Synthesis and biological evaluation of 1-(2-(adamantane-1-yl)-1: H -indol-5-yl)-3-substituted urea/thiourea derivatives as anticancer agents

Article abstract of DOI:10.1039/c7ra08149a

The indole ring, adamantane, and urea groups are important components of bioactive molecules. The orphan nuclear receptor Nur77 as a unique transcription factor encoded by an immediate early gene is a potential therapeutic target for cancer treatment. We synthesized a series of 1-(2-(adamantane-1-yl)-1H-indol-5-yl)-3-substituted urea/thiourea derivatives and identified which of these potential anticancer candidates could modulate the expression and activity of Nur77. The synthesized compounds were initially evaluated for their anti-proliferative activity against H460 lung cancer cells, HepG2 liver cancer cells, and MCF-7 breast cancer cells. Major compounds were found to be active against these tested cancer cell lines. The compounds with IC₅₀ values down to 20 μM exhibited selective cytotoxicity effects on the human lung cancer cell line (H460) and the normal lung cell line (MCR-5). Compounds 7n, 7s, and 7w induced Nur77-expression in a time- and dose-dependent manner in H460 cells. Compounds 7n and 7s strongly induced Parp cleavage in H460 cells, but 7w resulted in a slight induction of apoptosis. The apoptotic effect of 7s was largely inhibited when the Nur77 was knocked down by shRNA. This indicated that Nur77 served as a critical mediator for the anticancer action of 7s. The molecular docking study between Nur77 and 7s revealed that compound 7s exhibited a promising binding affinity with Nur77. These findings will provide a direction for the developing Nur77 regulator as anticancer agents.

Full text of DOI:10.1039/c7ra08149a

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PAPER
Synthesis and biological evaluation of 1-(2-
adamantane-1-yl)-1H-indol-5-yl)-3-substituted
urea/thiourea derivatives as anticancer agents†
(
Cite this: RSC Adv., 2017, 7, 51640
ab
a
a
a
a
Hongyu Hu,‡ Chunrong Lin,‡ Mingtao Ao,‡ Yufen Ji, Bowen Tang,
a
a
a
a
Xiaoxiao Zhou, Meijuan Fang, * Jinzhang Zeng* and Zhen Wu*
The indole ring, adamantane, and urea groups are important components of bioactive molecules. The
orphan nuclear receptor Nur77 as a unique transcription factor encoded by an immediate early gene is
a potential therapeutic target for cancer treatment. We synthesized a series of 1-(2-(adamantane-1-yl)-
1H-indol-5-yl)-3-substituted urea/thiourea derivatives and identified which of these potential anticancer
candidates could modulate the expression and activity of Nur77. The synthesized compounds were
initially evaluated for their anti-proliferative activity against H460 lung cancer cells, HepG2 liver cancer
cells, and MCF-7 breast cancer cells. Major compounds were found to be active against these tested
cancer cell lines. The compounds with IC50 values down to 20 mM exhibited selective cytotoxicity effects
on the human lung cancer cell line (H460) and the normal lung cell line (MCR-5). Compounds 7n, 7s,
and 7w induced Nur77-expression in a time- and dose-dependent manner in H460 cells. Compounds
7
n and 7s strongly induced Parp cleavage in H460 cells, but 7w resulted in a slight induction of
Received 24th July 2017
Accepted 18th October 2017
apoptosis. The apoptotic effect of 7s was largely inhibited when the Nur77 was knocked down by shRNA.
This indicated that Nur77 served as a critical mediator for the anticancer action of 7s. The molecular
docking study between Nur77 and 7s revealed that compound 7s exhibited a promising binding affinity
with Nur77. These findings will provide a direction for the developing Nur77 regulator as anticancer agents.
DOI: 10.1039/c7ra08149a
rsc.li/rsc-advances
1
7
18
19
acetylshikonin analogue SK07, DIM-C-pPhOCH
3
,
Vit K2,
1
. Introduction
20
21
diindolylmethane analog, Csn-B, cardenolides H9 and ATE-
22
23
10,24
25
Nur77 (also known as NR4A1, NGFI-B, or TR3) is an orphan i2-b4, AmB-NC, CD437,
n-butylidenephthalide, and
26
member of the nuclear receptor super-family that regulates the norcantharidin have been identied as critical modulators of
expression of genes involved in multiple physiological and the genomic or the nongenomic pathways of Nur77. These
1
pathological processes. As an immediate-early response gene, ndings suggest that Nur77 is a critical and meaningful drug
Nur77 expression could be induced by various stimuli, target. Thus, we are interested in exploring a novel Nur77
2–4
including mitogenic and apoptotic signaling. Nur77 plays regulator.
a role in cell survival, cell death, and is implicated in various
The indole ring has been proven to be a critical pharmaco-
3
–11
27–29
malignancies.
Nur77 is upregulated in gastric tumorsphere phore in many chemotherapeutics.
The introduction of
12
13
cells, non-small cell lung cancer (NSCLC) tissues and cells,
and colon tumors.
different side chains around the indole nucleus has resulted in
Previous studies have found that Nur77 is the development of various therapeutic drugs, including oba-
oen overexpressed in tumour tissues, when compared to the toclax, and sunitinib, as well as many lead compounds that
para-carcinoma normal tissues. Nur77 is a potentially prom- boast a wide variety of biological activities. Modifying the 2
14,15
30
31
nd
16
th
ising drug target for cancer treatment. A number of anticancer and 5 positions of the indole moiety has been shown to be
32
compounds with diverse chemical structures, such as crucial for receptor binding and activation. The adamantane
derivatives were initially used as an antiviral drug (adamantane)
33
to combat various strains of the u, to treat parkinson's
a
Fujian Provincial Key Laboratory of Innovative Drug Target Research, School of disease (adapalene, dopamantine, memantine, rimantadine
Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen, and tromantadine), and as dipeptidyl peptidase-4 (DPP-4)
3
61102, China. E-mail: wuzhen@xmu.edu.cn; jzzeng@xmu.edu.cn; fangmj@xmu.
inhibitors (saxagliptin, and vildagliptin) for the treatment of
type 2 diabetes. Some other clinical drugs like sorafenib are
urea derivatives of the indole pharmacophore.
Based on the biological proles of the indole ring, the ada-
mantane, the urea group, and the continuation of our research
edu.cn; Fax: +86-592-2189868; Tel: +86-592-2189868
34
b
College of Xingzhi, Zhejiang Normal University, Jinhua 321004, China
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/c7ra08149a
The rst two authors are co-rst authors.
