New developments for the design, synthesis and biological evaluation of potent SARS-CoV 3CLpro inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.03.118

A series of trifluoromethyl, benzothiazolyl or thiazolyl ketone-containing peptidic compounds as SARS-CoV 3CL protease inhibitors were developed and their potency was evaluated by in vitro protease inhibitory assays. Three candidates had encouraging resul

Full text of DOI:10.1016/j.bmcl.2009.03.118

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2722–2727
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New developments for the design, synthesis and biological evaluation of potent
SARS-CoV 3CLpro inhibitors
Thomas Regnier a, , Diganta Sarma a, , Koushi Hidaka a, Usman Bacha b, Ernesto Freire b, Yoshio Hayashi a,c,
,
*
Yoshiaki Kiso a,
*
a Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, 21st Century COE Program, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan
b Department of Biology, Johns Hopkins University, Baltimore, MD, USA
c Department of Medicinal Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of trifluoromethyl, benzothiazolyl or thiazolyl ketone-containing peptidic compounds as SARS-
CoV 3CL protease inhibitors were developed and their potency was evaluated by in vitro protease inhib-
itory assays. Three candidates had encouraging results for the development of new anti-SARS
compounds.
Received 24 February 2009
Revised 24 March 2009
Accepted 25 March 2009
Available online 28 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
SARS-CoV
Protease inhibitors
Drug design
Synthesis
In November 2002, it was reported an emergence of severe
acute respiratory syndrome (SARS) as a highly contagious and fatal
respiratory disease infecting more than 8000 individuals of which
9.6% patients died within a few months.1 Due to highly efficient
international cooperation, two groups rapidly reported that a novel
coronavirus (CoV) was the causative agent of SARS.2,3 CoV encodes
a chymotrypsin-like protease (3CLpro) that plays a pivotal role in
the replication of the virus.4 3CLpro, a cysteine protease, is function-
ally analogous to the main picornavirus protease 3Cpro with a cat-
alytic dyad (Cys-145 and His-41) in the active site. Cys acts as a
nucleophile, whereas His functions as a general base.5,6 In order
to find compounds that can inhibit SARS-CoV, numerous 3CLpro
inhibitors have been described, including C2-symmetric diols,7
bifunctional aryl boronic acids,8 keto-glutamine analogs,9 isatin
2006, the moderate activity can be the result of the formation of
a typical cyclic structure (Scheme 1, compounds 2a,b) that is not
expected to interact effectively with the active site of SARS-CoV
3CLpro 16
.
Herein, we report our results on improving the inhibitory activ-
ity of these compounds, by focusing on two strategies. First, keep-
ing the trifluoromethylketone moiety in place, we investigated
chemical modifications on the side chain of Glu or Gln residue at
the P1 position, in order to block the formation of the cyclic struc-
ture (Scheme 1) and modulate the hydrogen bonding ability of this
P1 position toward the active site, as well as modifying the amino
acid residues at the P2 and P3 positions. Second, we investigated a
replacement of the chemical warhead of the inhibitor, that is, the
trifluoromethyl unit, by other moieties such as electron-withdraw-
ing thiazolyl and benzothiazolyl groups. We believe that this mod-
ification would be valuable for enhancing the reactivity of the
covalent-adduct formation to the active site cysteine residue in
derivatives,10
a
,b-unsaturated esters,11 anilide,12 benzotriazole13
as well as glutamic acid and glutamine peptides possessing a tri-
fluoromethyl ketone group as reported by us and our collaborators
since 200614 and recently by another group.15 However, no effec-
tive therapy has been developed so far and it is still a matter of
necessity to discover new potent structures in case the disease
re-emerges.
SARS-CoV 3CLpro
.
From a synthetic point of view, the preparation of the target
compounds was envisioned following the synthetic routes illus-
trated in Schemes 2–4. Compounds 8a–e were prepared from
In our previous report, two compounds (Scheme 1, 1a,b) were
Cbz-L-Glu-OH (3) that was converted to the corresponding oxazo-
found to be moderate SARS-CoV 3CLpro inhibitors (Ki = 116 and
lidinone acid 4 under the conditions described by Moore et al.17
Amides 5a–d were next prepared by coupling compound 4 with
four kinds of amines using a standard HOBt–EDCꢀHCl coupling
method for peptides, resulting in excellent yields. Compounds
5a–d were then converted in a one-pot reaction to the correspond-
134 l
M, respectively).14a As mentioned by Cai and co-workers in
* Corresponding authors. Tel.: +81 75 595 4635; fax: +81 75 591 9900 (Y.K.).
