Substituted arylsulphonamides as inhibitors of perforin-mediated lysis

Article abstract of DOI:10.1016/j.ejmech.2017.05.048

The structure-activity relationships for a series of arylsulphonamide-based inhibitors of the pore-forming protein perforin have been explored. Perforin is a key component of the human immune response, however inappropriate activity has also been implicated in certain auto-immune and therapy-induced conditions such as allograft rejection and graft versus host disease. Since perforin is expressed exclusively by cells of the immune system, inhibition of this protein would be a highly selective strategy for the immunosuppressive treatment of these disorders. Compounds from this series were demonstrated to be potent inhibitors of the lytic action of both isolated recombinant perforin and perforin secreted by natural killer cells in vitro. Several potent and soluble examples were assessed for in vivo pharmacokinetic properties and found to be suitable for progression to an in vivo model of transplant rejection.

Full text of DOI:10.1016/j.ejmech.2017.05.048

European Journal of Medicinal Chemistry 137 (2017) 139e155
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Substituted arylsulphonamides as inhibitors of perforin-mediated
lysis
,
b
,
,
, b
Julie A. Spicer a *, Christian K. Miller a b, Patrick D. O'Connor a, Jiney Jose a
,
Kristiina M. Huttunen a c, Jagdish K. Jaiswal a b, William A. Denny a b, Hedieh Akhlaghi d,
,
,
,
Kylie A. Browne d, Joseph A. Trapani d
,
e
a Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New
Zealand
b Maurice Wilkins Centre for Molecular Biodiscovery, A New Zealand Centre for Research Excellence, Auckland, New Zealand
c School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland
d Cancer Immunology Program, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, Victoria 3000, Australia
e Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria 3052 Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure-activity relationships for a series of arylsulphonamide-based inhibitors of the pore-
forming protein perforin have been explored. Perforin is a key component of the human immune
response, however inappropriate activity has also been implicated in certain auto-immune and therapy-
induced conditions such as allograft rejection and graft versus host disease. Since perforin is expressed
exclusively by cells of the immune system, inhibition of this protein would be a highly selective strategy
for the immunosuppressive treatment of these disorders. Compounds from this series were demon-
strated to be potent inhibitors of the lytic action of both isolated recombinant perforin and perforin
secreted by natural killer cells in vitro. Several potent and soluble examples were assessed for in vivo
pharmacokinetic properties and found to be suitable for progression to an in vivo model of transplant
rejection.
Received 9 March 2017
Received in revised form
21 May 2017
Accepted 23 May 2017
Available online 26 May 2017
Keywords:
Perforin
Perforin inhibitor
Arylsulphonamide
Bioisostere
© 2017 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
Immunosuppressant
1. Introduction
plasma membrane, release their luminal contents into the synapse
[2,4]. Perforin performs a key role in this process because entry of
Perforin is a 67 kDa, calcium-dependent glycoprotein expressed
by only the natural killer (NK) cells and cytotoxic T lymphocytes
(CTLs) of the mammalian immune system [1,2]. These “killer lym-
phocytes” utilise the pore-forming ability of perforin as a critical
component of the granule exocytosis pathway; the principal
mechanism used by NK and CTL cells for tumour immuno-
surveillance and as a defence against viral infection and intracel-
lular pathogens [3]. Identification of a target cell by an effector cell
results in the formation of an immune synapse whereupon CTL (or
NK) secretory granules polarise to the site of contact. These gran-
ules contain both perforin and a group of pro-apoptotic serine
proteases known as granzymes, and upon fusion with the CTL
the granzymes required for cell death into the target cell cytosol is
solely dependent on its presence [1,5].
Although perforin is synthesized and secreted into the immune
synapse as a monomer, it rapidly binds to the target cell membrane
through its calcium-dependent C2 domain [6,7] and oligomerises
into large transmembrane pores composed of approximately 24
perforin monomers. This process was elucidated using a combi-
nation of the perforin X-ray crystal structure and cryoelectron
microscopy to reconstruct an entire perforin pore [8]. Electron
microscopy, X-ray crystallography and functional studies have also
shown that the process involves electrostatic interactions which
include a salt bridge formed between R213 on the ‘front’ surface of
one monomer interacting with E343 on the ‘back’ surface of the
adjacent monomer [9]. Similarly, mutational studies reveal that
D191, which is immediately adjacent to R213, makes interactions
that are key to oligomerisation and that substitution with a bulky
hydrophobic residue (D191V) abrogates this process [9].
* Corresponding author. Auckland Cancer Society Research Centre, Faculty of
Medical and Health Sciences, The University of Auckland, Private Bag 92019,
Auckland 1142, New Zealand.
E-mail address: j.spicer@auckland.ac.nz (J.A. Spicer).
http://dx.doi.org/10.1016/j.ejmech.2017.05.048
0223-5234/© 2017 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

140
J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
Until recently, the precise mechanism of granzyme entry into
physicochemical properties through variation of the sulphonamide
linker, linker position, and substitution on the central pyridine ring
and terminal benzene (Fig. 2).
the target cell was debated, but it is beyond any doubt that the
pore-forming activity of perforin is indispensable. In essence,
secreted perforin forms large (18 nm diameter) transmembrane
pores on the surface of the target cell, through which the granzyme
monomers (4 nm diameter) diffuse into the cytosol [10,11]. Once
internalised the granzymes cleave key substrates to initiate rapid
apoptotic death [5,10e13]. Unlike the granzymes, which are enco-
ded by many genes and are, therefore, subject to considerable
redundancy of function, the gene encoding perforin (PRF1) is pre-
sent as a single copy in all mammals. Gene-targeting studies in
mice [1] and naturally occurring disease-causing mutations in
humans [14,15] confirm that perforin deficiency cannot be
compensated by any other protein. This makes perforin an ideal
target for therapeutic intervention.
While perforin is a key component of the immune response,
inappropriate activity has also been implicated in a number of
human immunopathologies and therapy-induced conditions. These
include cerebral malaria, insulin-dependent diabetes, juvenile
idiopathic arthritis and postviral myocarditis [16e18], as well as
therapy-induced conditions such as allograft rejection and graft
versus host disease [19e21]. Our current goal is to seek small
molecule inhibitors of perforin as potential immunosuppressive
agents for the treatment of autoimmune diseases and other con-
ditions characterised by dysfunction of this pathway. This should be
a highly selective strategy since perforin is expressed exclusively by
CTL and NK cells, in contrast to approaches using conventional
immunosuppression treatments which indiscriminately depress
immune function [22e24].
Based on an initial hit from a mass screen [25], we have previ-
ously designed and optimised inhibitors of perforin that can; (i)
block recombinant purified perforin, (ii) block perforin delivered by
intact NK cells and, (iii) withstand incubation in serum (e.g. 1;
Fig. 1) [26e30].
While these compounds appeared highly promising, replace-
ment of the 2-thioxoimidazolidinone moiety that contained a po-
tential Michael acceptor and showed variable toxicity toward
perforin-producing NK cells proved problematic. This issue was
only overcome when we amalgamated our own finding that an aryl
sulphonamide could act as a bioisosteric replacement [30] with a
strategy implemented by GSK workers, where a thiazolidinedione
was replaced with a pyridyl-linked benzenesulphonamide to give 2
2. Results and discussion
2.1. Chemistry
The majority of the target compounds were constructed from
right to left starting with our previously published key iodide 75
[32] (Scheme 1). Suzuki reaction of 75 with a variety of commer-
cially available aminopyridine boronates under standard conditions
gave the required amine intermediates 76e79 which were subse-
quently reacted with a range of substituted aryl sulphonyl chlo-
rides. The 5-amino-3-pyridine derivative 78 [32] in particular was
employed in the preparation of all compounds in Table 3. One
exception was where the central pyridine ring was replaced with a
benzene; in this case the Suzuki step was carried out with 2-
methyl-5-nitrobenzeneboronic acid, the nitro compound (80) hy-
drogenated to give the amine (81), which was then reacted with
either
2,4-difluorobenzenesulphonyl
chloride
or
2-
pyridinesulphonyl chloride to afford 13 and 15 respectively.
Finally, amido-linked compound 8 was prepared by reaction of 78
with 2,4-difluorobenzoic acid chloride.
In a smaller number of cases, mostly those examples with
substitution on the central pyridine ring, the target compounds
were effectively synthesized from two halves; the fully elaborated
left-hand side benzenesulphonamide subunit (e.g 82e85) and key
iodide 75 as the right-hand side (Scheme 2). The intermediate
bromides 82e85 and 91e93 were prepared from a variety of
commercially available aryl sulphonyl chlorides and substituted 3-
aminopyridines (or anilines) under standard conditions. In the case
of 85, the sulphonamide NH was methylated with NaH and MeI in
DMF to give 86, and for 91e93 where protection of this NH was
required for the subsequent coupling to be successful, the alkyl-
ation was carried out with (chloromethoxy)ethane to give 94e96.
All bromides were converted to the corresponding boronates
87e90 and 97e99 under palladium-catalysed conditions using
bis(pinacolato)diboron and KOAc in DMSO, then finally reacted in a
Suzuki step with iodide 75 to introduce the thiophene-N-methyl-
isoindolinone subunit (6, 10e12, 17, 18, 20). Where required (for 17,
18, 20), deprotection was carried out under acidic conditions.
A limited number of “reverse” sulphonamides were also pre-
pared (Scheme 3). In the case of target compound 7, intermediate
bromide 100 was prepared from 2,4-difluoroaniline and 5-
bromopyridine-3-sulphonyl chloride. Protection of the sulphona-
mide was not required and conversion to the boronate 101 and
[31]. This approach resulted in
a
new series of
benzenesulphonamide-based perforin inhibitors, exemplified by 3,
which were potent, soluble and essentially non-toxic toward NK
cells [32]. In the following report we extend our study to explore
whether it is possible to further modulate activity and
Fig. 1. Perforin inhibitors and PI3K
a
clinical candidate GSK2126458.

J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
141
Fig. 2. Variations on 3 to give new benzenesulphonamide analogues.
Scheme 1. Reagents and conditions: (i) Boronate, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux; (ii) H2, 60 psi, 1:1:1 EtOH/EtOAc/THF, RT, 3 h; (iii) 2,4-Difluorobenzenesulfonyl
chloride or pyridine-2-sulfonyl chloride, pyridine, 0e45 ꢀC; (iv) 2,4-Difluorobenzenoic acid chloride, pyridine, 0e45 ꢀC, 16 h.
subsequent Suzuki coupling with 75 to give 7 proceeded smoothly.
Likewise, bromide 102 was prepared from 2-aminopyridine 3-
bromo-4-methylbenzenesulfonyl chloride, converted to the boro-
nate 107 and therein coupled with 75 to afford the final product 16.
For bromides 103 and 104, protection of the sulphonamide NH
(105, 106) was required for the sequential borylation (108, 109) and
Suzuki reactions to proceed in good yield, and afforded targets 14
and 19 respectively in good yield.
changing the position of the sulphonamide link to the pyridine, the
location of the pyridine nitrogen, and modification/replacement of
the sulphonamide itself.
By moving the sulphonamide link from the 5-position (3) to the
4-position (4), activity is abolished. If the sulphonamide is retained
in the preferred 5-position and the pyridine nitrogen moved to the
4-position (5), activity is lost 4-fold compared to the lead, 3
(IC50s ¼ 4.70 and 1.17
mM respectively). The requirement for a free
NH in the sulphonamide was probed through methylation of 3 to
give N-methyl compound 6. It appears likely that this acidic
hydrogen is required for interaction with the target protein since a
2.2. Inhibition of recombinant perforin-mediated lysis
14-fold drop in activity to IC50 ¼ 16.1
sulphonamide (7) is however still acceptable, with less than a 2-
M). Finally, replacement of
the sulphonamide with a carboxamide (8) results in complete loss
of potency, further supporting our hypothesis that the sulphona-
mide NH is required in the linker for optimal activity.
mM was observed. A “reverse”
In our initial report describing the discovery of
benzenesulphonamide-based inhibitors of perforin [32], analogue
design focussed on exploration of the thiophene and N-methyl-
isoindolinone subunits which comprise the right-hand side of the
molecule. For the current study we sought to further optimise
potency and physicochemical characteristics through manipulation
of the central pyridine and sulphonamide linker, as well as
employing a wide range of substituents on the left-hand benzene
(or aryl) ring.
fold reduction in activity (IC50 ¼ 2.24
m
The effect of variation about the central pyridine ring was
investigated next (Table 2). Direct replacement of the pyridine (3)
with a benzene (9) resulted in a loss of activity from IC50 ¼ 1.17
mM
to 5.74 M. Introduction of a 2-fluoro- (10) or 2-chloro-substituent
m
Table 1 shows a group of compounds that explore the effect of

142
J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
Scheme 2. Reagents and conditions: (i) a. NaH, DMF, 0ꢀ C, 0.5 h, b. MeI or (chloromethoxy)-ethane, DMF, 0ꢀ C-RT, 1.5 h; (ii) Bis(pinacolato)diboron, KOAc, Pd(dppf)Cl2, DMSO, 90 ꢀC,
3 h.; (iii) a. 75, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux, b. Deprotection if required: 3 M HCl/1,4-dioxane (1:1), 1 h.
Scheme 3. Reagents and conditions: (i) a. NaH, DMF, 0 ꢀC, 0.5 h, b. (Chloromethoxy)ethane, DMF, 0 ꢀC-RT, 1.5 h; (ii) Bis(pinacolato)diboron, KOAc, Pd(dppf)Cl2, DMSO, 90 ꢀC, 3 h.;
(iii) a. 75, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux, b. Deprotection if required: 3 M HCl/1,4-dioxane (1:1), 1 h.
(11) to the pyridine gave similar activity to 3 (IC50s ¼ 1.03 and
1.99 M respectively), while the electron-donating 2-methoxy-
substituent (12) gave an analogue which was somewhat poorer
(IC50 ¼ 3.56 M). Benzene-linked compounds (13e19) were then
without further detriment to the overall physicochemical proper-
ties. Although far from a comprehensive list, the introduction of
m
methyl (15; IC50 ¼ 15.4
m
M), fluoro (17; >20
mM), trifluoromethoxy
m
(18; >20 M) and methoxy (19; 9.68 m
m
M) groups proved unsuc-
explored to determine if substitution on the benzene ring could
improve activity. Introduction of a single methyl group on 9 to give
13 resulted in complete loss of potency, however at this point we
reasoned that the increased lipophilicity and reduced solubility
associated with the presence of two benzene rings (9 and 13) could
be improved if we effectively moved the central pyridine of 3 to the
other side of the sulphonamide link (2-pyridylsulphonamide
compounds 14e20). This enabled us to explore a small number of
compounds containing substitution on the central benzene ring,
cessful. The effect of reversing the sulphonamide orientation is
consistent with Table 1, appearing to be slightly detrimental,
although we only had a single matched pair for this comparison (15
vs 16; IC50s ¼ 15.4 vs > 20
mM). Lastly, compound 20 contains
pyridine rings on either side of the sulphonamide link. While the
solubility was significantly improved, the resulting activity
(IC50 ¼ 4.10
mM) was still no improvement over the lead 3.
Tables 1 and 2 are limited to either 2,4-difluorobenzene- or 2-
pyridyl-sulphonamides. Thus it remained for us to investigate the

