5-Hydroxyindole-based EZH2 inhibitors assembled via TCCA-catalyzed condensation and Nenitzescu reactions

Article abstract of DOI:10.3390/molecules25092059

5-Hydroxyindole derivatives have various demonstrated biological activities. Herein, we used 5-hydroxyindole as a synthetic starting point for structural alterations in a combinatorial process to synthesize 22 different compounds with EZH2 inhibitor pharmacophores. A series of 5-hydroxyindole-derived compounds were screened inhibitory activities against K562 cells. According to molecular modeling and in vitro biological activity assays, the preliminary structure-activity relationship was summarized. Compound L–04 improved both the H3K27Me3 reduction and antiproliferation parameters (IC₅₀ = 52.6 μM). These findings revealed that compound L–04 is worthy of consideration as a lead compound to design more potent EZH2 inhibitors. During the preparation of compounds, we discovered that trichloroisocyanuric acid (TCCA) is a novel catalyst which demonstrates condensation-promoting effects. To gain insight into the reaction, in situ React IR technology was used to confirm the reactivity. Different amines were condensed in high yields with β-diketones or β-ketoesters in the presence of TCCA to afford the corresponding products in a short time (10~20 min), which displayed some advantages and provided an alternative condensation strategy.

Full text of DOI:10.3390/molecules25092059

molecules
Article
5-Hydroxyindole-Based EZH2 Inhibitors Assembled
via TCCA-Catalyzed Condensation and
Nenitzescu Reactions
Fangyu Du 1,†, Qifan Zhou 1,†, Wenjiao Sun 1 , Cheng Yang 2, Chunfu Wu 2, Lihui Wang 2,* and
Guoliang Chen 1,
*
1
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenhe District,
Shenyang 110016, China; dufangyu1993@163.com (F.D.); zhouqifan921@126.com (Q.Z.);
swj912818@163.com (W.S.)
Department of Pharmacology, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenhe District,
Shenyang 110016, China; 18275366347@163.com (C.Y.); wucf@syphu.edu.cn (C.W.)
Correspondence: lhwang@syphu.edu.cn, (L.W.); chenguoliang@syphu.edu.cn, (G.C.)
Authors contributed equally to this work.
2
*
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 April 2020; Accepted: 23 April 2020; Published: 28 April 2020
Abstract: 5-Hydroxyindole derivatives have various demonstrated biological activities. Herein,
we used 5-hydroxyindole as a synthetic starting point for structural alterations in a combinatorial
process to synthesize 22 different compounds with EZH2 inhibitor pharmacophores. A series of
5-hydroxyindole-derived compounds were screened inhibitory activities against K562 cells. According
to molecular modeling and in vitro biological activity assays, the preliminary structure-activity
relationship was summarized. Compound L–04 improved both the H3K27Me3 reduction and
antiproliferation parameters (IC50 = 52.6 µM). These findings revealed that compound L–04 is
worthy of consideration as a lead compound to design more potent EZH2 inhibitors. During
the preparation of compounds, we discovered that trichloroisocyanuric acid (TCCA) is a novel
catalyst which demonstrates condensation-promoting effects. To gain insight into the reaction, in
situ React IR technology was used to confirm the reactivity. Different amines were condensed in
high yields with β-diketones or β-ketoesters in the presence of TCCA to afford the corresponding
products in a short time (10~20 min), which displayed some advantages and provided an alternative
condensation strategy.
Keywords: 5-hydroxyindole; EZH2 inhibitors; anti-tumor; trichloroisocyanuric acid
1. Introduction
The 5-hydroxyindole scaffold, ubiquitously present in natural products and pharmaceuticals,
with demonstrated diverse biological activities [
inhibitor LY31172 [ ], the antibiotic agent violacein [
neurotransmitter serotonin [ ], the anti-virus agent bufotenine [
indomethacin [ ] (Figure 1). To date, various protocols have been developed for its preparation [9–
1
,2
], such as the selective secretory phospholipase A2
], the anti-influenza virus drug arbidol [ ], the
], and the anti-inflammatory drug
13],
3
4
5
6
7
8
among which the Nenitzescu indole synthesis where 1,4-benzoquinone is condensed with enamines to
afford N-substituted 5-hydroxyindoles has been proven to be the simplest strategy [14]. The related
enamines are ubiquitous reagents in organic synthesis [15–17], which are generally obtained by
condensation reaction using amines, β-diketones or β-ketoesters as starting materials [18–21].
Our group is devoted to the study and development of histone methyltransferase inhibitors.
The enhancer of zeste homolog 2 (EZH2), known as the key catalyzed subunit of the polycomb repressor
Molecules 2020, 25, 2059; doi:10.3390/molecules25092059
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2059
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complex 2 (PRC2), can regulate trimethylated histone H3 lysine 27 (H3K27Me3) on chromatin, and
subsequently silence tumor suppressor genes [22]. It is reported that an abnormally high expression
of EZH2 was discovered in various malignant cells and other diverse biological processes [23].
In recent years, several EZH2 inhibitors (EZH2Is) have been the subject of clinical trials, summarized
in Table 1 [24–29].
Table 1. Representative EZH2Is in clinical trials.
Compound
Structure
Highest Phase
Tumor Type
Organization
HN
N
N
Me
Et
N
Solid tumors, relapsed
and refractory diffuse
large B-cell lymphoma
(DLBCL)
GSK–126 [24]
Discontinued
GlaxoSmithKline
Me
Me HN
O
Me
N
O
H
Metastatic / local
advanced epithelioid
sarcoma
Tazemetostat
(EPZ-6438) [25]
Launched
Epizyme
Metastatic
castration-resistant
prostate cancer
(mCRPC)
Constellation
Pharmaceuticals
CPI–1205 [26]
Phase I/II
Valemetostat
(DS–3201) [27]
Acute myeloid and
lymphocytic leukemia
Phase I
Daiichi Sankyo
O
Elapsed or refractory
small cell lung cancer
(SCLC), CRPC, DLBCL
and FL
O
O
Cl
PF–06821497 [28]
SHR–2554 [29]
HN
N
OMe
Phase I
Phase I
Pfizer
Me
OMe
Cl
Relapsed or refractory
mature lymphoid
neoplasms
undisclosed
Jiangsu Hengrui
Scheme 1. Synthetic route to target compounds via the key TCCA-catalyzed condensation and
Nenitzescu synthesis.

Molecules 2020, 25, 2059
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In 2020, tazemetostat was the first agent in its class to receive approved by U.S. Food and Drug
Administration (FDA) for the treatment of metastatic or locally advanced epithelioid sarcoma in adults
and pediatric patients aged 16 years and older [25]. Tazemetostat is also being investigated as a
chemotherapy agent for the treatment of several cancers, including follicular lymphoma, kidney cancer,
diffuse large B-cell lymphoma, solid tumor, etc. [25]. These observations stimulated numerous EZH2
drug discovery programs and greatly encouraged us to develop more novel EZH2Is for the treatment
of multiple cancer contexts.
O
Me
H
Me
NH2
N
N
N
H
O
HO
HO
HO
N
N
H
N
H
H
Bufotenine
Anti-virus agent
Serotonin (5-HT)
Neurotransmitters
Violacein
Antibiotic agent
Me
OH
Me
Me
N
N
O
O
P
Me
MeO
HO
COOEt
O
HO
Me
HO
Et
N
N
N
Me
S
Br
O
Cl
Arbidol
Anti-influenza virus drug
Indomethacin
Anti-inflammation drug
LY31172
Secretory phospholipase A2 inhibitor
Figure 1. Selected natural products and pharmaceuticals containing 5-hydroxyindole scaffold.
However, some agents’ relatively modest potency and pharmacokinetic properties remain
drawbacks for future studies as optimal EZH2Is; for example, GSK–126 is only administered by
injection due to its poor pharmacokinetic properties [30]. Moreover, the single administration dosage
of tazemetostat is up to 800 mg po bid [31]. Besides, the clinical therapeutic benefits of EZH2Is
remain unsatisfactory and their applications are limited to only certain hematological malignancies.
The abovementioned issues highlight the challenges that are associated with EZH2I therapy in the
context of cancer. Therefore, the development of novel EZH2I structures could be a way to overcome
these challenges. From the perspective of the structure of reported EZH2Is, while the initial structures
have given invaluable insight into small molecule EZH2Is, a number of open questions remain.
Most of the compounds take advantage of the structural characteristics of the pyridone moiety as an
active pharmacophore that binds to EZH2 structures. This feature may serve as a boon for future
inhibitor development.
Herein, based on the privileged 5-hydroxyindole scaffold and the main pyridone moiety
pharmacophore, a novel series of 5-hydroxyindole-based EZH2Is has been designed and synthesized
(Scheme 1), which then their antiproliferative effects against K562 cells in vitro and have been
assessed and their ability to reduce cellular H3K27Me3 levels further characterized. Further a
simple catalyst—1,3,5-trichloroisocyanuric acid (TCCA)—for promoting the condensation reactions of
β
-diketones or
discovered. Of note, TCCA has been previously reported as an inexpensive and relatively stable
chlorination [32 33] and oxidation reagent [34 35] and our results therefore further expand the
application scope of TCCA.
β-ketoesters and related amines during the preparation of compounds was accidentally
,
,

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2. Results and Discussion
2.1. Chemistry
2.1.1. Condensation of Amines with β-Diketones or β-Ketoesters Catalyzed by TCCA
Generally, the condensation reactions of carbonyl compounds with amines are carried out via
azeotropic removal of water due to their reversibility, which typically results in long reaction times,
low yields and harsh reaction conditions, and these drawbacks limit their practical application. It was
.
recently reported that some Lewis acids such as Zr(ClO4)2 6H2O, InBr3, Bi(O2CCF3)3, etc. could
catalyze this transformation under mild conditions [36–38]. Herein, we systematically investigated
the reaction conditions, including solvent, catalyst and molar ratio (seeing Supporting Information,
Table S1). In this process, TCCA (at 2 mol%) could effectively accelerate the condensation reaction.
The conversion of ethyl acetoacetate to the corresponding ethyl 3-(benzylamino)butyl-2-enoate was
monitored by in situ React IR technology. As seen in Figure 2, less than 1 min after the addition of
benzylamine to ethyl acetoacetate and TCCA in acetonitrile under ice bath conditions, a new sharp
peak belonging to ethyl 3-(benzylamino)but-2-enoate appeared at 1647 cm−1, and the intensity of the
peak increased gradually as the reaction proceeded. Of note also is the disappearance of the 1752 cm−1
band in parallel to the appearance of the 1647 cm−1 band. We believed that the new infrared absorption
at 1647 cm−1 is attributed to the formation of a C-N bonds. The conversion of ethyl acetoacetate was
less than 50% without any catalyst (Figure 2A), whereas ethyl acetoacetate was completely converted
in 15 minutes when the reaction was catalyzed by TCCA (Figure 2B).
Figure 2. 2D trends of the React IR experiment. (
A) Ethyl acetoacetate (1.0 g, 7.68 mmol), benzylamine
(0.82 g, 7.68 mmol) and MeCN (5 mL) were used; (
B
) Ethyl acetoacetate (1.0 g, 7.68 mmol), benzylamine
(0.82 g, 7.68 mmol), TCCA (34.8 mg, 0.15 mmol) and MeCN (5 mL) were used. The reaction was
conducted under ice bath conditions and allowed to naturally reach room temperature.
The reaction enjoys a wide substrate scope with respect to amine substrates, including primary
aliphatic amines and aromatic amines (Table 2). This facile method was successfully used for
β
-diketones or
The condensation reaction between aliphatic amines and
short reaction times of only 10–20 min (entries 1–9). A more sterically encumbered phenyl group at
the -position of the -ketoesters, and bulky amine substrate also reacted without incident (entries
β
-ketoesters, and excellent yield and short reaction times were observed in all cases.
β-ketoesters catalyzed by TCCA required
β
β
8, 16). Since the aromatic amines have weaker nucleophilic activity, a long reaction time was also
required (entries 3, 7, 8, 12–15). Moreover, electron-withdrawing groups on the benzene ring were not
beneficial to the reaction (entry 15); for example, nitro groups, resulted in longer reaction times and
lower conversion rates. Having established a facile method to the synthesis of intermediates, we next
turned our efforts toward the synthesis of 5-hydroxyindole-based EZH2Is.

Molecules 2020, 25, 2059
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Table 2. Scope with respect to substrates using TCCA as a catalyst a.
O
O
R3HN
O
TCCA
CH3CN
R
NH2
+
3
R1
R2
R2
R1
2
3a-p
Yield (%) b
Entry
R1
R2
R3
Product
Time (min)
O
BnHN
Me
1
Me
OEt
OEt
OEt
OEt
OEt
OEt
OEt
10
95
OEt
3a
O
BnCH2HN
Me
2
3
4
5
6
7
Me
Me
Me
Me
Me
Me
10
20
10
10
15
40
98
92
94
95
96
95
OEt
3b
O
PhHN
OEt
Me
3c
2-Furyl-CH2HN
Me
O
O
OEt
3d
O
CyHN
Me
OEt
3e
iPrHN
Me
O
OEt
3f
4-OEt-PhHN
Me
O
OEt
EtO
3g
2,6-iPr-PhHN
Me
O
8
Me
OEt
60
90
OEt
3h
O
(HOCH2)2HCHN
Me
HO
9
Me
Me
Me
Me
Me
Me
Me
Ph
OEt
OtBu
OtBu
OtBu
OtBu
Me
15
10
10
40
40
40
60
50
94
99
94
93
92
90
86
92
OEt
HO
3i
BnHN
Me
O
OtBu
10
11
12
13
14
15
3j
O
HO(CH2)2HN
Me
HO
HO
OtBu
3k
O
4-OEt-PhHN
Me
OtBu
EtO
3l
PhHN
Me
O
OtBu
3m
O
PhHN
Me
Me
3n
O
4-NO2-PhHN
Me
Me
Me
O2N
3o
O
BnHN
16
OEt
OEt
Ph
3p
a
Unless otherwise noted, β-diketones or β-ketoesters (7.68 mmol), amine (7.68 mmol), TCCA (0.15 mmol) and
CH3CN (5 mL) were used; the reactions were conducted under ice bath and naturally elevated to room temperature.
b
Crude yield.

