Benzenesulphonamide inhibitors of the cytolytic protein perforin

Article abstract of DOI:10.1016/j.bmcl.2016.12.057

The pore-forming protein perforin is a key component of mammalian cell-mediated immunity and essential to the pathway that allows elimination of virus-infected and transformed cells. Perforin activity has also been implicated in certain auto-immune conditions and therapy-induced conditions such as allograft rejection and graft versus host disease. An inhibitor of perforin activity could be used as a highly specific immunosuppressive treatment for these conditions, with reduced side-effects compared to currently accepted therapies. Previously identified first-in-class inhibitors based on a 2-thioxoimidazolidin-4-one core show suboptimal physicochemical properties and toxicity toward the natural killer (NK) cells that secrete perforin in vivo. The current benzenesulphonamide-based series delivers a non-toxic bioisosteric replacement possessing improved solubility.

Full text of DOI:10.1016/j.bmcl.2016.12.057

Accepted Manuscript
Benzenesulphonamide inhibitors of the cytolytic protein perforin
Julie A. Spicer, Christian K. Miller, Patrick D. O'Connor, Jiney Jose, Kristiina
M. Huttunen, Jagdish K. Jaiswal, William A. Denny, Hedieh Akhlaghi, Kylie
A. Browne, Joseph A. Trapani
PII:
S0960-894X(16)31336-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.057
BMCL 24546
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 September 2016
20 December 2016
21 December 2016
Please cite this article as: Spicer, J.A., Miller, C.K., O'Connor, P.D., Jose, J., Huttunen, K.M., Jaiswal, J.K., Denny,
W.A., Akhlaghi, H., Browne, K.A., Trapani, J.A., Benzenesulphonamide inhibitors of the cytolytic protein perforin,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.057
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Benzenesulphonamide inhibitors of the cytolytic protein perforin
Julie A. Spicera,b,*, Christian K. Millera,b, Patrick D. O’Connora,b, Jiney Josea,b, Kristiina M.
Huttunena,c, Jagdish K. Jaiswala,b, William A. Dennya,b, Hedieh Akhlaghid, Kylie A. Browned,
Joseph A. Trapanid,e
aAuckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The
University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
bMaurice Wilkins Centre for Molecular Biodiscovery, A New Zealand Centre for Research
Excellence, Auckland, New Zealand.
cSchool of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box
1627, FI-70211 Kuopio, Finland.
dCancer Immunology Program, Peter MacCallum Cancer Centre, 305 Grattan Street,
Melbourne, Victoria 3000, Australia.
eSir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville,
Victoria 3052 Australia.
Keywords
Perforin; Perforin inhibitor; Benzenesulphonamide; Bioisostere; Immunosuppressant
Graphical Abstract
Abstract
The pore-forming protein perforin is a key component of mammalian cell-mediated immunity
and essential to the pathway that allows elimination of virus-infected and transformed cells.
Perforin activity has also been implicated in certain auto-immune conditions and therapy-
induced conditions such as allograft rejection and graft versus host disease. An inhibitor of
perforin activity could be used as a highly specific immunosuppressive treatment for these
conditions, with reduced side-effects compared to currently accepted therapies. Previously
identified first-in-class inhibitors based on a 2-thioxoimidazolidin-4-one core show
suboptimal physicochemical properties and toxicity toward the natural killer (NK) cells that
secrete perforin in vivo. The current benzenesulphonamide-based series delivers a non-toxic
bioisosteric replacement possessing improved solubility.
The primary role of cytotoxic T lymphocytes (CTL) and NK cells is to eliminate virally
infected or oncogenic target cells.1 This process takes place via the granule exocytosis
pathway2 where, upon stable conjugation with a target cell, the contents of cytotoxic granules
contained in CTL or NK cells are secreted into the synaptic cleft formed between effector and

