Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: Structure-activity relationship study

Article abstract of DOI:10.1016/j.ejmech.2013.05.005

This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(-pyrrolidone-3-yl)-2-benzothiazole (8; K_i = 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CL^pro. In particular, compounds 26m and 26n exhibited good inhibitory activities with K_i values of 0.39 and 0.33 μM, respectively. These low-molecular weight compounds are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compound 26m with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1′ position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the N-arylglycine unit and a backbone hydrogen bond donor at the S3-position.

Full text of DOI:10.1016/j.ejmech.2013.05.005

European Journal of Medicinal Chemistry 65 (2013) 436e447
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, and biological evaluation of novel dipeptide-type
SARS-CoV 3CL protease inhibitors: Structureeactivity relationship
study
Pillaiyar Thanigaimalai a, Sho Konno a, Takehito Yamamoto a, Yuji Koiwai a,
Akihiro Taguchi a, Kentaro Takayama a, Fumika Yakushiji a, Kenichi Akaji b, Yoshiaki Kiso b,
Yuko Kawasaki c, Shen-En Chen c, Aurash Naser-Tavakolian c, Arne Schön c, Ernesto Freire c,
,
Yoshio Hayashi a
*
a Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan
b Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan
c Department of Biology, Johns Hopkins University, Baltimore, MD, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV
3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(-
pyrrolidone-3-yl)-2-benzothiazole (8; Ki ¼ 4.1 nM), in which the P3 valine unit was substituted with a
variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good
inhibitory activities against 3CLpro. In particular, compounds 26m and 26n exhibited good inhibitory
Received 11 March 2013
Received in revised form
20 April 2013
Accepted 7 May 2013
Available online 20 May 2013
activities with Ki values of 0.39 and 0.33 mM, respectively. These low-molecular weight compounds are
attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical
profiles. Docking studies were performed to model the binding interaction of the compound 26m with
the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent
inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a ben-
Keywords:
SARS
SARS-CoV 3CL protease
Peptidomimetics
Dipeptide
zothiazole warhead at the S10 position, (ii) a
g-lactam unit at the S1-position, (iii) an appropriately hy-
Cysteine protease inhibitors
Docking study
drophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the N-arylglycine unit
and a backbone hydrogen bond donor at the S3-position.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
complex activity [8]. This enzyme is indispensable for viral repli-
cation and infection processes, making it an ideal target for the
Since its first appearance in Southern China in late 2002, severe
acute respiratory syndrome (SARS) has been recognized as a global
threat [1,2]. Its rapid and unexpected spread to 32 countries has
affected more than 8000 individuals and caused nearly 800
(w10%) fatalities worldwide within a few months [1e3]. The
causative SARS pathogen is a novel coronavirus, SARS-CoV [4,5].
SARS-CoV is a positive-strand RNA virus with a genome sequence
that is only moderately homologous to other known coronaviruses
[6,7]. SARS-CoV encodes a chymotrypsin-like protease (3CLpro),
also referred to as the main protease (Mpro), which plays a pivotal
role in processing viral polyproteins and controlling replicase
design of antiviral therapies. The 3CLpro active site contains a
catalytic dyad in which a cysteine residue (Cys145) acts as a
nucleophile and a histidine (His41) residue acts as a general acid
or base [9,10]. The SARS epidemic was successfully controlled in
2003; however, the potential reemergence of pandemic SARS-CoV
continues to pose a risk, and new strains of SARS could potentially
be more virulent than the strains that contributed to the 2003
outbreak. Since 2003, two additional human coronaviruses, NL63
and HKU1, have been identified in patients around the world
[11,12]. Recently, a new SARS-like virus, HCoV-EMC, was identified
in at least two individuals, one of whom died [13]. Very recently,
the first case of a fatal respiratory illness similar to the deadly
SARS was confirmed in Britain [14]. The World Health Organiza-
tion (WHO) has announced that it is closely monitoring the situ-
ation and is working to “ensure a high degree of preparedness,
* Corresponding author. Tel./fax: þ81 42 676 3275.
E-mail address: yhayashi@toyaku.ac.jp (Y. Hayashi).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.005

P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
437
should the new virus be found to be sufficiently transmissible to
cause a community outbreak”. There is a significant need to
develop anti-SARS agents that are capable of treating this poten-
tially fatal respiratory illness. Several reports of crystalline forms
of the SARS-CoV 3CLpro protein bound to hexapeptidyl chlor-
omethyl ketone inhibitors have been reported [6,9], and numerous
peptidic structures have been reported in the context of targeted
antiviral drug design [15e22]. The reported protease inhibitors are
generally peptidic in nature, often five to three residues in length,
and bear a reactive warhead group at the C-terminus which forms
an interaction with the protease catalytic Cys145 (Fig. 1, 1e9). Two
of these compounds (8 and 9), recently described in a separate
report from our group [22], exhibited excellent potent inhibitory
activities with Ki values of 4.1 and 3.1 nM, respectively. These
peptidic inhibitors provided valuable insight into the design con-
straints for this system and quickly led to the development of non-
peptidic small molecule inhibitors (Fig. 1, 10e17) [23e30]. These
small molecular inhibitors generally showed moderate to good
activities.
Extending our studies toward the development of new anti-
SARS agents, we now report the design, synthesis, and evaluation
of a series of low-molecular weight dipeptide-type compounds in
which the P3 valine unit is removed from the previous lead Z-Val-
Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole compound (8, Fig. 1). A
preliminary SAR study led to the identification of inhibitors with
moderate to good inhibitory activities. In particular, compounds
26m and 26n exhibited potent inhibitory activities with Ki values of
0.39 and 0.33 mM, respectively. The binding interactions of 26m
were predicted using molecular modeling studies. We describe the
results of these extensive studies in detail, including the design,
synthesis, molecular modeling, and biological evaluation of a series
of SARS-CoV 3CLpro inhibitors.
2. Results and discussion
2.1. Synthesis
The synthesis of the title inhibitors was achieved through a
coupling reaction involving two key fragments, as shown in
Scheme 1. One of the key fragment intermediates (19) was syn-
thesized from the amino acid esters 18 with either corresponding
carboxylic acids via 1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide hydrochloridee1-hydroxybenzotriazole (EDC$HCleHOBt)
Recently, we performed a structureeactivity relationship study
based on the lead compound, Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-
thiazole (7) [21]. This study led to the discovery of the potent
compounds 8 and 9, with Ki values in the low nanomolar
range [22].
O
NH2
O
N
O
H2N
N
OH
OBn
O
O
O
O
O
O
O
O
O
H
N
H
N
H
H
N
H
N
H
N
H
O
N
O
CF3
N
N
H
N
H
N
H
Cl
N
H
N
H
N
H
N
H
N
HN
H
O
O
O
O
O
O
O
O
O
O
OH
NO2
NH
1 (CMK)
2
3
IC50 = 10 µM
IC50 = 0.60 µM
O
O
NH
NH2
OH
N
H
O
O
O
O
O
O
O
O
H
N
H
H
O
O
N
N
N
N
O
N
N
H
O
N
H
N
H
O
N
H
N
H
H
H
O
O
O
O
O
O
O
4
5
inact/K = 1900 M-1s-1
6
IC50 = 9.1 µM; K = 0.66 µM
K
i
i
IC50 = 0.065 µM
O
O
O
NH
NH
NH
O
O
O
O
O
O
H
S
N
S
S
N
H
N
H
N
N
O
N
N
O
N
H
N
H
O
N
N
H
N
N
H
H
H
O
O
O
O
O
O
7
8
9
K = 2.2 µM
i
IC50 = 1.7 µM; K = 0.0041 µM
K = 0.0031 µM
i
i
O
O
Cl
Cl
Cl
O
O
H2N
H
O
O
O
O
O
N
N
H
N
N
S
N
HN
10
11
IC50 = 323 µM
12
13
K
i
= 3.2 µM
K = 0.065 µM
IC50 = 0.37 µM
i
O
O
O
HO
NC
O
N
NH
N
N
H
N
O
O
N
F
N
N
S
N
H
O
N
H
N
Cl
NO2
14
= 0.0075 µM
15
IC50 = 6.1 µM
16
17
IC50 = 17.2 µM
K
i
IC50 = 6.8 µM
Fig. 1. Representative peptidomimetics (1e9) and small molecular (10e17) 3CLpro inhibitors highlighting reactive warhead groups (red).

438
P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
1.
H2N
O
O
O
i
H
N
H
N
ii
R1
R2
R1
R2
O
O
OH
R
O
R
O
R
18a-f
19a-u
20a-u
O
2.
O
NH
COOMe
NH
Ref. 20-22, 31
iii, iv
O
OMe
O
OMe
N
BocHN
BocHN
vi, vii
BocHN
O
O
22
O
23
21
O
NH
NH
v
O
H
N
R2
R3
R3
N
BocHN
H
O
R
O
O
25-27
24
Scheme 1. Reagents and conditions: i) EDC$HCl, HOBt$H2O, TEA/DMF (if reactant is a carboxylic acid) or Et3N/CH2Cl2 (if reactant is a benzyloxycarbonyl chloride), 0 ꢁCert; ii) TFA/
H2O, CH2Cl2 (if R1 ¼ tert-butyl unit), or LiOH$H2O, THF/H2O, (if R1 ¼ methyl unit); iii) 4M NaOH, MeOH; iv) HN(OMe)Me$HCl, EDC$HCl, HOBt$H2O, Et3N/DMF, 0 ꢁCert; v) n-BuLi,
THF, ꢀ78 ꢁC (if R3 ¼ benzothiazole) or LDA, THF, ꢀ78 ꢁC (if R3 ¼ 5-arylated thiazoles); vi) TFA/H2O (10:1), CH2Cl2; vii) 20, HBTU, DIPEA/DMF, 0 ꢁCert followed by HPLC purification.
Note: The substituents ReR3 are indicated in Tables 1e3
mediated coupling in the presence of triethylamine (TEA) in DMF or
acid chlorides in the presence of TEA in dichloromethane (CH2Cl2).
The resulting N-protected amino acid esters (19) were conse-
quently deprotected in the presence of TFA or lithium hydrox-
ide$water (LiOH$H2O) to afford the N-protected amino acids (20),
which were used directly in the subsequent step.
reaction was monitored by analytical HPLC, and the cleavage rates
were calculated from the decrease in the substrate peak area.
Tables 1e4 report the Ki or IC50 values as the mean of 3 independent
experiments.
In an effort to develop low-molecular weight peptidic SARS-CoV
3CLpro inhibitors, we designed a series of dipeptide-type inhibitors
based on the previous potent lead compound 8 (see Fig. 1). In a first
attempt, the P3 valine region was removed and the P4-
benzyloxycarbonyl (Cbz) in 8 was replaced with a series of small
moieties with sizes similar to that of the P3 valine group in 8. The
inhibitory activities of the resulting dipeptidic compounds bearing
The
approach similar to the syntheses reported previously [21,22,31].
Accordingly, the optically pure -glutamic acid ester 21 was con-
verted to the -lactam ester 22 by treatment with bromoacetoni-
g-lactam-thiazoles (24) were synthesized using an
L
g
trile, followed by reduction with PtO2 (5%) and cyclization. The
resulting ester 22 was hydrolyzed in the presence of 4 M NaOH in
methanol to yield the corresponding acid, which was coupled to
N,O-dimethylhydroxylamine via the EDCeHOBt method to afford
the Weinreb amide 23. The Weinreb amide 23 was then coupled to
the appropriate thiazoles in the presence of n-butyl lithium (n-
a valine mimic, isopentanoyl (25a; Ki ¼ 5.9
carbonyl (25b; Ki ¼ 23 M), were dramatically reduced compared to
the activity of 8 (Ki ¼ 0.0041 M) [22]; however, the introduction of
Cbz (25c; Ki and IC50 ¼ 0.46 and 21.0 M) as a P3 moiety resulted in
mM) or tert-butoxy
m
m
m
a 12-fold or 50-fold activity increase for 25a or 25b, respectively,
although the potency was reduced relative to the value for the
tripeptidic lead 8. This result suggested that the Cbz group, which
was introduced in place of the P3 scaffold in the dipeptidic 25c,
conveyed appreciable activity; therefore, compound 25c could
serve as a lead for further optimization steps. By retaining the P3
Cbz moiety in 25c, we examined the relevance of the leucine res-
idue (or isobutyl unit) for P2 substrate selectivity in comparison
with a variety of its congeners. Accordingly, a series of isosteres was
BuLi) or lithium diisopropylamide (LDA) at ꢀ78 ꢁC to afford the
g-
lactam-thiazoles 24. The Boc protecting group was then removed,
and the resulting intermediate was subsequently reacted with the
above-mentioned N-protected amino acids 20 in the presence of O-
benzotriazole-N,N,N0,N0-tetramethyluroniumhexafluoro phosphate
(HBTU) and DIPEA in DMF to afford the title compounds 25e27. All
compounds were purified by reverse phase HPLC and characterized
by 1H and 13C NMR, and mass spectrometry analysis. The purity of
each compound exceeded 90e95%.
introduced, including n-butyl (25d; Ki ¼ 1.60
Ki ¼ 1.71 M), sec-butyl (25f; Ki ¼ 29.0 M), 3-methyl(thio)ethyl
(25g; Ki ¼ 9.40 M), and benzyl (25h; Ki ¼ 1.20 M). The com-
mM), isopropyl (25e;
m
m
m
m
2.2. Biological activity
pounds bearing n-butyl (25d), isopropyl (25e), or benzyl (25h)
groups exhibited reasonable inhibitory activities, although the
potencies were reduced by a factor of 3e4 relative to the value for
25c. The inhibitory activity of the compound bearing sec-butyl (25f)
or 3-methyl(thio)ethyl (25g) was severely reduced compared to the
activity of 25c. Therefore, the leucine residue (or isobutyl group)
was more selective and appropriate as a P2 group in 25c, providing
enhanced inhibitory activity.
The compounds were subjected to a fluorometric protease
inhibitory assay against SARS-CoV 3CLpro, as described previously
[32,33]. Briefly, the kinetic parameters were determined at a con-
stant substrate concentration, and the inhibitor concentrations
were varied to assess the Ki values [22]. The IC50 values
were determined only for certain potent inhibitors, based on
the apparent decrease in the substrate concentration (H-Thr-Ser-
Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-NH2) upon digestion by
R188I SARS 3CLpro, as described previously [19,34]. The cleavage
The P3 moiety of the lead compound 25c was examined by
introducing a wide variety of substituents, such as aryl (or

