Identification of N-phenyl-3-methoxy-4-pyridinones as orally bioavailable H3 receptor antagonists and β-amyloid aggregation inhibitors for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2020.113096

Based on our previous work, a series of N-phenyl-3-methoxy-4-pyridinone derivatives were designed as orally bioavailable dual functional agents for therapy of Alzheimer's disease, through introducing alkyloxy moiety into 4-pyridinone ring to avoid the pos

Full text of DOI:10.1016/j.ejmech.2020.113096

Journal Pre-proof
Identification of N-phenyl-3-methoxy-4-pyridinones as orally bioavailable H receptor
3
antagonists and β-amyloid aggregation inhibitors for the treatment of Alzheimer's
disease
Minkui Zhang, Li Tang, Liu Jiang, Jun Wei, Yongzhou Hu, Rong Sheng
PII:
S0223-5234(20)31068-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.113096
EJMECH 113096
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 August 2020
Revised Date: 19 November 2020
Accepted Date: 8 December 2020
Please cite this article as: M. Zhang, L. Tang, L. Jiang, J. Wei, Y. Hu, R. Sheng, Identification of
N-phenyl-3-methoxy-4-pyridinones as orally bioavailable H receptor antagonists and β-amyloid
3
aggregation inhibitors for the treatment of Alzheimer's disease, European Journal of Medicinal
Chemistry, https://doi.org/10.1016/j.ejmech.2020.113096.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Elsevier Masson SAS. All rights reserved.

Graphical abstract
Identification of N-phenyl-3-methoxy-4-pyridinones as orally bioavailable H3
receptor antagonists and β-amyloid aggregation inhibitors for the treatment of
Alzheimer's disease
Minkui Zhang#, Li Tang#, Liu Jiang, Jun Wei, Yongzhou Hu, Rong Sheng*
O
O
N
N
O
7i
hH3R IC50 = 0.52 nM
Aβ1-40/Aβ1-42 aggregation inhibition
Oral bioavaliablity 47.9%
Good BBB permeability
Excellent subtypes selectivity
Low hERG channel inhibition risk
The binding pattern of 7i with H3R

Identification of N-phenyl-3-methoxy-4-pyridinones as
orally bioavailable H3 receptor antagonists and β-amyloid
aggregation inhibitors for the treatment of Alzheimer's
disease
Minkui Zhanga#, Li Tanga,b,#, Liu Jianga, Jun Weia, Yongzhou Hua, Rong Shenga*
a. College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China;
b.College of Health Science and Environmental Engineering, Shenzhen Technology
University, Shenzhen, China
#These authors contributed equally to this work.
*Corresponding author
Rong Sheng Tel/Fax: +86-571-8820-8458; E-mail address: shengr@zju.edu.cn.
Abstract
Based on our previous work, a series of N-phenyl-3-methoxy-4-pyridinone
derivatives were designed as orally bioavailable dual functional agents for therapy
of Alzheimer’s disease, through introducing alkyloxy moiety into 4-pyridinone ring to
avoid the possible phase II metabolism of 3-hydroxy-4-pyridinone in lead compound
3-hydroxy-2-methyl-1- (4-(3-(pyrrolidin-1-yl)propoxy)phenyl)-pyridin-4(1H)-one (4).
In vitro studies indicated that most of these compounds exhibit excellent H3 receptor
antagonistic activities and potent self-induced Aβ1-40/Aβ1-42 aggregation inhibitory
activities. In particular, 3-methoxy-1-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)-
pyridin-4(1H)-one (7i) demonstrated IC50 value of 0.52 nM in H3R antagonism and
good selectivity over other histamine receptor subtypes. The transmission electron
microscopy (TEM) images showed that compound 7i can inhibit self-mediated

Aβ1-40/Aβ1-42 aggregation efficiently. As expected, it exhibited desirable
pharmacokinetic properties in plasama and good BBB permeability. Furthermore,
compound 7i can efficiently block (R)-α-methylhistamine- induced dipsogenia and
reverse scopolamine-induced learning deficits of rats. All above results indicated that
compound 7i was a promising orally bioavailable dual functional agents with potenial
use in the treatment of Alzheimer’s disease.
Key words: Alzheimer’s disease; H3 receptor antagonists; β-amyloid aggregation
inhibition; Dual functional agents.
1. Introduction
Alzheimer’s disease (AD) afflicts more than 30 million people worldwide [1]. Although great
progress has been made in the treatment of AD in the past few decades, the etiology of this disease
remains unclear, and effective therapeutic interventions are still limited except
acetylcholinesterase (AChE) inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonist
memantine [2-5]. AD is a multifactorial disease, and diverse factors, including neurotransmitter
system dysfunction, β-amyloid (Aβ) aggregation, τ-protein hyper-phosphorylation, biometal
dyshomeostasis, oxidative stress, inflammation, and many more, seem to play significant roles in
the progress of AD [6-9]. This multifunctional nature of AD provides the logical foundation for the
development of novel drug design strategies, multi-target-directed-ligands (MTDLs), which can
simultaneously interfere with different causes of AD and may provide more efficient therapy
through
a synergistic effect [10-12]. Sodium Oligo-mannurarate (GV-971), a native
carbohydrate-based multi-targeting drug has specifically been approved in China for the treatment
of mild-to-moderate Alzheimer’s disease [13].
The H3 receptor functions as an auto- and hetero-receptor, negatively regulating the release of
histamine and other neurotransmitters, including acetylcholine (ACh), dopamine, noradrenaline,
and serotonin [14-17]. The role of AChE inhibitors in the clinic is to disrupt the hydrolysis of ACh
in the brain and, thus, palliate memory deficits. The antagonism of the H3 receptor can increase the
concentration of ACh released, as well as other attention-related neurotransmitters in the brain,
including serotonin, noradrenaline, and dopamine, and, therefore, may provide a better therapeutic

effect for AD patients. In the past few years, several H3 receptor antagonists have been approved
for clinical trials of various CNS diseases, including CEP-26401 (phase I for cognitive impairment,
Cephalon, Figure 1, 1), MK0249 (phase II for Alzheimer's Disease, Merck, Figure 1, 2) [18], and
Pitolisant, particularly approved by European Medicines Agency (EMA) in 2016 and by Food and
Drug Administration (FDA) in 2019 for the treatment of narcolepsy (Figure 1, 3) [19, 20].
Figure 1. Structures of representative H3 antagonists
Senile plaques formed by aggregated β-amyloid (Aβ) are the histopathological hallmarks of AD
[21-23]. Although the precise neurotic mechanism of aggregated β-amyloid is controversial,
numerous investigations have proven that it (both fibril and oligomer aggregated Aβ) is toxic to
neuronal cells [24-27]. Therefore, it is necessary to develop Aβ aggregation inhibitors that can
benefit AD therapy.
Recently, our group unveiled a series of 1-phenyl-3-hydroxy-4-pyridinone derivatives as
potential multi-target agents for AD therapy [28]. Specifically, 3-hydroxy-2-methyl-1-
(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)-pyridin-4(1H)-one (4) demonstrated very potent H3
receptor antagonistic activity (IC50 = 0.32 nM), efficient radical-scavenging effect, excellent
copper ion chelating activity, and Aβ aggregation inhibitory activity (Figure 2). However, the oral
bioavailability of the compound was relatively low in rats (F = 24 %, see SI), which limited its
further development. It’s reported that the market chelator Deferiprone (5) was metabolized by
UDP-glucuronosyl transferases (UGTs) into the corresponding 3-O-glucuronide conjugate (6)
(Figure 4) [29,30]. Based on the structure of 4, we deduced that the possible reason of low oral
bioavailability is the quick phase-II metabolism of the 3-hydroxy-4-pyridinone moiety. Therefore,
we try to modify the 3-hydroxyl moiety of the pyridinone ring to block the phase-II metabolism by
UGTs, thus to improve the bioavailability of the compound and its permeability through the
blood-brain barrier.

