Symmetric benzidine derivatives as anti-HCV agents: Insight into the nature, stereochemistry of the capping amino acid and the size of the terminal capping carbamates

Article abstract of DOI:10.1016/j.bioorg.2020.104089

Novel symmetric molecules, bearing a benzidine prolinamide core, two terminal carbamate caps of variable sizes and nature, including natural and unnatural amino acids were developed. Several terminal N-carbamate substituents of the core structure, ranging from linear methyl, ethyl and butyl groups to branching isobutyl group; and an aromatic substituent were also synthesized. Series 1 has hydrophobic AA residues, namely S and R phenylglycine and a terminal carbamate capping group, whereas Series 2 bears sulphur containing amino acids, specifically S and R methionine and the natural R methylcysteine. The novel compounds were tested for their inhibitory activity (EC₅₀) and their cytotoxicity (CC₅₀), using an HCV 1b (Con1) reporter replicon cell line. Compound 4 with the unnatural capping residue, bearing D-Phenylglycine amino acid residue and N-isobutyloxycarbonyl capping group, was the most active within the two series, with EC₅₀ = 0.0067 nM. Moreover, it showed high SI₅₀ > 14788524 and was not cytotoxic at the highest tested concentration (100 μΜ), indicating its safety profile. Compound 4 also inhibited HCV genotypes 2a, 3a and 4a. Compared to the clinically approved NS5A inhibitor Daclatasvir, compound 4 shows higher activity against genotypes 1b and 3a, as well as improved safety profile.

Full text of DOI:10.1016/j.bioorg.2020.104089

Journal Pre-proofs
Symmetric benzidine derivatives as anti-HCV agents: insight into the nature,
stereochemistry of the capping amino acid and the size of the terminal cap-
ping carbamates
Shereen E. Abdel Karim, Youssef H. Youssef, Mohammad Abdel-Halim,
Efseveia Frakolaki, Niki Vassilaki, Grigoris Zoidis, Nermin S. Ahmed,
Ashraf H. Abadi
PII:
S0045-2068(20)31386-9
DOI:
https://doi.org/10.1016/j.bioorg.2020.104089
YBIOO 104089
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
4 March 2020
30 June 2020
7 July 2020
Please cite this article as: S.E. Abdel Karim, Y.H. Youssef, M. Abdel-Halim, E. Frakolaki, N. Vassilaki, G.
Zoidis, N.S. Ahmed, A.H. Abadi, Symmetric benzidine derivatives as anti-HCV agents: insight into the nature,
stereochemistry of the capping amino acid and the size of the terminal capping carbamates, Bioorganic Chemistry
(2020), doi: https://doi.org/10.1016/j.bioorg.2020.104089
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Symmetric benzidine derivatives as anti-HCV agents: insight into
the nature, stereochemistry of the capping amino acid and the size
of the terminal capping carbamates
Shereen E. Abdel Karima, Youssef H. Youssefa, Mohammad Abdel-Halima, Efseveia Frakolakib,
Niki Vassilakib, Grigoris Zoidisc, Nermin S. Ahmeda, Ashraf H. Abadia*
aFaculty of Pharmacy and Biotechnology, Department of Pharmaceutical Chemistry, German
University in Cairo, 11835 Cairo, Egypt.
bMolecular Virology Laboratory, Hellenic Pasteur Institute, Vas. Sofias Avenue, 11521, Athens,
Greece.
cDepartment of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences,
National and Kapodistrian University of Athens, GR-15771, Athens, Greece.
*Correspondence: Prof. Ashraf H. Abadi, Faculty of Pharmacy and Biotechnology, Department of
Pharmaceutical Chemistry, German University in Cairo, 11835 Cairo, Egypt. Email:
ahabadi@yahoo.com; ashraf.abadi@guc.edu.eg
1

Abstract
Novel symmetric molecules, bearing a benzidine prolinamide core, two terminal carbamate
caps of variable sizes and nature, including natural and unnatural amino acids were developed.
Several terminal N-carbamate substituents of the core structure, ranging from linear methyl, ethyl
and butyl groups to branching isobutyl group; and an aromatic substituent were also synthesized.
Series 1 has hydrophobic AA residues, namely S and R phenylglycine and a terminal
carbamate capping group, whereas Series 2 bears sulphur containing amino acids, specifically S
and R methionine and the natural R methylcysteine. The novel compounds were tested for their
inhibitory activity (EC50) and their cytotoxicity (CC50), using an HCV 1b (Con1) reporter replicon
cell line. Compound 4 with the unnatural capping residue, bearing D-Phenylglycine amino acid
residue and N-isobutyloxycarbonyl capping group, was the most active within the two series, with
EC50=0.0067 nM. Moreover, it showed high SI50 >14788524 and was not cytotoxic at the highest
tested concentration (100 μΜ), indicating its safety profile. Compound 4 also inhibited HCV
genotypes 2a, 3a and 4a. Compared to the clinically approved NS5A inhibitor Daclatasvir,
compound 4 shows higher activity against genotypes 1b and 3a, as well as improved safety profile.
Keywords: NS5A inhibitors; Anti-HCV; capping groups.
2

1. Introduction
Hepatitis C is a hepatic disease induced by a blood borne virus called hepatitis C virus
(HCV). The severity of hepatitis ranges from mild illness that can be cured within a few weeks, to
a life-threatening disease, meanwhile 80% of the patients progress into a chronic infection [1].
Hepatitis C is a global health problem, as about 71 million people are infected chronically and at
risk of cirrhosis, liver failure, and hepatocellular carcinoma. To date, there is no available vaccine
for hepatitis C.
HCV belongs to family Flaviviridae, genus Hepacivirus [2] and it contains a positive sense
single strand RNA with length of 9600 nucleotides that encodes around 3000 amino acids
polyprotein.[3] The RNA polymerase of the virus is error-prone, so HCV shows high genetic
variability and classified into seven main genotypes and more than 100 subtypes that vary
regarding the risk factors of infections, response to treatment and geographic distribution [4].
The HCV genome consists of 5’ and 3’- terminal non translated regions (5` and 3`-NTR)
and a central open reading frame (ORF) which encodes a polyprotein processed into structural (S)
proteins (core [C], E1 and E2) and non-structural (NS) proteins (p7, NS2, NS3, NS4A, NS4B,
NS5A, and NS5B) [5,6]. NS5A is hydrophilic multifunctional phosphoprotein essential for HCV
replication and assembly. Moreover, it modulates cell signaling pathways and the interferon
response. Thus, NS5A is the target for current therapeutic strategies [7]. Until 2011, the standard
therapy of HCV was Pegylated interferon-α combined with Ribavirin for a duration of 24 or 48
weeks [8,9]. In 2011, a breakthrough in the treatment of HCV was accomplished with the
development of novel direct-acting antivirals (DAA) with pan-genotypic activity, high efficacy
and less side effects [10].
3

