Heterocycle-functional gramine analogues: Solvent- and catalyst-free synthesis and their inhibition activities against cell proliferation

Article abstract of DOI:10.1016/j.ejmech.2012.05.003

A series of novel gramine analogues were designed and synthesized via a convenient three-component reaction, and which were evaluated for their inhibition activities against cell proliferation. Their structures were confirmed by satisfactory spectra analyses mainly including ¹H NMR, and ESI-MS analyses. The preliminary assays indicated that some of the newly synthesized compounds displayed significantly good inhibition activities against human lung cancer (NCI-H460), hepatocellular liver carcinoma (HepG2), gastric cancer (SGC-7901 and BGC-823) cell lines compared with the control 5-Fluorouracil (5-FU), which might be developed as novel lead scaffold for potential anticancer agents.

Full text of DOI:10.1016/j.ejmech.2012.05.003

European Journal of Medicinal Chemistry 54 (2012) 248e254
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Heterocycle-functional gramine analogues: Solvent- and catalyst-free synthesis
and their inhibition activities against cell proliferation
, ,
,
Shaoyong Ke a 1, Liqiao Shi a 1, Xiufang Cao b, Qingyu Yang a, Ying Liang a, Ziwen Yang a
*
a National Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, No. 6 Nanhu Yaoyuan, Wuhan 430064, PR China
b College of Science, Huazhong Agricultural University, Wuhan 430070, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< Heterocycle-functional
gramine
R2
analogues.
Solvent-Free
Catalyst-Free
R4
< Significantly good inhibition activi-
ties compared with the 5-
Fluorouracil.
< Solvent- and catalyst-free three-
component reactions.
N
N
R3
R1
R3
HN
R4
N
R2 CHO
R1
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 gramine analogues were designed and synthesized via a convenient three-component
reaction, and which were evaluated for their inhibition activities against cell proliferation. Their
structures were confirmed by satisfactory spectra analyses mainly including 1H NMR, and ESI-MS
analyses. The preliminary assays indicated that some of the newly synthesized compounds displayed
significantly good inhibition activities against human lung cancer (NCI-H460), hepatocellular liver
carcinoma (HepG2), gastric cancer (SGC-7901 and BGC-823) cell lines compared with the control
5-Fluorouracil (5-FU), which might be developed as novel lead scaffold for potential anticancer agents.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 10 April 2012
Received in revised form
1 May 2012
Accepted 2 May 2012
Available online 12 May 2012
Keywords:
Gramine analogues
Synthesis
Inhibition activity
Cell proliferation
1. Introduction
kinds of nitrogen-containing heterocyclic compounds also play
crucial roles in medicinal chemistry and agrochemistry fields due to
Gramine, a natural indole alkaloid, has been isolated from
various raw plant and coal tar [1], and which exhibits wide phar-
maceutical activities similar to Ephedrine such as relaxation of
bronchial smooth muscle, vasorelaxation, blood pressure elevation,
relief of bronchitis nephritis and bronchial asthma [2]. Gramine
also plays important roles for the metabolite process of amino acid
in living organism. Up to now, gramine has been widely used as
a pharmaceutical lead scaffold for constructing various biologically
acitive indole-containing compounds [3] including heteroauxin, 5-
hydroxytryptamine (5-HT), and L-tryptophan. Recently, various
their extensive biological properties. Among them, indole skeleton
arouse many researchers’ interest [4], and many indole-type
analogues have been identified as potential antibacterial, anti-
cancer, antiviral agents and protein kinase inhibitors [5e10]. In
addition, some indole derivatives can also be used as important
synthons for further transformation to related fused-heterocycles
or polycyclic ring derivatives via appropriate reaction conditions
[11e18].
On the other hand, the discovery and identification of novel
highly effective anticancer agents with special characteristics than
the currently used are still an important endeavor in medicinal
chemistry [19e21], and so it was necessary to attempt neotype
chemical entities as potential chemotherapeutic agents with an
alternative mechanism of action.
* Corresponding author.
E-mail address: keshaoyong@163.com (S. Ke).
These two authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.003

S. Ke et al. / European Journal of Medicinal Chemistry 54 (2012) 248e254
249
Based on the aforementioned, the extensive bioactivity of gra-
mine mentioned above urged us to design and synthesize new
gramine analogues with simple and efficient method. As part of our
medicinal chemistry program aimed at the discovery of potential
anticancer agents, we wish to describe herein the convenient
synthesis, and biological evaluation of novel series of gramine
analogues. We utilized gramine scaffold as a prototype structural
unit and planned for the hybridization of the active heterocycles to
the core structure as shown in Fig. 1. Therefore, a series of gramine
analogues 4aeu were synthesized as shown in Scheme 1, and their
inhibition activities against various cancer cell lines (HepG2, SGC-
7901, BGC-823, NCI-H460) were also evaluated by MTT method.
little. Then we switched to alter the reaction temperature, and
investigate the various temperature effects on the conversion rate.
Among the reaction temperature tested, the temperature from 50
to 80 ꢀC gave the better results.
In order to explore the scope and limits of our solvent- and
catalyst-free formation process for diversity gramine analogues,
various substituted indoles 1aeb, aryl-aldehydes 2aeh, and het-
eroaryl amine or azole 3aed were selected and used as different
substrates to construct multi-substituted indole derivatives 4aeu,
the results are summarized in Table 1.
