Dihydrofuro[3,4-c]pyridinones as inhibitors of the cytolytic effects of the pore-forming glycoprotein perforin

Article abstract of DOI:10.1021/jm801063n

Dihydrofuro[3,4-c]pyridinones are the first class of small molecules reported to inhibit the cytolytic effects of the lymphocyte toxin perforin. A lead structure was identified from a high throughput screen, and a series of analogues were designed and prepared to explore structure-activity relationships around the core bicyclic thioxofuropyridinone and pendant furan ring. This resulted in the identification of a submicromolar inhibitor of the perforin-induced lysis of Jurkat T-lymphoma cells.

Full text of DOI:10.1021/jm801063n

7614
J. Med. Chem. 2008, 51, 7614–7624
Dihydrofuro[3,4-c]pyridinones as Inhibitors of the Cytolytic Effects of the Pore-Forming
Glycoprotein Perforin
Gersande Lena,† Joseph A. Trapani,‡ Vivien R. Sutton,‡ Annette Ciccone,‡ Kylie A. Browne,‡ Mark J. Smyth,‡
William A. Denny,† and Julie A. Spicer†,*
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The UniVersity of Auckland,
PriVate Bag 92019, Auckland 1142, New Zealand, and Cancer Immunology Program, Peter MacCallum Cancer Centre, St Andrew’s Place,
East Melbourne, Victoria 3002, Australia
ReceiVed August 25, 2008
Dihydrofuro[3,4-c]pyridinones are the first class of small molecules reported to inhibit the cytolytic effects
of the lymphocyte toxin perforin. A lead structure was identified from a high throughput screen, and a
series of analogues were designed and prepared to explore structure-activity relationships around the core
bicyclic thioxofuropyridinone and pendant furan ring. This resulted in the identification of a submicromolar
inhibitor of the perforin-induced lysis of Jurkat T-lymphoma cells.
Introduction
and other intracellular pathogens as well as the spontaneous
development of B-cell lymphoma as the mice aged.10 When
perforin-deficient mice were backcrossed with the nonobese
diabetes mouse strain, it was found that the incidence of
spontaneous diabetes over a one year period was reduced from
77% in a perforin +/+ control to 16% in perforin-deficient mice,
with onset of disease markedly delayed.11 In humans, defects
or mutations that impair perforin function result in familial
hemophagocytic lymphohistiocytosis syndrome, characterized
by severe anemia and hepatosplenomegaly, fever, and thrombo-
cytopenia.12-15 CTL and NK cells have also been implicated
in several autoimmune diseases (e.g., insulin-dependent dia-
betes)4,16 as well as therapy-induced conditions (e.g., allograft
rejection, graft-versus-host disease).17-19
To date, the only reported inhibitors of perforin function are
complex natural products, primarily concanamycin A and other
V-ATPase inhibitors such as bafilomycin A and prodigiosin 25-
Cs20 that inhibit cytoplasmic granule acidification.21 Other
reported inhibitors include cytochalasin D (an inhibitor of actin
polymerization), antimycin A, and oligomycin A (inhibitors of
cell respiration) and some protein kinase inhibitors (calphostin
C, herbimycin A, staurosporine).22 All of these classes of
inhibitors exert their effects on perforin processing, storage, or
release rather than by directly inhibiting perforin’s action on
the target cell. In this regard, small-molecule inhibitors of
perforin function are of potential interest as a new class of
therapeutic immunosuppressive agents, but to the best of our
knowledge there have been no reports of such compounds. We
report here studies on dihydrofuro[3,4-c]pyridinones as small-
molecule inhibitors of perforin-induced lysis.
The key cytotoxic effector cells, cytotoxic T lymphocytes
(CTLa) and natural killer (NK) cells, perform tumor surveillance
and provide a defense against viral infection and intracellular
pathogens. The granule exocytosis pathway used by CTLs and
NK cells is crucial for the induction of apoptosis in the
elimination of virus-infected or transformed cells.1-3 Two of
the main components found in the cytotoxic granules are a group
of serine proteases known as granzymes,4 and perforin, a
calcium-dependent pore-forming glycoprotein of about 67
kDa.5-8 Upon stable conjugation of the CTL or NK cell with
a target cell, the polarized release of the granule contents into
the synaptic cleft occurs, ultimately resulting in infiltration of
the target cell by the granzymes. Perforin is essential for this
process,7 due at least in part to its ability, upon binding calcium,
to assemble into highly ordered aggregates of 12-18 molecules
that form trans-membrane pores of 10-20 nm in diameter in
the plasma membrane.1,9 This can allow leakage of cell contents
and, probably more importantly, entry of the granzymes, which
promote both caspase-dependent and -independent apoptosis.4
This process was further elucidated recently with the publica-
tion of the crystal structure of a bacterial protein from Photo-
rhabdus luminescens which belongs to the same superfamily
of proteins as perforin.9 Both of these proteins contain the
membrane attack complex/perforin (MACPF) domain, and it
was concluded that the presence of this domain in both the
bacterial protein Plu-MACPF and perforin suggests the ability
to form a “pre-pore” oligomer, which binds to the cell membrane
and upon a conformational change converts into a giant ꢀ-barrel-
linked channel.
Deficiencies of many of the granule components are tolerated
in vivo;1 however, perforin is essential for protective immune
surveillance. In 1994, the generation of perforin-deficient mice
revealed a susceptibility to, and failure to clear, many viruses
Results and Discussion
Synthesis of Target Compounds. The lead compound (1)
was selected from a high throughput screen23,24 of approximately
100000 compounds sourced from commercial libraries using a
384-well plate format. Compounds were screened (at 20 µM)
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 the change in absorbance at 650 nm.
The lead structure 1 (Figure 1) is easily disconnected into
two subunits: the subunit A being the bicyclic thioxofuropyri-
dinone, with subunit B corresponding to the furan ring.
Analogues of 1 containing variations of both subunits were
* To whom correspondence should be addressed. Phone: +64 9 3737599.
Fax: +64 9 3737502. E-mail: j.spicer@auckland.ac.nz.
†
The University of Auckland;
Peter MacCallum Cancer Centre
‡
a Abbreviations: CTL, cytotoxic T lymphocyte; NK, natural killer;
MACPF, membrane attack complex/perforin; ROESY, rotational nuclear
Overhauser effect; PLO, pneumolysin; FasL, Fas-ligand; TNFR, tumor
necrosis factor R; TRAIL, TNF-related apoptosis inducing ligand; SAR,
structure-activity relationship.
10.1021/jm801063n CCC: $40.75
2008 American Chemical Society
Published on Web 11/14/2008

Inhibitors of the Pore-Forming Protein Perforin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7615
zothiophene-, and 3-indolecarboxaldehyde to afford the analo-
gous compounds linked through the 3-position (59, 60, 66, Table
2).
Within the set of compounds prepared from 7, the use of
commercially available aldehydes was not always possible.
Thus, it is indicated in Tables 1 and 2 when the required
aldehydes have been prepared. The preparation of 5-phenylth-
iophen-3-carboxaldehyde (71, Table 1 footnote) was achieved
according to known procedures.32 The synthesis of aldehydes
with substituents on the bicyclic aromatic rings (benzofurans
72-76, benzothiophenes 77, 78, 83-86, and indoles 79-82 in
Table 2) also involved the use of previously reported prepara-
tions.33-36 The characterization details of 71-86 are given in
the Supporting Information.
On the basis of the activity shown by compounds in the lead
thioxofuropyridinone (7) series, a smaller number of aldehydes
were then reacted with templates 5, 9, and 11. This afforded
the 5-methylfuran-(32), 5-phenylthiophene-(33), and pyrrolo-
(34) compounds (Table 1), as well as the 2-benzothiophene-
(51) and 3-benzothiophene (65) products (Table 2) in the
furopyridinedione (5) series. The 5-methylfuran-(36, 35) and
5-phenylthiophene-(38, 37) compounds were prepared in both
the pyrrolopyridinedione (9) and thioxopyrrolopyridinedione
(11) series, respectively (Table 1), whereas in Table 2, only
the optimal 3-benzothiophene-(69) compound was prepared by
reaction with template 11.
Figure 1
prepared (Tables 1 and 2). Modifications of subunit A involved
the preparation of the furopyridinedione (5), the furopyridinone
(8), the pyrrolopyridinedione (9), and the thioxopyrrolopyridi-
none (11) (Schemes 1 and 2). For the variations of subunit B,
a diverse range of aldehydes were either commercially available
or prepared where required. The two subunits can be assembled
via an aldolization-crotonization reaction.
A literature search showed that compounds 1, 12, 14, 26,
and 32 had been described previously.25-27 These compounds
were prepared by reaction of 7 (or 5 in the case of compound
32) with the relevant aldehydes. The preparation of 7 itself was
based on chemistry developed during the course of efforts to
determine the structure of vitamin B6,28,29 and we used this
method, with some minor modifications (Scheme 1) to prepare
7 and subsequently an array of 4-oxo and 4-thioxo-4,5-
dihydrofuro[3,4-c]pyridin-3(1H)-one analogues. Ethyl 2-ethoxy-
acetate (2) was reacted with acetone in the presence of sodium
hydride to give ethoxyacetylacetone (3). Standard reaction
conditions between 3 and 2-cyanoacetamide produced the
2-pyridone derivative 4. The lactonization was performed in
concentrated HCl and followed by reaction with phosphorus
pentachloride to afford the R-chloro-pyridine derivative 6.
Finally, displacement of the chlorine with thiourea yielded the
thioxofuropyridinone 7.
The furopyridinone template 8, however, reacted quite
differently under the same aldolization-crotonization conditions
and none of the desired products could be isolated. The main
product of the reaction was characterized by crystallography
and corresponds to ethyl 4-(hydroxymethyl)-6-methylnicoti-
nate,37 signifying opening of the lactone under the conditions
described in Scheme 3.
The stereoselectivity of the aldolization-crotonization reac-
tion has previously been reported to favor the formation of
Z-stereoisomers.25,31 Our observations for a sample of 12 in
DMSO-d6 in an experiment using a rotational nuclear Over-
hauser effect (ROESY) experiment are consistent with these
results. The olefinic proton (δ ) 7.08 ppm) and the pyridineth-
ione 7-H (δ ) 7.19 ppm) correlate strongly, indicating their
close proximity. In addition, no correlation between the pyridi-
nethione 7-H and the furan protons was observed (see Support-
ing Information for 1H and ROESY NMR spectra). These
observations confirmed that 12 had been prepared predominantly
as the Z-isomer. HPLC and LCMS analyses of the final
compounds (including 12) did show the presence of a minor
peak, the same molecular weight by LCMS, and most likely
being the E-isomer. Separation of the two isomeric forms of
23 by preparative HPLC was carried out, and although difficult
due to low solubility and very similar retention times, signifi-
cantly enriched samples were obtained. However, subsequent
HPLC analysis of these showed the same composition as before
separation, suggesting an interconversion of the two isomers in
solution.
The stability of 1 and 60 in DMSO was investigated. Three
separate studies were conducted; samples of the two compounds
were stored at room temperature or -20 °C, as well as a third
sample, which was frozen and defrosted for each HPLC analysis.
This last sample is an attempt to replicate conditions that are
commonly used for stock solutions in biological testing. Each
of the three samples was then subjected to HPLC analysis on a
weekly basis over four weeks. Results indicate that both
compounds gradually convert to the other geometrical isomer,
as after four weeks, 12%, 13%, and 10% of the E-isomer was
Intermediate 8 was prepared by catalytic hydrogenation of
compound 6, in accordance with previously reported results.30
The lactam 9 was obtained by reaction of 4 with concentrated
sulfuric acid (Scheme 2).31 Preparation of the corresponding
thioxo-analogue 11 involved the reaction of 2-thiocyanoaceta-
mide with 3 to give the thioxopyridine 10. The lactamization
then proceeded under the same conditions and afforded 11 in
moderate yields.
With five different bicyclic intermediates available, the
preparation of a series of analogues of compound 1 was
undertaken in a multiparallel fashion using a carousel. To a
suspension of starting material in ethanol was added each of
the various aldehydes and a catalytic amount of piperidine
(Scheme 3). The aldolization-crotonization reactions were
heated at reflux and monitored by mass spectrometry. Depending
on the reactivity of the aldehydes, the reaction time varied from
40 min to 20 h. The products precipitated out directly from the
reaction mixture and were simply collected by filtration, rinsed,
and dried.
