The discovery of indole derivatives as novel hepatitis C virus inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.03.062

In this study, a library of in-house small molecule was screened using a HCV cell-based assay and a compound (1) containing an N-protected indole scaffold (NINS) was identified as a novel anti-HCV inhibitor. Through structure activity relationship (SAR) study, it was observed that the racemic inhibitor (10m) displayed good anti-HCV activity (EC₅₀ = 1.02 ± 0.10 μM) with the excellent selectivity index (SI = 45.56). Interestingly, R-enantiomer ((R)-10m) showed better anti-HCV activity and lower cytotoxicity than S-enantiomer ((S)-10m). (R)-10m gave the best anti-HCV potency (EC₅₀ = 0.72 ± 0.09 μM) with the highest selectivity index (SI > 69.44). In addition, the mechanism of action study of NINS derivatives demonstrated that NINS derivatives interfere with the early step (viral entry) of the HCV life cycle.

Full text of DOI:10.1016/j.ejmech.2016.03.062

Accepted Manuscript
The discovery of indole derivatives as novel hepatitis C virus inhibitors
Zhiqiang Han, Xiao Liang, Yaxin Wang, Jie Qing, Lin Cao, Luqing Shang, Zheng Yin
PII:
S0223-5234(16)30245-8
10.1016/j.ejmech.2016.03.062
EJMECH 8487
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2016
Revised Date: 22 March 2016
Accepted Date: 23 March 2016
Please cite this article as: Z. Han, X. Liang, Y. Wang, J. Qing, L. Cao, L. Shang, Z. Yin, The discovery of
indole derivatives as novel hepatitis C virus inhibitors, European Journal of Medicinal Chemistry (2016),
doi: 10.1016/j.ejmech.2016.03.062.
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ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com
The discovery of indole derivatives as novel hepatitis C
virus inhibitors
Zhiqiang Han1, Xiao Liang1, Yaxin Wang1, Jie Qing2, Lin Cao2, Luqing Shang*1, and Zheng Yin*1
1. State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai
University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People’s Republic of China
2 Structural Biology Laboratory, Tsinghua University, Beijing 100084, People’s Republic of China
E-mail: zheng_yin@nankai.edu.cn; shanglq@nankai.edu.cn.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
In this study, a library of in-house small molecule was screened using a HCV cell-based
assay and a compound (1) containing an N-protected indole scaffold (NINS) was identified
as a novel anti-HCV inhibitor. Through structure activity relationship (SAR) study, it was
observed that the racemic inhibitor (10m) displayed good anti-HCV activity (EC50 = 1.02 ±
0.10 ꢀM) with the excellent selectivity index (SI = 45.56). Interestingly, R-enantiomer
((R)-10m) showed better anti-HCV activity and lower cytotoxicity than S-enantiomer ((S)-
10m). (R)-10m gave the best anti-HCV potency (EC50 = 0.72 ± 0.09 ꢀM) with the highest
selectivity index (SI > 69.44). In addition, the mechanism of action study of NINS
derivatives demonstrated that NINS derivatives interfere with the early step (viral entry) of
the HCV life cycle.
Available online
Keywords:
Hepatitis C virus (HCV),
Indole derivatives,
Structure–activity relationship (SAR) study,
Chiral effect.
2016 Elsevier Ltd. All rights reserved.
NS3/4A protease inhibitors, named direct acting antivirals
1. Introduction
Hepatitis C virus (HCV) infection is one of the primary
global public health problems and approximately 200
million people worldwide are at the risk of developing life-
threatening liver diseases, which may cause cirrhosis,
hepatocellular carcinoma, and ultimately death [1-5]. The
first standard of care for HCV infection was the
combination treatment with pegylated interferon alpha
(PEG-IFN-α) and Ribavirin (RBV) [6-8]. The combination
treatment was effective in only 40-50% of patients infected
with genotype 1 HCV, the most major (about 60%) all over
the world, and was associated with serious undesired side
effects (flu-like symptoms, headache, bone marrow
depression etc.) [9, 10]. In May 2011, the new standard of
care (SoC) for HCV infection, triple therapy, was
approved[11]. It involved a combination of PEG-IFN-
α, RVB and one of the protease inhibitors (PI), including
boceprevir [12] and telaprevir [13]. Both PIs are the viral
(DAAs). The sustained virologic response (SVR) rate for
patients infected with genotype 1 HCV increased from
50% to 70%. Compared with previous SoC, triple therapy
shortened the half of duration of treatment [14-16].
Unfortunately, the undesired side effects associated with
PEG-IFN-α and RVB can’t be eliminated [17, 18].
Sofosbuvir, the first nucleotide NS5B polymerase inhibitor
with pan-genotypic activity and a high barrier to resistance,
was approved by the FDA in December 2013 [19].
Sofosbuvir has significantly potent clinical antiviral
activity across all genotypes [20] and is the first INF-free
treatment option approved for patients unsuitable for INF
[21]. In addition, Sofosbuvir could achieve shorter duration
of treatment and higher sustained virologic response in
combination with other DAAs. Currently, a new era for
HCV treatment, all-oral treatment, has been opened by the
Gilead’s two-agent combo Havoni and Viekira’s four-

agent combo Viekira Pak [22, 23], which can achieve SVR
mechanism of action (MoA) different from 2, which
motivated us to investigate the structure activity
relationship (SAR) of NINS derivatives.
ACCEPTED MANUSCRIPT
in more 95% for genotype 1-infected patients with duration
as short as 8−12 weeks while avoiding the adverse events
produced by IFN. However, the high-cost of above
treatment is the barrier to its worldwide clinical application
[24]. To develop a safe and cost-effective treatment of
HCV infection, new chemical entities against HCV are still
highly needed. These reasons have driven us to
continuously screen small molecules for new, potent anti-
HCV inhibitors, ideally with mechanisms different from
current treatment.
Fig. 1. Structures of 1 and a known HCV inhibitor 2.
2. Results and discussion
In order to discover new chemical structures against
HCV replication, a phenotypic high throughput screening
(HTS) assay was established using the infectious cell
culture system of HCV (HCVcc). A hit (1) containing an
N-protected indole scaffold (NINS) was identified to have
inhibitory activity in the infectious HCVcc assay through
screening of in-house library of ~3000 compounds.
Interestingly, literature survey indicated that 2 [4], which
has similar chemical structure of 1, had been reported as an
inhibitor of HCV NS5B polymerase (Fig. 1). Nonetheless,
1 exhibited no ability of HCV RNA replication inhibition
in HCV replicon system, demonstrating that 1 has a novel
2.1. Chemistry
The general scheme for the synthesis of NINS
derivatives has been briefly summarized in Scheme 1.
Commercially available substituted hydrazines 3 was
treated with ethyl acetoacetate 4 to give intermediates 5,
which were condensed with 3-Indolealdehyde 6 and
subsequent dehydration to give intermediates 7. Reaction
of 7 with various electrophilic reagents, such as racemic
epibromohydrin 8, methyl iodide, benzyl bromide, and 3-
bromoproene, gave the corresponding N-protection
derivatives (1, 9a-m). The racemic NINS derivatives of
ring-opening (10a-o) were synthesized by coupling
Scheme 1 Reagents and conditions; a) MeOH, yield 80%; b) Piperidine, n-BuOH, reflux, yield 90%; c) NaH, DMSO, yield 70%; d) AlCl3,
amines, DCM, yield 50%;

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Table 1
The structures of NINS derivatives 1, 9a-m, and their in vitro activities against HCV.
Compound
R1
Ph
R2
EC50 (ꢀM)
CC50 (ꢀM)
SI[a]
8.02
5.78 ± 0.18
46.34 ± 0.53
1
p-Cl-Ph
p-CF3-Ph
m-CF3-Ph
p-Me-Ph
p-MeO-Ph
p-F-Ph
m-F-Ph
o-F-Ph
Me
6.82 ± 0.24
7.86 ± 0.39
5.94 ± 0.14
3.06 ± 0.09
2.06 ± 0.12
3.24 ± 0.16
2.65 ± 0.05
5.11 ± 0.17
>50
35.26 ± 0.27
40.43 ± 0.42
25.14 ± 0.15
13.18 ± 0.23
11.87 ± 0.35
>50
5.17
5.14
9a
9b
9c
9d
9e
9f
4.23
4.24
5.76
>15.43
9.67
25.63 ± 0.28
30.41 ± 0.49
>50
9g
9h
9i
5.95
N.A.[b]
>2.36
>2.88
>2.32
>2.03
Ph
Me
Bn
21.23 ± 0.38
17.35 ± 0.29
21.46 ± 0.56
24.53 ± 0.43
>50
9j
Ph
>50
9k
9l
Ph
>50
Ph
H
>50
9m
[a] The selectivity index (SI) is the ratio of CC50/EC50. [b] N.A.: not applicable.
intermediates (1, 9a-i) with various amines in the presence
of AlCl3/DCM. In addition, intermediates 7 was coupled
with the commercially available chiral epibromohydrin
(R)-8 and (S)-8 to afford the pure R and S enantiomers of
1, 9f, 9g, which were then treated with N-aminopropyl
morpholine to give the desired pure enantiomers ((R/S)-
10f, 10m, 10n) in the presence of AlCl3/DCM.
All synthesized NINS derivatives were evaluated for
their anti-HCV activity and cytotoxicity. The results were
summarized in Table 1-3. Firstly, the electron effects of R1
substitution were examined in comparison with inhibitor 1
(Table 1). Replacement of the phenyl group with p-Cl-
phenyl (9a), p-CF3-phenyl (9b) or m-CF3-phenyl group
(9c) showed similar anti-HCV potency (EC50 = 6.82 ±
0.24, 7.86 ± 0.39 and 5.94 ± 0.14 ꢀM, respectively) with
increased cytotoxicity (CC50=35.26 ± 0.27 ꢀM, 40.43 ±
0.42 ꢀM, 25.14 ± 0.15 ꢀM). p-Me-phenyl (9d) and p-MeO-
2.2. Biological activity and SAR study

