Design, synthesis and evaluation of chromone-2-carboxamido-alkylbenzylamines as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2015.01.042

A series of chromone-2-carboxamido-alkylbenzylamines were designed, synthesized and evaluated 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 49 displayed excellent inhibitory potency toward acetylcholinesterase (AChE), moderate anti-oxidative activity, selective biometal chelating, and possessed good inhibitory effects on self-induced and Cu²⁺-induced Aβ aggregation. Both kinetic analysis of AChE inhibition and molecular modeling study indicated that 49 was a mixed-type inhibitor, binding simultaneously to the catalytic active site and peripheral anionic site of AChE. These results suggested that 49 might be a potential multifunctional agent for AD treatment.

Full text of DOI:10.1016/j.bmc.2015.01.042

Accepted Manuscript
Design, synthesis and evaluation of chromone-2-carboxamido-alkylbenzyla-
mines as multifunctional agents for the treatment of Alzheimer’s disease
Qiang Liu, Xiaoming Qiang, Yan Li, Zhipei Sang, Yuxing Li, Zhenghuai Tan,
Yong Deng
PII:
S0968-0896(15)00066-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.01.042
BMC 12045
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
18 December 2014
20 January 2015
21 January 2015
Please cite this article as: Liu, Q., Qiang, X., Li, Y., Sang, Z., Li, Y., Tan, Z., Deng, Y., Design, synthesis and
evaluation of chromone-2-carboxamido-alkylbenzylamines as multifunctional agents for the treatment of
Alzheimer’s disease, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.01.042
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Design, synthesis and evaluation of chromone-2-carboxamido-alkylbenzylamines
as multifunctional agents for the treatment of Alzheimer’s disease
a,#
a,#
a
a
a
b,*
a,*
Qiang Liu , Xiaoming Qiang , Yan Li , Zhipei Sang , Yuxing Li , Zhenghuai Tan , Yong Deng
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
Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan Academy of
Chinese Medicine Sciences, Chengdu, 610041, China
#
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

Abstract
A series of chromone-2-carboxamido-alkylbenzylamines were designed, synthesized and evaluated
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 49
displayed excellent inhibitory potency toward acetylcholinesterase (AChE), moderate anti-oxidative
activity, selective biometal chelating, and possessed good inhibitory effects on self-induced and
2
+
Cu -induced Aβ aggregation. Both kinetic analysis of AChE inhibition and molecular modeling
study indicated that 49 was a mixed-type inhibitor, binding simultaneously to the catalytic active site
and peripheral anionic site of AChE. These results suggested that 49 might be a potential
multifunctional agent for AD treatment.
Keywords:
Alzheimer’s
disease,
Chromone-2-carboxamido-alkylbenzylamines,
Acetylcholinesterase inhibitors, Anti-oxidative activity, Biometal chelators, Aβ aggregation
inhibitors.
2

1. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that affects daily living
1
through memory loss and cognitive impairment. Current therapeutic options for the treatment of
AD, including cholinesterase inhibitors (donepezil, rivastigmine, and galantamine) and
N-methyl-D-aspartate (NMDA) receptor antagonist (memantine), have resulted in a modest
improvement in memory and cognitive function, but they do not prevent progressive
2
,3
neurodegeneration. Although the etiology of AD remain elusive, multiple factors, such as low
levels of acetylcholine (ACh), oxidative stress, accumulation of misfolded amyloid-β (Aβ) and
dyshomeostasis of biometals have been considered to play definitive roles in the pathophysiology of
AD, and several hypotheses based on these factors have been proposed to explain the mechanism of
4
,5
AD pathogenesis. According to the ‘‘cholinergic hypothesis’’, AChE acts primarily as a regulatory
enzyme at cholinergic synapses, while BuChE functions as an enzyme closely related to AChE and
6
,7
serves as a co-regulator of cholinergic neurotransmission by hydrolysing ACh. AChE and BuChE
inhibition have been documented as critical targets for the effective management of AD by an
8
increase the availability of ACh in the brain regions. However, BuChE is mainly localized in the
peripheral tissues including plasma and very small amount is present in the brain region. Many side
effects (e.g. gastrointestinal events, nausea, vomiting, diarrhoea, dizziness) mainly associated with
9
their peripheral AChE inhibitory activity. Therefore, the potential advantage of selective inhibition
of AChE over BChE may include lesser degree of associated side effects due to peripheral inhibition
8
of cholinesterase enzyme. So it may be a significative approach to develop specific AChE inhibitors
for the treatment of AD, with expectation for their fewer side effects.
Based on “amyloid hypothesis”, the production and accumulation of oligomeric aggregates of Aβ in
1
0
the brain are a central event in the pathogenesis of AD. Aβ40 and Aβ42 are the main isoforms of Aβ
peptides. In vivo studies show that Aβ42 is a major constituent of amyloid plaques and promulgate
that Aβ42 aggregation plays a critical role in the initiation of plaque formation and AD pathogenesis.
3

In vitro, Aβ42 displays lower solubility and more toxic and has the tendency to form fibrillar
1
1
aggregates at lower concentrations than Aβ40
.
Therefore, the prevention of Aβ42 aggregation
2
+
2+
2+
3+
attracts much current attention. Moreover, excess of metal ions such as Cu , Fe , Zn and Al
2
+
2+
have been found in Aβ plaques of AD brains, and Cu and Zn have been proved to promote Aβ
1
2
aggregation as described previously. Interestingly, the abnormally high levels of redox-active metal
2
+
2+
ions such as Cu , Fe or impaired regulation of redox-active metals which can induce the formation
1
3
of cytotoxic reactive oxygen species and neuronal damage. Thus, biometals chelators might be a
potential therapeutic strategy for the treatment of AD. In addition, Oxidative stress plays an
important role in neuronal degeneration. Oxidative damage marked by lipid peroxidation, nitration,
1
0
reactive carbonyls, and nucleic acid oxidation is increased in vulnerable neurons in AD. Therefore,
the successful protection of neuronal cells from oxidative damage could potentially prevent AD.
Obviously, there are many attractive targets for the development of anti-AD drugs, the compounds
possess two or more complementary biological activities have drawn considerable attention for their
advancement in the treatment of AD. In this regard, the multitarget-directed ligands strategy
14
(
MTDLs) has been performed by many research groups.
Figure 1. here >
<
Chromone (I), a privileged scaffold in medicinal chemistry, is the core fragment of several flavonoid
derivatives such as flavones and isoflavones (Figure 1). The derivatives of chromone have attracted
much attention in recent years because of their diverse pharmacological properties such as
1
5-17
anti-inflammatory effect, metal chelating ability, and neuroprotective effect.
Recent study has
indicated that a new family of tacrine-chromone hybrids showed interesting bioactivities for the
potential treatment of AD, such as inhibition of human AChE, BuChE, and β-secretase 1 (BACE-1),
1
8
as well as radical scavenger activity. However, whether this kind of tacrine-chromone hybrids
poccesses the effects on Aβ aggregation is unknown. Recently, our group has reported the synthesis
of genistein-O-alkylbenzylamines and discovered one 7,4′-O-modified genistein derivative (II) as a
4

1
9
potential multifunctional agent for the treatment of AD. However, this compound has low
inhibitory effects on self-induced Aβ1-42 aggregation.
Herein, chromone-2-carboxamide moiety was selected to combine with different length
alkylbenzylamine fragments to design a series of novel chromone-2-carboxamido-alkylbenzylamine
derivatives (Figure 1), to test whether these novel molecules might possess more potency in various
multifunctional activities and improve the shortcomings. In this paper,
a
series of
chromone-2-carboxamido-alkylbenzylamine derivatives were designed, synthesized and evaluated
biological activities based on the multitarget-directed ligands strategy (MTDLs). Their biological
activities including AChE and BuChE inhibition, the kinetics of enzyme inhibition, antioxidant
activities, biometal chelation, and effects on Aβ aggregation were also evaluated.
2
. Results and discussion
.1. Chemistry
2
The key intermediates 11a-d, 12a-h and 13a-d were prepared according to the route outlined in
Scheme 1. The secondary amines 5a-g were synthesized by reductive amination of methylamine,
ethylamine or propargylamine with the corresponding benzaldehydes 1-4 in the presence of
2
0
NaBH
salicylaldehyde 6 through reductive amination by using ethanol amine and NaBH
protection by using di-tert-butyl dicarbonate (Boc O) and Et N in one-pot reaction. Then 5h was
obtained through Mitsunobu Reaction and deprotection by the treatment with trifluoroacetic acid.
4
, while compound 5h was obtained from a different route. Compound 7 was prepared from
4
and then
2
3
2
1
The primary amine intermediates 11-13 were prepared by conventional method through Gabriel
syntheses starting from the commercially available ω-bromoalkylphthalimides and corresponding
secondary amines 5a-h.
<
Scheme 1. here >
The general procedure for the synthesis of the chromone-2-carboxamido-alkylbenzylamines 24-50
5

was depicted in Scheme 2. The chromone-2-carboxylic acid analogs 20-23 were prepared from
2
2
phloroglucinol 14 and 2,6-dihydroxyacetophenone 19. First, phloroglucinol 14 was acylated to
2
3
compound 15 by Fries rearrangement using BF
and K CO in acetone gave compound 16. In the reported method for the synthesis of
chromone-2-carboxylic acid involved three steps including condensation, saponification and
3 2 2 4
·Et O. Then treatment of the latter with Me SO
2
3
2
4
demethylation generally. But the workup was inconvenient. So we adopted a new one-pot strategy
by condensation of 16 with ethyl oxalate in the presence of EtONa in absolute EtOH and then
treatment with 40% HBr for 12 h or 48 h afforded compound 20 and 21 respectively. Similar
procedure was adopted in the preparation of compound 22 and the key intermediate compound 17
was synthesized by reacting compound 15 with dimethylamine hydrochloride in the presence of
3 2
NaHCO in H O under argon. More conveniently, for the synthesis of 7-H substituted chromone
moiety, it is easy to obtain the ester 23 from compound 19 directly by using excessive amounts of
EtONa in EtOH. Eventually, the chromone-2-carboxamido-alkylbenzylamines 24-50 were eventually
obtained by either condensation reaction or amination reaction with the appropriate primary amine
11a-d, 12a-h and 13a-d. All synthesized compounds were purified using recrystallization or
chromatography, and the analytical and spectroscopic data confirmed their structures, as detailed in
the experimental section.
<
Scheme 2. here >
2.2. Pharmacology
2.2.1. Inhibition studies on AChE and BuChE
Inhibitory activities toward AChE and BuChE of the synthesized compounds 24-50 and
intermediates 20-23 were evaluated according to the modified Ellman’s method. Their activities
were initially assayed on AChE from rat cortex homogenate (RatAChE) and BuChE from rat serum
(
RatBuChE). Moreover, the compounds were evaluated using electric eel AChE (EeAChE).
6

