Expanding the chemical space of anti-HCV NS5A inhibitors by stereochemical exchange and peptidomimetic approaches

Article abstract of DOI:10.1002/ardp.201800017

Here we report a series of potent anti-HCV agents bearing a symmetrical benzidine l-prolinamide backbone with different capping groups including alkyl/aryl carbamates of natural and unnatural valine and leucine amino acids. All compounds were investigated for their inhibitory activity in an HCV replicon assay on genotype 1b. The novel compounds share some chemical and clinical attributes of commercially available NS5A inhibitors. Compounds 5 and 6 with unnatural capping residue and ethyl and isobutyl carbamates showed EC₅₀ values in the picomolar range with a low toxicity profile and selectivity indices of several orders of magnitude. These findings enlarge the chemical space from which NS5A inhibitors may be discovered by adopting unnatural amino acids, amino acids other than valine and carbamates other than methyl as the capping groups.

Full text of DOI:10.1002/ardp.201800017

Received: 23 January 2018
Revised: 28 April 2018
Accepted: 2 May 2018
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DOI: 10.1002/ardp.201800017
FULL PAPER
Expanding the chemical space of anti-HCV NS5A inhibitors by
stereochemical exchange and peptidomimetic approaches
Triveena M. Ramsis1
Efseveia Frakolaki2
Nermin S. Ahmed1
Shereen E. Abdel Karim1
Niki Vassilaki2
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Ahmed A. M. Kamal3
Grigoris Zoidis4
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Ashraf H. Abadi1
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1 Faculty of Pharmacy and Biotechnology,
Department of Pharmaceutical Chemistry,
German University in Cairo, Cairo, Egypt
Abstract
Here we report a series of potent anti-HCV agents bearing a symmetrical benzidine
L-prolinamide backbone with different capping groups including alkyl/aryl carbamates
of natural and unnatural valine and leucine amino acids. All compounds were
investigated for their inhibitory activity in an HCV replicon assay on genotype 1b. The
novel compounds share some chemical and clinical attributes of commercially available
NS5A inhibitors. Compounds 5 and 6 with unnatural capping residue and ethyl and
isobutyl carbamates showed EC50 values in the picomolar range with a low toxicity
profile and selectivity indices of several orders of magnitude. These findings enlarge
the chemical space from which NS5A inhibitors may be discovered by adopting
unnatural amino acids, amino acids other than valine and carbamates other than methyl
as the capping groups.
2 Molecular Virology Laboratory, Hellenic
Pasteur Institute, Athens, Greece
3 Pharmaceutical and Medicinal Chemistry,
Department of Drug Design Optimization,
Saarland University and Helmholtz Institute
for Pharmaceutical Research Saarland (HIPS),
Saarbrücken, Germany
4 Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, School of Health
Sciences, National and Kapodistrian University
of Athens, Athens, Greece
Correspondence
Prof. Ashraf H. Abadi, Faculty of Pharmacy
and Biotechnology, Department of
Pharmaceutical Chemistry, German University
in Cairo, 11835 Cairo, Egypt.
K E Y W O R D S
Email: ashraf.abadi@guc.edu.eg
anti-HCV, bivalent ligands, NS5A inhibitors, peptidomimetics
Funding information
Science and Technology Development Fund
(Egypt), Grant number: 22848
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1
INTRODUCTION
with 31–33% nucleotide sequence divergence. Each genotype is
further divided into subtypes (designated by lower case letters), with
Hepatitis C virus (HCV) is the leading cause of chronic liver disease
worldwide as well as the primary cause of liver transplantations. The
World Health Organization (WHO) estimates that about 3% of the
world's population, i.e., 170 million people, is currently infected with
HCV.[1] Each year, 3 to 4 million people are newly infected with HCV.[2]
The pathogen causes significant morbidity, with approximately
399000 deaths per year as a direct result of complications involved
in end-stage liver diseases.[3] Most of the infected individuals (∼80%)
will develop chronic infection, the latter is often associated with the
development of hepatitis, steatosis, fibrosis, and liver cirrhosis. HCV is
currently classified into six main genotypes (identified as 1 through 6),
each subtype differing from one another by 20–25%.[4] The
combination of molecular cloning and development of a replicon
assay has enabled the discovery of novel DAAs targeting viral
proteins.[5,6] HCV is an enveloped positive-stranded RNA virus, the
RNA is translated into a polyprotein consisting of 10 proteins. This
polyprotein is then processed by both cellular and viral proteases to
form three structural (S) elements (core [C], E1, and E2), and seven non-
structural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B).
NS5A is a hydrophilic phosphoprotein containing an N-terminal
amphipathic α-helix that mediates virus association with the ER
membrane. This phosphoprotein exists in two forms: a basally
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Arch Pharm Chem Life Sci. 2018;1–10.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
1