2.2. Spectroscopy studies
All the structures of target compounds 4aeu were confirmed by
their 1H NMR and ESI-MS spectra analyses. For 1H NMR studies,
assignments of the signals are based on the chemical shifts and
intensity patterns. Their 1H NMR spectra indicated distinctive
signals of methine proton attached to amine, which presented
a singlet or doublet at about 5.13e6.77 ppm. For some N-methyl-
ation derivatives such as compounds 4f, 4g, 4h, 4i, 4k, 4m, 4o and
4r, the signals that appeared in their 1H NMR spectra in the ranges
3.67e3.71 ppm were attributed to the N-methyl protons of indole
ring. The several set of signals at lower fields in the 1H NMR spectra
of compounds 4aeu were assigned to the aromatic protons as
shown in the representative structures in Scheme 1. All the char-
acteristic peaks observed within the 1H NMR spectra for title
compounds are given in Materials and methods.
2. Results and discussion
2.1. Chemistry
In the present study, a series of novel heterocycle-functional
gramine analogues were constructed by convenient solvent- and
catalyst-free method. The general synthetic method for the prep-
aration of gramine derivatives 4aeu is outlined in Scheme 1.
The easily available substituted indoles 1aeb, aryl-aldehydes
2aeh, and heteroaryl amine or azole 3aed were selected as start-
ing materials, which were conveniently transferred to the corre-
sponding gramine analogues 4aeu by three components
condensation reaction under solvent- and catalyst-free conditions.
All the prepared azaheterocyclic gramine derivatives 4aeu gave
satisfactory chemical analyses, and the chemical structures and
physiochemical properties of the synthesized compounds were
summarized in Table 1.
2.3. Bioactivity evaluation
To achieve the above goal for the novel gramine analogues, the
initial experiment was conducted by heating a mixture of indole,
benzaldehyde and 2-aminopyridine in the presence of ethanol at
about 75 ꢀC (Table 2, Entry 1). However, this reaction was very slow
and the yield for model compound 4a was also relatively low. Then
various catalysts and solvents were screened for optimization of the
reaction conditions, and the normal acid catalysts and some Lewis
acids were all selected and used in the presence or absence of
appropriate protic solvents and aprotic solvents. From Table 2, we
observed that the formation of the desired compound 4a catalysed
by Lewis acids in the presence of aprotic solvent (Table 2, Entries 10,
12, 14) were relatively easier than the other conditions, but the
yields were just moderate. Whereafter, considering that solvent-
free technique has been widely used in modern organic synthesis
to improve the reaction efficiency, so we further use this condition
as an alternative method to explore the model reaction (Table 2,
Entry 17). As we can see from the results, under solvent- and
catalyst-free conditions, the yield for model compound 4a was
obviously elevated, which are indeed superior to other protocols.
Thus, solvent- and catalyst-free reactions under conventional
heating were found to be the better choice for this reaction.
Furthermore, the reaction time and temperature were also
selected to optimize the reaction conditions. Firstly the reaction
temperature was invariableness, and the reaction time was varied
from 0.5 to 3 h, however, the yields for target compound changed
2.3.1. Inhibitory effects of compounds 4aeu on the proliferation of
various cancer cells
The newly prepared gramine analogues 4aeu were evaluated
for their in vitro cytotoxic effects against HepG2 (hepatocellular
liver carcinoma), SGC-7901 (gastric cancer), BGC-823 (gastric
cancer), and NCI-H460 (lung cancer) cell lines by the standard MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
assay [22] using 5-FU (5-Fluorouracil) as a positive control. The
preliminary results were summarized in Fig. 2 and Table 3. The IC50
value represents the drug concentration required to inhibit cell
growth by 50%.
Generally, as shown in Fig. 2, the prepared gramine analogues
(4aeu) displayed moderate to good inhibition activities against five
human cancer cell lines. Notably, the compounds 4b, 4c, 4d, 4e, 4j
and 4k exhibited significant inhibitory activities against all four
tested cell lines with 71.4e83.7% growth inhibition at 40 mg/mL
concentration compared to the positive control 5-FU (66.8e80.0%).
Also, it is interesting to note that compound 4g showed cytotoxic
selectivity for a special human hepatocellular liver carcinoma and
gastric cancer cell types, and the inhibition against HepG2 (73.6%)
and SGC-7901 (70.8%) cell was obviously higher than 5-FU
respectively. However, from the results presented in Fig. 2, we
observed that the parent compound Gramine exhibited almost no
activity against all tested cancer cell lines, and the results of
preliminary antitumor assay indicated these novel heterocycle-
R2
R4
N
One-Pot
N
N
R3
R1
R3
N
N
H
Solvent-Free
Catalyst-Free
HN
R4
R1
R2 CHO
Gramine
Fig. 1. Design and synthetic strategy for gramine analogues.

