Structure-based design, synthesis, and biological evaluation of a series of novel and reversible inhibitors for the severe acute respiratory syndrome - Coronavirus papain-like protease

Article abstract of DOI:10.1021/jm900611t

We describe here the design, synthesis, molecular modeling, and biological evaluation of a series of small molecule, nonpeptide inhibitors of SARS-CoV PLpro. Our initial lead compound was identified via high-throughput screening of a diverse chemical library. We subsequently carried out structure - activity relationship studies and optimized the lead structure to potent inhibitors that have shown antiviral activity against SARS-CoV infected Vero E6 cells. Upon the basis of the X-ray crystal structure of inhibitor 24-bound to SARS-CoV PLpro, a drug design template was created. Our structure-based modification led to the design of a more potent inhibitor, 2 (enzyme IC₅₀ = 0.46 μM; antiviral EC₅₀ = 6 μM). Interestingly, its methylamine derivative, 49, displayed good enzyme inhibitory potency (IC₅₀ = 1.3 μM) and the most potent SARS antiviral activity (EC₅₀ = 5.2 μM) in the series. We have carried out computational docking studies and generated a predictive 3D-QSAR model for SARS-CoV PLpro inhibitors.

Full text of DOI:10.1021/jm900611t

5228 J. Med. Chem. 2009, 52, 5228–5240
DOI: 10.1021/jm900611t
Structure-Based Design, Synthesis, and Biological Evaluation of a Series of Novel and Reversible
Inhibitors for the Severe Acute Respiratory Syndrome-Coronavirus Papain-Like Protease
Arun K. Ghosh,*,† Jun Takayama,† Yoann Aubin,† Kiira Ratia,‡ Rima Chaudhuri,‡ Yahira Baez,‡ Katrina Sleeman,§
Melissa Coughlin, Daniel B. Nichols,§ Debbie C. Mulhearn,‡ Bellur S. Prabhakar, Susan C. Baker,§ Michael E. Johnson,‡ and
Andrew D. Mesecar‡
†Departments of Chemistry and Medicinal Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, ‡Center for
Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 South Ashland
Avenue, Chicago, Illinois 60607, §Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine,
Maywood, Illinois 60153, and Department of Microbiology and Immunology, University of Illinois, Chicago, Illinois 60607
Received May 8, 2009
We describe here the design, synthesis, molecular modeling, and biological evaluation of a series of small
molecule, nonpeptide inhibitors of SARS-CoV PLpro. Our initial lead compound was identified via
high-throughput screening of a diverse chemical library. We subsequently carried out structure-activity
relationship studies and optimized the lead structure to potent inhibitors that have shown antiviral
activity against SARS-CoV infected Vero E6 cells. Upon the basis of the X-ray crystal structure of
inhibitor 24-bound to SARS-CoV PLpro, a drug design template was created. Our structure-based
modification led to the design of a more potent inhibitor, 2 (enzyme IC50 = 0.46 μM; antiviral
EC50=6 μM). Interestingly, its methylamine derivative, 49, displayed good enzyme inhibitory potency
(IC50=1.3 μM) and the most potent SARS antiviral activity (EC50=5.2 μM) in the series. We have
carried out computational docking studies and generated a predictive 3D-QSAR model for SARS-CoV
PLpro inhibitors.
Introduction
targets for halting viral replication. One of the early and
essential processes is the cleavage of a multidomain, viral
polyprotein into 16 individual components termed nonstruc-
tural proteins, or nsps. These proteins assemble into com-
plexes to execute viral RNA synthesis.7 Two cysteine
proteases, a papain-like protease (PLpro) and a 3C-like
protease (3CLpro), reside within the polyprotein. They cata-
lyze their own release and that of the other nsps from the
polyprotein and initiate virus-mediated RNA replication.
Since 2003, numerous biochemical, structural, and inhibitor
development studies have been directed at the 3CLpro en-
zyme,8 which cleaves 11 sites within the polyprotein. Recently,
we reported potent inhibitors of 3CLpro that have shown
antiviral activity against SARS-CoV.9
Recent structural and functional studies directed at PLpro
have suggested potential roles for this protease beyond viral
peptide cleavage, including deubiquitination, deISGylation,
and involvement in virus evasion of the innate immune
response.10,11 Furthermore, studies have shown that the
homologous enzyme, PLP2, from the human coronavirus
229E, is essential for 229E viral replication.12 Therefore,
PLpro has emerged as a significant drug development target.
Our screening of a structurally diverse library of 50080
compounds led to the discovery of a noncovalent lead in-
hibitor 1 (7724772, Figure 1), with an IC50 value of 20 μM as a
racemic mixture.13 Subsequent SAR studies and lead optimi-
zation provided potent inhibitor 24 (IC50=600 nM), which
also inhibits SARS-CoV viral replication in Vero cells with an
EC50 value of 15 μM.13 In these studies, we also reported the
X-ray crystal structure of SARS-CoV PLpro bound to
Severe acute respiratory syndrome (SARS), a contagious
and fatal respiratory illness, was first reported in Guangdong
province, China, in November 2002.1 It rapidly spread to
other Asian countries, North America, and Europe, creating
panic to both the public and the World Health Organization
(WHOa). The emergence of SARS affected more than 8000
individuals and caused 774 deaths within a few months.6 Quite
remarkably, the spread of SARS-CoV was effectively halted
withinmonthsafter the initialoutbreaksthroughpublic health
measures. Through a concerted effort monitored by the
WHO, scientists determined that SARS is caused by a novel
coronavirus, SARS-CoV.2,3,3b The more recent isolation of
strains from zoonotic origins thought to be the reservoir for
SARS-CoV emphasizes the possibility of a reemergence.4,5 It
is quite alarming just how rapidly a contagious illness can
spread in the more mobile and highly interconnected world of
the 21st century. While there are no new reports of SARS
cases, there is no guarantee that this outbreak will not strike
again. Therefore, development of antivirals effective against
SARS-CoV is important for future outbreaks.
The identification of biochemical events critical to the
coronaviral lifecycle has provided a number of significant
*To whom correspondence should be addressed. Phone: (765)-494-
5323. Fax: (765)-496-1612. E-mail: akghosh@purdue.edu.
a Abbreviations: SARS, severe acute respiratory syndrome; SARS-
CoV, SARS-coronavirus 3CLpro, chymotrypsin-like protease; PLpro,
papain-like protease; WHO, World Health Organization; QSAR, quan-
titative structural-activity relationship; CoMSIA, comparative molec-
ular similarity indices analysis.
r
pubs.acs.org/jmc
Published on Web 07/31/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5229
Scheme 2a
Figure 1. Structure of inhibitors 1, 2, 24, and 49.
Scheme 1a
a Reagents and conditions: (a) Boc2O, Et3N, dioxane/H2O (2:1),
23 °C, 48 h; (b) (R)-(þ)-1-(2-naphthyl)ethylamine, EDCI, HOBT, DIPEA,
CH2Cl2, 23 °C, 16 h; (c) TFA, CH2Cl2, 23 °C, 2 h.
Scheme 3a
a Reagents and conditions: (a) EDCI, HOBT, DIPEA, 23 °C, 16 h.
inhibitor 24, which revealed important molecular insight into
the ligand-binding site interactions. We now describe the full
details of our significantly extended studies that include the
design, synthesis, molecular modeling, and biological evalua-
tion of a series of inhibitors of SARS-CoV PLpro.
Chemistry
As shown in Scheme 1, coupling of (R)-(þ)-1-(2-
naphthyl)ethylamine or (R)-(þ)-1-(1-naphthyl)ethylamine
with benzoic acid derivatives utilizing N-(3-dimethylaminopro-
pyl)-N0-ethylcarbodiimidehydrochloride (EDCI), 1-hydroxy-
benzotriazole hydrate (HOBT) in the presence of diisopro-
pylethylamine in CH2Cl2 at 23 °C gave the corresponding
inhibitor in excellent yield. Inhibitors 8 and 9 were synthesized
by general methods withN-Boc protected benzoic acid deriva-
tive followed by deprotection of the Boc group as shown in
Scheme 2. Protection of p-aminobenzoic acid 6 with di-t-butyl
dicarbonate in the presence of triethylamine in a mixture (2:1)
of dioxane and water at 23 °C for 16 h gave the corresponding
t-Bu carbamate 7.14 A coupling reaction utilizing EDCI and
HOBt provided inhibitor 8. Removal of the Boc group with
trifluoroacetic acid in CH2Cl2 at 23 °C for 2 h afforded
inhibitor 9.
Inhibitors 14 and 17 were racemic compounds (Scheme 3).
A Friedel-Crafts reaction of naphthalene 10 with propionyl
chloride 11 in the presence of aluminum chloride in 1,2-
dichloroethane at 35 °C for 4 h gave ethyl naphthyl ketone
12.15 Reductive amination16 with ammonium acetate and
sodium cyanoborohydride in methanol at 23 °C for 24 h
generated amine 13. Coupling of this amine with o-toluic acid
provided inhibitor 14. Reductive amination of naphthyl
phenyl ketone 15 by using similar conditions as the amine
a Reagent and conditions: (a) AlCl3, 1,2-dichloroethane, 35 °C, 4 h;
(b) NH4OAc, NaBH3CN, MeOH, 23 °C, 24 h; (c) o-toluic acid, EDCI,
HOBT, DIPEA, DMF, 23 °C, 16 h.
13 gave amine 16. Coupling of 16 with o-toluic acid furnished
inhibitor 17.
Inhibitor 21 with N-methyl amide was prepared as shown
in Scheme 4. Protection of (R)-(þ)-1-(1-naphthyl)-
ethylamine 18 with methyl chloroformate in the presence
of potassium carbonate in a mixture (1:1) of dioxane and
water at 0 °C for 1 h afforded carbamate 19. Reduction of 19
with lithium aluminum hydride in THF at reflux for 1 h
gave methylamine 20. A coupling reaction utilizing general
methods provided inhibitor 21. Inhibitor 23 was prepared
by coupling of optically active amine 18 with acid 7 as
shown.

5230 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ghosh et al.
Scheme 4a
Scheme 6a
a Reagents and conditions: (a) ClCO2Me, K2CO3, dioxane/H2O (1:1),
0 °C, 1 h; (b) LiAlH4, THF, reflux, 1 h; (c) o-toluic acid, EDCI, HOBT,
DIPEA, DMF, 23 °C, 16 h; (d) 7, EDCI, HOBT, DIPEA, DMF, 23 °C,
16 h; (e) TFA, CH2Cl2, 23 °C, 2 h.
Scheme 5a
a Reagents and conditions: (a) KI, NaIO4, conc H2SO4, 25-30 °C,
2 h; (b) (R)-(þ)-1-(1-naphthyl)ethylamine 18, EDCI, HOBT, DIPEA,
DMF/CH2Cl2 (1:1), 23 °C, 48 h; (c) CuCN, KCN, DMF, 130 °C, 16 h;
(d) SOCl2, MeOH, reflux, 4 h; (e) NBS, Bz2O2, CCl4, reflux, 24 h;
(f) NaH, NaOMe, MeOH, 50 °C, 4 h; (g) LiOH H2O, THF/H2O (5:1),
3
23 °C, 1.5 h; (h) (R)-(þ)-1-(1-naphthyl)ethylamine 18, EDCI, HOBT,
DIPEA, DMF/CH2Cl2 (1:1), 23 °C, 16 h; (i) H2, Pd-C, EtOAc, 23 °C,
10 h.
generated inhibitor 25. The synthesis of inhibitor 29 with a
gem-dimethyl group in the R-naphthyl side chain was carried
out by dimethylation of 1-cyanonaphthalene 26 with methyl
lithium in the presence of cerium(III) chloride in tetrahydro-
furan at 23 °C for 2 h to provide amine 27.17 A coupling
reaction using the general method described above provided
28, and hydrogenation in the presence of 5% Pd-C in a
mixture (1:1) ethyl acetate and methanol at 23 °C for 15 h
provided inhibitor 29.
The synthesis of 32 with 2-methyl-5-iodide and 33 with 2-
methyl-5-cyanide substituents on the benzamide moiety is
outlined in Scheme 6. Iodination of o-toluic acid 30 with
sodium periodate and potassium iodide in concentrated
sulfuric acid at 25-30 °C for 2 h afforded iodide 31.18 The
coupling reaction following the general method provided
inhibitor 32, and cyanation with copper cyanide and sod-
ium cyanide in DMF at 130 °C for 16 h furnished inhibitor
33. Inhibitor 40 was synthesized by esterification of 34 in
the presence of thionyl chloride in methanol at reflux for 4 h
to provide ester 35. Bromination of 35 with N-bromosucci-
nimide and benzoyl peroxide in carbon tetrachloride at
reflux for 24 h generated bromide 36.19 Reaction of 36 with
sodium hydride and sodium methoxide in methanol at 50 °C
for 4 h provided 37.20 Hydrolysis of 37 with lithium hydro-
xide monohydrate in a mixture (5:1) of tetrahydrofuran and
water at 23 °C for 16 h afforded acid 38. Coupling reaction
a Reagents and conditions: (a) H2, Pd-C, EtOAc/MeOH (1:1), 23 °C,
15 h; (b) Ac2O, Et3N, CH2Cl2, 23 °C, 18 h; (c) MeLi, CeCl3, THF, 23 °C,
2 h; (d) 2-methyl-5-nitrobenzoic acid, EDCI, HOBT, DIPEA, CH2Cl2,
23 °C, 16 h; (e) H2, Pd-C, EtOAc/MeOH (1:1), 23 °C, 15 h.
Inhibitors 24 and 25 containing 2-methyl-5-amino deriva-
tives were synthesized as outlined in Scheme 5. Reduction of
thenitro groupin5ibycatalytichydrogenationinthe presence
of 5% Pd-C in a mixture (1:1) of ethyl acetate and methanol
at 23 °C for 15 h provided inhibitor 24. Acetylation of 24 with
acetic anhydride and triethylamine in CH2Cl2 at 23 °C for 18 h

