Design and synthesis of l- and d-phenylalanine derived rhodanines with novel C5-arylidenes as inhibitors of HCV NS5B polymerase

Article abstract of DOI:10.1016/j.bmc.2013.03.041

Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV therapeutics development. Herein, we report the synthesis and in vitro evaluation of anti-NS5B polymerase activity of a molecular hybrid of our previously reported lead compounds 1 (IC₅₀ = 7.7 μM) and 2 (IC₅₀ = 10.6 μM) as represented by hybrid compound 27 (IC ₅₀ = 6.7 μM). We have explored the optimal substituents on the terminal phenyl ring of the 3-phenoxybenzylidene moiety in 27, by generating a set of six analogs. This resulted in the identification of compound 34 with an IC₅₀ of 2.6 μM. To probe the role of stereochemistry towards the observed biological activity, we synthesized and evaluated the d-isomers 41 (IC₅₀ = 19.3 μM) and 45 (IC₅₀ = 5.4 μM) as enantiomers of the l-isomers 27 and 34, respectively. The binding site of compounds 32 and 34 was mapped to palm pocket-I (PP-I) of NS5B. The docking models of 34 and 45 within the PP-I of NS5B were investigated to envisage the molecular mechanism of inhibition.

Full text of DOI:10.1016/j.bmc.2013.03.041

Accepted Manuscript
Design and synthesis of L- and D-phenylalanine derived rhodanines with novel
C5-arylidenes as inhibitors of HCV NS5B polymerase
Bhargav Patel, Ramalingam Krishnan, Nikhil Khadtare, K.R. Gurukumar,
Amartya Basu, Payal Arora, Aaditya Bhatt, Maulik R. Patel, Dibyendu Dana,
Sanjai Kumar, Neerja Kaushik-Basu, Tanaji T. Talele
PII:
S0968-0896(13)00259-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.03.041
BMC 10694
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
10 December 2012
1 March 2013
9 March 2013
Please cite this article as: Patel, B., Krishnan, R., Khadtare, N., Gurukumar, K.R., Basu, A., Arora, P., Bhatt, A.,
Patel, M.R., Dana, D., Kumar, S., Kaushik-Basu, N., Talele, T.T., Design and synthesis of L- and D-phenylalanine
derived rhodanines with novel C5-arylidenes as inhibitors of HCV NS5B polymerase, Bioorganic & Medicinal
Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.03.041
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Design and synthesis of L- and D-phenylalanine derived rhodanines with novel C5-
arylidenes as inhibitors of HCV NS5B polymerase
Bhargav Patela†, Ramalingam Krishnanb†, Nikhil Khadtarea†, K. R. Gurukumarb, Amartya Basub,
Payal Arorab, Aaditya Bhatta, Maulik R. Patelc, Dibyendu Danad, Sanjai Kumard, Neerja
Kaushik-Basub*, Tanaji T. Talelea*
a Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's
University, 8000 Utopia Parkway, Queens, NY 11439, USA
b Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185
South Orange Avenue, Newark, NJ 07103, USA
c Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York,
NY 10021, USA
d Department of Chemistry and Biochemistry, Queens College and the Graduate Center of the
City University of New York, 65-30 Kissena Blvd., Flushing, NY 11367, USA
* Corresponding author. Tel.: +1 718 990 5405; fax: +1 718 990 1877 (T.T.T)
Tel: +1 973 972 8653; fax: +1 973 972 5594 (N.K-B)
E-mail address: talelet@stjohns.edu ; kaushik@umdnj.edu
† These authors contributed equally
ABSTRACT: Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV
therapeutics development. Herein, we report the synthesis and in vitro evaluation of anti-NS5B
polymerase activity of a molecular hybrid of our previously reported lead compounds 1 (IC50 =
7.7 μM) and 2 (IC50 = 10.6 μM) as represented by hybrid compound 27 (IC50 = 6.7 μM). We
have explored the optimal substituents on the terminal phenyl ring of the 3-phenoxybenzylidene
moiety in 27, by generating a set of six analogs. This resulted in the identification of compound
34 with an IC50 of 2.6 μM. To probe the role of stereochemistry towards the observed biological
activity, we synthesized and evaluated the D-isomers 41 (IC50 = 19.3 μM) and 45 (IC50 = 5.4
μM) as enantiomers of the L-isomers 27 and 34, respectively. The binding site of compounds 32
and 34 was mapped to palm pocket-I (PP-I) of NS5B. The docking models of 34 and 45 within
the PP-I of NS5B were investigated to envisage the molecular mechanism of inhibition.
Keywords: L-Phenylalanine, D-Phenylalanine, Rhodanine, Knoevenagel condensation, Ullmann
condensation, HCV NS5B polymerase
1. Introduction
Hepatitis C virus (HCV) infection has emerged as one of the most significant disease1
and an estimated 200 million cases of HCV infections exist worldwide.2 Of those initially
infected with HCV, approximately 80% will progress to develop chronic liver disease and 20%

2
will eventually progress to liver cirrhosis and hepatocellular carcinoma.3 Until recently the
current therapy for treating HCV infection has been regular injections of pegylated interferon α
(PEG-IFN-α) in combination with daily oral administration of ribavirin (RBV). This
cumbersome process has found limited patient compliance due to severe adverse effects.4
Within the past year, two NS3/4 protease inhibitors, telaprevir5 and boceprevir6, in combination
with PEG-IFN/RBV have been approved for treating HCV genotype 1 infection. Although these
combination treatments have shown improved sustained virological response (SVR) in genotype
1 patients;7,8 they still harbor the severe side effects associated with IFN therapy. Therefore, the
search for novel, directly acting antiviral agents that specifically targets HCV and harbors
minimal adverse effects on the patient is an urgent medical necessity.
The HCV non-structural protein 5B (NS5B), a 66 kDa RNA-dependent RNA polymerase
(RdRp) is an important therapeutic target for its important role in replicating the HCV RNA
genome. This target is especially significant from the drug discovery point-of-view since humans
lacks its functional equivalent.9 The combination of crystallographic, biochemical and
mutagenesis studies have allowed the identification of at least five nonnucleoside inhibitor (NNI)
binding sites on NS5B enzyme.10 MK-3281 bound to thumb pocket (TP)-I11 and PF-868554
bound to TP-II12 of NS5B are located in the thumb domain, whereas acylpyrrolidine bound to
palm pocket (PP)-I13, HCV-796 bound to PP-II14 and GS9190 bound to PP-III15 of NS5B are
partially overlapped and are located between the thumb and the palm domains, in close
proximity to the active site. Several inhibitors targeting these binding sites on NS5B have
demonstrated strong efficacy in clinical trials.10
Compounds bearing the rhodanine scaffold have been previously reported as HCV NS5B
inhibitors, by us16 and others.17 Recently, the rhodanine scaffold has been a topic of debate in

3
regards to its promiscuous nature.18 While one group of researchers have considered rhodanines
as “frequent hitters” that interact with multiple targets and elicit broad inhibitory activity, others
believe rhodanines to be “privileged scaffolds”. The negative opinion primarily highlights its
insufficient selectivity. On the other hand, a more positive view considers rhodanine as a suitable
lead compound for tailoring its potency and selectivity at the step of advanced lead
optimization.18 Importantly, clinical success with the rhodanine analog, epalrestat, argues in
favor of its safety in humans. As part of our continued efforts towards development of more
potent rhodanine derivatives as anti-HCV agents, herein we report on the optimization of our two
previous rhodanine leads, compounds 1 and 216, through molecular hybridization, bioisosterism
and stereochemical optimization strategies that renders a rhodanine derivative with improved
NS5B inhibitory activity (Fig. 1).
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 23 and 27 was reported by us previously.19 The
benzaldehydes required for the preparation of target compounds were either commercially
available or prepared (3-11, 13-15) as shown in Scheme 1. The 2-phenoxybenzaldehyde (3) and
substituted 3-phenoxybenzaldehydes (4-9) were synthesized via Ullmann condensation using 2-
bromobenzaldehyde and 3-bromobenzaldehyde, respectively.20 Compounds 10 and 11 were
prepared by treating 3-formylphenyl boronic acid with benzyl bromide and bromobenzene,
respectively, by following Suzuki coupling procedure.21 The 3-benzoyl benzaldehyde (13) was
synthesized through the classical Weinreb amide approach.22 Weinreb amide was formed by the
coupling 3-benzoylbenzoic acid with N,O-dimethylhydroxylamine hydrochloride in presence of
EDC hydrochloride. Weinreb amide was then directly reduced by LiAlH4 to yield benzhydryl

4
alcohol (12) which when subjected to Dess-Martin periodinane oxidation yielded the desired 3-
benzoylbenzaldehyde (13). Aldehyde intermediates 14 and 15 were synthesized by alkylation of
3-hydroxybenzaldehyde with benzyl bromide and cinnamyl chloride, respectively, in the
presence of potassium carbonate. The synthesis of target compounds 17-39 and 41-45 is outlined
in Scheme 2. The phenylalanine-derived optically active rhodanine intermediates L-16 and D-40
were synthesized, respectively, from the corresponding L- and D-phenylalanine, as per the
previously reported procedure.19 Knoevenagel condensation of L-16 and D-40 with various
aromatic aldehydes at the nucleophilic C5 active methylene, respectively, resulted in target
compounds 17-39 (L-isomers) and 41-45 (D-isomers) bearing the Z-geometry as determined by
the chemical shift of the methine proton ranging from 7.7-8.2 ppm as a singlet (chemical shift for
corresponding methine proton of E-isomer is calculated to be 6.8 ppm).23,24 Owing to the fact
that the optical rotations for the L- and D-isomers were not equally opposite, we presumed that
partial racemization may have occurred during rhodanine ring construction and/or Knoevenagel
condensation which was further established by enantiomeric excess determination by chiral
HPLC methodology.
2.2. Structure-activity relationship
The compounds prepared in this study were evaluated utilizing a biochemical assay
against HCV NS5B polymerase as described previously.16 This assay involves quantification of
the amount of radioactive UMP incorporated into nascent RNA products employing
homopolymeric poly rA/U12 as template-primer and functionally active recombinant HCV
NS5BC∆21 (genotype 1b) protein. The activity of NS5B polymerase in the absence of the
inhibitor but containing an equivalent amount of DMSO (control reaction) was set at 100%. The
inhibitory activity of synthesized compounds was then quantified relative to this control.

