ), 1.52-1.46 (m, 2H, CH ), 1.39-1.35 (m, 2H, CH ), 1.11-1.07 (m, 3H, CH ); HR-ESI-MS:
2
2
2
3
+
Calcd. for C H NO [M+H] : 546.2856, found: 546.2848.
3
3
40
6
4.1.7.15. 4′-(6-((2-Methoxybenzyl)(methyl)amino)hexyloxy)-5,6,7-trimethoxyflavone (15o). Light
1
yellow oil, 71.0 % yield, 97.9 % HPLC purity. H NMR (400MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz,
3
2H, Ar-H), 7.38 (d, J = 7.2 Hz, 1H, Ar-H), 7.26 (t, J = 6.8 Hz, 1H, Ar-H), 6.99 (d, J = 8.8 Hz, 2H,
Ar-H), 6.94 (t, J = 7.2 Hz, 1H, Ar-H), 6.88 (d, J = 8.0 Hz, 1H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H,
Ar-H), 4.03 (t, J = 6.8 Hz, 2H, OCH ), 3.99 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 3.92 (s, 3H, OCH ),
2
3
3
3
3
.83 (s, 3H, OCH ), 3.65 (s, 2H, CH Ph), 2.54-2.50 (m, 2H, CH N), 2.30 (s, 3H, NCH ), 1.85-1.81
3 2 2 3
(
m, 2H, CH ), 1.68-1.64 (m, 2H, CH ), 1.51-1.47 (m, 2H, CH ), 1.43-1.39 (m, 2H, CH );
2 2 2 2
+
HR-ESI-MS: Calcd. for C H NO [M+H] : 562.2805, found: 562.2799.
33
40
7
4.1.7.16. 4′-(6-((Ethyl)(2-methoxybenzyl)amino)hexyloxy)-5,6,7-trimethoxyflavone (15p). Light
1
yellow oil, 76.5 % yield, 99.0 % HPLC purity. H NMR (400MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz,
3
2
H, Ar-H), 7.52 (brs, 1H, Ar-H), 7.29-7.25 (m, 1H, Ar-H), 6.98 (d, J = 8.8 Hz, 2H, Ar-H), 6.99-6.94
m, 1H, Ar-H), 6.88 (d, J = 8.0 Hz, 1H, Ar-H), 6.80 (s, 1H, =CH), 6.58 (s, 1H, Ar-H), 4.02 (t, J =
.4 Hz, 2H, OCH ), 4.00 (s, 3H, OCH ), 3.99 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 3.84 (s, 3H,
(
6
2
3
3
3
OCH ), 3.82 (s, 2H, CH Ph), 2.73-2.69 (m, 2H, CH N), 2.65-2,61 (m, 2H, CH N), 1.84-1.80 (m, 2H,
3
2
2
2
CH ), 1.70-1.66 (m, 2H, CH ), 1.52-1.48 (m, 2H, CH ), 1.40-1.36 (m, 2H, CH ), 1.21-1.17 (m, 3H,
2
2
2
2
+
CH ); HR-ESI-MS: Calcd. for C H NO [M+H] : 576.2961, found: 576.2972.
3
34 42
7
4.1.7.17. 4′-(6-(4-Benzylpiperazin-1-yl)hexyloxy)-5,6,7-trimethoxyflavone (15q). Light yellow oil,
2
6

ACCEPTED MANUSCRIPT
1
7
7
4
6.4 yield, 98.0% HPLC purity. H NMR (400MHz, CDCl ) δ 7.79 (d, J = 9.2 Hz, 2H, Ar-H),
3
.30-7.22 (m, 5H, Ar-H), 6.96 (d, J = 9.2 Hz, 2H, Ar-H), 6.78 (s, 1H, =CH), 6.56 (s, 1H, Ar-H),
.00 (t, J = 6.4 Hz, 2H, OCH ), 3.97 (s, 3H, OCH ), 3.96 (s, 3H, OCH ), 3.90 (s, 3H, OCH ), 3.51
2
3
3
3
(
s, 2H, CH Ph), 2.53-2.49 (m, 6H, CH N), 2.42-2.38 (m, 4H, CH N), 1.82-1.78 (m, 2H, CH ),
2 2 2 2
1
.54-1.50 (m, 2H, CH ), 1.50-1.45 (m, 2H, CH ), 1.38-1.34 (m, 2H, CH ); HR-ESI-MS: Calcd. for
2 2 2
+
C H N O [M+H] : 587.3121, found: 587.3120.