E-mail address: kiso@mb.kyoto-phu.ac.jp (Y. Kiso).
Equal contribution.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.118

T. Regnier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2722–2727
2723
COOH
O
O
O
H
N
H
N
O
CF3
Cbz
Cbz
N
H
N
H
Cbz
N
H
N
H
CF3
HO
O
O
O
2a
1a
CONH2
O
O
O
O
O
H
H
H
H
N
N
N
NH
CF3
Cbz
N
CF3
N
N
H
N
H
N
H
H
HO
O
O
O
2b
1b
Scheme 1. Previously reported trifluoromethyl ketone-containing peptides and their corresponding cyclic non-active counterparts.
COOH
COOH
COOH
O
CONR1R2
O
b
a
Cbz
N
H
Cbz
N
Cbz
5a-d
N
O
O
4
3
c
CONR1R2
CONR1R2
CONR1R2
d
CF3
O
e,f
CF3
OH
Cbz-AA
CF3
OH
7a-d
N
Cbz
N
H
H2N
H
8a-e
6a-d
Scheme 2. Reagents and conditions: (a) paraformaldehyde, p-TsOHꢀH2O, toluene, reflux, 2 h, 98%; (b) HNR1R2, HOBt, EDCꢀHCl, DMF, 0 °C–rt, overnight, 80–98%; (c) CsF,
CF3Si(CH3)3, THF, sonication, rt, 3 h then MeOH, rt, 30 min then NaBH4, rt, overnight, 48–61%; (d) H2, Pd/C (10%), MeOH, rt, overnight, 100%; (e) Cbz-AA-OH, HOBt, EDCꢀHCl,
DMF, 0 °C–rt, overnight; (f) Dess–Martin periodinane, CH2Cl2, rt, 16 h, EtOAc then filtration through Celite followed by HPLC purification.
CO2tBu
CO2tBu
COOH
CO2tBu
b
a
R3
N
O
Cbz
N
H
Cbz
N
H
Cbz
N
H
O
O
11a-b
10
9
c
CONR1R2
CONR1R2
R3
COOH
R3
R3
O
d
e,f
Cbz-AA
N
Cbz
N
Cbz
N
H
H
H
O
O
14a-f
13a-c
12a-b
S
N
S
N
R3 =
,
14e-f
14a-d
Scheme 3. Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochloride, EDCꢀHCl, HOBt, TEA, DMF, rt, 12 h, 90%; (b) thiazole or benzothiazole, n-BuLi, ꢁ78 °C,
2.5 h, 70%; (c) formic acid, rt, 12 h, 100%; (d) HNR1R2, EDCꢀHCl, HOBt, DMF, rt, 12 h, 90%; (e) triflic acid, DCM, rt, 5 min, 100% (f) Cbz-AA-OH, HOBt, EDCꢀHCl, DMF, rt, 12 h
followed by HPLC purification.
ing trifluoromethylalcohols 6a–d, whose Cbz group was de-pro-
tected after silica gel column chromatography, and the amino func-
tion in the resultant compounds 7a–d was coupled to the
appropriate peptide fragments.14 The peptide fragments were syn-
thesized according to known procedures.14,18 Finally, the resulting
peptides were directly engaged in the last oxidation step affording

2724
T. Regnier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2722–2727
O
O
NH
O
OMe
OMe
NH
a
Ref. 20
Boc
OH
Boc
OMe
Boc
N
H
N
H
N
H
O
O
O
O
O
17
16
15
b
O
NH
NH
NH
S
c
S
O
d
Boc
N
Boc
N
H
N
H
H2N
N
N
O
O
O
18
19
20
e
O
NH
S
N
Cbz-Val-Leu-HN
O
21
Scheme 4. Reagents and conditions: (a) LiOH, THF/H2O, 92%; (b) HN(OCH3)CH3, EDCꢀHCl, HOBt, DMF, rt, 12 h, 90%; (c) thiazole, n-BuLi, ꢁ78 °C, 2.5 h, 70%; (d) TFA/H2O, 4 h,
>99%; (e) Cbz-Val-Leu-OH, EDCꢀHCl, HOBt, DMF, rt, 12 h followed by HPLC purification.
pure target compounds 8a–e with moderate overall yields after
RP-HPLC purification by a CH3CN:(0.1% TFA/H2O) system.
