In silico identification, design and synthesis of novel piperazine-based antiviral agents targeting the hepatitis C virus helicase

Article abstract of DOI:10.1016/j.ejmech.2016.10.043

A structure-based virtual screening of commercial compounds was carried out on the HCV NS3 helicase structure, with the aim to identify novel inhibitors of HCV replication. Among a selection of 13 commercial structures, one compound was found to inhibit the subgenomic HCV replicon in the low micromolar range. Different series of new piperazine-based analogues were designed and synthesised, and among them, several novel structures exhibited antiviral activity in the HCV replicon assay. Some of the new compounds were also found to inhibit HCV NS3 helicase function in?vitro, and one directly bound NS3 with a dissociation constant of 570?±?270?nM.

Full text of DOI:10.1016/j.ejmech.2016.10.043

Accepted Manuscript
In silico identification, design and synthesis of novel piperazine-based antiviral agents
targeting the hepatitis C virus helicase
Marcella Bassetto, Pieter Leyssen, Johan Neyts, Mark M. Yerukhimovich, David N.
Frick, Matthew Courtney-Smith, Andrea Brancale
PII:
S0223-5234(16)30905-9
10.1016/j.ejmech.2016.10.043
EJMECH 9008
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 August 2016
Revised Date: 17 October 2016
Accepted Date: 18 October 2016
Please cite this article as: M. Bassetto, P. Leyssen, J. Neyts, M.M. Yerukhimovich, D.N. Frick, M.
Courtney-Smith, A. Brancale, In silico identification, design and synthesis of novel piperazine-based
antiviral agents targeting the hepatitis C virus helicase, European Journal of Medicinal Chemistry (2016),
doi: 10.1016/j.ejmech.2016.10.043.
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ACCEPTED MANUSCRIPT

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In silico identification, design and synthesis of novel
piperazine-based antiviral agents targeting the hepatitis C
virus helicase.
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Marcella Bassettoa , Pieter Leyssenb, Johan Neytsb, Mark M. Yerukhimovichc, David N. Frick ,
Matthew Courtney-Smitha, Andrea Brancalea
aCardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff, King Edward VII Avenue, Cardiff CF103NB, UK
bRega Institute for Medical Research, University of Leuven, Belgium
c
Department of Chemistry & Biochemistry, University of Wisconsin- Milwaukee, Milwaukee, Wisconsin 53211, United States
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Abstract
A structure-based virtual screening of commercial compounds was carried out on the HCV NS3
helicase structure, with the aim to identify novel inhibitors of HCV replication. Among a selection of
13 commercial structures, one compound was found to inhibit the subgenomic HCV replicon in the low
micromolar range. Different series of new piperazine-based analogues were designed and synthesized,
and among them, several novel structures exhibited antiviral activity in the HCV replicon assay. Some
of the new compounds were also found to inhibit HCV NS3 helicase function in vitro, and one directly
bound NS3 with a dissociation constant of 570 ± 270 nM.
Highlights
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Virtual screening studies on the HCV NS3 helicase.
Identification of a substituted piperazine as new anti-HCV scaffold.
Synthesis of different series of novel piperazine-based structures.
New inhibitors of the subgenomic HCV replicon 1b genotype identified.
Novel inhibitors of the HCV NS3 helicase discovered.
Key words
Structure-based virtual screening; HCV NS3-helicase; piperazine derivatives; anti-HCV activity; NS3
helicase inhibitors.
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1. Introduction
The hepatitis C virus (HCV) is a major cause of chronic liver disease because it infects approximately
3% of the global population [1]. HCV infection becomes chronic in 60-85% of patients, who are at
high risk of developing hepatic steatosis, fibrosis, cirrhosis and hepatocellular carcinoma [2, 3]. An
HCV vaccine is currently not available, while the standard of care for many years was a combination of
pegylated interferon (pegIFN) and ribavirin, a therapy that was not specific for HCV, was effective in
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Corresponding author
E-mail address: bassettom@cardiff.ac.uk (Dr. M. Bassetto)
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only 50% of HCV patients, and was associated with many side effects [4]. However, new interferon-
free combinations of direct acting antivirals (DAAs) have revolutionized HCV prognosis and
treatment.
HCV has a ~9,000 nt long single-stranded, positive-sense RNA genome, which encodes six non-
structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) essential for virus replication [5]. The
new FDA approved DAAs are highly potent inhibitors of the NS3 protease [6, 7], the NS5B
polymerase [8], and the NS5A protein [10]. Drugs still in development target other HCV proteins, like
NS4B [9]. All-oral combination therapies with NS5B polymerase, NS3 protease and/or NS5A
inhibitors are now the standard of HCV care, but they are associated with high costs [11, 12], and
resistant mutants have been reported for each approved DAA [13-17]. Due to these limitations, new
therapeutics that will reduce the costs of treatment and avoid the development of resistance are still
needed.
In the search for novel inhibitors of HCV replication, a still underexploited target remains the HCV
NS3 helicase, for which relatively few specific inhibitors have been reported so far, none reaching the
clinical evaluation stage [18]. The HCV helicase is essential for HCV replication [19, 20], and is most
likely needed for ATP-dependent unwinding of double-stranded RNA sequences formed during HCV
replication [21, 22]. An HCV helicase inhibitor was also recently shown to enhance the efficacy of the
latest generation of HCV protease inhibitors [23].
Due to its essential role and relative lack of selective inhibitors under development, the HCV NS3
helicase was chosen as a target for the in silico identification and synthesis of antivirals, through their
potential interference with the known RNA binding cleft.
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2 Results and discussion
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2.1 Molecular modelling
The HCV NS3 helicase occupies the C-terminal portion of the NS3 protein and is formed by three
domains, which define an ATP binding pocket in the cleft separating domain 1 from domain 2, and a
single-stranded nucleic acid binding site at the interface of the three domains (Figure 1) [24].
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Figure 1: NS3 helicase domains and binding sites within the 3KQN crystal structure [25].
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As revealed in several crystal structures available for the enzyme in complex with ssDNA or ssRNA
oligonucleotides [25, 26], the nucleic acid is bound within the closed conformation of the enzyme in a
narrow central space defined by the three main domains (Figure 1). Due to the essential role
demonstrated for Thr269, Arg393, Thr411 and Trp501 [27], computer-based studies were directed to
identify compounds binding the region defined by these residues. In particular, the sub-site defined by
Trp501 and Arg393 in the 3KQN crystal structure [25] was used for the virtual screening of the SPECS
library of commercial compounds [28]. The approximately 450,000 structures available were analysed
with MOE 2014.10 conformational search tool [29]; 500 low-energy conformations were kept for each
input molecule. The main interactions between the target residues and the co-crystallised substrate
were considered to build a pharmacophoric query, and the selection was restricted to five features
(Figure 2).
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Figure 2: Pharmacophoric model built on the 3KQN RNA binding cleft. The model consists of a hydrophobic/aromatic group to
interact with Trp501 (green), a hydrogen-bond acceptor or anion group to interact with Gly255 and Thr269 (orange), a H-bond
donor to target Asp296 (purple), a H-bond donor and acceptor to interact with Thr298 and Ser297 (yellow) and a hydrogen-bond
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acceptor to target Arg393 (blue). Exclusion volumes are hidden for clarity.
The 3,500 molecules matching the search criteria were further analysed with a molecular docking
procedure, using Glide in the standard precision SP mode [30]. The output poses were re-scored with
the Glide extra precision scoring function XP [31], FlexX [32] and Plants ChemPLP [33] scoring
functions. The rescoring results were combined with a consensus scoring procedure, in which for each
scoring function a pose is considered a hit if ranked in the top 25% of the score value range for all the
poses of the database. As a further analysis, the molecules matching this criterion for all three scoring
functions were re-docked with Glide SP [30] in the RNA binding pocket of the 3KQH crystal structure,
which corresponds to the high-affinity open conformation of the enzyme. A final selection of 13
derivatives was made after visual inspection of the docking results. These compounds were purchased
and tested in the HCV replicon assay (Table S1). Among them, 1a (Figure 3) showed an interesting
antiviral profile against HCV replication, with an EC50 value in the low micromolar range (Table 1).
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Cl
O
S
N
H
N
O
H
N
O
N
S
O
1a
Cl
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Figure 3: Structure and predicted binding mode of 1a in the 3KQH crystal structure.
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The predicted binding mode found for 1a indicates good spatial occupation of the target site, with one
aromatic ring in close proximity to Trp501, the piperazine linker filling the central space surrounding
Asn556, and the opportunity of hydrogen-bond formation between one sulfonamido group and Arg393
and Thr411 backbone (Figure 3). Given its potential to inhibit HCV replication, a series of derivatives
of 1a was designed and prepared for further investigations.
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2.2 Chemistry
Compound 1a is characterised by a symmetrical structure, with a central piperazine ring and two
aliphatic linkers connected by a sulfonamide group to two equal aromatic systems. The main
modifications designed included different aromatic substitutions, linker chain variations and disruption
of the molecule symmetry, as summarised in Figure 4.
Cl
O
S
N
H
N
O
H
N
O
N
S
O
1a
Cl
Aromatic
substituents
Piperazine
nucleus
Ar1
X
Y
N
H
N
H
n1
N
N
Y
X
n2
Ar2
Alifatic
linkers
Sulfonamide
group
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Figure 4: Proposed modifications on the scaffold of 1a.
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A first series of new derivatives was designed to explore the effect of the aromatic substituent on the
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original structure, by varying the substitutions on the phenyl rings while keeping the central piperazine
nucleus, maintaining the original three-carbon saturated linker or symmetrically shortening it to a two-
carbon chain. Different commercially available aromatic sulfonyl chlorides (2a-s) and sulphonamide 5
were used to obtain piperazine derivatives 1a-u and 8a-h according to an optimised two-step synthetic
pathway (Scheme 1). A further modification was designed to rigidify the three-carbon linker and study
the importance of the positive charge/H-bond acceptors of the piperazine amine groups, inserting an
amide group in correspondence of piperazine nitrogen atoms, using the scaffold of β-alanine (8) as new
linker for a third series of symmetrical compounds 11a, d, f-h (Scheme 1).
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Scheme 1: Reagents and conditions: a) i. Et3N, an. DCM, 0 °C, 10 min; ii. NaH, DMF, rt, 1h, followed by 1,3-dibromopropane,
0 °C, 10 min; iii. piperazine, NaHCO3, EtOH, reflux, 24h; iv. LiOH, THF, H2O, 80 °C, on. b) i. NaOH, H2O, 35 °C, 2h; ii.
TBTU, HOBt, DiPEA, an. THF, rt, 4h.
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The strategy to obtain amine compounds 1a-s and 8a-h began with the preparation of sulfonamides 6a-
s and 7a-h, all obtained with a nucleophilic displacement of amino alkylbromides 3-4 and the different
sulfonylchlorides. In order to avoid a self-reaction between two molecules of the nucleophile, the
amine group of the alkyl bromides was slowly released by the dropwise addition of triethylamine at 0
°C. 6u was obtained by treating sulfonamide 5 with sodium hydride, followed by the addition of 1,3-
dibromopropane. Final symmetrical products 1a-s, 1u and 8a-h were obtained through the
displacement of the intermediate bromide leaving groups by piperazine, using NaHCO3/ethanol as
base/solvent system and refluxing the reaction mixture for 24 h. Compound 1t, with a carboxylic
function on the aromatic rings, was obtained after basic hydrolysis of ester 1s. Symmetrical amide
products 11a, d, f-h were obtained reacting the different sulfonyl chlorides with β-alanine 9, with the
formation of carboxylic acid intermediates 10a-d, f-h, l, n, which were subsequently used for a
coupling reaction with piperazine, using TBTU as coupling agent. With the purpose to further explore
the role of the aliphatic linker, its elongation was also envisaged: compound 17 with a four-carbon
linker was obtained after optimisation of a four-step synthetic pathway (Scheme 2).
