Severe acute respiratory syndrome coronavirus papain-like novel protease inhibitors: Design, synthesis, protein-ligand X-ray structure and biological evaluation

Article abstract of DOI:10.1021/jm1004489

The design, synthesis, X-ray crystal structure, molecular modeling, and biological evaluation of a series of new generation SARS-CoV PLpro inhibitors are described. A new lead compound 3 (6577871) was identified via high-throughput screening of a diverse chemical library. Subsequently, we carried out lead optimization and structure-activity studies to provide a series of improved inhibitors that show potent PLpro inhibition and antiviral activity against SARS-CoV infected Vero E6 cells. Interestingly, the (S)-Me inhibitor 15h (enzyme IC₅₀ = 0.56 mn; antiviral EC₅₀ = 9.1 mn;) and the corresponding (R)-Me 15g (IC₅₀ = 0.32 mn; antiviral EC ₅₀ = 9.1 mn;) are the most potent compounds in this series, with nearly equivalent enzymatic inhibition and antiviral activity. A protein-ligand X-ray structure of 15g-bound SARS-CoV PLpro and a corresponding model of 15h docked to PLpro provide intriguing molecular insight into the ligand-binding site interactions.

Full text of DOI:10.1021/jm1004489

4968 J. Med. Chem. 2010, 53, 4968–4979
DOI: 10.1021/jm1004489
Severe Acute Respiratory Syndrome Coronavirus Papain-like Novel Protease Inhibitors: Design,
Synthesis, Protein-Ligand X-ray Structure and Biological Evaluation†
Arun K. Ghosh,*,‡ Jun Takayama,‡ Kalapala Venkateswara Rao,‡ Kiira Ratia,§ Rima Chaudhuri,§ Debbie C. Mulhearn,§
Hyun Lee,§ Daniel B. Nichols,^ Surendranath Baliji,^ 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 S.
Ashland, Chicago, Illinois 60607, and ^Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine,
Maywood, Illinois 60153
Received April 12, 2010
The design, synthesis, X-ray crystal structure, molecular modeling, and biological evaluation of a series
of new generation SARS-CoV PLpro inhibitors are described. A new lead compound 3 (6577871) was
identified via high-throughput screening of a diverse chemical library. Subsequently, we carried out lead
optimization and structure-activity studies to provide a series of improved inhibitors that show potent
PLpro inhibition and antiviral activity against SARS-CoV infected Vero E6 cells. Interestingly, the
(S)-Me inhibitor 15h (enzyme IC50 = 0.56 μM; antiviral EC50 = 9.1 μM) and the corresponding (R)-Me
15g (IC50 = 0.32 μM; antiviral EC50 = 9.1 μM) are the most potent compounds in this series, with nearly
equivalent enzymatic inhibition and antiviral activity. A protein-ligand X-ray structure of 15g-bound
SARS-CoV PLpro and a corresponding model of 15h docked to PLpro provide intriguing molecular
insight into the ligand-binding site interactions.
Introduction
discovery and design of a series of unprecedented noncovalent
SARS-CoV PLpro inhibitors displaying antiviral activity
against SARS-CoV with no associated cytotoxicity.10 Subse-
quently, a protein-ligand X-ray structure provided impor-
tant molecular insights for further design and optimization of
inhibitors.10 This initial work demonstrated that PLpro is a
viable target for the development of anti-SARS therapeutics.
Besides viral peptide cleavage, recent structural and func-
tional studies demonstrated that PLpro is involved in a
number of other important biochemical events, such as
deubiquitination, deISGylation, and involvement in the virus
evasion from the innate immune response.11,12 The homo-
logous enzyme PLP2, from the human coronavirus 229E, has
been shown to be critical to 229E viral replication.13 In
addition, recent studies have shown that human deubiquiti-
nating enzymes are potential anticancer drug-design targets.
Thus, PLpro is a significant target for development of drugs
against SARS andisa model for development of drugsagainst
other deubiquitinating enzymes involved in human diseases.
Recently, our primary screening of a library of 50 080
diverse, druglike compounds led to the identification of two
compounds after lead validation. Both leads reproducibly
inhibited PLpro in a dose dependent manner in the absence
and presence of Triton-X. Subsequently, our optimization
efforts of the most potent lead, 1 (7724772), containing a
benzamide scaffold (IC50 = 20.1 ( 1.1 μM) led to the design
of novel PLpro inhibitor 2 and related derivatives that
displayed antiviral activity against SARS-CoV. We recently
reported a detailed study describing synthesis, biological
studies, and X-ray structure of the protein-ligand complex
of 2-bound PLpro.10 In our continuing studies toward the
development of noncovalent/reversible PLpro inhibitors, we
Severe acute respiratory syndrome (SARSa) was first re-
ported in Guangdong province, China, in November 2002.1
SARS is a contagious respiratory illness with no effective treat-
ment to date. SARS affected three continents, infecting more
than 8000 individuals and causing nearly 800 deaths. Fortu-
nately, the spread of SARS-CoV was contained after the initial
outbreaks through public health measures. As it turned out, the
etiological agent of SARS is a novel coronavirus, SARS-CoV.2,3
There have been no known new cases of SARS since 2005.
However, recent isolation of strains from zoonotic origins
thought to be the reservoir for SARS-CoV raises the possibility
of a reemergence of SARS and related ailments.4,5 Consequently,
design and development of antivirals effective against SARS-
CoV should be an important priority against future outbreaks.
Biochemicaleventscritical tothe viral replication revealed a
number of important targets for therapeutic intervention of
SARS.6,7 Most notably, two cysteine proteases, a papain-like
protease (PLpro) and a 3C-like protease (3CLpro), play a
critical role in the virus-mediated RNA replication. Not
surprisingly, numerous studies related to the development of
SARS-CoV 3CLpro inhibitors have already been reported.8,9
In contrast, very few inhibitor design efforts against SARS-
CoV PLpro have been reported. We recently reported the
†The PDB accession code for 15g-bound PLpro X-ray structure is
3MJ5.
*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, severe acute respiratory syndrome coronavirus; 3CLpro, chymo-
trypsin-like protease; PLpro, papain-like protease; WHO, World Health
Organization.
r
pubs.acs.org/jmc
Published on Web 06/09/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4969
Scheme 1a
a Reagents and conditions: (a) 5a or 5b, EDCI, HOBT, NMM,
CH2Cl2, 23 ꢀC, 5 h; (b) TFA, 0-23 ꢀC, 6 h; (c) 1-naphthaldehyde,
Na(OAc)3BH, AcOH, CH2Cl2, 23 ꢀC, 12 h.
Scheme 2a
Figure 1. Structures of PLpro inhibitors 1-3 and 15g.
have now investigated the potential of the second and less
potent lead that evolved from our high-throughput screening
efforts. The second HTS lead, compound 3 (Figure 1), con-
tains a piperidine carboxamide scaffold and exhibited an IC50
value of 59 μM. Our subsequent lead optimization efforts led
to the design of potent inhibitor 15g (IC50 = 0.32 μM) which
inhibited SARS-CoV viral replication in Vero cells with an
EC50 value of 9.1 μM. The corresponding enantiomer 15h has
shown slightly less potent enzyme inhibitory activity (IC50
=
0.56 μM) and similar antiviral potency. A protein-ligand
X-ray structure of 15g-bound SARS-CoV PLpro was deter-
mined. Interestingly, this structure revealed a unique mode
of binding with SARS-CoV PLpro and that key molecular
interactions of inhibitor 15g are quite different from the
active-site interactions with inhibitor 2. Herein we describe
the design, synthesis, structure-activity studies, molecular
modeling, protein-ligand X-ray structure, and biological
evaluation of a series of novel and noncovalent inhibitors of
SARS-CoV PLpro.
a Reagents and conditions: (a) KOtBu, DMSO, 23 ꢀC, 48 h; (b) 10%
HCl, THF, 23 ꢀC, 18 h; (c) NaHCO3, 23 ꢀC, 16 h; (c) H2, PtO2, EtOAc,
23 ꢀC, 2 h.
Chemistry
To ascertain the importance of the position of the methoxy
substituent in lead inhibitor 3, we have synthesized the
corresponding 2-methoxy and 3-methoxybenzyl derivatives.
As shown in Scheme 1, Boc-piperidine-4-carboxylic acid 4
was coupled with 2- and 3-methoxybenzylamines 5a and 5b
using N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hy-
drochloride (EDCI) and 1-hydroxybenzotriazole hydrate
(HOBT) in the presence of N-methylmorpholine (NMM) in
CH2Cl2 to provide coupling products 6a and 6b in 92% and
94% yield, respectively. Removal of Boc-group by exposure
to trifluoroacetic acid (TFA) in CH2Cl2 at 0-23 ꢀC for 6 h
afforded the respective amine. Reductive amination of these
amines with 1-naphthaldehyde using Na(OAc)3BH in the
presence of acetic acid furnished inhibitors 7a and 7b in
70% and 71% yield, respectively.
For structure-activitystudies andoptimizationof potency,
weplannedtosynthesizederivatives of both1-and2-naphthy-
lethylpiperidin-4-carboxylic acids and coupled them with
various substituted benzylamine derivatives. The synthesis
of substituted piperidine-4-carboxylic acids is shown in
Scheme 2. Alkylation of dimethyl malonate 8 with commer-
cially available 2-bromomethyl-1,3-dioxolane 9 in the pre-
sence of KOtBu in DMSO at 23 ꢀC afforded malonate
derivative 10 as described previously.14 Deprotection of the
ketal functionalities was carried out by treatment of 10 with
10% aqueous HCl in THF at 23 ꢀC. The reaction was
quenched with solid NaHCO3, and the resulting crude dia-
ldehyde was used directly for the subsequent condensa-
tion reaction. Condensation of the dialdehyde with various

4970 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Ghosh et al.
Scheme 3a
Table 1. Structure and Activity of 1- and 2-Naphthylmethyl Deri-
vativesa
a Reagents and conditions: (a) NaCN, DMF, reflux, 16 h; (b)
LiOH H2O, THF/MeOH/H2O (3:1:1), 23 ꢀC, 16 h; (c) 5a-d, EDCI,
3
HOBT, DIPEA, CH2Cl2/DMF (9:1), 23 ꢀC, 15 h.
