Structure-activity relationship study of tricyclic necroptosis inhibitors

Article abstract of DOI:10.1021/jm061016o

Necroptosis is a regulated caspase-independent cell death mechanism that can be induced in multiple cell types and is characterized by morphological features resembling necrosis. Here we describe a series of tricyclic heterocycles (i.e., 3-phenyl-3,3a,4,5-tetrahydro-2H-benz[g]indazoles, 3-phenyl-2,3,3a,4-tetrahydro-[1]benzopyrano[4,3-c]pyrazoles, 3-phenyl-2,3,3a,4-tetrahydro[1]benzothiopyrano[4,3-c]pyrazoles, and 5,5-dioxo-3-phenyl-2,3,3a,4-tetrahydro[1]benzothiopyrano[4,3-c]pyrazoles], collectively termed Nec-3, that can potently inhibit necroptosis. For example, compounds 8, 22, 41, 53, and 55 inhibit necroptosis in an FADD-deficient variant of human Jurkat T cells treated for 24 h with TNF-α with EC₅₀ values in the range 0.15-0.29 μM. Distinct from the previously described series of hydantoin-containing indole derivatives (Nec-1), the Nec-3 series exhibits specificity in inhibiting TNF-α-induced necroptosis. A structure-activity relationship (SAR) study revealed that the (3R,3aR)-rel-diastereomers were more active than the (3R,3aS)-rel-diastereomers for all four ring systems. Introduction of fluorine or methoxy to the 8-position of the tricyclic ring and a methoxy to the 4-position of the pendent phenyl ring increased activity. Amides at the 2-position of the tricyclic ring were best. The Nec-3 series provides new tools for elucidating caspase-independent cell death pathways and potentially lead compounds for therapeutic development.

Full text of DOI:10.1021/jm061016o

1886
J. Med. Chem. 2007, 50, 1886-1895
Structure-Activity Relationship Study of Tricyclic Necroptosis Inhibitors
Prakash G. Jagtap,† Alexei Degterev,‡,§ Sungwoon Choi,† Heather Keys,§ Junying Yuan,‡ and Gregory D. Cuny*,†
Laboratory for Drug DiscoVery in Neurodegeneration, HarVard Center for Neurodegeneration and Repair, Brigham & Women’s Hospital and
HarVard Medical School, 65 Landsdowne Street, Cambridge, Massachusetts 02139, Department of Cell Biology, HarVard Medical School,
240 Longwood AVenue, Boston, Massachusetts 02115, and Department of Biochemistry, Tufts UniVersity Medical School, 136 Harrison
AVenue, Stearns 703, Boston, Massachusetts 02111
ReceiVed August 23, 2006
Necroptosis is a regulated caspase-independent cell death mechanism that can be induced in multiple cell
types and is characterized by morphological features resembling necrosis. Here we describe a series of
tricyclic heterocycles (i.e., 3-phenyl-3,3a,4,5-tetrahydro-2H-benz[g]indazoles, 3-phenyl-2,3,3a,4-tetrahydro-
[1]benzopyrano[4,3-c]pyrazoles, 3-phenyl-2,3,3a,4-tetrahydro[1]benzothiopyrano[4,3-c]pyrazoles, and 5,5-
dioxo-3-phenyl-2,3,3a,4-tetrahydro[1]benzothiopyrano[4,3-c]pyrazoles], collectively termed Nec-3, that can
potently inhibit necroptosis. For example, compounds 8, 22, 41, 53, and 55 inhibit necroptosis in an FADD-
deficient variant of human Jurkat T cells treated for 24 h with TNF-R with EC50 values in the range 0.15-
0.29 µM. Distinct from the previously described series of hydantoin-containing indole derivatives (Nec-1),
the Nec-3 series exhibits specificity in inhibiting TNF-R-induced necroptosis. A structure-activity relationship
(SAR) study revealed that the (3R,3aR)-rel-diastereomers were more active than the (3R,3aS)-rel-diastereomers
for all four ring systems. Introduction of fluorine or methoxy to the 8-position of the tricyclic ring and a
methoxy to the 4-position of the pendent phenyl ring increased activity. Amides at the 2-position of the
tricyclic ring were best. The Nec-3 series provides new tools for elucidating caspase-independent cell death
pathways and potentially lead compounds for therapeutic development.