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Scheme 2: Reagents and conditions: (i) Et3N, an. DCM, rt, 7h; (ii) Mesyl chloride, Et3N, an. DCM, rt, 2h; (iii) Piperazine,
NaHCO3, EtOH, reflux, 24h; (iv) TFA, H2O, rt, 30min, followed by 2a, Et3N, an. DCM, 0 °C to rt, 1h.
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The free amine in 4-aminobutanol 12 was first protected with BOC to give 14, and then the hydroxy
function was converted to mesylate ester in 15. Once displaced the mesylate groups with piperazine
(16), the two terminal amine groups were deprotected by hydrolysis of the carbamate ester with
trifluoroacetic acid, thus obtaining an intermediate salt, which was precipitated from the reaction
mixture and directly treated with an excess of 4-chloro-benzenesulfonyl chloride (2a) in basic
conditions to give 17.
The role of the sulfonamide groups was also explored by replacement with amide functions in 20
(Scheme 3a), obtained by reacting acyl chloride 18 with alkyl bromide 3, and then treating
intermediate 19 with piperazine at r.t. in THF. Moreover, an attempt to investigate the importance of
the piperazine central nucleus was made by replacing it with a para-phenylendiamine group in 21,
which maintains the overall length of the original scaffold (Scheme 3b).
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Scheme 3: Reagents and conditions: a) i. Et3N, an. DCM, 0 °C, 10 min; ii. Piperazine, Et3N, an. THF, rt, 48 h; b) i. p-
Phenylendiamine, Et3N, an. THF, rt, 72 h.
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Finally, the role of molecular symmetry for antiviral activity was also taken into consideration, and
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three small series of unsymmetrical compounds were designed and synthesised. All the compounds
prepared followed a rational approach guided by the biological results obtained for the previous series
of structures. In particular, the presence of two aromatic sulfonamide groups was to be maintained,
along with a para hydrophobic substituent in at least one phenyl ring, the central piperazine nucleus
and at least one three-carbon linker (Scheme 4). As a further confirmation of the importance of the
central disubstituted piperazine, derivative 39, in which only half of the original molecule is present,
was prepared by reacting intermediate 6a with piperidine 38, as shown in Scheme 4.
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Scheme 4: Reagents and conditions: a) i. Piperazine, NaHCO3, EtOH, reflux, 24h; ii. 6b-c, l-n, NaHCO3, EtOH, reflux, 24h. b) i.
NaHCO3, EtOH, reflux, 24h. c) i. TBTU, HOBt, DiPEA, an. THF, rt, 4h. d) i. NaHCO3, EtOH, reflux, 24h.
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A first series of unsymmetrical compounds 23-35 was designed to maintain the original scaffold while
introducing different para hydrophobic substituents in the two aromatic rings, combining together the
most successful substitutions previously found (4-chloro, 4-methyl, 4-tert-butyl and 4-trifluoromethyl),
along with the unsubstituted phenyl moiety and the biphenyl one. To achieve this result, the two amine
groups of piperazine had to be functionalised with two different alkyl bromides: first the pure
monosubstitution products were isolated, and then these intermediates were reacted with the second
alkyl bromide. A second small series of unsymmetrical derivatives 36a-b, l was designed to maintain
the same para hydrophobic substituent in the two aromatic rings, while reducing the length of one
linker from three to two methylene groups. Final products 36a-b, l were obtained by reacting mono-
substituted intermediates 22 with ethyl bromides 7a-b, l. A third and final series of unsymmetrical
structures 37a-c, l, n was planned to functionalise one piperazine amine group to amide, thus partially
rigidifying the scaffold, while keeping the overall length of the molecule and the same hydrophobic
substituent in the para position of the two aromatic rings. Mono-substituted intermediates 22a-c, l, n
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were reacted with carboxylic acids 10a-c, l, n, following a TBTU-assisted coupling reaction. Finally,
half-molecule derivative 39 was obtained by refluxing intermediate 6a with piperidine 38 in EtOH for
24 hours, using NaHCO3 as base.
2.3 Biological activity
2.3.1 HCV replicon and cytostatic assay
All the newly synthesised compounds were evaluated for their potential antiviral activity in the Huh5-2
replicon system (Table 1) [34]. The HCV protease inhibitor telaprevir (VX-950) was included as
positive control.
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Table 1: Antiviral effect of the test compounds on hepatitis C virus replication in the Huh5-2 replicon system and inhibition of
NS3 helicase unwinding activity.
SIe
Unwinding
IC50 (µM)f
>1000
n.d.
EC50(µM)a,d
EC90(µM)b,d
CC50(µM)c,d
Compound
1a
1b
1c
13.8±0.531
58.3
-
>182
>104
>197
>92.5
77.4
>13.2
-
>1.8
19.2
-
>10.3
n.d.
1d
1e
>92.5
18.1±1.57
82
>92.5
-
n.d.
61.1
4.4
n.d.
1f
>166
>166
>83.2
49.9
>2
n.d.
1g
1h
1i
>83.2
4.9
>83.2
-
n.d.
-
10
460±430
n.d.
76.9±18.2
8.7±5.03
6.5
173
>182
97.2±17.1
>162
4.9
>2.4
1l
42.9±34.1
11.2
310±190
>1000
630±270
>1000
>1000
n.d.
1m
1n
1o
1p
1q
1r
-
>25
1.2
-
3.9
>172
>172
>172
>172
>203
>207
>160
>176
>182
>95.9
>110
>104
>97.5
>184
>87.3
>87.3
>90.5
>86.6
>87.9
>159
>79.5
40.8
-
42.5±15.3
62±14.4
>207
>172
>4
156
>3.3
>207
-
n.d.
1s
56.2±3.2
>176
-
>2.8
>1000
>1000
n.d.
1t
-
-
1u
8a
8b
8c
39.6±9.03
>95.9
>110
>182
>4.6
-
-
250±110
n.d.
-
-
>104
>104
-
n.d.
8d
8e
>97.5
17.4±5.56
85.8
>97.5
-
n.d.
-
>10.6
n.d.
8f
>87.3
>1
n.d.
8g
8h
11a
11d
11f
11g
11h
>87.3
10.6
-
-
n.d.
-
>8.5
690±350
>1000
n.d.
-
-
-
-
-
-
>159
>159
-
n.d.
>79.5
12±4.24
-
-
-
n.d.
3.4
n.d.
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17
66.7±12.5
>209
164
>173
>2.5
-
790±270
n.d.
20
>209
>209
21
12.1
-
22
1.8
4.1
4.9
4
n.d.
23
19.9±6.07
9.93±3.34
2.21±0.747
10.1±0.799
2.12±0.722
3.74±0.878
3.5±0.664
6.62±2.34
1.3±0.777
26.3±6.25
1.98±0.307
4±2.49
>64
81.5±30.4
48.8±4.8
8.77±1.47
63.7±12.8
4.43±0.845
20.5±6.64
14.6±1.44
28.7±9.07
4.78±1.53
138
n.d.
24
36
n.d.
25
4.96
310±66
n.d.
26
33.5±5.72
6.3
2.1
5.5
4.2
4.3
3.7
5.3
3.3
3.5
5.8
4.9
>3.1
3.1
7.4
-
27
-
n.d.
28
-
n.d.
29
9.44
n.d.
30
13.8±6.02
>1000
90±30
n.d.
31
3.05±1.12
32
-
33
-
6.55±1.3
14±3.4
70.8±13
32.3±7.34
>214
n.d.
34
-
>1000
n.d.
35
12.1±1.36
6.59±2.03
69.2±21
1.7±0.17
9.32±5.44
>202
-
36a
-
n.d.
36b
212
n.d.
36l
-
5.21±1.51
69.1
170±30
>1000
n.d.
37a
25.9±6.32
37b
>202
>202
37c
64.3±2.24
3.84±1.34
3.03±0.226
61.9
173
>191
>3
5.4
22.1
2.3
58.8
-
n.d.
37l
-
-
-
-
-
-
20.8±10.7
66.9±50.2
145
>1000
>1000
n.d.
37n
39
(VX-950)
Primuline
Aurintricarboxylic
Acid
0.8±0.2
47
n.d.
-
-
10±2
0.3± 0.1
-
-
-
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203
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205
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a EC50 = 50% effective concentration (concentration at which 50% inhibition of virus replication is observed).
b EC90 = 90% effective concentration (concentration at which 50% inhibition of virus replication 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 2 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 Concentration needed to reduce rates of helicase-catalysed DNA unwinding by 50%
n.d. Not determined
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Considering the results obtained for 1a-t, most of the antiviral potential is associated with a
hydrophobic substituent in the para position of the two aromatic rings, as can be observed for 1a, 1c, 1l
and 1m. Moreover, moving the original 4-chloro group to position 3 (1i) or 2 (1u) is associated with
activity reduction. The lowest EC50 value is reached when a second phenyl group is placed in the
position 4 of the two aromatics (1n), although this modification also shows an increased cytotoxic
effect. Compounds 1e and 1h, with a para-nitrophenyl and 2-naphthyl aromatic moieties respectively,
are associated with an increased toxic effect in comparison with the other analogues in this series, while
the removal of the para-substituent or its replacement with methoxy groups leads to loss of activity
(1b, d, f, g). Replacement of the original phenyl rings with heteroaromatic moieties in 1p-r leads as
well to loss of activity, and the same effect can be observed for the substitution of the 2-naphthyl group
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(1h) with a 1-naphthyl in 1o: even though cytotoxicity is reduced, any activity against the viral
replication is lost. Trying to mimic the effect of the 4-nitro group of 1e, a 4-carboxylate function was
introduced as aromatic substituent in 1t: with this last modification, both cytotoxicity and antiviral
activity are abolished. A mild inhibition of the viral replication is observed for ethyl ester 1s, but its
antiviral potential is reduced in comparison with 1a. Symmetrical shortening of the aliphatic linker in
8a-h is associated with loss of activity, possibly indicating an important role of the linker for the viral
replication inhibition. An exception to this trend is represented by 8h and 8e, for which the presence of
a 4-nitrophenyl and a 2-naphthyl rings, respectively, results in activity retention in comparison with
their three-carbon counterparts, while the shortening of the linker appears to reduce the toxic effect
found for 1h and 1e. Antiviral results for symmetrical amides 11a, d, f-h indicate that either the
presence of the positive charge on the piperazine amine groups or the flexibility of the two linkers are
important for the viral replication inhibition, since this modification is correlated with loss of activity
for all seven derivatives prepared. A longer aliphatic linker of four methylene groups (17) is also
associated with loss of activity, confirming that the length of the molecule plays an important role in
HCV replication inhibition. Another feature that appears important is the presence of the two
sulfonamide groups: their replacement with amide groups in the symmetrical scaffold of 20 is
associated with a dramatic loss of activity. Biological results found for 21, where the 4-
phenylendiamine central ring both rigidifies the structure and removes the positive charge of the linker
compared to 1a, suggest an increased cytotoxicity, even if a low micromolar EC50 value is retained. The
loss of antiviral activity found for 39 indicates the importance of the overall length of the molecule, and
taken together with the results found for 21 this evidence suggests that the presence of a di-substituted
piperazine nucleus is essential for the antiviral activity of the novel scaffold.
Antiviral data found for compounds 23-35 suggest that symmetry is not essential for antiviral activity,
since the insertion of different para-hydrophobic substituents in the two aromatic rings is tolerated:
EC50 values for most of these compounds are in the range of 1-10 µM, suggesting activity retention as a
general trend. Nevertheless, in some cases the small modification carried out seems to strongly and
unexpectedly affect cytotoxicity, as revealed for 25 and 28-30. In the case of biphenyl products 27, 31,
33 and 34 this cytotoxic effect seems to be at least partially in line with the results obtained for 1n. In
the case of unsymmetrical compounds 36a-b, l, in which one aliphatic linker is shortened to two
methylene groups, the small change in the molecular structure is associated with a retained antiviral
effect, further confirming that symmetry is not required for antiviral activity. Nevertheless, with the
exception of 36b, also in this second series of unsymmetrical derivatives the small modification is
correlated with an increased cytotoxic effect. Finally, biological results for unsymmetrical compounds
37a-c, l and n, in which one piperazine nitrogen is functionalised to amide, suggest activity retention,
with 37a, 37l and 37n showing EC50 values in the low micromolar range, even though the toxic effect
seems to be enhanced, particularly for 37l. Data found for 37b, which, as expected, does not show any
antiviral activity, confirm the essential role played by a para-hydrophobic aromatic group for the viral
replication inhibition.
