Betulonic Acid Derivatives Interfering with Human Coronavirus 229E Replication via the nsp15 Endoribonuclease

Article abstract of DOI:10.1021/acs.jmedchem.0c02124

To develop antiviral therapeutics against human coronavirus (HCoV) infections, suitable coronavirus drug targets and corresponding lead molecules must be urgently identified. Here, we describe the discovery of a class of HCoV inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. Structure-activity relationship exploration of these 1,2,3-triazolo-fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC50: 0.6 μM) of HCoV-229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of HCoV-229E nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.

Full text of DOI:10.1021/acs.jmedchem.0c02124

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Article
Betulonic Acid Derivatives Interfering with Human Coronavirus
229E Replication via the nsp15 Endoribonuclease
Annelies Stevaert,∇ Besir Krasniqi,*,∇ Benjamin Van Loy, Tien Nguyen, Joice Thomas, Julie Vandeput,
Dirk Jochmans, Volker Thiel, Ronald Dijkman, Wim Dehaen, Arnout Voet, and Lieve Naesens*
Cite This: J. Med. Chem. 2021, 64, 5632−5644
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ABSTRACT: To develop antiviral therapeutics against human
coronavirus (HCoV) infections, suitable coronavirus drug targets
and corresponding lead molecules must be urgently identified.
Here, we describe the discovery of a class of HCoV inhibitors
acting on nsp15, a hexameric protein component of the viral
replication-transcription complexes, endowed with immune
evasion-associated endoribonuclease activity. Structure−activity
relationship exploration of these 1,2,3-triazolo-fused betulonic
acid derivatives yielded lead molecule 5h as a strong inhibitor
(antiviral EC50: 0.6 μM) of HCoV-229E replication. An nsp15
endoribonuclease active site mutant virus was markedly less
sensitive to 5h, and selected resistance to the compound mapped
to mutations in the N-terminal part of HCoV-229E nsp15, at an interface between two nsp15 monomers. The biological findings
were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors,
our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.
which inhibits the viral polymerase. Based on its interaction
with the highly conserved polymerase catalytic site, remdesivir
exhibits pan-coronavirus activity covering also HCoV-
229E.11−14 This broad CoV coverage also applies to the
clinical candidate GC376, a catalytic site inhibitor of the CoV
main protease (Mpro).15−17 Though less explored, the CoV
nsp15 endoribonuclease (EndoU) is a highly attractive drug
target since it has no cellular counterpart, its catalytic site is
conserved among CoVs, and it is amenable to structure-based
design based on available protein structures.18−22 Nsp15 is one
of the non-structural protein (nsp) components of the
replication-transcription complexes (RTCs), the site where
CoV RNA synthesis occurs.5,23,24 Although the functions of
nsp15 are not entirely understood, its EndoU function is
known to regulate viral RNA synthesis, limit the recognition of
viral dsRNA by cellular sensors, and prevent the dsRNA-
activated antiviral host cell response.25−29 The interferon type I
evading activity of nsp15 is well elaborated for mouse
coronavirus MHV-A5925 and HCoV-229E26 and was recently
also demonstrated for SARS-CoV-2.30 The concept to inhibit
INTRODUCTION
■
Four human CoVs (i.e., HCoV-229E, HCoV-HKU1, HCoV-
NL63, and HCoV-OC43) are endemic in the population and
account each year for 15 to 30% of common colds.1 These can
evolve into life-threatening lower respiratory tract infections in
elderly, children, and persons at risk.2,3 In addition, the current
SARS-CoV-2 pandemic is causing a major crisis in terms of
human health and socio-economic losses. Within a period of
∼20 years, SARS-CoV-2 is the third zoonotic coronavirus
(CoV) to enter the human species, coming after SARS (severe
acute respiratory syndrome) and MERS (Middle East
respiratory syndrome).4 Finally, the Coronaviridae family
contains several species causing serious disease in pets and
livestock.5
Somewhat similar to the respiratory illness caused by
endemic HCoVs, SARS-CoV-2 produces no or relatively
mild disease in most young persons.6 In contrast, in individuals
with comorbidities or higher age, the SARS-CoV-2 replication
phase is typically followed by a second phase that is
characterized by hyperinflammation, acute respiratory distress,
and multi-organ failure.7 Hence, management of COVID-19
most likely requires antiviral drugs to suppress initial virus
replication, plus anti-inflammatory medication, like cortico-
steroids, to treat severe cases.8 Several CoV proteins may be
suitable drug targets,9 but, at the moment, only two drug
classes have reached formal approval by the FDA, i.e., anti-
spike antibodies10 and the nucleotide analogue remdesivir,
Received: December 9, 2020
Published: April 20, 2021
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nsp15 is thus unique since it combines a direct antiviral effect
with the potential to revert viral evasion from host cell
immunity.
conditions.40 This yielded a series of 16 1,2,3-triazolo-fused
betulonic acids 5, most of which were isolated with high yield
(∼80%; Table 1). Diverse primary amines 3 were attached to
the 1,2,3-triazole ring to introduce a variety of aromatic or
aliphatic moieties.
We here report identification of a class of HCoV-229E
nsp15 inhibitors with a 1,2,3-triazolo-fused betulonic acid
structure. We describe their synthesis, structure−activity
relationship (SAR), and the mechanistic findings, in particular
resistance data, which corroborate nsp15 as the antiviral target
for HCoV-229E. These biological data accord with the binding
model that we obtained by compound docking in the
hexameric nsp15 protein structure. The model also explains
why the current lead is active against HCoV-229E, but not
other coronaviruses like SARS-CoV-2. In short, our study
validates the nsp15 protein, and particularly the interface
where the lead compound binds, as a druggable and pertinent
target for developing CoV inhibitors.
To establish the antiviral activity of the synthesized
compounds, the molecules were submitted to cell-based assays
with a broad range of DNA and RNA viruses. This phenotypic
screening indicated strong and selective activity in human
embryonic lung (HEL) fibroblast cells infected with HCoV-
229E. We used a viral cytopathic effect (CPE) reduction assay,
in which protection against CPE (expressed as the antiviral
EC50 value) was monitored by the MTS cell viability assay and
verified by microscopy. The MTS assay was also used to
quantify compound cytotoxicity (expressed as the CC50 value)
in mock-infected cells. Whereas the starting compounds
betulin 1 and betulonic acid 2 were virtually inactive, almost
all 1,2,3-triazolo-fused betulonic acid derivatives proved to be
highly effective CoV inhibitors (Table 1). Several compounds
in the series had EC50 values below 1 μM, which makes them
superior to two known CoV inhibitors, which we used as
reference compounds, i.e., K2247 and GS-441524, the
nucleoside form of remdesivir.13 Three analogues stood out
for having superior selectivity, i.e., 5g, 5h, and 5n, having a
selectivity index (ratio of CC50 to EC50) of 31, 76, and 27,
respectively. On the other hand, far lower activity was noted
for 5i (EC50 value: 13 μM), which bears a non-aromatic
cyclohexanemethyl substituent. Apparently, introducing this
bulky group caused a considerable reduction in antiviral
activity and selectivity. The capacity of 5h to fully suppress
HCoV-229E replication at non-toxic concentrations is evident
from the microscopic images in Figure 1A and the dose−
response curves in Figure 1B. Also, 5h fully prevented the
formation of dsRNA intermediates of CoV RNA synthesis, as
demonstrated by immunofluorescence staining of dsRNA in
HCoV-229E-infected human bronchial epithelial 16HBE cells
(Figure 1C).
RESULTS AND DISCUSSION
■
Compound Synthesis and Structure−Activity Rela-
tionship. Since the 1,2,3-triazole moiety has the unique
property to both accept and donate hydrogen bonds,
introducing this moiety can increase the potency of
pharmacologically active molecules.31−34 We decided to fuse
this group to betulonic acid, a pentacyclic triterpenoid
compound that was the starting point for a wide variety of
agents with potential pharmacological use.35−37 For instance,
the betulinic acid core is present in bevirimat, an HIV
maturation inhibitor that has undergone phase 2 clinical
evaluation.38,39 The 1,2,3-triazolo-fused betulonic acid deriva-
tives (Scheme 1) were synthesized by our recently developed
Scheme 1. Synthesis of 1,2,3-Triazole-Fused Betulonic Acid
a
Derivatives Starting from Betulin
To conduct a SAR exploration around the lead compound
5h (Scheme 2 and Table 2), we first investigated the
contribution of the α-methyl-phenylene moiety. Compound
5q, in which this entire moiety is missing, had ∼6-fold lower
antiviral activity than 5h. When only the α-methyl moiety was
missing (5r), the activity was not affected. Compound 5s,
which is the epimer at the 1,2,3-triazole substituent, displayed
almost the same EC50 value as 5h, indicating that isomerism
does not alter the activity. Cytotoxicity was however slightly
decreased, resulting in an even better selectivity index (≥90)
than that of 5h. In order to elucidate the role of the
isopropenyl side chain, we reduced this moiety by hydro-
genation, yielding compound 5t, which was 10- to 20-fold less
active. Replacement of the carboxylic acid moiety by a methyl
group (5u) proved deleterious.
