Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.08.082

Design, synthesis and biological evaluation of a series of 5-chloropyridine ester-derived severe acute respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors is described. Position of the carboxylate functionality is critical to potency. I

Full text of DOI:10.1016/j.bmcl.2008.08.082

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5684–5688
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and antiviral efficacy of a series of potent chloropyridyl
ester-derived SARS-CoV 3CLpro inhibitors
a
b
b
c
Arun K. Ghosh a, , Gangli Gong , Valerie Grum-Tokars , Debbie C. Mulhearn , Susan C. Baker ,
*
Melissa Coughlin d, Bellur S. Prabhakar d, Katrina Sleeman c, Michael E. Johnson b, Andrew D. Mesecar b
a Departments of Chemistry and Medicinal Chemistry, Purdue University, 560 Oval drive, West Lafayette, IN 47907, USA
b Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA
c Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, IL, USA
d Department of Microbiology and Immunology, University of Illinois at Chicago, IL 60607, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Design, synthesis and biological evaluation of a series of 5-chloropyridine ester-derived severe acute
respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors is described. Position of the car-
boxylate functionality is critical to potency. Inhibitor 10 with a 5-chloropyridinyl ester at position 4 of the
indole ring is the most potent inhibitor with a SARS-CoV 3CLpro IC50 value of 30 nM and an antiviral EC50
Received 15 July 2008
Revised 12 August 2008
Accepted 15 August 2008
Available online 28 August 2008
value of 6.9
l
M. Molecular docking studies have provided possible binding modes of these inhibitors.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
SARS 3CLpro
Ester
Antiviral
Inhibitor
Since its first appearance in southern China in late 2002, severe
acute respiratory syndrome (SARS) has been recognized as a global
threat.1 It has affected more than 8000 individuals in 32 countries
and caused nearly 800 fatalities worldwide within a few months.2
Its causative pathogen is a novel coronavirus and termed as SARS-
CoV.3,4 While SARS is contained in the world and no more cases
have been reported since April 2004, there is expectation that this
epidemic will strike again in an even more severe form. Further-
more, the nature of its unpredictable outbreak is a potential threat
to the global economy and public health. To date, no effective ther-
apy exists for this viral illness.
tors have been reported.7 In our continuing interest in the design
and development of SARS-CoV 3CLpro inhibitors, we recently re-
ported structure-based design of a number of potent peptidomi-
metic SARS-CoV 3CLpro inhibitors (1 and 2).8 The SARS-CoV
3CLpro active site contains a catalytic dyad where a cysteine resi-
due acts as a nucleophile and a histidine residue acts as the general
acid base.9 The inhibitors bind to SARS-CoV 3CLpro through a cova-
lent bond with the active site Cys-145 residue. These inhibitors
contain peptidomimetic scaffolds and lacked adequate potency,
particularly antiviral activity suitable for drug-development. Re-
cently, Wong and co-workers reported a new class of potent small
molecule benzotriazole ester-based 3CLpro inhibitors. Compound
3 is the most potent inhibitor among the benzotriazole esters.10
The mode of action involves acylation of the active site Cys-145 as-
sisted by the catalytic dyad. This irreversible enzyme acylation was
verified by electrospray ionization mass spectrometry of the inhib-
ited enzyme. While these inhibitors have shown very impressive
SARS-CoV 3CLpro enzyme inhibitory activity, their antiviral activ-
ity required improvement.11 It seems the indole-5-carboxylate
moiety plays an important role in binding with the enzyme active
site. Another class of hetereoaromatic ester inhibitors was also
identified and studied.12,13 The 5-chloropyridine moiety in 4
proved to be the key unit for the activity against 3CLpro. The report
however lacked antiviral data. We report herein the development
of 3-chloropyridyl ester-based SARS-CoV 3CLpro inhibitors that
exhibit potent enzyme inhibitory activity as well as very good
The SARS coronavirus is a positive-strand RNA virus. The 50 two-
thirds of the genome encodes two overlapping polyproteins, pp1a
and pp1ab, which are processed to generate the viral replication
complex. During viral replication, the replicase polyprotein under-
goes extensive processing by two viral proteases namely, chymo-
trypsin-like protease (3CLpro) and papain-like protease
(PLpro).5,6 Because of their essential roles in viral replication, both
proteases are recognized as attractive targets for development of
anti-SARS therapeutics.7 The structure and activity of the active
sites of both SARS-CoV 3CLpro and SARS-CoV PLpro have been elu-
cidated. Thus far, inhibitor design efforts are mostly limited to
SARS-CoV 3CLpro and numerous covalent and noncovalent inhibi-
* Corresponding author. Tel.: +1 765 494 5323; fax: +1 765 496 1612.