Scheme 4a
a Reagents and conditions: (a) NaBH3CN, MeOH/AcOH (50:1), 23
ꢀC, 48 h; (b) TFA, CH2Cl2, 23 ꢀC, 2 h; (c) 5c, N,N0-carbonyldiimidazole,
CH2Cl2, 23 ꢀC, 4 h.
optically active (S)- and (R)-1-methyl-1-naphthylmethyl-
amines, 1-methyl-2-naphthylmethylamines, 2-naphthylmethyl-
amine, 1-naphthylmethylamine, and dimethyl-1-naphthyl-
methylamine 11a-g10 in aqueous THF for 16 h afforded
dihydropyridines 12a-g in 39-62% yield.15,16 Catalytic hydro-
genation of dihydropyridines 12a-f in ethylacetate at 23 ꢀC
provided various piperidine derivatives 13a-f in 60-94%
yield.
The synthesis of various test inhibitors is shown in
Scheme 3. Treatment of diesters 13a-f with NaCN in DMF
at reflux for 16 h provided methyl esters 14a-f in 38-92%
yield. Dihydropyridine derivative 12g was similarly converted
to methyl ester 14g in a two-step sequence. Saponification
of 14a-g with aqueous LiOH in a mixture (3:1:1) of THF,
methanol, and water at 23 ꢀC for 16 h afforded the corre-
sponding carboxylic acids. Coupling of these resulting car-
boxylic acids with benzylamine derivatives 5a-d utilizing
EDCI in the presence of diisopropylethylamine as described
above furnished various inhibitors 15a-k in excellent yield
(80-99%).
a NA = not active.
of 4-methoxybenzylamine 5c in the presence of N,N0-carbo-
nyldiimidazole in CH2Cl2 followed by addition of 19 and
stirring of the resulting mixture at 23 ꢀC for 4 h afforded
piperazine derivative 20 in 90% yield.
To evaluate the effect of the corresponding piperazine
derivatives, we sought to synthesize racemic piperazine deri-
vative 20, and the synthesis is outlined in Scheme 4. Reductive
amination17 of Boc-piperazine 1618 with 1-acetonaphthone 17
using sodium cyanoborohydride in a mixture (50:1) of metha-
nol and acetic acid at 23 ꢀC for 48 h afforded 18 in 24% yield.
Removal of the Boc-group by treatment with trifluoroacetic
acid in CH2Cl2 at 23 ꢀC for 2 h provided amine 19.19 Treatment
Results and Discussion
The second HTS lead 3 is considerably weaker than the first
lead inhibitor 1, a benzamide derivative of 2-naphthylethyl-
amine. To enhance activity, we first investigated the effect of
2-methoxy and 3-methoxy derivatives 7a and 7b on PLpro
inhibitory activity. As shown in Table 1, 2-methoxy derivative
7a showed a very poor inhibitory activity. The 3-methoxy

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4971
Table 2. Structure and Activity of Benzodioxolane Derivartives
derivative, 7b, however, displayed slightly better activity than
the starting lead 3. Our previous structure-activity of lead 1
established that 1-naphthylethylamides were significantly
more potent than the corresponding 2-naphthyl derivative.
The X-ray structure of 2 bound to PLpro demonstrated that a
(R)-1-naphthylethylamide forms hydrophobic interactions
with the Tyr-265 and Tyr-269 aromatic rings and with side
chains of Pro-248 and Pro-249.10 The preference for (R)-
methyl was also documented, as it points into the interior of
the enzyme between Tyr-265 and Thr-302. On the basis of this
ligand-binding site interaction, we elected to incorporate
the (R)-methyl group. As shown in Table 1, the (R)-methyl
derivative 15a displayed an IC50 value of 1.2 μM. To ascertain
the importance of the position of the methoxy group, we
synthesized m-methoxy and p-methoxy derivatives. Interest-
ingly, m-methoxy derivative 15b exhibited improvement of
enzyme inhibitory activity with an IC50 value of 0.34 μM. The
corresponding p-methoxy derivative 15c has also shown
similar potency enhancement (>170-fold over 3). However,
the 2-methoxy derivative 15a showed a 3-fold reduction in
potency over 15b and 15c. We then examined the effect of
2-(R)-naphthylethyl derivatives on potency. As shown, both
m-methoxybenzylamide 15d and o-methoxybenzylamide 15e
displayed significant reductions in potency compared to the
1-(R)-naphthylethyl derivatives 15b and 15a, respectively.
Interestingly, the 2-(S)-naphthylethyl derivative 15f is 2-fold
more potent than the 2(R)-derivative 15d.
We next examined the effect of a piperazine ring in place of
piperidine in 15c by preparing compound 20. However, this
piperazine derivative showed no activity against PLpro. Most
likely, the piperazine derivative showed no activity against
PLpro because of the structural constraints imposed by the
carbon to nitrogen replacement on this ring. The new nitrogen
is then attached to the amide group, forming a urea moiety.
This urea moiety will tend to be planar, imposing a flexibility
constraint. GOLD docking shows the amide to rotate ∼90ꢀ
away from the optimal hydrogen-bonding orientation (data
not shown) of the other active compounds described here.
Our structure-activity studies established that both m-
methoxy and p-methoxy derivatives (15b and 15c) are equally
potent. Our preliminary modeling studies indicated that either
methoxy oxygen (meta or para) is within proximity to form a
hydrogen bond with the Gln-270 carboxamide side chain. On
the basis of these possible interactions, we incorporated a
benzodioxolane ring and examined its effect on inhibitory
potency. As shown in Table 2, dioxolane derivative 15g
exhibits potency comparable to the corresponding m- and
p-derivatives 15b and 15c. The corresponding (S)-derivative
15h also shows comparable enzyme inhibitory activity. To
examine the preference for a methyl group over a hydrogen at
the 1- and 2-naphthylmethyl positions, we have synthesized
and evaluated the corresponding unsubstituted derivatives 15i
and 15j. As shown, both compounds displayed significant
reduction in potency, indicating the importance of the methyl
group. We have also examined the corresponding gem-
dimethyl derivative 15k. Interestingly, this compound is in-
active, indicating that both methyl groups cannot be accom-
modated by the PLpro active site.
Table 3. Evaluation of Compounds as Inhibitors of SARS-CoV Repli-
cation in a Cell-Based Assay
compd
IC50 (μM)
EC50 (μM)
3
59.2 ( 7.8
1.21 ( 0.04
0.34 ( 0.01
0.34 ( 0.01
5.8 ( 0.1
NI
15a
15b
15c
15f
15g
15h
11.6 ( 0.6
9.7 ( 0.3
10.2 ( 0.5
>25
0.32 ( 0.01
0.56 ( 0.03
9.1 ( 0.5
9.1 ( 0.3
This protocol allows for the evaluation of both inhibitor
efficacy and cytotoxicity. As can be observed from the data
presented in Table 3, the original HTS lead (3) does not show
any antiviral activity. However, all 2-, 3-, and 4-methoxy
derivatives 15a-c show comparable antiviral activity. Inhibi-
tor 15f with a 2-naphthyl substituent displayed no antiviral
activity. While the (R)-methyl derivative 15g showed slightly
better enzyme activity than the (S)-methyl derivative 15h,
both inhibitors exhibited the same antiviral potency (EC50
=
9.1 μM). Interestingly, both dioxolane derivatives 15g and 15h
showed antiviral activity approximately comparable to the
activity of the corresponding methoxy or benzamide deriva-
tives reported in our previous studies.10
To obtain molecular insight into the ligand-binding site
interactions, the X-ray crystal structure of 15g bound to
PLpro was determined. Interestingly, the binding mode and
key molecular interactions of inhibitor 15g are quite different
than predicted and are different from the active-site interac-
tions with the benzamide-derived inhibitors we previously
reported. As shown in Figure 2, the inhibitor binds to a loop
adjacent to the active site via a series of interactions including
a hydrogen-bond formed between the carboxamide NH of the
inhibitor and the backbone carbonyl of Tyr-269, with 15g
wrapped around the β-turn. The 15g bound PLpro crystal
structure also confirms the presence of a few structural water
molecules conserved between the apo enzyme (PDB code
Antiviral activities of selected PLpro inhibitors were deter-
mined, and the results are shown in Table 3. The compounds
were assayed for their ability to rescue a Vero cell culture from
SARS-CoV infection. The viability of virus-infected Vero E6
cells as a function of inhibitor concentration was measured
relative to mock-infected cells using a luminescence assay.

4972 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Ghosh et al.
Figure 2. Stereorepresentation of 15g bound to PLpro, including the conserved waters adjacent to the binding site that may influence the
binding conformation, as described in the text.
Figure 3. X-ray structure of inhibitor 15g-bound (yellow) PLpro (gray) (PDB code 3MJ5) superimposed on the X-ray structure of inhibitor 2-
bound (cyan) PLpro (pink) (PDB code 3E9S).
