Rational design, synthesis and biological evaluation of novel multitargeting anti-AD iron chelators with potent MAO-B inhibitory and antioxidant activity

Article abstract of DOI:10.1016/j.bmc.2020.115550

A series of (3-hydroxypyridin-4-one)-coumarin hybrids were developed and investigated as potential multitargeting candidates for the treatment of Alzheimer's disease (AD) through the incorporation of iron-chelating and monoamine oxidase B (MAO-B) inhibition. This combination endowed the hybrids with good capacity to inhibit MAO-B as well as excellent iron-chelating effects. The pFe³⁺ values of the compounds were ranging from 16.91 to 20.16, comparable to more potent than the reference drug deferiprone (DFP). Among them, compound 18d exhibited the most promising activity against MAO-B, with an IC₅₀ value of 87.9 nM. Moreover, compound 18d exerted favorable antioxidant activity, significantly reversed the amyloid-β_1-42 (Aβ_1-42) induced PC12 cell damage. More importantly, 18d remarkably ameliorated the cognitive dysfunction in a scopolamine-induced mice AD model. In brief, a series of hybrids with potential anti-AD effect were successfully obtained, indicating that the design of iron chelators with MAO-B inhibitory and antioxidant activities is an attractive strategy against AD progression.

Full text of DOI:10.1016/j.bmc.2020.115550

Journal Pre-proofs
Rational design, synthesis and biological evaluation of novel multitargeting
anti-AD iron chelators with potent MAO-B inhibitory and antioxidant activity
Xiaoying Jiang, Jianan Guo, Yangjing Lv, Chuansheng Yao, Changjun
Zhang, Zhisheng Mi, Yuan Shi, Jinping Gu, Tao Zhou, Renren Bai, Yuanyuan
Xie
PII:
S0968-0896(20)30380-1
https://doi.org/10.1016/j.bmc.2020.115550
BMC 115550
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 January 2020
2 May 2020
5 May 2020
Please cite this article as: X. Jiang, J. Guo, Y. Lv, C. Yao, C. Zhang, Z. Mi, Y. Shi, J. Gu, T. Zhou, R. Bai, Y.
Xie, Rational design, synthesis and biological evaluation of novel multitargeting anti-AD iron chelators with
potent MAO-B inhibitory and antioxidant activity, Bioorganic & Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.bmc.2020.115550
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Rational design, synthesis and biological evaluation of novel
multitargeting anti-AD iron chelators with potent MAO-B inhibitory
and antioxidant activity
a
b
b
b
a
Xiaoying Jiang , Jianan Guo , Yangjing Lv , Chuansheng Yao , Changjun Zhang ,
b
b
b
c
b,
a,
Zhisheng Mi , Yuan Shi , Jinping Gu , Tao Zhou , Renren Bai *, Yuanyuan Xie
b,
*
a
Collaborative Innovation Centre of Yangtze River Delta Region Green
Pharmaceuticals, Zhejiang University of Technology, Hangzhou, P.R. China.
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou,
P.R. China.
b
c
School of Food Science and Biotechnology, Zhejiang Gongshang University,
Hangzhou, P.R. China
Correspondence:
Dr. Renren Bai, College of Pharmaceutical Sciences, Zhejiang University of
Technology,
Hangzhou
310014,
China.
Email:
renrenbai@zjut.edu.cn,
renrenbai@126.com
Dr. Yuanyuan Xie, Key Laboratory for Green Pharmaceutical Technologies and
Related Equipment of Ministry of Education, College of Pharmaceutical Sciences,
Zhejiang University of Technology, Hangzhou 310014, China. Email:
xyycz@zjut.edu.cn
1

Abstract: A series of (3-hydroxypyridin-4-one)-coumarin hybrids were developed and
investigated as potential multitargeting candidates for the treatment of Alzheimer's
disease (AD) through the incorporation of iron-chelating and monoamine oxidase B
(MAO-B) inhibition. This combination endowed the hybrids with good capacity to
3
+
inhibit MAO-B as well as excellent iron-chelating effects. The pFe values of the
compounds were ranging from 16.91 to 20.16, comparable to more potent than the
reference drug deferiprone (DFP). Among them, compound 18d exhibited the most
promising activity against MAO-B, with an IC50 value of 87.9 nM. Moreover,
compound 18d exerted favorable antioxidant activity, significantly reversed the
amyloid-β1-42 (Aβ1-42) induced PC12 cell damage. More importantly, 18d remarkably
ameliorated the cognitive dysfunction in a scopolamine-induced mice AD model. In
brief, a series of hybrids with potential anti-AD effect were successfully obtained,
indicating that the design of iron chelators with MAO-B inhibitory and antioxidant
activities is an attractive strategy against AD progression.
Keywords: Alzheimer’s disease; 3-Hydroxypyridin-4-one; Iron chelators; MAO-B
inhibitors; Multitarget agents
1
. Introduction
AD is an age-related chronic, fatal, devastatingly progressive and irreversible
1
neurodegenerative disorder. The symptoms of AD features memory loss and cognitive
impairments, including impaired judgment, decision-making, and orientation,
accompanied by behavioral and psychological symptoms of dementia.2 The
pathogenesis of AD is highly complicated, including deficits of acetylcholine (ACh),
abnormal Aβ deposition and accumulation, tau hyperphosphorylation,
neuroinflammation, oxidative stress and dyshomeostasis of biometals.3-8 However, the
exact etiology remains elusive, which has limited the development of pharmacotherapy
2

of AD based on only one single target. The current clinically available anti-AD drugs
just include donepezil, tacrine, rivastigmine, galantamine and memantine. Therefore,
the multitarget-directed ligands (MTDLs) strategy, aiming to this multifaceted disease,
has drawn much attention as a promising therapeutic approach.9-12
Among the various pathogeneses in AD, metal ion disorder, especially iron ion
and oxidative stress, has been indicated to be associated with the progressive
neurodegeneration and abnormal protein aggregation.13,14 The metal ion levels in AD
patients is 3-7 times higher than those in healthy individuals, leading to promoting tau
1
5
hyperphosphorylation and Aβ aggregation. Damage to iron homeostasis or defective
transport mechanisms will cause the accumulation of highly toxic metals, eventually
resulting in the formation of reactive oxygen species (ROS), oxidative stress,
mitochondrial damage and cell death through free radical reactions (such as the Fenton
reaction and Haber-Weiss reaction).16-18 Therefore, iron chelators can be used as
potential therapeutic agents for AD, which have also been shown to be effective against
neurodegenerative diseases in a series of animal models and applied in a number of
clinical trials.19-21 In addition, oxidative stress, as one of the earliest events in AD
pathogenesis, is also related to monoamine oxidase (MAO), particularly MAO-B.
MAO-B is considered as a potential target for β-phenethylamine such as dopamine with
2
2
the concomitant production of hydrogen peroxide. It is a key intermediate in the
Fenton reaction that produces ROS and increases oxidative stress implicated in the
progress of AD.23,24 Therefore, MAO-B inhibitors have attracted much attention in AD
treatment in recent years.25
The multifactorial points of AD have promoted the application of MTDLs strategy
in iron chelators.19,26,27 The challenge of designing pluripotent and/or multi-target drugs
is to combine multiple pharmacological fragments into one basic active molecule.28
DFP is an iron chelator widely used for the treatment of thalassemia and other systemic
iron overload diseases, showing high affinity and selectivity for iron.29,30 The
characteristics of nontoxicity, low molecular weight and suitable structure that make
DFP more easily to be optimized, and it has been considered as a lead compound to
3

design MTDLs for treating AD.31-35
Moreover, coumarin derivatives, with a wide range of biological activities, are
potential and valuable resources for the prevention and treatment of central nervous
system diseases. Structure-activity relationship (SAR) study demonstrated that the
MAO-B inhibitory activity of coumarin derivatives could be modulated by the
optimization at position C-3, C-4 and C-7.36-46
In this work, we rationally designed, synthesized and comprehensively biological
evaluated a series of novel multitargeting iron chelators with potent MAO-B inhibitory
and antioxidant activity, based on chemically hybridizing coumarin into the iron
chelator 3-hydroxypyridin-4-one (Fig. 1).44,45,47
Fig. 1. The design strategy for the multitargeting anti-AD hybrids.
2
2
. Results and discussion
.1 Chemistry
The synthetic strategies for the multitargeting agents 18a-f, 20a-z and 24 are
outlined in Schemes 1-7. Commercially available maltol 1 was used as the starting
material for the preparation of intermediates 3 and 4. Firstly, compound 2 was obtained
by protecting the 3-hydroxyl group of material 1 using iodomethane, benzyl bromide
or 4-methoxylbenzyl chloride. The resulting protected maltol was then reacted with
ethylenediamine or 3-aminopropanoic acid to produce intermediates 3 and 4,
respectively, under mild conditions (Scheme 1).48,49
Scheme 2 illustrates the synthetic route employed for the preparation of
4