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32
work on synthesis of indole derivatives, we initially synthe- treatment with the corresponding amine. The structures of the
1
13
sized a series of novel 1-(2-(adamantan-1-yl)-1H-indol-5-yl)-3- target compounds were conrmed with H-NMR, C-NMR, and
substituted urea/thiourea derivatives and evaluated their anti- high-resolution mass spectrometry (HRMS). The purities of the
cancer activity on cancer cell line (HepG2), and breast cancer target compounds were evaluated with high-performance liquid
cell line (MCF-7) by MTT assay. The compounds that exhibited chromatography with diode-array detection (HPLC-DAD) per-
prominent activity against tested cancer cell lines with IC50 formed at 254 nm and 280 nm.
down to 20 mM were subsequently tested for their cytotoxicity on
normal cell lines (MCR-5 and LO2) as well as their effect on
Nur77 activation in H460 cells. Those compounds that had 2.2. Anti-proliferative activities of synthesized compounds
Nur77 activation ability in H460 cells and also exhibited selec- in various cancer cells and their structure–activity
tive cytotoxicity on tumours and normal cell lines which were relationship
isolated from the same tissue, were used to further explore their
The synthesized compounds were evaluated for their anti-
induction of apoptosis in various tumour cell lines and normal
proliferative activities against various human cancer cell lines
NIH-3T3 cells. Compounds 7s, 7n and 7w induced apoptosis in
including H460, HepG2 and MCF-7. The concentration required
H460 cells. This ability for apoptosis induction was closely
for 50% inhibition of cell viability (IC ) was calculated. The IC
50
5
0
associated with their Nur77 activation. The RNA interference
technology and the molecular docking study were performed to
explore the molecular mechanisms that mediate the induction
of apoptosis by this specic class of compounds.
values of the compounds are summarized in Tables 1 and 2. The
results indicated that a majority of the synthesized compounds
had a moderate anti-proliferative activity against the three
human cancer cell lines. Exceptions were found in compounds
7c, 7d, 7e, 7g, 7h, 7i, 9c, and 9d. Attempts were made to
establish the structure–activity relationship (SAR) among the
tested compounds based on data collected from three inde-
pendent experiments.
2
. Results and discussion
2.1. Syntheses of the designed compounds
The general chemistry for the synthesis of 7a–7y and 9a–9o is
2.2.1. 1-(2-(Adamantan-1-yl)-1H-indol-5-yl)-3-substituted
outlined in Scheme 1. First, we synthesized 2-adamantane-1H- urea derivatives. Among the alkyl substituted compounds (7a–
indol-5-amine (5). The treatment of 1-adamantanecarboxylic 7j), compound 7f with a phenethyl group and 7a with a t-butyl
acid chloride (1) with o-toluidine resulted in N-o-tolylcycloada- group had the highest potency of anti-proliferative properties in
32
mantanecarboxamide (2), and 2.5 M of n-butyl lithium in H460 cells (IC50 of 7f, 0.42 ꢀ 0.09 mM), HepG2 cells (IC50 of 7f,
hexane was then added dropwise to obtain the corresponding 2- 0.28 ꢀ 0.08 mM) and MCF-7 cells (IC50 of 7a, 5.16 ꢀ 0.20 mM).
32
adamantane-1H-indole (3). Compound 5 was formed through The IC50 values of compounds 7c, 7d, 7g, and 7h were over 100
35
the nitrication and catalytic hydrogenation of compound 3.
mM for at least one of the three tested human cancer cell lines.
5
-Isocyanato-1H-indole-adamantane (6) was obtained by react- This indicated that the introduction of a n-alkyl group that had
36
ing compound 5 with triphosgene. The intermediate 6 was more than three carbon atoms to the pharmaceutical core was
subjected to reactions with various amines in order to produce unfavourable for anti-proliferative properties. Compound 7e
compounds 7a–7y. The reaction of compound 5 with triethyle- which contained a cycloheptyl group had no activity with IC50
nediamine (Dabco), CS
BTC) resulted in 5-isothiocyanato-1H-indole-adamantane (8).
Compound 8 was converted into compounds 9a–9o by 25.0 mM). This suggested that the substitution of the cycolakyl
2
, and bis(trichloromethyl)carbonate values up to 100 mM. Compound 7b which had a cyclobutyl
37
(
group demonstrated good anti-proliferative activity (IC50, 10.0–
Scheme 1 Synthesis of 1-(2-(substituted hydrazinecarbonyl group)-1H-indol-5-yl)-3-substituted urea. Reagents and conditions: (a) toluene, o-
ꢁ
ꢁ
ꢁ
toluidine, K
trimethylamine, 0 C, 4 h; (f) R–NH
2
CO
3
, r.t., 4 h; (b) n-BuLi, THF, 0 C, N
2
ꢁ
, 3 h; (c) NH
4
NO
3
, H
2
SO
4
, 0 C, 1 h; (d) 10% Pd/C, MeOH, 70 C, 2 h. (e) Triphosgene, THF,
ꢁ
ꢁ
2
, toluene, 60 C, 4 h. (g) Dabco, CS
2
, toluene, BTC, DCM. (h) R–NH
2
, toluene, 60 C, 4 h.
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a
Table 1 Anti-proliferative activity of 1-(2-(adamantan-1-yl)-1H-indol-5-yl)-3-substituted urea 7a–7y
IC50 (mM) ꢀ S.D.
Compound
R
H460
HepG2
MCF-7
7
7
a
5.77 ꢀ 0.12
25.25 ꢀ 1.45
5.93 ꢀ 0.15
15.19 ꢀ 1.00
5.16 ꢀ 0.20
13.78 ꢀ 0.98
b
7
7
c
>100
>100
34.06 ꢀ 1.25
>100
>100
d
>100
7
e
>100
>100
>100
7
f
0.42 ꢀ 0.09
0.28 ꢀ 0.08
1.42 ꢀ 0.10
6.96 ꢀ 0.12
7
g
13.21 ꢀ 0.15
>100
7
h
>100
>100
>100
7
7
7
i
>100
>100
23.11 ꢀ 0.52
16.01 ꢀ 1.02
8.07 ꢀ 0.85
j
11.75 ꢀ 0.85
11.99 ꢀ 0.95
22.10 ꢀ 1.00
13.06 ꢀ 1.10
k
7
7
l
41.82 ꢀ 2.55
60.89 ꢀ 4.55
75.74 ꢀ 2.65
18.32 ꢀ 1.45
40.47 ꢀ 2.89
13.96 ꢀ 0.65
m
7
n
o
12.89 ꢀ 1.41
54.13 ꢀ 2.40
22.24 ꢀ 1.41
17.22 ꢀ 1.36
18.1 ꢀ 2.18
7
11.60 ꢀ 0.65
7
p
50.33 ꢀ 2.70
72.46 ꢀ 3.40
51.09 ꢀ 1.70
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Table 1 (Contd.)
IC50 (mM) ꢀ S.D.
Compound
R
H460
HepG2
MCF-7
7
7
7
q
62.82 ꢀ 3.50
32.30 ꢀ 1.53
10.51 ꢀ 0.30
77.37 ꢀ 3.12
56.84 ꢀ 2.54
r
16.99 ꢀ 1.20
15.50 ꢀ 0.13
12.61 ꢀ 1.00
19.04 ꢀ 0.27
s
7
t
9.75 ꢀ 0.50
8.98 ꢀ 1.10
9.79 ꢀ 0.91
7
7
u
v
9.92 ꢀ 1.20
6.99 ꢀ 0.50
4.56 ꢀ 0.57
6.78 ꢀ 0.78
5.97 ꢀ 0.67
10.94 ꢀ 1.50
7
7
7
w
14.51 ꢀ 0.17
6.25 ꢀ 0.47
13.15 ꢀ 0.23
6.38 ꢀ 0.50
24.75 ꢀ 0.31
4.99 ꢀ 0.45
x
y
13.58 ꢀ 0.30
3.50 ꢀ 0.10
10.47 ꢀ 0.09
6.10 ꢀ 0.09
7.59 ꢀ 0.09
4.90 ꢀ 0.05
DDP
a
Each compound was tested in triplicate. All error bars represent mean ꢀ SD from three independent experiments.
