Rational modifications on a benzylidene-acrylohydrazide antiviral scaffold, synthesis and evaluation of bioactivity against Chikungunya virus

Article abstract of DOI:10.1016/j.ejmech.2018.02.054

Chikungunya virus is a re-emerging arbovirus transmitted to humans by Aedes mosquitoes, responsible for an acute febrile illness associated with painful and debilitating arthralgia, which can persist for several months or become chronic. Over the past few years, infection with this virus has spread worldwide with a previously unknown virulence. No specific antiviral treatments nor vaccines are currently available against this important pathogen. Starting from the structure of a class of selective anti-CHIKV agents previously identified in our research group, different modifications to this scaffold were rationally designed, and 69 novel small-molecule derivatives were synthesised and evaluated for their inhibition of Chikungunya virus replication in Vero cells. Further structure-activity relationships associated with this class of antiviral agents were elucidated for the original scaffolds, and novel antiviral compounds with EC₅₀ values in the low micromolar range were identified. This work provides the foundation for further investigation of these new structures as antivirals against Chikungunya virus.

Full text of DOI:10.1016/j.ejmech.2018.02.054

Accepted Manuscript
Rational modifications on a benzylidene-acrylohydrazide antiviral scaffold, synthesis
and evaluation of bioactivity against Chikungunya virus
Gilda Giancotti, Michela Cancellieri, Andrea Balboni, Mariateresa Giustiniano, Ettore
Novellino, Leen Delang, Johan Neyts, Pieter Leyssen, Andrea Brancale, Marcella
Bassetto
PII:
S0223-5234(18)30191-0
10.1016/j.ejmech.2018.02.054
EJMECH 10231
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 January 2018
Revised Date: 14 February 2018
Accepted Date: 16 February 2018
Please cite this article as: G. Giancotti, M. Cancellieri, A. Balboni, M. Giustiniano, E. Novellino, L.
Delang, J. Neyts, P. Leyssen, A. Brancale, M. Bassetto, Rational modifications on a benzylidene-
acrylohydrazide antiviral scaffold, synthesis and evaluation of bioactivity against Chikungunya virus,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.054.
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Rational modifications on a benzylidene-acrylohydrazide antiviral scaffold, synthesis
ACCEPTED MANUSCRIPT
and evaluation of bioactivity against Chikungunya virus
Gilda Giancottia, Michela Cancellieria, Andrea Balbonia, Mariateresa Giustinianob, Ettore Novellinob, Leen Delangc, Johan Neytsc,
1
Pieter Leyssenc, Andrea Brancalea, Marcella Bassettoa
aCardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff, King Edward VII Avenue, Cardiff CF103NB, UK
bDepartment of Pharmacy, University of Naples ‘Federico II’, Naples 80131, Italy
cRega Institute for Medical Research, University of Leuven, Belgium
Highlights
•
•
•
•
Rational modifications on a substituted benzylidene-acrylohydrazide scaffold with antiviral activity against CHIKV.
Synthesis of different series of novel analogues with improved stability and drug-like properties.
Different new inhibitors of the CHIKV replication identified.
Molecular modelling studies on the CHIKV nsP2 protease.
Keywords
Novel small-molecule antivirals; Chikungunya virus; structure-activity relationship studies; molecular modelling.
Abstract
Chikungunya virus is a re-emerging arbovirus transmitted to humans by Aedes mosquitoes, responsible for an acute febrile illness
associated with painful and debilitating arthralgia, which can persist for several months or become chronic. Over the past few
years, infection with this virus has spread worldwide with a previously unknown virulence. No specific antiviral treatments nor
vaccines are currently available against this important pathogen. Starting from the structure of a class of selective anti-CHIKV
agents previously identified in our research group, different modifications to this scaffold were rationally designed, and 69 novel
small-molecule derivatives were synthesised and evaluated for their inhibition of Chikungunya virus replication in Vero cells.
Further structure-activity relationships associated with this class of antiviral agents were elucidated for the original scaffolds, and
novel antiviral compounds with EC50 values in the low micromolar range were identified. This work provides the foundation for
further investigation of these new structures as antivirals against Chikungunya virus.
Graphical abstract
1. Introduction
1
Corresponding author
E-mail address: bassettom@cardiff.ac.uk (Dr. M. Bassetto)
1