DAAs are classified into three main classes: NS3/4A protease inhibitors, RNA-dependent
RNA-polymerase NS5B inhibitors, and NS5A protein inhibitors [11]. The first clinically approved
NS5A inhibitor was daclatasvir in 2014, followed by ledipasvir, ombitasvir, elbasvir, velpatasvir
and pibrentasvir. Although these inhibitors have extremely high potency, their clinical application
is still limited due to the high cost, their somewhat low efficacy against non-genotype 1 HCV
strains and a tendency to develop drug resistance[12]. Recently, in 2019, several modifications
were done to ombitasvir, the first generation NS5A inhibitor, where a dianiline or an aniline core
was used together with natural and unnatural amino acids. These compounds show more selectivity
toward GT 1b and 4a [13].
The efficacy of daclatasvir against the different HCV genotypes varies, with genotype 3
being the least sensitive [14]. Only the recently approved pibrentasvir and velpatasvir show similar
activity against all HCV genotypes [12]. As genotype 3 represents 30% of HCV infections
worldwide [15], there is still an urgent need for the development of more potent pan-genotypic
NS5A inhibitors with significant activity against genotype 3.
Different studies revealed the dimeric nature of domain I of NS5A and represented a
significant advance in the study of NS5A replication complex inhibitor (NS5A RCI)−target
interactions [16]. The roughly symmetrical nature of the most active NS5A inhibitors and the
symmetry of the crystallized protein dimer complements are consistent with resistance-associated
mutations suggesting that domain I is the binding site of daclatasvir and structurally related
compounds.
Our research group have recently worked on the design of symmetrical NS5A inhibitors.
[13, 17] Symmetrical analogues bearing a benzidine core were moderately active. [17] During the
course of our work, some monomeric benzidine analogues were prepared and tested, they showed
4

no significant anti-HCV activity on CON-1, Genotype 1b when compared to their symmetric
congeners. For example (diethyl ((2S,2'S)-((2S,2'S)-(([1,1'-biphenyl]-4,4' diylbis
(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-
diyl))dicarbamate), compound 9, reference 17, EC50 13.9 μM, versus (ethyl ((S)-3-methyl-1-oxo-
1-((S)-2-((4'-((S)-pyrrolidine-2-carboxamido)-[1,1'-biphenyl]-4-yl)carbamoyl)pyrrolidin-1-
yl)butan-2-yl)carbamate) EC50 >20 μM. This same finding was observed with other cores we
investigated (Abadi et al, unpublished data).
Therefore, we turned our attention only to this class of symmetric benzidine compounds.
Our aim was to expand the chemical space around this promising core and to develop potent pan
genotypic analogues with high safety margin.
In this study, symmetric NS5A inhibitors with benzidine L-prolinamide core, natural,
unnatural and sulphur-containing amino acids, were designed and synthesized. The key
pharmacophoric features of other reported NS5A inhibitors, such as the two hydrophobic aromatic
regions, a terminal hydrophobic chain and two or more hydrogen bond acceptors were maintained
[24] (Fig. 1).
5

Fig.1. Structural changes performed on the clinically approved NS5A inhibitor daclatasvir to
obtain compounds 1-25.
2. Results and discussion
2.1. Chemistry
Amide coupling of benzidine with N-Boc-L-Proline using coupling reagent EEDQ led to
N-Boc-protected compound, followed by deprotection with triflouroacetic acid (TFA) to give the
free amine, as (outlined in Scheme 1). Two series of capping groups were synthesized. The first
series 1-10 were synthesized by the reaction of D, L- phenylglycine and the corresponding
chloroformate in NaOH yielding the respective carbamate derivatives (outlined in Scheme 2). The
6

second series were synthesized by the reaction of sulphur containing amino acids (D, L-
methionine 11-20 and alkylated L-cysteine 21-25 respectively with the corresponding
chloroformate yielding the respective carbamate derivatives (outlined in Scheme 3 and 4).
Coupling each of these carbamate derivatives to the free amine using HBTU as a coupling reagent
was done (outlined in Scheme 1). The carbamate terminal groups varied from non-branching to
branching alkyl chains and from aliphatic to aromatic groups.
H
Boc
O
N
N
O
N
S
S
N
S
a
H2N
NH2
+
Boc
Boc
OH
N
O
H
I
S
O
H
H
O
N
N
b
S
S
NH
c
N
S
Cap
Cap
HN
N
S
N
O
N
O
H
H
II
Scheme 1. Reagents and Conditions: (a) N-Boc-L-Proline, EEDQ, CH2Cl2, 94%; (b) TFA,
CH2Cl2; (c) capping group (CG1-25), HBTU, DIPEA, CH2Cl2, 21-38%.
O
O
O
R,S
H
R,S
O
N
*
R1O
Cl
*
OH
+
R1
OH
NH2
O
For exact R, R1 and configuration of the capping AA, see Table 1
Scheme 2. Reagents and Conditions: aqueous NaOH, Dioxane.
7

O
O
O
R,S
*
R,S
*
S
S
R1O
Cl
OH
OH
+
O
NH
NH2
R1
O
For exact R1 and configuration of the capping AA, see Table 1
Scheme 3. Reagents and Conditions: Aqueous NaOH, Dioxane.
Finally, we also prepared thioether derivatives of L-Cysteine via S-alkylation to S-methyl in good
yield under basic conditions using 1,1,3,3-tetramethyl guanidine (TMG).
O
O
OH
O
O
R
a
b
R
R
HS
OH
S
R1
CH3-I
+
S
OH
N
O
NH2
H
NH2
For exact R1 and configuration of the capping AA, see Table 1
Scheme 4. Reagents and Conditions: (a) TMG, MeOH, Argon, 50°C; (b) Aqueous NaOH ,
Chloroformates, Dioxane.
The molecular masses and purity of the synthesized compounds were confirmed using
LC/MS. The charts of the compounds show molecular ion peak appear at (M+H) +, in addition to
a peak with m/z ratio half that of the molecular ion peak due to the symmetrical structures of the
compounds.
2.2. Compound screening against HCV genotype 1b replicon
Two series of novel dimeric analogues were synthesized. Series 1 has hydrophobic AA
residues and a terminal carbamate capping group, whereas Series 2 bears sulphur-containing
amino acids. The latter molecules have been designed to be active in their original form, as it has
been reported that they have the potential to be metabolized intracellularly via CYP450 to
8