E-mail address: akghosh@purdue.edu (A.K. Ghosh).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.082

A. K. Ghosh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5684–5688
5685
Table 1
SARS-CoV antiviral activity in cell culture assays. We have also car-
ried out molecular docking studies to obtain the potential binding
mode of these inhibitors.
Structures and activity of inhibitors
Compound structure
SARS 3CLpro IC50 (lM) SARS-CoV EC50 (l
M)a
The general synthetic method for 5-chloropyridyl ester inhibi-
tors is outlined in Scheme 1. Various chloro-3-pyridinyl esters 5,
9, 10, 12–14 (Table 1) were synthesized by esterification of 5-
chloro-3-pyridinol and the corresponding carboxylic acids14 medi-
ated by DCC and DMAP at 23 °C in CH2Cl2. The synthesis of 1-acet-
ylindolecarboxylate inhibitors were carried out by acetylation of
indole 5 and 10 with acetic anhydride and pyridine under reflux
to provide amide 6 and 11, respectively, in excellent yields.
The synthesis of 1-sulfonylindolecarboxylate inhibitors is out-
lined in Scheme 2. Direct sulfonamidation of the indole under reg-
ular TsCl/DMAP condition at 23 °C or higher temperatures could
not provide the desired product. To increase its reactivity, the in-
dole 15 was reduced to indoline 16 by sodium cyanoborohydride
in excellent yield.15 The resulting indoline readily reacted with to-
syl chloride or 3-nitrobenzenesulfonyl chloride to give sulfona-
mides 17 or 18 in good yields. Oxidation of indolines 17 and 18
to their corresponding indoles 19 and 20, respectively, was
achieved using manganese dioxide at high temperature.16 Hydro-
lysis of the methyl esters to the corresponding acids 21 or 22 using
sodium hydroxide followed by the general esterification method
described in Scheme 1 afforded the target compounds 7 or 8.
The structure and activity of inhibitors are shown in Table 1.
The enzyme inhibitory activity of the active esters against SARS-
CoV-3CLpro was determined using the full-length, authentic ver-
sion of the enzyme in a FRET-based, microplate assay described
by Grum-Tokars and co-workers.8,17 The assays were performed
N
N
N
O
O
O
0.2
NTb
N
H
3
N
0.31 0.05
0.40 0.06
0.37 0.06
24 0.9
O
Cl
N
H
5
N
N
O
O
Cl
NIc
N
6
O
Me
O
O
Cl
Cl
NT
N
S
7
O
O
O
in 96-well microplates using a reaction volume of 100 lL which
contained 50 mM HEPES, pH 7.5, 100 nM authentic SARS-CoV-
3CLpro enzyme, 1 mM DTT, 0.01 mg/mL BSA and varying concen-
trations of inhibitors. The reaction components, with the exception
of substrate, were incubated for 20 min and the reaction was initi-
ated by the addition of FRET-substrate HiLyte FluorTM 488-Glu-Ser-
Ala-Thr-Leu-Gln-Ser-Gly-Leu-Arg-Lys-Ala-Lys(QXL520TM)-NH2, giv-
N
O
O
0.089 0.014
NT
N
S
8
2
NO
O
ing a final substrate concentration of 2 l
M as described.8,17 The
IC50 values for inhibitors were determined by measuring the rates
of reaction with increasing inhibitor concentrations.
As shown in Table 1, the known10 benzotriazole ester inhibitor
3 was evaluated in our assay as a control. In inhibitor 5, the benzo-
triazole unit was replaced by a 5-chloropyridine unit. Inhibitor 5
has shown comparable enzymatic inhibitory potency (IC50
O
Cl
N
0.23 0.04
>25
H
O
N
9
0.31
viral activity while 5-chloropyridyl ester 5 exhibited antiviral
activity with an EC50 value of 24
M.18 When the indole nitrogen
was acetylated, the resulting compound 6 remained quite potent
(IC50 of 0.40 M) as did the tosylated indole 7 (IC50 of 0.37 M).
