Novel benzidine and diaminofluorene prolinamide derivatives as potent hepatitis C virus NS5A inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.06.033

Our study describes the discovery of a series of highly potent hepatitis C virus (HCV) NS5A inhibitors based on symmetrical prolinamide derivatives of benzidine and diaminofluorene. Through modification of benzidine, l-proline, and diaminofluorene derivatives, we developed novel inhibitor structures, which allowed us to establish a library of potent HCV NS5A inhibitors. After optimizing the benzidine prolinamide backbone, we identified inhibitors embedding meta-substituted benzidine core structures that exhibited the most potent anti-HCV activities. Furthermore, through a battery of studies including hERG ligand binding assay, CYP₄₅₀ binding assay, rat plasma stability test, human liver microsomal stability test, and pharmacokinetic studies, the identified compounds 24, 26, 27, 42, and 43 are found to be nontoxic, and are expected to be effective therapeutic anti-HCV agents.

Full text of DOI:10.1016/j.ejmech.2015.06.033

European Journal of Medicinal Chemistry 101 (2015) 163e178
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel benzidine and diaminofluorene prolinamide derivatives as
potent hepatitis C virus NS5A inhibitors
,
,
Il Hak Bae a 1, Hee Sun Kim b 1, Youngsu You a, Chieyeon Chough a, Weonu Choe a,
Min Kyung Seon a, Seung Gi Lee c, Gyochang Keum d, Sung Key Jang c *, B. Moon Kim a
,
, *
a Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, South Korea
b Department of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, South Korea
c Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, South Korea
d Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791,
South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Our study describes the discovery of a series of highly potent hepatitis C virus (HCV) NS5A inhibitors
Received 24 April 2015
Received in revised form
14 June 2015
Accepted 15 June 2015
Available online 24 June 2015
based on symmetrical prolinamide derivatives of benzidine and diaminofluorene. Through modification
of benzidine, L-proline, and diaminofluorene derivatives, we developed novel inhibitor structures, which
allowed us to establish a library of potent HCV NS5A inhibitors. After optimizing the benzidine proli-
namide backbone, we identified inhibitors embedding meta-substituted benzidine core structures that
exhibited the most potent anti-HCV activities. Furthermore, through a battery of studies including hERG
ligand binding assay, CYP450 binding assay, rat plasma stability test, human liver microsomal stability
test, and pharmacokinetic studies, the identified compounds 24, 26, 27, 42, and 43 are found to be
nontoxic, and are expected to be effective therapeutic anti-HCV agents.
Keywords:
HCV
NS5A inhibitor
Benzidine
© 2015 Elsevier Masson SAS. All rights reserved.
Diaminofluorene
Structureeactivity relationship
1. Introduction
3 to 4 million global population every year [1e5]. It has been
estimated that around 70e80% of those infested with HCV will
Hepatitis C virus (HCV) infection is reported in almost 170
million individuals worldwide (~2.4% of the population), including
approximately 5 million people in the United States with additional
progress to chronic hepatitis, which, if left untreated, may result in
liver cirrhosis and eventually liver cancer, with ultimately lethal
consequences (1e5%) [6,7]. It is well accepted that the HCV infec-
tion is one of the main reasons for liver transplantation in patients
[8]. Although vaccines are available for infection with other wide-
spread liver viruses such as hepatitis A virus (HAV) and hepatitis B
virus (HBV), no vaccines are available for HCV infection [8].
Recently, telaprevir (VX-950, Vertex Pharmaceuticals and Johnson
& Johnson) and boceprevir (Victrelis, Merck) were approved as
anti-HCV NS3-4A protease inhibitors by the US Food and Drug
Administration (FDA). The traditional therapy for patients with HCV
infection consists of oral doses of ribavirin (RBV) in combination
Abbreviations: HCV, hepatitis C virus; Peg-IFNa, pegylated interferon a; SVR,
sustained virological response; GT1a, hepatitis C virus genotype 1a, a subtype of
hepatitis C virus genotype 1; GT1b, hepatitis C virus genotype 1b, a subtype of
hepatitis C virus genotype 1; DAA, direct-acting antiviral; NS5A, hepatitis C virus
nonstructural protein 5A; pM, picomolar; RNA, ribonucleic acid; Boc, tert-butox-
ycarbonyl; TFA, trifluoroacetic acid; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide; DIPEA, N,N-diisopropylethylamine; DCM, dichloromethane; DMF, N,N-
dimethylformamide; NFSI, N-fluorobenzenesulfonimide; LiHMDS, lithium hexam-
with subcutaneous injections of PEGeIFNea and protease inhibitor
ethyldisilazide; EC50, concentration at which inhibition is half-maximal; mM,
for a total duration of 24e28 weeks [9e11]. However, this
interferon-centered therapy not only has serious side effects,
including anemia, rash and depression, but is also associated with a
restricted sustained virologic response (SVR), notably in those
infected with HCV genotype 1 (G-1) [12,13]. In G-1-infected pa-
tients, favorable results have been reported after the addition of
micromolar; nM, nanomolar; SAR, structureeactivity relationship; hERG, the hu-
man ether-a -go-go-related gene; CYP450, cytochrome P450; PK, pharmacokinetics;
HPBCD, (2-hydroxypropyl)-b-cyclodextrin; IV, intravenous; p.o., orally; WSTs, wa-
ter-soluble tetrazolium salts; DNA, deoxyribonucleic acid.
* Corresponding authors.
E-mail addresses: sungkey@postech.ac.kr (S.K. Jang), kimbm@snu.ac.kr (B. Moon
Kim).
1
These two individuals contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.06.033
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

164
I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
either boceprevir or telaprevir to the standard of care [14].
Although promising, advantageous outcome is expected from the
launching of direct acting antivirals (DAAs) into the therapeutic
regimen, their conceivable limitations include a limited genetic
barrier, which may be attributable to drug-resistant mutants
developed through long term medical care [10,15e20]. Therefore,
the discovery of safe and effective antiviral candidates aimed at
diverse HCV gene targets is greatly needed [21e28].
A polyprotein of almost 3200 amino acids is encoded from the
HCV genome, which contains three structural proteins (E1, E2, and
core) and six nonstructural proteins (NS2, NS3, NS4A-4B, and
NS5A-5B) [29e32]. Among the nonstructural proteins, NS5A has
been shown to have a direct role in viral replication, virus assembly,
virion production, virus persistence, and pathogenesis [33,34].
There are three domains in NS5A: domain 1 (37e213 residues),
containing the zinc-binding shape essential for HCV RNA replica-
tion; domain 2 (250e342 residues), which cooperates with cellular
proteins and NS5B; and domain 3 (356e447 residues), which has a
role in infectious HCV assembly, however not HCV RNA replication
[35e43].
carbamate L-valine moiety as a capping group (Fig. 2) [69]. In 2012,
Schinazi et al. reported nascent NS5A inhibitors containing a part of
biaryl core and some modifications on other parts through “Click”
and CeC bond cross coupling reactions [70,71]. We recently
developed a new class of NS5A inhibitors represented by BMK-
20113, which has benzidine (I) and L-proline (II) connected as an
amide functionality and a variety of capping groups (III) [72]. In this
paper, we report improvements in antiviral potency through the
introduction of new modifications to the backbone of BMK-20113:
L-proline to other proline isosteres (area I in Fig. 2), and benzidine
to substituted benzidine derivatives (area II in Fig. 2).
2. Chemistry
The symmetry in the benzidine-proline scaffold greatly
streamlined our strategy of synthesizing BMK-20113. Initially, we
wanted to evaluate the antiviral activity of the proline variation;
In 2010, a landmark NS5A inhibitor, daclatasvir (1), was reported
to present excellent anti-HCV activity, especially in patients with
HCV G-1 infection. This new class of inhibitor was approved by the
US FDA in 2014 [44e48]. The effective concentration (EC50) value of
daclatasvir was two-digit picomolar (pM) range in in vitro assay,
and treatment with a single 100 mg dose in clinical trials reduced
HCV RNA levels by an average of 3.3log10 without apparent toxicity
[49]. This result stimulated numerous research groups and phar-
maceutical companies to focus on the development of new in-
hibitors targeting NS5A [50e59]. Currently, there are many
candidate compounds in this series: ABT-267, ACH-2928, ACH-
3102, AZD-7295, BMS-346, BMS-665, BMS-824393, EDP-239, GS-
5885, GSK-2336805, IDX-719, MK-4882, MK-8742, PPI-461, and
PPI-1301 (Fig. 1) [60e67]. Most recently, interferon-free multi-class
drug combinations (daclatasvir and asunaprevier, Viekira Pak®, and
Harvoni®) have been reported as the most optimal therapy [68].
The structure of daclatasvir is characterized by a central biaryl core
unit linked to an imidazole and proline moiety, and lastly, a methyl
Fig. 2. Strategy for designing HCV NS5A inhibitors.
Fig. 1. Structure of NS5A inhibitors.

I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
165
therefore,
oxo- -proline,
-methyl- -proline, by using the standard peptide coupling proto-
L
-proline isosteres were employed, such as
D
-proline, 4-
with an 80% yield [76]. The nitro groups of both 30 and 31 were
efficiently reduced to amino functionality per our recent report on
commercially available Fe3O4 nanoparticles as a heterogeneous
catalyst for nitro group reduction in combination with hydrazine
monohydrate [77]. Extremely high yields (99%) of the desired dia-
mino compounds 32 and 33 were obtained. Moreover, a reasonable
yield (76%) of the diamino ketone compound 35 was obtained by
oxidizing the 9H-fluorene-2,7-diamine in the presence of a strong
base (Cs2CO3) in DMSO in open air [78].
L
L
-thioproline, -pipecolic acid, -pipecolic acid, and
L
D
a
L
col of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) in
dichloromethane (DCM). After purification of intermediates 2e8
through chromatographic columns, the Boc groups of the second-
ary amine site was removed with diluted trifluoroacetic acid (TFA)
in DCM [73]. Then methoxycarbonyl phenylglycine 9 was coupled
with the free amine with the aid of EDCI and N,N-diisopropyle-
thylamine (DIPEA) in DCM. In cases where solubility was a persis-
tent problem, we used N,N-dimethylformamide (DMF) as a solvent
[74,75]. The targeted benzidine proline derivatives 10e16 were
then prepared in low to reasonable yields (13e57%) (Scheme 1).
Next, structureeactivity relationship studies centered on the
benzidine core by employing various substituted benzidine com-
pounds were carried out using o- or m-substituted benzidine de-
rivatives (o-methyl, m-methyl, m-trifluoromethyl, m-fluoro, m-
chloro, and m-bromo). After a coupling reaction, almost pure N-
Boc-protected prolinamide products 17e22 were obtained in
excellent yields (92e97%). Using the same method described above,
capping groups were incorporated and the final products 23e28
were obtained in low to good yields (32e73%) (Scheme 2).
For the construction of the fluorene derivative core structures,
we first needed to synthesize 2,7-diaminofluorene derivatives
(Scheme 3). After the treatment of 2,7-dinitrofluorene with 1.00 M
lithium hexamethyldisilazide (LiHMDS) followed by N-fluo-
robenzenesulfonimide (NFSI) in tetrahydrofuran (THF), we ob-
tained 9,9-difluoro-2,7-dinitro-9H-fluorene 30 in 86% yield.
Dimethyl derivative was synthesized likewise, treating 2,7-
dinitrofluorene with NaOt-Bu followed by iodomethane in DMF
to furnish the desired 9,9-dimethyl-2,7-dinitro-9H-fluorene 31
After preparation of various 2,7-diaminofluorene derivatives,
inhibitors containing L-proline and the capping group were pre-
pared using the method described in Scheme 1. N-Boc-protected
fluorine prolinamides 36e39 were thus obtained in high yields
(91e97%), obviating column chromatographic purification. Depro-
tected amines and coupling of the (R)-phenylglycine derivative
gave us the desired fluorene derivatives 40e43 in moderate yields
(50e66%) (Scheme 4).
3. Results and discussion
To determine the inhibitory activities (EC50 values) of each
compound, we used assays based on the HCV cell culture system
(HCVcc) and HCV replicon system [79e81]. To determine EC50
values in the HCVcc system (genotype 2a), we used a modified JFH1
clone (JFH 5a-Rluc-ad34) containing the Renilla luciferase gene
(Rluc) in the NS5A region and cell culture adaptive mutations in the
E2 and p7 region [82]. Huh7.5.1 cells were infected with JFH5a-
Rluc-ad34 for 4 h. After infection, Huh7.5.1 cells were incubated
with compounds of interest at various concentrations for 3 d. Then,
the cells were harvested and luciferase activities were measured.
We determined EC50 values using the HCV replicon system to
Scheme 1. Synthesis of benzidine and proline derivatives from an amide skeleton. Reagents and conditions: (a) N-Boc-proline derivatives, EDCI, DCM, 31e99%; (b) TFA, DCM; (c) 9
(Capping Group), EDCI, DIPEA, DCM or DMF, 13e57% (2 steps).