Donepezil was used as reference compound. The IC50 values and selectivity index for the inhibition
of AChE and BuChE were summarized in Table 1.
<
Table 1. here >
All the target compounds showed significant AChE inhibitiory activities with IC50 values ranging
from submicromolar to micromolar, and demonstrated weak BuChE inhibitory activities, indicating
that these derivatives were selective inhibitors for AChE. In view of BuChE was mainly localized in
2
5
the peripheral tissues including plasma and very small amount was present in the brain region.
Thus, higher selectivity inhibitors toward AChEover BuChE, resulted in lesser degree of associated
side effects, might be beneficial for the treatment of AD. Therefore, it was identical to our design
strategy. Compound 49 exhibited the best inhibitory potency for AChE with IC50 value of 0.07 µM.
According to the above results, the properties of the substitutions at chromone nucleus 7 position had
significant effects on AChE inhibitory activities. The trend for AChE inhibitiory activities was 7-H >
7
-OCH
NR
the order N-ethyl-2-methoxy-benzenemethanamine
3
> 7-N(CH
of side chain effected the inhibitory activities, generally, the potency to inhibit AChE were in
2-(dimethylamino)-N-ethyl-benzenem
3 2
) > 7-OH. The screening data exhibited that the structure of terminal groups
1
R
2
≈
ethanamine > N-benzylethanamine > N-(2-methoxybenzy)methanamine > 4-(dimethylamino)-N-
ethylbenzylethanamine > N-benzylmethanamine > N-(2-methoxybenzyl)prop-2-yn-1-amine >
2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine. The compounds (25, 27, 29, 31, 37, 39) possessing a
N-ethyl group showed better AChE inhibitory activity than compounds (24, 26, 28, 30, 36, 38) with a
N-methyl group, in most cases, and the inhibitory activity dropped dramatically when the N-ethyl
1 2
group was replaced by N-propargyl (34). With the same scaffold and terminal groups NR R , the
inhibitory activity increased with the length of methylene linkers increased, except compound 49,
where the optimal chain length was four. Finally, compound 49 showed the most potent inhibition for
RatAChE with an IC50 value of 0.07 ± 0.002 µM, and 735.7-fold selectivity for AChE over BuChE.
Moreover, all the target compounds were determined by additional testing using EeAChE, it is
7

notable that all the tested compounds exhibited a higher inhibitory activity towards RatAChE than
EeAChE, except compounds 24, 28, 29, 36, 37 and 38 (Table 1). In addition, the choromone
scaffolds (20-23) showed low inhibitory activity and none selectivity toward AChE and BuChE,
indicating that the introduction of the O-alkylamines increased the ChEs inhibitory capacity and
improved the selectivity for AChE over BuChE, which is identical to our design strategy. Overall, the
results suggested that N-(2-methoxybenzyl)ethylamine and N-(2-dimethylaminobenzyl)ethylamine
might be the optimal substitution patterns for AChE inhibition. Almost all the compounds exhibited
very weak activity against BuChE (IC50 > 20 µM). This result showed that these target compounds
were potent AChE inhibitors with high selectivity toward AChE. This selectivity profile may be a
limitation, but this may also be beneficial to diminish peripheral cholinergic side effects and provide
lower toxicity. A typical case in point is tacrine, which had severe side effects because of its poor
2
6
selectivity. Based on the above assay results, we next selected the most potent AChE inhibitor, 49,
for kinetic analysis to investigate the type of inhibition.
2.2.2 Kinetic study for the inhibition of AChE
A kinetic study was carried out on compound 49 using EeAChE to gain further insight into the
mechanism of action on AChE of this family of compounds. The Lineweaver-Burk plots (Figure 2.)
m
showed both increased slopes (decreased Vmax) and intercepts (higher K ) at increasing
concentration of the inhibitor, which indicated a mixed-type inhibition. It revealed that compound 49
might be able to bind to the catalytic active site (CAS) as well as peripheral anionic site (PAS) of
AChE.
<
Figure 2. here >
2.2.3 Molecular modeling study
In order to obtain more information about the binding interactions between compound 49 and
1
1
TcAChE (PDB code: 1EVE), docking studies were carried out using the AUTODOCK 4.2 package
as shown in Figure 3.
8

<
Figure 3. here >
Results showed that compound 49 exhibited multiple binding modes with AChE. In the 49–TcAChE
complex, compound 49 occupied the entire enzymatic CAS, mid-gorge and PAS. The chromone
moiety occupied the PAS of AChE and showed π-π stacking with Phe288, Phe290, Phe331 and
Tyr334. Notably, a strong hydrogen bond was observed between the 4-carbonyl group at the
chromone moiety and Tyr121. Besides, the benzene ring of 49 displayed significant π-stacking
interaction in choline binding site represented by Trp84. The long chain of methylene folded in a
conformation in the gorge that allowed it to interact with Phe330 via the hydrophobic interaction.
The docking study showed that compound 49 exhibited a mixed type of inhibition, consistented with
our kinetic analysis result.
2.2.4 Antioxidant activity
The antioxidant activities of compounds 20-50 were evaluated by following the well-established
2
7,28
ORAC-FL method.
Peroxyl radicals were thermally generated from 2,2-azobis-(amidinopropane)
dihydrochloride and reacted with fluorescein to form nonfluorescent products at 535 nm. The
antioxidant capacity of compounds 20-50 was determined by their competition with fluorescein in
the radical capture, using a fluorescence microplate reader. Their ability to scavenge radicals was
provided as a Trolox (a vitamin E analog) equivalent, with their relative potency at 5 µM compared
with Trolox (Table 1). All the target compounds demonstrated moderate to good antioxidant
activity. Compounds 33, 40, 41 and 42 showed the most potent antioxidant activity of this family
with ORAC-FL values of 2.20, 2.10, 2.30 and 2.25 equivalents, respectively. The N-(2-dimethyl
aminobenzyl)ethylamine moiety seemed to be a potent substitution pattern for the radical scavenging
ability. It was noticeable that the free 7-OH of chromone derivatives was found to be crucial to the
radical scavenging ability when the ORAC-FL values were compared between that of the 7-H
derivatives (45-50) and that of the 7-OCH
3
derivatives (24-39).
2.2.5 Metal-chelating properties
9

2
+
2+
The chelating ability of compound 49 toward biologically relevant metal ions such as Cu , Fe ,
2
+
3+
Zn and Al was studied by UV–vis spectrometry. Electronic spectral of compound 49 in methanol
2
+
2+
2+
significantly changed in the presence of Cu ions while remained unchanged after adding Fe , Zn
3
+
and Al ions (Figure 4.). Upon the addition of CuCl
2
, the curve had a red shift (the peak at 340 nm
2
+
shift to 420 nm) suggesting the formation of complex 49-Cu . However, after adding FeSO
and AlCl no significant shift was observed in the UV spectrum.
Figure 4. here >
In order to determine the stoichiometry of the complex 49-Cu , the molar ration method was
employed by preparing the solution of compound 49 with ascending amounts of CuCl . The UV
spectrum was used to obtain the absorbance of the 49 complex and different concentrations of CuCl
at 420 nm. The results showed that absorbance linearly increased initially and then plateaued (Figure
.). The two straight lines intersected at a mole fraction of 0.98, revealing a 1:1 stoichiometry for
4
, ZnCl
2
,
3
<
2
+
2
2
5
2
+
complex 49-Cu .
<
Figure 5. here >
2.2.6 Effects on the Aβ Aggregation
2
+
The inhibition of self and Cu -induced Aβ1-42 aggregation by our synthetic derivatives was
26,29,30
determined by using thioflavin T fluorescence method
, with curcumin as reference
3
1
compound. The data for their effects on Aβ1-42 peptide aggregation at concentration of 25 µM was
summarized in Table 2. The marketed AD drug, donepezil, did not show any significant inhibitory
activity under the same experimental conditions. In the ThT assay screening for Aβ1-42 peptide
self-aggregation inhibition, the most potent compounds, similarly to AChE inhibition, were found to
be 31, 39, 49 and 50 featuring a N-(2-methoxybenzyl)ethylamine group on the terminal side of
carbon chain with respective inhibition ratio of 51.0%, 58.2%, 59.2% and 63.0%, which are higher
than that of curcumin (43.1%). Interestingly, these four compounds also exhibited the high inhibitory
activity for AChE. For example, compound 49, as the most potent AChE inhibitor, showed the
1
0

