Design, synthesis and lipid-lowering activities of penipyridone derivatives

Article abstract of DOI:10.1016/j.bmc.2021.116192

On the basis of our earlier discovered natural product penipyridone G with potential lipid-lowering utility, 35 penipyridone derivatives were designed, synthesized and characterized. Based on the oleic acid-induced HepG2 cell lipid accumulation model, compounds 12c, 14, 15f, 15k, 15o, 15p and 16f showed potent lipid-lowering activities among the synthetic compounds at 10 μM. In particular, compounds 4, 15k, 15o showed significant activities on inhibiting lipid accumulation in insulin resistant HepG2 cells, and these three compounds were safe and non-toxic within the concentration range of 400 μM. In comparison, 15o possessed the best lipid-lowering activity. Compared with the vehicle group, the triglyceride inhibition rate of 15o was about 30.2%, and the total cholesterol inhibition rate was about 14.8% at 20 μM, which was equipotent to Simvastatin. Our research indicates that 15o may serve as a promising lead compound for the development of hypolipidemic drugs.

Full text of DOI:10.1016/j.bmc.2021.116192

Bioorg. Med. Chem. 40 (2021) 116192
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and lipid-lowering activities of penipyridone derivatives
,
,
,
,*
Liping Lia 1, Zhongwei Duana 1, Donghui Baia 1, Fang Lua, Jiejie Haoa, Tianjiao Zhua
,
,b,c,*
Deihai Lia
a Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of
China
b Laboratory for Marine Drugs and Bioproducts of Qingdao Pilot National Laboratory for Marine Science and Technology, Qingdao 266237, People’s Republic of China
c Open Studio for Druggability Research of Marine Natural Products, Pilot National Laboratory for Marine Science and Technology, Qingdao 266237, People’s Republic
of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
On the basis of our earlier discovered natural product penipyridone G with potential lipid-lowering utility, 35
penipyridone derivatives were designed, synthesized and characterized. Based on the oleic acid-induced HepG2
cell lipid accumulation model, compounds 12c, 14, 15f, 15k, 15o, 15p and 16f showed potent lipid-lowering
Penipyridone derivatives
Synthesis
Lipid-lowering activity
No cytotoxicity
Molecular docking
activities among the synthetic compounds at 10 μM. In particular, compounds 4, 15k, 15o showed significant
activities on inhibiting lipid accumulation in insulin resistant HepG2 cells, and these three compounds were safe
and non-toxic within the concentration range of 400 M. In comparison, 15o possessed the best lipid-lowering
activity. Compared with the vehicle group, the triglyceride inhibition rate of 15o was about 30.2%, and the total
μ
cholesterol inhibition rate was about 14.8% at 20 μM, which was equipotent to Simvastatin. Our research in-
dicates that 15o may serve as a promising lead compound for the development of hypolipidemic drugs.
1. Introduction
hypolipidemic drugs with improved efficacy, safety and reliability.
Classically, natural products play a crucial role in drug discovery and
In recent years, the population of hyperlipidemia has increased
gradually around the world due to the unhealthy lifestyles, such as
smoking, high fatty acid intake and inherited genetic disorder.1,2
Hyperlipidemia is a common dyslipidemia characterized by increased
blood levels of total cholesterol (TC), triglycerides (TG), low-density
lipoprotein cholesterol (LDL-C) and decreased levels of high-density li-
poprotein cholesterol (HDL-C).3 Numerous epidemiological studies have
confirmed that hyperlipidemia can cause many complications, such as
atherosclerosis,4 cardiovascular diseases,5,6 hypertension and diabetes
mellitus,7 which poses a great threat to human health. Statins, as HMG-
CoA reductase inhibitors, are currently the first-line drugs for the
treatment of hyperlipidemia.8 In addition, fibrates, niacin, cholesterol
absorption inhibitors and bile acid sequestrants are also clinically used
to treat hyperlipidemia, because they can affect the metabolism of
cholesterol and triglycerides. However, their potential toxicities cause
great trouble to the patient, which limit their long-term applications.9
Therefore, it is always a hot research topic for medicinal chemists to seek
development. The contribution of natural products in the therapeutics of
dyslipidemia is phenomenal. Some promising natural compounds with
lipid-lowering activity are either in the clinical stage or in the devel-
opmental stage. Many other potential lead compounds deserve further
attentions.10 In 2016, we reported seven pyridone alkaloid natural
products A-G (Fig. 1A) (originally numbered 1–7), isolated from the
fermentation broth of an Antarctic moss-derived fungus, Penicillium
funiculosum GWT2-24. In vitro fat regulating activity test results showed
that compounds A, B, E and G possessed desirable inhibitory effects on
intracellular lipid accumulation at 10 μM, and had lowering effects on
total cholesterol and triglycerides. Especially, compound G exhibited
greater inhibitory activity on lipid accumulation in insulin resistant
HepG2 cells than other tested compounds.11 By comparing lipid-
lowering effects of compounds D with E and F with G, it seemed that
acetyl was an indispensable pharmacophore. In order to verify the role
of hydroxyethoxy group, compounds 1, 2, 3 and isomer 4 of compound E
were synthesized firstly (Fig.1B). Then according to the activity results
* Corresponding authors at: Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, People’s Republic of China (T. Zhu and D. Li).
E-mail addresses: zhutj@ouc.edu.cn (T. Zhu), dehaili@ouc.edu.cn (D. Li).
1
These first authors contributed equally to this article.
https://doi.org/10.1016/j.bmc.2021.116192
Received 8 March 2021; Received in revised form 25 April 2021; Accepted 26 April 2021
Available online 1 May 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

L. Li et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116192
Fig. 1. (A) The structures of penipyridones A-G; (B) The structures of pen-
ipyridone analogues 1–4.
of compounds A-G and 1–4, compounds G and 4 were selected as the
lead compounds. Consequently, thirty-one penipyridone derivatives
were designed and synthesized based on the lead compounds. Mean-
while, the lipid-lowering activities of all synthetic compounds were
evaluated and their structure–activity relationships (SARs) were also
discussed.
2. Results and discussion
2.1. Chemistry
Scheme 1. Structural optimization of Unit A. (i) Synthesis of compounds 11
and 12. Reagents and conditions: (I) LiHMDS, THF, ꢀ 78 ◦C, 30 min; (II)
NH4OAc, AcOH, 60 ◦C, 30 min; (III) saturated NH3 in MeOH, 48 h, 80 ◦C; (IV)
corresponding acid, EDCI, DMAP, CH2Cl2, 0 ◦C, 1 h. (V) 2-Bromoethanol,
K2CO3, acetone, 48 h, 80 ◦C. (ii) Synthesis of compounds of 13 and 14. Re-
agents and conditions: (I) CH3ONa, MeOH, 4 h; (II) Pd/C, H2, MeOH, 2 h.
2.1.1. Lead compounds
The lipid-lowering activities of compounds A-G and 1–4 were eval-
uated by the model of HepG2 adipose accumulation. As a result, com-
pound 4 showed comparable lipid-lowering activity with G, even
slightly better than G (Fig. 2A). Comparing compound G with compound
1 and compounds E or 4 with compound 3, the hydroxyethoxy group
was beneficial to improve lipid-lowering activity, and the Unit B was a
modifiable potential site. In addition, it seemed that the hydrogen of
amide group was essential, which was likely to interact with protein and
enhanced the lipid-lowering activity. Consequently, compounds G and 4
were selected as the lead compounds for further optimization. The Unit
A, B and C of compounds G and 4 were modified as followed (Fig. 2B).
commercially available methyl acetoacetate and N, N-dimethylforma-
mide dimethyl acetal as the starting materials.12 Compounds 6b and 6e
were synthesized using different commercially available acids and
oxalyl chloride as the starting materials.13 Compounds 6c and 6d were
purchased commercially.
In Scheme 1, compound 5 was treated with a series of different acyl
chlorides 6b–6e in the presence of lithium bis(trimethylsilyl) amide
(LiHMDS) and then acetic acid and NH4OAc were added to the reaction
mixture to afford the compound 2 and intermediates 7b-7d.14 After that
compounds 2 and 7b-7d were deacylated in the presence of saturated
2.1.2. Synthetic procedures
The synthetic protocols for the penipyridone derivatives were out-
lined in Schemes 1, 2 and 3. Compound 5 was synthesized using
Fig. 2. (A) The inhibition activity of compounds A-G and 1–4 on lipid accumulation in HepG2 cells in vitro (Simvastatin and tested compounds at 10 μM). Data were
expressed as the mean ± SD (n = 4) of each group of cells from at least three independent experiments; OA: Oleic Acid, **P < 0.01, compared with DMSO-treated
cells, #P < 0.05, compared with OA-treated cells. (B) Lead compounds with their structural units.
2

L. Li et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116192
Fig. 3. The inhibition activity on lipid accumulation in HepG2 cells in vitro
(Simvastatin and tested compounds at 10 μM). Data are expressed as the mean
Scheme 2. Structural optimization of Unit B: Synthesis of compounds 15 and
16. Reagents and conditions: (I) K2CO3, acetone, 80 ◦C, overnight. (II) mor-
pholine or thiomorpholine, K2CO3, acetone, 80 ◦C, overnight.
± SD (n = 6); OA: Oleic Acid, **P < 0.01, compared with DMSO-treated cells; #
P < 0.01, compared with OA-treated cells. Control (pink), Unit A (green), Unit B
(gold), Unit C(blue).
compounds in HepG2 cells by MTT assays (Table S1), none of them
showed cytotoxic effect under tested concentrations (100 μM).
SARs study revealed that the activities of compounds with pyridine
ring were better than that of pyridinone ring. In addition, Unit A was an
essential pharmacophore and S configuration of C-7 was preferred. R
configuration obviously impaired the lipid-lowering activity (11b).
Removel of acetyl group at C-7 (13), deacetylation (14) or increased the
length of C-16 (11a and 12a) all resulted in decreased activities than
those of compounds G and 4. When Unit A was replaced by phenyl (11c
and 12c) or tert-butyl (11d), lipid-lowering activities were lower than
that of compounds G and 4. Compound 15o with 4-ethylthiomorpholine
group at Unit B displayed the best lipid-lowering activity among all
compounds. The key factors affecting activities might be moderate size,
flexibility and hydrophilicity of Unit B. These results suggested that
either too large (bis(trifluoromethyl)benzyl) or too small (hydroxyethyl)
or too rigid (phenyl) R substituted groups was unfavorable. Compared
with compounds 15g, 16g and 16q, 15f and 16f showed better lipid-
lowering activities, possibly attributed to their better hydrophilicity.