5
Wedelolactone25 and aurintricarboxylic acid26, two validated NS5B inhibitors previously
characterized by us, were included as reference standards and yielded IC50 values (data not
shown) consistent with previously reported values. Depending on the nature of substituents on
benzylidene moiety, the tested compounds exhibited IC50 values ranging between 2 μM and 50
μM against NS5B polymerase as summarized in Table 1. At the onset, we prepared a set of nine
compounds to probe the effect of small electron withdrawing substituents at 3- and 4-positions of
the benzylidene moiety. This was important since in our previous studies the electron donating
substituents on benzylidene had proven detrimental for the NS5B RdRp activity.16 Replacement
of 2,4-dichlorobenzylidene in compound 2 (IC50 = 10.6 µM) with 3-chlorobenzylidene moiety
(compound 17, IC50 = 26.6 µM) proved deleterious for the compound’s anti-NS5B activity,
whereas substitution with 3-bromobenzylidene (compound 18, IC50 = 7.4 µM) exhibited near
comparable activity to compound 2. This suggested that groups with larger van der Waal’s radii
coupled with high lipophilicity are well-tolerated at the 3-position of the benzylidene moiety for
NS5B inhibition. This was also apparent from 3-cyanobenzylidene analog (compound 19, IC50 =
30.7 µM), which exhibited ~3-fold loss of activity. With the objective of investigating the
influence of mono-substitutions at the 4-position of benzylidene moiety, we synthesized 4-
bromobenzylidene analog (compound 20, IC50 = 11.7 µM), which showed comparable activity to
compound 2. The 4-fluorobenzylidene analog (compound 21, IC50 = 27.7 µM) and the 4-
chlorobenzylidene analog (compound 22, IC50 = 22.3 µM) exhibited ~2-3-fold loss in activity.
This data suggested that the larger size and lipophilicity of groups at 4-positon may be favorable
for NS5B inhibition. We next investigated the influence of disubstitutions on the benzylidene
ring.
Substituting the 2,4-dichlorobenzylidene moiety in compound 2 with the 3,4-
dichlorobenzylidene (compound 23, IC50 = 19.4 µM), 3,4-difluorobenzylidene (compound 24,

6
IC50 = 16.3 µM), and 3,5-difluorobenzylidene (compound 25, IC50 = 26.0 µM) resulted in 1.5- to
3-fold loss in activity. Thus, smaller substituents at varying positions of the benzylidene moiety
were unfavorable compared to lead compound 2, with the exception of 3-bromo and 4-bromo
analogs.
In our earlier work on glycine-derived rhodanines16, we observed that the 3-phenoxy
substitution on the benzylidene moiety enhanced NS5B inhibitory activity. To further explore the
role of this substituent, 2-phenoxy (compound 26, IC50 = 13.3 µM), 3-phenoxy (compound 27,
IC50 = 6.7 µM), and 4-phenoxy (compound 28, IC50 = 50.7 µM) groups were installed on the
benzylidene moiety. This data indicated that the phenoxy substituents are well tolerated at the 2-
position, and favor NS5B inhibitory activity at the 3-position of benzylidene. Introduction of 4-
phenoxy group on benzylidene; however, resulted in a dramatic loss in activity, suggesting the
possibility of a steric restriction in this region of the molecule. Capitalizing on these
observations of the importance of a phenoxy substituent at 3-position, we next explored the
chemical space around the optimal 3-phenoxy group of compound 27. Mono substitution with a
chloro group (compound 29, IC50 = 4.1 µM) at 3-position of phenoxy ring improved the NS5B
inhibitory activity by ~1.5-fold compared to compound 27, whereas substitution by a fluoro
group (compound 30, IC50 = 8.2 µM) resulted in a marginal loss of activity. Analogs with 4-
position substituents as the methoxy (compound 31, IC50 = 8.3 µM) or fluoro (compound 33,
IC50 = 12.5 µM) groups exhibited 1.5- to 2-fold loss in activity. Interestingly, the 4-chloro
substituent (compound 32, IC50 = 3.4 µM) was found to be 2-fold more potent relative to
compound 27. Since the 3-chloro and the 4-chloro substituents demonstrated improved NS5B
inhibitory activities, we next synthesized an analog carrying 3,4-dichloro substituent with the
expectation that it may further enhance NS5B inhibitory effect. Indeed, compound 34 (IC50 =

7
2.6 µM) bearing 3,4-dichlorophenoxy benzylidene moiety proved to be a better inhibitor than
compound 27, the unsubstituted analog and comparable to compounds 29 and 32 the mono-
substituted analogs. Together, these data suggest that the tail groups at 3- and 4-positions of the
phenoxy moiety tolerate halogens as well as methoxy group.
Since the concept of bioisosterism has been widely applied in lead optimization program
to achieve improved target inhibition as well as desired physicochemical profile, we sought to
optimize the heteroatom linkage between the two aryl rings through bioisoteric replacements.
Towards this goal, we prepared analogs replacing ether linkage with its classical isostere -CH2-
(compound 35, IC50 = 4.8 µM) that showed comparable activity to compound 27. Not
surprisingly, the non-classical isostere –C(=O)- bearing a carbonyl group (compound 36, IC50 =
11.1 µM) proved detrimental for the activity. From these IC50 values, it is apparent that correct
positioning of the terminal phenyl ring, rather than the hydrogen bond acceptor ether linkage
between the two phenyl rings, may be critical for NS5B inhibition.
We next sought to explore alternate strategies to access putative hydrophobic region in
palm pocket-I formed by residues Pro197, Leu384, Met414, Tyr415, Ile447, and Tyr448 by
displacing the terminal phenyl ring on the compounds using linkers of varying lengths. To
achieve this goal, we prepared a set of three compounds 37-39. These compounds represent no
spacer e.g., biphenyl compound 37 (IC50 = 16.6 µM); two atom methyleneoxy spacer e.g.,
compound 38 (IC50 = 21.8 µM) and a four atom allyloxy spacer e.g., compound 39 (IC50 = 6.4
µM). The ensuing data clearly underscores the importance of a longer spacer, particularly four
atoms, between the two phenyl rings for potent NS5B inhibition.
To understand the potential binding interactions of the aforementioned inhibitors within
the NNI binding site of HCV NS5B polymerase that could be exploited to improve efficacy of

8
our compounds, we performed docking studies using Glide v5.0 docking software (Schrodinger,
LLC., New York, NY), as previously described.16 To rule out any bias, each of the five reported
NS5B allosteric binding pockets TP-I (PDB ID: 2XWY),11 TP-II (PDB ID: 3FRZ),12 PP-I (PDB
ID: 3TYV)27, PP-II (PDB ID: 3FQL),14 and PP-III, that significantly overlaps with PP-II (large
grid box created around PP-II bound HCV-796 to obtain docking pose at PP-III), was examined
for inhibitor binding. Analysis of the binding energy data (Glidescores) for all single digit μM
inhibitors at each allosteric site revealed that they bind with affinity in the order PP-I>PP-II>PP-
III>TP-I>TP-II. We and other groups of researchers have previously shown that rhodanine core
containing inhibitors bind within PP-I domain of NS5B.16,17 Since all of the inhibitors
investigated in this report carry one chiral center, we also analyzed the binding models of L- and
D-isomers within the PP-I of NS5B. These analyses revealed that both the isomers had
comparable binding energy (Glidescore) which prompted us to hypothesize that both isomers
would exhibit comparable IC50 values. To test this hypothesis, we synthesized and tested
representative D-isomers 41 (IC50 = 19.3 µM, ee = 84%), 42 (IC50 = 4.1 µM, ee = 75%), 43 (IC50
= 5.1 µM, ee = 78%), 44 (IC50 = 10.3 µM, ee = 85%), and 45 (IC50 = 5.4 µM, ee = 93%) as
counterparts of compounds 27 (IC50 = 6.7 µM, ee = 85%), 29 (IC50 = 4.1 µM, ee = 93%), 32
(IC50 = 3.4 µM, ee = 86%), 33 (IC50 = 12.5 µM, ee = 84%), and 34 (IC50 = 2.6 µM, ee = 77%),
respectively, starting from the D-phenylalanine-derived rhodanine intermediate (40) as depicted
in Scheme 2. Taking enantiomeric purity into consideration these investigations indicated rather
comparable inhibitory activity of both the isomers. Attempts to correlate Glidescore energy data
(kcal/mol) with compound pIC50 (-log IC50) values resulted in r2 value of 0.48 with three outliers
(compounds 28, 37 and 38). This correlation is reasonable given the fact that there is still no
single scoring function that can correctly rank every protein-ligand complex in appropriate order.

