Design, Synthesis, and Structure-Activity Relationship Studies of Novel Indolyalkylpiperazine Derivatives as Selective 5-HT1A Receptor Agonists

Article abstract of DOI:10.1021/acs.jcim.9b00926

5-HT_1A receptor (5-HT_1AR) agonists have been implicated in the treatment of a variety of central nervous system (CNS) diseases such as depression and anxiety, et al. Based on our previously found compound FW01 (K_i = 51 ± 16 nM) obtained by virtual screening, a series of FW01 derivatives were designed and synthesized by the modification of the amide tail group as well as indole headgroup of FW01. SAR exploration found that amide tail group and indole headgroup play pivotal roles in determining the binding affinity and selectivity on dopamine and serotonin receptor subtypes. Among all tested compounds, 9_24 has a K_i value of 5 ± 0.6 nM with a good selectivity toward 5-HT_1AR. The [³⁵S] GTPγS assay showed that 9_24 is a full agonist toward 5-HT_1AR with an EC₅₀ value of 0.059 nM, which shows 266.2 and 146.4-fold selectivity to 5-HT_2A and D₃ respectively. Molecular dynamics simulations and molecular docking studies with 5-HT_1AR-9_24 were performed to disclose the mechanism of its high activity and selectivity. Finally, a detailed stepwise 9_24 induced signal transduction mechanism of 5-HT_1AR is proposed.

Full text of DOI:10.1021/acs.jcim.9b00926

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Computational Chemistry
Design, Synthesis and Structure-activity Relationship Studies of Novel
1A
Indolyalkylpiperazine Derivatives as Selective 5-HT Receptor Agonists
Wenli Wang, Lan Zheng, Wei Li, Chen Zhu, Weiqing Peng, Bing Han, and Wei Fu
J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00926 • Publication Date (Web): 08 Jan 2020
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Design, Synthesis and Structure-activity Relationship
Studies of Novel Indolyalkylpiperazine Derivatives as
Selective 5-HT1A Receptor Agonists
9
Wenli Wang#, Lan Zheng#, Wei Li, Chen Zhu, Weiqing Peng, Bing Han*, Wei Fu*
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School of Pharmacy ＆ Minhang Hospital, Fudan University, Shanghai 201301, P. R. China
Abstract
5-HT1A receptor (5-HT1AR) agonists have been implicated in the treatment of a
variety of central nervous system (CNS) diseases such as depression and anxiety et
al. Based on our previously compound FW01 (Ki = 51 ± 16 nM) obtained by virtual
screening, a series of FW01 derivatives were designed and synthesized by the
modification of the amide tail group as well as indole head group of FW01. SAR
exploration found that amide tail group and indole head group play pivotal roles in
determining the binding affinity and selectivity on dopamine and serotonin receptor
subtypes. Among all tested compounds, 9_24 has a Ki value of 5 ± 0.6 nM with a
good selectivity towards 5-HT1AR. The [35S] GTPγS assay showed that 9_24 is a full
agonist towards 5-HT1AR with an EC50 value of 0.059 nM, which shows 266.2 and
146.4-fold selectivity to 5-HT2A and D3 respectively. Molecular dynamics
simulations and molecular docking studies with 5-HT1AR-9_24 were performed to
disclose the mechanism of its high activity and selectivity. Finally, a detailed
stepwise 9_24 induced signal transduction mechanism of 5-HT1AR is proposed.
Keywords
Indolealkylpiperazine, selective agonist, 5-HT1A receptor, drug design, molecular
dynamics simulation
1. Introduction
Serotonin (5-hydroxytryptamine, 5-HT), an important neurotransmitter, is
involved in multiple physiological and pathological processes that are closely related
to behavior1,2. 5-HT mediates its physiological effects through at least 14 different
3-5
receptor subtypes, which are classified into seven subfamilies, 5-HT1 to 5-HT7
.
Among them, 5-HT1A receptor (5-HT1AR) was the first fully sequenced and has been
the target for various central nervous system (CNS) disorders, especially for anxiety
and depression6-9. Medicines like buspirone, gepirone, vilazodone and tandospirone
that act as 5-HT1AR partial agonists are representative anxiolytics and antidepressants
in clinical10-12. 5-HT1AR agonists were also found to be effective treatments for
relieving cognitive impairment and extrapyramidal side effects (EPS) associated with
antipsychotic therapy13,14. Besides, 5-HT1AR agonists are beneficial for alleviating
dyskinesia in Parkinson’s disease15. Recent findings showed that the 5-HT1AR agonists
could be potential treatment for ischemic stroke16,17. Due to the potential value of 5-
HT1AR agonists for the management of various health problems, it is important to
design novel 5-HT1AR agonists with high selectivity and improved pharmacological
profiles.
2. Design
In our previous studies, a series of potent 5-HT1AR agonists with novel scaffolds
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were obtained through the combination of pharmacophore-based virtual screening and
biological assays18. Among them, FW01 (Figure 1) possessed a high activity for 5-
HT1A receptor (Ki = 51 ± 16 nM, EC50 = 7 nM). The predicted binding mode of FW01
with 5-HT1AR (Figure 1) showed that FW01 adopted an extended conformation and
the protonated nitrogen atom formed a conserved salt bridge interaction with D3.32
(Figure 1). The indole head group was inserted into the pocket P1 formed by helix 3,
5, 6 and 7 of 5-HT1AR. The NH group and the F atom of the indole head formed a
hydrogen bond with T3.37 and T5.43, respectively. As for the amide tail group, the phenyl
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ring was inserted into the pocket P2 formed by residues F3.28, Y2.64 and W102ECL1
.
Meanwhile, the cyclohexyl ring stretched into the P3 pocket formed by helix 2, 6 and
7. Our previous work revealed that the head group of FW01 dynamically forms the
hydrogen bonds with the polar residues of either upper or lower pockets through its
nitrogen or fluorine atom by the flipping of the indole ring19. The binding mode of
FW01 with 5-HT1AR gave us hints that P1 upper and lower pockets, P2 and P3 binding
pockets are important for binding affinity and selectivity for 5-HT1AR (Figure 1).
Figure 1. The predicted binding mode of FW01 with 5-HT1AR. Ligands were shown in green sticks.
The binding residues of 5-HT1AR were shown in gray. Hydrogen bond interactions were represented
in dotted red line.
In this work, a series of FW01 derivatives were designed and synthesized by
modifying both the indole head and the amide tail group. Since the presence of polar
amino acid residues in P3 and the relatively larger size of P3, we replaced the
cyclohexyl ring with both polar and none-polar substituent groups with varying size. In
addition, different 5-substituted indole moieties (-OH, -OCH3, -CN) were further
introduced to enhance the hydrogen bonding interaction with surrounding polar
residues. Radio ligand binding assays and [35S] GTPγS assays were employed to assess
the in vitro binding affinity and the efficacy of synthesized compounds, respectively.
3. Results and Discussion
3.1. Chemistry
The synthetic route of the designed compounds was outlined in Schemes 1. 3-
indolepropanol moiety 2 was prepared from the corresponding phenylhydrazine
hydrochloride salts via Fischer indole synthesis20-22. The alcohol intermediate was
converted to bromo derivatives 3 with carbon tetrabromide and triphenylphosphine
in anhydrous methylene dichloride. The 5-cyanoindole propanol, 2d, was prepared
from 5-bromo compound 2c by the treatment with CuCN in DMF at 130 ℃.
Compound 3c was obtained from 3b via the demethylation reaction in the presence
of BBr3 in anhydrous methylene dichloride at -78 ℃.
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9
To synthesize intermediate 8, compound 5 was obtained by the reductive
amination of aniline derivatives 4 with a ketone derivative or commercially available
feedstock. Then, compound 5 was acylated with acyl chloride in anhydrous
methylene dichloride to obtain compound 6, followed by alkylation with N-Boc-
piperazine to yield compound 7. The deprotection of the N-Boc-piperazine moiety
of compound 7 led to the formation of intermediate 8. Compound 9 was synthesized
by the combination of 3 and 8 in the presence of potassium carbonate and acetonitrile.
Structures of all the compound 9 were confirmed by 1H NMR and ESI-MS spectra.
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Br
OH
R1
R1
R1
A
C
NH2
NH
N
H
N
H
a, R1=F
a, R1=F
a, R1=F
b, R1=OCH3
c, R1=Br
b, R1=OCH3
c, R1=OH
d, R1=CN
2
1
b, R1=OCH3
c, R1=Br
3
D
B
d, R1=CN
O
O
R2
O
R2
H
N
Cl
R2
N
N
N
R2
R3
H2N
CF3COO
N
BocN
N
R3
R3
R3
H
G
F
NH2
8(a-d)
6(a-d)
5(a-d)
7(a-d)
H
BocN
Cl
E
N
H2N
CF3COO
R4
R4
R4
N
N
R5
N
O
N
R4
N
R4
O
O
R5
R5
4
R5
8(e-q)
6(e-q)
5(e-q)
7(e-q)
N
R1
N
k, R4=H, R5=4-t-butylcyclohexyl
l, R4=H, R5=pyran
m, R4=H, R5=2-pentyl
n, R4=H, R5=3-pentyl
o, R4=H, R5=3-hexyl
a, R2=R3=phenyl
b, R2=R3=cyclohexyl
c, R2=H, R3=phenyl
d, R2=H, R3=cyclohexyl
e, R4=4-F, R5=cyclohexyl
f, R4=4-OCH3, R5=cyclohexyl
g, R4=4-SO2CH3, R5=cyclohexyl
h, R4=2-OCH3, R5=cyclohexyl
i, R4=H, R5=4-methylcyclohexyl
j, R4=H, R5=4-ethylcyclohexyl
R3
O
R2
N
N
O
R2
Br
H2N
R1
N
R3
N
H
I
CF3COO
R4
p, R4=H, R5=1-methoxypropane
q, R4=H, R5=cyclohexyl (
+
8(a-d)
N
R1
FW01
)
N
H
N
H2N
R4
N
N
R5
3
CF3COO
O
N
N
O
R5
H
8(e-q)
9
Scheme 1. Reagents and conditions: A) 3,4-dihydropyran, 4% H2SO4, DMF, reflux, 2 h; B) CuCN,
DMF, reflux, 6h; C) CBr4, PPh3, DCM, rt., 15 h; D) BBr3, DCM, -78℃, 2 h; E) ketones, Zn/AcOH,
reflux, 4 h; F) chloroacetyl chloride, TEA, DCM, rt., 5 h; G) Boc-piperazine, CH3CN, K2CO3, reflux,
8 h; H) TFA, DCM, rt., 3 h; I) K2CO3, CH3CN, reflux, 12 h.
