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.
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 VeroE6 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.
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 humancoronaviruses (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 anticoronaviral
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 origin[4] and closely related to bat coronaviruses from
the Betacoronavirus genus (group 2).[5] 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 (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] deubiquitination[11](the removal of ubiquitin),
and deISGylation[12] (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-CoVPLpro inhibitors (compounds 24(14) and 15g,h[15]), 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 24(14) and 15g(15) and their X-ray structures in complex with SARS-CoVPLpro
have been previously described.
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.
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 differentially
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-CoVPLpro 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]Chemical structures of
previously characterized SARS-CoVPLpro
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.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 apo[16] and
inhibitor-bound structures.[14,15] Specifically, there
is a highly mobile β-turn/loop (Gly267-Gly272) adjacent to the active site that closes upon 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-benzodioxole ring.Because the
antiviral potencies of these SARS-CoVPLpro 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-CoVPLpro to develop a second generation of PLpro
inhibitors. Here, we disclose SAR of a new series of potent SARS-CoVPLpro inhibitors, their selectivity, antiviral efficacy, biological
evaluation, and a detailed report of the molecular interactions between
SARS-CoVPLpro and inhibitors 3k and 3j aided
by X-ray crystallographic studies. Compound 3k was established
to be the most potent SARS-CoVPLpro 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-CoVPLpro over human homologue enzymes.
The improved resolution of the SARS-CoVPLpro–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.
Results
and Discussion
Chemistry
The preparation of analogues 1a–d bearing substitution off the α-methyl
group is summarized in Schemes 1–3.
Reagents and conditions: (a)
1-naphthyl-MgBr, ether, rt; (b) AqHCl; (c) LAH, THF, rt; (d) Ms2O, DIEA, DCM, 0 °C; (e) ethyl isonipecotate, rt; (f)
aqNaOH, EtOH, THF; (g) piperonylamine, EDCI, HOBt, DIEA, rt.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 12(18) was 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.
Reagents and conditions: (a)
LAH, THF, rt; (b) TBSCl, imidazole, ether, rt; (c) Ms2O,
DIEA, DCM, rt; (d) ethyl isonipecotate, rt; (e) aqNaOH, EtOH, 40
°C; (f) piperonylamine, EDCI, HOBt, DIEA, rt; (g) CsF, aqACN,
rt.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 described[19] provided amine 20. This 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.
Reagents and conditions: (a)
RNH2, EDCI or HATU, DIEA.Preparation
of (R)-amide analogues is summarized
in Scheme 4. The starting (R)-1-(1-(naphthalen-1-yl)ethyl)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.
Scheme 4
General Synthesis of Amide Analogues
Reagents and conditions: (a)
RNH2, EDCI or HATU, DIEA.
Synthesis of Quinolinyl-
and Isoquinolinylamides
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.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.
Synthesis of Conformationally Restricted
Analogue 7
Reagents and conditions:
(a)
NaBH3CN, NH4OAc, PrOH, 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) aqLiOH, THF/MeOH, rt; (g) 3-fluorobenzylamine, EDCI, HOBt,
DIEA, rt, 16 h.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 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.
Scheme 6
Synthesis of Conformationally Restricted
Analogue 7
Compound 15g is a competitive
inhibitor of SARS-CoVPLpro that displays
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-naphthalenylethyl and 1,3-benzodioxolylmethyl substituents
at the piperidinyl and carboxamidenitrogens, respectively. Previous
SAR has established the preference for the 1-naphthyl over the 2-naphthyl
substitution pattern and the requirement of a single 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-CoVPLpro–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 between these water molecules
and Asp165, Arg167, Tyr274, 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.