phenyl (9e) substituted compounds displayed two- and
displayed similar potency (EC50 = 5.11 ± 0.17 ꢀM ) and
lower selectivity index (SI = 5.95). Furthermore, the
effects of the alkyl substitutions were examined.
Interestingly, the methyl substituted compound (9i) had no
activity at the highest tested concentration (EC50 > 50 ꢀM),
showing that the aryl group is an indispensable structural
moiety for anti-HCV activity. With the phenyl group fixed
at the R1 position, the effects of R2 group were studied. N-
methyl (9j), N-benzyl (9k), and N-cylopropylmethyl (9l)
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three-fold improved anti-HCV potency (EC50 = 3.06 ± 0.09
and 2.06 ± 0.12ꢀM, respectively), but lower selectivity
index (SI = 4.24 and 5.76, respectively) due to the
increased cytotoxicity (CC50 = 13.18 ± 0.23 and 11.87 ±
0.35 ꢀM, respectively). With the introduction of fluorine
on the phenyl group, p-F-phenyl 9f and m-F-phenyl 9g
exhibited better potency (EC50 = 3.24 ± 0.16 and 2.65 ±
0.05 ꢀM, respectively) and selectivity index (SI > 15.43
and SI = 9.67, respectively), while o-F-phenyl 9h
Table 2
The structures of NINS derivatives 10a-o, and their in vitro activities against HCV.
SI[a]
N.A. [b]
N.A.
N.A.
> 2.29
7.76
Compound
10a
R1
Ph
R3
EC50( µM ) CC50( µM )
>50
>50
>50
>50
>50
> 50
Ph
10b
10c
Ph
>50
Ph
21.88 ± 0.22
10d
10e
Ph
5.46 ± 0.15 42.36 ± 0.24
2.04 ± 0.11 35.83 ± 0.25
Ph
17.56
4.00
10f
Ph
2.03 ± 0.06
8.13 ± 0.33
10g
p-Cl-Ph
p-CF3-Ph
m-CF3-Ph
p-Me-Ph
p-MeO-Ph
p-F-Ph
m-F-Ph
3.42 ± 0.26 17.52 ± 0.32
4.63 ± 0.19 11.28 ± 0.23
2.08 ± 0.12 17.83 ± 0.31
1.83 ± 0.20 36.21 ± 0.31
2.05 ± 0.13 15.43 ± 0.16
1.02 ± 0.10 46.47 ± 0.24
0.92 ± 0.06 21.46 ± 0.34
5.12
10h
10i
2.44
8.57
10j
19.79
7.53
10k
10l
45.56
23.33
10m
10n

2.79 ± 0.17 34.21 ± 0.43
o-F-PhACCEPTED MANUSCRIPT
12.26
10o
[a] The selectivity index (SI) is the ratio of CC50/EC50. [b] N.A.: not applicable.
showed significantly reduced inhibitory activity (EC50
=21.23 ± 0.38, 17.35 ± 0.29, and 21.46 ± 0.56 ꢀM).
Similarly, the one (9m) without protection gave
significantly reduced anti-HCV activity (EC50 = 24.53 ±
0.43 ꢀM) compared to 1 (EC50 = 5.78 ± 0.18 ꢀM). The
results may suggest that the glycidyl group is essential
chemical feature of NINS derivatives associated with anti-
HCV activity.
11.28 ± 0.23 and 17.83 ± 0.31 ꢀM, respectively). p-Me or
p-MeO substituted compounds (10k and 10l) showed
almost similar activities, but providing a much low
selectivity index due to the significantly increased
cytotoxicity (CC50 = 36.21 ± 0.31 and 15.43 ± 0.16 ꢀM,
respectively). Interestingly, p-F and m-F substituted
compounds (10m and 10n) exhibited two fold
improvement in anti-HCV potency (EC50 = 1.02 ± 0.10 and
0.92 ± 0.06 ꢀM, respectively), and 10m exhibited less
cytotoxicity (CC50 =46.47 ± 0.24 ꢀM) in comparison to
10n (CC50 = 21.46 ± 0.34 ꢀM). o-F substituted compound
(10r) displayed a less anti-HCV potency (EC50 = 2.79 ±
0.17 ꢀM) than other fluorinated compounds.
The glycidyl group may not be a favorable functional
group for drug due to its chemical reactivity that could be
attacked by the nucleophiles. Therefore, several NINS
derivatives of ring-opening of oxirane were synthesized. In
order to study SAR in an efficient and quick fashion,
racemic mixtures were used conveniently to evaluate their
anti-HCV activities (Table 2). Firstly, with the phenyl
group fixed at the R1 position, the effects of R3 groups
were studied in comparison with 1. When oxirane was
initially opened with various nucleophiles (10a-d), only the
morpholinyl substituted inhibitor (10d) gave anti-HCV
activities at a EC50 of 21.88 ± 0.22 ꢀM. Based on this
observation, we decided to further optimize 10d to
improve the potency. Thus the impact of the length of
carbon linker between amino group and morpholine moiety
was explored. As shown by 10e-g (EC50 = 5.46 ± 0.15,
2.04 ± 0.11 and 2.03 ± 0.06 ꢀM, respectively),
improvement of antiviral potency was found with increase
of the linker length (two carbon to four carbon). However,
the cytotoxicity was increase as well. (CC50= 42.36 ± 0.24,
35.83 ± 0.25, and 8.13 ± 0.33 ꢀM, respectively). As 10f
exhibited the highest SI, with the N-aminopropyl
morpholine group fixed at the R3 position, the electron
effects of R1 groups were studied (Table 2). Compared to
10f (EC50 = 2.04 ± 0.11 ꢀM), p-Cl-phenyl (10h), p-CF3-
phenyl (10i) or m-CF3-phenyl (10j) substituted compounds
displayed slightly reduced anti-HCV activity (EC50 = 3.42
± 0.26, 4.63 ± 0.19, and 2.08 ± 0.12 ꢀM, respectively) with
significantly increased cytotoxicity (CC50 = 17.52 ± 0.32,
The chirality of inhibitors could have important impacts
on their biological activity. Therefore, pure enantiomers of
NINS derivatives were synthesized and examined in order
to understand whether chirality was associated with anti-
HCV potency and cytotoxicity (Table 3). Interestingly, the
chirality of 10f has significantly influence on the anti-HCV
activity, with the (R)-enantiomer being 7-fold better
potency and less cytotoxicity (EC50 = 1.25 ± 0.07 ꢀM and
CC50 >50 ꢀM) than the corresponding (S)-enantiomer
(EC50 = 8.06 ± 0.13 ꢀM and CC50 = 21.53 ± 0.32 ꢀM).
Table 3 The structures of pure enantiomers R/S-10f, m, n
and their in vitro activities against HCV.
SI[a]
>40.00
2.67
Compound
(R)-10f
R1
Ph
EC50( µM )
1.25 ± 0.07
8.06 ± 0.13
0.72 ± 0.09
7.12 ± 0.21
0.74 ± 0.11
CC50( µM )
>50
Ph
21.53 ± 0.32
>50
(S)-10f
p-F-Ph
p-F-Ph
m-F-Ph
>69.44
4.86
(R)-10m
(S)-10m
(R)-10n
34.57 ± 0.53
31.06 ± 0.37
41.97

m-F-Ph
5.87 ± 0.18
23.31 ± 0.30
3.97
(S)-10n
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[a]. The selectivity index (SI) is the ratio of CC50/EC50.
Fig. 2. Evaluation of HCV entry and RNA replication in the presence of 1 (a) and 10m (b). Huh7.5.1 cells (3×104 per well in 100 µl 10% FBS-
DMEM medium) were cultured at 37 °C under 5% CO2 in 96-well plates overnight. Subsequently, the cells were treated with 5 µM 1, and 2
µM 10m either concurrent with 100 TCID50 of HCV(JFH-1)-Luc virus (0 h) or at intervals of -6, -4, -2, 0, 1, 2, 4, 6, 8 and 10 h post infection
(hpi),which 0 hpi indicated supplying inhibitors immediately after virus infection. After incubation at 37 °C for 24 h, luciferase activity was
determined by Rellia-GloTM Luciferase Assay System. Mock: control culture experiment (0.5% DMSO control).
Encouraged by the results, we next investigated the
antiviral activity of pure enantiomers of both 10m and 10n.
(R)-10m and (R)-10n displayed good anti-HCV activity
(EC50 = 0.72 ± 0.09, and 0.74 ± 0.11 ꢀM, respectively)
with low cytotoxicity (CC50 > 50 and 31.06 ± 0.37 ꢀM)
compared to their S-enantiomers (EC50 = 7.12 ± 0.21, and
5.87 ± 0.18 ꢀM, respectively). Among all of NINS
derivatives in this study, (R)-10m gave the best anti-HCV
activity (EC50 = 0.72 ± 0.09 ꢀM) and the highest selectivity
index (SI > 69.44).
activity compared with the control cultures. As shown in
Fig. 2, 1 and 10m all displayed good antiviral levels at
intervals -6, -4, -2, 0, and 1 hpi and unsatisfactory antiviral
levels at intervals 2, 4, 6, and 10 hpi, suggesting that the
selected compounds could only inhibit the viral entry step
and not at the replication process. Therefore, NINS
derivatives may exhibit anti-HCV activity by inhibiting
viral entry step of the viral cycle, whereas viral RNA
replication was not affected.
3. Conclusion
2.3. Mechanism of action (MoA) studies
In this study, NINS derivative was identified as an anti-
HCV inhibitor by a phenotypic high throughput screening
(HTS) assay using the infectious HCV cell culture system.
During our cell-based SAR study, the racemic inhibitor
10m displayed good anti-HCV activity (EC50 = 1.02 ± 0.10
ꢀM) with excellent selectivity index (SI = 45.56).
Interestingly, (R)-10m showed the better anti-HCV
activity and lower cytotoxicity than its S-enantiomer. Thus,
(R)-10m gave the best anti-HCV potency (EC50 = 0.72 ±
0.09 ꢀM) with the highest selectivity index (SI > 69.44). In
addition, the mechanism of action of NINS derivatives
were studied and demonstrated that NINS derivatives may
interfere with the early step (viral entry) of the HCV life
cycle. With its good anti-HCV activity and low
cytotoxicity, we believe the NINS scaffolds would have
NINS derivatives were identified using the target-free
infectious HCV cell culture system (HCVcc). Study was
performed to elucidate the MoA. In order to investigate
which step was blocked in the viral life cycle, we analyzed
the activity of selected compounds (1, 10m) using the
time-addition assay to monitor viral entry and RNA
replication. Huh7.5.1 cells were cultured at 37 °C under
5% CO2 in 96-well plates overnight. The Huh7.5.1 cells
were infected with HCV (JFH-1)-Luciferase-linker
reporter virus and treated with 1 and 10m at -6, -4, -2, 0, 1,
2, 4, 6 and 10 h post infection (hpi), in which 0 hpi
indicated supplying inhibitors immediately after virus
infection. After incubation at 37 °C for 24 h, antiviral
activity was determined by the reduction of luciferase