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2.1.2. Synthesis of 5-Hydroxyindole-Based EZH2Is
With the established protocol in hand, a series of 5-hydroxyindoles were obtained, as depicted
in Scheme 2. Initially, compounds 3a were reacted with benzoquinone or naphthalene to yield
5-hydroxyindoles 4a via an anhydrous ZnCl2-catalyzed Nenitzescu indole synthesis. The fragments
6a were prepared from 4a by alkylation and ammonolysis. Then, esters 6a were hydrolyzed
and acidified to afford carboxylic acids 7a . Meanwhile, 5-hydroxyindole 4a was transformed via
alkylation with brominated alkanes in the presence of NaH into ethers 6f h, respectively, which were
hydrolyzed and acidified to afford carboxylic acids 7f . On the other hand, carboxylic acids 7i
were obtained from 4a by direct hydrolysis and acidification. Finally, carboxylic acids 7a were
–
h
–
g
–
e
–e
–
e
–
–
h
–o
–
g
–o
assembled with pyridone derivatives in the presence of EDCI and HOBt to furnish the target compounds
L–01~L–22. The synthesis of the pyridone derivatives is described in the Supporting Information.
Scheme 2. Synthetic route to 5-hydroxyindole-based EZH2Is.
2.2. In Vitro Bioactivity Assay and SAR
Following the synthesis, the antiproliferative effects of the 5-hydroxyindole-based EZH2Is on K562
cells were then evaluated by a cell counting kit-8 (CCK-8) assay using tazemetostat as positive control.
The results showed that most of the target compounds exhibited a similar antiproliferative effect
compared with the control (Table 3). Different lengths of aliphatic chains were introduced to evaluate

Molecules 2020, 25, 2059
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the appropriate distance between the indole scafford and the basic functional group. The results
showed that the length of chain has no obvious effect on the inhibitory activity; for instance, compounds
L–01 (containing a two-carbon linker) and L–03 (containing a three-carbon linker) showed comparable
IC50 values. Additionally, it appeared that the terminal amine was essential for maintaining the activity.
The hydroxy group at the C5 position of an indole substituted with an amine side chain was beneficial
for the antiproliferative effect; for example, L–01 and L–06 had IC50 values of 83.8
µM and above
100 M, respectively. Furthermore, a hydrophobic piperidine group (compounds L–04 and L–05) had
µ
a beneficial effect on the IC50 value compared with that of hydrophilic morpholine moiety (compounds
L–01 and L–03).
As a refinement, some 5-hydroxylindoles bearing different substituents at N1 were synthesized
and investigated. Thus, all other N-substituted compounds (L–10
L–10), cyclohexyl (L–14) or (isopropyl, L–15) produced stronger inhibitory activities than benzyl (L–09
and phenyl (L–12). Particularly, it was noteworthy that the tricyclic compound L–11 showed inhibition
activity with an IC50 value of 55.2 M. Among the different aliphatic alkanes (methyl, ethyl, propyl,
, L–12~L–15), containing phenethyl
(
)
µ
isopropyl) on the pyridone ring, the results suggested a similar biochemical potency. Compounds L–20
and L–22 (with cyclohexane on the pyridone ring ) and L–18 (with a cyclopentane on pyridone ring)
showed less inhibition activities, while compounds L–19 and L–21 showed medium antiproliferative
effect, with IC50 values of 68 µM and 52 µM, respectively.
Table 3. Antiproliferative effects of 5-hydroxyindole-based inhibitors against K562 cells.
cLogP b
cLogP b
Compd.
IC50 (µM) ± SEM a
Compd.
IC50 (µM) ± SEM a
L–01
L–02
L–03
L–04
L–05
L–06
L–07
L–08
L–09
L–10
L–11
L–12
83.8 ± 1.8
72.6 ± 1.6
86.1 ± 2.7
52.6 ± 0.4
71.1 ± 3.2
>100
>100
>100
>100
3.94
5.10
4.33
5.10
5.49
4.80
5.02
4.63
3.94
4.13
5.29
3.88
L–13
L–14
L–15
L–16
L–17
L–18
L–19
L–20
L–21
>100
63.9 ± 3.2
56.3 ± 1.7
>100
>100
>100
3.53
4.40
3.47
5.02
4.72
4.89
5.27
5.28
5.60
5.59
4.73
68.1 ± 1.2
>100
51.8 ± 1.6
>100
59.2 ± 0.6
58.7 ± 3.8
55.2 ± 1.8
85.5 ± 1.0
L–22
Tazemetostat
a IC50: 50% inhibitory concentration (determined by standard CCK-8 assay) after treatment of 48 h. Each experiment
was carried out in triplicate. Predicted by pkCSM [39].
b
To correlate the K562 cell inhibition results with EZH2, we assessed the cellular H3K27Me3 levels
in K562 cells after being treated with compounds L–01
data showed that L–01 L–04 and tazemetostat significantly reduced cellular H3K27Me3 levels in K562
cells at concentrations of 5 M and 10 M in a dose-dependent manner. Thus, our results provide clear
~L–04 and tazemetostat (Figure 3). Western blot
~
µ
µ
evidence that these compounds could inhibit the EZH2 protein at low molarity, which is consistent
with the inhibition of K562 cell growth.

Molecules 2020, 25, 2059
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Figure 3. Effect of L–01~L–04 on cellular H3K27Me3 levels as determined by western blot analysis.
Western blot analysis of H3K27Me3 and Histone3 from whole cell extracts on K562 cells after treatment
of 72 h. The letter T is abbreviation of tazemetostat.
2.3. Molecular Docking
We used molecular modeling to determine a binding mode of some representative inhibitors
(L–04, L–19, L–22 and tazemetostat) in the binding site of EZH2 (PDB ID: 4W2R). Initially, we docked
L–04 and tazemetostat to their respective binding conformations. As indicated in Figure 4A, L–04
could maintain similar binding patterns compared with tazemetostat. The main pyridone moiety
pharmacophore could form a hydrogen bond with Trp521. The carbonyl oxygen of the amide linker
warhead engages EZH2 via tight hydrogen bonds with the catalytic tyrosine residue Tyr111. The indole
scaffold and phenyl ring form face-to-face contacts with Tyr111 and Tyr558. The C5 position of the
indole substituted with the amine side chain projects into the solvent region, where the protonated
piperidine did not form additional hydrogen bonds (Figure 4B,C).
Figure 4. Molecular modeling of 5-hydroxyindole-based inhibitors bound to EZH2 (Note: PDB ID
4W2R). (
04. Hydrogen bond contacts are shown with dashed lines and small molecules are shown as sticks;
) Binding interaction of 04; ( ) Overlay of 04 (light blue), 19 (pink) and 22 (light yellow)
docking poses.
A) Binding pocket surface of L–04 (light blue) and tazemetostat (yellow); (B) Binding pose of
L
–
(
C
L–
D
L
–
L–
L–

Molecules 2020, 25, 2059
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Therefore, the substituents at the C5 position of indole could be modified to improve the
pharmacokinetic properties; for example, compared with unsubstituted compound L–09 L–04 bearing
,
the basic chain showed lower binding energy. Small substituents such as C5 or C6-methyl or ethyl
groups on the pyridone ring were well compatible, while big substituents such as 5 or 6-membered
rings lost the hydrogen bond with Trp521, resulting in an invalid interaction mode with the EZH2
protein (Figure 4D). From the docking score perspective, L–18 and L–20 containing 5 or 6-membered
ring substituents on the pyridone ring resulted in a higher binding energy. In all, our data suggests
that L–04 is an ideal template to develop improved EZH2Is using molecular modeling to rationally
design new agents to treat multiple cancers.
3. Materials and Methods
3.1. General Information
All reagents and starting materials were obtained from commercial sources and used as received.
Melting points were measured with an X-4 melting point apparatus (Beijing Taike Instrument Co.,
Ltd., Beijing, China) and are uncorrected. 1H- and 13C-NMR spectra were recorded on an Ascent
600 MHz spectrometer (Bruker Billerica, MA, USA) using Me4Si (TMS) as the internal standard.
Electrospray ionization mass spectra (ESI-MS) was recorded using an 1100 Series MSD Trap SL (Agilent,
Santa Clara, CA, USA). High-resolution mass spectrometry (HRMS) results were recorded using an
Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS. The reactions were monitored by thin-layer
chromatography (TLC; HG/T2354-92, GF254), and terminated as judged by the consumption of starting
material. The TLC plates were visualized with a UV lamp (Gongyi Yuhua Instrument Co., Ltd,
Zhengzhou, China). React IR experiments were conducted using a ReactIR 15 (SN: 23267) equipped
with an MCT DiComp (Diamond) probe detector (SN: 23146) connected via an AgX
fiber (silver halide) detector (Mettler Toledo, Zurich, Switzerland. The iC IR 4.3 Reaction Analysis
Software was used during data collection and analysis.
×
9.5 mm
×
1.5 m
3.2. Syntheses
3.2.1. General Procedure for the Syntheses of Ethyl Enoates 3a–p
TCCA (714/34.8 mg, 31/0.15 mmol) was added to ethyl acetoacetate (20/1 g, 154/7.68 mmol) or
tert-butyl 3-oxobutanoate (1.2 g, 7.59 mmol) or acetylacetone (0.77 g, 7.69 mmol) or ethyl benzoylacetate
(1.48 g, 7.71 mmol) in acetonitrile (20 mL) under ice bath cooling, and the appropriate amine
(0.23/0.012 mol) was added dropwise. The mixture was stirred under ice bath conditions and allowed
to naturally warm up to room temperature for the indicated time. After the reaction was complete,
the mixture was poured into water, extracted with dichloromethane (3
×
50 mL), washed with water
(3 50 mL) and brine (50 mL). The organic solution was dried over magnesium sulfate, filtered,
×
evaporated under reduced pressure and used in the following reaction without any further purification.
Ethyl 3-(benzylamino)but-2-enoate (3a). Light yellow solid (32.0 g, 95%). M.p. 49–50 ◦C.1H-NMR
(CDCl3)
δ 8.95 (brs, 1H, NH), 7.34 (t, J = 7.6 Hz, 2H, ArH), 7.27–7.26 (m, 3H, ArH), 4.53 (s, 1H, C=C-H),
4.43 (d, J = 6.3 Hz, 2H, OCH2), 4.10 (q, J = 6.4 Hz, 2H, NCH2), 1.92 (s, 3H, CH3), 1.26 (t, J = 7.1 Hz, 3H,
CH3). HRMS (ESI) calcd for C13H17NO2Na [M+Na]+: 242.1151, found: 242.1151.
1
Ethyl 3-(phenethylamino)but-2-enoate (3b). Colorless liquid (35.1 g, 98%). H-NMR (CDCl3)
δ
8.65
(brs, 1H, NH), 7.30 (t, J = 7.3 Hz, 2H, ArH), 7.24–7.19 (m, 3H, ArH), 4.42 (s, 1H, C=C-H), 4.08 (q,
J = 7.1 Hz, 2H, OCH2), 3.45–3.42 (m, 2H, NCH2), 2.85 (t, J = 7.6 Hz, 2H, PhCH2), 1.82 (s, 3H, CH3), 1.25
(t, J = 7.1 Hz, 3H, CH3). HRMS (ESI) calcd for C14H19NO2Na [M+Na]+: 256.1308, found: 256.1307.