Table 1
In Vitro Analysis of SARS-CoV PLpro
Inhibition by 15g and Derivatives
compd
isomera
R1
R2
R3
IC50 (μM)b
15g
R
H
H
3,4-O-CH2-O
0.67 ± 0.03
1a
R,S
Me
H
3,4-O-CH2-O
17.2 ± 2.0
1b
R,S
OH
H
3,4-O-CH2-O
32.0 ± 4.5
1c
R,S
OMe
H
3,4-O-CH2-O
>100
1d
R,S
Ph
H
3,4-O-CH2-O
>100
2a
R
H
H
H
2.2 ± 0.1
2b
R
H
(R)-Me
H
13.5 ± 1.2
2c
R
H
(S)-Me
H
12.7 ± 0.3
2d
R
H
(R)-CH2-OMe
H
18.0 ± 1.9
2e
R
H
(S)-CH2-OMe
H
1.9 ± 0.1
3a
R
H
H
4-Et
0.47 ± 0.01
3b
R
H
H
4-CO-NH-Me
0.60 ± 0.02
3c
R
H
H
3-CO-NH-Me
0.63 ± 0.01
3d
R
H
H
4-NH-CO-Me
5.7 ± 0.5
3e
R
H
H
3-NH-CO-Me
0.39 ± 0.01
3f
R
H
H
3-CH2-NH-CO-Me
20.4 ± 1.2
3g
R
H
H
3-Cl
27.2 ± 4.1
3h
R
H
H
4-Cl
0.58 ± 0.02
3i
R
H
H
3,4-diF
29.2 ± 2.1
3j
R
H
H
4-F
0.49 ± 0.01
3k
R
H
H
3-F
0.15 ± 0.01
Chiral center.
Values are reported as mean
±
standard deviation based on a minimum of triplicate measurements.
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 comparable extent
(IC50 of 13.5 ± 1.2 and 12.7 ± 0.3 μM,
respectively), indicative of a steric clash with SARS-CoVPLpro. Interestingly,
a 10-fold stereopreference was observed for (S)-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 not engage the active site of the enzyme in favorable
interaction and are therefore unlikely to lead to activity improvement.Chiral center.Values are reported as mean
±
standard deviation based on a minimum of triplicate measurements.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-CoVPLpro–15g complex, one
of the 1,3-dioxolaneoxygens is within 3 Å of the Gln270 side chain amidenitrogen, 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-CoVPLpro,
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 inhibitory potency but not through interaction
with Gln270. 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 μM) and 3e (IC50 = 0.39 ± 0.01 μM), respectively. Further
extension of the acetamido group from the meta position, favored in
the previous pair, dramatically decreases activity of 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 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-CoVPLpro 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 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 SARSPLpro 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.
Table 2
Structures and Activities
of 3k Variants, Exploring Bioisosteric Replacements and
Scaffold
Perturbation
compd
isomera
R1
R2
IC50 (μM)b
3k
R
1-naphthyl
3-F-Ph-CH2
0.15 ± 0.01
4a
R,S
8-quinolinyl
3-F-Ph-CH2
7.0 ± 0.7
4b
R,S
5-quinolinyl
3-F-Ph-CH2
4.5 ± 0.2
4c
R,S
5-isoquinolinyl
3-F-Ph-CH2
6.8 ± 0.3
4d
R,S
1-isoquinolinyl
3-F-Ph-CH2
30.8 ± 2.6
5a
R
1-naphthyl
3-pyridinyl-CH2
26.3 ± 2.3
5b
R
1-naphthyl
4-pyridinyl-CH2
18.3 ± 0.9
5c
R
1-naphthyl
2-methoxy-4-pyridinyl-CH2
0.35 ± 0.02
6a
R
1-naphthyl
4-Cl-Ph-CH2CH2
1.6 ± 0.3
6b
R
1-naphthyl
3-F-Ph-CH2CH2
1.9 ± 0.1
Chiral center.
Values are reported as mean
±
standard deviation based on a minimum of triplicate measurements.
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-CoVPLpro in the
region bound by the benzene moiety.Chiral center.Values are reported as mean
±
standard deviation based on a minimum of triplicate measurements.Finally, tricyclic analogue 7 was prepared as a conformationally
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-CoVPLpro–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.
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.
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-CoVPLpro bound to 15g.