9
2.3. Mapping the inhibitor binding site on NS5B
With the goal of validating the presumed inhibitor binding pocket for the relevant
rhodanine derivatives on NS5B, we employed NS5B mutants P495L, M423T and M414T as
screens for TP-I, TP-II and PP-I site binders, respectively.28-30 These residues are critical
components of the aforementioned allosteric pockets on NS5B, thus mutations at these sites
results in loss of sensitivity to the inhibitor due to decrease in inhibitor binding.28-30 Consistent
with our screening strategy selection for PP-I binders, the inhibitory potency of representative
compounds 32 and 34 was impacted by mutation at M414 residue of NS5B, but not with P495L
and M423T mutants (Table 2). This was evident from the 16- to18-fold higher IC50 values for
compounds 32 and 34 against M414T NS5B relative to wild-type NS5B. By contrast, the IC50
values of the compounds against NS5B mutants P495L and M423T exhibited ≤1.5 fold change
relative to their corresponding wild-type NS5B values. This data thus suggests that the inhibitors
bind at PP-I site of NS5B.
2.4. Molecular docking
To gain insight into the molecular mechanism of inhibition, we analyzed the interactions
of the docked conformation of compound L-34 (panel A) and for comparison D-45 (panel B)
within the PP-I of NS5B (PDB ID: 3TYV)27 as shown in Fig. 2. The phenyl ring of the
phenylalanine may enter into cation-pi interaction (4.8 Å and 5.1 Å) with the guanidinium and ε-
NH3 groups of Arg158 and Lys141, respectively. Whereas one of the oxygen atoms of the
carboxylate group forms a water-mediated hydrogen bond with the backbone –NH of Ser556 (-
COO---H2O(598)---HN-Ser556), the other forms a water-mediated hydrogen bond with the side
chain hydroxyl group of Ser288 (-COO---H2O(790)---HO-Ser288). The electrophilic
benzylidene carbon atom is juxtaposed to initiate covalent chemistry with the nucleophilic –SH

10
group of Cys366 (-=C---SH-Cys366, 3.4 Å) as previously reported for related benzylidene
analog by Powers et al.17 The nucleophilicity of the –SH group of Cys366 may be enhanced by a
potential hydrogen bond with the guanidine group of Arg200 (-SH---N-guanidine-Arg200, 2.6
Å). The first phenyl ring of the benzylidene moiety is extensively stabilized by the hydrophobic
residues Pro197, Tyr448, Leu384, Met414, and the methylene groups of Arg200. The 3,4-
dichlorophenoxy ring is also stabilized through hydrophobic interactions with the side chains of
Met414, Tyr415, and Ile447. Whereas the 3-chloro group enter into a dipole interaction with the
side chain amide group of Asn411 (3-Cl---C=O, 3.4 Å), the 4-chloro group enter into two water-
mediated hydrogen bonding interactions with the amide group of Asn411 (4-Cl---HOH(1202)---
OH2677---O=C(NH2)-Asn411). The rhodanine ring acting as a scaffold for correct positioning
of the pharmacophore groups (the phenylalanine and the benzylidene moiety) is mainly
stabilized by Phe193 and Tyr448. The proposed binding mode is consistent with the observed
SAR: a) the requirement for correct positioning of the terminal phenyl ring, and b) the ability to
tolerate a variety of electron withdrawing hydrophobic substituents at the terminal phenyl ring.
The binding mode of D-45 was found to be relatively similar to that observed for L-34
with the subtle variations as described below. The phenyl ring of the phenylalanine portion is 5.6
and 6.3 Å away from the guanidinium and ε-NH3 groups of Arg158 and Lys141, respectively.
The electrophilic benzylidene carbon atom is located 3.5 Å away from the nucleophilic –SH
group of Cys366. Based on above observations it seems that cation-pi interaction may play a
positive role in NS5B inhibitory activity of the L-isomer.
Of the 28 rhodanine derivatives reported here, compound 35 seems to be the most
promising for futher optimization studies, as it exhibited no cellular toxicity and limited antiviral
activity (~30% inhibition of HCV RNA replication) at 100 μM concentration against HCV

11
replicon bearing hepatoma cells. We speculate that its ionizable carboxyl group may limit its
membrane permeability and thus its antiviral efficacy. We therefore propose to explore various
substitutions at the benzyl moiety of compound 35 for future optimization. Another possibility
would be replacement of the thiazolidinone core with alternate five membered scaffolds to
eliminate promiscuous nature of rhodanine ring. In addition, replacement of the carboxyl group
with ester and bioisosteric tetrazole ring may help improve cellular permeability and antiviral
potency. These proposed modifications would be guided by molecular docking investigations.
3. Conclusions
We have disclosed that properly functionalized benzylidene moiety at the C5-position of
the phenylalanine-derived rhodanine scaffold provides several single digit micromolar NS5B
inhibitors. Docking experiments have facilitated the interpretation of the inhibitory activity of
these rhodanines by predicting the molecular interactions of the compounds with NS5B. We
have also mapped the inhibitor binding site to PP-I of NS5B by counter screening against PP-I
site mutant, M414T NS5B. Recognizing the feasibility of improving the activity of 3-phenoxy
group by replacing it with 3-benzyl moiety and the preference for L-isomer, further SAR efforts
will be directed in future to explore the most optimal substituents at the benzyl moiety of the L-
isomer.
4. Experimental
4.1. General
Melting points (m.p.) were determined on a Thomas-Hoover capillary melting point
apparatus and are uncorrected. The reagents for organic synthesis were purchased from Aldrich
Chemical Co. (Milwaukee, WI), TCI America (Portland, OR), Alfa Aesar (Ward Hill, MA), and
Acros Organics (Antwerp, Belgium) and were used as received. All compounds were checked

12
for homogeneity by TLC using silica gel as a stationary phase. NMR spectra were recorded on a
Bruker 400 Avance DPX spectrometer (1H at 400 MHz and 13C at 100 MHz) outfitted with a z-
axis gradient probe. The chemical shifts for 1H and 13C are reported in parts per million (δ ppm)
downfield from tetramethylsilane (TMS) as an internal standard. The 1H NMR data are reported
as follows: chemical shift, multiplicity (s) singlet, (d) doublet, (dd) doublet of doublets, (t)
triplet, (dt) doublet of triplets and (m) multiplet, respectively. Optical rotation of the chiral
compounds was measured using PerkinElmer 241 Polarimeter with ethyl acetate as the solvent,
concentrations c are expressed as g/100 mL. The C, H, and N analyses were performed by
Atlantic Microlabs, Inc., (Norcross, GA) and the observed values were within 0.4% of
calculated values.
Chiral HPLC analysis
Chiral HPLC analysis was performed using Dionex Ultimate 3000 Series instrument. The
compounds were dissolved in ethyl acetate and injected (20 μL) into the chiralpak 1B column
(Daicel Corp., Fort Lee, NJ) with stationary phase as cellulose tris(3,5-
dimethylphenylcarbamate) immobilized on 5 µm silica-gel. Optimum resolution of the
enantiomers was achieved using an isocratic mobile phase (75:25 Hexane:Ethyl acetate with
0.1% TFA) eluting at a flow rate of 1 mL/min. The elutions were monitored at UV 370 nm in
form of major and minor peaks representing the respective enantiomers. The retention times (tR)
for major and minor peaks are given in minutes. The enantiomeric excess (ee) values were
calculated based on the area under peak for each enantiomers.
4.2. Synthesis
4.2.1. General procedure for preparation of phenoxybenzaldehydes (3-9)
Intermediates 3-9 were prepared following the procedure reported by Sasaki et al.20

