Novel N-substituted 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamide derivatives: Synthesis and biological evaluation

Article abstract of DOI:10.3390/molecules21050530

In this study, a series of novel N-substituted 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamide derivatives were synthesized, and evaluated for their cytotoxicity in human cell lines including Hela (cervical cancer), MCF7 (breast cancer ) and HepG2 (liver cancer). Several compounds were found to have potent anti-proliferative activity against those human cancer cell lines and compound 5r showed the most potent biological activity against HepG2 cells with an IC₅₀ value of 10.56 ± 1.14 μM. In addition, bioassays showed that compound 5r induced time-dependent and dose-dependent cleavage of poly ADP-ribose polymerase (PARP), and also induced a dose-dependent increase in caspase-3 and caspase-8 activity, but had little effect on caspase-9 protease activity in HepG2 cells. These results provide evidence that 5r-induced apoptosis in HepG2 cell is caspase-8-dependent.

Full text of DOI:10.3390/molecules21050530

molecules
Article
Novel N-Substituted 2-(2-(Adamantan-1-yl)-1H-Indol-
-yl)-2-Oxoacetamide Derivatives: Synthesis and
Biological Evaluation
3
†
†
Hong-Yu Hu , Xu-Dong Yu , Fei Wang, Chun-Rong Lin, Jin-Zhang Zeng, Ying-Kun Qiu,
Mei-Juan Fang * and Zhen Wu *
School of Pharmaceutical Sciences and the Key Laboratory for Chemical Biology of Fujian Province,
Xiamen University, South Xiang-An Road, Xiamen 361102, China; huhongyu22@126.com (H.-Y.H.);
yuxudong91@163.com (X.-D.Y.); tjdxwangfei@163.com (F.W.); reyiai@163.com (C.-R.L.);
jzzeng@xmu.edu.cn (J.-Z.Z.); qyk@xmu.edu.cn (Y.-K.Q.)
*
Correspondence: fangmj@xmu.edu.cn (M.-J.F.); wuzhen@xmu.edu.cn (Z.W.);
Tel./Fax: +86-592-218-9868 (M.-J.F. & Z.W.)
†
These authors contributed equally to this work.
Academic Editor: Jean Jacques Vanden Eynde
Received: 7 March 2016; Accepted: 16 April 2016; Published: 5 May 2016
Abstract: In this study, a series of novel N-substituted 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-
oxoacetamide derivatives were synthesized, and evaluated for their cytotoxicity in human cell
lines including Hela (cervical cancer), MCF7 (breast cancer ) and HepG2 (liver cancer). Several
compounds were found to have potent anti-proliferative activity against those human cancer
cell lines and compound 5r showed the most potent biological activity against HepG2 cells with
an IC50 value of 10.56
˘
1.14 µM. In addition, bioassays showed that compound 5r induced
time-dependent and dose-dependent cleavage of poly ADP-ribose polymerase (PARP), and also
induced a dose-dependent increase in caspase-3 and caspase-8 activity, but had little effect on
caspase-9 protease activity in HepG2 cells. These results provide evidence that 5r-induced apoptosis
in HepG2 cell is caspase-8-dependent.
Keywords: 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamidederivatives; poly ADP-ribose
polymerase (PARP); caspase-8; apoptosis
1
. Introduction
Despite continued research efforts, cancer remains one of the biggest threats to human health,
and it was estimated to be responsible for 15% of all global deaths in 2010 [1]. Current treatment for
tumors generally involves surgical resection (if possible), followed by radiotherapy and chemotherapy,
with the most common chemotherapies, according to Cancer Research UK, being temozolomide,
procarbazine, carmustine, lomustine and vincristine [
sufficient for long-term treatment because of the rapid chemoresistance developed by many cancers [
Multidrug resistance (MDR) is a major obstacle to successful cancer treatment [ ]. This has driven the
2
–5]. However these drugs alone simply aren’t
6
].
7,8
development of a variety of new anticancer agents with more potent, high specific and low cytotoxic
properties [9].
The indole moiety has been described as a privileged structure as it appears extensively in many
unrelated areas of biology and medicine, and depending on the substituents, can have a diverse
range of effects [10]. The indole ring system as one of the most ubiquitous heterocycles in Nature,
and has been becoming an important structural component in many pharmaceutical agents, such
as antidepressant [11], anticonvulsant [12], antifungal [13], antiviral [14] and anti-inflammatory [15],
and particularly new antitumor agents [16
–19]. Indole-3-glyoxylamide compounds, as a new class of
Molecules 2016, 21, 530; doi:10.3390/molecules21050530
www.mdpi.com/journal/molecules

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indole derivatives which have anti-tumor [20–22], anti-viral [23], anti-bacterial [24
,25], anti-HIV [26],
adenosine regulating receptor function [27] and other biological activities, have become one of the
most important chemical entities in the field of pharmaceutical research. Thus, we synthesized a novel
class of N-substituted 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamide derivatives and evaluated
their in vitro anti-proliferative activity against human breast (MCF7), cervical (Hela) and liver (HepG2)
cancer cells. To explain the molecular mechanisms mediating the induction of apoptosis by this class
of compounds, the activation of caspase-3, caspase-8 and caspase-9 was further examined using a
caspase activity assay kit in HepG2 cells after treatment with compound 5r.
2
. Results and Discussion
2
.1. Chemistry
The preparation of the N-substituted 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamide
derivatives 5a
). The general chemistry used in the synthesis of
methods [28 29]. Briefly, treatment of adamantane-1-carbonyl chloride (
N-o-tolylcycloadamantanecarboxamide ( ), followed by dropwise addition of 1.4 or 1.6 M n-BuLi in
hexane to get the compound . Then the intermediate 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetyl
chloride ( ) was easily formed by the reaction of and oxalyl chloride in anhydrous diethyl ether.
Finally, the reaction of compound with various substituted amines in toluene at 60 C for 4 h afforded
–
y
is outlined in Scheme 1. First, we synthesized 2-adamantane-1H-indol-5-amine
was adapted from reported previously
) with o-Toluidine resulted in
(3
3
,
1
2
3
4
3
˝
4
the title compounds 5a
data (HRMS, H-NMR and C-NMR).
–
y
. The structures of all the target compounds were confirmed by their spectral
1
13
Scheme 1. Synthesis of N-substituted 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamide. Reagents
˝
and conditions: (a) toluene, o-toluidine, K CO , r.t., 4 h; (b) n-BuLi, THF, 0 C, N , 3 h; (c) ether, oxalyl
2
3
2
˝
chloride, 0 C, 2 h; (d) toluene, o-toluidine, K CO , r.t., 4 h.
2
3
2
.2. Biological Evaluation
2
.2.1. Cytotoxicity in Human Tumor Cell Lines
All synthesized compounds were evaluated for their in vitro anti-proliferative activity against
three human cancer cell lines: MCF-7 (breast cancer cells), Hela (cervical cancer cells), HepG2 (liver
cancer cells). The concentration required for 50% inhibition of cell viability (IC ) was calculated and
50
the results are given in Table 1. Most compounds showed a moderate anti-proliferative activity against
all tested cell lines, whereas compounds 5a 5b 5e and 5 exhibited relatively weak activity against
one or all tested cell lines, with IC50 values over 100 M. On the other hand, 5i 5p 5r and 5y exhibited
1.54 M, was
,
,
j
µ
,
,
good cytotoxicity against HepG2, whereas compound 5f, with an IC50 value of 17.65
more active against Hela.
˘
µ

