X-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases

Article abstract of DOI:10.1021/jm401712t

Structure-guided design was used to generate a series of noncovalent inhibitors with nanomolar potency against the papain-like protease (PLpro) from the SARS coronavirus (CoV). A number of inhibitors exhibit antiviral activity against SARS-CoV infected Ve

Full text of DOI:10.1021/jm401712t

Article
pubs.acs.org/jmc
X‑ray Structural and Biological Evaluation of a Series of Potent and
Highly Selective Inhibitors of Human Coronavirus Papain-like
Proteases
Yahira M. Baez-Santos,† Scott J. Barraza,‡ Michael W. Wilson,‡ Michael P. Agius,‡ Anna M. Mielech,§
́
Nicole M. Davis,† Susan C. Baker,§ Scott D. Larsen,*,‡ and Andrew D. Mesecar*,†
†Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, Indiana 47907, United States
‡Vahlteich Medicinal Chemistry Core and Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann
Arbor, Michigan 48109, United States
§Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois 60153,
United States
ABSTRACT: Structure-guided design was used to generate a series of noncovalent
inhibitors with nanomolar potency against the papain-like protease (PLpro) from
the SARS coronavirus (CoV). A number of inhibitors exhibit antiviral activity
against SARS-CoV infected Vero E6 cells and broadened specificity toward the
homologous PLP2 enzyme from the human coronavirus NL63. Selectivity and
cytotoxicity studies established a more than 100-fold preference for the coronaviral
enzyme over homologous human deubiquitinating enzymes (DUBs), and no
significant cytotoxicity in Vero E6 and HEK293 cell lines is observed. X-ray
structural analyses of inhibitor-bound crystal structures revealed subtle differences
between binding modes of the initial benzodioxolane lead (15g) and the most
potent analogues 3k and 3j, featuring a monofluoro substitution at para and meta
positions of the benzyl ring, respectively. Finally, the less lipophilic bis(amide) 3e
and methoxypyridine 5c exhibit significantly improved metabolic stability and are
viable candidates for advancing to in vivo studies.
Reminiscent of the initial stages of SARS-CoV pandemic, global
travel has contributed to the spread of MERS coronavirus, with
a total of 178 laboratory-confirmed cases and a CFR of 43%.6
The infected individuals display SARS-like symptoms, including
a severe respiratory infection (SRI), and sometimes exhibit an
acute renal failure which is a unique signature of MERS
infection.2b,7 Today, a total of 6 human coronaviruses are
known, of which SARS-CoV and MERS-CoV are recognized as
highly pathogenic with the potential for human-to-human
transmission.8 Without an efficacious antiviral agent or vaccine,
the prevention of current and emerging coronaviruses
continues to rely strongly on public health measures to contain
outbreaks. Therefore, research toward the development of
anticoronaviral drugs continues to be of paramount importance.
The development of anticoronaviral drugs is challenging.
Although a number of coronaviral proteins have been identified
as potential drug targets,9 further development of drug
candidates has been compromised by the general lack of
antiviral data and biological evaluations, which can be done
only in BSL-3 facilities with select agent certification for
laboratories in the U.S. Two of the most promising drug targets
are the SARS-CoV-encoded cysteine proteases, 3CLpro
INTRODUCTION
■
More than 10 years after the pandemic caused by the SARS
(severe acute respiratory syndrome) coronavirus (CoV), no
anticoronaviral regimens have been developed for the treatment
of SARS-CoV or any other human coronaviruses (HCoV)
infection. SARS-CoV was established as the causative agent of
the fatal global outbreak of respiratory disease in humans
during 2002−2003 that resulted in a case-fatality rate (CFR) of
11%.1 In October 2012, the Centers for Disease Control and
Prevention (CDC) added SARS-CoV to the select agents list of
the Department of Health and Human Services (HHS). Among
many aspects that make SARS-CoV a potential threat to the
human population, the lack of effective vaccines or anticor-
onaviral drugs had a significant impact in its classification as a
select agent. However, even with the most extensive preventive
measures, the reemergence of SARS-CoV or other virulent
human coronaviruses poses a continuing threat. A powerful
reminder of this, as well as of the fatal repercussions of the
interspecies transmission potential of CoVs, was brought to the
forefront in September 2012 by the emergence of a new SARS-
like respiratory virus (previously termed HCoV-EMC, now
designated Middle East respiratory syndrome coronavirus,
MERS-CoV).2,3 As in the case of SARS-CoV, the MERS-CoV is
likely of zoonotic origin4 and closely related to bat
coronaviruses from the Betacoronavirus genus (group 2).5
Received: November 6, 2013
Published: February 25, 2014
© 2014 American Chemical Society
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Figure 1. Chemical structures of previously characterized SARS-CoV PLpro inhibitors: (A) hit (1) from a primary HTS from which lead 24 was
developed; (B) hit (2) from a primary HTS from which 15g and 15h were developed. The chiral center for the nearly equipotent isomers derived
from hit 2 is indicated with an asterisk.
(chymotrypsin-like protease) and PLpro (papain-like protease).
PLpro, in addition to playing an essential role during virus
replication, is proposed to be a key enzyme in the pathogenesis
of SARS-CoV. The well-established roles of PLpro enzymatic
activities include processing of the viral polyprotein,10
deubiquitination11(the removal of ubiquitin), and deISGyla-
tion12 (the removal of ISG15) from host-cell proteins. These
last two enzymatic activities result in the antagonism of the host
antiviral innate immune response.13 The SARS-CoV PLpro
inhibitors (compounds 2414 and 15g,h15), previously identified
in our lab via high-throughput screening (HTS), have low
micromolar inhibitory potency with minimal associated
cytotoxicity in SARS-CoV-infected Vero E6 cells and are
therefore viable leads for the development of drug candidates
(Figure 1). Detailed reports of the synthesis and biological
evaluation of inhibitors 2414 and 15g15 and their X-ray
structures in complex with SARS-CoV PLpro have been
previously described.
Compounds 24, 15h, and 15g share a number of chemical
and structural features (Figure 1), including the presence of a
naphthyl group adjacent to a stereogenic center containing a
methyl group and a nitrogen-centered hydrogen bond (H-
bond) donor (at a physiological pH). In addition, these leads
share carboxamide linkers of opposite orientation to differ-
entially substituted benzenoid groups. Structure−activity
relationships (SARs) have shown that in both inhibitor
subtypes the naphthyl ring is optimally substituted at C-1 and
the presence of the methyl group at the aforementioned
stereogenic center is important for potency.14,15 Interestingly,
SARS-CoV PLpro exhibited significant stereopreference for the
(R)-enantiomer of 24 (IC50 = 0.6 μM, EC50 = 15 μM),14 while
minimal stereochemical selectivity was observed between
enantiomers (R)-15g and (S)-15h (IC50 values of 0.67 and
0.56 μM, respectively; EC50 of 9.1 μM for both).15
inhibitor binding, thereby changing the orientation of the
enzyme’s backbone to allow for H-bonding with the inhibitor’s
core. However, relative to the conformation adopted with the
smaller inhibitor 24, compound 15g requires a different and
slightly more opened conformation to accommodate the longer
piperidine-4-carboxamide scaffold and the bulkier 1,3-benzo-
dioxole ring.
Because the antiviral potencies of these SARS-CoV PLpro
inhibitors are likely not yet sufficient to make them
therapeutically viable, further optimization of their inhibitory
potencies, as well as physicochemical properties, is necessary.
Toward this goal, we utilized our previous SAR analysis and the
X-ray structure of compound 15g in complex with SARS-CoV
PLpro to develop a second generation of PLpro inhibitors.
Here, we disclose SAR of a new series of potent SARS-CoV
PLpro inhibitors, their selectivity, antiviral efficacy, biological
evaluation, and a detailed report of the molecular interactions
between SARS-CoV PLpro and inhibitors 3k and 3j aided by
X-ray crystallographic studies. Compound 3k was established to
be the most potent SARS-CoV PLpro inhibitor identified thus
far in vitro. Furthermore, this compound displayed effective
perturbation of SARS-CoV replication in Vero E6 cells while
displaying low cytotoxicity and high selectivity for SARS-CoV
PLpro over human homologue enzymes. The improved
resolution of the SARS-CoV PLpro−3k and PLpro−3j X-ray
structures revealed novel factors accounting for the observed
high binding affinities of the piperidine-containing compounds
and the previously elusive molecular contributions of the benzyl
substituent of the carboxamide. Therefore, our data highlight
3k, as well as two slightly less potent but more metabolically
stable analogues 3e and 5c, as good candidates for advancing to
preclinical evaluations. In addition, we demonstrate the
effective inhibition of PLP2 from HCoV-NL63, a potentially
fatal pathogen in children and elderly, by compound 3k and
related analogues. Importantly, these compounds may also
present an opportunity for the development of broader-
spectrum antiviral drugs against infections caused by both
SARS-CoV and HCoV-NL63.
The structural bases for the high binding affinity of inhibitors
24 and 15g,h are also quite similar. The X-ray crystal structures
of PLpro−24 and PLpro−15g complexes revealed several
hydrophobic interactions resultant of the highly hydrophobic
naphthyl ring and few hydrogen bonds.13a,14 In addition,
significant conformational changes within the active site are
prominent between the apo16 and inhibitor-bound struc-
tures.14,15 Specifically, there is a highly mobile β-turn/loop
(Gly267-Gly272) adjacent to the active site that closes upon
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a
Scheme 1. Synthesis of α-Substituted Naphthyl Analogues
a
Reagents and conditions: (a) 1-naphthyl-MgBr, ether, rt; (b) Aq HCl; (c) LAH, THF, rt; (d) Ms2O, DIEA, DCM, 0 °C; (e) ethyl isonipecotate, rt;
(f) aq NaOH, EtOH, THF; (g) piperonylamine, EDCI, HOBt, DIEA, rt.
a
Scheme 2. Synthesis of α-Hydroxymethyl Analogue 1b
a
Reagents and conditions: (a) LAH, THF, rt; (b) TBSCl, imidazole, ether, rt; (c) Ms2O, DIEA, DCM, rt; (d) ethyl isonipecotate, rt; (e) aq NaOH,
EtOH, 40 °C; (f) piperonylamine, EDCI, HOBt, DIEA, rt; (g) CsF, aq ACN, rt.
reduced with LAH to the diol 13, and the primary alcohol was
selectively protected as the tert-butyldimethylsilyl (TBS) ether
to give 14. The free secondary alcohol was mesylated and
displaced by ethyl isonipecotate, and the resulting amino ester
16 was hydrolyzed to the corresponding acid and coupled with
piperonylamine to provide the TBS-protected compound 17.
The silyl ether was removed with CsF to afford the desired
hydroxy analogue 1b.
The displacement route used in Schemes 1 and 2 was not
successful with the more hindered phenyl analogue 1d, so we
had to employ the lengthier approach previously reported by
Ghosh et al. (Scheme 3).15 1-Naphthonitrile 18 was treated
with benzylmagnesium chloride, and the resulting imine was
hydrolyzed under acidic conditions to the ketone 19. Reductive
amination as previously described19 provided amine 20. This
RESULTS AND DISCUSSION
■
Chemistry. The preparation of analogues 1a−d bearing
substitution off the α-methyl group is summarized in Schemes
1−3.
The appropriate nitriles 8a,c were treated with 1-naphthyl
Grignard reagent, and the intermediary imines were hydrolyzed
to the ketones 9a,c under acidic conditions (Scheme 1).17 The
ketones were then reduced by the action of LAH to the
alcohols 10a,c, which were mesylated and displaced by
treatment with ethyl isonipecotate to provide the amino esters
11a,c. Saponification to the carboxylic acids was followed by
coupling with piperonylamine to give final racemic analogues
1a,c.
A similar displacement strategy was employed to obtain
hydroxyl analogue 1b (Scheme 2). Hydroxy ketone 1218 was
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a
Scheme 3. Synthesis of α-Phenylmethyl Analogue 1d
a
Reagents and conditions: (a) BnMgCl, ether, rt; (b) 1 M HCl, ether, 90 °C; (c) CH3ONH2, pyridine, rt; then BH3−THF, 80 °C;18 (d) dimethyl
2,2-bis((1,3-dioxolan-2-yl)methyl)malonate, HCl/THF; (e) 20, rt, 20 h; (f) H2, Pd/C, 40 psi, rt; (g) NaCN, DMF, 145 °C; (h) 1 M aq NaOH,
EtOH, 60 °C; (i) piperonylamine, EDCI, HOBt, DIEA, rt.
amine was then condensed with the bis-aldehyde derived from
dimethyl 2,2-bis((1,3-dioxolan-2-yl)methyl)malonate to give
the dihydropyridine dimethyl ester 21, which was readily
reduced to the piperidine 22 by heterogeneous hydrogenation.
The diester was converted into the monoester 23 by the
Krapcho method and was subsequently hydrolyzed to the acid
and coupled with piperonylamine to give the desired analogue
1d.
piperidine-4-carboxylic acid 25 was prepared according to the
method of Ghosh et al.15 Coupling of the acid to various
arylmethylamines and arylethylamines was effected either by
EDCI in the presence of DIEA or by the uronium-based
coupling reagent HATU.
The racemic quinolinyl and isoquinolinyl analogues (4a−c)
were generated from the corresponding aldehydes 26a−c by
treatment with methylmagnesium chloride to give the
methylcarbinols 27a−c (Scheme 5). Alternatively, carbinol
27d was prepared from 1-cyanoisoquinoline by addition of
methylmagnesium chloride followed by sodium borohydride
reduction of the resulting ketone, as described in the
Experimental Section. Displacement of the corresponding
mesylates with ethyl isonipecotate afforded esters 28a−d
(Scheme 5). Saponification followed by EDCI-mediated
coupling with 4-fluorobenzylamine provided the final amides
4a−d.
Preparation of (R)-amide analogues is summarized in
Scheme 4. The starting (R)-1-(1-(naphthalen-1-yl)ethyl)-
a
Scheme 4. General Synthesis of Amide Analogues
Conformationally restricted analogue 7 was prepared as
outlined in Scheme 6. Reductive amination of ketone 29 with
ammonium acetate and sodium cyanoborohydride provided
amine 30 in quantitative yield. Conversion to piperidine ester
33 was effected with a three-step sequence analogous to that
a
Reagents and conditions: (a) RNH2, EDCI or HATU, DIEA.
a
Scheme 5. Synthesis of Quinolinyl- and Isoquinolinylamides
a
Reagents and conditions: (a) MeMgCl, THF, −78 °C; (b) Ms2O, DIEA, DCM; (c) ethyl isonipecotate, DCM, rt; (d) 1 M NaOH, MeOH; (e)
EDCI, HOBt, DIEA, 3-fluorobenzylamine, THF.
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a
Scheme 6. Synthesis of Conformationally Restricted Analogue 7
a
Reagents and conditions: (a) NaBH3CN, NH4OAc, iPrOH, reflux, 44 h; (b) dimethyl 2,2-bis((1,3-dioxolan-2-yl)methyl)malonate, HCl/THF; (c)
30, NaHCO3, rt, 72 h ; (d) H2, PtO2, EA, 40 psi, 5 h; (e) NaCN, DMF, reflux, 16 h; (f) aq LiOH, THF/MeOH, rt; (g) 3-fluorobenzylamine, EDCI,
HOBt, DIEA, rt, 16 h.
Table 1. In Vitro Analysis of SARS-CoV PLpro Inhibition by 15g and Derivatives
a
b
compd
isomer
R1
R2
R3
IC50 (μM)
15g
1a
1b
1c
1d
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
3f
R
H
H
H
H
H
H
H
3,4-O-CH2-O
0.67 0.03
17.2 2.0
32.0 4.5
>100
R,S
R,S
R,S
R,S
R
Me
OH
OMe
Ph
H
3,4-O-CH2-O
3,4-O-CH2-O
3,4-O-CH2-O
3,4-O-CH2-O
>100
H
2.2 0.1
R
H
(R)-Me
H
13.5 1.2
12.7 0.3
18.0 1.9
1.9 0.1
R
H
(S)-Me
H
R
H
(R)-CH2-OMe
H
R
H
(S)-CH2-OMe
H
R
H
H
H
H
H
H
H
H
H
H
H
H
4-Et
0.47 0.01
0.60 0.02
0.63 0.01
5.7 0.5
R
H
4-CO-NH-Me
3-CO-NH-Me
4-NH-CO-Me
3-NH-CO-Me
3-CH2-NH-CO-Me
3-Cl
R
H
R
H
R
H
0.39 0.01
20.4 1.2
27.2 4.1
0.58 0.02
29.2 2.1
0.49 0.01
0.15 0.01
R
H
3g
3h
3i
R
H
R
H
4-Cl
R
H
3,4-diF
3j
R
H
4-F
3k
R
H
3-F
a
b
Chiral center. Values are reported as mean standard deviation based on a minimum of triplicate measurements.
employed in Scheme 3. Saponifiction with LiOH in a mixture
(3:1:1) of THF/MeOH/H2O furnished the free acid. Finally
amide coupling with 3-fluorobenzylamine, EDCI, HOBt, and
DIEA led to the production of the target analogue 7. It is worth
noting that the shorter ethyl isonipecotate displacement route
used in Schemes 1, 2, and 5 was not successful with the
mesylate derived from reduction/mesylation of 29, presumably
because of excessive steric hindrance.
potent enzymatic inhibition (IC50 = 0.67 μM) and low
micromolar antiviral activity against SARS-CoV. It evolved
from a small SAR study on the HTS hit 2 (Figure 1) and is
therefore an attractive lead for the development of anti-SARS
drug candidates.15 Compound 15g is composed of the central
piperidine-4-carboxamide core decorated with 1-naphthaleny-
lethyl and 1,3-benzodioxolylmethyl substituents at the piper-
idinyl and carboxamide nitrogens, respectively. Previous SAR
has established the preference for the 1-naphthyl over the 2-
naphthyl substitution pattern and the requirement of a single
Structure−Activity Relationship Analysis. Compound
15g is a competitive inhibitor of SARS-CoV PLpro that displays
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Table 2. Structures and Activities of 3k Variants, Exploring Bioisosteric Replacements and Scaffold Perturbation
a
b
compd
isomer
R1
R2
IC50 (μM)
3k
4a
4b
4c
4d
5a
5b
5c
6a
6b
R
1-naphthyl
3-F-Ph-CH2
3-F-Ph-CH2
3-F-Ph-CH2
3-F-Ph-CH2
3-F-Ph-CH2
0.15 0.01
7.0 0.7
4.5 0.2
6.8 0.3
30.8 2.6
26.3 2.3
18.3 0.9
0.35 0.02
1.6 0.3
1.9 0.1
R,S
R,S
R,S
R,S
R
8-quinolinyl
5-quinolinyl
5-isoquinolinyl
1-isoquinolinyl
1-naphthyl
3-pyridinyl-CH2
R
1-naphthyl
4-pyridinyl-CH2
R
1-naphthyl
2-methoxy-4-pyridinyl-CH2
4-Cl-Ph-CH2CH2
3-F-Ph-CH2CH2
R
1-naphthyl
R
1-naphthyl
a
b
Chiral center. Values are reported as mean standard deviation based on a minimum of triplicate measurements.
methyl substituent at the methylene linker for effective
inhibition (when compared to both unsubstituted and gem-
dimethyl derivatives), with minimal stereochemical preference.
