2-((4-Arylpiperazin-1-yl)methyl)benzonitrile Derivatives as Orally Available Inhibitors of Hepatitis C Virus with a Novel Mechanism of Action

Article abstract of DOI:10.1021/acs.jmedchem.0c00232

Although the direct-acting antivirals revolutionized the hepatitis C virus (HCV) infection treatment in the last decade, more efforts are needed to reach the elimination of HCV in the absence of a vaccine. 4-(Piperazin-1-yl)-2-((p-tolylamino)methyl)-benzonitrile (1) is a modest HCV inhibitor identified from an in-house screening using a HCV-infected Huh7.5 cell culture. Starting from it, the chemical optimization afforded a new 2-((4-arylpiperazin-1-yl)methyl)benzonitrile scaffold with significantly increased antiviral activity against HCV. A highly effective HCV inhibitor, 35 (L0909, EC50 = 0.022 μM, SI > 600), was identified by the structure-activity relationship study. The biological study revealed that L0909 could block HCV replication by acting on the HCV entry stage. The high sensitivity to clinical resistant HCV mutants and synergistic effect with clinical drugs were observed for this compound. The further pharmaceutical studies demonstrated that L0909 is long-lasting, is orally available, and has low toxicity in vivo. These results show L0909 as a promising HCV entry inhibitor for single or combinational therapeutic potential.

