Isatin compounds as noncovalent SARS coronavirus 3C-like protease inhibitors

Article abstract of DOI:10.1021/jm0602357

A series of isatin derivatives were synthesized and tested against SARS CoV 3C-like protease. Substitutions at the N-1 and C-5 positions were examined to elucidate the differences in substrate binding sites of the rhinovirus 3C protease and SARS CoV 3C-li

Full text of DOI:10.1021/jm0602357

3440
J. Med. Chem. 2006, 49, 3440-3443
Isatin Compounds as Noncovalent SARS
Coronavirus 3C-like Protease Inhibitors
Lu Zhou,† Ying Liu,*,† Weilin Zhang,† Ping Wei,†
Changkang Huang,† Jianfeng Pei,†,‡ Yaxia Yuan,† and
Luhua Lai*,†,‡
State Key Laboratory for Structural Chemistry of Unstable and
Stable Species, College of Chemistry and Molecular Engineering,
Peking UniVersity, Beijing 100871, China, and Center for
Theoretical Biology, Peking UniVersity, Beijing 100871, China
ReceiVed March 1, 2006
Abstract: A series of isatin derivatives were synthesized and tested
against SARS CoV 3C-like protease. Substitutions at the N-1 and C-5
positions were examined to elucidate the differences in substrate binding
sites of the rhinovirus 3C protease and SARS CoV 3C-like protease.
Compound 5f shows significant inhibition with an IC50 of 0.37 µM.
Further study showed that, unlike the irreversible covalent binding of
isatin derivatives to human rhinovirus 3C protease, the compounds
tested in this study are all noncovalent reversible inhibitors.
Severe acute respiratory syndrome (SARS), a life-threatening
form of atypical pneumonia, rapidly spread from its likely origin
in southern China to several other countries during late 2002
and early 2003.1 SARS is characterized by high fever (>38 °C),
malaise, rigor, headache, and nonproductive cough or dyspnea
and may progress to generalized interstitial infiltrates in the
lung.2 The disease had a quite high mortality rate (up to
15-19%) during the initial outbreak. In early 2003, the novel
human coronavirus named SARS coronavirus (SARS CoV) was
identified as the causative agent of SARS.1a Since SARS CoV
3C-like protease plays a very important role in the viral life
cycle, it has been proposed as a key target for the structure-
based drug design against SARS.3
SARS CoV 3CL protease inhibitors have been reported from
both screened compound libraries4-9 and designed compounds
based on substrate structure or active site properties.10-14 Their
scaffolds are diverse, including C2 symmetric peptidomimetic
compounds,4 3-quinolinecarboxylic acid derivatives,5 thiophene-
2-carboxylate derivatives,6 zinc-conjugated compounds,7 cin-
anserin,8 calmodulin,9 keto-glutamine analogues,10 anilide,11
bifunctional boronic acid compounds,12 isatin derivatives,13 and
etacrynic acid derivatives.14 With metal-conjugated structures,
some molecules might make a covalent bond with Cys145 at
the active site of SARS CoV 3CL protease.5-8 Some other
reported peptidyl molecules have complicated scaffolds and may
have low bioavailability.4,10,14 Till now, only a few candidates
have high inhibition activity against SARS CoV 3C-like protease
with IC50 values below the micromolar level.
Figure 1. (a) The complex structure of compound 5f and SARS CoV
3C-like protease from docking study. The figure was drawn by
PyMOL.21 The protein was shown in ribbons, and the compound 5f
and interacting residues in stick (blue: nitrogen, red: oxygen, orange:
sulfur). The carboxamide at C-5 makes H-bonds with Phe140 and
His163, and the oxygens at C-2 and C-3 make H-bonds with His41
and Cys145. The naphthyl ring fits into the hydrophobic S2 site formed
by Met 49 and Met 165. (b) The contacts of compound 5f and SARS
CoV 3C-like protease were shown by LIGPLOT.22
inhibitors of SARS CoV 3CL protease. Chen et al. reported
that N-substituted isatin derivatives which were HRV inhibitors
were effective SARS CoV 3C-like protease inhibitors.13 They
focused mainly on the isatin C-4, C-5, C-7, and N substituents,
aiming at the two sites (P1, P2) that were important for HRV
protease inhibitors. Most of their compounds have halogen
substituents at the C-5 position, and the most active compound
is an N-substituted 5-iodoisatin. As SARS CoV 3C-like protease
specifically cleaves peptide substrates with Gln at the P1 site,
a carboxamide group on the inhibitors may occupy the position
of the Gln side chain that forms specific hydrogen bonds with
Phe140 and His163. We have designed and synthesized a series
of N-substituted 5-carboxamide-isatin compounds and compared
their activities with 5-halogen-substituted compounds.13,16
Isatin compounds were docked into the substrate binding
pocket of SARS CoV 3C-like protease using the SYBYL
program (Tripos, Inc.) in a similar orientation as that of the
covalent binding substrate analogue as revealed in the recently
The active site of SARS CoV 3CL protease contains Cys145
and His41 to constitute a catalytic dyad, in which the cysteine
residue functions as the common nucleophile in the proteolytic
process.15 The catalytic domain is similar to other virus 3C
proteases, such as human rhinovirus (HRV) 3C protease.
