Cytotoxic 1,2,3-Triazoles as Potential Leads Targeting the S100A2-p53 Complex: Synthesis and Cytotoxicity

Article abstract of DOI:10.1002/cmdc.202000950

In silico screening predicted 1 (N-(4-((4-(3-(4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)piperazin-1-yl) sulfonyl)-phenyl)acetamide) as an inhibitor of the S100A2-p53 protein-protein interaction. S100A2 is a validated pancreatic cancer drug target.

Full text of DOI:10.1002/cmdc.202000950

Accepted Article
Title: Novel cytotoxic 1,2,3-triazoles as potential new leads targeting
the S100A2-p53complex
Authors: Jufeng Sun, Jennifer R Baker, Cecilia C Russell, Peter
J Cossar, Hong Ngoc Thuy Pham, Jennette A Sakoff,
Christopher J Scarlett, and Adam McCluskey
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000950
Link to VoR: https://doi.org/10.1002/cmdc.202000950
01/2020

10.1002/cmdc.202000950
ChemMedChem
FULL PAPER
Targeting the S100A2-p53 interactions with a series of novel 3,5-
bis(trifluoromethyl)benzene sulfonamides: Synthesis and cytotoxicity
Jufeng Sun,[a,b] Joey I Ambrus, [a] Cecilia C Russell,[a] Jennifer R Baker,[a] Peter J Cossar,[a] Melanie J Pirinen,[c]
Jennette A. Sakoff *[d] Christopher J Scarlett,[c] and Adam McCluskey *[a]
[a]
Ms J Sun (0000-0002-5165-9396), Dr J Ambrus (0000-0002-9560-301X), Dr CC Russell (0000-0002-4140-7785), Dr JR Baker (0000-0002-9560-301X), Dr PJ Cossar (0000-
0002-8260-5710) and Prof A McCluskey (0000-0001-7125-863X)
Chemistry, School of Environmental & Life Sciences
The University of Newcastle
University Drive, Callaghan, NSW 2308, Australia
E-mail: Adam.McCluskey@newcstle.edu.au
[b]
[c]
Medicinal Chemistry, School of Pharmacy,
Binzhou Medical University, Yantai, 264003, China
Ms MJ Predebon (0000-0002-2847-7818) and Prof CJ Scarlett (0000-0002-4140-7785)
School of Environmental & Life Sciences,
The University of Newcastle, Ourimbah NSW 2258, Australia.
Dr JA Sakoff (0000-000207009-5792)
[d]
Experimental Therapeutics Group, Department of Medical Oncology
Calvary Mater Newcastle Hospital
Edith Street, Waratah NSW 2298, Australia.
Supporting information for this article is given via a link at the end of the document.
Abstract: In silico approaches identified
1
, N-(6-((4-bromo-
endocrine cells that produce hormones. Of the two classes,
pancreatic ductal adenocarcinoma (PDAC) is the most
common cancer subtype, covering about 90% of all exocrine
tumours.3 Current combination chemotherapies for PDAC are
highly toxic and only a fraction of patients respond (~30%). As
a result, new targeted therapies which are effective against
PDAC are urgently needed.
One potentially promising new oncogenic target for the
treatment of PC is the S100 calcium binding protein A2
(S100A2), a member of the S100 protein family. S100A2 was
first isolated in 1989 and classified as a member of the S100
protein family.4 Since this time there have been over 25 S100
class protein identified. Of these S100A2 belongs to a highly
homologous sub-group that comprises A100A3, S100A4,
S100A5 and S100A6.5-7 The S100A2 gene encodes a protein of
98 amino acid residues, with a molecular mass of ~11 kDa, and
is characterised by four helices, two distinct calcium-binding
EF-hand motifs, a central hinge region and C- and N-terminal
variable domains.8 Like other S100 proteins, S100A2 occurs as
a homodimer in vitro and in vivo.6, 7 S100A2 displays a direct
interaction with the C-terminal of wildtype p53.9, 10
benzyl)amino)hexyl)-3,5-bis(trifluoromethyl)benzene sulfonamide,
as a potential inhibitor of S100A2-p53 protein-protein interaction, a
validated pancreatic cancer drug target. Subsequent cytotoxicity
screening revealed it to be a 2.97 µM cell growth inhibitor of the
MiaPaCa-2 pancreatic cell line. This is in keeping with our
hypothesis that inhibiting this interaction would have an anti-
pancreatic cancer effect with S100A2, the validated PC drug target.
A
combination of focused library synthesis (3 libraries, 24
compounds total) and cytotoxicity screening identified a propyl alkyl
diamine spacer as optimal; the nature of the terminal phenyl
substituent had limited impact on observed cytotoxicity, whereas N-
methylation was detrimental to activity. In total 15 human cancer
cell lines were examined, with most analogues showing broad
spectrum activity. Near uniform activity was observed against a
panel of six pancreatic cancer cell lines: MiaPaCa-2, BxPC-3, AsPC-1,
Capan-2, HPAC and PANC-1. In all cases there was good to excellent
correlation between the predicted docking pose in the S100A2-p53
binding groove and the observed cytotoxicity, especially in the
pancreatic cancer cell line with high endogenous S100A2 expression.
This supports S100A2 as a pancreatic cancer drug target.
The S100A2 protein forms homodimers and are regulated
by Ca2+; this enables the proteins to act as sensors which
respond to variations in Ca2+ concentrations.11 The binding of
calcium by S100A2 results in the activation of p53
transcriptional activity.9 S100A2 binds to residues 293-393 of
monomeric p53 in a ratio of one S100A2 dimer to one p53
monomer. In vitro, it also binds to tetrameric p53, but only
with low affinity.12-14
Introduction
Pancreatic cancer (PC) is one of the most lethal human
cancers. This cancer has a depressingly low five-year survival
rate of only 7%.1 It is also projected that pancreatic cancer will
be the second leading cause of cancer death by 2030.2 The
highly metastatic nature of PC presents a major challenge for
improving patient outcomes. These heterogeneous tumours
are composed of cancer cells with differing morphologies and
phenotypic profiles, making them extremely difficult to treat
as they evade targeted therapies. PC is divided into two main
classes: exocrine tumours deriving from the cells of the
exocrine pancreas and accounting for more than 95% of all
pancreatic tumours; and endocrine tumours coming from
It has been demonstrated that moderate and high levels of
S100A2 expression in pancreatic cancer is a poor prognosis marker
even after surgical resection. Patients displaying low levels of
S100A2 in pancreatic cancer had a survival benefit post-surgery,
even in the presence of lymph node metathesis.15 S100A2 is
upregulated in the aggressive and poor prognosis squamous
subtype of PDAC and is a predictive biomarker of this disease
1
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10.1002/cmdc.202000950
ChemMedChem
FULL PAPER
state.15, 16, 17 It is found in high concentrations in the nucleus of cells,
which is unique in the family of S100 proteins, and is associated
with the regulation of cell cycling.17 S100A2 modulates the tumour
suppressor p53 by binding with its transactivation domain. In
pancreatic cancer, over expression of S100A2 inhibits p53,
preventing p53 tumour suppression, resulting in aberrant cancer
cell proliferation.18 Importantly, the inhibition of the S100 protein
family binding to p53 has been the focus of drug discovery
campaigns.
family binding to p53 has been the focus of drug discovery
campaigns.
Results and Discussion
In silico analysis of the S100A2-p53 complex, using the
Molecular Operating Environment (MOE) software system
identified a surface pocket within S100A2, into which the p53
peptide showed good docking (Figure 1A). This “p53-groove”,
represents
a protein-protein ‘hot-spot area’ potentially
suitable for small molecule binding.23
S100A2 is upregulated in the aggressive and poor
prognosis squamous subtype of PDAC and is a predictive
Subsequent in silico screening of an in-house proprietary
20
biomarker of this disease state.19,
It is found in high
library of about 10,000 compounds identified sulfonamide
which was predicted to block the interaction between S100A2
and p53 (Figure 1). MTT cytotoxicity evaluation revealed to
1,
concentrations in the nucleus of cells, which is unique in the
family of S100 proteins, and is associated with the regulation
of cell cycling.21 S100A2 modulates the tumour suppressor p53
by binding with its transactivation domain. In pancreatic
cancer, over expression of S100A2 inhibits p53, preventing
p53 tumour suppression, resulting in aberrant cancer cell
proliferation.22 Importantly, the inhibition of the S100 protein
1
be a GI50 2.97 ± 0.03 µM inhibitor of the MiaPaCa-2 pancreatic
cancer cell line. These data, in conjunction with the synthetic
tractability of 1, made it an interesting lead in the potential
development of compounds active against pancreatic cancer.
The development and cytotoxicity of focused analogue
libraries of 1 is reported herein.
Figure 1. A. Overlay of
coloured: purple: hydrophilic area; green: hydrophobic area; B. The 2D MOE interaction plot of
(dashed lines) with S100A2. A and B in the amino acid identifiers refer to the 2 S100A2 chains in the homodimer sequence.
1
with p53 peptide (red) predicted by MOE and p53 groove (highlighted by the blue oval);
1 is coloured by atom type; protein surface
1
depicting the adjacent amino acids. Key
1
interactions are shown
Library
synthesis
was
envisaged
from
), a selection
3,5-
Br
bis(trifluoromethyl)benzene sulfonyl chloride (
2
O
O
H
N
of aminoalcohols (3a-c), and an aldehyde or amine (4a and/or
4b) (Figure 2). This proposed modular approach was effected
as shown in Scheme 1. Library 1 was accessed on treatment
F3C
S
N
1
H
of sulfonyl chloride (
alcohol ( ) followed by functional group interchange to install
the mesylate ( ). Nucleophilic displacement of the mesylate
with selected amines afforded Library 1 ( 12) with 25-72%
yields (Experimental). Library 1 possessed an ethyl linked
diamine moiety (from 3a).
2) with 2-aminoethan-1-ol (3a) to give
CF3
5
6
O
O
7
-
R2
R2
F3C
S
NH2
CHO
Cl
4a
4b
CF3
( )n
H2N
OH
2
3a, n = 0; 3b; n =1, 3c; n = 2
2
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10.1002/cmdc.202000950
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FULL PAPER
Figure 2. Chemical structure of Lead
1
; individual Library assembly
red); aminoalcohol (3a-c;
from 1.6 (BxPC-3) to 4.2 µM (PANC-1). However, 10 was also
highly active against the normal cell line, MCF10A (GI50 = 3.3
µM) indicating no cancer versus normal cell line selectivity in
this model system. As 10 shows significant potency
differences, at 5- to 10- fold greater against the pancreatic
fragments are coded by colour: sulfonyl chloride (
black) and aldehyde or amine (4a,b blue).
2
;
;
Analysis of the Library 1 MTT screening data at an initial 25
µM compound concentration (Supporting Information; Table
cancer cell line panel than
8 (3,4-di-OCH3), 9 (4-C(CH3)3) or 12
S1) resulted in analogues
response evaluation (Table 1). Analogues
8
-
11 proceeding to full dose
(4-BrPh) and 12 (4-
(4-CF3), the addition of the second phenyl moiety appears
responsible for the enhanced cytotoxicity. This effect was not
7
CF3) were insufficiently active to proceed to full dose response
evaluation.
exclusively steric as both the 4-C(CH3)3
9 and 1-naphthyl 11
were less active. In a similar manner the lone pair of electrons
associated with the di-OCH3
cytotoxicity (Table 1).
