Telescoped Synthesis of 2-Acyl-1-aryl-1,2-dihydroisoquinolines and Their Inhibition of the Transcription Factor NF-κB

Article abstract of DOI:10.1021/acscombsci.5b00001

(Chemical Equation Presented). A sequential single-flask multicomponent reactions is highly effective for the synthesis of 1,2-dihydroisoquinolines through amidealkylation from intermediate N-acylisoquinolinium salts under mild conditions. N-Acylisoquinol

Full text of DOI:10.1021/acscombsci.5b00001

Research Article
pubs.acs.org/acscombsci
Telescoped Synthesis of 2‑Acyl-1-aryl-1,2-dihydroisoquinolines and
Their Inhibition of the Transcription Factor NF-κB
Tsai-Wen Chung,† Yi-Tzu Hung,† Tushar Thikekar,† Vijaykumar V. Paike,† Fu Yuan Lo,‡ Pei-Hua Tsai,‡
Mei-Chih Liang,*,‡ and Chung-Ming Sun*,†,§
‡
†Department of Applied Chemistry, Department of Biological Science and Technology, National Chiao Tung University,
Hsinchu 300, Taiwan
§Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, 100 Shih-Chuan First Road, Kaohsiung 807,
Taiwan
S
* Supporting Information
ABSTRACT: A sequential single-flask multicomponent reactions is highly effective for the synthesis of 1,2-dihydroisoquinolines
through amidealkylation from intermediate N-acylisoquinolinium salts under mild conditions. N-Acylisoquinolinium ions and
trichloromethyl-1-(1H-indol-3-yl)isoquinoline-2(1H)-carboxylate have demonstrated their reactivity toward aromatic and ali-
phatic π-nucleophiles. One of the 1,2-dihydroisoquinoline derivatives was found to be a potent inhibitor for transcription factor
NF-κB by blocking IκBα degradation, p65 nuclear translocation, and NF-κB DNA binding in TNF-α-induced NIH 3T3 cells.
KEYWORDS: dihydroisoquinolines, transcription factor NF-κB, p65 nuclear translocation, amidealkylation, DNA binding, 3T3 cells
Wu et al. described silver-catalyzed tandem reactions of N-(2-
alkynylbenzylidene)-hydrazides with several alkynes to synthe-
size 1,2-dihydroisoquinolines.7 Larock and co-workers have syn-
thesized 1,2-dihydroquinolines through the one-pot, multicomponent
reaction of 2-(1-alkynyl)benzaldehydes, amines, and ketones in
the presence of AgOTf and L-proline.8
Nuclear factor-kappa B (NF-κB) is a transcription factor that
regulates many cellular processes, including immune responses
to infection and inflammation, apoptosis, and embryonic and
neuronal development.9 Constitutively active NF-κB has been
identified in malignant hematologic cells and NF-κB is thus a
promising target to develop anti-cancer therapeutics in
hematologic malignancies.9 A literature survey revealed that
isoquinoline and indole-based natural or synthetic inhibitors are
available for NF-κB activation, such as emetine, lestaurtinib,
tribromsalan, cycloepoxydon, and Sunitinib malate (Figure 1).10,11
The lestaurtinib and emetine are cytotoxic to various human acute
myeloid leukemia (AML) cell lines and primary human AML
INTRODUCTION
■
One of the challenging goals in synthetic chemistry is to develop
a new one-pot strategy to construct complex molecules through
simultaneous formation and isoquinolines from available starting
materials. Multicomponent reactions (MCRs) provide novel
methods for the formation of multiple carbon−carbon bonds in a
single operational step and represent an innovative approach to
the synthesis of polyfunctional molecular scaffolds.1 Single-flask
MCRs enable the rapid construction of small heterocycles that
play a significant role in drug discovery research to provide one of
the greatest sources of molecular diversity.2 Recently, one pot,
multistep, and successive addition reactions have shown
significant potential in the construction of pharmaceutically
interesting heterocycles.3 Among heterocycles, isoquinolines and
indoles are important structural motifs in natural products and
have received significant attention because of their broad
biological activities.4 Naturally occurring and synthetic analogs
of N-substituted indoles are pharmaceutically important because
they are inhibitors of enzymes and antihypertensive drugs.5
Recently, the stereoselective introduction of 1-arylation
to isoquinolines by using a acyl chloride and aryl Grignard
reagents was reported by Wanner and co-workers.6 Furthermore,
Received: January 2, 2015
Revised: June 18, 2015
© XXXX American Chemical Society
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Figure 1. Structure of NF-κB inhibitors.
