Synthesis, structure–activity relationship studies and biological evaluation of novel 2,5-disubstituted indole derivatives as anticancer agents

Article abstract of DOI:10.1111/cbdd.12808

Three novel series of 2,5-disubstituted indole derivatives were synthesized and evaluated in vitro for their antiproliferative activity against human cancer cells and HIV-1 inhibition activity used as a readout of cellular activity. Most compounds were found to have potent anticancer activity. In particular, 2c and 3b which showed effectively to repress HIV-1 transcription had a pan antiproliferative activity in cervical cancer cells (HeLa), breast cancer cells (MCF-7), liver cancer cells (HepG2), and lung cancer cells (H460 and A549). While 3b exhibited high sensitivity to A549 cells with the IC₅₀ value 0.48?±?0.15?μm, 2c showed high selectivity toward HepG2 cells with the IC₅₀ value 13.21?±?0.30?μm. With respect to the cellular mechanism of action, HepG2 cells treated with 2c and A549 cells treated with 3b for 24?h were studied by annexin V/PI staining and Western blot analysis, and results revealed that 2c and 3b may induce cancer cells apoptosis through inhibiting the phosphorylation at Ser2 of RNAPII CTD which can be phosphorylated by cyclin-dependent kinase 9. These studies indicated that 2c and 3b may develop as potent lead compounds in the therapy of cancer. However, determining their roles in preventing HIV-1 still requires further intensive study.

Full text of DOI:10.1111/cbdd.12808

Received Date : 24-Apr-2016
Revised Date : 05-Jun-2016
Accepted Date : 12-Jun-2016
Article type : Research Article
Synthesis, Structure-Activity Relationship Studies and Biological Evaluation of
Novel 2,5-Disubstituted Indole Derivatives as Anticancer Agents
a#
a#
a
a
a
a
Hongyu Hu , Jun Wu , Mingtao Ao , Huiru Wang , Tongtong Zhou , Yuhua Xue , Yingkun
a
a*
a*
Qiu , Meijuan Fang , Zhen Wu
a
School of Pharmaceutical Sciences and the Key Laboratory for Chemical Biology of Fujian Province,
Xiamen University, South Xiang-An Road, Xiamen, 361102, China.
#
These authors contribute equally to this paper
*
Corresponding authors. Tel.: +86-592-2189868; Fax: +86-592-2189868.
E-mail address: fangmj@xmu.edu.cn (Meijuan Fang); wuzhen@xmu.edu.cn (Zhen Wu)
Abstract
Three novel series of 2,5-disubstituted indole derivatives were synthesized and evaluated in vitro for
their anti-proliferative activity against human cancer cells and HIV-1 inhibition activity used as a
read-out of cellular activity. Most compounds were found to have potent anticancer activity. In
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12808
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particular, 2c and 3b which showed effectively to repress HIV-1 transcription had a pan anti-
proliferative activity in cervical cancer cells (Hela), breast cancer cells (MCF-7), liver cancer cells
(HepG2) and lung cancer cells (H460 and A549). While 3b exhibited high sensitivity to A549 cells with
the IC value 0.48±0.15 μM, 2c showed high selectivity toward HepG2 cells with the IC value
50
50
1
3.21±0.30 μM. With respect to the cellular mechanism of action, HepG2 cells treated with 2c and
A549 cells treated with 3b for 24 hours were studied by annexin V/PI staining and western blot
analysis, and results revealed that 2c and 3b may induce cancer cells apoptosis through inhibiting
the phosphorylation at Ser2 of RNAPII CTD which can be phosphorylated by cyclin-dependent kinase
9
. These studies indicated that 2c and 3b may develop as potent lead compounds in the therapy of
cancer. However, determining their roles in preventing HIV-1 still requires further intensive study.
Key words: 2,5-disubstituted indole derivatives; anticancer activity; HIV-1 inhibition activity;
phosphorylation; RNA polymerase II
Indole ring system as one of the most ubiquitous heterocycles in nature has been becoming an
important structural component in many pharmaceutical agents, such as antidepressant (1),
anticonvulsant (2,3), antifungal (4), antiviral (5) and anti-inflammatory (6), particularly in discovery of
new antitumor agents (7) or HIV inhibitors (3,8,9). In recent years, increasing numbers of researchers
from both industry and academia have embarked on the development of new indole-based small
molecules as potent antitumor agents (10) or HIV inhibitors (9). For example, small molecule Bcl-2
antagonist Obatoclax (phase III, Figure 1) could antagonize MCL-1 and overcome MCL-1-mediated
resistance to apoptosis (11). In addition, Sunitinib (Figure 1), a multi-targeted receptor tyrosine
kinase inhibitor containing indolin-2-one moiety, was approved by the FDA for the treatment of
advanced renal cell carcinoma (RCC) and gastrointestinal stromal tumors (GISTs) (12). Moreover,
benzyl-1H-indole derivatives also possessed prominent antitumor activity (13), such as a novel
synthetic microtubule inhibitor D-24851 (Phase I/II, Figure 1) (14). On the other hand, 3-aryl-
phospho-indole (API) was developed as novel HIV-1 non-nucleoside reverse transcriptase inhibitors
(
15,16). For example, API-18 (Figure 1) possessed excellent potency against wild-type HIV-1 with the
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EC value 0.1 nM (15,16). All these prompted us to investigate new potential anticancer agents and
50
nd
th
HIV inhibitors with the indole structure which had 2 and 5 position substitutions.
Figure 1. Structures of four compounds bearing indole-based moiety
Messenger RNA (mRNA) is a large family of RNA molecules that convey genetic information from
DNA to the ribosome, and inhibition of mRNA synthesis can reduce cancer cells apoptosis (17) which
is an attractive strategy for cancer treatment (18). mRNA synthesis by RNA polymerase II (RNAPII) is
regulated by the phosphorylation of RNAPII's C-terminal domain (CTD) in which Ser2 can be
phosphorylated by cyclin-dependent kinase 9 (CDK9) (19). CDK9 was also found to be a component
of the multiprotein complex TAK/P-TEFb, which is an elongation factor for RNAPII-directed
transcription and functions by phosphorylating the CTD of the largest subunit of RNAPII (16). Since
the discovery that the CDK inhibitor flavopiridol induces cell apoptosis by inhibiting CDK9 (20), the
enzyme has been a target for anticancer drug design. There have been some other CDK9's inhibitors
applied in cancer therapy, for example SCH727965 for breast cancer (21), AT7519 for refractory solid
tumors (22) and SNS-032 for chronic lymphocytic leukemia (23). On the other hand, P-TEFb
composed of Cdk9 and cyclin T1 is a required cellular cofactor for the human immunodeficiency
virus (HIV-1) transactivator, Tat. As CDK9 is a key component of P-TEFb, CDK9 inhibitors DRB,
seliciclib and flavopiridol have been reported to inhibit the HIV-1 virus transcription and replication
effectively (24). Over all, CDK9 inhibitors can be effective drugs for cancers, HIV-1 and some other
diseases.