J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
143
Table 1
Variation of the sulphonamide linker and position.
Compound
X (linker)
Linker Position
Y
Z
Inhibition of Jurkat Cell Lysis IC50 (mM)
3a
4
5
SO2NH
SO2NH
SO2NH
SO2NCH3
NHSO2
CONH
5
4
5
5
5
5
CH
C
N
CH
CH
CH
N
N
CH
N
N
1.17
>20
4.70
16.1
2.24
>20
6
7b
8
N
a
Included as a comparator; see Ref. [32].
Reverse sulphonamide.
b
Table 2
Variation of the pyridine ring.
Compound
R1
R2
Z
R3
Inhibition of Jurkat Cell Lysis IC50 (mM)
3a
9
2,4-diF-benzene
2,4-diF-benzene
2,4-diF-benzene
2,4-diF-benzene
2,4-diF-benzene
2,4-diF-benzene
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
H
H
F
Cl
OCH3
H
H
H
H
N
CH
N
N
N
CH
CH
CH
CH
CH
CH
CH
N
H
H
H
H
H
CH3
H
CH3
CH3
F
1.17
5.74
1.99
1.03
3.56
>20
8.92
15.4
>20
>20
>20
9.68
4.10
10
11
12
13
14b
15
16b
17
18
19b
20
H
OCF3
OCH3
F
H
H
H
2-pyridyl
2-pyridyl
a
Included as a comparator; see Ref. [32].
Reverse sulphonamide.
b
effect of alternative substituents on the left-hand side of the
molecule to determine if we could optimise potency and physico-
chemical properties further. A total of 52 new substituted benzene
or aryl sulphonamides are shown in Table 3.
perhaps because all these halogens are less electron-withdrawing
than fluorine, a concept that we explore in more detail below. A
different trend was observed when the electron-donating sub-
stituents methoxy (37e40) and trifluoromethoxy (41e43) were
employed; here the meta- position was favoured over ortho- and
Clearly substitution on the benzene ring is required, with
unsubstituted analogue 21 (IC50 ¼ 8.46
m
M) suffering a 7-fold loss
para-. The two meta-substituted examples 38 (2.56
mM) and 42
in activity. While 2,4-difluoro-compound (3) was the original lead
substitution pattern, it can be separated into the constituent mono-
fluoro- analogues 22e24. This reveals that the contribution of the
(1.42 M) showed significantly better activity in comparison to
m
other positional isomers. We then surveyed a variety of electron-
withdrawing substituents by preparing compounds 44e59. As a
group, these broadly paralleled the fluorine-substituted com-
pounds discussed above, where the ortho- or para- positional iso-
mers showed superior potency over the corresponding meta-
examples. More specifically, for the trifluoromethyl- (44e47),
ester- (51e54) and sulphonyl- (56, 57) substituted compounds, the
2- position is most important (IC50 ¼ 2.03
m
M), followed by 4-
M), and lastly the 3-position which is detrimental to activity
M). Accordingly, the 3,4-difluoro-substituted compound 25
M) while that of the 2,4,6-trifluoro-
M). This positional effect however, is
not apparent in the corresponding chloro- compounds (27e29;
IC50s 2.11e8.77 M) where the SAR between 2-, 3- and 4- is rela-
tively flat. This may help explain why the most potent compound of
the set is the 3,4-dichloro- 30 (1.32 M) and not the 2,4-dichloro-
target 31 (15.0 M) as might be expected from the fluorinated series
of analogues. The bromo- (32e35; IC50s 2.11e8.77 M) and iodo-
substituted (36; 5.00 M) compounds also displayed flat SAR,
(9.65
m
(>20
m
shows poor potency (18.6
m
analogue 26 is excellent (1.76
m
ortho isomer was best (IC50s ¼ 1.45, 2.90, 6.80
mM respectively),
while for the cyano- (48e50), carboxylic acid (55) and nitro- (58,
59) examples the para isomer showed high potency (5.17, 0.75 and
m
m
2.74 mM respectively). Compound 55 was particularly noteworthy,
m
being one of the few sub-micromolar inhibitors of perforin iden-
tified to date. This subset of results is consistent with an inductive
effect being exerted by electron-withdrawing substituents on the
m
m

Table 3
Modulation of activity through substitution on the sulphonamide.
benzene ring and through to the sulphonamide, enhancing in-
teractions with the protein and resulting in improved activity.
Hybrid compounds 60e65 were also prepared to investigate
whether the effects of individual substituents could be combined.
The resulting activities were neither synergistic nor additive,
bringing no further gain to the overall potency. A set of four com-
pounds (66e69) with a heterocycle (pyridine or thiophene) linked
to the sulphonamide were also prepared. The preference for the
heteroatom to be located directly next to the sulphonamide bond
was clear with the 2-pyridyl and 2-thiophenyl compounds 66 and
Compound R1
Inhibition of Jurkat Cell Lysis IC50 (mM)
21
22
23
24
3a
benzene
2-F-benzene
3-F-benzene
4-F-benzene
2,4-diF-benzene
3,4-diF-benzene
2,4,6-triF-benzene
2-Cl-benzene
3-Cl-benzene
4-Cl-benzene
3,4-diCl-benzene
2,4-diCl-benzene
2-Br-benzene
3-Br-benzene
4-Br-benzene
8.46
2.03
~20
68 (both IC50s ¼ 1.07
m
M) far superior to the corresponding 3-
9.65
1.17
18.6
1.76
4.01
2.42
5.39
1.32
15.0
2.66
2.11
8.77
2.87
5.00
14.8
2.56
6.27
13.8
17.7
1.42
>20
1.45
1.22
>20
>20
9.17
>20
5.17
2.90
7.77
8.16
linked isomers 67 and 69 (15.13 and 12.51
mM respectively).
Finally, a set of compounds containing a variety of substituted
heterocycles were prepared (70e74), but with the exception of the
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
4-oxazole 70 (IC50 ¼ 3.05
mM), none showed much promise.
2.3. Advanced assessment of selected compounds
Having shown that a range of benzenesulphonamides block lysis
by recombinant perforin, a subset of promising examples was
identified to test for inhibitory effect on the lytic action of whole NK
cells. Compounds were selected based on potency, and included
2,4-diBr-benzene
4-I-benzene
2-OCH3-benzene
3-OCH3-benzene
4-OCH3-benzene
3,4-diOCH3-benzene
2-OCF3-benzene
3-OCF3-benzene
4-OCF3-benzene
2-CF3-benzene
3-CF3-benzene
4-CF3-benzene
3,5-diCF3-benzene
2-CN-benzene
3-CN-benzene
4-CN-benzene
2-COOCH3-benzene
3-COOCH3-benzene
4-COOCH3-benzene
several for which the Jurkat IC50s were >20 mM, to further validate
our use of this higher throughput screen as our primary assay. The
inhibitors were co-incubated with KHYG1 human NK cells in me-
dium for 30 min at room temperature, 51Cr-labelled target cells
were added, and the resulting level of chromium release used to
determine residual lytic activity and thus degree of inhibition. The
use of whole NK cells to deliver perforin provides a more realistic
model of conditions in vivo compared to isolated recombinant
protein which acts indiscriminately. Recognition of aþ target cell,
formation of a synaptic cleft, and release of the granular contents
into the cavity between effector and target are all required ele-
ments for lysis to occur. Confirmation that the observed level of
inhibition is due to blocking the activity of perforin rather than
non-specific killing of the effector cell was also sought by
measuring the viability of the NK cells 24 h later. Our lead com-
pound for the current work and most potent compound from our
previous study [32], 2,4-difluorobenzene 3, is included as a refer-
ence point (Table 4). One notable omission from this table is the
potent 4-carboxylic acid-substituted compound 55 as this was toxic
4-COOCH2CH3-benzene 19.4
4-COOH-benzene
2-SO2CH3
0.75
6.80
>20
6.65
2.74
13.0
5.53
3.75
8.14
>20
>20
1.07
15.1
1.07
12.5
3.05
4-SO2CH3
2-NO2-benzene
4-NO2-benzene
2-F,3-Cl-benzene
2-CH3-4-F-benzene
3-CF3-4-F-benzene
3-Cl, 4-CH3-benzene
2-Cl, 4-CF3-benzene
3-CF3-5-Br-benzene
2-pyridyl
Table 4
Capacity of selected compounds to inhibit perforin delivered by KHYG1 NK cells.
Compound
Jurkat IC50
(
m
M)a
KHYG1 Inhibition
(% at 10
M)b
KHYG1 Viability
(% at 10
m
m
M)c
3-pyridyl
2-thiophenyl
3-thiophenyl
3
1.17
1.99
1.03
>20
>20
>20
1.76
1.45
1.22
>20
>20
5.17
>20
6.65
2.74
67.5 17.5
92.5 1.8
95.0 2.5
50.3 8.5
0
100
10
11
13
16
17
26
44
45
47
49
50
57
58
59
92.0 3.0
94.0 5.3
91.7 10.0
96.1 6.0
91.4 6.5
95.1 2.1
100
0
71
72
7.10
19.1
81.3 6.3
85.0 5.6
88.8 1.9
0
83.7 3.2
75.0 7.8
0
100
90.2 3.4
94.4 7.1
99.3 0.3
90.0 7.8
98.7 1.5
85.0 12.0
73
74
>20
95.6 1.9
77.5 5.6
a
Data given for compounds as determined by the Jurkat assay.
11.6
b
Inhibition by compound (10 m
M) of the perforin-induced lysis of 51Cr-labelled
K562 leukemia target cells when co-incubated with KHYG1 human NK cells. Percent
inhibition calculated compared to untreated control (n ¼ 4).
c
Viability of KHYG1 NK cells after 24 h by Trypan blue exclusion assay (n ¼ 3). See
a
Included as a comparator; see Ref. [32].
Experimental section for further details.

J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
145
to the NK cells and therefore the degree of inhibition was unable to
be determined.
Plasma pharmacokinetics were determined in male CD-1 mice for
compounds 3, 10, 11, 26, 50, 58 and 59 (Table 6). Blood samples
were collected at 5e8 time-points after dosing the compounds at
10 mg/kg in a solution of 20% hydroxypropyl-b-cyclodextrin by
intraperitoneal (IP) injection. For analysis of the samples, chro-
matographic conditions were optimised by HPLC for each com-
All of the compounds with Jurkat IC50s < 10
lent suppression of NK-cell mediated killing of labelled target cells
(68e96% at 10 M), however this activity did not correlate exactly
mM showed excel-
m
with their potency against isolated recombinant protein. This
finding may reflect the varying ability of the respective inhibitors to
access perforin located in a synaptic cleft within a complex bio-
logical milieu. Two examples with halogen on the central pyridine
ring (10, 11) and the 2-nitrobenzene compound 58 were particu-
larly effective in blocking NK cell action (93, 95 and 96% inhibition
respectively). Results from the set of compounds with
pound of interest and an internal standard.
A
liquid
chromatography with tandem mass spectrometry (LC-MS/MS)
method was then developed and validated for quantitation of each
analyte.
Compound 3 possessed the best half-life (12 h) but showed
much lower Cmax and AUC than other examples. While 10,11 and 26
had shorter half-lives (4.5e9.5 h) they also showed the highest Cmax
IC50s > 20 mM broadly validated our use of this assay as a primary
screen, with four of six examples showing no potency in either
assay (16, 17, 47, 57). Compound 13 demonstrated 50% inhibition at
(236, 149 and 124
m
mol/L) and exposures (AUC ¼ 2885, 2364 and
1019 mol/L*h respectively). 4-Cyanobenzene (50) and both
m
10
m
M, perhaps not surprisingly given that the only structural
nitrobenzene isomers (58, 59), had even shorter half-lives, however
the maximum concentration and overall exposure reached was still
superior to the compound with the overall longest half-life (3).
change from 3 was the replacement of a bridging pyridine with a
benzene ring. The one unexpected outlier was the 3-cyano com-
pound 49 which, although it had poor activity against isolated re-
combinant perforin, had excellent activity against perforin
produced by whole NK cells. The NK cells also retained excellent
viability across all examples, consistent with our previous findings
for this series [32] and in contrast to earlier reported classes
[26e28].
Preliminary physicochemical data was collected on the same
(active) subset of compounds in order to assess their potential for
progression to in vivo pharmacokinetic (PK) studies (Table 5).
Following conversion to the corresponding sodium salts the solu-
bility varied widely, with the 2,4,6-trifluorobenzene (26) and 4-
cyanobenzene (50) analogues being highly soluble, while the
presence of 2-fluoropyridine (10), 2-nitrobenzene or the more
lipophilic trifluoromethylbenzene group (44, 45) had a negative
impact on solubility. All examples tested showed good stability in
aqueous solution over 24 h, however results were more varied in
the presence of human, rat and mouse microsomes. While 10, 11,
and 58 showed acceptable stability (>70% parent after 30 min)
across all three species, the remaining compounds (3, 26, 45, 50, 59
and especially 44) showed moderate to poor stability with human
microsomes. This data in combination with poor solubility resulted
in the elimination of 44 and 45 from consideration for the in vivo PK
studies reported in section 2.4 below.
3. Conclusions
Between our previous report [32] and the current study, we
have explored in detail how changes made on
a
benzenesulphonamide-based template affects perforin inhibitory
activity. Analysis of the resulting SAR shows that although a range
of variations were explored throughout the molecule, the iso-
indolinone, thiophene, pyridine and sulphonamide link of 3 are
probably close to optimal, while there is some tolerance for sub-
stitution on the central pyridine ring and the terminal benzene
Table 6
In vivo Pharmacokinetics of selected compounds.a
Compound
T1/2
(h)
Cmax
mol/L)
AUC0-∞
mol/L*h)
(
m
(m
3
12.0
6.6
9.5
4.5
3.3
2.5
2.2
10
220
10
11
26
50
58
59
236
149
124
87
105
64
2885
2364
1019
642
415
383
a
Pharmacokinetic parameters derived from the plasma-concentration time
profiles for each compound following a 10 mg/kg i.p. dose. The results were pro-
cessed using a noncompartment model approach using Phoenix WinNonlin 6.2
(Pharsight Corporation, St. Louis, MO). The derived parameters are: maximum
plasma concentration (Cmax), the area under the curve (AUC) and plasma half-life
(T1/2). See Supplementary Material for further details of assay conditions.
2.4. In vivo pharmacokinetics
The in vivo PK parameters were measured for seven compounds
selected on the basis of the in vitro assessment described above.
Table 5
Physicochemical properties of selected compounds.
Compound
Solubilitya
g/mL)
cLogPb
Stability in Solutionc
(%)
Microsome Stability (%)d
(
m
Mouse
Rat
Human
3e
1080
428
4027
10674
88
574
12900
671
2.70 0.72
2.34 0.79
3.21 0.73
2.65 0.77
2.97 0.65
3.13 0.65
2.17 0.64
1.83 0.61
2.36 0.61
82
100
100
88
92
e
92
96
97
100
84
97
87
100
85
99
100
99
83
49
80
64
89
72
58
94
95
63
14
57
52
73
39
10
11
26
44
45
50
58
59
85
100
e
1680
a
Solubility of the sodium salt in water at room temperature; conversion to salt as described in experimental section 4.1.
cLogP calculated using ACD/PhysChem software v12.5.
b
c
Percentage of parent compound (as sodium salt) remaining after 24 h at 20 ꢀC in water.
d
e
Percentage of parent compound (as sodium salt) remaining after exposure to mouse, rat or human microsomes for 30 min.
Data for compound 3 from Ref. [32]. See Supplementary Material for further details of assay conditions.

