Exploration of a series of 5-arylidene-2-thioxoimidazolidin-4-ones as inhibitors of the cytolytic protein perforin

Article abstract of DOI:10.1021/jm401604x

A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte-expressed pore-forming protein perforin. Structure-activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by natural killer cells was determined. A number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells, and several were a significant improvement on previous classes of inhibitors, being substantially more potent and soluble. Representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations (≤2.5 μM) by surface plasmon resonance and prevented formation of perforin pores in target cells despite effective target cell engagement, as determined by calcium influx studies. Mouse PK studies of two analogues showed T_1/2 values of 1.1-1.2 h (dose of 5 mg/kg iv) and MTDs of 60-80 mg/kg (ip).

Full text of DOI:10.1021/jm401604x

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Article
Exploration of a Series of 5-Arylidene-2-thioxoimidazolidin-4-
ones as Inhibitors of the Cytolytic Protein Perforin
Julie A Spicer, Gersande Lena, Dani M Lyons, Kristiina Maria Huttunen, Christian Miller, Patrick
D. O'Connor, Matthew Bull, Nuala Helsby, Stephen Jamieson, William Alexander Denny, Annette
Ciccone, Kylie Browne, Jamie Lopez, Jesse Rudd-Schmidt, Ilia Voskoboinik, and Joeseph Trapani
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Nov 2013
Downloaded from http://pubs.acs.org on November 18, 2013
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Exploration of a Series of 5ꢀArylideneꢀ2ꢀ
thioxoimidazolidinꢀ4ꢀones as Inhibitors of the
Cytolytic Protein Perforin
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Julie A. Spicer , Gersande Lena , Dani M. Lyons , Kristiina M. Huttunen , Christian K.
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†,¶
†,¶
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Miller , Patrick D. O’Connor , Matthew Bull , Nuala Helsby , Stephen M. F. Jamieson ,
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,¶
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William A. Denny , Annette Ciccone , Kylie A. Browne , Jamie A. Lopez , Jesse Rudd-Schmidt ,
‡
‡,#
Ilia Voskoboinik , Joseph A. Trapani
†
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The
¶
University of Auckland, Private Bag 92019, Auckland 1142, New Zealand, Maurice Wilkins
Centre for Molecular Biodiscovery, A New Zealand Centre for Research Excellence, Auckland,
§
New Zealand, School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland,
‡
P.O. Box 1627, FIꢀ70211 Kuopio, Finland, Cancer Immunology Program, Peter MacCallum
#
Cancer Centre, St Andrew’s Place, East Melbourne, Victoria 3002, Australia, and Sir Peter
MacCallum Department of Oncology, The University of Melbourne, 3052 Australia.
A series of novel 5ꢀarylideneꢀ2ꢀthioxoimidazolidinꢀ4ꢀones were investigated as inhibitors of the
lymphocyteꢀexpressed poreꢀforming protein perforin. Structureꢀactivity relationships were
explored through variation of an isoindolinone or 3,4ꢀdihydroisoquinolinone subunit on a fixed
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ꢀthioxoimidazolidinꢀ4ꢀone/thiophene core. The ability of the resulting compounds to inhibit the
lytic activity of both isolated perforin protein and perforin delivered in situ by natural killer cells
was determined. A number of compounds showed excellent activity at concentrations that were
nonꢀtoxic to the killer cells and several were a significant improvement on previous classes of
inhibitors, being substantially more potent and soluble. Representative examples showed rapid
and reversible binding to immobilized mouse perforin at low concentrations (≤2.5 ꢁM) by
surface plasmon resonance, and prevented formation of perforin pores in target cells despite
effective target cell engagement, as determined by calcium influx studies. Mouse PK studies of
two analogues showed T1/2 values of 1.1ꢀ1.2 hr (dose of 5 mg/kg i.v.) and MTDs of 60ꢀ80 mg/kg
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Introduction
The key effector cells of the immune system, cytotoxic T lymphocytes (CTL) and natural killer
(NK) cells, eliminate virusꢀinfected and transformed cells principally through the granule
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exocytosis pathway. CTLs and NK cells contain secretory vesicles (granules) which are used
to store various cytotoxic proteins, including a group of proꢀapoptotic serine proteases
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(granzymes), and perforin, a poreꢀforming glycoprotein. Stable conjugation with a target cell
results in exocytic delivery of the granule contents into the immune synapse where perforin
facilitates entry of the granzymes into the target cell, triggering various apoptotic death
mechanisms.
The crystal structure of monomeric murine perforin has been determined to 2.75 Å resolution
and reveals a bent and twisted fourꢀstranded βꢀsheet MACPF domain flanked by two clusters of
αꢀhelices, an epidermal growth factor (EGF) domain, and a Cꢀterminal C2 domain which
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mediates initial, calciumꢀdependent membraneꢀbinding. The mechanism of pore formation has
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also been elucidated; upon exposure of perforin to the neutral, calciumꢀrich extraꢀcellular
environment of the immune synapse, deprotonation of key aspartate residues and subsequent
coordination of calcium takes place. This process triggers conformational changes which result
in the assembly of highly ordered oligomers of 19ꢀ24 subunits that form a pore with an internal
diameter of 130ꢀ200 Å. Site directed mutagenesis has established that direct ionic interactions
between the opposing faces of adjacent perforin monomers (Arg213 and Glu343 in particular)
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serve to assist pore selfꢀassembly and stabilisation of the resulting pore.
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Perforin is encoded on a singleꢀcopy gene in both mice and humans, and while many of the
granule components possess at least some degree of redundancy, perforin is absolutely essential
for protective immunosurveillance. Perforin knockꢀout mice show a reduced ability to reject
many tumor xenografts and when backcrossed with nonꢀobese diabetic mice, the incidence of
spontaneous diabetes is reduced from 77% in a perforin +/+ control to 16% in perforinꢀdeficient
mice, with onset of disease markedly delayed. This is because perforin is critical to deliver an
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autoimmune ‘lethal hit’ against the insulinꢀproducing pancreatic beta cells. Perforinꢀdeficient
mice also demonstrate increased susceptibility and failure to clear many viruses and other
intracellular pathogens, and highly aggressive disseminated B cell lymphomas occur in the
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majority of animals over the age of 12 months. In humans, complete loss of perforin function
results in familial hemophagocytic lymphohistiocytosis (FHL) syndrome, a severe
immunoregulatory disorder usually diagnosed in infancy and characterised by severe anemia and
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hepatosplenomegaly, fever, and thrombocytopenia.
There is abundant evidence to implicate inappropriate perforin activity in a number of human
pathologies, including cerebral malaria, insulinꢀdependent diabetes, juvenile idiopathic arthritis
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and postꢀviral myocarditis,
as well as therapyꢀinduced conditions such as allograft rejection
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and graftꢀversusꢀhost disease.
Immunosuppressive agents used to treat these diseases are
generally associated with a wide range of sideꢀeffects, many of which arise from the unintended
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impact of the drug treatment on nonꢀtherapeutic targets.
killer lymphocytes’, a selective inhibitor should result in fewer offꢀtarget effects, meaning a
small molecule inhibitor of perforin is of great interest as a new class of highly specific
As perforin is expressed only by
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immunosuppressive agents.
In our initial work in this area, we explored two series of compounds (1 and 2; Figure 1) which
showed appreciable perforinꢀinhibitory activity but possessed limitations such as poor aqueous
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solubility or reduced activity in serum, precluding further development.
We have
substantially overcome these issues in our current series of 5ꢀarylideneꢀ2ꢀthioxoimidazolidinꢀ4ꢀ
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oneꢀbased inhibitors, (3, 4)
and now seek to maximise inhibitory activity and optimise ‘drugꢀ
like’ properties. This class of rhodanineꢀrelated heterocycle has recently become the subject of
considerable debate in the medicinal chemistry community, due to several publications
classifying such subꢀstructures as either “privileged scaffolds” with potentially valuable
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biomolecular binding properties,
or “frequent/promiscuous hitters” and panꢀassay
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interference compounds (PAINS) which should not be pursued further.
While it is clear that
these chemotypes should be carefully monitored in development, recent literature contains many
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examples of selective inhibitors of various target proteins containing such substructures.