compounds 8 and 11 starting from commercially available kojic acid 5. The 2-
hydroxymethyl residue was chlorinated by sulfoxide chloride. Then the reduction of
chlorokojic acid with zinc dust and concentrated hydrochloric acid resulted in the
5
0
production of 6. Similarly, the protection of the 3-hydroxyl group of 5 and 6 was
achieved by methylation using iodomethane or benzylation using benzyl bromide. The
hydroxymethyl function was oxidized to a carboxylic acid by Jones reagent, which was
subsequently decarboxylated by heating in 1-methyl-2-pyrrolidinone to yield the
5
1
protected pyrones 10. The oxygen of 7 and 10 was substituted by nitrogen via reaction
with ethylenediamine to obtain intermediates 8 and 11, respectively.
The synthesis of coumarin-3-carboxylic acids was shown in Schemes 3-4. For
ethyl coumarin-3-carboxylates 13a-j, piperidine and glacial acetic acid were used as
catalysts in the reaction of substituted salicylaldehydes with diethyl malonate through
the Knoevenagel Condensation. While 15a-r were synthesized by O-alkylation or O-
benzylation of 13h-j with alkyl bromides or benzyl bromides in N,N-
dimethylformamide in the presence of potassium carbonate. The ethyl esters were
hydrolyzed with aqueous solution of sodium hydroxide to the corresponding coumarin-
5
2
3
-carboxylic acids 14a-g and 16a-r. Subsequently, coumarin-3-carboxylic acids were
coupled with 2-mercaptothiazoline using dicyclohexylcarbodiimide (DCC) and 4-
dimethylamino)pyridine (DMAP) to obtain the activated mercaptothiazoline coumarin
(
key intermediates, which could directly react with 3, 8 or 11 to provide amide
derivatives 17a-f and 19a-w. Finally, the designed compounds 18a-f and 20a-z were
obtained by boron tribromide (BBr ) or boron trichloride (BCl ) to remove the
3
3
protecting groups. One of the most difficulties was the selective debenzylation of 19f-
w, which should be strictly controlled the reaction conditions. We have found that
benzyl at position 6 and 4-methoxybenzyl at position 7 of coumarin were easier to be
removed than benzyl on hydroxypyridinone. Therefore, 3c was chosen to couple with
1
6a and 16n (Schemes 5-6).
Furthermore，compound 22 was synthesized by using salicylaldehyde and N-
5
3
acetylglycine followed by regioselective hydrolysis (Scheme 7). Then 22 and 4a were
5

directly condensed by DCC followed by demethylation to obtain aminoacyl compound
2
4. The aim was to compare the difference of activity between amides with aminoacyl.
O
OR
ii
3a, R= CH3
3
b, R= Bn
N
3c, R= 4-CH O-Bn
3
O
O
O
OH
OR
i
NH2
O
O
N
1
2a, R= CH3
OR
2b, R= Bn
iii
2c, R= 4-CH O-Bn
3
4a, R= CH3
COOH
Scheme 1. Reagents and conditions: (i) Iodomethane or benzyl bromide or 4-methoxylbenzyl
chloride, K CO , acetone, reflux, 3-8 h. (ii) Ethylenediamine, NaOH, ethanol, water, 70 ℃, 1.5 h.
2
3
(iii) 3-Aminopropanoic acid, 10N NaOH, ethanol, reflux, 8 h.
O
O
O
O
N
OH
OR
OR
i
ii
iii
8
8
a, R= CH3
b, R= Bn
O
O
6
7a, R= CH3
b, R= Bn
OH
7
NH2
HO
O
O
N
O
O
O
5
OR
OR
OR
iv
v
iii
1
1
1a, R= CH3
1b, R= Bn
HOOC
O
9
a, R= CH3
10a, R= CH3
10b, R= Bn
9b, R= Bn
NH2
Scheme 2. Reagents and conditions: (i) 1) SOCl , r.t., 2 h; 2) Zn/HCl, water, 75 ℃, 4 h. (ii)
2
Iodomethane or benzyl bromide, K CO , acetone, reflux, 3-8 h. (iii) Ethylenediamine, NaOH,
2
3
ethanol, water, 70 ℃, 1.5 h. (iv) 1) Iodomethane or benzyl bromide, K CO , acetone, reflux, 3-8 h;
2
3
2
) Jones reagent, acetone, reflux, 40 min. (v) 1-Methyl-2-pyrrolidinone, reflux, 4 h.
O
O
O
O
R1
R2
CHO
OH
R1
R2
R1
R2
OEt
OH
i
ii
O
O
R3
R3
R3
12a-j
13a-j
14a-g
1
2
3
1
2
3
1
1
1
1
1
2a, 13a, 14a, R = H, R = H, R = H
12f, 13f, 14f, R = H, R = CH3, R = H
1
2
3
1
2
3
2b, 13b, 14b, R = H, R = CH O, R = H
12g, 13g, 14g, R = H, R = H, R = CH3
3
1
2
3
1
2
3
2c, 13c, 14c, R = CH O, R = H, R = H
12h, 13h, R = OH, R = H, R = H
3
1
2
3
1
2
3
2d, 13d, 14d, R = H, R = H, R = CH O
12i, 13i, R = H, R = OH, R = H
3
1
2
3
1
2
3
2e, 13e, 14e, R = CH , R = H, R = H
12j, 13j, R = H, R = H, R = OH
3
Scheme 3. Reagents and conditions: (i) Diethyl malonate, piperidine, acetic acid, ethanol, reflux, 4-
6

3
0 h. (ii) 10% NaOH, ethanol, water, reflux, 15 min.
O
O
O
O
O
O
R1
R2
R4
R5
R4
R5
OEt
OEt
OH
i
ii
O
O
O
R3
R6
R6
13h-j
15a-r
16a-r
4
5
6
4
5
6
4
5
6
1
1
1
1
1
1
5a, 16a, R = OBn, R = H, R = H
15g, 16g, R = H, R = cyclohexylmethoxy, R = H 15m, 16m
,
R = H, R = 4-CF3-OBn, R = H
4
5
6
4
5
6
4
5
6
5b, 16b, R = H, R = OBn, R = H
15h, 16h, R = H, R = 3-Cl-OBn, R = H
15n, 16n
15o, 16o
15p, 16p
15q, 16q
,
,
R = H, R = 4-CH3O-OBn, R = H
4
5
6
4
5
6
4
5
6
5c, 16c, R = H, R = H, R = OBn
15i, 16i, R = H, R = 3-F-OBn, R = H
R = H, R = 2-NO2-OBn, R = H
4
5
6
4
5
6
4
5
6
5d, 16d, R = H, R = OCH CH , R = H
15j, 16j, R = H, R = 4-F-OBn, R = H
2
3
, R = H, R = 3-CH3-OBn, R = H
4
5
6
4
5
6
4
5
6
5e, 16e, R = H, R = OCH CH CH , R = H 15k, 16k, R = H, R = 2,5-F-OBn, R = H
2
2
3
, R = H, R = 4-iPr-OBn, R = H
4
5
6
4
5
6
4
5
6
5f, 16f, R = H, R = OH CC CH , R = H
15l, 16l, R = H, R = 3,5-F-OBn, R = H
15r, 16r
, R = H, R = 4-tBu-OBn, R = H
2
Scheme 4. Reagents and conditions: (i) Alkyl bromides or benzyl bromides, K CO , DMF, r.t., 48
2
3
h. (ii) 10% NaOH, ethanol, water, reflux, 15 min.
O
R3
O
R3
O
OR
O
O
O
O
O
N
N
R3
R2
OH
N
OR
N
OH
N
i
ii
+
H
H
R2
R2
R1
O
R1
R1
O
O
O
NH2
a-b, 8a-b, 11a-b
3
14a-b
17a-f
18a-f
1
3
2
1
3
2
1
1
1
1
1
1
7a, R= CH , R =R = H, R = CH
18a, R =R = H, R = CH
3
3
3
1
2
3
1
2
3
7b, R= CH , R =R = H, R = CH
18b, R =R = H, R = CH
3
3
3
1
2
3
1
2
3
7c, R= CH , R =R =R = H
18c, R =R =R = H
3
1
2
3
1
2
3
7d, R= Bn, R = CH O, R = CH , R = H
18d, R = CH O, R = CH , R = H
3
3
3 3
1
3
2
1
3
2
7e, R= Bn, R = CH O, R = CH , R = H
18e, R = CH O, R = CH , R = H
3 3
3
3
1
2
3
1
2
3
7f, R= Bn, R = CH O, R =R = H
18f, R = CH O, R =R = H
3
3
Scheme 5. Reagents and conditions: (i) 1) 2-Mercaptothiazoline, DCC, DMAP, DCM, r.t., 24 h; 2)
3
a-b, 8a-b or 11a-b, DCM, r.t., 24 h. (ii) BBr , anhydrous DCM, -48℃ to r.t., 12 h; or BCl ,
3
3
anhydrous DCM, -48℃ to r.t., 12 h.
O
O
O
OR
O
O
O
O
O
R1
R2
R1
R2
R1
R2
N
N
OH
N
H
OR
N
OH
N
i
ii
+
H
O
O
O
O
R3
R3
R3
NH2
b-c
3
14b-g, 16a-r
19a-w
20a-z
1
2
3
1
2
3
1
2
3
1
1
2
2
2
1
1
1
9a, 20a, R = CH O, R = H, R = H 19f, 20i, R = OBn, R = H, R = H
19o, 20r, R = H, R = 4-F-OBn, R = H
3
1
2
3
1
2
3
1
2
3
9b, 20b, R = H, R = H, R = CH O 19g, 20j, R = H, R = OBn, R = H
19p, 20s, R = H, R = 2,5-F-OBn, R = H
3
1
2
3
1
2
3
1
2
3
0c, R = OH, R = H, R = H
19h, 20k, R = H, R = H, R = OBn
19q, 20t, R = H, R = 3,5-F-OBn, R = H
1
2
3
1
2
3
1
2
3
0d, R = H, R = OH, R = H
19i, 20l, R = H, R = OCH2CH3, R = H
19r, 20u, R = H, R = 4-CF3-OBn, R = H
1
2
3
1
2
3
1
2
3
0e, R = H, R = H, R = OH
19j, 20m, R = H, R = OCH2CH2CH3, R = H
19s, 20v, R = H, R = 4-CH3O-OBn, R = H
1
2
3
1
2
3
1
2
3
9c, 20f, R = CH , R = H, R = H
19k, 20n, R = H, R = OH2CC CH, R = H
19t, 20w, R = H, R = 2-NO2-OBn, R = H
3
1
2
3
1
2
3
1
2
3
9d, 20g, R = H, R = CH , R = H
19l, 20o, R = H, R = cyclohexylmethoxy, R = H 19u, 20x, R = H, R = 3-CH3-OBn, R = H
3
1
2
3
1
2
3
1
2
3
9e, 20h, R = H, R = H, R = CH
19m, 20p, R = H, R = 3-Cl-OBn, R = H
19v, 20y, R = H, R = 4-iPr-OBn, R = H
3
1
2
3
1
2
3
1
9n, 20q, R = H, R = 3-F-OBn, R = H
19w, 20z, R = H, R = 4-tBu-OBn, R = H
Scheme 6. Reagents and conditions: (i) 1) 2-Mercaptothiazoline, DCC, DMAP, DCM, r.t., 24 h; 2)
7