Derivatives 14a–d with a thiazole-ketone and 14e,f with a ben-
zothiazole-ketone structure at the P1 residue were prepared as
shown in Scheme 3. Cbz-Glu(tBu)-OH 9 was converted to Weinreb
amide 10 and successively coupled to thiazole or benzothiazole in
the presence of n-BuLi as a base to afford ketones 11a,b.19 After
deprotection of the tert-butyl group by HCOOH, the resultant car-
boxyl group of compounds 12a,b was coupled to the amines to ob-
tain compounds 13a–c, followed by coupling of the peptide
fragments based on a similar approach depicted in Scheme 2. Com-
pounds 14a–f were obtained with moderate yields after HPLC
purification.
hydrolyzed to obtain the lactam acid 17, followed by the synthetic
steps depicted in Scheme 3 to get desired compound 21.
Dithiazolyl compound 26 was synthesized starting from Cbz-
Val-Leu-OH 22 (Scheme 5) that was first converted to succinimide
ester 23 followed by treatment with
pound 24. Dicarboxylate compound 24 was then converted to the
corresponding -double-Weinreb amide (25), followed by a
L-glutamic acid to obtain com-
a,c
treatment with 2 equiv of thiazole in the presence of n-BuLi to af-
ford the desired N-Cbz-protected dithiazolylketone compound
(26).
The inhibitory activities (Ki) of the first series of compounds
against SARS-CoV 3CLpro are shown in Table 1. The inhibition assay
was performed using a procedure mentioned earlier.14a In our pre-
vious report, compounds 1a,b (Scheme 1) were found to be moder-
Compound 21, which has a pyrrolidone structure at the P1 side
chain, was synthesized by a different approach as shown in
Scheme 4. Lactam ester 16 was prepared from commercially avail-
ate SARS-CoV 3CLpro inhibitors with Ki values of 116 and 135
lM,
respectively (Table 1, entries 1 and 2). These compounds were used
as leads for structural optimization in the development of potent
SARS-CoV 3CLpro inhibitors.
able N-Boc-L-(+)-glutamic acid dimethyl ester 15 by following a
previously published procedure.20 Lactam ester 16 was then
O
OH
OH
b
a
Cbz-Val-Leu-OSu
23
Cbz-Val-Leu-OH
Cbz-Val-Leu
N
H
22
O
24
c
S
S
O
O
N
N
N
O
d
N
Cbz-Val-Leu
26
N
H
Cbz-Val-Leu
N
H
O
O
O
25
Scheme 5. Reagents and conditions: (a) N-hydroxysuccinimide, DCC, THF, 0 °C–rt, 12 h, 93%; (b) L-glutamic acid, NaOH solution (aq), dioxane, 4 h, 0 °C–rt, 90%; (c)
HN(OCH3)CH3, EDCꢀHCl, HOBt, DMF, rt, 12 h, 90%; (d) thiazole, n-BuLi, ꢁ78 °C, 2.5 h followed by HPLC purification.

T. Regnier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2722–2727
2725
Table 1
than reference compound 1a. Next, to develop smaller sized inhib-
itors, we prepared CF3-ketone compounds 8d and 8e containing
the same P1 structure as compound 8a but different peptide chain
lengths, because peptide chain length plays a significant role in the
inhibition of enzymes by forming crucial hydrogen bonding inter-
actions. However, the observed change in activity was negligible
with either an increase or decrease of one residue in the peptide
chain length (compare entries 3, 6 and 7).
Structure and activity of the synthesized CF3-inhibitors
Entry
Compound
Structurea
Kib
(lM)
O
OH
1
1a
116 14
CF3
Cbz-Val-Leu-NH
O
In the commonly adopted mechanism of inhibition of SARS-CoV
3CLpro, a covalent bond is formed between the carbonyl group lo-
cated at the warhead of the inhibitor and the active site cysteine
residue in the enzyme. Modifying the electronegativity at this war-
head carbonyl moiety could accelerate the covalent bond forma-
O
NH2
CF3
2
3
4
5
1b
8a
8b
8c
135 32
Cbz-Ala-Val-Leu-NH
tion. Hence, as
a
second series of investigations, the
O
trifluoromethylketone moiety was replaced with an electron with-
drawing thiazolyl or benzothiazolyl counterpart.
