Expedited Approach toward the Rational Design of Noncovalent SARS-CoV-2 Main Protease Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.1c00509

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II, and XII, with eac

Full text of DOI:10.1021/acs.jmedchem.1c00509

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Expedited Approach toward the Rational Design of Noncovalent
SARS-CoV‑2 Main Protease Inhibitors
Naoya Kitamura, Michael Dominic Sacco, Chunlong Ma, Yanmei Hu, Julia Alma Townsend,
Xiangzhi Meng, Fushun Zhang, Xiujun Zhang, Mandy Ba, Tommy Szeto, Adis Kukuljac,
Michael Thomas Marty, David Schultz, Sara Cherry, Yan Xiang, Yu Chen,* and Jun Wang*
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ABSTRACT: The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been
reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II, and
XII, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design
with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A)
that is structurally distinct from the canonical Mpro inhibitor GC376. Significantly, 23R is highly selective compared with covalent
inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 Mpro with 23R revealed a
previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered 23R, one of the most
potent and selective noncovalent SARS-CoV-2 Mpro inhibitors reported to date, and a novel binding pocket in Mpro that can be
explored for inhibitor design.
09, and MI-3010 all belong to this category (Figure 1). PF-
07304814, an α-hydroxyl ketone prodrug, is being developed
INTRODUCTION
■
The COVID-19 pandemic had a significant impact on the
global economy and public health, and there is an urgent need
for therapeutic interventions. The viral polymerase inhibitor
by Pfizer and has optimal pharmacokinetic properties and
recently entered human clinical trials.6 GC376 has in vivo
antiviral efficacy in treating cats infected with lethal feline
infectious peritonitis virus.11,12 Recently, the GC376 analog 6j
was shown to protect mice from MERS-CoV infection.9 MI-09
and MI-30 were shown to protect mice from lethal SARS-
CoV-2 infection.10 These promising results highlight the
translational potential of Mpro inhibitors as potent SARS-
CoV-2 antivirals and validate Mpro as an antiviral drug target
for coronaviruses.
remdesivir gained FDA approval on October 22nd, 2020. The
combination therapy of remdesivir with the Janus kinase (JAK)
inhibitor baricitinib also received FDA emergency use
authorization.1 Among the other drug targets being pursued
at preclinical and clinical stages,2 the viral main protease
(Mpro), also called 3-chymotrypsin-like protease (3CLpro), is
one of the most extensively explored high profile antiviral drug
targets.3 Mpro is a cysteine protease encoded in the viral
polyprotein as nonstructural protein 5 (Nsp5) that cleaves the
viral polyproteins pp1a and pp1ab at more than 11 sites.
Despite its multiple proteolytic sites, Mpro was shown to have a
high substrate specificity of glutamine at the P1 position.4 As
such, the majority of the reported Mpro inhibitors were
designed to contain a 2-pyrrolidone at the P1 substitution as a
mimetic of the glutamine in the substrate.5 Most advanced
Mpro inhibitors including PF-07304814,6 GC376,7,8 6j,9 MI-
Special Issue: COVID-19
Received: March 19, 2021
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Figure 1. Promising SARS-CoV-2 Mpro inhibitors reported in the literature with translational potential.
Figure 2. Structure of Mpro with inhibitors. (A) X-ray crystal structure of SARS-CoV-2 Mpro in complex with calpain inhibitor XII (PDB: 6XFN).
(B) X-ray crystal structure of SARS-Co-V Mpro in complex with ML188 (R) (PDB: 3V3M). Hydrogen bonds are shown in dashed lines.
Drug discovery is a lengthy process involving iterative cycles
of design, synthesis, and pharmacological characterization. In
the event of the COVID-19 pandemic, an expedited approach
with a fast turnover of this development cycle is highly desired.
Using SARS-CoV-2 Mpro as a drug target, we report herein a
fast-track drug discovery approach by coupling structure-based
drug design and the Ugi four-component reaction (Ugi-4CR)
methodology. The design was based on the superimposed
structures of SARS-CoV or SARS-CoV-2 Mpro in complex with
inhibitors including GC376, calpain inhibitor XII, and
ML188 (R).7,8,13 The lead compound 23R from this study is
the most potent noncovalent SARS-CoV-2 Mpro inhibitor
reported to date in terms of enzymatic inhibition and cellular
antiviral activity. The target selectivity of the designed
inhibitors was profiled against a panel of viral proteases and
host proteases, and the noncovalent inhibitor 23R was found
to be highly selective compared to the covalent inhibitor
GC376. An X-ray crystal structure of SARS-CoV-2 Mpro in
complex with 23R was solved, revealing a drug-induced
conformational change and a previously unexplored binding
site in between the S2 and S4 pockets. Overall, this study led to
the discovery of the noncovalent Mpro inhibitor 23R with
potent enzymatic inhibition and in vitro cellular antiviral
activity with a novel mechanism of action.
inhibitors we recently discovered, calpain inhibitor XII has
an unexpected binding mode showing an inverted conforma-
tion in the active site (Figure 2A).8 Instead of projecting the
norvaline and leucine side chains into the S1 and S2 pockets as
one would expect from its chemical structure, the pyridinyl
substitution snuggly fits in the S1 pocket and forms a hydrogen
bond with the H163 imidazole (Figure 2A). This hydrogen
bond is essential, as replacing the pyridine with benzene led to
an analog UAWJ257 with a significant loss of enzymatic
inhibition.8 Examining the X-ray crystal structures of SARS-
CoV and SARS-CoV-2 Mpro in the PDB database revealed
another compound ML188 (R),13 which shares a similar
binding mode with calpain inhibitor XII. ML188 (R) is a
noncovalent SARS-CoV Mpro inhibitor derived from a high-
throughput screening hit.13 The pyridinyl from ML188 (R)
similarly fits in the S1 pocket and forms a hydrogen bond with
the H163 side chain imidazole (Figure 2B). In addition, the
furyl oxygen and its amide oxygen both form a hydrogen bond
with the G143 main chain amide amine. ML188 (R) was
reported to inhibit the SARS-CoV Mpro with an IC50 value of
1.5 0.3 μM and the SARS-CoV viral replication in Vero E6
cells with an EC50 value of 12.9 μM.13 Several follow up studies
have been conducted to optimize the enzymatic inhibition and
cellular antiviral activity of this series of compounds; however,
no significant improvement has been made.14,15
RESULTS AND DISCUSSION
The similar binding mode of ML188 (R) with calpain
inhibitor XII, coupled with the convenient synthesis through
the one pot Ugi-4CR, inspired us to design noncovalent SARS-
■
Rational Design of Noncovalent SARS-CoV-2 Mpro
Inhibitors. Among the noncanonical SARS-CoV-2 Mpro
B
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Figure 3. Design rationale for the noncovalent SARS-CoV-2 Mpro inhibitors. (A) Superimposed X-ray crystal structures of SARS-CoV Mpro
+
ML188 (R) (PDB: 3V3M, green) and SARS-CoV Mpro H41A mutant + peptide substrate (PDB: 2Q6G, yellow with backbone shown as ribbon
representation). (B) Superimposed X-ray crystal structures of SARS-CoV Mpro + ML188 (R) (PDB: 3V3M) and SARS-CoV-2 Mpro + GC376
(PDB: 6WTT). (C) Superimposed X-ray crystal structures of SARS-CoV Mpro + ML188 (R) (PDB: 3V3M) and SARS-CoV-2 Mpro + UAWJ247
(PDB: 6XBH). (D) Chemical structures of ML188 (R), peptide substrate VLQS, GC376, and UAWJ247. (E) Stepwise optimization of ML188
(R) toward potent noncovalent SARS-CoV-2 Mpro inhibitor.
CoV-2 Mpro inhibitors based on the ML188 (R) scaffold.
Specifically, we leverage our understanding of the Mpro
inhibition mechanism based on the X-ray crystal structures
of SARS-CoV-2 Mpro with multiple inhibitors to guide the lead
optimization (Figure 3A−D).7,8 Overlaying the X-ray crystal
structures of SARS-CoV Mpro + ML188 (R) (PDB: 3V3M)
and the SARS-CoV Mpro H41A mutant + the peptide substrate
(PDB: 2Q6G) revealed that the furyl, 4-tert-butylphenyl,
pyridinyl, and tert-butyl of ML188 (R) fit in the S1′, S2, S1,
and S3 pockets, respectively (Figure 3A, D). Therefore, the
furyl, 4-tert-butylphenyl, pyridinyl, and tert-butyl substitutions
in ML188 (R) were defined as P1′, P2, P1, and P3,
respectively. Next, overlaying the structure of SARS-CoV
3V3M) and SARS-CoV-2 Mpro + UAWJ247 (PDB: 6XBH)
suggested that the 4-tert-butyl at the P2 substitution of ML188
(R) can be replaced by phenyl to occupy the extra space in the
S2 pocket (Figure 3C, D). Overall, binding site analysis
suggests that extending the P2 and P3 substitutions of ML188
(R) might lead to better shape complementarity with the
SARS-CoV-2 Mpro (Figure 3E). In practice, we adopted a
stepwise optimization procedure in which the P3 and P2
substitutions were optimized individually in step 1 and then
the optimal P2/P3 substitutions were combined in step 2
(Figure 3E).
Guided by the design rationale elucidated above, a focused
library of ML188 analogs was designed and synthesized
(Figure 4). As the P1′ furyl and P1 pyridinyl both form a
critical hydrogen bond with the Mpro (Figure 4A, B), the P1′
and P1 substitutions were kept with minimal variations for the
design of noncovalent inhibitors (Figure 4C). All designed
compounds were synthesized using the one pot Ugi four-
component reaction and tested as enantiomer/diastereomer
mixtures (Figure 4C). To circumvent the need of relying on an
expensive chiral HPLC column for the separation of
Mpro + ML188 (R) (PDB: 3V3M) and SARS-CoV-2 Mpro
+
GC376 (PDB: 6WTT) suggested that the tert-butyl at the P3
substitution of ML188 (R) can be extended to fit in the S4
pocket (Figure 3B, D). Previous structure−activity relationship
studies of GC376 indicate that P4 substitution is important,
while P3 substitution does not contribute significantly to the
binding affinity, as it is solvent exposed.3,8,9,16 Similarly, the
overlaying structures of SARS-CoV Mpro + ML188 (R) (PDB:
C
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Figure 4. Design and synthesis of a focused library of noncovalent SARS-CoV-2 Mpro inhibitors. (A) X-ray crystal structure of SARS-CoV Mpro
+
ML188 (R) (PDB: 3V3M). (B) Binding interactions of ML188 (R) with SARS-CoV Mpro. (C) Synthesis of ML188 analogs using the Ugi four-
component reaction.
enantiomers, we strategically introduced the chiral isocyanide
so that the diastereomer product mixture can be separated by
convenient silica gel column or reverse phase HPLC column
purification.17
enantiomers, we therefore decided to fix the P3/P4
substitution as α-methylbenzyl substitution during the P2
optimization (Figure 5B). All compounds in Figure 5B were
designed to have extended substitutions at the 4-position of
benzyl to occupy the extra space in the S2 pocket (Figure 3C).
Consistent with the design hypothesis, several compounds
including 14, 17, 18, 19, 20, 21, and 23 had significantly
improved enzymatic inhibition (IC50 < 3 μM) compared to
compound 13. Replacing the tert-butyl in compound 13 with
the bulkier trimethylsilyl led to compound 14 with a 2.9-fold
increase in Mpro inhibition. Cyclohexyl (17), thienyl (19),
pyrrolyl (20), pyridinyl (21), and phenyl (23) were found to
be the most favorable substitutions at the S2 pocket.
Compound 16 with piperidyl substitution had similar potency
as compound 13, while compound 15 with O-tert butyl was
less active. Further extending the substitution to benzyl led to
compound 22 that was inactive, suggesting biphenyl might be
the longest substitution that can be accommodated at the S2
pocket.
Structure−Activity Relationship Studies of Non-
covalent SARS-CoV-2 Mpro Inhibitors. In total, 39
compounds were synthesized (Figure 5A−E) and all
compounds were initially tested as a mixture of enantiomers
or diastereomers in the FRET-based enzymatic assay against
SARS-CoV-2 Mpro at 20 μM (Figure 5F). Compounds showing
more than 50% inhibition at 20 μM were further titrated to
determine the IC50 values. Next, compounds with IC50 values
lower than 5 μM were selected for cellular cytotoxicity
profiling in Vero E6 cells, the cell line which was used for the
SARS-CoV-2 antiviral assay. The purpose was to prioritize lead
candidates for the in vitro cellular antiviral assay with infectious
SARS-CoV-2. As shown in Figure 5, the majority of the
designed compounds showed more than 50% inhibition when
tested at 20 μM. Specifically, Figure 5A lists compounds with
P4 variations. As a reference, ML188 (1) (racemic mixture)
inhibits SARS-CoV-2 Mpro with an IC50 value of 10.96 1.58
μM. It was found that compounds 2, 3, 5, 6, 7, 8, 10, and 13
had improved enzymatic inhibition compared to ML188 (1).
These results suggest that (a) isopropyl (2), cyclopropyl (3),
cyclopentyl (5), cyclohexyl (6), and phenyl (7 and 8) are the
more favorable substitutions at the P3 position than tert-butyl
and (b) compound 13 with the (S)-α-methylbenzyl sub-
stitution at the P3 position had improved potency, which
suggests that extending the substitutions to the S4 pocket
might improve the enzymatic inhibition (Figure 3B). Given the
advantage of convenient separation of diastereomers over
The P1′ and P1 substitutions (Figure 5C, D) were chosen to
retain the critical hydrogen bonds in ML188 (Figure 4A). It
was found that imidazole (24) was tolerated at the P1′ position
(IC50 = 0.96 0.09 μM), followed by isoxazole (25) (IC50
=
2.47 0.27 μM) and oxazole (26) (IC50 = 4.97 0.78 μM).
Pyrazine (27) was tolerated at the P1 position (IC50 = 4.93
0.79 μM); however, imidazole (28) and pyrimidine (29) were
not preferred (IC50 > 20 μM).
Next, the above identified favorable P1′, P2, P1, and P3/P4
substitutions were combined and the designed compounds
were shown in Figure 5E. Compounds 36, 37, and 38 were the
most potent leads with IC50 values of 0.81 0.24, 0.67 0.15,
D
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Figure 5. Structures of noncovalent and covalent SARS-CoV-2 Mpro inhibitors and the enzymatic inhibition against SARS-CoV-2 Mpro. (A)
Noncovalent analogs with P3/P4 modifications. (B) Noncovalent analogs with P2 modifications. (C) Noncovalent analogs with P1′ modifications.
