Analogs design, synthesis and biological evaluation of peptidomimetics with potential anti-HCV activity

Article abstract of DOI:10.1016/j.bmc.2013.03.017

Two series of peptidomimetics were designed, prepared and evaluated for their anti-HCV activity. One series possesses a C-terminal carboxylate functionality. In the other series, the electrophilic vinyl sulfonate moiety was introduced as a novel class of HCV NS3/4A protease inhibitors. In vitro based studies were then performed to evaluate the efficacies of the inhibitors using Human hepatoma cells, with the vinyl sulfonate ester (10) in particular, found to have highly potent anti-HCV activity with an EC₅₀ = 0.296 μM. Finally, molecular modeling studies were performed through docking of the synthesized compounds in the HCV NS3/4A protease active site to assess their binding modes with the enzyme and gain further insight into their structure-activity relationships.

Full text of DOI:10.1016/j.bmc.2013.03.017

Bioorganic & Medicinal Chemistry 21 (2013) 2742–2755
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Analogs design, synthesis and biological evaluation
of peptidomimetics with potential anti-HCV activity
Deena S. Lasheen a, Mohamed A.H. Ismail a, Dalal A. Abou El Ella a, Nasser S.M. Ismail a, Sameh Eid b,
Susan Vleck c, Jeffrey S. Glenn c, Andrew G. Watts d, Khaled A.M. Abouzid a,
⇑
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo 11566, Egypt
b Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
c Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, CA 94305-5187, USA
d Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of peptidomimetics were designed, prepared and evaluated for their anti-HCV activity. One
series possesses a C-terminal carboxylate functionality. In the other series, the electrophilic vinyl sulfo-
nate moiety was introduced as a novel class of HCV NS3/4A protease inhibitors. In vitro based studies
were then performed to evaluate the efficacies of the inhibitors using Human hepatoma cells, with the
vinyl sulfonate ester (10) in particular, found to have highly potent anti-HCV activity with an
Received 16 December 2012
Revised 23 February 2013
Accepted 3 March 2013
Available online 24 March 2013
EC50 = 0.296 lM. Finally, molecular modeling studies were performed through docking of the synthesized
compounds in the HCV NS3/4A protease active site to assess their binding modes with the enzyme and
gain further insight into their structure–activity relationships.
Keywords:
HCV NS3/4A protease inhibitors
Peptidomimetics
Ó 2013 Elsevier Ltd. All rights reserved.
Vinyl sulfonates
Molecular modeling
Covalent docking
1. Introduction
generation protease inhibitors, boceprevir or telaprevir, to this
SOC has dramatically changed the treatment of HCV-1 infected pa-
In 2007, the WHO declared Hepatitis C virus (HCV) infection as
a global health problem, with an estimate of more than 170 million
infected people worldwide.1 The disease has been described as a
‘silent epidemic’ and a ‘serious health crisis’. About 80% of the in-
fected cases develop chronic hepatitis, a condition that is incurable
in many patients, and without therapeutic intervention, it can lead
to morbidity or mortality within 10–20 years through either cir-
rhosis and hepatic failure or hepatocellular carcinoma.2 Despite
considerable reduction of the incidence of new infections, the
prevalence of HCV infection is predicted to remain constant in
the near future. Until recently, the standard of care (SOC) for
HCV infection has been based on a combination therapy of inject-
tients.6,7 Boceprevir or Telaprevir-based triple regimens have im-
proved SVR rates by at least 25–30% compared to the former
dual therapy. In addition, the duration of therapy can be reduced
in more than half of the patients to 24–28 weeks compared to
the conventional 48 weeks therapy.6–8
HCV is a Flavivirus, with a single stranded, positive sense RNA
genome of approximately 9600 nucleotides.9 This genome codes
for a single polyprotein that is co- and post-translationally cleaved
into 10 small proteins including structural (capsid, envelope glyco-
proteins) or (Core protein, E1 and E2), p7 and non-structural (NS2,
NS3, NS4A, NS4B, NS5A, and NS5B) proteins.10 Most of the viral
structural and non-structural proteins have been identified as po-
tential anti-HCV targets; however, the NS3 protease with its
NS4A cofactor remains one of the most promising targets due to
its crucial role in the viral polyprotein processing and hence viral
replication.11 Moreover, clinical data derived from several protease
inhibitors tested on HCV infected patients has clearly shown that
protease inhibition is associated with a rapid decline in viral
load.3,12 X-ray crystal structure of the full length NS3 protein has
revealed a C-terminal helicase domain, and an N-terminal protease
with a typical chymotrypsin-like fold, in addition to a structural a
zinc binding site. It has also revealed the non-covalent association
of the NS4A cofactor which is essential for the activation of the
able pegylated-interferon-a (peg-IFN-a) with oral ribavirin, which
resulted in sustained virological response (SVR) rates of only 50% in
patients with HCV-genotype 1 infection compared to almost 70–
80% for genotypes 2 and 3.1–4 Furthermore, this former SOC had
suffered from several drawbacks; being lengthy, expensive and
accompanied by significant adverse effects.1,2,4 Other factors such
as race, age, body weight, viral level and genotype also influenced
the success of treatment.5 However, The introduction of first
⇑
Corresponding author. Tel.: +20 1222165624; fax: +20 225080728.
E-mail address: Khaled.abouzid@pharm.asu.edu.eg (K.A.M. Abouzid).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.017

D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
2743
chosen to mimic the P1 amino acid at the natural NS3–NS4A sub-
strate cleavage site so was proposed to be better recognized by the
enzyme. The indoline ring was also replaced with a variety of ary-
lidene moieties to study their interaction pattern with the protease
binding site. A five-atom linker containing two hydrogen bond
acceptors and one hydrogen bond donor was retained to maintain
the hydrogen bonding interactions with the backbones of Ala157
and Arg155. An arylidene functionality was also introduced that
could also serve as an additional Michael acceptor group that
may be capable of interacting with the protease in a similar
pattern.
S
COOH
CHF2
CO2H
H
O
H
N
N
N
H
O
O
Figure 1. HCV protease inhibitor (I) IC50 = 0.7 lM.
protease. The protease active site has been shown to be situated at
the interface between the helicase and the protease domains, cre-
ating a well-defined binding cleft.13 Several peptidic and pepti-
domimetic NS3/4A protease inhibitors that possess either a C-
terminal acidic functionality or an electrophilic serine trap have
been reported.14–21 Ontoria et al.22 have proposed a structure–
activity relationship (SAR) and disclosed enzyme bound crystal
structure for indoline based peptidomimetic inhibitor (1) (Fig. 1),
however, further improvement of activity was still needed.
This design was further supported by molecular modeling stud-
ies, through docking of the designed molecules in the HCV NS3/4A
protease binding site to assess their binding modes with the prote-
ase active site.
3. Results and discussion
3.1. Chemistry
For the synthesis of the target esters (4a–f) and their corre-
sponding acids (5a–d), the following straight forward pathway
was pursued (Scheme 1). This involved the condensation of the
corresponding aldehydes (2a–c) with levulinic acid using catalytic
amounts of piperidine and acetic acid in refluxing benzene with
the azeotropic removal of water using a Dean–Stark trap to afford
the aryl oxohexenoic acids (3a–c) following reported methods.29
1H NMR confirmed the formation of E-isomers with J = 16 Hz.
Mixed anhydride approach using ethyl chloroformate and Et3N
was adopted for the condensation of 3a–c with the appropriate
2. Rationale and design
In this investigation, a structure-guided strategy was used to
design a series of peptidomimetic HCV NS3/4A protease inhibitors
that either possess a C-terminal carboxylate functionality to mimic
the product-based inhibitors, or an electrophilic vinyl sulfonate
moiety which is proposed to act as a Michael acceptor to be at-
tacked by the catalytic serine to form a covalent bond with the pro-
tease enzyme. The vinyl sulfonate group was chosen being a novel
class of HCV protease inhibitors that have never been reported be-
fore as serine protease inhibitors but have proven successful as Mi-
chael acceptors capable of covalently inhibiting several cysteine
and threonine proteases.23–27 Vinyl sulfonates have been reported
to be capable of inactivating several cysteine proteases through
irreversible addition of the thiol group of active site cysteine to
the electrophilic vinyl sulfonate moiety. This addition reaction is
catalyzed by histidine and aspartate residues present within the
active site of the protease.28
The design of these target molecules was based on modification
of the indoline-based peptidomimetic inhibitor (1),22 by perform-
ing specific bioisosteric replacements in the indoline lead. The car-
boxylic head, or the vinyl sulfonate functionality, was proposed to
occupy the oxyanion hole of the protease binding site. Several P1
amino acids were introduced replacing the difluoroethyl group of
(1). Amino acids with non-bulky side chains were selected, to be
accommodated within the rather small hydrophobic S1 pocket,13
such as alanine, valine, phenylalanine and threonine; which was
amino esters (viz.; L-alanine methyl ester HCl and L-valine methyl
ester HCl) to afford the ester derivatives (4a–f).30 The obtained es-
ters were hydrolyzed to the carboxylic acids (5a–d) under mild
conditions using lithium hydroxide at room temperature.31
The introduction of vinyl sulfonate moiety was achieved via the
sequence outlined in Scheme 2; Swern oxidation was employed for
the conversion of Boc-L-phenylalaninol to its corresponding
aldehyde (6) as previously reported.32 The Horner–Wadsworth–
Emmons (HWE) olefination reaction with ethyl (diethoxyphospho-
ryl)-methanesulfonate33 and n-ButylLithium (n-BuLi) at ꢀ78 °C
gave the corresponding vinyl sulfonate (7) with complete (E) stere-
oselectivity.34 Cleavage of the ethyl ester was effected by treat-
ment of the sulfonate (7) with n-tetrabutylammonium iodide (n-
Bu4NI) in refluxing acetone following the reported procedure.34
Boc deprotection of 7 was performed using 30% Trifluoroacetic acid
(TFA) in CH2Cl235 to afford the rather unstable amine (9) which was
used for the next step immediately after purification. Coupling of
the amine (9) with the 6-phenyl-4-oxohex-5-enoic acid (3a) via a
O
O
R1
R2
CHO
R1
R2
OH
b
a
OH
+
O
X
O
X
3a-c
2a-c
3a: R1=R2=H, X=CH;
2a: R1=R2=H, X=CH;
2b: R1=R2=H, X=N;
3b: R1=R2=H, X=N;
3c: R1,R2=-OCH2O-, X= CH
2c: R1,R2=-OCH2O-, X= CH
O
O
H
H
N
R1
R2
N
COOH
c
R1
COOMe
O
R3
O
R3
R2
X
4a-f
5a-d
Scheme 1. Synthesis of 4-oxo-6-arylhex-5-enamidopropanoates and butanoates and their corresponding acids: Reagents and conditions: (a) Piperidine, AcOH, benzene,
reflux, 14–16 h, 73–88%; (b) NH2CH(R3)COOMe, ethyl chloroformate, Et3N, CH2Cl2, rt, 2 days, 46–50%; (c) LiOHꢁH2O, THF/ethanol, rt, 2 h, 65–72%.

