Design and synthesis of novel 3-substituted-indole derivatives as selective H3 receptor antagonists and potent free radical scavengers

Article abstract of DOI:10.1016/j.bmc.2013.07.051

A series of novel 3-substituted-indole derivatives with a benzyl tertiary amino moiety were designed, synthesized and evaluated as H₃ receptor antagonists and free radical scavengers for Alzheimer's disease therapy. Most of these synthesized co

Full text of DOI:10.1016/j.bmc.2013.07.051

Bioorganic & Medicinal Chemistry 21 (2013) 5936–5944
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel 3-substituted-indole derivatives as
selective H3 receptor antagonists and potent free radical scavengers
a
b,
Li Tang a, Liying Zhao a, Lingjuan Hong b, Fenyan Yang a, Rong Sheng a, , Jianzhong Chen , Ying Shi
⇑
⇑
,
Naimin Zhou b, Yongzhou Hu a
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, School of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b Institute of Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 3-substituted-indole derivatives with a benzyl tertiary amino moiety were designed,
synthesized and evaluated as H3 receptor antagonists and free radical scavengers for Alzheimer’s disease
therapy. Most of these synthesized compounds exhibited moderate to potent antagonistic activities in
CREs driven luciferase assay. In particular, compound 2d demonstrated the most favorable H3 receptor
Received 17 May 2013
Revised 29 July 2013
Accepted 30 July 2013
Available online 8 August 2013
antagonistic activity with the IC50 value of 0.049 lM. Besides, it also displayed high binding affinity to
H3 receptor (Ki = 4.26 2.55 nM) and high selectivity over other three histamine receptors. Moreover,
2d and other two 3-substituted indole derivatives 1d and 3d exerted potent ABTS radical cation scaveng-
ing capacities similar to melatonin. Above results illustrate that 2d is an interesting lead for extensive
optimization to explore new drug candidate for AD therapy.
Keywords:
H3 receptor antagonists
Free radical scavengers
Melatonin
Ó 2013 Elsevier Ltd. All rights reserved.
Benzyl tertiary amine
1. Introduction
Fig. 1) and GSK-189254 (phase II, Fig. 1) act as wake-promoting
agents for narcoleptic patients. Clinical trials with JNJ-31001074
As one of four members of the G-protein coupled receptor
(GPCR) binding to histamine, H3 receptor was pharmacologically
identified by Arrang in 1983 and successfully cloned in 1999.1,2 It
is predominantly expressed in the central nervous system (CNS)
and functions as an autoreceptor that negatively regulates the re-
lease of histamine. More importantly, as a heteroreceptor, H3
receptor participates in modulating the release of key neurotrans-
mitters including acetylcholine, serotonin, noradrenaline, and
dopamine that are involved in attention, vigilance and cognition.3,4
Therefore, H3 receptor has been recognized as an effective thera-
peutic target for the treatment of CNS disorders, exemplified by
narcolepsy, Alzheimer’s disease (AD), attention deficit hyperactiv-
ity (ADHD), obesity, and schizophrenia.5
There are numerous of new H3 receptor antagonists with differ-
ent chemical scaffolds have been developed in the past few years,
most of these newly developed compounds are characterized by a
tertiary amine, a central core and a flexible part chosen from either
a polar group, a second basic amine or a lipophilic residue.6–13 Re-
cently, several of them being investigated in clinical trials for the
potential treatment of above mentioned CNS disorders, for exam-
ple, Pitolisant (BF2.649, phase III, Fig. 1), PF-03654746 (phase II,
(phase I, Fig. 1) are being carried out in adult and pediatric ADHD
groups. For Alzheimer’s disease (AD), MK-0249 (phase II, Fig. 1),
PF-03654746 (phase I, Fig. 1) and Irdabisant (CEP-26401, phase I,
Fig. 1) are used to alleviate the symptoms and slow the progression
of mild to moderate AD patients.14,15
Although the etiology of AD is not yet fully understood, there
are numerous studies prove that the imbalance between the gen-
eration of free radicals and reactive oxygen species (ROS) may be
involved in the pathogenesis of AD. Many free radical scavengers
have produced promising results for the treatment of AD in pre-
clinical stage and clinical trials. For example, melatonin protects
neuronal cells from Ab-induced oxidative damage in vitro and in-
creases survival in a transgenic model of AD.16 Moreover, in a con-
trolled clinical trial, vitamin E (a-tocopherol) delay clinically
important functional deterioration in patients with Alzheimer’s
disease.17
Based on above research results, we hypothesized that com-
pounds with dual activities of H3 receptor antagonism and free
radical scavenging may achieve better efficacy by a complemen-
tary manner.18 Our strategy to design such molecules was to com-
bine pharmacophores of H3 receptor antagonist and free radical
scavenger into one molecule.
The benzyl tertiary amine moiety was chosen as the pharmco-
phore of H3 receptor antagonists because it was frequently em-
ployed into H3 receptor antagonists, such as PF-03654746 and
JNJ-31001074. Furthermore, the molecular docking analysis of
⇑
Corresponding authors. Tel./fax: +86 571 8820 8458 (R.S.); tel./fax: +86 571
8820 6134 8000 (Y.S.).
E-mail addresses: shengr@zju.edu.cn (R. Sheng), shiying1991@yahoo.com (Y.
Shi).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.051

L. Tang et al. / Bioorg. Med. Chem. 21 (2013) 5936–5944
5937
Figure 1. Structures of non-imidazole H3 receptor antagonists in clinical trials.
PF-03654746 bound to H3 receptor suggested that the protonated
nitrogen of benzyl piperidine interact with crucial residue Asp114
in transmembrane helix 3 (TM3), which is highly conserved in G-
protein-coupled receptors (GPCRs).19,20
acid in the presence of HOBt/EDC resulted in amides 1a–i. Further
reduction of amide group of compounds 1a–1e and 1h with LiAlH4
afforded compounds 2a–f. Finally, condensation of phenol deriva-
tives 7a–d with indole-3-carboxylic acid, indole-3-acetic acid or
indole-3-propionic acid in the presence of DCC/DMAP provided
compounds 3a–i.
Melatonin was a potent free radical scavenger and a broad spec-
trum antioxidant, in which the indole moiety was validated as the
reactive center of interaction with cations and radicals due to its
high resonance stability and very low activation energy barrier to-
wards the free radical reactions.21 Moreover, this moiety was
widely present in various CNS-active drugs, such as anti-migraine
drug Rizatriptan, psychotherapeutic drug Oxypertine (Fig. 2).
Therefore, a new series of 3-substituted-indole derivatives are
designed, synthesized and evaluated as H3 receptor antagonists
and free radical scavengers through tethering of benzyl tertiary
amine to the 3-position of indole. In these designed target com-
pounds, the indole moiety not only serves as a free radical scaven-
ger pharmacophore, but also acts as the flexible part in H3 receptor
antagonists, and the benzyl tertiary amine is suppose to form crit-
ical hydrogen bond with the amino acid residue in H3 receptor.
Meanwhile, different kinds of linkers (alkyl amide, alkyl amino
and alkyl ester, Fig. 2) are employed to connect the two moieties
to explore the optimum one.
