Diarylthiophenes as inhibitors of the pore-forming protein perforin

Article abstract of DOI:10.1016/j.bmcl.2015.12.003

Evolution from a furan-containing high-throughput screen (HTS) hit (1) resulted in isobenzofuran-1(3H)-one (2) as a potent inhibitor of the function of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 NK cells. In the current study, structure-activity relationship (SAR) development towards a novel series of diarylthiophene analogues has continued through the use of substituted-benzene and -pyridyl moieties as bioisosteres for 2-thioxoimidazolidin-4-one (A) on a thiophene (B) -isobenzofuranone (C) scaffold. The resulting compounds were tested for their ability to inhibit perforin lytic activity in vitro. Carboxamide (23) shows a 4-fold increase(2) in lytic activity against isolated perforin and provides good rationale for continued development within this class.

Full text of DOI:10.1016/j.bmcl.2015.12.003

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diarylthiophenes as inhibitors of the pore-forming protein perforin
Christian K. Miller a,b, Kristiina M. Huttunen a,c, William A. Denny a,b, Jagdish K. Jaiswal a,
Annette Ciccone d, Kylie A. Browne d, Joseph A. Trapani d,e, Julie A. Spicer a,b,
⇑
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, PO Box 1627, FI-70211 Kuopio, Finland
d Cancer Immunology Program, Peter MacCallum Cancer Centre, St. Andrew’s Place, East Melbourne, Victoria 3002, 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:
Evolution from a furan-containing high-throughput screen (HTS) hit (1) resulted in isobenzofuran-1(3H)-
one (2) as a potent inhibitor of the function of both isolated perforin protein and perforin delivered in situ
by intact KHYG-1 NK cells. In the current study, structure–activity relationship (SAR) development
towards a novel series of diarylthiophene analogues has continued through the use of substituted-ben-
zene and -pyridyl moieties as bioisosteres for 2-thioxoimidazolidin-4-one (A) on a thiophene (B) -isoben-
zofuranone (C) scaffold. The resulting compounds were tested for their ability to inhibit perforin lytic
activity in vitro. Carboxamide (23) shows a 4-fold increase over (2) in lytic activity against isolated per-
forin and provides good rationale for continued development within this class.
Received 9 September 2015
Revised 29 November 2015
Accepted 4 December 2015
Available online xxxx
Keywords:
Perforin
Perforin inhibitor
Diarylthiophene
Bioisostere
Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
Immunosuppressant
Perforin (PRF) is a calcium-dependent pore-forming glycopro-
tein found within granules of cytotoxic T lymphocytes (CTL) and
natural killer (NK) cells.1,2 These cytotoxic effector cells play an
important role in immune-surveillance and homeostasis via a
granule exocytosis pathway responsible for the elimination of neo-
plastic/virus-infected cells and intracellular pathogens.3 Numerous
autoimmune diseases (e.g., insulin-dependent diabetes) and ther-
apy-induced conditions (e.g., allograft transplantation rejection,
graft-vs-host disease) adopt a synonymous biochemical path-
way.4,5 An integral part of this biological cascade is perforin
oligomerisation in which monomeric perforin binds calcium upon
entry into the immune synaptic cleft and forms highly ordered oli-
gomers with 19–24 adjacent perforin monomers. These in turn
form cylindrical-type pores which subsequently penetrate target
cell membranes, allowing rapid delivery of cytotoxic granzymes
into the cytosolic compartment, a process which culminates in
apoptosis of the target cell. Highly specific inhibitors of perforin
function are thus of interest as selective immunosuppressive
drugs.
