Design and synthesis of novel symmetric fluorene-2,7-diamine derivatives as potent hepatitis C virus inhibitors

Article abstract of DOI:10.3390/ph14040292

Hepatitis C virus (HCV) is an international challenge. Since the discovery of NS5A direct-acting antivirals, researchers turned their attention to pursue novel NS5A inhibitors with optimized design and structure. Herein we explore highly potent hepatitis C virus (HCV) NS5A inhibitors; the novel analogs share a common symmetrical prolinamide 2,7-diaminofluorene scaffold. Modification of the 2,7-diaminofluorene backbone included the use of (S)-prolinamide or its isostere (S,R)-piperidine-3-caboxamide, both bearing different amino acid residues with terminal carbamate groups. Compound 26 exhibited potent inhibitory activity against HCV genotype (GT) 1b (effective concentration (EC₅₀) = 36 pM and a selectivity index of >2.78 × 10⁶). Compound 26 showed high selectivity on GT 1b versus GT 4a. Interestingly, it showed a significant antiviral effect against GT 3a (EC₅₀ = 1.2 nM). The structure-activity relationship (SAR) analysis revealed that picomolar inhibitory activity was attained with the use of S-prolinamide capped with R- isoleucine or R-phenylglycine residues bearing a terminal alkyl carbamate group.

Full text of DOI:10.3390/ph14040292

pharmaceuticals
Article
Design and Synthesis of Novel Symmetric
Fluorene-2,7-Diamine Derivatives as Potent Hepatitis C
Virus Inhibitors
Mai H. A. Mousa 1, Nermin S. Ahmed 1,*, Kai Schwedtmann 2, Efseveia Frakolaki 3, Niki Vassilaki 3,
Grigoris Zoidis 4 , Jan J. Weigand 2 and Ashraf H. Abadi 1,
*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology,
German University in Cairo, Cairo 11835, Egypt; mai.abdelsadek@guc.edu.eg
Faculty of Chemistry and Food Chemistry, Technische Universität Dresden, 01062 Dresden, Germany;
Kai.Schwedtmann@chemie.tu-dresden.de (K.S.); jan.weigand@tu-dresden.de (J.J.W.)
Molecular Virology Laboratory, Hellenic Pasteur Institute, 11521 Athens, Greece; sevif@pasteur.gr (E.F.);
nikiv@pasteur.gr (N.V.)
Department of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences,
National and Kapodistrian University of Athens, 15771 Athens, Greece; zoidis@pharm.uoa.gr
Correspondence: nermin.salah@guc.edu.eg (N.S.A.); ashraf.abadi@guc.edu.eg (A.H.A.);
Tel.: +202-27590700 (ext. 3429) (N.S.A.); +202-27590700 (ext. 3400) (A.H.A.);
Fax: +202-27581041 (N.S.A. & A.H.A.)
2
3
4
*
Abstract: Hepatitis C virus (HCV) is an international challenge. Since the discovery of NS5A
direct-acting antivirals, researchers turned their attention to pursue novel NS5A inhibitors with
optimized design and structure. Herein we explore highly potent hepatitis C virus (HCV) NS5A
inhibitors; the novel analogs share a common symmetrical prolinamide 2,7-diaminofluorene scaffold.
Modification of the 2,7-diaminofluorene backbone included the use of (S)-prolinamide or its isostere
(S,R)-piperidine-3-caboxamide, both bearing different amino acid residues with terminal carbamate
groups. Compound 26 exhibited potent inhibitory activity against HCV genotype (GT) 1b (effective
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Mousa, M.H.A.; Ahmed,
N.S.; Schwedtmann, K.; Frakolaki, E.;
Vassilaki, N.; Zoidis, G.; Weigand, J.J.;
Abadi, A.H. Design and Synthesis of
Novel Symmetric
concentration (EC50) = 36 pM and a selectivity index of >2.78
×
106). Compound 26 showed high
Fluorene-2,7-Diamine Derivatives as
Potent Hepatitis C Virus Inhibitors.
Pharmaceuticals 2021, 14, 292.
selectivity on GT 1b versus GT 4a. Interestingly, it showed a significant antiviral effect against GT 3a
(EC50 = 1.2 nM). The structure-activity relationship (SAR) analysis revealed that picomolar inhibitory
activity was attained with the use of S-prolinamide capped with R- isoleucine or R-phenylglycine
residues bearing a terminal alkyl carbamate group.
https://doi.org/10.3390/ph14040292
Academic Editor: Serena Massari
Keywords: DAAs; 2,7-Diaminofluorene; HCV; NS5A inhibitors; prolinamide; replicon assay
Received: 18 February 2021
Accepted: 16 March 2021
Published: 25 March 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Hepatitis C Virus (HCV) infection represents a disease of major global impact that
afflicts around 71 million people worldwide. A minor portion of infected HCV cases
spontaneously resolve, whereas predominantly patients develop chronic hepatitis C. This
further leads to a varying number of complications, ranging from mild inflammation to
fibrosis and cirrhosis, conferring significant morbidity and mortality to diseased individu-
als. The World Health Organization (WHO) evaluates around 200 million individuals to be
chronically infected, with 3–4 million new cases prevailing annually. The standard treat-
ment for HCV infection over the past decade was a combinatorial treatment with pegylated
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
interferon alpha (PEG-IFNα) and the antiviral nucleoside analog, ribavirin (RBV). This
regimen suffered from many side effects, contraindications, and a low sustained virologic
response of 40–60% [1–3].
Since 2011, directly acting antivirals (DAAs), including the viral non-structural pro-
tein (NS) 3/4A protease inhibitors, the NS5B RNA-dependent RNA polymerase (RdRp)
nucleotide-like inhibitors, and NS5A replication complex inhibitors, have received clinical
Pharmaceuticals 2021, 14, 292. https://doi.org/10.3390/ph14040292
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Pharmaceuticals 2021, 14, 292
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approval for use as part of a combinatorial therapy for hepatitis C and chronic hepatitis C
infections [4].
Non-structural protein 5A (NS5A), which plays a crucial role in the virus replication, is
composed of 447 amino acids, segregated into multiple discrete domains. The first 33 amino
acid residues are highly conserved in all genotypes. They form an amphipathic alpha-helix
crucial for anchoring NS5A to the recruitment of endoplasmic reticulum membrane and
lipid droplets [
5
]. The rest of the monomer consists of three structural domains (domains
]. Each NS5A
I, II, and III), which are separated by low complexity sequences (LCSs) [5,6
domain binds separately to the RNA 30-untranslated region (UTR), suggesting that NS5A
plays diverse roles in many stages of the virus replication cycle. Reports indicated a
role of NS5A during RNA replication and virion assembly in vitro and in vivo. Domain I
coordinates a Zn2+ binding motif per protein molecule. The zinc atom is coordinated by four
NS5A cysteine residues (Cys39, Cys57, Cys59, and Cys80), which highlights the importance
of this ion in regards to NS5A protein folding, stability, and multiple NS5A related protein
functions [
6,
7]. Domain II (aa 250–342) and III (aa 355–447) are less conserved among HCV
genotypes relative to domain I and are natively unfolded [8,9
]. Domain II was found to
stimulate RNA binding and HCV replication through interaction with the cellular protein
cyclophilin A (CyPA) [10]. This confirms the observation that CyPA inhibitors, such as
cyclosporine A (CsA), contribute to the inhibition of HCV replication [11]. Domain III is
mainly crucial for HCV RNA assembly. This further emphasizes the importance of this
protein as a potential drug target [12,13].
The discovery of novel NS5A inhibitors requires the development of several in vitro
assays (such as binding, enzymatic, and cell-based assays), in addition to the rational
design of targeted inhibitors through enzyme-inhibitor co-crystallization.
The dimeric orientation of NS5A has elicited scientific debates; the crystal structure of
the N-terminal region showed some remarkable differences. On the other hand, the lack of
an inhibitor-protein complex renders a structural-based drug design approach unfeasible.
Moreover, the NS5A protein shows no clear enzymatic function. These challenges
intricate the efforts for optimizing the structures of novel NS5A inhibitors. Thus, most
of the efforts for the development of NS5A inhibitors are driven by cell-based replicon
activities and ligand-based approaches [14].
Against all odds, NS5A inhibitors carry unique characteristics that make them attrac-
tive candidates for combinatorial therapy. NS5A inhibitors are reported as pan-genotypic
with remarkable rapid initial viral RNA decline, while they do not elicit cross-resistance
with other direct-acting antivirals (DAAs).
In 2010, daclatasvir (a) was discovered and investigated for its ability to inhibit HCV
Genotype-1 infection. Daclatasvir was approved by the US FDA in 2014 [15]. Since
then, developing new inhibitors that can target NS5A became a hot research topic and an
industrial goal [16]. The Chemical structure of the clinical candidate Daclatasvir (
a) and a
highly active reported benzidine prolinamide derivative (b) [17], are shown below.
Daclatasvir (a) bears a central biphenyl backbone linked to bis-imidazole rings and
symmetrical bis-proline moieties; the proline is terminally capped with methylcarbamate L-
valine. We recently developed novel NS5A inhibitors with a benzidine core and connected
L-proline as an amide functionality and a variety of capping groups; those compounds

Pharmaceuticals 2021, 14, 292
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lacked the bis-imidazole rings of daclatasvir yet still showed high potency. Compound (
was the most active of this series (EC50 = 6.7 pM) [17].
b)
We aim to further investigate modifications to symmetrical NS5A inhibitors. Da-
clatasvir, which is the prototype for them, has been found to be the main NS5A inhibitor in
many combination therapies currently found on the market. Thus, here, novel analogs of da-
clatasvir (a) are reported, with significant anti-HCV potency through further rigidification
of the core part by changing the benzidine moiety to the tricyclic fluorene ring. Addi-
tionally, L-proline amide was replaced by proline isostere (S,R)-piperidine-3-carboxamide.
Several amino acid residues with both S and R configurations were used, namely: valine,
leucine, isoleucine, and phenylglycine. Lastly, the terminal carbamate group was modified
to a methyl, ethyl, butyl, or benzyl group (Figure 1).
Figure 1. Structures of the novel analogs (1–32).
2. Results and Discussion
2.1. Chemistry
Compounds
nitro groups of 2,7-dinitrofluorene were efficiently reduced by using hydrated stannous
chloride in HCl to afford 2,7-diaminofluorene ( ) at a yield of 65%. Compound (I) was
1–28 were prepared using 2,7-dinitrofluorene as a starting material. The
I
coupled with N-Boc-L-proline (N-tert-butyloxycarbonyl-L-proline) carboxylic acid in the
presence of HBTU/TEA (1-hydroxy-7-azabenzotriazole/triethylamine), which resulted in
compound II (80% yield) [18]. Deprotection of the latter using TFA (trifluoroacetic acid)
provided compound III (75% yield). Amino acid carbamate derivatives (i–xxviii) were
coupled with compound III using HBTU in the presence of TEA in dimethylformamide
(DMF) as a solvent for 3 h at room temperature to yield compounds 1–28 (Scheme 1). Amino
acid carbamate derivatives (i-xxviii) were synthesized by reacting commercially available
amino acids with various chloroformates using reported procedures (Scheme 2) [19]. The
exact stereochemical assignment and substituents are shown in Table 1.

