Design, Synthesis and Discovery of N,N’-Carbazoyl-aryl-urea Inhibitors of Zika NS5 Methyltransferase and Virus Replication

Article abstract of DOI:10.1002/cmdc.201900533

The recent outbreaks of Zika virus (ZIKV) infection worldwide make the discovery of novel antivirals against flaviviruses a research priority. This work describes the identification of novel inhibitors of ZIKV through a structure-based virtual screening approach using the ZIKV NS5-MTase. A novel series of molecules with a carbazoyl-aryl-urea structure has been discovered and a library of analogues has been synthesized. The new compounds inhibit ZIKV MTase with IC₅₀ between 23–48 μM. In addition, carbazoyl-aryl-ureas also proved to inhibit ZIKV replication activity at micromolar concentration.

Full text of DOI:10.1002/cmdc.201900533

Accepted Article
Title: Design, synthesis and discovery of novel N,N’-carbazoyl-
aryl-urea inhibitors of Zika NS5 methyltransferase and virus
replication.
Authors: Sharon Spizzichino, Giulio Mattedi, Kate Lauder, Coralie
Valle, Wahiba Aouadi, Bruno Canard, Etienne Decroly,
Suzanne J. F. Kaptein, Johan Neyts, Carl Graham, Zakary
Sule, David J. Barlow, Romano Silvestri, and Daniele
Castagnolo
This manuscript has been accepted after peer review and appears as an
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900533
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900533
A Journal of
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COMMUNICATION
Design, synthesis and discovery of novel N,N’-carbazoyl-aryl-urea
inhibitors of Zika NS5 methyltransferase and virus replication.
Sharon Spizzichino,[a,b] Giulio Mattedi,[a] Kate Lauder,[a] Coralie Valle,[c] Wahiba Aouadi,[c] Bruno
Canard,[c] Etienne Decroly,[c] Suzanne J.F. Kaptein,[d] Johan Neyts,[d] Carl Graham,[a] Zakary Sule,[a]
David J. Barlow,[a] Romano Silvestri[b] and Daniele Castagnolo*,[a]
[a]
[b]
Ms S. Spizzichino, Mr G. Mattedi, Ms K. Lauder, Mr C. Graham, Mr Z Sule, Dr D.J. Barlow, Dr D. Castagnolo
School of Cancer and Pharmaceutical Sciences
King's College London
London, SE1 9NH, United Kingdom
E-mail: daniele.castagnolo@kcl.ac.uk
Ms S. Spizzichino, Prof R. Silvestri
Department of Drug Chemistry and Technologies,
Sapienza University of Rome,
Laboratory Affiliated to Instituto Pasteur Italia – Fondazione Cenci Bolognetti,
Piazzale Aldo Moro 5, I-00185, Roma, Italy
[c]
[d]
Ms C. Valle, Dr. W. Aouadi, Dr. B. Canard, Dr. E. Decroly
AFMB, CNRS,
Aix-Marseille University,
UMR 7257, Case 925, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France
Dr S. J. F. Kaptein, Prof J Neyts
Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy,
KU Leuven, Leuven, Belgium
Supporting information and ORCID numbers for this article is given via a link at the end of the document.
Abstract: The recent outbreaks of Zika virus (ZIKV) infection
worldwide make the discovery of novel antivirals against flaviviruses
a research priority. This work describes the identification of novel
these diseases. Although a vaccine against DENV has recently
been commercialised (DENGVAXIA®),6 there is as yet no vaccine
against ZIKV available. In addition, there are no drugs available
to treat or prevent ZIKV infections, especially in the event of an
outbreak. A limited number of early-phase discovery studies have
identified few inhibitors of the flaviviruses DENV and ZIKV
replication,7-15 but there are no clinically approved drugs yet
available to target flaviviruses directly nor any that may serve as
vaccine adjuvants. The development of new antivirals thus
represents a research priority.
inhibitors of ZIKV through
a structure-based virtual screening
approach using the ZIKV NS5-MTase. A novel series of molecules
with a carbazoyl-aryl-urea structure has been discovered and a library
of analogues has been synthesized. The new compounds inhibit ZIKV
MTase with IC50 between 23-48 M. In addition, carbazoyl-aryl-ureas
also proved to inhibit ZIKV replication activity at micromolar
concentration.