In this work, we employed indole structural scaffold as a pharmaceutical core linker to design and
synthesize three series of structurally related indole derivatives (Scheme 1). All synthesized
compounds were subsequently evaluated in vitro for anti-proliferative activity against three human
cancer cell lines (Hela, HepG2 and A549) and HIV-1 inhibition activity used as a read-out of cellular
activity. Then, compounds 2c and 3b with good anti-proliferative activity against cancer cells and
excellent HIV-1 inhibition activity were selected for further cellular mechanism studies which
included testing the induction of cell apoptosis, and checking the level of cleaved PARP (the mark of
apoptosis), the phosphorylation level at Ser2 of RNAPII CTD and the expression level of CDK9 (25).
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Scheme 1. 2,5-Disubstituted indole derivatives
Experimental Section
Chemistry
All reagents were purchased and used without further purification, unless otherwise indicated.
Reactions were magnetically stirred and monitored by thin-layer chromatography (TLC) on Merck
silica gel 60F-254 by fluorescence. All of the final compounds were purified by column
1
13
chromatography. H-NMR and C-NMR spectra were obtained using a Bruker AV2 600 Ultra shield
spectrometer at 600 and 150 MHz respectively, 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 (dd), doublet-triplet (dt), triplet (t), triplet-triplet (tt),
triplet-doublet (td), quartet (q), quartet-doublet (qd), multiplet (m), and broad signal (brs). High-
resolution mass spectral (HRMS) data were acquired on a Q Exactive. Melting points were measured
on a SGW X-4 micro-melting point spectrometer and were uncorrected.
N-o-tolylcycloadamantanecarboxamide (5)
A mixture of o-toluidine (10.0 mmol, 1.07 g), 1-adamantanecarbonyl chloride (10.0 mmol, 1.98 g)
and anhydrous potassium carbonate (7.0 mmol, 0.97 g) in toluene (50 mL) was stirred at room
temperature for 4 hours. The resulting solid was filtered off and then stirred at room temperature
for 1.5 h with 50 mL of H O. The solid was filtered off and recrystallized from EtOH. White solid
2
1
(
75.5%). H-NMR (600 MHz, CDCl ): δ 7.88 (d, J = 8.07 Hz, 1H), 7.18-7.24 (m, 2H), 7.17 (d, J = 7.52 Hz,
3
1
1
H), 7.05 (dt, J = 1.10, 7.43 Hz, 1H), 2.26 (s, 3H), 2.11 (brs, 3H), 1.99 (d, J = 2.57 Hz, 6H), 1.77 (q, J =
+
+
2.29 Hz, 6H). ESI-HRMS (+): m/z calcd for C H NO [M+H] 270.1852, found 270.1853; calcd for
18 24
+
+
C H NONa [M+Na] 292.1672, found 292.1669.
1
8 23
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2
-Adamantane-1H-indole (6)
A stirred solution of amide 5 (10.0 mmol, 2.69 g) in THF (50 mL) under a N atmosphere was
2
maintained at an internal temperature of -5 to 5℃and treated dropwise with 0.1-0.15 mol of n-BuLi
as 2.5 M n-BuLi in hexane. The stirred mixture was kept at ambient temperature, cooled in an ice
bath, and treated dropwise with 2 M HCl (12 mL). The organic layer was separated and the aqueous
layer washed with C H . The combined organic layer was dried with anhydrous MgSO , filtered, and
6
6
4
concentrated. The residue was purified by column chromatography to get white solid with the yield
1
of 83%. H-NMR (600 MHz, CDCl ): δ 7.30 (d, J = 7.89 Hz, 1H), 7.18 (dd, J = 0.55, 8.07 Hz, 1H), 6.88
3
(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, 3H), 1.93
+
+
(
d, J = 2.93 Hz, 6H), 1.73 (brs, 6H). ESI-HRMS (+): m/z calcd for C H N [M+H] 252.1747, found
18 22
+
+
2
52.1748; calcd for C H NNa [M+Na] 274.1566, found 274.1568.
18 21
2
-Adamantane-5-nitro-1H-indole (7)
In a 250 mL round-bottom flask, 2-adamantane-1H-indole (10 mmol, 2.51 g) was dissolved in H SO
2
4
(
10 mL) after vigorous stirring. In a separate flask, NaNO (11 mmol, 0.94 g) was dissolved in H SO
3
2
4
(
10 mL), also after vigorous stirring, and added dropwise via addition funnel to the 2-adamantane-
1
H-indole. After addition, the reaction was stirred for another 10 min and then poured into ice water
(
200 mL), precipitating a yellow product. The product was isolated via filtration and washed with
1
cold water. After 12 h of drying under vacuum, 2.89 g of yellow product was isolated (97%). H-NMR
(
(
(
600 MHz, DMSO-d ): δ 11.65 (brs, 1H), 8.44 (d, J = 2.20 Hz, 1H), 7.93 (dd, J = 2.20, 8.99 Hz, 1H), 7.44
6
13
d, J = 8.80 Hz, 1H), 6.38 (d, J = 1.47 Hz, 1H), 2.07 (brs, 3H), 1.98 (brs, 6H), 1.67-1.81 (m, 6H). C-NMR
150 MHz, DMSO-d ): δ 154.0, 140.8, 139.9, 127.7, 116.9, 116.3, 111.4, 98.2, 41.9, 36.6, 34.1, 28.2.
6
+
+
+
ESI-HRMS (+): m/z calcd for C H N O [M+H] 297.1598, found 297.1599; calcd for C H N O Na
18
21
2
2
18 20
2
2
+
[
M+Na] 319.1417, found 319.1417.
