Design and synthesis of new tripeptide-type SARS-CoV 3CL protease inhibitors containing an electrophilic arylketone moiety

Article abstract of DOI:10.1016/j.bmc.2012.11.017

We describe here the design, synthesis and biological evaluation of a series of molecules toward the development of novel peptidomimetic inhibitors of SARS-CoV 3CL^pro. A docking study involving binding between the initial lead compound 1 and th

Full text of DOI:10.1016/j.bmc.2012.11.017

Bioorganic & Medicinal Chemistry 21 (2013) 412–424
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of new tripeptide-type SARS-CoV 3CL protease
inhibitors containing an electrophilic arylketone moiety
Sho Konno a, , Pillaiyar Thanigaimalai a, , Takehito Yamamoto a, Kiyohiko Nakada a, Rie Kakiuchi a,
Kentaro Takayama a, Yuri Yamazaki a, Fumika Yakushiji a, Kenichi Akaji b, Yoshiaki Kiso b, Yuko Kawasaki c,
Shen-En Chen 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:
We describe here the design, synthesis and biological evaluation of a series of molecules toward the
development of novel peptidomimetic inhibitors of SARS-CoV 3CLpro. A docking study involving binding
between the initial lead compound 1 and the SARS-CoV 3CLpro motivated the replacement of a thiazole
with a benzothiazole unit as a warhead moiety at the P10 site. This modification led to the identification
of more potent derivatives, including 2i, 2k, 2m, 2o, and 2p, with IC50 or Ki values in the submicromolar
to nanomolar range. In particular, compounds 2i and 2p exhibited the most potent inhibitory activities,
with Ki values of 4.1 and 3.1 nM, respectively. The peptidomimetic compounds identified through this
process are attractive leads for the development of potential therapeutic agents against SARS. The struc-
tural requirements of the peptidomimetics with potent inhibitory activities against SARS-CoV 3CLpro may
be summarized as follows: (i) the presence of a benzothiazole warhead at the S10-position; (ii) hydrogen
bonding capabilities at the cyclic lactam of the S1-site; (iii) appropriate stereochemistry and hydrophobic
moiety size at the S2-site and (iv) a unique folding conformation assumed by the phenoxyacetyl moiety
at the S4-site.
Received 1 October 2012
Revised 10 November 2012
Accepted 12 November 2012
Available online 24 November 2012
Keywords:
Peptidomimetics
SARS-CoV 3CL protease
Cysteine protease inhibitors
Docking study
SARS
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
nucleophile in the proteolytic process.9,10 Since SARS-CoV 3CLpro
plays an important role in the virus life cycle, it has been recog-
Severe acute respiratory syndrome (SARS), a typical form of
pneumonia, was first reported in southern China in November
2002. SARS rapidly spread to 32 countries, creating panic among
the public and in the World Health Organization (WHO).1–3 The
initial outbreak of SARS included more than 8000 individuals diag-
nosed with the disease. Of these, 774 lives were claimed.4 SARS is
characterized by a high fever (>38 °C), malaise, headache, rigor and
a non-productive cough.5 The causative agent of SARS has been
identified as a novel human coronavirus (SARS-CoV)6 that encodes
several viral proteases that proteolyze polyproteins to yield func-
tional proteins. One such highly conserved cysteine protease is
the main protease (Mpro), also known as the dimeric chymotryp-
sin-like protease (3CLpro).7–9 3CLpro mediates the majority of prote-
olytic processes involved in producing two large viral polyproteins,
replicases pb1a and pb1b.7–9 The active site of SARS-CoV 3CLpro
contains Cys145 and His41, which together constitute a catalytic
dyad in which the cysteine moiety functions as a common
nized as a key target for the discovery of anti-SARS agents. Numer-
ous SARS-CoV 3CLpro inhibitors have been identified through
chemical library screenings11–16 or rational design approaches
based on the active site properties.17–20 These protease inhibitors
include C2-symmetric peptidomimetics,11 3-quinolinecarboxylic
acid derivatives,12 bifunctional arylboronic acids,13 keto-glutamine
derivatives,14 isatin derivatives,15 thiophene-2-carboxylates,16 zinc
conjugated compounds,17 cinanserin,18 calmodulin,19 benzotriaz-
oles,20 ,b-unsaturated acids,21 anilide,22 and glutamic acid and
a
glutamine peptides possessing a trifluoromethyl ketone group.23,24
In our ongoing effort to develop anti-SARS agents, we previ-
ously identified a series of Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-thia-
zoles as SARS-CoV 3CLpro inhibitors.25 Among these compounds,
compound 1 (Fig. 1) was identified as a potent lead compound with
a Ki value of 2.20 l
M.25
Primary structure–activity relationship (SAR) studies of 1 have
revealed that the presence of a rigid cyclic amide (P1-site) and
the electron-withdrawing chemical warhead thiazolyl-2-ketone
(P10-moiety) are very important for the inhibitory activity. In the
present study, we performed a molecular modeling study involving
1 and the 3CLpro. The study revealed the presence of a space in the
⇑
Corresponding author. Tel./fax: +81 42 676 3275.
E-mail address: yhayashi@toyaku.ac.jp (Y. Hayashi).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.017

S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
413
O
B
A
NH
O
O
S
N
H
N
O
N
H
N
H
O
O
1
IC50 = 9.5
Ki = 2.20
µM
µM
Figure 1. Structure of a lead compound 1 (A) and their molecular stimulated representation (B).
S10-pocket that was larger than the size of the thiazole unit, and
the N-terminal P4-moiety protruded from the binding site
(Fig. 1B). Thus, P10 and P4 were sequentially optimized in a step-
by-step approach that involved testing a wide variety of substruc-
tural substitutions in 1. The P2 and P1- sites were focused in
parallel. As a result, some analogs were identified that exhibited
potent inhibitory activity on the submicromolar to nanomolar
range. Here 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. Chemistry
Scheme 2. Synthetic outline for the preparation of title compounds 2, 3 and 5.
Reagents and conditions: (a) HN(OMe)Me, EDCꢀHCl, HOBT, Et3N/DMF; (b) n-BuLi,
THF, ꢁ78 °C (if R2 = thiazole, 4,5-dimethylthiazole or benzothiazole) or LDA, THF,
ꢁ78 °C (if R2 = 5-arylated thiazoles); (c) TFA/H2O; (d) 9, HBTU, DIPEA/DMF followed
by HPLC purification. Note: The substituents R(acyl), R1 and R2 are indicated in
Tables 1–3.
3CLpro inhibitors are generally synthesized by assembling two
key fragments: dipeptidic 9 and the C-terminal thiazole derivatives
13 or 17. The dipeptides 9 were prepared via Fmoc-based solid-
phase peptide synthesis over Wang resin. The corresponding
Fmoc-amino acids were introduced onto the resin via diisopropyl-
carbodiimide (DIC)-mediated coupling in the presence of catalytic
amounts of 4-N,N0-dimethylaminopyridine (DMAP) in DMF
(Scheme 1). The resulting intermediate 6 was treated with 20%
piperidine in DMF to remove the Fmoc- group and coupled to
Fmoc-valine, yielding 7, via the DIC-HOBt (1-hydroxybenzotria-
zole) method in DMF. Further Fmoc-deprotection and the reaction
of 7 with various carboxylic acids or acid chlorides produced the
crucial P4-attached dipeptides 9 after treatment of resin 8 with tri-
fluoroacetic acid/water (10:1) for 1 h. The dipeptides 9 were used
directly in the next step without further purification.
reduction with PtO2 (5%), cyclization and hydrolysis. Further cou-
pling of 11 to N,O-dimethylhydroxylamine via the 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimideꢀhydrochloride (EDCꢀHCl)-HOBt
method yielded the Weinreb amide 12.26 The Weinreb amide
was then coupled to an appropriate thiazole in the presence of n-
butyl lithium (n-BuLi) or lithium diisopropylamide (LDA) at ꢁ78 °C
to afford the thiazoles 13, which were deprotected and subse-
quently reacted with the dipeptides 9 in the presence of O-benzo-
triazole-N,N,N0,N0-tetramethyluroniumhexafluoro phosphate (HBTU),
HOBt, and DIPEA in DMF to afford the title compounds 2, 3 and 5.
The syntheses of key intermediates on the path to the thiazoles
13, as well as the title inhibitors 2, 3 and 5, are indicated in Scheme
2. The optically pure glutamic acid ester 10 was converted to c-lac-
tam-acid 1126,27 by treatment with bromoacetonitrile, followed by
Scheme 1. Solid-phase synthesis of dipeptidic 9. Reagents and conditions:
(a) Fmoc-R1-OH, DIC, DMAP/DMF; (b) 20% piperidine/DMF; (c) Fmoc-Val-OH, DIC,
DMAP, HOBt/DMF; (d) 20% piperidine/DMF; (e) R(Acyl)-Cl, Et3N or R(Acyl)-OH, DIC,
HOBt/DMF; (f) TFA/H2O. Note: The substituents R (acyl) and R1 are indicated in
Tables 1–3.
Scheme 3. Synthetic outline for the preparation of imidazole type compounds 4.
Reagents and conditions: (a) HN(OMe)Me, EDCꢀHCl, HOBT, Et3N/DMF; (b) thiazole,
n-BuLi, THF, ꢁ78 °C; (c) TsCl, Et3N, THF; (d) TFA/H2O; (e) 9, HBTU, DIPEA/DMF
followed by HPLC purification. Note: The substituents R(acyl) and R1 are indicated
in Table 2.

414
S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
The imidazole-type compounds 4 were prepared as indicated in
parameters at a constant substrate concentration and different
inhibitor concentrations assessed Ki values. The initial rate
measurements were determined as previously described using a
fluorescence-based peptide cleavage assay with a fluorogenic sub-
strate, Dabcyl-KTSAVLQSGFRKME-Edans.24,30 The rate of substrate
cleavage was detected by the increase in fluorescent over time (see
Section 6.7). Tables 1–3 report the IC50 and/or Ki values as the
mean of 3 independent experiments.
Scheme 3. Another key imidazole thiazole 17 was prepared from
the commercially available Boc-His(Tos)-OH 14 using the same
reactions and conditions as indicated for the preparation of the
-lactam thiazoles 13. The tosyl-protected 17 was successfully
converted to the imidazole-type analogs 4 via deprotection and
c
coupling chemistry, as described for the preparation of 2, 3 and 5.
In our previous report, inhibitor 1 was found to be a moderate
3. Results and discussion
SARS-CoV 3CLpro inhibitor with an IC50 value of 9.5
l
M and a Ki
value of 2.20
lM. In a first attempt to investigate the effects of
The abilities of the synthetic compounds to inhibit the protease
activity of SARS-CoV 3CLpro were evaluated. Briefly, IC50 value of
each inhibitor was assessed from the apparent decrease of a sub-
strate (H-TSAVLQSGFRK-NH2) by the digestion with R188I SARS
3CLpro as described previously.28,29 Cleavage reaction was moni-
tored analytical HPLC and the cleavage rates were calculated from
the decrease of the substrate peak area. Determining the kinetic
the N-terminal substituents (P4-moiety) on the activity profile of
1 (Table 1), the inhibitory activities of a series of aromatic (2a &
2b) and aliphatic (2c & 2d) carbamates were evaluated. None of
these compounds showed inhibitory potency comparable to 1.
The same result was obtained for the acyl derivatives, such as 2e
and 2f. Interestingly, the derivative 2g (IC50 = 6.8 lM), a phenoxymethyl
Table 1
Inhibitory activities of compounds (2a–u)
Entry no.
R
R2
Inhibition
Entry no.
R
R2
Inhibition
IC50
(lM)
Ki (
lM)
IC50
(lM)
Ki (lM)
O
O
2a
36
NT
2l
1.9
NT
O2N
O
2b
2c
85
NT
NT
2m
2n
0.75
1.2
NT
O
250
0.011
2d
2e
280
13
NT
NT
2o
2p
0.65
NT
NT
0.003
2f
24
NT
2q
1.7
NT
2g
2h
6.8
2
NT
NT
2r
2s
3.4
2.9
NT
0.22
2i
2j
1.7
0.0041
0.022
0.014
2t
2u
1
1.5
7.5
9.5
0.22
NT
2.3
2k
0.92
NT
NT = not tested.