Scheme 1. A Novel Route toward the Synthesis of 2-Acyl-1-aryl-1,2-dihydroisoquinolines 5
blasts, through inhibition of NF-κB signaling in cancer cells.10
Epoxyquine A and cycloepoxydon were isolated from fermenta-
tion of a deuteromycete strain to inhibit activation of NF-κB that
regulates the expression of various cellular genes.11
observed that the electrophilic N-acylisoquinolinium intermedi-
ate 3 underwent selectively heteroarylation at the C-1 position by
nucleophilic attack of indoles 4{1}.15 The same reaction was
tested in different solvents (Table 1, entries 1−8), and we found
that a polar aprotic solvent such as dimethylformamide (DMF)
gave an 88% yield of the corresponding product (Table 1,
entry 7). An equimolar amount of sodium hydride gave a smooth
and clean reaction to afford the desired products 5{1,1,1} in
higher yields (94%) within 30 min at ambient temperature
(Table 1, entry 17). Although potassium carbonate (Table 1,
entry 7), 4-dimethylaminopyridine (Table 1, entry 15), and
sodium hydride (Table 1, entry 17) furnished the product
5{1,1,1} in more or less similar yields, sodium hydride was
chosen as a base for further reaction optimization because of its
higher yield (94%) and short reaction time (30 min) .
After confirming the viability of the proposed sequential one-
pot MCRs to construct diverse dihydroisoquinolines 5, we
explored the scope of this reaction with different isoquinolines
1{1−3}, indoles 4{1−6} and acyl chlorides 2{1−9} (Figure 3).
The reactions worked well, tolerating various functional groups
at C-4 and C-3 of the acyl chloride (Table 2, 5{1,1,5}−{1,1,8}).
The acyclic acid chloride 5{1,1,1} gave a better yield than the
cyclic acid chlorides (Table 2, 5{1,1,2} and 5{1,2,9}−{2,4,9}).
The absolute configuration of compound 5{1,1,2} was con-
firmed by X-ray diffraction (CCDC 935318),16 in Figure 2.
The crystallographic data reveal that the acid chloride and
indole moiety are linked to isoquinoline at the N2 and C1
position, respectively. The residue at the C1 indole group
distinctly occupies the space perpendicular to the basic skeleton
of isoquinoline.
As part of our continued interest in the synthesis of bio-
logically interesting small molecules,12 we have developed a
one-pot, telescoped synthesis for the construction of 2-acyl-1-
aryl-1,2-dihydroisoquinolines which represents a new class of
compounds to inhibit activation of transcription factor NF-κB.
Our initial effort was focused on one-pot MCRs through
N-carboxylation of isoquinoline, followed by C−C bond
formation between N-acylisoquinolinium ion and indole.
N-Acylisoquinolinium ions 3 were successfully used as electro-
philic reagents in an amidoalkylation reaction for the synthesis of
several isoquinolines derivatives and had high reactivity in the
heteroarylation reaction of aromatic π-nucleophiles of organo-
metallic compounds.13
Herein, we investigated MCRs of isoquinoline 1 with n-propyl
chloride 2{1} and 1H-indole 4 to 2-acyl-1-aryl-1,2-dihydroiso-
quinolines 5 using various bases and solvents at ambient tem-
perature (Scheme 1), and the results are summarized in Table 1.
To enhance the reaction efficiency and improve yields, we
conducted the sequential addition of reagents in the same pot
without any workup/purification of intermediates (Scheme 1).
A preliminary study shows potassium carbonate in toluene can
promote the C−C bond formation between N-acylisoquinoli-
nium ion 3 and indole 4{1} to afford the 2-acyl-1-aryl-1,2-
dihydroisoquinolines 5{1,1,1} in 15% yield (Table 1, entry 1).
The reaction occurs selectively at the C-3 position of indole even
when both the C-2 and C-3 positions are unoccupied.14 We
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a
alcohol and thiol nucleophiles performed well to afford resultant
products 8 and 9 in good yields. The selectivity was also observed
with 2-amino thiophenol, in which the thiol is more nucleophilic
than the aniline group, furnishing the corresponding compound
9{1,1,1,2} in high yield (92%). But thiol nucleophiles 14{3} gave
only a 45% yield for the same condition, and all of the phenols
nucleophiles did not react at all. Amine nucleophile 11{5} did
not deliver 7{1,1,1,5} but gave 1-(1H-indol-3-yl)isoquinoline. It
is clear that 4-aminopyridine plays a base instead of a nucleophile
because of the electron-withdrawing nature of pyridine N.