X-ray crystallographic studies of the SARS-CoV PLpro−15g
complex show that the methyl group of the (R)-enantiomer
extends into a small pocket that has both hydrophobic and
polar features and is filled with water molecules. To explore the
dimensions and availability of this pocket to substituents as well
as H-bond opportunities, the first set of analogues explores the
incorporation of larger or polar substituents (1a, -Me; 1b, -OH;
1c, -OMe; 1d, -Ph) at the methyl group (position R1 in Table
1). Addition of substituents to R1 resulted in higher IC50 values
for all substitutions, and loss of inhibitory potency was
proportional to substituent size. These trends indicate that
the corresponding pocket might be less accessible than
predicted by the crystal structure, with no clear opportunities
for extra H-bonding or lipophilic interactions. It also appears
that the potential entropic gain by displacing the water
molecules with larger R1 groups is not achieved likely because
of a larger enthalpic penalty of breaking the H-bonds formed
not engage the active site of the enzyme in favorable interaction
and are therefore unlikely to lead to activity improvement.
Previous SAR studies on the aromatic substitution pattern of
the benzyl group were limited to m- and p-methoxy analogues,
which were as potent as the lead benzodioxolane analogue,
15g.15 In the X-ray structure of the SARS-CoV PLpro−15g
complex, one of the 1,3-dioxolane oxygens is within 3 Å of the
Gln270 side chain amide nitrogen, suggesting the potential for
forming an H-bond. However, because of the poor electron
density for the Gln270 side chain, the oxygen’s contribution to
inhibitory potency was uncertain.15 Therefore, we mutated
Gln270 to an alanine, glutamate, or aspartate residue and
purified the mutant enzymes and determined the IC50 values
with 15g. We found that the IC50 values for the mutant
enzymes are the same as for wild type SARS-CoV PLpro,
indicating that Gln270 does not contribute to the binding of 15g
via interaction with the dioxolane ring (data not shown). As
observed with compound 2a, removal of the dioxolane ring
from 15g results in a ∼3-fold decrease in potency, suggesting
that the dioxolane group provides a significant contribution to
between these water molecules and Asp165, Arg167, Tyr274
,
inhibitory potency but not through interaction with Gln270.
Thr302, and Asp303. As a result, unlike the previously reported
predictions by fragment mapping program (FTMap) in which
all water molecules were removed before computational
analyses,20 here we demonstrate experimentally that this pocket
is unlikely to provide any extra ligand-binding sites or room for
larger substituents.
We next examined the effect of incorporating substituents at
the benzylic position R2 to form a second stereogenic carbon,
using unsubstituted compound 2a (IC50 = 2.2 0.1 μM) as the
comparator. Diastereoisomeric epimers 2b and 2c, with a
methyl group at R2, decreased the inhibitory activity by a
Therefore, to elucidate the nature of interactions provided by
the 1,3-benzodioxole group, benzene ring derivatives with
diverse substituents at position R3 were explored.
The incorporation of a p-ethyl group at R3 (3a) had a more
than 4-fold improvement in potency over unsubstituted
prototype 2a, surpassing that of the dioxolane lead (15g). A
comparable effect was achieved with both p- and m-
methylcarboxamide derivatives 3b (IC50 = 0.60
0.02 μM)
and 3c (IC50 = 0.63 0.01 μM) respectively displaying both H-
bonding donors and acceptors. Reversal of the amide direction
to the acetamido group resulted in significantly differing activity
levels for para and meta positional isomers 3d (IC50 = 5.7 0.5
comparable extent (IC50 of 13.5
1.2 and 12.7
0.3 μM,
respectively), indicative of a steric clash with SARS-CoV PLpro.
μM) and 3e (IC50 = 0.39
0.01 μM), respectively. Further
Interestingly, a 10-fold stereopreference was observed for (S)-
extension of the acetamido group from the meta position,
favored in the previous pair, dramatically decreases activity of
methoxymethyl 2e (IC50 = 1.9
0.1 μM), compared to
epimeric 2d (IC50 = 18.0 1.9 μM), which could be indicative
of a H-bond in that region or simply the ability of the more
active enantiomer 2e to retain the methoxy in a solvent-
exposed environment, thereby avoiding a desolvation penalty.
However, because the most active analogue in this series, 2e,
did not surpass the effectiveness of the initial unsubstituted
compound, 2a, we surmise that substitutions at position R2 do
the corresponding derivative (3f, IC50 = 20.4
1.2 μM). A
reversal in the meta vs para trend is seen with the
corresponding pair of chloro derivatives 3g (IC50 = 27.2
4.1 μM) and 3h (IC50 = 0.58 0.01 μM), with the halogen
tolerated only at the para position. Finally, derivatives 3j and
3k, featuring the monofluoro substitution at para and meta
positions, respectively, displayed a significant improvement
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over the parental 2a structure, with the 3-fluorobenzyl variant
possessing a more than 10-fold higher activity level (IC50 = 0.15
0.01 μM). Surprisingly, 3,4-difluoro substitution in 3i (IC50 =
29.2
2.1 μM) had a significantly detrimental effect,
decreasing the inhibitory potency by ∼200-fold when
compared to 3k. In general, neither steric nor electronic factors
can be invoked to rationalize systematically the effects of the
substitution pattern at R3 and R4 on the inhibitory activity, with
groups as diverse as 1,3-dioxolane, 4-ethyl, 3- and 4-
carboxamido, 3-acetamido, 3- and 4-fluoro, and 4-chloro
achieving a similar level of activity, despite presenting a wide
range in polarity, size, and H-bonding capacity. Significantly,
the monosubstitution at the meta position with the smallest
and yet the most electron-withdrawing fluoro group, capable of
inducing dramatic polarization effects in the π-system of the
associated benzene ring, resulted in the most potent SARS-CoV
PLpro inhibitor identified thus far.
In an attempt to improve the solubility of our most potent
compound, 3k, the bioisosteres of naphthalene and benzene
(quinoline and pyridine, respectively) were exploited by
synthesizing corresponding derivatives (Table 2). A detrimental
effect on activity is observed upon replacement of the naphthyl
ring system by quinolines (4a, IC50 = 7.0 0.7 μM and 4b,
IC50 = 4.5 0.2 μM) and isoquinolines, (4c, IC50 = 6.8 0.3
μM and 4d, IC50 = 30.8 2.6 μM), all tested as racemates. The
replacement of the phenyl ring in the 2a prototype by the
isosteric 3- and 4-pyridinyls (5a, IC50 = 26.3 2.3 μM and 5b,
IC50 = 18.3 0.9 μM) decreased potency by over an order of
magnitude. Interestingly, the activity was rescued by the
addition of a 3-methoxy group to the 4-pyridinyl ring of the
Figure 2. Chemical structure of compound 7. The conformationally
restricted analogue 7 was designed to lock the conformation of
compound 3k to the conformation observed in the X-ray crystal
structure of SARS-CoV PLpro bound to 15g.
that for 15g.15 Crystals for each SARS-PLpro complex resulted
after screening over 3000 crystallization conditions for
diffraction quality crystals. Complete X-ray data sets were
collected for the SARS-CoV PLpro−3k and PLpro−3j
complexes to resolutions of 2.1 and 2.5 Å, respectively (Table
3). Both SARS-CoV PLpro−inhibitor complexes crystallized in
space group C2 and contained two monomers per asymmetric
unit.
After identification of a molecular replacement solution and
performance of initial rounds of refinement of the SARS-CoV
PLpro structural model in the absence of any ligand or water,
strong (>3σ) residual electron density was observed in Fo − Fc
maps for both 3k and 3j in the active sites of each monomer
within the asymmetric unit. The strong and continuous
electron density for the inhibitors allowed for their unequivocal
positioning and modeling within the ligand-binding site (Figure
3a and Figure 3b). Both 3k and 3j bind to the SARS-CoV
PLpro active site in the same orientation (Figure 3c and Figure
3d).
5b analogue (5c, IC50 = 0.35
0.02 μM), confirming the
benefit of having a H-bonding group at C-3 of the aromatic
ring. We tested whether Gln270 was involved in an interaction
with compound 5c by determining its IC50 value with each of
the SARS PLpro mutant enzymes (Gln270Ala, Gln270Asp, and
Gln270Glu). We observed no differences in the IC50 values
within experimental error, indicating that no significant
interaction is involved (data not shown). Analysis of the active
site for other potential hydrogen-bonding residues produced no
obvious candidates.
Extending the separation of the two aromatic centers in
compounds 3h and 3k by one carbon atom resulted in
weakening of inhibition activity in corresponding variants 6a
(IC50 = 1.6 0.3 μM) and 6b (IC50 = 1.9 0.1 μM). The
effect is surprisingly minor, considering the nature of this
perturbation, and is perhaps indicative of a significant amount
of flexibility in the active site of SARS-CoV PLpro in the region
bound by the benzene moiety.
Finally, tricyclic analogue 7 was prepared as a conformation-
ally restricted analogue of our most potent inhibitor 3k (Figure
2). It was designed to lock the conformation of the bond
joining C-1 of the naphthyl ring to the piperidinylethyl group
to that observed in the X-ray structure of the SARS-CoV
PLpro−15g complex. This modification, however, led to a
significant loss of activity (IC50 = 5.1 0.5 μM). These results
suggest an inhibitor-induced-fit mechanism of association, in
which the size and conformational freedom of compound 3k
allow the optimal fit to be achieved.
X-ray Structural Analyses of SARS-CoV PLpro Bound
to 3k and 3j and Comparison to 15g. To gain structural
insight into the enhanced potency of compound 3k, SARS-CoV
PLpro was cocrystallized as a complex with 3k (PDB code
4OW0) and 3j (PDB code 4OVZ) using an approach similar to
As described previously for compounds 24 and 15g,
compounds 3k and 3j bind adjacent to the active site at the
enzyme S3−S4 subsites14,15 (Figure 3c and Figure 3d),
exclusive of any interactions with the catalytic triad (Cys112
-
His273-Asp287). Upon inhibitor binding, the β-turn/loop
(Gly267-Gly272) containing Tyr269 adopts a closed conformation
via an induced-fit mechanism to interact with the inhibitors.
This enables the formation of a 3 Å H-bond between the
backbone carbonyl of Tyr269 and the carboxyamide nitrogen of
the inhibitors. An additional and important interaction is
observed between the piperidine ring nitrogen and the side
chain carboxylate of Asp165. The puckering of the piperidine
ring positions the cationic nitrogen within a distance of 2.8 Å
from an oxygen of the carboxylate of Asp165, thereby forming a
charge-to-charge mediated H-bond.
A structural superimposition of the two SARS-CoV PLpro−
3k and PLpro−3j complexes (Figure 3e) shows that the 1-
naphthyl rings align identically and that they pack against the
two tandem prolines, Pro248 and Pro249, in a hydrophobic
pocket formed by the side chains of the prolines, Tyr265, Tyr269
,
and Thr302 (Figure 3c and Figure 3d). This pocket orients the
(R)-methyl group into a small cavity lined by hydrophobic and
hydrophilic side chains wherein some H-bond opportunities
exist with the side chains of Asp303, Thr302, and Tyr274. The
enhanced resolution of PLpro−3k and PLpro−3j complex
compared to PLpro−15g complex allowed for the better
placement of three conserved water molecules that are present
within this cavity (Figure 3e). The presence of these water
molecules increases the polarity and decreases the effective size
of the otherwise larger and mostly hydrophobic cavity. This
smaller cavity explains our observed SAR with hydrophobic or
polar extensions at the (R)-methyl (position R1 in Table 1)
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Table 3. Data Collection and Refinement Statistics
3k
3j
Data Collection
beamline
21-ID-F
0.98
21-ID-F
0.98
wavelength (Å)
space group
C2
C2
unit cell dimensions
a, b, c (Å)
119.4, 74.4, 98.3
90, 104, 90
100−2.10 (2.14−2.10)
514232
119.8, 73.5, 98.3
90, 104, 90
100−2.50 (2.54−2.50)
743761
α, β, γ (deg)
resolution (Å)
no. of reflections observed
no. of unique reflections
Rmerge (%)
49274
29021
6.2 (52.3)
6.5 (69.6)
I/σI
20.6 (1.9)
19.3 (1.8)
% completeness
redundancy
98.6 (96.1)
4.7 (3.9)
99.8 (100)
4.2 (4.2)
Refinement
resolution range (Å)
no. of reflections in working set
no. of reflections in test set
Rwork (%)
31.79−2.10
42169
35.75−2.50
24909
2134 (5.1%)
0.18
1265 (5.1%)
0.19
Rfree (%)
0.20
0.24
average B factor (Å2)
rmsd from ideal geometry
bond length (Å)
39
37
0.022
1.5
0.026
1.7
bond angle (deg)
Ramachandran plot
most favored (%)
allowed (%)
94.0
4.8
92.6
5.6
disallowed (%)
1.2
1.8
whereby the larger groups were all detrimental to binding
affinity. These observations suggest that the potential entropic
gain in binding energy by displacement of the water molecules
cannot be achieved by incorporation of larger or polar
substituents, as the enthalpy necessary to break the H-bonds
between water molecules and the side chains must be too large
to overcome.
specificity is an attractive approach for the treatment of
infections caused by current and future-emerging human
coronaviruses. However, to avoid drug-induced toxicity and
potential side effects, it is crucial to maintain high inhibitory
potency without cross-reactivity of critical homologues, the
cellular deubiquitinating enzymes (DUBs). First, to assess
compound selectivity, a set of the most potent analogues were
tested against a panel of human DUBs, including representative
ubiquitin specific proteases (USPs) with structural similarity to
PLpro, along with the human cysteine proteases caspase 3 and
cathepsin K (Table 4). Compounds were tested at 31 μM. If
the inhibitory activity was less than 10%, no inhibition is
reported. If the inhibitory activity was between 10% and 15% or
between 15% and 20%, then IC50 values of >100 or 31 μM are
reported. Importantly, we can detect no significant inhibition of
the human DUBs or cysteine protease enzymes tested above
20% at 31 μM, indicating that these PLpro inhibitors are
selective and are unlikely to have significant off-target activity.
Next, we evaluated the potential of this set of analogues for
the development of broader-spectrum coronaviral inhibitors. All
of the analogues in Tables 1 and 2 were counterscreened
against the viral orthologue PLP2 enzyme from the human
coronavirus NL63 (HCoV-NL63), a member of the α-
coronaviruses. HCoV-NL63 is one of the causative agents of
croup in children, and infection can result in hospitalization.21
Currently there are no specific treatments for individuals
infected with HCoV-NL63. For those analogues that showed
>30% inhibition at a single dose concentration of 100 μM, full
dose response curves of HCoV-NL63 PLP2 inhibition versus
increasing compound concentration up to 100 μM were
determined. Six compounds, including compound 3k, produced
A superimposition of PLpro in complex with 15g, 3k, and 3j
is shown in Figure 3f. As discussed above, previous structural
and computational analyses of the 15g-bound structure showed
that the 1,3-benzodioxole moiety can move within 3 Å of the
amide nitrogen of Gln270 side chain. However, mutation of
Gln270 to Ala, Glu, or Asp showed no significant change in
inhibitory potency when tested against 15g, 3k, and 3j,
indicative of no H-bond interactions with the side chain of
Gln270. Interestingly, while the superimposition between
PLpro−15g, PLpro−3k, and PLpro−3j complexes shows a
near perfect overlap of the 1-naphthyl rings, there is a 1 Å
difference in the position of the carboxamide nitrogen of 15g
when compared to 3k and 3j. This causes a slight tilt on the
orientations of the benzene rings to allow for the
accommodation of the different benzene substituents. As a
result, we conclude that the slightly enhanced inhibitory activity
of compound 3k compared to 3j and 15g is due to its ability to
form collectively stronger van der Waals’ interactions between
the m-fluorobenzene and the side chain of Tyr269, and slightly
stronger interactions with the backbone oxygen atoms of Tyr269
and Gln270 (Figure 3f).