Chikungunya virus (CHIKV) is an Arbovirus, transmitted to humans by mosquito Aedes Aegypti, which belongs to the Alphavirus
genus of the Togaviridae family, and it is associated with an acute pathology characterised by fever, rash and arthralgia. The last
ACCEPTED MANUSCRIPT
condition is often severe and has a tendency to persist for several months, becoming chronic in the 10% of infected individuals.1
Since its first description in Tanzania in 1952, infection with this virus has had recurrent outbreaks in tropical regions and, from
2005, it has re-emerged with a previously unknown virulence in different parts of Africa, Indian Ocean islands, India and South-
East Asia, also reaching Europe and the US.2-3 The virus ability to adapt to a new vector, the mosquito Aedes Albopictus, has
contributed to the worldwide spread of the infection.4 Different clinically approved compounds such as chloroquine, alpha-
interferon and ribavirin have been found to show some antiviral activity in vitro, but they demonstrated poor in vivo activity
against CHIKV infection in animal models 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 Even though research efforts on the identification and development of
selective treatments against CHIKV have been rapidly increasing in the last few years, only few selective anti-CHIKV agents
have been reported to date, and none of them has reached the clinical evaluation stage.7
As part of an ongoing project aiming to identify novel antiviral agents against Chikungunya virus, the structure of our previously
reported hit 1 (Figure 1), designed and synthesised as inhibitor of the CHIKV life cycle following a docking-based virtual
screening analysis of commercial compounds on the structure of the viral nsP2 protease,8 has been further modified to improve its
drug-like properties and gain further insights into the antiviral structure-activity relationships associated with its molecular
scaffold. These novel structural modifications allowed a deeper understanding of the functional groups required for antiviral
activity and the successful identification of novel anti-CHIKV agents with antiviral EC50 values in the low micromolar range and
better drug-like characteristics.
2. Results and discussion
2.1 Rational design of novel analogues
The structure of hit 1 is characterised by the presence of two differently substituted phenyl rings, ring A and ring B, a hydrazone
group directly attached to ring B, and an aliphatic spacer in the form of a trans double bond connecting ring A with the central
hydrazide function. While different close analogues of 1 have been prepared and evaluated mainly to determine the role of the
aromatic substituents and the presence of the trans double-bond and the hydrazone group,9 the present study focuses on a series of
69 novel molecular derivatives, designed to replace the reactive Michael’s acceptor in the structure of 1 and to susbtitute the
hydrazone linker, expected to be poorly stable in aqueous environments, with more water-stable polar groups. As summarised in
Figure 1, several new modifications to the original structures were carried out in order to: 1. explore different para hydrophobic
aromatic substituents on phenyl ring A (Series a); 2. include the reactive Michael’s acceptor trans double bond into a condensed
aromatic or hetero-aromatic ring (Series b); 3. replace the Michael’s acceptor trans double bond with a di-keto/enol function, thus
also introducing an extra hydroxyl group in the molecule and increasing its polarity (Series c), or with a hydrazine-carboxamide
or hydrazine-carbothioamide (Series d); 4. replace the hydrazone linker with either a urea function (Series e), or a double-
hydrazide (Series f); 5. include the hydrazone linker into an oxadiazole ring (Series g), while also exploring three different
spacers in the new molecular scaffold, the original trans double bond, a saturated ethylydene group (two-carbon spacer) or a
shorter methylene function (one-carbon spacer); 6. include the hydrazone linker into a triazole ring (Series h); 7. condense the
hydrazone linker and aromatic ring B in a terminal benzimidazole group (Series i), while maintaining the unsaturated double bond
or reducing it to a saturated ethylic spacer.
2

Series a
Series i
H
ACCEPTED MANUSCRIPT
X
R1
O
A
N
A
NH
O
N
HN
N
R3
B
B
R3
R2
X
X
= trans CH=CH
= CH2CH2
Series b
R1= CF3, OMe, i-Pr or Ph
O
Y
X
Series h
A
HN
N
R3
N
N
B
B
R2
N
b.1: X= (CH=CH), Y= (CH)
A
b.2: X
b.3: X
b.4: X
Y
= (N=CH), = (CH)
Y
= (NH), = (N)
= (CH=CH), = (N)
Previous hit scaffold
Y
Series g
b.5: X= (NH), Y= (CH)
R1
O
A
R3
Series c
X
O
B
O
A
HN
N
R3
N
A
N
O
B
R2
g.1: X= trans CH=CH
HN
N
R3
g.2: X
= CH2CH2
= CH2
1 R1= t-Bu, R2= H, R3= 3,4-OEt
B
g.3:X
R2
Series f
Series d
R2
R3
B
O
R3
H
N
B
N
H
HN
Y
N
A
O
HN
X
R1
A
Series e
R1
A
d.1: X= trans CH=CH,Y= O
d.2: X
NH
O
Y
= CH2CH2, = S
NH
R3
B
A
R1
NH
NH
O
R3
B
Figure 1. Structure of our previous antiviral hit and the different modifications carried out in the present study.
With the aim to take into account and to further confirm our previously reported structure-activity considerations,8-9 Series a was
designed to explore different hydrophobic substituents, other than a tert-butyl, a methyl or unsubstituted, in the para position of
aromatic ring A, while for all the other series of derivatives either a para tert-butyl or an unsubstituted ring A was maintained.
The substituents for ring B were instead chosen based on previous extensive modifications on this second phenyl group,
maintaining those substitutions that were found to be associated with a better antiviral profile.8-9 As depicted in Figure 2, Series
d.2 was envisaged in order to combine the structural features of our previous hit 1 with the left hand side portion of 2, a
structurally related in-house compound that, due to its similarities with 1, has been chosen for evaluation as potential anti-CHIKV
agent (Table 1). As reported in Table 1 and highlighted in Figure 2, the antiviral effect of 2 seems to be mainly associated with a
cytotoxic effect against the cells, possibly due to the presence of a rigid 2-tolyl-indole moiety on the right hand side of the
structure.
3

