Design, synthesis and evaluation against Chikungunya virus of novel small-molecule antiviral agents

Article abstract of DOI:10.1016/j.bmc.2018.01.002

Chikungunya virus is a re-emerging arbovirus transmitted to humans by mosquitoes, responsible for an acute flu-like illness associated with debilitating arthralgia, which can persist for several months or become chronic. In recent years, this viral infection has spread worldwide with a previously unknown virulence. To date, no specific antivirals treatments nor vaccines are available against this important pathogen. Starting from the structures of two antiviral hits previously identified in our research group with in silico techniques, this work describes the design and preparation of 31 novel structural analogues, with which different pharmacophoric features of the two hits have been explored and correlated with the inhibition of Chikungunya virus replication in cells. Structure-activity relationships were elucidated for the original scaffolds, and different novel antiviral compounds with EC₅₀ values in the low micromolar range were identified. This work provides the foundation for further investigation of these promising novel structures as antiviral agents against Chikungunya virus.

Full text of DOI:10.1016/j.bmc.2018.01.002

Accepted Manuscript
Design, synthesis and evaluation against Chikungunya virus of novel small-
molecule antiviral agents
Roberta Tardugno, Gilda Giancotti, Tine De Burghgraeve, Leen Delang, Johan
Neyts, Pieter Leyssen, Andrea Brancale, Marcella Bassetto
PII:
S0968-0896(17)32342-8
https://doi.org/10.1016/j.bmc.2018.01.002
BMC 14156
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
1 December 2017
3 January 2018
Please cite this article as: Tardugno, R., Giancotti, G., De Burghgraeve, T., Delang, L., Neyts, J., Leyssen, P.,
Brancale, A., Bassetto, M., Design, synthesis and evaluation against Chikungunya virus of novel small-molecule
antiviral agents, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.01.002
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Design, synthesis and evaluation against Chikungunya virus
of novel small-molecule antiviral agents
Roberta Tardugnoa, Gilda Giancottia, Tine De Burghgraeveb, Leen Delangb, Johan Neytsb, Pieter
1
Leyssenb, Andrea Brancalea, Marcella Bassettoa
aCardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff, King Edward VII Avenue, Cardiff CF103NB, UK
bRega Institute for Medical Research, University of Leuven, Belgium
Abstract
Chikungunya virus is a re-emerging arbovirus transmitted to humans by mosquitoes, responsible for an
acute flu-like illness associated with debilitating arthralgia, which can persist for several months or
become chronic. In recent years, this viral infection has spread worldwide with a previously unknown
virulence. To date, no specific antivirals treatments nor vaccines are available against this important
pathogen. Starting from the structures of two antiviral hits previously identified in our research group
with in silico techniques, this work describes the design and preparation of 31 novel structural
analogues, with which different pharmacophoric features of the two hits have been explored and
correlated with the inhibition of Chikungunya virus replication in cells. Structure-activity relationships
were elucidated for the original scaffolds, and different novel antiviral compounds with EC50 values in
the low micromolar range were identified. This work provides the foundation for further investigation
of these promising novel structures as antiviral agents against Chikungunya virus.
1. Introduction
Chikungunya virus (CHIKV) is an Arbovirus that belongs to the Alphavirus genus of the Togaviridae
family. Transmitted to humans by mosquito Aedes Aegypti, it is associated with an acute pathology
characterised by fever, rash and arthralgia. The last condition is often severe and may persist for
several months or become chronic in the 10% of infected individuals.1 CHIKV infection was first
described in Tanzania in 1952 and since 2005 it re-emerged with a previously unknown virulence in
Africa, Indian Ocean, India and South-East Asia, reaching Europe and the US.2-3 The ability to adapt to
a new vector, the mosquito Aedes Albopictus, has contributed to the worldwide spread of the infection.4
Clinically approved compounds such as chloroquine, alpha-interferon and ribavirin, even if showing
some antiviral effect in vitro, demonstrated poor in vivo activity against CHIKV infection and, to date,
no specific treatment is available, nor a vaccine is approved: the therapy is still limited to supportive
treatment of the symptoms.5-6 Research interest on CHIKV has been rapidly increasing in the last few
years. However, to date, only few selective anti-CHIKV agents have been reported, and none of them
has reached the clinical evaluation stage.7
As part of an ongoing project aiming to identify novel inhibitors of Chikungunya virus replication, the
structures of our previous hits 1 and 2 (Figure 1), identified as inhibitors of the CHIKV life cycle
1 Corresponding author
E-mail address: bassettom@cardiff.ac.uk (Dr. M. Bassetto)
1

following a docking-based virtual screening analysis of commercial compounds on the structure of the
viral nsP2 protease,8 have been modified to gain insights into the antiviral structure-activity
relationships associated with this molecular scaffold. These novel structural modifications led to a
deeper understanding of the pharmacophoric features required for antiviral activity and to the
identification of novel anti-CHIKV agents with antiviral EC50 values in the low micromolar range and
good selectivity indexes.
2. Results and discussion
2.1 Rational design of novel analogues
The scaffold of hits 1 and 2 is characterised by the presence of two differently substituted phenyl rings,
ring A and ring B, a hydrazyde group directly attached to ring B, and an aliphatic spacer (a cyclopropyl
group in the trans configuration or a trans double bond) connecting ring A with the central hydrazyde
function. In order to further explore the antiviral structure-activity relationships associated with these
agents, and taking into account our previously reported preliminary explorations of their structures,8 a
series of 31 novel molecular analogues was designed and synthesised. As summarised in Figure 1,
several new modifications to the original structures were carried out in order to: 1. explore different
aromatic substituents on the two phenyl rings, while maintaining a central linker characterised by a
cyclopropyl group in the trans configuration or a trans double bond linked to a hydrazide feature; 2.
replace the original hydrazide with a more stable amide function; 3. investigate the role of the rigidity
of the central linker by reducing the trans-double bond to an ethyl group.
Figure 1. Structure of our previous antiviral hits and their proposed modifications.
2

With the aim to take into account and to further confirm our previously reported structural
considerations,8 the novel analogues were designed to either maintain a hydrophobic substituent (tert-
butyl or methyl) in the para position of aromatic ring A, or to remove completely this substituent, in
order to assess its essential role for antiviral activity. The nature and presence of the two original
ethoxy groups in aromatic ring B was also extensively explored to understand their role in the
inhibition of the viral replication. Due to the easier purification associated with the presence of a trans
double bond replacing the trans-cyclopropilic ring of the original hit, and to the fact that 2 is not
associated with any cytotoxic effect in vitro,8 most of the planned modifications on the two aromatic
systems were carried out on the trans-ethylidene scaffold of 2. As a first attempt to improve the
stability of 2, which is not predicted to be highly stable in aqueous conditions due to the presence of a
hydrazone function, a small series of novel analogues was envisaged to replace this group with a more
stable amide bond. In the case of this last modification, in order to partially compensate for the reduced
overall length of the molecule, which appears to be detrimental for antiviral activity according to our
previous preliminary SAR data obtained for the original scaffold 1,8 the insertion of an amide linker
was only attempted with a bi-phenyl system replacing ring B, or with a para-trifluoromethyl
substituent on aromatic ring B.
2.2 Chemistry
All target compounds were synthesised according to an optimised two to four-step synthetic pathway,
as shown in Schemes 1-3.
Scheme 1: Reagents and conditions: i. ethyldiazoacetate, 125 °C, 4 h, r.t., o.n. (45%); hydrazine monohydrate, EtOH, reflux, o.n.
(85%); differently substituted benzaldehyde, EtOH, reflux, 24 h (54-76%).
Following our previously reported procedure,8 a small series of novel cyclopropyl derivatives was
prepared by reaction of 4-tert-butyl styrene with ethyldiazoacetate at 125 °C for 4h, followed by
stirring at r.t. o.n. In these conditions, a mixture of the two disatereomeric couples of enantiomers at the
cyclopropane ring is obtained (cis and trans), and the desired pure trans-substituted intermediate 4 was
isolated after chromatographic purification in a mixture of dichloromethane and diethyl ether. Ester 4
was then refluxed in EtOH with an excess of hydrazine monohydrate to yield hydrazide 5, which was
then treated with the appropriate differently substituted benzaldehyde to afford final hydrazones 6 and
7. These final products were envisaged to further investigate our previous findings suggesting that a
hydroxyl group in the para position of aromatic ring B is tolerated by its introduction together with the
tert-butyl substituent in ring A (7) and by its alkylation to methyl ether (6).
3