2.3.2 HCV NS3 helicase enzymatic assays
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About half of the newly synthesised compounds were tested for their ability to inhibit HCV NS3
helicase-catalysed nucleic acid unwinding. Somewhat surprisingly, most compounds tested did not
effect observed rates of DNA strand separation, even when supplied at concentrations as high as 1 mM
(Table 1). Primuline [18] and aurintricarboxylic acid [35] were included as positive controls.
The most active compound, 31 (IC50=90±30 µM), was selected for additional analysis along with a
compound with intermediate activity, 1l (IC50=310±190), and two compounds that did not inhibit
helicase-catalysed DNA unwinding, 1a and 11a (Figure 5A). Each compound was tested for its ability
to either dislodge HCV helicase from a bound oligonucleotide (Figure 5B) or prevent HCV helicase
from hydrolysing ATP (Figure 5C). Only 31 was able to fully displace NS3h from an oligonucleotide
at concentrations below 1 mM (Figure 5B). Both 31 and 1l inhibited ATP hydrolysis, but again 31
was a far more potent inhibitor in this assay (Figure 5C).
Interestingly, about 10 times less 31 was needed to inhibit NS3h-catalysed ATP hydrolysis than was
needed to inhibit nucleic acid binding to the same extent. Since small molecules can inhibit helicase-
catalysed ATP hydrolysis either by preventing ATP from binding or by preventing nucleic acids from
stimulating ATP hydrolysis, we next tested the ability of 31 to inhibit ATP hydrolysis in the absence
and presence of various concentrations of poly(U) RNA. In the absence of RNA, 31 did not inhibit
NS3h-catalysed ATP hydrolysis even at concentrations as high as 1 mM (data not shown). However,
31 inhibited ATP hydrolysis in the presence of RNA in a manner in which 31 appeared to compete
with RNA for a binding site on the enzyme (Figure 5D). Data best fit a kinetic model that assumes
that 31 affects the apparent dissociation constant for RNA (KRNA), but does not influence the Vmax
.
However, unlike what is seen with classic competitive inhibitors, the observed KRNA values were not
linearly dependent on the concentration of the inhibitor (i.e. 31). Instead, a plot of KRNA vs. 31
concentration reveals a sigmoidal relationship, indicative of a cooperative effect (Figure 5E).
To rule out the possibility that 31 might be acting by binding helicase substrates instead of NS3, we
also performed direct binding assays by monitoring the effect of 31 on intrinsic protein fluorescence.
Both NS3h and 31 fluoresce at 340 nm when excited at 280 nm. When each is alone, NS3h fluoresces
about 70 times more brightly than the same amount of 31 at these wavelengths. However, when a
solution of NS3h is titrated with 31, a new species forms that fluoresces twice as brightly as free NS3h,
and 140 times brighter than free 31. Titration data fit a model in which a 1:1 complex forms with a
dissociation constant of 0.57 µM. Importantly, this high affinity seems to best reflect the ability of 31
to inhibit replication of HCV replicons where the average observed EC50 value was 1.3 µM.
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B
C
100
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40
20
0
100
80
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40
20
0
1a
1l
11a
31
10
100 1000
10
100 1000
Compound (µM)
1a (>1,000)
Compound (µM)
1a (>1,000)
1l (590±150)
11a (>1,000)
31 (21±13)
1l (>1,000)
11a (>1,000)
31 (200±76)
D
E
F
8
6
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2
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600
31 (µM)
100
100
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44
30
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0
Kd = 0.57±0.27 µM
50
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0
5
10
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50 100 150
31 (µM)
31 (µM)
RNA (µM)
0.10 µM NS3h
0 µM NS3h
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Figure 5: Inhibition of HCV NS3 helicase activity for the newly synthesised piperazine derivatives. (A) Compounds selected for
analysis. (B) The ability of each compound to displace NS3h from a DNA oligonucleotide. Numbers in parenthesis are IC50
values with errors representing 95% confidence intervals seen in non-linear regression analysis of 2 independent titrations. (C)
Ability of each compound to inhibit NS3 helicase-catalysed ATP hydrolysis. Numbers in parenthesis are as in (B). (C) Rates of
ATP hydrolysis (nM ATP cleaved/s/nM NS3h) observed in the presence of various concentrations of poly(U) RNA and 31. Data
are globally fit to Eq. 1 (Methods) assuming 31 does not affect Vmax. (E) Amount of RNA needed to stimulate ATP hydrolysis to
50% maximum (KRNA) observed at various concentrations of 31. (F) Fluorescence observed with various concentrations of 31 in
the absence of NS3h (circles) or in the presence of 100 nM NS3h (squares). Data are globally fit to Eq. 2 (Methods) with the
noted dissociation constant and an Fe of 308, Fs of 4.5. and Fc of 641.
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These results suggest that enzymatic in vitro reactions might not be the best assays to detect antivirals
that target a helicase. The above observation that a compound that clearly binds directly to NS3h fails
to inhibit the enzyme at concentrations where it should be fully bound suggests that assay conditions
might prevent the enzyme-inhibitor interaction. For example, the helicase might form oligomers or
higher ordered structures when bound to nucleic acids or ATP such that ligand binding is blocked.
Alternatively, the rapid conformational changes that occur while the helicase acts as a molecular motor
in these assays might dislodge a bound inhibitor. Based on our results with 31, it is likely that other
compounds also bind NS3h with high affinity (examples: 1h, 1l-o, 1s-t, 8a, 8h, 17, 30-31, 34, 36l, 37a,
37l, and 37n). However, testing for at direct interaction between these other compounds and NS3h has
been challenging because they do not possess the same optical properties as 31. We are therefore still
exploring other possible assays to determine the affinity of other compounds generated in this project
for the HCV helicase, many of which are associated with less cytotoxicity in the replicon assay than 31
(Table 1).
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Based on the results obtained so far, some inhibition of the enzyme unwinding activity can be observed
with a naphthyl aromatic ring in the symmetrical structure (1h, 1o, 8h), both with a three or two-carbon
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linker, with a 4-carboxylic or 4-ethoxylate groups (1s-t), with a 4-chlorophenyl system and a two-
carbon or four-carbon linker (8a and 17), and with the original three-carbon symmetrical scaffold and a
4-tert-butylphenyl, 4-CF3-phenyl or a 4-biphenyl ring (1l-n). The trend for the enzymatic activity does
not seem to parallel the HCV replicon assay, since in this set of data the length of the linker does not
play an essential role, even if the presence of a bulky hydrophobic substituent in the para position of
the aromatic ring seems relevant for activity. Some ability to inhibit helicase-catalysed unwinding can
also be observed in all three series of unsymmetrical structures, in particular with a 4-tert-butyl group
in at least one aromatic (30-31, 36l, 37l), with at least one biphenyl moiety (31, 34, 37n), or with two
equal 4-chlorophenyl substituents in the unsymmetrical amide scaffold (37a).
This data would suggest that the antiviral effect of 31, should be at least in part due to its interference
with the NS3 helicase function, and that several commonly used helicase assays grossly underestimate
the potency of this, and likely related, compounds. The antiviral effect of the newly prepared structures
might alternatively result from the interference with an additional target, viral or cellular. Additional
studies aiming to improve the antiviral activity and understand the mechanism of action of these
compounds are ongoing and will be reported in due course.
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3 Conclusions
The application of computer-aided techniques to the study of the HCV NS3 helicase led to the
identification of one commercial piperazine-based analogue with an antiviral effect against HCV
replication in the low micromolar range. Starting from its structure, different modifications were
designed and carried out to explore the biological activity associated with the new scaffold. Several
analogues of 1a inhibited the HCV subgenomic replicon replication. Different structural modifications
were explored to understand the role of aromatic substituents, sulfonamide groups, linker chains,
piperazine central nucleus and symmetry of the original molecule. Two equal phenyl rings with a
hydrophobic substituent in the para position are essential for antiviral activity, along with the presence
of the two sulfonamide groups and the central piperazine nucleus. The length and nature of the two
linker chains is also important for activity retention: the presence of at least one three-carbon aliphatic
linker is essential, while shortening or elongating the two linkers at the same time is associated with
loss of activity. The same effect can be observed with the symmetrical functionalization of piperazine
amine groups to amide, by inserting a carbonyl group in the terminal methylene of the two linkers.
Replacement of the piperazine central ring with para-phenylendiamine is associated with loss of
activity, and activity is lost as well when piperazine central nucleus is replaced with piperidine,
maintaining only half of the original scaffold. The overall symmetry of the structure can tolerate small
perturbations such as two different para hydrophobic aromatic substituents, or different linker chains.
Unsymmetrical derivatives are in general associated with increased cytotoxicity in comparison with
their symmetrical counterparts.
In vitro evaluations of some of the newly synthesised compounds show that different inhibit the NS3
helicase activity. In particular, compound 31 bound free helicase with a sub-micromolar dissociation
constant, and it influenced the protein’s ability to bind nucleic acid substrates needed to stimulate ATP
hydrolysis. Even if the antiviral effect of the new structures could be at least partially correlated with
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inhibition of the NS3 helicase, other viral or cellular targets could still be involved, and the toxicity of
31 suggests it might also act against related cellular motor proteins. Further exploration of both
antiviral activity and biological targets of these compounds is the current focus of ongoing
investigations.
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4 Experimental
4.1 Materials and methods
All solvents used for chromatography were HPLC grade from Fisher Scientific (UK). 1H 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 compounds were more than 95% pure.
Intermediates 6-7, 10, 13, 14, 19, 22 were prepared according to literature procedures, described in
detail along with compound characterisation in the Supporting Information. Preparation and
characterisation details on the new target compounds 1a-s,u, 8a-h, 11a, d, f-h, 17, 20, 21, 23-35, 37a-c,
l, n are given below. 1H-NMR spectra of all final compounds are reported in the Supporting
Information.
4.1.1
General
method
for
the
preparation
of
N,N’-(piperazine-1,4-diyl)bis
(alkyl)diarylsulfonamides 1, 8
Piperazine (0.05 g, 0.6 mmol) and NaHCO3 (0.11 g, 1.3 mmol) were suspended in absolute ethanol (9
mL). The different alkyl bromide 6 or 7 (1.3 mmol) was then added portionwise to the suspension and
the reaction mixture was stirred under reflux for 24 h. The solvent was evaporated under reduced
pressure and the crude residue was purified by flash column chromatography.
4.1.1.1 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(4-chlorobenzene-sulfonamide) (1a)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 64% yield as a pale yellow solid. M.p. 168-170°C. TLC (9:1 DCM-
1
MeOH, Rf: 0.61). H-NMR (DMSO-d6),
δ
: 1.48-1,52 (m, 4H), 2.18-2.26 (m, 12H), 2.73-2.78 (m, 4H),
: 26.0, 40.8,
7.69 (d, J= 8.6 Hz, 4H), 7.71 (bs, 2H), 7.81 (d, J= 8.6 Hz, 4H). 13C-NMR (DMSO-d6),
δ
52.5, 54.7, 128.4, 129.3, 137.1, 139.3. Anal. Calcd for C22H30Cl2N4O4S2: C, 48.08; H, 5.50; N, 10.20.
Found: C, 47.95; H, 5.11; N, 10.07. MS [ESI, m/z]: 549.0, 551.0 [M+H].
4.1.1.2 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))dibenzene-sulfonamide (1b)
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Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 64% yield as a white solid. TLC (9:1 DCM-MeOH, Rf: 0.46). M.p. 136-
1
138°C. H-NMR (CDCl3),
δ
: 1.60 (m, 4H), 2.38 (m, 12H), 3.04 (t, J= 5.8 Hz, 4H), 7.10 (bs, 2H), 7.50
: 24.0, 44.0, 53.0, 57.9, 126.8, 129.0, 132.6,
(m, 4H), 7.56 (m, 2H), 7.83 (m, 4H). 13C-NMR (CDCl3),
δ
140.1. Anal. Calcd for C22H32N4O4S2: C, 54.98; H, 6.71; N, 11.66. Found: C, 54.85; H, 6.99; N, 11.48.
MS [ESI, m/z]: 481.1 [M+H].