We next evaluated 5h in cell culture assays with a panel of
other CoVs. The compound had no inhibitory effect on mouse
hepatitis virus A59 (MHV-A59) and feline infectious
peritonitis virus (FIPV), in CPE reduction assays in,
respectively, murine fibroblast L2 cells and Crandell−Rees
Feline Kidney cells (data not shown). HCoV-229E and FIPV
belong to the alpha genus, while MHV-A59 belongs to the beta
genus comprising also the highly pathogenic species SARS-
CoV, MERS-CoV, and SARS-CoV-2.48,49 In VeroE6-eGFP
cells infected with SARS-CoV-2, 5h and 5t were inactive [see
ref 50 for assay description]. Hence, though nicely active
a
Reagents and conditions: (i) Na2Cr2O7, H2SO4, acetone, H2O, 0 °C
to r.t., 1 h; (ii) betulonic acid 2 (1.0 equiv), primary amine 3 (1.4
equiv), 4-nitrophenyl azide 4 (1.0 equiv), toluene (0.4 mL), 100 °C, 4
Å MS, 24 h.
and convenient “triazolization” method to prepare 1,2,3-
triazoles from primary amines and ketones.40−42 First, Jones
oxidation was performed to convert betulin 1 into betulonic
acid 2 (Scheme 1).43 Betulin 1, a natural compound isolated
from the bark of Betula species, is commercially available.44−46
Next, the triazolization method was applied to betulonic acid 2
as the ketone source, using primary amines 3 and 4-
nitrophenyl azide 4, and the previously reported reaction
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a
Table 1. Anti-CoV Activity and Selectivity in Human HEL Cells Infected with HCoV-229E
a
b
c
HEL: human embryonic lung fibroblast cells. Yield after chromatographic purification. EC50: 50% effective concentration, i.e., compound
concentration producing 50% protection against viral cytopathic effect (CPE), as assessed by MTS cell viability assay or microscopic scoring of the
d
e
CPE. CC50: 50% cytotoxic concentration determined by MTS cell viability assay. Selectivity index or ratio of CC50 to EC50, both determined by
MTS assay. Values are the mean SEM (N = 3).
against HCoV-229E, 5h appeared, unfortunately, to be
confined to this CoV species. In addition, when tested against
a broad panel of DNA and RNA viruses, the 1,2,3-triazolo-
fused betulonic acid derivatives proved inactive against HIV,
herpes simplex virus, vaccinia virus, adenovirus, vesicular
stomatitis virus, Coxsackie B4 virus, respiratory syncytial virus,
parainfluenza-3 virus, reovirus-1, Sindbis virus, Punta Toro
virus, yellow fever virus, and influenza virus (data not shown).
Mechanistic Studies Establishing nsp15 as the Target
of 5h. Given the robust activity of the betulonic acid
derivatives against HCoV-229E, we used this virus to reveal
their mechanism of action and appreciate how their anti-CoV
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Figure 1. Activity of 5h against HCoV-229E. (A) Representative microscopic images showing protection against virus-induced cytopathic effect
(CPE) in human embryonic lung (HEL) cells. (B) Dose−response curves for inhibition of virus-induced CPE (solid squares) and for cytotoxicity
(open squares) in HEL cells, both determined by MTS cell viability assay. Data points are the mean SEM (N = 3). (C) Immunofluorescence
detection of viral dsRNA in HCoV-229E-infected human bronchial epithelial 16HBE cells at 24 h post-infection (p.i.). Green: dsRNA and blue:
nuclear DAPI staining. Compounds: 12 μM 5h or 12 μM GS-441524. Scale bar: 50 μm.
a
Scheme 2. SAR Study around Compound 5h
a
Reagents and conditions: (i) betulonic acid, NH4OAc, 4-nitrophenyl azide, DMF, 80 °C, 18 h, 4 Å MS, isolated yield 86%; (ii) betulonic acid,
benzyl amine, 4-nitrophenyl azide, toluene, 100 °C, 18 h, 4 Å MS, isolated yield 92%; (iii) betulonic acid, (R)-(+)-α-methylbenzylamine, 4-
nitrophenyl azide, toluene, 100 °C, 18 h, 4 Å MS, isolated yield 69%; (iv) dihydrobetulonic acid, (S)-(−)-α-methylbenzylamine, 4-nitrophenyl
azide, toluene, 100 °C, 18 h, 4 Å MS, isolated yield 90%; (v) lupenone, (S)-(−)-α-methylbenzylamine, 4-nitrophenyl azide, toluene, 100 °C, 18 h, 4
Å MS, isolated yield 83%.
activity spectrum may be expanded. A time-of-addition
experiment indicated that 5h acts post-entry at an early stage
in viral RNA synthesis since the molecule started to have
reduced activity when added at 6 h p.i. (Figure 2). For
comparison, the action point of the entry inhibitor bafilomycin,
an inhibitor of endosomal acidification, was situated before 2 h
p.i. K22 was still effective when added as late as 8 h p.i. This
CoV inhibitor targets nsp6-dependent anchorage of the viral
replication-transcription complexes (RTCs) to host cell-
derived double-membrane vesicles.47
Next, we performed two independent virus passaging
experiments to select 5h-resistant viruses and identify the
viral target. After three cell culture passages under increasing
concentrations (up to 40 μM) of 5h, HCoV-229E acquired
resistance. Whole virus genome sequencing revealed that this
was attributed to two substitutions in nsp15, K60R (first
selection) and T66I (second selection), located in the N-
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a
terminal part of this protein. For both mutants, 5h exhibited an
antiviral EC99 value (= concentration producing 100-fold
reduction in virus yield) of >40 μM, which is at least 14-fold
higher than the EC99 value of 2.9 μM measured for wild-type
(WT) virus (Figure 3A). Both mutant viruses remained fully
sensitive to GS-441524. The conclusion that 5h targets nsp15
was corroborated by determining its activity against a reverse-
engineered EndoU-deficient H250A-mutant HCoV-229E,
which lacks the catalytic His250 residue in the EndoU active
site.26 5h proved dramatically less active against this mutant
(Figure 3B), producing a maximal reduction in virus yield of
23-fold compared to 1479-fold for WT virus. Again, GS-
441524 proved equally active against nsp15-mutant and WT
virus. To conclude, we established 5h as an inhibitor of nsp15
and showed that its activity is linked to residues Lys60 and
Thr66 in the N-terminal part, plus His250 in the EndoU
catalytic site of nsp15. This inhibition of nsp15 accords with
the time-of-addition profile of 5h (see above), showing that the
compound interferes with an early stage in viral RNA synthesis.
Binding Model of 5h in the Hexameric nsp15 Protein.
To predict the possible binding site of 5h, the compound was
docked into the X-ray structure of the hexameric nsp15 protein
from HCoV-229E (PDB code: 4RS4). This hexameric
structure formed by two trimers is the functional form of
nsp15.18 First, a few sites in this PDB sequence were changed
to obtain the fully correct nsp15 sequence of HCoV-229E virus
(see Experimental Section for details). By using a pocket-
detection protocol implemented on the Site Finder module of
Molecular Operating Environment (MOE) software, we
identified a druggable binding pocket at the nsp15 dimer
interface, surrounded by the catalytic residue His250 in the
EndoU active site of one nsp15 monomer and residues Lys60
and Thr66 in the N-terminal domain of the other monomer
(Figure 4A). Next, ligand 5h was placed inside the pocket and
docked by using both MOE and GOLD software, and the
common top scoring binding mode was further analyzed. This
docked result indicates that the carboxylic acid moiety of 5h
Table 2. Activity of 5h Analogues in Human HEL Cells
Infected with HCoV-229E
b
c
antiviral activity (μM)
cytotoxicity (μM)
d
Code
EC50 (MTS) EC50 (CPE)
CC50
SI
1.9
14
91
7.6
5q
5r
5s
5t
5u
5h
4.3 0.6
0.85 0.05
1.1 0.2
3.4 0.4
0.71 0.04
0.67 0.02
6.1 1.7
>100
8.3 0.7
12
0
>100
≥99
>100
13
5
>100
0.65 0.08
0.60 0.18
49
2
76
a
b
HEL: human embryonic lung fibroblast cells. EC50: 50% effective
concentration, i.e., compound concentration producing 50% protec-
tion against viral cytopathic effect (CPE), as assessed by MTS cell
viability assay or microscopic scoring of the CPE. CC50: 50%
cytotoxic concentration determined by MTS cell viability assay.
Selectivity index or ratio of CC50 to EC50, both determined by MTS
c
d
assay. Values are the mean SEM (N = 3).
Figure 2. 5h acts post-entry at an early stage in viral RNA synthesis.
Compound addition was delayed until different time points after
infecting HEL cells with HCoV-229E, and viral RNA was quantified
at 16 h p.i. Compound concentrations: bafilomycin 6.3 nM; 5h and
K22: 15 μM. The Y axis shows the viral RNA copy number relative to
the virus control (mean SEM of two independent experiments).
Figure 3. Mutations in nsp15 confer resistance of HCoV-229E to 5h (left panels), but not to GS-441524 (right panels). The graphs show the effect
of the compounds on virus yield. (A) HEL cells infected with 5h-resistant mutants obtained by virus passaging under 5h and carrying substitution
K60R (first selection) or T66I (second selection) in nsp15. (B) 16HBE cells infected with EndoU-deficient mutant virus (H250Ansp15), obtained
by reverse genetics.26 Data points are the mean SEM (N = 3). An unpaired t-test (GraphPad Prism 8.4.3) was used to compare the mutant
̌
́
́
viruses to WT, and the resulting two-tailed P-values were adjusted for multiple comparisons using Holm−Sidak (α = 0.05). *, P < 0.05; **, P <
0.01; ***, P < 0.001.
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Figure 4. Binding mode of 5h in the HCoV-229E nsp15 hexameric protein (PDB 4RS4), as predicted by docking. (A) Hydrophobic pocket lies
adjacent to the EndoU catalytic center (catalytic triad consisting of His235, His250, and Lys291) and at an nsp15 dimer interface (monomers
depicted in a differently colored surface). The pocket is surrounded by His250, Lys60, and Thr66, explaining why 5h is inactive against HCoV-
229E viruses carrying mutations at these sites. (B) 5h occupies the pocket by making hydrophobic interactions with Val293 and side chain
fragments of Lys291 and Thr292. The molecule further engages in hydrogen-bonding interactions with Cys294 and Thr295 via the carboxylic acid
moiety and with Thr245 via the 1,2,3-triazole moiety. Additional hydrophobic interactions with Val63, Leu65, and Thr292 are made via the
aromatic ring-substituted 1,2,3-triazole moiety.