2FE8) and inhibitor 2 bound PLpro (PDB code 3E9S). One of
the conserved water molecules sits in the P5 pocket shown in
Figure 2 as spheres between residues Asp-165, Asp-303, and
Thr-302, preventing the inhibitor naphthyl rings from occu-
pying this pocket. In the stereoimage of 15g-bound PLpro we
also show twootherwatermolecules near residue Leu-163and
Lys-158 that may prevent the benzodioxolane ring from
flipping down toward Lys-158.
significantly between the two inhibitors. Interestingly, the
turn region between Tyr-269 and Gln-270 also shows signifi-
cant flexibility, particularly in the case of inhibitor 2 (PDB
code 3E9S), where the peptide bond between Tyr-269 and
Gln-270 flips by 180ꢀ to enable a hydrogen bond interaction
between the backbone nitrogen of Tyr-269 and the carbox-
amide oxygen in inhibitor 2. The carboxyamide nitrogen
makes a hydrogen bond with the side chain carboxylate
of Asp-165. The carboxy amide nitrogen of inhibitor 15g
(yellow) forms a hydrogen bond with the backbone carbonyl
Figure 3 superimposes 15g and our previously developed
inhibitor, 2,10 and demonstrates that the binding mode differs

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4973
Figure 4. (A) Superposition of enantiomer 15h (blue) with the crystal structure of 15g-bound (yellow) PLpro. (B) Docked alignment of the
gem-dimethyl substituted compound in the 15g-ligand removed PLpro crystal structure. The bumping collision of one of the methyl groups of
the gem-dimethyl (magenta) 15k with the Asp-165 carboxylate is noted.
oxygen of Tyr-269 (protein shown in gray). The naphthyl
rings of both inhibitors 2 and 15g align in a similar fashion in
the hydrophobic pocket formed by residues Tyr-269, Tyr-265,
Pro-248, Pro-249, and Thr-302. The overlapping position of
one conserved water molecule observed for both the inhibitor
2-boundPLpro (oxygen atom shownas sphere in pink) crystal
structure and inhibitor 15g-bound PLpro (oxygen atom
shown as sphere in red) crystal structure is shown as over-
lapping spheres.
one of the methyl groups exhibits a bumping collision with the
side chain of Asp-165. One of the methyl groups in 15k shifts
˚
almost 1.2 A toward residue Asp-165 when compared to the
single methyl substitution (R)-Me in 15g, as can be seen in
Figure 4B. It is important to note that the side chain of this Asp-
165 is locked in its position by a hydrogen bond with the
backbone NH of Arg-167. Hence, the gem-dimethyl substitu-
tion is not favorably accommodated in the active site because in
order to fit the hydrophobic methyl group near the hydrophilic
residue, the aspartic acid side chain would have to move out,
thereby breaking structural hydrogen bonding with Arg-167.
This hypothesis is further validated by the GoldScore
scoring function of GOLD, version 4.1, during the docking
study. Compound 15k is heavily penalized because of an
unfavorable internal energy term (-12 compared to about
-6 for both 15g and 15h) which is a sum of the internal
torsional strain and internal van der Waals energyterms of the
ligand. Docking with flexible residues also suggests that the
Gln-270 side chain may adopt conformations that might
enable hydrogen bonding interactions with one of the 1,3
Modeling Studies
To understand the SAR of the analogues of HTS hit com-
pound 3, we used computer modeling to explore the interac-
tionsof this series of inhibitors with PLpro. Theactivityof this
series of compounds is independent of stereoisomerism in
contrast to the series of compounds synthesized from the first
HTS hit compound 1.10 GOLD redocking of inhibitor 15g
into the PLpro crystal structure described above produces a
˚
heavy atom rmsd of 1.7 A with the crystal structure confor-
mation of 15g, indicating that docking satisfactorily repro-
duces the experimental structure. When the inhibitors 15g,
15h, and 15k are docked into the ligand removed 15g-bound
PLpro crystal structure (with residues Tyr-269 and Gln-270
flagged as flexible), the internal strain scores of the com-
pounds correlate very well with their enzymatic activities. The
conserved overlapping water molecules observed in both
chains A and B of the 15g-bound PLpro crystal structure
were included for all docking studies.
˚
benzodioxolane oxygens in 15g and 15h (within 3 A). How-
ever, all docked conformations generated for 15k show a loss
of this hydrogen bonding interaction. The closest benzodiox-
˚
olane oxygen of 15k is at least 4.8 A away from the side chain
of Gln-270 (not shown). Figure 4B highlights the potential
bumping collision of one of the methyl groups of 15k with
Asp-165, demonstrating that two methyl groups cannot be
accommodated favorably at this position.
In our previous study, we discussed the SAR of the
analogues of our first HTS hit 1 and the evolution of inhibitor
2 in great detail.10 In distinct contrast to the present work, that
series of compounds is extremely sensitive to the enantiomeric
form of the compound. From docking studies we concluded
that the (R)-Me form was active whereas the (S)-Me was
inactive because the (S)-Me conformation pushed the carbox-
amide group of the inhibitor away from the backbone NH of
Tyr-269, inhibiting hydrogen bond formation with the loop
residue.
To investigate the structural basis of the potency insensi-
tivity to the (R)-Me (15g) versus (S)-Me (15h) configuration,
we show the docked model of inhibitor 15h superimposed on
the crystal structure of 15g-bound PLpro in Figure 4A. From
this model, we observe an inversion of the piperidine ring
between the (R)-Me and (S)-Me binding modes that allows the
naphthyl rings of both isomers to be accommodated in the
active site in very similar orientations. The flexible piperidine
ring also acts as a spacer group that enables the carboxamide
NH of both 15h and 15g to hydrogen-bond with the backbone
carbonyl oxygen of Tyr-269 in a similar fashion, thereby
retaining the potency of both enantiomers. However, the gem-
dimethyl substitution in 15k decreases the freedom around the
carbon atom and locks the compound in a conformation where
Conclusion
We have designed, synthesized, and evaluated a novel series
of SARS-CoV PLpro inhibitors. Initial lead structure 3

4974 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Ghosh et al.
(IC50 = 59.2 μM) was discovered via high-throughput screen-
ing of a library of diverse compounds. Our preliminary
structure-activity studies and systematic modification guided
by X-ray crystal structure of 2-bound PLpro and subsequent
molecular modeling resulted in a potent inhibitor 15g with
enzyme inhibitory IC50 value of 320 nM and antiviral EC50
value of 9.1 μM in SARS-CoV-infected Vero E6 cells. Inter-
estingly, the corresponding (S)-isomer 15h is only slightly less
potent (IC50 = 560 nM) in PLpro inhibitory assays but equi-
potent in antiviral assays. The corresponding gem-dimethyl
derivative 15k is significantly less potent. A protein-ligand
mmol) in CH2Cl2 (3 mL), trifluoroacetic acid (0.15 mL) was
added at 0 ꢀC. The resulting mixture was stirred for 6 h at 23 ꢀC.
The reaction mixture was diluted with CH2Cl2 and basified by
slow addition of saturated NaHCO3 solution. The layers were
separated and the aqueous layer was extracted several times with
ethyl acetate. The combined organic layers were dried over
anhydrous Na2SO4. The solvent was removed under reduced
pressure to furnish the amine. To the crude amine in dry CH2Cl2
(5 mL), 1-naphthaldehyde (77 μL, 0.57 mmol), Na(OAc)3BH
(121 mg, 0.57 mmol), and AcOH (33 μL, 0.57 mmol) were added
successively at 23 ꢀC, and the resulting mixture was stirred for
12 h at 23 ꢀC. The reaction mixture was basified with 2 N NaOH
and diluted with CH2Cl2 and H2O. The organic layer was
separated and the aqueous layer extracted with CH2Cl2. The
combined organic layers were dried over anhydrous Na2SO4.
Solvent was removed under reduced pressure and the resulting
residue was purified by column chromatography over silica gel
(2% MeOH/CH2Cl2) to provide 1-[(1-naphthyl)methyl]-4-[(3-
methoxybenzylamino)carbonyl]piperidine as a viscous liquid
˚
X-ray structure of 15g-bound PLpro was determined to 2.6 A
resolution. This structure provided critical molecular insight
intothe ligandbinding siteinteractions. Itappearsthatthe key
active site interactions are quite different from the earlier
series of inhibitors. Further design of improved reversible
SARS-CoV PLpro inhibitors is currently underway in our
laboratories.
1
(79 mg, 71%). H NMR (400 MHz, CDCl3): δ 8.28-8.33 (m,
1H), 7.82-7.88 (m, 1H), 7.77 (dd, J = 2.2 and 7.1 Hz, 1H),
7.44-7.53 (m, 2H), 7.36-7.43 (m, 2H), 7.23 (t, J = 7.8 Hz, 1H),
6.77-6.86 (m, 3H), 5.79 (br, 1H), 4.40 (d, J = 5.7 Hz, 2H), 3.88
(s, 2H), 3.78 (s, 3H), 2.94-3.04 (m, 2H), 2.15 (tt, J = 4.2 and
11.4 Hz, 1 H), 2.06 (dt, J = 2.7 and 11.3 Hz, 2H), 1.72-1.88 (m,
4H). 13C NMR (100 MHz, CDCl3): δ 174.9, 159.8, 139.9, 134.3,
133.8, 132.5, 129.7, 128.3, 127.8, 127.2, 125.7, 125.6, 125.0,
124.8, 119.9, 113.3, 112.9, 61.3, 55.2, 53.3, 43.6, 43.3, 29.1. IR
(neat): 3290, 2922, 1644, 1598,1263 cm-1. MS (ESI): m/z 389
[M þ H]þ.
Experimental Section
Chemistry. 1H NMR and 13C NMR spectra were recorded on
Varian Oxford 300 and Bruker Avance 400 spectrometers.
Optical rotations were recorded on a Perkin-Elmer 341 polari-
meter. Anhydrous solvent was obtained as follows: CH2Cl2 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 a 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 in
the different solvent systems. All test compounds showed g95%
purity.
1-[(1-Naphthyl)methyl]-4-[(2-methoxybenzylamino)carbonyl]-
piperidine (7a). The title compound 7a was obtained as described
for compound 7b in 70% yield (viscous liquid). 1H NMR (400
MHz, CDCl3): δ 8.30 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 7.1 Hz,
1H), 7.77 (d, J = 7.1 Hz, 1H), 7.44-7.53 (m, 2H), 7.37-7.43 (m,
2H), 7.21-7.30 (m, 2H), 6.83-6.94 (m, 2H), 5.98 (br s, 1H), 4.43
(d, J = 5.6 Hz, 2H), 3.87 (s, 2H), 3.84 (s, 3H), 2.98 (d, J = 11.2
Hz, 2H), 2.01-2.20 (m, 3H), 1.68-1.84 (m, 4H). 13C NMR (100
MHz, CDCl3): δ 174.6, 157.5, 134.3, 133.8, 132.5, 129.8, 128.8,
128.3, 127.8, 127.2, 126.3, 125.7, 125.6, 125.1, 124.8, 120.7,
110.3, 61.3, 55.3, 53.4, 43.6, 39.3, 29.0. IR (neat): 3305, 1643,
1600, 1242 cm-1. MS (ESI): m/z 389 [M þ H]þ.