Therefore, HRV 3C protease inhibitors may also be potential
* To whom correspondence should be addressed. Phone: 86-10-
62757486. Fax: 86-10-62751725. E-mail: lhlai@pku.edu.cn (L.L.).
Phone: 86-10-62767284. Fax: 86-10-62751725. E-mail: liuying@pku.edu.cn
(Y.L.).
† State Key Laboratory for Structural Chemistry of Unstable and Stable
Species.
‡ Center for Theoretical Biology.
10.1021/jm0602357 CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/16/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3441
Table 1. Inhibition Activities of Isatin Derivatives against SARS CoV 3C-like Protease
IC50 or percentage (%)
of inhibition at 100 µM
Ki
Ki
pKi
pKi
name
R1
R2
(experimental)
(theoretical)
(experimental)
(theoretical)a
b
1e
2a
2b
2c
2d
2f
3a
3b
3c
3d
3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
H
CH3
CH3CH2CH2
n-C4H9
Ph CH2
â-C10H7CH2
CH3
CH3CH2CH2
n-C4H9
Ph CH2
â-C10H7CH2
CH3
CH3CH2CH2
n-C4H9
PhCH2
I
I
I
I
I
I
-
-
25% at 100 µM
66 ( 5 µM
6.6 × 10-5
5.0 × 10-5
1.1 × 10-6
8.5 × 10-5
3.0 × 10-5
1.2 × 10-6
4.18
4.3
5.96
4.07
4.53
5.93
50 ( 20 µM
1.1 ( 0.2 µM
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2H
CO2H
CO2H
CO2H
CO2H
-
-
-
-
-
-
-
-
-
-
H
â-C10H7CH2
CH3
CH3CH2CH2
n-C4H9
PhCH2
CO2H
46% at 100 µM
71 ( 6 µM
25 ( 2 µM
19 ( 2 µM
12.5 ( 0.5 µM
-
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
7.1 × 10-5
2.5 × 10-5
1.9 × 10-5
1.3 × 10-5
2.9 × 10-4
1.6 × 10-4
1.7 × 10-4
3.3 × 10-5
4.14
4.6
4.72
4.9
3.54
3.79
3.78
4.48
H
â-C10H7CH2
0.37 ( 0.03 µM
1.2 × 10-7
4.1 × 10-7
6.92
6.39
a The pKi values were calculated by SCORE program.18 b Inhibition less than 25% at 100 µM.
Scheme 1a
a Reagents and conditions: (a) NaH, DMF, R1X; (b) CO, MeOH, DMF,
Et3N, (Ph3P)2PdCl2; (c) K2CO3, MeOH, H2O; (d) N-hydroxysuccinimide,
EDC, CH2Cl2; (e) DMF, NH3(g).
Figure 2. Calculated pKi vs experimental pKi. The data were fit to a
straight line with an R2 of 0.85.
published complex crystal structure.17 The carbonyl oxygen
atoms at C-2 and C-3 make hydrogen bonds with the active
site residues, His41 and Cys145. The carboxamide at C-5 holds
the same position as that of the substrate P1 Gln side chain and
makes H-bonds with the backbone carbonyl of Phe140 and the
imidazole ring of His163. Alkyl groups at N-1 fit into the
hydrophobic S2 site formed by Met 49 and Met 165. The
binding orientation is similar to that in HRV-2 protease; where
Thr142 is replaced by Phe140 at the P1 pocket.16 Figure 1 shows
the orientation of compound 5f in the substrate binding pocket.