S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
415
Table 2
Inhibitory activities of compounds (3a–d & 4a–c)
O
NH
NH
N
O
O
S
S
H
N
H
N
R
R
N
H
N
H
N
H
N
H
N
N
O
R1
O
O
R1
3 (a-d)
O
4 (a-c)
Entry no.
R
R1
Inhibition IC50
>1600
(l
M)
Entry no.
R
R1
Inhibition IC50
260
(lM)
3a
4a
3b
3c
NT
4b
4c
1
210
O
NT
>1600
9.5
O
3d
>1600
NT = not tested.
Table 3
Inhibitory activities of compounds (5a–i)
Entry no.
R
R2
Inhibition Ki (
l
M)
Entry no.
R
R2
Inhibition Ki (lM)
S
S
5a
0.06
5f
22
N
N
S
S
5b
5c
5d
0.27
1.46
56
5g
5h
5i
0.47
0.17
0.33
O
N
N
S
S
N
N
O
S
S
N
N
Cl
S
5e
1.3
2i
0.0041
N
carbonyl, displayed a slightly higher activity than 1. Thus, the pres-
ence of the phenoxymethyl carbonyl at the N-terminal position ap-
peared to enhance the activity of 1.
and 2j bearing benzothiazole exhibited four- to fivefold higher
activities (2i; IC50 = 1.7 M, 2j; IC50 = 2.3 M) than 1 and 10- to
l
l
13-fold higher activities than 2h. Notably, 2i exhibited very potent
inhibition with a Ki value of 4.1 nM. This finding strongly suggested
that the benzothiazole unit in 1 was a suitable chemical warhead
group for occupying the S10-site. In the subsequent studies, inhib-
itors 2i and 2j were advanced as lead compounds for further
optimization.
A series of electron-donating or electron-withdrawing substitu-
ents were introduced onto the phenyl ring of the P4-moiety of 2j.
Indeed, compounds with an electron donating substituent such
as methoxy, hydroxyl, or N,N0-dimethylamino at the o-, p- or
m-positions (2k, 2l or 2m: p-, o- or m-methoxy, respectively;
The molecular modeling study involving 1 and the 3CLpro en-
zyme (Fig. 1) provided insight and understanding into the binding
of the inhibitor to the active site of the enzyme (See Section 4 and
Fig. 2). The introduction of a spacious warhead group (P10) was
suggested as a means for increasing the inhibitory activity; there-
fore, we modified inhibitor 1 to include larger units, such as 4,5-
dimethylthiazole, 5-methylthiazole, benzothiazole and a series of
5-arylated thiazoles at the P10 position.
The compound bearing 4,5-dimehtylthiazole, 2h, displayed a
lower inhibitory activity (IC50 = 22 lM) than 1. Compounds 2i

416
S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
Figure 2. Molecular dynamics stimulated pose of compound 2o, (yellow stick) bound to SARS-CoV 3CLpro (PDB ID: 1WOF (green stick) with blue molecular surface); with
lead compound 1 (red stick); (A) overlapped view of 2o with an original vinyl ester (green stick) and lead 1 (red stick); (B) contacted residues with hydrogen bonding
interactions (dotted lines).
2n: p-hydroxyl; and 2o or 2p: p- or m-N,N0-dimethylamino,
respectively) exhibited more potent inhibition than 2j. The m-
including 5b, 5g–i, were in the submicromolar range. These studies
confirmed that the benzothiazole unit was appropriate as a war-
head group for the P10-moiety.
methoxy (2m: IC50 = 0.75
IC50 = 0.65 M) analogs stood out in their inhibitory strengths.
l
M) and p-N,N0 dimethylamino (2o:
l
The most promising inhibitor was the m-N,N0-dimethylamino
derivative (2p), with a Ki value of 3.1 nM; however, analogs with
an electron-withdrawing substituent (2q: p-carboxyl or 2r: p-
nitro) displayed relatively low potencies. These results strongly
suggested that an electron-donating substituent on the phenyl
ring of the P4-moiety was important and favorable to enhance
4. Docking study
Previously, we demonstrated that the inhibitory activity of a
compound containing a thiazole residue for insertion into the
S10-pocket was not time-dependent, suggesting a reversible strong
binding interaction with the protease.25,27 Here, we examined the
molecular docking of the potent active compound, 2o, in compar-
ison with the lead compound 1 and a structurally similar ligand,31
the docking structure of which has been elucidated by x-ray crys-
inhibitory activity against 3CLpro
.
Isosteric replacement around the P4-scaffold in the context of 2j
yielded notable differences from the inhibitory activities of 2j.
Replacement of phenoxy (2j) with pyridinyloxy (2s) did not ham-
per the inhibitory potency. Interestingly, the potency was recov-
ered upon phenylamino substitution (2t: IC50 = 1.5 lM). The
chain length between the P4-carbonyl and the phenyl group con-
tributed significantly to the inhibitor potency, as indicated by a de-
creased in the activity of the analog 2u (IC50 = 7.5 lM).
A variety of substituent groups were introduced into the P2-
moiety (3a–d; Table 2). Initially, the stereochemistry of P2 and a
size-appropriate amino acid residue at the S2-position in 2i were
tallography (PDB ID: 1WOF, Ki = 10.7 l
M)31 (Fig. 2). Several mini-
mization processes were performed using the MMFF94X force
field to model the solvation environment surrounding the inhibi-
tor. A molecular simulation was subsequently performed. Figure
2A shows that the 2o moieties P1–P3 (yellow stick) interacted with
the same region of the enzyme as the lead 1. Interestingly, the ben-
zothiazole unit occupied the S10-pocket more tightly than the
smaller thiazole moiety. The minimized energies for 2o and 1 ob-
tained from the docking study were ꢁ43.65 and ꢁ37.56 kcal/mol,
respectively.32 Additionally, the 4-N,N0-dimethylaminophenoxy
acetyl group adopted a unique folding conformation by forming
tested. Compound 3a, in which L-leucine was replaced with D-leu-
cine, dramatically reduced the inhibitory activity. Replacement
with bulky hydrophobic side chains, such as cyclohexylmethyl
(3b) and benzyl (3c) resulted in solubility issue in the enzyme as-
say, and replacement with polar glutamic acid (3d) did not in-
new hydrophobic interactions with
site, as shown in Figure 2B.
a-carbon of Ala 191 at the P4
crease the inhibitory potency. L-leucine, therefore, appeared to
5. Conclusions
provide a suitable stereochemistry and appropriate size for the
P2-moiety to fit into the S2-pocket. This result also well correlated
with the docking study.
The hydrogen-bonding property of the pyrrolidone structure at
the P1-side chain of 1 was studied with an imidazole moiety. As
shown in Table 2, none of these compounds (4a–c) exhibited nota-
ble inhibitory activity against SARS-CoV 3CLpro. Thus, the cyclic
We describe here the design, synthesis and biological evalua-
tion of a series of tripeptide-type SARS-CoV 3CLpro inhibitors in a
SAR study. A docking study of the initial lead compound 1 bound
to the SARS-CoV 3CLpro provided a better understanding of the
inhibitor-active site structure and interactions. These studies led
to the development of several potent inhibitors, including 2i, 2k,
2m, 2o, and 2p with IC50 or Ki values in the submicromolar to
nanomolar range. Compounds 2i and 2p exhibited the most potent
inhibitory activity, with Ki values of 4.1 and 3.1 nM, respectively.
These results clearly indicated that the peptidomimetics were
promising inhibitors for the development of potential therapeutic
agents against SARS. The structural requirements of the peptidom-
imetics displaying potent inhibitory activity against SARS-CoV
3CLpro could be summarized as follows: (i) a benzothiazole unit
amide (c-lactam) at the P1 site was crucial for potent inhibitory
activity.
The P10-moiety was next examined by varying the 5-substituted
thiazoles (5a–i; Table 3). The inhibitory activities (Ki values) of
compounds 5a–i are listed in Table 3. Analog 5a (Ki = 60 nM)
showed 15-times lower activity than 2i; however, the other 5-ary-
lated thiazoles (5b–i) generally exhibited very low inhibitory activ-
ities compared to 5a, although the Ki values of some inhibitors,