250
S. Ke et al. / European Journal of Medicinal Chemistry 54 (2012) 248e254
R2
R2
Het NH2
X
H
N
X
X
Solvent-free
Catalyst-free
or
N
R2
CHO
or
X
N
N
Het
X
R1
N
N
H
N
N
R1
R1
1a-b
2a-h
3a-d
4a-u
2a R2 = H, X = C
2b R2 = 2-Cl, X = C
2c R2 = 4-Cl, X = C
2d R2 = 2-F, X = C
2e R2 = 4-F, X = C
2f R2 = 4-CF3, X = C
2g Picolinaldehyde
2h Isonicotinaldehyde
2-Aminopyridine
3a
R
R
1 = H
Benzo[d]thiazol-2-amine
1H-Pyrazole, X = C
1a
1b
3b
3c
3d
1 = Me
1H-1,2,4-Triazole, X = N
Scheme 1. The convenient synthesis of gramine analogues 4aeu.
functional Gramine analogues might be used as a high potential
active scaffold for optimization of anticancer agents.
Furthermore, the doseeresponse analysis of cell growth inhi-
bition activity for representative compounds 4q, 4t, 4u, and 5-FU
has been displayed in Fig. 3, which indicated that the cytotoxic
effects on cell lines of target compounds showed obvious
concentration-dependent manner. Especially, compound 4t
exhibited high potent growth inhibitory activities against HepG2,
SGC-7901, BGC-823 and NCI-H460 cell lines with the IC50 values of
In addition, the preliminary bioassay indicated most of the
target compounds (such as 4b, 4c, 4d, 4e, 4g, 4i, 4j, 4k, 4p, 4t and
4u) displayed good inhibitory activities compared to 5-FU, so in
order to further investigate the potential activities, the IC50 values
were evaluated and compared to those control compounds. The
inhibitory activities expressed as IC50 values for the target
compounds are presented in Table 3. The results further testify that
some of the designed Gramine analogues 4aeu exhibited higher
inhibition activity than the commercial 5-FU under the same
conditions. As indicated in Table 3, compounds 4p, 4t and 4u
showed the strongest inhibitory effect against HepG2, with an IC50
8.0 ꢂ 1.7, 8.5 ꢂ 2.6, 5.7 ꢂ 1.7 and 8.5 ꢂ 3.1
m
g mLꢁ1 (Table 3),
respectively, which was equivalent to or better than the control
compound 5-FU.
2.3.2. Effects of the compounds on the morphology of cancer cells
During the cytotoxicity experiments, significant morphological
changes of the cells have been observed under an inverted micro-
scope. So we present the selective morphological image of the cells
values of 9.0, 8.0 and 8.4
compounds 4t and 4u have the same inhibition activities as 5-FU,
and the IC50 values are 8.5, 8.2 and 8.7
g mLꢁ1 respectively.
m
g mLꢁ1, respectively. We also can find that
m
treated with compounds 4b, 4j, 4p and 4t at 20 mg/mL for 48 h in
Especially, compound 4t exhibited significant inhibition (Entry 20)
against all tested cancer cell lines compared to the positive control
5-FU.
the following pictures (Fig. 4). Contrast with the presented images
Table 2
Various catalysts and solvents effects on the reaction.a
Table 1
Chemical structure of synthesized gramine analogues (4aeu).
CHO
H
N
Entry Compd. Substrates
no.
Appearance
mp (ꢀC) Yielda
(%)
One-Pot
NH2
N
Indole Aldehyde Amine
N
H
N
1
2
3
4
4a
4b
4c
4d
1a
1a
1a
1a
2a
2b
2c
2d
3a
3a
3a
3a
White powder 143e145 92
White powder 184e186 91
White powder 126e127 86
Yellowish
powder
N
H
1a
2a
3a
4a
138e140 89
5
6
7
8
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
1a
1b
1b
1b
1b
1a
1b
1a
1b
1a
1b
1a
2e
2b
2c
2d
2e
2f
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3b
White powder 149e150 93
White powder 166e167 82
White powder 151e152 85
White powder 175e176 78
White powder 137e139 75
White powder 143e145 86
White powder 131e132 80
White powder 165e166 72
White powder 170e172 77
White powder 220e221 83
White powder 192e193 74
Entry
Catalyst
Solvent
Yieldb (%)
1
2
3
4
5
6
7
8
None
EtOH
EtOH
THF
Acetone
EtOH
Acetone
EtOH
Acetone
EtOH
Acetone
EtOH
Acetone
EtOH
28
Trace
52
46
21
31
25
Trace
10
71
39.6
71
26
73
59
MeSO3H
MeSO3H
MeSO3H
HCl
9
10
11
12
13
14
15
16
2f
2g
2g
2h
2h
2b
HCl
p-TSA
p-TSA
Zn(NO3)2
Zn(NO3)2
SnCl2
SnCl2
FeCl3
FeCl3
9
Yellowish
powder
160e161 84
10
11
12
13
14
15
16
17c
17
18
19
20
4q
4r
4s
4t
1a
1b
1a
1a
2d
2d
2g
2e
3b
3b
3b
3c
White powder 131e133 76
White powder 194e196 83
White powder 154e156 71
Acetone
EtOH
Acetone
None
Pale brown
powder
97e98 74
L
-Proline
-Proline
L
Trace
92
21
4u
1a
2e
3d
Pale brown
powder
100e101 78
None
a
Reaction conditions: conventional heating, 10% mol catalyst, 75 ꢀC, 2 h.
Isolated yields.
Solvent- and catalyst-free reaction under conventional heating.
22
Gramine 1a
(HCHO)n NHMe2 White powder 124e126 80
b
c
a
Isolated yield.