O
O
O
NH2
N
Ar
OH
i
ii
N
iii
N
O
H
H
R2
R1
R1
R1
R1
8a-c
9a-c
10a-c
11a R2= H, Ar= 4-OMe-Ph
R = H, Ar= 4-Me-Ph
12a-c
2
a R1= tert-Bu
b R1= Me
13a R2= H, Ar= 4-iPr-Ph
14a R2= H, Ar= 4-tert-Bu-Ph
iv
R = H
c
R = Me, Ar= 4-C H11-Ph
15a,c
16a R2= H, Ar= Ph
17a R2= H, Ar= NO2-Ph
R = Me, Ar= 4-Cl-Ph
1
2
6
O
18a
2
N
19a R2= H, Ar= 4-OH-Ph
20a R2= Me, Ar= Pyrazine
H
R1
R2
R = Me, Ar= 3,4-OMe-Ph
22a R2= H, Ar= 4-Ph-Ph
23a R2= Me, Ar= 4-Ph-Ph
21a
2
R = CF
25a,c
26a,c R2= Ph
2
3
R = H, Ar= 3,4-OEt-Ph
24c
2
Scheme 2: Reagents and conditions: i. malonic acid, cat. piperidine, pyridine, r.t., 3h (93-98%); ii. (a) oxalyl chloride, Et2O, r.t.,
3 h, (b) hydrazine monohydrate, r.t., 2 h (50-63%); iii. differently substituted benzaldehyde, EtOH, reflux, 24 h (65-77%); iv.
differently substituted benzylamine, TBTU, diisopropylethylamine, anhydrous DMF, r.t., o.n. (83-96%).
A series of 17 novel benzylidene-acrylohydrazides was instead prepared to further explore the effect of
the aromatic substituents on aromatic rings A and B on the scaffold of 2. Cinnamic acids 9a-c were
prepared following a Knoevenagel condensation- Doebner modification reaction between aldehydes
8a-c and malonic acid, in the presence of catalytic amounts of piperidine and refluxing the mixture in
9
pyridine. Acid intermediates 9a-c were then converted to the corresponding acyl chlorides using
oxalyl chloride,10 and treated in situ with hydrazine monohydrate to give acrylo-hydrazides 10a-c,11
which were finally reacted with differently substituted benzaldehydes or acetophenones to give target
compounds 11-24.12 Starting from intermediate acids 9a and 9c, a small family of 4 novel acrylamide
analogues was obtained following a TBTU-assisted coupling reaction with differently substituted
benzylamines in the presence of diisopropylamine.13 This last modification was envisaged as a means
to replace the hydrazone function on the final products with a more stable amide group. Due to the
overall reduced length of the scaffold, and according to some preliminary results obtained for the
corresponding amide product with a 3,4-dimethoxy substituent on aromatic ring B,8 this structural
change from hydrazone to amide was envisaged together with the insertion of a biphenyl system to
replace ring B (26a, c). 25a and 25c were prepared to compare the effect of the second phenyl ring with
a smaller hydrophobic group, namely the trifluoromethyl.
Scheme 3: Reagents and conditions: i. H2, 10% Pd/C, THF, r.t., 4h (93-98%); ii. (a) 1.25 M HCl in MeOH, reflux, o.n., (b)
hydrazine monohydrate, EtOH, reflux, o.n. (65-67%); differently substituted benzaldehyde, EtOH, reflux, 24 h (73-95%).
As a first attempt to investigate the role of the rigidity of the original linker (trans-
cyclopropylhydrazide or acrylohydrazide), a small family of 8 novel analogues was designed to replace
the two rigid systems in the original hits with a reduced ethyl group connecting aromatic ring A with
the hydrazide portion of the scaffold. The desired modification was achieved by catalytic
4

hydrogenation of cinnamic acids 9a and 9c to their unsaturated counterparts 27a and 27c, performed
under H2 atmosphere in the presence of 10% Pd/C catalyst.14 The unsaturated acid intermediates 27a, c
were then converted to their methyl ester counterparts by refluxing in 1.25M HCl in methanol, and
subsequently treated with hydrazine monohydrate in boiling EtOH to achieve the desired ester
displacement in hydrazides 28a, c.15 Final compounds 29-32 were obtained as reported for the other
two series of hydrazone compounds, by reacting hydrazides 28a, c with the appropriately substituted
benzaldehyde or acetophenone in refluxing EtOH for 24 hours.
2.3 Antiviral and cytotoxicity studies
All the newly synthesised compounds were evaluated for their potential anti-CHIKV activity in
Vero cells, investigating their ability to inhibit the cytopathogenic effect (CPE) induced by the virus,
and for their potential cytotoxicity. The known anti-CHIKV agent Chloroquine was included as
positive control.16
Table 1: Antiviral effect of the test compounds on CHIKV replication in Vero cells and cytotoxicity.
EC50(µM)a,d
EC90(µM)b,d
CC50(µM)c,d
Comp.
Scaffold
R1
R2
Ar
SIe
(ring A)
t-Bu
t-Bu
H
(ring B)
1af
a
a
a
a
a
b
a
a
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
d
H
3,4-OEt-Ph
4-CF3-Ph
4-OH-Ph
4-C6H11-Ph
4-OMe-Ph
3,4-OEt-Ph
4-Me-Ph
4-OH-Ph
4-OMe-Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
4-iPr-Ph
t-Bu-Ph
5.0 0.2
14 6
6.4 0.1
32 1
5.6 2
3.2 1.8
6.6
6.4 0.5
19 10
8.7 0.2
44 1
72 20
30 5
14
2.1
2.3
3.1
13
32
-
1bf
1cf
H
H
15 1
1df
1ef
H
Me
Me
H
101 1
72 2
H
12 6
2f
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
Me
11 4
101 50
n.d.
6
H
n.d.
7
H
2.7
3.8
n.d.
-
11a
12a
12b
12c
13a
14a
15a
15c
16a
17a
18a
19a
20a
21a
22a
23a
24b
25a
H
5.9 0.8
5.8 0.9
>359
93.3
38.8 16.4
10 3.4
>359
117 23.8
124 7.1
n.d.
19.8
21.4
-
H
H
H
H
>284
84.4 11.5
7.4 0.6
n.d.
0.9
3.5
-
t-Bu
t-Bu
t-Bu
H
H
2.1 2.4
>276
>248
>289
5.8 1.4
>285
>282
n.d.
n.d.
H
>276
Me
Me
H
4-C6H11-Ph
4-C6H11-Ph
Ph
>248
n.d.
-
>289
n.d.
-
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
Me
87.5 79.1
>285
22.1 6.8
n.d.
3.8
-
H
4-NO2-Ph
4-Cl-Ph
Me
H
>282
n.d.
-
4-OH-Ph
Pyrazine
3,4-OMe-Ph
4-Ph-Ph
4-Ph-Ph
3,4-OEt-Ph
-
n.d.
7.3 1.9
>310
-
Me
Me
H
103
122 32.1
>263
1.2
-
>263
n.d.
n.d.
n.d.
20.1 12.6
n.d.
-
Me
H
>252
113
>252
-
>284
>284
>2.5
0.9
t-Bu
CF3
125 91.1
47.9
115 11
5

25c
d
d
d
c
c
c
c
c
c
c
c
-
H
CF3
Ph
Ph
H
-
>246
2.9
>246
>271
>319
>11
n.d.
-
26a
t-Bu
H
-
4.3 1.1
n.d.
-
26c
-
>319
>11
-
29a
t-Bu
H
3,4-OEt-Ph
3,4-OEt-Ph
4-Me-Ph
4-Me-Ph
4-OH-Ph
4-Me-Ph
4-C6H11-Ph
4-C6H11-Ph
-
n.d.
-
29c
H
>294
>100
>375
36.4
>373
>82.4
>96
>294
>100
>375
n.d.
n.d.
-
30a
t-Bu
H
H
n.d.
-
30c
H
n.d.
-
31a
t-Bu
H
H
n.d.
-
31c
H
>373
>82.4
>96
n.d.
-
32a
t-Bu
H
Me
H
n.d.
-
32c
n.d.
-
Chloroquine
-
-
11 7
21 18
89 28
8.1
a EC50 = 50% effective concentration (concentration at which 50% inhibition of CPE is observed).
b EC90 = 90% effective concentration (concentration at which 50% inhibition of CPE is observed).
c CC50 = 50% cytostatic/cytotoxic concentration (concentration at which 50% adverse effect is observed on the host cell).
d The EC50, EC90 and CC50 values are the mean of at least 3 independent experiments, with standard deviations of 10% of the
value quoted unless otherwise stated (mean value standard deviations).
e SI = the ratio of CC50 to EC50.f Biological data as previously reported.8
n.d.= value could not be calculated.
Considering together both antiviral activity results and cytotoxicity associated with the novel
compounds, the small series of cyclopropyl derivatives 6-7 appears to be correlated with an overall
activity retention, confirming what already observed to some extent for the previously reported series
of analogues. Alkylation of this hydroxyl group to methoxy is also tolerated, suggesting that the
presence and nature of the substituent in ring B is not relevant for the biological potential of the
scaffold. In the acrylohydrazide series of structures (11-24), most of the antiviral potential appears to
be associated with the presence of a tert-butyl substituent at the para position in aromatic ring A,
together with an unsubstituted hydrazone linker and a small hydrophobic group (methyl or methoxy) at
the para position of aromatic ring B, as indicated by the fact that the best results in terms of antiviral
activity and SI index are achieved with 11a and 12a. Even though their selectivity index is lower in
comparison with 2 (the least toxic compound previously identified), the CC50 values obtained for these
two novel structures show a significantly reduced standard deviation in comparison with 2, allowing a
greater confidence in the evaluation of their safety in cells. The antiviral activity of the scaffold seems
to be simultaneously influenced by the nature of the two aromatic substituents, as the association of a
4-methyl substituent or of the original 3,4-diethoxy substituent in ring B with a smaller substituent as
the 4-methyl group in ring A is associated with loss of antiviral activity (12b, 24b), and the same effect
is obtained with an unsubstituted phenyl ring A (12c). Surprisingly given the structural features of 2,
increasing the size of the 4-methyl group in 12a to an isopropyl substituent (13a) is related to a
significantly increased cytotoxic effect, while a tert-butyl group in this position (14a) is associated with
a complete loss of both antiviral and cytotoxic effect. Trying to investigate the effect of a methyl-
substituted hydrazone linker and a 4-cyclohexyl substituent in ring B, previously observed as partially
tolerated with 1d, 15a and 15c have been prepared carrying this double modification. The complete
loss of activity associated with both these analogues suggests a different trend in antiviral effect
associated with the cyclopropyl scaffold of 1a-e and the acrylo structure of 2. The removal of the
6