4.1.1.3 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(4-methylbenzene-sulfonamide) (1c)
[36]
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 95:5 v/v. Obtained in 68% yield as a white solid. M.p. 176-178°C (lit. 181-183°C) [33]. TLC
1
(9:1 DCM-MeOH, Rf: 0.67). H-NMR (CDCl3),
δ
: 1.65 (m, 4H), 2.46 (bs, 18H), 3.07 (t, J= 5.6 Hz,
: 20.9,
4H), 7.12 (bs, 2H), 7.32 (d, J= 8.2 Hz, 4H), 7.74 (d, J= 8.2 Hz, 4H). 13C-NMR (DMSO-d6),
δ
26.0, 40.9, 52.6, 54.8, 126.5, 129.5, 137.8, 142.4. Anal. Calcd for C24H36N4O4S2: C, 56.67; H, 7.13; N,
11.01. Found: C, 56.89; H, 6.92; N, 11.14. MS [ESI, m/z]: 509.1 [M+H].
4.1.1.4 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(4-methoxybenzene-sulfonamide) (1d)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 91:9 v/v. Obtained in 60% yield as a white solid. M.p. 172-174°C. TLC (9:1 DCM-MeOH, Rf:
0.72). 1H-NMR (DMSO-d6),
Hz, 4H), 7.41 (t, J= 5.6 Hz, 2H), 7.70 (d, J= 8.3 Hz, 4H). 13C-NMR (DMSO-d6),
δ
: 1.47 (m, 4H), 2.18 (bs, 12H), 2.72 (m, 4H), 3.83 (s, 6H), 7.11 (d, J= 8.3
: 25.6, 39.7, 51.9,
δ
54.3, 56.0, 114.4, 126.5, 131.8, 165.2. Anal. Calcd for C24H36N4O6S2: C, 53.31; H, 6.71; N, 10.36.
Found: C, 53.44; H, 6.92; N, 10.40. MS [ESI, m/z]: 541.1 [M+H].
4.1.1.5 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(4-nitrobenzene-sulfonamide) (1e)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 91:9 v/v. Obtained in 43% yield as a pale yellow solid. M.p. 226-228°C. TLC (9:1 DCM-
1
MeOH, Rf: 0.59). H-NMR (DMSO-d6),
δ
: 1.49 (m, 4H), 2.18 (m, 12H), 2.82 (t, J= 6.8 Hz, 4H), 7.98
: 26.1, 40.8, 52.5,
(bs, 2H), 8.03 (d, J= 8.7 Hz, 4H), 8.42 (d, J= 8.7 Hz, 4H). 13C-NMR (DMSO-d6),
δ
54.6, 124.5, 128.0, 146.1, 149.5. Anal. Calcd for C22H30N6O8S2: C, 46.31; H, 5.30; N, 14.73. Found: C,
45.99; H, 5.63; N, 14.66. MS [ESI, m/z]: 571.1 [M+H].
4.1.1.6 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(3,4-dimethoxybenzene-sulfonamide)
(1f)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 95:5 v/v. Obtained in 65% yield as a yellow solid. M.p. 152-154 °C. TLC (9:1 DCM-MeOH,
Rf: 0.70). 1H-NMR (CDCl3),
(s, 6H), 6.53 (bs, 2H), 6.95 (d, J= 8.4 Hz, 2H), 7.33 (d, J= 2.1, 2H), 7.46 (dd, J1= 8.4 Hz, J2= 2.1 Hz,
2H). 13C-NMR (CDCl3),
: 24.1, 44.0, 53.0, 56.1, 56.2, 57.7, 109.7, 110.5, 120.7, 131.9, 149.0, 152.3.
Anal. Calcd for C26H40N4O8S2: C, 51.98; H, 6.71; N, 9.33. Found: C, 51.96; H, 6.94; N, 9.30. MS [ESI,
δ: 1.66 (m, 4H), 2.47 (m, 12H), 3.06 (t, J= 5.7 Hz, 4H), 3.93 (s, 6H), 3.96
δ
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m/z]: 601.1 [M+H].
4.1.1.7 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(2,5-dimethoxybenzene-sulfonamide)
(1g)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 95:5 v/v. Obtained in 71% yield as a white solid. M.p. 170-172 °C. TLC (9:1 DCM-MeOH, Rf:
0.73). 1H-NMR (CDCl3),
6H), 6.22 (bs, 2H), 7.00 (d, J= 8.9 Hz, 2H), 7.08 (dd, J1= 8.9 Hz, J2= 3.1 Hz, 2H), 7.47 (d, J= 3.1, 2H).
13C-NMR (CDCl3),
: 25.3, 44.4, 52.5, 55.4, 56.0, 57.2, 114.1, 114.8, 120.2, 132.0, 150.4, 153.3. Anal.
δ: 1.71 (m, 4H), 2.55 (bs, 12H), 3.00 (t, J= 6.2 Hz, 4H), 3.83 (s, 6H), 3.95 (s,
δ
Calcd for C26H40N4O8S2: C, 51.98; H, 6.71; N, 9.33. Found: C, 51.80; H, 7.02; N, 9.29. MS [ESI, m/z]:
601.1 [M+H].
4.1.1.8 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))dinaphthalene-2-sulfonamide (1h)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v.Obtained in 79% yield as a white solid. M.p. 186-188 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.62). H-NMR (CDCl3),
δ
: 1.66 (m, 4H), 2.46 (bs, 12H), 3.13 (t, J= 5.5 Hz, 4H), 7.32 (bs, 2H), 7.66
(m, 4H), 7.83 (dd, J1= 8.7 Hz, J2= 1.8 Hz, 2H), 7.95 (d, J= 7.9, 2H), 7.99 (m, 4H), 8.44 (s, 2H). 13C-
NMR (DMSO-d6), : 25.9, 40.8, 52.4, 54.7, 122.2, 127.3, 127.5, 127.7, 128.6, 129.1, 129.3, 131.7,
δ
134.0, 137.4. Anal. Calcd for C30H36N4O4S2: C, 62.04; H, 6.25; N, 9.65. Found: C, 61.89; H, 5.97; N,
9.48. MS [ESI, m/z]: 581.1 [M+H].
4.1.1.9 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(3-chlorobenzene-sulfonamide) (1i)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 79% yield as a white solid. M.p. 156-158 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.66). H-NMR (DMSO-d6),
δ
: 1.48 (m, 4H), 2.20 (m, 12H), 2.79 (m, 4H), 7.64 (t, J= 7.8 Hz, 2H),
: 26.0, 40.8, 52.5, 54.7, 125.1, 126.0,
7.74 (m, 6H), 7.78 (t, J= 1.8 Hz, 2H). 13C-NMR (DMSO-d6),
δ
131.3, 132.2, 133.8, 142.4. Anal. Calcd for C22H30Cl2N4O4S2: C, 48.08; H, 5.50; N, 10.20. Found: C,
47.90; H, 5.76; N, 10.08. MS [ESI, m/z]: 549.0, 551.0 [M+H].
4.1.1.10 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(4-tertbutylbenzene-sulfonamide) (1l)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 82% yield as a white solid. M.p. 188-190 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.59). H-NMR (DMSO-d6),
δ
: 1.31 (s, 18H), 1.46 (m, 4H), 2.16 (m, 12H), 2.75 (m, 4H), 7.49 (t, J=
: 26.0, 30.7,
5.8 Hz, 2H), 7.60 (d, J= 8.4 Hz, 4H), 7.70 (d, J= 8.4 Hz, 4H). 13C-NMR (DMSO-d6),
δ
34.7, 40.8, 52.5, 54.8, 125.9, 126.3, 137.6, 155.1. Anal. Calcd for C30H48N4O4S2: C, 60.78; H, 8.16; N,
9.45. Found: C, 60.96; H, 8.00; N, 9.51. MS [ESI, m/z]: 593.3 [M+H].
4.1.1.11
N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(4-(trifluoromethyl)-benzene
sulfonamide) (1m)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
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MeOH 96:4 v/v. Obtained in 61% yield as a white solid. M.p. 184-186 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.65). H-NMR (DMSO-d6),
8H). 13C-NMR (DMSO-d6),
δ
: 1.48 (m, 4H), 2.16 (bm, 12H), 2.81 (m, 4H), 7.49 (bs, 2H), 7.99 (m,
δ
: 26.0, 40.8, 52.5, 54.6, 123.6 (q, J= 272.7 Hz), 126.4 (q, J= 3.7 Hz),
127.4, 132.2 (q, J= 32.2 Hz), 144.5. Anal. Calcd for C24H30F6N4O4S2: C, 46.75; H, 4.90; N, 9.09.
Found: C, 46.91; H, 5.11; N, 9.18. MS [ESI, m/z]: 617.1 [M+H].
4.1.1.12 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(biphenyl-sulfonamide) (1n)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 87% yield as a white solid. M.p. 183-185 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.59). H-NMR (DMSO-d6),
δ
: 1.46-1.50 (m, 4H), 2.12-2.22 (m, 12H), 3.51-3.56 (m, 4H), 7.41-7.44
(m, 2H), 7.49-7.53 (m, 4H), 7.62 (bs, 2H), 7.71-7.75 (m, 4H), 7.84-7.89 (m, 8H). 13C-NMR (DMSO-
d6), : 26.0, 40.9, 52.5, 54.8, 127.0, 127.1, 127.3, 128.4, 129.0, 138.5, 139.2, 143.8. Anal. Calcd for
δ
C34H40N4O4S2: C, 64.53; H, 6.37; N, 8.85. Found: C, 64.24; H, 6.51; N, 8.78. MS [ESI, m/z]: 633.2
[M+H].
4.1.1.13 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))dinaphthalene-2-sulfonamide (1o)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 91% yield as a white solid. M.p. 216-218 °C. TLC (9:1 DCM-MeOH, Rf:
0.67). 1H-NMR (DMSO-d6),
(m, 6H), 7.91 (bs, 2H), 8.10 (m, 4H), 8.22 (d, J= 8.2 Hz, 2H), 8.64 (d, J= 8.4 Hz, 2H). 13C-NMR
(DMSO-d6), : 25.8, 40.7, 52.3, 54.6, 124.4, 124.6, 126.7, 127.5, 127.7, 128.5, 128.9, 133.6, 133.8,
δ: 1.36 (m, 4H), 1.93 (bs, 8H), 1.99 (t, J= 6.8 Hz, 4H), 2.79 (m, 4H), 7.67
δ
135.4. Anal. Calcd for C30H36N4O4S2: C, 62.04; H, 6.25; N, 9.65. Found: C, 61.91; H, 6.33; N, 9.63.
MS [ESI, m/z]: 581.2 [M+H].
4.1.1.14 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))diquinoline-8-sulfonamide (1p)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 76% yield as a white solid. M.p. 200-202 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.73). H-NMR (CDCl3),
δ
: 1.67 (m, 4H), 2.37 (bs, 12H), 2.95 (m, 4H), 6.79 (bs, 2H), 7.59 (dd, J1=
8.4, J2= 4.2, 2H), 7.68 (t, J= 7.6 Hz, 2H), 8.08 (d, J=8.0 Hz, 2H), 8.31 (d, J= 8.0 Hz, 2H), 8.46 (d, J=
7.1 Hz, 2H), 9.04 (m, 2H). 13C-NMR (CDCl3),
: 26.1, 42.4, 52.9, 56.0, 122.2, 125.7, 128.8, 131.4,
δ
133.1, 136.0, 136.9, 143.4, 151.1. Anal. Calcd for C28H34N6O4S2: C, 57.71; H, 5.88; N, 14.42. Found:
C, 57.55; H, 6.04; N, 14.34. MS [ESI, m/z]: 583.2 [M+H].