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Table 1. Inhibition of humor cancer cell lines (Hela, MCF7 and HepG2) by N-substituted
-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamide derivatives.
2
Structure
R
MTT (IC , µM)
50
Compound
Hela
MCF7
HepG2
5
a
>100
>100
>100
5
b
F
62.13 ˘ 3.45
43.21 ˘ 1.67
42.14 ˘ 2.61
>100
56.46 ˘ 2.71
37.87 ˘ 0.87
35.67 ˘ 1.69
5
c
46.12 ˘ 2.15
46.19 ˘ 1.34
N
S
5
d
5
e
>100
>100
61.12 ˘ 2.54
5
f
17.65 ˘ 1.54
42.12 ˘ 1.94
24.24 ˘ 1.09
40.67 ˘ 1.48
20.89 ˘ 0.87
35.65 ˘ 1.02
5
g
H CO
3
5
h
41.21 ˘ 1.54
38.91 ˘ 1.04
25.69 ˘ 2.01
CF3
5
i
j
28.23 ˘ 1.16
>100
26.11 ˘ 0.98
29.25 ˘ 1.90
36.15 ˘ 1.23
25.14 ˘ 0.61
21.34 ˘ 0.98
36.75 ˘ 1.85
5
OCH3
5
k
45.61 ˘ 1.67
5
l
65.43 ˘ 1.15
85.34 ˘ 1.54
54.32 ˘ 1.15
68.21 ˘ 2.78
76.34 ˘ 1.25
41.24 ˘ 1.12
43.21 ˘ 1.45
57.17 ˘ 2.15
40.13 ˘ 2.54
Cl
5
m
NO2
5
n
CO CH3
2

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Table 1. Cont.
Structure
R
MTT (IC , µM)
50
Compound
Hela
MCF7
HepG2
OCH3
5
o
35.13 ˘ 1.98
45.13 ˘ 2.32
43.12 ˘ 2.32
OCH3
OCH3
5
5
p
q
40.14 ˘ 1.56
36.56 ˘ 1.12
37.45 ˘ 0.67
35.65 ˘ 1.41
22.24 ˘ 1.68
38.65 ˘ 1.63
OCH3
OCH3
Cl
Cl
5
r
16.12 ˘ 1.54
34.35 ˘ 1.45
37.32 ˘ 1.78
47.32 ˘ 1.78
12.54 ˘ 1.15
45.83 ˘ 2.41
49.32 ˘ 2.16
56.14 ˘ 2.38
10.56 ˘ 1.14
35.67 ˘ 1.14
36.76 ˘ 0.79
42.15 ˘ 2.53
F
F
Br
5
s
F
5
t
Br
F
I
5
u
N
5
v
78.34 ˘ 0.56
32.15 ˘ 2.21
42.15 ˘ 2.23
56.34 ˘ 1.56
43.12 ˘ 2.10
53.12 ˘ 2.16
41.22 ˘ 2.19
46.14 ˘ 1.45
41.14 ˘ 1.92
N
5
w
N
N
5
x
5
y
31.47 ˘ 1.24
29.25 ˘ 1.90
21.34 ˘ 0.98
O
N
Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity
against these three cancer lines, with IC50 values of over 100 M, however introduction of single
ring aryl group at the N-position such as in compounds 5c 5d and 5f caused an increase in cytotoxic
activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic
activity against the Hela cell line, with an IC50 value of 17.65 1.54 M. These data suggested
µ
,
˘
µ
1
the presence of alkyl ring at the N -position had an important relationship with anti-proliferative
activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I),

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methyl (CH ), trifluoromethyl (CF ), methoxy (OCH ), cyano (CN), nitro (NO ) and/or methoxy
3
3
3
2
formyl (COOCH ) was introduced (compounds 5g
–
u). Substitution at the N-phenyl ring was
3
unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1.
In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted
in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group
(
compound 5e) or alkyl group (5b) between nitrogen atom and phenyl group was generally unfavorable
for anti-proliferative properties.
Based on data collected from three independent experiments, compound 5r showed the most
cytotoxic activity against Hela, MCF-7 and HepG2 cell lines with IC50 values of 16.12
2.54 1.15 and 10.56 1.14 M, respectively, so we used compound 5r for further biological
activity studies.
˘
1.54,
1
˘
˘
µ
2
.2.2. Growth Inhibitory Activity of 5r in HepG2
As cell proliferation depends on cell division which is regulated by the cell cycle, absence of normal
cell-cycle control is a hallmark of cancer [30]. Cell cycle-related proteins have been as the therapeutic
targets against cancer and lots of small molecules were developed as potent antitumor agents, such
as microtubule-targeting agents, and cyclin-dependent kinases, aurora kinases and polo-like kinases
inhibitors [31–34]. The present study sought to determine how compound 5r inhibited HepG2 cell
growth. Microscopic analysis indicated that the colonies of HepG2 cells decreased after compound 5r
treatment in a dose dependent manner, compared to the control group (Figure 1A). Furthermore, flow
cytometry was performed to examine cell cycle inhibition after 12 h of compound 5r treatment. The
results revealed a significant accumulation of cell-cycle arrest, with a decrease in G0/G1 phase and an
increase in G2/M phase arrest at 12 h, indicating that inhibitory activity of compound 5r was associated
with disruption of cell cycle (Figure 1B). Meanwhile, the effects of compound 5r on colony-formation
and cell-cycle distribution in Hela and MCF-7 cells were also examined (see Supplementary Materials).
Figure 1. Effects of compound 5r on colony-formation and cell-cycle distribution in HepG2 cells.
(
A
) Compound 5r inhibits the colony-forming activity of HepG2 cells. Cells were grown in 6-well
plates for 6 days and treated with compound 5r (1, 5 and 10 M). Colonies of HepG2 cells decreased
after compound 5r treatment in a dose dependent manner (** p < 0.01 and *** p < 0.001 compared with
the control, t-test); ( ) A flow cytometry assay was performed to examine cell cycle arrest. HepG2 cells
were treated with 10 M compound 5r for 12 h. Compound 5r reduced a significant accumulation of
µ
B
µ
cell-cycle arrest (*** p < 0.001 compared with the control, t-test).
2
.2.3. Inducing Apoptosis in HepG2 Cells
Apoptosis, as a fundamental biological process, plays an important role in cell growth,
development and tissue homeostasis [35
diseases, including cancer, neurodegenerative disorders and cardiovascular diseases [38
–
37]. Deregulation in apoptotic cell contributes to many
39]. Apoptosis
,