Introduction
by cell membrane and organelle disruption, cell swelling, and
mitochondrion impairment followed by cell lysis, which is
accompanied by a host inflammatory response.5 Although in
recent years multiple studies have demonstrated apoptosis
activation in various diseases, necrosis remains the prevalent
form of acute cell death in many pathologies, including stroke,6
myocardial infarction,7 trauma, and possibly some forms of
neurodegeneration.8 Very few attempts, however, have been
made to develop therapeutics specifically targeting necrosis
because of the conventional notion that, unlike apoptosis,
necrotic cell death is a nonregulated response to overwhelming
stress.
Cell death plays a prominent role in the pathophysiology of
many acute and chronic diseases. The complex mechanisms of
cell death are only beginning to be revealed. A complete
understanding of these mechanisms is essential to design
strategies for effectively treating many human diseases. Histori-
cally, cell death has been categorized morphologically as either
apoptosis or necrosis.1 However, the cellular mechanisms
leading to these two morphologically distinct cell death phe-
notypes are more interrelated than previously realized. The
ultimate manner of cell death following an insult is dependent
on many factors, including but not limited to the cell death signal
initiator and the signaling pathway existing in a particular cell
type.
Apoptosis is a genetically regulated process required for both
development and tissue homeostasis.2 Extensive studies of
apoptosis during the past decade revealed that it is characterized
by multiple highly regulated steps including caspase activation
and mitochondrial damage that leads to downstream events such
as concomitant nucleus and cytoplasm condensation, DNA
degradation, membrane blebbing, and caspase-mediated cleavage
of various cellular proteins.3,4 Apoptotic cell bodies are ef-
ficiently removed by engulfment processes that function to
eliminate the debris of dead cells and to prevent the activation
of inflammation. Abnormal regulation of apoptosis may lead
to inappropriate cell death, which may underlie the etiology of
many human diseases.
This notion is directly challenged by a number of recent
studies demonstrating the existence of regulated caspase-
independent cell death mechanisms9-16 with morphological
features resembling necrosis. Previously we have defined one
type of necrosis as necroptosis.17 Necroptosis represents a type
of regulated caspase-independent cell death pathway that may
offer an unprecedented opportunity to selectively target patho-
logical necrotic cell death.
Our laboratories have been seeking to identify and optimize
low molecular weight molecules capable of inhibiting necrop-
tosis to elucidate caspase-independent cell death pathways and
their roles in disease pathophysiology and to provide lead
compounds (i.e., necrostatins) for therapeutic development. A
series of hydantoin-containing indole derivatives (referred to
as the Nec-1 series) have recently been described as potent in
vitro necroptosis inhibitors (exemplified by 1, EC50 ) 0.05 µM)
that also were efficacious in an animal model of ischemic stroke
(Figure 1).17,18 Herein, we report the structure-activity relation-
ship (SAR) study for a new series of inhibitors (referred to as
the Nec-3 series) that block tumor necrosis factor R (TNF-R)
induced necroptosis. The Nec-3 series of compounds contain a
tricyclic heterocycle with an appended aryl substituent (i.e., 2).
Distinct from the Nec-1 compounds, the Nec-3 series show
Necrosis represents types of cell death morphologically and
mechanistically distinct from apoptosis. Necrosis is characterized
* To whom correspondence should be addressed. E-mail: gcuny@
rics.bwh.harvard.edu. Phone: (617) 768-8640. Fax: (617) 768-8606.
† Harvard Center for Neurodegeneration and Repair, Brigham &
Women’s Hospital and Harvard Medical School.
‡ Department of Cell Biology, Harvard Medical School.
§ Tufts University Medical School.
10.1021/jm061016o CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/16/2007

SAR Study of Tricyclic Necroptosis Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1887
Scheme 1. Synthesis of 3-Acetyl Tricyclic Derivativesa
Figure 1. Necrostatins.
specificity in inhibiting necroptosis induced by TNF-R and thus
may provide valuable tools in the elucidation of TNF-R-specific
signaling steps in caspase-independent cell death pathways.
a X ) CH2, O, or S. Reagents and conditions: (a) ArCHO, 8 N NaOH,
EtOH, rt, 2 h; (b) CH3CO2H, NH2NH2‚xH2O, 120 °C, 15 h.