Molecules 2020, 25, 2059
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1
Ethyl 3-(phenylamino)but-2-enoate (3c). Colorless liquid (29.0 g, 92%). H-NMR (DMSO-d6)
δ 10.38
(s, 1H, NH), 7.39–7.34 (m, 2H, ArH), 7.19–7.17 (m, 3H, ArH), 4.69 (d, J = 0.4 Hz, 1H, C=C-H), 4.06 (q,
J = 7.1 Hz, 2H, CH2), 2.01 (s, 3H, CH3), 1.20 (t, J = 7.1 Hz, 3H, CH3). HRMS (ESI) calcd for C12H16NO2
[M+H]+: 206.1176, found: 206.1170.
1
Ethyl 3-((furan-2-ylmethyl)amino)but-2-enoate (3d). Colorless liquid (30.2 g, 94%). H-NMR (CDCl3)
δ
8.80 (brs, 1H, NH), 7.35 (d, J = 1.4 Hz, 1H, ArH), 6.31–6.30 (m, 1H, ArH), 6.19 (d, J = 3.2 Hz, 1H, ArH),
4.52 (s, 1H, C=C-H), 4.37 (d, J = 6.3 Hz, 2H, NCH2), 4.08 (q, J = 7.1 Hz, 2H, OCH2), 1.99 (s, 3H, CH3),
1.24 (t, J = 7.1 Hz, 3H, CH3). HRMS (ESI) calcd for C11H15NO3Na [M+Na]+: 232.0949, found: 232.0949.
1
Ethyl 3-(cyclohexylamino)but-2-enoate (3e). Colorless liquid (30.8 g, 95%). H-NMR (CDCl3)
δ 8.63
(brs, 1H, NH), 4.39–4.37 (m, 1H, C=C-H), 4.10–4.05 (m, 2H, OCH2), 3.32–3.27 (m, 1H, NCH), 1.94–1.92
(m, 3H, CH3), 1.88–1.85 (m, 2H, CH2), 1.77–1.73 (m, 2H, CH2), 1.60–1.20 (m, 9H, CH2, CH3). HRMS
(ESI) calcd for C12H21NO2Na [M+Na]+: 234.1465, found: 234.1461.
1
Ethyl 3-(isopropylamino)but-2-enoate (3f). Colorless liquid (25.3 g, 96%). H-NMR (CDCl3)
δ
8.50
(brs, 1H, NH), 4.39 (s, 1H, C=C-H), 4.10–4.06 (m, 2H, OCH2), 3.70–3.66 (m, 1H, CH), 1.94 (s, 3H, CH3),
1.26–1.23 (m, 3H, CH3), 1.21-1.20 (m, 6H, 2
found: 172.1335.
´
CH3). HRMS (ESI) calcd for C9H18NO2 [M+H]+: 172.1332,
Ethyl 3-((4-ethoxyphenyl)amino)but-2-enoate (3g). White solid (1.82 g, 95%). M.p. 53–54 ◦C. 1H-NMR
(CDCl3) 10.14 (brs, 1H, NH), 7.01 (d, J = 8.8 Hz, 2H, ArH), 6.84 (d, J = 8.9 Hz, 2H, ArH), 4.64 (s, 1H,
δ
C=C-H), 4.16–4.12 (14.2 Hz, 7.1 Hz, 2 H, OCH2), 4.01 (q, J = 7.0 Hz, 2H, OCH2), 1.88 (s, 3H, CH3), 1.41
(t, J = 7.0 Hz, 3H, CH3), 1.28 (t, J = 7.1 Hz, 3H, CH3). HRMS (ESI) calcd for C14H19NO3Na [M+Na]+:
272.1257, found: 272.1252.
Ethyl 3-((2,6-diisopropylphenyl)amino)but-2-enoate (3h). White solid (2.0 g, 90%). M.p. 79–81 ◦C.
1H-NMR (CDCl3)
δ
9.84 (brs, 1H, NH), 7.30–7.26 (m, 1H, ArH), 7.17 (d, J = 7.7 Hz, 2H, ArH), 4.68
(brs, 1 H, C=C-H), 4.17 (q, J = 7.1 Hz, 2 H, OCH2), 3.13–3.07 (m, 2H, 2 CH), 1.62 (s, 3H, CH3), 1.31 (t,
J = 7.1 Hz, 3H, CH3), 1.22 (d, J = 6.9 Hz, 6H, 2 CH3), 1.12 (d, J = 6.8 Hz, 6H, 2 CH3). HRMS (ESI) calcd
for C18H27NO2Na [M+Na]+: 312.1934, found: 312.1933.
´
´
´
Ethyl 3-((1,3-dihydroxypropan-2-yl)amino)but-2-enoate (3i). White solid (1.45 g, 94%). M.p. 74–75 oC.
1H-NMR (CDCl3)
δ 8.76 (d, J = 9.4 Hz, 1H, NH), 4.53 (s, 1H, C=C-H), 4.09 (q, J = 7.1 Hz, 2H, OCH2),
3.79–3.64 (m, 4H, 2´CH2), 3.69–3.65 (m, 1H, CH), 1.98 (s, 3H, CH3), 1.25 (t, J = 7.1 Hz, 3H, CH3). HRMS
(ESI) calcd for C9H17NO4Na [M+Na]+: 226.1050, found: 226.1044.
tert-Butyl 3-(benzylamino)but-2-enoate (3j). White solid (1.96 g, 99%). M.p. 59~60 ◦C. 1H-NMR
(CDCl3)
δ 8.89 (brs, 1H, NH), 7.37–7.32 (m, 2H, ArH), 7.30–7.24 (m, 3H, ArH), 4.46 (s, 1H, C=C-H), 4.42
(d, J = 6.4 Hz, 2H, NCH2), 1.87 (s, 3H, CH3), 1.47 (s, 9H, 3
´CH3). HRMS (ESI) calcd for C15H21NO2Na
[M+Na]+: 270.1465, found: 270.1461.
tert-Butyl 3-((2-hydroxyethyl)amino)but-2-enoate (3k). White crystals (1.65 g, 94%). M.p. 51–53 ◦C.
1H-NMR (CDCl3)
8.59 (brs, 1H, NH), 4.43 (s, 1H, C=C-H), 3.74 (t, J = 5.3 Hz, 2H, CH2), 3.37 (q,
δ
J = 5.6 Hz, 2H, CH2), 1.92 (s, 3H, CH3), 1.46(s, 9H, 3
´CH3). HRMS (ESI) calcd for C10H19NO3 Na
[M+Na]+: 224.1257, found: 224.1252.
1
tert-Butyl 3-((4-ethoxyphenyl)amino)but-2-enoate (3l). White crystals (1.96 g, 93%). H-NMR (CDCl3)
10.10 (brs, 1H, NH), 7.01 (d, J = 8.8 Hz, 2H, ArH), 6.83 (d, J = 8.8 Hz, 2H, ArH), 4.58 (s, 1H, C=C-H),
δ
4.01 (q, J = 7.0 Hz, 2H, OCH2), 1.86 (s, 3H, CH3), 1.50 (s, 9H, 3
´CH3), 1.41 (t, J = 7.0 Hz, 3H, CH3).
HRMS (ESI): calcd for C16H23NO3Na [M+Na]+: 300.1576; found: 300.1567.

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1
tert-Butyl 3-(phenylamino)but-2-enoate (3m). White crystals (1.63 g, 92%). H-NMR (CDCl3)
δ 10.34
(brs, 1H, NH), 7.30 (t, J = 7.8 Hz, 2H, ArH), 7.13 (t, J = 7.4 Hz, 1H, ArH), 7.08 (d, J = 7.6 Hz, 2H, ArH),
4.62 (s, 1H, C=C-H), 1.50 (s, 9H, 3´
CH3). HRMS (ESI): calcd for C14H19NO2Na [M+Na]+: 256.1313,
found: 256.1307.
4-(Phenylamino)pent-3-en-2-one (3n). White crystals (1.21 g, 90%). M.p. 49–51 ◦C. 1H-NMR (CDCl3)
12.47 (brs, 1H, NH), 7.35–7.33 (m, 2H, ArH), 7.20 (t, J = 7.4 Hz, 1H, ArH), 7.11 (d, J = 7.5 Hz, 2H,
δ
ArH), 5.19 (brs, 1H, C=C-H), 2.10 (s, 3H, CH3), 2.00 (s, 3H, CH3). HRMS (ESI) calcd for C11H13NONa
[M+Na]+: 198.0889, found: 198.0885.
4-((4-Nitrophenyl)amino)pent-3-en-2-one (3o). Yellow solid (1.46 g, 86%). M.p. 143–144 ◦C. 1H-NMR
(CDCl3) δ 12.78 (brs, 1H, NH), 8.21–8.20 (dd, J = 7.2 Hz, 2.0 Hz, 2H, ArH), 7.19–7.18 (dd, J = 7.2 Hz,
2.0 Hz, 2H, ArH), 5.33 (brs, 1H, C=C-H), 2.19 (s, 3H, CH3), 2.15 (s, 3H, CH3). HRMS (ESI) calcd for
C11H13N2O3 [M+H]+: 221.0921, found: 221.0922.
Ethyl 3-(benzylamino)-3-phenylacrylate (3p). White solid (1.99 g, 92%). M.p. 71–72 ◦C. 1H-NMR
(CDCl3) δ 8.90 (brs, 1H, NH), 7.39–7.33 (m, 5H, ArH), 7.29 (t, J = 7.2 Hz, 2H, ArH), 7.23 (t, J = 7.3 Hz,
1H, ArH), 7.18 (d, J = 7.2 Hz, 2H, ArH), 4.67 (s, 1H, C=C-H), 4.26 (d, J = 6.5 Hz, 2H, NCH2), 4.15 (q,
J = 7.1 Hz, 2H, OCH2), 1.28 (t, J = 7.1 Hz, 3H, CH3). HRMS (ESI) calcd for C18H19NO2Na [M+Na]+:
304.1308, found: 304.1307.
3.2.2. General Procedure for the Syntheses of Ethyl 5-Hydroxyindolecarboxylates 4a–g
Anhydrous ZnCl2 (1.26 g, 9.25 mmol) was added to a stirred suspension of p-benzoquinone (10 g,
92.5 mmol) or naphthoquinone (14.6 g, 92.5 mmol) in dry DCM (50 mL). After heating at reflux, a
solution of crotonamine (92.5 mmol) in dry DCM (50 mL) was added into the mixture over 40 min and
stirred at reflux for a further 45 min. The mixture was cooled to room temperature and held at 4 ◦C
for 30 min to allow product precipitation. The solid was filtered and washed with DCM, water and
acetonitrile to give a corresponding products 4a–g.
Ethyl 1-benzyl-5-hydroxy-2-methyl-1H-indole-3-carboxylate (4a). The title product was obtained from
ethyl enoate 3a and p-benzoquinone according to the general procedure described above as an off white
1
solid (14.3 g, 50%). H-NMR (DMSO-d6)
δ 8.96 (s, 1H, OH), 7.40 (d, J = 2.3 Hz, 1H, ArH), 7.33–7.23
(m, 4H, ArH), 7.01–7.00 (d, J = 7.3 Hz, 2H, ArH), 6.63 (dd, J = 8.7 Hz, 2.4 Hz, 1H, ArH), 5.44 (s, 2H,
NCH2), 4.27 (q, J = 7.1 Hz, 2H, OCH2), 2.64 (s, 1H, CH3), 1.36 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR
(DMSO-d6)
127.7 (ArC), 127.6 (ArC), 126.6 (ArC), 112.1 (ArC), 111.3 (
46.3 (N H2benzyl), 15.0 (CH2 H3), 12.3 (
H3indole). HRMS (ESI) calcd for C19H19NO3Na [M+Na]+:
332.1257, found: 332.1255.
δ
165.6 (C=Oester), 153.3 (HOAr-
Cindole), 145.4 (ArC), 137.8 (ArC), 130.8 (ArC), 129.2 (ArC),
C
-C=O), 106.0 (ArC), 103.1 (ArC), 59.2 (O H2),
C
C
C
C
Ethyl 5-hydroxy-2-methyl-1-phenethyl-1H-indole-3-carboxylate (4b). The title product was obtained
from ethyl enoate 3b and p-benzoquinone according to the general procedure described above as an off
white solid (21.5 g, 72%). M.p. 175–177 ◦C. 1H-NMR (DMSO-d6)
δ 8.97 (s, 1H, OH), 7.38 (d, J = 2.2 Hz,
1H, ArH), 7.33 (d, J = 8.7 Hz, 1H, ArH), 7.27–7.20 (m, 3H, ArH), 7.12 (d, J = 7.1 Hz, 2H, ArH), 6.68–6.66
(dd, J = 8.6 Hz, 2.3 Hz, 1H, ArH), 4.32 (t, J = 7.3 Hz, 2H, OCH2), 4.24 (q, J = 7.1 Hz, 2H, NCH2), 2.95
(t, J = 7.3 Hz, 2H, CH2), 2.4 (s, 3H, CH3), 1.34 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
δ
165.6
(C=Oester), 153.1 (HOAr-Cindole), 145.5 (ArC), 138.7 (ArC), 130.1 (ArC), 129.4 (ArC), 128.8 (ArC), 127.7
(ArC), 127.0 (ArC), 111.8 (ArC), 111.0 ( -C=O), 106.0 (ArC), 102.4 (ArC), 59.1 (O H2), 44.8 (N H2), 35.6
H2Ph), 15.0 (CH2 H3), 11.7 (
H3indole). HRMS (ESI) calcd for C20H21NO3Na [M+Na]+: 346.1414,
found: 346.1403.
C
C
C
(C
C
C
Ethyl 5-hydroxy-2-methyl-1-phenyl-1H-indole-3-carboxylate (4c). The title product was obtained from
ethyl enoate 3c and p-benzoquinone according to the general procedure described above as a brown