In addition, many of these moieties also occur in drugs that are clinically useful, providing
templates with valuable structural features favoring protein binding which may be exploited in
an advantageous way. Nevertheless, our current lead of compound was subjected to, and passed,
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the PAINS filters (Professor Jonathon Baell, personal correspondence) possibly due to the
presence of an extra nitrogen in the core 5ꢀmembered ring.
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Herein we will show that we have identified an exciting new class of inhibitors which
unambiguously target perforin, offering up a novel mode of action and the potential to be further
developed as immunosuppressive agents for the effective treatment of transplant rejection and
selected autoimmune diseases.
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Results and Discussion
Lead compound 3 was selected from a small number of hits which showed reproducible
perforinꢀinhibitory activity in a high throughput screen of approximately 100,000 compounds
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sourced from commercial libraries. Compound 3 can be readily disconnected into three
subunits; a 2ꢀthioxoimidazolidinꢀ4ꢀone (A), a furan (B) and a benzofuranone (C), lending itself
to a study design where one unit can be independently varied in the presence of two other fixed
subunits. We have reported previously our study of the structureꢀactivity relationships (SAR)
26
resulting from variation of the Aꢀ and Bꢀsubunits, and here we focus on the effect of
introducing a wide range of Cꢀsubunits. Since thiophene 4 was one of the more potent
compounds in our initial work, the current study explores the effect of replacing the
benzofuranone Cꢀsubunit on a fixed 2ꢀthioxoimidazolidinꢀ4ꢀone/thiophene scaffold. The
requirement for substitution on the Cꢀsubunit aryl ring is explored, followed by a more detailed
investigation focusing on compounds containing a bicyclic moiety.
Synthesis of Target Compounds. The compounds of Tables 1, 2 and 3 were prepared by
heating a range of substituted aldehydes with 2ꢀthioxoimidazolidinꢀ4ꢀone in acetic acid in the
presence of βꢀalanine (Schemes 1ꢀ5). As a consequence, the majority of the chemistry which is
described here involves the preparation of key aldehyde intermediates. For the compounds of
Table 1, a small number of protected aldehydes (6ꢀ8) were prepared by Suzuki reaction of 2ꢀ(5ꢀ
bromothiophenꢀ2ꢀyl)ꢀ1,3ꢀdioxolane (5) with substituted phenylboronic acids, followed by
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deprotection (Scheme 1). The remaining aldehydes were commercially available, with the
exception of amides 34ꢀ42 which were prepared from carboxylic acid 28 using standard amide
coupling conditions. All the required aldehydes 9ꢀ42 were then converted to final compounds 43ꢀ
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Table 2 contains compounds where the benzofuranone of 3 has been replaced with a variety of
substituted isoindolinones (118ꢀ135, 165, 166) and 3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀones (167ꢀ
70). The isoindolinone targets were prepared by bromination of methyl ester 76, followed by
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iodides were then employed in a palladiumꢀcatalysed reaction using an AgNO /KF activator
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system, as originally described by Mori et al. Coupling of the iodides with either 2ꢀ(thiophenꢀ2ꢀ
yl)ꢀ1,3ꢀdioxolane or 2ꢀ(dimethoxymethyl)thiophene at the vacant 5ꢀposition of the thiophene
gave the desired protected aldehydes 82ꢀ86. Compound 82 was further modified through
NaH/RX alkylations of the isoindolinone NH to afford 87ꢀ92. The NMe derivative 87 was
alkylated a second time using LDA and methyl iodide to give methylation on the isoindolinone
CH (93) in 65% yield. Deprotections of 82ꢀ93 were carried out under acidic conditions to give
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the required aldehydes 94ꢀ106 which were then reacted with 2ꢀthioxoimidazolidinꢀ4ꢀone to give
final compounds 118ꢀ130 and 135. Likewise 136 (Table 3) was obtained by reaction of aldehyde
82 with imidazolidineꢀ2,4ꢀdione. A small set of aminoꢀsubstituted compounds (131ꢀ134) were
also prepared from the C3 alcohol 101. The aldehyde substituent of 101 was initially protected as
the dimethyl acetal 107 then the alcohol converted to the mesylate 108 using mesyl chloride and
TEA in THF. The mesylate was exchanged for iodide by heating with a large excess of NaI in
acetone, then the iodide of 109 displaced with various amines to give intermediates 110ꢀ113.
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Deprotection as described above gave the key aldehydes 114ꢀ117 which were reacted with 2ꢀ
thioxoimidazolidinꢀ4ꢀone to give final compounds 131ꢀ134.
Scheme 3 describes the preparation of ‘reverse’ and 5ꢀ and 6ꢀmembered lactams 165ꢀ170
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(
Table 2). The 5ꢀmembered lactamꢀboronates 147 and 148 were prepared from the corresponding
bromides 137 and 138 by Pdꢀcatalysed reaction with bis(pinacolato)diboron. The four 6ꢀ
membered lactamꢀboronates 149ꢀ152 were prepared in the same manner from the bromides 143ꢀ
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procedures.
Each of the boronates were then employed in a Suzuki reaction with bromide 5
to give compounds 153ꢀ158 which were deprotected and reacted with 2ꢀthioxoimidazolidinꢀ4ꢀ
one to afford final compounds 165ꢀ170. For comparison with the isoindolinones, a single indoleꢀ
based compound, 3ꢀacetylindole 178, was also prepared (Scheme 4). 5ꢀBromoindole 171 was
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protected and acetylated according to literature procedures,
followed by displacement of the
benzenesulphonylꢀprotecting group to give the corresponding Nꢀmethyl indole 174. Subsequent
Suzuki coupling, deprotection, and reaction with 2ꢀthioxoimidazolidinꢀ4ꢀone gave target
compound 178.
For the compounds of Table 3, protected bromothiopheneꢀ (179), bromobenzeneꢀ (180, 181),
bromopyridineꢀ (182), bromoindoleꢀ (183, 184) or bromoquinoloneꢀ (185) carboxaldehydes were
reacted with either commercially available 5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀ
yl)isoindolinꢀ1ꢀone or boronate 149 as described above, deprotected to aldehydes 195ꢀ203, and
converted to final products 204ꢀ212 (Scheme 5).
In vitro Activities of the 5ꢀArylideneꢀ2ꢀthioxoimidazolidinꢀ4ꢀones.
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In the first instance, the isobenzofuranꢀ1(3H)ꢀone Cꢀsubunit was replaced with a large range of
substitutedꢀbenzene derivatives (Table 1). Substitution on this ring is clearly required, with
phenyl (43) possessing no detectable activity. Each position was then surveyed with a range of
substituents, and while methyl and methoxy proved inactive (see Supporting Information), a
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variety of electronꢀwithdrawing substituents (halogen, CF , cyano, hydroxyl, carboxyl) did
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produce activity. The results for a series of chloroꢀ (44ꢀ46), fluoroꢀ (47, 48) and CF ꢀ (50, 51)
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compounds show a clear preference for substitution at the 4ꢀposition. Carboxyꢀbased examples
(59ꢀ61) were among the most potent, although in this instance the 3ꢀcarboxamide (60; IC =
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.79 ꢀM) was slightly better than its 4ꢀisomer (61; IC = 1.56 ꢀM). Methylation of the
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carboxamide NH of 61 resulted in a reduction of activity (67, 68), while amideꢀlinked weakly
basic sideꢀchains (69ꢀ71) were generally poor. The corresponding amideꢀlinked hydroxylated
sideꢀchains (72ꢀ75), however, exhibited significant activity (IC = 2.44ꢀ4.35 ꢀM) but were still
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.69 ꢀM), 4ꢀSMe (56; 1.15 ꢀM) and 4ꢀacetyl (57; 1.67 ꢀM). The ‘acyclic’ analogue of 4,
compound 62, showed a 4ꢀfold reduction in activity over its cyclic counterpart (IC = 3.03 ꢀM
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Table 1. Inhibitory Activities of CꢀSubunit Variants
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Inhibition
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Figure 1
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4ꢀCOOMe
3ꢀCONH
4ꢀCONH
2
2
3ꢀMe, 4ꢀCOOMe
4ꢀSO Me
4ꢀNHSO
3ꢀPyridyl
63
64
65
2
Me
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
7
7
7
7
7
7
6
7
8
9
0
1
2
3
4
5
4ꢀPyridyl
4ꢀCONHMe
4ꢀCONMe
4ꢀCOMorpholine
10.06
4.53
6.93
>20
>20
8.13
3.64
4.35
6.50
2.44
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4ꢀCONH(CH
4ꢀCONH(CH
4ꢀCONH(CH
4ꢀCONH(CH
)
2
)
3
)
2
)
3
Morpholine
Morpholine
OH
2
2
2
2
OH
4ꢀCONHCH
4ꢀCONHCH
CH(CH
)OH
2
3
CH(OH)CH
OH
2
2
a
Testing was carried out over a range of concentrations, with the IC50 being equal to the
concentration at which 50% inhibition of the lysis of Jurkat cells by purified recombinant
perforin was observed, as measured by 51Cr release. Values are the average of at least three
independent IC determinations.