S. Ke et al. / European Journal of Medicinal Chemistry 54 (2012) 248e254
251
Fig. 2. Inhibition activities against cell proliferation for compounds 4aeu at 40 mg/mL. Abbreviations: HepG2 e Human hepatocellular liver carcinoma cell line; SGC-7901 e Human
gastric cancer cell line; BGC-823 e Human gastric cancer cell line; NCI-H460 e Human large cell lung cancer cell line; Gramine e The parent compound used as a lead compound;
5-FU e 5-Fluorouracil, used as a positive control.
in Fig. 4, we found that the quantities of the cells treated with
compounds 4b, 4j, 4p and 4t were obviously decreased greatly for
the selective kinds of cells including SGC-7901 and NCI-H460 cell
lines. On the other hand, significant volume shrinkage and
changing to globular of some SGC-7901 cells have been observed. In
the cases of NCI-H460 cells, there were also similar morphological
changes such as volume shrink, changing to globular and fragment.
However, such morphological changes were not apparent in the
control cells (A and G).
been conveniently synthesized, and characterized by 1H NMR, and
MS spectra analyses. The preliminary bioassay results indicated
that some of the compounds displayed obviously good inhibition
activities against human lung cancer (NCI-H460), hepatocellular
liver carcinoma (HepG2), gastric cancer (SGC-7901 and BGC-823)
cell lines compared to 5-FU, which might be developed as novel
lead scaffold for potential anticancer agents. Further structural
optimization and activity profiles about the designed novel
heterocycle-functional gramine analogues are well ongoing in our
laboratory.
3. Conclusion
4. Materials and methods
In the present study, we have described the convenient solvent-
and catalyst-free synthesis and biological evaluation of a series of
gramine analogues. Twenty-one novel gramine derivatives have
4.1. Instrumentation and chemicals
All melting points (m.p.) were obtained with a digital model X-5
apparatus and are uncorrected. 1H NMR spectra were recorded on
a Brucker spectrometer at 400 MHz with CDCl3 as the solvent and
TMS as the internal standard. Chemical shifts are reported in
Table 3
Cytotoxic activity of the compounds against human tumor cells.
g mLꢁ1
)
a
Entry
Compd. no.
In vitro cytotoxicity IC50
(
m
d
(parts per million) values. Coupling constants nJ are reported in
HepG2b
SGC-7901b
BGC-823b
NCI-H460b
Hz. Standard abbreviations indicating multiplicity are used as
follows: s ¼ singlet, d ¼ doublet, dd ¼ doublet of doublets,
t ¼ triplet, q ¼ quadruplet, m ¼ multiplet and br ¼ broad. Mass
spectra were performed on a MicroMass Quattro microÔ API
instrument. Analytical thin-layer chromatography (TLC) was
carried out on precoated plates, and spots were visualized with
ultraviolet light. All chemicals or reagents used for syntheses were
commercially available, were of AR grade, and were used as
received. Anhydrous CH2Cl2 and CH3CN were dried according to
standard methods [23]. All other solvents and reagents were
analytical reagent and used directly without purification.
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
27.6 ꢂ 1.7
13.1 ꢂ 1.3
18.6 ꢂ 4.1
28.7 ꢂ 2.3
23.0 ꢂ 4.5
26.5 ꢂ 4.7
25.5 ꢂ 4.5
43.2 ꢂ 2.0
38.6 ꢂ 4.9
12.1 ꢂ 3.8
12.9 ꢂ 3.3
>100
>100
>100
42.5 ꢂ 3.6
9.0 ꢂ 2.3
7.0 ꢂ 1.6
>100
24.9 ꢂ 2.1
14.9 ꢂ 3.0
19.2 ꢂ 4.8
24.5 ꢂ 4.7
21.5 ꢂ 3.8
27.1 ꢂ 3.0
25.0 ꢂ 2.9
86.2 ꢂ 5.2
51.9 ꢂ 3.3
13.5 ꢂ 1.0
13.8 ꢂ 1.0
51.4 ꢂ 3.8
>100
29.4 ꢂ 3.2
22.7 ꢂ 1.9
22.8 ꢂ 1.9
37.6 ꢂ 0.9
21.8 ꢂ 2.3
39.2 ꢂ 5.2
30.8 ꢂ 6.3
64.2 ꢂ 5.1
51.6 ꢂ 5.9
10.1 ꢂ 1.3
16.8 ꢂ 1.2
46.9 ꢂ 1.0
>100
35.3 ꢂ 2.9
12.2 ꢂ 2.6
17.4 ꢂ 2.3
24.9 ꢂ 1.4
24.0 ꢂ 4.1
66.7 ꢂ 5.5
32.1 ꢂ 6.2
92.6 ꢂ 5.9
49.1 ꢂ 7.0
8.6 ꢂ 2.7
16.0 ꢂ 1.8
>100
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
>100
>100
>100
>100
37.9 ꢂ 3.9
10.5 ꢂ 0.7
9.4 ꢂ 2.9
>100
76.9 ꢂ 2.6
8.5 ꢂ 2.6
8.2 ꢂ 2.4
77.3 ꢂ 5.0
8.7 ꢂ 3.0
47.1 ꢂ 2.9
8.1 ꢂ 3.0
8.1 ꢂ 3.3
>100
74.9 ꢂ 0.1
5.7 ꢂ 1.7
10.3 ꢂ 2.7
62.8 ꢂ 0.4
8.2 ꢂ 3.1
56.0 ꢂ 2.5
13.2 ꢂ 5.6
12.1 ꢂ 5.2
>100
77.3 ꢂ 1.3
8.5 ꢂ 3.1
10.7 ꢂ 4.3
74.5 ꢂ 0.6
7.2 ꢂ 2.2
4.2. General synthetic procedure for gramine and its analogues
4aeu
4r
4s
4t
>100
The typical and optimum process for preparation of gramine
analogues 4aeu is shown as following: Appropriate substituted
aromatic aldehyde (5 mmol) and heteroaryl amine or azole
(5 mmol) were placed in a dried round-bottomed flask containing
a magnetic stirrer bar and stirred at about 75 ꢀC for 20e40 min.