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5231
Scheme 7a
Table 2. Structure and Activity of Naphthyl and Benzamide Deriva-
tives
compd
R
R0
2-Me
IC50 (μM)a
lead
5f
Me
Me
Me
Me
Me
8.7 ( 0.7
12.1 ( 0.7
NI
2,6-diMe
2-OH
5g
9
4-NH2
4-Boc-NH
2-Me
46.1 ( 13.0
NI
NI
8
14
17
Et (racemic)
Ph (racemic)
2-Me
NI
a IC50=enzyme inhibitory activity. NI=no inhibition.
Table 3. Structure and Activity of 1-, 2-Naphthalene and Benzamide
Derivatives
a Reagents and conditions: (a) H2, Pd-C, EtOAc, 23 °C, 16 h;
(b) NaNO2, conc HCl, CuCN, NaCN, H2O, 23 °C, 3 h; (c) Boc2O,
compd
R
R0
R”
IC50 (μM)a
NiCl2 6H2O, NaBH4, MeOH, 23 °C, 2 h; (d) MeI, KHMDS, THF,
3
lead
5h
2-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
H
2-Me
2-Me
2-Me
4-NH2
8.7 ( 0.7
2.3 ( 0.1
22.6 ( 6.9
24.8 ( 1.0
0.56 ( 0.03
2.64 ( 0.04
23 °C, 16 h; (e) LiOH H2O, THF/H2O (9:1), 23 °C, 16 h; (f) (R)-(þ)-1-(1-
3
H
naphthyl)ethylamine 18, EDCI, HOBT, DIPEA, CH2Cl2, 23 °C, 16 h;
(g) TFA, CH2Cl2, 23 °C, 2 h.
21
23
Me
H
24
25
H
2-Me and 5-AcNH
2-Me and 5-NH2
Table 1. Structure and Activity of Substituted Benzamide Derivatives
H
a IC50=enzyme inhibitory activity.
furan and water at 23 °C for 16 h generated acid 45.
Coupling reaction of 45 with amine 18 using conditions
described above provided inhibitor 47. Removal of Boc
group with trifluoroacetic acid in dichloromethane at 23 °C
for 2 h gave inhibitor 2. Inhibitor 49 was prepared from
benzylamine 44. Ester hydrolysis, coupling of the resulting
acid with amine 18, and subsequent removal of Boc-group
as described for 2 afforded inhibitor 49.
compd
R
IC50 (μM)a
lead
5a
2-Me
3-Me
4-Me
8.7 ( 0.7
14.8 ( 5.0
29.1 ( 3.8
90 ( 26
13.5 ( 6.8
149 ( 43
5b
5c
2-OMe
3-OMe
4-OMe
5d
5e
Results and Discussion
As described previously, screening of a library of 50080
diverse compounds identified 1 as an inhibitor of PLpro
activity.13 Because 1 is a racemic mix, we synthesized the
corresponding chiral compounds to evaluate the stereospeci-
fic recognition of these compounds by PLpro. Compound 1
with the R-configuration, was found to be twice as potent as
the racemic compound 1 (R-configuration compound, IC50=
8.7 μM; racemic compound 1, IC50=20.1 μM). We therefore
selected R-configuration compound for further optimization
as the lead compound.
As shown in Table 1, we modified the substituent on the
benzamide ring with methyl and methoxy groups. Com-
pounds 5a-5e turned out to be less potent than the lead
compound. Thus, a methyl group at the ortho position in lead
compound displayed the most potent activity (IC50=8.7 μM).
A methoxy group at the ortho position resulted in a 10-fold
reduction in potency compared to the lead compound. A
a IC50 = enzyme inhibitory activity.
with the general method gave 39 and hydrogenation in the
presence of 5% Pd-C in ethyl acetate at 23 °C for 10 h
provided inhibitor 40.
Inhibitors 47, 49, and 2 have been synthesized as shown in
Scheme 7. Reduction of nitro group in 35 by catalytic
hydrogenation in the presence of 5% Pd-C in ethyl acetate
at 23 °C for 15 h provided amine 41. Reaction of 41 with
sodium nitrite, copper cyanide, and sodium cyanide in
acidic condition at 23 °C for 3 h afforded corresponding
benzonitrile 42.21 Reduction and subsequent N-Boc protec-
tion with di-t-butyl dicarbonate and sodium borohydride in
the presence of nickel(II) chloride hexahydrate in metha-
nol at 23 °C for 2 h provided N-Boc-benzylamine 43.22
N-Methylation with methyl iodide and potassium bis-
(trimethylsilyl)amide in tetrahydrofuran at 23 °C for 16 h
afforded methylamine 44. Hydrolysis of 43 with lithium
hydroxide monohydrate in a mixture (9:1) of tetrahydro-
methoxy group in the meta position (compound 5f, IC50 =
13.5 μM) is the most potent analogue among the methoxy
substituted derivatives.

5232 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ghosh et al.
Figure 2. Docking in the presence of conserved water molecules is critical for replicating the binding conformation of inhibitors in the active
site of the bound form of SARS-CoV PLpro. (A) Docking of compound 2 in the absence of water molecules in the active site of the inhibitor-
bound form of SARS-CoV PLpro causes the naphthyl rings to flip down into a pocket, as shown by the docked conformation of compound 2
(in green) when compared to the crystal structure conformation of compound 24 (in cyan). In the X-ray structure of compound 24, the naphthyl
rings are flipped up in the opposite direction, holding the flexible loop in place. The yellow dotted lines show the possible interactions of the
docked compound 2 with residues Tyr269, Gln270, and Asp165 (catalytic domain residue numbering). (B) The three conserved water molecules
are marked; two of them are buried deep in the pocket (P5), whereas the third one lies in a groove between residues Lys158 and Glu168. The
position of these water molecules is integral to structure-based inhibitor design efforts. The crystal structure conformation of compound 24 is
shown in cyan, whereas the docked conformation of compound 2 in the presence of water molecules (red dots) is shown in green.
Table 4. Structure and Activity of Substituted Benzamide Derivatives
We next attempted further modification of the substituent
and an R-Disubstituted Naphthyl Derivative
on the benzamide as well as on the naphthyl rings, and the
results are summarized in Table 2. As shown, a 2,6-dimethyl
derivative, 5f, led to an increase in the IC50 value compared to
lead compound (compound 5f, IC50 = 12.1 μM). Bulky
substituents at the ortho and para positions of the benzamide
ring and R-naphthyl position also resulted in increased IC50
values.
compd
R
R0
IC50 (μM)a
In an attempt to further improve the potency of PLpro
inhibitors, we then examined 1-naphthalene derivatives and
also explored polar functionalities on the benzamide ring
baseduponmodelingstudies.TheresultsareshowninTable3.
Compound 5h, with a R-1-naphthylethylamide derivative,
shows improvement in potency compared to the lead compound
(compound 5h, IC50=2.3 μM). The importance of an amide
NH is demonstrated as the N-methyl derivative has signifi-
cantly attenuated potency (compound 21, IC50 = 22.6 μM;
compound 5h, IC50=2.3 μM). 5-Amino group resulted in an
even more potent inhibitor 24 with an IC50 value of 0.56 μM.
However, incorporation of a polar acetamide deriva-
24
29
33
40
32
47
49
2
H
2-Me and 5-NH2
2-Me and 5-NH2
0.56 ( 0.03
11.1 ( 1.3
5.2 ( 0.5
2.7 ( 0.1
1.4 ( 0.3
4.8 ( 0.4
1.3 ( 0.1
0.46 ( 0.03
Me
H
2-Me and 5-CN
2-CH2OMe and 5-NH2
2-Me and 5-I
H
H
H
2-Me and 5-CH2NHBoc
2-Me and 5-CH2NHMe
2-Me and 5-CH2NH2
H
H
a IC50=enzyme inhibitory activity.
dimethyl group in place of the (R)-methyl substituent, how-
ever, the corresponding inhibitor showed a reduction in
potency for SARS PLpro (compound 29, IC50 =11.1 μM).
Upon the basis of the X-ray structure, we then incorporated 5-
methylamine substituents on the benzamide ring. The result-
ing compound 2 showed a slight improvement in inhibitory
potency (compound 2, IC50=0.46 μM) and antiviral activity
(EC50 = 6 μM) as shown in Table 5. The addition of a
methyl group to the amine group of 2 provided compound
49, which showed slightly decreased enzyme activity (IC50=
1.3 μM) but significantly improved antiviral potency (EC50=
5.2 μM). These results illustrate the importance of testing
compounds not only for their ability to inhibit the purified
protein but also to assess their effects on cell viability and
inhibition of viral replication in a cell-based assay.
tive afforded a less potent derivative (compound 25, IC50
2.6 μM).
=
To obtain molecular insights into the ligand-binding site
interactions responsible for the inhibitory potency of com-
pound 24, the X-ray structure of PLpro complexed with 24
was determined previously to 2.5 A resolution.13,23 Compar-
˚
ison of the apoenzyme SARS-CoV PLpro structure to the
inhibitor 24-complexed structure reveals significant confor-
mational differences between these structures.23 Upon the
basis of this molecular insight, we further modified the
substituents on the benzamide ring. Particularly interesting
is the fact that the amine group in 24 is positioned at the
opening of the cleftwhere it appears to come within hydrogen-
bonding distance to the hydroxyl of Tyr269. Furthermore,
there are a number of water molecules present (see Figure 2)
that occupy the pocket accommodating the (R)-methyl sub-
stituent of 24. This suggested the potential for extending the
methyl branch further into the pocket through the addition of
polar substituents. As shown in Table 4, we incorporated a
To obtain molecular insights into the active site interactions
leading to improved inhibitory potency, we created an energy-
minimized model of 2 in the 24-inhibited SARS-CoV PLpro
active site (Figure 2B). The model reveals that the 5-methyl-
amine substituent of inhibitor 2 may be involved in hydrogen