P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
439
Table 1
SARS-CoV 3CLpro inhibitory activities (Ki) of 25aeh.
Entry no.
Inhibitors
Ki (m
M)
Entry no.
Inhibitors
Ki (mM)
25a
5.90
25e
1.71
25b
25c
23.0
25f
29.0
0.46
1.60
25g
9.40
1.20
25d
25h
heteroaryl) acetyls and propionyls, arylacrylyls, aryloxyacetyls, and
N-arylglycyls. Initially, the aryl (or heteroaryl) acetyls and pro-
pionyls were introduced, including phenylacetyl (26a;
activities compared to the lead 25c. The compound bearing an N-
(2-methoxyphenyl)glycyl (26n) was identified as the most potent
inhibitor in the present study. This result suggested that the
hydrogen bonding properties of the amino group on the N-aryl-
glycyl moiety might have contributed to the improvement in ac-
tivity (see Fig. 2B).
Ki ¼ 3.20
43
M), 4-methoxyphenylpropionyl (26c; Ki ¼ 0.62
idine-3-propionyl (26d; Ki ¼ 7.4 M). Among the compounds, a 4-
m
M), 4-methoxyphenylacetyl (26b; Ki and IC50 ¼ 0.42 and
m
m
M), and pyr-
m
methoxyphenylacetyl derivative (26b) exhibited potent inhibitory
activity; the phenylacetyl (26a) and pyridine-3-propionyl (26d)
derivatives in particular displayed low inhibitory activities. There-
fore, the 4-methoxyphenylacetyl moiety provided a good alterna-
tive to the P3 Cbz group in 25c. We next introduced the arylacrylyls,
The P10 moiety was examined next by varying the 5-substituted
thiazoles (27aed). The inhibitory activities of compounds 27ae
d are illustrated in Table 3. Inhibitor 27a exhibited an inhibitory
activity comparable to that of 25c. The other 5-arlylated thiazoles
(27bed) generally exhibited very low inhibitory activities
compared to 25c. These studies confirmed that the benzothiazole
unit was more suitable as a warhead group on the P10 moiety in 25c.
including cinnamoyl (26e; Ki ¼ 0.69
m
M), 4-methoxycinnamoyl
M), and 3,4-dimethoxycinnamoyl (26g;
M). These compounds showed moderate inhibitory ac-
(26f; Ki
Ki ¼ 1.30
¼
0.70 m
m
tivities relative to the lead 25c; thus, the acrylyls were not
considered further as P3 moieties in the context of 25c.
2.3. Molecular docking study
A previous report describing a SAR study of the tripeptidomi-
metic 7 (see Fig. 1) as a SARS inhibitor revealed that the introduc-
tion of an aryloxyacetyl at the N-terminal position (the P4 position)
appeared to significantly enhance the inhibitory activity
against SARS-CoV 3CLpro [22]. Therefore, we introduced a series of
aryloxyacetyls, including phenoxyacetyl (26h; Ki and IC50 ¼ 0.56
The binding mode of compound 26m with 3CLpro was simulated
using a molecular docking program as described previously [22].
We examined the molecular docking of the potent active com-
pound 26m in comparison with docking of the tripeptidic 9 and a
structurally similar ligand, the docking structure of which has been
elucidated previously by X-ray crystallography (PDB ID: 1WOF,
and 24
hydroxyphenoxyacetyl (26j; Ki
aminophenoxyacetyl (26k; Ki ¼ 0.84
m
M), 4-methoxyphenoxyacetyl (26i; Ki ¼ 1.56
8.4 M), and 3-dimethyl
M) as P3 groups in place of
m
M), 4-
Ki ¼ 10.7
mM) (Fig. 2) [35]. Several minimization processes were
¼
m
performed using the MMFF94X force field to model the solvation
environment surrounding the inhibitor. A molecular simulation
was then performed. As shown in Fig. 2A, the P10eP2 moieties in
26m, in the tripeptidic compound 9, and in the original ligand
interacted with the same region of the protease. Interestingly, the
aminoacetyl group on the N-(3-methoxyphenyl)glycyl group in
26m mimicked the P3 valine moieties of the tripeptidomimetic 9
and the reported ligand. The interactions of compound 26m with
the protein are described in detail in Fig. 2B. Particularly, a notable
hydrogen bonding involved between the amino group (eNH) of P3
N-(3-methoxyphenyl)glycine with a backbone amino acid residue
Glu166 of 3CLpro was observed to have relatively potent inhibition
than other inhibitors presented in the study against SARS-CoV
m
the Cbz group in 25c. Compounds 26h and 26k exhibited compa-
rable activities to 25c; however, the other derivatives (26i and 26j)
displayed reduced inhibitory activities relative to the lead com-
pound 25c. These results suggested that the introduction of an
aryloxyacetyl P3 group in the dipeptidic 25c did not appreciably
improve the inhibitory activity against 3CLpro. We also introduced
several N-arylglycyls, including N-(4-methoxyphenyl)glycyl (26l;
Ki ¼ 3.20
and 10.0
IC50 ¼ 0.33 and 14.0
compounds 26m and 26n displayed relatively potent inhibitory
m
M), N-(3-methoxyphenyl)glycyl (26m; Ki and IC50 ¼ 0.39
M), and N-(2-methoxyphenyl)glycyl (26n; Ki and
M). The results of these studies revealed that
m
m

440
P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
Table 2
SARS-CoV 3CLpro inhibitory activities (Ki) of 26aen.
Entry No.
Inhibitors
Ki (
m
M)
Entry No.
Inhibitors
Ki (mM)
26a
3.20
0.42
0.61
7.4
26h
0.56
1.56
8.4
26b
26c
26d
26e
26f
26i
26j
26k
26l
0.84
3.20
0.39
0.69
0.70
26m
26g
1.30
26n
0.33
3CLpro. The essential nature of this hydrogen bond will be explored
in further optimization studies of dipeptide-type SARS-CoV 3CLpro
inhibitors.
as appropriate P2 and P10 moieties, respectively, in 25c. Accord-
ingly, compounds 26m and 26n were recognized as potent in-
hibitors in the present study. These potent dipeptidic inhibitors
provide attractive leads, which are undergoing further structural
modifications in an effort to improve the pharmaceutical profiles.
These processes are underway and will be reported in the near
future.
3. Conclusion
In an effort to develop low-molecular weight peptidic anti-
SARS agents, we designed, synthesized, and evaluated a series of
dipeptide-type inhibitors in which the P3 valine unit in our potent
tripeptidomimetic lead compound 8 was replaced with a number
of distinct functionalities. In a preliminary study, the compound
25c, bearing a benzyloxycarbonyl (Cbz) P3 moiety, was identified
as a lead compound. The P3 moiety in 25c was systematically
modified with a view to improve the biological activity. This study
led to the identification of a compound bearing an N-arylglycyl as
a P3 moiety as having higher inhibitory activity due to the pres-
ence of a hydrogen bond with the backbone amino acid residue
Glu166 of 3CLpro. Leucine and benzothiazole units were identified
4. Experimental section
4.1. Materials and methods
Reagents and solvents were purchased from Wako Pure Chem-
ical Ind., Ltd. (Osaka, Japan), and Aldrich Chemical Co. Inc. (Mil-
waukee, WI) and were used without further purification. Analytical
thin-layer chromatography (TLC) was performed on Merck Silica
Gel 60F254 pre-coated plates. Preparative HPLC was performed
using a C18 reverse-phase column (19 ꢂ 150 mm; Sun-Fire Prep C18