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-CoVPLpro
was cocrystallized as a complex with 3k (PDB code 4OW0) and 3j (PDB code 4OVZ) using an approach similar to 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-CoVPLpro–3k and PLpro–3j complexes to resolutions of 2.1 and 2.5 Å, respectively
(Table 3). Both SARS-CoVPLpro–inhibitor
complexes crystallized in space group C2 and contained
two monomers per asymmetric unit.
Table 3
Data Collection and
Refinement Statistics
3k
3j
Data Collection
beamline
21-ID-F
21-ID-F
wavelength (Å)
0.98
0.98
space group
C2
C2
unit cell dimensions
a, b, c (Å)
119.4, 74.4, 98.3
119.8, 73.5, 98.3
α, β, γ (deg)
90, 104, 90
90, 104, 90
resolution (Å)
100–2.10 (2.14–2.10)
100–2.50 (2.54–2.50)
no. of reflections observed
514232
743761
no. of unique reflections
49274
29021
Rmerge (%)
6.2 (52.3)
6.5 (69.6)
I/σI
20.6 (1.9)
19.3 (1.8)
% completeness
98.6 (96.1)
99.8 (100)
redundancy
4.7 (3.9)
4.2 (4.2)
Refinement
resolution range (Å)
31.79–2.10
35.75–2.50
no. of reflections in working
set
42169
24909
no. of reflections in test
set
2134 (5.1%)
1265 (5.1%)
Rwork (%)
0.18
0.19
Rfree (%)
0.20
0.24
average B factor (Å2)
39
37
rmsd from ideal geometry
bond length (Å)
0.022
0.026
bond angle (deg)
1.5
1.7
Ramachandran plot
most favored (%)
94.0
92.6
allowed (%)
4.8
5.6
disallowed (%)
1.2
1.8
After identification of a molecular replacement
solution and performance
of initial rounds of refinement of the SARS-CoVPLpro 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-CoVPLpro active site in the same orientation (Figure 3c and Figure 3d).
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).
X-ray crystal structures
of SARS-CoVPLpro in complex with 3k and 3j. Stereoviews of SARS-CoVPLpro (blue
ribbon representation and gray surfaces) in complex with 3k (orange ball and sticks) are shown in (a) and (c), and SARS-CoVPLpro (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-CoVPLpro–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-CoVPLpro–15g complex (SARS-CoVPLpro displayed as a gray ribbon and 15g displayed as gray balls and sticks, pdb:3MJ5) with SARS-CoVPLpro–3k and PLpro–3j complex is shown in (f).As described previously for compounds 24 and 15g, compounds 3k and 3j bind adjacent
to the active site at the enzyme S3–S4 subsites[14,15] (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 carboxyamidenitrogen 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-CoVPLpro–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) 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.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 amidenitrogen 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 carboxamidenitrogen 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 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.
Table 4
Inhibitor Selectivity against Human
DUBs and Cysteine Proteases
IC50 (μM)a
15g
3k
3e
3j
human DUB
USP2
–
–
>100
–
USP7
–
–
>100
–
USP8
>100
>100
>100
>100
USP20
–
>100
>100
>100
USP21
>31
>31
>31
>31
DEN1
–
–
–
–
UCHL1
–
–
–
–
caspase 3
–
–
–
–
cathepsin K
–
–
–
–
–,
no inhibition.
–,
no inhibition.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-NL63PLP2 inhibition versus increasing compound concentration
up to 100 μM were determined. Six compounds, including compound 3k, produced 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-NL63PLP2 is achieved by this series of compounds,
albeit significantly weaker inhibition than for SARS-CoVPLpro (micromolar
versus nanomolar range), and suggest that SARS-CoVPLpro and HCoV-NL63PLP2 may display sufficient structural similarities at the active
site for the potential development of a dual-target inhibitor.
