Design, synthesis, and biological evaluation of 5-aminotetrahydroquinoline-based LSD1 inhibitors acting on Asp375

Article abstract of DOI:10.1002/ardp.202100102

The abnormal expression of lysine-specific histone demethylase 1 (LSD1) is associated with different cancer types, and LSD1?inhibitory activity seems to have high therapeutic potential in cancer treatment. Here, we report the design, synthesis, and biochemical evaluation of novel 5-aminotetrahydroquinoline-based LSD1 inhibitors. Among them, compounds A6, A8, B1–B5, and C4 showed preferable inhibitory effects on LSD1, with IC₅₀ = 0.19–0.82 μM. Several potent compounds were selected to evaluate their antiproliferative activity on A549 cells and MCF-7 cells with a high expression of LSD1. The potential binding modes of the compounds were revealed through molecular docking to rationalize the potency of compounds toward LSD1. Our data recognized that the 5-aminotetrahydroquinoline scaffold may serve as a starting point for developing potent LSD1 inhibitors for cancer therapy.

Full text of DOI:10.1002/ardp.202100102

Received: 14 March 2021
Revised: 6 April 2021
Accepted: 9 April 2021
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DOI: 10.1002/ardp.202100102
F U L L P A P E R
Design, synthesis, and biological evaluation of
5‐aminotetrahydroquinoline‐based LSD1 inhibitors
acting on Asp375
Jiangkun Yan1 | Yanting Gu2 | Yixiang Sun1 | Ziheng Zhang1
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Xiangyu Zhang1 | Xinran Wang1 | Tianxiao Wu1 | Dongmei Zhao1
|
Maosheng Cheng1
1Key Laboratory of Structure‐Based Drug
Design & Discovery of Ministry of Education,
Shenyang Pharmaceutical University,
Shenyang, China
Abstract
The abnormal expression of lysine‐specific histone demethylase 1 (LSD1) is asso-
ciated with different cancer types, and LSD1 inhibitory activity seems to have high
therapeutic potential in cancer treatment. Here, we report the design, synthesis, and
biochemical evaluation of novel 5‐aminotetrahydroquinoline‐based LSD1 inhibitors.
Among them, compounds A6, A8, B1–B5, and C4 showed preferable inhibitory
effects on LSD1, with IC50 = 0.19–0.82 µM. Several potent compounds were se-
lected to evaluate their antiproliferative activity on A549 cells and MCF‐7 cells with
a high expression of LSD1. The potential binding modes of the compounds were
revealed through molecular docking to rationalize the potency of compounds to-
ward LSD1. Our data recognized that the 5‐aminotetrahydroquinoline scaffold may
serve as a starting point for developing potent LSD1 inhibitors for cancer therapy.
2The School of Life Science and
Biopharmaceutical, Shenyang Pharmaceutical
University, Shenyang, China
Correspondence
Dongmei Zhao, Key Laboratory of Structure‐
Based Drug Design & Discovery of Ministry of
Education, Shenyang Pharmaceutical
University, 110016 Shenyang, China.
Email: medchemzhao@163.com
K E Y W O R D S
5‐aminotetrahydroquinoline, antitumor, drug design, epigenetic, LSD1 inhibitors
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1
INTRODUCTION
amine oxidative family, which can specifically remove the mono‐ and di‐
[5]
methylation of histones H3 lysine (H3K4). In addition, LSD1 can de-
[1]
In recent decades, great progress was made in epigenetics research.
Histone modification such as acetylation, phosphorylation, methylation,
and ubiquitination plays a pivotal role in the regulation of epigenetic
gene expression. For a long time, methylation was considered as an
irreversible and permanent histone marker. In 2004, the histone lysine‐
methylate many other nonhistone substrates. Tumor suppressor gene
p53, which plays an important role in DNA repair, cell proliferation,
differentiation, and apoptosis, is the first nonhistone identified as an
LSD1 substrate. LSD1 can specifically remove the methyl of K370 of
[6]
p53 and inhibit the expression of the target gene. LSD1 can also bind
[2]
[7]
[8]
[9]
[10]
[11]
specific demethylase 1 (LSD1) was discovered, indicating that histone
to DNMT1, E2F1, HIF‐1 α, AGO2,
and MYPT1,
thereby
methylation was a reversible process, which led to further research on
undermining proteins stability and biological activity. Acting on STAT3
[12]
LSD1. There are two main enzymes that regulated the methylation of
[3,4]
and Erα,
LSD1 will promote transcriptional activity and increase the
lysine residues: histone methyltransferase and histone demethylase.
incidence of cancer. Combining with MTA1, the activators and silence
[7]
LSD1 belongs to the flavin adenine dinucleotide (FAD)‐dependent
proteins are changed.
Jiangkun Yan and Yanting Gu contributed equally to this study.
© 2021 Deutsche Pharmazeutische Gesellschaft
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Arch Pharm. 2021;e2100102.
wileyonlinelibrary.com/journal/ardp
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https://doi.org/10.1002/ardp.202100102

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FIGURE 1 Structures of representative
LSD1 inhibitors
Studies have shown that LSD1 is highly expressed in lung cancer,
two amino acids play a critical role in the inhibition of LSD1. Efforts
have been made to develop compounds that act on Asp555; never-
[26–28]
acute myeloid leukemia, lymphoma, prostate cancer, bladder cancer,
[13–15]
and ER‐negative breast cancer cells.
Therefore, LSD1 is con-
theless, the role of Asp375 has not been revealed yet.
sidered to be a promising drug target for cancer intervention. A lot of
Therefore, we intended to design a novel type of compound that
could interact with Asp375 by changing the structure of aniline on
compound 8 and introducing the necessary groups.
LSD1 small molecule inhibitors currently under study have multiple
[16]
[17]
[18]
scaffolds, including cyclopropylamine,
[19]
amidoxime,
pyridine,
[20]
benzohydrazide,
thieno[3,2‐b]pyrrole,
and so forth. Specifically,
In this study, the small molecule LSD1 inhibitor 9 was selected as
the lead compound. A series of hybrids with 5‐aminotetrahydroquinoline
skeleton was designed and synthesized by using conformational re-
striction and fragment growth strategies. The enzyme inhibitory effects
of all compounds on LSD1 in vitro were tested. And most of the
potential compounds exhibited effective antiproliferative activity against
the selected cell lines; in particular, compound B4 displayed potent ac-
tivity against A549 (IC50 = 2.13 µM) and MCF‐7 (IC50 = 2.35 µM). The
structure–activity relationship (SAR) studies, molecular docking studies,
and the prediction of ADME (absorption, distribution, metabolism, and
elimination) have also been carried out.
trans‐2‐PCPA (1) can demethylate H3K4 and treat depression based
[21]
on its covalent inhibition of monoamine oxidases.
Many covalent
LSD1 inhibitors have been produced on the basis of 2‐PCPA
[3]
scaffold.
Encouragingly, ORY‐2001 (2), GSK2879552 (3), ORY‐
1001 (4), INCB059872 (5), and IMG‐7289 (6) (Figure 1) are under
clinical assessments for the treatment of acute myeloid leukemia, non‐
small‐cell lung carcinoma and neurodegenerative disorders, and so
[17,22,23]
forth.
In addition, it is worth noting that two reversible in-
hibitors, SP‐2577 (7) and CC‐90011 (8), are being evaluated for use in
[18,24]
humans alone or in combination with other therapeutic agents.
Also, there are currently no LSD1‐targeted drugs approved by the US
FDA for clinical use for cancer treatment. Thus, it is of great urgency
to develop more efficient LSD1 inhibitors of different chemotypes for
use in cancer treatment.
[25]
Recently, Sartori et al.
obtained compound 9 by high‐
throughput screening, which showed moderate inhibitory activity for
LSD1 (IC50 = 2.9 µM). It is worth mentioning that they obtained the
co‐crystal structure of compound 9, which foreshadowed the pro-
gress of the subsequent research work. From the protein cavity
diagram of compound 9, it can be seen that compound 9 is buried
deeply in a hydrophobic pocket in the protein and there are two
amino acid residues (Asp375 and Asp555) with strong electro-
negativity outside the cavity of the protein (Figure 2). Compound 10
was previously developed as an LSD1 inhibitor by our research team,
and it showed satisfactory pharmacological results. We researched
the binding mode of compound 10 and found that the terminal basic
fragment of compound 9 could form hydrogen bond interactions
with Asp555 (Figure 3b). The above results further prove that these
FIGURE 2 Protein cavity diagram of compound 9 (depicted in the
green model) in the active sites of LSD1 (red represents the acid
amino residues, and the distance from 8 to amino is depicted as
yellow lines)

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FIGURE 3 Design strategies of our target compounds based on
cocrystal structure of compound 9 (PDB ID: 5LGN). (a) Design
strategies for novel LSD1 inhibitors acting on Asp375. (b) Model of
action between compound 10 (depicted in the yellow model) and
LSD1 (flavin adenine dinucleotide [FAD] is identified by gray sticks).
(c) Model of action between compound B4 (depicted in the purple
model) and LSD1 (FAD is identified by gray sticks). The hydrogen
bonds are depicted as light green lines. The π–π interactions are
depicted as red lines
SCHEME 2 Synthesis of compounds B1–B5. Reagents and
conditions: (a) corresponding alcohols, PPh3, DIAD, THF, −20°C to
room temperature (r.t.), 52–75%; (b) NaBH4, MeOH, 0°C, 69–76%;
(c) I2, PPh3, DMAP, DCM, 0°C to r.t., 25–36%; (d) M6, K2CO3, DMF,
r.t., 72–83%; (e) 4 mol/l HCl/MeOH, r.t., 72–85%. DCM,
dichloromethane; DMAP, 4‐dimethylaminopyridine; DIAD,
diisopropyl azodicarboxylate; DMF, dimethylformamide; THF,
tetrahydrofuran
synthesized by a Mitsunobu reaction and their aldehyde group was
reduced to the hydroxyl to get M9a–M9m. Then, iodine‐mediated
halogenation was performed to obtain intermediates M10a–M0m
that were reacted with M6 in the presence of potassium carbonate in
DMF to gain target compounds A7, A14–A16 and compounds
M11a–M11i. The N‐Boc protecting group of M11a–M11i was re-
moved by utilizing 4 mol/l HCl/MeOH to obtain A1–A6, A8,
A10–A11, then through reductive amination reaction to gain A9,
A12, and A13.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Scheme 1 describes the synthetic routes of compounds A1–A16. In
short, ethyl 4H‐thieno[3,2‐b]pyrrole‐5‐carboxylate (M1) was used as
a starting material and was methylated with iodomethane to obtain
M2. M3 was obtained by hydrolysis of M2 under alkaline conditions.
5‐nitroquinoline (M4), another starting material, was reduced to gain
intermediate M5. M5 was treated with M3 in dry dimethylforma-
mide (DMF), in the presence of HATU, to give M6. M8a–M8m were
The synthetic routes of compounds B1–B5 are presented in
Scheme 2. The intermediates M13a–M13e were obtained by the
Mitsunobu reaction of tert‐butyl 4‐hydroxypiperidine‐1‐carboxylate
SCHEME 1 Synthesis of compounds A1–A16. Reagents and conditions: (a) NaH, CH3I, DMF, 0°C, 92%; (b) NaOH, MeOH, room
temperature (r.t.), 91%; (c) NiCl2·6H2O, NaBH4, MeOH, 0°C to r.t., 58%; (d) HATU, DIPEA, DMF, r.t., 75%; (e) corresponding alcohols, PPh3,
DIAD, THF, −20°C to r.t., 52–75%; (f) NaBH4, MeOH, 0°C, 69–76%; (g) I2, PPh3, DMAP, DCM, 0°C to r.t., 25–36%; (h) M6, K2CO3, DMF, r.t.,
72–83%; (i) 4 mol/l HCl/MeOH, r.t., 72–80%; (j) HCHO, HCOOH, NaBH4, DCM, r.t., 60–69%. DCM, dichloromethane; DIAD, diisopropyl
azodicarboxylate; DIPEA, N,N‐diisopropylethylamine; DMAP, 4‐dimethylaminopyridine; DMF, dimethylformamide; HATU, 1‐[bis
(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium 3‐oxide hexafluorophosphate; THF, tetrahydrofuran

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SCHEME 3 Synthesis of compounds C1–C14. Reagents and conditions: (a) HATU, DIPEA, DMF, room temperature (r.t.), 59–67%; (b) 10f,
K2CO3, DMF, r.t., 70–84%; (c) 4 mol/l HCl/MeOH, r.t., 70–82%. DIPEA, N,N‐diisopropylethylamine; DMAP, 4‐dimethylaminopyridine; DMF,
dimethylformamide; HATU, 1‐[bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium 3‐oxide hexafluorophosphate
TABLE 1 The inhibition data (IC50) of A1–A16 against LSD1 in vitro
1
a
1
a
Compound
A1
R
IC50 (μM)
Compound
A9
R
IC50 (μM)
1.02
2.13
A2
A3
3.55
1.24
A10
A11
1.92
1.52
A4
A5
A6
A7
A8
2.26
3.50
0.19
>50
0.39
2.9
A12
A13
A14
A15
A16
0.89
1.24
>50
>50
>50
b
b
9
2‐PCPA
40.25
a
The IC50 values are the average of at least two duplicate experiments. SEM < 20%.
b
Used as a positive control.