S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
417
was effectively accommodated as a chemical warhead by the S10-
pocket; (ii) hydrogen-bonding capabilities at the cyclic lactam at
the S1-position; (iii) appropriate stereochemistry and a size-appro-
priate L-leucine moiety in the S2-hydrophobic pocket and (iv) a un-
ique folding conformation assumed by the phenoxyacetyl moiety
at the S4-site.
To a solution of thiazole (1.373 g, 10.0 mmol) in THF at ꢁ78 °C
was added n-BuLi (2.0 mol in THF, 1.67 mL) dropwise over
15 min. After 1 h stirring, the Weinreb amide 12 (0.640 g,
2.0 mmol) in THF was slowly added dropwise over 20 min and
the solution was stirred for 3 h. The reaction was quenched with
sat. NH4Cl and allowed to stir at 0 °C for 20 min. The mixture
was evaporated and dissolved in ethyl acetate. This solution was
washed with water and brine, and then dried over Na2SO4. The or-
ganic layer was concentrated under reduced pressure and the
resulting residue was subjected to flash chromatography (EtOAc/
MeOH = 9:1) to obtain the pure compound 13a.
6. Experimental
6.1. Materials and methods
The data for the compound 12 & 13a has been reported in a pre-
Reagents and solvents were purchased from Wako Pure
Chemical Ind., Ltd. (Osaka, Japan), and Aldrich Chemical Co. Inc.
(Milwaukee, 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 100 mm; Sun-
vious article.25
Compounds 13b–d were prepared from 12 according to the pro-
cedure described for the synthesis of 13a.
6.2.2. tert-Butyl ((S)-1-(4,5-dimethylthiazol-2-yl)-1-oxo-3-((S)-
2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (13b)
Fire Prep C18 OBD™, 5 lm) with a binary solvent system: a linear
51% yield from 12; yellow solid; 1H NMR (400 MHz, CDCl3) d
5.89 (br s, 1H), 5.48–5.46 (m, 1H), 3.40–3.35 (m, 2H), 2.72–2.55,
(m, 5H), 2.41 (s, 3H), 2.18–2.00 (m, 3H), 1.44 (s, 9H); HRMS
gradient of CH3CN in 0.1% aqueous TFA at a flow rate of 6 mL/min,
detected at UV 254 and 222 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 spec-
trometer, a Varian Mercury 300 spectrometer (300 MHz) or a BRU-
KER 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 spectrometer. Mass spectra (ESI) were recorded on LCMS-
2010EV (SHIMADZU).
(ESI): m/z calcd for
C
17H25N3O4S [M+H]+ 367.1566, found
367.1568.
6.2.3. tert-Butyl ((S)-1-(benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)carbamate (13c)25
Compound 13c25 was prepared from 12 using a procedure sim-
ilar to that described for the preparation of 13a. The data for the
compound 13c has been reported in a previous article.25
6.2. General solid-phase synthetic procedure for the
preparation of the dipeptides (9)
6.2.4. tert-Butyl ((S)-1-(5-methylthiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)carbamate (13d)25
55% yield from 12; brown solid; 1H NMR (400 MHz, CDCl3) d
7.67 (s, 1H), 5.53–5.52 (m, 1H), 3.40–3.38 (m, 2H), 2.65–2.52 (m,
3H), 2.33 (s, 3H), 2.13–1.98 (m, 3H), 1.44 (s, 9H); HRMS (ESI): m/
z calcd for C16H24N3O4S [M+H]+ 354.1488, found 354.1491.
To the Wang resin (1.0 mmol) in DMF (5 mL) was added Fmoc-
amino acid (3 equiv), DIC (3 equiv) and DMAP (0.25 equiv), and the
mixture was stirred for 3 h. The resin was filtered, washed with
DMF and CH2Cl2 and dried under vacuum to yield 6. After removal
of the Fmoc-group using 20% piperidine in DMF over 20 min, the
next appropriate amino acid was coupled to the resin using the
coupling agents DIC (3 equiv) and HOBt (3 equiv) by solid-phase
synthesis techniques. The resulting intermediate 7 was then trea-
ted with 20% piperidine in DMF for 20 min to remove the Fmoc-
group and subsequently reacted with the corresponding carboxylic
acid (3 equiv) by the DIC-HOBt method or acid chloride (3 equiv) in
the presence of Et3N to produce 8. Finally, the resin was treated
with TFA/H2O (10:1, 2 mL), and the mixture was filtered after
1 h. The filtrate was removed under high vacuum to give the
desired dipeptides 9, which were used directly in the next step
without further purification or analysis.
6.2.5. tert-Butyl ((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-(5-
phenylthiazol-2-yl)propan-2-yl)carbamate (13e)
To a cooled solution of the commercially available 5-phenyl-
thiazole (4.78 mmol) in dry THF at ꢁ78 °C, a solution of LDA
(6.2 mmol) was slowly added. After 1 h, a pre-cooled solution of
Weinreb amide 12 in dry THF was slowly added and the reaction
was stirred at ꢁ78 °C for 2 h. The solution was allowed to warm
to room temperature, was quenched by the addition of water
(35 mL), and was extracted with ethyl acetate (3 ꢂ 50 mL). The
combined organic layers were dried over anhydrous Na2SO4, fil-
tered, and evaporated in vacuo. The crude mixture was then puri-
fied using flash chromatography (n-hexane/EtOAc = 3:7) to furnish
13e.
55% yield from 12; brown solid; 1H NMR (400 MHz, CDCl3) d
8.16 (s, 1H), 7.63–7.62 (d, J = 8.4 Hz, 2H), 7.47–7.42 (m, 3H),
5.53–5.52 (m, 1H), 3.40–3.38 (m, 2H), 2.65–2.52 (m, 2H), 2.13–
1.98 (m, 3H), 1.44 (s, 9H); HRMS (ESI): m/z calcd for C21H26N3O4S
[M+H]+ 416.1644, found 416.1653.
6.2.1. tert-Butyl ((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-
(thiazol-2-yl)propan-2-yl)carbamate (13a)25
Compound 11 was prepared through sequential reactions from
the well-known intermediate 10, as reported previously.27
To a solution of the acid 11 (5.0 g, 12.0 mmol in DMF, 80 mL)
was added EDCꢀHCl (1.85 g, 13.44 mmol), HOBt (1.56 g, 13.44
mmol) and N,O-dimethylhydroxylamine (1.31 g, 13.44 mol) at
ambient temperature. The solution was cooled to 0 °C and TEA
(1.87 mL, 13.44 mmol) was added slowly. After 2 h, the DMF was
evaporated and the resulting residue was dissolved in ethyl acetate
(100 mL). The organic phase was subsequently washed with 5% cit-
ric acid (50 mL), 5% NaHCO3 (50 mL) and brine (50 mL). This organ-
ic layer was then dried over Na2SO4 and concentrated under
reduced pressure to yield the Weinreb amide derivative 12, which
was purified by column chromatography (EtOAc/MeOH = 9.5:0.5).
Compounds 13f–j were prepared from 12 according to the pro-
cedure described for the synthesis of 13e.
6.2.6. tert-Butyl ((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-(5-(p-
tolyl)thiazol-2-yl)propan-2-yl)carbamate (13f)
51% yield from 12; brown solid; 1H NMR (400 MHz, CDCl3) d
8.12 (s, 1H), 7.52–7.50 (d, J = 8.0 Hz, 2H), 7.39–7.22 (d, J = 8.0 Hz,
2H), 5.45–5.32 (m, 1H), 3.40–3.26 (m, 2H), 2.65–2.50 (m, 2H),
2.39 (s, 3H), 2.12–1.94 (m, 3H), 1.43 (s, 9H); HRMS (ESI): m/z calcd
for C22H28N3O4S [M+H]+ 430.1801, found 430.1802.

418
S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
6.2.7. tert-Butyl ((S)-1-(5-(4-methoxyphenyl)thiazol-2-yl)-1-
oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (13g)
48% yield from 12; brown solid; 1H NMR (400 MHz, CDCl3) d
8.07–8.06 (d, J = 4.0 Hz, 1H), 7.57–7.55 (d, J = 8.8 Hz, 2H), 6.97–
6.95 (d, J = 8.4 Hz, 2H), 5.47–53 (m, 1H), 3.81 (s, 3H), 3.39–3.29
(m, 2H), 2.60–2.52 (m, 2H), 2.12–1.93 (m, 3H), 1.44 (s, 9H); HRMS
(ESI): m/z calcd for C22H28N3O5S [M+H]+ 446.1750, found 446.1754.
deprotected lactam residue (3 mmol) was coupled to the dipepti-
dic 9 (1.1 equiv) using the coupling agent HBTU (1.1 equiv) and
HOBt (1.1 equiv) in the presence of diisopropylethylamine (DIPEA,
1.1 equiv) in DMF (3 mL) at 0 °C. The reaction mixture was allowed
to stir for 2–3 h under ambient conditions. The solvent was then
evaporated under high vacuum, and the residue was dissolved in
ethyl acetate (50 mL). The organic layer was washed with 5% citric
acid (25 mL), 5% NaHCO3 (25 mL) and brine (25 mL). This solution
was dried over Na2SO4, filtered and evaporated under reduced
pressure to give a compound 2a.
6.2.8. tert-Butyl ((S)-1-(5-(4-chlorophenyl)thiazol-2-yl)-1-oxo-
3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (13h)
56% yield from 12; brown solid; 1H NMR (400 MHz, CDCl3) d
8.14–8.13 (d, J = 4.4 Hz, 1H), 7.56–7.54 (d, J = 8.8 Hz, 2H), 7.44–
7.42 (d, J = 8.4 Hz, 2H), 5.51–5.44 (m, 1H), 3.40–3.31(m, 2H),
2.61–2.36 (m, 2H), 2.12–1.93 (m, 3H), 1.44 (s, 9H); HRMS (ESI):
m/z calcd for C21H25ClN3O4S [M+H]+ 450.1254, found 450.1245.
Compounds 2f–u were prepared from 13a–c with 9 using a pro-
cedure similar to that described for the synthesis of 2a. Com-
pounds 2a–u were purified by reverse phase HPLC.
6.3.1. 4-Nitrobenzyl ((S)-3-methyl-1-(((S)-4-methyl-1-oxo-1-
(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-(thiazol-2-yl)propan-
2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)carbamate
(2a)
6.2.9. tert-Butyl ((S)-1-(5-(naphthalen-2-yl)thiazol-2-yl)-1-oxo-
3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (13i)
57% yield from 12; brown solid; 1H NMR (400 MHz, CDCl3) d
8.29–8.28 (d, J = 4.0 Hz, 1H), 8.11 (s, 1H), 7.92–7.85 (m, 3H),
7.76–7.74 (d, J = 8.4 Hz, 1H), 7.56–7.52 (m, 2H), 5.51–5.43 (m,
1H), 3.41–3.30 (m, 2H), 2.63–2.33 (m, 2H), 2.12–1.91 (m, 3H),
1.45 (s, 9H); HRMS (ESI): m/z calcd for C22H28N3O4S [M+H]+
466.1801, found 466.1798.
25% yield from 13a; white powder; 1H NMR (500 MHz, DMSO-
d6) d 8.63 (d, J = 7.6 Hz, 1H), 8.26 (d, J = 3.1 Hz, 1H), 8.22 (d,
J = 8.6 Hz, 2H), 8.18 (d, J = 3.1 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H),
7.65 (s, 1H), 7.62 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.9 Hz, 1H), 5.46–
5.39 (m, 1H), 5.18 (s, 2H), 4.38 (dd, J = 8.4, 14.8 Hz, 1H), 3.88 (dd,
J = 7.0, 8.6 Hz, 1H), 3.18 (dd, J = 8.7, 8.8 Hz, 1H), 3.11 (dd, J = 9.2,
16.3 Hz, 1H), 2.48–2.40 (m, 1H), 2.23–2.12 (m, 1H), 2.08–2.01 (m,
1H), 2.00–1.92 (m, 1H), 1.81–1.70 (m, 2H), 1.62–1.53 (m, 1H),
1.48–1.39 (m, 2H), 0.92–0.79 (m, 12H); 13C NMR (125 MHz,
DMSO-d6) d 191.3, 177.9, 172.2, 170.8, 164.4, 155.8, 146.9, 145.3,
145.2, 128.4, 127.9, 123.4, 64.1, 60.1, 53.0, 50.7, 40.8, 39.4, 37.8,
32.3, 30.4, 27.2, 24.0, 22.9, 21.7, 19.1, 18.1; HRMS (ESI): m/z calcd
for C29H39N6O8S [M+H]+ 631.2550, found 631.2551.
6.2.10. tert-Butyl ((S)-1-(5-(2-methoxyphenyl)thiazol-2-yl)-1-
oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (13j)
51% yield from 12; brown solid; 1H NMR (400 MHz, CDCl3) d
8.15–8.14 (d, J = 4.0 Hz, 1H), 7.38–7.34 (t, J = 8.0 Hz, 2H), 7.22–
7.20 (d, J = 8.0 Hz, 1H), 7.13–7.11 (d, J = 8.0 Hz, 1H), 6.99–6.95 (m,
1H), 5.45–533 (m, 1H), 3.83 (s, 3H), 3.40–3.29 (m, 2H), 2.62–2.33
(m, 2H), 2.10–1.89 (m, 3H), 1.44 (s, 9H); HRMS (ESI): m/z calcd
for C22H28N3O5S [M+H]+ 446.1750, found 446.1737.
6.3.2. Phenyl ((S)-3-methyl-1-(((S)-4-methyl-1-oxo-1-(((S)-1-
oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-(thiazol-2-yl)propan-2-
yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)carbamate (2b)
26% yield from 13a; white powder; 1H NMR (500 MHz, DMSO-
d6) d 8.66 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 3.2 Hz, 1H), 8.17 (d,
J = 3.2 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 8.9 Hz, 1H),
7.64 (s, 1H), 7.36 (t, J = 8.1 Hz, 2H), 7.19 (d, J = 7.5 Hz, 1H), 7.06
(d, J = 7.6 Hz, 2H), 5.46–5.38 (m, 1H), 4.40 (dd, J = 9.0, 15.5 Hz,
1H), 3.89 (dd, J = 7.3, 8.5 Hz, 1H), 3.16 (dd, J = 9.7, 8.5 Hz, 1H),
3.09 (dd, J = 9.1, 16.4 Hz, 1H), 2.48–2.41 (m, 1H), 2.22–2.12 (m,
1H), 2.08–1.93 (m, 2H), 1.81–1.69 (m, 2H), 1.68–1.54 (m, 1H),
1.48–1.35 (m, 2H), 0.93–0.80 (m, 12H); 13C NMR (125 MHz,
DMSO-d6) d 191.3, 177.9, 172.2, 170.6164.4, 154.3, 151.1, 145.3,
129.2, 128.4, 124.9, 121.6, 60.2, 53.0, 50.8, 40.8, 39.4, 37.8, 32.3,
30.3, 27.2, 24.0, 22.8, 21.8, 19.1, 18.2; HRMS (ESI): m/z calcd for
6.2.11. tert-Butyl ((2S)-3-(4,5-dihydro-1H-imidazol-4-yl)-1-(5-
methylthiazol-2-yl)-1-oxopropan-2-yl)carbamate (16)
The compound 16 was synthesized from 14 using a method
similar to that described for the preparation of 13a.
51% yield from 14; brown solid; 1H NMR (300 MHz, CDCl3) d
8.04 (d, J = 2.93 Hz, 1H), 7.71 (d, J = 2.90 Hz, 1H), 7.58 (s, 1H),
6.74 (s, 1H), 5.64 (br s, 1H), 3.32 (br s, 1H), 1.43 (s, 9H). HRMS
(ESI): m/z calcd for C14H18N4O3S [M+H]+ 323.1178, found 323.1183.
6.2.12. (S)-2,2-Dimethyl-1-(((1-oxo-1-(thiazol-2-yl)-3-(1-tosyl-
1H-imidazol-4-yl)propan-2-yl)amino)oxy)propan-1-one (17)
To a solution of 16 (1.6 mmol) in THF (20 mL) at 0 °C was added
tosyl chloride (1.5 equiv). After 10 min, Et3N (1.5 equiv) was added
slowly and allowed to stir for 3 h at the same condition. The sol-
vent was evaporated and the resulting residue was dissolved in
ethyl acetate (50 mL). This organic phase was washed with 5%
citric acid (25 mL), 5% NaHCO3 (25 mL) and brine (25 mL). This
layer was then dried over Na2SO4 and concentrated under reduced
pressure to yield 17, which was purified by column chromatogra-
phy (n-hexane/EtOAc = 5:5).
C
28H38N5O6S [M+H]+ 572.2543, found 572.2531.
6.3.3. Isobutyl ((S)-3-methyl-1-(((S)-4-methyl-1-oxo-1-(((S)-1-
oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-(thiazol-2-yl)propan-2-
yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)carbamate (2c)
38% yield from 13a; white powder; 1H NMR (500 MHz, DMSO-
d6) d 8.74 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 7.5 Hz, 1H), 8.10 (d,
J = 3.2 Hz, 1H), 8.03 (d, J = 3.2 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H),
5.46–5.39 (m, 1H), 5.18 (s, 2H), 4.38 (dd, J = 8.4, 14.8 Hz, 1H),
3.88 (dd, J = 7.0, 8.6 Hz, 1H), 3.18 (dd, J = 8.7, 8.8 Hz, 1H), 3.11
(dd, J = 9.2, 16.3 Hz, 1H), 2.48–2.40 (m, 1H), 2.23–2.12 (m, 2H),
2.08–2.01 (m, 1H), 2.00–1.92 (m, 1H), 1.81–1.70 (m, 2H), 1.62–
1.53 (m, 1H), 1.48–1.39 (m, 2H), 0.92–0.86 (m, 12H), 0.85–0.80
(m, 6H); 13C NMR (125 MHz, DMSO-d6) d 191.4, 173.9, 170.2,
169.0, 165.3, 157.1, 144.7, 128.1, 72.5, 60.9, 54.2, 51.6, 40.3, 39.4,
39.8, 33.2, 31.7, 31.0, 27.1, 25.9, 24.2, 23.1, 22.0, 19.1; HRMS
72% yield from 16; yellow solid; 1H NMR (300 MHz, CDCl3) d
8.02 (d, J = 3.2 Hz, 1H), 7.71 (d, J = 2.90 Hz, 1H), 7.60–7.57 (m,
3H), 7.43 (s, 1H), 7.33–7.31 (d, J = 8.1 Hz, 2H), 5.57 (br s, 1H),
2.37 (s, 3H), 1.44 (s, 9H). HRMS (ESI): m/z calcd for C21H24N4O5S2
[M+H]+ 477.1300, found 177.1312.
6.3. Synthetic procedure for the preparation of 2a
To a solution of 13a (2 mmol) in CH2Cl2 (2 mL) at 0 °C was
added TFA/H2O (10 mL) and the solution was stirred for 1 h. After
evaporating the solvent under reduced pressure, the corresponding
(ESI): m/z calcd for
C
26H41N5O6S [M+H]+ 551.2778, found
551.2780.