As expected, the reaction between 5, 7, and a selected set of
aldehydes proceeded efficiently to give a series of compounds
appended with both unsubstituted and substituted furan, thiophene,
pyrrole, imidazole, phenyl (Table 1), benzofuran, benzothiophene,
and indole (Table 2) rings.
The position of the link between subunits A and B was
explored by reaction of 7 with 3-furaldehyde and thiophene-3-
carboxaldehyde to give 3-linked compounds 39 and 40,
respectively (Table 1). For targets containing a bicyclic B-
subunit, 5 and 7 were reacted with 3-benzofuran-, 3-ben-

7616 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Lena et al.
Table 1. Structures and Inhibitory Activities of Dihydrofuro[3,4-c]pyridinones with a Monocyclic Substituent
compd
linka
X
Y
Zb
R2
Me
H
Ph
H
Me
Ph
H
IC50 (µM)c
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
O
O
O
S
O
S
O
S
S
S
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
NH
O
O
O
O
S
S
S
2.7
4.4
4.0
8.6
7.2
3.4
>20
>20
>20
>20
>20
>20
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
NH
NH (3-aza)d
NMe
H
H
4-F
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
O
4-Cl
4-Bre
4-Me
4-CF3
4-OMe
4-OCF3
4-SMe
4-NMe2
4-NHAc
4-Ph
Me
Ph
H
Me
Me
Ph
Ph
H
>20
>20
>20
>20
>20
>20
>20
>20
>20
5.0
>20
unstable
>20
>20
>20
>20
>20
>20
S
NH
O
O
S
S
O
S
O
O
H
S
Phf
a Position of link to substituent. b Templates 5, 7, 9, or 11 were reacted with commercially available aldehydes. c Testing was carried out over a range of
doses, with the IC50 being equal to the concentration at which 50% inhibition of the lysis of Jurkat cells by perforin was observed, as measured by 51Cr
release. Values are the average of at least two independent IC50 determinations. d Appended ring is 2-imidazolyl. e Used for preparative HPLC separation of
E- and Z-isomers but not tested. f The phenyl substituent is at the 5-position, relative to connection at the 3-position of the thiophene. This compound results
from reaction of 7 with novel aldehyde 71; see Supporting Information for preparation details and characterization of 71.
observed for compound 1 and 11%, 11%, and 8% for compound
60 at room temperature, -20 °C, and freeze-thaw, respectively
(see Figures 3 and 4 in Supporting Information for data).
Subunit B as a Monocyclic Substituent (Table 1). The
compounds were evaluated for inhibition of the lytic activity
of perforin on Jurkat T lymphoma cells using the release of
51Cr as a measure of cell lysis24 (see Experimental Section for
details). The results, as the concentration of drug to reduce the
level of perforin-induced lysis to 50% of controls (IC50), are
given in Tables 1 and 2.
The lead compound (1) showed significant inhibitory activity
in this assay, with an IC50 of 2.7 µM. Compounds 12-41 were
then explored for the nature of the appended ring (subunit B).
Removal of the furanyl 5-methyl group from 1 to give 12
resulted in decreased potency (IC50 4.4 µM), as did replacement
of the 5-methylfuran with a 5-phenylfuran group (13; IC50 4.0
µM). Because of concern about the mutagenic potential of the
furan (compounds 1 and 12 were positive in the Ames
Salmonella typhimurium TA100 tester strain38), compounds
14-16 explored replacement of the furan with thiophene. All
of these compounds were active in the lysis assay, with the best
being the 5-phenyl analogue (16; IC50 3.4 µM), although none
were as potent as the 5-methylfuran 1. The thiophene 14 and
the pyrrole 17, however, both tested negative in the Ames assay.
Compounds 17-19 employed various 5-membered nitrogen
heterocycles (pyrrole, imidazole, and N-methylpyrrole, respec-
tively) as furan replacements, but none of these compounds were
active. A series of compounds with a benzene ring bearing a
range of electron-donating and -withdrawing substituents at a
geometrically similar 4-position were also prepared (20-31).
No activity was observed for any of these analogues irrespective
of substitution. A limited number of other compounds with
ortho- or meta- substitution were synthesized but also had no
activity (data not shown). These data in general suggest the
desirability of a strong H-bond acceptor, but not a donor, in
the appended ring.
Compounds 32-34 study the replacement of the thioamide
sulfur by oxygen. For the 5-methylfuran pair (32/1), this resulted
in loss of activity, while for the pyrrole pair (34/17), both
compounds were inactive. Interestingly, for the 5-phenylth-
iophene pairing (33/16), only a slight loss of activity was
observed (IC50s 5.0 and 3.4 µM). Compounds 35-38 replaced
the potentially metabolically liable lactone moiety with a lactam.
One compound (35) was too unstable to test reliably, but both
36 and its lactone counterpart 32 were inactive and the pairs
37/16 and 38/33 clearly demonstrate the lack of utility of the
lactam. Finally, compounds 39-41 explored alternative linking
geometry to the appended heterocyclic ring; comparison of pairs

Inhibitors of the Pore-Forming Protein Perforin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7617
Table 2. Structures and Inhibitory Activities of Dihydrofuro[3,4-c]pyridinones with a Bicyclic Substituent
compd
linka
X
Y
Z
R3
starting aldehydeb
IC50 (µM)c
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
7d
S
S
S
S
S
S
S
S
S
O
S
S
S
S
S
S
S
S
S
S
S
S
S
O
S
S
O
S
S
O
O
O
O
O
O
O
S
S
S
S
NH
NH
NH
NH
NH
NH
NMe
O
S
S
S
S
H
8.2
6.7
2.4
7.2
3.0
5.1
9.5
13.1
5.4
4.6
1.3
4.6
3.1
>20
6.3
>20
>20
>20
0.97
3.8
5.1
4.7
7.5
8.5
>20
4.3
>20
>20
4.1
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
O
4-OMe
5-OMe
6-OMe
7-OMe
5-OMe, 6-OBn
H
5-OMe
6-OMe
H
H
5-OMe
6-OMe
5-OMe, 6-OBn
5-F, 6-OMe
5-OBn
H
H
H
4-OMe
5-OMe
6-OMe
5-Br
H
H
H
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
S
S
NH
NMe
NMe
S
H
H
H
NH
a Position of link to substituent. b Aldehydes which were reacted with 5, 7, 9, or 11 were commercially available unless stated otherwise; see Supporting
Information for preparation details and characterization of new aldehydes 72-86. c Testing was carried out over a range of doses, with the IC50 being equal
to the concentration at which 50% inhibition of the lysis of Jurkat cells by perforin was observed, as measured by 51Cr release. Values are the average of
at least two independent IC50 determinations. d Connection is at the 7-position of the indole.
Scheme 1a
Scheme 2a
a Reagents and conditions: (i) concd H2SO4, RT, 3 d.; (ii) 2-thiocy-
anoacetamide, piperidine (cat.), EtOH, 75 °C, 1 h.
benzofuran (42-47), benzothiophene (48-51), and indole
(52-58) is investigated in Table 2. Comparison of the 2-furan
(12) and 2-thiophene (14) with the 2-benzofuran (42) and
2-benzothiophene (48), respectively, saw a drop of potency in
both cases (12/42; IC50 4.4/8.2 µM and 14/48; IC50 8.6/9.5 µM).
More interesting, however, was the indole (52), which possessed
unexpectedly good activity (IC50 1.3 µM), especially given that
the corresponding 2-pyrrole 17 was inactive (IC50 >20 µM).
Tolerance for substitution on the benzene ring of the
benzofurans, benzothiophenes, and indoles was probed via the
a Reagents and conditions: (i) NaH, acetone, Et2O, -5 °C-RT; (ii)
2-cyanoacetamide, piperidine (cat.), EtOH, 75 °C, 1 h.; (iii) concd HCl,
120 °C, 10 h.; (iv) POCl3, PCl5, 115 °C, 0.5 h.; (v) thiourea, 1-BuOH, 110
°C, 10 h.; (vi) 10% Pd/C, Et3N, H2, 2.5 h.
39/12, 40/14, and 41/16 shows convincingly that this geometry
is not favorable.
Subunit B as a Bicyclic Substituent (Table 2). The impact
of replacing the furan of 12 with a bicyclic substituent such as

7618 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Lena et al.
Scheme 3a
Table 3. Capacity of Selected Compounds to Inhibit Perforin Delivered
by KHYG-1 NK Cells
viability of KHYG-1
inhibition of
target cell
killer cells 24 h
after exposure to
compoundsc,d (%)
Jurkat
IC50(µM)
compd
deatha,b (%)
1
2.7
3.4
2.4
4.6
1.3
0.97
50
100
100
65
95
63
72
0
0
100
0
88
16
44
51
52
60
a Reagents and conditions: (i) R1CHO, piperidine (cat.), EtOH, 75 °C,
40 min, -20 h.
a Inhibitor (to 20 µM final concentration) and medium were coincubated
with KHYG-1 human NK leukemia cells, 51Cr-labeled K562 target cells
were added, and target cell lysis measured by 51Cr release. Percent inhibition
calculated compared to untreated control. b Use of DMSO vehicle as control
resulted in no (0%) inhibition. c KHYG-1 viability was measured after 24 h.
by examining uptake/exclusion of the dye Trypan blue. Viable (clear) and
dead (blue) cells were counted, and percent viability calculated based on
total cell count. d Use of DMSO vehicle as control resulted in no effect
upon KHYG-1 cell viability (>95%).
synthesis of a series of methoxy-substituted compounds. For
the benzofurans, within the series, the most potent compound
was the 5-methoxy (44; IC50 2.4 µM), closely followed by the
7-methoxy (46; IC50 3.0 µM), whereas for the benzothiophenes,
the 6-methoxy (50; IC50 5.4 µM) was superior. In the indole
series, although the 5-methoxy (53) and 6-methoxy (54)
compounds had appreciable activity (IC50s 4.6 and 3.1 µM
respectively), neither were an improvement on the parent indole
52 (IC50 1.3 µM).
In a similar fashion to the compounds of Table 1, the position
of connection for subunit B was also explored. In contrast to
the monocyclic compounds (39-41), where a change of
geometry was disfavored, several of the 3-linked compounds
showed significant activity, including the N-methylindole 67
(IC50 ) 4.3 µM) and in particular, the 3-benzothiophene (60).
With an IC50 ) 0.97 µM, this compound was the most potent
in both series and additionally, was nonmutagenic in Ames
testing. The 4- (61), 5- (62), and 6-methoxy-3-benzothiophenes
(63) were all prepared, but none were as potent as the
unsubstituted parent compound 60. Perhaps surprisingly, the
3-linked indole showed poor activity (66; IC50 ∼ 20 µM), in
noticeable contrast to the 2-linked indole 52 (IC50 1.3 µM).
Replacement of the thioamide sulfur with oxygen (X ) S to
X ) O) in this series gave variable results. While for the
3-benzothiophene (65) and 3-(N-methylindole) (68), a reduction
in potency was observed when the respective pairs 65/60 and
68/67 are compared (IC50 8.5/0.97 µM and IC50 > 20/4.3 µM);
for the 2-linked benzothiophene (48, IC50 9.5 µM), this
modification resulted in an improvement in activity (51, IC50
4.6 µM).
Preparation of a single lactam, compound 69 (IC50 > 20 µM)
also confirmed the lack of utility found with this template in
the compounds of Table 1. One compound with a 7-linked
indole substituent (70) was also prepared and showed significant
activity (IC50 4.1 µM), although this has not been explored
further at this point in time.
Further Analysis of Compounds with Perforin Inhi-
bitory Activity. To determine whether the dihydrofuro[3,4-
c]pyridinone class of inhibitors are specific to perforin, or are
in fact more general inhibitors of other pore-forming proteins,
lead compound 1 was tested for its ability to inhibit the lytic
function of the pneumococcal toxin pneumolysin (PLO). PLO
is a bacterial pore-forming protein, which like perforin, is
released as soluble monomers before oligomerizing to form
active pores in the target cell membrane. At a concentration of
20 µM of compound 1, lysis of sheep red blood cells by PLO
was observed, showing that 1 specifically inhibits perforin but
not the mechanistically related PLO.