2.2. Biological activities and SAR
2.2.1. H3 receptor antagonistic activity and binding affinity
evaluation
cAMP-response elements (CREs) driven luciferase assay has
been widely utilized to measure the function of GPCR agonists
and antagonists. The H3 receptor antagonistic activities of these
newly synthesized 3-substituted indole derivatives have been
evaluated in HEK-293 cell lines expressing the human H3 receptor
and a reporter gene consisting of the firefly luciferase coding region
that is under the control of minimal promoter containing CREs.18,23
Thioperamide is employed as the positive control and results are
summarized in Table 1.
As shown in Table 1, most of the synthetic compounds demon-
strated moderate to potent H3 receptor antagonistic activities and
seven compounds displayed submicromolar IC50 values. Com-
pounds 2b (IC50 = 0.080
the most potent H3 receptor antagonistic activities, which were
more potent than that of Thioperamide (IC50 = 0.12
ven luciferase assay.
lM) and 2d (IC50 = 0.049 lM) exhibited
2. Results and discussion
2.1. Chemistry
lM) in CRE-dri-
The variation of linker between indole and benzene ring of ben-
zylamine moieties significantly influenced the H3 receptor antago-
nistic activities. Indole-3-alkylamino derivatives (2a–f) were more
potent than corresponding indole-3-amide derivatives (1a–e, 1h)
and indole-3-carboxylate derivatives (3a–e, 3h). For example,
The synthetic route for these new 3-substituted-indole deriva-
tives is outlined in Scheme 1. Reaction of 4-nitro-benzylbromide
4 with different secondary amines in refluxing acetonitrile gave
corresponding 4-nitro-benzyl amines 5a–d, which was followed
by catalytic hydrogenation with 5% Pd/C to furnish 4-amino-benzyl
tertiary amines 6a–d.22 Subsequent condensation of 6a–d with in-
dole-3-carboxylic acid, indole-3-acetic acid or indole-3-propionic
compound 2b (IC50 = 0.080
lM) displayed more potent H3 receptor
antagonistic activity than that of compounds 1b (IC50 = 2.84
lM)
Figure 2. CNS drugs containing indole moiety and our target 3-substituted-indole derivatives.

5938
L. Tang et al. / Bioorg. Med. Chem. 21 (2013) 5936–5944
Scheme 1. Reagents and conditions: (a) secondary amine, acetonitrile, reflux 2–4 h; (b) 5% Pd/C, methanol, rt 6–10 h; (c) indole-3-carboxylic acid or indole-3-acetic acid or
indole-3-propionic acid, HOBt/EDC, CH2Cl2/THF, rt overnight; (d) LiAlH4, THF, reflux 4–6 h; (e) indole-3-carboxylic acid or indole-3-acetic acid or indole-3-propionic acid,
DCC/DMAP, CH2Cl2/THF, rt over night.
Table 1
The H3 receptor antagonist activity and H3 receptor binding affinity of 3-substituted indole derivatives
linker
R1
N
N
H
R2
Compounds
Linker
n
NR1R2
H3R antagonistic activitya (IC50
, lM)
Thioperamide
0.12
1.21
2.84
1.37
2.19
3.50
>10
1a
1b
1c
1d
1e
1f
–(CH2)nCONH–
0
1
Piperidine
Piperidine
Pyrrolidine
Morpholine
Diethylamine
Piperidine
2
1g
1h
1i
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
Pyrrolidine
Morpholine
Diethylamine
Piperidine
>10
>10
>10
–(CH2)n+1NH–
–(CH2)nCOO–
0
1
0.54
0.080
0.18
0.049b
0.29
0.49
2.03
1.04
2.46
0.79
4.61
>10
Piperidine
Pyrrolidine
Morpholine
Diethylamine
Morpholine
Piperidine
2
0
1
Piperidine
Pyrrolidine
Morpholine
Diethylamine
Piperidine
2
3g
3h
3i
Pyrrolidine
Morpholine
Diethylamine
>10
>10
>10
a
Results are presented as the mean of at least three independent experiments.
H3 receptor binding affinity, Ki = 4.26 2.55 nM.
b
and 3b (IC50 = 1.04
erted H3 receptor antagonistic activities similar to indole-3-car-
boxylate derivatives (3a–e), with IC50 values ranging from 1.21 to
l
M). The indole-3-amide derivatives (1a–e) ex-
man H3 receptor in the presence of the compounds.23 As a result,
compound 2d showed high binding affinity to H3 receptor with
Ki value of 4.26 1.55 nM.
3.50
l
M and 0.79 to 4.61
lM, respectively. The compromised
activities of 1f–i and 3f–i (IC50 >10
lM) suggested that the intro-
2.2.2. Homology model and molecular docking
duction of longer linker with rigidity (propionamide, propionate)
was detrimental for H3 receptor antagonistic activities.
The most potent 3-indole ethylamino derivative 2d was se-
lected to perform H3 receptor binding affinity evaluation which
was measured in terms of displacement of [3H]-(R)-a-methyl-his-
tamine binding to membranes of HEK-293 cells expressing the hu-
To gain insight into the molecular determinants that modulate
the antagonistic activities of these compounds, molecular docking
study of PF-03654746, 2d, 1d and 1h with H3 receptor homology
model were performed, which was generated based on the crystal
structure of H1 receptor (PDB ID: 3RZE).24 The results are reported
in Figure 3.

L. Tang et al. / Bioorg. Med. Chem. 21 (2013) 5936–5944
5939
As expected, in Figure 3A, PF-3654746 binds to H3 receptor
through three key hydrogen-bond interactions with Asp114,
Glu206 and Tyr374, which are fully consistent with the reported
results.20 Figure 3B reveals that compound 2d has a favorable fit
into hydrophobic cavity in TMs 3-5-6 region of H3 receptor. The
benzyl morpholine and indole moiety of 2d endows two common
hydrogen bonds with crucial residues Asp114 and Glu206, respec-
tively. In addition, the nitrogen of ethyl amino linker forms the
third hydrogen bond with Tyr374. Although there is only minor
structural difference between compounds 1d (–CH2CONH– as lin-
ker) and 2d (–CH2CH2NH– as linker), Figure 3C reveals that the
direction of indole moiety in 1d is slightly different from 2d and
thereby result in the lost of hydrogen bond between indole and
Glu206, which may account for the 40-fold decrease of potency
for compound 1d. Figure 3D shows that compound 1h inserted
deeper than 2d into the hydrophobic cavity of TMs 3-5-6 region
in H3 receptor, which may be responsible for the loss of three
hydrogen bonds with crucial residues of H3 receptor.
Addition of antioxidants to the solution of pre-formed radical cat-
ion will reduce it, and the scavenging activities of the antioxidants
is determined by the degree of blanching of ABTSÅ+.25 Three 3-
substituted indole derivatives with different linkers (1d, 2d and
3d) were selected to evaluate their free radical scavenging capaci-
ties with Melatonin as positive control. The results are shown in
Figure 5.
The data of Figure 5 indicated that melatonin, 1d, 2d and 3d
scavenge ABTSÅ+ in a concentration dependent style, 1d, 2d and
3d exhibited potent ABTS radical cation scavenging capacities with
IC50 value of 4.71
lM, 3.05
l
M and 5.32
lM, respectively, similar
to that of Melatonin (IC50 = 1.92
lM), which confirmed our hypoth-
esis that compounds containing indole moiety was able to confer
potent ABTS radical cation scavenging capacities.
2.2.5. hERG patch-clamp assay
Many of reported H3 antagonists have shown hERG inhibitory
activities which may result in a potential safety liability. Three
most potent compounds (2b, 2c, 2d) and indole-3-amide derivative
1d, indole-3-ester derivative 3d were chosen to evaluate hERG ion
channel inhibitory activities. The results are shown in Table 2.