As shown in Table 2, when compared to trifluoromethylketone
containing compound 8b, corresponding compound 14a with thia-
O
Cbz-Val-Leu-NH
O
N
363 128
21.0 4.3
34.1 4.1
zolylketone
(Ki = 478 M; entry 2). However, a thiazolylketone derivative 14b
with N,N-diethylamide at the P1 side chain slightly increased
inhibitory activity (Ki = 112 M; entry 3) over the CF3-ketone
exhibited
drastically
decreased
inhibition
CF3
l
O
O
l
O
derivative 8a with a more bulky N,N-diisopropylamide structure.
Moreover, when the pyrrolidine structure was adapted to the P1
side chain, thiazolylketone derivative 21 (entry 4) drastically in-
N
CF3
creased inhibitory activity with a Ki value of 2.2 lM. Slightly smal-
Cbz-Val-Leu-NH
ler and rigid cyclic amide structure seemed to be well
accommodated by the S1 site. The introduction of a thiazolylke-
tone to the P1 side chain resulted in a compound (entry 5) that
O
N
exhibited decreased but relatively strong activity (Ki = 45.2 lM)
in comparison to pyrrolidine derivative 21. Inhibitory activity
was again decreased by reducing the peptide chain length of com-
pound 14b (compare entries 3, 6 and 7). A little improvement in
inhibitory activity was observed when the warhead thiazole was
replaced by a benzothiazole (compare entries 3, 7–9). Of the com-
pounds synthesized, inhibitor 21 possessing P1-pyrrolidone and
P10-thiazole was found to be the most potent SARS-CoV 3CLpro
inhibitor.
CF3
Cbz-Val-Leu-NH
O
O
N
6
8d
297 49
Our studies suggest that all the synthesized inhibitors are tight-
binding substrate mimetics that target the active site of SARS-
CF3
Cbz-Ala-Val-Leu-NH
3CL .
pro 22 Similar to the trifluoromethyl ketone compounds, the thi-
O
azole and benzothiazole ketone compounds did not show an in-
crease in inhibition after they were equilibrated with the
protease for 10 min. Thus, the inhibition of the compounds to the
protease was not time dependent indicating a reversible tight-
binding interaction with the protease. Moreover, to better under-
stand the potent activity of compound 21 with the enzyme, com-
putational molecular studies were performed (Fig. 1).
O
N
7
8e
584 167
CF3
Cbz-Leu-NH
O
A binding model of 21 with SARS-CoV 3CLpro was simulated
using a modeling package (MOE 2007.09, Chemical Computing
Group, Inc., Montreal, Canada). The initial conformation of 21
was built by changing a structurally similar ligand (Fig. 1) obtained
a
Cbz, benzyloxycarbonyl.
Ki, inhibition constant against SARS-CoV 3CLpro
.
b
Compound 8a was our first attempt at modifying the P1 residue
by converting the -carboxylic acid of compound 1a to a N,N-
from X-ray crystal data (PDB ID, 1WOF, Ki = 10.7 lM obtained from
c
Ref. 21). Several energy minimization processes with an MMFF94x
force field were additionally performed in a water soak environ-
ment around the inhibitor, followed by a molecular dynamics sim-
ulation. During the simulation, the moieties from P1 to P3
interacted in a similar manner as the original ligand except for
the thiazolyl ketone and benzyloxycarbonyl moieties (Fig. 1a).