Then indole or N-methylindole (5 mmol) was added in batches,
and the mixture was heated and detected by thin-layer chroma-
tography (TLC). After completion, the reaction mixture was cooled
to ambient temperature and a small quantity of EtOH was added.
The solution was poured into ice-water, and the precipitate formed
was filtered, washed with appropriate ice-ethanol and water. The
8.0 ꢂ 1.7
8.4 ꢂ 2.3
>100
9.3 ꢂ 2.7
4u
Graminec
5-FUd
The high activities data are represented in bold.
a
IC50 e Compound concentration required to inhibit tumor cell proliferation by
50%.
b
Abbreviations: HepG2 e Human hepatocellular liver carcinoma cell line; SGC-
7901 e Human gastric cancer cell line; BGC-823 e Human gastric cancer cell line;
NCI-H460 e Human large cell lung cancer cell line.
c
The parent compound used as a lead compound.
5-Fluorouracil, used as a positive control.
d

252
S. Ke et al. / European Journal of Medicinal Chemistry 54 (2012) 248e254
HepG2 Cell Line
BGC-823 Cell Line
90
80
70
60
50
40
30
20
10
0
90
80
70
60
50
40
30
20
10
0
4q
4q
4t
4t
4u
4u
5-FU
5-FU
1
5
10
20
40
80
1
5
10
20
40
80
Concentration (µg/mL)
Concentration (µg/mL)
Fig. 3. Doseeresponse analysis of cell growth inhibition activity for representative compounds 4q, 4t, 4u and 5-FU (positive control) against HepG2 (left) and BGC-823 (right) cell
lines.
crude products were purified by silica gel column-chromatography
(ethyl acetate/petroleum ether) or recrystallization to give target
compounds 4aeu as white solid or crystal. Based on the afore-
mentioned method, the gramine was also easily prepared by
reaction of indole, paraformaldehyde and dimethylamine. All the
compounds were characterized by ESI-MS, 1H NMR spectroscopic
data. Their physico-chemical properties and the spectra data are as
follows:
4.2.4. N-((2-fluorophenyl)(1H-indol-3-yl)methyl)pyridin-2-amine 4d
This compound was obtained following the above method as
yellowish powder, yield 89%, mp 138e140 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.30 (s, 1H), 8.09 (s, 1H), 7.65e7.33 (m, 5H), 7.23e7.02
(m, 4H), 6.84 (s, 1H), 6.60 (t, 1H), 6.45e6.35 (m, 2H), 5.46 (s, 1H);
MS (ESI) m/z 317.7 (M)þ, calcd. for C20H16FN3 m/z ¼ 317.1.
4.2.5. N-((4-fluorophenyl)(1H-indol-3-yl)methyl)pyridin-2-amine 4e
This compound was obtained following the above method as
white powder, yield 93%, mp 149e150 ꢀC; 1H NMR (400 MHz,
4.2.1. N-((1H-indol-3-yl)(phenyl)methyl)pyridin-2-amine 4a
This compound was obtained following the above method as
white powder, yield 92%, mp 143e145 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.23 (s, 1H), 8.10 (d, 4J ¼ 4 Hz, 1H), 7.52e7.35 (m, 5H),
7.21 (t, J ¼ 8 Hz, 1H), 7.10e6.95 (m, 3H), 6.78 (d, 4J ¼ 4 Hz, 1H), 6.60
(t, 1H), 6.34 (d, J ¼ 8 Hz, 1H), 6.14 (d, J ¼ 8 Hz, 1H), 5.18 (d, J ¼ 8 Hz,
1H); MS (ESI) m/z 224.1 (M ꢁ NH ꢁ Py)þ, calcd. for C20H16FN3 m/
z ¼ 317.1.
CDCl3):
d
¼ 8.18 (s, 1H), 8.10 (d, 4J ¼ 4 Hz, 1H), 7.55 (d, J ¼ 8 Hz, 1H),
7.48 (d, J ¼ 8 Hz, 2H), 7.38e7.28 (m, 5H), 7.20 (t, J ¼ 8 Hz,1H), 7.08 (t,
J ¼ 8 Hz, 1H), 6.79 (s, 1H), 6.58 (t, J ¼ 6 Hz, 1H), 6.35 (d, J ¼ 8 Hz, 1H),
6.13 (d, 4J ¼ 4 Hz, 1H), 5.23 (d, 4J ¼ 4 Hz, 1H); MS (ESI) m/z 321.9
(M þ Na)þ, calcd. for C20H17
N
3 m/z ¼ 299.1.