Isobenzofuran-1(3H)-one-based inhibitor 1 (Fig. 1) was a hit
selected from
a high-throughput screen of approximately
100,000 compounds sourced from commercial ‘lead discovery’
libraries.6 Subsequent SAR development led to the discovery of
potent inhibitor 2 in which an ꢀ8-fold improvement in inhibition
of isolated perforin protein function was achieved through replace-
ment of a central furan ring with a 2,5-thiophene moiety.7 In more
recently published work8 this template was elaborated further
through variation of the isobenzofuranone C-subunit where an
extensive SAR programme was conducted and the mechanism of
perforin inhibition investigated using real-time microscopy.8,9
The resulting isoindolinone-containing inhibitors were shown to
have improved lytic activity against perforin delivered in situ by
intact KHYG-1 NK cells and furthermore, were employed as probes
to elucidate the role of perforin in the granule exocytosis pathway.
This work resulted in the conclusion that this class of compounds
target perforin after its release into the immune synapse.
Abbreviations: HTS, high-throughput screen; SAR, structure–activity relation-
ships; PRF, perforin; CTL, cytotoxic T lymphocytes; NK, natural killer cells; PAINS,
pan-assay interference compounds; AgNO3, silver(I) nitrate; KF, potassium fluoride;
PFP, pentafluorophenyl; DCC, N,N0-dicyclohexylcarbodiimide; HOBt, hydroxyben-
zotriazole; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; DMF, dimethyl-
formamide; KOAc, potassium acetate; DMSO, dimethyl sulfoxide; THF,
tetrahydrofuran.
While substantial advances have been made in the develop-
ment and understanding of the thioxoimidazolidinone-based ser-
ies, more detailed in vitro and in vivo assessment7,10 revealed
several properties requiring further optimisation. Although well-
tolerated in healthy mice, even those compounds deemed suitable
⇑
Corresponding author. Tel.: +64 9 3737599; fax: +64 9 3737502.
E-mail address: j.spicer@auckland.ac.nz (J.A. Spicer).
http://dx.doi.org/10.1016/j.bmcl.2015.12.003
0960-894X/Ó 2015 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article in press as: Miller, C. K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.003

2
C. K. Miller et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
H
N
H
S
S
N
O
O
A
HN
HN
X
B
A
B
O
R
S
S
O
O
C
O
C
X = C or N
O
O
O
1
2
IC50 = 6.20 µM
(HTS hit)
New diarylthiophene inhibitors
IC50 = 0.78 µM
Figure 1. The development of new diarylthiophene inhibitors of perforin.
candidates for further in vivo studies showed some toxicity toward
whole NK cells.8 It is therefore likely that toxicity would also be
observed in the immunocompromised mice required for an effi-
cacy study. Also desirable would be the identification of new com-
pounds with significantly improved potency to deliver a lower
efficacious dose and better control of potential dose-related toxic-
ity. Finally, while the thioxoimidazolidinone series passed various
pan-assay interference compound (PAINS) filters,11 they still con-
tain a potentially reactive Michael acceptor and exist as an insep-
arable (and interconverting) mixture of E- and Z-isomers.
To address these aforementioned issues the need for a 2-thiox-
oimidazolidin-4-one bioisosteric replacement was apparent and
herein we report on the latest developments towards an exciting
new class of nontoxic inhibitors of perforin function that show
improved inhibition of the function of both isolated perforin pro-
tein and perforin delivered in situ by intact KHYG-1 NK cells.
Synthesis: Target compounds 6–23 (Scheme 1) were obtained in
a linear four-step sequence starting with the conversion of com-
mercially available 2-methyl-4-bromobenzoic acid to 5-bro-
moisobenzofuran-1(3H)-one 3.12 A sequential Finkelstein halogen
exchange reaction13 gave 5-iodoisobenzofuran-1(3H)-one 4 in
87% yield. This can also be prepared in high yield through diazoti-
zation of the corresponding 5-aminoisobenzofuran-1(3H)-one
using a literature procedure.14 Lactone derivative 4 was then uti-
lised in a palladium-catalysed arylation reaction with 2-bromoth-
iophene in the presence of a silver(I) nitrate/potassium fluoride
(AgNO3/KF) activator system15 to yield key intermediate bromide
5. Subsequent Suzuki couplings with a variety of commercially
available phenyl-boronates/boronic acids in the presence of cat-
alytic PdCl2(dppf), afforded the respective diarylthiophene targets
6–23 in generally good yields. Isoindolinone-based versions of 23
(23a and 23b) were also prepared in exactly the same manner,
using the isoindolin-1-one and 2-methylisoindolin-1-one-based
analogues16 of key bromide 5 (5a and 5b; Scheme 1).