Molecules 2016, 21, 530
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can be further characterized as cell death accompanied by the activation of a unique family of
cysteine-dependent specific proteases called caspases [40]. Two major signaling pathways induce
apoptotic cell death: the mitochondrial pathway and the death receptor pathway [41]. The former
relies on mitochondrial depolarization and permeability increase in response to a variety of cellular
stresses, including DNA damage, growth factor deprivation, ER stress, thus resulting in the release
of cytochrome C, then initiating formation of an APAF-1/caspase-9 complex and activation of
downstream executionary caspases, including caspase-3, caspase-6, and caspase-7, and finally leading
to cell death [42
ligands, including tumor necrosis factor-
,
43]. The second pathway is activated predominantly by the binding of death receptor
(TNF- ), fas ligand (CD95) and tumor necrosis factor-related
α
α
apoptosis inducing ligand (TRAIL) to their respective death receptors, then initiating the assembly of
large macromolecular complexes that recruit and activate caspase-8, which further cleave and activate
caspase-3 for apoptosis [44]. In our work, it was demonstrated that compound 5r could induce poly
ADP-ribose polymerase (PARP) cleavage, which served as a marker of cells undergoing apoptosis, in a
time- and dose-dependent manner (Figure 2A,B).
Figure 2. Apoptosis induction by compound 5r in HepG2. Western blot analysis of PARP in HepG2
cells treated with 10
µM compound 5r for different periods of time (0, 3, 6, 9, 12, 16 and 24 h) (A); and
different concentration of compound 5r (2, 4, 6, 8 and 10 µM) for 24 h (B).
The PARP is one of the important targets of caspase-3, which is also downstream of capase-8 and
caspase-9 [45]. Therefore, the activation of caspase-3, caspase-8 and caspase-9 was examined in HepG2
cells using a caspase activity assay kit after treatment with compound 5r. It was shown that compound
5r significantly stimulated caspase-3 and caspase-8 protease activities in HepG2 cells, yet had little
effect on caspase-9 protease activity (Figure 3). The results suggested that compound 5r resulted in the
caspases-3 activation and PARP cleavage by activating caspase-8, finally leading to cell death.
Figure 3. Induction of caspase-3, caspase-8 and caspase-9 by compound 5r in HepG2. Cells were
treated with different concentration of compound 5r (1, 2, 5 and 10
); caspase-8 ( ) and caspase-9 ( ) activity assay kit. As a positive control, HepG2 cells were treated
with (PTX, 1 M) and analyze. Compound 5r significantly stimulated caspase-3 and caspase-8 protease
µM) and analyzed by caspase-3
(
A
B
C
µ
activities in HepG2 cells, yet had little effect on caspase-9 protease activity (*** p < 0.001 and ns p > 0.05
compared with the control, t-test).

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3
. Materials and Methods
3
.1. General Information
All reagents were commercially available and used without further purification unless otherwise
indicated. Reaction mixtures were magnetically stirred and monitored by thin-layer chromatography
TLC) on Yantai Wish chemical products Co., Ltd. (Yantai, China) silica gel 60F-254 by fluorescence
quenching under UV light. All of the final compounds were purified by column chromatography.
(
1
13
H-NMR and C-NMR spectra were recorded on an AV2 600 MHz spectrometer (Bruker Biospin,
Swiss). Chemical shifts ( ) were given in parts per million (ppm) relative to tetramethylsilane (TMS) as
an internal standard. Multiplicities were abbreviated as follows: single (s), doublet (d), doublet-doublet
δ
(
(
dd), doublet-triplet (dt), triplet (t), triplet-triplet (tt), triplet-doublet (td), quartet (q), quartet-doublet
qd), multiplet (m), and broad signal (br s). Positive mode high-resolution mass spectral (HRMS) data
were acquired using electrospray ionization (ESI) on a Q Exactive LC-MS/MS instrument (Thermo
Fisher Scientific Inc., Waltham, MA, USA) with UV detection at 254 nm.
3
.2. Synthesis of N-o-Tolylcycloadamantanecarboxamide (2)
A mixture of o-toluidine (10.0 mmol), adamantane-1-carbonyl chloride (10.0 mmol) and anhydrous
potassium carbonate (7 mmol) in toluene (50 mL) was stirred at room temperature for 4 h. The resulting
solids was filtered off and stirred at room temperature for 1.5 h with H O (50 mL). After completion
2
of the reaction, the solid was filtered off and recrystallized from the appropriate solvent. White
1
solid product, yield 75.5%. H-NMR (CDCl ):
δ 7.88 (d, J = 8.07 Hz, 1H), 7.18–7.24 (m, 2H), 7.17 (d,
3
J = 7.52 Hz, 1H), 7.05 (dt, J = 1.10, 7.43 Hz, 1H), 2.26 (s, 3H), 2.11 (br s, 3H), 1.99 (d, J = 2.57 Hz, 6H),
+
+
1
2
.77 (q, J = 12.29 Hz, 6H). ESI-HRMS (+): m/z [M + H] calculated for C H NO , 270.1852, found
18 23
+
+
70.1853; [M + Na] calculated C H NONa , 292.1672, found 292.1669.
18
23
3
.3. Synthesis of 2-Adamantane-1H-indole (3)
A stirred solution of compound 2 (10 mmol) in 50 mL of tetrahydrofuran (THF) under a N
2
˝
atmosphere was maintained at an internal temperature of –5 to 5 C and treated dropwise with
.1–0.15 mol of 2.5 M n-BuLi in hexane. The mixture was stirred at ambient temperature for 3 h, cooled
in an ice bath, and treated dropwise with 2 M HCl (12 mL). Then, the organic layer was separated and
the aqueous layer washed with C H . The combined organic layer was dried with anhydrous MgSO ,
0
6
6
4
filtered, and concentrated in vacuo. The residue was recrystallized from the appropriate solvent. White
1
solid product, yield 56.5%. H-NMR (CDCl ):
δ 7.30 (d, J = 7.89 Hz, 1H), 7.18 (dd, J = 0.55, 8.07 Hz,
3
1
3
2
H), 6.88 (dt, J = 1.10, 7.52 Hz, 1H), 6.77–6.83 (m, 1H), 5.98 (d, J = 0.55 Hz, 1H), 1.97 (d, J = 2.57 Hz,
+
+
H), 1.93 (d, J = 2.93 Hz, 6H), 1.73 (br s, 6H). ESI-HRMS (+): m/z [M + H] calculated for C H N ,
18
21
+
+
52.1747, found 252.1748; [M + Na] calculated for C H NNa , 274.1566, found 274.1568.
18
21
3
.4. General Procedure for Synthesis of 5a–y [23,46]
A dry 50 mL capacity carousel reaction tube was charged with compound
starting material was dissolved in dry ether (12 mL). Oxalyl chloride (1.65 mmol) was added and
the mixture stirred at room temperature for 2 h, the solid was filtered off. Then compound was
used, without purification, for the next reaction. To a solution of (1 mmol) and a substituted amine
1 mmol) in toluene (10 mL) was added K CO (1.0 mmol), and the mixture stirred at room temp. After
3
(1.5 mmol), and this
4
4
(
2
3
completion of the reaction, the mixture was concentrated and purified by column chromatography
using appropriate mixtures of CH Cl /MeOH to give compounds 5a–y.
2
2
1
N-Cyclopropyl-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5a): yellow solid, yield 87.0%. H-NMR
DMSO-d6): 8.69 (d, J = 4.58 Hz, 1H), 7.53 (d, J = 7.52 Hz, 1H), 7.48 (d, J = 7.52 Hz, 1H), 7.24–7.27 (m,
(
δ
1
0
H), 2.89 (qt, J = 3.97, 7.61 Hz, 1H), 2.21 (br s, 6H), 2.08 (br s, 3H), 1.69–1.88 (m, 6H), 0.71–0.79 (m, 2H),
.52–0.61 (m, 2H). 13C-NMR (DMSO-d6):
δ 186.9, 170.3, 157.8, 134.7, 129.4, 122.3, 121.9, 119.8, 112.8,

