Inhibition of the pore-forming protein perforin by a series of aryl-substituted isobenzofuran-1(3H)-ones

Article abstract of DOI:10.1016/j.bmc.2011.12.011

An aryl-substituted isobenzofuran-1(3H)-one lead compound was identified from a high throughput screen designed to find inhibitors of the lymphocyte pore-forming protein perforin. A series of analogs were then designed and prepared, exploring structure-ac

Full text of DOI:10.1016/j.bmc.2011.12.011

Bioorganic & Medicinal Chemistry 20 (2012) 1319–1336
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Bioorganic & Medicinal Chemistry
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Inhibition of the pore-forming protein perforin by a series of aryl-substituted
isobenzofuran-1(3H)-ones
Julie A. Spicer a, , Kristiina M. Huttunen a,b, Christian K. Miller a, William A. Denny a, Annette Ciccone c,
⇑
Kylie A. Browne c, Joseph A. Trapani c,d
a Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
b School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, PO Box 1627, Kuopio FI-70211, Finland
c Cancer Immunology Program, Peter MacCallum Cancer Centre, St. Andrew’s Place, East Melbourne, Victoria 3002, Australia
d Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Victoria 3010, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
An aryl-substituted isobenzofuran-1(3H)-one lead compound was identified from a high throughput
screen designed to find inhibitors of the lymphocyte pore-forming protein perforin. A series of analogs
were then designed and prepared, exploring structure–activity relationships through variation of
2-thioxoimidazolidin-4-one and furan subunits on an isobenzofuranone core. The ability of the resulting
compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by
intact KHYG-1 natural killer effector cells was determined. Several compounds showed excellent activity
at concentrations that were non-toxic to the killer cells. This series represents a significant improvement
on previous classes of compounds, being substantially more potent and largely retaining activity in the
presence of serum.
Received 22 October 2011
Revised 5 December 2011
Accepted 7 December 2011
Available online 22 December 2011
Keywords:
Perforin
Perforin inhibitor
Isobenzofuran-1(3H)-one
Immunosuppressant
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
calcium-rich environment then allows perforin monomers to coor-
dinate calcium and oligomerize into highly ordered aggregates of
The granule exocytosis pathway is the principal mechanism
employed by natural killer (NK) cells and cytotoxic T lymphocytes
(CTL) to eliminate virus-infected and transformed cells.1–3 These
key effector cells contain secretory vesicles (granules) which are
used to store various cytotoxic proteins, including a group of pro-
apoptotic serine proteases known as granzymes,4,5 and perforin, a
67 kDa calcium-dependent pore-forming glycoprotein.6–10 CTL
and NK cells are protected from the inherent cytotoxicity of perfo-
rin during synthesis, trafficking and storage by a variety of mecha-
nisms,11 including the low pH present in the secretory granules
(typically 5.1–5.5).12 This results in protonation of key calcium-
binding aspartate residues, rendering perforin incapable of
calcium-triggered activation. Stable conjugation of the cytotoxic
effector cell with a target cell results in migration of granules to
the site of contact, followed by exocytic delivery of the granule
contents into the immune synapse. Exposure to this neutral,
19–24 subunits that form trans-membrane pores, 130–200 Å in
diameter, which penetrate the plasma membrane.13 This process
facilitates entry of the granzymes into the cytosol of the infected
or transformed cell where they initiate various apoptotic death
mechanisms.4
The membrane-attack complex/perforin (MACPF) domain that
is responsible for lytic activity in perforin is shared by a lytic pro-
tein toxin from Photorhabdus luminescens (Plu-MACPF), for which
the crystal structure has been published.14 This 2.0 Å resolution
structure revealed similarities with pore-forming cholesterol-
dependent cytolysins (CDCs) from gram-positive bacteria, suggest-
ing that lytic MACPF proteins may share a common mode of pore
formation. By analogy to well-characterised members of the CDC
family, such as pneumolysin (PLY) and streptolysin O, it appears
the mechanism consists of initial membrane binding through a
membrane proximal immunoglobulin domain. Upon further con-
formational changes, the monomers coalesce into a giant b-bar-
rel-linked channel which perforates the target cell membrane.
The detailed mechanism of this process has subsequently been
confirmed both through mutagenesis studies and determination
of the crystal structure of monomeric murine perforin (2.75 Å).13,15
Perforin is encoded on a single-copy gene in both mice and hu-
mans,5 and while there is a level of redundancy to many of the
granule components, perforin is indispensible for protective
Abbreviations: NK, natural killer; CTL, cytotoxic
T lymphocyte; MACPF,
membrane-attack complex/perforin; CDC, cholesterol-dependent cytolysin; PLY,
pneumolysin; EGF, epidermal growth factor; FLH, familial hemophagocytic
lymphohistiocytosis; SAR, structure–activity relationship; SEM, standard error of
the mean.
⇑
Corresponding author. Tel.: +649 923 6149; fax: +649 923 7502.
E-mail address: j.spicer@auckland.ac.nz (J.A. Spicer).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.011

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J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
immunosurveillance. Perforin knock-out mice demonstrate the ef-
fect of deficiency in vivo through increased susceptibility and fail-
ure to clear many viruses and other intracellular pathogens, as well
as the development of highly aggressive disseminated B cell lym-
phoma in the majority of animals over the age of 12 months.16 In
humans, complete loss of perforin function results in familial
hemophagocytic lymphohistiocytosis (FLH) syndrome which is
characterised by severe anemia and hepatosplenomegaly, fever,
and thrombocytopenia.17–20 Partial loss, depending upon the level
of residual activity, is still strongly associated with FLH (often late
onset) as well as a range of hematological malignancies, particu-
larly in adolescence.21
CTL and NK cells have also been implicated in several autoim-
mune diseases (e.g., insulin-dependent diabetes),4,22,23 therapy-
induced conditions (e.g., allograft rejection, graft-versus-host
disease),24–26 and potentially fatal complications of malarial and vir-
al infections(e.g., cerebralmalaria, post viral myocarditis).10 Current
accepted immunosuppressive treatments are associated with a
wide range of side-effects,27,28 many of which arise from the
non-specificity of therapies where multiple molecular targets are
affected indiscriminately. Given the pivotal role performed by perfo-
rin in CTL and NK cell induced lysis, a small molecule inhibitor of per-
forin is of potential interest as a new class of highly specific
immunosuppressive agents. In our initial work in this area, we have
already explored two series of compounds, the dihydrofuro
[3,4-c]pyridinones29 and the 1-amino-2,4-dicyanopyrido[1,2-a]
benzimidazoles,30 both of which showed appreciable perforin-
inhibitory activity. However we continue to seek improved potency
and drug-like properties, and thus report here our current work ona
series of aryl-substituted isobenzofuran-1(3H)-ones as small-mole-
cule inhibitors of perforin-induced lysis.
(C), a structure which lends itself to independent variation of each
subunit and subsequent analysis of the resulting structure–activity
relationships (SAR). In this study we explore the effect of varying
the 2-thioxoimidazolidin-4-one A-subunit and the furan B-subunit
on a fixed benzofuranone scaffold, on the ability of the resulting
compounds to inhibit perforin lytic activity.
The compounds of Table 1 were prepared through a key Suzuki
coupling with a variety of protected aldehydes (3–21) and boronate
22 (Scheme 1), to give intermediates 23–41. The boronate 22 is eas-
ily prepared in gram quantities from 4-bromo-2-methylbenzoic
acid32,33 and was employed in the synthesis of compounds in both
Tables 1 and 2. Many of the required aldehydes were commercially
available and the 5- and 6-bromobenzo[b]thiophene-2-carboxalde-
hydes were prepared according to published procedures.34,35
Deprotection of the Suzuki products 23–41 under acidic conditions
gave a range of heterocyclic aldehydes 42–60 which were reacted
with 2-thiohydantoin to give the target compounds 2, 94–100,
103–105, 109–116.
In order to expand our investigation into a range of heterocyclic
B-subunits based on non-commercially available substrates, a selec-
tion of key substituted aldehydes 64, 70, 72, 80, 84 were prepared as
described in Schemes 2–4. Pyrrole 64 was prepared by Suzuki
reaction of BOC-protected pyrrole boronic acid 61 with 5-bro-
moisobenzofuran-1(3H)-one (62) (Scheme 2). This afforded inter-
mediate 63 which was formylated under Vilsmeier conditions
(with concomitant deprotection of the carbamate) to give the
target aldehyde 64. Oxazole- and isoxazole-5-carboxaldehydes 70
and 72 were both prepared from 1-oxo-1,3-dihydroisobenzofuran-
5-carboxylic acid 65 (Scheme 3). In the case of the isoxazole, carbox-
ylic acid 65 was reduced to the benzyl alcohol 66 by activation with
CDI, followed by reduction with NaBH4. Oxidation with PCC on silica
gel gave the aldehyde 67 which was converted to the corresponding
oxime 68 by reaction with hydroxylamine hydrochloride. The oxime
68 was ring-closed through chlorination with N-chlorosuccinimide
and condensation with propargyl alcohol,36 giving hydroxymethyl
isoxazole 69. A second oxidation step with PCC/silica gave the
desired aldehyde 70. For the oxazole, the carboxylic acid 65 was con-
verted to the propargylic amide 71 by activation with pentafluor-
ophenyl trifluoroacetate and reaction with propargylamine.
Palladium-catalysed ring-closure to the oxazole using PdCl2(Bn)2
in the presence of CuCl2 and under an atmosphere of oxygen37 gave
aldehyde 72 in low (17%) yield. The isomeric isothiazolecarboxalde-
hydes 80 and 84 were prepared from 1-oxo-1,3-dihydroisobenzofu-
ran-5-carboxamide 75 (Scheme 4). The carboxamide 75 was reacted
with chlorocarbonylsulfenyl chloride by heating in dioxane, giving
1,3,4-oxathiazol-2-one 76, which in turn was condensed with ethyl
propiolate and decarboxylated38 to afford a mixture of isomeric
ethyl isothiazolecarboxylates (77, 81) which were separated by
chromatography. The esters 77 and 81 were hydrolysed under basic
conditions to the acids 78 and 82, then reduced to the corresponding
isoxazole alcohols 79 and 83. Finally, oxidation with PCC/silica gave
the desired aldehydes 80 and 84.
2. Results and discussion
2.1. Chemistry
A high throughput screen31 of approximately 100,000 com-
pounds, sourced from commercial libraries, was carried out using
a 384-well plate format. Compounds were screened (at 20 lM)
by incubation with sheep red blood cells, which in the absence of
an inhibitor are lysed by perforin, generating turbidity that can
be measured by a change in absorbance at 650 nm. This resulted
in the identification of a small number of hits which showed repro-
ducible perforin-inhibitory activity for recombinant mouse and
human perforin. These hits were then also screened for their ability
to inhibit the perforin-induced lysis of 51Cr-labelled Jurkat lym-
phoma cells and IC50s calculated. Compared to the original first-
in-class dihydrofuro[3,4-c]pyridinone perforin inhibitors,29 as
exemplified by compound 1 (IC50 = 0.97 lM), the new lead com-
pound
2
showed moderate, but reproducible activity
(IC50 = 6.20
lM; Fig 1).
Compound 2 can be readily disconnected into three subunits; a
With the target aldehydes 64, 70, 72, 80, 84 in hand, the
corresponding final products 101, 102, 106, 107, 108 were prepared
by reaction with 2-thiohydantoin in the presence of b-alanine
and refluxing AcOH. A single acyclic analogue (117) was also
prepared (Scheme 3); EDCI/HOBt-mediated coupling of the
carboxylic acid 65 with 3-amino-1-propanol gave alcohol 73 which
was oxidised to the aldehyde 74 with Dess–Martin periodinane.
Reaction with 2-thiohydantoin as above gave the alkylamido-linked
compound 117.
The compounds of Table 2 were synthesized by Suzuki reaction
of 2-bromothiophenes 4 and 85–87 with key boronate 22 (Scheme
5). This gave final products 118, 119, 120, 121 (121 from methyla-
tion of 120 with TMS-diazomethane) directly, and deprotection of
the cyclic acetal 24 gave key intermediate aldehyde 43, from which
2-thioxoimidazolidin-4-one (A), a furan (B) and a benzofuranone
S
H
N
O
A
S
O
HN
B
O
HN
C
S
O
O
1
2
IC50 = 0.97 µM
IC50 = 6.20 µM
O
Figure 1. Original and new perforin inhibitor leads.

J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
1321
M)
Table 1
Table 1 (continued)
Inhibitory activities of isobenzofuran-1(3H)-ones with various B-subunits
b
Number
Xa
Jurkat IC50
(l
H
S
N
S
S
O
O
X
110
0.49
HN
C
A
111
112
0.36
0.51
O
B
O
Number
Xa
Jurkat IC50
6.20
(lM)
b
H
N
113
0.36
2
O
H
N
94
95
0.78
1.05
114
115
0.82
1.72
S
N
N
S
96
97
>20
116
117
0.47
>20
CH2CH2NHCO
2.11
a
Structure of the B-subunit.
b
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 three
independent IC50 determinations.
98
99
1.80
2.00
0.37
N
the remaining compounds of Table 2 (122–134) were prepared.
Compounds 122–128 were prepared by simply heating aldehyde
43 with the appropriate heterocycle in the presence of b-alanine
and refluxing AcOH. The oxazole 129 was obtained by heating 43
with tosylmethyl isocyanate and K2CO3 in MeOH (Scheme 5), and
succinimide derivative 130 was isolated after reaction between
43 and triphenylphosphoranylidenesuccinimide39 89 (Scheme 6).
Scheme 7 describes the preparation of the remaining targets, a ser-
ies of five- and six-membered lactam derivatives (131–134). In the
first instance, the benzyl-protected diethyl (2-oxopyrrolidin-3-
yl)phosphonate 90 was prepared and reacted with 43 using
KHMDS and 18-crown-6 at low temperature.40 This gave com-
pound 131 from which the benzyl protecting group was unable
to be removed. These two steps were then repeated with the 4-
methoxybenzyl-protected compound 91, to give 132 which was
successfully deprotected to the unsubstituted lactam 133 using
TFA/anisole. Finally, this sequence was repeated to give the pro-
tected six-membered lactam 93 which was also successfully
deprotected, affording 134.
100
N
101
2.45
N
H
N
102
103
104
7.28
1.72
1.41
O
N
S
N
S
N
105
106
0.34
1.30
S
2.2. Structure–activity relationships
O
N
2.2.1. Effect of replacing the central furan ring (Table 1)
Lead compound 2 was employed as the starting point for the
design and synthesis of a new series of aryl-substituted isobenzofu-
ran-1(3H)-ones. All final products were then screened for their abil-
ity to inhibit the perforin-induced lysis of 51Cr-labelled Jurkat
lymphoma cells (for further details see Section 4). Our initial strat-
egy was to retain the 2-thioxoimidazolidin-4-one A- and isobenzof-
uranone C-subunits, while exchanging the central furan ring for a
variety of aromatic B-subunits. We first replaced the 2,5-furan of
lead 2 with a 2,5-thiophene (94). This modification resulted in a very
107
108
109
2.54
1.44
2.27
S
N
S
S
N