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RAMSIS ET AL.
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phosphorylated form and a hyperphosphorylated form.[7–10] NS5A is
also a multi-functional protein with no apparent enzymatic activity.
NS5A is proposed to be involved in resistance to the host immune
response, and is an essential component of the viral lifecycle with
involvement in replication and maturation/packaging of the viral
genome.[6] Until 2011, the combination of pegylated interferon-α
(PEG-IFN-α) and ribavirin regimen for a duration of 24 or 48 weeks has
been the standard of care for the treatment of HCV.[5] However, in
2014, daclatasvir (I) was the first NS5A inhibitor to be introduced to
the market and used in HCV treatment (Figure 1). Ombitasvir (II) and
ledipasvir were subsequently introduced to the market (Figure 1). Here
we report a class of symmetric bivalent ligands bearing a benzidine
L-prolinamide scaffold. The novel series is designed to keep the
previously reported pharmacophoric features of NS5A inhibitors,
namely the two aromatic hydrophobic regions, a hydrophobic terminal
chain and at least two hydrogen bond acceptors.[11]
triflouroacetic acid (TFA). A series of capping groups were coupled
to the free amine using HBTU. The capping groups are carbamate
derivatives prepared from a series of commercially available amino acid
enantiomers, using different chloroformates in NaOH and in high
yields (Schemes 1 and 2).
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2.2 Biology
Compounds 1–16 were tested for their EC50, EC90, and CC50 using
luciferase reporter replicon assay on genotype 1b, CON-1 strain. In
addition, selected compounds were also tested on genotype 2a, JFH-1
strain. The cell line used for virus replication was human hepatoma cell
line (Huh-7). EC50 is the compound concentration reducing the virus
replication in Huh-7 infected cells by 50%, CC50 is the compound
concentration decreasing the cell viability of non-infected Huh-7 by
50%, SI50 is the ratio of CC50/EC50 and it indicates the safety of the
compound as antiviral agent. The results are summarized in Tables 1
and 2.
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2
RESULTS AND DISCUSSION
All 16 compounds in Table 1 had the benzidine L-prolinamide part
unchanged while we adopted capping groups of R- and S-valine and
leucine containing different alkyl/aryl carbamate esters (Figure 1).
Although the molecule is structurally symmetric, the non-
coplanarity of the two phenyl rings of benzidine may lead to
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2.1 Chemistry
Coupling of benzidine with Boc-L-proline using EEDQ resulted in a
pure Boc-protected product which was deprotected using
FIGURE 1 Structures of the clinically available daclatasvir (I), ombitasvir (II) and the scaffold of newly synthesized compounds 1–16

RAMSIS ET AL.
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SCHEME 1 (a) N-Boc-L-proline, EEDQ, CH2Cl2; (b) TFA, CH2Cl2; (c) capping group, HBTU, DIPEA, CH2Cl2
asymmetric binding to the receptor and different projections of the
side chains in space, Figure 2.
or aromatic benzyl 12 rather than longer and bulkier isobutyl 10 and
butyl 11 terminal chains (Table 1).
For most of the clinically used NS5A inhibitors, the capping group
on the nitrogen of the proline/proline-like moiety is S-valine methyl
carbamate ester. The assay data indicate that the inhibitory activity of
these analogues is greatly dependent upon the structure of the
terminal capping groups.
Within the R-epimer series 1–8, the R-leucine containing
compounds have been more active than the R-valine ones. These
results indicate the success of our peptidiomimetic principle regarding
the use of unnatural capping residue and the development of active
analogues with capping groups bearing an R-configuration, both
approaches widen the scope for development of new clinically
effective DAAs.
Surprisingly, in the case of molecules with valine amino acids 1–4
and 9–12, the higher activity is limited to the R-epimers 1–4. This is a
new finding and indicates the value of testing both natural and
unnatural epimers of the same residue to decide about the more active
derivatives.
Most of the synthesized molecules were not cytotoxic at the
highest concentration tested; this leads to SI50s of several orders of
magnitude, for example compounds 5 and 6 showed SI50 values of
86523 and >71428 and SI90 values of 6200 and >6020, respectively.
Within the tested concentration ranges, the EC50s of compounds
5 and 6 as well as their CC50 and SI50 values against genotype 2a
displayed improved antiviral values in comparison to sofosbuvir which
was included as positive control in our assay.
Among the ethyl, isobutyl, butyl, and benzyl carbamates of 1–4
and 9–12, the most active derivatives are the benzyl carbamates 4 and
12. This indicates that this moiety is mostly involved in hydrophobic
interaction with the receptor.
Among the leucine derivatives 5–9 and 13–16, again the
R-epimers derivatives are more active than the S-epimers; this
confirms the crucial role of the stereocenter of the AA residue and
the important role of the projection plane upon activity.[12]
The higher activities of the R-epimers relative to the S-epimers for
those molecules doubles the chemical space from which new NS5A
inhibitors can be discovered. This also confirms the essential role of
stereochemical centre on the capping group, with an obvious favor to R
configuration.
Compounds 1–3 have shown nanomolar EC50 values on genotype
2a; this may indicate a promising pan genotypic activity of the novel
compounds.
To date all NS5A inhibitors were identified through cell-based
replicon assays. An X-ray of the NS5A protein co-crystallized with
daclatasvir (I), or any approved inhibitor, has never been established,
that is whydaclatasvir and its clinicallyrelated compounds are described
as NS5A replication complex inhibitors. Thus, we have defined the
binding pocket guided by previous literature,[11] reported resistance
mutations, and automatic site finder of the software MOE 2015.[13]
As per the carbamate moiety of the R-epimer derivatives 9–12, the
higher activity has been more associated with the small alkyls ethyl 9,
SCHEME 2 Synthesis of capping groups

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RAMSIS ET AL.
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TABLE 1 Replicon assay results for compounds 1–8 against HCV genotype 1b
Cpd #
1
Capping groups
Stereochemistry
EC50 (nM)
EC90 (nM)
CC50 (nM)
SI50
SI90
R
130
260
>20000
>154
>76
2
3
4
5
R
R
R
R
350
20
610
60
11250
6320
32
18
316
105
>174
6200
17.4
0.21
115
2.90
>20000
18170
>1149
86523
6
R
0.28
3.30
>20000
>71428
>6060
7
8
R
R
1.35
4.83
7.9
>20000
>20000
>14814
>4140
>2532
>533
37.5
9
S
3700
13000
>20000
>5.4
>1.5
10
11
S
S
>20000
>20000
>20000
>20000
>20000
1
1
1
1
>200000
12
13
S
S
580
4000
>100000
>20000
>170
>3
>25
1
7200
>20000
14
15
S
S
>20000
>20000
>20000
>20000
>20000
>20000
1
1
1
1
(Continues)