The oxoanion of the ketone generated from an attack of Cys145,
interacted with the amide backbone of Gly143, Ser144 and
Cys145 (Fig. 1b). The nitrogen in the thiazole was also in contact
with His41 through hydrogen bonding interactions. These interac-
tions indicate an acceptance of the unique ketone. On the other
hand, the benzyloxycarbonyl group barely made hydrophobic
diethylamide. However, compound 8a exhibited lower inhibitory
activity than lead compound 1a. A plausible reason for the ob-
served low activity could be attributed to a disruption of hydrogen
bonding interactions at the P1 position with the active site of the
enzyme, or an unfavorable steric hindrance to a covalent bond for-
mation at the CF3-ketone moiety with the active site cysteine res-
idue in the enzyme. On the other hand, replacements of P1 N,N-
diethylamide in compound 8a with N-morpholine- and N,N-meth-
ylbenzyl-amides increased the inhibitory activity (Table 1, entries
4 and 5). Indeed, the activity of compounds 8b and 8c with Ki val-
ues of 21.0 and 34.1 lM, respectively, was fivefold higher affinity

2726
T. Regnier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2722–2727
Table 2
a
Structure and activity of the synthesized thiazolyl and benzothiazolyl inhibitors
Entry
Compound
Structurea
Kib
(lM)
O
O
N
1
8b
21.0 4.3
478 193
112 34
2.20 0.8
CF3
Cbz-Val-Leu-NH
O
O
O
N
2
3
4
14a
14b
21
S
N
Cbz-Val-Leu-NH
O
b
O
N
S
N
Cbz-Val-Leu-NH
Cbz-Val-Leu-NH
O
H
N
O
S
N
O
S
O
N
5
6
26
45.2 7.8
S
N
Cbz-Val-Leu-NH
O
H
N
O
O
N
PDB ID, 1WOF
O
O
H
N
H
N
O
O
N
N
H
N
H
14c
462 145
S
O
O
O
N
N
Cbz-Leu-NH
H
N
O
O
O
O
S
H
N
O
N
Compound 21
O
N
N
H
H
O
O
7
8
14d
14e
14f
614 139
49.3 10
159 25
S
N
Cbz-Val-NH
O
Figure 1. Molecular dynamics simulated pose of compound 21 (green stick) bound
to SARS-CoV 3CLpro (PDB 1WOF, with red and cyan ribbons with molecular
surfaces). (a) Overlapped view with an original vinyl ester ligand (pink stick). (b)
Contacted residues with hydrogen bonding interactions (dotted line).
O
N
S
N
Cbz-Val-Leu-NH
O
SARS-CoV 3CLpro inhibitor. The measured inhibitory activity cou-
pled with possible structure modifications revealed by 3D docking
give us new directions for a fast development of much more potent
inhibitors. Further investigations on this new family of compounds
are currently in progress in our laboratory.
O
N
9
S
N
Cbz-Val-NH
O
Acknowledgements
a
Cbz, benzyloxycarbonyl.
b
Ki, inhibition constant against SARS-CoV 3CLpro
.
This research was supported by The Frontier Research Program;
The 21st Century COE Program from The Ministry of Education, Cul-
ture, Sports, Science and Technology, Japan; and Japan Society for
the Promotion of Science’s Post-Doctoral Fellowship for Foreign
Researchers. E.F. also acknowledges support from a grant from
the National Institutes of Health (GM 57144). The manuscript
was thoroughly revised by Dr. Jeffrey-Tri Nguyen.
interactions, suggesting a possibility for further optimizations of
the moiety.
In conclusion, we disclosed the inhibitory potency of compound
21, containing a P1-pyrrolidone and warhead thiazolyl unit, as a

T. Regnier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2722–2727
2727
9. Jain, R. P.; Petterson, H. I.; Zhang, J.; Aull, K. D.; Fortin, P. D.; Huitema, C.; Eltis, L.
D.; Parrish, J. C.; James, M. N. G.; Wishart, D. S.; Vederas, J. C. J. Med. Chem. 2004,
47, 6113.
References and notes
1. He, J.-F.; Peng, G.-W.; Min, J.; Yu, D.-W.; Liang, W.-L.; Zhang, S.-Y.; Xu, R.-H.;
Zheng, H.-Y.; Wu, X.-W.; Xu, J.; Wang, Z.-H.; Fang, L.; Zhang, X.; Li, H.; Yan, X.-
G.; Lu, J.-H.; Hu, Z.-H.; Huang, J.-C.; Wan, Z.-Y.; Hou, J.-L.; Lin, J.-Y.; Song, H.-D.;
Wang, S.-Y.; Zhou, X.-J.; Zhang, G.-W.; Gu, B.-W.; Zheng, H.-J.; Zhang, X.-L.; He,
M.; Zheng, K.; Wang, B.-F.; Fu, G.; Wang, X.-N.; Chen, S.-J.; Chen, Z.; Hao, P.;
Tang, H.; Ren, S.-X.; Zhong, Y.; Guo, Z.-M.; Liu, Q.; Miao, Y.-G.; Kong, X.-Y.; He,
W.-Z.; Li, Y.-X.; Wu, C.-I.; Zhao, G.-P.; Chiu, R. W. K.; Chim, S. S. C.; Tong, Y.-K.;
Chan, P. K. S.; Tam, J. S.; Lo, Y. M. D. Science 2004, 303, 1666.