P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
441
Table 3
SARS-CoV 3CLpro inhibitory activities (Ki) of 27aed.
Entry no.
Inhibitors
Ki (mM)
27a
0.66
27b
27c
37.0
52.0
27d
2.50
OBDÔ, 5 mm) with a binary solvent system: a linear gradient of
CH3CN in 0.1% aqueous TFA at a flow rate of 6 mL/min, detected at
UV 254 and 230 nm. All solvents used for HPLC were HPLC-grade.
All other chemicals were of analytical grade or better. 1H and 13C
NMR spectra were obtained using a JEOL 400 MHz spectrometer, a
Varian Mercury 300 spectrometer (300 MHz), or a BRUKER AV600
spectrometer (600 MHz) with tetramethylsilane as an internal
standard. High-resolution mass spectra (ESI or EI) were recorded on
a micromass Q-Tof Ultima API or a JEOL JMS-GCmate BU-20 spec-
trometer. Mass spectra (ESI) were recorded on an LCMS-2010EV
(SHIMADZU).
Fig. 2. Molecular dynamics stimulated pose of compound 26m (orange stick) bound to
SARS-CoV 3CLpro (PDB ID: 1WOF (black stick)). (A) Overlapped view of 26m with an
original vinyl ester (black stick) and lead 9 (light blue stick); (B) compound 26m was
shown as an orange stick. Dotted black lines represent the hydrogen bonding inter-
action. A notable hydrogen bonding interaction between the amino group (eNH) of N-
(3-methoxyphenyl)glycyl of 26m with a backbone amino acid residue Glu166 of 3CLpro
.
4.2. Synthetic procedures
dissolved in EtOAc (30 mL). The organic layer was washed with 5%
citric acid (20 mL ꢂ 2), 5% NaHCO3 (20 mL ꢂ 2), and brine (20 mL).
The solution was dried over Na2SO4, filtered, and evaporated under
reduced pressure to give compound 19a. The resulting crude
compound was purified by silica gel column chromatography using
hexaneeEtOAc as eluents. Yield 65%; white solid; 1H NMR
4.2.1. Synthetic procedures for the preparation of (S)-tert-butyl 2-
(3-methylbutanamido)-4-methylpentanoate (19a)
To a solution of the commercially available L-leucine tert-butyl
ester 18a (0.200 g, 0.89 mmol) in DMF (15 mL) was added an iso-
valeric acid (0.100 g, 0.98 mmol), HOBt$H2O (0.151 g, 0.98 mmol),
and EDC$HCl (0.189 g, 0.98 mmol). The resulting solution was
cooled to 0 ꢁC under ice bath conditions, and TEA was then added
dropwise. After 5 min, the ice bath was removed and the mixture
was allowed to stir for 2 h at ambient temperature. DMF was
removed under high vacuum, and the resulting residue was
(400 MHz, CD3OD):
d
4.20 (t, J ¼ 7.6 Hz, 1H), 2.03e1.90 (m, 3H),
1.57e1.42 (m, 3H), 1.34 (s, 9H), 0.86e0.79 (m, 12H). HRMS (ESI): m/z
calcd for C15H30NO3 [M þ H]þ 272.2226, found 272.2230.
The intermediates 19heu were prepared from L-leucine tert-
butyl ester 18a and various commercially available carboxylic acids
according to the procedure described for the synthesis of 19a.
Table 4
4.2.2. Synthesis of benzyl (S)-1-(tert-butoxycarbonyl)-3-methyl
butylcarbamate (19b)
Inhibitory activities (IC50) of selected inhibitors.
To a solution of the commercially available L-leucine tert-butyl
Entry no.
IC50 (mM)
ester 18a (0.500 g, 2.20 mmol) in CH2Cl2 (20 mL) was added ben-
zyloxycarbonyl chloride (0.410 mL, 2.5 mmol). The resulting solu-
tion was cooled to 0 ꢁC and TEA (0.380 mL, 2.5 mmol) was then
added dropwise. After 5 min, the ice bath was removed and the
mixture was allowed to stir for 2 h at room temperature. The
mixture was washed with H2O (20 mL) and brine (10 mL). The
25c
21.0
43.0
24.0
10.0
14.0
26b
26h
26m
26n

442
P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
resulting solution was dried over Na2SO4, filtered, and evaporated
under reduced pressure to give compound 19b, which was purified
by silica gel column chromatography using hexaneeEtOAc as elu-
4.2.10. (S)-tert-Butyl 2-(3-(4-methoxyphenyl)propanamido)-4-
methylpentanoate (19j)
Yield 79% from
L
-leucine tert-butyl ester (18a); colorless oil; 1
H
ents. Yield 85%; colorless oil; 1H NMR (400 MHz, CDCl3):
d
7.35e
NMR (400 MHz, CDCl3):
d
7.13 (d, J ¼ 8.8 Hz, 2H), 6.83 (d, J ¼ 8.0 Hz,
7.28 (m, 5H, merged with CDCl3), 5.10 (s, 2H), 4.29e4.23 (m, 1H),
1.74e1.67 (m, 2H), 1.62e1.58 (m, 1H), 1.44 (s, 9H), 0.95e0.93 (m,
6H). HRMS (ESI): m/z calcd for C18H27NO4Na [M þ Na]þ 344.1838,
found 344.1848.
2H), 4.54e4.48 (m, 1H), 3.79 (s, 3H), 2.94e2.90 (m, 2H), 2.55e2.44
(m, 2H), 1.56e1.44 (m, 3H), 1.44 (s, 9H), 0.90e0.88 (m, 6H). HRMS
(ESI): m/z calcd for C20H31NO4Na [M þ Na]þ 372.2151, found
372.2145.
The intermediates 19ceg were prepared from benzylox-
ycarbonyl chloride and various commercially available amino acid
esters 18bef according to the procedure described for the synthesis
of 19c.
4.2.11. (S)-tert-Butyl 2-(3-(pyridin-3-yl)propanamido)-4-methyl
pentanoate (19k)
Yield 56% from
L
-leucine tert-butyl ester (18a); yellow oil; 1H
NMR (400 MHz, CDCl3):
d
8.52 (s, 1H), 8.46 (d, J ¼ 3.6 Hz, 1H), 7.74e
4.2.3. Benzyl (S)-1-(methoxycarbonyl)pentylcarbamate (19c)
7.69 (t, J ¼ 8.0 Hz, 1H), 7.32e7.28 (m, 1H), 4.52e4.47 (m, 1H), 3.05e
3.01 (m, 2H), 2.62e2.52 (m, 2H), 1.58e1.48 (m, 3H), 1.46 (s, 9H),
0.91e0.89 (m, 6H). HRMS (ESI): m/z calcd for C18H29N2O3 [M þ H]þ
321.2178, found 321.2188.
Yield 67% from
L
-norleucine methyl ester (18b); colorless oil; 1H
NMR (400 MHz, CDCl3):
d
7.36e7.29 (m, 5H), 5.11 (s, 2H), 4.39e4.34
(q, J ¼ 5.6 Hz, 1H), 3.73 (s, 3H), 1.88e1.79 (m, 1H), 1.66e1.63 (m, 1H),
1.38e1.33 (m, 4H), 0.90e0.87 (t, J ¼ 6.4 Hz, 3H). HRMS (ESI): m/z
calcd for C15H22NO4 [M þ H]þ 280.1549, found 280.1545.
4.2.12. (S)-tert-Butyl 2-(cinnamamido)-4-methylpentanoate (19l)
Yield 79% from
L
-leucine tert-butyl ester (18a); colorless oil; 1
H
4.2.4. Benzyl (S)-1-(tert-butoxycarbonyl)-2-methylpropylcarbamate
NMR (400 MHz, CD3OD):
d
7.44 (d, J ¼ 21.0 Hz, 1H), 7.41 (d,
(19d)
J ¼ 7.4 Hz, 2H), 7.29e7.26 (m, 5H), 6.58 (d, J ¼ 21.0 Hz, 1H), 4.40e
4.30 (m, 1H), 1.86e1.50 (m, 3H), 1.36 (s, 9H), 0.95e0.79 (m, 6H).
HRMS (ESI): m/z calcd for C19H28NO3 [M þ H]þ 318.2069, found
318.2077.
Yield 71% from
NMR (400 MHz, CDCl3):
L
-valine tert-butyl ester (18c); colorless oil; 1H
7.39e7.31 (m, 5H), 5.10 (s, 2H), 4.20e4.17
d
(m, 1H), 2.15e2.12 (m, 1H), 1.46 (s, 9H), 0.96e0.87 (m, 6H). HRMS
(ESI): m/z calcd for C17H25NO4Na [M þ Na]þ 330.1681, found
330.1683.
4.2.13. (S)-tert-Butyl 2-((E)-3-(4-methoxyphenyl)acrylamido)-4-
methylpentanoate (19m)
4.2.5. Benzyl (1S,2R)-1-(tert-butoxycarbonyl)-2-methylbutylcarba
Yield 76% from
L
-leucine tert-butyl ester (18a); colorless oil; 1H
mate (19e)
NMR (400 MHz, CDCl3):
d
7.58 (d, J ¼ 15.6 Hz,1H), 7.44 (d, J ¼ 8.6 Hz,
Yield 66% from
L
-isoleucine tert-butyl ester (18d); colorless oil;
2H), 6.88 (d, J ¼ 8.6 Hz, 1H), 6.29 (d, J ¼ 15.6 Hz, 1H), 4.70e4.65 (m,
1H), 3.89 (s, 3H), 1.73e1.48 (m, 3H), 1.43 (s, 9H), 0.98e0.95 (m, 6H).
HRMS (ESI): m/z calcd for C20H31NO4 [M þ H]þ 348.2175, found
348.2183.
1H NMR (400 MHz, CDCl3):
d
7.36e7.29 (m, 5H), 5.30e5.28 (m, NH,
1H), 5.17 (s, 2H), 4.25e4.20 (m, 1H), 1.85e1.80 (m, 1H), 1.44 (s, 9H),
1.21e1.01 (m, 2H), 0.94e0.91 (m, 6H). HRMS (ESI): m/z calcd for
C
18H28NO4 [M þ H]þ 322.2018, found 322.2010.
4.2.14. (S)-tert-Butyl 2-((E)-3-(3,4-dimethoxyphenyl)acrylamido)-
4.2.6. Benzyl (S)-1-(tert-butoxycarbonyl)-3-(methylthio)propylcarba
mate (19f)
4-methylpentanoate (19n)
Yield 69% from
L
-leucine tert-butyl ester (18a); colorless oil; 1
H
Yield 72% from
L
-methionine tert-butyl ester (18e); colorless oil;
NMR (400 MHz, CDCl3):
d
7.53 (d, J ¼ 15.6 Hz,1H), 7.04 (d, J ¼ 8.0 Hz,
d
7.36e7.28 (m, 5H), 5.10 (s, 2H), 4.36 (q, J ¼ 6.0 Hz, 1H), 2.54e2.46
1H), 7.00 (s, 1H), 6.84 (d, J ¼ 8.0 Hz, 1H), 6.30 (d, J ¼ 15.6 Hz, 1H),
4.71e4.65 (m,1H), 3.90 (s, 6H),1.74e1.49 (m, 3H),1.43 (s, 9H), 0.98e
0.95 (m, 6H). HRMS (ESI): m/z calcd for C21H33NO5 [M þ H]þ
378.2280, found 378.2282.
(m, 2H), 2.08 (m, 5H), 1.94e1.92 (m, 2H), 1.46 (s, 9H). HRMS (ESI):
m/z calcd for C17H26NO4S [M þ H]þ 340.1583, found 340.1580.
4.2.7. Benzyl (S)-1-(methoxycarbonyl)-2-phenylethylcarbamate (19g)
Yield 82% from
solid; 1H NMR (400 MHz, CDCl3):
3H), 7.10e7.07 (m, 3H), 5.09 (s, 2H), 4.68e4.63 (m, 1H), 3.70 (s, 3H),
L
-phenylalanine methyl ester (18f): colorless
4.2.15. (S)-tert-Butyl 2-(2-phenoxyacetamido)-4-methylpentanoate
d
7.36e7.31 (m, 4H), 7.29e7.21 (m,
(19o)
Yield 77% from
L
-leucine tert-butyl ester (18a); colorless oil; 1
H
3.16e3.04 (m, 2H). HRMS (ESI): m/z calcd for
C
18H19NO4Na
NMR (400 MHz, CDCl3):
d
7.33 (t, J ¼ 8.4 Hz, 2H), 7.03 (t, J ¼ 8.0 Hz,
[M þ Na]þ 336.1212, found 336.1224.
1H), 6.96 (d, J ¼ 8.0 Hz, 2H), 4.63e4.60 (m, 1H), 4.52 (s, 2H), 1.68e
1.54 (m, 3H), 1.47 (s, 9H), 0.96e0.94 (m, 6H). HRMS (ESI): m/z calcd
for C18H27NO4Na [M þ Na]þ 344.1838, found 344.1835.
4.2.8. (S)-tert-Butyl 2-(2-phenylacetamido)-4-methylpentanoate
(19h)
Yield 59% from
L
-leucine tert-butyl ester (18a); colorless solid;
4.2.16. (S)-tert-Butyl 2-(2-(4-methoxyphenoxy)acetamido)-4-methyl
1H NMR (400 MHz, CDCl3):
d
7.35 (t, J ¼ 7.6 Hz, 2H), 7.31e7.28 (m,
pentanoate (19p)
Yield 79% from
NMR (400 MHz, CDCl3):
3H, merged with CDCl3), 4.52e4.47 (m, 1H), 3.59 (s, 2H), 1.57e1.52
(m, 3H), 1.42 (s, 9H), 0.88 (d, J ¼ 4.8 Hz, 6H). HRMS (ESI): m/z calcd
for C18H28NO3 [M þ H]þ 306.2069, found 306.2065.
L
-leucine tert-butyl ester (18a); yellow oil; 1H
6.90e6.84 (m, 4H), 4.62e4.59 (m, 1H),
d
4.46 (s, 2H), 3.78 (s, 3H), 1.68e1.54 (m, 3H), 1.47 (s, 9H), 0.96e0.94
(m, 6H). HRMS (ESI): m/z calcd for C18H29NO4Na [M þ Na]þ
374.1943, found 374.1953.
4.2.9. (S)-tert-Butyl 2-(2-(4-methoxyphenyl)acetamido)-4-methyl
pentanoate (19i)
Yield 69% from
L
-leucine tert-butyl ester (18a); colorless solid;
4.2.17. (S)-tert-Butyl 2-(2-(4-hydroxyphenoxy)acetamido)-4-methyl
pentanoate (19q)
1H NMR (400 MHz, CDCl3):
d
7.17 (d, J ¼ 8.4 Hz, 2H), 6.87 (d,
J ¼ 8.0 Hz, 2H), 4.51e4.46 (m, 1H), 3.79 (s, 3H), 3.53 (s, 2H), 1.57e
1.52 (m, 3H), 1.42 (s, 9H), 0.88e0.87 (m, 6H). HRMS (ESI): m/z calcd
for C19H27NO4 [M þ H]þ 336.2175, found 336.2173.
Yield 63% from
1H NMR (400 MHz, CD3OD):
2H), 4.36 (s, 2H), 4.32e4.29 (m, 1H), 1.56e1.50 (m, 3H), 1.35 (s, 9H),
L-leucine tert-butyl ester (18a); colorless solid;
d
6.75e6.72 (m, 2H), 6.63e6.60 (m,