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Scheme 1. Synthesis of compounds (1–28). Reagents and conditions: (i
) SnCl2.2H2O, HCl/CH3COOH, 65 ◦C, 5 h,
yield: 65%; (ii) HBTU (1-hydroxy-7-azabenzotriazole), TEA (triethylamine), DMF, room temperature, 30 min, yield: 80%;
(iii) TFA/CH2Cl2, 24 h, room temperature, yield: 75%; (iv) HBTU, TEA, DMF, 3 h, room temperature, yield: 16–29%.
Scheme 2. Synthesis of carbamate derivatives (i–xxviii) [17,20,21] Reagents and conditions: (i) NaOH in 1,4-dioxane,
overnight, room temperature, yield: 48–50%.

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Table 1. Effective concentration (EC50), cytotoxic concentration (CC50), and selectivity index (SI50) of compounds (
1
–28
)
tested on Hepatitis C virus (HCV) genotype (GT) 1b replicons.
Code
R
Stereo-Chemistry *
R’
HCV Replicon 1b
O
O
S
N
S
N
N
H
N
H
O
O
(1-28)
R
O
R
O
*
*
NH
HN
OR'
R'O
EC50
(nM)
CC50
(nM)
a
1
2
3
4
5
6
7
8
S
S
-CH3
-CH2CH3
-C4H9
SI50
ND b
ND
>1000
>1000
966.90
801.40
102
>100,000
>100,000
>100,000
>100,000
79,550
S
S
-CH2C6H5
-CH3
>103
>125
780
R
R
R
R
-CH2CH3
-C4H9
981.40
45.42
>100,000
>100,000
>102
-CH2C6H5
>2202
9
S
S
S
S
R
R
-CH3
-CH2CH3
-C4H9
27.50
0.137
976.60
>1000
0.081
2.55
>100,000
85,510
>3636
622,344
68
10
11
12
13
14
65,950
-CH2C6H5
-CH3
46,450
ND
>100,000
>100,000
>1,238,850
>39,185
-CH2CH3

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Table 1. Cont.
Code
15
R
Stereo-Chemistry *
R’
HCV Replicon 1b
R
-C4H9
>1000
34,220
ND
ND
16
17
R
S
-CH2C6H5
>1000
10.66
>100,000
-CH3
-CH2CH3
-C4H9
>100,000
>100,000
>100,000
98,730
>9381
>664
ND
18
19
20
21
22
S
S
S
S
S
150.60
>1000
>1000
8.50
-CH2C6H5
-CH3
ND
87,510
10,298
-CH2CH3
7.49
>100,000
>13,349
23
24
25
S
S
R
-C4H9
-CH2C6H5
-CH3
2.25
1.12
>100,000
>100,000
95,390
>44,444
>89,606
669,874
0.142
26
27
28
R
R
-CH2CH3
-C4H9
0.036
4.03
>100,000
>100,000
>100,000
>2,779,322
>24,795
R
-CH2C6H5
94.55
>1058
655,556
ND
Daclatasvir
IV
—
—
—
—
0.027
17,700
44990
—
—
>1000
a
b
SI50 = CC50/EC50
;
ND: Not Determined; * absolute configuration of terminal AA.
The second series was synthesized by reacting compound
-3-carboxylic acid to yield compound IV (41% yield), which was then deprotected to give
compound (51% yield). The latter was coupled with methylcarbamate of different amino
acids utilizing the same coupling procedures to yield compounds 29 32. (Scheme 3) [19].
The exact stereochemical assignment and substituents are shown in Table 2.
I with N-Boc-(S,R)-piperidine
V
–

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◦
Scheme 3. Synthesis of compounds (29–32). Reagents and conditions: (i) SnCl2.2H2O, HCl/CH3COOH, 65 C, 5 h, yield:
65%; (ii) HBTU, TEA, DMF, room temperature, 30 min, yield: 41%; (iii) TFA/CH2Cl2, 24 h, room temperature, yield:
51%; (iv) HBTU, TEA, DMF, 3 h, room temperature, yield: 15–18%. For details of compounds characterization, see the
experimental section.
Table 2. EC50, CC50, and SI50 of compounds (29–32) tested on HCV (GT)1b replicon.
HCV Replicon 1b
R
Code
EC50
(nM)
CC50
(nM)
a
SI50
O
O
N
N
H
N
H
N
R
R
S
S
29-32
O
O
O
O
NH
HN
O
O
29
30
342.20
289.10
295.50
>100,000
>100,000
>292
>346
31
98,280
333
32
304.50
>100,000
>328
a
SI50 = CC50/EC50.

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2.2. Biological Studies
All the synthesized compounds were evaluated for their anti-HCV 1b and their cyto-
toxicity, from which the selectivity index (SI50) was calculated. The most active compound
was tested for its antiviral activity on other genotypes and possible interactions with da-
clatasvir, and the coefficient of drug interaction was calculated. In addition, the effect
of the most active compound was tested for its effects upon the expression of the viral
RNA and NS5A proteins by qPCR and Western blot analysis, respectively. For the exact
methodologies, see the experimental section.
2.2.1. Cytotoxicity and Anti-HCV 1b Activity of the Synthesized Compounds
The HCV RNA replication inhibitory effect of the novel compounds and their impact
on cell viability was tested in the stable cell line Huh5-2 carrying an HCV subgenomic
reporter replicon of genotype 1b (strain Con1) [22]. The viral replication level was measured
in terms of the activity of the co-expressed firefly luciferase (F-Luc). Several concentrations
of the tested compounds were prepared in a cell culture medium and were used to treat
the cells for 72 h. The solvent DMSO was added as a control with a final concentration
of 0.2% so as not to impact viral replication (data not shown). First, different compound
concentrations of up to 100
reported as the median cytotoxic concentration (CC50). Next, all compounds were screened
for their anti-HCV activity at a concentration of 1 M, and compounds showing 50%
activity were further tested in several concentrations to calculate the median effective
concentration (EC50) values (Tables 1 and 2). The anti-HCV activity of the analogs 32 was
µM were evaluated for their effect on cell viability; results are
µ
≥
1
–
estimated by quantifying luciferase activity resulting from viral replication. Cytotoxicity
was determined by measuring the intracellular levels of ATP. The selectivity index (SI50)
was calculated as the ratio of CC50 to EC50 and was calculated as an indication of the
compounds’ safety. We used daclatasvir as a positive control.
The effect of the three major structural modifications on activity was investigated.
First, we turned our attention to the effect of the nature of the lipophilic amino acid in
the capping group, which includes size, chirality, branching and aromaticity. Second, we
evaluated the impact of changing the nature of the O- substituents of the terminal amino
acid capping group, which includes size and aromaticity, Table 1.
Finally, the effect of bioisoster replacement of L- proline with (S, R) 1,3-disubstituted
piperidine was tested, Table 2. We compared the fluorene to the benzidine core using a
congener previously reported by our research group (b) [17].
Both D- and L-epimers of valine, leucine, phenylglycine, whereas only L-isoleucine,
were employed as an amino acid residue of the capping group. The use of both natural
and unnatural amino acids can expand the pool of building blocks of NS5A inhibitors;
additionally, the use of unnatural amino acids can have an impact on compound stability
and resistance development.
For the valine residues, the R congeners were more active than their S epimers except
for compound 1. For the leucine residues, compound 9 was around 340 times less active
than its R congener compound 13. On the contrary, compound 10 bearing an L-leucine
was 18-fold more potent than its congener that has D-leucine moiety. For phenylglycine
residues, compounds 25
, 26 were 60-fold and 200-fold more active than their S counterparts
21 22, respectively. Analog 25 was 60-fold more potent than 21. The opposite was observed
,
comparing compound 28 and its R counterpart 24. It is worth mentioning that three out of
the four compounds showing picomolar activity have an R amino acid residue, indicating
that the interaction with the receptor is not stereo-specific.
It seems that the R configuration is generally preferred for compounds bearing a fluo-
rene prolinamide core, even though most of the NSSA inhibitors discovered incorporated
an S-valine residue. This expands the pool of amino acids that can be adopted in the design
of new NS5A inhibitors.
Next, we extended our investigation to what effect the size and aromaticity of the
amino acid residue of the capping group have on anti-HCV activity. It was found that the