target. This exposes the plasma membrane of the target cell to the combined action of
perforin, a 60 kDa calcium-dependent member of the membrane-attack complex/perforin
(MACPF) family of proteins, and a group of serine proteases known as granzymes. Perforin
is a pore-forming protein and is essential for this process since it facilitates infiltration of the
target cell by the granzymes, which in turn initiates apoptotic cell death. At high
concentrations recombinant perforin is also cytolytic in its own right and the physiological
means by which granzymes are delivered to the target cell cytosol has until recently been the
subject of hot debate.1-3 But recent studies utilising time-lapse microscopy have shown that
target cell death is invariably achieved through apoptosis (not lysis) and that perforin exerts
its biological effects by causing transient osmotic disruption of the target cell plasma
membrane, not endosomal vesicles. Accordingly, membrane perturbation by perforin pores is
sufficient to permit direct diffusion of granzymes into the target cell.4 The process is
remarkably rapid, with time-lapse microscopy revealing that perforin exocytosis and target
cell permeabilisation takes place within 30 seconds, while pore repair is initiated and
completed in another 80 seconds – sufficient time for the delivery of a lethal dose of
granzymes.4 Perforin is composed of an N-terminal MACPF domain and an EGF-like central
shelf, below which is located a membrane-interacting C2 domain.5 The protein binds
efficiently to cell membranes in the absence of calcium but requires binding to become
membranolytic.6,7 Upon exposure to calcium, perforin undergoes a conformational change
that allows it to assemble into highly ordered aggregates of 20-22 molecules where each
monomer contributes two β-hairpins to a β-barrel which spans the plasma membrane.5,8
Defective delivery and/or non-functional perforin within the granule exocytosis pathway is
known to be associated with various human disorders including familial haemophagocytic
lymphohistiocytosis (FHL), an inability to clear viral infections, and susceptibility to
haematological malignancies.3 Inappropriate perforin activity has also been implicated in a
variety of pathologies, including cerebral malaria, insulin-dependent diabetes, juvenile
idiopathic arthritis and postviral myocarditis9-11 as well as therapy-induced conditions such as
allograft rejection and graft versus host disease.2,12,13 Since perforin is expressed exclusively
by CTL and NK cells it is possible that a selective inhibitor of this protein could be used to
treat autoimmune diseases or therapy-induced conditions characterised by dysfunction of this
pathway. Unlike current immunosuppression therapies which have a wide range of side-
effects, an inhibitor that targets this mechanism could result in a potent immunosuppressive
therapy with greatly reduced side-effects.
The original lead for this programme arose from a high-throughput screen of approximately
100,000 compounds,14 and following an extensive SAR study,15,16 compound 1 (Figure 1)
was identified as one of the most potent inhibitors of recombinant perforin-induced lysis of
labelled Jurkat T lymphoma cells.

Figure 1. Historical inhibitors of perforin and PI3Kα clinical candidate GSK2126458
This work showed that while a thiophene B-subunit resulted in a significant increase in
activity, all variations explored as potential replacements for the 2-thioxoimidazolidin-4-one
A-subunit were either less potent or extremely insoluble.15 Introduction of an isoindolinone
C-subunit (in place of an isobenzofuranone) to give 1 gave greater potency (Jurkat IC50
=
0.51 µM) with improved solubility, however a major drawback for the entire series was
variable levels of toxicity when whole NK cells were used to deliver a lytic dose of
perforin.16 Although selected compounds were tested in vivo and found to be well-tolerated
with appropriate pharmacokinetics for future efficacy experiments, it was eventually
concluded that toxicity might still be observed in the immunocompromised mice required for
an efficacy study. Replacement of the 2-thioxoimidazolidin-4-one also remained a priority as
it contained a potentially reactive Michael acceptor and existed as an interconverting and
inseparable mixture of E- and Z-isomers. These issues were in part overcome through the
design and synthesis of a series of diarylthiophenes.17 Our rationale was that a substituted
aryl A-subunit could replicate the pattern of hydrogen bond donors and acceptors presented
by the 2-thioxoimidazolidin-4-one and presumably required for activity. We were successful
in identifying sub-micromolar inhibitors of isolated perforin (e.g., 2, 3) of which several had
activity against whole NK cells with minimal toxicity. One significant drawback of these
highly aromatic structures was their insolubility, which ranged between 0.023 – 16 µg/mL
making them unsuitable for further drug development. In order to improve potency and
particularly solubility, we emulated a strategy employed by GSK workers,18 where a
thiazolidinedione was replaced with a pyridyl-linked benzenesulphonamide (4) to give a
potent, orally bioavailable inhibitor of phosphoinositide 3-kinase α (PI3Kα) and mammalian
target of rapamycin (mTOR) with in vivo activity. Given that we had already successfully
identified an aryl sulphonamide (2) as a replacement for the closely related
thioxoimidazolidinone, this approach complemented our existing SAR and offered an
opportunity to target more potent, soluble perforin inhibitors.
Synthesis – The 2-thioxoimidazolidin-4-one subunit (A) of 1 was replaced with a pyridine-3-
yl-2,4-difluorobenzenesulphonamide which was linked through thiophene to a range of cyclic
amides and indoles (C), giving compounds 5-18 (Table 1). To connect the C-subunits and
thiophenes, Suzuki reactions were carried out for each halide and boronate pair to give target
compounds 23-34 (Scheme 1).