Fas-ligand (FasL), TNFR (tumor-necrosis factor R), or TRAIL
(TNF-related apoptosis inducing ligand), which trigger caspase
activation and ultimately cell death. Thus it is important to
establish that the mechanism of action of the dihydrofuro[3,4-
c]pyridinones is perforin-specific and does not also operate
through the death-receptor pathway. In a similar manner to the
perforin-induced lysis assay, Jurkat T lymphoma target cells
were labeled with 51Cr, preincubated with 1, then treated with
either anti-Fas agonistic monoclonal antibody CH11 or recom-
binant (rec)-TRAIL in order to induce cell death, which was
then measured by 51Cr release (see Experimental Section for
specific details). It was found that compound 1 does not block
cell death mediated through FasL or TRAIL (Figure 5, Sup-
porting Information). This result has therapeutic implications
because oversuppression of cell killing by cytotoxic lymphocytes
may have negative consequences such as interfering with the
capacity to down-regulate lymphocyte numbers following an
infection.4,39
A small subset of inhibitors (1, 16, 44, 51, 52, 60) were
selected on the basis of their activity in the perforin induced-
Jurkat lysis assay and evaluated for their ability to inhibit
perforin delivered by an intact NK cell line. The inhibitor (20
µM final concentration) and medium were coincubated with
KHYG-1 NK cells for 30 min at room temperature, 51Cr-labeled
K562 leukemia target cells added, and after 4 h cell lysis was
measured by 51Cr release. All compounds tested showed
inhibition of NK cell killing of the K562 targets, ranging from
50-100% (Table 3). To confirm the inhibitory activity exhibited
by the compounds against KHYG-1 NK cells was due to
blocking the action of perforin and not nonspecific toxicity
toward the effector cell, the viability of the NK cells in the
presence of the inhibitor was measured 24 h. later. Viable and
dead cells were counted and percent viability calculated based
on total cell count (for further details see Experimental Section).
Compounds 16, 44, and 52 were clearly toxic to the KHYG-1
cells, but importantly, lead compounds 1 and 60 (and also 51)
show it is possible to inhibit NK cell activity yet largely retain
NK cell viability.
Finally, lead compound 1 was tested to determine whether it
retained the ability to inhibit perforin activity in primary human
NK cells isolated from the buffy coats of healthy blood donors.
Using 51Cr-labeled K562 target cells, the release of 51Cr
resulting from cell lysis was blocked by >70%, showing that 1
Although the granule exocytosis pathway is crucial to the
operation of CTLs and NK cells, they may also exert their
cytotoxic effects through alternative death-receptor mediated
pathways.1,4 These pathways are activated by the binding of

Inhibitors of the Pore-Forming Protein Perforin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7619
and rinsed with EtOH (2 × 40 mL). Drying under vacuum afforded
4 (6.8 g, 56%) as a white powder. 1H NMR (400 MHz, DMSO-d6,
298 K) δ 1.17 (t, J ) 7.0 Hz, 3 H), 2.28 (s, 3 H), 3.53 (q, J ) 7.0
Hz, 2 H), 4.46 (s, 2 H), 6.28 (s, 1 H), 12.45 (s, 1 H). LCMS
(APCI+) calcd for C10H13N2O2, 193 (MH+), found 193.
is still able to inhibit perforin delivered by intact human NK
cells (Figure 6 in Supporting Information).
Conclusions
The dihydrofuro[3,4-c]pyridinones are the first class of small
molecules reported to inhibit the cytolytic effects of perforin in
cells. Molecules with this function are of potential use in the
treatment of some autoimmune diseases and therapy-induced
conditions characterized by undesired perforin secretion. The
preliminary data presented here includes some relatively potent
compounds (most notably the 2-indole 52 and the 3-ben-
zothiophene 60), but suggest fairly tight SAR. The lactone
oxygen and the preference for H-bond acceptor in the appended
ring appear to be a requirement for activity in the class. It is
not entirely clear whether it is necessary to have a thioamide
or an amide in the A-subunit, with variable activity in both sets
of examples. Although the 2-linked B-subunit is required for
activity in the monocycles, the point of connection is more
flexible for the bicyclic series. Substitution on the B-subunit
can also result in improved activity in some cases.
The inhibitory action of the dihydrofuro[3,4-c]pyridinones
is quite specific for perforin, with no effect observed on a
bacterial pore-forming protein with a similar mechanism of
action, or on death-receptor pathways mediated through FasL
and TNFR. Lead compounds 1 and 60 show inhibition of
KHYG-1 NK cell-mediated target cell killing while still
retaining NK cell viability. Compound 1 also shows excellent
inhibitory activity against intact primary human NK cells. This
initial study should therefore provide some substantial clues for
the further development of this class of compounds.
6-Methylfuro[3,4-c]pyridine-3,4(1H,5H)-dione (5). Compound
4 (6.4 g, 33 mmol) was dissolved in concd HCl (58 mL). The
mixture was stirred at 120 °C for 10 h then allowed to cool to RT.
A mixture of ice and water (∼100 mL) was added to the crude
mixture, and the resulting precipitate collected by filtration, washed
with water (3 × 20 mL), and dried to yield 5 (4.31 g, 79%) as a
1
white powder. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.29 (s,
3 H), 5.13 (s, 2 H), 6.30 (s, 1 H), 12.08 (s, 1 H). LCMS (APCI+)
calcd for C8H8NO3, 166 (MH+), found 166.
4-Chloro-6-methylfuro[3,4-c]pyridin-3(1H)-one (6). Com-
pound 5 (4.0 g, 24.2 mmol) was mixed with POCl3 (25 mL) and
PCl5 (7.3 g, 35.1 mmol) was added portionwise. The mixture was
stirred at 110 °C for 30 min then allowed to cool to RT. POCl3
was removed by evaporation, the residue was dissolved in CH2Cl2
(40 mL), and washed with water. The aqueous layer was extracted
with CH2Cl2 and the combined organic layers were dried over
MgSO4, which was then removed by filtration. After evaporation
of the solvent, the crude mixture was purified by silica gel column
chromatography (hexane, EtOAc, 65:35, v/v). The title compound
6 (2.99 g, 66%) was isolated as a white solid. 1H NMR (400 MHz,
DMSO-d6, 298 K) δ 2.59 (s, 3 H), 5.38 (s, 2 H), 7.60 (s, 1 H).
LCMS (APCI+) calcd for C8H7ClNO2, 184.5 (MH+), found 184.5.
6-Methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one
(7). Compound 6 (2.3 g, 12.6 mmol) was dissolved in butan-1-ol
(105 mL). Thiourea (958 mg, 12.6 mmol) was added, and the
mixture was stirred at 120 °C for 10 h. The reaction was allowed
to cool to RT and then stored at 4 °C for 16 h. The resulting solid
was collected by filtration and rinsed with EtOH (3 × 40 mL).
Drying under vacuum afforded 7 (2.01 g, 88%) as a beige solid.
1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.41 (s, 3 H), 5.14 (s, 2
H), 6.78 (s, 1 H), 13.48 (s, 1 H). LCMS (APCI+) calcd for
C8H8NO2S, 182 (MH+), found 182.
Experimental Section
Medicinal Chemistry. Analyses were performed by the Micro-
chemical 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 spectrometer and referenced to Me4Si. Mass spectra were
recorded either on a Varian VG 7070 spectrometer at nominal 5000
resolution or a Finnigan MAT 900Q spectrometer. All final
compound purities were determined to be >95% by HPLC.
Ethoxyacetylacetone (3). NaH (60% in mineral oil, 6.5 g, 162
mmol) was rinsed twice with hexane and then suspended in Et2O
(100 mL). The mixture was placed under an inert atmosphere and
cooled at -5 °C. Ethyl 2-ethoxyacetate (2) (20 mL, 147 mmol)
and a solution of acetone (12 mL, 162 mmol) in Et2O (50 mL)
were added. The mixture was stirred 3 h at -5 °C then 1 h at RT.
An ice cold solution of concd HCl (50 mL) in water (100 mL) was
added carefully. The two layers were separated, and the aqueous
phase was extracted with Et2O (100 mL). The combined organic
layers were dried over MgSO4, and the solvent was evaporated after
filtration. The crude product was purified by silica gel column
chromatography (hexane, EtOAc, 95:5, v/v) to afford 3 (15.08 g,
6-Methylfuro[3,4-c]pyridin-3(1H)-one (8). Compound 6 (730
mg, 3.97 mmol) was dissolved in EtOH (100 mL) and Et3N (6
mL), and 10% Pd/C (150 mg) were added. The reaction was carried
out in a pressure vessel under H2 (30 psi) at RT for 2.5 h. The
Pd/C was removed by filtration over celite, and the filtrate was
evaporated. The resulting residue was diluted with CH2Cl2 (20 mL)
and washed with water (20 mL). The organic layer was dried over
MgSO4, which was removed by filtration. Evaporation yielded 8
1
(440 mg, 74%) as a white foam. H NMR (400 MHz, DMSO-d6,
298 K) δ 2.62 (s, 3 H), 5.43 (s, 2 H), 7.58 (s, 1 H), 8.95 (s, 1 H).
LCMS (APCI+) calcd for C8H8NO2, 150 (MH+), found 150.
6-Methyl-1H-pyrrolo[3,4-c]pyridine-3,4(2H,5H)-dione (9). A
mixture of 4 (813 mg, 4.23 mmol) dissolved in concd H2SO4 (8
mL) was stirred at RT for 3 days. The flask was cooled to 0 °C
with ice/water and EtOH (32 mL) was added to the mixture which
produced a precipitate. This solid was collected by filtration and
dried. Dissolution in water was followed by neutralization by
addition of a saturated aqueous solution of NaHCO3. A solid
precipitated and was collected by filtration. Drying afforded the
1
71%) as a pale-yellow oil. H NMR (400 MHz, CDCl3, 298 K):
1
title compound 9 (564 mg, 81%) as a white solid. H NMR (400
1
The H NMR spectra shows two sets of signals corresponding to
MHz, DMSO-d6, 298 K) δ 2.28 (s, 3 H), 5.13 (s, 2 H), 6.28 (s, 1
H), 7.67 (s, 1 H), 12.18 (s, 1 H). LCMS (APCI+) calcd for
C8H9N2O2, 165 (MH+), found 165.
the two tautomeric forms of the molecule. Form 1: δ 1.23 (t, J )
7.0 Hz, 3 H), 2.26 (s, 3 H), 3.54 (q, J ) 7.0 Hz, 2 H), 3.63 (s, 2
H), 4.01 (s, 2 H); Form 2: δ 1.25 (t, J ) 7.0 Hz, 3 H), 2.09 (s, 3
H), 3.58 (q, J ) 7.0 Hz, 2 H), 4.01 (s, 2 H), 5.81 (s, 1 H), 15.23
(s, 1 H). LCMS (APCI+) calcd for C6H11O3, 131 (MH+), found
131.
4-(Ethoxymethyl)-6-methyl-2-oxo-1,2-dihydropyridine-3-car-
bonitrile (4). 2-Cyanoacetamide (5.75 g, 68.4 mmol) was suspended
in EtOH (60 mL) and the mixture was heated to 75 °C, at which
point 3 (8.9 g, 62.2 mmol) and piperidine (710 µL, 8.1 mmol) were
added and the mixture was stirred vigorously at 75 °C for 2 h. The
mixture, which contained a precipitate, was allowed to cool to RT
and stored 4 h at 4 °C. The resulting solid was collected by filtration
4-(Ethoxymethyl)-6-methyl-2-thioxo-1,2-dihydropyridine-3-
carbonitrile (10). In a flask wrapped with aluminum foil, 2-cyan-
othioacetamide (1.53 g, 15.3 mmol) was mixed with EtOH (20 mL).