146
J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
ring. A smaller panel of compounds was selected based on the
following criteria; in vitro potency against isolated recombinant
perforin and whole human NK cells, lack of toxicity against NK cells,
solubility and stability (aqueous and microsomal). For this group
the in vivo PK parameters were assessed to select potential candi-
dates for evaluation in a mouse model of transplant rejection. We
sought acceptable half-life (potentially impacts dosing frequency),
Cmax and AUC to ensure sufficient exposure to maximise our
chances of achieving efficacy. Other key parameters taken into
consideration for selection of the final in vivo candidates were
in vitro potency and solubility. While the in vivo mouse efficacy
studies are carried out using IP dosing, ultimately this product
would be administered to human patients intravenously, meaning
that sufficient solubility in a formulation close to physiological pH
is crucial.
Given the above criteria, one example from the panel that ap-
pears to possess a suitable balance of properties is the 2-chloro
pyridine 11. This compound shows excellent potency (without
toxicity) in vitro, good solubility, stability across all three species of
microsomes and acceptable PK properties. Compounds 58 and 3 are
also worth consideration; 58 because it is the most potent com-
pound of the set, and 3 based on a combination of potency, solu-
bility and half-life. The next step will be to conduct studies to
determine if these compounds are capable of preventing transplant
rejection in a mouse model.
4.1.2. General procedure B: 2,4-Difluoro-N-(5-(5-(2-methyl-1-
oxoisoindolin-5-yl)thiophen-2-yl)pyridin-2-yl)benzenesulfonamide
(4)
To a solution of 76 (120 mg, 0.37 mmol) in dry pyridine (12 mL)
under N2 at RT, was added 2,4-difluorobenzenesulphonyl chloride
(159 mg, 0.74 mmol) in CH2Cl2 (1.5 mL) dropwise over 5 min. The
mixture was stirred at 45 ꢀC under N2 for 16 , and the solvent then
removed under reduced pressure. The reaction was quenched with
a little water and the resulting solid collected by filtration and
washed with water and Et2O. Purification was carried out by trit-
uration with hot CH2Cl2/MeOH solution to give 4 as a pale brown
solid (65%); mp (CH2Cl2/MeOH) 269e272 ꢀC. 1H NMR [400 MHz,
(CD3)2SO] d 12.00 (bs, 1 H), 8.39 (bs, 1 H), 7.99e8.10 (m, 2 H), 7.89 (s,
1 H), 7.78 (dd, J ¼ 8.0, 1.3 Hz, 1 H), 7.69 (d, J ¼ 4.2 Hz, 1 H), 7.68 (d,
J ¼ 8.1 Hz,1 H), 7.57 (d, J ¼ 3.9 Hz,1 H), 7.44e7.53 (m,1 H), 7.26e7.33
(m, 1 H), 7.16e7.26 (m, 1 H) 4.50 (s, 2 H), 3.07 (s, 3 H). HRMS (ESIþ)
calcd for C24H18N3O3S2F2 498.0752 (MHþ), found 498.0746. Anal. C,
H, N. In some cases a bis-sulphonamide was also formed; here a
second step was introduced where the crude product was treated
with a 1:1 mixture of 1,4-dioxane and 2 M NaOH. The mono-
sulphonamide resulting from subsequent acidification of the re-
action mixture was isolated by filtration, washed well with water,
and dried. Purification was carried out by flash column chroma-
tography (MeOH/CH2Cl2 gradient).
4.1.3. 5-(5-(2-Aminopyridin-4-yl)thiophen-2-yl)-2-
methylisoindolin-1-one (77)
4. Experimental
Reaction of 75 with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)pyridin-2-amine according to general procedure A gave 77 as a
green-yellow solid (72%); mp (CH2Cl2/MeOH) 302e306 ꢀC. 1H NMR
4.1. Chemistry
[400 MHz, (CD3)2SO]
d
7.90e7.97 (m, 2 H), 7.82 (d, J ¼ 7.1 Hz, 1 H),
Elemental analyses were performed by the Microchemical
Laboratory, University of Otago, Dunedin, NZ; values are indicated
by the symbols of the elements and were within 0.4% of the
theoretical values. Melting points were determined using an Elec-
trothermal Model 9200 and are as read. Several compounds were
examined as sharp-melting solvates, on which elemental analyses
were determined. NMR spectra were measured on a Bruker
Advance 400 MHz spectrometer and referenced to Me4Si. Mass
spectra were recorded either on a Varian VG 7070 spectrometer at
nominal 5000 resolution or a Finnigan MAT 900Q spectrometer. All
final compound purities were determined to be >95% by HPLC on
7.67e7.73 (m, 2 H), 7.65 (d, J ¼ 3.4 Hz, 1 H), 6.83 (d, J ¼ 4.0 Hz, 1 H),
6.69 (s, 1 H), 6.05 (br s, 2 H) 4.52 (s, 2 H), 3.09 (s, 3 H). HRMS (ESIþ)
calcd for C18H16N3OS 322.1009 (MHþ), found 322.1002.
4.1.4. 2,4-Difluoro-N-(4-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-2-yl)benzenesulfonamide (5)
Amine 77 was reacted with 2,4-difluorobenzenesulfonyl chlo-
ride according to general procedure B to give 5 as a pale brown solid
(26%); mp (CH2Cl2/MeOH) 235e239 ꢀC. 1H NMR [400 MHz,
(CD3)2SO] d 13.27 (br s, 1 H), 7.99e8.09 (m, 2 H), 7.85e7.98 (m, 3 H),
an Alltech Alltima C18 column (3.2 ꢁ 150 mm, 5
m
m) eluting with
7.80 (d, J ¼ 3.8 Hz, 1 H), 7.72 (d, J ¼ 7.9 Hz, 1 H), 7.38e7.51 (m, 2 H),
5e80% MeCN/45 mM NH4HCO3.
7.19e7.32 (m, 2 H) 4.53 (s, 2 H), 3.09 (s, 3 H). HRMS (ESIþ) calcd for
C
24H18N3O3S2F2 498.0752 (MHþ), found 498.0754. Anal. C, H, N.
4.1.1. General procedure A: 5-(5-(6-Aminopyridin-3-yl)thiophen-2-
yl)-2-methylisoindolin-1-one (76)
4.1.5. N-(5-Bromopyridin-3-yl)-2,4-difluoro-N-
methylbenzenesulphonamide (86)
Iodide 75 [32] (500 mg, 1.41 mmol) and 5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)pyridin-2-amine (465 mg, 2.11 mmol)
were dissolved in a mixture of toluene (8 mL) and EtOH (4 mL). A
solution of 2 M Na2CO3 (2 mL) and Pd(dppf)Cl2.CH2Cl2 (57 mg,
0.07 mmol) were added and the entire mixture heated at reflux
under N2 for 2 h. Upon cooling, the desired product precipitated
from the reaction mixture, was isolated by filtration, and washed
with H2O, CH2Cl2 and MeOH. No further purification was required,
giving 76 as a green-yellow solid (326 mg, 72%); mp (MeOH/
2,4-Difluorobenzenesulfonyl chloride and 5-bromopyridin-3-
amine were reacted according to general procedure B. Without
further purification, the resulting crude sulphonamide 85 (540 mg,
15.5 mmol) was dissolved in dry DMF (20 mL) and cooled to 0 ꢀC.
NaH (41 mg, 17.0 mmol) was added and the mixture stirred for 0.5 ,
gradually being allowed to return to R.T. Methyl iodide (241 mg,
17.0 mmol) was then added dropwise and stirring continued for
1.5 h. After quenching with water the mixture was extracted with
CH2Cl2, dried with MgSO4 and evaporated to give a solid which was
purified by flash column chromatography (1e3% MeOH/CH2Cl2 as
eluant), yielding 86 as a brown solid (320 mg, 57%). 1H NMR
CH2Cl2) 252e254 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 8.28 (dd,
J ¼ 2.6, 0.6 Hz, 1 H), 7.86 (s, 1 H), 7.76 (dd, J ¼ 8.0, 1.5 Hz, 1 H), 7.70
(dd, J ¼ 8.6, 2.6 Hz, 1 H), 7.67 (d, J ¼ 7.9 Hz, 1 H), 7.62 (d, J ¼ 3.8 Hz,
1 H), 6.35 (d, J ¼ 3.8 Hz, 1 H), 6.51 (dd, J ¼ 8.6, 0.6 Hz, 1 H), 6.25 (br s,
2 H) 4.49 (s, 2 H), 3.08 (s, 3 H). HRMS (ESIþ) calcd for C18H16N3OS
322.1009 (MHþ), found 322.1007.
[400 MHz, (CD3)2SO]
d
8.65 (d, J ¼ 2.0 Hz, 1 H), 8.51 (d, J ¼ 2.2 Hz,
1 H), 8.04 (t, J ¼ 2.2 Hz, 1 H), 7.72e7.83 (m, 1 H), 7.57e7.68 (m, 1 H),
7.32 (dt, J ¼ 8.2, 2.4 Hz, 1 H), 3.26 (s, 3 H). LRMS (APCIþ) calcd for
C
12H9BrF2N2O2S 364 (MHþ), found 364.

J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
147
4.1.6. General procedure C: 2,4-Difluoro-N-methyl-N-(5-(5-(2-
methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-
4.1.10. 5-(5-(3-Aminophenyl)thiophen-2-yl)-2-methylisoindolin-1-
one (79)
benzenesulphonamide (6)
Iodide 75 was reacted with (3-aminophenyl)boronic acid ac-
cording to general procedure A to give 79 as a green solid (69%); mp
Bromide 86 (300 mg, 0.83 mmol), bis(pinacolato)diboron
(232 mg, 0.91 mmol), KOAc (243 mg, 2.48 mmol) and Pd(dppf)
Cl2.CH2Cl2 (34 mg, 0.04 mmol) were weighed into a flask, DMSO
(5 mL) added, and the entire mixture heated and stirred under N2
for 4 h. Upon cooling, the reaction was diluted with CH2Cl2 (25 mL)
and filtered through a pad of Celite®, washing well with additional
CH2Cl2. The filtrate and combined washings (ca 80 mL) were
washed with water (3 ꢁ 40 mL), brine (50 mL), dried (Na2SO4) and
filtered. Removal of the solvent under reduced pressure gave the
crude boronate 90 which was coupled directly to 75 according to
general procedure A, giving 6 as a cream solid (220 mg, 52%); mp
(CH2Cl2/MeOH) 244e247 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 7.89 (s,
1 H), 7.77 (dd, J ¼ 7.9, 1.3 Hz, 1 H), 7.68 (d, J ¼ 8.0 Hz, 1 H), 7.63 (d,
J ¼ 3.8 Hz, 1 H), 7.41 (d, J ¼ 3.8 Hz, 1 H), 7.08 (t, J ¼ 7.72 Hz, 1 H),
6.82e6.91 (m, 2 H), 6.55 (dd, J ¼ 7.9,1.3 Hz,1 H), 5.25 (br s, 2 H), 4.50
(s, 2 H), 3.08 (s, 3 H).
4.1.11. 2,4-Difluoro-N-(3-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)phenyl)benzene sulphonamide (9)
Amine 79 was reacted with 2,4-difluorobenzenesulphonyl
chloride according to general procedure B to give 9 as a yellow
solid (62%); mp (CH2Cl2/MeOH) 260e262 ꢀC. 1H NMR [400 MHz,
(CH2Cl2/MeOH) 200e202 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 8.87
(d, J ¼ 2.0 Hz, 1 H), 8.43 (d, J ¼ 2.3 Hz, 1 H), 7.96 (t, J ¼ 2.2 Hz, 1 H),
7.94 (s, 1 H), 7.83 (dd, J ¼ 7.8, 1.4 Hz, 1 H), 7.73e7.81 (m, 3 H), 7.71 (d,
J ¼ 7.9 Hz, 1 H), 7.63 (m, 1 H), 7.32 (dt, J ¼ 8.1, 2.0 Hz, 1 H), 4.52 (s,
2 H), 3.34 (s, 3 H), 3.09 (s, 3 H). Anal. C, H, N.
(CD3)2SO] d 10.84 (br s, 1 H), 7.93e8.02 (m, 1 H), 7.91 (s, 1 H), 7.81
(dd, J ¼ 8.0, 1.3 Hz, 1 H), 7.70 (d, J ¼ 8.0 Hz, 1 H), 7.67 (d, J ¼ 3.8 Hz,
1 H), 7.55 (dt, J ¼ 8.5, 2.5 Hz, 1 H), 7.47 (d, J ¼ 3.8 Hz, 1 H), 7.38e7.45
(m, 2 H), 7.25e7.36 (m, 2 H), 7.05 (d, J ¼ 7.4 Hz, 1 H), 4.51 (s, 2 H),
3.09 (s, 3 H). Anal. C, H, N.
4.1.7. 5-Bromo-N-(2,4-difluorophenyl)pyridine-3-sulphonamide
(100)
2,4-Difluoroaniline and 5-bromopyridine-3-sulphonyl chloride
were reacted according to general procedure B. The crude product
was recrystallised from 5% MeOH/CH2Cl2 and hexanes, and tritu-
rated with EtOAc to give 100 as an ivory solid (370 mg, 56%). 1H
4.1.12. N-(5-Bromo-2-fluoropyridin-3-yl)-2,4-
difluorobenzenesulphonamide (82)
5-Bromo-2-fluoropyridin-3-amine was reacted with 2,4-
difluorobenzenesulphonyl chloride according to general proced-
ure B to give 82 as a brown solid (17%). 1H NMR [400 MHz,
(CD3)2SO]
d
11.13 (br s, 1 H), 8.18 (s, 1 H), 8.01 (dd, J ¼ 8.6, 2.3 Hz,
NMR [400 MHz, (CD3)2SO]
d
10.52 (br s,1 H), 9.20 (d, J ¼ 2.2 Hz,1 H),
1 H), 7.81e7.92 (m, 1 H), 7.53e7.64 (m, 1 H), 7.22e7.32 (m, 1 H).
LRMS (APCIþ) calcd for C11H6BrF3N2O2S 368 (MHþ), found 368.
8.77 (d, J ¼ 2.0 Hz, 1 H), 8.26 (t, J ¼ 2.1 Hz, 1 H), 7.22e7.35 (m, 2 H),
7.02e7.15 (m, 1 H). LRMS (APCIþ) calcd for C11H7BrF2N2O2S 350
(MHþ), found 350.
4.1.13. 2,4-Difluoro-N-(2-fluoro-5-(5-(2-methyl-1-oxoisoindolin-5-
yl)thiophen-2-yl)pyridin-3-yl)benzenesulphonamide (10)
Bromide 82 was reacted with bis(pinacolato)diboron according
to general procedure C and the crude boronate 87 subsequently
coupled to 75 according to general procedure A to give 10 as a pale
green solid (32%); mp (MeOH/CH2Cl2) 237e239 ꢀC. 1H NMR
4.1.8. N-(2,4-Difluorophenyl)-5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridine-3-sulphonamide (7)
Bromide 100 was reacted with bis(pinacolato)diboron according
to general procedure C and the crude boronate 101 subsequently
coupled to 75 according to general procedure A, to give 7 as a
yellow solid (23%); mp (CH2Cl2/MeOH) 221e224 ꢀC. 1H NMR
[400 MHz, (CD3)2SO]
d
11.04 (br s, 1 H), 8.42 (s, 1 H), 8.06 (dd, J ¼ 9.1,
2.3 Hz, 1 H), 7.94 (s, 1 H), 7.84e7.92 (m, 1 H), 7.82 (dd, J ¼ 7.9, 1.5 Hz,
1 H), 7.74 (d, J ¼ 3.9 Hz, 1 H), 7.72 (d, J ¼ 8.0 Hz, 1 H), 7.68 (d,
J ¼ 3.9 Hz, 1 H), 7.57e7.65 (m, 1 H), 7.28 (dt, J ¼ 8.9, 2.4 Hz, 1 H), 4.52
(s, 2 H), 3.09 (s, 3 H). In this case the product was dissolved in 1,4-
dioxane and the sodium salt precipitated by slow addition of 2 M
NaOH to give a light-green solid (89%). 1H NMR [400 MHz,
[400 MHz, (CD3)2SO]
d
10.50 (br s, 1 H), 9.23 (d, J ¼ 2.2 Hz, 1 H), 8.71
(d, J ¼ 2.1 Hz, 1 H), 8.21 (t, J ¼ 2.2 Hz, 1 H), 7.96 (s, 1 H), 7.81e7.88 (m,
2 H), 7.78 (d, J ¼ 3.9 Hz, 1 H), 7.72 (d, J ¼ 8.0 Hz, 1 H), 7.23e7.35 (m,
2 H), 7.09 (ddt, J ¼ 9.2, 1.4 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C,
H, N.
(CD3)2SO]
d
7.91 (s, 1 H), 7.83e7.90 (m, 1 H), 7.79 (dd, J ¼ 7.9, 1.5 Hz,
1 H), 7.71 (d, J ¼ 2.3 Hz, 1 H), 7.67 (d, J ¼ 7.3 Hz, 1 H), 7.60e7.65 (m,
2 H), 7.33 (d, J ¼ 3.8 Hz, 1 H), 7.22 (dt, J ¼ 9.7, 2.5 Hz, 1 H), 7.11 (dt,
J ¼ 8.3, 2.5 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.
4.1.9. 2,4-Difluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzamide (8)
To 2,4-difluorobenzoic acid (99 mg, 0.62 mmol) in dry CH2Cl2
(2 mL) was added oxalyl chloride (193 mg,1.52 mmol) and 1 drop of
dry DMF. The whole mixture was refluxed for 2 , cooled to RT and
concentrated in vacuo to give 2,4-difluorobenzoic acid chloride. To
amine 78 [32] (100 mg, 0.31 mmol) in dry pyridine (10 mL) at 0 ꢀC
under N2 was added the acid chloride (110 mg, 0.62 mmol) in dry
CH2Cl2 (2 mL) dropwise over 4 min. The mixture was then left to
stir at 45 ꢀC for 16 , quenched with H2O and concentrated in vacuo.
The residue was taken up in citric acid, sonicated for 5 min, and the
precipitate formed was filtered and washed thoroughly with H2O,
MeOH, diethyl ether and dried on to silica gel. The crude material
was chromatographed (1e3% MeOH/CH2Cl2) to give 8 as a yellow
solid (52 mg, 36%); mp 267e269 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
4.1.14. N-(5-Bromo-2-chloropyridin-3-yl)-2,4-
difluorobenzenesulphonamide (83)
5-Bromo-2-chloropyridin-3-amine was reacted with 2,4-
difluorobenzenesulphonyl chloride according to general proced-
ure B, giving 83 as an off-white solid (32%). 1H NMR [400 MHz,
(CD3)2SO]
d
11.03 (br s, 1 H), 8.47 (d, J ¼ 2.3 Hz, 1 H), 8.04 (d,
J ¼ 2.3 Hz,1 H), 7.75e7.85 (m,1 H), 7.53e7.63 (m,1 H), 7.20e7.29 (m,
1 H). LRMS (APCIþ) calcd for C11H6BrF2ClN2O2S 385 (MHþ), found
385.
4.1.15. 2,4-Difluoro-N-(2-chloro-5-(5-(2-methyl-1-oxoisoindolin-
5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulphonamide (11)
d
10.76 (br s, 1 H), 8.77 (t, J ¼ 2.9 Hz, 2 H), 8.52 (t, J ¼ 2.0 Hz, 1 H),
Bromide 83 was reacted with bis(pinacolato)diboron according
to general procedure C and the crude boronate 88 subsequently
coupled to 75 according to general procedure A to give 11 as a
yellow solid (12%); mp (CH2Cl2/MeOH) 238e240 ꢀC. 1H NMR
7.96 (s, 1 H), 7.81e7.88 (m, 2 H), 7.75 (d, J ¼ 3.9 Hz, 1 H), 7.71 (d,
J ¼ 3.8 Hz, 1 H), 7.70 (s, 1 H), 7.49 (dt, J ¼ 9.4, 2.5 Hz, 1 H), 7.28 (dt,
J ¼ 8.6, 2.2 Hz, 1 H) 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIþ) calcd for
C
25H17N3O2F2S 462.5 (MHþ), found 462.8. Anal. C, H, N.
[400 MHz, (CD3)2SO]
d
10.92 (br s, 1 H), 8.68 (d, J ¼ 1.8 Hz, 1 H), 8.02