RAMSIS ET AL.
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TABLE 1 (Continued)
Cpd #
16
Capping groups
Stereochemistry
EC50 (nM)
EC90 (nM)
CC50 (nM)
SI50
SI90
S
6100
>10000
>10000
>1.6
1
Sofosbuvir
70
300
>5000
>71
>17
Docking of compounds 5 (EC50 = 0.21 nM), and daclatasvir (I),
using the closed conformer of NS5A (PDB ID: 3FQM), showed that the
two compounds bind to the binding pocket with nearly the same pose.
The 2D interactions of the two molecules showed hydrogen bonding
with Gln54 (Figure 3A–C).
uncorrected. 1H NMR spectra were recorded at 300 MHz using Varian
Mercury VX-300 MHz spectrometer. 13C NMR spectra were recorded
at 126 MHz using a Bruker DRX-126 MHz spectrometer. The solvents
used were DMSO-d6 or CDCl3. Chemical shifts are given in parts per
million (ppm), and all coupling constants (J) are given in Hz. The purities
of the tested compounds were determined by HPLC coupled with
mass spectrometry and were all higher than 95% purity unless
otherwise indicated. Mass spectrometric analysis (HPLC-ESI-MS) was
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3
CONCLUSION
performed on
a TSQ quantum (Thermo Electron Corporation)
The chemical space of NS5A inhibitors can be extrapolated beyond the
methylcarbamate of l-valine. The dynamic and kinetics of new
derivatives with unnatural amino acids is still to be investigated. To
further confirm that the novel compounds act via inhibiting NS5A
protein, more replicon assays are required using resistant NS5A
variant, this can validate NS5A as the potential target of the novel
bivalent compounds.
instrument equipped with an ESI source and a triple quadrupole
mass detector. All masses were reported as [M+H]+. Column
chromatography was performed using silica gel 70–230 mesh.
Reaction progress was monitored by TLC using fluorescent pre-
coated silica gel plates and detection of the components was made by
short UV light (λ = 254 nm). Reactions were carried out under argon
when needed.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
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4
EXPERIMENTAL
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4.1 Chemistry
4.1.2 General procedures for the preparation of the
amino acid carbamates[14]
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4.1.1 General
Distilled H2O (100 mL) and NaOH (100 mmol, 4.0 g) were added to a
500 mL round bottom flask equipped with a magnetic stirring bar and
the solution was cooled to 0°C in an ice bath. The appropriate amino
acid (35 mmol) was added and stirred until a homogeneous solution
All starting materials and solvents were obtained from commercial
suppliers, and were used without further purification. Melting points
were determined on a Büchi B-540 melting point apparatus and are
TABLE 2 Replicon assay results for compounds 1–3 against HCV genotype 2a
Cpd #
1
Capping groups
Stereochemistry
EC50 (nM)
EC90 (nM)
CC50 (nM)
SI50
SI90
R
610
5340
>20000
>33
>4
2
3
R
R
450
550
1730
>20000
>20000
>44
87
>12
20
11250