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largest set of active analogs (21
–
28) bore a phenylglycine residue indicating that activity
improves with aromatic amino acid residues. This can be attributed to both the lipophilicity
of these residues and their ability to be involved in van der Waals or stacking interac-
tions. Both D- and L-leucine compounds ( 16) exhibited better activity when compared
to their valine congeners ( ), indicating that more lipophilic residues are favored. This
π-π
9–
1–8
could encourage the preparation of commercial NS5A inhibitors from aromatic amino acid
residues rather than the commonly adopted S-valine.
The effect of the substituent on the terminal oxygen of the capping group was also
investigated. Results showed that the terminal capping group was not a detrimental factor
of activity compared to the amino acid residue’s chirality and lipophilicity. There was no
clear correlation between these groups and activity, yet we have generally noticed that a
methyl or a benzyl substituent positively affects the activity. Out of the nine most active
analogs, four compounds bore a methyl or benzyl terminal substituent (13
Comparing compound 26, the most active analog bearing an R-phenylglycine amino
acid residue with a terminal ethyl carbamate to its counterparts 25 27, and 28, we noticed
, 21, 24, and 25).
,
that compound 25 bearing a terminal methyl substituent was four times less potent, while
compound 27 bearing a butyl terminal group, showed 100 times less potency. A terminal
benzyl group reduced the activity by 2363 times. The introduction of two extra benzyl
groups increased the clog P from 5.7 in the case of methyl substituent to 7.4, which may
have affected both the ability of the compound to appropriately penetrate hepatoma cells
and its solubility during the assay and may have led to false lower potency.
The lack of a clear correlation pattern between terminal O-substituent and antiviral
activity is observed; we noticed in some cases that an aromatic lipophilic substituent is
favored compared to a small methyl substituent for compounds
8 and 24 compared to
compounds and 21. These findings indicate that the nature and conformation of the
5
entirety of the compound are crucial for activity rather than a single substitution.
We further investigated the optimal stereoelectronic features by replacing the com-
monly adopted L-proline with a six-membered common proline-memetic, namely (S,R)
piperidine. The latter was coupled at position three to the fluorene core with an amide
linkage. The terminal N-substituent was kept as methyl, and four S amino acid residues
were studied, namely valine, leucine, phenyl glycine, and isoleucine.
All compounds were less active than their congeners bearing L-proline, indicating
that a five-membered heterocycle enhances activity compared to a six-membered congener.
Compounds 1, 9, 17, and 21 were found to be five, 11, 28, and 36-fold more potent than
their six-membered congeners 29, 30, 31, and 32, respectively.
The racemic (S,R) center might have contributed negatively to the activity, compared
to a pure S or R chiral center. This can further be supported by the previously published
work of our group, where compounds bearing the D-proline were found to be inactive
relative to their L-proline congeners [20]. More research should be directed towards the
preparation of novel analogs with pure enantiomers of piperidine carboxylic acid and
its derivatives.
Compound 26, the most active analog of this series, bore an R-phenylglycine moiety
and ethyl carbamate terminal moiety.
Energy minimization of compound 26 (EC50 = 40 pM) using Chem3D ultra 7.0.0.
revealed a planar conformation of the tricyclic fused fluorene core, while the terminal two
ethyl moieties projected in the opposite direction, which may indicate that each of them
enters in one of the two chains of the NS5A protein (Figure 2).

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Figure 2. A planar fluorene core in compound 26 from which the two symmetric capping groups
are projected.
To assess the effect of compound core planarity on activity, compound
b
(EC50 = 6.7 pM) was energy minimized; the two phenyl cores showed non-coplanarity
and a dihyderal angle of 138.04◦. It seems that planarity is not favored for NS5A binding
(Figure 3) [17].
◦
Figure 3. A non-coplanar benzidine core with a dihyderal angle of 138.04 .
Based on the CC50 and SI50 values presented in Tables 1 and 2, all analogs (
1–32)
showed low cytotoxicity, even lower than daclatasvir (CC50 = 17.7 M) and high selectivity
µ
against virus replication. The most potent of them, compound 26, showed the highest
selectivity index (SI50 > 2779322), four-fold better than that of daclatasvir (SI50 = 655,556),
which indicates the very high safety profile. These promising results encourage adopting
this scaffold in the future for the development of NS5A inhibitors.
Finally, intermediate compound IV bearing a tertiary N-butoxy carbamate on the
piperidine amide moiety was tested, as a negative control, for its antiviral activity. Results
indicate the pivotal role of the terminal amino acid residue capped with a
substituted carbamate.
2.2.2. Activity of the Most Active Compound 26 Against HCV Genotypes 3a and 4a
Compound 26, showing the highest activity on genotype 1b was further tested against
HCV 3a and 4a replicons. The fact that genotype 4 is highly heterogeneous, of less response
to DAAs, and the most prevalent genotype in Egypt, made this genotype of specific interest.
Moreover, genotype 3 is the most difficult to treat genotype using DAAs, especially in
patients with liver cirrhosis [23]. Specifically, Huh7.5-3a and Huh7.5-4a stable cell lines
having HCV subgenomic replicons of genotypes 3a (strain S52) and 4a (strain ED43),
respectively, were treated with different concentrations of compound 26. Interestingly, a
significant effect was observed on GT 3a (EC50 = 1.2 nM), which was about seven-fold
higher than the one exerted by daclatasvir (EC50 = 8.3 nM). There was no significant activity
observed for GT 4a (EC50 = 117 nM). Further structural optimization is required, and testing
compound 26 in combination with other DAAs could improve the clinical outcome.

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2.2.3. Drug-Drug Interaction Studies of Compound 26 with the Approved HCV
Inhibitor Daclatasvir
Next, we investigated the effect of compound 26 in the presence of daclatasvir (DCV)
in Huh5-2 replicon cells. As shown in Figure 4, compound 26 and DCV had a significant
synergistic effect. More specifically, compound 26 showed a coefficient of drug interaction
(CDI
≈
0.7). It is worth noting that NS5A is a very flexible protein and can attain different
conformations (open conformer and closed conformer PDB ID: 1ZH1, 3FQM, respectively);
thus, there is the potential for different binding modes of daclatasvir and our molecules, at
the active center of the NS5A protein, leading to synergism. [24]
Figure 4. Synergistic anti-HCV activity of compound 26 with daclatasvir (DCV). Huh5-2 cells were
treated with 30 pM or 10 pM of DCV was used to in the presence or absence of the analog. F-Luc
activity was quantified as relative light units (RLU) per µg of total protein. Values derived from cells
treated with the solvent DMSO (control—C) were set to 100%. CDI: coefficient of drug interaction.
Bars show mean values derived from three independent experiments in triplicate. Error bars denote
standard deviation (SD).
This is supported by a previous manuscript that shows the synergistic effect between
the NS5A inhibitor daclatasvir and other NS5A inhibitors [25].
The coefficient of Drug interactions (CDI) is calculated as follows: CDI = AB / (A
×
B),
where AB is the ratio of the combination groups to the control group; A or B is the ratio of
the single-agent group to the control group. Thus, CDI value <1, = 1, or >1 indicates that the
drugs are synergistic, additive, or antagonistic, respectively.
2.2.4. Validation of Compound Activity with Additional Assays
The HCV RNA and NS5A protein levels were determined in cells treated with com-
pound 26 using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or
Western blot analysis, respectively (Figure 5A).
Compound 26 inhibited HCV RNA replication, with EC50 similar to that calculated
by luciferase assay (Table 1). Consistently, NS5A levels were reduced after treatment with
the inhibitor (Figure 5B).

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Figure 5. Anti-HCV activity of compound 26 at the level of viral RNA and protein. Different
concentrations of compound 26, or the solvent DMSO (control—C), were used to treat Huh5-2 cells.
(
A
) Quantification of (+) strand HCV RNA by RT-qPCR. Values are expressed as a percentage of
those obtained from control cells. mRNA levels of the housekeeping gene 14-3-3-zeta polypeptide
(YWHAZ) were used for normalization. ( ) (Top) Western blot analysis for NS5A. -actin was used
B
β
as a loading control. Numbers on the right refer to the positions of molecular mass marker proteins.
A representative experiment of three independent repetitions is shown. (Bottom) Densitometry
analysis data for NS5A (mean values from three independent repetitions) were normalized to
and to the values obtained with control cells (Figure S1).
β-actin
3. Material and Methods
3.1. Chemistry
Solvents and reagents were obtained from commercial suppliers and were used with-
out further purification. All solvents used were of pure analytical grade. Reactions that
needed anhydrous conditions were carried out in flame-dried glassware under a positive
pressure of dry N2 using the Schlenk line technique. Column chromatography was carried
out using silica gel 40–60
µM. 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); carbamates were detected using freshly prepared furfural/sulfuric acid detec-
tion reagent. Proton nuclear magnetic resonance (1H-NMR spectra and 13C-NMR spectra)
were recorded using a Bruker Fourier 300 using Varian Mercury 400 Plus. All spectra
were determined in the solvents indicated, and chemical shifts were reported in
Solvent signals were 2.50 for Dimethyl sulfoxide (DMSO-d6) and 7.2 for Chloroform
(CDCl3). 13C shifts were referenced to the deuterated solvent signal
39.5 for DMSO-d6
and 77.0 for CDCl3. Coupling constants (J) are given in hertz (Hz). Multiplicities patterns
δ units.
δ
δ
δ
δ
were as follows: s: singlet; d: doublet; t: triplet; q: quartet; m: multiplet; dd:doublet
of doublet; ddd: doublet of doublet of doublet. All NMR spectra were analyzed using
MestReNova software version 6.0.2–5475. Mass spectrometric analysis (UPLC-ESI-MS) was
performed using Waters ACQUITY Xevo TQD system, which consisted of an ACQUITY
UPLC H-Class system and XevoTM TQD triple-quadrupole tandem mass spectrometer
with an electrospray ionization (ESI) interface (Waters Corp., Milford, MA, USA). Acquity
BEH C18 100 mm
(Waters, Ireland). The solvent system consisted of water containing 0.1% TFA (A) and 0.1%
TFA in acetonitrile (B). HPLC-method: flow rate 200 L/min. The percentage of B started
×
2.1 mm column (particle size, 1.7 µm) was used to separate analytes
µ
at an initial of 5% and maintained for 1 minute, then increased up to 100% during 10 min,
kept at 100% for 2 min, and flushed back to 5% in 3 min then kept at 5% for 1 min. The MS
scan was carried out at the following conditions: capillary voltage 3.5 kV, cone voltage 20 V,
radio frequency (RF) lens voltage 2.5 V, source temperature 150 ◦C, and desolvation gas
temperature 500 ◦C. Nitrogen was used as the desolvation and cone gas at a flow rate of
1000 and 20 L/h, respectively. System operation and data acquisition were controlled using
Mass Lynx 4.1 software (Waters). The purities of the tested compounds were determined by
HPLC coupled with mass spectrometry and were higher than 95% purity unless otherwise