13
4.2.2. 2-Phenoxybenzaldehyde (3)
Starting with 2-bromobenzaldehyde (2.0 g, 10.81 mmol) and phenol (1.11 g, 11.89 mmol),
compound 3 (1.30 g, 61%) was obtained as colorless oil; Rf = 0.42 (n-hexane:ethyl acetate 95:5);
1H NMR (400 MHz; CDCl3; TMS) δ 10.52 (1H, s), 7.94 (1H, dd, J = 7.7 Hz, 1.6 Hz), 7.51 (1H,
t, J = 7.7 Hz), 7.39 (2H, m), 7.19 (2H, t, J = 7.4 Hz), 7.07 (2H, d, J = 8.1 Hz), 6.89 (1H, d, J =
8.3 Hz).
4.2.3. 3-(3-Chlorophenoxy)benzaldehyde (4)
Starting with 3-bromobenzaldehyde (2.0 g, 10.81 mmol) and 3-chlorophenol (1.53 g, 11.89
mmol), compound 4 (1.12 g, 45%) was obtained as yellow oil; Rf = 0.36 (n-hexane:ethyl acetate
95:5); 1H NMR (400 MHz; CDCl3; TMS) δ 9.98 (1H, s), 7.65 (1H, d, J = 7.5 Hz), 7.53 (1H, t, J
= 7.8 Hz), 7.48 (1H, s), 7.27-7.31 (2H, m), 7.14 (1H, d, J = 8.1 Hz), 7.01-7.02 (1H, m), 6.92 (1H,
13
d, J = 8.2 Hz); C NMR (100 MHz, CDCl3, TMS) δ 191.45, 157.48, 157.21, 138.15, 135.27,
130.80, 130.69, 125.54, 125.05, 124.19, 119.49, 118.54, 117.31.
4.2.4. 3-(3-Fluorophenoxy)benzaldehyde (5)
Starting with 3-bromobenzaldehyde (2.0 g, 10.81 mmol) and 3-fluorophenol (1.33 g, 11.89
mmol), compound 5 (0.92 g, 39%) was obtained as yellow oil; Rf = 0.35 (n-hexane:ethyl acetate
95:5); 1H NMR (400 MHz; CDCl3; TMS) δ 9.96 (1H, s), 7.65 (1H, d, J = 7.6 Hz), 7.49-7.54 (2H,
13
m), 7.27-7.33 (2H, m), 6.79-6.87 (2H, m), 6.73 (1H, dt, J = 9.9 Hz, 2.3 Hz); C NMR (100
MHz, CDCl3, TMS) δ 191.43, 171.21, 163.54 (d, J = 247.4 Hz), 157.77, 157.51, 138.24, 130.83,
130.68, 125.33, 118.75, 114.65, 110.90 (d, J = 21.2 Hz), 106.84 (d, J = 24.6 Hz).
4.2.5. 3-(4-Methoxyphenoxy)benzaldehyde (6)
Starting with 3-bromobenzaldehyde (2.0 g, 10.81 mmol) and 4-methoxyphenol (1.47 g, 11.89
mmol), compound 6 (1.64 g, 67%) was obtained as yellow oil; Rf = 0.31 (n-hexane:ethyl acetate

14
95:5); 1H NMR (400 MHz; CDCl3; TMS) δ 9.93 (1H, s), 7.54 (1H, d, J = 7.5 Hz), 7.46 (1H, t, J
= 7.8 Hz), 7.37 (1H, s), 7.23 (1H, d, J = 8.1 Hz), 7.00 (2H, d, J = 9.0 Hz), 6.91 (2H, d, J = 9.0
Hz), 3.82 (3H, s).
4.2.6. 3-(4-Chlorophenoxy)benzaldehyde (7)
Starting with 3-bromobenzaldehyde (2.0 g, 10.81 mmol) and 4-chlorophenol (1.53 g, 11.89
mmol), compound 7 (0.90 g, 36%) was obtained as yellow oil; Rf = 0.36 (n-hexane:ethyl acetate
95:5); 1H NMR (400 MHz; CDCl3; TMS) δ 9.95 (1H, s), 7.62 (1H, d, J = 7.5 Hz), 7.51 (1H, t, J
= 7.8 Hz), 7.44 (1H, s), 7.26-7.34 (3H, m), 6.96-6.98 (2H, m).
4.2.7. 3-(4-Fluorophenoxy)benzaldehyde (8)
Starting with 3-bromobenzaldehyde (2.0 g, 10.81 mmol) and 4-fluorophenol (1.33 g, 11.89
mmol), compound 8 (0.83 g, 36%) was obtained as yellow oil; Rf = 0.34 (n-hexane:ethyl acetate
95:5); 1H NMR (400 MHz; CDCl3; TMS) δ 9.93 (1H, s), 7.58 (1H, d, J = 7.6 Hz), 7.48 (1H, t, J
= 7.8 Hz), 7.40 (1H, s), 7.24 (1H, d, J = 8.1 Hz), 6.99-7.08 (4H, m).
4.2.8. 3-(3,4-Dichlorophenoxy)benzaldehyde (9)
Starting with 3-bromobenzaldehyde (2.0 g, 10.81 mmol) and 3,4-dichlorophenol (1.94 g, 11.89
mmol), compound 9 (0.82 g, 28%) was obtained as yellow oil; Rf = 0.34 (n-hexane:ethyl acetate
95:5); 1H NMR (400 MHz; CDCl3; TMS) δ 9.95 (1H, s), 7.65 (1H, d, J = 7.5 Hz), 7.53 (1H, t, J
= 7.8 Hz), 7.47 (1H, s), 7.39 (1H, d, J =8.7 Hz), 7.28 (1H, d, J = 8.0 Hz), 7.10 (1H, s), 6.88 (1H,
13
d, J = 8.7 Hz); C NMR (100 MHz, CDCl3, TMS) δ 191.64, 157.16, 155.45, 138.06, 133.43,
131.26, 130.80, 127.47, 125.91, 125.09, 120.96, 118.51, 118.49.
4.2.9. 3-Benzylbenzaldehyde (10)
Following the reported procedure21, starting with 3-formylphenyl boronic acid (1.75 g, 11.69
mmol) and benzyl bromide (2.0 g, 11.69 mmol), compound 10 (1.84 g, 80%) was prepared as

15
1
colorless oil; Rf = 0.50 (n-hexane:ethyl acetate 95:5); H NMR (400 MHz; DMSO-d6; TMS) δ
1H NMR (400 MHz; DMSO-d6; TMS) δ 9.99 (1H, s), 7.79 (1H, s), 7.76 (1H, d, J = 7.5 Hz),
7.58 (1H, d, J = 7.6 Hz), 7.51 (1H, t, J = 7.5 Hz), 7.26-7.32 (4H, m), 7.20 (1H, t, J = 6.8 Hz),
4.04 (2H, s).
4.2.10. 3-Phenylbenzaldehyde (11)
Following the reported procedure21, starting with 3-formylphenyl boronic acid (1.75 g, 11.69
mmol) and bromobenzene (1.83 g, 11.69 mmol), compound 11 (1.78 g, 84%) was prepared as
colorless oil; Rf = 0.40 (n-hexane:ethyl acetate 95:5); 1H NMR (400 MHz; CDCl3; TMS) δ 10.12
(1H, s), 8.14 (1H, s), 7.89 (2H, dd, J = 7.6 Hz, 1.7 Hz), 7.64-7.68 (3H, m), 7.51-7.41 (3H, m).
4.2.11. 3-Benzoylbenzaldehyde (13)
Intermediate 13 was prepared following a diminutive variation in the literature procedure.22 N,O-
dimethyl hydroxylamine HCl (1.36 g, 13.92 mmol), EDC.HCl (2.67 g, 13.92 mmol) and Et3N
(1.41 g, 13.92 mmol) were sequentially added to the solution of 3-benzoylbenzoic acid (3.0 g,
13.26 mmol) in DMF and stirred overnight at room temperature. Upon completion, the reaction
mass was diluted with ethyl acetate and washed thrice, each time with 10% citric acid, 10%
sodium bicarbonate and brine. The collected organic layers were dried over magnesium sulfate
and the solvent was evaporated under reduced pressure to give colorless viscous oil. The
colorless oil was further dissolved in anhydrous THF under nitrogen atmosphere. To it was
added LiAlH4 (0.23 g, 6.14 mmol) portion wise at -78°C. The mixture was stirred for 3 hours at
the same temperature. Upon completion, it was quenched with water. The quenched reaction was
diluted with ethyl acetate and filtered to remove the inorganic solid mass. The filtrate was
washed with brine, dried over magnesium sulfate and the solvent was removed in vacuo to give
crude product which was purified through flash chromatography (n-hexane:ethyl acetate 90:10)

16
to yield 3-(hydroxy(phenyl)methyl)benzaldehyde (12) (1.93 g, 69%) as colorless oil; Rf = 0.50
(n-hexane:ethyl acetate 90:10); 1H NMR (400 MHz; CDCl3; TMS) δ 9.95 (1H, s), 7.91 (1H, s),
7.76 (1H, d, J = 7.6 Hz), 7.65 (1H, d, J = 7.7 Hz), 7.48 (1H, t, J = 7.7 Hz), 7.27-7.37 (5H, m),
5.89 (1H, s), 2.83 (1H, s).
Intermediate 12 (1.0 g, 4.71 mmol) was dissolved in THF followed by addition of Dess-Martin
periodinane (3.0 g, 7.07 mmol) and stirred for 4 hours at room temperature and, upon completion
of the reaction, it was quenched by saturated NaHCO3 and saturated Na2S2O3. The quenched
reaction was extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed
with brine, dried over magnesium sulfate and the solvent was removed under reduced pressure.
The crude material was purified by flash chromatography (n-hexane:ethyl acetate 95:5) to yield
1
13 (0.8 g, 81%) as colorless oil; Rf = 0.45 (n-hexane:ethyl acetate 95:5); H NMR (400 MHz;
CDCl3; TMS) δ 10.09 (1H, s), 8.28 (1H, t, J = 1.6 Hz), 8.12 (1H, dt, J = 7.6 Hz, J = 1.3 Hz), 8.08
(1H, dt, J = 7.8 Hz, J = 1.5 Hz), 7.80 (2H, d, J = 7.9 Hz), 7.68 (1H, t, J = 7.7 Hz), 7.63 (1H, t, J
= 7.4 Hz), 7.50-7.53 (2H, m).
4.2.12. 3-Benzyloxybenzaldehyde (14)
To the solution of the 3-hydroxybenzaldehyde (1.43 g, 11.69 mmol) in acetonitrile were added
benzyl bromide (2.0 g, 11.69 mmol) and potassium carbonate (3.23 g, 23.38 mmol) and stirred
for 14 hours at room temperature. After the completion of reaction, as indicated by TLC, solvent
was removed in vacuo, the residue was diluted with water and extracted with ethyl acetate (3 x
20 mL). The separated organic layers were dried over magnesium sulfate and the solvent was
evaporated under reduced pressure. The residual oil was purified by flash chromatography (n-
hexane:ethyl acetate 95:5) to yield compound 14 (2.2 g, 89%) as white solid; Rf = 0.54 (n-