potential applications in new anti-HCV drug discovery and
1H NMR (400 MHz, CDCl3) δ 9.86 (s, 1H), 8.01 (dd, J =
8.6, 1.0 Hz, 2H), 7.90 – 7.84 (m, 1H), 7.76 (s, 1H), 7.57 –
7.50 (m, 1H), 7.44 (dd, J = 10.8, 5.2 Hz, 2H), 7.40 – 7.35
(m, 2H), 7.18 (t, J = 7.4 Hz, 1H), 4.59 (dd, J = 15.1, 3.0
Hz, 1H), 4.25 (dd, J = 15.1, 5.8 Hz, 1H), 3.35 (td, J = 5.8,
2.9 Hz, 1H), 2.87 (t, J = 4.3 Hz, 1H), 2.58 (dd, J = 4.6, 2.5
Hz, 1H), 2.40 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
163.25, 150.37, 140.51, 138.84, 137.00, 134.98, 128.99,
128.72, 124.47, 123.90, 122.71, 120.30, 119.21, 117.99,
112.18, 111.15, 77.32, 77.00, 76.68, 50.39, 49.41, 45.20,
13.15. HRMS (ESI) m/z 358.1553 (M+H+), calc. for
C22H20N3O2 358.1550.
ACCEPTED MANUSCRIPT
development.
4. Experimental section
4.1. Chemistry
4.1.1. General procedure
All of the commercial chemicals were analytical or best
available quality throughout. Solvents in this study were
dried with standard procedures. The proton nuclear
magnetic resonance (1H NMR) spectra and carbon nuclear
magnetic resonance (13C NMR) spectra were recorded on a
Bruker AVANCE-400 (400 MHz) (Bruker, Karlsruhe,
Germany) NMR spectrometer. Electrospray ionization
mass spectroscopy (ESI-MS) was performed on a
Shimadzu LCMS-2020 (Shimadzu, Kyoto, Japan). HRMS
were recorded on a high-resolution ESI-FTICR mass
spectrometer (Varian 7.0 T, Varian, USA). The products
from all of the reactions were purified by flash column
chromatography using silica gel or by preparative thin
layer chromatography using glass-backed silica gel plates,
unless otherwise indicated.
4.1.2.2. 1-(4-Chlorophenyl)-3-methyl-4-((1-(oxiran-2-ylme
thyl)-1H-indol-3-yl)methylene)-1H-pyrazol-5(4H)-one 9a
1H NMR (400 MHz, CDCl3) δ 9.86 (s, 1H), 8.00 (d, J =
8.7 Hz, 2H), 7.93 – 7.85 (m, 1H), 7.80 (s, 1H), 7.60 – 7.51
(m, 1H), 7.38 (t, J = 7.3 Hz, 4H), 4.63 (d, J = 15.1 Hz, 1H),
4.28 (dd, J = 15.1, 5.8 Hz, 1H), 3.38 (s, 1H), 2.89 (t, J =
4.2 Hz, 1H), 2.61 (d, J = 2.3 Hz, 1H), 2.41 (s, 3H). 13
C
NMR (101 MHz, CDCl3) δ 150.71, 140.64, 137.55,
137.08, 135.35, 129.39, 129.03, 128.73, 124.04, 122.88,
120.12, 120.07, 118.05, 112.30, 111.20, 50.40, 49.49,
45.24, 13.17. HRMS (ESI) m/z 392.1165 (M+H+), calc. for
C22H19N3O2Cl 392.1160.
4.1.2. General procedure for the coupling reaction for
synthesis of compounds 1, 9a-i.
The round-bottom flask was charged with 60%
suspension of NaH in mineral oil (1.0 mmol) and
compounds 7a-i (1.0 mmol) in dried DMSO (5 mL). After
stirring at the room temperature for 30 min, the mixture
solution of Epibromohydrin (2.0 mmol) in DMSO (1.0
mL) was added and the reaction mixture was stirred for 3h
at room temperature. The reaction mixture was filtered off
and washed with 2-Propanol. The mother liquor in DMSO
left after first filtration was diluted with water and
extracted with DCM (3*30 mL). The extract was twice
washed with water, dried over anhydrous Na2SO4, filtered,
and the solvent was concentrated on the rotary evaporator.
The residue was purified by column chromatography on
silica gel to give the desired products 9a-i.
4.1.2.3. 3-Methyl-4-((1-(oxiran-2-ylmethyl)-1H-indol-3-yl)
methylene)-1-(4(trifluoromethyl)phenyl)-1H-pyrazol-
5(4H)
-one 9b
1H NMR (400 MHz, CDCl3) δ 9.84 (s, 1H), 8.21 (d, J =
8.6 Hz, 2H), 7.92 – 7.84 (m, 1H), 7.80 (s, 1H), 7.66 (d, J =
8.6 Hz, 2H), 7.60 – 7.50 (m, 1H), 7.48 – 7.34 (m, 2H), 4.64
(dd, J = 15.1, 2.5 Hz, 1H), 4.27 (dd, J = 15.1, 5.8 Hz, 1H),
3.38 (dd, J = 5.6, 2.9 Hz, 1H), 2.90 (t, J = 4.2 Hz, 1H),
2.61 (dd, J = 4.3, 2.4 Hz, 1H), 2.42 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 163.67, 151.24, 141.73, 140.73, 137.08,
135.56, 129.01, 125.93, 125.89, 124.11, 122.96, 119.78,
118.17, 118.05, 112.34, 111.21, 50.37, 49.50, 45.23, 13.19.
4.1.2.1. 3-Methyl-4-((1-(oxiran-2-ylmethyl)-1H-indol-3-yl)
methylene)-1-phenyl-1H-pyrazol-5 (4H)-one 1