35
43
2
6
4.1.7.18. 5-Hydroxy-4′-(3-((2-Methoxybenzyl)(methyl)amino)propoxy)-6,7-dimethoxyflavone (16a).
1
Light yellow oil, 80.6% yield, 98.5% HPLC purity. H NMR (400MHz, CDCl ) δ 12.80 (s, 1H,
3
OH), 7.83 (d, J = 8.8 Hz, 2H, Ar-H), 7.32 (d, J = 7.6 Hz, 1H, Ar-H), 7.23 (d, J = 8.0 Hz, 1H, Ar-H),
7.00 (d, J = 8.8 Hz, 2H, Ar-H), 6.90 (t, J = 7.6 Hz, 1H, Ar-H), 6.86 (d, J = 8.0 Hz, 1H, Ar-H), 6.59
(
s, 1H, =CH), 6.55 (s, 1H, Ar-H), 4.14 (t, J = 6.4 Hz, 2H, OCH ), 3.97 (s, 3H, OCH ), 3.93 (s, 3H,
2
3
OCH ), 3.81 (s, 3H, OCH ), 3.58 (s, 2H, CH Ph), 2.68-2.62 (m, 2H, CH N), 2.29 (s, 3H, NCH ),
3
3
2
2
3
1
3
2
1
5
5
4
.12-2.08 (m, 2H, CH ); C NMR (100MHz, CDCl ) δ 182.5, 163.9, 162.1, 158.5, 157.6, 153.1,
2
3
52.9, 132.4, 130.5, 128.1, 127.8, 126.4, 123.0, 120.1, 114.9, 110.3, 105.9, 103.8, 90.5, 66.4, 60.7,
+
6.2, 55.3, 55.2, 53.8, 42.4, 26.9; HR-ESI-MS: Calcd. for C H NO [M+H] : 506.2179, found:
29
32
7
06.2176.
.1.7.19. 5-Hydroxy-4′-(3-((ethyl)(2-Methoxybenzyl) amino)propoxy)-6,7-dimethoxyflavone (16b).
1
Light yellow oil, 78.7% yield, 99.2% HPLC purity. H NMR (400MHz, CDCl ) δ 12.80 (s, 1H,
3
OH), 7.82 (d, J = 8.4 Hz, 2H, Ar-H), 7.40 (d, J = 6.4Hz, 1H, Ar-H), 7.22 (t, J = 7.6 Hz, 1H, Ar-H),
6.98 (d, J = 8.4 Hz, 2H, Ar-H), 6.91 (t, J = 7.6 Hz, 1H, Ar-H), 6.85 (d, J = 8.0 Hz, 1H, Ar-H), 6.59
(
s, 1H, =CH), 6.55 (s, 1H, Ar-H), 4.10 (t, J = 6.0 Hz, 2H, OCH ), 3.97 (s, 3H, OCH ), 3.93 (s, 3H,
2
3
OCH ), 3.82 (s, 3H, OCH ), 3.64 (s, 2H, CH Ph), 2.72-2.68 (m, 2H, CH N), 2.62-2.58 (m, 2H,
3
3
2
2
1
3
CH N), 2.04-2.00 (m, 2H, CH ), 1.08 (t, J = 7.2 Hz, 3H, CH ); C NMR (100MHz, CDCl ) δ 182.5,
2
2
3
3
164.0, 162.1, 158.5, 157.5, 153.0, 152.9, 132.4, 130.0, 127.8, 127.3, 126.7, 123.0, 120.2, 114.8,
110.1, 105.9, 103.7, 90.4, 66.4, 60.7, 56.2, 55.2, 51.3, 49.5, 47.7, 26.7, 11.7; HR-ESI-MS: Calcd.
+
for C H NO [M+H] : 520.2335, found: 520.2328.
3
0
34
7
2
7

ACCEPTED MANUSCRIPT
4
.1.7.20. 5-Hydroxy-4′-(3-((2-Methoxybenzyl)(methyl)amino)butoxy)-6,7-dimethoxyflavone (16c).