The mixture was heated at 75 °C, and then 3 (2.0 g, 13.8 mmol)
and piperidine (157 µL, 1.8 mmol) were added. The reaction was
stirred at 75 °C for 2 h and then allowed to cool to RT. After 2 h
at 4 °C, the precipitate was collected by filtration and rinsed with
EtOH (2 × 10 mL). Drying gave 10 (1.17 g, 41%) as a brown
1
solid. H NMR (400 MHz, DMSO-d6, 298 K) δ 1.18 (t, J ) 7.0
Hz, 3 H), 2.41 (s, 3 H), 3.56 (q, J ) 7.0 Hz, 2 H), 4.50 (s, 2 H),

7620 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Lena et al.
6.78 (s, 1 H), 13.42 (s, 1 H). LCMS (APCI+) calcd for C10H13N2OS,
209 (MH+), found 209.
12.17 (s, 1 H), 13.49 (s, 1 H). HRMS (EI+) calcd for C12H9N3O2S,
259.04155 (M+), found 259.04162.
6-Methyl-4-thioxo-4,5-dihydro-1H-pyrrolo[3,4-c]pyridin-3(2H)-
one (11). Compound 10 (362 mg, 1.74 mmol) was mixed with
concentrated H2SO4 (3.5 mL), and the reaction was performed in
exactly the same manner as for 9. The title compound 11 (172 mg,
55%) was obtained as a dark-green solid. 1H NMR (400 MHz,
DMSO-d6, 298 K) δ 2.43 (s, 3 H), 5.16 (s, 2 H), 6.84 (s, 1 H),
9.74 (s, 1 H), 13.68 (s, 1 H). LCMS (APCI+) calcd for C8H9N2OS,
181 (MH+), found 181.
General Procedure for the Aldolization-Crotonization Step.
A suspension of the appropriate bicyclic starting material (5, 7, 8,
9, or 11) in EtOH (0.07 mmol/mL) was heated at 60 °C. Piperidine
(0.1 equiv) and a solution of aldehyde (1.1 equiv) in EtOH (0.3
mmol/mL) were added. The mixture was heated to 75 °C. The
reaction was monitored by MS and stopped when the peak
corresponding to the starting material had disappeared (reaction
time varied from 40 min. to 20 h.). The mixture was allowed to
cool to RT and stored 2 h at 4 °C. The resulting solid was collected
by filtration, rinsed with EtOH (2 × 5 mL), Et2O (2 × 5 mL), and
dried to give the desired product.
(Z)-6-Methyl-1-((1-methyl-1H-pyrrol-2-yl)methylene)-4-thioxo-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (19). Yield 60%, dark-
red solid, mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K)
1
δ 2.41 (s, 3 H), 3.77 (s, 3 H) 6.25 (dd, J ) 2.6, 3.8 Hz), 6.91 (dd,
J ) 1.5, 4 Hz, 1 H), 7.0 (s, 1 H), 7.08 (t, J ) 2.0 Hz, 1 H), 7.22
(s, 1 H), 13.06 (s, 1 H). Anal. (C14H12N2O2S·0.5H2O) C, H, N.
(Z)-1-Benzylidene-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]py-
ridin-3(1H)-one (20). Yield 53%, yellow solid, mp 299-303 °C.
1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.45 (s, 3 H), 7.01 (s, 1
H), 7.19 (s, 1 H), 7.41 (t, J ) 7.3 Hz, 1 H), 7.48 (t, J ) 7.3 Hz, 2
H), 7.81 (d, J ) 7.3 Hz, 2 H), 13.52 (s, 1 H). HRMS (EI+) calcd
for C15H12NO2S, 270.05888 (MH+), found 270.05923.
(Z)-1-(4-Fluorobenzylidene)-6-methyl-4-thioxo-4,5-dihydro-
furo[3,4-c]pyridin-3(1H)-one (21). Yield 11%, yellow solid, mp
1
> 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2. 43 (s, 3
H), 7.00 (s, 1 H), 7.14 (s, 1 H), 7.34 (t, J ) 8.8 Hz, 2 H), 7.86 (dd,
J ) 5.7, 8.8 Hz, 2 H), 13.61 (s, 1 H). HRMS (EI+) calcd for
C15H11FNO2S, 288.04945 (MH+), found 288.04941.
(Z)-1-(4-Chlorobenzylidene)-6-methyl-4-thioxo-4,5-dihydro-
furo[3,4-c]pyridin-3(1H)-one (22). Yield 72%, yellow solid, mp
> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.44 (s, 3 H),
7.02 (s, 1 H), 7.16 (s, 1 H), 7.57 (d, J ) 8.6 Hz, 2 H), 7.82 (d, J
) 8.6 Hz, 2 H), 13.55 (s, 1 H). Anal. (C15H10ClNO2S) H, N. C:
calcd, 59.3; found 58.8.
(Z)-6-Methyl-1-((5-methylfuran-2-yl)methylene)-4-thioxo-4,5-
dihydrofuro[3,4-c]pyridin-3(1H)-one (1). Yield 59%, bright-
1
orange solid, mp 292-296 °C. H NMR (400 MHz, DMSO-d6,
298 K) δ 2.38 (s, 3 H), 2.40 (s, 3 H), 6.37 (d, J ) 3.3 Hz, 1 H),
6.91 (d, J ) 3.3 Hz, 1 H), 6.99 (s, 1 H), 7.13 (s, 1 H), 13.40 (s, 1
H). Anal. (C14H11NO3S) C, H, N.
(Z)-1-(4-Bromobenzylidene)-6-methyl-4-thioxo-4,5-dihydro-
furo[3,4-c]pyridin-3(1H)-one (23). Yield 44%, yellow solid, mp
> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.44 (s, 3 H),
7.00 (s, 1 H), 7.16 (s, 1 H), 7.74 (m, 4 H), 13.62 (s, 1 H). HRMS
(EI+) calcd for C15H1179BrNO2S, 347.96939 (MH+), found
347.97048; calcd for C15H1181BrNO2S, 349.96734 (MH+), found
349.96858.
(Z)-6-Methyl-1-(4-methylbenzylidene)-4-thioxo-4,5-dihydro-
furo[3,4-c]pyridin-3(1H)-one (24). Yield 73%, yellow solid, mp
> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.36 (s, 3 H),
2.44 (s, 3 H), 6.98 (s, 1 H), 7.17 (s, 1 H), 7.31 (d, J ) 8.1 Hz, 2
H), 7.72 (d, J ) 8.2 Hz, 2 H), 13.52 (s, 1 H). Anal. (C16H13NO2S)
C, H, N.
(Z)-6-Methyl-4-thioxo-1-(4-(trifluoromethyl)benzylidene)-4,5-
dihydrofuro[3,4-c]pyridin-3(1H)-one (25). Yield 17%, yellow
solid, mp 290-294 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ
2.45 (s, 3 H), 3.82 (s, 3 H), 7.09 (s, 1 H), 7.19 (s, 1 H), 7.85 (d, J
) 8.4 Hz, 2 H), 7.99 (d, J ) 8.4 Hz, 2 H), 13.64 (s, 1 H). Anal.
(C16H10F3NO2S·0.25H2O) C, H, N.
(Z)-1-(4-Methoxybenzylidene)-6-methyl-4-thioxo-4,5-dihydro-
furo[3,4-c]pyridin-3(1H)-one (26). Yield 84%, yellow solid, mp
> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.41 (s, 3 H),
3.82 (s, 3 H), 6.97 (s, 1 H), 7.08 (d, J ) 8.9 Hz, 2 H), 7.13 (s, 1
H), 7.78 (d, J ) 8.9 Hz, 2 H), 13.44 (s, 1 H). Anal. (C16H13NO3S)
C, H, N.
(Z)-1-(Furan-2-ylmethylene)-6-methyl-4-thioxo-4,5-dihydro-
furo[3,4-c]pyridin-3(1H)-one (12). Yield 49%, brown solid, mp
> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42 (s, 3 H),
6.72 (dd, J ) 1.7, 3.4 Hz, 1 H), 7.01 (d, J ) 3.4 Hz, 1 H), 7.08 (s,
1 H), 7.19 (s, 1 H), 8.04 (d, J ) 1.3 Hz, 1 H), 13.49 (s, 1 H). 13
C
NMR (100 MHz, DMSO-d6, 298 K) δ 19.13, 101.00, 102.98,
113.25, 115.91, 117.33, 139.40, 145.68, 148.43, 150.22, 154.95,
163.64, 175.97. HRMS (EI+) calcd for C14H12NO3S, 274.05379
(MH+), found 274.05404.
(Z)-6-Methyl-1-((5-phenylfuran-2-yl)methylene)-4-thioxo-4,5-
dihydrofuro[3,4-c]pyridin-3(1H)-one (13). Yield 96%, red solid,
1
mp 290-294 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42
(s, 3 H), 7.14 (m, 2 H), 7.38 (tt, J ) 1.1, 1.1, 1.7, 7.4 Hz, 1 H),
7.50 (t, J ) 7.7 Hz, 2 H), 7.81 (dd, J ) 1.1, 8.5 Hz, 2 H), 13.47
(s, 1 H). Anal. (C19H13NO3S) C, H, N.
(Z)-6-Methyl-1-(thiophen-2-ylmethylene)-4-thioxo-4,5-dihy-
drofuro[3,4-c]pyridin-3(1H)-one (14). Yield 81%, brown solid,
1
mp 280-284 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42
(s, 3 H), 7.12 (s, 1 H), 7.21 (dd, J ) 3.7, 5.1 Hz, 1 H), 7.40 (s, 1
H), 7.49 (d, J ) 3.5 Hz, 1 H), 7.87 (d, J ) 5.0 Hz, 1 H) 12.8 (s,
1 H). Anal. (C13H9NO2S2 ·1.25H2O) C, H, N.
(Z)-6-Methyl-1-((5-methylthiophen-2-yl)methylene)-4-thioxo-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (15). Yield 61%, brown
solid, mp 286-290 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ
2.41 (s, 3 H), 2.51 (s, 3 H), 6.92 (dd, J ) 1.1, 3.7 1 H), 7.10 (s, 1
H), 7.31 (m, 2 H), 13.36 (s, 1 H). Anal. (C14H11NO2S2) H, N. C:
calcd, 58.1; found 57.6.
(Z)-6-Methyl-4-thioxo-1-(4-(trifluoromethoxy)benzylidene)-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (27). Yield 8%, orange
solid, mp 279-282 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ
2.44 (s, 3 H), 7.04 (s, 1 H), 7.16 (s, 1 H), 7.49 (d, J ) 8.3 Hz, 2
H), 7.92 (d, J ) 8.8 Hz, 2 H), 13.62 (s, 1 H). HRMS (EI+) calcd
for C16H11F3NO3S, 354.04117 (MH+), found 354.04143.
(Z)-6-Methyl-1-((5-phenylthiophen-2-yl)methylene)-4-thioxo-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (16). Yield 77%, orange
1
solid, mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ
2.43 (s, 3 H), 7.13 (s, 1 H), 7.38 (t, J ) 7.3 Hz, 1 H), 7.41 (s, 1 H),
7.46 (t, J ) 7.2 Hz, 2 H), 7.50 (d, J ) 4.2 Hz, 1 H), 7.63 (d, J )
3.9 Hz, 1 H), 7.77 (m, 2 H), 13.48 (s, 1 H). Anal. (C19H13NO2S2)
H, N. C: calcd, 64.9; found 64.4.
(Z)-6-Methyl-1-(4-(methylthio)benzylidene)-4-thioxo-4,5-dihy-
drofuro[3,4-c]pyridin-3(1H)-one (28). Yield 43%, yellow solid,
1
mp 308-311 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.44
(Z)-1-((1H-Pyrrol-2-yl)methylene)-6-methyl-4-thioxo-4,5-di-
hydrofuro[3,4-c]pyridin-3(1H)-one (17). Yield 94%, red solid, mp
> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.40 (s, 3 H),
6.28 (m, 1 H), 6.72 (m, 1 H), 6.89 (s, 1 H), 7.02 (s, 1 H), 7.10 (m,
1 H), 11.26 (s, 1 H), 13.23 (s, 1 H, NH). Anal. (C13H10N2O2S·
0.5H2O) C, H, N.