P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
443
0.84e0.78 (m, 6H). HRMS (ESI): m/z calcd for C18H27NO5 [M þ H]þ
residue was neutralized with 2 M HCl and extracted with EtOAc
(20 mL ꢂ 4). The organic layer was washed with brine, dried over
Na2SO4, and evaporated under reduced pressure to yield the cor-
responding acid. To a solution of the acid (12.0 mmol) in DMF
(30 mL) were added EDC$HCl (0.540 g, 10.0 mmol), HOBt$H2O
(0.330 g, 11.0 mmol), and N,O-dimethylhydroxylamine (0.213 g,
11.0 mmol) at ambient temperature. The solution was cooled to
0 ꢁC, and TEA (0.240 mL,11.0 mmol) was added slowly. After 2 h, the
DMF was evaporated under high vacuum, and the resulting residue
was dissolved in EtOAc (100 mL). The resulting solution was sub-
sequently washed with 5% citric acid (20 mL ꢂ 2), 5% NaHCO3
(20 mL ꢂ 2), and brine (50 mL). The organic layer was then dried
over Na2SO4 and concentrated under reduced pressure to yield the
Weinreb amide derivative 23, which was purified by column
chromatography (EtOAc/MeOH ¼ 9.5:0.5).
338.1967, found 338.1961.
4.2.18. (S)-tert-Butyl 2-(2-(3-(dimethylamino)phenoxy)acetamido)-
4-methylpentanoate (19r)
Yield 62% from
L
-leucine tert-butyl ester (18a); yellow solid; 1H
NMR (400 MHz, CDCl3):
d
7.17 (t, J ¼ 8.0 Hz, 1H), 6.97 (d, J ¼ 8.0 Hz,
1H), 6.43 (d, J ¼ 8.0 Hz, 1H), 6.32 (s, 1H), 4.66e4.61 (m, 1H), 4.52 (s,
2H), 2.85 (s, 6H), 1.68e1.55 (m, 3H), 1.46 (s, 9H), 0.97e0.94 (m, 6H).
HRMS (ESI): m/z calcd for C20H33N2O4 [M þ H]þ 365.2440, found
365.2430.
4.2.19. (S)-tert-Butyl 2-(2-((4-methoxyphenyl)amino)acetamido)-
4-methylpentanoate (19s)
Yield 54% from
L
-leucine tert-butyl ester (18a); brown viscous
oil; 1H NMR (400 MHz, CDCl3):
d
6.78 (d, J ¼ 8.0 Hz, 2H), 6.58 (d,
The data for compounds 22 and 23 were reported previously
[20,21].
J ¼ 8.0 Hz, 1H), 4.56e4.49 (m, 1H), 3.77e3.71 (m, 5H), 1.55e1.38 (m,
12H), 0.96e0.86 (m, 6H). HRMS (ESI): m/z calcd for C19H30N2O4
[M þ H]þ 351.2284, found 351.2279.
4.4.1. tert-Butyl ((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxo
pyrrolidin-3-yl)propan-2-yl)carbamate (24a)
4.2.20. (S)-tert-Butyl 2-(2-((3-methoxyphenyl)amino)acetamido)-
4-methylpentanoate (19t)
To a solution of benzothiazole (1.37 g, 10.0 mmol) in THF
at ꢀ78 ꢁC was added n-BuLi (2.0 M in THF, 1.67 mL) dropwise over
20 min. After 1 h stirring, the Weinreb amide 23 (0.064 g, 2.0 mmol)
in THF was slowly added dropwise over 20 min and then the so-
lution was stirred for 3 h at same temperature. The reaction was
quenched with sat. NH4Cl and allowed to stir at 0 ꢁC for 20 min. The
mixture was evaporated and dissolved in EtOAc. This solution was
washed with water (100 mL) and brine (50 mL), and then dried over
Na2SO4. After filtration, the organic layer was concentrated under
reduced pressure and the resulting residue was subjected to flash
chromatography (EtOAc/MeOH ¼ 9:1) to obtain the pure com-
pound 24a. The data for the compound 24a has been reported in a
previous article [21].
Yield 57% from
L
-leucine tert-butyl ester (18a); brown viscous
oil; 1H NMR (400 MHz, CDCl3):
d
7.00 (d, J ¼ 8.0 Hz, 1H), 6.87e6.85
(m, 2H), 6.77 (s, 1H), 4.58e4.53 (m, 1H), 3.89e3.71 (m, 5H), 1.57e
1.51 (m, 2H), 1.48e1.46 (m, 1H), 1.43 (s, 9H), 0.95e0.91 (m, 6H).
HRMS (ESI): m/z calcd for C19H30N2O4 [M þ H]þ 351.2284, found
351.2297.
4.2.21. (S)-tert-Butyl 2-(2-((2-methoxyphenyl)amino)acetamido)-
4-methylpentanoate (19u)
Yield 60% from
L
-leucine tert-butyl ester (18a); light pink solid;
1H NMR (400 MHz, CDCl3):
d
6.87e6.74 (m, 3H), 6.51 (d, J ¼ 8.0 Hz,
1H), 4.58e4.53 (m, 1H), 3.87e3.75 (s, 5H), 1.57e1.52 (m, 2H), 1.48e
1.46 (m, 1H), 1.43 (s, 9H), 0.92e0.87 (m, 6H). HRMS (ESI): m/z calcd
for C19H30N2O4 [M þ H]þ 351.2284, found 351.2277.
4.4.2. tert-Butyl ((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-(5-phenyl
thiazol-2-yl)propan-2-yl)carbamate (24b)
To
a cooled solution of the commercially available 5-
4.3. Synthetic procedure for the preparation of 20
phenylthiazole (0.175 g, 10 mmol) in dry THF at ꢀ78 ꢁC was
slowly added a solution of LDA (1.0 mL, 14 mmol). After 1 h, a pre-
cooled solution containing the Weinreb amide 23 (0.329 g,
10 mmol) in anhydrous THF was slowly added, and the reaction was
stirred at ꢀ78 ꢁC over 2 h. The solution was allowed to warm to
room temperature, was quenched by the addition of water (35 mL),
and then extracted with EtOAc (3 ꢂ 50 mL). The combined organic
layers were dried over anhydrous Na2SO4, filtered, and evaporated
in vacuo. The crude mixture was then purified using flash chro-
matography (n-hexane/EtOAc ¼ 3:7) to furnish 24b. The data for
the compound 24b were reported previously [22].
Procedure A: To a solution of the corresponding tert-butyl ester
19 (0.110 g, 0.31 mmol) in CH2Cl2 (2 mL) at 0 ꢁC was added TFA/H2O
(10:1, 3 mL). After 5 min stirring, the reaction mixture was allowed
to stir at room temperature for 1 h. The solvent was completely
evaporated under reduced pressure to give the corresponding acids
20, which were used directly in the subsequent step without
further characterization.
Procedure B: To a solution of the corresponding methyl ester 19
(0.100 g, 0.35 mmol) in THF (3 mL) at room temperature was added
LiOH in water (0.102 g, 2.45 mmol). After 3 h stirring, the solvent
was completely evaporated under reduced pressure and the
resulting residue was neutralized with 2 M HCl. The solution was
extracted with EtOAc (20 mL ꢂ 2), dried over Na2SO4, filtered and
evaporated under reduced pressure to give the corresponding acids
20, which were directly used for next step without further
characterizations.
Compounds 24cee were prepared from 23 according to the
procedure described for the synthesis of 24b.
4.4.3. tert-Butyl ((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-(5-(p-tolyl)
thiazol-2-yl)propan-2-yl)carbamate (24c)
The data for the compound 24c has been reported in a previous
article [22].
4.4. Synthesis of tert-butyl (S)-1-(N-methoxy-N-methylcarbamoyl)-
2-((S)-2-oxopyrrolidin-3-yl)ethylcarbamate (23)
4.4.4. tert-Butyl ((S)-1-(5-(4-methoxyphenyl)thiazol-2-yl)-1-oxo-
3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (24d)
The data for the compound 24d has been reported in a previous
article [22].
Compound 22 was prepared through sequential reactions from
the well-known intermediate 21, as reported previously [20e
22,31].
To a solution of 22 (3.27 g, 8.0 mmol) in methanol (5 mL) was
added a 4 M NaOH solution (0.04 mol) at room temperature. The
mixture was stirred for 4 h and the methanol was evaporated. The
4.4.5. tert-Butyl ((S)-1-(5-(4-methoxyphenyl)thiazol-2-yl)-1-oxo-
3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (24e)
The data for the compound 24e has been reported in a previous
article [22].