As a further extension of this study, we found that the reac-
tion of N-sulfonylisoquinolinium 18 with indole 4 furnished the
corresponding 1-(1H-indol-3-yl)-2-(alkyl or aryl-sulfonyl)-1,2-
dihydroisoquinoline 19 in high yields (Scheme 3). We also
attempted to increase the diversity of compound 5 or 9 with
isothiocyanate 21 to 3-(2-butyryl-1,2-dihydroisoquinolin-1-yl)-
N-phenethyl-1H-indole-1-carbothioamide 22. Delighted with
this observation, we treated a number of 2-acyl-1-aryl-1,2-
dihydroisoquinolines 5{1,1,1} or 19{1,1,3}, 19{1,1,4} with
isothiocyanate 21{1−7} by this protocol. When the C-3 position
of the indole moiety is occupied by substituents other than
hydrogen, then the indole nitrogen is the most reactive toward
electrophiles.18 Thus, the nitrogen of indole was reacted with
isothiocyanate to afford the N-alkylated indole motif; for
example, substituted thioureas.
Table 1. Optimization of the Reaction Conditions
b
entry
base
solvent
yield (%)
1
2
3
4
5
6
7
8
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
Et3N
toluene
THF
15
18
N.R.
28
37
41
88
27
48
63
N.R.
60
38
10
86
43
94
MeOH
ether
dioxane
DMAC
DMF
EDC
c
9
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
d
10
11
12
13
14
15
16
Cs2CO3
CsF
DBU
DMAP
tBuOK
NaH
Initially, the reactions of 5{1,1,1} with (2-isothiocyanatoethyl)-
benzene 21{1} were carried out with NaH in THF at room
temperature to deliver substituted thiourea 22{1,1} in 82% yield
(Table 4). N-Alkylation reaction of 2-acyl-1-indole-1,2-dihy-
droisoquinoline with isothiocyanate was first reported in the
presence of base under mild reaction conditions. We tested the
reactions on 2-acyl-1-indole-1,2-dihydroisoquinoline 5{1,1,1}
(Table 4, entries 1−2) and 2-sulfonyl-1-indole-1,2-dihydroiso-
quinolin 19{1,1,3}, 19{1,1,4} to provide N-alkylated product
22{2,1}−{3,7} in good to moderate yields (Table 4, entries 3−10).
Earlier studies have shown that various isoquinoline- and
indole-containing compounds are inhibitors of NF-κB.7−10
Because NF-κB is a transcription factor involved in the regulation
of numerous genes,19 we proceeded to determine whether
2-acyl-1-aryl-1,2-dihydroisoquinolines could inhibit NF-κB activ-
ity. NIH 3T3 cells were either untreated (Figure 4A, lanes 1 and 2)
or treated with 20 μM 19{1,1,1} (lanes 3−7) and then stimulated
with TNF-α an indicated number of times. The level of IκBα was
then analyzed by Western blotting using a polyclonal antibody
against the carboxyl terminus of IκBα. Treatment with 20 μM
19{1,1,1} effectively inhibited IκBα degradation at various times
(5, 10, 15, and 40 min) post-TNF-α induction (Figure 4A, lanes
3−7). In contrast, treatment with TNF-α for 20 min led to
apparent IκBα degradation in the control cells (Figure 4A, lane 2).
Because phosphorylation of IκBα at serine residues 32 (S32) and
36 (S36) is necessary for TNFα-induced IκBα degradation,20 we
next examined the effect of 19{1,1,1} in IκBα phosphorylation
by a Western blot using an antibody specific for detecting S32
phosphorylation in IκBα. As shown in Figure 4B, phosphor-
ylation of IκBα peaked at 5 min following TNF-α stimulation in
untreated cells (lanes 2−4). Treatment with 20 μM 19{1,1,1} led
to effective inhibition of IκBα phosphorylation at various time
periods after stimulation (lanes 6−8). These results indicate that
19{1,1,1} is a potent inhibitor of NF-κB activation by blocking
phosphorylation and degradation of IκBα.