Molecules 2016, 21, 530
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+
+
108.2, 38.8, 36.5, 36.3, 28.3, 22.7, 21.5, 5.8.ESI-HRMS (+): m/z [M + H] calculated for C H N O ,
23
26
2
2
+
+
3
63.2067, found 363.2065; [M + Na] calculated for C H N O Na , 385.1886 found 385.1888.
23
26
2
2
N-(4-Fluorobenzyl)-2-(2-adamantane-1H-indol-3-yl)-2-oxoacetamide (5b): yellow solid, yield 87.5%.
1
H-NMR (DMSO-d6):
δ 9.17 (t, J = 6.05 Hz, 1H), 7.51 (d, J = 8.07 Hz, 1H), 7.42 (dd, J = 5.69, 8.44
Hz, 2H), 7.30 (d, J = 8.07 Hz, 1H), 7.18–7.23 (m, 2H), 7.09–7.13 (m, 1H), 6.92–6.96 (m, 1H), 4.46 (d,
J = 6.05 Hz, 2H), 2.22 (br s, 6H), 2.07 (br s, 3H), 1.73–1.84 (m, 6H). 13C-NMR (DMSO-d6):
186.5, 169.2,
61.8 (d, J = 243.2 Hz), 158.4, 135.4 (d, J = 3.3 Hz), 130.3 (d, J = 8.8 Hz), 129.4, 128.4, 125.8, 121.9 (d,
δ
1
+
J = 63.8 Hz), 119.8, 115.6, 115.4, 112.9, 108.0, 41.6, 38.8, 36.6, 36.4, 28.4. ESI-HRMS (+): m/z [M + H]
calculated for C H FN O , 431.2129, found 431.2126; [M + Na] calculated for C H FN O Na ,
+
+
+
27
27
2
2
27 27
2
2
4
53.1949, found 453.5194.
2
-(2-Adamantane-1H-indol-3-yl)-2-oxo-N-(2-(pyridin-3-yl)ethyl)acetamide (5c): yellow solid, yield 86.0%.
1
H-NMR (DMSO-d6):
.51 (d, J = 7.89 Hz, 1H), 7.34 (t, J = 8.16 Hz, 2H), 7.24 (dd, J = 5.23, 6.88 Hz, 1H), 7.12 (t, J = 7.43 Hz,
H), 7.04 (t, J = 7.52 Hz, 1H), 3.67 (q, J = 6.97 Hz, 2H), 3.00–3.06 (m, 2H), 2.20 (br s, 6H), 2.07 (br s, 3H),
δ 8.75 (t, J = 5.41 Hz, 1H), 8.53 (d, J = 4.22 Hz, 1H), 7.71 (dt, J = 1.47, 7.61 Hz, 1H),
7
1
1
1
.72–1.84 (m, 6H). 13C-NMR (DMSO-d6):
22.1, 121.9, 119.9, 112.7, 108.2, 40.0, 38.9, 38.7, 37.3, 36.5, 36.3, 28.4. ESI-HRMS (+): m/z [M + H]
δ
187.1, 169.0, 158.4, 149.6, 136.9, 134.6, 127.9, 123.7, 122.2,
+
+
+
+
calculated for C H N O , 428.2333, found 428.2330; [M + Na] calculated for C H N O Na ,
27
29
3
2
27 29
3
2
4
50.2152, found 450.2150.
2
-(2-Adamantane-1H-indol-3-yl)-2-oxo-N-(2-(thiophen-2-yl)ethyl)acetamide (5d): yellow solid, yield 88.9%.
1
H-NMR (DMSO-d6):
δ 11.69 (br s, 1H), 8.82 (t, J = 5.50 Hz, 1H), 7.51 (d, J = 8.07 Hz, 1H), 7.34–7.40
(
3
m, 2H), 7.13 (t, J = 7.52 Hz, 1H), 7.04–7.09 (m, 1H), 6.97 (d, J = 3.30 Hz, 2H), 3.55 (q, J = 6.85 Hz, 2H),
.05–3.13 (m, 2H), 2.21 (br s, 6H), 2.08 (br s, 3H), 1.73–1.83 (m, 6H). 13C-NMR (DMSO-d6):
δ 167.1,
1
8
49.1, 137.8, 121.9, 114.7, 108.0, 107.6, 106.0, 104.7, 102.2, 100.1, 92.8, 88.4, 20.2, 19.0, 16.7, 16.5, 9.5,
+
+
+
.5. ESI-HRMS (+): m/z [M + H] calculated for C H N O S , 433.1944, found 433.1942; [M + Na]
26
28
2
2
+
calculated for C H N O SNa , 455.1764, found 455.1761.
26
28
2
2
N-(2-(1H-Indol-2-yl)ethyl)-2-(2-adamantane-1H-indol-3-yl)-2-oxoacetamide (5e): yellow solid, yield 87.9%.
1
H-NMR (DMSO-d6):
δ 10.89 (brs, 1H), 8.79 (br s, 1H), 7.50–7.61 (m, 2H), 7.33–7.44 (m, 2H), 7.22–7.25
(
2
m, 1H), 7.16–7.19 (m, 1H), 7.09 (dd, J = 7.89, 16.14 Hz, 2H), 6.97–7.01 (m, 2H), 3.59 (d, J = 6.24 Hz,
H), 2.99 (t, J = 7.15 Hz, 2H), 2.22 (br s, 6H), 2.08 (br s, 3H), 1.70–1.85 (m, 6H). C-NMR (DMSO-d6): δ
13
1
1
4
87.3, 169.0, 157.6, 136.8, 129.4, 128.7, 127.9, 127.6, 125.8, 123.3, 122.2, 121.8, 121.4, 119.9, 118.7, 118.7,
+
+
12.7, 111.9, 41.6, 38.9, 36.6, 36.3, 28.4, 25.3. ESI-HRMS (+): m/z [M + H] calculated for C H N O ,
2
3
0
31
3
+
+
66.2489, found 466.2487; [M + Na] calculated for C H N O Na , 488.2308, found 488.2306.
30
31
3
2
1
2
-(2-Adamantane -3H-indol-3-yl)-2-oxo-N-phenylacetamide (5f): yellow solid, yield 91.9% . H-NMR
7.72-7.82 (m, 2H), 7.54 (d, J = 8.07 Hz, 1H), 7.35–7.44 (m, 3H), 7.15 (t, J = 7.43 Hz,
H), 7.10 (t, J = 7.52 Hz, 1H), 6.97-7.03 (m, 2H), 6.56 (dd, J = 1.10, 8.44 Hz, 1H), 6.44–6.52 (m, 1H),
.28 (br s, 6H), 2.10 (br s, 3H), 1.74-1.88 (m, 6H). 13C-NMR (DMSO-d6):
184.7, 167.9, 149.1, 139.1,
29.5, 129.2, 124.4, 122.0, 121.9, 120.2, 119.2, 116.1, 114.3, 107.7, 38.8, 36.6, 36.6, 28.4. ESI-HRMS
(DMSO-d6): δ
1
2
1
(
δ
+
+
+
+): m/z [M + H] calculated for C H N O , 399.2067, found 399.2063; [M + Na] calculated for
26 26 2 2
+
C H N O Na , 421.1886, found 421.1882.
26
26
2
2
2
(
1
2
1
-(2-Adamantane-3H-indol-3-yl)-2-oxo-N-p-tolylacetamide (5g): yellow solid, yield 86.5%. 1H-NMR
DMSO-d6): 10.68 (s, 1H), 7.66 (d, J = 8.25 Hz, 2H), 7.57 (d, J = 8.07 Hz, 1H), 7.41 (d, J = 8.07 Hz,
H), 7.21 (d, J = 8.25 Hz, 2H), 7.14 (t, J = 7.43 Hz, 1H), 7.02–7.06 (m, 1H), 2.30 (s, 3H), 2.27 (br s, 6H),
.11 (br s, 3H), 1.73–1.87 (m, 6H). 13C-NMR (DMSO-d6):
185.6, 167.4, 158.5, 136.4, 134.7, 133.6, 129.9,
29.4, 122.4, 122.2, 120.2, 119.4, 113.0, 107.9, 38.7, 36.5, 36.4, 28.4, 21.0. ESI-HRMS (+): m/z [M + H]+
δ
δ