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5233
Table 5. Evaluation of Compounds as Inhibitors of SARS-CoV Re-
plication in a Cell-Based Assaya
compd
IC50(μM)
EC50(μM)
1
20.1 ( 1.1
0.46 ( 0.03
8.7 ( 0.7
14.8 ( 5.0
0.56 ( 0.03
2.64 ( 0.04
11.1 ( 1.3
4.8 ( 0.4
NI
2
6.0 ( 0.1
NI
NI
lead
5a
24
25
29
47
49
14.5 ( 0.8
13.1 ( 0.7
NI
NI
5.2 ( 0.3
1.3 ( 0.1
a IC50=enzyme inhibitory activity; EC50=antiviral activity; NI=no
inhibition.
bonding with the side chain of residues Gln270 and Tyr269.
Similar tothe crystal structureconformation of compound24,
compound 2 appearstobeanchoredinthesitebytwo effective
hydrogen bonds made between the carboxamide group and
residues Asp165 and Gln 270. The three conserved water
molecules found in both the complex crystal structure of
compound 24-bound protein and the crystal structure of the
apo enzyme are retained for modeling studies and are shown
as red dots in the P5 binding pocket (see Figure 2B). In
docking studies, these water molecules are necessary for
obtaining the crystal-bound orientation of these compounds,
which positions the naphthyl ring of the inhibitor to be flipped
upward holding the loop (shown in cyan, Figure 2A). In the
absence of these three waters, the naphthyl ring tends to
occupy the P5 pocket, shown in green, Figure 2A.
To better quantitate and understand the contributions of
the inhibitor substituents to the inhibitory potency, we also
conducted an extensive quantitative structural activity rela-
tionship (QSAR) analysis. QSAR studies are often employed
as a standard method to determine valuable information for
designing novel and potent inhibitors. An alternative ap-
proach to labor-intensive chemical synthesis is to develop a
theory that quantitatively relates variations in biological
activity to changes in molecular descriptors for each com-
pound. The goal of our computational study was to develop a
robust QSAR model that can predict and differentiate inhibi-
tion values of this series of inhibitors against SARS-CoV
PLpro. Partial least-squares (PLS) methodology was used for
the 3D-QSAR analyses. The CoMSIA descriptors were used
as independent variables, and percent inhibition values at
100 μM were used as dependent variables in PLS regression
analyses to derive the models. The predictive value of the
models were evaluated by leave-one-out (LOO) cross-valida-
tion. To assist ligand-based structure design efforts, we used
docking in conjunction with 3D-QSAR (CoMSIA) to inves-
tigate the SAR of 46 inhibitors with considerable structural
diversity and a wide range of bioactivity against the SARS-
CoV PLpro.
Figure 3. Correlation plot between the predicted percent inhibition
of 41 compounds and the actual experimental percent inhibition
values obtained at 100 μM.
molecules made a substantial difference in replicating the
crystal structure binding geometry of compound 24 in dock-
ing studies. When docking was performed in the absence of
water molecules in the P5 pocket, the study suggested that the
naphthyl rings prefer to flip downward and probably find
lower energy conformations in this pocket as shown in
Figure 2A. In the inhibitor 24-bound crystal structure, two
of the conserved water molecules hinder placement of the
naphthyl rings in the P5 pocket and thereby flip the rings
upward. This position of the naphthyl rings fits nicely in the
hydrophobicpocket andforms favorableinteractions withthe
flexible loop residues. The five water molecules mentioned
above are clearly shown in Figure 2B and were retained for the
docking procedure prior to CoMSIA24 model generation. In
Figure 2B, the superimposition of the naphthyl rings of
modeled compound 2 (green) and X-ray structure bound
orientation of compound 24 (cyan) is clearly shown as obtai-
ned in the presence of the conserved water molecules.
Using the binding conformation of inhibitor 24 from the
crystal structure as a template, 45 other inhibitors were
docked. The docked geometry of these 45 inhibitors aligned
very well with the binding geometry of compound 24 in the
crystal structure. A predictive 3D-QSAR model was derived
from the alignment of the docked conformations of the
inhibitors as extracted directly from the 3D coordinates of
the docked complexes. A successful CoMSIA model was built
with a cross-validated q2 of 0.678 and bootstrap R2 of 0.984. It
is imperative to mention here that without the inclusion of at
least the three conserved water molecules, the docking geome-
try of the inhibitorsdoes notalign with the crystalstructure(as
shown in Figure 2A). In the absence of these water molecules,
the wrong ligand conformation is generated where the
naphthyl ring is flipped into the P5 pocket, consequently
resulting in an SAR with no correlation.
The crystal structures of the apo protein (PDB id: 2fe8)11
and the inhibitor 24-bound form (PDB id: 3e9s)13 were care-
fully studied, and it was determinedthat three water molecules
are conserved in the active sites of both the structures; two are
buried deep in the P5 pocket and one between residues Lys158
and Glu168. Apart from these three conserved water mole-
cules, two additional water molecules are located at the ridge
by the P5 pocket in the inhibitor-bound form of the protein.
As previously mentioned, by conducting extensive analyses
both with and without water molecules bound to the struc-
tures, we determined that retaining the three conserved water
The CoMSIA model at six components gave a cross-
validated q2 of 0.678, r2 of 0.984, F of 302.2, and a mean
standard error estimate of 4.72. The high r2 of 0.984 illustrates
that the physicochemical descriptors chosen were appropriate
to describe the binding interactions mode of the various
inhibitors with the PLpro active site. The plot shown in
Figure 3 shows the correlation between the experimental

5234 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ghosh et al.
Figure 4. Compound 24 is shown in cyan in its bound conformation as in the crystal structure aligned with the docked conformation of the
most active compound 2, in white, when docked in the presence of three conserved water molecules. (A) The electrostatic contour map for this
series of SARS-CoV PLpro inhibitors. Blue is the region of unfavorable negative charge, and red is of favorable negative charge. (B) The
hydrophobic contour map, where white is the region of unfavorable lipophilic interactions and magenta is of favorable lipophilicity. (C) A
steric contour map, with green denoting the region of favorable steric interactions and yellow denoting unfavorable regions.
percent inhibition values of 41 compounds and the inhibition
percentages predicted by the CoMSIA model. The contour
maps generated, shown in Figures 4A-C, were mapped back
to the topology of the active site to reveal that electrostatic,
hydrophobic, and steric fields were in accordance with the
distribution of the binding site residues. These contour maps
should prove to be helpful in determining the incorporation of
functional groups for the inhibitors that can extensively
interact with specific binding pocket residues. We conclude
that this robust 3D-QSAR model built using CoMSIA can be
used as a reliable guide for future structure-based drug design
efforts.
activity of 460 nM and antiviral EC50 value of 6 μM against
SARS. Interestingly, the corresponding methylamine deriva-
tive 49 (enzyme IC50=1.3 μM) has shown most potent antivi-
ral activity against SARS (EC50=5.2 μM). To obtain mole-
cular insight into the binding properties of 2 and its derivative,
we have created an active model based upon the X-ray
structure of 24-bound SARS PLpro. It appears that the
methylamine functionality is within proximity to hydrogen
bond with the side chain residues of Gln270 and Tyr269.
We have also carried out computational docking studies
and generated a predictive 3D-QSAR model for SARS-CoV
PLpro. Further design of reversible SARS-CoV PLpro inhi-
bitors is currently underway in our laboratory.
Conclusion
Experimental Section
In summary, a series of novel inhibitors of SARS-CoV
PLpro has been designed, synthesized, and evaluated in
enzyme inhibitory and antiviral assays. Initial lead compound
1 was discovered via high-throughput screening. This racemic
compound has shown enzyme IC50 value of 20 μM. The
stereochemical preference for the R-isomer was established
through synthesis and evaluation of optically pure inhibitors,
and kinetic studies showed that the optically pure lead in-
hibitor is a reversible inhibitor of SARS-CoV-PLpro enzyme.
In the absence of any structural information, initially our
structure-activity relationship studies and systematic modi-
fication guided by molecular modeling studies provided
potent inhibitor 24. This inhibitor displayed enzyme inhibi-
tory activity of 560 nM and antiviral EC50 value of 14.5 μM in
SARS-CoV infected Vero E6 cells. A protein-ligand X-ray
structure of 24-bound SARS-CoV PLpro provided detailed
molecular interaction in the active site of PLpro enzyme.
Upon the basis of the X-ray structural information, our
structure-based design led to identification of potent inhibi-
tors 2 and 49. Inhibitor 2 has shown potent enzyme inhibitory
Chemistry. 1H NMR and 13C NMR spectra were recorded on
Varian Oxford 300 and Bruker Avance 400 spectrometers.
Optical rotations were recorded on Perkin-Elmer 341 polari-
meter. Anhydrous solvent was obtained as follows: dichloro-
methane by distillation from CaH2, THF by distillation from Na
and benzophenone. All other solvents were reagent grade.
Column chromatography was performed with Whatman 240-
400 mesh silica gel under low pressure of 3-5 psi. TLC was
carried out with E. Merck silica gel 60-F-254 plates. Purity of all
test compounds was determined by HRMS and HPLC analysis
on an Agilent 1100 unit in two different solvent systems. All test
compounds showed g95% purity.
General Procedure for Coupling Reaction of Naphthylethyla-
mine and Benzoic Acid Derivative. 2-Methyl-N-[(R)-1-(1-
naphthyl)ethyl]benzamide (5h). To a solution of o-toluic acid
(16.2 mg, 0.12 mmol), N-(3-dimethylaminopropyl)-N0-ethylcar-
bodiimide hydrochloride (EDCI) (29.1 mg, 0.15 mmol), and 1-
hydroxybenzotriazole hydrate (HOBT) (20.5 mg, 0.15 mmol) in
dry CH2Cl2 was added
naphthyl)ethylamine 18 (20 mg, 0.12 mmol) and diisopropyl-
ethylamine (81.4 μL, 0.47 mmol) in dry CH2Cl2 at 0 °C under
a
solution of (R)-(þ)-1-(1-