sulfoxide and sulfone moieties, which have anti-HCV activity [25]. This synthetic approach could
possibly lead to a complementary resistance profile. The residues bear N-methoxycarbonyl, N-
ethoxycarbonyl, N-butyloxycarbonyl, N-isobutyloxycarbonyl or N-benzyloxycarbonyl carbamates
as terminal capping groups.
Compounds were tested for their effect on HCV RNA replication and cell viability in the
stable cell line Huh5-2 bearing a reporter replicon of HCV genotype 1b (strain Con1) [26]. In this
system, the level of viral replication corresponds to the level of the co-expressed firefly luciferase.
Cells were treated with serial dilutions of the compounds in cell culture medium for 72 hours,
where the final concentration of the solvent DMSO was 0.2%, so as not to affect viral replication
levels (data not shown). The activity (expressed as the 50% effective concentration [EC50]) and
the cytotoxicity (expressed as the 50% cytotoxic concentration [CC50]) of compounds 1-25 were
calculated by measuring luciferase activity derived from HCV replication and intracellular ATP
levels, respectively. The selectivity index (SI50) was calculated as ratio of CC50 versus EC50. The
results are summarized in (Table 1) and dose-response curve analysis for the most potent analogues
is presented (Fig. 2), using Daclatasvir as positive control.
Τhe benzidine L-prolinamide part is contained in all 25 compounds. Although most of the
commercially available NS5A inhibitors bear L-valine methyl carbamate ester as a capping group,
in our approach we adopted different capping groups of D, L-phenylglycine, D, L-methionine or
alkylated L-cysteine with different alkyl/aryl carbamate esters, which were designed to have higher
inhibitory activity.
Series 1 consists of carbamate derivatives with different capping groups that vary in length,
branching and aromaticity. It is worth mentioning that incorporation of a phenylglycine group on
both caps, show high inhibitory activities, regardless of the type of carbamate ester. Compounds
9

1-10 showed the highest potency among all analogues tested, with EC50 0.0067- 30 nM. This
indicated that the lipophilic features are essential for interaction with the nearby lipophilic residues
of NS5A and for better binding affinity for the target protein.
Compound 4 was the most active compound within the two series, with EC50=0.0067 nM.
Moreover, it showed high SI50 >14788524 and was not cytotoxic at the highest tested concentration
(100 μΜ), indicating its safety profile. It bears D-phenylglycine amino acid residue and N-
isobutyloxycarbonyl capping group. We conclude that these features are essential for interactions
with the receptor.
Surprisingly, within Series 1 compounds 1-5 and 6-10, containing the amino acid
phenylglycine, the D-epimers 1-5 showed the highest activity. This highlights the importance of
testing both natural and unnatural epimers of the same residue to figure out the more potent
derivatives and emphasizes the crucial role of the stereocenter of the amino acids.
Concerning the carbamate moiety in the D-phenylglycine containing molecules 1-5, we
observed that compounds 4 and 5 that bear an N-isobutyloxycarbonyl and N-benzyloxycarbonyl
capping group, respectively, were the most active, with EC50= 0.0067 nM and 0.018 nM. This
indicates that, in this series, the presence of aromatic or bulkier alkyl capping groups confers higher
activity rather than the small aliphatic ones.
Among L-phenylglycine carbamate derivatives 6-10, compound 10, bearing an N-
benzyloxycarbonyl capping group, was the most active one with EC50= 0.3 nM. This indicates that
aromaticity is essential for interactions with the target protein for this series of compounds.
In Series 2, a new approach was adopted. Instead of the commonly used lipophilic AA
residues, we investigated the role of sulphur containing AA namely D, L-methionine and L-
cysteine in the capping groups.
10

Among compounds 11-20 of Series 2, bearing a methionine amino acid residue, compound
18, with a N-butyloxycarbonyl group, was the most active analogue with EC50= 31.27 nM and
SI50>3198. However, upon comparing the rest of the S-epimers (L-methionine amino acids) to the
R- epimers (D-methionine amino acids) of the synthesized analogues, it was observed that the R-
epimers were more potent, confirming the importance of the stereocenter of the AA residue and
the crucial role of the projection plane for compound activity. These results expand the chemical
space from which new NS5A inhibitors can be discovered.
Among analogues 21-25 of Series 2, containing a S-methyl L-cysteine AA residue,
compound 21, that bears a N-methoxycarbonyl capping group, was the most active with
EC50=33.87 nM and SI50>2952. This indicates that for the analogues in this series, less bulky
aliphatic capping groups confer higher efficacy.
Interestingly, upon comparing the EC50 of L-cysteine compounds 21-25 with the respective
ones for methionine-bearing compounds 11-15, we noticed that compound 21 showed better
activity (EC50= 33.87 nM) relative to compound 11 (EC50= 443.1 nM), while the rest of methionine
derivatives 12-15, with EC50= 83 -508 nm, were more potent than compounds 22-25. This suggests
that the overall features of the molecule determine the anti-HCV efficacy.
11

Table 1: Activity, cytotoxicity and selectivity of the synthesized compounds (1-25) in HCV genotype 1b (Con1)
replicon assay.
H
O
N
S
N
S
Cap
Cap
N
N
H
O
Cpd #
1
Capping groups
Stereochemistry
EC50(nM)
CC50(nM)
SI50
R
0.0568
92510
1628697
O
O
N
H
O
2
3
4
R
R
R
0.03954
0.08061
0.006762
>100000
>100000
>100000
>2529084
>1240541
>14788524
O
O
N
H
O
O
O
N
H
O
O
O
N
H
O
5
6
R
S
0.01838
4
35330
1922198
>2500
O
O
N
H
O
>10000
O
O
N
H
O
7
8
S
S
4
>2500
>625
>333
O
O
N
H
O
30
>10000
O
O
N
H
O
12

9
S
S
30
>10000
88180
>333
O
O
N
H
O
10
0.32
274618
O
O
N
H
O
O O
HN
11
12
13
14
R
R
R
R
443.1
383.2
114.3
83.68
>100000
>100000
>100000
53740
>226
>261
>875
642
O
S
O O
O
HN
S
O
O
O
HN
S
O O
HN
O
S
15
16
R
S
508.1
192.6
58140
114
O
O
O
HN
S
O O
HN
>100000
>519
O
S
O O
HN
17
18
S
S
>1000
31.27
>1000
1
O
S
>100000
>3198
O O
HN
O
S
19
S
>1000
>1000
1
O O
HN
O
S
13

