Bioactivity-guided synthesis of gramine derivatives as new MT1 and 5-HT1A receptors agonists

Article abstract of DOI:10.1080/10286020.2017.1323885

Twenty-four gramine derivatives were synthesized and evaluated on MT₁ and 5-HT_1A receptors in vitro. Among them, seven derivatives (7, 8, 16, 19, 20, 21, and 24) exhibited higher agonisting activities on MT₁ or 5-HT_1A

Full text of DOI:10.1080/10286020.2017.1323885

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Bioactivity-guided synthesis of gramine derivatives
as new MT1 and 5-HT1A receptors agonists
Xiu-Juan Yin, Xiao-Yan Huang, Yun-Bao Ma, Chang-An Geng, Tian-Ze Li, Xing-
Long Chen, Tong-Hua Yang, Jun Zhou, Xue-Mei Zhang & Ji-Jun Chen
To cite this article: Xiu-Juan Yin, Xiao-Yan Huang, Yun-Bao Ma, Chang-An Geng, Tian-Ze Li,
Xing-Long Chen, Tong-Hua Yang, Jun Zhou, Xue-Mei Zhang & Ji-Jun Chen (2017): Bioactivity-
guided synthesis of gramine derivatives as new MT1 and 5-HT1A receptors agonists, Journal of
Asian Natural Products Research, DOI: 10.1080/10286020.2017.1323885
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1323885
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Date: 08 May 2017, At: 02:35

Journal of asian natural Products research, 2017
https://doi.org/10.1080/10286020.2017.1323885
Bioactivity-guided synthesis of gramine derivatives as new
MT1 and 5-HT1A receptors agonists
Xiu-Juan Yina,b,d, Xiao-Yan Huangb,c, Yun-Bao Mab,c, Chang-An Gengb,c, Tian-Ze Lib,c,
Xing-Long Chenb,d, Tong-Hua Yangb,c, Jun Zhoua,b, Xue-Mei Zhangb,c and Ji-Jun Chenb,c
aschoolofchemicalscienceandtechnology,Yunnanuniversity, Kunming 650091, china; bstateKey laboratory
of Phytochemistry and Plant resources in West china, Kunming institute of Botany, chinese academy of
sciences, Kunming 650201, china; cYunnan Key laboratory of natural Medicinal chemistry, Kunming institute
of Botany, chinese academy of sciences, Kunming 650201, china; dKunming institute of Botany, university of
chinese academy of sciences, Beijing 100049, china
ABSTRACT
ARTICLE HISTORY
received 25 february 2017
accepted 24 april 2017
Twenty-four gramine derivatives were synthesized and evaluated on
MT1 and 5-HT1A receptors in vitro. Among them, seven derivatives (7,
8, 16, 19, 20, 21, and 24) exhibited higher agonisting activities on
MT1 or 5-HT1A receptors. Compared with gramine, derivatives 7, 8, 16,
19, 20, 21, and 24 displayed 1.6–3.5-fold increase in agonistic rates
on 5-HT1A receptor. Particularly, derivatives 7, 19, and 21 exhibited
significant agonistic activities on MT1 and 5-HT1A receptors with EC50
values of 0.51, 0.39, 0.50 mΜ and 0.28, 0.46, 0.23 mΜ, respectively.
The preliminary structure–activity relationships of gramine derivatives
were summarized for further investigation on MT1 and 5-HT1A
receptors as new potential agonists.
KEYWORDS
Gramine; melatonin receptor;
5-hydroxy-tryptamine
receptor; agonistic
activity; structure–activity
relationships
MT1
5 -HT1A
1.5
1.36
0 .5
0.47
0.51
0.28
0 .4
0 .3
0 .2
0 .1
0 .0
0.39
0.50
1.0
0.5
0.0
0.46
0.23
Gramine
19
21
7
Gramin e
7
1 9
2 1
CONTACT Ji-Jun chen
chenjj@mail.kib.ac.cn
supplemental data for this article can be accessed https://doi.org/10.1080/10286020.2017.1323885
© 2017 informa uK limited, trading as taylor & francis Group

2
X.-J. YIN ET AL.