0
group, which had a small ring at the N -urea position, had against tested human cancer cell lines when compared with 7q
a greater effect on the compound's anti-proliferative activity. We or 7s, where F was introduced at the C-4 position of phenyl ring,
found that compounds 7k–7w, which contained the aryl groups as shown in Table 1. The remaining derivatives showed
0
at the N -urea position, generally had good anti-proliferative moderate activity. The presence of aromatic heterocyclic, such
properties. The impact of the substituent at the phenyl ring as pyridyl (7x) and pyrimidine (7y), at the urea end enhanced the
was investigated by introducing halogen (F, Br, or I), methyl anti-proliferative activity.
(
CH ), triuoromethyl (CF ), and/or methoxy (OCH ) groups.
2.2.2. 1-(2-(Adamantan-1-yl)-1H-indol-5-yl)-3-substituted
3
3
3
The results revealed that p-substitution at the phenyl ring might thiourea derivatives. We generated an additional series of
not be helpful for their anti-proliferative properties. Compound thiourea derivatives with the functional group adamant (Ad) at
7k had lower IC50 values than 7l or 7o. Compound 7t had lower the C2-position of the indole ring. This allowed us to explore the
IC50 values than 7q. Compounds with Br (7r or 7u) and I (7v) at effect of the urea group on the cancer cells' proliferation. These
the C-4 position of phenyl ring exhibited better inhibition thiourea derivatives were generally less active against H460,
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a
Table 2 Anti-proliferative activity (IC50, mM) of thiourea derivatives 9a–9o
IC50 (mM) ꢀ S.D.
Compound
R
H460
HepG2
MCF-7
9
a
20.23 ꢀ 0.12
27.25 ꢀ 1.40
15.93 ꢀ 0.17
25.89 ꢀ 0.20
9
b
18.02 ꢀ 1.00
21.13 ꢀ 0.98
9
9
c
>100
>100
>100
>100
>100
>100
d
9
e
30.20 ꢀ 0.88
35.50 ꢀ 0.85
25.00 ꢀ 0.20
9
9
9
f
14.52 ꢀ 0.10
25.36 ꢀ 0.15
22.23 ꢀ 0.18
22.20 ꢀ 0.18
26.12 ꢀ 0.10
22.36 ꢀ 0.15
16.96 ꢀ 0.17
19.62 ꢀ 0.10
26.36 ꢀ 0.19
g
h
9
9
9
9
i
38.54 ꢀ 0.18
31.80 ꢀ 0.85
25.19 ꢀ 0.95
21.80 ꢀ 1.55
32.56 ꢀ 0.25
22.10 ꢀ 1.00
29.06 ꢀ 1.10
15.74 ꢀ 0.65
28.11 ꢀ 0.52
26.01 ꢀ 1.00
18.07 ꢀ 0.85
20.47 ꢀ 1.89
j
k
l
9
9
m
n
25.80 ꢀ 1.55
41.89 ꢀ 1.20
28.48 ꢀ 1.45
22.20 ꢀ 1.40
23.80 ꢀ 0.65
28.1 ꢀ 2.10
9
o
20.13 ꢀ 1.40
3.50 ꢀ 0.10
17.22 ꢀ 1.30
6.10 ꢀ 0.09
21.60 ꢀ 0.60
4.90 ꢀ 0.05
DDP
a
Each compound was tested in triplicate. All error bars represent mean ꢀ SD from three independent experiments.
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HepG2 and MCF-7 cells than the urea derivatives (Table 2). activity for the cancer cells when compared to compounds 7f
Among the thiourea derivatives, compound 9f had the highest and 7a in the urea series. The follow-up studies focused on urea
anti-proliferative activity against H460 cells (14.52 ꢀ 0.10 mM) derivatives.
and MCF-7 cells (16.96 ꢀ 0.17 mM). Compound 9a had the
highest cellular anti-proliferative activity against HepG2 cells
2
.3. In vitro cytotoxicity of urea derivatives against two
(15.93 ꢀ 0.17 mM). Both displayed lower growth inhibition
normal cell lines
Twelve urea derivatives that showed prominent activity within
all three of the human cancer cell lines with IC50 values down to
a
Table 3 In vitro cytotoxicity of urea derivatives on normal cells
20 mM were assessed within the in vitro cytotoxic activity against
the human normal lung MCR-5 cell line and the normal hepa-
tocytes LO2 cell line. These urea derivatives' IC50 values for the
MCR-5 cells were above 45.0 mM. They exhibited good anti-
proliferative activity against the LO2 cells with IC50 values
MRC-5
LO2
IC50 (mM)
ꢀ S.D.
IC50 (mM)
ꢀ S.D.
b
c
Compound
RTI
RTI
(
aer 48 h of treatment) ranging from 4.0 mM to 35.0 mM, as
7
7
7
7
7
7
7
7
7
7
7
7
a
f
j
77.37 ꢀ 0.07
>100
13.41
>200
5.80
4.70
6.65
7.03
>10.26
5.07
5.82
>6.89
7.42
5.43 ꢀ 0.09
5.52 ꢀ 0.04
33.32 ꢀ 0.09
7.71 ꢀ 0.07
13.95 ꢀ 0.14
3.44 ꢀ 0.10
29.64 ꢀ 0.13
5.12 ꢀ 0.09
14.73 ꢀ 0.07
5.19 ꢀ 0.06
4.46 ꢀ 0.05
7.64 ꢀ 0.05
39.69 ꢀ 0.16
0.92
19.71
shown in Table 3. This demonstrated these compounds were
68.25 ꢀ 0.11
56.35 ꢀ 0.08
85.76 ꢀ 0.13
73.86 ꢀ 0.11
>100
50.34 ꢀ 0.12
63.71 ꢀ 0.06
>100
1.51 less toxic for the MCR-5 cells than for the LO2 cells. The IC50
k
n
s
t
u
v
w
x
y
0.59
0.63
0.22
3.30
0.73
ratio of the normal cells to the cancer cells was termed the
relative toxicity index” (RTI). The RTI was estimated to inves-
“
tigate their sensitivity between the normal and the tumour cells
in the two human tissue systems (lung and liver). In the lung
3.23 system, the tested urea derivatives were sensitivity to cancer
0.39
0.71
0.73
6.51
H460 cells, with RTI values up to 4.70. Most of the urea deriv-
atives in the liver system showed lower selective between the
human liver cancer HepG2 cells and the normal hepatocytes
LO2 cell line (RTI < 2). This excluded compounds 7f, 7t and 7v.
The results indicated that the normal lung MCR-5 cells were
more resistant to the toxic activity of these compounds than the
lung cancer H460 cells. A majority of the compounds did not
46.38 ꢀ 0.09
67.35 ꢀ 0.12
>100
5.96
>28.57
DDP
a
Each compound was tested in triplicate. All error bars represent mean
b
ꢀ
SD from three independent experiments. The IC50 ratio is between
c
lung normal MRC-5 cells to cancer H460 cells. The IC50 ratio is
between liver normal LO2 cells to cancer HepG2 cells.
Fig. 1 The Nur77 activation of compounds 7n, 7w, and 7s in H460 cells and their apoptosis induction in H460, HepG2, MCF-7 and NIH-3T3. (A)
Time-course analysis. H460 cancer cells treated with compounds (10 mM) for 0.5 h, 1 h, 3 h, 5 h, and 7 h were analysed to detect Nur77 levels via
western blotting. (B) Dose dependent effect of compounds to active Nur77. H460 cancer cells treated with compounds (5 mM, 10 mM, and 15 mM)
for 3 h were analysed via western blotting. (C) The apoptotic effects of compounds 7n, 7s, and 7w in H460, HepG2 and MCF-7 cancer cells. These
three cancer cell lines were initially treated with 10 mM compounds for 24 h and then analysed for the cleavage of Parp protein via western
blotting. (D) The apoptotic effects of compounds in NIH-3T3 cells.
