Synthesis and structure-activity relationship of novel indole acrylamide derivatives as hcv replication inhibitors

Article abstract of DOI:10.1002/bkcs.10021

Aseries of indole acrylamide derivatives were synthesized and evaluated for their inhibitory effects on hepatitis C virus (HCV) replication. Previously, we have identified (E)-N-(4- tert-butylphenyl)-3-(5-cyano-1H-indol-3-yl)-2-methylacrylamide (6c) as on

Full text of DOI:10.1002/bkcs.10021

Article
DOI: 10.1002/bkcs.10021
BULLETIN OF THE
S. Son et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Structure–Activity Relationship of Novel Indole
Acrylamide Derivatives as HCV Replication Inhibitors
†
†
*
Seohyun Son, Dahee Kim, Sungjin Lee, Guanghai Jin, Jin-Ah Park, Hyo-Kyung Han, Kyeong Lee,
*
and Choongho Lee
College of Pharmacy, Dongguk University-Seoul, Goyang 410-820, Ilsandong-gu, Korea.
*E-mail: kaylee@dongguk.edu; choongholee@dongguk.edu
Received August 11, 2014, Accepted September 19, 2014, Published online January 5, 2015
Aseries ofindole acrylamide derivatives weresynthesizedandevaluated fortheir inhibitoryeffects onhepatitis
C virus (HCV) replication. Previously, we have identified (E)-N-(4-tert-butylphenyl)-3-(5-cyano-1H-indol-3-
yl)-2-methylacrylamide (6c) as one of the promising leads for anti-HCV chemotherapy. Based on the structural
features of indole acrylamide, we have explored extended structure–activity relationship study using analogs
with substituted indoles, various amides, and N-substitution at the indole ring. Among the newly synthesized
series, 5-cyanoindole acrylamide analog with N-acetyl substitution (13c) (EC50 = 0.98 μM, CC50 = 40.74 μM,
and SI = 41.6) exhibited the most potent antiviral activity with reasonable cytotoxicity in a cell-based J6/JFH1
reporter assay using Huh7.5 cells. In addition, improved water solubility of 13c compared to 6c further merits
consideration of 13c as a valuable candidate for anti-HCV therapeutics development.
Keywords: Hepatitis C virus, HCV replication inhibitors, Indole derivatives, Structure–activity
relationship (SAR) study
Introduction
indole 2-methylacrylamide derivatives as novel HCV NS5B
polymerase inhibitors.5 In order to develop more potent
HCV replication inhibitors with improved water solubility,
we further synthesized a series of indoles with N-substitution
and evaluated their biological activities. Here, we report struc-
ture–activity relationship (SAR) of a novel series of indole
acrylamide derivatives. We hope that this work will lead to
highly potent HCV inhibitors for further development of a
new class of HCV replication inhibitors.
Hepatitis C virus (HCV) is a clinically important human path-
ogen affecting over 180 million people worldwide.1 Infection
with HCV has a high risk of developing chronic liver cirrhosis
and hepatocellular carcinoma.2 Approximately, 40–45% of all
liver transplantation occur due to HCV infection, and it is the
leading cause for chronic liver diseases in the United States.3
In spite of recent emergence of highly active, direct-acting
antivirals (DAAs) targeting specific HCV proteins, the current
standard therapy still relies on a combination of pegylated
interferon-α and ribavirin because of the DAA's unaffordable
pricetags.4 Inaddition, thisinterferon-basedtherapycancause
undesirable side effects such as flu-like symptoms, anemia,
and depression in patients. Therefore, the new class of anti-
HCV therapeutics with a novel mechanism of action is
urgently needed to develop a more cost-effective and safer
antiviral in the future.5
A number of small molecules targeting nonstructural viral
proteins have been identified and have progressed to various
stages of clinical development. They include inhibitors of NS3
protease6/helicase7, NS4B,8,9 NS5A,10,11 and NS5B polymer-
ase.12 Especially, the HCV NS5B polymerase has been widely
studied as an attractive target to develop HCV inhibitors
because this polymerase, which is an RNA-dependent RNA
polymerase, plays a critical role for viral replication in the
overall life cycle of the virus.
Results and Discussion
As continuing efforts from our ongoing program for the
identification of more effective HCV inhibitors, we have
chosen three compounds 6a–6c as the starting point for new
compound generation. As shown in Scheme 1, commercially
available reagents, such as unsubstituted or substituted indole-
3-carboxaldehyde 1a–1c were used for Wittig reaction with
(carbethoxyethylidene) triphenylphosphorane or triethyl
2-fluoro-2-phosphonoacetate to provide compounds 2a–2c
and 3a–3c. The ethyl esters 2a–2c and 3a–3c were then sub-
jected to hydrolysis to yield carboxylic acid compounds 4a–4c
and 5a–5c. Carboxylic acids 4a–4c and 5a–5c were used for
coupling by using standard coupling reagent and 4-tert-
butylaniline to provide a series of indole 2-mehtylacrylamide
derivatives 6a–6c and 7a–7c.
As shown in Scheme 2, we set out to explore the effect of
N-substitution at indole 2-methylacrylamide. By using same
method from Scheme 1, a series of indole 2-methylacrylamide
derivatives 6a–6c were prepared and further modified using
We have recently reported the discovery of a novel series of
† These two authors contributed equally to this work.
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Synthesis and SAR of Indole Acrylamides
KOREAN CHEMICAL SOCIETY
O
O
O
b
OH
H
OEt
a
R1
R1
R1
R2
R2
N
H
N
H
N
H
1a-1c
1: R1 = a: H
b: 5-OMe
2a-2c, 3a-3c
4a-4c, 5a-5c
4:
2:
3:
R2 = Me
R2 = F
R2 = Me
5: R2 = F
c: 5-CN
tBu
O
c
N
R1
H
R2
N
H
6a-6c, 7a-7c
6:
R2 = Me
7: R2 = F
Scheme 1. Reagents and conditions: (a) (Carbethoxyethylidene) triphenylphosphorane, CH2Cl2 for 2a–2c; Triethyl 2-fluoro-2-phosphonoace-
tate, DBU, LiCl, THF for 3a–3c. (b) NaOH, MeOH, THF, H2O, and (c) 4-tert-butylaniline, HBTU, DIPEA, DMF, CH2Cl2.
tBu
tBu
O
O
N
N
a or b
R1
H
R1
H
CH3
CH3
N
R3
N
H
6a-6c
8a-15a, 8b-15b, 8c-15c
O
6: R1 = a: H
10:
13:
R3 = 8: Me
9:
O
b: 5-OMe
c: 5-CN
O
11:
12:
O
O
S
O
14:
15:
O
N
N
Scheme 2. Reagents and conditions: (a) Alkyl halide, acyl halide or sulfonyl halide, NaH, DMF for 10a–15a, 10b–15b, 10c–15c; (b) TBAB,
20% NaOH, alkyl halide, CH2Cl2 for 8a–9a, 8b–9b, 8c–9c.
alkylation, acylation, or sulfonylation to give the desired com-
pounds 8a–15a, 8b–15b, and 8c–15c.
structure except for the substitution group in indole part, such
asH, 5-OMe, and5-CN. First, weexploredtheeffect offluoro-
substitutioninsteadofmethylgroupintheacrylamideportion,
which maintains the remaining parts. Compounds 7a–7c
All the newly synthesized compounds with N-substitution
of indole ring were evaluated for their antiviral activity in a
cell-based J6/JFH1 reporter assay using Huh7.5 cells. The
in-vitro EC50, CC50, and selective index values for each ana-
loguearelisted inTable 1, andclemizolewasusedasapositive
control. MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenylte-
trazolium Bromide) based cytotoxicity (CC50) was measured
in parallel, to confirm whether the observed antiviral effect
(EC50) of each compound was due to its specific inhibitory
action against HCV replication and not due to mere reduction
of cell numbers by its cytotoxic effect. On the basis of the pre-
vious SAR study on indole 2-methylacrylamides,5 we have
chosen three compounds, 6a–6c, as the most potent com-
pounds among the series. These compounds share a common
exhibited moderate to poor inhibitory activities (7a, EC50
4.7 μM, CC50 = 10.4 μM, and SI = 2.2; 7b, EC50 = 6.3 μM,
CC50 = 11.7 μM, and SI = 1.9; 7c, EC50 = 8.8 μM, CC50
=
=
24.0 μM, and SI = 2.7). This result suggested the importance
of the methyl acryl portion in comparison with the correspond-
ing fluoro acryl portion. Accordingly, we synthesized N-
substituted indole 2-methylacrylate derivatives for further
study, as shown in Table2.