166
I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
R1
H
R2
CH3
H
17 23
CH3
CF3
F
18 24
19 25
20 26
21 27
22 28
H
H
Cl
H
Br
H
Scheme 2. Synthesis of benzidine derivatives and prolinamide skeleton. Reagents and conditions: (a) N-Boc-L-Proline, EDCI, DCM, 92e97%; (b) TFA, DCM; (c) 9 (capping group),
EDCI, DIPEA, DCM, 32e73% (2 steps).
Scheme 3. Synthesis of fluorene-2,7-diamine derivatives. Reagents and conditions: (a) N-fluorobenzenesulfonimide, LiHMDS in THF (1.00 M solution), THF, 86%; (b) iodomethane,
NaOt-Bu, DMF, 80%; (c) Fe3O4, NH2NH2$H2O, DMF, 99%; (d) Cs2CO3, DMSO, 76%.
CH2
C=O
CMe2
CF2
36 40
37 41
38 42
39 43
Scheme 4. Synthesis of fluorine-containing prolinamide derivatives. Reagents and conditions: (a) N-Boc-L-Proline, EDCI, DCM, 91e97%; (b) TFA, DCM; (c) 3 (capping group), EDCI,
DIPEA, DCM 50e66% (2 steps).
0
evaluate the inhibitory activities of the synthesized compounds on
HCV replication. HCV replicon systems (genotype 1b) containing
HCV NS3e5B and the Renilla luciferase gene allow for productive
viral RNA replication in cell culture systems. HCV replicon-
containing cells were cultivated in the presence of serially diluted
compounds for 3 d. Cells were harvested after treatment with in-
hibitors for 3 d, and luciferase activities were measured. Luciferase
activities were normalized to those obtained from mock-treated
cells and EC50 values were calculated using a Sigma Plot analysis
based on luciferase activities in the cells. The antiviral activities
(EC50) of these compounds against the HCV GT-2a and GT-1b are
listed in Tables 1e3.
of the proline derivatives (Table 1). The EC50 s of our previously
reported inhibitor containing
1, compound 10), against GT-2a and GT-1b were reported to be 260
and 28 pM, respectively. The inhibitory activities of the -isomer 11
were 6500-fold and 2900-fold lower than those of the -epimer,
respectively, against the GT-2a and GT-1b genes (Table 1, entry 2).
Installation of a ketone at the 4-position of -proline 12 resulted in a
L-proline, namely BMK-20113 (entry
D
L
L
similar GT-2a inhibitory activity (EC50: 0.26 nM), and a slightly
decreased GT-1b inhibitory activity (EC50: 0.044 nM) compared to
those of the L-proline derivatives (Table 1, entry 3). When L-thio-
proline was incorporated into the benzidine 13, the activity was
slightly decreased in GT-2a, but there was a similar potency in GT-
1b (EC50 of GT-2a and GT-1b: 15.5 nM and 0.027 nM, respectively;
The SAR data indicates that the inhibitory activities of these
compounds are significantly dependent upon the structural feature
Table 1, entry 4). The inhibitor containing a six-membered L-

I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
167
Table 1
Structureeactivity relationships of inhibitors containing various proline isosteres against HCV type 2a and type 1b.
Entry
1
Compounds
R
Proline
-proline
HCVCC EC50 (type 2a) (nM)
0.26
Replicon EC50 (type 1b) (nM)
0.028
Cytotoxicity (mM)
10
L
>25
2
3
11
12
D
-proline
1770
0.26
83
>25
>25
4-oxo-
L-proline
0.044
4
5
6
7
13
14
15
16
L
-thioproline
15.5
180
419
3.3
0.027
2.1
>25
>25
>25
>25
L-pipecolic acid
D
-pipecolic acid
370
a
-methyl-
L-proline
2.2
pipecolic acid moiety 14 showed lower activity than its five-
membered ring counterpart (Table 1, entry 5). The opposite ste-
pronounced decrease in inhibitory activities (EC50 values all in the
nanomolar range; Table 1, entry 6). Lastly, the antiviral activities of
reoisomeric
D-pipecolic acid derivative 15 exhibited an even more
the a-methyl substituted L-proline derivative 16 were lower than
Table 2
Structureeactivity relationships of inhibitors containing substituted benzidine derivatives against HCV type 2a and type 1b.
Entry
Compound
R1
R2
HCVCC EC50 (type 2a) (nM)
Replicon EC50 (type 1b) (nM)
Cytotoxicity (mM)
1
2
3
4
5
6
7
10
23
24
25
26
27
28
H
H
CF3
CH3
F
H
CH3
H
H
H
0.26
193
0.025
0.32
0.16
0.01
0.028
7.4
>25
>25
>25
>25
>25
>25
>25
0.0025
0.035
0.005
0.007
0.007
Cl
Br
H
H
0.01

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I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
Table 3
Kinetic resolution of compound 24.
Entry
Compound 24
HCVCC EC50 (type 2a) (pM)
Replicon EC50 (type 1b) (pM)
Cytotoxicity (mM)
1
2
Isomer 1
Isomer 2
332
24.6
27.9
2.4
>25
>25
those of compound 10 (EC50 of GT-2a and GT-1b: 3.3 nM and
2.2 nM, respectively; Table 3, entry 7).
bis(trifluoromethyl)benzidine was successfully performed through
the use of a chiral preparative column on supercritical fluid chro-
matography (SFC) [96]. Then, each isomer of compound 24 was
synthesized using each enantiomeric diamine from the separation
following the method described in Scheme 2. Its effect on HCV
proliferation was measured. It was found that isomer 1 of com-
pound 24 showed a 13-fold less potent anti-viral activity than
isomer 2, which exhibited a similar activity to that of racemic 24
(Table 3, entries 1e2, and Table 2, entry 3, respectively). Therefore,
we concluded that the isomer 2 of 24 is the active inhibitor of HCV.
When we tested the anti-viral activities of compounds 23e28,
meta-substitution of the benzidine prolinamides made the in-
hibitors highly potent HCV inhibitors. Therefore, we envisioned
that the activity of m-,m-connected benzidine, i.e., fluorene de-
rivatives would exhibit high potency in the antiviral assays. Based
on the positive results of previously reported NS5A inhibitors
containing a fluorenyl core structure [66], we synthesized and
investigated inhibitors with a fluorenyl core. We were extremely
pleased to observe that these inhibitors exhibited exceptionally
high potencies, particularly in the replicon (GT-1b) assay (Table 4,
entries 1e4). Inhibitory activities (EC50) of compound 40 containing
the parent fluorene were 5.6 and 9 pM against GT-2a and GT-1b,
respectively (Table 4, entry 1). The flurorenone-containing inhibi-
tor 41 exhibited a similar potency to compound 40; EC50 of GT-2a
and GT-1b were 4.3 and 4 pM, respectively (Table 4, entry 2).
Furthermore, substituents at the methylene position of the fluorine
core showed a dramatic increase in anti-viral activity against the
GT-2a gene [97]. The inhibitory activity of compound 42 with a
dimethyl-substituted fluorene moiety was 2800-fold higher against
the GT-2a gene than that of compound 40 containing the parent
fluorene (EC50 of GT-2a and GT-1b: 20 and 4 pM, respectively;
Table 4, entry 3). Replacement of the dimethyl with difluorine 43
showed a similar effect (EC50 of GT-2a and GT-1b: 59 and 3 pM,
respectively; Table 4, entry 4) [98]. All of the inhibitors listed in
Next, after fixing the L-proline and capping groups at both ends,
we investigated the SAR studies of various benzidine-containing
derivatives by varying the middle biaryl portion (area I in Fig. 2).
We first tested the o-methyl substituted benzidine derivative 23 for
inhibitory effects and observed that low activities against the HCV
(EC50 of GT-2a and GT-1b: 193 nM and 7.4 nM, respectively; Table 2,
entry 2). However, a series of m-substituted benzidine derivatives
24e28 exhibited more enhanced inhibitory activities against HCV.
In detail, the EC50 of CF3-substituted compound 24 in GT-2a and GT-
1b were 25 and 2.5 pM, respectively (Table 2, entry 3), and the
values for CH3-substituted compound 25 were 320 and 35 pM,
respectively (Table 2, entry 4). After noticing the strong inhibitory
effect of the meta-substituted compound, we decided to explore a
heteroatom substitution in the m-position of the benzidine core
[83e86]. All three inhibitors containing m-fluoro-, m-chloro- and
m-bromo-substituted benzidine cores (compounds 26, 27, and 28,
respectively) exhibited powerful antiviral activities. EC50 values
against GT-2a and GT-1b were 160 and 5 pM, respectively, for
compound 26 (Table 2, entry 5) and 10 and 7 pM for compounds 27
and 28 (entries 6 and 7, respectively).
To see whether compound 24 has an additive or synergistic anti-
viral effect in combination with an NS5B polymerase inhibitor
(sofosbuvir), one of the top 10 blockbuster drugs in 2014 [87e91],
we treated NK/R2AN replicon cells with serially diluted compound
24 in the presence of an EC30 concentration (123 nM) of sofosbuvir,
and the luciferase activities in the cells were measured. Our data
demonstrated that the combination therapy of compound 24 and
sofosbuvir has a remarkably positive effect against HCV replicon
cells, probably due to the independent mechanisms of action of the
dual treatment [92e95] (Fig. 3).
Since compound 24 contains a core structure that can exist as
atropisomers, we were curious about the potentially different
inhibitory activities of each isomer. The separation of racemic 2,20-
Table 1, Table 2, Table 3, and Table 4 were not cytotoxic at 25 mM.
Since the compounds 24, 26, 27, 41, and 43 exhibited high
inhibitory activities both in the HCV GT-2a and the replicon (GT-1b)
systems (Table 2, entries 3, 6, 7, and Table 3, entries 3, 4, respec-
tively), we next focused on further evaluation of these selected
compounds [99]. First, to evaluate any probable cardiac toxicity, we
performed a hERG ligand binding assay, examining inhibition of the
inner rectifying voltage-gated fluorescence polarization potassium
ion channel which is encoded by hERG gene [100,101]. Compared to
astemizole, which was used as a control (99.9% inhibition at 10
the values of compounds 24, 26, 27, 42, and 43 (7.98%, 1%, 5.80%,
25.7%, 39.1% inhibition at 10 M, respectively) suggested that these
mM),
m
inhibitors bound poorly to the hERG membrane preparations,
reflecting minimal possible cardiac toxicity (Table 5).
Next, when the compounds were tested against rat plasma, our
data showed a high in vitro stability for compound 24, which was
unscathed after 2 h (99% of the compound remained; Table 6, entry
1), whereas after incubating for 30 min, compounds 26 and 27 had
Fig. 3. Effect of compound 24 in combination with EC30 of NS5B polymerase inhibitor
(sofosbuvir) on the HCV GT-1b replicon system. Measurements were carried out in
triplicate.