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stated (purity of compound 18 was >90%). Measurements for a melting point were not
changed and were done using capillary Büchi B-540 melting point apparatus.
3.1.1. General Procedure for Carbamates Synthesis (i–xxviii)
In a 250 mL round bottom flask equipped with a magnetic stirring bar, 25 mL of 1M
NaOH was cooled in an ice bath to 0 ◦C. The respective amino acid was added (8 mmol),
and the solution was stirred until it became completely homogenous. The corresponding
chloroformate (11 mmol), dissolved in 1,4-dioxane (10 mL), was added dropwise. The
reaction mixture was left to stir overnight at room temperature. The reaction mixture was
washed with diethyl ether (3
layer is placed in an ice bath and acidified dropwise to pH = 2 using concentrated HCl.
The respective carbamate is extracted from the aqueous layer by adding diethyl ether
×
50 mL), the organic layer was discarded. The aqueous
(3
×
50 mL). Organic layers were combined, dried over anhydrous Na2SO4, and concen-
trated under vacuum to give the crude product either as a clear, viscous oil or as a white
solid. No further purification was required (Scheme 2) [17,20,21] .
3.1.2. General Procedure for the Preparation of 2,7-diaminofluorene (I)
The starting material of 2,7-dinitrofluorene (4 mmol, 1 g) was placed into a 250 mL
round-bottom flask together with SnCl2.2H2O (80 mmol, 16 g) in concentrated HCl (12 mL)
◦
using acetic acid (24 mL) as a solvent. The mixture was refluxed for 5 h at 65 C. The
resulting suspension was cooled and poured into an aqueous solution of (313 mmol, 12.5 g
of NaOH in 500 mL of water. The precipitate was filtered, excess NaOH was added to
pH > 7 and extracted using diethyl ether (3 25 mL). Organic layers were combined, dried
)
×
over anhydrous Na2SO4, and concentrated under vacuum. The crude product was purified
by silica gel column chromatography using a mixture of methylene chloride: methanol
(10:0.3) and dried under vacuum to provide a light brown solid compound I (0.65 g, 65%).
Compound I appeared as a distinct blue spot using thin liquid chromatography [26].
3.1.3. General Procedure for Preparation of Compounds II and IV
Compound
I
(1 mmol, 0.5 g) was coupled with either N-Boc-L-proline or N-Boc-(S,R)-
was
piperidine-3-carboxylic acid (2 mmol) using HBTU (1 mmol, 0.967 g). Compound
I
added in one portion to the N-Boc derivative and TEA (2 mmol, 0.710 mL). The mixture
was stirred for 15 min in DMF in an ice bath. The reaction mixture was left to stir at room
temperature for 30 min. DMF was removed under vacuum, and the residue was extracted
using DCM and saturated sodium chloride solution. Organic layers were combined, dried
over anhydrous Na2SO4, and concentrated under vacuum to yield a dark red/brown oily
product compound II (80%) and dark red oily product compound IV (41%) [26].
3.1.4. General Procedure for Preparation of Compounds III and V
Compound II was dissolved in DCM (40 mL) and treated with TFA (6 mL) at room
temperature under an N2 atmosphere. The reaction was left to stir at room temperature for
24 h then both TFA and DCM were evaporated under vacuum to yield compounds III and V.
The reaction residue was dissolved in EtOAc, then 1M NaOH was added gradually
till pH = 12. The mixture was extracted using EtOAc (3
combined, dried over Na2SO4, and concentrated under vacuum. No further purification
was required [26].
×
25 mL). Organic layers were
3.1.5. General Procedure for Preparation of Compounds (1–32)
HBTU (1 mmol, 1.554 gm) was added in one portion to the respective amino acid
carbamate (3 mmol) and TEA (2 mmol, 0.571 mL), the mixture was stirred for 15 min in DMF
under ice-cooling. Compounds III and
V (1 mmol) were added portion-wise to the above
mixture. The reaction mixture was left to stir at room temperature for 3 h. The crude product
was purified by silica gel column chromatography using a mixture of DCM: MeOH.

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Di-tert-butyl3,3’-(((9H-fluorene-2,7diyl)bis(azanediyl))bis(carbonyl))bis
(piperidine-1-carboxylate) (IV)
Dark red Oil; Yield 41%; 1H NMR (400 MHz, DMSO-d6)
δ 9.77 (s, 2H), 7.61 (s, 2H),
7.44 (d, J = 8.2 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 3.69 (d, J = 48.0 Hz, 4H), 3.59 (s, 2H), 2.22
(d, J = 13.7 Hz, 4H), 2.19 (d, J = 10.9 Hz, 2H), 1.72–1.44 (m, 4H), 1.34 (dd, J = 23.9, 12.2 Hz,
4H), 1.14 (s, 18H); 13C NMR (101 MHz, DMSO-d6)
δ 172.19, 154.34, 143.95, 138.05, 136.74,
119.94, 118.39, 116.45, 85.18, 60.69, 59.11, 37.04, 28.04, 24.68, 24.54, and 22.89; MS (ESI) m/z:
619 [M + H] +.
N, N’-(9H-fluorene-2,7-diyl)bis(piperidine-3-carboxamide) (V)
Dark red oil; Yield 51%; 1H NMR (400 MHz, DMSO-d6)
δ 8.24 (s,2H), 7.91 (s, 2H), 7.69
(d, J = 8.4 Hz, 2H), 7.54–7.52 (m, 2H), 3.48 (s, 2H), 2.79 (d, J = 2.6 Hz, 4H), 2.71–2.69 (m, 4H),
2.58–2.55 (m, 2H), 1.90–1.85 (m, 4H), 1.71–1.67 (m, 2H), 1.48–1.41 (m, 4H); 13C NMR (101
MHz, DMSO-d6) δ 159.46, 125.55, 122.08, 118.91, 115.91, 115.32, 112.95, 63.05, 60.43, 31.17,
24.24, 22.44, and 21.60; MS (ESI) m/z: 419 [M+ H] +.
Dimethyl((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (1)
Yellow oil; Yield 20.6%; 1H NMR (500 MHz, DMSO-d6)
δ 10.07 (s, 2H), 7.89 (s, 2H),
7.72–7.69 (m, 2H), 7.47 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 4.05 (t, J = 8.5 Hz,
2H), 3.86 (s, 2H), 3.66–3.61 (m, 2H), 3.53 (s, 6H), 2.19–2.13 (m, 6H), 1.97–1.87 (m, 8H), 0.90
(d, J = 6.6 Hz, 12H); 13C NMR (126 MHz, DMSO-d6)
δ 170.85, 170.73, 157.26, 144.18, 138.11,
136.64, 119.96, 118.21, 116.28, 60.69, 58.43, 51.89, 47.71, 31.10, 29.98, 25.12, 19.39, and 19.12;
MS (ESI) m/z: 705.35 [M+ H]+.
Diethyl((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (2)
Yellow oil ; Yield 20%; 1H NMR (500 MHz, DMSO-d6)
δ 10.06 (s, 2H), 7.89 (s, 2H), 7.70
(d, J = 8.3 Hz, 2H), 7.49–7.46 (m, 2H), 7.23 (d, J = 8.3 Hz, 2H), 4.48 (dd, J = 8.1, 4.8 Hz, 2H),
4.04 (t, J = 8.4 Hz, 2H), 4.01–3.97 (m, 4H), 3.87–3.80 (m, 4H), 3.68–3.60 (m, 2H), 2.18–2.12
(m, 2H), 1.97–1.88 (m, 8H), 1.16 (t, J = 7.1 Hz, 6H), 0.93 (dd, J = 31.3, 6.7 Hz, 12H); 13C NMR
(126 MHz, DMSO-d6) δ 170.88, 170.75, 156.98, 144.02, 138.11, 136.64, 119.96, 118.21, 116.28,
60.70, 60.28, 58.33, 47.69, 38.71, 31.16, 25.14, 19.41, 19.11, and 15.11; MS (ESI) m/z: 733.38
[M+ H]+.
Dibutyl((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (3)
Yellow oil; Yield 23.5%; 1H NMR (500 MHz, DMSO-d6)
δ 10.05 (s, 2H), 7.89 (s, 2H), 7.70
(d, J = 8.3 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H), 4.48 (dd, J = 8.0, 4.5 Hz,
2H), 4.05 (t, J = 8.4 Hz, 2H), 3.93 (dt, J = 10.7, 5.4 Hz, 4H), 3.86 (s, 2H), 3.82 (d, J = 9.0 Hz, 2H),
3.64 (dd, J = 15.7, 6.3 Hz, 2H), 2.17 (dd, J = 12.8, 8.1 Hz, 2H), 2.00–1.85 (m, 8H), 1.56–1.47
(m, 4H), 1.35–1.28 (m, 4H), 0.96 (d, J = 6.6 Hz, 6H), 0.89 (t, J = 7.4 Hz, 12H); 13C NMR (101
MHz, DMSO-d6)
δ 170.87, 170.73, 156.94, 144.01, 138.10, 136.65, 119.94, 118.23, 116.29, 64.09,
58.34, 55.36, 47.70, 38.70, 31.21, 31.13, 29.94, 25.14, 19.40, 19.07, and 14.07; MS (ESI) m/z:
789.45 [M+ H]+.
Dibenzyl ((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7- diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (4)
Yellow oil; Yield 26%; 1H NMR (500 MHz, DMSO-d6)
δ 10(s, 2H), 7.89 (s, 2H), 7.70
(d, J = 8.3 Hz, 2H), 7.46 (t, J = 8.4 Hz, 2H), 7.35 (s, 10H), 7.32 (dd, J = 5.6, 3.4 Hz, 2H), 5.03
(d, J = 8.0 Hz, 4H), 4.51–4.44 (m, 2H), 4.08 (t, J = 8.4 Hz, 2H), 3.87–3.82 (m, 4H), 3.63 (dd, J =
15.3, 6 Hz, 2H), 2.21–2.17 (m, 2H), 2.02–1.89 (m, 8H), 0.97 (d, J = 6.7 Hz, 12H); 13C NMR
(101 MHz, DMSO-d6) δ 170.74, 170.73, 165.06, 156.70, 144.01, 138.09, 137.56, 136.65, 128.80,
128.11, 119.94, 118.23, 116.30, 65.86, 60.71, 58.46, 47.69, 38.70, 31.14, 25.11, 19.41, and 19.05;
MS (ESI) m/z: 857.42 [M+ H]+.
Dimethyl ((2R,2’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (5)
Yellow oil; Yield 19%; 1H NMR (500 MHz, DMSO-d6)
δ
9.72 (s, 2H), 7.88 (s, 2H), 7.71
(d, J = 8.2 Hz, 2H), 7.54 (d, J = 7.5 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 4.49–4.42 (m, 2H), 4.10 (t,