Molecular docking studies were performed using MOE for
analogues 8-11 (Library 1) and showed very similar docking
poses. The 3,5-bis-CF3 moiety in these compounds aligned in
the same direction. Additionally, the 3,4-diOCH3Ph in
7did not contribute strongly to
O
O
O
O
F3C
S
F3C
S
R
Cl
N
i, ii
H
CF3
CF3
5; R = OH
2
compound
8
, 4-C(CH3)3
9, 4-Ph-Ph 10, and 1-naphthyl 11 were
6; R = OMs
oriented in
a
similar direction/position (Supporting
iii
Information; Figure S1). The bulky 4-Ph-Ph 10 extended the
overall length of the molecule, which is thought to block the
interaction between S100A2 and p53 more efficiently
compared with the other compounds in Library 1 (Figure 3A),
whereas the 1-naphthyl 11 experiences a steric clash with the
top right hand side of the p53 groove resulting in the
decreased activity (Figure 3B).
O
O
H
N
R1
F3C
S
N
H
CF3
7-12
Table 1. GI50 (µM) determination of Library 1 compounds 8-11 against various
human cancer cell lines and the normal cell line MCF10A (normal breast). GI50 is the
compound concentration required to inhibit cell growth by 50% relative to an
untreated control.
Scheme 1. Reagents and Conditions: (i) 2-aminoethanol (3a), NaHCO3, H2O,
THF, RT, 4 h; (ii) CH3SO2Cl, pyridine, CH2Cl2, RT, 72 h; (iii) CH3CN, pyridine, 80 ºC,
11 h. For details of R1 see Tables 1 and 2.
Our human cancer cell line screening panel can be
considered as comprising two separate cohorts, the first
cohort (cohort-1) comprising: HT29 (colon), MCF-7 (breast),
A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate),
BEC-2 and U87 (glioblastoma), SJ-G2 (neuroblastoma), and
MCF10A (normal breast); with the second cohort (cohort-2)
made up of the pancreatic cancer cell lines: MiaPaCa-2, BxPC-
3, AsPC-1, Capan-2, PANC-1 and HPAC.
O
O
Cell Line
H
R1
F3C
S
N
N
H
CF3
8
9
10
4-Ph-Ph
11
R1
3,4-di-
OCH3Ph
4-C(CH3)3
1-naphthyl
GI50 (µM)
14 ± 0.58
13 ± 0.67
14 ± 1.0
HT29a
13 ± 0.58
25 ± 1.5
14 ± 2.3
14 ± 2.3
19 ± 3.2
25 ± 1.0
30 ± 1.2
23 ± 2.2
25 ± 1.5
29 ± 0.58
25 ± 2.4
29 ± 1.0
20 ± 8.8
20 ± 2.5
34 ± 2.6
34 ± 0.33
16 ± 0.33
2.7 ± 0.00
1.4 ± 0.19
2.6 ± 0.18
2.6 ± 0.18
2.6 ± 0.15
2.4 ± 0.15
2.5 ± 0.10
2.7 ± 0.22
1.4 ± 0.19
2.5 ± 0.18
3.3 ± 0.25
2.7 ± 0.15
1.6 ± 0.32
2.8 ± 0.10
2.4 ± 0.40
4.2 ± 0.61
2.8 ± 0.10
16 ± 1.0
18 ± 4.9
21 ± 2.0
21 ± 2.0
-
-
-
19 ± 0.67
-
-
22 ± 1.5
26 ± 2.2
23 ± 6.5
22 ± 0.58
21 ± 2.7
24 ± 3.9
18 ± 0.00
MCF-7b
A2780c
A2780c
H460d
MTT screening of Library 1 against cohort 1 cell lines
revealed, for analogues
response evaluation, a rank potency order of 10 (4-Ph-Ph) >>
(4-C(CH3)3Ph) > 11 (1-naphthyl) > (3,4-di-OCH3Ph). This
8-11 that proceeded to full dose
14 ± 1.0
14 ± 1.0
9
7
A431e
16 ± 0.67
16 ± 0.33
14 ± 0.58
13 ± 0.67
14 ± 0.00
14 ± 0.00
15 ± 0.33
8.8 ± 3.2
14 ± 1.2
Du145f
BE2-Cg
U87g
suggests that the combination of steric bulk and an additional
aromatic ring is beneficial to activity. The 1-naphthyl 11 adds
steric constraints not present with 10: and 9, while sterically
bulky, lacks the electron density associated with the second Ph
moiety of 10. The activity of 10 ranged from 1.4 (MCF-7 and
U87) to 2.7 µM (HT29 and BE2-C) (Table 1).
Continued evaluation of Library 1 in an expanded
pancreatic cancer cell line panel was undertaken (Table 1). As
with our standard cell line panel, the GI50 values of analogues
SJ-G2h
MCF10Ai
MiaPaCa-2j
BxPC-3j
AsPC-1j
Capan-2j
PANC-1j
HPACj
13 ± 0.33
13 ± 0.58
14 ± 0.58
8-11 were determined. Broad spectrum activity against
pancreatic cancer cell lines was observed, with 4-Ph 10 clearly
a colon; b breast; c ovarian; d lung; e skin; f prostate; g glioblastoma; h
neuroblastoma; i normal breast; j pancreas; ‘-‘ = not determined.
the most active with pancreatic cancer cell line GI50 values
3
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10.1002/cmdc.202000950
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Figure 3. The docking poses of selected compounds in the S100A2 binding site:
A
. Compound 10
;
B
. Compound 11. Compounds are coloured by atom type;
Protein surface: purple, hydrophilic region; green, hydrophobic area.
Examination of the binding poses of Library 1 analogues
suggested that while the 3,5-bis(trifluoromethyl)benzene
sulfonamide moiety engaged well with the p53 binding
pocket, the terminal phenyl moiety failed to fully engage
with the other side of the p53 binding groove. In turn this
suggested that increasing the alkyl spacer from ethyl to
propyl may have a positive effect on the observed
cytotoxicity. To this end, Library 2 was constructed using 3-
anti-pancreatic cancer effects (Table 2). The increase in
chain length resulted in a ca 10-fold potency increase for
the 4-Br 13, 5-fold increase for C(CH3)3 14, potency
retention for 4-Ph-Ph 15, a 10-fold potency for 1-naphthyl
17 and the 4-CF3 18 proceeded to GI50 determination with
GI50 values of 1.4 (MCF-7) to 3.2 µM (PANC-1). All Library 2
analogues displayed high levels of activity against the
pancreatic cancer cell lines examined. These outcomes are
consistent with the molecular modelling predictions.
The predicted docked poses of Library 2 analogues align
better with the terminus of the p53 groove (Figure 4A and
B). In the docked pose of all Library 2 compounds, better
access to the p53 groove is noted, and this is consistent with
the observed enhancement in cytotoxicity. This also
supports the action of these compounds through inhibition
of the S100A2-p53 interaction (but does not categorically
prove). This increase in potency and engagement with the
p53 groove is most evident for 1-naphthyl 17 and 4-CF3 18
with the extra flexibility afforded by the propyl chain
allowing better access to the p53 groove, reflected in
increased cytotoxicity. Further increase in alkyl spacer
length (butyl vs. propyl with 13 vs. 19) showed no additional
increase in potency. This suggests that a propyl alkyl spacer
is the optimal carbon chain length for inhibition of the
S100A2-p53 interaction.
aminopropan-1-ol (3b) to afford analogues 13-18 in 28-54%
yields, and analogue 19 was produced commencing with 4-
aminobutan-1-ol (3c) to examine the effect of further alkyl
spacer elongation in 36% yield. These analogues were
screened at 25 µM concentration with all analogues
proceeding to full dose response evaluation (Table 2).
Uniformly, Library 2 analogues were more active than
their Library 1 counterparts, supporting the hypothesis of
alkyl spacer elongation. The Library 2 compounds are all
broad spectrum cytotoxic (cohort-1 cell lines) with the
observed GI50 values generally <4 µM potent excepting 14
(7 µM, A431). This high level of activity also extended to
the cohort-2 analogues targeting pancreatic cancer.
Activity against the (cohort-2) pancreatic cancer lines, GI50
1.4 (15, BxPC-3) to 18 µM (14, PANC-1). In all cases, simple
halogen (13 and 19), alkyl (15), aromatic (16 and 17) and
electron withdrawing (18) moieties afforded high levels of
Table 2. GI50 (µM) determination of Library 2 compounds 13-19 against various human cancer cell lines and the normal cell line, MCF10A (normal breast). GI50 is the
compound concentration required to inhibit cell growth by 50% relative to an untreated control.
O
O
Cell Line
F3C
S
R1
N
( )n
N
H
H
CF3
13
4-BrPh
1
14
15
4-C(CH3)3
1
16
4-Ph-Ph
1
17
18
4-CF3
1
19
4-BrPh
2
R1
n
3,4-diOCH3Ph
1
1-naphthyl
1
GI50 (µM)
2.3±0.15
HT29a
MCF-7b
A2780c
H460d
A431e
2.2±0.10
1.9±0.05
2.6±0.00
2.5±0.22
2.5±0.20
2.6±0.17
2.7±0.03
2.7±0.20
1.9±0.00
3.8±0.10
3.4±0.79
7.0±0.46
4.0±0.32
3.6±0.33
2.0±0.00
2.1±0.15
2.9±0.03
2.5±0.21
2.8±0.13
2.7±0.20
2.5±0.07
2.6±0.15
1.4±0.17
3.1±0.15
-
-
-
2.9±0.21
1.4±0.20
2.8±0.35
-
-
-
1.6±0.38
1.5±0.19
2.1±0.12
-
-
-
1.7±0.35
2.6±0.03
2.5±0.17
2.6±0.18
2.7±0.09
2.6±0.12
Du145f
BE2-Cg
2.7±0.18
2.7±0.12
2.2±0.10
4
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FULL PAPER
U87g
2.1±0.09
2.4±0.12
3.1±0.19
2.3±0.27
1.5±0.13
2.8±0.09
2.0±0.31
2.2±0.17
2.9±0.13
3.4±0.12
18±0.33
5.3±0.62
2.8±0.18
13±1.2
6.4±2.4
15±1.3
18±0.88
3.5±0.24
2.3±0.10
2.4±0.12
3.2±0.15
2.3±0.13
1.4±0.35
2.7±0.09
2.1±0.10
2.5±0.15
2.3±0.30
2.2±0.10
2.3±0.07
2.8±0.12
2.4±0.12
1.7±0.24
2.5±0.10
2.2±0.06
2.5±0.17
2.7±0.17
-
-
-
-
-
-
SJ-G2h
MCF10Ai
MiaPaCa-2j
BxPC-3j
AsPC-1j
Capan-2j
PANC-1j
HPACj
3.2±0.03
2.8±0.10
2.7±1.2
3.0±0.06
2.7±0.27
2.7±0.07
2.4±0.21
3.3±0.09
2.5±0.21
1.7±0.033
2.7±0.23
2.0±0.21
3.2±0.66
2.7±0.13
3.2±0.07
2.1±0.15
1.7±0.12
2.9±0.09
1.9±0.27
2.2±0.00
2.0±0.23
a colon; b breast; c ovarian; d lung; e skin; f prostate; g glioblastoma; h neuroblastoma; i normal breast; j pancreas; ‘-‘ = not determined.
``
Figure 4. The highest scoring docking poses of selected Library 2 compounds: A. superimposition of analogues 13
red; compounds are coloured by atom type. Protein surface coloured: purple, hydrophilic region; green, hydrophobic region.
-
19
;
B. as for A with the p53 peptide shown in
. The MOE 2D interaction plots
C
depicting the compound adjacent amino acids,
S100A2 chains in the homodimer
C
. analogue 13 (4-Br); and
D
. As for
C
with analogue 16 (4-Ph). A and B in the amino acid identifiers refer to the 2
Compounds in Library 2 also displayed similar docking
poses (Figure 5) as compounds 11 in Library 1. The similar
and the binding pocket. This increased flexibility allowed
enhanced interference in blocking the interaction between
S100A2 and p53 peptide more effectively, leading to the
improved activity for compounds with a three-carbon
linker.