Scheme 1. Reagents and conditions: (i) Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux; (ii)
NIS, AcOH, CHCl3, RT; (iii) 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-
amine, tert-butyl (5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)carbamate or
3-aminopyridine-5-boronic acid, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux; (iv) TFA,
CH2Cl2 (1:1), RT; (v) 2,4-Difluorobenzenesulfonyl chloride, pyridine, 0-45 oC.
Selective iodination of the thiophene 2-position was then carried out with N-iodosuccinimide
for compounds 35-45 and 48, while for the indole targets (16, 17) the use of 2,5-
dibromothiophene in a Suzuki reaction gave the required intermediates with the bromide
already in place (46, 47). A second Suzuki step allowed the introduction of either the free
amino or BOC-protected pyridyl-3-amine subunit to compounds 49-62. Where required (55-
62) the carbamate was deprotected using a 1:1 mixture of trifluoroacetic acid in CH2Cl2 to
give amines 63-70. Finally, reaction of the amines with 2,4-difluorobenzenesulphonyl
chloride at low temperature gave the target products 5-18. In several cases the bis-
benzenesulphonamide was also formed; these were hydrolysed back to the mono-
sulphonamide by stirring with a 1:1 mixture of 1,4-dioxane and 2 M NaOH at room
temperature.
A small set of analogues where the thiophene of compound 5 was replaced with other
heterocycles was also prepared (compounds 19-22, Table 2 and Scheme 2).

Scheme 2. Reagents and conditions: (i) 2-Furanboronic acid or 2-bromothiazole,
Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux; (ii) NIS, AcOH, CHCl3, RT; (iii) 5-(4,4,5,5-
Tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-amine, Pd(dppf)Cl2, EtOH/toluene, 2 M
o
Na2CO3, reflux; (iv) 2,4-Difluorobenzenesulfonyl chloride, pyridine, 0-45 C; (v) 2,4-
o
Dibromothiazole, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux; (vi) a. Pyridine, 0-45 C,
b. (Chloromethoxy)ethane, NaH, DMF, RT; (vii) a. 78, Bis(pinacolato)diboron, KOAc,
o
Pd(dppf)Cl2, DMSO, 90 C, 3h.; b. 77, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux; c.
1:1 3 M HCl and 1,4-dioxane, reflux, 1 h., followed by 2 M NaOH to precipitate the sodium
salt.
For targets 19 and 20, the 2-furan- and 2-thiazoleboronic acids were coupled with 5-bromo-2-
methylisoindolin-1-one under standard conditions to give intermediates 71 and 72, which
were iodinated, coupled with pyridyl-3-amine-5-boronate, and reacted with 2,4-
difluorobenzenesulphonyl chloride as previously described (Scheme 2).
Unfortunately the isomeric thiazole 21 could not be prepared in this manner as the attempted
coupling of the thiazole bromide 77 and the pyridyl-3-amine boronate failed. In order to
circumvent this problem, the pyridine-sulphonamide 78 was introduced as a single subunit
via a Suzuki reaction. Protection of the sulphonamide NH with an ethoxymethyl group was
required for a successful coupling and this was removed under acidic conditions to furnish
the desired thiazole 21. Finally, the pyridyl analogue 22 was prepared through reaction of 5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine
and
5-bromo-2-
methylisoindolin-1-one to give the aminopyridine intermediate 79 (Scheme 3).