I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
169
Table 4
Structureeactivity relationship studies of inhibitors containing a fluorene skeleton against HCV type 2a and type 1b.
Entry
Compound
X
HCVCC EC50 (type 2a) (nM)
Replicon EC50 (type 1b) (nM)
Cytotoxicity (mM)
1
2
3
4
40
41
42
43
CH2
5.6
4.3
0.020
0.059
0.009
0.004
0.004
0.003
>25
>25
>25
>25
C]O
CMe2
CF2
Table 5
respectively; Table 8). However, there should still be a large safety
margin when we compare these inhibitions to their anti-viral ac-
tivities [106]. In summary, compounds 24, 26, 27, 42, and 43 do not
significantly inhibit these five representative CYP enzymes
(Table 8) [107].
Studies on the pharmacokinetics (PK) of 5 selected compounds
were carried out in rats, with the vehicles consisting of 5% DMSO,
10% solutol, and 85% (2-hydroxypropyl)-b-cyclodextrin (HPBCD)
Results of hERG ligand binding assay.a
Entry
Compound
% Inhibition (10 mM)
1
2
3
4
5
6
24
26
27
42
7.98 4.49
<1
5.80 3.22
25.7 4.90
39.1 2.93
99.9
43
Control (astemizole)
(Table 9). Despite high potencies in in vitro studies of HCV inhibi-
tion, the selected compounds provoked some concern due to the
symmetrical natures of their structures and their high molecular
weights, which might lead to undesirable PK properties [108e114].
Our data showed that the highest maximum concentration in
a
Fluorescence polarization assay.
Table 6
Stability in rat plasma (% remaining).
plasma (Cmax) was obtained with compound 42 (0.98
through oral administration, and compound 43 (18.64
m
m
mol/mL)
mol/mL)
Entry
Compound
0.5 h (%)
1 h (%)
2 h (%)
1
2
3
24
26
27
93
65
72
99
61
75
99
63
70
through intravenous administration (Table 9, entries 5 and 9,
respectively). The half-lives (t1/2) of these two compounds upon p.o.
administration were 1.7e6.4 h, and in IV administration were
0.9e2.9 h, respectively. When we checked the oral bioavailability (F
%), their low solubility and high lipophilicity contributed to a
moderate oral absorption and bioavailability [115,116].
degraded slightly (Table 6, entries 2 and 3). Consequently, it was
necessary to carry out additional stability tests.
Then, we tested some of our selected inhibitors (42 and 43)
against daclatasvir resistant mutants (L31V and Y93H) as described
in Materials and Method [18]. In vitro transcribed resistant mutant
RNAs (L31V and Y93H) were individually transfected to Huh7.5.1
cells [117,118]. After 4 h after transfection, the culture media were
replaced with DMEM media containing serially diluted compounds.
By comparing the luciferase activities of 42 and 43 with those of
daclatasvir, we concluded that 43 had a higher activity than 42 and
a similar anti-viral potency to daclatasvir, to single mutant viruses.
Therefore, we concluded that 43 could be a solution to the resis-
tance problems of current HCV drugs (Fig. 4).
In the human liver microsomal stability test, compound 24 was
diminished 21% in the plasma after 0.5 h (Table 7, entry 1). Com-
pounds 26, 27, 42, and 43 had lower levels of degradation after 0.5 h
(Table 7, entries 2, 3, 4, and 5, respectively). These results indicate
that these promising compounds had high microsomal stability in
the human liver [102,103].
Next, we outlined CYP450 inhibitory profiles to evaluate any
drugedrug interactions possible with the selected compounds 24,
26, 27, 42, and 43. In general, high EC50 values for the CYP450
enzyme inhibitor indicate decreased liability for drugedrug in-
teractions [104,105]. Our selected compounds exhibited low inhi-
bition profiles for various CYP450 isoforms. CYP1A2, CYP2D6,
CYP2C19, and CYP3A4 were rather weakly inhibited in the presence
of compounds 24, 26, 27, 42, and 43, while the inhibition was
Then, we tested the cytotoxicity of compound 43 against some
eukaryotic cells [119]. The cell counting kit-8 assay method was
used to measure the survival rate of the cells by using WST-8
higher for CYP2C9 (0.14, 0.0877, 0.116, 0.269, and 0.277
mM
Table 8
a
EC50 of compounds (mM) against various subtypes of CYP450.
Table 7
Entry
Compound
1A2
2C9
2D6
2C19
3A4
Human liver microsomal stability (% remaining).
1
2
3
5
4
24
26
27
42
43
78.8
>100
23.9
>100
>100
0.660
0.0877
0.116
0.269
0.277
>100
4.94
41.2
>100
>100
4.64
0.724
1.04
2.94
13.9
4.64
2.46
3.99
1.03
0.976
Entry
Compound
0.5 h (%)
1
2
3
4
5
24
26
27
42
43
79
97
96
95
92
a
Standard inhibitors for each CYP450 subzymes: 1A2:
a-naphthoflavone, 2C9:
sulfaphenazole, 2D6: quinidine, 2C19: miconazole 3A4: ketoconazole.

170
I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
Table 9
Pharmacokinetics features of selected inhibitors in rats.a
Entry
Compound
Administration route
p.o.
Dose (mg/kg)
Cmax
(
m
mol/mL)
t1/2 (h)
F (%)
1
2
3
5
4
6
7
8
10
9
24
26
27
42
43
24
26
27
42
43
10
10
10
10
10
5
5
5
5
5
0.44 0.14
0.11 0.07
0.17 0.07
0.98 0.02
0.39 0.20
13.76 4.1
16.63 2.57
15.88 1.62
13.26 1.13
18.64 19.4
3.80 0.11
2.34 0.37
6.37 3.39
4.45 4.02
1.68 0.43
2.90 0.62
0.88 0.34
1.55 0.25
1.62 0.78
0.90 0.02
11.74
3.12
4.99
13.8
7.6
iv
a
Vehicle: 5% DMSO/10% Solutol/85% HPBCD (20%, w/v), n ¼ 3.
tetrazolium salt. Hydrophilic WST-8 turns into orange formazan
dye by dehydrogenase in cell lines. The number of living cells is
determined by the activated dehydrogenase, which indicates the
eukaryotic cell survival rate. At the maximal concentration
(100 M) used in the experiment, compound 43 showed less than
50% inhibition (the limit) in monkey kidney, human embryonic
m
Fig. 4. Resistance profiles of inhibitors daclatasvir, 42, and 43. Measurements were carried out in triplicate.