(D) Noncovalent analogs with P1 modifications. (E) Noncovalent analogs with combined P1′, P1, P2, and P3/P4 modifications. Compounds with
potent enzymatic inhibition (IC50 < 5 μM) but moderate to high cellular cytotoxicity (CC50 < 100 μM) are labeled in red. Compounds with both
potent enzymatic inhibition (IC50 < 5 μM) and low cellular cytotoxicity (CC50 > 100 μM) are labeled in blue. (F) Percentage enzymatic activity of
SARS-CoV-2 Mpro in the presence of the designed compounds at 20 μM concentration.
E
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Figure 6. Enzymatic inhibition, cellular antiviral activity, and selectivity index of noncovalent Mpro inhibitors. (A) Antiviral activity and selectivity
index of noncovalent SARS-CoV-2 Mpro inhibitors. (Selection criteria IC50 < 1 μM, CC50 > 100 μM). Antiviral assay was performed using the
SARS-CoV-2 USA-WA1/2020 isolate in Vero E6 cells with an MOI of 0.05. (B) Chemical structures of the two diastereomers 23R and 23S. The
absolute stereochemistry of compound 23R was determined in the cocrystal structure of this diastereomer with SARS-CoV-2 Mpro (PDB: 7KX5).
(C and D) Antiviral activity of 23R against SARS-CoV-2 in Calu-3 cells. (C) Raw data of the percentage of immunofluorescence positive cells with
different concentrations of 23R. (D) Antiviral potency and cytotoxicity plots. N.T. = not tested. N.A. = not applicable.
and 0.28 0.07 μM, respectively. Compounds 39 and 40 were
also highly active with IC50 values of 1.48 0.56 and 1.05
0.09 μM, respectively.
was inactive in the antiviral assay (EC50 > 20 μM), probably
due to its incomplete inhibition of the Mpro in the cellular
content. Gratifyingly, compounds 20, 23, 37, 38, and 40 all
had potent cellular antiviral activity with EC50 values ranging
Among the active compounds with IC50 values lower than 5
μM, compounds 3, 6, 14, 17, 18, 19, 26, 34, and 36 had
moderate to high cellular cytotoxicity in Vero E6 cells (Figure
5A−E red), while compounds 5, 20, 21, 23, 24, 25, 27, 31, 32,
37, 38, 39, and 40 were well tolerated and the CC50 values
were greater than 100 μM (Figure 5A−E blue).
from 0.82 to 4.54 μM. Compound 24 was less active (EC50
=
13.06 2.30 μM), possibly due to the poor cellular membrane
permeability.
Given the potent antiviral activity and high selectivity index
of these potent lead compounds, we then selected the
noncovalent inhibitor 23 for further characterization. The
two diastereomers of 23 were separated by reverse phase
HPLC (Figure 6B). Both diastereomers were tested in the
FRET-based enzymatic assay. GC376 was included as a
positive control. It was found that 23R is the active
Cellular Antiviral Activity of Noncovalent SARS-CoV-
2 Mpro Inhibitors. Next, compounds with potent enzymatic
inhibition (IC50 ≤ 1 μM) and low cellular cytotoxicity (CC50
>
100 μM) were prioritized for the cellular antiviral assay with
infectious SARS-CoV-2 in Vero E6 cells using the immuno-
fluorescence assay as the primary assay (Figure 6A). ML188
(1) was included as a control. It was found that ML188 (1)
diastereomer with an IC50 value of 0.20
0.01 μM, while
the 23S diastereomer was not active (IC50 > 20 μM) (Figure
F
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Figure 7. Selectivity of noncovalent and covalent SARS-CoV-2 Mpro inhibitors shown in a color map. (A) Selectivity against viral cysteine proteases.
(B) Selectivity against host proteases. The values plotted were the IC50 values from the FRET-based enzymatic assay.
Figure 8. Characterization of the mechanism of action of 23R to SARS-CoV-2 Mpro using native mass spectrometry, thermal shift assay, and
enzyme kinetic studies. (A) Binding of 23R to SARS-CoV-2 Mpro using native mass spectrometry. Native mass spectra with the inset deconvolved
spectra revealing ligand binding with 10 μM or 30 μM GC376 added (middle panel) and 10 μM and 30 μM 23R (bottom panel) with 4 mM DTT
added. The peaks are annotated with the blue circle as the dimer, the green down triangle as the dimer with one ligand bound, and the purple up
triangle as the dimer with two ligands bound. (B) Dose-dependent melting temperature (Tm) shift in the thermal shift assay. 3 μM SARS-CoV-2
Mpro protein was incubated with various concentrations of ML188 or 23R in the presence of 4 mM DTT. Measured Tm was plotted against
compound concentration with one-site binding function in Prism 8. (C and D) Enzymatic kinetic assay with ML188 and compound 23R. Kinetic
parameters in the presence of various concentrations of ML188 or 23R were globally fitted with a Michaelis−Menten function in prism 8 (top
panels); double-reciprocal plots are shown in the right panels. The middle panels show the Morrison plots of compounds ML188 and 23R with 20
μM FRET substrate used.
6A). The stereochemistry of 23R was determined by the
cocrystal structure with SARS-CoV-2 Mpro as described in the
following section. Compared with the parent compound
ML188 (1), the optimized lead 23R had more than a 54-
fold increase in enzymatic inhibition against SARS-CoV-2
Mpro. Compound 23R also showed comparable potency
against SARS-CoV Mpro with an IC50 value of 0.27
0.03
μM. Neither ML188 (1) nor 23R inhibited the SARS-CoV-2
G
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Figure 9. X-ray crystal structure of SARS-CoV-2 Mpro in complex with 23R. (A) X-ray crystal structure of SARS-CoV-2 Mpro in complex with 23R
(PDB: 7KX5). (B) Superimposed structures of SARS-CoV-2 Mpro + 23R (PDB: 7KX5) and SARS-CoV Mpro + ML188 (R) (PDB: 3V3M). (C)
Superimposed structures of SARS-CoV-2 Mpro + 23R (PDB: 7KX5) and SARS-CoV-2 Mpro + calpain inhibitor XII (PDB: 6XFN). (D)
Superimposed structures of SARS-CoV-2 Mpro + 23R (PDB: 7KX5) and SARS-CoV-2 Mpro + GC376 (PDB: 6WTT).
papain-like protease (PLpro) (IC50 > 20 μM) (Figure 6A),
suggesting the inhibition of SARS-CoV-2 Mpro by 23R is
specific.
target selectivity.18,19 It was recently reported that GC376
inhibits host cathepsins B and L in addition to SARS-CoV-2
20
Mpro
,
and GC376 analogs compound_1 and zyy16 that
Next, the antiviral activity of 23R was tested against SARS-
CoV-2 (USA-WA1/2020 isolate) in Vero E6 cells using the
immunofluorescence assay. It was found that compound 23R
had an EC50 value of 1.27 μM (Figure 6A), which was similar
to the antiviral potency of the covalent inhibitor GC376 (EC50
= 1.51 μM). Compound 23R was also not cytotoxic to Vero
E6 cells at up to 100 μM. In contrast, the parent compound
ML188 (1) had no detectable antiviral activity when tested at
up to 20 μM. To further confirm the antiviral activity of
compound 23R, we performed a secondary antiviral assay in
the human lung epithelial Calu-3 cell line, which endogenously
expresses TMPRSS2 and is widely used as a physiological
relevant cell line for SARS-CoV-2 infection. It was found that
compound 23R inhibited SARS-CoV-2 (USA-WA1/2020
isolate) replication in Calu-3 cells with an EC50 value of 3.03
μM, and it was not cytotoxic at up to 100 μM (Figure 6C, D).
Profiling the Target Selectivity of 23R against Other
Viral Cysteine Proteases and Host Proteases. One of the
major challenges facing cysteine protease inhibitors is the
contained the aldehyde and cyanohydrin warheads, respec-
tively, inhibit both calpain 1 and cathepsin K.21 These results
raised the concern of the selectivity of this class of covalent
inhibitors. To profile the target selectivity of the lead
compounds developed in this study, we selected compound
23R as a representative example of the noncovalent inhibitor.
ML188 (1) and GC376 were included as controls. The
compounds were tested against a panel of viral cysteine
proteases including SARS-CoV Mpro, MERS-CoV Mpro
,
HCoV-OC43 Mpro, EV-A71 3Cpro, EV-D68 3Cpro, EV-A71
2Apro, EV-D68 2Apro, and SARS-CoV-2 PLpro, as well as host
cysteine proteases calpain 1, cathepsin K, cathepsin L, and
serine protease trypsin. It was found that GC376 inhibited all
the Mpro and 3Cpro tested including SARS-CoV-2 Mpro, SARS-
CoV Mpro, MERS-CoV Mpro, HCoV-OC43 Mpro, EV-A71
3Cpro, and EV-D68 3Cpro (IC50 ≤ 0.16 μM), but not the
unrelated EV-A71 2Apro, EV-D68 2Apro, and SARS-CoV-2
PLpro (IC50 > 50 μM) (Figure 7A and Table S1). In addition,
GC376 also inhibited host cysteine proteases calpain 1,
H
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cathepsin K, and cathepsin L (IC50 ≤ 0.074 μM) but not the
serine protease trypsin (IC50 > 50 μM) (Figure 7B and Table
S1). In contrast, the noncovalent inhibitor 23R developed in
this study only selectively inhibited SARS-CoV-2 Mpro and
SARS-CoV Mpro with high potency (IC50 ≤ 0.27 μM) but not
other viral proteases tested, which was similar to ML188 (1)
(Figure 7A and Table S1). Compound 23R showed weak
inhibition against calpain 1 and cathepsin L with IC50 values of
6.00 μM and 10.5 μM, respectively, corresponding to
selectivity indexes of 40 and 70 with regard to SARS-CoV-2
Mpro inhibition (Figure 7B and Table S1).
pyridinyl ring to the α-methylbenzyl moiety forms a hydrogen
bond with the main chain of Glu166. The benzyl ring of the α-
methylbenzyl moiety is partially positioned in both the S2 and
S4 pockets, a novel binding pose that has not been observed
with existing Mpro inhibitors. Normally, a substituent at this
position would be expected to flip away from the enzyme core
toward the solvent-exposed S3 pocket, which explains why P3
substitutions have little to no influence on the enzymatic
inhibition.4 However, the chirality and hydrophobic nature of
the benzyl ring in 23R cause it to project toward the core near
the S2 pocket, forcing Gln189 to rotate outward (Figure 9D).
This conformation is reinforced by π-stacking interactions with
the first phenyl of the biphenyl substituent. Notably, the
binding pose of 23R features continuous intramolecular π-
stacking, where the phenyl is sandwiched by furan and benzyl
Mechanism of Action of Noncovalent Inhibitor 23R in
Inhibiting SARS-CoV-2 Mpro. The mechanism of action was
characterized using native mass spectrometry, the thermal
shift-binding assay, and enzymatic kinetic studies (Figure 8). In
the native mass spectrometry binding assay, compound 23R
showed dose-dependent binding to SARS-CoV-2 Mpro, similar
to the positive control GC376, with a binding stoichiometry of
one drug per monomer (Figure 8A). Similarly, compound 23R
showed dose-dependent stabilization of the SARS-CoV-2 Mpro
in the thermal shift binding assay with an apparent Kd value of
9.43 μM, a 9.3-fold decrease compared to ML188 (1) (Figure
8B). In the enzymatic kinetic studies, 23R was shown to be a
noncovalent inhibitor with a Ki value of 0.07 μM (Figure 8C,
D top and middle panels). In comparison, the Ki for the parent
compound ML188 (1) is 2.29 μM. The Lineweaver−Burk or
double-reciprocal plot with different compound concentrations
yielded an intercept at the Y-axis, suggesting that 23R is a
competitive inhibitor similar to ML188 (1) (Figure 8C, D
bottom panel). Overall, the enzymatic kinetic studies
confirmed that compound 23R is a noncovalent inhibitor of
groups, potentially contributing to its potent inhibition of Mpro
.
Meanwhile, the S4 pocket remains largely unoccupied by 23R,
leaving room for further improvement. In summary, the X-ray
crystal structure of SARS-CoV-2 Mpro in complex with 23R
revealed two interesting structural features: (1) The P2
biphenyl is probably the longest substitution that can be
accommodated in the S2 pocket, which is consistent with our
design hypothesis. (2) The benzyl group from the terminal α-
methylbenzyl fits in a pocket in between the S2 and S4
pockets, and this is a ligand-induced binding site that has not
been previously explored. Although this is unexpected from the
design perspective, this novel binding mode suggests that the
new binding pocket in between S2 and S4 can be explored for
inhibitor design.
CONCLUSION
■
SARS-CoV-2 Mpro
.
The viral Mpro is a high profile antiviral drug target, and several
Mpro inhibitors are now in animal model studies and human
clinical trials.6 Among the known Mpro inhibitors, the majority
are covalent inhibitors such as GC376 analogs that contain a
pyrrolidone in the P1 position as a glutamine mimetic. Several
structurally distinct compounds including ebselen, disulfiram,
carmofur, PX-12, tideglusib, and shiknonin were claimed as
Mpro inhibitors22,23 but were later proved to be promiscuous
nonspecific cysteine protease inhibitors.24,25 In addition,
noncovalent inhibitors such as ML188 (R) were developed
and validated as SARS-CoV Mpro inhibitors.5,13 Several follow
up studies have been conducted to optimize the enzymatic
potency of this series of compounds against SARS-CoV Mpro
and SARS-CoV-2 Mpro. However, no significant improvement
has been made, and ML188 (R) remains the only noncovalent
inhibitor with moderate antiviral activity against SARS-CoV
(EC50 = 12.9 0.7 μM).14,15 Nevertheless, given the sequence
and structural similarities between SARS-CoV and SARS-CoV-
2 Mpro, and the similar binding mode of ML188 (R) and
calpain inhibitor XII, we hypothesize that ML188 (R) is a
promising scaffold for structure-based drug design. In this
study, we developed an expedited drug discovery approach for
the design of noncovalent SARS-CoV-2 Mpro inhibitors. The
design approach couples the superimposed X-ray crystal
structures with the one-pot Ugi 4CR synthetic methodology.
We were able to improve the enzymatic inhibition potency of
ML188 (1) by 54-fold from a focused library of 39
noncovalent inhibitors. This is a significant advantage
compared to covalent inhibitors such as GC376, which
involves at least a five-step synthesis. In addition, by
introducing the chiral isocyanide, the diastereomer product
can be conveniently separated by either silica gel column or
X-ray Crystal Structure of SARS-CoV-2 Mpro with 23R.