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solid (16.1 g, 59%). M.p. 203–205 ◦C. 1H-NMR (DMSO-d6)
δ
9.06 (s, 1H, OH), 7.63 (t, J = 7.4 Hz, 2H,
ArH), 7.57 (t, J = 7.3 Hz, 1H, ArH), 7.47 (d, J = 2.3 Hz, 1H, ArH), 7.45 (d, J = 7.3 Hz, 2H, ArH), 6.78 (d,
J = 8.7 Hz, 1H, ArH), 6.65–6.63 (dd, J = 8.8 Hz, 2.4 Hz, 1H, ArH), 4.32 (q, J = 7.1 Hz, 2H, OCH2), 2.49 (s,
3H, CH3), 1.38 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
δ
165.6 (C=Oester), 153.6 (HOAr-Cindole),
145.3 (ArC), 136.5 (ArC), 131.9 (ArC), 130.3 (ArC), 129.3 (ArC), 128.4 (ArC), 127.5 (ArC), 112.5 (ArC),
111.2 (ArC), 105.9 (ArC), 104.0 (ArC), 59.4 (O H2), 15.0 (CH2 H3), 13.3 ( H3indole). HRMS (ESI) calcd
for C18H17NO3Na [M+Na]+: 318.1101, found: 318.1100.
C
C
C
Ethyl 1-(furan-2-ylmethyl)-5-hydroxy-2-methyl-1H-indole-3-carboxylate (4d). The title product was
obtained from ethyl enoate 3d and p-benzoquinone according to the general procedure described
above as an off white solid (15.8 g, 57%). M.p. 207–211 ◦C. 1H-NMR (DMSO-d6)
δ 8.95 (s, 1H, OH),
7.55 (d, J = 0.8 Hz, 1H, ArH), 7.44 (d, J = 8.7 Hz, 1H, ArH), 7.36 (d, J = 2.2 Hz, 1H, ArH), 6.68–6.66 (dd,
J = 8.8 Hz, 2.3 Hz, 1H, ArH), 6.46 (d, J = 3.1 Hz, 1H, ArH), 6.39–3.38 (m, 1H, ArH), 5.39 (s, 2H, NCH2),
4.26 (q, J = 7.1 Hz, 2H, OCH2), 2.77 (s, 3H, CH3), 1.34 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
δ
165.5 (C=Oester), 153.2 (HOAr-
111.9 (ArC), 111.3 (ArC), 111.0 (ArC), 109.0 (ArC), 105.9 (ArC), 103.1 (ArC), 59.2 (O
12.2 (CH3indole). HRMS (ESI) calcd for C17H17NO4Na [M+Na]+: 322.1050, found: 322.1049.
C
indole), 150.5 (ArC), 145.3 (ArC), 143.5 (ArC), 130.4 (ArC), 127.6 (ArC),
C
H2), 15.0(CH2 H3),
C
Ethyl 1-cyclohexyl-5-hydroxy-2-methyl-1H-indole-3-carboxylate (4e). The title product was obtained
from ethyl enoate 3e and p-benzoquinone according to the general procedure described above as a
pink solid (16.3 g, 67%). M.p. 245–247 ◦C. 1H-NMR (DMSO-d6)
δ 8.90 (s, 1H, OH), 7.50 (d, J = 8.8 Hz,
1H, ArH), 7.40 (d, J = 2.3 Hz, 1H, ArH), 6.64–6.62 (dd, J = 8.8 Hz, 2.4 Hz, 1H, ArH), 4.28–4.24 (m, 3H,
NCH, OCH2), 2.73 (s, 3H, CH3), 2.22–2.20 (m, 2H, CH2), 1.86–1.84 (m, 2H, CH2), 1.76–1.75 (m, 2H,
CH2), 1.70–1.68 (m, 1H, CH2), 1.50–1.44 (m, 2H), 1.38–1.33 (m, 4H, CH3, CH2). 13C-NMR (DMSO-d6)
δ
165.7 (C=Oester), 152.5 (HOAr-
C
indole), 145.0 (ArC), 129.0 (ArC), 128.6 (ArC), 113.5 (ArC), 111.4 (ArC),
106.1 (ArC), 102.5 (ArC), 59.1 (O
C
H2), 55.5 (NCH2), 30.8 (CH2), 26.1 (CH2), 25.2 (CH2), 15.0 (CH2CH3),
12.4 (CH3indole). HRMS (ESI) calcd for C18H23NO3Na [M+Na]+: 324.1570, found: 324.1567.
Ethyl 5-hydroxy-1-isopropyl-2-methyl-1H-indole-3-carboxylate (4f). The title product was obtained
from ethyl enoate 3f and p-benzoquinone according to the general procedure described above as a
white solid (16.4 g, 68%). M.p. 168–170 ◦C. 1H-NMR (DMSO-d6)
δ
8.86 (s, 1H, OH), 7.42 (d, J = 8.8 Hz,
1H, ArH), 7.38 (d, J = 2.2 Hz, 1H, ArH), 6.63–6.61 (dd, J = 8.8 Hz, 2.3 Hz, 1H, ArH), 4.77–4.73 (m, 1H,
NCH), 2.69 (s, 3H, CH3), 1.57 (s, 6H, 2 165.3
CH3), 1.52 (d, J = 6.9 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(C=Oester), 152.5, 144.4 (ArC), 128.7 (ArC), 112.7 (ArC), 111.4 (ArC), 106.2 (ArC), 103.8 (ArC), 59.1
(O H2), 47.0 (N H2), 26.1 ( H3), 15.0 (CH2 H3), 12.5 ( H3indole). HRMS (ESI) calcd for C17H23NO3Na
[M+Na]+: 312.1570, found: 312.1571.
´
δ
C
C
C
C
C
Ethyl 5-hydroxy-2-methyl-1-phenethyl-1H-benzo[g]indole-3-carboxylate (4g). The title product was
obtained from ethyl enoate 3b and naphthoquinone according to the general procedure described above
1
for the ethyl 5-hydroxyindolecarboxylate as a white solid (21.4 g, 62%). H-NMR (DMSO-d6)
δ
9.77
(s, 1H, OH), 8.45 (d, J = 8.6 Hz, 1H, ArHnaphthalene), 8.32 (dd, J = 8.3 Hz, 0.8 Hz, 1H, ArHnaphthalene),
7.73 (s, 1H, ArHnaphthalene), 7.67–7.64 (m, 1H, ArHnaphthalene), 7.46 (t, J = 7.6 Hz, 1H, ArHnaphthalene),
7.31 (t, J = 7.0 Hz, 2H, PhH), 7.27–7.25 (m, 1H, PhH), 7.15 (d, J = 7.0 Hz, 2H, PhH), 4.77 (t, J = 7.3 Hz,
2H, NCH2), 4.30 (q, J = 7.1 Hz, 2H, OCH2), 3.14 (t, J = 7.3 Hz, 2H, PhCH2), 2.49 (s, 3H, CH3), 1.39 (t,
J = 7.1 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
δ 165.6 (C=Oester), 148.8 (HOAr-C), 143.6 (ArC), 138.3
(ArC), 129.3 (ArC), 129.1 (ArC), 127.2 (ArC), 126.9 (ArC), 125.0 (ArC), 124.0 (ArC), 123.6 (ArC), 123.1
(ArC), 123.0 (ArC), 122.7 (ArC), 120.7 (ArC), 104.2 (ArC), 102.3 (ArC), 59.4 (OCH2), 47.17 (NCH2), 35.7
(Ph
396.1564.
C
H2), 15.0 (CH3), 11.9 (CH3). HRMS (ESI) calcd for C24H23NO3Na [M+Na]+: 396.1570, found:

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3.2.3. General Procedure for the Syntheses of Compounds 5a–b
Ethyl 1-benzyl-5-(2-bromoethoxy)-2-methyl-1H-indole-3-carboxylate (5a). 1,2-Dibromoethane (32 g,
0.17 mol) was added to a stirred mixture of 4a (10 g, 32.3 mmol) and K2CO3 (28.2 g, 0.20 mol) in
ethanol (200 mL), and the reaction was refluxed for 15 h. The mixture was cooled to room temperature,
and filtered, washed with ethanol. The filtrate was evaporated under reduced pressure. The residue
mixture was added water, after stirring 10 min, the suspension solid was filtered, washed with water,
and dried to afford target product. The crude product was purified by column chromatography on
1
silica gel (EA:PE = 1:50) as a white solid (10.0 g, 71%). H-NMR (CDCl3)
δ
7.71 (d, J = 2.5 Hz, 1H, ArH),
7.29–7.08 (m, 4H, ArH), 6.96 (d, J = 6.9 Hz, 2H, ArH), 6.85 (dd, J = 8.8 Hz, 2.5 Hz, 1H, ArH), 5.33 (s,
2H, NCH2), 4.43–4.37 (m, 4H, 2
×
OCH2), 3.67 (t, J = 6.4 Hz, 2H, Br-CH2), 2.71 (s, 3H, CH3), 1.46 (t,
J = 7.1 Hz, 3H, CH3). ESI-MS m/z: 416.6 [M+H]+.
Ethyl 1-benzyl-5-(3-bromopropoxy)-2-methyl-1H-indole-3-carboxylate (5b). The title compound was
obtained as a white solid (12 g, 82%) by the same procedure described for compound 5a but using
1,3-dibromopropane instead of 1,2-dibromoethane.
3.2.4. General Procedure for Ammonolysis. Syntheses of 6a–e
To a solution of 5a (5 g, 12.0 mmol) or 5b (5.16 g, 12.0 mmol) in acetonitrile (20 mL), KI (4.0 g,
24.0 mmol) were added, and the reaction mixture was heated to reflux for 30 min. After cooling to
r.t., the amine (36 mmol) and anhydrous K2CO3 (6.6 g, 48.0 mmol) were added, and the mixture was
heated to reflux for the indicated time while monitoring the disappearance of starting material by TLC.
The cooled mixture was evaporated under reduced pressure, and water was added to the residue.
After stirring for 10 min, the solid suspension was filtered, washed with water, and dried to afford
target products 6a–e.
3.2.5. General Procedure for the Syntheses of O-Substituted 5-Hydroxylindoles 6f–h
5-Hydroxylindole 4a (0.8 g, 2.59 mmol) was added to a stirred suspension of NaH (60% dispersion
in mineral oil, 0.20 g, 517 mmol) in DMF (5 mL) under ice bath cooling. After 20 min, a haloalkane
(3.88 mmol) was added dropwise, and the mixture was allowed to react for 6 h. The mixture was
poured into water, and the resulting precipitate was filtered off, washed with water, and dried to afford
the intermediates 6f–h, which were used in the following reactions without any further purification.
3.2.6. General Hydrolysis Procedure: Syntheses of Compounds 7a–o
Carboxylate compound 4a–g or 6a–h (2.0 mmol) were suspended in a mixture of EtOH (10 mL)
and 60% NaOH aqueous (30 mmol), and refluxed for 3 h. The ethanol was removed under vacuum,
and the mixture was cooled and acidified. The solid was filtered and dried to afford intermediates
7a–o.
3.2.7. General Procedure for the Syntheses of the Target Compounds L–01~L–22
To a solution of indole derivatives (1 mmol), EDCI (0.29 g, 1.5 mmol), HOBt (0.20 g, 1.5 mmol) in
dry dichloromethane (10 mL) Et3N (0.76 g, 5 mmol) was added and the mixture was stirred at room
temperature for 30 min. Then, the corresponding pyridone derivatives (1 mmol) was added, and the
resulting mixture was reacted at room temperature for indicated time while monitoring by TLC. When
the reaction was complete the reaction mixture was poured into water, extracted with dichloromethane
(15
×
3 mL), and washed with water (3
×
15 mL) and brine (15 mL). The organic solution was dried
with anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The solid residue
was purified by column chromatography on silica gel.
1-Benzyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-5-(2-morpholinoethoxy)-1H-
indole-3-carboxamide (L–01). The title product was obtained from 7a and 3-(amino- methyl)-4,