Compounds 15l and 16l, methylated derivatives of compounds E and 4
with reduced hydrophilicity of Unit B, showed significantly decreased
activities. Bis (trifluoro-methyl) benzyl (15j and 16j), benzyl (15m and
16m) and methoxybenzyl(16r) at R-position reduced the activities,
whereas, relatively moderate size and flexibility substituent groups (15o
and 15p) had a positive effect on the potency, possibly because their
conformational effects enhanced the binding of compounds to the cor-
responding pockets of the active site. Methylation of the amino group
(Unit C) in amide (19) leaded to completely loss of the activity, possibly
that the hydrogen of amino group forms hydrogen bond with amino
acid, which enhanced the affinity with protein and enhances the lipid-
lowering activity (Fig. 4).
Scheme 3. Structural optimization of Unit C: Synthesis of compound 19. Re-
agents and conditions: (I) dimethylamine in MeOH, 48 h, 80 ◦C; (II) AcOH,
EDCI, DMAP, CH2Cl2, 0 ◦C, 1 h; (III) 2-bromoethanol, K2CO3, acetone, 48
h, 80 ◦C.
NH3 in MeOH to give the corresponding compounds 8c, 8d, (S)9 and (R)
10.15 Subsequently, under the condition of N-(3-dimethylaminopropyl)-
N’-ethyl-carbodiimide hydrochloride (EDCI) and 4-dimethylamino-pyr-
idine (DMAP), the compounds (S)9 and (R)10 were condensed with
corresponding carboxylic acid to obtained compounds 3, 8a, and 8b.16
According to the synthetic protocols in the Scheme 1 (i), compounds
4, 11a-11d and 12a-12c were synthesized in the presence of K2CO3 and
2-Bromoethanol.17 Compound 13 was prepared by hydrolysis reaction
of compound 4 in the presence of CH3ONa.18 Meanwhile, compound 4
was reduced by H2 in a methanol solution using palladium on carbon as
a catalyst to give compound 14 in Scheme 1 (ii).16
A series of compounds 15 and 16 were prepared from compound 3
which reacted with corresponding halogenated compounds in the
presence of potassium carbonate (Scheme 2).17
The compound 2 was treated with 2 M dimethylamine in MeOH to
give the compound 17, then 18 was obtained by acylation of compound
17. The compound 19 was prepared via similar procedures from com-
pounds 15 and 16, and the method was shown in Scheme 3.17,19
2.2. Structure-activity relationships of penipyridone derivatives
Taking compounds G and 4 as the lead compounds, we synthesized
31 penipyridone derivatives (Scheme 1, Scheme 2, Scheme 3). The lipid-
lowering activities of 31 compounds and Simvastatin were assessed on
oleic acid induced lipid accumulation in HepG2 cells in vitro at 10 μM.
The activity data were divided into three groups, according to the Unit
A, B and C of lead compounds (Fig.3).
The results demonstrated that compounds 12c, 14, 15f, 15k, 15o,
15p and 16f exhibited potent lipid-lowering activities at 10 μM, among
which, 15o displayed the greatest potency and decreased lipid concen-
tration. In addition, we also tested the cytotoxicity of the test
Fig. 4. Structure-activity relationship of penipyridone derivatives.
3

L. Li et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116192
A
B
C
Fig. 5. Effects of compounds 4, 15k and 15o on cytotoxicity of HepG2 cells determined by MTT assays. HepG2 cells were treated with different concentrations
compounds for 24 h. (A) Compound 4; (B) Compound 15k; (C) Compound 15o. Data were expressed as the mean ± SD (n = 6).
2.3. Biological evaluation
15o on TC at 5 μM (12.8%) was better than that of Simvastatin at 10 μM
(12.1%).
The cytotoxicity of compounds 4, 15k, 15o on HepG2 cells were
In order to observe the accumulation of lipid in HepG2 cells intui-
tively, we used oil red O staining to monitor the lipid-lowering effects.
Compared with the vehicle group, lipid accumulation was significantly
increased after OA treatment, while lipid content was significantly
decreased after drug treatment with different doses of compound 15o
(Fig. 6E). These results suggested that compound 15o could significantly
reduce intracellular lipid accumulation.
detected by MTT assays. The cell viability after treatment with the dose
of 400 μМ (4, 15k, 15o) was not significantly different from those in the
vehicle group, indicating that compounds 4, 15k, 15o had no significant
cytotoxicity up to 400 М(Fig. 5A-5C).
μ
To determine the lipid-lowering effects of compounds 4, 15k, 15o on
the lipid accumulation in insulin resistant cell model, we treated cells
with different concentrations of OA for 24 h to induce lipid accumula-
tion. The results showed that the lipid contents of HepG2 cells increased
significantly after treatment with 100 µM OA for 24 h (Fig. 6A).
Therefore, in the following experiments, we selected 100 µM OA to
induce hyperlipidemia model. Then the contents of triglyceride were
2.4. Molecular docking
The mechanism of lipid metabolism disorder caused by obesity is
complicated. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), an
intracellular glucocorticoid reactivating enzyme, has attracted wide
attention. It is a key enzyme to regulate glucocorticoids, which can
catalyze the conversion of inactive component cortisone into bioactive
glucocorticoids. Compared with subcutaneous adipose tissue, visceral
adipose tissue has higher glucocorticoid receptor, and glucocorticoids
play an important role in visceral adipose tissue differentiation. A large
number of experimental results showed that the activity and expression
of 11β-HSD1 was high in patients with hyperlipidemia and dyslipide-
mia.20 Veilleux A found that the mRNA level of 11β-HSD1 was different
in visceral and peripheral fat of obese patients, and the mRNA content of
11β-HSD1 was positively correlated with visceral fat. The differentiation
and maturation of adipocytes were related to the increased 11β-HSD1
mRNA level and oxidoreductase activity.21 Therefore, 11β-HSD1 can be
measured in HepG2 cells that were treated with 100 μM OA for 24 h with
or without compounds 4, 15k, 15o at 10 μM. The results suggested that
treated with compounds 4, 15k, 15o could significantly reverse the
triglyceride accumulation induced by OA in insulin resistant HepG2 cells
at 10 μM, while 15o had the best lipid-lowering effect among them
(Fig. 6B). After that, HepG2 cells induced by OA were treated with
different doses of compound 15o, and it was found that 15o had the best
triglyceride-lowering and cholesterol-lowering effect at 20 μM, proving
that the activity of 15o was dose-dependent (Fig. 6C and D). Compared
with the vehicle group, the triglyceride inhibition rate of 15o was about
30.2%, and the total cholesterol inhibition rate was about 14.8% at 20
μ
M, with the similar efficacy with the positive drug Simvastatin at 10 μM
(TG: 29.1%; TC: 12.1%). It should be noted that the inhibitory effect of
Fig. 6. (A) HepG2 cells treated with
different concentrations of OA promoted
intracellular lipid accumulation (100, 200
and 400 μM). (B) TG content in HepG2 cells.
HepG2 cells were treated with 100
for 24 h with or without compounds 4, 15k,
15o (10 M). (C-D) TG and TC content in
HepG2 cells. HepG2 cells were treated with
100 M OA for 24 h with different doses of
μM OA
μ
μ
15o. (E) Lipid accumulation in HepG2 cells
was observed by Oil Red O staining (40 X).
Data were expressed as the mean ± SD (n =
4). Significant difference compared with OA-
treated cells, **P < 0.01, ***P＜0.001, ****P
＜ 0.0001.
4

L. Li et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116192
Fig. 7. The predicted models of compounds 4, 15k, 15o with 11β-HSD1(PDB code: 3TFQ). The main protein residues are depicted in green sticks, while the ligands
are depicted as: (A) Compound 4 (cyan sticks); (B) Compound 15k (orange sticks); (C) Compound 15o (magenta sticks), respectively. (D) and (E) are the repre-
sentations of compounds 4, 15k, 15o docked into the binding site of 11β-HSD1. All pictures were prepared with PyMOL. For clarity, the NADP+ has been omitted
from this representation, and only a few of the surrounding residues are included in the figures.
a novel target for the treatment of central obesity.22
interactions (Fig. 7D and 7E). However, the binding features R group of
compounds 4, 15k and 15o were apparently different. These results
suggested that moderate size, flexibility and hydrophilicity (thio-
morpholine ring) R substituted groups of Unit B might be suitable to the
cavity, which was formed by Asn123, Thr124, Ser125, Tyr183, Tyr221
and other adjacent residues (Fig. 7E).
Further in, we performed a reverse docking to search the potential
lipid-metabolism related target by a designed computational approach
in the Supercomputing Center of Pilot National Laboratory for Marine
Science and Technology. By the analysis of the results, we found that
11β-hydroxysteroid dehydrogenase type 1 is the prime candidate target.
Now in order to visualize binding modes and to obtain an insight into
the relationships between penipyridone derivatives and their potencies,
molecular docking study was carried out with the highly potent com-
pounds 4, 15k, 15o within the active site of the crystal structure of 11β-
HSD1 complexed with a ligand, 3-[1-(4fluorophenyl)cyclopropyl]-4-(1-
methylethyl)-5-[4-(trifluorom-ethoxy)phenyl]-4H-1,2,4 triazole (PDB
code: 3TFQ), using Molecular Operating Environment (MOE®) 2016. 08
software. The favored poses from the docking calculations were shown
in Fig. 7, along with the residues in the binding pocket that contributed
most significantly to the binding energy.
3. Conclusion
In summary, a series of novel penipyridone derivatives were
designed and synthesized. The lipid-lowering activities were evaluated
by the adipose accumulation in the insulin resistant HepG2 cell models,
and compound 4, 15k, 15o displayed significant lipid-lowering activity
without obvious cytotoxicity. In particular, compound 15o showed the
similar effect with Simvastatin on decreasing the TG concentration in
HepG2 cells, and the inhibitory effect of 15o on TC at 5
μ
M was better
As seen in Fig. 7, the molecular docking results indicated that the
phenyl in Unit A was an excellent and privileged moiety for the cavity,
which was a hydrophobic cavity formed by Leu126, Val180, Val227,
Val231 and their adjacent lipophilic amino acids. In addition, hydrogen
of Ser170 formed interaction with the N atom of pyridine ring, which
could further stabilize the ligand–protein (Fig. 7A-7C). The carbonyl of
carbamoyl (C-13) and acetyl (C-16) formed hydrogen bonds with
Tyr183 and Leu217, respectively (Fig. 7B and 7C), which was non-
existent in compound 4. Moreover, both hydrogens of NH2 in Unit C
than that of Simvastatin at 10
μ
M. As the same time, molecular docking
analysis predicted that compound 15o showed higher binding affinity
with 11β-HSD1 than compounds 4 and 15k. Taken together, compound
15o as a potential 11β-HSD1 inhibitor could be considered as a prom-
ising lead compound for the treatment of hyperlipidemia.
4. Experimental section
4.1. Chemistry
–
formed hydrogen bonds with Leu215 (compound 4, N H⋅⋅⋅O 2.86 Å;
–
–
compound 15k, N H⋅⋅⋅O 2.04 Å) and Gly216 (compound 15o, N H⋅⋅⋅O
1.91 Å), and the lengths of hydrogen bonds in compounds 4 and 15k
were longer than that of 15o.
