Design, synthesis and biological activity of thiazolidine-4-carboxylic acid derivatives as novel influenza neuraminidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.02.019

A series of thiazolidine-4-carboxylic acid derivatives were synthesized and evaluated for their ability to inhibit neuraminidase (NA) of influenza A virus. All the compounds were synthesized in good yields starting from commercially available l-cysteine hydrochloride using a suitable synthetic strategy. These compounds showed moderate inhibitory activity against influenza A neuraminidase. The most potent compound of this series is compound 4f (IC₅₀ = 0.14 μM), which is about sevenfold less potent than oseltamivir and could be used to design novel influenza NA inhibitors that exhibit increased activity based on thiazolidine ring.

Full text of DOI:10.1016/j.bmc.2011.02.019

Bioorganic & Medicinal Chemistry 19 (2011) 2342–2348
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological activity of thiazolidine-4-carboxylic acid
derivatives as novel influenza neuraminidase inhibitors
Yu Liu a, Fanbo Jing b, Yingying Xu a, Yuanchao Xie a, Fangyuan Shi a, Hao Fang a, Minyong Li a, Wenfang Xu a,
⇑
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road, Jinan, Shandong 250012, China
b The Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shandong 266003, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thiazolidine-4-carboxylic acid derivatives were synthesized and evaluated for their ability to
inhibit neuraminidase (NA) of influenza A virus. All the compounds were synthesized in good yields start-
Received 9 January 2011
Revised 10 February 2011
Accepted 11 February 2011
Available online 17 February 2011
ing from commercially available
pounds showed moderate inhibitory activity against influenza A neuraminidase. The most potent
compound of this series is compound 4f (IC50 = 0.14 M), which is about sevenfold less potent than osel-
L-cysteine hydrochloride using a suitable synthetic strategy. These com-
l
tamivir and could be used to design novel influenza NA inhibitors that exhibit increased activity based on
thiazolidine ring.
Keywords:
Neuraminidase inhibitor
Influenza
Ó 2011 Elsevier Ltd. All rights reserved.
Thiazolidine
Biological activity
1. Introduction
tures of the group-1 neuraminidase (N1, N4, and N8) revealed a no-
vel cavity adjacent to the active site in group-1 but not in group-2
Influenza can cause serious public health and economic prob-
lems, which affects millions of people worldwide.1 Despite ad-
vances in the understanding of molecular and cellular aspects of
influenza, the disease remains the major cause of mortality and
morbidity among patients with respiratory diseases.2
proteins crystallography, suggesting new opportunities for drug
design that target this cavity in addition to the known active site.14
Earlier crystallographic and ensuing SAR studies have revealed
that the active site of NA could be divided into four major binding
sites. Steindl and Lange15 described the development of highly
selective pharmacophore models for inhibitors of viral NA within
the Catalyst software package. All NA inhibitors on the market or
in clinical phases possess strong structural resemblance in those
parts, which correspond to the fact that the four pockets are critical
for interaction with the active site of NA. The four pockets defining
the binding site are displayed in Figure 1.
The pocket C1 is comprised of the positively charged guanidino
groups of arginines 118, 292 and 371 and interacts with the car-
boxylate. In pocket C5, Arg 152 functions as the hydrogen-bond do-
nor. Trp 178 and Ile 222 comprise a small hydrophobic region. In
pocket C4, usually a guanidine or an amine group, participates in
charge–charge interactions and hydrogen bonds to Glu 119, Asp
151, and/or Glu 227. Compared with the 4-hydroxy analogues
developed earlier, these basic substituents are proved to increase
inhibitory activity. In pocket C6, Glu 276, the side chain of Arg
152, the amidic carbonyl of Trp 178 and Asp 151 form a new
hydrophobic binding pocket. Moreover, Glu277 and Tyr406 are be-
lieved to play a critical role in the catalytic activity of NA.16,17
According to the studies on NA active site and SAR of published
NA inhibitors, inhibition of the NA is mainly determined by the rel-
ative positions of the four substituents of the central ring.18 For
example, in oseltamivir, the four substituents are carboxyl ethyl
ester, amino, acetamino and alkyl. However, more and more recent
Influenza virus neuraminidase (NA) is thought to promote virus
entry and release of virion progeny, thereby enhances infection
efficiency. Rationally designed NA inhibitors (NAIs) that block the
viral life cycle are proved to be effective for the treatment of influ-
enza. The NAIs have currently emerged as promising therapeutics
for influenza. The first approved neuraminidase inhibitor zanami-
vir (Relenza) is rarely used because it is administered by inhala-
tion,3 which causes inconvenience. Subsequently discovered
oseltamivir (Tamiflu) is orally available.4 As the predominant
choice, oseltamivir is used worldwide for the treatment of influ-
enza. However, the generation and circulation of oseltamivir-resis-
tant mutants of seasonal influenza, as well as H5N1 avian
influenza, have become major concerns.5–11 Peramivir, the third
neuraminidase inhibitor, displays only limited oral bioavailabil-
ity.12,13 Intravenous peramivir is currently undergoing a pre-
emergency use authorization review for its use in cases of severe
influenza outbreaks.