20
S
>1000
>1000
1
O O
HN
O
S
21
22
H
N
R
R
33
>100000
>1000
>2952
1
O
S
O
O
H
>1000
N
O
S
O
O
H
N
23
24
25
R
R
R
>1000
>1000
>1000
>1000
>1000
>1000
1
1
1
O
S
O
O
H
N
O
O
S
O
H
N
O
S
O
O
Daclatasvir
0.0197
17700
898477
HCV 1b
120
100
80
60
40
20
0
4
5
Daclatasvir
C (pM)
0.1
1.0
10.0
100.0
1000.0
Fig. 2.Dose–response curve analysis for compounds 4 and 5 against HCV genotype 1b RNA replication. Serial
dilutions of the compounds were used to treat for 72 h Huh5-2 subgenomic replicon cells seeded at 30% confluency.
Viral RNA replication-derived firefly luciferase activity was determined and expressed as relative light units (RLU)
per μg of total protein. Values from cells treated with the solvent DMSO (control) were set to 100%. Daclatasvir was
used as a positive control. Bars represent mean values obtained from three separate experiments in triplicate. Error
bars represent standard deviation (SD).
14

2.3. Activity of compounds 4 and 5 against other HCV genotypes
Compounds 4 and 5 from Series 1, exhibiting the highest potency against HCV genotype 1b
among all synthesized compounds, were tested against other HCV genotypes. The effect of the
compounds on viral replication-derived luciferase activity was examined in Huh7-JFH1, Huh7.5-
3a and Huh7.5-4a stable cell lines containing subgenomic replicons of HCV GT 2a (strain JFH1),
GT 3a (strain S52) and GT 4a (strain ED43), respectively. The clinically approved NS5A inhibitor
daclatasvir was used for comparison.
Between the two molecules, compound 4 was more potent than compound 5 in all genotypes
tested (Table 2). Specifically, compound 4 showed significant activity against genotype 3a, with
EC50 1.365 nM. In addition, its activity against genotype 2a was slightly lower but still very strong,
with EC50=5.183 nM. Concerning genotype 4a, compound 4 had EC50=142.2 nM. It is worth
mentioning that the low cytotoxicity of the compound, in combination with its significant activity
in genotypes 2a and 3a, resulted in SI50 more than 5 orders of magnitude. Thus, compound 4 shows
high efficiency and against genotypes that are difficult to treat. Interestingly, compared to
daclatasvir, compound 4 was ~6 times more active in genotype 3a, while it also exhibited
significantly less cytotoxicity.
Table 2: Activity, cytotoxicity and selectivity of compounds 4 and 5, compared to Daclatasvir, HCV genotype 1b
(Con1), 2a (JFH1), 3a (S52) and 4a (ED43) replicon assays.
Genotype
GT 1b
GT 2a
GT 3a
GT 4a
CC50
(nM)
EC50
(nM)
EC50
(nM)
EC50
(nM)
EC50
(nM)
SI
SI
SI
SI
4
>100000 0.006762 >14788524
5.183
>19294 1.365 >73260
142.2
273.0
>703
5
35330
17700
0.01838
0.01965
1922198
900763
38.127
927
2.275
15530
2120
129
Daclatasvir
0.05784 306017 8.348
0.02119
835300
15

2.4. Effect of compound 4 on HCV RNA and protein levels
To confirm the anti-HCV activity of compound 4 and its mechanism of action by additional
assays other than measuring the virus-derived luciferase levels, viral HCV RNA and NS5A protein
levels in genotype 1b replicon cells (Huh5-2) were determined. Cells were treated with serial
dilutions of the compound and then viral RNA was quantified using reverse transcription -
quantitative polymerase chain reaction (RT-qPCR) and NS5A was evaluated using indirect
immunofluorescence. DMSO-treated cells were used as a negative control. We observed that
compound 4 reduced HCV RNA replication (Fig. 3A) with EC50=0.0089 pM, consistently to the
decrease in replication-derived luciferase activity (Table 1). In agreement, HCV NS5A levels, as
shown by indirect immunofluorescence analysis in replicon cells, were significantly reduced in the
highest compound concentration used (Fig. 3B).
16

Fig. 3.Effect of compound 4 on HCV RNA and protein expression levels in a subgenomic HCV genotype 1b (Con1)
replicon assay. Huh5-2 replicon cells were treated with serial dilutions of compound 4 or the solvent DMSO, for 72
hours. (A) (+) strand HCV RNA was quantified by RT-qPCR and normalized to the mRNA levels of the
housekeeping gene YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta).
Values from cells that were treated with DMSO (control) were set to 100. (B) HCV NS5A staining (left column),
using indirect immunofluorescence. Nuclei were stained with propidium iodide (PI; middle panels) as a cell viability
marker. Merged images are shown at the right. Scale bar, 20 μm.
17

2.5. Combinatory effect of compound 4 with DCV
To examine the interaction of compound 4 in combination with an approved NS5A inhibitor,
Huh5-2 replicon cells were treated with compound 4 in the presence or absence of daclatasvir
(DCV). Compound 4 and DCV had an antagonistic effect, which was determined by calculating
the coefficient of drug interaction (CDI≈1.3). This indicates that both compounds bind at the same
site in NS5A (Fig 4).
Combination of Compound 4 with Daclatasvir
120
100
80
60
40
20
0
Compound 4:
Daclatasvir:
8 pM
-
+
-
+
-
-
-
+
-
-
-
-
+
-
+
-
-
+
-
M
30
15
8
4
4 pM
30 pM
15 pM
-
-
-
-
-
-
-
+
+
Fig. 4. Combinatory activity of compound 4 (Cmp. 4) with Daclatasvir (DCV) against HCV replication. Huh5-2 cells
were treated or not with 30 pM or 15 pM DCV in the presence or absence of compound 4. F-Luc activity was
determined and calculated as relative light units (RLU) per μg of total protein. Values were expressed as percentage
of those obtained with cells treated with the solvent DMSO (control). CDI: coefficient of drug interaction,
CDI=AB/(AxB). Bars represent mean values obtained from three separate experiments in triplicate. Error bars
represent standard deviation (SD).
3. Conclusions
Despite the challenges that the discovery of pan-genotypic HCV NS5A inhibitors has to
face, due to the significant diversity among mutants/genotypes and the absent of protein/inhibitor
complexes X-ray structures for rational inhibitor design, we discovered a potent and almost pan-
18

genotypic NS5A inhibitor. Compound 4 with the unnatural capping residue, bearing D-
Phenylglycine amino acid residue and N-isobutyloxycarbonyl capping group, was the most potent
within the two series against genotype 1b, highlighting that these features are essential for
interactions with the receptor. These results expand the chemical space from which new NS5A
inhibitors may be discovered. Compound 4 inhibited HCV genotypes 2a, 3a and 4a, with
significant activity against genotype 3a with EC50 1.365 nM. Concerning genotype 4a, which is
the most prevalent in Egypt, compound 4 had EC50 142.2 nM. It is noteworthy that compound 4
compared to daclatasvir proved to be 16 and 35 more active against genotypes 1b and 3a
respectively. Results of combinatory effect of compound 4 and DCV indicate that both compounds
bind at the same site in NS5A.
Our findings suggest that the novel symmetric molecules, bearing a benzidine prolinamide
framework that we have developed, following mild and experimentally convenient protocols,
offers a promising core for further construction of new analogues with two terminal carbamate
caps of variable sizes and nature endowed with optimized anti-HCV properties through appropriate
terminal N-carbamate substituents. Future experiments with asymmetric scaffolds and further
modifications on the rigid biphenyl core should deliver more detailed SAR information to help
refine the requirements for optimal NS5A inhibitory activity.
19