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5235
argon atmosphere and it was allowed to stir for 15 h at 23 °C.
The reaction mixture was quenched with water and extracted
with CH2Cl2. The organic layers were dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography to furnish compound 5l
(33 mg, 98%) as a white solid, Rf=0.34 (hexane:EtOAc=3:1),
[R]20D -50.0 (c=1, CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.24
(d, 1H, J=8.5 Hz), 7.89 (d, 1H, J=8.0 Hz), 7.82 (d, 1H, J=
8.0 Hz), 7.60-7.51 (m, 3H), 7.46 (dd, 1H, J=7.6 and 7.7 Hz),
7.27-7.24 (m, 2H), 7.17 (d, 1H, J=7.7 Hz), 7.11 (dd, 1H, J=7.6
and 8.0 Hz), 6.15-6.07 (m, 2H), 2.44 (s, 3H), 1.79 (d, 3H, J=
6.4 Hz). 13C NMR (100 MHz, CDCl3): δ 168.9, 137.9, 136.3,
136.0, 133.9, 131.1, 130.9, 129.7, 128.7, 128.4, 126.5, 126.5,
129.5, 125.6, 125.1, 123.5, 122.5, 44.8, 20.5, 19.7. MS (EI): m/z
289.20 [M]þ. HRMS (EI), calcd for C20H19NO 289.1467, found
[M]þ 289.1468.
125.8, 124.8, 124.5, 113.6, 55.3, 49.1, 21.6. MS (EI): m/z
305.15 [M]þ. HRMS (EI), calcd for C20H19NO2 305.1416, found
[M]þ 305.1419.
2,6-Dimethyl-N-[(R)-1-(2-naphthyl)ethyl]benzamide (5f). The
title compound was obtained as described in the general proce-
dure in 94% yield (white solid). Rf=0.26 (hexane:EtOAc=3:1),
1
[R]20 þ32.9 (c=1, CHCl3). H NMR (300 MHz, CDCl3): δ
D
7.82-7.77 (m, 4H), 7.49-7.43 (m, 3H), 7.13 (dd, 1H, J=7.2 and
8.1 Hz), 6.98 (d, 2H, J=7.5 Hz), 6.17 (d, 1H, J=8.1 Hz), 5.56-
5.46 (m, 1H), 2.27 (s, 3H), 1.64 (d, 3H, J=6.3 Hz). 13C NMR
(75 MHz, CDCl3): δ 169.3, 140.1, 137.5, 134.1, 133.2, 132.7,
128.6, 128.4, 127.8, 127.5, 127.4, 126.2, 125.9, 124.8, 124.6, 48.6,
21.4, 19.0. MS (EI): m/z 303.05 [M]þ. HRMS (EI), calcd for
C21H21NO 303.1623, found [M]þ 303.1624.
2-Hydroxy-N-[(R)-1-(2-naphthyl)ethyl]benzamide (5g). The
title compound was obtained as described in the general proce-
dure in 97% yield (white solid). Rf=0.49 (hexane:EtOAc=3:1),
3-Methyl-N-[(R)-1-(2-naphthyl)ethyl]benzamide (5a). The ti-
tle compound was obtained as described in the general proce-
dure in 92% yield (white solid). Rf=0.35 (hexane:EtOAc=3:1),
1
[R]20 þ68.3 (c=1, CHCl3). H NMR (300 MHz, CDCl3): δ
D
12.39 (s, 1H), 7.85-7.80 (m, 4H), 7.51-7.33 (m, 5H), 6.97 (d,
1H, J=8.1 Hz), 6.81-6.76 (m, 2H), 5.49-5.39 (m, 1H), 1.67 (d,
3H, J=7.2 Hz). 13C NMR (75 MHz, CDCl3): δ 169.2, 161.5,
139.8, 134.2, 133.2, 132.7, 128.6, 127.8, 127.6, 126.3, 126.0,
125.4, 124.5, 124.5, 118.6, 118.5, 114.1, 49.1, 21.5. MS (EI): m/
z 291.10 [M]þ. HRMS (EI), calcd for C19H17NO2 291.1259,
found [M]þ 291.1261.
1
[R]20 þ39.5 (c=1, CHCl3). H NMR (300 MHz, CDCl3): δ
D
7.83-7.79 (m, 4H), 7.60-7.44 (m, 5H), 7.27 (d, 2H, J=5.4 Hz),
6.51 (d, 1H, J=6.9 Hz), 5.53-5.44 (m, 1H), 2.35 (s, 3H), 1.67 (d,
3H, J=6.6 Hz). 13C NMR (75 MHz, CDCl3): δ 166.8, 140.5,
138.3, 134.5, 133.3, 132.7, 132.2, 128.5, 128.4, 127.9, 127.6,
127.6, 126.2, 125.8, 124.8, 124.6, 123.9, 49.1, 21.5, 21.3. MS
(EI): m/z 289.15 [M]þ. HRMS (EI), calcd for C20H19NO
289.1467, found [M]þ 289.1468.
2-Methyl-5-nitro-N-[(R)-1-(1-naphthyl)ethyl]benzamide (5i).
The title compound was obtained as described in the general
procedure in 95% yield (white solid). Rf=0.24 (hexane:EtOAc=
3:1), [R]20D -53.0 (c=1, CHCl3). 1H NMR (300 MHz, CDCl3):
δ 8.18 (d, 1H, J=8.1 Hz), 8.11-8.06 (m, 2H), 7.87 (d, 1H, J=8.0
Hz), 7.81 (d, 1H, J=8.0 Hz), 7.60-7.43 (m, 4H), 7.32 (d, 1H, J=
8.4 Hz), 6.13-6.10 (bm, 2H), 2.49 (s, 3H), 1.80 (d, 3H, J=6.3
Hz). 13C NMR (75 MHz, CDCl3): δ 166.5, 144.3, 137.8, 137.3,
133.8, 131.9, 131.1, 128.9, 128.8, 126.7, 126.1, 125.2, 124.4,
123.2, 122.7, 122.6, 121.6, 45.2, 20.5, 20.0. MS (EI): m/z
334.20 [M]þ. HRMS (EI), calcd for C20H18N2O3 334.1317,
found [M]þ 334.1323.
4-Methyl-N-[(R)-1-(2-naphthyl)ethyl]benzamide (5b). The ti-
tle compound was obtained as described in the general proce-
dure in >99% yield (white solid). Rf=0.32 (hexane:EtOAc=
3:1), [R]20D þ19.7 (c=1, CHCl3). 1H NMR (300 MHz, CDCl3):
δ 7.82-7.79 (m, 4H), 7.68 (d, 2H, J=8.1 Hz), 7.50-7.42 (m,
3H), 7.17 (d, 2H, J=7.5 Hz), 6.59 (d, 1H, J=6.9 Hz), 5.52-5.42
(m, 1H), 2.36 (s, 3H), 1.64 (d, 3H, J=6.9 Hz). 13C NMR (75
MHz, CDCl3): δ 166.5, 141.8, 140.6, 133.3, 132.7, 131.6, 129.1,
128.5, 127.9, 127.6, 126.9, 126.2, 125.8, 124.8, 124.6, 49.1, 21.6,
21.4. MS (EI): m/z 289.10 [M]þ. HRMS (EI), calcd for
C20H19NO 289.1467, found [M]þ 289.1469.
2-Methoxy-N-[(R)-1-(2-naphthyl)ethyl]benzamide (5c). The
title compound was obtained as described in the general proce-
dure in >99% yield (white solid). Rf=0.23 (hexane:EtOAc=
3:1), [R]20D -30.7 (c=1, CHCl3). 1H NMR (300 MHz, CDCl3):
δ 8.28 (d, 1H, J=7.8 Hz), 8.22 (dd, 1H, J=1.8 and 8.1 Hz), 7.52
(dd, 1H, J=1.8 and 8.7 Hz), 7.49-7.40 (m, 3H), 7.07 (t, 1H, J=
7.7 Hz), 6.95 (d, 1H, J=9.0 Hz), 5.57-5.47 (m, 1H), 3.92 (s, 3H),
1.67 (d, 3H, J=6.3 Hz). 13C NMR (75 MHz, CDCl3): δ 164.4,
157.5, 141.2, 133.4, 132.7, 132.6, 132.3, 128.4, 127.8, 127.6,
126.1, 125.7, 124.7, 124.4, 121.6, 121.3, 111.3, 55.9, 49.1, 22.3.
MS (EI): m/z 305.15 [M]þ. HRMS (EI), calcd for C20H19NO2
305.1416, found [M]þ 305.1414.
3-Methoxy-N-[(R)-1-(2-naphthyl)ethyl]benzamide (5d). The
title compound was obtained as described in the general proce-
dure in >99% yield (white solid). Rf=0.24 (hexane:EtOAc=
3:1), [R]20D þ50.0 (c=1, CHCl3). 1H NMR (300 MHz, CDCl3):
δ 7.82-7.79 (m, 4H), 7.50-7.44 (m, 3H), 7.38-7.38 (m, 1H),
7.31-7.27 (m, 2H), 7.03-6.97 (m, 1H), 6.57 (d, 1H, J=7.8 Hz),
5.51-5.42 (m, 1H), 3.79 (s, 3H), 1.65 (d, 3H, J=7.2 Hz). 13C
NMR (75 MHz, CDCl3): δ 166.4, 159.8, 140.5, 136.0, 133.3,
132.7, 129.5, 128.5, 127.9, 127.6, 126.2, 125.9, 124.7, 124.6,
118.6, 117.7, 112.4, 55.4, 49.3, 21.5. MS (EI): m/z 305.20 [M]þ.
HRMS (EI), calcd for C20H19NO2 305.1416, found [M]þ
305.1417.
4-Methoxy-N-[(R)-1-(2-naphthyl)ethyl]benzamide (5e). The
title compound was obtained as described in the general proce-
dure in >99% yield (white solid). Rf=0.20 (hexane:EtOAc=
3:1), [R]20D þ3.0 (c=1, CHCl3). 1H NMR (300 MHz, CDCl3): δ
7.81-7.73 (m, 6H), 7.49-7.41 (m, 3H), 6.85 (d, 2H, J=8.7 Hz),
6.58 (d, 1H, J=7.8 Hz), 5.50-5.40 (m, 1H), 3.79 (s, 3H), 1.63 (d,
3H, J=6.9 Hz). 13C NMR (75 MHz, CDCl3): δ 166.1, 162.1,
140.7, 133.3, 132.7, 128.7, 128.4, 127.8, 127.5, 126.7, 126.1,
4-N-tert-Butoxycarbonylaminobenzoic Acid (7). To a solution
of 4-aminobenzoic acid 6 (520 mg, 3.8 mmol) in dioxane/H2O
(2:1) (13 mL) was added triethylamine (0.79 mL, 5.7 mmol) and
Boc2O (1.31 mL, 5.7 mmol) at 23 °C and it was allowed to stir for
48 h at same temperature. The solvent was removed under
reduced pressure, and 3 M HCl (5 mL) was added dropwise to
the residue at 0 °C. A precipitate was obtained, collected,
washed with water, and dried to give corresponding acid 7
(836 mg, 93%) as slightly yellow solid, Rf=0.78 (CH2Cl2:Me-
OH=9:1). 1H NMR (400 MHz, CDCl3): δ 9.25 (brs, 1H), 7.91
(d, 2H, J=8.7 Hz), 7.50 (d, 2H, J=8.7 Hz), 1.51 (s, 9H). 13C
NMR (100 MHz, CDCl3): δ 169.7, 154.8, 131.8, 125.3, 118.6,
118.5, 81.3, 28.6. MS (EI): m/z 237.10 [M]þ. HRMS (EI), calcd
for C12H15NO4 237.1001, found [M]þ 237.1004.
4-N0-tert-Butoxycarbonylamino-N-[(R)-1-(2-naphthyl)ethyl]-
benzamide (8). The title compound was obtained as described in
the general procedure in 60% yield (white solid). Rf = 0.76
(CH2Cl2:MeOH =9:1), [R]20 -91.6 (c =1, CHCl3:MeOH =
D
1:1). 1H NMR (300 MHz, CDCl3): δ 7.77-7.72 (m, 6H), 7.48-
7.37 (m, 5H), 5.40-5.33 (m, 1H), 1.60 (d, 3H, J=6.9 Hz), 1.47 (s,
9H). MS (EI): m/z 390.05 [M]þ. HRMS (EI), calcd for
C24H26N2O3 390.1943, found [M]þ 390.1942.
4-Amino-N-[(R)-1-(2-naphthyl)ethyl]benzamide (9). To a solu-
tion of Boc 8 (60 mg, 0.15 mmol) in CH2Cl2 (4 mL) was added
dropwise trifluoroacetic acid (0.6 mL) at 23 °C and it was
allowed to stir for 2 h at same temperature. The reaction was
concentrated under reduced pressure, and the residue was
treated with saturated NaHCO3 solution. The mixture was
extracted with CH2Cl2. The organic layers were dried over
Na2SO4 and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography to give
compound 9 (44 mg, 99%) as a white solid, Rf=0.60 (CH2Cl2:
MeOH = 9:1), [R]20 -58.0 (c = 1, CHCl3:MeOH = 4:1). H
1
D