We have calculated the binding affinity of the protein-ligand
complexes (Table 1) using the SCORE program.18 The experi-
mental pKi values correlate well with the calculated pKis (R2
of 0.85, Figure 2), verifying that the complex structures built
are reasonable.
Compounds 5a-d were synthesized by a five-step synthetic
route (Scheme 1).16 (1) Hydrogen at N-1 was substituted by
the alkyl groups using RX. (2) 5-Iodoisatin was changed into
5-carbomethoxyisatin via bis(triphenylphosphine)palladium chlo-
ride catalysis in a carbon monoxide atomosphere. (3) The
hydrolysis of the methyl ester to carboxylic acid was achieved
using potassium carbonate. (4) The activated 5-carboxylic acid
N-hydroxysuccinimide ester was made with N-hydroxysuccin-
imide. (5) Isatin-5-carboxamides were obtained by treating
5-carboxylic acid N-hydroxysuccinimide ester with ammonia.
Preparation of 5f was started from ethyl 4-aminobenzoate.
First, N-(4-ethoxycarbonylphenyl)-2-(hydroxyimino)acetamide
was obtained by reaction of ethyl 4-aminobenzoate, hydroxy-
lamine hydrochloride, and 2,2,2-trichloroacetaldehyde mono-
hydrate, and it was converted into isatin-5-carboxylic by
treatment with concentrated sulfuric acid at 90 °C; then 1H-
isatin-5-carboxylic acid was changed into 1H-isatin-5-carbox-
amide. Finally, 5f was obtained by reaction of 1H-isatin-5-
carboxamide with 2-(bromomethyl)naphthalene as shown in
Scheme 2. Compound structures were confirmed by 1H NMR,
MS, and elemental analysis, and the data are consistent with
previously published data.16
The inhibition activity was measured by continuous colori-
metric assay using colorimetric substrate Thr-Ser-Ala-Val-Leu-
Gln-pNA (HPLC purity, purchased from GL Biochemistry
Ltd).19 SARS 3C-like protease was cloned and expressed in
Escherichia coli and purified according to the published
procedure.20 The purified protease was stored with 5 mM DDT
and 50% glycerol in Tris-HCl buffer at -20 °C. Before the
inhibition test, the enzyme buffer was changed to PBS (20 mM,
pH 7.4). Colorimetric measurements of the protease activity

3442 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Letters
Scheme 2a
Table 2. Selective Inhibition of Compound 5f against Various Cysteine
and Serine Proteasesa
enzyme SARS 3CL papainb chymotrypsinc trypsind
IC50 (µM) 0.37 ( 0.03 11 ( 1 34 ( 2
352 ( 10 0.004 ( 0.00316
HRV-14
a The IC50 values were measured by HPLC except SARS 3CL protease
and concentration of substrate (S0120) was 400 µM. b The concentration of
papain was 4.5 µM. c The concentration of chymotrysin was 0.45 µM. d The
concentration of trypsin was 0.23 µM.
isatin inhibitors. It is more probable that the oxygen at C-3 may
form a hydrogen bond with Cys145 instead of forming a
covalent bond between the carbon and sulfur atoms.
a Reagents and conditions: (a) Cl3CCH(OH)2, NH2OH‚HCl, Na2SO4,
H2O; (b) H2SO4; (c) N-hydroxysuccinimide, EDC, CH2Cl2; (d) DMF,
NH3(g); (e) NaH, DMF, 2-(bromomethyl)naphthalene.
From the protein-inhibitor complex modeling study and the
inhibition data, we concluded the following: (1) A hydrophobic
group placed at the N-1 position can fit into the hydrophobic
pocket, and large, aromatic hydrophobic groups lead to high
activity. The naphthyl group also made the molecule more rigid
and fit into the pocket well. (2) Carboxamide substitution at
the C-5 position made the molecule three to four times more
active than iodic substitution in which iodine was demonstrated
to be a hydrophobic and electron affinitive group.13 However,
the carboxylic acid and ester derivatives showed no inhibition
at all, which was completely different from their binding with
HRV protease.16 The carboxamide group can form hydrogen
bonds with the side chain of His163 and the backbone of
Phe140, while the carboxylic acid and ester can only form one
hydrogen bond. The carboxylic acid and ester were also close
to Glu166 and encounter strong charge repulsion, thus making
them unsuitable for binding to the P1 site. (3) The C-3 oxygen
formed a hydrogen bond with the side chain of Cys145 that
was important for inhibition activity. If the oxygen atom was
changed into other groups, such as 4-methoxyphenylimino£ or
1-naphthylimino, the compounds lost activity (data not shown).
