Optimization of Triarylpyridinone Inhibitors of the Main Protease of SARS-CoV-2 to Low-Nanomolar Antiviral Potency

Article abstract of DOI:10.1021/acsmedchemlett.1c00326

Non-covalent inhibitors of the main protease (Mpro) of SARS-CoV-2 having a pyridinone core were previously reported with IC50 values as low as 0.018 μM for inhibition of enzymatic activity and EC50 values as low as 0.8 μM for inhibition of viral replication in Vero E6 cells. The series has now been further advanced by consideration of placement of substituted five-membered-ring heterocycles in the S4 pocket of Mpro and N-methylation of a uracil ring. Free energy perturbation calculations provided guidance on the choice of the heterocycles, and protein crystallography confirmed the desired S4 placement. Here we report inhibitors with EC50 values as low as 0.080 μM, while remdesivir yields values of 0.5-2 μM in side-by-side testing with infectious SARS-CoV-2. A key factor in the improvement is enhanced cell permeability, as reflected in PAMPA measurements. Compounds 19 and 21 are particularly promising as potential therapies for COVID-19, featuring IC50 values of 0.044-0.061 μM, EC50 values of ca. 0.1 μM, good aqueous solubility, and no cytotoxicity.

Full text of DOI:10.1021/acsmedchemlett.1c00326

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pubs.acs.org/acsmedchemlett
Letter
Optimization of Triarylpyridinone Inhibitors of the Main Protease of
SARS-CoV‑2 to Low-Nanomolar Antiviral Potency
∇
∇
Chun-Hui Zhang, Krasimir A. Spasov, Raquel A. Reilly, Klarissa Hollander, Elizabeth A. Stone,
Joseph A. Ippolito, Maria-Elena Liosi, Maya G. Deshmukh, Julian Tirado-Rives, Shuo Zhang,
Zhuobin Liang, Scott J. Miller, Farren Isaacs, Brett D. Lindenbach, Karen S. Anderson,*
and William L. Jorgensen*
Cite This: https://doi.org/10.1021/acsmedchemlett.1c00326
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*
sı Supporting Information
pro
ABSTRACT: Non-covalent inhibitors of the main protease (M ) of SARS-CoV-2 having a
pyridinone core were previously reported with IC values as low as 0.018 μM for inhibition
50
of enzymatic activity and EC50 values as low as 0.8 μM for inhibition of viral replication in
Vero E6 cells. The series has now been further advanced by consideration of placement of
pro
substituted five-membered-ring heterocycles in the S4 pocket of M and N-methylation of
a uracil ring. Free energy perturbation calculations provided guidance on the choice of the
heterocycles, and protein crystallography confirmed the desired S4 placement. Here we
report inhibitors with EC values as low as 0.080 μM, while remdesivir yields values of 0.5−
50
2
μM in side-by-side testing with infectious SARS-CoV-2. A key factor in the improvement is
enhanced cell permeability, as reflected in PAMPA measurements. Compounds 19 and 21
are particularly promising as potential therapies for COVID-19, featuring IC50 values of
0
.044−0.061 μM, EC50 values of ca. 0.1 μM, good aqueous solubility, and no cytotoxicity.
KEYWORDS: SARS-CoV-2, protease inhibitors, lead optimization, COVID-19 drugs
8
nfection by SARS-CoV-2, the coronavirus responsible for the
protein crystallography. The optimization rapidly delivered
inhibitors with IC50 values as low as 0.018 μM. Subsequent
testing using infected Vero E6 cells showed that several of the
inhibitors had similar antiviral potencies as the FDA-approved
drug remdesivir, with EC50 values of ca. 1 μM. Further
optimization is described here, with emphasis on placement of
1
I
COVID-19 pandemic, can be expected to be a serious world-
2
,3
health problem for many years. Though the rapid develop-
ment and efficacy of vaccines have been impressive, a substantial
fraction of the world’s population will remain unvaccinated, and
the continuing emergence of viral variants may add further
challenges. Thus, it is important to develop alternative therapies
pro
a five-membered-ring heterocycle in the S4 pocket of M and of
regulation of cell permeability for the antiviral activity. New
inhibitors are reported that have EC values as low as 0.08 μM
and auspicious pharmacological properties.