Therefore, we present here a series of N-phenyl-3-methoxy-4-pyridinones as orally bioavailable
H3 receptor antagonists and β-amyloid aggregation inhibitors. A summary of the drug design
strategy is shown in Figure 2, where several regions of the lead compound, 4, were modified to
explore the structure activity relationships (SAR) and find a promising candidate for further
development. 1) The modification of substituent groups on the pyridinone ring, the length of the
linker, and the cyclic amine moiety to produce compounds 7a-7j. 2) Replacement of the linear
alkyloxy linker with a piperidinyl-ether linker to manufacture compounds 8a-8f; 3) Replacement
of the alkoxy linker with a piperazine-carbonyl or piperazine-sulfonyl linker to furnish compounds
9a-9c and 10a-10c.
Figure 2. Design strategy for N-phenyl-3-methoxy-4-pyridinone derivatives
2. Results and discussion
2.1 Chemistry
The synthesis of N-phenyl-3-methoxy-4-pyridinone derivatives 7a-7j with a linear linker is
described in Scheme 1. Starting materials, compounds 11a-11e, were prepared as described

previously [28]. 11a-11e reacted with 4-aminophenol to form N-phenolpyridinone 12a-12e under
microwave conditions. 12a-12e were then alkylated with α,ω-dihaloalkane in the presence of
K2CO3 to yield compounds 13a-13g, which were condensed with various secondary amines to
obtain target compounds 7a-7j.
Scheme 1. Synthetic route to 7a-7j with linear linker
The synthesis of 8a-8f with a 4-piperidinyl-ether linker is described in Scheme 2.
4-fluoronitrobenzene, 14, reacted with N-Boc-4-hydroxypiperidine to yield phenoxy piperidine
derivative 15, followed by the deprotection of Boc, reductive amination, and the reduction of the
nitro-group to produce aniline derivatives 18a-18c. 18a-18c then reacted with 3-alkoxy-4-pyrone
to create target compounds 8a-8f.

Scheme 2. Synthetic route to 8a-8f with 4-piperidinyl-ether linker
The synthesis of 9a-9c with a piperazine-carbonyl linker is described in Scheme 3. Compound
11a reacted with 4-aminobenzoic acid in a sealed tube to give rise to intermediate 19. 19 was
condensed with N-Boc piperazine, after which Boc was deprotected to obtain key intermediate 21.
21 was subsequently subjected to reductive amination with various ketones to produce target
compounds 9a-9c.
Scheme 3. Synthetic route to 9a-9c with piperazine-carbonyl linker
The synthesis of derivatives 10a-10c with a piperazine-sulfonyl linker is described in Scheme 4.
4-nitrobenzenesulfonyl chloride, 22, was condensed with N-Boc-piperazine, followed by the
deprotection of Boc to obtain intermediate 24. 24 was reductively aminated with different ketones

to yield intermediates 25a-25c, whose nitro moieties were reduced by catalytic hydrogenation to
create aniline derivatives 26a-26c. 26a-26c were eventually condensed with 3-methyl-4-pyrone,
11a, to furnish target compounds 10a-10c.
Scheme 4. Synthetic route to 10a-10c with piperazine-sulfonyl linker
2.2 H3R antagonistic and Aβ1-40/Aβ1-42 aggregation inhibitory activity evaluation
The H3 receptor antagonistic activities of these N-phenyl-3-methoxy-4-pyridinones were
determined using the cAMP-response elements (CREs)-driven luciferase assay in hH3
receptor-expressed HEK-293 cell lines, and their Aβ1-40/Aβ1-42 aggregation inhibitory activities
were evaluated using the thioflavin T fluorescence assay [28].
The biological activities of these target compounds are shown in Table 1. Many of the
N-Phenyl-3-methoxy-4-pyridinone derivatives demonstrated potent hH3R antagonistic activities
and efficient Aβ1-40/Aβ1-42 aggregation inhibitory activities. 13 compounds (7a, 7b, 7f-7j, 8a-8f)
showed nanomolar or subnanomolar IC50 values against the H3 receptor. In addition, 20 µM of
each of 18 compounds (7a-7j, 8a-8f, 10b, 10c) exhibited 50.7-80.5% inhibition rates on Aβ1-40
aggregation, compared with the 54.8% inhibition rate of curcumin, and 12 compounds (7a, 7c-7e,
7g-7j, 8a, 9a, 10a, 10b) exhibited 50.9-67.5% inhibition rates on Aβ1-42 aggregation, compared
with the 65.7% inhibition rate of curcumin. Some compounds showed significantly different
activities on Aβ1-40 and Aβ1-42, suggesting that there may be different modes of action between
the compounds and Aβ1-40/Aβ1-42
.

The changes in the terminal cyclic amine group affected the H3 antagonistic activities of target
compounds. The pyrrolidine and piperidine derivatives, 7a and 7b, respectively, displayed similar
activities, while the morpholine and methyl piperazine analogs, 7c and 7d, respectively, showed a
drastic loss of activity in H3 antagonism, which is consistent with our previous results [28].
However, these changes did not affect the Aβ1-40/Aβ1-42 aggregation inhibitory activities of these
derivatives significantly.
7f, with four carbon length linkers, retained its hH3R activity and Aβ1-40 aggregation inhibitory
activity but lost its Aβ1-42 aggregation inhibitory activity, whereas, 7e, with a short length linker,
almost lost its hH3R inhibitory effect and bore a slight change in its Aβ1-40 and Aβ1-42 aggregation
inhibitory activities.
The changes in the alkyl group and their substitution positions on the 4-pyridinone ring resulted
in the production of compounds 7g-7j, all of which demonstrated potent H3 antagonistic activities,
with IC50 values ranging from 0.52 to 7.97 nM, and good Aβ1-40/Aβ1-42 inhibitory effects,
indicating that minor modifications on the pyridinone ring do not affect H3 antagonism or
Aβ1-40/Aβ1-42 inhibition. Compound 7i was the most potent hH3R antagonist, with a IC50 value of
0.52 nM, a Aβ1-40 inhibitory rate of 56.0% and a Aβ1-42 inhibitory rate of 63.4% at 20 μM.
Replacing the linear alkyoxy linker of 7a with 4-piperidinyl-ether, piperazine-carbonyl, or
piperazine-sulfonyl resulted in the creation of the other three series of compounds 8a-8f, 9a-9c,
and 10a-10c. Compounds 8a-8f retained potent H3R antagonistic activities, with IC50 values that
ranged from 0.46 to 8.92 nM, and good Aβ1-40 inhibitory activities (50.7-59.6% inhibition at 20
µM). In contrast, the other two serial compounds 9a-9c and 10a-10c only showed moderate H3R
antagonistic activities, with IC50 values that ranged from 22.11 to 350.5 nM, and decreased Aβ1-40
inhibitory activities (21.2-54.6%). Meanwhile, almost all of these compounds exhibited decreased
Aβ1-42 inhibitory activities, except isopropyl substituted compounds (8a, 9a, 10a). These findings
suggest that the linkers of target compounds play vital roles in H3 antagonism.

Table 1. Biological activities of N-phenyl-3-methoxy-4-pyridinone derivatives a
Inhibition of Aβ aggregation (%)b
hH3R
Cpd.
amine
linker
R1
R2
R3
IC50 (nM)
Aβ1-40
Aβ1-42
-O(CH2)3- Me
-O(CH2)3- Me
-O(CH2)3- Me
-O(CH2)3- Me
H
H
H
H
Me
Me
6.53±0.84
5.82±0.23
80.5±6.2
52.4±3.5
7a
7b
7c
7d
80.4±7.9
69.8±9.5
76.1±8.8
45.1±7.5
56.6±2.5
67.0±10.6
Me 107.8±12.5
Me 167.4±26.8
-O(CH2)2- Me
-O(CH2)4- Me
H
H
Me
Me
7e
7f
1787.1±104
4.33±0.30
51.6±7.4
77.3±5.1
59.1±1.9
26.9±4.2
-O(CH2)3-
-O(CH2)3-
-O(CH2)3-
Et
H
H
H
Me
1.11±0.08
1.94±0.49
0.52±0.07
7.97±0.88
64.9±2.9
60.1±6.8
56.0±8.4
52.7±3.7
62.4±3.2
55.2±2.7
63.4±9.9
67.5±1.9
7g
7h
7i
Me Me
H
H
Me
Et
-O(CH2)3- Me
7j
Me
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
8.92±0.54
1.67±0.14
2.43±0.18
0.74±0.59
1.39±0.37
0.46±0.06
54.4±3.1
58.5±6.4
59.6±4.9
52.5±7.2
57.9±2.8
50.7±4.9
63.5±5.3
41.4±6.1
7.5±3.4
9.3±1.7
1.5±2.5
N
8a
8b
8c
8d
8e
8f
Me
H
Me
H
9a
9b
Me
Me
H
H
Me 350.5±29.8
Me 68.30±0.06
28.5±7.9
43.6±6.7
50.9±1.6
44.3±6.1