148
J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
(d, J ¼ 2.4 Hz, 1 H), 7.96 (s, 1 H), 7.84 (dd, J ¼ 8.2, 1.8 Hz, 1 H),
7.74e7.85 (m, 3 H), 7.71 (d, J ¼ 8.0 Hz, 1 H), 7.55e7.65 (m, 1 H), 7.26
(dt, J ¼ 8.5, 2.0 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.
chromatography on silica gel (3:1 hexanes/EtOAc) giving 105 as a
colourless oil (373 mg, 63%). 1H NMR [400 MHz, CDCl3]
8.35 (ddd,
d
J ¼ 4.8, 1.9, 0.8 Hz, 1 H), 7.98 (dd, J ¼ 1.8, 1.8 Hz, 1 H), 7.69e7.74 (m,
3 H), 7.66 (ddd, J ¼ 8.1, 1.9, 1.0 Hz, 1 H), 7.48 (ddd, J ¼ 8.2, 0.8, 0.8 Hz,
1 H), 7.32 (dd, J ¼ 8.0, 8.0 Hz, 1 H), 7.16 (ddd, J ¼ 7.4, 4.9, 1.0 Hz, 1 H),
5.38 (s, 2 H), 3.68 (q, J ¼ 7.1 Hz, 2 H), 1.19 (t, J ¼ 7.1 Hz, 3 H). LRMS
(APCIþ) calcd for C12H10BrFN2O2S 325 (M-EtO)þ, found 325.
4.1.16. N-(5-Bromo-2-methoxypyridin-3-yl)-2,4-
difluorobenzenesulphonamide (84)
5-Bromo-2-methoxypyridin-3-amine was reacted with 2,4-
difluorobenzenesulphonyl chloride according to general proced-
ure B, giving 84 as an ivory solid (45%). 1H NMR [400 MHz,
4.1.21. General procedure E: sodium ((3-(5-(2-methyl-1-
(CD3)2SO]
d
10.44 (br s, 1 H), 8.12 (d, J ¼ 2.3 Hz, 1 H), 7.72e7.81 (m,
oxoisoindolin-5-yl)thiophen-2-yl)phenyl)sulfonyl)(pyridin-2-yl)
amide (14)
1 H), 7.75 (d, J ¼ 2.3 Hz, 1 H), 7.52e7.61 (m, 1 H), 7.18e7.27 (m, 1 H),
3.61 (s, 3 H). LRMS (APCIþ) calcd for C12H9BrF2N2O3S 380 (MHþ),
found 380.
Bromide 105 was reacted with bis(pinacolato)diboron and the
crude boronate 108 subsequently coupled to 75 according to gen-
eral procedure A. After extraction of the reaction mixture with
EtOAc and evaporation, the protected crude intermediate was
subjected to a one-pot deprotection and conversion to the sodium
salt as follows; the solid was taken up in a 1:1 solution of 3 M HCl
and 1,4-dioxane and then heated to reflux for 1 h. Upon cooling the
white precipitate, consisting of essentially pure sulfonamide, was
filtered and taken up in EtOH. Precipitation of the sodium salt was
accomplished by slow addition of 2 M NaOH to give 14 as a yellow
solid (68%, over 3 steps); mp (MeOH/CH2Cl2) 302e306 ꢀC. 1H NMR
4.1.17. 2,4-Difluoro-N-(2-methoxy-5-(5-(2-methyl-1-
oxoisoindolin-5-yl)-thiophen-2-yl)pyridin-3-yl)
benzenesulphonamide (12)
Bromide 84 was reacted with bis(pinacolato)diboron and the
crude boronate 89 subsequently coupled to 75 according to general
procedure A, to give 12 as a yellow powder (42%); mp (MeOH/
CH2Cl2) 224e227 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 10.35 (br s,
1 H), 8.35 (s, 1 H), 7.92 (s, 1 H), 7.85 (d, J ¼ 2.2 Hz, 1 H), 7.82 (dd,
J ¼ 8.0, 1.5 Hz, 1 H), 7.74e7.83 (m, 1 H), 7.70 (d, J ¼ 7.3 Hz, 1 H), 7.69
(d, J ¼ 4.0 Hz, 1 H), 7.51e7.61 (m, 1 H), 7.54 (d, J ¼ 3.7 Hz, 1 H), 7.22
(dt, J ¼ 8.3, 2.3 Hz, 1 H), 4.51 (s, 2 H), 3.66 (s, 3 H), 3.09 (s, 3 H). Anal.
C, H, N.
[400 MHz, (CD3)2SO]
d
8.09 (dd, J ¼ 1.6, 1.6 Hz, 1 H), 7.94 (br s, 1 H),
7.87 (ddd, J ¼ 4.9, 2.1, 0.7 Hz, 1 H), 7.83 (dd, J ¼ 8.0, 1.5 Hz, 1 H),
7.68e7.72 (m, 4 H), 7.56 (d, J ¼ 3.9 Hz, 1 H), 7.41 (dd, J ¼ 7.8, 7.8 Hz,
1 H), 7.19 (ddd, J ¼ 8.5, 7.0, 2.2 Hz,1 H), 6.59 (ddd, J ¼ 8.6, 0.9, 0.9 Hz,
1 H), 6.36 (ddd, J ¼ 7.0, 5.0, 1.0 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H).
Anal. C, H, N.
4.1.18. 2-Methyl-5-(5-(2-methyl-5-nitrophenyl)thiophen-2-yl)
isoindolin-1-one (80)
Iodide 75 was reacted with (2-methyl-5-nitrophenyl)boronic
acid according to general procedure A, followed by flash column
chromatography (CH2Cl2/MeOH 98:2 as eluant) to give 80 as a
4.1.22. N-(4-Methyl-3-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)phenyl)pyridine-2-sulfonamide (15)
Amine 81 was reacted with pyridine-2-sulfonyl chloride ac-
cording to general procedure B to give 15 as a cream solid (66 mg,
42%); mp (MeOH/CH2Cl2) 274e277 ꢀC. 1H NMR [400 MHz,
yellow solid (80%). 1H NMR [400 MHz, CDCl3]
d
8.32 (d, J ¼ 2.5 Hz,
1 H), 8.11 (dd, J ¼ 8.4, 2.5 Hz, 1 H), 7.87 (d, J ¼ 7.9 Hz, 1 H), 7.74 (dd,
J ¼ 7.9, 1.5 Hz, 1 H), 7.69 (dd, J ¼ 1.4, 0.7 Hz, 1 H), 7.46 (d, J ¼ 8.4 Hz,
1 H), 7.42 (d, J ¼ 3.8 Hz, 1 H), 7.16 (d, J ¼ 3.8 Hz, 1 H), 4.43 (s, 2 H),
3.23 (s, 3 H), 2.59 (s, 3 H). LRMS (APCIþ) calcd for C20H17N2O3S 365
(MHþ), found 365.
(CD3)2SO]
d
10.58 (s, 1 H), 8.74 (ddd, J ¼ 4.65, 1.6, 0.8 Hz, 1 H), 8.08
(dt, J ¼ 7.7, 7.7, 1.7 Hz, 1 H), 7.98 (td, J ¼ 7.9, 1.0, 1.0 Hz, 1 H), 7.90 (s,
1 H), 7.80 (dd, J ¼ 7.9, 1.6 Hz, 1 H), 7.70 (d, J ¼ 8.0 Hz, 1 H), 7.64e7.68
(m, 2 H), 7.26 (d, J ¼ 2.3 Hz,1 H), 7.16e7.20 (m, 2 H), 7.06 (dd, J ¼ 8.2,
2.3 Hz, 1 H), 4.51 (s, 2 H), 3.09 (s, 3 H), 2.32 (s, 3 H). Anal. C, H, N.
4.1.19. 2,4-Difluoro-N-(4-methyl-3-(5-(2-methyl-1-oxoisoindolin-
5-yl)thiophen-2-yl)phenyl)benzenesulfonamide (13)
4.1.23. Sodium (4-methyl-3-(5-(2-methyl-1-oxoisoindolin-5-yl)
A 200 mL Parr hydrogenation vessel was charged with 80
(388 mg, 1.06 mmol) which was dissolved in a 1:1:1 mixture of
ethanol, EtOAc and THF (90 mL). The mixture was agitated under 60
psi hydrogen at RT for 3 , before being filtered through Celite®. The
solvents were evaporated to give the crude aniline 81 (350 mg) of
which a subsample (111 mg, 0.33 mmol) was reacted with 2,4-
difluorobenzenesulfonyl chloride according to general procedure
B, giving 13 as a cream solid (51 mg, 30%); mp (MeOH/CH2Cl2)
thiophen-2-yl)phenylsulfonyl)(pyridin-2-yl)amide (16)
3-Bromo-4-methylbenzenesulfonyl
chloride
and
2-
aminopyridine were reacted according to general procedure B to
give 102. This was then reacted with bis(pinacolato)diboron ac-
cording to general procedure C and the crude boronate 107 was
coupled to 75 according to general procedure A to give 16. Con-
version to the sodium salt using general procedure E afforded a
yellow solid (55%, 4 steps). 1H NMR [400 MHz, (CD3)2SO]
d 7.93 (br
268e271 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d
10.68 (s, 1 H),
s, 1 H), 7.87e7.88 (m, 2 H), 7.83 (dd, J ¼ 8.0, 1.4 Hz, 1 H), 7.68e7.69
(m, 2 H), 7.64 (dd, J ¼ 7.9,1.8 Hz,1 H), 7.30 (d, J ¼ 8.0 Hz,1 H), 7.25 (d,
J ¼ 3.8 Hz, 1 H), 7.18 (ddd, J ¼ 8.6, 7.0, 2.2 Hz, 1 H), 6.58 (ddd, J ¼ 8.6,
1.0, 1.0 Hz, 1 H), 6.35 (ddd, J ¼ 7.0, 5.0, 1.0 Hz, 1 H), 4.51 (s, 2 H), 3.08
(s, 3 H), 2.44 (s, 3 H). Anal. C, H, N.
7.88e7.95 (m, 2 H), 7.80 (dd, J ¼ 8.0, 1.5 Hz, 1 H), 7.70 (d, J ¼ 8.0 Hz,
1 H), 7.67 (d, J ¼ 3.8 Hz,1 H), 7.56 (ddd, J ¼ 11.4, 9.2, 2.4 Hz,1 H), 7.29
(dt, J ¼ 8.5, 8.4, 2.1 Hz, 1 H), 7.18e7.24 (m, 2 H), 7.03 (dd, J ¼ 8.2,
2.4 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3H), 2.33 (s, 3 H). Anal. C, H, N.
4.1.20. General procedure D: 3-Bromo-N-(ethoxymethyl)-N-
(pyridin-2-yl)benzenesulfonamide (105)
4.1.24. N-{4-Fluoro-3-[5-(2-methyl-1-oxo-2,3-dihydro-1H-
isoindol-5-yl)-2-thienyl]phenyl}-2-pyridinesulfonamide (17)
Reaction of pyridine-2-amine and 3-bromobenzenesulfonyl
chloride was carried out according to general procedure B.
Without any further purification, to a solution of the crude sul-
phonamide 103 (500 mg, 1.60 mmol) and (chloromethoxy)ethane
(166 mg, 1.76 mmol) in DMF at RT was added NaH (42 mg,
1.76 mmol), then the reaction stirred for 1 h. After quenching with
water the mixture was extracted with CH2Cl2, dried with MgSO4
and evaporated to give a solid which was purified by flash
2-Pyridinesulfonyl chloride and 3-bromo-4-fluoroaniline were
reacted according to general procedure B to give 91. Protection of
the sulfonamide nitrogen was then carried out according to general
procedure D, giving 94 which was purified by column chromatog-
raphy eluting with hexanes/EtOAc 3:1. The protected sulphonamide
was then reacted directly with bis(pinacolato)diboron according to
general procedure C and the crude boronate 97 coupled to 75 ac-
cording to general procedure A. Deprotection according to general