substituent in ring B (16a) is associated with an increased cytotoxicity, while replacement of the para
substituent in this ring with a 4-nitro (17a) or a 4-chloro (18a) moiety led to complete loss of activity.
Introduction of a 4-hydroxy group in ring B (19a), which was envisaged as a means to increase the
polarity and therefore the water solubility of the scaffold, is associated with cytotoxicity, further
confirming the different trend for biological effects between the cyclopropylic and acrylo series of
compounds. Another attempt to obtain a more polar structure was carried out with the replacement of
ring B with a pyrazine group (20a), but this modification led to a significant activity reduction.
Insertion of an extra phenyl group at the para position of ring B in the unsubstituted hydrazone scaffold
(22a) leads to cytotoxicity, while the same modification on the methyl-hydrazone structure (23a) does
not show the same cytotoxic effect, but is associated with complete loss of activity. The same effect
can be observed for the presence of the substituted methyl-hydrazone linker and a 3,4-methoxy
substitution in ring B (21a), suggesting that the presence of an unsubstituted hydrazone central linker is
essential for antiviral activity retention in the acrylo-hydrazide family of structures. The replacement of
the hydrazone group with a more stable and more flexible benzylamide (25-26) is associated with
either loss of antiviral activity (25a, 25c, 26c) or with an increased cytotoxicity (26a).
Except for 31a, which seems to partially retain some antiviral effect with an EC50 in the micromolar
range, all the remaining unsaturated analogues 29-32 are associated with a complete abolishment of
antiviral effect, suggesting that the rigidity of the scaffold and the molecular geometry given by the
trans-cyclopropane or trans-double bond are essential for the antiviral effect of these molecules. A
general summary of the structure-activity relationships observed for the scaffold of 1 and 2 is depicted
in Figure 3.
Figure 3. Summary of the antiviral structure-activity relationships obtained with this work for the antiviral scaffold of 1 and 2.
2.4 Molecular modelling
As briefly explained in the Introduction, the original hit 1 was identified as a selective antiviral agent
against CHIKV following a docking-based virtual screening procedure of commercial compounds,
with which the structure of the CHIKV nsP2 protease, an essential enzyme for the viral replication, had
been chosen as target for the selection of potential inhibitors of the viral replication.8 Both hits 1 and 2
are predicted to exert their antiviral activity through the inhibition of this enzyme, by binding to its
catalytic pocket. Even though a recently published study suggests that 1 is not a strong inhibitor of
CHIKV nsP2 activity in vitro,17 three of our previously reported close analogues of 1 were instead
found, in the same study, to inhibit the protheolitical activity of CHIKV nsP2, along with several novel

closely related derivatives of 1, described in the same work, which all show inhibition of the CHIKV
protease activity in the reported enzymatic assay. Due to these indications, the predicted binding to the
protease active site was investigated for the novel compounds prepared, with the aim to possibly find a
rational explanation for the differences in activity found for the new structures. Glide SP docking
algorithm was used for all simulations carried out.18-19 The predicted binding to the nsP2 protease
found for our original hit 2 and representative novel analogues 12a, 25a and 29a is shown in Figure 4.
A)
B)
C)
D)
Figure 2. Predicted binding mode of 2 (A), 12a (B), 25a (C) and 29a (D) to the proteolitic site of CHIKV nsP2 (PDB ID 3TRK).
According to our molecular modelling predictions, the hydrazyde bond of 2 (Figure 2A) fits with high
affinity the proteolytic site of the enzyme and is involved, through its carbonyl oxygen, in a direct
hydrogen bond with the side chain of Trp650. The two aromatic portions, even though not involved in
direct interactions with the surrounding enzyme residues, appear important to anchor the central
portion of the molecule in the region defined by the catalytic triad. A very similar binding mode is
shared by 12a (Figure 2B), which retains the antiviral effect of 2 and is representative of the two first
series of novel compounds prepared, 6, 7 and 11-24. Docking results on the CHIKV nsP2 protein
catalytic pocket found for all the compounds in these two series (data not shown) do not reveal
significant differences in their predicted binding mode to the enzyme active site, possibly indicating
that the reason for their marked differences in antiviral activities might be related with cell-penetration
factors or with the additional interaction with different viral or host targets. A significant difference in
8

predicted binding is instead found for 25a, representative of the small amide series of compounds
prepared, for which the loss of activity can be correlated with its increased flexibility, which does not
allow an optimal fitting of the protein sub-site surrounding the catalytic residues, and to the overall
reduced size of the molecule. The same negative effect associated with the increased flexibility can be
speculated for 29a, where the saturation of the double bond in trans configuration is associated with a
different occupation of the target site, as in most of the predicted binding poses found the hydrogen
bond with Trp650 is lost in favour of a predicted interaction with Cys579.
3. Conclusions
Starting from the structures of two anti-CHIKV hits previously identified, different series of novel
analogues were designed and synthesised to explore the structure-activity relationships associated with
these antiviral agents. Different structural elements were identified as essential for antiviral properties,
such as the presence of a tert-butyl substituent on ring B, an unsubstituted hydrazone linker and the
overall length and rigidity of the acrylo-hydrazide scaffold, while the associated antiviral effect was
found to follow a different trend for the two original hit scaffolds 1 and 2. Different novel antiviral
agents with anti-CHIKV EC50 values in the low micromolar range and good selectivity indexes were
identified, while further structural modifications are the focus of present invetsigations and will be
reported in due course. In particular, present synthetic efforts are directed towards the insertion of the
trans-double bond of 2 in a condensed aromatic ring, on the replacement of the central linker with more
stable groups while maintaining the overall length of the molecule, and on the inclusion of the central
hydrazone group in a third aromatic ring.
Acknowledgements
This research is supported by the Sêr Cymru II programme, which is part-funded by Cardiff University
and the European Regional Development Fund through the Welsh Government. L.D. was funded by
the Research Foundation of Flanders (FWO). We thank Stijn Delmotte and Tom Bellon for excellent
technical assistance.
A. Supplementary data
All experimental procedures and compound characterisation data are reported and described in detail in
the Supporting Information.
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aided identification, design and synthesis of a novel series of compounds with selective antiviral
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9
Kale, S.C.; Kale, M.K.; Biyani, K.R. Synthesis of some new benzimidazole acetic acid
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54:5370-5373.
10
insertion of arynes. J Org Chem. 2016; 81:3542-3552.
11 Narasimhan, B.; Belsare, D.; Pharande, D.; Mourya, V.; Dhake, A. Esters, amides and
Zhang, L.; Geng, Y.; Jin, Z. Transition-metal-free synthesis of N-aryl hydroxamic acids via
substituted derivatives of cinnamic acid: synthesis, antimicrobial activity and QSAR investigations.
Eur J Med Chem. 2004, 39:827-834.
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Kumar, D.; Narang, R.; Judge, V.; Kumar, D.; Narasimhan, B. Antimicrobial evaluation of 4-
methylsulfanyl benzylidene/3-hydroxy benzylidene hydrazides and QSAR studies. Med Chem Res.
2012, 21: 382-394.
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synthesis of cyclic gramicidin S: starting amine acid is key to high yield. J Org Chem. 2006, 71:55-61.
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alkenes under ultramild conditions. Org Process Res Dev. 2012; 16:1230-1234.
15 Carvalho, S.A.; Feitosa, L.O.; Soares, M.; Costa, T.E.M.M.; Henriques, M.G.; Salomao, K.;
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A.D.; da Silva, E.F.; Fraga, C.A.M. Design and synthesis of new (E)-cinnamic N-acylhydrazones as
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18
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10

approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J
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Supporting Information
Design, synthesis and evaluation against Chikungunya virus
of novel small-molecule antiviral agents
Roberta Tardugnoa, Gilda Giancottia, Tine De Burghgraeveb, Leen Delangb, Johan Neytsb, Pieter
2
Leyssenb, Andrea Brancalea, Marcella Bassettoa
aCardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff, King Edward VII Avenue, Cardiff CF103NB, UK
bRega Institute for Medical Research, University of Leuven, Belgium
Contents
Chemistry materials and methods.
Preparation and characterisation of final products 3, 7-9.
Molecular modelling methodologies.
Biological assay protocols.
2 Corresponding author
E-mail address: bassettom@cardiff.ac.uk (Dr. M. Bassetto)
11