General information. Commercial reagents and solvents were pur-
chased from Sigma Aldrich, Fluka, and Alfa Aesar used as received,
without further purification. The 1H and 13C NMR spectra were recorded
at 500 MHz for 1H and at 125 MHz for 13C. The chemical shifts (δ) for 1H
and 13C are given in ppm relative to residual signals of the solvents
(CDCl3 at 7.26 ppm 1H NMR, 77.16 ppm 13C NMR. DMSO‑d6 at 2.50
The superimposed structures of compounds 4, 15k and 15o docked
into the11β-HSD1 binding site indicated that the three compounds were
well incorporated in the binding pocket and were able to form similar
5

L. Li et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116192
ppm 1H NMR, 39.52 ppm 13C NMR). Coupling constants are given in Hz.
The following abbreviations are used to indicate the multiplicity: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet. High-resolution
mass spectra (HRMS) were obtained from the Waters Q-Tof Ultima
109.32, 77.64, 52.82, 21.30. HRMS: [M+H]+ calcd. For C16H16NO5 m/z:
302.1023; found: 302.1020. [α] + 47 (c = 0.1 in CHCl3).
4.1.1.3. Methyl 4-oxo-6-phenyl-1,4-dihydropyridine-3-carboxylate (7c).
Compound 7c was prepared from 6c. Yellow solid, yield 67%. 1H NMR
(400 MHz, Acetone‑d6) δ 11.05 (s, 1H), 8.97 (s, 1H), 8.17 (d, J = 5.2 Hz,
3H), 7.50 (ddd, J = 6.8, 4.6, 1.8 Hz, 5H), 4.03 (s, 3H). 13C NMR (101
MHz, Acetone‑d6) δ 170.50, 168.39, 152.67, 138.86, 130.97, 129.60,
128.10, 109.83, 108.85, 53.14. HRMS: [M+H]+ calcd. For C13H12NO3
m/z: 230.0822; found: 230.0820.
Global. Optical rotations are reported as follows: [α] (c in g per 100 mL,
solvent: CHCl3, MeOH). All the reactions were set up under air and using
freshly distilled solvents, without any precautions to exclude moisture,
unless otherwise noted open air chemistry on the bench-top. Chro-
matographic purification of products was accomplished using force-flow
chromatography (FC) on silica gel (300–400 mesh). For thin layer
chromatography (TLC) analysis throughout this work, Merck pre-coated
TLC plates (silica gel 60 GF254, 0.25 mm) were used, using UV light as
the visualizing agent and phosphomolybdic acid or basic aqueous po-
tassium permanganate (KMnO4) as stain developing solutions. Organic
solutions were concentrated under reduced pressure on a Büchi rotary
evaporator.
4.1.1.4. Methyl
6-(tert-butyl)-4-oxo-1,4-dihydropyridine-3-carboxylate
(7d). Compound 7d was prepared from 6d. Yellow solid, yield 33%. 1H
NMR (400 MHz, Acetone‑d6) δ 10.91 (s, 1H), 8.84 (s, 1H), 6.94 (s, 1H),
4.00 (s, 3H), 1.33 (s, 9H). 13C NMR (101 MHz, Acetone‑d6) δ 176.86,
170.60, 168.08, 151.72, 108.63, 108.09, 52.99, 38.49, 29.93. HRMS:
[M+H]+ calcd. For C11H16NO3 m/z: 210.1125; found: 210.1129.
Note: NMR signals containing common solvent contaminants were
list⋅H2O in CDCl3 at 1.56 ppm 1H NMR, and in DMSO‑d6 at 3.33 ppm 1H
NMR; Ethyl acetate in CDCl3 at 2.05 (s), 4.12 (q), 1.26 (t) ppm 1H NMR;
Dichloromethane in CDCl3 at 5.30 (s) ppm 1H NMR.
4.1.2. General synthetic procedure of compounds 3, (S)9, (R)10 and
8a–8d.
To compound 2 and 7b-7d (0.33 mmol, 1.0 eq.) in a 50 mL dry seal
tube, 10 mL saturated ammonia methanol solution was added. The re-
action mixture was then heated to 80 ◦C and stirred for 48 h (monitored
by TLC). Upon completion, the reaction mixture was concentrated and
purified by column chromatography on silica gel (CH2Cl2/MeOH) to
obtain compounds 8c, 8d, (S)9 and (R)10.
4.1.1. General synthetic procedure of compounds 2 and 7b-7d.
Methyl acetoacetate (17.22 mmol, 1.0 eq.) and N, N-dime-
thylformamide dimethyl acetal (20.10 mmol, 1.2 eq.) were added to a
25 mL dry round bottom flask, then 500 μM DMF was dropped into the
flask at room temperature with stirring. After 20 h (monitored by TLC),
the mixture was concentrated and purified by column chromatography
on silica gel (ethyl acetate/petroleum ether) to obtain compound 5.
Corresponding acids (2.57 mmol, 1.0 eq.) was added 10 mL anhy-
drous dichloromethane in a 25 mL dry round bottom flask under ni-
trogen protection. When the solid was dissolved, oxalyl chloride (328
To a solution of compound (S)9 or (R)10 (0.41 mmol, 1.0 eq.), EDCI
(0.62 mmol, 1.5 eq.), DMAP (0.21 mmol, 0.50 eq.) and TEA (0.82 mmol,
2.0 eq.) in 10 mL anhydrous dichloromethane protected with nitrogen
and corresponding acid (0.82 mmol, 2.0 eq.) was slowly added under ice
bath, and stirred for 1 h (monitored by TLC). 1 N dilute hydrochloric
acid solution was added to the reaction mixture to quenching reaction.
The solution was extracted with CH2Cl2 (3 × 10 mL). The combined
organic phase was washed with brine, dried (Na2SO4), and concen-
trated. A crude solid that was purified by silica gel column chromatog-
raphy (CH2Cl2/MeOH) to obtain compounds 3, 8a and 8b.
μ
L, 3.86 mmol, 1.5 eq.) and 500 μM DMF were dropwise into the stirred
solution at 0 ◦C. Then the reaction was moved to at room temperature
for reaction. The reaction progress was monitored by TLC. Upon
completion, the reaction was concentrated under reduced pressure to
obtain product acyl chlorides 6b and 6e.
A solution of LiHMDS (1.0 M in THF, 5.20 mmol, 2.0 eq.) was cooled
to ꢀ 78 ◦C in 50 mL flask under nitrogen protection, and then freshly THF
solutions of prepared chlorides 6b-6e (2.60 mmol, 1.0 eq.) and 5 (3.12
mmol, 1.2 eq.) were added simultaneously slowly at ꢀ 78 ◦C. After
stirring for an additional 30 min, the mixture was moved at room tem-
perature. Glacial acetic acid (7.80 mmol, 3.0 eq.) and ammonium ace-
tate (5.20 mmol, 2.0 eq.) were added to the reaction mixture, which was
then heated to reflux and stirred for 30 min (monitored by TLC). The
mixture was poured into water and extracted with EtOAc (3 × 20 mL).
The combined organic phase was washed with brine, dried (Na2SO4),
and concentrated. The residue was purified by column chromate-graphy
on silica gel (ethyl acetate/petroleum ether) to obtain compounds 2, 7b-
7d.
4.1.2.1. (S)-(5-carbamoyl-4-oxo-1,4-dihydropyridin-2-yl) (phenyl)methyl
acetate (3). Compound 3 was prepared from (S)9. Colorless oil, yield
76%. 1H NMR (500 MHz, DMSO‑d6) δ 12.43 (s, 1H), 9.40 (s, 1H), 8.37 (s,
1H), 7.41 (ddd, J = 20.4, 16.5, 7.0 Hz, 6H), 6.64 (s, 1H), 6.39 (s, 1H),
2.18 (s, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 177.74, 169.76, 165.51,
149.58, 142.30, 137.13, 129.28, 129.21, 127.46, 118.58, 117.12, 72.85,
21.16. HRMS: [M+H]+ calcd. For C15H15N2O4 m/z: 287.1026; found:
287.1023. [α] + 18 (c = 2 in CHCl3).
4.1.2.2. (S)-6-(hydroxy(phenyl)methyl)-4-oxo-1,4dihydropyridine-3-car-
boxamide((S)9). Compound (S)9 was prepared from 2. White solid,
yield 75%. 1H NMR (500 MHz, CDCl3) δ 11.04 (s, 1H), 8.90 (s, 1H),
7.39–7.31 (m, 4H), 7.29 (d, J = 7.0 Hz, 1H), 6.80 (s, 1H), 5.69 (s, 1H),
3.98 (s, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 176.64, 167.17, 157.54,
146.32, 142.58, 129.54, 129.02, 127.31, 116.74, 116.56, 72.90, 52.64.
HRMS: [M+H]+ calcd. For C14H14NO4 m/z: 260.0917; found: 260.0913.
4.1.1.1. Methyl
(S)-6-(acetoxy(phenyl)methyl)-4-oxo-1,4-dihydropyr-
idine-3-carboxylate (2). Compound 2 was prepared from 6e. Colorless
oil, yield: 23%. 1H NMR (500 MHz, CDCl3) δ 11.13 (s, 1H), 8.89 (s, 1H),
7.42 (d, J = 7.3 Hz, 2H), 7.37–7.29 (m, 3H), 7.06 (s, 1H), 6.78 (s, 1H),
3.97 (s, 3H), 2.21 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 169.88, 169.80,
167.96, 165.61, 152.11, 138.27, 128.81, 128.64, 127.68, 109.34,
109.32, 77.64, 52.82, 21.31. HRMS: [M+H]+ calcd. For C16H16NO5 m/z:
[α] ꢀ 27 (c = 0.1 in CHCl3).
4.1.2.3. (R)-6-(hydroxy(phenyl)methyl)-4-oxo-1,4dihydropyridine-3-car-
boxamide((R)10). Compound (R)10 was prepared from 7b. Brown
solid, yield 60%. 1H NMR (500 MHz, DMSO‑d6) δ 11.91 (s, 1H), 9.49 (s,
1H), 8.30 (s, 1H), 7.44 (d, J = 7.6 Hz, 2H), 7.38 (dd, J = 14.1, 6.6 Hz,
302.1023; found: 302.1020. [α] + 55 (c = 0.1 in CHCl3).