J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
149
procedure E gave 17 (11%, 5 steps) 1H NMR [400 MHz, (CD3)2SO]
Intermediate 93 was then protected according to general procedure
D to give 96 as a colourless oil (32%, 2 steps). 1H NMR [400 MHz,
d
8.13 (br s, 1 H), 8.01 (br s, 1 H), 7.88e7.97 (m, 3 H), 7.83 (dd, J ¼ 8.0,
1.6 Hz, 1 H), 7.79e7.81 (m, 1 H), 7.75e7.77 (m, 1 H), 7.68e7.73 (m,
3 H), 7.61 (t, J ¼ 7.8 Hz, 1 H), 7.24 (d, J ¼ 8.4 Hz, 1 H), 6.87 (t,
J ¼ 6.9 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.
(CD3)2SO]
d
8.75 (ddd, J ¼ 4.7, 1.6, 0.9 Hz, 1 H), 8.22 (dd, J ¼ 2.4,
1.5 Hz, 1 H), 8.01 (dd, J ¼ 8.1, 2.4 Hz, 1 H), 7.84e7.92 (m, 2 H), 7.54
(ddd, J ¼ 7.4, 4.7, 1.4 Hz, 1 H), 5.21 (s, 2 H), 3.76 (q, J ¼ 7.1 Hz, 2 H),
1.20 (t, J ¼ 7.0 Hz, 3 H). LRMS (APCIþ) calcd for C11H8BrFN3O2S 346
(M-EtO)þ, found 346.
4.1.25. N-(5-Bromo-2-(trifluoromethoxy)phenyl)-N-(ethoxymethyl)
pyridine-2-sulfonamide (95)
Pyridine-2-sulfonyl chloride and 5-bromo-2-(trifluoromethoxy)
aniline were reacted according to general procedure B to give 92.
Protection of the sulfonamide according to general procedure D,
gave 95 which was purified by column chromatography eluting
with hexanes/EtOAc 3:1 and isolated as a colourless oil (89%, 2
4.1.30. N-(2-Fluoro-5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)pyridine-2-sulfonamide (20)
Bromide 96 was reacted with bis(pinacolato)diboron to afford
crude boronate 99 which was subsequently coupled to 75 accord-
ing to general procedure A. The intermediate from this step was
then deprotected according to general procedure E to give 20 as a
white solid (11%, 3 steps); mp (CH2Cl2/MeOH) 279e382 ꢀC. 1H NMR
steps).1H NMR [400 MHz, CDCl3]
d
8.75 (ddd, J ¼ 4.8, 1.6, 1.0 Hz, 1H),
7.82e7.90 (m, 2 H), 7.46e7.54 (m, 3 H), 7.10 (dddd, J ¼ 8.9, 1.9, 1.8,
1.8 Hz,1 H), 5.21 (br s, 2 H), 3.79 (q, J ¼ 7.0 Hz, 2 H),1.20 (t, J ¼ 7.0 Hz,
3 H). LRMS (APCIþ) calcd for C13H10BrF3N2O3S 409 (M-EtO)þ, found
409.
[400 MHz, (CD3)2SO]
d
10.9 (br s, 1 H), 8.76 (ddd, J ¼ 4.7, 1.6, 0.9 Hz,
1 H), 8.39 (dd, J ¼ 2.0, 1.3 Hz, 1 H), 8.21 (dd, J ¼ 9.2, 2.4 Hz, 1 H), 8.13
(dd, J ¼ 9.2, 2.4 Hz, 1 H), 8.02 (ddd, J ¼ 7.9, 1.0, 1.0 Hz, 1 H), 7.95 (br s,
1 H), 7.84 (dd, J ¼ 7.9, 1.6 Hz, 1 H), 7.70e7.74 (m, 3 H), 7.65
(J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.
4.1.26. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)-2-
(trifluoromethoxy)phenyl)pyridine-2-sulfonamide (18)
Bromide 95 was reacted with bis(pinacolato)diboron, to give the
crude boronate 98 which was subsequently coupled to 75 accord-
ing to general procedure A. The intermediate from this step was
then deprotected according to general procedure E giving 18 as a
yellow solid (51%, 3 steps); mp (MeOH/CH2Cl2) 200e201 ꢀC. 1H
4.1.31. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)benzenesulphonamide (21)
Amine 78 was reacted with benzenesulphonyl chloride ac-
cording to general procedure B to give 21 as a yellow solid (64%);
mp 300e303 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 10.77 (br s, 1 H),
NMR [400 MHz, (CD3)2SO]
d
10.61 (s, 1 H), 8.79 (ddd, J ¼ 4.6, 1.6,
8.67 (d, J ¼ 2.0 Hz, 1 H), 8.21 (d, J ¼ 2.4 Hz, 1 H), 7.94 (s, 1 H),
7.75e7.80 (m, 3 H), 7.68e7.76 (m, 3 H), 7.55e7.67 (m, 4 H), 4.52 (s,
2 H), 3.09 (s, 3 H). In this case the product was converted to its
sodium salt to give the desired product 21 as a yellow solid (94%).
0.8 Hz, 1 H), 8.11 (ddd, J ¼ 7.7, 7.7, 1.7 Hz, 1 H), 7.99 (ddd, J ¼ 7.9, 0.9,
0.9 Hz, 1 H), 7.93e7.95 (m, 1 H), 7.82 (dd, J ¼ 8.0, 1.5 Hz, 1 H), 7.79 (d,
J ¼ 2.3 Hz,1 H), 7.68e7.74 (m, 3 H), 7.61 (dd, J ¼ 8.6, 2.3 Hz,1 H), 7.53
(d, J ¼ 3.4 Hz, 1 H), 7.39 (dddd, J ¼ 8.5, 1.5, 1.5, 1.5 Hz, 1 H), 4.52 (s,
2 H), 3.09 (s, 3 H). Anal. C, H, N.
1H NMR [400 MHz, (CD3)2SO]
d
8.00 (d, J ¼ 2.1 Hz, 1 H), 7.90 (s, 1 H),
7.88 (d, J ¼ 2.4, 1 H), 7.80 (dd, J ¼ 7.9,1.5 Hz, 1 H), 7.71e7.77 (m,
J ¼ 8.0, 2 H), 7.68 (d, J ¼ 8.0, 1 H), 7.63 (d, J ¼ 3.8, 1 H), 7.10e7.30 (m,
5 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.
4.1.27. 5-Bromo-N-(ethoxymethyl)-2-methoxy-N-(pyridin-2-yl)
benzenesulfonamide (106)
Pyridine-2-amine and 5-bromo-2-methoxybenzenesulfonyl
chloride were reacted according to general procedure B. Protec-
tion of the sulfonamide 104 was then carried out according to
general procedure D, giving 106 as a colourless oil (99%, 2 steps). 1H
4.1.32. 2-Fluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)benzenesulfonamide (22)
Amine 78 was reacted with 2-fluorobenzenesulphonyl chloride
according to general procedure B to give 22 as a beige solid (82%);
mp (CH2Cl2/MeOH) 289e292 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
NMR [400 MHz, CDCl3]
d
8.76 (ddd, J ¼ 4.7, 1.6, 0.8 Hz, 1 H), 7.82 (dd,
J ¼ 7.7, 1.7 Hz, 1 H), 7.76 (ddd, J ¼ 7.9, 1.1, 1.1 Hz, 1 H), 7.48 (ddd,
J ¼ 7.6, 4.8, 1.3 Hz, 1 H), 7.37e7.40 (m, 2 H), 6.64e6.68 (m, 1 H), 5.19
(br s, 2 H), 3.80 (q, J ¼ 7.0 Hz, 2 H), 3.37 (s, 3 H), 1.21 (t, J ¼ 7.0 Hz,
3 H). LRMS (APCIþ) calcd for C13H13BrN2O3S 355 (M-EtO)þ, found
355.
d
11.11 (br s, 1 H), 8.67 (d, J ¼ 2.0 Hz, 1 H), 8.25 (d, J ¼ 2.4 Hz, 1 H),
7.95 (dt, J ¼ 7.4, 1.7 Hz, 2 H), 7.83 (dd, J ¼ 8.0, 1.5 Hz, 1 H), 7.68e7.76
(m, 4 H), 7.63 (d, J ¼ 3.8 Hz, 1 H), 7.38e7.49 (m, 2 H), 4.52 (s, 2 H),
3.09 (s, 3 H). LRMS (APCIꢂ) calcd for C24H18N3O3FS2 479 (M ꢂ H),
found 479. Anal. C, H, N.
4.1.28. Sodium ((2-methoxy-5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)phenyl)sulfonyl)(pyridin-2-yl)amide (19)
4.1.33. 3-Fluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)benzenesulfonamide (23)
Bromide 106 was reacted with bis(pinacolato)diboron according
to general procedure C, then the crude boronate 109 was subse-
quently coupled to 75 according to general procedure A. The in-
termediate from this step was deprotected and converted to the
sodium salt according to general procedure E, furnishing 19 as a
yellow solid (68%, 3 steps); mp (MeOH/CH2Cl2) 322e325 ꢀC. 1H
Amine 78 was reacted with 3-fluorobenzenesulphonyl chloride
according to general procedure B to give 23 as a beige solid (56%);
mp (CH2Cl2/MeOH) 292e294 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d
10.88 (br s, 1 H), 8.70 (d, J ¼ 2.0 Hz, 1 H), 8.22 (d, J ¼ 2.3 Hz, 1 H),
7.94 (s, 1 H), 7.83 (dd, J ¼ 7.9, 1.5 Hz, 1 H), 7.69e7.75 (m, 3 H),
7.61e7.68 (m, 4 H), 7.50e7.58 (m, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H).
Anal. C, H, N.
NMR [400 MHz, (CD3)2SO]
d
8.10 (br d, J ¼ 2.5 Hz, 1 H), 7.89 (br s,
1 H), 7.86 (ddd, J ¼ 4.9, 2.1, 0.8 Hz, 1 H), 7.79 (dd, J ¼ 8.0, 1.5 Hz, 1 H),
7.63e7.68 (m, 3 H), 7.36 (d, J ¼ 3.8 Hz, 1 H), 7.20 (ddd, J ¼ 8.4, 7.0,
2.1 Hz, 1 H), 7.04 (d, J ¼ 8.7 Hz, 1 H), 6.71 (br d, J ¼ 8.4 Hz, 1 H), 6.36
(ddd, J ¼ 6.9, 4.8, 1.0 Hz, 1 H), 4.50 (s, 2 H), 3.74 (s, 3 H), 3.08 (s, 3 H).
Anal. C, H, N.
4.1.34. 4-Fluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)benzenesulfonamide (24)
Amine 78 was reacted with 4-fluorobenzenesulphonyl chloride
according to general procedure B to give 24 as a yellow solid (86%);
mp (CH2Cl2/MeOH) 272e274 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
4.1.29. N-(5-Bromo-2-fluoropyridin-3-yl)-N-(ethoxymethyl)
pyridine-2-sulfonamide (96)
Pyridine-2-sulfonyl chloride and 5-bromo-2-fluoropyridin-3-
amine were reacted according to general procedure B.
d
10.79 (br s, 1 H), 8.69 (d, J ¼ 2.0 Hz, 1 H), 8.21 (d, J ¼ 2.3 Hz, 1 H),
7.94 (s, 1 H), 7.85e7.92 (m, 2 H), 7.83 (dd, J ¼ 7.9, 1.5 Hz, 1 H),
7.68e7.75 (m, 3 H), 7.66 (d, J ¼ 3.9 Hz, 1 H), 7.40e7.48 (m, 2 H), 4.52
(s, 2 H), 3.09 (s, 3 H). In this case the product was converted to its

150
J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
sodium salt to give the desired product as a yellow solid (90%). 1H
NMR [400 MHz, (CD3)2SO]
7.88 (d, J ¼ 2.5 Hz, 1 H), 7.73e7.84 (m, 3 H), 7.68 (d, J ¼ 7.9 Hz, 1 H),
7.64 (d, J ¼ 3.8 Hz, 1 H), 7.37e7.42 (m, 2 H), 7.15e7.23 (m, 2 H), 4.51
(s, 2 H) 3.08 (s, 3 H). Anal. C, H, N.
4.1.40. 3,4-Dichloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (30)
Amine 78 was reacted with 3,4-dichlorobenzenesulfonyl chlo-
ride according to general procedure B to give 30 as an orange-
brown solid (15%); mp (MeOH/CH2Cl2) 256e259 ꢀC. 1H NMR
d
8.03 (d, J ¼ 2.1 Hz, 1 H), 7.90 (s, 1 H),
[400 MHz, (CD3)2SO]
d
10.92 (br s, 1 H), 8.72 (d, J ¼ 2.0 Hz, 1 H), 8.23
(d, J ¼ 2.4 Hz, 1 H), 8.03 (d, J ¼ 2.2 Hz, 1 H), 7.94 (s, 1 H), 7.89 (d,
J ¼ 8.5 Hz,1 H), 7.83 (dd, J ¼ 8.0,1.6 Hz, 1 H), 7.70e7.77 (m, 4 H), 7.69
(d, J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). HRMS (ESIꢂ) calcd for
4.1.35. 3,4-Difluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (25)
Amine 78 was reacted with 3,4-difluorobenzenesulphonyl
chloride according to general procedure B to give 25 as a yellow
solid (18%); mp (CH2Cl2/MeOH) 282e285 ꢀC. 1H NMR [400 MHz,
C24H16Cl2N3O3S2 528.0016 (M ꢂ H), found 528.0048.
4.1.41. 2,4-Dichloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (31)
Amine 78 was reacted with 2,4-dichlorobenzenesulphonyl
chloride according to general procedure B to give 31 as a yellow
solid (55%); mp (CH2Cl2/MeOH) 282e285 ꢀC. 1H NMR [400 MHz,
(CD3)2SO]
d
10.88 (br s, 1 H), 8.72 (d, J ¼ 1.4 Hz, 1 H), 8.23 (d,
J ¼ 2.3 Hz, 1 H), 7.95 (s, 1 H), 7.90 (d, J ¼ 8.3 Hz, 1 H), 7.83 (d,
J ¼ 8.0 Hz,1 H), 7.66e7.76 (m, 6 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C,
H, N.
(CD3)2SO]
d
11.20 (br s, 1 H), 8.67 (d, J ¼ 2.0 Hz, 1 H), 8.26 (d,
4.1.36. 2,4,6-Trifluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (26)
Amine 78 was reacted with 2,4,6-trifluorobenzenesulphonyl
chloride according to general procedure B to give 26 as a beige
solid (16%); mp (CH2Cl2/MeOH) 272e275 ꢀC. 1H NMR [400 MHz,
J ¼ 2.4 Hz, 1 H), 8.14 (d, J ¼ 8.6 Hz, 1 H), 7.96 (s, 1 H), 7.91 (d,
J ¼ 2.0 Hz, 1 H), 7.83 (dd, J ¼ 8.0, 1.5 Hz, 1 H), 7.65e7.74 (m, 4 H), 7.64
(d, J ¼ 3.9 Hz,1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). In this case the product
was converted to its sodium salt to give the desired product as a
beige solid (89%). 1H NMR [400 MHz, (CD3)2SO]
d
8.08 (d, J ¼ 2.1 Hz,
(CD3)2SO]
d
11.43 (br s, 1 H), 8.28 (d, J ¼ 2.3 Hz, 1 H), 7.95 (s, 1 H),
1 H), 8.01 (d, J ¼ 8.4 Hz, 1 H), 7.88e7.92 (m, 2 H), 7.80 (dd, J ¼ 7.9,
1.5 Hz, 1 H), 7.68 (d, J ¼ 8.0, 1 H), 7.64 (d, J ¼ 3.8, 1 H), 7.54 (d, J ¼ 2.1,
1 H), 7.46 (dd, J ¼ 8.4, 2.2 Hz, 1 H), 7.41 (d, J ¼ 3.8 Hz, 1 H), 7.37 (t,
J ¼ 2.3 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.
7.83 (dd, J ¼ 7.9, 1.5 Hz, 1 H), 7.79 (t, J ¼ 2.2 Hz, 1 H), 7.73 (d,
J ¼ 3.9 Hz, 1 H), 7.71 (d, J ¼ 7.9 Hz, 1 H), 7.65 (d, J ¼ 3.9 Hz, 1 H), 7.47
(br t, J ¼ 9.4 Hz, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.
4.1.42. 2-Bromo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (32)
4.1.37. 2-Chloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)benzenesulfonamide (27)
Amine 78 was reacted with 2-bromobenzenesulfonyl chloride
according to general procedure B to give 32 as an orange solid
(30%); mp (MeOH/CH2Cl2) 289e293 ꢀC. 1H NMR [400 MHz,
Amine 78 was reacted with 2-chlorobenzenesulfonyl chloride
according to general procedure B to give 27 as a yellow solid (29%);
mp (MeOH/CH2Cl2) 299e303 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
(CD3)2SO]
d
11.15 (br s, 1 H), 8.63 (s, 1 H), 8.26 (d, J ¼ 2.3 Hz, 1 H),
d
11.14 (br s, 1 H), 8.64 (d, J ¼ 1.9 Hz, 1 H), 8.26 (d, J ¼ 2.3 Hz, 1 H),
8.20 (dd, J ¼ 7.9, 1.7 Hz, 1 H), 7.94 (s, 1 H), 7.80e7.88 (m, 2 H),
7.68e7.74 (m, 2 H), 7.67 (t, J ¼ 2.2 Hz, 1 H), 7.59e7.65 (m, 2 H), 7.54
(dt, J ¼ 7.6,1.7 Hz,1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIꢂ) calcd
for C24H17BrN3O3S2 539 (M ꢂ H), found 539. Anal. C, H, N.
8.17 (dd, J ¼ 7.3,1.2 Hz,1 H), 7.94 (s,1 H), 7.83 (dd, J ¼ 7.9,1.5 Hz,1 H),
7.55e7.73 (m, 7 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIꢂ) calcd for
C
24H17ClN3O3S2 495 (M ꢂ H), found 495. Anal. C, H, N.
4.1.38. 3-Chloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (28)
Amine 78 was reacted with 3-chlorobenzenesulfonyl chloride
according to general procedure B to give 28 as an orange solid
(49%); mp (MeOH/CH2Cl2) 291e295 ꢀC. 1H NMR [400 MHz,
4.1.43. 3-Bromo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (33)
Amine 78 was reacted with 3-bromobenzenesulfonyl chloride
according to general procedure B to give 33 as a yellow-orange solid
(30%); mp (MeOH/CH2Cl2) 303e307 ꢀC. 1H NMR [400 MHz,
(CD3)2SO]
d
10.88 (br s, 1 H), 8.71 (d, J ¼ 2.0 Hz, 1 H), 8.22 (d,
(CD3)2SO]
d
10.87 (br s, 1 H), 8.71 (d, J ¼ 1.9 Hz, 1 H), 8.21 (d,
J ¼ 2.4 Hz, 1 H), 7.95 (d, J ¼ 0.7 Hz, 1 H), 7.81e7.86 (m, 2 H),
7.69e7.79 (m, 5 H), 7.67 (d, J ¼ 3.9 Hz, 1 H), 7.63 (t, J ¼ 7.9 Hz, 1 H),
4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIꢂ) calcd for C24H17ClN3O3S2
495 (M ꢂ H), found 495. Anal. C, H, N.
J ¼ 2.3 Hz, 1 H), 7.98 (t, J ¼ 1.8 Hz, 1 H), 7.95 (d, J ¼ 0.7 Hz, 1 H),
7.78e7.90 (m, 3 H), 7.69e7.75 (m, 3 H), 7.67 (d, J ¼ 3.9 Hz, 1 H), 7.56
(t, J ¼ 8.0 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). HRMS (ESIþ) calcd for
C
N.
24H18BrN3O3S2Na 561.9865 (M þ Naþ), found 561.9862. Anal. C, H,
4.1.39. 4-Chloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (29)
Amine 78 was reacted with 4-chlorobenzenesulfonyl chloride
according to general procedure B to give 29 as a yellow solid (59%);
mp (MeOH/CH2Cl2) 281e284 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
4.1.44. 4-Bromo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (34)
Amine 78 was reacted with 4-bromobenzenesulfonyl chloride
according to general procedure B to give 34 as a yellow solid (60%);
mp (MeOH/CH2Cl2) 276e279 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d
10.84 (br s, 1 H), 8.70 (d, J ¼ 2.0 Hz, 1 H), 8.21 (d, J ¼ 2.3 Hz, 1 H),
7.94 (s, 1 H), 7.79e7.85 (m, 3 H), 7.65e7.74 (m, 6 H), 4.52 (s, 2 H),
3.09 (s, 3 H). LRMS (APCIꢂ) calcd for C24H17ClN3O3S2 495 (M ꢂ H),
found 495. Anal. C, H, N. In this case the product was converted to
its sodium salt to give the desired product as a yellow solid (77%),
d
10.84 (br s, 1 H), 8.69 (d, J ¼ 2.0 Hz, 1 H), 8.21 (d, J ¼ 2.4 Hz, 1 H),
7.94 (d, J ¼ 0.8 Hz, 1 H), 7.80e7.86 (m, 3 H), 7.69e7.76 (m, 5 H), 7.66
(d, J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIꢂ) calcd for
C
24H17BrN3O3S2 539 (M ꢂ H), found 539. Anal. C, H, N.
mp (EtOH) 240e244 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 8.04 (d,
J ¼ 2.1 Hz, 1 H), 7.91 (d, J ¼ 0.8 Hz, 1 H), 7.89 (d, J ¼ 2.4 Hz, 1 H), 7.80
(dd, J ¼ 7.9, 1.5 Hz, 1 H), 7.73 (d, J ¼ 8.6 Hz, 2 H), 7.68 (d, J ¼ 7.8 Hz,
1 H), 7.64 (d, J ¼ 3.8 Hz, 1 H), 7.43 (d, J ¼ 8.6 Hz, 2 H), 7.41 (d,
J ¼ 3.8 Hz, 1 H), 7.39 (t, J ¼ 2.3 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H).
Anal. C, H, N.
4.1.45. 2,4-Dibromo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamides (35)
Amine 78 was reacted with 2,4-dibromobenzenesulfonyl chlo-
ride according to general procedure B to give 35 as a cream solid
(81%); mp (MeOH/CH2Cl2) 276e279 ꢀC. 1H NMR [400 MHz,