1. Introduction
Melatonin (5-methoxy-N-acetyltryptamine, MT, Figure 1) is a neurohormone primarily
secreted from the vertebrate pineal gland in response to the central switch of the circadian
rhythm [1,2]. In 1917, the biological activity of melatonin was reported by McCord and
Allan to discover that extracts of bovine pineal gland caused blanching of Rana pipiens
tadpole skin in adjustment to environmental conditions [3,4]. Melatonin receptors were
differentiated into MT1, MT2, and MT3 subtypes based on their functions and pharmaco-
logical characteristics, which were viewed as potential targets for drug discovery [5–7]. e
human melatonin receptor subtypes showed 60% homology at the amino acid level and
distinct pharmacological profiles to partial agonists and antagonists. In mammals, MT1
and MT2 receptors had been cloned, which belonged to the G-protein-coupled receptor
superfamily, sharing some specific short amino-acid sequences [8]. e MT3 binding site
was purified to homogeneity and identified by partial peptide sequencing as the homolog
of the human quinone reductase 2 [9,10]. 5-Hydroxy-tryptamine (5-HT, Figure 1) was the
immediate precursor of melatonin, and the conversion was realized by the sequential cataly-
sis of two melatonin-synthesizing enzymes arylalkylamine N-acetyltransferase and hydrox-
yindole-O-methyl-transferase [11,12]. e known subtypes of 5-HT receptors, including
5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7, had been identified. Selective
agonists and antagonists for 5-HT receptor subtypes were characterized with modulating
drugs in a vast variety of diseases [13]. e melatonin could potentiate the 5-HT1A receptor
O
NHAc
NH2
N
H
O
HO
O
N
N
H
H
Agomelatine
5-Methoxy-N-acetyltryptamine
5-Hydroxy-tryptamine
O
NHAc
H
NHAc
N
O
O
Cl
O
O
N
N
H
N
H
H
Piromelatine
Luzindole
LY-156735
O2N
NHAc
NH
NO2
O
N
N
H
Cl
ML-23
UCM454
Figure 1. the clinical drugs and gramine.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
activation in the hypothalamus, which was modulated by melatonin and thermoregulatory
responses [14,15]. In recent years, several kinds of drugs which were capable of either selec-
tively stimulating or inhibiting MT and 5-HT receptors were reported, some of which had
been used for psychiatric purposes. For example, agomelatine (a MT1 and MT2 receptors
agonist with 5-HT2C antagonistic properties) was marketed for the treatment of depression
[16], and piromelatine (Neu-P11, a novel MT1, MT2, 5-HT1A, and 5-HT1D receptor agonist)
demonstrated efficacy for insomnia in the phase II clinical trial (Figure 1) [17].
Our previous studies proposed many natural small molecules and their derivatives had
activities on MT and 5-HT receptors using the fluo-8 calcium assay in vitro. Indole alkaloids
with privileged heterocyclic scaffolds provided tremendous opportunities to discover novel
drugs [18], Many drugs curing central nervous system diseases have been on market or in
clinical trial, including LY-156735, luzindole, ML-23 [19], UCM454, S-24014 (Figure 1) [20].
Gramine, a small molecule with fascinating indole ring, possessed many pharmaceutical
and therapeutic applications, such as vasodilatation [21], antioxidant [22], inhibiting the
early stage of enterovirus replication and treating neurodegenerative diseases [23]. However,
gramine and their derivatives as MT1 or 5-HT1A receptor agonists were not reported. Our
previous fluo-8 calcium assay suggested that gramine had activities on MT1 and 5-HT1A
receptors with agitating rates of 182.12 6.71 and 136.45 6.96% at the concentration of
1.00 mM, respectively. is encouraged us to synthesize derivatives 1–24 and study their
structure–activity relationships (SARs) in detail.
2. Results and discussion
2.1. Chemistry
In order to obtain new active compounds for MT1 and 5-HT1A receptors, the structure of
gramine was modified through a flexible synthetic method (Scheme 1). N-Methylation
derivative 1 was obtained from gramine and iodomethane in the presence of NaH. Derivative
2 with a single methyl group at N-1′ position was prepared by the reductive amination of
3-indole-carboxaldehyde with methylamine and NaBH(AcO)3. To investigate whether dif-
ferent alkyl chains or heterocyclic ring could affect the potential agonistic activities, deriv-
atives 3–24 were afforded by the reaction of gramine with secondary amines in anhydrous
toluene. e results showed that the synthetic routes had the advantages of simple reaction
and high yield.
2.2. Biological evaluation
All the synthesized derivatives (1–24) were evaluated on MT1 and 5-HT1A receptors at the
concentration of 1.00 mM (Table 1). Methylation derivative 1 possessed weak agonistic
activity on MT1 receptor with an agonistic rate of 61.55 13.89%, suggesting that NH (N-
1) in the indole ring was favorable for maintaining activity. Monomethylation derivative 2
had no activity, revealing that a trialkylamine group at N-1′ position played an important
role for maintaining activity. Derivative 3 with two ethyl groups had a similar agonistic
activity (184.19 5.0%) on MT1 receptor. e agonistic rates of homologs 3–6 decreased
on MT1 receptor in sequence from 184.19 5.0 to 20.70 2.99% (derivative 3 vs. 4 vs. 5 vs.
6). Derivative 7 with a pyrrolyl group at N-1′ position indicated similar agonistic activity

4
X.-J. YIN ET AL.