Figure 5. Comparison of the hydrophobic pocket, occupied by 5h, in the nsp15 proteins of HCoV-229E (left; PDB 4RS4) and SARS-CoV-2
(right; PDB 7K1O). The carboxylic acid moiety of 5h forms hydrogen bonds with both nsp15 binding pockets. On the other hand, the 1,2,3-
triazole group engages hydrogen-bonding interactions with the HCoV-229E nsp15 protein but is incompatible with the SARS-CoV-2 pocket.
forms hydrogen bonds with the backbone of residues Cys294
and Thr295 (Figure 4B). The importance of this interaction is
supported by the observation that 5u, the 5h analogue bearing
a methyl instead of carboxylic acid group, lacks antiviral
activity. Furthermore, the 1,2,3-triazole group of 5h engages in
hydrogen-bonding interactions with Thr245, explaining why
the parent compounds 1 and 2 are not active against HCoV-
229E. At the other side of the pocket, the aromatic ring of 5h
makes hydrophobic contacts with Val63 and Leu65. This may
explain why nearby mutations K60R and T66I yield resistance
to the compound since these substitutions may negatively
affect the interactions of Val63 and Leu65 or disturb the
conformation of the loop flanked by both residues. The
structure of the substituent attached to the 1,2,3-triazole ring
seems less determining since, in terms of antiviral activity, both
aromatic and smaller aliphatic substituents are tolerated (see
Tables 1 and 2). The one exception is the negative influence
from a bulky cyclohexanemethyl group (Table 1, compound
5i). Hence, the shape requirements for this substituent appear
not very strict and compatible with voluminous changes that
are sterically not clashing. This analysis concurs with the
shallow and surface-exposed nature of the binding pocket.
Analysis of the nsp15 sequence similarity between HCoV-
229E and SARS-CoV-2 showed that a few residues in the
pocket are not conserved (see Figure S1 in the Supporting
Information). This explains why docking 5h into the SARS-
CoV-2 nsp15 hexamer was unable to identify a similar pose
within the top solutions. The largest influence seems attributed
to the residue at position 244/245 since substituting Thr245
(present in HCoV-229E) by Gln244 (the corresponding
residue in SARS-CoV-2) abrogates a hydrogen-bond inter-
action with the 1,2,3-triazole group of 5h (Figure 5).
Additionally, the loop between Val/Ile63 and Leu/Pro65 has
a slightly different orientation in these two CoV nsp15
proteins. Both factors may explain why 5h is active against
HCoV-229E, but not SARS-CoV-2. Hence, one strategy to
improve the binding to SARS-CoV-2 nsp15 might be to
substitute the 1,2,3-triazole moiety by another type of ring
structure. At the other side of the molecule, the carboxylic acid
moiety probably needs to be kept since it forms hydrogen
bonds with both nsp15 proteins. The fact that most of the
pocket residues are conserved (Figure S1 in the Supporting
Information) underscores the relevance of this nsp15 interface
pocket for drug design. The role of this protein region in
forming inter-monomer interactions is evident from reports
that nsp15 exists as a monomer when key interactions in this
region (i.e., Arg61-Glu266 in SARS-CoV nsp15 and Tyr58-
Glu263 in MERS-CoV nsp15) are eliminated by mutation.22,51
When nsp15 is unable to hexamerize, the EndoU catalytic site
undergoes important structural changes that abolish RNA
binding and enzymatic activity.52
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crude product was dried in a vacuum oven, dissolved in Et2O (600
mL), and washed with water (300 mL), 7.5% hydrochloric acid (200
mL), water (200 mL), saturated aqueous NaHCO3 solution (200
mL), and water (200 mL). The crude reaction mixture was purified by
column chromatography (silica gel), whereas the eluent was used a
mixture of heptane and ethyl acetate (70:30) to afford betulonic acid
(23 g, 45% yield). Spectroscopic data for betulonic acid was
consistent with previously reported data for this compound.43
3-Oxo-lupan-28-oic Acid (Dihydrobetulonic Acid). Betulonic acid
(180 mg, 0.396 mmol, 1.0 equiv) was dissolved in a mixture of
MeOH/THF (2/6 mL). 10% Pd(OH)2 (30 mg) was added under a
N2 atmosphere. This atmosphere was replaced by a H2 atmosphere.
The reaction was stirred under a H2 atmosphere for 78 h, then filtered
through celite, and washed with CHCl3 to afford a white solid. The
residue obtained was purified by silica gel column chromatography
using 100% chloroform as an eluent to afford dihydrobetulonic acid
(quantitative yield) as a white amorphous powder. The spectra
proved the identity of the compound by comparing the data with the
literature.53
General Procedure. To a dried screw-capped reaction tube
equipped with a magnetic stirring bar was added betulonic acid,
amine, 4-nitrophenyl azide, and 4 Å molecular sieves (50 mg). The
mixture was dissolved in the proper solvent (toluene and DMF) and
stirred at 100 °C for 12−72 h. The reaction was monitored using
TLC with the plate first developed with DCM, and then different
ratios petroleum ether/ethyl acetate (7:3 and 6:4) were used
depending on the substrates. For visualization of TLC plates, 5%
H2SO4 in ethanol was used, and for more sensitive detection, cerium-
ammonium-molybdate after heating to 150−200 °C was used. The
crude reaction mixture was then directly purified by column
chromatography (silica gel), first with CH2Cl2 as an eluent to remove
all 4-nitroaniline formed during the reaction followed by using a
mixture of petroleum ether and ethyl acetate as an eluent to afford the
betulonic acid 1,2,3-triazole derivatives.
1′-(3,4,5-Trimethoxybenzyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-tria-
zole-28-oic Acid (5a). Betulonic acid (100 mg, 1 equiv, 0.220 mmol),
3,4,5-trimethoxybenzylamine (56 mg, 1.3 equiv, 0.286 mmol), 4-
nitrophenyl azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves
(50 mg), and toluene (0.5 mL). Reaction time is overnight. The
product was purified by flash column chromatography (first washed
with CH2Cl2 followed by petroleum ether:EtOAc 9:1 → 6:4)
affording 5a (122 mg, 84% yield) as off-white crystals. m.p. 152 °C.
1H NMR (400 MHz, CDCl3) δ 6.25 (s, 2H), 5.57 (s, 2H), 4.76 (s,
1H), 4.64 (s, 1H), 3.81 (s, 3H), 3.77 (s, 6H), 3.04 (m, 1H), 2.96 (d, J
= 15.4 Hz, 1H), 2.32−2.15 (m, 3H), 2.05−1.94 (m, 2H), 1.77−1.64
(m, 5H), 1.59−1.40 (m, 11H), 1.20 (s, 7H), 1.03 (s, 3H), 1.00 (s,
3H), 0.97 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
180.9, 153.5, 150.2, 141.9, 138.0, 137.4, 132.1, 109.8, 103.5, 60.8,
56.3, 56.1, 54.6, 52.8, 49.3, 49.1, 46.8, 42.4, 40.5, 38.9, 38.5, 37.0,
33.7, 33.3, 32.0, 30.5, 29.8, 29.7, 28.7, 25.5, 21.3, 21.3, 19.4, 18.9,
16.0, 15.7, 14.6. HRMS (ESI+): m/z calculated for C40H57N3O5H [M
+ H]+: 660.4370, found 660.4384.
To conclude, the nsp15 binding mode of 5h, predicted by
docking, nicely accords with the biological findings. Namely,
the binding model rationalizes the requirement of the 1,2,3-
triazolo function and carboxylate substituent; 5h resistance of
the nsp15-K60R, nsp15-T66I, and nsp15-H250A mutant
viruses; and lack of activity against SARS-CoV-2.
CONCLUSIONS
■
To conclude, we present the first prototype of CoV nsp15
inhibitors, having a 1,2,3-triazolo-fused betulonic acid
structure. The SAR analysis, resistance data, and docking
model provide strong evidence that the lead molecule 5h binds
to an inter-monomer nsp15 interface lying adjacent to the
EndoU catalytic core. This provides an excellent basis to
modify the substituents or betulonic acid scaffold and expand
the activity spectrum beyond HCoV-229E. Since 5h appears to
interact with the catalytic His250 residue in the EndoU
domain of nsp15, the molecule plausibly interferes with the
role of EndoU in regulating viral dsRNA synthesis.28 To
complement the findings in this report, obtained in non-
immune cells, we are currently evaluating the antiviral and
immunomodulatory effects of 5h in HCoV-229E-infected
human macrophages. This may validate the intriguing concept
that nsp15 inhibition could have a dual outcome, by inhibiting
CoV replication and promoting host cell antiviral immunity.
Since nsp15 is also considered to be an important immune
evasion factor for SARS-CoV-2, further exploration of our
betulonic acid skeleton and its nsp15 binding pocket is clearly
warranted.
EXPERIMENTAL SECTION
■
Chemistry. 1H and 13C NMR spectra were measured on
commercial instruments (Bruker Avance 300 MHz and Bruker
AMX 400 MHz and 600 MHz). Chemical shifts (δ) are reported in
parts per million (ppm) referenced to tetramethylsilane (1H) or the
internal (NMR) solvent signal (13C). Melting points were determined
using a Reichert Thermovar apparatus. For column chromatography,
70−230 mesh silica 60 (E. M. Merck) was used as the stationary
phase. Chemicals received from commercial sources were used
without further purification. Reaction dry solvents (toluene, DMF,
and THF) were used as received from commercial sources. TLC was
carried out on Kieselgel 60 F254 plates (Merck).
Exact mass was acquired on a quadrupole orthogonal acceleration
time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford,
MA). Samples were infused at 3 μL/min, and spectra were obtained
in positive (or: negative) ionization mode with a resolution of 15,000
(FWHM) using leucine enkephalin as the lock mass.