Neuraminidase remains an attractive anti-influenza drug target,
while the emergence of viruses resistant to the currently available
drugs has presented a new challenge. Noticeably, the crystal struc-
⇑
Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.019

Y. Liu et al. / Bioorg. Med. Chem. 19 (2011) 2342–2348
2343
L-cysteine hydrochloride in EtOH/H2O 1:1 to obtain the thiazoli-
dines 1 as diastereomers (anti and syn compounds). Then the thiaz-
olidines were alkylated with chloroacetyl chloride or phenylacetyl
chloride in the existence of NaHCO3 in H2O to yield chloroacetyl
thiazolidines 2 and phenylacetyl thiazolidines 3. Then the crude
chloroacetyl thiazolidines were aminated in ammonia solution to
get the crude compounds 4. Subsequently by Boc-protected reac-
tion and the Boc-group cleavage in HCl/EtOAc, compound 4 were
obtained as hydrochloride salts.
3. Result and discussion
All the target compounds (27 compounds) were tested for their
ability to inhibit NA. Preliminary result showed that most of the
compounds (24 compounds) displayed enhanced inhibitory activi-
ties (IC50 = 20.3–0.14 lM) compared to the lead compound 1a
(Table 1). The amino-acetyl-thiazolidines showed the best activi-
ties, and the order of increasing activity in R2 is: NH2– >
PhCH2CO– > ClCH2CO– > H.
The most potent compound is 4f (IC50 = 0.14 lM), which is the
Figure 1. Display of important amino acids forming four pockets of NA binding
site.15
mixture of 4fanti (the major product) and 4fsyn (the minor product).
In order to determine the interaction between thiazolidines and
the NA active site, compound 4fanti and 4fsyn were docked into
the active sites of NA separately.
R2
H
N
COOH
N
COOH
The binding of compound 4f in the active site of NA is shown in
Figure 3, and we find that the –COOH group of the target com-
pound interacts with the pocket C1 of NA active site by making
charge–charge interactions with Arg 292, Arg 371, Tyr 406 of this
subsite. The reason for which the –COOH group is important for
the development of novel inhibitors is that it is well established
that two or three Arg residues in the immediate vicinity of the car-
boxylic group of NA inhibitors play a key role in orienting and sta-
bilizing various inhibitors.30,31 The –NH2 group binds to the pocket
C4 by hydrogen bond interaction with Asp151. The –CO– group
forms hydrogen bond with Arg 118 and Arg 371 of pocket C1.
Moreover, for compound 4fanti, the –OCH3 group occupies the
hydrophobic region in pocket C6 formed by Glu 277, Glu 227 and
Glu 226. The –OH group forms hydrogen bond with Glu 277. The
hydrophobic region formed by Trp 178 and Ala 180 in pocket C5
accommodates the phenyl group.
R1
S
S
R1= different benzaldehyde, furaldehyde
R2=H, ClCH2CO-, PhCH2CO-, NH2CH2CO-
1a
Figure 2. Structure of compound 1 and the modified thiazolidine derivarives.
studies revealed some untypical neuramnidase inhibitors (includ-
ing flavonoids, diarylheptanoid, etc.) that beyond the ‘airplane’
model interaction with the NA active site,19–26 thus we suppose
that the aromatic group is suitable for the pocket of the NA active
site.
In previous studies,27–29 we have reported several kinds of novel
neuraminidase inhibitors based on pyrrolidine, benzyl and thiazole
scaffold. In our recent screening, we found that compound 1a
exhibited substantial NA inhibition (IC50 = 21.3 lM). Considering
For compound 4fsyn, the phenyl group together with the –OCH3
group of 4fsyn occupies the hydrophobic region formed by Arg 224
and Glu 277 of the C6 pocket. The –OH group does not form any
hydrogen bond with the active site, which may explain the reason
that the predicted activity of the 4fsyn is not so good as that of the
that no thiazolidine derivative has been reported as neuraminidase
inhibitors, we chose 1a as the lead compound the lead compound
for further modification and optimization (shown in Fig. 2).
2. Chemistry
4fanti.
After all, the occupation of the phenyl group in the active site
confirms our indication that the aromatic group is suitable for
the pocket of the NA active site. And the docking result of 4f com-
pared with oseltmivir shown in Figure 4 also supports this
suggestion.