S.1 Chemistry materials and methods
1
All solvents used for chromatography were HPLC grade from Fisher Scientific (UK). H and 13C NMR
spectra were recorded with a Bruker Avance DPX500 spectrometer operating at 500 and 125 MHz,
with Me4Si as internal standard. Mass spectra were determined with a Bruker microTOF spectrometer
using electrospray ionization (ESI source). For mass spectra, solutions were made in HPLC grade
methanol. Flash column chromatography was performed with silica gel 60 (230–400mesh) (Merck) and
TLC was carried out on precoated silica plates (kiesel gel 60 F254, BDH). Compounds were visualised
by illumination under UV light (254 nm). Melting points were determined on an electrothermal
instrument and are uncorrected. All solvents were dried prior to use and stored over 4 Å molecular
sieves, under nitrogen. Purity of prepared compounds was determined by HPLC-UV analysis (Thermo
HPLC connected with UV detector). All compounds tested in biological assays were >95% pure. The
purity of all final compounds was determined to be >95% by HPLC using the eluents water (eluent A),
acetonitrile (eluent B), at the following conditions: Varian Pursuit, 150 mm × 4.6 mm, 5.0 μm, 1.0
mL/min, gradient 30 min 10% → 100% eluent B in eluent A.
Intermediates 4, 5, 9a-c, 10a-c and 27-28 were prepared according to previously reported procedures.S1-
S2
Preparation and charecterisation of all final products are detailed below. Most hydrazone final
products show two sets of signals in NMR experiments.
S.1.1 General method for the preparation of hydrazones 6, 7, 11-24, 29-32
A mixture of the appropriate hydrazide (1 eq) and the differently substituted benzaldehyde or
.
acetophenone (1.2 eq) in EtOH (8.5 ml mmol/eq) was refluxed overnight or for 24 hours. The mixture
was then cooled to room temperature and the precipitate formed was filtered and washed with cold
EtOH and hexane. The solid obtained was purified by crystallization or flash column chromatography.
S.1.1.1 (E)-2-(4-(tert-Butyl)phenyl)-N'-(4-methoxybenzylidene)cyclopropanecarbohydrazide (6)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 70:30 v/v. Obtained in 76% yield as a white solid. M.p. 175-179 °C. TLC (8:2 n-
1
hexane-EtOAc, Rf: 0.35). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.25 (s, 9 H), 1.28 (s, 9H), 1.29-1.39 (m, 2H), 1.45-1.55 (m, 2H), 1.84-1.89 (m, 1H), 2.28-2.31 (m,
1H), 2.37-2.41 (m, 1H), 2.86-2.89 (m, 1H), 3.76 (s, 3H), 3.81 (s, 3H), 6.93 (d, J= 8.4 Hz, 2H), 7.01 (d,
J= 8.3 Hz, 2H), 7.10 (d, J= 8.2 Hz, 2H), 7.12 (d, J= 8.3 Hz, 2H), 7.28-7.32 (m, 4H), 7.55 (d, J= 8.0 Hz,
2H), 7.63 (d, J= 8.0 Hz, 2H), 7.95 (s, 1H), 8.07 (s, 1H), 11.30 (bs, 1H), 11.49 (bs, 1H). 13C-NMR
(DMSO-d6), δ: 14.8, 16.4, 21.5, 24.2, 24.8, 24.9, 31.1, 34.0, 55.2, 55.3, 114.2, 114.3, 125.0, 125.1,
125.5, 125.7, 126.7, 126.8, 128.1, 128.5, 137.6, 137.7, 143.0, 145.5, 148.5, 160.4, 160.6, 167.4, 172.6.
Anal. Calcd for C22H26N2O2: C, 75.40; H, 7.48; N, 7.99. Found: C, 75.47; H, 7.26; N, 7.84. MS [ESI,
m/z]: 351.2 [M+H].
S.1.1.2 (E)-2-(4-(tert-Butyl)phenyl)-N'-(4-hydroxybenzylidene)cyclopropanecarbohydrazide (7)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 90:10 v/v. Obtained in 54% yield as a pale yellow. M.p. 198-200 °C. TLC (5:5 n-
12

1
hexane-EtOAc, Rf: 0.41). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.24 (s, 9 H), 1.27 (s, 9H), 1.30-1.44 (m, 2H), 1.46-1.50 (m, 2H), 1.80-1.85 (m, 1H), 2.23-2.28 (m,
1H), 2.36-2.41 (m, 1H), 2.83-2.89 (m, 1H), 6.73-6.82 (m, 4H), 7.08 (d, J= 8.3 Hz, 2H), 7.11 (d, J= 8.2
Hz, 2H), 7.27-7.31 (m, 4H), 7.46 (d, J= 8.1 Hz, 2H), 7.52 (d, J= 8.1 Hz, 2H), 7.92 (s, 1H), 8.04 (s, 1H),
9.85 (bs, 2H), 11.22 (bs, 1H), 11.41 (bs, 1H). 13C-NMR (DMSO-d6), δ: 14.0, 16.3, 21.5, 24.1, 24.8,
24.9, 31.1, 34.0, 115.6, 115.6, 125.0, 125.1, 125.2, 125.3, 125.5, 125.7, 128.3, 128.6, 137.6, 137.7,
143.4, 145.9, 148.4, 159.0, 159.2, 167.2, 172.5. Anal. Calcd for C21H24N2O2: C, 74.97; H, 7.19; N,
8.33. Found: C, 75.13; H, 7.31; N, 8.11. MS [ESI, m/z]: 337.2 [M+H].
S.1.1.3 (2-E)-3-(4-(tert-Butyl)phenyl)-N'-(4-methoxybenzylidene)acrylohydrazide (11a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 71% yield as an off-white solid. M.p. 186-188 °C. TLC (5:5 n-
1
hexane-EtOAc, Rf: 0.40). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.29 (s, 9H), 1.30 (s, 9H), 3.81 (s, 6H), 6.67 (d, J= 15.7 Hz, 1H), 7.01 (d, J= 8.1 Hz, 4H), 7.44-7.48 (m,
4H), 7.54-7.58 (m, 4H), 7.60-7.66 (m, 2H), 7.67-7.72 (m, 5H), 8.02 (s, 1H), 8.20 (s, 1H), 11.36 (bs,
1H), 11.52 (bs, 1H). 13C-NMR (DMSO-d6), δ: 30.9, 34.5, 55.2, 55.3, 114.2, 116.5, 119.4, 125.7, 125.8,
126.8, 126.9, 127.5, 127.9, 128.4, 128.6, 131.9, 132.2, 140.1, 141.6, 142.8, 146.4, 152.6, 152.7, 160.6,
160.8, 161.4, 165.9. Anal. Calcd for C21H24N2O2: C, 74.97; H, 7.19; N, 8.33. Found: C, 74.81; H, 7.40;
N, 8.42. MS [ESI, m/z]: 359.1 [M+Na].
S.1.1.4 (2-E)-3-(4-(tert-Butyl)phenyl)-N'-(4-methylbenzylidene)acrylohydrazide (12a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 77% yield as an off-white solid. M.p. 200-202 °C. TLC (6:4 n-
1
hexane-EtOAc, Rf: 0.44). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.29 (s, 9H), 1.30 (s, 9H), 2.34 (s, 6H), 6.68 (d, J= 15.8 Hz, 1H), 7.26 (d, J= 7.9 Hz, 4H), 7.43-7.49 (m,
3H), 7.51-7.58 (m, 4H), 7.61-7.70 (m, 8H), 8.04 (s, 1H), 8.23 (s, 1H), 11.42 (bs, 1H), 11.59 (bs, 1H).
13C-NMR (DMSO-d6), δ: 21.0, 30.9, 34.5, 116.4, 119.4, 125.7, 125.8, 126.8, 127.1, 127.5, 127.9,
129.4, 131.5, 131.6, 131.9, 132.1, 139.5, 139.8, 140.2, 141.8, 143.1, 146.6, 152.6. Anal. Calcd for
C21H24N2O2: C, 74.97; H, 7.19; N, 8.33. Found: C, 74.81; H, 7.40; N, 8.42. MS [ESI, m/z]: 359.1
[M+Na].
S.1.1.5 (2E)-N'-(4-Methylbenzylidene)-3-(p-tolyl)acrylohydrazide (12b)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 67% yield as an off-white solid. M.p. 232-234 °C. TLC (5:5 n-
1
hexane-EtOAc, Rf: 0.32). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
2.32 (s, 6H), 2.37 (s, 6H), 6.65 (d, J= 15.7 Hz, 1H), 7.24-7.31 (m, 8H), 7.50-7.54 (m, 3H), 7.57 (d, J=
15.7 Hz, 1H), 7.60-7.68 (m, 7H), 8.03 (s, 1H), 8.22 (s, 1H), 11.40 (bs, 1H), 11.56 (bs, 1H). 13C-NMR
(DMSO-d6), δ: 20.9, 21.0, 116.1, 118.3, 119.2, 126.8, 127.0, 127.6, 127.7, 128.1, 129.3, 129.5, 131.4,
131.6, 131.9, 132.1, 139.5, 139.6, 139.8, 139.9, 140.4, 141.9, 143.1, 146.6, 161.4, 166.0. Anal. Calcd
for C18H18N2O: C, 77.67; H, 6.52; N, 10.06. Found: C, 77.51; H, 6.27; N, 10.24. MS [ESI, m/z]: 279.1
13