In the prior work, we reported two subseries of analogues, one
containing a cyanophenyl substituent, as in parampanel, and the
other a uracilyl group. Compounds 2−4 and 5−7 in Figure 1
are examples. For these compounds, model building and
crystallography demonstrated that the 3-pyridyl group resides
1
−3
for people who become infected with SARS-CoV-2. Among
the potential viral targets, the chymotrypsin-like or main
50
pro
4,5
protease, M , is particularly attractive. It is essential for
viral reproduction because of its cleavage of polyproteins that are
produced from ribosomal processing of the viral RNA, and its
8
mutation rate is low, with 96% sequence homology between the
6
2
002 SARS-CoV isoform and the 2019 SARS-CoV-2. In
contrast to the common design of peptide-like covalent
pro
4
,5
in the S1 pocket of M , the cyanophenyl or uracil ring in the S1′
inhibitors for cysteine proteases, including SARS-CoV-2, we
7
,8
pocket, the chlorophenyl group in the S2 pocket, and the alkoxy
substituent in the S3−S4 channel. Though parampanel is only a
weak inhibitor, with an IC value of >100 μM, 2−7 yielded IC
have pursued non-peptidic, non-covalent inhibitors. These
alternatives are anticipated to display reduced potential for
proteolytic degradation and induction of toxicities from off-
5
0
50
9
target modification of proteins or nucleic acids.
Received: June 7, 2021
Accepted: July 13, 2021
The research started from a virtual screen of ca. 2000 known,
approved drugs that led to the identification of 14 drugs as
pro
7
inhibitors of SARS-CoV-2 M with IC values as low as 5 μM.
50
One hit, the antiepileptic drug perampanel (1) (Figure 1), was
chosen for lead optimization using computer-aided design,
synthesis, a kinetic assay measuring enzyme inhibition, and
©
XXXX American Chemical Society
https://doi.org/10.1021/acsmedchemlett.1c00326
A
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Letter
Figure 1. Inhibitors of SARS-CoV-2 Mpro. 8−17 and 19−21 are new compounds.
Table 1. Measured Activities for Inhibition of SARS-CoV-2 Mpro
compd
IC50 (μM)
compd
IC50 (μM)
compd
IC50 (μM)
a
1
2
3
4
5
6
7
100−250
8
9
0.328 ± 0.061
0.083 ± 0.023
0.085 ± 0.005
0.131 ± 0.009
0.027 ± 0.004
0.042 ± 0.015
0.105 ± 0.013
15
16
17
18
19
20
21
0.038 ± 0.015
0.061 ± 0.007
0.059 ± 0.014
0.028 ± 0.007
0.044 ± 0.009
0.130 ± 0.018
0.061 ± 0.011
0.140 ± 0.020
0.170 ± 0.022
0.120 ± 0.006
0.120 ± 0.016
0.037 ± 0.004
0.025 ± 0.003
10
11
12
13
14
a
Fluorescence of the compound interfered with the assay.
values of 0.025−0.170 μM, as summarized in Table 1. The
μM in the uracil subseries; the corresponding compounds in the
cyanophenyl subseries are generally 2−3-fold less potent.
Subsequent modeling considered replacement of the R group
8
results were sensitive to the group that terminates in the S4 site:
3
,3,3-trifluoropropoxy and o-chlorobenzyloxy substituents
yielded the most potent inhibitors, with IC values near 0.020
in the alkoxy substituent with five-membered-ring heterocycles.
50
B
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Letter
Reduced torsional degrees of freedom and potential modulation
of solubility and metabolic stability were viewed as possible
benefits. The standard modeling consisted of structure building
expectation was that multiple heterocycles should be viable,
though there is some favoring of having an oxygen or sulfur atom
at position 5 (5O and 5S). The most promising compound
appeared to be the 4-methyl-5-thiazolyl analogue (3N5S).