Results and Discussion
Scheme 2. Synthesis of 5-Substituted Tricyclic Derivativesa
Chemistry. The 3-phenyl-3,3a,4,5-tetrahydro-2H-benz[g]-
indazoles (2, X ) CH2), 3-phenyl-2,3,3a,4-tetrahydro[1]-
benzopyrano[4,3-c]pyrazoles (2, X ) O), and 3-phenyl-2,3,3a,4-
tetrahydro[1]benzothiopyrano[4,3-c]pyrazoles (2, X ) S) were
prepared from 1-tetralones, 4-chromanones, and 4-thiochro-
manones, respectively, according to the procedures outlined in
Scheme 1.19 For example, compounds 3 (X ) CH2, O, or S)
were condensed with aromatic aldehydes in the presence of
sodium hydroxide to give chalcones 4. In the cases of 7-nitro-
1-tetralone and 7-methoxy-4-chromanone, condensations with
4-anisaldehyde were accomplished under acidic conditions.
Treatment of 4 with hydrazine hydrate in acetic acid gave a
mixture of readily separable diastereomers 5 (i.e., (3R,3aS)-rel-
isomer] and 6 (i.e., (3R,3aR)-rel-isomer].20-27 This general
procedure was used in the preparation of 7-41. The stereo-
1
chemical assignments were confirmed using H NMR, 1-D
NOE, and HH-COSY experiments. For example, 8 was assigned
as the (3R,3aR)-rel-isomer due to an NOE between the 3a-
position (δ 3.47-3.55) and the 3-position (δ 5.66) protons and
a large coupling constant (J ) 11.2 Hz) indicative of a syn-
relationship. The (3R,3aS)-rel-isomer, 7, lacked a similar NOE
between the protons at the 3a-position (δ 3.16-3.23) and at
the 3-position (δ 4.91) and had a smaller coupling constant (J
) 9.2 Hz), and the proton at the 3a-position (δ 3.20) was
shielded by the 4′-methoxyphenyl ring compared to the corre-
sponding proton in the (3R,3aR)-rel-isomer. The 5,5-dioxo-3-
phenyl-2,3,3a,4-tetrahydro[1]benzothiopyrano[4,3-c]pyrazoles
(2, X ) SO2), such as 42 and 43, were readily prepared by
oxidizing the corresponding 3-phenyl-2,3,3a,4-tetrahydro[1]-
benzothiopyrano[4,3-c]pyrazoles with m-chloroperoxybenzoic
acid (MCPBA).
a Reagents and conditions: (a) Me3SiN3, [CF3CO2]2PhI, rt, 24 h (29%
44 and 31% 45); (b) H2 (1 atm), 10% Pd-C, EtOH, rt, 24 h (59%); (c)
aq CH2O, HCO2H, THF, ∆, 24 h (47%).
Scheme 3. Synthesis of 3-Amido-Substituted Tricyclic
Derivativesa
The 5-position of the 3,3a,4,5-tetrahydro-2H-benz[g]indazole
ring system of 8 was modified as shown in Scheme 2.
Introduction of an azide to this benzylic position utilizing Me3-
SiN3 in the presence of [bis(trifluoroacetoxy)iodo]benzene was
accomplished following the procedure of Kita and co-workers
to give 44 in 29% yield.28 This reaction was also accompanied
by an elimination-oxidation reaction giving 45 in 31% yield.
The azide 44 was subsequently reduced by hydrogenation to
amine 46 and then converted to the tertiary amine 47 by
reductive amination with formaldehyde. The relative stereo-
a Reagents and conditions: (a) NH2NH2‚xH2O, t-BuCO2H, EtOH, ∆, 6
h; (b) RC(O)Cl, EtOAc, saturated aqueous NaHCO3, rt, 30 min.
hydrazine hydrate in the presence of trimethylacetic acid, as
shown in Scheme 3, amine 49 was isolated as a mixture of
diastereomers (the material was subsequently used without
purification). Unlike the case with acetic acid, utilizing a
sterically demanding acid such as trimethylacetic acid prevented
direct amide formation and allowed for the isolation of the
amine. Treatment of 49 with various acid chlorides yielded
amide derivatives 50 and 51. Amide 55 was subsequently
generated from 54 upon treatment with NaOH in methanol.