All HPLC data were acquired on an Agilent 1200 HPLC with a
quaternary pump, autosampler, and UV-DAD detector (set at 220,
230, and 240 nm). The injection volume was 5 μL of a dilution of 100
μg/mL (sample in mobile phase). The column was a Zorbax XBD
C18 column (5 μm; 4.6 mm x 150 mm), and the mobile phases were
as follows: (A) 0.1% formic acid in water, and (B) methanol. The
gradient consisted of: (1) 5 min from 50% A/50% B toward 25% A/
75% D; (2) 15 min toward 0% A/100% B; (3) 20 min 0% A/100% B.
Data collection and analysis were done with Agilent Chemstation
software. All tested compounds showed a purity of >95%.
3-Oxo-lup-20(29)-en-28-oic Acid (Betulonic Acid, 2). To a
solution of betulin (50.0 g, 113 mmol; purchased from Eburon
Organics BVBA) in acetone (1500 mL, use an ultra-sonic bath to
dissolve) was added freshly prepared Jones reagent [Na2Cr2O7, (66.5
g, 226 mmol) and H2SO4 (60 mL) in water (500 mL)] for 1 h in an
ice bath. The reaction mixture was allowed to warm to room
temperature, and stirring was continued for 6 h followed by TLC.
First, MeOH was added and then water to the reaction mixture. The
precipitate was filtered off and washed with water (500 mL). The
1′-(3,5-Dimethoxybenzyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-
28-oic Acid (5b). Betulonic acid (100 mg, 1 equiv, 0.220 mmol), 3,5-
dimethoxybenzylamine (48 mg, 1.3 equiv, 0.286 mmol), 4-nitro-
phenyl azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves (50
mg), and toluene (0.5 mL). Reaction time is overnight. The product
was purified by flash column chromatography (first washed with
CH2Cl2 followed by petroleum ether:EtOAc 9:1 → 6:4) affording 5b
(126 mg, 92% yield) as off-white crystals. m.p. 159 °C. 1H NMR (400
MHz, CDCl3) δ 6.34 (s, 1H), 6.16 (s, 2H), 5.57 (s, 2H), 4.76 (s, 1H),
4.64 (s, 1H), 3.71 (s, 6H), 3.08−2.99 (m, 1H), 2.95 (d, J = 15.4 Hz,
1H), 2.32−2.14 (m, 3H), 2.05−1.94 (m, 2H), 1.79−1.62 (m, 5H),
1.60−1.33 (m, 11H), 1.29−1.10 (m, 7H), 1.03 (s, 3H), 1.00 (s, 3H),
0.98 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.6,
161.1, 150.2, 141.8, 138.9, 138.0, 109.8, 104.4, 99.6, 56.4, 55.3, 54.5,
52.8, 49.2, 49.2, 46.9, 42.4, 40.5, 38.9, 38.5, 38.3, 37.0, 33.7, 33.3,
32.0, 30.6, 29.8, 28.7, 25.5, 21.3, 21.2, 19.4, 18.9, 16.0, 15.7, 14.6.
HRMS (ESI+): m/z calculated for C39H55N3O4H [M + H]+:
630.4265, found 630.4274.
5638
https://doi.org/10.1021/acs.jmedchem.0c02124
J. Med. Chem. 2021, 64, 5632−5644

Journal of Medicinal Chemistry
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Article
1′-(Pyridin-4-ylmethyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-
oic Acid (5c). Betulonic acid (100 mg, 1 equiv, 0.220 mmol), 4-
(aminomethyl)pyridine (31 mg, 1.3 equiv, 0.286 mmol), 4-nitro-
phenyl azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves (50
mg), and toluene (0.5 mL). Reaction time is overnight. The product
was purified by flash column chromatography (first washed with
CH2Cl2 followed by petroleum ether:EtOAc 9:1 → 6:4) affording 5c
30.5, 29.8, 29.7, 28.8, 28.4, 25.4, 23.8, 21.4, 21.3, 20.8, 20.5, 19.4,
18.8, 17.5, 17.3, 16.1, 15.7, 14.6, 7.9. HRMS (ESI+): m/z calculated
for C38H50F3N3O2H [M + H]+: 638.3927, found 638.3939.
1′-(4-Dimethylaminobenzyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-tria-
zole-28-oic Acid (5g). Betulonic acid (100 mg, 1 equiv, 0.220 mmol),
4-(dimethylamino)benzylamine (43 mg, 1.3 equiv, 0.286 mmol), 4-
nitrophenyl azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves
(50 mg), and toluene (0.5 mL). Reaction time is overnight. The
product was purified by flash column chromatography (first washed
with CH2Cl2 followed by petroleum ether:EtOAc 9:1 → 6:4)
affording 5g (112 mg, 80% yield) as off-white crystals. m.p. 190 °C.
1H NMR (400 MHz, CDCl3) δ 6.97 (d, J = 8.3 Hz, 2H), 6.64 (d, J =
8.3 Hz, 2H), 5.52 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.10−2.84 (m,
9H), 2.33−2.11 (m, 3H), 1.99 (m, 2H), 1.81−1.62 (m, 5H), 1.56−
1.34 (m, 11H), 1.25−1.15 (m, 7H), 1.06 (s, 3H), 0.99 (s, 3H), 0.97
(s, 3H), 0.76 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.4, 150.2,
150.1, 141.6, 137.7, 127.6, 123.9, 112.5, 109.8, 56.4, 54.6, 52.6, 49.2,
49.1, 46.8, 42.4, 40.5, 40.5, 38.9, 38.9, 38.4, 38.3, 37.0, 33.7, 33.3,
32.1, 30.5, 29.8, 28.7, 25.5, 23.8, 21.3, 21.3, 20.8, 19.4, 18.9, 16.0,
15.6, 14.6. HRMS (ESI+): m/z calculated for C39H56N4O2H [M +
H]+: 613.4475, found 613.4480.
1′-((S)-1-Phenylethyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-
oic Acid (5h). Betulonic acid (100 mg, 1 equiv, 0.220 mmol), (S)-
(−)-α-methylbenzylamine (35 mg, 1.3 equiv, 0.286 mmol), 4-
nitrophenyl azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular
sieves (50 mg), and toluene (0.5 mL). Reaction time is overnight.
The product was purified by flash column chromatography (first
washed with CH2Cl2 followed by petroleum ether:EtOAc 9:1 → 6:4)
affording 5h (107 mg, 84% yield) as off-white crystals. m.p. 327 °C.
1H NMR (400 MHz, CDCl3) δ 7.30−7.21 (m, 3H), 7.13 (d, J = 7.2
Hz, 2H), 5.73 (m, 1H), 4.76 (s, 1H), 4.64 (s, 1H), 3.00 (m, 2H),
2.33−2.12 (m, 3H), 2.07−1.92 (m, 5H), 1.81−1.62 (m, 5H), 1.58−
1.37 (m, 11H), 1.14 (m, 7H), 1.00 (s, 6H), 0.96 (s, 3H), 0.72 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 180.6, 150.2, 141.8, 141.1, 137.6,
1
(97 mg, 78% yield) as off-white crystals. m.p 172 °C. H NMR (400
MHz, CDCl3) δ 8.56 (d, J = 5.6 Hz, 2H), 6.93 (d, J = 5.6 Hz, 2H),
5.66 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.06 (m, 1H), 2.97 (d, J =
15.4 Hz, 1H), 2.36−2.15 (m, 3H), 2.06−1.95 (m, 2H), 1.83−1.64
(m, 5H), 1.60−1.37 (m, 11H), 1.32−1.21 (m, 7H), 1.16 (s, 3H), 1.01
(m, 3H), 1.00 (m, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
180.6, 150.4, 149.8, 146.1, 142.3, 138.3, 121.2, 109.7, 56.3, 54.4, 51.6,
49.2, 49.2, 46.9, 42.4, 40.5, 39.0, 38.4, 38.2, 37.0, 33.6, 33.2, 32.1,
30.6, 29.8, 29.6, 28.8, 25.4, 21.5, 21.4, 19.4, 18.8, 16.0, 15.7, 14.6.
HRMS (ESI+): m/z calculated for C36H50N4O2H [M + H]+:
571.4006, found 571.4013.
1′-(4-Methylbenzyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic
Acid (5d). Betulonic acid (100 mg, 1 equiv, 0.220 mmol), 4-
methylbenzylamine (35 mg, 1.3 equiv, 0.286 mmol), 4-nitrophenyl
azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves (50 mg),
and toluene (0.5 mL). Reaction time is overnight. The product was
purified by flash column chromatography (first washed with CH2Cl2
followed by petroleum ether:EtOAc 9:1 → 6:4) affording 5d (115
1
mg, 90% yield) as off-white crystals. m.p. 310 °C. H NMR (600
MHz, CDCl3) δ 7.09 (d, J = 7.9 Hz, 2H), 6.92 (d, J = 7.9 Hz, 2H),
5.59 (s, br, 2H), 4.75 (s, 1H), 4.63 (s, 1H), 3.03 (m, 1H), 2.95 (d, J =
15.4 Hz, 1H), 2.32−2.23 (m, 5H), 2.17 (d, J = 15.4 Hz, 1H), 2.05−
1.94 (m, 2H), 1.80−1.62 (m, 5H), 1.50 (m 11H), 1.22−1.09 (m,
7H), 1.03 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.77 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 181.8, 150.2, 141.7, 138.0, 137.4, 133.3, 129.3,
126.3, 109.8, 56.4, 54.5, 52.6, 49.2, 49.1, 46.8, 42.4, 40.8, 40.5, 38.9,
38.4, 38.2, 37.0, 33.7, 33.3, 32.0, 30.5, 29.7, 28.7, 28.4, 25.4, 23.8,
21.3, 21.3, 21.0, 20.8, 19.4, 18.9, 17.5, 17.2, 16.0, 15.6, 14.6. HRMS
(ESI+): m/z calculated for C38H53N3O2H [M + H]+: 584.4210, found
584.4217.