4. Experimental section
4.1. Chemistry
All starting materials, organic solvents and reagents were obtained from commercial
suppliers and used without purification. Column chromatography carried out using silica-gel 70-
230 μM mesh. The progress of the reactions was monitored by TLC using fluorescent pre-coated
silica gel plates and the compounds were detected by short UV light (λ= 254 nm). Reactions were
performed under argon when inert atmosphere was required. The detection of carbamates was done
using furfural/sulphuric acid solution.
1H-NMR spectra were done on Varian spectrometer, at 400 MHz and 13C-NMR spectra were run
at 101 MHz in Dimethyl Sulfoxide (DMSO). Chemical shifts (δ) were reported in parts per million
(ppm) downfield from TMS; and all coupling constants (J) were given in Hz. The purities of the
compounds were detected by HPLC coupled with mass spectrometer and were more than 90% for
all compounds.
The Mass spectrometric analysis (UPLC-ESI-MS) was carried out using Waters ACQUITY
XevoTQD system, which consisted of an ACQUITY UPLC H-Class system, XevoTM TQD triple-
quadrupole tandem mass spectrometer and an electrospray ionization (ESI) interface (Waters
Corp., Milford, MA, USA). Acquity BHE C18 100mm x 2.1 mm column (particle size, 1.7 μm was
used to separate analytes (Waters, Ireland). The solvent system was composed of water containing
0.1% TFA in acetonitrile (B). The masses were reported as protonated ions (M+H)+.
20

4.1.1. General procedures for the preparation of capping groups CG 1-20
[27]
A 500 mL round bottom flask was equipped with a magnetic stir bar, charged with distilled
H2O (100 mL) and NaOH (100 mmol, 4.0 g) and cooled in an ice bath to 0 °C. L-Phenylglycine/L-
Methionine/D-Phenylglycine/D-Methionine (35 mmol) was added, and stirred until the solution
was homogeneous. The corresponding carbonyl chloride (45.5 mmol) in 1,4-dioxane (40 mL) was
added dropwise using an addition funnel. The reaction mixture was left to stir overnight at room
temperature. The solution was extraction of the solution with Et2O (3 × 50 mL) was done, and the
organic layers were discarded. The aqueous layer was cooled in an ice bath to 0 °C, and
concentrated HCl was added dropwise until the pH = 2. The re-extraction of the aqueous solution
was done with Et2O (3 × 100 mL). The organic layers were combined, dried over anhydrous
MgSO4, filtered, and concentrated in vacuum to give the product as clear viscous oil. No further
purification was required.
4.1.2. General procedures for the preparation of alkylated L-Cysteine [28]
A solution of 1,1,3,3-tetramethyl guanidine (21 mmol) in 5 mL of MeOH was added
dropwise to a previously stirred mixture (under argon) of L-Cysteine (10 mmol) in 10 mL of
MeOH. To the homogenous reaction mixture was added alkylation reagent methyl iodide (10
mmol) in tetrahydrofuran dropwise and then was stirred for 1 h at 50°C. Then 10 mL of H2O
acidified with a stoichiometric quantity of AcOH (1:1) was added. The separated precipitate by
vacuum filtration was washed several times with ethanol to give white/off white solid. No further
purification was required.
21

4.1.3. General procedures for the preparation of capping groups CG 21-25
[27]
A 500 mL round bottom flask was equipped with a magnetic stir bar, charged with distilled
H2O (100 mL) and NaOH (100 mmol, 4.0 g) and cooled in an ice bath to 0 °C. S-methyl cysteine
(35 mmol) was added, and stirred until the solution was homogeneous. The corresponding
carbonyl chloride (45.5 mmol) in 1,4-dioxane (40 mL) was added dropwise using an addition
funnel. The reaction mixture was left to stir overnight at room temperature. The solution was
extraction of the solution with Et2O (3 × 50 mL) was done, and the organic layers were discarded.
The aqueous layer was cooled in an ice bath to 0 °C, and concentrated HCl was added dropwise
until the pH = 2. The re-extraction of the aqueous solution was done with Et2O (3 × 100 mL). The
organic layers were combined, dried over anhydrous MgSO4, filtered, and concentrated in vacuum
to give the product as clear viscous oil. No further purification was required.
4.1.4. General procedure for the preparation of (2S,2'S)-di-tert-butyl 2,2'-
(([1,1'-biphenyl]4,4'diylbis(azanediyl))bis(carbonyl)) bis(pyrrolidine-1-
carboxylate) (N-protected benzidine prolinamide) (I) [29]
EEDQ (22.58 g, 91.3 mmol) was added in one portion to a solution of benzidine (8.00 g,
38.1 mmol) and N-Boc-L-proline (18.83 g, 87.5 mmol) in dry CH2Cl2 (250 mL) and it was left to
stir for 16 hours at room temperature under argon. The resultant component was removed in
vacuum. The crude product was washed with Et2O and vacuum-filtered to provide compound I as
off white solid with 94% yield. No further purification was required.
22

4.1.5. General procedure for the preparation of (2S,2'S)-N,N'-([1,1'-
biphenyl]-4,4'-diyl)bis(pyrrolidine-2-carboxamide) (Deprotected
benzidine prolinamide) (II) [29]
CF3CO2H (60 mL) was added to the stirred solution of compound I (23.62 g, 40 mmol) in
CH2Cl2 at room temperature under argon. The mixture was left to stir for 3 hours before additional
portion of CF3CO2H (24 mL) was added. After stirring for an additional 4 hours at room
temperature, the component was evaporated in vacuum. The residue was dissolved in EtOAc,
washed with saturated NaHCO3 solution and brine. 1 N NaOH was added until pH= 12. The
residue was dried over anhydrous MgSO4 and concentrated in vacuum to give a pale yellow sticky
solid, which was dissolved in acetone and evaporated in vacuum. This step was repeated 2-3 times
to give pale yellow crystals. No further purification was required.
4.1.6. General procedure for the preparation of compounds 1-25 [30]
Capping groups CG 1-25 (3 mmol) were added to compound II (0.4 g, 1 mmol), HBTU
(1.13 g, 3 mmol) and TEA (0.7 mL) in CH2Cl2. The reaction mixture was left to stir for 2 hours
under argon at room temperature. The solvent was removed in vacuum and then the residue was
purified using silica gel column chromatography (98.5:1.5 CH2Cl2/ MeOH) to give compounds 1-
25.
4.1.6.1. Dimethyl ((1R,1'R)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-
diyl))dicarbamate (1)
1
Yellow semi-solid; yield: 29%; C42H44N6O8; LC/MS: [M+H]+ = 761.32; purity 100%; H-NMR
(400 MHz, DMSO) δ 7.69 – 7.60 (m, 9H), 7.43 – 7.32 (m, 9H), 5.49 (s, 2H), 4.56 – 4.37 (m, 2H),
3.53 (t, J = 3.7 Hz, 4H), 3.35 (s, 6H), 2.16 – 1.82 (m, 8H); 13C-NMR (101 MHz, DMSO)
23