e
17
a
Reactions were performed in the presence of base (1 equiv) at room
b
c
temp for 12 h. Isolated yield after column purification. Reaction tem-
perature 10 °C. Reaction temperature 40 °C. Reaction time 30 min;
N.R.- no reaction.
d
e
After reaction optimization, we examined synthesis of 1-(1H-
indol-3-yl)-N-phenylisoquinoline-2(1H)-carboxamide 7 by
using isocyanate 6, N,N′-carbonyldiimidazole (CDI) 10, and
triphosgene, illustrated in Scheme 2. The first approach using
pathway A (Scheme 2) did not produce the expected compound
in either DMF or toluene after prolonged reaction time at higher
temperatures. In path B, the reaction of 1 with CDI 10 and indole
4 followed by addition of amine 11 in DMF did not afford the
desired product 7{1,1,1}. Because acid chloride and sulfonyl
chloride are more reactive than isocyanate and CDI, the synthesis
of a Reissert type adduct is more favorable with more activated
electrophiles,17 therefore, when isoquinoline 1 was reacted with
triphosgene and indole 4 (path C), followed by addition of
various nucleophiles, such as amines, alcohols, or thiols, to
deliver the corresponding 1,2-dihydroisoquinolines 7 derivatives
smoothly (Table 3).
Isoquinoline activation by triphosgene followed by nucleo-
philic attack of indole afforded the corresponding key inter-
mediate trichloromethyl-1-(1H-indol-3-yl)isoquinoline-2(1H)-
carboxylate 16. The carbon NMR spectrum clearly shows two
extra quaternary carbon peaks at ∼148 and ∼132 ppm. Sub-
sequently, compound 16 was employed to expand the library
diversity, with various heteroatom nucleophiles, such as amines,
alcohols, and thiols, at room temperature, as illustrated in path C
(Scheme 2).17b For the scope of the nucleophiles, we reacted two
amines 11{1−7}, an alcohol 13{1−4}, and two thiols 14{1−3}
(Figure 3).
The utility of this stepwise one-pot and multistep synthesis
was demonstrated in Table 3. Although more-reactive nucleo-
philes afforded the highest yields (9), poor nucleophiles gave
modest yields (7 and 8). Not only amine nucleophiles but also
After TNF-α-induced IκB degradation, the transcription factor
NF-κB undergoes nuclear translocation and binds to targeted
genes.19b To further investigate whether NF-κB nuclear
C
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a
Table 2. Substrate Scope for One-Pot, Two-Step MCRs
a
Reactions were performed in the presence of NaH (1 equiv) at room temp (25 °C) for 30 min; in parentheses are isolated yield.
CONCLUSIONS
■
We report a telescopic type three-component synthesis of 2-acyl-
1-aryl-1,2-dihydroisoquinolines. The efficiency of this novel and
bioinspired MCR plays the key role for the synthesis of 2-acyl-1-
aryl-1,2-dihydroisoquinolines and 3-(2-butyryl-1,2-dihydroiso-
quinolin-1-yl)-N-phenethyl-1H-indole-1-carbothioamide deriva-
tives. The sequential one-pot MCRs show good functional group
tolerance and are high-yielding, and the final product isolation is
very straightforward. Multiple bonds were conveniently formed
Figure 2. ORTEP diagram of compound 5{1,1,2}.
in one pot during these telescopic processes and are useful for the
assembly of various N-heterocycles for pharmaceutical interests.
The biological experiments identified that the 1,2-dihydroiso-
translocation is effectively blocked by 19{1,1,1}, we analyzed
quinoline derivative 19{1,1,1} effectively inhibits NF-κB
nuclear extracts of samples by Western blotting using antibodies
activation by inhibition of IκBα degradation and NF-κB DNA
against the NF-κB subunit p65. As shown in Figure 4C, apparent
binding in TNF-α-stimulated NIH 3T3 cells. Further inves-
nuclear accumulation of p65 was observed in TNF-α-stimulated
tigation of these novel dihydroisoquinoline derivatives (5, 7, 8, 9,
3T3 cells (lane 2). In contrast, treatment of 19{1,1,1} resulted in
and 22) as potent inhibitors of the transcription factor NF-κB are
effective inhibition of p65 nuclear translocation (lane 4). In
in progress and will be reported in due course.
addition, 19{1,1,1} blocked NF-κB DNA binding activity as
measured by an electrophoretic mobility shift assay (EMSA)
EXPERIMENTAL PROCEDURES
(Figure 4C, lane 4 of upper panel). Taken together, these results
suggest that the novel 2-acyl-1-aryl-1,2-dihydroisoquinoline
derivative 19{1,1,1} is a potent inhibitor for NF-κB activation,
and the inhibition occurs via the inhibition of IκBα phosphor-
ylation and degradation, NF-κB nuclear translocation, and DNA
binding activity in TNF-α-induced NIH 3T3 cells (Figure 4).