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with the corresponding substituted p‐hydroxybenzaldehyde. Then
through reduction reaction with NaBH4 and the Appel reaction,
M15a–M15e were obtained. M15a–M15e and M3 were condensed
by HATU in dry DMF to gain M16a–M16e. Then the N‐Boc pro-
tecting group was removed by using 4 mol/l HCl/MeOH to get
B1–B5. Scheme 3 describes the synthesis routes of C1–C14. The
corresponding acid and M5 were condensed by HATU to obtain
M18a–M18n. Nucleophilic substitution reaction occurred between
M18a–M18n and intermediate 10f to gain M19a–M19n, which was
stripped of the N‐Boc protecting group to gain C1–C14. The
synthetic routes of compounds D1–D6 were consistent with those
described in Scheme 1.
TABLE 2 The inhibition data (IC50) of B1–B5 against LSD1 in
vitro
2
a
Compound
R
IC50 (μM)
B1
B2
B3
B4
B5
2‐F
0.41
0.21
0.26
0.46
0.45
2.9
3‐OMe
3‐Me
2‐NO2
4‐F
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2.2
Pharmacology/biology
|
2.2.1
Inhibitory activities against LSD1 and SAR
studies
b
9
All the synthetic compounds in this study were tested for enzyme
activity, and compounds 2‐PCPA and 9 were used as positive controls.
The LSD1 inhibitor screening kit was purchased from Cayman
Chemical Company. On the basis of the determination of the horse-
radish peroxidase assay, the enzyme inhibitory activities of all target
compounds were evaluated. The enzyme inhibitory activity of com-
pound A1 (IC50 = 1.02 µM) was twofold higher than that of compound
9, which was the first compound we designed. First, the alkaline
fragment A interacting with Asp375 was investigated, and the specific
replacement segments are shown in Table 1. When fragment A was
replaced by nitrogen‐containing aliphatic fragments, most compounds
showed superior inhibitory activity against LSD1, and the IC50 values
were all <4 µM, except for compound A7 (IC50 > 50 µM). These results
revealed that the nitrogen atom was an important factor for main-
taining the inhibitory activity. Among them, A6 and A8 showed potent
inhibitory activity, and their IC50 values were 0.19 and 0.39 µM, re-
spectively. Compared with A1, A10–A13 bearing the flexible chain
between the A‐terminal nitrogen atom and the oxygen atom on the
benzene ring had similar enzyme inhibitory activities. On the contrary,
when fragment A was replaced by aromatic groups, compounds
A14–A16 displayed weak inhibitory activities, indicating that the in-
troduction of aromatic rings resulted in potency loss.
b
2‐PCPA
40.25
a
The IC50 values are the average of at least two duplicate experiments.
SEM < 20%.
b
Used as a positive control.
Subsequently, we replaced the 4‐methyl‐4H‐thieno[3,2‐b]pyrrole
ring in part C with aromatic heterocycles and rigid benzene rings.
Most of the substitutions did not show exciting results, as shown in
Table 3. Benzofuran, benzothiophene, and indole skeletons showed
moderate inhibitory activities with IC50 values of 4.87, 3.56, and
3.67 µM, respectively. Interestingly, the activity of compound C4 was
significantly improved (IC50 = 0.82 µM) when the nitrogen atom in
the 2‐formyl indole fragment was modified by methyl, which may be
attributed to improve the interaction between the indole ring and
FAD. Compounds C5–C14 exhibited a slight inhibition effect on
LSD1 except C10 (IC50 = 1.02 µM), indicating that larger quinoline
ring, monoheterocyclic ring, as well as different substituted benzene
ring, were not conducive to maintain inhibitory activity against LSD1.
The introduction of indole ring could enhance the medicinal prop-
erties of the compounds; therefore, we designed and synthesized
compounds D1–D6. When the linker of the amide bond was at dif-
ferent positions, the IC50 values of compounds D1–D3 were 4.59,
5.23, and 3.82 µM, respectively, which may be explained as the amide
linker at the 2 position was conducive to the low‐energy conforma-
tion of the molecule to interact with protein. Then, the influence of
different substituents on nitrogen atoms was investigated, and D3,
D4 were obtained. The results of inhibition activities showed that the
steric hindrance was not conducive to the increase in inhibitory ac-
tivity. Considering the size of the protein cavity, we introduced
fluorine atoms into the indole ring, and synthesized compound D6
with an IC50 value of 1.24 µM, which revealed that the introduction
of substituents on the indole ring was disadvantageous to the
It is not difficult to find from the docking experimental result of
A6 (Figure 3a) that the benzene ring forms a hydrophobic interaction
with Cys360. Next, different substituents were introduced on the
benzene ring to examine the effect on the activity. The results of
enzyme inhibitory activity in vitro are shown in Table 2. B2 and B3,
whose benzene rings contained methyl or methoxy, exhibited similar
inhibitory activities as A6, and the IC50 values were 0.21 and
0.26 µM, respectively. When the fluorine atom and the nitro group
were introduced, the activities of B1, B4, and B5 decreased in
varying degrees, illustrating that the introduction of electron‐
withdrawing groups did not improve the inhibition activity. In gen-
eral, part B played a weak role in enhancing activity.

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TABLE 3 The inhibition data (IC50) of C1–C14, D1–D6 against LSD1 in vitro
a
a
3
3
Compound
R
IC (μM)
50
Compound
C11
R
IC (μM)
50
C1
4.87
3.56
3.67
>50
C2
C3
C12
C13
14.13
>50
C4
C5
C6
C7
C8
0.82
>50
C14
D1
D2
D3
D4
>50
4.59
5.23
3.82
1.92
16.57
>50
>50
C9
>50
D5
2.55
C10
1.02
D6
1.24
b
b
9
2.9
2‐PCPA
40.25
a
The IC50 values are the average of at least two duplicate experiments. SEM < 20%.
b
Used as a positive control.

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TABLE 4 Antiproliferative activity of
partial compounds
a
a
LSD1
IC50
LSD1
IC50 (µmol/l)
IC50 (µmol/l)
A549
IC50
Compound (µM) A549
MCF‐7
Compound (µM)
MCF‐7
A1
A2
A4
A6
A8
C4
A12
1.02
6.07 0.02
7.84 0.21 B1
0.41 5.72 0.15
0.21 6.57 0.09
0.26 6.36 0.14
0.46 2.13 0.56
0.45 6.85 0.24
1.02 8.49 0.13
8.79 1.17
7.17 0.18
7.76 0.22
2.35 0.52
6.96 0.09
0.35 0.05
35.98 3.41
1.24 17.53 0.46 14.18 0.99 B2
2.26
0.19
8.35 0.38
5.30 0.37
8.39 0.4
B3
6.91 0.04 B4
0.39 10.52 0.09 18.05 2.02 B5
0.82
0.89
7.61 0.28
7.02 0.05 C10
b
8.44 0.28 14.35 0.54
9
2.9
>100
Abbreviation: MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide.
a
MTT assay was used to determine the results after 72 h of administration; data are presented as the
mean SD of three independent experiments.
b
Used as a positive control.
improvement of activity. The results above manifested that π–π
stacking between the thieno[3,2‑b]pyrrole fragment and FAD was
essential for the compound to exert its inhibitory activity.
unclear, and possible explanations may be that the introduction of a
nitro group altered the physicochemical properties of compound B4.
Notably, compound C10 was able to potently inhibit the proliferation
of MCF‐7 cells with an IC50 value of 0.35 0.05 µM. The cell test
results have laid a solid foundation for our follow‐up work.
|
2.2.2
In vitro inhibition properties
|
Molecular docking
Next, we further studied the proliferation inhibitory ability of some
compounds with preferable enzyme inhibitory activity against two
tumor cell lines by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetra-
zolium bromide (MTT) assay, including lung cancer cells (A549) and
breast cancer cells (MCF‐7). As shown in Table 4, the tested com-
pounds showed satisfactory inhibitory activities against two cell
lines, indicating that such compounds were effective in inhibiting the
proliferation of cancer cells. Fortunately, B4 exhibited inspiring re-
sults at both the molecular and cellular levels, as it effectively
inhibited the proliferation of A549 cells and MCF‐7 cells with IC50
values of 2.13 0.56 and 2.35 0.52 µM, respectively. Although the
enzyme inhibitory activity of B4 was equivalent to that of A6, B4
displayed better results at the cellular level. The specific reason was
2.3
To rationalize the potency of compounds A6 and C4 against LSD1,
we used Glide 9.6 to conduct the molecular docking study. Crystal
structures of LSD1 (PDB ID: 5LGN) containing free cofactor FAD
were defined as the docking receptor. The docking results showed
that A6 occupied a preferable protein cavity (Figure 4a). The thieno
[3,2‐b]pyrrole fragment of A6 could form a strong π–π stacking ef-
fect with FAD, which was consistent with the action mode of 8
(Figure 4b). Meantime, the tetrahydroquinoline group attached to
the thieno[3,2‐b]pyrrole skeleton was surrounded by Trp695 and
formed hydrophobic interaction with this amino acid residue.
The benzene ring attached to the tetrahydroquinoline ring had a
FIGURE 4 The proposed binding mode of compound A6. (a) Model of action between compound A6 (depicted in the green model) and LSD1
(FAD is identified by gray sticks). (b) Superposition diagram of compound 9 (depicted in the blue model) and A6. The hydrogen bonds are
depicted as light green lines. The π–π interactions are depicted as red lines

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FIGURE 5 The proposed binding mode of compound C4. (a) Model of action between compound C4 (depicted in the purple model) and
LSD1 (flavin adenine dinucleotide [FAD] is identified by gray sticks). (b) Superposition diagram of compound 9 (depicted in the blue model) and
C4. The hydrogen bonds are depicted as light green lines. The π–π interactions are depicted as red lines
hydrophobic interaction with Cys360. In addition, the nitrogen atom
at the basic end of A6 could be protonated and the combined hy-
drogen could form a hydrogen bond with Asp375 (Figure 4b).
Compound C4 had the necessary hydrophobic effects, π–π stacking
with FAD, and a protein cavity that accommodated the molecule well
in a manner similar to A6 (Figure 5a,b). The docking score demon-
strated that compound A6 (−9.570 kcal/mol) was superior to com-
pound C4 (−9.327 kcal/mol). A significant difference between A6 and
C4 could be observed from Figures 4b and 5b, in which the basic
fragment of compound A6 was closer to Asp375, which might be the
reason why the activity of A6 was higher than that of C4. The
combination model of prediction provided a structural basis for
further structure‐based modifications.
the optimal range (c log P = 3). When the topological polar surface
2
area (TPSA) value was in the range of 60–140 Å , the drug had good
gastrointestinal permeability. Therefore, all the measured com-
pounds showed good permeability. In addition, the ligand efficiency
(LE), ligand lipophilic efficiency (LLE), and ligand efficiency‐
dependent lipophilicity (LELP) were essential for evaluating drugli-
keness. The properties of the tested compounds were far from those
of ideal drugs. Next, we need to optimize the physicochemical
properties of the compounds to improve their drug‐like properties.
|
3
CONCLUSION
In this study, we report 41 LSD1 inhibitors acting on Asp375 by using
conformational restriction and fragment growth strategies for the
first time, followed by evaluating their biochemical potency against
LSD1 according to our established protocol published before. Mo-
lecular docking experiments further predicted the binding modes of
the compounds we designed. Among them, compounds A6, A8,
B1–B5, and C4 displayed potent enzyme inhibitory activities against
|
2.3.1
Theoretical prediction of ADME properties
In recent years, small molecule drugs approved by the Food and Drug
Administration (FDA) of the United States have certain physico-
chemical properties. The druglikeness properties of compounds A6,
A8, B4, C4, and C10 were predicted by Sybyl software, and the
results are shown in Table 5. The molecular weight of the measured
compounds was approximately 500 g/mol, which was higher than the
ideal value. All c log P values were close to 6, which were higher than
LSD1. In particular, the inhibitory activity of compound A6 (IC50
=
0.19 μM) against LSD1 was nearly 15‐fold stronger than that of
compound 8 (IC50 = 2.9 μM). Further cellular assay indicated that B4
possesses comparable inhibitory activities against MCF‐7 cells and
A549 cells, whose IC50 values were 2.13 0.56 and 2.35 0.52 µM,
respectively. Besides, C10 showed potent inhibition against LSD1 in
MCF‐7 cells with an IC50 value of 0.35 0.05 μM and weak inhibitory
activity in A549 cells. In summary, these novel LSD1 inhibitors acting
on Asp375 deserve further investigation for cancer treatment.
TABLE 5 ADME properties of compounds A6, A8, B4, C4,
and C10
Compound M.W.
pIC50 c log P TPSA
LE
LLE
LELP
A6
A8
B4
500.66 6.72
5.77
5.76
5.73
5.85
5.81
86.77 0.26 0.95 22.19
85.67 0.26 0.65 22.15
132.59 0.22 0.61 26.04
58.53 0.23 0.24 25.43
53.60 0.23 0.18 26.13
486.63 6.41
545.21 6.34
494.64 6.09
509.23 5.99
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4
EXPERIMENTAL
Chemistry
C4
|
4.1
C10
Abbreviations: ADME, absorption, distribution, metabolism, and
elimination; c log P, partition coefficient; LE, ligand efficiency; LELP, ligand
efficiency‐dependent lipophilicity; LLE, ligand lipophilicity efficiency;
M.W., molecular weight; pIC50, −log [IC50(PAK4, mol/l)]; TPSA, topological
polar surface area.
|
4.1.1
General
The chemical raw materials and reagents used were purchased from
commercial channels and can be used directly without purification.