2744
D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
SO3- +NBu4
NHBoc
8
c
SO3Et
OH
NHBoc
a
b
O
d
NHBoc
NHBoc
6
7
O
H
N
SO3Et
SO3Et
e
NH2
O
10
9
Scheme 2. Synthesis of
L
-phenylalanine-derived vinyl sulfonates and their corresponding salts: Reagents and conditions: (a) (COCl)2, DMSO, ꢀ63 °C, 96%; (b) ethyl
(diethoxyphosphoryl)methane-sulfonate, n-BuLi, ꢀ78 °C, 1 h, 72%; (c) Bu4NI, acetone, reflux, 2 days, 94%; (d) TFA/CH2Cl2, rt, 1 h, 98%; (e) 3a, ethyl chloroformate, Et3N, CH2Cl2,
rt, 2 days, 44%.
OH
OH
c
a
b
O
NH2
NHAc
NHAc
11
12
SO3- +NBu4
SO3Et
d
NHAc
NHAc
14
13
Scheme 3a. Synthesis of acetylated
58%; (c) ethyl (diethoxyphosphoryl)methanesulfonate, n-BuLi, THF, ꢀ78 °C, 70%; (d) Bu4NI, acetone, reflux, 2 days, 78%.
L
-phenylalanine-derived vinyl sulfonates. Reagents and conditions: (a) Ac2O (1.1 equiv), pyridine, ꢀ20 °C, 4 h, 78%; (b) PDC, CH2Cl2, rt, 1 h,
OH
O
OTBDMS
COOMe
OTBDMS
OH
OH
O
c
a
b
OMe
OMe
NH2 . HCl
NHAc
NHAc
NHAc
16
15
17
OTBDMS
OH
OTBDMS
d
e
f
SO3Et
SO3Et
O
NHAc
NHAc
20
NHAc
18
19
Scheme 3b. Synthesis of
L
-threonine-derived vinyl sulfonates: Reagents and conditions: (a) Et3N, Ac2O, pyridine, ꢀ20 °C, 4 h, 88%; (b) TBDMS-Cl, imidazole, DMF, rt, 88%; (c)
LiBH4, THF/MeOH, rt, 2 days, 84%; (d) (COCl)2, DMSO, CH2Cl2, ꢀ63 °C, 100%; (e) ethyl (diethoxyphosphoryl)methanesulfonate, n-BuLi, THF, ꢀ78 °C, 59%; (f) 70% HF/pyridine,
3 h, rt, 86%.
mixed anhydride approach afforded the target
based vinyl sulfonate (10).
L
-phenylalanine-
a temperature not exceeding ꢀ20 °C, the more nucleophilic amino
group was selectively acetylated, with only traces of the diacetyl
side product which was easily purified by silica chromatography.
Oxidation using pyridinium dichromate (PDC) 37,38 followed by
HWE coupling afforded the vinyl sulfonate (13) which was subse-
The acetylated L-phenylalanine- and L-threonine-based vinyl
sulfonates were also prepared as probes to test the general ability
of vinyl sulfonates to inhibit the HCV serine protease. Threonine
was chosen to mimic the P1 amino acid in the natural NS3–NS4A
substrate cleavage site so was proposed to be well recognized by
the enzyme. The synthesis of these probes was pursued via the
chemical pathways outlined in Schemes 3a and 3b; Thus for pre-
quently converted to its tetrabutylammonium salt (14).34 The
L-
threonine based vinyl sulfonate (20) was prepared via initial N-
acetylation of
L
-threonine methyl ester HCl at ꢀ20 °C as previously
reported.39 The threonine hydroxyl group was then protected as its
tert-butyldimethylsilyl (TBDMS) ether following the reported pro-
cedure.40 LiBH4 in THF/MeOH (1:1)41 was employed for the reduc-
paring the acetylated L-phenylalanine-based vinyl sulfonate (13),
L
-phenylalaninol was selectively N-acetylated using acetic anhy-
dride in pyridine at ꢀ20 °C, modifying a reported procedure.36 It
is worth noting that performing the acetylation at 0 °C afforded
the diacetyl derivative, but when the reaction was carried out at
tion of the O-TBDMS-N-acetyl-L-threonine methyl ester (16) to
provide the desired threoninol product (17) in 84% yield, which
was then oxidized under Swern conditions to the corresponding

D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
2745
Table 1
EC50 and CC50 for the carboxylate series of compounds assayed for their anti-HCV activity and cell viability, together with their safety indices (CC50/EC50
)
O
H
R1
R2
N
COOR4
O
R3
X
Compd no
R1R2
X
R3
R4
EC50
0.8
(
lM)
CC50
(lM)
Safety index
Boceprevir
—
—
—
—
>100
—
4a
4b
4c
4d
4e
4f
H
H
H
H
CH
CH
N
CH3
CH(CH3)2
CH3
CH(CH3)2
CH3
CH(CH3)2
CH(CH3)2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
0.960
0.381
0.900
1.141
0.347
0.682
22.476
5.435
1.318
1.477
2.108
1.571
1.107
1.393
1.373
3.877
2.342
1.377
3.19
2.83
>4.45
2.224
N
–OCH2O–
–OCH2O–
H
CH
CH
CH
CH
5b
5c
>100
12.088
–OCH2O–
H
EC50: half the maximal effective concentration, CC50: 50% cytotoxicity concentration.
Table 2
EC50 and CC50 for vinyl sulfonate series of compounds assayed for their anti-HCV activity (genotype 2a) and cell viability, together with their safety indices (CC50/EC50
)
Compd no.
Structure
EC50
(lM)
CC50
(l
M)
Safety index
SO3Et
7
1.213
8.621
3.500
2.885
NHBoc
NHBoc
SO3- +NBu4
8
67.029
7.775
NA
10
0.296
ꢂ1
13
14
2.110
8.690
7.907
>100
3.747
>11.5
20
4.360
27.706
6.355
aldehyde (18) in quantitative yield. HWE coupling of 18 with ethyl
(EC50 = 0.296 lM), and the carboxylate esters 4b, 4e and 4f
(diethoxyphosphoryl)-methanesulfonate followed by silyl depro-
(EC50s = 0.381, 0.347 and 0.682, respectively) exhibited more po-
tent HCV RNA inhibitory activity on genotype 2a than the positive
tection using 70% HF in pyridine42,43 afforded the target
L-threo-
nine-based vinyl sulfonate (20).
control boceprevir (phase III clinical trials) (EC50 = 0.8 lM). How-
ever, alamarBlue viability assays performed on individual com-
pounds over the same concentration ranges demonstrated
reduced cell viability associated with most of the test compounds,
with the exception of compounds 5b and 14, which demonstrated
no observable cellular toxicity within the studied range.
Thus, it has been observed that the carboxylate esters were
much more potent than their corresponding acids (4b, 4e vs 5b,
5c) however, acids were much less toxic. The vinyl sulfonate ester
(10) exhibited the most potent anti-HCV activity, which was ex-
pected since it is more extended towards the S3 subsite of the en-
zyme. However, it demonstrated marked cellular toxicity. The
phenylalanine derived vinyl sulfonate esters (7 and 13) were gen-
3.2. Biological evaluation
The target compounds (4a–f, 5b, c, 7, 8, 10, 131, 4, 20) were then
evaluated for their ability to inhibit HCV replication in an in vitro
cell culture system using Huh7.5 (Human hepatoma) cells which
are highly permissive for the initiation of HCV replication.44 The
NS3 protease inhibitor, boceprevir (SCH503034)18,45 was used as
a positive control. J6/JFH HCV RNA (genotype 2a) harboring a Renil-
la luciferase reporter gene was transfected into Huh7.5 cells by
Liposome-mediated transfection using Lipofectamine 2000 (Invit-
rogen). Cells were grown in the presence of various nanomolar
and micromolar concentrations of each of the test compounds for
48 h, after which the cells were subjected to luciferase assays
and alamarBlue-based viability assays adopting reported
procedures.46,47
erally more potent (EC50s = 1.213 and 2.110
their corresponding tetrabutylammonium salts (8 and
(EC50s = 8.621 and 8.690 M, respectively). However, salts seem
l
M, respectively) than
14)
l
to be less toxic. The threonine derived vinyl sulfonate ester (20)
Luciferase assay performed revealed that all test compounds,
except for 5b, demonstrated potent inhibitory activity on HCV
RNA replication, with EC50s in submicromolar or low micromolar
range (Tables 1 and 2). Most notably, the vinyl sulfonate 10
exhibited significant inhibitory activity on HCV replication
(EC50 = 4.36 lM) as well as an acceptable safety profile
(CC50 = 27.706), hence, may be considered a good candidate for fur-
ther optimization, in order to increase the anti-HCV potency.