δ 170.66, 168.55, 165.03, 156.77, 138.64, 137.34, 134.83, 129.03, 128.46, 126.85, 119.88, 61.00,
57.05, 51.98, 47.32, 29.76, 25.11.
4.1.6.2. Diethyl ((1R,1'R)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-
diyl))dicarbamate (2)
1
Yellow semi-solid; yield: 20%; C44H48N6O8; LC/MS: [M+H]+ = 789.35; purity 91%; H-NMR
(400 MHz, DMSO) δ 7.69 – 7.54 (m, 9H), 7.47 – 7.32 (m, 9H), 5.73 (s, 2H), 4.64 – 4.32 (m, 2H),
4.05 – 3.48 (m, 8H), 2.05 – 1.73 (m, 8H), 1.13 (t, J = 7.1 Hz, 6H); 13C-NMR (101 MHz, DMSO)
δ 168.83, 168.60, 156.31, 130.08, 129.66, 129.02, 128.82, 128.47, 127.87, 126.85, 119.86, 55.34,
31.72, 29.46, 26.99, 25.11, 22.53, 15.08.
4.1.6.3. Dibutyl((1R,1'R)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-
diyl))dicarbamate (3)
1
Yellow semi-solid; yield: 22%; C48H56N6O8; LC/MS: [M+H]+ = 845.42; purity 91%; H-NMR
(400 MHz, DMSO) δ 7.90 – 7.39 (m, 10H), 7.39 – 7.27 (m, 8H), 5.76 (s, 2H), 4.98 – 4.37 (m, 4H),
13
4.03 – 3.93 (m, 8H), 3.28 – 2.69 (m, 4H), 1.37 – 1.25 (m, 10H), 1.24 – 0.9 (m, 6H); C-NMR
(101 MHz, DMSO) δ 172.54, 169.49, 156.44, 140.73, 138.16, 129.06, 128.77, 128.50, 128.08,
126.81, 120.01, 119.57, 70.27, 67.44, 64.27, 58.62, 55.36, 31.18, 30.41, 19.02, 14.07.
4.1.6.4. Diisobutyl((1R,1'R)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-
diyl))dicarbamate (4)
1
Yellow semi-solid; yield: 25%; C48H56N6O8; LC/MS: [M+H]+ = 845.42; purity 91%; H-NMR
(400 MHz, DMSO) δ 7.71 – 7.62 (m, 9H), 7.45 – 7.37 (m, 9H), 5.49 (s, 2H), 4.57 – 4.39 (m, 2H),
3.75 (d, J = 6.4 Hz, 4H), 3.27 – 3.01 (m, 4H), 2.01 – 1.76 (m, 10H), 0.85 (d, J = 5.5 Hz, 12H); 13C-
NMR (101 MHz, DMSO) 170.68, 168.88, 165.08, 137.61, 129.04, 128.85, 128.49, 128.22, 126.80,
120.10, 109.74, 70.64, 61.18, 57.09, 55.33, 29.77, 28.03, 24.70, 19.32.
24

4.1.6.5. Dibenzyl((1R,1'R)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-
diyl))dicarbamate (5)
1
Yellow semi-solid; yield: 25%; C54H52N6O8; LC/MS: [M+H]+ = 913.38; purity 92%; H NMR
(400 MHz, DMSO) δ 7.71 – 7.67 (m, 4H), 7.66 – 7.62 (m, 4H), 7.47 – 7.35 (m, 20H), 5.75 (s, 2H),
5.05 (s, 4H), 4.72 – 4.21 (m, 2H), 3.11 – 2.91 (m, 4H), 2.03 – 1.96 (m, 8H); 13C-NMR (101 MHz,
DMSO) δ 178.21, 162.30, 155.11, 130.11, 130.04, 129.82, 129.56, 129.43, 129.11, 129.03, 128.87,
128.79, 128.51, 128.23, 128.10, 70.24, 55.35, 31.74, 29.47, 27.00, 22.55.
4.1.6.6. Dimethyl ((1S,1'S)-((2S,2'S)-2,2'-(([1,1'-biphenyl]-4,4' diylbis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (6)
Yellow semi-solid; yield: 38%; C42H44N6O8; LC/MS: [M+H]+ = 761.59; purity 98.83%; 1H-
NMR (400 MHz, DMSO) δ 10.14 (s, 2H), 7.68 (d, J = 9.1 Hz, 4H), 7.63 (d, J = 3.4 Hz, 4H), 7.46
(d, J = 7.4 Hz, 4H), 7.38 (t, J = 3.7 Hz, 6H), 4.53 (dd, J = 8.2, 4.7 Hz, 2H), 3.55 (s, 8H), 3.17 (dd,
J = 13.2, 7.0 Hz, 2H), 2.18 (dd, J = 18.5, 7.9 Hz, 2H), 1.94 – 1.79 (m, 8H); 13C-NMR (101 MHz,
DMSO) δ 170.75, 167.39, 158.42, 138.76,137.44, 134.52, 128.81, 127.99, 126.72, 119.64, 78.70,
61.23,59.02, 57.94, 48.33, 31.02, 24.05.
4.1.6.7. Diethyl ((1S,1'S)-((2S,2'S)-2,2'-(([1,1'-biphenyl]-4,4'-diylbis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (7)
1
Light yellow semi-solid; yield: 30%; C44H48N6O8; LC/MS: [M+H]+ = 789.68; purity 92%; H-
NMR (400 MHz, DMSO) δ 10.12 (s, 2H), 7.61 (d, J = 9.6 Hz, 8H), 7.42 (d, J = 6.9 Hz, 4H), 7.34
(t, J = 9.4 Hz, 6H), 5.51 – 5.42 (m, 2H), 4.50 (s, 2H), 3.97 (d, J = 6.5 Hz, 4H), 3.09 (dd, J = 8.3,
5.8 Hz, 2H), 2.16 (dd, J = 10.7, 6.8 Hz, 2H), 1.88 – 1.82 (m, 4H), 1.21 (s, 4H), 1.13 (t, J = 6.9 Hz,
13
6H); C-NMR (101 MHz, DMSO) δ 170.75, 168.39, 156.42, 138.86, 137.44, 134.82, 128.81,
128.06, 126.88, 119.64, 79.70, 61.19, 60.02, 56.94, 47.34, 30.00, 25.05, 15.20.
25