To contain the different substituents to interact with the four
binding pockets of the NA active site, we used the following
chemical modifications as shown in Scheme 1. Benzaldehydes with
different substituents and 2-furaldehyde were used to couple with
Cl
COOH
O
NH2
COOH
O
N
b
N
d
R1
R1
S
2
S
H
COOH
HOOC
HS
NH2
+
N
a
4
Ph
R1CHO
O
R1
S
N
c
COOH
3
R1
1
S
Scheme 1. Reagents: (a) NaHCO3, EtOH/H2O; (b) ClCH2COCl, NaHCO3/H2O; (c) PhCH2COCl, NaHCO3/H2O; (d) NH2ÁH2O; BOC2O, THF; HCl/EtOAc.

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Y. Liu et al. / Bioorg. Med. Chem. 19 (2011) 2342–2348
Table 1
Table 1 (continued)
The structures and in vitro NA inhibitory activity of the target compounds and
oseltamivir
Compound
R1
R2
IC50 (lM)
R2
N
3g
4a
PhCH2CO–
NH2CH2CO–
2.89
0.53
O
COOH
R1
S
OH
Compound
R1
R2
H
IC50 (lM)
4b
NH2CH2CO–
0.21
1a
21.3
COOH
OH
4c
4d
4e
NH2CH2CO–
NH2CH2CO–
NH2CH2CO–
0.28
0.81
1.02
1b
H
20.2
COOH
NC
1c
1d
1e
H
H
H
18.1
23.4
22.5
NO2
OH
NC
NO2
OH
4f
NH2CH2CO–
NH2CH2CO–
0.14
H3CO
O
4g
0.98
0.02
1f
H
12.3
43.1
H3CO
O
Oseltamivir carboxylate
1g
2a
H
ClCH2CO–
7.53
5.84
4. Conclusion
OH
A series of novel influenza neuraminidase inhibitors based on
thiazolidine core were synthesized and evaluated for their ability
to inhibit neuraminidase (NA) of influenza A virus.
Several compounds were shown to possess moderate influenza
NA inhibitory activity, although in all cases, measured activities
were lower than that of oseltamivir. The most potent compound
2b
ClCH2CO–
COOH
2c
2d
2e
ClCH2CO–
ClCH2CO–
ClCH2CO–
10.7
8.64
7.65
NC
of the series is compound 4f (IC50 = 0.14 lM), about sevenfold less
potent than oseltamivir. The binding of compound 4f in the active
site of NA showed that the four pockets of the active site of NA
were all occupied, although not so well as oseltamivir to establish
a consistent binding orientation and more potent activity.
In summary, our study indicated that thiazolidine derivatives
could show potent NA inhibitory activity and this finding could
be used to design novel influenza NA inhibitors that exhibit in-
creased activity based on thiazolidine ring. Additionally, in the fol-
lowing study, we will perform the separation of anti and syn
diastereomers to find more active compounds.
NO2
OH
2f
ClCH2CO–
7.92
H3CO
O
2g
3a
ClCH2CO–
PhCH2CO–
12.9
1.21
0.65
OH
5. Experimental
3b
PhCH2CO–
5.1. Synthetic methods and spectroscopic details
COOH
3c
3d
3e
PhCH2CO–
PhCH2CO–
PhCH2CO–
1.92
2.47
1.87
All the reactions were carried out by the standard techniques
for the exclusion of moisture. Solvents were distilled prior to use
and flash chromatography was performed using silica gel (60 Å,
200 300 mesh). All the reactions were monitored by thin-layer
chromatography on 0.25 mm silica gel plates (60GF-254) and visu-
alized with UV light, or iodine vapour. Melting points were ob-
tained on an electrothermal melting point apparatus and are
uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra
were determined on a Brucker Avace 300 spectrometer using TMS
as an internal standard. Chemical shifts are reported in delta (d)
units, parts per million (ppm) downfield from TMS. High-resolu-
tion mass spectral (HRMS) data are reported as m/z (relative
intensity).
NC
NO2
OH
3f
PhCH2CO–
1.62
H3CO

Y. Liu et al. / Bioorg. Med. Chem. 19 (2011) 2342–2348
2345
(m, 1.1H, 5-H), 3.834(q, J = 6.9 Hz, J = 2.1 Hz, 0.45H, 4-H), 4.213 (t,
J = 6 Hz, 0.55H, 4-H) 5.649 (s, 0.45H, 2-H), 5.838 (s, 0.55H, 2-H),
7.033–7.360 (m, 4H). HRMS: m/z calcd for C10H11NO3S [M+H]+
226.0538; found: 226.0532.