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D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
3.3. Molecular modeling
crystal structure was that it has an Ile132 in place of Val132. In
HCV-1a and 1b genotypes, position 132 is a valine residue, while
in genotypes 2a and 2b it is replaced by Leu.49 Given that biological
assays were performed on HCV-2a, it was preferable to use a crys-
tal structure where position 132 is occupied by an amino acid clo-
ser in size to Leu; namely Ile, as none of the available crystal
structures contained a Leu132. However, a key residue in the S3
pocket of HCV protease, Cys159, is replaced by Ser159 in this crys-
tal structure. Ontoria et al. designed indoline-based peptide-
mimetics bearing a thiophene ring to establish favorable contacts
with the Cys159 residue in the lipophilic S3 pocket.22 On the other
hand, Hagel et al. successfully designed irreversible covalently-
bonded inhibitors targeting this non-catalytic cysteine, by means
of attaching an acrylamide terminal group (a low-reactivity Mi-
chael acceptor) to a carefully designed scaffold.50 For the purpose
of this study, though, we decided to keep Ser159 unchanged since
it is unlikely to have any detrimental effects on our docking
studies.
The proposed docking algorithm was validated by self-docking
of danoprevir, where it was removed from the complex (PDB:
3M5L) and then docked back into the binding site. Heavy-atom
root mean square deviation (RMSD) values between top-ranked
poses and the experimental crystal structure ranged from 0.4 to
1.4 Å (Fig. 2). Thus, Glide seemed to be an appropriate method
for reliable prediction of docking poses for our compounds.
The prepared final compounds (4a–f, 5b, c, 7, 8, 10, 13, 14, 20)
were subjected to detailed docking studies performed to predict
their binding modes to the HCV NS3/4A protease active site. These
studies have successfully identified reasonable binding poses for
all test molecules, with comparable docking scores, indicating that
the proposed compounds could potentially bind to the HCV prote-
ase active site with comparable strengths. Two docking runs were
done, an initial docking study, to assess the non-covalent binding
interactions of all the studied molecules with the oxyanion hole
and the other active site residues in the protease. This was fol-
lowed by another docking study to test the ability of the vinyl sul-
fonate ester (10) to covalently bind to the catalytic Ser139.
3.3.1. Validation of docking methodology
Crystal structure of HCV protease in complex with the non-
covalent macrocyclic inhibitor danoprevir (ITMN191) (PDB:
3M5L) was found to exhibit the highest resolution (1.25 Å) among
other solved structures.48 The co-crystallized ligand also showed
reasonable resemblance to the inhibitors developed here, being
peptidomimetic in nature, bearing an acidic head group, and a ter-
minal aryl moiety; namely the fluoroisoindoline substituent. This
crystal structure was, thus, selected for non-covalent docking
calculations. Another crucial reason for selecting this particular
F
N
O
O
O
O
S
O
N
NH
O
O
HN
O
O
N
H
Figure 2. (Top) Structure of danoprevir (ITMN191), IC50 <0.25 nM, EC50 = 2.1 nM. (Bottom) Results of self-docking of danoprevir (PDB code 3M5L); white carbons: crystal
structure pose, green carbons: top-ranked docking pose. Atom color codes: N: blue, O: red, S: yellow, F: turquoise, hydrogen atoms are not shown.

D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
2747
It has been shown that binding site residues of HCV NS3/4A pro-
tease exhibit induced-fit conformational changes to adapt to vari-
ous classes of ligands.51,48,52–54 Superposition of the active sites of
boceprevir crystal structure (PDB: 2OC8) and the protease model
employed in non-covalent docking (PDB: 3M5L) revealed a signif-
icant difference in side chain alignments (RMSD >3.7 Å). Docking of
boceprevir to the rigid structure of 3M5L, which was employed in
docking, would not be possible without allowing for readjustments
of protein side chains. Therefore, docking of boceprevir to 3M5L
has been a good challenge to test the induced-fit docking proce-
dure. Induced-fit docking of boceprevir to 3M5L structure success-
fully retrieved poses having RMSD values of ꢂ1.5 Å from the
original crystal structure geometry (Fig. 3). This result confirmed
the validity of the proposed induced-fit methodology in cases
where some degree of receptor adaptation was to be expected, that
is, with larger and/or more flexible ligands.
hypothesis. For some compounds however, different binding
modes were also observed where either the amide oxygen or en-
one oxygen of the inhibitor filled the oxyanion hole. These differ-
ences could be exemplified by comparing docking results for 4e
and 4f (Fig. 4). The alanine-based (4e) occupied the oxyanion hole
via its amide oxygen while in its valine-based analogue (4f) it was
filled by the enone oxygen. Additionally, both compounds were
predicted to extend deeper into the S3 pocket via their benzodiox-
ole tail interacting favorably with Ser159, which could in part ex-
plain their superior anti-HCV activities demonstrated in vitro.
Docking results of long flexible molecules, for example, 4c, 4e,
and 10, showed favorable interactions via their aromatic tail
groups with Ser159 in S3 pocket. Hagel et al. recently exploited a
similar scaffold to place a Michael acceptor in the vicinity of the
analogous non-catalytic Cys159 in S3 pocket of HCV NS3/4A prote-
ase.50 In our case, though, results from molecular docking did not
retrieve any poses where the Michael acceptor group was close en-
ough to the corresponding Ser159. This was not surprising, how-
ever, since the reactive groups in our molecules were fairly close
to the oxyanion hole binding motif. Placing the reactive groups
close to S3 pocket would, thus, result in breaking off all key inter-
actions in the oxyanion hole, and consequently the molecules
would not be able to bind to the active site.
3.3.2. Glide docking
Top-ranked poses for all studied compounds were found to oc-
cupy the oxyanion hole and exhibited favorable hydrogen bonding
and/or electrostatic interactions with backbone NH’s of Gly137 and
Ala139. In most cases, the acidic head groups (carboxylate or sulfo-
nate) occupied the oxyanion hole, which confirmed our design
O
H
N
NH2
N
H
N
H
N
O
O
O
O
Figure 3. (Top) Structure of boceprevir. (Bottom) Results of induced-fit docking of boceprevir; white carbons: crystal structure pose from PDB 2OC8, green carbons: top-
ranked docking pose to the receptor grid generated from PDB 3M5L. Atom color codes analogous to Figure 2.

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D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
O
O
H
N
H
COOMe
N
COOMe
O
O
O
O
O
O
Figure 4. Top-ranked docking poses for compounds 4e (left) and 4f (right). The bulky benzodioxole interacts favourably with Ile132, Ala157 and extends even deeper in the
S3 pocket reaching Ser159. Both compounds can also form H-bonds with the side chain of Lys136 via their carboxylate moieties.
Table 3
data). The relative strengths of S2/S3 interaction energies were,
Docking scores and interaction fingerprints for the docking poses of modelled
compounds; residues defining interaction subsites are given below the table
thus, reversed in favor of S2 in valine-based compounds in contrast
to their alanine-based counterparts (Fig. S2 in Supplementary
data).
Compd no.
Glide score
(kcal/mol)
Oxyanion
hole
S1
S2
S3
4a
4b
4c
4d
4e
4f
5b
5c
7
ꢀ3.00
ꢀ4.95
ꢀ3.42
ꢀ4.91
ꢀ4.65
ꢀ5.03
ꢀ4.99
ꢀ4.72
ꢀ4.61
ꢀ4.72
ꢀ5.91a
ꢀ3.70
ꢀ4.38
ꢀ5.11
ꢀ7.15
ꢀ2.88
ꢀ7.72
ꢀ2.64
ꢀ7.83
ꢀ6.21
ꢀ10.25
ꢀ15.08
ꢀ8.48
ꢀ8.71
ꢀ4.40a
ꢀ4.73
ꢀ9.75
ꢀ10.23
ꢀ5.62
ꢀ8.62
ꢀ6.46
ꢀ8.53
ꢀ5.95
ꢀ4.59
ꢀ3.97
ꢀ0.58
ꢀ6.20
ꢀ2.41
ꢀ6.37a
ꢀ3.72
0.17
ꢀ6.20
ꢀ14.56
ꢀ9.43
3.3.3. Induced-fit docking
ꢀ13.48
ꢀ4.79
Due to its larger size, compound (10) was also subjected to the
more time consuming induced-fit docking procedure. This com-
pound displayed the most extensive network of interactions in
the HCV protease binding site (Fig. 5). In agreement with the design
rationale, the oxyanion hole was occupied by the sulfonate head
group, which made two strong hydrogen bonds to backbone NHs
of Gly137 and Ala139. The phenylalanine moiety was situated be-
tween S1 and S3 pockets interacting with side chains of Ile132,
Phe154 and Ala157. The central part of the ligand wrapped around
His57 in S2 while the central amide NH was H-bonded to backbone
carbonyl of Agr155. The arylidene tail packed against the side chain
of Arg155 in the S2 subsite.
ꢀ20.77
ꢀ9.22
ꢀ13.05
ꢀ7.67
ꢀ16.04
ꢀ4.44
ꢀ13.74
ꢀ13.31
ꢀ22.76
ꢀ11.94
ꢀ12.55
ꢀ16.99a
ꢀ8.25
ꢀ66.04
ꢀ34.35
ꢀ10.78
ꢀ58.93
ꢀ8.65a
ꢀ11.21
ꢀ56.96
ꢀ9.29
8
10
13
14
20
ꢀ11.43
ꢀ3.72
ꢀ0.40
P
Oxyanion hole:
(Gly137, Ala139).
(Ala157, Ile132, Phe154).
(Ala157, Arg155, His57).
(Ile132, Ala157, Ser159, Lys136, Arg161).
P
S1:
S2:
S3:
P
P
3.3.4. Prime covalent docking
a
Score of the pose obtained from induced-fit docking.
As shown in Figure 5, the reactive vinyl group of (10) is situated
appropriately in the catalytic site. The distance between the b-car-
bon of Ala139 and the electropositive vinyl carbon was as low as
4.5 Å, which could facilitate Michael-type nucleophilic attack on
the labile carbon. Prime covalent docking of (10) study revealed
that upon covalent binding, the ligand could be well accommo-
dated within HCV protease active site. Most of the interactions ob-
served in the non-covalent docking were conserved upon covalent
addition of Ser139, although some active site residues had to un-
dergo significant re-arrangement, especially His57, Lys136 and
Arg155. The top-ranked poses showed that after covalent binding,
compound (10) could either fill S1 and S3 pockets or S2 and S3
pockets (Fig. 6).
Table 3 lists the interaction fingerprints for the studied com-
pounds (see Section 5.2.1 for details on calculation, and Figs. S2
and S3 inSupplementary data for plots). Analysis of the interaction
fingerprints for the carboxylate series revealed different subsite
preferences for the different structural classes. Although interac-
tions of the carboxylate derivatives with the oxyanion hole were
of comparable strengths; they differed with respect to interaction
with S2 and S3 subsites. The alanine-based derivatives (4a, 4c,
and 4e) tended to occupy the oxyanion hole via their amide oxy-
gen, which allowed the carboxymethyl group to make a hydrogen
bond with Lys136 in S3 pocket, or even a stronger salt bridge in
case of compound 5c. On the other hand, the oxyanion hole was
occupied via carboxylate or enone oxygen in case of valine-based
carboxylate derivatives (4b, 4d and 4f). Molecular orientation
was, thus, different from the alanine-based series since the
molecules’ tails generally packed against Arg155 side chain and
other carbonyl groups made H-bonds to Ala157 in S2 pocket (Com-
parison between 4a and 4b is given in Fig. S1 in Supplementary
4. Conclusion
In conclusion, vinyl sulfonates were introduced as a novel class
of peptidomimetic HCV NS3/4A protease inhibitors that has never
been reported before as serine protease inhibitors. The vinyl sulfo-
nate ester (10) exhibited highly potent anti-HCV activity in cell
culture based assay (EC50 = 0.296
by docking studies, which revealed its capability of forming an
lM). This was further justified