Having established the importance of the propyl linker,
further investigation of the terminal phenyl ring
substituents was undertaken with the synthesis of 20-30
from the corresponding aldehydes as per Scheme 1 in 39-
71% yields (see Experimental). Within Library 3 the effect
of N-methylation of the parent phenyl analogue 24 was also
explored. All Library 3 analogues were sufficiently active at
25 µM concentration to proceed to full dose response
evaluation (Table 3; Supporting Information, Table S3).
8
-
levels of inhibitory activity also can be explained via the
MOE 2D interaction plot. Examination of Figure 5 highlights
the similarity in binding poses for these compounds.
Specifically, compounds 13-17 and 19 engage with the
binding pocket through an arene-H interaction between the
aryl ring and amino PheA89 (e.g. Figure 4C, 13 (4-Br) and
4D, 16 (4-Ph); Supporting Information; Figure S2), and
analogue 18 with the binding pocket via the sulfonamide
nitrogen and amino AsnA87.
Library 2 analogues are more active than compounds in
Library 1 with the exception of compound 10. The increased
linker size for compounds in Library 2 is hypothesised to be
responsible for the increased activity. The increased linker
length may increase the flexibility of the molecule and
contribute to the enhanced binding between the molecules
5
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Table 3. GI50 (µM) determination of Library 3 compounds 20-30 against various human cancer cell lines and the normal cell line, MCF10A (normal breast). GI50 is the
compound concentration required to inhibit cell growth by 50% relative to an untreated control.
O
O
Cell Line
F3C
S
R1
N
N
H
R2
CF3
23
29
21
30
20
22
26
27
28
24
25
Cl
Cl
O
O
Cl
R1
R2
H3CO
Cl
Cl
OCH3
OCH3
CF3
Cl
H
H
H
H
CH3
H
H
H
H
H
H
GI50 (µM)
2.9±0.25
2.5±0.03
1.7±0.12
2.8±0.12
3.1±0.10
2.5±0.10
1.3±0.22
2.8±0.21
2.4±0.55
11±0.60
3.0±0.13
HT29a
10±3.5
12±1.2
4.7±0.95
>50
16±1.9
15±0.00
3.0±0.03
4.9±1.6
12±0.73
13±0.67
11±0.29
13±0.58
13±1.5
2.8±0.30
2.4±0.13
1.8±0.22
2.9±0.03
2.3±0.76
2.8±0.18
1.6±0.12
3.0±0.06
2.7±0.17
4.9±1.4
14±0.88
2.6±0.12 8.8±3.2
2.2±0.78 9.3±3.8
6.9±2.1
7.0±2.5
4.4±1.3
3.8±1.3
5.9±2.5
8.0±3.6
20±4.9
11±4.1
3.5±0.63 2.5±0.17
2.2±0.09
MCF-7b
A2780c
BE2-Cd
2.4±0.09
1.6±0.17
4.5±1.1
6.6±2.0
3.4±0.57
1.7±0.64
3.8±0.57
6.4±2.4
10±1.6
2.6±0.21 2.4±0.24
1.5±0.12 1.5±0.10
3.6±0.26 2.9±0.19
5.5±0.71 3.0±0.13
3.1±0.36 2.7±0.10
1.7±0.60 1.4±0.21
4.6±0.37 2.8±0.03
6.0±0.40 1.8±0.56
28±0.88
28±2.3
26±1.3
33±3.0
39±5.9
51±2.1
46±6.6
>50
5.9±2.4
8.6±2.4
7.9±2.4
7.4±2.4
7.8±3.3
9.6±3.4
9.1±3.5
10±3.1
1.5±0.15
2.8±0.00
3.2±0.03
2.6±0.22
1.2±0.35
2.9±0.15
2.5±0.13
3.4±0.9
15±5.5
11±3.6
11±4.0
11±4.6
13±5.0
13±4.8
MCF10Ae
MiaPaCa-2f
BxPC-3f
AsPC-1f
Capan-2f
PANC-1f
HPACf
14±1.0
16±2.1
3.2±0.24
12±0.58
2.4±0.25 2.5±0.17
5.6±1.30
2.7±0.13 15±4.3
12±0.67 10±3.9
2.4±0.19
30±1.5
2.8±0.07
3.0±0.10 2.7±0.27
a colon; b breast; c ovarian; dglioblastoma; e normal breast; f pancreas.
The panel of cancer cell lines was reduced in the evaluation
ring and PheA89 respectively, and compound 23 binds with
of Library 3, with analysis of the data presented in Table 3
S100A2 through an interaction between the nitrogen atom of
against HT29, MCF-7, A2780 and BEC-2 cell lines being benzene- sulfonamide and AsnA87 (sidechain acceptor)
representative of our broad-spectrum panel. In this instance, (Figure 6). The introduction of the N-CH3 moiety with 24 has
the observed activity was generally similar to, or slightly lower a profound impact on the docked conformation in the p53-
than, observed with Library 2. There are key outliers to this groove with the phenyl moiety twisting and the loss of a
data in particular the parent phenyl 25 with activity 5-fold critical H-bond donor interaction (Figure 5C and D). This is
lower, and a 10-fold potency reduction with N-CH3 24. These reflected in the significant loss in cytotoxicity observed with a
data support, with 25, the requirement for a phenyl 5- to 10- fold decrease in potency recorded. The effect was
substituent capable of accessing the upper area of the p53- most profound in the case of the panel of pancreatic cancer
groove; and the requirement for a H-bond donating capability cell lines with all activities in the GI50 range of >30 µM. This
with 24. In the main, the nature of the phenyl substituent has compares unfavourably with the corresponding free NH 25
little impact on the observed cytotoxicity if the analogue is
which is 3- to 5- fold more active; and 24 is 15-fold less active
capable of engaging with the p53-groove (Figure 5). Generally, than 30, the most active analogue against the pancreatic
Library 3 compounds displayed good to high levels of cancer cell lines with GI50 of 1.2 (BxPC-3) to 3.4 (PANC-1) µM.
cytotoxicity with GI50 values ranging from 1.5 (22
A2780); to >50 µM (24; HT29).
,
23 and 30
;
Of note it is known that the BxPC-3 cell line has high
endogenous levels of S100A2 (Figure 7). Thus, the cytotoxicity
Analogues 20, 26 and 30 are all predicted to engage with response is in line with the predicted effect of blocking the
S100A2 through an arene-H interaction between the benzene S100A2-p53 interaction.
6
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10.1002/cmdc.202000950
ChemMedChem
FULL PAPER
Figure 5. A. The best scoring binding pose of N- CH3 24; B. best scoring binding pose of N- CH3 25; Compounds coloured by atom type. Protein surface: purple, hydrophilic
region; green, hydrophobic region. C. The MOE 2D interaction plots depicting the compound adjacent amino acids, with analogue 24; D. As for C with analogue 25. A and B in
the amino acid identifiers refer to the 2 S100A2 chains in the homodimer
Figure 6. A. The MOE 2D interaction plots depicting the compound adjacent amino acids, with analogue 20
; B. As for C with analogue 23; C. As for C with
analogue 26 D. As for C with analogue 30. A and B in the amino acid identifiers refer to the 2 S100A2 chains in the homodimer.
;
7
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10.1002/cmdc.202000950
ChemMedChem
FULL PAPER
(
19F, 1H and 13C NMR) at 400, 400 and 100 MHz respectively or a Bruker Avance
III 600 MHz (1H and 13C NMR) spectra at 600 and 150 MHz respectively. All spectra
were recorded in deuterated dimethyl sulfoxide (DMSO-d6) obtained from
Cambridge Isotope Laboratories Inc. Chemical shifts (d) were measured in parts
per million (ppm) and referenced against the internal reference peaks. Coupling
constants (J) were measured in Hertz (Hz). NMR assignments were determined
through the interpretation of one- and two-dimensional spectra. Multiplicities
are denoted as singlet (s), broad singlet (bs), doublet (d), doublet of doublets (dd),
triplet (t), quartet (q), pentet (p), septet (sept) and multiplet (m). Peaks are listed
in decreasing chemical shift in the following format: chemical shift integration
(1H), multiplicity (1H), coupling constant (Hz).
N-[2-(4-Bromobenzylamino)-ethyl]-3,5-
bis(trifluoromethyl)benzenesulfonamide hydrochloride 7
Figure 7. High endogenous expression of S100A2 was observed in the PC
cell line BxPC-3 via Western blot.
To a solution of 2-aminoethanol (1.05 eq, 963 mg, 15.8 mmol) and
NaHCO3 (1.6 eq, 2.020 g, 24 mmol) in H2O (40 mL) was gradually added a solution
of 3,5-bis(trifluoromethyl)benzene sulfonyl chloride (1.0 eq, 4.689 g, 15 mmol)
in THF (40 m L). The reaction mixture was allowed to stir for 5 h at RT, monitored
by TLC (CH3OH/DCM: 10:1, Rf: 0.78). After the reaction, THF was removed under
vacuum. The residue was filtered and washed with water (500 mL) and hexanes
(200 mL) to afford the desired product as a white solid. Yield: 4.121 g, 82%, m.
p.: 88-90 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1H), 8.40 (s, 2H), 8.10 (s, 1H),
4.72 (t, J = 5.3 Hz, 1H), 3.39 (q, 5.2 Hz, 2H), 2.90 (t, J = 5.8 Hz, 2H); 13C NMR (101
MHz, DMSO-d6) δ 143.7, 131.3 (q, J = 33.7 Hz, 2C), 127.3 (unresolved q, J = 3.5
Hz), 126.3 (unresolved sept, J = 3.6 Hz, 2C), 122.7 (q, J = 271.7 Hz, 2C), 59.8, 45.3;
19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.1; IR max/cm-1: 3289 (N-H), 3166
(O-H), 1276 (S=O), 1155 (S=O), 1129 (C-F), 1107 (C-N); LRMS (ESI+) m/z: 338 [M
+H].
Conclusions
Virtual screening predicted sulfonamide
of the S100A2-p53 protein-protein interaction. S100A2 is a
validated pancreatic cancer drug target, and screening of in
the pancreatic cancer cell line, MiaPaCa-2, revealed it as a 2.97
µM potent cell growth inhibitor. The development of focused
compound libraries (Library 1, 2 and 3) demonstrated that a
propyl diamine linker gave rise to the highest level of both
broad-spectrum cytotoxicity and pancreatic cancer cell line
cytotoxicity. In total 15 human cell lines were evaluated. The
introduction of a N-CH3 substituent with 24 resulted in a 5- to
1 as an inhibitor
1
To
a
solution
of
N-(2-hydroxyethyl)-3,5-bis(trifluoromethyl)-
benzenesulfonamide (1 eq, 3.888 g, 11.5 mmol) and pyridine (2.5 eq, 2.275 g,
28.8 mmol) in CH2Cl2 (80 mL) was gradually added a solution of MsCl (2.2 eq,
2.885 g, 25.3 mmol) in CH2Cl2 (40 mL). The reaction mixture was allowed to stir
for 4 h at RT, monitored by UPLC-MS analysis. After the reaction, the solvent was
removed under vacuum. To the residue was added H2O (100 mL), and the
precipitate was collected and washed with water (300 mL) and hexanes (200 mL)
by filtration and dried under reduced pressure to give the desired product as a
white solid. Yield: 4.342 g, 91%, m. p.: 135-138 ºC. 1H NMR (400 MHz, DMSO-d6)
δ 8.49 (s, 1H), 8.45 (t, J = 5.8 Hz, 1H), 8.39 (s, 2H), 4.15 (t, J = 5.1 Hz, 2H), 3.25 (q,
5.4 Hz, 2H), 3.11 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 143.8, 131.9 (q, J = 33.6
Hz, 2C), 127.6 (unresolved quartet, J = 3.2 Hz, 2C), 127.0 (unresolved sept, J = 3.6
Hz), 122.7 (q, J = 271.7 Hz, 2C), 69.3, 42.2, 37.0; 19F NMR (376 MHz, DMSO-d6,
CF3CO2H) δ -58.8; IR max/cm-1: 3289 (N-H), 1277 (S=O), 1160 (S=O), 1133 (C-F),
1112 (C-N); LRMS (ESI+) m/z: 416 [M +H].