Assessing Potency and Selectivity of SARS-CoV PLpro
Inhibitors to Human and Viral USP Homologues. The
development of an enzyme inhibitor with broad-spectrum
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Figure 3. X-ray crystal structures of SARS-CoV PLpro in complex with 3k and 3j. Stereoviews of SARS-CoV PLpro (blue ribbon representation and
gray surfaces) in complex with 3k (orange ball and sticks) are shown in (a) and (c), and SARS-CoV PLpro (cyan ribbon representation and gray
surface) in complex with 3j (pink ball and sticks) are shown in (b) and (d). The corresponding Fo − Fc electron density omit maps (inhibitor atoms
omitted) contoured at 3σ are shown as gray mesh. Important amino acids for inhibitor binding are shown, and the H-bonds between inhibitor atoms
and amino acid residues are depicted as dotted lines. A superimposition of SARS-CoV PLpro−3k and PLpro−3j complexes is shown in (e) with
conserved water molecules displayed as blue and cyan spheres for the 3k− and 3j− complexes, respectively. A superimposition of SARS-CoV
PLpro−15g complex (SARS-CoV PLpro displayed as a gray ribbon and 15g displayed as gray balls and sticks, pdb:3MJ5) with SARS-CoV PLpro−
3k and PLpro−3j complex is shown in (f).
Table 4. Inhibitor Selectivity against Human DUBs and
Cysteine Proteases
Table 5. Compounds Displaying Dual-Target Inhibition of
SARS-CoV PLpro and HCoV-NL63 PLP2
a
IC50 (μM)
IC50 (μM)
PLpro
max I (%)
PLpro
15g
3k
3e
3j
compd
PLP2
PLP2
human DUB
USP2
15g
3k
5c
0.67
0.15
0.35
0.39
5.7
18
33
59
46
37
44
99
46
57
67
71
42
48
−
−
>100
−
−
>100
>100
>31
−
>100
>100
>100
>100
>31
−
−
−
>100
>100
>31
−
100
100
100
94
USP7
USP8
3e
3d
3i
USP20
−
>31
−
USP21
29
78
DEN1
UCHL1
caspase 3
cathepsin K
−
−
−
−
−
−
−
−
−
−
−
−
relative to the initial inhibitor 15g (tPSA = 50.8 Å2) and
therefore is proposed to enhance cell permeability and thus
antiviral activity relative to 15g. On the basis of these possible
improvements, compound 3k along with seven of the most
potent analogues and two quinolone derivatives were subjected
to antiviral assays using our well-established method and BSL-3
protocols.14 In this assay, compounds are titrated in both mock-
and SARS-CoV-infected Vero E6 cells, and the resulting dose−
response curves are fitted to the four-parameter logistic
equation. Curves were then compared to the mock-infected
cells to assess drug-induced cytotoxicity in Vero E6 and
HEK293 cell lines. The resultant EC50 values and cytotoxic
concentrations (CC50) are shown in Table 6. Because of their
greater potency, all tested compounds displayed low cytotox-
icity levels (CC50 > 68 μM) with improved therapeutic index
(TI) values in Vero E6 cell when compared to 15g.
Compounds with IC50 values of >2 μM (2a, 4a, and 4c) had
a
−, no inhibition.
typical dose response curves, and the inhibition data were fit to
determine the IC50 and maximum percent inhibition values
(Table 5). The results show that inhibition of HCoV-NL63
PLP2 is achieved by this series of compounds, albeit
significantly weaker inhibition than for SARS-CoV PLpro
(micromolar versus nanomolar range), and suggest that SARS-
CoV PLpro and HCoV-NL63 PLP2 may display sufficient
structural similarities at the active site for the potential
development of a dual-target inhibitor.
SARS-CoV Antiviral Activity and Cytotoxicity Evalua-
tion of the Most Potent Compounds. Compound 3k
exhibits lower topological polar surface area (tPSA = 32.2 Å2)
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Table 6. Compound Effect on Replication of SARS-CoV Virus, Cytotoxicity, and Therapeutic Indexes
15g
3k
3e
5c
3j
2e
Vero E6
0.39 0.01
8.3 0.6
>100
a
IC50
0.67 0.03
12.8 1.4
>100
0.15 0.01
5.4 0.6
>100
0.35 0.02
9.5 1.3
>100
0.49 0.01
11.6 0.8
>100
1.9 0.1
11.7 0.7
>100
a
EC50
CC50
TI
a
>7.8
>18.5
>12
>10.5
>8.6
>8.5
HEK293
68 15
a
CC50
>100
73 19
>100
73 29
>100
a
Values are reported in μM as mean standard deviation based on a minimum of triplicate measurements. EC50 values were derived from two
independent experiments. EC50, the half maximal effective concentration in SARS-CoV infected Vero E6 cells; CC50, 50% cytotoxic concentration;
TI, the therapeutic index defined as the ratio of CC50/EC50.
no antiviral activity displaying EC50 values of >50 μM (data not
shown). Compounds 3e, 5c, 3j, and 2e displayed similar to
slightly improved antiviral activity when compared to the initial
compound 15g. However, in the case of our best compound,
3k, which has a 4-fold improved IC50 value, an additional 2-fold
improvement in antiviral activity was observed (EC50 = 5.4
0.6 μM) when compared to 15g.
Metabolic Stability and Plasma Binding Studies. With
the ultimate goal of achieving protection from coronaviral
infection in vivo, we evaluated the stability of our best new
analogues to phase I metabolism by mouse liver microsomes
(Table 7). The lead compound 15g proved to be exceedingly
the plasma binding ability of the compounds in Table 6 by
measuring the shifts in their IC50 values in various
concentrations of human plasma protein (serum shift assays).25
We observed no significant changes in the IC50 values for the
compounds in the serum shift assays, which were performed in
the presence of 5%, 10%, and 20% human serum albumin
(HSA) (data not shown), the most abundant protein in human
plasma (40 mg/mL).26 This observation suggests that no
specific interactions between the compounds and HSA take
place.
CONCLUSIONS
■
Table 7. Stability of Selected Compounds to Metabolism by
Mouse Liver Microsomesa
A second-generation series of highly potent SARS-CoV PLpro
inhibitors was designed and evaluated biologically to further
advance anticoronavirus drug development. Four compounds
(3k, 3e, 3j, and 5c) were found to have more potent SARS-
CoV PLpro inhibition and SARS-CoV antiviral activity than the
most potent first generation inhibitor, 15g. None of these five
compounds exhibit cytotoxicity or off-target inhibitory activity
of a series of human DUB and cysteine protease enzymes, nor
do any of these five inhibitors bind to human serum albumin
(HSA). The second-generation compounds 3k, 3e, and 5c
exhibit significantly improved metabolic stability compared to
15g. Although compound 3k is the most potent SARS-CoV
PLpro inhibitor (IC50 = 0.15 μM) and the most effective
antiviral compound in cell culture (EC50 = 5.4 μM), it is
significantly less metabolically stable compared to compounds
3e and 5c, which are similarly effective in inhibiting SARS-CoV
PLpro (IC50 = 0.39 μM and IC50 = 0.35 μM) and inhibiting
SARS-CoV infected Vero cells (EC50 = 8.3 μM and EC50 = 9.5
μM). Thus, compounds 3e and 5c are likely to be the better
candidates to advance to animal efficacy models. Finally, the
high resolution of the inhibitor-bound crystal structures of
PLpro with 3k and 3j revealed novel aspects for the inhibitor
binding mode, providing guidance for the further optimization
of PLpro inhibitors.
a
b
compd
% rem (15 min)
T1/2 (min)
15g
3k
3e
0.2
1.2
20
1.6
2.8
7.0
8.0
5c
30
a
Percent of the parent compound remaining after 15 min incubation.
The initial concentration of the parent compound was 1 μM. Half-life
b
(minutes) of parent compound.
unstable, being completely consumed within 15 min. This was
not entirely unexpected because of the presence of the 3,4-
methylenedioxy moiety, which renders the benzyl aromatic ring
electron-rich and is itself a known target of cytochrome
P450s.22,23 Somewhat surprisingly, the much more electron-
poor 3-fluorobenzylamide 3k was still metabolized rapidly,
suggesting that most of the metabolism is occurring distal to
the benzylamide. Consistent with this hypothesis, the less
lipophilic bis(amide) 3e and methoxypyridine 5c were
significantly more stable, with 20% and 30% parent drug
remaining after 15 min, respectively, and half-lives 4- to 5-fold
longer than that of 15g.
Because the binding of an antiviral drug to human serum
proteins may result in reduced antiviral activity,24 we evaluated
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1H), 2.94−2.80 (m, 1H), 2.25 (bs, 1H), 2.06−1.66 (m, 8H), 0.68 (t, J
= 7.1 Hz, 3H). TOF ES+ MS: (M + H) 431.1. HPLC tR = 5.85 min,
>95% purity.
EXPERIMENTAL SECTION
■
General Synthetic Procedures. All reagents were used as
received from commercial sources unless otherwise noted. 1H and
13C spectra were obtained in DMSO-d6 or CDCl3 at room
(
)-N-(Benzo[d][1,3]dioxol-5-ylmethyl)-1-(2-hydroxy-1-
(naphthalen-1-yl)ethyl)piperidine-4-carboxamide (1b). Silyl
ether 17 (46 mg, 0.08 mmol) was dissolved in ACN (4 mL) and
H2O (2 mL). To this was added CsF (51 mg, 0.34 mmol), and the
mixture was stirred at room temperature for 24 h. The mixture was
then partitioned between 10% aqueous Na2CO3 (30 mL) and EtOAc
(50 mL) and extracted. The extract was dried with anhydrous MgSO4,
filtered, and concentrated in vacuo. The residue was then purified by
flash chromatography (4.7g amine-activated silica RediSep column,
temperature, unless otherwise noted, on Varian Inova 400 MHz,
Varian Inova 500 MHz, Bruker Avance DRX 500, or Bruker Avance
DPX 300 instrument. Chemical shifts for the 1H NMR and 13C NMR
spectra were recorded in parts per million (ppm) on the δ scale from
an internal standard of residual tetramethylsilane (0 ppm). Rotamers
are described as a ratio of rotamer A to rotamer B if possible.
Otherwise, if the rotamers cannot be distinguished, the NMR peaks
are described as multiplets. Mass spectrometry data were obtained on a
Waters Corporation LCT. Purity of all tested compounds was assessed
by HPLC using an Agilent 1100 series with an Agilent Zorbax Eclipse
Plus C18 column (254 nm detection) with the following gradient: 10%
ACN/water (1 min), 10−90% ACN/water (6 min), and 90% ACN/
water (2 min). Values for each compound are included at the end of
each experimental procedure, and all are over 95% pure. HPLC
retention times (tR) were recorded in minutes (min). Solvent
abbreviations used are the following: MeOH (methanol), DCM
(dichloromethane), EtOAc (ethyl acetate), Hex (hexanes), DMSO
(dimethylsulfoxide), DMF (dimethylformamide), H2O (water), THF
(tetrahydrofuran), ACN (acetonitrile). Reagent abbreviations used are
the following: HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetrame-
thyluronium hexafluorophosphate), HOAT (1-hydroxy-7-azabenzo-
triazole), HOBt (1-hydroxy-1,2,3-benzotriazole), EDCI (N-(3-dime-
thylaminopropyl)-N′-ethylcarbodiimide hydrochloride), DIEA (diiso-
propylethylamine), TFA (trifluoroacetic acid), MgSO4 (magnesium
sulfate), Na2SO4 (sodium sulfate), NaHCO3 (sodium bicarbonate),
Na2CO3 (sodium carbonate), Cs2CO3 (cesium carbonate), NH4Cl
(ammonium chloride), K2CO3 (potassium carbonate), KOH
(potassium hydroxide), HCl (hydrogen chloride), NaOH (sodium
hydroxide), LiOH (lithium hydroxide), LAH (lithium aluminum
hydride), EtOH (ethanol), NaCN (sodium cyanide), Et2O (diethyl
ether), CsF (cesium fluoride), NaCl (sodium chloride), TBSCl (tert-
butyldimethylsilyl chloride), Ms2O (methanesulfonic anhydride),
MsCl (methanesulfonyl chloride), AcOH (acetic acid), NaBH4
(sodium borohydride), NaBH3CN (sodium cyanoborohydride), H2
(hydrogen), N2 (nitrogen), MS (molecular sieves). Assay abbrevia-
tions are the following: LUC (luciferase), MTT ((3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide. Additional abbreviations
are the following: aq (aqueous), saturated (saturated), rt (room
temperature). All anhydrous reactions were run under an atmosphere
of dry nitrogen.
( )-N-(Benzo[d][1,3]dioxol-5-ylmethyl)-1-(1-(naphthalen-1-
yl)propyl)piperidine-4-carboxamide (1a). Ethyl 1-(1-(naphtha-
len-1-yl)propyl)piperidine-4-carboxylate 11a (480 mg, 1.48 mmol)
was dissolved in a 1:1:2 mixture of THF/EtOH/1 M aqueous NaOH,
total volume of 8 mL. This was stirred at 60 °C for 5 h. The mixture
was allowed to cool, and the organic solvents in the mixture were
mostly removed by rotary evaporation. The pH was then reduced by
addition of concentrated HCl to ∼pH 4, and the resulting white
precipitate was collected over a filter to give the desired carboxylic acid
(364 mg, 83%) without further purification. Then the following was
added sequentially to anhydrous DMF (1 mL): 3 Å molecular sieves,
1-(1-(naphthalen-1-yl)propyl)piperidine-4-carboxylic acid (50 mg,
0.17 mmol), DIEA (0.088 mL, 0.504 mmol), EDCI (36 mg, 0.19
mmol), HOBt (28 mg, 0.19 mmol), and finally piperonylamine (0.031
mL, 0.252 mmol). This was stirred at room temperature for 16 h, at
which time the material was partitioned between 10% Na2CO3
solution and EtOAc, and the organic layer was washed with 10%
Na2CO3 solution (3 × 20 mL) and brine (1 × 20 mL). The organic
layer was dried with anhydrous MgSO4, filtered, and concentrated in
vacuo. Purification was accomplished via flash chromatography (4 g
silica RediSep gold column on a CombiFlash, eluted with 70% EtOAc/
Hex) to give the desired product (34 mg, 47%). 1H NMR (500 MHz,
chloroform-d) δ 8.44 (bs, 1H), 7.91−7.84 (m, 1H), 7.77 (d, J = 8.1
Hz, 1H), 7.53−7.40 (m, 4H), 6.78−6.66 (m, 3H), 5.94 (s, 2H), 5.87−
5.81 (m, 1H), 4.32 (d, J = 5.6 Hz, 2H), 3.93 (bs, 1H), 3.39−3.26 (m,
1
50% EtOAc/Hex) to give the final product (28 mg, 77%). H NMR
(500 MHz, chloroform-d) δ 8.29 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 8.0
Hz, 1H), 7.87−7.81 (m, 1H), 7.58−7.45 (m, 4H), 6.81−6.64 (m, 3H),
5.95 (s, 2H), 5.69 (t, J = 4.7 Hz, 1H), 4.64−4.49 (m, 1H), 4.32 (d, J =
5.6 Hz, 2H), 4.09 (dd, J = 11.0, 7.8 Hz, 1H), 3.81 (dd, J = 10.9, 5.2 Hz,
1H), 3.27 (d, J = 11.2 Hz, 1H), 3.00 (d, J = 11.5 Hz, 1H), 2.32−1.75
(m, 7H). TOF ES+ MS: (M + H) 433.1, (M + Na) 455.1. HPLC tR =
5.43 min, >95% purity.
(
)-N-(Benzo[d][1,3]dioxol-5-ylmethyl)-1-(2-methoxy-1-
(naphthalen-1-yl)ethyl)piperidine-4-carboxamide (1c). Ethyl 1-
(2-methoxy-1-(naphthalen-1-yl)ethyl)piperidine-4-carboxylate 11c (80
mg, 0.23 mmol) was dissolved in EtOH (2 mL) and 6 M NaOH (5
mL). This was stirred at 50 °C for 3 h. The pH was dropped to 2 by
dropwise addition of concentrated HCl, and material was extracted
with 1:1 EtOAc/Et2O (3 × 10 mL). The solvent was then dried with
anhydrous MgSO4, filtered, and removed in vacuo to give 1-(2-
methoxy-1-(naphthalen-1-yl)ethyl)piperidine-4-carboxylic acid (30
mg, 41%) without further purification. Then the following was
added sequentially to anhydrous DMF (1 mL): 3 Å molecular sieves,
1-(2-methoxy-1-(naphthalen-1-yl)ethyl)piperidine-4-carboxylic acid
(20 mg, 0.06 mmol), DIEA (0.033 mL, 0.191 mmol), EDCI (14
mg, 0.07 mmol), HOBt (11 mg, 0.07 mmol), and finally piperonyl-
amine (0.012 mL, 0.096 mmol). This was stirred at room temperature
for 24 h, at which time the material was partitioned between 10%
aqueous Na2CO3 solution and EtOAc, and the organic layer was
washed with 10% aqueous Na2CO3 solution (3 × 10 mL) and brine (1
× 10 mL). The organic layer was dried with anhydrous MgSO4,
filtered, and concentrated in vacuo. The material was purified via
alumina flash chromatography (8 g basic alumina RediSep cartridge,
30% EtOAc/Hex) to give the final desired compound (13 mg, 46%).