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6-dimethylpyridin-2(1H)-one hydrochloride according to the general procedure described above as a
white solid (0.29 g, 55%). M.p.158–160 ◦C. 1H-NMR (DMSO-d6)
11.45 (s, 1H, NHpyridone), 7.65 (t,
δ
J = 5.1 Hz, 1H, NHCH2), 7.32–7.21 (m, 5H, ArH), 6.98 (d, J = 7.4 Hz, 2H, ArH), 6.74 (dd, J = 8.8 Hz,
2.3 Hz, 1H, ArH), 5.89 (s, 1H, ArHpyridone), 5.41 (s, 2H, NCH2benzyl), 4.33 (d, J = 5.4 Hz, 2H, NHCH2),
4.10 (t, J = 5.6 Hz, 2H, OCH2), 3.58 (t, J = 4.5 Hz, 4H, 2
3H, CH3), 2.49 (t, J = 10.0 Hz, 4H, 2
CH2), 2.26 (s, 3H, CH3), 2.12 (s, 3H, CH3). 13C-NMR (DMSO-d6)
165.1 ( =Oindole), 163.9 ( =Opyridone), 154.1 (Ar Hpyridone), 148.9 (Ar Hindole), 142.1 (Ar Hpyridone),
140.9 (N- =Cindole), 138.1 (Ar Hbenzyl), 131.4 (Ar Hbenzyl), 129.1 (Ar Hindole), 127.7 (Ar Hbenzyl),
126.6 (Ar Hindole), 126.5 (Ar Hpyridone), 123.0 (Ar Hbenzyl), 111.8 (Ar Hindole), 111.3 (N-C= indole),
108.7 (Ar Hpyridone), 108.1 (Ar Hindole), 103.1 (Ar Hindole), 66.7 ( H2O H2), 66.4 (NCH2CH2O), 57.7
(N H2CH2O), 54.2 ( H2N H2), 46.2 (N H2Ph), 35.6 (NH H2), 19.4 ( H3pyridone), 18.7 ( H3pyridone),
×
OCH2), 2.70 (t, J = 5.6 Hz, 2H, NCH2), 2.50 (s,
×
N
δ
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
11.9 (CH3indole). ESI-HRMS: m/z [M+Na]+ calcd for C31H36O4N4Na: 551.2629; found: 551.2621.
1-Benzyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-5-(3-(pyrrolidin-1-yl)propoxy)
-1H-indole-3-carboxamide (L–02). The title product was obtained from 7b and 3-(aminomethyl)-4,
6-dimethylpyridin-2(1H)-one hydrochloride according to the general procedure described above
◦
1
as a white solid (0.23 g, 34%). M.p.195-196 C. H-NMR (DMSO-d6)
δ 11.57 (brs, 1H, NHpyridone),
7.65 (t, J = 5.2 Hz, 1H, NHCH2), 7.32–7.18 (m, 5H, ArH), 6.98 (d, J = 7.4 Hz, 2H, ArH), 6.73–6.71 (dd,
J = 8.8 Hz, 2.2 Hz, 1H, ArH), 5.90 (s, 1H, ArHpyridone), 5.41 (s, 2H, NCH2benzyl), 4.33 (d, J = 5.3 Hz,
2H, NHCH2), 4.03 (t, J = 6.2 Hz, 2H, OCH2), 2.71 (brs, 2H, CH2), 2.63 (brs, 4H, 2
3H, CH3), 2.27 (s, 3H, CH3), 2.12 (s, 3H, CH3), 1.96–1.94 (m, 2H, CH2), 1.73 (brs, 4H, 2
13C-NMR (DMSO-d6)
165.1 ( =Oindole), 163.9 ( =Opyridone), 154.2 (Ar Hpyridone), 148.8 (Ar
143.1 (Ar Hpyridone), 140.8 (N- =Cindole), 138.1 (Ar Hbenzyl), 131.4 (Ar Hbenzyl), 129.1 (Ar
127.7 (Ar Hbenzyl), 126.6 (Ar Hindole), 126.3 (Ar Hpyridone), 123.0 (Ar Hbenzyl), 111.9 (Ar
111.3 (N-C=Cindole), 108.7 (Ar Hpyridone), 108.1 (Ar Hindole), 103.0 (Ar Hindole), 66.4 (O
(N H2CH2), 52.6 ( H2N H2tetrahydropyrrole), 46.2 (N
23.5 ( H2 H2tetrahydropyrrole), 19.4 ( H3pyridone), 18.7 (
×
CH2), 2.53 (s,
CH2).
Hindole),
×
δ
C
C
C
C
C
C
C
C
C
C
C
C
C
C
Hindole),
Hindole),
C
C
C
C
C
CH2), 53.9
C
C
C
C
C
H2Ph), 35.6 (NH
H3pyridone), 11.9 (
H2), 28.1 (CH2CH2CH2),
H3indole). ESI-HRMS: m/z
C
C
C
[M+H]+ calcd for C32H39O3N4: 527.3017; found: 527.3046.
1-Benzyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-5-(3-morpholinopropoxy)-1H-
indole-3-carboxamide (L–03). The title product was obtained from 7c and 3-(aminomethyl)-4,
6-dimethylpyridin-2(1H)-one hydrochloride according to the general procedure described above
as a yellow solid (0.30 g, 86%). M.p. 158-160 ◦C. 1H-NMR (DMSO-d6)
δ 11.53 (s, 1H, NHpyridone),
7.65 (t, J = 5.1 Hz, 1H, NHCH2), 7.31–7.21 (m, 5H, ArH), 6.98 (d, J = 7.5 Hz, 2H, ArH), 6.72 (dd,
J = 8.8 Hz, 2.3 Hz, 1H, ArH), 5.89 (s, 1H, ArHpyridone), 5.41 (s, 2H, NCH2benzyl), 4.33 (d, J = 5.3 Hz,
2H, NHCH2), 4.10 (t, J = 6.3 Hz, 2H, OCH2), 3.57 (t, J = 4.4 Hz, 4H, 2
2.40 (t, J = 7.2 Hz, 2H, CH2), 2.37 (s, 4H, 2 NCH2), 2.26 (s, 3H, CH3), 2.12 (s, 3H, CH3), 1.91–1.86
(m, 2H, CH2). 13C-NMR (DMSO-d6)
165.1 ( =Oindole), 163.9 ( =Opyridone), 154.3 (Ar Hpyridone),
148.9 (Ar Hindole), 143.0 (Ar Hpyridone), 140.8 (N- =Cindole), 138.2 (Ar Hbenzyl), 131.3 (Ar
Hbenzyl), 127.6 (Ar Hindole), 126.6 (Ar Hindole), 126.3 (Ar Hpyridone), 123.0 (Ar
Hindole), 111.3 (N-C=Cindole), 108.7 (Ar Hpyridone), 108.1 (Ar
H2O), 66.6 ( H2O H2), 55.5 ( H2N H2), 53.9 (N H2CH2CH2O), 46.2 (N
H2), 26.6 (NCH2 H2CH2O), 19.4 ( H3pyridone), 18.7 ( H3pyridone), 12.0 ( H3indole). ESI-HRMS:
m/z [M+Na]+ calcd for C32H38O4N4Na: 565.2785; found: 565.2797.
×
OCH2), 2.50 (s, 3H, CH3),
×
δ
C
C
C
C
C
C
C
C
C
C
C
C
C
C
Hbenzyl),
Hbenzyl),
129.1 (Ar
111.8 (Ar
C
C
Hindole), 103.0 (ArCHindole),
66.8 (CH2
(NH
C
C
C
C
C
C
C
C
CH2Ph), 35.6
C
C
C
1-Benzyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-5-(2-(piperidin-1-yl)ethoxy-
1H-indole-3-carboxamide (L–04). The title product was obtained from 7d and 3-(aminomethyl)-4,
6-dimethylpyridin-2(1H)-one hydrochloride according to the general procedure described above
◦
1
as a white solid (0.26 g, 50%). M.p.215–217 C. H-NMR (DMSO-d6)
δ 11.54 (s, 1H, NHpyridone),
7.65 (t, J = 4.9 Hz, 1H, NHCH2), 7.32–7.21 (m, 5H, ArH), 6.98 (d, J = 7.5 Hz, 2H, ArH), 6.73 (m,
1H, ArH), 5.89 (s, 1H, ArHpyridone), 5.41 (s, 2H, NCH2benzyl), 4.33 (d, J = 5.1Hz, 2H, NHCH2), 4.06

Molecules 2020, 25, 2059
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(t, J = 5.6 Hz, 2H, OCH2), 2.66 (t, J = 5.6 Hz, 2H, NCH2), 2.53 (s, 3H, CH3), 2.43 (s, 4H, 2
×
CH2),
2.26 (s, 3H, CH3), 2.12 (s, 3H, CH3), 1.49 (t, J = 5.3 Hz, 4H, 2
(DMSO-d6) 165.1 ( =Oindole), 163.9 ( =Opyridone), 154.2 (Ar
Hpyridone), 140.9 (N- =Cindole), 138.1 (Ar Hbenzyl), 131.3 (Ar
Hindole), 126.6 (Ar Hindole), 126.3 (Ar Hpyridone), 123.0 (Ar
Hpyridone), 108.1 (Ar Hindole), 103.1 (Ar
H2Ph), 46.2 (N H2CH2O), 35.6 (NH H2), 26.1 (
H2CH2piperidine), 19.4 ( H3pyridone), 18.7 ( H3pyridone), 11.9 (
H3indole). ESI-HRMS: m/z [M+H]+
calcd for C32H38O4N4Na: 527.3017; found: 527.3018.
×
CH2), 1.37 (m, 2H, CH2). 13C-NMR
δ
C
C
C
Hpyridone), 148.7 (Ar
Hbenzyl), 129.1 (Ar
Hbenzyl), 111.7 (Ar
CHindole), 143.1
(Ar
(Ar
C
C
C
C
C
C
C
Hbenzyl), 127.6
Hindole), 111.3
C
C
C
(N-C=Cindole), 108.7 (Ar
H2N H2), 54.9 (N
(CH2
C
C
C
Hindole), 66.6 (NCH2
CH2O), 58.1
(C
C
C
C
C
C
H2CH2 H2piperidine), 24.5
C
C
C
C
C
1-Benzyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-5-(3-(piperidin-1-yl)propoxy)-
1H-indole-3-carboxamide (L–05). The title product was obtained from 7e and 3-(aminomethyl)-4,
6-dimethylpyridin-2(1H)-one hydrochloride according to the general procedure described above as
a white solid (0.20 g, 41%). M.p. 210–212 ◦C. 1H-NMR (DMSO-d6)
δ 11.53 (s, 1H, NHpyridone), 7.68
(s, 1H, NHCH2), 7.31–7.21 (m, 5H, ArH), 6.98 (d, J = 7.5 Hz, 2H, ArH), 6.72 (dd, J = 8.8 Hz, 1.9 Hz,
1H, ArH), 5.89 (s, 1H, ArHpyridone), 5.41 (s, 2H, NCH2benzyl), 4.33 (d, J = 5.2 Hz, 2H, NHCH2), 3.99 (t,
J = 6.2 Hz, 2H, OCH2), 2.53 (s, 3H, CH3), 2.38 (t, J = 7.1 Hz, 2H, NCH2), 2.33 (s, 4H, 2
3H, CH3), 2.12 (s, 3H, CH3), 1.89–1.84 (m, 2H, CH2), 1.49 (t, J = 5.3 Hz, 4H, 2 CH2), 1.37 (s, 2H, CH2).
13C-NMR (DMSO-d6)
165.1 ( =Oindole), 163.9 ( =Opyridone), 154.3 (Ar Hpyridone), 148.8 (Ar
143.1 (Ar Hpyridone), 140.8 (N- =Cindole), 138.1 (Ar Hbenzyl), 131.3 (Ar Hbenzyl), 129.1 (Ar
127.6 (Ar Hindole), 126.6 (Ar Hindole), 126.3 (Ar Hpyridone), 123.0 (Ar Hbenzyl), 111.8 (Ar
111.3 (N-C=Cindole), 108.7 (Ar Hpyridone), 108.1 (Ar Hindole), 103.0 (Ar Hindole), 66.8 (
55.8 (N H2CH2CH2O), 54.6 ( H2N H2), 46.2 (N H2Ph), 35.6 (NH H2), 27.0 (NCH2 H2CH2O),
26.1 ( H3pyridone), 18.7 ( H3pyridone), 11.9
×
NCH2), 2.26 (s,
×
δ
C
C
C
CHindole),
C
C
C
C
CHbenzyl),
C
C
C
C
CHindole),
C
C
C
CH2O),
C
C
C
C
C
C
C
H2CH2CH2piperidine), 24.6 (CH2CH2CH2piperidine), 19.4 (
C
C
(CH3indole). ESI-HRMS: m/z [M+H]+ calcd for C33H41O3N4: 541.3173; found: 541.3179.
5-(Allyloxy)-1-benzyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-
3-carboxamide (L–06). The title product was obtained from 7f and 3-(amino- methyl)-4,6-dimethylpyridin
-2(1H)-one hydrochloride according to the general procedure described above as a white solid (0.35 g,
◦
1
78%). M.p. 245–47 C. H-NMR (DMSO-d6)
δ 11.58 (brs, 1H, NHpyridone), 7.69 (m, 1H, NHCH2),
7.33–7.21 (m, 5H, ArH), 6.98 (d, J = 7.4 Hz, 2H, ArH), 6.77–6.75 (dd, J = 8.9 Hz, 2.3 Hz, 1H, ArH),
6.10–6.04 (m, 1H, ArH), 5.89 (s, 1H, ArHpyridone), 5.41 (s, 2H, NCH2benzyl), 5.39 (d, J = 1.6 Hz, 1H,
ArH), 5.24–5.23 (m, ArH), 4.58 (d, J = 5.1 Hz, 2H, OCH2), 4.33 (d, J = 5.3 Hz, 2H, NHCH2), 2.54
(s, 3H, CH3), 2.26 (s, 3H, CH3), 2.12 (s, 3H, CH3). 13C-NMR (DMSO-d6)
δ
165.0 (
Hpyridone), 141.1 (N-
Hbenzyl), 127.7 (Ar Hindole),
H2=CH), 111.8 (ArCHindole),
C
=Oindole), 163.9
(
C
=Opyridone), 153.9 (Ar
138.1 (Ar Hbenzyl), 134.8 (CH2=
126.6 (Ar Hindole), 126.2 (Ar Hpyridone), 122.9 (Ar
111.3 (N-C=Cindole), 108.6 (Ar Hpyridone), 108.1 (Ar
(N H2Ph), 35.6 (NH H2), 19.4 ( H3pyridone), 18.7 (
[M+Na]+ calcd for C28H29O3N3Na: 478.2101; found: 478.2097.
C
Hpyridone), 148.8 (Ar
C
Hindole), 143.1 (Ar
C
C
=Cindole),
C
C
C
H), 131.4 (Ar
CHbenzyl), 129.1 (Ar
C
C
C
C
C
C
Hbenzyl), 117.5 (
CHindole), 103.4 (Ar
CHindole), 69.1 (
CH2O), 46.2
C
C
C
CH3pyridone), 11.9 (
C
H3indole). ESI-HRMS: m/z
1-Benzyl-5-(cyclopropylmethoxy)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1H
-indole-3-carboxamide (L–07). The titled product was obtained from 7g and 3-(aminomethyl)-4,
6-dimethylpyridin-2(1H)-one hydrochloride according to the general procedure described above as a
white solid (0.30 g, 64%). M.p.136–138 ◦C. 1H-NMR (DMSO-d6)
δ 11.53 (s, 1H, NHpyridone), 7.63 (t,
J = 5.2 Hz, 1H, NHCH2), 7.32–7.21 (m, 5H, ArH), 6.98 (d, J = 7.4 Hz, 2H, ArH), 6.74–6.73 (dd, J = 8.8 Hz,
2.3 Hz, 1H, ArH), 5.89 (s, 1H, ArHpyridone), 5.41 (s, 2H, NCH2benzyl), 4.33 (d, J = 8.6 Hz, 2H, NHCH2),
3.81 (d, J = 6.9 Hz, 2H, OCH2), 2.53 (s, 3H, CH3), 2.26 (s, 3H, CH3), 2.12 (s, 3H, CH3), 1.24–1.19
(m, 1H, CH), 0.58–0.55 (m, 2H, CH2), 0.33 (q, J = 5.2 Hz, 2H, CH2). 13C-NMR (DMSO-d6)
δ
165.1
Hpyridone), 140.9
Hbenzyl), 127.6 (Ar Hindole), 126.6
(C
=Oindole), 163.9 (
C=Opyridone), 154.3 (Ar
C
Hpyridone), 148.9 (Ar
CHindole), 143.1 (ArC
(N- =Cindole), 138.1 (Ar
C
C
Hbenzyl), 131.3 (Ar
C
Hbenzyl), 129.1 (Ar
C
C