4.2.6. N-((2-chlorophenyl)(1-methyl-1H-indol-3-yl)methyl)pyridin-
2-amine 4f
4.2.2. N-((2-chlorophenyl)(1H-indol-3-yl)methyl)pyridin-2-amine 4b
This compound was obtained following the above method as
white powder, yield 88%, mp 184e186 ꢀC; 1H NMR (400 MHz,
This compound was obtained following the above method as
white powder, yield 82%, mp 166e167 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.10 (d, 4J ¼ 4 Hz, 1H), 7.64 (q, 4J ¼ 4 Hz, J ¼ 8 Hz, 2H),
CDCl3):
d
¼ 8.11 (d, 4J ¼ 2 Hz, 2H), 7.64 (d, J ¼ 8 Hz, 3H), 7.39 (t,
7.42e7.23 (m, 6H), 7.10 (t, J ¼ 8 Hz, 1H), 6.60 (t, J ¼ 8 Hz, 1H), 6.56 (s,
1H), 6.43 (d, 4J ¼ 4 Hz, 1H), 6.29 (d, J ¼ 8 Hz, 1H), 5.27 (d, J ¼ 8 Hz,
1H), 3.70 (s, 3H); MS (ESI) m/z 346.5 (M)þ, calcd. for C21H18ClN3 m/
z ¼ 347.1.
J ¼ 8 Hz, 5H), 7.11 (t, J ¼ 8 Hz, 1H), 6.75 (s, 1H), 6.60 (t, J ¼ 8 Hz, 1H),
6.45 (d, J ¼ 8 Hz, 1H), 6.31 (d, J ¼ 8 Hz, 1H), 5.29 (s, 1H); MS (ESI) m/z
332.6 (M)þ, calcd. for C20H16ClN3 m/z ¼ 333.1.
4.2.3. N-((4-chlorophenyl)(1H-indol-3-yl)methyl)pyridin-2-amine
4c
4.2.7. N-((4-chlorophenyl)(1-methyl-1H-indol-3-yl)methyl)pyridin-
2-amine 4g
This compound was obtained following the above method as
This compound was obtained following the above method as
white powder, yield 86%, mp 126e127 ꢀC; 1H NMR (400 MHz,
white powder, yield 85%, mp 151e152 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.21 (s, 1H), 8.10 (d, 4J ¼ 4 Hz, 1H), 7.53 (d, J ¼ 8 Hz, 1H),
CDCl3):
d
¼ 8.10 (d, 4J ¼ 4 Hz, 1H), 7.53 (d, J ¼ 8 Hz, 1H), 7.45e7.22
7.42e7.28 (m, 5H), 7.21 (t, J ¼ 8 Hz, 1H), 7.09 (t, J ¼ 8 Hz, 1H), 6.78 (d,
4J ¼ 4 Hz, 1H), 6.60 (t, J ¼ 8 Hz, 1H), 6.33 (d, J ¼ 8 Hz, 1H), 6.15 (d,
4J ¼ 4 Hz, 1H), 5.18 (d, J ¼ 8 Hz, 1H); MS (ESI) m/z 332.5 (M)þ, calcd.
for C20H16ClN3 m/z ¼ 333.1.
(m, 7H), 7.09 (t, J ¼ 8 Hz, 1H), 6.61 (s, 1H), 6.59 (d, J ¼ 8 Hz, 1H), 6.32
(d, J ¼ 8 Hz, 1H), 6.14 (d, 4J ¼ 4 Hz, 1H), 5.15 (d, J ¼ 8 Hz, 1H), 3.71 (s,
3H); MS (ESI) m/z 254.4 (M ꢁ NH ꢁ Py)þ, calcd. for C21H18ClN3 m/
z ¼ 347.1.
Fig. 4. Morphology image of SGC-7901 (AeF) and NCI-H460 (GeL) cells treated with the compounds 4b, 4j, 4p, 4t and 5-FU for 48 h (200ꢃ). A and G are the control, the cells treated
with DMSO 0.1% (v/v) as a vehicle control. B and H, C and I, D and J, E and K, F and L are treated with the compounds 4b, 4j, 4p, 4t and 5-FU at concentration of 20 g/mL respectively.
m

S. Ke et al. / European Journal of Medicinal Chemistry 54 (2012) 248e254
253
4.2.8. N-((2-fluorophenyl)(1-methyl-1H-indol-3-yl)methyl)pyridin-
2-amine 4h
7H), 7.09 (t, J ¼ 8 Hz, 1H), 6.64e6.60 (m, 2H), 6.35e6.22 (m, 1H),
5.29 (d, J ¼ 8 Hz, 1H), 3.71 (s, 3H); MS (ESI) m/z 315.5 (M þ 1)þ,
calcd. for C20H18N4 m/z ¼ 314.2.
This compound was obtained following the above method as
white powder, yield 78%, mp 175e176 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.09 (d, 4J ¼ 4 Hz, 1H), 7.61e7.50 (m, 2H), 7.42e7.02 (m,
4.2.16. N-((2-chlorophenyl)(1H-indol-3-yl)methyl)benzo[d]thiazol-
2-amine 4p
7H), 6.68 (s, 1H), 6.59 (t, J ¼ 8 Hz, 1H), 6.43 (d, 4J ¼ 4 Hz, 1H), 6.36 (d,
J ¼ 8 Hz, 1H), 5.22 (d, J ¼ 8 Hz, 1H), 3.71 (s, 3H); MS (ESI) m/z 238.5
(M ꢁ NH ꢁ Py)þ, calcd. for C21H18FN3 m/z ¼ 331.1.