5236 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ghosh et al.
NMR (300 MHz, CDCl3): δ 7.82-7.80 (m, 4H), 7.60 (d, 2H, J=
8.1 Hz), 7.50-7.41 (m, 3H), 6.63 (d, 2H, J=8.7 Hz), 6.23 (d, 1H,
J=6.9 Hz), 5.52-5.42 (m, 1H), 1.66 (d, 3H, J=7.2 Hz). MS (EI):
m/z 290.15 [M]þ. HRMS (EI), calcd for C19H18N2O 290.1419,
found [M]þ 290.1424.
N-Methoxycarbonyl-(R)-(þ)-1-(2-naphthyl)ethylamine (19).
To a solution of (R)-(þ)-1-(2-naphthyl)ethylamine 18 (200 mg,
1.2 mmol) in a mixture (1:1) of dioxane and H2O was added
potassium carbonate (323 mg, 2.3 mmol) and methyl chloro-
formate (0.11 mL, 1.4 mmol) at 0 °C and it was allowed to stir for
1 h at 0 °C. The reaction was quenched with 10% HCl solution
and extracted with EtOAc. The organic layers were dried over
Na2SO4 and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography to furnish
1-(2-Naphthyl)propanone (12). To a solution of propionyl
chloride 11 (5.1 g, 55 mmol) and aluminum chloride (7.7 g, 58
mmol) in 1,2-dichloroethane (16 mL) was added dropwise a
solution of naphthalene 10 (7.9 g, 62 mmol) in 1,2-dichlor-
oethane (16 mL) over 3 h at 35 °C and it was allowed to stir for
1 h. The reaction was added 3 M HCl solution at 0 °C and then
separated a white solid. The filtrate was washed with water. The
organic layer was dried over Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography to furnish compound 12 (9.9 g, 98%) as a
colorless oil, Rf = 0.56 (hexane:EtOAc = 9:1). 1H NMR
(300 MHz, CDCl3): δ 8.58 (d, 1H, J=8.7 Hz), 7.94 (d, 1H, J=
8.1 Hz), 7.86-7.80 (m, 2H), 7.59-7.42 (m, 3H), 3.04 (q, 2H, J=
6.9 Hz), 1.27 (t, 3H, J=6.9 Hz). 13C NMR (75 MHz, CDCl3): δ
205.2, 136.0, 133.8, 132.2, 130.0, 128.3, 127.7, 127.1, 126.3,
125.7, 124.3, 35.2, 8.56. MS (EI): m/z 184.15 [M]þ. HRMS
(EI), calcd for C13H12O 184.0888, found [M]þ 184.0890.
compound 19 (268 mg, >99%) as a colorless oil, Rf = 0.36
1
(hexane:EtOAc=3:1), [R]20 þ96.8 (c=1, CHCl3). H NMR
D
(300 MHz, CDCl3): δ 7.82-7.74 (m, 4H), 7.50-7.40 (m, 3H),
5.14 (bm, 1H), 5.00 (bm, 1H), 3.66 (s, 3H), 1.54 (d, 3H, J =
6.9 Hz). 13C NMR (75 MHz, CDCl3): δ 156.2, 140.9, 133.1,
132.5, 128.1, 127.7, 127.4, 125.9, 125.5, 124.2, 124.1, 51.8, 50.5,
22.0. MS (EI): m/z 229 [M]þ. HRMS (EI), calcd for C14H15NO2
229.1103, found [M]þ 229.1103.
N-Methyl-(R)-(þ)-1-(2-naphthyl)ethylamine (20). To a sus-
pension of lithium aluminum hydride (93 mg, 2.4 mmol) in
THF (6 mL) was added dropwise a solution of carbamate 19
(268 mg, 1.2 mmol) in THF (1 mL) at 0 °C under argon
atmosphere and it was allowed to stir for 1 h at reflux tempera-
ture. The reaction was quenched with 1 M NaOH solution at
0 °C and the mixture was filtered through celite pad. The filtrate
was concentrated under reduced pressure and the residue was
purified by silica gel column chromatography to give amine 20
(186 mg, 86%) as a colorless oil, Rf =0.21 (CH2Cl2:MeOH=
9:1), [R]20D þ58.0 (c=1, CHCl3). 1H NMR (300 MHz, CDCl3):
δ 7.83-7.80 (m, 3H), 7.73 (s, 1H), 7.49-7.40 (m, 3H), 3.81 (q,
1H, J=6.6 Hz), 2.33 (s, 3H), 1.80 (bs, 1H), 1.43 (d, 3H, J=
6.6 Hz). 13C NMR (75 MHz, CDCl3): δ 142.4, 133.2, 132.6,
128.0, 127.5, 127.4, 125.7, 125.3, 125.1, 124.6, 60.1, 34.3, 23.7.
MS (EI): m/z 185.30 [M]þ. HRMS (EI), calcd for C13H15N
185.1204, found [M]þ 185.1205.
2-Methyl-N-[1-(2-naphthyl)propyl]benzamide (14). To a solu-
tion of ketone 12 (2.1 g, 11.4 mmol) in MeOH (50 mL) was added
ammonium acetate (8.8 g, 0.11 mol) and NaBH3CN
(528 mg, 8.0 mmol) at 23 °C and was stirred for 24 h. Conc. HCl
was added until pH <2, and the solvent was removed under reduced
pressure. The residue was taken up in water (15 mL) and extracted
once with Et2O. The aqueous layer was brought to pH >12 with
solid KOH and extracted with CH2Cl2. The organic layers were
dried over Na2SO4 and concentrated under reduced pressure to give
amine 13 as crude compound, MS (EI): m/z 185.20 [M]þ. HRMS
(EI), calcd for C13H15N 185.1204, found [M]þ 185.1206. Coupling
reaction was used general procedure with amine 13 (50 mg, mmol)
and o-toluic acid (37.5 mg, mmol) to give inhibitor 14 (21mg, 2 steps
26%) as a white solid, Rf=0.25 (hexane:EtOAc=3:1). 1H NMR
(300 MHz, CDCl3):δ7.84-7.78(m, 4H), 7.49-7.44(m, 3H), 7.35-
7.26(m, 2H), 7.20-7.14 (m, 2H), 6.12 (d, 1H, J=8.4 Hz), 5.28-5.20
(m, 1H), 2.39 (s, 3H), 2.04-1.95 (m, 2H), 0.99 (t, 3H, J=7.2 Hz).
13C NMR (75 MHz, CDCl3): δ 169.4, 139.4, 136.6, 136.0, 133.3,
132.7, 130.9, 129.8, 128.5, 127.8, 127.6, 126.5, 126.2, 125.8, 125.7,
125.3, 124.7, 55.2, 29.1, 19.7, 10.9. MS (EI): m/z 303.25 [M]þ.
HRMS (EI), calcd for C21H21NO 303.1623, found [M]þ 303.1624.
1-(2-Naphthyl)benzylamine (16). To a solution of naphthyl-
phenylketone 15 (600 mg, 2.6 mmol) in MeOH (15 mL) was
added ammonium acetate (2 g, 25.9 mmol) and NaBH3CN(120mg,
1.9 mmol) at 23 °C and it was allowed to stir for 24 h. Conc HCl
was added until pH <2, and the solvent was removed under
reduced pressure. The residue was taken up in water (4 mL) and
extracted once with Et2O. The aqueous layer was brought to
pH >12 with solid KOH and extracted with CH2Cl2. The
organic layers were dried over Na2SO4 and concentrated under
reduced pressure to give amine 16 (48 mg, 8%) as crude
2,N-Dimethyl-N-[(R)-1-(2-naphthyl)ethyl]benzamide (21).
The title compound was obtained as described in the general
procedure in 87% yield (white solid). Rf=0.26 (hexane:EtOAc=
3:1), [R]20D þ189.1 (c=1, CHCl3). 1H NMR (300 MHz, CDCl3):
δ 7.96-7.91 (m, 3.6H), 7.74-7.71 (m, 0.4H), 7.65-7.55 (m,
2.6H), 7.47-7.24 (m, 4.4H), 6.53 (q, 0.6H, J=7.2 Hz), 5.11-
5.08 (m, 0.4H), 3.04 (s, 0.7H), 2.97 (s, 0.4H), 2.54 (s, 2.6H), 2.43
(s, 2.3H), 1.82 (d, 2.1H, J=7.2 Hz), 1.79-1.72 (m, 0.9H). 13
C
NMR (75 MHz, CDCl3): δ 171.5, 137.7, 137.0, 133.6, 133.1,
132.7, 130.3, 128.7, 128.3, 127.9, 127.5, 126.4, 126.2, 126.1,
126.0, 125.6, 125.5, 125.0, 124.6, 56.6, 56.3, 50.0, 30.4, 27.5,
18.9, 18.1, 15.3. MS (EI): m/z 303.30 [M]þ. HRMS (EI), calcd
for C21H21NO 303.1623, found [M]þ 303.1627.
4-N0-tert-Butoxycarbonylamino-N-[(R)-1-(1-naphthyl)ethyl]-
benzamide (22). The title compound was obtained as described
in the general procedure in >99% yield (white solid). Rf =
0.73 (CH2Cl2:MeOH = 9:1), [R]20 -121.7 (c = 1, CHCl3:
D
MeOH=1:1). 1H NMR (300 MHz, CDCl3): δ 8.10 (d, 1H, J=
8.1 Hz), 7.80-7.73 (m, 2H), 7.59 (d, 2H, J=8.1 Hz), 7.55 (d,
1H, J=7.5 Hz), 7.49-7.34 (m, 2H), 7.42 (d, 1H, J=7.5 Hz),
7.31 (d, 2H, J=8.1 Hz), 7.04 (s, 1H), 6.52 (d, 2H, J=7.8 Hz),
1
compound, Rf =0.53 (CH2Cl2:MeOH=4:1). H NMR (300
MHz, CDCl3): δ 7.74-7.53 (m, 5H), 7.28-7.18 (m, 4H), 7.13-
7.01 (m, 3H), 5.14 (s, 1H), 1.89 (bs, 1H). 13C NMR (75 MHz,
CDCl3): δ 145.2, 142.8, 133.3, 132.5, 130.0, 128.5, 128.2, 127.9,
127.6, 127.0, 126.0, 125.7, 125.6, 124.9, 59.7. MS (EI): m/z
233.30 [M]þ. HRMS (EI), calcd for C17H15N 233.1204, found
[M]þ 233.1205.
2-Methyl-N-[1-(2-naphthyl)benzyl]benzamide (17). The title
compound was obtained as described in the general procedure
in 72% yield (white solid). Rf=0.39 (hexane:EtOAc=3:1). 1H
NMR (300 MHz, CDCl3): δ 7.88-7.80 (m, 5H), 7.55-7.34 (m,
9H), 7.29-7.24 (m, 2H), 6.66 (d, 1H, J=8.4 Hz), 6.57 (d, 1H, J=
8.4 Hz). 13C NMR (75 MHz, CDCl3): δ 169.1, 141.3, 138.7,
136.3, 136.0, 133.2, 132.7, 131.1, 130.0, 128.7, 128.7, 128.6,
128.0, 127.6, 127.5, 126.6, 126.3, 126.1, 126.0, 125.7, 125.5,
57.3, 19.8. MS (EI): m/z 351.40 [M]þ. HRMS (EI), calcd for
C25H21NO 351.1623, found [M]þ 351.1618.
6.10-6.01 (m, 1H), 1.71 (d, 3H, J=6.6 Hz), 1.47 (s, 9H). 13
C
NMR (75 MHz, CDCl3): δ 165.8, 152.4, 141.5, 138.3, 133.8,
131.1, 128.6, 128.3, 128.3, 127.9, 126.5, 125.7, 125.1, 123.4,
122.6, 117.6, 80.8, 45.1, 28.2, 20.7. MS (EI): m/z 390.25 [M]þ.
HRMS (EI), calcd for C24H26N2O3 390.1943, found [M]þ
390.1947.
4-Amino-N-[(R)-1-(1-naphthyl)ethyl]benzamide (23). The title
compound was obtained as described in the compound 9 in 95%
yield (slightly yellow solid). Rf =0.65 (CH2Cl2:MeOH=4:1),
[R]20D -137.8 (c=1, CHCl3:MeOH=4:1). 1H NMR (300 MHz,
CDCl3): δ 8.15 (d, 1H, J=7.5 Hz), 7.86-7.83 (m, 1H), 7.79 (d,
1H, J=8.1 Hz), 7.58-7.42 (m, 6H), 6.59 (d, 1H, J=8.1 Hz), 6.17
(d, 2H, J=7.5 Hz), 6.13-6.03 (m, 1H), 1.74 (d, 3H, J=6.6 Hz).
MS (EI): m/z 290.35 [M]þ. HRMS (EI), calcd for C19H18N2O
290.1419, found [M]þ 290.1422.