■
General Synthetic Procedures for 1-(1-(1H-Indol-3-yl)-
isoquinolin-2(1H)-yl)butan-1-one (5{1,1,1}). Isoquinoline
(0.77 mmol, 1 equiv) was dissolved in dimethylformamide
(5 mL), then butyryl chloride (0.85 mmol, 1.1 equiv) was added. The
reaction mixture was stirred under nitrogen atmosphere at ambient
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Scheme 2. Synthetic Route to the N-Ethyl-1-(1H-indol-3-yl)isoquinoline-2(1H)-carboxamide Derivatives 7, 8, and 9
a
Table 3. One-Flask, Three-Step MCRs Route toward 1-(1H-Indol-3-yl)-N-phenylisoquinoline-2(1H) Derivatives (7, 8, and 9)
a
Reactions were performed in the presence of NaH (10 equiv) at room temp (25 °C) for 35 min.
temperature for 5 min. The progress of the reaction was monitored
(0.77 mmol, 1 equiv) and sodium hydride (0.77 mmol, 1 equiv) were
by TLC. After complete consumption of isoquinoline, 1H-indole
added to the reaction, and the mixture was stirred for 30 min at room
E
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Figure 3. Diversity elements employed for library synthesis: isoquinoline 1{1−3}; indoles 4{1−4}; acyl chloride 2{1−9}; amine 11{1−7}; alcohol 13{1−4};
thiol 14{1−3}; sulfonyl chloride 17{1−4}; 2-acyl-1-aryl-1,2-dihydroisoquinolines 5{1,1,1}, 19{1,1,3}, 19{1,1,4}; and isothiocyanate 21{1−7}.
temperature. After reaction completion, the reaction mixture was
extracted with ethyl acetate (3 × 25 mL), and the collected organic
extracts were washed with aqueous sodium carbonate and dried over
magnesium sulfate. The solvent was removed by rotary evaporation,
and the residue was purified by silica column chromatography (eluent:
20% EA in hexane) to obtain the corresponding compound 1-(1-
(1H-indol-3-yl)isoquinolin-2(1H)-yl)butan-1-one 5{1,1,1} (94%).
1H NMR (300 MHz, CDCl3) δ 8.35 (s, 1H), 7.96 (d, J = 7.7 Hz,
1H), 7.37−7.10 (m, 8H), 6.63−6.58 (m, 2H), 6.13 (d, J = 7.6 Hz,
1H), 2.48−2.36 (m, 2H), 1.83−1.68 (m, 2H), 1.00 (t, J = 7.4 Hz,
3H); 13C NMR (75 MHz, CD3COCD3) δ 170.9, 137.2, 133.9,
131.3, 127.9, 127.5, 127.2, 126.3, 125.6, 125.3, 124.9, 121.8,
120.5, 119.4, 117.3, 111.6, 110.8, 49.4, 35.3, 18.4, 13.6; IR
(cm−1, neat) 3243, 2960, 1648, 1617, 746; ESI-MS m/z 339
[M + Na]+. HRMS m/z calcd for C21H20N2ONa, 316.1576;
found, 339.1475 [M + Na]+.
The Synthesis of N-Butyl-1-(1H-indol-3-yl)isoquinoline-
2(1H)-carboxamide (7{1,1,1}). A tetrahydrofuran (THF, 5 mL)
F
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Scheme 3. Synthetic Pathway for Compound 19
a
Table 4. Coupling Reaction of Various 1,2-Hydroquinolines with Thioisocyanate
a
b
Reactions were performed with base (10 equiv) in dry THF for 30 min. Isolated yield after column chromatography.
solution of isoquinoline (0.77 mmol, 1 equiv) and bis-
(trichloromethyl) carbonate (triphosgene, 1.16 mmol, 1.5 equiv)
was stirred at ambient temperature for 5 min. After complete
consumption of isoquinoline, 1H-indole (0.77 mmol, 1 equiv) and
potassium carbonate (1.55 mmol, 2 equiv) were added to the
reaction flask and stirred for 30 min, followed by addition of butan-
1-amine (11.6 mmol, 15 equiv). After the reaction was complete,
the solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography (eluent: 20% EA
in hexane) to get the corresponding compound N-butyl-1-(1H-
indol-3-yl)isoquinoline-2(1H)-carboxamide 7{1,1,1} (79%).