9c
Me
Me
Me
Me
H
H
H
H
Me 49.04±2.81
Me 385.3±25.8
Me 35.81±2.14
Me 22.11±3.56
49.0±3.4
21.2±4.1
51.5±6.9
54.6±5.0
23.9±7.9
64.0±4.1
54.4±8.9
N
10a
10b
10c
1.15±0.16
/
/
/
Clobenpropit
Curumin
54.8±6.5
65.7±5.3
a The values are expressed as the Mean±SD of three independent measurements.
b 20 µM of compounds and 25 µM of Aβ1-40 or Aβ1-42 were used.
c “N” means not determined.
2.3 Selectivity of the histamine receptor subtype
Based on hH3R antagonistic activities and Aβ1-40/Aβ1-42 aggregation inhibitory activities, the
most promising compound, 7i, was selected for further investigation. The antagonistic activities of
7i on three histamine receptor subtypes (H1R, H2R, and H4R) were evaluated using the
CRE-luciferase assay as previous described [28]. Table 2 reveals that compound 7i had no
antagonistic activity against H1R, H2R, or H4R, indicating the high selectivity of 7i for H3
receptors.
2.4 hERG channel inhibition
The development of many reported highly potent H3 antagonists have been stopped at the
preclinical trial stage because of their hERG channel inhibitory activities that is closely associated
with prolonging the QT interval [31]. As shown in Table 2, compound 7i (3.0 µM) only had a 6.3%
inhibitory effect on the hERG channel in the patch-clamp assay, indicating that it has a low risk of
prolonging the QT interval.

Table 2. Histamine Receptor Subtype Selectivity and hERG Inhibition of Compound 7i.
hH1R
hH2R
hH4R
Compound
hERG inhibition
(IC50, µM)
(IC50, µM)
(IC50, µM)
>10
/
>10
/
>10
/
6.3% ± 1.5%
44.1% ± 1.5%
7i (3.0 µM)
Cisapride (30 nM)
2.5 Study of the molecular docking of 7i with the H3 receptor
To assess the binding modes of this class of 1-phenyl-4-pyridinone derivatives with H3R,
compound 7i was selected for docking studies using previously established H3 receptor homology
model based on the H1 receptor crystal structure (PDB ID: 3RZE) [32]. The result showed that
compound 7i fitted well in the hydrophobic pocket formed by the TMs 3-5-6 region and three
hydrogen bonds between the amino acid residues of the H3 receptor formed. The methoxy moiety
of pyridinone of 7i formed a hydrogen bond with the OH group of Thr201, the alkoxy linker
formed another hydrogen bond with the oxygen of the phenol moiety of Tyr 374, and the
protonated nitrogen of the pyrrolidine moiety formed the third hydrogen bond with the carboxylic
acid of critical amino acid Asp114 (Figure 3). Additionally, the phenyl ring of 7i interacted with
the aromatic ring of Tyr 115 in a π−π stacking mode.
Figure 3. The binding pattern of 7i with H3R homology model.
2.6 The inhibition of Self-induced Aβ1-40 and Aβ1-42 aggregation by 7i

Besides using the ThT binding assay, the effect of 7i on self-induced Aβ1-40 and Aβ1-42 aggregation
was monitored using transmission electron microscopy (TEM) as described previously [28]. As
shown in Figure 4, after 24 h of incubation at 37 °C, Aβ1-42 self-aggregated into amyloid fibrils,
while Aβ1-40 self-aggregated into amyloid fibrils as well as plaque-like aggregates. However, when
curcumin or 7i was added (Figure 4c, 4d) under identical conditions only very few Aβ1-40 or Aβ1-42
fibrils were observed in TEM.
Figure 4. TEM images of samples of Aβ1-40 and Aβ1-42. ([Aβ] = 25 µM, 37°C, constant agitation).
(a) Aβ1-40 alone, 0h (b) Aβ1-40 alone, 24h (c) Aβ1-40+curcumin, 24h (d) Aβ1-40 +7i, 24h
(e) Aβ1-42 alone, 0h (f) Aβ1-42 alone, 24h (g) Aβ1-42+curcumin, 24h (h) Aβ1-42 +7i, 24h
2.7 Disaggregation of Self-induced Aβ1-40 and Aβ1-42 aggregation by 7i
Self-induced Aβ1-40 and Aβ1-42 aggregation fibrils were incubated with 7i to investigate the
disaggregation activity of 7i. Fresh Aβ1-40/Aβ1-42 was incubated for 24 h at 37 °C to yield
Aβ1-40/Aβ1-42 fibrils. 7i or curcumin was then added to the fibrils and incubated for another 24 h
under the same condition. Figure 5 shows that 7i disassembled the well-formed Aβ1-40/Aβ1-42
fibrils efficiently, as did curcumin.

Figure 5. TEM images of disaggregation experiments on Aβ1-40 and Aβ1-42 fibers.
([Aβ] = 25 µM, 37°C, constant agitation,24h).
(a) Aβ1-40 alone. (b) Aβ1-40+curcumin. (c) Aβ1-40 +7i.
(d) Aβ1-42 alone. (e) Aβ1-42+curcumin. (f) Aβ1-42 +7i.
2.8 The molecular docking of 7i with Aβ1-40 and Aβ1-42
To elucidate the binding modes of compound 7i with monomeric Aβ1-40 and Aβ1-42, molecular
docking was performed using Accelrys Discovery Studio 2.0, and the crystal structure of Aβ1-40
and Aβ1-42 (PDB code: 1BA4, 1IYT) was employed [33].
As shown in Figure 6, compound 7i bound slightly differently with Aβ1-40 and Aβ1-42. 7i was
located in the middle part of Aβ1-40 and interacted with residues of Asp23 and Lys28. A hydrogen
bond was formed between the carbonyl moiety of 4-pyridinone and the NH group of Lys28, and
salt bridges were erected between the basic pyrrolidine moiety of 7i and Asp23. The binding mode
of 7i with Aβ1-42 was similar to that of the lead compound 4, it bound to the molecular surface of
the C-terminus of the peptide chain to form hydrogen bonds and salt bridges with Lys28 and
Ala42, respectively. These results are consistent with the finding (Table 1) that 7i interact with
Aβ1-40 and Aβ1-42 in a different way.

Figure 6. The binding pattern of 7i with monomeric Aβ1-40 and Aβ1-42
2.9 In vivo pharmacokinetic study
We investigated the in vivo preliminary pharmacokinetic properties of compound 7i in rats (Figure
7, Table 3). After the intravenous administration of 2.0 mg/kg and intragastric administration of
5.0 mg/kg of 7i to rats, blood exposure to the compound (AUC0-t) reached 1412.5 ng•h/mL and
1266.4 ng•h/mL, respectively, with corresponding elimination half-lives of 1.59 hours and 4.64
hours. The oral bioavailability of 7i was 47.9%. Compared to compound 4 (p.o. 10 mg/kg; AUC0-t
1035.7 ng•h/mL; F value of 24.0%, Supplementary Table S1), 7i had significantly improved oral
exposure and bioavailability.
Additionally, brain exposure (AUC0-t) to 7i was 359.6 ng•h/mL and 277.2 ng•h/mL after
intravenous administration of 2.0 mg/kg and intragastric administration of 5.0 mg/kg, respectively,
with corresponding B/P values of 0.40 and 0.44.
Figure 7. The in vivo PK profile of 7i
Table 3. Pharmacokinetic Properties of Compound 7i

i.v. (2.0 mg/kg)
p.o. (5.0 mg/kg)
plasma
brain
plasma
brain
AUC0-t (ng·h/mL)
AUC0-∞ (ng·h/mL)
T1/2 (h)
1412.5
1412.6
1.59
359.6
570.8
5.64
3.54
28.5
1.00
77.8
1266.4
1693.4
4.64
277.2
737.8
3.77
6.89
36.5
3.33
49.9
Cl (L/h/kg)
Vd (L/kg)
Tmax (h)
1.43
3.26
3.27
18.9
0.16
2.00
Cmax (ng/mL)
B/P
510.5
429.6
0.40
-
0.44
47.9
F%
2.10 Dipsogenia model
The excellent H3 antagonistic activity in vitro and good PK profile of 7i prompted us to carry out
in vivo pharmacological experiments. The effect of compound 7i on the H3 receptor
agonist-induced dipsogenia model (SD rats) was tested at different times [34]. As shown in Figure
8 and Table S2, rats that were given (R)-α-Methylhistamine (2.5 mg/kg s.c.) for 2 h consumed
about 2.5-fold more water than vehicle control. Conversely, the three groups of rats that received
different doses of compound 7i (0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kg) consumed less water in a
dose-dependent manner, with an effective concentration of 0.3 mg/kg. These results demonstrate
that 7i can exert the function of an H3 receptor antagonist in vivo.