1H NMR (500 MHz, chloroform-d) δ 8.37 (d, J = 7.8 Hz, 1H), 7.88
(d, J = 7.2 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.60 (d, J = 7.0 Hz, 1H),
7.54−7.47 (m, 2H), 7.45 (t, J = 7.6 Hz, 1H), 6.79−6.71 (m, 3H), 5.96
(s, 2H), 5.73 (s, 1H), 4.34 (d, J = 5.6 Hz, 2H), 4.21 (s, 1H), 3.93 (dd, J
= 10.1, 6.3 Hz, 1H), 3.67 (dd, J = 10.3, 3.5 Hz, 1H), 3.36 (d, J = 11.1
Hz, 1H), 3.31 (s, 3H), 2.85 (d, J = 11.4 Hz, 1H), 2.24 (t, J = 10.8 Hz,
1H), 2.16−2.09 (m, 1H), 2.07−1.69 (m, 5H). TOF ES+ MS: (M + H)
447.1, (M + Na) 469.1. HPLC tR = 5.97 min, >95% purity.
( )-N-(Benzo[d][1,3]dioxol-5-ylmethyl)-1-(1-(naphthalen-1-
yl)-2-phenylethyl)piperidine-4-carboxamide (1d). Methyl 1-(1-
(naphthalen-1-yl)-2-phenylethyl)piperidine-4-carboxylate 23 (38 mg,
0.10 mmol) was dissolved in a 1:1:2 mixture of THF/EtOH/1 M
aqueous NaOH, total volume of 4 mL. This was stirred at 60 °C for 4
h. The mixture was allowed to cool, and the pH was reduced by
addition of concentrated HCl to ∼pH 4, and the resulting white
precipitate was collected over a filter to give 1-(1-(naphthalen-1-yl)-2-
phenylethyl)piperidine-4-carboxylic acid (30 mg, 82%) as a white
powder. Then the following was added sequentially to anhydrous
DMF (1 mL): 3 Å molecular sieves, 1-(1-(naphthalen-1-yl)-2-
phenylethyl)piperidine-4-carboxylic acid (30 mg, 0.08 mmol), DIEA
(44 μL, 0.25 mmol), EDCI (18 mg, 0.09 mmol), HOBt (14 mg, 0.09
mmol), and piperonylamine (16 μL, 0.13 mmol). This was stirred at
room temperature for 17 h, at which time the material was partitioned
between 10% aqueous Na2CO3 solution and EtOAc, and the organic
layer was washed with 10% aqueous Na2CO3 solution (3 × 20 mL)
and brine (1 × 20 mL). The organic layer was dried with anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue was then
purified by flash chromatography (10 g silica, 50% EtOAc/Hex) to
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give the final product (20 mg, 44%). 1H NMR (500 MHz, chloroform-
d) δ 8.47 (bs, 1H), 7.86−7.80 (m, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.46
(dt, J = 6.3, 3.3 Hz, 2H), 7.31 (bs, 2H), 7.12−7.00 (m, 3H), 6.89 (bs,
2H), 6.77−6.70 (m, 3H), 5.95 (s, 2H), 5.83−5.74 (m, 1H), 4.33 (d, J
= 5.7 Hz, 2H), 3.46 (dd, J = 13.6, 4.8 Hz, 1H), 3.41 (s, 1H), 3.20 (dd,
J = 13.6, 9.1 Hz, 1H), 2.95 (d, J = 9.8 Hz, 1H), 2.15−2.04 (m, 3H),
1.91−1.70 (m, 4H). TOF ES+ MS: (M + H) 493.1, (M + Na) 515.1.
HPLC tR = 6.22 min, >95% purity.
General Amide Coupling Method A. To a solution of HOAT
(0.07 g, 0.35 mmol) in dry DMF (5 mL) were added HATU (0.19 g,
0.49 mmol), (R)-1-(1-(naphthalen-1-yl)ethyl)piperidine-4-carboxylic
acid 25 (0.1 g, 0.35 mmol), and amine (0.42 mmol) followed by DIEA
(0.05 g, 0.35 mmol). The mixture was stirred overnight at room
temperature, then diluted with saturated aqueous NaHCO3 and
extracted with EtOAc (3×). The combined organic extracts were
washed with saturated NaCl (3×), dried (MgSO4), and concentrated.
The residue was purified by silica gel flash chromatography (1−4%
MeOH/DCM).
General Amide Coupling Method B. To a solution of (R)-1-(1-
(naphthalen-1-yl)ethyl)piperidine-4-carboxylic acid 25 (0.06 g, 0.21
mmol) in dry DMF (5 mL) were added EDCI (0.05 g, 0.28 mmol),
HOBt (0.04 g, 0.25 mmol), and DIEA (0.07 mL, 0.53 mmol),
followed by amine (0.21 mmol) and stirred overnight at room
temperature. After this time, the reaction mixture was diluted with
saturated NaHCO3 and extracted with EtOAc (3×). The combined
organic extracts were washed with saturated NaCl (3×), dried
(MgSO4), and concentrated. The residue was purified by silica gel
flash chromatography (1−5% MeOH/DCM).
(R)-N-Benzyl-1-(1-(naphthalen-1-yl)ethyl)piperidine-4-car-
boxamide (2a). Coupling method A: 42% yield. 1H NMR (400 MHz,
DMSO-d6) δ 8.44 (br s, 1H), 7.80−7.90 (m, 1H), 7.75−7.79 (m, 1H),
7.23−7.61 (m, 10H), 5.88 (s, 1H), 5.45 (br s, 1H), 4.32 (m, 2H), 4.20
(br s, 1H), 3.23−3.34 (m, 1H), 2.88−2.91 (m, 2H), 1.84−2.03 (m,
4H), 1.43 (m, 3H). TOF ES+ MS: (M + H) 373.2. HPLC tR = 5.8
min, >95% purity.
(R)-N-(4-Ethylbenzyl)-1-(1-(naphthalen-1-yl)ethyl)-
piperidine-4-carboxamide (3a). Coupling method B: 26% yield.
1H NMR (400 MHz, chloroform-d) δ 8.41 (d, J = 7.9 Hz, 1H), 7.82−
7.83 (m, 1H), 7.71−7.73 (m, 1H), 7.39−7.45 (m, 4H), 7.12−7.16 (m,
4H), 5.68 (m, 1H), 4.37 (d, J = 5.5 Hz, 2H), 3.99−4.20 (m, 1H), 3.22
(d, J = 11.2 Hz, 1H), 2.87 (d, J = 11.2 Hz, 1H), 2.61 (q, J = 7.6 Hz,
2H), 1.91−2.24 (m, 4H), 1.57−1.81 (m, 3H), 1.45 (d, J = 6.7 Hz,
3H), 1.20 (td, J = 7.6, 0.6 Hz, 3H). TOF ES+ MS: (M + H) 401.1.
HPLC tR = 5.9 min, 94% purity.
(R)-N-(4-(Methylcarbamoyl)benzyl)-1-(1-(naphthalen-1-yl)-
ethyl)piperidine-4-carboxamide (3b). Coupling method B: 50%
1
yield. H NMR (400 MHz, chloroform-d) δ 8.38 (s, 1H), 7.76−7.99
(m, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.59−7.70 (m, 2H), 7.53−7.32 (m,
4H), 7.20−7.32 (m, 2H), 6.20 (s, 2H), 4.42 (d, J = 6.0 Hz, 2H), 4.16
(d, J = 16.5 Hz, 1H), 3.22 (d, J = 10.8 Hz, 1H), 2.93−3.09 (m, 2H),
2.91 (d, J = 7.4 Hz, 1H), 1.32−2.28 (m, 9H). TOF ES+ MS: (M + H)
430.2. HPLC tR = 4.7 min, >95% purity.
(R)-N-(3-(Methylcarbamoyl)benzyl)-1-(1-(naphthalen-1-yl)-
ethyl)piperidine-4-carboxamide (3c). To a solution of tert-butyl 3-
(methylcarbamoyl)benzoic acid (0.3 g, 1.1 mmol) in dry DMF (15
mL) were added EDCI (0.26 g, 1.36 mmol), HOBt (0.21 g, 1.36
mmol), and DIEA (0.39 mL, 2.27 mmol), followed by methanamine
(0.03 g, 1.10 mmol). The mixture was stirred overnight at room
temperature. After dilution with saturated aqueous NaHCO3, the
aqueous layer was extracted with EtOAc (2×). The combined organic
layers were washed with saturated NaCl (3×) and dried (MgSO4),
concentrated in vacuo, and purified by flash chromatography (1−4%
1
MeOH/DCM) to provide the intermediate (0.1g, 54%). H NMR
(400 MHz, chloroform-d) δ 8.01 (s, 1H), 7.61−7.69 (m, 2H), 7.28−
7.41 (m, 3H), 4.32 (br s, 2H), 2.99 (d, J = 4.3 Hz, 3H), 1.49 (s, 9H).
TOF ES+ MS: (M + H) 165.1. This material was treated with 4 M
HCl in dioxane and stirred overnight at room temperature. After being
concentrated in vacuo, the crude 3-(aminomethyl)-N-methylbenza-
mide was coupled with 25 using method B to afford the desired
1
compound (0.02 g, 11%). H NMR (400 MHz, chloroform-d) δ 8.40
(s, 1H), 7.84 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.63 (d, J =
13.5 Hz, 2H), 7.37−7.54 (m, 2H), 7.35 (d, J = 4.6 Hz, 1H), 6.20 (m,
1H), 5.87 (m, 1H), 4.44 (d, J = 5.8 Hz, 2H), 4.11 (d, J = 7.2 Hz, 1H),
3.23 (s, 1H), 2.99 (d, J = 4.8 Hz, 2H), 2.91 (s, 1H), 0.75−2.24 (m,
13H). TOF ES+ MS: (M + H) 430.3. HPLC tR = 4.8 min, 92% purity.
(R)-N-(4-Acetamidobenzyl)-1-(1-(naphthalen-1-yl)ethyl)-
piperidine-4-carboxamide (3d). Coupling method B: 23% yield.
1H NMR (400 MHz, chloroform-d) δ 8.42 (d, J = 8.0 Hz, 1H), 7.76−
7.89 (m, 3H), 7.72 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7.33−
7.49 (m, 5H), 6.22 (m, 1H), 5.83 (m, 1H), 4.08 (q, J = 6.5 Hz, 1H),
3.64 (s, 3H), 3.12 (d, J = 11.4 Hz, 1H), 2.81 (d, J = 11.3 Hz, 1H),
2.13−2.40 (m, 1H), 1.95−2.15 (m, 2H), 1.84−1.97 (m, 1H), 1.64−
1.84 (m, 3H), 1.45 (d, J = 6.7 Hz, 3H). TOF ES+ MS: (M + H) 430.3.
HPLC tR = 5.19 min, >95% purity.
1-((R)-1-(Naphthalen-1-yl)ethyl)-N-((R)-1-phenylethyl)-
piperidine-4-carboxamide (2b). Coupling method A: 33% yield.
1H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J = 4.7 Hz, 1H), 7.86−7.88
(m, 1H), 7.76 (d, J = 6.48, 1H), 7.56 (br s, 1H), 7.42−7.48 (m, 3H),
7.32−7.34 (m, 5H), 5.68 (br s, 1H), 5.13−5.16 (m,1H), 4.13 (br s,
1H), 3.25 (d, J = 8.5 Hz, 1H), 2.29 (d, J = 6.4 Hz, 1H), 2.02−2.10 (m,
2H), 1.89−1.91 (m, 1H), 1.67−1.79 (m, 4H), 1.46−1.48 (m 3H).
TOF ES+ MS: (M + H) 387.2. HPLC tR = 5.9 min, >95% purity.
1-((R)-1-(Naphthalen-1-yl)ethyl)-N-((S)-1-phenylethyl)-
piperidine-4-carboxamide (2c). Coupling method A: 23% yield. 1H
NMR (400 MHz, DMSO-d6 and CDCl3) δ 8.22 (m, 1H), 7.86−8.1
(m, 2H), 7.72−7.81 (m, 2H), 7.32−7.48 (m, 4H), 7.32−7.38 (m, 3H),
6.96 (br s, 1H), 5.11 (br s, 1H), 4.89−4.91 (m, 1H), 5.13−5.16
(m,1H), 4.65−4.4.69 (m, 1H), 3.72−3.79 (m, 2H), 3.68−3.72 (m,
1H), 3.68−3.65 (m, 1H), 2.29 (m, 1H), 2.02−2.12 (m, 1H), 1.89−
1.91 (m, 1H),1.62−1.74 (m, 3H), 1.46−1.48 (m 3H). TOF ES+ MS:
(M + H) 387.2. HPLC tR = 5.9 min, >95% purity.
(R)-N-(3-Acetamidobenzyl)-1-(1-(naphthalen-1-yl)ethyl)-
piperidine-4-carboxamide (3e). Coupling method B: 90% yield.
1H NMR (400 MHz, chloroform-d) δ 8.41 (d, J = 7.8 Hz, 1H), 7.78−
7.93 (m, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.30−7.64 (m, 5H), 7.23 (d, J
= 6.6 Hz, 2H), 6.95 (d, J = 7.6 Hz, 1H), 5.80 (s, 1H), 4.36 (d, J = 5.7
Hz, 2H), 3.90−4.25 (m, 2H), 3.21 (d, J = 11.1 Hz, 1H), 2.87 (d, J =
11.1 Hz, 1H), 1.94−2.24 (m, 5H), 1.53−1.94 (m, 2H), 1.45 (d, J = 6.6
Hz, 3H), 1.24 (td, J = 7.1, 0.6 Hz, 2H). TOF ES+ MS: (M + H) 430.1.
HPLC tR = 5.0 min, >95% purity.
(R)-N-(3-(Acetamidomethyl)benzyl)-1-(1-(naphthalen-1-yl)-
ethyl)piperidine-4-carboxamide (3f). To a solution of 1,3-
phenylenedimethanamine (0.2 g, 1.4 mmol) in THF (20 mL) was
added acetic anhydride (0.07 mL, 0.73 mmol) in THF (10 mL)
dropwise. The resulting mixture was stirred overnight at room
temperature, then concentrated, and the residue was purified by silica
gel flash chromatography (1−5% MeOH/DCM), providing N-(3-
(aminomethyl)benzyl)acetamide (0.06 g, 23% yield, TOF ES+ MS,
(M + H) 178.1) which was subsequently coupled with 25 using
method B to afford the final compound (0.02 g, 12%). 1H NMR (400
MHz, chloroform-d) δ 8.42 (d, J = 7.4 Hz, 1H), 7.76−7.94 (m, 1H),
7.73 (m, 1H), 7.49−7.62 (m, 5H), 7.32−7.48 (m, 3H), 4.08 (m, 2H),
N-((R)-2-Methoxy-1-phenylethyl)-1-((R)-1-(naphthalen-1-yl)-
ethyl)piperidine-4-carboxamide (2d). Coupling method B: 60%
yield. 1H NMR (400 MHz, chloroform-d) δ 8.43 (d, J = 8.0 Hz, 1H),
7.82−7.85 (m, 1H), 7.73 (m, 1H), 7.56 (d, J = 7.2 Hz, 1H), 7.43−7.46
(m, 2H), 7.24−7.46 (m, 5H), 6.17 (d, J = 7.6 Hz, 1H), 5.15 (d, J = 7.2
Hz, 1H), 4.09−4.11 (m,1H), 3.63 (d, J = 4.7 Hz, 2H), 3.31 (s, 3H),
3.23 (d, J = 11.3 Hz, 1H), 2.89 (d, J = 11.4 Hz, 1H), 1.99−2.27 (m,
3H), 1.57−1.95 (m, 4H), 1.47 (d, J = 6.7 Hz, 2H). TOF ES+ MS: (M
+ H) 417.2. HPLC tR = 5.6 min, >95% purity.
N-((S)-2-Methoxy-1-phenylethyl)-1-((R)-1-(naphthalen-1-yl)-
ethyl)piperidine-4-carboxamide (2e). Coupling method B: 52%
yield. 1H NMR (400 MHz, chloroform-d) δ 8.43 (d, J = 8.0 Hz, 1H),
7.82−7.84 (m, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.56 (d, J = 7.2 Hz, 1H),
7.35−7.52 (m, 2H), 6.99−7.35 (m, 5H), 6.12 (d, J = 7.7 Hz, 1H), 5.14
(d, J = 7.1 Hz, 1H), 4.10 (m,1H), 3.63 (d, J = 4.7 Hz, 2H), 3.32 (s,
3H), 3.22 (d, J = 11.3 Hz, 1H), 2.88 (d, J = 11.6 Hz, 1H), 1.99−2.28
(m, 3H), 1.95−1.50 (m, 4H), 1.46 (d, J = 6.7 Hz, 2H). TOF ES+ MS:
(M + H) 417.2. HPLC tR = 5.6 min, >95% purity.