second highest inhibitory potency of self-induced Aβ1-42 aggregation with inhibitory percentage of
9.2%. Other target compounds showed moderate to good potencies ranging from 21.1% to 50.6%.
The results indicated that substitution on the 7 position of chromone moiety played a pivotal role in
inhibition of self-induced Aβ1-42 aggregation. The trend for Aβ1-42 inhibition was 7-H > 7-OCH
-N(CH > 7-OH (e.g. 49 > 31 > 43 > 42). Structure-activity relationship analysis showed that the
5
3
>
7
)
3 2
chain length turned out to play an important role, since the inhibitory potency rose as the length of
chain increased (e.g. 50 > 49 > 48).
<
Table 2. here >
2
+
Regarding the inhibition of Cu -induced Aβ1-42 aggregation, all compounds showed moderate
potencies ranging from 18.1% to 55.6%, which were lower than that of curcumin. The compounds
having potent inhibition of self-induced Aβ1-42 aggregation generally exhibited relatively good
2
+
inhibition of Cu -induced Aβ1-42 aggregation. For example, compound 50 showed the highest
inhibitory potency of self-induced Aβ1-42 aggregation (63.0%) and was also the most potent inhibitor
2
+
of Cu -induced Aβ1-42 aggregation (55.6%) among these compounds. Comparing to 7,4′-O-modified
genistein derivative (II), the highlight of this paper reported chromone-2-carboxamido-
alkylbenzylamines involves in the improvements of self-induced Aβ1-42 aggregation inhibition. The
most potent compounds 31, 39, 49 and 50 showed inhibition ratio with 51.0%, 58.2%, 59.2% and
19
63.0% respectively, which are much potent than 7,4′-O-modified genistein derivative (II) (35.0%).
What is more, according to ‘Lipinski’s rule of five’, chromone-2-carboxamido-alkylbenzylamines
derivatives are more likely to be drug candidates than 7,4′-O-modified genistein derivative (II) in
terms of their molecular weight.
Interestingly, the result of Aβ anti-aggregation activity is consistent with the outcome of inhibition
against AChE. These chromone derivatives, as was revealed in the kinetic study, were dual binding
site AChE inhibitors. This kind of inhibitors are often endowed with Aβ anti-aggregating
3
2
33-35
properties, which arise either from blockade of the AChE peripheral anionic site (PAS)
or from
11

a direct interaction with Aβ (blockade of spontaneous Aβ aggregation), in the latter case likely due to
the presence of aromatic planar moieties in the inhibitors. To be specific, comparing with
7,4′-O-modified genistein derivative (II), compounds 49 and 50 are structurally simpler since the
genistein 3-phenyl is removed and the linker is attached at the 2-position of chromone scaffold.
Without the existence of the bulky 3-phenyl on the chromone scaffold, 4-carbonyl group may be
more likely to form a hydrogen bond with the amino acid residues of Aβ and it is reported that this
kind of interaction between inhibitors and Aβ can finally increase the inhibitory activity towards the
3
6
Aβ aggregation.
3. Conclusion
We have designed, synthesized and evaluated 27 new chromone-2-carboxamido-alkylbenzylamines
as multifunctional agents against AD. Most of the compounds exhibited potent inhibitory activity
towards AChE and displayed high selectivity for AChE over BuChE. Compounds 49 showed the
most potent inhibition toward AChE with IC50 value of 0.07 ± 0.01 µM. The kinetic analysis
suggested that 49 showed mixed-type inhibition, and could bind to both CAS and PAS of AChE,
which was consistent with the molecular modeling study. Importantly, compound 50 and 49 showed
excellent self-induced Aβ aggregation inhibitory activity (63.0% and 59.2% repectively) and good
2
+
Cu -induced Aβ aggregation inhibitory activity (55.6% and 48.3% respectively). Moreover,
compound 49 also possessed the prospective property of acting as a metal chelator. Taken together,
compound 49 was a promising multifunctional agent for AD treatment.
4
. Experimental section
.1 Chemistry
4
Reagents were either purchased from common commercial sources. All solvents were purified and
dried by standard procedures. All the reactions were monitored by thin-layer chromatography (TLC)
1
2

on silica gel GF254 plates from Qingdao Haiyang Chemical Co. Ltd. (China), visualized in an iodine
chamber or with an UV lamp (254 nm). Column chromatography was performed using silica gel
(
230-400 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. (China). Melting points were
recorded on YRT-3 melting-point apparatus (China) and are uncorrected. NMR spectra were
recorded at room temperature in CDCl or DMSO-d solutions using a Varian INOVA 400 NMR
3
6
spectrometer or Varian INOVA 600 NMR spectrometer. Chemical shifts are reported in parts per
millions (ppm) relative to tetramethylsilane (TMS). Mass spectra were recorded on Agilent-6210
TOF LC-MS Spectrometer.
4.1.1 General procedure for the synthesis of secondary amines (5a-g)
2
0
Compounds 5a-g were prepared as previously described.
4
.1.2 2,3,4,5-Tetrahydrobenzo[f][1,4]oxazepine (5h)
.1.2.1 2-(((2-Hydroxyethyl)amino)methyl)phenol (7)
4
To a solution of salicylaldehyde (4.3 mL, 41.20 mmol) in MeOH was added ethanolamine (2.48 mL,
1.20 mmol). The mixture was stirred at room temperature for 6 h, and then NaBH (780 mg, 20.60
mmol) was added in portions within 2 h. The mixture is stirred at room temperature for 3.5 h, then
Et N (6.89 mL, 49.44 mmol) and Boc O (9.89 g, 45.32 mmol) was added. The mixture was stirred at
room temperature for 24 h. The solvent was evaporated under reduced pressure. H O (50 mL) was
4
4
3
2
2
added to the residue. The mixture was acidified with 3 N HCl to pH = 5 and white solid was formed
and collected by filtration. After being washed by water, compound 7 was obtained and used without
further purification; 94% yield; white solid; mp 108-109 °C.
4
.1.2.2 2,3,4,5-Tetrahydrobenzo[f][1,4]oxazepine (5h)
To a solution of Ph P (4.63 g, 22.44 mmol) in THF (15 mL) at -10 °C was added a solution of DIAD
4.4 mL, 22.44 mmol) in THF (10 mL) dropwise. Then a solution of compound 7 (3 g, 11.22 mmol)
in THF (20 mL) was added dropwise to the mixture. The reaction was stirred at room temperature for
2 h. The solvent was evaporated under reduced pressure. Dichloromethane (40 mL) and TFA (15
3
(
1
1
3

mL) were added to the residue and the mixture was stirred at room temperature for 5 h, then the
solvent was evaporated under reduced pressure. H O was added and the mixture was acidified with 3
2
N HCI to pH = 2. The aqueous phase was washed with dichloromethane (30 mL × 3). The aqueous
phase was then treated with sodium hydroxide until pH 12 was established. The mixture was then
extracted with dichloromethane (30 mL × 3). The combined organic phases were washed with
saturated aqueous NaCl (100 mL), dried over sodium sulfate, and filtered. The solvent was
evaporated to dryness under reduced pressure. Compound 5h was obtained and used without further
purification; 92.0% yield; light yellow oil.
4.1.3 General procedure for the synthesis of compounds 8-13
2
1
Compounds 8-13 were prepared as previously described.
4
.1.4 1-(2,4,6-Trihydroxyphenyl)ethanone (15)
Phloroglucinol 14 (10 g, 79.4 mmol) and acetic anhydride (18 mL, 190.0 mmol) were dissolved in
ethyl acetate (40 mL), and BF ·Et O (12 mL, 97.2 mmol) was added dropwise. The reaction mixture
was heated at 50 °C for 10 h. Then, H O (150 mL) was added and the reaction mixture was extracted
with ethyl acetate. After evaporation of the solvent the raw material was recrystallised from H O to
3
2
2
2
37
give compound 15; 64.9% yield; yellow crystal; mp 220-221 °C (lit. 221 °C).
.1.5 1-(2-Hydroxy-4,6-dimethoxyphenyl)ethanone (16)
To a mixture of 1-(2,4,6-trihydroxyphenyl)ethanone 15 (4.5 g, 24.2mmol) and anhydrous K
8.29 g, 55.6 mmol) in acetone (90 mL), Me SO (6.84 mL, 72.5 mmol) was added dropwise with
stirring. The resulting mixture was refluxed for 5 h and then filtered and concentrated under reduced
pressure. The residue was purified on a silica gel chromatography using CH Cl as eluent, obtaining
4
2
CO
3
(
2
4
2
2
3
8
compound 16; 84.2% yield; white solid; mp 78-79 °C (lit. 79-80 °C).
.1.6 1-(4-(Dimethylamino)-2,6-dihydroxyphenyl)ethanone (17)
To a mixture of dimethylamine hydrochloride (1.8 g, mmol) and NaHCO
O (4 mL), 1-(2,4,6-trihydroxyphenyl)ethanone (15) (200 mg, 1.07 mmol) was added. The reaction
4
3
(1.6 g, 19.05 mmol) in
H
2
1
4