Ultimately, it was decided to focus on the uracil subseries
10
of the protein−ligand complexes with the BOMB program
followed by conjugate-gradient optimization of the complexes
11
pro
using the MCPRO program with the OPLS-AA/M force field
since the cyanophenyl analogues are usually weaker M
1
2
13,14
8
for the protein, the OPLS/CM1A force field for the ligand,
inhibitors. Furthermore, in initial testing for off-target activity,
and a dielectric constant of 2. The results indicated that a methyl
group or a chlorine atom at the 2-position in the heterocycle
including inhibition of cytochrome P450 enzymes, the uracil-
containing compounds were superior. On the basis of the FEP
results and toxicity concerns for furans and thiophenes,
synthetic efforts were directed to oxazole and thiazole analogues.
An attempt to prepare the 2-imidazole analogue (2N5N) was
not successful. An overview of the syntheses of the uracil
analogues is provided in Scheme 1, and full details are provided
in the Supporting Information. Briefly, 5-bromo-2-fluoropyr-
pro
19,20
should project well into the S4 site of M , as for the terminal
8
carbon atom in the alkoxy groups of 2−7. Free energy
perturbation (FEP) calculations were then used to assess more
reliably relative binding affinities of alternative hetero-
1
0,15−17
cycles.
These calculations included the surrounding
water molecules and extensive configurational sampling of the
protein, ligand, and water. The FEP calculations were carried out
starting with the BOMB-built structures and using standard
protocols with the MCPRO program and the above-mentioned
idine underwent an S Ar reaction to afford S2, which after
N
Suzuki cross-coupling and deprotection yielded S4. Chan−
21
Evans−Lam coupling and bromination then yielded S6. This
key intermediate was coupled either with commercially available
arylboronic acids or with arylboronic acid pinacol esters, which
were prepared in a one-pot two-step sequence, to afford S7. The
target compounds 5−7, 10−15, and 18 were prepared by
demethylation of S7, or S7 was further methylated and
demethylated to yield 16, 17, 19, 20, and 21.
8
,10
force fields.
Relative free energies of binding, ΔΔG , were
b
obtained by mutating the ligand from structure A to structure B
for both the protein−ligand complex in water and the unbound
ligand. The configurational sampling for the systems was carried
out at 25 °C with Monte Carlo simulations including the 242
protein residues nearest to the active site and 1250 and 2000
18
TIP4P water molecules for the ligand-bound and ligand-free
The synthesized compounds were all initially tested for their
ability to inhibit the proteolytic activity of recombinant SARS-
8
calculations, respectively, as previously described.
pro
7,8,22,23
The FEP calculations were initiated from an N-methylpyrrol-
-ylmethoxy analogue, as summarized in Table 2. The
CoV-2 M , as previously described.
The substrate
2
(Dabcyl-KTSAVLQ↓SGFRKM-E(Edans-NH ); GL Biochem),
2
when cleaved, generates a product containing a free, fluorescent
Edans group. Fluorescence measurements used an excitation
wavelength of 360 nm and emission wavelength of 460 nm with
baseline subtraction for intrinsic fluorescence of each compound
and the uncleaved FRET substrate. All of the tested compounds
had purities of at least 95% based on HPLC, and all of the
measurements were performed in triplicate and averaged.
To begin, compound 8 with a cyclopentylmethoxy substituent
was prepared to continue the 2−4 series and to consider a
saturated five-membered ring in the S4 site. With an IC of
Table 2. Computed Changes in Free Energy of Binding (in
kcal/mol) for Conversion of Five-Membered-Ring
Heterocycles
a
5
0
0.328 μM (Table 1), it turned out to be 2-fold less potent than
the cyclopropylmethoxy analogue 3. Then, for the 5-methyl-4-
oxazolyl analogues, both the cyanophenyl (9) and uracilyl (10)
alternatives were prepared and yielded very similar IC values of
5
0
0
.083 and 0.085 μM, respectively. 11, the 4-methyl-5-oxazolyl
isomer of 10, was also prepared and was a little less active at
.131 μM. One thiophene-containing compound, 12, was
prepared because of the commercial availability of 2-chloro-3-
chloromethyl)thiophene; this compound was very potent, with
an IC50 of 0.027 μM. It was gratifying that the closely related
thiazole 13 was also very potent at 0.042 μM.