Full text of DOI:10.1021/acs.jmedchem.0c00232

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Article
2-((4-Arylpiperazin-1-yl)methyl)benzonitrile Derivatives as Orally
Available Inhibitors of HCV With a Novel Mechanism of Action
Xinbei Jiang, Jiali Tan, Yixuan Wang, Jinhua Chen, Jianrui LI, Zhi Jiang, Yanni Quan,
Jie Jin, Yu-Huan Li, Shan Cen, YANPING LI, Zong-Gen Peng, and Zhuo-Rong Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00232 • Publication Date (Web): 07 May 2020
Downloaded from pubs.acs.org on May 12, 2020
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2
-((4-Arylpiperazin-1-yl)methyl)benzonitrile Derivatives as
Orally Available Inhibitors of HCV With a Novel Mechanism of
Action
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†
,§
†,§
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†,+
†,‡
†
Xinbei Jiang, Jiali Tan, Yixuan Wang, Jinhua Chen, Jianrui Li, Zhi Jiang, Yanni
†
†
†,‡
†
*,†
*,†,‡
Zhuorong Li,*,†
Quan, Jie Jin, Yuhuan Li, Shan Cen, Yanping Li, Zonggen Peng,
†
CAMS Key Laboratory of Antiviral Drug Research, Institute of Medicinal Biotechnology,
Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050,
‡
China;
Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal
Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College,
Beijing 100050, China
KEYWORDS 2-((4-arylpiperazin-1-yl)methyl)benzonitrile, SAR, HCV entry inhibitor,
synergistic effect, bioavailability.
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ABSTRACT: Although the direct-acting antivirals revolutionized the HCV infection
treatment in last decade, more efforts are need to reach the elimination of HCV in the
absence of vaccine. 4-(Piperazin-1-yl)-2-((p-tolylamino)methyl)-benzonitrile (1) is a
modest HCV inhibitor identified from an in-house screening using HCV-infected Huh7.5
cells culture. Starting from it, the chemical optimization afforded a new 2-((4-
arylpiperazin-1-yl)methyl)benzonitrile scaffold with significantly increased antiviral
activity against HCV. A highly effective HCV inhibitor 35 (L0909, EC = 0.022 μM, SI >
0
1
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00) was identified by the SAR study. The biological study revealed that L0909 could
block HCV replication by acting on the HCV entry stage. The high sensitivity to clinical
resistant HCV mutants and synergistic effect with clinical drugs were observed for this
compound. The further pharmaceutical studies demonstrated L0909 is long-lasting, oral
available and low toxic in vivo. These results endowed L0909 a promising HCV entry
inhibitor for single or combinational therapeutic potential.
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INTRODUCTION
Hepatitis C virus (HCV) is an enveloped plus-stranded RNA virus, which belongs to the
family Flaviviridae and genus Hepacivirus. HCV infection is a major cause of chronic liver
disease, which may develop into chronic liver fibrosis, cirrhosis and even to hepatocellular
carcinoma. There are still 71 million HCV-infected patients globally and 1.75 million
individuals are newly infected with HCV in 2015 and an estimated 390,000 people died
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from HCV infection. The availability of all-oral and interferon-free direct-acting antivirals
(
DAAs) revolutionized the HCV infection treatment in last decade.2, 3 Peptidomimetics,
benzimidazole dimerics, and nucleoside analogs are major chemotypes that targeting HCV
4
-6
nonstructural (NS) protein 3/4A, 5A or 5B, respectively. For each type of DAAs, there
have been multiple new drugs (Table 1) approved by the US Food and Drug
7
Adiministration (FDA) and European Medicines Agency (EMA). The HCV DAA
combinations strongly inhibited HCV replication with over 95% of sustained virologic
response (SVR) rates and higher resistant barrier.8-10 However, a prophylactic vaccine
1
1
against HCV infection is still unavailable now. Due to the error-prone nature of the HCV
RNA polymerase, HCV will unavoidably develop drug-resistance during the treatment
with DAAs, even with the highly effective and pan-genotypic fixed-dose combination of
drugs.12, 13 Moreover, virus clearance by DAAs could not produce adaptive immunity to
HCV reinfection, which increased the risk of reinfection among people who inject drugs
1
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prisoners, and HIV-infected individuals. Therefore, development of new antivirals with
different antiviral mechanisms of action is still important to optimize current therapeutic
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regimens for increasing the overall SVR in the Real World and accelerating the global
eradication of HCV.
HCV subgenomic replicon system with deletion of HCV structural genes made an
important contribution to high-throughput screening of HCV replication inhibitors and the
success of DAAs.15-17 In comparison, the virus-infected cell-based approach contains a full
virus infectious cycle and allows the discovery of new molecules which targeting the virus
entry, package or release steps. Based on the spontaneous and efficient replication of the
full-length HCV genome JFH-1, the high-titer infectious HCV cell culture (HCVcc) system
was established for research and development of both vaccines and drugs against HCV.18,
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JFH-1 became the standard clone for in vitro studies of HCV, and then several chimeras
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of other genotypes were constructed based on the backbone of JFH-1. Besides of GS4.3
cells, a human hepatoma Huh-7 cell line carrying an HCV subgenomic replicon, we also