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J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
O
B
H
X
R1O
X
O
i
ii or iii
X
R1O
O
O
O
Br
+
O
OR2
OR2
O
O
O
22
23-41
42-60
O
O
3,4
iv or v
R1R2 =
H
O
N
5-21
R1 = R2 = Me
X = Heterocycles of Table 1
S
X
N
H
O
O
Compounds of Table 1;
2,94-100,103-105,109-116
Scheme 1. Reagents and conditions: (i) 2 M Na2CO3, toluene/EtOH, Pd(dppf)Cl2, reflux, 2 h; (ii) 1 M HCl, acetone, rt; (iii) p-TsOH, water, acetone, 50 °C; (iv) 2-thiohydantoin,
piperidine, EtOH, reflux, 15 h; (v) 2-thiohydantoin, b-alanine, AcOH, reflux, 15 h.
encouraging eightfold improvement in activity; IC50 = 0.78
l
M for
118, 119, 120 and 121 (no activity) suggest a requirement for a ring
as the A-subunit, the only exception being the aldehyde 43 which
the thiophene, compared to 6.20 M for the furan. The isomeric
l
2,4-thiophene (95) was also significantly more potent than the furan
2, suggesting some level of flexibility in the angle of connection be-
tween the A- and C-subunits. This was further investigated via the
synthesis of the three benzene-linked compounds 96–98. It was
established that the 1,4-linkage was optimal (98), with an
showed moderate activity (IC50 = 6.31 lM). Replacement of the 2-
thioxoimidazolidin-4-one of 94 with a variety of related five-mem-
bered heterocycles (122, 124, 128) is tolerated (IC50s = 1.12–
2.75 lM), but all examples show less activity. It is unclear why the
2-thioxothiazolidin-4-one 123 is so much poorer than the other
examples. Expansion of the thioxoimidazolidinone ring to a six-
membered ring is permissible, as illustrated by compounds 125
and 126 which have comparable activity to 94, but methylation of
the ring nitrogens (127) results in significant loss of activity. Oxazole
129 also shows moderate activity.
IC50 = 1.80
lM. The 1,3- (97) and 1,2- (96) linked compounds were
progressively less potent (IC50 = 2.11 and >20
lM, respectively).
The two pyridyl-linked isomers 99 and 100 showed a similar trend,
however in this case the position of the pyridine nitrogen also
appears important, with the 2,5-linked compound 100 (0.37
significantly more potent than its 3,6 counterpart 99 (2.00 M).
While 2,5-pyrrole 101 showed only moderate activity (IC50
2.45 M), the improvement in activity observed in the transition
from furan 2 to thiophene 94 is replicated in the corresponding oxa-
zole/thiazole pair 102 and 103 (IC50s = 7.28 and 1.72 M). 4-Thia-
zol-2-yl compound 104 also showed good activity (1.41 M), but
the outstanding isomer of the set was the 2-thiazol-4-yl analog
105 with an IC50 = 0.34 M. Isoxazole 106 and isothiazoles 107
and 108 also all showed appreciable activity (1.30–2.54 M).
lM)
l
Systematic deletion of NH and carbonyl groups from compound
=
122 (IC50 = 1.19
rolidine-2-one 133 results in significant loss of potency (IC50s = 6.97
and 6.81 M, respectively) which is compounded by expansion of
the ring to the six-membered analog 134 (IC50 = 15.2 M). Com-
lM) to give the pyrrolidine-2,5-dione 130 and pyr-
l
l
l
l
l
pounds 131 and 132 also reinforce the need for the amido nitrogens
to remain unsubstituted.
l
Another factor which contributes to the perforin-inhibitory
activity of aryl-substituted isobenzofuran-1(3H)-ones is the pres-
ence of a double bond fused to the A-unit, resulting in a mixture
of E- and Z-isomers. Reduction of this double bond in an active
compound, to give its saturated analogue, results in complete loss
of activity (data not shown). Analytical HPLC and 1H NMR studies
show gradual conversion of the predominant Z-isomer to the
corresponding E-isomer in solution, ruling out any possibility of
separation, and all compounds prepared have consequently been
synthesised as a mixture of E- and Z-isomers (see Supplementary
data for characterisation of isomers and stability studies).
l
Bicyclic B-units were then explored, with the benzothiophene
series 109–111 showing a similar dependence on the angle of con-
nection to the benzene series; the 2,5- and 2,6-isomers 110 and
111 (0.49 and 0.36
3,5-isomer 109 (2.27
phene pair of 2 and 94, the 2,5-benzofuran 112 and 2,5-benzothio-
phene 110 showed virtually identical activity (0.51 and 0.49 M,
respectively). Indoles 113, 114 and quinoline 116 all showed
sub-micromolar activity (0.36–0.82 M), with quinoline 115 a lit-
tle less potent at 1.72 M. Finally, a single acylic compound 117
lM) being significantly more potent than the
lM). Interestingly, unlike the furan/thio-
l
l
l
was prepared; it’s inactivity confirming that an aromatic B-subunit
is required, presumably by providing the additional rigidity needed
for an active conformation.
2.2.3. Further analysis of compounds with perforin-inhibitory
activity
Lead compound 2 was additionally tested for its ability to inhi-
bit the lytic function of the pneumococcal toxin pneumolysin
(PLY), a cholesterol-dependent pore-forming protein. PLY, like per-
forin, is released as a soluble monomer which assembles into olig-
omeric pores at the target cell membrane. Perforin lysis of 51Cr
labelled Jurkat cells is completely inhibited by 2 at a concentration
2.2.2. Exploring the influence of the thioxoimidazolidinone
(Table 2)
Having established a small group of preferred B-subunits, the
influence of A-subunit was then explored. For this study, the
B- and C-subunits were fixed as thiophene and isobenzofuran-
1(3H)-one, respectively. While thiophene 94 was not the most po-
tent compound of Table 1, it was selected based on a range of factors
including inhibitory activity, synthetic accessibility, cost of goods
and overall molecular weight of the final compound. Examples
of 20 lM. By substituting PLY for perforin under otherwise identi-
cal conditions, lysis was not inhibited by 2, showing that it specif-
ically inhibits perforin, but not the mechanistically-related PLY.
Those compounds which showed the ability to inhibit lysis of
labelled Jurkat cells by purified perforin were then also evaluated

J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
1323
M)
Table 2
Inhibitory activities of isobenzofuran-1(3H)-ones with various A-subunits
Table 2 (continued)
b
Number
Ra
Jurkat IC50 (l
A
B
PMB
R
C
O
S
N
132
>20
O
O
H
N
O
Number
Ra
Jurkat IC50 (lM)
b
133
134
6.81
15.2
118
119
43
120
121
H
Acetyl
CHO
CH2COOH
CH2COOMe
>20
>20
6.31
>20
>20
H
N
O
H
S
N
a
O
Structure of the A-subunit.
Testing was carried out over a range of doses, with the IC50 being equal to the
b
94
0.78
HN
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 three
independent IC50 determinations.
H
O
N
O
122
123
124
1.19
>10
for their inhibition of perforin delivered by an intact NK cell line.
The inhibitor (20 lM final concentration) and medium were co-
HN
H
N
incubated with KHYG-1 NK cells for 30 min at room temperature,
51Cr-labelled K562 leukemia target cells were then added, and cell
lysis evaluated after 4 h incubation at 37 °C by measuring 51Cr re-
lease. This assay can be regarded as a more stringent test of activity
than measurement of isolated perforin-mediated Jurkat cell lysis,
because the perforin is delivered by a whole cell and is thus more
representative of the situation in vivo. To confirm that the inhibi-
tory activity exhibited by the compounds against KHYG-1 NK cells
was due to blocking the action of perforin and not non-specific tox-
icity against the effector cell, the viability of the NK cells in the
presence of inhibitor was also measured 24 h later. Viable and dead
cells were counted and percent viability calculated based on total
cell count (for further details see Section 4). The key role played
by CTLs and NK cells in the overall immunological response means
that it is essential that any pharmacological intervention allows ra-
pid recovery of these cytotoxic effector cells, thus for the purposes
of this study, compounds are classified as toxic if NK cell viability
falls below 70%.
S
O
S
S
H
N
O
O
O
1.12
H
N
O
125
126
0.80
0.40
HN
O
H
S
N
O
O
HN
O
N
All compounds tested showed at least some suppression of NK
cell killing of the K562 targets, ranging from 11% to 100% inhibition
(Table 3), however most compounds also show an effect on the via-
bility of the KHYG-1 NK cells (10–68% viability), suggesting that
toxicity does make at least some contribution to the observed level
of inhibition for these examples. Of particular note in this regard
are indoles 113 and 114 which showed excellent activity against
O
127
128
3.96
2.75
N
O
H
N
isolated perforin (IC50 = 0.36 and 0.82 lM), but clearly the out-
HN
O
standing activity observed with whole NK cells (100% and 91%,
respectively) is significantly due to toxicity (10% and 23% viability).
Compared to dihydrofuro[3,4-c]pyridinone 1 (41% inhibition of
perforin-induced target cell killing), two compounds in the current
series; thiophene-linked 94 andimidazolidine-2,4-dione 122,
exhibited improved effectiveness in this assay (53% and 54% inhi-
bition, respectively) whilst demonstrating minimal KHYG-1 NK cell
toxicity (both around 90% viability).
N
O
129
130
3.18
6.97
N
O
H
N
O
The most significant drawback of the original series of dihydrof-
uro[3,4-c]pyridinones (e.g. 1) was that cellular activity was ad-
versely affected by increasing concentrations of serum. The
activity of 1 was reduced from 41% inhibition with no serum
Bn
N
O
131
>20

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J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
OHC
Br
+
N
H
i
iii
ii
N
BOC
101
O
O
O
B(OH)2
N
BOC
61
O
O
O
62
63
64
Scheme 2. Reagents and conditions: (i) 2 M Na2CO3, toluene/EtOH, Pd(dppf)Cl2, reflux, 2 h; (ii) POCl3, DMF, CH2Cl2, 0 °C to rt; (iii) 2-thiohydantoin, b-alanine, AcOH, reflux,
15 h.
O
HO
iv
H
R
O
ii
ii
HO
N
O
O
O
O
N
O
O
O
O
66
O
69
70
67
68
R = CHO
O
(iii)
v
R = CH=NOH
i
OHC
106
O
O
(vi)
(v)
(vii)
HO
HO
N
H
N
102
O
O
O
65
71
72
O
O
O
viii
O
O
O
ix
v
N
H
H
N
H
117
O
O
73
74
O
O
Scheme 3. Reagents and conditions: (i) (a) CDI, THF, rt; (b) NaBH4, H2O; (ii) PCC/silica, CH2Cl2; (iii) NH2OHꢀHCl, pyridine, MeOH, 60 °C, 1.5 h; (iv) (a) N-chlorosuccinimide,
pyridine, THF, 60 °C; (b) propargyl alcohol, 50 °C, 2 h; (v) 2-thiohydantoin, b-alanine, AcOH, reflux, 15 h; (vi) (a) PFP-TFA, pyridine, rt; (b) propargylamine, THF, rt; (vii)
Pd(Bn)2Cl2, O2, CuCl2, DMF, 100 °C; (viii) EDCI, HOBt, TEA, 3-amino-1-propanol, DMF, rt, 15 h; (ix) Dess–Martin periodinane, THF/DMF, rt, 3 h.
O
S
H
O
S
R
N
O
O
S
i
ii
v
H2N
N
N
O
O
O
O
80,84
O
O
O
O
75
76
vi
R = COOEt 77,81
78,82
iii
iv
R = COOH
R = CH2OH 79,83
107,108
Scheme 4. Reagents and conditions: (i) chlorocarbonylsufenyl chloride, dioxane, 100 °C; (ii) ethyl propiolate, 1,2-dichlorobenzene, 150 °C, 5 h; (iii) (a) 2 M NaOH, EtOH,
50 °C; (b) TFA/CH2Cl2, rt; (iv) (a) CDI, THF, rt; (b) NaBH4, H2O; (v) PCC/silica, CH2Cl2; (vi) 2-thiohydantoin, b-alanine, AcOH, reflux, 15 h.
i
118
R = H
R
R
Br
R = C(O)Me 119
or
S
S
O
R = H
R = C(O)Me
R = CH2COOH 87
85
86
R = CH2COOH 120
R = CH2COOMe
ii
O
121
O
O
O
24
R =
4
R =
iii
O
R = CHO 43
R = Heterocycles of Table 2; 122-129
iv or v or vi
Scheme 5. Reagents and conditions: (i) 22, 2 M Na2CO3, toluene/EtOH, Pd(dppf)Cl2, reflux, 2 h; (ii) TMS-diazomethane, Et2O/MeOH; (iii) 1 M HCl, acetone, rt; (iv) heterocycle
(R) from Table 2, piperidine, EtOH, reflux, 15 h; (v) heterocycle (R) from Table 2, b-alanine, AcOH, reflux, 15 h; (vi) tosylmethyl isocyanide, K2CO3, MeOH, reflux, 1 h.

J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
1325
H
N
O
O
O
O
Ph3P
i
ii
S
NH
NH
O
O
O
O
88
89
130
Scheme 6. Reagents and conditions: (i) Ph3P, AcOH, 110 °C, 30 min; (ii) 43, MeOH, reflux, 1 h.
R
N
O
O
P
OEt
HN
O
n
n
n
ii
i
N
OEt
R
S
S
O
O
O
n = 1, R = Bn; 90
n = 1, R = 4-OMeBn;
n = 2, R = 4-OMeBn;
n = 1, R = Bn; 131
n = 1, R = 4-OMeBn;
n = 2, R = 4-OMeBn;
n = 1; 133
134
n = 2;
O
O
91
92
132
93
Scheme 7. Reagents and conditions: (i) 43, KHMDS, 18-crown-6, THF, ꢁ78 °C to rt; (ii) TFA, anisole, reflux, 15 h.
Table 3
Capacity of selected compounds to inhibit perforin delivered by KHYG-1 NK cells
Number
Jurkat IC50
(lM)
KHYG-1 inhibition (% at 20
No seruma
l
M)
KHYG-1 viabilityc (%)
10% serumb
1
2
94
95
98
0.97
6.20
0.78
1.05
1.80
0.37
1.72
1.41
0.34
1.30
1.44
0.49
0.36
0.51
0.36
0.82
1.72
0.47
1.19
1.12
0.80
0.40
41 3.6
74 8.2
53 2.6
49 7.5
28 4.3
37 11.6
45 6.0
30 5.0
11 4.8
41 3.9
45 8.3
51 4.5
46 8.3
61 5.2
100 2.0
91 8.2
52 5.0
60 6.0
54 5.0
36 10.0
52 4.4
91 6.6
10.3 5.9
23 6.7
38.9 3.5
42 10.5
30 9.8
20 6.6
27 7.2
13 7.7
77 12.0
66 7.4
88 3.0
55 11.0
59 16.0
92 5.3
68 12.0
91 2.3
25 10.0
63 9.7
57 11.0
27 2.9
19.7 11.0
29.9 3.0
10 2.0
23.2 4.4
59 9.0
100
103
104
105
106
108
110
111
112
113
114
115
116
122
124
125
126
2
1.0
23 8.6
26 5.4
17 4.8
25 4.1
35 8.7
100 8.0
83 8.6
38 5.7
35 7.7
64 8.9
12 10.0
10 3.0
73 11.0
48.5 2.4
91.5 4.6
19 6.5
77.6 8.2
38 13.0
a
Inhibition by compound (20 lM) of the perforin-induced lysis of K562 target cells when co-incubated with KHYG-1 human NK cells (see Section 4). Percent inhibition
calculated compared to untreated control.
b
As for a, but in the presence of 10% mouse serum.
Viability of KHYG-1 NK cells after 24 h by Trypan blue exclusion assay (see Section 4). All results are the average of at least three separate determinations SEM.
c
(20
l
M concentration), to 10% in the presence of 10% mouse serum,
effects of perforin in cells. Molecules with this function are of poten-
tial interest as a new class of highly specific immunosuppressive
agents for the treatment of autoimmune diseases and therapy-in-
duced conditions characterized by undesired perforin secretion.
The SAR around this class of compounds was explored by inde-
pendent variation of the 2-thioxoimidazolidin-4-one A- and furan
B-subunits on an isobenzofuranone scaffold. The particular pattern
of hydrogen bond donors and acceptors present in the 2-thioxoim-
idazolidin-4-one appears optimal for inhibition of isolated perforin
protein, as none of the closely related five-membered replace-
ments were as potent and all other deletions or substitutions re-
sulted only in loss of activity. While the six-membered
heterocyclic ring analogs (125, 126) appeared at least as potent
severely limiting further development of the series. While this is
also evident for many of the compounds in the current series, of
the two most active compounds, 94 shows only limited loss of
activity, while 122 appears to demonstrate a small increase in
activity (from 54% inhibition in the absence of serum, to 64% in
its presence).
3. Conclusions
Starting with a high throughput screening lead of moderate
potency (2), a novel series of aryl-substituted isobenzofuran-
1(3H)-ones have been developed as potent inhibitors of the cytolytic