128.6, 127.5, 126.1, 109.8, 59.1, 56.3, 54.8, 49.3, 49.1, 46.8, 42.4, 40.5,
38.8, 38.4, 37.0, 33.6, 33.3, 32.0, 30.5, 29.7, 29.7, 28.6, 25.4, 23.3,
21.4, 21.3, 19.4, 19.0, 15.9, 15.6, 14.6. HRMS (ESI+): m/z calculated
for C38H53N3O2H [M + H]+: 584.4210, found 584.4218.
1′-(4-Fluorobenzyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic
Acid (5e). Betulonic acid (100 mg, 1 equiv, 0.220 mmol), 4-
fluorobenzylamine (36 mg, 1.3 equiv, 0.286 mmol), 4-nitrophenyl
azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves (50 mg),
and toluene (0.5 mL). Reaction time is overnight. The product was
purified by flash column chromatography (first washed with CH2Cl2
followed by petroleum ether:EtOAc 9:1 → 6:4) affording 5e (110 mg,
1′-(Cyclohexylmethyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-
oic Acid (5i). Betulonic acid (100 mg, 1 equiv, 0.220 mmol),
cyclohexanemethylamine (33 mg, 1.3 equiv, 0.286 mmol), 4-
nitrophenyl azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular
sieves (50 mg), and toluene (0.5 mL). Reaction time is overnight.
The product was purified by flash column chromatography (first
washed with CH2Cl2 followed by petroleum ether:EtOAc 9:1 → 6:4)
affording 5i (103 mg, 82% yield) as off-white crystals. m.p. 338 °C. 1H
NMR (600 MHz, CDCl3) δ 4.76 (s, 1H), 4.64 (s, 1H), 4.09 (m, 2H),
3.04 (m, 1H), 2.92 (m, 1H), 2.33−2.21 (m, 2H), 2.13 (d, J = 15.3 Hz,
1H), 2.07−1.95 (m, 2H), 1.79−1.64 (m, 10H), 1.61−1.34 (m, 11H),
1.32−1.19 (m, 10H), 1.18−1.08 (m, 5H), 1.00 (s, 3H), 0.99 (s, 3H),
0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.7, 150.2, 140.9,
137.8, 109.8, 56.4, 55.6, 54.7, 49.2, 49.2, 46.9, 42.4, 40.5, 38.8, 38.6,
38.5, 38.2, 37.0, 33.7, 33.3, 32.1, 31.0, 30.6, 29.8, 28.9, 26.3, 25.7,
25.4, 21.5, 21.3, 19.4, 18.9, 16.0, 15.7, 14.6. HRMS (ESI+): m/z
calculated for C37H57N3O2H [M + H]+: 576.4523, found 576.4529.
1′-(Benzo[d][1,3]dioxol-5-ylmethyl)-1H′-lup-2-eno-[2,3-d]-
[1,2,3]-triazole-28-oic Acid (5j). Betulonic acid (100 mg, 1 equiv,
0.220 mmol), piperonylamine (43 mg, 1.3 equiv, 0.286 mmol), 4-
nitrophenyl azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves
(50 mg), and toluene (0.5 mL). Reaction time is overnight. The
product was purified by flash column chromatography (first washed
with CH2Cl2 followed by petroleum ether:EtOAc 9:1 → 6:4)
affording 5j (105 mg, 78% yield) as off-white crystals. m.p. 313 °C. 1H
NMR (300 MHz, CDCl3) δ 6.72 (d, J = 7.9 Hz, 1H), 6.59−6.48 (m,
2H), 5.93 (d, J = 0.9 Hz, 2H), 5.53 (s, 2H), 4.76 (s, 1H), 4.64 (s,
1H), 3.10−2.89 (m, 2H), 2.35−2.11 (m, 3H), 1.99 (m, 2H), 1.83−
1.62 (m, 5H), 1.61−1.33 (m, 11H), 1.33−1.08 (m, 7H), 1.05 (s, 3H),
1.00 (s, 3H), 0.97 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 181.3, 150.2, 148.1, 147.2, 141.8, 137.9, 130.1, 119.9, 109.8,
108.3, 107.2, 101.1, 56.4, 54.5, 52.6, 50.8, 49.2, 49.2, 46.9, 42.4, 40.5,
1
85% yield) as off-white crystals. m.p. 309 °C. H NMR (400 MHz,
CDCl3) δ 7.00 (m, 4H), 5.60 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H),
3.08−2.99 (m, 1H), 2.95 (d, J = 15.3 Hz, 1H), 2.33−2.14 (m, 3H),
2.06−1.93 (m, 2H), 1.82−1.62 (m, 5H), 1.61−1.31 (m, 11H), 1.30−
1.10 (m, 7H), 1.08 (s, 3H), 1.02 (s, 3H), 1.00 (s, 3H), 0.77 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 181.1, 163.5, 161.0, 150.2, 142.0,
137.9, 132.2, 132.2, 128.2, 128.1, 115.8, 115.5, 109.8, 56.3, 54.5, 52.1,
49.2, 49.2, 46.9, 42.4, 40.5, 38.9, 38.5, 38.3, 37.0, 33.7, 33.3, 32.0,
30.5, 29.8, 28.7, 25.4, 21.3, 21.3, 19.4, 18.8, 16.0, 15.7, 14.6. HRMS
(ESI+): m/z calculated for C37H50FN3O2H [M + H]+: 588.3959,
found 588.3969.
1′-(4-Trifluoromethylbenzyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-tria-
zole-28-oic Acid (5f). Betulonic acid (100 mg, 1 equiv, 0.220 mmol),
4-(trifluoromethyl)benzylamine (50 mg, 1.3 equiv, 0.286 mmol), 4-
nitrophenyl azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves
(50 mg), and toluene (0.5 mL). Reaction time is overnight. The
product was purified by flash column chromatography (first washed
with CH2Cl2 followed by petroleum ether:EtOAc 9:1 → 6:4)
affording 5f (112 mg, 80% yield) as off-white crystals. m.p. 315 °C. 1H
NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0
Hz, 2H), 5.69 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.02 (m, 2H),
2.34−2.16 (m, 3H), 2.07−1.94 (m, 2H), 1.82−1.64 (m, 5H), 1.59−
1.38 (m, 11H), 1.18 (m, 7H), 1.02 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H),
0.78 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.4, 150.2, 142.1,
140.5, 138.2, 126.6, 125.7, 125.7, 109.8, 56.4, 54.5, 52.2, 49.3, 49.1,
46.9, 42.4, 40.8, 40.5, 38.9, 38.5, 38.2, 37.0, 33.8, 33.6, 33.3, 32.0,
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Journal of Medicinal Chemistry
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Article
38.9, 38.5, 38.3, 37.0, 33.7, 33.3, 32.0, 30.5, 29.8, 28.7, 25.4, 21.3,
21.3, 19.4, 18.9, 16.0, 15.7, 14.6. HRMS (ESI+): m/z calculated for
C38H51N3O4H [M + H]+: 614.3952, found 614.3951.
1.77−1.64 (m, 5H), 1.59−1.35 (m, 11H), 1.34−1.15 (m, 7H), 1.12
(d, J = 14.6 Hz, 3H), 0.99 (s, 3H), 0.96 (s, 3H), 0.78 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 180.4, 150.2, 141.9, 138.3, 135.7, 128.2,
125.2, 124.5, 122.1, 117.5, 110.6, 109.8, 100.0, 56.3, 54.6, 51.4, 49.2,
49.2, 46.9, 42.4, 40.5, 38.9, 37.0, 33.8, 33.3, 32.0, 30.5, 29.7, 29.7,
28.4, 25.5, 22.7, 21.3, 20.9, 19.4, 18.9, 16.0, 15.6, 14.6. HRMS (ESI+):
m/z calculated for C39H52N4O2H [M + H]+: 609.4162, found
609.4174.
1′-(Furan-2-ylmethyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-
oic Acid (5k). Betulonic acid (100 mg, 1 equiv, 0.220 mmol),
furfurylamine (28 mg, 1.3 equiv, 0.286 mmol), 4-nitrophenyl azide
(36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves (50 mg), and
toluene (0.5 mL). Reaction time is overnight. The product was
purified by flash column chromatography (first washed with CH2Cl2
followed by petroleum ether:EtOAc 9:1 → 6:4) affording 5k (66 mg,
1′-(2-Hydroxyethyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic
Acid (5o). Betulonic acid (100 mg, 1 equiv, 0.220 mmol),
ethanolamine (17 mg, 1.3 equiv, 0.286 mmol), 4-nitrophenyl azide
(36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves (50 mg), and
toluene (0.5 mL). Reaction time is overnight. The product was
purified by flash column chromatography (first washed with CH2Cl2
followed by CH2Cl2:MeOH 95:5) affording 5o (70 mg, 62% yield) as
1
53% yield) as a brown crystals. m.p. 227 °C. H NMR (400 MHz,
CDCl3) δ 7.35 (s, 1H), 6.34−6.31 (m, 1H), 6.23 (d, J = 3.1 Hz, 1H),
5.55 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.07−2.99 (m, 1H), 2.93 (d,
J = 15.3 Hz, 1H), 2.30−2.11 (m, 3H), 1.99 (d, J = 7.5 Hz, 2H), 1.78−
1.66 (m, 5H), 1.60−1.40 (m, 11H), 1.28 (t, J = 10.2 Hz, 7H), 1.15 (s,
3H), 1.00 (s, 3H), 0.98 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 179.9, 150.2, 148.8, 142.5, 141.6, 137.7, 110.7, 109.9, 109.0,
56.3, 54.6, 49.3, 49.2, 46.8, 46.4, 42.4, 40.6, 39.0, 38.4, 38.3, 37.0,
33.6, 33.3, 32.0, 30.5, 29.8, 29.7, 28.6, 19.4, 18.9, 16.1, 15.7, 14.6.
HRMS (ESI+): m/z calculated for C35H49N3O3H [M + H]+:
560.3846, found 560.3857.