Zika virus (ZIKV)1,2 is a single positive-strand RNA mosquito-
borne pathogen belonging to the family of Flaviviridae, genus
Flavivirus and it causes mild-severe diseases in humans and
animals. ZIKV is closely related to Dengue virus (DENV), and just
like DENV, is mainly transmitted to humans by bites of an infected
Aedes species mosquitoes (Ae. aegypti and Ae. albopictus).3
However, ZIKV can be also transmitted between humans through
contact with blood or other body fluids, sexual transmission2,4 and
by mother-to-foetus transmission during the pregnancy.6 While
global epidemics of DENV have spread over the past few decades
causing more than 20,000 demises per year, ZIKV infections have
emerged as a major public health concern only in the last few
years. Before 2007, only sporadic human disease cases were
reported in Africa and Asia. However, following the recent
outbreaks in the Americas in 2015, ZIKV was declared by the
World Health Organization (WHO) as a Public Health Emergency
of International Concern (PHEIC) and the first case of sexually-
transmitted ZIKV was reported in USA in 2008. While ZIKV
infections generally cause a mild fever, headache, malaise, skin
rashes and joint pain, they have been associated in several cases
with severe neurological and foetal complications leading to
microcephaly in new-borns4 and neurological diseases, such as
the Guillain–Barré syndrome (GBS), in adults.5 The recent
Flavivirus outbreaks, as well as the increasing number of cases
of ZIKV infections worldwide, have raised the attention of
pharmaceutical industries and healthcare providers toward the
identification and development of efficient treatments against
Figure 1. Examples of small molecules active against DENV and/or ZIKV, and
overview of this work.
1
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Within this context, viral proteins represent appealing targets for
the development of novel antiviral therapies. Examples of current
flavivirus inhibitors, such as 1-5 (Figure 1), have been designed
to target the DENV viral proteins NS3 (protease domain,16
helicase domain,17 and full-length NS3), NS5 (N-terminal
Methyltransferase domain (MTase)18-19 and C-terminal RNA-
dependent RNA polymerase (RdRp)20. The NS3 and NS5
proteins of DENV and ZIKV show a high degree of homology, and
their crystal structures have recently been determined.21-23 While
DENV NS5-polymerase has been investigated by a number of
research groups as a potential target for development of new
antivirals, there has been very little work carried out on DENV and
ZIKV NS5-MTase.23-24,18 The NS5-MTase is responsible for
maturation of the viral RNA cap and catalyses the methylation of
the N7 position of a guanine and the 2’-OH of the first
favourably interacting with the key residues of the binding pocket. c) Docking of
compound 6 in DENV NS5-MTase (PDB 5E9Q).24
Next, a library of analogues of compound 6 was designed and
synthesised to explore the chemical space around the urea
scaffold (Scheme 1). The carbazole derivatives 9a-f were first
synthesised through reaction of carbazole 7 with the appropriate
o
isocyanate 8 in toluene at 60 C with the aim to investigate the
effect of different substituents (electron-donating and electron-
withdrawing) on the phenyl ring bound to the urea moiety
(Scheme 1a). Similarly, thioureas 11a-b were synthesised from
10a-b to investigate the role of the isosteric sulphur in place of the
urea oxygen atom and to evaluate the influence of an extra
methylene group in 11b on MTase inhibition (Scheme 1b).
ribonucleotide to yield 7MeGpppA2′ Me-RNA.25 These methylations
O
of the RNA cap structure play a key role during virus replication
and are critical to virus survival in infected animals.26 Indeed,
biochemical studies and reverse genetic analysis have shown
that N7-MTase activity is essential for mRNA translation into viral
protein, and so viruses devoid of N7-MTase show a strongly
reduced replication phenotype.18 By contrast, 2′-O-MTase
defective viruses can replicate moderately well in infected cells,
but are highly attenuated in mice or rhesus monkey and induce a
strong antiviral response.27 Thus, the inhibition of both N7- and 2’-
O-MTase activities should restrain viral replication, making the
NS5-MTase a promising target for the development of new anti-
ZIKV, and potentially anti-DENV, antivirals.24
Herein, a structure-based virtual screening on a set of chemical
libraries using the ZIKV NS5-MTase was performed, with the aim
to identify structurally novel flaviviruses inhibitors.