S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
419
6.3.4. Neopentyl ((S)-3-methyl-1-(((S)-4-methyl-1-oxo-1-(((S)-1-
oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-(thiazol-2-yl)propan-2-
yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)carbamate (2d)
78% yield from 13a; white powder; 1H NMR (500 MHz, DMSO-
d6) d 8.61 (d, J = 7.3 Hz, 1H), 8.25 (d, J = 3.1 Hz, 1H), 8.17 (d,
J = 3.0 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.64 (s, 1H), 7.01 (d,
J = 8.9 Hz, 1H), 5.43–5.37 (m, 1H), 4.37 (dd, J = 8.0, 15.3 Hz, 1H),
3.83 (dd, J = 7.0, 8.8 Hz, 1H), 3.70–3.57 (m, 2H), 3.18 (dd, J = 9.4,
9.4 Hz, 1H), 3.09 (dd, J = 9.2, 16.4 Hz, 1H), 2.48–2.39 (m, 1H),
2.22–2.10 (m, 1H), 2.06–1.99 (m, 1H), 1.97–1.84 (m, 1H), 1.80–
1.67 (m, 2H), 1.62–1.51 (m, 1H), 1.47–1.33 (m, 2H), 0.93–0.78
(m, 21H); 13C NMR (125 MHz, DMSO-d6) d 191.2, 177.9, 172.2,
171.0, 164.4, 156.4, 145.2, 128.4, 72.8, 59.9, 53.0, 50.7, 40.9, 39.4,
37.8, 32.3, 31.4, 30.4, 27.2, 26.2, 24.0, 22.9, 21.7, 19.1, 18.2; HRMS
(ESI): m/z calcd for C27H44N5O6S [M+H]+ 566.3012, found 566.3008.
21.8, 19.1, 17.8; HRMS (ESI): m/z calcd for C29H40N5O6S [M+H]+
586.2699, found 586.2695.
6.3.8. Benzyl ((S)-1-(((S)-1-(((S)-1-(4,5-dimethylthiazol-2-yl)-1-
oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-
1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-
yl)carbamate (2h)
47% yield from 13b; white powder; 1H NMR (500 MHz, CD3OD)
d 8.09 (d, J = 7.5 Hz, 1H), 7.38–7.26 (m, 5H), 5.60–5.55 (m, 1H), 5.09
(s, 2H), 4.49–4.43 (m, 1H), 3.95 (d, J = 7.1 Hz, 1H), 3.36–3.25 (m,
2H, overlapping with MeOH), 2.68–2.59 (m, 1H), 2.47 (s, 1H),
2.40 (s, 1H), 2.39–2.33 (m, 1H), 2.15- 2.01 (m, 1H), 2.00–1.92 (m,
2H), 1.73–1.64 (m, 1H), 1.62–1.54 (m, 2H), 0.98–0.88 (m, 12H);
13C NMR (125 MHz, CD3OD) d 191.7, 181.8, 174.7, 174.2, 160.4,
158.6, 153.3, 138.6, 129.5, 129.0, 128.8, 67.7, 62.0, 54.5, 53.2,
41.9, 41.5, 40.0, 34.3, 32.0, 28.7, 25.8, 23.3, 22.2, 19.8, 18.6, 14.9,
11.9; HRMS (ESI): m/z calcd for C31H44N5O6S [M+H]+ 614.3012,
found 614.2993.
6.3.5. (S)-4-Methyl-2-((S)-3-methyl-2-(3-phenylpropanamido)-
butanamido)-N-((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-
(thiazol-2-yl)propan-2-yl)pentanamide (2e)
42% yield from 13a; white powder; 1H NMR (500 MHz, DMSO-
d6) d 8.57 (d, J = 7.5 Hz, 1H), 8.25 (d, J = 2.9 Hz, 1H), 8.17 (d,
J = 2.9 Hz, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.83 (d, J = 8.9 Hz, 1H),
7.63 (s, 1H), 7.26–7.09 (m, 1H), 5.46–5.38 (m, 1H), 4.32 (dd,
J = 8.8, 15.8 Hz, 1H), 4.16 (dd, J = 7.2, 8.7 Hz, 1H), 3.18 (dd, J = 9.2,
8.9 Hz, 1H), 3.08 (dd, J = 9.2, 16.4 Hz, 1H), 2.78 (t, J = 7.6 Hz, 2H),
2.55–2.37 (m, 3H, overlapping with DMSO-d6), 2.26–2.12 (m,
1H), 2.07–1.99 (m, 1H), 1.93–1.85 (m, 1H), 1.80–1.68 (m, 2H),
1.62–1.52 (m, 1H), 1.47–1.36 (m, 2H), 0.85 (dd, J = 6.5, 26.4 Hz,
6H), 0.76 (dd, J = 6.8, 12.7 Hz, 6H); 13C NMR (125 MHz, DMSO-d6)
d 191.3, 178.0, 172.2, 171.3, 170.9, 164.4, 145.3, 141.3, 128.4,
128.2, 125.8, 57.4, 53.0, 50.8, 40.7, 39.4, 37.8, 36.6, 32.4, 31.2,
30.6, 27.6, 24.1, 22.9, 21.8, 19.1, 18.1; HRMS (ESI): m/z calcd for
6.3.9. Benzyl ((S)-1-(((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)amino)-3-methyl-1-oxobutan-2-yl)carbamate
(2i)
24% yield from 13c; white powder; 1H NMR (500 MHz, CD3OD)
d 8.21 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.66–7.57 (m, 2H),
7.37–7.26 (m, 5H), 5.72 (d, J = 8.3 Hz, 1H), 5.09 (s, 2H), 4.52–4.42
(m, 1H), 3.97–3.92 (m, 1H), 3.39–3.32 (m, 2H, overlapping with
MeOH), 2.76–2.67 (m, 1H), 2.48–2.37 (m, 1H), 2.26–2.18 (m, 1H),
2.11–1.99 (m, 3H), 1.71–1.53 (m, 3H), 0.99–0.85 (m, 12H); 13C
NMR (125 MHz, CD3OD) d 193.5, 181.8, 174.9, 174.2, 165.5,
158.6, 154.8, 138.4, 138.2, 129.5, 129.3, 129.0, 128.8, 128.5,
126.5, 123.7, 67.7, 62.0, 55.2, 53.2, 41.9, 41.5, 40.0, 33.8, 32.0,
28.8, 25.8, 23.2, 22.2, 19.8, 18.6; HRMS (ESI): m/z calcd for
C
30H42N5O5S [M+H]+ 584.2907, found 584.2913.
C
33H42N5O6S [M+H]+ 636.2856, found 636.2843.
6.3.6. (S)-2-((S)-2-Acetamido-3-methylbutanamido)-4-methyl-
N-((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-(thiazol-2-
yl)propan-2-yl)pentanamide (2f)
6.3.10. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-((S)-3-methyl-2-
(2-phenoxyacetamido)butanamido)pentanamide (2j)
37% yield from 13a; white powder; 1H NMR (500 MHz, CD3OD)
d 8.74 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 7.5 Hz, 1H), 8.10 (d, J = 3.2 Hz,
1H), 8.02 (d, J = 3.2 Hz, 1H), 7.96 (d, J = 8.2 Hz, 1H), 5.68–5.60 (m,
1H), 4.44 (dd, J = 7.1, 15.5 Hz, 1H), 4.19–4.15 (m, 1H), 3.38–3.30
(m, 2H, overlapping with MeOH), 2.72–2.63 (m, 1H), 2.45–2.37
(m, 1H), 2.18–2.09 (m, 1H), 2.08–2.00 (m, 2H), 1.99–1.96 (s, 3H),
1.74–1.65 (m, 1H), 1.63–1.54 (m, 1H), 0.99–0.90 (m, 12H); 13C
NMR (125 MHz, CD3OD) d 192.0, 181.8, 174.9, 173.84, 173.7,
173.4, 165.9, 146.3, 128.6, 119.2, 60.3, 60.2, 55.1, 55.0, 53.3, 53.2,
41.9, 41.9, 41.5, 40.0, 34.1, 31.9, 28.7, 25.8, 23.3, 22.4, 22.2, 19.8,
18.7; HRMS (ESI): m/z calcd for C23H36N5O5S [M+H]+ 494.2437,
found 494.2424.
28% yield from 13c; white powder; 1H NMR (500 MHz, CD3OD)
d 8.31 (d, J = 7.2 Hz, 1H), 8.21 (d, J = 7.6 Hz, 1H), 8.11 (d, J = 7.5 Hz,
1H), 7.79 (d, J = 8.7 Hz, 1H), 7.66–7.55 (m, 2H), 7.30 (t, J = 6.5 Hz,
1H), 7.01–6.95 (m, 3H), 5.73 (dd, J = 3.3, 11.6 Hz, 1H), 4.58 (s,
2H), 4.44 (dd, J = 7.0, 15.7 Hz, 1H), 4.33 (dd, J = 7.2, 8.7 Hz, 1H),
3.41–3.30 (m, 2H, overlapping with MeOH), 2.78–2.70 (m, 1H),
2.45–2.41 (m, 1H), 2.25–2.18 (m, 1H), 2.11–2.01 (m, 3H), 1.69–
1.53 (m, 3H), 0.98–0.83 (m, 12H); 13C NMR (125 MHz, CD3OD) d
193.5, 181.8, 174.8, 173.3, 171.1, 165.5, 159.1, 154.8, 138.4,
130.7, 129.3, 128.5, 126.5, 123.7, 122.9, 115.9, 68.1, 59.4, 55.2,
53.4, 41.8, 41.5, 40.0, 33.8, 32.5, 28.8, 25.8, 23.2, 22.2, 19.8, 18.5;
HRMS (ESI): m/z calcd for C33H42N5O6S [M+H]+ 636.2856, found
636.2842.
6.3.7. (S)-4-Methyl-2-((S)-3-methyl-2-(2-phenoxyacetamido)-
butanamido)-N-((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-1-
(thiazol-2-yl)propan-2-yl)pentanamide (2g)
6.3.11. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-
2-oxopyrrolidin-3-yl)propan-2-yl)-2-((S)-2-(2-(4-
methoxyphenoxy)acetamido)-3-methylbutanamido)-
4-methylpentanamide (2k)
53% yield from 13a; white powder; 1H NMR (500 MHz, DMSO) d
8.64 (d, J = 7.7 Hz, 1H), 8.25 (d, J = 2.8 Hz, 1H), 8.17 (d, J = 2.8 Hz,
1H), 8.10 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 9.0 Hz, 1H), 7.64 (br s,
1H), 7.30–7.23 (m, 2H), 6.97–6.90 (m, 5H), 5.45–5.39 (m, 1H),
4.55 (dd, J = 14.7, 19.3 Hz, 2H), 4.35 (dd, J = 8.5, 14.9 Hz, 1H), 4.26
(dd, J = 6.5, 9.0 Hz, 1H), 3.18 (dd, J = 9.5, 8.9 Hz, 1H), 3.09 (dd,
J = 9.2, 16.4 Hz, 1H), 2.46–2.41 (m, 1H, overlapping with DMSO),
2.22–2.13 (m, 1H), 2.08–1.92 (m, 2H), 1.60–1.51 (m, 1H), 1.48–
1.36 (m, 2H), 0.85 (dd, J = 6.6, 25.7 Hz, 6H), 0.78 (dd, J = 6.5,
26.6 Hz, 6H); 13C NMR (125 MHz, DMSO) d 191.3, 178.0, 172.1,
172.0, 170.3, 167.4, 157.7, 145.3, 129.5, 128.4, 121.1, 114.6, 66.6,
56.8, 53.0, 52.9, 50.8, 40.7, 39.4, 37.8, 32.3, 31.0, 27.3, 24.1, 22.8,
11% yield from 13c; white powder; 1H NMR (500 MHz, CD3OD)
d 8.21 (d, J = 7.4 Hz, 1H), 8.11 (d, J = 6.9 Hz, 1H), 7.67–7.56 (m, 2H),
6.92 (d, J = 9.2 Hz, 2H), 6.85 (d, J = 9.2 Hz, 2H), 5.73 (dd, J = 3.3,
11.6 Hz, 1H), 4.52 (s, 2H), 4.44 (dd, J = 6.4, 8.8 Hz, 1H), 4.32 (d,
J = 7.2 Hz, 1H), 3.74 (s, 3H), 3.39–3.30 (m, 2H, overlapping with
MeOH), 2.78–2.69 (m, 1H), 2.48–2.42 (m, 1H), 2.25–2.18 (m, 1H),
2.12–2.01 (m, 3H), 1.68–1.53 (m, 3H), 0.99–0.83 (m, 12H); 13C
NMR (125 MHz, CD3OD) d 193.5, 181.8, 174.8, 173.2, 171.2,
165.5, 156.3, 154.8, 153.2, 138.4, 129.3, 128.5, 126.5, 123.7,