I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
171
lung, mouse fibroblast, and hamster ovary cell line (Table 10). Thus,
we concluded that 43 is nontoxic and safe in the tested eukaryotic
cell lines.
and safe lead warranting further study as a potential HCV drug
candidate. Further research on other pharmacological properties of
the selected inhibitors and new structure of the HCV NS5A in-
hibitors is currently under progress at our laboratories.
Next, we investigated the possibility that compound 43 might
have genetic toxicity using the Ames test [120,121]. For this pur-
pose, we used Salmonella typhimurium strains TA-98 and TA-100
that require several amino acids for growth to test whether com-
pound 43 functions as a mutagen. Growth of TA-98 and TA-100
strains in the presence of the compound of interest in the media
lacking histidine indicates that the compound is a potential
mutagen inducing substitution, addition, and/or deletion of DNA.
The result is considered to be positive when the number of colonies
on the compound-treated plate increases double or more of those
5. Experimental section
5.1. Chemical studies
5.1.1. General chemical methods
The 1H and 13C NMR-spectra were measured with an Agilent
400-MR DD2 Magnetic Resonance System (400 MHz) and a Varian/
Oxford As-500 (500 MHz) Spectrophotometer. The signals were
reported as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), or br s (broad singlet) and chemical shifts were
on the vehicle control plate. When we tested 200 mg/plate of 43, the
number of revertant colonies on the compound-treated plate was
not increased compared with that on the vehicle-treated plate. And
the number was much lower than those on 4 positive control
compound-treated plates (Table 11). Therefore, compound 43 is
considered to be non-genotoxic.
measured as parts per million (d values) from tetramethylsilane as
an internal standard at probe temperature in CDCl3 or DMSO-D6 for
neutral compounds. Reactions that needed anhydrous conditions
were carried out in flame-dried glassware under a positive pressure
of dry N2 using standard Schlenk line techniques. Evaporation of
solvents was performed at reduced pressure using a rotary evap-
orator. TLC was performed using silica gel 60F254 coated on an
aluminum sheet (E. Merck, Art.5554). Chromatogram was visual-
ized by UV-lamp (Vilber Lournat, VL-4LC) and/or colorized with
following solutions: (a) 20% ethanolic phosphomolybdic acid
(PMA), (b) potassium permanganate solution, and (c) 2% ninhydrin
ethanolic solution. Column chromatography was performed on
silica gel (Merck. 7734 or 9385 Kiesel gel 60), and the eluent was
mentioned in each procedure. High resolution mass spectra
(HRMS) were recorded on a ThermoFinnigan LCQ™ Classic, Quad-
rupole Ion-Trap Mass Spectrometer. HPLC analyses were carried out
on an Agilent HP1100 system (Santa Clara, CA, USA), composed of
an auto sampler, quaternary pump, photodiode array detector
(DAD), and HP Chemstation software. The separation was carried
4. Conclusion
In conclusion, we developed a series of extremely potent HCV
NS5A inhibitors using new benzidine and fluorene prolinamide
derivatives as core structures. Several of them have high potencies
that produce inhibition even at the single digit pM level. Through
the SAR studies using a variety of benzidine, proline, and fluorene
derivatives with a phenyl glycine capping group, we were able to
identify inhibitors possessing excessively high inhibitory activities.
Among the new inhibitors, compounds 24, 26, 27, 42, and 43 were
chosen for further evaluation since they were the most potent. A
synergistic effect with the NS5B polymerase inhibitor was seen
with compound 24. Moreover, subsequent studies demonstrated
that these compounds have a desirable low cardiac toxicity, high
human microsomal stability, and limited drugedrug interaction
possibilities. Compound 43 was revealed to be non-toxic for
eukaryotic cells and genetically non-toxic by the Ames test. The
results of this study suggest that compound 43 is a highly potent
out on a C18 Vydac 218TP54 column 250 ꢀ 4.6 mm i.d. (5
mm
particle size) with 0.1% TFA in water (A), acetonitrile (B), as a mobile
phase at a flow rate of 1 mL/min at 20 ꢁC. Method: 100% A and 0% B
(0 min), 0% A and 100% B (10 min), 0% A and 100% B (20 min),100% A
and 0% B (22 min), 100% A and 0% B (25 min). All materials were
purchased from a commercial supplier and used without further
purification unless otherwise noted.
Table 10
Cytotoxicity of 43 in eukaryotic cells.a
Compound
IC50 (mM)
5.1.1.1. (R)-2-((Methoxycarbonyl)amino)-2-phenylacetic acid (9).
Na2CO3 (0.55 g, 5.2 mmol) was added to an aq NaOH (10 mL of 1 M/
H2O, 10 mmol) solution of D-phenylglycine (1.500 g, 10.0 mmol) and
VERO
HFL-1
L929
NIH 3T3
CHOeK1
>100
43
>100
>100
>100
>100
the resulting solution was cooled in an ice-water bath. Methyl
chloroformate (0.85 mL, 11.0 mmol) was added dropwise, then the
cooling bath was removed and the reaction mixture was stirred at
a
VERO: African green monkey kidney cell line; HFL-1: human embryonic lung
cell line; L929: NCTC clone 929, mouse fibroblast cell line; NIH 3T3: mouse em-
bryonic fibroblast cell line; CHOeK1: Chinese hamster ovary cell line.
Table 11
Bacterial reverse mutation assay of 43.a
Tester strain
Compound
Dose (
m
g/plate)
g/plate)
Revertant colonies/plate (Mean SD) [Factor]b
Without S-9 mix
With S-9 mix
TA-98
Vehicle control
43
Vehicle control
43
Positive control
2-Nitrofluorene
Benzo[a]pyrene
Sodium azide
Benzo[a]pyrene
0
200
0
200
18
2
28
3
23 5[1.3]
117 10
106 3[0.9]
Without S-9 mix
187 14[10.2]
ND
30 2[1.1]
126 19
115 8[0.9]
With S-9 mix
ND
171 10[6.1]
ND
521 28[4.1]
TA-100
Dose (
m
TA-98
1
2
1
2
TA-100
728 15[6.2]
ND
ND: Non Determined.
a
(Mean SD, n ¼ 3).
b
No. of revertant colonies in the treated plate/No. of revertant colonies in the vehicle control plate.

172
I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
ambient temperature for 3.25 h. The reaction mixture was washed
with ether (3 ꢀ 18 mL), and the aqueous phase was cooled in an ice-
water bath and acidified with conc. HCl to a pH range of 1e2, and
extracted with CH2Cl2 (3 ꢀ 18 mL). The organic phase was dried
(MgSO4), filtered, and concentrated in vacuo, and the resulting oil
residue was treated with diethyl ether/hexanes (~5:4 ratio; 10 mL)
to provide a precipitate. The precipitate was filtered and washed
with diethyl ether/hexanes (~1:3 ratio) and dried in vacuo to pro-
vide 9 as a fluffy white solid (1.4 g, 67%). 1H NMR (DMSO-d6,
5.1.1.6. (2R,20R)-Di-tert-butyl 2,20-(([1,10-biphenyl]-4,40-diylbis(aza-
nediyl))bis(carbonyl))bis(piperidine-1-carboxylate) (7). Yield
190 mg (35%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 10.00 (s,
2H), 7.67-7.58 (dd, 8H), 4.69 (m, 2H), 3.81 (d, 2H), 3.26 (m, 2H), 2.07
(app br s, 6H), 1.64e1.58 (m, 6H), 1.37 (app br s, 22H). 13C NMR
(DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 170.8, 155.3, 138.1, 134.5,
126.4, 119.7, 78.9, 55.2, 53.9, 42.0, 28.0, 27.5, 24.2, 19.6. HRMS: Anal.
calcd. for [MþH]þ C34H46N4O6: 607.3490; found 607.3489.
d
¼ 2.5 ppm, 500 MHz): 12.79 (br s, 1H), 7.96 (d, J ¼ 12, 1H), 7.40-
5.1.1.7. (2S,20S)-Di-tert-butyl 2,20-(([1,10-biphenyl]-4,40-diylbis(aza-
7.29 (m, 5H), 5.13 (d, J ¼ 12, 1H), 3.55 (s, 3H).
nediyl))bis(carbonyl))bis(2-methylpyrrolidine-1-carboxylate)
(8).
Yield 86 mg (31%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 9.37
(app br s, 2H), 7.74-7.62 (m, 8H), 3.67 (m, 2H), 3.41 (m, 2H), 2.15 (m,
5.1.1.2. (2R,20R)-Di-tert-butyl 2,20-(([1,10-biphenyl]-4,40-diylbis(aza-
nediyl))bis(carbonyl))bis(pyrrolidine-1-carboxylate) (3). A mixture
of N-Boc-D-proline (771 mg, 3.6 mmol), EDCI (812 mg, 4.2 mmol),
and benzidine (300 mg, 1.63 mmol) in CH2Cl2 (4 mL) was stirred at
ambient temperature for 2 h. The resulting residue was divided
between CH2Cl2 and H2O. The organic soluble layer was washed
with 1 N aq HCl solution and brine, dried over MgSO4, filtered, and
concentrated in vacuo. Without any purification,17 was obtained as
2H), 1.90 (m, 6H), 1.51e1.26 (m, 24H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 173.0, 172.5, 153.0, 152.6, 138.4, 134.3,
126.1, 120.8, 120.6, 78.7, 78.6, 66.2, 65.9, 47.5, 47.1, 28.2, 28.0, 22.6,
22.1, 21.1. HRMS: Anal. calcd. for [MþH]þ C34H46N4O6: 607.3490;
found 607.3490.
5.1.1.8. Dimethyl ((1R,10R)-((2R,20R)-2,20-(([1,10-biphenyl]-4,40-diyl-
bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-
phenylethane-2,1-diyl))dicarbamate (11). Compound 17 (300 mg,
0.52 mmol) in CF3CO2H (2 mL) and CH2Cl2 (2 mL) was stirred at
room temperature for 4 h. The volatile component was removed in
vacuo, EDCI (258 mg, 0.620 mmol) and 9 (260 mg, 1.24 mmol) were
a solid (930 mg, 99%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz):
10.08 (s, 2H), 7.69-7.59 (dd, 8H), 4.29e4.20 (m, 2H), 3.44e3.35 (m,
4H), 2.21 (m, 2H), 1.90e1.78 (m, 6H), 1.40e1.28 (app br s, 18H). 13
NMR (DMSO-d6,
C
d
¼ 39.52 ppm, 100 MHz): 171.5, 153.2, 138.2,
134.4, 126.4, 119.6, 78.5, 60.4, 46.6, 31.0, 28.2, 28.0, 23.4. HRMS:
added in batches over 4 min to a solution of i-Pr2NEt (455
mL,
Anal. calcd. for [MþH]þ C32H42N4O6: 579.3177; found 579.3167.
2.6 mmol) in CH2Cl2 (3 mL) and the reaction mixture was stirred at
room temperature for 75 min. The residue was partitioned between
CH2Cl2 and H2O. The organic layer was washed with H2O and brine,
dried over MgSO4, filtered, and concentrated in vacuo. A silica gel
mesh was prepared from the residue and submitted to flash chro-
matography (silica gel: EtOAc/hexane as eluent) to provide 23 as a
5.1.1.3. (5S,50S)-Di-tert-butyl 5,50-(([1,10-biphenyl]-4,40-diylbis(aza-
nediyl))bis(carbonyl))bis(3-oxopyrrolidine-1-carboxylate)
(4).
Yield 320 mg (87%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz):
10.36 (d, 2H), 7.65 (m, 8H), 4.78e6.49 (m, 2H), 3.89-3.76 (m, 4H),
3.11 (m, 2H), 2.57 (m, 2H), 1.42e1.33 (app br s, 18H). 13C NMR
white solid (171 mg, 57%). 1H NMR (DMSO-d6,
400 MHz): 10.14 (s, 2H), 7.68-7.60 (m, 9H), 7.46e7.30 (m, 11H), 5.49
(d, 2H), 4.53 (m, 2H), 3.68 (m, 2H), 3.54 (s, 6H), 3.12 (m, 2H),
2.00e2.13 (m, 2H), 1.89e1.82 (m, 6H). 13C NMR (DMSO-d6,
d
¼ 2.5 ppm,
(DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 209.5, 208.9, 171.0, 170.7,
153.8, 153.4, 137.8, 134.8, 126.64, 126.58, 119.7, 79.9, 79.8, 57.8, 57.0,
53.1, 41.5, 41.2, 28.1, 28.0. HRMS: Anal. calcd. for [MþH]þ
C32H38N4O8: 607.2762; found 607.2756.
d
¼ 39.52 ppm, 100 MHz): 170.2, 168.1, 156.4, 138.2, 136.9, 134.4,
128.4, 128.4, 127.8, 126.4, 119.4, 60.6, 56.6, 51.6, 46.9, 29.4, 24.7.
HRMS: Anal. calcd. for [MþH]þ C42H44N6O8: 761.3293; found
761.3281.
5.1.1.4. (4R,40R)-Di-tert-butyl 4,40-(([1,10-biphenyl]-4,40-diylbis(aza-
nediyl))bis(carbonyl))bis(thiazolidine-3-carboxylate) (5). Yield 2.5 g
(64%). 1H NMR (DMSO-d6,
7.68-7.61 (dd, 8H), 4.72 (app br s, 1H), 4.63 (d, 2H), 4.55 (app br s,
d
¼ 2.5 ppm, 400 MHz): 10.15 (s, 2H),
5.1.1.9. Dimethyl ((1R,10R)-((5S,50S)-5,50-(([1,10-biphenyl]-4,40-diylbi-
s(azanediyl))bis(carbonyl))bis(3-oxopyrrolidine-5,1-diyl))bis(2-oxo-
1-phenylethane-2,1-diyl))dicarbamate (12). Yield 48 mg (23%). 1H
1H), 4.45 (d, 2H), 3.46 (m, 2H), 3.15 (m, 2H), 1.94e1.81 (m, 6H),
1.43e1.32 (app br s, 18H). 13C NMR (DMSO-d6,
100 MHz): 169.0, 152.6, 137.9, 134.7, 126.5, 119.8, 80.0, 63.0, 50.0,
35.2, 27.9. HRMS: Anal. calcd. for [MþH]þ C30H38N4O6S2: 615.2306;
found 615.2278.
d
¼ 39.52 ppm,
NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 10.38 (s, 2H), 7.92 (d, 2H),
7.70-7.10 (m, 18H), 5.46 (d, 2H), 4.94 (d, 2H), 4.25 (d, 2H) 3.89 (d,
2H), 3.54 (s, 6H), 3.06 (m, 2H), 2.54 (m, 2H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 208.5, 170.1, 169.4, 156.1, 137.8, 136.8,
134.7, 128.6, 128.2, 128.1, 126.5, 119.7, 57.4, 56.9, 53.1, 51.7, 40.4.
HRMS: Anal. calcd. for [MþH]þ C42H40N6O10: 789.2879; found
789.2877.
5.1.1.5. (2S,20S)-Di-tert-butyl 2,20-(([1,10-biphenyl]-4,40-diylbis(aza-
nediyl))bis(carbonyl))bis(piperidine-1-carboxylate) (6). A mixture of
N-Boc-L-pipecolic acid (400 mg, 1.75 mmol), EDCI (363 mg,
1.9 mmol), and benzidine (134 mg, 0.73 mmol) in CH2Cl2 (7 mL) was
stirred at ambient temperature for 2 h. The resulting residue was
partitioned between CH2Cl2 and H2O. The organic layer was
washed with 1.0 N aq HCl solution and brine, dried over MgSO4,
filtered, and concentrated in vacuo. A silica gel mesh was prepared
from the residue and submitted to flash chromatography (silica gel:
EtOAc/hexane as eluent) to provide 20 as a solid (186 mg, 42%). 1H
5.1.1.10. Dimethyl ((1R,10R)-((4R,40R)-4,40-(([1,10-biphenyl]-4,40-diyl-
bis(azanediyl))bis(carbonyl))bis(thiazolidine-4,3-diyl))bis(2-oxo-1-
phenylethane-2,1-diyl))dicarbamate (13). Yield 25 mg (14%). 1H
NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 9.89 (s, 2H), 7.93 (d, 2H),
7.73-7.59 (m, 8H), 7.46e7.13 (m, 10H), 5.64 (d, 2H), 4.87 (m, 4H),
4.53 (d, 2H), 3.58 (s, 6H), 3.36 (m, 2H), 3.19 (m, 2H). 13C NMR
(DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 168.3, 167.9, 156.4, 137.8,
NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 9.99 (s, 2H), 7.67-7.58 (dd,
136.3, 134.7, 128.7, 128.3, 128.1, 126.4, 119.8, 63.2, 56.8, 51.8, 49.0,
33.1. HRMS: Anal. calcd. for [MþH]þ C40H40N6O8S2: 797.2422;
found 797.2422.
8H), 4.70 (m, 2H), 3.82 (d, 2H), 3.34 (m, 2H), 2.08 (app br s, 2H),
1.72e1.58 (m, 6H), 1.37 (app br s, 22H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 170.8, 155.3, 138.1, 134.5, 126.4, 119.7
78.9, 55.2, 53.9, 42.0, 28.0, 27.5, 24.2, 19.3. HRMS: Anal. calcd. for
5.1.1.11. Dimethyl ((1R,10R)-((2S,20S)-2,20-(([1,10-biphenyl]-4,40-diyl-
bis(azanediyl))bis(carbonyl))bis(piperidine-2,1-diyl))bis(2-oxo-1-
[MþH]þ C34H46N4O6: 607.3490; found 607.3491.