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3.64 (s, 3H), 3.12 (d, J = 12.2 Hz, 1H), 2.80 (d, J = 12.2 Hz, 1H),
2.23−2.27 (m, 1H), 1.91−2.06 (m, 2H), 1.64−1.81 (m, 6H), 1.44 (dd,
J = 6.7, 3.7 Hz, 3H). TOF ES+ MS: (M + H) 444.3. HPLC tR = 5.4
min, 94% purity.
(R)-N-(3-Chlorobenzyl)-1-(1-(naphthalen-1-yl)ethyl)-
piperidine-4-carboxamide (3g). Coupling method A: 22% yield.
1H NMR (400 MHz, chloroform-d) δ 7.79−7.81 (m, 2H), 7.70 (s,
1H), 7.23−7.26 (m, 5H), 7.32−7.54 (m, 2H), 7.11−7.13 (m, 1H),
5.81 (br s, 1H), 4.40 (d, J = 5.8 Hz, 2H), 3.86 (s, 1H), 3.57−3.61 (m,
1H), 2.90−2.95 (m, 1H), 2.28−1.93 (m, 6H), 1.45 (d, J = 6.7 Hz,
3H). TOF ES+ MS: (M + H) 407.2. HPLC tR = 4.3 min, >95% purity.
(R)-N-(4-Chlorobenzyl)-1-(1-(naphthalen-1-yl)ethyl)-
piperidine-4-carboxamide (3h). Coupling method B: 29% yield.
1H NMR (400 MHz, chloroform-d) δ 8.39 (s, 1H), 7.79−7.96 (m,
1H), 7.73 (d, J = 8.2 Hz, 1H), 7.32−7.66 (m, 5H), 7.19−7.32 (m,
2H), 7.01−7.19 (m, 2H), 5.73 (br s, 1H), 4.36 (d, J = 5.9 Hz, 2H),
4.11 (br s, 1H), 3.22 (d, J = 10.9, 1H), 2.89 (br s, 1H), 1.73−2.09 (m,
6H), 1.47 (s, 3H). TOF ES+ MS: (M + H) 407.1. HPLC tR = 5.7 min,
>95% purity.
(R)-N-(3,4-Difluorobenzyl)-1-(1-(naphthalen-1-yl)ethyl)-
piperidine-4-carboxamide (3i). Coupling method A: 31% yield. 1H
NMR (400 MHz, chloroform-d) δ 7.79−7.81 (m, 3H), 7.70 (s, 1H),
7.43- 7.51 (m, 3H), 7.03−7.12 (m, 2H), 6.96 (br s, 1H), 5.83 (br s,
1H), 4.37 (d, J = 8.0 Hz, 2H), 3.59−3.64 (m, 1H), 3.32−3.39 (m,
1H), 2.91−2.94 (m, 1H), 1.93−2.28 (m, 3H), 1.75−1.89 (m, 4H),
1.43 (d, J = 6.7 Hz, 3H). TOF ES+ MS: (M + H) 409.2. HPLC tR =
5.78 min, >95% purity.
(R)-N-(4-Fluorobenzyl)-1-(1-(naphthalen-1-yl)ethyl)-
piperidine-4-carboxamide (3j). Coupling method B: 38% yield.
8.42−8.44 (m, 1H), 7.85−7.87 (m, 1H), 7.73−7.74 (d, J = 8.2 Hz,
1H), 7.55−7.56 (m, 1H), 7.46−7.54 (m, 3H), 7.20−7.22 (m, 3H),
7.01−7.11 (m, 2H), 5.73 (s, 1H), 4.38−4.39 (d, J = 4.8 Hz 2H), 4.09−
4.11 (m, 1H), 3.22 (d, J = 8.8 Hz, 1H), 2.89 (d, J = 9.2 Hz, 1H), 1.91−
2.06 (m, 3H), 1.71−1.84 (m, 3H), 1.47 (d, J = 5.3 Hz, 3H). TOF ES+
MS: (M + H) 391.2. HPLC tR = 5.9 min, >95% purity.
(R)-N-(3-Fluorobenzyl)-1-(1-(naphthalen-1-yl)ethyl)-
piperidine-4-carboxamide (3k). Coupling method B: 12% yield.
1H NMR (400 MHz, chloroform-d) δ 8.43 (s, 1H), 7.84−7.85 (m,
1H), 7.73 (d, J = 6.5 Hz, 1H), 7.56−7.57 (m, 1H), 7.41−7.48 (m,
2H), 7.21−7.27 (m, 1H), 7.02−7.06 (m, 1H), 6.93−6.95 (m, 1H),
5.75 (s, 1H), 4.42 (d, J = 4.6 Hz, 2H), 4.12 (br s, 1H), 3.24 (d, J = 7.8,
1H), 2.90 (d, J = 9.7, 1H), 1.99−2.14 (m, 2H), 1.90−1.93 (m, 1H),
1.57−1.80 (m, 5H), 1.46 (s, 3H). TOF ES+ MS: (M + H) 391.2.
HPLC tR = 5.7 min, >95% purity.
Hz, 3H). TOF ES+ MS: (M + H) 392.2. HPLC tR = 4.1 min, >95%
purity.
N-(3-Fluorobenzyl)-1-(1-(quinolin-5-yl)ethyl)piperidine-4-
carboxamide (4b). Ethyl 1-(1-(quinolin-5-yl)ethyl)piperidine-4-
carboxylate 28b (0.67 g, 2.15 mmol) was dissolved in methanol (20
mL), followed by 1 N sodium hydroxide (4.29 mL, 4.29 mmol) in one
portion. The resulting mixture was stirred 18 h at room temperature,
concentrated in vacuo, and partitioned between ether and a minimum
amount of water. The layers were separated, and the aqueous layer was
treated with AcOH dropwise until the solution measured acidic by pH
paper. The solution was concentrated in vacuo, dried under high
vacuum, and the resulting carboxylic acid was used without further
purification in the next step. To a solution of (R,S)-1-(1-(quinolin-5-
yl)ethyl)piperidine-4-carboxylic acid (0.100 g, 0.352 mmol) in dry
DMF (5 mL) were added EDCI (0.067 g, 0.350 mmol), HOBt (0.060
g, 0.420 mmol), and DIEA (0.060 mL, 0.35 mmol) followed by (3-
fluorophenyl)methanamine (0.040 g, 0.350 mmol) . The mixture was
stirred overnight at room temperature, diluted with saturated
NaHCO3, and extracted with EtOAc. The combined organic layers
were washed with saturated NaCl (3×) and dried (MgSO4), then
concentrated and purified by silica gel flash chromatography (1−5%
MeOH/DCM) to provide the target compound (0.05 g, 33%) as a
1
colorless oil. H NMR (400 MHz, chloroform-d) δ 8.84−8.89 (m,
1H), 7.96 (d, J = 8.4 Hz, 1H), 7.42−7.76 (m, 2H), 7.35 (dd, J = 8.6,
4.2 Hz, 1H), 7.13−7.32 (m, 2H), 6.74−7.07 (m, 3H), 6.02−5.97
(s,1H), 4.15−4.39 (q, J = 6.1 Hz, 2H), 4.02 (q, J = 6.7 Hz, 1H), 2.96−
3.23 (m, 1H), 2.65−2.96 (m, 1H), 1.94−2.17 (m, 6H), 1.54−1.88 (m,
4H), 1.44 (d, J = 6.7 Hz, 3H). TOF ES+ MS: (M + H) 392.1. HPLC
tR = 3.9 min, >95% purity.
N-(3-Fluorobenzyl)-1-(1-(isoquinolin-5-yl)ethyl)piperidine-4-
carboxamide (4c). Ethyl 1-(1-(isoquinolin-5-yl)ethyl)piperidine-4-
carboxylate 28c (0.50 g, 1.61 mmol) was dissolved in methanol (20
mL), followed by 1 N sodium hydroxide (3.2 mL, 3.2 mmol) in one
portion. The resulting mixture was stirred for 18 h at room
temperature, concentrated in vacuo, and partitioned between ether
and a minimum amount of water. The layers were separated, and the
aqueous layer was treated with AcOH dropwise until the solution
measured acidic by pH paper. The solution was concentrated in vacuo,
dried under high vacuum, and the resulting carboxylic acid was used
without further purification in the next step. To a solution of (R,S)-1-
(1-(isoquinolin-5-yl)ethyl)piperidine-4-carboxylic acid (0.100 g, 0.350
mmol) in dry DMF (5 mL) were added EDCI (0.067 g, 0.350 mmol),
HOBt (0.070 g, 0.420 mmol), and DIEA (0.061 mL, 0.352 mmol)
followed by (3-fluorophenyl)methanamine (0.044 g, 0.352 mmol).
The mixture was stirred overnight at room temperature, diluted with
saturated NaHCO3, and extracted with EtOAc (2×). The combined
organic extracts were washed with saturated NaCl (3×), dried
(MgSO4), then purified by silica gel flash chromatography (1−5%
MeOH/DCM) to afford the final compound (0.02 g, 16%). 1H NMR
(400 MHz, chloroform-d) δ 9.19 (s, 1H), 8.47 (d, J = 6.1 Hz, 1H),
8.18 (d, J = 6.1 Hz, 1H), 7.62−7.93 (m, 2H), 7.42−7.62 (m, 1H),
7.10−7.42 (m, 1H), 6.64−7.10 (m, 3H), 5.98 (t, J = 5.8 Hz, 1H), 4.39
(d, J = 5.9 Hz, 2H), 4.02 (q, J = 6.7 Hz, 1H), 3.14 (m, 1H), 2.56−3.04
(m, 1H), 1.89−2.37 (m, 3H), 1.53−1.89 (m, 3H), 1.42 (d, J = 6.6 Hz,
3H). TOF ES+ MS: (M + H) 392.1. HPLC tR = 4.0 min, >95% purity.
N-(3-Fluorobenzyl)-1-(1-(isoquinolin-1-yl)ethyl)piperidine-4-
carboxamide (4d). To a solution of 27d (50 mg, 0.16 mmol) in
MeOH (10 mL) was added NaOH (11 mg, 0.48 mmol), and the
mixture was allowed to stir at 50 °C for 6 h. The solution was
concentrated in vacuo and acidified with 4 M HCl in dioxane (5 mL).
The solution was again concentrated in vacuo and allowed to dry
overnight. The crude carboxylic acid was dissolved in DMF (10 mL),
and EDCI (49 mg, 0.38 mmol), HOBt (43 mg, 0.32 mmol), DIEA
(103 mg, 0.80 mmol), and 3-fluorobenzylamine (24 mg, 0.20 mmol)
were added and allowed to stir at 23 °C for 16 h. The reaction was
quenched with saturated NaHCO3 (25 mL), washed with H2O (2 ×
20 mL), brine (1 × 20 mL), dried (MgSO4), and concentrated in
vacuo. The residue was purified by flash chromatography (1−4%
MeOH/DCM) to furnish the desired material (23 mg, 37.1%) as a
dark brown oil. 1H NMR (400 MHz, chloroform-d) δ 8.66 (d, J = 8.5
N-(3-Fluorobenzyl)-1-(1-(quinolin-8-yl)ethyl)piperidine-4-
carboxamide (4a). Ethyl 1-(1-(quinolin-8-yl)ethyl)piperidine-4-
carboxylate 28a (0.38 g, 1.30 mmol) was dissolved in methanol (20
mL), followed by the addition of 1 N sodium hydroxide (2.6 mL, 2.6
mmol) in one portion. The resulting mixture was stirred for 18 h at
room temperature, then concentrated in vacuo and partitioned
between ether and a minimum amount of water. The layers were
separated, and the aqueous layer was treated with AcOH dropwise
until the solution was acidic by pH paper. The solution was then
concentrated in vacuo and dried under high vacuum overnight at room
temperature, and the resulting acid was used without further
purification. To a solution of (R,S)-1-(1-(quinolin-8-yl)ethyl)-
piperidine-4-carboxylic acid (0.1 g, 0.35 mmol) in dry DMF (5 mL)
was added EDCI (0.07 g, 0.35 mmol), HOBt (0.07 g, 0.42 mmol), and
DIEA (0.06 mL, 0.35 mmol) followed by (3-fluorophenyl)-
methanamine (0.04 g, 0.35 mmol). This reaction mixture was stirred
overnight at room temperature, after which it was diluted with
saturated NaHCO3 and extracted with EtOAc (1×). The combined
organic layers were washed with saturated NaCl (3×) and dried
(MgSO4), then concentrated and purified by silica flash chromatog-
1
raphy (1−5% MeOH/DCM) to obtain 4a (0.05 g, 33% yield). H
NMR (400 MHz, DMSO-d6) δ 8.75−8.98 (m, 2H), 8.35 (m, 1H),
8.27 (t, J = 6.0 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.60−7.72 (m, 1H),
7.42−7.59 (m, 2H), 7.21−7.42 (m, 2H), 7.00 (ddd, J = 23.3, 16.7, 9.5
Hz, 2H), 3.84−4.40 (m, 4H), 2.94 (d, J = 10.8 Hz, 1H), 2.76 (d, J =
10.8 Hz, 1H), 1.88−2.30 (m, 2H), 1.39−1.76 (m, 2H), 1.37 (d, J = 6.6
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Hz, 1H), 8.44 (d, J = 5.7 Hz, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.65 (t, J =
7.9 Hz, 1H), 7.59−7.49 (m, 2H), 7.09−6.96 (m, 2H), 6.92 (t, J = 8.3
Hz, 2H), 5.81 (dd, J = 7.5, 1.9 Hz, 1H), 4.40 (d, J = 5.8 Hz, 2H), 3.09
(d, J = 10.1 Hz, 1H), 2.91 (d, J = 11.5 Hz, 1H), 2.36−2.26 (m, 1H),
2.18−2.07 (m, 2H), 1.77 (dd, J = 8.5, 3.5 Hz, 3H), 1.53 (d, J = 6.7 Hz,
3H). TOF ES+ MS: (M + H) 392.2. HPLC tR = 5.2 min, >95% purity.
(R)-1-(1-(Naphthalen-1-yl)ethyl)-N-(pyridin-3-ylmethyl)-
piperidine-4-carboxamide (5a). Coupling method A: 50% yield.
1H NMR (400 MHz, chloroform-d) δ 8.48−8.51 (m, 2H), 7.83 (d, J =
7.8 Hz, 3H), 7.76 (s, 1H), 7.47−7.53 (m, 4H), 7.23−7.25 (m, 1H),
6.08 (s, 1H), 4.44−4.45 (m, 2H), 3.26 (d, J = 11.5 Hz, 1H), 3.04 (d, J
= 11.5 Hz, 1H), 2.11−2.48 (m, 2H), 1.63−2.11 (m, 7H), 1.48 (s, 3m).
TOF ES+ MS: (M + H) 374.2. HPLC tR = 4.6 min, >95% purity.
(R)-1-(1-(Naphthalen-1-yl)ethyl)-N-(pyridin-4-ylmethyl)-
piperidine-4-carboxamide (5b). Coupling method A: 27% yield.
1H NMR (400 MHz, chloroform-d) δ 8.51 (d, J = 5.1 Hz, 2H), 7.79−
7.83 (3H), 7.71 (s, 1H), 7.43−7.54 (m, 3H), 7.13 (d, J = 5.1 Hz, 2H),
6.06 (t, J = 6.2 Hz, 1H), 4.43 (d, J = 6.1 Hz, 2H), 3.63 (q, J = 6.7 Hz,
1H), 3.17−3.20 (m, 1H), 2.92- 2.95 (m, 1H), 1.96- 2.16 (m, 4H),
1.65−1.91 (m, 3H), 1.47 (d, J = 6.6 Hz, 3H). TOF ES+ MS: (M + H)
374.2. HPLC tR = 4.6 min, >95% purity.
(R)-N-((2-Methoxypyridin-4-yl)methyl)-1-(1-(naphthalen-1-
yl)ethyl)piperidine-4-carboxamide (5c). Coupling method B: 26%
yield. 1H NMR (400 MHz, chloroform-d) δ 8.38 (br s, 1H), 8.06 (d, J
= 5.3 Hz, 1H), 7.69−7.91 (m, 3H), 7.58 (br s, 1H), 7.45 (m, 3H), 6.72
(m, 1H), 6.56 (s, 2H), 5.85 (br s, 1H), 4.36 (d, J = 6.0 Hz, 2H), 4.10
(m, 1H), 3.89 (s, 3H), 3.25 (m, 1H), 2.94 (m, 1H), 1.58−2.15 (m,
5H), 1.41 (m, 3H). TOF ES+ MS: (M + H) 404.1. HPLC tR = 4.5
min, >95% purity.
2.08 (ddt, J = 11.7, 8.0, 3.9 Hz, 1H), 2.01−1.91 (m, 1H), 1.85 (d, J =
10.5 Hz, 1H), 1.74 (dd, J = 10.8, 5.5 Hz, 2H), 1.61 (d, J = 12.1 Hz,
1H), 1.42−1.30 (m, 1H). TOF ES+ MS: (M + H) 417.2. HPLC tR =
5.7 min, >95% purity.
2-Methoxy-1-(naphthalen-1-yl)ethanone (9c). 2-Methoxyace-
tonitrile 8c (0.25 mL, 3.33 mmol) was dissolved in a 250 mM solution
of naphthalen-1-ylmagnesium bromide (20 mL, 5.0 mmol) in THF
and stirred for 14 h at room temperature. 2 M HCl (10 mL) was
added, and this was stirred at room temperature for 8 h. At this time,
material was extracted with a 1:1 solution EtOAc/Et2O, dried with
anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo.