ACCEPTED MANUSCRIPT
Figure 2. Rational design of novel potential antiviral agents belonging to Series d.2.
2.2 Chemistry
All target compounds were synthesised according to an optimised two to four-step synthetic pathway, as shown in Schemes 1-5.
Scheme 1: Reagents and conditions: A: i. malonic acid, cat. piperidine, pyridine, r.t., 3h (80-92%); ii. oxalyl chloride, Et2O, r.t., 5 h (91-99%); iii. hydrazine
monohydrate, EtOH, reflux, 3h (76-94%); iv. NEt3, THF, r.t., o.n. (55-74%); B: i. (a) oxalyl chloride, Et2O, r.t., 3 h, (b) hydrazine monohydrate, r.t., 2 h (35%); vi.
EtOH, r.t., 72 h (42%).
A series of 12 novel benzylidene-acrylohydrazides was prepared to further explore the effect of the aromatic substituent in the
para position of aromatic ring A, by replacing the original tert-butyl with an isopropyl, trifluoromethyl, methoxy and phenyl
groups, respectively. Cinnamic acids 3a-d were prepared following a Knoevenagel condensation- Doebner modification reaction
between aldehydes 2a-d and malonic acid, in the presence of catalytic amounts of piperidine and refluxing the mixture in
pyridine.10 Acid intermediates 3a-d were then converted into the corresponding acyl chlorides 4a-d, using oxalyl chloride.11
Hydrazone intermediates 8-10 were prepared by refluxing aldehydes 5-7 with hydrazine monohydrate in ethanol, and these
hydrazones were finally reacted with acyl chlorides 4a-d at r.t. in THF, in the presence of triethylamine, thus yielding final
desired hydrazones 11a-d, 12a-d and 13a-d. This synthetic approach was also applied for the preparation of final hydrazones
bearing a para hydroxyl in ring B, as this modification had previously been associated with an interesting antiviral profile in the
original hit scaffold.8 While the preparation of the intermediate hydrazine with the 4-OH group was successful (data not shown),
the last step for the formation of the hydrazide bond did not work with this substituent in ring B, as a complex mixture of multiple
by-products was obtained, from which it was not possible to isolate the desired final compound. This might be due to the presence
of triethylamine in the reaction mixture, which might deprotonate the 4-hydroxyl group of the hydrazone intermediate, causing the
formation of a nucleophilic centre that could react with the acryloyl chloride, thus preventing the desired reaction to take place. In
order to obtain at least one final product carrying a 4-hydroxy group on aromatic ring B (16), an alternative synthetic strategy was
applied. As we had previously reported for the synthesis of 1,8 cinnamic acid 3a was converted into the corresponding chloride
using oxalyl chloride in DCM, and then treated in situ with hydrazine monohydrate to give acrylo-hydrazide 14,12 which was
4

finally reacted with 4-hydroxybenzaldehyde 15 to give the target compound 16.
Series b
ACCEPTED MANUSCRIPT
O
O
O
Y
X
Y
X
O
Y
O
Y
X
i
ii
iii
OH
R
X
HN NH2
HN
N
R3
17a-e
18a-e
19a-e
R2
20a-e
a
b
c
d
e
R2= H, Ar= 3,4-OEt
21a-e R2= H, Ar= 4-Me
22a-e
X= (CH=CH), Y= (CH)
X= (N=CH), Y= (CH)
X= (CH=CH), Y= (N)
X= (NH), Y= (CH)
X= (NH), Y= (N)
R2= H, Ar= 4-OH
23a-e R2= Me, R3= 4-C6H11
Series c
O
O
O
O
O
O
O
R3
N
NH2
i
ii
iii
N
OEt
N
H
H
R2
24
25
26
27
28
29
R2= H, Ar= 3,4-OEt
R2= H, Ar= 4-Me
R2= H, Ar= 4-OH
Series d
d.1
O
O
O
N
C
O
i
ii
iii
N3
OH
30
31
32
33
R2
NH2
iv
N
R3
8-10, 34
R2
H
H
N
N
N
R3
O
35
R2= H, Ar= 3,4-OEt
36 R2= H, Ar= 4-Me
37
R2
R2= H, Ar= 4-OH
38 R2= Me, Ar= 4-C6H11
NH2
N
R3
d.2
R2
H
H
8-10, 34
N
NH2
N
N
i
C
N
S
R3
S
ii
40
39
41
42
R2= H, Ar= 3,4-OEt
R2= H, Ar= 4-Me
43 R2= H, Ar= 4-OH
44
R2= Me, Ar= 4-C6H11
Scheme 2: Reagents and conditions: Series b: i. HCl, MeOH, reflux, o.n. (75-88%) or SOCl2, EtOH, reflux, 24h (82-84%); ii. hydrazine monohydrate, EtOH,
reflux, o.n. (79-87%); ii. differently substituted benzaldehyde, EtOH, reflux, 24 h (65-83%). Series c: i. diethyl oxalate, EtONa, EtOH, 0 °C, 30 min, r.t., 24 h
(55%); ii. hydrazine monohydrate, EtOH, reflux, o.n. (56%); iii. differently substituted benzaldehyde, EtOH, reflux, 24 h (63-87%). Series d.1: i. malonic acid, cat.
piperidine, pyridine, r.t., 3h (87%); ii. (a) ethyl chloroformate, Et2O, 0 °C, 1h, (b) NaN3, H2O, 0 °C, 7h (95%); iii. PhMe, 120 °C, 4h; iv. r.t., o.n. (37-50%). Series
d.2: i. CSCl2, NEt3, THF, 0 °C, 30 min, r.t., 30 min (100%); ii. PhMe, r.t., o.n. (35-44%).
A first attempt to modify the Michael’s acceptor in the structure of 1 was its insertion into a condensed aromatic or hetero-
aromatic ring (Series b). In particular, along with a naphthalene core, the hetero-aromatic moieties of 3- and 1-quinoline, 2-indole
and benzimidazole were chosen as a means to partially increase the polarity and water solubility of the structure at the same time.
A series of 20 final compounds was prepared with these modifications by converting starting acids 17a-e into their methyl or
ethyl ester counterparts, by reflux in 1.25M HCl in methanol (18a-d) or by reflux in EtOH in the presence of SOCl2 (18e), and
subsequently treating intermediate esters 18a-e with hydrazine monohydrate in boiling EtOH to achieve the desired ester
displacement in hydrazides 19a-e.13 Final compounds 20-23a-e were obtained by reacting hydrazides 19a-e with the appropriately
substituted benzaldehyde or acetophenone in refluxing EtOH for 24 hours.
A small series of 3 final products (Series c) was envisaged to mask the trans double bond in 1 with an extra carbonyl group in β-
position to the hydrazide carbonyl. As this β-di-keto group is expected to be in equilibrium with its enolic form, this modification
should preserve the rigid geometry of the molecule, previously found important for the antiviral activity of 1,8 while increasing its
polarity with the introduction of an extra hydroxyl group. To carry out this desired molecular alteration, acetophenone 24 was
converted into di-keto ester 25 according to a Claisen condensation,14 by treating it with diethyloxalate in EtOH, in the presence
of sodium ethoxide. Ester 25 was then converted into the corresponding hydrazide 26 as described above for Series b, and this
intermediate was reacted with the appropriate benzaldehyde to give final products 27-29.
5