Molecules 2016, 21, 530
9 of 15
+
+
+
calculated for C H N O , 413.2224, found 413.2226; [M + Na] calculated for C H N O Na ,
27
28
2
2
27 28
2
2
4
35.2043, found 435.2045.
N-(2-Methoxyphenyl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5h): yellow solid, yield 91.5%.
1
H-NMR (DMSO-d6):
δ 9.44 (br s, 1H), 8.11 (d, J = 7.70 Hz, 1H), 7.44 (dd, J = 8.07, 12.65 Hz, 2H),
7
(
.14–7.19 (m, 1H), 7.10 (d, J = 8.07 Hz, 1H), 7.00 (td, J = 7.45, 19.03 Hz, 2H), 6.91 (t, J = 7.34 Hz, 1H), 3.83
s, 3H), 2.26 (br s, 6H), 2.06 (br s, 3H), 1.71–1.85 (m, 6H). 1 C-NMR (DMSO-d6):
3
δ
185.2, 168.0, 150.5,
1
2
30.1, 127.2, 125.4, 122.1, 121.0, 120.9, 120.8, 119.4, 114.7, 114.3, 111.9, 111.0, 107.6, 56.2, 39.2, 36.9, 36.9,
8.7. ESI-HRMS (+): m/z [M + H] calculated for C H N O , 429.2173, found 429.2171; [M + Na]+
+
+
27
28
2
3
+
calculated for C H N O Na , 451.1992, found 451.1990.
27
28
2
3
2
8
-(2-Adamantane-3H-indol-3-yl)-2-oxo-N-(3-(trifluoromethyl)phenyl)acetamide (5i): yellow solid, yield
1
8.0%. H-NMR (DMSO-d ):
δ
8.22 (s, 1H), 8.02 (d, J = 8.07 Hz, 1H), 7.65 (t, J = 7.98 Hz, 1H), 7.51 (t,
6
J = 7.15 Hz, 2H), 7.35 (d, J = 8.07 Hz, 1H), 7.23–7.27 (m, 1H), 7.17 (d, J = 7.34 Hz, 1H), 7.09 (t, J = 7.52 Hz,
1
3
1
1
1
H), 6.98–7.02 (m, 1H), 2.27 (br s, 6H), 2.09 (br s, 3H), 1.73–1.86 (m, 6H). C-NMR (DMSO-d6): δ
83.8, 168.3, 140.0, 137.8, 130.8, 130.1(d, J = 31.9 Hz), 129.4, 128.7, 125.8, 123.1(q, J = 280.6 Hz), 119.0,
16.1(q, J = 3.3 Hz), 113.8, 107.5, 38.8, 36.6, 28.4, 21.5. ESI-HRMS (+): m/z [M + H] calculated for
+
+
+
+
C H F N O , 467.1941, found 467.1943; [M + Na] calculated for C H F N O Na , 489.1760,
27
25
3
2
2
27 25
3
2
2
found 489.1762.
N-(4-Methoxyphenyl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5j): yellow solid, yield 89.5%.
1
H-NMR (DMSO-d6):
δ 10.60 (s, 1H), 7.68 (d, J = 8.99 Hz, 2H), 7.54 (d, J = 7.89 Hz, 1H), 7.41 (d,
J = 8.25 Hz, 1H), 7.22–7.28 (m, 2H), 7.17 (d, J = 7.34 Hz, 2H), 7.10–7.16 (m, 2H), 7.01–7.07 (m, 1H), 6.97
13
(
(
5
d, J = 8.99 Hz, 2H), 3.76 (s, 3H), 2.30 (s, 3H), 2.26 (br s, 6H), 2.10 (br s, 3H), 1.74–1.87 (m, 6H). C-NMR
DMSO-d6): 185.6, 167.9,168.2, 158.6, 135.3, 134.7, 128.6,127.1, 126.6, 122.3, 122.1, 119.5, 113.1, 107.9,
5.7,38.7, 36.5, 36.5, 28.4, 19.05. ESI-HRMS (+): m/z [M + H] calculated for C H N O , 429.2173,
δ
+
+
27
28
2
3
+
+
found 429.2168; [M + Na] calculated for C H N O Na , 451.1992, found 451.1986.
27
28
2
3
N-(2,4-Dimethylphenyl)-2-(2-adamantane-1H-indol-3-yl)-2-oxoacetamide (5k): yellow solid, yield 81.5%.
1
H-NMR (DMSO-d6):
δ 10.08 (br s, 1H), 7.55 (m, 2H), 7.38 (d, J = 7.89 Hz ,1H), 7.16 (t, J = 7.34 Hz, 1H),
7
.11 (s, 1H ), 7.09 (d, J = 4.77 Hz,1H), 7.07 (s, 1H), 2.30 (s, 3H), 2.37 (s, 6H), 2.23 (s, 3H), 2.11 (s, 3H),
1
1
.83 (m, 6H). 13C-NMR (DMSO-d6):
δ
186.2, 167.9, 158.4, 135.5, 135.0, 133.0, 132.9, 131.6, 128.1, 127.2,
+
25.7, 122.3, 121.9, 119.7, 113.0, 108.0, 38.8, 36.6, 36.5, 28.4, 21.0, 18.1. ESI-HRMS (+): m/z [M + H]
+
+
+
calculated for C H N O , 427.238, found 427.2384; [M + Na] calculated for C H N O Na ,
28
30
2
2
28 30
2
2
4
49.2199, found 449.2201.
N-(4-Chloro-2-methylphenyl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5l): yellow solid, yield 87.5%.
1
H-NMR (DMSO-d6):
δ 7.56 (dd, J = 8.25, 13.57 Hz, 2H), 7.49 (d, J = 8.07 Hz, 1H), 7.39 (s, 1H), 7.34 (dd,
J = 2.02, 8.44 Hz, 1H), 7.15 (t, J = 7.61 Hz, 1H), 7.08–7.11 (m, 1H), 2.26 (s, 9H), 2.10 (br s, 3H), 1.75–1.87
(
1
m, 6H). 13C-NMR (DMSO-d6):
δ
185.6, 167.9, 168.2, 158.6, 135.3, 134.7, 130.6, 128.6, 127.1, 126.6, 122.3,
+
22.1, 119.5, 113.1, 107.9, 38.7, 36.5, 36.5, 28.4, 19.05, 18.3. ESI-HRMS (+): m/z [M + H] calculated
+
+
+
for C H ClN O , 447.1834, found 447.1832; [M + Na] calculated for C H ClN O Na , 469.1653,
27
27
2
2
27 27
2
2
found 469.1649.
2
-(2-Adamantane-3H-indol-3-yl)-N-(2-methyl-4-nitrophenyl)-2-oxoacetamide (5m): yellow solid, yield 90.5%.
1
H-NMR (DMSO-d6):
δ 8.20 (brs, 1H), 8.17 (d, J = 8.62 Hz, 1H), 8.02 (d, J = 7.70 Hz, 1H), 7.56 (d,
J = 8.07 Hz, 1H), 7.46 (d, J = 8.07 Hz, 1H), 7.16 (t, J = 7.52 Hz, 1H), 7.06–7.11 (m, 1H), 2.39 (s, 3H),
.27 (br s, 6H), 2.10 (br s, 3H), 1.75–1.87 (m, 6H); 13C-NMR (DMSO-d6):
185.1, 168.2, 158.7, 144.3,
42.9, 134.8, 133.0, 127.9, 126.1, 124.6, 122.5, 122.4, 122.2, 119.4, 113.1, 107.9, 38.7, 36.5, 36.5, 28.4, 18.3.
2
δ
1
+
+
+
ESI-HRMS (+): m/z [M + H] calculated for C H N O , 458.2074, found 458.2072; [M + Na]
calculated for C H N O Na , 480.1894, found 480.1892. methyl benzoate
27
27
3
4
+
27
27
3
4