J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
151
(CD3)2SO]
d
11.23 (br s, 1 H), 8.66 (d, J ¼ 1.9 Hz, 1 H), 8.25 (d,
521. In this case, the product was also converted to the sodium salt
according to general procedure E to give the title compound as a
yellow solid (96%); mp (EtOH) 240e244 ꢀC. 1H NMR [400 MHz,
J ¼ 2.4 Hz, 1 H), 8.16 (d, J ¼ 1.9 Hz, 1 H), 8.08 (d, J ¼ 8.5 Hz, 1 H), 7.94
(d, J ¼ 0.7 Hz, 1 H), 7.81e7.87 (m, 2 H), 7.67e7.74 (m, 2 H), 7.65 (t,
J ¼ 2.2 Hz, 1 H), 7.63 (d, J ¼ 3.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H).
LRMS (APCIꢂ) calcd for C24H17Br2N3O3S2 617, 619, 621 (M), found
617, 619, 621. Anal. C, H, N.
(CD3)2SO]
d
8.00 (d, J ¼ 2.1 Hz, 1 H), 7.89 (d, J ¼ 0.8 Hz, 1 H), 7.86 (d,
J ¼ 2.5 Hz, 1 H), 7.79 (dd, J ¼ 8.0, 1.6 Hz, 1 H), 7.68 (d, J ¼ 7.9 Hz, 1 H),
7.64 (d, J ¼ 3.8 Hz, 1 H), 7.37e7.41 (m, 2 H), 7.28e7.32 (m, 2 H), 6.92
(d, J ¼ 8.1 Hz, 1 H), 4.51 (s, 2 H), 3.76 (s, 3 H), 3.73 (s, 3 H), 3.08 (s,
3 H). Anal. C, H, N.
4.1.46. 4-Iodo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)benzenesulfonamides (36)
Amine 78 was reacted with 4-iodobenzenesulfonyl chloride
according to general procedure B to give 36 as a pale yellow solid
(59%); mp (CH2Cl2/Et2O) 295e298 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
4.1.51. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-2-(trifluoromethoxy)benzenesulfonamide (41)
Amine
78
was
reacted
with
2-
d
10.80 (br s, 1 H), 8.70 (d, J ¼ 2.0 Hz, 1 H), 8.20 (d, J ¼ 2.4 Hz, 1 H),
trifluoromethoxybenzenesulphonyl chloride according to general
procedure B to give 41 as a pale yellow solid (42%); mp (CH2Cl2/
7.99 (d, J ¼ 8.6 Hz, 2 H), 7.95 (d, J ¼ 0.70 Hz, 1 H), 7.84 (dd, J ¼ 8.0,
1.5 Hz, 1 H), 7.70e7.74 (m, 2 H), 7.68 (t, J ¼ 2.2 Hz, 1 H), 7.66 (d,
J ¼ 3.9 Hz, 1 H), 7.56 (d, J ¼ 8.6 Hz, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H).
LRMS (APCIꢂ) calcd for C24H18IN3O3S2 587 (M), found 587. Anal. C,
H, N.
MeOH) 251e254 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 11.03 (br s,1 H),
8.67 (d, J ¼ 1.7 Hz, 1 H), 8.23 (d, J ¼ 2.3 Hz, 1 H), 8.09 (dd, J ¼ 7.8,
1.6 Hz, 1 H), 7.94 (s, 1 H), 7.75e7.85 (m, 2 H), 7.68e7.74 (m, 3 H), 7.63
(d, J ¼ 3.8 Hz, 1 H), 7.55e7.62 (m, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H).
Anal. C, H, N.
4.1.47. 2-Methoxy-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (37)
Amine 78 reacted with 2-methoxybenzenesulfonyl chloride
according to general procedure B to give 37 as a yellow solid (26%);
mp (MeOH/CH2Cl2) 273e276 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
4.1.52. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-3-(trifluoromethoxy)benzenesulfonamide (42)
Amine
78
was
reacted
with
3-
d
10.49 (br s, 1 H), 8.61 (d, J ¼ 2.0 Hz, 1 H), 8.24 (d, J ¼ 2.4 Hz, 1 H),
trifluoromethoxybenzenesulphonyl chloride according to general
procedure B to give 42 as a light brown solid (22%); mp (MeOH/
7.94 (s, 1 H), 7.86 (dd, J ¼ 7.9, 1.7 Hz, 1 H), 7.82 (dd, J ¼ 8.0, 1.6 Hz,
1 H), 7.67e7.73 (m, 3 H), 7.56e7.62 (m, 2 H), 7.19 (d, J ¼ 7.9 Hz, 1 H),
7.08 (dd, J ¼ 7.6, 0.7 Hz, 1 H), 4.52 (s, 2 H), 3.88 (s, 3 H), 3.09 (s, 3 H).
LRMS (APCIꢂ) calcd for C25H20N3O4S2 491 (M ꢂ H), found 491. Anal.
C, H, N.
CH2Cl2) 230e232 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 10.90 (br s,
1 H), 8.70 (d, J ¼ 2.0 Hz, 1 H), 8.20 (d, J ¼ 2.3 Hz, 1 H), 7.94 (s, 1 H),
7.80e7.87 (m, 2 H), 7.67e7.79 (m, 6 H), 7.65 (d, J ¼ 3.8 Hz, 1 H), 4.52
(s, 2 H), 3.09 (s, 3 H). HRMS (APCIþ) calcd for C25H18F3N3O4S2
546.0764 (MHþ), found 546.0747.
4.1.48. 3-Methoxy-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (38)
Amine 78 was reacted with 3-methoxybenzenesulfonyl chloride
according to general procedure B to give 38 as a pale yellow solid
(33%); mp (MeOH/CH2Cl2) 278e280 ꢀC. 1H NMR [400 MHz,
4.1.53. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-4-(trifluoromethoxy)benzenesulfonamide (43)
Amine
78
was
reacted
with
4-
trifluoromethoxybenzenesulphonyl chloride according to general
procedure B to give 43 as a pale yellow solid (41%); mp (CH2Cl2/
(CD3)2SO]
d
10.76 (br s, 1 H), 8.67 (d, J ¼ 2.0 Hz, 1 H), 8.21 (d,
J ¼ 2.3 Hz, 1 H), 7.94 (d, J ¼ 0.7 Hz, 1 H), 7.83 (dd, J ¼ 8.0, 1.5 Hz, 1 H),
7.68e7.75 (m, 3 H), 7.65 (d, J ¼ 3.9 Hz, 1 H), 7.51 (t, J ¼ 8.0 Hz, 1 H),
7.36e7.41 (m, 1 H), 7.32 (t, J ¼ 2.1 Hz, 1 H), 7.21 (ddd, J ¼ 8.3, 2.6,
0.8 Hz, 1 H), 4.52 (s, 2 H), 3.79 (s, 3 H), 3.09 (s, 3 H). HRMS (ESIþ)
calcd for C25H22N3O4S2 492.1046 (MHþ), found 492.1033. Anal. C, H,
N.
MeOH) 293e296 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 10.88 (br s,
1 H), 8.70 (d, J ¼ 2.0 Hz, 1 H), 8.22 (d, J ¼ 2.3 Hz, 1 H), 7.92e7.98 (m,
3 H), 7.82 (dd, J ¼ 8.0, 1.5 Hz, 1 H), 7.72 (m, 3 H), 7.66 (d, J ¼ 3.9 Hz,
1 H), 7.57e7.63 (m, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.
4.1.54. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-2-(trifluoromethyl)benzenesulfonamide (44)
Amine 78 was reacted with 2-(trifluoromethyl)benzenesulfonyl
chloride according to general procedure B to give 44 as a pale
yellow solid (67%); mp (CH2Cl2/Et2O) 271e275 ꢀC. 1H NMR
4.1.49. 4-Methoxy-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (39)
Amine 78 was reacted with 4-methoxybenzenesulfonyl chloride
according to general procedure B to give 39 as a dark yellow solid
(37%); mp (MeOH/CH2Cl2) 254e258 ꢀC. 1H NMR [400 MHz,
[400 MHz, (CD3)2SO]
d
11.15 (br s, 1 H), 8.68 (d, J ¼ 1.8 Hz, 1 H), 8.25
(CD3)2SO]
d
10.62 (br s, 1 H), 8.66 (d, J ¼ 1.8 Hz, 1 H), 8.20 (d,
(d, J ¼ 2.3 Hz, 1 H), 8.21 (d, J ¼ 7.7 Hz, 1 H), 8.03 (d, J ¼ 7.4 Hz, 1 H),
7.84e7.96 (m, 3 H), 7.82 (dd, J ¼ 8.0, 1.4 Hz, 1 H), 7.68e7.74 (m, 3 H),
7.63 (d, J ¼ 3.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIꢂ)
calcd for C25H18F3N3O3S2 529 (M), found 529. Anal. C, H, N.
J ¼ 2.2 Hz, 1 H), 7.94 (s, 1 H), 7.83 (d, J ¼ 8.3 Hz, 1 H), 7.76 (d,
J ¼ 8.9 Hz, 2 H), 7.67e7.74 (m, 3 H), 7.64 (d, J ¼ 3.8 Hz, 1 H), 7.10 (d,
J ¼ 8.9 Hz, 2 H), 4.52 (s, 2 H), 3.79 (s, 3 H), 3.09 (s, 3 H). HRMS (ESIþ)
calcd for C25H22N3O4S2 492.1046 (MHþ), found 492.1033.
4.1.50. 3,4-Dimethoxy-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide, sodium salt (40)
Amine 78 was reacted with 3,4-dimethoxybenzenesulfonyl
chloride according to general procedure B to give 40 as a pale or-
4.1.55. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-3-(trifluoromethyl)benzenesulfonamides (45)
Amine 78 was reacted with 3-(trifluoromethyl)benzenesulfonyl
chloride according to general procedure B to give 45 as a pale
yellow solid (65%); mp (1,4-dioxane) 262e265 ꢀC. 1H NMR
ange solid (31%). 1H NMR [400 MHz, (CD3)2SO]
d 10.57 (br s, 1 H),
8.66 (d, J ¼ 2.0 Hz, 1 H), 8.21 (d, J ¼ 2.4 Hz, 1 H), 7.94 (d, J ¼ 0.7 Hz,
1 H), 7.83 (dd, J ¼ 7.9, 1.5 Hz, 1 H), 7.69e7.75 (m, 3 H), 7.65 (d,
J ¼ 3.9 Hz, 1 H), 7.39 (dd, J ¼ 8.5, 2.2 Hz, 1 H), 7.32 (d, J ¼ 2.2 Hz, 1 H),
7.10 (d, J ¼ 8.6 Hz, 1 H), 4.52 (s, 2 H), 3.79 (s, 3 H), 3.78 (s, 3 H), 3.09
(s, 3 H). LRMS (APCIꢂ) calcd for C26H22N3O5S2 521 (M ꢂ H), found
[400 MHz, (CD3)2SO]
d
10.92 (br s, 1 H), 8.71 (d, J ¼ 2.0 Hz, 1 H), 8.20
(d, J ¼ 2.3 Hz, 1 H), 8.05e8.12 (m, 3 H), 7.93 (d, J ¼ 0.7 Hz, 1 H), 7.86
(d, J ¼ 7.9 Hz, 1 H), 7.82 (dd, J ¼ 7.9, 1.6 Hz, 1 H), 7.68e7.74 (m, 3 H),
7.66 (d, J ¼ 3.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIꢂ)
calcd for C25H18F3N3O3S2 529 (M), found 529. Anal. C, H, N.