Of note, compound 6c, the most potent inhibitor in the pre-
vious study, had poor water solubility, which may cause dif-
ficulties in further study. To develop a better analog with
improved water solubility as well as high potency, we
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Synthesis and SAR of Indole Acrylamides
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Table 1. Structure–activity relationships of indole 2-methylacrylamides (6a–6c) or 2-fluoroacrlyamides (7a–7c).
tBu
O
N
R1
H
R2
N
H
Structure
No.
R1
R2
Anti-HCV (EC50, μM)
Cytotoxicity (CC50, μM)
Selectivity index (CC50/EC50)
6a
6b
6c
7a
7b
7c
Clemizole
H
Me
Me
Me
F
F
F
2.2
3.0
1.1
4.7
6.3
8.8
8.2
17.3
16.4
61.8
10.4
11.7
24.0
34.6
7.9
5.5
56.1
2.2
1.9
2.7
5-OMe
5-CN
H
5-OMe
5-CN
N/A
N/A
4.4
envisioned that theN-alkylated compounds6a–6c might show
desired profiles. As shown in Table 2, a series of N-substituted
indole ring derivatives 8a–15a, 8b–15b, and 8c–15c, which
were derived from the leading compound 6a–6c, were evalu-
ated for their biological activity. Among the N-substituted
indoles based on the 6a derivative, the sulfonyl analog 11a
(EC50 = 1.56 μM, CC50 = 54.1 μM, and SI = 34.7) exhibited
enhanced potency and selectivity index compared to the orig-
inal compound 6a. The corresponding benzyl (9a), acetyl
(13a), and morpholine-4-ethyl (14a) substituted compounds
showed moderate activity profiles, whereas methyl (8a),
CBZ (carboxybenzyl) (10a), and benzoyl (12a) substituted
compounds lost the activity. The N,N-dimethylaminoethyl
analog 15a showed moderate antiviral activity, but it was
found to be toxic with the SI value of 0.4. In the case of 5-
methyoxy indoles 8b–15b, which were derived from 6b, it
was found that N-substitution at the indole ring was not toler-
able on HCV inhibition, even though benzyl sulfonyl com-
pound 11b exhibited better selectivity (EC50 = 5.3 μM,
CC50 = 66 μM, and SI = 12.5) than the original 6b. As shown
in Table 2, 5-cyano indoles 8c–15c were also evaluated as
potential HCV agents. Of note, indole 13a and 5-OMe indole
13b, which aresubstituted withthe acetylgroup, showed mod-
erate potency, but the corresponding 5-CN indole 13c exhib-
ited enhanced potency (13a, EC50 = 4.0 μM, CC50 = 25.5 μM,
and SI = 6.40; 13b, EC50 = 8.9 μM, CC50 ≥100 μM, and SI
≥11.2; 13c, EC50 = 0.98 μM, CC50 = 40.74 μM, and SI =
41.6). Interestingly, the benzoyl compound 12c had improved
potency, while compounds 12a, and 12b, which were substi-
tuted with benzoyl, were less potent (12a, EC50 = 33.3 μM,
CC50 ≥100 μM, and SI ≥3.0; 12b, EC50 ≥100 μM, CC50
≥100 μM, and SI ≥1.0; 12c, EC50 = 1.16 μM, CC50 = 69.0 μ
M, and SI = 59.5). In Table 3, especially, Compound 6c had
poor solubility in water, even though its antiviral activity
was the best. Solubility in water is a general issue with syn-
thetic compounds for further study, and therefore the kinetic
solubilities of compounds 6c, 12c, and 13c were measured
by using an established nephelometry method. As in Table
3, an aromatic carbonyl-containing compound 12c was not
improved compared to the original compound 6c. Interest-
ingly, an alkyl carbonyl-containing compound 13c exhibited
much better water solubility at the three different pH condi-
tions than original compound 6c.
From the SAR study, we found that compounds 12c and 13c
exhibited potent anti-HCV activities. Therefore, we further
evaluated them for their in-vitro water solubility under various
pH conditions. The kinetic solubility of compounds 6c, 12c,
and 13c were measured using an established nephlometry
method. Of note, the solubility of benzoyl-substituted com-
pound 12c was not improved over the original compound
6c (Table 3). Interestingly, the acetyl-substituted compound
13cexhibited muchbetterwater solubility atthethreedifferent
pH conditions than the original compound 6c.
In order to confirm the antiviral effect of compound 13c on
HCV replication in a reporter-free system, we transfected in-
vitro-transcribed genotype 2a J6/JFH1 RNAs into Huh7.5
cells and treated these cells with increasing amounts of com-
pound 13c for 72 h. Then, the viral as well as the host GAPDH
RNA levels were measured by real-time polymerase chain
traction (RT-PCR) analysis. As shown in Figure 1(a), a nearly
threefold higher EC50 value (3.2 μM) was observed in this sys-
tem compared with the one (0.98 μM) measured previously
using the renilla luciferase-linked reporter virus. In addition,
when J6/JFH1 RNA-transfected cells were treated with com-
pound 13c at a concentration of 10 μM for different time per-
iods, thelengthof time required forreducing HCV RNA levels
by half (T1/2) turned out to be 26.8 h (Figure 1(a)). We also
wanted to test the inhibitory activity of compound 13c against
the more clinically relevant genotype 1b HCV to see whether
compound 13c had any cross-genotype antiviral activity. For
this purpose, we used a Bart79I subgenomic replicon system,
inwhichgenotype1bHCVRNAsencodingonlythenonstruc-
tural components of stably replicating viral genes under selec-
tion pressure of G418 due to expression of a neomycin
phosphotransferase gene were inserted in front of HCV genes.
In this system, compound 13c showed a 1.3-fold higher
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Table 2. Structure–activity relationships of N-substituted indole 2-methylacrylamides (8a–15a, 8b–15b, and 8c–15c).
tBu
O
N
H
R1
CH3
N
R3
Structure
No.
R1
R2
Anti-HCV (EC50, μM)
Cytotoxicity (CC50, μM)
Selectivity index (CC50/EC50)
8a
9a
H
H
Me
21.7
6.8
19.3
>100
0.9
>14.7
O
10a
H
>100
63.7
>0.6
O
O
S
11a
12a
H
H
1.56
33.3
54.1
34.7
>3.0
O
O
>100
O
13a
14a
H
H
4.0
7.9
25.5
19.7
6.4
2.5
O
N
15a
H
18.5
6.9
0.4
N
8b
9b
5-OMe
5-OMe
Me
8.9
>100
26.5
>100
3.0
>1.0
O
10b
5-OMe
14.2
>100
7.0
O
O
S
11b
12b
5-OMe
5-OMe
5.3
66
12.5
>1.0
O
O
>100
>100
O
13b
14b
5-OMe
5-OMe
8.9
>100
30.9
>11.2
2.0
O
O
15.6
N
15b
5-OMe
>100
>100
>1.0
N
8c
9c
5-CN
5-CN
Me
9.5
>100
27.2
>100
2.9
>1.0
O
10c
5-CN
33.0
21.8
0.7
O
O
S
11c
12c
5-CN
5-CN
10.4
1.16
17.0
69.0
1.6
O
O
59.5
O
13c
14c
5-CN
5-CN
0.98
25.0
40.74
>100
41.6
>4.0
O
N
15c
5-CN
N/A
1.8
8.2
2.1
1.2
4.4
N
Clemizole
N/A
34.6
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potency against HCV replication activity (EC50 = 2.4 μM)
than that in the genotype 2a infectious clone system (EC50
= 3.2 μM); accordingly, compound 13c needed a 1.8-fold
purpose, J6/JFH1 RNA-transfected Huh7.5 cells were treated
with increasing concentrations of compound 13c for 120 h.
Then, levelsofviralNS3aswellashostGAPDHproteinswere
measured by Western blot analyses. As shown in Figure 2(a),
lower EC50 as well as shorter T1/2 values (0.5 μM and 16.1 h,
respectively) were determined by this protein stability-based
method when compared with those determined previously
by the RNA stability-based method (3.2 μM and 26.8 h,
respectively). WhenHuh7.5cellsstablymaintaininggenotype
1b subgenomic Bart79I RNAs were used to assess the dose-
and time-dependent effects of compound 13c on viral protein
levels, a similar length of time was necessary to reduce HCV
NS3 protein levels by half (T1/2 = 14.2 h) in spite of a lower
antiviral potency (EC50 = 1.7 μM) (Figure 2(b)).
shorter time for reducing HCV RNA levels by half (T1/2
=
14.6 h) than in the genotype 2a infectious clone system (T1/
2 = 26.8 h) (Figure 1(b)). These data demonstrated the cross-
genotype potency of compound 13c against both genotype
1b and genotype 2a HCV replicons in a reporter-free system.