Figure 8. Evaluation of 7i in (R)-α-methylhistamine-induced dipsogenia model in 2 hours (rats, n
= 8 per group). White bar shows the vehicle model, black bars shows groups taken
(R)-α-methylhistamine and different dose of 7i (*, p<0.05; **, p<0.01 compared with
(R)-α-methylhistamine alone group).
2.11 Morris Water Maze Testing
To further investigate the effect of 7i on an AD model, we performed the Morris water maze of
scopolamine-induced spatial learning and memory deficits study in rats [35]. The difference in the
latency time taken by rats to find the platform was used as the evaluation parameter. Results
indicate that the anticholinergic agent, scopolamine, caused a significant learning impairment in
rats (Figure 9, Table S3), while pretreatment with 7i (1.0 or 3.0 mg/kg for 2 h before each training
session) significantly reversed these memory deficits, confirming the fact that H3 antagonist 7i
could improve the cholinergic function of rats.
Figure 9. Evaluation of compound 7i on Morris water maze. (##, p<0.05 versus vehicle; **,
p<0.01 versus scopolamine, n = 8 per group.)
3. Conclusion
In summary, to avoid the possible phase II metabolism of lead compound 4, the
3-hydroxy-4-pyridinone moiety was changed to alkyloxy moiety and
a
series of
N-phenyl-3-methoxy-4-pyridinone derivatives were synthesized and evaluated as orally
bioavailable H3 receptor antagonists with β-amyloid aggregation inhibitory activities. Further
biological evaluations on the most promising compound 7i, indicate that it possesses a potent and
high selective H3 receptor antagonistic activity in vitro and in vivo, a good PK profile

accompanied with good BBB permeability and low hERG inhibition risk. These results suggest
that 7i has therapeutic potential as a novel multi-target agent for the treatment of Alzheimer’s
disease.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (21172193) and
Zhejiang Provincial Natural Foundation of China (No.R2110297). We thank Jianyang Pan
(Research and Service Center, College of Pharmaceutical Sciences, Zhejiang University) for
performing NMR spectrometry for structure elucidation
4. Experimental section
4.1 Biology
General procedure of biological evaluation is presented in Supplementary Information.
4.2 Chemistry
General Methods. All Reagents and solvents were purchased from common commercial of
1
analytical grade. H NMR were recorded on a BRUKER AVANCE III 500 M spectrometer, 13C
NMR were recorded on a 125 MHz spectrometer (chemical shifts are given in ppm relative to
TMS as internal standard). Mass spectra (ESI-MS) were performed on an FINNIGAN
LCQ-DECAXP spectrometer.
General Synthetic Procedure for compound 12a-12e
A mixture of compound 11a-11e (2.0 mmol), aminophenol (4.4 mmol), 2.5 mL diluted HCl
solution and 2.0 mL ethanol was placed in a MW tube (10mL) containing a magnetic stirring bar.
The reaction tube was sealed and irradiated in the cavity of a microwave apparatus at 155 ℃ for
25 minutes. After completion of the reaction, the tube was removed, cooled to ambient
temperature, and the precipitate was filtered and rinsed with hot water to afford 12a-12e.
3-Methoxy-1-(4-hydroxyphenyl)-2-methylpyridin-4(1H)-one (12a)

1
Pale-yellow solid, yield: 74%. H NMR (500 MHz, DMSO-d6): δ 10.03 (s, 1H), 7.53 (d, J = 7.5
Hz, 1H), 7.25 (d, J = 9.0 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 6.16 (d, J = 7.5 Hz, 1H), 3.73 (s, 3H),
1.96 (s, 3H). ESI-MS: m/z=232 [M+H]+.
3-Methoxy-1-(4-hydroxyphenyl)-2-ethylpyridin-4(1H)-one (12b)
1
Yellow solid, yield: 63%. H NMR (500 MHz, DMSO-d6): δ 10.02 (s, 1H), 7.48 (d, J = 7.5 Hz,
1H), 7.27 (d, J = 9.0 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 6.16 (d, J = 7.5 Hz, 1H), 3.80 (s, 3H), 2.39
(q, J = 6.0 Hz, 2H), 0.91 (q, J = 6.0 Hz, 3H). ESI-MS: m/z=246 [M+H]+.
5-Methoxy-1-(4-hydroxyphenyl)-2-methylpyridin-4(1H)-one (12c)
1
Yellow soild, yield: 73%, H NMR (400 MHz, DMSO-d6): δ 10.00 (s, 1H), 7.25-7.23 (m, 3H),
6.89 (d, J = 8.4 Hz, 2H), 6.14 (s, 1H), 3.62 (s, 3H), 1.93 (s, 3H). ESI-MS: m/z=232 [M+H]+.
3-Methoxy-1-(4-hydroxyphenyl)pyridin-4(1H)-one (12d)
Yellow soild, yield: 67%, 1H NMR (400 MHz, DMSO-d6): δ 9.88 (s, 1H), 7.77 (d, J = 7.2 Hz, 1H),
7.57 (s, 1H); 7.40 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 6.21 (d, J = 7.2 Hz, 1H), 3.71 (s,
3H). ESI-MS: m/z=218 [M+H]+.
3-Ethoxy-1-(4-hydroxyphenyl)-2-methylpyridin-4(1H)-one (12e)
1
Yellow soild, yield 71%. H NMR (500 MHz, DMSO-d6): δ 10.03 (s, 1H), 7.52 (d, J = 7.5 Hz,
1H), 7.24 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 6.16 (d, J = 7.5 Hz, 1H), 4.07 (q, J = 7.0
Hz, 2H), 1.97 (s, 3H), 1.24 (t, J = 7.0 Hz, 3H). ESI-MS: m/z=246 [M+H]+.
General Synthetic Procedure for compound 13a-13g
A
mixture of compound 12a-12e (5.0 mmol), K2CO3 (1.38g, 10.0 mmol), and
1-bromo-3-chloropropane (or 1-bromo-2-chloroethane, or 1-bromo-4-chlorobutane, 1.0 mL, 10.0
mmol) was refluxed in 25 mL acetonitrile for 6-8 hours. The mixture was cooled to ambient
temperature, filtered, washed with acetonitrile and concentrated to dryness. The crude product was
purified by silica gel chromatography to give pure 13a-13g.

3-Methoxy-1-(4-(3-chloropropoxy)phenyl)-2-methylpyridin-4(1H)-one (13a)
Yellow soild, yield 80%. 1H NMR (500 MHz, CDCl3): δ 7.20 (d, J = 7.5 Hz, 1H), 7.12 (d, J = 9.0
Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.42 (d, J = 7.5 Hz, 1H), 4.13 (t, J = 6.0 Hz, 2H), 3.86 (s, 3H),
3.72 (t, J = 6.0 Hz, 2H), 2.24-2.19 (m, 2H), 2.00 (s, 3H). ESI-MS: m/z=308 [M+H]+ .
3-Methoxy-1-(4-(2-chloroethoxy)phenyl)-2-methylpyridin-4(1H)-one (13b)
Pale-yellow soild, yield 59%. 1H NMR (500 MHz, CDCl3): δ 7.26 (d, J = 7.5 Hz, 1H), 7.20 (d, J =
9.0 Hz, 2H), 7.03 (d, J = 9.0 Hz, 2H), 6.47 (d, J = 7.5 Hz, 1H), 4.29 (t, J = 6.0 Hz, 2H), 3.90 (s,
3H), 3.86(t, J = 6.0 Hz, 2H), 2.05 (s, 3H). ESI-MS: m/z=294 [M+H]+.
3-Methoxy-1-(4-(4-chlorobutoxy)phenyl)-2-methylpyridin-4(1H)-one (13c)
Pale-yellow soild, yield 59%. 1H NMR (500 MHz, CDCl3): δ 7.29 (d, J = 7.5 Hz, 1H), 7.20 (d, J =
9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.54 (d, J = 7.5 Hz, 1H), 4.06 (t, J = 6.0 Hz, 2H), 3.92 (s,
3H), 3.65(t, J = 6.0 Hz, 2H), 2.07 (s, 3H), 2.00-1.98 (m, 4H). ESI-MS: m/z=322 [M+H]+.
3-Methoxy-1-(4-(3-chloropropoxy)phenyl)-2-ethylpyridin -4(1H)-one (13d)
1
Pale-yellow soild, yield 76%. H NMR (500 MHz, CDCl3): δ 7.16-7.13 (m, 3H), 6.94 (d, J = 9.0
Hz, 2H), 6.40 (d, J = 7.5 Hz, 1H), 4.13 (t, J = 6.0 Hz, 2H), 3.90 (s, 3H), 3.72 (t, J = 6.0 Hz, 2H),
2.44 (q, J = 7.5 Hz, 2H), 2.24-2.19 (m, 2H), 0.94 (t, J = 7.5 Hz, 3H). ESI-MS: m/z=322 [M+H]+.
5-Methoxy-1-(4-(3-chloropropoxy)phenyl)-2-methylpyridin-4(1H)-one (13e)
Pale-yellow soild, yield: 85%, 1H NMR (500 MHz, CDCl3): δ 7.23 (d, J = 9.0 Hz, 2H), 7.04 (d, J
= 9.0 Hz, 2H), 6.98 (s, 1H), 6.44 (s, 1H), 4.20 (t, J = 6.0 Hz, 2H), 3.79 (t, J = 6.0 Hz, 2H), 3.76 (s,
3H), 2.31-2.26 (m, 2H), 2.02 (s, 3H). ESI-MS: m/z=308 [M+H]+.
3-Methoxy-1-(4-(3-chloropropoxy)phenyl)- pyridin-4(1H)-one (13f)
1
Pale-yellow soild, yield 66%. H NMR (500 MHz, CDCl3): δ 7.57-7.56 (m, 1H), 7.38 (d, J = 9.0
Hz, 2H), 7.32-7.31 (m, 1H), 7.03 (d, J = 9.0 Hz, 2H), 6.57 (d, J = 7.0 Hz, 1H), 4.18 (t, J = 6.0 Hz,
2H), 3.88 (s, 3H), 3.78 (t, J = 6.0 Hz, 2H), 2.33-2.25 (m, 2H). ESI-MS: m/z=294 [M+H]+.