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2
-Adamantane-1H-indol-5-amine (8)
Compound 7 (10 mmol, 2.96 g) was dissolved in ethanol (150 mL), 10% Pd/C was added (300 mg),
and the mixture was subjected to H (38 psi) using a Parr hydrogenator for 3.5 h. The mixture was
2
filtered over Celite, which was washed with methanol. After concentration and drying under
1
vacuum, 2.5 g of a brown powder (94%) was isolated. H-NMR (600 MHz, CDCl ): δ 7.80 (brs, 1H),
3
7
2
.10 (d, J = 8.44 Hz, 1H), 6.86 (d, J = 2.20 Hz, 1H), 6.57 (dd, J = 2.20, 8.44 Hz, 1H), 6.05 (dd, J = 0.73,
13
.20 Hz, 1H), 3.23-3.65 (m, 2H), 2.09 (brs, 3H), 1.96 (d, J = 2.38 Hz, 6H), 1.78 (q, J = 12.17 Hz, 6H). C-
NMR (150 MHz, CDCl ): δ 149.9, 139.3, 130.3, 129.4, 111.6, 110.8, 105.3, 95.5, 42.6, 36.8, 33.7, 28.5.
3
+
+
ESI-HRMS (+): m/z calcd for C H N [M+H] 267.1856, found 267.1858.
18
23
2
N-(2-adamantane-1H-indol-5-yl)-benzamide (1a)
A mixture of 2-adamantane-1H-indol-5-amine 8 (10.0 mmol, 2.66 g), benzoyl chloride (10.0 mmol,
1
.41 g) and anhydrous potassium carbonate (7.0 mmol, 0.97 g) in toluene (50 mL) was stirred at
room temperature for 4 h. The resulting solids was filtered off and then stirred at room temperature
for 1.5 h with 50 mL of H O. The solid was filtered off and recrystallized from EtOH. White solid
2
1
(
78.7%). Mp: 263-265
℃
. H-NMR (600 MHz, DMSO-d ): δ 10.82 (s, 1H), 10.04 (s, 1H), 7.98 (d, J = 7.34
6
Hz, 2H), 7.86 (d, J = 0.92 Hz, 1H), 7.55-7.59 (m, 1H), 7.50-7.54 (m, 2H), 7.32 (dd, J = 1.74, 8.53 Hz, 1H),
.25 (d, J = 8.62 Hz, 1H), 6.07 (d, J = 1.65 Hz, 1H), 2.07 (brs, 3H), 1.98 (d, J = 2.20 Hz, 6H), 1.73-1.81
7
13
(
m, 6H). C-NMR (150 MHz, DMSO-d ): δ 165.4, 150.8, 135.9, 133.5, 131.6, 131.1, 128.7, 128.0,
6
+
1
28.0, 115.6, 112.3, 110.8, 95.6, 42.3, 36.8, 33.9, 28.4. ESI-HRMS (+): m/z calcd for C H N O
25
27
2
+
+
+
[
M+H] 371.2118, found 371.2111; calcd for C H N ONa [M+Na] 393.1937, found 393.1929.
25
26
2
At the Series A, the other target compounds were synthesized following the general procedure as
described above with the yield of 84–89%. Their structures were also confirmed (see ‘Supporting
Information’).
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5
-Isocyanato-1H-indole-adamantane (9)
A solution of 2-adamantane-1H-indol-5-amine 8 (10.0 mmol, 2.66 g) dissolved in THF (80 mL) was
slowly dropped into a stirred solution of triphosgene (10.0 mmol, 2.98 g) in THF (10 mL) by using a
constant-pressure dropping funnel. NEt (21.0 mmol, 3 mL) was then added slowly to the reaction
3
mixture after 8 were added. The reacton mixture was stirred at 0
℃for 2 h, After completion of the
reaction, the mixture was concentrated and purified by column chromatography using appropriate
1
mixtures of EtOAc and PE to yield the titled compound. White solid (61.5%). H NMR (600 MHz,
CDCl ): δ 8.01 (brs, 1H), 7.21 (d, J = 8.44 Hz, 1H), 6.85 (dd, J = 2.02, 8.44 Hz, 1H), 6.17 (d, J = 1.47 Hz,
3
1
3
1
1
H), 2.07-2.15 (m, 3H), 1.98 (brs, 6H), 1.74-1.85 (m, 6H). C-NMR (150 MHz, CDCl ): δ 151.0, 133.2,
3
29.0, 125.1, 123.6, 118.1, 115.5, 111.1, 96.4, 42.5, 36.7, 33.8, 28.4.
1
-(4-Chloro-2-methoxyphenyl)-3-(1H-indol-2-adamantine-5-yl)urea (2c)
A mixture of 5-isocyanato-1H-indole-adamantine 9 (0.4 mmol, 0.12 g) in toluene (5 mL) and 4-
chloro-2-methoxybenzenamine (0.4 mmol) was heated at 60 for 4 h. After completion of the
℃
reaction, the mixture was concentrated and purified by column chromatography using appropriate
1
mixtures of CH Cl and MeOH to get the titled compound. White solid (84.2%). Mp: 253-255℃. H-
2
2
NMR (600 MHz, DMSO-d ): δ 10.73 (s, 1H), 8.69 (s, 1H), 8.38 (s, 1H), 7.56 (d, J = 1.28 Hz, 1H), 7.45 (d,
6
J = 2.02 Hz, 1H), 7.27 (d, J = 8.62 Hz, 1H), 7.21 (d, J = 8.62 Hz, 1H), 7.00 (dd, J = 1.83, 8.44 Hz, 1H),
6
1
1
.94 (dd, J = 2.11, 8.53 Hz, 1H), 6.03 (d, J = 1.47 Hz, 1H), 3.83 (s, 3H), 2.05 (brs, 3H), 1.96 (brs, 6H),
13
.70-1.80 (m, 6H). C-NMR (150 MHz, DMSO-d ): δ 155.0, 153.4, 150.7, 141.0, 132.9, 131.2, 130.1,
6
28.4, 114.3, 113.4, 111.1, 110.4, 103.1, 95.4, 56.2, 42.3, 36.8, 33.9, 28.4. ESI-HRMS (+): m/z calcd
+
+
+
+
for C H ClN O [M+H] 450.1943, found 450.1942; calcd for C H ClN O Na [M+Na] 472.1762,
26
29
3
2
26 28
3
2
found 472.1760.