The results demonstrated that compound 1d and 3d almost did
2.2.3. Histamine receptor selectivity
Provided the distinct biological function of the four subtypes of
histamine GPCRs, the selectivity is an important issue in designing
H3 receptor antagonist. Indole-3-ethylamino derivative 2d was se-
lected to evaluate for its biological activities against other three
subtypes of histamine receptors (H1R, H2R and H4R) using the same
CRE-driven luciferase assay as H3 receptor. As shown in Figure 4,
not exhibit any hERG inhibition at 3 lM as measured by patch
clamp assay, while all three 3-indole ethylamino derivatives 2b,
2c and 2d showed moderate hERG inhibition at the same condi-
tion, these results implies that more work need to be done to find
a promising new drug candidate with satisfactory hERG profile.
compound 2d did not influence the responses induced by 5 lM
histamine in the luciferase activity in cells expressing H1R, H2R
or H4R. These results demonstrated that compound 2d is an H3
receptor antagonist with preferable selectivity versus H1, H2 and
H4 receptors.
3. Conclusion
With the goal of identifying dual-acting compounds with potent
H3 receptor antagonistic activities and free radical scavenging
properties, a series of novel 3-substituted-indole derivatives were
designed through incorporating benzyl tertiary amine into 3-posi-
tion of indole skeleton with different linkers. Most of these synthe-
sized compounds exhibited moderate to potent H3 receptor
2.2.4. ABTS radical cation scavenging capacity assay
ABTS [2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)]
radical cation (ABTSÅ+) was a blue-colored cation with absorption
maxima at wavelengths 415 nm, 645 nm, 734 nm and 815 nm.
Figure 3. The predicted best configuration of PF-03654746, 2d, 1d and 1h bound to H3 receptor. PF-03654746 (A) and 2d (B) bound to H3 receptor with similar mode, while
1d (purple in C) and 1h (blue in D) exhibited different binding mode with H3 receptor in comparison with 2d (gold in C and D).

5940
L. Tang et al. / Bioorg. Med. Chem. 21 (2013) 5936–5944
Figure 4. The selectivity of compound 2d for human histamine receptors by CRE-luciferase transcription assay. HEK-293 cells stably expressing human H1 receptor (A),
human H2 receptor (B), or human H4 receptor (C) were treated with different concentrations of compound 2d in the presence of 5 M histamine, single histamine response
l
curve used as the control separately. The presented data points are the mean SE of triplicate values from a single experiment and are representative of three separate
experiments.
with TMS as the internal standard. Proton Chemical shifts are ex-
pressed in parts per million (ppm) and coupling constants in Hz.
Mass spectra (ESI-MS) were performed on a Finnigan LCQ DecaXP
ion trap mass spectrometry.
4.1.1. General procedure for preparation of compounds 6a–d
To a solution of 1-(bromomethyl)-4-nitrobenzene 4 (1.0 g, 4.6
mmol) in 15 mL acetonitrile, was added a solution of piperidine
in 5 mL acetonitrile at room temperature. The mixture was re-
fluxed for 2 h and the solvent was removed under vacuum to
nearly dryness. The residue was acidified with 20 mL 2 mol/L
hydrochloric acid and extracted with ethyl acetate (10 mL ꢀ 3).
Concentrated ammonium hydroxide was added to aqueous solu-
tion up to clearly basic, and the product was extracted with ethyl
acetate (20 mL ꢀ 3). The combined organic layer was washed with
brine, dried over anhydrous Na2SO4, and removed solvent under
Figure 5. Concentration dependence in scavenging of ABTSÅ+ by 1d, 2d, 3d and
Melatonin with the results being obtained at the end of a 6-min incubation.
reduced pressure to give 5a which was used in the following reac-
tion without further purification. A mixture of 5a (1.0 mmol) in
methanol (20 mL) and 5% palladium on carbon (20 mg) was stirred
under hydrogen (balloon) for 6–10 h. The mixture was then filtered
Table 2
through celite and then concentrated to obtain crude product,
which was purified by silica gel column chromatography eluting
with petroleum, ethyl acetate and triethylamine (20:20:1) to afford
6a.
The hERG inhibitory activities of 3-substituted indole derivatives
Compounds
1d
2b
2c
2d
3d
Cisepride (100 nM)
89
hERG (%Inh.,3
l
M)
6
87
73
54
12
4.1.1.1. 4-(Piperidin-1-ylmethyl)-aniline (6a).
Yellow solid
(yield: 59.0%); mp: 115–117 °C (lit. 120–123 °C).26
antagonistic activities. The preliminary SAR reveals that alkyl-
amino linker is favorable for H3 receptor antagonistic activity.
The most potent H3 antagonist 2d (IC50 = 0.049 lM) also shows
4.1.1.2. 4-(Pyrrolidin-1-ylmethyl)-aniline (6b).
Yellow oil
(yield: 63.0%); 1H NMR (500 MHz, CDCl3): d 7.11 (d, J = 7.5 Hz,
2H), 6.64 (d, J = 7.5 Hz, 2H), 3.62 (s, 2H), 3.51 (s, 2H), 2.47–2.48
(m, 4H), 1.78 (m, 4H).
high binding affinity to H3 receptor with Ki value of
4.26 2.55 nM, and good selectivity over other three histamine
receptors. Molecular docking analysis demonstrates that 2d bind
to H3 receptor via three key hydrogen-bond interactions with
Asp114, Glu206, and Tyr374, respectively. Moreover, three 3-
substituted indole derivatives with different linkers (1d, 2d and
3d) all exerted potent ABTS radical cation scavenging activities
similar to melatonin. All these results suggested that 2d can be ex-
plored as an interesting dual-functional lead for the development
of AD therapeutic drugs.
4.1.1.3. 4-(Morpholinomethyl)-aniline (6c).
Yellow solid
Yellow solid
(yield 56.0%); mp: 99–100 °C (lit. 100.8–102.2 °C).22
4.1.1.4. 4-(Diethylaminomethyl)-aniline (6d).
(yield 53.0%); mp: 40–42 °C (lit. 46–47 °C).27
4.1.2. General procedure for preparation of compounds 1a–1i
To a mixture of 4-amino benzyl amine 6a (0.42 mmol) and in-
dole-3-carboxylic acid (0.50 mmol) in 8 mL CH2Cl2 and 2 mL
THF, was added HOBt (1.0 mmol), EDC (1.0 mmol) and triethyl
amine (1.0 mmol). The mixture was stirred over night at room
temperature and stopped by addition of 10 mL 5% NaHCO3 solu-
tion, then, the mixture was extracted with CH2Cl2 (15 ꢀ 3 mL)
and organic layers were combined, washed with brine, dried over
Na2SO4. The solvent was evaporated under vacuum to give crude
4. Experimental section
4.1. Synthesis
General, all reagents and solvents used were purchased from
common commercial of analytical grade. Melting points were re-
corded on a B-540 Buchi melting-point apparatus and uncorrected,
1H NMR were recorded on a BRUKER AVIII 500 M spectrometer

L. Tang et al. / Bioorg. Med. Chem. 21 (2013) 5936–5944
5941
product, which was purified by silica gel column chromatography
eluting with petroleum, ethyl acetate and triethylamine (20:20:1)
to afford 1a.
piperidine-CH2, H-3, H-5), 1.43–1.41 (m, 2H, piperidine-CH2, H-
4); MS (ESI) m/z = 362.3 [M+H]+.