O
O
O
N
N
(i)
(iii)
(v)
N
22
R2HN
Br
N
R1
N
81
R2 = BOC
R1 = NH2 79
R1 = Br
N
(iv)
(ii)
82
R2 = H
80
Scheme 3. Reagents and conditions: (i) 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-
yl)pyridin-2-amine, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux; (ii) a. 47% HBr,
NaNO2, -10 oC; b. Br2, -10 oC-RT; (iii) tert-Butyl (5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)pyridin-3-yl)carbamate, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux; (iv) TFA,
CH2Cl2 (1:1), RT; (v) 2,4-Difluorobenzenesulfonyl chloride, pyridine, 0-45 oC.
The amine was diazotized with NaNO2 in HBr, treated with Br2 to convert to the bromide 80,
then subjected to the same steps as the other compounds of Scheme 2 to give the final
compound 22.
Structure–activity relationships: Our strategy involved linking a pyridine-3-yl-2,4-
difluorobenzenesulphonamide subunit via a thiophene to a variety of bicyclic templates A-N
(Table 1). These templates were selected as those most likely to display activity, having been
previously optimised in our thioxoimidazolidinone lead series.16 In the present case, we
elected to concentrate on isoindolinone-based analogues rather than the isobenzofuranones
we had studied previously due to the tendency of the lactone to ring-open in the presence of
human plasma.17 Thus the first compound to be targeted was the N-methylisoindolinone 5,
the benzenesulphonamide analogue of the most potent compound in the
thioxoimidazolidinone series (1). Gratifyingly, the activity of this compound (IC50 = 1.17
µM) was only ~2-fold less than 1 (0.51 µM), demonstrating that it is possible to replace the
thioxoimidazolidinone with a benzenesulphonamide moiety and retain potency.
Table 1. Variation of the C-subunit

Inhibition of Jurkat Cell
Lysis IC50a (µM)
Number
R
A
B
C
D
E
F
1.17
5
6.87
6
4.15
7
10.4
8
>20
9
>20
10
11
12
13
14
15
16
17
18
G
H
I
>20
13.6
>20
J
5.18
K
L
M
N
10.3
5.80
7.58
11.2
aTesting was carried out over a range of doses, with the IC50 being equal to the concentration
at which 50% inhibition of the lysis of Jurkat cells by perforin was observed, as measured by
51Cr release. Values are the average of at least two independent IC50 determinations.
The NH-isoindolinones 6 and 7 also showed appreciable activity (IC50s = 6.87 and 4.15 µM),
although the isomeric N-methylisoindolinone 8 was ~10-fold poorer. In contrast to the
thioxoimidazolidinone series, for the benzenesulphonamides the 6-membered cyclic amides

9-12 all showed poor activity, as did the methyl-substituted analogue 13. Given the activity
displayed by the 5-membered examples 5-7, we also elected to explore the isomeric
oxindoles 14 and 15. These too demonstrated significant activity with the NH-oxindole 14
(IC50 = 5.18 µM) ~2-fold superior to its N-methyl partner 15 (10.3 µM). Finally, indoles 16-
18 were investigated. Targets 16 and 17 showed good activity with IC50s = 5.80 and 7.58 µM
respectively, however acetyl-substitution at the 3-position of the indole (18) was detrimental
to activity. Overall, the N-methylisoindolinone-containing 5 was clearly the most potent
compound which is consistent with our findings in the thioxoimidazolidinone series.
On the assumption that the SAR of the B-subunit would also follow that of the
thioxoimidazolidinones, we selected a small set of heterocyclic replacements for the
thiophene subunit based on our previous work (Table 2).15
Table 2. Thiophene replaced with other heterocycles
Inhibition of Jurkat Cell Lysis
IC50a (µM)
Compound
Y
1.17
5
15.0
17.1
>20
8.17
19
20
21
22
S
N
aTesting was carried out over a range of doses, with the IC50 being equal to the concentration
at which 50% inhibition of the lysis of Jurkat cells by perforin was observed, as measured by
51Cr release. Values are the average of at least two independent IC50 determinations.
Our objectives here were to both improve the solubility of the target compounds as well as
mitigate any potential metabolic risk presented by the thiophene.19 As anticipated,
introduction of a furan resulted in a loss of activity from IC50 = 1.17 µM to 15.0 µM. For the
remaining thiazole (20, 21) and pyridyl (22) analogues the activity was unexpectedly
disappointing (IC50s = 17.1, >20, 8.17 µM respectively). In our historic series these
heterocycles possessed activity similar to the lead thiophene, however, in the present case the