3
b or 3c, DCM, r.t., 24 h. (ii) BCl ,anhydrous DCM, -78℃ to r.t., 12 h.
3
H
CHO
OH
N
NH2
O
i
ii
O
O
O
O
12a
21
22
O
O
H
N
H
N
N
N
iii
O
iv
OH
O
O
O
O
O
O
23
24
Scheme 7. Reagents and conditions: (i) N-acetylglycine, anhydrous sodium acetate, acetic
anhydride, reflux, 8 h. (ii) 70% H SO , 120 ℃, 3 h. (iii) 4a, DCC, DCM, r.t., 24 h. (iv) BBr ,
2
4
3
anhydrous DCM, -48℃ to r.t., 12 h.
2
.2 Iron-chelating activity test
The pKa values and iron affinity constants for iron(III) of all of the compounds
were determined by the automatic spectrophotometric titration system and the results
were presented in Table 1 with deferiprone as the reference drug.51 All of these
3
+
synthesized compounds exhibited excellent pFe values ranging from 16.91 to 20.16
Table 1). Compounds 18d, 20y and 20z displayed the most potent iron-chelating
(
3
+
activity with pFe values of 18.93, 18.98 and 20.16, respectively, superior to that of
3
+
deferiprone (pFe = 17.50). The superior affinity of deferiprone analogs for iron results
from the extensive delocalization of electrons in its resonance structures (Scheme 8)
2
9
because of the competition effect at different pH values in aqueous solution. In
general, all hydroxypyridinone derivatives show preferable iron-chelating ability.
Because of the amide bond, the spectrophotometric analysis of the titrations of all
the compounds were fitted to three pKa values, such as compounds 18d and 20w (Fig.
2
). However, only the pK value (< 3.40) assigned to the protonation of the 4-oxo
a1
group and the pK value in the range of 8.74-10.05 assigned to the dissociation of the
a2
3
1
3
-hydroxyl group determine the iron chelation of the compounds. The UV profiles of
8d and 20w over the pH range of 2.0-10.5 on analysis yielded two main pKa values,
.27, 9.70 and 3.24, 9.74, respectively, which show the pH dependence of the ligand
8

ionization equilibrium (Fig. 2). Obviously, almost all pKa values of the hybrids are
lower than the corresponding value of deferiprone, because of substituents on nitrogen
influence the delocalization of the negative charge on 4-oxygen.
By analyzing the absorption spectra of iron(III)/ligand solution at different pH
values, the concentration of the species (FeL, FeL , and FeL ) can be calculated as a
2
3
function of pH values, obtaining the stability constant: logβ (14.91), logβ (26.24) and
1
2
logβ (35.29). As found with typical 3-hydroxypyridin4-one ligands, there are three
3
iron(III) complexes with the neutral 3:1 complex dominating in the neutral pH range
3
+
(
6-9) (Figure 3). The pFe value of 18d was calculated to be 18.93, which is higher
than that of DFP (17.50) under the same experimental conditions.
OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
H
N
H
OH
N
H
O
O
O
O
O
O
O
O
O
O
O
O
pKa1
pKa2
OH
OH
O
O
O
N
H
N
H
OH
N
H
Scheme 8. Proton equilibria and resonance structures of 18d
3
+
a
Table 1. The pKa and pFe values of the target compounds
Compound
8a
8b
8c
8d
8e
8f
0a
0b
0c
0d
0e
pKa1
3.22
3.30
3.00
3.27
3.34
3.07
3.27
3.22
3.33
2.75
3.35
pKa2
9.59
9.05
8.74
9.70
9.07
8.74
9.58
9.55
10.05
9.92
9.98
Logβ1b
14.02
12.92
12.93
14.91
13.90
12.91
14.16
14.20
15.08
13.81
14.44
Logβ2c
25.02
23.34
23.43
26.24
24.48
23.36
25.31
25.36
26.49
25.19
26.03
Logβ3c
33.90
31.77
31.77
35.29
32.96
31.85
32.83
34.11
34.12
34.19
34.77
pFe3+
17.88
17.34
18.24
18.93
18.47
18.31
17.10
18.21
17.19
17.19
17.62
1
1
1
1
1
1
2
2
2
2
2
9

2
0f
0g
0h
3.33
3.32
3.30
3.25
3.25
3.28
3.30
3.31
3.32
3.29
3.26
3.32
3.27
3.20
3.25
3.37
3.24
3.24
3.27
3.18
3.17
3.29
3.67
3.67
3.61
9.67
9.75
9.75
9.32
9.70
9.58
9.75
9.75
9.68
9.76
9.56
9.65
9.76
9.74
9.78
9.83
9.77
9.74
9.72
9.79
9.73
9.48
9.84
9.84
9.78
14.10
14.16
14.43
13.68
14.09
14.02
14.20
14.57
14.18
14.27
14.39
14.13
14.17
14.12
14.26
14.04
14.11
15.14
14.20
14.56
14.75
13.80
14.43
15.08
15.03
25.37
25.44
25.76
24.26
24.93
25.39
25.57
26.04
25.50
25.13
24.95
24.67
25.11
24.89
24.96
25.12
24.81
25.91
24.45
25.55
26.09
24.78
25.68
27.40
27.42
34.15
34.28
35.23
32.04
33.24
32.87
34.72
34.59
34.13
34.66
34.55
33.30
33.51
33.65
33.46
34.87
33.30
34.62
35.20
35.61
36.63
33.34
34.23
36.98
37.35
17.90
17.79
18.71
16.91
16.95
17.16
18.21
18.12
17.86
18.11
18.60
17.12
17.04
17.19
16.92
18.11
16.97
18.17
18.77
18.98
20.16
17.65
17.50
20.20
20.74
2
2
2
0i
2
0j
2
0k
0l
0m
0n
2
2
2
2
0o
0p
0q
2
2
2
0r
0s
0t
0u
0v
0w
2
2
2
2
2
2
2
0x
0y
2
0z
2
4
DFP
DFPa
DFPd
aThecompounds were measured in a 0.1 M KCl solution.
bThe compounds were tested in DMSO: KCl (0.1 M) = 1:4 (v/v) to address the solubility issue.
cThe compounds were tested in DMSO: KCl (0.1 M) = 1:1.5 (v/v) to address the solubility issue.
dThe data from reference tested in a 0.1 M KCl solution.45
1
0