HRMS (ESI) m/z 426.1426 (M+H+),
for
1H), 3.83 (s, 4H), 3.35 (dd, J = 5.7, 2.8 Hz, 1H), 2.87 (t, J
ACCcEaPlcT. ED MANUSCRIPT
C23H19N3O2F3 426.1424.
= 4.3 Hz, 1H), 2.59 (dd, J = 4.5, 2.5 Hz, 1H), 2.41 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 162.98, 156.75, 150.01,
140.49, 137.04, 134.95, 132.28, 129.04, 123.89, 122.71,
121.28, 120.45, 118.03, 113.96, 112.22, 111.15, 55.48,
50.41, 49.43, 45.24, 13.12. HRMS (ESI) m/z 388.1653
(M+H+), calc. for C23H22N3O3 388.1656.
4.1.2.4. 3-Methyl-4-((1-(oxiran-2-ylmethyl)-1H-indol-3-yl)
methylene)-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-
5(4H)-one 9c
1H NMR (400 MHz, CDCl3) δ 9.85 (d, J = 11.5 Hz,
1H), 8.39 (s, 1H), 8.29 (d, J = 8.3 Hz, 1H), 7.88 (dd, J =
8.3, 4.3 Hz, 1H), 7.81 (s, 1H), 7.59 – 7.49 (m, 2H), 7.39
(dt, J = 6.9, 5.9 Hz, 3H), 4.66 (dd, J = 15.1, 2.9 Hz, 1H),
4.29 (dd, J = 15.1, 5.8 Hz, 1H), 3.39 (td, J = 6.0, 2.9 Hz,
1H), 2.90 (t, J = 4.3 Hz, 1H), 2.62 (dd, J = 4.6, 2.5 Hz,
1H), 2.43 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 163.53,
151.04, 140.77, 139.43, 137.11, 135.55, 129.21, 129.03,
124.09, 122.94, 121.56, 120.64, 120.60, 119.87, 118.05,
115.50, 115.46, 112.33, 111.25, 50.42, 49.54, 45.25, 13.18.
HRMS (ESI) m/z 426.1422 (M+H+), calc. for
C23H19N3O2F3 426.1424.
4.1.2.7. 1-(4-Fluorophenyl)-3-methyl-4-((1-(oxiran-2-yl
methyl)-1H-indol-3-yl)methylene)-1H-pyrazol-5(4H)-one
9f
1H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H), 8.01 – 7.91
(m, 2H), 7.85 (dd, J = 6.3, 2.6 Hz, 1H), 7.74 (s, 1H), 7.58 –
7.48 (m, 1H), 7.37 (dt, J = 5.5, 3.9 Hz, 2H), 7.10 (t, J = 8.7
Hz, 2H), 4.58 (dd, J = 15.1, 2.8 Hz, 1H), 4.22 (dd, J =
15.1, 5.9 Hz, 1H), 3.35 (dd, J = 5.9, 3.0 Hz, 1H), 2.87 (t, J
= 4.3 Hz, 1H), 2.58 (dd, J = 4.5, 2.5 Hz, 1H), 2.37 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 163.04, 160.80, 158.38,
150.39, 140.55, 136.97, 135.22, 135.03, 135.00, 128.93,
123.93, 122.76, 120.88, 120.81, 119.95, 117.97, 115.43,
115.20, 112.20, 111.13, 50.34, 49.39, 45.18, 13.09. HRMS
(ESI) m/z 376.1457 (M+H+), calc. for C22H19N3O2F
376.1456.
4.1.2.5. 3-Methyl-4-((1-(oxiran-2-ylmethyl)-1H-indol-3-yl)
methylene)-1-p-tolyl-1H-pyrazol-5(4H)-one 9d
1H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 7.88 (t, J =
8.0 Hz, 3H), 7.78 (s, 1H), 7.59 – 7.50 (m, 1H), 7.43 – 7.32
(m, 2H), 7.23 (d, J = 8.3 Hz, 2H), 4.61 (dd, J = 15.1, 2.9
Hz, 1H), 4.28 (dd, J = 15.1, 5.7 Hz, 1H), 3.36 (d, J = 2.8
Hz, 1H), 2.87 (t, J = 4.3 Hz, 1H), 2.60 (dd, J = 4.5, 2.5 Hz,
1H), 2.41 (s, 3H), 2.36 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 163.14, 150.14, 140.47, 137.06, 136.45, 134.87,
134.12, 129.27, 129.06, 123.89, 122.71, 120.57, 119.35,
118.03, 112.21, 111.16, 50.44, 49.44, 45.25, 20.97, 13.15.
HRMS (ESI) m/z 372.1709 (M+H+), calc. for C23H22N3O2
372.1707.
4.1.2.8. 1-(3-Fluorophenyl)-3-methyl-4-((1-(oxiran-2-ylme
thyl)-1H-indol-3-yl)methylene)-1H-pyrazol-5(4H)-one 9g
1H NMR (400 MHz, CDCl3) δ 9.82 (s, 1H), 7.86 (d, J =
9.0 Hz, 3H), 7.74 (s, 1H), 7.59 – 7.48 (m, 1H), 7.44 – 7.29
(m, 3H), 6.93 – 6.79 (m, 1H), 4.60 (dd, J = 15.1, 2.6 Hz,
1H), 4.24 (dd, J = 15.1, 5.8 Hz, 1H), 3.41 – 3.31 (m, 1H),
2.88 (t, J = 4.3 Hz, 1H), 2.59 (dd, J = 4.5, 2.5 Hz, 1H),
2.38 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 164.13,
163.33, 161.71, 150.75, 140.60, 140.41, 140.30, 137.02,
135.23, 129.88, 129.79, 128.97, 123.98, 122.81, 119.95,
117.98, 114.04, 114.01, 112.23, 111.15, 110.91, 110.70,
106.17, 105.90, 50.35, 49.45, 45.20, 13.10. HRMS (ESI)
m/z 376.1459 (M+H+), calc. for C22H19N3O2F 376.1456.
4.1.2.6. 1-(4-Methoxyphenyl)-3-methyl-4-((1-(oxiran-2-yl
methyl)-1H-indol-3-yl)methylene)-1H-pyrazol-5(4H)-one
9e
1H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 7.87 (dd, J
= 11.6, 7.5 Hz, 3H), 7.78 (d, J = 5.2 Hz, 1H), 7.57 – 7.52
(m, 1H), 7.40 – 7.35 (m, 2H), 6.96 (d, J = 9.0 Hz, 2H),
4.60 (dd, J = 15.1, 2.9 Hz, 1H), 4.27 (dd, J = 15.1, 5.7 Hz,
4.1.2.9. 1-(2-Fluorophenyl)-3-methyl-4-((1-(oxiran-2-ylme
thyl)-1H-indol-3-yl)methylene)-1H-pyrazol-5(4H)-one 9h

1H NMR (400 MHz, CDCl3) δ 9.85 (d, J = 11.5 Hz,
4.1.3.1. 3-Methyl-4-((1-methyl-1H-indol-3-yl)methylene)-
ACCEPTED MANUSCRIPT
1H), 8.39 (s, 1H), 8.29 (d, J = 8.3 Hz, 1H), 7.88 (dd, J =
8.3, 4.3 Hz, 1H), 7.81 (s, 1H), 7.59 – 7.49 (m, 2H), 7.39
(dt, J = 6.9, 5.9 Hz, 3H), 4.66 (dd, J = 15.1, 2.9 Hz, 1H),
4.29 (dd, J = 15.1, 5.8 Hz, 1H), 3.39 (td, J = 6.0, 2.9 Hz,
1H), 2.90 (t, J = 4.3 Hz, 1H), 2.62 (dd, J = 4.6, 2.5 Hz,
1H), 2.43 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 163.04,
160.80, 158.38, 150.39, 140.55, 136.97, 135.22, 135.03,
128.93, 123.93, 122.76, 120.88, 120.81, 119.95, 117.97,
115.43, 115.20, 112.20, 111.13, 50.34, 49.39, 45.18, 13.09.
HRMS (ESI) m/z 376.1458 (M+H+), calc. for
C22H19N3O2F 376.1456.
1-phenyl-1H-pyrazol-5(4H)-one 9j
1H NMR (400 MHz, CDCl3) δ 9.84 (s, 1H), 8.07 – 7.98
(m, 2H), 7.89 (dd, J = 6.0, 2.7 Hz, 1H), 7.80 (s, 1H), 7.51 –
7.34 (m, 5H), 7.18 (t, J = 7.4 Hz, 1H), 3.94 (s, 3H), 2.42 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 163.40, 150.44,
141.41, 138.95, 137.35, 135.24, 129.09, 128.74, 124.44,
123.70, 122.63, 119.52, 119.25, 118.00, 111.78, 110.61,
34.04, 13.19. HRMS (ESI) m/z 316.1447 (M+H+), calc. for
C20H18N3O 316.1444.
4.1.3.2. 4-((1-Benzyl-1H-indol-3-yl)methylene)-3-methyl-
1-phenyl-1H-pyrazol-5(4H)-one 9k
4.1.2.10. 1,3-Dimethyl-4-((1-(oxiran-2-ylmethyl)-1H-indol
1H NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 7.98 – 7.89
(m, 2H), 7.80 (d, J = 7.8 Hz, 1H), 7.71 (s, 1H), 7.35 (t, J =
8.0 Hz, 2H), 7.29 – 7.16 (m, 6H), 7.10 (t, J = 8.1 Hz, 3H),
5.35 (s, 2H), 2.32 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
163.28, 150.38, 140.96, 138.85, 136.62, 135.36, 135.09,
129.32, 128.92, 128.72, 128.10, 126.70, 124.45, 123.76,
122.61, 119.95, 119.21, 118.02, 112.08, 111.39, 51.52,
13.14. HRMS (ESI) m/z 392.1751 (M+H+), calc. for
C26H22N3O 392.1757.
-3-yl)methylene)-1H-pyrazol-5(4H)-one 9i
1H NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 7.89 – 7.83
(m, 1H), 7.73 (s, 1H), 7.58 – 7.48 (m, 1H), 7.41 – 7.31 (m,
2H), 4.57 (dd, J = 15.0, 3.0 Hz, 1H), 4.26 (dd, J = 15.0, 5.8
Hz, 1H), 3.45 (s, 3H), 3.36 (dd, J = 5.9, 2.9 Hz, 1H), 2.88
(t, J = 4.3 Hz, 1H), 2.60 (dd, J = 4.5, 2.5 Hz, 1H), 2.32 (s,
3H).13C NMR (101 MHz, CDCl3) δ 164.16, 148.75,
140.20, 136.98, 134.73, 128.92, 123.77, 122.59, 119.94,
118.00, 112.18, 111.05, 50.37, 49.37, 45.29, 31.19, 13.01.
HRMS (ESI) m/z 296.1397 (M+H+), calc. for C17H18N3O2
296.1394.
4.1.3.3. 4-((1-(Cyclopropylmethyl)-1H-indol-3-yl)meth
ylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one 9l
1H NMR (400 MHz, CDCl3) δ 9.93 (s, 1H), 7.99 (d, J =
8.0 Hz, 2H), 7.87 – 7.81 (m, 1H), 7.74 (d, J = 9.9 Hz, 1H),
7.41 (dd, J = 16.3, 8.4 Hz, 3H), 7.32 (dt, J = 12.1, 5.9 Hz,
2H), 7.14 (t, J = 7.4 Hz, 1H), 4.06 (d, J = 7.0 Hz, 2H), 2.37
(s, 3H), 0.84 (d, J = 6.9 Hz, 1H), 0.67 (q, J = 5.4 Hz, 2H),
0.43 (q, J = 5.0 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ
163.34, 150.40, 140.42, 138.93, 136.77, 135.24, 129.21,
128.78, 128.69, 124.36, 123.51, 122.51, 119.33, 119.25,
118.02, 111.83, 110.87, 51.96, 13.16, 10.91, 4.35. HRMS
(ESI) m/z 356.1753 (M+H+), calc. for C23H22N3O
356.1757.
4.1.3. General procedure for the coupling reaction for
synthesis of compounds 9j-l.
The round-bottom flask was charged with 60%
suspension of NaH in mineral oil (1.0 mmol) and
compounds 5a (1.0 mmol) in dried DMSO (5 mL). After
stirring at the room temperature for 30 min, the mixture
solution of R2X (2.0 mmol) in DMSO (1.0 mL) was added
and the reaction mixture was stirred for 3 h at room
temperature. The reaction was diluted with water and
extracted with DCM (3*30 mL). The extract was twice
washed with water, dried over anhydrous Na2SO4, filtered,
and the solvent was concentrated on the rotary evaporator.
The residue was purified by column chromatography on
silica gel to give the desired products 9j-l.
4.1.4. General procedure for the coupling reaction for
synthesis of compound 9m
To a solution of 3-methyl-1-phenyl-2-pyrazolin-5-one 5
(1.0 mmol) and Indole-3-carbaldehyde 6 (1.0 mmol) in