Molecules 2020, 25, 2059
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(Ar
(Ar
19.4 (
C
C
Hindole), 126.3 (Ar
Hpyridone), 108.1 (Ar
H3pyridone), 18.7 (
[M+Na]+ calcd for C29H31O3N3Na: 492.2258; found: 492.2266.
C
Hpyridone), 122.9 (Ar
Hindole), 103.8 (Ar
H3pyridone), 11.9 (
C
C
C
Hbenzyl), 111.8 (Ar
Hindole), 72.9 (
H3indole), 10.9 (
C
Hindole), 111.3 (N-C=Cindole), 108.6
C
C
C
C
H2O), 46.2 (N H2Ph), 35.6 (NH
H), 3.6 ( H2CH2). ESI-HRMS: m/z
C
CH2),
C
C
1-Benzyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethoxy-2-methyl-1H-indole-3-
carboxamide (L–08). The title product was obtained from 7h and 3-(amino-methyl)-4,6-dimethylpyridin
-2(1H)-one hydrochloride according to the general procedure described above as a pink solid (0.30 g,
43%). M.p. 263–264 ◦C. 1H-NMR (DMSO-d6)
δ 11.54 (s, 1H, NHpyridone), 7.65 (s, 1H, NHCH2), 7.32–7.21
(m, 5H, ArH), 6.98 (d, J = 7.6 Hz, 2H, ArH), 6.73 (d, J = 8.8 Hz, 1H, ArH), 5.89 (s, 1H, ArHpyridone),
5.41 (s, 2H, NCH2benzyl), 4.33 (d, J = 5.3 Hz, 2H, NHCH2), 4.03 (q, J = 6.8 Hz, 2H, CH2), 2.54 (s, 3H,
CH3), 2.26 (s, 3H, CH3), 2.12 (s, 3H, CH3), 1.34 (t, J = 6.9 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
=Oindole), 164.0 ( =Opyridone), 154.2 (Ar Hpyridone), 148.7 (Ar Hindole), 143.2 (Ar Hpyridone), 141.0
=Cindole), 138.1 (Ar Hbenzyl), 131.3 (Ar Hbenzyl), 127.6 (Ar Hindole), 126.6
Hindole), 126.2 (Ar Hpyridone), 122.9 (Ar Hindole), 111.3 (N-C=Cindole), 108.6
Hpyridone), 108.1 (Ar H2O), 46.2 (N H2Ph), 35.7 (NH H2),
H3pyridone), 18.7 ( H3pyridone), 15.4 (CH2CH3), 11.9 (
H3indole). ESI-HRMS: m/z [M+Na]+ calcd
for C27H29O3N3Na: 466.2101; found: 466.2107.
δ 165.1
(C
C
C
C
C
(N-
(Ar
(Ar
C
C
C
C
C
C
Hbenzyl), 129.1 (Ar
Hbenzyl), 111.8 (Ar
Hindole), 63.8 (
C
C
C
C
C
Hindole), 102.8 (Ar
C
C
C
C
19.4 (C
C
C
1-Benzyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-hydroxy-2-methyl-1H-indole-3-
carboxamide (L–09). The title product was obtained from 7i and 3-(aminomethyl)-4,6-dimethylpyridin
-2(1H)-one hydrochloride according to the general procedure described above as a white solid (0.40,
◦
1
61%). M.p. 288–290 C. H-NMR (DMSO-d6)
δ 11.55 (s, 1H, NHpyridone), 8.85 (s, 1H, OH), 7.44 (t,
J = 5.3 Hz, 1H, NHCH2), 7.29–7.20 (m, 4H, ArH), 7.13 (d, J = 2.0 Hz, 1H, ArH), 6.99 (d, J = 7.6 Hz, 2H,
ArH), 6.61–6.59 (dd, J = 8.6 Hz, 2.0 Hz, 1H, ArH), 5.89 (s, 1H, ArHpyridone), 5.37 (s, 2H, NCH2benzyl),
4.33 (d, J = 5.4 Hz, 2H, NHCH2), 2.52 (s, 3H, CH3), 2.26 (s, 3H, CH3), 2.12 (s, 3H, CH3). 13C-NMR
(DMSO-d6)
(Ar Hpyridone), 140.7 (N-
127.6 (Ar Hindole), 126.6 (Ar
108.1 (Ar Hpyridone), 108.0 (Ar
H3pyridone), 18.7 (
δ
165.4 (
C
=Oindole), 163.9 (
=Cindole), 138.2 (Ar
Hindole), 122.9 (Ar
C
=Opyridone), 152.6 (Ar
Hbenzyl), 130.7 (Ar
Hbenzyl), 111.6 (Ar
Hindole), 104.4 (Ar Hindole), 46.1 (N
H3indole). ESI-HRMS: m/z [M+Na]+ calcd for C25H25O3N3Na:
C
Hpyridone), 148.9 (Ar
Hbenzyl), 129.1 (Ar
Hindole), 111.0 (N-C=Cindole),
H2Ph), 35.6 (NH H2), 19.4
C
Hindole), 143.1
C
C
C
C
C
C
C
CHbenzyl),
C
C
C
C
C
C
(C
C
H3pyridone), 12.0 (C
438.1788; found: 438.1798.
N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-hydroxy-2-methyl-1-phenethyl-1H-indole-3
-carboxamide (L–10 The title product was obtained from 7j and 3-(amino- methyl)-4,6-dimethylpyridin
-2(1H)-one hydrochloride according to the general procedure described above as a white solid (0.25 g,
58%). M.p.242–245 ◦C. 1H-NMR (DMSO-d6)
11.54 (brs, 1H, NHpyridone), 8.84 (brs, 1H, OH), 7.32 (t,
).
δ
J = 5.0 Hz, 1H, NHCH2), 7.28–7.19 (m, 4H, ArH), 7.16 (d, J = 7.1 Hz, 2H, ArH), 7.10 (d, J = 1.7 Hz,
1H, ArH), 6.65–6.63 (dd, J = 8.6 Hz, 1.9 Hz, 1H, ArH), 5.89 (s, 1H, ArHpyridone), 4.30 (d, J = 5.3 Hz,
2H, NHCH2), 4.26 (t, J = 7.3 Hz, 2H, NCH2), 2.91 (t, J = 7.3 Hz, 2H, PhCH2), 2.37 (s, 3H, CH3), 2.25
(s, 3H, CH3), 2.12 (s, 3H, CH3). 13C-NMR (DMSO-d6)
δ
165.4 (
Hpyridone), 140.8 (N-
Hindole), 126.9 (Ar Hindole), 126.5 (Ar
Hindole), 110.8 (N-C=Cindole), 108.1 (Ar Hpyridone), 107.4 (Ar Hindole), 104.3 (Ar
H2), 19.4 ( H3pyridone), 18.6 ( H3pyridone), 11.6
C
=Oindole), 163.9 (
=Cindole), 138.9 (Ph
Hpyridone), 123.0 (Ph
Hindole),
C
=Opyridone), 152.4
H), 130.1
H),
(ArC
Hpyridone), 148.8 (Ar
C
Hindole), 143.1 (Ar
C
C
C
(PhC
H), 129.4 (Ph H), 128.8 (Ar
C
C
C
C
C
111.4 (Ar
C
C
C
C
44.6 (N
C
H2CH2Ph), 35.8 (NCH2
C
H2Ph), 35.6 (NH
C
C
C
(CH3indole). ESI-HRMS: m/z [M+Na]+ calcd for C26H27O3N3Na: 452.1945; found: 452.1980.
N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-hydroxy-2-methyl-1-phenethyl-1H-benzo-
[g]indole-3-carboxamide (L–11). The title product was obtained from 7k and 3-(amino- methyl)-4,
6-dimethylpyridin-2(1H)-one hydrochloride according to the general procedure described above
◦
as a white solid (0.35 g, 64%). M.p. 240 C (decomposes). 1H-NMR (DMSO-d6)
δ 11.36 (brs, 1H,

Molecules 2020, 25, 2059
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NHpyridone), 8.42 (d, J=8.6 Hz, 1H, ArH), 8.29 (d, J=8.3 Hz, 1H, ArH), 7.62 (t, J=7.5 Hz, 1H, NHCH2),
7.44-7.41 (m, 2H, ArH), 7.37 (s, 1H, ArH), 7.34-7.29 (m, 2H, ArH), 7.27-7.25 (m, 1H, ArH), 7.21 (d,
J=7.4 Hz, 2H, ArH), 5.89 (s, 1H, ArHpyridone), 4.70 (t, J=7.4 Hz, 2H, NCH2), 4.34 (d, J=5.0 Hz, 2H,
NHCH2), 3.08 (t, J=7.3 Hz, 2H, PhCH2), 2.37 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.12 (s, 3H, CH3).
13C-NMR (DMSO-d6)
143.1 (Ar Hpyridone), 138.5 (N-
δ
165.6 (
C
=Oindole), 163.9 (
C
C
=Opyridone), 149.2 (Ar
=Cindole), 137.6 (Ph H), 129.3 (Ph
H), 124.0 (Ar H), 123.5 (Ar
H), 110.3 (N-C=Cindole), 108.1 (Ar
H2CH2Ph), 36.0 (NCH2 H2Ph), 35.6 (NH H2), 19.4 ( H3pyridone), 18.7
H3indole). ESI-HRMS: m/z [M+Na]+ calcd for C30H29O3N3Na: 502.2101; found:
C
C
Hpyridone), 148.0 (Ar
H), 129.1 (Ar Hindole), 127.2
H), 122.9 (Ar H), 122.8
Hpyridone),
CHindole),
C
C
C
(Ar
C
Hindole), 126.7 (Ar
H), 122.7 (Ar
Hindole), 47.1 (N
H3pyridone), 11.7 (
502.2124.
C
Hpyridone), 124.0 (Ar
C
C
C
C
(ArC
C
H), 122.6 (Ar
C
H), 120.6 (Ar
C
C
101.2 (Ar
C
C
C
C
C
(C
C
N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-hydroxy-2-methyl-1-phenyl-1H-indole-3-
carboxamide (L–12). The title product was obtained from 7l and 3-(amino- methyl)-4,6-dimethylpyridin
-2(1H)-one hydrochloride according to the general procedure described above as a white solid (0.15 g,
38%). M.p. 285–287 ◦C. 1H-NMR (DMSO-d6)
δ 11.56 (brs, 1H, NHpyridone), 8.94 (s, 1H, OH), 7.62–7.59
(m, 2H, ArH), 7.54–7.50 (m, 2H, ArH), 7.40 (d, J = 7.3 Hz, 2H, ArH), 7.18 (d, J = 2.1 Hz, 1H), 6.78 (d,
J = 8.7 Hz, 1H, ArH), 6.61–6.59 (dd, J = 8.7 Hz, 2.2 Hz, 1H, ArH), 5.90 (s, 1H, ArHpyridone), 4.35 (d,
J = 5.4 Hz, 2H, NHCH2), 2.38 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.12 (s, 3H, CH3). 13C-NMR (DMSO-d6)
δ
165.3 (
140.3 (N-
126.7 (Ar
108.1 (Ar
C
C
=Oindole), 163.9 (
=Cindole), 137.0 (Ph
Hpyridone), 122.9 (Ph
Hpyridone), 104.4 (Ar
C
=Opyridone), 152.9 (Ar
H), 131.7 (Ar Hindole), 130.2 (Ph
H), 122.1 (Ar Hindole), 111.0 (N-C=Cindole), 109.1 (Ar
Hindole), 35.6 (NH
C
Hpyridone), 149.0 (Ar
C
Hindole), 143.1 (Ar
Hindole), 128.4 (Ph
Hindole),
CH2), 19.4 (CH3pyridone), 18.7 (CH3pyridone), 13.0
C
Hpyridone),
C
C
C
H), 128.8 (Ar
C
CH),
C
C
C
C
C
C
(CH3indole). ESI-HRMS: m/z [M+Na]+ calcd for C24H23O3N3Na: 424.1632; found: 424.1662.
N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(furan-2-ylmethyl)-5-hydroxy-2-methyl-1H-
indole-3-carboxamide (L–13). The title product was obtained from 7m and 3-(amino- methyl)-4,
6-dimethylpyridin-2(1H)-one hydrochloride according to the general procedure described above as
a white solid (0.30 g, 75%). M.p. 290–292 ◦C. 1H-NMR (DMSO-d6)
δ 11.54 (s, 1H, NHpyridone), 8.85
(s, 1H, OH), 7.53 (s, 1H, NHCH2), 7.39 (dd, J = 13.6 Hz, 5.2 Hz, 2H, ArH), 7.08 (d, J = 2.0 Hz, 1H,
ArH), 6.64-6.63 (dd, J = 8.6 Hz, 2.0 Hz, 1H), 6.40–6.36 (m, 2H, ArH), 5.88 (s, 1H, ArHpyridone), 5.32 (s,
2H, NCH2), 4.31 (d, J = 5.3 Hz, 2H, NHCH2), 2.64 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.11 (s, 3H, CH3).
13C-NMR (DMSO-d6)
148.9 (Ar Hindole), 143.3 (Ar
126.5 (Ar Hindole), 122.9 (Ar
(Ar Hpyridone), 108.0 (Ar
18.6 ( H3pyridone), 11.9 (
found: 428.1587.
δ
165.3 (
C
=Oindole), 163.9 (
Hpyridone), 143.1 (Ar
Hfuran), 111.5 (Ar Hindole), 111.0 (N-C=
Hindole), 104.3 (Ar Hindole), 46.2 (N H2), 35.5 (NH
H3indole). ESI-HRMS: m/z [M+Na]+ calcd for C23H23O4N3Na: 428.1581;
C
=Opyridone), 152.5 (Ar
C
C
Hpyridone), 151.0 (Ar
C
Hfuran),
C
C
C
C
CHfuran), 140.5 (N-
=Cindole), 130.4 (ArCHfuran),
C
C
indole), 108.6 (Ar
C
Hindole), 108.1
H3pyridone),
C
C
C
C
C
C
H2), 19.4 (C
C
1-Cyclohexyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-hydroxy-2-methyl-1H-indole-3-
carboxamide (L–14). The title product was obtained from 7n and 3-(amino- methyl)-4,6-dimethylpyridin
-2(1H)-one hydrochloride according to the general procedure described above as a white solid (0.36
g, 90%). M.p. 268–270 ◦C. 1H-NMR (DMSO-d6)
δ 11.53 (s, 1H, NHpyridone), 8.80 (s, 1H, OH), 7.44 (t,
J = 5.3 Hz, 1H, NHCH2), 7.32 (t, J = 5.3 Hz, 1H, ArH), 7.05 (d, J = 1.9 Hz, 1H, ArH), 6.60–6.58 (dd,
J = 2.0 Hz, 1H, ArH), 5.88 (s, 1H, ArHpyridone), 4.31 (d, J = 5.3 Hz, 2H, NHCH2), 4.20 (brs, 1H, NCH),
2.57 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.11 (s, 3H, CH3), 2.19–2.17 (m, 2H, CH2), 1.86 (d, J = 13.0 Hz, 2H,
CH2), 1.74–1.67 (m, 3H, CH2+1/2CH2), 1.48–1.30 (m, 3H, CH2+1/2CH2). 13C-NMR (DMSO-d6)
δ
165.7
Hpyridone), 140.1
Hindole), 113.1 (Ar Hindole), 111.0
H), 35.5 (NH H2),
(
C
=Oindole), 163.9 (
(N- =Cindole), 129.0 (Ar
(N-C= indole), 108.1 (Ar
C
=Opyridone), 151.8 (Ar
Hindole), 127.5 (Ar
Hpyridone), 107.9 (Ar
C
Hpyridone), 148.9 (Ar
Hpyridone), 122.9 (Ar
Hindole), 104.2 (Ar
CHindole), 143.0 (ArC
C
C
C
C
C
C
C
C
C
Hindole), 55.2 (N
C
C