Using X-ray crystallography, we successfully determined the
binding pose of 23R with SARS-CoV-2 Mpro at 2.6 Å
resolution (Figure 9A, Table S2). Electron density reveals
the body of 23R extends throughout the substrate binding
channel, with side chains occupying S1′, S1, S2, and a
previously unexplored subpocket in between S2 and S4. The
binding pose is similar to the previously solved structure of
SARS-CoV Mpro with ML188 (R) (PDB: 3V3M),13 consistent
with the similarities between the two compounds and between
the two proteins (Figure 9B). The furyl moiety of 23R binds to
a portion of the P1′ site, which normally accommodates small
hydrophobic residues. While the furylamide carbonyl group of
23R does not insert into the oxyanion hole, it does form a
bifurcated hydrogen bond with the apical residue of this
oxyanion hole, Gly143. However, the furan ring oxygen is
likely a weaker hydrogen bond acceptor than the amide
oxygen, and it lies outside of the plane of Gly143’s amide NH.
Directly attached to the furylamide moiety is a P2 biphenyl
group and a P1 pyridinyl ring. The P2 biphenyl group projects
directly into the S2 pocket, which prefers hydrophobic residues
such as leucine and phenylalanine. As expected, the P1
pyridinyl ring occupies the S1 pocket, which is known for its
strict preference for glutamine. While most Mpro inhibitors bear
a pyrrolidinone glutamine mimetic at the P1 position, we
determined that more hydrophobic residues can also bind to
the S1 site and that hydrogen bond formation with His163 is
critical for inhibition.8 In this instance, the pyridinyl ring of
23R is nearly superimposable with the same moiety from
calpain inhibitor XII (Figure 9C) forming a close (2.9 Å)
hydrogen bond with His163. An amide bond connecting the
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trifluoroacetic acid in 90% CH3CN and 10% H2O. The gradients
are 0−2 min 10% B, 2−15 min 10%−100% B, 15−18 min, 100% B,
18.1−20 min 10% B. All compounds submitted for testing were
confirmed to be >95.0% purity by HPLC traces. All final products
were characterized by proton and carbon NMR, HPLC, and HRMS.
General Procedure for Ugi-4CR Reaction.
reverse phase HPLC column, bypassing the need for an
expensive chiral HPLC column. Target selectivity profiling
showed that the noncovalent inhibitor 23R only selectively
inhibits SARS-CoV-2 and SARS-CoV Mpro, but not other viral
proteases and host proteases including calpain 1, cathepsins L
and K, and trypsin. In contrast, the covalent Mpro inhibitor
GC376 is not selective and inhibits host cysteine proteases,
which might result in potential side effects. Furthermore, the
X-ray crystal structure of SARS-CoV-2 Mpro in complex with
23R reveals a ligand-induced binding pocket in between S2
and S4 sites that can be explored for drug design. Overall,
using the expedited drug discovery approach, this study
revealed a promising noncovalent Mpro inhibitor 23R with a
well characterized mechanism of action and potent cellular
antiviral activity for further development.
Ug-4CR reaction was performed according to the reported procedure
with modifications.13 Amine (1.0 equiv) and aldehyde (1.0 equiv)
were mixed in methanol (10 mL) and stirred at room temperature for
30 min. Then carboxylic acid (1.0 equiv) and isocyanide (1.0 equiv)
were added sequentially and the resulting mixture was stirred at room
temperature overnight. After that, the solvent was removed under
reduced pressure and the crude product was purified with flash silica
gel chromatography (methanol in dichloromethane 1−5% or acetone
in hexane 30−80%).
General Procedures for the Synthesis of Compounds 24, 28, 33,
35, and 37 by TFA Deprotection. To a solution of N-trityl-protected
Ugi-4CR compound in dichloromethane (5 mL) was added TFA (1
mL). The mixture was stirred at room temperature for 2 h, and the
solvent was removed under reduced pressure. The crude mixture was
diluted in dichloromethane and purified by silica gel flash column
chromatography (ammonia 10% methanol in dichloromethane 10−
15%) to give the final product.
MATERIALS AND METHODS
■
Cell Lines and Viruses. VERO E6 cells (ATCC, CRL-1586) were
cultured in Dulbecco’s modified Eagle’s medium (DMEM),
supplemented with 5% heat inactivated FBS in a 37 °C incubator
with 5% CO2. SARS-CoV-2, isolate USA-WA1/2020 (NR-52281),
was obtained through BEI Resources and propagated once on VERO
E6 cells before it was used for this study. Studies involving SARS-
CoV-2 were performed at the UTHSCSA biosafety level-3 laboratory
by personnel wearing powered air purifying respirators.
Protein Expression and Purification. SARS CoV-2 main
protease (Mpro or 3CL) gene from strain BetaCoV/Wuhan/
WIV04/2019 and SARS-CoV main protease from strain
CDC#200301157 in the pET29a(+) vector with E. coli codon
optimization were ordered from GenScript (Piscataway, NJ). The
Mpro gene was then subcloned into pE-SUMO vector as described
previously.7,8 The expression and purification of SARS-CoV and
SARS-CoV-2 Mpro with unmodified N- and C-termini were detailed in
our previous publication.8
Procedure for the Synthesis of Compound 18 by Suzuki−
Miyamura Cross-coupling.
The expression and purification of SARS CoV-2 papain-like
protease (PLpro) were also described in our previous publications.7,8,24
The expression and purification of EV-A71 2Apro and 3Cpro, EV-
D68 2Apro and 3Cpro were described in our previous publications.24,26
Cathepsin K and Cathepsin L were purchased from EMD Millipore
(catalog #. 219461 and 219402, respectively).
Calpain 1 and trypsin were purchased from Sigma-Aldrich (catalog
#. C6108 and T6763, respectively).
To solution of 2-[N-(4-bromophenyl)-1-(furan-2-yl)formamido]-N-
[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide (1 mmol) and furan-
2-boronic acid (1 mmol) in 1,4-dioxane in a microwave reaction vial
was added an aqueous solution of K2CO3 (4 mmol). The resulting
solution was purged with N2 for 5 min. The catalyst, Pd(PPh3)4 (0.1
mmol), was added in one portion. The vial was capped and heated to
140 °C for 30 min with microwave irradiation. After cooling down to
room temperature, the reaction solution was diluted with dichloro-
methane and extracted with water, followed by brine. The organic
layer was dried over MgSO4, filtrated, and concentrated under
reduced pressure. The crude product was purified by silica gel flash
column chromatography (methanol in dichloromethane 1−5%) to
give the final product.
Peptide Synthesis. The SARS-CoV-2 Mpro FRET substrate
Dabcyl-KTSAVLQ/SGFRKME(Edans) was synthesized as described
before.7 The SARS-CoV-2 PLpro, EV-A71 2Apro and 3Cpro, and EV-
D68 2Apro and 3Cpro FRET substrates were listed in our previous
publication24 and were synthesized by solid-phase synthesis through
iterative cycles of coupling and deprotection using the previously
optimized procedure.27
General Chemical Methods. All chemicals were purchased from
commercial vendors and used without further purification unless
otherwise noted. 1H and 13C NMR spectra were recorded on a
Bruker-400 or -500 NMR spectrometer. Chemical shifts are reported
in parts per million referenced with respect to residual solvent
(CD3OD) 3.31 ppm, (DMSO-d6) 2.50 ppm, and (CDCl3) 7.26 ppm
or from internal standard tetramethylsilane (TMS) 0.00 ppm. The
following abbreviations were used in reporting spectra: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets;
ddd, doublet of doublet of doublets. All reactions were carried out
under N2 atmosphere, unless otherwise stated. HPLC-grade solvents
were used for all reactions. Flash column chromatography was
performed using silica gel (230−400 mesh, Merck). High resolution
mass spectra were obtained using an OrbitrapTM for all the
compounds, obtained in an Ion Cyclotron Resonance (ICR)
spectrometer. The purity was assessed by using Shimadzu UPLC
with Shimdazu C18-AQ column (4.6 × 150 mm P/N #227-30767-
05) at a flow rate of 1 mL/min; λ = 254 and 220 nm; mobile phase A,
0.1% trifluoroacetic acid in H2O, and mobile phase B, 0.1%
Procedure for the Synthesis of 4-(Thiophen-2-yl)aniline by
Suzuki−Miyamura Cross-coupling.
The starting material 4-(thiophen-2-yl)aniline used for the synthesis
of compound 19 was prepared using the following procedure. To a
suspension of thiophene-2-boronic acid (1.0 mmol) and sodium
carbonate (1.0 mmol) in toluene/methanol (4:1, 40 mL) was added
4-bromoaniline (1.0 mmol). The resulting solution was purged with
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N2 for 10 min. The catalyst, Pd(PPh3)4 (0.1 mmol), was added in one
portion. The reaction mixture was stirred for 16 h at 100 °C. After
cooling to room temperature, the reaction mixture was diluted with
ethyl acetate (100 mL) and washed with water (100 mL). The
organic layer was dried over MgSO4, filtrated, and concentrated under
reduced pressure. The crude product was purified by silica gel flash
column chromatography (ethyl acetate in hexane 20−40%) to give
the final product.
gel chromatography (methanol in dichloromethane 1−5%) to give the
final product.
a. N-tert-Butyl-2-[N-(4-tert-butylphenyl)-1-(furan-2-yl)-
formamido]-2-(pyridin-3-yl)acetamide (1). Light yellow solid, 81%
yield. 1H NMR (400 MHz, DMSO-d6) δ 8.38−8.24 (m, 2H), 7.86 (s,
1H), 7.69−7.66 (m, 1H), 7.37 (dt, J = 7.9, 2.0 Hz, 1H), 7.26−7.17
(m, 2H), 7.26−7.02 (br s, 1H), 7.14−7.04 (m, 2H), 6.31 (dd, J = 3.6,
1.7 Hz, 1H), 6.15 (s, 1H), 5.24 (d, J = 3.5 Hz, 1H), 1.22 (s, 9H), 1.19
(s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 168.1, 158.1, 151.1,
150.9, 148.5, 146.4, 145.2, 137.3, 136.5, 131.2, 130.8, 125.2, 122.7,
115.8, 111.2, 62.3, 50.4, 34.3, 31.0, 28.3. C26H31N3O3, HRMS
calculated for m/z [M + H]+: 434.24437, found 434.2438. HPLC
purity: 97%.
1
4-(Thiophen-2-yl)aniline. White solid. 31% yield. H NMR (500
MHz, CDCl3) δ 7.43−7.38 (m, 2H), 7.17−7.12 (m, 2H), 7.01 (dd, J
= 5.0, 3.6 Hz, 1H), 6.69−6.63 (m, 2H), 3.68 (s, 2H). 13C NMR (126
MHz, CDCl3) δ 146.10, 145.11, 127.93, 127.25, 125.24, 123.17,
121.36, 115.39.
The isocyanide used for the synthesis of compound 40 was
prepared and used in the method below:
b. 2-[N-(4-tert-Butylphenyl)-1-(furan-2-yl)formamido]-N-(prop-
1
an-2-yl)-2-(pyridin-3-yl)acetamide (2). White solid. 82% yield. H
NMR (400 MHz, DMSO-d6) δ 8.37−8.28 (m, 2H), 8.10 (d, J = 7.5
Hz, 1H), 7.76−7.61 (m, 1H), 7.37 (dt, J = 7.9, 2.0 Hz, 1H), 7.30−
7.17 (m, 2H), 7.24−7.00 (br s, 1H), 7.17−7.07 (m, 2H), 6.30 (dd, J =
3.6, 1.7 Hz, 1H), 6.15 (s, 1H), 5.26 (d, J = 3.6 Hz, 1H), 3.99−3.76
(m, 1H), 1.20 (s, 9H), 1.07 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.5 Hz,
3H). 13C NMR (101 MHz, DMSO-d6) δ 167.6, 158.2, 151.1, 151.0,
148.6, 146.3, 145.2, 137.4, 136.5, 131.0, 130.7, 125.2, 122.8, 115.8,
111.2, 62.1, 40.8, 34.3, 31.0, 22.0, 22.0. C25H29N3O3, HRMS
calculated for m/z [M + H]+: 420.2287 (calculated), 420.2282
(found). HPLC purity: 98%.
c. 2-[N-(4-tert-Butylphenyl)-1-(furan-2-yl)formamido]-N-cyclo-
propyl-2-(pyridin-3-yl)acetamide (3). White solid. 80% yield. 1H
NMR (400 MHz, DMSO-d6) δ 8.34−8.28 (m, 3H), 7.67 (dd, J = 1.7,
0.7 Hz, 1H), 7.37 (dt, J = 8.0, 1.9 Hz, 1H), 7.26−7.19 (m, 2H), 7.25−
6.94 (br s, 1H), 7.16−7.09 (m, 2H), 6.31 (dd, J = 3.6, 1.7 Hz, 1H),
6.09 (s, 1H), 5.28 (dd, J = 3.6, 0.8 Hz, 1H), 2.71−2.60 (m, 1H), 1.20
(s, 9H), 0.67−0.53 (m, 2H), 0.40−0.25 (m, 2H).13C NMR (101
MHz, DMSO-d6) δ 169.8, 158.2, 151.1, 148.7, 146.3, 145.2, 137.4,
136.4, 130.7, 125.3, 122.8, 115.4, 111.2, 62.0, 34.3, 31.0, 22.0, 5.1.
C25H27N3O3, HRMS calculated for m/z [M + H]+: 418.2131
(calculated), 418.2125 (found). HPLC purity: 96%.
Procedure for Synthesis of N-(3-Phenylpropyl)formamide.
3-Phenyl propylamine (2 mmol) in a microwave reaction vial was
mixed with ethyl formate (6 mL). The vial was capped and heated to
70 °C overnight. After reaction residual ethyl formate was evaporated
under reduced pressure. The crude product was purified by silica gel
flash column chromatography (methanol in dichloromethane 1−3%)
to give the product N-(3-phenylpropyl)formamide. Oil, 98% yield. 1H
NMR (500 MHz, CDCl3) δ 8.15−7.96 (m, 1H), 7.33−7.24 (m, 2H),
7.24−7.12 (m, 3H), 6.18−5.73 (m, 1H), 3.35−3.17 (m, 2H), 2.69−
2.60 (m, 2H), 1.89−1.78 (m, 2H). 13C NMR (126 MHz, CDCl3) δ
170.33, 164.88, 161.44, 141.51, 141.27, 140.65, 128.67, 128.55,
128.52, 128.42, 128.41, 126.32, 126.13, 126.07, 41.15, 39.39, 37.84,
33.36, 33.22, 32.60, 32.54, 31.17, 31.16, 23.31.