1-(tert-Butoxycarbonyl)-4-[(3-methoxybenzylamino)carbonyl]-
piperidine (6b). To a solution of 1-(tert-butoxycarbonyl)piper-
idine-4-carboxylic acid (344 mg, 1.5 mmol) in dry CH2Cl2
(5 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-
drochloride (EDC HCl) (287 mg, 1.5 mmol), 1-hydroxybenzo-
triazole hydrate (HOBt H2O) (203 mg, 1.5 mmol), N-methyl-
3
3
morpholine (NMM) (0.16 mL, 1.5 mmol), and 3-methoxyben-
zylamine (0.13 mL, 1 mmol) were added successively at 23 ꢀC
under argon atmosphere, and the resulting reaction mixture was
stirred for 5 h at the same temperature. The reaction mixture was
quenched with aqueous NaOH solution and extracted with
CH2Cl2. The organic layers were dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (40% EtOAc/
1-[(R)-1-(1-Naphthyl)ethyl]-4,4-bis(methoxycarbonyl)-1,4-
dihydropyridine (12a). A solution of malonate 10 (1.8 g, 5.92
mmol) in 10% hydrochloric acid solution (35 mL) and THF (35
mL) was stirred for 18 h at 23 ꢀC. The solution was neutralized
with powdered sodium hydrogen carbonate, and then 1-(R)-
naphthylmethylamine 11a (1.0 g, 5.84 mmol) in THF (5 mL) was
added. After the mixture was stirred for 16 h at 23 ꢀC, the
aqueous layer was extracted with EtOAc and dried over
Na2SO4. Removal of the solvent afforded the residue, which
was purified by silica gel column chromatography to furnish
1
hexanes) to furnish 6b (327 mg, 94%) as a viscous liquid. H
NMR (400 MHz, CDCl3): δ 7.24 (t, J = 7.6 Hz, 1H), 6.76-6.86
(m, 3H), 5.78 (br, 1H), 4.41 (d, J = 5.6 Hz, 2H), 4.12 (br, 2H),
3.79 (s, 3H), 2.73 (br t, J = 11.2 Hz, 2H), 2.25 (tt, J = 4.0 and
11.6 Hz, 1H), 1.82 (br d, J = 12.0 Hz, 2H), 1.65 (ddd, J = 4.1,
12.2, and 24.8 Hz, 2H), 1.45 (s, 9H). 13C NMR (100 MHz,
CDCl3): δ 174.1, 159.9, 154.6, 139.7, 129.8, 119.9, 113.4, 112.9,
79.6, 55.2, 43.5, 43.4, 28.6, 28.4.
1-(tert-Butoxycarbonyl)-4-[(2-methoxybenzylamino)carbonyl]-
piperidine (6a). The title compound 6a was obtained as described
for compound 1-(tert-butoxycarbonyl)-4-[(3-methoxybenzyla-
mino)carbonyl]piperidine in 92% yield (viscous liquid). 1H
NMR (400 MHz, CDCl3): δ 7.22 (br t, J = 7.2 Hz, 2H),
6.83-6.92 (m, 2H), 6.09 (br, 1H), 4.41 (d, J = 5.8 Hz, 2H),
4.09 (br, 2H), 3.83 (s, 3H), 2.70 (br t, J = 11.1 Hz, 2 H), 2.20 (tt,
J = 3.7 and 11.6 Hz, 1H), 1.77 (br d, J = 12.0 Hz, 2H), 1.59
(ddd, J = 4.4, 12.0, and 24.8 Hz, 2H), 1.43 (s, 9H). 13C NMR
(100 MHz, CDCl3): δ 173.9, 157.5, 154.6, 129.6, 128.8, 126.1,
120.6, 110.3, 79.5, 55.3, 43.2, 39.2, 28.5, 28.3.
compound 12a (1.1 g, 54%) as a colorless oil. Rf = 0.74 (hexane/
1
EtOAc = 1:1). [R]20 -58 (c 1, CHCl3). H NMR (300 MHz,
D
CDCl3): δ 7.90 (d, 1H, J = 7.8 Hz), 7.84 (d, 1H, J = 7.8 Hz),
7.80-7.75 (m, 1H), 7.54-7.40 (m, 4H), 6.21 (d, 2H, J = 8.3 Hz),
5.16 (q, 1H, J = 6.6 Hz), 4.77 (d, 2H, J = 8.3 Hz), 3.69 (s, 6H),
1.67 (d, 3H, J = 6.6 Hz). 13C NMR (75 MHz, CDCl3): δ 171.4,
136.2, 133.7, 130.8, 129.2, 128.7, 128.4, 126.3, 125.5, 124.9,
123.7, 122.8, 95.3, 56.8, 54.0, 52.4, 19.4. IR (neat): 2951, 1736,
1249, 1069 cm-1. MS (EI): m/z 352 [M þ H]þ. HRMS (EI), calcd
for C21H22NO4 352.1549, found 352.1553.
1-[(R)-1-(2-Naphthyl)ethyl]-4,4-bis(methoxycarbonyl)-1,4-
dihydropyridine (12b). The title compound was obtained as
described in compound 12a in 58% yield (colorless oil). Rf =
0.79 (hexane/EtOAc = 1:1). [R]20D þ32 (c 1, CHCl3). 1H NMR
(300 MHz, CDCl3): δ 7.84-7.78 (m, 3H), 7.66 (s, 1H),
7.49-7.43 (m, 2H), 7.33 (dd, 1H, J = 1.5 and 8.7 Hz), 6.21 (d,
2H, J = 8.3 Hz), 4.78 (d, 2H, J = 8.3 Hz), 4.59 (q, 1H, J = 6.9
Hz), 3.72 (s, 6H), 1.64 (d, 3H, J = 6.9 Hz). 13C NMR (75 MHz,
1-[(1-Naphthyl)methyl]-4-[(3-methoxybenzylamino)carbonyl]-
piperidine (7b). To a solution of 1-(tert-butoxycarbonyl)-4-
[(3-methoxybenzylamino)carbonyl]piperidine (100 mg, 0.287

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4975
CDCl3): δ 171.6, 139.2, 133.1, 132.6, 129.6, 128.4, 127.9, 127.7,
127.5, 126.2, 125.9, 124.8, 95.3, 60.4, 54.1, 52.6, 19.5. IR (neat):
2952, 1732, 1253, 1069 cm-1. MS (EI): m/z 292 [M - CO2Me]þ.
HRMS (EI), calcd for C19H18NO2 292.1337, found [M -
CO2Me]þ 292.1345.
1-[(S)-1-(2-Naphthyl)ethyl]-4,4-bis(methoxycarbonyl)-1,4-
dihydropyridine (12c). The title compound was obtained as
described in compound 12a in 54% yield (colorless oil). Rf =
0.73 (hexane/EtOAc = 1:1). [R]20D -32 (c 1, CHCl3). MS (EI):
m/z 351 [M]þ. HRMS (EI), calcd for C21H21NO4 351.1471,
found [M]þ 351.1477.
1-[(S)-1-(1-Naphthyl)ethyl]-4,4-bis(methoxycarbonyl)-1,4-
dihydropyridine (12d). The title compound was obtained as
described in compound 12a in 42% yield (colorless oil). Rf =
0.77 (hexane/EtOAc = 1:1). [R]20D þ57 (c 1, CHCl3). MS (ESI):
m/z 374 [M þ Na]þ. HRMS (ESI), calcd for C21H21NO4Na
374.1368, found 374.1371.
1-(1-Naphthylmethyl)-4,4-bis(methoxycarbonyl)-1,4-dihydro-
pyridine (12e). The title compound was obtained as described in
compound 12a in 39% yield (colorless oil). Rf = 0.82 (hexane/
EtOAc = 1:1). 1H NMR (300 MHz, CDCl3): δ 7.86-7.80 (m,
2H), 7.77 (d, 1H, J = 8.7 Hz), 7.54-7.48 (m, 2H), 7.42 (t, 1H,
J = 8.3 Hz), 7.30 (d, 1H, J = 6.9 Hz), 6.15 (d, 2H, J = 8.3 Hz),
4.82 (d, 2H, J = 8.3 Hz), 4.74 (s, 2H), 3.73 (s, 6H). 13C NMR (75
MHz, CDCl3): δ 171.6, 133.5, 132.6, 131.1, 130.7, 128.7, 128.2,
126.4, 125.8, 125.4, 125.1, 122.5, 95.3, 54.5, 53.7, 52.7. IR (neat):
2951, 1735, 1253, 1067 cm-1. MS (EI): m/z 278 [M - CO2Me]þ.
HRMS (EI), calcd for C18H16NO2 278.1181, found 278.1185.
1-(2-Naphthylmethyl)-4,4-bis(methoxycarbonyl)-1,4-dihydro-
pyridine (12f). The title compound was obtained as described in
compound 12a in 62% yield (colorless oil). Rf = 0.80 (hexane/
EtOAc = 1:1). 1H NMR (300 MHz, CDCl3): δ 7.80-7.77 (m,
3H), 7.60 (s, 1H), 7.48-7.41 (m, 2H), 7.28 (d, 1H, J = 1.8 Hz),
6.16 (d, 2H, J = 8.0 Hz), 4.81 (d, 2H, J = 8.0 Hz), 4.41 (s, 2H),
3.73 (s, 6H). 13C NMR (75 MHz, CDCl3): δ 171.5, 134.9, 133.1,
132.6, 131.2, 128.5, 127.7, 127.5, 126.2, 125.9, 125.8, 124.8, 95.3,
56.9, 53.6, 52.6. IR (neat): 2950, 1731, 1253, 1066 cm-1. MS (EI):
m/z 278 [M - CO2Me]þ. HRMS (EI), calcd for C18H16NO2
278.1181, found 278.1184.
compound 13a in 74% yield (colorless oil). Rf = 0.48 (hexane/
1
EtOAc = 1:1). [R]20 þ23 (c 1, CHCl3). H NMR (400 MHz,
D
CDCl3): δ 7.83-7.80 (m, 3H), 7.72 (s, 1H), 7.53 (dd, 1H, J = 1.3
and 8.7 Hz), 7.49-7.42 (m, 2H), 3.73 (s, 6H), 3.49 (q, 1H, J = 6.7
Hz), 2.57-2.55 (bm, 2H), 2.47-2.42 (m, 2H), 2.24-2.13 (m, 4H),
1.42 (d, 3H, J = 6.7 Hz). 13C NMR (100 MHz, CDCl3): δ 171.7,
142.1, 133.3, 132.7, 128.0, 127.7, 127.5, 125.8, 125.8, 125.4, 64.8,
53.2, 52.5, 47.9, 31.1, 19.6. IR (neat): 2951, 1731, 1250, 1073 cm-1
.