4.1.6.8. Dibutyl ((1S,1'S)-((2S,2'S)-2,2'-(([1,1'-biphenyl]-4,4'-diylbis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (8)
Light green semi-solid; yield: 23%; C48H56N6O8; LC/MS: [M+H]+ = 845.74; purity 94%; 1H-NMR
(400 MHz, DMSO) δ 10.14 (s, 2H), 7.67 (s, J = 8.8 Hz, 4H), 7.58 (d, J = 8.4 Hz, 4H), 7.45 (d, J =
7.1 Hz, 4H), 7.39 – 7.34 (m, 6H), 5.48 (s, 2H), 4.53 (dd, J = 8.0, 4.5 Hz, 2H), 3.96 (t, J = 5.9 Hz,
4H), 3.14 (dd, J = 11.1, 3.9 Hz, 2H), 2.17 (dd, J = 12.0, 6.5 Hz, 2H), 1.91 – 1.85 (m, 4H), 1.55 –
1.49 (m, 4H), 1.36 – 1.24 (m, 8H), 0.89 (t, J = 7.2 Hz, 6H); 13C-NMR (101 MHz, DMSO) δ 171.02,
169.59, 157.22, 139.56, 138.44, 135.88, 129.00, 128.08, 125.48, 120.10, 77.70, 62.29, 61.02,
57.55, 48.24, 32.02, 24.05, 19.98, 15.00, 14.07.
4.1.6.9. Diisobutyl ((1S,1'S)-((2S,2'S)-2,2'-(([1,1'-biphenyl]-4,4'-diylbis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (9)
1
Light green semi-solid; yield: 28%; C48H56N6O8; LC/MS: [M+H]+ = 845.74; purity 100%; H-
NMR (400 MHz, DMSO) δ 10.16 (s, 2H), 7.66 (d, J = 8.7 Hz, 4H), 7.62 (d, J = 9.0 Hz, 4H), 7.46
(d, J = 7.3 Hz, 4H), 7.38 (t, J = 7.2 Hz, 6H), 5.50 (s, 2H), 4.53 (dd, J = 7.5, 4.4 Hz, 2H), 3.74 (d,
J = 6.1 Hz, 4H), 3.15 (dd, J = 36.1, 8.8 Hz, 2H), 2.18 (dd, J = 19.0, 12.7 Hz, 2H), 1.96 – 1.76 (m,
13
8H), 1.29 – 1.20 (m, 2H), 0.88 (d, J = 6.7 Hz, 12H); C-NMR (101 MHz, DMSO) δ 170.06,
169.00, 158.32, 139.00, 137.44, 134.00, 128.88, 127.05, 124.48, 121.13, 78.70, 64.33, 62.02,
58.23, 46.24, 29.89, 25.10, 18.98, 14.00.
4.1.6.10. Dibenzyl ((1S,1'S)-((2S,2'S)-2,2'-(([1,1'-biphenyl]-4,4'-diylbis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (10)
1
Yellow semi-solid; yield: 30%; C54H52N6O8; LC/MS: [M+H]+ = 913.59; purity 96%; H-NMR
(400 MHz, DMSO) δ 10.11 (s, 2H), 7.61 (dd, J = 17.4, 7.9 Hz, 10H), 7.31 (dd, J = 18.7, 7.2 Hz,
18H), 5.50 (s, 2H), 5.02 (s, 4H), 4.50 (dd, J = 7.7, 4.9 Hz, 2H), 3.10 (dd, J = 15.5, 7.1 Hz, 2H),
2.16 (dd, J = 15.3, 8.2 Hz, 2H), 2.04 – 1.91 (m, 4H), 1.89 – 1.83 (m, 4H); 13C-NMR (101 MHz,
26

DMSO) δ 171.94, 170.75, 168.63, 156.41, 144.67, 137.44, 128.84, 128.76, 128.20, 128.09, 119.84,
65.94, 60.97, 57.17, 29.52, 27.04, 23.63, 22.85, 14.35, 11.27, 10.59.
4.1.6.11.Dimethyl((2R,2'R)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-diylbis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(4-(methylthio)-1-oxobutane-1,2-diyl))dicarbamate (11)
Yellow semi-solid; yield: 30%; C36H48N6O8S2; LC/MS: [M+H]+ =757.30; purity 91%;1H-NMR
(400 MHz, DMSO) δ 7.70 – 7.63 (m, 8H), 4.50 – 4.41 (m, 4H), 3.58 (s, 6H), 3.56 – 3.53 (m, 4H),
2.88 (t, J = 8.2 Hz, 4H), 2.07 (s, 6H), 2.00 – 1.80 (m, 12H); 13C-NMR (101 MHz, DMSO) δ 170.76,
157.24, 134.69, 130.60, 124.68, 114.94, 112.22, 55.24, 52.11, 34.91, 31.04, 30.08, 24.77, 22.59,
15.07, 14.85.
4.1.6.12. Diethyl((2R,2'R)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-(methylthio)-1-oxobutane-1,2-
diyl))dicarbamate (12)
Yellow semi-solid; yield: 20%; C38H52N6O8S2; LC/MS: [M+H]+ =785.33; purity 91%; 1H-NMR
(400 MHz, DMSO) δ 7.69 – 7.60 (m, 8H), 4.49 – 4.36 (m, 4H), 4.03 (q, J = 12.5 Hz, 4H), 3.82 –
3.48 (m, 4H), 2.71 – 2.68 (m, 4H), 2.06 (s, 6H), 2.00 – 1.74 (m, 12H), 1.16 (t, J = 7.0 Hz, 6H);
13C-NMR (101 MHz, DMSO) δ 171.40, 170.76, 156.79, 138.45, 134.89, 126.81, 119.99, 61.04,
60.62, 55.34, 51.93, 47.31, 31.08, 30.10, 24.80, 22.60, 15.04.
4.1.6.13. Dibutyl((2R,2'R)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-(methylthio)-1-oxobutane-1,2-
diyl))dicarbamate (13)
Yellow semi-solid; yield: 30%; C42H60N6O8S2; LC/MS: [M+H]+ = 841.39; purity 96%; 1H-NMR
(400 MHz, DMSO) δ 7.86 – 7.25 (m, 8H), 4.43 (m, 4H), 3.99 – 3.95 (m, 4H), 3.84 – 3.57 (m, 4H),
2.57 – 2.53 (m, 4H), 2.06 (s, 6H), 1.94 – 1.74 (m, 12H), 1.51 (m, 8H), 0.85 (t, J = 4.8 Hz, 6H);
13C-NMR (101 MHz, DMSO) δ 171.41, 170.82, 156.93, 138.45, 135.46, 126.84, 120.04, 64.36,
61.05, 51.97, 31.14, 30.26, 27.00, 24.78, 22.55, 19.01, 15.14, 14.86, 14.04.
27