1. Procedure for the Synthesis of Compound 40.
d. 2-[N-(4-tert-Butylphenyl)-1-(furan-2-yl)formamido]-2-(pyri-
din-3-yl)-N-(2,4,4-trimethylpentan-2-yl)acetamide (4). Light yellow
1
solid. 60% yield. H NMR (500 MHz, DMSO-d6) δ 8.36−8.28 (m,
2H), 7.79−7.59 (m, 2H), 7.43−7.35 (m, 1H), 7.32-6.97 (br s, 1H),
7.30−7.18 (m, 2H), 7.12 (dd, J = 8.0, 4.8 Hz, 1H), 6.36−6.26 (m,
1H), 6.18 (s, 1H), 5.25 (d, J = 3.6 Hz, 1H), 1.67 (dd, J = 75.5, 14.6
Hz, 2H), 1.35−1.23 (m, 7H), 1.21 (s, 9H), 0.86 (s, 9H). 13C NMR
(101 MHz, DMSO-d6) δ 167.6, 158.0, 151.1, 150.9, 148.5, 146.3,
145.1, 137.4, 136.6, 131.0, 130.8, 125.2, 122.5, 115.7, 111.2, 62.4,
54.3, 50.6, 34.2, 31.1, 31.1, 31.1, 31.0, 28.9, 28.3. C30H39N3O3, HRMS
calculated for m/z [M + H]+: 490.3069 (calculated), 490.3064
(found). HPLC purity: 97%.
e. 2-[N-(4-tert-Butylphenyl)-1-(furan-2-yl)formamido]-N-cyclo-
pentyl-2-(pyridin-3-yl)acetamide (5). White solid. 85% yield. 1H
NMR (400 MHz, DMSO-d6) δ 8.36−8.28 (m, 2H), 8.18 (d, J = 7.0
Hz, 1H), 7.70−7.65 (m, 1H), 7.37 (dt, J = 7.9, 2.0 Hz, 1H), 7.28−
7.18 (m, 2H), 7.25−6.94 (br s, 1H) 7.18−7.06 (m, 2H), 6.30 (dd, J =
3.6, 1.7 Hz, 1H), 6.16 (s, 1H), 5.26 (d, J = 3.6 Hz, 1H), 4.02 (h, J =
6.9 Hz, 1H), 1.86−1.67 (m, 2H), 1.65−1.31 (m, 5H), 1.30−1.21 (m,
1H), 1.20 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 168.1, 158.2,
151.1, 151.0, 148.6, 146.3, 145.2, 137.3, 136.5, 131.0, 130.7, 125.2,
122.8, 115.8, 111.2, 62.0, 50.6, 34.3, 32.0, 31.8, 31.0, 23.5, 23.4.
C27H31N3O3, HRMS calculated for m/z [M + H]+: 446.2443
(calculated), 446.2438 (found). HPLC purity: 100%.
f. 2-[N-(4-tert-Butylphenyl)-1-(furan-2-yl)formamido]-N-cyclo-
hexyl-2-(pyridin-3-yl)acetamide (6). White solid. 81% yield. 1H
NMR (500 MHz, DMSO-d6) δ 8.36−8.30 (m, 2H), 8.09 (d, J = 7.7
Hz, 1H), 7.67 (dd, J = 1.7, 0.7 Hz, 1H), 7.38 (dt, J = 7.9, 2.0 Hz, 1H),
7.28−7.02 (br s, 1H), 7.25−7.19 (m, 2H), 7.17−7.10 (m, 2H), 6.31
(dd, J = 3.6, 1.7 Hz, 1H), 6.18 (s, 1H), 5.28 (d, J = 3.6 Hz, 1H),
3.63−3.52 (m, 1H), 1.86−1.40 (m, 4H), 1.32−1.14 (m, 13H), 1.14−
0.92 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 167.6, 157.9, 151.1,
151.0, 148.6, 146.3, 145.2, 137.3, 136.5, 131.0, 130.7, 125.2, 122.7,
116.2, 111.2, 62.0, 47.9, 34.2, 32.0, 31.0, 25.1, 24.5, 24.3. C28H33N3O3,
To solution of N-(3-phenylpropyl)formamide (2 mmol) in dichloro-
methane (50 mL) was added triethylamine (4.8 mmol). The solution
was cooled to approximately −10 °C using an ethanol ice bath.
Triphosgene (0.4 mmol) was added to the stirring mixture. The
resulting mixture was stirred at −10 °C for 10 min. In a separate 250
mL round-bottomed flask, 4-aminobiphneyl (2 mmol) and 3-
pyridinecarboxaldehyde (2 mmol) were mixed in methanol (100
mL) and stirred at room temperature for 30 min. Then carboxylic acid
(2 mmol) and the resulting isocyanide solution (1.0 equiv) were
added sequentially, and the resulting mixture was stirred at room
temperature overnight. After that, the solvent was removed under
reduced pressure and the crude product was purified with flash silica
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HRMS calculated for m/z [M + H]+: 460.2600 (calculated),
460.2595 (found). HPLC purity: 96%.
(101 MHz, DMSO) δ 168.9, 158.2, 151.3, 151.1, 148.8, 146.3, 145.3,
139.1, 137.6, 136.5, 130.7, 128.1, 127.1, 126.7, 125.3, 122.7, 115.9,
111.2, 62.4, 42.3, 34.3, 31.0. C29H29N3O3, HRMS calculated for m/z
[M + H]+: 468.2287 (calculated), 468.2282 (found). HPLC purity:
98%.
g. 2-[N-(4-tert-Butylphenyl)-1-(furan-2-yl)formamido]-N-(4-fluo-
rophenyl)-2-(pyridin-3-yl)acetamide (7). Brown solid. 86% yield. 1H
NMR (400 MHz, MeOD-d4) δ 8.43 (d, J = 2.3 Hz, 1H), 8.34 (dd, J =
4.9, 1.6 Hz, 1H), 7.63 (dt, J = 8.0, 2.0 Hz, 1H), 7.59−7.53 (m, 2H),
7.52 (d, J = 1.8 Hz, 1H), 7.45−6.60 (br s, 2H), 7.30 (s, 2H), 7.25−
7.17 (m, 1H), 7.08−6.98 (m, 2H), 6.37 (s, 1H), 6.25 (dd, J = 3.6, 1.7
Hz, 1H), 5.46 (d, J = 3.6 Hz, 1H), 1.26 (s, 9H). 13C NMR (101 MHz,
MeOD-d4) δ 169.5, 161.4, 153.8, 152.2, 149.9, 147.4, 146.7, 140.3,
139.4, 137.4, 134.5, 132.2, 131.9, 127.1, 125.5, 123.1, 123.0, 118.4,
116.4, 116.2, 112.3, 65.5, 35.5, 31.6. C28H26FN3O3, HRMS calculated
for m/z [M + H]+: 472.2036 (calculated), 472.2031 (found). HPLC
purity: 98%.
m. 2-[N-(4-tert-Butylphenyl)-1-(furan-2-yl)formamido]-N-[(1S)-
1-phenylethyl]-2-(pyridin-3-yl)acetamide (13). Light yellow solid.
84% yield. 1H NMR (400 MHz, DMSO−D6) δ 8.68 (dd, J = 7.8, 2.7
Hz, 1H), 8.41−8.23 (m, 2H), 7.67 (ddd, J = 5.7, 1.7, 0.7 Hz, 1H),
7.46−7.24 (m, 3H), 7.28−6.94 (br s, 1H), 7.23−7.11 (m, 5H), 7.11−
7.01 (m, 2H), 6.34−6.24 (m, 2H), 5.28 (dd, J = 3.6, 0.8 Hz, 0.5H),
5.22 (dd, J = 3.6, 0.8 Hz, 0.5H), 5.04−4.91 (m, 1H), 1.37 (d, J = 7.0
Hz, 1.5H), 1.25 (d, J = 7.0 Hz, 1.5H), 1.19 (d, J = 6.0 Hz, 9H). 13C
NMR (101 MHz, DMSO) δ 168.1, 167.9, 158.2, 158.1, 151.3, 151.2,
151.0, 148.7, 146.3, 146.2, 145.2, 145.2, 144.3, 144.0, 137.5, 137.4,
136.4, 136.3, 130.8, 130.4, 128.1, 128.0, 126.6, 126.5, 126.1, 125.7,
125.2, 122.8, 122.5, 115.8, 115.8, 111.2, 111.2, 62.0, 48.3, 48.2, 34.2,
31.0, 30.9, 22.2, 22.1. C30H31N3O3, HRMS calculated for m/z [M +
H]+: 482.2443 (calculated), 482.2438 (found). HPLC purity: 97%.
n. 2-[1-(Furan-2-yl)-N-[4-(trimethylsilyl)phenyl]formamido]-N-
[(1S)-1-phenylethyl]-2-(pyridinyl)acetamide (14). Light yellow
solid. 50% yield. 1H NMR (500 MHz, CDCl3) δ 8.50−8.41 (m,
2H), 7.47 (dd, J = 49.6, 8.2 Hz, 1H), 7.39−7.16 (m, 8H), 7.10−6.81
(m, 4H), 6.25 (d, J = 13.4 Hz, 1H), 6.18−6.13 (m, 1H), 5.50−5.45
(m, 0.5H), 5.44−5.39 (m, 0.5H), 5.22−5.12 (m, 1H), 1.54 (d, J = 6.9
Hz, 1.5H), 1.48 (d, J = 7.0 Hz, 1.5H), 0.23 (s, 9H). 13C NMR (126
MHz, CDCl3) δ 168.0, 167.7, 159.8, 159.7, 151.5, 151.4, 149.8, 149.7,
146.2, 146.2, 145.1, 145.1, 143.1, 142.1, 142.0, 139.7, 139.5, 138.4,
138.1, 134.1, 134.1, 130.2, 130.1, 130.0, 129.8, 128.7, 128.6, 127.4,
127.3, 126.3, 126.1, 122.9, 122.9, 117.4, 117.4, 111.3, 111.3, 63.4,
62.9, 49.5, 49.5, 22.0, 21.9, 0.1. C29H31N3O3Si, HRMS calculated for
m/z [M + H]+: 498.2212 (calculated), 498.2220 (found). HPLC
purity: 96%.
h. 2-[N-(4-tert-Butylphenyl)-1-(furan-2-yl)formamido]-N-(4-me-
thoxyphenyl)-2-(pyridin-3-yl)acetamide (8). Brown solid. 79%
1
yield. H NMR (400 MHz, DMSO-d6) δ 10.22 (s, 1H), 8.42 (d, J
= 2.3 Hz, 1H), 8.34 (dd, J = 4.8, 1.6 Hz, 1H), 7.72−7.67 (m, 1H),
7.54−7.46 (m, 2H), 7.43 (dt, J = 8.0, 2.0 Hz, 1H), 7.35−7.05 (br s,
2H), 7.27−7.21 (m, 2H), 7.17−7.10 (m, 1H), 6.92−6.83 (m, 2H),
6.32 (dd, J = 3.6, 1.7 Hz, 1H), 6.30 (s, 1H), 5.29 (d, J = 3.6 Hz, 1H),
3.71 (s, 3H), 1.20 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 167.3,
158.3, 155.3, 151.3, 151.2, 149.0, 146.2, 145.4, 137.5, 136.3, 131.9,
130.8, 130.2, 125.4, 122.9, 120.7, 116.0, 113.9, 111.8, 62.7, 55.1, 34.3,
31.0. C29H29N3O4, HRMS calculated for m/z [M + H]+: 484.2236
(calculated), 484.2231 (found). HPLC purity: 96%.
i. 2-[N-(4-tert-Butylphenyl)-1-(furan-2-yl)formamido]-N-(2,6-di-
methylphenyl)-2-(pyridin-3-yl)acetamide (9). Light yellow solid.
1
75% yield. H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.48 (s,
1H), 8.37 (d, J = 4.9 Hz, 1H), 7.70 (dd, J = 1.7, 0.7 Hz, 1H), 7.50 (dt,
J = 8.0, 2.0 Hz, 1H), 7.28−7.08 (br s, 2H), 7.28−7.21 (m, 2H), 7.21−
7.13 (m, 1H), 7.10−6.99 (m, 3H), 6.35 (s, 1H), 6.32 (dd, J = 3.6, 1.7
Hz, 1H), 5.28 (dd, J = 3.6, 0.8 Hz, 1H), 2.07 (s, 6H), 1.21 (s, 9H).
13C NMR (101 MHz, DMSO-d6) δ 167.7, 158.3, 151.4, 151.2, 149.0,
146.2, 145.3, 137.7, 136.4, 135.4, 134.6, 130.8, 130.2, 127.6, 126.5,
125.4, 122.7, 115.9, 111.2, 62.6, 34.3, 31.0, 17.9. C30H31N3O3, HRMS
calculated for m/z [M + H]+: 482.2443 (calculated), 482.2438
(found). HPLC purity: 98%.
o. 2-{N-[4-(tert-Butoxy)phenyl]-1-(furan-2-yl)formamido}-N-
[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide (15). Yellow solid.
1
75% yield. H NMR (500 MHz, DMSO-d6) δ 8.72 (t, J = 8.17 Hz,
1H), 8.31 (dd, J = 55, 2.4 Hz, 1H), 8.33 (ddd, J = 13.5, 4.8, 1.6 Hz,
1H), 7.69 (ddd, J = 7.0, 1.7, 0.8 Hz, 1H), 7.50-7.00 (br s, 1H), 7.42-
7.06 (m, 3H), 7.43−7.32 (m, 1H), 7.31−7.12 (m, 3H), 6.76 (s, 3H),
6.34 (ddd, J = 8.2, 3.6, 1.7 Hz, 1H), 6.27 (d, J = 13.2 Hz, 1H), 5.46
(d, J = 3.6 Hz, 0.5H), 5.41 (d, J = 3.6 Hz, 0.5H), 5.05−4.94 (m, 1H),
1.38 (d, J = 7.1 Hz, 1.5H), 1.25 (d, J = 7.0 Hz, 1.5H), 1.19 (d, J = 9.5
Hz, 9H). 13C NMR (126 MHz, DMSO-d6) δ 168.3, 168.1, 166.3,
158.2, 158.1, 154.7, 154.7, 151.4, 151.4, 148.7, 146.4, 146.3, 145.3,
145.3, 144.3, 144.0, 137.6, 137.6, 134.1, 134.1, 132.0, 130.7, 130.3,
128.2, 128.1, 126.7, 126.6, 126.2, 125.7, 123.7, 122.9, 122.6, 116.1,
116.0, 111.2, 111.2, 78.3, 62.0, 48.4, 48.2, 28.4, 22.3, 22.1.