Analogous methodology was adopted to generate secondary
and tertiary carboxamide targets 29 and 30, by first reacting iodide
24 with 2-bromothiophene in the presence of AgNO3/KF
(Scheme 2). The afforded bromide 25 was then converted to car-
boxamide intermediates 26 and 2717 through activation of the car-
boxylic acid to a pentafluorophenyl (PFP) ester followed by
subsequent addition of the corresponding amine (methylamine
or dimethylamine) in pyridine for 2–3 h at room temperature.
Key boronate 28 was prepared in multi-gram quantities from bro-
mide 318 (Scheme 1) and subsequently used in a Suzuki coupling
with intermediates 26 and 27 to furnish carboxamide targets 29
(75%) and 30 (57%), respectively.
Compounds 31 and 32, where the carboxamide side-chain was
elaborated further by incorporating a series of basic side-chains,
were accessed by applying the same chemistry as summarised in
Scheme 1. A final step Suzuki coupling of key intermediate bro-
mide 5 with the corresponding phenyl boronate (as according to
the compounds of Table 1) gave 31 and 32 in good to modest
yields, respectively, (61% and 42%).
With the chemistry now established for the mono-substituted
A-ring targets shown in Schemes 1 and 2, a series of di-substituted
targets were investigated. Phenyl iodides 33–35 were prepared via
O
B
O
O
O
O
S
O
28
O
O
B
71
(Table 1)
5'
O
O
R
iii
4'
3'
R = Substituents according
to the compounds of Table 1
X
O
O
i
ii
I
Br
N
S
Br
O
O
O
iii
4
3
5
X = O
X = NH 5a
5b
X = NMe
X
Br
iv
S
5'
S
O
R
N
ii
4'
68
69
3'
Compounds of Table 1;
6-23 , 31, 32, 58-63 (X = O)
O
23a
23b
(X = NH)
S
O
(X = NMe)
N
70
(Table 1)
Scheme 1. Reagents and conditions: (i) sealed tube, 1,4-dioxane, CuI (5.0 mol %), NaI, racemic trans-N,N0-dimethyl-1,2-cyclohexanediamine (10 mol %), 110 °C, N2, 18 h; (ii)
2-bromothiophene or 69, DMSO, KF, PdCl2(PPh3)2, AgNO3, 100 °C, N2, 2 h; (iii) toluene/EtOH, 2 M Na2CO3, PdCl2(dppf)ꢁCH2Cl2, 85–90 °C, N2, 2–6 h; (iv) thiophene-2-boronic
acid, toluene/EtOH, 2 M Na2CO3, PdCl2(dppf)ꢁCH2Cl2, 85 °C, N2, 2 h.
Please cite this article in press as: Miller, C. K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.003

C. K. Miller et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Br
I
Br
S
ii a - b
i
S
R
HO
HO
O
O
26
R = NHMe
O
24
25
R = NMe2 27
O
O
O
B
O
O
S
O
iii
28
R
O
29
R = NHMe
(Table 1)
R = NMe2 30 (Table 1)
Scheme 2. Reagents and conditions: (i) 2-bromothiophene, DMSO, KF, PdCl2(PPh3)2, AgNO3, 100 °C, N2, 2 h; (ii) (a) THF, pyridine, pentafluorophenyltrifluoroacetate, rt, 2 h;
(b) THF, methylamine (40% soln.) or dimethylamine, rt, 2–3 h; (iii) toluene/EtOH, 2 M Na2CO3, PdCl2(dppf)ꢁCH2Cl2, 85–90 °C, N2, 2–6 h.