[M+H].
S.1.1.6 N'-(4-Methylbenzylidene)cinnamohydrazide (12c)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 71% yield as an off-white solid. M.p. 204-206 °C. TLC (5:5 n-
hexane-EtOAc, Rf: 0.34). One set of signals observed in NMR experiments. 1H-NMR (CDCl3), δ: 2.43
(s, 3H), 7.34-7.19 (m, 2H), 7.48-7.37 (m, 3H), 7.73-7.54 (m, 5H), 7.86 (s, 1H), 7.90 (d, J= 16.0 Hz,
1H); 9.47 (bs, 1H).13C-NMR (CDCl3), δ: 21.5, 116.5, 127.2, 128.3, 128.8, 128.9, 129.5, 130.0, 131.0,
135.1, 140.5, 143.7, 167.3. Anal. Calcd for C17H16N2O: C, 77.25; H, 6.10; N, 10.60. Found: C, 77.12;
H, 6.39; N, 10.37. MS [ESI, m/z]: 265.1 [M+H].
S.1.1.7 (2E)-3-(4-(tert-Butyl)phenyl)-N'-(4-isopropylbenzylidene)acrylohydrazide (13a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 77% yield as an off-white solid. M.p. 223-225 °C. TLC (5:5 n-
1
hexane-EtOAc, Rf: 0.55). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.22 (d, J= 6.8 Hz, 12H), 1.29 (s, 9H), 1.30 (s, 9H), 3.01-2.85 (m, 2H), 6.68 (d, J= 15.8 Hz, 1H), 7.29-
7.35 (m, 4H), 7.43-7.50 (m, 4H), 7.52-7.64 (m, 5H), 7.65-7.72 (m, 6H), 8.05 (s, 1H), 8.24 (s, 1H),
11.43 (s, 1H), 11.59 (s, 1H). 13C-NMR (DMSO-d6), δ: 23.6, 30.9, 33.3, 33.4, 34.5, 116.4, 119.3, 125.7,
125.7, 126.7, 126.9, 127.1, 127.5, 127.9, 131.9, 132.0, 132.1, 140.2, 141.7, 143.0, 146.5, 150.2, 150.6,
152.6, 152.7, 161.4, 166.0. Anal. Calcd for C23H22N2O: C, 77.27; H, 8.10; N, 8.04. Found: C, 77.49; H,
8.25; N, 7.87. MS [ESI, m/z]: 349.2 [M+H].
S.1.1.8 (2E)-N'-(4-(tert-Butyl)benzylidene)-3-(4-(tert-butyl)phenyl)acrylohydrazide (14a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 68% yield as an off-white solid. M.p. 223-225 °C. TLC (6:4 n-
1
hexane-EtOAc, Rf: 0.52). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.29 (s, 18H), 1.31 (s, 18H), 6.69 (d, J= 15.7 Hz, 1H), 7.39-7.50 (m, 7H), 7.52-7.58 (m, 3H), 7.58-7.62
(m, 2H), 7.62-7.71 (m, 7H), 8.05 (s, 1H), 8.25 (s, 1H), 11.46 (bs, 1H), 11.60 (bs, 1H). 13C-NMR
(DMSO-d6), δ: 30.8, 30.9, 31.0, 31.2, 34.5, 116.4, 119.3, 125.5, 125.6, 125.7, 126.6, 126.9, 127.5,
127.9, 131.5, 131.9, 132.1, 140.3, 141.7, 142.8, 146.5, 152.4, 152.6, 152.7, 161.5, 166.0. Anal. Calcd
for C24H30N2O: C, 79.52; H, 8.34; N, 7.73. Found: C, 79.45; H, 8.51; N, 7.87. MS [ESI, m/z]: 363.2
[M+H].
S.1.1.9 (2E)-3-(4-(tert-Butyl)phenyl)-N'-(1-(4-cyclohexylphenyl)ethylidene)acrylohydrazide (15a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 65% yield as a pale yellow solid. M.p. 258-260 °C. TLC (6:4 n-
1
hexane-EtOAc, Rf: 0.45). One set of signals observed in NMR experiments. H-NMR (CDCl3), δ:
1.35-1.30 (m, 2H), 1.38 (s, 9H), 1.47-1.45 (m, 4H), 2.32 (s, 3H), 2.66-2.51 (m, 1H), 7.31 (d, J= 8.3 Hz,
2H), 7.46 (d, J= 8.5 Hz, 2H), 7.60-7.65 (m, 3H), 7.76 (d, J= 8.3, 2H), 7.89 (d, J= 15.9 Hz, 1H), 9.10
(bs, 1H, NH).13C-NMR (CDCl3), δ: 12.8, 26.1, 26.8, 31.1, 34.3, 34.9, 44.4, 116.0, 125.8, 126.2, 126.6,
14

127.0, 128.2, 132.6, 135.8, 147.3, 149.6, 153.5, 167.9. Anal. Calcd for C27H34N2O: C, 80.55; H, 8.51;
N, 6.96. Found: C, 80.66; H, 8.37; N, 7.13. MS [ESI, m/z]: 403.3 [M+H].
S.1.1.10 (1-(4-Cyclohexylphenyl)ethylidene)cinnamohydrazide (15c)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 69% yield as a pale yellow solid. M.p. 240-242 °C. TLC (5:5 n-
1
hexane-EtOAc, Rf: 0.40). One set of signals observed in NMR experiments. H-NMR (CDCl3), δ:
1.36-1.26 (m, 1H), 1.54-1.38 (m, 4H, ), 1.80 (m, 1H), 1.99-1.84 (m, 4H), 2.31 (s, 3H), 2.64-2.53 (m,
1H), 7.31 (d, J= 8.1 Hz, 2H), 7.47-7.40 (m, 3H), 7.70-7.63 (m, 3H), 7.75 (d, J= 8.1, 2H), 7.89 (d, J=
15.8 Hz, 1H), 8.91 (s, 1H, NH). 13C-NMR (CDCl3), δ: 26.1, 26.8, 34.3, 44.4, 116.8, 126.2, 127.0,
128.3, 128.8, 130.0, 132.2, 135.6, 143.7, 149.4, 149.7, 167.5. Anal. Calcd for C23H26N2O: C, 79.73; H,
7.56; N, 8.09. Found: C, 79.49; H, 7.41; N, 8.30. MS [ESI, m/z]: 347.2 [M+H].
S.1.1.11 (2E)-N'-Benzylidene-3-(4-(tert-butyl)phenyl)acrylohydrazide (16a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 73% yield as an off-white solid. M.p. 227-229 °C. TLC (5:5 n-
1
hexane-EtOAc, Rf: 0.45). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.29 (s, 9H), 1.30 (s, 9H), 6.68 (d, J= 15.7 Hz, 1H), 7.41-7.48 (m, 10H), 7.54-7.59 (m, 3H), 7.62 (d, J=
15.7, 1H), 7.65-7.70 (m, 3H), 7.72-7.78 (m, 4H), 8.08 (s, 1H), 8.27 (s, 1H), 11.49 (bs, 1H), 11.66 (bs,
1H). 13C-NMR (DMSO-d6), δ: 30.9, 34.53, 116.3, 119.2, 125.7, 125.8, 126.8, 127.0, 127.5, 127.9,
128.7, 129.7, 129.9, 131.9, 132.1, 134.2, 134.3, 140.4, 141.9, 143.0, 146.6, 152.7, 152.8, 161.6, 166.1.
Anal. Calcd for C20H22N2O: C, 78.40; H, 7.24; N, 9.14. Found: C, 78.67; H, 6.99; N, 8.88. MS [ESI,
m/z]: 307.2 [M+H].
S.1.1.12 (2E)-3-(4-(tert-Butyl)phenyl)-N'-(4-nitrobenzylidene)acrylohydrazide (17a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 70% yield as a pale yellow solid. M.p. 230-232 °C. TLC (5:5 n-
1
hexane-EtOAc, Rf: 0.35). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.30 (s, 18H), 6.68 (d, J= 15.7 Hz, 1H), 7.41-7.48 (m, 4H), 7.53-7.60 (m, 3H), 7.73-7.81 (m, 4H), 7.88-
8.07 (m, 4H), 8.16 (s, 1H), 8.24-8.31 (m, 4H), 8.36 (s, 1H), 11.79 (bs, 1H), 11.94 (bs, 1H). 13C-NMR
(DMSO-d6), δ: 30.9, 34.5, 115.9, 118.91, 123.9, 125.6, 125.7, 125.8, 127.7, 127.8, 127.9, 128.1, 131.8,
132.0, 140.5, 140.6, 140.7, 141.1, 142.6, 144.0, 147.6, 147.7, 152.9, 153.0, 161.9, 166.5. Anal. Calcd
for C20H21N3O3: C, 68.36; H, 6.02; N, 11.96. Found: C, 68.12; H, 5.89; N, 12.21. MS [ESI, m/z]: 352.2
[M+H].
S.1.1.13 (2E)-3-(4-(tert-Butyl)phenyl)-N'-(1-(4-chlorophenyl)ethylidene)acrylohydrazide (18a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 72% yield as an off-white solid. M.p. 244-246 °C. TLC (5:5 n-
1
hexane-EtOAc, Rf: 0.41). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.30 (s, 18H), 2.30 (s, 3H), 2.33 (s, 3H), 7.02 (d, J= 15.6 Hz, 1H), 7.45-7.51 (m, 8H), 7.53-7.60 (m,
15