5.1.3. (2RS,4R)-2-(2-Carboxy-phenyl)-thiazolidine-4-carboxylic
acid (1c)
White solid. Yield: 90%. Mp 160–161 °C. 1H NMR (DMSO-d6,
300 MHz): 2.870–3.022 (m, 1.5H, 5-H), 3.124–3.360 (m, 0.25H, 5-
H), 3.906 (q, J = 6.9 Hz, J = 3 Hz, 0.75H, 4-H), 4.080 (t, J = 6.9 Hz,
0.25H, 4-H) 6.158 (s, 0.25H, 2-H), 6.508 (s, 0.75H, 2-H), 7.296–
7.780 (m, 4H). HRMS: m/z calcd for
C
11H11NO4S [M+H]+
254.0487; found: 254.0486.
5.1.4. (2RS,4R)-2-(4-Cyano-phenyl)-thiazolidine-4-carboxylic
acid (1d)
White solid. Yield: 96%. Mp 145–147 °C. 1H NMR (DMSO-d6,
300 MHz): 3.065–3.133 (m, 0.75H, 5-H), 3.284–3.396 (m, 1.25H,
5-H), 3.902 (q, J = 6.6 Hz, J = 2.1 Hz, 0.375H, 4-H), 4.140 (t,
J = 6 Hz, 0.625H, 4-H) 5.606 (s, 0.375H, 2-H), 5.807 (s, 0.625H, 2-
H), 7.598–7.854 (m, 4H). HRMS: m/z calcd for
C11H10N2O2S
[M+H]+ 235.0541; found: 235.0534.
5.1.5. (2RS,4R)-2-(2-Nitro-phenyl)-thiazolidine-4-carboxylic
acid (1e)
White solid. Yield: 96%. Mp 145–147 °C. 1H NMR (DMSO-d6,
300 MHz): 3.324–3.810 (m, 2H, 5-H), 5.088–5.198 (m, 1H, 4-H),
6.324 (s, 0.7H, 2-H), 6.768 (s, 0.3H, 2-H) 7.729–8.285 (m, 4H).
HRMS: m/z calcd for
C
10H10N2O4S [M+H]+ 255.0439; found:
255.0437.
5.1.6. (2RS,4R)-2-(2-Hydroxy-3-methoxy-phenyl)-thiazolidine-
4-carboxylic acid (1f)
Light yellow solid. Yield: 92%. Mp 137–138 °C. 1H NMR (DMSO-
d6, 300 MHz): 2.931–3.040 (m, 1H, 5-H), 3.171–3.368 (m, 1H, 5-H),
3.777–3.858 (m, 3H, –OCH3), 3.836 (t, 0.46H, 4-H), 4.207 (t,
J = 5.4 Hz, 0.54H, 4-H) 5.671 (s, 0.46H, 2-H), 5.865 (s, 0.54H, 2-H),
6.704–6.971 (m, 3H). HRMS: m/z calcd for C11H13NO4S [M+H]+
256.0645; found: 256.0643.
5.1.7. (2RS,4R)-Furan-2-yl-thiazolidine-4-carboxylic acid (1g)
Brown solid. Yield: 98%. Mp 140–142 °C. 1H NMR (DMSO-d6,
300 MHz): 2.965–3.381 (m, 2H, 5-H), 3.876 (q, J = 6.9 Hz,
J = 2.1 Hz, 0.33H, 4-H), 4.114 (t, J = 6.3 Hz, 0.67H, 4-H) 5.607 (s,
0.33H, 2-H), 5.742 (s, 0.67H, 2-H), 6.348 (d, J = 3.3 Hz, 0.67H),
6.382 (dd, J = 1.8 Hz, 3.3 Hz, 0.67H), 6.447 (dd, J = 1.8 Hz, 3.3 Hz,
0.33H), 6.508 (d, J = 3.3 Hz, 0.33H), 7.591 (d, J = 1.8 Hz, 0.67H),
7.663 (d, J = 1.8 Hz, 0.33H). HRMS: m/z calcd for C8H9NO3S
[M+H]+ 200.0381; found: 200.0388.
Figure 3. Detailed view of the docking result of compound 4f with some key amino
acid residues in the active site of neuraminidase (PDB ID: 2hu4) through the
Surflex-Dock module of Sybyl 8.1. The yellow lines and numbers show the potential
hydrogen bonds and bond length. The first one is 4fanti, and the second one is 4fsyn
.
5.1.1. (2RS,4R)-2-Phenyl-thiazolidine-4-carboxylic acid (1a)
To the solution of L-cysteine hydrochloride hydrate and NaHCO3
(1.1 equiv) in water (200 mL), benzaldehyde (1.1 equiv) in 95% eth-
anol (200 mL) was added in one portion. The reaction mixture was
stirred for 6 h. The product was filtered, washed with ethanol, and
dried to afford as a white solid. Yield: 95%. Mp 159–160 °C (lit.