17
1
hexane:ethyl acetate 95:5); H NMR (400 MHz; CDCl3; TMS) δ 9.97 (1H, s), 7.32-7.48 (8H,
m), 7.23-7.26 (1H, m), 5.12 (2H, s).
4.2.13. 3-Cinnamyloxybenzaldehyde (15)
Following the procedure as described for 14, starting with 3-hydroxybenzaldehyde (1.60 g, 13.10
mmol) and cinnamyl chloride (2.0 g, 13.10 mmol), compound 15 (2.46 g, 79%) was obtained as
pale yellow solid; Rf = 0.62 (n-hexane:ethyl acetate 95:5); 1H NMR (400 MHz; CDCl3; TMS) δ
9.98 (1H, s), 7.41-7.48 (6H, m), 7.34 (3H, m), 6.76 (1H, d, J = 15.9 Hz), 6.42 (1H, dt, J = 16.1
13
Hz, 5.8 Hz), 4.77 (2H, d, J = 5.8 Hz); C NMR (100 MHz, CDCl3, TMS) δ 192.22, 159.14,
137.76, 136.21, 133.49, 130.13, 128.62, 128.05, 126.60, 123.68, 123.64, 122.15, 113.12, 68.64.
4.2.14. Synthesis of 2-(5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic
acid derivatives (17-39)
Intermediate L-16 ([α]25 –80.79° (c 0.1) was prepared and subjected to Knoevenagel
D
condensation with aromatic aldehydes adhering to the procedures described in our previous
reports;16,19 to obtain target compounds 17-39.
4.2.15. (L,Z)-2-(5-(3-Chlorobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic
acid (17)31
Starting with L-16 (0.30 g, 1.07 mmol) and 3-chlorobenzaldehyde (0.15 g, 1.07 mmol),
compound 17 (0.30 g, 72%) was obtained as brown solid; m.p. 146-148 °C; Rf = 0.60
1
(DCM:MeOH 95:5); H NMR (400 MHz, DMSO-d6, TMS) δ 13.51 (1H, s), 7.81 (1H, s), 7.74
13
(1H, s), 7.54-7.58 (3H, m), 7.16-7.20 (5H, m), 5.88 (1H, s), 3.50 (2H, s); C NMR (100 MHz,
DMSO-d6, TMS) δ 193.03, 169.12, 166.78, 136.95, 135.24, 134.57, 132.74, 131.82, 131.27,
129.46, 128.92, 128.84, 128.79, 127.25, 58.68, 33.52; Anal. Calcd. for C19H14ClNO3S2·1/4C6H14:
C, 57.87; H, 4.15; N, 3.29. Found: C, 58.13; H, 4.29; N, 3.66.

18
4.2.16. (L,Z)-2-(5-(3-Bromobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic
acid (18)
Starting with L-16 (0.30 g, 1.07 mmol) and 3-bromobenzaldehyde (0.19 g, 1.07 mmol),
compound 18 (0.23 g, 48%) was obtained as brown solid; m.p. 128-130 °C; Rf = 0.64
1
(DCM:MeOH 95:5); H NMR (400 MHz, DMSO-d6, TMS) δ 13.51 (1H, s), 7.88 (1H, s), 7.81
(1H, s), 7.72 (1H, d, J = 7.7 Hz), 7.58 (1H, d, J = 7.4 Hz), 7.50 (1H, t, J = 7.7 Hz), 7.15-7.23
13
(5H, m), 5.89 (1H, s), 3.51 (2H, s); C NMR (100 MHz, DMSO-d6, TMS) δ 193.06, 169.10,
166.77, 136.98, 135.52, 134.13, 132.68, 132.02, 129.46, 129.17, 128.79, 127.24, 123.07, 122.89,
58.72, 33.56; Anal. Calcd. for C19H14BrNO3S2·1/10H2O: C, 50.69; H, 3.18; N, 3.11. Found: C,
50.83; H, 3.56; N, 3.15.
4.2.17. (L,Z)-2-(5-(3-Cyanobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic
acid (19)
Starting with L-16 (0.30 g, 1.07 mmol) and 3-cyanobenzaldehyde (0.14 g, 1.07 mmol),
compound 19 (0.12 g, 29%) was obtained as yellow solid; m.p. 103-105 °C; Rf = 0.67
1
(DCM:MeOH 95:5); H NMR (400 MHz, DMSO-d6, TMS) δ 13.53 (1H, s), 8.12 (1H, s), 7.96
(1H, d, J = 7.7 Hz), 7.88 (1H, d, J = 7.8 Hz), 7.84 (1H, s), 7.73 (1H, t, J = 7.8 Hz), 7.14-7.25
13
(5H, m), 5.89 (1H, s), 3.50 (2H, s); C NMR (100 MHz, DMSO-d6, TMS) δ 192.97, 169.09,
166.78, 136.93, 135.14, 134.55, 134.49, 134.30, 132.00, 131.16, 129.46, 128.79, 127.26, 123.63,
118.48, 113.10, 58.72, 33.52; Anal. Calcd. for C20H14N2O3S2.1/10H2O: C, 60.62; H, 3.61; N,
7.07. Found: C, 60.49; H, 3.47; N, 7.09.
4.2.18. (L,Z)-2-(5-(4-Bromobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic
acid (20)32

19
Starting with L-16 (0.30 g, 1.07 mmol) and 4-bromobenzaldehyde (0.19 g, 1.07 mmol),
compound 20 (0.38 g, 79%) was obtained as yellow solid; m.p. 175-180 °C; Rf = 0.62
1
(DCM:MeOH 95:5); H NMR (400 MHz, DMSO-d6, TMS) δ 13.49 (1H, s), 7.79 (1H, s), 7.74
13
(2H, d, J = 8.5 Hz), 7.56 (2H, d, J = 8.5 Hz), 7.15-7.23 (5H, m), 5.88 (1H, s), 3.51 (2H, s); C
NMR (100 MHz, DMSO-d6, TMS) δ 193.20, 169.12, 166.88, 136.95, 133.14, 133.01, 132.33,
129.45, 128.76, 127.23, 125.56, 125.44, 121.90, 58.66, 33.79; Anal. Calcd. for
C19H14BrNO3S2·1/4H2O: C, 50.39; H, 3.23; N, 3.09. Found: C, 50.11; H, 3.63; N, 3.07.
4.2.19. (L,Z)-2-(5-(4-Fluorobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic
acid (21)
Starting with L-16 (0.30 g, 1.07 mmol), and 4-fluorobenzaldehyde (0.13 g, 1.07 mmol),
compound 21 (0.34 g, 83%) was obtained as brown solid; m.p. 146-148 °C; Rf = 0.60
1
(DCM:MeOH 95:5), H NMR (400 MHz, DMSO-d6, TMS) δ 13.43 (1H, s), 7.83 (1H, s), 7.69-
13
7.73 (2H, m), 7.38-7.42 (2H, m), 7.14-7.22 (5H, m), 5.88 (1H, s), 3.51 (2H, s); C NMR (100
MHz, DMSO-d6, TMS) δ 193.79, 169.14, 166.91, 163.77 (d, J = 251.8 Hz), 136.97, 133.91,
133.31, 129.87, 129.45, 128.75, 127.21, 120.81, 117.24 (d, J = 22.2 Hz), 58.65, 33.54; Anal.
Calcd. for C19H14FNO3S2: C, 58.90; H, 3.64; N, 3.62. Found: C, 59.18; H, 3.70; N, 3.60.
4.2.20. (L,Z)-2-(5-(4-Chlorobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic
acid (22)
Starting with L-16 (0.30 g, 1.07 mmol) and 4-chlorobenzaldehyde (0.15 g, 1.07 mmol),
compound 22 (0.22 g, 51%) was obtained as yellow solid; m.p. 173-176 °C; Rf = 0.60
1
(DCM:MeOH 95:5), H NMR (400 MHz, DMSO-d6, TMS) δ 13.63 (1H, s), 7.81 (1H, s), 7.60-
13
7.66 (4H, m), 7.14-7.22 (5H, m), 5.88 (1H, s), 3.52 (2H, s); C NMR (100 MHz, DMSO-d6,
TMS) δ 193.13, 169.21, 166.94, 137.13, 136.38, 132.97, 132.92, 132.04, 130.08, 129.44, 128.76,