1326
J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
against isolated perforin, extreme insolubility precluded further
development. The area where the greatest gains in potency were
found was in the linking B-subunit. Simply replacing 2,5-furan 2
with 2,5-thiophene 94 resulted in an eightfold increase in activity.
Several compounds containing bicyclic B-subunits (105, 110–113)
were also potent inhibitors of perforin (IC50s = 0.34–0.51 lM),
showing between 12- and 18-fold more activity than the original
lead 2.
Although a wide range of compounds showed excellent activity
toward isolated perforin protein, unfortunately many also proved
toxic to the effector cell when whole NK cells were employed to
deliver perforin. The overall best compound in this study, there-
fore, was imidazolidine-2,4-dione 122 which showed good inhibi-
a suspension of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)iso-
benzofuran-1(3H)-one 22 (527 mg, 2.03 mmol) in EtOH (10 mL).
This boronate ester was prepared in turn from 5-bromoisobenzofu-
ran-1(3H)-one33 62 according to a literature procedure.34 A solution
of 2 M Na2CO3 (6.6 mL) and Pd(dppf)Cl2 (90 mg, 0.10 mmol) were
added and the entire mixture was heated at reflux under nitrogen
for 2 h. Upon cooling, all solvents were removedunder reduced pres-
sure and the resulting residue was partitioned between water
(50 mL) and CH2Cl2 (50 mL). Two further CH2Cl2 (50 mL) extractions
were performed, then the combined organic fractions dried
(Na2SO4), filtered, and the solvent removed under reduced pressure
to afford a residue which was purified by flash column chromatogra-
phy on silica gel (10% EtOAc/hexanes as eluant). The title compound
23 was isolated as a beige solid (416 mg, 75%). 1H NMR [(CD3)2SO] d
7.98 (s, 1H), 7.92 (dd, J = 8.1, 1.2 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.21
(d, J = 3.5 Hz, 1H), 6.73 (d, J = 3.4 Hz, 1H), 5.98 (s, 1H), 5.44 (s, 2H),
4.05–4.14 (m, 2H), 3.94–4.03 (m, 2H).
tory activity against isolated perforin (IC50 = 1.19 lM) and
demonstrated excellent suppression of KHYG-1 NK cell mediated
lysis in the presence of serum (64%) without toxicity toward the
effector NK cells (>90% survival).
While it is noted that moieties similar to the 2-thioxoimidazoli-
din-4-one and imidazolidine-2,4-dione (thiazolidinediones, rhoda-
nines) are widely reported in the literature and have been
described as ‘frequent (or promiscuous) hitters’,41 there remain
many examples of such substructures being present in clinical
drugs.42 As observed above, lead compound 2 is a specific inhibitor
of perforin, and at least in the case of PLY, is not a more general
inhibitor of a mechanistically related pore-forming protein. In the
present study, the 2-thioxoimidazolidin-4-one subunit is present
in the majority of compounds synthesised, yet a wide range of
activities is still observed against isolated perforin protein (IC50s
4.1.2. General procedure B: 5-(1-oxo-1,3-dihydroisobenzofuran-
5-yl)furan-2-carbaldehyde (42)
Compound 23 (416 mg, 1.53 mmol) was dissolved in acetone
(20 mL), to which was added 1 M HCl (4 mL). The resulting solution
was stirred at rt for 3 h., at which point an off-white solid had precip-
itated. The mixture was diluted with water (100 mL) and extracted
with CH2Cl2 (4 ꢂ 100 mL). The combined CH2Cl2 fractions were
dried (Na2SO4), filtered, and the solvent removed under reduced
pressure to give a solid which was triturated with Et2O to afford 42
as a beige solid upon filtration (292 mg, 84%); mp (Et2O) 229–
231 °C. 1H NMR [(CD3)2SO] d 9.68 (s, 1H), 8.17 (br s, 1H), 8.09 (dt,
J = 8.0, 0.7 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 3.8 Hz, 1H),
7.53 (d, J = 3.8 Hz, 1H), 5.48 (s, 2H). Anal. (C13H8O4) H, C; +0.5.
from 0.34 to >20 lM) as well as cell killing induced by intact NK
cells (KHYG-1 inhibition for non-toxic active compounds tested
range = 10–64%). These data demonstrate that the compounds pos-
sess the capability to specifically target perforin-dependent CTL
and NK cell induced lysis.
4.1.3. General procedure C: (E,Z)-5-((5-(1-oxo-1,3-dihydroiso-
benzofuran-5-yl)furan-2-yl)methylene)-2-thioxoimidazolidin-
4-one (2)
Our original publication29 described the first class of small mol-
ecules reported to inhibit the cytolytic effects of perforin in cells,
but the SAR was relatively flat (<3-fold improvement over the ori-
ginal lead) and further development was precluded by inactivity in
the presence of serum. The current series shows significant
improvement in activity over original hit 2, but more importantly,
activity is retained in the presence of serum. Further work to elu-
cidate the detailed mechanism of inhibition is on-going, including
attempts to obtain a co-crystal structure of drug bound in the tar-
get protein. The results from this study now underpin our current
work to further refine the aryl-substituted isobenzofuran-1(3H)-
one series in order to identify a suitable in vivo candidate.
Compound 42 (50 mg, 0.22 mmol) was suspended in EtOH
(10 mL), to which was added 2-thiohydantoin (26 mg, 0.23 mmol)
and piperidine (1 drop). This mixture was stirred at rt for 72 h. The
title compound 2 was collected by filtration, directly from the reac-
tion mixture, as a brown solid (57 mg, 80%); mp (EtOH) >300 °C. 1H
NMR [(CD3)2SO] d 12.20 (br s, 1 H), 12.02 (br s, 1H), 8.18 (br s, 1H),
8.15 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.44 (d, J = 3.8 Hz,
1H), 7.38 (d, J = 3.8 Hz, 1H), 6.44 (s, 1H), 5.49 (s, 2H). LRMS (APCI+)
calcd for
C
16H11N2O4S 327 (MH+), found 327. Anal.
(C16H10N2O4Sꢀ0.75H2O) C, H, N.
4.1.4. 5-(5-(1,3-Dioxolan-2-yl)thiophen-2-yl)isobenzofuran-
1(3H)-one (24)
4. Experimental section
4.1. Chemistry
5-Bromo-2-thiophenecarboxaldehyde was protected as the cyc-
lic acetal 4 according to a literature procedure43 then reacted with
22 according to general procedure A. Purification by flash column
chromatography on silica gel (CH2Cl2 as eluant) gave 24 as a cream
solid (56%). 1H NMR [(CD3)2SO] d 7.95 (br s, 1H), 7.84–7.90 (m, 2H),
7.63 (d, J = 3.8 Hz, 1H), 7.29 (d, J = 3.7 Hz, 1H), 6.08 (s, 1H), 5.43 (s,
2H), 4.01–4.11 (m, 2H), 3.92–4.00 (m, 2H). LRMS (APCI+) calcd for
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 Advance 400 MHz spectrometer and
referenced to Me4Si. Mass spectra were recorded either on a Varian
VG 7070 spectrometer at nominal 5000 resolution or a Finnigan
MAT 900Q spectrometer. All final compound purities were deter-
mined to be >95% by HPLC on an Alltech Alltima C18 column
C
15H13O4S 289 (MH+), found 289.
4.1.5. 5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thiophene-2-
carbaldehyde (43)
(3.2 ꢂ 150 mm, 5
lm) eluting with 5–80% MeCN/45 mM NH4HCO3.
Compound 24 was deprotected according to general procedure
B. Trituration with Et2O gave 43 as a beige solid (89%); mp (Et2O)
224–226 °C. 1H NMR [(CD3)2SO] d 9.96 (s, 1H), 8.12 (br s, 1H),
8.11 (d, J = 4.0 Hz, 1H), 8.02 (dd, J = 8.1, 1.5 Hz, 1H), 7.93 (d,
J = 7.9 Hz, 1H), 7.33 (d, J = 4.0 Hz, 1H), 5.47 (s, 2H). LRMS (APCI+)
calcd for C13H9O3S 245 (MH+), found 245. Anal. (C13H8O3) H, C; +0.5.
4.1.1. General procedure A: 5-(5-(1,3-dioxolan-2-yl)furan-2-yl)
isobenzofuran-1(3H)-one (23)
5-Bromo-2-furaldehyde was protected as the cyclic acetal 3
according to a literature procedure.43 The cyclic acetal (666 mg,
3.04 mmol) was dissolved in toluene (27 mL), to which was added

J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
1327
4.1.6. General procedure D: (E,Z)-5-((5-(1-oxo-1,3-dihydroiso
benzofuran-5-yl)thiophen-2-yl)methylene)-2-thioxoimidazoli
din-4-one (94)
1H), 7.99 (dd, J = 7.8, 1.2 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.81 (td,
J = 7.5, 1.4 Hz, 1H), 7.76 (d, J = 0.7 Hz, 1H), 7.63–7.70 (m, 2H),
7.55 (dd, J = 7.7, 0.8 Hz, 1H), 5.49 (s, 2H). LRMS (APCI+) calcd for
Compound 43 (50 mg, 0.21 mmol), 2-thiohydantoin (26 mg,
0.23 mmol) and b-alanine (20 mg, 0.23 mmol) were suspended in
AcOH (5 mL) and the mixture heated at reflux for 15 h. Upon cool-
ing, a yellow-brown solid crystallized out of solution and was col-
lected by filtration. This material did not require further
purification. The title compound 94 was isolated as an orange-
brown solid after drying under vacuum (52 mg, 78%); mp (AcOH)
>300 °C. 1H NMR [(CD3)2SO] d 12.40 (br s, 1H), 12.02 (br s, 1H),
7.99 (br s, 1H), 7.93 (dd, J = 8.1, 1.4 Hz, 1H), 7.83–7.91 (m, 3H),
6.65 (s, 1H), 5.45 (s, 2H). Anal. (C16H10N2O3S2) H, N, C; +0.5.
C
15H11O3 239 (MH+), found 239.
4.1.12. (E,Z)-5-(2-(1-Oxo-1,3-dihydroisobenzofuran-5-
yl)benzylidene)-2-thioxoimidazolidin-4-one (96)
Reaction of 45 with 2-thiohydantoin according to general pro-
cedure D gave a solid which was isolated by filtration from the
reaction mixture and did not require further purification. The de-
sired product 96 was obtained as a yellow solid (95%); mp (AcOH)
>300 °C. 1H NMR [(CD3)2SO] d 12.25 (br s, 2H), 7.92 (d, J = 7.9 Hz,
1H), 7.81–7.87 (m, 1H), 7.68 (d, J = 0.5 Hz, 1H), 7.49–7.57 (m,
3H), 7.41–7.46 (m, 1H), 6.15 (s, 1H), 5.45 (s, 2H). Anal.
(C18H12N2O3S) C, H, N.
4.1.7. 5-(5-(Dimethoxymethyl)thiophen-3-yl)isobenzofuran-
1(3H)-one (25)
4-Bromo-2-thiophenecarboxaldehyde was protected as the di-
methyl acetal 5 according to a literature procedure,44 then reacted
with 22 according to general procedure A. Purification by flash col-
umn chromatography on silica gel (20% EtOAc/hexanes as eluant)
gave 25 as a white solid (44%). 1H NMR [(CD3)2SO] d 8.11 (d,
J = 1.6 Hz, 1H), 8.01 (d, J = 0.6 Hz, 1H), 7.94 (dd, J = 8.0, 1.4 Hz,
1H), 7.85 (d, J = 8.0 Hz, 1H), 7.62 (dd, J = 1.5, 0.8 Hz, 1H), 5.69 (s,
1H), 5.43 (s, 2H), 3.28 (s, 6H). LRMS (APCI+) calcd for C15H15O4S
291 (MH+), found 291.
4.1.13. 5-(3-(Dimethoxymethyl)phenyl)isobenzofuran-1(3H)-
one (27)
3-Bromobenzaldehyde was protected as the dimethyl acetal 7
according to a literature procedure,44 then reacted with 22 accord-
ing to general procedure A. Purification by flash column chroma-
tography on silica gel (20% EtOAc/hexanes as eluant) gave 27 as a
white solid (59%). 1H NMR [(CD3)2SO] d 7.97 (d, J = 0.5 Hz, 1H),
7.93 (d, J = 8.0 Hz, 1H), 7.87 (dd, J = 8.0, 1.4 Hz, 1H), 7.72–7.77 (m,
2H), 7.52–7.58 (m, 1H), 7.47 (d, J = 7.7 Hz, 1H), 5.48 (s, 1H), 5.47
(s, 2H), 3.30 (s, 6H). LRMS (APCI+) calcd for C17H17O4 285 (MH+),
found 285.
4.1.8. General procedure E: 4-(1-oxo-1,3-dihydroisobenzofuran-
5-yl)thiophene-2-carbaldehyde (44)
Compound 25 (98 mg, 0.34 mmol) was dissolved in a mixture of
acetone (6 mL) and water (2 mL), then toluenesulfonic acid
(20 mg) added, and the mixture stirred at 50 °C for 3 h. After cool-
ing, the reaction mixture was partitioned between satd NaHCO3
solution (50 mL) and CH2Cl2 (50 mL). An additional extraction
was carried out with CH2Cl2 (50 mL), then the combined organic
extracts dried (Na2SO4), filtered, and the solvent removed under
reduced pressure to afford 44 which did not require further purifi-
cation and was isolated as a white solid (87%). 1H NMR [(CD3)2SO] d
10.00 (d, J = 1.3 Hz, 1H), 8.67 (t, J = 1.4 Hz, 1H), 8.59 (d, J = 2.0 Hz,
1H), 8.08 (d, J = 0.6 Hz, 1H), 8.01 (dd, J = 8.1, 1.4 Hz, 1H), 7.93 (d,
J = 7.9 Hz, 1H), 5.48 (s, 2H).
4.1.14. 3-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzaldehyde
(46)
Compound 27 was deprotected according to general procedure
E, giving 46 which did not require further purification and was iso-
lated as a cream solid (86%). 1H NMR [(CD3)2SO] d 10.13 (s, 1H),
8.30 (t, J = 1.6 Hz, 1H), 8.12 (dq, J = 7.7, 1.9, 1.1 Hz, 1H), 8.06 (t,
J = 0.9 Hz, 1H), 8.00 (dt, J = 7.7, 1.3 Hz, 1H), 7.97 (d, J = 1.1 Hz,
2H), 7.77 (t, J = 7.7 Hz, 1H), 5.49 (s, 2H). LRMS (APCI+) calcd for
C
15H11O3 239 (MH+), found 239.
4.1.15. (E,Z)-5-(3-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzyl
idene)-2-thioxoimidazolidin-4-one (97)
Reaction of 46 with 2-thiohydantoin according to general pro-
cedure D, followed by trituration with Et2O gave 97 as a yellow so-
lid (65%); mp (AcOH) 285–288 °C. 1H NMR [(CD3)2SO] d 12.33 (br s,
2H), 8.04 (br s, 2H), 7.92–8.00 (m, 2H), 7.81 (br d, J = 7.8 Hz, 1H),
7.74 (br d, J = 8.0 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 6.58 (s, 1H),
5.50 (s, 2H). LRMS (APCIꢁ) calcd for C18H11N2O3S 335 (MꢁH), found
335. Anal. (C18H12N2O3Sꢀ0.5H2O) C, H, N.
4.1.9. (E,Z)-5-((4-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thiophen-
2-yl)methylene)-2-thioxoimidazolidin-4-one (95)
Reaction of 44 with 2-thiohydantoin according to general pro-
cedure D, followed by trituration with dioxane gave 95 as a yellow
solid (80%); mp (AcOH) >300 °C. 1H NMR [(CD3)2SO] d 12.39 (s, 1H),
12.21 (s, 1H), 8.30–8.35 (m, 2H), 8.07 (br d, J = 8.3 Hz, 2H), 7.94 (d,
J = 8.1 Hz, 1H), 6.56 (s, 1H), 5.49 (s, 2H). LRMS (APCIꢁ) calcd for
C
16H9N2O3S2 341 (MꢁH), found 341. Anal. (C16H10N2O3S2ꢀ0.25H2O)
4.1.16. 5-(4-(Dimethoxymethyl)phenyl)isobenzofuran-1(3H)-
one (28)
C, H, N.
4-Bromobenzaldehyde was protected as the dimethyl acetal 8
according to a literature procedure,44 then reacted with 22 accord-
ing to general procedure A. Purification by flash column chroma-
tography on silica gel (20% EtOAc/hexanes as eluant) gave 28 as a
white solid (51%). 1H NMR [(CD3)2SO] d 7.97 (d, J = 0.4 Hz, 1H),
7.93 (dd, J = 8.0, 0.5 Hz, 1H), 7.88 (dd, J = 8.0, 1.4 Hz, 1H), 7.78 (d,
J = 8.4 Hz, 2H), 7.53 (d, J = 8.2 Hz, 2H), 5.47 (s, 2H), 5.46 (s, 1H),
3.30 (s, 6H). LRMS (APCI+) calcd for C17H17O4 285 (MH+), found 285.
4.1.10. 5-(2-(Dimethoxymethyl)phenyl)isobenzofuran-1(3H)-
one (26)
2-Bromobenzaldehyde was protected as the dimethyl acetal 6
according to a literature procedure,44 then reacted with 22 accord-
ing to general procedure A. Purification by flash column chroma-
tography on silica gel (20% EtOAc/hexanes as eluant) gave 26 as a
white solid (53%). 1H NMR [(CD3)2SO] d 7.92 (d, J = 7.8 Hz, 1H),
7.61–7.66 (m, 2H), 7.55 (dd, J = 7.9, 1.3 Hz, 1H), 7.44–7.51 (m,
2H), 7.27–7.33 (m, 1H), 5.47 (s, 2H), 5.14 (s, 1H), 3.28 (s, 3H),
3.16 (s, 3H). LRMS (APCI+) calcd for C17H17O4 285 (MH+), found 285.
4.1.17. 4-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzaldehyde
(47)
Compound 28 was deprotected according to general procedure
E to give 47 as a white solid (87%). 1H NMR [(CD3)2SO] d 10.10 (s,
1H), 8.03–8.08 (m, 3H), 7.95–8.02 (m, 4H), 5.49 (s, 2H). LRMS
(APCI+) calcd for C15H11O3 239 (MH+), found 239.
4.1.11. 2-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzaldehyde (45)
Compound 26 was deprotected according to general procedure
E to give 45 as a cream solid (93%). 1H NMR [(CD3)2SO] d 9.91 (s,