444
P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
4.5. Synthesis of (S)-N-((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-(3-methylbutanamido)
pentanamide (25a)
31.5, 28.1, 27.4, 25.9, 23.6, 21.7, 21.5, 13.6. HRMS (ESI): m/z calcd for
C
28H33N4O5S [M þ H]þ 537.2172 found 537.2166.
4.5.4. Benzyl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)
carbamate (25e)
To a solution of 24a (0.200 g, 0.5 mmol) in CH2Cl2 (3 mL) at 0 ꢁC
was added TFA/H2O (10:1, 5 mL), and the solution was stirred for
1 h. After evaporating the solvent under reduced pressure, the
corresponding deprotected lactam residue (0.100 g, 0.53 mmol)
was coupled to the carboxylic acid 20a (0.136 g, 0.38 mmol) using
the coupling agent HBTU (0.147 g, 0.38 mmol) in the presence of
diisopropylethylamine (0.050 mL, 0.38 mmol) in DMF (3 mL) at
Yield 39% from 24a; yellow solid; 1H NMR (400 MHz, CDCl3):
d
8.18 (d, J ¼ 8.0 Hz, 1H), 7.98 (d, J ¼ 8.0 Hz, 1H), 7.59e7.51 (m, 2H),
7.39e7.30 (m, 5H), 5.74e5.65 (m, 1H), 5.15e5.09 (m, 2H), 4.40e4.20
(m, 1H), 3.39e3.36 (m, 2H), 2.65e2.52 (m, 2H), 2.30e2.16 (m, 3H),
2.03e1.96 (m, 1H), 1.10e0.89 (m, 6H). 13C NMR (400 MHz, CD3OD):
0
ꢁC. The reaction mixture was allowed to stir for 2e3 h under
d 193.5, 181.8, 174.6, 165.4, 158.5, 154.7, 138.4, 129.5, 129.0, 128.7,
ambient conditions. The solvent was then evaporated under a high
vacuum, and the residue was dissolved in EtOAc (50 mL). The
organic layer was washed with 5% citric acid (20 mL ꢂ 2), 5%
NaHCO3 (20 mL ꢂ 2), and brine (25 mL). This solution was dried
over Na2SO4, filtered, and evaporated under reduced pressure to
give compound 25a. Yield 35%; light yellow solid; 1H NMR
126.6, 123.7, 79.5, 78.9, 67.6, 61.1, 55.3, 41.6, 40.0, 38.2, 38.1, 33.9,
33.2, 32.7, 28.7, 25.9, 23.7. HRMS (ESI): m/z calcd for C27H31N4O5S
[M þ H]þ 523.2015 found 523.2003.
4.5.5. Benzyl ((2S,3R)-1-(((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-
2-oxopyrrolidin-3-yl)propan-2-yl)amino)-3-methyl-1-oxopentan-2-
yl)carbamate (25f)
(400 MHz, CD3OD):
d
8.21 (d, J ¼ 8.0 Hz, 1H), 8.11 (d, J ¼ 8.0 Hz, 1H),
7.65e7.51 (m, 2H), 5.72e5.69 (m, 1H), 4.61e4.42 (m, 1H), 3.38e3.29
(m, 4H, merged with CD3OD), 2.73e2.66 (m, 1H), 2.46e2.40 (m,
1H), 2.22e2.00 (m, 4H),1.67e1.53 (m, 3H),1.28e1.21 (m, 2H), 0.95e
Yield 42% from 24a; light yellow solid; 1H NMR (400 MHz,
CD3OD):
d
8.19 (d, J ¼ 8.4 Hz, 1H), 8.08 (d, J ¼ 8.4 Hz, 1H), 7.63e7.56
(m, 2H), 7.33e7.27 (m, 5H), 5.75e5.73 (m, 1H), 5.11e5.03 (s, 2H),
4.12e4.05 (m, 1H), 3.35e3.25 (m, 2H, merged with CD3OD), 2.70e
2.50 (m, 1H), 2.49e2.39 (m, 1H), 2.38e2.01 (m, 2H), 2.00e1.85 (m,
1H), 1.80e1.75 (m, 1H), 1.67e1.49 (m, 2H), 1.08e0.86 (m, 6H). HRMS
(ESI): m/z calcd for C28H33N4O5S [M þ H]þ 537.2178 found 537.2178.
0.86 (m, 12H). 13C NMR (400 MHz, CD3OD):
d 193.3, 181.8, 175.8,
165.5, 154.8, 138.4, 129.3, 126.5, 123.7, 55.3, 53.1, 52.9, 46.1, 45.9,
41.8, 41.5, 40.0, 39.8, 29.4, 28.8, 27.5, 25.8, 23.4, 22.8, 21.9. HRMS
(ESI): m/z calcd for C25H35N4O4S [M þ H]þ 487.2379 found
487.2373.
Compounds 25beh were prepared from 24a with 20aeh using a
procedure similar to that described for the synthesis of 25a.
4.5.6. Benzyl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)amino)-4-(methylthio)-1-oxobutan-
2-yl)carbamate (25g)
4.5.1. tert-Butyl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-
2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-
2-yl)carbamate (25b)
Yield 45% from 24a; white solid; 1H NMR (400 MHz, CDCl3):
d
8.19 (d, J ¼ 8.4 Hz, 1H), 8.00 (d, J ¼ 8.0 Hz, 1H), 7.59e7.52 (m, 2H),
7.35e7.25 (m, 5H), 5.78e5.61 (m, 1H), 5.20e5.10 (m, 2H), 4.60e4.44
(m, 1H), 3.40e3.34 (m, 2H), 2.69e2.50 (m, 1H), 2.49e2.38 (m, 2H),
2.38e2.09 (m, 2H), 2.08e2.00 (m, 5H), 1.99e1.89 (m, 2H). 13C NMR
Yield 37% from 24a; colorless solid; 1H NMR (400 MHz,
CDCl3):
d
8.18 (d, J ¼ 8.0 Hz, 1H), 7.99 (d, J ¼ 7.6 Hz, 1H), 7.60e7.52
(m, 2H), 5.75e5.66 (m, 1H), 4.24e4.04 (m, 1H), 3.40e3.37 (m,
2H), 2.66e2.45 (m, 2H), 2.17e1.95 (m, 2H), 1.94e1.72 (m, 1H),
1.70e1.53 (m, 3H), 1.44 (s, 9H), 0.96e0.88 (m, 6H). HRMS (ESI):
m/z calcd for
525.2145.
(400 MHz, CD3OD): d 193.5, 181.8, 174.8, 165.7, 165.4, 158.4, 154.7,
138.4, 129.4, 129.3, 128.8, 127.3, 126.5, 123.7, 67.8, 56.0, 41.5, 40.0,
33.8, 32.8, 31.7, 31.0, 30.7, 29.3, 28.7, 24.1, 15.2. HRMS (ESI): m/z
calcd for C27H31N4O5S2 [M þ H]þ 555.1736 found 555.1743.
C
25H35N4O5SNa [M
þ
Na]þ 525.2148, found
4.5.7. Benzyl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)amino)-1-oxo-3-phenylpropan-2-
yl)carbamate (25h)
4.5.2. Benzyl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)
carbamate (25c)
Yield 43% from 24a; colorless solid; 1H NMR (400 MHz, CDCl3):
Yield 43% from 24a; colorless solid; 1H NMR (400 MHz, CDCl3):
d
8.19 (d, J ¼ 7.6 Hz, 1H), 7.99 (d, J ¼ 7.6 Hz, 1H), 7.61e7.55 (m, 2H),
d
8.16 (d, J ¼ 8.4 Hz, 1H), 7.97 (d, J ¼ 8.4 Hz, 1H), 7.58e7.51 (m, 2H),
7.51e7.17 (m, 10H, merged with CDCl3), 5.82e5.79 (m, 1H), 5.11e
5.08 (m, 2H), 4.43e4.30 (m, 1H), 3.39e3.31 (m, 2H), 3.20e3.15 (m,
2H), 2.65e2.45 (m, 2H), 2.39e2.14 (m, 3H). 13C NMR (400 MHz,
7.32e7.27 (m, 5H, merged with CDCl3), 5.70e5.58 (m, 1H), 5.12e
5.08 (m, 2H), 4.39e4.14 (m, 1H), 3.30e3.27 (m, 2H), 2.64e2.49 (m,
2H), 2.24e2.10 (m, 2H), 2.09e1.89 (m, 1H), 1.86e1.67 (m, 2H), 1.66e
1.48 (m, 1H), 0.93e0.85 (m, 6H). 13C NMR (400 MHz, CD3OD):
CDCl3):
d 192.9, 181.2, 174.3, 165.6, 163.8, 161.8, 154.8, 141.9, 139.2,
129.3, 128.9, 127.4, 127.0, 126.2, 125.9, 125.5, 123.3, 117.9, 114.1, 63.1,
55.2, 54.4, 41.6, 44.3, 41.4, 39.7, 38.2, 33.0, 27.8, 24.7, 22.8. HRMS
(ESI): m/z calcd for C31H32N4O5S [M þ H]þ 571.2015 found 571.2003.
d
193.5, 181.8, 175.8, 165.5, 158.4, 154.8, 138.4, 129.4, 128.7, 127.3,
126.5, 123.7, 67.7, 67.5, 56.0, 55.1, 54.9, 42.0, 41.9, 39.9, 33.9, 32.7,
29.3, 28.7, 25.8, 23.7, 22.0, 21.8. HRMS (ESI): m/z calcd for
C
28H33N4O5S [M þ H]þ 537.2172 found 537.2153.
4.6. Synthesis of 26
4.5.3. Benzyl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)amino)-1-oxohexan-2-yl)carbamate
(25d)
Compounds 26aer were prepared from 24a with 20ieu using a
procedure similar to that described for the synthesis of 25a.
Yield 45% from 24a; light yellow solid; 1H NMR (400 MHz,
4.6.1. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro
lidin-3-yl)propan-2-yl)-4-methyl-2-(2-phenylacetamido)
pentanamide (26a)
CD3OD): d 8.37e8.28 (m, 2H), 7.69e7.64 (m, 2H), 7.40e7.30 (m, 5H),
5.56e5.47 (m, 1H), 5.04e4.99 (m, 2H), 4.07e4.01 (m, 1H), 3.22e3.11
(m, 2H, merged with CD3OD), 2.51e2.11 (m, 3H), 1.97e1.75 (m, 2H),
1.67e1.49 (m, 2H), 1.21e1.08 (m, 4H), 0.85e0.72 (m, 3H). 13C NMR
Yield 47% from 24a; white solid; 1H NMR (400 MHz, CD3OD):
d
8.09 (d, J ¼ 8.0 Hz, 1H), 8.01 (d, J ¼ 8.0 Hz, 1H), 7.55e7.47 (m, 2H),
(400 MHz, CD3OD):
d
202.2, 192.8, 178.0, 172.4, 164.3, 155.8, 152.8,
7.19e7.11 (m, 5H), 5.61e5.57 (m, 1H), 4.35e4.32 (m, 1H), 3.44 (s,
2H), 3.40e3.36 (m, 1H), 3.19e3.07 (m, 1H), 2.57e2.52 (m, 1H),
137.0, 136.3, 77.2, 65.2, 64.2, 42.1, 41.9, 41.5, 37.8, 36.6, 32.0, 31.6,