C30H31N3O4, HRMS calculated for m/z [M + H]+: 498.2392
(calculated), 498.2387 (found). HPLC purity: 97%.
j. N-[(1H-1,2,3-Benzotriazol-1-yl)methyl]-2-[N-(4-tert-butylphen-
yl)-1-(furan-2-yl)formamido]-2-(pyridin-3-yl)acetamide (10). Yel-
1
low solid. 22% yield. H NMR (500 MHz, CDCl3) δ 8.80 (t, J =
6.7 Hz, 1H), 8.50−8.46 (m, 1H), 8.40−8.36 (m, 1H), 8.10 (d, J = 8.3
Hz, 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.63−7.55 (m, 1H), 7.52−7.38 (m,
3H), 7.24 (s, 1H), 7.12−6.83 (m, 3H), 6.38 (s, 1H), 6.33−6.20 (m,
3H), 5.47−5.40 (m, 2H), 1.37 (s, 9H). 13C NMR (126 MHz, CDCl3)
δ 169.8, 159.8, 152.7, 151.6, 149.7, 146.0, 145.9, 145.3, 138.3, 136.1,
132.5, 130.1, 129.6, 127.9, 126.2, 124.4, 122.9, 119.6, 117.5, 111.3,
111.0, 63.0, 51.4, 34.7, 31.3. C29H28N6O3, HRMS calculated for m/z
[M + H]+: 509.2301 (calculated), 509.2296 (found). HPLC purity:
96%.
p. 2-[1-(Furan-2-yl)-N-[4-(piperidin-1-yl)phenyl]formamido]-N-
[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide (16). Light brown
k. 2-[N-(4-tert-Butylphenyl)-1-(furan-2-yl)formamido]-N-[(4-
1
methylbenzenesulfonyl)methyl]-2-(pyridin-3-yl)acetamide (11).
solid. 78% yield. H NMR (400 MHz, DMSO-d6) δ 8.65 (dd, J =
1
Yellow solid. 49% yield. H NMR (400 MHz, DMSO-d6) δ 9.28−
7.8, 3.6 Hz, 1H), 8.40−8.19 (m, 2H), 7.70 (ddd, J = 6.3, 1.7, 0.7 Hz,
1H), 7.64-7.01 (br s, 1H), 7.48−7.29 (m, 3H), 7.28−7.03 (m, 4H),
6.68 (s, 3H), 6.35−6.28 (m, 1H), 6.25 (d, J = 7.8 Hz, 1H), 5.30−5.23
(m, 0.5H), 5.22−5.13 (m, 0.5H), 4.98 (h, J = 7.1 Hz, 1H), 3.11−3.02
(m, 4H), 1.53 (s, 6H), 1.37 (d, J = 7.0 Hz, 1.5H), 1.24 (d, J = 7.0 Hz,
1.5H). 13C NMR (101 MHz, DMSO-d6) δ 168.3, 168.1, 158.5, 158.5,
151.3, 151.2, 150.8, 148.7, 146.4, 146.3, 145.1, 145.1, 144.3, 144.0,
137.6, 137.6, 131.7, 128.8, 128.7, 128.1, 128.0, 126.6, 126.5, 126.1,
125.7, 115.7, 115.7, 114.5, 111.3, 62.0, 54.9, 48.6, 48.3, 48.1, 30.6,
25.7, 24.9, 24.9, 24.1, 23.7, 22.3. C31H32N4O3, HRMS calculated for
m/z [M + H]+: 509.2552 (calculated), 509.2547 (found). HPLC
purity: 97%.
9.20 (m, 1H), 8.37 (dd, J = 4.3, 2.2 Hz, 1H), 8.23 (s, 1H), 7.69 (dd, J
= 1.8, 0.7 Hz, 1H), 7.56−7.49 (m, 2H), 7.30−7.26 (m, 2H), 7.24−
7.17 (m, 2H), 7.15−7.05 (m, 2H), 6.96 (s, 2H), 6.31 (dd, J = 3.6, 1.7
Hz, 1H), 6.23 (s, 1H), 5.26 (d, J = 3.6 Hz, 1H), 4.93 (dd, J = 14.1, 7.5
Hz, 1H), 4.61 (dd, J = 14.1, 5.6 Hz, 1H), 2.34 (s, 3H), 1.20 (s, 9H).
13C NMR (101 MHz, DMSO-d6) δ 169.3, 158.1, 151.4, 151.2, 148.9,
146.1, 145.4, 144.4, 137.7, 136.0, 134.4, 130.7, 129.5, 128.4, 125.3,
122.6, 116.0, 111.8, 61.6, 60.1, 34.3, 30.9, 21.0. C30H31N3O5S, HRMS
calculated for m/z [M + H]+: 546.2062 (calculated), 546.2057
(found). HPLC purity: 96%.
l. N-Benzyl-2-[N-(4-tert-butylphenyl)-1-(furan-2-yl)formamido]-
2-(pyridin-3-yl)acetamide (12). Light yellow solid. 49% yield. 1H
NMR (400 MHz, DMSO-d6) δ 8.74 (t, J = 5.9 Hz, 1H), 8.37−8.31
(m, 2H), 7.72−7.66 (m, 1H), 7.39 (dt, J = 8.0, 1.9 Hz, 1H), 7.33−
7.18 (m, 7H), 7.21−7.05 (br s, 1H), 7.17−7.08 (m, 2H), 6.31 (dd, J =
3.6, 1.7 Hz, 1H), 6.22 (s, 1H), 5.28 (d, J = 4.21 Hz, 1H), 4.42−4.27
(m, 2H), 1.21 (s, 9H). 13C NMR (101 MHz, DMSO-d6) 13C NMR
q. 2-[N-(4-Cyclohexylphenyl)-1-(furan-2-yl)formamido]-N-[(1S)-
1-phenylethyl]-2-(pyridin-3-yl)acetamide (17). White solid. 83%
1
yield. H NMR (400 MHz, DMSO-d6) δ 8.68 (dd, J = 7.7, 3.4 Hz,
1H), 8.32 (dd, J = 10.5, 4.7 Hz, 1H), 8.31 (d, J = 48 Hz, 1H), 7.70−
7.64 (m, 1H), 7.44−7.28 (m, 3H), 7.28−7.11 (m, 4H), 7.10−6.98
(m, 4H),6.33−6.24 (m, 2H), 5.25 (d, J = 3.6 Hz, 0.5H), 5.20 (d, J =
L
https://doi.org/10.1021/acs.jmedchem.1c00509
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
3.6 Hz, 0.5H), 5.05−4.91 (m, 1H), 2.48−2.31 (m, 1H), 1.84−1.57
(m, 5H), 1.39-1.10 (m, 5H), 1.31 (dd, J = 41.17, 7.02 Hz, 3H) 13C
NMR (101 MHz, DMSO-d6) δ 168.1, 167.9, 158.2, 158.1, 151.3,
151.2, 148.7, 147.8, 146.3, 146.2, 145.2, 145.2, 144.3, 144.0, 137.5,
136.6, 136.6, 131.1, 128.1, 128.0, 126.7, 126.6, 126.5, 126.1, 125.7,
115.8, 115.8, 111.2, 62.0, 48.3, 48.2, 43.1, 33.8, 33.8, 33.7, 26.1, 25.4,
22.2, 22.1. C32H33N3O3, HRMS calculated for m/z [M + H]+:
508.2600 (calculated), 508.2595 (found). HPLC purity: 98%.
r. 2-[1-(Furan-2-yl)-N-[4-(furan-2-yl)phenyl]formamido]-N-[(1S)-
1-phenylethyl]-2-(pyridin-3-yl)acetamide (18). Light brown solid.
35% yield. 1H NMR (500 MHz, DMSO-d6) δ 8.80 (dd, J = 13.9, 7.8
Hz, 1H), 8.66−8.45 (m, 2H), 7.82 (dt, J = 8.1, 1.9 Hz, 1H), 7.71
(dddd, J = 24.9, 6.9, 1.8, 0.8 Hz, 2H), 7.64−7.46 (m, 3H), 7.45−7.07
(m, 7H), 6.96 (ddd, J = 5.9, 3.4, 0.8 Hz, 1H), 6.62−6.55 (m, 1H),
6.38 (d, J = 11.7 Hz, 1H), 6.35 (ddd, J = 8.2, 3.6, 1.7 Hz, 1H), 5.66
(dd, J = 2.8 Hz, 0.8 Hz, 0.5H), 5.62 (d, J = 3.6, 0.8 Hz, 0.5H), 5.06−
4.94 (m, 1H), 1.37 (d, J = 7.0 Hz, 1.5H), 1.29 (d, J = 7.0 Hz, 1.5H).
13C NMR (126 MHz, DMSO-d6) δ 167.4, 167.2, 158.2, 158.2, 151.9,
1H), 8.44−8.23 (m, 2H), 7.66 (ddd, J = 5.5, 1.7, 0.7 Hz, 1H), 7.45−
7.31 (m, 3H), 7.31−7.11 (m, 7H), 7.10−6.96 (m, 6H), 6.39−6.18
(m, 2H), 5.43−5.38 (m, 0.5H), 5.36−5.30 (m, 0.5H), 5.06−4.89 (m,
1H), 3.86 (d, J = 6.6 Hz, 2H), 1.37 (d, J = 7.0 Hz, 1.5H), 1.25 (d, J =
7.0 Hz, 1.5H). 13C NMR (101 MHz, DMSO-d6) δ 168.1, 167.9,
158.2, 158.1, 151.4, 151.3, 148.8, 146.3, 146.3, 145.2, 145.2, 144.2,
143.9, 141.1, 140.9, 137.5, 137.1, 137.0, 131.3, 130.7, 130.3, 129.2,
128.4, 128.4, 128.3, 128.3, 128.2, 128.0, 126.6, 126.6, 126.1, 125.9,
125.9, 125.7, 122.9, 122.6, 116.0, 116.0, 111.1, 62.0, 48.3, 48.2, 40.2,
22.3, 22.1. C33H29N3O3, HRMS calculated for m/z [M + H]+:
516.2287 (calculated), 516.2282 (found). HPLC purity: 98%.
w. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(furan-2-yl)formamido)-N-[(1S)-
1-phenylethyl]-2-(pyridin-3-yl)acetamide (23). White solid. 89%
1
yield. H NMR (400 MHz, DMSO-d6) δ 8.75 (dd, J = 7.8, 5.0 Hz,
1H), 8.53−8.21 (m, 2H), 7.72−7.66 (m, 1H), 7.66−7.58 (m, 2H),
7.58−7.24 (m, 10H), 7.24−7.05 (m, 4H), 6.47−6.23 (m, 2H), 5.63−
5.53 (m, 0.5H), 5.52−5.43 (m, 0.5H), 5.10−4.92 (m, 1H), 1.39 (d, J
= 7.0 Hz, 1.5H), 1.27 (d, J = 7.0 Hz, 1.5H). 13C NMR (101 MHz,
DMSO-d6) δ 168.1, 167.9, 158.2, 158.1, 151.3, 151.3, 148.9, 146.3,
146.2, 145.3, 145.3, 144.2, 143.9, 139.4, 138.55, 138.50, 138.4, 137.6,
131.8, 128.95, 128.93, 128.2, 128.0, 127.8, 126.6, 126.6, 126.5, 126.4,
126.1, 125.7, 116.2, 116.2, 111.3, 62.1, 48.4, 48.3, 22.2, 22.1.
C32H27N3O3, HRMS calculated for m/z [M + H]+: 502.2130
(calculated), 502.2132 (found). HPLC purity: 97%.
148.3, 148.2, 146.2, 146.1, 145.5, 145.5, 144.1, 143.8, 143.4, 141.3,
141.1, 138.1, 138.0, 132.4, 132.0, 131.6, 131.6, 130.0, 128.2, 128.1,
126.7, 126.6, 126.1, 125.8, 124.4, 124.1, 123.5, 116.6, 116.6, 112.2,
111.4, 111.4, 107.0, 61.9, 48.5, 48.3, 22.2, 21.9. C30H25N3O4, HRMS
calculated for m/z [M + H]+: 492.1923 (calculated), 492.1918
(found). HPLC purity: 98%.
x. (2R)-2-(N-{[1,1′-Biphenyl]-4-yl}-1-(furan-2-yl)formamido)-N-
[(1S)-1-phenylethyl]-2-(pyridin- 3-yl)acetamide (23R, Jun8-76-
3A). White solid, 45% yield. 1H NMR (500 MHz, DMSO-d6) δ
8.74 (d, J = 7.9 Hz, 1H), 8.43 (d, J = 2.3 Hz, 1H), 8.35 (dd, J = 4.8,
1.6 Hz, 1H), 7.71−7.66 (m, 1H), 7.66−7.61 (m, 2H), 7.60−7.52 (m,
2H), 7.50 (dt, J = 8.0, 2.0 Hz, 1H), 7.46−7.32 (m, 8H), 7.30−7.15
(m, 3H), 6.36 (s, 1H), 6.32 (dd, J = 3.6, 1.7 Hz, 1H), 5.50 (d, J = 3.6
Hz, 1H), 5.02 (p, J = 7.1 Hz, 1H), 1.27 (d, J = 7.0 Hz, 3H). 13C NMR
(126 MHz, DMSO-d6) δ 168.1, 158.1, 151.3, 148.9, 146.2, 145.3,
143.9, 139.4, 138.5, 138.5, 137.6, 131.8, 130.7, 128.9, 128.2, 127.8,
126.6, 126.5, 126.4, 126.1, 123.0, 116.2, 111.3, 62.1, 48.2, 22.1.
C32H27N3O3, HRMS calculated for m/z [M + H]+: 502.2130
(calculated), 502.2132 (found). HPLC purity: 100%.
s. 2-[1-(Furan-2-yl)-N-[4-(thiophen-2-yl)phenyl]formamido]-N-
[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide (19). Light yellow
1
solid. 81% yield. H NMR (500 MHz, DMSO-d6) δ 8.79−8.71 (m,
1H), 8.48−8.23 (m, 2H), 7.74−7.64 (m, 1H), 7.58−7.45 (m, 4H),
7.44−7.31 (m, 3H), 7.30−7.14 (m, 4H), 7.14−7.05 (m, 3H), 6.40−
6.28 (m, 2H), 5.63−5.69 (m, 0.5H), 5.58−5.54 (m, 0.5H), 5.07−4.96
(m, 1H), 1.39 (d, J = 7.1 Hz, 1.5H), 1.26 (d, J = 7.0 Hz, 1.5H). 13C
NMR (126 MHz, DMSO-d6) δ 168.1, 167.9, 158.2, 158.1, 151.4,
151.3, 148.9, 146.3, 146.2, 145.4, 145.3, 144.2, 143.9, 142.0, 138.2,
138.2, 137.6, 137.6, 133.4, 133.3, 133.3, 131.9, 130.6, 130.2, 128.6,
128.5, 128.2, 128.1, 126.6, 126.6, 126.2, 126.1, 126.1, 125.7, 125.1,
124.4, 124.4, 123.0, 122.8, 116.3, 116.3, 111.3, 111.3, 62.02, 62.00,
48.3, 48.2, 22.3, 22.1. C30H25N3O3S, HRMS calculated for m/z [M +
H]+: 508.1694 (calculated), 508.1689 (found). HPLC purity: 96%.
t. 2-[1-(Furan-2-yl)-N-[4-(1H-pyrrol-1-yl)phenyl]formamido]-N-
[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide (20). Yellow solid.
y. (2S)-2-(N-{[1,1′-biphenyl]-4-yl}-1-(furan-2-yl)formamido)-N-
[(1S)-1-phenylethyl]-2-(pyridin-3- yl)acetamide (23S). White solid,
1
45% yield. H NMR (500 MHz, DMSO-d6) δ 8.75 (d, J = 7.6 Hz,
1
1H), 8.38−8.25 (m, 2H), 7.73−7.66 (m, 1H), 7.66−7.59 (m, 2H),
7.53 (d, J = 8.0 Hz, 2H), 7.47−7.31 (m, 4H), 7.30−7.13 (m, 5H),
7.12−7.06 (m, 3H), 6.36−6.32 (m, 2H), 5.54 (d, J = 3.6 Hz, 1H),
5.00 (p, J = 7.1 Hz, 1H), 1.38 (d, J = 7.0 Hz, 3H). 13C NMR (126
MHz, DMSO) δ 168.0, 158.2, 151.4, 148.9, 146.3, 145.4, 144.3,
139.4, 138.5, 138.5, 137.6, 131.8, 130.3, 128.9, 128.1, 127.8, 126.6,
126.5, 126.5, 125.8, 122.7, 116.3, 111.4, 62.1, 48.4, 22.3. C32H27N3O3,
HRMS calculated for m/z [M + H]+: 502.2130 (calculated),
502.2132 (found). HPLC purity: 97%.