5
0
Although the above study (Table 1) produced some potent compounds, none were an
improvement on lead compound 4. An alternative approach where the entire isobenzofuranꢀ
1
(3H)ꢀone unit was replaced with an isoindolinꢀ1ꢀone was therefore implemented (Table 2),
resulting in a series of compounds considered less susceptible to hydrolysis (amide versus ester)
and where the nitrogen atom provides an additional position at which a sideꢀchain can be
appended.
Isoindolinꢀ1ꢀone 118 showed a reduction of activity over lactone 4 (from IC = 0.78 ꢀM to
50
2
.55 ꢀM). Activity was restored through substitution on the nitrogen atom, with both methylꢀ
(
119; IC = 0.51 ꢀM) and ethylꢀ (120; IC = 0.60 ꢀM) substituted compounds more potent than
50
50
the lead compound 4. A more bulky substituent such as isopropyl (123; IC = 4.42 ꢀM) was less
5
0
acceptable, but the acetate 128 and carbamate 129 both showed excellent activity (IC50 = 0.93
and 0.55 ꢀM respectively). In this series it also appears possible to introduce various sideꢀchains
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off the isoindolinone and maintain activity, with the two alcohols 125 and 126, amongst the most
potent compounds of the class (IC s = 0.78 and 0.53 ꢀM). Diol 127 and Oꢀacetate 124 also
5
0
demonstrated good activity, but when a basic amino substituent was introduced in an attempt to
improve solubility, activity was virtually abolished with only morpholine (130, not
reproducible), piperidine 132, and pyrrolidine 134 (IC s = 11.78 and 8.93 ꢀM) showing any
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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
5
0
evidence of activity. Introduction of a methyl substituent on the lactam ring (135), resulted in
poorer activity than the corresponding unsubstituted analogue (135; IC = 3.21 ꢀM vs 119; IC
5
0
50
=
0.51 ꢀM).
Since the 3ꢀcarboxamide 60 was 2ꢀfold more potent than 4ꢀcarboxamide 61 in the substituted
benzene series (Table 1), the effect of reversing the orientation of the lactam ring was
investigated (165). The same result was observed; the IC50 for the compound with the lactam
amide in the 3ꢀposition 165 (relative to the thiophene) was around 2ꢀfold better than the
corresponding lactam amide in the 4ꢀposition (118) (IC50 = 1.38 ꢀM compared to 2.55 ꢀM).
Expansion from a 5ꢀ (118) to a 6ꢀmembered lactam ring (167) also resulted in a significant
increase in activity (from IC = 2.55 to 1.14 ꢀM), and improved a further 2ꢀfold (to 0.64 ꢀM)
5
0
through introduction of the “reverse” 3ꢀlactam amide (168). NꢀMethylation of 165, 167 and 168
to give 166, 169 and 170 respectively did not result in the same increase in activity seen when
118 was alkylated to give the analogous Nꢀmethyl compound 119.
Table 2. Inhibitory Activities of Lactam CꢀSubunits
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Inhibition
of
Cmpd Structure X
R
Jurkat cell lysis:
a
IC (ꢀM)
5
0
b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
118
119
I
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
H
2.55
0.51
0.60
1.52
1.18
4.42
1.17
0.78
0.53
1.26
0.93
0.55
variable
>20
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
I
Me
Et
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
165
166
167
I
I
nPr
nBu
iPr
I
I
I
CH
CH
CH
CH
Ac
CH
CH
CH
OAc
OH
2
2
2
2
2
2
2
I
I
CH OH
2
I
CH(OH)CH OH
2
I
I
C(O)OEt
I
CH
CH
CH
CH
CH
) Me
H
CH
CH
CH
CH
CH
morph
2
2
2
2
2
2
2
2
2
2
I
CH
CH
CH
CH
NMe
2
2
2
2
2
I
piperidine
11.78
I
NMepiperazine >20
I
pyrrolidine
8.93
3.21
1.38
1.89
1.14
0.64
2.33
4.76
I
CH(CH
3
c
II
II
I
CH
CH
2
2
Me
H
(CH
(CH
(CH
(CH
)
2
)
2
)
2
)
2
2
2
2
2
168
169
170
II
I
H
Me
Me
II
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d
1
78
5.30
a
Testing was carried out over a range of concentrations, with the IC50 being equal to the
concentration at which 50% inhibition of the lysis of Jurkat cells by purified recombinant
perforin was observed, as measured by Cr release. Values are the average of at least three
independent IC determinations. Preparation of compounds 118ꢀ135 described in Scheme 2.
5
1
b
5
0
c
d
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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
Preparation of compounds 165ꢀ170 described in Scheme 3. Compound 178 contains a 3ꢀ
acetylindole Cꢀsubunit, and its synthesis is described in Scheme 4.
Compounds 136 and 204ꢀ212 (Table 3) combine those Bꢀsubunits which were previously
2
6
identified as having excellent activity in the isobenzofuranoneꢀbased series, with selected
isoindolinones and 3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀones from the current series. Compound 136
contains a imidazolidineꢀ2,4ꢀdione Aꢀsubunit and is the only example to show an improvement,
being 3ꢀfold better than the analogous isobenzofuranone reported in our initial paper (IC = 0.40
5
0
ꢀM compared to 1.19 ꢀM). All other compounds (204ꢀ212) contain a 2ꢀthioxoimidazolidinꢀ4ꢀ
one Aꢀsubunit and are 1.5ꢀ4ꢀfold poorer than the corresponding isobenzofuranones (see Figure 1,
Supporting Information).
Table 3. Inhibitory Activities of Optimised Aꢀ, Bꢀ, and CꢀSubunits
Inhibition
of
a
Cmpd
X
n
Jurkat cell lysis:
b
IC (ꢀM)
5
0
c
136
204
2,5ꢀthiophene
2,4ꢀthiophene
1,4ꢀbenzene
1,3ꢀbenzene
3,6ꢀpyridine
1
1
1
1
1
0.40
3.00
5.55
3.44
0.63
205
206
207
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
2
2
2
08
09
10
11
12
2,6ꢀ(1Hꢀindole)
2,5ꢀ(1Hꢀindole)
2,6ꢀ(1Hꢀindole)
2,5ꢀ(1Hꢀindole)
1
1
2
2
2
1.58
2.50
1.66
1.45
2.55
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2,6ꢀquinoline
a
b
Structure of the Bꢀsubunit. Testing was carried out over a range of concentrations, with the
IC50 being equal to the concentration at which 50% inhibition of the lysis of Jurkat cells by
purified recombinant perforin was observed, as measured by Cr release. Values are the average
5
1
c
of at least three independent IC determinations. The 2ꢀthioxoimidazolidinꢀ4ꢀone Aꢀsubunit is
5
0
replaced with a imidazolidineꢀ2,4ꢀdione.
Those compounds which showed good inhibition of the purified protein were then also
evaluated for their ability to inhibit perforin produced by an intact NK cell line (KHYGꢀ1; see
experimental for details); this assay is a better model for in vivo studies as the perforin is
delivered by a functional NK cell. To confirm that inhibition of NK cell function was due to
blocking the action of perforin and not nonꢀspecific toxicity against the effector cell, viability
o
was also measured 24 h after the NK cells were exposed to the compounds (4 h, 37 C), then
washed and reꢀplated in culture. Elimination of any nonꢀspecific toxicity is important because
CTLs and NK cells play a key role in the overall immunological response, meaning that any
potential perforinꢀtargeted treatment must allow rapid recovery of these cytotoxic effector cells.