20
127.20, 121.89, 58.84, 33.58; Anal. Calcd. for C19H14ClNO3S2: C, 56.50; H, 3.49; N, 3.47.
Found: C, 56.42; H, 3.42; N, 3.54.
4.2.21.
(L,Z)-2-(5-(3,4-Dichlorobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (23)
Experimental procedure and spectral characterization data is same as reported previously.19
4.2.22.
(L,Z)-2-(5-(3,4-Difluorobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (24)
Starting with L-16 (0.30 g, 1.07 mmol) and 3,4-difluorobenzaldehyde (0.15 g, 1.07 mmol),
compound 24 (0.31 g, 72%) was obtained as yellow solid; m.p. 158-162 °C; Rf = 0.52
1
(DCM:MeOH 95:5); H NMR (400 MHz, DMSO-d6, TMS) δ 13.53 (1H, s), 7.74-7.79 (2H, m),
13
7.58-7.65 (1H, m), 7.46-7.48 (1H, m), 7.14-7.22 (5H, m), 5.88 (1H, s), 3.50 (2H, s); C NMR
(100 MHz, DMSO-d6, TMS) δ 193.20, 169.01, 166.79, 151.21 (dd, J = 253.6, 12.96 Hz), 150.11
(dd, J = 248.9, 12.96 Hz), 136.96, 132.10, 130.90, 129.43, 128.76, 128.19, 127.22, 122.47,
120.46 (d, J = 17.9 Hz), 119.26 (d, J = 17.9 Hz), 58.72, 33.54; Anal. Calcd. for
C19H13F2NO3S2·CH2Cl2: C, 48.99; H, 3.08; N, 2.86. Found: C, 48.79; H, 2.78; N, 3.07.
4.2.23.
(L,Z)-2-(5-(3,5-Difluorobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (25)
Starting with L-16 (0.30 g, 1.07 mmol) and 3,5-difluorobenzaldehyde (0.15 g, 1.07 mmol),
compound 25 (0.29 g, 67%) was obtained as yellow solid; m.p. 156-160 °C; Rf = 0.50
1
(DCM:MeOH 95:5); H NMR (400 MHz, DMSO-d6, TMS) δ 13.53 (1H, s), 7.80 (1H, s), 7.45
(1H, tt, J = 9.1 Hz, 2.3 Hz), 7.35 (2H, d, J = 6.6 Hz), 7.15-7.23 (5H, m), 5.89 (1H, s), 3.51 (2H,
13
s); C NMR (100 MHz, DMSO-d6, TMS) δ 192.94, 169.04, 166.72, 162.99 (dd, J = 247.7 Hz,
13.2 Hz), 161.79, 136.92, 136.35, 131.62, 129.43, 128.78, 127.24, 124.33, 113.86 (d, J = 26.5

21
Hz), 106.75 (d, J = 20.2 Hz), 58.74, 33.53; Anal. Calcd. for C19H13F2NO3S2: C, 56.29; H, 3.23;
N, 3.45. Found: C, 56.09; H, 3.26; N, 3.36.
4.2.24.
(L,Z)-2-(5-(2-Phenoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (26)
Starting with L-16 (0.30 g, 1.07 mmol) and 3 (0.21 g, 1.07 mmol), compound 26 (0.16 g, 33%)
was obtained as yellow solid; m.p. 65-70 °C; Rf = 0.62 (DCM:MeOH 95:5); 1H NMR (400 MHz,
DMSO-d6, TMS) δ 13.51 (1H, s), 7.91 (1H, s), 7.57 (1H, d, J = 7.8 Hz), 7.44-7.53 (3H, m), 7.24-
13
7.32 (2H, m), 7.09-7.22 (7H, m), 6.90 (1H, d, J = 8.3 Hz), 5.87 (1H, s), 3.49 (2H, s); C NMR
(100 MHz, DMSO-d6, TMS) 193.64, 169.14, 166.99, 156.70, 156.13, 137.03, 133.88, 130.87,
129.45, 128.75, 128.31, 127.19, 125.01, 124.74, 123.91, 123.40, 122.44, 119.76 , 118.76, 58.74,
33.56; Anal. Calcd. for C25H19NO4S2·1/10H2O: C, 64.80; H, 4.18; N, 3.02. Found: C, 64.57; H,
4.04; N, 2.95.
4.2.25.
(L,Z)-2-(5-(3-Phenoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (27)
Experimental procedure and spectral characterization data is same as reported previously19
except the new information provided in regards to specific rotation: [α]25D -155.09° (c 10); ee =
85% (tR (major) = 13.58, tR (minor) = 11.32).
4.2.26.
(L,Z)-2-(5-(4-Phenoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (28)
Starting with L-16 (0.30 g, 1.07 mmol) and 4-phenoxybenzaldehyde (0.21 g, 1.07 mmol),
compound 28 (0.42 g, 86%) was obtained as yellow solid; m.p. 137-139 °C; Rf = 0.8
1
(DCM:MeOH 95:5), H NMR (400 MHz, DMSO-d6, TMS) δ 13.48 (1H, s), 7.79 (1H, s), 7.64
(2H, d, J = 8.4 Hz), 7.46 (2H, t, J = 7.6 Hz), 7.08-7.27 (10H, m), 5.88 (1H, s), 3.52 (2H, s); 13C

22
NMR (100 MHz, DMSO-d6, TMS) δ 193.22, 169.21, 166.99, 160.17, 155.35, 137.01, 133.89,
133.75, 130.83, 129.45, 128.74, 127.80, 127.19, 125.31, 120.47, 119.29, 118.75, 58.61, 33.56;
Anal. Calcd. for C25H19NO4S2·1/6C6H14: C, 65.62; H, 4.52; N, 2.94. Found: C, 65.48; H, 4.89; N,
2.79.
4.2.27.
(L,Z)-2-(5-(3-(3-Chlorophenoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (29)
Starting with L-16 (0.30 g, 1.07 mmol) and 4 (0.25 g, 1.07 mmol), compound 29 (0.32 g, 60%)
was obtained as yellow solid, m.p. 55-58 °C; Rf = 0.50 (DCM:MeOH 95:5); [α]25 -171.06° (c
D
10); ee = 93% (tR (major) = 14.54, tR (minor) = 11.99); 1H NMR (400 MHz, DMSO-d6, TMS) δ
13.52 (1H, s), 7.81 (1H, s), 7.57 (1H , t, J = 7.8 Hz), 7.39-7.45 (2H, m), 7.31 (1H, s), 7.15-7.26
13
(8H, m), 7.04 (1H, d, J = 7.8 Hz), 5.87 (1H, s), 3.50 (2H, s); C NMR (100 MHz, DMSO-d6,
TMS) δ 193.14, 169.12, 166.81, 157.57, 157.24, 137.03, 135.19, 134.63, 133.55, 132.15, 131.91,
129.44, 128.76, 127.23, 126.31, 124.50, 122.25, 121.98, 121.30, 119.46, 118.10, 58.73, 33.53;
Anal. Calcd. for C25H18ClNO4S2: C, 60.54; H, 3.66; N, 2.82. Found: C, 60.29; H, 3.68; N, 2.81.
4.2.28.
(L,Z)-2-(5-(3-(3-Fluorophenoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (30)
Starting with L-16 (0.30 g, 1.07 mmol) and 5 (0.23 g, 1.07 mmol), compound 30 (0.40 g, 78%)
was obtained as yellow solid; m.p. 58-60 °C; Rf = 0.45 (DCM:MeOH 95:5); 1H NMR (400 MHz,
DMSO-d6, TMS) δ 13.54 (1H, s), 7.82 (1H, s), 7.58 (1H, t, J = 8.0 Hz), 7.39-7.47 (2H , m), 7.31
(1H, s), 7.15-7.23 (6H, m), 7.03 (1H, t, J = 8.4 Hz), 6.98 (1H, d, J = 10.4 Hz), 6.90 (1H, d, J =
13
8.3 Hz), 5.87 (1H, s), 3.50 (2H, s); C NMR (100 MHz, DMSO-d6, TMS) δ 193.20, 169.13,
166.81, 163.40 (d, J = 245.3 Hz), 157.84, 157.19, 137.00, 135.15, 133.55, 132.01, 131.88,
129.44, 128.76, 127.21, 126.24, 122.21, 121.97, 121.21, 115.29, 111.26 (d, J = 20.9 Hz), 107.05

23
(d, J = 24.5 Hz), 58.73, 33.53; Anal. Calcd. for C25H18FNO4S2.4/5H2O: C, 60.79; H, 4.00; N,
2.84. Found: C, 60.41; H, 3.73; N, 2.83.
4.2.29.
(L,Z)-2-(5-(3-(4-Methoxyphenoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (31)
Starting with L-16 (0.30 g, 1.07 mmol) and 6 (0.24 g, 1.07 mmol), compound 31 (0.38 g, 73%)
was obtained as yellow solid; m.p. 68-72 °C; Rf = 0.48 (DCM:MeOH 95:5); 1H NMR (400 MHz,
DMSO-d6, TMS) δ 13.74 (1H, s), 7.76 (1H, s), 7.50 (1H, t, J = 8.0 Hz), 7.29 (1H, d, J = 7.8 Hz),
7.17-7.20 (2H, m), 7.11-7.14 (4H, m), 7.05-7.07 (3H, m), 6.98-7.00 (2H, m), 5.82 (1H, s), 3.75
13
(3H, s), 3.50 (2H, s); C NMR (100 MHz, DMSO-d6, TMS) δ 193.33, 166.92, 159.40, 156.59,
148.95, 137.44, 134.89, 133.54, 131.57, 129.38, 128.74, 127.16, 125.11, 122.10, 121.73, 120.20,
119.02, 116.02, 115.74, 59.35, 55.92, 33.73; Anal. Calcd. for C26H21NO5S2·2/5H2O: C, 62.61; H,
4.41; N, 2.81. Found: C, 62.22; H, 4.16; N, 2.82.
4.2.30.
(L,Z)-2-(5-(3-(4-Chlorophenoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (32)
Starting with L-16 (0.30 g, 1.07 mmol) and 7 (0.25 g, 1.07 mmol), compound 32 (0.42 g, 79%)
was obtained as yellow solid; m.p. 132-135 °C; Rf = 0.48 (DCM:MeOH 95:5); [α]25D -159.16° (c
10); ee = 86% (tR (major) = 13.88, tR (minor) = 11.44); 1H NMR (400 MHz, DMSO-d6, TMS) δ
13.53 (1H, s), 7.80 (1H, s), 7.56 (1H, t, J = 8.0 Hz), 7.47 (2H, d, J = 8.4 Hz), 7.38 (1H, d, J = 7.6
Hz), 7.28 (1H, s), 7.09-7.19 (8H, m), 5.87 (1H, s), 3.49 (2H, s); 13C NMR (100 MHz, DMSO-d6,
TMS) δ 193.13, 169.12, 166.81, 157.62, 155.32, 137.00, 135.12, 133.57, 131.82, 130.54, 129.44,
128.76, 128.37, 127.21, 125.92, 122.16, 121.64, 121.33, 120.93, 58.71, 33.55; Anal. Calcd. for
C25H18ClNO4S2: C, 60.54; H, 3.66; N, 2.82. Found: C, 60.33; H, 3.44; N, 2.65.