A further modification of the original linker of 1 was envisaged to insert an extra nitrogen between the trans double bond and the
carbonyl group, thus obtaining an hydrazine-carboxamide spacer in the final products 35-38 (Series d.1). This alteration should
ACCEPTED MANUSCRIPT
alter the nature and the reactivity of the Michael’s acceptor and increase the polarity of the final molecules; moreover, the linker
length would be increased of one atomic position in comparison with 1 and, while the effect of shortening the spacer between the
two phenylic portions of the molecule has already been explored,9 the effect of an increased molecular size on the antiviral
activity of the scaffold had not been evaluated so far. To obtain this modification, substituted cinnamic acid 31 was prepared
through a Knoevenagel condensation between benzaldehyde 30 and malonic acid,15 followed by conversion of this acid into the
corresponding anhydride using ethyl chloroformate in the presence of triethylamine.16 The anhydride was not isolated, but treated
in situ with sodium azide in water, giving desired intermediate 32. Isocyanate 33, which was not isolated, was prepared through a
Curtius rearrangement by refluxing azide 32 in toluene for 4 hours.17 The reaction mixture was allowed to cool to r.t., and then the
appropriate hydrazine 8-10 or 34 was added, thus giving the final desired products 35-38.
A final modification of the original Michael’s acceptor group was designed, as described above, to combine the scaffold of 1 with
hit 2, a potential in-house antiviral candidate with toxicity issues possibly related with the presence of a tolyl-naphthyl substituent
on its hydrazine-thioamide group (Series d.2). Final products 41-44 were obtained by converting phenylethylamine 39 into the
corresponding isothiocyanate 40, using thiophosgene in THF in the presence of triethylamine. Intermediate 40 was then treated
with the appropriate benzaldehyde or acetophenone in toluene at r.t. to afford the target compounds 41-44 in moderate yields.
Scheme 3: Reagents and conditions: Series e: i. (a) ethyl chloroformate, Et2O, 0 °C, 1h, (b) NaN3, H2O, 0 °C, 7h (70-95%); ii. PhMe, 120 °C, 4h; iii. r.t., o.n. (39-
47%); iv. r.t., o.n. (42-55%). Series f: i. H2SO4, EtOH, reflux, o.n. (85-90%); ii. hydrazine monohydrate, EtOH, reflux, o.n. (78-86%); iii. oxalyl chloride, DCM, 0
°C to r.t., 5h. (99%); iv. NEt3, THF, 0 °C to r.t., o.n. (25-50%).
A first attempt to replace the hydrazone bond in 1 with a more stable group in aqueous conditions was made by inserting a urea
function as spacer between the trans double bond and aromatic ring B (Series e). This alteration was envisaged also as a means to
mitigate the reactivity of the Michael’s acceptor, and it was carried out both with a methylene spacer between the urea and ring B
(51-55), thus maintaining the same overall length of the scaffold, and with the urea feature directly linked to ring B (58-61), to
further assess the importance of the linker length for antiviral activity. The desired final compounds were obtained by reacting
isocyanates 33 and 47, not isolated but obtained in situ as described above for Scheme 2, with the corresponding benzylamine 48-
50 or aniline 56-57, at room temperature in toluene.
The hydrazone bond was also replaced with a more water stable double hydrazide in compounds 69-70, while maintaining the
original overall length of the spacer between the two aromatic portions of the molecule (Series f). This modification was achieved
by reacting the appropriate hydrazide 66-67, obtained by hydrazynolysis of the intermediate esters 64-65, with acyl chloride 68,
isolated from the reaction of substituted cinnamic acid 31 with oxalyl chloride.
6

Series g.1
N
ACCEPTED MANUSCRIPT
O
O
N
R3
i
NH2
O
OH
N
+
R3
H
31
66 R3= 3,4-OEt
67 R3= 4-Me
R = 3,4-OEt
71
72
3
R = 4-Me
3
Series g.2
O
O
O
N
N
R3
NH2
i
iii
ii
N
OH
OH
O
H
O
R1
R1
R1
R1
OH
R3
75 R1= t-Bu
76 R1= H
31 R1= t-Bu
45 R1= H
73 R1= t-Bu
74 R1= H
77 R1= t-Bu, R3= 3,4-OEt
78 R1= t-Bu, R3= 4-Me
79 R1= H, R3= 3,4-OEt
80 R1= H, R3= 4-Me
62-63
O
iii
NH2
N
H
O
OH
75
N
N
HO
81
OH
O
iv
82
O
O
OH
O
O
83
vi
N
NH2
N
H
iii
75
O
O
N
v
iii
OH
O
OH
O
O
75
HO
81
84
85
Series g.3
H
O
N
ii
62-63
i
N
N
NH2
R3
O
O
O
88 R3= 3,4-OEt
89 R3= 4-Me
86
87
Series h
N3
Br
i
ii
N
N
CN
N
93
90
91
92
Scheme 4: Reagents and conditions: Series g.1: i. POCl3, reflux, o.n. (52-69%). Series g.2: i. 10% Pd/C, H2, THF, r.t., 4h (93-98%); ii. a) HCl, MeOH, reflux, o.n.
(89-93%); b) hydrazine monohydrate, EtOH, reflux, o.n. (80-88%); iii. POCl3, reflux, o.n. (63-75%); iv. acetic anhydride, H2SO4, 80 °C, 2h (95%); v. benzyl
bromide, KOH, 9:1 EtOH: H2O, reflux, 20h (99%); vi. 10% Pd/C, H2, MeOH/THF, o.n. (51%). Series g.3: i. hydrazine monohydrate, EtOH, reflux, o.n. (88%); ii.
POCl3, reflux, o.n. (72-74%). Series h: i. NaN3, DMF, 80°C, 24h (73%); ii. sodium ascorbate, CuSO4, H2O, DCM, r.t., 24h (37%).
The desired inclusion of the hydrazone bond of 1 in an oxadiazole ring was achieved by refluxing substituted cinnamic acid 31
with hydrazides 66-67 in POCl3, obtaining final products 71-72 with a trans double bond between aromatic ring A and the newly
introduced oxadiazole (Series g.1). As this modification is associated with a significant extra rigidity in the final structure, the
contemporary reduction of the trans double bond was also envisaged in final products 77-80, in which the extra rigidity of the
oxadiazole ring is compensated with the increased flexibility of the aliphatic linker (Series g.2). This double modification was
achieved by reducing cinnamic acids 31 and 45 under catalytic hydrogenation conditions. Reduced acids 73-74 were then
converted to their methyl ester derivatives and then displaced with hydrazine monohydrate to give hydrazides 75-76, which were
finally reacted with acids 62-63 in refluxing phosphorus oxychloride to give the desired final products 77-80. One final product in
this series was designed with a 4-hydroxy function on aromatic ring A (82), however when refluxing 75 with 81 in phosphorus
oxychloride, no trace of the desired product 82 could be observed, while a mixture of different impurities was formed. The reason
for this undesired reactivity might rely on the interference of the free phenolic group with the reaction mechanism, via a potential
7