10-fold potency reduction relative to the free NH analogue 25
.
Analysis of the cytotoxicity screening data revealed a poor
correlation between the terminal phenyl substituent and
cytotoxicity provided that the substituent could fully access
the S100A2-p53 binding groove. As a consequence, it was
noted that 4-substituted phenyl analogues were generally
more active. In all instances there was good to excellent
correlation between the predicted binding pose in the
S100A2-p53 binding groove and the observed cytotoxicity.
This was particularly evident upon examination of the
cytotoxicity of Library 2 and Library 3 compounds against the
six pancreatic cancer cell lines examined (MiaPaCa-2, BxPC-3,
AsPC-1, Capan-2, HPAC and PANC-1). Combined, the data
presented herein is consistent with the inhibition of the
S100A2-p53 protein-protein interaction with an anti-
pancreatic cancer drug target.
To a solution of 4-bromobenzylamine (2.0 eq, 371 mg, 2 mmol) and
pyridine (1.0 eq, 80 mg, 1.0 mmol) in MeCN (30 mL) was added 2-(3,5-
bis(trifluoromethyl)phenylsulfonamido)ethyl methanesulfonate (1.0 eq, 415 mg,
1.0 mmol). The reaction mixture was stirred for 11 h at 80 ºC, monitored by LC-
MS analysis. Following the reaction, the mixture was filtered, then the filtrate
was concentrated under reduced pressure. To the residue was added 2 mL
acetone and 2 mL 10% HCl and dried under compressed air. The residue was
washed with water (20 mL) and hexanes (20 mL) and then recrystallized using
acetone (5 mL) and ether (10 mL) to give compound 7 as a hydrochloride salt, a
white solid. Yield: 190 mg, 35%, m. p.: dec. >213 ºC. 1H NMR (400 MHz, DMSO-
d6) δ 9.36 (s, 2H), 8.63 (s, 1H), 8.54 (s, 1H), 8.42 (s, 2H), 7.64 (d, J = 8.4 Hz, 2H),
7.49 (d, J = 8.4 Hz, 2H), 4.14 (s, 2H), 3.17 (s, 2H), 3.00 (s, 2H); 13C NMR (101 MHz,
DMSO-d6) 142.5, 132.4 (2C), 131.5 (2C), 131.4 (q, J = 33.7, 2C), 131.2, 127.5
(unresolved quartet, J = 3.3 Hz), 126.9 (overlapping q, J = 3.4 Hz, 2C), 122.7 (q, J
= 271.8 Hz, 2C), 122.4, 49.1, 45.8, 38.6; 19F NMR (376 MHz, DMSO-d6, CF3CO2H)
δ -58.1; IR max/cm-1: 2989 (N-H), 1278 (S=O), 1163 (S=O), 1137 (C-F), 1095 (C-N),
681 (C-Br); LRMS (ESI+) m/z: 505[M-HCl+H, 79Br]/507 [M-HCl+H, 81Br]; HRMS
calculated for C17H16BrClF6N2O2S [M-HCl+H, 79Br]: 505.0014/[M-HCl+H, 81Br]:
506.9991; found: 504.9942/506.9942.
Experimental
Chemistry
General Methods
All reactions were performed using standard laboratory equipment and
glassware. Solvents and reagents were purchased from Sigma Aldrich, Alfa Aesar
or AK Scientific and used as received. Organic solvent extracts were dried over
magnesium sulfate (MgSO4), and dried under reduced pressure with either Büchi
or Heidolph rotary evaporators. Melting points were recorded in open capillaries
on a Büchi 565 Melting Point Apparatus. Where available, literature values are
provided and appropriately referenced. Electrospray mass spectra were
recorded using H2O (with 1% formic acid, solvent A) and 90% MeCN in H2O (with
1% formic acid, solvent B) as carrier solvents on an Agilent Technologies 1260
Infinity UPLC system with a 6120 Quadrupole LC/MS in electrospray ionization
(ESI) positive and negative modes. TLC was performed on Merck silica gel 60 F254
precoated aluminium plates with a thickness of 0.2 mm. Nuclear magnetic
resonance (NMR) spectroscopy was performed on a Bruker Avance III 400 MHz
N-[2-(3,4-Dimethoxybenzylamino)-ethyl]-3,5-bis(trifluoromethyl)benzene
sulfonamide hydrochloride 8
Synthesized using the general procedure as described for 23, 2-(3,5-
bis(trifluoromethyl)phenylsulfonamido)ethylmethanesulfonate (1.0 eq, 415 mg,
1.0 mmol) and 3,5-dimethoxybenzylamine (2.0 eq, 332 mg, 2.0 mmol) reacted
(monitored by IPLC-MS analysis) to afford compound 8 as a hydrochloride salt, a
white solid. Yield: 130 mg, 25%, m. p.: dec. >160 ºC. 1H NMR (400 MHz, DMSO-
d6) δ 9.23 (s, 2H), 8.63 (s, 1H), 8.54 (s, 1H), 8.43 (s, 2H), 7.23 (d, J = 1.6 Hz, 1H),
7.02 - 6.96 (m, 2H), 4.07 (s, 2H), 3.77 (d, J = 4.3 Hz, 6H), 3.17 (s, 2H), 2.97 (s, 2H);
13C NMR (101 MHz, DMSO-d6) 149.3, 148.6, 142.5, 131.5 (q, J = 33.7 Hz, 2C), 127.5
8
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10.1002/cmdc.202000950
ChemMedChem
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(unresolved q, J = 3.1 Hz), 126.9 (overlapping q, J = 3.5 Hz, 2C), 123.8, 122.7, 122.6
(q, J = 271.7 Hz, 2C), 113.7, 111.5, 55.5 (2C), 49.8, 45.5, 38.7; 19F NMR (376 MHz,
DMSO-d6, CF3CO2H) δ -58.1; IR max/cm-1: 2976 (N-H), 1277 (S=O), 1267 (C-O),
1162 (S=O), 1133 (C-F), 1107 (C-N), 1025 (C-O); LRMS (ESI+) m/z: 487 [M-HCl+H];
HRMS calculated for C19H21ClF6N2O4S [M-HCl+H]: 487.1121; found: 487.1048.
reacted (monitored by TLC CH3OH/DCM: 10:1, Rf: 0.80) to afford the desired
product as a white solid. Yield: 2.811 g, 80%, m. p.: 95-97 ºC. 1H NMR (400 MHz,
DMSO-d6) δ 8.50 (s, 1H), 8.37 (s, 2H), 7.99 (s, 1H), 4.40 (s, 1H), 3.35 (d, J = 6.2 Hz,
2H), 2.88 (t, J = 7.2 Hz, 2H), 1.55 – 1.49 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ
143.4, 131.5 (q, J = 33.7 Hz, 2C), 127.2 (unresolved q, J = 3.5 Hz, 2C), 126.5
(unresolved sept, J = 3.5 Hz), 122.8 (q, J = 271.7 Hz, 2C), 57.8, 39.9, 32.4; 19F NMR
(376 MHz, DMSO-d6, CF3CO2H) δ -58.9; IR max/cm-1:3483 (N-H), 3120 (O-H), 1276
(S=O), 1157 (S=O), 1135 (C-F), 1107 (C-N); LRMS (ESI+) m/z: 352 [M+H].
N-[2-(4-tert-Butylbenzylamino)-ethyl]-3,5-bis(trifluoromethyl)benzene
sulfonamide hydrochloride 9
Synthesized using the general procedure as described for 7, 2-(3,5-
bis(trifluoromethyl)phenylsulfonamido)ethylmethanesulfonate (1.0 eq, 415 mg,
1.0 mmol) and 4-tert-butylbenzylamine (1.5 eq, 250 mg, 1.5 mmol) were reacted
(monitored by UPLC-MS analysis) to afford compound 9 as a hydrochloride salt,
a white solid. Yield: 150 mg, 29%, m. p.: dec. >186 ºC. 1H NMR (400 MHz, DMSO-
d6) δ 9.23 (s, 2H), 8.64 (br s, 1H), 8.54 (s, 1H), 8.43 (s, 2H), 7.44 (s, 4H), 4.10 (s,
2H), 3.17 (t, J = 6.4 Hz, 2H), 3.00 (t, J = 6.4 Hz, 2H), 1.28 (s, 9H); 13C NMR (101
MHz, DMSO-d6) 151.5, 142.5, 131.5 (q, J = 33.7 Hz, 2C), 129.9 (2C), 128.9, 127.5
(unresolved q, J = 3.3 Hz), 126.9 (m, 2C), 125.4 (2C), 122.6 (q, J = 271.8 Hz, 2C),
49.6, 45.8, 38.7, 34.4, 31.0 (3C); 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.1;
IR max/cm-1: 2970 (N-H), 1279 (S=O), 1164 (S=O), 1128 (C-F), 1094 (C-N); LRMS
(ESI+) m/z: 483 [M-HCl+H]; HRMS calculated for C21H25ClF6N2O2S [M-HCl+H]:
483.1534; found: 483.1463.
3-(3,5-bis(trifluoromethyl)phenylsulfonamido)propyl methane sulfonate
was
obtained
from
N-(3-hydroxypropyl)-3,5-bis(trifluoromethyl)-
benzenesulfonamide (1 eq, 4.436 g, 12.64 mmol) and MsCl (2.0 eq, 2.886 g, 25.28
mmol) (monitored by UPLC-MS analysis) as a white solid. Yield: 4.887 g, 90%, m.
p.: 121-124 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 8.38 (s, 2H), 8.16 (t, J
= 5.8 Hz, 1H), 4.19 (t, J = 6.2 Hz, 2H), 3.14 (s, 3H), 2.93 (dd, J = 12.9, 6.7 Hz, 2H),
1.80 (p, J = 6.6 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) δ 143.0, 131.5 (q, J = 33.6
Hz, 2C), 127.2 (q, J = 3.3 Hz, 2C), 126.7 (unresolved sept, J = 3.5 Hz), 122.6 (q, J =
271.7 Hz, 2C), 67.5, 38.8, 36.5, 28.8; 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -
58.6; IR max/cm-1: 3263 (N-H), 1277 (S=O), 1164 (S=O), 1131 (C-F), 1106 (C-N);
LRMS (ESI+) m/z: 430 [M+H].