420
S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
117.0, 115.8, 69.0, 59.3, 56.1, 55.2, 53.4, 41.8, 41.5, 40.0, 33.8, 32.5,
1.70–1.52 (m, 3H), 0.99–0.82 (m, 12H); 13C NMR (125 MHz,
CD3OD) d 193.5, 181.8, 174.8, 173.3, 173.2, 170.7, 165.5, 154.8,
143.3, 138.4, 129.3, 128.5, 126.5, 123.8, 120.2, 117.3, 68.6, 59.4,
55.2, 53.4, 45.2, 41.8, 416, 40.0, 33.9, 32.5, 28.8, 25.8, 23.2, 22.2,
28.8, 25.8, 23.2, 22.2, 19.8, 18.5; HRMS (ESI): m/z calcd for
C
34H44N5O7S [M+H]+ 666.2961, found 666.2993.
6.3.12. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-
2-oxopyrrolidin-3-yl)propan-2-yl)-2-((S)-2-(2-(2-
19.8, 18.5; HRMS (ESI): m/z calcd for C
35H47N6O6S [M+H]+
669.3278, found 669.3320.
methoxyphenoxy)acetamido)-3-methylbutanamido)-
4-methylpentanamide (2l)
6.3.16. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-
2-oxopyrrolidin-3-yl)propan-2-yl)-2-((S)-2-(2-(3-
(dimethylamino)phenoxy)acetamido)-3-methylbutanamido)-
4-methylpentanamide (2p)
18% yield from 13c; white powder; 1H NMR (500 MHz, CD3OD)
d 8.29 (d, J = 7.2 Hz, 1H), 8.21 (d, J = 7.4 Hz, 1H), 8.11 (d, J = 8.0 Hz,
1H), 7.99 (d, J = 8.5 Hz, 1H), 7.67–7.57 (m, 2H), 7.02 (d, J = 7.2 Hz,
3H), 6.92–6.86 (m, 1H), 5.78–5.70 (m, 1H), 4.57 (d, J = 3.5 Hz,
2H), 4.44 (dd, J = 6.5, 14.1 Hz, 1H), 4.34 (dd, J = 7.1, 8.6 Hz, 1H),
3.88 (s, 3H), 3.41–3.32 (m, 2H, overlapping with MeOH), 2.79–
2.69 (m, 1H), 2.48–2.41 (m, 1H), 2.26–2.17 (m, 1H), 2.15–1.99
(m, 3H), 1.72–1.53 (m, 3H), 1.00–0.82 (m, 12H); 13C NMR
(125 MHz, CD3OD) d 193.5, 181.8, 174.9, 173.2, 171.5, 165.5,
154.8, 151.5, 148.9, 138.4, 129.3, 128.5, 126.5, 124.5, 123.7,
122.2, 117.5, 113.6, 70.8, 59.3, 56.5, 55.1, 53.4, 41.8, 41.5, 40.0,
33.9, 32.6, 28.8, 25.8, 23.2, 22.2, 19.7, 18.4; HRMS (ESI): m/z calcd
for C34H43N5O7S [M+H]+ 665.2883, found 665.2881.
37% yield from 13c; light green solid; 1H NMR (500 MHz, CDCl3)
d 8.21 (br s, 1H, NH), 8.18–8.16 (d, J = 8.0 Hz, 1H), 7.99–7.97 (d,
J = 8.0 Hz, 1H), 7.61–7.58 (m, 2H), 7.43–7.40 (m, 1H), 6.50–6.48
(d, J = 8.0 Hz, 1H), 6.44 (s, 1H), 6.38–6.36 (d, J = 8.0 Hz, 1H), 5.67–
5.62 (m, 1H), 4.57–4.53 (m 1H), 4.52 (s, 2H), 4.34–4.31 (m, 1H),
3.40–3.32 (m, 2H), 3.01 (s, 6H), 2.60–2.53 (m, 2H), 2.17–2.02 (m,
3H), 2.00–1.89 (m, 1H), 1.72–1.52 (m, 3H), 0.96–0.89 (m, 12H);
13C NMR (125 MHz, DMSO-d6) d 191.4, 178.2, 172.2, 171.3, 170.9,
164.1, 145.7, 141.1, 128.4, 128.3, 125.8, 57.4, 53.1, 50.8, 40.7,
39.4, 37.8, 36.6, 32.4, 31.2, 30.6, 27.5, 24.1, 22.9, 21.7, 19.2, 18.0;
HRMS (ESI): m/z calcd for C35H48N6O6S [M+H]+ 679.3278, found
679.3287.
6.3.13. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-
2-oxopyrrolidin-3-yl)propan-2-yl)-2-((S)-2-(2-(3-
methoxyphenoxy)acetamido)-3-methylbutanamido)-
4-methylpentanamide (2m)
6.3.17. 4-(2-(((S)-1-(((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)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-
oxoethoxy)benzoic acid (2q)
14% yield from 13c; white powder; 1H NMR (500 MHz, CD3OD)
d 8.22 (d, J = 7.4 Hz, 1H), 8.13 (d, J = 6.9 Hz, 1H), 7.65–7.54 (m, 2H),
7.06 (d, J = 9.2 Hz, 1H), 6.87 (d, J = 9.2 Hz, 2H), 6.73 (s, 1H), 5.71 (dd,
J = 3.3, 11.2 Hz, 1H), 4.54 (s, 2H), 4.41 (dd, J = 6.2, 8.8 Hz, 1H), 4.31
(d, J = 7.0 Hz, 1H), 3.71 (s, 3H), 3.36–3.27 (m, 2H), 2.77–2.68 (m,
1H), 2.51–2.44 (m, 1H), 2.25–2.18 (m, 1H), 2.13–2.03 (m, 3H),
1.71–1.56 (m, 3H), 0.98 - 0.85 (m, 12H); 13C NMR (125 MHz,
CD3OD) d 193.5, 181.8, 174.8, 173.2, 171.2, 165.5, 162.5, 160.3,
154.8, 138.4, 131.2, 129.3, 128.5, 126.5, 123.7, 108.7, 107.8,
102.4, 68.2, 59.3, 55.8, 55.2, 53.3, 41.5, 40.0, 33.8, 32.5, 23.2,
22.2, 19.8, 18.5; HRMS (ESI): m/z calcd for C34H43N5O7S [M+H]+
665.2883, found 665.2882.
19% yield from 13c; white powder; 1H NMR (500 MHz, DMSO-
d6) d 12.57 (br s, 1H), 8.70 (d, J = 7.4 Hz, 1H), 8.20 (d, J = 7.5 Hz,
1H), 8.16 (d, J = 7.2 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.89 (d,
J = 8.9 Hz, 1H), 7.80 (d, J = 8.9 Hz, 2H), 7.63–7.55 (m, 2H), 6.93 (d,
J = 8.9 Hz, 2H), 5.48–5.41 (m, 1H), 4.60 (dd, J = 14.8, 19.1 Hz, 1H),
4.29 (dd, J = 8.1, 14.7 Hz, 1H), 4.19 (dd, J = 6.6, 8.8 Hz, 1H), 3.14
(dd, J = 9.3, 8.9 Hz, 1H), 3.05 (dd, J = 7.4, 16.4 Hz, 1H), 2.42–2.37
(m, 1H, overlapping with DMSO), 2.22–2.14 (m, 1H), 2.10–2.02
(m, 1H), 1.94–1.84 (m, 1H), 1.82–1.71 (m, 2H), 1.50–1.41 (m,
1H), 1.38–1.28 (m, 2H), 0.75 (dd, J = 3.0, 6.6 Hz, 6H), 0.71 (d,
J = 6.6 Hz, 6H); 13C NMR (125 MHz, DMSO-d6) d 192.8, 178.0,
172.1, 170.3, 166.9, 166.8, 164.3, 161.3, 152.8, 136.3, 131.2,
128.1, 127.4, 125.2, 123.5, 123.1, 114.4, 66.6, 56.9, 53.2, 50.8,
40.7, 39.4, 37.8, 32.1, 30.9, 27.4, 24.0, 22.7, 21.7, 19.0, 17.9; HRMS
6.3.14. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-
2-oxopyrrolidin-3-yl)propan-2-yl)-2-((S)-2-(2-(4-
hydroxyphenoxy)acetamido)-3-methylbutanamido)-
4-methylpentanamide (2n)
(ESI): m/z calcd for
C
34H41N5O8S [M+H]+ 679.2676, found
21% yield from 13c; white powder; 1H NMR (500 MHz, CD3OD)
d 8.21 (d, J = 8.1 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.66–7.56 (m, 2H),
6.84 (d, J = 9.1 Hz, 2H), 6.72 (d, J = 9.0 Hz, 2H), 5.72 (dd, J = 3.4,
11.5 Hz, 1H), 4.49 (s, 2H), 4.48–4.41 (m, 1H), 4.33 (d, J = 7.0 Hz,
1H), 3.37–3.30 (m, 2H, overlapping with MeOH), 2.78–2.70 (m,
1H), 2.49–2.37 (m, 1H), 2.36–2.18 (m, 1H), 2.15–1.99 (m, 3H),
1.71–1.52 (m, 3H), 0.96–0.81 (m, 12H); 13C NMR (125 MHz,
CD3OD) d 193.5, 181.8, 174.8, 173.2, 171.3, 165.5, 154.8, 153.4,
152.4, 138.4, 129.3, 128.5, 126.5, 123.7, 117.1, 117.0, 69.1, 59.2,
55.2, 53.3, 41.8, 41.5, 40.0, 33.8, 32.6, 28.8, 25.8, 23.2, 22.2, 19.8,
18.5; HRMS (ESI): m/z calcd for C33H42N5O7S [M+H]+ 652.2805,
found 652.2839.
679.2674.
6.3.18. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-((S)-3-methyl-2-
(2-(4-nitrophenoxy)acetamido)butanamido)pentanamide (2r)
12% yield from 13c; white powder; 1H NMR (500 MHz, CD3OD)
d 8.89 (d, J = 7.9 Hz, 1H), 8.29 (d, J = 7.3 Hz, 1H), 8.26–8.20 (m, 3H),
8.12 (d, J = 7.6 Hz, 1H), 7.67–7.58 (m, 2H), 7.15 (d, J = 9.4 Hz, 2H),
5.77–5.70 (m, 1H), 4.75 (s, 2H), 4.44 (dd, J = 6.1, 14.4 Hz, 1H),
4.30 (d, J = 7.3 Hz, 1H), 3.41–3.32 (m, 2H, overlapping with MeOH),
2.77–2.69 (m, 1H), 2.49–2.42 (m, 1H), 2.25–2.18 (m, 1H), 2.13–2.02
(m, 3H), 1.68–1.53 (m, 3H), 0.99–0.83 (m, 12H); 13C NMR
(125 MHz, CD3OD) d 193.5, 181.8, 174.8, 173.2, 169.8, 154.8,
143.6, 138.4, 129.3, 128.5, 126.8, 126.5, 123.7, 116.2, 68.3, 59.7,
55.2, 53.3, 41.8, 41.5, 40.0, 33.9, 32.3, 28.8, 25.8, 23.2, 22.2, 19.8,
18.7; HRMS (ESI): m/z calcd for C33H40N6O8S [M+H]+ 680.2628,
found 680.2627.
6.3.15. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-
2-oxopyrrolidin-3-yl)propan-2-yl)-2-((S)-2-(2-(4-
(dimethylamino)phenoxy)acetamido)-3-methylbutanamido)-
4-methylpentanamide (2o)
18% yield from 13c; white powder; 1H NMR (500 MHz, CD3OD)
d 8.31 (d, J = 7.0 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.11 (d, J = 7.0 Hz,
1H), 7.67–7.57 (m, 2H), 7.25 (d, J = 8.7 Hz, 2H), 7.07 (d, J = 8.1 Hz,
2H), 5.73 (dd, J = 3.4, 11.6 Hz, 1H), 4.60 (s, 2H), 4.48–4.41 (m,
1H), 4.32 (d, J = 7.0 Hz, 1H), 3.42–3.32 (m, 2H), 2.78–2.69 (m,
1H), 2.50–2.42 (m, 1H), 2.24–2.17 (m, 1H), 2.14–2.03 (m, 3H),
6.3.19. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-((S)-3-methyl-2-
(2-(pyridin-3-yloxy)acetamido)butanamido)pentanamide (2s)
33% yield from 13c; white powder; 1H NMR (500 MHz, CD3OD)
d 8.54 (br s, 1H), 8.38 (d, J = 5.0 Hz, 1H), 8.31 (d, J = 7.3 Hz, 1H), 8.21