Cyclization of 48 with various semicarbazides and thiosemi-
carbazides gave 58-60 (Scheme 4). In all cyclization reactions
reported herein where both the (3R,3aR)-rel- and (3R,3aS)-rel-
isomers were formed, they could be separated by column
chromatography on silica gel. Compound 59 was subsequently
converted to 61 by a sequence of reactions involving alkylation
1
chemistry at the 5-position of 47 was established by H NMR
and HH-COSY experiments. The dimethylamine protons of 47
(δ 2.21) had a coupling interaction with the proton at the 3a-
position (δ 4.03-4.09) indicative of a syn-relationship.
Derivatives with various substituents on the 2-position of the
tricyclic ring were also prepared. Compounds 52 and 53 were
synthesized according to Scheme 1, except that formic acid and
propionic acid, respectively, were used as the solvent in place
of acetic acid. When chalcone 48 was allowed to react with

1888 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Jagtap et al.
Scheme 4. Synthesis of 3-Non-Amido-Substituted Tricyclic
of the pendent phenyl group appeared to be comparable between
the two ring systems. Replacing the 3,3a,4,5-tetrahydro-2H-
benz[g]indazole ring with a 2,3,3a,4-tetrahydro[1]benzothiopyrano-
[4,3-c]pyrazole (8 vs 41) was well tolerated, but replacement
with a 5,5-dioxo-3-phenyl-2,3,3a,4-tetrahydro[1]benzothiopyrano-
[4,3-c]pyrazole ring resulted in a slight decrease in activity (8
vs 43).
Derivativesa
Next, the SAR of various nitrogen substituents on the
2-position of the 3,3a,4,5-tetrahydro-2H-benz[g]indazole ring
was examined (Table 3). In general, amides were best, with
the ethyl and hydroxymethyl amides (i.e., 53 and 55) being more
active than 8. Introduction of ureas or thioureas to the 2-position
of the tricyclic ring was detrimental to activity. Likewise,
replacing the amide with an alkyl group (62) resulted in
complete loss of necroptosis inhibitory activity.
a Reagents and conditions: (a) NH2NHC(O)NHR or NH2NHC(S)NHR,
EtOH, concd HCl, ∆, 6 h; (b) MeI, EtOH, ∆, 2 h (85%); (c) NH3 (2.0 N
in EtOH), 60 °C, 16 h (88%).
In the FADD-deficient variant of human Jurkat T cells treated
for 24 h with TNF-R, representative compounds from the Nec-1
series, (()-1, and the Nec-3 series, 8, had very similar inhibitory
activities (EC50 ) 0.21 ( 0.0218 and 0.29 ( 0.07 µM,
respectively). Similar to FADD-deficient Jurkat cells, mouse
fibrosarcoma L929 cells were previously shown to undergo
necroptosis upon treatment with TNF-R.17 Both (()-1 and 8 at
30 µM completely protected L929 cells from TNF-R-induced
necroptosis (Figure 2). In addition to TNF-R, the pan-caspase
inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluorometh-
ylketone (zVAD.fmk) has also been found to induce necrosis
in L929 cells,29,30 which was efficiently inhibited by (()-1
(Figure 2). However, 8 was ineffective against zVAD.fmk-
induced necroptosis in L929 cells. Parenthetically, zVAD.fmk
was incapable of inducing necroptosis in the FADD-deficient
variant of human Jurkat T cells. These results further demon-
strate that the mechanism(s) resulting in necroptotic cell death
are dependent on both the initiator of cell death and the cell
type. Furthermore, these results suggest that the Nec-1 and Nec-3
series of compounds most likely modulate different molecular
targets in the necroptosis pathway. As previously demonstrated,
the Nec-1 series of inhibitors have worked in a board range of
cell types utilizing various cell death initiators.17 In contrast,
the Nec-3 series exhibit specificity toward necroptosis induced
by TNF-R. More selective compounds, such as the Nec-3 series,
may be advantageous in treating certain conditions.
with methyl iodide to give intermediate 65 followed by treatment
with ammonia in ethanol. Finally, 62 was prepared from 8 by
reduction with lithium aluminum hydride.