mixture was warmed to 70 °C and stirred for 10 h under an argon atmosphere. H
added and the reaction mixture was extracted with ethyl acetate (5 mL × 3), the combined organic
phases were washed successively with saturated aqueous Na CO (5 mL × 3) and saturated aqueous
NaCl (10 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 CH Cl as eluent,
obtaining compound 17; 67.83% yield; white solid; mp 192-193 °C. H NMR (400MHz, CDCl ) δ
2
O (10 mL) was
2
3
2
2
1
3
5.65 (s, 2H), 3.06 (s, 6H), 2.63 (s, 3H).
4.1.7 1-(4-(Dimethylamino)-2-hydroxy-6-methoxyphenyl)ethanone (18)
To a mixture of 1-(4-(dimethylamino)-2,6-dihydroxyphenyl)ethanone 17 (810 mg, 4.15 mmol) and
anhydrous K CO (745 mg, 5.40 mmol) in acetone (16 mL), Me SO (0.47 mL, 4.98 mmol) was
2
3
2
4
added dropwise with stirring. The resulting mixture was refluxed for 5 h and then filtered and
concentrated under reduced pressure. The residue was purified on a silica gel chromatography using
1
CH
2
Cl
2
as eluent, obtaining compound 18, 97.1% yield. H NMR (400MHz, CDCl
3
) δ 14.17 (s, 1H),
5.77-5.76 (d, J = 2.4 Hz, 1H), 5.62-5.61 (d, J = 2.4 Hz, 1H), 3.87 (s, 3H), 3.04 (s, 6H), 2.56 (s, 3H).
4.1.8 General procedure for the synthesis of compounds 20-22
A solution of compound 16 or 18 (6.1 mmol) in ethyl oxalate (25.7 mmol) was added to a freshly
prepared solution of EtONa in EtOH (prepared by addition of 872 mg of sodium to 25 mL of
absolute EtOH). The mixture was refluxed for 10 h. After which, the reaction mixture was cooled to
room temperature, ethanol was evaporated and 40% aqueous HBr (18 mL) was added. The mixture
was refluxed for 12 h or 48 h. After being cooled to room temperature, the mixture was treated with
10% sodium hydroxide solution until pH 12 was established. The mixture was filtered and the filtrate
was acidified with 12 N HCI to pH < 2. The resulting solid was collected and was recrystallized from
acetic acid to give the corresponding compounds 20-22.
4.1.8.1 5-Hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20)
1
5

It was synthesized from 1-(2-hydroxy-4,6-dimethoxyphenyl)ethanone (16) according to the general
1
procedure (treatment with 40% aqueous HBr for 12 h); 57% yield, brown solid; mp > 240 °C. H
NMR (400MHz, DMSO-d
.1.8.2 5,7-Dihydroxy-4-oxo-4H-chromene-2-carboxylic acid (21)
It was synthesized from 1-(2-hydroxy-4,6-dimethoxyphenyl)ethanone (16) according to the general
6
) δ 12.41 (s, 1H), 6.86 (s, 1H), 6.71 (s, 1H), 6.44 (s, 1H), 3.87 (s, 3H).
4
3
9
procedure (treatment with 40% aqueous HBr for 48 h); 61% yield; brown solid, mp > 240 °C (Lit.
305–308 °C).
4.1.8.3 7-(Dimethylamino)-5-hydroxy-4-oxo-4H-chromene-2-carboxylic acid (22)
It was synthesized from 1-(4-(dimethylamino)-2-hydroxy-6-methoxyphenyl)ethanone (18) according
to the general procedure (treatment with 40% aqueous HBr for 12h); 65% yield; brown solid; mp >
1
2
40 °C. H NMR (400MHz, DMSO-d
6
) δ 12.78 (s, 1H), 6.54 (s, 1H), 6.17 (s, 1H), 6.05 (s, 1H), 3.00
(
s, 6H), 1.88 (s, 1H).
4.1.9 Ethyl 5-hydroxy-4-oxo-4H-chromene-2-carboxylate (23)
A solution of 2,6-dihydroxyacetophenone (19) (1.4 g, 9.2 mmol) in ethyl oxalate (5 mL, 36.8 mmol)
was added to a freshly prepared solution of EtONa in EtOH (prepared by addition of 1.06 g of
sodium to 60 mL of absolute EtOH). The mixture was refluxed for 10 h and the mixture is cooled to
room temperature, and 4 ml of 12 N HCl is added dropwise. The mixture is heated at 90 °C for 30
minutes and cooled, and the solvent is removed under reduced pressure. H
the residue and the mixture was extracted with EtOAc (20 mL × 3). The combined organic phases
were washed with saturated aqueous NaCl (40 mL), dried over Na SO , and filtered. The solvent was
2
O (40 mL) was added to
2
4
evaporated to dryness under reduced pressure. The residue was purified on a silica gel
chromatography using mixtures of petroleum ether/acetone (15:1) as eluent, obtaining compound 27;
4
0
3
8% yield, yellow solid; mp 143-146 °C. (Lit. 148 °C)
4.1.10 General procedure for the synthesis of compounds 24-44
1
6

To a mixture of the corresponding carboxylic acid derivatives 20-22 (0.21 mmol), EDCI (0.32 mmol)
and HOBt (0.21 mmol) in THF were added the appropriate intermediates 11a-d, 12a-h, and 13a-d
(
0.25 mmol). The reaction mixture was stirred for 24 h at room temperature. The solvent was
evaporated under reduced pressure. H O (10 mL) was added to the residue and the mixture was
extracted with dichloromethane (8 mL × 3). The combined organic phases were washed successively
with saturated aqueous NaHCO (5 mL × 2) and saturated aqueous NaCl (10 mL), dried over sodium
2
3
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 (12:1) as eluent to obtain the
corresponding compounds 24-44.
4.1.10.1
N-(3-(Benzyl(methyl)amino)propyl)-5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-
carboxamide (24)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-benzyl-N-methylpropane-1,3-diamine (11a) according to the general procedure and gave
1
compound 24 as a light yellow oil; 57% yield. H NMR (400 MHz, CDCl
3
) δ 12.51 (s, 1H), 9.11 (brs,
1
4
1
9
3
4
H), 7.31-7.18 (m, 5H), 7.02 (s, 1H), 0.94 (d, J = 2 Hz, 1H), 5.95 (s, 1H), 3.74 (s, 3H), 3.59-3.54 (m,
1
3
H), 2.66-2.63 (t, J = 5.6 Hz, 2H), 2.36 (s, 3H), 1.84 (brs, 2H). C NMR (100 MHz, CDCl ) δ
3
82.34, 165.77, 162.12, 158.37, 156.56, 155.59, 137.71, 129.08, 128.38, 127.34, 110.30, 106.02,
+
8.24, 92.73, 62.94, 56.77, 55.60, 41.81, 40.46, 24.60. HRMS-ESI [M+H] Calcd for C22
24 2 5
H N O :
97.1685, found 397.1688.
.1.10.2
N-(3-(Benzyl(ethyl)amino)propyl)-5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-
carboxamide (25)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-benzyl-N-ethylpropane-1,3-diamine (11b) according to the general procedure and gave compound
1
2
5 as a light yellow oil; 59% yield. H NMR (400 MHz, CDCl
3
) δ 12.50 (s, 1H), 9.10 (brs, 1H),
7.32-7.30 (m, 2H ), 7.21-7.15 (m, 3H), 7.01 (s, 1H), 6.36-6.35 (d, J = 2 Hz, 2H), 6.00 (s, 1H), 3.74 (s,
1
7

3
7
1
H), 3.63 (s, 2H), 3.56-3.52, (q, J = 5.6 Hz, 2H), 2.70-2.65 (m, 4H), 1.84 (brs, 2H), 1.18-1.15 (t, J =
1
3
3
.2 Hz, 3H). C NMR (100 MHz, CDCl ) δ 182.36, 165.79, 162.16, 158.44, 156.61, 155.60, 137.82,
29.20, 128.35, 127.29, 110.42, 106.11, 98.36, 92.70, 58.49, 55.61, 52.40, 47.24, 40.54, 24.41, 11.11.
+
HRMS-ESI [M+H] Calcd for C23
H
26
N
2
5
O 411.1842, found 411.1851.
4.1.10.3
5-Hydroxy-7-methoxy-N-(3-((2-methoxybenzyl)(methyl)amino)propyl)-4-oxo-4H-
chromene-2-carboxamide (26)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-(2-methoxybenzyl)-N-methylpropane-1,3-diamine (11c) according to the general procedure and
1
gave compound 26 as a light yellow oil; 61% yield. H NMR (400 MHz, CDCl
3
) δ 12.49 (s, 1H),
9.72 (s, 1H), 7.23-7.21 (d, J = 6.8 Hz, 1H), 7.15-7.11 (t, J = 7.2 Hz, 1H), 6.98 (s, 1H), 6.86-6.83 (t, J
=
7.2 Hz, 1H), 6.67-6.65 (d, J = 8 Hz, 1H), 6.32-6.31 (d, J = 2 Hz, 1H), 5.74 (s, 1H), 3.72 (s, 3H),
13
3
.61-3.55 (m, 5H), 2.73-2.72 (t, J = 4.8 Hz, 2H), 2.36 (s, 3H), 1.86 (brs, 2H). C NMR (100 MHz,
CDCl ) δ 182.42, 165.74, 162.00, 158.40, 157.90, 156.61, 156.01, 131.43, 129.04, 125.36, 120.11,
10.36, 110.07, 105.98, 98.24, 92.70, 57.68, 57.28, 55.59, 54.84, 41.71, 41.09, 24.18. HRMS-ESI
3
1
+
[
M+H] Calcd for C23
H
26
N
2
O
6
427.1791, found 427.1796.
4.1.10.4 N-(3-(Ethyl(2-methoxybenzyl)amino)propyl)-5-hydroxy-7-methoxy-4-oxo-4H-
chromene-2-carboxamide (27)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-ethyl-N-(2-methoxybenzyl)propane-1,3-diamine (11d) according to the general procedure and
1
gave compound 27 as a light yellow oil; 60% yield. H NMR (400 MHz, CDCl
3
) δ 12.49 (s, 1H),
9
6
4
1
.72 (brs, 1H), 7.26 (s, 1H), 7.13-7.10 (t, J = 7.2 Hz, 1H), 7.00 (s, 1H), 6.85-6.81 (t, J = 7.6 Hz, 1H),
.66-6.64 (d, J = 7.6 Hz), 6.33 (s, 1H), 5.64 (br, 1H), 3.71 (s, 3H), 3.63-3.56 (m,7H), 2.74-2.69 (m,
1
3
H), 1.91 (brs, 2H), 1.14 (brs, 3H). C NMR (100 MHz, CDCl
3
) δ 182.40, 165.71, 161.95, 158.38,
57.89, 156.57, 155.94, 131.52, 128.94, 125.42, 120.08, 110.32, 110.09, 106.00, 98.33, 92.56, 55.54,
1
8