Scheme 1. synthesis of the gramine derivatives. reagents and conditions: (a) nah, ch3i, dMf, 0 °c, 50–
70%. (b) Methylamine, ch3oh, r.t.; naBh(aco)3, etoh, r.t. 65%. (c) corresponding amines, toluene, reflux,
75–97%. (d) 2-amino-ethyl-piperazine, toluene, reflux, 70%.
on MT1 receptor, while the agonistic activities of piperidyl derivative 8 and morpholinyl
derivative 9 decreased significantly. Piperazinyl derivative 10 exhibited moderate activity
on MT1 receptor. ese results enlightened us to further explore the effects of derivatives
11–19 by introducing a piperazinyl group. Compared with derivative 10, derivative 11
with a N-methylation piperazine group, and derivative 12 with a N-ethylation piperazine
group obviously reduced activities on MT1 receptor; derivatives 13, 14, and 15 with OH,
OMe, and NH2 groups, respectively, on C-2 position of N-ethyl piperazine possessed weak
activities; derivative 16 with a N-isopropylation piperazine group and acylated derivatives
17–18 had no activities on MT1 receptor; derivative 19 with bisindole structure showed a

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 1. agonistic activities of gramine derivatives on Mt1 and 5-ht1a receptors.a
Agonistic rate (%)b
Compound
R
MT1
5-HT1A
agomelatine
5-hydroxy-tryptamine
Gramine
1
2
–
–
–
–
100 7.83
–
182.12 6.71
61.55 13.89
72.73 14.59
–
100 3.43
136.45 6.96
0.77 3.25
1.84 0.54
NHMe
NEt2
3
4
5
6
7
184.19 5.0
122.25 5.17
53.03 3.83
20.70 2.99
193.66 15.02
275.40 9.65
161.56 20.38
165.71 16.98
−16.24 2.20
357.35 17.47
N(n-Pr)2
N(n-Bu)2
N(n-Hex)2
R
N
N
H
8
9
34.32 20.16
71.59 0.09
220.93 2.88
65.93 1.91
N
N
N
N
O
10
11
148.07 7.32
83.26 9.09
65.58 2.01
72.38 1.57
NH
N
12
13
14
15
106.38 4.93
49.52 3.54
68.99 4.91
110.08 1.21
116.12 21.38
56.94 0.59
N
N
N
N
OH
63.84 3.12
N
N
N
N
OMe
NH2
166.60 21.82
N
16
17
18
19
127.11 9.31
−9.34 20.16
−2.44 1.84
220.97 7.64
243.78 11.46
21.30 4.97
17.98 4.61
467.55 3.12
N
N
N
N
NCOCH3
NBoc
N
N
H
20
21
22
175.23 7.63
204.10 11.72
62.63 4.24
237.02 15.38
486.60 17.62
49.98 0.71
NH
N
N
N
N
NCHO
(Continued)

6
X.-J. YIN ET AL.
Table 1. (continued).
Agonistic rate (%)b
Compound
R
MT1
5-HT1A
23
78.66 2.45
54.81 2.78
NCOCH3
N
24
78.50 3.02
270.0 23.32
NBoc
N
aagomelatine and 5-hydroxy-tryptamine were tested at the concentration of 1.11 and 6.67 μM, respectively. other com-
pounds were tested at the concentration of 1.00 mM.
bagonist data on melatonin and 5-hydroxy-tryptamine receptors were the mean of triplicate experiments.
1.2-fold increase in agonistic activity on MT1 receptor; derivative 20 with a homopiperzinyl
group at N-1′ position proposed activity with an agonistic rate of 175.23 7.63% on MT1
receptor, indicating that the homopiperzinyl group was suitable for maintaining agonistic
activity. Enlightened by these results, derivatives 21–24 with different substituents on the
homopiperzinyl group were synthesized. e agonistic rates of derivatives 21–24 displayed
from 204.10 11.72 to 62.63 4.24% on MT1 receptor. e methylation derivative 21
exhibited significantly agonistic activity on MT1 receptor with agonistic rate of 204.10%
on MT1 receptor. N-formyl (22), N-acetyl (23) and N-tert-butoxycarbonyl (24) showed
weak activities on MT1 receptor. e above analyses suggested that pyrrolyl, morpholinyl,
piperidyl, piperazinyl, and homopiperazinyl groups at N-1′ position had a different effect
for agonistic activities.
In Table 1, methylation derivative 1 had no agonistic activity on 5-HT1A receptor, sug-
gesting that NH was preferable for activity. Derivative 2 with a single methyl group at N-1′
position displayed no activity on 5-HT1A receptor. Derivative 3 with two ethyl groups at
N-1′ position increased activity with an agonistic rate of 275.40 9.65% on 5-HT1A recep-
tor. e agonistic activities of homologs 3–6 decreased gradually with agonistic rates from
275.40 9.65 to −16.24 2.20% (derivative 3 vs. 4 ≈ 5 vs. 6). e above analyses suggested
that the trialkylamine moiety at N-1′ position was crucial for agonistic activity. Heterocyclic
derivatives displayed attractive agonistic activities on 5-HT1A receptor. Derivative 7 with a
pyrrolyl group and derivative 8 containing a piperazinyl group at N-1′ position exhibited
2.7- and 1.6-fold increase in agonistic rates, respectively. Derivative 9 with a morpholinyl
group and 10 with a piperazinyl group at N-1′ position showed no agonistic activities.