4.1.1.15 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))dithiophene-2-sulfonamide (1q)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 83% yield as a white solid. TLC (9:1 DCM-MeOH, Rf: 0.45). M.p. 138-
140 °C. 1H-NMR (DMSO-d6),
δ: 1.52 (m, 4H), 2.24 (m, 12H), 2.86 (m, 4H), 7.18 (dd, J1= 5.1, J2= 3.7,
2H), 7.57 (dd, J1= 3.7, J2= 1.3, 2H), 7.79 (bs, 2H), 7.92 (dd, J1= 5.1, J2= 1.3, 2H). 13C-NMR (DMSO-
d6), δ: 25.8, 41.2, 52.6, 54.9, 127.6, 131.3, 132.2, 141.4. Anal. Calcd for C18H28N4O4S4: C, 43.88; H,
5.73; N, 11.37. Found: C, 43.59; H, 5.96; N, 11.31. MS [ESI, m/z]: 493.0 [M+H].
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4.1.1.16 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))dipyridine-3-sulfonamide (1r)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 91:9 v/v. Obtained in 63% yield as a pale yellow solid. M.p. 140-142 °C. TLC (9:1 DCM-
1
MeOH, Rf: 0.52). H-NMR (DMSO-d6),
δ
: 1.50 (m, 4H), 2.20 (bs, 12H), 2.82 (bs, 4H), 7.65 (dd, J1=
8.0 Hz, J2= 4.9 Hz, 2H), 7.85 (bs, 2H), 8.16 (dt, J1= 8.0 Hz, J2= 1.9 Hz, 2H), 8.82 (dd, J1= 4.9 Hz, J2=
1.9 Hz, 2H), 8.94 (d, J= 2.2 Hz, 2H). 13C-NMR (DMSO-d6),
: 26.0, 40.8, 52.5, 54.6, 124.2, 134.4,
δ
136.8, 146.9, 152.9. Anal. Calcd for C20H30N6O4S2: C, 49.77; H, 6.27; N, 17.41. Found: C, 49.55; H,
6.07; N, 17.23. MS [ESI, m/z]: 483.1 [M+H].
4.1.1.17
Ethyl
3-(N-(3-(4-(3-(4-carboxyphenylensulfonamido)propyl)
piperazin-1-yl)propyl)
sulfamoyl)benzoate (1s)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 41% yield as a white solid. M.p. 170-172 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.56). H-NMR (CDCl3),
δ
: 1.44 (t, J= 7.1 Hz, 6H), 1.67 (m, 4H), 2.47 (m, 12H), 3.11 (t, J= 5.6 Hz,
4H), 4.44 (q, J= 7.1 Hz, 4H), 7.45 (bs, 2H), 7.92 (d, J= 8.5 Hz, 4H), 8.19 (d, J= 8.5 Hz, 4H).
13C-NMR (CDCl3),
: 14.2, 23.8, 44.3, 53.0, 57.9, 61.6, 126.8, 130.2, 133.9, 144.1, 165.2. Anal. Calcd
δ
for C28H40N4O8S2: C, 53.83; H, 6.45; N, 8.97. Found: C, 53.69; H, 6.71; N, 8.76. MS [ESI, m/z]: 625.2
[M+H].
4.1.1.18 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(2-chlorobenzene-sulfonamide) (1u)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 98:2 v/v. Obtained in 80% yield as a white solid. M.p. 162-164 °C. TLC (9:1 DCM-MeOH, Rf:
0.79). 1H-NMR (DMSO-d6),
Hz, 2H), 7.65 (m, 4H), 7.88 (t, J= 5.1 Hz, 2H), 7.96 (dd, J1= 7.9 Hz, J2= 1.3 Hz, 2H). 13C-NMR
(DMSO-d6), : 25.7, 41.0, 52.5, 54.9, 127.6, 130.5, 130.5, 131.7, 133.9, 137.8. Anal. Calcd for
δ: 1.49 (m, 4H), 2.17 (m, 12H), 2.85 (m, 4H), 7.54 (td, J1= 7.4 Hz, J2= 1.5
δ
C22H30Cl2N4O4S2: C, 48.08; H, 5.50; N, 10.20. Found: C, 48.27; H, 5.34; N, 10.32. MS [ESI, m/z]:
549.1, 551.1 [M+H].
4.1.1.19 N,N’-(2,2’-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(4-chlorobenzene sulfonamide) (8a)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 95:5 v/v. Obtained in 45% yield as a white solid. M.p. 218-220 °C. TLC (9:1 DCM-MeOH, Rf:
0.66). 1H-NMR (CDCl3),
8.5 Hz, 4H), 7.82 (d, J= 8.5 Hz, 4H). 13C-NMR (DMSO-d6),
δ
: 2.17, (bs, 8H), 2.43 (t, J= 5.5, 4H), 3.08 (m, 4H), 5.14 (bs, 2H), 7.51 (d, J=
: 40.9, 52.8, 54.9, 128.6, 129.7, 137.9,
δ
139.8. Anal. Calcd for C20H26Cl2N4O4S2: C, 46.06; H, 5.03; N, 10.74. Found: C, 46.23; H, 5.31; N,
10.87. MS [ESI, m/z]: 521.0, 523.0 [M+H].
4.1.1.20 N,N’-(2,2’-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))dibenzenesulfonamide (8b)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 53% yield as a white solid. M.p. 158-160 °C. TLC (9:1 DCM-MeOH, Rf:
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0.40). 1H-NMR (CDCl3),
δ
: 2.17, (s, 8H, H-3’), 2.39 (t, J= 5.8, 4H, H-2’), 3.00 (m, 4H, H-1’), 5.13 (bs,
2H, NH), 7.53 (m, 4H, H-3), 7.60 (tt, J1= 7.3 Hz, J2= 2.1 Hz, 2H, H-4), 7.88 (m, 4H, H-2). 13C-NMR
(CDCl3), : 39.1, 52.2, 55.4, 127.0, 129.0, 132.6, 139.6. Anal. Calcd for C20H28N4O4S2: C, 53.08; H,
δ
6.24; N, 12.38. Found: C, 52.89; H, 6.53; N, 12.32. MS [ESI, m/z]: 453.1 [M+H].
4.1.1.21 N,N’-(2,2’-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(4-methylbenzene-sulfonamide) (8c)
[37]
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 98:2 v/v. Obtained in 89% yield as a white solid. M.p. 182-184°C (lit. 185-187°C) [34]. TLC
1
(9:1 DCM-MeOH, Rf: 0.59). H-NMR (CDCl3),
δ
: 2.24, (bs, 8H), 2.39 (t, J= 5.7, 4H), 2.45 (s, 6H),
2.98 (m, 4H), 5.10 (bs, 2H), 7.32 (d, J= 8.2 Hz, 4H), 7.57 (d, J= 8.2 Hz, 4H). 13C-NMR (DMSO-d6),
δ:
20.9, 40.0, 52.4, 56.7, 126.4, 129.5, 137.6, 142.4. Anal. Calcd for C22H32N4O4S2: C, 54.98; H, 6.71; N,
11.66. Found: C, 54.93; H, 6.95; N, 11.64. MS [ESI, m/z]: 481.1 [M+H].
4.1.1.22 N,N’-(2,2’-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(4-methoxy-benzenesulfonamide) (8d)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 87% yield as a white solid. M.p. 170-172 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.45). H-NMR (DMSO-d6),
δ
: 1.45 (t, J= 5.4, 4H), 2.24, (bm, 8H), 2.71-2.75 (m, 4H), 3.89 (s, 6H),
: 39.1,
7.09 (d, J= 8.8 Hz, 4H), 7.41 (t, J= 5.5 Hz, 2H), 7.81 (d, J= 8.8 Hz, 4H). 13C-NMR (CDCl3),
δ
52.3, 55.4, 55.6, 114.1, 129.2, 131.1, 162.8. Anal. Calcd for C22H32N4O6S2: C, 51.54; H, 6.29; N,
10.93. Found: C, 51.39; H, 6.47; N, 10.84. MS [ESI, m/z]: 513.1 [M+H].
4.1.1.23 N,N’-(2,2’-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(4-nitrobenzene-sulfonamide) (8e)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 63% yield as a pale yellow solid. M.p. 212-214 °C. TLC (9:1 DCM-
MeOH, Rf: 0.68). 1H-NMR (DMSO-d6),
7.92 (bs, 2H), 8.05 (d, J= 8.8 Hz, 4H), 8.40 (d, J= 8.8 Hz, 4H). 13C-NMR (DMSO-d6),
δ
: 2.18, (bs, 8H), 2.55 (t, J= 6.6, 4H), 2.98 (t, J= 6.6 Hz, 4H),
: 40.0, 52.3,
δ
56.8, 124.4, 127.9, 146.4, 149.4. Anal. Calcd for C20H26N6O8S2: C, 44.27; H, 4.83; N, 15.49. Found: C,
44.17; H, 4.66; N, 15.27. MS [ESI, m/z]: 543.0 [M+H].
4.1.1.24 N,N’-(2,2’-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(3,4-dimethoxy-benzenesulfonamide)
(8f)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 74% yield as a white solid. M.p. 152-154 °C. TLC (9:1 DCM-MeOH, Rf:
0.53). 1H-NMR (CDCl3),
5.11 (bs, 2H), 6.94 (m, 2H), 7.34 (m, 2H), 7.49 (m, 2H). 13C-NMR (CDCl3),
δ
: 2.26, (bs, 8H), 2.40 (t, J= 5.6, 4H), 2.99 (m, 4H), 3.94 (s, 6H), 3.96 (s, 6H),
: 39.1, 52.3, 56.2, 56.3,
δ
56.4, 109.7, 110.5, 121.0, 131.2, 149.1, 152.5. Anal. Calcd for C24H36N4O8S2: C, 50.33; H, 6.34; N,
9.78. Found: C, 50.19; H, 6.50; N, 9.62. MS [ESI, m/z]: 573.1 [M+H].
4.1.1.25 N,N’-(2,2’-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(2,5-dimethoxy-benzenesulfonamide)
(8g)
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Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 95:5 v/v. Obtained in 77% yield as a white solid. M.p. 176-178 °C. TLC (9:1 DCM-MeOH, Rf:
0.47). 1H-NMR (CDCl3),
5.58 (t, J= 4.9 Hz, 2H), 6.96 (d, J= 9.1 Hz, 2H), 7.08 (dd, J1=9.1 Hz, J2= 3.1 Hz, 2H), 7.47 (d, J= 3.1
Hz, 2H). 13C-NMR (CDCl3),
: 39.8, 52.6, 56.0, 56.1, 57.1, 113.7, 114.8, 120.3, 127.8, 150.2, 153.4.
δ: 2.31, (bs, 8H), 2.42 (t, J= 5.6, 4H), 2.98 (m, 4H), 3.83 (s, 6H), 3.92 (s, 6H),
δ
Anal. Calcd for C24H36N4O8S2: C, 50.33; H, 6.34; N, 9.78. Found: C, 50.26; H, 6.65; N, 9.76. MS [ESI,
m/z]: 573.1 [M+H].
4.1.1.26 N,N’-(2,2’-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))dinaphthalene-2-sulfonamide (8h)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 82% yield as a white solid. M.p. 176-178 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.56). H-NMR (CDCl3),
δ
: 2.18, (bs, 8H), 2.37 (t, J= 5.8, 4H), 3.01 (m, 4H), 5.22 (bs, 2H), 7.66 (m,
4H), 7.82 (dd, J1= 8.8 Hz, J2= 1.8 Hz, 2H), 7.92 (d, J= 8.1 Hz, 2H), 7.97 (m, 4H), 8.45 (s, 2H).
13C-NMR (CDCl3),
: 39.2, 52.3, 55.5, 122.2, 127.7, 127.9, 128.6, 128.8, 129.2, 129.4, 131.9, 134.7,
δ
137.3. Anal. Calcd for C28H32N4O4S2: C, 60.85; H, 5.84; N, 10.14. Found: C, 60.71; H, 6.03; N, 10.06.
MS [ESI, m/z]: 553.1 [M+H].