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RAMSIS ET AL.
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and DIPEA (0.7 mL) in CH2Cl2. The reaction mixture was stirred for 2 h
at room temperature under N2. The volatile component was removed
in vacuo. The residue was then purified using silica gel column
chromatography (98.5:1.5 CH2Cl2/MeOH) to provide compounds
1–16.
Diethyl((2R,2′R)-((2S,2′S)-2,2′-(([1,1′-biphenyl]4,4′diylbis(azane-
diyl))bis(carbonyl))bis(pyrrolidine-2,1diyl))bis(3-methyl-1-
oxobut-ane-2,1-diyl))dicarbamate (1)
FIGURE 2 Showing non-coplanarity of the two core phenyls with
dihyderal angle of 135.7°
Dark brown semi-solid; yield: 26.4%; C38H52N6O8; FT-IR (cm−1): 3210
+
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was obtained. The corresponding chloroformate (45.5 mmol) in 1,4-
dioxane (40 mL) was added dropwise through an addition funnel. The
reaction mixture was then allowed to stir at room temperature
overnight. The solution was extracted with Et2O (3 × 50 mL), and the
organic layers were discarded. The aqueous layer was again cooled to
0°C in an ice bath, and concentrated HCl was added dropwise until
pH = 2. The aqueous solution was again extracted with
Et2O (3 × 50 mL). The aqueous layer was discarded and the organic
layers were combined, dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo to give the crude product as clear viscous oil.
Further purification was not required.
(N-H), 1692 (C O), 1640 (C O), 1602 (CO); LC/MS: [M+H] = 721;
purity 95%; 1H NMR (300 MHz, CDCl3) δ 9.25 (s, 2H), 7.58 (d,
J = 8.2 Hz, 4H), 7.38 (d, J = 8.4 Hz, 4H), 5.35 (d, J = 8.2 Hz, 2H), 4.76 (d,
J = 6.4 Hz, 2H), 4.26 (t, J = 7.9 Hz, 2H), 4.17–3.95 (m, 6H), 3.70–3.56
(m, 2H), 2.49 (s, 2H), 2.17 (s, 2H), 2.10–1.97 (m, 6H), 1.20 (t, J = 7.1 Hz,
6H), 1.03 (dd, J = 6.7, 3.3 Hz, 12H); 13C NMR (126 MHz, CDCl3): δ
179.13, 174.58, 156.57, 136.00, 126.53, 126.38, 120.34, 61.36,
58.81, 46.95, 31.10, 19.11, 17.44, 14.58, 8.80.
Diisobutyl((2R,2′R)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-
methyl-1-oxobutane-2,1-diyl))dicarbamate (2)
Dark brown semi-solid; yield: 25%; C42H60N6O8; FT-IR (cm−1): 3210
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4.1.3 General procedures for the N-protected
+
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(N-H), 1676 (C O), 1635 (C O), 1610 (C O); LC/MS: [M+H] = 777;
purity 95%; 1H NMR (300 MHz, CDCl3): δ 9.07 (s, 2H), 7.65 (d,
J = 8.4 Hz, 4H), 7.45 (d, J = 8.2 Hz, 4H), 5.28 (s, 2H), 4.77 (d, J = 6.9 Hz,
2H), 4.47 (s, 2H), 4.01 (s, 2H), 3.81 (d, J = 6.5 Hz, 4H), 3.53 (d, J = 8.0 Hz,
2H), 2.09 (s, 8H), 1.90–1.71 (m, 2H), 1.63 (dd, J = 11.5, 7.1 Hz, 2H), 1.00
(dd, J = 6.5, 2.6 Hz, 12H), 0.86 (t, J = 7.4 Hz, 12H); 13C NMR (126 MHz,
CDCl3): δ 173.22, 169.00, 157.30, 137.31, 136.49, 126.99, 120.53,
61.32, 51.52, 47.26, 40.85, 27.98, 25.56, 24.25, 22.41, 21.87, 19.26,
19.01.
benzidine prolinamide[12]
EEDQ (91.3 mmol) was added to a stirred solution of N-Boc-L-proline
(18.83 g, 87.5 mmol) and benzidine (38.1 mmol) in dry CH2Cl2
(250 mL). The mixture was stirred at room temperature for 16 h under
N2. The volatile component was removed in vacuo. The residue was
washed with Et2O and suction-filtered to provide the respective
compounds as white solid with >95% yield. Further purification was
not required.
Dibutyl((2R,2′R)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-
methyl-1-oxo-butane-2,1-diyl))dicarbamate (3)
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4.1.4 General procedures for the deprotected
benzidine prolinamide[15]
N-Boc-proline intermediates (0.04 mol) in CH2Cl2 were treated with
CF3CO2H (60 mL) at room temperature under N2. The mixture was
stirred for 3 h before additional CF3CO2H (24 mL) was added. After
being stirred for an additional 4 h period at room temperature, the
volatile component was evaporated in vacuo. The residue was then
dissolved in EtOAc, washed with saturated NaHCO3 solution and brine.
Some 1 N NaOH was added till pH reached 12. After that, the residue
was dried over anhydrous Na2SO4. EtOAc was evaporated in vacuo to
give a pale yellow sticky solid. This semi-solid was dissolved in acetone
and evaporated in vacuo. This step was repeated two to three times to
give a pale yellow crystalline solid. Further purification wasnotrequired.
Off-white solid; yield: 25%; mp: 81–82°C; C42H60N6O8; FT-IR (cm−1):

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3211 (N-H), 1689 (C O), 1631 (C O), 1600 (C O); LC/MS:
[M+H]+ = 777; purity 95%; 1H NMR (300 MHz, CDCl3): δ 9.07 (s,
2H), 7.66 (d, J = 8.3 Hz, 4H), 7.44 (d, J = 8.3 Hz, 4H), 5.39 (s, 2H), 4.76 (d,
J = 6.8 Hz, 2H), 4.46 (s, 2H), 4.02 (dd, J = 14.5, 7.0 Hz, 6H), 3.14 (s, 6H),
2.51 (s, 2H), 2.07 (s, 4H), 1.52 (td, J = 13.7, 6.7 Hz, 8H), 0.99 (dd, J = 6.3,
2.8 Hz, 12H), 0.86 (t, J = 7.3 Hz, 6H); 13C NMR (126 MHz, CDCl3) δ
172.96, 169.01, 157.59, 136.96, 127.56, 126.98, 120.65, 65.55,
61.28, 58.68, 47.02, 28.60, 24.57, 19.03, 8.79.
Dibenzyl((2R,2′R)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-
methyl-1-oxo-butane-2,1-diyl))dicarbamate (4)
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4.1.5 General procedures for the preparation of the
Pale yellow solid; yield: 34.5%; mp: 109–111°C; C48H56N6O8;
final compounds (1–16)[12]
FT-IR (cm−1): 3300 (N-H), 1694 (C O), 1631 (CO), 1605 (C O);
LC/MS: [M+H]+ = 845; purity 95%; 1H NMR (300 MHz, CDCl3): δ
9.19 (s, 2H), 7.60 (d, J = 8.6 Hz, 4H), 7.41 (d, J = 8.6 Hz, 4H), 7.28


Deprotected benzidine prolinamide (0.003 mol) was added to the
amino acid carbamates (0.4 g, 0.001 mol), HBTU (1.13 g, 0.003 mol)

RAMSIS ET AL.
7
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FIGURE 3 (A) An overlay of the best docked poses of compound 5 (cyan) and daclatasvir (pink), in the receptor binding pocket. The
receptor and polar hydrogens were removed for clarity. (B and C) The 2D ligand interaction shows interaction by hydrogen bonding between
the amide or imidazole with residue Gln54
(d, J = 3.0 Hz, 10H), 5.41 (s, 2H), 5.06 (dd, J = 32.7, 12.3 Hz, 4H),
4.78 (d, J = 7.7 Hz, 2H), 4.26 (s, 2H), 3.60 (s, 4H), 2.55 (s, 2H), 2.12–
1.99 (m, 8H), 1.05–1.01 (m, 12H); 13C NMR (126 MHz, CDCl3) δ
172.52, 168.93, 156.82, 137.28, 136.32, 128.29, 127.61,
126.83, 120.29, 67.41, 61.19, 58.42, 47.70, 30.89, 24.67, 19.50,
18.26.