(m, 1H), 7.30 – 7.25 (m, 2H), 7.19 (dd, J = 5.3, 3.8 Hz,
butanol (10 mL) was added piperidine (1.1AmCmColE) uPnTdeEr Dan MANUSCRIPT
N2 atmosphere and the mixture was stirred at reflux for 1 h.
On cooling the precipitate was filtered off to give the
desired product 9m.
3H), 7.15 (dd, J = 7.4, 4.8 Hz, 4H), 7.06 (d, J = 7.4 Hz,
1H), 5.19 (s, 1H), 4.16 – 4.03 (m, 2H), 4.02 – 3.95 (m,
1H), 3.59 (q, J = 13.2 Hz, 2H), 2.63 (dd, J = 12.1, 3.8 Hz,
1H), 2.48 (dd, J = 12.1, 7.9 Hz, 1H), 2.24 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 163.13, 150.36, 141.11,
139.27, 138.83, 137.15, 135.09, 128.94, 128.64, 128.47,
128.12, 127.21, 124.38, 123.62, 122.53, 119.53, 119.19,
117.86, 111.94, 111.21, 68.65, 53.55, 51.87, 51.44, 13.06.
HRMS (ESI) m/z 465.2287 (M+H+), calc. for C29H28N4O2
465.2285.
4.1.4.1. 4-((1H-indol-3-yl)methylene)-3-methyl-1-phenyl-
1H-pyrazol-5(4H)-one 9m
1H NMR (400 MHz, DMSO) δ 9.82 (s, 1H), 8.18 (dd, J
= 6.1, 2.9 Hz, 1H), 8.12 (s, 1H), 8.01 (d, J = 7.8 Hz, 2H),
7.62 – 7.57 (m, 1H), 7.43 (t, J = 7.9 Hz, 2H), 7.35 – 7.29
(m, 2H), 7.16 (t, J = 7.3 Hz, 1H), 2.42 (s, 3H). 13C NMR
(101 MHz, DMSO) δ 162.85, 151.05, 138.94, 138.32,
137.39, 136.45, 128.78, 128.19, 123.99, 123.61, 122.18,
118.74, 118.36, 118.07, 113.00, 112.32, 13.09. HRMS
(ESI) m/z 302.1291 (M+H+), calc. for C19H16N3O
302.1288.
4.1.5.2. 4-((1-(3-(Butylamino)-2-hydroxypropyl)-1H-indol
-3-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-
one 10b
1H NMR (400 MHz, CDCl3) δ 9.78 (s, 1H), 7.93 (d, J =
7.9 Hz, 2H), 7.82 – 7.76 (m, 1H), 7.67 (s, 1H), 7.46 (d, J =
8.2 Hz, 1H), 7.37 (t, J = 7.9 Hz, 2H), 7.32 – 7.27 (m, 2H),
7.14 (t, J = 7.3 Hz, 1H), 4.29 – 4.16 (m, 2H), 4.14 (s, 1H),
3.52 (s, 1H), 2.73 (dd, J = 12.1, 3.2 Hz, 1H), 2.54 (ddd, J =
18.4, 11.5, 6.5 Hz, 3H), 2.35 (s, 3H), 1.45 – 1.35 (m, 2H),
1.26 (t, J = 7.3 Hz, 2H), 0.86 (t, J = 7.3 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 163.18, 150.39, 141.10, 138.82,
137.20, 135.16, 128.95, 128.66, 124.46, 123.67, 122.56,
119.55, 119.31, 117.86, 111.99, 111.26, 99.94, 68.24,
52.29, 51.45, 49.27, 31.57, 20.20, 13.83, 13.08. HRMS
(ESI) m/z 431.2447 (M+H+), calc. for C26H31N4O2
431.2442.
4.1.5. General procedure for the coupling reaction for
synthesis of compounds 10a-o.
To a solution of compounds 1 (1.0 mmol) and amino
complex (1.5 mmol) in dried DCM (5 mL) was added
AlCl3 (0.1 mmol) under the N2 atmosphere and the mixture
was stirred at room temperature for 10 h. The reaction
mixture was filtered off and filtrate was removed in vacuo
to give a residue. The residue was purified by column
chromatography on silica gel to give the desired products
10a-g.
To a solution of compounds 9a-h (1.0 mmol) and 4-
Aminopropyl morpholine (1.5 mmol) in dried DCM (5
mL) was added AlCl3 (0.1 mmol) under the N2 atmosphere
and the mixture was stirred at room temperature for 10 h.
The reaction mixture was filtered off and filtrate was
removed in vacuo to give a residue. The residue was
purified by column chromatography on silica gel to give
the desired products 10h-o.
4.1.5.3. 4-((1-(3-(Dibutylamino)-2-hydroxypropyl)-1H-ind
ol-3-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-
one 10c
1H NMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 8.00 (dd, J
= 8.6, 1.0 Hz, 2H), 7.90 – 7.84 (m, 1H), 7.80 (s, 1H), 7.62
– 7.54 (m, 1H), 7.42 (dd, J = 10.8, 5.1 Hz, 2H), 7.38 – 7.33
(m, 2H), 7.17 (t, J = 7.4 Hz, 1H), 4.26 (dd, J = 5.4, 2.4 Hz,
2H), 4.10 – 4.01 (m, 1H), 2.55 – 2.46 (m, 3H), 2.44 – 2.35
(m, 6H), 1.45 – 1.36 (m, 4H), 1.25 – 1.16 (m, 4H), 0.86 (t,
J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 163.36,
150.40, 141.12, 138.95, 137.37, 135.22, 129.09, 128.70,
4.1.5.1. 4-((1-(3-(Benzylamino)-2-hydroxypropyl)-1H-ind
ol-3yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-
one 10a
1H NMR (400 MHz, CDCl3) δ 9.68 (s, 1H), 7.85 (d, J =
7.7 Hz, 2H), 7.73 – 7.66 (m, 1H), 7.55 (s, 1H), 7.34 – 7.31