Table 5
Compounds Displaying Dual-Target Inhibition
of SARS-CoV PLpro and HCoV-NL63 PLP2
IC50 (μM)
max I (%)
compd
PLpro
PLP2
PLpro
PLP2
15g
0.67
18
99
46
3k
0.15
33
100
57
5c
0.35
59
100
67
3e
0.39
46
100
71
3d
5.7
37
94
42
3i
29
44
78
48
SARS-CoV Antiviral
Activity and Cytotoxicity Evaluation of the
Most Potent Compounds
Compound 3k exhibits lower
topological polar surface area (tPSA = 32.2 Å2) 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 VeroE6 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 cytotoxicity
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 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.
Table 6
Compound Effect on
Replication of
SARS-CoV Virus, Cytotoxicity, and Therapeutic Indexes
15g
3k
3e
5c
3j
2e
Vero E6
IC50a
0.67 ± 0.03
0.15 ± 0.01
0.39 ± 0.01
0.35 ± 0.02
0.49 ± 0.01
1.9 ± 0.1
EC50a
12.8 ± 1.4
5.4 ± 0.6
8.3 ± 0.6
9.5 ± 1.3
11.6 ± 0.8
11.7 ± 0.7
CC50a
>100
>100
>100
>100
>100
>100
TI
>7.8
>18.5
>12
>10.5
>8.6
>8.5
HEK293
CC50a
>100
73 ± 19
68 ± 15
>100
73 ± 29
>100
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.
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 VeroE6 cells; CC50, 50% cytotoxic
concentration; TI, the therapeutic index defined as the ratio of CC50/EC50.
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 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.
Table 7
Stability of Selected
Compounds to
Metabolism by Mouse Liver Microsomes
compd
% rem (15 min)a
T1/2 (min)b
15g
0.2
1.6
3k
1.2
2.8
3e
20
7.0
5c
30
8.0
Percent
of the parent compound remaining
after 15 min incubation. The initial concentration of the parent compound
was 1 μM.
Half-life
(minutes) of parent compound.
Percent
of the parent compound remaining
after 15 min incubation. The initial concentration of the parent compound
was 1 μM.Half-life
(minutes) of parent compound.Because the binding of an antiviral drug to human serum proteins
may result in reduced antiviral activity,[24] we evaluated 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% humanserum 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
A second-generation
series of highly potent SARS-CoVPLpro 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-CoVPLpro 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 humanserum 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-CoVPLpro 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-CoVPLpro (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.
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 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′-tetramethyluronium
hexafluorophosphate), HOAT (1-hydroxy-7-azabenzotriazole), HOBt (1-hydroxy-1,2,3-benzotriazole),
EDCI (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride), DIEA (diisopropylethylamine),
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 abbreviations
are the following: LUC (luciferase), MTT ((3-(4,5-dimethylthiazol-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.
Ethyl 1-(1-(naphthalen-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, 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.
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 piperonylamine (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.
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 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).
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 chromatography (1–5% MeOH/DCM)
to obtain 4a (0.05 g, 33% yield). 1H 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 Hz, 3H). TOF ES+ MS: (M + H) 392.2.
HPLC tR = 4.1 min, >95% purity.
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 colorless oil. 1H 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.
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.
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 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.
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 (40–60% EtOAc/Hex) to furnish 7 (26 mg, 30%) as a dark oil. 1H 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), 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-Methoxyacetonitrile 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% EtOAc/Hex) to give the desired
product (118 mg, 18%). 1H 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).
(±)-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 workup[5] was employed. Precipitate was filtered off and the filtrate concentrated
in vacuo to give the desired product as an oil (104 mg, 87%) used
without further purification. 1H 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).
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, 84%) as a slightly
yellow oil. 1H 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).
2-Methoxy-1-(naphthalen-1-yl)ethanol10c (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 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% EtOAc/Hex gradient) to give the desired
product (91 mg, 52%). 1H 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).
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).
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).
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, 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).
Dimethyl 2,2-bis((1,3-dioxolan-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-phenylethanamine 20 (446 mg, 1.80 mmol), prepared previously by the literature
method[19] 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).