The residue was purified by flash chromatography (20 g silica, 5−20%
1
EtOAc/Hex) to give the desired product (118 mg, 18%). H NMR
(500 MHz, chloroform-d) δ 8.66 (d, J = 8.6 Hz, 1H), 8.05 (d, J = 8.2
Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.86 (d, J = 7.2 Hz, 1H), 7.64 (t, J =
7.7 Hz, 1H), 7.61−7.55 (m, 1H), 7.53 (t, J = 7.7 Hz, 1H), 4.74 (s,
2H), 3.57 (s, 3H).
( )-1-(Naphthalen-1-yl)propan-1-ol (10a). 1-(Naphthalen-1-
yl)propan-1-one 9a (100 mg, 0.54 mmol), prepared from nitrile 8a
by the literature method,17 was dissolved in anhydrous THF (1 mL) in
dry glassware and cooled to −78 °C. LAH (1 M in THF, 0.27 mL,
0.27 mmol) was added dropwise to the solution. The mixture was
allowed to warm to room temperature and stirred for 4 h or until
complete by TLC. The reaction was then worked up according to the
Fieser method,5 and the resulting precipitate was filtered off. The
filtrate was collected and concentrated in vacuo. The residue was then
purified by flash chromatography (15 g silica, 20% EtOAc/Hex) to
provide the desired product (85 mg, 85%). 1H NMR (500 MHz,
chloroform-d) δ 8.13 (d, J = 7.8 Hz, 1H), 7.94−7.89 (m, 1H), 7.81 (d,
J = 8.2 Hz, 1H), 7.65 (d, J = 7.1 Hz, 1H), 7.57−7.47 (m, 3H), 5.44−
5.34 (m, 1H), 2.32 (s, 1H), 2.09−2.00 (m, 1H), 1.99−1.89 (m, 1H),
1.06 (t, J = 7.4 Hz, 3H).
(R)-N-(4-Chlorophenethyl)-1-(1-(naphthalen-1-yl)ethyl)-
piperidine-4-carboxamide (6a). Coupling method B: 33% yield.
1H NMR (400 MHz, chloroform-d) δ 8.41 (d, J = 7.5 Hz, 1H), 7.83
(dd, J = 7.1, 2.1 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.54 (m, 1H),
7.29−7.50 (m, 3H), 7.13−7.33 (m, 3H), 6.86−7.18 (m, 2H), 5.41 (s,
1H), 4.08 (s, 1H), 3.45 (q, J = 6.8 Hz, 1H), 3.18 (d, J = 11.2 Hz, 1H),
2.76−2.98 (m, 1H), 2.38−2.76 (m, J = 6.8 Hz, 2H), 1.52−2.16 (m,
7H), 1.45 (d, J = 6.8 Hz, 3H). TOF ES+ MS: (M + H) 421.1. HPLC
tR = 6.0 min, >95% purity.
(R)-N-(3-Fluorophenethyl)-1-(1-(naphthalen-1-yl)ethyl)-
piperidine-4-carboxamide (6b). Coupling method B: 36% yield.
1H NMR (400 MHz, chloroform-d) δ 8.42 (d, J = 7.8 Hz, 1H), 7.77−
8.04 (m, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.48−7.65 (m, 1H), 7.32−7.48
(m, 3H), 7.18−7.32 (m, 1H), 6.65−6.99 (m, 3H), 5.42 (s, 1H), 4.08
(d, J = 5.9 Hz, 1H), 3.37−3.56 (m, 1H), 3.19 (d, J = 11.2 Hz, 1H),
2.78−2.90 (m, 1H), 2.77 (s, 1H), 1.87−2.09 (m, 5H), 1.54−1.86 (m,
4H), 1.45 (d, J = 6.6 Hz, 3H). TOF ES+ MS: (M + H) 405.2. HPLC
tR = 5.8 min, >95% purity.
( )-2-Methoxy-1-(naphthalen-1-yl)ethanol (10c). 2-Methoxy-
1-(naphthalen-1-yl)ethanone 9c (118 mg, 0.59 mmol) was dissolved in
anhydrous Et2O (6 mL) in an ice bath, and a 1 M (in THF) solution
of LAH (0.59 mL, 0.59 mmol) was added under N2. This was stirred at
room temperature for 3 h, at which time the Fieser workup5 was
employed. Precipitate was filtered off and the filtrate concentrated in
vacuo to give the desired product as an oil (104 mg, 87%) used
1
without further purification. H NMR (500 MHz, chloroform-d) δ
8.09 (d, J = 8.3 Hz, 1H), 7.93−7.89 (m, 1H), 7.83 (d, J = 8.2 Hz, 1H),
7.79 (d, J = 7.1 Hz, 1H), 7.58−7.50 (m, 3H), 5.75 (dd, J = 8.9, 2.5 Hz,
1H), 3.80 (dd, J = 10.1, 2.8 Hz, 1H), 3.57 (dd, J = 9.9, 9.0 Hz, 1H),
3.50 (s, 3H), 3.30 (bs, 1H).
( )-Ethyl 1-(1-(Naphthalen-1-yl)propyl)piperidine-4-carbox-
ylate (11a). 1-(Naphthalen-1-yl)propan-1-ol 10a (326 mg, 1.75
mmol) was dissolved in DCM (4 mL) at 0 °C with 3 Å molecular
sieves. DIEA (0.92 mL, 5.25 mmol) was added, and with continuous
stirring at 0 °C, Ms2O (396 mg, 2.27 mmol) was added dropwise. The
solution was stirred for 40 min at 0 °C, at which point ethyl piperidine-
4-carboxylate (1.62 mL, 10.5 mmol) was added. The mixture was then
stirred for 48 h at room temperature. At that time, EtOAc was added
and washed with 10% aqueous Na2CO3 (3 × 25 mL). The organic
layer was then dried (MgSO4), filtered, and removed in vacuo.
Purification was accomplished via flash chromatography (20 g silica,
gradient 0−40% EtOAc/Hex) to give the desired product (480 mg,
N-(3-Fluorobenzyl)-1-(1,2,3,4-tetrahydrophenanthren-4-yl)-
piperidine-4-carboxamide (7). To a solution of 33 (97 mg, 0.23
mmol) in THF/MeOH/H2O (3:1:1) (7 mL) at 0 °C was added LiOH
(19 mg, 0.45 mmol), and the mixture was allowed to stir at room
temperature for 16 h. The reaction mixture was concentrated in vacuo,
acidified using HCl−dioxane, and crystallized with the addition of
EtOAc (5 mL). The product was filtered to furnish the carboxylic acid
(65 mg, 70%) as a white solid. This acid was carried forward without
purification and was dissolved (63 mg, 0.2 mmol) in dry DMF (5 mL),
followed by EDCI (41 mg, 0.27 mmol), HOBt (37 mg, 0.27 mmol),
DIEA (133 mg, 1.03 mmol), and 3-fluorobenzylamine (28 mg, 0.23
mmol), and the reaction mixture was allowed to stir at room
temperature for 16 h. The reaction mixture was then diluted with
EtOAc, quenched with saturated NaHCO3, washed with H2O (2 × 10
mL), saturated NaCl (1 × 10 mL), dried (MgSO4), and concentrated
in vacuo. The residue was purified by silica gel flash chromatography
1
84%) as a slightly yellow oil. H NMR (500 MHz, chloroform-d) δ
8.48 (bs, 1H), 7.90−7.86 (m, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.54−7.43
(m, 4H), 4.14 (q, J = 7.1 Hz, 2H), 3.94 (bs, 1H), 3.29−3.16 (m, 1H),
2.87−2.75 (m, 1H), 2.27 (tt, J = 11.3, 4.1 Hz, 1H), 2.12−1.93 (m,
5H), 1.85−1.66 (m, 3H), 1.26 (t, J = 7.1 Hz, 3H), 0.70 (t, J = 7.3 Hz,
3H).
( )-Ethyl 1-(2-Methoxy-1-(naphthalen-1-yl)ethyl)piperidine-
4-carboxylate (11c). 2-Methoxy-1-(naphthalen-1-yl)ethanol 10c
(104 mg, 0.51 mmol) was dissolved in DCM (4 mL) at 0 °C, and
the following was added sequentially: 3 Å molecular sieves, DIEA
(0.27 mL, 1.54 mmol), and Ms2O (116 mg, 0.67 mmol). This was
stirred for 40 min at 0 °C, at which time ethyl piperidine-4-carboxylate
(0.48 mL, 3.09 mmol) was added. This was stirred for 48 h at room
temperature. The material was extracted with EtOAc, and this was
1
(40−60% EtOAc/Hex) to furnish 7 (26 mg, 30%) as a dark oil. H
NMR (400 MHz, chloroform-d): δ 8.37 (d, J = 8.4 Hz, 1H), 7.74 (d, J
= 8.0 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.40 (dt, J = 21.1, 7.2 Hz,
2H), 7.24 (d, J = 4.5 Hz, 2H), 7.16 (d, J = 8.4 Hz, 1H), 6.95 (dd, J =
28.2, 8.1 Hz, 3H), 5.68 (s, 1H), 4.46 (t, J = 4.4 Hz, 1H), 4.39 (d, J =
5.8 Hz, 2H), 3.02−2.87 (m, 2H), 2.84−2.74 (m, 1H), 2.58 (ddd, J =
14.2, 9.9, 4.3 Hz, 2H), 2.33 (t, J = 10.7 Hz, 1H), 2.27−2.14 (m, 1H),
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washed with 10% Na2CO3 (3 × 25 mL). The organic layer was dried
over MgSO4, filtered, and the filtrate was concentrated in vacuo. The
residue was purified by flash chromatography (20 g silica, 0−10%
0.338 mmol), EDCI (42.2 mg, 0.220 mmol), and HOBt (33.7 mg,
0.220 mmol) in anhydrous DMF (1 mL) over 3 Å molecular sieves.
Piperonylamine (0.032 mL, 0.254 mmol) was added, and the mixture
was allowed to stir for 32 h at room temperature. After this time, a 1:1
solution of Et2O/EtOAc (25 mL) was added and washed with 10%
aqueous Na2CO3 (3 × 20 mL) and brine (1 × 10 mL), dried with
anhydrous MgSO4, and concentrated in vacuo. The crude oil was
purified by flash chromatography (25 g silica, 10% EtOAc/Hex) to
give the desired product (46 mg, 50%) as an oil. 1H NMR (500 MHz,
chloroform-d) δ 8.39 (d, J = 7.4 Hz, 1H), 7.87 (d, J = 9.1 Hz, 1H),
7.77 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 6.9 Hz, 1H), 7.53−7.39 (m, 3H),
6.80−6.69 (m, 3H), 5.96 (s, 2H), 5.73 (bs, 1H), 4.35 (d, J = 5.6 Hz,
2H), 4.17−4.05 (m, 2H), 3.88 (dd, J = 10.5, 4.0 Hz, 1H), 3.46 (d, J =
11.1 Hz, 1H), 2.85 (d, J = 11.3 Hz, 1H), 2.28 (t, J = 10.7 Hz, 1H),
2.19−2.09 (m, 1H), 2.09−2.01 (m, 1H), 1.95−1.72 (m, 4H), 0.81 (s,
9H), −0.12 (s, 3H), −0.15 (s, 3H).
1-(Naphthalen-1-yl)-2-phenylethanone (19). 1-Naphthonitrile
18 (1.00 g, 6.53 mmol) was dissolved in anhydrous Et2O (20 mL) in
dry glassware, followed by the addition of benzylmagnesium chloride
(7.83 mL, 7.83 mmol) under N2. This was stirred at room temperature
for 18 h. At this time, 1 M HCl was added (5 mL) and the mixture was
stirred at 90 °C for 35 min (during which time it was necessary to add
more Et2O), then allowed to cool to room temperature, at which point
material was extracted by EtOAc/Et2O, dried with anhydrous MgSO4
and the filtrate was concentrated in vacuo. The resulting residue was
purified by flash chromatography (20 g silica, 10% EtOAc/Hex) to
give the desired ketone (1.35 g, 84%). 1H NMR (500 MHz,
chloroform-d) δ 8.60 (d, J = 8.8 Hz, 1H), 8.00 (ddd, J = 9.8, 7.1, 1.1
Hz, 2H), 7.90 (d, J = 7.3 Hz, 1H), 7.62−7.50 (m, 3H), 7.38−7.28 (m,
5H), 4.41 (s, 2H).
1
EtOAc/Hex gradient) to give the desired product (91 mg, 52%). H
NMR (500 MHz, chloroform-d) δ 8.41 (d, J = 8.2 Hz, 1H), 7.92−7.86
(m, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.63 (d, J = 7.0 Hz, 1H), 7.57−7.44
(m, 3H), 4.33−4.19 (m, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.95 (dd, J =
10.3, 6.2 Hz, 1H), 3.69 (dd, J = 10.3, 3.7 Hz, 1H), 3.33 (s, 3H), 3.32−
3.25 (m, 1H), 2.86−2.77 (m, 1H), 2.38−2.23 (m, 2H), 2.14 (t, J =
10.2 Hz, 1H), 2.03−1.93 (m, 1H), 1.91−1.69 (m, 3H), 1.27 (t, J = 7.1
Hz, 3H).
(
)-1-(Naphthalen-1-yl)ethane-1,2-diol (13). 2-Hydroxy-1-
(naphthalen-1-yl)ethanone 12 (200 mg, 1.07 mmol), prepared
previously by the literature method,18 was dissolved in anhydrous
THF (2 mL) and was cooled in an ice bath. 1 M LAH in THF (1.07
mL, 1.07 mmol) was slowly added dropwise to the solution. The
mixture was then allowed to warm to room temperature and was
stirred for 2 h. After this time, the reaction was worked up by the
Fieser method,27 the aluminum was filtered off, and the filtrate solvent
was removed in vacuo to give a white solid. This was purified by flash
chromatography (10 g silica, 50% EtOAc/Hex) to give the desired
product (168 mg, 83%) as a white solid. 1H NMR (500 MHz, DMSO-
d6) δ 8.16 (d, J = 8.3 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.1
Hz, 1H), 7.66 (d, J = 7.1 Hz, 1H), 7.57−7.48 (m, 3H), 5.44 (d, J = 4.2
Hz, 1H), 5.33 (dt, J = 7.7, 4.0 Hz, 1H), 4.87 (t, J = 5.8 Hz, 1H), 3.66
(ddd, J = 10.1, 6.0, 4.0 Hz, 1H), 3.50 (ddd, J = 11.3, 7.4, 5.9 Hz, 1H).
(
)-2-((tert-Butyldimethylsilyl)oxy)-1-(naphthalen-1-yl)-
ethanol (14). 1-(Naphthalen-1-yl)ethane-1,2-diol 13 (280 mg, 1.49
mmol) was dissolved in anhydrous DMF (8 mL), followed by
imidazole (253 mg, 3.72 mmol) and TBSCl (247 mg, 1.64 mmol).
This was stirred at room temperature for 4 h, after which time the
mixture was partitioned between H2O (25 mL) and EtOAc (25 mL)
and extracted. The extract was washed with H2O (1 × 20 mL) and
brine (1 × 20 mL) and dried over MgSO4 and concentrated in vacuo.
The crude residue was then purified by flash chromatography (10%
EtOAc/Hex) to give the silyl ether (445 mg, 99%) as a nearly colorless
oil. 1H NMR (500 MHz, methanol-d4) δ 8.12 (d, J = 8.3 Hz, 1H), 7.82
(d, J = 8.1 Hz, 1H), 7.76−7.69 (m, 2H), 7.49−7.39 (m, 3H), 5.55 (dd,
J = 6.6, 4.5 Hz, 1H), 4.94 (d, J = 4.2 Hz, 1H), 3.96−3.83 (m, 2H), 0.84
(s, 9H), −0.04 (s, 3H), −0.09 (s, 3H).
(
)-Dimethyl 1-(1-(Naphthalen-1-yl)-2-phenylethyl)-
pyridine-4,4(1H)-dicarboxylate (21). Dimethyl 2,2-bis((1,3-dioxo-
lan-2-yl)methyl)malonate (578 mg, 1.90 mmol) was dissolved in a 1:1
mixture of THF (10 mL) to 10% aqueous HCl (10 mL) and stirred at
room temperature for 18 h. At this time, the reaction was neutralized
with solid NaHCO3 and a solution of 1-(naphthalen-1-yl)-2-phenyl-
ethanamine 20 (446 mg, 1.80 mmol), prepared previously by the
literature method19 from 19, in THF (6 mL) was added. The mixture
was stirred at room temperature for 20 h. At this time, the solution was
extracted with EtOAc (2 × 50 mL) and the combined extracts were
dried over anhydrous MgSO4 and the filtrate was concentrated in
vacuo. The residue was then purified via flash chromatography (15 g
silica, 15% EtOAc/Hex) to give the desired condensed product (470
mg, 58%) as a yellow oil. 1H NMR (300 MHz, chloroform-d) δ 7.92−
7.85 (m, 3H), 7.66 (d, J = 0.9 Hz, 1H), 7.57−7.48 (m, 3H), 7.35−7.26
(m, 5 H), 6.08 (d, J = 8.5 Hz, 2H), 5.21 (dd, J = 4.8, 9.7 Hz, 1H), 4.69
(d, J = 8.5 Hz, 2 H), 3.72 (s, 6H), 3.56−3.35 (m, 2 H).