Molecules 2016, 21, 530
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Methyl 3-methyl-4-(2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamido)benzoate (5n): yellow solid, yield 89.5%.
1
H-NMR (DMSO-d6):
δ
11.82 (s, 1H), 10.39 (s, 1H), 7.90 (s, 1H), 7.88 (d, J = 1.65 Hz, 1H), 7.82–7.87 (m,
1
3
H), 7.56 (d, J = 7.89 Hz, 1H), 7.49 (d, J = 8.07 Hz, 1H), 7.12–7.16 (m, 1H), 7.08–7.12 (m, 1H), 3.86 (s,
13
H), 2.34 (s, 3H), 2.24–2.29 (m, 6H), 2.11 (br s, 3H), 1.75–1.87 (m, 6H); C-NMR (DMSO-d6):
δ 185.4,
1
1
4
67.8, 166.4, 158.5, 140.4, 134.7, 132.2, 132.0, 129.4, 128.7, 127.9, 126.8, 125.8, 124.7, 122.5, 122.2, 119.5,
+
+
13.0, 107.9, 52.5, 38.7, 36.5, 28.4, 21.5, 18.2. ESI-HRMS (+): m/z [M + H] calculated for C H N O ,
4
2
9
30
2
+
+
71.2278, found 471.2278; [M + Na] calculated for C H N O Na , 493.2098, found 493.2096.
29
30
2
4
N-(3,4-Dimethoxyphenyl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5o): yellow solid, yield 87.9%.
1
H-NMR (DMSO-d6):
δ 10.45 (br s, 1H), 7.49 (d, J = 8.07 Hz, 1H), 7.40 (d, J = 2.38 Hz, 1H), 7.35–7.37 (m,
1
1
1
H), 7.05 (t, J = 7.52 Hz, 1H), 6.94–6.99 (m, 2H), 3.75 (d, J = 8.25 Hz, 6H), 2.27(br s, 6H), 2.08 (br s, 3H),
.72–1.86 (m, 6H). 13C-NMR (DMSO-d6):
δ
167.7, 149.2, 145.8, 137.8, 132.8, 129.4, 128.7, 125.8, 121.6,
+
21.5, 111.9, 113.8, 112.7, 112.2, 107.7, 105.2, 56.2, 55.8, 39.6, 38.9, 36.7, 28.5. ESI-HRMS (+): m/z [M + H]
+
+
+
calculated for C H N O , 459.2278, found 459.2277; [M + Na] calculated for C H N O Na ,
28
30
2
4
28 30
2
4
4
81.2098, found 481.2098.
N-(3,5-Dimethoxyphenyl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5p): yellow solid, yield 89.5%.
1
H-NMR (DMSO-d6):
δ 10.68 (s, 1H), 7.55 (d, J = 8.07 Hz, 1H), 7.39 (d, J = 8.07 Hz, 1H), 7.12–7.16 (m,
1
2
H), 7.04-7.08 (m, 1H), 7.01 (s, 1H), 7.01 (s, 1H), 6.33 (t, J = 2.20 Hz, 1H), 3.74 (s, 6H), 2.26 (br s, 6H),
.10 (br s, 3H), 1.74–1.86 (m, 6H). 13C-NMR (DMSO-d6):
δ
185.3, 167.6, 161.1, 158.6, 140.6, 134.8, 129.4,
+
1
27.9, 122.4, 122.3, 119.4, 113.0, 107.8, 98.7, 96.2, 55.6, 38.7, 36.5, 36.5, 28.4. ESI-HRMS (+): m/z [M + H]
+
+
+
calculated for C H N O , 459.2278, found 459.2276; [M + Na] calculated for C H N O Na ,
28
30
2
4
28 30
2
4
4
81.2098, found 481.2096.
N-(4-Chloro-3-methoxyphenyl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5q): yellow solid, yield
1
8
5.7%. H-NMR (DMSO-d ):
δ
7.59 (s, 1H), 7.54 (d, J = 7.89 Hz, 1H), 7.43 (s, 2H), 7.38 (d, J = 8.07 Hz,
6
1
(
1
H), 7.15–7.19 (m, 1H), 7.13 (t, J = 7.70 Hz, 1H), 7.02–7.07 (m, 1H), 3.79–3.88 (m, 3H), 2.26 (br s, 6H), 2.10
br s, 3H), 1.74–1.86 (m, 6H). 1 C-NMR (DMSO-d6):
3
δ
184.8, 167.7, 155.1, 139.2, 137.8, 130.5, 129.0, 125.8,
+
22.2, 119.3, 116.3, 113.3, 112.9, 107.7, 104.8, 56.3, 40.1, 38.8, 36.6, 28.4, 21.5. ESI-HRMS (+): m/z [M + H]
+
+
+
calculated for C H ClN O , 463.1783, found 463.1781; [M + Na] calculated for C H ClN O Na ,
27
27
2
3
27 27
2
3
4
85.1602, found 485.1601.
N-(3-Chloro-4-fluorophenyl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5r): yellow solid, yield 85.9%.
1
H-NMR (DMSO-d6):
H), 7.56 (d, J = 8.07 Hz, 1H), 7.47 (t, J = 9.08 Hz, 1H), 7.36 (d, J = 8.07 Hz, 1H), 7.13–7.16 (m, 1H),
.05–7.10 (m, 1H), 2.26 (br s, 6H), 2.10 (br s, 3H), 1.75–1.85 (m, 6H). 13C-NMR (DMSO-d6):
184.9,
δ 11.85 (br s, 1H), 11.01 (br s, 1H), 8.07 (dd, J = 2.57, 6.79 Hz, 1H), 7.68–7.72 (m,
1
7
δ
1
67.4, 158.8 (d, J = 244.3 Hz), 154.1, 136.2, 134.7, 129.4, 128.7, 125.8, 122.5 (d, J = 15.4 Hz), 121.5, 120.6 (d,
+
J = 6.6 Hz), 119.3, 117.8, 113.1, 107.7, 38.7, 36.5, 28.3, 21.5. ESI-HRMS (+): m/z [M + H] calculated for
C H ClFN O , 451.1583, found 451.1584; [M + Na] calculated for C H ClFN O Na , 473.1403,
+
+
+
26
24
2
2
26 24
2
2
found 473.1405.
N-(4-Bromo-2-fluorophenyl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5s): yellow solid, yield 88.5%.
1
H-NMR (DMSO-d6):
δ 7.88 (br s, 1H), 7.68 (d, J = 9.90 Hz, 1H), 7.55 (d, J = 8.07 Hz, 1H), 7.43–7.52
(
m, 2H), 7.14 (t, J = 7.15 Hz, 1H), 7.04–7.09 (m, 1H), 2.26 (br s, 6H), 2.10 (br s, 3H), 1.71–1.87 (m, 6H).
1
3
C-NMR (DMSO-d ) :
δ
184.6, 168.0, 155.0 (d, J = 249.8 Hz), 129.4, 128.7, 128.2, 128.2, 128.1, 126.9,
6
1
25.8, 122.1(d, J = 29.7 Hz), 119.8(d, J = 23.1Hz), 119.3, 113.2, 107.8, 38.7, 36.6, 36.5, 28.4. ESI-HRMS
+
+
+
(
+): m/z [M + H] calculated for C H BrFN O , 495.1078, found 495.1079; [M + Na] calculated for
26 24 2 2
+
C H BrFN O Na , 517.0897, found 517.0896.
26
24
2
2
N-(5-Bromo-2-fluorophenyl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5t): yellow solid, yield 85.6%.
1
H-NMR (DMSO-d6):
δ 8.17 (br s, 1H), 7.53 (d, J = 8.07 Hz, 1H), 7.46 (d, J = 8.07 Hz, 1H), 7.38 (br s,