1328
J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
4.1.18. (E,Z)-5-(4-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzyl
idene)-2-thioxoimidazolidin-4-one (98)
2H). LRMS (APCIꢁ) calcd for C17H10N3O3S 336 (MꢁH), found 336.
Anal. (C17H11N3O3S) C, H, N.
Reaction of 47 with 2-thiohydantoin according to general pro-
cedure D gave 98 as a yellow solid (89%); mp (AcOH) >300 °C. 1H
NMR [(CD3)2SO] d 12.29 (s, 2H), 8.03 (br s, 1H), 7.88–7.97 (m,
4H), 7.83 (d, J = 8.5 Hz, 2H), 6.54 (s, 1H), 5.48 (s, 2H). LRMS (APCIꢁ)
calcd for C18H11N2O3S 335 (MꢁH), found 335. Anal. (C18H12N2O3S)
C, H, N.
4.1.25. 5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-1H-pyrrole-2-
carbaldehyde (64)
Compound 62 (1.01 g, 4.74 mmol) and 1-(t-butoxycar-
bonyl)pyrrole-2-boronic acid 61 were reacted according to general
procedure A and partially purified by flash column chromatogra-
phy on silica gel (10% EtOAc/hexanes as eluant). The crude product,
tert-butyl 2-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-1H-pyrrole-1-
carboxylate 63, was obtained as a pale yellow oil (704 mg, 50%)
and used directly in the next step.
4.1.19. 5-(6-(Dimethoxymethyl)pyridin-3-yl)isobenzofuran-
1(3H)-one (29)
2-Bromopyridine-5-carboxaldehyde was protected as the di-
methyl acetal 9 according to a literature procedure,44 then reacted
with 22 according to general procedure A. Purification by flash col-
umn chromatography on silica gel (CH2Cl2, followed by 10% ace-
tone/CH2Cl2 as eluant) gave 29 as a white solid (34%). 1H NMR
[(CD3)2SO] d 8.79 (s, 1H), 8.19 (s, 1H), 8.13 (d, J = 8.1 Hz, 1H),
8.01 (d, J = 8.0 Hz, 1H), 7.91 (dd, J = 8.2, 2.0 Hz, 1H), 7.82 (d,
J = 8.2 Hz, 1H), 5.52 (s, 1H), 5.45 (s, 2H), 3.38 (s, 6H). LRMS (APCI+)
calcd for C16H16NO4 286 (MH+), found 286.
DMF (150 mg, 2.06 mmol) was added to a flask and cooled to
0 °C. POCl3 (316 mg, 2.06 mmol) was added dropwise and the mix-
ture stirred without cooling for 15 min. 1,2-Dichloroethane
(1.0 mL) was added and the mixture cooled back to 0 °C. A solution
of 63 (559 mg, 1.87 mmol) in dichloroethane (1.0 mL) was added
dropwise and the resulting bright orange solution heated at reflux
for 15 min. Upon cooling to room temperature, NaOAc (845 mg,
10.3 mmol) in water (3.7 mL) was added and heating at reflux con-
tinued for a further 15 min. The mixture was allowed to cool, then
extracted with CH2Cl2 (4 ꢂ 50 mL) and the combined organic frac-
tions washed with satd NaHCO3 (100 mL) and dried (Na2SO4). Fil-
tration and removal of the solvent under reduced pressure gave a
crude product (with the BOC deprotected) which was purified by
flash column chromatography on silica gel (5% acetone/CH2Cl2 as
eluant), followed by trituration with Et2O. The title compound 64
was isolated as a beige solid (95 mg, 22%); mp (Et2O) 275–
278 °C. 1H NMR [(CD3)2SO] d 12.64 (s, 1H), 9.57 (s, 1H), 8.16 (s,
1H), 8.11 (dd, J = 1.8, 1.4 Hz, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.13 (dd,
J = 3.9, 2.2 Hz, 1H), 6.99 (dd, J = 4.0, 2.4 Hz, 1H), 5.45 (s, 2H). LRMS
(APCI+) calcd for C13H10NO3 228 (MH+), found 228. Anal.
(C13H9NO3) C, H, N.
4.1.20. 5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)picolinalde
hyde (48)
Compound 29 was deprotected according to general procedure
E to give 48 as a white solid (41%). 1H NMR [(CD3)2SO] d 10.18 (s,
1H), 9.22 (dd, J = 2.1, 0.8 Hz, 1H), 8.48 (br s, 1H), 8.37–8.42 (m,
2H), 8.33 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 8.2 Hz, 1H), 5.52 (s, 2H).
LRMS (APCI+) calcd for C14H10NO3 240 (MH+), found 240.
4.1.21. (E,Z)-5-((6-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)pyri
dine-3-yl)methylene (99)
Reaction of 48 with 2-thiohydantoin according to general pro-
cedure D gave 99 as a yellow solid (89%); mp (AcOH) >300 °C. 1H
NMR [(CD3)2SO] d 12.38 (br s, 2H), 9.00 (d, J = 2.2 Hz, 1H), 8.45 (s,
1H), 8.38 (d, J = 8.1 Hz, 2H), 8.17 (d, J = 8.4 Hz, 1H), 7.97 (d,
J = 8.1 Hz, 1H), 6.57 (s, 1H), 5.50 (s, 2H). LRMS (APCIꢁ) calcd for
4.1.26. (E,Z)-5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-1H-
pyrrol-2-yl)methylene)-2-thioxoimidazolidin-4-one (101)
Reaction of 64 with 2-thiohydantoin according to general pro-
cedure D gave 101 as a dark red solid (58%); mp (dioxane)
>300 °C. 1H NMR [(CD3)2SO] d 13.01 (s, 1H), 12.51 (br s, 1H),
12.19 (br s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.88 (br s, 1H), 7.85 (dd,
J = 8.0, 1.3 Hz, 1H), 7.05 (br dd, J = 3.8, 2.5 Hz, 1H), 6.99 (br dd,
J = 3.8, 2.0 Hz, 1H), 6.65 (s, 1H), 5.45 (s, 2H). Anal.
(C16H11N3O3Sꢀ0.25AcOH) C, H, N.
C17H10N3O3S 336 (MꢁH), found 336. Anal. (C17H11N3O3S) C, H, N.
4.1.22. 5-(6-(Dimethoxymethyl)pyridin-3-yl)isobenzofuran-
1(3H)-one (30)
5-Bromopyridine-2-carboxaldehyde was protected as the di-
methyl acetal 10 according to a literature procedure,44 then re-
acted with 22 according to general procedure A. Purification by
flash column chromatography on silica gel (CH2Cl2, followed by
10% acetone/CH2Cl2 as eluant) gave 30 as a off-white solid (68%).
1H NMR (CDCl3) d 8.87 (d, J = 1.9 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H),
7.97 (dd, J = 8.1, 2.4 Hz, 1H), 7.74 (d, J = 8.1 Hz, 1H), 7.66–7.72
(m, 2H), 5.45 (s, 1H), 5.40 (s, 2H), 3.45 (s, 6H). LRMS (APCI+) calcd
for C16H16NO4 286 (MH+), found 286.
4.1.27. 1-Oxo-N-(prop-2-ynyl)-1,3-dihydroisobenzofuran-5-car
boxamide (71)
1-Oxo-1,3-dihydroisobenzofuran-5-carboxylic acid 65 (370 mg,
2.08 mmol) was dissolved in THF (15 mL). To this solution was
added pyridine (1.64 g, 20.8 mmol), followed by pentafluorophe-
nyltrifluoroacetate (2.91 g, 10.4 mmol) and the resulting mixture
stirred at rt for 2 h. All solvent was removed under reduced pres-
sure to give an oil which was dissolved in EtOAc (100 mL) and
washed with 1 M HCl (2 ꢂ 50 mL), water (50 mL), satd NaHCO3
(2 ꢂ 50 mL) and brine (50 mL). The organic layer was dried
(Na2SO4), filtered, and the solvent removed under reduced pressure
to give a crude product which was purified by flash column chro-
matography on silica gel (20% EtOAc/hexanes as eluant) to give
the intermediate pentafluorophenyl ester (666 mg, 93%) which
was used immediately in the next step.
4.1.23. 5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)picolinalde
hyde (49)
Compound 30 was deprotected according to general procedure
E to give 49 as a off-white solid (88%). 1H NMR [(CD3)2SO] d 10.06
(s, 1H), 9.23 (dd, J = 2.3, 0.7 Hz, 1H), 8.44 (ddd, J = 8.1, 2.3, 0.7 Hz,
1H), 8.16 (d, J = 0.7 Hz, 1H), 7.98–8.09 (m, 3H), 5.51 (s, 2H). LRMS
(APCI+) calcd for C14H10NO3 240 (MH+), found 240.
4.1.24. (E,Z)-5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)pyri
din-2-yl)methylene)-2-thioxoimidazolidin-4-one (100)
The above ester (666 mg, 1.94 mmol) was dissolved in THF
(15 mL), to which was added propargylamine (160 mg, 2.90 mmol).
This mixture was stirred at rt for 0.5 h, then all solvent removed un-
der reduced pressure to yield a crude solid which was purified by
flash column chromatography on silica gel (CH2Cl2 followed by 5%
MeOH/CH2Cl2 as eluant). The title compound 71 was isolated as
white needles (350 mg, 84%); mp (Et2O) 242–245 °C dec. 1H NMR
Reaction of 49 with 2-thiohydantoin according to general pro-
cedure D, gave 100 as a yellow-green solid (80%); mp (AcOH)
>300 °C. 1H NMR [(CD3)2SO] d 12.54 (br s, 1H), 11.52 (br s, 1H),
9.15 (d, J = 2.4 Hz, 1H), 8.29 (dd, J = 8.2, 2.5 Hz, 1H), 8.09 (s, 1H),
7.96–8.02 (m, 2H), 7.87 (d, J = 8.2 Hz, 1H), 6.69 (s, 1H), 5.50 (s,

J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
1329
[(CD3)2SO] d 9.19 (t, J = 5.5 Hz, 1H), 8.10 (s, 1H), 8.01 (dd, J = 8.0,
1.4 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 5.47 (s, 2H), 4.10 (dd, J = 5.5,
2.5 Hz, 2H), 3.14 (t, J = 2.5 Hz, 1H). LRMS (APCI+) calcd for
chromatography on silica gel (20% EtOAc/hexanes as eluant) gave
32 as a off-white solid (16%). 1H NMR [(CD3)2SO] d 8.27 (d,
J = 0.6 Hz, 1H), 8.14 (dt, J = 8.0, 0.8 Hz, 1H), 7.96 (dd, J = 8.0,
0.4 Hz, 1H), 7.84 (d, J = 0.7 Hz, 1H), 5.59 (d, J = 0.7 Hz, 1H), 5.48
(s, 2H), 3.34 (s, 6H). LRMS (APCI+) calcd for C14H14NO4S 292
(MH+), found 292.
C
12H10NO3 216 (MH+), found 216. Anal. (C12H9NO3) C, H, N.
4.1.28. 2-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)oxazole-5-carb
aldehyde (72)
PdCl2(BnCN)2 (9 mg, 0.02 mmol) and CuCl2.2H2O (8 mg,
0.05 mmol) in DMF were stirred at rt for 1 h. A solution of 71
(100 mg, 0.47 mmol) was added and the entire mixture stirred at
100 °C under an atmosphere of oxygen for 2 h. Upon cooling, the
reaction mixture was partitioned between EtOAc (50 mL) and brine
(50 mL). A precipitate formed which was collected by filtration.
The EtOAc fraction was dried (Na2SO4) and the solvent removed
under reduced pressure to give a solid which was combined with
the above precipitate. The crude product was purified by flash col-
umn chromatography on silica gel (5% MeOH/CH2Cl2as eluant) to
give 72 as a cream solid (18 mg, 17%). 1H NMR [(CD3)2SO] d 9.87
(s, 1H), 8.42 (s, 2H), 8.28 (dd, J = 8.0, 1.4 Hz, 1H), 8.05 (d,
J = 8.1 Hz, 1H), 5.52 (s, 2H). HRMS (FAB+) calcd for C12H8NO4
230.0453 (MH+), found 230.0451.
4.1.34. 4-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thiazole-2-carb
aldehyde (51)
Compound 32 was deprotected according to general procedure
E to give 51 as a white solid (100%). 1H NMR [(CD3)2SO] d 10.02 (s,
1H), 8.88 (s, 1H), 8.35 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.00 (d,
J = 8.0 Hz, 1H), 5.50 (s, 2H). HRMS (ESI-TOF) calcd for C12H8NO3SNa
268.0044 (M+Na), found 268.0039.
4.1.35. (E,Z)-5-((4-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thia
zol-2-yl)methylene)-2-thioxoimidazolidin-4-one (104)
Reaction of 51 with 2-thiohydantoin according to general pro-
cedure D gave 104 as a yellow solid (73%); mp (AcOH) >295 °C.
1H NMR [(CD3)2SO] d 12.45 (br s, 1H), 11.26 (br s, 1H), 8.43 (s,
1H), 8.38 (dd, J = 8.0, 1.4 Hz, 1H), 8.35 (s, 1H), 8.02 (d, J = 7.9 Hz,
1H), 6.66 (s, 1H), 5.53 (s, 2H). LRMS (APCIꢁ) calcd for C15H8N3O3S2
342 (MꢁH), found 342. Anal. (C15H9N3O3S2) C, H, N.
4.1.29. (E,Z)-5-((2-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)oxa
zol-5-yl)methylene)-2-thioxoimidazolidin-4-one (102)
Reaction of 72 with 2-thiohydantoin according to general pro-
cedure D gave 102 as a bright yellow solid (71%); mp (dioxane)
>295 °C. 1H NMR [(CD3)2SO] d 12.54 (br s, 1H), 12.32 (br s, 1H),
8.45–8.51 (m, 2H), 8.06 (dd, J = 8.0, 0.4 Hz, 1H), 8.00 (d, J = 0.4 Hz,
1H), 6.54 (s, 1H), 5.54 (s, 2H). LRMS (APCIꢁ) calcd for C15H8N3O4S
326 (MꢁH), found 326. Anal. (C15H9N3O4S) C, H, N.
4.1.36. 5-(4-(Dimethoxymethyl)thiazol-2-yl)isobenzofuran-
1(3H)-one (33)
2-Bromo-1,3-thiazole-4-carboxaldehyde was protected as the
dimethyl acetal 13 according to a literature procedure,44 then re-
acted with 22 according to general procedure A. Purification by
flash column chromatography on silica gel (20% EtOAc/hexanes
as eluant) gave 33 as a pale pink solid (51%). 1H NMR [(CD3)2SO]
d 8.45 (s, 1H), 8.26 (s, 1H), 8.18 (dd, J = 8.1, 1.2 Hz, 1H), 7.92 (d,
J = 8.0 Hz, 1H), 5.72 (s, 1H), 5.47 (s, 2H), 3.41 (s, 6H). LRMS (APCI+)
calcd for C14H14NO4S 292 (MH+), found 292.
4.1.30. 5-(5-(Dimethoxymethyl)thiazol-2-yl)isobenzofuran-
1(3H)-one (31)
2-Bromo-1,3-thiazole-5-carboxaldehyde was protected as the
dimethyl acetal 11 according to a literature procedure,44 then re-
acted with 22 according to general procedure A. Purification by
flash column chromatography on silica gel (20% EtOAc/hexanes
as eluant) gave 31 as a white solid (25%). 1H NMR [(CD3)2SO] d
8.25 (s, 1H), 8.15 (dt, J = 8.0, 0.6 Hz, 1H), 7.98 (d, J = 0.7 Hz, 1H),
7.95 (d, J = 8.0 Hz, 1H), 5.81 (d, J = 0.7 Hz, 1H), 5.48 (s, 2H), 3.35
(s, 6H). LRMS (APCI+) calcd for C14H14NO4S 292 (MH+), found 292.
4.1.37. 2-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thiazole-4-
carbaldehyde (52)
Compound 33 was deprotected according to general procedure
E to give 52 as a white solid (100%). 1H NMR [(CD3)2SO] d 10.05 (d,
J = 1.2 Hz, 1H), 8.92 (d, J = 1.2 Hz, 1H), 8.34–8.36 (m, 1H), 8.26–8.29
(m, 1H), 7.98 (d, J = 7.9 Hz, 1H), 5.50 (s, 2H). HRMS (ESI-TOF) calcd
for C12H7NO3SNa 268.0044 (M+Na), found 268.0039.
4.1.31. 2-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thiazole-5-carb
aldehyde (50)
4.1.38. (E,Z)-5-((2-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thia
zol-4-yl)methylene)-2-thioxoimidazolidin-4-one (105)
Compound 31 was deprotected according to general procedure
E to give 50 as a cream solid (100%). 1H NMR [(CD3)2SO] d 10.13 (s,
1H), 8.86 (s, 1H), 8.40 (d, J = 0.7 Hz, 1H), 8.27 (dt, J = 8.0, 0.7 Hz,
1H), 8.01 (d, J = 8.2 Hz, 1H), 5.51 (s, 2H). HRMS (FAB+) calcd for
Reaction of 52 with 2-thiohydantoin according to general pro-
cedure D gave 105 as a yellow-brown solid (88%); mp (AcOH)
>295 °C. 1H NMR [(CD3)2SO] d 12.62 (br s, 1H), 11.43 (br s, 1H),
8.83 (s, 1H), 8.54 (s, 1H), 8.37 (dd, J = 8.1, 1.2 Hz, 1H), 7.97 (d,
J = 8.0 Hz, 1H), 6.84 (s, 1H), 5.52 (s, 2H). LRMS (APCIꢁ) calcd for
C
12H8NO3S 246.0225 (MH+), found 246.0224.
4.1.32. (E,Z)-5-((2-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thia
zol-5-yl)methylene)-2-thioxoimidazolidin-4-one (103)
C15H8N3O3S2 342 (MꢁH), found 342. Anal. (C15H9N3O3S2) C, H, N.
Reaction of 50 with 2-thiohydantoin according to general pro-
cedure D gave 103 as a yellow solid (89%); mp (AcOH) >300 °C.
4.1.39. General procedure F: 5-(hydroxymethyl)isobenzofuran-
1(3H)-one (66)
1H NMR [(CD3)2SO] Observe E- and Z-isomers separately.
d
Compound 65 (3.00 g, 16.8 mmol) was dissolved in THF
(300 mL), to which was added carbonyldiimidazole (CDI; 5.46 g,
33.7 mmol). After stirring at rt for 2 h., this reaction mixture was
slowly added to a solution of NaBH4 (3.18 g, 84.0 mmol) in water
(80 mL), stirred for a further 5 minutes, then quenched with concd
HCl to pH 2. This aqueous mixture was extracted with EtOAc
(2 ꢂ 50 mL), the fractions combined, dried (Na2SO4) and filtered,
then the solvent removed under reduced pressure to afford a crude
solid which was purified by flash column chromatography on silica
gel (EtOAc as eluant). The title compound 66 was isolated as a
white solid (2.59 g, 94%). 1H NMR in agreement with literature
values.45
12.14–12.57 (m, 2H), 8.58 (d, J = 0.6 Hz, 0.7H), 8.48 (d, J = 0.5 Hz,
0.3H), 8.26–8.30 (m, 1H), 8.12–8.19 (m, 1H), 7.95–8.01 (m, 1H),
6.94 (s, 0.3H), 6.82 (s, 0.7H), 5.50 (s, 1.4H), 5.49 (s, 0.6H). LRMS
(APCIꢁ) calcd for C15H8N3O3S2 342 (MꢁH), found 342. Anal.
(C15H9N3O3S2ꢀ0.25H2O) C, H, N.
4.1.33. 5-(2-(Dimethoxymethyl)thiazol-4-yl)isobenzofuran-
1(3H)-one (32)
4-Bromo-2-formyl-1,3-thiazole was protected as the dimethyl
acetal 12 according to a literature procedure,44 then reacted with
22 according to general procedure A. Purification by flash column