3-Ethoxy-1-(4-(3-chloropropoxy)phenyl)-2-methylpyridin-4(1H)-one (13g)
Pale-yellow soild, yield 77%. 1H NMR (500 MHz, CDCl3): δ 7.19 (d, J = 7.5 Hz, 1H), 7.12 (d, J =
9.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.38 (d, J = 7.5 Hz, 1H), 4.18 (q, J = 7.0 Hz, 2H), 4.12 (t, J
= 6.0 Hz, 2H), 3.72 (t, J = 6.0 Hz, 2H), 2.24-2.19 (m, 2H), 2.00 (s, 3H), 1.30 (t, J = 7.0 Hz, 1H).
ESI-MS: m/z=322 [M+H]+.
General Synthetic Procedure for compound 7a-7j
A mixture of 13a-13g (0.5 mmol), secondary amine (pyrrolidine, or piperidine, or morpholine, or
N-methylpiperazine, 1.5 mmol) and triethylamine (0.4 mL, 2.5 mmol) was refluxed overnight in
10 mL acetonitrile. The mixture was concentrated to dryness and the residue was partitioned
between ethyl acetate (20 mL) and 2.0 mol/L hydrochloric acid (20 mL). The organic layers were
discarded and the aqueous layer was basified with 2.0 mol/L NaOH (pH > 10) and extraction with
EtOAc (20 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, and
concentrated to dryness in vacuo. The residue was purified by silica gel chromatography to give
7a-7j.
3-Methoxy-2-methyl-1-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)pyridin-4(1H)-one (7a)
Pale-yellow solid, yield 61%, mp 89℃. IR (KBr, cm−1) 3029, 2995, 2949, 2878, 1621, 1557, 1508,
1288, 1239, 1052, 840. 1H NMR (500 MHz, CDCl3): δ 7.19 (d, J = 7.5Hz, 1H), 7.09 (d, J = 9.0 Hz,
2H), 6.93 (d, J = 9.0 Hz, 2H), 6.38 (d, J = 7.5 Hz, 1H), 4.05 (t, J = 6.5 Hz, 2H), 3.84 (s, 3H), 2.70
(t, J = 7.0 Hz, 2H), 2.62-2.59 (m, 4H), 2.07-2.01 (m, 2H), 1.99 (s, 3H), 1.82-1.78 (m, 4H). 13C
NMR (125 MHz, CDCl3): δ 173.74, 159.54, 147.36, 141.11, 139.01, 134.46, 127.82, 116.84,
+
115.42, 66.49, 59.41, 54.24, 52.96, 28.67, 23.44, 14.07. HRMS (ESI) m/z Calcd. for C20H27N2O3
[M+H]+ 343.2016, Found 343.2024. HPLC purity: 98.5%.
3-Methoxy-2-methyl-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)pyridin-4(1H)-one (7b)
Pale-yellow solid, yield 82%, mp 89℃. IR (KBr, cm−1) 3071, 2967, 2929, 2883, 1623, 1573, 1508,
1286, 1248, 1045, 836. 1H NMR (500 MHz, CDCl3): δ 7.20 (d, J = 7.5Hz, 1H), 7.10 (d, J = 9.0 Hz,
2H), 6.93 (d, J = 9.0 Hz, 2H), 6.38 (d, J = 7.5 Hz, 1H), 4.03 (t, J = 6.5 Hz, 2H), 3.84 (s, 3H), 2.58
(t, J = 7.0 Hz, 2H), 2.53-2.47 (m, 4H), 2.07-2.01 (m, 2H), 1.99 (s, 3H), 1.64-1.61 (m, 4H),

1.44-1.42 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 173.74, 159.54, 147.34, 141.20, 139.04,
134.43, 127.81, 116.80, 115.42, 66.92, 59.41, 55.77, 54.60, 26.57, 25.81, 24.29, 14.07. HRMS
(ESI) m/z Calcd. for C21H29N2O3+ [M+H]+ 357.2173, Found 357.2182. HPLC purity: 97.5%.
3-Methoxy-2-methyl-1-(4-(3-morpholinopropoxy)phenyl)pyridin-4(1H)-one (7c)
Pale-yellow solid, yield 81%, mp 87℃. IR (KBr, cm−1) 3065, 2974, 2936, 2897, 1620, 1556, 1508,
1288, 1238, 1052, 843. 1H NMR (500 MHz, CDCl3): δ 7.21 (d, J = 7.5Hz, 1H), 7.10 (d, J = 9.0 Hz,
2H), 6.93 (d, J = 9.0 Hz, 2H), 6.41 (d, J = 7.5 Hz, 1H), 4.03 (t, J = 6.5 Hz, 2H), 3.84 (s, 3H),
3.69-3.65 (m, 4H), 2.51 (t, J = 7.0 Hz, 2H), 2.47-2.41 (m, 4H), 2.00 (s, 3H), 1.98-1.93 (m, 2H).
13C NMR (125 MHz, CDCl3): δ 173.73, 159.51, 147.35, 141.38, 139.14, 134.50, 127.84, 116.80,
115.43, 66.74, 66.52, 59.49, 55.37, 53.64, 26.12 , 14.09. HRMS (ESI) m/z Calcd. for
C20H27N2O4+ [M+H]+ 359.1965, Found 359.1971. HPLC purity: 98.4%.
3-Methoxy-2-methyl-1-(4-(3-(4-methyl-1-piperazinyl)propoxy)phenyl)pyridin-4(1H)-one (7d)
1
Pale-yellow solid, yield: 64%, mp 95℃. H NMR (400 MHz, CDCl3): δ 7.27 (d, J = 7.6Hz, 1H),
7.17 (d, J = 8.8Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.46 (d, J = 7.6 Hz, 1H), 4.09 (t, J = 6.0 Hz, 2H),
3.91 (s, 3H), 2.60-2.56 (m, 6H), 2.48-2.40 (m, 4H), 2.34 (s, 3H), 2.06 (s, 3H), 2.05-2.00 (m, 2H).
13C NMR (100 MHz, CDCl3): δ 173.77, 159.51, 147.40, 141.15, 139.01, 134.55, 127.86, 116.86,
+
115.43, 66.66, 59.44, 54.89, 52.84, 45.77, 26.55, 14.07. HRMS (ESI) m/z Calcd. for C21H30N3O3
[M+H]+ 372.2282, Found 372.2294. HPLC purity: 97.4%.
3-Methoxy-2-methyl-1-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)pyridin-4(1H)-one (7e)
1
Pale-yellow solid, yield: 70%, mp 90℃. H NMR (500 MHz, CDCl3): δ 7.24 (d, J = 7.5Hz, 1H),
7.15 (d, J = 9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.43 (d, J = 7.5 Hz, 1H), 4.17 (t, J = 6.5 Hz, 2H),
3.89 (s, 3H), 2.97 (t, J = 7.0 Hz, 2H), 2.68-2.66 (m, 4H), 2.04 (s, 3H), 1.83-1.80 (m, 4H). 13C
NMR (100 MHz, CDCl3): δ 173.78, 159.34, 147.39, 141.18, 139.01, 134.67, 127.87, 116.85,
+
115.52, 67.42, 59.44, 54.82, 54.68, 23.47, 14.07. HRMS (ESI) m/z Calcd. for C19H25N2O3
[M+H]+ 329.1860, Found 329.1867. HPLC purity: 97.1%.
3-Methoxy-2-methyl-1-(4-(4-(pyrrolidin-1-yl)butoxy)phenyl)pyridin-4(1H)-one (7f)