Following this, 4-bromobenzylamine (2.0 eq, 0.37 g, 2.0 mmol) and 3-(3,5-
bis(trifluoromethyl)phenylsulfonamido)propylmethane sulfonate (1.0 eq, 429
mg, 1 mmol) were reacted (monitored by UPLC-MS analysis) to afford compound
7-29 as a hydrochloride salt, a white solid. Yield: 301 mg, 54%, m. p.: dec. >235
ºC.1H NMR (400 MHz, DMSO-d6) δ 9.21 (br s, 2H), 8.52 (s, 1H), 8.33 (br s, 1H),8.39
(s, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 4.09 (s, 2H), 2.90 (t, J = 7.0
Hz, 4H), 1.8 –1.78 (m, 2H); 13C NMR (101 MHz, DMSO-d6) 143.0, 132.3 (2C), 131.5
(2C), 131.5 (q, J = 33.6 Hz, 2C), 131.4, 127.3 (unresolved q, J = 3.3 Hz), 126.6
(overlapping q, J = 3.4 Hz, 2C), 122.6 (q, J = 271.8 Hz, 2C), 122.3, 49.2, 44.0, 39.9,
26.0; 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.1; IR max/cm-1: 2941 (N-H),
1275 (S=O), 1158 (S=O), 1134 (C-F), 1088 (C-N), 680 (C-Br); LRMS (ESI+) m/z: 519
[M-HCl+H, 79Br]/521 [M-HCl+H, 81Br]; HRMS calculated for C18H18ClF6N2O2S [M-
HCl+H, 79Br]: 519.0171/[M-HCl+H, 81Br]: 521.0148; found: 519.0098/521.0098.
N-(2-(([1,1'-biphenyl]-4-ylmethyl)amino)ethyl)-3,5-
bis(trifluoromethyl)benzene sulfonamide 10
Synthesized using the general procedure as described for 7, 2-(3,5-
bis(trifluoromethyl)phenylsulfonamido)ethylmethanesulfonate (1.0 eq, 415 mg,
1.0 mmol) and 4-phenylbenzylamine (1.5 eq, 273 mg, 1.5 mmol) were reacted
(monitored by UPLC-MS analysis) to afford compound 10 as a white solid. Yield:
210 mg, 42%, m. p.: 129-131 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1H), 8.39
(s, 2H), 7.64 (d, J = 7.4 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.34
(dd, J = 14.9, 7.6 Hz, 3H), 3.65 (s, 2H), 2.97 (t, J = 6.3 Hz, 2H), 2.55 (t, J = 6.1 Hz,
2H). No signals of NH; 13C NMR (101 MHz, DMSO-d6) 143.6, 140.0, 139.2, 138.7,
131.4 (q, J = 33.7 Hz, 2C), 128.9 (2C), 128.6 (2C), 127.3, 127.2 (unresolved q, J =
3.1 Hz), 126.6 (2C), 126.4 (4C), 122.7 (q, J = 271.7 Hz, 2C), 52.0, 47.7, 42.5; 19F
NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.1; IR max/cm-1: 2920 (N-H), 1277 (S=O),
1157 (S=O), 1117 (C-F), 1097 (C-N); LRMS (ESI+) m/z: 503 [M-HCl+H]; HRMS
calculated for C23H20F6N2O2S [M-HCl+H]: 503.1220; found: 503.1150.
N-[3-(3,4-Dimethoxybenzylamino)-propyl]-3,5-bis(trifluoromethyl)benzene
sulfonamide hydrochloride 14
Synthesized using the general procedure as described for 13, 3,5-
N-(2-((Naphthalen-1-ylmethyl)amino)ethyl)-3,5-
bis(trifluoromethyl)benzene sulfonamide hydrochloride 11
dimethoxybenzylamine (2.0 eq, 330 mg,
2
mmol) and 3-(3,5-
bis(trifluoromethyl)phenylsulfonamido)propylmethane sulfonate (1.0 eq, 429
mg, 1 mmol) were reacted (monitored by UPLC-MS analysis) to afford compound
14 as a hydrochloride salt, a white solid. Yield: 270 mg, 50%, m. p.: dec. >207 ºC.
1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 2H), 8.52 (s, 1H), 8.40 (s, 2H), 8.33 (br s,
1H), 7.24 (d, J = 1.7 Hz, 1H), 7.03 – 7.00 (m, 2H), 4.03 (s, 2H), 3.78 (s, 3H), 3.76 (s,
3H), 2.90 (s, 4H), 1.86 – 1.79 (m, 2H); 13C NMR (101 MHz, DMSO-d6) 149.2, 148.6,
143.1, 131.5 (q, J = 33.6 Hz, 2C), 127.3 (unresolved q, J = 3.1 Hz), 126.6
(overlapping q, J = 3.5 Hz, 2C), 124.0, 122.7 (q, J = 272.1Hz, 2C), 122.6, 113.8,
111.5, 55.6, 55.5, 49.9, 43.7, 39.9, 26.0; 19F NMR (376 MHz, DMSO-d6, CF3CO2H)
δ -58.1; IR max/cm-1: 2934 (N-H), 1275 (S=O), 1267 (C-O), 1159 (S=O), 1133 (C-F),
1085 (C-N), 1027 (C-O); LRMS (ESI+) m/z: 501 [M-HCl+H]; HRMS calculated for
C20H23ClF6N2O4S [M-HCl+H]: 501.1275; found: 501.1204.
Synthesized using general procedure as described for 7, 2-(3,5-
bis(trifluoromethyl)-phenylsulfonamido)ethylmethane sulfonate (1.0 eq, 415 mg,
1.0 mmol) and 1-naphthylmethylamine (1.5 eq, 240 mg, 1.5 mmol) were reacted
(monitored by UPLC-MS analysis) to afford the desired compound 11 as a
hydrochloride salt, an off-white solid. Yield: 220 mg, 43%, m. p.: dec. >235 ºC. 1H
NMR (400 MHz, DMSO-d6) δ 9.26 (s, 2H), 8.66 (s, 1H), 8.55 (s, 1H), 8.43 (s, 2H),
8.23 (d, J = 8.1 Hz, 1H), 8.02 (d, J = 7.9 Hz, 2H), 7.74 (d, J = 7.0 Hz, 1H), 7.67 – 7.55
(m, 3H), 4.67 (s, 2H), 3.22 (s, 4H); 13C NMR (101 MHz, DMSO-d6) 142.5, 133.3,
131.5 (q, J = 33.7, 2C), 131.1, 129.7, 129.1, 128.7, 128.0, 127.5 (unresolved q, J =
2.9 Hz, 2C), 126.9 (unresolved sept, J = 3.6 Hz), 126.8, 126.3, 125.3, 123.8, 122.6
(q, J = 271.8 Hz, 2C), 46.8, 46.5, 38.7; 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -
58.1; IR max/cm-1: 2855 (N-H), 1278 (S=O), 1164 (S=O), 1107.0 (C-F), 1097 (C-N);
LRMS (ESI+) m/z: 477 [M-HCl+H]; HRMS calculated for C24H23ClF6N2O2S [M-HCl+H]:
477.1065; found: 477.0993.
N-[3-(4-tert-Butylbenzylamino)-propyl]-3,5-bis(trifluoromethyl)benzene
sulfonamide hydrochloride 15
Synthesized using the general procedure as described for 13, 4-tert-
butylbenzylamine (2.0 eq, 330 mg, 2.0 mmol) and 3-(3,5-
bis(trifluoromethyl)phenylsulfonamido)propylmethane sulfonate (1.0 eq, 429
mg, 1 mmol) were reacted (monitored by UPLC-MS analysis) to afford compound
15 as a hydrochloride salt, a white solid. Yield: 231 g, 43%, m. p.: dec. >241 ºC. 1H
NMR (400 MHz, DMSO-d6) δ 9.12 (br s, 2H), 8.52 (s, 1H), 8.34 (br s, 1H), 8.40 (s,
2H), 7.45 (s, 4H), 4.05 (s, 2H), 2.93 – 2.89 (m, 4H), 1.86 – 1.79 (m, 2H), 1.28 (s,
9H); 13C NMR (101 MHz, DMSO-d6) 151.5, 143.1, 131.5 (q, J = 33.6 Hz, 2C), 129.9
(2C), 129.1, 127.3 (unresolved q, J = 3.2 Hz), 126.7 (overlapping q, J = 3.4 Hz, 2C),
125.4 (2C), 122.7 (q, J = 271.7 Hz, 2C), 49.7, 44.1, 39.9, 34.4, 31.0 (3C), 26.0; 19F
NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.1; IR max/cm-1: 2934.0 (N-H), 1280.1
(S=O), 1164.9 (S=O), 1136.9 (C-F), 1082.1 (C-N); LRMS (ESI+) m/z: 497.2 [M-
HCl+H]; HRMS calculated for C22H27ClF6N2O2S [M-HCl+H]: 497.1691; found:
497.1619.
3,5-Bis(trifluoromethyl)-N-[2-(4-trifluoromethylbenzylamino)-ethyl]-
benzene sulfonamide hydrochloride 12
Synthesized using the general procedure as described for 7, 2-(3,5-
bis(trifluoromethyl)phenylsulfonamido)ethylmethanesulfonate (1.0 eq, 415 mg,
1.0 mmol) and 4-(trifluoromethyl)benzylamine (2 eq, 352 mg, 2 mmol) were
reacted (monitored by UPLC-MS analysis) to afford compound 12 as
a
hydrochloride salt, a white solid. Yield: 381 mg, 72%, m. p.: dec. >212 ºC. 1H NMR
(400 MHz, DMSO-d6) δ 9.46 (s, 2H), 8.63 (s, 1H), 8.53 (s, 1H), 8.42 (s, 2H), 7.81 (d,
J = 8.3 Hz, 2H), 7.76 (d, J = 8.2 Hz, 2H), 4.25 (s, 2H), 3.19 (t, J = 6.3 Hz, 2H), 3.02 (t,
J = 6.3 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) 142.5, 136.5, 131.5 (q, J = 33.7 Hz,
2C), 131.0 (2C), 129.4 (q, J = 31.7 Hz), 127.5 (unresolved q, J = 3.1 Hz, 2C), 126.8
(unresolved sept, J = 3.4 Hz), 125.4 (q, J = 3.7 Hz, 2C), 124.1 (q, J = 270.5 Hz),
122.6 (q, J = 271.8 Hz, 2C), 49.2, 46.0, 38.6; 19F NMR (376 MHz, DMSO-d6,
CF3CO2H) δ -58.8, -59.0; IR max/cm-1: 2982 (N-H), 1279 (S=O), 1162 (S=O), 1125
(C-F), 1096 (C-N); LRMS (ESI+) m/z: 495 [M-HCl+H]; HRMS calculated for
C18H16ClF9N2O2S [M-HCl+H]: 495.0785; found: 495.0710.
N-(3-(([1,1'-biphenyl]-4-ylmethyl)amino)propyl)-3,5-
bis(trifluoromethyl)benzene sulfonamide hydrochloride 16
Synthesized using the general procedure for 13, 4-phenylbenzylamine (2.0
eq, 369 mg, 2.0 mmol) and 3-(3,5-bis(trifluoromethyl)phenylsulfonamido)propyl-
methanesulfonate (1.0 eq, 429 mg, 1.0 mmol) were reacted (monitored by UPLC-
MS analysis) to afford compound 16 as a hydrochloride salt, a white solid. Yield:
160 mg, 29%, m. p.: dec. >239 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 2H),
N-(3-((4-bromobenzyl)amino)propyl)-3,5-bis(trifluoromethyl)benzene
sulfonamide hydrochloride 13
Synthesized using the general procedure as described for 7, 3-
aminopropanol (1.05 eq, 0.788 mg, 10.5 mmol) and 3,5-
bis(trifluoromethyl)benzene-1-sulfonyl chloride (1.0 eq, 3.121 g, 10 mmol) were
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000950
ChemMedChem
FULL PAPER
8.52 (s, 1H), 8.37 (br s, 1H), 8.40 (s, 2H), 7.74 – 7.68 (m, 4H), 7.63 (d, J = 8.2 Hz,
2H), 7.49 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.3 Hz, 1H), 4.15 (s, 2H), 2.97 – 2.90 (m,
4H), 1.89 –1.82 (m, 2H);13C NMR (101 MHz, DMSO-d6) 143.1, 140.6, 139.4, 131.4
(q, J = 33.6, 2C), 131.1, 130.7 (2C), 129.0 (2C), 127.8, 127.3 (unresolved q, J = 3.2
Hz), 126.8 (2C), 126.7 (2C), 126.6 (overlapping q, J = 3.4 Hz, 2C), 122.6 (q, J = 271.7
Hz, 2C), 49.6, 44.1, 39.9, 26.0; 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.2; IR
79Br]/535 [M-HCl+H, 81Br]; HRMS calculated for C23H29ClF3N5O5S [M-HCl+H, 79Br]:
533.0326/[M-HCl+H, 81Br]: 535.0304; found: 533.0255/535.0255.