1330
J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
4.1.40. General procedure G: 1-oxo-1,3-dihydroisobenzofuran-
5-carbaldehyde (67)
as eluant) to give 76 as a cream solid (244 mg, 80%); mp (CH2Cl2/
hexane) 195–198 °C. 1H NMR [(CD3)2SO]
8.24 (dd, J = 1.1,
d
Compound 66 (1.0 g, 6.10 mmol) was dissolved in CH2Cl2
(200 mL), pyridinium chlorochromate on silica gel46 (6.71 g of
1 mmol/g silica) added, and the mixture stirred at rt for 3 h. All sol-
vent was then removed under reduced pressure and the resulting
powder loaded onto a column of flash silica gel which was eluted
with CH2Cl2 to obtain 67 as a white solid (620 mg, 63%). 1H NMR
in agreement with literature values.45,47
0.7 Hz, 1H), 8.11 (dt, J = 8.0, 0.7 Hz, 1H), 8.02 (dd, J = 8.1, 0.4 Hz,
1H), 5.50 (s, 2H). LRMS (APCI+) calcd for C10H6NO4S 236 (MH+),
found 236. Anal. (C10H5NO4S) C, H, N.
4.1.46. Ethyl 3-(1-oxo-1,3-dihydroisobenzofuran-5-yl)isothiaz
ole-4-carboxylate (77)
A solution of 76 (240 mg, 1.02 mmol) and ethyl propiolate
(400 mg, 4.08 mmol) in 1,2-dichlorobenzene (2 mL) was heated
at 150 °C for 15 h. All solvent was removed under reduced pressure
and the resulting mixture of two isomers (273 mg, 93% total yield)
partially separated by flash column chromatography on silica gel
(20% EtOAc/hexanes as eluant). The title compound 77 was isolated
as a white solid (53 mg, 18%). 1H NMR [(CD3)2SO] d 9.78 (s, 1H),
7.91 (d, J = 8.0 Hz, 1H), 7.88 (br s, 1H), 7.77 (dt, J = 7.9, 0.6 Hz,
1H), 5.48 (s, 2H), 4.21 (q, J = 7.1 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H).
LRMS (APCI+) calcd for C14H12NO4S 290 (MH+), found 290.
4.1.41. (E,Z)-1-Oxo-1,3-dihydroisobenzofuran-5-carbaldehyde
oxime (68)
Compound 67 (445 mg, 2.75 mmol) was dissolved in MeOH
(40 mL), to which was added pyridine (1.09 g, 13.7 mmol) and
NH2OHꢀHCl (954 mg, 13.7 mmol). The reaction mixture was stirred
at 60 °C for 1.5 h, then upon cooling was diluted with water
(100 mL), with the resulting white precipitate collected by filtra-
tion and dried to give 68 (325 mg, 67%). 1H NMR in agreement with
literature values.47
4.1.47. General procedure H: 3-(1-oxo-1,3-dihydroisobenzofu
ran-5-yl)isothiazole-4-carboxylic acid (78)
4.1.42. 5-(5-(Hydroxymethyl)isoxazol-3-yl)isobenzofuran-1(3H)-
one (69)
Compound 77 (53 mg, 0.18 mmol) was suspended in EtOH
(10 mL), to which was added 2 M NaOH (10 mL). This mixture
was stirred at 50 °C for 4 h., allowed to cool, and partitioned be-
tween 1 M HCl (20 mL) and EtOAc (20 mL). The aqueous layer
was further extracted with EtOAc (2 ꢂ 20 mL), then the combined
organic fractions dried (Na2SO4), filtered, and the solvent removed
under reduced pressure. The resulting crude solid was dissolved in
1:1 TFA/CH2Cl2 (4 mL) and stirred at rt for 4 h. Removal of the reac-
tion solvent under reduced pressure gave 78 as a white solid
(47 mg, 98%) which was isolated without further purification. 1H
NMR [(CD3)2SO] d 13.15 (br s, 1H), 9.72 (s, 1H), 7.90 (d, J = 8.0 Hz,
1H), 7.87 (s, 1H), 7.77 (dt, J = 7.9, 0.7 Hz, 1H), 5.47 (s, 2H). LRMS
(APCI+) calcd for C12H8NO4S 262 (MH+), found 262.
Compound 68 (320 mg, 1.81 mmol) was dissolved in THF
(10 mL) then pyridine (1 drop) and N-chlorosuccinimide (266 mg,
1.99 mmol) added. This mixture was heated at 60 °C for 0.5 h.
Upon cooling to rt, propargyl alcohol (132 mg, 2.35 mmol) and tri-
ethylamine (201 mg, 1.99 mmol) were added and a white precipi-
tate formed. The reaction was then heated at 50 °C for 2 h. The
solvent was removed under reduced pressure and the residue ta-
ken up into CH2Cl2 (100 mL), which was then washed with water
(2 ꢂ 100 mL), brine (100 mL) and dried (Na2SO4). Removal of the
solvent afforded a white solid which was purified by flash column
chromatography on silica gel (10% acetone/CH2Cl2 as eluant) to
give 69 as a fluffy white solid (56%). 1H NMR [(CD3)2SO] d 8.19
(d, J = 0.4 Hz, 1H), 8.04 (dt, J = 8.0, 0.7 Hz, 1H), 7.97 (d, J = 8.1 Hz,
1H), 7.05 (s, 1H), 5.74 (t, J = 6.0 Hz, 1H), 5.49 (s, 2H), 4.65 (d,
J = 5.7 Hz, 2H). LRMS (APCI+) calcd for C12H10NO4 232 (MH+), found
232.
4.1.48. 5-(4-(Hydroxymethyl)isothiazol-3-yl)isobenzofuran-
1(3H)-one (79)
Compound 78 was reduced according to general procedure F to
give 79 as a pale yellow waxy solid (37 mg, 84%). 1H NMR
[(CD3)2SO] d 9.05 (s, 1H), 8.06 (s, 1H), 8.00 (dt, J = 8.0, 0.7 Hz,
1H), 7.95 (d, J = 7.9 Hz, 1H), 5.49 (br s, 3H), 4.64 (d, J = 0.6 Hz,
2H). LRMS (APCI+) calcd for C12H10NO3S 248 (MH+), found 248.
4.1.43. 3-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)isoxazole-5-car
baldehyde (70)
Compound 69 was oxidized to the corresponding aldehyde
according to general procedure G. Purification by flash column
chromatography on silica gel (CH2Cl2 as eluant) gave 70 as a white
solid (65%). 1H NMR [(CD3)2SO] d 10.00 (s, 1H), 8.30 (s, 1H), 8.18
(dt, J = 8.0, 0.7 Hz, 1H), 8.08 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 5.52
(s, 2H). LRMS (APCI+) calcd for C12H8NO4 230 (MH+), found 230.
4.1.49. 3-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)isothiazole-4-
carbaldehyde (80)
Compound 79 was oxidized to the corresponding aldehyde
according to general procedure G to give 80 as a white solid
(84%). 1H NMR [(CD3)2SO] d 10.01 (s, 1H), 9.98 (s, 1H), 8.00–8.02
(m, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.92 (dt, J = 7.9, 0.7 Hz, 1H), 5.50
(s, 2H). LRMS (APCI+) calcd for C12H8NO3S 246 (MH+), found 246.
4.1.44. (E,Z)-5-((3-(1-Oxo-1,3-dihydroisobenzofuran-5-
yl)isoxazol-5-yl)methylene)-2-thioxoimidazolidin-4-one (106)
Reaction of 70 with 2-thiohydantoin according to general pro-
cedure D gave 106 as a yellow solid (86%); mp (AcOH) >300 °C.
1H NMR [(CD3)2SO] Observe E- and Z-isomers separately. d 12.65
(br s, 1H), 12.29 (br s, 1H), 8.19 (br s, 0.1H), 8.17 (br s, 0.9H),
8.03–8.09 (m, 2H), 7.95 (s, 0.1H), 7.88 (s, 0.9H), 6.50 (s, 0.1H),
6.44 (s, 0.9H), 5.52 (s, 1.8H), 5.50 (s, 0.2H). LRMS (APCIꢁ) calcd
for C15H8N3O4S 326 (MꢁH), found 326. Anal. (C15H9N3O4Sꢀ0.5H2O)
C, H, N.
4.1.50. (E,Z)-5-((3-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)isot
hiazol-4-yl)methylene)-2-thioxoimidazolidin-4-one (107)
Reaction of 80 with 2-thiohydantoin according to general pro-
cedure D gave 107 as a yellow solid (86%); mp (AcOH) >300 °C.
1H NMR [(CD3)2SO] d 12.32 (br s, 2H), 9.70 (d, J = 0.5 Hz, 1H),
8.00 (d, J = 7.9 Hz, 1H), 7.93 (s, 1H), 7.81 (dt, J = 8.0, 0.7 Hz, 1H),
6.25 (s, 1H), 5.49 (s, 2H). LRMS (APCIꢁ) calcd for C15H8N3O3S2
342 (MꢁH), found 342. Anal. (C15H9N3O3S2) C, H, N.
4.1.45. 5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-1,3,4-oxathia
zol-2-one (76)
4.1.51. Ethyl 3-(1-oxo-1,3-dihydroisobenzofuran-5-yl)isothiaz
ole-5-carboxylate (81)
The mixture of two isomers (273 mg, 93% total yield) isolated
above was partially separated by flash column chromatography
on silica gel (20% EtOAc/hexanes as eluant). The title compound
81 was isolated as a white solid (119 mg, 40%). 1H NMR [(CD3)2SO]
Compound 75 (230 mg, 1.30 mmol) was suspended in dioxane
(20 mL), chlorocarbonylsulfenyl chloride (340 mg, 2.60 mmol)
added, and the mixture heated at reflux for 15 h. Upon cooling,
all solvent was removed under reduced pressure and the resulting
solid purified by flash column chromatography on silica gel (CH2Cl2