Biology. Evaluation of necroptosis inhibitory activity was
performed using an FADD-deficient variant of human Jurkat T
cells treated for 24 h with TNF-R as previously described.17
Under these conditions the cells underwent necroptosis (the
DMSO control had ∼40% viability), which was inhibited by
(()-1 (EC50 ) 0.21 ( 0.2 µM) as a positive control.18 For EC50
value determinations, cells were treated with 10 ng/mL human
TNF-R in the presence of increasing concentrations of test
compounds (0.029, 0.058, 0.12, 0.23, 0.46, 0.93, 1.9, 3.7, 11.1,
33, and 100 µM) in duplicate for 24 h followed by ATP-based
viability assessment. EC50 values ( SD were determined from
at least two independent experiments.
In four cases where both isomers were examined with the
3,3a,4,5-tetrahydro-2H-benz[g]indazole ring system, the
(3R,3aR)-rel-isomers were more active than the corresponding
(3R,3aS)-rel-isomers at inhibiting necroptosis (Table 1). For
example, 8 was 22 times more potent than 7. In addition,
introduction of the methoxy group at the 8-position of the
tricyclic ring gave increased activity (e.g., 9 vs 11 and 13 vs
8).
One model of the necroptosis cell death pathway to explain
the observed results described herein is shown in Figure 3. Cell
death can be activated via different initiators, including TNF-R
agonism of the TNF-R receptor, followed by initial signal
transduction through various pathways, some of which are cell
type dependent. These pathways may then converge downstream
into a common signaling pathway leading to the characteristic
necrotic morphology, accompanied by plasma membrane per-
meability, mitochondrion dysfunction, and autophagy. The
Nec-1 series may target a step in the universal part of the
pathway, resulting in broad activity, whereas the Nec-3 series
may target a more upstream step that is not activated by
zVAD.fmk in L929 cells, but that is activated by TNF-R in
both the FADD-deficient variant of human Jurkat T cells and
L929 cells. Identification of the molecular targets for both series
of compounds will assist the elucidation of this emerging
complex cell death pathway and may help guide compound
selection for treating various diseases.
Translocation of the methoxy group to the 6-, 7-, or 9-position
of the tricyclic ring (e.g., 14, 16, and 18) resulted in loss of or
reduced activity. Increasing the size (19) or introduction of an
amine onto the ether (20) at the 8-position of the tricyclic ring
also resulted in diminished activity. An electron-withdrawing
fluorine (22) was well tolerated at the 8-position of the tricyclic
ring. However, replacing the fluorine with a nitro group (24)
diminished inhibitory activity. An electron-donating methoxy
group on the 4′-position of the pendent phenyl ring resulted in
increased activity compared to a fluorine or nitro substituent (8
vs 11 and 25). However, increasing the size of the ether (26),
transposition to the 2′-position (27), or replacing it with a
hydroxyl (28) or a thioether (29) was detrimental to activity to
varying degrees. Introduction of a primary amine (46) or a
tertiary amine (47) on the 5-position of the tricyclic ring also
resulted in activity erosion.