D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
2749
O
H
N
SO3Et
O
Figure 5. Induced-fit docking pose for compound (10). The distance between the beta carbon of Ala139 and the electropositive carbon susceptible to Michael-type attack is
marked. Re-adjustments in active site residues allowed better interactions with HCV protease S1 (Ala157 and Phe154), S2 (Arg155 and His57), and S3 (Ile132 and Lys136)
subsites.
extensive network of interactions with the oxyanion hole, and
other non-prime pockets in the HCV protease binding site. Also,
the predicted poses placed the electropositive vinyl carbon in close
vicinity to the catalytic OH of Ser139, which could facilitate a Mi-
chael-type addition reaction. In addition, another series of pepti-
domimetic carboxylate-based HCV NS3/4A protease inhibitors
have also been introduced. All the prepared carboxylate esters
demonstrated potent anti-HCV inhibitory activity, with com-
pounds (4b and 4e) being the most potent (EC50s = 0.381 and
on Stuart Scientific apparatus and were uncorrected. 1H NMR spec-
tra were recorded in d scale given in ppm on a Joel 270 MHz spec-
trophotometer, or a Varian 400 MHz spectrophotometer, or a
Varian 300 MHz and referred to TMS. 13C NMR spectra were re-
corded on Varian spectrophotometer operating at 100 MHz. High
resolution mass spectroscopy (HRMS) was performed on a Bruker
MicroOTOF™ instrument coupled with an electrospray source
(ESI-TOF). FT-IR spectra were recorded on a Perkin–Elmer spectro-
photometer. Elemental analyses were performed at the Microana-
lytical Center, Cairo University.
0.347 lM, respectively). The above apparent antiviral potencies,
however, must be interpreted in light of corresponding relatively
low CC50s, which could account for a significant component of
the EC50s. Several compounds, however, exhibited considerably
more favorable therapeutic indices (e.g., compounds 8 and 14).
5.1.2. (E)-4-Oxo-6-(pyridin-3-yl)hex-5-enoic acid (3b)
To a solution of pyridine-3-carbaldehyde (nicotinaldehyde)
(20 mmol), and levulinic acid (2.32 g, 20 mmol) in dry benzene
(60 mL), piperidine (0.25 mL, 2.5 mmol) and glacial acetic acid
(0.8 mL, 14 mmol) were added. The solution was refluxed using a
Dean–Stark apparatus until the disappearance of starting material
as judged by TLC (14 h). The reaction was allowed to cool to rt, then
the solvent was evaporated in vacuo. The residue was dissolved in
EtOAc (50 mL), washed with 20% AcOH (2 ꢃ 20 mL), brine
(2 ꢃ 20 mL), dried over anhydrous MgSO4, and filtered. The filtrate
was evaporated in vacuo then recrystallized from hot ethanol to af-
ford the titled product (3b), as yellow crystals, (3 g, 73%); mp 176–
178 °C; 1H NMR (300 MHz, CDCl3): d 8.76 (s, 1H, pyridyl), d 8.63–
8.62 (d, 1H, pyridyl), d 7.88–7.85 (d, 1H, pyridyl), d 7.62–7.58 (d,
J = 16 Hz, 1H, py-CH@CH), d 7.35–7.31 (m, 2H, pyridyl), d 6.85–
6.81 (d, J = 16 Hz, 1H, py-CH@CH), d 3.06–2.99 (m, 2H, CO–CH2–
5. Experimental
5.1. Chemistry
5.1.1. Materials and instrumentation
Starting materials and reagents were purchased from Sigma–Al-
drich or Acros Organics and used without further purification.
Anhydrous methylene chloride was dried by distillation over cal-
cium hydride. Anhydrous THF was dried by distillation over so-
dium/benzophenone. Anhydrous benzene was dried by
distillation over Na metal. Other solvents are purchased from Fish-
er scientific or Sigma–Aldrich and used without further purifica-
tion. Analytical thin layer chromatography (TLC) was performed
on silica gel 60 F254 packed on Aluminium sheets, purchased from
Merck. Column chromatography was performed on silica 60 (35–
70 microns) purchased from Fisher. Melting points were recorded
CH2–COOH),
(
d 2.60–2.54 (m, 2H, CO–CH2–CH2–COOH); FT-IR
m0max, cmꢀ1): 3400–2900 (OH carboxylic), 1715, 1694 (2 C@O),
MS (Molecular formula: C11H11NO3, Mwt.: 205.07): m/z 205.05
(M+, 31.11%), 103.65 (78.08%).

2750
D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
Figure 6. Covalent docking poses for compound (10): first top-ranked pose (left), second top-ranked pose (right). Covalent bond with catalytic Ser139 is marked with red
arrow.
5.1.3. (S,E)-Methyl 2-(4-oxo-6-arylhex-5-enamido)propanoates
ꢀ1): 3318 (NH), 3069 (CH@CH), 2932, 2912 (CH aliphatic), 1735
and (S,E)-Methyl 3-methyl-2-(4-oxo-6-arylhex-5-enamido)but-
(C@O ester), 1692 (C@O ketone), 1665 (C@O amide); MS (Mwt.:
anoates (4a–f)
5.1.3.1. General procedure.
acid (3a–c) (3 mmol) and triethylamine (0.708 g, 7 mmol) in meth-
ylene chloride (10 mL) stirred at ꢀ10 °C in an ice-salt bath, ethyl
chloroformate (0.29 mL, 3 mmol) was added dropwise, and stirring
was continued for 10 min, after which a solution of the respective
317.38): m/z 317.60 (M+, 37.19%), 187.50 (100%). Anal. Calcd for
To an ice-cooled solution of the
C18H23NO4: C, 68.12; H, 7.30; N, 4.41. Found: C, 68.36; H, 7.44;
N, 4.51.
5.1.3.4
(S,E)-Methyl 2-(4-oxo-6-(pyridin-3-yl)hex-5-enami-
do)propanoate (4c).
amino acid ester hydrochloride salt (viz.;
L-alanine methyl ester
This compound was purified by column chromatography (gra-
dient elution starting from EtOAc then 1% MeOH/EtOAc and
increasing polarity to 5% MeOH/EtOAc) to afford 4c as pale yellow
crystals (0.42 g, 48%); mp 116 °C; 1H NMR (300 MHz, CDCl3): d 8.77
(s, 1H, pyridyl), d 8.64–8.62 (d, 1H, pyridyl), d 7.89–7.85 (d, 1H, pyr-
idyl), d 7.62–7.57 (d, J = 16 Hz, 1H, py-CH@CH), d 7.34–7.32 (m, 1H,
pyridyl), d 6.86–6.80 (d, J = 16 Hz, 1H, py-CH@CH), d 6.24 (br s, 1H,
NH), d 4.62–4.57 (m, 1H, NH–CH–CO2Me), d 3.76 (s, 3H, CO2–CH3), d
3.15–3.00 (t, 2H, CO–CH2–CH2–CO), d 2.65–2.58 (t, 2H, CO–CH2–
CH2–CO), d 1.44–1.36 (d, 3H, NHCH–CH3); FT-IR (m0max, cmꢀ1):
3321 (NH), 3056 (CH@CH), 1733 (C@O ester), 1694 (C@O ketone),
1670 (C@O amide); MS (Mwt.: 290.31): m/z 290.35 (M+, 1.05%),
188.00 (42.05%), 131.95 (100%). Anal. Calcd for C15H18N2O4: C,
62.06; H, 6.25; N, 9.65. Found: C, 62.09; H, 5.95; N, 9.72.
HCl or -valine methyl ester HCl) in CH2Cl2 (3 mL) was added drop-
L
wise. The cooling bath was then removed and the reaction was
stirred at rt for 2 days until the disappearance of the starting mate-
rials as judged by TLC. The solvent was evaporated in vacuo; the
residue was dissolved in EtOAc (20 mL). The organic layer was
washed with 10% HCl (2 ꢃ 15 mL) then saturated Na2CO3 solution
(2 ꢃ 15 mL), dried over anhydrous MgSO4 and filtered. The filtrate
was evaporated in vacuo to afford the crude products (4a–f) which
were purified by column chromatography.
5.1.3.2 (S,E)-Methyl 2-(4-oxo-6-phenylhex-5-enamido)propano-
ate (4a).
The titled compound was purified by column chromatography
(gradient elution starting from 1% EtOAc/CH2Cl2 and increasing
polarity to 10% EtOAc/CH2Cl2) to afford 4a as buff crystals (0.4 g,
46%); mp 74–75 °C; 1H NMR (300 MHz, CDCl3): d 7.64–7.60 (d,
J = 16 Hz, 1H, Ph-CH@CH), d 7.59–7.54 (m, 3H, Ar), d 7.42–7.39
(m, 2H, Ar), d 6.79–6.74 (d, J = 16 Hz, 1H, Ph-CH@CH), d 6.25 (br
s, 1H, NH), d 4.62–4.55 (q, 1H, NH–CH–CO2Me), d 3.76 (s, 3H,
CO2–CH3), d 3.11–3.00 (t, 2H, CO–CH2–CH2–CO), d 2.64–2.55 (t,
2H, CO–CH2–CH2–CO), d 1.44–1.39 (d, 3H, NHCH–CH3); FT-IR ce:i-
5.1.3.5
(S,E)-Methyl 3-methyl-2-(4-oxo-6-(pyridin-3-yl)hex-5-
enamido)butanoate (4d).
The titled compound was purified by column chromatography
(gradient elution starting from EtOAc then 1% MeOH/EtOAc and
increasing polarity to 5% MeOH/EtOAc) to afford 4d as pale yellow
crystals (0.45 g, 47%); mp 76–77 °C; 1H NMR (300 MHz, CDCl3): d
8.76 (s, 1H, pyridyl), d 8.62–8.61 (d, 1H, pyridyl), d 7.88–7.84 (d,
1H, pyridyl), d 7.62–7.56 (d, J = 16 Hz, 1H, py-CH@CH), d 7.36–
7.33 (m, 1H, pyridyl), d 6.85–6.80 (d, J = 16 Hz, 1H, py-CH@CH), d
6.21 (br s, 1H, NH), d 4.58–4.53 (m, 1H, NH–CH–CO2Me), d 3.74
(s, 3H, CO2–CH3), d 3.12–3.03 (m, 2H, CO–CH2–CH2–CO), d 2.68–
2.62 (m, 2H, CO–CH2–CH2–CO), d 2.18–2.15 (m, 1H, NHCH–CH), d
0.97–0.92 (m, 6H, CH-(CH3)2); FT-IR (m0max, cmꢀ1): 3325 (NH),
3059 (CH@CH), 2966, 2910 (CH aliphatic), 1740 (C@O ester),
1684 (C@O ketone), 1665 (C@O amide); MS (Mwt.: 318.37): m/z
319.00 (M+, 34.19%), 259.15 (49.26%), 132.15 (100%). Anal. Calcd
for C17H22N2O4: C, 64.13; H, 6.97; N, 8.80. Found: C, 64.21; H,
6.72; N, 8.86.
talic>max , cmꢀ1): 3315 (NH), 3055 (CH@CH), 1739 (C@O ester),
max
1689 (C@O ketone), 1672 (C@O amide); MS (Mwt.: 289.33): m/z
289.75 (M+, 28.18%), 187.20 (100%). Anal. Calcd for C16H19NO4: C,
66.42; H, 6.62; N, 4.84. Found: C, 66.75; H, 6.42; N, 4.82.
5.1.3.3. (S ,E)-Methyl 3-methyl-2-(4-oxo-6-phenylhex-5-enami-
do)butanoate (4b).
The titled compound was purified by col-
umn chromatography (gradient elution starting from 1% EtOAc/
CH2Cl2 and increasing polarity to 10% EtOAc/CH2Cl2) to afford 4b
as buff crystals (0.48 g, 50%); mp 84–85 °C; 1H NMR (300 MHz,
CDCl3): d 7.64–7.58 (d, J = 16 Hz, 1H, Ph-CH@CH), d 7.57–7.54 (m,
3H, Ar), d 7.42–7.39 (m, 2H, Ar), d 6.79–6.74 (d, J = 16 Hz, 1H, Ph-
CH@CH), d 6.22–6.20 (br s, 1H, NH), d 4.58–4.54 (m, 1H, NH–CH–
CO2Me), d 3.75 (s, 3H, CO2–CH3), d 3.13–3.04 (m, 2H, CO–CH2–
CH2–CO), d 2.67–2.61 (m, 2H, CO–CH2–CH2–CO), d 2.18–2.16 (m,
1H, NHCH–CH), d 0.97–0.92 (m, 6H, CH-(CH3)2); FT-IR (m0max, cm
5.1.3.6
(S,E)-Methyl 2-(6-(benzo[d][1,3]dioxol-5-yl)-4-oxohex-
5-enamido)propanoate (4e).
This titled compound was purified by column chromatography
(gradient elution starting from 1% EtOAc/CH2Cl2 and increasing