Piperazinyl derivative 10 displayed weak activity on 5-HT1A receptor. Compared with deriv-
ative 10, the agonistic activities of derivative 11 with a N-methylation piperazine group
and derivative 12 with a N-ethylation piperazine group decreased; derivatives 13, 14, and
15 with OH, OMe, and NH2 groups at C-2 position of N-ethyl piperazine possessed weak
activities; derivative 16 with a N-isopropylation piperazine group exhibited about 2-fold
activity on 5-HT1A receptor. Acylated derivatives 17 and 18 had no activities; derivative 19
with bis(indole) structure demonstrated an approximately 3.5-fold increase in agonistic
activity on 5-HT1A receptor. Derivative 20 with a homopiperzinyl group at N-1′ position
proposed better agonistic activity on 5-HT1A receptors (237.02 15.38%, at 1.00 mM),
indicating that the homopiperzinyl group was preferable for gaining high agonistic activity.
e agonistic rates of homologs 21–24 with different substituents on the homopiperzinyl
group were varied from 486.60 17.62 to 49.98 0.71% on 5-HT1A receptor. Interestingly,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
the N-methylation derivative 21 exhibited higher agonistic activity on 5-HT1A receptor
with an agonistic rate of 486.60% on 5-HT1A receptor. Agonistic activities of N-formyl and
N-acetyl substituted analogs 22 and 23 resulted in an obvious decrease on 5-HT1A receptor.
N-tert-butoxycarbonyl analog 24 displayed an approximate 2-fold increase in agonistic
activity on 5-HT1A receptor. e above analyses suggested the trialkylamine moiety at N-1′
position was important for agonistic activity.
e agonistic activities of derivatives 7, 19, and 21 were higher than other derivatives,
which prompted us to evaluate the EC50 values of derivatives 7, 19, and 21 on MT1 and
5-HT1A receptors. It was noted that derivatives 7, 19, and 21 exhibited significant activi-
ties on MT1 and 5-HT1A receptors with EC50 values of 0.51, 0.39, 0.50 mΜ and 0.28, 0.46,
0.23 mΜ, respectively. e compared results were summarized in Table 2.
ese results indicated the following SARs (Figure 2): (1) a secondary amine on indole
ring played a significant role; (2) the single methylation at N-1′ position resulted in the
decrease of agonistic activities; (3) the side chains at N-1′ position (less than 3 carbons)
were crucial for agonistic activities; (4) the pyrrolyl, piperazinyl, and homopiperazinyl
Table 2. the ec50 (mM) values of derivatives 7, 19, and 21 on Mt1-heK293 and 5-ht1a- heK293 cells in
vitro.
EC50 (mM)
Compound
MT1
5-HT1A
Gramine
7
19
21
1.36
0.51
0.39
0.50
0.47
0.28
0.46
0.23
notes: dose-response of agonistic activity was performed in triplicate. ec50 values for the derivatives 7, 19, 21, and gramine
were determined from the dose-response curves obtained with seven concentrations from the range of 0.02–1.52 mM,
and calculated by the software of Graphpad Prism 5.0. the activity data were marked in bold font.
Figure 2. structure–activity relationships of gramine derivatives.

8
X.-J. YIN ET AL.
groups were preferred for agonistic activities; (5) the piperidyl and morpholinyl groups
were not favorable to maintain agonisting activities. is investigation will provide val-
uable information for further developing gramine derivatives as potential MT and 5-HT
receptors agonists.
3. Conclusion
In summary, twenty-four gramine derivatives were synthesized and examined for their
agonistic activities in vitro, of which, derivatives 7, 8, 16, 19, 20, 21, and 24 displayed higher
agonisting activities on MT1 and HT1A receptors. In particular, derivatives 7, 19, and 21
exhibited significant agonistic activities on MT1 and 5-HT1A receptors with EC50 values of
0.51, 0.39, 0.50 mΜ and 0.28, 0.46, 0.23 mΜ, respectively. In addition, the SARs analyses
indicated that a secondary amine on indole ring and the trialkylamine moiety at N-1′
position played significant roles for agonistic activities in the current series of compounds.
4. Experimentation
4.1. General experimental procedures
Melting points were measured on a micro-melting point apparatus SGW®X-4B (Shanghai
Precision & Scientific Instrument Co., Ltd., Shanghai, China). 1H NMR and 13C NMR data
were recorded in CDCl3 on a 400 MHz spectrometer (Bruker, Bremerhaven, Germany) with
tetramethylsilane as an internal standard. Low-resolution mass spectra and high-resolution
mass spectra data were measured on Shimadzu liquid chromatography-mass spectrome-
try-ion trap-time of flight (Shimadzu, Kyoto, Japan). e purity of the target compounds
was checked by three different solvent systems and HPLC methods. e solvents were
dried according to standard procedures. Organic solvents were analytical grade reagents,
purchased from Tianjin Chemical Reagent Co., Ltd (Tianjin, China). All reactions were
carried out under an air atmosphere and monitored by using thin-layer chromatography
(TLC, 200-300 mesh, Qingdao Makall Group Co., Ltd; Qingdao, China). Gramine and cor-
responding amines were purchased from Alfa Aesar or J&K Scientific Ltd (Bingjing, China).