152
J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
4.1.56. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-4-(trifluoromethyl)benzenesulfonamide (46)
Amine 78 was reacted with 4-trifluoromethylbenzenesulphonyl
chloride according to general procedure B to give 46 as a pink solid
(55%); mp (CH2Cl2/MeOH) 282e284 ꢀC. 1H NMR [400 MHz,
d
10.74 (br s, 1 H), 8.66 (d, J ¼ 2.0 Hz, 1 H), 8.22 (d, J ¼ 2.4 Hz, 1 H),
7.95e7.99 (m, 1 H), 7.93 (br d, J ¼ 0.7 Hz, 1 H), 7.82 (dd, J ¼ 8.0,
1.6 Hz, 1 H), 7.68e7.76 (m, 5 H), 7.63e7.67 (m, 2 H), 4.52 (s, 2 H),
3.85 (s, 3 H), 3.09 (s, 3 H). LRMS (APCIþ) calcd for C26H22N3O5S2 520
(MHþ), found 520. Anal. C, H, N.
(CD3)2SO]
d
10.99 (br s, 1 H), 8.72 (d, J ¼ 2.0 Hz, 1 H), 8.22 (d,
J ¼ 2.3 Hz, 1 H), 7.97e8.07 (m, 4 H), 7.94 (s, 1 H), 7.83 (dd, J ¼ 8.0,
1.6 Hz, 1 H), 7.68e7.76 (m, 3 H), 7.67 (d, J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H),
3.09 (s, 3 H). Anal. C, H, N.
4.1.62. Methyl 3-(N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)sulfamoyl)benzoate (52)
Amine 78 was reacted with methyl 3-(chlorosulfonyl)benzoate
according to general procedure B giving 52 as a beige solid (24%);
mp (CH2Cl2/MeOH) 250e254 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
4.1.57. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-3,5-bis(trifluoromethyl)benzenesulphonamide (47)
Amine 78 was reacted with 3,5-bis(trifluoromethyl)benzene-1-
sulphonyl chloride according to general procedure B to give 47 as a
beige solid (63%); mp (MeOH/CH2Cl2) 299e302 ꢀC. 1H NMR
d
10.92 (s, 1 H), 8.70 (d, J ¼ 8.7 Hz, 1 H), 8.38 (dd, J ¼ 1.6, 1.6 Hz, 1 H),
8.19e8.21 (m, 2 H), 8.07 (ddd, J ¼ 7.9, 1.9, 1.1 Hz, 1 H), 7.94 (br s, 1 H),
7.77 (d, J ¼ 7.9 Hz, 1 H), 7.70e7.75 (m, 3 H), 7.66 (d, J ¼ 3.4 Hz, 1 H),
4.52 (s, 2 H), 3.87 (s, 3 H), 3.09 (s, 3 H). HRMS (ESIþ) calcd for
[400 MHz, (CD3)2SO]
d
11.04 (br s, 1 H), 8.75 (d, J ¼ 2.0 Hz, 1 H), 8.53
C
26H22N3O5S2 520.0995 (MHþ), found 520.1004.
(s, 1 H), 8.37 (s, 2 H), 8.22 (d, J ¼ 2.4 Hz, 1 H), 7.93 (s, 1 H), 7.82 (dd,
J ¼ 7.9, 1.4 Hz, 1 H), 7.72e7.76 (m, 2 H), 7.71 (d, J ¼ 7.9 Hz, 1 H), 7.68
(d, J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.
4.1.63. Methyl 4-(N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)sulfamoyl)benzoate (53)
Amine 78 was reacted with methyl 4-(chlorosulfonyl)benzoate
according to general procedure B to give 53 as a pale yellow solid
(60%); mp (CH2Cl2/Et2O) 269e272 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
4.1.58. 2-Cyano-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)benzenesulfonamide (48)
Amine 78 was reacted with 2-cyanobenzenesulfonyl chloride
according to general procedure B to give 48 as a pale mustard-
yellow solid (43%); mp (MeOH/CH2Cl2) >310 ꢀC. 1H NMR
d
10.95 (br s,1 H), 8.67 (d, J ¼ 1.9 Hz,1 H), 8.19 (d,J ¼ 2.3 Hz,1 H), 8.13
(d, J ¼ 8.6 Hz, 2 H), 7.92e7.97 (m, 3 H), 7.82 (dd, J ¼ 8.0, 1.6 Hz, 1 H),
7.68e7.73 (m, 3 H), 7.65 (d, J ¼ 3.9 Hz,1 H), 4.52 (s, 2 H), 3.85 (s, 3 H),
3.09 (s, 3 H). LRMS (APCIþ) calcd for C26H22N3O5S2 520 (MHþ),
found 520. Anal. C, H, N.
[400 MHz, (CD3)2SO]
d
11.16 (br s, 1 H), 8.95 (d, J ¼ 1.8 Hz, 1 H), 8.89
(d, J ¼ 1.5 Hz, 1 H), 8.57 (t, J ¼ 2.1 Hz, 1 H), 8.47 (d, J ¼ 7.4 Hz, 1 H),
8.13 (d, J ¼ 7.0 Hz, 1 H), 7.90e8.00 (m, 3 H), 7.85 (dd, J ¼ 7.9, 1.3 Hz,
1 H), 7.79 (d, J ¼ 3.9 Hz, 1 H), 7.76 (d, J ¼ 3.9 Hz, 1 H), 7.72 (d,
J ¼ 8.0 Hz, 1 H), 4.53 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIꢂ) calcd for
4.1.64. Ethyl 4-(N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)sulfamoyl)benzoate (54)
Amine 78 was reacted with ethyl 4-(chlorosulfonyl)benzoate
according to general procedure B to give 54 as a pale yellow solid
(52%); mp (CH2Cl2/Et2O) 272e275 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
C
25H18N4O3S2 486 (M), found 486. Anal. C, H, N. In this case the
product was also converted to its sodium salt according to general
procedure E to give the desired product as a pale yellow solid
(100%). 1H NMR [400 MHz, (CD3)2SO]
(t, J ¼ 2.2 Hz, 1 H), 7.91e7.96 (m, 2 H), 7.85 (dd, J ¼ 8.0, 1.4 Hz, 1 H),
7.67e7.73 (m, 3 H), 7.68 (d, J ¼ 3.9 Hz, 1 H), 7.55e7.61 (m, 2 H), 4.52
(s, 2 H), 3.09 (s, 3 H).
d
8.47 (t, J ¼ 2.5 Hz, 2 H), 8.32
d
10.93 (br s, 1 H), 8.69 (d, J ¼ 2.0 Hz, 1 H), 8.20 (d, J ¼ 2.4 Hz, 1 H),
8.13 (d, J ¼ 8.6 Hz, 2 H), 7.93e7.98 (m, 3 H), 7.83 (dd, J ¼ 8.0, 1.6 Hz,
1 H), 7.69e7.74 (m, 3 H), 7.66 (d, J ¼ 3.9 Hz,1 H), 4.52 (s, 2 H), 4.31 (q,
J ¼ 7.1 Hz, 2 H), 3.09 (s, 3 H), 1.29 (t, J ¼ 7.1 Hz, 3 H). LRMS (APCIþ)
calcd for C27H24N3O5S2 534 (MHþ), found 534. Anal. C, H, N.
4.1.59. 3-Cyano-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)benzenesulfonamide (49)
Amine 78 was reacted with 3-cyanobenzenesulfonyl chloride
according to general procedure B to give 49 as a cream solid (39%);
mp (CH2Cl2/Et2O) 282e285 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
4.1.65. 4-(N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)sulfamoyl)benzoic acid (55)
Amine 78 was reacted with 4-chlorosulphonyl benzoic acid
according to general procedure B to give 55 as a pale pink solid
(6%); mp (MeOH/CH2Cl2) >310 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d
10.96 (br s, 1 H), 8.71 (d, J ¼ 2.0 Hz, 1 H), 8.30 (t, J ¼ 1.5 Hz, 1 H),
8.22 (d, J ¼ 2.3 Hz,1 H), 8.15 (dt, J ¼ 7.9,1.2 Hz,1 H), 8.10 (ddd, J ¼ 8.1,
1.9, 1.1 Hz, 1 H), 7.95 (d, J ¼ 0.8 Hz, 1 H), 7.79e7.86 (m, 2 H),
7.67e7.74 (m, 4 H), 4.51 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIþ) calcd for
d
13.46 (v br s, 1 H), 10.91 (v br s, 1 H), 8.68 (d, J ¼ 1.9 Hz, 1 H), 8.21
(d, J ¼ 2.3 Hz, 1 H), 8.11 (d, J ¼ 8.6 Hz, 2 H), 7.91e7.95 (m, 3 H), 7.83
(dd, J ¼ 8.0, 1.5 Hz, 1 H), 7.69e7.73 (m, 3 H), 7.65 (d, J ¼ 3.9 Hz, 1 H),
4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIꢂ) calcd for C25H19N3O5S2 505
(M), found 505. Anal. C, H, N.
C
25H19N4O3S2 487 (MHþ), found 487. Anal. C, H, N.
4.1.60. 4-Cyano-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)benzenesulfonamide (50)
Amine 78 was reacted with 4-cyanobenzenesulphonyl chloride
according to general procedure B to give 50 as a yellow solid (10%);
mp (CH2Cl2/MeOH) 282e285 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
4.1.66. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-2-(methylsulfonyl)benzenesulfonamide (56)
Amine 78 was reacted with 2-(methanesulfonyl)benzene-
sulfonyl chloride according to general procedure B to give 56 as a
pale orange solid (77%); mp (Et2O/CH2Cl2) 269e272 ꢀC. 1H NMR
d
11.02 (br s, 1 H), 8.71 (d, J ¼ 2.0 Hz, 1 H), 8.21 (d, J ¼ 2.4 Hz, 1 H),
8.08 (d, J ¼ 8.6 Hz, 2 H), 7.98 (d, J ¼ 8.6 Hz, 2 H), 7.95 (s, 1 H), 7.83
(dd, J ¼ 8.0, 1.6 Hz, 1 H), 7.74 (d, J ¼ 4.0 Hz, 1 H), 7.72 (s, 1 H), 7.71 (d,
J ¼ 8.4 Hz, 1 H), 7.68 (d, J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H).
Anal. C, H, N.
[400 MHz, (CD3)2SO]
d
10.22 (br s, 1 H), 8.68 (d, J ¼ 1.5 Hz, 1 H),
8.22e8.27 (m, 2 H), 8.14e8.18 (m, 1 H), 7.89e7.97 (m, 3 H), 7.82 (dd,
J ¼ 8.0, 1.5 Hz, 1 H), 7.76 (t, J ¼ 2.2 Hz, 1 H), 7.68e7.73 (m, 2 H), 7.65
(d, J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.53 (s, 3 H), 3.09 (s, 3 H). LRMS
(APCIꢂ) calcd for C25H21N3O5S3 539 (M), found 539. Anal. C, H, N.
4.1.61. Methyl 2-(N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)sulfamoyl)benzoate (51)
Amine 78 was reacted with methyl 2-(chlorosulfonyl)benzoate
according to general procedure B to give 51 as a pale orange solid
(23%); mp (CH2Cl2/Et2O) 209e212 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
4.1.67. Sodium (5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-
yl)pyridin-3-yl)((4-(methylsulfonyl)phenyl)sulfonyl)amide (57)
Amine 78 was reacted with 4-(methanesulfonyl)benzene-
sulfonyl chloride according to general procedure B and the

J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
153
resulting crude product converted directly to the sodium salt ac-
cording to general procedure E. The salt was recrystallised from
EtOH to give 57 as a cream solid (27%); mp (EtOH) > 300 ꢀC. 1H NMR
benzene-1-sulphonyl chloride according to general procedure B to
give 62 as a yellow solid (40%); mp 250e252 ꢀC. 1H NMR [400 MHz,
(CD3)2SO]
d
10.93 (br s, 1 H), 8.20 (d, J ¼ 2.4 Hz, 1 H), 8.11e8.19 (m,
[400 MHz, (CD3)2SO]
d
8.07 (d, J ¼ 2.1 Hz, 1 H), 7.91e7.98 (m, 5 H),
2 H), 7.93 (d, J ¼ 0.6 Hz, 1 H), 7.82 (dd, J ¼ 8.4, 1.6 Hz, 1 H), 7.77 (d,
J ¼ 9.9 Hz, 1 H), 7.68e7.75 (m, 2 H), 7.70 (d, J ¼ 7.9 Hz, 1 H), 7.66 (d,
J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). HRMS (APCIþ) calcd for
7.90 (d, J ¼ 0.7 Hz, 1 H), 7.81 (dd, J ¼ 8.0, 1.5 Hz, 1 H), 7.68 (d,
J ¼ 8.2 Hz, 1 H), 7.65 (d, J ¼ 3.8 Hz, 1 H), 7.42e7.45 (m, 2 H), 4.52 (s,
2 H), 3.19 (s, 3 H), 3.09 (s, 3 H). LRMS (APCIꢂ) calcd for C25H21N3O5S3
539 (M ꢂ Na), found 539. HRMS (ESIþ) calcd for C25H21N3NaO5S3
562.0536 (MHþ), found 562.0522.
C
25H18F4N3O3S2 548.0720 (MHþ), found 548.0743.
4.1.73. 3-Chloro-4-methyl-N-(5-(5-(2-methyl-1-oxoisoindolin-5-
yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (63)
4.1.68. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-2-nitrobenzenesulfonamide (58)
Amine
78
was
reacted
with
3-chloro-4-
methylbenzenesulphonyl chloride according to general procedure
Amine 78 was reacted with 2-nitrobenzenesulfonyl chloride
according to general procedure B to give 58 as a yellow solid (32%);
mp (MeOH/CH2Cl2) 270e273 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
B to give 63 as an off-white solid (50%); mp (CH2Cl2/MeOH)
277e279 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 10.81 (br s, 1 H), 8.70
(d, J ¼ 2.0 Hz, 1 H), 8.22 (d, J ¼ 2.3 Hz, 1 H), 7.94 (s, 1 H), 7.80e7.86
(m, 2 H), 7.64e7.75 (m, 5 H), 7.58 (d, J ¼ 8.2 Hz, 1 H), 4.52 (s, 2 H),
3.09 (s, 3 H), 2.36 (s, 3 H). Anal. C, H, N.
d
11.21 (bs, 1 H), 8.71 (d, J ¼ 1.9 Hz, 1 H), 8.26 (d, J ¼ 2.4 Hz, 1 H),
8.06e8.10 (m, 1 H), 7.98e8.01 (m, 1 H), 7.94 (d, J ¼ 0.8 Hz, 1 H),
7.86e7.90 (m, 2 H), 7.83 (dd, J ¼ 8.0, 1.6 Hz, 1 H), 7.72e7.75 (m, 2 H),
7.71 (d, J ¼ 8.0 Hz, 1 H), 7.66 (d, J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s,
3 H). LRMS (APCIꢂ) calcd for C24H17N4O5S2 506 (M ꢂ H), found 506.
Anal. C, H, N.
4.1.74. 2-Chloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)-4-(trifluoromethyl)
benzenesulfonamide (64)
Amine
78
was
reacted
with
2-chloro-4-
4.1.69. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-4-nitrobenzenesulfonamide (59)
Amine 78 was reacted with 4-nitrobenzenesulfonyl chloride
according to general procedure B to give 59 as a pale yellow solid
(56%); mp (MeOH/CH2Cl2) 272e275 ꢀC. 1H NMR [400 MHz,
trifluoromethylbenzenesulphonyl chloride according to general
procedure B to give 64 as a beige solid (61%); mp (CH2Cl2/MeOH)
292e295 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 11.39 (br s, 1 H), 8.68
(d, J ¼ 2.0 Hz, 1 H), 8.35 (d, J ¼ 5.0 Hz, 1 H), 8.28 (d, J ¼ 2.4 Hz, 1 H),
8.17 (s, 1 H), 7.97 (dd, J ¼ 8.4, 1.2 Hz, 1 H), 7.93 (s, 1 H), 7.82 (dd,
J ¼ 7.9, 1.4 Hz, 1 H), 7.67e7.75 (m, 1 H), 7.64 (d, J ¼ 3.9 Hz, 1 H), 4.52
(s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.
(CD3)2SO]
d
11.09 (br s, 1 H), 8.71 (d, J ¼ 1.9 Hz,1 H), 8.40 (dq, J ¼ 9.0,
5.0 Hz, 2 H), 8.22 (d, J ¼ 2.3 Hz, 1 H), 8.07 (dq, J ¼ 8.9, 5.0 Hz, 2 H),
7.95 (s, 1 H), 7.83 (dd, J ¼ 7.9, 1.4 Hz, 1 H), 7.76 (t, J ¼ 2.2 Hz,1 H), 7.73
(d, J ¼ 4.0 Hz, 1 H), 7.71 (d, J ¼ 8.0 Hz, 1 H), 7.68 (d, J ¼ 3.9 Hz, 1 H),
4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCIꢂ) calcd for C24H17N4O5S2 506
(M ꢂ H), found 506. HRMS (APCIþ) calcd for C24H18N4O5S2 507.0791
(MHþ), found 507.0792. In this case the product was also converted
to its sodium salt according to general procedure E, giving an or-
4.1.75. 3-Bromo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)-5-(trifluoromethyl)
benzenesulphonamide (65)
Amine 78 was reacted with 3-bromo-5-(trifluoromethyl)ben-
zene-1-sulphonyl chloride according to general procedure B to give
65 as a yellow solid (26%); mp 280e283 ꢀC. 1H NMR [400 MHz,
ange solid (89%). 1H NMR [400 MHz, (CD3)2SO]
d
8.24 (d, J ¼ 8.8 Hz,
2 H), 8.11 (d, J ¼ 1.6 Hz, 1 H), 7.96 (d, J ¼ 8.8 Hz, 2 H), 7.94 (d,
J ¼ 2.4 Hz, 1 H), 7.91 (s, 1 H), 7.81 (dd, J ¼ 8.0, 1.4 Hz, 1 H), 7.68 (d,
J ¼ 8.0 Hz, 1 H), 7.65 (d, J ¼ 3.9 Hz, 1 H), 7.47 (t, J ¼ 2.2 Hz, 1 H), 7.45
(d, J ¼ 3.8 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H).
(CD3)2SO] d 11.01 (br s, 1 H), 8.69 (s, 1 H), 8.34 (s, 1 H), 8.24 (s, 1 H),
8.20 (d, J ¼ 2.2 Hz, 1 H), 8.05 (s, 1 H), 7.93 (s, 1 H), 7.82 (d, J ¼ 8.4 Hz,
1 H), 7.63e7.77 (m, 4 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.
4.1.76. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)pyridine-2-sulfonamide (66)
4.1.70. 3-Chloro-2-fluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (60)
Amine 78 was reacted with 3-chloro-2-fluorobenzenesulphonyl
chloride according to general procedure B to give 60 as a pale
yellow solid (63%); mp (CH2Cl2/MeOH) 269e272 ꢀC. 1H NMR
Amine 78 with pyridine-2-sulphonyl chloride according to
general procedure B to give 66 as a cream solid (20%); mp
272e275 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 11.01 (br s, 1 H),
8.73e8.78 (m, 1 H), 8.66 (d, J ¼ 2.0, Hz, 1 H), 8.30 (d, J ¼ 2.4 Hz, 1 H),
8.11 (td, J ¼ 7.8, 1.7 Hz, 1 H), 8.60 (dt, J ¼ 7.6, 1.0 Hz, 1 H), 7.94 (s, 1 H),
7.81e7.87 (m, 2 H), 7.73 (d, J ¼ 3.9 Hz, 1 H), 7.71 (d, J ¼ 8.1 Hz, 1 H),
7.66e7.70 (m,1 H), 7.64 (d, J ¼ 3.8 Hz,1 H), 4.52 (s, 2 H), 3.09 (s, 3 H).
In this case the product was also converted to its sodium salt ac-
cording to general procedure E giving a light-brown solid (89%). 1H
[400 MHz, (CD3)2SO]
d
11.30 (br s, 1 H), 8.71 (d, J ¼ 1.9 Hz, 1 H), 8.27
(d, J ¼ 2.3 Hz, 1 H), 7.87e7.98 (m, 3 H), 7.83 (dd, J ¼ 7.9, 1.2 Hz, 1 H),
7.68e7.76 (m, 3 H), 7.66 (d, J ¼ 3.9 Hz, 1 H), 7.45 (t, J ¼ 8.0 Hz, 1 H),
4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.
4.1.71. 4-Fluoro-2-methyl-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl) benzenesulphonamide (61)
Amine 78 was reacted with 4-fluoro-2-methylbenzene-1-
sulphonyl chloride according to general procedure B to give 61 as
a light-brown solid (50%); mp 280e283 ꢀC. 1H NMR [400 MHz,
NMR [400 MHz, (CD3)2SO]
d
8.53 (td, J ¼ 4.7, 1.4 Hz, 1 H), 8.04 (d,
J ¼ 2.1 Hz, 1 H), 7.93 (d, J ¼ 2.4 Hz, 1 H), 7.91 (s, 1 H), 7.83e7.87 (m,
2 H), 7.80 (dd, J ¼ 8.0, 1.5 Hz, 1 H), 7.68 (d, J ¼ 7.9 Hz, 1 H), 7.65 (d,
J ¼ 3.8 Hz, 1 H), 7.60 (t, J ¼ 2.2 Hz, 1 H), 7.41 (d, J ¼ 3.8 Hz, 1 H),
7.32e7.38 (m, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). HRMS (APCIþ) calcd
for C23H17N4NaO3S2 485.0713 (MHþ), found 485.0710.
(CD3)2SO]
d
10.95 (br s, 1 H), 8.65 (d, J ¼ 2.0 Hz, 1 H), 8.23 (d,
J ¼ 2.4 Hz, 1 H), 8.05 (dd, J ¼ 5.8, 3.1 Hz, 1 H), 7.95 (s, 1 H), 7.83 (dd,
J ¼ 7.9, 1.4 Hz, 1 H), 7.68e7.75 (m, 2 H), 7.66 (t, J ¼ 2.2 Hz, 1 H), 7.63
(d, J ¼ 3.9 Hz, 1 H), 7.33 (dd,J ¼ 9.9, 2.5 Hz, 1 H), 7.27 (dt, J ¼ 8.4,
2.6 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.
4.1.77. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)pyridine-3-sulfonamide (67)
Amine 78 was reacted with pyridine-3-sulphonyl chloride ac-
cording to general procedure B to give 67 as a light brown solid
(45%); mp (CH2Cl2/MeOH) 283e286 ꢀC. 1H NMR [400 MHz,
4.1.72. 4-Fluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-
2-yl)pyridin-3-yl)-3-(trifluoromethyl)benzenesulphonamide (62)
Amine 78 was reacted with 4-fluoro-3-(trifluoromethyl)
(CD3)2SO]
d
10.98 (br s,1 H), 8.97 (d, J ¼ 2.0 Hz,1 H), 8.83 (dd, J ¼ 4.8,
1.4 Hz, 1 H), 8.70 (d, J ¼ 1.9 Hz, 1 H), 8.22 (d, J ¼ 2.2 Hz, 1 H), 8.20 (dt,