At the Series B, the other target compounds were synthesized following the general procedure as
described above with the yield of 81–89%. Their structures were also confirmed (see ‘Supporting
Information’).
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(
E)-ethyl 2-(2-(4-nitrophenyl)hydrazono)propanoate (11)
A solution of 1-(4-nitrophenyl) hydrazine (65 mmol, 10.0 g) in EtOH (100 mL) was treated with ethyl
-oxopropanoate (72 mmol, 8.4 g), The mixture has been stirred at 78 for 2 h, when TLC indicated
2
℃
that the reaction had gone to completion. On cooling, Precipitated complex was filtered, washed
with EtOH, and dried to afford (E)-ethyl 2-(2-(4-nitrophenyl)hydrazono)propanoate (11) as a yellow
1
solid. Yield: 14.5 g (88.9%). H-NMR (600 MHz, CDCl ): δ 8.21 (d, J = 8.99 Hz, 2H), 8.09 (s, 1H), 7.28 (d,
3
J = 8.99 Hz, 2H), 4.32-4.39 (m, 2H), 2.17 (s, 3H), 1.40 (t, J = 7.06 Hz, 3H).
Ethyl 5-nitro-1H-indole-2-carboxylate (12)
The solution of (E)-ethyl 2-(2-(4-nitrophenyl)hydrazono)propanoate 11 (42 mmol, 10 g) in
polyphosphoric acids was stirred at 100℃for 2 h, and monitored by TLC analysis. After completion of
the reaction, the mixture was diluted with a lot of water. Precipitated complex was filtered, washed
with water, and dried to afford ethyl 5-nitro-1H-indole-2-carboxylate (12) as a green solid. Yield: 8.1
1
g (82.4%). H-NMR (600 MHz, DMSO-d ): δ 12.58 (brs, 1H), 8.71 (d, J = 2.02 Hz, 1H), 8.12 (dd, J =
6
2
=
.20, 9.17 Hz, 1H), 7.61 (d, J = 9.17 Hz, 1H), 7.42 (d, J = 0.92 Hz, 1H), 4.37 (q, J = 7.09 Hz, 2H), 1.35 (t, J
7.15 Hz, 3H).
Ethyl 5-amino-1H-indole-2-carboxylate (13)
Compound 12 (10 mmol, 2.34 g) was dissolved in ethanol (150 mL), 10% Pd/C was added (300 mg),
and the mixture was subjected to H (38 psi) using a Parr hydrogenator for 3.5 h. The mixture was
2
filtered over Celite, which was washed with methanol. After concentration and drying under
1
vacuum, 1.86 g of a brown powder (91.2%) was isolated. H-NMR (600 MHz, DMSO-d ): δ 11.38 (brs,
6
1
7
1
H), 7.16-7.19 (m, 1H), 6.84 (dd, J = 0.83, 2.11 Hz, 1H), 6.69-6.73 (m, 2H), 4.63 (brs, 2H), 4.29 (q, J =
13
.09 Hz, 2H), 1.31 (t, J = 7.15 Hz, 3H). C-NMR (150 MHz, DMSO-d ): δ 161.9, 142.5, 132.0, 128.2,
6
27.1, 117.2, 113.2, 106.6, 103.6, 60.6, 14.8.
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Ethyl 5-benzamido-1H-indole-2-carboxylate (14)
A mixture of ethyl 5-amino-1H-indole-2-carboxylate 13 (10.0 mmol, 2.04 g), benzoyl chloride (10.0
mmol, 1.41 g) and anhydrous potassium carbonate (7.0 mmol, 0.97 g) in toluene (50 mL) was stirred
at room temperature for 4 h. The resulting solid was filtered off and then stirred at room
temperature for 1.5 h with 50 mL of H O. The solid was filtered off and recrystallized from EtOH.
2
1
White solid (75.5%). H-NMR (600 MHz, DMSO-d ): δ 11.87 (brs, 1H), 10.23 (brs, 1H), 8.16 (s, 1H),
6
7
4
1
.99 (d, J = 7.34 Hz, 2H), 7.56-7.61 (m, 2H), 7.50-7.55 (m, 2H), 7.43 (d, J = 8.80 Hz, 1H), 7.15 (s, 1H),
13
.35 (q, J = 7.09 Hz, 2H), 1.35 (t, J = 7.15 Hz, 3H); C-NMR (150 MHz, DMSO-d ): δ 165.7, 161.7,
6
35.7, 135.0, 132.6, 131.8, 128.8, 128.3, 128.1, 127.0, 120.6, 113.7, 112.9, 108.2, 60.9, 14.8; ESI-
+
+
+
HRMS (+): m/z calcd for C H N O [M+H] 309.1234, found 309.1225; calcd for C H N O Na
18
17
2
3
18 16
2
3
+
[
M+Na] 331.1053, found 331.1053.
5
-Benzamido-1H-indole-2-carbohydrazide (15)
To a solution of ethyl 5-benzamido-1H-indole-2-carboxylate (5 mmol, 1.54 g) in 95% ethyl alcohol (10
mL) hydrazine hydrate (10 mL) was added, and the reaction mixture was heated under reflux for 8 h.
The hydrazide was obtained in quantitative yields directly after evaporation of the reaction solution
1
without any additional purification step. White solid (85.5%). H-NMR (600 MHz, DMSO-d ): δ 11.58
6
(s, 1H), 10.14 (s, 1H), 9.76 (brs, 1H), 8.07 (s, 1H), 7.97 (d, J = 7.15 Hz, 2H), 7.56-7.60 (m, 1H), 7.51-
7
.55 (m, 2H), 7.47 (dd, J = 1.65, 8.80 Hz, 1H), 7.39 (d, J = 8.80 Hz, 1H), 7.07 (d, J = 1.47 Hz, 1H), 4.50
13
(
s, 2H). C-NMR (150 MHz, DMSO-d ): δ 165.6, 161.6, 135.8, 133.9, 132.2, 131.7, 131.6, 128.8,
6
+
+
1
28.0, 127.3, 118.9, 113.3, 112.5, 102.4. ESI-HRMS (+): m/z calcd for C H N O [M+H] 295.119,
16
15
4
2
+
+
found 295.1182; calcd for C H N O Na [M+Na] 317.1009, found 317.1004.