After confirming suppression of HCV replication by com-
pound 13c at the RNA level, we wanted to see whether this
compound13c-inducedinhibition ofHCVreplicationcanalso
lead to the downregulation of viral protein levels. For this
Table 3. In-vitro water solubility of 6c, 12c, and 13c.
Solubility (μM)a
Conclusion
No.
pH 2
pH 7.4
pH 9
In conclusion, we have designed a novel series of indole acryl-
amide derivatives as potential HCV replication inhibitors
using a cell-based J6/JFH1 reporter assay using Huh7.5 cells.
WehavecarriedoutSARexploration basedonstructural mod-
ification using substituted indoles, various amides, and N-
6c
12c
13c
58.0
65.0
103.0
38.0
35.0
101.3
11.3
57.0
35.7
a Values are the mean of three experiments.
Figure1. Effectofcompound13conHCVreplication. (a)Dose–responsegraphdeterminedbymeasuringrelativeHCVaswellasGAPDHRNA
levels via real-time qRT-PCR analysis of J6/JFH1 RNA-transfected Huh7.5 cells treated with increasing concentrations of compound 13c for 72
h. The time–response graph was determined by measuring relative HCV as well as GAPDH RNA levels via real-time qRT-PCR analysis of J6/
JFH1RNA-transfectedHuh7.5cellstreatedwith10 μMofcompound13c forincreasingperiodsoftime. (b)Dose–responsegraphdeterminedby
measuring relative HCV well as GAPDH RNA levels via real time qRT-PCR analysis of Bart79I RNA-transfected Huh7.5 cells treated with
increasing concentrations of compound 13c for 72 h. The time–response graph was determined by measuring relative HCV as well as GAPDH
RNA levels via real time qRT-PCR analysis of Bart79I RNA replicon Huh7.5 cells treated with 10 μM ofcompound 13c for increasing periods of
time. Average values from triplicate experiments are depicted. EC50 and T1/2 values were determined based on each response curve. Error bars
represent standard deviation values.
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Figure 2. Effect of compound 13c on levels of HCV proteins. (a) Dose–response determined by measuring relative levels of HCV NS3 as well as
host GAPDH proteins via Western blot analyses of J6/JFH1 RNA-transfected Huh7.5 cells treated with increasing concentrations of compound
13c for 120 h. The time–response curve was determined by measuring relative levels of HCV NS3 as well as host GAPDH proteins via Western
blot analyses of J6/JFH1 RNA-transfected Huh7.5 cells treated with 10 μM of compound 13c for increasing periods of time. (b) Dose–response
curve determined by measuring relative levels of HCV NS3 as well as host GAPDH proteins via Western blot analyses of Bart79I replicon
Huh7.5 cells treated with increasing concentrations of compound 13c for 120 h. The time–response was determined by measuring relative levels
ofHCV NS3aswellashost GAPDHproteinsviaWestern blotanalyses ofBart79I repliconHuh7.5 cellstreated with 10 μMof compound13cfor
increasing periods of time. Numbers below GAPDH blots represent relative quantitation of intensity of viral protein after normalization to levels
of GAPDH proteins.
substitution of the indole ring. Among the synthesized com-
pounds, N-acetyl-substituted indole acrylamide 13c displayed
the most potent antiviral activity with good selectivity and bet-
ter water solubility compared to the previously identified 6c.
As a continuation of our study toward the identification of
effective HCV inhibitors, we concluded that compound 13c
was the most potent inhibitor among the series. Compound
13c may serve as a promising platform for further develop-
mentofanewclassofHCVNS5BRNApolymerase inhibitors
in the future.
Medical Systems, Inc. Palo Alto, CA, USA). Chemical shifts
are provided in parts per million (ppm) downfield with cou-
pling constants in hertz (Hz) from tetramethylsilane (internal
standard). Mass spectra were recorded with high-resolution
mass spectrometry (HRMS) (electrospray ionization MS,
ESI-MS) obtained on a Waters, Xevo, G2, QTof (Wilford,
MA,USA) mass spectrometer or MS (electron ionization
MS, EI-MS)obtained onaJMS-700, Jeol(Tokyo, Japan) mass
spectrometer. Purities of the final products were checked by
reversed phase high-pressure liquid chromatography (RP-
HPLC), which was performed on a Waters Corp. HPLC sys-
tem equipped with an ultraviolet (UV) detector set at 254 nm.
The mobile phases were (A) H2O containing 0.05% trifluoroa-
cetic acid and (B) CH3CN. HPLC had a YMC Hydrosphere
C18 (HS-302) column (5-μM particle size, 12-nm pore size)
that was 4.6 mm in diameter × 150 mm in size with a flow rate
of 1.0 mL/min. Compound purity was assessed with (Method
A) a gradient of 20% B to 100% B in 35 min or (Method B) a
gradient of 25% B to 100% B in 35 min. Melting points were
determined on a Fisher Scientific apparatus (Waltham, MA,
USA; CAT No.: 12-144).
Experimental
General. All the reagents used were commercial grade che-
micals and used without further purification. All reactions
were carried out under the atmosphere of dried argon in
flame-dried glassware. All reaction products were purified
by flash column chromatography (CC) with silica gel
60 (230–400 mesh Kieselgel 60). In addition, reactions were
monitored by thin-layer chromatography (TLC) using glass-
backed plates coated with 0.25 mm silica gel (E. Merck, silica
gel 60 F254). The chromatograms were visualized using ultra-
violet illumination, exposure to iodine vapors, and staining
with PMA or Hanessian's solution. Proton nuclear magnetic
resonance (1H NMR) spectra were determined on a Varian
(400 MHz) or a Bruker (500 MHz) spectrometer (Varian
Cell Culture. Cells of the human hepatoma cell line Huh7.5
werecultured inmonolayers asdescribed previously,13,14 with
media consisting of DMEM (Sigma-Aldrich, St.Louis, MO,
USA) supplemented with 1% L-glutamine (Hyclone, GE
Healthcare Bio-Sciences Corp., Piscataway, NJ, USA), 1%
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penicillin–streptomycin (Hyclone), 1% nonessential amino
acid (Hyclone), and 10% fetal bovine serum (JR Scientific,
CA, USA).
48, 72 h after treating HCV RNA-transfected cells with 10 μ
M of the compound.
Quantitative RT-PCR (qRT-PCR) Analysis. Onto a 6-well
plate (Costar 3610), 3× 105 cells (Huh7.5-J6/JFH1or Bart79I)
were plated and supplemented with DMSO or compound 13c
at the indicated concentrations. At 3 days after incubation,
total cellular RNA was extracted using the RNeasy® mini
kit (Qiagen) in accordance with the manufacturer's instruc-
tions. The yield of extracted RNA was assessed spectrophoto-
metrically. The expression of HCV subgenomic RNA and
cellular RNA was measured by quantitative RT-PCR (qRT-
PCR) analysis as previously described. Each sample was nor-
malized using the endogenous reference gene glyceraldehyde-
3-phosphate dehydrogenase (GAPDH). The cDNA quantifi-
cation was performed by the CFX384 RT PCR detection sys-
tem (Bio-Rad, Hercules, CA, USA). The primers used for the
qRT-PCR were as follows: FW-J6/JFH1-CTCCGCCAT-
Plasmids. Rluc-J6/JFH1 (FL-J6/JFH-50C19Rluc2AUbi) 15 is
a monocistronic, full-length HCV genome that expresses
renilla luciferase and was derived from the previously
described infectious genotype 2a HCV genome J6/JFH1.16
Bart79I is a high-efficiency bicistronic subgenomic replicon
of HCV derived from the HCV genotype 1b Con1 sequence
that harbors the neomycin phosphotransferase gene in the first
cistron and the HCV nonstructural proteins in the second cis-
tron under the translational control of an EMCV IRES.14 This
plasmid also has an adaptive mutation (S2204I) in NS5A,
which increases the replication efficiency. FL-J6/JFH-
50C19Rluc2AUbi and Bart79I were gifts from Dr. Charles
Rice of Rockefeller University.
In-vitro Transcription for Production of HCV RNA Gen-
omes. In-vitro transcription for production of HCV RNA
genomes was performed as previously described.17 Briefly,
Wild-type Bart79I, J6/JFH1, or RLuc-J6/JFH1 plasmids were
linearized by ScaI for Bart79I or XbaI for J6/JFH1 (NEB)
digestion. The T7 promoter-driven in-vitro transcription of
the digested plasmid was performed to produce the wild type
HCV RNA genomes by using a MEGAscript kit (Ambion).