used the HCVcc system with the chimeric genotype 2a (J6/JFH-1) to investigate the anti-
HCV activity of synthetic compounds with expectation to find some molecules with novel
mechanism of action (MoA) from that of DAAs.21,
22
4-(piperazin-1-yl)-2-((p-
tolylamino)methyl)-benzonitrile (1, Figure 2) was identified from our in-house screening
as a moderate HCV inhibitor in HCVcc system assay while it was not active in GS4.3
replicon assay. This indicated that its antiviral activity depends on the different MoA from
the existing HCV drugs. Encouraged by this feature, we further performed a deep chemical
optimization taken 1 as a hit. Here we described the design, synthesis, and SAR (structure-
activity relationship) study of a new class of 2-((4-arylpiperazin-1-yl)methyl)benzonitriles
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against HCV propagation for the first time. Meanwhile, the pharmacological and
preclinical evaluation led to the identification of a highly effective, low toxic, and oral
available HCV entry inhibitor.
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RESULTS AND DISCUSSION
Design of new 2-((4-arylpiperazin-1-yl)methyl)benzonitrile scaffold with antiviral
activity against HCV in HCVcc system. In order to evaluate the substitution effect of hit
compound 1 on the anti-HCV activity, a small set of 4-substituted-2-
(phenylaminomethyl)benzonitrile analogs 8–13 (Figure 1) was firstly designed and
synthesized by bioisosteres replacement strategy. The 50% effective concentration (EC )
5
0
of these compounds was initially calculated by determining the HCV RNA level in HCVcc
using a real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
method. The 50% cytotoxic concentration (CC ) was determined by MTT method in naïve
5
0
4
Huh7.5 cells culture. As shown in Figure 1, N -methylation of piperazine ring afforded the
comparable antiviral activity (8, EC = 1.62 µM) to the naked NH group (1, EC = 1.68
5
0
50
µM). However, when the piperazin-1-yl was changed into the morpholin-4-yl (9) or
4
piperidin-1-yl (10), the antiviral activity was lost. These results indicated the N atom of
piperazine made the obvious contribution to the antiviral activity. In addition, para-chloro
substitution on aniline ring (12, EC = 0.42 µM) is more active than the methyl (8, EC
5
0
50
= 1.62 µM) or methoxy substitution (11, EC = 2.14 µM). Meanwhile, comparing to 8,
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both antiviral activity and the selectivity index (SI) was dramatically increased when the
NH group of aniline was methylated (13a, EC = 0.89 µM, SI = 92) and the piperazine
5
0
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was replaced by a flexible chain of alkyldiamine (13b, EC = 0.18 µM, SI = 86). Therefore,
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beneficial to antiviral potency.
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Unfortunately, poor bioavailability (F < 5%) was observed for 8 and 13b when either of
which was administered to rats by oral gavage (Supporting information). We estimated that
the low bioavailability was resulted from the scaffold but not the substituted groups.
Therefore, we attempted to insert a piperazine between benzonitrile and aniline group of
1
3b to afford a new 2-((4-arylpiperazin-1-yl)methyl)benzonitrile scaffold (Figure 1). In the
meanwhile, a SAR (structure-activity relationship) study upon the new scaffold was
performed to discover novel preclinical candidate compounds.
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Figure 1. The chemical optimization from hit compound 1 led to a new 2-((4-
arylpiperazin-1-yl)methyl)benzonitrile scaffold. The antiviral activity was determined in
HCV-infected Huh7.5 cells. EC was calculated at RNA level detected with qRT-PCR
5
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and CC was detected with MTT assay. F, bioavailability.
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Chemistry. As shown in Scheme 1A, starting from commercially available 4-fluoro-2-
methylbenzonitrile, bromination of methyl group was done using N-Bromosuccinimide
(NBS) in the presence of catalytic amount of p-toluenesulfonic acid (p-TSA) in CCl to
4
afford benzyl bromide intermediate 2 by yield above 80%. Then potassium carbonate
(
K CO ) mediated nucleophilic substitution of 2 with different aromatic amine in dimethyl
2 3
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sulfoxide (DMSO) at room temperature afforded the intermediate 3–7. The resulted
mixture was directly underwent nucleophilic aromatic substitution (SNAr) reaction in one-
pot
with
corresponding
alkylamine
to
afford
4-substituted-
2-
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(phenylaminomethyl)benzonitrile analogs 8–13 in the presence of K CO in DMSO at
2
3
1
20°C by yield range of 42–64% (Synthesis procedure was shown in Supporting
information).
Scheme 1. Preparation procedure of compounds 8–13 and 32–64a
A
CN
CN
CN
CN
Br
R2
R2
a
b1
N
c
N
F
R3
F
CH3
F
R1
R1
2
3 R1 = Me, R2 = H
R1 = OMe, R2 = H
5 R1 = Cl, R2 = H
R1 = Me, R2 = Me
8 R1 = Me, R2 = H, R3 = 4-methyl-piperazin-1-yl
9 R1 = Me, R2 = H, R3 = morphorlin-4-yl
10 R1 = Me, R2 = H, R3 = piperidin-1-yl
11 R1 = OMe, R2 = H, R3 = 4-methyl-piperazin-1-yl
12 R1 = Cl, R2 = H, R3 = 4-methyl-piperazin-1-yl
4
6
7 R1 = Cl, R2 = Me
13a R1 = Me, R2 =Me, R3 = 4-methyl-piperazin-1-yl
13b R1 = Cl, R2 = Me, R3 = dimethylaminoethyl(methyl)amino
B
N
CN
N
R1
N
N
CN
CN
Br
R1
CN
a
b2
c
F
N
N
F
CH3
F
32-43
CN
2
Cl
1
4 R1 = H
20 R1 = 4-CN
1
1
1
1
1
5 R1 = 4-Me
6 R1 = 4-OMe
7 R1 = 4-Cl
8 R1 = 4-Br
9 R1 = 4-F
21 R1 = 4-NO2
R2
N
N
22 R1 = 2-F-4-Cl
23 R1 = 2,4-2Cl
24 R1 = 2-Me-4-Cl
25 R1 = 2-NO2-4-Cl
b2
46-60
CN
N
N
CN
R3
X
c