I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
173
phenylethane-2,1-diyl))dicarbamate (14). Compound 20 (73 mg,
0.12 mmol) in CF3CO2H (1 mL) and CH2Cl2 (1 mL) was stirred at
room temperature for 5 h. The volatile component was removed in
vacuo, EDCI (60 mg, 0.31 mmol) and compound 9 (60 mg,
0.29 mmol) were added in batches over 4 min to a solution of i-
d
¼ 2.5 ppm, 400 MHz): 10.44 (d, 2H), 8.21 (s, 1H), 8.14 (s, 1H), 7.92-
7.82 (dd, 2H), 7.33 (m, 2H), 4.26 (m, 2H), 3.50e3.38 (m, 4H), 2.22 (m,
2H), 1.94e1.80 (m, 6H), 1.42 (s, 9H), 1.30 (s, 9H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 171.9, 171.6, 153.8, 153.2, 139.0, 132.0,
131.0, 128.19, 128.15, 127.9, 124.9, 122.1, 121.2, 121.0, 116.34, 116.28,
116.2, 116.1, 78.7, 78.6, 60.5, 60.1, 54.1, 46.6, 46.4, 31.0, 29.9, 27.8,
Pr2NEt (105 mL, 0.60 mmol) in DMF (1 mL) and the reaction mixture
was stirred at room temperature for 75 min. The residue was par-
titioned between CH2Cl2 and H2O. The organic soluble layer was
washed with H2O and brine, dried over MgSO4, filtered, and
concentrated in vacuo. A silica gel mesh was prepared from the
residue and submitted to flash chromatography (silica gel: EtOAc/
hexane as eluent) to provide 26 as a solid (12 mg, 13%). 1H NMR
27.7, 24.0, 23.3. 19F NMR (DMSO-d6, 377 MHz):
d
ꢂ57.37. HRMS:
Anal. calcd. for [MþH]þ C34H40F6N4O6: 715.2925; found 715.2919.
5.1.1.16. (2S,20S)-Di-tert-butyl 2,20-(((2,20-dimethyl-[1,10-biphenyl]-
4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-1-carboxylate)
(19). Yield 2.75 g (96%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz):
(DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 9.99-9.84 (s, 2H), 7.85e7.30 (m,
9.97 (s, 2H), 7.57-7.41 (m, 4H), 6.97 (app br s, 2H), 4.26 (m, 2H),
20H), 5.73e5.64 (m, 2H), 5.17/4.85 (m, 2H), 4.45/3.77 (m, 2H), 3.55
3.42e3.35 (m, 4H), 2.21 (m, 2H), 1.97 (s, 6H), 1.89e1.78 (m, 6H),
(app br s, 6H), 3.18/2.83 (m, 2H), 2.15 (m, 2H), 1.76 (m, 2H),
1.40e1.31 (app br s, 18H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm,
1.63e1.24 (m, 8H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz):
100 MHz): 171.4, 171.0, 153.6, 153.2, 138.0, 138.0, 135.69, 135.65,
129.52, 120.48, 120.47, 120.4, 120.3, 116.7, 116.5, 78.6, 78.4, 60.3,
60.0, 46.7, 46.5, 31.3, 31.1, 30.2, 28.1, 28.0, 23.9, 23.3, 19.7. HRMS:
Anal. calcd. for [MþH]þ C34H46N4O6: 607.3490; found 607.3489.
170.0, 169.3, 168.5, 156.2, 138.0, 137.2, 134.5, 128.6, 128.4, 128.2,
127.7, 126.5, 120.2, 119.7, 67.0, 55.5, 52.8, 51.6, 43.2, 27.5, 25.1, 24.6,
19.7. HRMS: Anal. calcd. for [MþH]þ C44H48N6O8: 789.3606; found
789.3605.
5.1.1.17. (2S,20S)-Di-tert-butyl
4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-1-carboxylate)
(20). Yield 2.6 g (93%). 1H NMR (DMSO-d6,
2,20-(((2,20-difluoro-[1,10-biphenyl]-
5.1.1.12. Dimethyl ((1R,10R)-((2R,20R)-2,20-(([1,10-biphenyl]-4,40-diyl-
bis(azanediyl))bis(carbonyl))bis(piperidine-2,1-diyl))bis(2-oxo-1-
phenylethane-2,1-diyl))dicarbamate (15). Yield 23 mg (15%). 1H
d
¼ 2.5 ppm, 400 MHz):
10.33 (s, 2H), 7.72-7.68 (m, 2H), 7.46e7.36 (m, 4H), 4.28e4.19 (m,
2H), 3.45e3.32 (m, 4H), 2.22 (m, 2H), 1.99e1.79 (m, 6H), 1.40e1.29
NMR (DMSO-d6,
7.84e7.28 (m, 20H), 5.72e5.64 (m, 2H), 5.16/4.85 (m, 2H), 4.45/3.75
d
¼ 2.5 ppm, 400 MHz): 9.98-9.84 (s, 2H),
(app br s, 18H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz):
(m, 2H), 3.55 (app br s, 6H), 3.17/2.83 (m, 2H), 2.15 (m, 2H), 1.76 (m,
172.0, 171.6, 160.1, 157.7, 153.6, 153.1, 140.5, 140.4, 131.58, 131.55,
117.0, 116.9, 115.1, 115.0, 106.2, 106.0, 78.74, 78.57, 60.4, 60.1, 46.7,
46.6, 31.0, 30.2, 28.1, 28.0, 24.0, 23.4. 19F NMR (DMSO-d6, 377 MHz):
2H), 1.63e1.23 (m, 8H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm,
100 MHz): 170.0, 169.3, 168.5, 156.2, 138.0, 137.2, 134.5, 128.6, 128.4,
128.2, 127.7, 126.5, 120.2, 119.7, 67.0, 55.5, 52.8, 51.6, 43.2, 27.5, 25.1,
24.6, 19.7. HRMS: Anal. calcd. for [MþH]þ C44H48N6O8: 789.3606;
found 789.3605.
d
ꢂ113.53. HRMS: Anal. calcd. for [MþH]þ C32H40F2N4O6: 647.2398;
found 647.2887.
5.1.1.18. (2S,20S)-Di-tert-butyl
2,20-(((2,20-dichloro-[1,10-biphenyl]-
5.1.1.13. Dimethyl ((1R,10R)-((2S,20S)-2,20-(([1,10-biphenyl]-4,40-diyl-
bis(azanediyl))bis(carbonyl))bis(2-methylpyrrolidine-2,1-diyl))bis(2-
oxo-1-phenylethane-2,1-diyl))dicarbamate (16). Yield 77 mg (41%).
4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-1-carboxylate)
(21). Yield 2.8 g (95%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz):
10.31 (app br s, 2H), 7.97-7.87 (m, 2H), 7.63e7.52 (m, 2H), 7.29e7.25
(m, 2H), 4.29e4.20 (m, 2H), 3.47e3.41 (m, 2H), 3.38e3.32 (m, 2H),
1H NMR (DMSO-d6,
7.77-7.57 (m, 10H), 7.40e7.32 (m, 10H), 5.46 (m, 2H), 3.99 (m, 1H),
d
¼ 2.5 ppm, 400 MHz): 9.03 (s,1H), 8.89 (s,1H),
2.25e2.19 (m, 2H), 1.92e1.82 (m, 6H), 1.41e1.31 (app br s, 18H). 13
C
3.76 (m, 1H), 3.56 (s, 3H), 3.54 (s, 3H), 3.48 (m, 1H), 3.21 (m, 1H),
NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 171.9, 171.5, 153.6, 153.1,
2.18e2.08 (m, 2H), 1.91e1.80 (m, 6H), 1.55 (s, 3H), 1.43 (s, 3H). 13
C
139.9,132.5,132.0,131.6,119.2,117.6, 78.7, 78.5, 60.4, 60.1, 46.7, 46.5,
31.3, 31.0, 30.1, 28.1, 28.0, 23.9, 23.3. HRMS: Anal. calcd. for [MþH]þ
C32H40Cl2N4O6: 647.2398; found 647.2394.
NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 171.8, 171.7, 168.2, 167.7,
156.4, 156.2, 138.2, 138.0, 137.2, 136.5, 134.7, 134.3, 128.7, 128.4,
128.22, 128.17, 127.70, 127.68, 126.09, 126.05, 120.9, 120.3, 67.6, 67.5,
57.2, 57.0, 51.7, 51.6, 47.7, 47.5, 23.5, 23.1, 20.6, 20.5. HRMS: Anal.
calcd. for [MþH]þ C44H48N6O8: 789.3606; found 789.3600.
5.1.1.19. (2S,20S)-Di-tert-butyl 2,20-(((2,20-dibromo-[1,10-biphenyl]-
4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-1-carboxylate)
(22). Yield 990 mg (92%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm,
5.1.1.14. (2S,20S)-Di-tert-butyl 2,20-(((3,30-dimethyl-[1,10-biphenyl]-
4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-1-carboxylate)
(17). A mixture of N-Boc-L-proline (9.47 g, 44.0 mmol), EDCI
400 MHz): 10.26 (s, 2H), 8.12-8.01 (m, 2H), 7.66e7.55 (m, 2H), 7.23
(app br s, 2H), 4.26e4.20 (m, 2H), 3.39e3.35 (m, 4H), 2.22 (m, 2H),
1.89e1.81 (m, 6H), 1.40e1.30 (app br s, 18H). 13C NMR (DMSO-d6,
(9.97 g, 52.0 mmol), and ortho-tolidine (4.25 g, 20.0 mmol) in
CH2Cl2 (30 mL) was stirred at ambient temperature for 2 h. The
resulting residue was partitioned between CH2Cl2 and H2O. The
organic layer was washed with 1.0 N aq HCl solution and brine,
dried over MgSO4, filtered, and concentrated in vacuo. Without any
purification, 3 was obtained as a solid (11.3 g, 93%). 1H NMR (DMSO-
d
¼ 39.52 ppm, 100 MHz): 172.0, 171.6, 153.7, 153.1, 139.9, 135.9,
131.4, 123.0, 122.3, 122.2, 118.2, 118.1, 78.8, 78.6, 60.5, 60.2, 46.8,
46.6, 31.0, 30.2, 28.2, 28.0, 24.0, 23.4. HRMS: Anal. calcd. for [MþH]þ
C32H40Br2N4O6: 735.1387; found 735.1373.
5.1.1.20. Dimethyl
biphenyl]-4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-
diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (23).
Compound 3 (144 mg, 0.238 mmol) in CF3CO2H (1 mL) and CH2Cl2
(1 mL) was stirred at room temperature for 5 h. The volatile
component was removed in vacuo, EDCI (119 mg, 0.620 mmol), and
9 (100 mg, 0.572 mmol) were added in batches over 4 min to a
((1R,10R)-((2S,20S)-2,20-(((3,30-dimethyl-[1,10-
d6,
d
¼ 2.5 ppm, 400 MHz): 9.35 (d, 2H), 7.51 (s, 2H), 7.48-7.42 (m,
4H), 4.34 (m, 2H), 3.45 (m, 2H), 3.55 (m, 2H), 2.17 (s, 6H), 2.16 (m,
2H), 1.94e1.81 (m, 6H), 1.42e1.37 (app br s, 18H). 13C NMR (DMSO-
d6,
d
¼ 39.52 ppm, 100 MHz): 171.4, 153.3, 138.5, 135.4, 132.2, 128.3,
125.3,124.0, 78.5, 59.9, 46.6, 31.4, 28.1, 23.3,17.9. HRMS: Anal. calcd.
for [MþH]þ C34H46N4O6: 607.3490; found 607.3480.
solution of i-Pr2NEt (208 mL, 1.192 mmol) in CH2Cl2 (1 mL). The
5.1.1.15. (2S,20S)-Di-tert-butyl 2,20-(((2,20-bis(trifluoromethyl)-[1,10-
reaction mixture was stirred at room temperature for 75 min. The
residue was divided between CH2Cl2 and H2O. The organic layer
was washed with H2O and brine, dried over MgSO4, filtered, and
biphenyl]-4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-1-
carboxylate) (18). Yield 4.3
g
(96%). 1H NMR (DMSO-d6,