Replacing the 3,3a,4,5-tetrahydro-2H-benz[g]indazole ring
with a 2,3,3a,4-tetrahydro[1]benzopyrano[4,3-c]pyrazole ring
(Table 2) only resulted in a slight decrease in activity (8 vs 37
and 22 vs 39). However, the difference in activities between
the (3R,3aR)-rel- and (3R,3aS)-rel-isomers in some cases
appeared to be more dramatic in the 2,3,3a,4-tetrahydro[1]-
benzopyrano[4,3-c]pyrazole ring system (38 vs 39). The SAR
Conclusions
A series of tricyclic heterocycles were found to inhibit TNF-
R-induced necroptosis in both the FADD-deficient variant of
human Jurkat T cells and mouse fibrosarcoma L929 cells. An

SAR Study of Tricyclic Necroptosis Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1889
Table 1. EC50 Determinations for Inhibition of Necroptosis in FADD-Deficient Jurkat T Cells Treated with TNF-R
compd
R1
R2
4′-OMe
4′-OMe
4′-F
4′-F
4′-F
X
isomer
EC50a,b (µM)
7
8
9
8-OMe
8-OMe
H
8-OMe
8-OMe
8-OMe
H
6-OMe
7-OMe
7-OMe
7,8-di-OMe
9-OMe
8-OBn
8-O(CH2)2Rc
8-F
CH2
A
B
B
A
B
B
B
B
A
B
B
B
B
B
A
B
A
B
B
B
B
B
B
B
B
B
B
B
6.4 ( 2
0.29 ( 0.07
5.4 ( 1
3.7 ( 0.8
0.96 ( 0.09
0.68 ( 0.01
0.84 ( 0.3
>100
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CHNH2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
46e
47
3′-F
4′-OMe
4′-OMe
4′-F
4′-F
4′-F
>100
>100
>100
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-NO2
4′-OBn
2′-OMe
4′-OH
4′-SMe
4′-OH
3′,4′-OCH2O-
3′,4′-O(CH2)2O-
4′-OMe
4′-OMe
>50
5.0 ( 0.7
>50
2.7 ( 0.8
0.28 ( 0.03
>100
8-F
8-NO2
8-NO2
8-OMe
8-OMe
8-OMe
8-OMe
8-OMe
8-OH
8-OMe
8-OMe
8-OMe
8-OMe
3.5 ( 2
1.8 ( 0.4
>100
>100
5.9 ( 1
1.1 ( 0.7
>100
2.6 ( 1d
2.1 ( 0.7
1.2 ( 0.4
5.1 ( 2
f
f
CHNMe2
a Positive control: (()-1, EC50 ) 0.21 ( 0.02 µM, N ) 3. b EC50 ( SD values were determined from two independent experiments, unless otherwise
noted. c R ) morpholine. d EC50 ( SD values were determined from three independent experiments. e Oxalate salt. f (5S)-rel-Isomer.
Table 2. EC50 Determinations for Inhibition of Necroptosis in
FADD-Deficient Jurkat T Cells Treated with TNF-R
Table 3. EC50 Determinations for Inhibition of Necroptosis in
FADD-Deficient Jurkat T Cells Treated with TNF-R
a,b
a,b
EC50
EC50
compd
R
(µM)
compd
R
(µM)
52
53
54
55
56
57
HCdO
C(dO)Et
C(dO)CH2OAc
C(dO)CH2OH 0.16 ( 0.02
C(dO)CH2OMe 0.44 ( 0.2
C(dO)CF3
0.92 ( 0.09
0.15 ( 0.01
3.1 ( 0.2
58
59
60
61
62
C(dO)NH2
C(dS)NH2
C(dS)NHMe 7.9 ( 2
C(dNH)NH2 >100
Et
4.3 ( 3c
8.8 ( 3
compd
R1
R2
X
isomer
EC50a,b (µM)
33
34
35
36
37
38
39
40
41
42
43
Me
Me
OMe
OMe
OMe
F
F
F
F
O
O
O
O
O
O
O
S
A
B
B
A
B
A
B
A
B
A
B
>100
11.6 ( 6c
4.8 ( 1
>100
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
9.9 ( 0.1
0.46 ( 0.001
>100
0.39 ( 0.2
a Positive control: (()-1, EC50 ) 0.21 ( 0.02 µM, N ) 3. b EC50 ( SD
values were determined from two independent experiments, unless otherwise
noted. c EC50 ( SD values were determined from three independent
experiments.
F
0.70 ( 0.1
5.1 ( 2
OMe
OMe
OMe
OMe
S
SO2
SO2
0.28 ( 0.07
>100
0.75 ( 0.1
group to the 4-position of the pendent phenyl ring increased
activity, while placing the methoxy at the 2-position of the
phenyl ring eliminated activity. Amides at the 2-position of the
tricyclic ring systems were best. The tricyclic necroptosis
inhibitors were not effective at inhibiting zVAD.fmk-induced
necroptosis in L929 cells. This is in contract to a hydantoin-
indole necrostatin, suggesting that the two series of compounds
are mechanistically distinct. Overall, in this paper we report a
second novel class of small-molecule necroptosis inhibitors that
can further assist in the elucidation of caspase-independent cell
a Positive control: (()-1, EC50 ) 0.21 ( 0.02 µM, N ) 3. b EC50 ( SD
values were determined from two independent experiments, unless otherwise
noted. c EC50 ( SD values were determined from three independent
experiments.