159–160 °C). 1H NMR (DMSO-d6, 300 MHz): 3.049–3.166 (m,
0.9H, 5-H), 3.294–3.406 (m, 1.1H, 5-H), 3.902 (q, J = 7.2 Hz,
J = 1.5 Hz, 0.45H, 4-H), 4.234 (t, J = 4.8 Hz, J = 2.4 Hz, 0.55H, 4-H),
5.504 (s, 0.45H, 2-H), 5.669 (s, 0.55H, 2-H), 7.242–7.533 (m,
5H).HRMS: m/z calcd for C12H14N2O3S [M+H]+ 210.0588; found:
210.0587.
5.1.8. 3-(2-Chloro-acetyl)-(2RS,4R)-2-phenylthiazolidine-4-
carboxylic acid (2a)
To the solution of compound 1a with NaHCO3 (2 equiv) in
150 mL H2O, ClCH2COCl (1.2 equiv) was added dropwise to the
solution and stirred at room temperature until no insoluble mate-
rial existed. The mixture was adjusted to pH 8–9 with NaHCO3, and
washed with DCM (50 mL Â 3). The aqueous layer was then acidi-
fied with citric acid to pH 2–3, and extracted with DCM
(50 mL Â 3). The organic phase was dried and evaporated to give
the crude product, which was purified by column chromatography
to give the title compound.
Compounds 1b–1g were prepared following the general proce-
dure as described above.
White solid. Yield: 72.0%. Mp 121–123 °C, 1H NMR (CDCl3,
300 MHz): 3.389–3.410 (m, 2H, 5-H), 3.729–3.883 (m, 2H, Cl–
CH2), 5.151 (t, 1H, 4-H), 6.215 (s, 1H, 2-H), 7.354–7.630 (m, 5H).
HRMS: m/z calcd for C12H12ClNO4S [M+H]+ 286.0304; found:
286.0297.
5.1.2. (2RS,4R)-2-(2-Hydroxy-phenyl)-thiazolidine-4-carboxylic
acid (1b)
White solid. Yield: 95%. Mp 160–161 °C (lit. 167 °C). 1H NMR
(DMSO-d6, 300 MHz): 2.946–3.046 (m, 0.9H, 5-H), 3.176–3.367

2346
Y. Liu et al. / Bioorg. Med. Chem. 19 (2011) 2342–2348
Figure 4. Docking result of compound 4f and oseltamivir (4f is displayed by atom type while oseltamivir is shown in lime line). The active pocket is displayed using Channel
pattern (Type: Cavity Depth) with the program MOLCAD Surfaces of Sybyl8.1. The first one is 4fanti, and the second one is 4fsyn
.
Compounds 2b–2g were prepared following the general proce-
dure as described above.
6.632 (d, 1H), 7.428 (d, 1H). HRMS: m/z calcd for C13H14NO5S
[M+H]+ 329.0125; found: 329.0117.
5.1.9. 3-(2-Chloro-acetyl)-(2RS,4R)-2-(2-hydroxy-phenyl)-thiaz-
olidine-4-carboxylic acid (2b)
5.1.11. 3-(2-Chloro-acetyl)-(2RS,4R)-2-(4-cyano-phenyl)-thiazo-
lidine-4-carboxylic acid (2d)
Whitle solid. Yield: 78.5%. Mp 139–141 °C, 1H NMR (CDCl3,
300 MHz): 2.998–3.482 (m, 4H, 5-H, Cl–CH2), 4.656–5.223 (m,
1H, 4-H), 6.332 (s, 0.25H, 2-H), 6.427 (s, 0.75H, 2-H), 6.667–6.862
(m, 2H), 7.016–7.175 (m, 1H), 7.474 (d, J = 7.2 Hz, 0.25H), 7.885
(d, J = 7.2 Hz, 0.75H). HRMS: m/z calcd for C12H12ClNO4S [M+H]+
302.0254; found: 302.0252.
Whitle solid. Yield: 71.5%. Mp 68–70 °C, 1H NMR (CDCl3,
300 MHz): 3.276–3.588 (m, 2H, 5-H), 4.096–4.167 (m, 2H, Cl–
CH2–), 5.088–5.111 (m, 0.7H, 4-H), 5.133–5.209 (m, 0.3H, 4-H),
6.273 (s, 1H, 2-H), 7.639–7.816 (m, 4H). HRMS: m/z calcd for
C
13H11ClN2O3S [M+H]+ 311.0257; found: 311.0249.