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RAMSIS ET AL.
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Diethyl((2R,2′R)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-
methyl-1-oxo-pentane-2,1-diyl))dicarbamate (5)
Diethyl((2S,2′S)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-
methyl-1-oxo-butane-2,1-diyl))dicarbamate (9)
Yellow oil; yield: 20%; C38H52N6O8; FT-IR (cm−1): 3248 (N-H), 1680
Pale yellow solid; yield: 28%; mp: 127–129°C; C40H56N6O8; FT-IR
+
(cm−1): 3275 (N-H), 1689 (CO), 1632 (C O), 1600 (C O); LC/MS:
[M+H]+ = 748; purity 99%; 1H NMR (300 MHz, CDCl3): δ 9.09 (s, 2H),
7.65 (d, J = 8.2 Hz, 4H), 7.45 (d, J = 8.4 Hz, 4H), 5.32 (s, 2H), 4.77 (d,
J = 6.5 Hz, 2H), 4.48 (s, 2H), 4.17–3.94 (m, 6H), 3.53 (d, J = 7.8 Hz, 2H),
1.99 (s, 8H), 1.76 (s, 2H), 1.63 (t, J = 11.7 Hz, 2H), 1.47 (s, 2H), 1.20 (t,
J = 7.1 Hz, 6H), 1.00 (dd, J = 6.4, 4.2 Hz, 12H); 13C NMR (126 MHz,
CDCl3): δ 173.21, 169.00, 157.11, 137.34, 136.39, 126.98, 120.44,
61.35, 51.48, 47.44, 40.98, 28.39, 24.66, 21.84, 14.57.
(CO), 1635 (C O), 1619 (CO); LC/MS: [M+H] = 777; purity 98%;



1H NMR (300 MHz, CDCl3) δ 9.50 (s, 2H), 7.40 (d, J = 8.2 Hz, 4H), 7.30
(d, J = 8.2 Hz, 4H), 5.28 (s, 2H), 4.78 (d, J = 5.3 Hz, 2H), 4.36 (d,
J = 6.6 Hz, 2H), 4.16–4.04 (m, 4H), 3.80 (d, J = 8.6 Hz, 2H), 3.66 (s, 2H),
2.45 (s, 2H), 2.18 (s, 2H), 2.09–1.99 (m, 4H), 1.95 (s, 2H), 1.24 (t,
J = 7.0 Hz, 12H), 1.00 (d, J = 6.7 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ
175.22, 173.58, 155.55, 136.33, 126.53, 126.56, 121.05, 62.36,
59.07, 47.13, 31.10, 19.45, 18.14, 15.52, 9.00.
Diisobutyl((2R,2′R)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-
methyl-1-oxo-pentane-2,1-diyl))dicarbamate (6)
Diisobutyl((2S,2′S)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-
methyl-1-oxo-butane-2,1-diyl))dicarbamate (10)
Pale yellow solid; yield: 25%; mp: 117–118°C; C44H64N6O8; FT-IR
Dark yellow semi-solid; yield: 22%; C42H60N6O8; FT-IR (cm−1): 3225
(N-H), 1680 (CO), 1632 (CO), 1622 (CO); LC/MS: [M+H]+ = 777; purity
98%; 1H NMR (300 MHz, DMSO) δ 10.08 (s, 2H), 7.62 (d, J = 8.6 Hz,
4H), 7.56 (d, J = 8.6 Hz, 4H), 4.41 (d, J = 23.8 Hz, 2H), 4.02 (t, J = 8.3 Hz,
2H), 3.86–3.76 (m, 4H), 3.71 (d, J = 6.7 Hz, 4H), 3.62 (s, 2H), 2.05–1.94
(m, 4H), 1.91–1.85 (m, 4H), 1.84–1.76 (m, 4H), 0.88–0.84 (m, 24H).
13C NMR (126 MHz, DMSO) δ 170.73, 157.01, 138.68, 134.72,
126.88, 119.83, 70.42, 60.69, 59.95, 58.20, 46.37, 30.28, 29.95,
29.88, 28.11, 25.12, 19.59, 19.31, 19.04, 18.50.
(cm−1): 3282 (N-H), 1689 (C O), 1633 (C O), 1603 (C O); LC/MS:
[M+H]+ = 804; purity 100%; 1H NMR (300 MHz, CDCl3): δ 9.07 (s, 2H),
7.65 (d, J = 8.4 Hz, 4H), 7.45 (d, J = 8.2 Hz, 4H), 5.28 (s, 2H), 4.77 (d,
J = 6.9 Hz, 2H), 4.47 (s, 2H), 4.01 (s, 2H), 3.81 (d, J = 6.5 Hz, 4H), 3.53 (d,
J = 8.0 Hz, 2H), 2.09 (s, 8H), 1.90–1.71 (m, 6H), 1.63 (dd, J = 11.5,
7.1 Hz, 2H), 1.00 (dd, J = 6.5, 2.6 Hz, 12H), 0.86 (t, J = 7.4 Hz, 12H);
13C NMR (126 MHz, CDCl3): δ 173.22, 169.00, 157.30, 137.31,
136.49, 127.17, 120.53, 61.32, 51.52, 47.26, 40.85, 27.98, 24.25,
23.37, 22.41, 19.01.