24
4.2.31.
(L,Z)-2-(5-(3-(4-Fluorophenoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (33)
Starting with L-16 (0.30 g, 1.07 mmol) and 8 (0.23 g, 1.07 mmol), compound 33 (0.35 g, 69%)
was obtained as yellow solid; m.p. 58-62 °C; Rf = 0.42 (DCM:MeOH 95:5); [α]25 -168.45° (c
D
1
9); ee = 84% (tR (major) = 14.11, tR (minor) = 11.43); H NMR (400 MHz, DMSO-d6, TMS) δ
13.55 (1H, s), 7.79 (1H, s), 7.53 (1H, t, J = 8.0 Hz), 7.34 (1H, d, J = 7.6 Hz), 7.27 (2H, t, J = 8.7
Hz), 7.13-7.19 (9H, m), 5.87 (1H, s), 3.49 (2H, s); 13C NMR (100 MHz, DMSO-d6, TMS) δ
193.14, 169.15, 166.84, 159.05 (d, J = 239.9 Hz), 158.52, , 152.23, 137.03, 135.03, 133.72,
131.76, 129.51, 128.77, 127.23, 125.64, 122.05, 121.90, 120.92, 119.99, 117.30 (d, J = 23.8
Hz), 58.72, 33.53; Anal. Calcd. for C25H18FNO4S2: C, 62.62; H, 3.78; N, 2.92. Found: C, 62.35;
H, 3.85; N, 2.90.
4.2.32. (L,Z)-2-(5-(3-(3,4-dichlorophenoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (34)
Starting with L-16 (0.30 g, 1.07 mmol) and 9 (0.29 g, 1.07 mmol), compound 34 (0.30 g, 53%)
was obtained as yellow solid; m.p. 182-185 °C; Rf = 0.49 (DCM:MeOH 95:5); [α]25D -144.69° (c
10); ee = 77% (tR (major) = 15.55, tR (minor) = 13.03); 1H NMR (400 MHz, DMSO-d6, TMS) δ
13.54 (1H, s), 7.82 (1H, s), 7.66 (1H, d, J = 8.9 Hz), 7.58 (1H, t, J = 8.0 Hz), 7.39-7.41 (2H, m),
7.35 (1H, s), 7.15-7.26 (6H, m), 7.08 (1H, dd, J = 8.8 Hz, 2.6 Hz), 5.87 (1H, s), 3.50 (2H, s); 13C
NMR (100 MHz, DMSO-d6, TMS) δ 193.21, 169.21, 166.86, 157.01, 156.04, 137.10, 135.25,
133.43, 132.62, 132.18, 131.91, 129.42, 128.75, 127.19, 126.53, 126.25, 122.35, 121.96, 121.48,
121.42, 119.76, 58.83, 33.63; Anal. Calcd. for C25H17Cl2NO4S2: C, 56.61; H, 3.23; N, 2.64.
Found: C, 56.51; H, 3.14; N, 2.69.

25
4.2.33. (L,Z)-2-(5-(3-Benzylbenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic
acid (35)
Starting with L-16 (0.30 g, 1.07 mmol) and 10 (0.21 g, 1.07 mmol), compound 35 (0.19 g, 39%)
1
was obtained as yellow solid; m.p. 122-125 °C; Rf = 0.54 (DCM:MeOH 95:5); H NMR (400
MHz, DMSO-d6, TMS) δ 13.54 (1H, s), 7.75 (1H, s), 7.44-7.46 (3H, m), 7.40 (1H, s), 7.25-7.32
(4H, m), 7.13-7.21 (6H, m), 5.87 (1H, s), 4.01 (2H, s), 3.50 (2H, s); 13C NMR (100 MHz,
DMSO-d6, TMS) δ 193.36, 169.16, 166.90, 143.33, 141.10, 137.03, 134.31, 133.32, 132.19,
131.14, 130.17, 129.44, 129.23, 129.03, 128.75, 127.19, 126.66, 121.14, 58.71, 41.10, 33.55;
Anal. Calcd. for C26H21NO3S2: C, 67.95; H, 4.61; N, 3.05. Found: C, 68.08; H, 4.60; N, 2.99.
4.2.34.
(L,Z)-2-(5-(3-Benzoylbenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (36)
Starting with L-16 (0.30 g, 1.07 mmol) and 13 (0.22 g, 1.07 mmol), compound 36 (0.17 g, 34%)
was obtained as yellow solid; m.p. 70-74 °C; Rf = 0.46 (DCM:MeOH 95:5); 1H NMR (400 MHz,
DMSO-d6, TMS) δ 13.53 (1H, s), 7.90-7.94 (3H, m), 7.86 (1H, d, J = 7.6 Hz), 7.78 (2H, d, J =
7.8 Hz), 7.70-7.75 (2H, m), 7.57-7.60 (2H, m), 7.15-7.20 (5H, m), 5.88 (1H, s), 3.51 (2H, s); 13C
NMR (100 MHz, DMSO-d6, TMS) δ 195.37, 193.12, 169.10, 166.78, 138.47, 137.16, 137.02,
136.87, 134.69, 133.62, 133.46, 132.23, 132.06, 130.41, 130.29, 129.45, 129.17, 128.77, 127.23,
122.51, 58.76, 33.56; Anal. Calcd. for C26H19NO4S2·1/3H2O: C, 65.12; H, 4.13; N, 2.92. Found:
C, 64.98; H, 3.99; N, 2.93.
4.2.35. (L,Z)-2-(5-(3-Phenylbenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic
acid (37)
Starting with L-16 (0.30 g, 1.07 mmol) and 11 (0.19 g, 1.07 mmol), compound 37 (0.24 g, 51%)
was obtained as yellow solid; m.p. 75-77 °C; Rf = 0.42 (DCM:MeOH 95:5); 1H NMR (400 MHz,

26
DMSO-d6, TMS) δ 13.64 (1H, s), 7.91 (1H, s), 7.83 (1H, d, J = 7.6 Hz), 7.73 (2H, d, J = 7.7 Hz),
7.64 (1H, t, J = 7.7 Hz), 7.59 (1H, d, J = 7.7 Hz), 7.51 (2H, t, J = 7.5 Hz), 7.42 (1H, t, J = 7.3
Hz), 7.15-7.23 (6H, m), 5.87 (1H, s), 3.53 (2H, s); 13C NMR (100 MHz, DMSO-d6, TMS) δ
193.38, 169.40, 166.97, 141.73, 139.41, 137.32, 134.27, 133.90, 130.67, 129.93, 129.90, 129.59,
129.43, 128.76, 128.68, 128.55, 127.16, 127.31, 121.72, 59.05, 33.70; Anal. Calcd. for
C25H19NO3S2.4/5H2O: C, 65.28; H, 4.51; N, 3.05. Found: C, 64.97; H, 4.39; N, 3.12.
4.2.36.
(L,Z)-2-(5-(3-Benzyloxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (38)
Starting with L-16 (0.30 g, 1.07 mmol) and 14 (0.23 g, 1.07 mmol), compound 38 (0.22 g, 43%)
was obtained as yellow solid; m.p. 70-73 °C; Rf = 0.45 (DCM:MeOH 95:5); 1H NMR (400 MHz,
DMSO-d6, TMS) δ 13.78 (1H, s), 7.76 (1H, s), 7.46-7.48 (3H, m), 7.40 (2H, t, J = 7.2 Hz), 7.35
13
(1H, d, J = 6.8 Hz), 7.16-7.24 (8H, m), 5.83 (1H, s), 5.17 (2H, s), 3.52 (2H, s); C NMR (100
MHz, DMSO-d6, TMS) δ 193.60, 167.00, 159.29, 137.46, 137.09, 134.55, 133.94, 131.17,
129.39, 128.97, 128.74, 128.45, 128.24, 127.11, 123.46, 121.74, 118.63, 117.05, 69.87, 59.31,
33.79; Anal. Calcd. for C26H21NO4S2.2/5H2O: C, 64.68; H, 4.55; N, 2.90. Found: C, 64.57; H,
4.51; N, 2.88.
4.2.37.
(L,Z)-2-(5-(3-Cinnamyloxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (39)
Starting with L-16 (0.30 g, 1.07 mmol) and 15 (0.25 g, 1.07 mmol), compound 39 (0.30 g, 56%)
1
was obtained as yellow solid; m.p. 137-140 °C; Rf = 0.58 (DCM:MeOH 95:5); H NMR (400
MHz, DMSO-d6, TMS) δ 13.62 (1H, s), 7.79 (1H, s), 7.45-7.50 (3H, m), 7.35 (2H, t, J = 7.4 Hz),
7.14-7.29 (9H, m), 6.80 (1H, d, J = 16.0 Hz), 6.52 (1H, dt, J = 15.95 Hz, 5.83 Hz), 5.88 (1H, s),
4.80 (2H, d, J = 5.6 Hz), 3.52 (2H, s); 13C NMR (100 MHz, DMSO-d6, TMS) δ 193.37, 169.29,