P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
445
2.39e2.20 (m, 2H), 2.12e1.88 (m, 2H), 1.79e1.48 (m, 3H), 0.88e0.76
(m, 6H). 13C NMR (400 MHz, CD3OD):
193.5, 181.8, 175.1, 165.5,
154.8,138.4,136.9,130.2,129.5,128.4,127.8,126.5,124.2,123.7, 55.9,
55.2, 53.3, 43.7, 41.8, 39.9, 39.2, 33.7, 32.8, 29.4, 28.7, 25.8, 23.7, 22.1.
HRMS (ESI): m/z calcd for C28H33N4O4S [M þ H]þ 521.2223 found
521.2216.
7.42 (m, 3H), 6.95 (d, J ¼ 8.8 Hz, 2H), 6.90e6.88 (m, 1H), 6.61e6.50
(m, 2H), 5.80e5.62 (m, 1H), 4.62e4.50 (m, 1H), 3.84 (s, 3H), 3.40e
3.29 (m, 2H, merged with CD3OD), 2.80e2.65 (m, 1H), 2.63e2.45
(m, 1H), 2.43e2.20 (m, 1H), 2.19e1.99 (m, 1H), 1.92e1.78 (m, 1H),
1.77e1.50 (m, 3H), 1.05e0.88 (m, 6H). 13C NMR (400 MHz, CD3OD):
d
d
191.9, 180.2, 173.3, 166.6, 163.7, 160.8, 153.2, 140.9, 137.1, 129.3,
127.9, 127.4, 127.0, 125.5, 122.3, 117.9, 117.7, 114.1, 55.2, 54.4, 51.6,
51.4, 41.6, 40.4, 38.7, 33.0, 27.6, 24.6, 22.7, 21.6. HRMS (ESI): m/z
calcd for C30H34N4O5S [M þ H]þ 563.2328 found 563.2328.
4.6.2. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro
lidin-3-yl)propan-2-yl)-2-(2-(4-methoxyphenyl)acetamido)-4-
methylpentanamide (26b)
Yield 37% from 24a; yellow solid; 1H NMR (400 MHz, CD3OD):
4.6.7. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro
lidin-3-yl)propan-2-yl)-2-((E)-3-(3,4-dimethoxyphenyl)acrylamido)-
4-methylpentanamide (26g)
d
8.20 (d, J ¼ 8.0 Hz, 1H), 8.11 (d, J ¼ 8.0 Hz, 1H), 7.64e7.58 (m, 2H),
7.20 (d, J ¼ 8.0 Hz, 2H), 6.84 (d, J ¼ 8.4 Hz, 2H), 5.69e5.67 (m, 1H),
4.45e4.41 (m, 1H), 3.73 (s, 3H), 3.51e3.21 (m, 4H, merged with
CD3OD), 2.63e2.52 (m, 1H), 2.51e2.21 (m, 2H), 2.10e1.88 (m, 2H),
1.86e1.55 (m, 3H), 0.92e0.81 (m, 6H). 13C NMR (400 MHz, CD3OD):
Yield 42% from 24a; yellow solid; 1H NMR (400 MHz, CDCl3):
d
8.20e8.14 (m, 1H), 8.00e7.94 (m, 1H), 7.60e7.50 (m, 3H), 7.10e
6.99 (m, 2H), 6.84 (d, J ¼ 8.0 Hz, 1H), 6.30 (d, J ¼ 16.0 Hz, 1H), 5.85e
5.60 (m, 1H), 4.78e4.66 (m, 1H), 3.90 (s, 6H), 3.42e3.30 (m, 2H),
2.70e2.45 (m, 2H), 2.40e2.25 (m, 2H), 2.20e1.85 (m, 2H),1.80e1.64
d
193.5, 181.8, 175.1, 174.4, 165.5, 160.1, 154.8, 138.4, 131.4, 129.3,
128.4, 126.5, 123.7, 114.9, 55.6, 55.2, 42.8, 42.6, 41.6, 41.5, 39.9, 33.7,
29.4, 28.7, 25.8, 25.4, 24.2, 23.2, 22.1. HRMS (ESI): m/z calcd for
(m, 2H), 1.10e0.88 (m, 6H). 13C NMR (400 MHz, CD3OD):
d 193.5,
C
29H34N4O5SNa [M þ Na]þ 573.2148 found 573.2148.
182.4, 176.2, 169.7, 155.5, 152.3, 149.5, 142.8, 138.3, 135.3, 131.9,
129.3, 127.3, 125.7, 123.5, 119.3, 112.7, 117.7, 56.5, 54.2, 53.6, 42.3,
41.6, 40.0, 33.8, 30.7, 29.3, 28.8, 25.8, 23.6, 22.1. HRMS (ESI): m/z
calcd for C31H36N4O6SNa [M þ Na]þ 615.2253 found 615.2253.
4.6.3. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro
lidin-3-yl)propan-2-yl)-2-(3-(4-methoxyphenyl)propanamido)-4-
methylpentanamide (26c)
Yield 43% from 24a; colorless solid; 1H NMR (400 MHz, CDCl3):
4.6.8. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro
lidin-3-yl)propan-2-yl)-4-methyl-2-(2-phenoxyacetamido)pentana
mide (26h)
d
8.17 (d, J ¼ 8.4 Hz, 1H), 7.98 (d, J ¼ 8.0 Hz, 1H), 7.60e7.51 (m, 2H),
7.10 (d, J ¼ 8.4 Hz, 2H), 6.81 (d, J ¼ 8.4 Hz, 2H), 5.67e5.55 (m, 1H),
4.59e4.50 (m, 1H), 3.79 (s, 3H), 3.40e3.37 (m, 2H), 2.93e2.89 (m,
2H), 2.66e2.43 (m, 4H), 2.22e1.99 (m, 3H),1.77e1.57 (m, 2H), 1.46e
Yield 45% from 24a; colorless solid; 1H NMR (400 MHz, CDCl3):
d
8.17 (d, J ¼ 8.0 Hz,1H), 8.00e7.97 (m,1H), 7.60e7.51 (m, 2H), 7.31e
1.40 (m, 1H), 0.91e0.86 (m, 6H). 13C NMR (400 MHz, CDCl3):
d
192.2,
7.27 (m, 2H, merged with CDCl3), 7.10e6.85 (m, 3H), 5.70e5.65 (m,
1H), 4.76e4.66 (m, 1H), 4.55e4.50 (m, 2H), 3.40e3.21 (m, 2H),
2.70e2.50 (m, 2H), 2.35e2.19 (m, 1H), 2.34e2.20 (m, 2H), 2.19e1.85
(m, 1H), 1.80e1.65 (m, 2H), 1.00e0.88 (m, 6H). 13C NMR (400 MHz,
180.3, 172.2, 162.9, 162.5, 158.0, 155.7, 155.4, 147.6, 140.6, 132.7,
129.7, 129.3, 127.3, 113.8, 55.2, 54.8, 50.0, 41.5, 40.6, 38.6, 38.4, 34.8,
33.4, 30.0, 28.7, 27.9, 24.7, 22.8, 21.9. HRMS (ESI): m/z calcd for
C
30H36N4O5SNa [M þ Na]þ 587.2034 found 587.2032.
CD3OD): d 193.6, 181.8, 174.8, 174.5, 171.5, 165.5, 159.2, 154.7, 138.4,
130.6, 129.3, 126.7, 123.7, 115.8, 68.1, 55.9, 52.9, 41.9, 40.7, 39.8, 33.7,
4.6.4. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro
lidin-3-yl)propan-2-yl)-4-methyl-2-(3-(pyridin-3-yl)propanamido)
pentanamide (26d)
29.2, 28.8, 25.7, 23.5, 22.0, 21.7, 21.2. HRMS (ESI): m/z calcd for
C
28H33N4O5S [M þ H]þ 537.2174 found 537.2174.
Yield 27% from 24a; brown solid; 1H NMR (400 MHz, CDCl3):
4.6.9. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro
lidin-3-yl)propan-2-yl)-2-(2-(4-methoxyphenoxy)acetamido)-4-
methylpentanamide (26i)
d
8.30e8.28 (m, 2H), 7.88e7.86 (d, J ¼ 8.0 Hz, 1H), 7.66e7.64 (m,
2H), 7.51e7.26 (m, 2H), 7.20e7.16 (m, 1H), 5.61e5.57 (m, 1H), 4.49e
4.45 (m, 1H), 3.35e3.29 (m, 2H), 3.01e2.97 (m, 2H), 2.83e2.80 (m,
2H), 2.71e2.69 (m, 2H), 2.58e2.42 (m, 3H), 2.41e2.25 (m, 2H),
1.90e1.84 (m, 1H), 1.73e1.53 (m, 2H), 0.96e0.85 (m, 6H). HRMS
(ESI): m/z calcd for C28H34N5O4S [M þ H]þ 536.2332 found
536.2327.
Yield 45% from 24a; yellow solid; 1H NMR (400 MHz, CDCl3):
d
8.18 (d, J ¼ 7.6 Hz, 1H), 7.99 (d, J ¼ 7.6 Hz, 1H), 7.60e7.52 (m, 2H),
6.90e6.80 (m, 4H), 5.81e5.65 (m, 1H), 4.70e4.61 (m, 1H), 4.60e
4.45 (m, 2H), 3.76 (s, 3H), 3.40e3.28 (m, 2H), 2.75e2.40 (m, 2H),
2.39e1.90 (m, 3H), 1.89e1.58 (m, 3H), 1.00e0.89 (m, 6H). 13C NMR
(400 MHz, CD3OD):
d 192.5, 180.2, 172.4, 169.4, 168.7, 163.8, 154.7,
4.6.5. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro
lidin-3-yl)propan-2-yl)-2-cinnamamido-4-methylpentanamide (26e)
Yield 41% from 24a; yellow solid; 1H NMR (400 MHz, CDCl3):
151.5, 145.3, 137.2, 128.0, 127.1, 125.6, 122.4, 115.9, 114.7, 68.1, 55.6,
53.8, 41.7, 40.6, 38.0, 37.8, 32.9, 28.8, 25.8, 23.5, 22.0, 21.8. HRMS
(ESI): m/z calcd for C29H35N4O6S [M þ H]þ 567.2277, found
567.2268.
d
8.17e8.15 (m, J ¼ 8.0 Hz,1H), 7.97e7.93 (m,1H), 7.70e7.50 (m, 5H),
7.49e7.45 (m, 2H), 6.47e6.43 (m, 1H), 5.70e5.60 (m, 1H), 4.90e4.72
(m, 1H), 3.40e3.27 (m, 2H), 2.60e2.45 (m, 2H), 2.40e1.99 (m, 2H),
1.95e1.80 (m, 1H), 1.76e1.42 (m, 3H), 0.96e0.83 (m, 6H). 13C NMR
4.6.10. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyr
rolidin-3-yl)propan-2-yl)-2-(2-(4-hydroxyphenoxy)acetamido)-4-
methylpentanamide (26j)
(400 MHz, CD3OD):
d 193.5, 181.2, 176.3, 165.7, 154.7, 142.4, 138.3,
136.2, 130.9, 129.9, 128.4, 126.5, 123.7, 121.3, 56.1, 55.6, 54.4, 53.7,
42.1, 41.6, 40.1, 33.8, 30.7, 29.52, 28.9, 26.0, 25.9, 23.3, 22.1. HRMS
(ESI): m/z calcd for C29H33N4O4S [M þ H]þ 533.2223 found
533.2243.
Yield 40% from 24a; yellow solid; 1H NMR (400 MHz, CD3OD):
d
8.21 (d, J ¼ 8.1 Hz, 1H), 8.11 (d, J ¼ 8.1 Hz, 1H), 7.65e7.58 (m, 2H),
6.82e6.78 (m, 2H), 6.70e6.65 (m, 2H), 5.78e5.66 (m, 1H), 4.55e
4.50 (m, 1H), 4.60e4.45 (m, 2H), 3.35e3.18 (m, 2H, merged with
CD3OD) 2.75e2.45 (m, 1H), 2.44e2.20 (m, 2H), 2.19e1.89 (m, 2H),
1.88e1.61 (m, 1H), 1.60e1.35 (m, 2H), 0.99e0.97 (m, 6H). 13C NMR
4.6.6. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro
lidin-3-yl)propan-2-yl)-2-((E)-3-(4-methoxyphenyl)acrylamido)-4-
methylpentanamide (26f)
(400 MHz, CD3OD):
d 192.9, 181.5, 173.2, 165.3, 154.7, 138.4, 132.3,
128.5, 127.2, 126.5, 124.3, 123.7, 116.8, 113.1, 69.2, 56.0, 54.2, 42.0,
40.0, 39.8, 34.0, 32.6, 29.2, 28.8, 25.7, 23.4, 22.0, 21.7. HRMS (ESI):
m/z calcd for C28H32N4O6SNa [M þ Na]þ 575.1940 found 575.1926.
Yield 33% from 24a; yellow solid; 1H NMR (400 MHz, CD3OD):
d
8.26 (d, J ¼ 7.0 Hz,1H), 8.18e8.10 (m,1H), 7.70e7.55 (m, 2H), 7.54e