For the purposes of this study, compounds were classified as toxic if NK cell viability fell below
70% at 24 h.
The data contained in Table 4 was used to identify compounds suitable for advancement into in
vivo pharmacokinetic studies, as a prelude to in vivo efficacy testing. In the absence of serum, the
majority of compounds possessed similar or improved inhibitory activity compared to previous
lead 4, notably 118, 135 and 167 which all showed excellent activity in this more demanding
2
6
system (56%, 90%, 83% compared to 53%). As reported with previous series, a number of
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compounds were adversely affected in the presence of serum, precluding their further
consideration (125, 126, 129, 168, 199). The solubility of all compounds was substantially
improved by their conversion to sodium salts, resulting in an enhanced drug profile, although
some examples (118, 119, 129, 136, 168) still proved too insoluble in this form for
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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
administration in vivo. Compounds 119 and 167 also showed some evidence of toxicity toward
the NK cells (28% and 38% survival at 20 ꢀM concentration respectively). The compound with
the best overall profile for an in vivo candidate was 135; combining superior activity in the
presence and absence of serum (90% and 67% respectively), with good viability (74%) and
aqueous solubility (1408 ꢀg/mL). In addition, when 167 was tested at a lower concentration of 5
ꢀM, the resultant lack of NK toxicity suggested a possible therapeutic window, meaning this
compound was also selected as an in vivo candidate.
Table 4. Additional Testing on Selected Compounds
KHYGꢀ1 inhibition
Jurkat
KHYGꢀ1 Solubility
(
% at 20 ꢁM)
Cmpd IC50
(ꢁM)
viability
(ꢀg/mL)
10%
c
a
no serum
b
(%)
[sodium salt]
serum
4
1
1
1
1
1
0.78
2.55
0.51
0.78
0.53
0.55
3.21
0.40
53±2.6
56±7.2
42±5.5
43±5.8
48±7.9
46±5.9
90±4.0
49±9.8
38.9±3.5 88±3.0
23.0 [43.0]
0.54 [140]
0.13 [151]
43.0 [3429]
1.78 [10187]
0.18 [397]
1.32 [1408]
11.6 [136]
18
19
25
26
29
55±6.5
37±2.4
24±3.4
17±3.0
35±9.5
67±6.1
50±7.1
95±3.5
28±1.5
81±0.5
90±3.0
93±4.1
74±8.3
93±3.0
135
136
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
1
2
67
67
68
07
1.17
ꢀ
83±4.6
51±4.2
51±4.0
43±5.4
81±5.3
34±6.4
23±7.1
13±3.8
38±7.8
82±3.6
87±4.1
92±2.6
0.53 [1573]
ꢀ
d
0.64
0.63
0.13 [37.2]
e
0.59 [ꢀ]
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Inhibition by compounds (20 ꢀM) of the perforinꢀinduced lysis of K562 target cells when coꢀ
incubated with KHYGꢀ1 human NK cells (see Experimental Section). Percent inhibition
calculated compared to untreated control. As for a, but in the presence of 10% mouse serum.
Viability of KHYGꢀ1 NK cells after 24 h by Trypan blue exclusion assay (see Experimental
Section). All results are the average of at least three separate determinations ±SEM. Compound
67 was also tested at 5 ꢀM concentration to test for activity under less toxic conditions.
Insufficient material to prepare sodium salt.
b
c
d
1
e
In vivo Pharmacokinetics of Selected Compounds. Compounds 135 and 167 were evaluated
for plasma pharmacokinetics in male CDꢀ1 mice. Blood samples were collected at multiple timeꢀ
points after dosing at 5 mg/kg in a solution of 20% hydroxypropylꢀβꢀcyclodextrin by intravenous
injection. Both compounds were determined to have acceptable drug exposures (AUC = 1151
h.ng/mL and 955 h.ng/mL, respectively), despite high clearance and volume of distribution
values (Table 5). On this basis, 135 and 167 were progressed to maximum tolerated dose (MTD)
studies by single or multiple doses (intraperitoneally) to establish an appropriate level for
efficacy studies. Both compounds were found to be well tolerated after a single dose of 80 and
60 mg/kg respectively, while when administered twice daily over 3 days, 60 and 40 mg/kg were
found to be suitable for multiple dosing.
a
Table 5. in vivo Pharmacokinetics of Compounds 135 and 167
T1/2
(h)
Cmax
AUC0ꢀ∞
V
Cl
Cmpd
(ng/mL) (h.ng/mL) (mL/kg)
(mL/h/kg)
4346
1
1
35
67
1.15
1.30
4488
4513
1151
955
1478
2750
5237
a
Pharmacokinetic parameters derived from the plasmaꢀconcentration time profiles for each
compound following a 5 mg/kg i.v. dose. The results were processed using a bolus i.v. dose, twoꢀ
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compartmental model with Phoenix WinNonlin 6.2 (Pharsight Corporation, St. Louis, MO). The
derived parameters are: maximum plasma concentration (Cmax), the area under the curve (AUC),
plasma halfꢀlife (T ), volume of distribution (V) and clearance (Cl).
1/2
Binding Affinity of Inhibitors to Perforin Protein. While several series of potent inhibitors
2
3ꢀ26
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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
have now been identified,
little is known about the mechanism by which the cytolytic
activity of perforin action is blocked. In order to find out more about the nature and degree of
inhibitor binding, recombinant purified mouse perforin was immobilized on a surface plasmon
resonance (SPR) Sꢀseries CM5 sensor chip using standard amide coupling methodology, then
solutions of in vivo candidates 135 and 167 in buffer passed over the covalentlyꢀbound protein,
and relative binding affinities determined using a Biacore T200 SPR machine.
Inhibitors 135 and 167 were found to bind reversibly to the immobilized perforin and during
injection a steadyꢀstate response was achieved within seconds (Figures 2 and 3, Supporting
Information). At the end of the injection the compounds dissociated rapidly with an almost
immediate return to baseline resonance response. Any nonꢀspecific binding to the sensor chip
surface was accounted for through the response in the reference flow cell. Due to the relative
insolubility of the compounds in the running buffer, low analyte concentrations were required
(
≤2.5 ꢁM) and the relative resonance responses at steady state were typically below 10 response
units (RU). However, although the absolute response values were low, it is clear that 135 and
67 can bind to perforin even at low concentrations. This data demonstrates for the first time that
1
these inhibitors possess the ability to bind reversibly to perforin monomers. Prior to this
experiment it was also not known whether immobilization of perforin monomers to the sensor
chip surface via (random) amide couplings could render potential binding sites for inhibitors
inaccessible. Combined, these findings show that SPR biosensor analysis can be a useful
technique to determine perforin protein–drug binding interactions.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Investigation of the Mechanism of Perforin Inhibition. In order to investigate whether 167
inhibited perforin directly in the context of the physiological immune synapse, we also used realꢀ
time microscopy to analyze the functional interaction between primary human natural killer cells
and their cognate model targets, HeLa cells. When the killer cell engages with its target cell, a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
+
cascade of downstream signaling events leads to the influx of extracellular Ca (as detected by
4
2
2+
the calcium ionophore Fluoꢀ4). As shown recently, Ca influx directly precedes degranulation
and the release of perforin into the synaptic cleft. Perforin then forms pores in the target cell
membrane, which can be detected as an influx of extracellular propidium iodide (PI), a dye that
42
gains fluorescence upon binding to the cytosolic RNA. Calcium influx into the killer cell and
PI uptake by the target cell thus serve as indicators of a functional immune synapse. In the
DMSOꢀtreated control cells, every natural killer cell that engaged with its target cell (n=50 cells),
delivered functional perforin to the target (Figure 2). By contrast, in the presence of 20 ꢁM 167,
only 60% of killer cells delivered functional perforin to the target. Formation of the perforin pore
was blocked in the remaining 40% of synapses, despite effective target cell engagement (Figure
2). These data demonstrate that 167 directly inhibits perforinꢀinduced lysis through reduction of
cell membrane binding and/or prevention of transmembrane pore formation, thus preventing
target cell death.