1H NMR (400 MHz, CDCl3) δ 9.49 (s, 1H), 7.71 (d, J =
124.42, 123.64, 122.56, 119.81, 119.34, A11C7.C90E, P1T12E.0D6, MANUSCRIPT
111.51, 66.59, 57.95, 54.12, 51.74, 29.44, 26.61, 22.52,
13.98, 13.16. HRMS (ESI) m/z 487.3072 (M+H+), calc. for
C30H39N4O2 487.3068.
8.0 Hz, 2H), 7.61 (d, J = 7.6 Hz, 1H), 7.44 (s, 1H), 7.39 (d,
J = 7.9 Hz, 1H), 7.26 – 7.08 (m, 5H), 7.02 (t, J = 7.3 Hz,
1H), 4.52 (s, 1H), 4.28 – 4.13 (m, 2H), 3.51 (s, 4H), 2.98
(t, J = 10.2 Hz, 2H), 2.87 (dd, J = 20.9, 9.1 Hz, 1H), 2.79
(dd, J = 12.0, 6.3 Hz, 1H), 2.42 – 2.26 (m, 6H), 2.23 – 2.16
(m, 3H), 1.87 (dd, J = 14.1, 6.7 Hz, 1H), 1.79 – 1.68 (m,
1H). 13C NMR (101 MHz, CDCl3) δ 162.86, 150.30,
140.64, 138.57, 136.98, 134.97, 128.67, 128.58, 124.57,
123.94, 122.69, 119.52, 119.42, 117.73, 112.04, 111.37,
66.24, 65.98, 57.04, 53.26, 50.87, 50.81, 48.61, 21.47,
12.99. HRMS (ESI) m/z 502.2811 (M+H+), calc. for
C29H36N5O3 502.2813.
4.1.5.4. 4-((1-(2-Hydroxy-3-morpholinopropyl)-1H-indol-
3-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one
10d
1H NMR (400 MHz, CDCl3) δ 9.79 (s, 1H), 7.96 (dd, J
= 8.6, 0.9 Hz, 2H), 7.83 – 7.76 (m, 1H), 7.65 (s, 1H), 7.47
– 7.43 (m, 1H), 7.42 – 7.36 (m, 2H), 7.34 – 7.30 (m, 2H),
7.15 (t, J = 7.4 Hz, 1H), 4.28 (dd, J = 14.2, 3.6 Hz, 1H),
4.18 (dd, J = 14.2, 6.7 Hz, 1H), 4.10 (t, J = 4.7 Hz, 1H),
3.69 – 3.62 (m, 4H), 2.59 – 2.51 (m, 2H), 2.43 – 2.30 (m,
7H). 13C NMR (101 MHz, CDCl3) δ 163.12, 150.33,
141.15, 138.81, 137.21, 135.01, 128.92, 128.61, 124.35,
123.55, 122.47, 119.56, 119.15, 117.82, 111.94, 111.20,
66.79, 65.99, 61.48, 53.60, 51.23, 13.00. HRMS (ESI) m/z
445.2232 (M+H+), calc. for C26H29N4O3 445.2234.
4.1.5.7. 4-((1-(2-Hydroxy-3-(4-morpholinobutylamino)pro
pyl)-1H-indol-3-yl)methylene)-3-methyl-1-phenyl-1H-
pyrazol-5(4H)-one 10g
1H NMR (400 MHz, CDCl3) δ 9.73 (s, 1H), 7.87 (d, J =
8.1 Hz, 2H), 7.80 (d, J = 8.0 Hz, 1H), 7.69 (s, 1H), 7.55 (d,
J = 7.5 Hz, 1H), 7.32 (dt, J = 8.1, 5.7 Hz, 4H), 7.13 (t, J =
7.3 Hz, 1H), 4.59 (s, 1H), 4.33 (d, J = 5.0 Hz, 2H), 3.56 (d,
J = 5.0 Hz, 4H), 2.98 (d, J = 11.4 Hz, 1H), 2.87 (d, J =
10.0 Hz, 1H), 2.77 (ddd, J = 26.3, 12.2, 6.0 Hz, 2H), 2.41 –
2.24 (m, 9H), 1.76 – 1.66 (m, 2H), 1.51 (dd, J = 13.2, 6.6
Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 162.95, 150.39,
140.73, 138.66, 137.07, 135.06, 128.76, 128.67, 124.66,
124.03, 122.78, 119.61, 119.51, 117.82, 112.13, 111.46,
66.33, 66.07, 60.38, 57.13, 53.35, 50.90, 48.70, 21.56,
13.08. HRMS (ESI) m/z 516.2965 (M+H+), calc. for
C30H38N5O3 516.2969.
4.1.5.5. 4-((1-(2-Hydroxy-3-(2-morpholinoethylamino)pro
pyl)-1H-indol-3-yl)methylene)-3-methyl-1-phenyl-1H-
pyrazol-5(4H)-one 10e
1H NMR (400 MHz, CDCl3) δ 9.86 (s, 1H), 7.98 (d, J =
8.3 Hz, 2H), 7.86 (dd, J = 6.2, 2.7 Hz, 1H), 7.77 (s, 1H),
7.56 – 7.50 (m, 1H), 7.41 (t, J = 7.9 Hz, 2H), 7.38 – 7.31
(m, 2H), 7.17 (t, J = 7.4 Hz, 1H), 4.29 (d, J = 5.8 Hz, 2H),
4.08 (dd, J = 8.1, 3.4 Hz, 1H), 3.66 (t, J = 4.5 Hz, 4H),
2.78 (dd, J = 12.6, 3.4 Hz, 1H), 2.70 (t, J = 5.8 Hz, 2H),
2.58 (dd, J = 12.6, 8.1 Hz, 1H), 2.41 (d, J = 5.5 Hz, 9H).
13C NMR (101 MHz, CDCl3) δ 163.20, 150.37, 141.07,
138.84, 137.20, 135.12, 128.96, 128.64, 124.42, 123.61,
122.54, 119.59, 119.23, 117.88, 111.95, 111.26, 68.71,
66.74, 57.79, 53.44, 52.14, 51.36, 45.49, 13.07. HRMS
(ESI) m/z 488.2654 (M+H+), calc. for C28H34N5O3
488.2656.
4.1.5.8. 1-(4-Chlorophenyl)-4-((1-(2-hydroxy-3-(3-morph-
ol Inopropylamino)propyl)-1H-indol-3-yl)methylene)-3-
methyl-1H-pyrazol-5(4H)-one 10h
1H NMR (400 MHz, MeOD) δ 9.75 (s, 1H), 7.93 (dd, J
= 16.0, 7.0 Hz, 4H), 7.64 (d, J = 8.0 Hz, 1H), 7.37 (dd, J =
14.8, 7.7 Hz, 4H), 4.49 – 4.29 (m, 3H), 3.68 (d, J = 4.3 Hz,
4H), 3.29 – 3.19 (m, 2H), 3.11 (ddd, J = 30.5, 17.4, 9.2 Hz,
2H), 2.64 – 2.42 (m, 6H), 2.34 (s, 3H), 1.87 (dd, J = 13.7,
6.7 Hz, 2H). 13C NMR (101 MHz, MeOD) δ 164.53,
152.91, 142.78, 138.93, 138.59, 138.01, 130.52, 129.65,
4.1.5.6. 4-((1-(2-Hydroxy-3-(3-morpholinopropylamino)pr
opyl)-1H-indol-3-yl)methylene)-3-methyl-1-phenyl-1H-
pyrazol-5(4H)-one 10f

125.16, 124.14, 121.31, 119.97, 119.51, A11C3.C75E, P1T12E.3D8, MANUSCRIPT
propyl)-1H-indol-3-yl)methylene)-3-methyl-1-p-tolyl-1H-
67.45, 67.09, 58.44, 54.57, 52.23, 51.27, 49.60, 22.63,
13.07. HRMS (ESI) m/z 536.2421 (M+H+), calc. for
C29H35N5O3Cl 536,2423.
pyrazol-5(4H)-one 10k
1H NMR (400 MHz, MeOD) δ 9.80 (s, 1H), 8.03 – 7.94
(m, 2H), 7.76 (d, J = 8.3 Hz, 2H), 7.65 – 7.59 (m, 1H),
7.36 (p, J = 6.6 Hz, 2H), 7.22 (d, J = 8.2 Hz, 2H), 4.42 (dd,
J = 13.9, 3.6 Hz, 1H), 4.29 (dd, J = 14.0, 7.2 Hz, 1H), 4.25
– 4.19 (m, 1H), 3.66 (t, J = 4.4 Hz, 4H), 2.95 – 2.75 (m,
4H), 2.45 (t, J = 6.2 Hz, 6H), 2.36 (d, J = 4.5 Hz, 6H), 1.81
– 1.69 (m, 2H). 13C NMR (101 MHz, MeOD) δ 164.42,
152.51, 142.94, 138.72, 138.12, 137.65, 135.78, 130.59,
130.23, 125.01, 124.03, 120.92, 119.93, 119.45, 113.61,
112.44, 68.87, 67.60, 58.30, 54.70, 52.86, 52.61, 49.30,
25.27, 21.01, 12.98. HRMS (ESI) m/z 516.2965 (M+H+),
calc. for C30H38N5O3 516.2969.
4.1.5.9. 4-((1-(2-Hydroxy-3-(3-morpholinopropylamino)pr
opyl)-1H-indol-3-yl)methylene)-3-methyl-1(4(trifluorom
ethyl)phenyl)-1H-pyrazol-5(4H)-one 10i
1H NMR (400 MHz, MeOD) δ 9.78 (s, 1H), 8.18 (d, J =
8.5 Hz, 2H), 7.98 – 7.88 (m, 2H), 7.63 (t, J = 7.9 Hz, 3H),
7.36 (dd, J = 11.6, 6.0 Hz, 2H), 4.45 (d, J = 10.6 Hz, 1H),
4.32 (d, J = 10.3 Hz, 2H), 3.67 (d, J = 3.7 Hz, 4H), 3.24 –
3.11 (m, 2H), 3.09 – 2.93 (m, 2H), 2.52 (dd, J = 16.8, 11.1
Hz, 6H), 2.35 (s, 3H), 1.84 (dd, J = 12.6, 6.3 Hz, 2H). 13C
NMR (101 MHz, MeOD) δ 164.96, 153.43, 143.29,
142.95, 138.61, 138.07, 130.53, 126.86, 126.82, 125.16,
124.15, 119.71, 119.48, 119.21, 113.76, 112.40, 67.60,
67.53, 58.43, 54.61, 52.36, 51.71, 49.57, 23.40, 13.14.
HRMS (ESI) m/z 570.2691 (M+H+), calc. for
C30H35N5O3F3 570.2687.
4.1.5.12. 4-((1-(2-Hydroxy-3-(3-morpholinopropylamino)
propyl)-1H-indol-3-yl)methylene)-1-(4-methoxyphenyl)-3-
methyl-1H-pyrazol-5(4H)-one 10l
1H NMR (400 MHz, MeOD) δ 9.80 (s, 1H), 8.02 (d, J =
8.5 Hz, 2H), 7.74 (d, J = 9.0 Hz, 2H), 7.67 (d, J = 8.4 Hz,
1H), 7.39 (p, J = 7.6 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H),
4.46 (t, J = 8.4 Hz, 1H), 4.43 – 4.32 (m, 2H), 3.82 (s, 3H),
3.66 (d, J = 4.4 Hz, 4H), 3.29 – 3.19 (m, 2H), 3.10 (ddd, J
= 27.9, 17.6, 9.4 Hz, 2H), 2.63 – 2.45 (m, 6H), 2.37 (s,
3H), 1.86 (dd, J = 13.3, 6.7 Hz, 2H). 13C NMR (101 MHz,
MeOD) δ 164.28, 158.73, 152.39, 142.69, 138.66, 138.23,
133.20, 130.56, 125.16, 124.14, 122.97, 120.21, 119.56,
114.95, 113.77, 112.38, 67.46, 67.10, 58.41, 55.95, 54.57,
52.18, 51.25, 49.55, 22.63, 12.96. HRMS (ESI) m/z
532.2920 (M+H+), calc. for C30H38N5O4 532.2918.
4.1.5.10. 4-((1-(2-Hydroxy-3-(3-morpholinopropylamino)
propyl)-1H-indol-3-yl)methylene)-3-methyl-1-(3(trifleoro
methyl)phenyl)-1H-pyrazol-5(4H)-one 10j
1H NMR (400 MHz, MeOD) δ 9.80 (s, 1H), 8.44 (s,
1H), 8.19 (d, J = 8.2 Hz, 1H), 8.03 – 7.94 (m, 2H), 7.64 (d,
J = 8.4 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.38 (dt, J = 11.6,
7.8 Hz, 3H), 4.46 (d, J = 10.5 Hz, 1H), 4.36 (dd, J = 19.7,
7.7 Hz, 2H), 3.75 – 3.62 (m, 4H), 3.30 – 3.20 (m, 2H), 3.11
(ddd, J = 30.9, 17.5, 9.3 Hz, 2H), 2.62 – 2.43 (m, 6H), 2.38
(s, 3H), 1.88 (dt, J = 12.7, 6.5 Hz, 2H). 13C NMR (101
MHz, MeOD) δ 164.88, 153.28, 142.93, 140.91, 138.63,
138.16, 130.64, 130.52, 125.19, 124.17, 122.58, 121.46,
121.39, 119.88, 119.51, 115.88, 115.84, 113.81, 112.41,
67.43, 67.14, 58.42, 54.57, 52.20, 51.26, 49.58, 22.62,
13.11. HRMS (ESI) m/z 570.2685 (M+H+), calc. for
C30H35N5O3F3 570.2687.
4.1.5.13. 1-(4-Fluorophenyl)-4-((1-(2-hydroxy-3-(3-mor
pholinopropylamino)propyl)-1H-indol-3-yl)methylene)-3-
methyl-1H-pyrazol-5(4H)-one 10m
1H NMR (400 MHz, MeOD) δ 9.77 (s, 1H), 8.01 – 7.96
(m, 1H), 7.94 (s, 1H), 7.91 (dd, J = 8.9, 4.9 Hz, 2H), 7.65
(d, J = 8.3 Hz, 1H), 7.42 – 7.32 (m, 2H), 7.13 (t, J = 8.7
Hz, 2H), 4.48 – 4.42 (m, 1H), 4.42 – 4.29 (m, 2H), 3.75 –
3.61 (m, 4H), 3.29 – 3.19 (m, 2H), 3.19 – 3.01 (m, 2H),
2.63 – 2.43 (m, 6H), 2.35 (s, 3H), 1.87 (dd, J = 14.3, 6.5
4.1.5.11. 4-((1-(2-Hydroxy-3-(3-morpholinopropylamino)