3.2. In vitro Biological activity evaluation
All the synthesized compounds were subjected to competitive binding assays
for the human dopamine (D1, D2 and D3) and serotonin (5-HT1A and 5-HT2A)
receptors to assess the binding affinity and also to test the selectivity for 5-HT1AR
over the other receptors.
Table 1 Binding affinities (Ki or percentage displacement of radio ligand at 10 μM) of compounds
for dopamine receptors (D1, D2 and D3) and serotonin receptors (5-HT1A and 5-HT2A).
N
N
X
Y
F
N
O
N
H
Ki ± SEM (nM) or inhibition rate at 10 M
Compounds
X
Y
D1
D2
D3
5-HT1A
5-HT2A
FW01
9_1
34.1%
31.7%
2161±2
51±16
206.7±7
32.64%
1.74%
85.24%
80.41%
89.75%
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9
9_2
9_3
9_4
9_5
9_6
9_7
NDa
NDa
NDa
21.11%
103±3
783±4
55±7
14.16%
75.30%
85.21%
83.63%
40.50%
58.83%
H
18.08%
NDa
9.13%
NDa
52.99%
NDa
H
F
14.78%
14.82%
8.53%
20.47%
41.01%
12.73%
51.65%
76.06%
53.38%
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O
112±3
195±1
O
S
O
O
9_8
-2.41%
43.28%
139±115
103±16
354±26
9_9
34.0%
52.81
24.4%
76.95
409±53
778±104
78.55%
45.29%
255 ±34
170±18
280±7
88±12
95±4
163±13
1353±77
27.63%
83.48%
160±15
165±2
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9_11
9_12
9_13
9_14
9_15
9_16
24.48%
2.14%
43.7%
25.74%
29.42%
25.6%
10.43%
37.27%
46.9%
77.53%
776±18
38.1%
63.13%
9±1
O
6.5±0.1
14±1
5±2
725±27
153±15
532 ±16
3.4±1
O
a. ND: no determined
The value of Ki was determined when inhibition rate is more than 90% at 10 M
As shown in Table 1, the tail is highly sensitive to the activity and selectivity
of dopamine and serotonin receptor subtypes. When X was phenyl group, the
substitute Y was set to be the cyclohexyl’s derivatives and the alkyl substitutes to
examine the activity and selectivity of this series of compounds towards DA and 5-
HT receptor subtypes. Comparing to FW01, the activity of 9_1 and 9_3, in which
the phenyl group and H atom were used to replace the cyclohexyl group in Y position,
were slightly decreased, indicating that cyclohexyl group was superior to phenyl and
hydrogen. We assumed that a relatively large pocket (P3) exists within this region.
Then, the methyl, ethyl and isopropyl groups were used to substitute the para-
position of cyclohexyl group and designed 9_9, 9_10 and 9_11. Data showed that
their activities for 5-HT1A were kept but slightly decreased compared with FW01.
Furthermore, when the polar O atom was introduced to the cyclohexyl group and
designed 9_12 with pyran ring, the binding affinity for 5-HT1A increased
dramatically and reached to 9 nM. The increased activity was attributed to the
hydrogen bonding interaction with the polar residues in P3, indicating that P3 pocket
has partial polar characteristic. The alkyl groups were also used to replace the
cyclohexyl group and designed compounds 9_13, 9_14 and 9_15, the activities of
these compounds against 5-HT1A were significantly improved. Due to the flexibility
of the carbon chain, the alkyl group could form a favorable hydrophobic interaction
with the hydrophobic residues in P3 pocket. Notably, the 2-hexyl substituent (9_15)
showed superior affinity among the three compounds, which was attributed to the
more flexible carbon chain which posed a stretched conformation to form a more
favorable hydrophobic interaction with P3. In addition, the polar O atom was
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introduced into the alkyl group and 9_16 was designed. It is not surprising to find
that 9_16 with an oxygen atom in the alkyl-chain displayed excellent affinity for 5-
HT1AR (Ki = 3.4 ± 1 nM).
By examining the pocket around X group, it shows a pocket formed by an
aromatic cluster, indicating that the ligand should contain the aromatic group in X
region to keep or increase the activity. To verify it, the cyclohexyl ring was used to
replace the phenyl group of FW01 and designed 9_2. The binding activity of 9_2
was greatly reduced towards 5-HT1AR and D1-D3. Furthermore, H atom was used to
replace the phenyl group and designed 9_4 whose binding affinity towards 5-HT1AR
was also significantly reduced. It demonstrates the importance of phenyl ring in P2
pocket for maintaining the binding affinity. Based on above findings, modifications
were further focused on phenyl ring to examine the SAR by replacement with
different sizes of substituents at the para-position. Fluorine atom and sterically larger
groups like methoxyl and methylsulfonyl groups were introduced to design 9_5, 9_6
and 9_7. The activity of 9_5 with F atom was kept, while that of 9_6 and 9_7 by the
introduction of large-size groups were slightly decreased, showing that there is a
small hydrophobic pocket at X position. It is worth noting that compound 9_8 with
a methoxyl group at ortho-positon of the phenyl ring showed slightly improved
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affinity for D3R and 5-HT2AR, indicating that P2 pocket is important for selectivity.
Table 2 Binding affinities (Ki or percentage displacement of radio ligand at 10 μM) of compounds
for dopamine receptors (D1, D2 and D3) and serotonin receptors (5-HT1A and 5-HT2A), perhaps due
to hydrogen receptor property of oxygen. With the increase of affinity for 5-HT1AR and relatively
small change of binding affinity for other receptor, selectivity was greatly improved.
N
N
R1
N
R2
O
N
H
Ki ± SEM (nM)
Compounds
R1
R2
D1
D2
D3
5-HT1A
5-HT2A
19.88%
871±71
325±6
0.6±0.1
0.4±0.2
5±1
711±101
9_17
9_18
9_19
9_20
9_21
9_22
9_23
9_24
9_25
OCH3
OCH3
OCH3
OH
4.01%
3.81%
29.97%
10.18%
8.92%
36.56%
4.72%
2.14%
41.34%
39.34%
52.22%
39.25%
46.53%
144±19
59.16%
37.27%
357±63
62.72%
255±23
521±76
71.05%
580±12
732±81
45.29%
801±12
735±159
187±12
O
O
0.8±0.1
0.1±0.3
3±0.4
6±1
531±21
OH
O
521±21
O
OH
1553±156
1331±17
1583±62
CN
5±0.6
9±1
CN
O
O
CN
Encouraged by the above results, we next extended our efforts to enhance the
hydrogen bonding interaction between the indole head group and the polar residues
in P1. For this purpose, we designed three more FW01 derivatives with -OCH3, -
OH, -CN substituents at the original F atom position. As shown in Table 2, these
compounds showed high affinity and selectivity for 5-HT1AR, with the order of 5-
HT1AR affinity as -F < -CN < -OCH3 < -OH, indicating that the activity of ligand
with hydrogen bond donor in this region is better than that of ligand with hydrogen
acceptor. Consistent with the findings above, the alkyl-chain substituent groups with
the oxygen atom showed superior affinity, especially the methoxyl-isopropyl
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substituent group (9_18, 9_21, 9_24).
Table 3 [35S]GTPγS assays of specific compounds for 5-HT1AR
5-HT1AR
Compounds
EC50 (nM)
1.88
Emax%
96.2±6.8
88.0±11.4
110.4±7.8
124.3±7.3
135.7±10.2
100
9_18
9_20
9_21
9_23
9_24
5-HT
4.61
2.44
0.76
0.059
3.34
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5-HT1AR activation abilities of the selected compounds were performed by
employing [35S] GTPγS assays. The curve of EC50 for positive compound 5-HT was
shown in Figure S1 in the supporting information. Emax of 5-HT is set as 100%. The
compounds were defined as partial agonists when Emax is lower than 100%, while they
were defined as full agonists when Emax is more than 100%. As shown in Table 3, all
compounds produced full or partial agonistic activity for 5-HT1AR. Among them, the
5-CN indole derivatives 9_24 was the most potent compound with EC50 of 0.059 nM
and displayed strong agonistic activity with Emax of 135.7%.
3.3. Binding Mode
Figure 2. The predicted binding mode of 9_24 with 5-HT1AR. Ligand was shown in green sticks.
5-HT1AR and surrounding residues in gray. Hydrogen bond interactions were represented in dotted
red.
We next studied the binding mode of 9_24 with active 5-HT1A receptor. We used
the GOLD 5.1 program23 to dock 9_24 into the binding site of the 5-HT1A receptor
which was obtained by homology modeling by taking X-ray structure of 5-HT1B
receptor as template (PDB code: 4iar, resolution:2.7 Å) 24. The results of molecular
modeling were shown in Figure 2. The protonated nitrogen atom of 9_24 formed strong
salt bridge with D3.32 in 5-HT1AR, and the distance of these two atoms is 1.5 Å. The
cyano group and the NH group of the indole ring formed hydrogen bonding with the
hydroxyl oxygen atom of T5.43 and T3.37. The phenyl ring of 9_24 was inserted into the
pocket P2 and formed π-π interactions with F3.28, W102EL1 and Y2.64, which is in
accordance with the binding mode of FW01 with 5-HT1AR.
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Figure 3. (A) The sequences of 5-HT1AR, 5-HT2AR and D3R at binding site. (B) The electrostatic
potential energy diagram of 9_24 and 5-HT2AR at binding site. (C) The electrostatic potential
energy diagram of 9_24 and D3R at binding site.
To explain the high selectivity of 9_24 towards 5-HT1A receptor, the sequences of
5-HT1A receptor, 5-HT2A receptor and D3 receptor at the binding site were aligned. The
3D model of 5-HT2A receptor was built by taking X-ray structure of 5-HT2C receptor
(PDB code: 6bqh, resolution:2.7 Å) as a template by Discovery Studio 3.5 program.