4H), 7.63-7.71 (m, 3H), 7.80-7.95 (m, 4H), 10.62 (bs, 1H), 10.67 (bs, 1H). 13C-NMR (DMSO-d6), δ:
13.5, 13.9, 30.9, 34.5, 116.6, 119.7, 125.7, 127.4, 127.5,127.8, 127.9, 128.1, 128.3, 132.1, 133.8, 137.0,
140.3, 142.0, 152.6. Anal. Calcd for C21H23ClN2O: C, 71.07; H, 6.53; N, 7.89. Found: C, 71.15; H,
6.50; N, 8.02. MS [ESI, m/z]: 355.2, 357.2 [M+H].
S.1.1.14 (2E)-3-(4-(tert-Butyl)phenyl)-N'-(4-hydroxybenzylidene)acrylohydrazide (19a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 0:100 v/v. Obtained in 69% yield as a pale yellow solid. M.p. 268-270 °C. TLC (3:7 n-
1
hexane-EtOAc, Rf: 0.30). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.29 (s, 9H), 1.30 (s, 9H), 6.66 (d, J= 16.0 Hz, 1H), 6.82-6.86 (m, 4H), 7.43-7.48 (m, 5H), 7.53-7.60
(m, 7H), 7.62-7.69 (m, 3H), 7.97 (s, 1H), 8.15 (s, 1H), 9.87 (bs, 1H), 9.91 (bs, 1H), 11.28 (bs, 1H),
11.45 (bs, 1H). 13C-NMR (DMSO-d6), δ: 30.9, 34.5, 115.6, 116.6, 118.5, 119.5, 125.2, 125.3, 125.7,
125.8, 127.4, 127.9, 128.5, 128.8, 131.9, 132.2, 139.9, 141.4, 143.2, 146.8, 152.5, 152.7, 159.1, 159.3,
161.2, 165.8. Anal. Calcd for C20H22N2O2: C, 74.51; H, 6.88; N, 8.69. Found: C, 74.62; H, 6.59; N,
8.54. MS [ESI, m/z]: 323.2 [M+H].
S.1.1.15 (2E)-3-(4-(tert-Butyl)phenyl)-N'-(1-(pyrazin-2-yl)ethylidene)acrylohydrazide (20a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 0:100 v/v. Obtained in 74% yield as an off-white solid. M.p. 230-232 °C. TLC (4:6 n-
1
hexane-EtOAc, Rf: 0.28). Two sets of signals observed in NMR experiments. H-NMR (CDCl3), δ:
1.31 (s, 9H), 1.37 (s, 9H), 2.46 (s, 6H), 6.94 (d, J= 15.7 Hz, 1H), 7.33 (d, J= 8.4 Hz, 2H), 7.41-7.49 (m,
3H), 7.52 (d, J= 8.4, 2H), 7.57-7.69 (m, 4H), 7.76 (d, J= 15.7, 1H), 7.93 (d, J= 15.7, 1H), 8.56
(apparent s, 3H), 9.29 (s, 1H), 9.45 (bs, 1H), 10.98 (bs, 1H). 13C-NMR (CDCl3), δ: 10.4, 31.1, 34.8,
115.0, 117.2, 125.8, 127.8, 128.2, 132.0, 132.2, 141.6, 142.8, 143.0, 143.7, 144.7, 146.3, 150.6, 153.9,
161.1, 167.8. Anal. Calcd for C19H22N4O: C, 70.78; H, 6.88; N, 17.38. Found: C, 70.94; H, 6.61; N,
17.42. MS [ESI, m/z]: 323.2 [M+H].
S.1.1.16 (2E)-3-(4-(tert-butyl)phenyl)-N'-(1-(3,4-dimethoxyphenyl)ethylidene)acrylohydrazide (21a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 40:60 v/v. Obtained in 75% yield as a pale yellow solid. M.p. 200-202 °C. TLC (5:5 n-
1
hexane-EtOAc, Rf: 0.23). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.30 (s, 18H), 2.28 (s, 3H), 2.32 (s, 3H), 3.80 (s, 6H), 3.81 (s, 3H), 3.87 (s, 3H), 6.97-7.04 (m, 3H),
7.32-7.38 (m, 2H), 7.41-7.50 (m, 6H), 7.52-7.61 (m, 4H), 7.62-7.68 (m, 3H), 10.50 (bs, 1H), 10.58 (bs,
1H). 13C-NMR (DMSO-d6), δ: 13.3, 14.1, 30.9, 34.5, 55.3, 55.5, 108.9, 109.4, 111.1, 111.2, 117.0,
118.5, 119.1, 119.7, 120.0, 125.7, 127.4, 127.7, 130.8, 132.2, 139.8, 141.4, 147.2, 148.4, 149.9, 150.1,
151.7, 152.5, 152.5, 161.8, 163.2. Anal. Calcd for C20H22N2O2: C, 74.51; H, 6.88; N, 8.69. Found: C,
74.62; H, 6.59; N, 8.54. MS [ESI, m/z]: 323.2 [M+H].
S.1.1.17 (2E)-N'-([1,1'-Biphenyl]-4-ylmethylene)-3-(4-(tert-butyl)phenyl)acrylohydrazide (22a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
16

hexane-EtOAc 50:50 v/v. Obtained in 65% yield as a pale yellow solid. M.p. 218-220 °C. TLC (5:5 n-
1
hexane-EtOAc, Rf: 0.30). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.30 (s, 9H), 1.31 (s, 9H), 6.69 (d, J= 15.7 Hz, 1H), 7.38-7.43 (m, 2H), 7.46-7.52 (m, 8H), 7.56-7.61
(m, 4H), 7.63-7.71 (m, 3H), 7.72-7.75 (m, 4H), 7.76-7.80 (m, 4H), 7.82-7.87 (m, 4H), 8.11 (s, 1H),
8.30 (s, 1H), 11.52 (bs, 1H), 11.68 (bs, 1H). 13C-NMR (DMSO-d6), δ: 30.9, 34.5, 116.4, 119.3, 125.7,
125.8, 126.6, 126.9, 127.4, 127.5, 127.6, 127.8, 128.0, 129.0, 139.3, 140.4, 141.3, 141.5, 141.9, 142.6,
146.1, 152.7, 152.8, 160.3, 161.5, 166.2. Anal. Calcd for C26H26N2O: C, 81.64; H, 6.85; N, 7.32.
Found: C, 81.51; H, 6.99; N, 7.14. MS [ESI, m/z]: 383.2 [M+H].
S.1.1.18 (2E)-N'-(1-([1,1'-Biphenyl]-4-yl)ethylidene)-3-(4-(tert-butyl)phenyl)acrylohydrazide (23a)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 68% yield as an off-white solid. M.p. 254-256 °C. TLC (6:4 n-
1
hexane-EtOAc, Rf: 0.30). Two sets of signals observed in NMR experiments. H-NMR (DMSO-d6), δ:
1.31 (s, 18H), 2.34 (s, 3H), 2.37 (s, 3H), 7.04 (d, J= 15.7 Hz, 1H), 7.39-7.44 (m, 2H), 7.47-7.52 (m,
8H), 7.57-7.60 (m, 3H), 7.62-7.69 (m, 4H), 7.72-7.78 (m, 8H), 7.92-7.96 (m, 4H), 10.60 (bs, 1H),
10.68 (bs, 1H). 13C-NMR (DMSO-d6), δ: 14.6, 30.9, 34.5, 117.6, 119.8, 125.8, 126.4, 126.5, 126.9,
127.5, 127.7, 127.9, 128.9, 137.2, 139.2, 140.1, 140.5, 141.3, 142.5, 144.9, 145.7, 146.5, 147.6, 161.4,
164.9, 166.2. Anal. Calcd for C27H28N2O: C, 81.78; H, 7.12; N, 7.06. Found: C, 81.99; H, 7.35; N,
6.84. MS [ESI, m/z]: 397.2 [M+H].
S.1.1.19 (2E)-N'-(3,4-Diethoxybenzylidene)-3-(p-tolyl)acrylohydrazide (24c)
Purified by flash column chromatography eluting with n-hexane-EtOAc 100:0 v/v increasing to n-
hexane-EtOAc 50:50 v/v. Obtained in 66% yield as a pale yellow solid. M.p. 170-172 °C. TLC (5:5 n-
1
hexane-EtOAc, Rf: 0.30). Two sets of signals observed in NMR experiments. H-NMR (CDCl3), δ:
1.33-1.48 (m, 6H), 1.48-1.59 (m, 6H), 2.37 (s, 3H), 2.42 (s, 3H), 4.06-4.13 (m, 4H), 4.17 (q, J= 6.9 Hz,
2H), 4.22 (q, J= 6.9 Hz, 2H), 6.48 (d, J= 15.5 Hz, 1H), 6.80 (d, J= 6.9 Hz, 1H), 6.93 (d, J= 8.0 Hz, 1H),
7.01-7.11 (m, 3H), 7.34-7.42 (m, 3H), 7.55-7.64 (m, 3H), 7.87 (s, 1H), 7.89 (s, 2H), 8.15 (s, 1H), 9.34
(bs, 1H), 9.99 (bs, 1H). 13C-NMR (CDCl3), δ: 14.7, 14.8, 21.5, 64.5, 64.7, 111.1, 112.7, 115.7, 121.6,
126.7, 128.3, 129.6, 132.5, 140.4, 143.5, 143.9, 148.9, 150.8, 167.7. Anal. Calcd for C21H24N2O3: C,
71.57; H, 6.86; N, 7.95. Found: C, 71.33; H, 6.99; N, 8.04. MS [ESI, m/z]: 353.2 [M+H].
S.1.1.20 3-(4-(tert-Butyl)phenyl)-N'-(3,4-diethoxybenzylidene)propanehydrazide (29a)
Purified by crystallization from EtOH. Obtained in 79% yield as a white solid. M.p. 108-112 °C. TLC
(5:5 n-hexane-EtOAc, Rf: 0.61). Two sets of signals observed in NMR experiments. 1H-NMR (DMSO-
d6), δ: 1.25 (s, 9H), 1.26 (s, 9H), 1.31-1.37 (m, 12H), 2.46-2.49 (m, 2H), 2.83-2.89 (m, 6H), 4.02-4.08
(m, 8H), 6.96-7.00 (m, 2H), 7.10-7.14 (m, 2H), 7.15 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 7.24
(d, J = 1.8 Hz, 1H), 7.26 (d, J = 1.8 Hz, 1H), 7.27-7.31 (m, 4H), 7.87 (s, 1H), 8.04 (s, 1H), 11.16 (bs,
1H), 11.23 (bs, 1H). 13C-NMR (DMSO-d6), δ: 14.6, 14.7, 31.2, 29.7, 33.9, 63.6, 63.7, 109.6, 112.5,
120.7, 121.5, 124.9, 127.8, 142.6, 146.0, 126.9, 137.9, 148.1, 149.7, 167.5, 173.3. Anal. Calcd for
C24H32N2O3: C, 72.70; H, 8.13; N, 7.06. Found: C, 72.95; H, 8.17; N, 6.88. MS [ESI, m/z]: 397.1
17