16
14
4
2
(
E)-N'-(4-Methylbenzylidene)-5-benzamido-1H-indole-2-carbohydrazide (3b)
To a solution of 5-benzamido-1H-indole-2-carbohydrazide 15 (0.25 mmol ,0.075 g) in ethanol (5 mL),
-methylbenzaldehyde (0.3 mmol, 0.036 g) was added, and the reaction mixture was heated at
4
reflux for 8 h, and monitored by TLC analysis. After completion of the reaction, the mixture was
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concentrated. Then the residue was purified by column chromatography to get white solid with the
1
yield of 83.6%. Mp: 297-298℃. H-NMR (600 MHz, DMSO-d ): δ 11.83 (brs, 1H), 11.79 (brs, 1H),
6
1
7
0.18 (s, 1H), 8.44 (brs, 1H), 8.18 (brs, 1H), 8.00 (d, J = 7.15 Hz, 2H), 7.66 (d, J = 7.52 Hz, 2H), 7.57-
13
.61 (m, 1H), 7.51-7.56 (m, 3H), 7.45 (d, J = 8.62 Hz, 1H), 7.30 (d, J = 7.89 Hz, 3H), 2.36 (s, 3H). C-
NMR (150 MHz, DMSO-d ): δ 165.7, 158.0, 147.7, 140.3, 135.7, 134.5, 132.5, 132.1, 131.8, 131.2,
6
1
29.9, 128.8, 128.0, 127.5, 127.3, 119.8, 113.5, 112.7, 104.1, 21.5. ESI-HRMS (+): m/z calcd for
+
+
+
+
C H N O [M+H] 397.1659, found 397.1651; calcd for C H N O Na [M+Na] 419.1478, found
24
21
4
2
24 20
4
2
4
19.1470.
At the Series C, the other target compounds were synthesized following the general procedure as
described above with the yield of 84–90%. Their structures were also confirmed (see ‘Supporting
Information’).
Biological assays
Luciferase assay
Inhibition of HIV-1 transcription was measured in NH2 cell line using luciferase assay. NH2 was a
stable cell line based on Hela cells with an integrated HIV-1 LTR-luciferase reporter gene and stably
expressing PCDNA3-Tat-flag, the expression level of report gene luciferase could reveal the HIV-1
transcription level. In this experiment, NH2 cells in 48-well plate were treated with compounds (0. 2
μM, 2M and 20 μM) for 8 hours and 0.2 μM flavopiridol as the positive control respectively, after
treatment cell lysates were normalized and measured luciferase activity using luciferase kit from
Promega.
MTT toxicity assay
All the cancer cells used in MTT assay were cultured in DMEM medium (Life Technological) with 10%
FBS. The MTT reagent 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide was
purchased from sigma (M2128). Cells were cultured in 96-well plate with 100 L medium and the
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4
total amount of cells in each well was about 1×10 . Concentration gradients (5-7 different
concentrations) of each compound were then added to cells (six repeats), cisplatin (DDP) as the
positive control and dimethyl sulfoxide (DMSO) as the negative control. After 48 hours treatment, 10
μL MTT (5 mg/mL) was added to each wells staining for 4 hours in cell incubator. Then in each well,
medium was discarded and 100 μL DMSO was used, after short time shaking, the light intensity was
measured at a wavelength of 490 nm. At last, the IC values (concentrations required to inhibit 50%
50
of cell growth) were calculated using GraphPad Prism 5 software.
Annexin V/PI staining and cell cycle analysis
5
Cells were cultured in six-well with about 1×10 cells in each well and treated with varying
concentrations of 2c or 3b for 24 hours respectively. Then the cell apoptosis analysis was performed
following the protocols (BD Bioscience). After stained with FITC annexin V/PI, the apoptosis status
was analyzed using a Beckman Epics Altra Culter and data were analyzed with EXP032 software. The
status and percentage of cells undergoing apoptosis were defined as early apoptosis (annexin V-
positive and PI-negative) and late apoptosis (annexin V- positive and PI-positive).
Western blot analysis
To check the proteins changes after compounds treatment, the western blot analysis was performed.
After 24 hours treatment with varying concentrations of 2c and 3b, cells on plate were washed with
1× PBS and added with 1×SDS loading buffer for lysing. The samples were boiled (100℃, 10 min)
and used for western blot analysis. The phosphorylation level of CTD ser2 (p-Ser2, A300-654A,
Bethyl), total CDK9 (11705-1-AP, proteintech) protein level and cleaved PARP (9542, CST) were
tested, and a-Tubulin (T-6074, sigma) was used as a control. Both anti-rabbit and anti-mouse IgG
were purchased from sigma.
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Result and Discussion
Chemistry
In this work, we synthesized three series of novel chemically modified indole compounds including
nd
an adamantane group or a N'-(substituted methylene)-carbohydrazide group at the 2 position and
th
a substituted urea group or a substituted benzamide group at the 5 position in the indole system.
First, we synthesized 2-adamantane-1H-indol-5-amine (8). The general chemistry for the synthesis of
8
was adapted from the methods reported previously (26-28) and outlined in Scheme 2. Briefly,
treatment of 1-adamantanecarboxylic acid chloride (4) with o-toluidine resulted in N-o-
tolylcycloadamantanecarboxamide (5), followed by dropwise add of 1.4 or 1.6 M n-Butyllithium in
hexane to get the corresponding 2-adamantane-1H-indole (6). 8 can be easily formed through
nitrification and catalytic hydrogenation of compound 6. 8 was converted to Series A (1a-1e, Scheme
3
) by reaction with benzoyl chloride and 5-isocyanato-1H-indole-adamantane (9) was obtained by
reaction with 8 and triphosgene. 9 converted to Series B (2a-2p, Scheme 3) by reaction with
corresponding amine (29).
Scheme 2. Synthesis of 2-(adamantan-1-yl)-1H-indol-5-amine. Reagents and conditions: (a) toluene,
o-toluidine, K CO , r.t., 4 h; (b) n-BuLi, THF, 0℃, N , 3 h; (c) NH NO , H SO , 0℃, 1 h; (d) Fe, AcOH,
2
3
2
4
3
2
4
H O, EtOH, 70℃, 2 h.