3H), 7.30 (t, J = 7.2 Hz, 1H), 6.60 (s, 1H), 6.35 (s, 1H), 5.66 (s, 1H). 13
C
4.1.1.2. Methyl (R)-6-(acetoxy(phenyl)methyl)-4-oxo-1,4-dihydropyr-
idine-3-carboxylate (7b). Compound 7b was prepared from 6b. Colorless
oil, yield: 30%. 1H NMR (500 MHz, CDCl3) δ 11.13 (s, 1H), 8.89 (s, 1H),
7.42 (d, J = 7.3 Hz, 2H), 7.37–7.29 (m, 3H), 7.06 (s, 1H), 6.78 (s, 1H),
3.97 (s, 3H), 2.20 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 169.88, 169.80,
167.96, 165.61, 152.11, 138.27, 128.81, 128.64, 127.68, 109.34,
NMR (125 MHz, DMSO‑d6) δ 177.43, 165.36, 153.37, 141.81, 141.48,
128.43, 127.88, 126.38, 117.79, 115.89, 70.76. HRMS: [M+H]+ calcd.
For C13H13N2O3 m/z: 245.0921; found: 245.0924. [α] ꢀ 27 (c = 0.1 in
CHCl3).
6

L. Li et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116192
4.1.2.4. (S)-(5-carbamoyl-4-oxo-1,4-dihydropyridin-2-yl)(phenyl)methyl
3-methylbutanoate (8a). Compound 8a was prepared from (S)9 and 3-
methylbutanoic acid. Colorless oil, yield 72%. 1H NMR (500 MHz,
CDCl3) δ 11.76 (s, 1H), 10.05 (d, J = 4.8 Hz, 1H), 8.52 (s, 1H), 7.40–7.33
(m, 5H), 6.79 (s, 1H), 6.47 (s, 1H), 5.90 (d, J = 4.9 Hz, 1H), 2.31 (d, J =
7.2 Hz, 2H), 2.15–2.07 (m, 1H), 0.91 (dd, J = 6.6, 3.7 Hz, 6H). 13C NMR
(125 MHz, CDCl3) δ 178.58, 172.29, 167.07, 149.00, 142.69, 135.61,
129.61, 129.26, 127.43, 119.13, 117.91, 72.77, 43.30, 25.87, 22.49,
22.46. HRMS: [M+H]+ calcd. For C18H21N2O4 m/z: 329.1496; found:
chromatography on silica gel (ethyl acetate/petroleum ether) to obtain
compounds 4, 11a-11d, 12a-12c.
4.1.4.1. (S)-(5-carbamoyl-4-(2-hydroxyethoxy) pyridin-2-yl) (phenyl)
methyl acetate (4). Compound 4 was prepared from 3. Colorless oil,
yield 53%. 1H NMR (500 MHz, DMSO‑d6) δ 8.71 (s, 1H), 7.72 (s, 1H),
7.60 (s, 1H), 7.46 (d, J = 7.3 Hz, 2H), 7.32 (tt, J = 14.6, 7.2 Hz, 4H), 6.66
(s, 1H), 5.16 (t, J = 5.3 Hz, 1H), 4.28 (t, J = 4.3 Hz, 2H), 3.77 (dd, J =
9.2, 4.9 Hz, 2H), 2.18 (s, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 170.06,
165.09, 163.67, 163.61, 152.03, 139.01, 128.88, 128.58, 127.75,
118.27, 105.58, 77.63, 71.40, 59.41, 21.33. HRMS: [M+H]+ calcd. For
329.1491. [α] ꢀ 43 (c = 0.1 in CHCl3).
4.1.2.5. (R)-(5-carbamoyl-4-oxo-1,4-dihydropyridin-2-yl)(phenyl)methyl
acetate (8b). Compound 8b was prepared from (S)10 and glacial acetic
acid. Colorless oil, yield 86%. 1H NMR (500 MHz, CDCl3) δ 11.86 (s, 1H),
10.03 (d, J = 4.5 Hz, 1H), 8.52 (s, 1H), 7.36 (s, 5H), 6.75 (s, 1H), 6.46 (s,
1H), 5.99 (d, J = 4.5 Hz, 1H), 2.16 (s, 3H). 13C NMR (125 MHz, CDCl3) δ
178.56, 170.10, 167.15, 149.07, 142.76, 135.52, 129.66, 129.27,
127.50, 118.82, 117.85, 73.06, 21.09. HRMS: [M+H]+ calcd. For
C17H19N2O5 m/z: 331.1288; found: 331.1285. [α] + 43 (c = 1 in CHCl3).
4.1.4.2. (S)-(5-carbamoyl-4-(2-hydroxyethoxy) pyridin-2-yl) (phenyl)
methyl 3-methylbutanoate1(11a). Compound 11a was prepared from 8a.
White solid, yield 51%. H NMR (500 MHz, DMSO‑d6) δ 8.72 (s, 1H),
7.75–7.71 (m, 1H), 7.59 (s, 1H), 7.47 (d, J = 7.4 Hz, 2H), 7.32 (dq, J =
14.4, 7.2 Hz, 4H), 6.70 (s, 1H), 5.09 (t, J = 4.8 Hz, 1H), 4.27–4.20 (m,
2H), 3.79 (d, J = 4.4 Hz, 2H), 2.37 (dd, J = 7.1, 3.3 Hz, 2H), 2.06 (tt, J =
13.5, 6.8 Hz, 1H), 0.91 (d, J = 6.7 Hz, 7H). 13C NMR (125 MHz,
DMSO‑d6) δ 171.35, 164.64, 163.25, 163.19, 151.62, 138.62, 128.42,
128.12, 127.27, 117.84, 105.08, 77.01, 70.92, 59.00, 42.56, 25.27,
22.09. HRMS: [M+H]+ calcd. For C20H25N2O5 m/z: 373.1758; found:
C
15H15N2O4 m/z: 287.1026; found: 287.1029. [
α] ꢀ 30 (c = 0.1 in
CHCl3).
4.1.2.6. 4-oxo-6-phenyl-1,4-dihydropyridine-3-carboxamide (8c). Com-
pound 8c was prepared from 7c. White solid, yield 60%. 1H NMR (500
MHz, DMSO‑d6) δ 12.42 (s, 1H), 9.55 (s, 1H), 8.42 (s, 1H), 7.78 (s, 2H),
7.55 (s, 5H), 6.76 (s, 1H). 13C NMR (125 MHz, DMSO‑d6) δ 177.51,
165.43, 148.23, 142.06, 132.46, 130.59, 129.13, 126.83, 117.76,
116.62. HRMS: [M+H]+ calcd. For C12H11N2O2 m/z: 215.0815; found:
215.0817.
373.1754. [α] ꢀ 8 (c = 0.1 in CHCl3).
4.1.4.3. (R)-(5-carbamoyl-4-(2-hydroxyethoxy) pyridin-2-yl) (phenyl)
methyl acetate (11b). Compound 11b was prepared from 8b. White
solid, yield 50%. 1H NMR (500 MHz, DMSO‑d6) δ 8.72 (s, 1H), 7.74–7.71
(m, 1H), 7.59 (s, 1H), 7.46 (d, J = 7.4 Hz, 2H), 7.37–7.28 (m, 4H), 6.67
(s, 1H), 5.09 (t, J = 5.3 Hz, 1H), 4.28 (t, J = 4.6 Hz, 2H), 3.79 (dd, J =
9.3, 5.1 Hz, 2H), 2.18 (s, 3H). 13C NMR (125 MHz, DMSO‑d6)δ 169.60,
164.66, 163.24, 163.18, 151.62, 138.57, 128.43, 128.13, 127.30,
117.83, 105.13, 77.19, 70.96, 59.02, 20.87. HRMS: [M+H]+ calcd. For
4.1.2.7. 6-(tert-butyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (8d).
Compound 8d was prepared from 7d: white solid, 76%. 1H NMR (500
MHz, DMSO‑d6) δ 11.73 (s, 1H), 9.52 (s, 1H), 8.27 (s, 1H), 7.39 (s, 1H),
6.26 (s, 1H), 1.26 (s, 9H). 13C NMR (125 MHz, DMSO‑d6) δ 177.80,
165.36, 158.40, 141.55, 116.80, 114.58, 34.44, 28.32. HRMS: [M+H]
+ calcd. For C10H15N2O2 m/z: 195.1128; found: 195.1123.
C
17H19N2O5 m/z: 331.1288; found: 331.1287. [
α] ꢀ 37 (c = 0.1 in
CHCl3).
4.1.3. Synthetic procedure of compound 1
4.1.4.4. 4-(2-hydroxyethoxy)-6-phenylnicotinamide (11c). Compound
11c was prepared from 8c. White solid, yield 16%. 1H NMR (500 MHz,
DMSO‑d6) δ 8.92 (s, 1H), 8.17 (d, J = 6.9 Hz, 2H), 7.76 (s, 1H), 7.67 (d, J
= 3.3 Hz, 1H), 7.66 (d, J = 3.2 Hz, 1H), 7.49 (dq, J = 14.0, 6.8 Hz, 3H),
5.14 (t, J = 5.3 Hz, 1H), 4.44–4.36 (m, 2H), 3.83 (dd, J = 9.4, 4.9 Hz,
2H). 13C NMR (125 MHz, DMSO‑d6) δ 164.80, 163.50, 160.09, 152.19,
131.49, 129.72, 128.65, 127.12, 116.85, 104.79, 70.98, 59.11. HRMS:
[M+H]+ calcd. For C14H15N2O3 m/z: 259.1077; found: 259.1076.
To a solution of compound 3 (0.10 mmol, 1.0 eq.), DMAP (0.05
mmol, 0.50 eq.) and TEA (0.20 mmol, 2.0 eq.) in10 mL anhydrous THF
protected with nitrogen, isovaleryl chloride (0.30 mmol, 3.0 eq.) was
slowly added at room temperature for 6 h (monitored by TLC). The
solution was extracted with ethyl acetate (3 × 10 mL). The combined
organic phase was washed with brine, dried (Na2SO4), and concen-
trated. A crude solid that was purified by silica gel column chromatog-
raphy using CH2Cl2 and MeOH as the eluent to obtain colorless oil
compound 1.
4.1.4.5. 6-(tert-butyl)-4-(2-hydroxyethoxy) nicotinamide (11d). Com-
pound 11d was prepared from 8d. White solid, yield 55%. 1H NMR (500
MHz, DMSO‑d6) δ 8.77 (s, 1H), 7.66 (s, 1H), 7.58 (s, 1H), 7.09 (s, 1H),
5.10 (t, J = 5.2 Hz, 1H), 4.29–4.24 (m, 2H), 3.78 (dd, J = 8.8, 4.5 Hz,
2H), 1.31 (s, 9H). 13C NMR (125 MHz, DMSO‑d6) δ 173.36, 165.01,
162.96, 151.08, 115.80, 103.57, 70.60, 59.09, 37.71, 29.81. HRMS:
[M+H]+ calcd. For C12H19N2O3 m/z: 239.1390; found: 239.1397.