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possess a selective resistance against the HepG2 cancer cells or 2.6. Compound 7s induced apoptosis depending the Nur77
the LO2 normal hepatocytes cells.
activation and the best growth inhibition of H460 cancer cells
We used RNA interference technology to conrm that the
apoptosis effect of these compounds was related with the acti-
vation of Nur77. The H460 cancer cells were transfected with
2
.4. The Nur77 activation ability of compounds with IC50
values below 20 mM in H460
A popular research model has showed that H460 cancer cells are shNur77 or shctr for 24 h and then treated with 10 mM of the
10,40
sensitive to the Nur77 dependent apoptotic effect.
Our compounds for an additional 24 h. The cleavage of PARP and
synthesized urea derivatives had a good sensitivity between the the expression of Nur77 were examined (Fig. 2(A)). The
normal MCR-5 cells and the tumour H460 cells that were iso- compounds still induced Parp cleavage in cells transfected with
lated from human lung tissue. So, we investigated whether our the shctr. In cells transfected with the shNur77, the Nur77 was
synthesized urea derivatives with IC50 values below 20 mM could effectively blocked. The degree of PARP cleavage of 7s and 7n
activate Nur77 in the H460 cancer cells. There were three was signicantly down regulated, particularly in 7s. There was
compounds (7n, 7s, and 7w) that activated Nur77. Results little change for 7w. This suggested that Nur77 could play a key
showed that Nur77 was activated upon treatment with 10 mM of role in the apoptosis effect of 7s in H460 cells.
the compounds (7n, 7s, and 7w) in a time-dependent manner
Fig. 1(A)). Nur77 activation was observed in H460 cells treated cells was conrmed with MTT assays. The effects of these
The cell growth inhibition of 7n, 7s, and 7w in H460 cancer
(
with 7n/7s for 0.5 h. This activation was delayed for 1 h in compounds on the growth of H460 cells at various concentra-
compound 7w. Nur77 was activated further when treated for 3 h tions (5 mM, 10 mM, and 15 mM) were tested at 24 h, 48 h, 72 h,
and 5 h with any of the compounds. The activation was main- and 96 h (Fig. 2(B)). The results showed that these compounds
tained at a high level at 7 h. We examined whether Nur77 inhibited the proliferation of the H460 cell line in a dose- and
activation was also dose-dependent. H460 cancer cells were time-dependent manner. Compound 7s demonstrated superior
treated with various concentrations of each compound for 3 h (5 growth inhibition on H460 cells. At exposure times up to 96 h,
mM, 10 mM, and 15 mM) (Fig. 1(B)). As the concentration of each
compound increased, the activation of Nur77 improved. Note,
when equal amounts of protein lysates (40 mg) was added, 7s
was more effective in up regulating Nur77 than 7n and 7w. 7w
was the weakest compound.
2.5. The apoptosis induction of 7n, 7w and 7s in H460,
HepG2, MCF-7, and NIH-3T3
Apoptosis is a popular approach used by a majority of the
38
anticancer drugs to kill tumour cells. Apoptosis stimulation is
considered standard and is the best strategy for anticancer
39
therapy. Compounds 7n, 7s, and 7w were selected to study the
apoptotic effects in various human cancer cells. These cancer
cell lines were treated with the three compounds at 10 mM for
24 h to induce apoptosis. The cleavage of PARP protein was used
as a sensitive apoptotic marker that occurred early in the
apoptotic response. It was then examined by western blotting.
The PARP cleavage was observed in various degrees in the tested
cell lines (Fig. 1(C)). The induction of PARP cleavage of all three
compounds was apparent in H460 cancer cells. Compounds 7n
and 7s had better activity than 7w. Compounds 7n and 7s
induced the cleavage of PARP protein in HepG2 cancer cells, but
7w did not have an effect. Compound 7s was the only compound
that slightly induced PARP cleavage in MCF-7 cancer cells.
These results suggested that 7n, 7s, and 7w were more potent
inducers of apoptosis in H460 cancer cell line than the other
two cell lines. Compound 7s showed higher apoptosis against
these three human cancer cell lines than compounds 7w and
Fig. 2 The compounds induced apoptosis and inhibited growth of
H460 cells. (A) Nur77 expression was required for the apoptotic effect
of 7n, 7s, and 7w in H460 cells. H460 cancer cells were transfected
with control or Nur77 shRNA for 24 h, then treated with or without 10
7
n. The apoptotic effect of these compounds at various mM of the compounds for an additional 24 h. The Parp cleavage and
Nur77 expression were analysed via western blotting. (B) Growth
inhibition activities of compounds on H460 cells were assessed with
MTT assay. H460 cancer cells treated with these three compounds at
various concentrations (5 mM, 10 mM and 15 mM) and tested at 24 h,
concentrations was tested in NIH-3T3 mouse embryo cells over
2
4 h. The results showed that all of the three compounds did
not induce PARP cleavage at various concentrations for 24 h
Fig. 1(D)). This indicated that these three compounds had little
effect on normal cells.
(
48 h, 72 h, and 96 h time points. DMSO was used as the negative
control.
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the inhibition rates of 7n at the various concentrations (5 mM, 2.7. Molecular docking study of 7s and Nur77
1
0 mM and 15 mM) were 35.02%, 70.73%, and 86.50%. The
The in vitro bioassay revealed the high ability of compound 7s
when activating Nur77 in H460 cancer cells. We performed
a molecular docking study for 7s and Nur77. The docking study
revealed that compound 7s exhibited a promising binding
inhibition rates for 7w were 18.02%, 34.47%, and 51.71%. The
inhibition rates for 7s were 75.70%, 95.72%, and 96.05%. The
anti-proliferation abilities of these three compounds were
similar to their apoptosis induction and Nur77 activation effects
in H460 cells.
affinity with Nur77 with
a docking binding energy of
ꢂ1
ꢂ7.30 kcal mol . The specic non-bonded interactions
between 7s and the residues of Nur77 in 3D and 2D are shown
in Fig. 3. The urea group of compound 7s formed a hydrogen
bonding interaction with the backbone's carbonyl group of
amino acid LEU175. The moiety of diuorobenzene inserted
deeply into the hydrophobic pocket formed by PHE172,
LEU224, VAL167, PHE158, LEU162 and LEU118. The adaman-
tine group extended out of the pocket and interacted with the
surface of ALA201, ASN198, and GLN197 via hydrophobic
interactions. The hydrogen bond between 7s and Nur77 could
still exist even though the LEU175 mutated to other amino acids
(like an alanine). This indicated that the hydrogen bond
between 7s and the backbone of LEU175 could be important for
the affinity and selection of the complex 7s–Nur77.
3
. Conclusion
In this study, we synthesized and conducted a biological eval-
uation of two new series of 1-(2-(adamantan-1-yl)-1H-indol-5-yl)-
3-substituted urea/thiourea derivatives as potential anticancer
agents. The synthetic method was relatively simple. The target
compounds were easily puried and produced in high yields.
Among of synthesized compounds, most showed moderate
anti-proliferative activity against all three of the tested cancer
cell lines. The compounds with prominent anticancer activity
also exhibited good sensitivity between the normal (MRC-5) and
the tumour (H460) cell lines that were isolated from lung tissue.