2
N to
ΔΔGb
2N to
ΔΔGb
2
3
4
5
3
4
5
N
O
O
O
S
0.0
−0.24
0.42
−1.61
−0.16
0.76
2N3N
2N4N
2N5N
3N5O
3N5S
5N3O
5N3S
1.33
−0.05
−1.99
−1.74
−3.20
−1.15
0.28
0
(
S
S
Importantly, it was possible to obtain a high-resolution (1.9
Å) X-ray crystal structure for the complex of thiazole 13 with
−2.11
a
Statistical uncertainties from independent runs were ±1 kcal/mol.
pro
SARS-CoV-2 M . As shown in Figure 2, the crystal structure
confirmed the expectations from the modeling, with clear
placement of the methyl group in the S4 site between the side
chains of Met165 and Leu167. There are five protein−ligand
hydrogen bonds: the pyridinone carbonyl oxygen with Glu166
N (2.81 Å), the pyridine nitrogen with His163 (2.99 Å), and the
uracil O−NH−O edge with the NH of Cys145 (3.47 Å) and the
backbone O and NH of Thr26 (3.50 and 3.29 Å, respectively).
An edge-to-face aryl−aryl interaction between the chlorophenyl
ring and His41 and a short (2.93 Å) contact between the uracil
O4 and the nitrogen atom of Gly143 are also notable. However,
the angular aspects of the latter contact are not consistent with a
cyanophenyl series was modeled with the expectation that the
relative results for the uracil series would be similar. The
reference compound was converted to the six possible furan and
thiophene analogues, a pyrazole, and the 2- and 5-imidazoles in
an 11-window perturbation series. The 2- and 3-furan (5O and
3
O) and thiophene (5S and 3S) analogues were then converted
to the corresponding oxazoles and thiazoles. In all, 14 possible
heterocycles were considered (Table 2). Since the uncertainties
in the results from independent runs were ca. ±1 kcal/mol, the
results in Table 2 showed modest variation. Thus, the
C
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a
Scheme 1. General Synthesis of the Uracil-Containing Inhibitors
a
Reagents and conditions: (a) benzyl alcohol, NaH, anhydrous THF, 0 to 70 °C; (b) Cs CO , PdCl (PPh ) , DMF, 80 °C, N ; (c) 10% Pd/C,
2
3
2
3
2
2
MeOH/H O (10/1 v/v), H , 40 °C; (d) 3-pyridylboronic acid, Cu(OAc) , TMEDA, anhydrous DMF, air bubbled, rt; (e) NBS, dry DMF, rt; (f)
2
2
2
arylboronic acid, K CO , PdCl (PPh ) , DMF, 120 °C, N ; (g) (i) aryl bromide, KOAc, PdCl (PPh ) , B Pin , anhydrous DMF, 80 °C, N ; (ii)
2
3
2
3
2
2
2
3
2
2
2
2
MeOH, DMF, K CO , 120 °C, N ; (h) LiCl, pTsOH, anhydrous DMF, 80 °C; (j) MeI, MeCN, 60 °C; (k) TBAB, diglyme, 130 °C.
2
3
2
hydrogen bond; rather, the NH of Gly143 is hydrogen-bonded
with the side-chain carbonyl oxygen atom of Asn142 (2.68 Å).
From a force-field optimization of the 13−M complex, the
uracilyl group and also to monitor the effects with parallel
artificial membrane permeation assay (PAMPA) measurements
using hydrophobic poly(vinylidene difluoride) (PVDF) mem-
pro
24
most favorable raw interactions between the inhibitor and
protein residues are in the order Glu166 (best), Met165,
Gln189, His41, and Cys145, i.e., the S2−S4 region. Net effects
on binding would require consideration of desolvation.
branes.