Conclusions
The current study has resulted in further optimisation of a novel new series of smallꢀmolecule
2
6
inhibitors of the poreꢀforming protein perforin. By building on our previous studies, we have
designed compounds which possess enhanced drugꢀlike properties compared to earlier structures.
We also report new mechanistic evidence which reveals a specificity for the granule exocytosis
pathway, of which perforin is an integral component.
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Structureꢀactivity relationships for variation of the Cꢀsubunit on a 2ꢀthioxoimidazolidinꢀ4ꢀ
one/thiophene scaffold showed a need for substitution, especially at the 4ꢀposition, for simple
substitutedꢀbenzene derivatives (Table 1). In this series the 3ꢀ and 4ꢀcarboxamides 60 and 61
proved most potent, although this was limited to primary amides, as the introduction of Nꢀ
substitution and extended hydroxyalkylꢀ or aminoalkylꢀ sidechains (67ꢀ75) resulted in a loss of
activity. The ‘acyclic’ analogue of the lead compound (62) also showed an almost 4ꢀfold
reduction in activity, suggesting retention of a bicyclic Cꢀsubunit to be the best approach. The
isobenzofuranone of 4 was therefore replaced with a variety of isomeric isoindolinones and 3,4ꢀ
dihydroisoquinolinꢀ1(2H)ꢀones. NꢀSubstitution on the isoindolinone nitrogen was beneficial,
with methylꢀ (119), ethylꢀ (120), hydroxyethylꢀ (125) and hydroxypropylꢀ (126) substituents all
demonstrating excellent activity (IC s = 0.51, 0.60, 0.78, 0.53 ꢀM respectively). Comparable
10
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15
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54
55
56
57
58
59
60
5
0
substitution on the 3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀone nitrogen did not improve activity
(comparing 167/169 and 168/170 pairings), although these compounds (167, 168) still showed
potent inhibitory activity in their own right. Lastly, a series of hybrid compounds were prepared
2
6
(Table 3); combining Bꢀsubunits which were previously identified as conferring good activity,
with the above optimized Cꢀsubunits. In this instance, the isoindolinones were all poorer than the
corresponding isobenzofuranones (Figure 1, Supporting Information).
A smaller subset of active compounds were then screened for their ability to inhibit perforinꢀ
dependent lysis induced by whole NK cells, resulting in the selection of 135 and 167 as suitable
candidates for in vivo studies. Problems observed previously, such as poor aqueous solubility,
inactivity in serum, and toxicity have been substantially overcome in the present series.
The mechanism of perforin inhibition by these compounds has also been investigated further,
and for the first time, compounds with in vitro inhibitory activity have been shown to reversibly
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bind perforin by SPR detection. Realꢀtime imaging of natural killerꢀtarget cell interaction
demonstrates that the compounds block perforin within the immunological synapse and prevent
target cell death, but have no effect on the stability of immune synapse formation between the
two cells. These inhibitors almost certainly target perforin after its release into the immune
synapse, consistent with the fact that the original screen through which the inhibitors were
identified featured the use of purified perforin protein rather than perforin delivered by an intact
killer cell.
0
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Together, the data we have amassed demonstrate that this series of compounds are selective
inhibitors of perforin, and not nonꢀspecific promiscuous binders. We have previously shown that
these compounds do not inhibit the mechanisticallyꢀrelated poreꢀforming protein pneumolysin
and that we observe a wide range of activities (0.36ꢀ>20 ꢀM) against both isolated perforin and
2
6
perforin produced by whole NK cells. Furthermore, the membraneꢀpiercing terminal
components of the mammalian membrane attack complex of complement (MAC), which share
significant structural similarity to perforin and alternative, receptorꢀmediated proꢀapoptoic
pathways used by cytotoxic lymphocytes, are not inhibited (data not shown). These observations
are recapitulated in the current report by the clear SAR demonstrated by the Cꢀsubunit (IC s =
5
0
0.51ꢀ>20 ꢀM), irrespective of the presence of a 5ꢀarylideneꢀ2ꢀthioxoimidazolidinꢀ4ꢀone as the Aꢀ
subunit. A range of activities was also observed against perforin released by intact NK cells, a far
more rigorous model which requires the compound to block perforin released into the
4
3
immunological synapse that transiently forms between the killer cell and its target. Our live cell
imaging analyses clearly show for the first time that perforin operating within a functional
immune synapse, an intricate microꢀenvironment comprising many proteins taking part in
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various complex signaling pathways, can be selectively targeted to inhibit perforinꢀdependent
apoptosis of the target cell.
In summary, we have taken significant steps toward elucidating the role of perforin in the
granule exocytosis pathway, using smallꢀmolecule inhibitors to investigate binding affinity and
probe the mechanism of membrane interaction and subsequent pore formation. We have found
compounds 135 and 167 to be both potent and soluble inhibitors of perforin in vitro. These
compounds also possess appropriate in vivo pharmacokinetic characteristics and toxicity profiles,
for their selection as in vivo efficacy candidates in our pursuit of a novel class of perforin
inhibitors suitable for the effective treatment of autoimmuneꢀbased diseases and transplant
rejection.
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59
60
Experimental Section
Medicinal Chemistry. Analyses were performed by the Microchemical Laboratory,
University of Otago, Dunedin, NZ. Melting points were determined using an Electrothermal
Model 9200 and are as read. NMR spectra were measured on a Bruker Avance 400 spectrometer
and referenced to Me Si. Mass spectra were recorded either on a Varian VG 7070 spectrometer
4
at nominal 5000 resolution or a Finnigan MAT 900Q spectrometer. All final compound purities
were determined to be >95% by HPLC.
Representative general procedures are given below, full experimental for all remaining final
compounds and intermediates is given in Supporting Information.
General Procedure A: (4ꢀ(5ꢀ(1,3ꢀDioxolanꢀ2ꢀyl)thiophenꢀ2ꢀyl)phenyl)methanol (6)
(
Scheme 1; R = CH OH). 5ꢀBromoꢀ2ꢀthiophenecarboxaldehyde was protected as the cyclic
2
4
4
acetal 5 according to a literature procedure. The acetal 5 (278 mg, 1.18 mmol) was then
dissolved in toluene (12 mL), to which was added
a
suspension of 4ꢀ
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2
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2
2
2
2
2
2
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3
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3
3
3
3
3
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5
5
6
(
hydroxymethyl)benzeneboronic acid (150 mg, 0.99 mmol) in EtOH (4 mL). A solution of 2 M
Na CO (3 mL) and Pd(dppf)Cl (40 mg, 0.05 mmol) were added and the entire mixture was
2
3
2
heated at reflux under nitrogen for 2 h. Upon cooling, all solvents were removed under reduced
pressure and the resulting residue was partitioned between water (50 mL) and CH Cl (50 mL).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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7
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9
0
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6
7
8
9
0
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2
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4
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6
7
8
9
0
2
2
Two further CH Cl (50 mL) extractions were performed, then the combined organic fractions
2
2
dried (Na SO ), filtered, and the solvent removed under reduced pressure to afford a residue
2
4
which was purified by flash column chromatography on silica gel (20% EtOAc/hexanes as
1
eluant) to give 6 as a pale yellow solid (171 mg, 66%). H NMR [400 MHz, (CD ) SO] δ 7.60
3
2
(d, J = 8.3 Hz, 2 H), 7.36 (d, J = 3.6 Hz, 1 H), 7.35 (d, J = 8.4 Hz, 2 H), 7.20 (d, J = 3.8 Hz, 1 H),
6.04 (s, 1 H), 5.19 (t, J = 5.6 Hz, 1 H), 4.51 (d, J = 5.5 Hz, 2 H), 4.10ꢀ4.07 (m, 2 H), 3.93ꢀ4.00
+
+
(m, 2 H). LRMS (APCI ) calcd for C H O S 263 (MH ), found 263. This material contained ca
14 15 3
5
% of deprotected aldehyde which was carried into the next step.