R3
X
F
N
N
R4
R4
14-19
44, 45, 61-64
2
2
2
2
3
3
6, 44 X = N, R3 = 2-chloropyridin-5-yl, R4 = H
7, 45 X = N, R3 = pyridin-4-yl, R4 = H
8, 61 X = CH, R3 = 4-chlorophenyl, R4 = H
9, 62 X = N, R3 = 4-chlorophenyl, R4 = Me
0, 63 X = N, R3 = 4-chlorobenzyl, R4 = H
1, 64a X = N, R3 = Boc, R4 = H
d
64 X = N, R3 = H, R4 = H
a
Reagents and conditions: (a) NBS, p-TSA, CCl , reflux; (b1) substituted aniline, K CO ,
4
2
3
DMSO; (b2) substituted piperazine or piperidine, K CO , Acetone or ACN; (c) primary or
2
3
second amine, K CO , DMSO, 120-140°C; (d) 2M HCl in ether, methanol.
2
3
New 2-((4-arylpiperazin-1-yl)methyl)benzonitrile compounds were easily prepared
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from 2 by the similar method in Scheme 1A. Firstly, different substituted piperazine or
piperidine was reacted with 2 in the presence of K CO in acetone or acetonitrile (ACN) to
2
3
afford the intermediates 14–31 in satisfied yield about 70%. Then, the SNAr reaction of
1
1
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intermediates 14–27 with N ,N ,N -trimethyl-1,2-ethyldiamine was performed under
heating or microwave irradiation at 120–140°C in the presence of K CO in DMSO to
2
3
afford diverse analogs 32–45 in the moderate yield about 40% (Scheme 1B). These analogs
were designed and synthesized to investigate the effect of R at benzene ring or benzene
1
self on anti-HCV activity. Similarly, compounds 46–60 were prepared by reaction of
intermediate 17 with different nucleophilic amines in similar yield as 32–45 in order to
compare the superiority of R . In addition, analog 61 and 62 were designed and synthesized
2
through intermediates 28 and 29 in which the piperazine ring was replaced by piperidine
or 3-methyl-piperazine, respectively. Compound 63 was correspondingly obtained from
the reaction of 2 with 4-chlorobenzylpiperazine and the following SNAr reaction through
1
1
2
intermediate 30 with N ,N ,N -trimethyl-1,2-ethyldiamine. Title compounds 64 were
designed to investigate the contribution of phenyl group to the antiviral potency.
Compound 64 was obtained in the presence of hydrochloride acid in methanol from the
deprotection of Boc group from 64a, which was prepared through the intermediate 31.
Scheme 2. Synthesis of compound 65a
N
N
CN
N
CN
N
NO2
a
NH2
N
N
N
N
39
65
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6
a
Reagents and conditions: (a) SnCl , HCl, methanol.
2
In Scheme 2, reduction of nitro compound 39 conveniently afforded corresponding
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
amino product 65 in the presence of stannous chloride and hydrochloric acid in methanol.
In Scheme 3, compound 35 was hydrolyzed in the presence of hydrogen peroxide and
sodium hydroxide in ethanol to afford amide derivative 66 by yield of 36%.
Scheme 3. Synthesis of compound 66a
N
N
CN
N
CONH2
N
Cl
a
Cl
N
N
N
N
35
66
a
Reagents and conditions: (a) NaOH, H O , ethanol/DMSO.
2
2
Taking 4-fluoro-3-formylbenzonitrile (67) as start material, NaBH CN mediated
3
reductive amination of aldehyde with 1-(4-chlorophenyl)piperazine afforded intermediate
1
1
2
68 by yield of 78%. The followed SNAr reaction of 68 with N ,N ,N -trimethyl-1,2-
ethyldiamine afforded a 3-(4-arylpiperazin-1-yl)methylbenzonitrile derivative 69 by yield
2
3
of 36.1% (Scheme 4). .In Scheme 5, reaction of 1-(bromomethyl)-3-iodobenzene (70)
with 1-(4-chlorophenyl)piperazine easily afforded the intermediate 71 by yield of 79%,
1
1
2
which reacted with N ,N ,N -trimethyl-1,2-ethyldiamine by Ullmann coupling in the
presence of K CO , L-proline and CuI afforded cyano-absent compound 72 by yield of
2
3
4
1%.24
Scheme 4. Synthesis of compound 69a
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9
Cl
CN
Cl
CN
F
CN
F
N
a
b
N
N
N
N
CHO
N
67
68
69
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) 1-(4-chlorophenyl)piperazine, NaBH CN, acetic acid,
3
1
1
2
methanol; (b) N ,N ,N -trimethylethane-1,2-diamine, K CO , DMSO, 120°C.
2
3
Scheme 5. Synthesis of compound 72a
Cl
Cl
N
a
b
N
N
N
N
Br
N
I
I
70
71
72
a
Reagents and conditions: (a) 1-(4-chlorophenyl)piperazine, K CO , acetone; (b)
2
3
1
1
2
N ,N ,N -trimethylethane-1,2-diamine, K CO , L-proline, CuI, DMSO, 90°C.
2
3
SAR study of 2-((4-arylpiperazin-1-yl)methyl)benzonitrile derivatives. In order to
facilitate the rapid screening of a large number of compounds, we employed an In-Cell
Western analysis instrument, which could automatically quantify the intracellular HCV
protein level in 96-well plate, to determine the inhibitory effect of newly synthetic
2
5
compounds on viral replication in HCVcc assay. In-Cell Western analysis is much more
labor-saving and cost-effective than classical qRT-PCR method. Both telaprevir (VX-950)
and sofosbuvir was taken as positive drugs in this assay. Novel 2-((4-
substitutedphenylpiperazin-1-yl)methyl)benzonitrile derivatives 32–45 as well as 65 were
firstly designed and synthesized to investigate the substituent effect of Aryl group on
antiviral potency when Aryl is benzene ring. These derivatives maintained the same
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dimethylaminoethyl(methyl)amino side chain on the para-site of benzonitrile ring.
Table 1. In vitro inhibitory activity of 2-cyanobenzyl piperazine derivatives against
HCV replication in Huh7.5 cellsa
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CN
N
N
N Aryl
N
Compd.
Aryl
EC (μM)
CC (μM)
SI
5
0
50
1
3
3
3b
2
/
0.506±0.220
0.902±0.315
0.399±0.169
1.435±0.294
0.083±0.085
0.104±0.047
0.749±0.156
1.869±1.454
0.314±0.253
6.427±3.566
15.498±2.538
50.219±31.293
36.195±50.873
45.536±25.585
12.761±4.908
13.315±3.362
52.104±34.318
9.054±6.815
30.6
55
90
31
154
128
70
4