Hz, 2H). 13C NMR (101 MHz, MeOD) δ 164.40, 162.35,
4.1.6. General procedure for the coupling reaction for
synthesis of compounds(R/S)-10f, (R/S)-10m, and (R/S)-
10n.
ACCEPTED MANUSCRIPT
159.94, 152.69, 142.78, 138.60, 138.15, 136.44, 136.42,
130.52, 125.16, 124.14, 122.36, 122.29, 119.98, 119.51,
116.33, 116.11, 113.75, 112.40, 67.45, 67.09, 58.44, 54.56,
52.22, 51.27, 49.60, 22.63, 13.03. HRMS (ESI) m/z
520.2721 (M+H+), calc. for C29H35N5O3F 520.2718.
To a solution of compounds (R/S)-1, (R/S)-9f and
(R/S)-9g (1.0 mmol) and 4-Aminopropyl morpholine (1.5
mmol) in dried DCM (5 mL) was added AlCl3 (0.1 mmol)
under the N2 atmosphere and the mixture was stirred at
room temperature for 10 h. The reaction mixture was
filtered off and filtrate was removed in vacuo to give a
residue. The residue was purified by column
chromatography on silica gel to give the desired products
(R/S)-10f, (R/S)-10m, and (R/S)-10n.
4.1.5.14. 1-(3-Fluorophenyl)-4-((1-(2-hydroxy-3-(3-morph
olinopropylamino)propyl)-1H-indol-3-yl)methylene)-3-
methyl-1H-pyrazol-5(4H)-one 10n
1H NMR (400 MHz, MeOD) δ 9.80 (s, 1H), 8.04 – 7.94
(m, 2H), 7.81 (dd, J = 17.1, 9.9 Hz, 2H), 7.65 (d, J = 8.3
Hz, 1H), 7.43 – 7.33 (m, 3H), 6.88 (td, J = 8.4, 2.1 Hz,
1H), 4.48 (d, J = 10.3 Hz, 1H), 4.43 – 4.32 (m, 2H), 3.75 –
3.62 (m, 4H), 3.30 – 3.19 (m, 2H), 3.11 (ddd, J = 29.0,
17.5, 9.3 Hz, 2H), 2.65 – 2.44 (m, 6H), 2.37 (s, 3H), 1.95 –
1.79 (m, 2H). 13C NMR (101 MHz, MeOD) δ 164.74,
153.04, 142.80, 138.65, 138.10, 131.22, 131.13, 130.54,
125.18, 124.16, 120.04, 119.53, 115.21, 113.78, 112.39,
111.81, 111.59, 106.85, 106.58, 67.44, 67.12, 58.44, 54.58,
52.20, 51.27, 49.58, 22.63, 13.06. HRMS (ESI) m/z
520.2716 (M+H+), calc. for C29H35N5O3F 520.2718.
4.1.6.1. 4-((1-(2-Hydroxy-3-(3-morpholinopropylamino)
propyl)-1H-indol-3-yl)methylene)-3-methyl-1-phenyl-1H-
pyrazol-5(4H)-one (R)-10f.
1H NMR (400 MHz, CDCl3) δ 9.49 (s, 1H), 7.71 (d, J =
8.0 Hz, 2H), 7.61 (d, J = 7.6 Hz, 1H), 7.44 (s, 1H), 7.39 (d,
J = 7.9 Hz, 1H), 7.26 – 7.08 (m, 5H), 7.02 (t, J = 7.3 Hz,
1H), 4.52 (s, 1H), 4.28 – 4.13 (m, 2H), 3.51 (s, 4H), 2.98
(t, J = 10.2 Hz, 2H), 2.87 (dd, J = 20.9, 9.1 Hz, 1H), 2.79
(dd, J = 12.0, 6.3 Hz, 1H), 2.42 – 2.26 (m, 6H), 2.23 – 2.16
(m, 3H), 1.87 (dd, J = 14.1, 6.7 Hz, 1H), 1.79 – 1.68 (m,
1H). 13C NMR (101 MHz, CDCl3) δ 162.86, 150.30,
140.64, 138.57, 136.98, 134.97, 128.67, 128.58, 124.57,
123.94, 122.69, 119.52, 119.42, 117.73, 112.04, 111.37,
66.24, 65.98, 57.04, 53.26, 50.87, 50.81, 48.61, 21.47,
4.1.5.15. 1-(2-Fluorophenyl)-4-((1-(2-hydroxy-3-(3-morph
olinopropylamino)propyl)-1H-indol-3-yl)methylene)-3-
methyl-1H-pyrazol-5(4H)-one 10o
1H NMR (400 MHz, MeOD) δ 9.75 (s, 1H), 8.12 – 8.01
(m, 2H), 7.73 – 7.64 (m, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.42
(ddd, J = 14.1, 10.4, 5.6 Hz, 3H), 7.31 (t, J = 8.6 Hz, 2H),
4.46 (t, J = 8.4 Hz, 1H), 4.36 (t, J = 8.3 Hz, 2H), 3.73 –
3.59 (m, 4H), 3.23 (dt, J = 12.6, 3.8 Hz, 2H), 3.16 – 2.99
(m, 2H), 2.62 – 2.43 (m, 6H), 2.40 (s, 3H), 1.95 – 1.77 (m,
2H). 13C NMR (101 MHz, MeOD) δ 164.95, 159.74,
157.25, 153.46, 142.91, 139.14, 138.73, 130.78, 130.70,
130.50, 129.29, 125.75, 125.72, 125.29, 124.29, 119.59,
118.56, 117.69, 117.49, 113.96, 112.48, 67.42, 67.04,
58.38, 54.54, 52.17, 51.21, 49.51, 22.60, 12.99. HRMS
(ESI) m/z 520.2719 (M+H+), calc. for C29H35N5O3F
520.2718.
12.99. [α]25 +63.4 (c 0.28, MeOH);HRMS (ESI) m/z
D
502.2811 (M+H+), calc. for C29H36N5O3 502.2813.
4.1.6.2. 4-((1-(2-Hydroxy-3-(3-morpholinopropylamino)
propyl)-1H-indol-3-yl)methylene)-3-methyl-1-phenyl-1H-
pyrazol-5(4H)-one (S)-10f.
1H NMR (400 MHz, CDCl3) δ 9.51 (s, 1H), 7.73 (d, J =
8.0 Hz, 2H), 7.63 (d, J = 7.6 Hz, 1H), 7.46 (s, 1H), 7.41 (d,
J = 7.9 Hz, 1H), 7.28 – 7.10 (m, 5H), 7.04 (t, J = 7.3 Hz,
1H), 4.54 (s, 1H), 4.30 – 4.15 (m, 2H), 3.53 (s, 4H), 3.00
(t, J = 10.2 Hz, 2H), 2.89 (dd, J = 20.9, 9.1 Hz, 1H), 2.81
(dd, J = 12.0, 6.3 Hz, 1H), 2.44 – 2.28 (m, 6H), 2.25 – 2.18
(m, 3H), 1.89 (dd, J = 14.1, 6.7 Hz, 1H), 1.81 – 1.70 (m,