e calcium flow assay was performed on MT1-HEK293 and 5-HT1A-HEK293 cell lines
by Wash Free Fluo-8 Calcium Assay Kit (HD Biosciences Co. Ltd, Shanghai, China, HD03-
0010), and the results were read with a FlexStation 3 Benchtop Multi-Mode Microplate
Reader (Molecular Devices, Sunnyvale, California, U.S.A.) at room temperature. CO2
incubator was purchased from ermo Forma Co., Ltd (U.S.). MT1 and 5-HT1A receptor
cell lines were purchased from HD Biosciences Co. Ltd (Shanghai, China). Agomelatine
(CAS: 138112-76-2) and 5-hydroxytryptamine (CAS: 50-67-9) were purchased from
Sahn Chemical Technology Co., Ltd (Shanghai, China) and Yunnan Zehao Trade Co., Ltd
(Yunnan, China), respectively, which was used as the positive control.
4.2. Chemical synthesis
4.2.1. General procedure for preparation of compound 1
Gramine (3.0 mmol) was dissolved in N, N-dimethyl formamide (15.0 mL), and NaH
(3.3 mmol) was slowly added into the above solution at 0 °C. e mixture were dropped by
CH3I (3.0 mmol) at room temperature, and monitored by TLC. Subsequently, the reaction

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
mixture were suspended in saturated NaCl (30.0 ml), extracted with EtOAc (3 × 30 ml)
and washed with saturated NaCl. e organic layer was dried over anhydrous Na2SO4 and
concentrated to dryness in vacuo. Purification by column chromatography on silica gel
using Et2NH/MeOH/CHCl3 (2/5/93, v/v/v) provided a target derivative.
N-Methylgramine (1): White amorphous powder, mp 113–114 °C, yield 70%.
4.2.2. General procedure for preparation of compound 2
CH3NH2 (6.0 mmol, MeOH 5 ml) methanol solution was slowly dropped into indole-3-car-
boxaldehyde (2.0 mmol, MeOH 10 ml) methanol solution. e mixture was stirred at room
temperature for 3 h, and then concentrated to a light yellow oil. is oil and NaBH(AcO)3
(2.2 mmol) were subsequently dissolved in EtOH (10 ml), and the mixture was stirred at
room temperature for 6 h. e solvent was evaporated in vacuo to give crude products
which were poured into 1 mol/L NaOH (1.5 ml), extracted with EtOAc (3 × 30 ml) and
washed with saturated NaCl (30 ml). e organic layer was dried over anhydrous Na2SO4,
concentrated to dryness in vacuo and purified by column chromatography on silica gel
using Et2NH/MeOH/CHCl3 (2/4/94, v/v/v).
N-Methyl-3-indolylmethylamine (2): Yellow oil, yield 65%.
4.2.3. General procedure for preparation of compounds 3–18 and 20–24
e gramine (2 mmol) and the corresponding amine (2–10 mmol) were dissolved in dry
toluene (15 ml), and the mixture was refluxed for 8–14 h and monitored by TLC. e sol-
vent was condensed subsequently in vacuo to obtain crude products which was purified by
column chromatography on silica gel using Et2NH/MeOH/CHCl3 (2/4/94–3/5/92, v/v/v).
N, N-Diethyl-3-indolylmethylamine (3): White amorphous powder, mp 105-106 °C,
yield 97%.
N, N-di(n-Propyl)-3-indolylmethylamine (4): Yellow oil, yield 98%.
N, N-di(n-Butyl)-3-indolylmethylamine (5): Yellow oil, yield 96%.
N, N-di(n-Hexyl)-3-indolylmethylamine (6): Yellow oil, yield 97%. 1H NMR (400 MHz,
CDCl3) δH: 8.75 (s, 1H, NH), 7.86-7.02 (m, 5H, H-2, 4, 5, 6, 7), 3.93 (s, 2H, CH2 N), 2.64-
2.60 (m, 4H, H-1′, 1″), 1.68-1.66 (m, 4H, H-2′, 2″), 1.45-1.38 (m, 12H, H-3′, 3″, 4′, 4″, 5′,
5″), 1.01-0.98 (m, 6H, H-6′, 6″). 13C NMR (100 MHz, CDCl3) δ: 136.3 (C-8), 128.2 (C-9),
123.8 (C-2), 121.7 (C-6), 119.5 (C-5), 119.2 (C-4), 113.1 (C-3), 111.2 (C-7), 53.8 (C-1′, 1″),
49.1 (CH2 N), 32.0 (C-2′, 2″), 27.5 (C-4′, 4″), 27.0 (C-3′, 3″), 22.8 (C-5′, 5″), 14.2 (C-6′,
6″). ESIMS: m/z 315 [M + H]+; HRESIMS: m/z 315.2688 [M + H]+ (calcd for C21H34N2,
315.2795).
N-Pyrrolyl-3-indolylmethylamine (7): White amorphous powder, mp 123–124 °C, yield
96%.