3
Me
34
OMe
3
3
3
3
5
6
7
8
Cl
Br
F
CN
NO2
NH2
39
12.831±4.810
55.231±41.138
40
8
6
4
5
0

F
0.205±0.102
0.166±0.131
0.203±0.173
13.154±4.443
9.849±6.073
10.091±5.664
64
59
49

Cl
Cl
Cl
Cl

4
4
1
2
Me

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9
O N
2
4
4
3
4
0.217±0.292
12.538±4.483
52.618±33.381
57
21

Cl
N
2.511±0.486


Cl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
45
N
2.737±0.653
0.146±0.040
0.104±0.031
44.708±26.494
55.024±27.164
>200
16
VX-950
Sofosbuvir
377
>1917
a
The EC50 and CC50 values were indicated as mean ± SD values which were calculated from three independent
experiments. The EC50 was determined by In-cell western method with treatment time of 72 h.
As shown in Table 1, lipophilic substituents such as methyl (33, EC = 0.399 µM) and
5
0
halogen substitution (EC = 0.082, 0.104, and 0.749 µM for 35-chloro, 36-bromo, and 37-
5
0
fluoro, respectively) afforded enhanced activity comparing to unsubstituted derivative 32
EC = 0.902 µM). Among them, 35 demonstrated the most superior selectivity index (SI)
(
5
0
of 154. Moreover, the obvious decreased activity of 4-fluoro derivatives than chloro and
bromo-substitution indicated that the halogen bond played a role to the antiviral potency.
Conversely, those derivatives with lipophobic substituents like methoxy, cyano, or amino
group exhibited weaker potency (EC = 1.435, 1.869, and 6.427 µM for 32, 38, and 65,
50
respectively) in comparison to 32. Surprisingly, compound 39 with substitution of strong
electron withdrawing nitro group exerted comparable strong antiviral activity (EC =
5
0
0
.314 µM) as methyl substitution compound 33. When the benzene was changed into
pyridine, the antiviral activity of corresponding derivatives was decreased of more than 20-
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
fold comparing to 35 no matter the chloro-substitution was reserved (44) or not (45). Based
on above results, we designed to introduce another substituent into C2-position of the 4-
chlorophenyl fragment with expectation to further increase the efficacy. However, the
higher EC and lower SI values of these derivatives (40–43) were observed comparing to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
3
5 although the selected substituent was validated as beneficial one in para-position of
benzene ring. Therefore, we intended to maintain the 4-chlorophenyl for Aryl group within
the chemical structure of subsequent compounds.
When we fixed the scaffold as shown in Table 2, a series of novel 2-((4-
chlorophenyl)piperazin-1-yl)methylbenzonitrile derivatives (46–60) was designed and
synthesized to explore the influence of para-substituent on the benzonitrile on the antiviral
activity. Comparing to dimethylaminoethyl(methyl)amino group of 35, more rigid cyclic
piperazine group (46, 47) decreased the activity by 5–6 folds. When ethyl-1,2-diamine was
lengthened into propyl-1,3-diamine (48), a 3-fold more decreased antiviral activity was
observed. However, the activity of compound 49 is between 35 and 48, which probably
indicated a requirement for the length between two nitrogen atoms of R group. The activity
2
was almost lost when ethyl-1,2-diamino was replaced by dimethylamino 50 or
methylbutylamino 51. This indicated that the terminal N atom of alkyldiamino group is
important for antiviral potency. The removal of methyl group from the initial N of ethyl-
1,2-diamine (52) or replacing this N atom with O atom (53) resulted in a folds of decreasing
in both antiviral activity and SI value, which might reveal the role of basicity of this N
atom. Meanwhile, the comparison among 54–56 demonstrated that the bulky substituents
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on the terminal N atom would damage the biological activity. The removal of methyl
substitution from the terminal N atom of ethyl-1,2-diamine led to a slightly decreased
potency (57, EC = 0.289±0.156 µM) against HCV replication in Huh7.5 cell culture.
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
However, modifying the terminal N atom of R into the amide (58, 59) significantly
2
reduced the antiviral activity. Hence, we referred that the nucleophilicity of terminal N
atom is beneficial to the drug-target interaction. Interestingly, the antiviral activity was
regained when six-membered piperazine (47, EC = 0.783±0.292 µM) was replaced with
5
0
seven-membered diazacycle (60, EC = 0.153±0.057 µM).
5
0
Table 2. In vitro inhibitory activity of 4-substituted 2-((4-(4-chlorophenyl)piperazin-
1
-yl)methyl)benzonitriles against HCV replication in Huh7.5 cellsa
Cl
CN
N
N
R2
Compd.
R2
EC (μM)
CC (μM)
SI
50
50
3
5
 N
N
0.125±0.094
0.657±0.401
0.783±0.292
0.423±0.078
0.318±0.042
57.519±26.727
13.918±3.923
13.749±4.889
16.103±3.262
15.576±2.199
13.477±4.017
>200
111
20
20
36
42
>3
46
 N
 N
NH
4
4
4
5
7
8
9
0
N
N
N

 N
N
N

H
N
5
1
118.954±31.514
>200
>1

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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H
N
5
5
2
3
0.952±0.102
0.814±0.193
14.819±4.735
16.607±2.170
15
20


N
O
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
N
5
4


N
0.454±0.224
7.641±2.997
16
H
N
5
5
5
5
5
6
5
6
7
8
9
0
N
4.694±1.141
13.705±6.934
0.289±0.156
79.380±36.266
7.293±2.295
0.153±0.057
76.309±34.987
>200
16
>14
54
>2
5
O
N
H
N