5.1.12. 3-(2-Chloro-acetyl)-(2RS,4R)-2-(2-nitroh-phenyl)-thiazo-
lidine-4-carboxylic acid (2e)
5.1.10. 3-(2-Chloro-acetyl)-(2RS,4R)-2-(2-carboxy-phenyl)-thia-
zolidine-4-carboxylic acid (2c)
Whitle solid. Yield: 70.1%. Mp 71–73 °C, 1H NMR (CDCl3,
300 MHz): 3.324–4.161 (m, 4H, 5-H, Cl–CH2–), 5.088–5.109 (m,
0.65H, 4-H), 5.198–5.132 (m, 0.35H, 4-H), 6.324 (s, 1H, 2-H),
7.729–7.75 (m, 0.7H), 7.863–7.889 (m, 1.3H), 8.161–8.187 (m,
Whitle solid. Yield: 70.8%. Mp 117–118 °C, 1H NMR (CDCl3,
300 MHz): 3.112–3.434 (m, 2H, 5-H), 4025–4.223 (m, 2H, Cl–
CH2–), 4.964 (t, J = 8.1 Hz, 1H, 4-H), 6.273 (s, 1H), 6.353 (dd, 1H),

Y. Liu et al. / Bioorg. Med. Chem. 19 (2011) 2342–2348
2347
0.7H), 8.258–8.285 (m, 1.3H). HRMS: m/z calcd for C12H11ClN2O5S
5-H), 5.041 (t, 1H, J = 8.1 Hz, 4-H), 6.094 (s, 0.75H, 2-H), 6.296 (s,
0.25H, 2-H), 7.041–7.715 (m, 9H). HRMS: m/z calcd for
C
[M+H]+ 331.0155; found: 331.0149.
18H16N2O5S [M+H]+ 373.0858; found: 373.0847.
5.1.13. 3-(2-Chloro-acetyl)-(2RS,4R)-2-(2-hydroxy-3-methoxy-
phenyl)-thiazolidine-4-carboxylic acid (2f)
5.1.20. 3-Phenylacetyl-(2RS,4R)-2-(2-hydroxy-3-methoxy-phen-
yl)-thiazolidine-4-carboxylic acid (3f)
Whitle solid. Yield: 70.5%. Mp 130–132 °C, 1H NMR (CDCl3,
300 MHz): 3.325–3.464 (m, 2H, 5-H), 3.773–3.818 (d, 1H,
J = 13.5 Hz, Cl–CH2–), 3.916 (s, 3H, –OCH3), 4.010–4.055 (d, 1H,
J = 13.5 Hz, Cl–CH2–), 5.043 (t, 1H, J = 6.9 Hz, 4-H), 6.486 (s, 1H,
2-H), 6.849–6.942 (m, 2H), 7.352 (dd, 1H). HRMS: m/z calcd for
Whitle solid. Yield: 88.9%. Mp 148–150 °C, 1H NMR (CDCl3,
300 MHz): 3.244–3.685 (m, 4H, 5-H, Ph–CH2–), 3.914 (s, 3H, –
OCH3), 4.981 (t, 1H, J = 6.9 Hz, 4-H), 5.977(s, 0.5H, 2-H), 6.473 (s,
0.5H, 2-H), 6.840–7.316 (m, 8H). HRMS: m/z calcd for C19H19NO5S
[M+H]+ 374.1062; found: 374.1053.
C
13H14ClNO5S [M+H]+ 332.0359; found: 332.0357.
5.1.14. 3-(2-Chloro-acetyl)-2-(2RS,4R)-furan-2-yl-thiazolidine-
4-carboxylic acid (2g)
5.1.21. 3-Phenylacetyl-2-(2RS,4R)-furan-2-yl-thiazolidine-4-
carboxylic acid (3g)
White solid. Yield: 71.2%. Mp 140–142 °C, 1H NMR (CDCl3,
300 MHz): 3.385–3.406 (m, 2H, 5-H), 3.729–3.885 (m, 2H, Cl–
CH2–), 5.125–5.167 (m, 1H, 4-H), 6.353 (s, 1H, 2-H), 6.273 (dd,
1H, J = 1.8 Hz, 3.3 Hz), 6.632 (d, J = 3.3 Hz, 1H), 7.428(d, J = 1.8 Hz,
1H). HRMS: m/z calcd for C10H10ClNO4S [M+H]+ 271.0097; found:
276.0094.
Whitle solid. Yield: 89.2%. Mp 163–165 °C, 1H NMR (CDCl3,
300 MHz): 3.302–3.366 (m, 2H, Ph–CH2–), 3.747–3.776 (m, 2H,
5-H), 4.941 (t, 1H, J = 8.1 Hz, 4-H), 6.162 (s, 1H, 2-H), 6.335 (dd,
J = 3 Hz, J = 3.3 Hz, 1H), 6.632 (d, J = 3 Hz, 1H), 7.177–7.411 (m,
6H). HRMS: m/z calcd for C16H15NO4S [M+H]+ 318.0800; found:
318.0793.