lM) as that of 3. However, inhibitor 3 did not exhibit any anti-
l
O
O
Cl
l
l
0.03 0.01
6.9 0.9
N
10
Interestingly, its nitrobenzenesulfonamide analog 8 shows an im-
proved IC50 value (89 nM). We then investigated the importance
N
H
O
O
Cl
N
N
O
O
DCC, DMAP
+
CH2Cl2, 23 ºC
N
11
R
OH
R
O
Cl
HO
O
Cl
1.08 0.24
NI
70-90%
5 9 10 12-14
N
,
,
,
O
Me
N
N
Ac2O, Py
reflux
O
O
O
Cl
Cl
95%
N
N
H
N
N
H
O
Me
0.08 0.02
12.1 1.6
5
indole-5-carboxylate
6
indole-5-carboxylate
O
O
Cl
10 indole-4-carboxylate
11 indole-4-carboxylate
12
(continued on next page)
Scheme 1. Synthesis of inhibitors 5, 6, 9–14.

5686
A. K. Ghosh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5684–5688
Ph
O
Table 1 (continued)
CO2Et
O
N
O
H
N
H
N
SARS 3CLpro IC50 (lM) SARS-CoV EC50 (l
M)a
Me
Compound structure
H
H
O
Boc N
S
1
N
N
O
>100
NT
N
H
O
O
O
Cl
HO
CO2Et
13
O
H
H
N
N
Boc
N
H
N
H
O
O
N
O
2
N
H
O
O
O
Cl
0.14 0.017
NT
N
N
O
N
N
N
14
O
Me
O
O
Cl
a
b
c
For assay protocol, see Ref. 18.
NI = no inhibtion.
N
H
3
S
4
NT = not tested.
Figure 1. Structures of SARS-CoV 3CLpro inhibitors.
of the carboxylic position on the benzene ring of indole. Accord-
ingly, carboxylate substitution on indole rings at 5, 6, 4 and 7 posi-
tions resulted in chloropyridinyl esters 5, 9, 10 and 12,
respectively. These inhibitors were evaluated and as it turned
out, inhibitor 10, with a carboxylate at the 4-position, was the
most potent inhibitor with an IC50 of 30 nM, a 10-fold potency
enhancement over 5 containing a carboxylate at the 5-position.
Compound 10 also shows the best SARS-CoV antiviral activity with
an EC50 value of 6.9 lM. Acylation of 10–11 resulted in a 30-fold
loss of potency. Penicillin-derived chloropyridine 13 did not show
any appreciable activity. Tetrahydroisoquinoline derivative 14,
however, exhibited an enzyme IC50 value of 0.14 lM. In general,
positioned near the more hydrophobic S2 pocket, with the indole
nitrogen likely interacting with the imidazole group of His-41,
see Figure 2.
Next, we analyzed the interaction between 10 and 3CLpro in the
‘post-reaction’ or covalently modified state. The product of the
reaction of 10 with 3CLpro was docked using a more recently re-
leased 3CLpro crystal structure, (PDBID: 2V6N).20 This crystal
structure contains a benzotriazole ester molecule which has re-
acted with the thiol of Cys-145, forming a covalently bound ligand
similar to the compounds presented in this paper. We have used
this crystal structure for the post-reaction complex due to the
notable movement of the His-41 in 2V6N, which flips and is able
an indole with a free nitrogen is more potent than its correspond-
ing protected analogue.
to
p stack with the aromatic moiety of the smaller covalently
bound ligand.20 GOLD3.221 was chosen again for generating this
To confirm that 3CLpro is covalently modified by 10, we deter-
mined enzyme modification using MALDI-TOF.19 Authentic SARS-
CoV 3CLpro was incubated with compound 10 for 20 min and then
analyzed in comparison with untreated enzyme. A shift of approx-
imately 217 Da was observed after treatment of SARS-CoV 3CLpro
with the inhibitor confirming covalent modification. Covalent
modification by similar reactive esters has also been reported.13a,20
To obtain molecular insight into the binding properties of these
active ester-based inhibitors, we conducted docking studies in the
3CLpro active site. GOLD3.221 was used to dock our most active
compound, 10 (GRL-0496), into the active site of the authentic
SARS-CoV 3CLpro structure (PDBID: 2HOB).22 In search of obtain-
ing a model of the associated complex between the unreacted ester
and protein, (i.e. prior to nucleophilic attack by Cys-145), the dis-
tance between the carbonyl carbon atom of 10 and the sulfur atom
of Cys-145 was constrained to be in the range of 2.5–3.5 Å. This
pre-reaction or ‘collision complex’ is shown in Figures 1 and 2,
and resulted in a distance of 2.8 Å between the carbonyl carbon
of 10 and the sulfur of Cys-145. This orientation of the ligand has
the chloropyridyl group situated in the S1 pocket, with the chloro
group pointing towards the surface of the protein. The nitrogen of
the chloropyridinyl leaving group is in close proximity (2.4 Å) to
the imidazole nitrogen of His-163. The carbonyl oxygen is situated
between three backbone nitrogens, forming three hydrogen bonds.