GAATCACTC,
RV-J6/JFH1-ACGACACTCATAC-
TAACGC, FW-Bart79I-AGAGCCATAGTGGTCT, RV-
Bart79I-CCAAATCTCCAGGCATTGAGC, FW-GAPDH-
TGGTCTCCTCTGACTTCA,
and
RV-GAPDH-
CGTTGTCATACCAGGAAATG. A time–response curve
was also determined by measuring renilla luciferase activities
as well as cell viabilities at 24, 48, 72 h after treating HCV
RNA-transfected cells with 10 μM of compound 13c.
Generation of Stable HCV Replicon Cell Lines. The estab-
lishment of Huh7.5 cells stably maintaining a Bart79I subge-
nomic replicon in the presence of G418 selection has been
described elsewhere.9 Briefly, in-vitro transcribed Bart79I
RNAs were transfected into Huh7.5 cells by using a lipofec-
tamine 2000 transfection reagent. The transfected cells were
supplemented with G418 at a final concentration of 750 μg/
mL. This selection medium was replaced every 3 days for 3
weeks. The establishment of Huh7.5 cells stably maintaining
a Bart79I-YFP subgenomic replicon in the presence of G418
selection was performed similarly. The establishment of
Huh7.5 cells stably maintaining a J6/JFH1 infectious clone
was performed by transfecting in-vitro transcribed J6/JFH1
RNAs into Huh7.5 cells by using a lipofectamine 2000 trans-
fection reagent. The establishment of Huh7.5 cells stably
maintaining an Rluc-J6/JFH1 clone was also performed
similarly.
Western Blot Analysis. Onto a 6-well plate (Costar 3610),
1.5 × 105 cells (Huh7.5-J6/JFH1 or Bart79I) were plated
and supplemented with DMSO or compound 13c at the indi-
cated concentrations. At 5 days after incubation, whole-cell
extracts werepreparedinRIPAbuffer (150 mM NaCl, 1% Tri-
ton X-100, 1% deoxycholic acid sodium salt, 0.1% sodium
dodecyl sulfate, 50 mM Tris-HCl , pH 7.5, 2 mM EDTA; gen-
DEPOT) containing a cocktail of protease inhibitors
(Complete, Roche Diagnostic at a final concentration of 1 tab-
let per 50 mL RIPA buffer) and quantitated by the Bradford
assay (Bio-Rad). Equal amounts of protein were electrophor-
esed on an SDS polyacrylamide gel, subsequently transferred
to a polyvinylidene difluoride membrane (Immobilon-P;
Millipore, Bedford, MA, USA), and probed with a mouse
anti-NS3 monoclonal antibodies (1878 for NS3, Virostat).
A time–response curve was also determined by performing
similar Western blot analyses at 24, 48, 72 h after treating cells
with 10 μM of compound 13c.
General Procedure for Wittig Reaction. A solution of
indole aldehyde (1.0 equiv) and (carbethoxyethylidene) tri-
phenylphosphorane (1.3 equiv) in CH2Cl2 was refluxed for
18 h and the excess solvent was evaporated under reduced
pressure. The reaction mixture was partitioned between
CH2Cl2 and water. The organic layer was dried over magne-
sium sulfate (MgSO4) and purified by CC on silica gel using
0–40% EtOAc in hexanes as eluent to obtain the desired com-
pounds 2a–2c as yellow solid. The mixture of indole aldehyde
(1.0 equiv) and triethyl 2-fluoro-2-phosphonoacetate was
dissolved in THF. DBU and LiCl were added to the mixture,
and the resulting mixture was stirred overnight at room tem-
perature. After the reaction was complete, the reaction mixture
Cell Viability and Anti-HCV Replication Analysis Using a
Luciferase Assay. In-vitro transcribed RLuc-J6/JFH1 RNAs
were transfected into Huh7.5 cells by using a lipofectamine
2000 transfection reagent (Invitrogen) as described by the
manufacturer. Transfected cells were plated onto a white
96-well plate (Costar 3610) and supplemented with DMSO
or 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM of the
compounds. At 3 days after incubation, cells were incubated
for 3 h at 37 ꢀC in the presence of EZ-CYTOX (10% tetrazo-
lium salt; Dogen) reagent to assess the cytotoxicity. Renilla
luciferase activities were measured by using a luciferase rea-
gent (1 mM coelenterazine in methanol-HCL; Goldbio).
A time–response curve was also determined by measuring
renilla luciferase activities as well as cell viabilities at 24,
Bull. Korean Chem. Soc. 2015, Vol. 36, 88–98
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
94

BULLETIN OF THE
Article
Synthesis and SAR of Indole Acrylamides
KOREAN CHEMICAL SOCIETY
was quenched by 10% citric acid and extracted with CH2Cl2.
The organic layer was dried over MgSO4 and purified by col-
umn chromatography on silica gel using 0–40% EtOAc in
hexanes as eluent to yield compounds 3a–3c as a green solid.
(E)-Ethyl3-(1H-indol-3-yl)-2-methylacrylate(2a):white
(d, J = 1.0 Hz, 3H); 13C-NMR (CD3OD, 100 Hz) δ 172.91,
137.47, 132.35, 129.20, 127.99, 123.60, 121.33, 119,10,
113.40, 112.62, 15.19; MS (EI) m/z 201 (M+); HRMS (EI)
m/z calcd. for C12H11NO2 201.0790, found: 201.0791; purity
>99.9 % (as determined by RP-HPLC, tR = 7.9 min).
1
solid, yield 65%. m.p. 138–139 ꢀC; H-NMR (CDCl3, 500
(E)-3-(5-Methoxy-1H-indol-3-yl)-2-methylacrylic
acid
1
MHz) δ 8.50 (brs, 1H), 8.05 (s, 1H), 7.83 (d, J =7.8 Hz, 1H),
7.51 (d, J = 2.4 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.22–7.30
(m, 2H), 4.31 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H);
13C-NMR (CDCl3, 100Hz) δ 168.11, 134.40, 128.93, 126.60,
124.54, 122.19, 122.07, 119.69, 117.91, 112.22, 110.22,
59.61, 14.03, 13.43; MS (EI) m/z 229 (M+), HRMS (ESI) m/z
calcd. for C14H16NO2 [(M + H)+] 230.1181, found: 230.1178;
purity 100 % (as determined by RP-HPLC, tR = 16.5 min).
(E)-Ethyl 3-(5-methoxy-1H-indol-3-yl)-2-methylacrylate
(4b): white solid, yield 78%. H-NMR (CDCl3, 400 MHz) δ
8.43 (brs, 1H), 8.12 (s, 1H), 7.54 (d, J = 2.8 Hz, 1H), 7.31 (d, J
= 8.8 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 6.94 (dd, J = 2.4, 8.8
Hz, 1H), 3.90 (s, 3H), 2.21 (s, 3H); MS (ESI) m/z 232 (M + H)+.
(E)-3-(5-Cyano-1H-indol-3-yl)-2-methylacrylic
acid
1
(4c): brown solid, yield 99%. H-NMR (DMSO-d6, 400
MHz) δ 12.21 (brs, 1H), 8.27 (s, 1H), 7.9 (d, J = 16.0 Hz,
2H), 7.60 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 2.08
(s, 3H); MS (ESI) m/z 227 (M + H)+.
1
(2b): white solid, yield 99%. H-NMR (CDCl3, 400 MHz) δ
(E)-2-Fluoro-3-(1H-indol-3-yl)acrylic acid (5a): green
1
8.38 (brs, 1H), 7.99 (s, 1H), 7.48 (d, J = 2.8 Hz, 1H), 7.30 (d,
J = 8.8 Hz, 1H), 7.23 (d, J = 2.4 Hz, 1H), 6.93 (dd, J = 2.4,
8.4 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 3.90 (s, 3H), 2.19
(s,3H), 1.38 (t, J = 7.2 Hz, 3H); MS (ESI) m/z 260 (M + H)+,
258 (M – H)–.
solid, yield 99%. H-NMR (CD3OD, 400 MHz) δ 8.42 (s,
1H), 7.77 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 0.8
Hz, 1H), 7.37–7.41 (m, 3H), 7.10–7.17 (m, 5H); HRMS
(ESI) m/z calcd. for C11H9NO2F [(M + H)+] 206.0617, found:
206.0617.