esterification from their native carboxylic acids in the presence of
EtOH and catalytic H2SO4, using conventional methodology
(Scheme 3). The sequential step was carried out as for carboxylic
acid analogue 25 (refer to Scheme 2) affording ethyl esters 36–
38, these in turn were then coupled with key boronate ester 28
via a Suzuki reaction to give diarylthiophene esters 39–41 in good
to excellent yields. Hydrolysis of the resulting esters with 2 M
NaOH in MeOH under reflux conditions, gave bis-carboxylic acid
intermediates 42–44, respectively. Subsequent dehydration to
isobenzofuran-1(3H)-one precursors 45–47 was achieved quanti-
tatively using a mixture of TFA/CH2Cl2 (1:1) at room temperature
for approximately 3–4 h. Target compounds 48–57 were then syn-
thesised utilising a variety of amide-coupling strategies (Scheme 3).
Carboxamide 48 was prepared successfully in 57% yield going via
the analogous PFP-active ester intermediate as employed for car-
boxamides 26 and 27 (refer to Scheme 2). Compounds 49–54,
which contain various extended neutral and basic side-chains
appended to the 4-carboxamide moiety, were accessed through
treatment of the corresponding acid precursor with DCC and cat-
alytic HOBt at elevated temperatures. Target compounds 55
(48%), 56 (80%), and 57 (38%), all of which contain a 3-substi-
tuted-phenyl primary amine, were accessed through an EDCI/
HOBt-mediated amide-coupling in the presence of anhydrous
DMF at 0 °C to room temperature over 1–2 h.
activity.8 This approach is illustrated in Figure 2 and highlights
key objectives which include removal of the Michael acceptor
and elimination of isomeric mixtures.
For the current study we elected to retain the isobenzofuranone
C-subunit of 2 as this class is generally more potent than the cor-
responding isoindolinones8 which were originally introduced on
the assumption they would be less susceptible to hydrolysis. How-
ever subsequent stability studies showed no clear advantage with
both the isoindolinones and isobenzofuranones showing variable
half-lives in vitro (microsome stability) and in vivo.11 Parent com-
pound (6) was employed as a start point for the replacement of the
2-thioxoimidazolidin-4-one A-subunit, but due to its lack of
potency (Jurkat IC50 >20 lM; Table 1) a series of simple substituted
phenyl based derivatives were designed and investigated.
Compounds 7–15 (Table 1) follow an independent 2, 3, 4 substi-
tution pattern on a benzene A-ring with derivative 15 (4-OMe)
demonstrating activity against isolated perforin protein in our Jur-
kat assay (IC50 = 9.36 lM). The remaining methoxy isomers (13,
14) showed no detectable inhibition, suggesting that substitution
para to the connecting thiophene B-subunit is essential for activity.
Prompted by this encouraging result and with derivative 15 pro-
viding an H-bond acceptor moiety, we looked to introduce an
NH2, OH and CN group at positions 3 and 4 accordingly. Isomers
16 (3-NH2) and 17 (4-NH2) both have comparable activity
Compounds 58–63 (Scheme 1) encompass a variety of A-ring
moieties (such as an alcohol, a methanethiol and various sulfon-
amides). These were all accessed through a key Suzuki coupling
step from key intermediate 5 with the corresponding substituted
boronate, as according to the compounds of Table 1. Isobenzofu-
ran-1(3H)-one dimer (71) was prepared in the same manner.
Bromide 64 was commercially available and was converted to
iodide 65 via a copper-mediated halogen exchange reaction as
adapted from literature procedure13 (Scheme 4). Boronate 66 was
synthesised from corresponding bromide intermediate 5 in the
presence of bis(pinacolato)diboron and KOAc and subsequently
coupled with iodide 65 utilising Suzuki methodology to yield pyr-
idyl carboxamide 67 in low yield.