+
5
4
4
4.80, 53.10, 47.33, 41.24, 23.81, 10.77. HRMS-ESI [M+H] Calcd for C24
H
28
N
2
O
6
441.1947, found
41.1943.
.1.10.5
N-(4-(Benzyl(methyl)amino)butyl)-5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-
carboxamide (28)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-benzyl-N-methylbutane-1,4-diamine (12a) according to the general procedure and gave compound
1
2
7
2
8 as a light yellow oil; 57% yield. H NMR (400 MHz, CDCl
3
) δ 12.45 (s, 1H), 7.89 (brs, 1H),
.38-7.27 (m, 5H), 7.00 (s, 1H), 6.36-6.33 (m, 2H), 3.79 (s, 1H), 3.62 (s, 2H), 3.50-3.49 (m, 2H),
1
3
3
.55-2.52 (t, J = 6 Hz, 2H), 2.27 (s, 3H), 1.74-1.72 (t, J = 2.8 Hz, 4H). C NMR (100 MHz, CDCl )
δ 182.24, 165.96, 162.03, 158.72, 156.73, 155.39, 136.88, 129.26, 128.40, 127.53, 110.49, 106.08,
+
9
8.57, 92.74, 61.63, 56.22, 55.76, 41.80, 39.51, 26.51, 24.18. HRMS-ESI [M+H] Calcd for
C
24
H
28
N
2
O
5
425.1998, found 425.1990.
4.1.10.6
N-(4-(Benzyl(ethyl)amino)butyl)-5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-
carboxamide (29)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-benzyl-N-ethylbutane-1,4-diamine (12b) according to the general procedure and gave compound
1
2
9 as a light yellow oil; 51% yield. H NMR (400 MHz, CDCl
3
) δ 12.46 (s, 1H), 7.44 (brs, 1H), 7.37
-
7.23 (m, 5H), 7.01 (s, 1H), 6.39-6.37 (m, 2H), 3.83 (s, 1H), 3.64 (s, 2H), 3.48-3.43 (q, J = 6 Hz, 2H),
1
3
2
1
9
3
.62-2.52 (m, 4H), 1.71-1.62 (m, 4H), 1.10-1.06 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, CDCl ) δ
82.33, 166.08, 162.30, 158.71, 156.80, 155.28, 129.10, 128.33, 137.23, 110.76, 106.25, 98.64,
+
2.86, 57.76, 55.87, 52.28, 47.21, 39.69, 26.92, 24.34, 10.95. HRMS-ESI [M+H] Calcd for
C
25
H
30
N
2
O
5
439.2155, found 439.2148.
4.1.10.7
5-Hydroxy-7-methoxy-N-(4-((2-methoxybenzyl)(methyl)amino)butyl)-4-oxo-4H-
chromene-2-carboxamide (30)
1
9

It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-(2-methoxybenzyl)-N-methylbutane-1,4-diamine (12c) according to the general procedure and
1
gave compound 30 as a light yellow oil; 50% yield. H NMR (400 MHz, CDCl
3
) δ 12.40 (br, 1H),
8.38 (brs, 1H), 7.31-7.29 (d, J = 7.2 Hz, 1H), 7.273-7.234(m, 1H), 6.96 (s, 1H), 6.94-6.90 (t, J = 7.6
Hz, 1H), 6.87-6.85 (d, J = 8.4 Hz, 1H), 6.32-6.31 (d, J = 2 Hz, 1H), 6.18-6.17 (d, J = 2 Hz, 1H), 3.79
(
s, 3H), 3.77 (s, 3H), 3.62 (s, 2H), 3.49-3.46 (m, 2H), 2.55-2.52 (m, 2H), 2.25 (s, 3H), 1.77-1.71 (m,
1
3
4
1
3
4
3
H). C NMR (100 MHz, CDCl ) δ 182.28, 165.91, 162.05, 158.70, 157.77, 156.74, 155.68, 131.27,
28.87, 125.04, 120.24, 110.41, 110.35, 106.09, 98.42, 92.76, 56.74, 55.74, 55.49, 55.05, 42.26,
+
9.43, 26.46, 24.27. HRMS-ESI [M+H] Calcd for C25
H
30
N
2
O
6
455.2104, found 455.2109.
.1.10.8
N-(4-(Ethyl(2-methoxybenzyl)amino)butyl)-5-hydroxy-7-methoxy-4-oxo-4H-
chromene-2-carboxamide (31)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-ethyl-N-(2-methoxybenzyl)butane-1,4-diamine (12d) according to the general procedure and gave
1
compound 31 as a light yellow oil; 62% yield. H NMR (400 MHz, CDCl
3
) δ 12.45 (s, 1H), 8.04 (brs,
1
6
3
H), 7.43-7.41 (d, J = 7.2 Hz, 1H), 7.28-7.24 (m, 1H), 6.99 (s, 1H), 6.95-6.91 (t, J = 7.6 Hz, 1H),
.87-6.85 (d, J = 8.4 Hz, 1H), 6.47 (brs, 1H), 6.37-6.36 (d, J = 1.6 Hz, 1H), 3.83-3.80 (m, 8H),
13
.50-3.47 (m, 2H), 2.75-2.66 (m, 4H), 1.77-1.71 (m, 4H), 1.74-1.14 (t, J = 7.2 Hz, 3H). C NMR
) δ 182.41, 166.06, 162.10, 158.87, 157.81, 156.92, 155.49, 131.30, 129.20, 120.52,
(
100 MHz, CDCl
3
110.60, 110.48, 106.27, 98.70, 92.93, 55.89, 55.27, 52.23, 51.22, 47.47, 39.28, 26.60, 23.74, 10.27.
+
HRMS-ESI [M+H] Calcd for C26
H
32
N
2
O
6
469.2260, found 469.2271.
4.1.10.9
N-(4-((2-(Dimethylamino)benzyl)(ethyl)amino)butyl)-5-hydroxy-7-methoxy-4-oxo-
4
H-chromene-2-carboxamide (32)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-(2-(dimethylamino)benzyl)-N-ethylbutane-1,4-diamine (12e) according to the general procedure
1
and gave compound 32 as a light yellow oil; 66% yield. H NMR (400 MHz, CDCl
3
) δ 12.38 (s, 1H),
2
0

8
7
3
1
1
9
.79 (brs, 1H), 7.85-7.83 (d, J = 7.6 Hz, 1H), 7.41-7.37 (t, J = 6.8 Hz, 1H), 7.28-7.24 (m, 1H),
.20-7.16 (t, J = 7.6 Hz, 1H), 7.03 (brs, 1H), 7.00 (s, 1H), 6.35-6.35 (d, J = 2 Hz, 1H), 4.35 (s, 2H),
.89 (s, 3H), 3.56-3.51 (q, J = 5.6 Hz, 2H), 3.11-3.02 (m, 4H), 2.66 (s, 6H), 2.06-2.01 (m, 2H),
1
3
.77-1.74 (m, 2H), 1.37-1.33 (t, J = 7.6 Hz, 3H). C NMR (100 MHz, CDCl ) δ 182.50, 166.12,
3
61.59, 159.24, 157.21, 155.41, 153.81, 131.83, 130.81, 125.05, 124.44, 120.80, 110.19, 106.25,
+
8.94, 93.34, 55.96, 51.41, 50.68, 46.53, 45.54, 37.81, 25.60, 21.17, 8.11. HRMS-ESI [M+H] Calcd
for C27
H
35
N
3
O
5
482.2577, found 482.2580.
N-(4-((4-(Dimethylamino)benzyl)(ethyl)amino)butyl)-5-hydroxy-7-methoxy-4-oxo-
H-chromene-2-carboxamide (33)
4
.1.10.10
4
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-(4-(dimethylamino)benzyl)-N-ethylbutane-1,4-diamine (12f) according to the general procedure
1
and gave compound 33 as a light yellow oil; 64% yield. H NMR (400 MHz, CDCl
3
) δ 12.43 (s, 1H),
8.31 (brs, 1H), 7.27-7.25 (m, 2H), 6.99 (s, 1H), 6.69-6.64 (m, 3H), 6.35-6.35 (d, J = 2 Hz, 1H), 3.85
(
s, 3H), 3.81 (s, 2H), 3.52-3.48 (q, J = 4 Hz, 2H), 2.93 (s, 6H), 2.83-2.72 (m, 4H), 1.87-7.72 (m, 4H),
1
3
1
1
3
4
4
.26-1.23 (m, 3H). C NMR (100 MHz, CDCl ) δ 182.35, 165.97, 161.79, 158.95, 156.93, 155.41,
3
50.34, 130.94, 119.75, 112.10, 110.32, 106.15, 98.67, 93.05, 56.36, 55.84, 51.24, 46.45, 40.19,
+
8.85, 29.13, 26.26, 22.79, 9.70. HRMS-ESI [M+H] Calcd for C27
35 3 5
H N O 482.2577, found
82.2584.
.1.10.11 N-(4-(Ethyl(2-(prop-2-yn-1-yl)benzyl)amino)butyl)-5-hydroxy-7-methoxy-4-oxo-4H-
chromene-2-carboxamide (34)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-ethyl-N-(2-(prop-2-yn-1-yl)benzyl)butane-1,4-diamine (12g) according to the general procedure
1
and gave compound 34 as a light yellow oil; 59% yield. H NMR (400 MHz, CDCl
3
) δ 12.48 (s, 1H),
7.72 (brs, 1H), 7.36-7.34 (d, J = 6.8 Hz, 1H), 7.27-7.23 (m, 1H), 6.99 (s, 1H), 6.94-6.92 (t, J = 7.2
Hz, 1H), 6.87-6.85 (d, J = 8 Hz, 1H), 6.35 (s, 1H), 6.13 (s, 1H), 3.80 (s, 3H), 3.69 (s, 2H), 3.51-3.49
2
1