(s, 3 H), 2.49 (s, 3 H), 6.99 (s, 1 H), 7.16 (s, 1 H), 7.37 (d, J ) 8.6
Hz, 2 H), 7.75 (d, J ) 8.6 Hz, 2 H), 13.48 (s, 1 H). Anal.
(C16H13NO2S2) C, H, N.
(Z)-1-(4-(Dimethylamino)benzylidene)-6-methyl-4-thioxo-4,5-
dihydrofuro[3,4-c]pyridin-3(1H)-one (29). Yield 25%, dark-red
solid, mp 281-284 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ
2.40 (s, 3 H), 3.02 (s, 6 H), 6.81 (d, J ) 9.1 Hz, 2 H), 6.89 (s, 1
H), 7.07 (s, 1 H), 7.68 (d, J ) 9.0 Hz, 2 H), 13.28 (s, 1 H). Anal.
(C17H16N2O2S·0.8CH2Cl2) C, H, N.
(Z)-1-((1H-Imidazol-2-yl)methylene)-6-methyl-4-thioxo-4,5-di-
hydrofuro[3,4-c]pyridin-3(1H)-one (18). Yield 64%, orange solid,
1
mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42 (s,
3 H), 7.00 (s, 1 H), 7.19 (br s, 1 H), 7.28 (s, 1 H), 7.33 (br s, 1 H),

Inhibitors of the Pore-Forming Protein Perforin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7621
(Z)-N-(4-((6-Methyl-3-oxo-4-thioxo-4,5-dihydrofuro[3,4-c]py-
ridin-1(3H)-ylidene)methyl)phenyl)acetamide (30). Yield 33%,
yellow solid, mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298
K) δ 2.07 (s, 3 H), 2.43 (s, 3 H), 6.95 (s, 1 H), 7.16 (s, 1 H), 7.71
(d, J ) 8.9 Hz, 2 H), 7.76 (d, J ) 8.9 Hz, 2 H), 13.47 (s, 1 H).
Anal. (C17H14N2O3S) C, H, N.
7.69 (dd, J ) 2.9, 5.0 Hz, 1 H), 7.99 (dd, J ) 0.8, 2.9 Hz, 1 H)
13.48 (s, 1 H). Anal. (C13H9NO2S2) C, H. N: calcd, 5.1; found 4.4.
(Z)-6-Methyl-1-((5-phenylthiophen-3-yl)methylene)-4-thioxo-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (41). Yield 73%, yellow
1
1
solid, mp > 295 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ
2.43 (s, 3 H), 7.05 (s, 1 H), 7.12 (s, 1 H), 7.37 (t, J ) 7.3 Hz, 1 H),
7.47 (t, J ) 7.4 Hz, 2 H), 7.68 (d, J ) 7.2 Hz, 2 H), 7.80 (d, J )
(Z)-1-(Biphenyl-4-ylmethylene)-6-methyl-4-thioxo-4,5-dihy-
drofuro[3,4-c]pyridin-3(1H)-one (31). Yield 70%, yellow solid,
1.1 Hz,
1 H), 7.98 (s, 1 H), 13.51 (s, 1 H). Anal.
1
(C19H13NO2S2 ·1.2H2O) C, H, N.
mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.46 (s,
(Z)-1-(Benzofuran-2-ylmethylene)-6-methyl-4-thioxo-4,5-di-
hydrofuro[3,4-c]pyridin-3(1H)-one (42). Yield 70%, orange solid,
3 H), 7.07 (s, 1 H), 7.21 (s, 1 H), 7.41 (t, J ) 7.4 Hz, 1 H), 7.50
(t, J ) 7.3 Hz, 2 H), 7.75 (d, J ) 8.5 Hz, 2 H), 7.83 (d, J ) 8.5
Hz, 2 H), 7.91 (d, J ) 8.5 Hz, 2 H), 13.62 (s, 1 H). Anal.
(C21H15NO2S·0.75H2O) C, H, N.
1
mp 291-295 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.44
(s, 3 H), 7.02 (s, 1 H), 7.25 (s, 1 H), 7.31 (t, J ) 7.1 Hz, 1 H), 7.41
(t, J ) 7.2 Hz, 1 H), 7.43 (s, 1 H), 7.65 (dd, J ) 0.7, 8.3 Hz, 1 H),
7.76 (d J ) 7.5 Hz, 1 H), 13.60 (s, 1 H). Anal. (C17H11NO3S) C,
H, N.
(Z)-6-Methyl-1-((5-methylfuran-2-yl)methylene)furo[3,4-c]py-
ridine-3,4(1H,5H)-dione (32). Yield 64%, yellow solid, mp >290
1
°C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.30 (s, 3 H), 2.37
(Z)-1-((4-Methoxybenzofuran-2-yl)methylene)-6-methyl-4-
(s, 3 H), 6.35 (d, J ) 3.3 Hz, 1 H), 6.70 (s, 1 H), 6.87 (d, J ) 3.3
Hz, 1 H), 6.89 (s, 1 H), 12.15 (s, 1 H). Anal. (C14H11NO4 ·0.25H2O)
C, H, N.
thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (43). Yield 27%,
1
orange solid, mp 290-294 °C. H NMR (400 MHz, DMSO-d6,
298 K) δ 2.44 (s, 3 H), 3.94 (s, 3 H), 6.84 (d, J ) 7.8 Hz, 1 H),
7.15 (s, 1 H), 7.21 (s, 1 H), 7.24 (d, J ) 8.3 Hz, 1 H), 7.33 (s, 1
H), 7.36 (t, J ) 7.9 Hz, 1 H), 13.57 (s, 1 H). Anal. (C18H13NO4S)
C, H, N.
(Z)-6-Methyl-1-((5-phenylthiophen-2-yl)methylene)furo[3,4-
c]pyridine-3,4(1H,5H)-dione (33). Yield 89%, yellow solid, mp
> 295 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.33 (s, 3 H),
6.69 (s, 1 H), 7.31 (s, 1 H), 7.36 (t, J ) 7.3 Hz, 1 H), 7.46 (m, 3
H), 7.60 (d, J ) 3.9 Hz, 1 H), 7.57 (d, J ) 7.2 Hz, 2 H), 12.11 (s,
1 H). Anal. (C19H13NO3S) C, H, N.
(Z)-1-((1H-Pyrrol-2-yl)methylene)-6-methylfuro[3,4-c]pyridine-
3,4(1H,5H)-dione (34). Yield 68%, brown solid, mp > 300 °C.
1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.30 (s, 3 H), 6.26 (t, J
) 2.8 Hz, 1 H), 6.58 (d, J ) 0.5 Hz, 1 H), 6.69 (d, J ) 3.3 Hz, 1
H), 6.80 (s, 1 H), 7.07 (s, 1 H), 11.24 (s, 1 H), 11.97 (s, 1 H).
Anal. (C13H10N2O3) C, H, N.
(Z)-1-((5-Methoxybenzofuran-2-yl)methylene)-6-methyl-4-
thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (44). Yield 38%,
1
orange solid, mp > 295 °C. H NMR (400 MHz, DMSO-d6, 298
K) δ 2.44 (s, 3 H), 3.80 (s, 3 H), 7.02 (dd, J ) 2.6, 9 Hz, 1 H),
7.17 (s, 1 H), 7.24 (s, 1 H), 7.25 (d, J ) 2.5 Hz, 1 H), 7.36 (s, 1
H), 7.55 (d,
(C18H13NO4S·0.75H2O) C, H, N.
(Z)-1-((6-Methoxybenzofuran-2-yl)methylene)-6-methyl-4-
thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (45). Yield 21%,
red solid, mp > 295 °C. H NMR (400 MHz, DMSO-d6, 298 K)
J ) 9 Hz, 1 H), 13.56 (s, 1 H). Anal.
(Z)-6-Methyl-1-((5-methylfuran-2-yl)methylene)-4-thioxo-4,5-
dihydro-1H-pyrrolo[3,4-c]pyridin-3(2H)-one (35). Yield 31%,
1
δ 2.43 (s, 3 H), 3.85 (s, 3 H), 6.94 (dd, J ) 2.2, 8.6 Hz, 1 H), 7.16
(s, 1 H), 7.23 (s, 1 H), 7.29 (d, J ) 1.2 Hz, 1 H), 7.36 (s, 1 H),
7.62 (d, J ) 8.6 Hz, 1 H), 13.52 (s, 1 H). Anal. (C18H13NO4S·0.5
H2O) C, H, N.
1
brown solid, mp > 290 °C. H NMR (400 MHz, DMSO-d6, 298
K) δ 2.36 (s, 3 H), 2.42 (s, 3 H), 6.35 (d, J ) 3.3 Hz, 1 H), 6.84
(s, 1 H), 6.87 (d, J ) 3.3 Hz, 1 H), 7.19 (s, 1 H), 9.80 (s, 1 H),
13.66 (s, 1 H). HRMS (EI+) calcd for C14H13N2O2S, 273.06977
(MH+), found 273.07023.
(Z)-1-((7-Methoxybenzofuran-2-yl)methylene)-6-methyl-4-
thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (46). Yield 10%,
(Z)-6-Methyl-1-((5-methylfuran-2-yl)methylene)-1H-pyrrolo[3,4-
c]pyridine-3,4(2H,5H)-dione (36). Yield 22%, green solid, mp
200-204 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.30 (s, 3
H), 2.33 (s, 3 H), 6.33 (d, J ) 0.3 Hz, 1 H), 6.66 (s, 1 H), 6.74 (s,
1 H), 6.85 (d, J ) 2.9 Hz, 1 H), 8.59 (s, 1 H), 12.14 (s, 1 H).
HRMS (EI+) calcd for C14H13N2O3, 257.09262 (MH+), found
257.09273.
1
yellow solid, mp 286-290 °C. H NMR (400 MHz, DMSO-d6,
298 K) δ 2.44 (s, 3 H), 3.97 (s, 3 H), 7.01 (d, J ) 7.8 Hz, 1 H),
7.22 (m, 3 H), 7.31 (d, J ) 7.7 Hz, 1 H), 7.40 (s, 1 H), 13.59 (s,
1 H). HRMS (EI+) calcd for C18H14NO4S, 340.06435 (MH+), found
340.06469.
(Z)-1-((6-(Benzyloxy)-5-methoxybenzofuran-2-yl)methylene)-
6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (47).
Yield 73%, red solid, mp 278-282 °C. 1H NMR (400 MHz,
DMSO-d6, 298 K) δ 2.43 (s, 3 H), 3.82 (s, 3 H), 5.19 (s, 2 H),
7.13 (s, 1 H), 7.20 (s, 1 H), 7.26 (s, 1 H), 7.31 (s, 1 H), 7.34 (t, J
) 7.2 Hz, 1 H), 7.71 (t, J ) 7.5 Hz, 2 H), 7.43 (s, 1 H), 7.48 (d,
J ) 7.0 Hz, 1 H), 13.48 (s, 1 H). Anal. (C25H19NO5S) C, H, N.
(Z)-1-(Benzo[b]thiophen-2-ylmethylene)-6-methyl-4-thioxo-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (48). Yield 86%, yellow
(Z)-6-Methyl-1-((5-phenylthiophen-2-yl)methylene)-4-thioxo-
4,5-dihydro-1H-pyrrolo[3,4-c]pyridin-3(2H)-one (37). Yield 9%,
1
dark-brown solid, mp > 290 °C. H NMR (400 MHz, DMSO-d6,
298 K) (E and Z isomers coexist in this spectra but are not
distinguished) δ 2.46 (s, 3 H), 2.49 (s, 3 H), 6.85 (s, 1 H), 7.13 (s,
1 H), 7.25 (m, 3 H), 7.37 (m, 3 H), 7.45 (m, 4 H), 7.60 (d, J ) 3.8
Hz, 1 H), 7.64 (d, J ) 7.6 Hz, 2 H), 7.74 (d, J ) 7.5 Hz, 2 H),
8.92 (s, 1 H), 9.89 (s, 1 H), 10.38 (s, 1 H), 13.81 (s, 2 H). Anal.
(C19H14N2OS2 ·1.5H2O) C, H, N.
