A novel small-molecule inhibitor of IL-6 signalling

Article abstract of DOI:10.1016/j.bmcl.2010.09.117

A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.

Full text of DOI:10.1016/j.bmcl.2010.09.117

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7029–7032
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel small-molecule inhibitor of IL-6 signalling
Giovanna Zinzalla a, Mohammad R. Haque a, B. Piku Basu b, John Anderson b, Samantha L. Kaye a,
Shozeb Haider c, Fyeza Hasan b, Dyeison Antonow a, Samantha Essex a, Khondaker M. Rahman a,
Jonathan Palmer a, Daniel Morgenstern b, Andrew F. Wilderspin a, Stephen Neidle c, David E. Thurston a,
⇑
a Cancer Research UK Protein–Protein Interactions Drug Discovery Research Group, The School of Pharmacy, University of London, 29-39 Brunswick Sq., London WC1N 1AX,
United Kingdom
b UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom
c Cancer Research UK Biomolecular Structure Group, The School of Pharmacy, University of London, 29-39 Brunswick Sq., London WC1N 1AX, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route,
and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have
promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit
phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell
lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells
suggesting STAT3-specific inhibitory properties.
Received 11 August 2010
Revised 21 September 2010
Accepted 23 September 2010
Available online 29 September 2010
Keywords:
Small molecule
IL-6 signalling
Inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
STAT3
Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates
hematopoiesis, inflammation and the immune system. It is impli-
cated in the pathogenesis of various inflammatory disorders such
as rheumatoid arthritis, inflammatory bowel disease, osteoarthri-
tis, psoriasis, endotoxemia and toxic shock syndrome.1,2 IL-6 also
plays a key role in the pathophysiology of several cancers, and its
over-expression has been implicated in the tumourigenesis of mul-
tiple myeloma, ovarian, renal cell, prostate, cervical and breast car-
cinomas.1,3–5 Therefore, the identification of inhibitory agents to
target IL-6 signalling is currently of interest in both anti-inflamma-
tory and anticancer drug discovery.
IL-6 signals mainly through activation of the STAT3 (signal
transducer and activator of transcription-3) protein.6,7 It initially
complexes with its membrane-bound receptor, gp130, inducing
dimerisation and activating bound JAK (Janus Associated Kinase)
protein which autophosphorylates and in-turn phosphorylates
STAT3 on its tyrosine 705 residue. This triggers homodimerisation
of the STAT3 protein followed by movement to the nucleus where
it binds to cognate DNA sequences and activates transcription in a
range of genes associated with proliferation and survival. Although
no selective small-molecule IL-6 inhibitors have been reported to
date, there are examples of molecules in the patent literature that
inhibit IL-6 signalling in combination with a number of other
signalling pathways. Currently, several approaches are being
investigated to selectively inhibit the IL-6 signalling pathway
including targeting formation of the IL-6/gp130/JAK complex and
inhibition of formation of the STAT3 homodimer.8–10
As part of a programme to discover novel inhibitors of signalling
pathways known to be important for tumour cell growth and sur-
vival, we have identified the novel IL-6 pathway inhibitor 6a
through screening a small library of arylsulphonamidyl thiophene
amides (Table 1) prepared in-house11 based on preliminary in sil-
ico studies. The library was synthesised via a 4-step synthetic route
(Scheme 1) starting with chlorosulfonation of commercially avail-
able 1, followed by formation of the sulfonylamide derivatives
3a,b. The next step involved attachment of the R1 substituents
(4a–e) via an amide bond using EDC/DMAP to provide interme-
diates 5a–f. The final step involved Suzuki reaction with
a
range of boronic acids employing tetrakis(triphenylphosphine)
palladium(0) as catalyst at 100 °C under microwave irradiation
(Table 1). All reactions proceeded within a short time period (i.e.,
5–20 min) and in good yields to afford the final products 6a–v,
all of which were typically >98% pure.12
The library members were initially evaluated in an MTS cell via-
bility assay using both MDA-MB-231 breast (STAT3-dependent)
and A4 (STAT3-null) cancer cells in which STAT3 signalling had
been stimulated with IL-6. Both cell lines were treated with up
to 125 lM of each library member, and then monitored for 24 h.
Compound 6a emerged as a potential STAT3-specific inhibitor,13
leading to a reduction of ꢀ40% viable cells in the MDA-MB-231 line
compared to A4 (Fig. 1). An accompanying Trypan Blue exclusion
⇑
Corresponding author. Tel.: +44 (0)207 753 5932; fax: +44 (0)207 753 5964.
E-mail address: david.thurston@pharmacy.ac.uk (D.E. Thurston).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.117