4.1.2.7. 3-(1H-Indol-3-yl)-N-(4-(pyrrolidin-1-ylmethyl)phenyl)
4.1.2.1.
boxamide (1a).
IR (KBr) 3434, 3369, 3109, 2947, 1632, 1598, 1531, 1411, 748
cmꢁ1 1H NMR (500 MHz, CDCl3): d 9.54 (br s, 1H, H-1), 8.09–
8.07 (m, 1H, H-4), 7.89 (s, 1H, H-2), 7.78 (s, 1H, H-7), 7.61 (d,
J = 8.5 Hz, 2H, H-30 and H-50), 7.45–7.43 (m, 1H, H-5), 7.30–7.26
(m, 3H, H-20, H-60, H-6), 3.49 (s, 2H, benzylic-CH2), 2.43–2.41 (m,
4H, piperidine-CH2, H-2, H-6), 1.62–1.57 (m, 4H, piperidine-CH2,
H-3, H-5), 1.44–1.42 (m, 2H, piperidine-CH2, H-4); MS (ESI) m/
z = 334.4 M+H]+.
N-(4-(Piperidin-1-ylmethyl)phenyl)-1H-indole-3-car-
propanamide (1g).
135–137 °C; IR (KBr) 3434, 3284, 3054, 2959, 1656, 1600, 1544,
1411, 734 cmꢁ1 1H NMR (500 MHz, CDCl3): d 8.24 (br s, 1H, H-
1), 7.65 (d, J = 8.0 Hz, 1H, H-4), 7.39 (d, J = 8.0 Hz, 1H, H-7), 7.34
(d, 2H, H-30, H-50), 7.25–7.20 (m, 4H, CONH, H-20, H-60, H-5),
7.16–7.13 (m, 1H, H-6), 7.00 (s, 1H, H-2), 3.62 (s, 2H, benzylic-
CH2), 3.23 (t, J = 7.5 Hz, 2H, CH2), 2.78 (t, J = 7.5 Hz, 2H, CH2),
2.57–2.54 (m, 4H, pyrrolidine-CH2, H-2, H-5), 1.81–1.79 (m, 4H,
pyrrolidine-CH2, H-3, H-4); MS (ESI) m/z = 348.4 [M+H]+.
Slightly yellow solid (yield: 36.5%); mp:
Yellow solid (yield 19.8%); mp: 108–110 °C;
;
;
4.1.2.8. 3-(1H-Indol-3-yl)-N-(4-(morpholinomethyl)phenyl)pro-
4.1.2.2.
acetamide (1b).
IR (KBr) 3298, 3261, 3067, 2937, 1661, 1607, 1536, 1413, 741
cmꢁ1 1H NMR (500 MHz,CDCl3): d 8.43 (br s, 1H, H-1), 7.65 (d,
J = 8.0 Hz, 1H, H-4), 7.46 (d, J = 8.0 Hz, 1H, H-7), 7.40 (s, 1H, H-2),
7.29–7.26 (m, 2H, H-30, H-50), 7.24–7.18 (m, 4H, H-20, H-60, H-5,
H-6), 3.91 (s, 2H, CH2CO), 3.42 (s, 2H, benzylic-CH2), 2.35–2.30
(m, 4H, piperidine-CH2, H-2, H-6), 1.58–1.53 (m, 4H, piperidine-
CH2, H-3, H-5), 1.42–1.40 (m, 2H, piperidine-CH2, H-4); MS (ESI)
m/z = 348.3 M+H]+.
2-(1H-Indol-3-yl)-N-(4-(piperidin-1-ylmethyl)phenyl)
panamide (1h).
IR (KBr) 3435, 3288, 3054, 2958, 1656, 1600, 1543, 1411, 1114,
740 cmꢁ1 1H NMR (500 MHz, CDCl3): d 8.05 (br s, 1H, H-1), 7.64
(d, J = 7.5 Hz, 1H, H-4), 7.38 (d, J = 8.0 Hz, 1H, H-7), 7.32 (d,
J = 8.5 Hz, 2H, H-30, H-50), 7.23–7.20 (m, 3H, CONH, H-2, H-5),
7.15 (m, 1H, H-6), 7.07–7.04 (m, 2H, H-20, H-60), 3.73–3.69 (m,
4H, morpholine-CH2, H-2, H-6), 3.46 (s, 2H, benzylic-CH2), 3.22
(t, J = 7.0 Hz, 2H, CH2), 2.76 (t, J = 7.0 Hz, 2H, CH2), 2.42–2.40 (m,
4H, morpholine-CH2, H-3, H-5); MS (ESI) m/z = 364.4 [M+H]+.
White solid (yield 41.2%); mp: 114–116 °C;
White solid (yield: 24.4%). Mp: 185–187 °C;
;
;
4.1.2.9. N-(4-((Diethylamino)methyl)phenyl)-3-(1H-indol-3-yl)
4.1.2.3. 2-(1H-Indol-3-yl)-N-(4-(pyrrolidin-1-ylmethyl)phenyl)
propanamide (1i).
73–75 °C; IR (KBr) 3454, 3300, 3054, 2970, 1656, 1600, 1540,
1410, 742 cmꢁ1 1H NMR (500 MHz, CDCl3): d 8.50 (br s, 1H, H-
1), 8.33 (br s, 1H, CONH), 7.60 (d, J = 7.5 Hz, 1H, H-4), 7.32–7.30
(m, 3H, H-7, H-30, H-50), 7.22–7.17 (m, 3H, H-2, H-20, H-60), 7.12–
7.10 (m, 1H, H-5), 6.90 (m, 1H, H-6), 3.53 (s, 2H, benzylic-CH2),
3.17 (t, J = 7.0 Hz, 2H, CH2), 2.71 (t, J = 7.0 Hz, 2H, CH2), 2.53 (q,
J = 7.0 Hz, 4H, CH2 ꢀ 2), 1.08 (t, J = 7.5 Hz, 6H, CH3 ꢀ 2); MS (ESI)
m/z = 350.4 [M+H]+.
Slightly yellow solid (yield 39.5%); mp:
acetamide (1c).
IR (KBr) 3292, 3255, 3052, 2968, 1655, 1605, 1536, 1412, 738
cmꢁ1 1H NMR (500 MHz, CDCl3): d 8.40 (br s, 1H, H-1), 7.65 (d,
White solid (yield 28.1%); mp: 177–178 °C;
;
;
J = 8.0 Hz, 1H, H-4), 7.46 (d, J = 8.5 Hz, 1H, H-7), 7.40 (s, 1H, H-2),
7.30–7.27 (m, 2H, H-30, H-50), 7.24–7.18 (m, 4H, H-20, H-60, H-5,
H-6), 3.91 (s, 2H, CH2CO), 3.57 (s, 2H, benzylic-CH2), 2.49–2.45
(m, 4H, pyrrolidine-CH2, H-2, H-5), 1.78–1.76 (m, 4H, pyrroli-
dine-CH2, H-3, H-4); MS (ESI) m/z = 334.3 [M+H]+.
4.1.3. General procedure for preparation of compounds 2a–f
To a solution of compound 1a (0.50 mmol) in 15 mL anhydrous
THF, LiAlH4 (1.0 mmol) was added in one portion. The resulting
mixture was refluxed for 4 h and carefully quenched with H2O
(5.0 mL). The organic layer was separated and the aqueous solution
was extracted with EtOAc (20 mL ꢀ 3). The combined organic layer
was dried (Na2SO4) and evaporated to dryness to obtain crude
product, which was purified by silica gel column chromatography
eluting with petroleum, ethyl acetate and triethylamine (20:20:1)
to afford 2a.