Molecules 2016, 21, 530
11 of 15
1
1
1
H), 7.30 (t, J = 9.44 Hz, 1H), 7.12 (t, J = 7.24 Hz, 1H), 7.03–7.08 (m, 1H), 2.25 (br s, 6H), 2.08 (br s, 3H),
.74-1.85 (m, 6H). 13C-NMR (DMSO-d6):
170.3, 168.6, 154.1(d, J = 247.6 Hz), 137.8, 129.4, 128.7, 128.2,
27.3, 125.8, 122.0(d, J = 28.6 Hz), 119.4, 118.3(d, J = 21.0 Hz), 116.2, 113.2, 107.9, 38.7, 36.6, 36.5, 28.4.
δ
ESI-HRMS (+): m/z [M + H] calculated for C H BrFN O , 495.1078, found 495.1079; [M + Na]+
+
+
26
24
2
2
+
calculated for C H BrFN O Na , 517.0897, found 517.0893.
26
24
2
2
N-(2-Fluoro-4-iodophenyl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5u): yellow solid, yield 86.5%.
1
H-NMR (DMSO-d6):
δ 7.73 (d, J = 9.72 Hz, 2H), 7.61 (d, J = 7.89 Hz, 1H), 7.51 (d, J = 7.89 Hz, 1H), 7.45
(
(
1
(
d, J = 8.07 Hz, 1H), 7.06–7.12 (m, 1H), 7.02 (t, J = 7.43 Hz, 1H), 2.26 (br s, 6H), 2.08 (br s, 3H), 1.74–1.86
m, 6H). 13C-NMR (DMSO-d6):
170.3, 168.6, 154.9 (d, J = 253.1 Hz), 137.8, 134.0, 129.4, 128.7, 127.2,
25.8, 125.0 (d, J = 22.0 Hz), 121.7 (d, J = 22.1 Hz), 119.3, 113.6, 107.8, 38.8, 36.7, 36.6, 28.5. ESI-HRMS
δ
+
+
+
+): m/z [M + H] calculated for C H FIN O , 543.0939, found 543.0939; [M + Na] calculated for
26 24 2 2
+
C H FIN O Na , 565.0759, found 565.0757.
26
24
2
2
1
2
(
-(2-Adamantane-3H-indol-3-yl)-2-oxo-N-(pyridin-3-yl)acetamide (5v): yellow solid, yield 88.6%. H-NMR
DMSO-d6): 11.93 (br s, 1H), 11.05 (s, 1H), 8.93 (d, J = 2.02 Hz, 1H), 8.38 (d, J = 4.03 Hz, 1H), 8.21 (d,
J = 8.25 Hz, 1H), 7.59 (d, J = 7.89 Hz, 1H), 7.45 (dd, J = 4.77, 8.07 Hz, 1H), 7.39 (d, J = 8.07 Hz, 1H),
.15 (t, J = 7.61 Hz, 1H), 7.04–7.09 (m, 1H), 2.27 (br s, 6H), 2.10 (br s, 3H), 1.74–1.86 (m, 6H). 13C-NMR
DMSO-d6): 185.0, 167.8, 158.8, 145.5, 141.8, 135.7, 134.8, 127.8, 127.3, 124.4, 122.5, 122.4, 119.3, 113.2,
δ
7
(
1
4
δ
+
+
07.7, 38.7, 36.5, 36.5, 28.3. ESI-HRMS (+): m/z [M + H] calculated for C H N O , 400.202, found
25 25 3 2
+
+
00.2017; [M + Na] calculated for C H N O Na , 422.1839, found 422.1836.
25
25
3
2
1
2
(
-(2-Adamantane-3H-indol-3-yl)-2-oxo-N-(pyridin-2-yl)acetamide (5w): yellow solid, yield 90.6%. H-NMR
DMSO-d6): 11.93 (brs, 1H), 11.04 (s, 1H), 8.92 (d, J = 2.20 Hz, 1H), 8.38 (d, J = 4.59 Hz, 1H), 8.21 (d,
J = 8.25 Hz, 1H), 7.59 (d, J = 8.07 Hz, 1H), 7.45 (dd, J = 4.68, 8.16 Hz, 1H), 7.39 (d, J = 8.25 Hz, 1H),
.15 (t, J = 7.61 Hz, 1H), 7.04–7.09 (m, 1H), 2.27 (brs, 6H), 2.10 (brs, 3H), 1.74–1.87 (m, 6H). 13C-NMR
DMSO-d6): 185.0, 167.8, 158.8, 145.5, 141.8, 135.7, 134.8, 127.8, 127.3, 124.4, 122.5, 122.4, 119.3, 113.2,
δ
7
(
1
4
δ
+
+
07.7, 38.7, 36.5, 36.5, 28.3. ESI-HRMS (+): m/z [M + H] calculated for C H N O , 400.202, found
25 25 3 2
+
+
00.2016; [M + Na] calculated for C H N O Na , 422.1839, found 422.1835.
25
25
3
2
2
-(2-Adamantane-3H-indol-3-yl)-2-oxo-N-(pyrimidin-5-yl)acetamide (5x): yellow solid, yield 90.1%.
1
H-NMR (DMSO-d ): δ 9.16 (s, 2H), 8.97 (s, 1H), 7.54–7.59 (m, 1H), 7.38 (d, J = 8.25 Hz, 1H), 7.11–7.15
6
13
(m, 1H), 7.03–7.08 (m, 1H), 2.26 (br s, 6H), 2.09 (br s, 3H), 1.75–1.85 (m, 6H). C-NMR (DMSO-d6):
δ
184.5, 168.2, 153.9, 148.4, 147.67, 129.3, 128.7, 128.1, 125.8, 122.3, 119.3, 113.4,107.6, 38.7, 36.5, 36.5, 28.4,
9.1. ESI-HRMS (+): m/z [M + H] calculated for C H N O , 401.1972, found 401.1969; [M + Na]+
+
+
1
24 24 4 2
+
calculated for C H N O Na , 423.1791, found 423.1785.
24
24
4
2
N-(5-tert-Butylisoxazol-3-yl)-2-(2-adamantane-3H-indol-3-yl)-2-oxoacetamide (5y): yellow solid, yield.
1
8
6.8%. H-NMR (DMSO-d ): δ 11.79 (br s, 2H), 7.56 (d, J = 7.89 Hz, 1H), 7.38 (d, J = 8.07 Hz, 1H), 7.16
6
(
(
1
4
t, J = 7.52 Hz, 1H), 7.08–7.12 (m, 1H), 6.72 (s, 1H), 2.24 (br s, 6H), 2.10 (br s, 3H), 1.75–1.86 (m, 6H), 1.35
s, 9H). 13C-NMR (DMSO-d6):
δ
184.2, 181.5, 167.7, 158.9, 157.8, 134.8, 127.7, 122.7, 122.5, 122.4, 119.0,
+
+
13.2, 107.5, 99.8, 38.5, 36.5, 33.1, 28.9, 28.3. ESI-HRMS (+): m/z [M + H] calculated for C H N O ,
27 31 3 3
+
+
46.2438, found 446.2433; [M + Na] calculated for C H N O Na , 468.2258, found 468.2256.
27
31
3
3
3
.5. Cell Culture
All of the human cancer cell lines were obtained from the American Type Culture Collection
ATCC, Manassas, VA, USA) and grown in DMEM culture medium containing 10% fetal bovine serum
(
(
˝
v/v) in 5% CO at 37 C.
2