1
Pale-yellow solid, yield: 76%, mp 82℃. H NMR (500 MHz, CDCl3): δ 7.27 (d, J = 7.5Hz, 1H),
7.16 (d, J = 9.0 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 6.45 (d, J = 7.5 Hz, 1H), 4.05 (t, J = 6.5 Hz, 2H),
3.91 (s, 3H), 2.58-2.54 (m, 6H), 2.06 (s, 3H), 1.90-1.84 (m, 2H), 1.82-1.78 (m, 4H), 1.77-1.72 (m,
2H). 13C NMR (100 MHz, CDCl3): δ173.77, 159.58, 147.38, 141.20, 139.03, 134.45, 127.83,
116.83, 115.40, 68.20, 59.43, 56.00, 54.12, 27.20, 25.31, 23.39, 14.06. HRMS (ESI) m/z Calcd.
for C21H29N2O3+ [M+H]+ 357.2173, Found 357.2171. HPLC purity: 96.5%.
2-Ethyl-3-methoxy-1-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)pyridin-4(1H)-one (7g)
Pale-yellow solid, yield 58%, mp 93℃. IR (KBr, cm−1) 3034, 2956, 2882, 2817, 1633, 1583, 1506,
1
1487, 1289, 1248, 1015, 846. H NMR (500 MHz, CDCl3): δ 7.15-7.12 (m, 3H), 6.93 (d, J = 9.0
Hz, 2H), 6.37 (d, J = 7.5 Hz, 1H), 4.08 (t, J = 6.5 Hz, 2H), 3.90 (s, 3H), 2.94-2.88 (m, 2H),
2.87-2.84 (m, 4H), 2.43 (q, J = 7.5 Hz, 2H), 2.19-2.16 (m, 2H), 1.95-1.90 (m, 4H), 0.93 (t, J =
7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 173.37, 159.00, 146.98, 146.24, 136.37, 134.77,
128.51, 117.17, 115.54, 71.74, 65.66, 53.93, 52.69, 26.37, 23.38, 20.13, 8.75. HRMS (ESI) m/z
Calcd. for C21H29N2O3+ [M+H]+ 357.2173, Found 357.2182. HPLC purity: 98.7%.
5-Methoxy-2-methyl-1-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)pyridin-4(1H)-one (7h)
Pale-yellow solid, yield 64%, mp 133℃. IR (KBr, cm−1) 3046, 2966, 2951, 2843, 1626, 1583,
1554, 1509, 1286, 1243, 1018, 849. 1H NMR (500 MHz, CDCl3): δ 7.12 (d, J = 9.0 Hz, 2H), 6.95
(d, J = 9.0 Hz, 2H), 6.88 (s, 1H), 6.31 (s, 1H), 4.06 (t, J =6.5 Hz, 2H), 3.68 (s, 3H), 2.78-2.74 (m,
2H), 2.68-2.64 (m, 4H), 2.11-2.06 (m, 2H), 1.93 (s, 3H), 1.86-1.80 (m, 4H). 13C NMR (125 MHz,
CDCl3): δ 172.85, 159.32, 148.88, 145.93, 134.97, 127.94, 123.14, 116.15, 115.52, 66.39, 56.23,
+
54.17, 52.94, 27.78, 23.46, 20.12. HRMS (ESI) m/z Calcd. for C20H27N2O3 [M+H]+ 343.2016,
Found 343.2013. HPLC purity: 98.5%.
3-Methoxy-1-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)pyridin-4(1H)-one (7i)
Pale-yellow solid, yield 78%, mp 93℃. IR (KBr, cm−1) 3037, 2974, 2952, 2883, 1627, 1557, 1509,
1
1286, 1243, 1020, 830. H NMR (500 MHz, CDCl3): δ 7.47-7.45 (dd, J1 = 7.5 Hz, J2 = 2.5 Hz,
1H), 7.27 (d, J = 9.0 Hz, 2H), 7.25 (d, J = 2.5 Hz, 1H), 6.87 (d, J = 9.0 Hz, 2H), 6.45 (d, J = 7.5
Hz, 1H), 4.07 (t, J = 5.5 Hz, 2H), 3.79 (s, 3H), 3.38-3.35 (m, 4H), 3.10-3.07 (m, 2H), 2.35-2.31 (m,

2H), 2.16-2.12 (m, 4H). 13C NMR (125 MHz, CDCl3): δ 172.31, 158.72, 150.43, 136.93, 136.80,
124.45, 121.31, 115.93, 115.72, 66.30, 56.33, 54.12, 52.91, 31.23, 29.67, 27.57, 23.43. HRMS
(ESI) m/z Calcd. for C19H25N2O3+ [M+H]+ 329.1860, Found 329.1863. HPLC purity: 99.0%.
3-Ethoxy-2-methyl-1-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)pyridin-4(1H)-one (7j)
Pale-yellow solid, yield 85%, mp 89℃. IR (KBr, cm−1) 3425, 3138, 3053, 2965, 2929, 2876, 2698,
1
1621, 1565, 1509, 1479, 1285, 1239, 1050, 845. H NMR (500 MHz, CDCl3): δ 7.17 (d, J = 7.5
Hz, 1H), 7.09 (d, J = 9.0 Hz, 2H), 6.92 (d, J = 9.0 Hz, 2H), 6.37 (d, J = 7.5 Hz, 1H), 4.18 (q, J =
7.0 Hz, 2H), 4.03 (t, J = 6.0 Hz, 2H), 2.68 (d, J = 7.5 Hz, 2H), 2.60-2.58 (m, 4H), 2.04-2.00 (m,
2H), 1.99 (s, 3H), 1.80-1.76 (m, 4H), 1.30 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ
173.88, 159.42, 146.38, 141.05, 138.84, 134.64, 127.84, 116.71, 115.40, 67.19, 66.78, 54.23,
+
52.96, 28.57, 23.46, 15.65, 14.30. HRMS (ESI) m/z Calcd. for C21H29N2O3 [M+H]+ 357.2173,
Found 357.2174. HPLC purity: 97.7%.
N-(tert-Butoxycarbonyl)-4-(4-nitrophenoxy)piperidine (15)
N-(tert-Butoxycarbonyl)-4-hydroxypiperidine (1.0 g, 4.97 mmol) was dissolved in anhydrous
DMF (5 mL) and cooled to 0 °C. NaH (60%, 300 mg, 7.5 mmol) was added portionwise in 15 min,
and then the mixture was stired for a further 30 min at ambient temperature. The mixture was then
cooled to 0 °C, and a solution of 4-fluoronitrobenzene 14 (771 mg, 5.5 mmol) in DMF (10 mL)
was added dropwise. The reaction mixture was stirred overnight at ambient temperature. After
completion of the reaction, water was added, and the aqueous layer was extracted with EtOAc.
The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to afford
1
1.3 g 15 as a pale-yellow solid, yield 81%. H NMR (500 MHz, CDCl3): δ 8.22 (d, J = 9.0 Hz,
2H), 6.97 (d, J = 9.0 Hz, 2H), 4.63-4.59 (m, 2H), 3.73-3.68 (m, 2H), 3.41-3.36 (m, 2H), 1.99-1.94
(m, 2H), 1.82-1.76 (m, 2H), 1.48 (s, 9H). ESI-MS: m/z =323.2 [M+H]+.
4-(4-Nitrophenoxy)piperidine (16)
A mixture of 15 (1 g, 3.1 mmol) in CH2Cl2 (10 mL) was added trifluoroacetic acid (2.5 mL), and
stirred overnight at ambient temperature. The mixture was concentrated to dryness and the residue
was partitioned between ethyl acetate (20 mL) and 2.0 mol/L NaOH (20 mL). The combined