Fig. 2. The pH-dependent UV spectra of compounds18d and 20w. A-1. The pH-dependence of the
titration spectrum of compound 18d. A-2. The pKa values of compound 18d over the pHrange of
2
.0-10.5 in 0.1 M KCl at 25 °C. B-1. The pH-dependence of the titration spectrum of compound
0w. B-2. The pKa values of compound 20w over the pH range of 2.0-10.5 in 0.1 M KCl at 25 °C.
2
Fig. 3. The pH-dependent UV spectra of compound 18d. A. The pH-dependence of the spectrum of
compound 18d in the presence of Fe3+ over thepH range 0.955 and 1.925 in 0.1M KCl : DMSO =
3
+
4
:1 (v/v) at 25 °C, [Fe ] = 1.0 μM, [18d] = 1.1 μM. B. The pH-dependence of the spectrum of
3
+
compound 18d in the presence of Fe over the pH range 2.572 and 9.730 in 0.1 M KCl : DMSO =
3
+
3+
3
:2 (v/v) at 25 °C, [Fe ] = 1.0 μM, [18d] = 5.0 μM. C. Speciation plot of Fe /18d as measured by
the percentage formation relative to [iron(III)]total as a function of pH. This plot was calculated
from the affinityconstants reported in Table 1 and the iron(III) hydrolysis constants were as follows:
1
1

FeOH = -2.563, Fe(OH) = -6.205, Fe(OH) = -15.100, Fe (OH) = -2.843, Fe (OH) = -6.059,
2
3
2
2
3
4
Fe(OH) = -21.883.
4
2
.3 MAO-B inhibition assay
The MAO-B inhibitory activities of all the targeted compounds were detected.
They were preliminarily evaluated at 100 nM and pargyline (a MAO-B inhibitor) was
selected as the positive control. As shown in Table 2, almost all of the compounds
exhibited significant MAO-B inhibitory effects with around 50% inhibition at the
concentration of 100 nM. Compound 18d showed the most potent inhibitory effect and
was higher than the reference drug, pargyline. The corresponding IC50 values of
compounds with more than 40% inhibition were tested and the results were shown in
Table 3. There was no doubt that compound 18d was proved to be the most potent
hybrid exhibiting the highest MAO-B inhibitory activity (IC = 87.9 nM), showing
5
0
more efficient than that of pargyline (IC = 107.3 nM). The results of compounds 18a-c
5
0
and 18d-f indicated that 2-methyl substituted 3-hydroxypyridin-4-one was the most
optimal. In comparison to the compounds modified at C-6 and C-8 of the coumarin
ring, C-7 modified ones exhibited better MAO-B inhibitory activities. Compounds 20p-
z were slightly more potent than that of the alkoxy C-7 substituted hybrids (20l-o).
Moreover, compounds possessing electron-withdrawing groups (Cl, F, CF and NO )
3
2
at ortho-, meta- or para-position of the benzyloxy phenyl ring displayed better MAO-
B inhibition. While compound 20w showed a lower IC value of 98.6 nM. In addition,
5
0
the amide bond inversion did not increase the inhibitory activity (24).
Table 2. The MAO-B inhibitory activities of the targeted compounds
Inhibitory rate ± SEM
%, 100 nM)
Inhibitory rate ± SEM
Compound
Compound
(
(%, 100 nM)
1
8a
8b
8c
8d
8e
46.88 ± 2.40
44.78 ± 1.86
46.10 ± 2.24
65.45 ± 2.76
35.45 ± 0.48
20l
20m
20n
20o
20p
46.25 ± 0.07
45.90 ± 0.22
20.68 ± 1.81
44.60 ± 0.87
47.37 ± 0.79
1
1
1
1
1
2

1
8f
0a
0b
0c
0d
0e
0f
0g
0h
31.34 ± 0.75
38.53 ± 2.47
46.00 ± 1.30
46.41 ± 1.04
29.52 ± 1.99
46.20 ± 1.85
34.79 ± 1.69
44.32 ± 1.61
43.96 ± 3.33
47.66 ± 0.37
30.11 ± 1.63
46.54 ± 0.29
20q
20r
49.07 ± 0.66
48.13 ± 0.79
49.09 ± 0.20
47.38 ± 0.27
47.43 ± 0.19
47.56 ± 0.27
50.56 ± 0.36
44.63 ± 3.29
45.69 ± 0.75
45.28 ± 0.47
43.54 ± 1.55
53.00 ± 0.98
2
2
20s
2
20t
2
20u
2
20v
2
20w
20x
2
2
20y
2
0i
20z
2
0j
24
2
0k
Pargyline
Table 3. The IC values of the selected compounds
5
0
Compound
8a
8b
8c
IC (nM)
Compound
20p
IC (nM)
5
0
50
1
173.3
129.3
194.6
87.9
135.8
107.9
127.8
114.9
125.3
118.5
142.9
98.6
1
20q
1
20r
1
2
8d
0b
20s
110.7
187.8
157.8
162.7
156.5
100.2
130.7
108.1
134.6
142.7
20t
2
2
0c
0e
20u
20v
2
0g
20w
20x
2
0h
143.7
117.3
122.1
159.6
107.3
2
0i
20y
2
0k
20z
2
0l
0m
0o
24
2
Pargyline
2
2
.4 Molecular modeling
To evaluate the binding mode of compound 18d with MAO-B, molecular
modeling simulation was performed, using the CDOCKER program of the Discovery
Studio 2016 software based on the crystal structure of human MAO-B (PDB entry
2
V5Z). As shown in Fig. 4, the ligand can pass through both the entrance and substrate
cavities of the enzyme. The 7-methoxy group located in the entrance cavity and
interacted with PRO104. The coumarin moiety of 18d has occupied the substrate cavity
and had several lipophilic interactions with ILE 199, ILE 316, CYS 172, TYR 326 and
1
3

LEU 171. While the hydroxypyridinone moiety was toward the FAD cofactor and had
a hydrogen bond interaction between the carbonyl and FAD. Pi-Pi interaction was
observed between the pyridinone and TYR 398, and Pi-alkyl interaction was observed
between the methyl of pyridinone and TYR 326. Moreover, the carbonyl of amide
formed a hydrogen bond with residue GLN 206. The binding mode showed important
interactions between hydroxypyridinone moiety with residues around it and FAD,
indicating that hydroxypyridinone moiety not only had iron chelation ability but also
worked as a key fragment in binding with MAO-B. Those observations might explain
the activity of the 18d.
Fig. 4. Best docking poses of compound 18d at the active site of MAO-B.The most relevant
interacting residues are presented in gray carbons polytube, FAD cofactor is shown as orange
carbons polytube and ligands as yellow carbons polytube. A. The 3D interaction diagram of 18d
1
4

with MAO-B. B.The planar interaction diagram of 18d with MAO-B.
2
.5 Cytotoxicity and antioxidant activity assays
The in vitro cell viability of the compound 18d was further determined by MTT
assay against neural pheochromocytoma-derived PC12 cell line. As shown in Fig. 5A,
in all of the concentrations of 18d (3.13, 6.25, 12.5, 25, 50 μM), its cell viability was
higher than 90% except at 50 μM. More than 98% of the cell viability was obtained
when the concentration was lower than 12.5 μM. Therefore, the concentration of 10 μM
was chosen as the working concentration in the following tests.
After Aβ1-42 induced cellular damage, the cell viability of PC12 cell line treated
with compound 18d at 10 μM was also determined with memantine as the reference
drug (Fig. 5B). Obviously, compared with the model group, the cell viability was hardly
changed when treated with memantine (55.23% vs. 54.88%). However, when cells
treated with 18d, the cell viability increased to 88.15%, comparable to the negative
control group, indicating that 18d had cytoprotective activity against Aβ1-42 induced
cellular damage.
The production of ROS in cells can significantly lead to oxidative stress and
oxidative damage. Therefore, the antioxidant activity of 18d was further investigated.
Intracellular ROS production was detected by using the peroxide-sensitive fluorescent
probe 2, 7-dichlorofluorescein (DCFH-DA). As shown in Fig. 5C and Fig. 6, DCFH
staining data displayed that 18d can remarkablely reduce the intracellular ROS levels
with a significant decrease of fluorescence compared with model group (8.20% vs.
3
0.7%), while memantine showed no antioxidant activity. Consequently, these results
indicated that 18d had cytoprotective activity against antagonizing oxidative stress.
1
5

Fig. 5. The cell viability and antioxidant activity measured by the MTT assay and DCFH-
DArespectively. A.The cytotoxicity at various concentrations of 18d. B. The cell viability of Aβ1-42
induced cells at 10 μM of 18d. C. Inhibition of intracellular ROS production at 10 μM of
1
8dwithmemantine as the positive referenceagent. The concentration of Aβ1-42 was 40 μM and the
concentration of compound 18d and memantine was 10 μM.
1
6