2. 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.; Rota, P.; Fields, B.;
DeRisi, J.; Yang, J.-Y.; Cox, N.; Hughes, J. M.; LeDuc, J. W.; Bellini, W. J.;
Anderson, L. J. N. Eng. J. Med. 2003, 348, 1953.
3. Frosten, C.; Günther, S.; Preiser, W.; Van Der Werf, S.; Brodt, H.-R.; Backer, S.;
Rabenau, H.; Panning, M.; Kolesnikova, L.; Fouchier, R. A. M.; Berger, A.;
Burguiére, A.-M.; Cinatl, J.; Eickmann, M.; Escriou, N.; Grywna, K.; Kramme, S.;
Manuguerra, J.-C.; Müller, S.; Rickerts, V.; Stürmer, M.; Vieth, S.; Klenk, H.-D.;
Osterhaus, A. D. M. E.; Schmitz, H.; Doerr, H. W. N. N. Eng. J. Med. 2003, 348,
1967.
4. Rota, P. A.; Oberste, M. S.; Monroe, S. S.; Nix, W. A.; Campagnoli, R.; Icenogle, J.
P.; Peñaranda, S.; Bankamp, B.; Maher, K.; Chen, M.; Tong, S.; Tamin, A.; Lowe,
L.; Frace, M.; DeRisi, J. L.; Chen, Q.; Wang, D.; Erdman, D. D.; Peret, T. C. T.;
Burns, C.; Ksiazek, T. G.; Rollin, P. E.; Sanchez, A.; Liffick, S.; Holloway, B.; Limor,
J.; McCaustland, K.; Olsen-Rasmussen, M.; Fouchier, R.; Günther, S.; Osterhaus,
A. D. M. E.; Drosten, C.; Pallansch, M. A.; Anderson, L. J.; Bellini, W. J. Science
2003, 300, 1394.
5. Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J. R.; Hilgenfeld, R. Science 2003,
300, 1763.
6. Snijder, E. J.; Bredenbeek, P. J.; Dobbe, J. C.; Thiel, V.; Ziebuhr, J.; Poon, L. L. M.;
Guan, Y.; Rozanov, M.; Spaan, W. J.; Gorbalenya, A. E. J. Mol. Biol. 2003, 331,
991.
7. (a) Wu, C.-Y.; Jan, J.-T.; Ma, S.-H.; Kuo, C.-J.; Juan, H.-F.; Cheng, E. Y.-S.; Hsu, H.-
H.; Huang, H.-C.; Wu, D.; Brik, A.; Liang, F.-S.; Liu, R.-S.; Fang, J.-M.; Chen, S.-T.;
Liang, P.-H.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10012; (b) Shao,
Y.-M.; Yang, W.-B.; Peng, H.-P.; Hsu, M.-F.; Tsai, K.-C.; Kuo, T.-H.; Wang, A. H.-J.;
Liang, P.-H.; Lin, C.-H.; Yang, A.-S.; Wong, C.-H. ChemBioChem 2007, 8, 1654.
8. Bacha, U.; Barrila, J.; Velasquez-Campoy, A.; Leavitt, S. A.; Freire, E. Biochemistry
2004, 43, 4906.
10. Chen, L.-R.; Wang, Y.-C.; Lin, Y.-W.; Chou, S.-Y.; Chen, S.-F.; Liu, L.-T.; Wu, Y.-T.;
Kuo, C.-J.; Chen, T. S.-S.; Juang, S.-H. Bioorg. Med. Chem. Lett. 2005, 15, 3058.