The structure of the ZIKV NS5 MTase was built by homology
modelling using SWISS-MODEL28-29 and the compounds in the
NCI Diversity Set V database docked in the S-adenosyl
methionine (SAM) binding site in order to identify molecules that
might disrupt the activity of the enzyme. A set of 40 best ranking
molecules were initially identified, among which the urea 6
emerged as the best candidate due to its favourable interactions
with the SAM binding site. The urea 6 was thus evaluated in
enzymatic inhibition assays against ZIKV NS5-MTase, showing
an IC50 of 35 M (Table 1). A similar IC50 of 38 M was observed
when 6 was assayed against DENV NS5-MTase. Due to the
novelty of the structure when compared to known flavivirus
inhibitors, the urea 6 was thus selected as a hit compound for
further studies. Figure 2b shows the most favourable binding pose
of the hit compound 6 in the ZIKV NS5-MTase binding pocket.
The urea spacer connects the carbazole with a 2-methyl-4-nitro-
phenyl group, which occupies the region that interacts with the
methionine backbone of SAM (Figure 2a). The nitro group of 6
interacts with the side chain of S62 in a similar way to the SAM
carboxyl group, possibly stabilising the interaction of the
compound with the protein. The high sequence and structural
identity between ZIKV and DENV NS5-MTases lead to the
identical binding of compound 6 in both proteins (Figure 2c).
Scheme 1. Synthesis of urea derivatives 9a-f and 11a,b
The role of the carbazole ring on the antiviral activity was also
explored through its replacement with various phenyl moieties
(derivatives 12a-e) and indole ring (derivatives 14a-d). Ureas
12a-e and 14a-d were obtained from appropriate isocyanates 8
and 10 in toluene according to Scheme 2.
Scheme 2. Synthesis of urea derivatives 12a-e and 14a-d
Figure 2. a) SAM bound ZIKV NS5 MTase in PDB 5M5B.23 b) Docking of
compound 6 in ZIKV NS5 MTase. The carbazole pocket is accommodated in
the hydrophobic environment that binds the adenine portion of SAM. The p-nitro
group interacts with Ser62, mimicking the carboxy moiety of SAM. The
carbamide spacer allows the two domains to be spaced appropriately for
In order to evaluate the role of the nitro group on antiviral activity,
the amino derivatives 15 and 16 were also synthesised;
compounds 6 was treated with Fe(0) in conc. HCl leading to 15 in
high yields, while carbazole 7 was reacted under microwave
irradiation with Cbz,Boc-lysine to give, after deprotection in
HCl/AcOEt, the pure 16 (Scheme 3a). Finally, we also tried to
2
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modify the N-ethyl substituent of 6 in order to increase the
selectivity of our inhibitors targeting the MTase SAM binding site.
For this purpose, we compared the structure of the human N7
MTase (hRNMT) involved in the capping of cellular mRNA, with
that of both viral MTases. The X-ray structure revealed structural
similarities between the viral and the human MTases (NS5 and
hRNMT). However, both ZIKV and DENV NS5-MTase possess a
hydrophobic pocket located near the exocyclic amide of SAM,18
which is absent in the hRNMT. In the ZIKV MTase, the region is
defined by the amino acids F139, I153, G164, E155, R166, and
V170, compared to residues F133, I147, G148, E149, R160, and
V164 in the DENV MTase. We anticipated that the absence of this
hydrophobic pocket in hRNMT could help to increase compound
selectivity for the viral MTases and, hence, limit the interference
of inhibitors with host MTases. In the docking snapshot, the ethyl
group of compounds 6 is located in proximity of this pocket, clearly
showing that its replacement with a benzyl or a phenylethyl moiety
could lead to derivatives able to selectively recognise and inhibit
only the viral enzymes over the host enzymes (Figure 3).
The compound 21e and 21f showed the more potent reduction of
ZIKV NS5-MTase activity of ~70%.
Figure 3. a) SAH bound to hRNTM (PDB 5E8J).30 b) Docking of compound 21b
in ZIKV NS5 MTase. The ethyl spacer on the carbazole nitrogen allows the
phenyl ring to interact effectively with the hydrophobic pocket near the SAM
binding site. The absence of the region in hRNMT is a potential determinant of
target selectivity.