N
15.806±3.003
>200
N
H
O
NH
 N
O
N
40.006±16.199
12.501±5.547

N
N
81
N

a
The EC50 and CC50 values were indicated as mean ± SD values which were calculated from three independent
experiments. The EC50 was determined by In-cell western method with treatment time of 72 h.
Replacement of the piperazine with either piperidine (61) or 3-methylpiperazine (62)
didn’t afford a favorable potency comparing to the corresponding 35 (Table 3). In addition,
the obvious decreased antiviral activity of compound 63 and 64 comparing to 35 also
demonstrated that 4-phenylpiperazine fragment was crucial to the high potency against
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HCV. Alternatively, hydrolysis of cyano into amide (66) and the removal of cyano (72) led
to a dramatic drop of antiviral potency. As we predicted, both the activity and SI of
compound 69 was low when the 4-phenylpiperazine fragment was shifted from ortho- into
meta-position of benzonitrile.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. In vitro inhibitory activity of compounds 61–64, 66, 69, and 72 against HCV
replication in Huh7.5 cellsa
Compd.
Chemical structure
EC (μM)
CC (μM)
SI
5
0
50
CN
N
N
N
Cl
3
6
6
6
6
6
5
1
2
3
4
6
N
N
0.125±0.094
0.142±0.115
0.321±0.166
1.051±0.354
13.918±3.923
3.750±1.650
13.727±4.368
15.638±3.331
111
26
42
14
>5
13
CN
N
N
N
Cl
CN
N
N
Cl
N
Cl
CN
N
N
N
N
CN
N
N
NH
35.011±11.124 >200
N
CONH2
N
N
N
Cl
N
1.155±0.872
1.326±0.468
0.936 ±0.408
15.485±1.936
CN
6
7
9
2
14.612±2.373
13.552±4.912
11
14
N
N
N
Cl
N
N
N
N
Cl
N
H
a
The EC50 and CC50 values were indicated as mean ± SD values which were calculated from three independent
experiments. The EC50 was determined by In-cell western method with treatment time of 72 h.
The inhibitory effect on HCV RNA level in HCVcc assay. To further evaluate the
anti-HCV potential of this new chemotype, the intracellular HCV RNAs were quantified
with qRT-PCR method after treatment of the HCVcc cells with active compounds 35 and
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
36. As shown in Table 4, the calculated EC against HCV RNA content was 22, 31 and
5
0
4
6 nM for compound 35, 36, and positive drug sofosbuvir, respectively. 35 exhibited the
most potent activity among the three test compounds. However, a dramatically higher SI
was presented for sofosbuvir than our synthetic compounds because the cytotoxicity of
sofosbuvir was overwhelmingly low (CC > 200 μM). Notably, compound 35 and 36
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
significantly inhibited HCV RNA synthesis in a dose-dependent manner (Figure 2).
Moreover, both of them exerted the stronger suppression on HCV RNA at lower
concentration level than sofosbuvir. In view of drug-like ability, we chose compound 35
as a potential candidate for further study.
Table 4. The effect of compound 35 and 36 at HCV RNA level in Huh7.5 cellsa
Compd.
CC (μM)
EC (μM)
SI
50
50
3
5
14.77±2.07
15.60±1.00
>200
0.022±0.005
0.031±0.007
0.046±0.014
671
36
503
Sofosbuvir
> 4347
a
The EC50 and CC50 values were indicated as mean ± SD values which were calculated from three independent
experiments. The EC50 was determined by qRT-PCR method with treatment time of 72 h.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Compound 35 and 36 possessed high potency against HCV RNA replication in
Huh7.5 cells. Intracellular HCV RNA was extracted and detected by qRT-PCR method
after treatment of HCV-infected cells for 72 h with test compounds or reference drug
sofosbuvir.
Investigation the effect of compound 35 (L0909) on HCV life cycle in Huh7.5 cells.
The altered scaffold of compound 35 (named L0909 hereafter) from original hit 1 made us
perform a further investigation to confirm its effectiveness in HCV replicon system. When
we treated GS4.3 HCV replicon cells with L0909 in a serial of concentrations for 72 h, no
inhibition was observed for all treatment groups, even at a concentration of 5.0 μM while
the reference drug VX-950 (telaprevir), an HCV NS3 protease inhibitor, showed a strong
inhibitory effect (Figure 3A). Moreover, L0909 was ineffective in HCV replicon assay
even though it was treated for 14 days at concentration of 0.1 or 0.5 μM (Figure 3B). These
results suggested that L0909 might not interrupt the intracellular HCV RNA replication in
the replicon GS4.3 cells. The mechanism of L0909 against HCV replication is different for
those of known DAAs, and might involve the viral attachment / entry steps or late stages
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in the HCV life cycle.
A
B
0
1
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0
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. L0909 was ineffective against HCV replication in HCV replicon (GS4.3) cells
after exposure time for 72 h (A) or 14 days (B). Intracellular proteins were extracted and
detected with Western Blot assay, and VX-950 was taken as a positive control. The protein
bands showed the results of a representative experiment. The data presented are mean ±
standard deviation. Student’s t-test was used. *p< 0.05 and **p < 0.01 vs solvent control.
For further investigating which stages of HCV life cycle was interrupted by L0909,
Huh7.5 cells were treated with L0909 prior to, during or after HCV viral incubation (Figure
4A). At 72 h, the proteins were detected with Western Blot. Firstly, pre-treatment of L0909
is ineffective (Figure 4B). The most dramatic anti-HCV activity was observed when L0909
was simultaneously administered during the inoculation of HCV (Figure 4C).
Comparatively, post-treatment only induced relative weak effect on HCV replication
(Figure 4D). The results suggested that L0909 might act on the early stage of the viral life
cycle or virus entry stage.
A
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0
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9
0
B
C
D
Figure 4. Time-of-addition experiment of L0909 in HCV-infected Huh7.5 cells. (A)
Schematic illustration of the experiment. (B-D) Huh7.5 cells were treated with L0909 at 2
h prior to infection with HCV (B), during the inoculation of HCV (C), or 2 h after HCV