D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
2751
polarity to 20% EtOAc/CH2Cl2) to afford 4e as orange buff crystals
(0.48 g, 48%); mp 121–122 °C; 1H NMR (300 MHz, CDCl3): d 7.55–
7.49 (d, J = 16 Hz, 1H, Ph-CH@CH), d 7.06 (s, 1H, Ph), d 7.05–7.02
(d, J = 8 Hz, 1H, Ph), d 6.84–6.82 (d, J = 8 Hz, 1H, Ph), d 6.63–6.57 (d,
J = 16 Hz, 1H, Ph-CH@CH), d 6.26 (br s, 1H, NH), d 6.00 (s, 2H, OCH2O),
d4.62–4.57 (m, 1H, NH–CH–CO2Me), d 3.76 (s, 3H, CO2–CH3), d3.07–
3.00 (t, 2H, CO–CH2–CH2–CO), d 2.62–2.58 (t, 2H, CO–CH2–CH2–CO),
d1.43–1.40 (d, 3H, NHCH–CH3); FT-IR (m0max, cmꢀ1): 3310 (NH), 3052
(CH@CH), 2966 (CH aliphatic), 1729(C@O ester), 1689(C@O ketone),
1652 (C@O amide); MS (Mwt.: 333.34): m/z 333.55 (M+, 49.61%),
231.25 (100%). Anal. Calcd for C17H19NO6: C, 61.25; H, 5.75; N,
4.20. Found: C, 61.47; H, 5.43; N, 4.51.
J = 16 Hz, 1H, Ph-CH@CH), d 7.57–7.54 (m, 3H, Ar), d 7.41–7.39
(m, 2H, Ar), d 6.79–6.74 (d, J = 16 Hz, 1H, Ph-CH @CH), d 6.48–
6.45 (d, 1H, NH), d 4.56–4.51 (m, 1H, NH–CH–CO2H), d 3.10–3.06
(t, 2H, CO–CH2–CH2–CO), d 2.68–2.64 (t, 2H, CO–CH2–CH2–CO), d
2.25–2.24 (q, 1H, NHCH–CH), d 1.00–0.97 (m, 6H, CH–(CH3)2); FT-
IR (m0max, cmꢀ1): 3400–2800 (OH carboxylic), 1720, 1705 (2 C@O)
1655 (C@O amide); MS (Mwt.: 303.35): m/z 303.10 (M+, 6.57%),
186.80 (53.78%), 102.60 (55.87%), 54.30 (51.89%). Anal. Calcd for
C17H21NO4: C, 67.31; H, 6.98; N, 4.62. Found: C, 67.48; H, 7.15;
N, 4.38.
5.1.4.4 (S,E)-2-(6-(Benzo[d][1,3]dioxol-5-yl)-4-oxohex-5-enami-
do)propanoic acid (5c).
5.1.3.7
(S,E)-Methyl 2-(6-(benzo[d][1,3]dioxol-5-yl)-4-oxohex-
The above compound was separated as buff crystals, (160 mg,
72%); mp 143–145 °C; 1H NMR (300 MHz, CDCl3): d 7.56–7.50 (d,
J = 16 Hz, 1H, Ph-CH @CH), d 7.06 (s, 1H, Ph), 7.06–7.03 (d,
J = 8 Hz, 1H, Ph), d 6.84–6.82 (d, J = 8 Hz, 1H, Ph), d 6.62–6.57 (d,
J = 16 Hz, 1H, Ph-CH@CH), d 6.42 (d, 1H, NH), d 6.02 (s, 2H, OCH2O),
d 4.55 (t, 1H, NH–CH–CO2Me), d 3.06–3.02 (t, 2H, CO–CH2–CH2–
CO), d 2.64–2.60 (t, 2H, CO–CH2–CH2–CO), d 1.48–1.46 (d, 3H,
NHCH–CH3); FT-IR (mm0 ax, cmꢀ1): 3400–3100 (OH carboxylic),
1705, 1695 (2 C@O) 1672 (C@O amide); MS (Mwt.: 319.31): m/z
319 (M+, 7.46%), 256.80 (100%), 134.75 (73.44%). Anal. Calcd for
5-enamido)-3-methyl-butanoate (4f).
The titled compound was purified by column chromatography
(gradient elution starting from 1% EtOAc/CH2Cl2 and increasing polar-
ity to 20% EtOAc/CH2Cl2) to afford 4f as buff crystals (0.48 g, 48%); mp
77–78 °C; 1H NMR (300 MHz, CDCl3): d 7.55–7.49 (d, J = 16 Hz, 1H,
Ph-CH@CH), d 7.05 (s, 1H, Ph), d 7.05–7.02 (d, J = 8 Hz, 1H, Ph), d
6.84–6.81 (d, J = 8 Hz, 1H, Ph), d 6.63–6.57 (d, J = 16 Hz, 1H, Ph-CH
@CH), d 6.26–6.23 (br s, 1H, NH), d 6.02 (s, 2H, OCH2O), d 4.57–4.53
(m, 1H, NH–CH–CO2Me), d 3.74–3.73 (s, 3H, CO2–CH3), d 3.09–3.00
(m, 2H, CO–CH2–CH2–CO), d 2.64–2.62 (m, 2H, CO–CH2–CH2–CO),
2.18–2.15 (q, 1H, NHCH–CH), d 0.96–0.92 (m, 6H, CH-(CH3)2); FT-IR
C16H17NO6: C, 60.18; H, 5.37; N, 4.39. Found: C, 60.44; H, 5.18;
(
m0max, cmꢀ1): 3305 (NH), 3058 (CH@CH), 2957, 2910 (CH aliphatic),
N, 4.42.
1727 (C@O ester), 1690 (C@O ketone), 1615 (C@O amide); MS
(Mwt.: 361.39): m/z 361.65 (M+, 53.72%), 174.70 (90.12%), 87.60
(100%). Anal. Calcd for C19H23NO6: C, 63.15; H, 6.41; N, 3.88. Found:
C, 63.06; H, 6.67; N, 3.70.
5.1.4.5 (S,E)-2-(6-(Benzo[d][1,3]dioxol-5-yl)-4-oxohex-5-enami-
do)-3-methylbutanoic acid (5d).
The titled compound was separated as orange oil, (165 mg,
68%); 1H NMR (300 MHz, CDCl3): d 7.56–7.50 (d, J = 16 Hz, 1H,
Ph-CH@CH), d 7.05 (s, 1H, Ph), 7.05–7.02 (d, J = 8 Hz, 2H, Ph), d
6.83–6.81 (d, J = 8 Hz, 1H, Ph), d 6.62–6.57 (d, J = 16 Hz, 1H, Ph-
CH@CH), d 6.55 (d, 1H, NH), d 6.01 (s, 2H, OCH2O), d 4.56–4.51
(m, 1H, NH–CH–CO2Me), d 3.06–3.02 (m, 2H, CO–CH2–CH2–CO), d
2.65–2.63 (t, 2H, CO–CH2–CH2–CO), 2.24–2.22 (q, 1H, NHCH–CH),
d 0.99–0.95 (m, 6H, CH-(CH3)2); FT-IR (m0max, cmꢀ1): 3400–2900
(OH carboxylic), 1710, 1701 (2 C@O) 1672 (C@O amide); MS
(Mwt.: 347.36): m/z 347.35 (M+, 18.40%), 174.30 (82.24%). Anal.
Calcd for C18H21NO6: C, 62.24; H, 6.09; N, 4.03. Found: C, 62.45;
H, 5.88; N, 4.15.
5.1.4. (S,E)-2-(4-Oxo-6-arylhex-5-enamido)propanoic acids and
(S,E)-3-Methyl-2-(4-oxo-6-arylhex-5-enamido)butanoic acids
(5a–d)
5.1.4.1. General procedure.
To a stirred solution of the
respective ester (4a,b,e and f) (0.7 mmol) in a 2:1 mixture of
THF/ethanol (15 mL) at rt, aqueous solution of LiOH (70 mg,
3.5 mmol, in 6 mL H2O) was added portionwise and the resulting
solution was stirred at rt for 2–3 h until TLC (2% MeOH/EtOAc) re-
vealed the disappearance of starting material. The solvent was par-
tially removed in vacuo; EtOAc (20 mL) was then added followed
by 1 N HCl solution (10 mL). The layers were separated, the aque-
ous phase was extracted with EtOAc (3 ꢃ 15 mL), the combined or-
ganic phase was dried over anhydrous MgSO4 and filtered. The
filtrate was evaporated in vacuo to afford the crude acid (5a–d)
which was further purified by column chromatography (using gra-
dient elution starting from EtOAc then 1% MeOH/EtOAc and
increasing polarity to 5% MeOH/EtOAc).
5.1.4.6. (S,E)-Ethyl 3-(tert-butyloxycarbonylamino)-4-phenyl-
but-1-ene-1-sulfonate (7).
To a stirred solution of ethyl
(diethoxyphosphoryl)methanesulfonate (1.56 g, 6 mmol) in anhy-
drous THF (25 mL) at ꢀ78 °C, n-BuLi (2.5 M in hexane) (3.12 mL,
7.8 mmol) was added. The solution was stirred for 30 min, before
a solution of 6 (1.5 g) in anhydrous THF (4 mL) was added, and
the mixture was stirred at ꢀ78 °C for 1 h. The reaction was then
quenched by pH 7 phosphate buffer (5 mL). The aqueous phase
was extracted with ether (3 ꢃ 20 mL), the combined organic phase
was washed with brine (2 ꢃ 15 mL), dried over anhydrous MgSO4,
and filtered. The filtrate was concentrated in vacuo to afford a
crude yellow oil, which was purified by flash column chromatogra-
phy (cyclohexane/EtOAc 3:1) (TLC stained with KMnO4) to afford 7
as yellow crystals, (1.53 g, 72%); mp 64–65 °C; 1H NMR (400 MHz,
CDCl3): d 7.34–7.28 (m, 3H, Ar), d 7.17–7.15 (d, J = 7 Hz, 2H, Ar), d
6.86–6.81 (dd, J = 5 and 15 Hz, 1H, CH@CH–SO3), d 6.23–6.19 (dd,
J = 1 and 15 Hz, 1H, CH@CH–SO3), d 4.65 (br s, 1H, CH –NH), d
4.55 (br s, 1H, NH), d 4.09–4.03 (m, 2H, SO3–CH2), d 2.94–2.91 (d,
2H, CH2-Ph), d 1.41 (s, 9H, t-Bu), d 1.34–1.3 (t, J = 7 Hz, 3H, SO3–
CH2–CH3); 13C NMR (100 MHz, CDCl3): d 154.73 (C@O), d 147.81
(CH@CH–SO3), d 135.52, 129.3, 128.85, 127.28 (Ar), d 125.05
(CH@CH–SO3), d 66.99 (SO3–CH2), d 51.97 (CH–NH), d 40.3 (CH2-
Ph), d 28.23 ((CH3)3), d 26.91 (C–(CH3)3), d 14.77 (SO3–CH2–CH3);
5.1.4.2
(S,E)-2-(4-Oxo-6-phenylhex-5-enamido)propanoic acid
(5a).
The titled acid was separated as yellow oil, (125 mg, 65%); 1H NMR
(300 MHz, CDCl3): d 7.64–7.59 (d, J = 16 Hz, 1H, Ph-CH@CH), d 7.57–
7.54 (m, 3H, Ar), d 7.41–7.39 (m, 2H, Ar), d 6.79–6.73 (d, J = 16 Hz,
1H, Ph-CH@CH), d 6.54–6.52 (d, 1H, NH), d 4.59–4.51 (m, 1H, NH–CH
–CO2H), d 3.11–3.06 (t, 2H, CO–CH2–CH2–CO), d 2.65–2.63 (t, 2H, CO–
CH2–CH2–CO), d 1.47–1.45 (d, 3H, NHCH–CH3); FT-IR (m0max, cmꢀ1):
3400–2900 (OH carboxylic), 1710, 1701 (2 C@O) 1665 (C@O amide);
MS (Mwt.: 275.30): m/z 275.40 (M+, 1.12%), 131 (24.46%), 102.45
(50.96), 54.05 (100%). Anal. Calcd for C15H17NO4: C, 65.44; H, 6.22; N,
5.09. Found: C, 65.32; H, 6.11; N, 5.22.
5.1.4.3
(S,E)-3-Methyl-2-(4-oxo-6-phenylhex-5-enamido)buta-
noic acid (5b).
The titled compound was separated as buff crystals, (145 mg,
FT-IR (c
max, cmꢀ1): 3365 (NH), 3062 (CH@CH), 2980, 2932 (CH ali-
68%); mp 129–131 °C; 1H NMR (300 MHz, CDCl3): d 7.63–7.59 (d,