General Procedure B: 5ꢀ(4ꢀ(Hydroxymethyl)phenyl)thiopheneꢀ2ꢀcarbaldehyde (24)
(Scheme 1; R = CH OH). Compound 6 (171 mg, 0.65 mmol) was dissolved in acetone (10 mL),
2
to which was added 1 M HCl (2 mL). This mixture was stirred at RT for 6 h, then concentrated
under reduced pressure to afford a pale yellow suspension which was extracted into CH Cl2
2
(2x50 mL). The combined organic fractions were evaporated down to give 24 as a yellow solid
1
(142 mg, 100%). H NMR [400 MHz, (CD ) SO] δ 9.90 (s, 1 H), 8.03 (d, J = 3.9 Hz, 1 H), 7.76
3
2
(d, J = 8.3 Hz, 2 H), 7.72 (d, J = 4.0 Hz, 1 H), 7.42 (d, J = 8.4 Hz, 2 H), 5.26 (t, J = 5.7 Hz, 1 H),
+
+
4
.54 (d, J = 5.6 Hz, 2 H). LRMS (APCI ) calcd for C H O S 219 (MH ), found 219.
12 11 2
General
Procedure
C:
(E,Z)ꢀ4ꢀ(5ꢀ((5ꢀOxoꢀ2ꢀthioxoimidazolidinꢀ4ꢀ
ylidene)methyl)thiophenꢀ2ꢀyl)benzyl acetate (58) (Scheme 1; R = CH OAc). 5ꢀ(4ꢀ
2
(Hydroxymethyl)phenyl)thiopheneꢀ2ꢀcarbaldehyde (24) (140 mg, 0.64 mmol), 2ꢀ
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thioxoimidazolidinꢀ4ꢀone (89 mg, 0.77 mmol) and βꢀalanine (69 mg, 0.77 mmol) were
suspended in AcOH (5 mL) and the mixture heated at reflux for 15 h. Upon cooling an orange
solid crystallized out of solution which was collected by filtration and purified by flash column
chromatography on silica gel (20% THF/hexanes), gave 58 as an orange solid (18%), mp
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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
o
1
(
THF/nꢀpentane) 230ꢀ234 C. H NMR [400 MHz, (CD ) SO] δ 12.37 (s, 1 H), 11.95 (s, 1 H),
3
2
7
.83 (d, J = 4.0 Hz, 1 H), 7.72 (d, J = 8.3 Hz, 2 H), 7.65 (d, J = 4.0 Hz, 1 H), 7.44 (d, J = 8.4 Hz,
+
2 H), 6.63 (s, 1 H), 5.10 (s, 2 H), 2.08 (s, 3 H). LRMS (APCI ) calcd for C H N O S 359
1
7
15
2
3 2
+
(
MH ), found 359. Anal. (C H N O S ) C, H, N.
17 14 2 3 2
General Procedure D: 4ꢀ(5ꢀFormylthiophenꢀ2ꢀyl)ꢀNꢀmethylbenzamide (34) (Scheme 1; R
= CONR R , where NR R = NHMe). 4ꢀ(5ꢀFormylthiophenꢀ2ꢀyl)benzoic acid (28) (85 mg,
1
2
1
2
0
.37 mmol) was dissolved in THF (5 mL), to which was added pyridine (289 mg, 3.67 mmol),
followed by pentafluorophenyltrifluoroacetate (PFPꢀTFA). This mixture was stirred at RT for 15
h, then all solvent removed under reduced pressure to afford a crude solid which was dissolved
in EtOAc (25 mL) and washed with 1 M HCl (2x25 mL), water (25 mL), sat. NaHCO (25 mL)
3
and brine (25 mL). The organic layer was then dried (Na SO ), filtered, and the solvent removed
2
4
under reduced pressure to give the crude intermediate ester (146 mg, 0.37 mmol) which was
immediately dissolved in THF (5 mL) and treated with 2 M methylamine in MeOH (1.85 mL,
3.70 mmol). After 1 h stirring at RT, 1 M HCl (5 mL) was added to hydrolyse the undesired
imine. The reaction mixture was then diluted with water (20 mL) and extracted with EtOAc
(
2x20 mL) and then the combined organic fractions dried (Na SO ), filtered and evaporated
2 4
1
down to give a crude solid. Trituration with Et O gave 34 as a pale yellow solid (80%). H NMR
2
[
400 MHz, (CD ) SO] δ 9.93 (s, 1 H), 8.47ꢀ8.53 (br m, 1 H), 8.07 (d, J = 4.0 Hz, 1 H), 7.93 (d, J
3
2
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2
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=
8.7 Hz, 2 H), 7.89 (d, J = 8.7 Hz, 2 H), 7.84 (d, J = 4.0 Hz, 1 H), 2.80 (d, J = 4.5 Hz, 3 H).
+
+
LRMS (APCI ) calcd for C H NO S 246 (MH ), found 246.
13 12
2
(E,Z)ꢀNꢀMethylꢀ4ꢀ(5ꢀ((5ꢀoxoꢀ2ꢀthioxoimidazolidinꢀ4ꢀylidene)methyl)thiophenꢀ2ꢀ
0
1
2
3
4
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7
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9
0
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2
3
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0
yl)benzamide (67) (Scheme 1; R = CONR R , where NR R = NHMe). Reaction of 34 with 2ꢀ
1
2
1
2
thiohydantoin according to general procedure C gave 67 as a dark orange solid (76%), mp
o
1
(AcOH) >300 C. H NMR [400 MHz, (CD ) SO] δ 12.37 (br s, 1 H), 11.97 (br s, 1 H), 8.47 (br
3
2
q, J = 4.5 Hz, 1 H), 7.90 (d, J = 8.5 Hz, 2 H), 7.84 (d, J = 4.0 Hz, 1 H), 7.80 (d, J = 8.5 Hz, 2 H),
ꢀ
7.74 (d, J = 4.0 Hz, 1 H), 2.79 (d, J = 4.5 Hz, 3 H). LRMS (APCI ) calcd for C H N O S 342
16
12
3
2 2
(MꢀH), found 342. Anal. (C H N O S ) C, H, N.
16 13 3 2 2
General Procedure E: 5ꢀ(5ꢀ(1,3ꢀDioxolanꢀ2ꢀyl)thiophenꢀ2ꢀyl)isoindolinꢀ1ꢀone (82)
(Scheme 2; R = H, R R = dioxolane). 2ꢀThiophenecarboxaldehyde was protected as the cyclic
1
2
3
4
4
acetal according to a literature procedure. 2ꢀ(Thiophenꢀ2ꢀyl)ꢀ1,3ꢀdioxolane was then reacted
with 5ꢀiodoisobenzofuranꢀ1(3H)ꢀone (77) using a procedure adapted from a literature
37
reference. The iodide (1.23 g, 4.75 mmol), PdCl (PPh ) (333 mg, 0.48 mmol) and KF (1.10 g,
2
3 2
19.0 mmol) were weighed into a flask and dissolved in DMSO (35 mL). The mixture was placed
under an atmosphere of N , 2ꢀ(thiophenꢀ2ꢀyl)ꢀ1,3ꢀdioxolane (2.22 g, 14.2 mmol) and AgNO
2
3
o
(807 mg, 4.75 mmol) added, then the resulting suspension stirred for 0.5 h at 100 C. Further
portions of AgNO (3x807 mg) were added at 0.5 h intervals, to give a total reaction time of 2 h.
3
Upon cooling, the mixture was filtered through a plug of celite which was washed well with
CHCl . The resulting CHCl solution (ca 200 mL) was washed with water (3x100 mL), dried
3
3
(Na SO ), filtered, and the solvent removed under reduced pressure to give a crude solid which
2
4
was purified by flash column chromatography on silica gel (5% acetone/CH Cl as eluant).
2
2
1
Trituation with Et O gave 82 as a pale yellow solid (61%). H NMR [400 MHz, (CD ) SO] δ
2
3 2
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.53 (br s, 1 H), 7.85 (d, J = 0.6 Hz, 1 H), 7.76 (dd, J = 7.9, 1.6 Hz, 1 H), 7.68 (d, J = 7.9 Hz, 1
H), 7.53 (d, J = 3.7 Hz, 1 H), 7.25 (d, J = 3.6 Hz, 1 H), 6.07 (s, 1 H), 4.41 (s, 2 H), 4.02ꢀ4.09 (m,
+
+
2
H), 3.94ꢀ4.01 (m, 2 H). LRMS (APCI ) calcd for C H NO S 288 (MH ), found 288.