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G. Zinzalla et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7029–7032
Table 1
Structures and yields of library members 6a–v12
SM
n
R1
R2
Product
Yielda (%)
5a
5b
5c
5d
5e
5a
5a
5a
5a
5a
5a
5a
5a
5a
5a
5a
5a
5a
5a
5a
5a
5f
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
Thiophene-2-yl
Furan-2-yl
1-Methyl-1H-pyrrol-2-yl
Thiazol-2-yl
Tolyl
Tolyl
Tolyl
Tolyl
6a
6b
6c
6d
6e
6f
6g
6h
6i
75
75
79
73
88
29
44
70
88
88
86
55
75
95
64
82
83
78
87
65
49
75
Thiazol-5-yl
Tolyl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
Thiophene-2-yl
4-Methoxyphenyl
4-Fluorophenyl
Pyridin-4-yl
4-Hydroxyphenyl
4-Mercaptophenyl
4-Chlorophenyl
Thiophen-2-yl
Furan-2-yl
4-(Trifluoromethyl) phenyl
4-Carboxyphenyl
4-Nitrophenyl
1H-Indol-5-yl
4-Alaninylphenylb
Naphthalen-1-yl
4-Aminophenylc
5-Pyrimidinyl
Tolyl
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
a
b
c
>98% purity based on LC–MS and NMR analysis.
Reaction carried out at 130 °C.
As the pinacol ester.
Pyrrolidine or
piperidine
(3 eq)
O
Neat ClSO3H
S
O
Br
O
(12 eq)
S
HO
Br
-5 oC to rt, 24 h,
83%
HO
MeOH,
S
0 oC to rt,1h,
68 - 74%
Cl
O
2
1
R1
NH
2 (1.1 - 1.2 eq)
4a: R1 = thiophene-2-yl
O
4b: R1 = furan-2-yl
S
Br
O
4c: R1 = 1-methyl-1H-pyrrol-2-yl
4d: R1 = thiazol-2-yl
HO
S
N
4e: R1 = thiazol-5-yl
O
n
EDC.HCl (1.2 eq), DMAP (0.1 eq),
DCM, 0 oC to rt, 5-20 h,
50 - 79%
3a: n = 1
3b: n = 2
R2-B(OH)2 (1.2 - 1.5 eq),
[P(Ph)3]4Pd(0) (0.1 eq),
O
O
Figure 1. MTS cell viability assay profiles for 6a in MDA-MB-231 (STAT3-depen-
dent) and A4 (STAT3-null) cells after 24 h exposure.
S
S
R2
O
Br
O
N
H
R1
N
R1
K2CO3 (2 eq)
H
MW, 100 oC, 5-20 min
(see Table 1 for R2
and yields)
S
S
N
N
O
O
n
n
6a-v
treated with Oncostatin M to activate STAT3 signalling via the IL-6/
gp130 receptor prior to addition of 6a which was shown to selec-
tively inhibit STAT3 transcriptional activity with an EC50 of 15 lM
(Fig. 2). Interestingly, although some molecules of similar structure
to 6a (see Table 1) had a range of activities in the MTS assay in the
MDA-MB-231 cell line (i.e., 5–100 lM) and modest activity in the
STAT3-reporter assay, 6a was the only molecule in the library with
5a-f
5a: n = 1, R1 = thiophene-2-yl
5b: n = 1, R1 = furan-2-yl
5c: n = 1, R1 = 1-methyl-1H-pyrrol-2-yl
5d: n = 1, R1 = thiazol-2-yl
5e: n = 1, R1 = thiazol-5-yl
5f: n = 2, R1 = thiophene-2-yl
STAT3-selective properties.
Scheme 1. Synthesis of library members 6a–v.
Further confirmation of the specificity of 6a for STAT3 inhibition
was obtained by investigating its effect on phosphorylation of the
tyrosine 705 residue within the STAT3 protein (i.e., to give P-
STAT3) in serum-starved MDA-MB-231 and HeLa cells 20 min after
the addition of IL-6 to induce phosphorylation.16 Compound 6a
was found to inhibit STAT3 phosphorylation downstream from
IL-6 in both HeLa and MDA-MB-231 cells at concentrations of
assay using the same MDA-MB-231 cells and with identical expo-
sure times to 6a resulted in a high percentage of viable cells, thus
confirming a cytostatic rather than a cytotoxic effect for this mol-
ecule (data not shown).14
10–30 and 30–100 lM, respectively (Fig. 3). IL-6 did not induce
phosphorylation of STAT1 in either cell line, although some phos-
phorylation was observed in HeLa cells in the presence of higher
concentrations (30 and 100 lM) of 6a. This could be due to diver-
Compound 6a was progressed into a luciferase reporter assay
based on HeLa cells containing plasmids constructed with either
a STAT3 minimal promoter (stable line) or a SV40 control promoter
(transient transfection) upstream from luciferase.15 The cells were