infection (D). The proteins were detected with Western Blot at 72 h. The protein bands
showed the results of a representative experiment. The data presented are mean ± standard
deviation. Student’s t-test was used. *p< 0.05 and **p < 0.01 vs solvent control.
To further confirm above putative target step, the antiviral effect was investigated when
Huh7.5 cells were infected with HCV and simultaneously treated with L0909 for 4 h.
Consequently, L0909 exerted a significant anti-HCV efficacy in a dose-dependent manner
(Figure 5A). However, VX-950 did not inhibit HCV replication. Furthermore, when
shortened the incubation time less than 2 h to 15 minutes, L0909 (1.0 μM) still displayed
strong anti-HCV activity (Figure 5B). This further suggested that L0909 might block the
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HCV replication at the stage of viral entry into cells.
A
B
0
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0
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0
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2
3
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6
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8
9
0
1
2
3
4
5
6
7
8
9
0
C
Figure 5. L0909 interrupted HCV replication at viral entry stage. Huh7.5 cells were
infected with HCV and simultaneously incubated with the corresponding compound for 4
h (A) or incubated with compound (1.0 μM) for the indicated time (B). The supernatant
was removed and replaced by fresh culture medium at the indicated time, and intracellular
proteins were detected by In-Cell Western at 72 h. L0909 inhibited genotype 1b (C, left)
and 2a (C, right) HCVpp entry into Huh7.5 cells in a dose-dependent manner. *p < 0.05
and **p < 0.01 vs solvent control.
We also tested the inhibitory activity of L0909 in HCV pseudoparticle (HCVpp) model.
HCVpp is a viral-like particle with HCV glycoproteins incorporated into the viral envelope
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and thus only the early steps of virus entry are similar to that in the HCVcc system.26
2
7
Dasatinib is an HCV entry inhibitor targeting EphA2 protein. Dasatinib showed slight
inhibitory activity against genotype 1b and 2a HCVpp (Figure 5C) while the negative
control telaprevir (VX-950) showed no activity in the HCVpp system even at a
concentration of 10 μM (Figure 5C, left). L0909 exhibited the inhibitory effect on HCVpp
infection in a dose-dependent manner in two HCVpp assays (Figure 5C, left and right),
suggesting that L0909 indeed is an HCV entry inhibitor.
1
1
1
1
1
1
1
1
1
1
2
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0
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9
0
1
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4
5
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8
9
0
To explore whether L0909 would directly affect the cell-free virions to abolish
subsequent HCV infection, HCV viral stock was pre-incubated with L0909 for 2 h and
then incubated with Huh7.5 cells, intracellular proteins were extracted and quantified by
western blot at 72 h. However, the additional pre-incubation of L0909 with HCV virions
did not significantly affect the HCV infectivity (Figure 6A), suggesting that L0909 might
not directly inactivate the viral particles.
Besides, to further investigate the mechanism of action of L0909, we carried out a
2
8
resistance selection experiment. Wild-type (WT) HCV-infected cells were treated with
L0909 by the stepwise increasing concentrations from 0.02 μM to 5.0 μM. Over a period
of 10 weeks passages, L0909-resistant HCV was induced. After sequencing, we found a
potential mutant F291V (T1211G in genome) in HCV structural glycoprotein E1 (Figure
6B), which plays a key role in the process of HCV entry into cells. Meanwhile, synonymous
nucleotide mutation in Core G511A and E2 G2000C, T2246C in genome were also
detected. In order to precisely identify whether the F291V mutant is associated with drug-
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resistance, F291V site mutant plasmid was constructed accordingly and the mutant
infectious virus was prepared. Then the antiviral activity of L0909 was detected using qRT-
PCR. Indeed, L0909 displayed an approximate 20-fold increasing of EC value against
5
0
0
1
2
3
4
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6
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9
0
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2
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0
1
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9
0
mutant virus comparing to that against WT virus (Figure 6C), suggesting that F291V
mutation will impact the antiviral activity of L0909. The results indicated that L0909
inhibited the HCV glycoprotein E1-mediated entry.
A
B
C
Figure 6. L0909 inhibited HCV glycoprotein E1-mediated entry. (A) L0909 did not
inactive HCV particle. Huh7.5 cells were incubated with HCV viral stock that was pre-
treated with 1 μM of L0909 for 2 h following diluted 20 times leading to a concentration
of 0.05 μM of L0909. As controls, HCV viral stock was treated with 0.05 μM or 1 μM of
L0909. Intracellular proteins were extracted and quantified by western blot at 72 h. (B)
Schematic diagram of amino acid residues in glycoprotein E1 in wild type (wtJFH1/E1F291
)
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and L0909-resistant (mtJFH1/E1F291V) virus. (C) Huh7.5 cells were incubated with wild-
type (WT) or F291V mutant virus and simultaneously treated with L0909, the intracellular
RNA was detected with qRT-PCR at 72 h.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To determine whether L0909 is a broad-spectrum antiviral agent or not, we tested the
inhibitory activity of L0909 towards other viruses, such as Influenza virus, Coxsakie virus,
and Zika virus, of all which infection process include viral entry steps in cell culture models.
As Table 5 indicated, L0909 only displayed modest antiviral potency against Coxsakie and
Zika viruses at micromole concentration. Moreover, no inhibition was found when L0909