Thus, N-methylated analogues (16, 17, and 19) of some of
the previously reported potent inhibitors (6, 7, and 18) were
prepared along with 20 and 21, the N-methylated analogues of
14 and 15. The synthetic route to 21, which is outlined in
Scheme 2, culminates in a Mitsunobu−Suzuki sequence to yield
the protected uracil, which is methylated and demethylated as in
Scheme 1. As listed in Table 1, N-methylation increased the IC50
values by 2−3-fold, though 16, 17, 19, and 21 remain very
potent at 0.061, 0.059, 0.044, and 0.061 μM, respectively. The
details of the PAMPA assay are provided in the Supporting
Information; however, theophylline, diclofenac, and chloram-
phenicol were used as controls and yielded permeability results
8
In the previous work, replacement of the terminal methyl
group in 2 and 5 with trifluoromethyl (4 and 7) produced
significant gains in activity, and some benefits from reduced
metabolism might also be expected. Thus, 14, the trifluor-
omethyl analogue of oxazole 11, was prepared and did yield a
small increase in activity, with an IC of 0.105 μM. It was also
50
possible to synthesize the desired (Table 2, 3N5S) 5-thiazolyl
analogue with a CF group at the 4-position, 15. Indeed, this
3
pro
compound is a very potent inhibitor of M , with an IC of
5
0
−
6
0
.038 μM.
consistent with literature ranges of (0.3−0.7) × 10 , (3−5) ×
8
−6
−6
pro
By this point and from the previous work, it had become
10 , and (5−5.5) × 10 cm/s, respectively. Results for 11 M
−
6
apparent that although some of the inhibitors had IC50 values
below 0.1 μM, including 15, they showed little or no activity in
virally infected Vero E6 cellular assays. A likely possibility
seemed to be that the uracilyl group rendered the compounds
sufficiently polar that there might be problems with low cell
permeability. To test this idea, it was decided to reduce the
polarity and hydrogen-bonding needs by methylating N1 of the
inhibitors are recorded in Table 3 and range from 0.24 × 10 to
4.27 × 10 cm/s. The permeabilities of the three cyanophenyl-
containing compounds (3, 4, and 9) are good (>3 × 10 cm/s),
while they are low (<0.8 × 10 cm/s) for the three
unmethylated uracil-containing compounds (11, 14, and 18).
N-Methylation significantly improved the permeabilities, such
that those of 16, 17, 19, 20, and 21 are all above 1 × 10− cm/s.
−
6
−
6
−
6
6
D
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Table 3. Measured PAMPA Permeabilities (in 10− cm/s)
6
and Aqueous Solubilities (in μg/mL)
compd
PAMPA
aq. sol.
3 ± 2
25.0 ± 0.1
13.1 ± 0.3
7.6 ± 0.7
167.7 ± 1.1
ND
14.2 ± 0.7
57.0 ± 0.4
21.8 ± 0.9
116.5 ± 8.8
34.5 ± 0.3
24.8 ± 1.4
3
4
9
4.27 ± 0.07
4.0 ± 1.9
3.04 ± 0.09
ND
0.24 ± 0.02
0.57 ± 0.09
3.25 ± 0.46
2.26 ± 0.14
0.73 ± 0.05
1.50 ± 0.59
1.03 ± 0.18
1.16 ± 0.32
10
11
14
16
17
18
19
20
21
25,26
Britton−Robinson buffer at pH 6.5.
With the exception of 3,
27
the results are all in the range observed for oral drugs. 11 is the
most soluble compound but has low permeability, while the final
compounds 16, 17, and 19−21 show both acceptable solubility
and permeability. The curiously much greater solubility of 11
versus its isomer 10 was fully reproducible and consistent with
observations from handling the compounds; it may arise from
significantly different arrangements in the crystalline state with
intermolecular hydrogen bonding retarded by the methyl group
adjacent to the azole nitrogen atom in 11.
Figure 2. (A) Crystal structure of the complex of 13 with Mpro. Carbon
atoms of the ligand are shown in light yellow. Hydrogen bonds between
the ligand and protein are noted with dashed lines. The resolution is 1.9
Å. Deposited in the PDB with ID 7N44. (B) Close-up of the S3−S4
channel with placement of the methyl group in the S4 site. (C) Surface
view with coloring of the protein by atom type.