J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
1331
d 8.65 (s, 1H), 8.42 (s, 1H), 8.33 (dt, J = 8.1, 0.7 Hz, 1H), 7.96 (d,
J = 8.1 Hz, 1H), 5.49 (s, 2H), 4.40 (q, J = 7.1 Hz, 2H), 1.36 (t,
J = 7.1 Hz, 3H). LRMS (APCI+) calcd for C14H12NO4S 290 (MH+),
found 290.
4.1.58. 5-Bromo-2-(dimethoxymethyl)benzo[b]thiophene (15)
5-Bromo-2-methylbenzo[b]thiophene35 was oxidized to 5-bro-
mobenzo[b]thiophene-2-carbaldehyde using a previously reported
method.36 This aldehyde was then protected as the dimethyl acetal
according a literature procedure44 to give 15 as a light orange solid
(44%). 1H NMR (CDCl3) d 7.89 (d, J = 1.8 Hz, 1H), 7.67 (d, J = 8.6 Hz,
1H), 7.41 (dd, J = 8.6, 1.8 Hz, 1H), 7.25 (s, 1H), 5.68 (d, J = 1.0 Hz,
1H), 3.41 (s, 6H). LRMS (APCI+) calcd for C10H8BrOS 255
(M+ꢁMeOH), found 255.
4.1.52. 5-(5-(Hydroxymethyl)isothiazol-3-yl)isobenzofuran-
1(3H)-one (83)
Compound 81 was deprotected to the corresponding carboxylic
acid, 3-(1-oxo-1,3-dihydroisobenzofuran-5-yl)isothiazole-5-car-
boxylic acid 82, according to general procedure H. Without further
purification, the crude acid was then reduced according to general
procedure F to give 83 as a yellow-white solid (49%). 1H NMR
[(CD3)2SO] d 8.28 (s, 1H), 8.20 (dt, J = 8.0, 0.7 Hz, 1H), 7.94 (d,
J = 8.1 Hz, 1H), 7.87 (t, J = 1.0 Hz, 1H), 5.97 (t, J = 5.6 Hz, 1H), 5.48
(s, 2H), 4.90 (dd, J = 5.6, 1.0 Hz, 2H). LRMS (APCI+) calcd for
4.1.59. 5-(2-(Dimethoxymethyl)benzo[b]thiophen-5-yl)isoben
zofuran-1(3H)-one (35)
Compound 15 was reacted with 22 according to general proce-
dure A. The title compound 35 was obtained as a white solid (56%).
1H NMR (CDCl3) d 8.00 (d, J = 8.6 Hz, 1H), 7.98 (s, 1H), 7.93 (d,
J = 8.4 Hz, 1H), 7.80 (dt, J = 8.0, 0.7 Hz, 1H), 7.72 (dd, J = 1.2,
0.7 Hz, 1H), 7.57 (dd, J = 8.4, 1.8 Hz, 1H), 7.40 (s, 1H), 5.73 (d,
J = 1.0 Hz, 1H), 5.39 (s, 2H), 3.44 (s, 6H). LRMS (APCI+) calcd for
C19H17O4S 341 (MH+), found 341.
C
12H10NO3S 248 (MH+), found 248.
4.1.53. 3-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)isothiazole-5-
carbaldehyde (84)
Compound 83 was oxidized according to general procedure G to
give 84 as a white solid (48%). 1H NMR [(CD3)2SO] d 10.21 (s, 1H),
8.78 (s, 1H), 8.38 (t, J = 0.7 Hz, 1H), 8.29 (dt, J = 8.0, 0.7 Hz, 1H), 8.00
(d, J = 8.0 Hz, 1H), 5.51 (s, 2H). LRMS (APCI+) calcd for C12H8NO3S
246 (MH+), found 246.
4.1.60. 5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzo[b]thio
phene-2-carbaldehyde (54)
Compound 35 was deprotected according to general procedure
E to give 54 as a white solid (0.19 g, 99%). 1H NMR (CDCl3) d 10.16
(s, 1H), 8.17 (d, J = 1.5 Hz, 1H), 8.11 (s, 1H), 8.03 (d, J = 7.9 Hz, 1H),
8.02 (d, J = 8.5 Hz, 1H), 7.81 (dt, J = 8.0, 0.7 Hz, 1H), 7.76 (dd, J = 8.5,
1.8 Hz, 1H), 7.74 (dd, J = 1.4, 0.7 Hz, 1H), 5.41 (s, 2H). LRMS (APCI+)
calcd for C17H11O3S 295 (MH+), found 295.
4.1.54. (E,Z)-5-((3-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)isot
hiazol-5-yl)methylene)-2-thioxoimidazolidin-4-one (108)
Reaction of 84 with 2-thiohydantoin according to general pro-
cedure D gave 108 as a mustard-yellow solid (79%); mp (AcOH)
>300 °C. 1H NMR [(CD3)2SO] Observe E- and Z-isomers separately.
d 12.59 (br s, 1H), 12.42 (br s, 0.3H), 12.33 (br s, 0.7H), 8.60 (s,
0.7H), 8.42 (s, 0.3H), 8.34 (s, 0.7H), 8.28–8.33 (m, 1H), 8.21 (dd,
J = 8.1, 1.2 Hz, 0.3H), 8.02 (d, J = 8.0 Hz, 0.7H), 7.96 (d, J = 8.0 Hz,
0.3H), 6.97 (s, 0.3H), 6.83 (s, 0.7H), 5.52 (s, 1.4H), 5.49 (s, 0.6H).
LRMS (APCIꢁ) calcd for C15H8N3O3S2 342 (MꢁH), found 342. Anal.
(C15H9N3O3S2) C, H, N.
4.1.61. (E,Z)-5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzo
[b]thiophen-2-yl)methylene)-2-thioxoimidazolidin-4-one (110)
Reaction of 54 with 2-thiohydantoin was carried out according
to general procedure D to give 110 as a bright yellow solid (90%);
mp (AcOH) >305 °C. 1H NMR [(CD3)2SO] Observe E- and Z-isomers
separately. d12.03–12.60 (br m, 2H), 8.20 (d, J = 1.6 Hz, 1H), 8.19 (s,
1H), 8.14 (d, J = 8.5 Hz, 1H), 8.11 (s, 0.1H), 8.04 (s, 0.9H), 7.95–7.99
(m, 2H), 7.79 (dd, J = 8.5, 1.8 Hz, 1H), 6.92 (s, 0.1H), 6.66 (s, 0.9H),
5.50 (s, 2H). HRMS (ESIꢁ) calcd for C20H11N2O3S2 391.0217
(MꢁH), found 391.0218. Anal. (C20H12N2O3S2) C, H, N.
4.1.55. 5-(3-(Dimethoxymethyl)benzo[b]thiophen-5-yl)isoben
zofuran-1(3H)-one (34)
5-Bromobenzothiophene-3-carboxaldehyde was protected as
the dimethyl acetal 14 according to a literature procedure,44 then
reacted with 22 according to general procedure A. Purification by
flash column chromatography on silica gel (20% EtOAc/hexanes
as eluant) gave 34 as a white solid (56%). 1H NMR [(CD3)2SO] d
8.25 (d, J = 1.5 Hz, 1H), 8.16 (d, J = 8.5 Hz, 1H), 8.01 (d, J = 0.4 Hz,
1H), 7.96 (d, J = 8.1 Hz, 1H), 7.92 (dd, J = 8.1, 1.4 Hz, 1H), 7.84 (s,
1H), 7.77 (dd, J = 8.4, 1.8 Hz, 1H), 5.84 (s, 1H), 5.50 (s, 2H), 3.30
(s, 6H). LRMS (APCI+) calcd for C19H17O4S 341 (MH+), found 341.
4.1.62. 6-Bromo-2-(dimethoxymethyl)benzo[b]thiophene (16)
6-Bromo-2-methylbenzo[b]thiophene35 was oxidized to 6-bro-
mobenzo[b]thiophene-2-carbaldehyde using a previously reported
method.36 This aldehyde was then protected as the dimethyl acetal
according a literature procedure44 to give 16 as a yellow solid
d 7.96 (d, J = 1.8 Hz, 1H), 7.59 (d,
J = 8.5 Hz, 1H), 7.44 (dd, J = 8.5, 1.8 Hz, 1H), 7.27 (s, 1H), 5.67 (d,
J = 1.0 Hz, 1H), 3.41 (s, 6H).
(61%). 1H NMR (CDCl3)
4.1.56. 5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzo[b]thio
phene-3-carbaldehyde (53)
4.1.63. 5-(2-(Dimethoxymethyl)benzo[b]thiophen-6-yl)isoben
zofuran-1(3H)-one (36)
Compound 34 was deprotected according to general procedure E
to give 53 as a white solid (100%). 1H NMR [(CD3)2SO] d 10.19 (s, 1H),
9.07 (s, 1H), 8.88 (d, J = 1.5 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.04 (br s,
1H), 7.92–8.00 (m, 2H), 7.89 (dd, J = 8.5, 1.9 Hz, 1H), 5.51 (s, 2H).
LRMS (APCI+) calcd for C17H11O3S 295 (MH+), found 295.
Compound 16 was reacted with 22 according to general proce-
dure A to give 36 as a white solid (72%). 1H NMR (CDCl3) d 8.08 (d,
J = 0.8 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.81
(dd, J = 8.0, 0.7 Hz, 1H), 7.73 (s, 1H), 7.60 (dd, J = 8.3, 1.6 Hz, 1H),
7.37 (s, 1H), 5.73 (s, 1H), 5.39 (s, 2H), 3.44 (s, 6H). LRMS (APCI+)
calcd for C19H17O4S 341 (MH+), found 341.
4.1.57. (E,Z)-5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzo
[b]thiophen-3-yl)methylene)-2-thioxoimidazolidin-4-one (109)
Reaction of 53 with 2-thiohydantoin according to general pro-
cedure D gave 109 as a yellow solid (97%); mp (AcOH) >300 °C.
1H NMR [(CD3)2SO] d 12.28 (br s, 2H), 8.56 (s, 1H), 8.41 (d,
J = 1.5 Hz, 1H), 8.22 (d, J = 8.5 Hz, 1H), 8.16 (d, J = 0.7 Hz, 1H),
8.07 (dd, J = 8.0, 1.4 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.86 (dd,
J = 8.5, 1.7 Hz, 1H), 6.95 (s, 1H), 5.50 (s, 2H). Anal.
(C20H12N2O3S2ꢀ0.5H2O) C, H, N.
4.1.64. 6-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzo[b]thio
phene-2-carbaldehyde (55)
Compound 36 was deprotected according to general procedure
E to give 55 as a white solid (76%). 1H NMR (CDCl3) d 10.15 (s, 1H),
8.15 (d, J = 1.5 Hz, 1H), 8.08 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 8.03 (d,
J = 8.0 Hz, 1H), 7.83 (dd, J = 8.0, 1.2 Hz, 1H), 7.76 (s, 1H), 7.70 (dd,
J = 8.4, 1.6 Hz, 1H), 5.40 (s, 2H). LRMS (APCI+) calcd for C17H11O3S
295 (MH+), found 295.

1332
J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
4.1.65. (E,Z)-5-((6-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzo
[b]thiophen-2-yl)methylene)-2-thioxoimidazolidin-4-one (111)
Reaction of 55with 2-thiohydantoin was carried out according
to general procedure D to give 111 as a brown solid (40%); mp
(AcOH) >310 °C. 1H NMR [(CD3)2SO] Observe E- and Z-isomers sep-
arately. d 12.05–12.58 (br m, 2H), 8.44 (d, J = 1.5 Hz, 1H), 8.17 (s,
1H), 8.07 (s, 1H), 7.94–8.01 (m, 3H), 7.82 (dd, J = 8.4, 1.7 Hz, 1H),
6.93 (s, 0.05H), 6.64 (s, 0.95H), 5.49 (s, 2H). HRMS (ESIꢁ) calcd
7.81 (d, J = 7.5 Hz, 1H), 7.73 (s, 1H), 7.67 (s, 1H), 7.46 (dd, J = 8.4,
1.5 Hz, 1H), 7.32 (q, J = 0.9 Hz, 1H), 5.39 (s, 2H). LRMS (APCI+) calcd
for C17H12NO3 278 (MH+), found 278.
4.1.73. (E,Z)-5-((6-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-1H-
indol-2-yl)methylene)-2-thioxoimidazolidin-4-one (113)
Reaction of 57 with 2-thiohydantoin was carried out according
to general procedure D to give 113 as a bright red solid (74%); mp
(AcOH) >310 °C. 1H NMR [(CD3)2SO] Observe E- and Z-isomers sep-
arately. d 12.10–12.79 (br m, 2H), 12.03 (s, 0.8H), 11.591 (s, 0.2H),
7.90–8.02 (m, 4H), 7.74 (s, 0.2H), 7.73 (d, J = 8.4 Hz, 0.8H), 7.48 (dd,
J = 8.4, 1.6 Hz, 0.8H), 7.47 (dd, J = 8.3, 1.7 Hz, 0.2H), 7.38 (s, 0.2H),
7.16 (s, 0.8H), 6.72 (s, 0.8H), 6.59 (s, 0.2H), 5.47 (s, 2H). HRMS
(ESIꢁ) calcd for C20H12N3O3S 374.0605 (MꢁH), found 374.0608.
Anal. (C20H13N3O3Sꢀ0.25AcOH) C, H, N.
for
C
20H11N2O3S2 391.0217 (MꢁH), found 391.0213.Anal.
(C20H12N2O3S2ꢀ0.2AcOH) C, H, N.
4.1.66. 5-Bromo-2-(dimethoxymethyl)benzofuran (17)
5-Bromobenzofuran-2-carbaldehydewas protected as the di-
methyl acetal according to a literature procedure44 to give 17 as
a yellow solid (98%). 1H NMR (CDCl3) d 7.70 (d, J = 1.5 Hz, 1H),
7.35–7.40 (m, 2H), 6.76 (s, 1H), 5.55 (s, 1H), 3.41 (s, 6H). LRMS
(APCI+) calcd for C10H8BrO2 239 (M+ꢁMeOH), found 239.
4.1.74. 5-Bromo-2-(dimethoxymethyl)-1H-indole (19)
5-Bromo-1H-indole-2-carbaldehyde was protected as the di-
methyl acetal according to a literature procedure44 to give 19 as
an orange solid (95%). 1H NMR (CDCl3) d 8.42 (br s, 1H), 7.73 (t,
J = 0.9 Hz, 1H), 7.20–7.28 (m, 2H), 6.45–6.48 (m, 1H), 5.62 (d,
J = 0.6 Hz, 1H), 3.40 (s, 6H). LRMS (APCI+) calcd for C10H8BrO2 239
(M+ꢁMeOH), found 239.
4.1.67. 5-(2-(Dimethoxymethyl)benzofuran-5-yl)isobenzofu
ran-1(3H)-one (37)
Compound 17 was reacted with 22 according to general procedure
A to give 37 as a white solid (53%). 1H NMR (CDCl3) d 7.99 (d, J = 8.0 Hz,
1H), 7.81 (d, J = 1.8 Hz, 1H), 7.77 (dd, J = 8.0, 1.4 Hz, 1H), 7.68 (d,
J = 0.6 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.54 (dd, J = 8.6, 1.9 Hz, 1H),
6.76 (t, J = 0.8 Hz, 1H), 5.60 (d, J = 0.8 Hz, 1H), 5.38 (s, 2H), 3.45 (s,
6H). LRMS (APCI+) calcd for C19H17O5 325 (MH+), found 325.
4.1.75. 5-(2-(Dimethoxymethyl)-1H-indol-5-yl)isobenzofuran-
1(3H)-one (39)
Compound 19 was reacted with 22 according to general proce-
dure A to give 39 as a pale red solid (67%). 1H NMR (CDCl3) d 8.50
(br s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.86 (s, 1H), 7.80 (d, J = 8.0 Hz,
1H), 7.71 (s, 1H), 7.47 (s, 2H), 6.61 (d, J = 1.8 Hz, 1H), 5.67 (s, 1H),
5.37 (s, 2H), 3.41 (s, 6H). LRMS (APCI+) calcd for C19H18NO4 324
(MH+), found 324.
4.1.68. 5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzofuran-2-
carbaldehyde (56)
Compound 37 was deprotected according to general procedure
E to give 56 as a white solid (80%). 1H NMR (CDCl3) d 9.93 (s, 1H),
7.98–8.03 (m, 2H), 7.69–7.79 (m, 4H), 7.63 (d, J = 0.8 Hz, 1H), 5.40
(s, 2H). LRMS (APCI+) calcd for C17H11O4 279 (MH+), found 279.
4.1.76. 5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-1H-indole-2-
carbaldehyde (58)
4.1.69. (E,Z)-5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzo
furan-2-yl)methylene)-2-thioxoimidazolidin-4-one (112)
Reaction of 56 with 2-thiohydantoin was carried out according
to general procedure D to give 112 as a bright yellow solid (87%);
mp (AcOH) >310 °C. 1H NMR [(CD3)2SO] d 12.23 (br s, 2H), 8.06 (d,
J = 0.9 Hz, 1H), 8.00 (s, 1H), 7.93 (d, J = 0.8 Hz, 2H), 7.77–7.82 (m,
2H), 7.50 (s, 1H), 6.56 (s, 1H), 5.48 (s, 2H). HRMS (ESIꢁ) calcd for
Compound 39 was deprotected according to general procedure
E to give 58 as a pale yellow solid (94%). 1H NMR (CDCl3) d 12.12
(br s, 1H), 9.90 (s, 1H), 8.17 (d, J = 1.2 Hz, 1H), 7.98 (s, 1H), 7.92
(s, 2H), 7.76 (dd, J = 8.7, 1.8 Hz, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.49
(d, J = 0.8 Hz, 1H), 5.47 (s, 2H). LRMS (APCI+) calcd for C17H12NO3
278 (MH+), found 278.
C
20H11N2O4S
375.0445 (MꢁH),
found
375.0435. Anal.
(C20H12N2O4Sꢀ0.10AcOH) C, H, N.
4.1.77. (E,Z)-5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-1H-
indol-2-yl)methylene)-2-thioxoimidazolidin-4-one (114)
Reaction of 58 with 2-thiohydantoin was carried out according
to general procedure D to give 114 as a orange-brown solid (78%);
mp (AcOH) >300 °C. 1H NMR [(CD3)2SO] Observe E- and Z-isomers
separately. d 12.09–12.74 (br m, 2H), 12.03 (s, 0.8H), 11.591 (s,
0.2H), 7.90–8.03 (m, 4H), 7.74 (s, 0.2H), 7.72 (d, J = 8.4 Hz, 0.8H),
7.48 (dd, J = 8.4, 1.6 Hz, 0.8H), 7.46 (dd, J = 8.3, 1.6 Hz, 0.2H), 7.37
(s, 0.2H), 7.16 (s, 0.8H), 6.72 (s, 0.8H), 6.59 (s, 0.2H), 5.47 (s, 2H).
HRMS (ESIꢁ) calcd for C20H12N3O3S 374.0605 (MꢁH), found
374.0597. Anal. (C20H13N3O3Sꢀ0.20AcOH) C, H, N.
4.1.70. 6-Bromo-2-(dimethoxymethyl)-1H-indole (18)
6-Bromo-1H-indole-2-carbaldehydewas protected as the di-
methyl acetal according to a literature procedure44 to give 18 as
a yellow solid (81%). 1H NMR (CDCl3) d 8.36 (br s, 1H), 7.51 (s,
1H), 7.45 (d, J = 8.4 Hz, 1H), 7.20 (dd, J = 8.4, 1.7 Hz, 1H), 6.48–
6.51 (m, 1H), 5.61 (d, J = 0.6 Hz, 1H), 3.38 (s, 6H). LRMS (APCI+)
calcd for C10H9BrNO 238 (M+ꢁMeOH), found 238.
4.1.71. 5-(2-(Dimethoxymethyl)-1H-indol-6-yl)isobenzofuran-
1(3H)-one (38)
Compound 18 was reacted with 22 according to general proce-
dure A to give 38 as a pale yellow solid (29%). 1H NMR (CDCl3) d
8.53 (br s, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.68–
7.73 (m, 2H), 7.61 (t, J = 0.8 Hz, 1H), 7.39 (dd, J = 8.2, 1.6 Hz, 1H),
6.58 (t, J = 1.0 Hz, 1H), 5.67 (d, J = 0.5 Hz, 1H), 5.37 (s, 2H), 3.41 (s,
6H). LRMS (APCI+) calcd for C19H18NO4 324 (MH+), found 324.
4.1.78. 7-Bromo-2-(dimethoxymethyl)quinoline (20)
7-Bromoquinoline-2-carbaldehyde was protected as the di-
methyl acetal according to a literature procedure44 to give 20 as
a brown solid (90%). 1H NMR (CDCl3) d 8.35 (d, J = 1.8 Hz, 1H),
8.17 (d, J = 8.5 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.5 Hz,
1H), 7.64 (dd, J = 8.7, 1.9 Hz, 1H), 5.46 (s, 1H), 3.47 (s, 6H). LRMS
(APCI+) calcd for C12H13BrNO2 283 (MH+), found 283.
4.1.72. 6-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-1H-indole-2-
carbaldehyde (57)
Compound 38 was deprotected according to general procedure
E to give 57 as a pale yellow solid (69%). 1H NMR (CDCl3) d 9.89 (s,
1H), 9.12 (br s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H),
4.1.79. 5-(2-(Dimethoxymethyl)quinolin-7-yl)isobenzofuran-
1(3H)-one (40)
Compound 20 was reacted with 22 according to general proce-
dure A to give 40 as a yellow solid (62%). 1H NMR (CDCl3) d 8.44 (t,