1
off-white crystals. m.p. 236 °C. H NMR (400 MHz, DMSO) δ 4.72
(s, 1H), 4.59 (s, 1H), 4.36 (m, 2H), 3.86 (m, 2H), 2.98 (m, 1H), 2.70
(d, J = 15.2 Hz, 1H), 2.30 (m, 1H), 2.11 (m, 2H), 1.82 (d, J = 6.9 Hz,
2H), 1.64 (s,br, 5H), 1.58−1.33 (m, 11H), 1.33−1.22 (m, 7H), 1.17
(s, 3H), 0.98 (s, 3H), 0.93 (s, 3H), 0.71 (s, 3H). 13C NMR (101
MHz, DMSO) δ 177.7, 150.7, 140.2, 138.0, 110.1, 79.6, 60.7, 55.9,
54.5, 51.4, 48.9, 47.0, 42.5, 38.9, 38.1, 36.7, 33.6, 33.3, 32.0, 30.5,
29.8, 28.7, 25.5, 21.4, 19.4, 18.8, 16.2, 15.8, 14.8. HRMS (ESI+): m/z
calculated for C32H49N3O3H [M + H]+: 524.3846, found 524.3853.
1′-Heptyl-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic Acid (5p).
Betulonic acid (100 mg, 1 equiv, 0.220 mmol), 1-heptylamine (33 mg,
1.3 equiv, 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv, 0.220
mmol), 4 Å molecular sieves (50 mg), and toluene (0.5 mL).
Reaction time is overnight. The product was purified by flash column
chromatography (first washed with CH2Cl2 followed by petroleum
ether:EtOAc 9:1 → 6:4) affording 5p (72 mg, 57% yield) as off-white
1′-((1H-Indol-3-yl)methyl)-1H′-Lup-2-eno-[2,3-d]-[1,2,3]-tria-
zole-28-oic Acid (5l). Betulonic acid (100 mg, 1 equiv, 0.220 mmol),
tryptamine (46 mg, 1.3 equiv, 0.286 mmol), 4-nitrophenyl azide (36
mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves (50 mg), and toluene
(0.5 mL). Reaction time is overnight. The product was purified by
flash column chromatography (first washed with CH2Cl2 followed by
petroleum ether:EtOAc 9:1 → 6:4) affording 5l (115 mg, 84% yield)
1
as off-white crystals. m.p. 196 °C. H NMR (400 MHz, DMSO) δ
10.87 (s, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H),
7.10−7.02 (m, 2H), 6.96 (m, 1H), 4.72 (s, 1H), 4.72 (s, 1H), 4.59 (s,
1H), 4.54 (s, 2H), 3.17 (s, 1H), 3.03−2.92 (m, 1H), 2.70 (d, J = 15.2
Hz, 1H), 2.29 (m, 1H), 2.14−2.06 (m, 2H), 1.81 (d, J = 6.9 Hz, 2H),
1.63 (d, J = 29.9 Hz, 5H), 1.57−1.24 (m, 11H), 1.25−1.01 (m, 7H),
0.96 (s, 3H), 0.93 (s, 3H), 0.90 (s, 3H), 0.63 (s, 3H). 13C NMR (101
MHz, DMSO) δ 177.7, 150.7, 140.3, 137.8, 136.5, 127.4, 123.7,
121.4, 118.9, 118.3, 111.8, 110.7, 110.1, 55.9, 54.4, 50.1, 49.0, 48.9,
47.0, 42.5, 38.8, 38.1, 36.7, 33.5, 33.3, 32.0, 31.1, 30.5, 29.7, 28.6,
26.9, 25.5, 21.4, 21.1, 19.4, 18.8, 16.1, 15.7, 14.8. HRMS (ESI+): m/z
calculated for C40H54N4O2H [M + H]+: 623.4319, found 623.4317.
1′-(2-(1H-Indol-3-yl)ethyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-
28-oic Acid (5m). Betulonic acid (100 mg, 1 equiv, 0.220 mmol), 5-
methoxytryptamine (54 mg, 1.3 equiv, 0.286 mmol), 4-nitrophenyl
azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves (50 mg),
and toluene (0.5 mL). Reaction time is overnight. The product was
purified by flash column chromatography (first washed with CH2Cl2
followed by petroleum ether:EtOAc 9:1 → 6:4) affording 5m (126
1
crystals. m.p. 273 °C. H NMR (600 MHz, CDCl3) δ 4.76 (s, 1H),
4.64 (s, 1H), 4.33−4.23 (m, 2H), 3.04 (m, 1H), 2.91 (d, J = 15.3 Hz,
1H), 2.33−2.22 (m, 2H), 2.13 (d, J = 15.3 Hz, 1H), 2.00−1.96 (m,
2H), 1.79−1.64 (m, 5H), 1.61−1.35 (m, 13H), 1.35−1.19 (m, 14H),
1.17 (s, 3H), 1.00 (s, 3H), 0.99 (s, 3H), 0.88 (m, 3H), 0.77 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 181.7, 150.2, 141.0, 137.3, 109.8,
56.4, 54.6, 49.6, 49.2, 49.2, 46.9, 42.4, 40.5, 38.9, 38.5, 38.2, 37.0,
33.6, 33.3, 32.1, 31.6, 30.8, 30.6, 29.8, 29.7, 28.8, 28.6, 26.9, 25.4,
22.5, 21.3, 19.4, 18.9, 16.0, 15.7, 14.6, 14.0. HRMS (ESI+): m/z
calculated for C37H59N3O2H [M + H]+: 578.4679, found 578.4687.
1H′-Lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic Acid (5q). Betulonic
acid (100 mg, 1 equiv, 0.220 mmol), ammonium acetate (85 mg, 5
equiv, 1.100 mmol), 4-nitrophenyl azide (51 mg, 1.4 equiv, 0.308
mmol), 80 °C, 4 Å molecular sieves (50 mg), and DMF (0.8 mL).
Reaction time is overnight. The product was purified by flash column
chromatography (first washed with CH2Cl2 followed by
CH2Cl2:MeOH 95:5) affording 5q (90 mg, 86% yield) as off-white
crystals. m.p. 158 °C. Spectroscopic data for compound 5q was
consistent with previously reported data for this compound.27 1H
NMR (400 MHz, CDCl3) δ 4.77 (s, 1H), 4.64 (s, 1H), 3.03 (d, J =
10.1 Hz, 1H), 2.90 (d, J = 15.5 Hz, 1H), 2.37−2.22 (m, 2H), 2.12 (d,
J = 15.5 Hz, 1H), 2.00 (dd, J = 19.9, 11.7 Hz, 2H), 1.83−1.66 (m,
5H), 1.57 (dd, J = 38.5, 24.6 Hz, 12H), 1.36−1.24 (m, 7H), 1.21 (d, J
= 7.0 Hz, 4H), 1.01 (s, 3H), 1.00 (s, 3H) 0.78 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 181.0, 150.3, 150.1, 140.5, 109.8, 56.3, 53.4,
49.2, 49.0, 46.9, 42.5, 40.7, 39.0, 38.4, 37.3, 37.0, 33.3, 33.3, 32.1,
31.0, 30.6, 29.8, 25.5, 23.7, 21.4, 19.4, 19.1, 16.2, 15.6, 14.6. HRMS
(ESI+): m/z calculated for C30H45N3O2H [M + H]+: 480.3584, found
480.3585.
1
mg, 88% yield) as off-white crystals. m.p. 240 °C. H NMR (400
MHz, DMSO) δ 10.69 (s, 1H), 7.19 (d, J = 8.7 Hz, 1H), 7.05 (s, 1H),
6.76 (s, 1H), 6.67 (d, J = 8.7 Hz, 1H), 4.72 (s, 1H), 4.59 (s, 1H),
4.57−4.45 (m, 2H), 3.71 (s, 2H), 3.03−2.92 (m, 1H), 2.69 (d, J =
15.3 Hz, 1H), 2.28 (m, 1H), 2.09 (m, 2H), 1.81 (d, J = 6.9 Hz, 2H),
1.73−1.55 (m, 5H), 1.32 (m, 11H), 1.19−1.02 (m, 7H), 0.96 (s, 3H),
0.89 (s, 3H), 0.86 (s, 3H), 0.58 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 177.7, 153.5, 150.7, 140.3, 137.9, 131.5, 127.8, 124.3,
112.4, 111.7, 110.7, 110.1, 99.9, 55.9, 55.5, 54.4, 50.2, 48.9, 47.0, 42.5,
38.8, 38.1, 36.7, 33.5, 33.3, 32.0, 30.5, 29.7, 28.5, 26.9, 25.5, 22.5,
21.4, 20.9, 19.4, 18.8, 16.0, 15.7, 14.8. HRMS (ESI+): m/z calculated
for C41H56N4O3H [M + H]+: 653.4424, found 653.4418.
1′-((1H-Indol-4-yl)methyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-
28-oic Acid (5n). Betulonic acid (100 mg, 1 equiv, 0.220 mmol), 4-
(aminomethyl) indole (42 mg, 1.3 equiv, 0.286 mmol), 4-nitrophenyl
azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves (50 mg),
and toluene (0.5 mL). Reaction time is overnight. The product was
purified by flash column chromatography (first washed with CH2Cl2
followed by petroleum ether:EtOAc 9:1 → 6:4) affording 5n (98 mg,
1′-Benzyl-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic Acid (5r).
Betulonic acid (100 mg, 1 equiv, 0.220 mmol), benzylamine (31 mg,
1.3 equiv, 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv, 0.220
mmol), 4 Å molecular sieves (50 mg), and toluene (0.5 mL).