1
3
(
m, 2H), 3.36 (s, 2H), 2.70-2.37 (t, J = 6 Hz, 2H), 2.24 (brs, 1H), 1.79-1.69 (m, 4H). C NMR (100
MHz, CDCl ) δ 182.30, 166.00, 162.28, 158.64, 157.88, 156.71, 155.50, 131.02, 128.77, 125.70,
20.32, 110.63, 110.48, 106.16, 98.43, 92.74, 78.35, 73.49, 55.82, 55.20, 53.00, 51.54, 42.17, 39.71,
3
1
2
4
4
+
6.70, 24.68. HRMS-ESI [M+H] Calcd for C27
.1.10.12 N-(4-(2,3-Dihydrobenzo[f][1,4]oxazepin-4(5H)-yl)butyl)-5-hydroxy-7-methoxy-
-oxo-4H-chromene-2-carboxamide (35)
H
30
N
2
O
6
479.2104, found 479.2112.
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
4
-(2,3-dihydrobenzo[f][1,4]oxazepin-4(5H)-yl)butan-1-amine (12h) according to the general
1
procedure and gave compound 35 as a light yellow oil; 58% yield. H NMR (400 MHz, CDCl
3
) δ
12.46 (s, 1H), 7.27 (brs, 1H), 7.20-7.12 (m, 2H), 7.01-6.97 (m, 3H), 6.44-6.38 (m, 2H), 4.08-4.06 (t,
J = 4 Hz, 2H), 3.87 (s, 5H), 3.13-3.47 (q, J = 6 Hz, 2H), 3.13-3.11 (t, J = 4 Hz, 2H), 2.56-2.52 (t, J =
1
3
6
1
5
3
.8 Hz, 2H), 1.73-1.62 (m, 4H). C NMR (100 MHz, CDCl ) δ 182.27, 166.11, 162.38, 159.72,
58.69, 156.74, 155.20, 130.92, 130.55, 128.74, 123.46, 120.76, 110.77, 106.21, 98.59, 92.90, 69.48,
+
7.99, 57.97, 55.89, 52.89, 39.75, 26.94, 24.67. HRMS-ESI [M+H] Calcd for C25
H
28
N
2
6
O 453.1947,
found 453.1940.
.1.10.13 N-(6-(Benzyl(methyl)amino)hexyl)-5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-
4
carboxamide (36)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-benzyl-N-methylhexane-1,6-diamine (13a) according to the general procedure and gave compound
1
3
7
3
6 as a light yellow oil; 50% yield. H NMR (400 MHz, CDCl
3
) δ 12.46 (s, 1H), 7.36-7.27 (m, 4H),
.03 (brs, 2H), 6.49-6.49 (d, J = 1.6 Hz, 1H), 6.39-6.38 (d, J = 2.4 Hz, 1H), 3.87 (s, 3H), 3.59 (s, 2H),
.49-3.44 (q, J = 6.8 Hz, 2H), 2.48-2.44 (t, J = 7.2 Hz, 2H), 2.27 (s, 3H), 1.66-1.60 (m, 4H), 1.39
1
3
(
brs, 4H). C NMR (100 MHz, CDCl
3
) δ 182.23, 166.01, 162.11, 158.52, 156.67, 155.14, 137.09,
1
2
29.28, 128.24, 127.33, 110.50, 106.05, 98.53, 92.86, 61.76, 56.71, 55.81, 41.63, 39.78, 29.08, 26.65,
+
6.49. HRMS-ESI [M+H] Calcd for C25
H
30
N
2
5
O 439.2155, found 439.2162.
2
2

4.1.10.14
N-(6-(Benzyl(ethyl)amino)hexyl)-5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-
carboxamide (37)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-benzyl-N-ethylhexane-1,6-diamine (13b) according to the general procedure and gave compound
1
3
7
3
7 as a light yellow oil; 66% yield. H NMR (400 MHz, CDCl
3
) δ 12.45 (s, 1H), 7.37-7.23 (m, 5H),
.01 (brs 2H), 6.47-6.46 (d, J = 2.4 Hz, 1H), 6.38-6.37 (d, J = 2.4 Hz, 1H), 3.86 (s, 3H), 3.63 (s, 2H),
.47-3.42(q, J = 6.8 Hz 2H), 2.61-2.55 (q, J = 7.2 Hz, 2H), 2.51-2.47 (t, J = 7.2 Hz, 2H), 1.66-1.62
1
3
(
m, 2H), 1.56-1.53 (m, 2H), 1.37-1.35 (m, 4H), 1.10-1.06 (t, J = 7.2 Hz, 3H). C NMR (100 MHz,
CDCl ) δ 182.21, 166.00, 162.11, 158.48, 156.64, 155.14, 138.15, 129.01, 128.12, 127.01, 110.47,
06.03, 98.52, 92.83, 57.60, 55.79, 52.56, 47.02, 39.81, 29.10, 26.73, 26.22, 11.08. HRMS-ESI
3
1
+
[
M+H] Calcd for C26
32 2 5
H N O 453.2311, found 453.2303.
4.1.10.15 5-Hydroxy-7-methoxy-N-(6-((2-methoxybenzyl)(methyl)amino)hexyl)-4-oxo-4H-
chromene-2-carboxamide (38)
It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-(2-methoxybenzyl)-N-methylhexane-1,6-diamine (13c) according to the general procedure and
1
gave compound 38 as a light yellow oil; 55% yield. H NMR (400 MHz, CDCl
3
) δ 12.40 (br, 1H),
7
6
3
1
1
5
.36-7.34 (d, J = 7.6 Hz, 1H), 7.27-7.21 (m, 2H), 6.99 (s, 1H), 6.94-6.92 (t, J = 7.2 Hz, 1H),
.88-6.86 (d, J = 8.4 Hz, 1H), 6.49-6.48 (d, J = 2 Hz, 1H), 6.35-6.34 (d, J = 2.0 Hz, 1H), 3.85 (s, 3H),
.82 (s, 3H), 3.62 (s, 2H), 3.48-3.43 (q, J = 6.4 Hz, 2H), 2.51-2.47 (t, J = 7.6 Hz, 2H), 2.28 (s, 3H),
1
3
3
.68-1.60 (m, 4H), 1.39 (brs, 4H). C NMR (100 MHz, CDCl ) δ 182.23, 165.98, 162.10, 158.50,
57.68, 155.13, 130.93, 128.50, 125.14, 120.18, 110.49, 110.30, 106.05, 98.53, 92.83, 57.11, 55.80,
+
5.27, 54.98, 41.86, 39.78, 29.09, 26.73, 26.53. HRMS-ESI [M+H] Calcd for C26
H
32
N
2
O
6
469.2260,
found 469.2255.
.1.10.16
chromene-2-carboxamide (39)
4
N-(6-(Ethyl(2-methoxybenzyl)amino)hexyl)-5-hydroxy-7-methoxy-4-oxo-4H-
2
3