The published D3R crystal structure of homo sapiens was bound to risperidone (PDB
code: 3pbl, resolution:2.89 Å). As shown in Figure 3A, the residues of the active site
in three receptors differed greatly and thus the conformations of the three receptors at
the binding site were different. The 3D structures of the active site of 5-HT1AR complex
and the 5-HT2AR were superimposed to explain the selectivity of 9_24 for 5-HT1AR
and 5-HT2AR. As shown in Figure 3B, the indole head, the protonated nitrogen and the
phenyl group had steric hindrance with residues W3.28, D3.32 and I4.56 of active site in
the 5-HT2A receptor, resulting in the reduced affinity of 9_24 for 5-HT2AR. The same
phenomenon was found in the case of D3R when superposing 5-HT1AR and D3R. The
protonated nitrogen and the benzene ring of indole head group had steric collapse with
residues D3.32 and S5.43 It explains why 9_24 shows high selectivity towards 5-HT1AR
over 5-HT2AR and D3R. The binding free energy of (S)-configuration of compounds is
lower than that of (R)- configuration, indicating these compounds mainly exists in its S
configuration (Table S1).
3.4. 9_24 induced activation mechanism of 5-HT1AR
3.4.1. The dynamic characteristic of active site.
The molecular dynamic simulations were performed to investigate how 9_24
activates the 5-HT1AR receptor at the molecular level. Two simulation systems, 9_24
bound to the active-state 5-HT1AR and the inactive-state 5-HT1AR without ligand (apo),
were performed for 100-ns molecular simulations triple times, respectively. Each MD
simulation for a system got similar conformational motion features (Figure S2), one
representative trajectory was selected and used in further analysis. The structure of
inactive state 5-HT1AR was built by taking X-ray structure of 5-HT1BR as a template
(PDB code: 5v54). Gromos conformational cluster analysis was used to cluster the
snapshots and obtain the representative structures for 5-HT1AR-9_24 and apo-5-HT1AR
systems within the course of 100 ns simulation. It can be seen that the orientation of P2
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is perpendicular to P3’s (Figure 4). Phenyl ring exactly matches the pocket of broader
P2 and long alkoxyl chain stretches into the narrow pocket of P3. P2 is located in the
hydrophobic environment and a few amino acids with positive charge form P3 pocket
during MD simulations.
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Figure 4. The electrostatic potential diagram of 9_24 and 5-HT1AR at binding site.
3.4.2. The dynamic binding mode and helix motion.
A conserved hydrogen bond of the NH group of the indole ring in 9_24 with the
oxygen atom of hydroxyl in T3.37 is formed (Figure 5A). The cyano group of indole
head group is located in a hydrophilic cavity formed by T5.39, S5.42 and T5.43. Within the
course of 100 ns simulation, the cyano group flips in this cavity by forming alternative
hydrogen bonds with T5.39, S5.42 and T5.43 (Figure 5B and C): the cyano forms a
hydrogen bond (HB) with T5.39 at 0.65 ns, while it forms HB with S5.42 at 52.38 ns and
forms HB with S5.43 at 73.01 ns. The protonated nitrogen atom of 9_24 forms a
bifurcated hydrogen bond network with the carboxylic acid of D3.32 and the phenolic
hydroxyl group of Y7.43 (Figure 5A and D). Therefore, the 3-7 lock in 9_24 activated
5-HT1AR-complex system keeps lock, while that in inactive-state apo system is broken
(Figure 5A and E). This phenomenon is in an agreement with that of active and inactive
system in 5-HT1B 24. In addition, the phenyl group of 9_24 forms π-π interaction with
F3.28 (Figure 5A).
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Figure 5. (A) The binding mode of 9_24 with 5-HT1AR at binding site. Ligand was shown in green
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sticks. 5-HT1AR and surrounding residues in gray. Hydrogen bond interactions were represented in
dotted red. (B) The cyano group of 9_24 formed hydrogen bonds with residues in helix 5 of 5-
HT1AR. (C) The distances of ligand with T5.43, S5.42 and T5.39. The distances of ligand with (D) D3.32
and Y7.43. (E) The distance between D3.32 and Y7.43, also known as 3-7 lock.
In terms of 9_24-5-HT1AR simulation system, the strong interactions of 9_24 with
5-HT1AR result in the significant motion in helixes 3, 6 and 7: helix 3 moves towards
binding pocket and helix 7 bends to helix3 (Figure 6A). The hydrophobic packing
interaction of I3.40, P5.50 and F6.44 stabilizes the relative positions of H3, H5 and H6 in
its inactive state. The side chain of I3.40 flips up due to the interaction of 9_24 with D3.32
and T3.37. As a result, the side chains of P5.50 and F6.44 are repositioned, which causes
corresponding swing in H5 and H6 (Figure 6B). In the meantime, W6.48 acts as a
“rotamer toggle switch” to adjust the rotation of H6 (Figure 6C)26. Finally, H3, H5 and
H6 undergo significant motions in a synergistic way which leaves a space for the
binding of Gα/s protein. It is noteworthy that H6 dramatically moves outward 8.9 Å.
3.4.3. Proposed activation mechanism.
Based on the structural modeling and MD simulations, a stepwise 9_24 induced
signal transduction process of 5-HT1AR is proposed. Typically, the 5-HT1AR adopts the
inactive state through its intramolecular interaction. As the protonated 9_24 approaches
to the extracellular mouth of the binding cavity of 5-HT1AR, the electrostatic attraction
between the negatively charged region of the 5-HT1AR and positively charged 9_24
motivates the ligand recognition and binding process. The binding of 9_24 with the
residues of active site in 5-HT1AR results in the rearrangement of side chains in P5.50,
I3.40 and F6.44 and formation of 3-7 lock. In the meantime, W6.48 acts as a toggle switch
to adjust the rotation of H6. H3, H5 and H6 undergoes significant motion in a synergic
way, and the synergic helixes movement in H5, H6 and H7 makes space for the binding
of Gα/s protein. Finally, 5-HT1AR is fully activated, leading to the downstream signal
transduction (Figure 6D).
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Figure 6. (A) The helixes movements of 9_24 bond to 5HT1AR compared with apo system. (B) The
superimposed structures of apo and 5-HT1AR-9_24 systems and the rearrangements of I3.40, P5.50 and
F6.44 when 9_24 bond to 5HT1AR. (C) W6.48 and F6.44 cause the movements of H6 in apo and 5HT1AR-
9_24 systems. (D) Proposed activation process of 5HT1AR. R: inactive state; R´: electrostatically
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induced binding of an agonist; R ´´: agonist binding induced major structural rearrangement; R*:
fully activated 5HT1AR. Ligand was shown in green balls and sticks. 5-HT1AR-9_24 systems and
surrounding residues in teal. Apo system and surrounding residues in yellow. Hydrogen bond
interactions were represented in dotted red.
4. Conclusion
In summary, a series of novel indolyalkylpiperazine derivatives exhibiting high
affinity and good selectivity for 5-HT1AR were designed and synthesized. The
substituents on the amide tail group of these compounds influence the affinity and
selectivity towards 5-HT1AR. An unsubstituted phenyl ring and a flexible alkyl-chain
with a polar atom were beneficial to the activity and selectivity for 5-HT1AR. Among
these compounds, compound 9_24 with a 5-CN indole substituent showed relatively
high binding affinity (Ki = 5 ± 0.6 nM) and high potent agonistic activity (EC50 = 0.0593
nM) with perfect selectivity for 5-HT1AR. Molecular docking results suggested that
9_24 formed salt-bridge interaction with D3.32 of 5-HT1AR. The indole head group
formed a hydrogen bond with T3.37 and T5.43. The high selectivity of 9_24 towards 5-
HT1AR is due to the non-conserved residues in P1 pocket in 5-HT1AR, 5-HT2AR and
D3R, which determines the pharmacological difference in three receptors. A stepwise
9_24 induced activation model of 5-HT1AR was proposed: The electrostatic interaction
between negatively charged active site of the 5-HT1AR and the positively protonated
charged 9_24 is the initial driving force for the ligand recognition. Then, 9_24 binds
with 5-HT1AR by forming hydrogen bond and hydrophobic interaction with the residues
of active site in 5-HT1AR. It leads to the rearrangement of P5.50, I3.40 and F6.44 and
formation of 3-7 lock. W6.48 acts as a toggle switch to adjust the rotation of TM6. The
synergic helixes movement in H5, H6 and H7 makes space for the binding of Gα /s
protein. Finally, 5-HT1AR is activated by 9_24, thus 9_24 can serve as a lead compound
for further pharmacological studies.
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5. Experimental protocols
5.1. Chemistry
Reagents and solvents were purchased from Adamas-beta and used without
further purification. Analytical thin-layer chromatography was performed on HSGF
254 (0.15-0.2 mm thickness, Yantai Jiangyou Company, Yantai, Shandong, China).
1
Column chromatography was carried out on silica gel (200-300 mesh). H spectra
was recorded on a Brucker AMX-400/600 instrument, using TMS as an internal
standard and the chemical shifts were reported in parts per million (ppm). Proton-
coupling patterns were described as singlet, doublet, triplet, quartet, multiplet, and
coupling constants (J) values are given in hertz (Hz). Mass spectra were given with
an electric ionization (ESI) produced by HP5973N analytical mass spectrometer. All
tested compounds had a minimal purity of 95% determined by HPLC.
5.1.1. General procedures for the preparation of compounds 2a-c.
To a solution of corresponding phenylhydrazine hydrochloride (30.0 mmol) in 4%
H2SO4 (aq) (50 ml) and N,N-dimethylformamide (50 ml) at 100°C was added
dihydrofuran (36.0 mmol) dropwise over 2 min. The reaction mixture was stirred for 2
h and then poured into water, extracted with ethyl acetate (5×40 ml). The organic phases
were combined, washed with saturated brine, dried over anhydrous Na2SO4, filtered
and evaporated. The residue was purified by chromatography (petroleum ether: ethyl
acetate 20:1) to yield 2a-c as oily liquid.
5.1.1.1. 3-(5-fluoro-1H-indol-3-yl)propan-1-ol (2a); 1H NMR (400 MHz, CDCl3)
δ 8.07 (s, 1H), 7.25 – 7.17 (m, 1H), 7.03 (d, J = 2.9 Hz, 1H), 6.98 – 6.88 (m, 1H), 3.72
(d, J = 6.4 Hz, 2H), 2.81 (t, J = 7.5 Hz, 2H), 1.97 (p, J = 6.9 Hz, 2H). ESI-MS m/z
216.1 [M+Na] +; 191.9 [M-H] -.