Dibutyl((2S,2′S)-((2S,2′S)-(([1,1′-biphenyl]-4,4′-
Dibutyl((2R,2′R)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-
methyl-1-oxo-pentane-2,1-diyl))dicarbamate (7)
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-
methyl-1-oxo-butane-1,2-diyl))dicarbamate (11)
White solid; yield: 24%; mp: 100–103; C42H60N6O8; FT-IR (cm−1):
Pale yellow solid; yield: 24%; mp: 85–87°C; C44H64N6O8; FT-IR (cm−1):
3250 (N-H), 1689 (C O), 1630 (CO), 1609 (C O); LC/MS:
[M+H]+ = 777; purity 96%; 1H NMR (300 MHz, DMSO) δ 10.02 (d,
J = 22.5 Hz, 2H), 8.80 (s, 2H), 7.62 (d, J = 8.8 Hz, 4H), 7.56 (d, J = 8.8 Hz,
4H), 4.43 (dt, J = 15.4, 7.8 Hz, 2H), 4.08–3.98 (m, 2H), 3.97–3.85 (m,
4H), 3.78 (t, J = 11.6 Hz, 2H), 3.67–3.56 (m, 2H), 2.67 (s, 2H), 2.14 (dd,
J = 12.6, 8.1 Hz, 2H), 1.99 (dd, J = 14.3, 8.2 Hz, 2H), 1.88 (dd, J = 15.4,
10.0 Hz, 4H), 1.57–1.40 (m, 4H), 1.38–1.24 (m, 4H), 0.93 (d, J = 6.7 Hz,
6H), 0.85 (dd, J = 15.1, 7.9 Hz, 12H). 13C NMR (126 MHz, DMSO) δ
170.84, 156.91, 138.68, 134.75, 126.82, 119.77, 64.06, 60.64, 58.30,
47.65, 46.21, 38.68, 31.18, 30.26, 29.88, 25.13, 19.36, 19.00, 14.06,
9.08.



3300 (N-H), 1688 (CO), 1631 (CO), 1601 (C O); LC/MS:
[M+H]+ = 804; purity 99%; 1H NMR (300 MHz, CDCl3): δ 9.07 (s,
2H), 7.66 (d, J = 8.3 Hz, 4H), 7.44 (d, J = 8.3 Hz, 4H), 5.39 (s, 2H), 4.76 (d,
J = 6.8 Hz, 2H), 4.46 (s, 2H), 4.02 (dd, J = 14.5, 7.0 Hz, 6H), 3.14 (s, 6H),
2.51 (s, 2H), 2.07 (s, 6H), 1.76 (s, 2H), 1.52 (td, J = 13.7, 6.7 Hz, 8H),
0.99 (dd, J = 6.3, 2.8 Hz, 12H), 0.86 (t, J = 7.3 Hz, 6H); 13C NMR
(126 MHz, CDCl3): δ 173.28, 169.08, 157.30, 137.29, 136.47, 126.97,
120.53, 65.52, 61.35, 51.51, 46.90, 40.78, 30.97, 28.25, 24.69, 23.46,
21.83, 19.04, 13.72, 9.00.
Dibenzyl((2R,2′R)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-
methyl-1-oxo-pentane-2,1-diyl))dicarbamate (8)
Dibenzyl((2S,2′S)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-
methyl-1-oxo-butane-2,1-diyl))dicarbamate (12)
Pale yellow solid; yield: 25%; mp: 81–83°C; C50H60N6O8; FT-IR (cm−1):
Yellow oil; yield: 25%; C48H56N6O8; FT-IR (cm−1): 3285 (N-H), 1697


3300 (N-H), 1693 (C O), 1631 (CO), 1600 (C O); LC/MS:
[M+H]+ = 872; purity: 95%; 1H NMR (300 MHz, CDCl3): δ 9.06 (s,
2H), 7.63 (d, J = 8.6 Hz, 4H), 7.43 (d, J = 8.6 Hz, 4H), 7.28 (d, J = 4.7 Hz,
10H), 5.42 (s, 2H), 5.07 (q, J = 12.2 Hz, 4H), 4.75 (d, J = 6.9 Hz, 2H), 4.50
(s, 2H), 4.00 (s, 2H), 3.53 (s, 2H), 2.99 (s, 2H), 2.55 (s, 4H), 2.09 (s, 6H),
1.76 (s, 2H), 1.00 (t, J = 5.4 Hz, 12H); 13C NMR (126 MHz, CDCl3): δ
173.19, 169.13, 156.30, 137.25, 128.29, 127.06, 120.52, 67.27,
61.28, 51.63, 46.94, 40.97, 28.26, 24.65, 8.79.
+


(C O), 1625 (C O), 1600 (CO); LC/MS: [M+H] = 845; purity 95%;
1H NMR (300 MHz, DMSO) δ 10.05 (d, J = 28.6 Hz, 2H), 7.62 (d,
J = 8.7 Hz, 4H), 7.56 (d, J = 8.7 Hz, 4H), 7.39–7.22 (m, 10H), 5.19–4.85
(m, 4H), 4.45 (dd, J = 8.6, 5.3 Hz, 2H), 4.06 (t, J = 8.3 Hz, 2H), 3.80 (d,
J = 6.2 Hz, 2H), 3.62 (d, J = 8.6 Hz, 2H), 2.10 (d, J = 32.2 Hz, 2H), 2.04–
1.79 (m, 8H), 1.21 (s, 2H), 0.89 (dt, J = 30.8, 10.9 Hz, 12H). 13C NMR
(126 MHz, DMSO) δ 170.71, 156.54, 138.85, 137.47, 134.87, 128.78,