interaction with the carbonyl group of the hydrazide 75. The free hydroxyl group of 81 was therefore acetylated into intermediate
83, but also in this case the desired product was not obtained and the formation of a mixture of impurities was observed. The
ACCEPTED MANUSCRIPT
desired product 82 was finally obtained by protection of the free hydroxyl in 81 to benzyl ether in 84, which was reacted with
hydrazide 75 giving the desired intermediate 85 with moderate yield (52%). 85 was finally deprotected into the target molecule 82
by catalytic hydrogenation. A small series of two novel products, 88-89, was obtained, following the same strategy, with a shorter
aliphatic spacer of one methylene group between the oxadiazole and aromatic ring A, by converting methyl ester 86 into the
corresponding hydrazide 87, and then refluxing this hydrazide in POCl3 in the presence of benzoic acid 62 or 63 (Series g.3).
Lastly, one final product with a triazole ring replacing the hydrazone bond (93, Series h) was obtained starting from 2-
bromoethylbenzene 90, which was treated with sodium azide to achieve the nucleophilic displacement of the bromide leaving
group by the azide in intermediate 91.18 Intermediate azide 91 was then reacted with nitrile 92 according to a variation of Huisgen
1,3-dipolar cycloaddition, in which 1,4-disubstituted 1,2,3-triazole are formed in a region-specific manner by a copper(I)-
catalysed reaction.19-20 The catalyst is formed in situ by reduction of the Cu(II) salt (CuSO4), using sodium ascorbate as the
reducing agent.
Series h
R3
O
N
ii
N
N
H
O
H
OH
98 R3= Me
99 R3= H
R3
NH2
NH2
R3
i
N
NH2
31
N
H
R3
96 R3= Me
97 R3= H
94 R3= Me
95 R3= H
O
N
ii
N
N
O
H
H
OH
100 R3= Me
101 R3= H
73
Scheme 5: Reagents and conditions: i. BrCN, 1:1 H2O:MeOH, 50 °C, 1h (87-89%); ii. TBTU, DiPEA, THF, r.t., o.n. (35-53%).
One last attempt to replace the hydrazone bond in the original scaffold was made by condensing it with aromatic ring B in a
benzimidazole heterocycle (Series h). This last modification was carried out both maintaining the trans double bond feature in the
linker (98-99), and reducing it to a saturated ethylic spacer (100-101), in order to compensate for the higher rigidity of the new
moiety in comparison with the original phenyl-hydrazone. A small series of 4 final products was obtained by reacting ortho
phenylendiamines 94 and 95 with cyanogen bromide at 50 °C in a water-methanol mixture, thus isolating amino-benzimidazoles
96 and 97. The final target products were then obtained with a coupling reaction between aromatic amines 96-97 and the
appropriate acid 31 or 73, using TBTU as coupling reagent in the presence of diisopropyethylamine.21
2.3 Antiviral and cytotoxicity studies
The newly prepared 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.22
Table 1: Antiviral effect of the test compounds on CHIKV replication in Vero cells and cytotoxicity.
Comp.
Scaffold
R1
R2
R3
SIe
Max.
inhib.f
100%
100%
30%
75%
8%
47%
Max at
[µM]f
12.5
2.5
8
69
EC50(µM)a,d
EC90(µM)b,d
CC50(µM)c,d
(ring A)
t-Bu
-
i-Pr
CF3
OMe
Ph
(ring B)
3,4-OEt
-
3,4-OEt
3,4-OEt
3,4-OEt
3,4-OEt
1g
2
11a
11b
11c
11d
a
-
a
a
a
a
H
-
H
H
H
H
3.2±1.8
1.8±0.9
>29.2
48.9±1.2
>271
11±4
2.5±0.8
>29.2
n.d.
>271
>80.2
101±50
7.8±0.3
n.d.
84.1±16.8
n.d.
32
4.4
-
1.7
-
45
120
>80.2
n.d.
-
8