Finally, the preparation of 4-pyridyl-containing target 70 began
with the Suzuki-coupling of 4-bromopyridine 68 with thiophene-
2-boronic acid, as described by Effenberger et al.,19 to furnish 4-
(2-thienyl)pyridine 69 in high yield. Isobenzofuran-1(3H)-one 4
was then installed through treatment with AgNO3 and KF in the
presence of a palladium-complex, as previously discussed, to afford
4-pyridyl derivative 70 in 27% yield (Scheme 1).
(IC50s = 13.97 and 11.82 lM, respectively), showing that substitu-
tion at the 3-position is tolerated in this particular case. This is con-
firmed further by 3-hydroxyl-containing compound 18 in which a
2-fold increase in potency is achieved (IC50 = 5.89 lM). Looking at
3-CN derivative 20 in comparison, activity is lost altogether sug-
gesting that an H-bond donor may be required in this position
for activity. In contrast, 4-OH containing compound 19 loses all
activity whilst 4-CN containing compound 21 (IC50 = 6.87 lM)
retains potency similar to that of 18. This reinforces the argument
in that an H-bond acceptor at position 4 is more favourable and
perhaps necessary for activity to exist in this series (15 and 21 vs
19).
Compounds 22 and 23 were designed with a carboxamide moi-
ety installed at positions 3 and 4, respectively. Substitution at posi-
tion 3 was well tolerated giving rise to an IC50 = 2.97 lM—2-fold
greater than our previously most active compound 18. However
when the primary amide is moved to position 4, a dramatic
increase in the ability to inhibit perforin lytic activity is seen. Ben-
zene-4-carboxamide 23 has
greater than lead thioxoimidazolidinone 2 (Figs. 1 and 2) against
isolated perforin (0.18 M vs 0.78 M, respectively). Furthermore,
the corresponding pyridine-4-carboxamide 67 is also one of our
most potent compounds (IC50 = 0.92 M). Although at the outset
a potency approximately 4-fold
Structure–activity relationships: Based on our previous studies
described above, we hypothesised that selected substitutions on
a benzene or pyridyl ring could potentially occupy the same space
as the thioxoimidazolidinone pharmacophore, thereby mimicking
the network of hydrogen bond donors/acceptors required for
l
l
l
a decision was made to retain the more potent isobenzofuranone
C-subunit, for completeness the analogous isoindolin-1-one (23a)
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4
C. K. Miller et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 1
and 2-methylisoindolin-1-one (23b) derivatives were also pre-
pared. As expected this modification resulted in a loss of activity.
Conversion of the primary amide of 23 to a secondary derivative
(29) results in a loss of efficacy, while the presence of a tertiary
amide (30, 31) abolishes activity completely. Ester 39 showed lim-
Inhibitory activities of isobenzofuran-1(3H)-ones with various A-subunits
O
B
A
X
C
S
ited activity (10.97 lM), while compounds 40, 46, 48 and 55 were
O
designed in an effort to combine the outstanding potency of 23
with the preferred 3-OH of 18 or 3-NH2 of 16, thereby introducing
an H-bond donor at the 3-position and adding an ionisable centre
to assist solubility. While this approach did generate potent (and
slightly more soluble—see Supplementary data) compounds, none
were an improvement on 23. In an extension of this strategy, sol-
ubilising sidechains were introduced in examples 32, 48–54 and
56, 57. Disappointingly, compounds with strongly or weakly basic
sidechains (32, 49, 50, 56, 57) displayed poor activity or were inac-
tive, while neutral compounds 51 and 53 showed only moderate
Number Scheme Xa
Inhibition of
Jurkat cell
lysis IC50
b
(lM)
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
Ph
>20
>20
>20
>20
>20
>20
>20
>20
>20
9.36
13.97
11.82
5.89
>20
20
6.87
2.97
0.18
6.04
>20
10.7
>20
2-Me Ph
3-Me Ph
4-Me Ph
2-Cl Ph
3-Cl Ph
4-Cl Ph
10
11
12
13
14
15
16
17
18
19
20
21
22
23
23ac
23bd
29
30
activity (IC50s 3.31 and 2.65 lM), respectively.