Fig. 6. Theintracellular ROS generation of compound 18d. The concentration of compound 18d and
memantine was 10 μM. The fluorescence of cell nucleus is blue and the cytoplasm is green. In each
column, the first photo means the fluorescence of cell nucleus; the second means the fluorescence
of cytoplasm and the third meansthe superposition of the nucleus and cytoplasm.
2
.6 In vivo behavioral studies
The research and development of anti-AD agents in behavioral research are of
great guiding significance. Based on the above multiple evaluations, compound 18d
with the best multipotent activities was selected for in vivo behavior study using a
Morris water maze model.
The animal model was built on the basis of scopolamine-induced cognition-
impaired adult ICR mice and applied for the cognitive improvement effects evaluation
of 18d (15 mg/kg). Pargyline, memantine and tacrine (15 mg/kg) were used as the
reference drugs. Pargyline, memantine, tacrine and compound 18d were
intraperitoneally injected to the ICR mice 30 min before intraperitoneal administration
of scopolamine (5 mg/kg) or phosphate-buffered saline solution for 10 consecutive days.
The following test included 2 days of learning and memory training, 3 days cognitive
evaluation and a probe trial on the fifth day with the above intraperitoneal
1
7

administration sequentially. The data of all the groups on the fifth day are shown in Fig.
7
.
Compared to the control group, scopolamine led to a remarkable delay of the
#
latency to target (13.91 ± 1.85 s vs. 29.54 ± 5.77 s, p < 0.05) and the prolonged distance
#
(
2.69 ± 0.33 m vs. 6.56 ± 1.27 m, p < 0.05). Moreover, the entries significantly
decreased (4.00 ± 0.33 vs. 2.19 ± 0.16, ###p < 0.001), indicating that the cognitive
impairment mice model was successfully built. Pargyline, memantine and tacrine all
ameliorated the cognitive impairment and the latency to target was reduced to 20.85 ±
4
.45 s, 13.42 ± 3.71 s and 18.03 ± 2.78 s respectively. In comparison to pargyline and
tacrine, 18d markedly reduced the latency to target (14.94 ± 3.01 s, *p < 0.05).
Compound 18d considerably ameliorated the cognitive impairment of the treated mice
and was much better than pargyline and tacrine, but less effective than memantine.
The distance and entries to target and the trajectories of the mice in each group
were also analyzed (Fig. 6-7). Compared to the model group, pargyline slightly
shortened the distance (4.37 ± 0.90 m) and increased the entries to target (3.81 ± 0.38,
*
*p < 0.01), while tacrine worked better (4.28 ± 0.70 m; 4.63 ± 0.32, ****p < 0.0001).
Memantine was proved to be the most potent anti-AD agent (3.52 ± 0.77 m; 5.19 ±
.64, ***p < 0.001). When treated with 18d, the distance to target was significantly
shortened (3.87 ± 0.98 m) and the entries to target were almost the same as tacrine (4.63
0.56, ***p < 0.001). These results also supported by trajectory analysis. As shown in
0
±
Fig. 8, the trajectory of the mice in the scopolamine model group was very long and
disordered, while pargyline and tacrine groups (Fig. 8C and E) showed shortened
distances, but still longer than the control group (Fig. 8A). Mice treated with 18d (Fig.
8
F) was proved to be better than pargyline and tacrine but less effective than memantine
(Fig. 8D), which showed a similar distance to that of the normal mice. Taken together,
these results supported that 18d remarkably ameliorated the cognition impairment
induced by scopolamine.
1
8

-1
Fig. 7. Anti-AD effects of intraperitoneal injection of pargyline (15 mg kg ), memantine (15 mg
-1
-1
-1
kg ), tacrine (15 mg kg ),18d (15 mg kg ) on scopolamine-induced cognitive impairment in ICR
mice determined by the Morriswater maze test. A. The latency to the target. B. The distance to
thetarget. C. The number of times enters target quadrant. Data were presented as the mean ± SEM
n = 8; p < 0.05, p < 0.01, ###p < 0.001, Control group vs. scopolamine model group; *p < 0.05,
#
##
(
*
*p <0.01, ***p < 0.001,****p < 0.0001, 18d or pargyline or memantine or tacrine group vs.
scopolamine model group).
Fig. 8. The trajectories of mice in control (A), model (B), pargyline (C), memantine (D), tacrine (E)
and 18d (F) groups in the Morris water maze test.
3
. Conclusion
Based on the multitargeting drug development strategy for the treatment of AD, a
novel series of hybrids by conjugating 3-hydroxypyridin-4-one amide derivatives with
1
9

coumarin derivatives were designed, synthesized and pharmaceutically evaluated. They
were designed by combining an anti-MAO-B pharmacophore (coumarin) with an iron-
chelating drug (deferiprone) through an amide linker with an appropriate chain length
to enable a dual-binding mode.
The designed compounds all displayed preferable iron-chelating ability and
showed promising anti-MAO-B activities in the in vitro assays. Compounds 18d, 20y
3
+
and 20z displayed the most potent iron-chelating activity with pFe values of 18.93,
3
+
1
8.98 and 20.16, respectively, superior to that of deferiprone (pFe = 17.50). Moreover,
compound 18d (IC = 87.9 nM) and 20w (IC = 98.6 nM) was proved to be the most
5
0
50
effective MAO-B inhibitor, superior to that of the reference drug pargyline (IC =
5
0
1
07.3 nM). Additionally, compound 18d exerted cytoprotective activity against Aβ1-42
induced cell damage and oxidative stress. Most importantly, compound 18d
significantly ameliorated the cognition impairment of mice in the Morris water maze
test. All in all, 18d was proved to be a promising multifunctional agent with potential
anti-AD activity.
4
4
4
. Experimental section
.1. Chemistry
.1.1 General procedures
All the reagents were obtained from commercial suppliers and used without further
purification unless otherwise indicated. TLC analysis was performed using pre-coated
glass plates. Silica gel for column chromatography was purchased from Qingdao
Haiyang Chemical Co., Ltd. Melting points were determined using a Büchi B-540
1
13
capillary melting point apparatus. H NMR and C NMR were recorded with Bruker
and Varian instrument at 600, 400 and 150, 100 MHz, respectively, where CDCl or
3
DMSO-d was used as the solvent. Tetramethylsilane (TMS) was used as an internal
6
standard. The following abbreviations (or combinations thereof) were used to explain
multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet
of doublets, dt = doublet of triplets, br = broad. Coupling constants, J was reported in
2
0

Hertz unit (Hz). Mass spectra were measured with Thermo Finnigan LCQ-Advantage
in positive polarity. High-resolution mass spectra (HRMS) were measured on a Bruker
micro OTOF-Q II instrument or Shimadzu LCMSIT-TOF mass spectrometer using ESI
techniques.
4
.1.2 General procedures for the preparation of compounds 2a-c and 7a-b
A solution of maltol 1 or 6 (60 mmol), iodomethane or benzyl bromide or 4-
methoxylbenzyl chloride (90 mmol), anhydrous K CO (12.42 g, 90 mmol) and acetone
2
3
(150 mL) was refluxed for 6 h. After the reaction was completed, the reaction mixture
was evaporated under vacuum, and the residual was resolved in water (100 mL),
extracted by DCM (4×100 mL) and washed by saturated brine (2×100 mL). The
organic solution was evaporated to dryness to obtain 2a-c as yellow oil, 7a-b as white
solid.
4
.1.2.1 3-Methoxy-2-methyl-4H-pyran-4-one (2a)
1
Yellow oil, yield 99%; H NMR (400 MHz, CDCl ) δ 7.62 (d, J = 5.6Hz, 1H),
3
6
4
.34 (d, J = 5.6Hz, 1H), 3.85 (s, 3H), 2.33 (s, 3H); MS (ESI) m/z 141 [M+H]+.
.1.2.2 3-(Benzyloxy)-2-methyl-4H-pyran-4-one (2b)
1
Yellow oil; yield 99%; H NMR (400 MHz, CDCl ) δ 7.59 (d, J = 6.0 Hz, 1H),
3
7
3
4
.42-7.36 (m, 2H), 7.36-7.31 (m, 3H), 6.37 (d, J = 5.6 Hz, 1H), 5.16 (s, 2H), 2.09 (s,
H).
.1.2.3 3-((4-Methoxybenzyl)oxy)-2-methyl-4H-pyran-4-one (2c)
1
Yellow oil, yield 97%; H NMR (400 MHz, CDCl ) δ 7.58 (d, J = 5.6 Hz, 1H),
3
7
3
1
4
.30 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.36 (d, J = 5.6 Hz, 1H), 5.10 (s, 2H),
1
3
.80 (s, 3H), 2.06 (s, 3H); C NMR (100 MHz, CDCl ) δ 175.3, 160.0, 159.9, 153.5,
3
43.8, 130.9, 129.2, 117.3, 113.9, 73.25, 55.4, 15.0.
.1.2.4 5-Methoxy-2-methyl-4H-pyran-4-one (7a)
o
1
White solid, yield 95%, mp 60-62 C; H NMR (400 MHz, CDCl ) δ 7.44 (s, 1H),
3
1
3
6
.13 (d, J = 0.4 Hz, 1H), 3.68 (s, 3H), 2.21 (d, J = 0.4 Hz, 3H); C NMR(100 MHz,
CDCl ) δ 173.5, 164.6, 147.9, 137.3, 113.4, 56.7, 20.3.
3
2
1