4.1.2.4.
acetamide (1d).
IR (KBr) 3290, 3256, 3064, 2962, 1658, 1607, 1535, 1415, 1116,
743 cmꢁ1 1H NMR (500 MHz, CDCl3): d 8.34 (br s, 1H, H-1), 7.63
(d, J = 7.5 Hz, 1H, H-4), 7.45 (d, J = 8.5 Hz, 1H, H-7), 7.39 (s, 1H,
CONH), 7.30–7.26 (m, 3H, H-2, H-30, H-50), 7.23–7.16 (m, 4H, H-
20, H-60, H-5, H-6), 3.90 (s, 2H, CH2CO), 3.68 (m, 4H, morpholine-
CH2, H-2, H-6), 3.41 (s, 2H, benzylic-CH2), 2.40 (m, 4H, morpho-
line-CH2, H-3, H-5); MS (ESI) m/z = 350.3 [M+H]+.
2-(1H-Indol-3-yl)-N-(4-(morpholinomethyl)phenyl)
White solid (yield 23.2%); mp: 128–130 °C;
;
4.1.2.5. N-(4-((Diethylamino)methyl)phenyl)-2-(1H-indol-3-yl)
4.1.3.1. N-((1H-Indol-3-yl)methyl)-4-(piperidin-1-ylmethyl)ani-
acetamide (1e).
133–135 °C; IR (KBr) 3291, 3256, 3051, 2967, 1657, 1605, 1535,
1412, 740 cmꢁ1 1H NMR (500 MHz, CDCl3): d 8.30 (br s, 1H, H-
Slightly yellow solid (yield 23.9%); mp:
line (2a).
Yellow oil (yield 65.4%); IR (KBr) 3410, 3055, 2934,
1616, 1522, 1411, 742 cmꢁ1; 1H NMR (500 MHz, CDCl3): d 8.22 (br
s, 1H, H-1), 7.69 (d, J = 8.0 Hz, 1H, H-4), 7.40 (d, J = 8.0 Hz, 1H, H-7),
7.24 (m, 1H, H-5), 7.19–7.10 (m, 3H, H-2, H-30, H-50), 6.68 (d, J = 8.0
Hz, 2H, H-20, H-60), 4.48 (s, 2H, CH2NH), 3.46 (s, 2H, benzylic-CH2),
2.44–2.40 (m, 4H, piperidine-CH2, H-2, H-6), 1.64–1.60 (m, 4H,
piperidine-CH2, H-3, H-5), 1.44–1.42 (m, 2H, piperidine-CH2, H-
4); MS (ESI) m/z = 320.2 [M+H]+.
;
1), 7.64 (d, J = 8.0 Hz, 1H, H-4), 7.44 (d, J = 8.0 Hz, 1H, H-7), 7.36
(s, 1H, CONH), 7.28–7.25 (m, 3H, H-2, H-30, H-50), 7.23–7.10 (m,
4H, H-20, H-60, H-5, H-6), 3.90 (s, 2H, CH2CO), 3.49 (s, 2H, ben-
zylic-CH2), 2.52 (t, J = 7.5 Hz, 4H, CH2 ꢀ 2), 1.01 (t, J = 7.5 Hz, 6H,
CH3 ꢀ 2); MS (ESI) m/z = 336.3 [M+H]+.
4.1.2.6.
propanamide (1f).
154 °C; IR (KBr) 3437, 3295, 3053, 2933, 1657, 1600, 1542, 1410,
744 cmꢁ1 1H NMR (500 MHz, CDCl3): d 8.24 (br s, 1H, H-1), 7.65
(d, J = 8.0 Hz, 1H, H-4), 7.38 (d, J = 8.0 Hz, 1H, H-7), 7.31 (d,
J = 8.5 Hz, 2H, H-30, H-50), 7.22–7.20 (m, 3H, CONH, H-20, H-60),
7.15–7.13 (m, 2H, H-5, H-6), 7.00 (s, H-2), 3.43 (s, 2H, benzylic-
CH2), 3.22 (t, J = 7.5 Hz, 2H, CH2), 2.76 (t, J = 7.5 Hz, 2H, CH2),
2.37–2.32 (m, 4H, piperidine-CH2, H-2, H-6), 1.58–1.55 (m, 4H,
3-(1H-Indol-3-yl)-N-(4-(piperidin-1-ylmethyl)phenyl)
4.1.3.2. N-(2-(1H-Indol-3-yl)ethyl)-4-(piperidin-1-ylmethyl)ani-
White solid (yield 43.5%); mp: 152–
line (2b).
Yellow solid (yield 69.3%); mp: 126–128 °C; IR
(KBr) 3404, 3204, 2961, 1617, 1526, 1413, 820, 744 cmꢁ1
;
1H
;
NMR (500 MHz, CDCl3): d 8.56 (br s, 1H, H-1), 8.47 (br s, 1H,
CONH), 7.69 (d, J = 8.0 Hz, 1H, H-4), 7.42 (d, J = 8.0 Hz, 1H, H-7),
7.28 (m, 1H, H-5), 7.20–7.15 (m, 3H, H-6, H-30, H-50), 7.07 (s, 1H,
H-2), 6.62 (d, J = 8.0 Hz, 2H, H-20, H-60), 3.79 (br s, 1H, NH) 3.54
(t, J = 6.5 Hz, 2H, NCH2), 3.45 (s, 2H, benzylic-CH2), 3.16 (t, J = 6.5
Hz, 2H, CH2), 2.45–2.42 (m, 4H, piperidine-CH2, H-2, H-6), 1.65–

5942
L. Tang et al. / Bioorg. Med. Chem. 21 (2013) 5936–5944
1.61 (m, 4H, piperidine-CH2, H-3, H-5), 1.49–1.47 (m, 2H, piperi-
1.49 (m, 4H, piperidine-CH2, H-3, H-5), 1.35–1.32 (m, 2H, piperi-
dine-CH2, H-4); MS (ESI) m/z = 334.5 [M+H]+.
dine-CH2, H-4); MS (ESI) m/z = 335.3 [M+H]+.
4.1.3.3.
aniline (2c).
(KBr) 3394, 3136, 2966, 1614, 1528, 1453, 820, 742 cmꢁ1
N-(2-(1H-Indol-3-yl)ethyl)-4-(pyrrolidin-1-ylmethyl)
Yellow solid (yield 63.2%); mp: 78–80 °C; IR
1H
4.1.4.2. 4-(Piperidin-1-ylmethyl)phenyl-2-(1H-indol-3-yl)ace-
tate (3b).
Yellow oil (yield 28.1%); IR (KBr) 3407, 3016,
;
2932, 2852, 1751, 1625, 1592, 1505, 1197, 1133, 741 cmꢁ1
;
1H
NMR (500 MHz, CDCl3): d 8.13 (br s, 1H, H-1), 7.64 (d, J = 10.0 Hz,
1H, H-4), 7.40 (d, J = 10.0 Hz, 1H, H-7), 7.23 (m, 1H, H-5), 7.15–
7.11 (m, 3H, H-30, H-50, H-6), 7.06 (d, J = 2.5 Hz, 1H, H-2), 6.58 (d,
J = 10.5 Hz, 2H, H-20, H-60), 3.56 (s, 2H, benzylic-CH2), 3.49 (t,
J = 8.5 Hz, 2H, NCH2), 3.11 (t, J = 8.5 Hz, 2H, CH2), 2.56–2.54 (m,
4H, pyrrolidine-CH2, H-2, H-5), 1.80–1.78 (m, 4H, pyrrolidine-
CH2, H-3, H-4); MS (ESI) m/z = 320.3 M+H]+.