2752
D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
phatic), 1690 (C@O carbamate), 1354, 1165 (SO2); HRMS (ESI+):
(Mwt. 355); m/z found 378.1031 [M+Na]+, C17H25NO5SNa requires
378.1351.
C
11H16NO2 requires 194.1181 and 216.0987 [M+Na]+, C11H15NO2-
Na requires 216.1000.
5.1.8. (S)-2-Acetamido-3-phenylpropan-1-al (12)
5.1.5. (S,E)-Ethyl 3-amino-4-phenylbut-1-ene-1-sulfonate (9)
A solution of 7 (100 mg, 0.28 mmol) in 30% TFA/CH2Cl2 (10 mL)
was stirred at rt for 1 h after which TLC (2% MeOH/EtOAc) (stained
with KMnO4) showed no starting material. The mixture was con-
centrated to dryness to afford the crude amine which was purified
by column chromatography (gradient elution starting from EtOAc
then 1% MeOH/EtOAc and increasing polarity to 5% MeOH/EtOAc)
to give 9 as yellow oil, (70 mg, 98%); 1H NMR (270 MHz, CDCl3):
d 9.8 and 7.83 (2br s, 2H, NH2), d 7.33–7.30 (m, 3H, Ar), d 7.16–
7.14 (d, 2H, Ar), d 6.88–6.8 (dd, 1H, CH@CH–SO2), d 6.4–6.34 (d,
1H, CH@CH–SO2), d 4.29 (br s, 1H, CH–NH2), d 4.00–3.83 (m, 2H,
SO3–CH2), d 3.21–3.00 (dt, 2H, CH2-Ph), d 1.27–1.21 (t, 3H, SO3–
CH2–CH3).
A mixture of 11 (0.193 g, 1 mmol), PDC (0.752 g, 1 mmol), acti-
vated molecular sieves (3 Å) (1 g) in anhydrous methylene chloride
(5 mL) was stirred at rt for 1.5 h, after which TLC (EtOAc/CH2Cl2/
MeOH 6:3:1) (stained with ninhydrin) showed no starting mate-
rial. The mixture was diluted with ether (15 mL), filtered twice
over celite, concentrated in vacuo to afford a crude yellowish oil
of 12, (0.11 g, 58%), which was used in the next step without fur-
ther purification.
5.1.9. (S,E)-Ethyl 3-acetamido-4-phenylbut-1-ene-1-sulfonate
(13)
To a stirred solution of ethyl (diethoxyphosphoryl)methanesul-
fonate (0.15 g, 0.58 mmol) in anhydrous THF (3 mL) at ꢀ78 °C, n-
BuLi (2.5 M in hexane) (0.3 mL, 0.75 mmol) was added, the solution
was stirred for 30 min, before a solution of 12 (0.11 g) in anhydrous
THF (2 mL) was added, and the mixture was stirred at ꢀ78 °C for
1 h. The reaction was then quenched by pH 7 phosphate buffer
(2 mL), the aqueous phase was extracted with ether (3 ꢃ 5 mL).
The combined organic phase was washed with brine (2 ꢃ 10 mL),
dried over MgSO4 and filtered. The filtrate was evaporated in vacuo
to afford the crude product which was purified by flash column
chromatography (CH2Cl2/EtOAc 6:4) (TLC stained with KMnO4) to
afford 13 as pale yellow crystals, (120 mg, 70%); mp 82–83 °C; 1H
NMR (270 MHz, CDCl3): d 7.29–7.28 (m, 3H, Ar), d 7.16 (d, 2H,
Ar), d 6.81–6.79 (dd, 1H, CH@CH–SO3), d 6.2 (dd, 1H, CH@CH–
SO3), d 5.65 (br s, 1H, NH), d 5.28–4.97 (m, 1H, CH–NH), d 4.06–
4.01 (q, 2H, SO3–CH2), d 2.94–2.89 (d, 2H, CH2-Ph), d 2.03 (s, 3H,
5.1.6. (S,E)-Ethyl 3-((E)-4-oxo-6-phenylhex-5-enamido)-4-
phenylbut-1-ene-1-sulfonate (10)
To an ice-cooled solution of 3a (56 mg, 0.27 mmol) and triethyl-
amine (70 mg, 0.675 mmol) in methylene chloride (5 mL) stirred at
ꢀ10°C in an ice-salt bath, ethyl chloroformate (30
lL, 0.27 mmol)
was added dropwise, while stirring for 10 min, after which a solu-
tion of the amine (9) (70 mg, 0.27 mmol) in CH2Cl2 (2 mL) was
added dropwise. The cooling bath was then removed and the reac-
tion was stirred at rt for 2 days until the disappearance of the start-
ing materials as judged by TLC (EtOAc/CH2Cl2 1:1) (stained with
KMnO4). The solvent was evaporated in vacuo; the residue was dis-
solved in EtOAc (10 mL). The organic layer was washed with 10%
HCl (2 ꢃ 5 mL), then saturated Na2CO3 solution (2 ꢃ 5 mL), dried
over anhydrous MgSO4 and filtered. The filtrate was evaporated
in vacuo to afford the crude product which was purified by column
chromatography (gradient elution starting from CH2Cl2 then 1%
EtOAc/CH2Cl2 and increasing polarity till 8% EtOAc/CH2Cl2) to final-
ly give the titled product (10) as orange yellow crystals (52 mg,
44%); mp 142–143; 1H NMR (300 MHz, CDCl3): d 7.61–7.57 (d,
J = 16 Hz, 1H, Ph-CH@CH), d 7.56–7.54 (m, 3H, Ar), d 7.44–7.39
(m, 2H, Ar), d 7.34–7.30 (m, 3H, Ar), d 7.25–7.18 (d, 2H, Ar), d
6.90–6.83 (dd, J = 5 and 15 Hz, 1H, CH@CH–SO3), d 6.76–6.70 (d,
J = 16 Hz, 1H, Ph-CH@CH), d 6.37–6.31 (d, J = 15 Hz, 1H, CH@CH–
SO3), d6.06–6.04 (m, 1H, NH), d5.01–4.99 (m, 1H, NH–CH–CH@CH),
d 4.09–4.05 (q, 2H, SO3–CH2), d 3.41–3.39 (m, 2H, CH2-Ph), d 2.99–
2.96 (m, 2H, CO–CH2–CH2–CO), d 2.54–2.50 (m, 2H, CO–CH2–CH2–
CO), d 1.34–1.27 (m, 3H, SO3–CH2–CH3); FT-IR (m0max, cmꢀ1): 3362
(NH), 3062 (CH@CH), 3022 (CH aromatic), 2977 (CH aliphatic),
1702 (C@O ketone), 1665 (C@O amide), 1365, 1175 (SO2); MS
(Mwt.: 441.54): m/z 441.80 (M+, 1.11%), 91 (100%). Anal. Calcd
for C24H27NO5S: C, 65.28; H, 6.16; N, 3.17. Found: C, 65.17; H,
6.07; N, 3.32.
Ac), d 1.33–1.31 (t, 3H, SO3–CH2–CH3); b (c
max, cmꢀ1): 3295
(NH), 3085 (CH@CH), 2973, 2895 (CH aliphatic), 1672 (C@O
amide), 1352, 1170 (SO2); HRMS (ESI+): (Mwt. 297); m/z found:
298.1111 [M+H]+, C14H20NO4S requires 298.1113 and 320.0938
[M+Na]+, C14H19NO4SNa requires 320.0932.
5.1.10. Tetrabutylammonium (S,E)-3-acetamido-4-phenylbut-
1-ene-1-sulfonate (14)
A solution of 13 (60 mg, 0.2 mmol) in anhydrous acetone
(10 mL) was treated with tetrabutylammonium iodide (75 mg,
0.2 mmol) and the mixture was refluxed for 2 days till the disap-
pearance of starting material as judged by TLC (EtOAc/CH2Cl2/
MeOH 6:3:1) (stained with KMnO4). The solvent was evaporated
in vacuo, and the crude product was purified by flash column chro-
matography (EtOAc/CH2Cl2/MeOH 6:2:2) to afford 14 as yellow
crystals, (90 mg, 88%); mp 157–158 °C; 1H NMR (270 MHz,
CD3OD): d 7.24–7.2 (m, 5H, Ar), d 6.46–6.44 (dd, 1H, CH@CH–
SO3), d 6.38 (d, 1H, CH@CH–SO3), d 4.22–4.17 (m, 1H, CH–NH), d
3.3–3.29 (m, 8H, N–(CH2)4), d 2.92–2.77 (dt, 2H, CH2-Ph), 1.87 (s,
3H, Ac), d 1.7–1.6 (m, 8H, bu4), d 1.5–1.2 (m, 8H, bu4), d 1.05–
5.1.7. (S)-2-Acetamido-3-phenylpropan-1-ol (11)
0.99 (m, 12H, bu4); FT-IR (c
max, cmꢀ1): 3269 (NH), 3168 (CH@CH),
To a solution of
L
-phenylalaninol (1 g, 6.6 mmol) in pyridine
2963, 2873 (CH aliphatic), 1672 (C@O amide), 1363, 1180 (SO2);
(20 mL) at ꢀ20 °C, acetic anhydride (0.7 mL, 7.26 mmol) was added
dropwise, and the solution was stirred at ꢀ20 °C for 2 h after which
TLC (EtOAc/CH2Cl2/MeOH 6:2:2) (stained with ninhydrin) showed
no starting material. The reaction was quenched with methanol
(3 mL) then evaporated in vacuo. The residue was dissolved in
EtOAc (15 mL), washed with water (2 ꢃ 15 mL), brine
(2 ꢃ 15 mL), dried over anhydrous MgSO4 and filtered. The filtrate
was evaporated to dryness to afford 11 as white needles which was
recrystallized from EtOAc, (1.1 g, 78%); mp 98–99 °C; 1H NMR
(270 MHz, CDCl3): d 7.25–7.21 (m, 5H, Ar), d 5.7 (br s, 1H, NH), d
4.15–4.12 (m, 1H, CH–NH), d 3.66–3.58 (m, 2H, CH2–OH), d 2.87–
2.84 (d, 2H, CH2-Ph), d 2.64–2.62 (t, 1H, OH), d 2.03 (s, 3H, Ac);
HRMS (ESI+): (Mwt. 193); m/z found: 194.1168 [M+H]+,
HRMS (ESIꢀ): (Mwt. 510); m/z found: 268.0630 [MꢀNBu4]ꢀ,
C12H14NO4S requires 268.0644.
5.1.11. (2R,3R)-2-Acetamido-3-(tert-butyldimethylsilyloxy)butan-
1-ol (17)
To a stirred solution of 16 (2.3 g, 7.96 mmol) in a 1:1 mixture of
THF and MeOH (30 mL) at 0 °C, LiBH4 (0.7 g, 31.82 mmol) was
added portionwise, the solution was stirred at rt for 2 days, during
which additional LiBH4 (0.35 g, 15.92 mmol) in methanol (10 mL)
was added. Upon disappearance of starting material as judged by
TLC (EtOAc/CH2Cl2/MeOH 5:4.5:0.5) (stained with H2SO4), the
reaction was quenched by addition of glacial acetic acid (7 mL)
with continued stirring for 30 min. The solvent was evaporated