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5.1.2. Procedures for the preparation of compound 2d.
For intermediate 2d, it can be prepared from 3-(5-bromo-1H-indol-3-yl)propan-1-
ol (2c). To a solution of 2c (1.00 g, 3.94 mmol) in N,N-dimethylformamide (10 ml) was
added CuCN (1.10 g, 11.82 mmol). The reaction mixture was stirred at 130℃ for 6 h.
After reaction, the reaction mixture was poured into water, extracted with ethyl acetate
(5×10 ml). The organic phases were combined, washed with saturated brine, dried over
anhydrous Na2SO4, filtered and evaporated. The residue was purified by
chromatography (petroleum ether: ethyl acetate 20:1) to yield 0.71 g of 2d as yellowish
oily liquid. (90.0% yield)
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5.1.3. General procedures for the preparation of compounds 3a, 3b, 3d.
To a solution of 2 (3.0 mmol) in anhydrous dichloromethane (6 ml) was added
carbon tetrabromide (9.0 mmol) and triphenylphosphine (9.0 mmol) at 0℃ and then
transferred to room temperature, stirred for 15 h. After reaction, 30 ml water was added
and the aqueous phase was washed with dichloromethane (3×20 ml). The organic
phases were combined, washed with saturated brine, dried over anhydrous Na2SO4,
filtered and evaporated. The residue was purified by chromatography (petroleum ether:
ethyl acetate 20:1) to yield 3a, 3b, 3d.
5.1.3.1. 3-(3-bromopropyl)-5-fluoro-1H-indole (3a); 1H NMR (400 MHz, CDCl3)
δ 8.07 (s, 1H), 7.29 – 7.24 (m, 1H), 7.03 (d, J = 2.9 Hz, 1H), 6.98 – 6.87 (m, 1H), 3.72
(d, J = 6.4 Hz, 2H), 2.81 (t, J = 7.5 Hz, 2H), 1.97 (p, J = 6.9 Hz, 2H). ESI-MS m/z
255.9, 257.9 [M+H] +.
5.1.4. Procedures for the preparation of compound 3c.
For intermediate 3c, it can be prepared from 3-(3-bromopropyl)-5-methoxy-1H-
indole (3b). Boron tribromide (0.82 ml, 14.6 mmol) in anhydrous dichloromethane (30
ml) was added dropwise to the mixture of 3b (1.0 g, 4.9 mmol) in anhydrous
dichloromethane (15 ml) under N2 protection at -78°C. Two hours later, the reaction
mixture was poured into water, extracted with ethyl acetate (3×30 ml). The organic
phases were combined, washed with saturated brine, dried over anhydrous Na2SO4,
filtered and evaporated. The residue was purified by chromatography (petroleum ether:
ethyl acetate 10:1) to yield 0.77g of 3c as black green oily liquid. (81.0% yield)
5.1.5. General procedures for the preparation of compounds 5e-q.
To a solution of corresponding aniline (30 mmol) and corresponding ketone (30
mmol) in acetic acid (25 ml) and water (2.5 ml) was added Zn powder (30 mmol), the
reaction mixture was stirred at 75°C for 4 h. After cooling to room temperature, crushed
ice was added and ammonia solution was used to adjust the pH to about 10. The aqueous
phase was washed with dichloromethane (3×40 ml). The organic phases were combined,
washed with saturated brine, dried over anhydrous Na2SO4, filtered and evaporated.
The residue was purified by chromatography (petroleum ether: ethyl acetate 10:1) to
yield 5e-q as light yellow oily liquid.
5.1.5.1. N-cyclohexylaniline (5q); 1H NMR (400 MHz, CDCl3) δ 7.21 – 7.11 (m,
2H), 6.63 (dd, J = 30.6, 7.6 Hz, 3H), 3.31 – 3.20 (m, 1H), 2.06 (d, J = 12.2 Hz, 2H),
1.77 (dt, J = 13.7, 4.2 Hz, 2H), 1.66 (d, J = 13.0 Hz, 1H), 1.36 (t, J = 12.5 Hz, 2H), 1.29
– 1.21 (m, 1H), 1.15 (q, J = 12.8, 11.7 Hz, 2H). ESI-MS m/z 176.0 [M+H] +.
5.1.6. General procedures for the preparation of compounds 6a-q
To a solution of 5a-q (30.0 mmol) and triethylamine (36.0 mmol) in anhydrous
dichloromethane (50 ml), chloroacetyl chloride (36.0 mmol) was added at 0°C under
N2 atmosphere and then stirred at room temperature for 5 h. After reaction, 50 ml water
was added and the aqueous phase was washed with dichloromethane (3×40 ml). The
organic phases were combined, washed with saturated brine, dried over anhydrous
Na2SO4, filtered and evaporated. The residue was purified by chromatography
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(petroleum ether: ethyl acetate 30:1) to yield 6a-q as off-white solid.
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5.1.6.1. 2-chloro-N-cyclohexyl-N-phenylacetamide (6q); H NMR (300 MHz,
DMSO) δ 7.44-7.49 (m, 3H), 7.25-7.28 (m, 2H), 4.31-4.42 (m, 1H), 3.78 (s, 2H), 1.66-
1.79 (m, 4H), 1.49-1.53 (m, 1H), 1.23-1.36 (m, 2H), 0.92-1.01 (m, 3H). ESI-MS m/z
252.1 [M+H] +.
5.1.7. General procedures for the preparation of compounds 7a-q.
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To a suspension of 6a-q (20.0 mmol), potassium carbonate (24.0 mmol), and
catalytic amount of KI (0.03 mmol) in acetonitrile (50 ml) was added tert-butyl
piperazine-1-carboxylate (24.0 mmol). The reaction mixture was refluxed for 8 h at
75°C and then acetonitrile was evaporated. 50 ml water was added and the aqueous
phase was washed with dichloromethane (3×30 ml). The organic phases were combined,
washed with saturated brine, dried over anhydrous Na2SO4, filtered and evaporated.
The residue was purified by chromatography (dichloromethane: methanol 50:1) to yield
7a-q as yellowish oily liquid.
5.1.7.1. Tert-butyl 4-(2-(cyclohexyl(phenyl)amino)-2-oxoethyl)piperazine-1-
carboxylate (7q); 1H NMR (400 MHz, CDCl3) δ 7.39 (s, 3H), 7.08 (d, J = 3.7 Hz, 2H),
4.58 (t, J = 11.7 Hz, 1H), 3.40 (s, 4H), 2.76 (s, 2H), 2.37 (s, 4H), 1.82 (d, J = 11.3 Hz,
2H), 1.71 (d, J = 12.7 Hz, 2H), 1.56 (d, J = 13.6 Hz, 1H), 1.43 (s, 9H), 1.35 (d, J = 13.9
Hz, 1H), 1.26 (s, 1H), 1.01 (q, J = 12.1 Hz, 2H), 0.89 (d, J = 13.2 Hz, 1H). ESI-MS m/z
402.0 [M+H]+
5.1.8. General procedures for the preparation of compounds 8a-q.
To a solution of 7a-q (15.0 mmol) in dichloromethane was added trifluoroacetic
acid (15 mL). The mixture was stirred at room temperature for 3 h, then concentrated,
washed with petroleum ether and ethyl acetate separately to yield 8a-q as off-white
solid.
5.1.8.1. N-cyclohexyl-N-phenyl-2-(piperazin-1-yl)acetamide trifluoroacetate (8q);
1H NMR (400 MHz, CDCl3) δ 7.47 (s, 3H), 7.12 (s, 2H), 4.46 (s, 1H), 3.49 (d, J = 28.7
Hz, 8H), 3.33 (s, 2H), 1.77 (dd, J = 23.1, 13.0 Hz, 4H), 1.57 (d, J = 11.9 Hz, 1H), 1.31
(t, J = 19.9 Hz, 3H), 1.12 – 0.95 (m, 2H), 0.90 (d, J = 13.0 Hz, 1H). ESI-MS m/z 302.0
[M+H]+
5.1.9. General procedures for the preparation of compounds 9_1-9_25 and FW01.
To a solution of 3a-d (3.0 mmol) in acetonitrile was added potassium carbonate
(4.5 mmol) and 8a-q (3.6 mmol). The reaction mixture was refluxed for 12 h at 85℃
and then acetonitrile was evaporated. 50 ml water was added and the aqueous phase
was washed with dichloromethane (3×30ml). The organic phases were combined,
washed with saturated brine, dried over anhydrous Na2SO4, filtered and evaporated.
The residue was purified by chromatography (dichloromethane: methanol 50:1) to yield
9_1-9_25 as white solid.
5.1.9.1.
2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N,N-
diphenylacetamide (9_1). Compound 9_1 was prepared from 3-(3-bromopropyl)-5-
fluoro-1H-indole 3a and N,N-diphenyl-2-(piperazin-1-yl)acetamide trifluoroacetate
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8a as a white solid. Yield 77.6%; H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H, NH),
7.35-7.20 (m, 12H, Ar-H), 6.96 (s, 1H), 6.89-6.81 (m, 1H), 3.10 (s, 2H), 2.68 (t, J =
7.4 Hz, 2H), 2.55-2.39 (m, 10H), 1.87-1.84 (m, 2H). 13C NMR (100 MHz, CDCl3) δ
171.00, 155.51, 138.42, 133.03, 131.18, 131.18, 130.53, 130.53, 129.78, 129.78,
129.18, 129.18, 128.53, 128.53, 127.73, 127.26, 126.08, 114.57, 112.77, 109.96,
106.83, 60.16, 56.23, 53.50, 53.50, 52.93, 52.93, 26.93, 24.93. ESI-MS m/z 471.3
[M+H] +.