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, 20% EtOAc/Hex)
to give the desired monoester (38 mg, 43%). 1H NMR (500
MHz, chloroform-d) δ 8.49 (s, 1H), 7.83 (dt, J = 6.9, 3.5 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 compound (0.435 g,
79%). 1H 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 afford the target
compound (0.77 g, 86%) as a yellow oil. 1H NMR (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-cyanoisoquinoline (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).
To a 0 °C solution of 1-(quinolin-8-yl)ethanol27a (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, 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).
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
chromatography (10–50% EtOAc/Hex) to give the desired compound
(0.70 g, 83%) as a clear oil. 1H 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).
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 (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,
which was sufficiently pure to use in the next step. 1H
NMR (400 MHz, chloroform-d) δ 8.18 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 8.1 Hz, 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).
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).
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-CoVPLpro(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. Humanserum albumin (HSA, catalog no.
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. coliBL21(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-CoVPLpro 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-NL63PLP2(1565–1894) was purified by the same protocol as SARS-CoVPLpro 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-CoVPLpro 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-CoVPLpro were concentrated to 6
and 12 mg/mL.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-CoVPLpro and HCoV-NL63PLP2, 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
Humanserum 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 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]
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.
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-CoVPLpro 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-CoVPLpro–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-CoVPLpro–15g inhibitor complex structure (pdb:3MJ5) as a search model and the MolRep[31] 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.
Authors: Naina Barretto; Dalia Jukneliene; Kiira Ratia; Zhongbin Chen; Andrew D Mesecar; Susan C Baker Journal: J Virol Date: 2005-12 Impact factor: 5.103
Authors: A Bermingham; M A Chand; C S Brown; E Aarons; C Tong; C Langrish; K Hoschler; K Brown; M Galiano; R Myers; R G Pebody; H K Green; N L Boddington; R Gopal; N Price; W Newsholme; C Drosten; R A Fouchier; M Zambon Journal: Euro Surveill Date: 2012-10-04
Authors: Sander van Boheemen; Miranda de Graaf; Chris Lauber; Theo M Bestebroer; V Stalin Raj; Ali Moh Zaki; Albert D M E Osterhaus; Bart L Haagmans; Alexander E Gorbalenya; Eric J Snijder; Ron A M Fouchier Journal: MBio Date: 2012-11-20 Impact factor: 7.867
Authors: Eveline Kindler; Hulda R Jónsdóttir; Doreen Muth; Ole J Hamming; Rune Hartmann; Regulo Rodriguez; Robert Geffers; Ron A M Fouchier; Christian Drosten; Marcel A Müller; Ronald Dijkman; Volker Thiel Journal: MBio Date: 2013-02-19 Impact factor: 7.867
Authors: Lia van der Hoek; Klaus Sure; Gabriele Ihorst; Alexander Stang; Krzysztof Pyrc; Maarten F Jebbink; Gudula Petersen; Johannes Forster; Ben Berkhout; Klaus Uberla Journal: PLoS Med Date: 2005-08-23 Impact factor: 11.069
Authors: Miklós Békés; Wioletta Rut; Paulina Kasperkiewicz; Monique P C Mulder; Huib Ovaa; Marcin Drag; Christopher D Lima; Tony T Huang Journal: Biochem J Date: 2015-06-01 Impact factor: 3.857
Authors: Anna M Mielech; Xufang Deng; Yafang Chen; Eveline Kindler; Dorthea L Wheeler; Andrew D Mesecar; Volker Thiel; Stanley Perlman; Susan C Baker Journal: J Virol Date: 2015-02-18 Impact factor: 5.103
Authors: Xufang Deng; Sudhakar Agnihothram; Anna M Mielech; Daniel B Nichols; Michael W Wilson; Sarah E StJohn; Scott D Larsen; Andrew D Mesecar; Deborah J Lenschow; Ralph S Baric; Susan C Baker Journal: J Virol Date: 2014-08-06 Impact factor: 5.103
Figure 1
Scheme 1
Scheme 3
Scheme 2
Scheme 4
Scheme 5
Scheme 6
Figure 2
Figure 3