80% yield. H NMR (400 MHz, DMSO-d6) δ 8.76 (dd, J = 7.8, 4.0
Hz, 1H), 8.37 (d, J = 51.9 Hz 1H), 8.34 (dd, J = 12.0, 4.4 Hz, 1H),
7.72−7.66 (m, 1H), 7.57−7.30 (m, 7H), 7.30−7.12 (m, 4H), 7.11−
7.05 (m, 2 H), 6.38−6.30 (m, 2H), 6.23 (dt, J = 5.2, 2.2 Hz, 2H),
5.58 (d, J = 3.6 Hz, 0.5H), 5.53 (d, J = 3.6 Hz, 0.5H), 5.01 (h, J = 7.3
Hz, 1H), 1.39 (d, J = 7.0 Hz, 1.5H), 1.26 (d, J = 7.0 Hz, 1.5H). 13C
NMR (101 MHz, DMSO-d6) δ 168.3, 168.1, 158.3, 158.2, 151.5,
151.4, 148.9, 146.3, 146.3, 145.5, 145.4, 144.3, 144.0, 139.1, 139.1,
137.7, 137.6, 135.8, 135.7, 132.6, 128.2, 128.1, 126.7, 126.7, 126.2,
125.8, 118.7, 118.3, 116.4, 116.4, 111.4, 110.9, 110.9, 61.9, 54.9, 48.4,
48.3, 22.3, 22.2. C30H26N4O3, HRMS calculated for m/z [M + H]+:
491.2083 (calculated), 491.2078 (found). HPLC purity: 100%.
u. 2-[1-(Furan-2-yl)-N-[4-(pyridin-2-yl)phenyl]formamido]-N-
[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide (21). Yellow solid.
z. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(1H-imidazol-4-yl)formamido)-N-
[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide (24). White solid.
1
38% yield. H NMR (500 MHz, DMSO-d6) δ 12.76 (s, 1H), 8.74
(d, J = 7.9 Hz, 1H), 8.44 (d, J = 2.4 Hz, 1H), 8.36 (dd, J = 4.8, 1.7 Hz,
1H), 7.73−7.32 (br s, 1H), 7.68−7.60 (m, 3H), 7.60−7.49 (m, 3H),
7.48−7.23 (m, 9H), 7.19 (ddd, J = 7.8, 4.8, 0.8 Hz, 1H), 6.40 (s, 1H),
5.53 (s, 1H), 5.03 (p, J = 7.2 Hz, 1H), 1.26 (d, J = 7.0 Hz, 3H). 13C
NMR (126 MHz, DMSO-d6) δ 168.3, 151.3, 148.8, 144.0, 139.6,
138.6, 137.6, 137.0, 132.1, 130.9, 128.9, 128.2, 127.8, 126.6, 126.5,
126.1, 123.0, 62.0, 48.2, 22.2. C31H27N5O2, HRMS calculated for m/z
[M + H]+: 502.2242 (calculated), 502.2238 (found). HPLC purity:
96%.
1
88% yield. H NMR (500 MHz, DMSO-d6) δ 8.77 (dd, J = 7.8, 5.8
Hz, 1H), 8.65−8.59 (m, 1H), 8.46-8.27 (m, 2H), 7.97−7.88 (m, 3H),
7.88−7.78 (m, 1H), 7.68 (ddd, J = 7.6, 1.7, 0.8 Hz, 1H), 7.57−7.00
(m, 10H), 6.36 (d, J = 14.4 Hz, 1H), 6.32 (ddd, J = 9.0, 3.6, 1.7 Hz,
1H), 5.59 (d, J = 3.6, 0.8 Hz, 0.5H), 5.54 (d, J = 3.6, 0.8 Hz, 0.5H),
5.09−4.94 (m, 1H), 1.39 (d, J = 7.0 Hz, 1.5H), 1.27 (d, J = 7.0 Hz,
1.5H). 13C NMR (126 MHz, DMSO-d6) δ 168.2, 168.0, 158.2, 158.1,
154.6, 151.4, 151.3, 149.5, 149.5, 149.0, 148.9, 146.3, 146.3, 145.4,
145.4, 144.3, 144.0, 139.9, 139.9, 138.0, 137.7, 137.7, 137.3, 137.3,
131.6, 130.6, 130.3, 128.2, 128.1, 126.7, 126.6, 126.5, 126.2, 125.8,
123.1, 122.9, 122.8, 120.3, 120.3, 116.4, 116.4, 111.43, 111.41, 62.1,
48.4, 48.3, 22.3, 22.1. C31H26N4O3, HRMS calculated for m/z [M +
H]+: 503.2083 (calculated), 503.2078 (found). HPLC purity: 98%.
v. 2-[N-(4-Benzylphenyl)-1-(furan-2-yl)formamido]-N-[(1S)-1-
phenylethyl]-2-(pyridin-3-yl)acetamide (22). White solid. 80%
aa. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(1,2-oxazol-5-yl)formamido)-N-
[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide (25). Light yellow
1
solid. 82% yield. H NMR (400 MHz, DMSO-d6) δ 8.83 (dd, J =
7.7, 5.7 Hz, 1H), 8.56−8.26 (m, 3H), 7.65−7.45 (m, 5H), 7.44−7.06
(m, 11H), 6.35 (d, J = 11.2 Hz, 1H), 5.88−5.77 (m, 1H), 5.13−4.94
(m, 1H), 1.40 (d, J = 7.0 Hz, 1.5H), 1.28 (d, J = 7.0 Hz, 1.5H). 13C
NMR (101 MHz, DMSO-d6) δ 167.5, 167.3, 161.7, 161.6, 157.0,
157.0, 151.4, 151.3, 150.7, 149.1, 144.1, 143.8, 139.7, 138.4, 137.6,
137.4, 137.3, 131.4, 130.1, 129.7, 128.9, 128.9, 128.2, 128.1, 127.9,
1
yield. H NMR (400 MHz, DMSO-d6) δ 8.71 (dd, J = 7.8, 4.2 Hz,
M
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126.7, 126.7, 126.56, 126.54, 126.1, 125.8, 123.1, 122.8, 107.11,
107.10, 62.3, 48.5, 48.4, 22.3, 22.1. C31H26N4O3, HRMS calculated for
m/z [M + H]+: 503.2083 (calculated), 503.2078 (found). HPLC
purity: 98%.
31.1, 29.0, 28.4. C32H35N3O3, HRMS calculated for m/z [M + H]+:
510.2756 (calculated), 510.2751 (found). HPLC purity: 100%.
ah. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(furan-2-yl)formamido)-N-cy-
clopentyl-2-(pyridin-3-yl)acetamide (32). White solid. 78% yield.
1H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J = 20.6 Hz, 2H), 8.24 (d,
J = 7.0 Hz, 1H), 7.68 (dd, J = 1.7, 0.7 Hz, 1H), 7.66−7.60 (m, 2H),
7.55 (d, J = 8.1 Hz, 2H), 7.51-7.12 (br s, 1H), 7.50−7.29 (m, 5H),
7.16 (dd, J = 7.9, 4.7 Hz, 1H), 6.33 (dd, J = 3.6, 1.7 Hz, 1H), 6.24 (s,
1H), 5.53 (d, J = 3.6 Hz, 1H), 4.06 (h, J = 6.6 Hz, 1H), 1.90−1.70
(m, 2H), 1.68−1.37 (m, 5H), 1.33−1.19 (m, 1H). 13C NMR (101
MHz, DMSO-d6) δ 168.2, 158.2, 151.2, 148.8, 146.3, 145.3, 139.4,
138.5, 137.5, 131.7, 130.9, 128.9, 127.8, 126.5, 126.4, 123.0, 116.2,
111.3, 62.0, 50.7, 32.1, 31.8, 23.5, 23.4. C29H27N3O3, HRMS
calculated for m/z [M + H]+: 466.2130 (calculated), 466.2125
(found). HPLC purity: 96%.
ab. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(1,3-oxazol-5-yl)formamido)-N-
[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide (26). Yellow solid.
1
85% yield. H NMR (400 MHz, DMSO-d6) δ 8.80 (dd, J = 7.8, 4.7
Hz, 1H), 8.57−8.23 (m, 3H), 7.69−7.49 (m, 5H), 7.48−7.32 (m,
6H), 7.31−7.07 (m, 5H), 6.34 (d, J = 9.3 Hz, 1H), 5.87 (d, J = 16.3
Hz, 1H), 5.02 (h, J = 7.6 Hz, 1H), 1.40 (d, J = 7.0 Hz, 1.5H), 1.27 (d,
J = 7.0 Hz, 1.5H). 13C NMR (101 MHz, DMSO-d6) δ 167.8, 167.6,
156.9, 156.8, 153.69, 153.65, 151.4, 151.3, 149.1, 144.2, 144.24,
144.21, 143.9, 140.1, 138.4, 137.6, 137.6, 137.5, 137.4, 131.8, 130.4,
130.3, 129.9, 128.9, 128.9, 128.2, 128.1, 127.9, 126.8, 126.7, 126.65,
126.64, 126.1, 125.7, 123.1, 122.8, 62.1, 48.4, 48.3, 22.2, 22.1.
C31H26N4O3, HRMS calculated for m/z [M + H]+: 503.2083
(calculated), 503.2078 (found). HPLC purity: 97%.
ai. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(1H-imidazol-4-yl)formamido)-
N-cyclopentyl-2-(pyridin-3-yl)acetamide (33). White solid. 44%
1
ac. N-tert-Butyl-2-[N-(4-tert-butylphenyl)-1-(furan-2-yl)-
yield. H NMR (500 MHz, DMSO-d6) δ 8.38 (d, J = 2.3 Hz, 1H),
formamido]-2-(pyrazin-2-yl)acetamide (27). Light yellow solid.
8.33 (dd, J = 4.8, 1.7 Hz, 1H), 8.24 (d, J = 7.1 Hz, 1H), 7.82-7.32 (br
s, 1H), 7.73−7.55 (m, 5H), 7.53−7.29 (m, 5H), 7.23−7.12 (m, 1H),
6.24 (s, 1H), 5.54 (s, 1H), 4.12−4.00 (m, 1H) 1.90−1.70 (m, 2H),
1.67−1.40 (m, 5H), 1.32−1.19 (m, 1H). 13C NMR (126 MHz,
DMSO-d6) δ 168.4, 151.2, 148.8, 139.6, 138.7, 138.6, 137.5, 132.1,
131.1, 129.0, 127.9, 126.6, 123.0, 62.0, 50.7, 32.1, 31.9, 23.5, 23.4.
C28H27N5O2, HRMS calculated for m/z [M + H]+: 466.2242
(calculated), 466.2238 (found). HPLC purity: 98%.
aj. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(furan-2-yl)formamido)-N-cyclo-
hexyl-2-(pyridin-3-yl)acetamide (34). White solid. 83% yield. 1H
NMR (500 MHz, CDCl3/ MeOD-d4) δ 8.43 (s, 1H), 8.38−8.31 (m,
1H), 7.55−7.48 (m, 3H), 7.48−7.27 (m, 6H), 7.26−6.95 (br s, 1H),
7.16−7.05 (m, 2H), 6.22 (s, 1H), 6.16−6.08 (m, 1H), 5.55 (d, J = 3.6
Hz, 1H), 3.79 (s, 1H), 3.77−3.69 (m, 1H), 2.04−1.46 (m, 5H),
1.43−0.9 (m, 5H). 13C NMR (126 MHz, CDCl3/ MeOD-d4) δ 168.1,
168.0, 159.8, 150.9, 148.9, 145.9, 145.1, 141.5, 139.3, 138.5, 137.9,
131.3, 130.8, 128.8, 127.8, 127.4, 126.8, 123.3, 117.5, 111.3, 62.7,
62.6, 32.5, 32.4, 25.3, 24.8, 24.7. C30H29N3O3, HRMS calculated for
m/z [M + H]+: 480.2287 (calculated), 480.2282 (found). HPLC
purity: 97%.