174
I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
concentrated in vacuo. A silica gel mesh was prepared from the
12H), 5.51 (d, 2H), 4.39 (m, 2H), 3.84 (m, 2H), 3.55 (s, 6H), 3.52 (m,
residue and submitted to flash chromatography (silica gel: EtOAc/
2H), 3.19 (m, 2H), 2.05e1.81 (m, 8H). 13C NMR (DMSO-d6,
hexane as eluent) to provide 11 as a white solid (77 mg, 41%). 1
H
d
¼ 39.52 ppm, 100 MHz): 170.7, 168.5, 156.2, 139.8, 137.1, 135.9,
NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 9.31 (s, 2H), 7.74 (d, 2H),
131.3, 128.6, 128.1, 127.9, 123.0, 122.3, 118.1, 60.8, 56.7, 51.7, 47.0,
29.3, 24.3. HRMS: Anal. calcd. for [MþH]þ C42H42Br2N6O8:
917.1504; found 917.1521.
7.54-7.23 (m, 16H), 5.52 (d, 2H), 4.52 (m, 2H), 3.85 (m, 2H), 3.52 (s,
6H), 3.18 (m, 2H), 2.28 (s, 6H), 2.00e1.82 (m, 8H). 13C NMR (DMSO-
d6,
d
¼ 39.52 ppm, 100 MHz): 170.1, 168.8, 156.1, 137.1, 136.5, 135.4,
132.2, 128.6, 128.2, 128.1, 128.0, 125.2, 123.9, 60.6, 56.8, 51.6, 46.9,
29.1, 24.3, 17.9. HRMS: Anal. calcd. for [MþH]þ C44H48N6O8:
789.3606; found 789.3597.
5.1.1.26. 9,9-Difluoro-2,7-dinitro-9H-fluorene (30).
2,7-Nitro-9H-fluorene (100 mg, 0.39 mmol) and N-fluo-
robenzenesulfonimide (NFSI) (369 mg, 1.17 mmol) were dissolved
in DMF and cooled to ꢂ20 ꢁC. LiHMDS (1.0 M in THF, 1.17 mL,
1.17 mmol) was added dropwise over 5 min. After an additional
2 h at 0 ꢁC, TLC indicated the completion of the reaction, and the
excess base was quenched by addition of MeOH. The suspension
was filtered over Celite and concentrated in vacuo. A silica gel mesh
was prepared from the residue and submitted to flash chroma-
tography (silica gel: CH2Cl2/hexane as eluent) to provide 30 as a
5.1.1.21. Dimethyl
((1R,10R)-((2S,20S)-2,20-(((2,20-bis(tri-
fluoromethyl)-[1,10-biphenyl]-4,40-diyl)bis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarba-
mate (24). Yield 145 mg (32%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm,
400 MHz): 10.29 (s, 2H), 8.21 (d, 2H), 7.83 (m, 2H), 7.75 (d, 2H), 7.43-
7.05 (m, 12H), 5.51 (d, 2H), 4.41 (m, 2H), 3.85 (app br s, 2H), 3.54 (s,
6H), 3.20 (app br d, 2H), 2.06e1.82 (m, 8H). 13C NMR (DMSO-d6,
yellow solid (98 mg, 86%). 1H NMR (DMSO-d6,
400 MHz): 8.63 (d, 2H), 8.53 (d, 1H), 8.56 (d, 1H), 8.36 (s, 1H), 8.34
(s, 1H). 13C NMR (DMSO-d6,
¼ 39.52 ppm, 100 MHz): 149.1, 142.5,
138.5, 129.2, 144.3, 120.8, 119.6. 19F NMR (DMSO-d6, 377 MHz):
ꢂ110.3.
d
¼ 2.5 ppm,
d
¼ 39.52 ppm, 100 MHz): 171.26, 168.75, 156.42, 139.30, 137.16,
132.79, 131.36, 128.89, 128.35, 128.25, 125.07, 122.88, 121.81, 116.37,
d
61.05, 56.97, 51.87, 47.22, 29.45, 24.51. 19F NMR (DMSO-d6,
377 MHz):
897.3041; found 897.3046.
d
ꢂ57.28. HRMS: Anal. calcd. for [MþH]þ C44H42F6N6O8:
d
5.1.1.27. 9,9-Dimethyl-2,7-dinitro-9H-fluorene (31). A mixture of
2,7-nitro-9H-fluorene (100 mg, 0.39 mmol) and NaOt-Bu (75 mg,
0.78 mmol) were dissolved in DMF at ice bath under N2. Then, CH3I
5.1.1.22. Dimethyl
((1R,10R)-((2S,20S)-2,20-(((2,20-dimethyl-[1,10-
biphenyl]-4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-
diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate
(25).
¼ 2.5 ppm, 400 MHz):
(49 mL, 0.78 mmol) was slowly added to the mixture, and stirred for
Yield 292 mg (73%). 1H NMR (DMSO-d6,
d
2 h. The solution was poured into water and a precipitate was
formed. The product was filtered, washed with water, and air-dried.
Without any purification, 31 (89 mg, 80%) was obtained as a yellow
9.84 (s, 2H), 7.73 (d, 2H), 7.59 (s, 2H), 7.48-7.14 (m, 12H), 6.87 (d,
2H), 5.51 (d, 2H), 4.42 (m, 2H), 3.84 (m, 2H), 3.55 (s, 6H), 3.20 (m,
2H), 1.98 (s, 6H), 1.97e1.78 (m, 8H). 13C NMR (DMSO-d6,
solid. 1H NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 8.59 (d, 2H), 8.33
d
¼ 39.52 ppm, 100 MHz): 170.2, 168.4, 156.1, 137.9, 137.1, 135.8,
(m, 4H), 1.60 (s, 6H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz):
135.8, 129.58, 128.62, 128.1, 127.9, 120.5, 116.7, 60.7, 56.8, 51.7, 47.0,
29.4, 24.3, 19.8. HRMS: Anal. calcd. for [MþH]þ C44H48N6O8:
789.3606; found 789.3605.
156.2, 148.1, 142.7, 123.6, 122.9, 118.7, 47.9, 25.6.
5.1.1.28. 9,9-Difluoro-9H-fluorene-2,7-diamine (32). A magnetic
stirrer bar, Fe3O4 (purchased from Aldrich, 4 mg, 0.015 mmol), and
DMF (0.5 mL) were added to an oven-dried Schlenk tube, and the
mixture was sonicated in an ultrasound bath for 1 min under argon.
Compound 31 (22 mg, 0.075 mmol) and hydrazine monohydrate
5.1.1.23. Dimethyl
biphenyl]-4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-
diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (26).
Yield 134 mg (40%). 1H NMR (DMSO-d6,
((1R,10R)-((2S,20S)-2,20-(((2,20-difluoro-[1,10-
d
¼ 2.5 ppm, 400 MHz):
(29 mL, 0.60 mmol) were then added to the mixture. The reaction
10.43 (s, 2H), 7.74-7.71 (m, 3H), 7.46e7.11 (m,15H), 5.51 (d, 2H), 4.43
(m, 2H), 3.83 (m, 2H), 3.54 (s, 6H), 3.19 (m, 2H), 2.05e1.77 (m, 8H).
mixture was stirred at 80 ꢁC under an argon atmosphere until the
reaction was completed. After magnetic separation of the catalyst,
the organic layer was concentrated in vacuo. The residue was par-
titioned between CH2Cl2 and H2O. The organic layer was washed
with brine, dried over MgSO4, filtered, and concentrated in vacuo.
Without any purification, 32 (17 mg, 98%) was obtained as a yellow
13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 170.8, 168.4, 160.0,
158.0, 156.1, 140.5, 137.2, 131.6, 128.6, 128.5, 128.1, 127.9, 127.6, 117.1,
115.1, 106.3, 106.1, 60.8, 56.7, 51.7, 47.0, 29.3, 24.3. 19F NMR (DMSO-
d6, 377 MHz):
d
ꢂ73.45. HRMS: Anal. calcd. For [MþH]þ
C42H42F2N6O8: 797.3105; found 797.3112.
solid. 1H NMR (DMSO-d6,
(s, 1H), 6.8 (d, 2H), 6.61 (d, 1H), 6.59 (d, 1H), 5.3 (s, 4H). 13C NMR
(DMSO-d6,
¼ 39.52 ppm, 100 MHz): 148.0, 137.3, 137.1, 127.7, 123.7,
d
¼ 2.5 ppm, 400 MHz): 7.19 (s, 1H), 7.17
5.1.1.24. Dimethyl
((1R,10R)-((2S,20S)-2,20-(((2,20-dichloro-[1,10-
d
biphenyl]-4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-
119.8, 116.45, 109.1. 19F NMR (DMSO-d6, 377 MHz):
d
ꢂ106.7. HRMS:
diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate
(27).
¼ 2.5 ppm, 400 MHz):
Anal. calcd. for [MþH]þ C13H10F2N2: 233.0885; found 233.0885.
Yield 146 mg (44%). 1H NMR (DMSO-d6,
10.14 (s, 2H), 7.97-7.91 (m, 2H), 7.75 (d, 2H), 7.65e7.55 (m, 2H),
7.43e7.12 (m, 12H), 5.51 (d, 2H), 4.39 (m, 2H), 3.84 (m, 2H), 3.55 (s,
6H), 3.20 (m, 2H), 2.06e1.79 (m, 8H). 13C NMR (DMSO-d6,
d
5.1.1.29. 9,9-Dimethyl-9H-fluorene-2,7-diamine (33). Yield of 69 mg
(98%) was obtained as
¼ 2.5 ppm, 400 MHz): 7.21 (s, 1H), 7.19 (s, 1H), 6.6 (d, 2H), 6.47 (d,
1H), 6.45 (d, 1H), 4.9 (s, 4H), 1.3 (s, 6H). 13C NMR (DMSO-d6,
a
yellow solid. 1H NMR (DMSO-d6,
d
d
¼ 39.52 ppm, 100 MHz): 170.8, 168.5, 156.2, 139.9, 137.1, 132.6,
132.1, 131.7, 128.6, 128.1, 127.9, 119.3, 117.7, 60.8, 56.73, 51.68, 47.0,
29.3, 24.3. HRMS: Anal. calcd. for [MþH]þ C42H42Cl2N6O8:
829.2514; found 829.2518.
d
¼ 39.52 ppm, 100 MHz): 153.5, 146.7, 128.4, 118.6, 112.6, 108.5,
45.6, 27.7. HRMS: Anal. calcd. for [MþH]þ C15H16N2: 225.1386;
found 225.1383.
5.1.1.25. Dimethyl
biphenyl]-4,40-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-
diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (28).
Yield 110 mg (42%). 1H NMR (DMSO-d6,
¼ 2.5 ppm, 400 MHz):
10.13 (s, 2H), 8.32e8.12 (m,2H), 7.92e7.61 (m, 4H), 7.41e7.13 (m,
((1R,10R)-((2S,20S)-2,20-(((2,20-dibromo-[1,10-
5.1.1.30. 2,7-Diamino-9H-fluoren-9-one (35). A mixture of 9H-flu-
orene-2,7-diamine (294 mg,1.5 mmol) and Cs2CO3 (1.5 g, 4.5 mmol)
in DMSO (7 mL) was stirred under an atmosphere of air. When TLC
showed that no starting material remained, the solution was
poured into water and a precipitate was formed. The product was
d