27
166.93, 159.18, 137.14, 136.50, 134.51, 134.26, 133.28, 131.17, 129.43 129.15, 128.75, 128.42,
127.18, 126.99, 125.00, 123.45, 121.57, 118.65, 116.92, 68.79, 58.86, 33.62; Anal. Calcd. for
C28H23NO4S2. 1/4H2O: C, 66.45; H, 4.68; N, 2.77. Found: C, 66.09; H, 4.63; N, 2.70.
4.2.38. (D)-2-(4-Oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic acid (40)
It was prepared following the same procedure as for compound L-16, except substituting D-
phenylalanine for L-phenylalanine; obtained as yellow oil (3 g, 35%); Rf = 0.70 (DCM:MeOH
1
95:5) [α]25D +60.77° (c 10); H NMR (400 MHz, DMSO-d6, TMS) δ 13.32 (1H, s), 7.15-7.25
(5H, m), 5.68 (1H, s), 4.21-4.36 (2H, m), 3.42-3.47 (2H, m).
4.2.39.
(D,Z)-2-(5-(3-Phenoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (41)
Starting with D-40 (0.30 g, 1.07 mmol) and 3-phenoxybenzaldehyde (0.21 g, 1.07 mmol),
compound 41 (0.17 g, 35%) was obtained as yellow solid with slight variations from the reported
procedure33; m.p. 70-75 °C; Rf = 0.60 (DCM:MeOH 95:5); [α]25D +166.83° (c 10); ee = 84% (tR
(major) = 11.09, tR (minor) = 13.34); 1H NMR (400 MHz, DMSO-d6, TMS) δ 13.70 (1H, s), 7.77
(1H, s), 7.54 (1H, t, J = 7.9 Hz), 7.44 (2H, t, J = 7.8 Hz), 7.35 (1H, d, J = 7.7 Hz), 7.15-7.22 (8H,
m), 7.08 (2H, d, J = 7.8 Hz), 5.81 (1H, s), 3.52 (2H, s); 13C NMR (100 MHz, DMSO-d6, TMS) δ
192.66, 168.70, 166.36, 157.63, 155.79, 140.76, 136.52, 134.54, 133.26, 131.74, 130.74, 129.00,
128.32, 126.78, 125.35, 124.23, 123.03, 121.64, 121.06, 119.91, 119.32, 58.22, 33.10; Anal.
Calcd. for C25H19NO4S2: C, 65.06; H, 4.15; N, 3.03. Found: C, 64.78; H, 4.38; N, 2.96.
4.2.40.
(D,Z)-2-(5-(3-(3-Chlorophenoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (42)
Starting with D-40 (0.30 g, 1.07 mmol) and 4 (0.25 g, 1.07 mmol), compound 42 (0.17 g, 32%)
was obtained as yellow solid; m.p. 55-57 °C; Rf = 0.50 (DCM:MeOH 95:5); [α]25 +126.61° (c
D

28
10); ee = 75% (tR (major) = 11.97, tR (minor) = 14.51); 1H NMR (400 MHz, DMSO-d6, TMS) δ
13.69 (1H, s), 7.80 (1H, s), 7.58 (1H, t, J = 7.6 Hz), 7.39-7.46 (2H, m), 7.31 (1H, s), 7.15-7.27
13
(8H, m), 7.04 (1H, d, J = 7.9 Hz), 5.82 (1H, s), 3.51 (2H, s); C NMR (100 MHz, DMSO-d6,
TMS) δ 193.16, 169.21, 166.83, 157.52, 157.18, 137.08, 135.20, 134.60, 133.49, 132.09, 131.88,
129.45, 128.76, 127.20, 126.27, 124.46, 122.28, 121.98, 121.23, 119.46, 118.04, 58.82, 33.59;
Anal. Calcd. for C25H18ClNO4S2.1/4H2O: C, 59.99; H, 3.73; N, 2.80. Found: C, 59.60; H, 3.72;
N, 2.86.
4.2.41.
(D,Z)-2-(5-(3-(4-Chlorophenoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (43)
Starting with D-40 (0.30 g, 1.07 mmol) and 7 (0.25 g, 1.07 mmol), compound 43 (0.19 g, 36%)
was obtained as yellow solid; m.p. 68-72 °C; Rf = 0.48 (DCM:MeOH 95:5); [α]25 +139.20° (c
D
10); ee = 78% (tR (major) = 11.47, tR (minor) = 13.69); 1H NMR (400 MHz, DMSO-d6, TMS) δ
13.62 (1H, s), 7.80 (1H, s), 7.57 (1H, t, J = 8.0 Hz), 7.47 (2H, d, J = 8.7 Hz), 7.38 (1H, d, J = 7.7
Hz), 7.28 (1H, s), 7.10-7.22 (8H, m), 5.83 (1H, s), 3.50 (2H, s); 13C NMR (100 MHz, DMSO-d6,
TMS) δ 193.19, 169.10, 166.86, 157.63, 155.32, 137.06, 135.13, 133.54, 131.83, 130.55, 129.43,
128.76, 128.37, 127.20, 125.92, 122.21, 121.64, 121.34, 120.93, 58.84, 33.57; Anal. Calcd. for
C25H18ClNO4S2: C, 60.54; H, 3.66; N, 2.82. Found: C, 60.26; H, 3.84; N, 2.87.
4.2.42.
(D,Z)-2-(5-(3-(4-Fluorophenoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (44)
Starting with D-40 (0.30 g, 1.07 mmol) and 8 (0.23 g, 1.07 mmol), compound 44 (0.15 g, 29%)
was obtained as yellow solid; m.p. 73-76 °C; Rf = 0.42 (DCM:MeOH 95:5); [α]25 +144.59° (c
D
1
9); ee = 85% (tR (major) = 11.28, tR (minor) = 13.47); H NMR (400 MHz, DMSO-d6, TMS) δ
13.67 (1H, s), 7.77 (1H, s), 7.54 (1H, t, J = 8.0 Hz), 7.35 (1H, d, J = 7.7 Hz), 7.28 (2H, t, J = 8.7

29
Hz), 7.14-7.20 (9H, m), 5.80 (1H, s), 3.50 (2H, s); 13C NMR (100 MHz, DMSO-d6, TMS) δ
193.16, 169.20, 166.85, 159.07 (d, J = 239.7 Hz), 158.53, , 152.18, 137.17, 135.04, 133.58,
131.75, 129.44, 128.79, 127.21, 125.58, 122.10, 121.90, 120.95, 119.98, 117.28 (d, J = 23.4 Hz),
58.89, 33.63; Anal. Calcd. for C25H18FNO4S2.4/5H2O: C, 60.79; H, 4.00; N, 2.84. Found: C,
60.83; H, 3.75; N, 2.79.
4.2.43. (D,Z)-2-(5-(3-(3,4-Dichlorophenoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (45)
Starting with D-40 (0.30 g, 1.07 mmol) and 9 (0.29 g, 1.07 mmol), compound 45 (0.23 g, 40%)
was obtained as yellow solid with slight variations from the reported procedure33; m.p. 175-180
°C; Rf = 0.49 (DCM:MeOH 95:5); [α]25 +157.54° (c 10); ee = 93% (tR (major) = 12.89, tR
D
(minor) = 15.24); 1H NMR (400 MHz, DMSO-d6, TMS) δ 13.60 (1H, s), 7.81 (1H, s), 7.66 (1H,
d, J = 8.9 Hz), 7.59 (1H, t, J = 8.0 Hz), 7.39-7.41 (2H, m), 7.34 (1H, s), 7.15-7.26 (6H, m), 7.08
13
(1H, dd, J = 8.8 Hz, 2.6 Hz), 5.86 (1H, s), 3.51 (2H, s); C NMR (100 MHz, DMSO-d6, TMS)
193.13, 169.15, 166.82, 157.04, 156.05, 136.98, 135.25, 133.53, 132.64, 132.20, 131.94, 129.46,
128.78, 127.24, 126.55, 126.27, 122.33, 122.00, 121.52, 121.45, 119.80, 58.69, 33.54; Anal.
Calcd. for C25H17Cl2NO4S2: C, 56.61; H, 3.23; N, 2.64. Found: C, 56.89; H, 3.21; N, 2.61.
4.3. Determination of NS5B inhibitory activity
HCV NS5B polymerase inhibition was evaluated essentially as reported previously.16
4.4. Mutant counter screen assay
The binding site of the inhibitors on NS5B was investigated employing a mutant counter-
screen assay as described previously.28-30 Recombinant NS5BC∆21 mutant proteins P495L,
M423T and M414T were employed to screen for TP-I, TP-II and PP-I site binders, respectively.