Development of ciclesonide analogues that block SARS-CoV-2 RNA replication

Article abstract of DOI:10.1016/j.bmcl.2021.128052

Ciclesonide is an inhaled corticosteroid used to treat asthma and is currently undergoing clinical trials for treatment of coronavirus disease 2019 (COVID-19). An active metabolite of ciclesonide, Cic2, was recently reported to repress severe acute respir

Full text of DOI:10.1016/j.bmcl.2021.128052

Bioorg. Med. Chem. Lett. 43 (2021) 128052
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of ciclesonide analogues that block SARS-CoV-2
RNA replication
,
,
,
Genichiro Tsujia 1, Kenzo Yonemitsub 1, Takahito Itoa, Yuta Yanasea c, Masashi Uemab,
Nobumichi Ohokad, Takao Inoued, Hiroshi Asakurab, Yosuke Demizua
,c,*
a Division of Organic Chemistry, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki, Kanagawa 210-9501, Japan
b Division of Biomedical Food Research, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki, Kanagawa 210-9501, Japan
c Graduate School of Medical Life Science, Yokohama City University, 1-7-29, Yokohama, Kanagawa 230-0045, Japan
d Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki, Kanagawa 210-9501, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ciclesonide is an inhaled corticosteroid used to treat asthma and is currently undergoing clinical trials for
treatment of coronavirus disease 2019 (COVID-19). An active metabolite of ciclesonide, Cic2, was recently re-
ported to repress severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomic RNA replication.
Herein, we designed and synthesized a few types of ciclesonide analogues. Cic4 (bearing an azide group) and
Cic6 (bearing a chloro group) potently decreased SARS-CoV-2 viral replication and had low cytotoxicity
compared with Cic2 (bearing a hydroxy group). These compounds are promising as novel therapeutic agents for
COVID-19 that show significant antiviral activity.
SARS-CoV-2
COVID-19
Endonuclease
Ciclesonide
Viral growth inhibition
Introduction
metabolite with other functional groups Cic3–Cic10 would improve
efficacy. In addition, viral growth inhibition and cytotoxicity of the
At the end of 2019, an outbreak of coronavirus disease 2019 (COVID-
19) occurred in Wuhan city and rapidly spread across the world. The
causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-
CoV-2), has produced more than 117 million infections and 2.5 million
deaths as of March 2021.1 Numerous therapies and vaccines for SARS-
CoV-2 infection are currently being developed around the world, and
several drugs have been suggested to be effective for treatment of
COVID-19.2 Antiviral drugs commonly work by inhibiting proteases,
viral polymerases or nucleases. Remdesivir has been approved in some
countries for treatment of COVID-19, and decreases viral replication by
inhibiting the polymerase.3 Although no nuclease inhibitors against
SARS-CoV-2 have yet been approved for COVID-19 treatment, it was
recently reported that ciclesonide (Cicle), an approved drug for treat-
ment of steroidal asthma, is its promising candidate.4,5 Cicle activity
depends on hydrolysis by esterases to yield the active metabolite Cic2
(Fig. 1), and Cic2 was reported to repress SARS-CoV-2 genomic RNA
replication.4
synthesized analogues were evaluated.
The synthetic route for those analogues is shown in Scheme 1.
Briefly, Cic2 with was converted to the mesylated Cic3.5 Then, Cic4
(bearing an azide group) and Cic10 (bearing an amino group) were
obtained from Cic3. The analogue Cic5, in which the ester group of Cic1
was replaced with an amide group, was synthesized from Cic10. The
compounds Cic6 (bearing a chloro group), Cic7 (bearing a bromo
group), and Cic8 (bearing an iodo group) were synthesized from the
mesylated Cic3.5 The Cic9 analogue, bearing a dimethylamino group,
was prepared by treating Cic7 with dimethylamine.
The synthesized compounds were incubated with Vero E6/TMPRSS2
cells for 18 h and cytotoxicity was evaluated via release of lactate de-
hydrogenase (LDH).6 The prodrug Cicle, Cic4 (bearing an azide group),
Cic5 (an amide-type Cicle), and Cic6 (bearing a chloro group) did not
show significant cytotoxicity at concentrations up to 40 µM. In contrast,
the active metabolite Cic2, Cic3 (bearing a methanesulfonyl group), and
Cic10 (bearing an amino group) were cytotoxic at concentrations above
20 µM. Cic7 (bearing a bromo group), Cic8 (bearing an iodo group), and
Cic9 (bearing a dimethyl amino group) were cytotoxic at concentrations
In this report, we investigated the potential of Cic2 derivatives with
the expectation that replacing the primary hydroxy group of the active
* Corresponding author at: Division of Organic Chemistry, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki, Kanagawa 210-9501, Japan.
E-mail address: demizu@nihs.go.jp (Y. Demizu).
1
These authors contributed equally to this study.
https://doi.org/10.1016/j.bmcl.2021.128052
Received 8 March 2021; Received in revised form 11 April 2021; Accepted 14 April 2021
Available online 20 April 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