S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
421
(d, J = 7.4 Hz, 1H), 8.11 (d, J = 7.0 Hz, 1H), 7.98 (dd, J = 2.8, 8.7 Hz,
1H), 7.79 (dd, J = 5.3, 8.7 Hz, 1H), 7.67–7.58 (m, 2H), 5.73 (dd,
J = 3.5, 11.6 Hz, 1H), 4.85 (s, 2H), 4.45 (dd, J = 6.6, 14.2 Hz, 1H),
4.33–4.26 (m, 1H), 3.41–3.32 (m, 2H, overlapping with MeOH),
2.77–2.68 (m, 1H), 2.49–2.42 (m, 1H), 2.25–2.16 (m, 1H), 2.14–
2.01 (m, 3H), 1.68–1.54 (m, 3H), 1.00–0.84 (m, 12H); 13C NMR
(125 MHz, CD3OD) d 193.5, 181.8, 174.9, 173.3, 169.4, 165.5,
157.6, 154.8, 138.5, 138.4, 134.3, 129.8, 129.3, 128.5, 128.0,
126.5, 123.8, 68.5, 59.9, 55.2, 53.3, 41.8, 41.5, 40.0, 33.9, 32.2,
28.8, 25.8, 23.2, 22.2, 19.8, 18.7; HRMS (ESI): m/z calcd for
3H), 1.71–1.53 (m, 3H), 0.99–0.85 (m, 12H); HRMS (ESI): m/z calcd
for C33H42N5O6S [M+H]+ 636.2856, found 636.2851.
6.4.2. Benzyl ((2S)-1-(((2S)-1-(((2S)-1-(benzo[d]thiazol-2-yl)-1-
oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)amino)-3-cyclohexyl-
1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-
yl)carbamate (3b)
26% yield from 13c; white powder; 1H NMR (500 MHz, DMSO-
d6) d 8.74 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 7.5 Hz, 1H), 8.10 (d,
J = 3.2 Hz, 1H), 8.03 (d, J = 3.2 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H),
5.46–5.39 (m, 1H), 5.18 (s, 2H), 4.38 (dd, J = 8.4, 14.8 Hz, 1H),
3.88 (dd, J = 7.0, 8.6 Hz, 1H), 3.18 (dd, J = 8.7, 8.8 Hz, 1H), 3.11
(dd, J = 9.2, 16.3 Hz, 1H), 2.48–2.40 (m, 1H), 2.23–2.12 (m, 2H),
2.08–2.01 (m, 1H), 2.00–1.92 (m, 1H), 1.81–1.70 (m, 2H), 1.62–
1.53 (m, 1H), 1.48–1.39 (m, 2H), 0.92–0.86 (m, 12H), 0.85–0.80
C
32H41N6O6S [M+H]+ 637.2808, found 637.2809.
6.3.20. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-((S)-3-methyl-2-
(2-(phenylamino)acetamido)butanamido)pentanamide (2t)
17% yield from 13c; slightly yellow powder; 1H NMR (500 MHz,
CD3OD) d 8.24 (d, J = 7.2 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.11 (d,
J = 7.8 Hz, 1H), 7.67–7.57 (m, 2H), 7.19 (dd, J = 6.5, 8.4 Hz, 2H),
6.82 (t, J = 7.3 Hz, 1H), 6.75 (d, J = 7.9 Hz, 2H), 5.72 (dd, J = 3.4,
11.8 Hz, 1H), 4.41 (dd, J = 5.3, 14.0 Hz, 1H), 4.28 (d, J = 6.8 Hz,
1H), 3.85 (dd, J = 7.1, 10.9 Hz, 1H), 3.38–3.31 (m, 2H, overlapping
with MeOH), 2.75–2.67 (m, 1H), 2.46–2.39 (m, 1H), 2.24–2.17 (m,
1H), 2.11–1.98 (m, 3H), 1.68–1.50 (m, 3H), 0.98–0.77 (m, 12H);
13C NMR (125 MHz, CD3OD) d 193.5, 181.8, 174.8, 173.3, 172.7,
165.5, 154.8, 147.2, 138.4, 130.4, 129.3, 128.5, 126.7, 126.5,
123.7, 121.1, 115.6, 59.5, 55.1, 53.3, 41.8, 41.5, 39.9, 33.8, 32.5,
28.8, 25.8, 23.2, 22.2, 19.8, 18.3; HRMS (ESI): m/z calcd for
(m, 6H); HRMS (ESI): m/z calcd for
C
36H45N5O6S [M+H]+
676.3169, found 676.3173.
6.4.3. Benzyl ((2S)-1-(((2S)-1-(((2S)-1-(benzo[d]thiazol-2-yl)-
1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)amino)-1-oxo-
3-phenylpropan-2-yl)amino)-3-methyl-1-oxobutan-
2-yl)carbamate (3c)
28% yield from 13c; white powder; 1H NMR (500 MHz, DMSO-
d6) d 8.27 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 7.7 Hz, 1H), 7.70–7.61 (m,
2H), 7.33–7.07 (m, 10H), 5.71 (d, J = 8.3 Hz, 1H), 5.10 (s, 2H),
4.53–4.42 (m, 1H), 3.99–3.93 (m, 1H), 3.39–3.32 (m, 4H), 2.80–
2.71 (m, 1H), 2.60–2.55 (m, 1H), 2.30–2.22 (m, 1H), 2.11–1.99
(m, 3H), 1.00–0.89 (m, 6H); HRMS (ESI): m/z calcd for C36H40N5O5S
[M+H]+ 670.2699, found 670.2685.
C
33H43N6O5S [M+H]+ 635.3016, found 635.3009.
6.3.21. (S)-N-((S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-
2-oxopyrrolidin-3-yl)propan-2-yl)-2-((S)-2-(2-
(benzylamino)acetamido)-3-methylbutanamido)-
6.4.4. (4S)-5-(((2S)-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-(2-oxopyrr
olidin-3-yl)propan-2-yl)amino)-4-((S)-2-(((benzyloxy)carb
onyl)amino)-3-methylbutanamido)-5-oxopentanoicacid (3d)
22% yield from 13c; white powder; 1H NMR (500 MHz, DMSO-
d6) d 8.25 (d, J = 8.4 Hz, 1H), 8.23 (d, J = 7.7 Hz, 1H), 7.69–7.62 (m,
2H), 7.35–7.27 (m, 5H), 5.54 (d, J = 8.3 Hz, 1H), 5.04 (s, 1H), 4.45–
4.39 (m, 1H), 3.37–3.31 (m, 2H), 3.15–3.14 (m, 2H), 2.27–2.12
(m, 2H), 1.98–1.84 (m, 3H), 1.65–1.40 (m, 3H), 1.24–1.02 (m,
2H), 0.86–0.82 (m, 6H); HRMS (ESI): m/z calcd for C32H37N5O8S
[M+H]+ 652.2441, found 652.2432.
4-methylpentanamide (2u)
22% yield from 13c; yellow powder; 1H NMR (500 MHz,
CD3OD) d 8.21 (d, J = 7.2 Hz, 1H), 8.18 (d, J = 7.2 Hz, 1H), 8.09 (d,
J = 7.8 Hz, 1H), 7.64–7.51 (m, 2H), 7.21 (dd, J = 6.5, 8.0 Hz, 2H),
6.83 (t, J = 7.3 Hz, 1H), 6.79 (d, J = 7.9 Hz, 2H), 5.73 (dd, J = 3.4,
11.8 Hz, 1H), 4.44 (dd, J = 5.1, 14.0 Hz, 1H), 4.31 (d, J = 6.4 Hz,
1H), 3.81 (s, 2H), 3.78 (dd, J = 7.1, 10.9 Hz, 1H), 3.40–3.34 (m,
2H, overlapping with MeOH), 2.75–2.70 (m, 1H), 2.46–2.40 (m,
1H), 2.24–2.20 (m, 1H), 2.10–2.00 (m, 3H), 1.70–1.50 (m, 3H),
1.00–0.77 (m, 12H); 13C NMR (125 MHz, CD3OD) d 193.5, 181.8,
174.8, 173.3, 171.1, 165.5, 159.1, 154.8, 147.1, 138.4, 130.7,
129.3, 128.4, 125.3, 126.5, 123.7, 122.9, 121.1, 115.9, 68.1, 59.4,
55.2, 53.3, 41.8, 41.5, 40.0, 33.8, 32.5, 28.8, 25.8, 23.4, 22.2, 19.8,
18.3; HRMS (ESI): m/z calcd for C34H45N6O5S [M+H]+ 649.3172,
found 649.3156.
6.5. Synthetic procedure for the preparation of 4a–c
Compounds 4a–c was prepared from 17 (2 mmol) with an
appropriate dipeptidic 9 (1.1 equiv) using a procedure similar to
that described for the preparation of 2a. Compounds 4a–c were
purified by reverse phase HPLC.
6.4. Synthetic procedure for the preparation of 3a–d
6.5.1. Benzyl ((S)-1-(((S)-1-(((S)-3-(1H-imidazol-4-yl)-1-oxo-
1-(thiazol-2-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-
2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (4a)
36% yield from 17; colorless solid; 1H NMR (300 MHz, DMSO-
d6) d 8.81 (d, J = 9.60 Hz, 1H), 8.66 (d, J = 13.6 Hz, 1H), 8.27 (br s,
1H), 8.16–8.13 (m, 1H), 7.81 (t, J = 19.6 Hz, 1H), 7.31–7.24 (br s,
2H), 6.98–6.91 (m, 3H), 4.56 (s, 1H), 431–4.18 (m, 2H), 3.25 (br s,
1H), 1.93–191 (m, 1H), 1.36–1.24 (m, 3H), 0.84–0.70 (m, 12H);
HRMS (ESI): m/z calcd for C28H36N6O5S [M+H]+ 569.2535, found
569.2546.
Compounds 3a–d was prepared from 13c (2 mmol) with an
appropriate dipeptidic 9 (1.1 equiv) using a procedure similar to
that described for the preparation of 2a. Compounds 3a–d were
purified by reverse phase HPLC.
6.4.1. Benzyl ((2S)-1-(((2R)-1-(((2S)-1-(benzo[d]thiazol-2-yl)-1-
oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-
oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate
(3a)
22% yield from 13c; White powder; 1H NMR (500 MHz, DMSO-
d6) d 8.21 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.66–7.57 (m,
2H), 7.37–7.26 (m, 5H), 5.72 (d, J = 8.3 Hz, 1H), 5.09 (s, 2H), 4.52–
4.42 (m, 1H), 3.97–3.92 (m, 1H), 3.39–3.32 (m, 2H), 2.76–2.67
(m, 1H), 2.48–2.37 (m, 1H), 2.26–2.18 (m, 1H), 2.11–1.99 (m,
6.5.2. (S)-N-((S)-3-(1H-imidazol-4-yl)-1-oxo-1-(thiazol-
2-yl)propan-2-yl)-4-methyl-2-((S)-3-methyl-2-(2-
phenoxyacetamido)butanamido)pentanamide (4b)
43% yield from 17; colorless solid; 1H NMR (300 MHz, DMSO-
d6) d 8.89 (d, J = 4.65 Hz, 1H), 8.70–8.69 (m, 1H), 8.28 (br s, 1H),