2
Scheme 3. Synthesis of series A and series B. Reagents and conditions: (a) benzoyl chloride, toluene,
0℃, 8 h; (b) triphosgene, THF, trimethylamine, 0℃,4 h; (c) R-NH , toluene, 60℃, 4 h.
6
2
We synthesized ethyl 5-amino-1H-indole-2-carboxylate (13). The general chemistry for the synthesis
of 13 was adapted from the methods reported previously (28) and outlined in Scheme 4. 13 was
acylated to get the ethyl 5-benzamido-1H-indole-2- carboxylate (14) by reacting with benzoyl
chloride in toluene at 60℃for 4 h. 5-Benzamido-1H-indole- 2-carbohydrazid (15) were obtained by
dropping hydrazine hydrate in 14 ethanol (30). The desired analogs (3a-3s, Scheme 4) were obtained
by dropwise addition of corresponding aldehyde in 15 ethanol solution.
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Scheme 4. Synthesis of series C. Reagents and conditions: (a) ethyl 2-oxopropanoate, EtOH, reflux, 2
h; (b) polyphosphoric acid, 100℃, 1 h; (c) Fe, AcOH, H O, EtOH, 70℃, 2 h. (d) benzoyl chloride,
2
toluene, 60℃, 8 h; (e) Hydrazine hydrate, EtOH, reflux, 6 h; (f) RCHO, EtOH, reflux, 8 h.
Anti-proliferative activity in human cancer cell lines based on MTT assay and structure-activity
relationship
All synthesized compounds were first evaluated the anti-proliferative activity against Hela cancer
cells. Their anti-proliferative activity was summarized in table 1. The result indicated that most
compounds showed a moderate anti-proliferative activity against Hela cells such as 2c with the IC50
value 15.54 μM and 3b with the IC value 8.70 μM.
50
Considering the whole result in Hela cells, attempts were made to establish the structure-activity
relationship (SAR) among the tested compounds. Firstly, we found that in the 2-adamantane-5-
substituted benzamide-indole series (series A), all compounds had good anti-proliferative activity
against Hela cells. Substitutions at phenyl ring had little effect on compound's anti-proliferative
activity, and replacement of phenyl ring with pyridyl ring showed similar cellular anti-proliferative
activity (Table 1, 1d vs 1e). Among the 2-adamantane-5-substituted urea-indole derivatives (series
B), aryl substituted compounds (2a~2g and 2n~2p) were generally more active against Hela cells
than alkylated derivatives (2h~2m). Among aryl substituted compounds, substitution at C4-phenyl
ring played an important role in enhancing cellular anti-proliferative activity, but substitution at C3-
phenyl ring was unhelpful for anti-proliferative activity (Table 1, 2a~2d vs 2e~2g). In addition,
compared with compound 2d with hydrophobic group (CH ) at o- position of phenyl ring, compound
3
2
c bearing o- hydrogen bond acceptor function group (OCH ) exhibited weak anti-proliferative
3
activity. In the 2-[N'-(substituted methylene)-carbohydrazide]-5-benzamide-indole derivatives (series
C), different substituted groups and positions of the phenyl ring introduced at the N'-methylene
position displayed an important relationship with anti-proliferative activity. Compared with
compound 3a (phenyl, IC >100 M), compounds 3b, 3c and 3d which had methyl (CH ), carboxyl
50
3
(
COOH) and yilthiomethyl (SCH ) group at the C-4 position of phenyl ring respectively enhanced
3
cellular anti-proliferative activity with an over 10-fold gain in cellular toxicity, suggesting that the
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impact of the substituent at the C-4 position of the phenyl ring was significant. However, compound
g with the 4-OCH substitution at phenyl ring was no active with IC value up to 100 μM. To further
3
3
50
evaluate the importance of substituted position at the phenyl ring, compounds 3h (2-OCH ), 3i (3,5-
3
OCH ), 3j (3,4-OCH ), 3k (2,3-OCH ) and 3l (2,3,4-OCH ) were subsequently synthesized. Biological
3
3
3
3
evaluation revealed that two methoxy groups were introduced at m- position (3i, IC , 1.67 μM) to
50
be a determining factor for anti-proliferative activities against Hela cells. Additionally, compounds 3q
IC , 24.7 μM) and 3r (IC , 73.5 μM) both possessing a bicyclic aromatic ring system at the N'-
(
50
50
methylene position showed increased activities against Hela cells compared with the compounds 3o
IC , over 100 μM) and 3h (IC , over 100 μM) both bearing a single aromatic cyclic ring system at
(
50
50
the N'-methylene position, respectively. The IC value of compound 3s was over 100 μM, indicating
50
that the introduction of n-alkyl group to the pharmaceutical core may be unfavorable for anti-
proliferative properties, together with the results based upon B series.
Furthermore, all compounds with IC value below 20 μM were evaluated for their in vitro anti-
50
proliferation activities against another two cancer cell lines, which were HepG2 and A549, with the
positive contrast drug DDP. As shown in Table 1, the results indicated that most of the target
compounds could also effectively inhibit the proliferation of HepG2 and A549 cells. Interestingly,
nd
compound 2d with adamantane substitution at the 2 position of indole ring and (4-chloro-2-
methyl) phenyl substitution at urea end showed minimum IC value (0.80 μM) with highest
50
inhibition among the compounds in the series against HepG2 cells. Against A549 cells, compound 3d
showed the highest inhibition with IC value of 0.16 μM, compounds 2a (IC , 0.50 μM) and 3b (IC ,
50
50
50
0
.48 μM) showed more inhibition than the standard drug DDP (IC , 1.83 μM).
50
Table 1. Anti-proliferative activity (IC , M) against cancer cells of 2,5-disubstituted indole
50
derivatives
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HIV-1 inhibition activity
It is reported that phosphorylation of the RNA polymerase II carboxyl-terminal domain (RNAPII CTD)
by CDK9 is directly responsible for human immunodeficiency virus type 1 tat-activated
transcriptional elongation (31). So the HIV activity may be used as a read-out of cellular activity.
After evaluating compounds' anti-proliferative activity against cancer cells by MTT assay, we then
used luciferase assay to test whether these compounds can inhibit HIV-1 transcription. Interestingly,
2
0 kinds among all tested compounds had the HIV-1 inhibition activity as shown in table 2, besides
2
c and 3b were more potential than others. These compounds bearing both anticancer activity and
anti-HIV activity may have dual utility.