(E)-Ethyl 3-(5-cyano-1H-indol-3-yl)-2-methylacrylate
(2c): white solid, yield 99%. H-NMR (CD3OD, 400 MHz)
(E)-2-Fluoro-3-(5-methoxy-1H-indol-3-yl)acrylic acid
(5b): green solid, yield 85%. H-NMR (CD3OD, 400 MHz)
1
1
δ 8.15 (s, 1H), 7.98 (s, 1H), 7.81 (s, 1H), 7.58 (d, J = 8.0
Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 4.28 (q, J = 6.0 Hz, 2H),
2.18 (s, 3H), 1.37 (t, J = 8.0 Hz, 3H); MS (ESI) m/z 255 (M
+ H)+, 253 (M – H).
δ 8.37 (s, 1H), 7.16–7.31 (m, 4H), 6.82–6.85 (m, 2H), 3.85
(s, 3H);HRMS (ESI) m/z calcd. for C12H11NO3F [(M + H)+]
236.0723, found: 236.0730.
(E)-3-(5-Cyano-1H-indol-3-yl)-2-fluoroacrylic
acid
1
(E)-2-Fluoro-3-(1H-indol-3-yl)acrylic acid (3a): green
(5c): green solid, yield 72%. H-NMR (CD3OD, 400 MHz)
δ 8.54 (s, 1H), 8.16 (s, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.47
(dd, J = 1.4, 8.6 Hz, 1H), 7.25–7.31 (m, 1H); HRMS (ESI)
m/z calcd. for C12H8N2O2F [(M + H)+] 231.0570, found:
231.0570.
1
solid, yield 99%. H-NMR (CD3OD, 400 MHz) δ 8.42 (s,
1H), 7.77 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 0.8
Hz, 1H), 7.37–7.41 (m, 3H), 7.10–7.17 (m, 5H); HRMS
(ESI) m/z calcd. for C11H9NO2F [(M + H)+] 206.0617, found:
206.0617.
General Procedure for Preparation of Substituted Indole
2-methylacrylamide and 2-fluoroacrylamide Analogs.
The mixture of substituted (E)-3-(1H-indol-3-yl)-2-
methylacrylic acid (1 equiv.) or (E)-2-fluoro-3-(1H-indol-3-
yl)acrylic acid (1 equiv) was dissolved in a mixture of DMF
and CH2Cl2 (v/v, 1:1). To the resulting mixture was added
DIPEA (1.5 equiv) and HBTU (1.5 equiv) and stirred for 30
min at room temperature. After stirring, the amine (1.0 equiv)
wasaddedtothesolutionatroomtemperature andstirredover-
night at room temperature. The mixture was partitioned with
EtOAc and water. The organic layer was dried over MgSO4
and purified by column chromatography on silica gel using
0–60% EtOAc in hexanes as eluent to provide compounds
6a–6c and 7a–7c.
(E)-N-(4-tert-Butylphenyl)-3-(1H-indol-3-yl)-2-methy-
lacrylamide (6a): yellow solid, yield 77%. m.p. 178–179 ꢀC;
1H-NMR (CD3OD, 400 MHz) δ 7.80 (d, J = 8.4 Hz, 1H), 7.76
(s, 1H), 7.60 (s, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.37–7.43 (m,
3H), 7.13–7.19 (m, 3H), 2.26 (s, 3H), 1.33 (s, 9H); 13C-NMR
(CD3OD, 100Hz) δ 146.82, 136.13, 136.00, 127.73, 126.32,
125.73, 125.08, 122.00, 120.81, 119.61, 117.99, 111.91,
111.08, 33.78, 30.41, 14.43; MS (EI) m/z 332 (M+); HRMS
(EI) m/z calcd. for C22H24N2O 332.1889, found: 332.1888;
purity >99.9% (as determined by RP-HPLC, tR = 21.2 min).
(E)-2-Fluoro-3-(5-methoxy-1H-indol-3-yl)acrylic acid
1
(3b): green solid, yield 85%. H-NMR (CD3OD, 400 MHz)
δ 8.37 (s, 1H), 7.16–7.31 (m, 4H), 6.82–6.85 (m, 2H), 3.85
(s, 3H); HRMS (ESI) m/z calcd. for C12H11NO3F [(M +
H)+] 236.0723, found: 236.0730.
(E)-3-(5-Cyano-1H-indol-3-yl)-2-fluoroacrylic acid (3c):
green solid, yield 72%. 1H-NMR (CD3OD, 400 MHz) δ 8.54
(s, 1H), 8.16 (s, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.47 (dd, J =
1.4, 8.6 Hz, 1H), 7.25–7.31 (m, 1H); HRMS (ESI) m/z calcd.
for C12H8N2O2F [(M + H)+] 231.0570, found: 231.0570.
General Synthetic Procedure for Hydrolysis. A solution of
the acrylate (1.0 equiv.) in THF/H2O/MeOH was treated with
sodium hydroxide (10.0 equiv.) in water at 5 ꢀC and then
heated in an oil bath and refluxed for 2–4 h. The reaction mix-
ture was acidified with 2 N HCl to pH ~2. The mixture was
partitioned between EtOAc and water. The organic layer
was dried over MgSO4, filtered, and concentrated in-vacuo
to give crude products 4a–4c and 5a–5c, which were used
without further purification.
(E)-3-(1H-Indol-3-yl)-2-methylacrylic acid (4a): white
1
solid, yield 61%. m.p. 237–238 ꢀC; H-NMR (acetone-d6,
500 MHz) δ 10.84 (brs, 1H), 8.10 (s, 1H), 7.79 (d, J = 8.0
Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.16–7.24 (m, 3H), 2.17
Bull. Korean Chem. Soc. 2015, Vol. 36, 88–98
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
95

BULLETIN OF THE
Article
Synthesis and SAR of Indole Acrylamides
KOREAN CHEMICAL SOCIETY
(E)-N-(4-tert-Butylphenyl)-3-(5-methoxy-1H-indol-3-yl)-
equiv) was added to the solution at room temperature. The
resulting mixture was stirred for 1 h at room temperature
and transferred to an oil bath. Alkyl halide was added to the
solution, and the resulting mixture was refluxed for 1.5 h.
The reaction was quenched by ice-water and partitioned with
EtOAc and water. The organic layer dried over MgSO4, fil-
tered, and the excess solvent was evaporated under reduced
pressure. Theresiduewaspurifiedbycolumnchromatography
on silica gel using 0–30% EtOac in hexanes as eluent to yield
products 14a–15a, 14b–15b, and 14c–15 c. A solution of the
amide 6a–6c (1.0 equiv) and tetrabutylammonium bromide
(TBAB, 0.5 equiv) in CH2Cl2 was added 20% NaOH and
methyl iodide at room temperature. The resulting mixture
was stirred overnight at room temperature and quenched by
water. The mixture was extracted with CH2Cl2 and water.
The organic layer was dried over MgSO4, filtered, and evapo-
rated. The residue was purified by column chromatography on
silica gel using 0–30% EtOAc in hexanes as eluent to give
compounds 8a–9a, 8b–9b, and 8c–9c.
2-methylacrylamide (6b): yellow solid, yield 61%. m.
p. 160–161 ꢀC; 1H-NMR (CD3OD, 400 MHz) δ 7.74 (s, 1H),
7.53–7.57 (m, 3H), 7.39 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4
Hz, 2H), 6.84 (dd, J = 2.4, 8.4 Hz, 1H), 3.86 (s, 3H), 2.25 (s,
3H), 1.33 (s, 9H); 13C-NMR (CD3OD, 100 Hz) δ 171.95,
156.10, 148.28, 137.60, 132.50, 129.79, 127.96, 127.74,
126.54, 122.34, 113.83, 113.27, 101.20, 56.21, 35.26, 31.88,
15.84; HRMS (ESI) m/z calcd. for C23H27N2O2 [(M + H)+]
363.2073, found: 363.2068; purity >99.9 % (as determined
by RP-HPLC, tR = 20.4 min).
(E)-N-(4-tert-Butylphenyl)-3-(5-cyano-1H-indol-3-yl)-2-
methylacrylamide (6c): yellow solid, yield 25%. m.
p. 268–269 ꢀC; 1H-NMR (CD3OD, 400 MHz) δ 8.18 (s, 1H),
7.68 (s, 1H), 7.60(s, 1H), 7.45–7.49 (m, 3H), 7.39 (dd, J =
1.6, 8.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 2.16 (s, 3H), 1.24
(s, 9H); 13C-NMR (CD3OD, 100 Hz) δ 171.48, 148.41,
139.26, 137.47, 129.89, 129.33, 129.05, 126.57, 126.22,
125.83, 125.56, 122.24, 121.61, 114.12, 113.86, 103.81,
35.27, 31.87, 15.88; HRMS (ESI) m/z calcd. for C23H24N3O
[(M + H)+] 358.1919, found: 358.1922; purity >99.9 % (as
determined by RP-HPLC, tR = 20.1 min).