Article
5-Amino-2-methyl-N-[(R)-1-(1-naphthyl)ethyl]benzamide (24).
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5237
(hexane:EtOAc=1:1). 1H NMR (400 MHz, CDCl3): δ 8.56 (d,
1H, J=8.7 Hz), 7.88 (d, 1H, J=7.0 Hz), 7.75 (d, 1H, J=8.1 Hz),
7.65 (d, 1H, J=7.3 Hz), 7.49-7.42 (m, 3H), 6.90 (d, 1H, J=
8.0 Hz), 6.60 (s, 1H), 6.53 (d, 1H, J=8.0 Hz), 6.21 (s, 1H), 2.21 (s,
3H), 2.08 (s, 6H). 13C NMR (100 MHz, CDCl3): δ 169.1, 143.9,
141.2, 138.0, 135.0, 131.7, 130.2, 129.7, 128.7, 125.8, 125.2,
125.2, 125.0, 123.7, 116.3, 113.1, 57.5, 28.5, 18.6. MS (EI): m/z
318.45 [M]þ. HRMS (EI), calcd for C21H22N2O 318.1732, found
[M]þ 318.1729.
To a stirred solution of nitro 5m (37 mg, 0.11 mmol) in
EtOAc/MeOH (1:1) (3 mL) was added 5% Pd-C (4 mg) and
it was allowed to stir for 15 h at 23 °C under H2 atmosphere. The
reaction was filtered through a celite pad and the filtrate was
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography to furnish compound 24
(27 mg, 80%) as a white solid, Rf=0.29 (hexane:EtOAc=1:1),
[R]20D -76.8 (c=1, CHCl3). 1H NMR (300 MHz, CDCl3): δ 8.20
(d, 1H, J=8.4 Hz), 7.85 (d, 1H, J=8.0 Hz), 7.78 (d, 1H, J=
8.0 Hz), 7.57-7.40 (m, 4H), 6.89 (d, 1H, J=8.0 Hz), 6.70 (bd,
2H, J=13.5 Hz), 6.10-6.07 (bm, 2H), 3.25 (bs, 2H), 2.27 (s, 3H),
1.73 (d, 3H, J=6.0 Hz). 13C NMR (75 MHz, CDCl3): δ 169.0,
143.9, 138.0, 136.9, 133.8, 131.7, 131.1, 128.7, 128.3, 127.2,
126.5, 125.8, 125.1, 123.5, 122.5, 116.6, 113.3, 44.7, 20.5, 18.6.
MS (EI): m/z 304.30 [M]þ.
5-N-Acetylamino-2-methyl-N-[(R)-1-(1-naphthyl)ethyl]benza-
mide (25). To a stirred solution of amine 24 (14 mg, 0.05 mmol)
in CH2Cl2 (0.5 mL) was added dropwise triethylamine (9.6 μL,
0.07 mmol) and acetic anhydride (5.2 μL, 0.06 mmol) at 0 °C and
it was allowed to stir for 18 h at 23 °C. The reaction was
quenched with saturated NH4Cl solution and extracted with
CH2Cl2. The organic layers were dried over Na2SO4 and con-
centrated under reduced pressure. The residue was purified
by silica gel column chromatography to furnish compound 25
(5.4 mg, 34%) as a white solid, Rf=0.60 (CH2Cl2:MeOH=9:1).
1H NMR (300 MHz, CDCl3): δ 8.19 (d, 1H, J=8.1 Hz), 7.85 (d,
1H, J=7.5 Hz), 7.77 (d, 1H, J=8.1 Hz), 7.56-7.39 (m, 4H),
7.35-7.32 (m, 2H), 7.04 (d, 1H, J=7.5 Hz), 6.22 (d, 1H, J=
8.1 Hz), 6.12-6.03 (m, 1H), 2.33 (s, 3H), 2.05 (s, 3H), 1.74 (d,
3H, J=6.6 Hz). MS (EI): m/z 346.30 [M]þ. HRMS (EI), calcd
for C22H22N2O2 346.1681, found [M]þ 346.1682.
5-Iodo-2-methylbenzoic Acid (31). NaIO4 (295 mg, 1.38 mmol)
and KI (685 mg, 4.13 mmol) were added over 45 min slowly
portionwise to stirred 95% H2SO4 (15 mL). Stirring was con-
tinued for 1 h at 25-30 °C to give a dark-brown iodinating
solution at 25-30 °C. To a stirred solution of 2-toluic acid 30
(680 mg, 5 mmol) in 95% H2SO4 (5 mL), the iodinating solution
was added dropwise over 45 min while maintaining the tempera-
ture at 25-30 °C. Stirring was continued for 2 h, and the
iodination reaction was quenched by slowly pouring the final
reaction mixture into stirred ice water. The mixture was extracted
with AcOEt and dried over anhydrous Na2SO4. The solvent was
evaporated under reduced pressure and purification by silica gel
flash column chromatography to afford compound 31 in 63%
yield. 1H NMR (400 MHz, CDCl3): δ 8.38 (d, 1H, J=1.8 Hz),
7.75(dd, 1H, J=8.1, 1.8 Hz), 7.02 (d, 1H, J=8.1 Hz), 2.59 (s, 3H).
5-Iodo-2-methyl-N-[1-methyl-1-(1-naphthyl)ethyl]benzamide
(32). The title compound was obtained as described in the
general procedure using DMF:CH2Cl2 (1:1) as a solvent in
1
87% yield (white solid). H NMR (400 MHz, CDCl3): δ 8.20
(d, 1H, J=8.5 Hz), 7.89 (d, 1H, J=8.0 Hz), 7.83 (d, 1H, J=
8.1 Hz), 7.64-7.44 (m, 6H), 6.92 (d, 1H, J=7.8 Hz), 6.12 (m,
1H,), 5.94 (bd, 1H, J=8.3 Hz), 2.36 (s, 3H), 1.80 (d, 3H, J=
6.7 Hz). 13C NMR (100 MHz, CDCl3): δ 167.1, 138.6, 138.4,
137.6, 135.6, 135.0, 133.9, 132.7, 131.1, 128.8, 128.6, 126.6,
125.9, 125.1, 123.3, 122.6, 90.0, 44.9, 20.5, 19.3. MS (ESI): m/z
438.0 [M þ Na]þ. HRMS (ESI), calcd for C20H18INONa
438.0331; found [M þ Na]þ 438.0333.
1-Methyl-1-(1-naphthyl)ethylamine (27). CeCl3 7H2O (3.77 g,
3
10.1 mmol) was dried while stirring at 160 °C under reduced
pressure for 3 h. Argon was added slowly, and the flash was
cooled in an ice bath. THF (20 mL) was added and the suspen-
sion was stirred at 23 °C for 2 h. Methyl lithium ( 1.5 M)in THF
(6.7 mL, 10.1 mmol) was added below -50 °C. The mixture was
stirred for 30 min at -78 °C and a solution of 1-cyanonaphtha-
lene 26 (500 mg, 3.3 mmol) in THF (2 mL) was added. Stirring at
23 °C was continued for 2 h. Conc NH4OH (6.5 mL) was added
at -78 °C, and the mixture was warmed to 23 °C and filtered
with a celite pad. The solid was washed with CH2Cl2. The filtrate
was extracted with CH2Cl2 and the organic layers were dried
over Na2SO4 and concentrated under reduced pressure. The
residue was taken up in toluene (10 mL) and stirred with 3%
H3PO4 (10 mL) for 15 min. The toluene layer was extracted with
water (ꢀ2), and the combined water layers were washed with
toluene and made basic with conc NH4OH solution. The
mixture was extracted with CH2Cl2, and the organic layers were
dried over Na2SO4 and concentrated under reduced pressure to
furnish compound 27 (368 mg, 61%) as a colorless oil, Rf=0.25
(CH2Cl2:MeOH=9:1). 1H NMR (300 MHz, CDCl3): δ 9.03 (d,
1H, J=9.0 Hz), 7.98 (d, 1H, J=8.1 Hz), 7.86 (d, 1H, J=8.4 Hz),
7.71 (dd, 1H, J=1.2 and 7.5 Hz), 7.65-7.48 (m, 3H), 1.89 (s,
6H). 13C NMR (75 MHz, CDCl3): δ 144.5, 135.0, 131.2, 129.2,
129.0, 128.1, 127.6, 124.9, 124.8, 122.8, 53.9, 33.3.
5-Cyano-2-methyl-N-[1-methyl-1-(1-naphthyl)ethyl]benza-
mide (33). Compound 32 (29 mg, 0.07 mmol) was dissolved in
dry DMF (2 mL). CuCN (62 mg, 0.7 mmol) and a crystal of
KCN were added. The mixture was flushed with nitrogen and
stirred at 80 °C for 1 h then 130 °C for 10 h. CuCN (62 mg,
0.7 mmol) was added again. The mixture was flushed with
nitrogen and stirred at 130 °C for 6 h. After this time, NH4OH
solution was poured into reaction mixture, and the mixture was
extracted with AcOEt and dried over anhydrous Na2SO4. The
solvent was evaporated under reduced pressure and purification
by silica gel flash column chromatography to afford compound
33 in 78% yield as a white solid. 1H NMR (400 MHz, CDCl3): δ
8.19 (d, 1H, J=8.4 Hz), 7.87 (d, 1H, J=8.3 Hz), 7.84 (d, 1H, J=
8.2 Hz), 7.63-7.44 (m, 6H), 7.29 (d, 1H, J=7.8 Hz), 6.12 (m,
1H), 6.08-5.99 (bs, 1H), 2.48 (s, 3H), 1.81 (d, 3H, J=6.6 Hz).
13C NMR (100 MHz, CDCl3): δ 166.6, 141.9, 137.4, 137.2,
133.9, 133.0, 131.8, 131.0, 130.1, 128.9, 128.7, 126.7, 126.0,
125.1, 123.1, 122.6, 118.1, 109.7, 45.0, 20.4, 20.1. MS (EI): m/z
314.10 [M]þ. HRMS (EI), calcd for C21H18N2O 314.1419, found
[M]þ 314.1424.
2-Methyl-5-nitrobenzoic Acid Methyl Ester (35). To a stirring
MeOH (4 mL) in a round-bottom flask was added dropwise
thionyl chloride (0.24 mL, 3.3 mmol) at 0 °C. The mixture was
added 2-methyl-5-nitrobenzoic acid 34 (300 mg, 1.7 mmol) at
0 °C and it was allowed to stir for 4 h at reflux temperature. The
reaction was concentrated under reduced pressure, and the
residue was purified by silica gel column chromatography to
give corresponding compound 35 (320 mg, 99%) as a colorless
oil, Rf = 0.85 (hexane:EtOAc = 1:1). 1H NMR (400 MHz,
CDCl3): δ 8.52 (s, 1H), 8.05 (s, 1H), 7.30 (s, 1H), 3.83 (s, 3H),
2.56 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 165.4, 147.6, 145.6,
132.5, 130.1, 125.8, 125.3, 52.1, 21.5. MS (EI): m/z 195 [M]þ.
HRMS (EI), calcd for C9H9NO4 195.0532, found [M]þ
195.0539.
2-Methyl-5-nitro-N-[1-methyl-1-(1-naphthyl)ethyl]benzamide
(28). The title compound was obtained as described in the general
procedure in 91% yield (white solid). Rf = 0.26 (hex-
ane:EtOAc=3:1). 1H NMR (300 MHz, CDCl3): δ 8.50 (d, 1H,
J=8.1 Hz), 8.07 (d, 1H, J=2.4 Hz), 7.95 (dd, 1H, J=2.4 and 8.4
Hz), 7.87 (d, 1H, J=8.4 Hz), 7.76 (d, 1H, J=8.1 Hz), 7.59 (d, 1H,
J=7.5 Hz), 7.54-7.39 (m, 3H), 7.17 (d, 1H, J=8.7 Hz), 6.87 (bs,
1H), 2.24 (s, 3H), 1.95 (s, 6H). 13C NMR (75 MHz, CDCl3): δ
166.2, 145.3, 143.9, 140.6, 138.0, 134.9, 131.3, 131.4, 129.9, 129.8,
128.7, 125.3, 125.2, 125.1, 123.7, 123.7, 121.4, 57.5, 28.5, 19.5.
5-Amino-2-methyl-N-[1-methyl-1-(1-naphthyl)ethyl]benza-
mide (29). The title compound was obtained as described for
compound 24 in 75% yield (slightly yellow solid). Rf = 0.18