2-OMe Ph
3-OMe Ph
4-OMe Ph
3-NH2 Ph
4-NH2 Ph
3-OH Ph
4-OH Ph
3-CN Ph
4-CN Ph
3-CONH2 Ph
4-CONH2 Ph
4-CONH2 Ph
4-CONH2 Ph
4-CONHMe Ph
4-CONMe2 Ph
In order to further increase diversity and enrich the SAR of the
diarylthiophene series, a range of commercially available boro-
nates were deployed in Suzuki reactions with key intermediate 5
(Scheme 1), generating analogues 58–63. Results were mixed, with
the methyl alcohol 59 and methyl sulfonamide 63 (IC50s 0.92 and
1.09 lM, respectively) the best of this set. Finally, given similarities
in SAR between the A- and C-subunits7 the symmetrical analogue
71 was synthesised but proved only moderately potent as well as
extremely insoluble.
Biological activity and stability: The five most potent inhibitors of
isolated recombinant perforin (23, 48, 59, 63, and 67 from Table 1)
were then subjected to more advanced assessment. Preliminary
stability studies were carried out by incubation in human or mouse
plasma at 37 °C with the percentage parent remaining measured at
24 h (Table 2). All five inhibitors were significantly more stable in
mouse plasma compared to human. This result is not unexpected,
as the anticancer agent camptothecin which also contains a lactone
moiety has been shown to co-exist in both the closed and ring-
opened form and that this equilibrium is distinctly different for
human and mouse plasma. In mouse plasma the ratio of open to
closed is 50:50%, while in human plasma this shifts to 90:10 due
to the strong affinity of human serum albumin for the ring-opened
form.20 It is likely that the same phenomenon is operating in the
present case and while not necessarily an issue for mouse studies,
will need to be addressed in future work.
The compounds were then evaluated for their ability to block
the effect of perforin delivered by whole KHYG-1 NK cells (Table 2).
Employing whole NK cells is a more rigorous model of conditions
in vivo than the use of isolated recombinant protein since effector
cell identification of the labelled target cell and formation of an
immune synapse are required for perforin release. Any putative
inhibitor must also possess the ability to access perforin released
into the synaptic cleft. After co-incubation of inhibitor with
KHYG-1 NK cells in medium for 30 min at room temperature,
51Cr-labelled K562 leukaemia target cells were added and cell lysis
evaluated after 4 h incubation at 37 °C by measuring 51Cr release.
The viability of the NK cells in the presence of inhibitor was also
assessed 24 h. later to confirm that any inhibitory activity exhib-
ited by the compounds was due to blocking the action of perforin
and not nonspecific killing of the effector cells. Viable and dead
cells were counted and percent viability calculated based on total
cell count (for further details see Supplementary data). Of the five
compounds selected for further testing, three showed poor or no
activity; the 3-OH, 4-CONH2 compound 48, 4-methyl alcohol 59
and methanesulfonamide 63. Gratifyingly, the most potent com-
pound against isolated perforin (23) showed comparable activity
N
4-
Ph
31e
1
>20
N
O
32
39
40
46
48
1
3
3
3
3
4-CONH(CH2)3NMe2 Ph
4-COOEt Ph
3-OH, 4-COOEt Ph
3-NH2, 4-COOH Ph
3-OH, 4-CONH2 Ph
>20
10.97
>20
5.84
0.67
O
O
49e
3
N
12.75
3-OH, 4-
Ph
Ph
N
H
50e
51
52
53
54
55
3
3
3
3
3
3
3-OH, 4-CONH(CH2)2NMe2 Ph
3-OH, 4-CONHCH2CH2OH Ph
3-OH, 4-CONHCH2CH(OH)CH3 Ph
3-OH, 4-CONHCH2CONH2 Ph
3-OH, 4-CONHCH2CH2CONH2 Ph
3-NH2, 4-CONH2 Ph
>20
3.31
>20
2.65
7.12
1.20
O
O
56e
3
N
14.71
3-NH2, 4-
N
H
57e
58
59
60
61
62
63
67
70
71
3
1
1
1
1
1
1
4
1
1
3-NH2, 4-CONH(CH2)2NMe2 Ph
4-SMe Ph
4-CH2OH Ph
4-SO2Me Ph
3-SO2NH2 Ph
4-SO2NHtert-Bu Ph
4-NHSO2Me Ph
4-CONH2, 3-pyridyl
4-Pyridyl
>20
>20
0.90
>20
4.85
6.09
1.09
0.92
4.53
6.24
5-Isobenzofuran-1(3H)-one
a
Structure of the A-subunit.