12a
12b
12c
12d
13a
13b
13c
13d
16
20a
20b
20c
20d
20e
21a
21b
21c
21d
21e
22a
22b
22c
22d
22e
23a
23b
23c
23d
23e
a
a
a
a
a
a
a
a
i-Pr
CF3
OMe
Ph
i-Pr
CF3
OMe
Ph
i-Pr
-
-
-
-
H
H
H
H
Me
Me
Me
Me
H
H
H
H
H
4-Me
4-Me
4-Me
25.9
96.3±16.1
n.d.
>150
25±5.9
203±41
1
2.1
-
>1.3
-
-
-
-
-
-
-
-
-
-
50%
61%
13%
78%
13%
12%
9%
15%
46%
24%
3%
20
99
ACCEPTED MANUSCRIPT
>112
230
>112
>294
>193
>241
>87.7
>29.6
>324
>276
>275
>275
>213
>284
>347
>346
>346
>361
>120
>344
>343
>343
>358
>357
>270
>202
>269
>209
n.d.
n.d.
>294
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
6.2±0.04
112
294
193
241
66
4-Me
4-C6H11
4-C6H11
4-C6H11
4-C6H11
4-OH
3,4-OEt
3,4-OEt
3,4-OEt
3,4-OEt
3,4-OEt
4-Me
4-Me
4-Me
4-Me
4-Me
4-OH
4-OH
4-OH
4-OH
>193
>241
>87.7
>29.6
>324
>276
>275
>275
>213
>284
>347
>346
>346
>361
>120
>344
>343
>343
348
30
36
a
b.1
b.2
b.3
b.4
b.5
b.1
b.2
b.3
b.4
b.5
b.1
b.2
b.3
b.4
b.5
b.1
b.2
b.3
b.4
b.5
276
275
275
213
284
347
346
346
361
120
344
343
343
358
357
270
202
269
209
4.3
8%
50%
6%
-
H
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
H
H
H
H
H
H
H
H
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.4
10%
27%
3%
45%
14%
2%
<1%
<1%
51%
33%
14%
19%
13%
41%
83%
H
H
4-OH
>357
>270
>202
>269
>209
2.6
Me
Me
Me
Me
Me
4-C6H11
4-C6H11
4-C6H11
4-C6H11
4-C6H11
Comp.
Scaffold
R1
(ring A)
R2
R3
SIe
Max.
inhib.f
4%
Max at
[µM]f
244
74
EC50(µM)a,d
EC90(µM)b,d
CC50(µM)c,d
(ring B)
3,4-OEt
4-Me
4-OH
3,4-OEt
4-Me
4-OH
C6H11
3,4-OEt
4-Me
4-OH
C6H11
3,4-OMe
4-Me
27
28
29
35
36
37
38
41
42
43
44
51
52
53
54
55
58
59
60
61
69
70
c
c
c
-
-
-
-
-
-
-
-
H
H
H
H
H
H
Me
H
H
H
Me
-
-
-
-
-
-
-
-
-
>244
>74
7.2±1.3
>100
246±52.1
5.6±0.4
42.3±15.7
5.9±1.1
>336
48.3±19.9
4.3±0.5
11.6
>103
>107
>320
>376
>61
9.1
>396
>393
>244
>74
8
>100
>298
7.5
81±53
84±54
14.7±3.3
n.d.
192±38.7
10.4±2.3
123±34.7
109±29.5
n.d.
87.8±44
52.9±11.6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
-
-
2.3
-
0.8
1.9
2.9
18
13%
87%
49%
50%
89%
90%
90%
12%
67%
90%
100%
10%
8%
13%
18%
16%
100%
21%
2%
7.9
d.1
d.1
d.1
d.1
d.2
d.2
d.2
d.2
e.1
e.1
e.1
e.1
e.1
e.2
e.2
e.2
e.2
f
100
187
7.4
79
9.3
56
51
6
17
103
107
320
376
61
78.8
9.8±2.8
>336
n.d.
5.9±0.5
14.8
>103
>107
>320
>376
>61
-
-
-
-
1.8
12.2
t-Bu
t-Bu
t-Bu
H
-
-
-
-
-
-
-
-
-
-
-
H
3,4-OMe
4-Me
4-Me
4-OH
4-Me
4-OH
3,4-OEt
4-Me
H
t-Bu
t-Bu
H
H
-
11.9
4.1
>396
>393
>244
>99.1
396
197
6.3
-
-
>244
>99.1
11%
9%
f
-
99.1
9