Molecules 2016, 21, 530
12 of 15
3
.6. Cytotoxicity against Cancer Cell Lines
3
Confluent cancer cells in good state were cultured in 96-well plates (5–6
ˆ
10 cells/well) and
˝
treated with various concentrations of compounds at 37 C for 24 h. Then, the cells were incubated
with 20
µ
L of 5 mg/mL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT,
˝
(
Sigma-Aldrich, Saint Louis, MO, USA) reagent at 37 C for 4 h. The supernatant was removed, and
cells were dissolved in 150
µL dimethyl sulfoxide and shaken for 5 min. Finally, the light absorption
(OD) of the dissolved cells was measured at 490 nm.
3
.7. Western Blot Analysis
Equal amounts of the lysates were electrophoresed on 8% SDS-PAGE gel and transferred onto
PVDF membranes (Roche, Shanghai, China). After blocked with 5% nonfat milk in TBST (20 mM
Tris-HCl (pH 7.4), 150 mM NaCl and 0.1% Tween 20) for 1 h, The membranes were incubated
with various primary antibodies overnight and secondary antibodies for 2 h, finally detected
using ECL system. Proteins were detected with the following antibodies: Rabit anti-Parp mAb
(
46D11, Cell Signaling Technology, Shanghai, China), Mouse anti-β-actin mAb (sc-8432, Santa Cruz,
Shanghai, China).
3
.8. A Flow Cytometry Assay
Hela cells were cultured in 6-well plate and treated with various compounds in serum free
medium for 24 h. Then, the cells were detached by trypsin and fixed in 70% cold ethanol overnight
˝
at 4 C. The next day, the cells were centrifuged in 3000 rpm for 53 min, washed twice in PBS, and
incubated with DNase A (100 g/mL) and propidium iodide (PI) solution (50 g/mL) at room
µ
µ
temperature for 30 min. The cell cycle was detected by flow cytometry (Beckman Coulter, Pasadena,
CA, USA).
3
.9. Colony Formation Assay
Hela cells were cultured in 6-well plate (200 cells/well) and treated with various compounds in 1%
serum medium for 6 days. Then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.
3
.10. Caspase Activity Assay
The activities of caspase-3, caspase-8 and caspase-9 were measured using the caspase activity
kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly,
testis lysates were prepared after treatment. 50 L testis lysate, 50 L reaction buffer and 5 L caspases
substrate were added, incubated at 37 C for 3 h. Samples were measured with an ELISA Reader
Bio-Rad instrument Group, Hercules, CA, USA) at an absorbance of 405 nm. All the experiments were
carried out in triplicates.
µ
µ
µ
˝
(
4
. Conclusions
In this paper, we synthesized and conducted a biological evaluation of a new series of
N-substituted 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamide derivatives as potential anticancer
agents. The synthetic method was relatively simple, and the compounds were produced in high yields
and easily purified. Compound 5r showed more significant inhibitory activity against HepG2 cells
than other compounds, with an IC50 value of 10.56
˘
1.14 µM, as well as excellent selectivity toward
HepG2 over HeLa and MCF-7 cells. Western blot analysis and flow cytometry assay demonstrated that
compound 5r could arrest the cell cycle, activate caspase-8 and caspase-3 and induce cell apoptosis.
However, determining its roles in preventing cancer still require further intensive study.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
2
1/5/530/s1.

Molecules 2016, 21, 530
13 of 15
Acknowledgments: This work was supported by the National Natural Science Foundation of China (No. 81273400,
No. 81302652, No. 31471273 and 31461163002), the Project of South Center for Marine Research (14GYY023NF23).
This research was also financially supported by Fujian Science and Technology project (Grant No.2014N5012) and
the 10th Singapore-China Joint Research Program (S2014GR0448).
Author Contributions: H.H., F.W. and Y.Q. performed the synthesis and structure elucidation. X.Y., C.L. and
J.Z. contributed in the biological activity. Z.W. and M.F. designed all molecules, prepared the manuscript and
supervised whole research project.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all compounds are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).