4.1.2 Synthesis of 3-(N-(3-(4-(3-(4-Carboxyphenylensulfonamido)propyl)piperazin-1-yl)-propyl)-
sulfamoyl) benzoic acid (1t)
Compound 1s (0.15 g, 0.3 mmol) was dissolved 3 mL of THF. LiOH monohydrate (0.07 g, 1.7 mmol)
was dissolved in 4 mL of distilled water and added to the THF solution. The reaction was stirred o.n. at
80 °C. The organic solvent was then removed at reduced pressure and the water residue was acidified
to pH 5 with 1M HCl solution. The resulting precipitate was filtered, washed with water and dried
1
under vacuum to afford a pure white solid in 88% yield. M.p. > 300 °C. H-NMR (D2O),
δ
: 1.48 (m,
4H), 2.15 (t, J= 7.8 Hz, 4H), 2.31 (bm, 8H), 2.69 (t, J= 7.1 Hz, 4H), 7.72 (d, J= 8.1 Hz, 4H), 7.88 (d,
J= 8.1 Hz, 4H). 13C-NMR (D2O),
: 27.8, 43.5, 51.5, 55.6, 126.2, 129.1, 138.7, 145.2, 174.5. Anal.
δ
Calcd for C24H32N4O8S2: C, 50.69; H, 5.67; N, 9.85. Found: C, 50.53; H, 5.45; N, 9.71. MS [ESI, m/z]:
591.1 [M+Na].
4.1.3 General method for the preparation of N,N’-(piperazine-1,4-diyl)bis(3-oxopropane-)diaryl
sulfonamides 11
The different 3-arylsulfonylamino propionic acid 10 (1.3 mmol), TBTU (0.45 g, 1.4 mmol) and HOBt
(0.19 g, 1.4 mmol) were suspended in anhydrous THF (9 mL) at r.t. DiPEA (0.7 mL, 4.2 mmol) was
then added to the reaction mixture, followed by piperazine (41) (0.05 g, 0.6 mmol). The reaction
mixture was left stirring at r.t. for 4 h. The organic solvent was then removed at reduced pressure and
the residue was suspended in EtOAc (100 mL). The organic layer was washed with saturated NaHCO3
solution (2 x 70 mL), then with saturated NH4Cl solution (2 x 70 mL) and finally with brine (70 mL).
The organic solvent was removed under vacuum after drying over MgSO4. The crude residue was
purified by flash column chromatography.
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4.1.3.1 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))bis(4-chlorobenzene-sulfonamide)
(11a)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 98:2 v/v. Obtained in 41% yield as a white solid. M.p. 206-208 °C. TLC (9:1 DCM-MeOH, Rf:
0.76). 1H-NMR (CDCl3),
2H), 7.52 (d, J= 8.5 Hz, 4H), 7.83 (d, J= 8.5 Hz, 4H). 13C-NMR (DMSO-d6),
δ
: 2.62 (t, J= 5.2 Hz, 4H), 3.33 (m, 4H), 3.43 (m, 4H), 3.56 (m, 4H), 7.1 (bs,
: 32.5, 38.8, 40.6, 40.9,
δ
44.3, 44.6, 128.4, 129.3, 137.2, 139.2, 168.6. Anal. Calcd for C22H26Cl2N4O6S2: C, 45.75; H, 4.54; N,
9.70. Found: C, 45.61; H, 4.81; N, 9.52. MS [ESI, m/z]: 598.9, 600.9 [M+Na].
4.1.3.2
N,N’-(3,3’-(Piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))bis(4-methoxybenzene-
sulfonamide) (11d)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 98:2 v/v. Obtained in 46% yield as a white solid. M.p. 178-180 °C. TLC (9:1 DCM-MeOH, Rf:
0.65). 1H-NMR (CDCl3),
6H), 5.42 (bs, 2H), 7.00 (d, J= 8.8 Hz, 4H), 7.82 (d, J= 8.8 Hz, 4H). 13C-NMR (CDCl3),
δ
: 2.59 (t, J= 5.6 Hz, 4H), 3.23 (m, 4H), 3.42 (m, 4H), 3.60 (m, 4H), 3.89 (s,
: 33.0, 33.1,
δ
39.0, 39.1, 41.2, 44.7, 44.9, 55.6, 114.3, 129.1, 131.7, 162.8, 168.8. Anal. Calcd for C24H32N4O8S2: C,
50.69; H, 5.67; N, 9.85. Found: C, 50.67; H, 5.92; N, 9.76. MS [ESI, m/z]: 591.1 [M+Na].
4.1.3.3
N,N’-(3,3’-(Piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))bis(3,4-dimethoxy-benzene
sulfonamide) (11f)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 98:2 v/v. Obtained in 47% yield as a white solid. M.p. 102-104 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.65). H-NMR (CDCl3),
δ
: 2.80 (bs, 4H), 3.21 (m, 4H), 3.55 (m, 4H), 3.95 (m, 12H), 4.04 (bs, 4H),
: 33.0,
5.25 (bs, 1H), 5.54 (bs, 1H), 6.97 (m, 2H), 7.33 (m, 2H), 7.49 (m, 2H). 13C-NMR (CDCl3),
δ
33.2, 38.8, 39.0, 41.1, 41.3, 44.9, 45.1, 56.2, 56.3, 56.4, 56.5, 109.6, 109.7, 110.6, 110.7, 120.8, 120.9,
129.9, 131.6, 149.2, 149.5, 152.0, 152.7, 169.8, 171.8. Anal. Calcd for C26H36N4O10S2: C, 49.67; H,
5.77; N, 8.91. Found: C, 49.51; H, 6.03; N, 8.80. MS [ESI, m/z]: 651.1 [M+Na].
4.1.3.4
N,N’-(3,3’-(Piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))bis(2,5-dimethoxy-benzene
sulfonamide) (11g)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 52% yield as a white solid. M.p. 90-92 °C. TLC (9:1 DCM-MeOH, Rf:
0.63). 1H-NMR (CDCl3),
6H), 3.97 (s, 6H), 5.92 (t, J= 6.1 Hz, 2H), 7.00 (d, J= 9.0 Hz, 2H), 7.09 (dd, J1= 9.0 Hz, J2= 3.0 Hz,
2H), 7.46 (d, J= 3.0 Hz, 2H). 13C-NMR (DMSO-d6),
: 32.2, 40.5, 40.8, 44.2, 44.4, 55.7, 56.5, 114.2,
δ: 2.55 (t, J= 6.0 Hz, 4H), 3.22 (m, 4H), 3.37 (m, 4H), 3.60 (m, 4H), 3.84 (s,
δ
114.3, 119.5, 128.2, 150.1, 152.3, 168.9. Anal. Calcd for C26H36N4O10S2: C, 49.67; H, 5.77; N, 8.91.
Found: C, 49.61; H, 5.93; N, 8.89. MS [ESI, m/z]: 651.1 [M+Na].
4.1.3.5 N,N’-(3,3’-(Piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))dinaphthalene-2-sulfonamide (11h)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
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MeOH 97:3 v/v. Obtained in 54% yield as a white solid. M.p. 228-230 °C. TLC (9:1 DCM-MeOH, Rf:
0.79). 1H-NMR (CDCl3),
(dd, J1= 8.7 Hz, J2= 1.7 Hz, 2H), 7.93 (d, J= 7.9 Hz, 2H), 7.99 (m, 4H), 8.45 (s, 2H). 13C-NMR
(DMSO-d6), : 32.5, 38.9, 40.5, 40.8, 44.2, 44.4, 122.2, 127.3, 127.5, 127.7, 128.6, 129.1, 129.3, 131.6,
δ: 2.55 (m, 4H), 3.28 (m, 8H), 3.52 (m, 4H), 5.61 (bs, 2H), 7.66 (m, 4H), 7.86
δ
134.11, 137.3, 168.5. Anal. Calcd for C30H32N4O6S2: C, 59.19; H, 5.30; N, 9.20. Found: C, 58.84; H,
5.61; N, 9.96. MS [ESI, m/z]: 631.0 [M+Na].
4.1.4 Synthesis of {4-[4-(4-tert-Butoxycarbonylamino-butyl)-piperazin-1-yl]-butyl}-carbamic acid
tert-butyl ester (16)
Piperazine (0.17 g, 2.1 mmol) and NaHCO3 (0.38, 4.5 mmol) were suspended in 3 mL of absolute
ethanol and added dropwise of 79 (1.21 g, 4.5 mmol) diluted in 5 mL of ethanol. The reaction mixture
was stirred under reflux for 24 h. The solvent was evaporated under vacuum, and the residue was
suspended in DCM (30 mL) and washed with saturated NaHCO3 solution (2 x 30 mL) and brine (30
mL). The organic phase was evaporated at reduced pressure after drying over MgSO4 to give a yellow
1
solid in 70% yield. H-NMR (CDCl3),
δ
: 1.37 (m, 18 H), 1.63 (m, 8H), 2.32 (bs, 4H), 2.56 (bs, 8H),
3.33 (t, J= 5.3 Hz, 4H), 5.56 (bs, 2H). 13C-NMR (CDCl3),
δ: 24.4, 25.7, 28.4, 28.5, 40.5, 53.0, 58.0,
78.8, 156.0. MS [ESI, m/z]: 397.3 [M+H].
4.1.5 N,N’-(4,4’-(Piperazine-1,4-diyl)bis(butane-4,1-diyl))bis(4-chlorobenzene-sulfonamide) (17)
Compound 16 (0.63 g, 1.5 mmol) was dissolved at 0 °C in a mixture of 2 mL of TFA and 0.2 mL of
distilled water. The reaction mixture was left stirring at r.t for 30 min., then the volume was reduced
under vacuum and the remaining suspension was poured dropwise into 5 mL of ice-cooled diethyl
ether. The resulting white precipitate was left in the fridge o.n. before being filtered and washed with
cold diethyl ether. The white solid was suspended with 4-chlorobenzenesulfonyl chloride (2a) (0.63 g,
3.0 mmol) in 7 mL of anhydrous DCM; the resulting mixture was treated dropwise with triethylamine
(1.3 mL, 9.5 mmol) under ice-cooling and stirred for 1 h at r.t. The reaction mixture was then diluted
with DCM and washed with water (2 x 30 mL) and brine (30 mL). The organic solvent was removed
under vacuum after drying over MgSO4 and the crude residue was purified by flash column
chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-MeOH 91:9 v/v to give a
1
white solid in 15% yield. M.p. 182-184 °C. TLC (9:1 DCM-MeOH, Rf: 0.64). H-NMR (CDCl3),
δ
:
1.63 (m, 8H), 2.50 (bm, 4H), 2.77 (bm, 8H), 2.96 (t, J= 5.3 Hz, 4H), 7.49 (d, J= 8.6 Hz, 4H), 7.81 (d,
J= 8.6 Hz, 4H). 13C-NMR (CDCl3),
: 24.1, 28.2, 42.8, 51.8, 57.7, 128.4, 129.2, 138.7, 139.2. Anal.
δ
Calcd for C24H34 Cl2N4O4S2: C, 49.91; H, 5.93; N, 9.70. Found: C, 49.67; H, 6.11; N, 9.61. MS [ESI,
m/z]: 577.1, 579.1 [M+H].
4.1.6 Synthesis of N,N’-(3,3’-(piperazine-1,4-diyl)bis(propane-3,1-diyl))bis(4-chlorobenzamide)
(20) [38]
A mixture of 19 (0.5 g, 1.8 mmol), piperazine (0.07 g, 0.8 mmol) and triethylamine (0.24 mL, 1.7
mmol) in 7 mL of anhydrous THF was stirred under nitrogen atmosphere for 48 h at r.t. The reaction
mixture was then diluted with DCM (20 mL), washed with saturated NaHCO3 solution (2 x 30 mL) and
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brine (30 mL) and dried over MgSO4. The organic solvent was removed under vacuum and the crude
residue was purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing
to DCM-MeOH 91:9 v/v to give a white solid in 36% yield. M.p. 180-182 °C. TLC (9:1 DCM-MeOH,
1
Rf: 0.63). H-NMR (CDCl3),
δ
: 1.82 (m, 4H), 2.56 (m, 12H), 3.59 (m, 4H), 7.42 (d, J= 8.5 Hz, 4H),
: 26.1, 37.9, 52.7, 55.6, 128.2, 129.0,
7.78 (d, J= 8.5 Hz, 4H), 8.05 (bs, 2H). 13C-NMR (DMSO-d6),
δ
133.3, 135.7, 164.9. Anal. Calcd for C24H30Cl2N4O2: C, 60.38; H, 6.33; N, 11.74. Found: C, 60.58; H,
6.51; N, 11.90. MS [ESI, m/z]: 477.1, 479.1 [M+H].