Molecules 2020, 25, 2059
18 of 24
31.0 (
C
H2cyclohexyl), 26.2 (CH2cyclohexyl), 25.3 (CH2cyclohexyl), 19.4 (
C
H3pyridone), 18.6 (
C
H3pyridone), 12.5
(CH3indole). ESI-HRMS: m/z [M+Na]+ calcd for C24H29O3N3Na: 430.2101; found: 430.2116.
N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-hydroxy-1-isopropyl-2-methyl-1H-indole-3-
carboxamide (L–15). The titled product was obtained from 7o and 3-(amino- methyl)-4,6-dimethylpyridin
-2(1H)-one hydrochloride according to the general procedure described above as a white solid
(0.24 g, 65%). M.p. 234–236 ◦C. 1H-NMR (DMSO-d6)
δ 11.53 (s, 1H, NHpyridone), 8.80 (s, 1H, OH),
7.39 (d, J = 8.8 Hz, 1H, ArH), 7.32 (t, J = 5.2 Hz, 1H, NHCH2), 7.06 (d, J = 2.1 Hz, 1H, ArH),
6.61–6.59 (dd, J = 8.8 Hz, 2.2 Hz, 1H, ArH), 5.88 (s, 1H, ArHpyridone), 4.71–4.66 (m, 1H, NCH), 4.31 (d,
J = 5.3 Hz, 2H, NHCH2), 2.56 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.11 (s, 3H, CH3), 1.50 (d, J = 7.0 Hz,
6H, 2
148.9 (Ar
122.9 (Ar
104.3 (Ar
×
CH3). 13C-NMR (DMSO-d6)
Hindole), 143.0 (Ar Hpyridone), 139.9 (N-
Hindole), 112.5 (Ar Hindole), 111.1 (N-C=Cindole), 108.1 (Ar
Hindole), 46.8 (N
δ
165.6 (
C
=Oindole), 163.8 (
C
=Opyridone), 151.9 (Ar
C
Hpyridone),
Hindole), 127.5 (Ar Hindole),
Hpyridone),
C
C
C
C
C
C
=Cindole), 128.7 (Ar
C
C
CHpyridone), 107.7 (ArC
C
H), 35.5 (NH
CH2), 21.5 (CH3), 19.4 (
C
H3pyridone), 18.6 (
CH3pyridone), 12.4
(CH3indole). ESI-HRMS: m/z [M+Na]+ calcd for C21H25O3N3Na: 390.1788; found: 390.1821.
N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-2-methyl-5-(3-(piperidin-1-yl)-
propoxy)-1H-indole-3-carboxamide (L–16). The title product was obtained from 7e and
3 according to the
general procedure described above as a white solid (80 mg, 40%). M.p. 200–202 ◦C. 1H-NMR (CDCl3)
δ
12.06 (brs, 1H, NHpyridone), 7.46 (t, J = 5.7 Hz, 1H, NHCH2), 7.34–7.32 (m, 2H, ArH), 6.73 (dd, J = 7.1 Hz,
2.2 Hz, 1H, ArH), 5.87 (s, 1H, ArHpyridone), 4.70–4.65 (m, 1H, NCH), 4.58 (d, J = 6.0 Hz, 2H, NHCH2),
4.12 (t, J = 6.6 Hz, 2H, OCH2), 2.72 (brs, 2H, CH2), 2.71 (s, 3H, CH3), 2.60 (brs, 4H, CH2
(s, 3H), 2.23 (s, 3H, CH3), 2.08–2.06 (m, 2H, CH2), 1.71–1.70 (m, 4H, 2 CH2), 1.58 (d, J = 7.0 Hz,
6H, 2 165.9 ( =Oindole), 165.0 ( =Opyridone), 153.8
CH3), 1.48 (brs, 2H, CH2). 13C-NMR (CDCl3)
(Ar Hpyridone), 149.3 (Ar Hindole), 142.3 (Ar Hpyridone), 141.0 (N- =Cindole), 129.3 (Ar Hindole),
126.7 (Ar Hindole), 123.8 (Ar Hindole), 112.3 (Ar Hindole), 111.6 (N-C=Cindole), 109.5 (Ar Hpyridone),
108.0 (Ar Hpyridone), 102.2 (Ar Hindole), 66.9 ( H2O), 55.8 (N H2CH2CH2O), 54.2 ( H2N H2), 46.9
(N H), 35.6 (NH H2), 26.1 (NCH2 H2CH2O), 24.8 (CH2piperidine), 23.7 (CH2piperidine), 21.4 (CH3),
19.6 ( H3pyridone), 19.0 ( H3pyridone), 12.1 (
H3indole). ESI-HRMS: m/z [M+H]+ calcd for C29H41O3N4:
493.3173; found: 493.3174.
NCH2), 2.41
×
×
δ
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
1-Isopropyl-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-5-(3-(piperidin-1-yl)
propoxy)-1H-indole-3-carboxamide (L–17). The title product was obtained from 7e and pyridone 25
according to the general procedure described above as a white solid (100 mg, 48%). M.p. 181–188 ◦C.
1H-NMR (CDCl3)
δ 7.66 (t, J = 5.0 Hz, 1H, NHCH2), 7.42 (d, J = 1.9 Hz, 1H, ArH), 7.35 (d, J = 8.9 Hz,
1H, ArH), 6.74–6.72 (dd, J = 8.9 Hz, 2.2 Hz, 1H, ArH), 5.90 (s, 1H, ArHpyridone), 4.71–4.67 (m, 1H,
NCH), 4.63 (d, J = 5.5 Hz, 2H, OCH2), 4.15 (t, J = 6.1 Hz, 2H, NCH2), 3.88 (s, 3H, OCH3), 2.90
(brs, 2H, CH2), 2.74–2.62 (m, 6H, CH2), 2.32 (s, 3H, CH3), 2.14 (t, J = 6.8 Hz, 2H, CH2), 1.77 (brs,
4H, 2
×
CH2), 1.59 (d, J = 7.0 Hz, 6H, 2
×
CH3), 1.50 (brs, 2H, CH2). 13C-NMR (CDCl3)
OCH3pyridone), 165.4 ( =Opyridone), 153.5 (Ar Hpyridone), 146.0 (Ar
Hindole), 126.7 (Ar Hindole), 112.3 (Ar
Hindole), 103.0 (Ar Hindole), 94.5 (Ar
H2CH2CH2O), 53.9 ( H2N H2), 46.9 (N
24.1 (CH2piperidine), 23.2 (CH2piperidine), 21.4 (CH3), 19.5 ( H3pyridone), 12.2 (
[M+H]+ calcd for C29H41O4N4: 509.3122; found: 509.3127.
δ
165.8
Hindole),
Hindole), 111.4 (N-C=Cindole),
Hpyridone), 66.8 ( H2O), 56.1
H2), 25.4 (NCH2 H2CH2O),
H3indole). ESI-HRMS: m/z
(C
=Oindole), 165.7 (Ar
C
C
C
C
C
C
141.5 (N-
108.2 (Ar
C=Cindole), 129.5 (Ar
C
C
C
C
Hpyridone), 107.5 (Ar
C
C
C
(OCH3), 55.6 (N
C
C
C
C
H), 32.7 (NH
C
C
C
1-Isopropyl-2-methyl-N-((2-oxo-2,5,6,7-tetrahydro-1H-cyclopenta[b]pyridin-3-yl)methyl)-5-(3-(piperidin-1
-yl)propoxy)-1H-indole-3-carboxamide (L–18). The title product was obtained from 7e and pyridone 17
according to the general procedure described above as a white solid (65 mg, 31%). M.p. 141–144 ◦C.
1H-NMR (CDCl3)
δ
7.45 (s, 1H, OH), 7.43 (t, J = 5.5 Hz, 1H, NHCH2), 7.35–7.33 (m, 2H, ArH), 6.73–6.71
(dd, J = 8.9 Hz, 1.9 Hz, 1H, ArH), 4.70–4.66 (m, 1H, NCH), 4.50 (d, J = 5.9 Hz, 2H, OCH2), 4.20 (t,

Molecules 2020, 25, 2059
19 of 24
J = 6.7 Hz, 2H, NCH2), 2.95 (brs, 2H, CH2), 2.85 (t, J = 7.2 Hz, 2H, CH2), 2.82 (brs, 2H, CH2), 2.71
(s, 3H, CH3), 2.68 (t, J = 7.3 Hz, 2H, CH2), 2.22 (t, J = 6.4 Hz, 2H, CH2), 2.10–2.05 (m, 2H, CH2),
1.82 (brs, 4H, 2
166.0 ( =Oindole), 164.5 (
137.2 (N- =Cindole), 129.3 (Ar
112.4 (N-C= indole), 111.8 (Ar
(N H2CH2CH2O), 54.0 ( H2N
25.6 (NCH2
×
CH2), 1.58 (d, J = 7.0 Hz, 6H, 2
=Opyridone), 153.7 (Ar Hpyridone), 147.9 (Ar
Hindole), 126.6 (Ar Hindole), 126.3 (Ar
Hpyridone), 108.1 (Ar Hpyridone), 102.2 (Ar
H2), 46.9 (N H), 40.5 (NH H2), 31.5 ( H2pyridone), 29.6 (
H2CH2O), 24.2 (CH2piperidine), 23.3 (CH2piperidine), 23.0 (CH2pyridone), 21.4 (CH3), 12.0
×
CH3), 1.55 (brs, 2H, CH2). 13C-NMR (CDCl3)
Hindole), 141.3 (Ar Hpyridone),
Hindole), 119.0 (Ar Hindole),
Hindole), 66.9 ( H2O), 55.6
H2pyridone),
δ
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
(CH3indole). ESI-HRMS: m/z [M+H]+ calcd for C30H41O3N4: 505.3173; found: 505.3188.
N-((6-Ethyl-4-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-2-methyl-5-(3-(piperidin-1-yl)
propoxy)-1H-indole-3-carboxamide (L–19). The title product was obtained from 7e and pyridone 22
according to the general procedure described above as a yellow solid (75 mg, 36%). M.p. 180–182 oC.
1H-NMR (CDCl3)
δ
7.42 (t, J = 5.5 Hz, 1H, NHCH2), 7.34–7.33 (m, 2H, ArH), 6.74–6.72 (dd, J = 8.9 Hz,
1.9 Hz, 1H, ArH), 5.90 (s, 1H, ArHpyridone), 4.71–4.64 (m, 1H, NCH), 4.59 (d, J = 6.1 Hz, 2H, OCH2),
4.10 (t, J = 6.2 Hz, 2H, NCH2), 2.71 (s, 3H, CH3), 2.69 (brs, 2H, CH2), 2.55 (brs, 4H, 2 CH2), 2.52 (q,
J = 7.6 Hz, 2H, CH2), 2.43 (s, 3H), 2.03 (brs, 2H, CH2), 1.67 (brs, 4H, 2 CH2), 1.58 (s, 6H, 2 CH3), 1.45
(brs, 2H, CH2), 1.18 (t, J = 7.5 Hz, 3H, CH3). 13C-NMR (CDCl3)
165.9 ( =Oindole), 165.1 ( =Opyridone),
153.9 (Ar Hpyridone), 149.5 (Ar Hindole), 148.1 (Ar Hpyridone), 141.0 (N- =Cindole), 129.3 (Ar Hindole),
126.7 (Ar Hindole), 123.7 (Ar Hindole), 112.3 (Ar Hindole), 111.5 (N-C=Cindole), 108.0 (Ar Hpyridone),
107.8 (Ar Hpyridone), 102.4 (Ar H2O), 55.9 (N H2CH2CH2O), 54.2 ( H2N H2),
46.9 (N H), 35.7 (NH H2CH2O), 26.0 ( H2CH3pyridone), 24.9 (CH2piperidine), 23.7
H3pyridone), 12.2 ( H3indole). ESI-HRMS: m/z
[M+H]+ calcd for C30H43O3N4: 507.3330; found: 507.3330.
×
×
×
δ
C
C
C
C
C
C
C
C
C
C
C
C
C
Hindole), 66.9 (
C
C
C
C
C
C
H2), 26.1 (NCH2
C
C
(CH2piperidine), 21.4 (CH3), 19.8 (
C
H3pyridone), 12.7 (CH2
C
C
1-Isopropyl-2-methyl-N-((2-oxo-1,2,5,6,7,8-hexahydroquinolin-3-yl)methyl)-5-(3-(piperidin-1-yl)propoxy)
-1H-indole-3-carboxamide (L–20). The title product was obtained from 7e and pyridone 16 according to
the general procedure described above as a white solid (52 mg, 25%). M.p. 127–130 ◦C. 1H-NMR
(CDCl3)
δ 11.61 (brs, 1H, NHpyridone), 7.41 (brs, 2H, ArH), 7.36–7.34 (m, 2H, ArH), 6.74–6.73 (dd,
J = 8.9 Hz, 1.9 Hz, 1H, ArH), 4.73–4.67 (m, 1H, NCH), 4.49 (d, J = 5.8 Hz, 2H, OCH2), 4.21 (t, J = 5.8 Hz,
2H, NCH2), 3.06 (brs, 2H, CH2), 2.86 (brs, 3H, CH2), 2.73 (s, 3H, CH3), 2.65 (brs, 2H, CH2), 2.46–2.45
(m, 3H, CH2), 2.25 (brs, 2H, CH2), 1.85 (brs, 4H, CH2), 1.71–1.70 (m, 5H, CH2), 1.61 (brs, 3H, CH2),
1.59 (d, J = 7.0 Hz, 6H, 2
(Ar Hpyridone), 141.9 (Ar
(Ar Hindole), 125.9 (Ar Hpyridone), 113.9 (Ar
(Ar Hpyridone), 102.9 (Ar Hindole), 67.0 ( H2O), 55.6 (N
40.2 (NH H2), 29.7 (CH2pyridone), 26.7 (CH2pyridone), 26.1 (NCH2
×
CH3). 13C-NMR (CDCl3)
Hindole), 141.6 (Ar Hpyridone), 141.5 (N-
Hindole), 112.4 (Ar
H2CH2CH2O), 53.8 (
H2CH2O), 23.8 (CH2piperidine), 22.4
H3indole). ESI-HRMS: m/z [M+H]+ calcd for
δ
165.9 (
C
=Oindole), 163.7 (
=Cindole), 129.5 (Ar
Hindole), 111.8 (N-C=
H2N H2), 46.9 (N
C
=Opyridone), 153.6
Hindole), 126.6
indole), 107.9
H),
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
(CH2pyridone), 21.7 (CH2pyridone), 21.4 (CH3), 12.1 (
C
C31H43O3N4: 519.3330; found: 519.3329.
N-((6-Isobutyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-2-methyl-5-(3-(piperidin-1-yl)propoxy)
-1H-indole-3-carboxamide (L–21). The title product was obtained from 7e and pyridone 20 according
to the general procedure described above as a white solid (26 mg, 12%). M.p. 99–101 ◦C. 1H-NMR
(CDCl3)
δ 12.23 (brs, 1H, NHpyridone), 7.47 (d, J = 6.9 Hz, 1H, ArH), 7.37–7.35 (m, 2H, ArH), 7.31 (t,
J = 5.5 Hz, 1H, ArH), 6.75–6.74 (dd, J = 8.9 Hz, 2.0 Hz, 1H, ArH), 6.00 (d, J = 6.1 Hz, 1H, ArHpyridone),
4.71–4.65 (m, 1H, NCH), 4.53 (d, J = 5.8 Hz, 2H, OCH2), 4.09 (t, J = 6.1 Hz, 2H, NCH2), 2.74 (brs, 2H,
CH2), 2.72 (s, 3H, CH3), 2.61 (brs, 4H, 2 × CH2), 2.40 (d, J = 7.2 Hz, 2H, CH2), 2.08–2.07 (m, 2H, CH2),
2.00–1.93 (m, 1H, CH), 1.71 (brs, 4H, 2
0.85 (d, J = 6.6 Hz, 6H, 2
(Ar Hpyridone), 148.1 (Ar
125.7 (Ar Hindole), 111.5 (N-C=Cindole), 108.0 (Ar
×
CH2), 1.59 (d, J = 7.0 Hz, 6H, 2
166.1 ( =Oindole), 165.0 (
=Cindole), 139.5, 129.4 (Ar Hindole), 126.8 (Ar
Hpyridone), 105.9 (Ar Hpyridone), 102.7 (Ar
×
CH3), 1.47 (brs, 2H, CH2),
×
CH3). 13C-NMR (CDCl3)
Hpyridone), 141.1 (N-
δ
C
C
=Opyridone), 153.8
C
C
C
C
C
C
Hindole),
Hindole),
C
C
C