Thus, the synthesis of derivatives 21a-f bearing a phenyl-alkyl
substituent on the carbazole nitrogen was planned. Carbazole 17
was treated with HNO3 and converted into the nitro derivative 18,
which was in turn alkylated on the aromatic nitrogen with
appropriate benzyl or alkyl halide in the presence of NaH to give
compounds 19a-b. Reduction of the nitro group of 19 with SnCl2
led to amino derivatives 20a-b. The latter were then treated with
Scheme 3. Synthesis of urea derivatives 15, 16 and 21a-f
Conversely the compound 15 and 16 and those of series 11 and
14 barely inhibited the ZIKV MTase. Interestingly compound 6 and
those of the series 9 showed a similar inhibition profile on DENV
NS5-MTase, but the compounds of series 21 barely inhibit the
DENV MTase suggesting some specificity of this family. A dose-
response assay was then performed for the most promising
compounds and the IC50 values deduced from titration curves
after curve fitting are shown in Table 1. The results indicate that
the replacement of the methyl group of 6 with a chlorine atom in
9a did not affect the inhibitory activity of the compound against
ZIKV MTase, whilst it proved to be detrimental for inhibiting the
DENV MTase. Similarly, the replacement of the electron
withdrawing nitro group of 6 with other substituents (i.e. the
electron donating iodine or methoxy in 9c-d, or electron
withdrawing -CN or -CF3 in 9b and 9f) affected negatively the
compound’s inhibitory activity mainly against the ZIKV MTase.
The different activity of compounds 6 and 9 can be explained
taking in consideration the crucial H-bond interaction between
nitro groups of 6 with the hydroxyl group of S62, which is not
possible for derivatives 9b-f. The low activity of compounds 11b
can be attributed to the lack of appropriate substituents on the
phenyl ring able to interact with S62. On the other hand, the
carbazole ring proved to be crucial for the antiviral activity since
its replacement with other rings in 12 and 14 led to inactive
compounds. Derivatives 21c, 21e and 21f showed good activity
against ZIKV MTase at concentrations similar to 6 (IC50 = 23-48
M). It is likely that the N-benzyl or N-phenylethyl substituents in
compounds 21 are helping these compounds to bind to the viral
o
4-I-phenyl- or 2,4-(MeO)2-phenyl-isocyanate in toluene at 60 C
affording ureas 21c-f in high yields. A different strategy was
adopted to synthesise the derivatives 21a-b, due to the
commercial lack of the appropriate isocyanate reagent. The 2-
o
methyl-4-nitro-aniline was treated with triphosgene at 0 C in the
presence of Et3N leading to the formation of the corresponding
isocyanate, which was reacted in situ with carbazole 7 to give the
desired ureas 21a-b in good yields (Scheme 3).
The library of compounds 9, 11, 12, 14, 15, 16 and 21 was then
assessed on different purified recombinant MTases. All
compounds were initially screened at 50 µM against ZIKV NS5-
MTase and hRNMT as well as against DENV NS5-MTase due to
its similarity with ZIKV protein. The SAM mimetic sinefungin was
included as control.
The MTases were incubated with radiolabelled [3H]-SAM together
with the GpppAC4 RNA substrate and the different compounds at
50µM. The reaction was stopped after 30 mins at 30 °C. The
sample products were filtered on a DEAE membrane to remove
non-incorporated [3H]-SAM, and radioactivity transferred on the
RNA substrate was counted. Compounds 6, 9a, 9d, 21b, 21c, 21e
and 21f showed inhibition of ZIKV NS5-MTase higher than ~30%.
3
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10.1002/cmdc.201900533
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proteins even if no substantial improvement from 6 was detected.
A dose-response curve for hit compound 6 is reported in Figure 4.
activity against ZIKV in the cell-based assay (EC50 = 4.78 M).
Interestingly, derivative 15,31 which did not show any activity
against ZIKV NS5-MTase, was able to inhibit ZIKV replication with
an EC50 of 1.67 M. It is plausible that 15 inhibits ZIKV replication
via a different mode of action than via NS5-MTase inhibition, or
that this compound is partially metabolized in the treated cells.
Table 1. IC50 values of compounds 6, 9, 11 and 21 against host and flavivirus
MTases
hRNMT
ZIKV NS5-MTase
IC50 (M)
35 ± 5
DENV NS5-MTase
Cmpd
Table 2. EC50 and CC50 values against ZIKV of the most active compounds of
the library
ZIKV
6
13.9 ± 0.7
38 ± 6.7
≈ 133
Cmpd
9a
9b
9c
9d
≈ 122
≈ 253
46 ± 1.1
≈ 144
EC50 (M)
>3.99
>50
CC50 (M)
3.99
30.7
>10
≈ 267
6
27.4 ± 1.9
5.3 ± 0.4
NAb
≈ 62
9a
9b
>10
≈ 397
60 ± 32
9c
>10
>10
9f
6.8 ± 0.5
NAb
NAb
≈ 236
NAb
9d
>50
>50
11b
21b
≈ 112
70 ± 1.3
114 ± 1.3
11b
15
4.78
1.67
12.5
25
19.57
>12.5
20
nda
nda
nda
nda
nda
23 ± 1.2
48 ± 1.3
NAb
NAb
21c
21e
21b
21d
21f
100
> 20
20
26 ± 1.2
NAb
21f
DENV
Sinefungin
1.18 ± 0.05
0.63 ± 0.04
Cmpd
15
EC50 (M)
CC50 (M)
aActivity not determined. bNo activity observed above 50M
9.75
20
Finally, the ability of all the derivatives synthesised in this work to
inhibit ZIKV replication was assessed. Results of the most active
compounds, as well as their effect on cell viability, are reported in
Table 2. Compound 6 and 9a-d, which showed MTase inhibitory
activity, were devoid of any antiviral activity against ZIKV.