Finally, multiple compounds were tested for inhibition of
SARS-CoV-2 replication in Vero E6 cells. Inhibition of viral
replication was tested in several cellular assays, as detailed in the
Supporting Information. These include a rapid screening
replicon assay using baby hamster kidney (BHK) cells with
noninfectious SARS-CoV-2, in which the spike protein in the
viral genome is replaced with a nanoluciferase reporter, allowing
the ability to multiplex in 96-well plates and assess inhibition of
viral replication in 24 h. In parallel, the compound general
cytotoxicity in either Vero E6 cells or normal human bronchial
epithelial (NHBE) cells can be assessed using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
The results for 11, 14, and 20 are interesting: the permeability of
1
1 doubles upon replacement of the methyl group by a
trifluoromethyl group on the oxazole ring and then doubles
again upon N-methylation of the uracil ring to a value of 1.03 ×
−
6
1
1
0
cm/s for 20. Similarly, the permeability of 18 doubles to
−
6
.50 × 10 cm/s for N-methylated 19. As discussed below, a
−
6
PAMPA result above ca. 0.7 × 10 cm/s appears to be necessary
for a compound to show activity in the Vero E6 cell assays.
Aqueous solubilities were also measured for 11 compounds, as
reported in Table 3, using a standard shake-flask procedure in
8
,28
28
(MTT)
or alamarBlue dyes as readouts. Also, assays
using infectious SARS-CoV-2 were performed to examine the
a
Scheme 2. Synthesis of the Precursor of 21
a
Reagents and conditions: (a) DMF, 120 °C; (b) isoamyl nitrite, anhydrous 1,4-dioxane, 85 °C; (c) LiAlH , THF, 0 °C to rt, N ; (d) ADDP, n-
4
2
Bu P, toluene, 60 °C; (e) (i) KOAc, PdCl (PPh ) , B Pin , anhydrous DMF, 80 °C, N ; (ii) MeOH, DMF, K CO , 120 °C, N ; (f) MeI, MeCN,
6
3
2
3
2
2
2
2
2
3
2
0 °C; (g) LiCl, pTsOH, anhydrous DMF, 80 °C; (h) TBAB, diglyme, 130 °C.
E
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Table 4. Enzyme Inhibition (IC ), Anti-SARS-CoV-2 Activity (EC ), and Cell Cytotoxicity (CC ) (in μM)
50
50
50
EC50
CC50
compd
IC50
replicon
infectious virus
Vero E6
NHBE
a
b
g
g
remdesivir
−
0.59 ± 0.25
1.51 ± 0.01
1.76 ± 0.31
1.20 ± 0.20
0.73 ± 0.26
>40
0.77, 2.1
1.5
0.98
72 ± 28
41 ± 2
20 ± 2
>100
g
g
g
c
,
g
g
g
2
3
4
9
0.140 ± 0.020
0.170 ± 0.020
0.120 ± 0.006
0.083 ± 0.023
0.042 ± 0.015
0.105 ± 0.013
0.038 ± 0.015
0.061 ± 0.007
0.059 ± 0.014
0.028 ± 0.007
0.044 ± 0.009
0.061 ± 0.011
22 ± 7.2
c
,
g
g
g
>100
g
g
g
1.1 ± 0.5
1.7 ± 0.1
22 ± 8
ND
25 ± 5
h
e
e
ND
e
13
14
15
16
17
18
19
21
ND
>100
>100
>100
82
22
d
e
NA
NA
ND
>100
>100
>100
ca. 95
>95
d
e
ND
0.53 ± 0.23
0.82 ± 0.28
3.2 ± 1.0
0.175 ± 0.005
1.08
1.2
ND
e
>100
>80
>32.5
ca. 1
b
f
f
0.08
>32.5
e
ND
>100
>100
a
b
c
d
e
f
g
h
From ref 30. alamarBlue. Plaque Assay. NA = not active. ND = not determined. Limited by the solubility in DMSO. From ref 8. MTT.
cytopathic effect in Vero E6 cells in the lower-throughput viral
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00326.
■
2
9
plaque assay or using MTT or alamarBlue readout. The results
from repeated individual assays are summarized in Table 4; only
the plaque assay and CC results for remdesivir, 2, 3, and 4 were
sı
*
50
8
previously reported. It should be noted that comparisons with
remdesivir are complicated by the fact that it is a prodrug that
requires activation by enzymatic hydrolysis and intracellular
phosphorylation.