4
7
.1.2.5 5-(Benzyloxy)-2-methyl-4H-pyran-4-one (7b)
o
1
White solid, yield 96%, mp 88-90 C; H NMR (400 MHz, CDCl ) δ 7.46 (s, 1H),
3
1
3
.41-7.28 (m, 5H), 6.20 (s, 1H), 5.05 (s, 2H), 2.21 (s, 3H); C NMR (100 MHz, CDCl )
3
δ 174.9, 165.1, 146.8, 141.7, 136.0, 128.7, 128.4, 127.9, 114.4, 71.9, 19.7.
4
.1.3 General procedure for the preparation of compound 6
kojic acid 5 (21.3 g, 150 mmol) was dissolved in thionyl chloride (85 mL) and
stirred for 2 h at room temperature, after which time a pale yellow crystalline mass
formed. The product was collected by filtration and washed with petroleum ether to
afford colorless amorphous powder chlorokojic acid. Then chlorokojic acid (21.7 g,
1
35 mmol) was added to 100 mL of distilled water and heated to 50 °C with stirring.
Zinc dust (17.6 g, 0.271 mol) was added followed by the dropwise addition of
concentrated hydrochloric acid (40.9 mL) over 1 h with vigorous stirring maintaining
the temperature between 70 and 80 °C. The reaction mixture was stirred for a further
4
h at 75 °C. The excess zinc was removed by hot filtration, and the filtrate was extracted
with DCM (5×200 mL). The combined organic extracts were dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo to yield the crude product.
Recrystallization from 2-propanol afforded colorless plates 6.
o
5
-Hydroxy-2-methyl-4H-pyran-4-one (6): White solid, yield 67%, mp 151-153 C;
1
13
H NMR (400 MHz, CDCl ) δ 7.73 (s, 1H), 6.61 (s, 1H), 6.24 (s, 1H), 2.29 (s, 3H); C
3
NMR (100 MHz, CDCl ) δ 173.5, 165.6, 144.7, 137.5, 111.1, 20.4.
3
4
.1.4 General procedure for the preparation of compounds 9a-b and 10a-b
A solution of kojic acid 5 (8.52 g, 60 mmol), iodomethane or benzyl bromide (90
mmol), anhydrous K CO (12.42 g, 90 mmol) and acetone (150 mL) was refluxed for
2
3
6
h. After the reaction was completed, the reaction mixture was evaporated under
vacuum and the residual was resolved in water (100 mL), extracted by DCM (4×100
mL) and washed by saturated brine (2×100 mL). The organic solution was evaporated
to dryness to obtain white solid. Then the white solid (40 mmol) was refluxed in acetone
2
2

(600 mL). Jones-reagent (120 mmol, 45 mL) was added to the solution and the reaction
mixture was refluxed for 40 min (or stirred below 20 °C for 3 h). The reaction mixture
was cooled to room temperature. The precipitate was collected by filtration and washed
with water to give white solids 9a-b.
9
a-b (60 mmol) and 1-methyl-2-pyrrolidinone (150 mL) were refluxed for 4 h.
The solvent was removed azeotropically with N,N-dimethylformamide (200 mL) in
high vacuum. The residue was extracted into DCM and washed with 5% NaOH (3×80
mL). The organic layer was dried over Na SO , concentrated and recrystallized from
2
4
toluene to give the yellow solids 10a-b.
.1.4.1 5-Methoxy-4-oxo-4H-pyran-2-carboxylic acid (9a)
4
o
1
White solid, yield 70%, mp 262-264 C; H NMR (400 MHz, DMSO-d ) δ 8.21
6
1
3
(
s, 1H), 6.88 (s, 1H), 3.70 (s, 3H); C NMR (100 MHz, DMSO-d ) δ 172.1, 160.0,
6
1
4
52.0, 148.9, 139.7, 116.4, 56.3.
.1.4.2 5-(Benzyloxy)-4-oxo-4H-pyran-2-carboxylic acid (9b)
o
1
White solid, yield 80%, mp 196-198 C; H NMR (400 MHz, DMSO-d ) δ 8.35
6
1
3
(
s, 1H), 7.33-7.43 (m, 5H), 6.93 (s, 1H), 4.96 (s, 2H); C NMR (DMSO-d ) δ 173.7,
6
1
4
61.5, 153.2, 148.9, 142.0, 136.5, 129.2, 129.0, 128.9, 117.8, 71.3.
.1.4.3 3-Methoxy-4H-pyran-4-one (10a)
o
1
Yellow solid, yield 24%, mp 89-91 C; H NMR (400 MHz, DMSO-d ) δ 7.73 (d,
6
1
3
J = 5.6 Hz, 1H), 7.59 (s, 1H), 6.40 (d, J = 5.6 Hz, 1H), 3.77 (s, 3H); C NMR (100
MHz, DMSO-d ) δ 173.2, 154.2, 149.2, 138.2, 115.8, 56.3.
6
4
.1.4.4 3-(Benzyloxy)-4H-pyran-4-one (10b)
o
1
White solid, yield 50%, mp 80-81 C; H NMR (400 MHz, CDCl ) δ 7.65 (dd, J =
3
5
5
.6, 0.8 Hz, 1H), 7.54 (d, J = 0.4 Hz, 1H), 7.41-7.29 (m, 5H), 6.41 (d, J = 5.6 Hz, 1H),
.08 (s, 2H).
4
.1.5 General procedure for the preparation of compounds 3a-c, 8a-b and 11a-b
A mixture of 2a-c, 7a-b or 10a-b (20 mmol), ethylene diamine (1.26 g, 21 mmol)
and NaOH (0.72 g, 18 mmol) in ethanol (20 mL) and water (18 mL) was heated to
2
3

7
0 °C for 1.5 h. The mixture was evaporated and purified by column chromatography
(
DCM:MeOH:25% NH = 10:1:0.1). The solvents were removed in vacuo to give 3a-
3
c, 8a-b or 11a-b as yellow oil.
4
.1.5.1 1-(2-Aminoethyl)-3-methoxy-2-methylpyridin-4(1H)-one (3a)
1
Yellow oil, yield 99%; H NMR (400 MHz, CDCl ) δ 7.27 (d, J = 7.6 Hz, 1H),
3
6
2
1
4
.23-6.16 (m, 1H), 3.81 (t, J = 6.4 Hz, 2H), 3.68 (t, J = 5.8 Hz, 3H), 2.90 (t, J = 5.6 Hz,
1
3
H), 2.25 (t, J = 4.0 Hz, 3H); C NMR (100 MHz, CDCl ) δ 173.1, 147.6, 140.7, 139.2,
3
16.6, 59.2, 56.0, 41.9, 12.1.
.1.5.2 1-(2-Aminoethyl)-3-(benzyloxy)-2-methylpyridin-4(1H)-one (3b)
1
Yellow oil, yield 51%; H NMR (400 MHz, CDCl ) δ 7.39 (d, J = 2.0 Hz, 1H),
3
7
1
.36 (d, J = 1.6 Hz, 1H), 7.33-7.30 (m, 2H), 7.29-7.27 (m, 2H), 6.38 (d, J = 7.6 Hz,
1
3
H), 5.17 (s, 2H), 3.83 (t, J = 6.4 Hz, 2H), 2.94 (t, J = 6.4 Hz, 2H), 2.09 (s, 3H); C
NMR (100 MHz, CDCl ) δ 173.5, 146.1, 141.1, 139.0, 137.5, 129.2, 128.3, 128.1, 117.0,
3
7
4
3.1, 56.1, 42.0, 12.7.
.1.5.3 1-(2-Aminoethyl)-3-((4-methoxybenzyl)oxy)-2-methylpyridin-4(1H)-one (3c)
1
Yellow oil, yield 52%; H NMR (400 MHz, DMSO-d ) δ 7.53 (d, J = 7.6 Hz, 1H),
6
7
3
.31 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 6.11 (d, J = 7.2 Hz, 1H), 4.95 (s, 2H),
1
3
.80 (t, J = 6.8 Hz, 2H), 3.75 (s, 3H), 2.73 (t, J = 6.8 Hz, 2H), 2.14 (s, 3H); C NMR
(
100 MHz, DMSO-d ) δ 171.8, 158.9, 145.0, 140.6, 139.6, 130.1, 129.8, 115.5, 113.5,
6
7
4
1.3, 55.3, 55.0, 41.9, 12.0.
.1.5.4 1-(2-Aminoethyl)-5-methoxy-2-methylpyridin-4(1H)-one (8a)
1
Yellow oil, yield 97%; H NMR (400 MHz, CDCl ) δ 6.99 (s, 1H), 6.07 (d, J =
3
4
2
1
4
.8 Hz, 1H), 3.78 (d, J = 4.0 Hz, 2H), 3.60 (d, J = 4.0 Hz, 2H), 2.90 (d, J = 4.4 Hz, 2H),
1
3
.16 (d, J = 4.0 Hz, 2H); C NMR (100 MHz, CDCl ) δ 172.0, 149.0, 145.1, 122.9,
3
16.5, 56.0, 55.5, 41.8, 19.0.
.1.5.5 1-(2-Aminoethyl)-5-(benzyloxy)-2-methylpyridin-4(1H)-one (8b)
1
Yellow oil, yield 57%; H NMR (400 MHz, CDCl ) δ 7.25 (s, 1H), 7.21-7.14 (m,
3
4
H), 7.01 (s, 1H), 6.14 (s, 1H), 4.94 (s, 2H), 3.65 (t, J = 5.6 Hz, 2H), 2.75 (t, J = 6.4
1
3
Hz, 2H), 2.10 (s, 3H); C NMR (100 MHz, CDCl ) δ 172.7, 147.0, 145.3, 136.5, 128.3,
3
2
4