4.1.3.1. (S)-(5-((3-methylbutanoyl) carbamoyl)-4-oxo-1,4-dihydropyridin-
2-yl) (phenyl)methyl acetate (1). Colorless oil, yield 66%. 1H NMR (500
MHz, DMSO‑d6) δ 13.09 (s, 1H), 8.48 (s, 1H), 7.45 (d, J = 7.3 Hz, 2H),
7.43–7.34 (m, 3H), 6.63 (s, 1H), 6.48 (s, 1H), 2.60 (dd, J = 6.9, 2.4 Hz,
2H), 2.42 (td, J = 8.2, 3.0 Hz, 1H), 2.18 (d, J = 2.4 Hz, 3H), 0.91 (dd, J =
6.7, 2.5 Hz, 7H). 13C NMR (125 MHz, DMSO‑d6) δ 177.59, 174.04,
169.76, 163.57, 152.31, 145.67, 137.41, 129.19, 129.17, 127.55, 116.99,
116.31, 73.61, 47.04, 24.93, 22.75, 21.18. HRMS: [M+H]+ calcd. For
4.1.4.6. (S)-(5-carbamoyl-1-(2-hydroxyethyl)-4-oxo-1,4-dihydropyridin-
2-yl) (phenyl)methyl3-methyl Butanoate(12a). Compound 12a was pre-
1
C20H23N2O5 m/z: 371.1601; found: 371.1606. [α] + 40 (c = 0.5 in
pared from 8a. Colorless oil, yield 31%. H NMR (500 MHz, CDCl3) δ
CHCl3).
9.87 (d, J = 4.7 Hz, 1H), 8.46 (s, 1H), 7.42–7.37 (m, 3H), 7.31 (dt, J =
5.9, 3.0 Hz, 2H), 6.93 (s, 1H), 6.78 (s, 1H), 5.93 (d, J = 4.3 Hz, 1H),
4.06–3.98 (m, 2H), 3.92–3.84 (m, 2H), 2.30 (d, J = 7.2 Hz, 2H), 2.10 (dt,
J = 13.6, 6.8 Hz, 1H), 0.95–0.91 (m, 6H). 13C NMR (125 MHz, CDCl3) δ
177.44, 171.98, 167.85, 149.66, 147.92, 135.08, 129.50, 128.97,
128.15, 120.24, 118.44, 71.51, 61.26, 55.77, 43.26, 25.81, 22.49,
22.44. HRMS: [M+H]+ calcd. For C20H25N2O5 m/z: 373.1758; found:
4.1.4. General synthetic procedure of compounds 4, 11 and 12
The reaction of 3 and 8a–8d (0.12 mmol, 1.0 eq.) and K2CO3 (0.24
mmol, 2.0 eq.) and bromoethanol (0.24 mmol, 2.0 eq.) in 5 mL anhy-
drous acetone was heated 60–80 ◦C and stirred for 12–48 h (monitored
by TLC). The reaction mixture was extracted with ethyl acetate (3 × 5
mL). The combined organic phase was washed with brine, dried by
Na2SO4, and concentrated. The residue was purified by column
373.1757. [α] + 7 (c = 0.1 in CHCl3).
7

L. Li et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116192
4.1.4.7. (R)-(5-carbamoyl-1-(2-hydroxyethyl)-4-oxo-1,4-dihydropyridin-
2-yl) (phenyl)methyl acetate(12b). Compound 12b was prepared from
8b. Colorless oil, yield 20%. 1H NMR (500 MHz, DMSO‑d6) δ 9.40 (d, J
= 4.5 Hz, 1H), 8.47 (s, 1H), 7.49 (d, J = 4.5 Hz, 1H), 7.45–7.39 (m, 5H),
6.95 (s, 1H), 6.44 (s, 1H), 5.15 (t, J = 5.1 Hz, 1H), 4.08–4.01 (m, 2H),
3.61 (dt, J = 16.3, 5.5 Hz, 1H), 3.54–3.47 (m, 1H), 2.18 (s, 3H). 13C NMR
(125 MHz, DMSO‑d6) δ 176.35, 169.27, 164.87, 149.55, 147.95, 135.68,
129.00, 128.64, 127.68, 119.18, 118.14, 71.04, 59.95, 54.92, 20.65.
HRMS: [M+H]+ calcd. For C17H19N2O5 m/z: 331.1288; found:
4.1.6.1. (S)-(5-carbamoyl-4-(2-(dimethylamino) ethoxy) pyridin-2-yl)
(phenyl)methyl acetate(15f). Compound 15f was prepared from com-
pound 3 and 2-chloro-N,N-dimethylethan-1-amine. Colorless oil, yield
29%. 1H NMR (500 MHz, DMSO‑d6) δ 8.53 (s, 1H), 7.63 (s, 1H), 7.48 (s,
1H), 7.30 (t, J = 8.2 Hz, 2H), 7.23–7.09 (m, 4H), 6.51 (d, J = 6.7 Hz,
1H), 4.16 (d, J = 3.1 Hz, 2H), 3.10 (s, 2H), 2.34 (s, 3H), 2.03 (d, J = 13.6
Hz, 8H). 13C NMR (125 MHz, DMSO‑d6) δ 169.59, 164.69, 163.03,
151.43, 138.59, 128.43, 128.14, 127.32, 118.04, 105.23, 77.17, 66.60,
57.07, 45.18, 20.89. HRMS: [M+H]+ calcd. For C19H24N3O4 m/z:
331.1286. [
α] ꢀ 68 (c = 0.1 in CHCl3).
358.1761; found: 358.1766. [
α
] + 15 (c = 1.2 in CHCl3).
4.1.4.8. 1-(2-hydroxyethyl)-4-oxo-6-phenyl-1,4-dihydropyridine-3-car-
boxamide(12c). Compound 12c was prepared from 8c. Colorless oil,
yield 33%. 1H NMR (500 MHz, DMSO‑d6) δ 9.54 (d, J = 4.4 Hz, 1H), 8.57
(s, 1H), 7.54–7.51 (m, 3H), 7.51–7.47 (m, 3H), 6.26 (s, 1H), 4.98 (t, J =
5.2 Hz, 1H), 3.94 (t, J = 5.2 Hz, 2H), 3.40 (dd, J = 10.2, 5.1 Hz, 2H). 13C
NMR (125 MHz, DMSO‑d6) δ 176.21, 165.18, 151.83, 146.89, 131.67,
131.50, 129.65, 128.85, 128.71, 128.64, 121.36, 118.12, 59.58, 55.55.
HRMS: [M+H]+ calcd. For C14H15N2O3 m/z: 259.1077; found:
259.1078.
4.1.6.2. (S)-(5-carbamoyl-1-(2-(dimethylamino)
ethyl)-4-oxo-1,4-dihy
dropyridin-2-yl) (phenyl)methyl acetate(16f). Compound 16f was pre-
pared from compound 3 and 2-chloro-N,N-dimethylethan-1-amine.
Colorless oil, yield 62%. 1H NMR (500 MHz, DMSO‑d6) δ 9.40 (s, 1H),
8.42 (s, 1H), 7.50 (s, 1H), 7.43 (d, J = 4.9 Hz, 6H), 6.87 (s, 1H), 6.49 (s,
1H), 4.16–4.06 (m, 1H), 3.98 (d, J = 14.2 Hz, 1H), 2.47–2.39 (m, 1H),
2.37–2.29 (m, 1H), 2.19 (s, 3H), 2.10 (s, 6H). 13C NMR (125 MHz,
DMSO‑d6) δ 176.27, 169.44, 164.85, 149.29, 147.84, 135.44, 129.29,
129.08, 127.82, 118.98, 118.10, 71.15, 58.39, 50.56, 45.20, 20.65.
HRMS: [M+H]+ calcd. For C19H24N3O4 m/z: 358.1761; found:
4.1.5. General synthetic procedure of compounds 13 and 14
The reaction of compound 4 (0.10 mmol, 1.0 eq.) and MeONa (0.15
mmol, 1.5 eq.) in 10 mL MeOH, was stirred reaction at room tempera-
ture for 4 h, monitoring by TLC. The reaction mixture was extracted with
ethyl acetate (3 × 10 mL). The combined organic phase was washed with
brine, dried (Na2SO4), and concentrated. The residue was purified by
column chromatography on silica gel (CH2Cl2/MeOH) to obtain com-
pound 13.
358.1768. [α] ꢀ 26 (c = 0.1 in CHCl3).
4.1.6.3. (S)-(5-carbamoyl-4-(2-(pyrrolidin-1-yl) ethoxy) pyridin-2-yl)
(phenyl)methyl acetate(15g). Compound 15g was prepared from com-
pound 3 and 1-(2-chloroethyl) pyrrolidine. White solid, yield 22%. 1H
NMR (500 MHz, DMSO‑d6) δ 8.34 (s, 1H), 7.57 (s, 1H), 7.31 (s, 1H), 7.12
(d, J = 7.4 Hz, 2H), 7.04–6.93 (m, 4H), 6.33 (d, J = 7.5 Hz, 1H), 4.00 (s,
2H), 2.16 (d, J = 1.7 Hz, 6H), 1.84 (s, 3H), 1.53 (s, 1H), 1.33 (s, 3H). 13C
NMR (125 MHz, DMSO‑d6) δ 169.60, 164.74, 163.06, 151.44, 138.60,
128.44, 128.14, 127.32, 118.23, 105.38, 77.17, 67.63, 53.48, 53.40,
23.10, 20.90. HRMS: [M+H]+ calcd. For C21H26N3O4 m/z: 384.1918;
The reaction of compound 4 (0.1 mmol, 1.0 eq.) and palladium
carbon (6.60 mg, 20%) in 6 mL MeOH using H2 gas as the hydrogen
source, was put at room temperature and stirred for 2 h (monitored by
TLC). The reaction solution was filtered with diatomite and concen-
trated. The residue was purified by column chromatography on silica gel
(CH2Cl2/MeOH) to obtain compounds 14.
found: 384.1911. [α] + 10 (c = 1.4 in CHCl3).