446
P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
4.6.11. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyr
rolidin-3-yl)propan-2-yl)-2-(2-(3-(dimethylamino)phenoxy)
acetamido)-4-methylpentanamide (26k)
4.7.1. Benzyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyr
rolidin-3-yl)-1-(5-phenylthiazol-2-yl)propan-2-yl)amino)pentan-
2-yl)carbamate (27a)
Yield 35% from 24a; light green solid; 1H NMR (400 MHz,
Yield 55% from 24b; colorless solid; 1H NMR (400 MHz, CD3OD):
CDCl3):
d
8.18 (d, J ¼ 7.6 Hz, 1H), 7.99e7.97 (d, J ¼ 8.0 Hz, 1H), 7.60e
d
8.32e8.30 (m, 1H), 7.72 (d, J ¼ 7.20 Hz, 2H), 7.48e7.40 (m, 3H),
7.52 (m, 2H), 7.29e7.22 (m, 1H, merged with CDCl3), 7.11 (d,
J ¼ 8.4 Hz, 1H), 6.77e6.70 (m, 1H), 6.56 (dd, 8.1, 8.4 Hz, 2H), 5.70e
5.66 (m, 1H), 4.79e4.67 (m, 1H), 4.66e4.50 (m, 2H), 3.41e3.28 (m,
2H), 3.04 (s, 3H), 3.01 (s, 3H), 2.75e2.60 (m, 1H), 2.59e2.49 (m, 1H),
2.48e2.35 (m, 1H), 2.34e2.10 (m, 2H), 2.05e1.85 (m, 1H), 1.79e1.57
7.37e7.24 (m, 5H), 5.65e5.61 (m, 1H), 5.15e5.03 (m, 2H), 4.30e4.20
(m, 1H), 3.40e3.23 (m, 2H, merged with CD3OD), 2.75e2.65 (m,
1H), 2.64e2.06 (m, 2H), 2.05e1.80 (m, 2H), 1.79e1.60 (m, 1H), 1.59e
1.52 (m, 2H), 0.99e0.89 (m, 6H). 13C NMR (400 MHz, CD3OD):
d
192.0, 181.8, 175.8, 164.2, 158.4, 148.9, 141.9, 131.5, 130.9, 130.7,
(m, 2H), 0.99e0.90 (m, 6H). 13C NMR (400 MHz, CD3OD):
d
193.6,
129.4, 128.9, 128.7, 128.3, 67.7, 61.5, 55.7, 54.8, 42.4, 41.5, 40.1, 38.9,
181.9, 178.0,172.3, 171.6, 171.3, 165.7, 142.6,138.4,131.2, 129.3, 128.5,
126.8, 123.7, 121.9, 90.7, 77.2, 66.7, 57.4, 52.8, 41.5, 40.0, 33.7, 28.8,
25.7, 23.4, 22.0, 21.7. HRMS (ESI): m/z calcd for C30H38N5O5S
[M þ H]þ 580.2594 found 580.2563.
34.2, 29.3, 28.6, 25.9, 24.2, 23.4, 22.1, 21.8. HRMS (ESI): m/z calcd for
C
30H35N4O5S [M þ H]þ 563.2328 found 563.2308.
4.7.2. Benzyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyr
rolidin-3-yl)-1-(5-(p-tolyl)thiazol-2-yl)propan-2-yl)amino)pentan-
2-yl)carbamate (27b)
4.6.12. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro
lidin-3-yl)propan-2-yl)-2-(2-((4-methoxyphenyl)amino)acetamido)-
4-methylpentanamide (26l)
Yield 51% from 24c; colorless solid; 1H NMR (400 MHz, CD3OD):
d
8.32e8.29 (m, 1H), 7.62 (d, J ¼ 8.4 Hz, 2H), 7.40e7.20 (m, 7H),
Yield 45% from 24a; light yellow solid; 1H NMR (400 MHz,
5.70e5.57 (m, 1H), 5.15e5.01 (m, 2H), 4.30e4.18 (m, 1H), 3.40e3.25
(m, 2H, merged with CD3OD), 2.75e2.40 (m, 1H), 2.50e2.30 (m,
2H), 2.38 (s, 3H), 2.29e2.09 (m, 1H), 2.08e1.75 (m, 2H), 1.74e1.65
(m, 1H), 1.63e1.49 (m, 2H), 0.97e0.90 (m, 6H). HRMS (ESI): m/z
calcd for C31H37N4O5S [M þ H]þ 577.2485 found 577.2484.
CD3OD):
d
8.24 (d, J ¼ 8.0 Hz, 1H), 8.14 (d, J ¼ 8.0 Hz, 1H), 7.68e7.60
(m, 2H), 7.06 (t, J ¼ 8.4 Hz, 1H), 6.30e6.17 (m, 3H), 5.72e5.68 (m,
1H), 4.53e4.50 (m, 1H), 3.84e3.65 (m, 2H), 3.82 (s, 3H), 3.37e3.28
(m, 2H, merged with CD3OD), 2.72e2.55 (m, 1H), 2.54e2.29 (m,
1H), 2.28e1.83 (m, 3H), 1.62e1.41 (m, 3H), 0.90e0.83 (m, 6H). 13
C
NMR (400 MHz, CD3OD):
d
193.4, 181.8, 175.4, 165.6, 164.0, 155.8,
4.7.3. Benzyl ((S)-1-(((S)-1-(5-(4-methoxyphenyl)thiazol-2-yl)-1-
oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-
oxopentan-2-yl)carbamate (27c)
154.7, 138.3, 132.0, 129.3, 129.2, 128.4, 127.1, 126.4, 124.2, 122.9,
116.7, 113.9, 105.2, 103.2, 56.2, 53.4, 41.8, 40.0, 39.4, 33.9, 29.3, 28.8,
26.1, 22.1, 21.9. HRMS (ESI): m/z calcd for C29H36N5O5S [M þ H]þ
566.2437 found 566.2428.
Yield 47% from 24d; light yellow solid; 1H NMR (400 MHz,
CDCl3):
d 8.05e8.03 (m, 1H), 7.60e7.52 (m, 2H), 7.60e7.27 (m, 5H),
7.00e6.95 (m, 2H), 5.64e5.56 (m, 1H), 5.20e5.08 (m, 1H), 4.40e
4.25 (m, 1H), 3.86 (s, 3H), 3.35e3.34 (m, 2H), 2.67e2.42 (m, 2H),
2.22e2.04 (m, 2H), 2.02e1.96 (m, 1H), 1.80e1.52 (m, 3H), 0.97e0.86
(m, 6H). HRMS (ESI): m/z calcd for C31H37N4O6S [M þ H]þ 593.2434
found 593.2414.
4.6.13. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyr
rolidin-3-yl)propan-2-yl)-2-(2-((3-methoxyphenyl)amino)
acetamido)-4-methylpentanamide (26m)
Yield 41% from 24a; light yellow solid; 1H NMR (400 MHz,
CD3OD):
d
8.21 (d, J ¼ 7.6 Hz, 1H), 8.12 (d, J ¼ 7.6 Hz, 1H), 7.66e
7.58 (m, 2H), 7.10e6.89 (m, 4H), 5.80e5.65 (m, 1H), 4.60e4.45 (m,
1H), 3.94e3.72 (m, 2H), 3.76 (s, 3H), 3.36e3.29 (m, 2H, merged
with CD3OD), 2.75e2.55 (m, 1H), 2.49e2.25 (m, 1H), 2.20e1.80
(m, 3H), 1.70e1.45 (m, 3H), 0.99e0.84 (m, 6H). 13C NMR
4.7.4. Benzyl ((S)-1-(((S)-1-(5-(2-methoxyphenyl)thiazol-2-yl)-1-
oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-
oxopentan-2-yl)carbamate (27d)
Yield 51% from 24e; light yellow solid; 1H NMR (400 MHz,
(400 MHz, CD3OD):
d
193.4, 181.8, 177.4, 162.3, 160.2, 157.8, 141.2,
CDCl3):
d
8.12e8.10 (m, 1H), 7.37e7.27 (m, 6H), 7.19 (d, J ¼ 7.6 Hz,
138.4, 133.9, 129.3, 128.4, 126.5, 123.7, 119.3, 116.4, 109.9, 66.0,
55.6, 55.4, 53.0, 42.0, 41.7, 41.5, 34.3, 28.8, 25.7, 23.3, 21.9, 21.5.
HRMS (ESI): m/z calcd for C29H36N5O5S [M þ H]þ 566.2437 found
566.2415.
1H), 7.13e7.11 (m, 1H), 6.95 (dd, 1.8, 8.2 Hz, 1H), 5.70e5.53 (m, 1H),
5.15e5.08 (m, 2H), 4.40e4.30 (m, 1H), 3.85 (s, 3H), 3.50e3.25 (m,
2H), 2.70e2.37 (m, 2H), 2.35e2.05 (m, 2H), 2.04e1.80 (m, 1H),
1.79e1.60 (m, 2H), 1.59e1.45 (m, 1H), 0.99e0.80 (m, 6H). 13C NMR
(400 MHz, CDCl3):
d 190.8, 180.3, 172.8, 162.9, 160.1, 156.1, 147.4,
4.6.14. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyr
rolidin-3-yl)propan-2-yl)-2-(2-((2-methoxyphenyl)amino)
acetamido)-4-methylpentanamide (26n)
140.6, 136.4, 131.5, 130.4, 128.4, 127.9, 119.7, 115.1, 113.1, 66.8, 55.4,
53.4, 42.2, 41.7, 40.7, 38.9, 33.5, 31.9, 28.2, 24.7, 24.6, 23.0, 22.9, 22.0.
HRMS (ESI): m/z calcd for C31H37N4O6S [M þ H]þ 593.2434 found
593.2427.
Yield 45% from 24a; light yellow solid; 1H NMR (400 MHz,
CD3OD):
d
8.11 (d, J ¼ 7.6 Hz, 1H), 8.02 (d, J ¼ 7.6 Hz, 1H), 7.56e7.48
(m, 2H), 6.85e6.82 (m, 1H), 6.79e6.87 (m, 2H), 6.56e6.54 (m, 1H),
5.65e5.56 (m,1H), 4.43e4.39 (m,1H), 3.81e3.73 (m, 5H), 3.31e3.25
(m, 2H, merged with CD3OD), 2.58e2.45 (m, 2H), 2.44e2.10 (m,
4.8. Molecular docking studies
The crystal structure of the SARS-CoV 3CL protease in complex
with a substrate analog inhibitor (coded 1WOF) [35] was obtained
from the Protein Data Bank (PDB; http://www.rcsb.org/pdb/home/
home.do). Initially, a binding model of 26m with 3CLpro was
simulated to form the basis of a comparison with our previous
potent lead tripeptidomimetic 9 and a substrate analog, using
molecular operating environment (MOE) software. Several mini-
mization processes were performed using the MMFF94X force
field to model the solvation environment surrounding the inhib-
itor. Structures having a relatively low binding free energy and a
high number of cluster members were selected for the subsequent
docking conformation optimization step. The minimized energies
1H), 2.09e1.68 (m, 3H), 1.40e1.38 (m, 3H), 0.80e0.71 (m, 6H). 13
C
NMR (400 MHz, CD3OD):
d 193.4, 181.8, 174.9, 173.3, 165.5, 159.8,
154.4,138.4,133.3,130.2,129.3,128.0,126.9,123.7,123.2,116.3, 65.4,
56.1, 55.4, 53.0, 42.0, 41.6, 40.1, 34.3, 33.7, 28.8, 25.7, 23.3, 21.9, 21.2.
HRMS (ESI): m/z calcd for C29H36N5O5S [M þ H]þ 566.2437 found
566.2426.
4.7. Synthesis of 27
Compounds 27aed were prepared from 24bed with 20a using a
similar procedure to that described for the synthesis of 25a.