MS (EI): m/z 355 [M]þ. HRMS (EI), calcd for C21H25NO4
355.1784, found 355.1781.
1-[(S)-1-(2-Naphthyl)ethyl]-4,4-bis(methoxycarbonyl)piperi-
dine (13c). The title compound was obtained as described in
compound 13a in 67% yield (colorless oil). Rf = 0.48 (hexane/
EtOAc = 1:1). [R]20D -24 (c 1, CHCl3). MS (EI): m/z 355 [M]þ.
HRMS (EI), calcd for C21H25NO4 355.1784, found 355.1786.
1-[(S)-1-(1-Naphthyl)ethyl]-4,4-bis(methoxycarbonyl)piperi-
dine (13d). The title compound was obtained as described in
compound 13a in 87% yield (colorless oil). Rf = 0.7 (hexane/
EtOAc = 1:1). [R]20D -9 (c 1, CHCl3). MS (ESI): m/z 356 [M þ
H]þ. HRMS (ESI), calcd for C21H26NO4 356.1862, found
356.1865.
1-(1-Naphthylmethyl)-4,4-bis(methoxycarbonyl)piperidine (13e).
The title compound was obtained as described in compound 13a in
60% yield (colorless oil). Rf = 0.70 (hexane/EtOAc = 1:1). H
1
NMR (300 MHz, CDCl3): δ 8.29-8.26 (m, 1H), 7.84-7.81 (m,
1H), 7.77-7.73 (m, 1H), 7.52-7.43 (m, 2H), 7.38-7.36 (m, 2H),
3.84 (s, 2H), 3.72 (s, 6H), 2.48 (t, 4H, J = 5.4 Hz), 2.13 (t, 4H, J =
5.4 Hz). 13C NMR (75 MHz, CDCl3): δ 171.7, 134.1, 133.8, 132.5,
128.3, 127.9, 127.2, 125.6, 125.5, 125.0, 124.8, 61.2, 53.3, 52.5, 50.6,
31.0. IR (neat): 2950, 1732, 1254, 1072 cm-1. MS (EI): m/z 341
[M]þ. HRMS (EI), calcd for C20H23NO4 341.1627, found
341.1630.
1-(2-Naphthylmethyl)-4,4-bis(methoxycarbonyl)piperidine (13f).
The title compound was obtained as described in compound 13a in
94% yield (colorless oil). Rf = 0.48 (hexane/EtOAc = 1:1). H
1
NMR (400 MHz, CDCl3): δ 7.83-7.78 (m, 3H), 7.72 (s, 1H),
7.50-7.42 (m, 3H), 3.74 (s, 6H), 3.61 (s, 2H), 2.48 (bm, 4H), 2.19
(t, 4H, J = 5.5 Hz). 13C NMR (100 MHz, CDCl3): δ 171.7, 135.9,
133.2, 132.7, 127.8, 127.6, 127.6, 127.5, 127.2, 125.9, 125.5, 63.2,
53.2, 52.5, 50.5, 30.9. IR (neat): 2950, 1732, 1254, 1073 cm-1. MS
(EI): m/z 341 [M]þ. HRMS (EI), calcd for C20H23NO4 341.1627,
found [M]þ 341.1626.
1-[1-Methyl-1-(1-naphthyl)ethyl]-4,4-bis(methoxycarbonyl)piperi-
dine (13g). The title compound 13g was obtained as described in
compound 13a in 93% yield (colorless oil). Rf = 0.29 (hexane/
EtOAc = 9:1). 1H NMR (400 MHz, CDCl3):δ9.59 (d, 1H, J = 7.9
Hz), 7.84 (dd, 1H, J = 2.2 and 7.0 Hz), 7.74 (d, 1H, J = 8.0 Hz),
7.52-7.44 (m, 3H), 7.37 (t, 1H, J = 7.7 Hz), 3.74 (s, 6H), 2.63 (bm,
4H), 2.14 (bm, 4H), 1.59 (s, 6H). 13C NMR (100 MHz, CDCl3): δ
171.8, 143.7, 134.9, 132.0, 128.6, 128.2, 128.0, 125.1, 124.7, 124.4,
123.6, 62.2, 53.6, 52.4, 43.4, 31.8, 23.6. IR (neat): 2973, 1735, 1251,
1124, 780 cm-1. MS (ESI): m/z 370 [M þ H]þ. HRMS (ESI), calcd
for C22H28NO4 370.2018, found 370.2013.
1-[1-Methyl-1-(1-naphthyl)ethyl]-4,4-bis(methoxycarbonyl)-
1,4-dihydropyridine (12g). The title compound was obtained as
described in compound 12a in 41% yield (colorless oil). Rf =
1
0.77 (hexane/EtOAc = 1:1). H NMR (300 MHz, CDCl3): δ
8.20-8.16 (m, 1H), 7.89 (d, 1H, J = 7.8 Hz), 7.84-7.80 (m, 1H),
7.77 (d, 1H, J = 7.8 Hz), 7.52-7.36 (m, 4H), 6.27 (d, 2H, J = 8.1
Hz), 4.77 (d, 2H, J = 8.1 Hz), 3.69 (s, 6H), 1.77 (s, 6H). 13C
NMR (75 MHz, CDCl3): δ 171.6, 140.1, 134.7, 130.5, 129.1,
129.0, 127.7, 126.1, 126.0, 125.3, 124.7, 124.0, 96.0, 61.9, 53.8,
52.5, 28.7. IR (neat): 2951, 1736, 1252, 1062 cm-1. MS (ESI):
m/z 388 [M þ Na]þ. HRMS (ESI), calcd for C22H23NO4Na
388.1525, found 388.1529.
1-[(R)-1-(1-Naphthyl)ethyl]-4,4-bis(methoxycarbonyl)pipe-
ridine (13a). To a stirred solution of dihydropyridine 12a (1.1 g, 3.1
mmol) in EtOAc (75 mL) was added platinum(IV) oxide (100 mg),
and the mixture was allowed to stir for 2 h at 23 ꢀC under H2
atmosphere. The mixture was filtered through Celite pad, and the
filtrate was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography to furnish compound
1-[(R)-1-(1-Naphthyl)ethyl]-4-methoxycarbonylpiperidine (14a).
To a stirred solution of dimethyl malonate 13a (917 mg, 2.58
mmol) in DMF (25 mL) was added sodium cyanide (190 mg, 3.88
mmol) at 23 ꢀC, and the mixture was allowed to stir for 16 h at
reflux temperature. The mixture was diluted with EtOAc and
washed with water. The organic layer was dried over Na2SO4,
filtered, and concentrated. The residue was purified by silica gel
column chromatography to furnish compound 14a (704 mg, 92%)
13a (942 mg, 88%) as a colorless oil. Rf = 0.7 (hexane/EtOAc =
1
1:1). [R]20 þ9 (c 1, CHCl3). H NMR (300 MHz, CDCl3): δ
D
8.44-8.41 (m, 1H), 7.85-7.81 (m, 1H), 7.73 (d, 1H, J = 8.1 Hz),
7.56 (d, 1H, J = 6.9 Hz), 7.50-7.39 (m, 3H), 4.03 7.73 (q, 1H, J =
6.3 Hz), 3.72 (s, 6H), 2.58-2.56 (m, 2H), 2.47-2.40 (m, 2H),
2.20-2.04 (m, 4H), 1.44 (d, 3H, J = 6.3 Hz). 13C NMR (75 MHz,
CDCl3): δ 171.8, 140.7, 134.0, 131.5, 128.6, 127.3, 125.4, 125.3,
125.2, 124.5, 124.1, 61.7, 53.3, 52.4, 47.9, 31.2, 18.7. IR (neat): 2952,
1734, 1251 cm-1.MS(ESI):m/z356 [M þ H]þ.HRMS(ESI),calcd
for C21H26NO4 356.1862, found 356.1866.
as a colorless oil. Rf = 0.56 (CH2Cl2/MeOH = 9:1). [R]20 þ2
D
(c 1, CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.48 (dd, 1H, J =
1.2 and 7.6 Hz), 7.87 (d, 1H, J = 7.1Hz), 7.76 (d, 1H, J = 8.1 Hz),
7.60 (d, 1H, J = 7.1 Hz), 7.53-7.43 (m, 3H), 4.12 (q, 1H, J = 6.7
Hz), 3.68 (s, 3H), 3.17-3.15 (m, 1H), 2.87-2.84 (m, 1H),
2.35-2.27 (m, 1H), 2.10 (ddd, 2H, J = 2.6, 11.2, and 19.8 Hz),
1.97-1.92 (m, 1H), 1.83-1.71 (m, 3H), 1.49 (d, 3H, J = 6.7 Hz).
13C NMR (100 MHz, CDCl3): δ 175.8, 140.8, 134.0, 131.6, 128.6,
127.2, 125.4, 125.3, 125.3, 124.5, 124.2, 61.6, 51.5, 49.1, 41.3, 28.7,
1-[(R)-1-(2-Naphthyl)ethyl]-4,4-bis(methoxycarbonyl)piperi-
dine (13b). The title compound was obtained as described in

4976 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Ghosh et al.
28.6, 18.6. IR (neat): 2950, 1732, 1169, 780 cm-1. MS (EI):m/z 297
[M]þ. HRMS (EI), calcd for C19H23NO2 297.1729, found
297.1730.