4.1.6.4. (S)-(5-carbamoyl-4-oxo-1-(2-(pyrrolidin-1-yl)
ethyl)-1,4-dihy
dropyridin-2-yl) (phenyl)methyl acetate(16g). Compound 16g was pre-
pared from compound 3 and 1-(2-chloroethyl) pyrrolidine. Colorless oil,
yield 72%. 1H NMR (500 MHz, DMSO‑d6) δ 9.39 (d, J = 3.6 Hz, 1H), 8.44
(s, 1H), 7.49 (d, J = 3.9 Hz, 1H), 7.43 (d, J = 8.2 Hz, 5H), 6.89 (s, 1H),
6.47 (s, 1H), 4.10 (dd, J = 13.9, 7.0 Hz, 1H), 4.05–3.95 (m, 1H), 2.63
(dt, J = 12.7, 6.2 Hz, 1H), 2.54 (d, J = 6.2 Hz, 1H), 2.40 (d, J = 17.0 Hz,
4H), 2.19 (s, 3H), 1.63 (s, 4H). 13C NMR (125 MHz, DMSO‑d6) δ 176.26,
169.40, 164.80, 149.29, 147.75, 135.51, 129.26, 129.06, 127.76,
119.02, 118.16, 71.15, 54.84, 53.58, 51.59, 23.20, 20.65. HRMS:
4.1.5.1. (S)-6-(hydroxy(phenyl)methyl)-4-(2-hydroxyethoxy)
nicotin-
1
amide (13). White solid, yield 81%. H NMR (500 MHz, DMSO‑d6) δ
8.69 (s, 1H), 7.69 (s, 1H), 7.58 (s, 1H), 7.40 (t, J = 7.0 Hz, 2H), 7.35 (s,
1H), 7.29 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.3 Hz, 1H), 6.19 (d, J = 4.3 Hz,
1H), 5.68 (d, J = 4.1 Hz, 1H), 5.09 (t, J = 5.4 Hz, 1H), 4.29–4.22 (m,
2H), 3.79 (dd, J = 9.6, 4.7 Hz, 2H). 13C NMR (125 MHz, DMSO‑d6) δ
168.69, 164.83, 163.21, 151.29, 143.84, 128.04, 127.06, 126.67,
116.95, 103.89, 75.46, 70.72, 58.98. HRMS: [M+H]+ calcd. For
[M+H]+ calcd. For C21H26N3O4 m/z: 384.1918; found: 384.1914. [
ꢀ 46 (c = 0.1 in CHCl3).
α
]
C
15H17N2O4 m/z: 289.1183; found: 289.1186. [
α] + 37 (c = 1.1 in
CHCl3).
4.1.5.2. 6-benzyl-4-(2-hydroxyethoxy) nicotinamide (14). White solid,
yield 88%. 1H NMR (500 MHz, DMSO‑d6) δ 8.74 (s, 1H), 7.69 (s, 1H),
7.56 (s, 1H), 7.32–7.25 (m, 4H), 7.21–7.17 (m, 1H), 7.16 (s, 1H), 5.09 (t,
J = 5.4 Hz, 1H), 4.23–4.18 (m, 2H), 4.06 (s, 2H), 3.77 (dd, J = 9.5, 5.1
Hz, 2H). 13C NMR (125 MHz, DMSO‑d6) δ 165.12, 164.91, 162.97,
151.88, 139.38, 128.89, 128.37, 126.21, 116.30, 107.51, 70.72, 59.01,
43.80. HRMS: [M+H]+ calcd. For C15H17N2O3 m/z: 273.1234; found:
273.1236.
4.1.6.5. (S)-(4-(allyloxy)-5-carbamoylpyridin-2-yl) (phenyl)methyl ace-
tate(15h). Compound 15h was prepared from compound 3 and 3-bro-
1
moprop-1-ene. White solid, yield 33%. H NMR (500 MHz, DMSO‑d6)
δ 8.60 (s, 1H), 7.66 (s, 1H), 7.57 (s, 1H), 7.47–7.43 (m, 2H), 7.37–7.32
(m, 2H), 7.32–7.28 (m, 1H), 7.26 (s, 1H), 6.65 (s, 1H), 6.06 (ddt, J =
17.2, 10.6, 5.4 Hz, 1H), 5.45 (dd, J = 17.3, 1.6 Hz, 1H), 5.32 (dd, J =
10.6, 1.4 Hz, 1H), 4.84 (d, J = 5.3 Hz, 2H), 2.18 (s, 3H). 13C NMR (125
MHz, DMSO‑d6) δ 169.57, 165.03, 162.70, 162.38, 150.74, 138.58,
132.43, 128.44, 128.15, 127.30, 119.09, 118.74, 105.05, 77.22, 69.11,
20.87. HRMS: [M+H]+ calcd. For C18H19N2O4 m/z: 327.1339; found:
4.1.6. General synthetic procedure of compounds 15 and 16
The reaction of compound 3 (0.12 mmol, 1.0 eq.) and K2CO3 (0.24
mmol, 2.0 eq.) and corresponding halides (0.24 mmol, 2.0 eq.) in 5 mL
anhydrous acetone was heated 60–80 ◦C and stirred for 12–48 h
(monitored by TLC). The reaction mixture was extracted with ethyl
acetate (3 × 5 mL). The combined organic phase was washed with brine,
dried (Na2SO4), and concentrated. The residue was purified by column
chromatography on silica gel (ethyl acetate/petroleum ether) to obtain a
series of compounds 15 and 16.
327.1331. [α] + 27 (c = 1.0 in CHCl3).
4.1.6.6. (S)-(1-allyl-5-carbamoyl-4-oxo-1,4-dihydropyridin-2-yl) (phenyl)
methyl acetate(16h). Compound 16h was prepared from compound 3
1
and 3-bromoprop-1-ene. Colorless oil, yield 50%. H NMR (500 MHz,
DMSO‑d6) δ 9.37 (d, J = 4.4 Hz, 1H), 8.44 (s, 1H), 7.56 (d, J = 4.4 Hz,
1H), 7.48–7.37 (m, 5H), 6.84 (s, 1H), 6.48 (s, 1H), 5.80 (ddd, J = 15.5,
10.4, 5.1 Hz, 1H), 5.22 (d, J = 10.5 Hz, 1H), 5.02 (d, J = 17.2 Hz, 1H),
8

L. Li et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116192
4.72 (dd, J = 16.8, 4.9 Hz, 1H), 4.64–4.52 (m, 1H), 2.17 (s, 3H). 13C
NMR (125 MHz, DMSO‑d6) δ 176.34, 169.26, 164.66, 149.40, 147.24,
135.51, 132.85, 129.23, 129.07, 127.62, 119.54, 118.90, 118.20, 71.07,
54.62, 20.66. HRMS: [M+H]+ calcd. For C18H19N2O4 m/z: 327.1339;
found: 373.1398. [α] ꢀ 12 (c = 0.1 in CHCl3).
4.1.6.12. Ethyl (S)-2-(2-(acetoxy(phenyl)methyl)-5-carbamoyl-4-oxopyr-
idin-1(4H)-yl) acetate (16k). Compound 16k was prepared from com-
1
found: 327.1336. [α] + 66 (c = 0.2 in CHCl3).
pound 3 and ethyl 2-bromoacetate. Colorless oil, yield 52%. H NMR
(500 MHz, DMSO‑d6) δ 9.31 (d, J = 4.5 Hz, 1H), 8.51 (s, 1H), 7.55 (d, J
= 4.5 Hz, 1H), 7.45–7.37 (m, 3H), 7.37–7.33 (m, 2H), 6.91 (s, 1H), 6.49
(s, 1H), 5.00 (q, J = 18.1 Hz, 2H), 3.94 (qd, J = 7.0, 1.3 Hz, 2H), 2.14 (s,
3H), 1.11 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 176.58
(s), 168.96 (s), 167.28 (s), 164.57 (s), 149.07 (s), 148.66 (s), 134.97 (s),
128.94 (d, J = 17.0 Hz), 127.19 (s), 120.27 (s), 118.53 (s), 71.54 (s),
61.50 (s), 53.27 (s), 20.47 (s), 13.81 (s). HRMS: [M+H]+ calcd. For
4.1.6.7. (S)-(4-(2-(1,3-dioxolan-2-yl) ethoxy)-5carbamoylpyridin-2-yl)
(phenyl)methyl acetate(15i). Compound 15i was prepared from com-
pound 3 and 2-(2-chloroethyl)-1,3-dioxolane. Colorless oil, yield 52%.
1H NMR (500 MHz, CDCl3) δ 9.18 (s, 1H), 7.95 (s, 1H), 7.41 (d, J = 7.3
Hz, 2H), 7.36–7.27 (m, 3H), 6.97 (s, 1H), 6.82 (s, 1H), 6.06 (s, 1H), 5.04
(t, J = 3.9 Hz, 1H), 4.30 (ddd, J = 9.5, 6.9, 2.9 Hz, 2H), 3.97 (dd, J = 8.7,
5.2 Hz, 2H), 3.87 (dd, J = 8.7, 5.1 Hz, 2H), 2.27 (dd, J = 9.7, 5.2 Hz,
2H), 2.20 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 169.90, 165.38, 164.21,
163.49, 154.24, 138.35, 128.75, 128.55, 127.44, 115.98, 104.01,
102.44, 77.79, 65.07, 64.29, 32.57, 21.31. HRMS: [M+H]+ calcd. For
C19H21N2O6 m/z: 373.1394; found: 373.1390. [
α] + 17 (c = 0.1 in
CHCl3).
4.1.6.13. (S)-(5-carbamoyl-4-(2-methoxyethoxy) pyridin-2-yl) (phenyl)
methyl acetate(15l). Compound 15l was prepared from compound 3 and
1-bromo-2-methoxyethane. White solid, yield 46%. 1H NMR (500 MHz,
DMSO‑d6) δ 8.68 (s, 1H), 7.70 (s, 1H), 7.52 (s, 1H), 7.46 (d, J = 7.4 Hz,
2H), 7.32 (dq, J = 14.5, 7.2 Hz, 4H), 6.67 (s, 1H), 4.38 (dd, J = 5.1, 3.3
Hz, 2H), 3.78–3.70 (m, 2H), 3.31 (s, 3H), 2.18 (s, 3H). 13C NMR (125
MHz, DMSO‑d6) δ 169.59, 164.72, 163.07, 162.95, 151.27, 138.56,
128.44, 128.14, 127.32, 118.13, 105.02, 77.20, 69.75, 68.05, 58.21,
20.88. HRMS: [M+H]+ calcd. For C18H21N2O5 m/z: 345.1445; found:
C
20H23N2O6 m/z: 387.1551; found: 387.1553. [α] + 3 (c = 0.1 in
CHCl3).
4.1.6.8. (S)-(1-(2-(1,3-dioxolan-2-yl)
ethyl)-5-carbamoyl-4-oxo-1,4-
dihydropyridin-2-yl) (phenyl)methyl acetate(16i). Compound 16i was
prepared from compound
3 and 2-(2-chloroethyl)-1,3-dioxolane.
Colorless oil, yield 28%. 1H NMR (500 MHz, CDCl3) δ 9.85 (d, J = 4.2
Hz, 1H), 8.45 (s, 1H), 7.43–7.39 (m, 3H), 7.34 (dd, J = 6.5, 3.1 Hz, 3H),
6.82 (s, 1H), 6.81 (s, 1H), 5.98 (d, J = 4.2 Hz, 1H), 4.90 (t, J = 3.5 Hz,
1H), 4.04–3.93 (m, 4H), 3.93–3.82 (m, 4H), 2.17 (s, 3H). 13C NMR (125
MHz, CDCl3) δ 177.44, 169.68, 166.15, 149.36, 147.21, 134.84, 130.04,
129.50, 128.40, 119.92, 119.18, 101.16, 71.41, 65.33, 65.28, 48.58,
33.85, 21.07. HRMS: [M+H]+ calcd. For C20H23N2O6 m/z: 387.1551;
345.1441. [α] + 31 (c = 0.2 in CHCl3).