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|
The reaction was monitored with thin‐layer chromatography (TLC)
using silica gel (Yantai Jiangyou Silica Gel Development Co. Ltd).
Column chromatography was performed on silica gel (200–300
methanol (100 ml), sodium borohydride (4.34 g, 114.80 mmol) was ad-
ded in batches within 2 h. The solution, turned black, was stirred at
room temperature until consumption of the M4, as determined by TLC.
The mixture was concentrated under vacuum, and the black solid
was dissolved in ethyl acetate (100 ml), stirred for 10 min, and the solid
was filtered out. The filtrate was dried with anhydrous Na2SO4, eva-
porated under reduced vacuum, purified on silica gel column (petroleum
ether/ethyl acetate = 4:1), and M5 (1.00 g, 58%) as yellow solid was
1
mesh) for the purification of compounds. The H nuclear magnetic
13
resonance (NMR) and
C NMR spectra (see the Supporting In-
formation) were recorded on a Bruker 400 or 600 MHz spectro-
meter. Electrospray ionization–high‐resolution mass spectrometry
(ESI‐HRMS) spectra were obtained on a Bruker micromass mass
spectrometer. The purity of all biologically evaluated compounds was
determined to be >95% by reverse‐phase high‐performance liquid
chromatography analysis. The method conditions were as follows:
A mixture of solvents H2O (A) and MeOH (B) (VA:VB = 5:95) was
used as the eluent, and the flow rate was 1.0 ml/min. The peaks were
detected at λ = 254 nm.
1
obtained. H NMR (400 MHz, DMSO‐d6) δ 6.54 (t, J = 7.8 Hz, 1H), 5.84
(d, J = 7.6 Hz, 1H), 5.73 (d, J = 7.8 Hz, 1H), 5.21 (s, 1H), 4.46 (s, 2H),
3.10–3.01 (m, 2H), 2.32 (t, J = 6.4 Hz, 2H), 1.83–1.75 (m, 2H).
|
4.1.5
Synthesis of 4‐methyl‐N‐(1,2,3,4‐
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
tetrahydroquinolin‐5‐yl)‐4H‐thieno‐
[3,2‐b]pyrrole‐5‐carboxamide (M6)
Yield 75%, yellow solid. A mixture of 4‐methyl‐4H‐thiopheno[3,2‐b]
pyrrole‐5‐carboxylic acid (0.10 g, 0.55 mmol) and HATU (0.21 g,
0.55 mmol) in anhydrous DMF (2 ml) was stirred at room tempera-
ture for 1 h, and then the intermediate M5 (0.07 g, 0.50 mmol) and
N,N‐diisopropylethylamine (0.07 g, 0.55 mmol) were added. The
mixture was stirred at room temperature until the reaction was
complete as indicated by TLC. The mixture was poured into water
(20 ml) and extracted with ethyl acetate (20 ml × 3). The combined
organic phase was washed with water (20 ml × 2), dried over anhy-
|
4.1.2
Synthesis of ethyl 4‐methyl‐4H‐thieno[3,2‐
b]pyrro‐5‐carboxylate (M2)
Yield 92%, gray solid. A mixture of M1 (2.00 g, 10.24 mmol) in anhydrous
DMF containing sodium hydride (0.49 g, 20.48 mmol) was stirred under
ice bath conditions for 10 min. Then the methyl iodide (2.90 g,
20.48 mmol) was added and the mixture was stirred for 30 min. The
reaction mixture was poured into 20 ml water and the mixture was
extracted with ethyl acetate (20 ml × 3). The combined organic phase
was washed with water (20 ml × 2), dried over anhydrous Na2SO4, and
drous Na2SO4, and concentrated under vacuum to afford M6 (0.13 g,
1
75%).
H NMR (400 MHz, DMSO‐d6) δ 9.22 (s, 1H), 7.48
1
concentrated under vacuum to afford the crude product. H NMR
(d, J = 5.3 Hz, 1H), 7.31 (s, 1H), 7.22 (d, J = 5.3 Hz, 1H), 6.98 (d,
J = 7.4 Hz, 1H), 6.79 (d, J = 7.2 Hz, 1H), 6.48 (t, J = 7.6 Hz, 1H), 4.00 (s,
3H), 3.25–3.20 (m, 2H), 2.72 (t, J = 6.2 Hz, 2H), 1.79 (p,
J = 6.2 Hz, 2H).
(400 MHz, chloroform‐d) δ 7.32 (d, J = 5.4 Hz, 1H), 7.25 (s, 1H), 7.18
(s, 1H), 4.33 (q, J = 7.1 Hz, 2H), 4.05 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H).
|
4.1.3
Synthesis of 4‐methyl‐4H‐thieno[3,2‐b]-
|
General procedure for the synthesis of
compounds M8a–M8m
pyrrole‐5‐carboxylic acid (M3)
4.1.6
Yield 91%, gray solid. The intermediate M2 (1.89 g, 9.04 mmol) was
dissolved in MeOH (6 ml), and then a solution of NaOH (1.44 g,
36.16 mmol) in water (3 ml) was added and stirred at room tem-
perature for 2 h. The solvent was concentrated under reduced
pressure, water (30 ml) was added, and the pH was adjusted to 1–2
with a 3 M aqueous HCl solution. The product was extracted by ethyl
acetate and dried over anhydrous sodium sulfate. Filtration and
Triphenylphosphine (15.00 mmol), 4‐hydroxybenzaldehyde M7
(10.00 mmol), and corresponding alcohol (10.00 mmol) were dis-
solved in dry THF (10 ml). After cooling to −20°C, a solution of
diethyl azodicarboxylate (15.00 mmol) in anhydrous tetrahydrofuran
(30 ml) was added. The mixture was stirred at −20°C for 30 min and
transferred to room temperature until reaction was complete as
indicated by TLC. The organic solvent was removed in vacuo, and the
residue was diluted with ethyl acetate (50 ml) and washed with sa-
turated aqueous brine. The organic phase was dried over Na2SO4,
evaporated to dryness, and purified with chromatography on silica
gel to give products M8a–M8m.
1
concentration in vacuo gave the product M3. H NMR (400 MHz,
dimethyl sulfoxide [DMSO]‐d6) δ 12.45 (s, 1H), 7.55 (d, J = 5.4 Hz,
1H), 7.22 (d, J = 5.4 Hz, 1H), 7.13 (s, 1H), 4.00 (s, 3H).
|
4.1.4
Synthesis of 1,2,3,4‐tetrahydroquinolin‐5‐
amine (M5)
tert‐Butyl 3‐(4‐formylphenoxy)pyrrolidine‐1‐carboxylate (M8g)
1
Yield 66%, off‐white oil. H NMR (400 MHz, DMSO‐d6) δ 9.85 (s, 1H),
Yield 58%, yellow solid. To a solution of 5‐nitroquinoline (1.74 g,
7.83 (d, J = 8.7 Hz, 2H), 7.13 (d, J = 8.7 Hz, 2H), 4.44 (dt, J = 9.8,
5.7 Hz, 1H), 2.11–2.03 (m, 2H), 1.87–1.79 (m, 2H), 1.39 (s, 9H).
11.48 mmol) and nickel chloride hexahydrate (13.64 g, 57.42 mmol) in

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|
4.1.7
General procedure for the synthesis of
6.84 (s, 1H), 6.78 (d, J = 8.6 Hz, 2H), 4.84 (s, 1H), 3.96 (s, 2H), 3.93
(s, 3H), 3.33–3.22 (m, 2H), 2.99–2.92 (m, 2H), 2.78 (t, J = 6.5 Hz, 2H),
1.83–1.73 (m, 2H), 1.40 (d, J = 7.9 Hz, 9H).
compounds M9a–M9m
M8a–M8m (10.00 mmol) were added into methanol (20 ml) and stir-
red in an ice bath for 10 min, and then sodium borohydride
(20.00 mmol) was added to the mixture within 30 min. Next, the
mixture was stirred at room temperature until the reaction was
complete as indicated by TLC. The solvent was vacuum‐
concentrated, and the off‐white oil‐like M9a–M9m were obtained.
|
4.1.10
General procedure for the synthesis of
compounds B1–B5, C1–C14, and D1–D6
The synthetic routes of these compounds are similar to that de-
scribed in Scheme 1, and please refer to the above synthetic methods
for detailed synthetic steps.
|
4.1.8
General procedure for the synthesis of
compounds M10a–M10m
4‐Methyl‐N‐{1‐[4‐(piperidin‐4‐ylmethoxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A1)
Triphenylphosphine
(4.88 mmol)
and
4‐dimethylaminopyridine
1
(1.30 mmol) were added to dichloromethane (40 ml) solution, stirred
in ice bath for 10 min, and I2 (1.24 g, 4.88 mmol) was added. The mix-
ture was stirred at room temperature for 1 h, intermediates M9a–M9m
were added, and the mixture was stirred continuously at room tem-
perature for 1–4 h until reaction was complete as indicated by TLC. The
mixture was concentrated under vacuum and the condensate was
purified by silica gel chromatography (petroleum ether/ethyl acetate =
20:1) to obtain M10a–M10m.
Yield 74%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 9.45 (s, 1H),
7.49 (d, J = 5.2 Hz, 1H), 7.28 (s, 1H), 7.23 (d, J = 5.2 Hz, 1H), 7.16
(d, J = 8.2 Hz, 2H), 6.90 (t, J = 7.9 Hz, 1H), 6.85 (d, J = 8.3 Hz, 2H), 6.53
(d, J = 7.6 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H), 4.80 (s, 1H), 4.44 (s, 2H),
4.00 (s, 3H), 3.69–3.39 (m, 4H), 3.33 (s, 2H), 2.92–2.75 (m, 3H), 2.65
(t, J = 5.6 Hz, 2H), 1.98 (dt, J = 12.9, 6.0 Hz, 2H), 1.90–1.86 (m, 2H),
13
1.85 (s, 1H). C NMR (151 MHz, DMSO‐d6) δ 175.1, 160.6, 158.1,
146.3, 144.8, 136.1, 130.9, 128.3, 128.1, 126.4, 120.9, 118.8, 114.9,
114.7, 111.6, 109.3, 105.1, 73.0, 54.5, 49.5, 46.2, 36.6, 34.7, 30.3,
+
tert‐Butyl 3‐[4‐(iodomethyl)phenoxy]pyrrolidine‐1‐
carboxylate (M10g)
25.9, 23.6, 21.9. HR‐MS (ESI): C30H34N4O2S, [M+H] : 515.2481;
found: 515.2479.
1
Yield 30%, yellow solid. H NMR (400 MHz, DMSO‐d6) δ 7.41 (d,
J = 8.7 Hz, 2H), 7.11 (d, J = 8.7 Hz, 2H), 5.35 (s, 2H), 5.06 (s, 1H), 3.57
(d, J = 12.4 Hz, 2H), 3.48–3.42 (m, 2H), 2.25–2.08 (m, 2H), 2.10–1.94
(m, 2H), 1.40 (s, 10H).
N‐[1‐(4‐{[(1s,4s)‐4‐Aminocyclohexyl]oxy}benzyl)‐1,2,3,4‐
tetrahydroquinolin‐5‐yl]‐4‐methyl‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A2)
1
Yield 73%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 9.47 (s, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.30 (s, 1H), 7.23 (d, J = 5.3 Hz, 1H), 7.20 (d,
J = 8.2 Hz, 2H), 6.91 (d, J = 8.4 Hz, 3H), 6.58 (s, 1H), 6.48 (s, 1H), 4.53
(s, 1H), 4.45 (s, 2H), 4.00 (s, 3H), 3.37–3.28 (m, 4H), 2.66 (t, J = 6.3 Hz,
4H), 2.00–1.97 (m, 2H), 1.91–1.86 (m, 3H), 1.78–1.74 (m, 3H), 1.63
|
4.1.9
General procedure for the synthesis of
compounds A7, A14–A16, M11A–M11i
13
A mixture of potassium carbonate (0.07 g, 0.48 mmol), inter-
mediate M6 (0.12 g, 0.40 mmol), and intermediates M10a–M10m
(0.48 mmol) in dry DMF (2 ml) was stirred at room temperature for
2–4 h until the reaction was complete as indicated by TLC. The
reaction mixture was poured into 20 ml water and the mixture was
extracted with ethyl acetate (20 ml × 3). The combined organic
phase was washed with water (20 ml × 2), dried over anhydrous
Na2SO4, and concentrated under vacuum to afford the crude
product. The crude product was chromatographed on silica gel
(petroleum ether/ethyl acetate = 8:1) to give A7, A14–A16,
M11A–M11i.
(d, J = 13.1 Hz, 2H). C NMR (151 MHz, DMSO‐d6) δ 160.6, 156.3,
144.8, 136.2, 132.4, 130.7, 130.1, 128.6, 128.2, 126.5, 120.9, 111.6,
105.2, 70.2, 49.5, 48.7, 44.0, 34.8, 27.4, 25.2, 23.5. HR‐MS (ESI):
+
C
30H34N4O2S, [M+H] : 515.2481; found: 515.2472.
N‐[1‐(4‐{[(1r,4r)‐4‐Aminocyclohexyl]oxy}benzyl)‐1,2,3,4‐
tetrahydroquinolin‐5‐yl]‐4‐methyl‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A3)
1
Yield 72%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 9.49 (s, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.30 (s, 1H), 7.23 (d, J = 5.3 Hz, 1H), 7.20
(d, J = 8.2 Hz, 2H), 6.92 (d, J = 8.6 Hz, 3H), 6.58 (s, 1H), 6.49 (s, 1H),
4.53 (s, 1H), 4.46 (s, 2H), 4.00 (s, 3H), 3.34–3.32 (m, 2H), 2.67 (t,
J = 6.3 Hz, 2H), 1.93 (d, J = 13.2 Hz, 2H), 1.91–1.87 (m, 2H), 1.79–1.76
(m, 1H), 1.77–1.73 (m, 2H), 1.69 (d, J = 13.4 Hz, 2H), 1.63
tert‐Butyl‐3‐(4‐{[5‐(4‐methyl‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamido)‐3,4‐dihydroquinolin‐1(2H)‐yl]methyl}phenoxy)-
pyrrolidine‐1‐carboxylate (M11g)
13
(d, J = 13.3 Hz, 2H). C NMR (151 MHz, DMSO‐d6) δ 160.6, 156.3,
1
Yield 30%, yellow solid. H NMR (400 MHz, DMSO‐d6) δ 8.91 (s, 1H),
144.8, 136.2, 130.7, 130.1, 128.6, 128.2, 126.5, 120.9, 116.5, 111.6,
7.61 (d, J = 7.6 Hz, 1H), 7.45 (d, J = 5.3 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H),
7.16 (d, J = 5.3 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.90 (d, J = 6.0 Hz, 1H),
105.2, 70.2, 48.7, 44.0, 27.4, 25.2, 25.2, 23.5, 22.6. HR‐MS (ESI):
+
C30H34N4O2S, [M+H] : 515.2481; found: 515.2465.