1
4
27.9, 127.8, 127.7, 117.7, 71.3, 55.2, 41.6, 18.9.
.1.5.6 1-(2-Aminoethyl)-3-methoxypyridin-4(1H)-one (11a)
1
Yellow oil, yield 94%; H NMR (400 MHz, CDCl ) δ 7.24 (d, J = 2.0 Hz, 1H),
3
7
3
1
4
.03 (d, J = 2.0 Hz, 1H), 6.27 (d, J = 7.2 Hz, 1H), 3.81 (t, J = 5.8 Hz, 2H), 3.69 (s, 3H),
1
3
.01 (t, J = 5.8 Hz, 2H); C NMR (100 MHz, CDCl ) δ 172.0, 150.4, 137.5, 121.4,
3
15.3, 60.2, 56.1, 42.3.
.1.5.7 1-(2-Aminoethyl)-3-(benzyloxy)pyridin-4(1H)-one (11b)
1
Yellow oil, yield 98%; H NMR (400 MHz, CDCl ) δ 7.29 (s, 1H), 7.25-7.18 (m,
3
5
2
1
H), 7.05 (d, J = 2.0 Hz, 1H), 6.28-6.23 (m, 1H), 4.95 (s, 2H), 3.65 (t, J = 5.6 Hz, 2H),
1
3
.79 (d, J = 5.6 Hz, 2H); C NMR (100 MHz, CDCl ) δ 172.3, 148.4, 148.3, 137.6,
3
36.2, 136.1, 128.3, 127.9, 127.8, 125.9, 116.1, 71.2, 59.5, 41.9.
4
.1.6 General procedure for the preparation of compound 4a
To a solution of 2a (2.80 g, 20 mmol) and 3-aminopropanoic acid (2.14 g, 24 mmol)
was added in ethanol (40 mL). Aq NaOH (10 N) was added to the reaction until pH 13
was attained. The reaction mixture was heated to reflux for 8 h. The solvent was reduced
half and the pH was adjusted 4 with aq HCl. The aqueous solution was concentrated to
afford yellow solid, then washed by acetone. The residue was dissolved in methanol,
filtered, and concentrated to afford solid which washed by anhydrous ethanol to obtain
4
a as pale yellow solid.
-(3-Methoxy-2-methyl-4-oxopyridin-1(4H)-yl)propanoic acid (4a): pale yellow
3
o
1
solid, yield 98%, mp 183-185 C; H NMR (600 MHz, DMSO-d ) δ 12.77 (s, 1H), 8.48
6
(d, J = 6.6 Hz, 1H), 7.53 (d, J = 7.2 Hz, 1H), 4.53 (t, J = 7.2 Hz, 2H), 3.81 (s, 3H), 2.90
1
3
(
t, J = 7.2 Hz, 2H), 2.60 (s, 3H); C NMR (150 MHz, DMSO-d ) δ 171.4, 164.0, 149.4,
6
1
44.0, 142.3, 112.4, 60.4, 51.6, 33.7, 12.8; ESI-HRMS: m/z calcd for C H NO
10 14 4
+
[
M+H] : 212.0917; found: 212.0908.
4
.1.7 General procedures for the preparation of compounds 13a-j
Diethyl malonate (3.84 g, 24 mmol), piperidine (0.2 mL) and glacial acetic acid (2
2
5

drops) were added to a solution of the appropriate salicylaldehyde derivatives 12a-j (20
mmol) in ethanol (24 mL) and the resulting mixture was refluxed for 4-30 h until the
disappearance of the starting materials (monitored by TLC). To the reaction mixture
was added 50 mL of ice water and the crystalline solid was filtered and washed by 50%
cold ethanol. Recrystallization from 50% EtOH gave solid crystals 13a-j.
4
.1.7.1 Ethyl 2-oxo-2H-chromene-3-carboxylate (13a)
o
1
White solid, yield 84%, mp 92-94 C; H NMR (600 MHz, DMSO-d ) δ 8.74 (s,
6
1
H), 7.92-7.90 (m, 1H), 7.73 (t, J = 8.4 Hz, 1H), 7.41 (dd, J = 17.4, 6.6 Hz, 2H), 4.30
1
3
(
q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H); C NMR (150 MHz, DMSO-d ) δ 162.6,
6
1
4
56.0, 154.5, 148.7, 134.5, 130.3, 124.8, 117.8, 117.7, 116.1, 61.2, 14.1.
.1.7.2 Ethyl 7-methoxy-2-oxo-2H-chromene-3-carboxylate (13b)
o
1
White solid, yield 87%, mp 134-135 C; H NMR (600 MHz, CDCl ) δ 8.49 (s,
3
1
4
H), 7.51 (d, J = 13.8 Hz, 1H), 6.90 (d, J = 13.8 Hz, 1H), 6.82 (d, J = 22.2 Hz, 1H),
1
3
.40 (dd, J = 22.2, 6.6 Hz, 2H), 3.89 (s, 3H), 1.41 (t, J = 7.2 Hz, 3H); C NMR (150
MHz, CDCl ) δ 165.3, 163.5, 157.7, 157.3, 149.1, 130.8, 114.2, 113.8, 111.7, 100.5,
3
6
4
1.8, 56.1, 14.4.
.1.7.3 Ethyl 6-methoxy-2-oxo-2H-chromene-3-carboxylate (13c)
o
1
Yellow solid, yield 95%, mp 136-137 C; H NMR (600 MHz, CDCl ) δ 8.47 (s,
3
1
4
4
H), 7.28 (d, J = 9.0 Hz, 1H), 7.21 (dd, J = 9.0, 3.0 Hz, 1H), 7.00 (d, J = 3.0 Hz, 1H),
.40 (q, J = 7.2 Hz, 2H), 3.85 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H).
.1.7.4 Ethyl 8-methoxy-2-oxo-2H-chromene-3-carboxylate (13d)
o
1
Yellow solid, yield 85%, mp 96-97 C; H NMR (600 MHz, CDCl ) δ 8.51 (s, 1H),
3
7
.28 (dd, J = 7.2, 1.2 Hz, 1H), 7.20 (d, J = 1.2 Hz, 1H), 7.18 (s, 1H), 4.43 (q, J = 7.2
Hz, 2H), 3.99 (s, 3H), 1.42 (t, J = 7.2 Hz, 3H).
.1.7.5 Ethyl 6-methyl-2-oxo-2H-chromene-3-carboxylate (13e)
4
o
1
White solid, yield 97%, mp 104-105 C; H NMR (600 MHz, DMSO-d ) δ 8.65
6
(s, 1H), 7.67 (s, 1H), 7.54 (dd, J = 8.4, 2.4 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 4.29 (q, J
1
3
=
7.2 Hz, 2H), 2.36 (s, 3H), 1.30 (t, J = 7.2 Hz, 3H); C NMR (150 MHz, DMSO-d )
6
δ 162.7, 156.1, 152.7, 148.5, 135.4, 134.1, 129.7, 117.6, 117.5, 115.9, 61.2, 14.1.
2
6

4
1
.1.7.6 Ethyl 7-methyl-2-oxo-2H-chromene-3-carboxylate (13f)
o
1
White solid, yield 85%, mp 103-105 C; H NMR (400 MHz, CDCl ) δ 8.49 (s,
3
H), 7.48 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 8.0 Hz, 2H), 4.40 (q, J = 7.2 Hz, 2H), 2.48
(s, 3H), 1.40 (t, J = 7.2 Hz, 3H).
4
.1.7.7 Ethyl 8-methyl-2-oxo-2H-chromene-3-carboxylate (13g)
o
1
White solid, yield 72%, mp 82-84 C; H NMR (400 MHz, CDCl ) δ 8.49 (s, 1H),
3
7
7
4
.48 (d, J = 7.2 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 4.41 (q, J =
.2 Hz, 2H), 2.46 (s, 3H), 1.41 (t, J = 7.2 Hz, 3H).
.1.7.8 Ethyl 6-hydroxy-2-oxo-2H-chromene-3-carboxylate (13h)
o
1
Brown solid, yield 58%, mp 183-185 C; H NMR (400 MHz, DMSO-d ) δ 9.88
6
(s, 1H), 8.65 (s, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.20 (d, J = 2.4 Hz, 1H), 7.15 (dd, J =
1
3
9
.2, 2.8 Hz, 1H), 4.28 (q, J = 7.2 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H); C NMR (100 MHz,
DMSO-d ) δ 162.8, 156.3, 154.0, 148.4, 147.9, 122.6, 118.2, 117.8, 117.0, 113.7, 61.2,
6
1
4
4.0.
.1.7.9 Ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (13i)
o
1
White solid, yield 76%, mp 165-167 C; H NMR (600 MHz, DMSO-d ) δ 11.07
6
(s, 1H), 8.65 (s, 1H), 7.74 (d, J = 9.0 Hz, 1H), 6.83 (dd, J = 8.4, 2.4 Hz, 1H), 6.72 (d, J
1
3
=
2.4 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H); C NMR (150 MHz,
DMSO-d ) δ 164.1, 163.0, 157.1, 156.4, 149.4, 132.1, 114.0, 112.1, 110.4, 101.8, 60.8,
6
1
4
4.1.
.1.7.10 Ethyl 8-hydroxy-2-oxo-2H-chromene-3-carboxylate (13j)
o
1
Brown solid, yield 51%, mp 176-178 C; H NMR (400 MHz, CDCl ) δ 8.53 (s,
3
1
1
1
H), 7.27 (d, J = 7.6 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H), 6.16 (s,
1
3
H), 4.42 (q, J = 7.2 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ
3
63.0, 156.0, 149.4, 143.5, 143.0, 125.4, 120.9, 120.7, 118.4, 118.3, 62.2, 14.4.
4
.1.8 General procedures for the preparation of compounds 15a-r
Potassium Carbonate (1.66 g, 12 mmol) and 13h-j (10 mmol) were dissolved in
DMF (25 mL) and the mixture was stirred at room temperature for several minutes,
2
7