1
solid, mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ
2.41 (s, 3 H), 7.19 (s, 1 H), 7.44 (m, 2 H), 7.49 (s, 1 H), 7.80 (s,
1 H), 7.95 (m, 1 H), 8.03 (m, 1 H), 13.48 (s, 1 H). Anal.
(C17H11NO2S2) C, H, N.
(Z)-6-Methyl-1-((5-phenylthiophen-2-yl)methylene)-1H-pyr-
rolo[3,4-c]pyridine-3,4(2H,5H)-dione (38). Yield 24%, brown
1
solid, mp > 295 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ
(Z)-1-((5-Methoxybenzo[b]thiophen-2-yl)methylene)-6-meth-
yl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (49). Yield
47%, red solid, mp > 290 °C. 1H NMR (400 MHz, DMSO-d6,
298 K) δ 2.44 (s, 3 H), 3.86 (s, 3 H), 7.04 (dd, J ) 2.2, 8.7 Hz, 1
H), 7.16 (s, 1 H), 7.43 (s, 1 H), 7.61 (d, J ) 1.7 Hz, 1 H), 7.71 (s,
1 H), 7.85 (d, J ) 8.6 Hz, 1 H), 13.49 (s, 1 H). Anal.
(C18H13NO3S2 ·0.75H2O) C, H, N.
(Z)-1-((6-Methoxybenzo[b]thiophen-2-yl)methylene)-6-meth-
yl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (50). Yield
46%, orange solid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6,
298 K) δ 2.44 (s, 3 H), 3.83 (s, 3 H), 7.06 (dd, J ) 2.5, 8.8 Hz, 1
H), 7.19 (s, 1 H), 7.47 (s, 1 H), 7.48 (d, J ) 2.4 Hz, 1 H), 7.72 (s,
1 H), 7.89 (d, J ) 8.8 Hz, 1 H), 13.43 (s, 1 H). Anal.
(C18H13NO3S2 ·H2O) C, H, N.
2.35 (s, 3 H), 6.35 (s, 1 H), 7.21 (m, 2 H), 7.38 (m, 3 H), 7.61 (d,
J ) 7.3 Hz, 2 H), 8.23 (m, 2 H), 12.50 (s, 1 H). HRMS (EI+)
calcd for C19H15N2O2S, 335.08542 (MH+), found 335.08558.
(Z)-1-(Furan-3-ylmethylene)-6-methyl-4-thioxo-4,5-dihydro-
furo[3,4-c]pyridin-3(1H)-one (39). Yield 60%, yellow solid, mp
290-294 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42 (s, 3
H), 6.89 (d, J ) 1.8 Hz, 1 H), 6.97 (s, 1 H), 7.11 (s, 1 H), 7.81 (t,
J ) 1.5 Hz, 1 H), 8.13 (d, J ) 0.7 Hz, 1 H), 13.48 (s, 1 H). Anal.
(C13H9NO3S) C, H, N.
(Z)-6-Methyl-1-(thiophen-3-ylmethylene)-4-thioxo-4,5-dihy-
drofuro[3,4-c]pyridin-3(1H)-one (40). Yield 46%, brown solid,
1
mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42 (s,
3 H), 7.10 (s, 1 H), 7.13 (s, 1 H), 7.53 (dd, J ) 1.1, 5.1 Hz, 1 H),

7622 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Lena et al.
(Z)-1-(Benzo[b]thiophen-2-ylmethylene)-6-methylfuro[3,4-
c]pyridine-3,4(1H,5H)-dione (51). Yield 74%, yellow solid, mp
> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.35 (s, 3 H),
6.77 (s, 1 H), 7.42 (m, 3 H), 7.44 (s, 1 H), 7.94 (m, 1 H), 8.02 (m,
1 H), 12.20 (s, 1 H). Anal. (C17H11NO3S·0.7H2O) C, H, N.
(Z)-1-((1H-Indol-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihy-
drofuro[3,4-c]pyridin-3(1H)-one (52). Yield 16%, dark-brown
1 H), 8.39 (s, 1 H), 13.48 (br s, 1 H). HRMS (FAB+) calcd for
C18H14NO3S2, 356.04151(MH+), found 356.04162.
(Z)-1-((5-Methoxybenzo[b]thiophen-3-yl)methylene)-6-meth-
yl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (62). Yield
28%, yellow solid, mp 237-241 °C. 1H NMR (400 MHz, DMSO-
d6, 298 K) δ 2.48 (s, 3 H), 3.92 (s, 3 H), 7.12 (dd, J ) 2.3, 8.8 Hz,
1 H), 7.39 (s, 1 H), 7.42 (s, 1 H), 7.83 (d, J ) 2.4 Hz, 1 H), 7.94
(d, J ) 8.8 Hz, 1 H), 8.42 (s, 1 H), 13.26 (s, 1 H). HRMS (EI+)
calcd for C18H14NO3S2, 356.04151 (MH+), found 356.04112.
(Z)-1-((6-Methoxybenzo[b]thiophen-3-yl)methylene)-6-meth-
yl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (63). Yield
84%, yellow solid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6,
298 K) δ 2.46 (s, 3 H), 3.86 (s, 3 H), 7.15 (dd, J ) 2.4, 8.9 Hz, 1
H), 7.38 (s, 1 H), 7.42 (s, 1 H), 7.65 (d, J ) 2.3 Hz, 1 H), 8.20 (d,
J ) 8.9 Hz, 1 H), 8.22 (s, 1 H), 13.51 (s, 1 H). Anal.
(C18H13NO3S2 ·0.5H2O) C, H, N.
(Z)-1-((5-Bromobenzo[b]thiophen-3-yl)methylene)-6-methyl-
4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (64). Yield
32%, orange solid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6,
298 K) δ 2.47 (s, 3 H), 7.43 (s, 1 H), 7.44 (s, 1 H), 7.61 (dd, J )
1.8, 8.6 Hz, 1 H), 8.06 (d, J ) 8.6 Hz, 1 H), 8.48 (s, 1 H), 8.62 (d,
J ) 1.8 Hz, 1 H), 13.49 (s, 1 H). Anal. (C17H10BrNO2S2 ·0.7H2O)
C, H, N: calcd, 3.4, found 4.4.
1
solid, mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ
2.44 (s, 3 H), 7.03 (m, 3 H), 7.17 (m, 3 H), 7.55 (d, J ) 8.3, 1 H),
7.31 (d, J ) 8.0, 1 H), 11.23 (s, 1 H), 13.02 (s, 1 H). Anal.
(C17H12N2O2S·0.25H2O) C, H, N.
(Z)-1-((5-Methoxy-1H-indol-2-yl)methylene)-6-methyl-4-thioxo-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (53). Yield 41%, dark
1
brown solid, mp > 295 °C. H NMR (400 MHz, DMSO-d6, 298
K) δ 2.43 (s, 3 H), 3.76 (s, 3 H), 6.85 (dd, J ) 2.5, 8.9 Hz, 1 H),
6.91 (d, J ) 1.5 Hz, 1 H), 7.04 (s, 1 H), 7.08 (d, J ) 2.5 Hz, 1 H),
7.14 (s, 1 H), 7.45 (d, J ) 8.9 Hz, 1 H), 11.10 (s, 1 H), 13.11 (s,
1 H). Anal. (C18H14N2O3S.0.25H2O) C, H, N.
(Z)-1-((6-Methoxy-1H-indol-2-yl)methylene)-6-methyl-4-thioxo-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (54). Yield 97%, dark-
1
red solid, mp 250-254 °C. H NMR (400 MHz, DMSO-d6, 298
K) δ 2.42 (s, 3 H), 3.82 (s, 3 H), 6.71 (dd, J ) 2.3, 8.7 Hz, 1 H),
6.92 (d, J ) 1.6 Hz, 1 H), 7.04 (s, 1 H), 7.08 (d, J ) 2.1 Hz, 1 H),
7.12 (s, 1 H), 7.49 (d, J ) 8.7 Hz, 1 H), 11.10 (s, 1 H), 13.27 (s,
1 H). Anal. (C18H14N2O3S·0.25H2O) C, H, N.
(Z)-1-(Benzo[b]thiophen-3-ylmethylene)-6-methylfuro[3,4-
c]pyridine-3,4(1H,5H)-dione (65). Yield 75%, yellow solid, mp
> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.37 (s, 3 H),
7.03 (s, 1 H), 7.35 (s, 1 H), 7.46 (t, J ) 7.9 Hz, 1 H), 7.54 (t, J )
7.9 Hz, 1 H), 8.08, (d, J ) 7.9 Hz, 1 H), 8.34 (d, J ) 7.9 Hz, 1 H),
8.39 (s, 1 H), 12.17 (s, 1 H). Anal. (C17H11NO3S) C, H, N.
(Z)-1-((1H-Indol-3-yl)methylene)-6-methyl-4-thioxo-4,5-dihy-
drofuro[3,4-c]pyridin-3(1H)-one (66). Yield 53%, orange solid,
(Z)-1-((6-(Benzyloxy)-5-methoxy-1H-indol-2-yl)methylene)-6-
methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (55).
1
Yield 83%, brown solid, mp 240-244 °C. H NMR (400 MHz,
DMSO-d6, 298 K) δ 2.44 (s, 3 H), 3.79 (s, 3 H), 5.13 (s, 2 H),
6.88 (s, 1 H), 7.01 (s, 1 H), 7.10 (d, J ) 7.3 Hz, 2 H), 7.18 (s, 1
H), 7.34 (t, J ) 7.3 Hz, 1 H), 7.41 (t, J ) 7.1 Hz, 2 H), 7.49 (d,
J ) 7.0 Hz, 1 H), 10.95 (s, 1 H) 13.27 (s, 1 H). Anal. (C25H20N2O4S)
C, H, N.
1
mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.43 (s,
3 H), 7.21 (m, 2 H), 7.24 (s, 1 H), 7.44 (s, 1 H), 7.49 (dd, J ) 1.7,
6 Hz, 1 H), 8.01 (dd, J ) 1.9, 7 Hz, 1 H), 8.04 (s, 1 H), 11.97 (s,
1 H), 13.23 (s, 1 H). Anal. (C17H12N2O2S·0.15H2O) C, H, N.
(Z)-6-Methyl-1-((1-methyl-1H-indol-3-yl)methylene)-4-thioxo-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (67). Yield 92%, orange
(Z)-1-((5-Fluoro-6-methoxy-1H-indol-3-yl)methylene)-6-meth-
yl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (56). Yield
28%, red solid, mp > 280 °C. 1H NMR (400 MHz, DMSO-d6,
298 K) δ 2.43 (s, 3 H), 3.88 (s, 3 H), 6.92 (d, J ) 1.7 Hz, 1 H),
7.05 (s, 1 H), 7.12 (s, 1 H), 7.24 (d, J ) 7.6 Hz, 1 H), 7.40 (d, J
) 11.7 Hz, 1 H), 11.15 (s, 1 H), 13.39 (s, 1 H). Anal.
(C18H13FN2O3S·0.75H2O) C, H, N.
(Z)-1-((5-(Benzyloxy)-1H-indol-2-yl)methylene)-6-methyl-4-
thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (57). Yield 70%,
brown solid, mp 247-251 °C. 1H NMR (400 MHz, DMSO-d6,
298 K) δ 2.43 (s, 3 H), 5.10 (s, 2 H), 6.90 (d, J ) 1.6 Hz, 1 H),
6.92 (dd, J ) 2.4, 8.9 Hz, 1 H), 7.03 (s, 1 H), 7.14 (s, 1 H), 7.18
(d, J ) 2.4 Hz, 1 H), 7.33 (t, J ) 7.25 Hz, 2 H), 7.39 (d, J ) 7.1
Hz, 2 H), 7.46 (m, 2 H) 11.11 (s, 1 H) 13.28 (s, 1 H). Anal.
(C24H18N2O3S) C, H, N.
1
solid, mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ
2.47 (s, 3 H), 3.93 (s, 3 H), 7.23 (s, 1 H), 7.25 (m, 1 H), 7.30 (t,
J ) 7.9 Hz, 1 H), 7.42 (s, 1 H), 7.54 (d, J ) 7.9 Hz, 1 H), 8.02 (d,
J ) 7.4 Hz, 1 H), 8.07 (s, 1 H), 13.08 (s, 1 H). Anal.
(C18H14N2O2S·0.5H2O) C, H, N.