NMR (500 MHz, CDCl3): d 8.43 (br s, 1H, H-1), 8.37 (br s, 1H,
CONH), 7.72 (d, J = 8.0 Hz, 1H, H-4), 7.36 (d, J = 8.0 Hz, 1H, H-7),
7.30 (d, J = 8.5 Hz, 2H, H-30, H-50), 7.24–7.21 (m, 2H, H-2, H-5),
7.18 (m, 1H, H-6), 7.02 (d, J = 8.5 Hz, 2H, H-20, H-60), 4.02 (s, 2H,
CH2CO), 3.46 (s, 2H, benzylic-CH2), 2.38–2.36 (m, 4H, piperidine-
CH2, H-2, H-6), 1.60–1.55 (m, 4H, piperidine-CH2, H-3, H-5),
1.44–1.42 (m, 2H, piperidine-CH2, H-4); MS (ESI) m/z = 349.3
[M+H]+.
4.1.3.4. N-(2-(1H-Indol-3-yl)ethyl)-4-(morpholinomethyl)ani-
line (2d).
Yellow oil (yield 59.8%); IR (KBr) 3407, 3106,
4.1.4.3. 4-(Pyrrolidin-1-ylmethyl)phenyl-2-(1H-indol-3-yl)ace-
2955, 1614, 1521, 1113, 742 cmꢁ1
;
1H NMR (500 MHz, CDCl3): d
tate (3c).
Slightly yellow oil, yield 24.7%; IR (KBr) 3408,
8.26 (br s, 1H, H-1), 7.65 (d, J = 8.0 Hz, 1H, H-4), 7.37 (d, J = 8.0
Hz, 1H, H-7), 7.23 (t, J = 7.5 Hz, 1H, H-5), 7.17–7.13 (m, 3H, H-30,
H-50, H-6), 7.03 (s, 1H, H-2), 6.60 (d, J = 8.5 Hz, 2H, H-20, H-60),
3.74 (m, 4H, morpholine-CH2, H-2, H-6), 3.50 (t, J = 7.0 Hz, 2H,
NCH2), 3.43 (s, 2H, benzylic-CH2), 3.12 (t, J = 7.0 Hz, 2H, CH2),
2.46–2.44 (m, 4H, morpholine-CH2, H-3, H-5); MS (ESI) m/
z = 336.4 [M+H]+.
3022, 2929, 1751, 1619, 1596, 1505, 1193, 1112, 742 cmꢁ1
;
1H
NMR (500 MHz, CDCl3): d 8.32 (br s, 1H, H-1), 7.71 (d, J = 8.0 Hz,
1H, H-4), 7.37 (d, J = 8.0 Hz, 1H, H-7), 7.31 (d, J = 8.5 Hz, 2H, H-30,
H-50), 7.23–7.20 (m, 2H, H-2, H-5), 7.17 (m, 1H, H-6), 7.01 (d,
J = 8.5 Hz, 2H, H-20, H-60), 4.01 (s, 2H, CH2CO), 3.62 (s, 2H, ben-
zylic-CH2), 2.55–2.52 (m, 4H, pyrrolidine-CH2, H-2, H-5), 1.82–
1.80 (m, 4H, pyrrolidine-CH2, H-3, H-4); MS (ESI) m/z = 335.4
[M+H]+.
4.1.3.5.
aniline (2e).
2968, 1614, 1519, 1455, 741 cmꢁ1
8.14 (br s, 1H, H-1), 7.68 (d, J = 8.0 Hz, 1H, H-4), 7.42 (d, J = 8.0
Hz, 1H, H-7), 7.28 (m, 1H, H-5), 7.19–7.15 (m, 3H, H-6, H-30, H-
50), 7.10 (d, J = 2.0 Hz, 1H, H-2), 6.63 (d, J = 8.0 Hz, 2H, H-20, H-60),
3.53 (m, 4H, NCH2, benzylic-CH2), 3.15 (t, J = 6.5 Hz, 2H, CH2),
2.57–2.55 (q, 4H, CH2 ꢀ 2), 1.11 (t, J = 7.0 Hz, 6H, CH3 ꢀ 2); MS
(ESI) m/z = 322.4 [M+H]+.
N-(2-(1H-Indol-3-yl)ethyl)-4-((diethylamino)methyl)
Yellow oil (yield 70.8%); IR (KBr) 3410, 3102,
1H NMR (500 MHz, CDCl3): d
4.1.4.4. 4-(Morpholinomethyl)phenyl-2-(1H-indol-3-yl)acetate
;
(3d).
Slightly yellow oil, yield 30.5%; IR (KBr) 3405, 3057,
2956, 1751, 1608, 1596, 1505, 1196, 1116, 865, 743 cmꢁ1
;
1H
NMR (500 MHz, CDCl3): d 8.18 (br s, 1H, H-1), 7.71 (d, J = 9.5 Hz,
1H, H-4), 7.38 (d, J = 10.0 Hz, 1H, H-7), 7.31 (d, J = 10.0 Hz, 2H, H-
30, H-50), 7.24–7.20 (m, 2H, H-2, H-5), 7.18–7.14 (m, 1H, H-6),
7.02 (d, J = 10.0 Hz, 2H, H-20, H-60), 4.01 (s, 2H, CH2CO), 3.70 (m,
4H, morpholine-CH2, H-2, H-6), 3.46 (s, 2H, benzylic-CH2), 2.43
(m, 4H, morpholine-CH2, H-3, H-5); MS (ESI) m/z = 351.5 [M+H]+.
4.1.3.6. N-(3-(1H-Indol-3-yl)propyl)-4-(morpholinomethyl)ani-
line (2f).
Slightly yellow oil (yield 70.8%); IR (KBr) 3410,
4.1.4.5. 4-((Diethylamino)methyl)phenyl)-2-(1H-indol-3-yl)ace-
3105, 2956, 1614, 1521, 1113, 863, 742 cmꢁ1
;
1H NMR
tate (3e).
Slightly yellow oil (yield 40.8%); IR (KBr) 3407,
(500 MHz, CDCl3): d 8.05 (br s, 1H, H-1), 7.62 (d, J = 8.0 Hz, 1H,
H-4), 7.42 (d, J = 8.0 Hz, 1H, H-7), 7.22 (t, J = 7.5 Hz, 1H, H-5),
7.14–7.10 (m, 3H, H-6, H-30, H-50), 6.98 (d, J = 2.0 Hz, 1H, H-2),
6.56 (d, J = 8.0 Hz, 2H, H-20, H-60), 3.72 (m, 4H, morpholine-CH2,
H-2, H-6), 3.40 (s, 2H, benzylic-CH2), 3.22 (t, J = 7.0 Hz, 2H,
NCH2), 2.91 (t, J = 7.5 Hz, 2H, CH2), 2.44–2.42 (m, 4H, morpho-
line-CH2, H-3, H-5), 2.08–2.02 (m, 2H, CH2); MS (ESI) m/z = 350.4
[M+H]+.