5.1.9.2. N,N-dicyclohexyl-2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-
yl)acetamide (9_2). Compound 9_2 was prepared from 3-(3-bromopropyl)-5-fluoro-
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1H-indole 3a and N,N-dicyclohexyl-2-(piperazin-1-yl)acetamide trifluoroacetate 8b as
a white solid. Yield 72.9%; 1H NMR (400 MHz, CDCl3) δ 7.95 (s, 1H), 7.28 (s, 1H),
7.18 (s, 1H), 6.97-6.89 (m, 2H), 4.57-4.48 (m, 2H), 2.85 (s, 2H), 2.73-2.69 (m, 2H),
2.61-2.48(m, 10H), 1.94-1.87 (m, 4H), 1.75-1.70 (m, 6H), 1.65-1.58 (m, 8H), 1.31-
1.24 (m, 4H). 13C NMR (100 MHz, CDCl3 ) δ 169.77, 155.51, 133.03, 127.73, 126.08,
114.57, 112.77, 109.96, 106.83, 60.40, 56.23, 53.50, 53.50, 52.93, 52.93, 52.68, 31.37,
31.37, 26.93, 26.93, 25.92, 25.92, 24.93, 24.93, 24.76, 24.76. ESI-MS m/z 483.1
[M+H] +.
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5.1.9.3.
2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-
phenylacetamide (9_3). Compound 9_3 was prepared from 3-(3-bromopropyl)-5-
fluoro-1H-indole 3a and N-phenyl-2-(piperazin-1-yl)acetamide trifluoroacetate 8c as
a white solid. Yield 86.4%; 1H NMR (400 MHz, CDCl3) δ 9.13 (s, 1H), 8.06 (s, 1H),
7.58-7.56 (m, 2H), 7.34 (t, J = 7.9 Hz, 2H), 7.27-7.23 (m, 2H), 7.11 (t, J = 7.4 Hz, 1H),
7.03 (d, J = 1.8 Hz, 1H), 6.93 (td, J = 9.1, 2.4 Hz, 1H), 3.14 (s, 2H), 2.75 (t, J = 7.5
13
Hz, 2H), 2.68 (s, 4H), 2.56 (s, 4H), 2.47 (t, J = 8.0 Hz, 2H), 1.94-1.87 (m, 2H). C
NMR (100 MHz, CDCl3) δ 179.65, 155.37, 137.95, 135.32, 128.90, 128.90, 127.73,
126.08, 123.70, 120.89, 120.89, 114.57, 112.77, 109.96, 106.83, 59.99, 56.23, 53.50,
53.50, 52.93, 52.93, 26.93, 24.93. ESI-MS m/z 395.2 [M+H] +.
5.1.9.4.
N-cyclohexyl-2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-
yl)acetamide (9_4). Compound 9_4 was prepared from 3-(3-bromopropyl)-5-fluoro-
1H-indole 3a and N-cyclohexyl-2-(piperazin-1-yl)acetamide trifluoroacetate 8d as a
white solid. Yield 83.8%; H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 7.78 (s, 1H),
7.21 (s, 1H), 7.00-6.93 (m, 2H), 6.67 (s, 1H), 4.95-4.87 (m, 1H), 3.01 (s, 2H), 2.87 (t,
J = 12.0 Hz, 2H), 2.72-2.68 (m, 8H), 2.19-2.10 (m, 2H), 1.85-1.68 (m, 6H), 1.62 (s,
1
13
1H), 1.43–1.13 (m, 5H). C NMR (100 MHz, CDCl3) δ 173.45, 155.51, 133.03,
127.73, 126.08, 114.57, 112.77, 109.96, 106.83, 59.91, 56.23, 53.25, 53.25, 52.75,
52.75, 49.43, 33.63, 33.63, 26.93, 25.92, 24.93, 24.73, 24.73. ESI-MS m/z 401.5
[M+H] +.
5.1.9.5. N-cyclohexyl-2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-
(4-fluorophenyl)acetamide (9_5). Compound 9_5 was prepared from 3-(3-
bromopropyl)-5-fluoro-1H-indole 3a and N-cyclohexyl-N-(4-fluorophenyl)-2-
(piperazin-1-yl)acetamide trifluoroacetate 8e as a white solid. Yield 80.2%; 1H NMR
(400 MHz, CDCl3) δ 8.05 (s, 1H), 7.25-7.20 (m, 2H), 7.11-7.07 (m, 4H), 7.01 (s, 1H),
6.91-6.85 (m, 1H), 4.57-4.52 (m,1H), 2.72 (s, 2H), 2.70-2.68 (m, 4H), 2.46-2.39 (m,
8H), 1.90-1.75 (m, 2H), 1.69-1.60 (m, 2H), 1.41-1.36 (m, 4H), 1.27-1.20 (m, 2H),
13
1.01-0.92 (m, 2H). C NMR (100 MHz, CDCl3) δ 171.25, 159.13, 136.01, 133.03,
127.73, 126.62, 126.62, 126.08, 117.15, 117.15, 114.57, 112.77, 109.96, 109.96,
106.83, 59.71, 56.23, 54.28, 53.21, 53.21, 52.93, 52.93, 31.72, 31.72, 26.93, 25.92,
24.93, 24.76, 24.76. ESI-MS m/z 495.2 [M+H] +.
5.1.9.6. N-cyclohexyl-2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-
(4-methoxyphenyl)acetamide (9_6). Compound 9_6 was prepared from 3-(3-
bromopropyl)-5-fluoro-1H-indole 3a and N-cyclohexyl-N-(4-methoxyphenyl)-2-
(piperazin-1-yl)acetamide trifluoroacetate 8f as a white solid. Yield 83.8%; 1H NMR
(400 MHz, CDCl3) δ 8.04 (s, 1H), 7.25-7.20 (m, 2H), 7.01-6.87 (m, 6H), 4.55-4.50(m,
1H), 3.84 (s, 3H), 2.74 (s, 2H), 2.69 (t, J = 7.2 Hz, 2H), 2.55 (t, J = 7.2 Hz, 2H), 2.47-
2.38 (m, 8H), 1.89-1.69 (m, 2H), 1.71-1.65 (m, 2H), 1.37-1.31 (m, 4H), 1.25-1.21 (m,
2H), 0.89-0.83 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 175.18, 159.30, 155.51, 132.99,
132.99, 127.73, 126.00, 126.00, 115.60, 115.60, 114.57, 112.77, 112.01, 109.96,
106.83, 59.71, 56.16, 55.31, 54.15, 53.21, 53.21, 52.87, 52.87, 31.57, 31.57, 26.93,
25.92, 24.93, 24.58, 24.58. ESI-MS m/z 507.2 [M+H] +.
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5.1.9.7. N-cyclohexyl-2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-
(4-(methylsulfonyl)phenyl)acetamide (9_7). Compound 9_7 was prepared from 3-(3-
bromopropyl)-5-fluoro-1H-indole
3a
and
N-cyclohexyl-N-(4-
(methylsulfonyl)phenyl)-2-(piperazin-1-yl)acetamide trifluoroacetate 8g as a white
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solid. Yield 70.8%; H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 7.27-7.18 (m, 2H),
7.10-6.83 (m, 6H), 4.48-4.42(m,1H), 3.32(s, 3H), 2.72(s, 2H), 2.62 (t, J = 7.5 Hz, 2H),
2.51-2.42 (m, 10H), 1.91-1.83 (m, 2H), 1.51 (d, J = 12.8 Hz, 2H), 1.33-1.18 (m, 2H),
1.00-0.95 (m, 4H), 0.89-0.81 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 179.31, 155.51,
144.58, 144.58, 135.18, 133.03, 130.21,130.21, 127.73, 126.08, 121.93, 114.57,
112.77, 109.96, 106.83, 60.71, 58.76, 56.21, 55.31, 52.89, 52.89, 50.76, 50.76, 43.80,
31.72, 31.72, 26.93, 25.92, 24.93, 24.76, 24.76. ESI-MS m/z 555.1 [M+H] +.
5.1.9.8. N-cyclohexyl-2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-
(2-methoxyphenyl)acetamide (9_8). Compound 9_8 was prepared from 3-(3-
bromopropyl)-5-fluoro-1H-indole 3a and N-cyclohexyl-N-(2-methoxyphenyl)-2-
(piperazin-1-yl)acetamide trifluoroacetate 8h as a white solid. Yield 74.8%; 1H NMR
(400 MHz, CDCl3) δ 8.07 (s, 1H), 7.26-7.20 (m, 3H), 7.11-7.02 (m, 5H), 4.57-4.49
(m, 1H), 3.81 (s, 3H), 2.76 (s, 2H), 2.71 (t, J = 7.2 Hz, 2H), 2.63-2.42 (m, 10H), 1.89-
1.72 (m, 2H), 1.70-1.65 (m, 2H), 1.42-1.38 (m, 4H), 1.26-1.20 (m, 2H), 0.91-0.85 (m,
2H). 13C NMR (100 MHz, CDCl3) δ 176.23, 159.35, 155.51, 132.99, 132.99, 127.73,
126.05, 126.05, 115.60, 115.60, 114.41, 112.77, 112.01, 109.96, 106.85, 59.71, 56.23,
55.31, 54.15, 53.21, 53.21, 52.87, 52.87, 31.57, 31.57, 26.93, 25.92, 24.92, 24.73,
24.73. ESI-MS m/z 507.5 [M+H] +.
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5.1.9.9.
2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-(4-
methylcyclohexyl)-N-phenylacetamide (9_9). Compound 9_9 was prepared from 3-
(3-bromopropyl)-5-fluoro-1H-indole 3a and N-(4-methylcyclohexyl)-N-phenyl-2-
(piperazin-1-yl)acetamide trifluoroacetate 8i as a white solid. Yield 81.5%; 1H NMR
(400 MHz, CDCl3) δ 8.44 (s, 1H), 7.39-7.30 (m, 3H), 7.25-7.23 (m, 1H), 7.18 (dd, J =
9.6, 2.4 Hz, 1H), 7.10-7.08 (m, 2H), 7.01 (s, 1H), 6.89-6.83 (m , 1H), 4.51-4.43 (m,
1H), 2.75 (s, 2H), 2.68 (t, J = 7.2 Hz, 2H), 2.54-2.44 (m, 10H), 1.93-1.86 (m, 2H),
1.67-1.55 (m, 5H), 1.46 (d, J = 12.8 Hz, 2H), 1.32-1.26 (m, 2H), 0.72 (d, J = 7.2 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 178.31, 159.35, 140.30, 136.21, 129.76, 129.76,
127.73, 127.21, 126.30, 126.10, 114.57, 113.45, 112.77, 109.96, 106.83, 59.35, 55.21,
52.75, 52.21, 52.21, 50.93, 50.93, 32.93, 32.93, 32.28, 29.39, 26.93, 24.93, 21.89,
21.89. ESI-MS m/z 491.4 [M+H] +.
5.1.9.10.