1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 8.06−7.97 (m,
1H), 7.34−7.27 (m, 1H), 7.21−7.14 (m, 6H), 6.82−6.73 (m, 2
H), 6.78 (s, 1H), 6.74 (d, J = 7.5 Hz, 1H), 6.02 (d, J = 7.5 Hz,
1H), 4.85 (m, 1H), 3.32−3.23 (m, 2H), 1.51−1.37 (m, 2H),
1.31−1.19 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 155.4, 136.7, 133.1, 130.7, 127.9, 127.4, 127.1, 125.7,
124.9, 124.9, 123.9, 122.7, 120.5, 120.3, 118.4, 111.6, 109.3, 51.9,
41.2, 32.4, 20.4, 14.2; IR (cm−1, neat) 3216, 2925, 1616, 1517,
742; ESI-MS m/z 368 [M + Na]+. HRMS m/z calcd for
C22H23N3O, 345.1841; found, 368.1741 [M + Na]+.
The Synthesis of 3-(2-Butyryl-1,2-dihydroisoquinolin-
1-yl)-N-phenethyl-1H-indole-1-carbothioamide (22{1,1}).
Sodium hydride (6.32 mmol, 20 equiv) was dissolved in
tetrahydrofuran (THF, 10 mL), followed by addition of
compound 1-(1-(1H-indol-3-yl)isoquinolin-2(1H)-yl)butan-
1-one 5{1,1,1} (0.32 mmol, 1 equiv) with slow addition of
(2-isothiocyanatoethyl)benzene (1.58 mmol, 1.5 equiv) in a
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Figure 4. Novel 2-acyl-1-aryl-1,2-dihydroisoquinoline derivative 19 is a potent inhibitor of the transcription factor NF-κB. NIH 3T3 cells were untreated
or treated with 20 μM 19{1,1,1} for 2 h and stimulated with or without TNF-α (5 ng/mL) for indicated times before harvesting. Equal amounts of whole
cell lysates were analyzed by Western blotting with the indicated antibodies. 19{1,1,1} inhibited TNF-α induced IκBα degradation (A) and
phosphorylation (B) in 3T3 cells. (C) 19{1,1,1} inhibited p65 nuclear translocation, and NF-κB DNA binding activity induced by TNF-α. 3T3 cells
were either untreated or treated with 19{1,1,1} (20 μM) and stimulated with or without TNF-α (5 ng/mL) as described in parts A and B. EMSA analysis
using a biotin-labeled NF-κB binding sequence and Western blots for p65 and TFIID (nuclear protein loading control) were performed using equal
amounts of nuclear extracts.
round-bottom flask at 0 °C. The resultant reaction mixture was
stirred at 0 °C for 30 min. After the reaction was complete, the
solvent was evaporated under reduced pressure. The resulting
crude residue was purified by column chromatography (eluent:
10% EA in hexane) to obtain the corresponding 3-(2-butyryl-1,2-
dihydroisoquinolin-1-yl)-N-phenethyl-1H-indole-1-carbothioa-
mide 22{1,1} (82%).
Samples containing equal amounts of protein were analyzed by
SDS−polyacrylamide gel electrophoresis and transferred to
PVDF membranes. Western blotting primary antibodies were
purchased from the following companies: rabbit polyclonal anti-
IκBα (SC-371, Santa Cruz Biotechnology, Dallas, TX); rabbit
monoclonal antiphospho-IκBα (Ser32; 2859S, Cell Signaling
Technology, Beverly, MA); rabbit polyclonal anti-NF-κB p65
subunit (SC-372, Santa Cruz Biotechnology); rabbit polyclonal
anti-TFIID (SC-204, Santa Cruz Biotechnology); goat poly-
clonal anti-β-actin (SC-1616, Santa Cruz Biotechnology), and
rabbit polyclonal anti-α-tubulin (SC-12462-R, Santa Cruz
Biotechnology). The blots with bound primary antibodies were
then incubated with horseradish peroxidase (HRP)-conjugated
secondary antibodies (Thermo Scientific, Waltham, MA) and
visualized using SuperSignal West Dura Chemiluminescent
Subtract (Thermo Scientific).