N-Piperidyl-3-indolylmethylamine (8): White amorphous powder, mp 126–127 °C, yield
97%.
N-Morpholinyl-3-indolylmethylamine (9): White amorphous powder, mp 123.5–
124.5 °C, yield 98%.
N-Piperazinyl-3-indolylmethylamine (10): White amorphous powder, mp 122–123 °C,
yield 90%.
N-Methyl-N-Piperazinyl-3-indolylmethylamine (11): White amorphous powder, mp
137–138 °C, yield 92%.

10
X.-J. YIN ET AL.
1
N-Ethyl-N-Piperazinyl-3-indolylmethylamine (12): Yellow oil, yield 93%. H NMR
(400 MHz, CDCl3) δH: 9.60 (s, 1H, NH), 7.75-7.01 (m, 5H, H-2, 4, 5, 6, 7), 3.74 (s, 2H,
CH2 N), 2.96-2.70 (m, 4H, H-2′, 6′), 1.83-1.75 (m, 2H, CH2CH3), 1.83–1.75 (m, 4H, H-3′,
5′), 1.17 (d, 3H, CH2CH3). 13C NMR (100 MHz, CDCl3) δ: 136.3 (C-8), 128.2 (C-9), 124.3
(C-2), 121.7 (C-6), 119.4 (C-5), 119.3 (C-4), 111.5 (C-3), 111.2 (C-7), 61.1 (C-3′, 5′), 53.7
(CH2 N), 53.4 (C-2′, 6′), 50.6 (C-1″), 20.1 (C-2″). ESIMS: m/z 244 [M + H]+; HRESIMS:
m/z 244.1705 [M + H]+ (calcd for C15H21N3, 244.1808).
N-(2″-Hydroxyethyl)-N-Piperazinyl-3-indolylmethylamine (13): White amorphous
powder, mp 86–87 °C, yield 83%.
N-(2″-Methoxyethyl)-N-Piperazinyl-3-indolylmethylamine (14): Yellow oil, yield 85%.
1H NMR (400 MHz, CDCl3) δH: 9.71 (s, 1H, NH), 7.73-6.94 (m, 5H, 5H, H-2, 4, 5, 6, 7),
3.76 (s, 2H, CH2 N), 3.51-3.49 (m, 2H, CH2CH2OCH3), 3.32 (s, 3H, CH2CH2OCH3), 2.69-
2.57 (m, 10H, H-1″, 2′, 3′, 5′, 6′). 13C NMR (100 MHz, CDCl3) δ: 136.3 (C-8), 128.3 (C-9),
124.6 (C-2), 121.6 (C-6), 119.3 (C-5), 119.2 (C-4), 111.4 (C-3), 111.1 (C-7), 70.0 (C-2″),
58.8 (OCH3), 57.8 (C-3′, 5′), 53.5 (CH2 N), 53.1 (C-2′, 6′), 52.6 (C-1″). ESIMS: m/z 274
[M + H]+; HRESIMS: m/z 274.1810 [M + H]+ (calcd for C16H23N3O, 274.1914).
1
N-Aminoethyl-N-Piperazinyl-3-indolylmethylamine (15): Yellow oil, yield 80%. H
NMR (400 MHz, CDCl3) δH: 8.38 (s, 1H, NH), 7.73-7.10 (m, 5H, H-2, 4, 5, 6, 7), 3.74
(s, 2H, CH2 N), 2.79-2.17 (m, 14H, 2-aminoethylpiperazinyl-H). 13C NMR (100 MHz,
CDCl3) δ: 136.1 (C-8), 128.0 (C-9), 123.8 (C-2), 121.9 (C-6), 119.4 (C-5), 119.4 (C-4),
112.1 (C-3), 111.0 (C-7), 60.7 (CH2CH2 NH2), 53.2 (CH2N), 53.2 (C-3′, 5′), 52.9 (C-2′, 6′),
38.6 (CH2CH2NH2). ESIMS: m/z 259 [M + H]+; HRESIMS: m/z 259.1841 [M + H]+ (calcd
for C15H22N4, 259.1917).
N-Isopropyl-N-piperazinyl-3-indolylmethylamine (16): White amorphous powder, mp
1
118-119 °C, yield 93%. H NMR (400 MHz, CDCl3) δH: 9.57 (s, 1H, NH), 7.73-6.94 (m,
5H, H-2, 4, 5, 6, 7), 3.78 (s, 2H, CH2 N), 2.69-2.60 (m, 9H, H-1″, 2′, 3′, 5′, 6′), 1.05 (d, 6H,
Me). 13C NMR (100 MHz, CDCl3) δ: 136.2 (C-8), 128.3 (C-9), 124.4 (C-2), 121.7 (C-6),
119.3 (C-5), 119.3 (C-4), 111.3 (C-3), 111.2 (C-7), 54.5 (C-1″), 53.1 (CH2 N), 53.1 (C-3′, 5′),
48.7 (C-2′, 6′), 18.7 (C-Me). ESIMS: m/z 258 [M + H]+; HRESIMS: m/z 258.1862 [M + H]+
(calcd for C16H23N3, 258.1965).