N-(4-ethylcyclohexyl)-2-(4-(3-(5-fluoro-1H-indol-3-
yl)propyl)piperazin-1-yl)-N-phenylacetamide (9_10). Compound 9_10 was prepared
from 3-(3-bromopropyl)-5-fluoro-1H-indole 3a and N-(4-ethylcyclohexyl)-N-phenyl-
2-(piperazin-1-yl)acetamide trifluoroacetate 8j as a white solid. Yield 78.3%; 1H NMR
(400 MHz, CDCl3) δ 8.66 (s, 1H), 7.45-7.41(m, 3H), 7.32-7.26 (m, 1H), 7.16 (s, 1H),
7.12-7.02 (m, 3H), 6.92-6.85 (m, 1H), 4.46-4.41 (m, 1H), 3.33 (s, 2H), 2.95-2.78 (m,
10H), 2.71 (t, J = 7.2 Hz, 2H), 2.25 – 2.14 (m, 2H), 1.77 (d, J = 15.2 Hz, 2H), 1.69–
1.50 (m, 6H), 1.45 (s, 1H), 1.26–1.16 (m, 2H), 0.74 (t, J = 7.2 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ 176.31, 158.21, 141.19, 137.05, 129.55, 129.55, 127.73, 127.21,
126.56, 126.21, 114.21, 113.45, 111.59, 109.96, 107.35, 57.21, 53.56, 52.71, 52.03,
52.03, 50.75, 50.75, 31.25, 31.25, 30.75, 29.41, 27.55, 24.72, 23.13, 20.81, 19.27. ESI-
MS m/z 505.3 [M+H] +.
5.1.9.11.
N-(4-(tert-butyl)cyclohexyl)-2-(4-(3-(5-fluoro-1H-indol-3-
yl)propyl)piperazin-1-yl)-N-phenylacetamide (9_11). Compound 9_11 was prepared
from 3-(3-bromopropyl)-5-fluoro-1H-indole 3a and N-(4-(tert-butyl)cyclohexyl)-N-
phenyl-2-(piperazin-1-yl)acetamide trifluoroacetate 8k as a white solid. Yield 73.3%;
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1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.39-7.37 (m, 3H), 7.25-7.20 (m, 3H),
7.00 (s, 1H), 6.91-6.85 (m, 1H), 4.70-4.67 (m, 1H), 2.74 (s, 2H), 2.69 (t, J = 7.6 Hz,
2H), 2.47-2.37 (m, 10H), 1.96-1.81 (m, 4H), 1.63-1.58 (m, 2H), 1.38-1.33 (m, 2H),
13
1.02-0.97 (m, 1H), 0.87-0.78 (m, 2H), 0.67 (s, 9H). C NMR (100 MHz, CDCl3) δ
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178.76, 158.49, 140.35, 140.35, 133.03, 129.76, 127.73, 127.73, 126.30, 126.10,
126.10, 114.57, 112.77, 109.96, 106.83, 58.57, 56.23, 56.23, 53.90, 53.50, 52.93,
47.49, 32.69, 28.02, 28.02, 27.22, 26.97, 26.97, 24.93, 24.93, 24.93. ESI-MS m/z
533.3[M+H] +.
5.1.9.12. 2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-phenyl-N-
(tetrahydro-2H-pyran-4-yl)acetamide (9_12). Compound 9_12 was prepared from 3-
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(3-bromopropyl)-5-fluoro-1H-indole
3a
and
N-phenyl-2-(piperazin-1-yl)-N-
(tetrahydro-2H-pyran-4-yl)acetamide trifluoroacetate 8l as a white solid. Yield 81.4%;
1H NMR (400 MHz, DMSO-d6) δ 7.81 (s, 1H), 7.54 (s, 1H), 7.38-7.25 (m, 5H), 7.05
(s, 1H), 6.78 (d, J = 6.4 Hz, 2H), 4.58-4.53 (m, 1H), 3.78-3.66 (m, 4H), 2.65 (s ,2H),
2.53-2.43 (t, J = 9.2 Hz, 2H), 2.32 (s, 10H), 1.78-1.64 (m, 4H), 1.29-1.19 (m, 2H). 13C
NMR (100 MHz, DMSO-d6) δ 174.29, 155.23, 143.32, 135.21, 132.45, 132.45, 127.50,
126.51, 126.10, 126.10, 119.21, 114.43, 113.87, 111.63, 105.21, 61.34, 60.38, 58.65,
56.36, 54.21, 53.40. 52.27, 52.27, 47.32, 30.48, 31.25, 25.43, 23.21. ESI-MS m/z
479.5 [M+H] +.
5.1.9.13. 2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-(pentan-2-
yl)-N-phenylacetamide (9_13). Compound 9_13 was prepared from 3-(3-
bromopropyl)-5-fluoro-1H-indole 3a and N-phenyl-2-(piperazin-1-yl)-N-(pentan-2-
yl)acetamide trifluoroacetate 8m as a white solid. Yield 86.5%; 1H NMR (400 MHz,
CDCl3) δ 8.48 (s, 1H), 7.40-7.36 (m, 3H), 7.25-7.18 (m, 2H), 7.08 (d, J = 6.3 Hz, 2H),
6.98 (s, 1H), 6.92-6.89 (m, 1H), 4.90-4.85 (m, 1H), 2.74 (s, 2H), 2.67 (t, J = 7.4 Hz,
2H), 2.46-2.37 (m, 10H), 1.88-1.80 (m, 2H), 1.46-1.32 (m, 3H), 1.24-1.15 (m, 1H),
1.0 (d, J = 6.7 Hz, 3H), 0.90 (t, J = 6.7 Hz, 7.0, 3H). 13C NMR (100 MHz, CDCl3) δ
168.92, 158.61, 156.36, 137.91, 132.75, 130.54, 129.14, 128.27, 127.88, 127.73,
123.10, 116.11, 111.65, 110.19, 103.77, 60.40, 58.13, 53.89, 53.27, 52.91, 52.76, 50.1,
36.9, 27.0, 22.8, 19.9, 19.0, 14.1. ESI-MS m/z 465.2 [M+H] +.
5.1.9.14. 2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-(pentan-3-
yl)-N-phenylacetamide (9_14). Compound 9_14 was prepared from 3-(3-
bromopropyl)-5-fluoro-1H-indole 3a and N-phenyl-2-(piperazin-1-yl)-N-(pentan-3-
1
yl)acetamide trifluoroacetate 8n as a white solid. Yield 84.9%; H NMR (400 MHz,
CDCl3) δ 8.58 (s, 1H), 7.45–7.37 (m, 3H), 7.30–7.24 (m, 1H), 7.14-7.09 (m, 1H), 7.12–
7.08 (m, 2H), 7.07 (d, J = 2.1 Hz, 1H), 6.90 (td, J = 9.0, 2.5 Hz, 1H), 4.60-4.52 (m,
1H), 2.85 (s, 2H), 2.82 (t, J = 7.3 Hz, 2H), 2.81–2.52 (m, 10H), 2.07-1.98 (m, 2H),
13
1.42–1.29 (m, 4H), 0.97 (t, J = 7.2 Hz, 6H). C NMR (100 MHz CDCl3) δ 170.53,
156.57, 155.65, 137.83, 132.86, 130.76, 129.61, 129.40, 128.75, 127.70, 124.35,
116.53, 112.03, 110.94, 102.63, 61,33, 56.46, 55.56, 55.32, 52.93, 51.31, 50.32, 34.57,
29.56, 27.88, 26.79, 23.74, 14.51. ESI-MS m/z 465.1 [M+H] +.
5.1.9.15.
2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-(hexan-3-
yl)-N-phenylacetamide (9_15). Compound 9_15 was prepared from 3-(3-
bromopropyl)-5-fluoro-1H-indole 3a and N-phenyl-2-(piperazin-1-yl)-N-(hexan-3-
1
yl)acetamide trifluoroacetate 8o as a white solid. Yield 77.8%; H NMR (400 MHz,
CDCl3) δ 7.99 (s, 1H), 7.45–7.38 (m, 3H), 7.25 (d, J = 8.8 Hz, 1H), 7.13 (s, 2H), 7.02
(d, J = 2.4 Hz, 1H), 6.99 (s, 1H), 6.86-6.75 (m, 1H), 4.77–4.68 (m, 1H) , 2.81 (s, 2H),
2.75 (t, J = 8.4 Hz, 2H), 2.46-2.51 (m, 10H), 1.97 (m, 2H), 1.44–1.29 (m, 6H), 1.00 (t,
J = 7.3 Hz, 3H), 0.93 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz CDCl3) δ 170.52, 153.46,
151.37, 137.67, 134.93, 130.91, 128.83, 127.56, 125.54, 121.30, 120.89, 117.55,
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115.99, 113.83, 103.60, 61.22, 57.10, 56.67, 55.30, 55.07, 52.75, 52.39, 34.84, 28.74,
25.59, 22.36, 20.71, 14.56, 12.95. ESI-MS m/z 479.1 [M+H] +.
5.1.9.16.
2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1-yl)-N-(1-
methoxypropan-2-yl)-N-phenylacetamide (9_16). Compound 9_16 was prepared from
3-(3-bromopropyl)-5-fluoro-1H-indole 3a and N-phenyl-2-(piperazin-1-yl)-N-(1-
methoxypropan-2-yl)acetamide trifluoroacetate 8p as a white solid. Yield 79.5%; 1H
NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 7.21-7.14 (m, 3H), 7.05-6.83 (m, 2H), 6.76
(d, J = 6.3 Hz, 2H), 6.71 (s, 1H), 6.68-6.53 (m, 1H), 4.90-4.85 (m, 1H), 3.21(s, 3H),
3.18 (d, J = 7.2Hz, 2H), 2.74 (s, 2H), 2.67 (t, J = 7.4 Hz, 2H), 2.46-2.37 (m, 10H),
1.88-1.80 (m, 2H), 0.98-0.75 (d, J = 8.4Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 168.95,
159.35, 157.21, 141.85, 132.65, 132.65, 131.28, 129.55, 129.51, 128.73, 127.89,
115.43, 113.87, 109.25, 105.67, 65.21, 59.25, 58.13, 56.89, 53.71, 53.71, 52.43, 52.43,
51.07, 35.21, 25.75, 12.13. ESI-MS m/z 467.5 [M+H] +.