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J = 8.1 Hz, 2H), 3.85 (t, J = 10.4 Hz, 4H), 3.57 (s, 6H), 3.52–3.42 (m, 2H), 2.16–2.10 (m, 4H),
1.98–1.92 (m, 4H), 1.45 (s, 2H), 0.93–0.86 (m, 12H); 13C NMR (126 MHz, DMSO-d6)
δ 172.26,
170.79, 157.51, 143.95, 136.77, 134.06, 119.97, 118.34, 116.48, 60.82, 58.54, 52.06, 47.54, 30.16,
27.32, 24.82, 19.50, and 18.84; MS (ESI) m/z: 705.35 [M+ H]+.
Diethyl((2R,2’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (6)
Yellow oil ; yield 24.3%; 1H NMR (500 MHz, DMSO-d6)
δ 9.67 (s, 2H), 7.88 (s, 2H), 7.71
(d, J = 6.2 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 7.9 Hz, 2H), 4.45 (d, J = 6.5 Hz, 2H),
4.02–3.98 (m, 4H), 3.85 (s, 2H), 3.62 (dd, J = 16.6, 7.5 Hz, 2H), 3.00–2.95 (m, 2H), 2.19–2.08
(m, 4H), 1.99–1.89 (m, 8H), 1.19–1.11 (m, 12H), 0.91–0.85 (m, 6H); 13C NMR (126 MHz,
DMSO-d6)
δ 170.92, 170.67, 157.08, 143.92, 137.85, 136.80, 119.94, 118.40, 116.49, 60.51, 58.47,
47.56, 46.06, 38.71, 30.11, 24.73, 19.50, 15.08, and 9.48; MS (ESI) m/z: 733.38 [M+ H]+.
Dibutyl((2R,2’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (7)
Yellow oily ; Yield 18.5%; 1H NMR (500 MHz, DMSO-d6)
δ 9.66 (s, 2H), 7.88 (d, J =
5.5 Hz, 2H), 7.71 (dd, J = 8.3, 2.3 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H),
4.48–4.42 (m, 2H), 4.08 (t, J = 8.1 Hz, 2H), 3.98–3.90 (m, 4H), 3.87–3.80 (m, 4H), 3.62 (dd, J =
16.7, 7.5 Hz, 2H), 2.18–2.10 (m, 2H), 2.00–1.91 (m, 8H), 1.54–1.48 (m, 4H), 1.35–1.27 (m, 4H),
0.91–0.83 (m, 18H); 13C NMR (126 MHz, DMSO-d6)
δ 170.95, 170.65, 165.06, 157.20, 143.97,
136.81, 119.91, 118.44, 116.51, 64.31, 60.86, 58.51, 47.56, 38.71, 31.19, 30.09, 24.73, 19.50, 19.02,
18.88, and 14.05; MS (ESI) m/z: 789.45 [M+ H]+.
Dibenzyl((2R,2’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (8)
Yellow oil ; Yield 22%; 1H NMR (500 MHz, DMSO-d6)
δ 9.73 (s, 2H), 7.88 (d, J = 9.5 Hz,
2H), 7.73–7.68 (m, 2H), 7.53 (dd, J = 8.4, 3.6 Hz, 2H), 7.37–7.27 (m, 12H), 5.11–5.00 (m, 4H),
4.51–4.41 (m, 2H), 4.13 (t, J = 8.2 Hz, 2H), 3.83 (d, J = 11.2 Hz, 4H), 3.52–3.46 (m, 2H), 3.09
(qd, J = 7.3, 4.9 Hz, 2H), 1.99–1.92 (m, 4H), 1.17 (t, J = 7.3 Hz, 4H), 0.94–0.82 (m, 12H); 13
C
NMR (101 MHz, DMSO-d6) δ 170.75, 170.66, 156.90, 144.03, 143.86, 138.04, 137.43, 137.04,
128.90, 128.08, 119.93, 118.44, 116.50, 70.37, 65.98, 60.85, 58.61, 38.70, 30.13, 24.76, 19.51, and
18.83; MS (ESI) m/z: 857.42 [M+ H]+.
Dimethyl((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxopentane-1,2-diyl))dicarbamate (9)
Yellow oil; Yield 19.4%; 1H NMR (400 MHz, CDCl3)
δ 9.61 (s, 2H), 7.26 (s, 2H), 7.16
(d, J = 7.9 Hz, 4H), 7.12 (d, J = 8.0 Hz, 2H), 5.22 (d, J = 8.8 Hz, 2H), 4.68 (dd, J = 7.6, 4.4 Hz,
2H), 4.56–4.50 (m, 2H), 3.80–3.68 (m, 6H), 3.59 (s, 4H), 3.17 (s, 2H), 2.53 (dd, J = 11.6, 2.2
Hz, 4H), 2.27–1.99 (m, 4H), 1.76–1.43 (m, 4H), 0.94 (dd, J = 11.9, 6.6 Hz, 6H), 0.89–0.82 (m,
6H);13C NMR (126 MHz, CDCl3)
δ 172.10, 170.03, 165.52, 156.91, 143.12, 136.76, 118.78,
117.80, 115.65, 61.02, 52.26, 47.70, 41.53, 38.65, 36.72, 29.05, 25.15, 24.60, 23.39, and 21.77; MS
(ESI) m/z: 733.38 [M + H]+.
Diethyl((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxopentane-1,2-diyl))dicarbamate (10)
Yellow oil; Yield 29%; 1H NMR (500 MHz, DMSO-d6)
δ 9.67 (s, 2H), 7.36 (d, J = 7.7 Hz,
2H), 7.25 (s, 2H), 7.06 (d, J = 8.5 Hz, 2H), 6.90 (s, 2H), 4.40–4.29 (m, 2H), 4.15–4.03 (m, 2H),
3.88–3.80 (m, 2H), 3.57–3.48 (m, 2H), 2.32–2.13 (m, 4H), 2.00–1.96 (m, 4H), 1.89–1.79 (m, 4H),
1.65–1.56 (m, 8H), 1.43–1.35 (m, 6H), 0.88 (dd, J = 12.7, 4.9 Hz, 12H); 13C NMR (126 MHz,
DMSO-d6)
δ 172.06, 171.54, 156.82, 143.92, 137.87, 136.80, 119.94, 118.41, 116.56, 60.45, 60.19,
53.32, 51.33, 31.13, 24.74, 24.64, 23.67, 23.49, 21.86, 15.07, and 9.09; MS (ESI) m/z: 761.42
[M + H]+.
Dibutyl ((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxopentane-1,2-diyl))dicarbamate (11)
Yellow oil; Yield 28.4%; 1H NMR (500 MHz, DMSO-d6)
δ 10.03 (s, 2H), 7.88 (s, 2H),
7.70 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 1.2 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 4.48 (dd, J = 8.0,
4.6 Hz, 2H), 4.32–4.26 (m, 2H), 3.91 (dd, J = 24.2, 17.6 Hz, 8H), 3.77–3.68 (m, 2H), 3.57 (dt,
J = 9.2, 5.7 Hz, 2H), 2.69 (s, 8H), 2.18–2.00 (m, 4H), 1.96–1.89 (m, 4H), 1.58–1.44 (m, 4H),