15 14 3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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33
34
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41
42
43
44
45
46
47
48
49
50
51
52
53
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59
60
General Procedure F: 5ꢀ(5ꢀ(1,3ꢀDioxolanꢀ2ꢀyl)thiophenꢀ2ꢀyl)ꢀ2ꢀmethylisoindolinꢀ1ꢀone
(87) (Scheme 2; R1 = Me, R R = dioxolane). 5ꢀ(5ꢀ(1,3ꢀDioxolanꢀ2ꢀyl)thiophenꢀ2ꢀ
2
3
yl)isoindolinꢀ1ꢀone (82) (150 mg, 0.52 mmol) was dissolved in dry DMF (10 mL) and the
o
resulting solution cooled to 0 C (ice/water). NaH (23 mg, 0.58 mmol) was added and the mixture
stirred for 0.5 h at this temperature. Methyl iodide (82 mg, 0.58 mmol) was added dropwise to
o
the solution of anion and the reaction stirred for 0.25 h at 0 C, followed by 1 h at RT. The
mixture was diluted with water (50 mL) and extracted with EtOAc (3x50 mL). The combined
EtOAc fractions were washed with water (3x50 mL), brine (50 mL) and dried (Na SO ).
2
4
Filtration and removal of the solvent under reduced pressure gave a crude yellow solid which
was purified by flash column chromatography on silica gel (5% acetone/CH Cl as eluant) to
2
2
1
give 87 as a yellow solid (82%). H NMR [400 MHz, (CD ) SO] δ 7.87 (d, J = 0.7 Hz, 1 H),
3
2
7.76 (dd, J = 7.9, 1.6 Hz, 1 H), 7.67 (d, J = 8.0 Hz, 1 H), 7.53 (d, J = 3.8 Hz, 1 H), 7.25 (d, J =
3.8 Hz, 1 H), 6.07 (s, 1 H), 4.49 (s, 2 H), 4.02ꢀ4.09 (m, 2 H), 3.94ꢀ3.99 (m, 2 H), 3.08 (s, 3 H).
+
+
LRMS (APCI ) calcd for C H NO S 302 (MH ), found 302.
16
16
3
5
ꢀ(2ꢀMethylꢀ1ꢀoxoisoindolinꢀ5ꢀyl)thiopheneꢀ2ꢀcarbaldehyde (95) (Scheme 2; R = Me, R
1
5
1
=
H). Deprotection of 87 according to general procedure B gave 95 as a yellow solid (89%). H
NMR [400 MHz, (CD ) SO] δ 9.94 (s, 1 H), 8.08 (d, J = 4.0 Hz, 1 H), 8.04 (d, J = 0.8 Hz, 1 H),
3
2
7.91 (dd, J = 7.9, 1.6 Hz, 1 H), 7.86 (d, J = 4.0 Hz, 1 H), 7.74 (d, J = 7.9 Hz, 1 H), 4.53 (s, 2 H),
+
+
3
.09 (s, 3 H). LRMS (APCI ) calcd for C H NO S 258 (MH ), found 258.
14 12 2
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2
2
2
2
2
2
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3
3
3
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5
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5
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6
(
E,Z)ꢀ2ꢀMethylꢀ5ꢀ(5ꢀ((5ꢀoxoꢀ2ꢀthioxoimidazolidinꢀ4ꢀylidene)methyl)thiophenꢀ2ꢀ
yl)isoindolinꢀ1ꢀone (119) (Scheme 2; R = Me, R = H). Reaction of 95 with 2ꢀthiohydantoin
1
5
o
1
according to general procedure C gave 119 as an orange solid (92%), mp (AcOH) >300 C. H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NMR [400 MHz, (CD ) SO] Observe E- and Z- isomers separately. δ 11.90ꢀ12.38 (m, 2 H), 7.92
3
2
(br d, J = 0.7 Hz, 1 H), 7.82 (br d, J = 4.0 Hz, 0.8 H), 7.80ꢀ7.84 (m, 1 H), 7.75ꢀ7.78 (m, 1 H),
ꢀ
7
.67ꢀ7.73 (m, 1.2 H), 6.82 (s, 0.2 H), 6.65 (s, 0.8 H), 4.51 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI )
calcd for C H N O S 354 (MꢀH), found 354.
17 12
3
2 2
General Procedure G: 2ꢀ(2ꢀHydroxyethyl)ꢀ5ꢀiodoisoindolinꢀ1ꢀone (78) (Scheme 2; R =
1
45
CH CH OH). Methyl 4ꢀiodoꢀ2ꢀmethylbenzoate (76) (860 mg, 3.12 mmol) was dissolved in
2
2
benzene (10 mL), to which was added Nꢀbromosuccinimide (NBS) (666 mg, 3.74 mmol) and
,2’ꢀazobis(2ꢀmethylpropionitrile) (51 mg, 0.31 mmol). This mixture was heated at reflux
2
temperature for 6 h, the reaction allowed to cool, filtered and the resulting filtrate diluted with
Et O (100 mL). This solution was washed with sat. sodium metabisufite (50 mL) and brine (50
2
mL), dried (Na SO ) and filtered. Removal of the solvent under reduced pressure gave a
2
4
transparent oil which was purified by filtration through a plug of silica gel (5% EtOAc/hexanes
as eluant). The resulting oil solidified under high vacuum to afford a white solid (1.08 g), shown
1
to be 93% desired monoꢀbromide and 7% unreacted starting material by H NMR. This solid was
used directly in the next step:
The bromide (1.08 mg, 3.04 mmol) was dissolved in THF (10 mL) and 2ꢀaminoethanol (928
mg, 15.2 mmol) added. The reaction mixture was stirred at RT for 15 h, then all solvent removed
under reduced pressure. The resulting residue was purified by flash column chromatography on
silica gel (10% acetone/CH Cl as eluant) to give 78 as a crystalline white solid (562 mg, 59%
2
2
1
over 2 steps). H NMR [400 MHz, (CD ) SO] δ 8.02 (d, J = 0.7 Hz, 1 H), 7.84 (dd, J = 7.9, 1.4
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Hz, 1 H), 7.45 (d, J = 7.9 Hz, 1 H), 4.82 (t, J = 5.4 Hz, 1 H), 4.52 (s, 2 H), 3.58ꢀ3.64 (m, 2 H),
+
+
3
.32ꢀ3.57 (m, 2 H). LRMS (APCI ) calcd for C H INO 304 (MH ), found 304.
10 11 2
General
Procedure
H:
5ꢀ(2ꢀ(2ꢀHydroxyethyl)ꢀ1ꢀoxoisoindolinꢀ5ꢀyl)thiopheneꢀ2ꢀ
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11
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carbaldehyde (100) (Scheme 2; R = CH CH OH, R = H). 2ꢀThiophenecarboxaldehyde was
1
2
2
5
4
6
protected as the dimethyl acetal according to a literature procedure, then reacted with 78
according to general procedure E to give 83. Deprotection directly to the aldehyde was then
carried out as follows; compound 83 (430 mg, 1.29 mmol) was dissolved in acetone (32 mL), to
which was added water (8 mL) and pꢀtoluenesulfonic acid (100 mg). This mixture was stirred at
o
5
0 C for 5 h. Upon cooling, the reaction mixture was diluted with water (100 mL) and extracted
with CH Cl (3x50 mL). Purification by flash column chromatography on silica gel (5%
2
2
1
MeOH/CH Cl as eluant) gave 100 as a yellow solid (378 mg, 71% over 2 steps). H NMR [400
2
2
MHz, (CD ) SO] δ 9.94 (s, 1 H), 8.08 (d, J = 4.0 Hz, 1 H), 8.04 (d, J = 0.8 Hz, 1 H), 7.92 (dd, J
3
2
= 7.9, 1.6 Hz, 1 H), 7.85 (d, J = 4.0 Hz, 1 H), 7.75 (d, J = 8.0 Hz, 1 H), 4.83 (t, J = 5.4 Hz, 1 H),
+
+
4
.62 (s, 2 H), 3.56ꢀ3.67 (m, 4 H). LRMS (APCI ) calcd for C H NO S 288 (MH ), found 288.