G. Zinzalla et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7029–7032
7031
Figure 2. Effect of 6a in a luciferase reporter assay in HeLa cells containing either
STAT3 or SV40 promoters upstream of luciferase. Error bars indicate mean SEM
from three independent experiments.
Figure 4. Potential binding sites of 6a at the protein–protein interfaces of the IL-6/
gp130 complex. Best docks of 6a (represented as sticks) are shown at positions A
and B. The model (PDB 1P9M) of the IL-6
a-receptor (red)/gp130 (yellow)/IL-6
(green) complex is coloured distinctly to highlight the protein–protein interfaces.
Acknowledgments
Cancer Research UK is acknowledged for providing financial
support for these studies (C180/A9327 to D.E.T. and C129/A4489
to S.N.), and the Commonwealth Commission is thanked for a
Scholarship (BDCS-2007-44) for M.R.H.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.117.
References and notes
1. Hodge, D. R.; Hurt, E. M.; Farrar, W. L. Eur. J. Cancer 2005, 41, 2502.
2. Scheller, J.; Ohnesorge, N. Scand. J. Immun. 2006, 63, 321.
3. Ara, T.; DeClerck, Y. A. Eur. J. Cancer 2010, 46, 1223.
Figure 3. Western Blots showing the effect of 6a on the expression of STAT3, P-
4. Blanchard, F.; Duplomb, L.; Baud’huin, M.; Brounais, B. Cytokine Growth Factor
Rev. 2009, 20, 19.
STAT3, STAT1, P-STAT1 and GAPDH in IL-6-stimulated HeLa and MDA-MB-231 cells.
5. David, S. H.; Laura, S. A.; Razelle, K. Cancer 2007, 110, 1911.
6. Hirano, T.; Ishihara, K.; Hibi, M. Oncogene 2000, 19, 2548.
7. Yu, H.; Pardoll, D.; Jove, R. S. Nat. Rev. Cancer 2009, 9, 798.
8. Adachi, Y.; Yoshio-Hoshino, N.; Nishimoto, N. Curr. Pharm. Des. 2008, 14, 1217.
9. Mitsuyama, K.; Matsumoto, S.; Masuda, J.; Yamasaki, H.; Kuwaki, K.; Takedatsu,
H.; Sata, M. Anticancer Res. 2007, 27, 3749.
10. Rose-John, S.; Waetzig, G. H.; Scheller, J.; Gratzinger, J.; Seegert, D. Expert Opin.
Ther. Targets 2007, 11, 613.
sion of IL-6 signalling through STAT1 due to STAT3 signalling
blockade at these higher concentrations of 6a, a phenomenon that
has been previously described.17
There is no information as yet on a possible site of binding of 6a,
although there is a crystal structure available (PDB 1P9M) of part of
11. General procedure for library synthesis. Preparation of 2: Chlorosulfonation of 1
was carried out with an excess of chlorosulfonic acid (99%, 12 equiv) in neat
conditions for 40 h at room temperature. The reaction mixture was poured into
an ice bath and 2 (P98% pure) was collected as a white solid (83% yield).
Preparation of 3a and 3b: Pyrrolidine or piperidine was added in excess
(3 equiv) to a solution of 2 in methanol, and the mixture stirred for 1 h at room
temperature. The reaction mixture was then extracted with water and acidified
to pH ꢀ1 to precipitation 3a or 3b as white solids (68–83% yields). Preparation
of 5a–f: Amide coupling was achieved by adding an excess (1.1–1.2 equiv) of
one of the amines 4a–e into a solution of 3a or 3b, EDCÁHCl (1.2 equiv) and
DMAP (0.1 equiv) in dichloromethane, followed by stirring for 16–20 h at room
temperature. The resulting amides (5a–f) were isolated in 50–79% yields and
>98% purity by column chromatography on silica gel. Preparation of 6a–v:
Suzuki cross-coupling between 5a–f and the respective boronic acids (1.2–
1.5 equiv) in CH3CN/H2O (3:1) was achieved by addition of tetrakis(triphenyl-
phosphine)palladium (0.1 equiv) and K2CO3 (2 equiv) followed by heating for
5–20 min at 100 °C under microwave irradiation. The products (6a–v) were
isolated in 29–95% yields and >98% purity by column chromatography on silica
gel.
the IL-6/a
-receptor/gp130 complex.18 We used this as a starting-
point for an in silico modelling study,19 which has so far located
two possible low-energy sites. In one site (labelled B in Fig. 4),
6a is shown binding in a cavity on the surface of the IL-6/a-recep-
tor interface. We speculate that this could potentially prevent
effective interaction of IL-6 with its receptors. In the other site (la-
belled A), 6a is shown buried in a cavity at the IL-6/gp130 interface,
and is thus less likely to interfere with receptor binding. The inter-
action energies for the dockings were calculated as À8.82 kcal/mol
and À8.25 kcal/mol for sites A and B, respectively.
In summary, a novel synthetic IL-6 signalling inhibitor (6a) has
been identified. Preliminary cellular studies indicate that, at low
micromolar concentrations, the molecule is cytostatic and capable
of selectively inhibiting STAT3 activation following IL-6 stimula-
tion at both phosphorylation and transcriptional levels. Further
studies are underway to identify the precise site of action of 6a fol-
lowed by the design of more-potent molecules in this series.
12. Full characterisation data for 6a–v are provided in Supplementary data.
13. General procedure for the MTS assay: 20,000 MDA-MB-231 or A4 cells were
plated into a 96-well plate, and ꢀ80% confluency achieved by incubating
overnight. Inhibitors were added and the cells incubated for a further 24 h.
MTS reagent (CellTiter 96Ò Aqueous One Solution Cell Proliferation Assay MTS