This compound was obtained following the above method as
yellowish powder, yield 84%, mp 160e161 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 9.51 (bs, 1H), 8.39 (d, J ¼ 8 Hz, 1H), 8.19 (s, 1H), 8.02 (d,
4.2.9. N-((4-fluorophenyl)(1-methyl-1H-indol-3-yl)methyl)pyridin-
2-amine 4i
J ¼ 8 Hz, 1H), 7.86 (d, J ¼ 8 Hz, 1H), 7.67e7.37 (m, 6H), 7.13e7.07 (m,
2H), 6.72 (s, 1H), 6.54 (s, 1H), 5.31 (s, 1H); MS (ESI) m/z 390.2
(M þ 1)þ, calcd. for C22H16ClN3S m/z ¼ 389.1.
This compound was obtained following the above method as
white powder, yield 75%, mp 137e139 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.09 (d, 4J ¼ 4 Hz, 1H), 7.55e7.20 (m, 6H), 7.12e6.95 (m,
4.2.17. N-((2-fluorophenyl)(1H-indol-3-yl)methyl)benzo[d]thiazol-
2-amine 4q
3H), 6.63 (s, 1H), 6.59 (t, J ¼ 8 Hz, 1H), 6.33 (d, J ¼ 8 Hz, 1H), 6.13 (d,
4J ¼ 4 Hz, 1H), 5.16 (d, J ¼ 8 Hz, 1H), 3.71 (s, 3H); MS (ESI) m/z 238.5
(M ꢁ NH ꢁ Py)þ, calcd. for C21H18FN3 m/z ¼ 331.1.
This compound was obtained following the above method as
white powder, yield 76%, mp 131e133 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.16 (bs, 1H), 7.98 (bs, 1H), 7.67e7.50 (m, 8H), 7.20e7.10
4.2.10. N-((1H-indol-3-yl)(4-(trifluoromethyl)phenyl)methyl)pyridin-
2-amine 4j
(m, 5H), 6.42 (s, 1H); MS (ESI) m/z 397.2 (M þ Na)þ, calcd. for
C22H16FN3S m/z ¼ 373.1.
This compound was obtained following the above method as
white powder, yield 86%, mp 143e145 ꢀC; 1H NMR (400 MHz,
4.2.18. N-((2-fluorophenyl)(1-methyl-1H-indol-3-yl)methyl)benzo
[d]thiazol-2-amine 4r
CDCl3):
d
¼ 8.26 (s, 1H), 8.10 (s, 1H), 7.60e7.53 (m, 5H), 7.41e7.35
(m, 2H), 7.25e7.22 (m, 1H), 7.10 (d, J ¼ 8 Hz, 1H), 6.75 (s, 1H), 6.61
(t, J ¼ 8 Hz, 1H), 6.34 (d, J ¼ 8 Hz, 1H), 6.27 (d, 4J ¼ 4 Hz, 1H), 5.20 (s,
1H); MS (ESI) m/z 366.5 (M)þ, calcd. for C21H16F3N3 m/z ¼ 367.1.
This compound was obtained following the above method as
white powder, yield 83%, mp 194e196 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 7.63e7.52 (m, 3H), 7.40e7.19 (m, 6H), 7.16e7.02 (m, 4H),
6.62 (s, 1H), 6.47 (s, 1H), 3.67 (s, 3H); MS (ESI) m/z 386.9 (M)þ, calcd.
for C23H18FN3S m/z ¼ 387.1.
4.2.11. N-((1-methyl-1H-indol-3-yl)(4-(trifluoromethyl)phenyl)
methyl)pyridin-2-amine 4k
This compound was obtained following the above method as
4.2.19. N-((1H-indol-3-yl)(pyridin-2-yl)methyl)benzo[d]thiazol-2-
amine 4s
white powder, yield 80%, mp 131e132 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.10 (d, 4J ¼ 4 Hz, 1H), 7.63e7.54 (m, 5H), 7.38e7.31 (m,
This compound was obtained following the above method as
3H), 7.11 (t, J ¼ 8 Hz, 1H), 6.60 (t, J ¼ 8 Hz, 2H), 6.32 (d, J ¼ 8 Hz, 1H),
6.26 (d, 4J ¼ 4 Hz, 1H), 5.18 (d, 4J ¼ 4 Hz, 1H), 3.71 (s, 3H); MS (ESI)
m/z 380.7 (M)þ, calcd. for C22H18F3N3 m/z ¼ 381.1.
white powder, yield 71%, mp 154e156 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.55 (s, 1H), 7.73 (s, 2H), 7.60e7.48 (m, 5H), 7.28 (d,
J ¼ 12 Hz, 5H), 7.12 (t, J ¼ 8 Hz, 2H), 6.77 (s, 1H); MS (ESI) m/z 379.8
(M þ Na)þ, calcd. for C21H16N4S m/z ¼ 356.1.