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1.37–1.26 (m, 6H), 0.93–0.86 (m, 12H); 13C NMR (126 MHz, DMSO-d6)
δ
171.33, 170.75,
156.83, 143.99, 138.06, 136.68, 119.95, 118.27, 116.35, 64.03, 60.65, 51.19, 47.19, 38.71, 37.05,
31.21, 29.80, 25.16, 24.61, 23.68, 21.79, 19.04, and 14.08; MS (ESI) m/z: 817.48 [M + H]+.
Dibenzyl((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxopentane-1,2-diyl))dicarbamate (12)
Yellow oil; Yield 23.8%; 1H NMR (500 MHz, DMSO-d6)
δ 10.04 (s, 2H), 7.88 (s, 2H),
7.71 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 5.8 Hz, 2H), 7.39–7.29 (m, 12H), 5.02 (s, 4H), 4.48 (dd,
J = 8.0, 4.6 Hz, 2H), 4.32 (t, J = 6.6 Hz, 2H), 3.90–3.74 (m, 2H), 3.70–3.49 (m, 4H), 2.21–2.12
(m, 4H), 1.98–1.84 (m, 4H), 1.55–1.34 (m, 6H), 0.91 (d, J = 6.6 Hz, 12H); 13C NMR (126 MHz,
DMSO-d6)
δ 171.19, 170.74,156.58, 143.99, 138.06, 137.55, 136.68, 128.79, 128.26, 128.15,
119.95, 118.28, 116.35, 65.84, 60.68, 51.32, 47.22, 38.80, 31.15, 29.80, 25.16, 24.62, 23.68, and
21.79; MS (ESI) m/z: 885.45 [M+ H]+.
Dimethyl((2R,2’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(4-methyl-1-oxopentane-1,2-diyl))dicarbamate (13)
Yellow oil; Yield 19%; δ 9.12 (s, 2H), 7.76–7.65 (m, 2H), 7.42
1H NMR (400 MHz, CDCl3)
(d, J = 23.6 Hz, 6H), 5.65 (d, J = 5.8 Hz, 2H), 4.67 (d, J = 6.4 Hz, 2H), 4.47 (s, 2H), 3.69–3.57
(m, 6H), 2.00 (dd, J = 11.2, 4.2 Hz, 6H), 1.77–1.64 (m, 4H), 1.60–1.52 (m, 4H), 1.49–1.39 (m,
4H), 1.28 (t, J = 7.3 Hz, 6H), 0.93 (dd, J = 12.1, 6.6 Hz, 6H); 13C NMR (126 MHz, CDCl3)
δ
172.79, 172.22, 169.16, 165.71, 157.36, 143.80, 136.84, 119.22, 118.68, 116.87, 52.39, 51.45,
47.33, 46.75, 36.93, 28.66, 24.64, 23.39, 21.78, and 8.68; MS (ESI) m/z: 733.38 [M + H]+.
Diethyl ((2R,2’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(4-methyl-1-oxopentane-1,2-diyl))dicarbamate (14)
Yellow oil; Yield 18.7%; 1H NMR (500 MHz, DMSO-d6)
δ 8.85 (s, 2H), 7.39 (dd, J = 26.3,
7.7 Hz, 2H), 7.25 (s, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.90 (s, 2H), 4.00–3.95 (m, 6H), 3.92–3.78
(m, 4H), 3.48–3.41 (m, 4H), 3.35 (d, J = 2.9 Hz, 6H), 2.04–1.93 (m, 4H), 1.59 (ddd, J = 31.4,
15.6, 9.0 Hz, 4H), 1.49–1.35 (m, 6H), 0.86 (dd, J = 11.9, 6.6 Hz, 12H); 13C NMR (126 MHz,
DMSO-d6)
δ 175.06, 171.56, 170.66, 156.83, 156.54, 143.94, 119.95, 118.42, 116.49, 70.25, 60.19,
53.33, 46.26, 31.11, 24.73, 23.79, 23.48, 21.85, 15.05, and 9.07; MS (ESI) m/z: 761.42 [M + H]+.
Dibutyl((2R,2’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(4-methyl-1-oxopentane-1,2-diyl))dicarbamate (15)
Yellow oil; Yield 21.4%; 1H NMR (500 MHz, DMSO-d6)
δ 10.03 (s, 2H), 7.88 (s, 2H),
7.70 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 4.47 (dd, J = 8.0,
4.6 Hz, 2H), 4.29 (t, J = 7.2 Hz, 2H), 3.93 (dd, J = 13.9, 7.1 Hz, 4H), 3.86 (s, 2H), 3.74–3.53 (m,
6H), 2.25–2.00 (m, 8H), 1.92–1.83 (m, 4H), 1.56–1.45 (m, 8H), 1.36–1.26 (m, 6H), 0.91–0.84
(m, 12H); 13C NMR (126 MHz, DMSO-d6)
δ 171.33, 170.75, 165.06, 156.83, 143.99, 138.06,
119.95, 118.27, 116.34, 64.03, 60.65, 52.25, 51.19, 31.21, 29.79, 25.16, 24.61, 23.68, 23.23, 21.79,
19.04, and 14.08; MS (ESI) m/z: 817.48 [M + H]+.
Dibenzyl((2R,2’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(4-methyl-1-oxopentane-1,2-diyl))dicarbamate (16)
Yellow oil; Yield 25.5%; 1H NMR (400 MHz, DMSO-d6)
δ 10.02 (s, 2H), 7.88 (s, 2H),
7.70 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.40–7.26 (m, 12H), 5.02 (s, 4H), 4.48 (dd,
J = 7.8, 4.5 Hz, 2H), 4.39–4.27 (m, 2H), 3.58 (dd, J = 14.3, 7.8 Hz, 2H), 2.18–1.99 (m, 4H),
1.96–1.84 (m, 4H), 1.78–1.62 (m, 4H), 1.55–1.41 (m, 4H), 1.40–1.29 (m, 2H), 0.91 (d, J = 6.5
Hz, 12H); 13C NMR (101 MHz, DMSO-d6)
δ 171.19, 170.73, 156.57, 143.98, 138.05, 137.55,
136.68, 128.80, 128.25, 128.14, 119.94, 118.28, 116.36, 65.84, 60.67, 55.37, 51.32, 38.71, 29.79,
25.15, 24.61, 23.67, and 21.79; MS (ESI) m/z: 885.45 [M + H]+.
Dimethyl((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis (carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(4-methyl-1-oxopentane-1,2-diyl))dicarbamate (17)
Yellow oil; Yield 18.7%; 1H NMR (400 MHz, CDCl3)
δ 9.77 (s, 2H), 7.42–7.34 (m, 4H),
7.21 (d, J = 6.9 Hz, 2H), 7.12 (d, J = 8.1 Hz, 2H), 5.33–5.30 (m, 2H), 4.78 (dd, J = 7.7, 4.9 Hz,
2H), 4.47–4.42 (m, 2H), 3.92 (t, J = 6.7 Hz, 4H), 3.69 (d, J = 14.7 Hz, 6H), 2.31–2.19 (m,
4H), 2.13–2.00 (m, 4H), 1.86–1.62 (m, 4H), 1.26–1.19 (m, 2H), 1.17–1.07 (m, 12H); 13C NMR
(
126 MHz, CDCl3) δ 171.77, 170.03, 157.02, 143.05, 136.77, 128.95, 118.78, 117.78, 115.65,

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61.11, 56.93, 52.33, 48.13, 38.64, 26.91, 25.19, 15.53, 11.15, and 10.96; MS (ESI) m/z: 733.38
[M + H]+.
Diethyl((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7 diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(4-methyl-1-oxopentane-1,2-diyl))dicarbamate (18)
Yellow oil ; Yield 19.5%; 1H NMR (500 MHz, DMSO-d6)
δ 10.05 (s, 2H), 7.89 (s, 2H),
7.71 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 4.48 (dd, J = 8.1, 4.6
Hz, 2H), 4.09 (t, J = 8.8 Hz, 2H), 3.97 (tt, J = 10.8, 5.3 Hz, 4H), 3.88–3.78 (m, 4H), 3.68–3.56
(m, 2H), 2.26–2.07 (m, 4H), 1.96–1.67 (m, 6H), 1.57–1.45 (m, 2H), 1.20–1.09 (m, 8H), 0.96–0.80
(m, 12H);13C NMR (101 MHz, DMSO-d6)
δ 171.03, 170.72, 156.77, 144.01, 138.10, 136.65,
119.95, 118.23, 116.29, 60.72, 60.28, 56.87, 47.73, 36.31, 29.97, 25.10, 24.86, 15.29, 15.10, and
11.02; MS (ESI) m/z: 761.42 [M + H]+.
Dibutyl((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(4-methyl-1-oxopentane-1,2-diyl))dicarbamate (19)
Yellow oil ; Yield 24.7%; 1H NMR (500 MHz, DMSO-d6) δ 10.05 (s, 2H), 7.89 (s, 2H),
7.70 (d, J = 4.0 Hz, 2H), 7.47 (d, J = 1.4 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 4.48 (dd, J = 8.0,
4.6 Hz, 2H), 4.09 (t, J = 8.8 Hz, 2H), 2.69 (s, 2H), 2.22–2.02 (m, 4H), 1.99–1.87 (m, 4H), 1.73
(d, J = 8.7 Hz, 4H), 1.55–1.45 (m, 8H), 1.36–1.26 (m, 6H), 1.20–1.07 (m, 4H), 0.93–0.78 (m,
18H); 13C NMR (101 MHz, DMSO-d6)
δ 171.03, 170.72, 156.88, 144.01, 138.10, 136.65, 119.95,
118.23, 116.29, 64.09, 60.71, 56.88, 47.70, 36.30, 31.21, 29.96, 25.10, 24.85, 19.03, 15.29, 14.08,
and 11.02; MS (ESI) m/z: 817.48 [M + H]+.
Dibenzyl((2S,2’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(4-methyl-1-oxopentane-1,2-diyl))dicarbamate (20)
Yellow oil ; Yield 27.4%; 1H NMR (500 MHz, DMSO-d6) δ 10.05 (s, 2H), 7.89 (s, 2H),
7.71 (d, J = 8.3 Hz, 2H), 7.50 (dd, J = 13.0, 8.6 Hz, 4H), 7.40–7.28 (m, 10H), 5.06–4.96 (m, 4H),
4.49 (dd, J = 7.9, 4.6 Hz, 2H), 4.13 (t, J = 8.8 Hz, 2H), 3.86 (s, 4H), 3.70–3.60 (m, 4H), 2.69 (s,
2H), 2.19–1.99 (m, 4H), 1.94–1.86 (m, 4H), 1.59–1.46 (m, 2H), 0.93 (d, J = 6.7 Hz, 12H); 13
C
NMR (101 MHz, DMSO-d6) δ 170.90, 170.70, 165.06, 164.78, 156.64, 144.00, 138.10, 136.65,
128.80, 128.24, 128.09, 119.94, 118.23, 116.30, 65.87, 60.73, 57.02, 47.75, 36.34, 29.97, 25.09,
24.84, 15.29, and 11.03; MS (ESI) m/z: 885.45 [M + H]+.
Dimethyl((1S,1’S)-((2S,2’S)-(((9H-fluorene-2,7diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (21)
Yellow oil; Yield 16%; 1H NMR (500 MHz, CDCl3)
δ 9.34 (s, 2H), 8.23 (s, 2H), 7.79 (s,
2H), 7.59 (d, J = 15.7 Hz, 2H), 7.42 (d, J = 10.2 Hz, 2H), 7.28–7.05 (m, 10H), 5.53 (d, J = 7.6
Hz, 2H), 4.50–4.32 (m, 2H), 3.50 (s, 2H), 2.21–2.07 (m, 6H), 1.95–1.80 (m, 4H), 1.59–1.31 (m,
4H), 1.23–1.03 (m, 4H); 13C NMR (126 MHz, CDCl3)
δ 172.92, 170.38, 169.49, 168.84, 156.38,
143.77, 137.42, 129.34, 128.88, 127.87, 119.37, 118.34, 116.34, 58.50, 56.90, 52.22, 47.76, 29.43,
24.78, and 22.25; MS (ESI) m/z: 773.32 [M + H]+.
Diethyl((1S,1’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (22)
Yellow oil; Yield 24%; 1H NMR (500 MHz, CDCl3)
δ 9.46 (s, 2H), 7.78 (s, 2H), 7.50 (d, J
= 14.6 Hz, 6H), 7.41 (d, J = 6.8 Hz, 10 H), 5.91 (s, 2H), 5.66–5.51 (m, 2H), 5.00–4.73 (m, 4H),
3.72 (s, 2H), 3.31–3.12 (m, 4H), 2.14–2.00 (m, 8H), 1.43–1.29 (m, 6H); 13C NMR (126 MHz,
CDCl3)
δ 168.24, 167.20, 163.40, 154.04, 141.67, 135.29, 134.64, 134.39, 127.37, 125.99, 117.32,
116.33, 114.29, 59.32, 54.86, 45.32, 36.85, 26.11, 23.09, 12.63, and 6.79; MS (ESI) m/z: 801.35
[M + H] +.
Dibutyl((1S,1’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (23)
Yellow oil; Yield 23.5%; 1H NMR (500 MHz, DMSO-d6) δ 10.07 (s, 2H), 7.87 (s, 2H),
7.71 (d, J = 2.6 Hz, 2H), 7.55 (d, J = 8.3 Hz, 2H), 7.44 (d, J = 7.2 Hz, 2H), 7.40–7.29 (m, 10H),
5.47 (d, J = 8.3 Hz, 2H), 4.52 (dd, J = 8.1, 4.6 Hz, 2H), 3.95 (tt, J = 6.5, 3.2 Hz, 4H), 3.88 (s,
2H), 3.72–3.61 (m, 4H), 2.23–2.09 (m, 4H), 1.91–1.85 (m, 4H), 1.55–1.47 (m, 4H), 1.35–1.26
(m, 4H), 0.89–0.85 (m, 6H); 13C NMR (126 MHz, DMSO-d6)
δ 170.55, 168.62, 156.46, 144.02,
138.03, 137.43, 136.70, 128.85, 128.54, 128.23, 120.01, 118.30, 116.37, 64.22, 61.03, 57.02, 55.38,
38.72, 31.19, 29.82, 25.12, 19.02, and 14.08; MS (ESI) m/z: 857.42 [M + H]+.