(Z)-6-Methyl-1-((1-methyl-1H-indol-3-yl)methylene)furo[3,4-
c]pyridine-3,4(1H,5H)-dione (68). Yield 66%, yellow solid, mp
> 290 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.32 (s, 3 H),
3.90 (s, 3 H), 6.81 (s, 1 H), 7.24 (t, J ) 7 Hz, 1 H), 7.29 (t, J )
7 Hz, 1 H), 7.32 (s, 1 H), 7.53 (d, J ) 7.7 Hz, 1 H), 8.02 (m, 2 H),
11.88 (s, 1 H). HRMS (EI+) calcd for C18H14N2O3 306.10044 (M+),
found 306.10025.
(Z)-6-Methyl-1-((1-methyl-1H-indol-2-yl)methylene)-4-thioxo-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (58). Yield 78%, red
solid, mp > 295 °C.1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.45
(s, 3 H), 3.99 (s, 3 H), 7.08 (t, J ) 7.7 Hz, 1 H), 7.25 (m, 2 H),
7.29 (s, 1 H), 7.38 (s, 1 H), 7.50 (d, J ) 8 Hz, 1 H), 7.65 (d, J )
8.0, 1 H), 13.52 (s, 1 H). Anal. (C18H14N2O2S·0.5H2O) C, H, N.
(Z)-1-(Benzofuran-3-ylmethylene)-6-methyl-4-thioxo-4,5-di-
hydrofuro[3,4-c]pyridin-3(1H)-one (59). Yield 76%, yellow solid,
(Z)-1-(Benzo[b]thiophen-3-ylmethylene)-6-methyl-4-thioxo-
4,5-dihydro-1H-pyrrolo[3,4-c]pyridin-3(2H)-one (69). Yield 26%,
brown solid, mp 272-275 °C. 1H NMR (400 MHz, DMSO-d6,
298 K) δ 2.48 (s, 3 H), 7.30 (s, 1 H), 7.46 (s, 1 H), 7.48 (m, 1 H),
7.53 (t, J ) 7.1 Hz, 1 H), 8.07, (d, J ) 7.9 Hz, 1 H), 8.31 (d, J )
7.9 Hz, 1 H), 8.39 (s, 1 H), 9.31 (s, 1 H) 13.74 (s, 1 H). Anal.
(C17H12N2OS2 ·0.75H2O) C, H, N.
1
mp > 290 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.46 (s,
(Z)-1-((1H-Indol-7-yl)methylene)-6-methyl-4-thioxo-4,5-dihy-
drofuro[3,4-c]pyridin-3(1H)-one (70). Yield 79%, orange solid,
3 H), 7.27 (s, 1 H), 7.32 (s, 1 H), 7.43 (m, 1 H), 7.68 (m, 2 H),
8.07, (t, J ) 4.1 Hz, 1 H) 8.49 (s, 1 H), 13.55 (s, 1 H). HRMS
(EI+) calcd for C17H12NO3S, 310.05379 (MH+), found 310.05463.
(Z)-1-(Benzo[b]thiophen-3-ylmethylene)-6-methyl-4-thioxo-
4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (60). Yield 23%, yellow
1
mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ 2.46 (s,
3 H), 7.01 (s, 1 H), 7.23 (t, J ) 7.8 Hz, 1 H), 7.35 (s, 1 H), 7.47
(m, 2 H), 7.51 (t, J ) 2.8 Hz, 1 H), 7.85 (d, J ) 7.5 Hz, 1 H),
11.37 (s, 1 H), 13.30 (s, 1 H). Anal. (C17H12N2O2S·0.5H2O) C, H,
N.
1
solid, mp > 300 °C. H NMR (400 MHz, DMSO-d6, 298 K) δ
2.47 (s, 3 H), 7.48 (m, 3 H), 7.55 (td, J ) 1.1, 7.1 Hz, 1 H), 8.08
(d, J ) 7.9 Hz, 1 H), 8.34 (d, J ) 8 Hz, 1 H), 8.43 (s, 1 H), 13.51
(s, 1 H). Anal. (C17H11NO2S2 ·0.75H2O) C, H, N.
(Z)-1-[(4-Methoxy-1-benzothien-3-yl)methylene]-6-methyl-4-
thioxo-4,5-dihydrofuro[3,4-c]pyridine-3(1H)-one (61). Yield 42%,
yellow solid. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.50 (s, 3
H), 4.06 (s, 3 H), 7.05 (d, J ) 7.9 Hz, 1 H), 7.15 (s, 1 H), 7.41 (t,
J ) 8.0 Hz, 1 H), 7.62 (d, J ) 8.0 Hz, 1 H), 7.72 (s, 1 H), 8.39 (s,
Inhibition of Perforin-Mediated Lysis of Sheep Red Blood
Cells (SRBC). A commercial library of ∼100000 compounds was
screened for the ability to reproducibly inhibit perforin-mediated
lysis of SRBC at a compound concentration of 100 µM.23
Compounds were assayed as single points using 384-well plate
methodology measuring change in turbidity. Lysis of the SRBC
results in a change of the turbidity of the reaction mixture, whereas
inhibition of cell lysis results in reduction or abolition of the change

Inhibitors of the Pore-Forming Protein Perforin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7623
in turbidity reading. A solution of the compound in DMSO was
added to 10 µL of 0.5 µg/mL perforin in buffer or controls,
respectively, with at least 30 min preincubation with compound
routinely. 0.04 mL of SRBC was then added in RBC buffer.
Samples were initially read (t ) 0 min) at an absorbance of 650
nM (in Envision; using an Envision reader, automation ABS@650
nm), incubated for 15 min. at 37 °C, then read at an absorbance of
650 nm (in Envision) to assess the change in turbidity of the reaction
mixture. As the inhibitor compounds were routinely dissolved in
DMSO, the same concentration of DMSO was used as a negative
control for the inhibition of perforin. Secondary screening was
carried out using the same methodology, employing a series of
concentrations; 100, 20, 4, 0.8, and 0.16 µM to determine an IC50.
Inhibition of PLO-Mediated Lysis of SRBC. To determine
whether the active inhibitors identified in the above screen (Perforin/
SRBC) were also able to block the lytic function of PLO, the
inhibitors were then tested at a concentration of 20 µM in the
presence PLO instead of perforin.
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 labeled by
incubation in medium with 100 µCi 51Cr for one hour. The cells
were then washed three times to remove unincorporated isotope
and resuspended at 1 × 105 cells per mL in RPMI buffer
supplemented with 0.1% BSA. Each test compound was preincu-
bated to concentrations of 20, 10, 5, 2.5, and 1.25 µM with
recombinant perforin for 30 min with DMSO as a negative control.
51Cr labeled Jurkat cells were then added and cells were incubated
at 37 °C for 4 h. The supernatant was collected and assessed for
its radioactive content on a γ counter (Wallac Wizard 1470
automatic γ 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 µM were titrated
down to lower concentrations in the same manner as above to
determine an accurate IC50.
Inhibition of Fas Ligand- or TRAIL-Mediated Lysis of
Jurkat Cells. Fifty thousand 51Cr-labeled Jurkat cells (0.05 mL)
were preincubated with inhibitor to a final concentration of 20 µM
for 30 min at 37 °C in the wells of a round-bottom, sterile 96-well
plate in a humidified CO2 incubator. Two negative controls were
used: (i) the diluent (DMSO) used to dissolve the inhibitors was
added at the same concentration as used in the other wells, and (ii)
a compound known not to inhibit perforin (designated B3) was
also used at the same concentration as the perforin inhibitor. Each
data point was performed in triplicate. Each plate was set up in
duplicate so that the capacity to block both Fas- and TRAIL-
mediated cell death could be evaluated independently. To each well
of the duplicate plate was added either anti-Fas agonistic mono-
clonal antibody CH11 or recombinant (rec)-TRAIL to initiate cell
death. Each agent was used at a final concentration of 250 ng/mL,
and the reaction was carried out in a final volume of 200 µL RPMI
medium containing 1% BSA. As further controls, three wells
contained Jurkat cells in which the detergent SDS was added, and
these wells were used to estimate the maximum release of 51Cr.
After 2 h, the cells were pelleted by centrifugation and 50 µL of
the assay supernatant was collected and assessed for its radioactive
content on a γ counter. The results in Figure 5 (Supporting
Information) show the mean 51Cr release ((standard error mean).
KHYG-1 Cytotoxicity Assay. KHYG-1 cells were washed and
resuspended in RPMI + 0.1% BSA at 4 × 105 cells/ml and 50 µL
of KHYG-1 cells were dispensed to each well of a 96-well
V-bottom plate. Test compounds were added to KHYG-1 cells at
various concentrations up to 20 µM and incubated at RT for 20
min. One × 106 K562 target cells were labeled with 75 µCi 51Cr
in 200 µL RPMI for 90 min at 37 °C, cells were washed as
described above, and resuspended in 5 mL RPMI + 0.1% BSA.
50 µL of 51Cr labeled K562 leukemia 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 γ counter (Turku, Finland). The percentage of specific
cytotoxicity was calculated by the formula:
% specific lysis )
(experimental release - spontaneous release)
(maximum release - spontaneous release)
× 100 (1)
and expressed as the mean of triplicate assays ( standard error of
the mean.
Toxicity to KHYG-1 NK Cells. The toxicity assay was carried
out in exactly the same manner as the killing assay above, but
instead of adding the labeled K562 target cells, 100 µL of RPMI
0.1% BSA was added. Cells were incubated for 4 h at 37 °C then
washed ×3 in RPMI + 0.1% BSA. Cells were then resuspended
in 200 µL of complete medium and incubated for 18 to 24 h at 37
°C. Trypan blue was added to each well and viable (clear) cells
counted as a percentage of total (clear + blue) cell number (%
viability).
Inhibition of Perforin Secreted by Primary Human NK Cells.
Human CD3-CD56+ NK cells were isolated from the buffy coats
of healthy blood donors (Australian Red Cross Blood Bank). The
mononuclear cell fraction was initially isolated by centrifugation
on a Hypaque-Ficoll cushion. NK cells were then isolated using a
MACS human NK isolation kit (Miltenyi Biotec). Non-NK cells
were labeled, and NK cells were purified by negative selection using
an AutoMACS separator. The cells were washed three times in
RPMI buffer containing 0.1% BSA, then used on the same day or
cultured in media containing 100 U/mL IL-2 for up to 3 days. K562
leukemia cells were washed and labeled with 51Cr as described
above and used as target cells in the NK killing assay. NK cells
(up to 2 × 105) were placed in triplicate wells of a V-bottom 96
well plate, together with each of the inhibitors indicated, or with
DMSO diluent or with medium alone, which were used as the
negative controls. The reaction volume was 200 µL, in RPMI buffer
containing 0.1% BSA. The plate was incubated at RT for 30 min,
then labeled K562 cells (1 × 104 in 50 µL of the same solution)
were then added to each well, and the plate was incubated for 4 h
at 37 °C as before. 51Cr released into the supernatant was harvested
using a Skatron harvesting press and radioactivity estimated on a
Wallac Wizard 1470 automatic γ counter and % specific lysis
calculated as previously.
Acknowledgment. J.A.T. is supported by a Program Grant
and a Senior Principal Research Fellowship from the National
Health and Medical Research Council of Australia. This work
was supported by Incitive Limited (Australia) and the Auckland
Division of the Cancer Society of New Zealand. We also thank
Pamela Murray for conducting the Ames testing of selected
compounds, Sisira Kumara for the HPLC studies, and Maruta
Boyd for NMR studies.
Supporting Information Available: Experimental for the
preparation of selected intermediates, elemental analyses, NMR,
HRMS, HPLC data on final compounds and selected figures as
indicated in the text. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) Voskoboinik, I.; Smyth, M. J.; Trapani, J. A. Perforin-mediated target-
cell death and immune homeostasis. Nat. ReV. Immunol. 2006, 6, 940–
952.
(2) Lieberman, J. Mechanisms of granule-mediated cytotoxicity. Curr.
Opin. Immunol. 2003, 15, 513–515.
(3) Ashton-Rickardt, P. G. The granule pathway of programmed cell death.