N-[3-(4-Methoxybenzylamino)-propyl]-3,5-
bis(trifluoromethyl)benzenesulfonamide hydrochloride 20
Synthesized using the general procedure as described for 13, 4-methoxy-

max/cm-1: 2940 (N-H), 1281 (S=O), 1158 (S=O), 1128 (C-F), 1092 (C-N); LRMS (ESI+)
benzylamine
(2.5
eq,
340
mg,
2.5
mmol)
and
3-(3,5-
bis(trifluoromethyl)phenylsulfonamido)propylmethane sulfonate (1.0 eq, 429
mg, 1 mmol) were reacted (monitored by UPLC-MS analysis) to afford compound
20 as a hydrochloride salt, a white solid. Yield: 310 mg, 61%, m. p.: 224-227 ºC.
1H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 2H), 8.51 (s, 1H), 8.39 (s, 2H), 8.35 (s, 1H),
7.45 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 4.03 (s, 2H), 3.76 (s, 3H), 2.88 (dd,
J = 14.0, 7.1 Hz, 4H), 1.85 – 1.78 (m, 2H); 13C NMR (101 MHz, DMSO-d6) 159.7,
143.1, 131.7 (2C), 131.4 (q, J = 33.7 Hz, 2C), 127.3 (unresolved q, J = 3.2 Hz, 2C),
126.6 (unresolved sept, J = 3.5 Hz), 123.8, 122.6 (q, J = 271.8 Hz, 2C), 113.9 (2C),
55.2, 49.4, 43.7, 39.9, 24.9; 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.0; IR
m/z: 517 [M-HCl+H]; HRMS calculated for C24H23ClF6N2O2S [M-HCl+H]: 517.1377;
found: 517.1306.
N-(3-((naphthalen-1-ylmethyl)amino)propyl)-3,5-
bis(trifluoromethyl)benzene sulfonamide hydrochloride 17
Synthesized using the general procedure as described for 13, 1-
naphthylmethylamine (1.5 eq, 240 mg, 1.5 mmol) and 3-(3,5-
bis(trifluoromethyl)phenylsulfonamido)propylmethane sulfonate (1.0 eq, 429
mg, 1.0 mmol) were reacted (monitored by LC-MS analysis) to afford compound
17 as a hydrochloride salt, a white solid. Yield: 201m g, 38%, m. p.: dec. >242 ºC.
1H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 2H), 8.53 (s, 1H), 8.41 (s, 2H), 8.36 (br s,
1H), 8.23 (d, J = 8.2 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 6.7 Hz, 1H), 7.68
– 7.56 (m, 3H), 4.63 (s, 2H), 3.10 ( t, J =7.7 Hz, 2H), 2.92 (t, J = 6.7 Hz, 2H), 1.92 –
1.85 (m, 2H); 13C NMR (101 MHz, DMSO-d6) 143.1, 133.3, 131.5 (q, J = 33.6 Hz,
2C), 131.1, 129.6, 129.1, 128.7, 128.1, 127.3 (unresolved q, J = 2.9 Hz, 2C), 126.8,
126.6 (unresolved sept, J = 3.3 Hz), 126.3, 125.3, 123.8, 122.7 (q, J = 271.7 Hz,
2C), 46.9, 44.9, 39.9, 26.0; 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.6; IR

max/cm-1: 2942 (N-H), 1280 (S=O), 1249 (C-O), 1164 (S=O), 1133 (C-F), 1076 (C-
N); LRMS (ESI+) m/z: 471 [M-HCl+H]; HRMS calculated for C19H21ClF6N2O3S [M-
HCl+H]: 471.1166; found: 471.1099.
N-[3-(3-Methoxybenzylamino)-propyl]-3,5-
bis(trifluoromethyl)benzenesulfonamide hydrochloride 21
Synthesized using the general procedure as described for 13, 3-methoxy-
benzylamine (2.5 eq, 340 mg, 2.5 mmol) and 3-(3,5-bis(trifluoromethyl)phenyl-
sulfonamido)propylmethane sulfonate (1.0 eq, 429 mg, 1.0 mmol) were reacted
(monitored by UPLC-MS analysis) to afford compound 21 as a hydrochloride salt,
a white solid. Yield: 270 mg, 53%, m. p.: 213-216 ºC. 1H NMR (400 MHz, DMSO-
d6) δ 9.27 (s, 2H), 8.50 (s, 1H), 8.40 (s, 2H), 8.18 (s, 1H), 7.32 (t, J = 7.9 Hz, 1H),
7.21 (s, 1H), 7.08 (d, J = 7.3 Hz, 1H), 6.96 (d, J = 7.5 Hz, 1H), 4.06 (s, 2H), 3.77 (s,
3H), 2.90 (t, J = 6.7 Hz, 4H), 1.89 – 1.79 (m, 2H); 13C NMR (101 MHz, DMSO-d6)
159.3, 143.1, 133.5, 131.4 (q, J = 33.6 Hz, 2C), 129.7, 127.3 (unresolved q, J = 3.1
Hz, 2C), 126.6 (unresolved sept, J = 3.4 Hz), 122.6 (q, J = 271.8 Hz, 2C), 122.0,
115.5, 114.5, 55.2, 49.9, 44.0, 39.9, 26.0; 19F NMR (376 MHz, DMSO-d6, CF3CO2H)
δ -58.1; IR max/cm-1: 2940 (N-H), 1273 (S=O), 1265 (C-O), 1164 (S=O), 1138 (C-F),

max/cm-1: 2842 (N-H), 1279 (S=O), 1159 (S=O), 1131 (C-F), 1117 (C-N); LRMS (ESI+)
m/z: 491.2 [M-HCl+H]; HRMS calculated for C21H19ClF6N2O2S [M-HCl+H]:
491.1221; found: 491.1105.
3,5-Bis-trifluoromethyl-N-[3-(4-trifluoromethylbenzylamino)-propyl]-
benzene sulfonamide hydrochloride 18
Synthesized using the general procedure as described for 13, 4-
(trifluoromethyl)benzylamine (2.5 eq, 440 mg, 2.5 mmol) and 3-(3,5-
bis(trifluoromethyl)phenylsulfonamido propylmethane sulfonate (1.0 eq, 429
mg, 1.0 mmol) were reacted (monitored by UPLC-MS analysis) to afford
compound 18 as a hydrochloride salt, a white solid. Yield: 150 mg, 28%, m. p.:
dec. >207 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.33 (s, 2H), 8.51 (s, 1H), 8.39 (s,
2H), 8.33 (s, 1H),7.82 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.3 Hz, 2H), 4.22 (s, 2H), 2.95
– 2.89 (m, 4H), 1.87 – 1.80 (m, 2H); 13C NMR (101 MHz, DMSO-d6) 143.1, 136.7,
131.5 (q, J = 33.7 Hz, 2C), 131.0 (2C), 129.3 (q, J = 31.6 Hz), 127.3 (unresolved q,
J = 3.2 Hz, 2C), 126.6 (unresolved sept, J = 3.5 Hz), 125.4 (q, J = 3.8 Hz, 2C), 124.1
(q, J = 270.5 Hz), 122.6 (q, J = 271.7 Hz, 2C), 49.3, 44.3, 39.9, 26.0; 19F NMR (376
MHz, DMSO-d6, CF3CO2H) δ -57.8, -58.0; IR max/cm-1: 2981 (N-H), 1275 (S=O),
1159 (S=O), 1133 (C-F), 1068 (C-N); LRMS (ESI+) m/z: 509 [M-HCl +H]; HRMS
calculated for C19H18ClF9N2O2S M-HCl+H]: 509.0942; found: 509.0867.
1093 (C-N); LRMS (ESI+) m/z: 471 [M-HCl+H]
C19H21ClF6N2O3S [M-HCl+H]: 471.1168; found: 471.1099.
; HRMS calculated for
N-[3-(2-Methoxybenzylamino)-propyl]-3,5-bis(trifluoromethyl)benzene
sulfonamide hydrochloride 22
Synthesized using the general procedure as described for 13, 2-methoxy-
benzylamine
(2.5
eq,
340m
g,
2.5
mmol)
and
3-(3,5-
bis(trifluoromethyl)phenylsulfonamido)propylmethane sulfonate (1.0 eq, 429
mg, 1.0 mmol) were reacted (monitored by UPLC-MS analysis) to afford
compound 22 as a hydrochloride salt, a white solid. Yield: 270 mg, 53%, m. p.:
208-211 ºC1H NMR (400 MHz, DMSO-d6) δ 8.93 (s br, 2H), 8.52 (s, 1H), 8.40 (s,
2H), 8.35 (s br , 1H), 7.45 – 7.39 (m, 2H), 7.09 (d, J = 8.0 Hz, 1H), 6.99 (td, J = 7.5,
0.8 Hz, 1H), 4.05 (s, 2H), 3.83 (s, 3H), 2.90 (t, J = 6.8 Hz, 4H), 1.87 – 1.79 (m, 2H);
13C NMR (101 MHz, DMSO-d6) 157.5, 143.1, 131.5, 131.5 (q, J = 33.6 Hz, 2C),
130.8, 127.3 (unresolved q, J = 3.1 Hz, 2C), 126.6 (unresolved sept, J = 3.5 Hz),
122.6 (q, J = 271.8 Hz, 2C), 120.3, 119.7, 111.1, 55.6, 44.9, 44.1, 39.9, 25.8; 19F
NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.1; IR max/cm-1: 2963 (N-H), 1281
(S=O), 1251 (C-O) 1181 (S=O), 1134 (C-F), 1112 (C-N); LRMS (ESI+) m/z: 471 [M-
HCl+H]; HRMS calculated for C19H21ClF6N2O3S [M-HCl+H]: 471.1169; found:
471.1099.
N-[4-(4-Bromo-benzylamino)-butyl]-3,5-
bis(trifluoromethyl)benzenesulfonamide hydrochloride 19
A solution of 4-bromobenzaldehyde (0.5 eq, 912 mg, 5.0 mmol) and
butane-1,4-diamine (1.0 eq, 881 mg, 1.0 mmol) in methanol (40 mL) was stirred
for 20 h at RT. Then NaBH4 was added at 0 oC and the reaction mixture was stirred
for another 1 h (monitored by UPLC-MS analysis). The crude was washed with 1
M NaOH 60 mL and extracted with DCM (3 x 30 mL). The organic layer was dried
with anhydrous MgSO4 and concentrated under reduced pressure. The residue
was triturated with methanol (3 x 10 mL) and cold ether (3 x 20 mL) to afford the
desired product as a white solid. Yield: 520 mg, 41%, purity > 90% by NMR and
LC-MS analysis. The product used directly in the next step without purification.