YAN ET AL.
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(S)‐4‐Methyl‐N‐{1‐[4‐(pyrrolidin‐3‐ylmethoxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A4)
4‐Methyl‐N‐(1‐{4‐[(tetrahydro‐2H‐pyran‐4‐yl)methoxy]benzyl}‐
1,2,3,4‐tetrahydroquinolin‐5‐yl)‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A7)
1
1
Yield 78%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 9.43 (s, 1H),
Yield 74%, white solid. H NMR (600 MHz, DMSO‐d6) δ 9.44 (s, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.28 (s, 1H), 7.22 (d, J = 5.3 Hz, 1H), 7.18
(d, J = 8.5 Hz, 2H), 6.89 (dt, J = 7.8, 3.6 Hz, 3H), 6.53 (d, J = 7.6 Hz, 1H),
6.44 (d, J = 8.3 Hz, 1H), 4.44 (s, 2H), 4.00 (s, 3H), 3.94–3.86 (m, 4H),
3.34–3.32 (m, 2H), 3.03 (qd, J = 7.2, 5.4 Hz, 3H), 2.65 (t, J = 6.5 Hz,
2H), 2.46 (q, J = 7.2 Hz, 2H), 2.00–1.92 (m, 2H), 1.57 (dd, J = 12.8,
7.48 (d, J = 5.3 Hz, 1H), 7.28 (s, 1H), 7.22 (d, J = 5.3 Hz, 1H), 7.17 (d,
J = 8.5 Hz, 2H), 6.88 (dd, J = 8.4, 3.3 Hz, 3H), 6.54 (d, J = 7.7 Hz, 1H),
6.44 (d, J = 8.3 Hz, 1H), 4.43 (s, 2H), 4.00 (s, 3H), 3.86 (dd, J = 11.1,
3.2 Hz, 2H), 3.79 (d, J = 6.4 Hz, 2H), 3.32 (m, 7H), 2.65 (t, J = 6.5 Hz,
2H), 1.97 (dt, J = 6.1, 2.4 Hz, 1H), 1.88–1.85 (m, 2H), 1.67–1.64 (m,
13
13
6.9 Hz, 1H). C NMR (151 MHz, DMSO‐d6) δ 169.2, 160.5, 157.8,
2H). C NMR (151 MHz, DMSO‐d6) δ 169.2, 160.5, 157.8, 146.2,
146.2, 144.8, 136.1, 131.2, 130.6, 128.3, 128.2, 126.5, 120.8, 118.83,
115.0, 114.7, 111.6, 109.3, 104.7, 69.6, 55.1, 49.6, 48.3, 46.5, 45.3,
144.8, 136.1, 131.0, 130.7, 128.3, 126.4, 120.9, 118.8, 114.9, 114.7,
111.6, 109.3, 105.1, 69.8, 54.5, 49.5, 48.6, 46.1, 45.6, 34.8, 33.7, 23.6,
+
+
34.8, 33.74, 23.6, 23.1. HR‐MS (ESI): C29H32N4O2S, [M+H] :
23.1. HR‐MS (ESI): C30H34N4O2S, [M+Na] : 538.2140; found:
501.2324; found: 501.2305.
538.2137.
(R)‐4‐Methyl‐N‐{1‐[4‐(pyrrolidin‐3‐ylmethoxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A5)
4‐Methyl‐N‐{1‐[4‐(pyrrolidin‐3‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A8)
1
1
Yield 77%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 9.44 (s, 1H),
Yield 79%, brown oil. H NMR (600 MHz, DMSO‐d6) δ 9.45 (s, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.28 (s, 1H), 7.23 (d, J = 5.4 Hz, 1H), 7.18
(d, J = 8.6 Hz, 2H), 6.90 (d, J = 3.2 Hz, 4H), 6.53 (d, J = 7.7 Hz, 1H), 6.43
(d, J = 8.3 Hz, 1H), 4.44 (s, 2H), 4.00 (s, 3H), 3.34–3.32 (m, 2H),
3.12–3.07 (m, 2H), 2.93 (t, J = 7.6 Hz, 2H), 2.77 (d, J = 6.2 Hz, 2H),
2.65 (t, J = 6.4 Hz, 2H), 2.54 (d, J = 7.4 Hz, 1H), 1.98–1.91 (m, 2H),
7.49 (d, J = 5.2 Hz, 1H), 7.28 (s, 1H), 7.23 (d, J = 5.2 Hz, 1H), 7.16
(d, J = 8.2 Hz, 2H), 6.90 (t, J = 7.9 Hz, 1H), 6.85 (d, J = 8.3 Hz, 2H), 6.53
(d, J = 7.6 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H), 4.80 (s, 1H), 4.44 (s, 2H),
4.00 (s, 3H), 3.62–3.38 (m, 4H), 3.33 (s, 2H), 2.84–2.75 (m, 2H), 2.65
(t, J = 5.6 Hz, 2H), 1.98 (dd, J = 13.7, 6.5 Hz, 1H), 1.89–1.86 (m, 2H).
13
13
1.57–1.54 (m, 1H). C NMR (151 MHz, DMSO‐d6) δ 169.2, 160.5,
C NMR (151 MHz, DMSO‐d6) δ 160.5, 156.8, 146.3, 144.8, 136.1,
157.8, 146.2, 144.8, 136.1, 131.3, 130.7, 128.3, 128.2, 126.4, 120.9,
130.9, 130.7, 128.4, 128.1, 126.5, 120.9, 118.8, 115.8, 114.7, 111.6,
118.8, 115.0, 114.7, 111.6, 109.3, 105.0, 69.8, 49.6, 48.7, 46.1, 45.6,
+
109.3, 105.1, 78.5, 54.5, 53.3, 49.6, 45.9, 39.6, 33.6, 23.6, 21.9. HR‐
+
34.8, 33.7, 23.6, 23.1. HR‐MS (ESI):
C
29H32N4O2S, [M+H] :
MS (ESI): C28H30N4O2S, [M+H] : 487.2168; found: 487.2161.
501.2324; found: 501.2314.
|
4.1.12
Synthesis of 4‐methyl‐N‐(1‐{4‐[(1‐
|
4.1.11
Synthesis of 4‐methyl‐N‐{1‐[4‐(piperidin‐4‐
methylpiperidin‐4‐yl)oxy]benzyl}‐1,2,3,4‐
tetrahydroquinolin‐5‐yl)‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A9)
yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}‐4H‐
thieno[3,2‐b]pyrrole‐5‐carboxamide (A6)
Yield 79%, yellow solid. M11f (0.2 g, 0.34 mmol) was added to HCl/
MeOH (3 ml, 4 mol/l), and the mixture was stirred for 30 min at room
temperature for 2 h. The solvent was evaporated and the pH
was adjusted to 10 with sodium carbonate. Then the mixture was
extracted with ethyl acetate (20 ml × 3), and the combined organic
phase was washed with water (20 ml × 2), dried over anhydrous
Na2SO4, and concentrated under vacuum to afford A6 (0.16 g,
Yield 60%, yellow oil. A mixture of A6 (0.2 g, 0.4 mmol), 40% for-
maldehyde aqueous solution (0.75 ml), and formic acid (2 ml) was
stirred for 4 h. The reaction mixture was poured into 20 ml water and
saturated sodium carbonate solution was added to adjust the pH to
10. The mixture was extracted with ethyl acetate (10 ml × 3), com-
bined with ethyl acetate phase, dried with anhydrous sodium sulfate,
and concentrated. Chromatography on silica gel (petroleum ether/
ethyl acetate = 5:1) was carried out to afford A9 (0.13 g, 60%) as a
1
79.4%) as a yellow solid. H NMR (600 MHz, DMSO‐d6) δ 9.48
1
(s, 1H), 7.48 (d, J = 5.3 Hz, 1H), 7.30 (s, 1H), 7.22 (d, J = 5.3 Hz, 1H),
7.15 (d, J = 8.5 Hz, 2H), 6.91–6.88 (m, 3H), 6.54 (d, J = 7.6 Hz, 1H),
6.44 (d, J = 8.4 Hz, 1H), 4.43 (s, 2H), 4.00 (s, 3H), 3.33–3.31 (m, 2H),
yellow solid. H NMR (600 MHz, DMSO‐d6) δ 9.45 (s, 1H), 7.49
(d, J = 5.3 Hz, 1H), 7.29 (s, 1H), 7.22 (d, J = 5.3 Hz, 1H), 7.16
(d, J = 8.6 Hz, 2H), 6.91 (d, J = 3.1 Hz, 1H), 6.89 (t, J = 3.9 Hz, 2H), 6.53
(d, J = 7.7 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H), 4.43 (s, 2H), 4.31 (dt,
J = 7.8, 4.1 Hz, 1H), 4.00 (s, 3H), 3.33–3.31 (m, 2H), 2.75 (q, J = 7.1 Hz,
2H), 2.65 (t, J = 6.3 Hz, 4H), 2.20 (s, 3H), 1.88–1.85 (m, 2H), 1.77 (s,
2.94–2.90 (m, 2H), 2.66 (t, J = 6.3 Hz, 2H), 2.54 (d, J = 10.7 Hz, 2H),
13
1.89–1.86 (m, 4H), 1.62 (d, J = 1.0 Hz, 1H), 1.57–1.51 (m, 3H).
C
NMR (151 MHz, DMSO‐d6) δ 170.8, 160.6, 156.4, 146.3, 144.8,
136.1, 131.0, 130.7, 128.3, 128.0, 126.4, 120.8, 118.8, 116.3, 111.6,
109.3, 105.1, 73.9, 54.5, 49.5, 44.2, 34.8, 32.8, 23.6, 21.9, 21.1. HR‐
13
2H), 1.65–1.59 (m, 2H). C NMR (151 MHz, DMSO‐d6) δ 169.2,
160.6, 156.3, 146.3, 144.8, 136.1, 131.2, 130.7, 128.3, 128.1, 126.5,
120.9, 118.8, 116.3, 111.6, 109.3, 105.1, 72.0, 54.5, 52.6, 49.5, 45.8,
+
MS (ESI): C29H32N4O2S, [M+H] : 501.2324; found: 501.2319.