3147, 2925, 1751, 1618, 1504, 1457, 1198, 743 cmꢁ1
;
1H NMR
(500 MHz, CDCl3): d 8.30 (br s, 1H, H-1), 7.73 (d, J = 8.0 Hz, 1H,
H-4), 7.35–7.31 (m, 3H, H-7, H-30, H-50), 7.22–7.16 (m, 3H, H-5,
H-6, H-2), 7.01 (d, 2H, J = 8.0 Hz, H-20, H-60), 4.02 (s, 2H, CH2CO),
3.55 (s, 2H, benzylic-CH2), 2.54 (q, J = 7.0 Hz, 4H, CH2 ꢀ 2), 1.05
(t, J = 7.5 Hz, 6H, CH3 ꢀ 2); MS (ESI) m/z = 337.5 [M+H]+.
4.1.4.6. 4-(Piperidin-1-ylmethyl)phenyl-3-(1H-indol-3-yl)pro-
panoate (3f).
White solid (yield 48.3%); mp: 89–90 °C; IR
4.1.4. General procedure for preparation of compounds 3a–i
To a solution of indole-3-carboxylic acid (1.55 mmol) in 10 mL
dichloromethane and 2 mL THF, was added DCC (1.71 mmol),
DMAP (1.55 mmol) and 4-piperidin-1-yl-methyl-phenol 7a (1.55
mmol). The mixture was stirred at room temperature over night
and stopped by addition of 4.0 mL water, followed by extracting
with dichloromethane (20 mL ꢀ 3), the combined organic layer
was washed with NaHCO3, saline, dried over anhydrous Na2SO4
and evaporated to dryness to get crude product, which was puri-
fied by silica gel column chromatography eluting with petroleum,
ethyl acetate and triethylamine (20:20:1) to afford 3a.
(KBr) 3403, 3137, 2925, 1751, 1629, 1543, 1280, 1157, 736 cmꢁ1
;
1H NMR (500 MHz, CDCl3): d 8.01 (br s, 1H, H-1), 7.66 (d, J = 7.5
Hz, 1H, H-4), 7.38 (d, J = 8.0 Hz, 1H, H-7), 7.30 (d, J = 8.5 Hz, 2H,
H-30, H-50), 7.23 (t, J = 7.0 Hz, 1H, H-5), 7.16 (t, J = 7.5 Hz, 1H, H-
6), 7.08 (d, J = 2.0 Hz, 1H, H-2), 6.96 (d, J = 8.5 Hz, 2H, H-20, H-60),
3.44 (s, 2H, benzylic-CH2), 3.25 (t, J = 7.5 Hz, 2H, CH2CO), 2.98 (t,
J = 7.5 Hz, 2H, CH2), 2.36–2.34 (m, 4H, piperidine-CH2, H-2, H-6),
1.60–1.54 (m, 4H, piperidine-CH2, H-3, H-5), 1.44–1.42 (m, 2H,
piperidine-CH2, H-4); MS (ESI) m/z = 363.5 [M+H]+.
4.1.4.7. 4-(Pyrrolidin-1-ylmethyl)phenyl-3-(1H-indol-3-yl)pro-
panoate (3g).
White solid (yield 50.6%); mp: 98–100 °C; IR
4.1.4.1. 4-(Piperidin-1-ylmethyl)phenyl-1H-indole-3-carboxyl-
(KBr) 3403, 3137, 2925, 1751, 1629, 1543, 1280, 1157, 736 cmꢁ1
;
ate (3a).
Yellow oil (yield 27.1%); IR (KBr) 3287, 3052, 2928,
1H NMR (500 MHz, CDCl3): d 8.05 (s, 1H, H-1), 7.70 (d, J = 8.0 Hz,
1H, H-4), 7.42 (d, J = 8.0 Hz, 1H, H-7), 7.36 (d, J = 8.5 Hz, 2H, H-30,
H-50), 7.23 (t, J = 7.5 Hz, 1H, H-5), 7.17 (t, J = 7.5 Hz, 1H, H-6),
7.09 (d, J = 2.0 Hz, 1H, H-2), 7.00 (d, J = 8.5 Hz, 2H, H-20, H-60),
3.63 (s, 2H, benzylic-CH2), 3.28 (t, J = 7.5 Hz, 2H, CH2CO)), 3.02 (t,
J = 7.5 Hz, 2H, CH2), 2.54–2.52 (m, 4H, pyrolidine-CH2, H-2, H-5),
2852, 1677, 1611, 1440, 1170, 1010, 749 cmꢁ1
;
1H NMR (500
MHz, CDCl3): d 9.46 (br s, 1H, H-1), 8.01 (m, 1H, H-4), 7.80 (s,
1H, H-2), 7.70 (s, 1H, H-7), 7.53 (d, J = 8.0 Hz, 2H, H-30, H-50),
7.36–7.34 (m, 1H, H-5), 7.22–7.19 (m, 3H, H-20, H-60), 3.40 (s, 2H,
benzylic-CH2), 2.35–2.33 (m, 4H, piperidine-CH2, H-2, H-6), 1.53–

L. Tang et al. / Bioorg. Med. Chem. 21 (2013) 5936–5944
5943
1.83–1.81 (m, 4H, pyrolidine-CH2, H-3, H-4); MS (ESI) m/z = 349.3
the presence of various compound concentrations in the buffer
(50 mM Tris–HCl, pH 7.4). The reaction mixtures were incubated
for 90–120 min at room temperature to achieve binding equilib-
rium. After washing, the membrane-bound radioactivity was mea-
sured on a Topcount (PerkinElmer) at 20 °C. Non-specific binding
was determined in the presence of excessive cold histamine (final
[M+H]+.
4.1.4.8. 4-(Morpholinomethyl)phenyl-3-(1H-indol-3-yl)propan-
oate (3h).
Slightly yellow oil, yield 45.7%; IR (KBr) 3406, 3058,
2956, 1750, 1609, 1543, 1196, 1116, 863, 738 cmꢁ1
;
1H NMR
(500 MHz, CDCl3): d 8.11 (br s, 1H, H-1), 7.66 (d, J = 7.5 Hz, 1H,
H-4), 7.38 (d, J = 8.0 Hz, 1H, H-7), 7.31 (d, J = 8.5 Hz, 2H, H-30, H-
50), 7.22 (t, J = 7.0 Hz, 1H, H-5), 7.15 (t, J = 7.0 Hz, 1H, H-6), 7.06
(d, J = 2.5 Hz, 1H, H-2), 6.97 (d, J = 8.5 Hz, 2H, H-20, H-60), 3.74
(m, 4H, morpholine-CH2, H-2, H-6), 3.48 (s, 2H, benzylic-CH2),
3.24 (t, J = 7.5 Hz, 2H, CH2CO), 2.98 (t, J = 7.5 Hz, 2H, CH2), 2.44–
2.42 (m, 4H, morpholine-CH2, H-3, H-5); MS (ESI) m/z = 365.4
[M+H]+.
concd, 300 lM). IC50 values were converted to Ki values using the
Cheng–Prusoff equation. Each experiment was performed in dupli-
cate and repeated for three to six times.