As shown in Figure 2: the first hydrogen bond is from Cys-145(NH)
(2.3 Å), the second is from Ser-144(NH) (2.4 Å), and the third is
from Gly-143(NH) (2.8 Å). This suggests that a fairly strong hydro-
gen bonding network is present within the active site which likely
aids in positioning and stabilizing the carbonyl group of the ester
for nucleophilic attack by the Cys-145. The indole group of 10 is
O
O
NaCNBH3,
OMe
OMe
OMe
16
HOAc, 10 °C
N
H
N
H
15
96%
TsCl or
3-NO2C6H4SO2Cl
DMAP, CH2Cl2,
23 ºC
90%
O
O
OMe
N
S
O
MnO2, toluene,
100 °C
R2
N
S
O
R2
O
75%
R1
O
R1
1
2
19
R = Me; R = H
20 R1 = H; R2 = NO2
17
1
2
R = Me; R = H
18 R1 = H; R2 = NO2
aq. NaOH,
95%
EtOH, 80 ºC
N
O
O
OH
O
Cl
DCC, DMAP,
N
S
O
5-chloro-3-pyridinol
N
S
O
R2
R1
O
CH2Cl2, 23 ºC
60%
R2
R1
O
1
2
1
2
21
R = Me; R = H
22 R1 = H; R2 = NO2
7
R = Me; R = H
8 R1 = H; R2 = NO2
Scheme 2. Synthesis of inhibitors 7 and 8.

A. K. Ghosh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5684–5688
5687
Figure 2. Relaxed stereoview of the GOLD conformation of an associated complex between 10 (green) and 3CLpro (PDBID: 2HOB), with residues shown in magenta.
Figure 3. GOLD docked conformation of 10 (green), covalently linked to Cys-145 of 3CLpro based on the 2V6N 3CLpro structure.20
model, as it has the capability for docking covalently bound
ligands.
shown in Figure 3, such as Asp-187 and Gln-189 are more than
5 Å away from the indolyl, but might come into play with larger
substituents, such as in compound 11.
Figure 3 is a docked model of 10 covalently attached to the Cys-
145 sulfur atom. The model suggests that the indole group of 10
shifts and positions itself where the leaving group was in the com-
plex, more towards the S1 pocket. This positioning of inhibitor 10
in the complex is not surprising as a similar orientation has been
proposed before by James’ group for similar esters.23,24 More
The results of the docking studies presented here suggest that
the indole group of 10 can potentially occupy two different binding
pockets during the course of the reaction. Dynamic enzymatic rear-
rangement in the vicinity of Cys-145 have recently been suggested
from the X-ray structure of SARS-CoV 3CLpro that had been reacted
with 1-(4-dimethylaminobenzoyloxyl)-benzotriazole,20 and sug-
gests that after covalently linking to Cys-145, the indolyl of 10
shifts towards the S1 pocket and stacks with the shifted imidazole
ring of His-41, locking the orientation, with implications of where
substitutions on the indolyl ring would be most beneficial. By hav-
ing both of these models now available, structural modifications of
importantly though is the obvious
p–p stacking of the indolyl of
10 with the imidazole ring of His-41, which is also seen in the ben-
zotriazole group of the referenced crystal structure (2V6N).20 There
is approximately 4 Å between the aromatic rings of the indolyl and
the imidazole of His-41. This interaction clearly determines the po-
sition of the indolyl group of our compounds. Other residues

5688
A. K. Ghosh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5684–5688
9. Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J. R.; Hilgenfeld, R. Science 2003,
300, 1763.
10. 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, 261.
11. Thus far, no antiviral activity has been reported for these active ester-derived
inhibitors. Inhibitor 3 did not exhibit any antiviral activity in our assay.