Three compounds that could active Nur77 were obtained: 7n,
7s, and 7w. They were evaluated for their abilities to induce
apoptosis in various cells and the inhibition of cell growth in
H460 cancer cells. The results showed that 7n and 7s achieved
better anticancer activity than 7w. The shRNA experiment
revealed that the apoptosis effect of 7n and 7s was dependent on
Nur77, particularly in 7s. Compound 7w could have an alter-
native mechanism. The molecular docking suggested that
compound 7s exhibited a promising binding affinity with
Fig. 3 Molecular docking 3D and 2D models of 7s and Nur77. (A) The
interactions between Nur77 are displayed on the grey surface Nur77. These results are helpful in the development of a Nur77
presentation. The 7s is represented in the yellow stick. The protein
surface conformation is the crystal structure of homodimer of the
targeted drug. Further studies are also required to determine
their roles in preventing cancer.
orphan nuclear receptor Nur77 (PDB ID: 4RE8). The moiety of
difluorobenzene of ligand inserted deeply into the hydrophobic
pocket. (B) Close-up view depiction of the superposition of Nur77 LBP
bound to 7s. The Nur77 binding site is presented with a coloured
ribbon. Neighbouring amino acids were displayed in lines within
a distance of 5 A˚ approximately to 7s (the hydrogen bond between 7s
and LEU 175 are shown as yellow dots). (C) 2D binding model of 7s with
4. Experimental section
4.1. Chemistry
All reagents were commercially available and used without
Nur77: hydrogen bonds are indicated with solid arrows, colour lines further purication, unless otherwise indicated. Reactions were
around 7s stand for the binding pocket and the residues in colours
nearby established the pocket. The green colour denotes the hydro-
phobic nature of amino acids, the red colour denotes the acid amino
acids, the purple denotes the alkalinity of amino acids, the cyan
magnetically stirred and monitored by thin-layer chromatog-
raphy (TLC) on Merck silica gel 60F-254 by uorescence. The
target compounds were puried by column chromatography.
The purities of the target compounds were assessed by high
performance liquid chromatography (HPLC) with a COSMOSIL
denotes the polar amino acids, and the grey points of ligand atoms
denotes the solvent accessibility.
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C18-MS-II column (250 mm ꢃ 4.6 mm i.d., 5 mm). The HPLC a yellow product. The product was isolated via ltration and
analysis was performed on an Agilent Technologies 1100 Series washed with cold water. Aer 12 h of drying under vacuum,
1
HPLC system. The mobile phase was acetonitrile (A) and water 2.89 g of yellow product was isolated (97%). H NMR (600 MHz,
(
B), eluted as the following gradient program: A from 80% to DMSO-d ): d 11.65 (br s, 1H, NH), 8.44 (d, J ¼ 2.2 Hz, 1H, Ar–H),
6
1
1
2
00% and B from 20% to 0% during 0–20 min. The ow rate was 7.93 (dd, J ¼ 2.2, 9.0 Hz, 1H, Ar–H), 7.44 (d, J ¼ 8.8 Hz, 1H, Ar–
ꢂ1
1
0
mL min and the detection wavelengths were 254 nm and H), 6.38 (d, J ¼ 1.5 Hz, 1H, H-3 ), 2.04–2.08 (m, 3H, Ad–H), 1.96–
13
13
80 nm. H-NMR and C-NMR spectra were obtained using 1.99 (m, 6H, Ad–H), 1.72–1.80 (m, 6H, Ad–H); C NMR (150
0
a Bruker AV2 600 Ultra shield spectrometer at 600 and 150 MHz MHz, DMSO-d
6
): d 154.0 (C-2 ), 140.8 (C–NO
2
), 139.9 (Ar–CH),
respectively. Chemical shis were given in parts per million 127.7 (Ar–C), 116.9 (Ar–C), 116.3 (Ar–CH), 111.4 (Ar–CH), 98.2
0
(
ppm) relative to tetramethyl-silane (TMS) as an internal stan- (C-3 ), 41.9 (Ad–C), 36.6 (s, 3C, Ad–C), 34.1 (s, 3C, Ad–C), 28.2 (s,
+
dard. Multiplicities were abbreviated as follows: single (s), 3C, Ad–C); ESI-HRMS (+): m/z [M + H] calculated for
doublet (d), doublet–doublet (dd), doublet–triplet (dt), triplet C H N O , 297.1598, found 297.1599; [M + Na] calculated
t), triplet–triplet (tt), triplet–doublet (td), quartet (q), quartet- for C18
+
+
1
8
21
2
2
+
(
H
20
N
2
O
2
Na , 319.1417, found 319.1417.
doublet (qd), multiplet (m), and broad signal (br s). High-
4.1.4. 2-Adamantane-1H-indol-5-amine (5). Compound 4
resolution mass spectral (HRMS) data were acquired on a Q (2.96 g, 10 mmol) was dissolved in 150 mL of ethanol. 10% Pd/C
Exactive. Melting points were measured on a SGWX-4 micro- was added (300 mg), and the mixture was subjected to H (38
melting point spectrometer and were uncorrected. psi) using a Parr hydrogenator for 3.5 h. The mixture was
.1.1. N-o-Tolylcycloadamantanecarboxamide ltered over Celite, which was washed with methanol. Aer
2
4
(2).
A
mixture of 10.0 mmol of o-toluidine, 10.0 mmol of 1-ada- concentration and drying under vacuum, 2.5 g of a brown
1
mantanecarbonyl chloride, and 7.0 mmol of anhydrous potas- powder (94%) was isolated. H NMR (600 MHz, CDCl ): d 7.80 (s,
3
sium carbonate in 50 mL of toluene was stirred at room 1H, NH), 7.10 (d, J ¼ 8.4 Hz, 1H, Ar–H), 6.86 (d, J ¼ 2.2 Hz, 1H,
temperature for 4 h. The resulting solid was rst ltered off and Ar–H), 6.57 (dd, J ¼ 2.2, 8.4 Hz, 1H, Ar–H), 6.05 (dd, J ¼ 2.2 Hz,
0
stirred at room temperature for 1.5 h with 50 mL of H
2
O. The 1H, H-3 ), 3.45 (br s, 2H, NH
2
), 2.06–2.12 (m, 3H, Ad–H), 1.94–
1
3
solid was ltered off and recrystallized from the appropriate 1.98 (m, 6H, Ad–H), 1.75–1.81 (m, 6H, Ad–H); C NMR (150
1
0
solvent. White solid (75.5%). H NMR (600 MHz, CDCl
3
): d 7.88 MHz, CDCl
3
): d 149.9 (C-2 ), 139.3 (C–NH
2
), 130.3 (Ar–C), 129.4
0
(
d, J ¼ 8.1 Hz, 1H, Ar–H), 7.18–7.24 (m, 2H, C]ONH and Ar–H), (Ar–C), 111.6 (Ar–CH), 110.8 (Ar–CH), 105.3 (Ar–CH), 95.5 (C-3 ),
7
2
.17 (d, J ¼ 7.5 Hz, 1H, Ar–H), 7.05 (dt, J ¼ 1.1, 7.4 Hz, 1H, Ar–H), 42.6 (Ad–C), 36.8 (s, 3C, Ad–C), 33.7 (s, 3C, Ad–C), 28.5 (s, 3C,
+
+
.26 (s, 3H, Ar–CH ), 1.99–2.13 (m, 3H, Ad–H), 1.97–2.01 (m, 6H, Ad–C); ESI-HRMS (+): m/z [M + H] calculated for C H N ,
3
18 23 2
+
Ad–H), 1.73–1.80 (m, 6H, Ad–H); ESI-HRMS (+): m/z [M + H]
267.1856, found 267.1858.