RAMSIS ET AL.
9
|
128.46, 128.21, 128.08, 126.83, 126.81, 119.94, 65.83, 60.64, 58.41,
30.29, 29.89, 25.12, 19.36, 19.02.
(dd, J = 19.4, 12.3 Hz, 2H), 3.60–3.48 (m, 2H), 2.22–2.07 (m, 2H), 2.07–
1.95 (m, 2H), 1.89 (ddd, J = 17.2, 11.9, 5.9 Hz, 4H), 1.65 (d, J = 9.7 Hz,
4H), 1.53–1.33 (m, 4H), 0.88 (d, J = 6.6 Hz, 12H). 13C NMR (126 MHz,
DMSO) δ 171.14, 170.83, 156.60, 138.65, 137.57, 134.61, 128.77,
128.46, 128.22, 128.12, 126.78, 119.80, 65.64, 63.23, 60.47, 51.25,
47.39, 29.66, 25.23, 24.47, 23.55, 21.71.
Diethyl((2S,2′S)-((2S,2′S)-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-
methyl-1-oxo-pentane-1,2-diyl))dicarbamate (13)
Yellow oil; yield: 30%; C40H56N6O8; FT-IR (cm−1): 3275 (N-H), 1686
+


(C O), 1633 (C O), 1601 (CO); LC/MS: [M+H] = 748; purity 95%;
1H NMR (300 MHz, DMSO) δ 10.01 (s, 2H), 8.18 (s, 2H), 7.62 (d,
J = 8.8 Hz, 4H), 7.58–7.54 (m, 4H), 4.50–4.39 (m, 2H), 4.27 (s, 2H), 3.95
(dt, J = 6.7, 3.4 Hz, 4H), 3.62–3.57 (m, 4H), 2.18–2.09 (m, 2H), 2.02 (dd,
J = 12.2, 6.7 Hz, 2H), 1.96–1.83 (m, 4H), 1.66 (s, 2H), 1.48–1.38 (m,
4H), 1.15–1.11 (m, 6H), 0.91–0.86 (m, 12H). 13C NMR (126 MHz,
DMSO) δ 174.92, 170.85, 156.74, 138.70, 134.75, 126.80, 119.77,
60.20, 54.02, 52.41, 51.15, 47.12, 42.27, 24.73, 23.33, 21.56, 18.50,
17.17, 15.04, 12.91.
|
4.2 Biology
|
4.2.1 HCV anti-viral activity using genotype 1b,
CON-1 strain replicon and genotype 2a, JFH-1 strain
replicon[16–18]
Huh-7 cells were cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% heat-inactivated fetal bovine
serum (FBS), 0.1 mM nonessential amino acids, and 1× penicillin-
streptomycin-glutamine.
Diisobutyl((2S,2′S)-((2S,2′S)-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-
methyl-1-oxo-pentane-1,2-diyl))dicarbamate (14)
The cells were infected with replicons of HCV genotype 1b, BM4-
5 FEO or genotype 2a, JFH-1 (GenBank accession number AB047639),
containing a firefly luciferase-neomycompounds cin phosphotransfer-
ase fusion (FEO) protein. This has been accomplished by electropora-
tion as previously described.[16] Compound activity assays were
carried out using 10000 replicon cells per well on 96-well plates.[16]
Compounds were added 24–120 h after transfection or infection with
supernatant. Cells and compounds were then incubated for 72 or
120 h with all conditions run in triplicate. Each experiment was
performed on three separate occasions.
Brownish oil; yield: 24.4%; C44H64N6O8; FT-IR (cm−1): 3282 (N-H),
+

1686 (CO), 1630 (CO), 1605 (C O); LC/MS: [M+H] = 804; purity
100%; 1H NMR (300 MHz, CDCl3) δ 10.07 (s, 2H), 7.65 (d, J = 8.4 Hz,
4H), 7.35 (d, J = 8.3 Hz, 4H), 5.40 (s, 2H), 4.64 (d, J = 6.6 Hz, 2H), 4.40 (s,
2H), 4.01 (s, 2H), 3.77 (d, J = 6.4 Hz, 4H), 3.47 (d, J = 8.0 Hz, 2H), 2.11 (s,
8H), 1.92–1.77 (m, 6H), 1.66 (dd, J = 11.0, 7.1 Hz, 2H), 1.00 (dd, J = 6.3,
2.6 Hz, 12H), 0.88 (t, J = 7.6 Hz, 12H). 13C NMR (126 MHz, DMSO) δ
170.34, 156.33, 138.14, 134.26, 126.31, 125.05, 119.31, 76.39,
69.78, 60.13, 50.66, 45.72, 29.23, 27.60, 24.65, 24.06, 23.13, 21.27,
18.80, 8.58.
4.2.2 Determination of compounds cytotoxicity[19]
|
The cytotoxicity of compounds 1–16 on uninfected Huh-7 cells was
determined using MTT method as described.[19] The 50% cytotoxicity
concentration (CC50) was calculated from the optical density data
using nonlinear regression curve fitting to the 4-parameter logistic
equation and GraphPad Prism 4 software.
Dibutyl((2S,2′S)-((2S,2′S)-2,2′-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-
methyl-1-oxo-pentane-2,1-diyl))dicarbamate (15)
Pale white solid; yield: 26%; mp: 105–107°C; C44H64N6O8; FT-IR
(cm−1): 3280 (N-H), 1685 (C O), 1636 (C O), 1610 (CO); LC/MS:
[M+H]+ = 804; purity 98%; 1H NMR (300 MHz, CDCl3) δ 9.16 (s, 2H),
7.76 (d, J = 8.2 Hz, 4H), 7.54 (d, J = 8.5 Hz, 4H), 5.36 (s, 2H), 4.56 (d,
J = 6.6 Hz, 2H), 4.30 (s, 2H), 4.02 (dd, J = 14.4, 7.2 Hz, 6H), 3.22 (s, 6H),
2.60 (s, 2H), 2.10 (s, 6H), 1.78 (s, 2H), 1.45 (td, J = 13.5, 6.4 Hz, 8H),
1.01 (dd, J = 6.2, 2.6 Hz, 12H), 0.86 (t, J = 7.2 Hz, 6H). 13C NMR
(126 MHz, CDCl3) δ 174.42, 168.68, 157.25, 138.09, 136.57, 127.07,
121.33, 65.74, 61.58, 52.48, 46.98, 41.64, 31.20, 27.09, 24.80, 23.23,
22.00, 18.88, 13.58, 8.90.