DTT may react with the C-3 ketone group and inactivate the
compounds.16
We also tested the inhibition activity of compound 5f against
other proteases (Table 2). Compound 5f was a more selective
inhibitor against SARS CoV 3C-like protease than papain (30
times), chymotrypsin (92 times), and trypsin (950 times). As
5f was also a potent inhibitor against HRV-14 3C protease,16 it
may be used as a broad-spectrum antiviral agent.
In conclusion, a series of N-substituted isatin derivatives were
synthesized and tested against SARS CoV 3C-like protease
using a colorimetric assay and confirmed by HPLC. The
compounds were shown to be noncovalent reversible inhibitors
of SARS CoV 3C-like protease. The C-5 position was found to
favor a carboxamide group and the N-1 position to favor large
hydrophobic substituents. The lowest IC50 value (0.37 µM) was
observed with compound 5f, and it was selective for SARS CoV
3C-like protease over other proteases.
were performed in 96-well microtiter plates using a multiwell
ultraviolet spectrometer (Spectra Max 190; Molecular Device).
Substrate (final concentration of 200 µM) and inhibitors (final
concentration 0-5.5 µM) were mixed together in 20 mM PBS
buffer (pH ) 7.4) in the presence of 5% (vol/vol) DMSO, and
the reaction was initiated by adding SARS CoV 3C-like protease
(final concentration of 1 µM). Increase in absorbance at 390
nm was measured, and the IC50 value was calculated by the
following equation:
V0/V ) 1 + [I]/IC50
where V0 is the initial rate of the reaction without inhibitor, V
is the initial rate of the reaction with the inhibitor at various
concentrations, [I] is the concentration of the inhibitor, and IC50
is the concentration resulting in 50% inhibition.
Before the reaction, the enzyme was exchanged to a fresh
phosphate buffer without dithiothreitol (DTT), as DTT reacted
with isatin derivatives and caused an increase in the absorbance
at 390 nM. An HPLC-based enzyme activity assay was also
used to verify the reaction. HPLC assay showed that the
inhibition of these isatin derivatives decreased if DTT was added
to the mixture. Out of the 23 compounds, 10 were found to be
active at 100 µM. The IC50 values of 8 compounds are lower
than 100 µM, the best being 0.37 ( 0.03 µM (Ki ) 0.12 µM,
compound 5f). Table 1 lists the inhibition activities of the
compounds. The Ki values of the 8 compounds were calculated
according to the literature.19 The pKi (-log Ki) values are also
listed in Table 1.
Compound 5f is a potent inhibitor with an IC50 of 0.37 (
0.03 µM against SARS CoV 3C-like protease. As this compound
was reported to be a covalent binding inhibitor of human
rhinovirus 3C protease,16 it is interesting to know whether it
will bind covalently or noncovalently to SARS CoV 3C-like
protease.
SARS CoV 3C-like protease was mixed with compound 5f
and incubated at 37 °C under nitrogen atmosphere for 72 h.
The mixture was measured by a MALDI-TOFF mass spec-
trometer. The protein peak remained at the same position as
that in the pure enzyme, indicating that the inhibitor did not
form a covalent bond with the protease (data not shown).
We also preincubated the inhibitor and the protease for 24 h
at 4 °C and examined the enzyme activity of the solution. The
activity of the protease that was mixed with the inhibitor for
24 h was the same as that of the mixture which was tested
immediately after mixing. This confirms again that the protease
did not change after incubation with the inhibitor for a long
period of time. The UV-vis spectrum also showed little change
after the protease was mixed with the inhibitor. All this evidence
indicates that Cys145 did not form a covalent bond with the
Acknowledgment. We thank the National Natural Science
Foundation of China (20473001, 10345004, 30490245), the
Ministry of Science and Technology of China, and the Ministry
of Education of China for financial support. We also thank
Professor Jianguo Chen for providing the clones of SARS CoV
3C-like protease.
Supporting Information Available: Experiemntal procedure
for the synthesis of target compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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