1H). 13C NMR (101 MHz, CDCl3) δ 162.82, 150.26,
4.1.6.5. 1-(3-Fluorophenyl)-4-((1-(2-hydroxy-3-(3-morp
ACCEPTED MANUSCRIPT
140.60, 138.53, 136.94, 134.93, 128.63, 128.54, 124.53,
123.90, 122.65, 119.48, 119.38, 117.69, 112.00, 111.33,
66.20, 65.94, 57.00, 53.22, 50.83, 50.77, 48.57, 21.43,
holinopropylamino)propyl)-1H-indol-3-yl)methylene)-3-
methyl-1H-pyrazol-5(4H)-one (R)-10n.
1H NMR (400 MHz, MeOD) δ 9.80 (s, 1H), 8.04 – 7.94
(m, 2H), 7.81 (dd, J = 17.1, 9.9 Hz, 2H), 7.65 (d, J = 8.3
Hz, 1H), 7.43 – 7.33 (m, 3H), 6.88 (td, J = 8.4, 2.1 Hz,
1H), 4.48 (d, J = 10.3 Hz, 1H), 4.43 – 4.32 (m, 2H), 3.75 –
3.62 (m, 4H), 3.30 – 3.19 (m, 2H), 3.11 (ddd, J = 29.0,
17.5, 9.3 Hz, 2H), 2.65 – 2.44 (m, 6H), 2.37 (s, 3H), 1.95 –
1.79 (m, 2H). 13C NMR (101 MHz, MeOD) δ 164.74,
153.04, 142.80, 138.65, 138.10, 131.22, 131.13, 130.54,
125.18, 124.16, 120.04, 119.53, 115.21, 113.78, 112.39,
111.81, 111.59, 106.85, 106.58, 67.44, 67.12, 58.44, 54.58,
12.95. [α]25 – 43.4 (c 0.32, MeOH);HRMS (ESI) m/z
D
502.2815 (M+H+), calc. for C29H36N5O3 502.2813.
4.1.6.3. 1-(4-Fluorophenyl)-4-((1-(2-hydroxy-3-(3 morph
olinopropylamino)propyl)-1H-indol-3-yl)methylene)-3-
methyl-1H-pyrazol-5(4H)-one (R)-10m.
1H NMR (400 MHz, MeOD) δ 9.75 (s, 1H), 7.99 – 7.95
(m, 1H), 7.93 (s, 1H), 7.89 (dd, J = 8.9, 4.9 Hz, 2H), 7.63
(d, J = 8.3 Hz, 1H), 7.40 – 7.31 (m, 2H), 7.12 (t, J = 8.7
Hz, 2H), 4.47 – 4.41 (m, 1H), 4.41 – 4.28 (m, 2H), 3.74 –
3.60 (m, 4H), 3.28 – 3.18 (m, 2H), 3.17 – 2.99 (m, 2H),
2.62 – 2.42 (m, 6H), 2.33 (s, 3H), 1.86 (dd, J = 14.3, 6.5
Hz, 2H). 13C NMR (101 MHz, MeOD) δ 164.43, 162.38,
159.97, 152.72, 142.81, 138.63, 138.18, 136.47, 136.45,
130.55, 125.19, 124.17, 122.39, 122.32, 120.01, 119.54,
116.36, 116.14, 113.78, 112.43, 67.48, 67.12, 58.47, 54.59,
52.20, 51.27, 49.58, 22.63, 13.06.[α]25 + 36.4 (c 0.34,
D
MeOH);HRMS (ESI) m/z 520.2716 (M+H+), calc. for
C29H35N5O3F520.2718.
4.1.6.6. 1-(3-Fluorophenyl)-4-((1-(2-hydroxy-3-(3-morp
holinopropylamino)propyl)-1H-indol-3-yl)methylene)-3-
methyl-1H-pyrazol-5(4H)-one (S)-10n.
52.25, 51.30, 49.63, 22.66, 13.06. [α]25 + 54.4 (c 0.48,
D
1H NMR (400 MHz, MeOD) δ 9.81 (s, 1H), 8.05 – 7.94
(m, 2H), 7.82 (dd, J = 17.1, 9.9 Hz, 2H), 7.66 (d, J = 8.3
Hz, 1H), 7.44 – 7.34 (m, 3H), 6.89 (td, J = 8.4, 2.1 Hz,
1H), 4.48 (d, J = 10.3 Hz, 1H), 4.44 – 4.32 (m, 2H), 3.76 –
3.63 (m, 4H), 3.30 – 3.20 (m, 2H), 3.12 (ddd, J = 29.0,
17.5, 9.3 Hz, 2H), 2.66 – 2.44 (m, 6H), 2.38 (s, 3H), 1.96 –
1.80 (m, 2H). 13C NMR (101 MHz, MeOD) δ 164.76,
153.06, 142.82, 138.67, 138.12, 131.24, 131.15, 130.56,
125.20, 124.18, 120.06, 119.55, 115.23, 113.80, 112.41,
111.83, 111.61, 106.87, 106.60, 67.46, 67.14, 58.46, 54.60,
MeOH);HRMS (ESI) m/z 520.2715 (M+H+), calc. for
C29H35N5O3F 520.2718.
4.1.6.4. 1-(4-Fluorophenyl)-4-((1-(2-hydroxy-3-(3-morp
holinopropylamino)propyl)-1H-indol-3-yl)methylene)-3-
methyl-1H-pyrazol-5(4H)-one (S)-10m.
1H NMR (400 MHz, MeOD) δ 9.77 (s, 1H), 8.01 – 7.96
(m, 1H), 7.94 (s, 1H), 7.91 (dd, J = 8.9, 4.9 Hz, 2H), 7.65
(d, J = 8.3 Hz, 1H), 7.42 – 7.32 (m, 2H), 7.13 (t, J = 8.7
Hz, 2H), 4.48 – 4.42 (m, 1H), 4.42 – 4.29 (m, 2H), 3.75 –
3.61 (m, 4H), 3.29 – 3.19 (m, 2H), 3.19 – 3.01 (m, 2H),
2.63 – 2.43 (m, 6H), 2.35 (s, 3H), 1.87 (dd, J = 14.3, 6.5
Hz, 2H). 13C NMR (101 MHz, MeOD) δ 164.40, 162.35,
159.94, 152.69, 142.78, 138.60, 138.15, 136.44, 136.42,
130.52, 125.16, 124.14, 122.36, 122.29, 119.98, 119.51,
116.33, 116.11, 113.75, 112.40, 67.45, 67.09, 58.44, 54.56,
52.22, 51.29, 49.60, 22.65, 13.08. [α]25 – 25.4 (c 0.41,
D
MeOH);HRMS (ESI) m/z 520.2720 (M+H+), calc. for
C29H35N5O3F 520.2718.
4.2. Biological evaluation
4.2.1. HCVcc assay
The HCV virus assay was performed using a previously
52.22, 51.27, 49.60, 22.63, 13.03. [α]25 – 46.4 (c 0.43,
D
described method with some modifications.[25]
A
MeOH);HRMS (ESI) m/z 520.2721 (M+H+), calc. for
humanized Renilla luciferase reporter gene was introduced
into the C-terminus of the plasmid of pJFH-1 (a gift from
C29H35N5O3F 520.2718.

Apath, L.L.C) between amino acids 399 aAndC4C00EoPf TNES5DA MANUSCRIPT
This work was supported by the National Basic
in the JFH-1 genome. The resulting plasmid phRluc-JFH-1
was digested with XbaI and used as a template for RNA
transcription. The transcripts were prepared in vitro using
the Ambion MEGAscript Kit; subsequently, 10 ꢀg of RNA
was mixed with 400 ꢀlHuh7.5.1 cells (1×107 cells/ml).
Following electroporation, Huh7.5.1 cells containing virus
transcripts were seeded onto a 10-cm dish. After the cells
were cultured for 10 days and passaged every 3 days, the
supernatant was collected and filtered to obtain the HCV
(JFH-1)-Rluc virus stock solution.
Research Program of China (973 program, Grant No.
2013CB911104), the National Natural Science Foundation
of China (Grant No. 21572116), the Tianjin Science and
Technology Program (Grant No. 13JCYBJC24300,
13JCQNJC13100), the "111" Project of the Ministry of
Education of China (Project No. B06005).
References
[1] C.W. Shepard, L. Finelli, M.J. Alter, Global
epidemiology of hepatitis C virus infection, The
Lancet infectious diseases, 5 (2005) 558-567.
The antiviral activity and cytotoxicity of NINS
derivatives was determined using HCV (JFH-1)-Rluc-
infected Huh7.5.1 cells. Huh7.5.1 cells were seeded in 96-
well plates and incubated at 37 °C with 5% CO2. After 24
h, Huh7.5.1 cells were treated with serially diluted
compounds and infected with the Renilla luciferase-linked
HCV (JFH-1)-Luc reporter virus. The luciferase levels
were quantified by measuring the firefly luciferase activity
in relative luminescence units at 48 h post infection (hpi).
Antiviral activity was determined by Rellia-GloTM
Luciferase Assay System. EC50 and CC50 were calculated
by using the Renilla-GloTM345 Luciferase reagent
(Promega) and a Glomax plate reader (Promega). All
results were presented as the average results of three
independent experiments and SDs (n = 3) are presented.
[2] M.J. Alter, D. Kruszon-Moran, O.V. Nainan, G.M.
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List of captions
Fig. 1. Structures of 1 and a known HCV inhibitor 2.
Scheme 1 Reagents and conditions; a) MeOH, yield 80%; b) Piperidine, n-BuOH, reflux, yield 90%; c)
NaH, DMSO, yield 70%; d) AlCl3, amines, DCM, yield 50%;
Table 1 The structures of NINS derivatives 1, 9a-m, and their in vitro activities against HCV.
Table 2 The structures of NINS derivatives 10a-o, and their in vitro activities against HCV.
Table 3 The structures of pure enantiomers R/S-10f, m, n and their in vitro activities against HCV.
Fig. 2. Evaluation of HCV entry and RNA replication in the presence of 1 (a) and 10m (b). Huh7.5.1 cells
(3×104 per well in 100 µl 10% FBS-DMEM medium) were cultured at 37 °C under 5% CO2 in 96-well
plates overnight. Subsequently, the cells were treated with 5 µM 1, and 2 µM 10m either concurrent with
100 TCID50 of HCV(JFH-1)-Luc virus (0 h) or at intervals of -6, -4, -2, 0, 1, 2, 4, 6, 8 and 10 h post
infection (hpi),which 0 hpi indicated supplying inhibitors immediately after virus infection. After
incubation at 37 °C for 24 h, luciferase activity was determined by Rellia-GloTM Luciferase Assay System.
Mock: control culture experiment (0.5% DMSO control).

ACCEPTED MANUSCRIPT
Highlights:
1. Indole derivatives were discovered as HCV inhibitors.
2. SAR studies resulted in the most potent and the least cytotoxic compound (R) -10m.
3. R-enantiomer showed better anti-HCV activity compared with S-enantiomer.
4. NINS derivatives interfere with the viral entry step.