|
4.3 Molecular modeling
Docking experiments were implemented to dock compound 5 and
daclatasvir (I) into the proposed active site of NS5A with the program
MOE version 2015.10.12 Compounds were constructed into the MOE
window, then a conformational search was done for each of them using
systematic method, and the databases were saved. The Protein Data
Bank (PDB) crystal structure of NS5A co-crystallized with any NS5A
inhibitor was not available therefore the binding pocket was defined.[11]
The PDB crystal structure of NS5A (PDB ID: 3FQM) was imported into
MOE. The protein structure was protonated and atomic charges were
assigned. The binding pocket was defined using the site finder feature,
other residues involved in viral mutation were also included in the
defined pocket namely Gln54, Tyr93, Thr95, and Pro97, additionally
residues previously claimed to be involved in H-bonding/hydrophobic
interaction with daclatasvir (I) were included. The binding pocket was
selected and extended 4.5 Å around the pocket. A database of
Dibenzyl((2S,2′S)-((2S,2′S)-(([1,1′-biphenyl]-4,4′-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-
methyl-1-oxo-pentane-1,2-diyl))dicarbamate (16)
Yellow oil; yield: 29%; C38H52N6O8; FT-IR (cm−1): 3294 (N-H), 1689
+

(CO), 1636 (C O), 1608 (CO); LC/MS: [M+H] = 872; purity 95%;
1H NMR (300 MHz, DMSO) δ 10.11 (s, J = 61.3 Hz, 2H), 7.62 (d,
J = 8.7 Hz, 4H), 7.56 (d, J = 8.7 Hz, 4H), 7.35–7.28 (m, 10H), 4.96 (d,
J = 29.6 Hz, 4H), 4.45 (dd, J = 8.2, 4.9 Hz, 2H), 4.34–4.17 (m, 2H), 3.68

10
RAMSIS ET AL.
|
compound 5 and daclatasvir (I) was prepared as .mdb file then docking
into the defined pocket was launched. The poses of the ligand
conformation were generated using Triangle Matcher; the scoring
function used was London dG, with refinement using Force field.
[9] M. J. Evans, C. M. Rice, S. P. Goff, Proc. Natl. Acad. Sci. 2004, 101, 13038.
[10] P. Neddermann, M. Quintavalle, C. Di Pietro, A. Clementi, M.
Cerretani, S. Altamura, L. Bartholomew, R. De Francesco, J. Virol.
2004, 78, 13306.
[11] K. H. Barakat, A. Anwar-Mohamed, J. A. Tuszynski, M. J. Robins, D. L.
Tyrrell, M. Houghton, J. Chem. Inf. Model. 2015, 55, 362.
[12] I. H. Bae, J. K. Choi, C. Chough, S. J. Keum, H. Kim, S. K. Jang, B. M.
Kim, ACS Med. Chem. Lett. 2014, 5, 255.
ACKNOWLEDGMENTS
[13] M. O. E., Chemical Computing Group Inc., 1010 Sherbooke St. West,
Suite #910, Montreal, QC, Canada, H3A 2R7, 2017.
This project is partially financed by the Science and Technology
Development Fund (STDF), Egypt, Grant No: 22848, to Ashraf H.
Abadi. The authors are grateful to the authority of the National
Institute of Allergic and Infectious Diseases (NIAID), Division of
Microbiology and Infectious Diseases (DMID), Rockville, MD 20852,
USA, and the Southern Research Institute (SRI), Alabama for
performing the biological testing in part.
[14] K. M. Engle, D.-H. Wang, J.-Q. Yu, J. Am. Chem. Soc. 2010, 132,
14137.
[15] D. R. St Laurent, M. H. Serrano-Wu, M. Belema, M. Ding, H. Fang, M.
Gao, J. T. Goodrich, R. G. Krause, J. A. Lemm, M. Liu, O. D. Lopez, V. N.
Nguyen, P. T. Nower, D. R 2nd O'Boyle, B. C. Pearce, J. L. Romine, L.
Valera, J. H. Sun, Y. K. Wang, F. Yang, X. Yang, N. A. Meanwell, L. B.
Snyder, J. Med. Chem. 2014, 57, 1976.
[16] D. L. Wyles, K. A. Kaihara, F. Vaida, R. T. Schooley, J. Virol. 2007, 81, 3005.
[17] T. Kato, T. Date, M. Miyamoto, A. Forsake, K. Tokushige, M.
Mizokami, T. Wakita, Gastroenterology 2003, 125, 1808.
[18] S. Hopkins, B. Scorneaux, Z. Huang, M. G. Murray, S. Wring, C.
Smitley, R. Harris, F. Erdmann, G. Fisher, Y. Ribeill, Antimicrob. Agents
Chemother. 2010, 54, 660.
ORCID
Ashraf H. Abadi
http://orcid.org/0000-0002-7433-261X
[19] C. J. Halfman, Methods Enzymol. 1981, 74, 481.
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Additional supporting information may be found online in the
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