12. Blanchard, J. E.; Elowe, N. H.; Huitema, C.; Fortin, P. D.; Cechetto, J. D.; Eltis, L.
D.; Brown, E. D. Chem. Biol. 2004, 11, 1445.
13. (a) Zhang, J.; Pettersson, H. I.; Huitema, C.; Niu, C.; Yin, J.; James, M. N.; Eltis, L.
D.; Vederas, J. C. J. Med. Chem. 2007, 50, 1850; (b) Niu, C.; Yin, J.; Zhang, J.;
Vederas, J. C.; James, M. N. Bioorg. Med. Chem. 2008, 16, 293.
14. The indolecarboxylic acids are either from a commercial source or made from
hydrolysis of corresponding esters or oxidation of corresponding aldehydes.
The starting material for 13 is from Boc protection of 6-aminopenicillanic acid.
For preparation of the starting material of 14, 2-acetyl-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid, please see: Grunewald, G. L.;
Romero, F. A.; Criscione, K. R. J. Med. Chem. 2005, 48, 134.
the indole group can be tailored to enhance interactions with the
S2 pocket for the complex formation to readily occur, but at the
same time, consider that the ligand must be mobile enough to then
occupy the S1 pocket post-reaction.
In conclusion, our design strategies by combining the key parts
of two mechanism-based inhibitors led to a series of 5-chloropy-
ridinyl indolecarboxylate inhibitors with enzymatic potency at
submicromolar levels. The position of the carboxylic acid ester is
critical to its potency. Indolecarboxylate 10 with a carboxylate
functionality at the 4-position is the most potent inhibitor with
an enzyme inhibitory activity against SARS-CoV 3CLpro with an
IC50 of 30 nM and antiviral potency with an EC50 value of 6.9 lM.
Further design and synthesis of more effective inhibitors are in
progress in our laboratories.
15. Gribble, G. W.; Hoffman, J. H. Synthesis 1977, 859.
16. Chandra, T.; Zou, S.; Brown, K. L. Tetrahedron Lett. 2004, 45, 7783.
17. Grum-Tokars, V.; Ratia, K.; Begaye, A.; Baker, S. C.; Mesecar, A. D. Virus Res.
2008, 133, 63.
Acknowledgment
18. SARS-CoV antiviral activity assays.Vero E6 cells were maintained in Minimal
Essential Media (MEM) (Gibco) supplemented with 100 U/mL penicillin,
The financial support of this work is provided by the National
Institute of Health (NIAID, P01 A1060915).
100
lg/mL streptomycin (Gibco) and 10% fetal calf serum (FCS) (Gemini Bio-
Products). The SARS-CoV Urbani strain used in this study was provided by the
Centers for Disease Control and Prevention.4 All experiments using SARS-CoV
were carried out in a Biosafety Level 3 facility using approved biosafety
protocols.Vero E6 cells were seeded onto flat-bottom, 96-well plates at a
density of 9 ꢀ 103 cells/well. Cells were either mock infected with serum-free
References and notes
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MEM or infected with 300 TCID50/well of SARS-CoV Urbani in 100 lL of serum-
free MEM and incubated for 1 h at 37 °C with 5% CO2. Following the 1 h
incubation period, the viral inoculum was removed and, 100 of MEM
supplemented with 2% FCS and containing the inhibitor compound of interest
at the desired concentration (serial twofold dilutions from 50 to 0.1 M) was
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l
added. Cells were incubated for a period of 48 h at 37 °C with 5% CO2. Each
condition was set up in triplicate and antiviral assays were performed
independently on at least two separate occasions. Cell viability was
measured 48 h post infection using the CellTiter-Glo Luminescent Cell
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(RLU).
19. Authentic SARS-CoV 3CLpro was diluted to 1
mM HEPES pH 7.5. Five micrometers of compound 10 was added using a 1:400
dilution into the enzyme solution. The final volume was 200 L and contained
lM concentration using cold 20
l
a final DMSO concentration of 0.25%. Control reactions were performed in the
same buffer in the presence of 0.25% DMSO in the absence of inhibitor.
Incubation was performed at room temperature for 20 min. Sinapinic acid was
prepared as a saturating solution in 50% acetonitrile and 1% TFA. The sandwich
method of preparation was used for MALDI. One microliters of the matrix was
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spotted onto
a
plate followed by
1 lL of the enzyme inhibitor complex
followed by a top layering of 1
l
L of matrix. A Voyager DE Pro Time of Flight
(TOF) mass spectrometer was used for determining the molecular weights.
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