D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
2753
in vacuo. The residue was dissolved in CH2Cl2 (20 mL), washed
17.92 (C(CH3)3), d 14.83 (SO3–CH2–CH3), d
(2CH3); FT-IR (c
ꢀ4.48 and ꢀ4.9
with saturated NaHCO3 (2 ꢃ 15 mL), brine (2 ꢃ 15 mL), dried over
anhydrous MgSO4 and filtered. The filtrate was evaporated to dry-
ness to afford the crude product which was purified by flash col-
umn chromatography (EtOAc/CH2Cl2/MeOH 5:4.5:0.5) to afford
17 as white needles, (1.75 g, 84%); mp 72–73 °C; 1H NMR (400
MHz, CDCl3): d 5.95 (br s, 1H, NH), d 4.13–4.08 (dq, J = 2 and
6 Hz, 1H, CH–O), d 3.87–3.81 (dt, J = 2 and 6 Hz, 1H, CH–NH), d
3.67–3.54 (dt, J = 6 and 9 Hz, 2H, CH2–OH), d 2.99 (br s, 1H, OH),
d 2.03 (s, 3H, Ac), d 1.16–1.14 (d, J = 6 Hz, 3H, CH3–CH–O), d 0.89
(s, 9H, t-Bu), d 0.08 (2s, 6H, 2CH3); 13C NMR (100 MHz, CDCl3): d
171.25 (C@O), d 67.28 (CH–O), d 64.11 (CH2–OH), d 56.53 (CH–
NH), d 25.78 ((CH3)3), d 23.37 (Ac), d 21.14 (CH3), d 17.92
max, cmꢀ1): 3354 (NH), 3062 (CH@CH), 2963,
2943, 2865 (CH aliphatic), 1667 (C@O amide), 1355, 1175 (SO2);
HRMS (ESI+): (Mwt. 365); m/z found: 366.1782 [M+H]+,
C
C
15H32NO5SSi requires 366.1770
15H31NO5SSiNa requires 388.1590.
and 388.1595 [M+Na]+,
5.1.14. (3R,4R,E)-Ethyl 3-acetamido-4-hydroxypent-1-ene-1-
sulfonate (20)
To a solution of 19 (0.25 g, 0.685 mmol) in THF (10 mL), 70% HF/
pyridine (0.7 mL) was added, and the resulting mixture was stirred
at rt for 3 h after which TLC (2% MeOH/EtOAc) (stained with
KMnO4) revealed the disappearance of starting material. The reac-
tion was quenched by addition of saturated NaHCO3 solution
(5 mL). The mixture was concentrated in vacuo, the residue was
dissolved in EtOAc (10 mL), washed with saturated NaHCO3
(3 ꢃ 15 mL), H2O (2 ꢃ 10 mL), brine (2 ꢃ 15 mL), dried over anhy-
drous MgSO4 and filtered. The filtrate was evaporated to dryness
to afford the crude product which was purified by flash column
chromatography (1% MeOH/EtOAc) to give 20 as white needles,
(148 mg, 86%); mp 112–114 °C; 1H NMR (270 MHz, CD3OD):
6.95–6.88 (dd, 1H, CH@CH–SO3), d 6.51–6.44 (d, 1H, CH@CH–
SO3), d 4.62–4.61 (br s, 1H, CH–NH), d 4.20–4.12 (q, 2H, SO3–
CH2), d 3.96–3.94 (m, 1H, CH –OH), d 2.04–1.99 (s, 3H, Ac), d
1.36–1.31 (t, 3H, SO3–CH2–CH3), d 1.17–1.09 (d, 3H, CH3–CH–O);
(C(CH3)3), d ꢀ4.27 and ꢀ5.04 (2CH3); FT-IR (
c
max, cmꢀ1): 3367
(NH), 3421 (OH), 2954, 2928, 2858 (CH aliphatic), 1677 (C@O
amide); HRMS (ESI+): (Mwt. 261); m/z found: 262.1828 [M+H]+,
C12H28NO3Si requires 262.1838, 284.1649 [M+Na]+, C12H27NO3SiNa
requires 284.1658.
5.1.12. (2S,3R)-2-Acetamido-3-(tert-butyldimethylsilyloxy)butan-
1-al (18)
To a stirred solution of oxalyl chloride (0.19 mL, 2.25 mmol) in
anhydrous methylene chloride (4 mL) at ꢀ63 °C, anhydrous DMSO
(0.213 mL, 3 mmol) in CH2Cl2 (4 mL) was added over 10 min.
Immediately following, a solution of 17 (0.392 g, 1.5 mmol) in
CH2Cl2 (6 mL) was added over 10 min, the reaction was stirred at
ꢀ63 °C for 30 min. Triethylamine (0.84 mL, 6 mmol) was then
added over 5 min, and the mixture was stirred at ꢀ63 °C for an
additional 30 min. The reaction was then allowed to warm to rt,
20% aqueous KHSO3 (5 mL) was added, then cyclohexane
(10 mL), and the reaction was stirred vigorously at rt till the sepa-
ration of 2 phases. The layers were separated, the aqueous phase
was extracted with CH2Cl2 (3 ꢃ 10 mL), the combined organic
phase was washed with saturated NaHCO3 solution (2 ꢃ 10 mL),
brine (2 ꢃ 10 mL), dried over anhydrous MgSO4 and filtered. The
filtrate was evaporated to dryness to afford the crude aldehyde
(18) as yellowish oil (0.39 g, 100%), which was used in the next
step without further purification. 1H NMR (270 MHz, CDCl3): d
9.59 (s, 1H, CH@O), d 6.25 (br s, 1H, NH), d 4.54–4.49 (m, 2H, CH
@NH and CH@O), d 2.11 (s, 3H, Ac), d 1.17 (d, 3H, CH3–CH–O), d
0.89 (s, 9H, t-Bu), d 0.08–0.06 (2s, 6H, 2CH3).
FT-IR (c
max, cmꢀ1): 3475 (OH), 3285 (NH), 3115 (CH@CH), 2958,
2885 (CH aliphatic), 1672 (C@O amide), 1365, 1172 (SO2); HRMS
(ESI+): (Mwt. 251); m/z found: 252.0906 [M+H]+, C9H18NO5S re-
quires 252.0906 and 274.0721 [M+Na]+, C9H17NO5SNa requires
274.0725.
5.2. Biological evaluation
5.2.1. Materials and methods
5.2.1.1 Cell culture and culture media.
Human hepatoma cells (Huh7.5) were maintained in Dulbecco’s
modified eagle’s medium (DMEM) (Gibco) supplemented with 1%
L-glutamine (Gibco), 1% penicillin, 1% streptomycin (Gibco), 1ꢃ
nonessential amino acids (Gibco) and 10% fetal bovine serum
(FBS) (Omega Scientific). Cell lines were passaged twice weekly
after treatment with 0.05% trypsin–0.02% EDTA and seeding at a
dilution of 1:5. Subconfluent Huh7.5 cells were trypsinized and
collected by centrifugation at 700g for 5 min. The cells were then
washed three times in ice-cold RNase-free phosphate-buffered sal-
ine (PBS) (BioWhittaker) and resuspended at 1.5 ꢃ 107 cells/mL in
PBS.
5.1.13. (3R,4R,E)-Ethyl 3-acetamido-4-(tert-butyldimethylsilyloxy)
pent-1-ene-1-sulfonate (19)
To a stirred solution of ethyl (diethoxyphosphoryl)methanesul-
fonate (0.38 g, 1.46 mmol) in anhydrous THF (6 mL) at ꢀ78 °C,
n-BuLi (2.5 M in hexane) (0.88 mL, 2.19 mmol) was added. The
solution was stirred for 30 min, after which a solution of 18
(0.39 g) in anhydrous THF (4 mL) was added, and the reaction
was stirred at ꢀ78 °C for 1 h. The reaction was then quenched by
pH 7 phosphate buffer (5 mL), allowed to warm to rt, then concen-
trated in vacuo. The residue was dissolved in CH2Cl2 (15 mL),
washed with brine (2 ꢃ 10 mL), dried over MgSO4 and filtered.
The filtrate was evaporated in vacuo to afford the crude product
which was purified by flash chromatography (EtOAc/CH2Cl2/cyclo-
hexane 3:4:2) (TLC stained with KMnO4) to afford 19 as white nee-
dles, (0.314 g, 59%); mp 86–87 °C; 1H NMR (400 MHz, CDCl3): d
6.85–6.8 (dd, J = 5 and 15 Hz, 1H, CH@CH–SO3), d 6.31–6.27 (dd,
J = 2 and 15 Hz, 1H, CH@CH–SO3), d 5.91–5.89 (d, 1H, NH), d
4.59–4.57 (m, 1H, CH–NH), d 4.18–4.10 (q, J = 7 Hz, 2H, SO3–CH2),
d 4.08–4.03 (dq, J = 2 and 6 Hz, 1H, CH–O–), d 2.08 (s, 3H, Ac), d
1.38–1.34 (t, J = 7 Hz, 3H, SO3–CH2–CH3), d 1.19–1.18 (d, J = 6 Hz,
3H, CH3 –CH–O), d 0.88 (s, 9H, t-Bu), d 0.07 (2s, 6H, 2CH3); 13C
NMR (100 MHz, CDCl3): d 169.98 (C@O), d 147.05 (CH@CH–SO3),
d 125.76 (CH@CH–SO3), d 69.1 (CH–OH), d 66.9 (SO3–CH2), d
55.13 (CH–NH), d 25.74 ((CH3)3), d 23.21 (Ac), d 20.98 (CH3), d
5.2.1.2 Transfection and drug treatment.
The plasmid FL-J6/JFH-5’C19Rluc2AUbi that consists of the full-
length HCV genome (genotype 2a) and expresses Renilla luciferase
(Rluc) was transfected into Huh7.5 cells using Lipofectamine 2000
(Invitrogen) following manufacturer’s specifications. After 24 h,
cells were pooled and seeded in 96-well plates (2–3 ꢃ 104 cells/
well). Cells were grown in quadruplicates in the presence of serial
dilutions of the test compounds (4a–f, 5b,c, 7, 8, 10, 13, 14,20) dis-
solved in DMSO. DMSO was used as a negative control, while the
NS3 protease inhibitor boceprevir (SCH503034) was used as posi-
tive control. After 48 h, cells were subjected to luciferase assays
and alamarBlue-based viability assays.
5.2.1.3 Assay protocols.
(i) Luciferase assay: Viral RNA replication was determined using
Renilla luciferase assays (Promega). Cells were washed with PBS
and shaked in lysis buffer according to the manufacturer’s protocol.
After 15 min incubation at ꢀ80 °C and thawing, luciferase assay
buffer containing the assay substrate was injected, and luciferase