1
Light yellow oil, 71.2% yield, 98.2% HPLC purity. H NMR (400MHz, CDCl ) δ 12.79 (s, 1H,
3
OH), 7.83 (d, J = 8.8 Hz, 2H, Ar-H), 7.37 (d, J = 6.0 Hz, 1H, Ar-H), 7.28-7.24 (m, 1H, Ar-H), 6.99
(d, J = 8.8 Hz, 2H, Ar-H), 6.94 (t, J = 7.6 Hz, 1H, Ar-H), 6.88 (d, J = 8.4 Hz, 1H, Ar-H), 6.59 (s, 1H,
=
CH), 6.55 (s, 1H, Ar-H), 4.06 (t, J = 6.0 Hz, 2H, OCH ), 3.97 (s, 3H, OCH ), 3.93 (s, 3H, OCH ),
2
3
3
3
.83 (s, 3H, OCH ), 3.62 (s, 2H, CH Ph), 2.59-2.54 (m, 2H, CH N), 2.31 (s, 3H, NCH ), 1.89-1.85
3 2 2 3
1
3
(
m, 2H, CH ), 1.83-1.79 (m, 2H, CH ); C NMR (100MHz, CDCl ) δ 182.5, 163.9, 162.0, 158.6,
2 2 3
1
57.7, 153.1, 152.9, 132.4, 130.8, 128.3, 127.8, 125.8, 123.1, 120.2, 114.8, 110.3, 105.9, 103.8,
0.5, 67.9, 60.7, 56.9, 56.2, 55.3, 55.1, 42.0, 26.8, 23.4; HR-ESI-MS: Calcd. for C H NO
7
9
3
0
34
+
[
M+H] : 520.2335, found: 520.2325.
4.1.7.21. 5-Hydroxy-4′-(3-((ethyl)(2-Methoxybenzyl) amino)butoxy)-6,7-dimethoxyflavone (16d).
1
Light yellow oil, 71.5% yield, 97.9% HPLC purity. H NMR (400MHz, CDCl ) δ 12.80 (s, 1H,
3
OH), 7.83 (d, J = 8.8 Hz, 2H, Ar-H), 7.44 (d, J = 6.8 Hz, 1H, Ar-H), 7.23 (t, J = 7.2 Hz, 1H, Ar-H),
6.98 (d, J = 8.8 Hz, 2H, Ar-H), 6.93 (t, J = 7.2 Hz, 1H, Ar-H), 6.86 (d, J = 8.0 Hz, 1H, Ar-H), 6.59
(
s, 1H, =CH), 6.55 (s, 1H, Ar-H), 4.02 (t, J = 6.4 Hz, 2H, OCH ), 3.97 (s, 3H, OCH ), 3.93 (s, 3H,
2
3
OCH ), 3.83 (s, 3H, OCH ), 3.65 (s, 2H, CH Ph), 2.60-2.56 (m, 4H, CH N), 1.87-1.83 (m, 2H,
3
3
2
2
13
CH ), 1.74-1.70 (m, 2H, CH ), 1.12-1.08 (m, 3H, CH ); C NMR (100MHz, CDCl ) δ 182.5, 164.0,
2
2
3
3
162.1, 158.6, 157.6, 153.1, 152.9, 132.4, 130.2, 127.8, 127.6, 126.6, 123.0, 120.2, 114.8, 110.2,
105.9, 103.8, 90.5, 68.0, 60.7, 56.2, 55.2, 52.7, 51.1, 47.5, 26.9, 23.2, 11.5; HR-ESI-MS: Calcd. for
+
C H NO [M+H] : 534.2492, found: 534.2489.
31
36
7
4.1.7.22. 5-Hydroxy-4′-(3-((2-Methoxybenzyl)(methyl)amino)hexyloxy)-6,7-dimethoxyflavone (16e).
1
Light yellow oil, 72.6% yield, 98.8% HPLC purity. H NMR (400MHz, CDCl ) δ 12.80 (s, 1H,
3
OH), 7.84 (d, J = 8.8 Hz, 2H, Ar-H), 7.39 (d, J = 7.2 Hz, 1H, Ar-H), 7.26 (t, J = 7.2 Hz, 1H, Ar-H),
7.00 (d, J = 8.8 Hz, 2H, Ar-H), 6.95 (t, J = 7.6 Hz, 1H, Ar-H), 6.88 (d, J = 8.0 Hz, 1H, Ar-H), 6.59
(
s, 1H, =CH), 6.55 (s, 1H, Ar-H), 4.04 (t, J = 6.0 Hz, 2H, OCH ), 3.97 (s, 3H, OCH ), 3.93 (s, 3H,
2
3
OCH ), 3.84 (s, 3H, OCH ), 3.66 (s, 2H, CH Ph), 2.56-2.52 (m, 2H, CH N), 2.32 (s, 3H, NCH ),
3
3
2
2
3
2
8