ACCEPTED MANUSCRIPT
Comp.
Scaffold
R1
(ring A)
R2
R3
SIe
Max.
inhib.f
100%
8%
61%
100%
4%
Max at
[µM]f
127.4
157
13.5
26
EC50(µM)a,d
EC90(µM)b,d
CC50(µM)c,d
(ring B)
3,4-OEt
4-Me
3,4-OEt
4-Me
3,4-OEt
4-Me
4-OH
3,4-OEt
4-Me
-
71
72
77
78
79
80
82
88
89
93
98
99
g.1
g.1
g.2
g.2
g.2
g.2
g.2
g.3
g.3
h
i.1
i.1
i.2
i.2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
29
88.2
>157
n.d.
n.d.
n.d.
15.5±3.1
n.d.
n.d.
n.d.
191±47.1
n.d.
n.d.
200±47.5
n.d.
-
-
1.2
-
-
-
1.8
-
-
1.6
-
-
-
-
8.1
>157
12.4±2.1
17.2
>98.4
>378
108±31.7
39.7
t-Bu
t-Bu
H
H
21.7
>98.4
>378
>103
n.d.
22.7
n.d.
>300
>157
>298
>311
21±18
73.9
1
2%
t-Bu
69%
15%
100%
74%
47%
38%
8%
103
131.4
27.2
163
300
157
1.6
-
-
-
-
-
-
-
-
17.9
122±15
>300
>157
>298
>311
11±7
Me
H
Me
H
n.d.
n.d.
n.d.
89±28
100
101
Chloroquine
41%
-
311
-
-
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).
f Maximum observed inhibition of the virus-induced CPE and concentration (µM) at which maximum inhibition is reached.
g 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 presence of a para
tert-butyl substituent in the original scaffold of 1, as partially observed previously,8 appears to be important for antiviral activity,
as its replacement with different groups (iso-propyl, trifluoromethyl, methoxy or phenyl, Series a) in compounds 11-13a-d and 16
is associated with a complete loss of activity. The only derivatives which retain some antiviral effect in the micromolar range are
11b, 12a and 12b, but for these compounds the selectivity index SI is low, indicating that their antiviral activity is associated with
a toxic effect on the cells. Also the attempted enclosure of the trans double bond of 1 into a condensed aromatic or heteroaromatic
ring in 20-23a-e (Series b) is associated with a complete abolishment of antiviral effect, even if these modifications are not
associated with cytotoxicity. The only analogue in this series which still retains some activity is 23e, but also in this case the
antiviral effect of the molecule appears to be correlated to cytotoxicity. The attempted replacement of the Michael’s acceptor with
a β-di-carbonyl group in 27-29 (Series c) is associated with loss of antiviral activity and increased cytotoxicity for 27 and 28,
while 29 still retains some antiviral potential, but its toxic effect is increased in comparison with 1, and its SI value low.
Replacement of the hydrazide spacer with a hydrazine-carboxamide (Series d.1) is associated with a complete loss of activity for
35 and 36, while 37 and, to less extent, 38 still maintain antiviral EC50 values in the low micromolar (37) or micromolar (38)
range, but they are also associated with increased cytotoxicity. A more positive effect is observed for the combination of the
structures of 1 and 2 (Series d.2), as analogues 41 and 44 in this series are associated with retained antiviral activity in
comparison with 1, even if with lower SI values, and reduced cytotoxicity in comparison with 2. Replacement of the hydrazone
spacer with a urea function while maintaining the original length of the molecule (Series e.1) is associated with antiviral activitity
retention in analogue 51, but this modification appears to be tolerated only with the concomitant presence of a 3,4-dimethoxy
substituent on aromatic ring B, as the other analogues in this series (52-53) do not show any residual antiviral effect. In the case of
the novel urea analogues with a shorter linker (Series e.2), derivative 59 is associated with activity retention in comparison with 1,
but also in this case, the other analogues belonging to this series (58, 60-61) are associated with loss of antiviral activity.
Conversion of the hydrazide linker to a more stable double hydrazide in 69 and 70 (Series f) is associated with complete loss of
antiviral activity. Inclusion of the hydrazone bond in an oxadiazole ring (Series g.1, g.2 and g.3) appears to be tolerated in all
three sub-series of structures (with a trans-double bond, saturated ethylene and shorter methylene spacer, respectively), but with a
different effect found for the substituent in aromatic ring B: in Series g.1, a 3,4-diethoxy feature is associated with activity
retention (71), while the 4-methil analogue 72 is inactive; in Series g.2, while the presence of the tert-butyl substituent in ring A is
10

further confirmed as important for activity retention, a 4-methyl group in ring B is associated with antiviral activity retention (78),
while the presence of a 3,4-diethoxy substituent is linked to increased cytotoxicity; in Series g.3, 89 (4-methyl group in ring B)
ACCEPTED MANUSCRIPT
retains the original antiviral activity without showing relevant cytotoxicity, while a 3,4-diethoxy feature in ring B is correlated
with loss of antiviral effect. Finally, inclusion of the hydrazone group in a triazole ring in 93 (Series h) is associated with partial
antiviral activity retention, even if at high concentrations, while the attempted condensation of the hydrazone group with ring B in
a benzimidazole ring in 98-101 (Series i.1 and i.2) is associated with a complete loss of antiviral activity.
2.4 Molecular docking studies on CHIKV nsP2 protease
As hit 1 was identified as selective antiviral agents against CHIKV following docking-based studies on the catalytic pocket of the
CHIKV nsP2 protease, and a recent study has shown that three of our previously reported close analogues of 1 inhibit the
protheolitical activity of CHIKV nsP2 in vitro,23 the predicted binding to the nsP2 protease active site was investigated for the
novel compounds prepared, aiming to identify a rational explanation for the differences in activity found for the new structures.
The Glide SP docking algorithm was used.24-25 The predicted binding to the nsP2 protease found for original hit 1 and
representative novel analogues of each series explored in this work is shown in Figure 3.
1
20a
20e
23e
27
29
37
41
51
59
69
71
78
89
93
11