( )-Ethyl 1-(2-((tert-Butyldimethylsilyl)oxy)-1-(naphthalen-
1-yl)ethyl)piperidine-4-carboxylate (16). 2-((tert-
Butyldimethylsilyl)oxy)-1-(naphthalen-1-yl)ethanol 14 (320 mg, 1.06
mmol) was dissolved in DCM (6 mL) with 3 Å molecular sieves,
followed by DIEA (0.28 mL, 1.59 mmol). MsCl (0.10 mL, 1.27 mmol)
was added dropwise, and the mixture was allowed to stir at room
temperature for 3 h. Ethyl piperidine-4-carboxylate (0.82 mL, 5.29
mmol) was then added dropwise and the mixture stirred for 23 h at
room temperature. A 1:1 solution of Et2O/EtOAc (30 mL) was added.
The solution was washed with aqueous 10% aqueous Na2CO3 (3 × 20
mL) and brine (1 × 15 mL), dried with anhydrous MgSO4, filtered,
and the filtrate was concentrated in vacuo. The crude residue was then
purified by flash chromatography (20 g silica, 10% EtOAc/Hex) to
give the desired product (276 mg, 59%) as an oil. 1H NMR (500 MHz,
chloroform-d) δ 8.41 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 7.2 Hz, 1H),
7.77 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 7.0 Hz, 1H), 7.51−7.41 (m, 3H),
4.17−4.11 (m, 3H), 4.11−4.07 (m, 1H), 3.91−3.86 (m, 1H), 3.42−
3.33 (m, 1H), 2.85−2.76 (m, 1H), 2.35−2.24 (m, 2H), 2.12 (t, J =
11.3 Hz, 1H), 2.01−1.92 (m, 1H), 1.88−1.64 (m, 3H), 1.26 (t, J = 7.1
Hz, 3H), 0.81 (s, 9H), −0.12 (s, 3H), −0.15 (s, 3H).
(
)-Dimethyl 1-(1-(Naphthalen-1-yl)-2-phenylethyl)-
piperidine-4,4-dicarboxylate (22). Dimethyl 1-(1-(naphthalen-1-
yl)-2-phenylethyl)pyridine-4,4(1H)-dicarboxylate 21 (80 mg, 0.187
mmol) was dissolved in N2-sparged MeOH (5 mL), and 10% Pd/C
was added. This was mixed under 40 psi of H2 for 8 h at room
temperature. This was then filtered through Celite and the filtrate
concentrated in vacuo to give the desired product (57 mg, 71%)
1
without further purification. H NMR (500 MHz, chloroform-d) δ
8.51 (bs, 1H), 7.87−7.79 (m, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.50−7.43
(m, 2H), 7.29 (bs, 2H), 7.10−7.01 (m, 3H), 6.82 (bs, 2H), 4.13 (s,
1H), 3.74 (s, 6H), 3.44 (dd, J = 13.5, 4.6 Hz, 1H), 3.17 (dd, J = 13.5,
9.3 Hz, 1H), 2.72 (bs, 2H), 2.51 (bs, 2H), 2.29−2.08 (m, 4H).
(
)-Methyl 1-(1-(Naphthalen-1-yl)-2-phenylethyl)-
piperidine-4-carboxylate (23). Dimethyl 1-(1-(naphthalen-1-yl)-2-
phenylethyl)piperidine-4,4-dicarboxylate 22 (103 mg, 0.24 mmol) was
dissolved in anhydrous DMF (3 mL), and NaCN (17.6 mg, 0.36
mmol) was added to the solution. This was stirred at 145 °C for 16 h.
The mixture was allowed to cool. H2O was added, and material was
extracted with EtOAc (3 × 50 mL). The combined organic extracts
were dried with anhydrous MgSO4 and concentrated in vacuo. The
resulting residue was purified via flash chromatography (10 g silica,
(
)-N-(Benzo[d][1,3]dioxol-5-ylmethyl)-1-(2-((tert-
butyldimethylsilyl)oxy)-1-(naphthalen-1-yl)ethyl)piperidine-4-
carboxamide (17). Ethyl 1-(2-((tert-butyldimethylsilyl)oxy)-1-
(naphthalen-1-yl)ethyl)piperidine-4-carboxylate 16 (100 mg, 0.23
mmol) was dissolved in ethanol (6 mL) and 7 M aqueous NaOH
(3 mL) and stirred at 40 °C for 3 h. The mixture was then cooled to 0
°C and acidified (∼pH 2) with concentrated HCl. The resulting white
precipitate was collected over filter and dried under high vacuum
overnight to give the desired carboxylic acid (70 mg, 75%) as a white
powder. The crude solid was added to a solution of DIEA (0.059 mL,
1
20% EtOAc/Hex) to give the desired monoester (38 mg, 43%). H
NMR (500 MHz, chloroform-d) δ 8.49 (s, 1H), 7.83 (dt, J = 6.9, 3.5
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Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.47 (dt, J = 6.4, 3.3 Hz, 2H), 7.37−
7.22 (m, 2H), 7.11−6.99 (m, 3H), 6.89 (s, 2H), 4.27 (s, 1H), 3.68 (s,
3H), 3.46 (dd, J = 13.6, 4.9 Hz, 1H), 3.31 (s, 1H), 3.20 (dd, J = 13.5,
9.1 Hz, 1H), 2.90 (d, J = 11.5 Hz, 1H), 2.28 (tt, J = 11.2, 4.0 Hz, 1H),
2.22−2.04 (m, 2H), 1.93 (d, J = 13.1 Hz, 1H), 1.84−1.66 (m, 3H).
1-(Quinolin-8-yl)ethanol (27a). To a −78 °C solution of
quinoline-8-carbaldehyde 26a (0.5 g, 3.18 mmol) in dry THF under
N2 was added methylmagnesium chloride (3.82 mL, 3.82 mmol)
dropwise. The resulting mixture was stirred 30 min before warming to
room temperature. NH4Cl was added, and the mixture was stirred for
10 min before diluting with EtOAc/Et2O and washing with saturated
NaCl and drying over MgSO4. The residue was purified by flash
chromatography (10−40% EtOAc/Hex) to afford the desired
1.27 mmol, 47% yield) was added in one portion. The resulting
mixture was stirred overnight at room temperature, after which time
the mixture was diluted with aqueous NaHCO3 and extracted with
DCM. The extracts were dried over MgSO4, concentrated, and
purified by flash chromatography (10−50% EtOAc/Hex) to provide
28a as a clear oil (0.39 g, 47%). 1H NMR (400 MHz, chloroform-d) δ
8.87 (d, J = 4.2, 1H), 8.53 (d, J = 8.6 Hz, 1H), 7.90−8.13 (m, 1H),
7.60−7.81 (m, 2H), 7.40 (dd, J = 8.6, 4.2 Hz, 1H), 5.59 (q, J = 6.5 Hz,
1H), 3.28−3.30 (m. 1H), 2.77−2.81 (m, 1H), 2.22−2.30 (m, 1H),
1.92−1.30 (m, 8H), 1.40 (d, J = 6.5 Hz, 3H), 1.23 (dd, J = 6.6, 0.4 Hz,
3H).
Ethyl 1-(1-(Quinolin-5-yl)ethyl)piperidine-4-carboxylate
(28b). To a 0 °C solution of 1-(quinolin-5-yl)ethanol 27b (0.47 g,
2.70 mmol) in dry DCM was added DIEA (1.0 g, 8.14 mmol) followed
by Ms2O (0.71 g, 4.07 mmol). The resulting mixture was stirred for 40
min at 0 °C, and then ethyl piperidine-4-carboxylate (0.70 g, 83%
yield) was added in one portion. The resulting mixture was stirred
overnight at room temperature, after which time the mixture was
diluted with NaHCO3 and extracted with DCM. The extracts were
dried over MgSO4, concentrated, and purified by flash chromatog-
raphy (10−50% EtOAc/Hex) to give the desired compound (0.70 g,
1
compound (0.435 g, 79%). H NMR (400 MHz, chloroform-d) δ
8.84 (dd, J = 4.3, 1.9 Hz, 1H), 8.18 (dd, J = 8.3, 1.9 Hz, 1H), 7.72 (dd,
J = 8.1, 1.6 Hz, 1H), 7.32−7.62 (m, 3H), 6.09 (s, 1H), 5.44 (q, J = 6.7
Hz, 1H), 1.73 (dd, J = 6.6, 0.4 Hz, 3H).
1-(Quinolin-5-yl)ethanol (27b). To a −78 °C solution of
quinoline-5-carbaldehyde 26b (0.82 g, 5.2 mmol) in dry THF under
N2 was added methylmagnesium chloride (6.3 mL, 6.3 mmol)
dropwise. The resulting mixture was stirred for 30 min before warming
to room temperature. Saturated aqueous NH4Cl was added, and the
mixture was stirred for 10 min before being diluted with EtOAc/Et2O,
washed with saturated NaCl, and dried over MgSO4. The residue was
purified by silica flash chromatography (10−40% EtOAc/Hex) to
1
83%) as a clear oil. H NMR (400 MHz, chloroform-d) δ 8.87−8.91
(m, 1H), 7.98 (d, J = 8.5 Hz, 1H), 7.47−7.67 (m, 2H), 7.36 (m, 2H),
3.87−4.33 (m, 2H), 3.02 (d, J = 11.2 Hz, 1H), 2.76 (d, J = 11.2 Hz,
1H), 2.24 (m, 1H), 1.92−2.19 (m, 2H), 1.52−1.92 (m, 5H), 1.45 (dd,
J = 6.7, 0.7 Hz, 3H), 1.21 (td, J = 7.1, 0.7 Hz, 3H).
1
afford the target compound (0.77 g, 86%) as a yellow oil. H NMR
Ethyl 1-(1-(Isoquinolin-5-yl)ethyl)piperidine-4-carboxylate
(28c). To a 0 °C solution of 1-(isoquinolin-5-yl)ethanol 27c (0.47
g, 2.71 mmol) in dry DCM was added DIEA (1.05 g, 8.14 mmol)
followed by Ms2O (0.71 g, 4.07 mmol). The resulting mixture was
stirred for 40 min at 0 °C, and then ethyl piperidine-4-carboxylate
(0.52 g, 1.67 mmol, 61.3% yield) was added in one portion. The
resulting mixture was stirred overnight at room temperature, diluted
with saturated NaHCO3, and extracted with DCM. The extracts were
dried over MgSO4, concentrated, and purified by flash chromatog-
raphy (10−50% EtOAc/Hex) to provide 27c as a clear oil (0.52 g,
61% yield). 1H NMR (400 MHz, chloroform-d) δ 9.22 (d, J = 0.9 Hz,
1H), 8.49 (dd, J = 6.1, 0.7 Hz, 1H), 8.20 (dd, J = 6.1, 1.0 Hz, 1H),
7.67−7.95 (m, 2H), 7.46−7.67 (m, 1H), 3.87−4.21 (m, 3H), 3.05 (d, J
= 11.2 Hz, 1H), 2.74 (d, J = 11.2 Hz, 1H), 2.25 (tt, J = 11.2, 4.2 Hz,
1H), 1.95−2.17 (m, 2H), 1.57−1.95 (m, 4H), 1.43 (dd, J = 6.7, 0.7
Hz, 3H), 1.21 (t, J = 7.1 Hz, 3H).
Ethyl 1-(1-(Isoquinolin-1-yl)ethyl)piperidine-4-carboxylate
(28d). To a solution of 27d (215 mg, 1.24 mmol) in dry DCM
were added DIEA (482 mg, 3.73 mmol) and Ms2O (326 mg, 1.87
mmol), and the mixture was allowed to stir for 40 min at 0 °C. To the
solution, ethyl isonipecotate (586 mg, 3.73 mmol) was added
dropwise, and the mixture was allowed to stir at 23 °C for 18 h.
The mixture was acidified using 6 M HCl (6 mL) and extracted with
DCM (2 × 15 mL). The aqueous layer was then basified using
saturated NaHCO3 (15 mL) and extracted with DCM (3 × 15 mL).
The organic layer was dried under MgSO4 and concentrated in vacuo.
The residue was purified by flash chromatography (10−50% EtOAc/
Hex) to furnish the desired compound (62 mg, 16%) as a yellow oil.
1H NMR (400 MHz, chloroform-d) δ 8.69 (d, J = 8.5 Hz, 1H), 8.45
(400 MHz, chloroform-d) δ 8.87 (d, J = 1.7 Hz, 1H), 8.53 (d, J = 8.6
Hz, 1H), 7.90−8.13 (m, 1H), 7.60−7.68 (m, 2H), 7.40 (dd, J = 8.6,
4.2 Hz, 1H), 5.59 (q, J = 6.5 Hz, 1H), 2.32 (br s, 1H), 1.65 (d, J = 6.5
Hz, 3H).
1-(Isoquinolin-5-yl)ethanol (27c). To a −78 °C solution of
isoquinoline-5-carbaldehyde 26c (1.13 g, 7.19 mmol) in dry THF
under N2 was added methylmagnesium chloride (8.63 mL, 8.63
mmol) dropwise. The resulting mixture was stirred for 30 min before
warming to room temperature. Saturated aqueous NH4Cl was added,
and the mixture was stirred 10 min before diluting with EtOAc/Et2O.
The organic layer was washed with saturated NaCl and dried over
MgSO4. Concentration provided a white solid which was triturated in
ether, filtered, and dried under high vacuum to give 1-(isoquinolin-5-
yl)ethanol 26c (1.1 g, 88% yield). 1H NMR (400 MHz, chloroform-d)
δ 8.88 (d, J = 1.4 Hz, 1H), 8.48 (d, J = 7.8 Hz, 1H), 7.65 (m, 2H), 7.58
(m, 1H), 7.28−7.41 (m, 2H), 4.69 (q, J = 6.3 Hz, 1H), 1.49 (d, J = 6.3
Hz, 3H).
1-(Isoquinolin-1-yl)ethanol (27d). To a solution of 1-cyanoiso-
quinoline (301 mg, 1.96 mmol) in dry Et2O (10 mL) at 0 °C, 3.92 mL
of 1 M MeMgBr (3.92 mmol) was added and allowed to stir for 3 h to
produce a bright orange solution. The reaction was quenched with
H2O (15 mL) and 6 M HCl (5 mL) and stirred for 1 h at 80 °C. The
solution was basified with saturated NaHCO3, extracted with Et2O (2
× 25 mL), dried over MgSO4, and concentrated in vacuo. The residue
was purified by silca flash chromatography (30% EtOAc/Hex) to
furnish 1-(isoquinolin-1-yl)ethanone (304 mg, 91%) as a yellow oil.
1H NMR (400 MHz, chloroform-d) δ 8.95 (d, J = 8.4 Hz, 1H), 8.57
(d, J = 5.5 Hz, 1H), 7.88−7.82 (m, 1H), 7.80 (d, J = 5.5 Hz, 1H), 7.69
(pd, J = 6.9, 1.3 Hz, 2H), 2.86 (s, 3H). This compound (304 mg, 1.78
mmol) was then dissolved in MeOH (6 mL), and NaBH4 was added
and the mixture was allowed to stir at 0 °C for 18 h. The mixture was
concentrated in vacuo and treated with saturated aqueous NH4Cl. The
solution was extracted with DCM (3 × 10 mL), dried under MgSO4,
and concentrated in vacuo. The residue was purified by silica flash
chromatography (30% EtOAc/Hex) to furnish the desired compound
(226.5 mg, 73.6%) as a clear oil. 1H NMR (400 MHz, chloroform-d) δ
8.43 (d, J = 5.7 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.86 (d, J = 8.2 Hz,
1H), 7.70 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.66−7.54 (m, 2H), 5.58 (p,
J = 5.7 Hz, 1H), 5.37−5.23 (m, 1H), 1.59 (d, J = 6.5 Hz, 3H).
Ethyl 1-(1-(Quinolin-8-yl)ethyl)piperidine-4-carboxylate
(28a). To a 0 °C solution of 1-(quinolin-8-yl)ethanol 27a (0.47 g,
2.71 mmol) in dry DCM was added DIEA (1.05 g, 8.14 mmol)
followed by Ms2O (0.71 g, 4.10 mmol). The resulting mixture was
stirred for 40 min at 0 °C. Then ethyl piperidine-4-carboxylate (0.39 g,
(d, J = 5.7 Hz, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.67−7.61 (m, 1H),
7.57−7.47 (m, 2H), 4.36 (q, J = 6.7 Hz, 1H), 4.08 (q, J = 7.1 Hz, 2H),
2.25 (dtt, J = 22.2, 8.2, 3.5 Hz, 2H), 2.11 (td, J = 11.3, 2.7 Hz, 1H),
1.88−1.75 (m, 2H), 1.75−1.67 (m, 2H), 1.65−1.61 (m, 1H), 1.52 (d, J
= 6.7 Hz, 3H), 1.20 (t, J = 7.1 Hz, 3H).