Interestingly, compound 15 also showed some activity against
DENV (EC50 = 9.75 M), even if the antiviral activity is clearly
linked to an adverse toxic effect on the host cell (CC50 = 20 M).
Finally, compound 21b which showed no enhanced activity
against ZIKV MTases, proved to inhibit ZIKV replication at good
concentration (EC50 = 12.5 M). However, compound 21b
showed toxicity in cellular assay as well as 21f which was found
active on ZIKV MTase instead. This could suggest that these
compounds might target another MTase (i.e. cellular MTase)
involved in virus replication. Interestingly, compound 21d, which
showed poor inhibition of ZIKV MTase, inhibits ZIKV with good
EC50 = 25 M and no toxicity (CC50 = 100 M).
In summary, a structure-based virtual screening protocol on a set
of chemical libraries using the ZIKV NS5-MTase was performed
leading to the identification of a novel class of carbazoyl-urea
inhibitors. Compounds 6 and 21c, 21e, 21f showed inhibitory
activity against ZIKV MTase as well as the related DENV MTase.
Conversely, the urea derivative 15 showed an antiviral effect
against ZIKV with EC50 of 1.67 M, despite its poor inhibitory
activity toward the NS5-MTase in vitro. Studies to identify novel
MTase inhibitors, to optimise the structure-activity relationships of
carbazoyl-urea analogues of 6 and to fully unravel their mode of
action are currently in progress in our laboratories.
Figure 4. Dose-response curve for hit compound 6
Possible reasons for the lack of antiviral activity may be the
inability of the molecules to enter the cell, or their conversion into
inactive metabolites in infected cells. However, since compound
6 proved to be cytotoxic (CC50 > 3.99 M), it is likely that this
compound and analogues thereof can enter the cells. Moreover,
the inhibitory activity of 6 and 9a-d against the host MTase (Table
1) hints towards an antimetabolic effect. Another possible
explanation is that compounds 6 and 9a-d mainly inhibit the 2’-O
MTase activity without affecting the N7 MTase activity. This could
explain the very poor inhibition of ZIKV replication in the cell-
based assay, as the 2’-O methylation of the cap is not essential
for viral replication in most cell lines. Derivative 11b, which
showed some MTase inhibitory activity, also exhibited antiviral
Experimental Section
Experimental details are reported in the Supporting Information.
4
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Acknowledgements
This work was supported by the European program H2020 under
the ZIKAlliance project (grant agreement 734548), the EVAg
Research Infrastructure (grant agreement 653316) and by the
French research agency ANR (VMTaseIn, grant ANR-ST14-
ASTR-0026). DC gratefully acknowledges EPSRC (Global
Challenges Competition King’s College London) and Royal
Society (RG160870) for research funding and financial support.
We thank Caroline Collard, Charlotte Vanderheydt and Elke Maas
for their assistance in the acquisition of the antiviral data. C.V. get
a fellowship from the National Research Agency ANR under the
program RAB-Cap and W.A. from the infectiopole Sud.
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Keywords: Zika • flavivirus • methyltransferase • antiviral • urea
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[28] When the work was carried out, the 3D structure of ZIKV NS5-MTase
was not elucidated yet. Thus, a homology model based on the DENV
NS5 MTase was built. The homology model was then compared to the
3D structure of ZIKV NS5MTase once it was released, showing full
similarity.
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10.1002/cmdc.201900533
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Novel antivirals targeting ZIKA virus. The recent outbreaks of Zika virus (ZIKV) worldwide make the discovery of novel antivirals a
priority. Using a structure-based virtual screening approach we were able to identify and develop a novel series of carbazoyl-urea
derivatives able to inhibit the ZIKV NS5-MTase as well as ZIKV replication at micromolar concentration.
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This article is protected by copyright. All rights reserved.