Crit. ReV. Immunol. 2005, 25, 161–182.
(4) Russell, J. H.; Ley, T. J. Lymphocyte-mediated cytotoxicity. Annu.
ReV. Immunol. 2002, 20, 323–370.
(5) Lichtenheld, M. G.; Olsen, K. J.; Lu, P.; Lowrey, D. M.; Hameed,
A.; Hengartner, H.; Podack, E. R. Structure and function of human
perforin. Nature 1988, 335, 448–451.
(6) Podack, E. R. Perforin: Structure, function and regulation. Curr. Top.
Microbio. Immun. 1992, 178, 175–185.

7624 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Lena et al.
(7) Trapani, J. A.; Smyth, M. J. Functional significance of the perforin/
granzyme cell death pathway. Nat. ReV. Immunol. 2002, 2, 735–747.
(8) Voskoboinik, I.; Trapani, J. A. Addressing the mysteries of perforin
function. Immunol. Cell Biol. 2006, 84, 66–71.
structure of hetarylmethylene-4-thiofuro[3,4-c]pyridin-3-ones. Russ.
J. Org. Chem. 1995, 31, 1650–1652.
(26) Kaigorodova, E. A.; Mikhailichenko, S. N.; Vasilin, V. K.; Terekhov,
V. I.; Kul’nevich, V G. Investigation of furopyridines. Part 7. Synthesis
and antibacterial activity of 1-arylidene-3-oxo-4-thioxofuro[3,4-c]-
pyridines. Pharm. Chem. J. 1998, 32, 194–196.
(27) Arustamova, I. S.; Kaigorodova, E. A.; Kul’nevich, V. G.; Ugrak,
B. I. Research in the field of furopyridines. 2. Synthesis and steric
structure of 6-methyl-1-hetarylidene-3,4-dioxo[3,4-c]pyridines. Chem.
Heterocycl. Compds. 1993, 29, 539–544.
(28) Bruce, W. F.; Coover, H. W. Pyridine Derivatives. I. 3-Cyano-4-
ethoxymethyl-6-methyl-2-pyridone and some related transformation
products. J. Am. Chem. Soc. 1944, 66, 2092–2094.
(9) Rosado, C. J.; Buckle, A. M.; Law, R. H. P.; Butcher, R. E.; Kan,
W.-T.; Bird, C. H.; Ung, K.; Browne, K. A.; Baran, K.; Bashtannyk-
Puhalovich, T. A.; Faux, N. G.; Wong, W.; Porter, C. J.; Pike, R. N.;
Ellisdon, A. M.; Pearce, M. C.; Bottomley, S. P.; Emsley, J.; Smith,
A. I.; Rossjohn, J.; Hartland, E. L.; Voskoboinik, I.; Trapani, J. A.;
Bird, P. I.; Dunstone, M. A.; Whisstock, J. C. A common fold mediates
vertebrate defence and bacterial attack. Science 2007, 317, 1548–1551.
(10) Kagi, D.; Ledermann, B.; Burki, K.; Seiler, P.; Odermatt, B.; Olsen,
K. J.; Podack, E. J.; Zinkernagel, R. M.; Hengartner, H. Cytotoxicity
mediated by T cells and natural killer cells is greatly impaired in
perforin-defficient mice. Nature 1994, 369, 31–37.
(29) Harris, S. A.; Stiller, E. T.; Folkers, K. Structure of Vitamin B6. II.
J. Am. Chem. Soc. 1939, 61, 1242–1244.
(30) Prostenik, M.; Butula, I. Selective reduction of some halogen-
substituted pyridines of the vitamin B6 series. Chem. Ber. 1977, 110,
2106–2113.
(11) Kagi, D.; Odermatt, B.; Seiler, P.; Zinkernagel, R. M.; Mak, T. W.;
Hengartner, H. Reduced incidence and delayed onset of diabetes in
perforin-deficient nonobese diabetic mice. J. Exp. Med. 1997, 186,
989–997.
(31) Shul’ga, N. A.; Balyakina, M. V.; Gunar, V. I. Peculiarities of the
nitration of 2-methyl-4-methoxymethyl-5-cyano-6-pyridone in a mix-
ture of HNO3 and H2SO4. Pharm. Chem. J. 1983, 17, 665–669.
(32) (a) Preparation of 71: Karminski-Zamola, G.; Dogan, J.; Bajic, M.;
Blazevic, J.; Malesevic, M. Synthesis of some furyl- and thienylacry-
lates or diacrylates and acrylic acids by the palladium catalysed
vinylation of substituted bromofurans and bromothiophenes. Hetero-
cycles 1994, 38, 759–767. (b) Hassan, J.; Se´vignon, M.; Gozzi, C.;
Schulz, E.; Lemaire, M. Aryl-aryl bond formation one century after
the discovery of the Ullmann reaction. Chem. ReV. 2002, 102, 1359–
1469.
(33) Preparation of benzofurans: Chapleo, C. B.; Myers, P. L.; Butler,
R. C. M.; Davis, J, A.; Doxey, J. C.; Higgins, S. D.; Myers, M.; Roach,
A. G.; Smith, C. F. C.; Stillings, M. R.; Welbourn, A. P. R-Adreno-
receptor reagents. 2. Effects of modifications of the 1,4-benzodioxan
ring system on R-adrenoreceptor activity. J. Med. Chem. 1984, 27,
570–576.
(34) (a) Preparation of benzothiophenes: Bridges, A. J.; Lee, A.; Schwartz,
C. E.; Towle, M. J.; Littlefield, B. A. The synthesis of three
4-substituted benzo[b]thiophene-2-carboxamidines as potent and selec-
tive inhibitors of urokinase. Bioorg. Med. Chem. 1993, 1, 403–410.
(b) Titus, R. L.; Titus, C. F. 2-Amino-3-(6-methoxybenzo[b]thien-3-
yl)propanoic acid. J. Heterocycl. Chem. 1973, 10, 679–681. (c)
Campaigne, E.; Rogers, R. B. Benzo[b]thiophene derivatives. XXI.
On the proof of structure of the sulfur isostere of Psilocin. J. Het.
Chem. 1973, 10, 963–966.
(12) Katano, H.; Cohen, J. I. Perforin and lymphohistiocytic proliferative
disorders. Br. J. Haematol. 2005, 128, 739–750.
(13) Henter, J.-I.; Arico, M.; Elinder, G.; Imashuku, S.; Janka, G. Familial
hemophagocytic lymphohistiocytosis. Primary hemophagocytic lym-
phohistiocytosis. Hematol. Oncol. Clin. North Am. 1998, 12, 417–
433.
(14) Voskoboinik, I.; Thia, M.-C.; De Bono, A.; Browne, K.; Cretney, E.;
Jackson, J. T.; Darcy, P. K.; Jane, S. M.; Smyth, M. J.; Trapani, J. A.
The functional basis for hemophagocytic lymphohistiocytosis in a
patient with co-inherited missense mutations in the perforin (PFN1)
gene. J. Exp. Med. 2004, 200, 811–816.
(15) Catalfamo, M.; Henkart, P. A. Perforin and the granule exocytosis
cytotoxicity pathway. Curr. Opin. Immunol. 2003, 15, 522–527.
(16) Pearl-Yafe, M.; Kaminitz, A.; Yolcu, E. S.; Yaniv, I.; Stein, J.;
Askenasy, N. Pancreatic islets under attack: cellular and molecular
effectors. Curr. Pharm. Des. 2007, 13, 749–760.
(17) Veale, J. L.; Liang, L. W.; Zhang, Q.; Gjertson, D. W.; Du, Z.;
Bloomquist, E. W.; Jia, J.; Qian, L.; Wilkinson, A. H.; Danovitch,
G. M.; Pham, P.-T. T.; Rosenthal, J. T.; Lassman, C. R.; Braun, J.;
Reed, E. F.; Gritsch, H. A. Noninvasive diagnosis of cellular and
antibody-mediated rejection by perforin and granzyme B in renal
allografts. Hum. Immunol. 2006, 67, 777–786.
(18) Choy, J. C.; Kerjner, A.; Wong, B. W.; McManus, B. M.; Granville,
D. J. Perforin mediates endothelial cell death and resultant transplant
vascular disease in cardiac allografts. Amer. J. Pathol. 2004, 165, 127–
133.
(19) Barry, M.; Bleackley, R. C. Cytotoxic T lymphocytes: All roads lead
to death. Nature ReV. Immunol. 2002, 2, 401–409.
(20) Togashi, K.; Kataoka, T.; Nagai, K. Characterization of a series of
vacuolar type H+-ATPase inhibitors on CTL-mediated cytotoxicity.
Immunol. Lett. 1997, 55, 139–144.
(21) Kataoka, T.; Shinohara, N.; Takayama, H.; Takaku, K.; Kondo, S.;
Yonehara, S.; Nagai, K. Concanamycin A, a powerful tool for
characterization and estimation of contribution of perforin- and Fas-
based lytic pathways in cell-mediated cytotoxicity. J. Immunol. 1996,
156, 3678–3686.
(22) Kataoka, T.; Taniguchi, M.; Yamada, A.; Suzuki, H.; Hamada, S.;
Magae, J.; Nagai, K. Identification of low molecular weight probes
of perforin- and Fas-based killing mediated by cytotoxic T-lympho-
cytes. Biosci. Biotechnol. Biochem. 1996, 60, 1726–1728.
(23) Holloway, G. A.; Baell, J. B.; Fairlamb, A. H.; Novello, P. M.; Parisot,
J. P.; Richardson, J.; Watson, K. G.; Street, I. P. Discovery of
2-iminobenzimidazoles as a new class of trypanothione reductase
inhibitor by high-throughput screening. Bioorg. Med. Chem. Lett. 2007,
17, 1422–1427.
(24) Trapani, J. A. and Smyth, M. J. Retroviral vectors encoding recom-
binant mouse perforin, its expression and therapeutic uses thereof.
Patent WO 2005083098 A1, March, 1, 2005.
(25) Kaigodorova, E. A.; Kvak, S. N.; Ugrak, B. I.; Zaplishnyi, V. N.;
Kul’nevich, V. G. Studies of furopyridines. VI. Synthesis and steric
(35) Preparation of indoles Marco, J. L. Improved preparation of N-pro-
pargyl-2-(5-benzyloxy-indolyl)methylamine. J. Heterocycl. Chem.
1998, 35, 475–476.
(36) (a) Reduction-oxidation reactions Mitsumori, S.; Tsuri, T.; Honma,
T.; Hiramatsu, Y.; Okada, T.; Hashizume, H.; Inagaki, M.; Arimura,
A.; Yasui, K.; Asanuma, F.; Kishino, J.; Ohtani, M. Synthesis and
biological activity of various derivatives of a novel class of potent,
selective, and orally active prostaglandin D2 receptor antagonists. 1.
Bicyclo[2.2.1]heptane derivatives. J. Med. Chem. 2003, 46, 2436–
2445. (b) Chapman, N. B.; Clarke, K.; Saraf, S. D. Pharmacologically
active benzo[b]thiophen derivatives. Part III. 2-Substituted compounds.
J. Chem. Soc. C 1967, 731–735. (c) Ogiso, A.; Kurabayashi, M.;
Takahashi, S.; Mishima, H.; Woods, M. C. The structure and total
synthesis of Futoenone, a constituent of Piper futokadzura. Chem.
Pharm. Bull. 1970, 18, 105–114. (d) Zaidlewicz, M.; Chechlowska,
A.; Prewysz-Kwinto, A.; Wojtczak, A. Enantioselective synthesis of
2- and 3-benzofuryl ꢀ-amino alcohols. Heterocyles 2000, 55, 569–
577.
(37) Boyd, P. D. W.; Lena, G.; Spicer, J. A. Ethyl 4-hydroxymethyl-2-
methylpyridine-5-carboxylate. Acta Crystallogr., Sect. E 2008, 64,
o883.
(38) Maron, D. M.; Ames, B. N. Revised methods for the Salmonella
mutagenicity test. Mutat. Res. 1983, 113, 173–215.
(39) Trapani, J. A. Dual mechanisms of apoptosis induction by cytotoxic
lymphocytes. Int. ReV. Cytol. 1998, 182, 111–192.
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