5238 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ghosh et al.
2-Bromomethyl-5-nitrobenzoic Acid Methyl Ester (36). Com-
pound 35 (100 mg, 0.53 mmol) was dissolved in CCl4 (4 mL),
followed by addition of NBS (100 mg, 0.58 mmol) and a
catalytic amount of benzoyl peroxide. The mixture was stirred
at reflux for 24 h. Another portion, of dibenzoyl peroxide
(40 mg, 0.23 mmol) was added and then the mixture was stirred
and heated at reflux for another 10 h. The mixture was allowed
to cool to 23 °C and was filtered. The filtrate was washed with
NaHCO3, dried over Na2SO4, and the solvent evaporated in
vacuo. The residue was purified by silica gel column chroma-
tography to afford compound 36 in 89% yield. 1H NMR
(400 MHz, CDCl3): δ 8.81 (d, 1H, J=2.5 Hz), 8.33 (dd, 1H,
J=8.5, 2.5 Hz), 7.68 (d, 1 H, 8.5 Hz), 5.00 (s, 2H), 4.01 (s, 3H).
2-Methoxymethyl-5-nitrobenzoic Acid Methyl Ester (37).
NaH (44 mg, 1.1 mmol) was added to a round-bottomed flask
containing methanol (2 mL) at 0 °C. The sodium methoxide
solution was added to a cold solution of compound 36 (60 mg,
0.22 mmol) in methanol (2 mL) at 0 °C. The resulting solution
was stirred at 50 °C for 4 h. After this time, NH4Cl solution was
poured into reaction mixture at 0 °C, and the mixture was
extracted with AcOEt and dried over anhydrous Na2SO4. The
solvent was evaporated under reduced pressure and purification
by silica gel flash column chromatography to afford corre-
sponding compound 37 in 72% yield. 1H NMR (400 MHz,
CDCl3): δ 8.82 (d, 1H, J=2.4 Hz), 8.38 (dd, 1H, J=8.7, 2.4 Hz),
7.94 (d, 1 H, 8.7 Hz), 4.94 (s, 2H), 3.96 (s, 3H), 3.53 (s, 3H).
2-Methoxymethyl-5-nitrobenzoic Acid (38). To a stirring solu-
tion of compound 37 (36 mg, 0.75 mmol) in THF/H2O mixture
and 8.1 Hz), 3.82 (s, 3H), 3.64 (s, 2H), 2.43 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 168.1, 144.1, 132.3, 129.8, 129.5, 118.8,
116.7, 51.6, 20.6. MS (EI): m/z 165.20 [M]þ. HRMS (EI), calcd
for C9H11NO2 165.0790, found [M]þ 165.0787.
2-Methyl-5-cyanobenzoic Acid Methyl Ester (42). CuCN (228
mg, 2.5 mmol) was suspended in distilled water (2 mL). NaCN
(353 mg, 7.2 mmol) was added with vigorous stirring, and the
internal temperature was kept below 40 °C until all the CuCN
went into solution. A suspension of amine 41 (350 mg, 2.1 mmol)
in water (4 mL) and conc HCl (0.7 mL) was stirred and cooled in
an ice bath. When the temperature reach 5 °C, a solution of
NaNO2 (190 mg, 2.8 mmol) in water (0.6 mL) was added
dropwise at 5 °C. When all the NaNO2 was added, the solution
was added dropwise the NaCN/CuCN solution at 0 °C. A few
drops of methanol were added to keep the foaming under
control. Stirring was continued for 3 h at 23 °C. The suspension
was extracted with EtOAc, and the organic layers were dried
over Na2SO4 and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography to
give compound 42 (115 mg, 31%) as a colorless oil, Rf=0.63
(hexane:EtOAc=1:1). 1H NMR (400 MHz, CDCl3): δ 8.08 (d,
1H, J=1.7 Hz), 7.56 (dd, 1H, J=1.7 and 7.9 Hz), 7.28 (d, 1H, J=
7.9 Hz), 3.83 (s, 3H), 2.56 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 165.7, 145.5, 134.4, 134.1, 132.4, 130.3, 117.8, 109.7, 52.1, 21.8.
5-N-tert-Butoxycarbonylmethylamino-2-methylbenzoic Acid
Methyl Ester (43). To a solution of nitrile 42 (40 mg, 0.23 mmol)
in MeOH (1.5 mL) was added Boc2O (0.1 mL, 0.46 mmol) and
NiCl2 6H2O (5.4 mg, 0.022 mmol) at 0 °C. NaBH4 (61 mg, 1.6
3
(5 mL:1 mL) at 0 °C was added solid LiOH H2O (120 mg,
mmol) was then added in small portions over 15 min. The
reaction was allowed to stir for 2 h at 23 °C. At this point,
diethylenetriamine (25 μL, 0.23 mmol) was added. The mixture
was allowed to stir for 15 min. The solvent was removed, and the
residue was dissolved with EtOAc. The organic layer was
washed with saturated NaHCO3 solution and dried over
Na2SO4. The solvent was removed under reduced pressure to
give a residue, which was purified by silica gel column chroma-
tography to furnish compound 43 (54 mg, 85%) as a colorless
oil, Rf = 0.49 (hexane:EtOAc = 3:1). 1H NMR (400 MHz,
CDCl3): δ 7.78 (s, 1H), 7.29 (d, 1H, J=7.8 Hz), 7.17 (d, 1H,
J=7.8 Hz), 4.92 (bs, 1H), 4.26 (d, 2H, J=5.7 Hz), 3.85 (s, 3H),
2.53 (s, 3H), 1.42 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 167.8,
155.8, 139.2, 136.5, 132.0, 131.3, 129.4, 129.5, 79.5, 51.8, 44.0,
28.3, 21.3. MS (CI): m/z 278.30 [M]þ. HRMS (CI), calcd for
C15H20NO4 278.1392, found [M - H]þ 278.1398.
3
5 mmol), and the resulting solution was stirred at 23 °C for 1.5 h.
After this period, the reaction mixture was evaporated until
1 mL, and the mixture was extracted with toluene to remove
organic impurities. The aqueous layer was cooled to 0 °C,
acidified with 25% aqueous citric acid until pH 3-4, extracted
with AcOEt, and dried over anhydrous Na2SO4. The solvent
was evaporated and purification by silica gel flash column
chromatography to furnish compound 37 in 90% yield. 1H
NMR (400 MHz, CD3OD and CDCl3): δ 8.73 (d, 1H, J =
2.4 Hz), 8.26 (dd, 1H, J=8.6, 2.4 Hz), 7.80 (d, 1 H, 8.6 Hz), 4.88
(s, 2H), 3.43 (s, 3H).
2-Methoxymethyl-5-nitro-N-[1-methyl-1-(1-naphthyl)ethyl]-
benzamide (39). The title compound was obtained as described
in the general procedure using DMF/CH2Cl2 (1:1) as a solvent in
73% yield (white solid). 1H NMR (400 MHz, CD3OD and
CDCl3): δ 8.55 (d, 1H, J=2.4 Hz), 8.22 (d, 1H, J=8.4 Hz), 8.21
(d, 1 H, 8.4 Hz), 7.90 (d, 1H, J=7.9 Hz), 7.83 (d, 1H, J=8.1 Hz),
7.64-7.46 (m, 5H), 7.43 (br d, J=8.2 Hz), 6.16 (m, 1H), 4.38 and
4.32 (AB, 2H, J=11.5 Hz), 2.92 (s, 3H), 1.81 (d, 3H, J=6.8 Hz).
5-Amino-2-methoxymethyl-N-[1-methyl-1-(1-
naphthyl)ethyl]benzamide (40). The title compound was obt-
ained as described for compound 24 in 82% yield (slightly
yellow solid). 1H NMR (400 MHz, CDCl3): δ 8.24 (d, 1H, J=
8.3 Hz), 7.97 (br d, 1H, J=7.7 Hz), 7.88 (d, 1H, J=8.0 Hz), 7.80
(d, 1H, J=8.1 Hz), 7.60-7.43 (m, 4H), 7.15 (d, 1H, J=2.5 Hz),
7.00 (d, 1H, J=8.1 Hz), 6.65 (dd, 1H, J=8.1, 2.5 Hz), 6.15 (m,
1H), 4.08 and 4.02 (AB, 2H, J=10.2 Hz), 3.80 (br s, 2H), 2.70 (s,
3H), 1.77 (d, 3H, J=6.8 Hz). 13C NMR (100 MHz, CDCl3): δ
167.1, 146.9, 138.5, 138.0, 133.9, 132.8, 131.2, 128.7, 128.2,
126.5, 125.8, 125.2, 123.6, 123.2, 122.6, 116.2, 73.2, 56.8, 44.8,
20.4. MS (EI): m/z 334.20 [M]þ. HRMS (EI), calcd for
C21H22N2O2 334.1681, found [M]þ 334.1679.
5-(N,N-tert-Butoxycarbonylmethyl)methylamino-2-methyl-
benzoic Acid Methyl Ester (44). To a solution of N-Boc amine 43
(60 mg, 0.21 mmol) in THF (3 mL) was added dropwise 0.5 M
KHMDS in toluene (0.64 mL, 0.32 mmol) at 0 °C under argon
atmosphere and it was allowed to stir for 30 min at 0 °C. The
mixture was added dropwise MeI (21 μL, 0.34 mmol) at 0 °C and
it was allowed to stir for 16 h at 23 °C. The reaction was
quenched with saturated NH4Cl solution and extracted with
EtOAc. The organic layers were dried over Na2SO4 and con-
centrated under reduced pressure. The residue was purified by
silica gel column chromatography to give compound 44 (52 mg,
1
83%) as a colorless oil, Rf = 0.60 (hexane:EtOAc = 3:1). H
NMR (400 MHz, CDCl3): δ 7.75 (s, 1H), 7.24 (bs, 1H), 7.17 (d,
1H, J=7.8 Hz), 4.36 (bs, 2H), 3.85 (s, 3H), 2.80 and 2.74 (each s,
3H), 2.54 (s, 3H), 1.45 (s, 9H). 13C NMR (100 MHz, CDCl3): δ
167.8, 155.6, 139.1, 135.6, 131.9, 131.2, 130.8, 129.5, 79.8, 51.7,
33.8, 28.3, 21.3.
5-Amino-2-methylbenzoic Acid Methyl Ester (41). To a solu-
tion of nitro 35 (635 mg, 3.3 mmol) in EtOAc (10 mL) was added
10% Pd-C (30 mg) and it was allowed to stir for 16 h at 23 °C
under H2 atmosphere. The reaction was filtered through a celite
pad, and the filtrate was concentrated under reduced pressure.
The residue was purified by silica gel column chromatography to
furnish compound 41 (536 mg, >99%) as a colorless oil, Rf=
0.57 (hexane:EtOAc=1:1). 1H NMR (400 MHz, CDCl3): δ 7.21
(d, 1H, J=2.5 Hz), 6.97 (d, 1H, J=8.1 Hz), 6.69 (dd, 1H, J=2.5
5-N-tert-Butoxycarbonylmethylamino-2-methylbenzoic Acid
(45). To a solution of ester 43 (54 mg, 0.19 mmol) in a mixture
(9:1) of THF and water (2 mL) was added LiOH H2O (12 mg,
0.29 mmol) at 0 °C and it was allowed to stir for 16 h at 23 °C.
The reaction was concentrated under reduced pressure, and the
residue was diluted with saturated NaHCO3 solution. The
mixture was extracted with Et2O, and the aqueous layer was
acidified with 1 M HCl solution to pH 4. The white solid was
extracted with EtOAc, and the organic layers were dried over
3