4.2.12. N-((1H-indol-3-yl)(pyridin-2-yl)methyl)pyridin-2-amine 4l
This compound was obtained following the above method as
white powder, yield 72%, mp 165e166 ꢀC; 1H NMR (400 MHz,
4.2.20. 3-((4-fluorophenyl)(1H-pyrazol-1-yl)methyl)-1H-indole 4t
This compound was obtained following the above method as
pale brown powder, yield 74%, mp 97e98 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.59 (d, 4J ¼ 4 Hz, 1H), 8.47 (s, 1H), 8.10 (d, 4J ¼ 4 Hz, 1H),
7.64 (d, J ¼ 8 Hz, 2H), 7.50 (d, J ¼ 8 Hz, 1H), 7.34e7.30 (m, 2H),
7.17e7.06 (m, 3H), 6.95 (s, 1H), 6.56 (t, J ¼ 8 Hz,1H), 6.41 (d, J ¼ 8 Hz,
1H), 6.31 (d, J ¼ 4 Hz, 1H), 5.91 (d, J ¼ 4 Hz, 1H); MS (ESI) m/z 207.4
(M ꢁ NH ꢁ Py)þ, calcd. for C19H16N4 m/z ¼ 300.1.
CDCl3):
d
¼ 7.94 (s, 2H), 7.37 (d, J ¼ 8 Hz, 4H), 7.18 (t, J ¼ 8 Hz, 2H),
7.03e6.94 (m, 3H), 6.65 (s, 2H), 5.88 (s, 1H); MS (ESI) m/z 339.5
(M þ 2Na þ 1)þ, calcd. for C18H14FN3 m/z ¼ 291.1.
4.2.21. 3-((4-fluorophenyl)(1H-1,2,4-triazol-1-yl)methyl)-1H-
indole 4u
4.2.13. N-((1-methyl-1H-indol-3-yl)(pyridin-2-yl)methyl)pyridin-
2-amine 4m
This compound was obtained following the above method as
This compound was obtained following the above method as
pale brown powder, yield 78%, mp 100e101 ꢀC; 1H NMR (400 MHz,
white powder, yield 77%, mp 170e172 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 7.95 (s, 2H), 7.37 (d, J ¼ 8 Hz, 2H), 7.19 (d, J ¼ 8 Hz, 2H),
CDCl3):
d
¼ 8.61 (d, 4J ¼ 4 Hz,1H), 7.58 (t, J ¼ 8 Hz,1H), 7.40e7.12 (m,
7.03e6.96 (m, 4H), 6.65 (s, 2H), 5.88 (s, 1H); MS (ESI) m/z 339.5
(M þ 2Na)þ, calcd. for C17H13FN4 m/z ¼ 292.1.
9H), 7.00 (t, J ¼ 8 Hz, 2H), 6.68 (s, 1H), 6.04 (s, 1H), 3.68 (s, 3H); MS
(ESI) m/z 221.5 (M ꢁ NH ꢁ Py)þ, calcd. for C20H18N4 m/z ¼ 314.2.
4.3. In vitro cytotoxicity assays
4.2.14. N-((1H-indol-3-yl)(pyridin-4-yl)methyl)pyridin-2-amine 4n
This compound was obtained following the above method as
white powder, yield 83%, mp 220e221 ꢀC; 1H NMR (400 MHz,
The in vitro cytotoxicity of the synthesized compounds against
different human cancer cell lines was measured with the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay [22]. Briefly, HepG2 (hepatocellular liver carcinoma), SGC-
7901 (gastric cancer), BGC-823 (gastric cancer), and NCI-H460
(lung cancer) cells were seeded at 2 ꢃ 104 cells per well in 96-
well plates and grown to subconfluence. After removal of the
growth medium, six serial dilutions of each tested compound in
CDCl3):
1H), 6.65 (d, 1H), 6.40e6.25 (m, 2H), 5.13 (s, 1H); MS (ESI) m/z 300.5
(M)þ, calcd. for C19H16
4 m/z ¼ 300.1.
d
¼ 8.57 (s, 3H), 8.11 (s, 2H), 7.44 (d, 3H), 7.15 (s, 2H), 6.82 (s,
N
4.2.15. N-((1-methyl-1H-indol-3-yl)(pyridin-4-yl)methyl)pyridin-
2-amine 4o
This compound was obtained following the above method as
200
a humidified atmosphere containing 5% CO2. After 72 h of exposure,
the culture medium was removed and 30 L of the MTT solution
m
L test medium were added. Plates were incubated at 37 ꢀC in
white powder, yield 74%, mp 192e193 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 8.55 (d, J ¼ 4 Hz, 2H), 8.08 (d, J ¼ 4 Hz,1H), 7.54e7.31 (m,
m

254
S. Ke et al. / European Journal of Medicinal Chemistry 54 (2012) 248e254
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(5 mg/mL in PBS) was added to each well. The plate was further
incubated for 4 h to allow MTT formazan formation. To dissolve the
resulting MTT formazan, 50 mL of DMSO was added to each well,
followed by thorough mixing with a microplate shaker. Absorbance
at 570 nm was measured on a microplate reader (Thermo Scientific,
MK3). All the data of the experiment were analyzed with SPSS
software, and the 50% inhibitory concentrations (IC50) of each
compound for the different cell lines were determined. All assays
were performed in triplicate on three independent experiments,
and measurement data were expressed as the mean ꢂ S.D.
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We gratefully acknowledge the support of this work by the
Projects in the Youth Science Foundation of Hubei Academy of
Agricultural Sciences (2011NKYJJ19) and the National Natural
Science Foundation of China (31000867). The authors also grate-
fully acknowledge the partial support from Hubei Biopesticide
Engineering Research Centre.
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