4.1.5. 5-Isocyanato-1H-indole-adamantane (6). To a solu-
tion of ethyl 5-amino-1H-indole-2-adamantane 2.66 g (10.0
+
+
calculated for C18
calculated for C18
H
H
24NO , 270.1852, found 270.1853; [M + Na]
+
23NONa , 292.1672, found 292.1669.
4
.1.2. 2-Adamantane-1H-indole (3). A stirred solution of mmol) dissolved in THF (80 mL) was slowly dropped into a stir-
0 mmol of N-o-tolylcycloadamantanecarboxamide (2) in 50 mL red solution of triphosgene 2.98 g, (10.0 mmol) in THF (10 mL)
of THF under a N atmosphere was maintained at an internal by using a constant-pressure dropping funnel. NEt (3 mL, 21.0
1
2
3
ꢁ
temperature of ꢂ5 to 5 C and treated dropwise with 0.1– mmol) was then added slowly to the reaction mixture aer the
0
.15 mol of n-BuLi as 2.5 M n-BuLi in hexane. The stirred ethyl 5-amino-1H-indole-2-adamantane was added. The reaction
ꢁ
mixture was kept at ambient temperature, cooled in an ice bath, mixture was stirred at 0 C for 2 h. Aer completion of the
and treated dropwise with 12 mL of 2 M HCl. The organic layer reaction, the mixture was concentrated and puried by column
was separated and the aqueous layer washed with C
combined organic layer was dried with anhydrous MgSO
ltered, and concentrated in vacuo. Recrystallization of the MHz, CDCl
6 6
H . The chromatography using appropriate mixtures of EtOAc and PE to
1
4
,
yield the titled compound. White solid (61.5%). H NMR (600

3
): d 8.01 (s, 1H, NH), 7.21 (d, J ¼ 8.4 Hz, 1H, Ar–H),
0
residue was from the appropriate solvent. White solid (56.5%). 6.85 (dd, J ¼ 2.0, 8.4 Hz, 1H, Ar–H), 6.17 (d, J ¼ 1.5 Hz, 1H, H-3 ),
1
H NMR (600 MHz, CDCl ): d 7.30 (d, J ¼ 7.9 Hz, 1H, Ar–H), 7.18 2.07–2.15 (m, 3H, Ad–H), 1.95–2.00 (m, 6H, Ad–H), 1.74–1.85 (m,
3
1
3
0
(
dd, J ¼ 0.6, 8.1 Hz, 1H, Ar–H), 6.88 (dt, J ¼ 1.1, 7.5 Hz, 1H, Ar– 6H, Ad–H); C NMR (150 MHz, CDCl ): d 151.0 (C-2 ), 133.2 (Ar–
3
0
H), 6.77–6.83 (m, 1H, Ar–H), 5.98 (d, J ¼ 0.6 Hz, 1H, H-3 ), 1.96– C), 129.0 (Ar–C), 125.1 (Ar–C), 123.6 (N]C]O), 118.1 (Ar–CH),
0
2
.00 (m, 3H, Ad–H), 1.91–1.94 (m, 6H, Ad–H), 1.70–1.78 (m, 6H, 115.5 (Ar–CH), 111.1 (Ar–CH), 96.4 (C-3 ), 42.5 (Ad–C), 36.7 (s, 3C,
+
+
Ad–H); ESI-HRMS (+): m/z [M + H] calculated for C18
2
2
H
22N , Ad–C), 33.8 (s, 3C, Ad–C), 28.4 (s, 3C, Ad–C).
+
+
52.1747, found 252.1748; [M + Na] calculated for C18
74.1566, found 274.1568.
H
21NNa ,
4.1.6. General method for the synthesis of compounds 7a–
7y. To a solution of 5-isocyanato-1H-indole-adamantine 6
4.1.3. 2-Adamantane-5-nitro-1H-indole (4). In a 250 mL (0.12 g, 0.4 mmol) in toluene (5 mL) aliphatic, aromatic or
round-bottom ask, 2.51 g of 2-adamantane-1H-indole (10 heteroaromatic amine (0.4 mmol) was added, and the reaction
ꢁ
mmol) was dissolved in 10 mL of H SO aer vigorous stirring. mixture was heated at 60 C for 4 h. Aer completion of the
2
4
In a separate ask, 0.94 g of NaNO
3
(1.1 ꢃ 10 mmol) was dis- reaction, the mixture was concentrated and puried by column
solved in 10 mL of H SO , also aer vigorous stirring, and then chromatography using appropriate mixtures of CH Cl and
2
4
2
2
was added dropwise (via addition funnel) to the above mixture. MeOH.
Aer addition, the reaction mixture was stirred for another 4.1.6.1 Compound 7n. White solid; yield: 87.3%; HPLC
0 min and then poured into 200 mL of ice water, precipitating purity: 95.5% (t
ꢁ
1
1
R
¼ 21.05 min); mp: 265–267 C; H NMR (600
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0
MHz, DMSO-d
6
): d 10.74 (s, 1H, NH-1 ), 8.90 (s, 1H, C]ONH-1), mmol) and triethylenediamine (2.08 g, 18.6 mmol) in 40 mL of
0
8
.46 (s, 1H, C]ONH-3), 8.04 (s, 1H, H-2 ), 7.56 (d, J ¼ 1.5 Hz, 1H, toluene, CS (3.5 g, 46.5 mmol) was added slowly. The resulting
2
0
H-4 ), 7.54–7.56 (m, 1H, Ar–H), 7.46–7.51 (m, 1H, Ar–H), 7.27 (d, mixture was stirred at room temperature for 8 h. Aer ltration,
0
J ¼ 7.7 Hz, 1H, Ar–H), 7.21 (d, J ¼ 8.4 Hz, 1H, H-7 ), 7.01 (dd, J ¼ the lter cake was washed with toluene and dried. Obtained
0
0
2
3
.0, 8.4 Hz, 1H, H-6 ), 6.03 (d, J ¼ 1.7 Hz, 1H, H-3 ), 2.03–2.08 (m, intermediate was suspended in 40 mL of DCM. Then, a 25 mL of
13
H, Ad–H), 1.92–1.99 (m, 6H, Ad–H), 1.72–1.80 (m, Ad–H);
C
DCM solution of BTC (5.0 g, 17.0 mmol) was added slowly under
0
ꢁ
NMR (150 MHz, DMSO-d
6
): d 153.4 (C]O), 150.8 (C-2 ), 141.5 0–5 C. The mixture was stirred for 2 h at room temperature,
00
0
0
00
(
C-1 ), 133.0 (C-8 ), 131.1 (C-5 ), 130.3 (C-5 ), 129.96 (q, J ¼ and then was reuxed for 1.5–2 h. Aer ltration, the ltrate was
0
3
(
4
0.8 Hz, CF
3
C), 128.4 (C-9 ), 124.8 (q, J ¼ 271.8 Hz, CF
3
), 122.0 concentrated in vacuo. The mixture was concentrated and
00
00
0
C-6 ), 118.0 (q, J ¼ 4.4 Hz, C-2 ), 114.35 (C-6 ), 114.30 (q, J ¼ puried by column chromatography using appropriate mixtures
00
0
0
0
.4 Hz, C-4 ) 111.1 (C-7 ), 110.5 (C-4 ), 95.4 (C-3 ), 42.3 (Ad–C), of EtOAc and PE to yield the titled compound. White solid
1
3
6.8 (s, 3C, Ad–C), 33.9 (s, 3C, Ad–C), 28.4 (s, 3C, Ad–C); ESI- (62.5%). H NMR (600 MHz, CDCl ): d 8.12 (br s, 1H, NH), 7.40
3
+
+
0
0
0
HRMS (+): m/z [M
+
H] calculated for
C
26
H
27
F
3
N
3
O , (d, J ¼ 1.8 Hz, 1H, H-4 ), 7.23 (d, J ¼ 8.4 Hz, 1H, H-7 ), 6.98 (dd, J
+
0
4
N
54.2101, found 454.2101; [M + Na] calculated for C26
H
26
F
3
-
¼ 2.0, 8.4 Hz, 1H, H-6 ), 6.20 (d, J ¼ 1.5 Hz, 1H, H-3 ), 2.08–2.14
(m, 3H, Ad–H), 1.94–1.99 (m, 6H, Ad–H), 1.74–1.85 (m, 6H, Ad–
+
3
ONa , 476.192, found 476.1922.