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5239
Na2SO4, and concentrated under reduced pressure to provide
corresponding acid 45 (39 mg, 76%) as a white solid, Rf=0.51
(CH2Cl2:MeOH=9:1). 1H NMR (300 MHz, CDCl3): δ 7.78 (s,
1H), 7.27 (d, 1H, J=7.8 Hz), 7.15 (d, 1H, J=7.8 Hz), 4.78 (bs,
1H), 4.19 (s, 2H), 2.50 (s, 3H), 1.40 (s, 9H). 13C NMR (75 MHz,
CDCl3): δ 170.8, 158.0, 139.5, 135.3, 132.5, 131.4, 130.2, 129.7,
80.1, 44.2, 28.7, 21.6.
5-N-tert-Butoxycarbonylmethylamino-2-methyl-N0-[(R)-1-(1-
naphthyl)ethyl]benzamide (47). The title compound was ob-
tained as described in the general procedure in 91% yield
(white solid). Rf = 0.20 (hexane:EtOAc = 3:1). 1H NMR
(300 MHz, CDCl3): δ 8.20 (d, 1H, J=8.4 Hz), 7.85 (d, 1H, J=
7.5 Hz), 7.78 (d, 1H, J=8.4 Hz), 7.58-7.40 (m, 4H), 7.13 (d, 2H,
J=7.5 Hz), 7.08 (d, 1H, J=7.8 Hz), 6.15-6.06 (bm, 2H), 4.86
(bs, 1H), 4.14 (d, 2H, J=5.1 Hz), 2.36 (s, 3H), 1.75 (d, 3H, J=
6.0 Hz), 1.39 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 168.7,
155.8, 137.9, 136.5, 136.5, 134.9, 133.9, 131.1, 128.7, 128.7,
128.4, 127.2, 126.5, 125.9, 125.5, 125.1, 123.5, 122.5, 79.5,
44.8, 43.9, 28.3, 20.6, 19.3. MS (EI): m/z 418.45 [M]þ. HRMS
(EI), calcd for C26H30N2O3 418.2256, found [M]þ 418.2252.
5-(N,N-tert-Butoxycarbonylmethyl)methylamino-2-meth-
yl-N0-[(R)-1-(1-naphthyl)ethyl]benzamide (48). The title com-
pound was obtained as described for compound 45 and
46 ligands were drawn using Chem 3D-Ultra 9.0. These
structures were minimized in Sybyl7.3 by molecular mechanics
with the Tripos force field to a gradient of 0.005 kcal/(mol A)
after addition of hydrogen atoms to the ligands. The crystal
structure coordinates of the bound ligand, 24, were used as a
reference molecule for docking the other 45 compounds in
GOLDv3.2, which allows full ligand flexibility and automatic
consideration of cavity bound water molecules. The genetic
algorithm parameters were all set to default except the number
of the operations were increased to 500000 and the population
size was incremented to 500. If the top three conformations
generated by the docking software for the ligands did not differ
˚
from each other by at least 1.5A, early termination was
allowed, ensuring nonredundant ligand conformations. The
five water molecules were explicitly defined in the docking
configuration file and were integral to the docking procedure
and results. The top three conformations were saved for
further analysis. The best scoring ligand conformation was
determined as the final docked conformation for each of the 46
ligands and was used for the alignment in the proceeding
QSAR analysis.
CoMSIA. The final top scoring docked conformations of
the 46 molecules were retrieved from the GOLDv3.2 run.
Mulliken charges were calculated for these fully optimized
structures based on the AM1 method in the Sybyl distribution
of MOPAC. The resulting charged 46 molecules were aligned
to each other for the CoMSIA analyses to explore the specific
contributions of the effect of physicochemical properties on
their bioactivities. By virtue of their similar docked confor-
mations in the active site of SARS-PLpro, the overall align-
ment did not need much manual tuning. Using the QSAR
module in Sybyl, a spreadsheet was created for the 46 aligned
molecules. Five physicochemical properties namely, steric,
electrostatic, hydrophobic fields, and hydrogen bond donor
and acceptor were evaluated. Similarity indices were com-
general procedure in 98% yield as two steps (white solid). Rf=
1
0.20 (hexane:EtOAc = 3:1), [R]20 -46.3 (c = 1, CHCl3). H
D
NMR (300 MHz, CDCl3): δ 8.22 (d, 1H, J=8.1 Hz), 7.85 (d, 1H,
J=7.5 Hz), 7.78 (d, 1H, J=8.4 Hz), 7.58-7.40 (m, 4H), 7.11 (d,
1H, J=7.2 Hz), 6.16-6.05 (m, 1H), 6.04 (bs, 1H), 4.28 (s, 2H),
2.71 (s, 3H), 2.38 (s, 3H), 1.76 (d, 3H, J=6.3 Hz), 1.39 (s, 9H).
13C NMR (75 MHz, CDCl3): δ 168.8, 155.5, 137.8, 136.6, 135.6,
134.9, 133.0, 131.1, 128.8, 128.7, 128.4, 127.2, 126.5, 125.9,
125.6, 125.1, 123.5, 122.5, 79.6, 51.9, 44.7, 33.8, 28.3, 20.5,
19.4. MS (ESI): m/z 455.99 [M þ Na]þ. HRMS (ESI), calcd
for C27H32N2O3Na 455.2311, found [M þ Na]þ 455.2312.
N-Methyl-5-methylamino-2-methyl-N0-[(R)-1-(1-
˚
puted using a probe with a charge of þ1, radius of 1 A, and a
naphthyl)ethyl]benzamide (49). The title compound was ob-
tained as described for compound 9 in 76% yield (white solid).
Rf=0.27 (CH2Cl2:MeOH=9:1), [R]20D -71.5 (c=1, MeOH). 1H
NMR (300 MHz, CDCl3 plus a small amount of CD3OD): δ
8.25 (d, 1H, J=8.1 Hz), 7.88 (d, 1H, J=8.4 Hz), 7.79 (d, 1H, J=
8.4 Hz), 7.63 (d, 1H, J=7.3 Hz), 7.59-7.44 (m, 3H), 7.25 (d, 2H,
J=7.8 Hz), 7.17 (d, 1H, J=7.8 Hz), 6.05 (q, 1H, J=6.9 Hz), 3.61
hydrophobicity of þ1. The default value of 30 kcal/mol was
set as the maximum cutoff for steric and electrostatic energies.
With standard options for variable scaling, SAMPLE meth-
od, leave-one-out cross validations were performed to deter-
mine the optimal principal component number. The cross-
validation runs were carried out for 20 groups and the 10
round bootstrap run followed. The final CoMSIA model was
obtained with this optimal number of components and non-
cross validated conventional correlations. The training set
was further tuned to delete five outlier compounds; this
improved the model’s q2 value considerably. Hence, the
resulting CoMSIA model training set consisted of 41 mole-
cules including compound 24.
(s, 2H), 2.32 (s, 3H), 2.31 (s, 3H), 1.69 (d, 3H, J=6.9 Hz). 13
C
NMR (75 MHz, CDCl3 plus a small amount of CD3OD): δ
171.9, 140.3, 138.1, 137.8, 135.7, 135.5, 132.4, 131.8, 130.9,
129.9, 129.0, 128.2, 127.3, 126.7, 126.5, 124.3, 123.8, 55.7,
46.3, 35.5, 21.4, 19.4. MS (EI): m/z 332.30 [M]þ. HRMS (EI),
calcd for C22H24N2O 332.1889, found [M]þ 332.1891.
5-Methylamino-2-methyl-N-[(R)-1-(1-naphthyl)ethyl]benzam-
ide (2). The title compound was obtained as described for
compound 9 in 56% yield (white solid). Rf = 0.11 (CH2Cl2:
MeOH=9:1). 1H NMR (400 MHz, CDCl3 plus a small amount
of CD3OD): δ 8.14 (d, 1H, J=8.5 Hz), 7.78 (d, 1H, J=8.0 Hz),
7.69 (d, 1H, J=8.2 Hz), 7.52 (d, 1H, J=7.1 Hz), 7.47-7.34 (m,
3H), 7.16-7.15 (m, 2H), 7.06 (d, 1H, J=8.2 Hz), 5.93 (q, 1H, J=
SARS-CoV PLpro Purification and IC50 Value Determina-
tion. The SARS-CoV PLpro enzyme (residues 1541-1855) was
expressed and purified from E. coli according to our previously
reported procedures.11 IC50 values for all inhibitors were deter-
mined using a 96-well plate based assay similar to our previously
described methods.11 The substrate used in the assay was the
fluorogeneic peptide Arg-Leu-Arg-Gly-Gly-AMC (RLRGG-
AMC), which was purchased from Bachem Bioscience. Reac-
tions were performed in a total volume of 50 μL, which
contained the following components: 50 mM HEPES, pH 7.5,
0.1 mg/mL BSA, 5 mM DTT, 50 μM RLRGG-AMC, 2%
DMSO, and varying concentrations of inhibitor (0-200 μM).
Reactions were initiated with the addition of PLpro to produce a
final enzyme concentration of 125 nM. Reaction progress was
monitored continuously on a Tecan Genios Pro microplate
reader (λexcitation=360 nm; λemission=460 nm; gain=40). Initial
rate data were fit to the equation vi=vo/(1 þ [I]/IC50) using the
Enzyme Kinetics module of SigmaPlot (v. 9.01 Systat Software,
Inc.) where vi is the reaction rate in the presence of inhibitor, vo is
the reaction rate in the absence of inhibitor, and [I] is the
inhibitor concentration.
6.8 Hz), 3.61 (s, 2H), 2.21 (s, 3H), 1.59 (d, 3H, J=6.8 Hz). 13
C
NMR (100 MHz, CDCl3 plus a small amount of CD3OD): δ
172.0, 140.9, 140.3, 138.1, 135.5, 135.3, 132.4, 131.9, 129.9,
130.0, 129.0, 127.3, 127.1, 126.7, 126.5, 124.3, 123.7, 46.3,
46.0, 21.4, 19.3. MS (EI): m/z 318.30 [M]þ. HRMS (EI), calcd
for C21H22N2O 318.1732, found [M]þ 318.1734.
Computational Material and Methods. Docking. A genetic
algorithm implemented in GOLD v3.225 was used to dock 46
inhibitors into the active site of the ligand bound form of the
SARS-CoV PLpro crystal structure (PDB id: 3e9s).13 All
ligand and water molecules except five, namely HOH2555,
HOH2563 (conserved), HOH2590 (conserved), HOH2574,
and HOH2595 (conserved) were deleted from the crystal
structure of the ligand-bound form of the protease. Polar
hydrogen atoms were added using the Sybyl package.26 The

5240 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ghosh et al.
Antiviral assay. SARS-CoV antiviral assays were performed
as previously described.13 Briefly, Vero E6 cells (approximately
1 ꢀ 104 cells/well) in a 96-well plate were either mock-infected or
infected with 100-fold the median tissue culture infective dose
of SARS-CoV-Urbani for 1 h at 37 °C. The viral inoculum was
removed and replaced with 100 μL of culture medium contain-
ing serial dilutions of the inhibitor compound, from 50 μM to
0.1 μM. Cells were incubated for 48 h at 37 °C and evaluated for
cell viability using the CellTiter-Glo Luminescent Cell Viability
Assay (Promega) and output expressed as relative luciferase
units (RLU).
Structure-based design, synthesis, and biological evaluation of
peptidomimetic SARS-CoV 3CLpro inhibitors.. Bioorg. Med.
Chem. Lett. 2007, 17, 5876–5880. (b) Jain, R. P.; Pettersson, 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. Synthesis and
Evaluation of Keto-Glutamine Analogues as Potent Inhibitors of Severe
Acute Respiratory Syndrome 3CLpro. J. Med. Chem. 2004, 47, 6113–
6434. (c) Vederas, J. C.; Jain, R. P. Structural variations in keto-
glutamines for improved inhibition against hepatitis A virus 3C protei-
nase. Bioorg. Med. Chem. Lett. 2004, 14, 3655.
(10) (a) Devaraj, S. G.; Wang, N.; Chen, Z.; Chen, Z.; Tseng, M.;
Barretto, N.; Lin, R.; Peters, C. J.; Tseng, C.-T. K.; Baker, S. C.; Li,
K. Regulation of IRF-3-dependent Innate Immunity by the Pa-
pain-like Protease Domain of the Severe Acute Respiratory Syn-
drome Coronavirus. J. Biol. Chem. 2007, 282, 32208–32221. (b)
Ratia, K.; Saikatendu, K. S.; Santarsiero, B. D.; Barretto, N.; Baker, S.
C.; Stevens, R. C.; Mesecar, A. D. Severe acute respiratory syndrome
coronavirus papain-like protease: structure of a viral deubiquitinating
enzyme. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 5717–5722. (c)
Barretto, N.; Jukneliene, D.; Ratia, K.; Chen, Z.; Mesecar, A. D.; Baker,
S. C. The Papain-Like Protease of Severe Acute Respiratory Syndrome
Coronavirus Has Deubiquitinating Activity. J. Virol. 2005, 79, 15189–
15198.
Acknowledgment. This work was supported by the Public
Health Service Research Grant P01 AI060915 (“Development
of Novel Protease Inhibitors as SARS Therapeutics”).
Supporting Information Available: HRMS and HPLC data of
inhibitors and a stereoview of the X-ray structure of inhibitor
24-bound SARS-CoV PLpro. This material is available free of
charge via the Internet at http://pubs.acs.org.
(11) (a) Lindner, H. A.; Fotouhi-Ardakani, N.; Lytvyn, V.; Lachance,
ꢀ
P.; Sulea, T.; Menard, R. The Papain-Like Protease from the
Severe Acute Respiratory Syndrome Coronavirus Is a Deubiqui-
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