then appropriate alkyl bromides or benzyl bromides derivatives (15 mmol) were added
dropwise to the mixture and stirred for 48 h. Upon completion, water is added to the
mixture and the produced precipitate was filtrated, washed with water to afford solid
powder 15a-r.
4
.1.8.1 Ethyl 6-(benzyloxy)-2-oxo-2H-chromene-3-carboxylate (15a)
o
1
Yellow solid, yield 98%, mp 154-156 C; H NMR (400 MHz, CDCl ) δ 8.44 (s,
3
1
2
1
4
H), 7.43-7.35 (m, 5H), 7.29 (s, 2H), 7.07 (s, 1H), 5.11 (s, 2H), 4.41 (q, J = 7.2 Hz,
1
3
H), 1.40 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ 163.3, 157.0, 155.5, 150.0,
3
48.4, 136.2, 128.9, 128.5, 127.5, 123.4, 118.7, 118.3, 118.0, 112.2, 70.7, 62.1, 14.3.
.1.8.2 Ethyl 7-(benzyloxy)-2-oxo-2H-chromene-3-carboxylate (15b)
o
1
White solid, yield 93%, mp 152-154 C; H NMR (600 MHz, CDCl ) δ 8.49 (s,
3
1
H), 7.50 (d, J = 9.0 Hz, 1H), 7.41 (q, J = 7.2 Hz, 4H), 7.37-7.35 (m, 1H), 6.96 (dd, J
=
9.0, 2.4 Hz, 1H), 6.87 (d, J = 1.8 Hz, 1H), 5.15 (s, 2H), 4.38 (q, J = 7.2 Hz, 2H), 1.39
1
3
(
t, J = 7.2 Hz, 3H); C NMR (150 MHz, CDCl ) δ 164.3, 163.6, 157.6, 157.2, 149.0,
3
1
4
35.4, 130.9, 128.9, 128.7, 127.7, 114.4, 111.9, 101.6, 70.9, 61.8, 14.4.
.1.8.3 Ethyl 8-(benzyloxy)-2-oxo-2H-chromene-3-carboxylate (15c)
o
1
Brown solid, yield 99%, mp 134-136 C; H NMR (400 MHz, CDCl ) δ 8.48 (s,
3
1
3
H), 7.46 (d, J = 7.2 Hz, 2H), 7.38 (t, J = 7.2 Hz, 2H), 7.39-7.30 (m, 1H), 7.19-7.16 (m,
1
3
H), 5.25 (s, 2H), 4.41 (d, J = 7.2 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H); C NMR (100
MHz, CDCl ) δ 163.2, 156.3, 148.8, 146.1, 145.5, 136.1, 128.8, 128.3, 127.5, 124.7,
3
1
4
21.3, 118.8, 118.7, 118.5, 77.5, 62.1, 14.3.
.1.8.4 Ethyl 7-ethoxy-2-oxo-2H-chromene-3-carboxylate (15d)
o
1
White solid, yield 93%, mp 119-121 C; H NMR (600 MHz, CDCl ) δ 8.49 (s,
3
1
4
7
4
H), 7.48 (d, J = 9.0 Hz, 1H), 6.87 (dd, J = 8.4, 2.4 Hz, 1H), 6.78 (d, J = 1.8 Hz, 1H),
.39 (q, J = 7.2 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H), 1.39 (t, J =
.2 Hz, 3H).
.1.8.5 Ethyl 2-oxo-7-propoxy-2H-chromene-3-carboxylate (15e)
o
1
White solid, yield 98%, mp 105-107 C; H NMR (600 MHz, CDCl ) δ 8.49 (s,
3
1
H), 7.48 (d, J = 9.0 Hz, 1H), 6.87 (dd, J = 8.4, 2.4 Hz, 1H), 6.79 (d, J = 2.4 Hz, 1H),
2
8

4
.39 (q, J = 7.2 Hz, 2H), 4.00 (t, J = 6.6 Hz, 2H), 1.90-1.76 (m, 2H), 1.39 (t, J = 7.2
Hz, 3H), 1.05 (t, J = 7.2 Hz, 3H).
4
.1.8.6 Ethyl 2-oxo-7-(prop-2-yn-1-yloxy)-2H-chromene-3-carboxylate (15f)
o
1
White solid, yield 96%, mp 153-156 C; H NMR (600 MHz, CDCl ) δ 8.50 (s,
3
1
7
4
H), 7.52 (d, J = 9.0 Hz, 1H), 6.97-6.92 (m, 2H), 4.78 (d, J = 2.4 Hz, 2H), 4.39 (q, J =
.2 Hz, 2H), 2.59 (t, J = 2.4 Hz, 1H), 1.40 (t, J = 7.2 Hz, 3H).
.1.8.7 Ethyl 7-(cyclohexylmethoxy)-2-oxo-2H-chromene-3-carboxylate (15g)
o
1
White solid, yield 78%, mp 115-117 C; H NMR (400 MHz, CDCl ) δ 8.49 (s,
3
1
4
H), 7.47 (d, J = 8.8 Hz, 1H), 6.85 (dd, J = 8.4, 2.0 Hz, 1H), 6.78 (d, J = 2.0 Hz, 1H),
.40 (q, J = 7.2 Hz, 2H), 3.83 (d, J = 6.0 Hz, 2H), 1.88-1.69 (m, 6H), 1.39 (t, J = 6.8
1
3
Hz, 3H), 1.36-1.16 (m, 3H), 1.12 (q, J = 11.2 Hz, 2H); C NMR (100 MHz, CDCl ) δ
3
1
2
4
65.1, 163.7, 157.8, 149.1, 130.8, 114.2, 114.0, 111.6, 101.0, 74.4, 61.8, 37.6, 29.9,
6.5, 25.8, 14.4.
.1.8.8 Ethyl 7-((3-chlorobenzyl)oxy)-2-oxo-2H-chromene-3-carboxylate (15h)
o
1
White solid, yield 97%, mp 149-150 C; H NMR (600 MHz, CDCl ) δ 8.49 (s,
3
1
1
7
4
H), 7.51 (d, J = 8.4 Hz, 1H), 7.43 (s, 1H), 7.34-7.33 (m, 2H), 7.30 (dd, J = 7.2, 3.0 Hz,
H), 6.95 (dd, J = 9.0, 2.4 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H), 5.12 (s, 2H), 4.39 (q, J =
.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H).
.1.8.9 Ethyl 7-((3-fluorobenzyl)oxy)-2-oxo-2H-chromene-3-carboxylate (15i)
o
1
White solid, yield 95%, mp 157-159 C; H NMR (600 MHz, CDCl ) δ 8.49 (s,
3
1
7
6
4
H), 7.51 (d, J = 9.0 Hz, 1H), 7.37 (td, J = 7.8, 6.0 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H),
.14 (d, J = 9.6 Hz, 1H), 7.04 (td, J = 8.4, 2.4 Hz, 1H), 6.96 (dd, J = 9.0, 2.4 Hz, 1H),
.85 (d, J = 2.4 Hz, 1H), 5.14 (s, 2H), 4.39 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 7.2 Hz, 3H).
.1.8.10 Ethyl 7-((4-fluorobenzyl)oxy)-2-oxo-2H-chromene-3-carboxylate (15j)
o
1
White solid, yield 95%, mp 153-155 C; H NMR (600 MHz, CDCl ) δ 8.49 (s,
3
1
6
H), 7.50 (d, J = 9.0 Hz, 1H), 7.40 (dd, J = 8.4, 5.4 Hz, 2H), 7.09 (t, J = 8.4 Hz, 2H),
.94 (dd, J = 8.4, 2.4 Hz, 1H), 6.86 (s, 1H), 5.10 (s, 2H), 4.39 (q, J = 7.2 Hz, 2H), 1.39
(t, J = 7.2 Hz, 3H).
4
.1.8.11 Ethyl 7-((2,5-difluorobenzyl)oxy)-2-oxo-2H-chromene-3-carboxylate (15k)
2
9