5.1.15. 3-Phenylacetyl-(2RS,4R)-2-phenylthiazolidine-4-carbo-
xylic acid (3a)
5.1.22.3-(2-Amino-acetyl)-(2RS,4R)-2-phenylthiazolidine-4-car-
boxylic acid (4a)
To the solution of compound (2a) with NaHCO3 (2 equiv) in
150 mL H2O, PhCH2COCl (1.2 equiv) was added dropwise to the
solution and stirred at room temperature until no insoluble mate-
rial existed. The mixture was adjusted to pH 8–9 with NaHCO3, and
washed with DCM (50 mL Â 3). The aqueous layer was then acidi-
fied with citric acid to pH 2–3, and extracted with DCM
(50 mL Â 3). The organic phase was dried and evaporated to give
the crude product, which was purified by column chromatography
to give compound. White solid. Yield: 77.1%. Mp 163–165 °C, 1H
NMR (CDCl3, 300 MHz): 3.241–3.470 (m, 2H, 5-H), 3.354 (m, 2H,
Ph–CH2–), 5.078 (t, 1H, J = 6.9 Hz, 4-H), 6.087 (s, 1H, 2-H), 7.063–
The crude product 2a was dissolved in 50 mL NH3ÁH2O and
mixed for 3 days. Then the mixture was evaporated and dissolved
in 50 mL distilled water with NaHCO3 (2 equiv), then BOC2O
(1.1 equiv) in 50 mL THF was added dropwise to it. Stir at room
temperature for 12 h. The mixture was evaporated and acidified
with 1 M citric acid to pH 2–3, then extracted with EtOAC. The
combined organic phase was dried, evaporated and purified by col-
umn chromatography to give the Boc-protected thiazolidine,
which was added to 20 mL HCl/EtOAc, and stirred overnight. The
precipitate was collected through filtration and washed with EtOAc
to obtain the product.
7.507 (m, 9H). HRMS: m/z calcd for
C
18H17NO3S [M+H]+
Yield: 45.3%. Mp 159–161 °C, 1H NMR (H2O-d6, 300 MHz):
3.171–3.295 (m, 1.2H, 5-H), 3.371–3.941 (m, 2.8H, 5-H, NH2–
CH2–), 4.954–4.997 (m, 1H, 4-H), 6.137, 6.145 (2s, 1H, 2-H),
7.220–7.404 (m, 3H), 7.447–7.467 (m, 1H), 7.571–7.593 (m, 1H).
HRMS: m/z calcd for C12H14N2O3S [M+H]+ 267.0785; found:
267.0793.
328.1007; found: 328.1005.
Compounds 3b–3g were prepared following the general proce-
dure as described above.
5.1.16. 3-Phenylacetyl-(2RS,4R)-2-(2-hydroxy-phenyl)-thiazoli-
dine-4-carboxylic acid (3b)
Compounds 4b–4g were prepared following the general proce-
dure as described above.
White solid. Yield: 82.5%. Mp 152–154 °C, 1H NMR (CDCl3,
300 MHz): 3.209–3.668 (m, 4H, 5-H, Ph–CH2–), 4.990–5.032 (m,
1H, 4-H), 6.338, 6.405 (2s, 1H, 2-H), 6.821–6.929 (m, 2H), 7.024–
7.260 (m, 6H), 7.563 (d, J = 7.5 Hz, 1H). HRMS: m/z calcd for
5.1.23. 3-(2-Amino-acetyl)-(2RS,4R)-2-(2-hydroxy-phenyl)-thia-
zolidine-4-carboxylic acid (4b)
C
18H17NO4S [M+H]+ 344.0957; found: 344.0959.
Yield: 41.2%. Mp 125–127 °C, 1H NMR (H2O-d6, 300 MHz):
3.732–3.810 (m, 2H, 5-H), 3.981–4.188 (m, 2H, NH2–CH2–),
5.401–5.414 (m, 1H, 4-H), 6.343, 6.357 (2s, 1H, 2-H), 6.838–6.932
(m, 2H), 7.144–7.171 (m, 1H), 7.417–7.447 (m, 1H). HRMS: m/z
calcd for C12H14N2O4S [M+H]+ 283.0753; found: 283.0746.
5.1.17. 3-Phenylacetyl-(2RS,4R)-2-(2-carboxy-phenyl)-thiazoli-
dine-4-carboxylic acid (3c)
Whitle solid. Yield: 86.8%. Mp 185–187 °C, 1H NMR (CDCl3,
300 MHz): 3.165–3.627 (m, 4H, 5-H, Ph–CH2), 4.565–4.181 (m,
1H, 4-H), 6.153, 6.216 (2s, 1H, 2-H), 6.990–8.081 (m, 9H). HRMS:
m/z calcd for C19H17NO5S [M+H]+ 372.0906; found: 372.0910.