Details for the synthetic procedures, compound charac-
terization, computations, assays, and crystallographic
results (PDF)
Accession Codes
Coordinates for the crystal structure (Figure 2) have been
deposited in the Protein Data Bank with ID 7N44.
The prior cyanophenyl-containing protease inhibitors 2−4
and the polymerase inhibitor remdesivir have similar activities
against SARS-CoV-2, with EC50 values of 0.6−2 μM. The
cyanophenyl-containing oxazole 9 showed similar activity in the
plaque assay, though greater potency was indicated in the
replicon results. Azoles 14 and 15 are not active in the replicon
assay in spite of their IC values of 0.105 and 0.038 μM (Table
AUTHOR INFORMATION
Corresponding Authors
■
Karen S. Anderson − Department of Pharmacology and
Department of Molecular Biophysics and Biochemistry, Yale
University School of Medicine, New Haven, Connecticut
50
1
). This can now be attributed with confidence to poor cell
0
6520-8066, United States; orcid.org/0000-0003-3433-
permeability, as reflected in the PAMPA result for 14 (Table 3).
The other unmethylated uracil, 18, is a little more permeable
and gives an EC50 of 3.2 μM in the replicon assay. Improved
results are found for the N-methylated compounds 16, 17, 19,
and 21. In particular, 19 is strikingly potent, with EC values of
0780; Email: karen.anderson@yale.edu
William L. Jorgensen − Department of Chemistry, Yale
University, New Haven, Connecticut 06520-8107, United
States; orcid.org/0000-0002-3993-9520;
Email: william.jorgensen@yale.edu
50
0
.175 μM in the replicon assay and 0.08 μM with infectious
Authors
SARS-CoV-2. These potent compounds also show negligible
cytotoxicity in both Vero E6 and NHBE cells.
Chun-Hui Zhang − Department of Chemistry, Yale University,
New Haven, Connecticut 06520-8107, United States
Krasimir A. Spasov − Department of Pharmacology, Yale
University School of Medicine, New Haven, Connecticut
In conclusion, the prior work on pyridinone inhibitors of the
main protease of SARS-CoV-2 has been expanded to include
five-membered-ring heterocycles as substituents for filling the
S3−S4 channel. Thirteen new compounds were reported, with
eight giving IC50 results for enzyme inhibition in the range
06520-8066, United States
Raquel A. Reilly − Department of Pharmacology, Yale
University School of Medicine, New Haven, Connecticut
06520-8066, United States
Klarissa Hollander − Department of Pharmacology and
Department of Molecular Biophysics and Biochemistry, Yale
University School of Medicine, New Haven, Connecticut
0
.027−0.085 μM. Projection of a methyl substituent on the
azoles into the S4 site was confirmed by the crystal structure for
the complex of thiazole 13 with M . Cell permeability was
pro
recognized as a problem for antiviral activity in Vero E6 cells for
some of the potent enzyme inhibitors. This was well-reflected in
PAMPA measurements and remedied by N-methylation of the
uracilyl group. The outcome was notable improvement in
antiviral activity for 16, 17, 19, and 21. Compound 19 is
particularly compelling; it is an order of magnitude more potent
than remdesivir with no apparent cytotoxicity and high aqueous
solubility.
06520-8066, United States
Elizabeth A. Stone − Department of Chemistry, Yale University,
New Haven, Connecticut 06520-8107, United States;
orcid.org/0000-0002-2253-3452
Joseph A. Ippolito − Department of Chemistry, Yale University,
New Haven, Connecticut 06520-8107, United States
Maria-Elena Liosi − Department of Chemistry, Yale University,
New Haven, Connecticut 06520-8107, United States
F
https://doi.org/10.1021/acsmedchemlett.1c00326
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Maya G. Deshmukh − Department of Pharmacology and
M.D.−Ph.D. Program, Yale University School of Medicine,
New Haven, Connecticut 06520-8066, United States;
orcid.org/0000-0002-3704-3229
Julian Tirado-Rives − Department of Chemistry, Yale
University, New Haven, Connecticut 06520-8107, United
States; orcid.org/0000-0001-7330-189X
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∇
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