1
3
0
0
4
.1.6.2 Compound 7s. White solid; yield: 89.6%; HPLC H); C-NMR (150 MHz, CDCl ): d 151.4 (C-2 ), 134.1 (C-8 ), 131.8
3
ꢁ
1
0
0
0
0
purity: 96.4% (t
R
¼ 17.89 min); mp: 178–181 C; H NMR (600 (C-9 ), 128.7 (C-5 ), 122.6 (N]C]S), 119.0 (C-6 ), 117.3 (C-7 ),
0
0
0
MHz, DMSO-d ): d 10.73 (d, J ¼ 1.3 Hz, 1H, NH-1 ), 8.72 (s, 1H, 111.2 (C-4 ), 96.8 (C-3 ), 42.5 (Ad–C), 36.7 (s, 3C, Ad–C), 33.9 (s,
6
C]ONH-1), 8.36 (d, J ¼ 2.0 Hz, 1H, C]ONH-3), 8.12–8.17 (m, 3C, Ad–C), 28.4 (s, 3C, Ad–C).
0
0
0
00
1
H, 6 ), 7.56 (d, J ¼ 1.8 Hz, 1H, H-4 ), 7.26–7.30 (m, 1H, 5 ), 7.20
4.1.8. General method for the synthesis of compounds 9a–
0
00
(
8
d, J ¼ 8.4 Hz, 1H, 7 ), 7.01–7.05 (m, 1H, H-3 ), 6.99 (dd, J ¼ 2.1, 9o. To a solution of 5-isocyanato-1H-indole-adamantane (8)
0
0
.5 Hz, 1H, H-6 ), 6.02 (d, J ¼ 1.7 Hz, 1H, H-3 ), 2.03–2.08 (m, 3H, (0.12 g, 0.4 mmol) in toluene (5 mL), aliphatic, aromatic, or
13
Ad–H), 1.93–1.98 (m, 6H, Ad–H), 1.72–1.80 (m, 6H, Ad–H);
C
heteroaromatic amine (0.4 mmol) was added, and the reaction
ꢁ
NMR (150 MHz, DMSO-d
6
): d 156.8 (dd, J ¼ 12.1, 241.0 Hz, CF- mixture was heated at 60 C for 4 h. Aer completion of the
0
0
0
00
2
2
1
), 153.1 (C]O), 152.3 (dd, J ¼ 12.1, 241.5 Hz, CF-4 ), 150.8 (C- reaction, the mixture was concentrated and puried by column
0
0
0
), 132.9 (C-8 ), 131.2 (C-5 ), 128.4 (C-9 ), 125.1 (dd, J ¼ 3.3, chromatography using appropriate mixtures of CH Cl2 and
2
00
00
0
1.0 Hz, C-1 ), 121.9 (dd, J ¼ 3.3, 8.8 Hz, C-6 ), 113.9 (C-6 ), 111.4 MeOH.
0
0
0
0
(
dd, J ¼ 23.1, 24.2 Hz, C-5 ), 111.1 (C-7 ), 110.0 (C-4 ), 104.1 (dd, J
4.1.8.1 Compound 9a. White solid; yield: 84.6%; mp: 285–
00
0
ꢁ
1
¼
3.3, 21.3 Hz, C-3 ), 95.4 (C-3 ), 42.3 (Ad–C), 36.8 (s, 3C, Ad–C), 287 C; HPLC purity: 97.3% (t
R
¼ 21.07 min); H NMR (600
+
0
3
3.9 (s, 3C, Ad–C), 28.4 (s, 3C, Ad–C); ESI-HRMS (+): m/z [M + H]
MHz, DMSO-d
6
): d 10.89 (s, 1H, NH-1 ), 9.15 (s, 1H, C]SNH-1),
+
0
0
calculated for C25
H
26
F
2
N
3
O
422.2038, found 422.2030; [M + 7.32 (d, J ¼ 1.7 Hz, 1H, H-4 ), 7.25 (d, J ¼ 8.4 Hz, 1H, H-7 ), 6.87
+
+
0
Na] calculated for C25
H
25
F
2
N
3
ONa , 444.1850, found 444.1852. (dd, J ¼ 1.9, 8.5 Hz, 1H, H-6 ), 6.73 (br s, 1H, C]SNH-3), 6.07 (d,
0
(ESI Fig. S1†)
J ¼ 1.5 Hz, 1H, H-3 ), 2.03–2.09 (m, 3H, Ad–H), 1.93–1.98 (m, 6H,
1
3
4
.1.6.3 Compound 7w. White solid; yield: 85.8%; HPLC Ad–H), 1.72–1.80 (m, 6H, Ad–H), 1.45 (s, 9H, (CH ) C); C NMR
3
3
ꢁ
1
0
0
purity: 94.8% (t ¼ 20.80 min); mp: 271–273 C; H NMR (600 (150 MHz, DMSO-d ): d 180.3 (C]S), 151.1 (C-2 ), 134.3 (C-8 ),
R
6
0
0
0
0
0
0
MHz, DMSO-d ): d 10.74 (s, 1H, NH-1 ), 8.76 (s, 1H, C]ONH-1), 130.2 (C-5 ), 128.4 (C-9 ), 118.9 (C-6 ), 116.5 (C-7 ), 111.3 (C-4 ),
6
0
0
0
8
1
1
.55 (s, 1H, C]ONH-3), 7.88–7.98 (m, 1H, H-6 ), 7.56 (d, J ¼ 95.7 (C-3 ), 53.0 ((CH
3
)
3
C), 42.2 (Ad–C), 36.8 (s, 3C, Ad–C), 33.9
0
00
.7 Hz, 1H, H-4 ), 7.23–7.27 (m, 1H, H-5 ), 7.21 (d, J ¼ 8.6 Hz, (s, 3C, Ad–C), 29.2 (s, 3C, (CH C), 28.4 (s, 3C, Ad–C); ESI-HRMS
3 3
)
0
0
+
+
H, 7 ) 6.99 (dd, J ¼ 2.0, 8.6 Hz, 1H, H-6 ), 6.03 (d, J ¼ 1.7 Hz, 1H, (+): m/z [M + H] calculated for C23
32 3
H N S , 382.2311, found
0
+
+
31 3
H-3 ), 2.03–2.08 (m, 3H, Ad–H), 1.93–1.98 (m, 6H, Ad–H), 1.72– 382.2307; [M + Na] calculated for C23H N SNa , 404.2131,
1
3
1
.80 (m, 6H, Ad–H); C NMR (150 MHz, DMSO-d
6
): d 152.9 (C] found 404.2126.