It was synthesized from 5-hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (20) and
N-ethyl-N-(2-methoxybenzyl)hexane-1,6-diamine (13d) according to the general procedure and gave
1
compound 39 as a light yellow oil; 59% yield. H NMR (400 MHz, CDCl
3
) δ 12.44 (s, 1H),
7.66-7.61 (m, 2H), 7.39-7.35 (m, 1H), 7.03 (s, 1H), 7.00-6.96 (dt, J = 7.2 Hz, 0.8 Hz, 1H), 6.94-6.91
(
(
d, J = 8.4 Hz, 1H) 6.70-6.69 (d, J = 2 Hz, 1H), 6.38-6.37 (d, J = 2.4 Hz, 1H), 4.16 (s, 2H), 3.88-3.87
m 6H), 3.49-3.44 (q, J = 6.8 Hz, 2H), 3.03-2.97 (q, J = 7.2 Hz, 2H), 2.29-2.85 (m, 2H), 1.85 (br,
2
H), 1.71-1.67 (m, 2H), 1.43-1.41 (t, J = 3.6 Hz, 4H), 1.38-1.34 (t, J = 7.2 Hz, 3H). HRMS-ESI
+
[
M+H] Calcd for C27
H
34
N
2
O
6
483.2417, found 483.2415.
4.1.10.17
N-(3-(ethyl(2-methoxybenzyl)amino)propyl)-5,7-dihydroxy-4-oxo-4H-chromene-2-
carboxamide (40)
It was synthesized from 5,7-dihydroxy-4-oxo-4H-chromene-2-carboxylic acid (21) and
N-ethyl-N-(2-methoxybenzyl)propane-1,3-diamine (11d) according to the general procedure and
1
gave compound 40 as a light yellow oil; 50% yield. H NMR (400 MHz, CDCl
3
) δ 12.31 (br, 1H),
9.27 (brs, 1H), 7.29-7.20 (m, 2H), 6.92-6.83 (m, 2H), 6.78-6.76 (d, J = 8 Hz, 1H), 6.18 (s, 1H), 5.98
(
s, 1H), 5.77 (br, 1H), 3.86 (s, 2H), 3.72 (s, 3H), 3.49 (s, 2H), 2.86-2.78 (m, 4H), 1.96 (brs, 2H),
1
3
1
1
4
4
4
.16-1.13 (t, J = 6.8 Hz, 3H). C NMR (100 MHz, CDCl ) δ 181.52, 168.12, 161.73, 159.60, 157.76,
3
57.15, 154.59, 131.75, 130.13, 121.61, 120.56, 110.55, 109.80, 104.27, 100.74, 95.26, 55.18, 51.15,
+
7.44, 38.58, 39.15, 24.49, 9.66. HRMS-ESI [M+H] Calcd for C23
26 2 6
H N O 427.1791, found
27.1802.
.1.10.18
N-(4-(Ethyl(2-methoxybenzyl)amino)butyl)-5,7-dihydroxy-4-oxo-4H-chromene-2-
carboxamide (41)
It was synthesized from 5,7-dihydroxy-4-oxo-4H-chromene-2-carboxylic acid (21) and
N-ethyl-N-(2-methoxybenzyl)butane-1,4-diamine (12d) according to the general procedure and gave
1
compound 41 as a light yellow oil; 53% yield. H NMR (400 MHz, CDCl
3
) δ 12.23 (br, 1H), 8.52
(
brs, 1H), 7.41-7.39 (d, J = 6.8 Hz, 1H), 7.31-7.27 (m, 1H), 6.90-6.83 (m, 2H), 6.77 (s, 1H), 6.48 (s,
2
4

1
H), 6.13 (1s, 1H), 5.93 (br, 1H), 4.10 (s, 2H), 3.79 (s, 3H), 3.40 (brs, 2H), 2.98-2.96 (m, 4H), 1.86
+
(
brs, 2H), 1.62 (brs, 2H), 1.29-1.25 (t, J = 6.4 Hz, 3H). HRMS-ESI [M+H] Calcd for C24
H
28
N
2
O
6
4
41.1947, found 441.1948.
.1.10.19 N-(6-(Ethyl(2-methoxybenzyl)amino)hexyl)-5,7-dihydroxy-4-oxo-4H-chromene-2-
4
carboxamide (42)
It was synthesized from 5,7-dihydroxy-4-oxo-4H-chromene-2-carboxylic acid (21) and
N-ethyl-N-(2-methoxybenzyl)hexane-1,6-diamine (13d) according to the general procedure and gave
1
compound 42 as a light yellow oil; 52% yield. H NMR (400 MHz, CDCl
3
) δ 12.29 (br, 1H), 7.75
(
brs, 1H), 7.37-7.36 (d, J = 6.8 Hz, 1H), 7.30-7.26 (m, 1H), 6.93-6.90 (t, J = 7.2 Hz, 1H), 6.86-6.83
m, 2H), 6.32 (s, 1H), 6.07 (s, 1H), 4.36 (br, 1H), 3.89 (s, 2H), 3.79 (s, 3H), 3.33-3.31 (m, 2H),
(
1
3
2
.82-2.77 (m, 2H), 2.69-2.64 (m, 2H), 1.59-1.47 (m, 4H), 1.26-1.17 (m, 7H). C NMR (100 MHz,
) δ 181.51, 169.42, 161.70, 159.30, 157.84, 157.37, 154.81, 131.62, 130.04, 120.69, 110.64,
09.79, 104.00, 101.14, 95.50, 55.40, 52.50, 50.85, 47.18, 39.76, 28.94, 26.64, 26.45, 24.87, 9.90.
CDCl
3
1
+
HRMS-ESI [M+H] Calcd for C26
H
32
N
2
O
6
469.2260, found 469.2263.
7-(Dimethylamino)-N-(4-(ethyl(2-methoxybenzyl)amino)butyl)-5-hydroxy-4-oxo-
H-chromene-2-carboxamide (43)
4.1.10.20
4
It was synthesized from 7-(dimethylamino)-5-hydroxy-4-oxo-4H-chromene-2-carboxylic acid (22)
and N-ethyl-N-(2-methoxybenzyl)butane-1,4-diamine (12d) according to the general procedure and
1
gave compound 43 as a light yellow oil; 51% yield. H NMR (600 MHz, CDCl
3
) δ 12.42 (s, 1H),
7
1
3
3
1
3
.82 (brs, 1H),7.44-7.43 (d, J = 7.2 Hz, 1H), 7.27-7.24 (m, 1H), 6.93-6.91 (t, J = 7.8 Hz, 1H), 6.89 (s,
H), 6.86-6.85 (d, J = 7.8 Hz, 1H), 6.18 (s, 1H), 6.05-6.04 (d, J = 1.8 Hz, 1H), 3.83 (s, 2H), 3.81 (s,
H), 3.01 (s, 6H), 2.76-2.72 (m, 2H), 2.69-2.67 (m, 2H), 1.77-1.67 (m, 4H), 1.18-1.16 (t, J = 6.6 Hz,
13
3
H). C NMR (100 MHz, CDCl ) δ 180.95, 161.16, 159.15, 157.70, 156.97, 155.37, 154.40, 131.23,
29.16, 123.41, 120.41, 110.36, 110.11, 102.61, 94.84, 89.34, 55.19, 52.04, 50.86, 47.28, 39.89,
+
9.10, 26.55, 23.33, 10.19. HRMS-ESI [M+H] Calcd for C26
H
33
N
3
5
O 468.2420, found 468.2426.
2
5

4
.1.10.21 7-(Dimethylamino)-N-(4-((2-(dimethylamino)benzyl)(ethyl)amino)butyl)-5-hydroxy-
-oxo-4H-chromene-2-carboxamide (44)
4
It was synthesized from 7-(dimethylamino)-5-hydroxy-4-oxo-4H-chromene-2-carboxylic acid (22)
and N-(2-(dimethylamino)benzyl)-N-ethylbutane-1,4-diamine (12e) according to the general
1
procedure and gave compound 44 as a light yellow oil; 55% yield. H NMR (400 MHz, CDCl
3
) δ
1
1
3
2.43 (s, 1H), 7.65-7.63 (d, J = 7.6 Hz, 1H), 7.52 (s, 1H), 7.27-7.22 (m, 1H), 7.13-7.11 (d, J = 7.6 Hz,
H), 7.09-7.06 (t, J = 7.2 Hz, 1H), 6.91 (s, 1H), 6.18 (s, 1H), 6.07-6.08 (d, J = 2Hz, 1H), 3.85 (s, 2H),
.46-3.44 (m, 2H), 3.04 (s, 6H), 2.70-2.66 (m, 10H), 1.69-1.68 (m, 4H), 1.14-1.11 (t, J = 6.8 Hz, 3H).
1
3
3
C NMR (100 MHz, CDCl ) δ 181.01, 161.31, 159.17, 157.03, 155.46, 154.36, 153.17, 131.20,
1
3
4
30.43, 128.11, 123.46, 119.30, 110.30, 102.72, 94.98, 89.31, 52.43, 51.92, 47.33, 45.20, 39.98,
+
9.34, 26.73, 23.70, 10.55. HRMS-ESI [M+H] Calcd for C27
H
36
N
4
O
4
481.2737, found 481.2738.
.1.11 General procedure for the synthesis of compounds 45-50
To a solution of ethyl 5-hydroxy-4-oxo-4H-chromene-2-carboxylate (23) (30 mg, 0.13 mmol) in
EtOH (3 mL), were added the appropriate intermediates 11a-d, 12a-h, and 13a-d (0.17 mmol). The
mixture was refluxed for 10 h. The solvent was evaporated under reduced pressure. H
was added to the residue and the mixture was extracted with dichloromethane (8 mL × 3). The
4
combined organic phases were washed with saturated aqueous NaCl (30 mL), dried over Na SO ,
2
O (10 mL)
2
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 obtain the
corresponding compounds 45-50.
4.1.11.1
N-(3-(Benzyl(methyl)amino)propyl)-5-hydroxy-4-oxo-4H-chromene-2-carboxamide
(
45)
It was synthesized from N-benzyl-N-methylpropane-1,3-diamine (11a) according to the general
1
procedure and gave compound 45 as a light yellow oil; 78% yield. H NMR (400 MHz, CDCl
3
) δ
12.29 (s, 1H), 9.25 (s, 1H), 7.46-7.23 (m, 6H), 7.07 (s, 1H), 6.81-6.79 (d, J = 8 Hz, 1H), 6.49-6.46 (d,
2
6