5.1.24. 3-(2-Amino-acetyl)-(2RS,4R)-2-(2-carboxy-phenyl)-thia-
zolidine-4-carboxylic acid (4c)
Yield: 43.5%. Mp 156–158 °C, 1H NMR (H2O-d6, 300 MHz):
3.739–3.807 (m, 2H, 5-H), 3.974–4.082 (m, 2H, NH2–CH2–),
5.128–5.324 (m, 1H, 4-H), 6.306, 6.357 (2s, 1H, 2-H), 6.807–8.200
(m, 4H). HRMS: m/z calcd for C13H14N2O5S [M+H]+ 311.0701;
found: 311.0703.
5.1.18. 3-Phenylacetyl-(2RS,4R)-2-(4-cyano-phenyl)-thiazoli-
dine-4-carboxylic acid (3d)
White solid. Yield: 89.0%. Mp 117–119 °C, 1H NMR (CDCl3,
300 MHz): 3.234–3.833 (m, 4H, 5-H, Ph–CH2–), 5.035–5.086 (m,
1H, 4-H), 6.141 (s, 0.75H, 2-H), 6.337 (s, 0.25H, 2-H), 7.041–7.772
(m, 9H). HRMS: m/z calcd for C19H16N2O3S [M+H]+ 353.0960;
found: 353.0966.
5.1.25. 3-(2-Amino-acetyl)-(2RS,4R)-2-(4-cyano-phenyl)-thiaz-
olidine-4-carboxylic acid (4d)
Yield: 42.9%. Mp 131–134 °C, 1H NMR (H2O-d6, 300 MHz):
3.245–3.313 (m, 0.8H, 5-H) 3.851–4.053 (m, 2.2H, 5-H, NH2–
CH2–), 4.869–4.981 (m, 1H, 4-H), 6.234, 6.264 (2s, 1H, 2-H),
7.693–7.919 (m, 4H). HRMS: m/z calcd for C13H13N3O3S [M+H]+
292.0756; found: 292.0752.
5.1.19. 3-Phenylacetyl-(2RS,4R)-2-(2-nitroh-phenyl)-thiazoli-
dine-4-carboxylic acid (3e)
White solid. Yield: 87.5%. Mp 124–126 °C, 1H NMR (CDCl3,
300 MHz): 3.221–3.370 (m, 2H, Ph–CH2–), 3.519–3.821 (m, 2H,

2348
Y. Liu et al. / Bioorg. Med. Chem. 19 (2011) 2342–2348
5.1.26. 3-(2-Amino-acetyl)-(2RS,4R)-2-(2-nitroh-phenyl)-thiazo-
lidine-4-carboxylic acid (4e)
and created based on the previous ligand oseltamivir by the Sur-
flex-Dock Protomol Generation Programme, and other parameters
were set as default.
Yield: 43.8%. Mp 111–113 °C, 1H NMR (DMSO-d6, 300 MHz):
3.194–3.296 (m, 1.1H, 5-H), 3.383–3.997 (m, 2.9H, 5-H, NH2–
CH2–), 4.923–4.971 (m, 0.45H, 4-H), 5.011–5.041 (m, 0.55H, 4-H),
6.173 (s, 0.45H, 2-H), 6.227 (s, 0.55H, 2-H), 7.533–7.795 (m, 4H).
Acknowledgments
HRMS: m/z calcd for
C
12H13N3O5S [M+H]+ 312.0654; found:
We thank Yumei Yuan, Zhaopeng Liu and Xinguang Liu for their
contributions to this work. This work was supported by the Na-
tional Nature Science Foundation of China (Grant No. 36072541).
312.0650.
5.1.27. 3-(2-Amino-acetyl)-(2RS,4R)-2-(2-hydroxy-3-methoxy-
phenyl)-thiazolidine-4-carboxylic acid (4f)
References and notes
Yield: 44.5%. Mp 152–155 °C, 1H NMR (H2O-d6, 300 MHz):
3.204–3.305 (m, 2H, 5-H), 3.702 (s, 3H), 3.996–4.107 (m, 2H,
NH2–CH2–), 5.407 (m, 1H, 4-H), 6.280, 6.331 (2s, 1H, 2-H), 6.829–
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The binding modes were clarified by docking based on the co-
crystal complex of N1 neuraminidase in complex with correspond-
ing ligand oseltamivir downloaded from PDB database (http://
www.rcsb.org/pdb/home/home.do), the Protein Data Bank ID
codes were 2hu4.pdb. Before docking, the pre-existing ligand was
extracted out and hydrogen atoms and charges were added. The
docking studies were performed using the Surflex-Dock module
of Sybyl 8.1, and the maximum number of poses per ligand was
set to 10. The active site of the protein was automatically explored
33. http://www.nisn.org/documents/Zambon—VIRGIL IC50 SOP.pdf.