[M+H].
S.1.1.21 N'-(3,4-Diethoxybenzylidene)-3-phenylpropanehydrazide (29c)
Purified by crystallization from EtOH. Obtained in 77% yield as a white solid. M.p. 94-98 °C. TLC
(3:7 n-hexane-EtOAc, Rf: 0.72). Two sets of signals observed in NMR experiments. 1H-NMR (DMSO-
d6), δ: 1.31-1.36 (m, 12H), 2.52-2.55 (m, 2H), 2.87-2.92 (m, 6H), 4.01-4.08 (m, 8H), 6.96-7.00 (m,
2H), 7.10-7.14 (m, 2H), 7.16-7.21 (m, 2H), 7.23-7.25 (m, 3H), 7.26-7.31 (m, 7H), 7.88 (s, 1H), 8.04 (s,
1H), 11.17 (bs, 1H), 11.24 (bs, 1H). 13C-NMR (DMSO-d6), δ: 14.6, 14.7, 30.1, 30.7, 33.8, 35.7, 109.7,
112.6, 120.7, 121.5, 125.8, 125.9, 126.8, 142.7, 146.1, 126.9, 141.0, 148.2, 149.9, 173.2. Anal. Calcd
for C20H24N2O3: C, 70.56; H, 7.11; N, 8.23. Found: C, 70.69; H, 7.33; N, 8.05. MS [ESI, m/z]: 341.0
[M+H].
S.1.1.22 3-(4-(tert-Butyl)phenyl)-N'-(4-methylbenzylidene)propanehydrazide (30a)
Purified by crystallization from EtOH. Obtained in 81% yield as a white solid. M.p. 113-115 °C. TLC
(5:5 n-hexane-EtOAc, Rf: 0.66). Two sets of signals observed in NMR experiments. 1H-NMR (DMSO-
d6), δ: 1.33 (s, 18H), 2.39 (s, 3H), 2.41 (s, 3H), 2.60-2.64 (m, 2H), 3.04-3.08 (m, 4H), 3.10-3.14 (m,
2H), 7.19-7.20 (m, 2H), 7.23 (d, J = 8.1 Hz, 4H), 7.26 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.3 Hz, 4H),
7.57 (d, J = 8.1 Hz, 4H), 7.62 (d, J = 8.1 Hz, 2H), 7.79 (s, 1H), 7.96 (s, 1H), 8.48 (bs, 1H), 9.73 (bs,
1H). 13C-NMR (DMSO-d6), δ: 21.5, 31.0, 30.2, 34.6, 34.4, 125.3, 125.5, 127.1, 127.6, 143.7, 128.1,
138.1, 140.4, 148.9, 175.4. Anal. Calcd for C21H26N2O: C, 78.22; H, 8.13; N, 8.69. Found: C, 77.97; H,
8.26; N, 8.88. MS [ESI, m/z]: 323.1 [M+H].
S.1.1.23 N'-(4-Methylbenzylidene)-3-phenylpropanehydrazide (30c)
Purified by crystallization from EtOH. Obtained in 92% yield as a white solid. M.p. 91-95 °C. TLC
(3:7 n-hexane-EtOAc, Rf: 0.65). Two sets of signals observed in NMR experiments. 1H-NMR (DMSO-
d6), δ: 2.32 (s, 3H), 2.33 (s, 3H), 2.52-2.53 (m, 2H), 2.88-2.95 (m, 6H), 7.16-7.20 (m, 2H), 7.21-7.26
(m, 6H), 7.27-7.31 (m, 6H), 7.52 (d, J = 7.9 Hz, 2H), 7.56 (d, J = 7.8 Hz, 2H), 7.95 (s, 1H), 8.10 (s,
1H), 11.22 (bs, 1H), 11.31 (bs, 1H). 13C-NMR (DMSO-d6), δ: 20.9, 29.9, 33.7, 125.8, 126.5, 126.9,
128.2, 128.3, 129.3, 129.4, 145.8, 131.5, 139.3, 141.0, 173.3. Anal. Calcd for C17H18N2O: C, 76.66; H,
6.81; N, 10.52. Found: C, 76.43; H, 6.59; N, 10.74. MS [ESI, m/z]: 267.0 [M+H].
S.1.1.24 3-(4-(tert-butyl)phenyl)-N'-(4-hydroxybenzylidene)propanehydrazide (31a)
Purified by crystallization from EtOH. Obtained in 95% yield as a white solid. M.p. 165-169 °C. TLC
(5:5 n-hexane-EtOAc, Rf: 0.51). Two sets of signals observed in NMR experiments. 1H-NMR (DMSO-
d6), δ: 1.25 (s, 18H), 2.47 (t, J = 7.6 Hz, 2H), 2.83-2.91 (m, 6H), 6.78-6.82 (m, 4H, 7.15 (d, J = 8.1Hz,
2H), 7.18 (d, J = 8.1 Hz, 2H), 7.28-7.31 (m, 4H), 7.46 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H),
7.86 (s, 1H), 8.03 (s, 1H), 9.88 (bs, 2H), 11.06 (bs, 1H), 11.16 (bs, 1H). 13C-NMR (DMSO-d6), δ: 29.5,
33.7, 31.2, 35.7, 115.7, 127.9, 128.2, 128.6, 146.1, 124.9, 137.9, 148.1, 159.2, 173.1. Anal. Calcd for
C20H24N2O2: C, 74.04; H, 7.46; N, 8.64. Found: C, 73.85; H, 7.71; N, 8.47. MS [ESI, m/z]: 325.0
[M+H].
18

S.1.1.25 N'-(4-hydroxybenzylidene)-3-phenylpropanehydrazide (31c)
Purified by crystallization from EtOH. Obtained in 88% yield as a white solid. M.p. 135-139 °C. TLC
(3:7 n-hexane-EtOAc, Rf: 0.60). Two sets of signals observed in NMR experiments. 1H-NMR (DMSO-
d6), δ: 2.53-2.62 (m, 2H), 2.86-2.95 (m, 6H), 6.78-6.83 (m, 4H), 7.16-7.21 (m, 2H), 7.22-7.31 (m, 8H),
7.46 (d, J = 8.1 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.87 (s, 1H), 8.02 (s, 1H), 9.88 (bs, 2H), 11.08 (bs,
1H), 11.16 (bs, 1H). 13C-NMR (DMSO-d6), δ: 30.0, 33.7, 115.5, 115.6, 125.8, 125.9, 128.2, 128.3,
128.4, 128.7, 142.8, 146.1, 125.2, 141.4, 159.2, 173.1. Anal. Calcd for C16H16N2O2: C, 71.62; H, 6.01;
N, 10.44. Found: C, 71.90; H, 5.98; N, 10.35. MS [ESI, m/z]: 269.0 [M+H].
S.1.1.26 3-(4-(tert-Butyl)phenyl)-N'-(1-(4-cyclohexylphenyl)ethylidene)propanehydrazide (32a)
Purified by crystallization from EtOH. Obtained in 86% yield as a white solid. M.p. 142-144 °C. TLC
1
(7:3 n-hexane-EtOAc, Rf: 0.66). One set of signals observed in NMR experiments. H-NMR (CDCl3),
δ: 1.33 (s, 9H), 1.41-1.47 (m, 4H), 1.59-1.61 (m, 1H), 1.76-1.81 (m, 1H), 1.86-1.91 (m, 4H), 2.20 (s,
3H), 2.53-2.58 (m, 1H), 3.04-3.08 (m, 2H), 3.10-3.14 (m, 2H), 7.23-7.27 (m, 4H), 7.35 (d, J = 6.7 Hz,
2H), 7.68 (d, J = 6.9 Hz, 2H), 8.55 (bs, 1H). 13C-NMR (CDCl3), δ: 12.4, 21.4, 26.1, 26.8, 34.3, 34.7,
30.1, 44.3, 125.3, 126.1, 126.9, 128.1, 135.4, 138.2, 146.7, 148.8, 149.6, 175.1. Anal. Calcd for
C27H36N2O: C, 80.15; H, 8.97; N, 6.92. Found: C, 79.93; H, 9.15; N, 6.97. MS [ESI, m/z]: 405.2
[M+H].
S.1.1.27 N'-(1-(4-Cyclohexylphenyl)ethylidene)-3-phenylpropanehydrazide (32c)
Purified by crystallization from EtOH. Obtained in 90% yield as a white solid. M.p. 119-121 °C. TLC
1
(6:4 n-hexane-EtOAc, Rf: 0.61). One set of signals observed in NMR experiments. H-NMR (DMSO-
d6), δ: 1.18-1.27 (m, 2H), 1.31-1.44 (m, 6H), 1.67-1.73 (m, 4H), 1.74-1.81 (m, 8H), 2.20 (s, 6H), 2.46-
2.48 (m, 2H), 2.62-2.66 (m, 2H), 2.88-2.93 (m, 4H), 2.94-2.98 (m, 2H), 7.16-7.21 (m, 2H), 7.22-7.31
(m, 12H), 7.65 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.2 Hz, 2H), 10.27 (bs, 1H), 10.43 (bs, 1H). 13C-NMR
(DMSO-d6), δ: 13.4, 25.5, 26.3, 30.0, 30.8, 33.7, 34.3, 35.5, 43.4, 125.8, 125.9, 126.2, 126.5, 126.6,
128.2, 128.3, 141.1, 147.1, 148.7, 174.2. Anal. Calcd for C23H28N2O: C, 79.27; H, 8.10; N, 8.04.
Found: C, 79.41; H, 8.02; N, 7.91. MS [ESI, m/z]: 349.1 [M+H].
S.1.2 General method for the preparation of amides 25, 26
A mixture of the appropriate acid 9a or 9c (1 eq) and TBTU (1.2 eq.) was suspended in anhydrous THF
(5 ml . mmol/eq.) at r.t. under a N2 atmosphere. DiPEA (2.5 eq.) was then added to the reaction,
followed by the appropriate benzyl amine (1 eq.). The reaction was stirred at r.t. for 4-8 hours
(monitored by T.L.C.), then diluted with EtOAc (50 ml . mmol/eq). The organic phase was washed with
.
0.5M aq. citric acid solution (2x 30 ml mmol/eq), sat. aq. NaHCO3 solution (2x 30 ml . mmol/eq), and
finally with brine (30 ml . mmol/eq). The organic layer was dried over Na2SO4 concentrated under
vacuum. The solid obtained was purified by crystallization or flash column chromatography.
S.1.2.1 (E)-3-(4-(tert-Butyl)phenyl)-N-(4-(trifluoromethyl)benzyl)acrylamide (25a)
19