1
86% yield. H NMR (500 MHz, DMSO-d6) δ 8.53 (dd, J = 2.5, 1.5
Hz, 1H), 8.47 (d, J = 1.5 Hz, 1H), 8.43 (d, J = 2.6 Hz, 1H), 8.00 (s,
1H), 7.68 (dd, J = 1.7, 0.7 Hz, 1H), 7.35−7.16 (m, 4H), 6.33 (dd, J =
3.6, 1.7 Hz, 1H), 6.28 (s, 1H), 5.39 (d, J = 3.6 Hz, 1H), 1.22 (s, 9H),
1.14 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 166.0, 158.3, 152.0,
151.0, 146.2, 145.3, 145.2, 143.6, 143.0, 136.9, 130.2, 125.2, 116.0,
111.2, 65.1, 50.5, 34.3, 31.0, 28.1. C25H30N4O3, HRMS calculated for
m/z [M + H]+: 435.2396 (calculated), 435.2391 (found). HPLC
purity: 96%.
ad. N-tert-Butyl-2-[N-(4-tert-butylphenyl)-1-(furan-2-yl)-
formamido]-2-(1H-imidazol-4-yl)acetamide (28). Light yellow
1
solid. 60% yield. H NMR (400 MHz, DMSO-d6) δ 8.91 (d, J =
1.3 Hz, 1H), 8.02 (s, 1H), 7.73−7.66 (m, 1H), 7.36−7.29 (m, 2H),
7.23 (dd, J = 1.3, 0.7 Hz, 3H), 6.34 (dd, J = 3.6, 1.7 Hz, 1H), 6.27 (s,
1H), 5.34 (d, J = 3.6 Hz, 1H), 1.24 (s, 9H), 1.23 (s, 9H). 13C NMR
(101 MHz, DMSO-d6) δ 165.4, 158.2, 151.4, 146.0, 145.5, 136.4,
134.5, 129.9, 128.3, 125.5, 119.6, 116.2, 111.4, 56.2, 50.7, 34.4, 31.0,
28.2. C24H30N4O3, HRMS calculated for m/z [M + H]+: 423.2396
(calculated), 423.2391 (found). HPLC purity: 97%.
ae. N-tert-Butyl-2-[N-(4-tert-butylphenyl)-1-(furan-2-yl)-
ak. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(1H-imidazol-4-yl)formamido)-
formamido]-2-(pyrimidin-5-yl)acetamide (29). Yellow solid. 78%
N-cyclohexyl-2-(pyridin-3-yl)acetamide (35). White solid. 42%
1
1
yield. H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.46 (s, 2H),
yield. H NMR (500 MHz, DMSO-d6) δ 8.40 (d, J = 2.3 Hz, 1H),
7.95 (s, 1H), 7.68 (dd, J = 1.7, 0.8 Hz, 1H), 7.33-7.12 (br s, 2H)7.27
(d, J = 8.0 Hz, 2H), 6.32 (dd, J = 3.6, 1.7 Hz, 1H), 6.16 (s, 1H), 5.32
(d, J = 3.6 Hz, 1H), 1.22 (s, 9H), 1.21 (s, 9H). 13C NMR (101 MHz,
DMSO-d6) δ 167.1, 158.1, 157.8, 157.2, 151.3, 146.2, 145.3, 136.4,
130.7, 129.6, 125.5, 116.0, 111.3, 60.5, 50.5, 34.3, 30.9, 28.2.
C25H30N4O3, HRMS calculated for m/z [M + H]+: 435.2396
(calculated), 435.2391 (found). HPLC purity: 98%.
af. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(furan-2-yl)formamido)-N-cyclo-
propyl-2-(pyridin-3-yl)acetamide (30). Light yellow solid. 89% yield.
1H NMR (500 MHz, DMSO-d6) δ 8.46−8.26 (m, 3H), 7.69 (dd, J =
1.7, 0.8 Hz, 1H), 7.66−7.62 (m, 2H), 7.62−7.50 (m, 2H), 7.49-7.14
(br s, 1H), 7.48−7.31 (m, 5H), 7.17 (dd, J = 7.9, 4.8 Hz, 1H), 6.34
(dd, J = 3.6, 1.7 Hz, 1H), 6.18 (s, 1H), 5.55 (dd, J = 3.6, 0.8 Hz, 1H),
2.74−2.65 (m, 1H), 0.73−0.55 (m, 2H), 0.46−0.26 (m, 2H). 13C
NMR (126 MHz, DMSO-d6) δ 169.9, 158.2, 151.1, 148.9, 146.3,
145.4, 139.5, 138.5, 138.5, 137.5, 131.7, 130.6, 128.9, 127.8, 126.5,
126.5, 123.0, 116.3, 111.3, 62.0, 22.5, 5.6, 5.5. C27H23N3O3, HRMS
calculated for m/z [M + H]+: 438.1817 (calculated), 438.1812
(found). HPLC purity: 98%.
8.33 (dd, J = 4.8, 1.7 Hz, 1H), 8.17 (d, J = 7.7 Hz, 1H), 7.89 (s, 1H),
7.69−7.63 (m, 2H), 7.59 (d, J = 7.2 Hz, 2H), 7.54-6.99 (br s, 1 H),
7.51−7.32 (m, 5H), 7.17 (ddd, J = 7.9, 4.8, 0.8 Hz, 1H), 6.26 (s, 1H),
5.56 (s, 1H), 3.67-3.57 (m, 1H), 1.86−1.48 (m, 5H), 1.35−1.16 (m,
3H), 1.15−0.96 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 167.8,
159.8, 151.2, 148.8, 139.8, 138.5, 138.4, 137.6, 136.8, 132.0, 130.9,
129.0, 127.9, 126.7, 126.6, 123.0, 62.1, 48.0, 32.1, 25.1, 24.5, 24.4.
C29H29N5O2, HRMS calculated for m/z [M + H]+: 480.2399
(calculated), 480.2394 (found). HPLC purity: 96%.
al. 2-[N-(4-Cyclohexylphenyl)-1-(1,2-oxazol-5-yl)formamido]-N-
[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide (36). Yellow solid.
1
76% yield. H NMR (400 MHz, DMSO-d6) δ 8.69 (dd, J = 7.8, 3.1
Hz, 1H), 8.32 (ddd, J = 10.3, 4.8, 1.6 Hz, 1H), 8.31 (dd, J = 46.3, 2.2
Hz, 1H), 7.74−7.60 (m, 1H), 7.46−7.29 (m, 3H), 7.28−6.96 (m,
7H), 6.35−6.19 (m, 2H), 5.26−5.22 (m, 0.5H), 5.21−5.15 (m,
0.5H), 5.08−4.87 (m, 1H), 2.48−2.34 (m, 1H), 1.87−1.59 (m, 5H),
1.47−1.07 (m, 5H), 1.31 (dd, J = 47.0 7.0 Hz, 3 H) 13C NMR (101
MHz, DMSO-d6) δ 168.2, 167.9, 158.2, 158.1, 151.3, 151.3, 148.8,
147.8, 146.3, 146.2, 145.3, 145.2, 144.3, 144.0, 137.5, 136.6, 136.6,
131.1, 130.7, 130.4, 128.2, 128.0, 126.7, 126.6, 126.6, 126.1, 125.7,
122.8, 122.5, 115.8, 115.8, 111.2, 62.0, 54.9, 48.3, 48.2, 43.1, 33.8,
33.8, 33.7, 26.1, 25.4, 22.3, 22.1. C31H32N4O3, HRMS calculated for
m/z [M + H]+: 509.2552 (calculated), 509.2547 (found). HPLC
purity: 97%.
ag. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(furan-2-yl)formamido)-2-(pyri-
din-3-yl)-N-(2,4,4-trimethylpentan-2-yl)acetamide (31). Light yel-
1
low solid. 62% yield. H NMR (400 MHz, DMSO-d6) δ 8.48−8.22
(m, 2H), 7.75−7.66 (m, 2H), 7.66−7.60 (m, 2H), 7.60−7.49 (m,
2H), 7.51-7.09 (br s, 1H), 7.49−7.30 (m, 5H), 7.15 (ddd, J = 7.9, 4.8,
0.8 Hz, 1H), 6.32 (dd, J = 3.6, 1.7 Hz, 1H), 6.25 (s, 1H), 5.49 (dd, J =
3.6, 0.8 Hz, 1H), 1.68 (dd, J = 75.4, 14.5 Hz, 2H), 1.29 (d, J = 16.4
Hz, 6H), 0.87 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 167.7,
158.0, 151.3, 148.6, 146.3, 145.2, 139.4, 138.6, 138.6, 137.6, 131.9,
130.9, 128.9, 127.8, 126.5, 126.4, 122.8, 116.1, 111.3, 62.4, 50.7, 31.2,
am. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(1H-imidazol-4-yl)formamido)-
N-[(1S)-1-phenylethyl]-2-(pyrazin-2-yl)acetamide (37). Yellow solid.
1
42% yield. H NMR (500 MHz, DMSO-d6) δ 8.89−8.37 (m, 5H),
7.70−7.61 (m, 3H), 7.57 (dd, J = 16.7, 8.1 Hz, 2H), 7.52−7.41 (m,
3H), 7.40−7.29 (m, 4H), 7.29−7.16 (m, 3H), 6.61−6.47 (m, 1H),
N
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5.07−4.93 (m, 1H), 1.33 (d, J = 7.0 Hz, 1.5H), 1.30 (d, J = 7.0 Hz,
1.5H). 13C NMR (126 MHz, DMSO-d6) δ 166.7, 166.5, 151.4, 151.3,
146.1, 146.0, 144.2, 144.1, 143.8, 143.7, 143.4, 138.7, 131.6, 131.5,
129.0, 128.2, 128.1, 127.9, 126.6, 126.6, 126.1, 126.0, 64.5, 48.4, 48.3,
22.2, 22.2. C30H26N6O2, HRMS calculated for m/z [M + H]+:
503.2195 (calculated), 503.2190 (found). HPLC purity: 99%.
an. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(furan-2-yl)formamido)-N-[(1S)-
1-phenylethyl]-2-(pyrazin-2-yl)acetamide (38). Yellow solid. 83%
HEPES, pH7.5, 0.01% triton-100, and 5 mM DTT. The percentage of
inhibition and enzymatic IC50 values were calculated as previously
described.7,8 Briefly, the assay was performed in 96-well plates with
100 μL of 100 nM Mpro protein or 200 nM PLPro protein in their
respective reaction buffers. Then 1 μL testing compound at various
concentrations was added to each well and incubated at 30 °C for 30
min. The enzymatic reaction was initiated by adding 1 μL of 1 mM
corresponding FRET substrate (the final substrate concentration is 10
μM). The reaction was monitored in a Cytation 5 image reader with
filters for excitation at 360/40 nm and emission at 460/40 nm at 30
°C for 1 h. The initial velocity of the enzymatic reaction with and
without testing compounds was calculated by linear regression for the
first 15 min of the kinetic progress curve.
1
yield. H NMR (500 MHz, DMSO-d6) δ 8.79 (dd, J = 24.5, 7.8 Hz,
1H), 8.68−8.34 (m, 3H), 7.72−7.67 (m, 1H), 7.66−7.61 (m, 2H),
7.60−7.51 (m, 2H), 7.49-7.26 (br s, 1H), 7.48−7.41 (m, 2H), 7.39−
7.30 (m, 4H), 7.29−7.16 (m, 3H), 6.56−6.46 (m, 1H), 6.40−6.31
(m, 1H), 5.66−5.64 (m, 0.5H), 5.62−5.59 (m, 0.5H), 5.10−4.90 (m,
1H), 1.39−1.24 (m, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.5,
166.3, 158.3, 158.3, 151.2, 151.1, 146.2, 146.1, 146.0, 145.5, 145.4,
144.1, 144.0, 143.8, 143.7, 143.4, 139.6, 139.5, 138.8, 138.8, 138.6,
138.6, 131.2, 131.1, 129.0, 128.2, 128.1, 127.9, 126.7, 126.6, 126.1,
126.0, 116.6, 116.5, 111.4, 111.4, 64.5, 64.4, 48.5, 48.4, 22.2.
C31H26N4O3, HRMS calculated for m/z [M + H]+: 503.2083
(calculated), 503.2078 (found). HPLC purity: 100%.
For the Morrison plot, 10 μL of 100 nM SARS-CoV-2 Mpro protein
was added to 190 μL of Mpro reaction buffer containing testing
compound and the FRET substrate, and the reaction was monitored
for 2 h. The final FRET substrate concentration in this assay is 20 μM.
Detailed curve fitting and KI determination were described
previously.7,8
For Michaelis−Menten and Lineweaver−Burk plots, assay was
carried out as follows: 50 μL of 50 μM Mpro protein was added to 50
μL of reaction buffer containing testing compound and various
concentrations of FRET substrate to initiate the enzyme reaction. The
initial velocity of the enzymatic reaction with and without testing
compounds was calculated by linear regression for the first 15 min of
the kinetic progress curve, plotted against substrate concentrations in
Prism 8 with the Michaelis−Menten equation.
Cathepsin K and cathepsin L enzymatic assay was carried out as
follows: cathepsin K or cathepsin L was activated by incubating in
reaction buffer [20 mM sodium acetate, 1 mM EDTA, and 5 mM
DTT (pH 5.5)] for 30 min at 30 °C. Upon activation, the assay was
assembled in 96-well plates with 100 μL of cathepsin K at 200 pM or
cathepsin L at 300 pM in reaction buffer. Then, 1 μL of testing
compound at various concentrations was added to each well and
incubated at 30 °C for 30 min. The enzymatic reaction was initiated
by adding 1 μL of FRET substrate Z-Phe-Arg-AMC (BACHEM,
catalog #. 4003379.0050) (the final substrate concentration is about 5
μM for cathepsin K and 1 μM for cathepsin L). The reaction was
monitored in a Cytation 5 image reader with filters for excitation at
360/40 nm and emission at 460/40 nm at 30 °C for 1 h. The IC50
values were calculated as described in the previous section.
Calpain 1 enzymatic assay was carried out as follows: 1 μL of 1 μg/
μL calpain 1 protein was added to 100 μL of calpain I reaction buffer
[50 mM HEPES, 50 mM NaCl, and 10 mM DTT and 5 mM CaCl2
(pH 7.5)]; the enzymatic reaction was iniated by adding 1 μL of 4
mM N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin sub-
strate (Sigma-Aldrich catalog #. S6510). The reaction was monitored
with a Cytation 5 image reader with filters for excitation at 360/40 nm
and emission at 460/40 nm at 30 °C for 1 h. The IC50 values were
calculated as described in the previous section.
Trypsin enzymatic assay was carried out in a 50 μL volume
containing 100 nM trypsin in 50 mM HEPES (pH7.6) reaction buffer,
100 μM Bz-Arg-AMC·HCl (BACHEM, catalog no. 4002540.0050),
and serial concentrations of test compounds in duplicate. The
reaction was monitored with a Cytation 5 image reader with filters for
excitation at 360/40 nm and emission at 460/40 nm at 30 °C for 1 h.
The IC50 values were calculated as described in the previous section.
Differential Scanning Fluorimetry (DSF). The thermal shift
binding assay (TSA) was carried out using a Thermal Fisher
QuantStudio 5 Real-Time PCR System as described previously.7,8,29
Briefly, 3 μM SARS-CoV-2 Mpro protein in Mpro reaction buffer was
incubated with various concentrations of compound ML188 or 23R
at 30 °C for 30 min. 1X SYPRO orange dye was added, and the
fluorescence of the well was monitored under a temperature gradient
range from 20 to 90 °C with a 0.05 °C/s incremental step. Measured
Tm was plotted against compound concentration with one-site binding
function in Prism 8.
ao. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(furan-2-yl)formamido)-N-(2,2-
diphenylethyl)-2-(pyrazin-2-yl)acetamide (39). Yellow solid. 65%
1
yield. H NMR (500 MHz, DMSO-d6) δ 8.39−8.19 (m, 3H), 7.69
(dd, J = 1.7, 0.7 Hz, 1H), 7.67−7.60 (m, 2H), 7.58−7.50 (m, 2H),
7.46−7.42 (m, 2H), 7.38−7.33 (m, 1H), 7.32−7.10 (m, 12H), 7.05
(dt, J = 7.9, 2.0 Hz, 1H), 6.96 (dd, J = 8.0, 4.8 Hz, 1H), 6.33 (dd, J =
3.6, 1.7 Hz, 1H), 6.21 (s, 1H), 5.52 (dd, J = 3.6, 0.8 Hz, 1H), 4.23 (t,
J = 7.9 Hz, 1H), 4.06−3.90 (m, 1H), 3.73−3.57 (m, 1H). 13C NMR
(126 MHz, DMSO-d6) δ 168.7, 158.2, 151.4, 148.5, 146.3, 145.3,
142.7, 142.6, 139.4, 138.5, 138.5, 137.4, 131.6, 130.5, 129.0, 128.4,
128.3, 127.9, 127.8, 127.8, 126.5, 126.5, 126.3, 126.3, 122.7, 116.3,
111.3, 62.1, 50.0, 43.6. C38H31N3O3, HRMS calculated for m/z [M +
H]+: 578.2443 (calculated), 578.2438 (found). HPLC purity: 96%.
ap. 2-(N-{[1,1′-Biphenyl]-4-yl}-1-(furan-2-yl)formamido)-N-(3-
phenylpropyl)-2-(pyridin-3-yl)acetamide (40). Light yellow solid.