I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
175
filtered, washed with water, and air-dried. Without any purifica-
tion, 35 (239 mg, 76%) was obtained as a solid. 1H NMR (DMSO-d6,
removed in vacuo. EDCI (253 mg, 1.3 mmol) and 9 (255 mg,
1.3 mmol) were added in batches over 4 min to a solution of i-
d
¼ 2.5 ppm, 400 MHz): 7.10 (s,1H), 7.08 (s,1H), 6.70 (d, 2H), 6.57 (d,
Pr2NEt (441 mL, 2.5 mmol) in CH2Cl2 (2 mL), and the reaction
1H), 6.57 (d, 1H) 5.30 (s, 4H). 13C NMR (DMSO-d6,
100 MHz): 194.9, 148.2, 134.6, 133.3, 119.9, 118.6, 109.7. HRMS: Anal.
calcd. for [MþH]þ C13H10N2O: 211.0866; found 211.0867.
d
¼ 39.52 ppm,
mixture was stirred at room temperature for 75 min. The residue
was partitioned between CH2Cl2 and H2O. The organic layer was
washed with H2O and brine, dried over MgSO4, filtered, and
concentrated in vacuo. A silica gel mesh was prepared from the
residue and submitted to flash chromatography (silica gel: EtOAc/
hexane as eluent) to provide 40 as a white solid (198 mg, 50%). 1H
5.1.1.31. (2S,20S)-Di-tert-butyl 2,20-(((9H-fluorene-2,7-diyl)bis(aza-
nediyl))bis(carbonyl))bis(pyrrolidine-1-carboxylate) (36). A mixture
of N-Boc-
L
-proline (323 mg, 1.5 mmol), EDCI (312 mg, 1.63 mmol),
NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 9.92 (s, 2H), 7.91 (s, 2H),
and 2,7-diaminofluorene (123 mg, 0.63 mmol) in CH2Cl2 (2 mL) was
stirred at ambient temperature for 2 h. The resulting residue was
partitioned between CH2Cl2 and H2O. The organic layer was
washed with 1.0 N aq HCl solution and brine, dried over MgSO4,
filtered, and concentrated in vacuo. Without any purification, 36
was obtained as a solid (359 mg, 97%). 1H NMR (DMSO-d6,
7.75-7.69 (m, 4H), 7.56 (d, 2H), 7.44e7.13 (m, 10H), 5.52 (d, 2H), 4.43
(m, 2H), 3.88e3.83 (m, 2H), 3.55 (s, 6H), 3.21 (m, 2H), 2.04e1.78 (m,
8H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 170.2, 168.5,
156.2, 143.6, 137.5, 137.2, 136.4, 128.7, 128.1, 127.9, 119.6, 118.1, 116.2,
60.8, 56.8, 51.7, 47.0, 36.7, 29.4, 24.3. HRMS: Anal. calcd. for [MþH]þ
C43H44N6O8: 773.3293; found 773.3296.
d
¼ 2.5 ppm, 400 MHz): 10.0 (d, 2H), 7.69 (d, 2H), 7.72 (d, 2H), 7.57-
7.51 (m, 2H), 4.31e4.21 (m, 2H), 3.88 (s, 2H), 3.45e3.37 (m, 4H),
2.23 (m, 2H), 1.94e1.80 (m, 6H), 1.41 (app br s, 9H), 1.29 (app br s,
5.1.1.36. Dimethyl ((1R,10R)-((2S,20S)-2,20-(((9-oxo-9H-fluorene-2,7-
diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(2-oxo-
1-phenylethane-2,1-diyl))dicarbamate (41). Yield 12 mg (66%). 1H
9H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 171.4, 153.2,
143.5,137.6, 136.3,119.5, 118.0, 116.1, 78.6, 78.5, 60.4, 46.6, 31.1, 28.2,
28.0, 24.0, 23.4. HRMS: Anal. calcd. for [MþH]þ C33H42N4O6:
591.3177; found 591.3168.
NMR (DMSO-d6,
7.76 (t, 4H), 7.64 (d, 2H), 7.43-7.10 (m, 10H), 5.51 (d, 2H), 4.39 (m,
2H), 3.85 (m, 2H), 3.55 (s, 6H), 3.20 (m, 2H), 2.05e1.79 (m, 8H). 13
NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 10.10 (s, 2H), 7.91 (s, 2H),
C
d
¼ 39.52 ppm, 100 MHz): 192.8, 170.6, 168.5,
5.1.1.32. (2S,20S)-di-tert-butyl
bis(azanediyl))bis(carbonyl))bis(pyrrolidine-1-carboxylate)
Yield 131 mg (91%). 1H NMR (DMSO-d6,
10.24 (s, 2H), 7.91 (m, 2H), 7.91 (m, 2H), 7.62 (m, 2H), 4.26-4.16 (m,
2H), 3.46e3.40 (m, 2H), 3.37e3.31 (m, 2H), 2.24e2.16 (m, 2H),
1.94e1.76 (m, 6H), 1.40 (app br s, 9H), 1.27 (app br s, 9H). 13C NMR
2,20-(((9-oxo-9H-fluorene-2,7-diyl)
(37).
¼ 2.5 ppm, 400 MHz):
156.2, 139.5, 138.8, 137.1, 134.2, 128.6, 128.1, 127.9, 125.0, 121.1, 115.0,
60.8, 56.7, 51.7, 47.0, 29.3, 24.3. HRMS: Anal. calcd. for [MþH]þ
C43H42N6O9: 787.3086; found 787.3089.
d
5.1.1.37. Dimethyl ((1R,10R)-((2S,20S)-2,20-(((9,9-dimethyl-9H-fluo-
rene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))
bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (42). Yield 135 mg
(DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 192.9, 171.9, 171.4, 153.6,
153.1, 139.6, 138.8, 134.2, 125.0, 121.1, 115.0, 78.8, 78.6, 60.5, 60.1,
46.6, 46.1, 31.0, 30.2, 28.2, 28.0, 24.0, 23.4. HRMS: Anal. calcd. for
[MþH]þ C33H40N4O7: 605.2970; found 605.2980.
(53%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz): 9.96 (s, 2H), 7.86
(s, 2H), 7.74 (d, 2H), 7.69 (d, 2H), 7.51 (dd, 2H), 7.43-7.10 (m, 10H),
5.52 (d, 2H), 4.43 (m, 2H), 3.83 (m, 2H), 3.55 (s, 6H), 3.20 (m, 2H),
2.05e1.78 (m, 8H), 1.41 (s, 6H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm,
5.1.1.33. (2S,20S)-di-tert-butyl 2,20-(((9,9-dimethyl-9H-fluorene-2,7-
diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-1-carboxylate) (38).
100 MHz): 170.1, 168.3, 156.1, 153.7, 138.0, 137.2, 133.7, 128.6, 128.1,
127.8, 119.8, 118.3, 113.7, 60.7, 56.7, 51.6, 47.0, 46.5, 29.3, 27.2, 24.3.
HRMS: Anal. calcd. for [MþH]þ C45H48N6O8: 801.3606; found
801.3611.
Yield 201 mg (97%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz):
10.10 (app br s, 2H), 7.84 (s,1H), 7.79 (s,1H), 7.66 (d, 2H), 7.51 (t, 2H),
4.31-4.28 (m, 1H), 4.24e4.21 (m, 1H), 3.44e3.40 (m, 2H), 3.37e3.31
(m, 2H), 2.21e2.16 (m, 2H), 1.95e1.80 (m, 6H), 1.40 (app br s, 9H),
5.1.1.38. Dimethyl
((1R,10R)-((2S,20S)-2,20-(((9,9-difluoro-9H-fluo-
1.34 (s, 6H), 1.28 (app br s, 9H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm,
rene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))
100 MHz): 174.3, 173.9, 171.4, 171.0, 153.6, 153.2, 138.1, 138.0, 133.7,
133.6, 119.7, 118.4,118.2,113.8,113.6, 78.6, 78.5, 60.4, 60.0, 46.6, 46.1,
42.2, 42.1, 31.0, 30.3, 28.2, 28.1, 27.9, 27.1, 24.0, 23.4. HRMS: Anal.
calcd. for [MþH]þ C35H42N4O6: 619.3490; found 619.3496.
bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (43). Yield 18 mg
(52%). 1H NMR (DMSO-d6,
8.05 (s, 2H), 7.77 (d, 2H), 7.71 (s, 4H), 7.43-7.10 (m,10H), 5.51 (d, 2H),
d
¼ 2.5 ppm, 400 MHz): 10.14 (s, 2H),
4.39 (m, 2H), 3.85 (m, 2H), 3.55 (s, 6H), 3.21 (m, 2H), 2.06e1.79 (m,
8H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz): 170.6, 168.5,
5.1.1.34. (2S,20S)-Di-tert-butyl 2,20-(((9,9-difluoro-9H-fluorene-2,7-
diyl)bis(azanediyl))bis(carbonyl))bis(pyrrolidine-1-carboxylate) (39).
156.2,139.3,137.3,137.0,128.6,128.4,128.1,127.9,127.6,122.8,121.2,
114.5, 60.8, 56.7, 51.6, 47.0, 29.3, 24.3. HRMS: Anal. calcd. for
[MþH]þ C43H42F2N6O8: 809.3105; found 809.3109.
Yield 28 mg (95%). 1H NMR (DMSO-d6,
d
¼ 2.5 ppm, 400 MHz):
10.29 (app br s, 2H), 8.06 (s, 1H), 8.02 (s, 1H), 7.69-7.66 (m, 4H),
4.30e4.25 (m, 1H), 4.21e4.18 (m, 1H), 3.45e3.41 (m, 2H), 3.37e3.34
(m, 2H), 2.23e2.16 (m, 2H), 1.92e1.80 (m, 6H), 1.40 (app br s, 9H),
5.2. Biological studies
1.27 (app br s, 9H). 13C NMR (DMSO-d6,
d
¼ 39.52 ppm, 100 MHz):
5.2.1. Measurement of the anti-HCV activities of compounds using
HCVcc
174.3, 173.9, 171.9, 171.5, 153.6, 153.1, 139.4, 133.56, 133.49, 122.8,
122.7, 121.2, 114.5, 114.4, 78.8, 78.6, 60.5, 59.3, 58.6, 46.6, 46.2, 46.1,
31.0, 30.3, 28.2, 28.0, 27.9, 27.7, 24.0, 23.4. 19F NMR (DMSO-d6,
377 MHz):
C33H40F2N4O6: 627.2989; found 627.2997.
- Cell line and cell culture
d
ꢂ108.9, -109.0. HRMS: Anal. calcd. for [MþH]þ
Huh 7.5.1 cells were grown in Dulbecco0s Modified Eagle0s me-
dium (DMEM; Gibco) supplemented with antibiotics (100 U/mL
5.1.1.35. Dimethyl
((1R,10R)-((2S,20S)-2,20-(((9H-fluorene-2,7-diyl)
penicillin, 10
bovine serum (
m
g/mL streptomycin) and 10% heat-inactivated fetal
D
bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-
phenylethane-2,1-diyl))dicarbamate (40). A mixture of biphenyl 36
(300 mg, 0.51 mmol) in CF3CO2H (2 mL) and CH2Cl2 (2 mL) was
stirred at room temperature for 5 h. The volatile component was
FBS; Thermo) at 37 ꢁC in a humidified 6.0% CO2
incubator.
- Virus production