(E)-N-(4-tert-Butylphenyl)-2-fluoro-3-(1H-indol-3-yl)
acrylamide (7a): yellow solid, yield 16%. m.p. 214–215 ꢀC;
1H-NMR (CD3OD, 400 MHz) δ 8.56 (s, 1H), 7.68 (d, J = 7.2
Hz, 1H), 7.57–7.60 (m, 2H), 7.39–7.41 (m, 3H), 7.24 (s, 1H),
7.11–7.19 (m, 3H), 1.33 (s, 9H); HRMS (ESI) m/z calcd. for
C21H22N2OF [(M + H)+] 337.1716, found: 337.1720.
(E)-N-(4-tert-Butylphenyl)-2-fluoro-3-(5-methoxy-1H-
indol-3-yl)acrylamide (7b): yellow solid, yield 48%. m.
p. 198–199 ꢀC; 1H-NMR (CD3OD, 400 MHz) δ 8.5 (s, 1H),
7.87 (s, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz,
2H), 7.29 (d, J = 9.2 Hz, 1H), 7.21 (s, 1H), 7.17 (d, J = 2.4
Hz, 1H), 7.13–7.15 (m, 2H), 6.82 (dd, J = 2.4, 8.8 Hz, 1H),
6.67 (d, J = 6.8 Hz, 1H), 3.85 (s, 3H), 1.33 (s, 9H); HRMS
(ESI) m/z calcd. for C21H22N2OF [(M + H)+] 337.1716,
found: 337.1720.
(E)-N-(4-tert-Butylphenyl)-2-methyl-3-(1-methyl-1H-
1
indol-3-yl)acrylamide (8a): yellow oil, yield 37%. H-
NMR (CDCl3, 400 MHz) δ 7.78 (d, J = 8.0 Hz, 1H), 7.71
(s, 1H), 7.52–7.56 (m, 3H), 7.30–7.39 (m, 5H), 7.23 (t, J
= 7.0 Hz, 2H), 3.88 (s, 3H), 2.80 (s, 3H), 1.33 (s, 9H); HRMS
(ESI) m/z calcd. for C23H26N2O [(M + H)+] 347.2123,
found: 347.2129; purity >99.6% (as determined by RP-
HPLC, tR = 23.2 min).
(E)-3-(1-Benzyl-1H-indol-3-yl)-N-(4-tert-butylphenyl)-2-
methylacrylamide (9a): yellow solid, yield 34%.m.p.
1
165–166 ꢀC; H-NMR (CDCl3, 400 MHz) δ 7.20–7.35 (m,
7H), 7.15 (t, J = 7.4 Hz, 1H), 7.10 (s, 1H), 7.00–7.08 (m, 6H),
5.07 (s, 2H), 1.86 (s, 3H), 1.26 (s, 9H); HRMS (ESI) m/z calcd.
for C29H30N2O [(M – H)–] 421.2258, found: 421.2262; purity
>99.9% (as determined by RP-HPLC, tR = 28.4 min).
(E)-benzyl 3-[3-(4-tert-Butylphenylamino)-2-methyl-3-
oxoprop-1-enyl]-1H-indole-1-carboxylate (10a): yellow
1
solid, yield 32%. m.p. 197–198 ꢀC; H-NMR (CDCl3, 400
(E)-N-(4-tert-Butylphenyl)-3-(5-cyano-1H-indol-3-yl)-2-
fluoroacrylamide (7c): yellow solid, yield 16%. m.
p. 271–272 ꢀC; 1H-NMR (CD3OD, 400 MHz) δ 8.68 (s, 1H),
8.17 (s, 1H), 7.39–7.60 (m, 7H), 7.21 (d, J = 26.4, 2H), 1.33
(s, 9H); HRMS (ESI) m/z calcd. for C21H22N2OF [(M + H)+]
337.1716, found: 337.1720.
General Procedure for N-Substitution of Indole Ring.
Sodium hydride (3.0 equiv) was added slowly to the amide
6a–6c (1.0 equiv.) in DMF at 0 ꢀC. The resulting mixture
was stirred for 1 h in an ice bath and transferred to room tem-
perature. To the solution was added alkyl halide or acyl halide
at room temperature, and stirred for 1 h at room temperature.
The reaction was quenched by ice-water and partitioned with
EtOAc and water. The organic layer was dried over MgSO4,
filtered, and evaporated under reduced pressure. The residue
was purified by column chromatography on silica gel using
0–30% EtOAc in hexanes as eluent to give compounds
10a–13a, 10b–13b, and 10c–13c. The amides 6a–6c (1.0
equiv) were dissolved in DMF, and sodium hydride (3.0
MHz) δ 8.17 (s, 1H), 7.79 (s, 1H), 7.67 (d, J = 7.6 Hz, 1H),
7.50–7.55 (m, 6H), 7.37–7.46 (m, 6H), 7.26–7.37 (m, 1H),
5.51 (s, 2H), 2.28 (s, 3H), 1.32 (s, 9H);HRMS (ESI) m/z calcd.
for C30H30N2O3 [(M – H)–] 465.2178, found: 465.2171;
purity >99.9% (as determined by RP-HPLC, tR = 27.9 min).
(E)-3-[1-(Benzylsulfonyl)-1H-indol-3-yl]-N-(4-tert-butyl-
phenyl)-2-methylacrylamide (11a): yellow solid, yield 29%.
m.p. 151–152 ꢀC; 1H-NMR (CDCl3, 400 MHz) δ 7.87 (dd, J =
1.8, 7.0 Hz, 1H), 7.70 (dd, J = 2.4, 6.8 Hz, 1H), 7.53 (d, J = 8.4
Hz, 3H), 7.46 (s, 1H), 7.35–7.41 (m, 4H), 7.30 (d, J = 7.2 Hz,
1H), 7.17 (t, J = 7.6 Hz, 2H), 7.04 (s, 1H), 6.85 (d, J = 7.6 Hz,
2H), 4.54 (s, 2H), 1.97 (s, 3H), 1.33 (s, 9H); HRMS (ESI) m/
z calcd. for C29H30N2O3S [(M – H)] 485.1899, found:
485.1902; purity >99.9% (as determined by RP-HPLC, tR =
25.5 min).
(E)-3-(1-Benzoyl-1H-indol-3-yl)-N-(4-tert-butylphenyl)-
2-methylacrylamide (12a): yellow solid, yield 31%.m.
1
p. 187–188 ꢀC; H-NMR (CDCl3, 400 MHz) δ 8.35 (d, J =
8.0 Hz, 1H), 7.80 (s, 1H), 7.78 (d, J = 1.6 Hz, 1H), 7.64–7.73
Bull. Korean Chem. Soc. 2015, Vol. 36, 88–98
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
96

BULLETIN OF THE
Article
Synthesis and SAR of Indole Acrylamides
KOREAN CHEMICAL SOCIETY
(m, 2H), 7.52–7.59 (m, 6H), 7.48 (s, 1H), 7.42–7.48 (m, 2H),
7.40 (s, 1H), 7.26 (s, 1H), 2.19 (s, 3H), 1.32 (s, 9H); HRMS
(ESI) m/z calcd. for C29H28N2O2 [(M – H)–] 435.2073, found:
435.2092; purity >99.9% (as determined by RP-HPLC, tR =
26.4 min).
3.0, 9.0 Hz, 1H), 5.49 (s, 2H), 3.82 (s, 3H), 2.28 (s, 3H), 1.33
(s, 9H); HRMS (ESI) m/z calcd. for C31H32N2O4 [(M – H)-]
495.2284, found: 495.2267; purity >99.9% (as determined by
RP-HPLC, tR = 27.9 min).
(E)-3-[1-(Benzylsulfonyl)-5-methoxy-1H-indol-3-yl]-N-
(4-tert-butylphenyl)-2-methylacrylamide (11b): yellow
(E)-3-(1-Acetyl-1H-indol-3-yl)-N-(4-tert-butylphenyl)-
2-methylacrylamide (13a): yellow solid, yield 38%.m.