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+
30.7, 23.6, 21.9, 21.7. HR‐MS (ESI): C30H34N4O2S, [M+H] :
118.8, 114.9, 111.6, 109.3, 105.1, 67.6, 65.5, 54.5, 50.8, 49.6, 36.6,
30.5, 25.8.
515.2481; found: 515.2470.
N‐{1‐[4‐(2‐Aminoethoxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}‐4‐
methyl‐4H‐thieno[3,2‐b]pyrrole‐5‐carboxamide (A10)
N‐(1‐{4‐[(3‐Aminobenzyl)oxy]benzyl}‐1,2,3,4‐tetrahydroquinolin‐5‐
yl)‐4‐methyl‐4H‐thieno[3,2‐b]pyrrole‐5‐carboxamide (A14)
1
1
Yield 76%, white solid. H NMR (600 MHz, DMSO‐d6) δ 9.53 (s, 1H),
Yield 79%, brown oil. H NMR (600 MHz, DMSO‐d6) δ 9.44 (s, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.34 (s, 1H), 7.23 (d, J = 5.3 Hz, 1H), 7.21
(d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.6 Hz, 2H), 6.90 (t, J = 8.0 Hz, 1H), 6.53
(d, J = 7.7 Hz, 1H), 6.43 (d, J = 8.3 Hz, 1H), 4.46 (s, 2H), 4.19 (t,
J = 5.2 Hz, 2H), 4.00 (s, 3H), 3.16 (t, J = 5.3 Hz, 3H), 2.66 (t, J = 6.5 Hz,
7.48 (s, 1H), 7.28 (s, 1H), 7.22 (d, J = 5.3 Hz, 1H), 7.17 (d, J = 8.5 Hz,
2H), 7.00 (t, J = 7.7 Hz, 1H), 6.93 (d, J = 8.6 Hz, 2H), 6.90 (t, J = 8.0 Hz,
1H), 6.62 (s, 1H), 6.54 (t, J = 8.3 Hz, 2H), 6.49 (d, J = 7.8 Hz, 1H), 6.44
(d, J = 8.3 Hz, 1H), 5.09 (s, 2H), 4.91 (s, 2H), 4.44 (s, 2H), 4.00 (s, 3H),
13
13
2H), 1.89–1.86 (m, 2H). C NMR (151 MHz, DMSO) δ 160.5, 157.2,
3.33 (d, J = 5.3 Hz, 2H), 2.65 (t, J = 6.2 Hz, 2H), 1.90–1.85 (m, 2H).
C
146.2, 144.8, 136.2, 131.8, 130.6, 130.4, 130.1, 128.4, 128.2, 126.4,
120.8, 118.9, 115.2, 114.8, 111.6, 109.3, 105.2, 64.7, 54.5, 49.6, 38.6,
34.8, 23.6, 21.9.
NMR (151 MHz, DMSO‐d6) δ 160.5, 157.8, 149.2, 144.8, 138.1,
136.1, 131.1, 130.7, 130.1, 129.3, 128.3, 126.5, 118.8, 115.3, 115.2,
114.7, 113.8, 113.3, 111.6, 109.3, 105.0, 70.0, 54.5, 49.5, 34.7, 23.5,
+
21.8. HR‐MS (ESI):
C31H30N4O2S, [M+Na] : 555.1987; found:
N‐{1‐[4‐(3‐Aminopropoxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}‐
4‐methyl‐4H‐thieno[3,2‐b]pyrrole‐5‐carboxamide (A11)
555.1982.
1
Yield 78%, off‐white solid. H NMR (600 MHz, DMSO‐d6) δ 9.44
N‐(1‐{4‐[(3‐Cyanobenzyl)oxy]benzyl}‐1,2,3,4‐tetrahydroquinolin‐5‐
yl)‐4‐methyl‐4H‐thieno[3,2‐b]pyrrole‐5‐carboxamide (A15)
(s, 1H), 7.49 (d, J = 5.2 Hz, 1H), 7.28 (s, 1H), 7.22 (d, J = 5.2 Hz, 1H),
7.16 (d, J = 8.2 Hz, 2H), 6.90 (t, J = 7.9 Hz, 1H), 6.85 (d, J = 8.3 Hz,
2H), 6.53 (d, J = 7.6 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H), 4.79 (s, 1H),
4.44 (s, 2H), 4.00 (s, 3H), 3.49 (d, J = 15.3 Hz, 2H), 3.33 (s, 2H),
2.85–2.75 (m, 2H), 2.65 (t, J = 5.6 Hz, 2H), 1.98 (dd, J = 13.7, 6.5 Hz,
1
Yield 80%, white solid. H NMR (600 MHz, DMSO‐d6) δ 9.44 (s, 1H),
7.90 (s, 1H), 7.79 (t, J = 8.4 Hz, 2H), 7.61 (t, J = 7.8 Hz, 1H), 7.49
(d, J = 5.3 Hz, 1H), 7.28 (s, 1H), 7.22 (d, J = 5.3 Hz, 1H), 7.20 (d,
J = 8.6 Hz, 2H), 6.99 (d, J = 8.6 Hz, 2H), 6.90 (t, J = 8.0 Hz, 1H), 6.54 (d,
J = 7.7 Hz, 1H), 6.43 (d, J = 8.3 Hz, 1H), 5.14 (s, 2H), 4.45 (s, 2H), 4.00
(s, 3H), 3.33–3.31 (m, 2H), 2.66 (t, J = 6.4 Hz, 2H), 1.89–1.86 (m, 2H).
13
1H), 1.89–1.86 (m, 2H). C NMR (151 MHz, DMSO‐d6) δ: 172.3,
160.2, 157.9, 144.8, 141.8, 131.3, 130.2, 129.1, 128.4, 126.1,
13
122.3, 122.0, 120.9, 114.6, 111.5, 104.7, 65.5, 57.2, 55.1, 34.8,
+
C NMR (151 MHz, DMSO‐d6) δ 160.5, 158.0, 146.2, 144.8, 136.1,
27.3, 25.4, 21.7, 17.1. HR‐MS (ESI):
C
27H30N4O2S, [M+H] :
131.0, 130.7, 128.3, 128.1, 126.4, 120.9, 118.8, 114.9, 114.7, 111.6,
475.2168; found: 475.2159.
109.2, 105.0, 72.5, 67.1, 54.5, 49.5, 34.9, 34.8, 29.7, 23.6, 21.9. HR‐
+
MS (ESI): C32H28N4O2S, [M+Na] : 555.1831; found: 555.1827.
4‐Methyl‐N‐(1‐{4‐[3‐(methylamino)propoxy]benzyl}‐1,2,3,4‐
tetrahydroquinolin‐5‐yl)‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A12)
4‐Methyl‐N‐(1‐{4‐[(3‐nitrobenzyl)oxy]benzyl}‐1,2,3,4‐
tetrahydroquinolin‐5‐yl)‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A16)
1
Yield 79%, white solid. H NMR (600 MHz, DMSO‐d6) δ 9.49 (s, 1H),
1
7.49 (d, J = 5.3 Hz, 1H), 7.29 (s, 1H), 7.23 (s, 1H), 7.17 (d, J = 8.6 Hz,
2H), 6.91–6.88 (m, 3H), 6.53 (d, J = 7.6 Hz, 1H), 6.44 (d, J = 8.3 Hz,
1H), 4.45 (s, 2H), 4.23 (t, J = 6.6 Hz, 1H), 4.00 (s, 3H), 3.97
Yield 81%, brown solid. H NMR (600 MHz, DMSO‐d6) δ 9.44 (s, 1H),
8.31 (s, 1H), 8.19 (d, J = 9.7 Hz, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.69
(t, J = 7.9 Hz, 1H), 7.49 (d, J = 5.3 Hz, 1H), 7.29 (s, 1H), 7.23–7.20
(m, 3H), 7.01 (d, J = 8.6 Hz, 2H), 6.90 (t, J = 8.0 Hz, 1H), 6.54 (d,
J = 7.7 Hz, 1H), 6.44 (d, J = 8.4 Hz, 1H), 5.24 (s, 2H), 4.45 (s, 2H), 4.00
(s, 3H), 3.33 (d, J = 5.6 Hz, 2H), 2.66 (t, J = 6.4 Hz, 2H), 1.89–1.86 (m,
(t, J = 5.6 Hz, 2H), 3.34–3.31 (m, 4H), 2.80 (t, J = 5.6 Hz, 2H), 2.65
13
(t, J = 6.3 Hz, 2H), 2.32 (s, 3H), 1.90–1.85 (m, 2H).
C NMR
(151 MHz, DMSO) δ 160.6, 157.9, 146.3, 144.8, 136.1, 132.0, 131.0,
13
130.7, 129.1, 128.2, 126.4, 120.9, 118.8, 114.9, 111.6, 109.3, 105.1,
2H). C NMR (151 MHz, DMSO‐d6) δ 160.5, 157.3, 148.3, 146.2,
67.6, 65.4, 50.8, 49.5, 36.6, 34.8, 30.5, 23.6, 21.9. HR‐MS (ESI):
144.8, 136.1, 134.4, 130.7, 131.7, 130.5, 128.4, 128.2, 126.5, 123.1,
+
C
28H32N4O2S, [M+H] : 489.2324; found: 489.2158.
122.4, 120.9, 118.8, 115.3, 114.7, 111.6, 109.3, 105.1, 68.4, 54.5,
+
49.5, 34.8, 23.6, 21.9. HR‐MS (ESI):
C31H28N4O2S, [M+Na] :
4‐Methyl‐N‐(1‐{4‐[2‐(methylamino)ethoxy]benzyl}‐1,2,3,4‐
tetrahydroquinolin‐5‐yl)‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (A13)
575.1729; found 575.1735.
N‐{1‐[2‐Fluoro‐4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐4‐methyl‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (B1)
1
Yield 81%, white solid. H NMR (600 MHz, DMSO‐d6) δ 9.49 (s, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.29 (s, 1H), 7.23 (d, J = 5.3 Hz, 1H), 7.17 (d,
J = 8.3 Hz, 2H), 6.89 (d, J = 7.0 Hz, 3H), 6.53 (d, J = 7.7 Hz, 1H), 6.44 (d,
J = 8.4 Hz, 1H), 4.45 (s, 2H), 4.00 (s, 3H), 3.97 (t, J = 5.5 Hz, 2H), 2.80
(d, J = 11.3 Hz, 2H), 2.65 (t, J = 6.5 Hz, 2H), 2.32 (s, 3H), 1.88 (t,
1
Yield 81%, white solid. H NMR (600 MHz, DMSO‐d6) δ 9.50 (s, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.29 (s, 1H), 7.23 (d, J = 5.3 Hz, 1H), 7.15
(t, J = 8.5 Hz, 1H), 7.06 (d, J = 12.1 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H),
6.91 (t, J = 8.0 Hz, 1H), 6.55 (d, J = 7.7 Hz, 1H), 6.41 (d, J = 8.3 Hz, 1H),
4.45 (s, 2H), 4.33 (dt, J = 9.0, 4.8 Hz, 1H), 4.00 (s, 3H), 3.35–3.33 (m,
13
J = 5.8 Hz, 2H). C NMR (151 MHz, DMSO) δ 167.4, 160.6, 157.9,
146.2, 144.8, 132.2, 131.0, 130.7, 129.1, 128.4, 128.1, 126.4, 120.9,