N-Acetyl-N-Piperazinyl-3-indolylmethylamine (17): White amorphous powder, mp
116–116.5 °C, yield 97%.
N-tert-Butyloxycarbonyl-N-Piperazinyl-3-indolylmethylamine (18): White amor-
1
phous powder, mp 160–161 °C, yield 96%. H NMR (400 MHz, CDCl3) δH: 8.93 (s,
1H, NH), 7.77-7.07 (m, 5H, H-2, 4, 5, 6, 7), 3.77 (s, 2H, CH2 N), 3.48 (m, 4H, H-3′,
5′), 2.51-2.49 (m, 4H, H-2′, 6′), 1.50 (s, 9H, Me-Boc). 13C NMR (100 MHz, CDCl3) δ:
154.9 (CO), 136.3 (C-8), 127.9 (C-9), 124.1 (C-2), 121.9 (C-6), 119.4 (C-5), 119.4 (C-4),
111.6 (C-3), 111.2 (C-7), 79.7 (C-Boc), 53.5 (CH2 N), 52.7 (C-2′, 6′), 45.0 (C-3′, 5′),
28.4 (C-Boc). ESIMS: m/z 316 [M + H]+; HRESIMS: m/z 316.1952 [M + H]+ (calcd for
C18H25N3O2, 316.2020).
N-Homopiperazinyl-3-indolylmethylamine (20): Yellow oil, yield 76%.
N-Methyl-N-homopiperazinyl-3-indolylmethylamine (21): Yellow oil, yield 80%.1H
NMR (400 MHz, CDCl3) δH: 10.05 (s, 1H, NH), 7.80-7.02 (m, 5H, H-2, 4, 5, 6, 7), 3.89 (s,
2H, CH2 N), 2.89-2.85 (m, 4H, H-2′, 4′), 2.75-2.69 (m, 4H, H-6′, 7′), 2.40 (s, 3H, NMe),
1.89-1.85 (m, 2H, H-3′). 13C NMR (100 MHz, CDCl3) δ: 136.4 (C-8), 128.1 (C-9), 124.3
(C-2), 121.6 (C-6), 119.4 (C-5), 119.1 (C-4), 112.6 (C-3), 111.3 (C-7), 57.5 (C-6′), 56.8 (C-7′),

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11
54.3 (C-4′), 54.0 (C-2′), 53.6 (CH2 N), 46.9 (NMe), 27.0 (C-3′). ESIMS: m/z 244 [M + H]+;
HRESIMS: m/z 244.1742 [M + H]+ (calcd for C15H21N3, 244.1808).
N-Formyl-N-homopiperazinyl-3-indolylmethylamine (22): White amorphous powder,
mp 108–109.5 °C, yield 88%. 1H NMR (400 MHz, CDCl3) δH: 8.71 (s, 1H, NH), 8.08-7.05
(m, 5H, H-2, 4, 5, 6, 7), 7.72 (m, 1H, CHO), 3.83 (s, 2H, CH2 N), 3.54-3.39 (m, 4H, H-2′,
4′), 2.75-2.66 (m, 4H, H-6′, 7′), 1.90-1.88 (m, 2H, H-3′). 13C NMR (100 MHz, CDCl3) δ:
162.8 (CHO), 136.3 (C-8), 127.6 (C-9), 123.6 (C-2), 121.8 (C-6), 119.4 (C-5), 119.2 (C-4),
112.6 (C-3), 111.1 (C-7), 56.7 (C-7′), 54.2 (C-2′), 53.4 (CH2 N), 48.0 (C-6′), 43.7 (C-4′), 29.2
(C-3′). ESIMS: m/z 258 [M + H]+; HRESIMS: m/z 258.1524 [M + H]+ (calcd for C15H19N3O,
258.1601).
N-Acetyl-N-homopiperazinyl-3-indolylmethylamine (23): White amorphous powder,
1
mp 116-117 °C, yield 86%. H NMR (400 MHz, CDCl3) δH: 8.71 (s, 1H, NH), 7.75-7.02
(m, 5H, H-2, 4, 5, 6,7), 3.82 (s, 2H, CH2 N), 3.67-3.61 (m, 4H, H-2′, 4′), 2.72-2.66 (m, 4H,
H-6′, 7′), 2.09 (s, 3H, CH3), 1.85–1.84 (m, 2H, H-3′). 13C NMR (100 MHz, CDCl3) δ: 170.5
(CO), 136.3 (C-8), 127.6 (C-9), 123.8 (C-2), 121.6 (C-6), 119.2 (C-5), 119.0 (C-4), 112.3
(C-3), 111.1 (C-7), 55.2 (C-7′), 54.0 (C-2′), 53.3 (CH2 N), 48.5 (C-6′), 44.9 (C-4′), 28.0
(C-3′), 21.6 (Me). ESIMS: m/z 272 [M + H]+; HRESIMS: m/z 272.1688 [M + H]+ (calcd for
C16H21N3O, 272.1757).