predicted activity did not eventuate, suggesting that the SAR is tighter for this class of
compounds.
Biological activity, solubility & stability: The most potent inhibitors of recombinant perforin-
mediated lysis of labelled Jurkat cells were then tested for their ability to block whole NK
cells from lysing 51Cr-labelled (K562 leukaemia) target cells (Table 3).
Table 3. Capacity of selected compounds to inhibit perforin delivered by KHYG-1 NK cells
Jurkat
IC50
(µM)a
KHYG-1
Inhibition
(% at 20 µM)b
88 3.8
KHYG-1
Viability
(% at 20 µM)c
93 3.3
Number
1.17
5
6
6.87
86 1.3
94 4.0
4.15
91 1.3
96 6.0
7
5.18
80 13.1
96 1.0
14
b
aData given for the most potent compounds as determined by the Jurkat assay. Inhibition by
compound (20 µM) of the perforin-induced lysis of 51Cr-labelled K562 leukemia target cells
when co-incubated with KHYG-1 human NK cells. Percent inhibition calculated compared to
untreated control (n = 4). cViability of KHYG-1 NK cells after 24 h by Trypan blue exclusion
assay (n = 3). All results are the average of at least three separate determinations SEM. (see
Supplementary Data for details).
The use of NK cells to secrete perforin rather than employing isolated recombinant protein is
a more stringent test of a compound’s ability to block lytic activity, requiring the inhibitor to
access the synaptic cleft between effector (NK) and target cells where perforin is released. In
this assay, inhibitor is co-incubated with KHYG-1 NK cells in medium for 30 minutes at
room temperature, 51Cr-labelled target cells added and cell lysis evaluated after 4 hours
incubation at 37 oC by measuring 51Cr release. The viability of the NK cells in the presence of
inhibitor was also assessed 24 hours later to confirm that any inhibitory activity exhibited by
the compounds was due to blocking the action of perforin and not non-specific killing of the
effector cells.
Exemplars 5, 6, 7 and 14 from Table 1 were therefore selected as potent inhibitors of
recombinant perforin. Indole 16 (IC50 = 5.80 µM) was excluded from this analysis due to its
activity being adversely affected in the presence of serum. When 16 was tested at 20 µΜ
concentration in a Jurkat assay containing 10% mouse serum, inhibitory activity was reduced
from 88% (0.1% BSA alone) to 32%. This phenomenon has been observed in previous series
we have studied,15,16 however in the cases of 5, 6, 7 and 14 the activity in the presence of
10% mouse serum was similar to that obtained in its absence.
When tested for their ability to block NK cell-induced lysis, the four preferred compounds
showed excellent levels of inhibition (80-91%) at a concentration of 20 µM and significantly,
there was virtually no effect on NK cell viability (93-96% survival at 20 µM). This is an