Reaction time is overnight. The product was purified by flash column
chromatography (first washed with CH2Cl2 followed by petroleum
ether:EtOAc 9:1 → 6:4) affording 5r (115 mg, 92% yield) as off-white
1
73% yield) as off white crystals. m.p. 260 °C. H NMR (400 MHz,
1
CDCl3) δ 8.32 (s, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.22 (m, 1H), 7.07
(m, 1H), 6.52−6.40 (m, 2H), 5.93 (m, 2H), 4.76 (s, 1H), 4.65 (s,
1H), 3.07−2.93 (m, 2H), 2.31−2.16 (m, 3H), 2.05−1.93 (m, 2H),
crystals. m.p. 290 °C. H NMR (400 MHz, CDCl3) δ 7.33−7.24 (m,
3H), 7.02 (d, J = 7.2 Hz, 2H), 5.64 (s, 2H), 4.76 (s, 1H), 4.64 (s,
1H), 3.09−3.00 (m, 1H), 2.96 (d, J = 15.4 Hz, 1H), 2.33−2.13 (m,
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3H), 2.06−1.93 (m, 2H), 1.82−1.63 (m, 5H), 1.61−1.37 (m, 11H),
1.27−1.15 (m, 7H), 1.04 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H) , 0.77 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 181.5, 150.2, 141.8, 138.0,
136.4, 128.7, 127.7, 126.3, 109.8, 56.4, 54.5, 52.8, 49.2, 49.1, 46.9,
42.4, 40.5, 38.9, 38.5, 38.3, 37.0, 33.7, 33.3, 32.0, 30.5, 29.7, 28.7,
25.4, 23.8, 21.3, 21.3, 19.4, 18.9, 16.0, 15.7, 14.6. HRMS (ESI+): m/z
calculated for C37H51N3O2H [M + H]+: 570.4053, found 570.4064.
1′-((R)-1-Phenylethyl)-1H′-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-
oic Acid (5s). Betulonic acid (100 mg, 1 equiv, 0.220 mmol), (R)-
(+)-α-methylbenzylamine (35 mg, 1.3 equiv, 0.286 mmol), 4-
nitrophenyl azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular
sieves (50 mg), and toluene (0.5 mL). Reaction time is overnight.
The product was purified by flash column chromatography (first
washed with CH2Cl2 followed by petroleum ether:EtOAc 9:1 → 6:4)
affording 5s (88 mg, 69% yield) as off white crystals. m.p. 327 °C. 1H
NMR (600 MHz, CDCl3) δ 7.30−7.20 (m, 3H), 7.15−7.10 (m, 2H),
5.73 (m, 1H), 4.75 (s, br, 1H), 4.64 (s,br, 1H), 3.02 (m, 1H), 2.95 (d,
J = 15.4 Hz, 1H), 2.32−2.22 (m, 2H), 2.17 (d, J = 15.4 Hz, 1H),
2.05−1.95 (m, 5H), 1.80−1.64 (m, 5H), 1.64−1.39 (m, 11H), 1.38−
1.25 (m, 7H), 1.00 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H) 0.72 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 181.5, 150.2, 141.7, 141.1, 137.6,
128.6, 127.5, 126.1, 109.8, 59.1, 56.4, 54.8, 49.3, 49.1, 46.8, 42.4, 40.5,
38.8, 38.4, 38.3, 37.0, 33.6, 33.3, 32.0, 30.5, 29.7, 28.6, 25.4, 23.3,
21.4, 21.3, 19.4, 19.0, 15.9, 15.6, 14.6. HRMS (ESI+): m/z calculated
for C38H53N3O2H [M + H]+: 584.4210, found 584.4214.
1′-((S)-1-Phenylethyl)-1H′-lupano-[2,3-d]-[1,2,3]-triazole-28-oic
Acid (5t). Dihydrobetulonic acid (100 mg, 1 equiv, 0.219 mmol), (S)-
(−)-α-methylbenzylamine (34 mg, 1.3 equiv, 0.285 mmol), 4-
nitrophenyl azide (36 mg, 1 equiv, 0.219 mmol), 4 Å molecular
sieves (50 mg), and toluene (0.5 mL). Reaction time is overnight.
The product was purified by flash column chromatography (first
washed with CH2Cl2 followed by petroleum ether:EtOAc 9:1 → 6:4)
affording 5t (115 mg, 90% yield) as off-white crystals. m.p. 173 °C.
1H NMR (400 MHz, CDCl3) δ 7.33−7.27 (m, 2H), 7.22 (m, 3H),
5.73 (m, 1H), 2.98 (d, J = 15.2 Hz, 1H), 2.32−2.22 (m, 3H), 2.17 (d,
J = 15.3 Hz, 1H), 2.03 (m, 3H), 1.96−1.63 (m, 5H), 1.45 (m, 11H),
1.29−1.22 (m, 7H), 1.10 (s, 3H), 0.97 (m, 6H), 0.87 (s, 3H), 0.82 (s,
3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.5, 141.7,
141.0, 137.5, 128.6, 127.5, 126.2, 59.2, 56.8, 54.7, 49.1, 48.7, 44.1,
42.6, 40.6, 38.8, 38.3, 38.3, 37.4, 33.8, 33.4, 31.9, 29.8, 29.7, 28.8,
26.8, 23.7, 23.0, 22.7, 21.3, 21.3, 18.9, 16.2, 15.7, 14.7, 14.5. HRMS
(ESI+): m/z calculated for C38H55N3O2H [M + H]+: 586.4366, found
586.4370.
1′-((S)-1-Phenylethyl)-28-methyl-1H′-lup-2-eno-[2,3-d]-[1,2,3]-
triazole (5u). Lupenone (100 mg, 1 equiv, 0.235 mmol; provided by
Milan Urban), (S)-(−)-α-methylbenzylamine (37 mg, 1.3 equiv,
0.306 mmol), 4-nitrophenyl azide (39 mg, 1 equiv, 0.235 mmol), 4 Å
molecular sieves (50 mg), and toluene (0.5 mL). Reaction time is
overnight. The product was purified by flash column chromatography
(first washed with CH2Cl2 followed by petroleum ether:EtOAc 9:1 →
6:4) affording 5u (107 mg, 83% yield) as an off-white solid. m.p. 283
°C. 1H NMR (600 MHz, CDCl3) δ 7.29 (t, J = 7.7 Hz, 2H), 7.23 (t, J
= 7.3 Hz, 1H), 7.15 (d, J = 7.8 Hz, 2H), 5.75 (m, 1H), 4.71 (s, 1H),
4.60 (s, 1H), 2.95 (d, J = 15.3 Hz, 1H), 2.39 (m, 1H), 2.17 (d, J =
15.4 Hz, 1H), 2.03 (d, J = 7.0 Hz, 3H), 1.93 (m, 1H), 1.74−1.68 (m,
5H), 1.68−1.40 (m, 11H), 1.40−1.27 (m, 9H), 1.06 (s, 3H), 0.98 (s,
3H), 0.96 (s, 3H), 0.80 (s, 3H), 0.73 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 150.7, 141.9, 141.2, 137.6, 128.6, 127.5, 126.1, 109.5, 77.2,
77.0, 76.8, 59.1, 54.9, 49.2, 48.2, 47.9, 43.0, 42.8, 40.7, 39.9, 38.8,
38.5, 38.2, 35.5, 33.6, 33.3, 29.8, 28.6, 27.5, 25.1, 23.4, 21.5, 19.3,
19.0, 18.0, 15.9, 15.6, 14.5. HRMS (ESI+): m/z calculated for
C38H55N3H [M + H]+: 554.4468, found 554.4462.
In parallel, the compounds were added to a mock-infected plate to
assess cytotoxicity. Besides the test compounds, two references were
included, i.e., K22 [(Z)-N-[3-[4-(4-bromophenyl)-4-hydroxypiperi-
din-1-yl]-3-oxo-1-phenylprop-1-en-2-yl]benzamide;47 from Chem-
Div] and GS-441524 (the nucleoside form of remdesivir; from
Carbosynth). After 5 days incubation at 35 °C, microscopy was
performed to score virus-induced CPE. To next perform the
colorimetric MTS cell viability assay, the reagent (CellTiter 96
AQueous MTS Reagent from Promega) was added to the wells, and 24
h later, absorbance at 490 nm was measured in a plate reader.
Antiviral activity was calculated from three independent experiments
and expressed as EC50 or concentration showing 50% efficacy in the
MTS or microscopic assay (see ref 56 for calculation details).
Cytotoxicity was expressed as 50% cytotoxic concentration (CC50) in
the MTS assay.
Immunofluorescence Detection of Viral dsRNA. Semiconfluent
cultures of human bronchial epithelial 16HBE cells (a gift from P.
Hoet, Leuven, Belgium) in 8-well chamber slides (Ibidi) were infected
with HCoV-229E (MOI: 1000) in the presence of 12 μM 5h or GS-
441524. After 4 h incubation at 35 °C, the inoculum was removed, the
compound was added again, and the slides were further incubated. At
24 h p.i., the cells were subjected to immunostaining for dsRNA (all
incubations at room temperature). After cell fixation with 3.7%
formaldehyde in PBS for 15 min and permeabilization with 0.2%
Triton X-100 in PBS for 10 min, unspecific binding sites were blocked
with 1% BSA in PBS for 30 min. Next, 1 h incubation was done with
mouse monoclonal anti-dsRNA antibody (J2, SCICONS English &
Scientific Consulting Kft; diluted 1:1000 in PBS with 1% BSA,)
followed by 1 h incubation with goat anti-mouse AlexaFluor488
(A21131, Invitrogen; 1:1000 in PBS with 1% BSA). Cell nuclei were
stained with DAPI (4′,6-diamidino-2-phenylindole, Invitrogen) in
PBS for 20 min at RT. Microscopic images were acquired using the
Leica TCS SP5 confocal microscope (Leica Microsystems) with a
HCX PL APO 63x (NA 1.2) water immersion objective. DAPI and
AlexaFluor488 were detected with excitation lines at 405 nm (blue)
and 488 nm (green) and emission lines of 410−480 nm (blue) and
495−565 nm (green).