N-[3-(3,4-Dichlorobenzylamino)-propyl]-3,5-bis(trifluoromethyl)benzene
sulfonamide hydrochloride 23
Synthesized using the general procedure as described for 13, 3,4-dichloro-
benzylamine (2.5 eq, 441 mg, 2.5 mmol) and 3-(3,5-bis(trifluoromethyl)phenyl-
sulfonamido)propylmethane sulfonate (1.0 eq, 429 mg, 1.0 mmol) were reacted
(monitored by UPLC-MS analysis) to afford compound 23 as a hydrochloride salt,
a white solid. Yield: 341 mg, 62%, m. p.: 231-234 ºC. 1H NMR (400 MHz, DMSO-
d6) δ 9.27 (s, 2H), 8.52 (s, 1H), 8.39 (s, 2H), 8.32 (s, 1H), 7.87 (d, J = 1.9 Hz, 1H),
7.72 (d, J = 8.3 Hz, 1H), 7.53 (dd, J = 8.3, 2.0 Hz, 1H), 4.13 (s, 2H), 2.91 (dd, J =
11.9, 6.1 Hz, 4H), 1.85 – 1.78 (m, 2H); 13C NMR (101 MHz, DMSO-d6) 143.0, 133.1,
132.3, 131.6, 131.4 (q, J = 33.6 Hz, 2C), 131.1, 130.7, 130.6, 127.3 (unresolved q,
J = 3.1 Hz, 2C), 126.6 (unresolved sept, J = 3.5 Hz), 122.6 (q, J = 271.7 Hz, 2C), 48.6,
44.1, 39.9, 26.0; 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.0; IR max/cm-1:
2930 (N-H), 1275 (S=O), 1159 (S=O), 1134 (C-F), 1088 (C-N), 681 (C-Cl); LRMS
(ESI+) m/z: 509 [M-HCl+H, 35Cl]/510 [M-HCl+H, 37Cl]; HRMS calculated for
C18H17Cl3F6N2O2S [M-HCl+H, 35Cl]: 509.0282/[M-HCl+H, 37Cl]: 511.0252; found:
509.0214.
A solution of N1-(4-bromobenzyl)-butane-1,4-diamine (1.0 eq, 130 mg, 0.5
mmol) in methanol (10 mL) was added 3,5-bis(trifluoromethyl)benzenesulfonyl
chloride (1.1. eq, 169 mg, 0.55 mmol) slowly over 1 h at 0 oC, then stirred for
another 2 h (monitored by UPLC-MS analysis). The crude was washed using
aqueous NH4Cl (30 mL). The organic was concentrated. The residue was dissolved
using 2 mL acetone and 4 mL 10% HCl was added. The mixture was filtered and
triturated three times using cold ether (3 x 10 mL) to afford the desired
compound 19 as a hydrochloride salt, a white solid. Yield: 100 mg, 36%, m. p.:
202-206 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 2H), 8.50 (s, 1H), 8.38 (s, 2H),
8.19 (t, J = 5.8 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 4.08 (t, J =
16.8 Hz, 2H), 2.86 – 2.78 (m, 4H), 1.69-1.61 (m, 2H), 1.49 – 1.41 (m, 2H); 13C NMR
(101 MHz, DMSO-d6) 143.2, 132.4 (2C), 131.6 (2C), 131.5 (q, J = 33.7 Hz, 2C),
131.4, 127.3 (unresolved q, J = 3.2 Hz, 2C), 126.6 (unresolved sept, J = 3.5 Hz),
122.7 (q, J = 271.8 Hz, 2C), 122.4, 49.2, 46.0, 42.0, 26.3, 22.6; 19F NMR (376 MHz,
DMSO-d6, CF3CO2H) δ -58.5; IR max/cm-1: 2972.8 (N-H), 1279.8 (S=O), 1167.5
(S=O), 1135.2 (C-F), 1108.9 (C-N), 682.3 (C-Br); LRMS (ESI+) m/z: 533 [M-HCl+H,
N-[3-(Benzylmethylamino)-propyl]-3,5-
bis(trifluoromethyl)lbenzenesulfonamide hydrochloride 24
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000950
ChemMedChem
FULL PAPER
Synthesized using the general procedure as described for 13, benzyl-
7.5 Hz, 2H), 2.92 (t, J = 6.8 Hz, 2H), 1.89 – 1.82 (m, 2H); 13C NMR (101 MHz, DMSO-
d6) 143.1, 133.6, 132.0, 131.5 (q, J = 33.6 Hz, 2C), 130.8, 129.8, 129.6, 127.5,
127.3 (unresolved q, J = 3.1 Hz, 2C), 126.6 (unresolved sept, J = 3.3 Hz), 122.6 (q,
J = 271.7 Hz, 2C), 47.0, 44.5, 39.9, 25.9; 19F NMR (376 MHz, DMSO-d6, CF3CO2H)
δ -58.1; IR max/cm-1: 2921 (N-H), 1276 (S=O), 1159 (S=O), 1134 (C-F), 1087 (C-N),
681 (C-Cl); LRMS (ESI+) m/z: 475 [M-HCl+H]; HRMS calculated for
C18H18Cl2F6N2O2S [M-HCl+H]: 475.0673/[M-HCl+H, 37Cl]: 477.0641; found:
475.0603.
methyl-amine (2.5 eq, 300 mg, 2.5 mmol) and 3-(3,5-bis(trifluoromethyl)phenyl-
sulfonamido)propylmethane sulfonate (1.0 eq, 429 mg, 1.0 mmol) were reacted
(monitored by UPLC-MS analysis) to afford compound 24 as a hydrochloride salt,
a white solid. Yield: 0.35 g, 71%, m. p.: 124-127 ºC. 1H NMR (400 MHz, DMSO-d6)
δ 10.53 (s, 1H), 8.52 (s, 1H), 8.39 (s, 2H), 8.33 (t, J = 5.9 Hz, 1H), 7.57 (dd, J = 6.0,
2.9 Hz, 2H), 7.46 – 7.45 (m, 3H), 4.35 (dd, J = 13.0, 4.2 Hz, 1H), 4.21 (dd, J = 12.9,
5.9 Hz, 1H), 3.15 – 3.08 (m, 1H), 3.02 – 2.95 (m, 1H), 2.87 (q, J = 6.5 Hz, 2H), 2.60
(d, J = 4.6 Hz, 3H), 1.94 – 1.87 (m, 2H); 13C NMR (101 MHz, DMSO-d6) 142.9, 131.6,
131.3, 131.4 (q, J = 33.4 Hz, 2C), 130.0, 129.4, 128.7 (2C), 127.3 (unresolved q, J
= 3.1 Hz, 2C), 126.6 (unresolved sept, J = 3.4 Hz), 122.6 (q, J = 271.7 Hz, 2C), 58.2,
52.2, 39.9, 38.6, 23.8; 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.0; IR max/cm-
1: 2948 (N-H), 1282 (S=O), 1160 (S=O), 1132 (C-F), 1080 (C-N); LRMS (ESI+) m/z:
455 [M-HCl+H]; HRMS calculated for C19H21ClF6N2O2S [M-HCl+H]: 455.1221;
found: 455.1150.
N-{3-[(2,3-Dihydrobenzo[1,4]dioxin-2-ylmethyl)-amino]-propyl}-3,5-
bis(trifluoromethyl)benzene sulfonamide hydrochloride 29
Synthesized using the general procedure as described for 13, 2-
aminomethyl-1,4-benzodioxane (2.5 eq, 411 mg, 2.5 mmol) and 3-(3,5-
bis(trifluoromethyl)phenylsulfonamido)propylmethane sulfonate (1.0 eq, 429
mg, 1.0 mmol) were reacted (monitored by UPLC-MS analysis) to afford
compound 29 as a hydrochloride salt, an off-white solid. Yield: 281 mg, 53%, m.
p.: dec. >220 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.33 (s br, 1H), 9.11 (s br, 1H),
8.52 (s, 1H), 8.40 (s, 2H), 8.35 (t, J = 5.5 Hz, 1H), 6.91 – 6.87 (m, 4H), 4.61 (t, J =
7.3 Hz, 1H), 4.36 (dd, J = 11.6, 2.2 Hz, 1H), 4.05 (dd, J = 11.7, 6.8 Hz, 1H), 3.28 (s,
1H), 3.19 – 3.14 (m, 1H), 3.01 (d, J = 6.2 Hz, 2H), 2.92 (dd, J = 11.1, 5.8 Hz, 2H),
1.88 – 1.81 (m, 2H); 13C NMR (101 MHz, DMSO-d6) 143.1, 142.7, 141.9, 131.5 (q,
J = 33.7 Hz, 2C), 127.3 (unresolved q, J = 3.0 Hz, 2C), 126.6 (unresolved sept, J =
3.7 Hz), 122.6 (q, J = 271.7 Hz, 2C), 121.72, 121.68, 117.4, 117.1, 69.1, 64.8, 46.4,
45.0, 39.8, 25.9; 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.0; IR max/cm-1:
2970 (N-H), 1276 (S=O), 1167 (S=O), 1134 (C-F), 1108 (C-O), 1080 (C-N); LRMS
(ESI+) m/z: 499 [M-HCl+H]; HRMS calculated for C20H21ClF6N2O4S [M-HCl+H]:
499.1114; found: 499.1048.
N-(3-Benzylaminopropyl)-3,5-bis(trifluoromethyl)benzene
hydrochloride 25
Synthesized using the general procedure as described for 13, benzylamine
eq, 274 mg, 2.5 mmol) and 3-(3,5-
sulfonamide
(2.5
bis(trifluoromethyl)phenylsulfonamido)propylmethane sulfonate (1.0 eq, 429
mg, 1.0 mmol) were reacted (monitored by UPLC-MS analysis) to afford
compound 25 as a hydrochloride salt, a white solid. Yield: 300 mg, 63%, m. p.:
214-217 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 2H), 8.52 (s, 1H), 8.39 (s, 2H),
8.34 (s, 1H), 7.53 (dd, J = 7.5, 2.0 Hz, 2H), 7.45 – 7.40 (m, 3H), 4.10 (s, 2H), 2.91
(dd, J = 13.4, 8.0 Hz, 4H), 1.87 – 1.79 (m, 2H); 13C NMR (101 MHz, DMSO-d6) 143.1,
132.0, 131.5 (q, J = 33.6, 2C), 130.1 (2C), 128.9, 128.6 (2C), 127.3 (unresolved q,
J = 3.1 Hz, 2C), 126.6 (unresolved sept, J = 3.4 Hz), 122.6 (q, J = 271.7 Hz, 2C),
50.0, 44.1, 39.9, 25.9; 19F NMR (376 MHz, DMSO-d6, CF3CO2H) δ -58.0; IR max/cm-
1: 2927 (N-H), 1278 (S=O), 1167 (S=O), 1128 (C-F), 1107 (C-N); LRMS (ESI+) m/z:
441 [M-HCl+H] ; HRMS calculated for C18H19ClF6N2O2S [M-HCl+H]: 441.1059;
found: 441.0993.