YAN ET AL.
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|
4H), 2.93 (dd, J = 8.6, 4.1 Hz, 2H), 2.66 (t, J = 6.2 Hz, 2H), 2.53 (s, 2H),
13
4.46 (s, 2H), 4.39 (dt, J = 8.8, 4.8 Hz, 1H), 4.00 (s, 3H), 3.34–3.31 (m,
2H), 2.93 (d, J = 12.3 Hz, 2H), 2.65 (t, J = 6.2 Hz, 2H), 2.57 (t,
13
1.90–1.86 (m, 4H).
C NMR (151 MHz, DMSO) δ 174.9, 160.6,
152.5, 146.1, 144.8, 143.8, 136.2, 133.1, 130.6, 128.2, 126.5, 123.1,
120.9, 118.9, 118.3, 115.0, 111.6, 109.2, 105.1, 76.0, 54.2, 49.6, 44.0,
34.8, 32.8, 25.7, 23.6, 21.9.
J = 10.4 Hz, 2H), 1.93–1.81 (m, 6H). C NMR (151 MHz, DMSO) δ
162.2, 160.6, 157.9, 146.0, 144.8, 136.2, 130.7, 129.6, 128.1, 126.5,
120.9, 118.9, 114.9, 112.2, 111.6, 109.1, 105.1, 103.7, 103.6, 74.1,
49.4, 48.7, 43.9, 34.8, 32.4, 23.6, 21.8.
N‐{1‐[2‐Methoxy‐4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐4‐methyl‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (B2)
N‐{1‐[4‐(Piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}-
benzofuran‐2‐carboxamide (C1)
1
1
Yield 79%, white solid. H NMR (600 MHz, DMSO‐d6) δ 9.46 (s, 1H),
Yield 75%, yellow oil. H NMR (600 MHz, DMSO‐d6) δ 10.23 (s, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.28 (s, 1H), 7.23 (d, J = 5.3 Hz, 1H), 6.92–6.88
(m, 3H), 6.73 (d, J = 8.0 Hz, 1H), 6.54 (d, J = 7.7 Hz, 1H), 6.45
(d, J = 8.3 Hz, 1H), 4.43 (s, 2H), 4.22 (t, J = 6.5 Hz, 1H), 4.00 (s, 3H),
9.42 (s, 1H), 9.34 (s, 1H), 8.40 (s, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.97
(d, J = 7.3 Hz, 1H), 7.47 (t, J = 7.3 Hz, 2H), 7.25 (d, J = 6.4 Hz, 2H), 6.98
(d, J = 8.0 Hz, 3H), 6.64 (d, J = 35.6 Hz, 2H), 4.48 (s, 2H), 3.44
(q, J = 7.0 Hz, 1H), 3.33 (s, 2H), 3.17 (s, 2H), 3.03 (s, 2H), 2.70 (s, 2H),
3.72 (s, 3H), 3.35–3.31 (m, 4H), 2.92 (d, J = 11.7 Hz, 2H), 2.66
13
13
(t, J = 6.5 Hz, 2H), 2.47 (d, J = 17.4 Hz, 2H), 1.90–1.83 (m, 4H).
C
2.10 (s, 2H), 1.91 (s, 2H), 1.88–1.83 (m, 2H). C NMR (151 MHz,
NMR (151 MHz, DMSO) δ 167.4, 160.5, 154.4, 146.4, 144.8, 136.1,
130.7, 129.6, 128.2, 127.3, 126.5, 125.4, 120.9, 118.8, 114.6, 113.9,
111.6, 109.3, 105.1, 65.5, 54.7, 49.6, 43.8, 34.8, 30.5, 23.6, 21.9, 16.8.
DMSO‐d6) δ 160.7, 156.4, 146.3, 140.8, 139.7, 135.7, 130.9, 128.3,
126.7, 126.6, 125.9, 125.7, 125.4, 123.2, 116.3, 114.6, 109.6, 73.9,
+
54.5, 49.5, 44.1, 32.8, 23.6, 21.8. HR‐MS (ESI): C30H31N3O3, [M+H] :
482.2444; found 482.2437.
4‐Methyl‐N‐{1‐[2‐methyl‐4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (B3)
N‐{1‐[4‐(Piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}-
benzo[b]thiophene‐2‐carboxamide (C2)
1
1
Yield 77%, white solid. H NMR (600 MHz, DMSO‐d6) δ 9.44 (s, 1H),
Yield 80%, yellow oil. H NMR (600 MHz, DMSO‐d6) δ 10.14 (s, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.28 (s, 1H), 7.23 (d, J = 5.3 Hz, 1H), 7.05
(s, 1H), 7.01–6.99 (m, 1H), 6.90 (dd, J = 8.1, 6.2 Hz, 2H), 6.53
(d, J = 7.6 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H), 4.39 (s, 2H), 4.35 (dt,
J = 8.1, 4.3 Hz, 1H), 4.00 (s, 3H), 3.35–3.28 (m, 4H), 2.95–2.91 (m,
9.31 (s, 1H), 9.22 (s, 1H), 8.35 (s, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.98
(d, J = 7.2 Hz, 1H), 7.48 (t, J = 7.5 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H), 6.97
(d, J = 8.5 Hz, 2H), 6.94 (t, J = 8.1 Hz, 1H), 6.54 (d, J = 49.1 Hz, 2H),
4.63–4.60 (m, 1H), 4.47 (s, 2H), 3.36–3.33 (m, 2H), 3.20–3.17 (m, 2H),
3.06–3.01 (m, 2H), 2.68 (t, J = 6.1 Hz, 2H), 2.12–2.08 (m, 2H), 1.90 (d,
2H), 2.65 (t, J = 6.4 Hz, 2H), 2.58 (t, J = 10.4 Hz, 2H), 2.13 (s, 3H),
13
13
1.89–1.85 (m, 4H).
C NMR (151 MHz, DMSO) δ 167.4, 160.5,
J = 5.2 Hz, 2H), 1.87–1.83 (m, 2H). C NMR (151 MHz, DMSO‐d6) δ
156.6, 150.8, 146.3, 145.5, 144.8, 136.1, 132.0, 130.7, 129.1, 128.1,
126.5, 120.9, 118.9, 114.7, 111.6, 109.3, 105.0, 68.3, 65.5, 56.0, 54.9,
49.6, 44.2, 34.8, 30.4, 23.6, 22.3, 21.9.
160.6, 156.3, 146.3, 140.5, 139.7, 135.6, 130.9, 126.8, 126.6, 126.1,
125.7, 125.4, 123.3, 118.9, 116.3, 114.5, 109.6, 73.8, 54.5, 49.5, 32.6,
+
23.6, 21.8. HR‐MS (ESI): C30H31N3O2S, [M+H] : 498.2215; found
498.2208.
4‐Methyl‐N‐{1‐[2‐nitro‐4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (B4)
N‐{1‐[4‐(Piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}‐
1H‐indole‐2‐carboxamide (C3)
1
1
Yield 80%, white solid. H NMR (600 MHz, DMSO‐d6) δ 9.49 (s, 1H),
Yield 80%, yellow oil. H NMR (600 MHz, DMSO‐d6) δ 11.76 (s, 1H),
7.70 (d, J = 2.1 Hz, 1H), 7.50–7.47 (m, 2H), 7.38 (d, J = 9.0 Hz, 1H),
7.29 (s, 1H), 7.23 (d, J = 5.3 Hz, 1H), 6.92 (t, J = 8.0 Hz, 1H), 6.57
(d, J = 7.7 Hz, 1H), 6.42 (d, J = 8.3 Hz, 1H), 4.66–4.61 (m, 1H), 4.51
9.81 (s, 1H), 9.26 (s, 1H), 9.17 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.45
(d, J = 8.2 Hz, 1H), 7.33 (s, 1H), 7.26–7.18 (m, 4H), 7.06 (t, J = 7.5 Hz,
1H), 6.97 (d, J = 8.7 Hz, 2H), 6.56 (d, J = 67.6 Hz, 2H), 4.61
(d, J = 3.5 Hz, 1H), 4.47 (s, 2H), 3.35–3.33 (m, 2H), 3.20–3.17 (m, 2H),
3.05–3.02 (m, 2H), 2.69 (t, J = 6.3 Hz, 2H), 2.11–2.08 (m, 2H),
(s, 2H), 4.00 (s, 3H), 3.38–3.33 (m, 4H), 2.93–2.85 (m, 2H), 2.66 (t,
13
J = 6.3 Hz, 2H), 2.59–2.52 (m, 2H), 1.89 (dd, J = 11.4, 6.2 Hz, 4H).
C
13
NMR (151 MHz, DMSO) δ 167.4, 160.5, 149.0, 145.9, 144.8, 140.9,
136.2, 132.0, 129.1, 128.2, 126.5, 120.8, 119.0, 117.3, 115.2, 111.6,
105.2, 75.5, 65.5, 53.9, 49.7, 43.3, 34.8, 32.0, 23.5, 21.9, 19.1.
1.91–1.88 (m, 2H), 1.85–1.82 (m, 2H). C NMR (151 MHz, DMSO‐
d6) δ 160.2, 156.4, 146.3, 137.2, 136.0, 132.3, 130.9, 128.3, 127.5,
126.5, 123.9, 122.0, 120.2, 118.8, 116.3, 112.9, 109.3, 104.0, 73.7,
54.5, 49.6, 43.9, 32.6, 30.7, 23.6, 21.9. HR‐MS (ESI): C30H32N4O2S,
+
N‐{1‐[3‐Fluoro‐4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐4‐methyl‐4H‐thieno[3,2‐b]pyrrole‐5‐
carboxamide (B5)
[M+H] : 481.2604; found: 481.2584.
1‐Methyl‐N‐{1‐[4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐1H‐indole‐2‐carboxamide (C4)
1
Yield 76%, white solid. H NMR (600 MHz, DMSO‐d6) δ 9.46 (s, 1H),
1
7.49 (d, J = 5.3 Hz, 1H), 7.28 (s, 1H), 7.23 (d, J = 5.3 Hz, 1H), 7.10
(t, J = 8.8 Hz, 1H), 6.92 (t, J = 8.0 Hz, 1H), 6.86 (d, J = 12.3 Hz, 1H),
6.73 (d, J = 8.4 Hz, 1H), 6.55 (d, J = 7.7 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H),
Yield 78%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 9.85 (s, 1H),
9.34 (s, 1H), 9.26 (s, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.56 (d, J = 8.4 Hz,
1H), 7.33–7.29 (m, 2H), 7.26 (d, J = 7.5 Hz, 2H), 7.13 (t, J = 7.4 Hz,