11. (a) Ghosh, A. K.; Xi, K.; Ratia, K.; Santarsiero, B. D.; Fu, W.; Harcourt, B. H.; Rota,
P. A.; Baker, S. C.; Johnson, M. E.; Mesecar, A. D. J. Med. Chem. 2005, 48, 6767; (b)
Shie, J.-J.; Fang, J.-M.; Kuo, T.-H.; Kuo, C.-J.; Liang, P.-H.; Huang, H.-J.; Wu, Y.-T.;
Jan, J.-T.; Cheng, E. Y.-S.; Wong, C.-H. Bioorg. Med. Chem. 2005, 13, 5240; (c)
Ghosh, A. K.; Xi, K.; Grum-Tokars, V.; Xu, X.; Ratia, K.; Fu, W.; Houser, K. V.; Baker,
S. C.; Johnson, M. E.; Mesecar, A. D. Bioorg. Med. Chem. Lett. 2007, 17, 5876.
12. Shie, J.-J.; Fang, J.-M.; Kuo, T.-H.; Kuo, C.-J.; Liang, P.-H.; Huang, H.-J.; Yang, W.-
B.; Lin, C.-H.; Chen, J.-L.; Wu, Y.-T.; Wong, C.-H. J. Med. Chem. 2005, 48, 4469.
13. Wu, C.-Y.; King, K.-Y.; Kuo, C.-J.; Fang, J.-M.; Wu, Y.-T.; Ho, M.-Y.; Liao, C.-L.;
Shie, J.-J.; Liang, P.-H.; Wong, C.-H. Chem. Biol. 2006, 13, 4469.
14. (a) Sydnes, M. O.; Hayashi, Y.; Sharma, V. K.; Hamada, T.; Bacha, U.; Barrila, J.;
Freire, E.; Kiso, Y. Tetrahedron 2006, 62, 8601; (b) Bacha, U.; Barrila, J.; Gabelli,
B.; Kiso, Y.; Amzel, L. M.; Freire, E. Chem. Biol. Drug Des. 2008, 72, 34.
15. Shao, Y.-M.; Yang, W.-B.; Kuo, T.-H.; Tsai, K.-C.; Lin, C.-H.; Yang, A.-S.; Liang, P.-
H.; Wong, C.-H. Bioorg. Med. Chem. 2008, 16, 4652.
16. Zhang, H.-Z.; Zhang, H.; Kemnitzer, W.; Tseng, B.; Cinatl, J., Jr.; Michaelis, M.;
Doerr, H. W.; Cai, S. X. J. Med. Chem. 2006, 49, 1198.
17. Luesch, H.; Hoffman, D.; Hevel, J. M.; Becker, J. E.; Golakoti, T.; Moore, R. E. J.
Org. Chem. 2003, 68, 83.
18. Kuo, C. J.; Chi, Y. H.; Hsu, J. T. A.; Liang, P. H.; Leavitt, S. A.; Freire, E. Biochem.
Biophys. Res. Commun. 2004, 318, 862.
19. Maynard, G. D.; Cheng, H. C.; Kane, J. M.; Staeger, M. A. Bioorg. Med. Chem. Lett.
1993, 4, 753.
20. Tian, Q.; Nayyar, N. K.; Babu, S.; Chen, L.; Tao, J.; Lee, S.; Tibbetts, A.; Moran, T.;
Liou, J.; Guo, M.; Kennedy, T. P. Tetrahedron Lett. 2001, 42, 6807.
21. Yang, H.; Xie, W.; Xue, X.; Yang, K.; Ma, J.; Liang, W.; Zhao, Q.; Zhou, Z.; Pei, D.;
Ziebuhr, J.; Hilgenfeld, R.; Yuen, K. Y.; Wong, L.; Gao, G.; Chen, S.; Chen, Z.; Ma,
D.; Bartlam, M.; Rao, Z. PLoS Biol. 2005, 3, 1742.
22. Briefly, the inhibitors were equilibrated with the enzyme for 10 min before the
addition of the substrate to determine their activity respectively. The kinetic
reaction was initiated by the addition of the substrate which has a sequence
reminiscent of the N-terminal auto-cleavage site of the protease, flanked by the
fluorescent groups, dabcyl and edans. The rate of substrate cleavage can be
detected by an increase in fluorescence that can be monitored over time. The Ki
or the inhibition constant is an indication of the compound as potency.