4.1.7
Synthesis
of
N,N’-(2,2’-(1,4-phenylenebis(azanediyl))bis(ethane-2,1-diyl))bis(4-
chlorobenzenesulfonamide) (21)
A mixture of 7a (1.05 g, 3.5 mmol), 4-phenylendiamine (0.16 g, 1.5 mmol) and triethylamine (0.47
mL, 3.4 mmol) in 13 mL of anhydrous THF was stirred under nitrogen atmosphere for 72 h at r.t. The
reaction mixture was then diluted with DCM (35 mL), washed with saturated NaHCO3 solution (2 x 30
mL) and brine (30 mL) and dried over MgSO4. The organic solvent was removed under vacuum and
the crude residue was purified by flash column chromatography eluting with n-hexane-EtOAc 100:0
v/v increasing to n-hexane-EtOAc 20:80 v/v to afford a light brown solid in 17% yield. M.p. 162-164
1
°C. TLC (8:2 EtOAc-nhexane, Rf: 0.46). H-NMR (DMSO-d6),
δ
: 2.87 (t, J= 6.5 Hz, 4H), 2.97 (m,
4H), 4.70 (t, J= 6.1 Hz, 2H), 6.32 (s, 4H), 7.65 (d, J= 8.6 Hz, 4H), 7.79 (d, J= 8.6 Hz, 4H), 7.81 (bs,
2H). 13C-NMR (DMSO-d6),
: 41.8, 43.6, 113.7, 128.3, 129.3, 137.2, 139.3, 139.6. Anal. Calcd for
δ
C22H24Cl2N4O4S2: C, 48.62; H, 4.45; N, 10.31. Found: C, 48.81; H, 4.39; N, 10.38. MS [ESI, m/z]:
543.0, 545.0 [M+H].
4.1.8 General method for the preparation of unsymmetrical sulfonamides 23-36
A mixture of the different N-(3-piperazin-1-yl-propyl)-arylsulfonamide 22 (1.0 mmol), N-(3-
bromopropyl)arylsulfonamide 3 or N-(2-bromoethyl)arylsulfonamide 2 (1.5 mmol) and NaHCO3 (0.16
g, 2 mmol) in absolute ethanol (7 mL) was stirred under reflux for 24 h. The solvent was evaporated
under reduced pressure and the crude residue was purified by flash column chromatography.
4.1.8.1
N-{3-[4-(3-Benzenesulfonylamino-propyl)-piperazin-1-yl]-propyl}-4-chloro-benzene
sulfonamide (23)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 58% yield as a white solid. M.p. 128-130 °C. TLC (9:1 DCM-MeOH, Rf:
1
0.59). H-NMR (DMSO-d6),
δ
: 1.44-1.51 (m, 4H), 2.11-2.26 (m, 12H), 2.74-2.79 (m, 4H), 7.56-7.62
: 26.0,
(m, 3H), 7.61-7.64 (m, 1H), 7.66-7.70 (m, 3H), 7.78 (d, J= 8.3 Hz, 2H). 13C-NMR (DMSO-d6),
δ
26.1, 40.8, 40.9, 52.5, 54.7, 54.8, 126.4, 128.4, 129.1, 129.3, 132.2, 137.1, 139.3, 140.5. Anal. Calcd
for C22H31ClN4O4S2: C, 51.30; H, 6.07; N, 10.88. Found: C, 51.19; H, 6.35; N, 10.67. MS [ESI, m/z]:
515.1, 517.1 [M+H].
4.1.8.2 4-Chloro-N-(3-(4-(3-(4-methylbenzenesulfonylamido)propyl)-piperazin-1-yl)-propyl)benzene
sulfonamide (24)
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Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 86% yield as a white solid. M.p. 172-174 °C. TLC (DCM-MeOH 9:1 v/v,
Rf: 0.57). 1H-NMR (DMSO-d6),
4H), 7.39 (d, J= 8.1 Hz, 2H),7.48 (t, J= 5.6 Hz, 1H), 7.64-7.71 (m, 5H), 7.79 (d, J= 8.7 Hz, 2H). 13C-
NMR (DMSO-d6), : 20.9, 26.0, 40.8, 40.9, 52.5, 54.7, 54.8, 126.5, 128.4, 129.3, 129.5, 137.1, 137.6,
δ: 1.43-1.51 (m, 4H), 2.10-2.28 (m, 12H), 2.38 (s, 3H), 2.69-2.81 (m,
δ
139.4, 142.4. Anal. Calcd for C23H33ClN4O4S2: C, 52.21; H, 6.29; N, 10.59. Found: C, 52.09; H, 5.94;
N, 10.58. MS [ESI, m/z]: 529.1, 531.1 [M+H].
4.1.8.3 4-tert-Butyl-N-(3-(4-(3-(4-chlorophenylsulfonylamido)propyl)-piperazin-1-yl)-propyl)benzene
sulfonamide (25)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 60% yield as a white solid. M.p. 130-132 °C. TLC (DCM-MeOH 9:1 v/v,
Rf: 0.44). 1H-NMR (DMSO-d6),
4H), 7.49 (t, J= 5.5 Hz, 1H), 7.60 (d, J= 8.5 Hz, 2H), 7.65-7.72 (m, 5H), 7.78 (d, J= 8.5 Hz, 2H). 13C-
NMR (DMSO-d6), : 26.0, 30.7, 34.7, 40.8, 40.9, 52.5, 52.6, 54.7, 54.8, 125.9, 126.3, 128.4, 129.3,
δ: 1.30 (s, 9H), 1.41-1.53 (m, 4H), 2.11-2.35 (m, 12H), 2.72-2.80 (m,
δ
137.1, 137.7, 139.4, 155.1. Anal. Calcd for C26H39ClN4O4S2: C, 54.67; H, 6.88; N, 9.80. Found: C,
54.51; H, 6.49; N, 9.61. MS [ESI, m/z]: 571.2, 573.2 [M+H].
4.1.8.4 4-Chloro-N-(3-(4-(3-(4-(trifluoromethyl)phenylsulfonylamido)propyl)-piperazin-1-yl)-propyl)
benzenesulfonamide (26)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 85% yield as a white solid. M.p. 158-160 °C. TLC (DCM-MeOH 9:1 v/v,
Rf: 0.61). 1H-NMR (DMSO-d6),
J= 8.5 Hz, 2H), 7.69 (bs, 1H), 7.78 (d, J= 8.5 Hz, 2H), 7.86 (bs, 1H), 7.94-8.03 (m, 4H). 13C-NMR
(DMSO-d6), : 26.0, 40.7, 40.8, 52.5, 54.6, 54.7, 126.4 (q, J= 3.7 Hz), 127.4, 128.4, 129.2, 131.8,
δ: 1.44-1.51 (m, 4H), 2.11-2.27 (m, 12H), 2.74-2.83 (m, 4H), 7.67 (d,
δ
131.9, 137.1, 139.4, 144.5. Anal. Calcd for C23H30ClF3N4O4S2: C, 47.38; H, 5.19; N, 9.61. Found: C,
47.32; H, 5.17; N, 9.48. MS [ESI, m/z]: 583.1, 585.1 [M+H].
4.1.8.5
N-(3-(4-(3-(4-Chlorophenylsulfonylamido)propyl)-piperazin-1-yl)-propyl)biphenyl-4-
sulfonamide (27)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 71% yield as a white solid. M.p. 138-140 °C. TLC (DCM-MeOH 9:1 v/v,
1
Rf: 0.63). H-NMR (DMSO-d6),
δ
: 1.42-1.54 (m, 4H), 2.15-2.31 (m, 12H), 2.72-2.81 (m, 4H), 7.44 (t,
J= 5.7 Hz, 1H), 7.50-7.54 (m, 2H), 7.62 (t, J= 5.7 Hz, 1H), 7.65-7.79 (m, 3H), 7.74 (d, J= 7.1 Hz, 2H),
7.78 (d, J= 8.6 Hz, 2H), 7.85 (d, J= 8.6 Hz, 2H), 7.89 (d, J= 8.6 Hz, 2H). 13C-NMR (DMSO-d6),
δ
:
26.0, 26.1, 40.8, 40.9, 52.5, 54.7, 54.8, 127.0, 127.1, 127.3, 128.4, 129.0, 129.3, 137.1, 138.5, 139.2,
139.3, 143.8. Anal. Calcd for C28H35ClN4O4S2: C, 56.89; H, 5.97; N, 9.48. Found: C, 56.73; H, 6.08; N,
9.30. MS [ESI, m/z]: 591.1, 593.1 [M+H].
4.1.8.6
4-tert-Butyl-N-(3-(4-(3-(phenylsulfonylamido)propyl)-piperazin-1-yl)-propyl)benzene
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sulfonamide (28)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 54% yield as a white solid. M.p. 134-136 °C. TLC (DCM-MeOH 9:1 v/v,
Rf: 0.55). 1H-NMR (DMSO-d6),
4H), 7.49 (t, J= 5.6 Hz, 1H), 7.58-7.62 (m, 5H), 7.66-7.69 (m, 1H), 7.70 (d, J= 8.5 Hz, 2H), 7.78 (d, J=
7.8 Hz, 2H). 13C-NMR (DMSO-d6),
: 26.0, 26.1, 30.7, 34.7, 40.8, 40.9, 52.5, 52.6, 54.8, 54.9, 125.9,
δ: 1.30 (s, 9H), 1.43-1.49 (m, 4H), 2.11-2.26 (m, 12H), 2.72-2.78 (m,
δ
126.3, 126.4, 129.1, 132.2, 137.6, 140.5, 155.2. Anal. Calcd for C26H40N4O4S2: C, 58.18; H, 7.51; N,
10.44. Found: C, 58.35; H, 7.46; N, 10.47. MS [ESI, m/z]: 537.2 [M+H].
4.1.8.7 4-tert-Butyl-N-(3-(4-(3-(4-methylphenylsulfonylamido)propyl)-piperazin-1-yl)-propyl)benzene
sulfonamide (29)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 83% yield as a white solid. M.p. 155-157 °C. TLC (DCM-MeOH 9:1 v/v,
1
Rf: 0.64). H-NMR (DMSO-d6),
δ
: 1.30 (s, 9H), 1.43-1.49 (m, 4H), 2.09-2.24 (m, 12H), 2.38 (s, 3H),
2.70-2.78 (m, 4H), 7.39 (d, J= 8.1 Hz, 2H), 7.49 (bs, 2H), 7.60 (d, J= 8.5 Hz, 2H), 7.66 (d, J= 8.1 Hz,
2H), 7.70 (d, J= 8.5 Hz, 2H). 13C-NMR (DMSO-d6),
: 20.9, 26.0, 30.7, 34.7, 40.8, 40.9, 52.5, 52.6,
δ
54.8, 54.9, 125.9, 126.3, 126.5, 129.5, 137.6, 137.7, 142.4, 155.1. Anal. Calcd for C27H42N4O4S2: C,
58.88; H, 7.69; N, 10.17. Found: C, 59.07; H, 7.92; N, 10.23. MS [ESI, m/z]: 573.2 [M+Na].