J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
1333
J = 0.9 Hz, 1H), 8.26 (d, J = 8.3 Hz, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.96
(d, J = 8.5 Hz, 1H), 7.91 (dt, J = 8.0, 0.8 Hz, 1H), 7.82–7.85 (m, 2H),
7.73 (d, J = 8.5 Hz, 1H), 5.52 (s, 1H), 5.41 (s, 2H), 3.50 (s, 6H). LRMS
(APCI+) calcd for C20H18NO4 336 (MH+), found 336.
propanol (338 mg, 4.49 mmol) was then added in one portion and
the whole mixture stirred at rt for 16 h. The reaction mixture was
concentrated, diluted with NaHCO3 (100 mL), and extracted with
EtOAc (3 ꢂ 100 mL). The combined organic fractions were dried
(Na2SO4), filtered, and the solvent removed under reduced pressure
togiveacrudeoil,whichwasdry-loadedontosilicagel. Flashcolumn
chromatography (EtOAc followed by 0.5–1% MeOH/EtOAc as eluant)
yielded73asawhitecrystallinesolid(343 mg, 52%);mp130–132 °C.
1H NMR [(CD3)2SO] d 8.68 (br t, J = 5.1 Hz, 1H), 8.07 (s, 1H), 8.00 (d,
J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 5.46 (s, 2H), 4.45 (t,
J = 5.2 Hz, 1H), 3.44–3.52 (m, 2H), 3.29–3.40 (m, 2H), 1.65–1.74 (m,
2H). LRMS (APCI+) calcd for C12H13NO4 236 (MH+), found 236.
4.1.80. 7-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)quinoline-2-
carbaldehyde (59)
Compound 40 was deprotected according to general procedure E
to give 59 as a pale yellow solid (61%). 1H NMR (CDCl3) d 10.25 (s,
1H), 8.52 (t, J = 0.9 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.09 (d,
J = 8.4 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.97
(dd, J = 8.5, 1.8 Hz, 1H), 7.94 (d, J = 8.6 Hz, 1H), 7.87 (s, 1H), 5.43
(s, 2H). LRMS (APCI+) calcd for C18H12NO3 290 (MH+), found 290.
4.1.87. 1-Oxo-N-(3-oxopropyl)-1,3-dihydroisobenzofuran-5-
carboxamide (74)
4.1.81. (E,Z)-5-((7-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)quino
line-2-yl)methylene (115)
To a solution of 73 (118 mg, 0.50 mmol) in THF/DMF 1:1 (5 mL),
Dess–Martin periodinane (319 mg, 0.75 mmol) was added. After
stirring for 3 h. at rt, the solution was diluted with CH2Cl2
(15 mL), and washed with satd aq NaS2O3 (10 mL), NaHCO3
(10 mL), and brine (10 mL). The organic extract was evaporated
and further dried under high vacuum. Flash column chromatogra-
phy (50% EtOAc/hexanes as eluant) yielded 74 as a off-white solid
(101 mg, 86%) which was used directly in the next step. 1H NMR
[(CD3)2SO] d 9.70 (t, J = 1.6 Hz, 1H), 8.81 (br t, J = 5.1 Hz, 1H), 8.06
(s, 1H),7.97 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 5.46 (s, 2H),
3.53–3.61 (m, 2H), 2.69–2.75 (m, 2H). LRMS (APCI+) calcd for
Reaction of 59 with 2-thiohydantoin was carried out according
to general procedure D to give 115 as a yellow-green solid (85%);
mp (AcOH) >310 °C. 1H NMR [(CD3)2SO] d 11.81 (br s, 2H), 8.77 (s,
1H), 8.47 (d, J = 8.4 Hz, 1H), 8.20 (s, 1H), 8.12 (dd, J = 8.6, 2.7 Hz,
2H), 8.03 (dd, J = 8.5, 2.9 Hz, 2H), 7.87 (d, J = 8.4 Hz, 1H), 6.74 (s,
1H), 5.54 (s, 2H). HRMS (ESIꢁ) calcd for C21H12N3O3S 386.0605
(MꢁH), found 386.0600. Anal. (C21H13N3O3Sꢀ0.30AcOH) C, H, N.
4.1.82. 6-Bromo-2-(dimethoxymethyl)quinoline (21)
6-Bromoquinoline-2-carbaldehyde was protected as the di-
methyl acetal according to a literature procedure44 [except using
HCl (g) as catalyst instead of p-TsOH] to give 21 as a dark orange
solid (70%). 1H NMR (CDCl3) d 8.11 (d, J = 8.6 Hz, 1H), 8.02 (d,
J = 9.0 Hz, 1H), 7.99 (d, J = 2.2 Hz, 1H), 7.79 (dd, J = 9.0, 2.2 Hz,
1H), 7.69 (d, J = 8.6 Hz, 1H), 5.46 (s, 1H), 3.47 (s, 6H). LRMS (APCI+)
calcd for C12H13BrNO2 283 (MH+), found 283.
C
12H11NO4 234 (MH+), found 234.
4.1.88. (E,Z)-1-Oxo-N-(3-(5-oxo-2-thioxoimidazolidin-4-yli
dene)propyl)-1,3-dihydroisobenzofuran-5-carboxamide (117)
Reaction of 74 with 2-thiohydantoin was carried out according
to general procedure D to give a crude product which was purified
by preparative HPLC [Synergi-Max RP C12 column, eluting with a
gradient composed of mobile phases A (1% water/TFA) and B
(90% MeCN/water)]. The title compound 117 was isolated as a
brown solid (10%); mp (AcOH) 239–243 °C. 1H NMR [(CD3)2SO] d
12.10 (br s, 1H), 11.97 (br s, 1H), 8.86 (s, 1H), 8.06 (s, 1H), 7.98
(d, J = 8.1 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 5.74 (t, J = 7.8 Hz, 1H),
5.47 (s, 2H), 3.38–3.48 (m, 2H), 2.52–2.60 (m, 2H).HRMS (ESIꢁ)
calcd for C20H11N2O4S 330.0554 (MꢁH), found 330.0546.
4.1.83. 5-(2-(Dimethoxymethyl)quinolin-6-yl)isobenzofuran-
1(3H)-one (41)
Compound 21 was reacted with 22 according to general proce-
dure A to give 41 as a pale yellow solid (84%). 1H NMR (CDCl3) d
8.28 (dd, J = 8.6, 1.6 Hz, 2H), 8.07 (d, J = 2.0 Hz, 1H), 8.05 (d,
J = 8.0 Hz, 1H), 7.99 (dd, J = 8.8, 2.1 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H),
7.80 (s, 1H), 7.74 (d, J = 8.5 Hz, 1H), 5.52 (s, 1H), 5.41 (s, 2H), 3.50
(s, 6H). LRMS (APCI+) calcd for C20H18NO4 336 (MH+), found 336.
4.1.89. 5-(Thiophen-2-yl)isobenzofuran-1(3H)-one (118)
Reaction of 22 with 2-bromothiophene 85 according to general
procedure A, followed by purification by flash column chromatog-
raphy on silica gel (20% EtOAc/hexanes as eluant), gave 118 as a
crystalline cream solid (61%); mp (EtOAc/n-pentane) 180–182 °C.
1H NMR [(CD3)2SO] d 7.95 (d, J = 0.6 Hz, 1H), 7.90 (dd, J = 8.1,
1.5 Hz, 1H), 7.85 (dd, J = 8.1, 0.5 Hz, 1H), 7.74 (dd, J = 3.7, 1.1 Hz,
1H), 7.71 (dd, J = 5.1, 1.1 Hz, 1H), 7.22 (dd, J = 5.1, 3.7 Hz, 1H),
5.44 (s, 2H). LRMS (APCI+) calcd for C12H9O2S 217 (MH+), found
217. Anal. (C12H8O2S) C, H, N.
4.1.84. 6-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)quinoline-2-
carbaldehyde (60)
Compound 41 was deprotected according to general procedure
E to give 60 as a pale yellow solid (78%). 1H NMR (CDCl3) d 10.26 (s,
1H), 8.40 (d, J = 8.4 Hz, 1H), 8.39 (d, J = 8.7 Hz, 1H), 8.04–8.14 (m,
4H), 7.91 (dt, J = 8.0, 0.6 Hz, 1H), 7.83 (s, 1H), 5.43 (s, 2H). LRMS
(APCI+) calcd for C18H12NO3 290 (MH+), found 290.
4.1.85. (E,Z)-5-((6-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)quino
lin-2-yl)methylene)-2-thioxoimidazolidin-4-one (116)
Reaction of 60 with 2-thiohydantoin was carried out according
to general procedure D to give 116 as a yellow-green solid (82%);
mp (AcOH) >310 °C. 1H NMR [(CD3)2SO] d 12.50 (br s, 1H), 11.86
(br s, 1H), 8.48 (t, J = 8.2 Hz, 2H), 8.41 (d, J = 2.1 Hz, 1H), 8.23 (dd,
J = 8.9, 2.1 Hz, 1H), 8.16 (s, 1H), 8.09 (dd, J = 8.1, 1.3 Hz, 1H), 7.99
(d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H), 6.74 (s, 1H), 5.51 (s,
2H). HRMS (ESIꢁ) calcd for C21H12N3O3S 386.0605 (MꢁH), found
386.0609. Anal. (C21H13N3O3S) C, H, N.
4.1.90. 5-(5-Acetylthiophen-2-yl)isobenzofuran-1(3H)-one (119)
2-Acetyl-5-bromothiophene 86 was reacted with 22 according
to general procedure A, followed by flash column chromatography
on silica gel (CH2Cl2 as eluant), to give 119 as a pale yellow solid
(24%); mp (EtOAc/n-hexane) 193–196 °C. 1H NMR [(CD3)2SO] d
8.08 (br s, 1H), 7.97–8.02 (m, 2H), 7.92 (d, J = 8.1 Hz, 1H), 7.84 (d,
J = 4.0 Hz, 1H), 5.46 (s, 2H), 2.57 (s, 3H). LRMS (APCI+) calcd for
C14H11O3S 259 (M+H), found 259.Anal. (C14H10O3S) C, H, N.
4.1.86. N-(3-Hydroxypropyl)-1-oxo-1,3-dihydroisobenzofuran-
5-carboxamide (73)
4.1.91. 2-(5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thiophen-2-
yl)acetic acid (120)
To a stirred solution of 65 (500 mg, 2.81 mmol) in dry DMF
(30 mL) was added triethylamine (398 mg, 3.93 mmol), EDCIꢀHCl
(700 mg, 3.65 mmol) and HOBt (493 mg, 3.65 mmol). 3-Amino-1-
5-Bromo-2-thiopheneacetic acid 87 was reacted with 22
according to general procedure A. The crude product was isolated
by acidification of the basic aqueous layer with concd HCl upon