1
solid, yield 25%. m.p. 201–202 ꢀC; H-NMR (CDCl3, 400
1
p. 202–203 ꢀC; H-NMR (CDCl3, 400 MHz) δ 8.44 (d, J =
MHz) δ 7.75 (d, J = 9.2 Hz, 1H), 7.53 (d, J = 8.8 Hz, 2H),
7.46 (d, J = 10.4 Hz, 2H), 7.39 (d, J = 8.8 Hz, 2H), 7.27–7.29
(m, 1H), 7.18 (t, J = 7.6 Hz, 2H), 7.10 (d, J = 2.4 Hz, 1H),
6.98–7.01 (m, 2H), 6.87 (d, J = 8.0 Hz, 2H), 4.52 (s, 2H),
3.88 (s, 3H), 1.97 (s, 3H), 1.34 (s, 9H); HRMS (ESI) m/z calcd.
for C30H32N2O4S[(M – H)–] 515.2005, found: 515.2003; purity
>99.7% (as determined by RP-HPLC, tR = 25.5 min).
(E)-3-(1-Benzoyl-5-methoxy-1H-indol-3-yl)-N-(4-tert-
butylphenyl)-2-methylacrylamide (12b): yellow solid,
yield 32%.m.p. 180–181 ꢀC; 1H-NMR (CDCl3, 400 MHz) δ
8.25 (d, J = 9.2 Hz, 1H), 7.78 (s, 1H), 7.76 (d, J = 1.2 Hz,
1H), ; HRMS (ESI) m/z calcd. for C30H30N2O3 [(M – H)–]
403.2022, found: 403.2018; purity >99.7% (as determined
by RP-HPLC, tR = 26.3 min).
(E)-3-(1-Acetyl-5-methoxy-1H-indol-3-yl)-N-(4-tert-
butylphenyl)-2-methylacrylamide (13b): yellow solid,
yield 33%.m.p. 141–142 ꢀC; 1H-NMR (CDCl3, 400 MHz) δ
8.32 (d, J = 10.0 Hz, 1H), 7.54 (t, J = 8.4 Hz, 5H), 7.4 (dd,
J = 1.8, 6.6 Hz, 2H), 7.09 (d, J = 2.4 Hz, 1H), 7.00–7.03
(m, 1H), 3.88 (s, 3H), 2.63 (s, 3H), 2.30 (s, 3H), 1.34 (s,
9H); HRMS (ESI) m/z calcd. for C25H28N2O3 [(M – H)–]
465.2178, found: 465.2173; purity >99.8% (as determined
by RP-HPLC, tR = 22.8 min).
8.4 Hz, 1H), 7.67 (d, J = 7.2 Hz, 1H), 7.52–7.55(m, 5H),
7.34–7.45 (m, 4H), 2.71 (s, 3H), 2.30 (s, 3H), 1.33 (s, 9H);
HRMS (ESI) m/z calcd. for C24H26N2O2 [(M – H)–]
373.1916, found: 373.1922; purity >99.7% (as determined
by RP-HPLC, tR = 22.9 min).
(E)-N-(4-tert-Butylphenyl)-2-methyl-3-[1-(2-morpholi-
noethyl)-1H-indol-3-yl]acrylamide (14a): yellow solid,
1
yield 35%. m.p. 151–152 ꢀC; H-NMR (CDCl3, 400 MHz)
δ 7.78 (d, J = 8.0 Hz, 1H), 7.70 (s, 1H), 7.54–7.56 (m, 3H),
7.48 (s, 1H), 7.38 (d, J = 6.8 Hz, 2H), 7.27 (t, J = 7.6 Hz,
1H), 7.24 (t, J = 7.4 Hz, 2H), 4.30 (t, J = 6.8 Hz, 2H), 3.71
(t, J = 4.0 Hz, 4H), 2.80 (t, J = 7.7 Hz, 2H), 2.50 (t, J = 4.4
Hz, 4H), 2.29 (s, 3H), 1.26 (s, 9H); HRMS (ESI) m/z calcd.
for C28H35N3O2 [(M – H)–] 444.2670, found: 444.2659;
purity >99.7% (as determined by RP-HPLC, tR = 11.3 min).
(E)-N-(4-tert-Butylphenyl)-3-{1-[2-(dimethylamino)
ethyl]-1H-indol-3-yl}-2-methylacrylamide (15a): yellow
1
solid, yield 36%. m.p. 128–129 ꢀC; H-NMR (CDCl3, 400
MHz) δ 7.77 (d, J = 8.0 Hz, 1H), 7.69 (s, 1H), 7.52–7.57 (m,
3H), 7.43 (s, 1H), 7.37 (d, J = 18,4 Hz, 3H), 7.29 (t, J = 7.4
Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 4.29 (t, J = 7.0 Hz, 2H),
2.75 (t, J = 7.2 Hz, 2H), 2.28 (s, 6H), 2.28 (s, 3H), 1.35 (s,
9H); HRMS (ESI) m/z calcd. for C26H33N3O [(M + H)+]
404.2702, found: 404.2692; purity >99.6% (as determined by
RP-HPLC, tR = 11.0 min).
(E)-N-(4-tert-Butylphenyl)-3-[5-methoxy-1-(2-morpho-
linoethyl)-1H-indol-3-yl]-2-methylacrylamide (14b):yel-
1
low solid, yield 28%. m.p. 185–186 ꢀC; H-NMR (CDCl3,
(E)-N-(4-tert-Butylphenyl)-3-(5-methoxy-1-methyl-1H-
indol-3-yl)-2-methylacrylamide (8b): yellow solid, yield
400 MHz) δ7.70 (s, 1H), 7.53–7.58 (m, 3H), 7.43 (s, 1H),
7.37–7.39 (m, 2H), 7.25–7.27 (m, 1H), 7.19 (d, J = 2.4 Hz,
1H), 6.93 (dd, J = 2.4, 8.8 Hz, 1H), 4.25 (t, J = 6.4 Hz, 2H),
3.88 (s, 3H), 3.70 (t, J = 4.8 Hz, 4H), 2.77 (t, J = 6.6 Hz,
2H), 2.49 (t, J = 4.4 Hz, 4H), 2.28 (s, 3H), 1.33 (s, 9H); HRMS
(ESI) m/z calcd. for C29H37N3O3 [(M – H)–] 474.2757, found:
474.2753; purity >99.7% (as determined by RP-HPLC, tR =
11.1 min).
(E)-N-(4-tert-Butylphenyl)-3-{1-[2-(dimethylamino)
ethyl]-5-methoxy-1H-indol-3-yl}-2-methylacrylamide (15b):
yellow solid, yield 29%. m.p. 144–145 ꢀC; 1H-NMR
(CDCl3, 400 MHz) δ 7.70 (s, 1H), 7.58 (s, 1H), 7.53–7.57
(m, 2H), 7.37–7.39 (m, 3H), 7.25–7.27 (m, 1H), 7.19 (d, J
= 2.4 Hz, 1H), 6.93 (dd, J = 2.4, 8.8 Hz, 1H), 4.24 (t, J =
7.0 Hz, 2H), 3.87 (s, 3H), 2.73 (t, J = 6.8 Hz, 2H), 2.30
(s, 6H), 2.27 (s, 3H), 1.33 (s, 9H); HRMS (ESI) m/z
calcd. for C27H35N3O2 [(M + H)+] 434.2808, found:
434.2809; purity >99.8% (as determined by RP-HPLC, tR =
10.9 min).
1
33%. m.p. 149–150 ꢀC; H-NMR (CDCl3, 400 MHz) δ7.71
(s, 1H), 7.54 (t, J = 8.0 Hz, 3H), 7.38 (d, J = 8.8 Hz, 2H),
7.30 (s, 1H), 7.23 (s, 1H), 7.20 (d, J = 2.4 Hz, 1H), 6.95
(dd, J = 2.4, 9.2 Hz, 1H), 3.89 (s, 3H), 3.85 (s, 3H), 2.28 (s,
3H), 1.33 (s, 9H); HRMS (ESI) m/z calcd. for C24H28N2O2
[(M – H)–] 375.2073, found: 375.2081; purity >99.8% (as
determined by RP-HPLC, tR = 22.7 min).
(E)-3-(1-Benzyl-5-methoxy-1H-indol-3-yl)-N-(4-tert-
butylphenyl)-2-methylacrylamide (9b): yellow solid, yield
31%.m.p. 119–120 ꢀC; 1H-NMR (CDCl3, 400 MHz) δ
7.27–7.35 (m, 5H), 7.24 (s, 1H), 7.09 (s, 1H), 7.03–7.07
(m, 4H), 7.01 (s, 1H), 6.93 (d, J = 2.0 Hz, 1H), 6.81 (dd, J =
2.4, 8.8 Hz, 1H), 5.06 (s, 2H), 3.84 (s, 3H), 1.76 (s, 3H),
1.24 (s, 9H); HRMS (ESI) m/z calcd. for C30H32N2O2 [(M
+ H)+] 453.2515, found: 453.2551; purity >99.9% (as deter-
mined by RP-HPLC, tR = 27.7 min).