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Dibenzyl((1S,1’S)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (24)
Yellow oil; Yield 20.3%; 1H NMR (500 MHz, CDCl3)
δ 9.68–9.46 (m, 2H), 7.52 (d, J = 6.9
Hz, 2H), 7.47 (dd, J = 12.8, 6.7 Hz, 2H), 7.40 (dd, J = 14.8, 7.5 Hz, 4H), 7.31 (d, J = 6.7 Hz, 10
H), 6.21–5.90 (m, 2H), 5.65–5.51 (m, 2H), 5.15–5.03 (m, 4H), 4.92–4.63 (m, 2H), 3.81–3.60 (m,
2H), 3.49–3.29 (m, 4H), 3.03–2.87 (m, 8H), 2.23–1.93 (m, 12H); 13C NMR (126 MHz, CDCl3)
δ
169.68, 169.24, 165.62, 155.70, 143.42, 137.06, 136.58, 136.18, 129.27, 128.52, 128.19, 128.04,
119.09, 118.08, 116.00, 66.99, 61.22, 56.89, 47.46, 29.70, 28.49, and 25.01; MS (ESI) m/z: 925.38
[M + H]+.
Dimethyl((1R,1’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) bis
(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (25)
Yellow oil; Yield 17.75%; 1H NMR (500 MHz, DMSO-d6)
δ 8.13–8.00 (m, 6H), 7.86–7.78
(m, 2H), 7.67–7.57 (m, 2H), 7.46–7.19 (m, 10H), 3.75–3.43 (m, 4H), 2.63–2.50 (m, 2H), 2.36
(dt, J = 3.6, 1.8 Hz, 6H), 1.67–1.51 (m, 4H), 1.21–0.99 (m, 4H), 0.96–0.61 (m, 4H); 13C NMR
(126 MHz, DMSO-d6) δ 177.29, 171.77, 168.82, 156.86, 142.77, 136.78, 129.95, 129.02, 128.81,
128.24, 125.11, 120.02, 110.60, 66.76, 62.17, 58.47, 52.72, 52.15, 49.83, and 21.68; MS (ESI)
m/z: 773.32 [M + H]+.
Diethyl((1R,1’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (26)
Yellow oil; Yield 18%; 1H NMR (500 MHz, CDCl3-d)
δ 9.43 (d, J = 11.9 Hz, 2H), 7.74 (d,
J = 16.3 Hz, 2H), 7.65 (s, 2H), 7.47 (d, J = 8.4 Hz, 4H), 7.42–7.35 (m, 10H), 6.05 (dd, J = 13.9,
6.7 Hz, 2H), 5.58–5.52 (m, 2H), 4.89 (s, 2H), 3.69 (dd, J = 16.8, 7.4 Hz, 4H), 3.34 (dd, J = 17.3,
9.2 Hz, 4H), 2.38 (dd, J = 19.5, 11.9 Hz, 4H), 2.13–2.01 (m, 4H), 1.25–1.21 (m, 6H); 13C NMR
(126 MHz, CDCl3-d) δ 170.43, 170.19, 169.03, 155.97, 143.71, 136.74, 136.30, 129.22, 128.83,
127.96, 119.34, 116.26, 109.60, 61.60, 61.33, 56.78, 47.47, 36.98, 29.70, 27.47, and 14.66; MS
(ESI) m/z: 801.35 [M + H]+.
Dibutyl((1R,1’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (27)
Yellow oil; Yield 20.6%; 1H NMR (400 MHz, CDCl3-d)
δ 9.22 (s, 2H), 7.90 (s, 2H), 7.82
(s, 2H), 7.62 (d, J = 7.9 Hz, 2H), 7.56–7.36 (m, 10H), 7.24–7.13 (m, 2H), 5.90 (s, 2H), 5.50 (s,
2H), 4.93–4.76 (m, 2H), 3.67–3.53 (m, 4H), 3.35–3.17 (m, 4H), 2.44–2.32 (m, 4H), 2.12–1.88 (m,
4H), 1.41–1.31 (m, 8H), 0.98–0.86 (m, 6H),13C NMR (126 MHz, CDCl3-d)
δ 170.31, 169.76,
164.89, 162.51, 147.97, 143.78, 130.27, 129.46, 127.88, 125.85, 119.35, 110.75, 94.04, 66.00, 45.68,
32.12, 31.09, 29.77, 25.58, 22.99, 19.08, 14.66, and 13.89; MS (ESI) m/z: 857.42 [M + H]+.
Dibenzyl((1R,1’R)-((2S,2’S)-(((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl)) Bis
(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (28)
Yellow oil; Yield 22%; 1H NMR (500 MHz, DMSO-d6)
δ 10.11 (s, 2H), 7.89 (d, J = 16.4
Hz, 2H), 7.79 (s, 2H), 7.72 (d, J = 3.4 Hz, 2H), 7.55 (d, J = 7.4 Hz, 2H), 7.51–7.41 (m, 20H),
5.51–5.46 (m, 2H), 5.04 (s, 4H), 3.88 (s, 2H), 3.13 (dd, J = 18.0, 11.0 Hz, 2H), 2.25–2.01 (m,
4H), 1.93–1.77 (m, 8H); 13C NMR (126 MHz, DMSO-d6)
δ 170.56, 169.18, 168.57, 156.30,
155.98, 149.61, 144.06, 138.43, 137.49, 137.33, 136.70, 129.06, 128.55, 127.88, 120.06, 118.32,
116.36, 66.11, 61.05, 57.15, 47.36, 37.07, 29.82, 25.13; MS (ESI) m/z: 925.38 [M + H]+.
Dimethyl ((((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis(piperidine-3,1-diyl))
bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (29)
Yellow oil; Yield 18%;1H NMR (400 MHz, CDCl3-d)
δ 7.85 (d, J = 11.0 Hz, 2H), 7.65–7.61
(m, 2H), 7.45 (s, 2H), 7.33 (d, J = 13.4 Hz, 2H), 7.26 (s, 2H), 5.12 (s, 2H), 4.32 (s, 2H), 3.84 (s,
6H), 3.69–3.62 (m, 4H), 3.58–3.44 (m, 4H), 2.58 (t, J = 12 Hz, 2H), 2.37–2.22 (m, 2H), 2.09–1.96
(m, 4H), 1.41–1.24 (m, 4H), 1.23 (s, 12H); 13C NMR (126 MHz, CDCl3-d)
δ 171.03, 170.65,
167.73, 132.47, 128.87, 119.69, 118.91, 116.94, 68.17, 49.14, 39.09, 38.74, 37.01, 30.37, 28.93,
23.76, 22.99, 14.05, and 10.96; MS (ESI) m/z: 733.38 [M + H]+.
Dimethyl ((((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis(piperidine-3,1-diyl))
bis(4-methyl-1-oxopentane-1,2-diyl))dicarbamate (30)
Yellow oil; Yield 17.5%; 1H NMR (500 MHz, CDCl3-d)
δ
7.75–7.71 (m, 4H), 7.57–7.53
(m, 6H), 4.27–4.19 (m, 4H), 3.79–3.74 (m, 4H), 3.73–3.67 (m, 6H), 1.73–1.71 (m, 2H), 1.70–1.67