Testing 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.
Isobenzofuran-1(3H)-one C-subunit replaced with an isoindolin-1-one.
Isobenzofuran-1(3H)-one C-subunit replaced with an 2-methylisoindolin-1-
one.
b
c
d
(60% inhibition of lytic activity at 20 lM concentration) to many
e
Detailed structures for compounds, 31, 49, 50, 56 and 57 can be found in the
Supplementary information (Experimental Section).
of the active compounds contained in our previous report on the
thioxoimidazolidinone-based series.8 In addition, pyridinecarbox-
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C. K. Miller et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
O
O
B
O
O
O
R1
O
I
28
ii
R1
O
R1
O
Br
S
S
O
i
EtO
EtO
EtO
R
1 = H 39 (Table 1)
R1 = H 33
R1 = OH 40 (Table 1)
R
1 = H 36
R1 = NH2 37
R1 = OH 38
R
1 = NH2 34
R
1 = NH2 41
R1 = OH 35
iii
OH
OH
O
R1
R1
iv
S
S
O
O
HO
HO
R1 = H 42
O
R
1 = H 45
O
R1 = NH2 43
R1 = OH 44
v
R1 = NH2 46 (Table 1)
R1 = OH 47
O
R1
S
O
R2
R1 and R2 = Substituents according
48 57
O
to the compounds of Table 1;
-
Scheme 3. Reagents and conditions: (i) 2-bromothiophene, DMSO, KF, PdCl2(PPh3)2, AgNO3, 100 °C, N2, 2–4 h; (ii) toluene/EtOH, 2 M Na2CO3, PdCl2(dppf)ꢁCH2Cl2, 85–90 °C,
N2, 1–6 h; (iii) MeOH, 2 M NaOH, 100 °C, 2 h, HCl-workup; (iv) TFA–DCM (1:1), rt, 3–4 h; (v) THF, pyridine, pentafluorophenyltrifluoroacetate, rt, 2 h, R2-NH2, rt, 2 h, 48; or
pyridine, DCC, HOBt, 75 °C, 5–6 h, 49–54; or anhydrous-DMF, EDCIꢁHCl, HOBt, rt, 2 h, R2-NH2, 0 °C ? rt, 1–2 h, 55–57.
X
X = I 65
H2N
i
N
X = Br 64
O
iii
O
O
O
ii
O
S
S
B
O
S
O
O
Br
O
H2N
5
66
N
O
67 (Table 1)
Scheme 4. Reagents and conditions: (i) sealed tube, 1,4-dioxane, CuI (5.0 mol %), NaI, racemic trans-N,N0-dimethyl-1,2-cyclohexanediamine (10 mol %), 110 °C, N2, 24 h; (ii)
DMSO, bis(pinacolato)diboron, KOAc, PdCl2(dppf)ꢁCH2Cl2, 90 °C, N2, 4 h; (iii) toluene/EtOH, 2 M Na2CO3, PdCl2(dppf)ꢁCH2Cl2, 85–90 °C, N2, 1.5 h.