1,2,3,4-Tetrahydrophenanthren-4-amine (30). To a solution
of 2,3-dihydrophenanthren-4(1H)-one 29 (305 mg, 1.55 mmol) in
isopropanol (50 mL) were added NaCNBH3 (677 mg, 10.8 mmol)
and NH4(OAc) (4.831 g, 62.68 mmol), and the mixture was allowed
to stir at reflux for 72 h under N2 atmosphere. Approximately 50% of
the solvent was evaporated, and the reaction mixture was extracted
with DCM (3 × 40 mL), dried under MgSO4, and concentrated in
vacuo to afford the desired compound (301 mg, 98%) as a dark oil,
1
which was sufficiently pure to use in the next step. H NMR (400
MHz, chloroform-d) δ 8.18 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 8.1 Hz,
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1H), 7.64 (d, J = 8.5 Hz, 1H), 7.53 (t, J = 7.3 Hz, 1H), 7.42 (t, J = 7.4
Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 4.72 (s, 1H), 2.92 (dd, J = 10.4, 5.7
Hz, 2H), 2.15−1.84 (m, 4H).
A9511, purity 97−99%) for serum shift assays was obtained from
Sigma-Aldrich.
SARS-CoV PLpro and NL63-CoV PLP2 Expression and
Purification. The expression plasmids containing SARS-CoV pET-
15b-PLpro(1541−1855), SARS-CoV pET-11a-PLpro(1541−1855), and
HCoV-NL63 pET-15b-PLP2(1565−1894) were transformed into E. coli
BL21(DE3) for protein expression. All purifications were performed at
4 °C with slight variations in the purification procedures. One liter of
cells containing SARS-CoV pET-15b-PLpro(1541−1855) wild type and
mutants, SARS-CoV pET-11a-PLpro(1541−1855), or HCoV-NL63 pET-
15b-PLP2(1565−1894) were grown for 24 h at 25 °C in medium
containing 3 g of KH2PO4, 6 g of Na2HPO4, 20 g of tryptone, 5 g of
yeast extract, 5 g of NaCl, pH 7.2, supplemented with 0.2% lactose,
0.6% glycerol, 0.05% glucose, and 50 μg/mL carbenicilin. After
growth, the cells were pelleted by centrifugation (18500g, 30 min at 4
°C) and an amount of ∼12 g of cell pellet was resuspended in 50 mL
of buffer A (20 mM Tris, pH 7.5, 500 mM NaCl, 10 mM imidazole)
containing lysozyme and DNase I. The resuspended cells were lysed
on ice via sonication using a 600 W model VCX ultrasonicator (7−8
min at 70% amplitude; 5.5 pulse on, 9.9 pulse off) or a 400 W 450
model digital sonifier cell disruptor (10 min at 60% amplitude; 5.5
pulse on, 9.9 pulse off). Cells debris were pelleted by centrifugation
(30000g, 25 min, 4 °C).
For SARS-CoV PLpro WT and mutants (pET-15b-PLpro(1541−1855)
and pET-11a-PLpro(1541−1855)), the clarified lysate was loaded at 2 mL/
min onto a 5 mL HiTrap chelating HP column (GE Healthcare)
charged with Co2+ and equilibrated with buffer A. Unbound proteins
were washed with five column volumes (CV) of buffer A. Bound
proteins were eluted with a linear gradient of 0−100% buffer B (20
mM Tris, pH 7.5, 500 mM NaCl, 500 mM imidazole), at 2 mL/min,
followed by a 5× CV 100% buffer B wash. Fractions containing SARS-
CoV PLpro were exchanged and concentrated into buffer C (20 mM
Tris, pH 7.5, 20% glycerol, 10 mM DTT) to 5−10 mg/mL. Aliquots
of 500 μL of the concentrated protein were flash frozen in liquid
nitrogen for 10 min, then stored at −80 °C. HCoV-NL63
PLP2(1565−1894) was purified by the same protocol as SARS-CoV
PLpro except that a 5 mL HisTrap FF column (GE Healthcare)
precharged with Ni2+ was used and 10 mM β-mercaptoethanol was
included in the purification buffers.
For crystallization, the eluted fractions of the pET-11a-
PLpro(1541−1855) from the Co2+ column were concentrated and
supplemented with 1 mg of TEV protease per 30 mg of PLpro and
dialyzed for 18 h at 4 °C against 1 L (∼1000× dilution) of dialysis
buffer (20 mM Tris, pH 7.5, 10% glycerol, 10 mM β-
mercaptoethanol). Following dialysis the β-mercaptoethanol was
removed by buffer exchange and the His-tagged TEV protease was
removed by loading the sample onto a 5 mL HiTrap chelating HP
column. Cleaved untagged SARS-CoV PLpro was obtained in the
flow-through and concentrated to 25 mg/mL prior to loading onto a
Superdex 200 26/60 gel filtration column equilibrated with buffer D
(20 mM Tris, pH 7.5, 100 mM NaCl, and 10 mM DTT). The protein
was eluted at a flow rate of 0.5 mL/min with equilibrium buffer, and
the fractions containing SARS-CoV PLpro were concentrated to 6 and
12 mg/mL.
Dimethyl 1-(1,2,3,4-Tetrahydrophenanthren-4-yl)pyridine-
4,4(1H)-dicarboxylate (31). To a solution of dimethyl 2,2-
bis((1,3-dioxolan-2-yl)methyl)malonate (464 mg, 1.53 mmol) in
THF (10 mL) was added a 10% HCl solution (9.3 mL) dropwise.
The reaction mixture was allowed to stir at room temperature for 16 h.
The reaction mixture was neutralized with aqueous Na2CO3. Amine
30 (301 mg, 1.53 mmol) was dissolved in THF (3 mL) and added
dropwise to the reaction mixture. The pH was adjusted to ∼5 using
AcOH, and the reaction mixture was allowed to stir at room
temperature for 72 h. The reaction mixture was quenched with
saturated aqueous NaHCO3, extracted with EtOAc (2 × 15 mL), dried
over MgSO4, and concentrated in vacuo. The residue was purified by
silica flash chromatography (20% EtOAc/Hex) to furnish 31 (221 mg,
38%) as an oil. 1H NMR (400 MHz, chloroform-d) δ 7.77 (d, J = 7.9
Hz, 2H), 7.71 (d, J = 8.5 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.41 (t, J =
7.3 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.05 (d, J = 7.7 Hz, 2H), 4.97 (s,
1H), 4.66 (d, J = 7.8 Hz, 2H), 3.68 (s, 5H), 3.13−2.74 (m, 4H), 2.28−
2.08 (m, 2H).
Dimethyl 1-(1,2,3,4-Tetrahydrophenanthren-4-yl)-
piperidine-4,4-dicarboxylate (32). To a solution of 31 (221 mg,
0.59 mmol) in EtOAc (25 mL) was added PtO2 (20 mg), and N2 was
bubbled through for 15 min. The mixture was placed under H2 (40
psi) at room temperature for 5 h, and reaction progress was monitored
by HPLC. The reaction mixture was filtered through Celite and
concentrated in vacuo. The residue was then purified by silica flash
chromatography (5−10% EtOAc/Hex) to furnish the desired
compound (150 mg, 67%) as a yellow oil. 1H NMR (400 MHz,
chloroform-d) δ 8.37 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 7.7 Hz, 1H),
7.63 (d, J = 8.3 Hz, 1H), 7.45−7.35 (m, 2H), 7.15 (d, J = 8.4 Hz, 1H),
4.43 (t, J = 5.4 Hz, 1H), 3.69 (s, 6H), 2.88 (t, J = 5.4 Hz, 1H), 2.82−
2.76 (m, 1H), 2.54 (dd, J = 7.3, 3.6 Hz, 2H), 2.19−2.11 (m, 1H), 2.02
(dd, J = 7.0, 3.4 Hz, 1H), 1.94 (dd, J = 7.3, 3.0 Hz, 2H), 1.71 (dq, J =
9.8, 5.3, 4.7 Hz, 2H), 1.30−1.20 (m, 2H), 0.92−0.77 (m, 2H).
Methyl 1-(1,2,3,4-Tetrahydrophenanthren-4-yl)piperidine-4-
carboxylate (33). To a solution of 32 (150 mg, 0.39 mmol) in DMF
(10 mL) was added NaCN (29 mg, 0.59 mmol), and the mixture was
allowed to stir at reflux for 16 h under N2 atmosphere. The reaction
mixture was cooled and diluted with EtOAc (10 mL), quenched with
aqueous NaHCO3, and washed with saturated NaCl (2 × 10 mL). The
extract was dried over MgSO4, concentrated in vacuo, and the residue
was purified by silica flash chromatography (0−10% EtOAc/Hex) to
furnish the desired compound (97 mg, 76%) as a yellow oil. 1H NMR
(400 MHz, chloroform-d) δ 8.40 (d, J = 8.2 Hz, 1H), 7.74 (d, J = 7.8
Hz, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.45−7.32 (m, 2H), 7.16 (d, J = 8.4
Hz, 1H), 4.44 (t, J = 4.9 Hz, 1H), 3.62 (s, 3H), 2.96−2.86 (m, 2H),
2.83−2.75 (m, 1H), 2.60−2.49 (m, 2H), 2.38−2.27 (m, 1H), 2.24−
2.15 (m, 2H), 2.05−1.90 (m, 1H), 1.84 (d, J = 14.0 Hz, 1H), 1.74 (td,
J = 12.0, 10.7, 4.7 Hz, 3H), 1.65 (d, J = 14.5 Hz, 1H), 1.34 (qd, J =
12.1, 4.1 Hz, 1H).
General Materials for Enzyme Purification and Kinetic
Assays. SARS-CoV pET-15b-PLpro(1541−1855) (cloned between the
restriction sites BamH I and Bpu1102 I) and the HCoV-NL63 pET-
15b-PLP2(1565−1894) (cloned between the restriction sites BamH I and
Bpu1102 I) were obtained from Dr. Susan Baker’s lab at Loyola
University Chicago, Stritch School of Medicine. For crystallization,
SARS-CoV PLpro(1541−1855) fused at the N-terminus to TEV protease
cleavage sites and a 6 histidine tag was synthetized and codon
optimized and cloned into pET11a using the restriction sites Nde I and
Bpu1102 I by BioBasic Inc. Costar 96-well black microplates were
purchased from Corning. Synthetic peptide substrate, Z-RLRGG-
AMC, used for IC50 determination was purchased from Bachem. The
E. coli expression strain BL21(DE3) was purchased from Novagen. LB
medium components were purchased from BD Biosciences. The 5 mL
HiTrap chelating HP column and the Superdex 200 were obtained
from GE Healthcare, Life Sciences. Bradford reagent and BSA
standard solution used for quantification of protein concentration were
purchased from Bio-Rad. Human serum albumin (HSA, catalog no.
For all proteins, the protein concentration was determined by
cuvette-based Bradford assay and purity was monitored by SDS−
PAGE analysis and by calculating the specific activity at every step of
the purification.
Determination of IC50 Values for Synthesized Compounds.
The 100 μL inhibition assays were performed in triplicate in a 96-well
plate format as previously described.14b The final enzymes
concentrations were 0.14 and 0.4 μM for SARS-CoV PLpro and
HCoV-NL63 PLP2, respectively. The assays were performed at 25 °C,
and the enzyme activity was monitored by measuring the PLP-
mediated release of AMC from the RLRGG-AMC peptide substrate
(50 μM), using the EnVision multimode plate reader from
PerkinElmer.
Serum Shift Assays. Human serum albumin (HSA) was dissolved
to a final concentration of 50 mg/mL in buffer containing 50 mM
HEPES, pH 7.5, 2.5 mM DTT. Serum shift assays were performed by
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Article
determining the IC50 values for selected compounds as described
above except that the assays contained 5%, 10%, or 20% HSA, where
40 mg/mL is 100%.25,26,28
ASSOCIATED CONTENT
Accession Codes
The atomic coordinates and structure factors have been
deposited in the Protein Data Bank under code 4OW0 for
PLpro−3k complex and code 4OVZ for PLpro−3j complex.
■
Counterscreen Assays for Selectivity against DUBs, USPs,
and Cysteine Proteases. The NL63-HCoV PLP2 counterscreening
assay was performed in a 100 μL reaction volume including 400 nM
enzyme, 100 μM compound, and 50 μM RLRGG-AMC substrate in
assay buffer (50 mM HEPES, pH 7.5, 0.1 mg/mL BSA, 2 mM DTT).
The assay was performed at 25 °C in triplicate for each inhibitor, and
the enzyme activity was monitored as described above. The
counterscreens with human USP and cysteine proteases were
determined using purified enzymes available from Progenra, Inc. as
part of their DUB screening service. The assays were performed in
triplicate using 31.6 μM compounds and Ub-rhodamine 110 as a
substrate for USPs, Nedd8-EKL for DEN1, and commercially available
fluorogenic peptide substrates for caspase 3 and cathepsin K.
AUTHOR INFORMATION
Corresponding Authors
*S.D.L.: phone, (734) 615-0454; e-mail, sdlarsen@med.umich.
■
edu.
*A.D.M.: phone, (765) 494-1924; e-mail, amesecar@purdue.
edu.
Notes
The authors declare no competing financial interest.
Antiviral and Cytotoxicity Assays. SARS-CoV antiviral activity
in infected Vero E6 cells was performed as previously described in a
BSL-3 laboratory.14b Briefly, Vero E6 cells were seeded into 96-well
culture plates at 1 × 103 cells per well 24 h prior to infection with
SARS-CoV. Cells were mock or SARS-CoV infected for 48 h in the
presence of increasing concentrations of compounds. Upon
incubation, cell viability was determined using Cell Titer-Glo
luminescent cell viability assay (Promega). Experiments were
performed twice in triplicate. Both the half maximal effective
concentration (EC50) and the 50% cytostatic concentration (CC50)
values were calculated using a four-parameter logistic equation by
means of Sigma Plot 10 or GraphPad Prism software. Culturing and
cell viability measurements of HEK 293 cells were performed as
described above, in triplicate, 48 h after compound addition.
Liver Metabolism Studies. Compounds (1 μM) were incubated
with mouse liver microsomes (0.5 mg/mL) and NADPH in 0.1 M
phosphate buffer at 37 °C as previously described.29
Mutagenesis of Gln270. The mutagenesis of Gln270 to Ala, Glu, or
Asp was performed by QuickChange site-directed mutagenesis as
described by Zhen et al.30 Mutant enzymes were purified as described
above for the wild-type SARS-PLpro enzyme.
Crystallization of SARS-CoV PLpro in Complex with
Inhibitors. Prior to crystallization, untagged SARS-CoV PLpro in
25 mM Tris, pH 7.5, 100 mM NaCl, 10 mM DTT at concentrations of
6 or 12 mg/mL was incubated with 2 mM inhibitor (dissolved in
DMSO or ethanol) added to a 100× dilution and incubated overnight
at 4 °C. Crystallization was performed at 20 °C using the sitting-drop
vapor-diffusion method by mixing equal amounts of protein/inhibitor
with reservoir solution containing 100 mM sodium citrate, pH 5.5,
40% (v/v) PEG 600. Crystals were soaked in cryosolution consisting
of reservoir solution, 2 mM inhibitor, and 20% glycerol. Crystals were
flash-frozen in liquid nitrogen and stored until synchrotron time was
available. Crystals were transferred from liquid nitrogen into a stream
of dry nitrogen gas at 100 K for X-ray data collection.
X-ray data were collected at the Life Sciences-Collaborative Access
Team (LS-CAT) on beamline 21-ID-F at the Advanced Photon
Source, Argonne National Laboratory. Data were processed and scaled
using HKL2000. The SARS-CoV PLpro−3k and PLpro−3j complex
crystallized as two monomers in the asymmetric unit. Crystals belong
to space group C2 with unit cell dimensions of a = 119 Å, b = 74 Å, c =
98 Å, β = 104°. The inhibitor-bound structures diffracted to
resolutions of 2.1 and 2.5 Å for 3k and 3j, respectively. The initial
phases were determined from molecular replacement solutions using
the SARS-CoV PLpro−15g inhibitor complex structure (pdb:3MJ5) as
a search model and the MolRep31 program of the CCP4 suite.32
Model building and refinement was performed using the programs
Refmac,33 Phenix,34 and Coot.35 The final X-ray data processing and
refinement statistics are summarized in Table 3. The final coordinates
have been deposited in the Protein Data Bank under PDB code 4OW0
for PLpro−3k complex and PDB code 4OVZ for PLpro−3j complex.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the synchrotron beamline personnel
at the Advanced Photon Source (LS-CAT) 21-ID-F beamline.
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 DE-AC02-06CH11357. Use of the
LS-CAT Sector 21 was supported by the Michigan Economic
Development Corporation and the Michigan Technology Tri-
Corridor (Grant 085P1000817). This work was partially
supported by National Institutes of Health Grant AI085089
to A.D.M. and S.C.B. A.D.M. is also supported by a grant from
the Walther Cancer Foundation. S.J.B. and M.P.A. were
supported by University of Michigan Rackham Merit Fellow-
ships. The authors also acknowledge support from the Purdue
Center for Cancer Research DNA Sequencing and Macro-
molecular Crystallography shared resources.
ABBREVIATIONS USED
■
SARS, severe acute respiratory syndrome; CoV, coronavirus;
SARS-CoV, severe acute respiratory syndrome coronavirus;
HCoV, human coronavirus; MERS, Middle East respiratory
syndrome; PLpro, papain-like protease; PLP2, papain-like
protease 2; 3CLpro, chymotrypsin-like protease; HTS, high-
throughput screening; Ub, ubiquitin; ISG15, interferon-induced
stimulated gene 15; DUB, deubiquitinating enzyme; USP,
ubiquitin specific protease; HSA, human serum albumin; PDB,
Protein Data Bank
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