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G. Zinzalla et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7029–7032
Solution; Promega) was added in accordance with the manufacturer’s protocol.
were probed with the following antibodies: STAT3, P-STAT3, STAT1, P-STAT1
and GAPDH (Cell Signalling Inc.).
17. Regis, G.; Pensa, S.; Boselli, D.; Novelli, F.; Poli, V. Semin. Cell Dev. Biol. 2008, 19,
351.
18. Boulanger, M. J.; Chow, D. C.; Brevnova, E. E.; Garcia, K. C. Science 2003, 300,
2104.
19. Molecular modeling methodology: The ligand was generated via the PRODRG
server (http://davapc1.bioch.dundee.ac.uk/prodrg/). The non-polar hydrogen
atoms were merged, rotatable bonds assigned and partial atomic charges
calculated as implemented in the AutoDockTools package (Morris, G. M.;
Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew R. K.; Olsen, A. J. J.
Comp. Chem. 1998, 19, 1639). After removing IL-6, Grid maps were then
After 3 h, absorbance readings were taken at 490 nm and assumed to be
directly proportional to the number of living cells. Absorbance values for cells
treated with either vehicle or media alone were used as controls. The MTS data
(calculated as percentage of control values) were calculated from triplicate
measurements in at least two separate experiments to allow SDs to be
calculated.
14. General procedure for Trypan Blue assay: MDA-MB-231 cells were plated into 24
well plates and allowed to incubate overnight to achieve 80% confluency.
Inhibitors were then added, and the cells incubated for a further 24 h. After
addition of Trypan Blue, unstained (viable) and stained (non-viable) cells were
counted and calculated as a percentage of total cells using a haemocytometer.
15. General procedure for the Luciferase Reporter assay: HeLa cells containing the
relevant plasmid were plated-out and incubated overnight to achieve 80%
generated on the receptor interfaces where the IL-6
a-receptor and gp130
should reside. The grid was large enough to encompass the entire IL-6 binding
interface on the receptors. The grid points were thus chosen to be
118 Â 118 Â 118 Å with a grid spacing of 0.375 Å. Automated docking was
carried out using Autodock 4.0. A Lamarckian genetic algorithm (LGA) was
applied to investigate plausible interactions between the ligand and receptor.
The Solis and Wets algorithm (Solis, F. J.; Wets, J. B. Math. Oper. Res. 1981, 6, 19)
was employed to carry out the local search. Values for all other docking
parameters were kept as standard. Multiple docking runs can enhance the
performance of docking programs (Wang, R.; Lu, Y.; Wang, S. J. Med. Chem.
2003, 46, 2287), and so 100 independent docking runs were carried out. The
best docked conformation at each interface was then extracted based on
interaction energies.
confluency. The medium was then changed to
a serum-free type, and
Oncostatin M (100 ng/ml) was added at the same time as an inhibitor. The
cells were allowed to incubate for 8 h, and then cell lysates were prepared and
luciferase activities measured using dual luciferase kits (Promega) in
accordance with the manufacturer’s protocol.
16. General procedure for Western Blots: 1 Â 106cells were plated into 2% FCS media
in 6-well plates and allowed to incubate overnight. The medium was then
changed to a serum-free type, and IL-6 (20 ng/ml) was added at the same time
as an inhibitor. Whole cell extracts were then prepared using RIPA buffer
(Thermo Scientific) with protease and phosphatase inhibitors. Extracts were
dissolved in SDS buffer and run on a 10% PAGE gel for 1 h at 100 V (4 °C). Blots