4.1.6.14. Diisobutyl ((2R,2'R)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-(methylthio)-1-oxobutane-1,2-
diyl))dicarbamate (14)
Yellow semi-solid; yield: 21%;C42H60N6O8S2; LC/MS: [M+H]+ = 841.39; purity 91%; 1H-NMR
(400 MHz, DMSO) δ 7.70 – 7.50 (m, 8H), 4.46 – 4.39 (m, 2H), 4.13 – 3.94 (m, 2H), 3.73 – 3.69
(m, 8H), 2.20 – 2.04 (m, 8H), 2.01 (s, 6H), 1.84 – 1.76 (m, 8H), 1.24 – 1.09 (m, 2H), 0.85 (d, J =
13
2.8 Hz, 12H); C-NMR (101 MHz, DMSO) δ 174.22, 170.81, 156.89, 138.63, 134.79, 126.80,
119.81, 70.30, 60.64, 53.08, 51.72, 47.36, 30.82, 30.25, 28.10, 25.13, 19.31, 14.92.
4.1.6.15.Dibenzyl((2R,2'R)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-diylbis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(4-(methylthio)-1-oxobutane-1,2-diyl))dicarbamate (15)
1
Off-white semi-solid; yield: 32%; C48H56N6O8S2; LC/MS: [M+H]+ = 909.36; purity 97%; H-
NMR (400 MHz, DMSO)δ 7.80 – 7.69 (m, 9H), 7.62 – 7.55 (m, 9H), 5.02 (s, 4H), 4.54 – 4.40 (m,
4H), 4.23 – 4.06 (m, 4H), 2.43 – 2.19 (m, 4H), 2.06 (s, 6H), 2.01 – 1.78 (m, 12H); 13C-NMR (101
MHz, DMSO) δ 170.72, 167.42, 156.62, 137.33, 132.19, 131.99, 129.09, 128.78, 128.15, 126.77,
120.03, 67.84, 66.05, 55.42, , 30.27, 28.83, 23.71, 22.86, , 14.32, 11.23.
4.1.6.16.Dimethyl((2S,2'S)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-diylbis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(4-(methylthio)-1-oxobutane-1,2-diyl))dicarbamate (16)
Yellow semi-solid; yield: 35% ; C36H48N6O8S2; LC/MS: [M+H]+ = 757.30; purity 100%; 1H-NMR
(400 MHz, DMSO) δ 7.64 – 7.56 (m, 8H), 4.48 – 4.37 (m, 4H), 3.65 – 3.59 (m, 4H), 3.51 (s, 6H),
2.54 (t, J = 7.2 Hz, 4H), 2.05 (s, 6H), 1.98 – 1.72 (m, 12H); 13C-NMR (101 MHz, DMSO) δ 170.83,
170.61, 157.04, 138.63, 134.78, 126.81, 119.84, 60.67, 55.28, 51.87, 47.39, , 31.16, 29.80, 25.10,
22.59, 15.09.
28

4.1.6.17. Diethyl((2S,2'S)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-(methylthio)-1-oxobutane-1,2-
diyl))dicarbamate (17)
Yellow semi-solid; yield: 33%; C38H52N6O8S2; LC/MS: [M+H]+ = 785.33; purity 100%; 1H-NMR
(400 MHz, DMSO) δ 7.66 – 7.55 (m, 8H), 4.42 (m, 4H), 3.96 (q, J = 7.0 Hz, 4H), 3.77 – 3.60 (m,
13
4H), 2.54 (t, J = 7.1 Hz, 4H), 2.04 (s, 6H), 1.98 – 1.70 (m, 12H), 1.13 (t, J = 3.5 Hz, 6H); C-
NMR (101 MHz, DMSO) δ 174.25, 170.82, 156.62, 138.63, 134.80, 126.81, 119.82, 60.31, 55.32,
53.03, 51.69, 47.37, 46.16, 31.11, 29.85, 25.13, 15.12.
4.1.6.18. Dibutyl((2S,2'S)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-(methylthio)-1-oxobutane-1,2-
diyl))dicarbamate (18)
Yellow semi-solid; yield:25%; C42H60N6O8S2; LC/MS: [M+H]+ = 841.39; purity 91%; 1H-NMR
(400 MHz, DMSO) δ 7.65 – 7.56 (m, 8H), 4.47 – 4.37 (m, 4H), 3.93 (t, J = 4.0 Hz, 4H), 3.77 –
3.64 (m, 4H), 2.54 (t, J = 7.8 Hz, 4H), 2.05 (s, 6H), 1.97 – 1.78 (m, 12H), 1.54 – 1.47 (m, 8H),
0.87 (t, J = 7.2 Hz, 6H); 13C-NMR (101 MHz, DMSO) δ 174.20, 170.81, 156.73, 138.62, 134.81,
126.80, 119.83, 64.09, 60.64, 55.32, 53.13, 51.72, 47.36, 31.16, 29.86, 25.13, 19.01, 15.13, 14.04.
4.1.6.19. Diisobutyl((2S,2'S)-((2S,2'S)-(([1,1'-biphenyl]-4,4'-
diylbis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(4-(methylthio)-1-oxobutane-1,2-
diyl))dicarbamate (19)
Yellow semi-solid; yield: 26%; C42H60N6O8S2; LC/MS: [M+H]+ = 841.39; purity 92%; 1H-NMR
(400 MHz, DMSO) δ 7.65 – 7.56 (m, 8H), 4.52 – 4.39 (m, 4H), 4.05 (d, J = 4.7 Hz, 4H), 3.74 –
3.66 (m, 4H), 2.53 (t, J = 7.1 Hz, 4H), 2.01 (s, 6H), 1.97 – 1.85 (m, 2H), 1.85 – 1.77 (m, 12H),
0.85 (d, J = 2.8 Hz, 12H); 13C NMR (101 MHz, DMSO) δ 174.22, 170.67, 156.89, 138.63, 134.79,
126.80, 119.81, 70.30, 60.64, 52.95, 51.77, 47.17, 30.82, 30.25, 28.10, 25.13, 19.31, 15.27.
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