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1H), 6.98 (s, 3H), 6.66 (d, J = 62.2 Hz, 2H), 4.64–4.62 (m, 1H), 4.49
(s, 2H), 4.01 (s, 3H), 3.35–3.32 (m, 2H), 3.21–3.17 (m, 2H), 3.06–3.02
(m, 2H), 2.72 (t, J = 6.1 Hz, 2H), 2.11 (dd, J = 13.4, 5.8 Hz, 2H), 1.92
N‐{1‐[4‐(Piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}-
thiophene‐2‐carboxamide (C8)
1
Yield 75%, brown solid. H NMR (600 MHz, DMSO‐d6) δ 9.77 (s, 1H),
13
(s, 2H), 1.87–1.83 (m, 2H). C NMR (151 MHz, DMSO‐d6) δ 169.2,
7.95 (s, 1H), 7.81 (d, J = 4.9 Hz, 1H), 7.21–7.19 (m, 1H), 7.15 (d,
J = 8.5 Hz, 2H), 6.92–6.88 (m, 3H), 6.49 (d, J = 7.6 Hz, 1H), 6.46 (d,
J = 8.3 Hz, 1H), 4.43 (s, 2H), 4.33 (dt, J = 8.9, 4.7 Hz, 1H), 3.33–3.31
164.6, 156.2, 150.3, 147.8, 146.2, 136.5, 131.1, 128.3, 126.4, 123.1,
118.9, 116.3, 114.8, 109.4, 108.4, 108.1, 102.2, 72.9, 54.5, 49.5, 46.1,
43.1, 33.7, 31.5, 23.6, 23.1, 21.9. HR‐MS (ESI): C31H34N4O2S,
(m, 2H), 2.93 (dd, J = 8.7, 3.9 Hz, 2H), 2.62 (t, J = 6.3 Hz, 2H), 2.54 (t,
13
+
[M+H] : 495.2760; found 495.2753.
J = 11.3 Hz, 2H), 1.87 (dt, J = 10.9, 6.9 Hz, 4H), 1.44–1.40 (m, 2H).
C
NMR (151 MHz, DMSO‐d6) δ 160.2, 156.4, 146.3, 140.5, 135.8,
131.7, 130.9, 129.2, 128.5, 128.3, 126.6, 119.0, 116.3, 114.7, 109.6,
73.8, 54.5, 49.5, 44.0, 32.6, 23.6, 21.8. HR‐MS (ESI): C26H29N3O2S,
N‐{1‐[4‐(Piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}-
quinoline‐3‐carboxamide (C5)
1
+
Yield 58%, yellow solid H NMR (600 MHz, DMSO‐d6) δ 10.25 (s,
[M+H] : 448.2059; found: 448.2040.
1H), 9.39 (s, 1H), 9.04 (s, 1H), 8.14 (d, J = 7.9 Hz, 1H), 8.11
(d, J = 8.4 Hz, 1H), 7.90 (t, J = 7.4 Hz, 1H), 7.72 (t, J = 7.0 Hz, 1H), 7.19
(d, J = 8.2 Hz, 2H), 6.97–6.92 (m, 3H), 6.58 (d, J = 7.2 Hz, 1H), 6.48 (d,
J = 8.3 Hz, 1H), 4.54–4.49 (m, 1H), 4.46 (s, 2H), 3.35 (t, J = 5.6 Hz, 2H),
3.10–3.05 (m, 2H), 2.83 (t, J = 9.6 Hz, 2H), 2.71 (t, J = 5.5 Hz, 2H),
2.05–2.01 (m, 2H), 1.89 (d, J = 9.9 Hz, 2H), 1.87 (s, 1H), 1.69 (d,
4‐Methyl‐N‐{1‐[4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}nicotinamide (C9)
1
Yield 76%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 10.16 (s,
1H), 9.29 (s, 1H), 9.21 (s, 1H), 8.95 (s, 1H), 8.76 (s, 1H), 7.81 (s, 1H),
7.20 (d, J = 8.3 Hz, 2H), 6.97 (s, 2H), 6.93 (d, J = 8.0 Hz, 1H), 6.65 (s,
1H), 6.51–6.42 (m, 1H), 4.64–4.58 (m, 1H), 4.46 (s, 2H), 3.36–3.34 (m,
4H), 3.18 (d, J = 5.5 Hz, 2H), 3.05–3.02 (m, 2H), 2.72 (t, J = 6.1 Hz,
13
J = 8.4 Hz, 2H). C NMR (151 MHz, DMSO‐d6) δ 164.3, 156.1, 149.7,
148.9, 146.3, 136.5, 131.7, 131.3, 129.7, 129.2, 128.4, 127.9, 127.0,
13
126.5, 118.8, 116.4, 114.6, 109.5, 71.4, 54.5, 49.6, 42.1, 29.7, 23.7,
+
2H), 2.60 (s, 3H), 2.10 (d, J = 8.4 Hz, 2H), 1.86–1.82 (m, 2H). C NMR
21.9. HR‐MS (ESI):
C
31H32N4O2, [M+H] : 493.2603; found:
(151 MHz, DMSO‐d6) δ 160.5, 156.3, 146.2, 144.7, 136.1, 130.9,
493.2591.
128.2, 126.4, 120.8, 118.8, 116.2, 114.7, 111.5, 109.2, 105.1, 73.9,
+
54.5, 49.5, 44.2, 30.6, 25.8, 21.9. HR‐MS (ESI): C28H32N4O2, [M+H] :
N‐{1‐[4‐(Piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}-
quinoline‐2‐carboxamide (C6)
457.2604; found: 457.2600.
1
Yield 76%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 10.38 (s,
N‐{1‐[4‐(Piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}‐
4‐(trifluoromethyl)benzamide (C10)
1H), 9.28 (s, 1H), 9.20 (s, 1H), 8.66 (d, J = 8.4 Hz, 1H), 8.25 (d,
J = 8.4 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.92
(t, J = 7.5 Hz, 1H), 7.77 (t, J = 7.4 Hz, 1H), 7.26 (d, J = 7.2 Hz, 2H),
7.23–7.17 (m, 1H), 7.02 (s, 1H), 6.98 (d, J = 8.1 Hz, 2H), 6.57 (s, 1H),
4.66 (s, 1H), 4.49 (s, 2H), 3.35 (s, 2H), 3.18 (s, 2H), 3.04 (s, 2H), 2.82
(t, J = 5.6 Hz, 2H), 2.12–2.08 (m, 2H), 2.00 (s, 2H), 1.86–1.82 (m,
1
Yield 76%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 10.06 (s,
1H), 9.22 (s, 1H), 9.14 (s, 1H), 8.17 (d, J = 7.7 Hz, 2H), 7.93–7.87 (m,
2H), 7.23 (d, J = 8.0 Hz, 2H), 6.97 (t, J = 7.9 Hz, 3H), 6.57 (d,
J = 33.4 Hz, 2H), 4.63–4.60 (m, 1H), 4.47 (s, 2H), 3.36–3.33 (m, 2H),
3.18 (d, J = 11.0 Hz, 2H), 3.06–3.02 (m, 2H), 2.66 (t, J = 6.1 Hz, 2H),
13
13
2H). C NMR (151 MHz, DMSO‐d6) δ 162.9, 161.3, 156.2, 152.9,
2.11–2.08 (m, 2H), 1.92–1.88 (m, 2H), 1.86–1.82 (m, 2H). C NMR
147.2, 145.9, 135.2, 131.9, 129.0, 128.5, 128.0, 127.6, 126.4,
(151 MHz, DMSO‐d6) δ 167.4, 164.5, 156.3, 146.3, 138.9, 132.0,
125.3, 122.6, 116.1, 115.9, 112.9, 103.8, 73.9, 54.8, 49.8, 32.8,
130.9, 129.0, 128.3, 126.6, 125.8, 116.3, 114.6, 109.6, 73.9, 54.5,
+
+
25.9, 23.9, 22.8. HR‐MS (ESI): C31H32N4O2, [M+H] : 493.2603;
49.5, 44.1, 32.8, 23.6, 21.8. HR‐MS (ESI): C29H30F3N3O2, [M+H] :
found: 493.2589.
510.2368; found: 510.2356.
N‐{1‐[4‐(Piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}-
benzo[d][1,3]dioxole‐5‐carboxamide (C7)
N‐{1‐[4‐(Piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}-
benzamide (C11)
1
1
Yield 70%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 9.87 (s, 1H),
Yield 77%, yellow solid. H NMR (600 MHz, DMSO‐d6) δ 9.73 (s, 1H),
9.19 (s, 1H), 9.11 (s, 1H), 8.02–7.98 (m, 1H), 7.82 (d, J = 5.0 Hz, 1H),
7.23 (d, J = 8.2 Hz, 2H), 7.21–7.19 (m, 1H), 6.97 (d, J = 8.6 Hz, 2H),
6.65–6.50 (m, 2H), 4.62 (dt, J = 7.4, 3.9 Hz, 1H), 4.47 (s, 2H), 4.08 (s,
4H), 3.34–3.31 (m, 2H), 3.22–3.18 (m, 2H), 3.06–3.02 (m, 2H), 2.65 (t,
J = 6.3 Hz, 2H), 2.12–2.08 (m, 2H), 1.91–1.87 (m, 2H), 1.86–1.81 (m,
7.96 (d, J = 7.4 Hz, 2H), 7.57 (t, J = 7.3 Hz, 1H), 7.51 (t, J = 7.5 Hz, 2H),
7.20 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 6.90 (t, J = 8.0 Hz, 1H),
6.52 (d, J = 7.7 Hz, 1H), 6.45 (d, J = 8.3 Hz, 1H), 4.59 (dt, J = 7.5,
3.9 Hz, 1H), 4.45 (s, 2H), 3.21–3.18 (m, 2H), 3.03 (d, J = 5.6 Hz, 4H),
2.63 (t, J = 6.4 Hz, 2H), 2.10–2.05 (m, 2H), 1.88–1.85 (m, 2H),
13
13
2H). C NMR (151 MHz, DMSO‐d6) δ 160.2, 156.3, 146.3, 140.5,
1.83–1.79 (m, 2H). C NMR (151 MHz, DMSO‐d6) δ 160.5, 156.3,
135.8, 131.7, 130.9, 129.2, 128.4, 128.3, 119.0, 116.3, 114.7, 109.6,
146.2, 144.8, 136.1, 131.1, 128.3, 128.1, 126.4, 120.8, 118.7, 116.3,
114.7, 111.6, 109.2, 105.0, 72.1, 54.5, 49.5, 45.9, 34.8, 23.0, 21.8.
73.8, 54.5, 49.5, 44.0, 32.7, 23.6, 21.8. HR‐MS (ESI): C30H33N3O3, [M
+
+
+H] :486.2600; found 486.2395.
HR‐MS (ESI): C29H31N3O2, [M+H] : 442.2495; found: 442.2487.