J = 8.4 Hz, 1H), 3.72 (s, 3H), 3.62-3.58 (q, J = 5.2 Hz, 2H), 2.77-2.74 (t, J = 5.6 Hz, 2H), 2.43 (s,
1
3
3
1
3
H), 1.95-1.91 (m, 2H). C NMR (100 MHz, CDCl ) δ 183.79, 160.70, 158.56, 155.99, 155.34,
36.04, 129.39, 128.66, 128.02, 111.94, 111.26, 110.46, 107.12, 62.67, 56.33, 41.20, 39.93, 24.43.
+
HRMS-ESI [M+H] Calcd for C21
H
22
N
2
4
O 367.1580, found 367.1584.
4.1.11.2
5-Hydroxy-N-(3-((2-methoxybenzyl)(methyl)amino)propyl)-4-oxo-4H-chromene-2-
carboxamide (46)
It was synthesized from N-(2-methoxybenzyl)-N-methylpropane-1,3-diamine (11c) according to the
1
general procedure and gave compound 46 as a light yellow oil; 82% yield. H NMR (400 MHz,
CDCl
3
) δ 12.29 (s, 1H), 9.82 (s, 1H), 7.35-7.30 (t, J = 8.4 Hz, 1H), 7.26-7.25 (m, 1H), 7.18-7.14 (m,
H), 7.05 (s, 1H), 6.90-6.86 (t, J = 7.2 Hz, 1H), 6.77-6.74 (d, J = 8.4 Hz, 1H), 6.69-6.67 (d, J = 8 Hz,
H), 6.02-6.00 (d, J = 8 Hz, 1H), 3.64-3.57 (m, 7H), 2.76-2.73 (t, J = 5.6 Hz, 2H), 2.35 (s, 3H),
1
1
1
1
4
4
13
3
.89-1.88 (m, 2H). C NMR (100 MHz, CDCl ) δ 183.76, 160.48, 158.28, 158.02, 156.37, 155.18,
35.84, 131.64, 129.22, 125.31, 120.04, 111.65, 111.06, 110.43, 110.12, 107.04, 57.84, 57.31, 54.84,
+
1.58, 41.11, 29.14, 24.20. HRMS-ESI [M+H] Calcd for C22
H
24
N
2
5
O 397.1685, found 397.1688.
.1.11.3
5-Hydroxy-N-(4-((2-methoxybenzyl)(methyl)amino)butyl)-4-oxo-4H-chromene-2-
carboxamide (47)
It was synthesized from N-(2-methoxybenzyl)-N-methylbutane-1,4-diamine (12c) according to the
1
general procedure and gave compound 47 as a light yellow oil; 77% yield. H NMR (400 MHz,
CDCl
H), 6.98-6.94 (t, J = 7.6 Hz, 1H), 6.90-6.88 (d, J = 8 Hz, 1H), 6.79-6.77 (d, 8.4 Hz, 1H), 6.61-6.59
d, 8.8 Hz, 1H), 3.81 (s, 3H), 3.74 (s, 2H), 3.52-3.51 (m, 2H), 2.66-2.65 (m, 2H), 2.32 (s, 3H),
3
) δ 12.28 (brs, 1H), 8.74(brs, 1H), 7.47-7.43 (t, J = 8.4 Hz, 1H), 7.37-7.28 (m, 2H), 7.04 (s,
1
(
1
3
1
1
5
4
3
.81-1.80 (m, 4H). C NMR (100 MHz, CDCl ) δ 183.70, 160.54, 158.72, 157.89, 156.20, 155.36,
35.97, 131.92, 131.92, 129.56, 123.28, 120.38, 111.75, 111.19, 110.53, 110.34, 107.29, 56.27,
+
5.12, 54.97, 41.62, 39.04, 26.00, 23.62. HRMS-ESI [M+H] Calcd for C23
H
26
N
2
5
O 411.1842, found
11.1836.
2
7

4.1.11.4
N-(3-(Ethyl(2-methoxybenzyl)amino)propyl)-5-hydroxy-4-oxo-4H-chromene-2-
carboxamide (48)
It was synthesized from N-benzyl-N-methylpropane-1,3-diamine (11a) according to the general
1
procedure and gave compound 48 as a light yellow oil; 75% yield. H NMR (400 MHz, CDCl
3
) δ
12.29 (s, 1H), 9.25 (s, 1H), 7.46-7.23 (m, 6H), 7.07 (s, 1H), 6.81-6.79 (d, J = 8 Hz, 1H), 6.49-6.46 (d,
J = 8.4 Hz, 1H), 3.72 (s, 3H), 3.62-3.58 (q, J = 5.2 Hz, 2H), 2.77-2.74 (t, J = 5.6 Hz, 2H), 2.43 (s,
1
3
3
1
3
H), 1.95-1.91 (m, 2H). C NMR (100 MHz, CDCl ) δ 183.79, 160.70, 158.56, 155.99, 155.34,
36.04, 129.39, 128.66, 128.02, 111.94, 111.26, 110.46, 107.12, 62.67, 56.33, 41.20, 39.93, 24.43.
+
HRMS-ESI [M+H] Calcd for C23
H
26
N
2
O
5
411.1842, found 411.1840.
4.1.11.5
N-(4-(Ethyl(2-methoxybenzyl)amino)butyl)-5-hydroxy-4-oxo-4H-chromene-2-
carboxamide (49)
It was synthesized from N-ethyl-N-(2-methoxybenzyl)butane-1,4-diamine (12d) according to the
1
general procedure and gave compound 49 as a light yellow oil; 78% yield. H NMR (400 MHz,
CDCl
.28-7.23 (m, 1H), 7.05 (s, 1H), 6.95-6.91 (t, J = 7.2 Hz, 1H), 6.86-6.84 (d, J = 8 Hz, 1H), 6.81-6.79
d, J = 8 Hz, 1H), 6.77-6.75 (d, J = 8.4 Hz, 1H), 3.81 (s, 3H), 3.75 (s, 2H), 3.49-3.48 (m, 2H),
3
) δ 12.28 (brs, 1H), 8.26 (s, 1H), 7.52-7.48 (t, J = 8.4 Hz, 1H), 7.41-7.39 (d, J = 7.6 Hz, 1H),
7
(
1
3
2
1
1
3
.70-2.63 (m, 4H), 1.75 (brs, 4H), 1.13-1.09 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, CDCl ) δ
83.71, 160.66, 158.71, 157.78, 156.07, 155.39, 136.03, 131.26, 128.94, 120.37, 111.89, 111.26,
+
10.53, 110.40, 107.23, 55.17, 52.21, 51.18, 47.37, 39.41, 26.55, 23.95, 10.21. HRMS-ESI [M+H]
Calcd for C24
.1.11.6
carboxamide (50)
H
28
N
2
O
5
425.1998, found 425.1992.
4
N-(6-(ethyl(2-methoxybenzyl)amino)hexyl)-5-hydroxy-4-oxo-4H-chromene-2-
It was synthesized from N-ethyl-N-(2-methoxybenzyl)hexane-1,6-diamine (13d) according to the
1
general procedure and gave compound 50 as a light yellow oil; 80% yield. H NMR (400 MHz,
CDCl ) δ 12.29 (s, 1H), 7.61-7.57 (t, 1H), 7.42-7.40 (d, J = 7.2 Hz, 1H), 7.23-7.19 (t, J = 8 Hz, 1H),
3
2
8

7.11 (s, 1H), 6.96-6.84 (m, 5H), 3.82 (s, 3H), 3.60 (s, 2H), 3.49-3.44 (q, J = 6.8 Hz, 2H), 2.57-2.45
1
3
(
m, 4H), 1.67-1.63 (m, 2H), 1.56-1.51 (m, 2H), 1.38-1.37 (m, 4H), 1.07-1.05 (t, J = 6.8 Hz, 3H). C
NMR (100 MHz, CDCl ) δ 183.62, 160.79, 158.49, 157.62, 155.65, 155.34, 136.14, 130.53, 128.24,
20.32, 112.11, 111.24, 110.62, 110.22, 107.09, 55.27, 52.85, 50.99, 47.46, 39.84, 29.08, 26.79,
3
1
2
4
4
+
6.51, 26.19, 11.12. HRMS-ESI [M+H] Calcd for C26
H
32
N
2
5
O 453.2311, found 453.2315.
.2 Biological evaluation
.2.1 Inhibition Experiments of AChE and BuChE
1
9
AChE and BuChE activities were measured by Ellman’s method with slight modification. Rat
cortex homogenate and rat serum were used as resource of RatAChE and BuChE respectively and
EeAChE derived from Electrophorus electricus (Sigma-Aldrich Co.). The brain homogenate was
preincubated 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 (1mmol/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 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 and changes in
absorbance were detected at 405 nm in a Varioskan Flash Multimode Reader (Thermo Scientific).
For Electrophorus electricus AChE inhibition assay, EeAChE (0.05 U/mL, final concentration) was
used and the assay was carried out in a phosphate buffer (0.01 mmol/L, pH = 8.0). Changes in
41
absorbance were detected at 412 nm. The other procedure was the same as above. Thus, IC50 values
were calculated as the concentration of compound that produces 50% AChE or BuChE activity
inhibition. Donepezil was applied as positive drug. All samples were assayed in triplicate.
4.2.2 Kinetic Characterization of AChE Inhibition
2
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