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(m, 4H), 1.47–1.43 (m, 4H), 1.44–1.41 (m, 2H), 1.40–1.37 (m, 4H), 1.01 (dd, J = 12.1, 5.2 Hz,
4H), 0.92 (s, 12H); 13C NMR (126 MHz, CDCl3-d)
δ 171.03, 170.65, 167.73, 132.47, 130.89,
128.87, 119.69, 118.91, 116.94, 68.17, 49.14, 39.09, 38.74, 37.01, 30.37, 28.93, 23.76, 22.99, 14.05,
and 10.96; MS (ESI) m/z: 761.42 [M + H]+.
Dimethyl ((((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis(piperidine-3,1-diyl))
bis(3-methyl-1-oxopentane-1,2-diyl))dicarbamate (31)
Yellow oil; Yield 16%; 1H NMR (500 MHz, DMSO-d6)
δ 10.05 (s, 2H), 7.88 (s, 2H), 7.70
(d, J = 7.4 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.7 Hz, 2H), 4.12 (s, 2H), 3.86 (s,
2H), 3.17–3.00 (m, 8H), 2.85–2.74 (m, 4H), 2.62 (dd, J = 18.1, 16.4 Hz, 6H), 2.07–1.94 (m,
8H), 1.78–1.65 (m, 4H), 1.46 (d, J = 7.4 Hz, 6H), 0.87–0.79 (m, 6H); 13C NMR (126 MHz,
DMSO- d6)
δ 173.50, 168.73, 161.01, 152.03, 130.20, 120.21, 115.08, 110.60, 81.39, 66.64, 62.46,
54.79, 47.70, 44.82, 44.17, 37.73, 33.62, 29.42, 24.58, 22.69, and 12.71; MS (ESI) m/z: 761.42
[M + H]+.
Dimethyl((((9H-fluorene-2,7-diyl)bis(azanediyl))bis(carbonyl))bis(piperidine-3,1-diyl))
bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (32)
Yellow oil; Yield 15%; 1H NMR (400 MHz, CDCl3-d)
δ
7.45–7.36 (m, 10H), 7.15–6.99
(m, 5H), 6.80–6.67 (m, 5H), 4.05–3.86 (m, 4H), 3.81–3.57 (m, 8H), 1.39–1.27 (m, 2H), 1.23
(s, 6H), 0.89–0.77 (m, 8H); 13C NMR (126 MHz, CDCl3-d)
171.47, 171.25, 165.77, 161.64,
δ
153.89, 145.04, 138.20, 129.97, 129.27, 127.73, 116.82, 115.53, 115.27, 71.13, 63.75, 46.76, 40.13,
31.93, 29.70, 22.70, and 14.12; MS (ESI) m/z: 801.35 [M + H]+.
3.2. Biological Assays
3.2.1. Cell Culture
The stable replicon cell line Huh5-2 (genotype 1b; Con1) (kindly provided by R. Barten-
schlager, Heidelberg University, Germany) has been described previously [22]. The stable
cell lines Huh7.5-3a and Huh7.5-4a have been constructed [27] with the subgenomic re-
porter replicons of HCV 3a (S52) S52-SG(Feo)(AII) and HCV 4a (ED43) ED43-SG(Feo)(VYG)
(kindly provided by C.M. Rice, The Rockefeller University, NY) [28]. Cells were cultured
in high glucose (25 mM) Dulbecco’s modified minimal essential medium (Invitrogen),
supplemented with 2 mM L-glutamine, 0.1 mM non-essential amino acids, 100 U/mL
penicillin, 100 µg/mL streptomycin, and 10% (v/v) fetal calf serum (referred to as com-
plete DMEM). Complete DMEM was supplemented with G418 at 500 µg/mL for Huh5-2,
750 µg/mL for Huh7.5-3a, and 350 µg/mL for Huh7.5-4a.
3.2.2. Cell-Based Antiviral and Cytotoxicity Assays
Cells were treated with serial dilutions of the test compounds or the solvent DMSO
for 72 h. Antiviral activity and cytotoxicity were determined by measuring virus-derived
luciferase activity or intracellular ATP levels, respectively. The median effective concen-
tration (EC50) of the compounds, reducing luciferase signal by 50%, and their median
cytotoxic concentration (CC50), causing 50% cell death, were determined by nonlinear
regression analysis using the Prism 6.0 software (GraphPad Software Inc.). Daclatasvir was
kindly provided by Dr. Marc Windisch (Institut Pasteur Korea).
3.2.3. Luciferase and Bradford Assays
Firefly luciferase activity was measured using Luciferase Assay System (Promega),
according to the manufacturer’s instructions. Measurements were performed with a
GloMax 20/20 single tube luminometer (Promega) for 10 s. Values were normalized to
total protein amounts as quantified by Bradford assay (Pierce).
3.2.4. Measurement of Intracellular ATP Levels
ATP was measured using the ViaLight HS BioAssay kit (Lonza) according to the
manufacturer’s protocol in a GloMax 20/20 single-tube luminometer (Promega) for 1 s.
Values were normalized to total protein amounts.

Pharmaceuticals 2021, 14, 292
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3.2.5. Gel Electrophoresis and Western Blot Analysis
Denaturing SDS-polyacrylamide gel electrophoresis and Western blotting were per-
formed as described previously [29]. Dilutions of 1:2000 for HCV NS5A (9E10) monoclonal
antibody (kindly provided by C. Rice), 1:6000 for β-actin monoclonal antibody (Merck-
Millipore), and 1:2000 for the secondary anti-mouse horseradish peroxidase-conjugated
antibody (Cell Signaling) were used. Image quantification analysis was performed with
Quantity I Bio-Rad software.
3.2.6. Total RNA Extraction and Quantification of Viral Replicons
Total RNA was extracted from Huh5-2 cells with Nucleozol reagent (Macherey-Nagel),
according to the manufacturer’s instructions, and used to perform reverse-transcription
(RT) and quantitative real-time polymerase chain reaction (qPCR) for replicon RNA. RT was
performed using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Promega)
and reverse primers specific for Con1 IRES (5’-GGATTCGTGCTCATGGTGCA-3’) and the
housekeeping gene YWHAZ (5’- GGATGTGTTGGTTGCATTTCCT-3’). For qPCR, primers
specific for the Con1 IRES (forward: 5’-GGCCTTGTGGTACTGCCTGATA-3’and reverse:
5’-GGATTCGTGCTCATGGTGCA-3’) and KAPA SYBR FAST qPCR Master Mix (Kapa
Biosystems) were used. YWHAZ mRNA was used as a normalization control (forward: 5’-
GCTGGTGATGACAAGAAAGG-3’ and reverse: 5’- GGATGTGTTGGTTGCATTTCCT-3’).
3.2.7. Statistical Analysis
In all diagrams, bars represent the mean values of three independent experiments
in triplicate. Error bars represent standard deviation. Only results subjected to statistical
analysis using Student’s t-test with p
≤
0.05 were considered as statistically significant and
presented. Statistical calculations were performed with Excel Microsoft Office®.
4. Conclusions
Novel 2,7-diaminofluorene-S-prolinamide or 2,7-diaminofluorene-(S,R)-piperidine-
3-caboxamide bearing several capping groups were prepared and evaluated for their
anti-HCV activity. We concluded that the 2,7-diaminofluorene-S-prolinamide core analogs
exhibited better activity over the 2,7-diaminofluorene-(S,R)-piperidine-3-carboxamide core
counterparts, a fact that can be attributed to the extra methylene carbon in the piperi-
dine carboxamide analogs, which forces a conformation and changes the relative orien-
tation of some essential groups in the binding site. Additionally, the derivatives with
R-phenylglycine as a terminal capping group were more potent than the frequently tested
S-valine analogs. This can help to design of novel NS5A inhibitors with great diversity. Our
findings encourage the development of new peptidomimetic analogs. They support the
successful implementation of the peptidomimetic approach for the development of novel
NS5A inhibitors with improved physicochemical and pharmacokinetic properties. This
work will be extended to investigate the conformational and stereoelectronic aspects of the
core scaffold and obtain more SAR information to help refine the critical requirements for
optimal activity.
Supplementary Materials: The following are available online at https://www.mdpi.com/1424-824
1
7/14/4/292/s1. Figure S1: H-NMR and 13C-NMR spectra of selected compounds.
Author Contributions: M.H.A.M., did all the experimental chemistry, explained the spectra, and
wrote the drafted manuscript. N.S.A., contributed to the idea, revised all the chemistry and biology
results, and helped in writing the manuscript. K.S. assisted with experimental chemistry and helped
in explaining the spectra. E.F. did the biological testing and helped in writing that section. N.V. did
the biological testing part and helped in writing it. G.Z. helped in explaining chemistry and biology
figures and results, and helped in writing the manuscript and optimizing it. J.J.W., supervised the
chemistry done in Germany and revised the chemistry part of the manuscript. A.H.A. coordinated
the whole study and its conceptualization, helped in explaining chemistry and biology figures and

Pharmaceuticals 2021, 14, 292
21 of 22
results, and helped in writing the manuscript. All authors have read and agreed to the published
version of the manuscript.
Funding: This paper is based upon work supported by the Science and Technology Develop-
ment Fund (STDF), Egypt, Funding Authority, under grant 22848 to Ashraf Abadi. Mai Moussa
thanks the German Academic Exchange Service (DAAD) for an Erasmus plus (ERA+) stipend
awarded to Nermin Ahmed. Grigoris Zoidis thanks Asklepios Gilead Hellas Grants Program for the
financial support.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or supplementary material.
Acknowledgments: We are grateful to Ralf Bartenschlager (Heidelberg University, Germany) for
kindly providing the Huh5-2 cell line and Charles M. Rice for kindly providing replicon plasmids
S52-SG (Feo) (AII) and ED43-SG (Feo) (VYG), as well as the 9E10 HCV NS5A-specific antibody.
Conflicts of Interest: The authors have declared no conflict of interest.
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