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5.1.9.17. N-(hexan-3-yl)-2-(4-(3-(5-methoxy-1H-indol-3-yl)propyl)piperazin-1-
yl)-N-phenylacetamide (9_17). Compound 9_17 was prepared from 3-(3-
bromopropyl)-5-methoxy-1H-indole 3b and N-phenyl-2-(piperazin-1-yl)-N-(hexan-3-
1
yl)acetamide trifluoroacetate 8o as a white solid. Yield 79.4%; H NMR (400 MHz,
CDCl3) δ 7.99 (s, 1H), 7.45–7.38 (m, 3H), 7.25 (d, J = 8.8 Hz, 1H), 7.13 (s, 2H), 7.02
(d, J = 2.4 Hz, 1H), 6.99 (s, 1H), 6.86-6.79 (m, 1H), 4.77–4.68 (m, 1H), 3.87 (s, 3H),
2.81 (s, 2H), 2.75 (t, J = 7.2 Hz, 2H), 2.46-2.38 (m, 10H), 1.97-1.83 (m, 2H), 1.44–
1.29 (m, 6H), 1.00 (t, J = 8.4 Hz, 3H), 0.93 (t, J = 7.2 Hz, 3H). 13C NMR (100MHz
CDCl3) δ 169.32, 153.88, 137.88, 131.56, 129.98, 129.40, 129.25, 128.52, 127.71,
122.38, 114.71, 114.08, 112.08, 111.93, 100.65, 60.16, 57.71, 56.81, 56.78, 56.02,
52.45, 51.70, 51.60, 34.92, 29.60, 29.63, 22.68, 21.15, 14.13, 11.45. ESI-MS m/z
491.2 [M+H] +.
5.1.9.18.
2-(4-(3-(5-methoxy-1H-indol-3-yl)propyl)piperazin-1-yl)-N-(1-
methoxypropan-2-yl)-N-phenylacetamide (9_18). Compound 9_18 was prepared from
3-(3-bromopropyl)-5-methoxy-1H-indole 3b and N-phenyl-2-(piperazin-1-yl)-N-(1-
methoxypropan-2-yl)acetamide trifluoroacetate 8p as a white solid. Yield 72.6%; 1H
NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.34-7.20 (m, 3H), 7.15-7.01 (m, 2H), 6.95
(d, J = 6.4Hz, 2H), 6.71-6.65 (m, 2H), 4.85-4.73 (m, 1H), 3.38 (s, 3H), 3.25(s, 3H),
3.18-3.13 (m, 2H), 2.75 (s, 2H), 2.61(t, J = 7.4 Hz, 2H), 2.51-2.43 (m, 10H), 1.85-1.79
(m, 2H), 1.01-0.89 (d, J = 8.4Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 171.72, 156.21,
155.43, 140.73, 131.58, 131.58, 130.28, 129.63, 129.63, 128.75, 127.26, 113.57,
112.76, 110.75, 108.75, 68.35, 59.73, 58.49, 57.35, 55.69, 53.15, 53.15, 52.43, 52.43,
50.21, 30.41, 28.76, 18.51. ESI-MS m/z 479.5 [M+H] +.
5.1.9.19. 2-(4-(3-(5-methoxy-1H-indol-3-yl)propyl)piperazin-1-yl)-N-phenyl-N-
(tetrahydro-2H-pyran-4-yl)acetamide (9_19). Compound 9_19 was prepared from 3-
(3-bromopropyl)-5-methoxy-1H-indole 3b and N-phenyl-2-(piperazin-1-yl)-N-
( tetrahydro-2H-pyran-4-yl)acetamide trifluoroacetate 8l as a white solid. Yield 75.1%;
1H NMR (400 MHz, DMSO-d6) δ 7.83 (s, 1H), 7.56 (s, 1H), 7.25-7.13 (m, 5H), 7.03
(s, 1H), 6.89 (d, J = 6.4 Hz, 2H), 4.58-4.53 (m, 1H), 3.80-3.69 (m, 4H), 3.32 (s, 3H),
2.66 (s ,2H), 2.51-2.42 (t, J = 9.2 Hz, 2H), 2.30 (s, 10H), 1.77-1.65 (m, 4H), 1.26-1.20
(m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 172.31, 155.23, 145.28, 135.21, 132.52,
132.52, 128.40, 126.43, 126.10, 126.10, 118.21, 115.34, 113.87, 110.78, 105.21, 67.12,
67.10, 58.65, 57.32, 56.36, 53.40, 53.40. 52.27, 52.27, 48.53, 30.48, 30.29, 25.43,
25.21. ESI-MS m/z 491.3 [M+H] +.
5.1.9.20. N-(hexan-3-yl)-2-(4-(3-(5-hydroxy-1H-indol-3-yl)propyl)piperazin-1-
yl)-N-phenylacetamide (9_20). Compound 9_20 was prepared from 3-(3-
bromopropyl)-5-hydroxy-1H-indole 3c and N-phenyl-2-(piperazin-1-yl)-N-(hexan-3-
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yl)acetamide trifluoroacetate 8o as a white solid. Yield 83.8%; H NMR (600 MHz,
CDCl3) δ 8.87 (s, 1H), 7.4-7.38 (m, 3H), 7.14 (d, J = 8.6 Hz, 1H), 7.11-7.03 (m, 2H),
6.92 (s, 1H), 6.75 (d, J = 8.4 Hz, 2H), 4.71-4.53 (m, 1H), 2.82 (s, 2H), 2.70-2.61 (m,
6H), 2.53 (t, J = 6.8 Hz, 2H), 2.37 (t, J = 6.4 Hz, 2H), 1.85 (s, 2H), 1.42-1.36 (m, 2H),
1.39-1.27 (m, 6H), 0.98 (t, J = 7.2 Hz, 3H), 0.92 (t, J = 7.2 Hz, 3H). 13C NMR (100
MHz CDCl3) δ 169.30, 150.01, 137.40, 131.40, 129.78, 129.66, 129.63, 128.81,
127.80, 127.72, 122.89, 112.84, 112.24, 112.08, 103.37, 59.62, 57.01, 56.94, 56.83,
51.63, 51.57, 50.12, 34.83, 29.70, 25.89, 22.70, 20.01, 14.08, 11.52. ESI-MS m/z
477.2 [M+H] +.
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5.1.9.21.
2-(4-(3-(5-hydroxy-1H-indol-3-yl)propyl)piperazin-1-yl)-N-(1-
methoxypropan-2-yl)-N-phenylacetamide (9_21). Compound 9_21 was prepared from
3-(3-bromopropyl)-5-hydroxy-1H-indole 3c and N-phenyl-2-(piperazin-1-yl)-N-(1-
methoxypropan-2-yl)acetamide trifluoroacetate 8p as a white solid. Yield 70.7%; 1H
NMR (400 MHz, CDCl3) δ 7.71 (s, 1H), 7.36-7.25 (m, 3H), 7.15-7.06 (m, 2H), 6.89
(d, J = 8.4 Hz, 2H), 6.79-6.73 (m, 2H), 4.89-4.80 (m, 1H), 3.31 (s, 3H), 3.20-3.14 (m,
2H), 2.73 (s, 2H), 2.65 (t, J = 7.2 Hz, 2H), 2.62-2.53 (m, 10H), 1.85-1.78 (m, 2H),
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1.02-0.95 (m, 3H). C-NMR (100 MHz, CDCl3) δ 173.89, 155.23, 153.21, 141.58,
130.52, 130.52, 129.54, 128.71, 128.71, 126.31, 125.87, 114.75, 112.89, 111.63,
109.83, 70.21, 59.75, 58.63, 57.81, 52.87, 52.87, 51.35, 51.35, 50.21, 28.33, 27.65,
19.21. ESI-MS m/z 465.3 [M+H] +.
5.1.9.22. 2-(4-(3-(5-hydroxy-1H-indol-3-yl)propyl)piperazin-1-yl)-N-phenyl-N-
(tetrahydro-2H-pyran-4-yl)acetamide (9_22). Compound 9_22 was prepared from 3-
(3-bromopropyl)-5-hydroxy-1H-indole 3c and N-phenyl-2-(piperazin-1-yl)-N-
( tetrahydro-2H-pyran-4-yl)acetamide trifluoroacetate 8l as a white solid. Yield 76.3%;
1H NMR (400 MHz, DMSO-d6) δ 7.67 (s, 1H), 7.32 (s, 1H), 7.25-7.13 (m, 5H), 7.06
(s, 1H), 6.89 (d, J = 6.4 Hz, 2H), 4.79 (s, 1H), 4.63-4.51 (m, 1H), 3.80-3.65 (m, 4H),
2.66 (s ,2H), 2.53-2.42 (t, J = 9.2 Hz, 2H), 2.31 (s, 10H), 1.75-1.63 (m, 4H), 1.26-1.21
(m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 172.31, 155.23, 145.28, 135.21, 132.52,
132.52, 128.40, 126.43, 126.10, 126.10, 118.21, 115.34, 113.87, 110.78, 105.21, 67.12,
67.10, 58.65, 56.36, 53.40, 53.40. 52.27, 52.27, 48.53, 30.48, 30.29, 25.43, 25.21. ESI-
MS m/z 477.21 [M+H] +.
5.1.9.23. 2-(4-(3-(5-cyano-1H-indol-3-yl)propyl)piperazin-1-yl)-N-(hexan-3-yl)-
N-phenylacetamide (9_23). Compound 9_23 was prepared from 3-(3-bromopropyl)-
5-cyano-1H-indole 3d and N-phenyl-2-(piperazin-1-yl)-N-(hexan-3-yl)acetamide
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trifluoroacetate 8o as a white solid. Yield 81.1%; H NMR (400 MHz, DMSO-d6) δ
9.15 (s, 1H), 7.91 (s, 1H), 7.39 (t, J = 7.2 Hz, 5H), 7.09 (d, J = 8.0 Hz, 3H), 4.76–4.67
(m, 1H), 2.79 (s, 2H), 2.77–2.70 (m, 2H), 2.46-2.31 (m, 10H), 1.88 (s, 2H), 1.42–1.25
(m, 6H), 0.97 (t, J = 7.2 Hz, 3H), 0.90 (t, J = 8.4 Hz, 3H). 13C NMR (100 MHz, DMSO-
d6) δ 164.34, 132.73, 131.55, 124.75, 124.04, 123.17, 122.05,121.89, 119.28, 118.42,
115.78, 111.26, 106.86, 105.72, 96.52, 55.21, 52.89, 52.52, 51.76, 51.35, 47.55, 44.91,
29.61, 27.25, 21.42, 20.64, 17.17, 14.72, 8.84, 6.25. ESI-MS m/z 486.5 [M+H] +.