N-[3-(4-Chloro-3-trifluoromethylbenzylamino)-propyl]-3,5-
bis(trifluoromethyl)benzenesulfonamide hydrochloride 30
Synthesized using the general procedure as described for 13, 4-chloro-3-
trifluoromethylbenzylamine (2.5 eq, 520m g, 2.5 mmol) and 3-(3,5-bis(trifluoro-
methyl)phenylsulfonamido)propylmethane sulfonate (1.0 eq, 429 mg, 1.0 mmol)
were reacted (monitored by UPLC-MS analysis) to afford compound 30 as a
hydrochloride salt, an off-white solid. Yield: 309 mg, 53%, m. p.: dec. >230 ºC. 1H
NMR (400 MHz, DMSO-d6) δ 9.25 (s br, 2H), 8.52 (s, 1H), 8.39 (s, 2H), 8.32 (s br,
1H), 8.10 (s, 1H), 7.87 – 7.80 (m, 2H), 4.21 (s, 2H), 2.95-2.89 (m, 4H), 1.85 – 1.78
(m, 2H); 13C NMR (101 MHz, DMSO-d6) 143.0, 136.1, 132.1, 131.9, 131.5 (q, J =
33.7, 2C), 131.3 (unresolved q, J = 1.8 Hz), 129.9 (q, J = 5.1 Hz), 127.3 (unresolved
q, J = 3.3 Hz, 2C), 126.63 (q, J = 30.8 Hz), 126.6 (unresolved sept, J = 3.5 Hz), 122.7
(q, J = 271.5 Hz), 122.6 (q, J = 271.7 Hz, 2C), 48.7, 44.2, 39.8, 26.1; 19F NMR (376
MHz, DMSO-d6, CF3CO2H) δ -58.0, 58.1; IR max/cm-1: 2960 (N-H), 1275 (S=O),
1176 (S=O), 1133 (C-F), 1089 (C-N), 680 (C-Cl); LRMS (ESI+) m/z: 543 [M-HCl+H,
35Cl]/545 [M-HCl+H, 37Cl]; HRMS calculated for C19H17Cl2F9N2O2S [M-HCl+H, 35Cl]:
543.0546/[M-HCl+H, 37Cl]: 545.0516; found: 543.0477.
N-[3-(4-Chlorobenzylamino)-propyl]-3,5-bis(trifluoromethyl)benzene
sulfonamide hydrochloride 26
Synthesized using the general procedure as described for 13, 4-chloro-
benzylamine (2.5 eq, 349 mg, 2.5 mmol) and 3-(3,5-bis(trifluoromethyl)phenyl-
sulfonamido)propylmethane sulfonate (1.0 eq, 429 mg, 1.0 mmol) were reacted
(monitored by UPLC-MS analysis) to afford compound 26 as a hydrochloride salt,
a white solid. Yield: 201 mg, 39%, m. p.: 228-231 ºC. 1H NMR (400 MHz, DMSO-
d6) δ 9.23 (s, 2H), 8.51 (s, 1H), 8.39 (s, 2H), 8.33 (s, 1H), 7.57 – 7.55 (m, 2H), 7.52
– 7.49 (m, 2H), 4.11 (s, 2H), 2.89 (d, J = 6.1 Hz, 4H), 1.85 – 1.78 (m, 2H); 13C NMR
(101 MHz, DMSO-d6) 143.1, 133.7, 132.1 (2C), 131.8 (q, J = 33.7 Hz, 2C), 131.0,
128.6 (2C), 127.3 (unresolved q, J = 3.2 Hz, 2C), 126.6 (unresolved sept, J = 3.4
Hz), 122.6 (q, J = 271.7 Hz, 2C), 49.1, 44.0, 39.9, 26.0; 19F NMR (376 MHz, DMSO-
d6, CF3CO2H) δ -58.0; IR max/cm-1: 2937 (N-H), 1276 (S=O), 1159 (S=O), 1135 (C-
F), 1088 (C-N), 681 (C-Cl); LRMS (ESI+) m/z: 475 [M-HCl+H, 35Cl]/477 [M-HCl+H,
37Cl]; HRMS calculated for C18H18Cl2F6N2O2S [M-HCl+H, 35Cl]: 475.0673/[M-HCl+H,
37Cl]: 477.0641; found: 475.0603.
Biology
N-[3-(3-Chlorobenzylamino)-propyl]-3,5-
bis(trifluoromethyl)benzenesulfonamide hydrochloride 27
Cell culture and stock solutions
Synthesized using the general procedure as described for 13, 3-chloro-
benzylamine (2.5 eq, 350 mg, 2.5 mmol) and 3-(3,5-bis(trifluoromethyl)phenyl-
sulfonamido)propylmethane sulfonate (1.0 eq, 429 mg, 1.0 mmol) were reacted
(monitored by UPLC-MS analysis) to afford compound 27 as a hydrochloride salt,
a white solid. Yield: 230 mg, 45%, m. p.: 227-230 ºC. 1H NMR (400 MHz, DMSO-
d6) δ 9.24 (s, 2H), 8.51 (s, 1H), 8.39 (s, 2H), 8.34 (s, 1H), 7.67 (s, 1H), 7.51 – 7.44
(m, 3H), 4.12 (s, 2H), 2.91 (dd, J = 13.2, 6.6 Hz, 4H), 1.86 – 1.79 (m, 2H); 13C NMR
(101 MHz, DMSO-d6) 143.1, 134.4, 133.1, 131.5 (q, J = 33.6 Hz, 2C), 130.4, 130.0,
128.9, 128.8, 127.3 (unresolved quartet, J = 3.1 Hz, 2C), 126.6 (unresolved sept,
J = 3.3 Hz), 122.6 (q, J = 271.7 Hz, 2C), 49.3, 44.2, 39.9, 26.0; 19F NMR (376 MHz,
DMSO-d6, CF3CO2H) δ -58.0; IR max/cm-1: 2937 (N-H), 1281 (S=O), 1159 (S=O),
1134 (C-F), 1084 (C-N), 682 (C-Cl); LRMS (ESI+) m/z: 475 [M-HCl+H, 35Cl]/477 [M-
HCl+H, 37Cl]; HRMS calculated for C18H18Cl2F6N2O2S [M-HCl+H, 35Cl]:
475.0673/[M-HCl+H, 37Cl]: 477.0641; found: 475.0603.
Stock solutions were prepared as follows and stored at -20 °C: drugs were stored
as 40 mM solutions in DMSO. All cell lines were cultured in a humidified
atmosphere 5% CO2 at 37 ºC. The cancer cell lines were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) (Trace Biosciences, Australia) supplemented
with 10% foetal bovine serum, 10 mM sodium bicarbonate, penicillin (100 IU/mL),
streptomycin (100 μg/mL), and glutamine (4 mM). The non-cancer MCF10A cell
line was cultured in DMEM:F12 (1:1) cell culture media, 5% heat inactivated
horse serum, supplemented with penicillin (50 IU/mL), streptomycin (50 μg/mL),
20mM Hepes, L-glutamine (2mM), epidermal growth factor (20ng/mL),
hydrocortisone (500ng/mL), cholera toxin (100ng/mL), and insulin (10 μg/mL).
In vitro growth inhibition MTT assay
Cells in logarithmic growth were transferred to 96-well plates. Cytotoxicity was
determined by plating cells in duplicate in 100 μL medium at a density of 2500-
4000 cells/well. On day 0, (24 h after plating) when the cells were in logarithmic
growth 100 μL medium with or without the test agent was added to each well.
After 72 h drug exposure growth inhibitory effects were evaluated using the MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl- tetrazolium bromide) assay and
absorbance read at 540 nm. Percentage growth inhibition was determined at a
fixed drug concentration of 25 μM. A value of 100% is indicative of complete cell
growth inhibition. Those analogues showing appreciable percentage growth
inhibition underwent further dose response analysis allowing for the calculation
of a GI50 value. This value is the drug concentration at which cell growth is 50%
N-[3-(2-Chlorobenzylamino)-propyl]-3,5-bis(trifluoromethyl)benzene
sulfonamide hydrochloride 28
Synthesized using the general procedure as described for 13, 2-chloro-
benzylamine (2.5 eq, 350 mg, 2.5 mmol) and 3-(3,5-bis(trifluoromethyl)phenyl-
sulfonamido)propylmethane sulfonate (1.0 eq, 429 mg, 1.0 mmol) were reacted
(monitored by UPLC-MS analysis) to afford compound 28 as a hydrochloride salt,
a white solid. Yield: 228 mg, 45%, m. p.: 237-240 ºC. 1H NMR (400 MHz, DMSO-
d6) δ 9.29 (s br, 2H), 8.52 (s, 1H), 8.40 (s, 2H), 8.35 (s br, 1H), 7.72 (dd, J = 6.8, 2.5
Hz, 1H), 7.55 (dd, J = 7.3, 2.0 Hz, 1H), 7.48 – 7.42 (m, 2H), 4.22 (s, 2H), 2.99 (t, J =
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000950
ChemMedChem
FULL PAPER
inhibited based on the difference between the optical density values on day 0
and those at the end of drug exposure.24, 25
[15] Biankin, A. V.; Kench, J. G.; Colvin, E. K.; Segara, D.; Scarlett, C. J.;
Nguyen, N. Q.; Chang, D. K.; Morey, A. L.; Lee, C. -S.; Pinese, M.; Kuo,
S. C. L.; Susanto, J. M.; Cosman, P. H.; Lindeman, G. J.; Visvader, J. E.;
Nguyen, T. V.; Merrett, N. D.; Warusavitarne, J.; Musgrove, E. A.;
Henshall, S. M.; Sutherland, R. L.; NSW Pancreatic Cancer Network.
Expression of S100A2 calcium-binding protein predicts response to
pancreatomy for pancreatic cancer. Gastroenterology, 2009, 137,
558-568.
[16] Ohuchida, K.; Mizumoto, K.; Ishikawa, N.; Fujii, K.; Konomi, H.; Nagai,
E.; Yamaguchi, K.; Tsuneyoshi, M.; Tanaka, M. The role of S100A6 in
pancreatic cancer development and its clinical implications as a
diagnostic marker and therapeutic target. Clin. Cancer Res. 2005, 11,
7785-7793.
Molecular docking
Molecular docking simulations were performed using the default settings
of Molecular Operating Environment (MOE). The human S100A2 crystal structure
(RCSB ID: 2RGI) was prepared in MOE using the protonate 3D function: the
system was protonated, partial charges added, and minimization performed. The
designed 3D structures were built using the software molecular builder and were
subjected to conformational analysis using a stochastic search approach. Each
conformational library was docked into the p53 binding groove of the S100A2
homodimer using “Triangle Matcher” as the placement method and “London
dG” as the scoring function for the initial ligand placement. The top 20 poses
were then refined with the GBVI/WSA scoring function. The highest ranked pose
for each compound was relaxed in the binding groove using LigX energy
minimisation. Analysis and visualisation of the docking output, such as
identification of hydrogen bonds, steric clashes, hydrophobic interactions, or π-
π interactions, were performed in MOE.24, 25
[17] Ohuchinda, K.; Ohuchida, K.; Mizumoto, K.; Miyasaka, Y.; Yu, J.; Cui,
L.; Yamaguchi, H.; Toma, H.; Takahata, S.; Sato, N.; Nagai, E.;
Yamaguchi, K.; Tsuneyoshi, M.; Tanaka, M. Over-expression of
S100A2 in pancreatic cancer corelates with progression and poor
prognosis. J. Pathol. 2007, 213, 275-282.
[18] Hountis, P.; Mattaios, D.; Froudarkis, M.; Bouros, D.; Kakolyris, S.;
S100A2 protein and non-small cell lung cancer. The dual role
concept. Tumour Biol. 2014, 35, 7327-7333.
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12
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10.1002/cmdc.202000950
ChemMedChem
FULL PAPER
Acknowledgements
JS is the recipient of a joint scholarship of UON UPRS and
Binzhou Medical University (China), and this project has
received funding from the UON Priority Research Centre for
Drug Development. AM is the recipient of funding from the
Australian Cancer Research Foundation and the Ramaciotti
Foundation. This work was also supported by the University
of Newcastle Priority Research Centre for Drug Development.
13
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10.1002/cmdc.202000950
ChemMedChem
FULL PAPER
Targeting the S100A2-p53 complex reveals novel pancreatic cancer cytotoxic specific small molecule inhibitors. Enhanced potency is noted against
S100A2 containing PC cell lines MiaPaCa-2 and BxPC-3.
14
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