G. Tsuji et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128052
assays following infection with SARS-CoV-2 JPN/TY/WK-521 strains for
18 h in media containing different concentrations of these compounds.6
As shown in Fig. 4, Cicle, Cic2, Cic4, and Cic6 showed concentration-
dependent inhibition of viral growth. The concentrations of Cicle and
Cic2 required to reduce viral titres by 1000-fold compared with control
dimethyl sulfoxide treatment (approximately 4.6 × 105 plaque-forming
units/mL) were greater than 80 µM for Cicle and 60.95 µM for Cic2.
By contrast, Cic4 and Cic6 were able to reduce viral titres by 1000-fold
at concentrations of 7.36 µM and 5.95 µM, respectively, indicating that
Cic4 and Cic6 were more effective in inhibiting viral growth than Cicle
or Cic2.
In conclusion, we developed Cicle analogues (Cic3–Cic10) those
inhibited SARS-CoV-2 genomic RNA replication and growth. In the
Cic3–Cic10 analogues, the primary hydroxy group of Cic2, the active
metabolite of the prodrug Cicle, was replaced with various functional
groups. Among the synthesized analogues, Cic4 (bearing an azide
group) and Cic6 (bearing a chloro group) showed potent reduction of
viral replication and low cytotoxicity compared with Cic2. The low
cytotoxicity and strong antiviral activity of these compounds is a new
discovery, and we hope that the results of our study will serve as a
stepping stone for the development of effective therapeutics against
SARS-CoV-2 and other viruses. The detailed structure–activity rela-
tionship between Cic4 and Cic6 is difficult to describe at this stage
because target molecules (proteins) of Cicle compounds are not yet
found. However, Cic4 and Cic6 are less polar than Cic2, Cic9 and Cic10,
therefore, they may enter the cells more easily. Cic4 and Cic6 are also
less reactive than Cic3, Cic7 and Cic8, so they may be less degraded and
less likely to react with intracellular molecules. Recently, it was reported
that Cicle inhibited SARS-CoV-2 replication but resistance mutations
mapped to viral nonstructural protein 3 (nsp3) and nsp4.4 Considering
the report, further biological assays are needed to understand the potent
activity of the ligands Cic4 and Cic6, including search for their target
proteins and evaluation of their binding activity, and evaluation of their
chemical stability. Elucidation of those evaluation and further structural
expansion starting from the azide group of Cic4 (e.g., using click
chemistry) are underway.
Fig. 1. Chemical structures of ciclesonide (Cicle) and its active metabo-
lite Cic2.
of 40 µM (Fig. 2). The prodrug Cicle and its amide analogue Cic5, as well
as the less reactive Cic4 (bearing an azide group), Cic9 (bearing a
dimethylamino group), and Cic6-Cic8 with halogen atoms tended to be
less cytotoxic. On the other hand, Cic2 (bearing a hydroxy group) and
Cic10 (bearing an amino group) with proton donors and the O-sulfo-
nylated Cic3 tended to show slightly higher cytotoxicity.
Next, Vero E6/TMPRSS2 cells were infected with SARS-CoV-2 JPN/
TY/WK-521 strains for 18 h in medium containing 10 µM of each com-
pound. Viral growth inhibition was assessed by measuring the amount of
viral RNA in supernatants using quantitative reverse transcription-PCR.6
In the absence of any compounds, supernatants contained 4.8 × 1010
viral RNA copies/well. In the presence of Cicle, viral RNA in superna-
tants was significantly reduced to 5.2 × 108 copies/well. Cic4 and Cic6,
which showed no cytotoxicity (Fig. 2), had more pronounced effects on
reduction of the viral RNA than Cicle (2.1 × 105 copies/well and
4.6 × 105 copies/well, respectively, Fig. 3). The compounds Cic2, Cic3,
and Cic7 also showed significant reductions of viral RNA levels
compared with Cicle (Fig. 3), but Cic3 and Cic7 except for the active
metabolite Cic2 were excluded from subsequent evaluations because of
their cytotoxicity at high concentrations (Fig. 2).
To further evaluate the inhibitory effects of Cic4 and Cic6 on viral
growth, viral titres in cell supernatants were assessed using plaque
Scheme 1. Synthetic route for Cicle analogues Cic3–Cic10.6
2

G. Tsuji et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128052
Fig. 2. LDH toxicity of the synthesized Cicle analogues against Vero E6/TMPRSS2 cells. LDH, lactate dehydrogenase. Each independent experiment was done
in triplicate.
Fig. 3. Evaluation of viral RNA levels in Vero E6/TMPRSS2 cells at 18 h after SARS-CoV-2 infection in the presence of Cicle and Cicle analogues. DMSO, dimethyl
sulfoxide. Each independent experiment was done in triplicate.
3

G. Tsuji et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128052
ac) for editing a draft of this manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128052.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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This study was supported by grants from the Japan Agency for
Medical Research and Development (JP20fk0108297j to N.O., M.U., and
Y.D.). We thank Edanz Group (https://en-author-services.edanz.com/
4