0
00
O), 150.8 (C-2 ), 145.3 (ddd, J ¼ 2.2, 9.9, 242.1 Hz, CF-2 ), 141.7
At the series of thiourea derivatives, the other nal
0
0
(
ddd, J ¼ 3.3, 11.0, 245.4 Hz, CF-4 ), 139.5 (dt, J ¼ 14.3, 246.5 Hz, compounds were synthesized following the general procedure
00
00
0
0
CF-3 ), 126.5 (dd, J ¼ 3.3, 7.7 Hz, C-6 ), 133.0 (C-8 ), 131.0 (C-5 ), as described above with the yield of 84–92%. Their structures
0
00
0
1
28.4 (C-9 ), 115.2 (m, C-1 ), 114.0 (C-6 ), 112.1 (dd, J ¼ 3.3, were characterized by NMR, ESI-HRMS and HPLC analyses. (see
00
0
0
0
1
7.1 Hz, C-5 ), 111.1 (C-7 ), 110.1 (C-4 ), 95.4 (C-3 ), 42.3 (Ad–C), ‘ESI†’).
3
6.8 (s, 3C, Ad–C), 33.9 (s, 3C, Ad–C), 28.4 (s, 3C, Ad–C); ESI-
+
+
HRMS (+): m/z [M
+
H] calculated for
C
25
H
25
F
3
N
3
O ,
+
4.2. Biology materials and methods
440.1944, found 440.1938; [M + Na] calculated for C25
H
24
F
3
-
+
N ONa , 462.1764, found 462.1760.
4.2.1. Reagents and antibodies. 3-(4,5-Dimethylthiazol-2-
3
At the series of urea derivatives, the other target compounds yl)-2,5-diphenyltetrazoliumbromide (MTT) and DMSO were
were synthesized following the general procedure as described purchased from Sigma-Aldrich. TurboFect Transfection
above with the yield of 84–90%. Their structures were conrmed Reagent, goat anti-rabbit and anti-mouse secondary antibody
1
13
by the spectroscopic data including H NMR, C NMR, and ESI- conjugated to horseradish peroxidase were from Thermo Fisher
HRMS (see ‘ESI†’). Scientic. Anti-Nur77 (3960S), anti–poly (ADP ribose) poly-
.1.7. 5-Isocyanato-1H-indole-adamantane (8). To a solu- merase (Parp) (9542) and anti-b-actin (4970) were from Cell
tion of ethyl 5-amino-1H-indole-2-adamantane (4.1 g, 15.5 Signal Technology. Enhanced chemiluminescence reagents
4
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40
from GE Healthcare and a cocktail of proteinase inhibitors from Gaussian 09 package at the B3LYP/6-31G (d) level to serve as
Roche were used in this study. All other chemicals used were the docking ligand. The X-ray crystal structure of the Nur77 was
commercial products of analytic grade obtained from Sigma.
downloaded from the protein Data Bank (PDB ID: 4RE8). All of
4.2.2. Cell culture. All of the human cancer cell lines were the water molecules and other subunits (GOL, 3 MJ) were then
obtained from the American Type Culture Collection (ATCC). removed, missing side chains were lled and hydrogen atoms
MCR-5 normal lung cells were cultured in minimum essential were added, using the Schr ¨o dinger 2016-1 soware (LLC, New
medium (MEM) supplemented with 10% fetal bovine serum York, NY, USA, 2016). The resulting hydrogen-Nur77 was used
and kept in a CO
atmosphere of 5% CO
2
incubator which consisted of a humidied as the target receptor for molecular docking.
ꢁ
2
at 37 C with 90% humidity. NCI-H460
4.3.2. The docking experiments. Molecular docking was
lung cancer cells and LO2 normal hepatocytes cells were carried out using the Glide programme (Small-Molecule Drug
maintained in RPMI 1640 medium supplemented with 10% Discovery Suite 2016-1: Glide, version 6.6, Schr ¨o dinger, LLC,
ꢁ
fetal bovine serum, at 37 C under 5% CO2/95% air. MCF-7 NY, 2016.) in the standard precision (SP) mode to investigate the
breast cancer cells, HepG2 liver cancer cells, and NIH-3T3 mechanism of interaction between the compound 7s and
mouse embryo cells were grown in Dulbeccos Modifed Eagles protein Nur77. The binding site of native ligand 3 MJ was
Medium (DMEM) supplemented with 10% fetal bovine serum chosen for docking site. Throughout the docking process, the
ꢁ
in a humidied atmosphere containing 5% CO
2
at 37 C.
protein Nur77 was held rigid; only the torsional bonds of 7s
4.2.3. MTT assays. Cells were cultured in 96-well plate with were permitted to be free. Default values were used for all the
1
00 mL medium and the total amount of cells in each well was parameters during docking. The docking results presented here
4
41
about 1 ꢃ 10 . Concentration gradients (5–7 different concen- were analysed by using PyMOL soware and Maestro, version
42
trations) of each compound were then added to cells (three 10.1.
repeats), cisplatin (DDP) as the positive control and dimethyl
sulfoxide (DMSO) as the negative control. Aer 48 h treatment,
ꢂ1
Conflicts of interest
10 mL MTT (5 mg mL ) was added to each wells staining for 4
hours in cell incubator. Then in each well, medium was dis-
carded and 100 mL DMSO was used, aer short time shaking,
the light intensity was measured at a wavelength of 490 nm in
There are no conicts to declare.
Microplate Reader (Thermo). At last, the IC50 values (concen- Acknowledgements
trations required to inhibit 50% of cell growth) were calculated
using GraphPad Prism 5 soware and cell inhibition curves
were conducted.
This work was supported by the National Natural Science
Foundation of China (No. 81773600, 81302652, 31471273,
1376172 and 31461163002), the Project of South Center for
Marine Research (No. 14GYY023NF23). This research was also
nancially supported by Fujian Science and Technology project
No. 2014N5012). We also thank the College of Pharmaceutical
Sciences, Xiamen University and the Department of Chemistry,
College of Chemistry and Chemical Engineering, Xiamen
University for providing us with the Shrodinger 2015-1 soware
and Gaussian 09 soware.
4
4.2.4. Western blotting. Equal amounts of cell lysates were
electrophoresed on 10% SDS polyacrylamide gel electrophoresis
and transferred to the polyvinylidene diuoride (PVDF)
membranes (0.22 mM). The membranes were blocked with 5%
nonfat milk in TBST buffer (50 mM Tris-HCl, pH7.4, 150 mM
NaCl and 0.1% Tween 20) for 1 h at room temperature and

(
ꢁ
incubated with various primary antibodies overnight at 4 C.
Aer three TBST washes, the membranes were incubated with
HRP-conjugated anti-mouse or anti-rabbit antibody for 2 h at
room temperature. The nal immunoreactive products were
Notes and references
detected with an enhanced chemiluminescence (ECL) system.
0
4
.2.5. shRNA transfection. The shNur77 (5 -ACAGTC-
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