1334
J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
workup. 1H NMR showed a mixture of two products; the desired
tricylic compound and a product resulting from ring-opening of
the lactone. This mixture was stirred with TFA to ring-close back
to a single desired product. Removal of the TFA under reduced
pressure afforded a residue which was dissolved in CH2Cl2 and
then also removed under reduced pressure. This procedure was re-
peated twice, then the resulting solid triturated with Et2O and col-
lected by filtration to give 120 as a beige solid (75%); mp (Et2O)
211–214 °C. 1H NMR [(CD3)2SO] d 12.66 (br s, 1H), 7.91 (br s,
1H), 7.82–7.86 (m, 2H), 7.58 (d, J = 3.7 Hz, 1H), 7.03 (d, J = 3.8 Hz,
1H), 5.43 (s, 2H), 3.89 (s, 2H). HRMS (ESI+) calcd for C14H10NaO4S
297.0192 (M+Na), found 297.0197.
4.1.97. 5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thiophen-
2-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione
(126)
Reaction of 43 with 4,6-dihydroxy-2-mercaptopyrimidine
according to general procedure D gave 126 as a dark orange solid
(100%); mp (AcOH) >300 °C. 1H NMR [(CD3)2SO] d 12.42 (s, 2H),
8.55 (s, 1H), 8.29 (d, J = 4.4 Hz, 1H), 8.18 (d, J = 0.7 Hz, 1H), 8.07
(dd, J = 1.5, 8.0 Hz, 1H), 7.98 (d, J = 4.4 Hz, 1H), 7.94 (d, J = 8.0 Hz,
1H), 5.47 (s, 2H). Anal. (C17H10N2O4S2ꢀ0.5H2O) C, H, N.
4.1.98. 1,3-Dimethyl-5-((5-(1-oxo-1,3-dihydroisobenzofuran-5-
yl)thiophen-2-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione
(127)
4.1.92. Methyl 2-(5-(1-oxo-1,3-dihydroisobenzofuran-5-yl)thio
phen-2-yl)acetate (121)
Reaction of 43 with 1,3-dimethylbarbituric acid according to
general procedure D gave 127 as a orange solid (99%); mp (AcOH)
>300 °C. 1H NMR (CF3COOD) d 9.06 (s, 1H), 8.10–8.17 (m, 4H), 7.83
(d, J = 4.2 Hz, 1H), 5.61 (s, 2H), 3.62 (s, 3H), 3.60 (s, 3H). LRMS
(APCI+) calcd for C19H15N2O5S 383 (MH+), found 383. Anal.
(C19H14N2O5S) C, H, N.
Compound 120 (140 mg, 0.51 mmol) was dissolved in a mixture
of Et2O (4 mL) and MeOH (2 mL), then treated dropwise with 2 M
trimethylsilyldiazomethane in Et2O (0.38 mL, 0.77 mmol). After
stirring for 1 h. at rt, AcOH was added dropwise to quench the reac-
tion. All solvent was then removed under reduced pressure, the
resulting residue dissolved in EtOAc (50 mL) and then washed with
satd NaHCO3 solution (3 ꢂ 50 mL), water (50 mL), brine (50 mL),
dried (Na2SO4) and filtered. After removal of the solvent under re-
duced pressure the crude product was purified by flash column
chromatography on silica gel (10% acetone/CH2Cl2 as eluant) to
give 121 as a beige solid (87 mg, 59%); mp (Et2O) 119–121 °C. 1H
NMR [(CD3)2SO] d 7.91 (br s, 1H), 7.82–7.86 (m, 2H), 7.60 (d,
J = 3.7 Hz, 1H), 7.06 (d, J = 3.7 Hz, 1H), 5.43 (s, 2H), 4.01 (s, 2H),
3.67 (s, 3H). LRMS (APCI+) calcd for C15H13O4S 289 (M+H), found
289. Anal. (C15H12O4S) C, H, N.
4.1.99. (E,Z)-2-Imino-1-methyl-5-((5-(1-oxo-1,3-dihydroisobenzo
furan-5-yl)thiophen-2-yl)methylene)imidazolidin-4-one (128)
Reaction of 43 with creatine according to general procedure D
gave 128 as a yellow-orange solid (95%); mp (AcOH) >300 °C. 1H
NMR [(CD3)2SO] d 11.91 (v br s, 1H), 7.94 (br s, 1H), 7.96 (br s,
1H), 7.84–7.91 (m, 2H), 7.67 (d, J = 3.9 Hz, 1H), 7.63 (d, J = 4.2 Hz,
1H), 6.61 (s, 1H), 5.44 (s, 2H), 3.21 (s, 3H). HRMS (FAB+) calcd for
C
17H14N3O3S 340.0756 (MH+), found 340.07550. Anal.
(C17H13N3O3Sꢀ0.75H2O) C, H, N.
4.1.100. 5-(5-(Oxazol-5-yl)thiophen-2-yl)isobenzofuran-1(3H)-
one (129)
4.1.93. (E,Z)-5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thio
phen-2-yl)methylene)imidazolidine-2,4-dione (122)
The title compound was prepared by reaction of 43 with hydan-
toin according to general procedure C to give 122 as a yellow solid
(44%); mp (EtOH) >300 °C. 1H NMR [(CD3)2SO] d 10.20–11.50 (v br
s, 2H), 7.96 (br s, 1H), 7.86–7.92 (m, 2H), 7.81 (d, J = 4.0 Hz, 1H),
7.65 (d, J = 4.0 Hz, 1H), 6.60 (s, 1H), 5.49 (s, 2H). Anal.
(C16H10N2O4S) C, H, N.
Compound 43 (100 mg, 0.41 mmol), tosylmethyl isocyanide
(88 mg, 0.45 mmol) and K2CO3 (113 mg, 0.82 mmol) were sus-
pended in MeOH (10 mL), stirred at rt for 5 min., then heated at re-
flux for 1 h. The reaction mixture was diluted with CH2Cl2 (50 mL)
and the resulting solution evaporated onto a small quantity of silica
under reduced pressure. Purification was carried out by flash col-
umn chromatography on silica gel (50% EtOAc/hexanes as eluant)
to give 129 as a pale yellow solid (93 mg, 80%); mp (Et2O) 229–
231 °C. 1H NMR [(CD3)2SO] d 8.47 (s, 1H), 8.00 (s, 1H), 7.93 (dd,
J = 8.0, 1.4 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 3.9 Hz, 1H),
7.63 (s, 1H), 7.57 (d, J = 3.9 Hz, 1H), 5.45 (s, 2H). LRMS (APCI+) calcd
for C15H10NO3S 284 (MH+), found 284. Anal. (C15H9NO3S) C, H, N.
4.1.94. (E,Z)-5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-
yl)thiophen-2-yl)methylene)-2-thioxothiazolidin-4-one (123)
Reaction of 43 with 2-thioxo-4-thiazolidinone according to gen-
eral procedure D gave 123 as a dark orange solid (95%); mp (AcOH)
>300 °C. 1H NMR [(CD3)2SO] d 13.85 (br s, 1H), 8.10 (br s, 1H), 8.01
(dd, J = 8.1, 1.4 Hz, 1H), 7.87–7.95 (m, 3H), 7.76 (br d, J = 3.4 Hz,
1H), 5.46 (s, 2H). LRMS (APCI+) calcd for C16H10NO3S3 360 (MH+),
found 360. Anal. (C16H9NO3S3) C, H, N.
4.1.101. (E,Z)-3-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thio
phen-2-yl)methylene)pyrrolidine-2,5-dione (130)
Compound 43 (100 mg, 0.41 mmol) was suspended in MeOH
(5 mL) and triphenylphosphoranylidenesuccinimide 89 (147 mg,
0.41 mmol) (which was in turn prepared from succinimide 88
according to a literature procedure39) added. This mixture was
heated at reflux for 1 h. Upon cooling, the resulting crude product
was collected by filtration from the reaction mixture and purified
by trituration with acetone. The title compound 130 was isolated
as a yellow solid (83 mg, 62%); mp (acetone) >295 °C. 1H NMR
[(CD3)2SO] d 11.43 (s, 1H), 8.05 (s, 1H), 7.97 (dd, J = 8.1, 1.5 Hz,
1H), 7.90 (d, J = 8.1 Hz, 1H), 7.85 (d, J = 3.9 Hz, 1H), 7.64 (m, 2H),
5.46 (s, 2H), 3.55 (s, 2H). LRMS (APCI+) calcd for C17H12NO4S 326
(MH+), found 326. Anal. (C17H11NO4S) C, H, N.
4.1.95. (E,Z)-5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thio
phen-2-yl)methylene)thiazolidine-2,4-dione (124)
Reaction of 43 with 2,4-thiazolidinedione according to general
procedure D gave 124 as a bright orange solid (96%); mp (AcOH)
>300 °C. 1H NMR [(CD3)2SO] d 12.50 (br s, 1H), 8.03–8.88 (m, 2H),
7.98 (dd, J = 8.1, 1.5 Hz, 1H), 7.87–7.93 (m, 2H), 7.74 (d,
J = 4.1 Hz, 1H), 5.46 (s, 2H). LRMS (APCI+) calcd for C16H10NO4S2
344 (MH+), found 344. Anal. (C16H9NO4S2) C, H, N.
4.1.96. 5-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)thiophen-
2-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (125)
Reaction of 43 with barbituric acid according to general proce-
dure D gave 125 as a yellow-brown solid (100%); mp (AcOH)
>300 °C. 1H NMR [(CD3)2SO] d 11.31 (br s, 2H), 8.52 (s, 1H), 8.23
(d, J = 4.2 Hz, 1H), 8.15 (br s, 1H), 8.05 (dd, J = 8.1, 1.4 Hz, 1H),
7.95 (s, 1H), 7.93 (d, J = 3.6 Hz, 1H), 5.47 (s, 2H). Anal.
(C17H10N2O5S) C, H, N.
4.1.102. General procedure I: (E,Z)-1-benzyl-3-((5-(1-oxo-1,3-
dihydroisobenzofuran-5-yl)thiophen-2-yl)methylene)pyrroli
din-2-one (131)
Diethyl
1-(benzyl)-2-oxopyrrolidin-3-ylphosphonate40
90
(212 mg, 0.68 mmol) was dissolved in THF (6 mL), to which was
added 18-crown-6 (902 mg, 3.41 mmol). This mixture was cooled

J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
1335
to -78 °C under N2, then a 0.5 M solution of potassium bis(trimeth-
ylsilyl)amide in toluene (1.50 mL, 0.75 mmol) added dropwise. After
30 min, a solution of 43 (200 mg, 0.82 mmol) in THF (10 mL) was
added and the solution allowed to warm to rt and stir for 18 h. NH4Cl
(satd, 50 mL) was added to the reaction mixture which was ex-
tracted with CH2Cl2 (3 ꢂ 50 mL). The combined organic fractions
were dried (Na2SO4), filtered, and the solvent removed under re-
duced pressure to yield a solid which was purified by flash column
chromatography on silica gel (5% acetone/CH2Cl2 as eluant). The title
compound 131 was isolated as a yellow solid (114 mg, 42%); mp
(Et2O) 205–209 °C. 1H NMR [(CD3)2SO] d 8.01 (s, 1H), 7.93 (dd,
J = 8.1, 1.4 Hz, 1H), 7.88 (d, J = 7.0 Hz, 1H), 7.82 (d, J = 3.9 Hz, 1H),
7.52 (d, J = 4.0 Hz, 1H), 7.46 (t, J = 2.8 Hz, 1H), 7.33–7.39(m, 2H),
7.25–7.32 (m, 3H), 5.44 (s, 2H), 4.56 (s, 2H), 3.45 (t, J = 6.3 Hz, 2H),
2.96–3.02 (m, 2H). LRMS (APCI+) calcd for C24H20NO3S 402 (MH+),
found 402. Anal. (C24H19NO3S) C, H, N.
compound 93 was isolated as a yellow solid (29%). 1H NMR
[(CD3)2SO] d 8.02 (s, 1H), 7.95 (dd, J = 8.1, 1.4 Hz, 1H), 7.88 (d,
J = 8.2 Hz, 1H), 7.86 (br s, 1H), 7.82 (d, J = 3.9 Hz, 1H), 7.51 (d,
J = 4.0 Hz, 1H), 7.22 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H),
5.44 (s, 2H), 4.57 (s, 2 H), 3.74 (s, 3H), 3.34 (br t, J = 5.7 Hz, 2H),
2.84 (br t, J = 5.8 Hz, 2H), 1.86 (pentet, J = 6.0 Hz, 2H). LRMS (APCI+)
calcd for C26H23NO4S 446 (MH+), found 446.
4.1.107. (E,Z)-3-((5-(1-Oxo-1,3-dihydroisobenzofuran-5-
yl)thiophen-2-yl)methylene)piperidin-2-one (134)
Compound 93 was deprotected according to general procedure J
to give 134 as a yellow solid (85%). 1H NMR [(CD3)2SO] d 8.02 (br s,
1H), 7.95 (dd, J = 8.1, 1.5 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.83 (br s,
1H), 7.81 (d, J = 4.0 Hz, 1H), 7.76 (br t, J = 2.0 Hz, 1H), 7.50 (d,
J = 4.0 Hz, 1H), 5.44 (s, 2H), 3.22–3.28 (m, 2H), 2.78–2.84 (m, 2H),
1.85 (pentet, J = 6.0 Hz, 2 H). HRMS (FAB+) calcd for C18H16NO3S
326.0851 (MH+), found 326.0846.
4.1.103. (E,Z)-1-(4-Methoxybenzyl)-3-((5-(1-oxo-1,3-dihydroiso
benzofuran-5-yl)thiophen-2-yl)methylene)pyrrolidin-2-one
(132)
4.2. Biology
Diethyl
1-(4-methoxybenzyl)-2-oxopyrrolidin-3-ylphospho-
4.2.1. Inhibition of perforin-mediated lysis of sheep red blood
cells (SRBC)
nate 91 (prepared by adapting a literature procedure40) was re-
acted with 43 according to general procedure I. Purification was
carried out by flash column chromatography on silica gel (5% ace-
tone/CH2Cl2 as eluant) to give 132 as a yellow solid (17%). 1H NMR
[(CD3)2SO] d 8.00 (d, J = 0.6 Hz, 1H), 7.93 (dd, J = 8.1, 1.5 Hz, 1H),
7.87 (d, J = 8.1 Hz, 1H), 7.82 (d, J = 3.9 Hz, 1H), 7.51 (d, J = 4.0 Hz,
1H), 7.44 (t, J = 2.7 Hz, 1H), 7.21 (dt, J = 8.7, 2.4 Hz, 2H), 6.92 (dt,
J = 8.7, 2.5 Hz, 2H), 5.44 (s, 2H), 4.48 (s, 2H), 3.74 (s, 3H), 3.41 (t,
J = 6.2 Hz, 2H), 2.97 (m, 2H). HRMS (FAB+) calcd for C25H22NO4S
432.1270 (MH+), found 432.1269.
As reported previously29 compound 2 was identified from
screening a commercial library of ꢃ100,000 compounds for the
ability to reproducibly inhibit perforin-mediated lysis of SRBC at
a compound concentration of 100 lM.
4.2.2. Inhibition of perforin-mediated lysis of Jurkat cells
The ability of the compounds to inhibit the lysis of nucleated
(Jurkat T lymphoma) cells in the presence of 0.1% BSA, as measured
by release of 51Cr was measured. Jurkat target cells were labelled
by incubation in medium with 100 l
Ci 51Cr for one hour. The cells
4.1.104. General procedure J: (E,Z)-3-((5-(1-oxo-1,3-dihydroiso
benzofuran-5-yl)thiophen-2-yl)methylene)pyrrolidin-2-one
(133)
were then washed three times to remove unincorporated isotope
and re-suspended at 1 ꢂ 105 cells per mL in RPMI buffer supple-
mented with 0.1% BSA. Each test compound was pre-incubated to
Compound 132 (71 mg, 0.17 mmol) was dissolved in TFA
(5 mL), to which was added anisole (5 drops). This mixture was
heated at reflux for 15 h. then the solvent removed under reduced
pressure. The resulting residue was dissolved in 5% MeOH/CH2Cl2
(50 mL) and washed with satd NaHCO3 (50 mL). The solution was
dried over Na2SO4, filtered, and evaporated onto silica prior to puri-
fication by flash column chromatography (5% MeOH/CH2Cl2 as elu-
ant) to afford 133 as a yellow solid (39 mg, 71%), mp (Et2O/CH2Cl2)
224–227 °C. 1H NMR [(CD3)2SO] d 8.17 (br s, 1H), 3.01 (br s, 1H),
7.93 (dd, J = 8.1, 1.5 Hz, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.81 (d,
J = 3.9 Hz, 1H), 7.48 (d, J = 4.0 Hz, 1H), 7.34 (t, J = 2.8 Hz, 1H), 5.45
(s, 2H), 3.44 (t, J = 6.3 Hz, 2H), 3.01 (td, J = 6.4, 2.8 Hz, 2H). LRMS
(APCI+) calcd for C17H14NO3S 312 (MH+), found 312. Anal.
(C17H13NO3S) C, H, N.
concentrations of 20, 10, 5, 2.5 and 1.25 lM with recombinant per-
forin for 30 min with DMSO as a negative control. 51Cr labeled Jur-
kat cells were then added and cells were incubated at 37 °C for 4 h.
The supernatant was collected and assessed for its radioactive con-
tent on a gamma counter (Wallac Wizard 1470 automatic gamma
counter). Each data point was performed in triplicate and an IC50
was calculated from the range of concentrations described to
above. Compounds with an IC50 <1
concentrations in the same manner as above, to determine an
accurate IC50
lM were titrated down to lower
.
4.2.3. Inhibition of PLY-mediated lysis of Jurkat cells
In order to determine whether perforin inhibitor 2 was also able
to block the lytic function of PLY, it was tested at a concentration of
20 l
M in the presence PLY instead of perforin with 51Cr labeled
4.1.105. Diethyl 1-(4-methoxybenzyl)-2-oxopiperidin-3-ylphos
phonate (92)
Jurkat target cells as described above.
The title compound 92 was prepared by adapting a literature
procedure40 and was isolated as a pale yellow oil (27%). 1H NMR
[(CD3)2SO] d 7.20 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 4.63
(d, J = 14.6 Hz, 1H), 4.47 (d, J = 14.6 Hz, 1H), 4.12–4.29 (m, 4H),
3.79 (s, 3H), 3.21–3.29 (m, 1H), 3.12–3.20 (m, 1H), 3.08 (t,
J = 6.5 Hz, 0.5H), 3.02 (t, J = 6.5 Hz, 0.5H), 2.15–2.27 (m, 1H),
2.01–2.14 (m, 2H), 1.34 (q, J = 7.0 Hz, 6H). LRMS (APCI+) calcd for
C17H26NO5P 356 (MH+), found 356.
4.2.4. KHYG-1 cytotoxicity assay
KHYG-1 cells were washed and resuspended in RPMI + 0.1% BSA
at 4 ꢂ 105 cells/mL and 50
l
L of KHYG-1 cells were dispensed to
each well of a 96-well V-bottom plate. RPMI (50
lL) + 0.1% BSA
or 10% (final concentration) of serum was added to each well then
test compounds were added to KHYG-1 cells at various concentra-
tions up to 20
l
M and incubated at rt for 20 minutes. 1 ꢂ 106 K562
target cells were labelled with 75 l lL RPMI for
Ci 51Cr in 200
90 min at 37 °C, cells were washed as described above and resus-
4.1.106. (E,Z)-1-(4-Methoxybenzyl)-3-((5-(1-oxo-1,3-dihydroiso
benzofuran-5-yl)thiophen-2-yl)methylene)piperidin-2-one (93)
Compound 92 was reacted with 43 according to general proce-
dure I. Purification was carried out by flash column chromatogra-
phy on silica gel (5% acetone/CH2Cl2 as eluant). The title
pended in 5 ml RPMI + 0.1% BSA. 50
l
L of 51Cr labelled K562 leuke-
mia target cells were added to each well of the KHYG-1 plate
(Effector:Target 2:1) and incubated at 37 °C for 4 h. 51Cr release
was assayed using a Skatron Harvesting Press and radioactivity
estimated on a Wallac Wizard 1470 Automatic Gamma counter

1336
J. A. Spicer et al. / Bioorg. Med. Chem. 20 (2012) 1319–1336
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This work was supported by the Wellcome Trust (Grant 092717)
and the Auckland Division of the Cancer Society of New Zealand.
K.M.H. thanks the Academy of Finland (Grant 135439), the Finnish
Cultural Foundation, and Jenny and Antti Wihuri Foundation for
financial support. We also thank Sisira Kumara and Karin Tan for
HPLC studies, and Maruta Boyd and Shannon Black for NMR studies.
Supplementary data
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