P. Thanigaimalai et al. / European Journal of Medicinal Chemistry 65 (2013) 436e447
447
[8] V. Thiel, K.A. Ivanov, A.A. Putics, T. Hertzig, B. Schelle, S. Bayer, B. Weiabrich,
E.J. Snijder, H. Rabenau, H.W. Doerr, J. Ziebuhr, J. Gen. Virol. 84 (2003) 2305e
2315.
[9] H. Yang, M. Yang, Y. Ding, Y. Liu, Z. Lou, Z. Zhou, L. Sun, L. Mo, S. Ye, H. Pang,
G.F. Gao, K. Anand, M. Bartlam, R. Hilgenfeld, Z. Rao, Proc. Natl. Acad. Sci. U. S.
A. 100 (2003) 13190e13195.
of 26m, obtained from the docking study, were ꢀ40.67
and ꢀ35.52 kcal/mol.
Acknowledgments
[10] K. Chou, D. Wei, W. Zhong, Biochem. Biophys. Res. Commun. 308 (2003)
148e151.
[11] K. Pyrc, B. Berkhout, L. van der Hoek, J. Virol. 81 (2007) 3051e3057.
[12] B.C. Fielding, Future Microbiol. 6 (2011) 153e159.
[13] A.M. Zaki, S. van Boheemen, T.M. Bestebroer, A.D.M.E. Osterhaus,
R.A.M. Fouchier, N. Engl. J. Med. 367 (2012) 1814e1820.
[14] http://www.independent.co.uk/life-style/health-and-families/health-news/
sarslike-virus-spreads-persontoperson-in-the-uk-8492750.html.
[15] Y.M. Shao, W.B. Yang, T.H. Kuo, K.C. Tsai, C.H. Lin, A.S. Yang, P.H. Liang,
C.H. Wong, Bioorg. Med. Chem. 16 (2008) 4652e4660.
This research was supported by Grants from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) of
Japan, including a Grant-in-aid for Young scientist (Tokubetsu
Kenkyuin Shorei-hi) 23$01104 and a Grant-in-aid for Scientific
Research 23659059 and 23390029. E.F. acknowledges support from
the National Institutes of Health (grant GM57144).
[16] R.P. Jain, H.I. Pettersson, J. Zhang, K.D. Aull, P.D. Fortin, C. Huitema, L.D. Eltis,
J.C. Parrish, M.N. James, D.S. Wishart, J.C. Vederas, J. Med. Chem. 47 (2004)
6113e6116.
Appendix A. Supplementary data
[17] S. Yang, S.J. Chen, M.F. Hsu, J.D. Wu, C.T. Tseng, Y.F. Liu, H.C. Chen, C.W. Kuo,
C.S. Wu, L.W. Chang, W.C. Chen, S.Y. Liao, T.Y. Chang, H.H. Hung, H.L. Shr,
C.Y. Liu, Y.A. Huang, L.Y. Chang, J.C. Hsu, C.J. Peters, A.H. Wang, M.C. Hsu,
J. Med. Chem. 49 (2006) 4971e4980.
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.05.005.
[18] T.W. Lee, M.M. Cherney, C. Huitema, J. Liu, K.E. James, J.C. Powers, L.D. Eltis,
M.N.G. James, J. Mol. Biol. 353 (2005) 1137e1151.
[19] K. Akaji, H. Konno, H. Mitsui, K. Teruya, Y. Shimamoto, Y. Hattori, T. Ozaki,
M. Kusunoki, A. Sanjoh, J. Med. Chem. 54 (2011) 7962e7973.
[20] M.O. Sydnes, Y. Hayashi, V.K. Sharma, T. Hamada, U. Bacha, J. Barrila, E. Freire,
Y. Kiso, Tetrahedron 62 (2006) 8601e8609.
[21] T. Regnier, D. Sharma, K. Hidaka, U. Bacha, E. Freire, Y. Hayashi, Y. Kiso, Bioorg.
Med. Chem. Lett. 19 (2009) 2722e2727.
[22] S. Konno, P. Thanigaimalai, K. Nakada, T. Yamamoto, Y. Yamazaki, F. Yakushiji,
K. Akaji, Y. Kiso, Y. Kawasaki, E. Freire, Y. Hayashi, Bioorg. Med. Chem. 21
(2013) 412e424.
[23] U. Kaeppler, N. Stiefl, M. Schiller, R. Vicik, A. Breuning, W. Schmitz,
D. Rupprecht, C. Schmuck, K. Baumann, J. Ziebuhr, T. Schirmeister, J. Med.
Chem. 48 (2005) 6832e6842.
[24] L. Chen, C. Gui, X. Luo, Q. Yang, S. Gunther, E. Scandella, C. Drosten, D. Bai,
X. He, B. Ludewig, J. Chen, H. Luo, Y. Yang, Y. Yang, J. Zou, V. Thiel, K. Chen,
J. Shen, X. Shen, H. Jiang, J. Virol. 79 (2005) 7095e7103.
References
[1] C. Drosten,S. Gunther, W.Preiser, S.VenderWerf,H.R. Brodt, S.Becker, H.Rabenau,
M. Panning, L. Kolensnikova, R.A.M. Fouchier, A. Berger, A.M. Burguiere, J. Cinatl,
M. Eickmann, N. Escriou, K. Grywna, S. Kramme, J. Manuguerra, S. Muller,
V. Rickerts, M. Sturmer, S. Vieth, H.D. Klenk, A.D.M.E. Osterhaus, H. Schmitz,
H.W. Doerr, N. Engl. J. Med. 348 (2003) 1967e1976.
[2] N. Lee, D. Hui, A. Wu, P. Chan, P. Cameron, F.M. Joynt, A. Ahuja, M.Y. Yung,
C.B. Leung, K.F. To, M.D. Leu, C.C. Szeto, S. Chung, J.J.Y. Sung, N. Engl. J. Med.
348 (2003) 1986e1994.
[3] T.G. Ksiazek, D. Erdman, C.S. Goldsmith, S.R. Zaki, T. Peret, S. Emery, S. Tong,
C. Urbani, J.A. Comer, W. Lim, P.E. Rollin, S.F. Dowell, A.E. Ling, C.D. Humphrey,
W.J. Shieh, J. Guarner, C.D. Paddock, P. Rota, B. Fields, J. DeRisi, J.Y. Yang,
N. Cox, J.M. Hughes, J.W. LeDuc, W.J. Bellini, L.J. Anderson, N. Engl. J. Med. 348
(2003) 1953e1966.
[25] J.E. Blanchard, N.H. Elowe, C. Huitema, P.D. Fortin, J.D. Cechetto, L.D. Eltis,
E.D. Brown, Chem. Biol. 11 (2004) 1445e1453.
[4] K. Anand, J. Ziebuhr, P. Wadhwani, J.R. Mesturs, R. Hilgenfeld, Science 300
(2003) 1763e1767.
[26] L. Zhou, Y. Liu, W. Zhang, P. Wei, C. Huang, J. Pei, Y. Yuan, L. Lai, J. Med. Chem.
49 (2006) 3440e3443.
[27] C.Y. Wu, K.Y. King, C.J. Kuo, J.M. Fang, Y.T. Wu, M.Y. Ho, C.L. Liao, J.J. Shie,
P.H. Liang, C.H. Wong, Chem. Biol. 13 (2006) 261e268.
[28] R. Ramajayam, K.P. Tan, H.G. Liu, P.H. Liang, Bioorg. Med. Chem. Lett. 20 (2010)
3569e3572.
[29] R. Ramajayam, K.P. Tan, H.G. Liu, P.H. Liang, Bioorg. Med. Chem. 18 (2010)
7849e7854.
[5] J.S. Peiris, S.T. Lai, L.L. Poon, Y. Guan, L.Y. Yam, W. Lim, J. Nicholls, W.K. Yee,
W.W. Yan, M.T. Cheung, V.C. Cheng, K.H. Chan, D.N. Tsang, R.W. Yung, T.K. Ng,
K.Y. Yuen, Lancet 361 (2003) 1319e1325 (and references therein).
[6] P.A. Rota, M.S. Oberste, S.S. Monroe, W.A. Nix, R. Campagnoli, J.P. Icenogle,
S. Penaranda, B. Bankamp, K. Maher, M.H. Chem, W. Tong, A. Tamin, L. Lowe,
M. Frace, J.L. DeRisi, Q. Chen, D. Wang, D.D. Erdman, T.C. Peret, C. Burns,
T.G. Ksiazek, P.E. Rollin, A. Sanchez, S. Liffick, B. Holloway, J. Limor,
K. McCaustland, M. Olsen-Rasmussen, R. Fouchier, S. Gunther, A.D. Osterhaus,
C. Drosten, M.A. Pallansch, L.J. Anderson, W.J. Bellini, Science 300 (2003)
1394e1399.
[30] P. Mukherjee, P. Desai, L. Ross, E.L. White, M.A. Avery, Bioorg. Med. Chem. 7
(2008) 4138e4149.
[31] Q. Tian, N.K. Nayyar, S. Babu, L. Chen, J. Tao, S. Lee, A. Tibbetts, T. Moran, J. Liou,
M. Guo, T.P. Kennedy, Tetrahedron Lett. 42 (2001) 6807e6809.
[32] U. Bacha, J. Barrila, B. Gabelli, Y. Kiso, L.M. Amzel, E. Freire, Chem. Biol. Drug
Des. 72 (2008) 34e39.
[33] J. Barrila, U. Bacha, E. Freire, Biochemistry 45 (2006) 14908e14916.
[34] K. Akaji, H. Konno, M. Onozuka, A. Makino, H. Saito, K. Nosaka, Bioorg. Med.
Chem. 16 (2008) 9400e9408.
[35] H. Yang, W. Xie, X. Xue, K. Yang, J. Ma, W. Liang, Q. Zhao, Z. Zhou, D. Pei,
J. Ziebuhr, R. Hilgenfeld, K.Y. Yuen, L. Wong, G. Gao, S. Chen, Z. Chen, D. Ma,
M. Bartlam, Z. Rao, PLoS Biol. 3 (2005) 1742e1752.
[7] M.A. Marra, S.J. Jones, C.R. Astell, R.A. Holt, A. Brooks-Wilson, Y.S. Butterfield,
J. Khattra, J.K. Asano, S.A. Barber, S.Y. Chan, A. Cloutier, S.M. Coughlin,
D. Freeman, N. Girn, O.L. Griffith, S.R. Leach, M. Mayo, H. McDonald,
S.B. Montgomery, P.K. Pandoh, A.S. Petrescu, A.G. Robertson, J.E. Schein,
A. Siddiqui, D.E. Smailus, J.M. Sott, G.S. Yang, F. Plummer, A. Andonov,
H. Artsob, N. Bastien, K. Bermard, T.F. Booth, D. Bowness, M. Czub, M. Drebot,
L. Fernando, R. Flick, M. Garbutt, M. Gray, A. Grolla, S. Jones, H. Feldmann,
A. Meyers, A. Kabani, Y. Li, S. Normand, U. Stroher, G.A. Tipples, S. Tyler,
R. Vogrig, D. Ward, B. Watson, R.C. Brunham, M. Krajden, M. Petric,
D.M. Skowronski, C. Upton, R.L. Roper, Science 300 (2003) 1399e1404.