(E)-Benzyl
3-[3-(4-tert-butylphenylamino)-2-methyl-3-
oxoprop-1-enyl]-5-methoxy-1H-indole-1-carboxylate (10b):
(E)-N-(4-tert-Butylphenyl)-3-(5-cyano-1-methyl-1H-
indol-3-yl)-2-methylacrylamide (8c): yellow solid, yield
38%. m.p. 188–189 ꢀC; 1H-NMR (CDCl3, 400 MHz) δ 8.08
(t, J = 0.8 Hz, 1H), 7.69 (s, 1H), 7.52–7.57 (m, 3H), 7.50
1
yellow solid, yield 26%. m.p. 146–147 ꢀC; H-NMR (CDCl3,
400 MHz) δ 8.04 (s, 1H), 7.76 (s, 1H), 7.49–7.55 (m, 6H),
7.37–7.43 (m, 5H), 7.15 (d, J = 44.4 Hz, 1H), 6.98 (dd, J =
Bull. Korean Chem. Soc. 2015, Vol. 36, 88–98
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
97

BULLETIN OF THE
Article
Synthesis and SAR of Indole Acrylamides
KOREAN CHEMICAL SOCIETY
(dd, J = 1.4, 8.6 Hz, 1H), 7.36–7.39 (m, 4H), 3.90 (s, 3H), 2.26
(s, 3H), 1.33 (s, 9H); HRMS (ESI) m/z calcd. for C24H25N3O
[(M–H)-] 370.1919, found: 370.1908; purity >99.9% (as
determined by RP-HPLC, tR = 22.0 min).
J = 4.4 Hz, 4H), 2.28 (s, 3H), 1.33 (s, 9H); HRMS (ESI) m/z
calcd. for C29H34N4O2 [(M – H)–] 469.2604, found: 469.2586;
purity >99.6% (as determined by RP-HPLC, tR = 11.1 min).
(E)-N-(4-tert-Butylphenyl)-3-{5-cyano-1-[2-(dimethyla-
(E)-3-(1-Benzyl-5-cyano-1H-indol-3-yl)-N-(4-tert-butyl-
phenyl)-2-methylacrylamide(9c):yellowsolid, yield39%.m.
mino)ethyl]-1H-indol-3-yl}-2-methylacrylamide
(15c):
yellow solid, yield 38%. m.p. 179–180 ꢀC; 1H-NMR
(CDCl3, 400 MHz) δ 8.07 (d, J = 0.8 Hz, 1H), 7.54 (s, 1H),
7.54–7.57 (m, 3H), 7.53 (s, 1H), 7.47 (dd, J = 1.2, 8.4 Hz,
1H), 7.36–7.42 (m, 3H), 4.27 (t, J = 6.6 Hz, 2H), 2.73 (t, J
= 6.6 Hz, 2H), 2.33 (s, 6H), 2.23 (s, 3H), 1.32 (s, 9H); HRMS
(ESI) m/z calcd. for C27H32N4O [(M – H)–] 427.2498, found:
427.2490; purity >99.9% (as determined by RP-HPLC, tR =
10.8 min).
1
p. 175–176 ꢀC; H-NMR (CDCl3, 400 MHz) δ 7.51 (s, 1H),
7.37 (dd, J = 1.6, 8.8 Hz, 1H), 7.31–7.33 (m, 4H), 7.29 (s,
3H), 7.18 (s, 1H), 7.00–7.06 (m, 4H), 6.79 (s, 1H), 5.06 (s,
2H), 1.90 (s, 3H), 1.25 (s, 9H); HRMS (ESI) m/z calcd. for
C30H29N3O [(M – H)-] 446.2232, found: 446.2214; purity
>99.9% (as determined by RP-HPLC, tR = 26.7 min).
(E)-Benzyl 3-[3-(4-tert-butylphenylamino)-2-methyl-3-
oxoprop-1-enyl]-5-cyano-1H-indole-1-carboxylate (10c):
yellow solid, yield 36%. m.p. 180–181 ꢀC; 1H-NMR
(CDCl3, 400 MHz) δ 8.30 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H),
7.87 (s, 1H), 7.48–7.56 (m, 4H), 7.39–7.41 (m, 3H),
7.30–7.39 (m, 1H), 7.22 (s, 1H), 7.17 (t, J = 7.8 Hz, 2H),
6.87 (d, J = 7.2 Hz, 2H), 4.58 (s, 2H), 2.03 (s, 3H), 1.33 (s,
9H); HRMS (ESI) m/z calcd. for C31H29N3O3 [(M – H)–]
490.2131, found: 490.2130; purity >99.8% (as determined
by RP-HPLC, tR = 27.1 min).
Acknowledgments. This research was supported by the
National Research Foundation (NRF) funded by the Ministry
of Science (No. 2012M3A9C1053532 and 2014 R1A2A2A
01005455), Korea, and by the Basic Science Research Pro-
gram through the NRF funded by the Ministry of Education,
Science and Technology (2013R1A1A1011851).
References
(E)-3-[1-(Benzylsulfonyl)-5-cyano-1H-indol-3-yl]-N-(4-
tert-butylphenyl)-2-methylacrylamide (11c): yellow solid,
yield 28%.m.p. 237–238 ꢀC; 1H-NMR (CDCl3, 400 MHz) δ
8.01 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.48–7.56 (m, 4H),
7.39–7.41 (m, 3H), 7.28–7.32 (m, 1H), 7.22 (s, 1H), 7.17 (t,
J = 7.8 Hz, 2H), 6.87 (d, J = 7.2 Hz, 2H), 4.58 (s, 2H), 2.03
(s, 3H), 1.33 (s, 9H); HRMS (ESI) m/z calcd. for
C30H29N3O3S [(M – H)–] 510.1851, found: 510.1859; purity
>99.9% (as determined by RP-HPLC, tR = 25.0 min).
(E)-3-(1-Benzoyl-5-cyano-1H-indol-3-yl)-N-(4-tert-butyl-
phenyl)-2-methylacrylamide (12c): yellow solid, yield 42%.
m.p. 170–171 ꢀC; 1H-NMR (CDCl3, 400 MHz) δ 8.44 (dd, J =
0.8, 8.4 Hz, 1H), 8.05 (d, J=1.2 Hz, 1H), 7.80(s, 1H), 7.86 (d, J
= 12.0 Hz, 1H), 7.69 (dd, J = 1.4, 9.0 Hz, 2H), 7.62 (s, 1H), 7.59
(d, J = 5.6 Hz, 2H), 7.54 (s, 1H), 7.47–7.52 (m, 3H), 7.38–7.41
(m, 2H), 2.23 (s, 3H), 1.33 (s, 9H); HRMS (ESI) m/z calcd. for
C30H27N3O2 [(M – H)–] 460.2025, found: 460.2023; purity
>99.7% (as determined by RP-HPLC, tR = 20.1 min).
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(E)-3-(1-Acetyl-5-cyano-1H-indol-3-yl)-N-(4-tert-butyl-
phenyl)-2-methylacrylamide (13c): yellow solid, yield 37%.
m.p. 206–207ꢀC; 1H-NMR (CDCl3, 400 MHz) δ 8.58 (d, J =
8.4 Hz, 1H), 8.00 (s, 1H), 7.68 (dd, J = 1.2, 8.8 Hz, 1H), 7.65
(s, 1H), 7.53–7.55 (m, 1H), 7.48 (s, 1H), 7.41 (d, J = 8.8 Hz,
2H), 2.69 (s, 3H), 2.29 (s, 3H), 1.34 (s, 9H); HRMS (ESI) m/z
calcd. forC25H25N3O2 [(M – H)-]398.1869, found:398.1867;
purity >99.6% (as determined by RP-HPLC, tR = 20.0 min).
(E)-N-(4-tert-Butylphenyl)-3-[5-cyano-1-(2-morpholi-
noethyl)-1H-indol-3-yl]-2-methylacrylamide (14c): yellow
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1
solid, yield 31%. m.p. 180–181 ꢀC; H-NMR (CDCl3, 400
MHz) δ 8.10 (d, J = 0.8 Hz, 1H), 7.54–7.60 (m, 4H), 7.51 (dd,
J = 1.6, 8.8 Hz, 1H), 7.38–7.42 (m, 3H), 4.30 (t, J = 6.4 Hz,
2H), 3.69 (t, J = 4.4 Hz, 4H), 2.79 (t, J = 6.4 Hz, 2H), 2.50 (t,
Bull. Korean Chem. Soc. 2015, Vol. 36, 88–98
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
98