4.2.4. ABTS radical cation scavenging activity assay25
2,20-Azino-bis-2-ethybenz-thiazoline-6-sulfonic acid (ABTS)
was dissolved in purified water to a 7 mM concentration. ABTS rad-
ical cation (ABTSÅ+) was produced by reacting ABTS stock solution
with 2.45 mM potassium persulfate (final concentration) and
allowing the mixture to stand in the dark at room temperature
for at least 18 h before use. The stock solution of ABTSÅ+ was serially
4.1.4.9. 4-((Diethylamino)methyl)phenyl-3-(1H-indol-3-yl)pro-
panoate (3i).
Slightly yellow oil (yield 51.2%); IR (KBr) 3406,
3145, 2928, 1750, 1619, 1505, 1456, 1196, 742 cmꢁ1
;
1H NMR
diluted with sodium phosphate buffer (50 mM, pH 7.4) to 100
Melatonin and 1d, 2d and 3d, at different concentrations (total vol-
ume of 50 L) were added to 150 L of 100
M ABTSÅ+ solution,
lM.
(500 MHz, CDCl3): d 8.04 (s, 1H, H-1), 7.66 (d, J = 8.0 Hz, 1H, H-
4), 7.38 (d, J = 8.0 Hz, 1H, H-7), 7.32 (d, J = 8.5 Hz, 2H, H-30, H-50),
7.23 (m, 1H, H-5), 7.16 (t, J = 7.5 Hz, 1H, H-6), 7.08 (d, J = 1.5 Hz,
1H, H-2), 6.96 (d, J = 8.5 Hz, 2H, H-20, H-60), 3.54 (s, 2H, benzylic-
CH2), 3.25 (t, J = 7.5 Hz, 2H, CH2CO), 2.98 (t, J = 7.5 Hz, 2H, CH2),
2.55 (q, J = 7.0 Hz, 4H, CH2 ꢀ 2), 1.05 (t, J = 7.0 Hz, 6H, CH3 ꢀ 2);
MS (ESI) m/z = 351.2 [M+H]+.
l
l
l
respectively. After the addition of either melatonin or another anti-
oxidant to the ABTSÅ+ solution, complete mixing of reactants was
achieved by bubbling three to four times using plastic pipettes.
The optical absorbance of ABTSÅ+ at 415 nm was measured at 6
min after addition and equilibrated at 30 °C. Each individual treat-
ment was repeated for three times and the results of the experi-
ments were compared.
4.2. Pharmacology
4.2.1. Constructs, cell culture and generation of stable cell
lines23
4.2.5. hERG patch-clamp assay
HEK 293 cells stably expressing the hERG ion channel were
used. hERG current was recorded and analysed using the suite of
pCLAMP programs (version 10, Molecular Devices, Union City,
CA, USA). Onset and steady-state block of hERG tail current due
to the test compounds were measured by a depolarizing test pulse
depolarization at ꢁ40 mV for 2 s after a prepulse to 40 mV for 4 s
from a holding potential of ꢁ80 mV. This pulse pattern was re-
peated at 20 s intervals. Peak tail currents were measured from
the test pulse and monitored during repetitive pulsing to reach a
steady state. Patch pipettes were made from glass capillaries using
a PC-10 micropipette puller (Narishige, Japan). The pipette solution
contained (in mM): K Aspartate, 130; MgCl2, 5; EGTA 5; HEPES, 10;
Tris-ATP 4; pH 7.2 (adjusted with KOH).
The human H1 receptor and human H2 receptor genes were
cloned from HEK-293 genomic DNA by PCR. The human H3 gene
was cloned using human thalamus poly-A RNA (Clontech, Palo
Alto, CA, USA) with RT-PCR methods. The human H4 receptor gene
was cloned from human bone marrow Marathon-Ready cDNAs
(Clontech, Basingstoke, UK) by PCR. Primers were designed accord-
ing to the published human histamine receptor gene sequences
(GenBank accession nos. X76786, M64799, AF140538, and
AB044934).
HEK-293 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum,
100 U/mL penicillin, and 100 lL streptomycin in a humidified
atmosphere of 95% air and 5% CO2 at 37 °C. For transfection, plas-
mid constructs were transfected or co-transfected into HEK-293
cells using Lipofectamine 2000 (Invitrogen) according to the man-
ufacturer’s instructions. Stable transfectants were selected in the
4.3. Computational chemistry
4.3.1. Homology modeling
presence of 800
l
g/mL G418.
The H3 receptor homology model was constructed starting from
the preliminary transmembrane models based on the recently re-
ported 3.1 Å crystal structure of the histamine H1 receptor (PDB
ID: 3RZE)24 H1 receptor has a sequence identity of 22.09% in overall
and 32.12% in transmembrane regions to H3 receptor. The primary
sequence of human histamine H3 receptor was obtained from the
Universal Protein Resource (UniProt ID: Q9Y5N1). To align the se-
quence of H3 receptor with that of H1 receptor, the ClustalW pro-
gram was employed and the same alignment was got as Soo-
Kyung Kim reported.20 In this study, COMPOSER program in SYB-
YL6.9 software (Tripos Inc., St. Louis, MO, USA) was applied to build
a three-dimensional model of the histamine H3 receptor structure.
4.2.2. CRE-driven reporter gene assay18,23
Stable HEK-293 cells co-transfected with H3 receptor (or H1, H2,
H4 receptor) and pCRE-Luc were seeded in a 48-well plate over-
night and were grown to 90–95% confluence. Afterwards, the cells
were stimulated with 10 lM forskolin or 10 lM forskolin plus dif-
ferent concentrations of histamine or compound in serum-free
DMEM, and the cells were incubated for 5 h at 37 °C. Luciferase
activity was detected using a firefly luciferase kit (Promega, Madi-
son, WI, USA).
4.2.3. Human histamine H3 receptor binding assay23
HEK-293 cells stably expressing human H3 receptor were har-
vested and membrane was gained by centrifugal classification.
4.3.2. Molecular docking
The docking study was performed by using the FlexiDock pro-
gram in SYBYL6.9 software. The binding pocket of H3 receptor
was defined to cover all residues within 5 Å of the ligand in the
homology modeling. Ligand with the most basic moiety was man-
ually placed in the active site to interact with Glu206 in TM5 and/
or Asp114 in TM3 and then, the ligand–H3 receptor complex was
The membranes (50
-methylhistamine at various concentrations (from 0.03 nM to
50 nM) for saturation binding assays. For competitive binding as-
says, the membranes (50 g of protein) were incubated with
2 nM [3H] N-
-methylhistamine for recombinant hH3 receptor in
l
g of protein) were incubated with [3H] N-
a
l
a

5944
L. Tang et al. / Bioorg. Med. Chem. 21 (2013) 5936–5944
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further optimized. All single bonds of residue side chains inside the
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was allowed to rotate on all the single bonds and move flexibly
within the tentative binding pocket. Kollman all-atom charges
were loaded to the protein, and Gasteiger–Huckel charges were as-
signed to the ligand atoms. The structural optimization was per-
formed for 100,000 generations using a TRIPOS forcefield and
genetic algorithm. As a result, 20 best scoring ligand–protein com-
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Acknowledgements
The authors thank the National Natural Science Foundation of
China (No. 21172193), the Ministry of Science and Technology
(2012CB910402 and 2012AA020303) and the Zhejiang Provincial
Natural Foundation of China (No. R2110297) for financial support.
We also thank Jianyang Pan (Pharmaceutical Informatics Institute,
Zhejiang University) for performing NMR and mass spectrometry
for structure elucidation.
18. Huang, W.; Tang, L.; Shi, Y.; Huang, S.; Xu, L.; Sheng, R.; Wu, P.; Li, J.; Zhou, N.;
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