176
I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
In vitro transcription and RNA electroporation were performed
5.2.4. Observation of the effects of compounds on drug-resistant
HCV mutants
as described in the previous report [122]. In vitro transcribed RNA of
JFH5a-Rluc-ad34, which is a derivative of JFH1 with adaptive mu-
tations in the E2 and p7 regions [82], was transfected into Huh7.5.1
cells by electroporation. JFH5a-Rluc-ad34 virus contains a reporter
Renilla luciferase (Rluc) for convenient assay of virus proliferation
[82]. Cell culture media containing HCV were collected 3~5 days
We selected HCV mutants showing resistance against BMK-
20113 by continuous cultivation of Huh 7.5.1 cells containing HCV
replicon NK/R2AN in the presence of BMK-20113. We found that the
majority of mutants had mutations at the N-terminal region of
NS5A protein (L31V and/or Y93H in NS5A) similarly to the dacla-
tasvir resistant mutants described previously [117,123]. We used
NK/R2AN derivatives containing single mutations (L31V or Y93H in
NS5A) to investigate the drug resistance of lead compounds. Briefly,
Huh 7.5.1 cells were electroporated with RNAs containing resistant
mutations and cultivated on 12-well plates. At 4 h after trans-
fection, media were replaced with DMEM containing serially
diluted compounds and further cultivated. At 3 days after com-
pound treatment, the cells were harvested and luciferase activities
were measured. Luciferase activities were normalized to those
obtained from mock-treated cells.
after electroporation and then filtered through a 0.45
filter.
mM pore size
- Antiviral activity test with infectious HCV particles
Huh 7.5.1 cells were inoculated with a JFH5a-Rluc-ad34 virus
stock and cultivated for 3 h. At 3 h after the virus inoculation, HCV-
infected Huh7.5.1 cells were cultivated with media containing
serially diluted compounds. At 3 days after chemical treatment, the
cells were harvested and luciferase activities in the cells were
measured using a Renilla luciferase assay system (Promega) ac-
cording to the manufacturer0s direction. Finally the luciferase ac-
tivities were normalized to those obtained from mock-treated cells.
5.2.5. hERG ligand binding assay [99]
The hERG ligand binding assay was performed by using the
hERG Fluorescence Polarization Assay Kit (Cat No. PV5365), con-
taining predictor™ hERG tracer red, predictor™ hERG membrane,
5.2.2. Measurement of anti-HCV activities of compounds using HCV
replicon
and predictor™ hERG FP assay buffer. Test compounds (10
mM) and
positive control (Astemizole) were added to the 384-well plate
(5
10
m
m
L each; low-volume polystyrene plate, corning #3677). Then,
L of membrane and 5 L of tracer were added to each plate by
L,
- Cell line and cell culture
m
using a multi-pipette (Multi 16 channel pipette type 5e50
m
Huh 7.5.1 cells containing a bicistronic HCV replicon NK/R2AZ
(genotype 1b) were used to test the anti-HCV activities of the
compounds (Fig. 5). The first and second open reading frames
(ORFs) of the replicon contain the Renilla luciferase gene fused with
the neomycin phosphotransferase gene and the HCV nonstructural
genes NS3-5, respectively. The replicon-containing cells were
cultivated under the same conditions as described above with an
additional antibiotic G418 (0.5 mg/mL, Calbiochem).
Thermo). The plate was then covered with aluminum foil and was
kept for 2e3 h at room temperature. Lastly, the fluorescence po-
larization of the hERG red tracer was measured using a microplate
reader (molecular devices, SpectraMax M5e, excitation (540 nm)
emission (585 nm) filters).
5.2.6. Plasma stability measurement
Rat plasma (297
water bath. The plasma and 100 mM test compounds (3 mL) were
m
L) was pre-incubated for 5 min at 37 ꢁC in a
- NK/R2AN
- Antiviral activity assay test with HCV replicon
mixed using a vortex, and incubated for 37 ꢁC in a water bath. Then
the mixture was sampled multiple times (0, 15, 30, 60, and
Huh 7.5.1 cells containing HCV replicon (NK/R2AN) were plated
on a 12-well plate (5 ꢀ 104 cells per well). At 16 h after cultivation,
NK/R2AN-containing cells were incubated with media containing
serially diluted compounds for 3 days. After the chemical treat-
ment, the cells were harvested and luciferase activities were
measured. Luciferase activities were measured using the Renilla
luciferase assay system (Promega) according to the manufacturer0s
direction, and then normalized to those obtained from mock-
treated cells.
120 min). A mixture of crude sample (50 mL) and acetonitrile
(100
mL) was then treated by centrifugation at 10,000 rpm for
10 min at 25 ꢁC. Finally, supernatant was isolated and analyzed
using an LC-MS/MS.
5.2.7. Method to determine stability in rat and human liver
microsomes [103]
The metabolic stability of the compounds was evaluated in
pooled rat (BD Gentest® Cat No. 452501) or human livers (BD
GentestCat. 452161) microsomal fractions with
b-NADPH over 30 m
using 1 M of compounds. The compounds in the supernatants
were analyzed by LC-MS/MS.
m
5.2.3. Observation of the effects of co-treatment of two compounds
Huh 7.5.1 cells containing HCV replicon (NK/R2AN) were plated
on a 12-well plate (5 ꢀ 104 cells per well). At 16 h after cultivation,
NK/R2AN cells were treated with serially diluted compounds in the
presence of 123 nM of sofosbuvir (EC30). At 3 days after treatment,
luciferase activities were measured using the Renilla luciferase
assay system (Promega) according to the manufacturer0s direction.
5.2.8. Method of CYP450 inhibition
The interaction of human cytochrome CYP450 with compounds
was studied on a cytochrome panel consisting of CYP1A2, CYP2C9,
CYP2C19, CYP2D6, and CYP3A4 (P450-glo, promega kit) according
Fig. 5. The structure of replicon NKR2AN.

I.H. Bae et al. / European Journal of Medicinal Chemistry 101 (2015) 163e178
177
to the manufacturer0s protocols. The inhibition effects were
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We thank Prof. Nakcheol Jung of Korea Basic Science Institute
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