Purified by crystallisation from MeOH. Obtained in 67% yield as a white solid. M.p. 150-154 °C. TLC
(5:5 n-hexane-EtOAc, Rf: 0.72). 1H-NMR (CDCl3), δ: 1.34 (s, 9H), 4.64 (d, J= 5.9 Hz, 2H), 6.15 (t, J=
5.9 Hz, 1H), 6.44 (d, J= 15.5 Hz, 1H), 7.40 (d, J= 8.4 Hz, 2H), 7.44-7.47 (m, 4H), 7.61 (d, J= 8.1 Hz,
2H), 7.69 (d, J= 15.5 Hz, 1H). 13C-NMR (CDCl3), δ: 31.1, 34.8, 43.2, 119.1, 125.5, 125.7, 126.8,
127.7, 127.9, 129.6, 129.9, 131.8, 141.8, 142.4, 166.2. Anal. Calcd for C21H22F3NO: C, 69.79; H, 6.14;
N, 3.88. Found: C, 69.60; H, 5.97; N, 4.03. MS [ESI, m/z]: 384.1 [M+Na].
S.1.2.2 N-(4-(Trifluoromethyl)benzyl)cinnamamide (25c)
Purified by crystallisation from MeOH. Obtained in 86% yield as a white solid. M.p. 130-132 °C. TLC
(5:5 n-hexane-EtOAc, Rf: 0.69). 1H-NMR (CDCl3), δ: 4.63 (d, J= 5.9 Hz, 2H), 6.28 (t, J= 5.9 Hz, 1H),
6.48 (d, J= 15.6 Hz, 1H), 7.36-7.38 (m, 3H), 7.44 (d, J= 8.1 Hz, 2H), 7.48-7.51 (m, 2H), 7.58 (d, J= 8.1
Hz), 7.70 (d, J= 15.6 Hz, 1H). 13C-NMR (CDCl3), δ: 43.2, 120.0, 123.0, 125.5, 125.6, 125.7, 127.9,
128.8, 129.9, 134.6, 141.9, 142.4, 166.0. Anal. Calcd for C17H14F3NO: C, 66.88; H, 4.62; N, 4.59.
Found: C, 67.02; H, 4.55; N, 4.37. MS [ESI, m/z]: 306.1 [M+H].
S.1.2.3 (E)-N-([1,1'-Biphenyl]-4-ylmethyl)-3-(4-(tert-butyl)phenyl)acrylamide (26a)
Purified by crystallisation from MeOH. Obtained in 94% yield as a white solid. M.p. 140-144 °C. TLC
(5:5 n-hexane-EtOAc, Rf: 0.79). 1H-NMR (CDCl3), δ: 1.35 (s, 9H), 4.64 (d, J= 5.7 Hz, 2H), 5.97 (t, J=
5.7 Hz, 1H), 6.42 (d, J= 15.5 Hz, 1H), 7.37 (m, 1H), 7.40-7.45 (m, 4H), 7.45-7.48 (m, 4H), 7.58-7.61
(m, 4H), 7.70 (d, J= 15.5 Hz, 1H). 13C-NMR (CDCl3), δ: 31.1, 35.2, 43.5, 119.5, 125.8, 127.1, 127.3,
127.5, 127.6, 128.4, 128.8, 132.0, 137.3, 140.6, 140.7, 141.3, 153.2, 166.0. Anal. Calcd for C26H27NO:
C, 84.51; H, 7.37; N, 3.79. Found: C, 84.77; H, 7.41; N, 3.65. MS [ESI, m/z]: 392.2 [M+Na].
S.1.2.4 N-([1,1'-Biphenyl]-4-ylmethyl)cinnamamide (26c)
Purified by crystallisation from MeOH. Obtained in 83% yield as a white solid. M.p. 162-166 °C. TLC
(6:4 n-hexane-EtOAc, Rf: 0.48). 1H-NMR (CDCl3), δ: 4.65 (d, J= 5.8 Hz, 2H), 5.96 (t, J= 5.8 Hz, 1H),
7.37-7.40 (m, 4H), 7.43 (d, J= 8.2 Hz, 2H), 7.44-7.48 (m, 2H), 7.45 (d, J= 15.6 Hz, 1H), 7.52-7.54 (m,
2H), 7.59-7.61 (m, 4H), 7.72 (d, J= 15.6 Hz, 1H). 13C-NMR (CDCl3), δ: 43.6, 120.3, 127.1, 127.3,
127.5, 127.8, 128.4, 128.8, 128.9, 129.7, 134.8, 137.2, 140.6, 140.7, 141.5, 165.7. Anal. Calcd for
C22H19NO: C, 84.31; H, 6.11; N, 4.47. Found: C, 84.15; H, 5.87; N, 4.51. MS [ESI, m/z]: 314.2
[M+H].
S.2 Molecular Modelling
All molecular modeling studies were performed on a MacPro dual 2.66 GHz Xeon running Ubuntu
14.04. CHIKV nsP2 protease crystal structure was downloaded from the PDB data bank
(http://www.rcsb.org/; PDB code 3TRK). Hydrogen atoms were added to the protein, using the
Protonate 3D routine of the Molecular Operating Environment (MOE2015.10).S3 Ligand structures
were built with MOE and minimized using the MMFF94x force field until a RMSD gradient of 0.05
kcal mol_1 was reached. The
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Maestro LigPrep toolS4 was used to prepare the ligands using the default settings, whereas the protein
was prepared using the preparation wizard tool.S5 The docking simulations were performed using
Maestro Glide SP using the default parameters.S6
S.3 Virus cell-based CPE reduction assay
Chikungunya virus, Indian Ocean strain 889, isolated in 2006, was cultured on African green monkey
kidney (Vero) cells (ATCC CCL-81) in minimum essential medium MEM Rega3 (Invitrogen,
Belgium) supplemented with 10% Foetal Bovine Serum (FBS; Integro, The Netherlands), 1% L-
glutamine and 1% sodium bicarbonate (Invitrogen). Antiviral assays were performed in MEM Rega-3
medium supplemented with 2% FBS.
Vero cells were seeded in 96-well tissue culture plates (Becton Dickinson Falcon 96-Well Cell Culture
Plate) at a density of 2.5 x 104 cells/well in 100 µl assay medium and were allowed to adhere overnight
in an incubator (37 °C, 5% CO2, 95-99% RH). Next, a compound dilution series was added after which
the cultures were infected with 0.01 MOI of CHIKV 899 in 100 µl assay medium and returned to the
incubator. Each assay was performed in multiplicate in the same test and assays were repeated
independently. On day 5 post-infection (p.i.), the cell viability in each assay well was measured using
the MTS/PMS method as described by the manufacturer (Promega, The Netherlands). Chloroquine was
included in the assay as a reference compound.S7 The 50% effective concentration (EC50) is defined as
the compound concentration that is required to inhibit virus-induced cytopathogenic effect (CPE) by
50% and was determined using logarithmic interpolation employing a custom-designed database-
coupled interface (Accelrys, United Kingdom). The anti-metabolic effect of the compounds was
evaluated in uninfected, compound-treated cells, also by means of the MTS/PMS method. The 50%
cytostatic/cytotoxic concentration (CC50) is defined to be the concentration of compound that reduces
the overall metabolic activity of the uninfected, compound-treated cells by 50%, and was calculated
using logarithmic interpolation. All assay wells were checked microscopically for minor signs of virus-
induced CPE or alterations of the cell or monolayer morphology.
References
S1 Bassetto, M.; De Burghgrave, T.; Delang, L.; Massarotti, A.; Coluccia, A.; Zonta, N.; Gatti, V.;
Colombano, G.; Sorba, G.; Silvestri, R.; Tron, G.C.; Neyts, J.; Leyssen, P.; Brancale, A. Computer-
aided identification, design and synthesis of a novel series of compounds with selective antiviral
activity against chikungunya virus. Antiviral Res. 2013, 98, 12-18.
S2 Cheung, S.Y.; Chow, H.F.; Ngai, T.; Wei, X. Synthesis of organometallic poly(dendrimer)s by
macromonomer polymerization: effect of denrimer size and structural rigidity on the polymerization
efficiency. Chem. Eur. J. 2009, 15, 2278-2288.
S3 Molecular Operating Environment (MOE 2015.10); Chemical Computing Group, Inc.: Montreal,
Quebec, Canada; URL <http://www.chemcomp.com>; 2015.
S4 Schrödinger Release 2017-1: LigPrep, New York, NY: Schrödinger, LLC; 2017.
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S5 Schrödinger Release 2017-1: Schrödinger Suite 2017-1 Protein Preparation Wizard; Epik, New
York, NY: Schrödinger, LLC; 2017; Impact, New York, NY: Schrödinger, LLC; 2017; Prime, New
York, NY: Schrödinger, LLC; 2017.
S6 Schrödinger Release 2017-1: Glide, Schrödinger, LLC, New York, NY, 2017.
S7 Khan, M.; Santosh, S.R.; Tiwari, M.; Lakshmana Rao, P.V.; Parida, M. Assessment of in vitro
prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in vero cells. J. Med.
Virol. 2010, 82, 817–824.
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