5.1.9.24.
2-(4-(3-(5-cyano-1H-indol-3-yl)propyl)piperazin-1-yl)-N-(1-
methoxypropan-2-yl)-N-phenylacetamide (9_24). Compound 9_24 was prepared from
3-(3-bromopropyl)-5-cyano-1H-indole 3d and N-phenyl-2-(piperazin-1-yl)-N-(1-
methoxypropan-2-yl)acetamide trifluoroacetate 8p as a white solid. Yield 71.8%; 1H
NMR (400 MHz, CDCl3) δ 8.92 (s, 1H), 7.82-7.76 (m, 3H), 7.35-7.21 (m, 2H), 6.95
(d, J = 12.0Hz, 2H), 6.79-6.68(m, 2H), 4.79-4.58(m, 1H), 3.35 (s, 3H), 3.27-3.18 (m,
2H), 2.65 (s, 2H), 2.58 (t, J = 8.4 Hz, 2H), 2.66-2.49 (m, 10H), 1.88-1.80 (m, 2H),
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1.05-0.97 (m, 3H). C NMR (100 MHz, CDCl3) δ 175.21, 156.83, 155.19, 143.21,
131.35, 131.35, 129.48, 129.03, 128.63, 127.53, 127.12, 126.57, 118.72, 113.56,
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110.56, 108.21, 71.34, 57.23, 56.82, 55.78, 53.15, 53.15, 52.49, 52.49, 50.71, 29.51,
28.32, 17.55. ESI-MS m/z 474.5 [M+H] +.
5.1.9.25. 2-(4-(3-(5-cyano-1H-indol-3-yl)propyl)piperazin-1-yl)-N-phenyl-N-
(tetrahydro-2H-pyran-4-yl)acetamide (9_25). Compound 9_25 was prepared from 3-
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3d
and
N-phenyl-2-(piperazin-1-yl)-N-
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( tetrahydro-2H-pyran-4-yl)acetamide trifluoroacetate 8l as a white solid. Yield 77.5%;
1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 8.05 (s, 1H), 7.52–7.36 (m, 5H), 7.31
(s, 1H), 7.20 (d, J = 6.8 Hz, 2H), 4.63 (t, J = 11.2 Hz, 1H), 3.80 (d, J = 7.6 Hz, 2H),
3.40-3.32 (m, 2H), 2.66 (d, J = 9.8 Hz, 4H), 2.28 (s, 10H), 1.78–1.56 (m, 4H), 1.24-
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1.18 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 171.25, 141.30, 139.34, 134.34,
129.76, 128.39, 126.30, 126.10, 118.82, 113.43, 112.77, 101.83, 67.12, 59.71, 56.23,
53.50, 52.93, 48.48, 30.48, 26.93, 24.93. ESI-MS m/z 486.5 [M+H] +.
5.1.9.26. N-cyclohexyl-1-(3-(5-fluoro-1H-indol-3-yl)propyl)-N-phenylpiperidine-
4-carboxamide (FW01). Compound FW01was prepared from 3-(3-bromopropyl)-5-
fluoro-1H-indole 3a and N-cyclohexyl-N-phenyl-2-(piperazin-1-yl)acetamide 8q as a
foamed solid. Yield 35.7%; 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.40 (d, J = 5.1
Hz, 3H), 7.24 (dd, J = 13.6, 8.6 Hz, 2H), 7.11 – 7.09 (m, 2H), 7.02 (s, 1H), 6.92 (t, J =
9.0 Hz, 1H), 4.59 (t, J = 12.0 Hz, 1H), 2.75 (s, 2H), 2.70 (t, J = 7.5 Hz, 2H), 2.44 (m, J
= 22.1, 14.7 Hz, 10H), 1.91 – 1.81 (m, 6H), 1.72 (d, J = 13.6 Hz, 3H), 1.57 (d, J = 12.6
Hz, 1H), 1.39 (dd, J = 26.0, 12.9 Hz, 2H), 0.98 (m, 4H). ESI-MS m/z 477.2 [M+H]+
5.2. Biological evaluation
5.2.1. In vitro binding assays
The in vitro binding affinity were measured by radioligand binding assays using
HEK-293 cells expressing human D1R, D2R, D3R, 5-HT1AR and 5-HT2AR. The initial
screening was conducted with 10 μM compound to test its inhibition on the binding of
a tritiated radioligand to each receptor. A compound that inhibited binding by more
than 85% was further assayed for IC50. The Ki value was calculated based on this
relation: Ki = IC50 / (1 + C/Kd). [3H]-SCH23390, [3H]-Spiperone, [3H]-8-OH-DPAT and
[3H]-Ketanserin were used as standard radioligands for D1, D2, D3, 5-HT1A, and 5-HT2A
receptors, respectively. The binding reaction was conducted in duplicated tubes in 200
μL binding buffer containing 50 mM Tris, 4 mM MgCl2 (pH 7.4) at 30 °C for 50 min
with increasing concentrations (within the range of 1 nM ~ 100 μM) of the testing
compound in the presence of 0.7 nM [3H]SCH23390, [3H]Spiperone, [3H]-8-OH-DPAT
or [3H]-Ketanserin. The reaction was started by addition of membranes (15 μg/tube),
stopped by rapid filtration through a Whatman GF/B glass fiber filter and subsequently
washed with cold buffer. Nonspecific binding was determined by parallel incubations
with 10 μM SCH23390 for D1R, 10 μM Spiperone for D2R/D3R, 10 μM 5-HT for 5-
HT1AR, 10 μM butaclmol for 5-HT2AR. Scintillation cocktail was added, and the
radioactivity was determined in a MicroBeta liquid scintillation counter. The IC50 and
Ki values were calculated by nonlinear regression using a sigmoidal function.
5.2.2. [35S] GTPγS Assay
The [35S] GTPγS assay was performed to evaluate the function of synthesized
compounds to 5-HT1AR. The [35S] GTPγS reaction was conducted at 30 °C for 30 min
with 30 μg of membrane protein in a final volume of 200 μl with various concentration
of each of the tested compound. The natural substrate of 5-HT1AR, serotonin, was used
as a positive control. The binding buffer contains 50 mM Tris (pH 7.4), 5 mM MgCl2,
1 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, 1 mM DL-dithiothreitol
(DTT), and 40 μM guanosine triphosphate (GDP). The [35S] GTPγS reaction was
initiated by adding of [35S] GTPγS (final concentration of 0.1 nM). Non-specific
binding was measured in the presence of 100 μM 5 ′ -guanylimidodiphosphate
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(Gpp(NH)p). The [35S] GTPγS reaction was terminated by the addition of 1 ml of ice-
cold washing buffer, and was rapidly filtered with GF/C glass fiber filters (Whatman),
and then washed for three times. Radioactivity was determined by liquid scintillation
counting.
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5.3. Molecular Modeling
5.3.1. Homology Modeling
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The amino acid sequence of 5-HT1AR was downloaded from the UniProtKB
database (Entry code: P08908), and sequence similarity search was performed using
NCBI BLAST server27. The lately disclosed structures (PDB code: 4IAR and 5V54)24
of 5-HT1B active-state and inactive-state receptors were selected as the templates to
construct the agonistic and antagonist conformations of 5-HT1AR respectively.
Sequence alignment of 5-HT1AR and 5-HT1BR was carried out using Discovery Studio
3.5 (hereafter abbreviated to DS). Homology modeling was performed with DS. Ten
models were generated after loop refinement and the one with the lowest Discrete
Optimized Protein Energy (DOPE) score was submitted to energy minimization (100
steps steepest descent with backbone constrained). The PROCHECK program27 was
used to evaluate the stereochemical quality of 5-HT1AR.
5.3.2. Molecular Docking
Molecular docking was carried out using GOLD 5.123. Residues were numbered
according to the generalized numbering scheme proposed by Ballesteros and Weinstein.
The binding site was defined to include all residues within a 15.0 Å radius of the
conserved D3.32 Cγ carbon atom. A hydrogen-bond constraint was set between the
protonated nitrogen atom (N1) of ligand and D3.32 side chain. Ten conformations were
produced for each ligand, and Gold-Score was used as scoring function. Other
parameters were set as standard defaults. High-scoring complexes were inspected
visually to select the most reasonable solution.
5.3.3. Molecular dynamics simulations
The molecular dynamics simulations (hereafter abbreviated to MD) were
performed using the GROMACS 5.1.2. package. Two simulation systems of 9_24
bound to active state 5-HT1AR and inactive state 5-HT1AR without ligand(apo) were
embedded in hydrated POPC membrane via CHARMM-GUI29,30 server. A neutralized
system with an ionic concentration of 0.15 mol/L was reached by replacing water
molecules with proper number of Na+ and Cl﹣. Then both systems were minimized and
gradually equilibrated in an NPT ensemble at 310 K and 1 bar. Periodic boundary
conditions were applied to these simulation systems. Bonds containing hydrogen atoms
were restrained with the LINCS algorithm to allow an integration time step of 2 fs.
Electrostatic interactions were calculated using the particle mesh Ewald (PME)
summation algorithm. Finally, the production MD simulations of two systems were
performed for 100 ns and both systems were performed 3 times. All analyses of MD
trajectories were performed using the tools implemented in the GROMACS 5.1.2
package.
Author Contributions
#Wenli Wang and Lan Zheng contributed equally to this work.
Author Information
Corresponding Authors
*E-mail: wfu@fudan.edu.cn. Phone: +86-21-51980096
*E-mail: Hbshcn@163.com.
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Conflict of interests
The authors declared no conflict of interests.
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Acknowledgments
This work was supported by the grants from National Natural Science Foundation of
China (NO. 81773635) and Shanghai Science and Technology Development Funds
(NO. 14431900500).
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