1
76% yield. H NMR (500 MHz, DMSO-d6) δ 8.55 (d, J = 2.2 Hz,
1H), 8.49 (dd, J = 5.2, 1.6 Hz, 1H), 8.32 (t, J = 5.5 Hz, 1H), 7.89−
7.79 (m, 1H), 7.64 (d, J = 1.5 Hz, 1H), 7.63−7.51 (m, 4H), 7.49−
7.42 (m, 1H), 7.42−7.21 (m, 5H), 7.21−7.13 (m, 2H), 7.12−7.06
(m, 3H), 6.30 (dd, J = 3.6, 1.7 Hz, 1H), 6.19 (s, 1H), 5.58 (d, J = 3.5
Hz, 1H), 3.08 (q, J = 6.6 Hz, 2H), 2.48−2.39 (m, 2H), 1.69−1.55 (m,
2H). 13C NMR (101 MHz, DMSO-d6) δ 167.7, 158.3, 146.1, 145.5,
141.6, 139.7, 138.8, 138.5, 132.8, 131.2, 128.9, 128.2, 128.2, 127.9,
126.8, 126.6, 125.7, 124.5, 116.6, 111.4, 62.6, 38.6, 32.4, 30.7.
C33H29N3O3, HRMS calculated for m/z [M + H]+: 516.2287
(calculated), 516.2282 (found). HPLC purity: 98%.
Native Mass Spectrometry. Prior to analysis, the protein was
buffer exchanged into 0.2 M ammonium acetate (pH 6.8) and diluted
to 10 μM. DTT was dissolved in water and prepared at a 400 mM
stock. Each ligand was dissolved in ethanol and diluted to 10× stock
concentrations. The final mixture was prepared by adding 4 μL of
protein, 0.5 μL of DTT stock, and 0.5 μL of ligand stock for a final
concentration of 4 mM DTT and 70 μM protein. The final ligand
concentrations were 10 μM and 30 μM. The mixtures were then
incubated for 10 min at room temperature prior to analysis. Each
sample was mixed and analyzed in triplicate.
Native mass spectrometry (MS) was performed using a Q-Exactive
HF quadrupole-Orbitrap mass spectrometer with the Ultra-High Mass
Range research modifications (Thermo Fisher Scientific). Samples
were ionized using nanoelectrospray ionization in positive ion mode
using 1.0 kV capillary voltage at a 150 °C capillary temperature. The
samples were all analyzed with a 1,000−25,000 m/z range, the
resolution set to 30,000, and a trapping gas pressure set to 3. Between
10 and 50 V of source fragmentation was applied to all samples to aid
in desolvation. Data were deconvolved and analyzed with UniDec.28
Enzymatic Assays. The main protease (Mpro) enzymatic assays
were carried out in Mpro reaction buffer containing 20 mM HEPES
pH 6.5, 120 mM NaCl, 0.4 mM EDTA, 20% glycerol, and 4 mM
DTT, and the SARS-CoV-2 papain-like protease (PLpro) enzymatic
assays were carried out in PLPro reaction buffer containing 50 mM
Cytotoxicity Measurement. Evaluation of the cytotoxicity of
compounds was carried out using the neutral red uptake assay.30,31
Briefly, 80,000 cells/mL of the tested cell lines was dispensed into 96-
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well cell culture plates at 100 μL/well. Twenty-four hours later, the
growth medium was removed and washed with 150 μL PBS buffer.
200 μL of fresh serum-free medium containing serial diluted
compounds was added to each well. After incubating for 5 days at
37 °C, the medium was removed and replaced with 100 μL DMEM
medium containing 40 μg/mL neutral red and incubated for 2−4 h at
37 °C. The amount of neutral red taken up was determined by
measuring the absorbance at 540 nm using a Multiskan FC
Microplate Photometer (Fisher Scientific). The CC50 values were
calculated from best-fit dose response curves with variable slope in
Prism 8.
Immunofluorescence Assay. Antiviral immunofluorescence
assay was carried out as previously described.8 Briefly, Vero E6 cells
in 96-well plates (Corning) were infected with SARS-CoV-2 (USA-
WA1/2020 isolate) at MOI of 0.05 in DMEM supplemented with 1%
FBS. Immediately before the viral inoculation, the tested compounds
in a 3-fold dilution concentration series were also added to the wells
in triplicate. The infection proceeded for 48 h without the removal of
the viruses or the compounds. The cells were then fixed with 4%
paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with
DMEM containing 10% FBS, and stained with a rabbit monoclonal
antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an
Alexa Fluor 488-conjugated goat antimouse secondary antibody
(ThermoFisher Scientific). Hoechst 33342 was added in the final step
to counterstain the nuclei. Fluorescence images of approximately ten
thousand cells were acquired per well with a 10× objective in a
Cytation 5 (BioTek). The total number of cells, as indicated by the
nuclei staining, and the fraction of the infected cells, as indicated by
the NP staining, was quantified with the cellular analysis module of
the Gen5 software (BioTek).
(APS) in Argonne, IL, and processed with the HKL3000 software
suite. The CCP4 versions of MOLREP32 was used for molecular
replacement using a previously solved SARS-CoV-2 Mpro structure,
6YB7. Structural refinement was performed using REFMAC533 and
COOT.34 The crystallographic statistics is shown in Supporting
Information Table S2. The complex structure for SARS-CoV-2 Mpro
with 23R has been deposited in the Protein Data Bank with the
accession ID of 7KX5 (SARS-CoV-2 MPro + Jun8-76-3A).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00509.
Target selectivity of noncovalent inhibitor 23R;
crystallographic statistics; H NMR and C NMR spectra
for final products; HPLC traces for representative
compounds (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
■
Corresponding Authors
Yu Chen − Department of Molecular Medicine, Morsani
College of Medicine, University of South Florida, Tampa,
Florida 33612, United States; Phone: 813-974-7809;
Email: ychen1@usf.edu
Antiviral Assay in Calu-3 Cells. Calu-3 cells (ATCC, HTB-55)
grown in Minimal Eagles Medium supplemented with 1%
nonessential amino acids, 1% penicillin/streptomycin, and 10% FBS
are plated in 384 well plates. The next day, 50 nL of drug suspended
in DMSO is added as an 8-pt dose response with 3-fold dilutions
between test concentrations in triplicate, starting at 40 μM final
concentration. The negative control (DMSO, n = 32) and positive
control (10 μM Remdesivir, n = 32) are included on each assay plate.
Calu3 cells are pretreated with controls and test drugs (in triplicate)
for 2 h prior to infection. In BSL3 containment, SARS-CoV-2 (isolate
USA-WA1/2020) diluted in serum free growth medium is added to
plates to achieve an MOI = 0.5. Cells are incubated continuously with
drugs and SARS-CoV-2 for 48 h. Cells are fixed and then
immunstained with anti-dsRNA (J2), and nuclei are counterstained
with Hoechst 33342 for automated microscopy. Automated image
analysis quantifies the number of cells per well (toxicity) and the
percentage of infected cells (dsRNA+ cells/cell number) per well.
SARS-CoV-2 infection at each drug concentration was normalized to
aggregated DMSO plate control wells and expressed as percentage-of-
control (POC = % Infection sample/Avg % Infection DMSO cont). A
nonlinear regression curve fit analysis (GraphPad Prism 8) of POC
infection and cell viability versus the log10 transformed concentration
values to calculate EC50 values for infection and CC50 values for cell
viability. Selectivity index (SI) was calculated as a ratio of drug’s CC50
and EC50 values (SI = CC50/IC50).
Jun Wang − Department of Pharmacology and Toxicology,
College of Pharmacy, The University of Arizona, Tucson,
Arizona 85721, United States; orcid.org/0000-0002-
4845-4621; Phone: 520-626-1366; Email: junwang@
pharmacy.arizona.edu; Fax: 520-626-0749
Authors
Naoya Kitamura − Department of Pharmacology and
Toxicology, College of Pharmacy, The University of Arizona,
Tucson, Arizona 85721, United States
Michael Dominic Sacco − Department of Molecular
Medicine, Morsani College of Medicine, University of South
Florida, Tampa, Florida 33612, United States
Chunlong Ma − Department of Pharmacology and
Toxicology, College of Pharmacy, The University of Arizona,
Tucson, Arizona 85721, United States
Yanmei Hu − Department of Pharmacology and Toxicology,
College of Pharmacy, The University of Arizona, Tucson,
Arizona 85721, United States
Julia Alma Townsend − Department of Chemistry and
Biochemistry, The University of Arizona, Tucson, Arizona
85721, United States
Xiangzhi Meng − Department of Microbiology, Immunology
and Molecular Genetics, University of Texas Health Science
Center at San Antonio, San Antonio, Texas 78229, United
States
Fushun Zhang − Department of Microbiology, Immunology
and Molecular Genetics, University of Texas Health Science
Center at San Antonio, San Antonio, Texas 78229, United
States
Xiujun Zhang − Department of Molecular Medicine, Morsani
College of Medicine, University of South Florida, Tampa,
Florida 33612, United States
Mandy Ba − Department of Pharmacology and Toxicology,
College of Pharmacy, The University of Arizona, Tucson,
Arizona 85721, United States
Mpro Crystallization and Structure Determination. 23R was
added to 20 mg/mL SARS-CoV-2 Mpro to a final concentration of
1.75 mM and incubated overnight at 4°C. This mixture was then
diluted 4-fold with protein stock buffer (20 mM Tris pH 7.5, 200 mM
NaCl, 1 mM DTT) and then spun down at 13,000g for 1 min to
remove precipitate. Crystals were grown by mixing the protein-
inhibitor sample with an equal volume of crystallization buffer (20%
PEG 3350, 0.2 M NaF) in a vapor diffusion, hanging drop apparatus.
Crystals were then transferred to a drop with crystallization buffer
containing 5 mM 23R for 1 h, followed by a brief soaking in a
cryoprotectant solution of 30% PEG 3350 and 15% glycerol with 2
mM 23R. Crystals were then flash frozen in liquid nitrogen for X-ray
diffraction.
X-ray diffraction data for the SARS-CoV-2 Mpro structures were
collected on the SBC 19-ID beamline at the Advanced Photon Source
P
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Tommy Szeto − Department of Pharmacology and
Toxicology, College of Pharmacy, The University of Arizona,
Tucson, Arizona 85721, United States
Adis Kukuljac − Department of Molecular Medicine, Morsani
College of Medicine, University of South Florida, Tampa,
Florida 33612, United States
by a COVID-19 pilot grant from UTHSCSA and NIH grant
AI151638 to Y.X. SARS-Related Coronavirus 2, Isolate USA-
WA1/2020 (NR-52281) was deposited by the Centers for
Disease Control and Prevention and obtained through BEI
Resources, NIAID, NIH.
Michael Thomas Marty − Department of Chemistry and
Biochemistry, The University of Arizona, Tucson, Arizona
85721, United States
David Schultz − Department of Biochemistry and Biophysics,
University of Pennsylvania, Philadelphia, Pennsylvania
19104, United States
Sara Cherry − Department of Pathology and Laboratory
Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania 19104, United States
Yan Xiang − Department of Microbiology, Immunology and
Molecular Genetics, University of Texas Health Science
Center at San Antonio, San Antonio, Texas 78229, United
States
ABBREVIATIONS USED
■
SARS-CoV-2, severe acute respiratory syndrome coronavirus
2; JAK, janus kinase; Ugi-4CR, Ugi four-component reaction;
Mpro, main protease; 3CLpro, 3-chymotrypsin-like protease;
Nsp5, nonstructural protein 5; 2Apro, 2A protease; 3Cpro, 3C
protease; PLpro, papain-like protease
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00509
Author Contributions
J.W. conceived and designed the study; N.K. synthesized and
characterized the compounds; M.S. carried out Mpro
crystallization and structure determination with the assistance
of X.Z. and analyzed the data with Y.C.; C.M. expressed the
Mpro and PLpro and performed the IC50 determination, thermal
shift-binding assay, and enzymatic kinetic studies; Y.H.
performed the cytotoxicity assay; X.M. and F.Z. performed
the SARS-CoV-2 immunofluorescence assay in Vero E6 cells
under the guidance of Y.X. D.S. performed the SARS-CoV-2
immunofluorescence assay in Calu-3 cells under the guidance
of S.C. J.T. performed the native mass spectrometry experi-
ments with the guidance from M.T.M.; J.W. and Y.C. secured
funding and supervised the study; J.W., Y.C., and M.S. wrote
the manuscript with input from the others.
Notes
The authors declare the following competing financial
interest(s): Naoya Kitamura, Chunlong Ma, Yanmei Hu, and
Jun Wang are inventors of a filed patent claiming the use of
23R and related compounds as potential COVID-19 antiviral
drugs.
ACKNOWLEDGMENTS
■
This research was partially supported by the National Institutes
of Health (NIH) (Grants AI147325 and AI157046) and the
Arizona Biomedical Research Centre Young Investigator grant
(ADHS18-198859) to J.W. The antiviral assay in Calu-3 cells
was conducted through the NIAID preclinical service under a
nonclinical evaluation agreement. J.A.T. and M.T.M. were
funded by the National Institute of General Medical Sciences
and National Institutes of Health (Grant R35 GM128624 to
M.T.M.). We thank Michael Kemp for assistance with
crystallization and X-ray diffraction data collection. We also
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Argonne National Laboratory, particularly those at the
Structural Biology Center (SBC), with X-ray diffraction data
collection. SBC-CAT is operated by UChicago Argonne, LLC,
for the U.S. Department of Energy, Office of Biological and
Environmental Research under contract DE-AC02-
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This paper was published ASAP on April 23, 2021, with errors
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