important result in the context of this work because we have observed varying degrees of
toxicity toward the KHYG-1 cells in all the other series we have studied to date.15-17
Preliminary physicochemical data was also collected on 5, 6, 7 and 14 (Table 4) in order to
assess their potential for progression to in vivo pharmacokinetic studies.
Table 4. Physicochemical properties of selected compounds
Microsome Stability (%)c
Stability
Plasma
Solubilitya
in
Solutionb
(%)
Protein
Bindingd
(%)
Number
(µg/mL)
Mouse
Rat
Human
1080
10565
12940
392
81.5
92
92
42
84
99
88
82
79
58
85
83
71
5
6
99.89
99.70
99.97
99.91
82.0
97.3
7
88.7
14
aSolubility of the sodium salt in water at room temperature; conversion to salt as described in
b
General Procedure D (Supplementary Data). Percentage of parent compound (sodium salt)
o
c
remaining after 24 h. at 20 C in water. Percentage of parent compound (sodium salt)
d
remaining after exposure to mouse, rat or human microsomes for 30 minutes. Percentage of
o
parent compound bound to mouse plasma after 6 h. incubation at 37 C as determined by
equilibrium dialysis.
The N-methylisoindolinone 5 showed moderate solubility (1.1 mg/mL) and stability in
solution (82% over 24 h.) and was stable in the presence of mouse (92%) and rat (99%)
microsomes, but some instability was observed for human microsomes (58%). The two NH-
isoindolinones 6 and 7 showed excellent solubility, at 10.6 and 12.9 mg/mL respectively.
Compound 6 also showed moderate stability in aqueous solution and good stability over all
three species of microsomes (92, 88, 85%). In contrast, the isomeric isoindolinone 7 showed
excellent stability in water (97%), rat and human microsomes but was metabolised
extensively in the presence of mouse microsomes (42% remaining after 0.5 h.). The oxindole
14 was stable in solution and in the presence of mouse, rat and human microsomes but
possessed disappointing solubility at 0.39 mg/mL. Finally, all four compounds showed very
high levels of plasma protein binding.
Given that the pyridine-3-yl-2,4-difluorobenzenesulphonamide subunit is derived from a
structure that was optimised to target PI3Kα, it was also considered prudent to counter-screen
for off-target effects related to this pathway. To that end, compound 5 was included in a high-
throughput screen to test for PI3Kα activity (for details see Supplementary Data) where it
was found that the IC50 was >6.25 µM. Thus any activity that lead compound 5 possesses
toward PI3Kα is a minimum of 6-fold less than that observed against perforin. While this
result still requires more detailed investigation, it at least provides some reassurance that

these compounds are optimised as potent inhibitors of the desired target perforin rather than
PI3Kα.
In previous studies we sought to design and optimise potent and “drug-like” inhibitors of
perforin based on an initial hit from a mass screen. The most difficult obstacle to overcome
proved to be replacement of a problematic 2-thioxoimidazolidinone moiety containing a
potential Michael acceptor and showing variable toxicity toward the perforin-producing NK
cells. Our overall approach is based on selectively inhibiting perforin to facilitate allograft
transplantation, while at the same time preserving the NK cells so that withdrawal of the drug
would be expected to restore NK cell killing within hours. Toxicity exhibited toward the NK
cells is therefore an issue for both in vivo efficacy investigations and any potential clinical
studies. In the current series the 2,4-difluorobenzenesulphonamide has been successfully
employed as a bioisosteric replacement for the 2-thioxoimidazolidinone to give compounds
with activity comparable to the best of our previous series. In addition, all four of the most
potent inhibitors (5, 6, 7, 14) showed >90% viability of the NK cells, a greater rate of
survival than all preceding series and essentially non-toxic.
Compound 1 was the most potent inhibitor of perforin, while 6 had the best overall balance of
solubility and stability. Although we had previously identified a set of diarylthiophene
alternatives to the 2-thioxoimidazolidinones, these were extremely insoluble compared to the
current series which shows far superior solubility. Although no single compound possessed
an ideal profile, the overall combination of potency, solubility, non-toxicity and selectivity
obtained in this study gives us confidence that further refinement will furnish an appropriate
candidate for in vivo studies. For example, one area for further exploration will be whether it
is possible to further modulate activity and physicochemical properties through substitution
on the benzene ring of the sulphonamide. Our future objectives will therefore be to retain the
benefits of the 2,4-difluorobenzenesulphonamide at the same time as further optimising this
highly promising series of perforin inhibitors towards a potential clinical candidate.
Acknowledgements
This work was supported by the Wellcome Trust (Grant 097767) and the Auckland Division
of the Cancer Society of New Zealand. The authors thank Sisira Kumara for HPLC and
solubility work, Karin Tan for HPLC and Maruta Boyd for NMR studies.
Supplementary data
Supplementary data associated with this manuscript can be found in the online version at:
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