4.1.8.8
4-tert-Butyl-N-(3-(4-(3-(4-(trifluoromethyl)phenylsulfonylamido)propyl)-piperazin-1-yl)-
propyl)benzenesulfonamide (30)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 95:5 v/v. Obtained in 70% yield as a white solid. M.p. 156-158 °C. TLC (DCM-MeOH 9:1 v/v,
Rf: 0.76). 1H-NMR (DMSO-d6),
2H), 2.78-2.82 (m, 2H), 7.49 (t, J= 5.5 Hz, 1H), 7.60 (d, J= 8.4 Hz, 2H), 7.70 (d, J= 8.4 Hz, 2H), 7.86
(bs, 1H), 7.97-9.01 (m, 4H). 13C-NMR (DMSO-d6),
: 26.0, 30.7, 34.7, 40.8, 40.9, 52.5, 52.6, 54.6,
δ: 1.30 (s, 9H), 1.42-1.51 (m, 4H), 2.09-2.26 (m, 12H), 2.73-2.77 (m,
δ
54.8, 125.9, 126.3, 126.4, 126.5, 127.4, 132.2, 137.7, 144.5, 155.1. Anal. Calcd for C27H39F3N4O4S2: C,
53.62; H, 6.50; N, 9.26. Found: C, 53.46; H, 6.71; N, 9.25. MS [ESI, m/z]: 605.2 [M+H].
4.1.8.9
N-(3-(4-(3-(4-tert-Butylphenylsulfonylamido)propyl)-piperazin-1-yl)-propyl)
biphenyl-4-
sulfonamide (31)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 58% yield as a white solid. M.p. 146-148 °C. TLC (DCM-MeOH 9:1 v/v,
Rf: 0.62). 1H-NMR (DMSO-d6),
4H), 7.44 (t, J= 7.5 Hz, 1H), 7.47-7.53 (m, 3H), 7.58-7.64 (m, 3H), 7.70 (d, J= 8.5 Hz, 2H), 7.74 (d, J=
7.2 Hz, 2H), 7.85 (d, J= 8.5 Hz, 2H), 7.89 (d, J= 8.5 Hz, 2H). 13C-NMR (DMSO-d6),
: 26.0, 26.1,
δ: 1.29 (s, 9H), 1.41-1.53 (m, 4H), 2.07-2.25 (m, 12H), 2.71-2.83 (m,
δ
30.7, 34.7, 40.8, 40.9, 52.5, 52.6, 54.8, 125.9, 126.3, 127.0, 127.2, 127.3, 128.4, 129.1, 137.7, 138.5,
139.2, 143.7, 155.1. Anal. Calcd for C32H44N4O4S2: C, 62.71; H, 7.24; N, 9.14. Found: C, 62.69; H,
7.32; N, 9.09. MS [ESI, m/z]: 613.2 [M+H].
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4.1.8.10
4-Methyl-N-(3-(4-(3-(phenylsulfonylamido)propyl)-piperazin-1-yl)-propyl)benzene
sulfonamide (32)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 95:5 v/v. Obtained in 52% yield as a white solid. M.p. 137-139 °C. TLC (DCM-MeOH 9:1 v/v,
Rf: 0.65). 1H-NMR (DMSO-d6),
4H), 7.39 (d, J= 8.1 Hz, 2H), 7.48 (t, J= 5.4 Hz, 1H), 7.56-7.62 (m, 3H), 7.66 (d, J= 8.1 Hz, 2H), 7.61-
7.64 (m, 1H), 7.78 (d, J= 7.5 Hz, 2H). 13C-NMR (DMSO-d6),
: 20.9, 26.0, 26.1, 40.9, 52.6, 54.8,
δ: 1.43-1.51 (m, 4H), 2.13-2.26 (m, 12H), 2.38 (s, 3H), 2.70-2.79 (m,
δ
126.4, 126.5, 129.2, 129.5, 132.2, 137.6, 140.5, 142.4. Anal. Calcd for C23H34N4O4S2: C, 55.84; H,
6.93; N, 11.33. Found: C, 55.73; H, 7.18; N, 11.20. MS [ESI, m/z]: 495.2 [M+H].
4.1.8.11
N-(3-(4-(3-(4-Methylphenylsulfonylamido)propyl)-piperazin-1-yl)-propyl)biphenyl-4-
sulfonamide (33)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 67% yield as a white solid. M.p. 134-136 °C. TLC (DCM-MeOH 9:1 v/v,
Rf: 0.58). 1H-NMR (DMSO-d6),
δ: 1.42-1.54 (m, 4H), 2.13-2.25 (m, 12H), 2.37 (s, 3H), 2.69-2.74 (m,
2H), 2.78-2.83 (m, 2H), 7.38 (d, J= 8.1 Hz, 2H), 7.44 (t, J= 7.3 Hz, 1H), 7.47 (t, J= 5.5 Hz, 1H), 7.49-
7.53 (m, 2H), 7.63 (t, J= 5.5 Hz, 1H), 7.66 (d, J= 8.2 Hz, 2H), 7.72-7.75 (m, 2H), 7.86 (d, J= 8.5 Hz,
2H), 7.89 (d, J= 8.5 Hz, 2H). 13C-NMR (DMSO-d6),
δ: 20.9, 26.0, 26.1, 40.9, 52.6, 54.8, 54.9, 126.5,
127.0, 127.2, 127.3, 128.4, 129.1, 129.6, 137.6, 138.5, 139.3, 142.5, 143.8. Anal. Calcd for
C29H38N4O4S2: C, 61.03; H, 6.71; N, 9.82. Found: C, 60.86; H, 6.52; N, 9.74. MS [ESI, m/z]: 571.2
[M+H].
4.1.8.12 N-(3-(4-(3-(Phenylsulfonamido)propyl)-piperazin-1-yl)-propyl)biphenyl-4-sulfonamide (34)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 71% yield as a white solid. M.p. 124-126 °C. TLC (DCM-MeOH 9:1 v/v,
1
Rf: 0.47). H-NMR (DMSO-d6),
δ
: 1.42-1.54 (m, 4H), 2.11-2.25 (m, 12H), 2.73-2.77 (m, 2H), 2.78-
2.82 (m, 2H), 7.44 (t, J= 7.3 Hz, 1H), 7.50-7.53 (m, 2H), 7.56-7.61 (m, 3H), 7.61-7.66 (m, 2H), 7.70
(d, J= 7.4 Hz, 2H), 7.77-7.79 (m, 2H), 7.85 (d, J= 8.5 Hz, 2H), 7.89 (d, J= 8.5 Hz, 2H). 13C-NMR
(DMSO-d6), δ: 26.0, 40.9, 52.5, 54.8, 54.9, 126.4, 127.0, 127.2, 127.4, 128.4, 129.0, 129.1, 132.3,
138.5, 139.3, 142.5, 143.8. Anal. Calcd for C28H36N4O4S2: C, 60.41; H, 6.52; N, 10.06. Found: C,
60.27; H, 6.81; N, 9.87. MS [ESI, m/z]: 557.2 [M+H].
4.1.8.13 N-(3-(4-(3-(Phenylsulfonamido)propyl)-piperazin-1-yl)-propyl)-4-(trifluoromethyl) benzene
sulfonamide (35)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 50% yield as a white solid. M.p. 119-121 °C. TLC (DCM-MeOH 9:1 v/v,
1
Rf: 0.72). H-NMR (DMSO-d6),
δ
: 1.43-1.51 (m, 4H), 2.11-2.23 (m, 12H), 2.73-2.77 (m, 2H), 2.78-
2.82 (m, 2H), 7.58-7.61 (m, 4H), 7.62-7.66 (m, 2H), 7.77-7.79 (m, 2H), 7.88 (bs, 1H), 8.00 (s, 4H).
13C-NMR (DMSO-d6),
: 26.0, 40.8, 40.9, 52.5, 54.6, 54.8, 126.4, 126.5, 127.4, 129.1, 132.2, 132.3,
137.6, 144.5. Anal. Calcd for C23H31F3N4O4S2: C, 50.35; H, 5.70; N, 10.21. Found: C, 50.51; H, 5.53;
δ
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N, 10.34. MS [ESI, m/z]: 549.2 [M+H].
4.1.8.14 4-Chloro-N-(3-(4-(2-(4-chlorophenylsulfonamido)ethyl)-piperazin-1-yl)-propyl) benzene
sulfonamide (36a)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 72% yield as a white solid. M.p. 133-135 °C. TLC (DCM-MeOH 9:1 v/v,
1
Rf: 0.46). H-NMR (DMSO-d6),
δ
: 1.43-1.51 (m, 2H), 2.07-2.27 (m, 12H), 2.73-2.80 (m, 2H), 2.82-
: 26.0, 40.0, 40.8, 52.4,
2.87 (m, 2H), 7.58-7.73 (m, 6H), 7.76-7.83 (m, 4H). 13C-NMR (DMSO-d6),
δ
52.5, 54.7, 56.7, 128.4, 128.4, 129.2, 129.3, 137.0, 137.1, 139.3, 139.6. Anal. Calcd for
C21H28Cl2N4O4S2: C, 47.10; H, 5.27; N, 10.46. Found: C, 47.23; H, 5.54; N, 10.50. MS [ESI, m/z]:
535.1, 537.1 [M+H].
4.1.8.15 N-(3-(4-(2-(Phenylsulfonamido)ethyl)-piperazin-1-yl)-propyl) benzenesulfonamide (36b)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 97:3 v/v. Obtained in 47% yield as a white solid. M.p. 135-137 °C. TLC (DCM-MeOH 9:1 v/v,
1
Rf: 0.69). H-NMR (DMSO-d6),
δ
: 1.43-1.49 (m, 2H), 2.14-2.28 (m, 12H), 2.73-2.78 (m, 2H), 2.81-
2.86 (m, 2H), 7.49 (bs, 1H), 7.55-7.62 (m, 5H), 7.61-7.66 (m, 2H), 7.76-7.82 (m, 4H). 13C-NMR
(DMSO-d6), : 26.0, 40.0, 40.9, 52.4, 52.5, 54.8, 56.7, 126.4, 129.1, 129.2, 132.2, 140.4, 140.5. Anal.
δ
Calcd for C21H30N4O4S2: C, 54.05; H, 6.48; N, 12.01. Found: C, 53.82; H, 6.45; N, 12.13. MS [ESI,
m/z]: 467.1 [M+H].
4.1.8.16 4-tert-Butyl-N-(3-(4-(2-(4-tert-butylphenylsulfonamido)ethyl)-piperazin-1-yl)-propyl) benzene
sulfonamide (36l)
Purified by flash column chromatography eluting with DCM-MeOH 100:0 v/v increasing to DCM-
MeOH 96:4 v/v. Obtained in 53% yield as a pale yellow solid. M.p. 86-88 °C. TLC (DCM-MeOH 9:1
1
v/v, Rf: 0.55). H-NMR (DMSO-d6),
δ
: 1.30 (s, 9H), 1.31 (s, 9H), 1.41-1.49 (m, 2H), 2.05-2.21 (m,
12H), 2.71-2.77 (m, 2H), 2.78-2.84 (m, 2H), 7.40 (bs, 1H), 7.49 (t, J= 5.2 Hz, 1H), 7.58-7.62 (m, 4H),
7.68-7.73 (m, 4H). 13C-NMR (DMSO-d6),
: 26.0, 30.7, 39.0, 40.0, 40.8, 52.4, 52.5, 54.8, 56.7, 125.8,
δ
125.9, 126.0, 126.3, 137.6, 137.7, 155.1. Anal. Calcd for C29H46N4O4S2: C, 60.17; H, 8.01; N, 9.68.
Found: C, 60.11; H, 8.22; N, 9.69. MS [ESI, m/z]: 579.2 [M+H].
4.1.9
General method for the preparation of amides 37
The different 3-arylsulfonylamino propionic acid 10a-c, l, n (1.1 mmol) and TBTU (0.38 g, 1.2 mmol)
were suspended in anhydrous THF (6 mL) at r.t. DiPEA (0.4 mL, 2.4 mmol) was then added to the
reaction mixture, followed by the different N-(3-piperazin-1-yl-propyl)-arylsulfonamide 22a-c, l, n (1
mmol). The reaction mixture was left stirring at r.t. for 4 h. The organic solvent was then removed at
reduced pressure and the residue was diluted with EtOAc (30 mL). The organic layer was washed with
saturated NaHCO3 solution (2 x 30 mL) and brine (30 mL). The organic solvent was removed under
vacuum after drying over MgSO4. The crude residue was purified by flash column chromatography.
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