Table 2. HIV-1 inhibition activity
Cellular mechanism of action
According to the MTT assay and HIV-1 inhibition assay, compounds 2c and 3b were selected for
further cellular mechanism studies. Firstly, a range of human cancer cell lines were treated with
these two compounds for 48 hours, including breast cancer cells (MCF-7), liver cancer cells (HepG2)
and lung cancer cells (H460 and A549). The MTT assay's results showed that 2c and 3b had a pan
anti-proliferative activity in these cancer cell lines, but A549 cells were relative sensitive to 3b with
the IC value 0.48 μM and HepG2 cells to 2c with the IC value 13.21 μM as shown in Table 3.
50
50
Nextly, we studied whether the growth inhibition of cancer cells was caused by cellular apoptosis.
Table 3. Anti-proliferative activity of 2c and 3b against a panel of human cancer cell lines by MTT
assay.
To test the induction of apoptosis, A549 cells treated with 3b and HepG2 cells treated with 2c for 24
hours were doubly stained with Annexin V/PI (propidium iodide). As shown in figure 2, compounds
3
b and 2c could effectively induce apoptosis of A549 and HepG2 cells respectively and the effects
were does-dependent. At the 0.5 μM concentration 3b causes 4.0% late apoptosis annexin V-positive
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and PI-positive cells, and the percentage increases to 50.1% at 2.5 μM (Figure 2A). On the other
hand, the treatment of HepG2 with 2c at 10 μM and 20 M concentrations resulted in 13.9% and
2
3.7% apoptotic cells respectively (Figure 2B).
Figure 2. A549 cells (A) and HepG2 cells (B) were treated with 3b and 2c respectively at the indicated
concentrations for 24 hours and analyzed by annexin V/PI staining. The cells undergoing apoptosis
were defined as two kinds of apoptosis (early apoptosis annexin V-positive cells and late apoptosis
annexin V-positive and PI-positive cells).
mRNA synthesis and transcription in cells are regulated by a series of phosphorylation events
involving the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII)
(
19). Three cyclin-dependent kinases (CDK7, CDK8 and CDK9) which are known to phosphorylate the
RNAPII CTD during transcription, primarily phosphorylate the RNAPII CTD at Ser5. Furthermore, CDK9
plays a key role in the phosphorylation at Ser2 of RNAPII CTD. Based on our experiments’ data, 2c
and 3b could induce cancer cells apoptosis and effectively inhibit HIV-1 transcription, so we checked
cleaved PARP (the mark of apoptosis), phosphorylated Ser2 (p-Ser2) level and the expression level of
CDK9 by using western blot analysis. The western blot analysis showed that the p-Ser2 decreased
and the cleaved PARP increased slightly when the concentration of 3b was about 1×IC value, but
50
the changes were obvious when the concentration was about 5×IC value in A549 cells after 24
50
hours treatment (Figure 3A). Analogous results were obtained with 2c in HepG2 cells, with inhibition
of the phosphorylation at Ser-2 of RNAPII CTD and induction of cleaved PARP being observed (Figure
3
1
B). However, both the treatment of A549 cells with 3b and the treatment of HepG2 with 2c at
×IC , 2×IC and 5×IC concentration for a period of 24 h had little effect on the level of CDK9's
50
50
50
expression (Figure 3). These data supported the conclusion that 3b and 2c may not affect the
expression of CDK9, but could inhibit the phosphorylation at Ser2 of RNAPII CTD and then induce
cancer cells apoptosis.
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Figure 3. Western blot analysis of A549 cells after 3b treatment (A) and HepG2 cells after 2c
treatment (B) for 24 hours, a-Tubulin was used as internal control.
Conclusion
Now, significant numbers of anticancer or anti-HIV lead compounds are indole-based small
molecules (32,33). In this study, three novel series of 2,5-disubstituted indole derivatives were
designed and efficiently synthesized with good yield. Furthermore, all synthesized compounds were
assessed for both their anti-proliferative activity against human cancer cells and HIV-1 inhibition
activity which was used as a read-out of cellular activity. Interestingly, many compounds showed
both anticancer activity and HIV-1 inhibition activity, so they may have dual utility. Among them,
compounds 2c and 3b possessing good anticancer activity and excellent HIV-1 inhibition activity
were further examined in more detail regarding its cellular mode of action. Annexin V/PI staining
and cell cycle analysis indicated that compounds 2c and 3b successfully inhibited cell cycle
progression and displayed good apoptosis in HepG2 cells and A549 cells respectively. The western
blot analysis clearly showed the p-Ser2 decreased, the cleaved PARP increased, and the expression
level of CDK9 neither decreased nor increased both in the HepG2 cells treated with 2c and the A549
cells treated with 3b. These results implied that 3b and 2c may induce cancer cells apoptosis through
inhibiting the phosphorylation at Ser2 of RNAPII CTD which is essential in mRNA synthesis. However,
further studies are required to determine their roles in preventing HIV-1. Additionally, in our lab
nd
th
further detailed structural modifications at 2 an 5 positions of indole moiety with various group
are being underway to getter better analogs with improve efficacy.
Acknowledgements
The project was supported by the National Natural Science Foundation of China (No. 81273400 and
No. 81302652). This research was also financially supported by Fujian Science and Technology
project (Grant No. 2014N5012).
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Conflict of Interest
The authors confirm that this article content has no conflict of interests.
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Spectra data of compounds 1b~1e, 2a~2b, 2d~2p, 3a and 3c~3s.
Figure 1. Structures of four compounds bearing indole-based moiety
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(A)
DMSO
0.5 M
2.5 M
PI
AnnexinV
(B)
DMSO
10 M
20 M
PI
AnnexinV
Figure 2. A549 cells (A) and HepG2 (B) cells were treated with 3b and 2c respectively at the indicated
concentrations for 24 hours and analyzed by annexin V/PI staining. The cells undergoing apoptosis were
defined as two kinds of apoptosis (early apoptosis annexin V-positive cells and late apoptosis annexin V-
positive and PI-positive cells).
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(B)
(A)
Figure 3. Western blot analysis of A549 cells after 3b treatment (A) and HepG2 cells after 2c treatment
B) for 24 hours, a-Tubulin was used as internal control.