422
S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
8.15 (t, J = 6.27 Hz, 1H), 7.37–7.33 (m, 2H), 7.20 (t, J = 13.7 Hz, 2H),
7.08 (d, J = 8.1 Hz, 2H), 4.36–4.34 (m, 1H), 3.92–3.86 (m, 1H), 3.26
(br s, 1H), 1.98–1.96 (m, 1H), 1.37–1.23 (m, 3H), 0.84–0.81 (m,
12H); HRMS (ESI): m/z calcd for C28H36N6O5S [M+H]+ 569.2527,
found 569.2546.
30.6, 28.1, 27.3, 24.0, 22.8, 21.7, 20.8, 19.1, 19.0, 17.8; HRMS
(ESI): m/z calcd for C37H49N5O7S [M+H]+ 706.3274, found 706.3275.
6.6.4. (2S)-2-((S)-2-(2-(4-Methoxyphenoxy)acetamido)-
3-methylbutanamido)-N-((2S)-1-(5-(4-methoxyphenyl)thiazol-
2-yl)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)-
6.5.3. Phenyl ((S)-1-(((S)-1-(((S)-3-(1H-imidazol-4-yl)-1-oxo-
1-(thiazol-2-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-
2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (4c)
38% yield from 17; colorless solid; 1H NMR (300 MHz, CDCl3) d
8.57 (t, J = 13.6 Hz, 1H), 7.20 (t, J = 14.7 Hz, 1H), 7.10 (d, J = 7.73 Hz,
2H), 4.59 (br s, 1H), 4.11 (br s, 1H), 1.64 (br s, 2H), 1.26 (t, J = 4.1 Hz,
2H), 0.9–0.93 (m, 9H); HRMS (ESI): m/z calcd for C27H34N6O5S
[M+H]+ 569.2527, found 569.2546.
4-methylpentanamide (5d)
40% yield from 13g; yellow solid; 1H NMR (400 MHz, CDCl3) d
8.09 (s, 1H), 7.58–7.56 (d, J = 8.0 Hz, 2H), 6.99–6.97 (d, J = 8.4 Hz,
2H), 6.92–6.84 (m, 4H), 5.66–5.62 (m, 1H), 4.51–4.50 (m, 1H),
4.48 (s, 2H), 4.33–4.30 (m, 1H), 3.89 (s, 3H), 3.77 (s, 3H), 3.38–
3.31 (m, 2H), 2.60–2.53 (m, 2H), 2.30–2.01 (m, 4H), 1.77–1.56
(m, 3H), 0.96–0.87 (m, 12H); 13C NMR (400 MHz, DMSO-d6) d
191.0, 178.0, 172.1, 170.3, 167.7, 161.4, 160.5, 153.7, 151.6,
146.7, 140.2, 128.7, 122.2, 115.2, 114.8, 67.3, 56.8, 55.3, 52.6,
50.8, 32.4, 30.9, 30.6, 28.1, 27.2, 24.1, 24.0, 22.8, 21.7, 21.5, 19.1,
17.8; HRMS (ESI): m/z calcd for C37H49N5O8S [M+H]+ 722.3224,
found 722.3237.
6.6. Synthetic procedure for the preparation of 5a–i
Compound 5a–i was prepared from 13d–i (2 mmol) with an
appropriate dipeptidic 9 (1.1 equiv) using a procedure similar to
that described for the preparation of 2a. Compounds 5a–i were
purified by reverse phase HPLC.
6.6.5. (2S)-N-((2S)-1-(5-(4-Chlorophenyl)thiazol-2-yl)-1-oxo-
3-(2-oxopyrrolidin-3-yl)propan-2-yl)-2-((S)-2-(2-(4-
methoxyphenoxy)acetamido)-3-methylbutanamido)-
4-methylpentanamide (5e)
6.6.1. Benzyl ((2S)-3-methyl-1-(((2S)-4-methyl-1-oxo-1-(((2S)-1-
oxo-3-(2-oxopyrrolidin-3-yl)-1-(5-phenylthiazol-2-yl)propan-
2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)carbamate
(5a)
47% yield from 13h; yellow solid; 1H NMR (400 MHz, CDCl3) d
8.06 (s, 1H), 7.48–7.46 (d, J = 8.0 Hz, 2H), 7.34–7.32 (d, J = 8.4 Hz,
2H), 6.82–6.73 (m, 4H), 5.57–5.52 (m, 1H), 4.41–4.40 (m, 1H),
4.38 (s, 2H), 4.22–4.17 (m, 1H), 3.67 (s, 3H), 3.30–3.19 (m, 2H),
2.53–2.38 (m, 2H), 2.04–1.90 (m, 4H), 1.57–1.42 (m, 3H), 0.96–
0.87 (m, 12H); 13C NMR (125 MHz, DMSO-d6) d 191.4, 178.2,
172.1, 170.4, 167.9, 163.1, 153.7, 151.5, 145.0, 141.7, 134.4,
129.4, 128.8, 128.6, 115.5, 114.5, 67.2, 57.2, 55.2, 53.6, 52.7, 50.7,
32.2, 30.8, 30.5, 27.2, 24.0, 22.7, 21.6, 21.5, 19.0, 18.9, 17.7; HRMS
33% yield from 13e; colorless solid; 1H NMR (400 MHz, CDCl3) d
8.15 (s, 1H), 7.62–7.61 (m, 2H), 7.47–7.40 (m, 2H), 7.37–7.24 (m,
5H), 5.41–5.33 (m, 1H), 5.15 (s, 1H), 4.58–4.52 (m, 2H), 4.07–4.03
(m, 1H), 3.36–3.29 (m, 2H), 2.61–2.55 (m, 2H), 2.16–1.96 (m,
3H), 1.81–1.70 (m, 2H), 1.62–1.53 (m, 1H), 1.48–1.39 (m, 2H),
0.99–0.88 (m, 12H); 13C NMR (400 MHz, DMSO-d6) d 191.5,
177.9, 172.2, 170.8, 162.9, 156.1, 146.4, 141.3, 137.1, 129.8,
129.7, 128.2, 127.6, 127.1, 65.3, 60.3, 53.6, 52.7, 60.7, 32.3, 30.3,
30.2, 28.1, 27.2, 24.0, 22.8, 21.7, 19.1, 18.0; HRMS (ESI): m/z calcd
for C35H44N5O6S [M+H]+ 662.3012, found 662.3018.
(ESI): m/z calcd for
C
36H46N5O7SCl [M+H]+ 726.2728, found
726.2723.
6.6.6. (2S)-2-((S)-2-(2-(4-Methoxyphenoxy)acetamido)-
3-methylbutanamido)-4-methyl-N-((2S)-1-(5-(naphthalen-
2-yl)thiazol-2-yl)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-
2-yl)pentanamide (5f)
6.6.2. (2S)-2-((S)-2-(2-(4-Methoxyphenoxy)acetamido)-
3-methylbutanamido)-4-methyl-N-((2S)-1-oxo-3-(2-
oxopyrrolidin-3-yl)-1-(5-phenylthiazol-2-yl)propan-
2-yl)pentanamide (5b)
42% yield from 13i; yellow solid; 1H NMR (400 MHz, CDCl3) d
8.29–8.28 (d, J = 4.6 Hz, 1H), 8.10 (s, 1H), 7.91–7.87 (m, 3H),
7.71–7.69 (t, J = 8.4 Hz, 1H), 7.60–7.53 (m, 1H), 7.36–7.33 (m,
1H), 6.92–6.83 (m, 4H), 7.70–5.67 (m, 1H), 4.55–4.51 (m, 1H),
4.48 (s, 2H), 4.37–4.33 (m, 1H), 3.76 (s, 3H), 3.39–3.29 (m, 2H),
2.64–2.2.50 (m, 2H), 2.39–2.33 (m, 1H), 2.19–2.04 (m, 2H), 1.91–
1.86 (m, 1H), 1.71–1.55 (m, 3H), 0.96–0.89 (m, 12H); 13C NMR
(400 MHz, DMSO-d6) d 191.0, 178.2, 172.2, 170.4, 167.9, 162.6,
153.8, 151.5, 146.6, 141.5, 133.1, 129.0, 128.2, 127.6, 127.2,
127.1, 124.3, 115.4, 114.5, 67.2, 56.9, 55.2, 52.7, 50.9, 48.5, 32.3,
30.5, 27.2, 24.0, 22.7, 21.6, 19.0, 17.7; HRMS (ESI): m/z calcd for
45% yield from 13e; colorless solid; 1H NMR (400 MHz, CDCl3) d
8.16 (s, 1H), 7.64–7.61 (d, J = 8.4 Hz, 2H), 7.48–7.40 (m, 3H), 6.92–
6.83 (m, 4H), 5.68–5.62 (m, 1H), 4.55–4.50 (m 1H), 4.48 (s, 2H),
4.34–4.30 (m, 1H), 3.77 (s, 3H), 3.40–3.29 (m, 2H), 2.61–2.44 (m,
2H), 2.19–2.02 (m, 3H), 1.91–1.85 (m, 1H), 1.71–1.53 (m, 3H),
0.96–0.88 (m, 12H); 13C NMR (400 MHz, DMSO-d6) d 191.0,
185.7, 178.7, 174.3, 172.3, 168.2, 164.0, 162.4, 156.8, 153.8,
151.4, 146.6, 140.9, 139.2, 129.8, 129.4, 127.0, 122.2, 115.5,
114.4, 67.2, 57.4, 55.3, 52.7, 51.0, 48.9, 47.1, 32.7, 30.4, 27.1,
24.0, 22.5, 21.4, 21.2, 18.8, 17.5; HRMS (ESI): m/z calcd for
C
40H4N5O5SNa [M+Na]+ 764.3094, found 764.3095.
C
36H47N5O7S [M+H]+ 692.3118, found 692.3145.
6.6.7. (2S)-2-((S)-2-(2-(4-Methoxyphenoxy)acetamido)-
3-methylbutanamido)-N-((2S)-1-(5-(2-methoxyphenyl)thiazol-
2-yl)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)-4-
6.6.3. (2S)-2-((S)-2-(2-(4-Methoxyphenoxy)acetamido)-
3-methylbutanamido)-4-methyl-N-((2S)-1-oxo-3-(2-
oxopyrrolidin-3-yl)-1-(5-(p-tolyl)thiazol-2-yl)propan-
2-yl)pentanamide (5c)
methylpentanamide (5g)
39% yield from 13j; colorless solid; 1H NMR (400 MHz, CDCl3) d
8.14 (s, 1H), 8.08 (br s, 1H, NH), 7.38–7.34 (t, J = 8.0 Hz, 1H), 7.21–
7.19 (d, J = 7.2 Hz, 1H), 7.13–7.09 (m, 1H), 6.99–6.87 (m, 2H), 6.83–
6.80 (m, 2H), 5.70–5.56 (m, 1H), 4.62–4.52 (m, 1H), 4.48 (s, 2H),
4.31–4.30 (m, 1H), 3.86 (s, 3H), 3.77 (s, 3H), 3.41–3.37 (m, 2H),
2.63–2.50 (m, 2H), 2.33–2.21 (m, 1H), 2.20–2.09 (m, 2H), 1.91–
1.86 (m, 2H), 1.71–1.55 (m, 3H), 0.95–0.86 (m, 12H); 13C NMR
(400 MHz, DMSO-d6) d 191.1, 177.9, 171.3, 167.6, 162.6, 159.6,
153.7, 151.6, 146.2, 141.6, 131.0, 130.6, 127.1, 119.6, 115.5,
47% yield from 13f; colorless solid; 1H NMR (400 MHz, CDCl3) d
8.12 (s, 1H), 7.51–7.49 (d, J = 8.4 Hz, 2H), 7.26–7.24 (d, J = 8.0 Hz,
2H), 6.89–6.83 (m, 4H), 5.64–5.59 (m, 1H), 4.57–4.52 (m, 1H),
4.48 (s, 2H), 3.77 (s, 3H), 3.38–3.29 (m, 2H), 2.59–2.53 (m, 2H),
2.40 (s, 3H), 2.29–2.01 (m, 4H), 1.75–1.56 (m, 3H), 0.96–0.87 (m,
12H); 13C NMR (400 MHz, DMSO-d6) d 191.4, 177.7, 176.3, 172.1,
170.3, 167.7, 162.3, 153.7, 151.5, 146.6, 140.8, 139.7, 129.9,
127.0, 115.5, 114.54, 67.3, 57.1, 55.3, 53.6, 52.6, 50.8, 38.87, 32.4,