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
(EDCI) (138.0 mg, 0.72 mmol) and 1-hydroxybenzotriazole
hydrate (HOBT) (97.3 mg, 0.72 mmol) in dry CH2Cl2/DMF
(9:1) (8 mL) were added 2-methoxybenzylamine 5a (52.7 μL,
0.40 mmol) and diisopropylethylamine (0.38 mL, 2.2 mmol) at
0 ꢀC under argon atmosphere. The mixture 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, filtered, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography to
furnish compound 15a (143 mg, 99%) as a white amorphous
solid. Rf = 0.42 (CH2Cl2/MeOH = 9:1). [R]20D -2 (c 1, CHCl3).
1H NMR (400 MHz, CDCl3): δ 8.46 (d, 1H, J = 7.8 Hz),
7.86-7.84 (m, 1H), 7.74 (d, 1H, J = 8.1 Hz), 7.58 (d, 1H, J =
7.0 Hz), 7.51-7.41 (m, 3H), 7.23 (d, 1H, J = 7.3 Hz), 6.90 (t, 1H,
J = 7.3 Hz), 6.85 (d, 1H, J = 8.4 Hz), 6.17 (bt, 1H, J = 5.8 Hz),
4.44 (d, 2H, J = 5.8 Hz), 4.10 (q, 1H, J = 6.6 Hz), 3.81 (s, 3H),
3.23-3.20 (m, 1H), 2.87-2.85 (m, 1H), 2.12-1.95 (m, 3H),
1.89-1.68 (m, 4H), 1.47 (d, 3H, J = 6.6 Hz). 13C NMR (100
MHz, CDCl3): δ 174.9, 157.5, 140.9, 134.1, 131.7, 129.6, 128.7,
128.7, 127.3, 126.5, 125.5, 125.4, 125.3, 124.5, 124.3, 120.7, 110.3,
61.7, 55.3, 52.0, 49.2, 43.7, 39.1, 29.3, 18.7. IR (neat): 3302, 2938,
1650, 1243, 753 cm-1. MS(ESI):m/z403 [M þ H]þ. HRMS(ESI),
calcd for C26H31N2O2 403.2386, found 403.2388.
1-[(R)-1-(1-Naphthyl)ethyl]-4-(3-methoxybenzylamino)carbo-
nylpiperidine (15b). The title compound was obtained as de-
scribed in compound 15a in 95% yield (white amorphous solid).
Rf = 0.49 (CH2Cl2/MeOH = 9:1). [R]20D -2 (c 1, CHCl3). 1H
NMR (400 MHz, CDCl3): δ 8.45 (d, 1H, J = 7.7 Hz), 7.86-7.84
(m, 1H), 7.74 (d, 1H, J = 8.1 Hz), 7.58 (d, 1H, J = 7.1 Hz),
7.51-7.41 (m, 3H), 6.83-6.78 (m, 3H), 6.12 (bt, 1H, J = 5.7
Hz), 4.37 (d, 2H, J = 5.7 Hz), 4.10 (q, 1H, J = 6.6 Hz), 3.76 (s,
3H), 3.23-3.20 (m, 1H), 2.90-2.85 (m, 1H), 2.14-1.95 (m, 3H),
1.89-1.69 (m, 4H), 1.47 (d, 3H, J = 6.6 Hz). 13C NMR (100
MHz, CDCl3): δ 175.2, 159.8, 140.7, 140.1, 134.0, 131.6, 129.6,
128.6, 127.2, 125.4, 125.3, 125.3, 124.5, 124.2, 119.8, 113.2,
112.7, 61.6, 55.1, 51.8, 49.1, 43.5, 43.2, 29.3, 18.6. IR (neat):
3293, 2937, 1644, 1263, 781 cm-1. MS (EI): m/z 402 [M]þ.
HRMS (EI), calcd for C26H30N2O2 402.2307, found 402.2303.
1-[(R)-1-(1-Naphthyl)ethyl]-4-(4-methoxybenzylamino)carbonyl-
piperidine (15c). The title compound was obtained as described in
compound 15a in 88% yield (white amorphous solid). Rf = 0.56
(CH2Cl2/MeOH = 9:1). [R]20D -2 (c 1, CHCl3). 1H NMR (400
MHz, CDCl3): δ 8.45 (d, 1H, J = 7.7 Hz), 7.86-7.84 (m, 1H),
7.74 (d, 1H, J = 8.1 Hz), 7.57 (d, 1H, J = 7.1 Hz), 7.51-7.41 (m,
3H), 7.27 (d, 2H, J = 8.5 Hz), 6.84 (d, 2H, J = 8.5 Hz), 5.97 (bt,
1H, J = 5.6 Hz), 4.33 (d, 2H, J = 5.6 Hz), 4.10 (q, 1H, J = 6.7
Hz), 3.77 (s, 3H), 3.23-3.20 (m, 1H), 2.89-2.86 (m, 1H),
2.13-1.95 (m, 3H), 1.89-1.68 (m, 4H), 1.47 (d, 3H, J = 6.7
Hz). 13C NMR (100 MHz, CDCl3): δ 175.0, 158.9, 140.7, 134.0,
131.6, 130.5, 129.0, 128.6, 127.2, 125.4, 125.3, 125.3, 124.5,
124.2, 114.0, 61.6, 55.2, 51.8, 49.1, 43.5, 42.8, 29.3, 18.6. IR
(neat): 3292, 2932, 1513, 1644, 1249, 781 cm-1. MS (EI): m/z 402
[M]þ. HRMS (EI), calcd for C26H30N2O2 402.2307, found
402.2299.
1-[(R)-1-(2-Naphthyl)ethyl]-4-methoxycarbonylpiperidine (14b).
The title compound was obtained as described incompound 14a in
78% yield (colorless oil). Rf = 0.43 (CH2Cl2/MeOH = 9:1).
[R]20 þ16 (c 1, CHCl3). 1H NMR (400 MHz, CDCl3): δ
D
7.84-7.80 (m, 3H), 7.72 (s, 1H), 7.53 (dd, 1H, J = 1.1 and 8.4
Hz), 7.50-7.43 (m, 2H), 3.67 (s, 3H), 3.57 (q, 1H, J = 6.8 Hz),
3.08-3.06 (m, 1H), 2.86-2.83 (m, 1H), 2.29-2.22 (m, 1H),
2.09-1.91 (m, 3H), 1.87-1.71 (m, 3H), 1.45 (d, 3H, J = 6.8
Hz). 13C NMR (100 MHz, CDCl3): δ 175.7, 141.7, 133.3, 132.7,
127.8, 127.7, 127.5, 126.0, 125.9, 125.8, 125.4, 64.7, 51.5, 50.5, 49.6,
41.2, 28.5, 19.3. IR (neat): 2949, 1732, 1258, 1172 cm-1. MS (EI):
m/z 297 [M]þ. HRMS (EI), calcd for C19H23NO2 297.1729, found
[M]þ 297.1732.
1-[(S)-1-(2-Naphthyl)ethyl]-4-methoxycarbonylpiperidine (14c).
The title compound was obtained as described incompound 14a in
90% yield (colorless oil). Rf = 0.47 (CH2Cl2/MeOH = 9:1).
[R]20 -15 (c 1, CHCl3). MS (EI): m/z 297 [M]þ. HRMS (EI),
D
calcd for C19H23NO2 297.1729, found 297.1731.
1-[(S)-1-(1-Naphthyl)ethyl]-4-methoxycarbonylpiperidine (14d).
The title compound was obtained as described incompound 14a in
76% yield (colorless oil). Rf = 0.57 (CH2Cl2/MeOH = 9:1).
[R]20D -2 (c 1, CHCl3). MS (EI): m/z 297 [M]þ. HRMS (EI), calcd
for C19H23NO2 297.1729, found 297.1729.
1-(1-Naphthylmethyl)-4-methoxycarbonylpiperidine (14e). The
title compound was obtained as described in compound 14a in
38% yield (colorless oil). Rf = 0.53 (CH2Cl2/MeOH = 9:1). 1H
NMR (400 MHz, CDCl3): δ 8.31 (d, 1H, J = 7.8 Hz), 7.86 (dd,
1H, J = 1.6 and 7.1 Hz), 7.78 (d, 1H, J = 7.1 Hz), 7.53-7.47 (m,
2H), 7.44-7.39 (m, 2H), 3.89 (s, 2H), 3.68 (s, 3H), 2.94-2.88 (m,
2H), 2.37-2.30 (m, 1H), 2.12 (dt, 2H, J = 1.6 and 11.2 Hz),
1.90-1.86 (m, 2H), 1.81-1.72 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ 175.7, 134.2, 133.7, 132.5, 128.3, 127.8, 127.2, 125.6,
125.5, 125.0, 124.7, 61.3, 53.1, 51.5, 41.1, 28.3. IR (neat): 2949,
1736, 1167, 788 cm-1. MS (EI): m/z 283 [M]þ. HRMS (EI), calcd
for C18H21NO2 283.1572, found 283.1569.
1-(2-Naphthylmethyl)-4-methoxycarbonylpiperidine (14f). The
title compound was obtained as described in compound 14a in
47% yield (colorless oil). Rf = 0.44 (CH2Cl2/MeOH = 9:1). 1H
NMR (400 MHz, CDCl3): δ 7.84-7.80 (m, 3H), 7.73 (s, 1H),
7.51-7.43 (m, 3H), 3.68 (s, 3H), 3.65 (s, 2H), 2.93-2.88 (m, 2H),
2.36-2.28 (m, 1H), 2.08 (dt, 2H, J = 2.2 and 11.4 Hz), 1.94-1.75
(m, 4H). 13C NMR (100 MHz, CDCl3): δ 175.6, 135.9, 133.2,
132.7, 127.8, 127.6, 127.6, 127.5, 127.3, 125.9, 125.5, 63.3, 52.9,
51.6, 41.0, 28.2. IR (neat): 2948, 1736, 1167 cm-1. MS (ESI): m/z
284 [M þ H]þ. HRMS (ESI), calcd for C18H22NO2 284.1651,
found 284.1652.