Electrophoretic Mobility Shift Assay. NF-κB activity was
measured by an electrophophoretic mobility shift assay (EMSA).
Equal amounts of nuclear extracts were incubated with the
26-base-pair biotin end-labeled κB binding site (κB site:
5-GGGAAATTCC-3)22 using the LightShift Chemiluminescent
EMSA Kit (Thermo Scientific) according to the manufacturer’s
instructions. The complexes were separated by gel electro-
phoresis using a 5% nondenaturing polyacrylamide gel and
transferred to a nylon membrane. The transferred DNA was
cross-linked to membrane by UV light and then incubated with
streptavidin−horseradish peroxidase conjugate. The biotin-
labeled DNA−protein complexes were detected by chemilumi-
nescence.
1H NMR (300 MHz, CDCl3) δ 7.96 (d, J = 7.9 Hz, 1H), 7.41−
7.05 (m, 14H), 6.59 (d, J = 7.5 Hz, 1H), 6.13 (d, J = 7.5 Hz, 1H),
4.04 (q, J = 6.5 Hz, 2H), 3.06 (t, J = 6.5 Hz, 2H), 2.38 (t, J = 7.4
Hz, 2H), 1.77−1.62 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ 179.9, 171.7, 138.4, 134.5, 132.6, 130.5,
129.8, 129.5, 129.2, 129.0, 129.0, 128.5, 128.2, 127.5, 127.3,
125.5, 124.8, 124.5, 123.2, 121.8, 121.3, 112.8, 112.2, 48.9, 47.4,
35.9, 34.2, 18.6, 14.3; IR (cm−1, neat) 3239, 2958, 1656, 1621,
1344, 1174, 1043; ESI-MS m/z 479.32 (M+). HRMS m/z calcd
for C30H29N3OS, 479.2031; found, 478.1974 [M − H]+.
Cell Culture and Treatment. NIH 3T3 fibroblasts were
maintained in Dulbecco’s modified Eagle’s medium (DMEM)
(Biowest, Nuaille, France) supplemented with 10% fetal bovine
serum (FBS; Biowest), 100 U/mL penicillin, and 100 μg/mL
streptomycin. Cells were maintained at 37 °C in a fully
humidified incubator with 5% CO2.
Stock solutions of 2-acyl-1-aryl-1,2-dihydroisoquinoline de-
rivative 19{1,1,1} were dissolved in DMSO and stored at −80 °C
before use. Twenty-four hours before drug treatment, 3T3 cells
were serum-starved in medium containing 0.5% FBS. After
serum starvation, the cells were treated with the vesicle control
DMSO or 20 μM 19{1,1,1} for 2 h and stimulated with 5 ng/mL
recombinant human TNF-α (R&D Systems, Minneapolis, MN)
for the indicated amount of time.
Protein Extraction and Western Blotting. After the
indicated treatments, 3T3 cells were washed with ice-cold PBS
twice before harvesting. Whole cell lysates or nuclear extracts
were prepared as previously described.21 The protein concen-
tration was then determined by the method of Bradford using the
Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) according to
the manufacturer’s instructions.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional preparation procedures; 1H, 13C, ESI, HRMS, IR data,
and spectra for the compounds; X-ray information. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acscombsci.5b00001.
H
DOI: 10.1021/acscombsci.5b00001
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ACS Combinatorial Science
Research Article
1272−1280. (b) Bonnesen, C.; Eggleston, I. M.; Hayes, J. D. Dietary
indoles and isothiocyanates that are generated from cruciferous
vegetables can both stimulate apoptosis and confer protection against
DNA damage in human colon cell lines. Cancer Res. 2001, 61, 6120−
6130.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: cmsun@mail.nctu.edu.tw.
*E-mail: mcliang@cc.nctu.edu.tw.
Notes
■
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the National Science Council of Taiwan for
financial support and the authorities of the National Chiao Tung
University for providing the laboratory facilities. This study was
particularly supported by the “Centre for bioinformatics research
of aiming for the Top University Plan” of the National Chiao
Tung University and Ministry of Education, Taiwan.
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