(
Scheme 1. 2,5-Disubstituted indole derivatives
Scheme 2. Synthesis of 2-(adamantan-1-yl)-1H-indol-5-amine. Reagents and conditions: (a) toluene, o-
toluidine, K CO , r.t., 4 h; (b) n-BuLi, THF, 0℃, N , 3 h; (c) NH NO , H SO , 0℃, 1 h; (d) Fe, AcOH, H O,
2
3
2
4
3
2
4
2
EtOH, 70℃, 2 h.
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Scheme 3. Synthesis of series A and series B. Reagents and conditions: (a) benzoyl chloride, toluene,
0℃, 8 h; (b) triphosgene, THF, trimethylamine, 0℃,4 h; (c) R-NH , toluene, 60℃, 4 h.
6
2
Scheme 4. Synthesis of series C. Reagents and conditions: (a) ethyl 2-oxopropanoate, EtOH, reflux, 2 h;
b) polyphosphoric acid, 100℃, 1 h; (c) Fe, AcOH, H O, EtOH, 70℃, 2 h. (d) benzoyl chloride, toluene,
(
2
6
0℃, 8 h; (e) Hydrazine hydrate, EtOH, reflux, 6 h; (f) RCHO, EtOH, reflux, 8 h.
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Table 1. Anti-proliferative activity (IC , M) against cancer cells of 2,5-disubstituted indole derivatives
50
IC (M) ± S.D.
50
Compound
R /R /R
1
2
3
Hela
HepG2
A549
1
a
b
2.59±0.08
10.89±0.12
10.55±0.05
9.32±0.07
11.16±0.09
21.54±0.05
1
6.96±0.10
2.40±0.05
1.34±0.08
15.92±0.12
20.62±0.11
8.34±0.10
OCH3
Cl
1
c
1
d
e
1
Cl
3.33±0.11
22.23±0.12
27.12±0.13
N
2
a
b
1.44±0.05
2.21±0.09
0.99±0.08
3.90±0.06
0.50±0.06
CN
2
18.02±0.10
2
c
15.54±0.12
13.21±0.11
15.92±0.09
2
d
1.54±0.05
0.80±0.04
3.87±0.09
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2
e
34.16±0.11
27.80±0.13
nd
nd
nd
nd
2
f
2
2
g
26.30±0.11
nd
nd
h
31.27±0.11
>100
nd
nd
nd
nd
2
2
i
j
>100
nd
nd
nd
nd
2
k
33.21±0.15
2
l
82.56±0.10
>100
nd
nd
nd
nd
2
m
2
n
o
2.77±0.08
3.64±0.09
6.41±0.10
2
12.58±0.12
3.65±0.05
3.04±0.08
2
p
a
9.42±0.10
>100
6.93±0.09
nd
13.92±0.12
nd
3
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3
b
8.70±0.11
2.93±0.09
0.48±0.05
3
c
10.88±0.15
7.30±0.13
9.08±0.10
1.41±0.08
2.58±0.09
0.16±0.05
3
d
e
S
3
46.06±0.13
58.30±0.14
nd
nd
nd
nd
OH
3
f
3
3
g
>100
>100
nd
nd
nd
nd
OCH3
h
H CO
3
3
3
i
j
OCH3
1.67±0.07
3.22±0.05
1.41±0.05
OCH3
OCH3
>100
>100
nd
nd
nd
nd
nd
nd
OCH3
3
k
H CO
OCH3
OCH3
3
3
l
22.16±0.13
H CO
3
OCH3
3
m
O
18.91±0.11
>100
24.08±0.16
nd
12.08±0.17
nd
3
n
S
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3
3
o
p
>100
nd
nd
N
N
N
18.41±0.15
11.24±0.10
6.26±0.09
3
q
24.70±0.08
73.50±0.20
nd
nd
nd
nd
3
r
H CO
3
3
s
>100
nd
nd
DDP
4.52±0.10
6.10±0.09
1.83±0.05
Each compound was tested in sextuplicate, nd: not determined. The error bars in all panels represent
mean ± SD from three independent experiments.
Table 2. HIV-1 inhibition activity
Concentration (M)
Concentration (M)
Compound
Compound
0
.2
2.0
20
0.2
2.0
20
1
2
2
a
b
d
0.84±0.02 0.74±0.05 0.70±0.02
1.00±0.09 0.91±0.08 0.45±0.05
0.86±0.03 0.86±0.05 0.67±0.03
1.00±0.09 0.84±0.07 0.32±0.04
2a
2c
2e
2h
1.10±0.10 0.67±0.06 0.44±0.02
0.86±0.02 0.56±0.02 0.29±0.01
1.01±0.10 1.00±0.09 0.30±0.04
1.04±0.10 0.77±0.07 0.11±0.03
2
f
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2
k
0.95±0.08 0.90±0.07 0.28±0.04
0.94±0.10 0.92±0.08 0.18±0.03
1.05±0.11 1.04±0.11 0.25±0.02
0.96±0.08 0.55±0.04 0.27±0.03
0.96±0.10 0.64±0.05 0.33±0.02
1.04±0.09 1.01±0.11 0.35±0.02
2n
2s
3b
3i
0.90±0.09 0.88±0.05 0.59±0.04
1.05±0.08 1.02±0.10 0.15±0.03
0.76±0.06 0.64±0.05 0.26±0.03
0.97±0.09 0.59±0.04 0.43±0.03
0.89±0.09 0.72±0.04 0.47±0.03
1.00±0.09 0.46±0.03 0.23±0.03
2
2
r
t
3
f
3
l
3n
3q
3
p
Each compound was tested in triplicate. The error bars in all panels represent mean ± SD from three
independent experiments.
Table 3. Anti-proliferative activity of 2c and 3b against a panel of human cancer cell lines by MTT assay.
Compound
Human cell line 48h –MTT IC (μM) ± S.D.
50
HeLa
MCF-7
HePG2
H460
A549
2
c
15.54±0.08
8.70±0.06
19.30±0.35
5.10±0.11
13.21±0.30
2.93±0.45
31.92±0.35
1.90±0.55
15.92±0.25
0.48±0.15
3b
Each compound was tested in sextuplicate. The error bars in all panels represent mean ± SD from three
independent experiments.
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