15 14 3
(E,Z)ꢀ2ꢀ(1ꢀOxoꢀ5ꢀ(5ꢀ((5ꢀoxoꢀ2ꢀthioxoimidazolidinꢀ4ꢀylidene)methyl)thiophenꢀ2ꢀ
yl)isoindolinꢀ2ꢀyl)ethyl acetate (124) (Scheme 2; R = CH CH OAc, R = H). Reaction of 100
1
2
2
5
with 2ꢀthiohydantoin according to general procedure C gave 124 as a red powder (57%), mp
o
1
(AcOH) 261ꢀ264 C. H NMR [400 MHz, (CD ) SO ] δ 12.39 (s, 1 H), 11.98 (s, 1 H), 7.92ꢀ7.96
3
2
(m, 1 H), 7.87 (d, J = 4.0 Hz, 1 H), 7.84 (dd, J = 8.0, 1.5 Hz, 1 H), 7.77 (d, J = 4.0 Hz, 1 H),
7.73 (d, J = 7.9 Hz, 1 H), 6.65 (s, 1 H), 4.59 (s, 2 H), 4.26 (t, J = 5.4, Hz, 2 H), 3.78 (t, J = 5.3,
Hz, 2 H), 2.00 (s, 3 H).
General Procedure I: (E,Z)ꢀ2ꢀ(2ꢀHydroxyethyl)ꢀ5ꢀ(5ꢀ((5ꢀoxoꢀ2ꢀthioxoimidazolidinꢀ4ꢀ
ylidene)methyl)thiophenꢀ2ꢀyl)isoindolinꢀ1ꢀone (125) (Scheme 2; R = CH CH OH, R = H).
1
2
2
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Hydrolysis of 124 (161 mg, 0.42 mmol) was carried out by removing the previous reaction
solvent (AcOH; 4 mL) from the reaction mixture under reduced pressure and treating the crude
Oꢀacetate with K CO (289 mg, 2.09 mmol) in a mixture of MeOH (10 mL) and water (2 mL) at
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3
0
1
2
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0
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0
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0
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0
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0
RT for 1 h. The MeOH was removed under reduced pressure to give an orangeꢀblack oil which
o
was cooled to 0 C (ice/water) and acidified with 1 M HCl. The resulting solid was collected by
filtration, allowed to airꢀdry, then stirred in warm MeOH until a homogenous suspension was
obtained. This was collected by filtration and dried under vacuum to give 125 as an orange solid
o
1
(78 mg, 48% over 2 steps), mp (AcOH) 285 C (dec.). H NMR [400 MHz, (CD ) SO] Observe
3
2
E- and Z- isomers separately. δ 11.98ꢀ12.39 (m, 2 H), 7.93 (br s, 1 H), 7.87 (d, J = 4.0 Hz, 1 H),
7.83 (dd, J = 8.0, 1.5 Hz, 0.9 H), 7.77 (d, J = 4.0 Hz, 1 H), 7.72 (d, J = 8.0 Hz, 1 H), 7.68 (d, J =
4.0 Hz, 0.1 H), 6.84 (s, 0.1 H), 6.65 (s, 0.9 H), 4.82 (t, J = 5.2 Hz, 1 H), 4.60 (s, 2 H), 3.62ꢀ3.67
ꢀ
(
m, 2 H), 3.56ꢀ3.61 (m, 2 H). LRMS (APCI ) calcd for C H N O S 384 (MꢀH), found 384.
1
8
14
3
3 2
General Procedure J: (E,Z)ꢀ2ꢀ(2ꢀMorpholinoethyl)ꢀ5ꢀ(5ꢀ((5ꢀoxoꢀ2ꢀthioxoimidazolidinꢀ4ꢀ
ylidene)methyl)thiophenꢀ2ꢀyl)isoindolinꢀ1ꢀone (130) (Scheme 2; R = CH CH morpholine,
1
2
2
R = H). Reaction of 105 with 2ꢀthiohydantoin according to general procedure C gave 130. In
5
this case (and others where the target compound contained an amine) the AcOH solvent was
removed under reduced pressure to afford an orangeꢀblack oil which gave an orange solid when
suspended and stirred in a mixture of acetone (5 mL) and sat. NaHCO (10 mL). This solid was
3
collected by filtration, dried under vacuum and triturated with MeOH to afford 130 as an orange
o
1
solid (72%), mp (MeOH) 261ꢀ264 C. H NMR [400 MHz, (CD ) SO] Observe E- and Z-
3
2
isomers separately. δ 11.97ꢀ12.29 (m, 2 H), 7.94 (s, 1 H), 7.80ꢀ7.87 (m, 2 H), 7.75ꢀ7.79 (m, 1 H),
.72 (d, J = 8.0 Hz, 0.85 H), 7.68 (d, J = 3.9 Hz, 0.15 H), 6.84 (s, 0.15 H), 6.64 (s, 0.15 H), 4.61
7
(s, 2 H), 3.66 (t, J = 6.2 Hz, 2 H), 3.55 (t, J = 4.5 Hz, 4 H), 2.57 (t, J = 6.2 Hz, 2 H), 2.43 (br s, 4
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+
+
H). LRMS (APCI ) calcd for C H N O S 455 (M+H), found 455. HRMS (ESI ) calcd for
2
2
23
4
3 2
+
C H N O S 455.1206 (MH ), found 455.1201.
22 23
4
3 2
5
ꢀ(5ꢀ(Dimethoxymethyl)thiophenꢀ2ꢀyl)ꢀ2ꢀ(3ꢀhydroxypropyl)isoindolinꢀ1ꢀone
(107)
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(
Scheme 2; R = OH). Compound 101 (1.20 g, 3.98 mmol) was dissolved in a mixture of MeOH
4
(50 mL) and trimethylorthoformate (5 mL). pꢀToluenesulfonic acid (100 mg) was added, along
with 4 Å molecular sieves (5.0 g), then the reaction mixture heated at reflux for 15 h. Upon
cooling, the reaction mixture was diluted with CH Cl (100 mL) and filtered through celite
2
2
which was washed well with CH Cl . The solvent was removed from the combined filtrate under
2
2
reduced pressure to give an oil which was dissolved in EtOAc (150 mL) and washed with sat.
NaHCO (2x100 mL), water (100 mL) and brine (100 mL). The organic phase was dried
3
(Na SO ), filtered, and the solvent removed to afford 107 as a waxy, pale yellow solid (100%).
2
4
1
H NMR [400 MHz, (CD ) SO] δ 7.86 (d, J = 0.8 Hz, 1 H), 7.75 (dd, J = 8.0, 1.5 Hz, 1 H), 7.67
3
2
(d, J = 7.9 Hz, 1 H), 7.54 (d, J = 3.7 Hz, 1 H), 7.02 (dd, J = 3.8, 0.8 Hz, 1 H), 5.66 (d, J = 0.7 Hz,
1
H), 4.51 (s, 2 H), 4.49 (t, J = 5.1 Hz, 1 H), 3.57 (t, J = 7.2 Hz, 2 H), 3.45 (q, J = 5.9 Hz, 2 H),
+
+
3
.32 (s, 6 H), 1.76 (pentet, J = 6.7 Hz, 2 H). LRMS (APCI ) calcd for C H NO S 348 (MH ),
18 22 4
found 348.
3ꢀ(5ꢀ(5ꢀ(Dimethoxymethyl)thiophenꢀ2ꢀyl)ꢀ1ꢀoxoisoindolinꢀ2ꢀyl)propyl methanesulfonate
(108) (Scheme 2; R = OMs). Alcohol 107 (1.29 g, 3.72 mmol) was dissolved in dry THF (40
4
mL). Triethylamine (3.76 g, 37.2 mmol) and methanesulfonyl chloride (1.70 g, 14.9 mmol) were
added and the reaction mixture stirred at RT for 3.5 h. The mixture was diluted with EtOAc (200
mL), which was washed with water (100 mL), sat. NaHCO (100 mL) and brine (100 mL). The
3
organic layer was dried (Na SO ), filtered, and the solvent removed under reduced pressure to
2
4
give a residue which was purified by flash column chromatography on silica gel (5%
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