4.1.6.14. (S)-(5-carbamoyl-1-(2-methoxyethyl)-4-oxo-1,4-dihydropyr-
idin-2-yl) (phenyl)methyl acetate(16l). Compound 16l was prepared
from compound 3 and 1-bromo-2-methoxyethane. Colorless oil, yield
19%. 1H NMR (500 MHz, DMSO‑d6) δ 9.37 (d, J = 4.4 Hz, 1H), 8.45 (s,
1H), 7.52 (d, J = 4.4 Hz, 1H), 7.43 (dt, J = 11.6, 6.9 Hz, 5H), 6.94 (s,
1H), 6.44 (s, 1H), 4.22 (ddd, J = 14.3, 7.1, 3.4 Hz, 1H), 4.17–4.09 (m,
1H), 3.54 (ddd, J = 10.5, 7.2, 3.1 Hz, 1H), 3.48–3.38 (m, 1H), 3.20 (s,
3H), 2.18 (s, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 176.32, 169.35,
164.76, 149.62, 147.85, 135.60, 129.22, 129.04, 127.68, 119.19,
118.34, 71.03, 70.52, 58.40, 52.37, 21.05. HRMS: [M+H]+ calcd. For
found: 387.1555. [α] + 35 (c = 0.1 in CHCl3).
4.1.6.9. (S)-(4-((3,5-bis(trifluoromethyl)benzyl) oxy)-5-carbamoylpyridin-
2-yl) (phenyl)methyl acetate(15j). Compound 15j was prepared from
compound
3
and 1-(bromomethyl)-3,5-bis(trifluoromethyl)benzene.
1
Yellow solid, yield 42%. H NMR (500 MHz, DMSO‑d6) δ 8.58 (s, 1H),
8.27 (s, 2H), 8.11 (s, 1H), 7.72 (d, J = 5.9 Hz, 1H), 7.72–7.67 (m, 1H),
7.41 (d, J = 7.1 Hz, 2H), 7.35–7.27 (m, 4H), 6.66 (s, 1H), 5.57 (s, 2H),
2.16 (s, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 169.48, 165.20, 162.74,
161.95, 150.43, 139.51, 138.51, 131.70, 131.48, 130.54, 130.28, 128.64,
128.46, 128.44, 128.39, 128.15, 127.18, 120.02, 104.90, 77.23, 68.21,
20.77. HRMS: [M+H]+ calcd. For C24H19F6N2O4 m/z: 513.1244; found:
C
18H21N2O5 m/z: 345.1445; found: 345.1448. [
α] + 104 (c = 0.2 in
CHCl3).
4.1.6.15. (S)-(4-(benzyloxy)-5-carbamoylpyridin-2-yl)(phenyl)methyl ac-
etate(15m). Compound 15m was prepared from compound 3 and
(bromomethyl)benzene. White solid, yield 56%. 1H NMR (500 MHz,
DMSO‑d6) δ 8.60 (s, 1H), 7.65 (s, 1H), 7.59 (s, 1H), 7.51 (d, J = 7.1 Hz,
2H), 7.44–7.38 (m, 4H), 7.38–7.27 (m, 5H), 6.63 (s, 1H), 5.39 (s, 2H),
2.15 (s, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 169.50, 165.01, 162.74,
162.44, 150.70, 138.49, 135.70, 128.58, 128.42, 128.22, 128.13,
127.87, 127.27, 119.22, 105.18, 77.22, 70.02, 20.84. HRMS: [M+H]+
513.1242. [α] + 17 (c = 0.8 in CHCl3).
4.1.6.10. (S)-(1-(3,5-bis(trifluoromethyl)benzyl)-5-carbamoyl-4-oxo-1,4-
dihydropyridin-2-yl) (phenyl)methyl acetate(16j). Compound 16j was
prepared from compound
3
and 1-(bromomethyl)-3,5-bis(tri-
fluoromethyl)benzene. Colorless oil, yield 15%. 1H NMR (500 MHz,
DMSO‑d6) δ 9.34 (d, J = 4.5 Hz, 1H), 8.59 (s, 1H), 7.98 (s, 1H),
7.63–7.57 (m, 3H), 7.30 (ddd, J = 13.1, 6.1, 3.0 Hz, 6H), 6.83 (s, 1H),
6.63 (s, 1H), 5.65 (d, J = 17.2 Hz, 1H), 5.54 (d, J = 17.2 Hz, 1H), 2.03 (s,
3H). 13C NMR (125 MHz, DMSO‑d6) δ 176.44, 169.20, 164.43, 149.75,
147.81, 139.49, 134.76, 131.69, 131.49, 130.53, 130.26, 129.06,
128.75, 128.64, 127.80, 127.12, 119.46, 119.35, 71.29, 54.38, 20.33.
HRMS: [M+H]+ calcd. For C24H19F6N2O4 m/z: 513.1244; found:
calcd. For C22H21N2O4 m/z: 377.1496; found: 377.1493. [α] + 31 (c =
0.5 in CHCl3).
4.1.6.16. (S)-(1-benzyl-5-carbamoyl-4-oxo-1,4-dihydropyridin-2-yl)
(phenyl)methyl acetate(16m). Compound 16m was prepared from
1
compound 3 and (bromomethyl)benzene. Colorless oil, yield 53%. H
NMR (500 MHz, DMSO‑d6) δ 9.35 (d, J = 4.5 Hz, 1H), 8.53 (s, 1H), 7.58
(d, J = 4.5 Hz, 1H), 7.39 (ddd, J = 6.2, 4.6, 2.0 Hz, 3H), 7.36 (d, J = 7.6
Hz, 2H), 7.32 (dd, J = 5.5, 2.2 Hz, 3H), 7.03 (d, J = 7.3 Hz, 2H), 6.85 (s,
1H), 6.46 (s, 1H), 5.40–5.26 (m, 2H), 1.94 (s, 3H). 13C NMR (125 MHz,
DMSO‑d6) δ 176.34, 169.14, 164.59, 149.68, 147.74, 136.02, 135.49,
129.10, 128.99, 128.93, 127.94, 127.31, 126.35, 120.27, 118.95, 71.11,
55.40, 20.24. HRMS: [M+H]+ calcd. For C22H21N2O4 m/z: 377.1496;
513.1240. [α] + 22 (c = 0.1 in CHCl3).
4.1.6.11. Ethyl (S)-2-((2-(acetoxy(phenyl)methyl)-5-carbamoylpyridin-4-
yl) oxy) acetate (15k). Compound 15k was prepared from compound 3
and ethyl 2-bromoacetate. Colorless oil, yield 48%. 1H NMR (500 MHz,
DMSO‑d6) δ 8.76 (s, 1H), 7.78 (d, J = 14.6 Hz, 2H), 7.45 (d, J = 7.2 Hz,
2H), 7.32 (dt, J = 23.7, 7.2 Hz, 3H), 7.25 (s, 1H), 6.65 (s, 1H), 5.11 (s,
2H), 4.23–4.18 (m, 2H), 2.17 (s, 3H), 1.22 (t, J = 7.1 Hz, 4H). 13C NMR
(125 MHz, CDCl3) δ 169.86, 167.09, 164.83, 164.58, 162.23, 154.62,
138.13, 128.84, 128.68, 127.43, 116.27, 104.38, 77.65, 65.46, 62.46,
21.32, 14.23. HRMS: [M+H]+ calcd. For C19H21N2O6 m/z: 373.1394;
found: 377.1491. [α] + 43 (c = 0.1 in CHCl3).
4.1.6.17. (S)-(4-(2-bromoethoxy)-5-carbamoylpyridin-2-yl) (phenyl)methyl
acetate(15n). Compound 15n was prepared from compound 3 and 1,2-
dibromoethane. White solid, yield 55%. 1H NMR (500 MHz, DMSO‑d6) δ
9

L. Li et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116192
8.71 (s, 1H), 7.80 (s, 1H), 7.51–7.44 (m, 3H), 7.35 (t, J = 7.3 Hz, 2H),
7.32–7.26 (m, 2H), 6.67 (s, 1H), 4.62–4.58 (m, 2H), 3.96–3.91 (m, 2H),
2.18 (s, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 169.60, 164.45, 163.22,
162.28, 151.53, 138.49, 128.43, 128.16, 127.35, 117.87, 104.90, 77.15,
68.67, 30.90, 20.90. HRMS: [M+H]+ calcd. For C17H18BrN2O4 m/z:
MeOH) to obtain compound 17.
To a solution of compound 17 (0.10 mmol, 1.0 eq.), DMAP (0.05
mmol, 0.5 eq.) and TEA (0.20 mmol, 2.0 eq.) in 5 mL anhydrous
dichloromethane protected with nitrogen, and corresponding acid (0.20
mmol, 2.0 eq.) was slowly added under ice bath and stirred for 1 h
(monitored by TLC). 1 N dilute hydrochloric acid solution was added to
the reaction mixture to quenching reaction. The solution was then
extracted with CH2Cl2 (3 × 5 mL). The combined organic phase was
washed with brine, dried (Na2SO4), and concentrated. A crude solid that
was purified by silica gel column chromatography (CH2Cl2/MeOH) to
obtain compound 18.
393.0444; found: 393.0448. [α] + 17 (c = 1.0 in CHCl3).
4.1.6.18. (S)-(5-carbamoyl-4-(2-thiomorpholinoethoxy)
pyridin-2-yl)
(phenyl)methyl acetate(15o). The reaction of compound 15n (0.12
mmol, 1.0 eq.) and potassium carbonate (0.24 mmol, 2.0 eq.) and thi-
omorpholine (0.36 mmol, 3.0 eq.) in 5 mL anhydrous acetone was
◦
The reaction of compound 18 (0.15 mmol, 1.0 eq.) and K2CO3 (0.30
mmol, 2.0 eq.) and corresponding halides (0.30 mmol, 2.0 eq.) in 5 mL
anhydrous acetone was heated 60–80 ◦C and stirred for 12 h (monitored
by TLC). The reaction mixture was extracted with ethyl acetate (3 × 5
mL). The combined organic phase was washed with brine, dried by
Na2SO4, and concentrated. The residue was purified by column chro-
matography on silica gel (ethyl acetate/petroleum ether) to obtain
compound 19.
heated 60 C to reflux and stirred for 12 h (monitored by TLC). The
reaction mixture was extracted with ethyl acetate (3 × 5 mL). The
combined organic phase was washed with brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatography on
silica gel (ethyl acetate/petroleum ether) to obtain compound 15o,
colorless oil, yield 67%. 1H NMR (500 MHz, CDCl3) δ 9.18 (s, 1H), 8.02
(s, 1H), 7.42 (d, J = 7.2 Hz, 2H), 7.31 (ddd, J = 10.9, 9.7, 5.7 Hz, 3H),
6.95 (s, 1H), 6.82 (s, 1H), 6.08 (s, 1H), 4.24 (dd, J = 9.4, 4.9 Hz, 2H),
2.83 (t, J = 5.4 Hz, 2H), 2.81–2.75 (m, 4H), 2.67–2.60 (m, 4H), 2.21 (s,
3H). 13C NMR (125 MHz, CDCl3) δ 169.94, 165.45, 164.27, 163.49,
154.21, 138.26, 128.80, 128.61, 127.44, 116.34, 104.81, 77.76, 65.22,
57.09, 55.01, 28.04, 21.34. HRMS: [M+H]+ calcd. For C21H26N3O4S m/
4.1.7.1. (S)-6-(hydroxy(phenyl)methyl)-N,N-dimethyl-4-oxo-1,4-dihy-
dropyridine-3-carboxamide (17). Yellow solid, yield 37%. 1H NMR (500
MHz, DMSO‑d6) δ 11.64 (s, 1H), 7.61 (s, 1H), 7.44 (d, J = 7.4 Hz, 2H),
7.35 (t, J = 7.4 Hz, 2H), 7.28 (t, J = 6.9 Hz, 1H), 6.48 (s, 1H), 6.16 (s,
1H), 5.58 (s, 1H), 2.84 (d, J = 48.2 Hz, 6H). 13C NMR (101 MHz,
DMSO‑d6) δ 174.8, 167.2, 153.3, 142.6, 137.4, 128.8, 128.3, 126.9,
114.7, 71.5, 38.0, 34.8. HRMS: [M+H]+ calcd. For C15H17N2O3 m/z:
z: 416.1639; found: 416.1637. [α] ꢀ 2 (c = 0.2 in CHCl3).