YAN ET AL.
15 of 17
|
4‐Methyl‐N‐{1‐[4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}benzamide (C12)
1‐Methyl‐N‐{1‐[4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐1H‐indole‐6‐carboxamide (D2)
1
1
Yield 82%, brown solid. H NMR (600 MHz, DMSO‐d6) δ 9.66 (s, 1H),
Yield 79%, brown solid. H NMR (600 MHz, DMSO‐d6) δ 9.14 (s, 1H),
8.18 (s, 1H), 8.15 (d, J = 7.9 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.24 (t,
J = 7.6 Hz, 1H), 7.17 (d, J = 7.0 Hz, 3H), 6.90 (d, J = 8.5 Hz, 3H), 6.59 (d,
J = 7.7 Hz, 1H), 6.42 (d, J = 8.4 Hz, 1H), 4.44 (s, 2H), 4.35 (dt, J = 9.0,
4.8 Hz, 1H), 3.87 (s, 3H), 3.34–3.32 (m, 2H), 2.94 (dt, J = 12.7, 4.4 Hz,
2H), 2.68 (t, J = 6.3 Hz, 2H), 2.64–2.52 (m, 2H), 1.86 (d, J = 23.2 Hz,
7.87 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 8.6 Hz, 2H),
6.91–6.87 (m, 3H), 6.51 (d, J = 7.6 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H),
4.43 (s, 2H), 4.40–4.36 (m, 1H), 3.33–3.30 (m, 2H), 3.03 (d, J = 1.8 Hz,
2H), 2.99–2.94 (m, 2H), 2.62 (t, J = 6.4 Hz, 4H), 2.37 (s, 3H), 1.94–1.89
13
(m, 2H), 1.88–1.84 (m, 2H). C NMR (151 MHz, DMSO‐d6) δ 173.9,
13
169.2, 165.4, 156.2, 146.2, 141.7, 136.5, 132.3, 129.3, 128.3, 128.0,
6H). C NMR (151 MHz, DMSO) δ 167.4, 160.6, 156.4, 146.3, 135.8,
126.4, 116.3, 114.7, 109.3, 73.0, 54.5, 49.5, 43.2, 33.7, 31.6, 23.6,
+
134.2, 132.0, 130.9, 129.1, 128.3, 126.5, 118.8, 116.3, 114.6, 112.8,
21.8. HR‐MS (ESI):
C
29H33N3O2, [M+H] : 456.2651; found:
112.4, 109.5, 105.4, 73.9, 65.5, 54.5, 49.5, 44.1, 32.8, 32.1, 30.5, 23.6,
+
456.2635.
21.9. HR‐MS (ESI): C31H34N4O2, [M+H] : 495.2760; found:
495.2901.
N‐{1‐[4‐(Piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐tetrahydroquinolin‐5‐yl}‐
3‐(trifluoromethyl)benzamide (C13)
1‐Methyl‐N‐{1‐[4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐1H‐indole‐3‐carboxamide (D3)
1
Yield 81%, brown solid. H NMR (400 MHz, DMSO‐d6) δ 10.03 (s,
1
1H), 8.30 (s, 2H), 7.99–7.91 (m, 1H), 7.81–7.73 (m, 1H), 7.21–7.12
(m, 2H), 6.91 (s, 3H), 6.52 (d, J = 6.0 Hz, 1H), 6.47 (d, J = 6.5 Hz,
Yield 76%, brown solid. H NMR (600 MHz, DMSO‐d6) δ 9.74 (s, 1H),
8.22 (s, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.52
(d, J = 2.9 Hz, 1H), 7.20 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H), 6.91
(t, J = 8.0 Hz, 1H), 6.55 (d, J = 7.7 Hz, 1H), 6.50 (d, J = 2.8 Hz, 1H), 6.45
(d, J = 8.3 Hz, 1H), 4.62 (dt, J = 7.4, 4.0 Hz, 1H), 4.46 (s, 2H), 3.88 (s,
1H), 4.43 (s, 3H), 3.76–3.51 (m, 2H), 3.33 (s, 4H), 2.63 (s, 2H), 2.50
13
(s, 2H), 2.02–1.78 (m, 4H).
C NMR (151 MHz, DMSO‐d6) δ
164.1, 156.3, 146.3, 136.0, 132.2, 131.0, 130.2, 128.3, 126.6,
124.6, 118.9, 116.3, 114.6, 109.6, 72.9, 60.2, 54.5, 49.6, 33.8,
+
3H), 3.35–3.32 (m, 2H), 3.17–3.14 (m, 2H), 3.03–3.00 (m, 2H), 2.68 (t,
13
23.6, 21.8. HR‐MS (ESI):
C
29H30F3N3O2, [M+H] : 510.2368;
J = 6.2 Hz, 2H), 2.11 (dd, J = 13.3, 3.5 Hz, 2H), 1.90–1.84 (m, 4H).
C
found: 510.2363.
NMR (151 MHz, DMSO‐d6) δ 163.2, 156.3, 146.2, 137.2, 136.7,
132.7, 131.1, 128.3, 127.1, 126.4, 122.5, 121.6, 121.2, 118.4, 116.3,
114.5, 110.7, 110.0, 108.8, 73.5, 65.5, 58.2, 54.5, 49.5, 33.5, 32.4,
4‐Fluoro‐N‐{1‐[4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}benzamide (C14)
+
27.0, 23.7, 21.9. HR‐MS (ESI): C31H34N4O2, [M+H] : 495.2760;
1
Yield 72%, brown solid. H NMR (600 MHz, DMSO‐d6) δ 9.83 (s,
found: 495.2739.
1H), 8.07–8.03 (m, 2H), 7.33 (t, J = 8.8 Hz, 2H), 7.17 (d, J = 8.4 Hz,
2H), 6.90 (dd, J = 15.4, 8.2 Hz, 3H), 6.50 (d, J = 7.7 Hz, 1H), 6.45 (d,
J = 8.3 Hz, 1H), 4.47–4.40 (m, 3H), 3.33–3.29 (m, 2H), 3.03 (s, 2H),
1‐Ethyl‐N‐{1‐[4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐1H‐indole‐2‐carboxamide (D4)
1
2.73 (t, J = 13.0 Hz, 2H), 2.62 (t, J = 6.3 Hz, 2H), 1.98–1.94 (m, 2H),
13
Yield 74%, brown solid. H NMR (600 MHz, DMSO‐d6) δ 9.77 (s, 1H),
1.88–1.84 (m, 2H), 1.59 (dd, J = 10.9, 6.4 Hz, 2H).
C NMR
7.68 (d, J = 2.9 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.31–7.27 (m, 2H),
7.16 (d, J = 8.5 Hz, 2H), 7.12 (t, J = 7.4 Hz, 1H), 6.93–6.88 (m, 3H),
6.56 (d, J = 7.7 Hz, 1H), 6.46 (d, J = 8.3 Hz, 1H), 4.58 (q, J = 7.1 Hz,
2H), 4.44 (s, 2H), 4.34 (dt, J = 9.5, 5.0 Hz, 1H), 3.35–3.32 (m, 2H), 2.93
(d, J = 12.5 Hz, 2H), 2.69 (t, J = 6.4 Hz, 2H), 2.55 (t, J = 10.4 Hz, 2H),
1.91–1.87 (m, 4H), 1.64 (dd, J = 8.4, 6.4 Hz, 2H), 1.29 (t, J = 7.0 Hz,
(151 MHz, DMSO‐d6) δ 164.5, 163.6, 156.1, 146.2, 136.3, 131.5,
130.8, 130.7, 128.3, 126.5, 118.9, 116.4, 116.3, 115.8, 115.6,
114.8, 109.4, 72.0, 54.5, 49.5, 46.0, 42.4, 33.7, 30.5, 23.6,
+
21.8. HR‐MS (ESI): C28H30FN3O2, [M+H] : 460.2400; found:
460.2386.
13
3H). C NMR (151 MHz, DMSO) δ 168.6, 160.0, 156.3, 145.5, 137.9,
1‐Methyl‐N‐{1‐[4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐1H‐indole‐5‐carboxamide (D1)
135.9, 135.3, 132.0, 131.0, 128.3, 127.1, 126.2, 123.9, 122.2, 120.6,
119.2, 114.7, 110.8, 107.1, 75.5, 68.4, 54.9, 50.9, 44.9, 32.6, 30.4,
1
+
Yield 75%, brown solid. H NMR (600 MHz, DMSO‐d6) δ 9.59 (s, 1H),
23.5, 21.0, 16.2. HR‐MS (ESI): C32H35N4O2, [M+H] : 509.2917;
8.26 (s, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.52 (d, J = 8.6 Hz, 1H), 7.43 (d,
J = 3.0 Hz, 1H), 7.18 (d, J = 8.5 Hz, 2H), 6.91 (dd, J = 17.9, 8.3 Hz, 3H),
6.57–6.54 (m, 2H), 6.44 (d, J = 8.3 Hz, 1H), 4.44 (s, 3H), 3.84 (s, 3H),
3.34–3.32 (m, 2H), 3.03 (dd, J = 12.6, 5.4 Hz, 2H), 2.73 (t, J = 10.8 Hz,
found: 509.2921.
1‐Isopropyl‐N‐{1‐[4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐1H‐indole‐2‐carboxamide (D5)
1
2H), 2.67 (t, J = 6.3 Hz, 2H), 2.49–2.46 (m, 2H), 1.98–1.94 (m, 2H),
13
Yield 75%, brown solid. H NMR (600 MHz, DMSO‐d6) δ 9.83 (s, 1H),
1.90–1.86 (m, 2H).
C NMR (151 MHz, DMSO) δ 166.5, 156.2,
7.70 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.23 (q, J = 1.6 Hz, 1H),
7.20 (d, J = 8.6 Hz, 2H), 7.11– 7.08 (m, 2H), 6.96 (d, J = 8.6 Hz, 2H),
6.91 (t, J = 8.0 Hz, 1H), 6.59 (d, J = 7.7 Hz, 1H), 6.46 (d, J = 8.4 Hz, 1H),
5.41 (p, J = 7.0 Hz, 1H), 4.61 (dt, J = 7.5, 3.9 Hz, 1H), 4.46 (s, 2H), 3.18
(dd, J = 10.4, 6.0 Hz, 3H), 3.04 (d, J = 7.3 Hz, 4H), 2.70 (t, J = 6.3 Hz,
146.2, 138.4, 131.5, 131.3, 128.4, 127.8, 126.4, 126.1, 121.4, 121.1,
118.9, 116.4, 114.9, 109.8, 109.1, 102.0, 72.2, 54.5, 49.6, 46.1, 42.7,
+
33.1, 30.7, 23.6, 22.4, 21.9. HR‐MS (ESI): C31H34N4O2, [M+H] :
495.2760; found: 495.2763.

16 of 17
YAN ET AL.
|
2H), 2.29 (s, 1H), 2.10 (dt, J = 10.1, 5.4 Hz, 2H), 1.92–1.89 (m, 2H),
13
cell lines were cultured in minimal necessary medium supplemented
with 10% fetal bovine serum. Cells were seeded into approximately
1.84 (dd, J = 13.4, 3.9 Hz, 2H), 1.59 (d, J = 7.0 Hz, 6H). C NMR
4
(151 MHz, DMSO) δ 161.6, 155.8, 146.2, 139.4, 136.8, 134.5, 133.5,
131.7, 130.5, 128.4, 127.0, 126.5, 123.5, 122.3, 120.2, 118.5, 116.4,
114.5, 113.0, 105.8, 69.5, 54.5, 49.6, 48.0, 45.7, 27.6, 23.5, 21.9, 20.4.
5 × 10 well 96‐well plates and incubated in 5% CO2 at 37°C for
24 h. Three wells were treated with different concentrations of
compound and medium. Subsequently, the test compound was added
to the medium and incubated for 72 h. Fresh MTT was added to each
well at 5 μg/ml. After 4 h of incubation, the MTT medium was re-
moved and 100 μl DMSO was added to each well. Results were
measured using a microplate reader (MK3; Thermo Fisher Scientific).
All compounds were tested three times, with the IC50 value defined
as the concentration of the vehicle that reduced the absorbance of
the negative wells by 50% in the MTT assay.
+
HR‐MS (ESI): C33H38N4O2, [M+H] : 523.3073; found: 523.3126.
5‐Fluoro‐1‐methyl‐N‐{1‐[4‐(piperidin‐4‐yloxy)benzyl]‐1,2,3,4‐
tetrahydroquinolin‐5‐yl}‐1H‐indole‐2‐carboxamide (D6)
1
Yield 78%, brown solid. H NMR (600 MHz, DMSO‐d6) δ 9.82 (s, 1H),
7.60–7.57 (m, 1H), 7.46 (dd, J = 9.5, 2.5 Hz, 1H), 7.26 (s, 1H), 7.15 (dd,
J = 9.0, 2.7 Hz, 3H), 6.90 (dd, J = 17.8, 8.4 Hz, 3H), 6.56 (d, J = 7.7 Hz,
1H), 6.46 (d, J = 8.3 Hz, 1H), 4.43 (s, 2H), 4.32 (dt, J = 9.1, 4.9 Hz, 1H),
4.00 (s, 3H), 3.36–3.31 (m, 4H), 2.92 (dt, J = 12.2, 3.8 Hz, 2H), 2.68 (t,
13
|
Molecular docking
J = 6.3 Hz, 2H), 2.53 (t, J = 10.1 Hz, 2H), 1.90–1.86 (m, 4H). C NMR
4.3
(151 MHz, DMSO‐d6) δ 172.4, 166.4, 155.4, 146.8, 136.9, 135.6,
132.8, 131.7, 130.7, 128.4, 127.7, 126.4, 120.2, 119.4, 116.4, 114.9,
110.4, 108.5, 102.7, 69.6, 60.2, 54.5, 49.6, 46.3, 33.1, 27.5, 21.2, 14.5.
All computational experiments were conducted on a Dell PowerEdge
R900 workstation under an RHEL 5.3 platform. Chemical structures
were prepared by Sybyl 6.9.1 (Tripos Inc.) (Tripos Associates).
The protein structure was prepared in the Discovery Studio 3.0
software package (BIOVIA Inc.). Ligand preparation: The compounds
were sketched and optimized in Sybyl 6.9.1 with a Tripos force field
and saved in mol2 format. Protein preparation: The X‐ray crystal
structures of LSD1 were downloaded from the RCSB Protein Data
Bank, PDB code 5LHI (http://www.rcsb.org/pdb/).
+
HR‐MS (ESI): C31H33FN4O2, [M+H] : 513.2666; found: 513.2656.
|
4.2
Biological assays
|
4.2.1
LSD1 activity assay
LSD1 Inhibitor Screening Assay Kits were purchased from Cayman, and
the LSD1 enzyme inhibition assay was performed with an LSD1 In-
hibitor Screening Assay Kit (Item No. 700120) according to the man-
ufacturer's protocol. Briefly, the test protocol included setting 100%
initial active wells, background wells, positive control wells, and in-
hibitor wells. First, 120 μl of assay buffer was added to the 100% initial
active wells, positive control wells, and inhibitor wells, and 140 μl of
assay buffer was added to the background well. The 100% initial active
wells and background wells had 10 μl of the DMSO solution, and the
inhibitor wells and the positive control wells required the addition of a
10 μl DMSO solution of the inhibitor. Then, 20 μl of LSD1 was added to
the wells used, and 20 μl of the peptide was added to the wells other
than the background well. During the process of adding the solutions,
the 96‐well plate was cooled on an ice pack to prevent enzymatic
reactions. After the addition of the solutions, it was incubated for
30 min at room temperature in the dark. Then, 20 μl of HRP and 10 μl
of the fluorogenic substrate were added to each well and incubated at
room temperature for 10 min in the dark. The fluorescence intensity
was measured at 530 nm excitation and 590 nm emission. We graphed
either the percent inhibition or percent initial activity as a function of
the inhibitor concentration to determine the IC50 value (concentration
at which there was 50% inhibition).
Docking: Crystal structures of LSD1 (PDB code: 5LHI) were
imported into Glide 9.6, defined as the receptor structure and the
location of the active site with a box of size 15 × 15 × 15 Å. The OPLS
2005 force field was used for grid generation. The standard precision
was set for docking studies with two crucial residues, Asp375 and
Asp555, in constrained binding to get accurate results. All other
parameters were maintained as the default.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the Program for Innovative
Research Team of the Ministry of Education and the Program for
Liaoning Innovative Research Team in University.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
Dongmei Zhao
http://orcid.org/0000-0001-5156-6125
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Additional Supporting Information may be found online in the
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How to cite this article: J. Yan, Y. Gu, Y. Sun, Z. Zhang, X.
Zhang, X. Wang, T. Wu, D. Zhao, M. Cheng, Design, synthesis,
and biological evaluation of 5‐aminotetrahydroquinoline‐
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