a
b
= Double bond required
Table 2
Michael
= Angle of connection important
acceptor
Capacity of selected compounds to inhibit perforin delivered by KHYG-1 NK cells
c = Position of nitrogen important
H
N
S
Number Jurkat
KHYG-1
KHYG-1
viabilityc
(%)
Plasma stabilityd
(% at 24 h)
H
N
O
A
a
IC50
inhibitionb (% at
HN
a
S
O
c
B
S
(
l
M)
20 lM)
N
A
Mouse Human
R1
B
H
O
C
4
23
48
59
63
67
0.18
0.67
0.90
1.09
0.92
63
20
13
90
92
79
—
75
75
87
80
66
46
49
73
70
39
S
3
2
O
R2
C
O
E/Z Isomeric
mixtures
R1 & R2 = H-bond
acceptors/donors
2
b
O
No activity
80
IC50 = 0.78 µM
82
Figure 2. Rationale for the development of new diarylthiophene inhibitors.
a
Data given for five most potent compounds as determined by the Jurkat assay.
Inhibition by compound (20 lM) of the perforin-induced lysis of K562 target
b
amide 67 was essentially equivalent to our previously most potent
in vivo candidates (80% inhibition of lysis). Most importantly, this
activity was observed in the absence of toxicity toward the effector
cells (90% and 82% viability, respectively). For the purposes of this
work, compounds are classified as toxic if NK cell viability falls
below 70%. This is due to the importance of CTLs and NK cells in
the overall immunological response which means that it is
essential that any pharmacological intervention allows rapid
recovery of these cytotoxic effector cells for normal immunological
function.
cells when co-incubated with KHYG-1 human NK cells (see Supplementary data).
Percent inhibition calculated compared to untreated control.
c
Viability of KHYG-1 NK cells after 24 h by Trypan blue exclusion assay (see
Supplementary data).
d
Data given as % parent measured at 24 h in mouse and human plasma (see
Supplementary data for further details).
thioxoimidazolidinone-based series. Several of these compounds
showed improved inhibition of perforin-mediated lysis, with ben-
zenecarboxamide 23 demonstrating a 4-fold increase (0.18
over the corresponding thioxoimidazolidinone inhibitor
(0.78
l
M)
2
In summary a new series of benzene- and pyridine-carboxam-
ides have been designed as isosteric replacements for our previous
l
M). Compound 23 and its pyridine analogue 67 also showed
Please cite this article in press as: Miller, C. K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.003

6
C. K. Miller et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
excellent activity against the lytic action of whole human NK cells,
at a level comparable to our previously identified in vivo candi-
dates.8 Use of an isostere in place of the thioxoimidazolidinone
A-subunit has also enabled removal of a potentially reactive
Michael acceptor moiety and elimination of E- and Z-isomeric mix-
tures. Finally, these compounds do not appear to possess the mar-
ginal toxicity associated with many of the thioxoimidazolidinones.
We can conclude that we have identified viable replacements
for the thioxoimidazolidinone subunit while improving potency.
We have also overcome several key concerns in relation to this his-
toric series and now seek to expand on these findings to identify a
suitable in vivo candidate. The mechanism of action of these inhi-
bitors is still under investigation, however given our previous con-
clusion that this class of compounds target perforin after its release
into the immune synapse it is most likely that inhibition occurs by
either (1) blocking the monomers from association with the target
cell membrane, (2) counteracting the oligomerisation process that
forms the pre-pore, or (3) by blocking of the conformational
changes required for pore formation. This work is currently ongo-
ing and further developments will be reported in the future.
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Acknowledgments
This work was supported by the Wellcome Trust (Grant
097767) and the Auckland Division of the Cancer Society of New
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and the Orion-Farmos Research Foundation for financial support.
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Maruta Boyd and Shannon Black for NMR studies.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.12.
003.
Please cite this article in press as: Miller, C. K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.003