N-tert-Butyloxycarbonyl-N-homopiperazinyl-3-indolylmethylamine (24): Yellow amor-
phous powder, mp 95–96 °C, yield 82%. 1H NMR (400 MHz, CDCl3) δH: 8.91 (s, 1H, NH),
7.76-7.03 (m, 5H, H-2, 4, 5, 6, 7), 3.83 (s, 2H, CH2 N), 3.55-3.43 (m, 4H, H-2′, 4′), 2.72-
2.66 (m, 4H, H-6′, 7′), 1.85-1.82 (m, 2H, H-3′), 1.49 (s, 9H, Me-Boc). 13C NMR (100 MHz,
CDCl3) δ: 155.7 (CO), 136.4 (C-8), 127.9 (C-9), 123.8 (C-2), 121.8 (C-6), 119.5 (C-5), 119.3
(C-4), 112.8 (C-3), 111.2 (C-7), 79.4 (C-Boc), 55.77 (C-7′), 54.7 (C-2′), 53.5 (CH2 N), 46.8
(C-6′), 46.2 (C-4′), 28.6 (C-Me-Boc), 27.9 (C-3′). ESIMS: m/z 330 [M + H]+; HRESIMS:
m/z [M + H]+ 330.1284 (calcd for C19H27N3O2, 330.2176).
4.2.4. General procedure for preparation of compound 19
e gramine (4 mmol) and 2-amino-ethyl-piperazine (2 mmol) were dissolved in dry tolu-
ene (15 ml). e mixture were refluxed for 10 h monitored by TLC. e solvent was removed
in vacuo to afford crude products which were purified by column chromatography on silica
gel using Et2NH /MeOH/CHCl3 (3:5: 92, v/v/v).
N-[N-(3-Indolylmethyl)-aminoethyl]-N-piperazinyl-3-indolylmethylamine (19): Yellow
oil, yield 70%. 1H NMR (400 MHz, CDCl3) δH: 8.88 (s, 1H, NH), 8.85 (s, 1H, NH), 7.72-7.01
(m, 10H, H-2, 2′, 4, 4′, 5, 5′, 6, 6′, 7, 7′), 3.98 (s, 2H, CH2 N), 3.72 (s, 2H, CH2CH2NHCH2),
2.80–2.28 (m, 13H, 2-aminoethylpiperazinyl-H). 13C NMR (100 MHz, CDCl3) δ: 136.4
(C-8, 8′), 128.1 (C-9), 127.0 (C-9′), 124.1 (C-2), 122.8 (C-2′), 122.0 (C-6), 121.8 (C-6′),
119.4 (C-5), 119.4 (C-5′), 118.7 (C-4, 4′), 114.5 (C-3), 111.9 (C-3′), 111.3 (C-7), 111.1 (C-7′),
57.4 (CH2 N), 53.1 (C-3″, 5″), 53.0 (CH2 N), 52.7 (C-2″, 6″), 45.6 (CH2CH2NHCH2), 44.5
(CH2CH2NHCH2). ESIMS: m/z 388 [M + H]+; HRESIMS: m/z 388.2477 [M + H]+ (calcd
for C24H29N5, 388.2496).
4.3. Biological test methods
e agonistic rates of the tested compounds were performed according to the previous
reports, [24,25] and the details of fluo-8 calcium assay were listed as follows. HEK293 cell

12
X.-J. YIN ET AL.
lines stably expressing the human MT1 or 5-HT1A receptor were cultivated in Dubecco’s
modified Eagle’s medium supplemented with 10% fetal bovine serum, and cultured with
95% O2/5% CO2 at 37 °C. e cells were seeded in a matrigel coated 96-well black plate with
a plating volume of 100 μl/well at a density of 4 × 104/well, and incubated in CO2 incuba-
tor (ermo Forma 3310, Gaithersburg, U.S.) for overnight. en the cells were dyed by
HDB wash free calcium assay kit, and placed in CO2 incubator for 1 h. Tested compounds
and the positive drug were dissolved in 10 μl dimethyl sulfoxide and 990 μl HBSS buffer,
respectively, and extracted a plating volume of 100 μl/well in a Matrigel coated 96-well
clear bottom plate. Two 96-well plates were put into Flexstation 3 Benchtop Multi-Mode
Microplate Reader. e absorption values were read by Flexstation 3 Benchtop Multi-Mode
Microplate Reader at room temperature with wavelength (excitation: 485 nm; emission:
525 nm; emission cut-off: 515 nm). e agonistic activity was performed in triplicate. EC50
values for the derivatives 7, 19, 21, and gramine were determined from the dose-response
curves obtained with seven concentrations from the range of 0.02 to 1.52 mM. e results
were calculated by the sofware of Graphpad Prism 5.0.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was financially supported by the Hundred Talents Program of the Chinese Academy of
Sciences (CAS), the Program of Yunling Scholar, the National Natural Science Foundation of China
[grant number 81573322], the Youth Innovation Promotion Association (CAS), and the West Light
Foundation of the Chinese Academy of Sciences (Western Youth Scholars “A”).
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