154
J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
J ¼ 8.1, 1.8 Hz, 1 H), 7.94 (s, 1 H), 7.84 (d, J ¼ 7.9 Hz, 1 H), 7.75 (t,
J ¼ 2.2 Hz, 1 H), 7.73 (d, J ¼ 3.9 Hz, 1 H), 7.71 (d, J ¼ 8.0 Hz, 1 H), 7.69
(d, J ¼ 3.9 Hz, 1 H), 7.65 (dd, J ¼ 5.2, 2.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s,
3 H). HRMS (APCIþ) calcd for C23H18N4O3S2 463.0893 (MHþ), found
463.0891.
(d, J ¼ 3.9 Hz, 1 H), 6.12 (s, 1 H), 4.51 (s, 2 H), 3.09 (s, 3 H), 2.34 (s,
3 H), 2.15 (s, 3 H). LRMS (APCIꢂ) calcd for C29H25N5O3S2 555 (M),
found 555. Anal. C, H, N.
4.1.83. N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide (73)
Amine 78 was reacted with 2,3-dihydrobenzo[b][1,4]dioxine-6-
sulfonyl chloride according to general procedure B to give 73 as a
4.1.78. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)thiophene-2-sulfonamide (68)
Amine 78 was reacted with thiophene-2-sulphonyl chloride
according to general procedure B to give 68 as a cream solid (71%);
mp (CH2Cl2/MeOH) 300e304 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
yellow solid (78%). 1H NMR [400 MHz, (CD3)2SO]
d 10.64 (s, 1 H),
8.68 (d, J ¼ 2.0 Hz, 1 H), 8.22 (d, J ¼ 2.3 Hz, 1 H), 7.95 (br s, 1 H), 7.84
(dd, J ¼ 8.0, 1.4 Hz, 1 H), 7.74e7.70 (m, 3 H), 7.66 (d, J ¼ 3.9 Hz, 1 H),
7.28e7.31 (m, 2 H), 7.04 (d, J ¼ 8.5 Hz, 1 H), 4.52 (s, 2 H), 4.28 (m,
4 H), 3.09 (s, 3 H). In this case the product was also converted to its
sodium salt according to general procedure E giving a yellow solid
d
10.91 (br s, 1 H), 8.72 (d, J ¼ 2.0, Hz, 1 H), 8.24 (d, J ¼ 2.3 Hz, 1 H),
7.93e7.98 (m, 2 H), 7.83 (dd, J ¼ 8.0, 1.5 Hz, 1 H), 7.78 (t, J ¼ 2.2 Hz,
1 H), 7.74 (d, J ¼ 3.9 Hz,1 H), 7.68 (d, J ¼ 3.9 Hz,1 H), 7.65 (dd, J ¼ 3.8,
1.3 Hz, 2 H), 7.15 (dd, J ¼ 4.9, 3.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H).
In this case the product was also converted to its sodium salt ac-
cording to general procedure E giving a pale yellow solid (90%). 1H
(58%). 1H NMR [400 MHz, (CD3)2SO]
d
8.00 (d, J ¼ 2.1 Hz, 1 H), 7.90
(br s, 1 H), 7.85 (d, J ¼ 2.4, 1 H), 7.80 (dd, J ¼ 8.0, 1.5 Hz, 1 H),
7.65e7.70 (m, 2 H), 7.37e7.41 (m, 2 H), 7.17e7.21 (m, 2 H), 6.82 (d,
J ¼ 8.3, 1 H), 4.51 (s, 2H), 4.21 (br s, 4H), 3.08 (s, 3H). HRMS calcd for
NMR [400 MHz, (CD3)2SO]
d
8.08 (d, J ¼ 2.1, Hz, 1 H), 7.92 (d,
J ¼ 2.4 Hz, 1 H), 7.90 (s, 1 H), 7.80 (dd, J ¼ 7.9, 1.5 Hz, 1 H), 7.68 (d,
J ¼ 8.0 Hz, 1 H), 7.65 (d, J ¼ 3.8 Hz, 1 H), 7.46e7.53 (m, 2 H), 7.42 (d,
J ¼ 3.9 Hz, 1 H), 7.27 (dd, J ¼ 3.6, 1.3 Hz, 1 H), 6.93 (dd, J ¼ 5.0, 3.6 Hz,
1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.
C
26H20N3NaO5S2, 541.0742 (Mꢂ þ Naþ); found, 541.0744.
4.1.84. 4-Methyl-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)thiazole-5-sulphonamide (74)
Amine 78 was reacted with 4-methylthiazole-5-sulphonyl
chloride according to general procedure B, giving 74 as a brown
4.1.79. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)thiophene-3-sulfonamide (69)
Amine 78 was reacted with thiophene-3-sulphonyl chloride
according to general procedure B to give 69 as a yellow solid (47%);
solid (12%); mp 277e280 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 11.15
(br s, 1 H), 9.20 (s, 1 H), 8.78 (d, J ¼ 2.0 Hz, 1 H), 8.25 (d, J ¼ 2.4 Hz,
1 H), 7.95 (d, J ¼ 0.6 Hz, 1 H), 7.83 (dd, J ¼ 7.9, 1.5 Hz, 1 H), 7.77 (t,
J ¼ 2.2 Hz, 1 H), 7.74 (d, J ¼ 3.9 Hz, 1 H), 7.71 (d, J ¼ 8.0 Hz, 1 H), 6.69
(d, J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H), 2.52 (s, 3 H). HRMS
calcd for C22H18N4O3S3, 483.06138 (Mþ); found, 483.06251.
mp (CH2Cl2/MeOH) > 300 ꢀC. 1H NMR [400 MHz, (CD3)2SO]
d 10.72
(br s, 1 H), 8.68 (d, J ¼ 1.9, Hz, 1 H), 8.31 (q, J ¼ 1.3 Hz, 1 H), 8.24 (d,
J ¼ 2.3 Hz, 1 H), 7.95 (s, 1 H), 7.83 (dd, J ¼ 8.0, 1.4 Hz, 1 H), 7.73e7.78
(m, 3 H), 7.70 (d, J ¼ 8.0 Hz, 1 H), 7.66 (d, J ¼ 3.9 Hz, 1 H), 7.32 (dd,
J ¼ 5.2, 1.4 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.
4.2. Inhibition of perforin-mediated lysis of Jurkat cells
4.1.80. N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)
pyridin-3-yl)-4-(oxazol-5-yl)benzenesulfonamides (70)
Amine 78 was reacted with 4-(1,3-oxazol-5-yl)benzenesulfonyl
chloride according to general procedure B to give 70 as a pale
yellow solid (81%); mp (MeOH/CH2Cl2) 278e281 ꢀC. 1H NMR
The ability of the compounds to inhibit the lysis of labelled
nucleated (Jurkat T lymphoma) cells in the presence of 0.1% BSA
was measured by the release of 51Cr. Jurkat target cells were
labelled by incubation in medium with 100 m
Ci 51Cr for 1 h. The cells
[400 MHz, (CD3)2SO]
d
10.80 (br s, 1 H), 8.69 (d, J ¼ 2.0 Hz, 1 H), 8.53
were then washed three times to remove unincorporated isotope
and re-suspended at 1 ꢁ 105 cells per mL in RPMI buffer supple-
mented with 0.1% BSA. Each test compound was pre-incubated to
(s, 1 H), 8.23 (d, J ¼ 2.3 Hz, 1 H), 7.86e7.96 (m, 6 H), 7.80 (dd, J ¼ 7.9,
1.4 Hz, 1 H), 7.68e7.73 (m, 3 H), 7.66 (d, J ¼ 3.9 Hz, 1 H), 4.52 (s, 2 H),
3.09 (s, 3 H). LRMS (APCIꢂ) calcd for C27H20N4O4S2 528 (M), found
528. Anal. C, H, N.
concentrations of 20, 10, 5, 2.5 and 1.25
mM with recombinant
perforin for 30 min with DMSO as a negative control. 51Cr labelled
Jurkat cells were then added and cells were incubated at 37 ꢀC for
4 h. The supernatant was collected and assessed for its radioactive
content on a gamma counter (Wallac Wizard 1470 automatic
gamma counter). Each data point was performed in triplicate and
an IC50 was calculated from the range of concentrations described
above.
4.1.81. 5-(Isoxazol-5-yl)-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)
thiophen-2-yl)pyridin-3-yl)thiophene-2-sulfonamide (71)
Amine 78 was reacted with 5-(5-isoxazyl)thiophene-2-sulfonyl
chloride according to general procedure B to give 71 as a pale pink
solid (20%); mp (MeOH/1,4-dioxane) 245e249 ꢀC. 1H NMR
[400 MHz, (CD3)2SO]
d 11.15 (br s, 1 H), 8.76 (br s, 1 H), 8.72 (d,
J ¼ 2.0 Hz, 1 H), 8.29 (d, J ¼ 2.3 Hz, 1 H), 7.93 (d, J ¼ 0.7 Hz, 1 H), 7.82
(dd, J ¼ 7.9, 1.5 Hz, 1 H), 7.79 (t, J ¼ 2.2 Hz, 1 H), 7.71e7.75 (m, 4 H),
7.70 (d, J ¼ 3.6 Hz, 1 H), 7.11 (d, J ¼ 1.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s,
3 H). LRMS (APCIꢂ) calcd for C25H18N4O4S3 534 (M), found 534.
Anal. C, H, N.
4.3. KHYG1 inhibitory assay
KHYG1 cells are washed and re-suspended in RPMI/0.1% BSA at
16 ꢁ 105 cells/mL and 100
mL of the cell suspension is dispensed to
each well of a 96-well V-bottom plate. Test compounds are then
4.1.82. 4-(3,5-Dimethyl-1H-pyrazol-1-yl)-N-(5-(5-(2-methyl-1-
oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide
(72)
Amine 78 was reacted with 4-(3,5-dimethyl-1H-pyrazol-1-yl)
benzenesulfonyl chloride according to general procedure B to give
72 as a beige solid (14%); mp (Et2O/CH2Cl2) 265e268 ꢀC. 1H NMR
added (50
m
L) at a final concentration of 20
m
M and incubated at RT
for 20 min. 51Cr-labelled K562 leukemia target cells (50
mL,
2 ꢁ 105 cells/mL) are then added to each well and incubated at 37 ꢀC
for 4 h. 51Cr release is assayed using a Skatron Harvesting Press and
radioactivity estimated on a Wallac Wizard 1470 Automatic Gamma
counter (Turku, Finland). The inhibitory function is then deter-
mined by identifying the number of untreated or inhibitor treated
effector cells required to kill the same number of targets. The
percent inhibition is calculated by the formula:
[400 MHz, (CD3)2SO]
d 10.81 (br s, 1 H), 8.70 (s, 1 H), 8.25 (d,
J ¼ 2.2 Hz, 1 H), 7.94 (br s, 1 H), 7.91 (d, J ¼ 8.7 Hz, 2 H), 7.82 (dd,
J ¼ 8.0, 1.3 Hz, 1 H), 7.77 (d, J ¼ 8.7 Hz, 2 H), 7.68e7.74 (m, 3 H), 7.61

J.A. Spicer et al. / European Journal of Medicinal Chemistry 137 (2017) 139e155
155
Fig. 3. Calculation of inhibitory function in the KHYG1 assay.
N. Lukoyanova, E.W.W. Leung, R.S. Norton, J.A. Lopez, K.A. Browne, H. Yagita,
G.J. Lloyd, A. Ciccone, S. Verschoor, J.A. Trapani, J.C. Whisstock, I. Voskoboinik,
Biochem. J. 456 (2013) 323.
ꢀ
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ðXÞ
100 ꢂ
ꢁ 100
ð16Þ
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where x is the point the inhibitor intersects with the DMSO on a
curve. In the example shown (Fig. 3) “x” is the x-intercept corre-
sponding to the point on the DMSO control curve that yields the
same level of 51Cr release as the test compound at an E/T ratio of
16:1.
4.4. Toxicity to KHYG1 NK cells
The toxicity assay was carried out in exactly the same manner as
the killing assay above, but instead of adding the labelled K562
target cells, 100 mL of RPMI 0.1% BSA was added. Cells were incu-
bated for 4 h at 37 ꢀC and then washed ꢁ3 in RPMI þ 0.1% BSA. Cells
were then re-suspended in 200 mL of complete medium and incu-
bated for 18e24 h at 37 ꢀC. Trypan blue was added to each well.
Viable (clear) cells and total (clear þ blue) cells were counted, and
the percentage of viable cells was calculated compared to DMSO
treated cell control (% viability).
Acknowledgements
This work was supported by the Wellcome Trust (Grant 097767),
the Auckland Division of the Cancer Society of New Zealand, the
Maurice & Phyllis Paykel Trust (MPPT; Grant Number: 8.2.29) and
the Genesis Oncology Trust (Grant Number: GOT-1616-PDA). The
authors thank Sisira Kumara for HPLC and solubility work, Karin
Tan for HPLC, Maruta Boyd and Shannon Black for NMR studies.
[24] S.P. Cobbold, Immunotherapy 1 (2009) 447e460.
[25] J.A. Trapani and M.J. Smyth, WO 2005083098 A1, March 1, 2005.
[26] G. Lena, J.A. Trapani, V.R. Sutton, A. Ciccone, K.A. Browne, M.J. Smyth,
W.A. Denny, J.A. Spicer, J. Med. Chem. 51 (2008) 7614.
[27] D.M. Lyons, K.M. Huttunen, K.A. Browne, A. Ciccone, J.A. Trapani, W.A. Denny,
J.A. Spicer, Bioorg. Med. Chem. 19 (2011) 4091.
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J.A. Trapani, Bioorg. Med. Chem. 20 (2012) 1319.
Appendix A. Supplementary data
[29] J.A. Spicer, G. Lena, D.M. Lyons, K.M. Huttunen, C.K. Miller, P.D. O'Connor,
M. Bull, N.A. Helsby, S.M.F. Jamieson, W.A. Denny, A. Ciccone, K.A. Browne,
J.A. Lopez, J. Rudd-Schmidt, I. Voskoboinik, J.A. Trapani, J. Med. Chem. 56
(2013) 9542.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.05.048.
[30] C.K. Miller, K.M. Huttunen, W.A. Denny, J.K. Jaiswal, A. Ciccone, K.A. Browne,
J.A. Trapani, J.A. Spicer, Bioorg. Med. Chem. Lett. 26 (2016) 355.
[31] S.D. Knight, N.D. Adams, J.L. Burgess, A.M. Chaudhari, M.G. Darcy,
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P.A. Elkins, C.M. Gardiner, E. Garver, S.A. Gilbert, R.R. Gontarek, J.R. Jackson,
K.L. Kershner, L. Luo, K. Raha, C.S. Sherk, C.eM. Sung, D. Sutton, P.J. Tummino,
R.J. Wegrzyn, K.R. Auger, D. Dhanak, ACS Med. Chem. Lett. 1 (2010) 39.
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W.A. Denny, H. Akhlaghi, K.A. Browne, J.A. Trapani, Bioorg. Med. Chem. Lett.
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