ACCEPTED MANUSCRIPT
98
Figure 3. Predicted binding mode of 1a and representative analogues of each series of novel compounds to the proteolitic site of CHIKV nsP2 (PDB ID 3TRK).
According to our molecular docking studies summarised in Figure 3, the hydrazone bond of 1 fits the central part of the
proteolytic site of the enzyme and is involved, through its carbonyl oxygen, in a direct hydrogen bond with the side chain of
Trp650, which forms the enzyme catalytic triad together with Cys579 and His649. The two aromatic portions, even though not
involved in direct interactions with the surrounding enzyme residues, appear important to anchor the central part of the molecule
in the region defined by the catalytic triad. Even if, as expected, a very similar predicted binding mode is shared by all the novel
analogues of Series a (11-13a-d, 16, data not shown), in which different para aromatic substituents have been explored for ring
A, the presence of a tert-butyl in this position appears to be key to an optimal fitting of the catalytic pocket, being all the
attempted modifications associated with loss of antiviral activity. The predicted binding for Series b, where the trans double bond
of the Michael’s acceptor is included in a condensed aromatic ring, reveals for most of the derivatives in this series, exemplified in
Figure 3 by 20a and 20e, an altered occupation of the target site, where the space optimally fitted by ring B of 1a is not
completely reached and the hydrogen bond with Trp650 is lost. These observations seem to be in line with the loss of antiviral
activity found for this series. An exception to this trend is represented by 23e, for which the presence of a para-cyclohexyl
substituent on ring B combined with a condensed benzimidazole as ring A appears to be associated with a conserved overall
fitting of the target site, and this observation might explain the residual activity found for this analogue (Table 1). As represented
by the docking results found for 27, the replacement of the Michael’s acceptor with a β-dicarbonyl group in Series c is associated
with a perturbed occupation of the catalytic pocket, with ring A shifted towards the catalytic triad and an incomplete fitting of the
spatial portion on the left hand side of Cys579. This effect appears to be compensated by the presence of a 4-hydroxy substituent
in ring B, which is expected to stabilise the same occupation of the target site found for 1a by forming an extra hydrogen bond
with the backbone of Gln807, thus providing a rational explanation to its residual antiviral effect. The same observation can be
made for Series d.1, where the perturbed fitting of the pocket due to the presence of a longer linker is compensated for 37 by the
formation of an extra hydrogen bond with Gln807. In Series d.2, the presence of the hydrazine-carbothioamide linker associated
with a saturated ethylene spacer and an unsubstituted ring A appears to be associated with a much less consistent occupation of
the target pocket, due to the extra flexibility in the structure, which can be compensated by a retained hydrogen bond with Trp650
and the presence of a bulky substituent (41) in ring B, which still allows to anchor the molecule on the sub-pocket on the right
hand side of the catalytic triad, thus explaining the retention of antiviral activity. The same effect of a bulky ring B substituent can
be observed in fact for 44 (data not shown). This same observation of the necessity of a bulky substituent in ring B for binding and
activity retention, can be extended to Series e.1, where the concomitant presence of a 3,4-dimethoxy group in ring B and a 4-tert-
butyl in ring A appears to be essential to anchor the molecule on the target site, thus preserving the potential for hydrogen bond
formation with Trp650 and the antiviral activity (51). In Series e.2, this effect of stabilisation of the binding mode is achieved
with the presence of a 4-hydroxy substituent (59), which is predicted to form an extra hydrogen bond with the lateral chain of
Gln807. As exemplified by the docking results found for 69, the presence of a double-hydrazide linker in Series f is associated
with a distorted occupation of the target site and the loss of the hydrogen bond with Trp650, thus providing a reasonable
explanation to the loss of antiviral activity found for this series. As can be observed for the predicted binding mode found for 71,
78 and 89, the replacement of the hydrazone linker with an oxadiazole ring in Series g.1, g.2 and g.3, respectively, appears to be
associated with a retained potential occupation of the target site, with the predicted maintenance of the hydrogen bond with
Trp560 and with a similar fitting of the binding pocket for all three sub-series of structures, which are associated, at least for the
three analogues shown in Figure 3, with antiviral activity retention, even if with higher EC50 values in comparison with 1a-b.
Analogue 93 (Series h) is expected to only partially fit the catalytic pocket of the protease, due to the absence of substituent in
12

ring A and to the flexibility of its linker: also in this case, the docking results obtained appear to support the experimental data,
which highlight loss of antiviral activity for this compound. Finally, Series i, as shown in Figure 3 for the predicted binding
ACCEPTED MANUSCRIPT
found for 98, is not expected to allow an optimal, linear occupation of the target pocket, as the hydrazone bond is rigidly
condensed with ring B into a benzimidazole group: this observation is consistent with the biological results, which demonstrate
loss of antiviral activity for this series of novel structures. The correlation between the results obtained with docking simulations
and the experimental data appears to represent a useful validation of the predicted binding mode of these agents to the CHIKV
nsP2 protease, and such docking studies might now be used with relative confidence to make an estimation on the potential
activity of novel analogues, possibly directing future synthetic efforts.
3. Conclusions
Starting from the structures of two anti-CHIKV hits, several series of novel analogues were designed and 69 new derivatives were
synthesised to expand the structure-activity relationships associated with these antiviral agents, and to improve their drug-like and
water-stability properties. Different structural elements were confirmed as essential for antiviral properties, such as the presence
of a tert-butyl substituent on ring B, the successful replacement of the Michael’s acceptor present in the original hit structures was
achieved by the insertion of a hydrazine-carbothioamide (41, 44), and a viable replacement of the original hydrazone linker has
been achieved with a urea (51, 59) or an oxadiazole linker (71, 78, 89). The differences in biological activities found for the
various series of novel analogues were rationally explained and correlated with the predicted binding to the CHIKV nsP2 protease
with a series of molecular docking studies, and these considerations might now be used to direct future synthetic efforts. Different
novel antiviral agents with anti-CHIKV EC50 values in the low micromolar range were identified, and these findings provide the
foundation for further investigation of these new structures as antivirals against Chikungunya virus. Further structural
improvements towards the identification of preclinical antiviral candidates are the focus of present investigations and will be
reported in due course, along with the confirmation of the mechanism of action of these compounds.
4. Experimental
4.1 Synthetic chemistry 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. All final compounds were more than 95% pure.
Details for the preparation and full characterisation of the new target final compounds are described in the Supporting
Information.
All intermediates were generally prepared according to literature procedures, which are described in detail along with compound
characterisation in the Supporting Information.
4.2 Virus cell-based CPE reduction assay
Chikungunya virus, Indian Ocean strain 899, 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.25
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.
13

4.3 Molecular Modelling
ACCEPTED MANUSCRIPT
All molecular modelling 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).26 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
Maestro LigPrep tool27 was used to prepare the ligands using the default settings, whereas the protein was prepared using the
preparation wizard tool.28 The docking simulations were performed using Maestro Glide SP using the default parameters.24
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. M.C. was funded by the European Union Grant EUVIRNA [Grant No.
264286]. L.D. was funded by the Research Foundation of Flanders (FWO). We thank Stijn Delmotte and Tom Bellon for
excellent technical assistance.
Appendix A. Supplementary data
All experimental procedures and compound characterisation data for the reaction intermediates are reported and described in
detail in the Supporting Information.
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