1-[1-Methyl-1-(1-naphthyl)ethyl]-4-methoxycarbonylpiperidine
(14g). The title compound was obtained as described in com-
pound 14a in 87% yield (colorless oil). Rf = 0.43 (hexane/
1
EtOAc = 9:1). H NMR (400 MHz, CDCl3): δ 9.63-9.60 (m,
1H), 7.87-7.84 (m, 1H), 7.76 (d, 1H, J = 8.0 Hz), 7.51-7.46 (m,
3H), 7.39 (t, 1H, J = 7.8 Hz), 3.69 (s, 3H), 2.96 (bs, 2H), 2.34-2.28
(m, 3H), 1.84-1.75 (bm, 4H), 1.61 (s, 6H). 13C NMR (100 MHz,
CDCl3): δ 175.9, 144.1, 134.8, 132.0, 128.6, 128.4, 127.9, 125.1,
124.7, 124.4, 123.5, 62.3, 51.5, 45.9, 41.9, 29.2, 22.7. IR (neat): 2950,
1736, 1171, 780 cm-1. MS (EI): m/z 311 [M]þ. HRMS (EI), calcd
for C20H25NO2 311.1885, found 311.1891.
1-[(R)-1-(2-Naphthyl)ethyl]-4-(3-methoxybenzylamino)carbo-
nylpiperidine (15d). The title compound was obtained as de-
scribed in compound 15a in 94% yield (white amorphous solid).
Rf = 0.43 (CH2Cl2/MeOH = 9:1). [R]20D þ10 (c 1, CHCl3). 1H
NMR (400 MHz, CDCl3): δ 7.82-7.79 (m, 3H), 7.70 (s, 1H),
7.52 (dd, 1H, J = 1.2 and 8.4 Hz), 7.49-7.42 (m, 2H), 7.22 (t,
1H, J = 7.6 Hz), 6.83-6.79 (m, 3H), 5.95 (bt, 1H, J = 5.7 Hz),
4.38 (d, 2H, J = 5.7 Hz), 3.77 (s, 3H), 3.56 (q, 1H, J = 6.7 Hz),
3.16-3.14 (m, 1H), 2.90-2.88 (m, 1H), 2.11-1.91 (m, 3H),
1.89-1.70 (m, 4H), 1.44 (d, 3H, J = 6.7 Hz). 13C NMR (100
MHz, CDCl3): δ 175.0, 159.8, 141.7, 140.0, 133.2, 132.6, 129.6,
127.8, 127.7, 127.5, 125.9, 125.9, 125.8, 125.4, 119.8, 113.2,
112.8, 64.7, 55.1, 50.8, 49.7, 43.4, 43.2, 29.1, 19.3. IR (neat):
3296, 2932, 1645, 1264 cm-1. MS (ESI): m/z 403 [M þ H]þ.
HRMS (ESI), calcd for C26H31N2O2 403.2386, found 403.2390.
1-[(R)-1-(1-Naphthyl)ethyl]-4-(2-methoxybenzylamino)carbonyl-
piperidine (15a). To a stirred solution of ester 14a (106 mg, 0.36
mmol) inTHF/MeOH/H2O(3:1:1) (8mL) wasaddedLiOH H2O
3
(22.4 mg, 0.53 mmol) at 0 ꢀC, and the mixture was allowed to stir
for 16 h at 23 ꢀC. The mixture was concentrated, and to it was
added a saturated NaHCO3 solution. The mixture was extracted
with Et2O. The aqueous layer was adjusted to pH 2 with 10% HCl
solution and extracted with EtOAc. The organic layers were dried
over Na2SO4, filtered, and concentrated to give the correspond-
ing acid as a colorless oil. To a solution of acid (0.36 mmol),

Article
1-[(R)-1-(2-Naphthyl)ethyl]-4-(2-methoxybenzylamino)carbo-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4977
1040 cm-1. MS (ESI): m/z 403 [M þ H]þ. HRMS (ESI), calcd for
C25H27N2O3 403.2022, found 403.2025.
nylpiperidine (15e). The title compound was obtained as de-
scribed in compound 15a in 98% yield (white amorphous solid).
Rf = 0.47 (CH2Cl2/MeOH = 9:1). [R]20D þ12 (c 1, CHCl3). 1H
NMR (400 MHz, CDCl3): δ 7.82-7.79 (m, 3H), 7.70 (s, 1H),
7.51 (d, 1H, J = 8.4 Hz), 7.49-7.43 (m, 2H), 7.28-7.24 (m, 2H),
6.93-6.85 (m, 2H), 6.02 (bt, 1H, J = 5.7 Hz), 4.43 (d, 2H, J =
5.7 Hz), 3.84 (s, 3H), 3.57 (q, 1H, J = 6.7 Hz), 3.16-3.14 (m,
1H), 2.90-2.88 (m, 1H), 2.08-1.86 (m, 4H), 1.84-1.64 (m, 3H),
1.45 (d, 3H, J = 6.7 Hz). 13C NMR (100 MHz, CDCl3):
δ 174.6, 157.5, 141.6, 133.2, 132.6, 129.7, 129.0, 128.8,
128.2, 127.8, 127.7, 127.5, 126.3, 126.0, 125.8, 125.4, 120.6,
110.2, 64.7, 55.2, 50.8, 49.8, 43.5, 39.2, 29.1, 19.3. IR (neat):
3306, 2933, 1645, 1243, 751 cm-1. MS (ESI): m/z 403 [M þ
H]þ. HRMS (ESI), calcd for C26H31N2O2 403.2386, found
403.2394.
1-[1-Methyl-1-(1-naphthyl)ethyl]-4-[3,4-(methylenedioxy)benzyl-
amino]carbonylpiperidine (15k). The title compound was obtained
as described in compound 15a in 90% yield (white amorphous
1
solid). Rf = 0.51 (hexane/EtOAc = 1:1). H NMR (400 MHz,
CDCl3): δ 9.56-9.53 (m, 1H), 7.82-7.79 (m, 1H), 7.72 (d, 1H, J =
7.9 Hz), 7.46-7.41 (m, 3H), 7.36 (t, 1H, J= 7.6 Hz), 6.73-6.71 (m,
2H), 6.67 (dd, 1H, J = 1.1 and 8.2 Hz), 6.00 (bt, 1H, J = 5.7 Hz),
5.90 (s, 2H), 4.29 (d, 2H, J = 5.7 Hz), 2.92 (bs, 2H), 2.21 (bm, 2H),
2.09-2.01 (m, 1H), 1.72 (bm, 4H), 1.56 (s, 6H). 13C NMR (100
MHz, CDCl3): δ 175.2, 147.8, 146.8, 144.0, 134.8, 132.4, 132.0,
128.5, 128.3, 127.9, 125.1, 124.7, 124.4, 123.5, 120.8, 108.2, 100.9,
62.3, 46.1, 44.0, 43.1, 29.8, 24.7. IR (neat): 3294, 2974, 1642, 1490,
1253, 1041, 780 cm-1. MS (ESI): m/z 311 [M þ H]þ. HRMS (ESI),
calcd for C27H31N2O3 431.2335, found 431.2330.
1-[(S)-1-(2-Naphthyl)ethyl]-4-(3-methoxybenzylamino)carbo-
nylpiperidine (15f). The title compound was obtained as de-
scribed in compound 15a in 83% yield (white amorphous solid).
Rf = 0.37 (CH2Cl2/MeOH = 9:1). [R]20D -10 (c 1, CHCl3). MS
(ESI): m/z 403 [M þ H]þ. HRMS (ESI), calcd for C26H31N2O2
403.2386, found 403.2392.
1-[1-(1-Naphthyl)ethyl]-4-tert-butoxycarbonylpiperazine (18).
To a solution of N-Boc-piperazine 16 (107 mg, 0.58 mmol) and
1-acetonaphthone 17 (0.10 mL, 0.69 mmol) in MeOH/AcOH
(50:1) (4 mL) was added sodium cyanoborohydride (38 mg, 0.58
mmol) at 0 ꢀC. The mixture was allowed to stir for 48 h at 23 ꢀC.
The reaction was quenched with saturated NaHCO3 solution,
and the organic layer was extracted with CH2Cl2. The organic
layer was dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography to furnish compound 18 (46 mg, 24%) as a
colorless oil. Rf = 0.75 (hexane/EtOAc = 1:1). 1H NMR (300
MHz, CDCl3): δ 8.42 (d, 1H, J = 7.2 Hz), 7.86-7.83 (m, 1H),
7.74 (d, 1H, J = 10.8 Hz), 7.57 (d, 1H, J = 7.2 Hz), 7.50-7.39
(m, 3H), 4.09 (q, 1H, J = 6.6 Hz), 3.45-3.33 (m, 4H), 2.53 (bm,
2H), 2.42-2.35 (m, 2H), 1.47 (d, 3H, J = 6.6 Hz), 1.44 (s, 9H).
13C NMR (75 MHz, CDCl3): δ 154.7, 140.1, 134.0, 131.5, 128.7,
127.4, 125.5, 125.3, 125.3, 124.6, 124.0, 79.4, 61.5, 50.5, 43.8,
28.4, 18.7.
1-[(R)-1-(1-Naphthyl)ethyl]-4-[3,4-(methylenedioxy)benzyl-
amino]carbonylpiperidine (15g). The title compound was ob-
tained as described in compound 15a in 93% yield (white
amorphous solid). Rf = 0.47 (CH2Cl2/MeOH = 9:1). [R]20
D
1
-2 (c 1, CHCl3). H NMR (400 MHz, CDCl3): δ 8.45 (d, 1H,
J = 7.5 Hz), 7.86-7.83 (m, 1H), 7.73 (d, 1H, J = 8.1 Hz), 7.56
(d, 1H, J = 6.9 Hz), 7.51-7.40 (m, 3H), 6.73-6.66 (m, 3H), 6.20
(bt, 1H, J = 5.7 Hz), 5.89 (s, 2H), 4.29 (d, 2H, J = 5.7 Hz), 4.08
(q, 1H, J = 6.7 Hz), 3.22-3.19 (m, 1H), 2.88-2.85 (m, 1H),
2.13-1.94 (m, 3H), 1.87-1.67 (m, 4H), 1.46 (d, 3H, J = 6.7 Hz).