4.1.6.19. (S)-(5-carbamoyl-4-(2-morpholinoethoxy) pyridin-2-yl) (phenyl)
methyl acetate(15p). The synthetic method was similar to that of com-
1
273.1234; found: 273.1237. [α] ꢀ 5 (c = 0.2 in CHCl3).
pound 15o, and starting from morpholine. Colorless oil, yield 95%. H
NMR (500 MHz, CDCl3) δ 9.17 (s, 1H), 8.11 (s, 1H), 7.41 (d, J = 7.2 Hz,
2H), 7.36–7.26 (m, 3H), 6.96 (s, 1H), 6.82 (s, 1H), 6.19 (s, 1H), 4.32–4.20
(m, 2H), 3.70–3.62 (m, 4H), 2.81 (t, J = 5.4 Hz, 2H), 2.51 (s, 4H), 2.20 (s,
3H). 13C NMR (125 MHz, CDCl3) δ 169.91, 165.50, 164.23, 163.50,
154.14, 138.25, 128.77, 128.58, 127.41, 116.39, 104.81, 77.73, 66.91,
65.05, 56.73, 53.42, 21.31. HRMS: [M+H]+ calcd. For C21H26N3O5 m/z:
4.1.7.2. (S)-(5-(dimethylcarbamoyl)-4-oxo-1,4-dihydropyridin-2-yl)
(phenyl)methyl acetate (18). Colorless oil, yield 65%. 1H NMR (500
MHz, CDCl3) δ 8.02 (s, 1H), 7.40–7.36 (m, 2H), 7.35–7.27 (m, 3H), 6.67
(s, 2H), 3.05 (s, 6H), 2.14 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ
180.1, 178.2, 149.4, 139.6, 139.5, 138.3, 137.7, 50.8, 50.6, 50.4, 31.1,
11.6. HRMS: [M+H]+ calcd. For C17H19N2O4 m/z: 315.1339; found:
400.1867; found: 400.1869. [α] ꢀ 21 (c = 0.1 in CHCl3).
315.1333. [α] –22 (c = 0.1 in CHCl3).
4.1.6.20. (S)-(5-carbamoyl-4-oxo-1-(2-(piperidin-1-yl) ethyl)-1,4-dihydrop
yridin-2-yl) (phenyl)methyl acetate(16q). Compound 16q was prepared
from 1-(2-chloroethyl) piperidine. Colorless oil, yield 73%. 1H NMR (500
MHz, DMSO‑d6) δ 9.40 (d, J = 4.7 Hz, 1H), 8.41 (s, 1H), 7.46–7.42 (m,
6H), 6.87 (s, 1H), 6.48 (s, 1H), 2.35–2.30 (m, 3H), 2.26 (d, J = 5.1 Hz,
2H), 2.18 (s, 3H), 1.98 (s, 2H), 1.91 (s, 8H). 13C NMR (125 MHz,
DMSO‑d6) δ 176.37, 169.45, 164.91, 149.24, 148.54, 135.49, 129.31,
129.06, 127.91, 118.64, 117.57, 71.22, 59.75, 57.33, 53.87, 25.41,
21.05, 20.65. HRMS: [M+H]+ calcd. For C22H28N3O4 m/z: 398.2074;
4.1.7.3. (S)-(5-(dimethylcarbamoyl)-4-(2-hydroxyethoxy)pyridin-2-yl)
(phenyl)methyl acetate(19). Colorless oil, yield 72%. 1H NMR (500 MHz,
DMSO‑d6) δ 8.20 (s, 1H), 7.47 (d, J = 7.3 Hz, 2H), 7.38–7.29 (m, 3H),
7.28 (s, 1H), 6.68 (s, 1H), 4.92 (t, J = 5.2 Hz, 1H), 4.20 (t, J = 4.8 Hz,
2H), 3.72(dd, J = 9.7, 5.0 Hz, 2H), 2.96 (s, 3H), 2.78 (s, 3H), 2.18 (s,
3H). 13C NMR (125 MHz, DMSO‑d6) δ 169.61, 165.52, 161.42, 161.24,
148.17, 138.81, 128.44, 128.09, 127.27, 121.68, 104.63, 77.28, 70.25,
59.22, 37.60, 34.24, 20.91. HRMS: [M+H]+ calcd. For C19H23N2O5 m/z:
found: 398.2077. [
α
] + 55 (c = 0.3 in CHCl3).
359.1601; found: 359.1607. [α] + 10 (c = 0.4 in CHCl3).
4.2. Biological assays
4.1.6.21. (S)-(5-carbamoyl-1-(4-methoxybenzyl)-4-oxo-1,4-dihydropyr-
idin-2-yl)(phenyl)methyl acetate(16r). Compound 16r was prepared
from 1-(bromomethyl)-4-methoxybenzene. Colorless oil, yield 84%. 1H
NMR (500 MHz, DMSO‑d6) δ 9.36 (d, J = 4.6 Hz, 1H), 8.49 (s, 1H), 7.57
(d, J = 4.6 Hz, 1H), 7.45–7.39 (m, 3H), 7.38–7.32 (m, 2H), 7.01 (d, J =
8.7 Hz, 2H), 6.94–6.90 (m, 2H), 6.89 (s, 1H), 6.47 (s, 1H), 5.22 (q, J =
15.9 Hz, 2H), 3.73 (s, 3H), 2.03 (s, 3H). 13C NMR (125 MHz, DMSO‑d6) δ
176.32, 169.22, 164.63, 159.05, 149.67, 147.32, 135.54, 129.14,
129.03, 128.27, 127.55, 127.44, 119.95, 118.90, 114.37, 71.12, 55.17,
54.91, 20.40. HRMS: [M+H]+ calcd. For C23H23N2O5 m/z: 407.1601;
4.2.1. HepG2 cell culture and treatments
The HepG2 cells were obtained from ATCC and grown in Dulbecco
Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA) sup-
plemented with 10% fetal bovine serum and antibiotics (100
μg/L
penicillin, 100 μ
g/L streptomycin) and maintained at 37 ◦C in a hu-
midified atmosphere of 95% air ꢀ 5% carbon dioxide. Culture medium
was changed every 2 days, and the number of viable cells was deter-
mined using the MTT method and approximately 2.5 × 104 cells were
plated in each well. Control cells received only with the solvent (DMSO)
found: 407.1609. [α] + 48 (c = 0.3 in CHCl3)
and the OA cells treated only with 100 μM oleic acid (Sigma, USA) (OA-
treated cells). In parallel, the experiment cells were treated with or
4.1.7. Synthetic of compounds 17–19
without different target compounds (10 μM) dissolved in DMSO and
To compound 2 (0.33 mmol, 1.0 eq.) in a 25 mL dry seal tube, 10 mL
of 2 M dimethylamine methanol solution was added. The reaction
oleic acid.23,24 Simvastatin was used as a positive control drug (10
μM).
◦
A single experiment was repeated six times to calculate the standard
deviation.
mixture was then heated to 80 C and stirred for 48 h (monitored by
TLC). Upon completion, the reaction was concentrated under reduced
pressure and purified by column chromatography on silica gel (CH2Cl2/
10

L. Li et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116192
4.2.2. Oil red O staining
Acknowledgments
For quantification, The HepG2 cells were fixed with 10% neutral
formalin for 1 h at room temperature, washed with phosphate-buffered
saline and then stained for 1 h with 0.5% oil red O in 60% isopropanol.
After washing with distilled water, the stained cells were observed under
a microscope (N-MSI-vectra).24 The lipid droplets in cells were stained
with oil red O for 30 min and then washed with 70% ethanol for
quantification by measuring its absorbance at 358 nm.
This work was financially supported by the National Key R&D Pro-
gram of China (grants 2018YFC1406705), the NSFC-Shandong Joint
Fund (U1906212), the National Science and Technology Major Project
for Significant New Drugs Development (2018ZX09735004), the Marine
S&T Fund of Shandong Province for Qingdao Pilot National Laboratory
for Marine Science and Technology (2018SDKJ0401-2), the Funda-
mental Research Funds for the Central Universities (201941001), the
Taishan Scholar Youth Expert Program in Shandong Province
(tsqn201812021) and the Youth Innovation Plan of Shandong province
(2019KJM004).
4.2.3. Cell viability assay
The cytotoxic effect was reported as percentage of viable cells after
exposure to myclobutanil with respect to vehicle-treated cells.25 HepG2
cells (human hepatoma cell line), (seeded at a density of 1.2 × 104 cells
per well, 100 μL per well) were grown in 96-well plates for 24 h and then
Appendix A. Supplementary material
treated with serial dilutions of compounds 4, 15k, 15o and DMSO as a
control, in DMEM supplemented with 10% FBS. A stock solution of each
complex was prepared in DMSO and filtered with Minisart filters (0.22
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116192.
μ
m) and the final concentration of the compounds were 10 μM. After
incubation at 37 ◦C for 48 h, 5 mg/mL of MTT in PBS was added into
each well and incubated at 37 ◦C for further 4 h. Successively, a solu-
bilizing solution (10% SDS, 0.01 N HCl) was added to dissolve the for-
mazan salt and lyse the cells and incubated 5% CO2, 95% air at 37 ◦C.
Finally, absorbance was read at the wavelength of 490 nm using an
ELISA microplate reader. Values obtained from the wells treated with
only DMSO were set as 100% of viable cells.26
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