was administered to Influenza virus. The data indicated that L0909 is a specific HCV entry
inhibitor.
Table 5. Inhibitory activity of L0909 against Influenza, Coxsakie, and Zika virus in
vitroa
Virus
Compound
L0909
CC (μM)
EC (μM)
SI
5
0
50
4.28
> 2.47
2.14
7.41
0.002
5.15
33
/
Influenza A
Oseltamivir
L0909
> 640
22.22
>1.05
18.39
> 200
> 299
3
Coxsakie B3
Zika
Pleconaril
L0909
> 525
3
Ribavirin
> 6
a
The EC50 was detected with CPE method and calculated with Reed-Muench method.
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Inhibitory effect on mutant HCV variants. Distinct mechanism of action from DAAs
might endow L0909 less inclination of cross-resistance with current DAAs. We further
confirmed this assumption by measuring the EC value of L0909 in both wildtype and
5
0
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
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0
1
2
3
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9
0
drug-resistant HCV infected cell cultures. A156T and D168V mutants in the NS3 protein,
and S282T mutant in the NS5B protein are commonly HCV variants resistant to VX-950,
simeprevir, and sofosbuvir, respectively. As expected, L0909 kept similar potency against
these drug-resistant viruses to the wild-type one (Table 6).
Table 6. The high potency of L0909 against drug-resistant HCV mutantsa.
EC (μM)
5
0
Drugs
WT
A156T
0.0019
1.213
D168V
S282T
L0909
0.003
0.03
0.0044
VX-950
0.0078
0.0092
0.0294
Simeprevir
Sofosbuvir
>1
0.1479
a
the inhibitory activity was detected with qRT-PCR and calculated with Reed-Muench method. WT, Wild-Type.
Synergistic efficacy of L0909 with DAAs. The antiviral therapy needs the combination
use of different type of antiviral agents due to high frequency of viral mutation. As a viral
entry inhibitor, the antiviral mechanism of L0909 is different from current DAAs. Taking
simeprevir (NS3/4A protease inhibitor), daclatasvir (NS5A inhibitor) and sofosbuvir
(NS5B polymerase inhibitor) as the representatives for current DAA types, we investigated
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their antiviral activity of both single use of L0909 (0.1 μM) and combining with these
DAAs by measuring the HCV core protein level. The q value calculated by Webb method
between single-drug treatment and combinational treatment was 1.05, 1.27 and 1.08 for
simeprevir, daclatasvir and sofosbuvir, respectively (Figure 7). It suggested that L0909
possessed the synergistic anti-HCV activity when combining with these DAAs.
1
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1
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1
1
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9
0
A
B
C
Figure 7. L0909 enhanced the inhibitory activities against HCV of DAAs. HCV-infected
Huh7.5 cells were treated with L0909 (0.1 μM) combined with 0.025 μM of simeprevir
(A), 0.1 μM of sofosbuvir (B), or 0.016 nM of daclatasvir (C). At 72 h, intracellular proteins
were analyzed with Western Blot assay. *p<0.05, **p<0.01, combination group vs single
#
##
L0909 group; p<0.05, p<0.01, combination group vs single positive drug group.
In vivo pharmacokinetic study. To investigate the pharmacokinetics (PK) of L0909 in
vivo, we determined the plasma drug concentration at a serial of time points after single
dose of L0909 were administered to Sprague−Dawley (SD) rats or beagle dogs by vein
injection (iv) or oral gavage (po) (Figure 8). L0909 could be well absorbed by both rats
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and dogs with Tmax of 3 h after oral dose (Table 7). The oral Cmax value of L0909 after
administration at 15 mg/kg dose to rats or dogs, was 500.72 ng/mL and 94.51 ng/mL,
respectively, which is 54 or 10-fold of the antiviral EC value of L0909 (EC = 22 nM)
5
0
50
0
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0
in vitro. Meanwhile, the in vivo elimination of L0909 was slow with long half-life time
T ) of more than 10 h for both rats and dogs. Moreover, the clearance (Cl) of L0909 was
(
1
/2
significantly slower in rats than in dogs. Although the clearance of L0909 was much
quicker in the dogs comparing to in rats, the AUC value in dogs was acceptable due to its
higher V value. The bioavailability (F) of L0909 was calculated as 59% and 39%,
z
respectively, for rats and dogs (Table 7). To be noted, the plasma concentration of L0909
in both rats and dogs was still higher than the in vitro EC value at 24 h after oral
5
0
administration. Moreover, L0909 was still observed in rats with a concentration (C =
7
2
1
3.66 ng/mL) comparable to the in vitro EC value at 72 h after treatment.
50
Rat 5 mg/kg (iv)
Rat 15 mg/kg (po)
Dog 5 mg/kg (iv)
Dog 15 mg/kg (po)
6
5
00.00
00.00
400.00
3
2
1
00.00
00.00
00.00
0
.00
0
10
20
30
40
50
60
70
80
Time (h)
Figure 8. The concentration-time curve of L0909 in SD rats and beagle dogs after
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administration by tail vein injection (iv) or oral gavage (po).
Table 7. PK parameters of L0909 in rats and dogsa
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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0
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0
Animal
Parameters
Dose
T1/2
Rats (n = 12)
Dogs (n = 6)
Unit
mg/kg
h
iv
po
iv
po
5
15
5
15
14.34
/
12.83
3.08
7.28
12.72
3
Tmax
h
/
Cmax
ng/mL
616.99
500.72
9589.46
531.93
1072.46
94.51
1275.09
238116.97
12025.86
15.62
0.69
AUC (0-72h)
Vz
h × ng/mL 5415.2
mL/kg
mL/h/kg
ng/mL
ng/mL
%
20057.05
34834.07 49181.42
CL
1088.11
76.96
9.2
2246.3
118.78
13.66
4752.98
C24h
6.16
/
C72h
F
59
39
a
the parameters were calculated according to non-compartment model. F is bioavailability.
In vivo toxicity. To determine the safety of L0909 in vivo, normal Kunming mice were
divided into 4 groups (n = 6) and treated with blank solvent or single dose (250, 500, or
000 mg/kg) of L0909 by oral gavage for each group and were followed up for 7 days.
1
None of the mice died and the body weight did not change in the L0909 treated groups
(Figure 9A). Blood samples were collected at the end of the experiment and examined for
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