13C NMR (100 MHz, CDCl3): δ 175.2, 147.8, 146.8, 140.7,
134.0, 132.4, 131.6, 128.6, 127.2, 125.4, 125.3, 125.3, 124.5,
124.2, 120.8, 108.2, 100.9, 61.6, 51.8, 49.1, 43.5, 43.0, 29.3,
1-[1-(1-Naphthyl)ethyl]-4-(4-methoxybenzylamino)carbonyl-
piperazine (20). To a solution of Boc 18 (32 mg, 0.094 mmol) in
dry CH2Cl2 (2 mL) was added trifluoroacetic acid (0.3 mL) at
0 ꢀC. The mixture was allowed to stir for 1 h at 23 ꢀC and then
was concentrated under reduced pressure. To the residue was
added toluene, and the mixture was concentrated under re-
duced pressure to give crude compound 19. To a solution of
1,10-carbonyldiimidazole (18.6 mg, 0.11 mmol) in dry CH2Cl2
(2 mL) was added dropwise 4-methoxybenzylamine 5c
(15.7 μL, 0.12 mmol) at 0 ꢀC under argon atmosphere, and
the mixture was allowed to stir for 4 h at 23 ꢀC. The mixture
was added dropwise to a solution of 19 in dry CH2Cl2 (1 mL).
The mixture was allowed to stir for 24 h at 23 ꢀC and then was
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography to furnish compound 20
(34 mg, 90%) as a white amorphous solid, Rf = 0.57 (CH2Cl2/
MeOH = 9:1). 1H NMR (400 MHz, CDCl3): δ 8.32 (d, 1H, J =
7.5 Hz), 7.81-7.78 (m, 1H), 7.69 (d, 1H, J = 8.1 Hz), 7.52 (d,
1H, J = 7.1 Hz), 7.44-7.36 (m, 3H), 7.13 (d, 2H, J = 8.6 Hz),
6.78 (d, 2H, J = 8.6 Hz), 4.22 (s, 2H), 4.07 (q, 1H, J = 6.6 Hz),
3.72 (s, 3H), 3.34-3.21 (m, 4H), 2.55-2.50 (m, 2H), 2.39-2.34
(m, 2H), 1.42 (d, 3H, J = 6.6 Hz). 13C NMR (100 MHz,
CDCl3): δ 160.1, 160.0, 159.4, 141.1, 135.4, 132.9, 130.2, 130.1,
130.0, 128.9, 127.0, 126.8, 126.0, 125.2, 115.2, 62.6, 56.6, 51.7,
18.6. IR (neat): 3294, 2924, 1644, 1489, 1252, 1040, 781 cm-1
.
MS (ESI): m/z 417 [M þ H]þ. HRMS (ESI), calcd for
C26H29N2O3 417.2178, found 417.2178.
1-[(S)-1-(1-Naphthyl)ethyl]-4-[3,4-(methylenedioxy)benzyl-
amino]carbonylpiperidine (15h). The title compound was ob-
tained as described in compound 15a in 80% yield (white
amorphous solid). Rf = 0.56 (CH2Cl2/MeOH = 9:1). [R]20
D
þ2 (c 1, CHCl3). MS (EI): m/z 417 [M þ H]þ. HRMS (EI),
calcd for C26H29N2O3 417.2178, found 417.2173.
1-(1-Naphthylmethyl)-4-[3,4-(methylenedioxy)benzylamino]-
carbonylpiperidine (15i). The title compound was obtained as
described in compound 15a in >99% yield (white amorphous
solid). Rf = 0.48 (CH2Cl2/MeOH = 9:1). 1H NMR (400 MHz,
CDCl3): δ 8.30-8.28 (m, 1H), 7.84 (d, 1H, J = 7.2 Hz), 7.77 (d,
1H, J = 7.8 Hz), 7.52-7.45 (m, 2H), 7.41-7.37 (m, 2H),
6.74-6.67 (m, 3H), 5.91 (bm, 1H), 5.91 (s, 2H), 4.30 (d, 2H,
J = 5.7 Hz), 3.87 (s, 2H), 2.99-2.96 (m, 2H), 2.35-2.29 (m,
1H), 2.18-2.01 (m, 3H), 1.88-1.72 (m, 3H). 13C NMR (100
MHz, CDCl3): δ 174.9, 147.8, 146.8, 134.1, 133.7, 132.5, 132.2,
128.3, 127.8, 127.2, 126.0, 125.6, 125.5, 125.0, 124.7, 120.9,
108.2, 101.0, 61.2, 53.3, 43.4, 43.1, 28.9. IR (neat): 3307,
2924, 1645, 1490, 1252, 1040 cm-1. MS (ESI): m/z 403 [M þ
H]þ. HRMS (ESI), calcd for C25H27N2O3 403.2022, found
403.2025.
1-(2-Naphthylmethyl)-4-[3,4-(methylenedioxy)benzylamino]-
carbonylpiperidine (15j). The title compound was obtained as
described in compound 15a in 88% yield (white amorphous
solid). Rf = 0.42 (CH2Cl2/MeOH = 9:1). 1H NMR (400 MHz,
CDCl3): δ 7.82-7.78 (m, 3H), 7.72 (s, 1H), 7.49-7.42 (m, 3H),
6.74-6.68 (m, 3H), 6.01 (t, 1H, J = 5.6 Hz), 5.91 (s, 2H), 4.31 (d,
2H, J = 5.6 Hz), 3.63 (s, 2H), 2.97-2.94 (m, 2H), 2.15-2.07 (m,
1H), 2.04-1.98 (m, 2H), 1.83-1.77 (m, 4H). 13C NMR (100
MHz, CDCl3): δ 174.9, 147.8, 146.8, 135.9, 132.7, 132.3, 127.8,
127.6, 127.6, 127.4, 127.3, 125.9, 125.5, 120.9, 108.2, 101.0, 63.2,
53.1, 43.3, 43.1, 28.9. IR (neat): 3293, 2923, 1643, 1490, 1252,
45.4, 45.2, 20.0. IR (neat): 3340, 2925, 1618, 1512, 1248 cm-1
.
MS (ESI): m/z 404 [M þ H]þ. HRMS (ESI), calcd for C25H30-
N3O2 404.2338, found 404.2336.
Molecular Modeling. Computational analyses utilized the
Sybyl 8.1 suite (Tripos, Inc.) and the GOLD docking program
(CCDC) following previously described protocols.10
X-ray Crystallography. The complex of inhibitor 15g with
purified PLpro was formed prior to crystallization by incubat-
ing 10 mg/mL PLpro (in 20 mM Tris, pH 7.5, 10 mM DTT) with
2 mM 15g at 4 ꢀC for 16 h. Diffraction-quality crystals grew
from a sitting drop containing 5 mg/mL PLpro, 1 mM 15g, 1 M
(NH4)2SO4, 50 mM MES, pH 6.5, and 2.5% PEG 400. Crystals

4978 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Ghosh et al.
were flash-frozen in liquid nitrogen and then transferred into a
dry nitrogen stream at 100 K for X-ray data collection. The data
set of the complex was collected at the Southeast Regional
Collaborative Access Team (SER-CAT) beamline at the Ad-
vanced Photon Source, Argonne National Laboratory. Data
were processed and scaled using the HKL2000 program suite.
Crystals belonged to the space group C2, with two monomers in
the asymmetric unit. The inhibitor-complexed structure was
X.-N.; Chen, S.-J.; Chen, Z.; Hao, P.; Tang, H.; Ren, S.-X.; Zhong,
Y.; Guo, Z.-M.; Liu, Q.; Miao, Y.-G.; Kong, X.-Y.; He, W.-Z.; Li,
Y.-X.; Wu, C.-I.; Zhao, G.-P.; Chiu, R. W. K.; Chim, S. S. C.;
Tong, Y.-K.; Chan, P. K. S.; Tam, J. S.; Lo, Y. M. D. Molecular
evolution of the SARS-coronavirus during the course of the SARS
epidemic in China. Science 2004, 303, 1666–1669.
(7) Baker, S. C. Coronaviruses: Molecular Biology. In Encyclopedia of
Virology, 3rd ed.; Mahy, B. W. J., Van Regenmortel, M. H. V., Eds.;
Academic Press: Amsterdam, 2008; Vol. 1, pp 554-562.
(8) Ghosh, A. K.; Xi, K.; Johnson, M. E.; Baker, S. C.; Mesecar, A. D.
Progress in anti-SARS coronavirus chemistry, biology and che-
motherapy. Annu. Rep. Med. Chem. 2006, 41, 183–196. (b) Yang, H.;
Bartlam, M.; Rao, Z. Drug design targeting the main protease, the
Achilles' heel of coronaviruses. Curr. Pharm. Des. 2006, 12, 4573–
4590.
˚
solved to 2.63 A by molecular replacement using the SARS-
CoV PLpro apoenzyme structure (PDB entry 2FE8) as a search
model in the AMoRe program of the CCP4 suite. Manual model
building was performed using Wincoot, and iterative rounds
of positional and B-factor refinement and map building were
performed using CNS. The structure was deposited under PDB
code 3MJ5.
SARS-CoV Antiviral and PLpro Inhibition Assays. SARS-
CoV antiviral assays and PLpro inhibition assays were per-
formed as previously described.10
(9) (a) Ghosh, A. K.; Xi, K.; Grum-Tokars, V.; Xu, X.; Ratia, K.; Fu,
W.; Houser, K. V.; Baker, S. C.; Johnson, M. E.; Mesecar, A. D.
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
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Acknowledgment. This work was supported by a National
Institutes of Health ResearchGrant P01AI060915 (“Develop-
ment of Novel Protease Inhibitors as SARS Therapeutics”).
The authors gratefully acknowledge the synchrotron beam-
line personnel atAdvanced Photon Source (SER-CAT) 22-ID
and 22-BM beamlines. Use of the Advanced Photon Source
was supported by the U.S. Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Contract No.
W-31-109-Eng-38.
Supporting Information Available: HPLC and HRMS data
of inhibitors. This material is available free of charge via the
Internet at http://pubs.acs.org.
(11) (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 papain-
like protease domain of the severe acute respiratory syndrome
coronavirus. J. Biol. Chem. 2007, 282, 32208–32221. (b) Ratia, K.;
Saikatendu, K. S.; Santarsiero, B. D.; Barretto, N.; Baker, S. C.;
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