organic layers were washed with brine, dried over Na2SO4, and concentrated to afford 665 mg 16
as pale-yellow liquid, yield 92%. 1H NMR (500 MHz, CDCl3): δ 8.21 (d, J = 9.0 Hz, 2H), 6.96 (d,
J = 9.0 Hz, 2H), 4.56-4.52 (m, 2H), 3.21-3.16 (m, 2H), 2.86-2.82 (m, 2H), 2.11-2.06 (m, 2H),
1.81-1.74 (m, 2H). ESI-MS: m/z =223.1 [M+H]+.
General Synthetic Procedure for compound 17a-17c
A mixture of 16 (1 g, 4.5 mmol) and sodium cyanoborohydride (566 mg, 9 mmol) was dissolved
in CH3OH (10 mL), and acetone (or cyclobutanone, or cyclopentanone, 5 mL) and acetic acid
(270 mg) were added and stirred at ambient temperature for 2h. After completion of the reaction,
the mixture was concentrated to dryness and the residue was partitioned between ethyl acetate (20
mL) and 2.0 mol/L NaOH (20 mL). The combined organic layers were washed with brine, dried
over Na2SO4, and concentrated to afford 17a-17c.
1-Isopropyl-4-(4-nitrophenoxy)piperidine (17a)
Yellow solid, yield 89%. 1H NMR (500 MHz, CDCl3): δ 8.19 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0
Hz, 2H), 4.41-4.45 (m, 1H), 2.80-2.75 (m, 3H), 2.46-2.41 (m, 2H), 2.02-1.99 (m, 2H), 1.87-1.85
(m, 2H), 1.07 (d, J = 6.5Hz, 6H). ESI-MS: m/z =265.2 [M+H]+.
1-Cyclobutyl -4-(4-nitrophenoxy)piperidine (17b)
Yellow solid, yield 80%. 1H NMR (500 MHz, CDCl3): δ 8.19 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0
Hz, 2H), 4.49-4.47 (m, 1H), 2.77-2.75 (m, 1H), 2.64-2.61 (m, 2H), 2.24-2.22 (m, 2H), 2.07-2.00
(m, 4H), 1.90-1.87 (m, 4H), 1.70-1.68 (m, 2H). ESI-MS: m/z =277.2 [M+H]+.
1-Cyclopentyl -4-(4-nitrophenoxy)piperidine (17c)
Yellow solid, yield 86%. 1H NMR (500 MHz, CDCl3): δ 8.20 (d, J = 9.0 Hz, 2H), 6.96 (d, J = 9.0
Hz, 2H), 4.48-4.46 (m, 1H), 2.84-2.82 (m, 2H), 2.60-2.59 (m, 1H), 2.46-2.44 (m, 2H), 2.11-2.08
(m, 2H), 1.91-1.88 (m, 4H), 1.72-1.71 (m, 2H), 1.56-1.54 (m, 2H), 1.48-1.46 (m, 2H). ESI-MS:
m/z =291.2 [M+H]+.
General Synthetic Procedure for compound 8a-8f

A mixture of compound 17a-17c (3.8 mmol) in methanol (10mL) was added 10%Pd/C (200mg),
stirred overnight at ambient temperature under hydrogen atmosphere. After filtered off the catalyst,
the filtrate was concentrated to give 18a-18c, which were used directly without further purification.
Then 18a-18c (1.3 mmol) and 11a (or 11d, 1 mmol) was dissolved in 0.5N hydrochloric acid (3
mL), stirred at 170 °C for 30 min using a microwave reactor. After cooling, the mixture was
extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4
and evaporated. The residue was purified by silica gel chromatography (EtOAc: CH3OH: TEA =
20: 1: 0.5) to give 8a-8f.
1-(4-((1-Isopropylpiperidin-4-yl)oxy)phenyl)-3-methoxy-2-methylpyridin-4(1H)-one (8a)
Yellow solid, yield 56%, mp 142℃. 1H NMR (500 MHz, CDCl3): δ 7.25 (d, J = 7.5Hz, 1H), 7.15
(d, J = 9.0 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 6.44 (d, J = 7.5 Hz, 1H), 4.37-4.35 (m, 1H), 3.90 (s,
3H), 2.81-2.76 (m, 3H), 2.47-2.43 (m, 2H), 2.08-2.06 (m, 2H), 2.05 (s, 3H), 1.89-1.82 (m, 2H),
1.08 (s, 3H), 1.07 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 173.77, 158.11, 147.34, 141.26, 139.07,
134.36, 127.91, 116.84, 116.59, 73.34, 59.46, 54.54, 45.58, 30.88, 18.38, 14.14. HRMS (ESI) m/z
Calcd. for C21H29N2O3+ [M+H]+ 357.2173, Found 357.2174.
1-(4-((1-Isopropylpiperidin-4-yl)oxy)phenyl)-3-methoxypyridin-4(1H)-one (8b)
Yellow solid, yield 48%, mp 97℃. 1H NMR (500 MHz, CDCl3): δ 7.38 (d, J = 7.5Hz, 1H), 7.25 (d,
J = 9.0 Hz, 2H), 7.20 (s, 1H), 6.95 (d, J = 9.0 Hz, 2H), 6.42 (d, J = 7.5 Hz, 1H), 4.31-4.29 (m, 1H),
3.80 (s, 3H), 2.71-2.66 (m, 3H), 2.37-2.33 (m, 2H), 2.03-2.01 (m, 2H), 1.84-1.82 (m, 2H), 1.05 (s,
3H), 1.04 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 172.33, 157.50, 150.43, 136.79, 136.74, 124.50,
121.19, 117.04, 115.92, 73.42, 56.26, 54.56, 45.55, 30.85, 18.38. HRMS (ESI) m/z Calcd. for
C21H29N2O3+ [M+H]+ 343.2016, Found 343.2020.
1-(4-((1-Cyclobutylpiperidin-4-yl)oxy)phenyl)-3-methoxy-2-methylpyridin-4(1H)-one (8c)
Yellow solid, yield 53%, mp 153℃. 1H NMR (500 MHz, CDCl3): δ 7.25 (d, J = 7.5Hz, 1H), 7.15
(d, J = 9.0 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 6.44 (d, J = 7.5 Hz, 1H), 4.38-4.36 (m, 1H), 3.92 (s,
3H), 2.77-2.76 (m, 1H), 2.66-2.64 (m, 2H), 2.21-2.20 (m, 2H), 2.06-2.00 (m, 7H), 1.88-1.86 (m,
4H), 1.75-1.66 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 173.75, 158.03, 147.36, 141.08, 139.00,

134.42, 127.95, 116.90, 116.57, 72.66, 60.28, 59.45, 46.68, 30.26, 27.45, 14.15. HRMS (ESI) m/z
Calcd. for C22H29N2O3+ [M+H]+ 369.2173, Found 369.2176.
1-(4-((1-Cyclobutylpiperidin-4-yl)oxy)phenyl)-3-methoxypyridin-4(1H)-one (8d)
Yellow solid, yield 51%, mp 152℃. 1H NMR (500 MHz, CDCl3): δ 7.38 (d, J = 7.5Hz, 1H), 7.25
(d, J = 9.0 Hz, 2H), 7.20 (s, 1H), 6.95 (d, J = 9.0 Hz, 2H), 6.42 (d, J = 7.5 Hz, 1H), 4.31-4.29 (m,
1H), 3.74 (s, 3H), 2.73-2.66 (m, 1H), 2.57-2.55 (m, 2H), 2.14-2.12 (m, 2H), 1.96-1.92 (m, 4H),
1.84-1.78(m, 4H), 1.65-1.63 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 172.35, 157.40, 150.47,
136.85, 136.76, 124.53, 121.08, 117.05, 115.98, 73.07, 60.28, 56.26, 46.54, 30.07, 27.36, 14.16.
HRMS (ESI) m/z Calcd. for C21H27N2O3+ [M+H]+ 355.2016, Found 355.2025.
1-(4-((1-Cyclopentylpiperidin-4-yl)oxy)phenyl)-3-methoxy-2-methylpyridin-4(1H)-one (8e)
Yellow solid, yield 51%, mp 151℃. 1H NMR (500 MHz, CDCl3): δ 7.25 (d, J = 7.5Hz, 1H), 7.15
(d, J = 9.0 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 6.44 (d, J = 7.5 Hz, 1H), 4.38-4.36 (m, 1H), 3.91 (s,
3H), 2.84-2.82 (m, 2H), 2.59-2.54 (m, 1H), 2.42-2.41 (m, 2H), 2.06-2.00 (m, 5H), 1.89-1.87 (m,
4H), 1.72-1.71 (m, 2H), 1.57-1.56 (m, 2H), 1.55-1.52 (m, 2H). 13C NMR (125 MHz, CDCl3): δ
173.76, 158.03, 147.37, 141.12, 139.01, 134.45, 127.96, 116.90, 116.57, 72.90, 67.47, 59.46,
+
49.38, 30.58, 30.47, 24.21, 14.16. HRMS (ESI) m/z Calcd. for C23H31N2O3 [M+H]+ 383.2329,
Found 383.2339.
1-(4-((1-Cyclopentylpiperidin-4-yl)oxy)phenyl)-3-methoxypyridin-4(1H)-one (8f)
Yellow solid, yield 44%, mp 157℃. 1H NMR (500 MHz, CDCl3): δ 7.38 (d, J = 7.5Hz, 1H), 7.25
(d, J = 9.0 Hz, 2H), 7.20 (s, 1H), 6.95 (d, J = 9.0 Hz, 2H), 6.42 (d, J = 7.5 Hz, 1H), 4.31-4.29 (m,
1H), 3.75 (s, 3H), 2.74-2.72 (m, 2H), 2.55-2.50 (m, 1H), 2.40-2.38 (m, 2H), 2.02-2.00 (m, 2H),
1.85-1.82 (m, 4H), 1.67-1.64 (m, 2H), 1.54-1.53 (m, 2H), 1.52-1.50 (m, 2H). 13C NMR (125 MHz,
CDCl3): δ 172.31, 157.46, 150.44, 136.77, 124.50, 121.10, 117.04, 115.92, 73.05, 67.44, 56.24,
+
49.42, 30.65, 30.55, 24.21. HRMS (ESI) m/z Calcd. for C22H29N2O3 [M+H]+ 369.2173, Found
369.2167.
4-(3-Methoxy-2-methyl-4-oxopyridin-1(4H)-yl)benzoic acid (19)