Time-of-Addition Assay. Confluent HEL cells were infected with
HCoV-229E (MOI: 100) in a 96-well plate, and the compounds [5h,
bafilomycin A1 (from Cayman), or K22] were added at −0.5, 0.5, 2, 4,
6, or 8 h post-infection (p.i.). At 16 h p.i., the supernatant was
discarded and each well was washed twice with ice-cold PBS. The
cells were lysed on ice for 10 min with 22 μL of lysis mix, consisting of
lysis enhancer and resuspension buffer at a 1:10 ratio (both from the
CellsDirect One-Step RT-qPCR kit; Invitrogen). Next, the lysates
were incubated for 10 min at 75 °C and treated with DNase
(Invitrogen) to remove interfering cellular DNA. The number of viral
RNA copies in each sample was determined by one-step RT-qPCR.
Five microliters of lysate was transferred to a qPCR plate containing
9.75 μL of RT-qPCR mix (CellsDirect One-Step RT-qPCR) and 0.25
μL of Superscript III RT/Platinum Taq enzyme and HCoV-229E N-
gene specific primers and probe.57 The RT-qPCR protocol consisted
of 15 min at 50 °C; 2 min at 95 °C; and 40 cycles of 15 s at 95 °C and
45 s at 60 °C. An N-gene plasmid standard was included to allow
absolute quantification of viral RNA genome copies. The data from
two independent experiments were expressed as the number of viral
RNA copies at 16 h p.i. relative to the virus control receiving no
compound.
Selection of Resistant Coronavirus Mutants. HEL cells were
infected with HCoV-229E virus (MOI: 25), and 5h was added at
different concentrations. After 5 days incubation at 35 °C, the CPE
was scored microscopically to estimate the EC50 value. From the
highest compound concentration conditions showing virus-induced
CPE, the supernatants plus cells were frozen at −80 °C. These
harvests were further passaged in HEL cells under gradually increasing
compound concentrations, until a manifest increase in EC50 was
observed. A no compound control condition was passaged in parallel.
The final virus passages were submitted to RNA extraction; reverse
transcription; high-fidelity PCR; and cycle sequencing on the entire
viral genome using a set of 39 primers (sequences available upon
Biology. Anti-coronavirus Evaluation in Cell Culture. HCoV-
229E was purchased from ATCC (VR-740) and expanded in human
embryonic lung fibroblast cells (HEL; ATCC CCL-137). The titers of
virus stocks were determined in HEL cells and expressed as TCID50
(50% tissue culture infective dose).54 The cytopathic effect (CPE)
reduction assay was performed in 96-well plates containing confluent
HEL cell cultures, as previously described.55 Serial compound
dilutions were added together with HCoV-229E at an MOI of 100.
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request). After sequence assembly with CLC Main Workbench 7.9.1
(Qiagen), the sequences of the viruses passaged in the absence and
presence of 5h were aligned in order to identify the 5h resistance sites
in the HCoV-229E genome.
Benjamin Van Loy − Laboratory of Virology and
Chemotherapy, Rega Institute, KU Leuven, 3000 Leuven,
Belgium
Tien Nguyen − Biochemistry, Molecular and Structural
Biology, Department of Chemistry, KU Leuven, 3001 Leuven,
Belgium
Joice Thomas − Molecular Design and Synthesis, Department
of Chemistry, KU Leuven, 3001 Leuven, Belgium;
orcid.org/0000-0001-5517-4663
Julie Vandeput − Laboratory of Virology and Chemotherapy,
Rega Institute, KU Leuven, 3000 Leuven, Belgium
Dirk Jochmans − Laboratory of Virology and Chemotherapy,
Rega Institute, KU Leuven, 3000 Leuven, Belgium
Volker Thiel − Institute of Virology and Immunology (IVI),
3012 Bern, Switzerland; Department of Infectious Diseases
and Pathobiology, Vetsuisse Faculty, University of Bern, 3012
Bern, Switzerland
Ronald Dijkman − Institute of Virology and Immunology
(IVI), 3012 Bern, Switzerland; Department of Infectious
Diseases and Pathobiology, Vetsuisse Faculty and Institute for
Infectious Diseases (IFIK), University of Bern, 3012 Bern,
Switzerland
Virus Yield Assay. The virus yield assay was performed in 96-well
plates with semiconfluent cultures of 16HBE cells or confluent HEL
cells. Serial dilutions of compound 5h were added, and the cells were
infected (MOI: 100) with wild-type HCoV-229E (229E-WT),
EndoU-deficient HCoV-229E (229E-H250Ansp15),26 or the mutant
viruses obtained by passaging under 5h (229E-K60Rnsp15 or 229E-
T66Insp15). After 4 h incubation at 35 °C, the inoculum was removed,
the compound dilutions were added again, and the plates were further
incubated. At 3 days p.i., the supernatants were collected, and 2 μL of
each supernatant was lysed on ice by adding 11 μL of lysis mix
containing lysis enhancer and resuspension buffer at a 1:10 ratio. The
lysates were incubated for 10 min at 75 °C, and the viral RNA copy
number was determined by RT-qPCR as described above for the
time-of-addition assay. The data were collected in three independent
experiments and expressed as the fold reduction in viral RNA
compared to the virus control receiving no compound.
Computational Work. Starting from the published X-ray structure
of the hexameric nsp15 protein from HCoV-229E (PDB code: 4RS4),
we first changed a few amino acid residues, i.e., S1Q, S17G, A142T,
M219I, and S252L, to obtain the fully correct nsp15 protein sequence
of HCoV-229E virus. The structures of HCoV-229E nsp15 and
SARS-CoV-2 nsp 15 (PDB code: 7K1O) were prepared using MOE
(Chemical Computing Group, Montreal, Canada). Hydrogen
addition and optimization of protonation state and rotamers of the
mutations were conducted using the AMBER-EHT force field, and
identification of the potential binding sites in the multimeric complex
was performed using MOE. Docking of betulonic acid derivatives was
carried out by means of both MOE and GOLD with default settings,
where GBVI/WSA score and Goldscore functions were used,
respectively. The common top scoring solution was selected for
further research.
Wim Dehaen − Molecular Design and Synthesis, Department
of Chemistry, KU Leuven, 3001 Leuven, Belgium
Arnout Voet − Biochemistry, Molecular and Structural
Biology, Department of Chemistry, KU Leuven, 3001 Leuven,
Belgium; orcid.org/0000-0002-3329-2703
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02124
Author Contributions
∇A.S. and B.K. contributed equally. A.S. and L.N. designed,
performed, and interpreted the biological experiments. B.K.
and J.T. performed compound synthesis and analysis. B.V.L.,
J.V., and D.J. performed antiviral experiments. T.N. and A.V.
performed and interpreted the in silico study. V.T. and R.D.
provided the materials. A.S., B.K., W.D., A.V., and L.N. co-
wrote the manuscript. All authors gave approval to the final
version of the manuscript.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02124.
(Figure S1) Alignment of nsp15 protein sequences,
HPLC traces of key compounds, and H NMR and 13C
1
NMR spectra of the synthesized compounds (PDF)
Notes
PDB coordinates for computational models: 5h in
complex with nsp15 from HCoV-229E (PDB)
The authors declare no competing financial interest.
PDB coordinates for computational models: 5h in
complex with nsp15 from SARS-CoV-2 (PDB)
ACKNOWLEDGMENTS
■
B.K. acknowledges receipt of an Erasmus Mundus Western
Balkan Action 2 and doctoral fellowship from KU Leuven.
B.V.L. holds an SB-PhD fellowship from the FWO Research
Foundation Flanders (project: 1S66321N). Mass spectrometry
was made possible by the support of the Hercules Foundation
of the Flemish Government (grant: 20100225-7). We
acknowledge the support of NVIDIA Corporation for donating
the Titan Xp GPU used for this study. This research work was
supported by funding from the KU Leuven (IDO/12/020 and
project C14/19/78); the European Union’s Innovative
Medicines Initiative (IMI) under grant agreement
101005077 [Corona Accelerated R&D in Europe (CARE)
project]; and Fundació La Marató de TV3, Spain (project no.
201832-30). We thank Amy Swinnen and Leentje Persoons for
dedicated technical assistance and Els Vanstreels for providing
confocal microscopy training. We also thank Milan Urban for
providing lupenone.
Molecular formula strings (CSV)
AUTHOR INFORMATION
■
Corresponding Authors
Besir Krasniqi − Molecular Design and Synthesis, Department
of Chemistry, KU Leuven, 3001 Leuven, Belgium;
Phone: (+32)496798402; Email: besir.krasniqi@
kuleuven.be
Lieve Naesens − Laboratory of Virology and Chemotherapy,
Rega Institute, KU Leuven, 3000 Leuven, Belgium;
orcid.org/0000-0001-9742-9302; Phone: (+32)
16322098; Email: lieve.naesens@kuleuven.be
Authors
Annelies Stevaert − Laboratory of Virology and
Chemotherapy, Rega Institute, KU Leuven, 3000 Leuven,
Belgium; orcid.org/0000-0003-3316-2826
5642
https://doi.org/10.1021/acs.jmedchem.0c02124
J. Med. Chem. 2021, 64, 5632−5644

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Cihlar, T.; Jordan, R.; Denison, M. R.; Baric, R. S. Broad-spectrum
antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.
Sci. Transl. Med. 2017, 9, No. eaal3653.
ABBREVIATIONS USED
■
CC50, 50% cytotoxic concentration; CoV, coronavirus;
COVID-19, coronavirus disease 2019; CPE, cytopathic effect;
DAPI, 4′,6-diamidino-2-phenylindole; EndoU, endoribonu-
clease; FIPV, feline infectious peritonitis virus; HEL, human
embryonic lung; MERS, Middle East respiratory syndrome;
MHV-A59, mouse hepatitis virus A59; MTS, 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium; nsp, non-structural protein; p.i., post-
infection; RTC, replication-transcription complex; SARS,
severe acute respiratory syndrome; SARS-CoV-2, severe
acute respiratory syndrome coronavirus 2; TCID50, 50% tissue
culture infective dose
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