2754
D. S. Lasheen et al. / Bioorg. Med. Chem. 21 (2013) 2742–2755
activity was measured using a Berthold LB96V luminometer. Signal
was normalized relative to samples grown in the presence of
DMSO as a negative control. Experiments were repeated 3 times,
each time with 4 replicates.
(ii) Viability assay: Cells were incubated for 2 h at 37 °C in the
presence of either 10% alamarBlue reagent (Invitrogen) or for 1 h
in the presence of 10% Prestoblue reagent (Invitrogen) to assess
cytotoxicity. Fluorescence was detected using FLEXstationII 384
(Molecular Devices). Signal was normalized as described above.
der Waals, Columbic and H-bond terms) for all residues within
12.0 Å distance from any found pose. Interactions fingerprints were
calculated for studied compounds by summing per-residue inter-
action energies for amino acids comprising HCV protease oxyanion
hole (Gly137 + Ala139) and the three sub-pockets S1(Ala157 + I-
le132 + Phe154), S2 (Ala157 + Arg155 + His57) and S3 (Ile132 + A-
la157 + Ser159 + Lys136 + Arg161). Interaction fingerprints for
studied compounds are given in Table 3.
Due to its relatively larger size and higher flexibility, compound
(10) was docked using a specialized induced-fit docking protocol58
through a combination of Glide (version 5.7, Schrödinger, LLC, NY,
2011) and Prime (version 3.0, Schrödinger, LLC, NY, 2011). After the
initial Glide docking, amino acids within 5.0 Å radius around any
suggested pose were considered as flexible, and their side chain
conformations were optimized by Prime. Up to 50 poses were re-
tained for each calculation within an energy window of 40.0 kcal/
mol. Poses were finally energy minimized, re-scored and priori-
tized by Glide SP scoring.
5.2.1.4 Statistical analysis.
EC50 and CC50 values were measured by fitting data to a 3-
parameter logistic curve using the formula:
Y ¼ a þ ðb ꢀ aÞ=ð1 þ 10ðXꢀcÞÞ
where a, b, and c represent minimum binding, maximum binding,
and log EC50 or log CC50
Bioscience).
, respectively) (BioDataFit; Chang
5.3.4. Covalent Docking for (10)
5.3. Molecular modeling
Docking of vinyl sulfonate (10) was performed using the cova-
lent docking facility in Prime. Crystal structure of (1) covalently
bound to catalytic Ser139 of HCV NS3/4A protease was used
(PDB: 1W3C).22 Downloaded crystal structure was processed into
a full-atom optimized model as described previously. Ligand struc-
ture was deleted, Ser139 hydroxyl hydrogen was added, Val132
was mutated into Ile and the protein alone was energy minimized
again to a gradient of 0.05 kJ/mol Å. The Ser139 terminal O–H bond
was specified as the ‘receptor bond to break’. Vinyl sulfonate dou-
ble bonds were reduced to single bonds to match the proposed li-
gand structure upon binding; then modified structures were re-
minimized. The ligand reactive group was set up to match the pat-
tern H2C–C–S; such that any of the two hydrogens furthest from
sulfur could be the leaving atom. Finally, protein residues within
8.0 Å from Ser139 were included in Prime conformational sam-
pling and optimization.
5.3.1. Preparing ligand structures
All the studied compounds were using Maestro Interface (ver-
sion 9.2, Schrödinger, LLC, NY, 2011) and geometries were opti-
mized with using OPLS_2005, the implementation of the OPLS-
AA forcefield55 in MacroModel (version 9.9, Schrödinger, LLC, NY,
2011), combined with GB/SA implicit solvent model.56 Carboxylate
esters and vinyl sulfonate esters were modeled as the neutral
species while vinyl sulfonate tetrabutylammonium salts were
modeled as the negatively charged de-protonated species.
Compounds 5b and 5c were modeled as the ionized carboxylate
form as predicted by Epik.57
5.3.2. Preparing enzyme structures
Complexes of HCV NS3/4A protease with various ligands were
downloaded from the Protein Data Bank. For each crystal structure,
hydrogens were added, water molecules were removed and bond
orders for proteins and ligand were corrected using Maestro Pro-
tein Preparation Wizard. Partial charges were calculated from
OPLS-AA55 forcefield while protonation states and oxidation states
for metals were assigned by Epik.57 Orientations of added hydro-
gens were extensively sampled for optimal H-bond formation
and the model was then refined by minimization to heavy-atom
RMSD of 0.3 Å to the crystal structure geometry. Eventually, an
all-atom ligand–protein complex with properly assigned ionization
states and atomic charges was available that could be used in sub-
sequent modeling steps.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.017.
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