A mixture of methoxymaltol 11a (1.40 g, 10 mmol), 4-aminobenzoic acid (2.74 g, 20 mmol) was
dissolved in C2H5OH (10 mL) and H2O (10 mL), and the reaction was sealed at 150-160 ° C for
12 h. After completion of the reaction, the mixture was cooled, and ethanol was removed under
reduced pressure. The precipitate was then filtered and wash with cold ethanol to afford 19 (1.83g)
as a whith solid, yield 71%. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 8.10 (d, J = 8.4 Hz,
2H), 7.64-7.60 (m, 3H), 6.23 (d, J = 7.6 Hz, 2H), 3.76 (s, 3H), 2.00 (s, 3H). ESI-MS: m/z =260
[M+H]+.
N-Boc-3-methoxy-2-methyl-1-(4-(piperazine-1-carbonyl)phenyl)pyridin-4(1H)-one (20)
A mixture of 19 (520 mg, 2 mmol), N-Boc-piperazine (744 mg, 4 mmol) and EDC (460 mg, 2.4
mmol) in dry DMF (5 mL) was added trimethylamine, stirred at ambient temperature for 2h. After
completion of the reaction, water (50 mL) was added, and the aqueous layer was extracted with
EtOAc (20 mL×3). The combined organic layers were washed with brine, dried over Na2SO4 and
evaporated to afford 20 as a white solid, which were used directly without further purification.
3-Methoxy-2-methyl-1-(4-(piperazine-1-carbonyl)phenyl)pyridin-4(1H)-one (21)
A mixture of 20 (855 mg, 2 mmol) in CH2Cl2 (5 mL) was added trifluoroacetic acid (1 mL), and
refluxed for 2h. The mixture was partitioned between CH2Cl2 and 10% NaOH. The combined
organic layers were washed with brine, dried over Na2SO4, and concentrated to afford 370 mg 21
as white solid, yield 57%. 1H NMR (500 MHz, CDCl3): δ 7.60 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.5
Hz, 2H), 7.28 (d, J = 7.5 Hz, 1H), 6.51 (d, J = 7.5 Hz, 1H), 3.93 (s, 3H), 3.90-3.84 (m, 2H),
3.56-3.50 (m, 2H), 3.08-2.94 (m, 4H), 2.10 (s, 3H). ESI-MS: m/z =328 [M+H]+.
General Synthetic Procedure for compound 9a-9c
A mixture of 21 (0.31 mmol) and sodium cyanoborohydride (29 mg, 0.46 mmol) in CH3OH2 (3
mL) was added acetone (or cyclobutanone, or cyclopentanone, 0.11mL) and acetic acid (17 µL),
and stirred at ambient temperature for 2h. After completion of the reaction, water was added, and
the aqueous layer was extracted with EtOAc. The combined organic layers were washed with
brine, dried over Na2SO4, and concentrated to dryness in vacuo. The residue was purified by silica
gel chromatography (EtOAc: CH3OH: TEA = 10: 1: 0.5) to give 9a-9c.

1-(4-(4-Isopropylpiperazine-1-carbonyl)phenyl)-3-methoxy-2-methylpyridin-4(1H)-one(9a)
White solid, yield 68%, mp 150℃. 1H NMR (500 MHz, CDCl3): δ 7.59 (d, J = 8.5 Hz, 2H), 7.35
(d, J = 8.5 Hz, 2H), 7.26 (d, J = 7.5 Hz, 1H), 6.47 (d, J = 7.5 Hz, 1H), 3.93 (s, 3H), 3.87 (s, 2H),
3.52 (m, 2H), 2.83 (t, J = 5.5 Hz, 1H), 2.68-2.56 (m, 4H), 2.09 (s, 3H), 1.11-1.10 (m, 6H). 13C
NMR (125 MHz, CDCl3): δ173.73, 167.74, 147.46, 141.79, 140.68, 138.60, 138.10, 131.37,
129.99, 129.59, 116.99, 65.58, 59.49, 52.66, 30.57, 19.19, 14.16. HRMS (ESI) m/z Calcd. for
C21H28N3O3+ [M+H]+ 370.2125, Found 370.2130.
1-(4-(4-Isopropylpiperazine-1-carbonyl)phenyl)-3-methoxy-2-methylpyridin-4(1H)-one (9b)
White solid, yield 58%, mp 216℃. 1H NMR (500 MHz, CDCl3): δ 7.58 (d, J = 8.5 Hz, 2H), 7.35
(d, J = 8.5 Hz, 2H), 7.25 (d, J = 7.5 Hz, 1H), 6.47 (d, J = 7.5 Hz, 1H), 3.93 (s, 3H), 3.87 (s, 2H),
3.51 (s, 2H), 2.86-2.80 (m, 1H), 2.49-2.38 (m, 4H), 2.09-2.05 (m, 5H), 1.97-1.94 (m, 2H),
1.80-1.69(m, 2H). 13C NMR (125 MHz, CDCl3): δ173.82, 168.53, 147.59, 142.69, 140.23, 138.29,
136.99, 130.93, 128.88, 127.19, 117.27, 65.59, 60.01, 59.50, 26.72, 19.19, 14.26, 14.22. HRMS
(ESI) m/z Calcd. for C22H28N3O3+ [M+H]+ 382.2125, Found 382.2138.
1-(4-(4-Isopropylpiperazine-1-carbonyl)phenyl)-3-methoxy-2-methylpyridin-4(1H)-one (9c)
White solid, yield 61%, mp 234℃. 1H NMR (400 MHz, CDCl3): δ 7.60 (d, J = 8.4 Hz, 2H), 7.36
(d, J = 8.4 Hz, 2H), 7.31 (d, J = 7.6 Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 3.92 (s, 3H), 3.86-3.80 (m,
2H), 3.54-3.46 (m, 2H), 2.66-2.48 (m, 5H), 2.10 (s, 3H), 1.90-1.85 (m, 2H), 1.74-1.69 (m, 2H),
1.60-1.56 (m, 2H), 1.46-1.40 (m, 2H). 13C NMR (125 MHz, CDCl3): δ173.75, 168.32, 147.52,
142.53, 140.12, 138.21, 137.14, 128.80, 127.07, 117.21, 67.19, 59.42, 52.50, 51.76, 30.28, 24.00,
14.18. HRMS (ESI) m/z Calcd. for C23H30N3O3+ [M+H]+ 396.2282, Found 396.2289.
N-Boc-4-((4-nitrophenyl)sulfonyl)piperazine-1-carboxylate (23)
A mixture of N-Boc-piperazine (3.05 g, 16.4 mmol) and DIPEA (3.14 mL, 18 mmol) in dry
CH2Cl2 (20 mL) was added 22 (3.99 g, 18 mmol) in portions, stired under nitrogen atmosphere at
ambient temperature for 1h. After completion of the reaction, water was added, and the aqueous
layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over