S. Konno et al. / Bioorg. Med. Chem. 21 (2013) 412–424
423
114.5, 112.4, 67.3, 56.8, 60.8, 52.7, 50.8, 32.4, 27.2, 24.0, 22.8, 21.6,
19.1, 17.8; HRMS (ESI): m/z calcd for C37H47N5O8SNa [M+Na]+
744.3043, found 744.3051.
phosphate, 10 mM sodium chloride, 1 mM EDTA, 1 mM TCEP and
2% DMSO (pH 7.4). While varying inhibitor concentrations (0–
200
centration 100 nM) to a solution of substrate at final concentration
of 10 M to a total volume of 120 L in a microcuvette. The data
lM), the reaction was initiated by adding protease (final con-
l
l
6.6.8. (2S)-2-((S)-2-(2-(3-(Dimethylamino)phenoxy)acetamido)-
3-methylbutanamido)-4-methyl-N-((2S)-1-oxo-3-(2-
from these assays were analyzed by using the non-linear regres-
sion analysis software Origin 7.
oxopyrrolidin-3-yl)-1-(5-phenylthiazol-2-yl)propan-2-
yl)pentanamide (5h)
36% yield from 13e; light green solid; 1H NMR (400 MHz, CDCl3)
d 8.16 (s, 1H), 8.10 (br s, 1H, NH), 7.61–7.58 (m, 2H), 7.44–7.41 (m,
3H), 7.22–7.16 (m, 1H), 6.52–6.50 (d, J = 8.0 Hz, 1H), 6.45 (s, 1H),
6.38–6.36 (d, J = 8.0 Hz, 1H), 5.68–5.63 (m, 1H), 4.58–4.53 (m
1H), 4.54 (s, 2H), 4.35–4.31 (m, 1H), 3.37–3.31 (m, 2H), 2.97 (s,
6H), 2.61–2.57 (m, 2H), 2.18–2.03 (m, 3H), 1.99–1.89 (m, 1H),
1.71–1.54 (m, 3H), 0.96–0.89 (m, 12H); 13C NMR (400 MHz,
DMSO-d6) d 191.1, 177.9, 170.3, 167.5, 162.6, 158.6, 146.4, 141.3,
129.4, 127.1, 102.6, 66.5, 56.7, 53.8, 32.4, 27.1, 24.0, 22.8, 21.7,
Acknowledgments
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 Ken-
kyuin Shorei-hi) 23ꢀ01104 and a Grant-in-aid for Scientific Research
23659059.
References and notes
19.0 17.8; HRMS (ESI): m/z calcd for
705.3434, found 705.3429.
C
37H50N6O6S [M+H]+
1. Ksiazek, T. G.; Erdman, D.; Goldsmith, C. S.; Zaki, S. R.; Peret, T.; Emery, S.; Tong,
S.; Urbani, C.; Comer, J. A.; Lim, W.; Rollin, P. E.; Dowell, S. F.; Ling, A. E.;
Humphrey, C. D.; Shieh, W. J.; Guarner, J.; Paddock, C. D.; Roca, P.; Fields, B.;
DeRisi, J.; Yang, J. Y.; Cox, N.; Hughes, J. M.; LeDuc, J. W.; Bellini, W. J.; Anderson,
L. J. N. Engl. J. Med. 2003, 348, 1953.
6.6.9. (2S)-2-((S)-2-(2-(3-(Dimethylamino)phenoxy)acetamido)-
3-methylbutanamido)-4-methyl-N-((2S)-1-(5-methylthiazol-2-
yl)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)pentanamide
(5i)
2. Drosten, C.; Gunther, S.; Preiser, W.; van der Werf, S.; Brodt, H. R.; Becker, S.;
Rabenau, H.; Panning, M.; Kolesnikova, L.; Fouchier, R. A. M.; Berger, A.;
Burguiere, A. M.; Cinatl, J.; Eickmann, M.; Escriou, N.; Grywna, K.; Kramme, S.;
Manuguerra, J.-C.; Muller, S.; Rickerts, V.; Sturmer, M.; Vieth, S.; Klenk, H. D.;
Osterhaus, A. D. M. E.; Schmitz, H.; Doerr, H. W. N. Engl. J. Med. 2003, 348, 1967.
3. Peiris, J. S.; Lai, S. T.; Poon, L. L.; Guan, Y.; Yam, L. Y.; Lim, W.; Nicholls, J.; Yee,
W. K.; Yan, W. W.; Cheung, M. T.; Cheng, V. C.; Chan, K. H.; Tsang, D. N.; Yung, R.
W.; Ng, T. K.; Yuen, K. Y. Lancet 2003, 361, 1319. and references therein.
4. World Health Organization, Communicable Disease Surveillance & Response,
website: http://www.who.int/csr/sars/archive/2003_05_07a/en and http://
www.who.int/csr/sars/country/en/Country2003_08_15.pdf. Summary table of
SARS cases by country (1 November 2002 to 7 August 2003).
5. Lee, N.; Hui, D.; Wu, A.; Chan, P.; Cameron, P.; Joynt, G. M.; Ahuja, A.; Yung, M.
Y.; Leung, C. B.; To, K. F.; Lui, S. F.; Szeto, C. C.; Chung, S.; Sung, J. J. Y. N. Engl. J.
Med. 2003, 348, 1986.
35% yield from 13d; light green solid; 1H NMR (400 MHz, CDCl3)
d 8.10 (br s, 1H, NH), 7.66 (s, 1H), 7.43–7.23 (t, J = 8.4 Hz, 1H), 6.88–
6.85 (m, 2H), 6.70 (s, 1H), 5.60–5.58 (m, 1H), 4.58–4.53 (m, 1H),
4.50 (s, 2H), 4.37–4.33 (m, 1H), 3.36–3.27 (m, 2H), 3.08 (s, 6H),
2.61–2.44 (m, 5H), 2.15–2.01 (m, 3H), 1.91–1.85 (m, 1H), 1.64–
1.53 (m, 3H), 0.94–0.86 (m, 12H); 13C NMR (400 MHz, DMSO-d6)
d 191.4, 177.9, 172.0, 170.3, 167.6, 162.7, 162.4, 158.6, 150.9,
143.6, 129.7, 106.6, 103.4, 99.9, 66.6, 57.0, 53.5, 50.8, 32.5, 31.0,
28.1, 27.2, 24.0, 22.8, 21.7, 21.6, 19.1, 19.0, 17.8; HRMS (ESI): m/z
calcd for C32H48N6O6S [M+H]+ 643.3278, found 643.3259.
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IC50 value of each inhibitor was assessed from the apparent de-
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R188I SARS 3CL protease as described previously.30,29 Briefly, the
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cease of the substrate peak area. Each IC50 value was obtained from
the sigmoidal dose–response curve. Each experiment was repeated
3 times and the results were averaged.
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