Molecules 2020, 25, 2059
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66.9 (
H(CH3)2), 28.5 (CH2piperidine), 25.9 (NCH2
(CH( H3)2), 21.4 (CH3), 12.2 (
H3indole). ESI-HRMS: m/z [M+H]+ calcd for C31H45O3N4: 521.3486;
found: 521.3488.
C
H2O), 55.8 (N
C
H2CH2CH2O), 54.1 (
C
H2NC
H2), 46.9 (N
CH), 42.1 (NH
CH2), 39.9 (
CH2CH), 29.7
(C
CH2CH2O), 24.7 (CH2piperidine), 23.6 (CH2piperidine), 22.1
C
C
1-Isopropyl-2-methyl-N-((4-methyl-2-oxo-1,2,5,6,7,8-hexahydroquinolin-3-yl)methyl)-5-(3-(piperidin-1-yl)
propoxy)-1H-indole-3-carboxamide (L–22). The title product was obtained from 7e and pyridone 15
according to the general procedure described above as a white solid (38 mg, 17%). M.p. 194–196 ◦C.
1H-NMR (CDCl3)
δ 13.10 (brs, 1H, NHpyridone), 7.55 (t, J = 5.8 Hz, 1H, NHCH2), 7.34–7.31 (m, 2H,
ArH), 6.74–6.72 (dd, J = 8.9 Hz, 2.2 Hz, 1H, ArH), 4.70–4.65 (m, 1H, NCH), 4.63 (d, J = 5.9 Hz, 2H,
OCH2), 4.00 (t, J = 6.4 Hz, 2H, NCH2), 3.02 (t, J = 5.9 Hz, 2H, CH2), 2.71 (s, 3H, CH3), 2.42–2.34 (m, 8H,
4
2
×
CH2), 2.14 (s, 3H, CH3), 1.86–1.84 (m, 2H, CH2), 1.75–1.71 (m, 4H, 2
CH3), 1.56–1.54 (m, 4H, 2
CH2), 1.39 (brs, 2H, CH2). 13C-NMR (CDCl3)
Hpyridone), 150.1 (Ar Hpyridone), 140.7 (N- =Cindole), 140.4 (Ar
Hindole), 128.8 (Ar Hindole), 126.8 (Ar Hindole), 122.8 (Ar Hpyridone), 114.4 (Ar
111.3 (N-C=Cindole), 108.1 (Ar Hpyridone), 102.3 (Ar H2O), 56.0 (N
54.4 ( H2N H2), 46.9 (N H), 35.0 (NH H2CH2O), 26.7 (CH2pyridone), 25.5
×
CH2), 1.58 (d, J = 7.0 Hz, 6H,
×
×
δ
165.9 ( =Oindole), 163.7
C
(C
=Opyridone), 154.0 (Ar
C
C
C
C
C
C
Hindole),
Hindole),
130.9 (Ar
C
C
C
C
C
Hindole), 66.9 (
C
CH2CH2CH2O),
C
C
C
C
H2), 27.4 (NCH2C
(CH2piperidine), 25.1 (CH2piperidine), 24.2 (CH2piperidine), 22.4 (CH2pyridone), 22.3 (CH2pyridone), 21.4 (CH3),
19.2 ( H3pyridone), 16.7 (CH2pyridone), 12.2 (
H3indole). ESI-HRMS: m/z [M+H]+ calcd for C32H45O3N4:
C
C
533.3486; found: 533.3495.
3.2.8. React IR Experiment
The ReactIR 15 DiComp probe was inserted in a 50 mL, three-neck flask equipped with a magnetic
stirrer. IR spectra were obtained every 15 s. Data collection began at the start of the experiment.
The solution of ethyl acetoacetate (1.0 g, 7.68 mmol) and TCCA (34.8 mg, 0.15 mmol) in MeCN (5 mL)
was cooled to 0 ◦C, and benzylamine (0.82 g, 7.68 mmol) was added dropwise. The resulting solution
was stirring under ice bath and naturally elevated to at room temperature for indicated time. After the
reaction was completed, the workup was consistent with the general procedure 3.1.1.
3.3. In Vitro Biological Activity Assays
3.3.1. Cell Growth Inhibition Assay
The human chronic myeloid leukemia cell line K562 was purchased from the American Type
Culture Collection (ATCC, Perry Pkwy, Gaithersburg, USA). The cells were cultured in RPMI 1640
medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin at 37 ◦C in a
humid atmosphere containing 5% CO2 in air. In the cell growth experiments, cells were seeded with
5000 cells/well density in the culture medium containing 100
10, 1, 0.1 M) in 96 well cell culture plates. After treatment for 48 hours, cell growth was measured
by WST-8 assay based on lactate dehydrogenase using the Spectramax Paradox Multimode detection
platform. The CCK-8 reagent was added to each well of 10 L, and the cells were incubated for another
µL diluent series of test compounds (100,
µ
µ
1–4 hours and read at 450 nm. The readings were standardized relative to the cells treated by the
carrier, and IC50 was calculated by nonlinear regression analysis using SPSS statistics 20.0 software
(IBM, Chicago, Michigan, USA).
3.3.2. Western Blot Analysis
The cells were treated with test compounds (10 and 5 µM), and the extract was prepared by
adding radio immunoprecipitation assay (RIPA) lysis buffer to the cells under ice bath. The protein
concentrations were determined by the bicinchoninic acid (BCA) assay. Protein samples were
separated by sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to
a polyvinylidene fluoride (PVDF) membrane. Cell membranes were blocked with 5% milk for 1 hour at

Molecules 2020, 25, 2059
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room temperature and overnight at 4 ◦C with designated primary antibodies. After washing with tris
buffered saline Tween (TBST) three times for 30 min, incubated with horseradish peroxidase-labeled
secondary antibody for 1 h at room temperature, and then washing with TBST three times for 30 min to
enhance chemiluminescence. The band intensity in Figure 3 was quantified by the Odyssey software
(Li-Cor, Lincoln, Nebraska, USA) and histone H3 was used as a housekeeping protein for normalization.
3.4. Molecular Docking
We processed EZH2 protein (PDB code: 4W2R) using the protein preparation wizard in Schrödinger
suite [40–42]. First, the complex’s protonation states were adjusted for consistency with pH 7.4. Second,
all hydrogen atoms and the total structure for the protein and small molecules were evaluated for
energy accuracy using an all-atom force field, OPLS_2005; restrained minimization; and heavy atom
convergence to a 0.3 Å RMSD. Third, the above prepared EZH2 protein and co-crystallized molecule
were used to generate a receptor grid file. The position of co-crystallized molecule was used to determine
the active site location (x = 30.57, y = 18.29, and z = 54.62) and size (inner box = 10 Å
×
10 Å
×
10 Å;
outer box = 20 Å 20 Å 20 Å). The grid was generated using the OPLS_2005 force field. The glide
×
×
docking was then carried out between the prepared protein and molecules. The docking result was
exported and analyzed using Discovery Studio 2019 and PyMol.
4. Conclusions
In summary, we have synthesized a series of 5-hydroxyindole scaffold compounds bearing as main
pharmacophore a pyridone moiety. According to molecular modeling and in vitro biological activity
assays, the preliminary structure-activity relationships were determined and summarized. Compound
L–04 improved both the H3K27Me3 reduction and antiproliferation parameters (IC50 = 52.6
K562 cells. It bears mentioning that L–21 had comparable inhibitory effect (IC50 = 51.8 M) to L–04
however, molecular docking indicated that L–21 (with the N1 position substituted with an isopropyl
group) had reduced binding energy due to the absence of interactions created by the benzyl group.
µM) on
µ
;
π-π
In all, we believe that L–04 should remain a potential candidate to design more EZH2Is, and future
in vivo studies using patient-derived tumor xenograft models are required to further assess the utility
of this compound.
During the preparation of compounds, we discovered a novel catalytic activity of TCCA in
condensation reactions. Different amines were condensed with β-diketones or β-ketoesters in the
presence of TCCA to afford the corresponding product in short times with high yields, which displayed
some significant advantages and provided an alternative condensation strategy. In addition, we
established a model for L–04 binding to EZH2 using molecular docking, which will be utilized to
direct future analogue design and synthesis. These findings further support the therapeutic potential
of EZH2Is as anti-cancer agents.
Supplementary Materials: The following are available online, 1H-NMR and HR-MS spectra of
and
-aminopropionates; Preparation of pyridone derivatives; 1H-NMR, 13C-NMR and HR-MS spectra of
intermediates and EZH2 inhibitors; Table S1: Reaction conditions.
β-aminopropenones
β
Author Contributions: Conceptualization, F.D. and Q.Z.; methodology, F.D. and Q.Z.; software, W.S. and C.Y.;
validation, L.W. and C.Y.; formal analysis, F.D. and Q.Z.; investigation, W.S. and C.Y.; resources, G.C., L.W. and
C.W.; data curation, W.S.; writing—original draft preparation, F.D. and Q.Z.; writing—review and editing, F.D.
and Q.Z.; visualization, F.D. and Q.Z.; supervision, G.C. and L.W.; project administration, G.C. and L.W.; funding
acquisition, C.G, L.W. and C.W. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (81973365), and Liaoning
Revitalization Talents Program (XLYC1908031, XLYC1902008). The APC was funded by Liaoning Revitalization
Talents Program (XLYC1908031, XLYC1902008).
Acknowledgments: The authors are indebted to Testing Center of School of Pharmaceutical Engineering for
providing the HPLC-MS data for this study.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2020, 25, 2059
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©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).