SAR study revealed that the (3R,3aR)-rel-diastereomers were
more active then the corresponding (3R,3aS)-rel-diastereomers.
Introduction of fluorine or methoxy to the 8-position of the
tricyclic ring increased activity, while substitution at the 6-, 7-,
and 9-positions was detrimental. Also, introduction of a methoxy

1890 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Jagtap et al.
and again with ethanol. The solid was dried in vacuo to give 2-(4-
fluorobenzylidene)-7-methoxy-1-tetralone (2.485 g, 88%): mp
1
129-30 °C; H NMR (500 MHz, CDCl3) δ 2.88-2.91 (m, 2H),
3.06-3.10 (m, 2H), 3.87 (s, 3H), 7.06-7.13 (m, 3H), 7.17 (d, J )
8.4 Hz, 1H), 7.42 (d, J ) 5.2 Hz, 1H), 7.44 (d, J ) 5.6 Hz, 1H),
7.61 (d, J ) 3.2 Hz, 1H), 7.81 (s, 1H); HFABMS m/z 283.1134
(calcd for C18H16FO2, MH+, 283.1134).
For characterization of other 2-arylidene-1-tetralones, 2-(arylidene)-
chroman-4-ones, and 2-(arylidene)thiochroman-4-ones, see the
Supporting Information.
Figure 2. L929 cells were treated with 10 ng/mL human TNF-R (black
bars) or 100 µM zVAD.fmk (white bars) in the presence of DMSO
(control), 30 µM (()-1, or 8. After 24 h, cell viability was determined
using a luminescent ATP-based assay. Average cell viabilities (%) (
SD for two independent experiments are shown.
General Procedure for the Synthesis of 2-Arylidene-1-
tetralones and 2-(Arylidene)chroman-4-ones under Acidic Con-
ditions. Exemplified for 2-(4-Methoxybenzylidene)-7-nitro-1-
tetralone. A mixture of 7-nitro-1-tetralone (0.382 g, 2.0 mmol),
4-methoxybenzaldehyde (0.243 mL, 2.0 mmol), and concentrated
HCl (10 mL) in methanol (5.4 mL) was heated at reflux for 4 h.
The mixture was allowed to cool to room temperature and then
filtered. The residue was washed with a small amount of methanol
and dried in vacuo to give 2-(4-methoxybenzylidene)-7-nitro-1-
tetralone (2.485 g, 88%) as a pale yellow solid: 1H NMR (500
MHz, CDCl3) δ 3.04-3.9 (m, 2H), 3.19-3.22 (m, 2H), 3.87 (s,
3H), 6.96-6.99 (m, 2H), 7.44-7.47 (m, 3H), 7.93 (s, 1H), 8.32
(dd, J1 ) 8.5 Hz, J2 ) 2.5 Hz, 1H), 8.94 (d, J ) 2.0 Hz, 1H).
General Procedure for the Preparation of (3R,3aS)-rel- and
(3R,3aR)-rel-2-Acetyl-3,3a,4,5-tetrahydro-3-(aryl)-2H-benz[g]-
indazoles, 2-Acetyl-2,3,3a,4-tetrahydro[1]benzopyrano[4,3-c]-
pyrazoles, and 2-Acetyl-2,3,3a,4-tetrahydro[1]benzothiopyrano-
[4,3-c]pyrazoles. Exemplified for (3R,3aS)-rel-2-Acetyl-3,3a,4,5-
tetrahydro-3-(4-fluorophenyl)-8-methoxy-2H-benz[g]indazole (10)
and (3R,3aR)-rel-2-Acetyl-3,3a,4,5-tetrahydro-3-(4-fluorophe-
nyl)-8-methoxy-2H-benz[g]indazole (11). A solution of 2-(4-
fluorobenzylidene)-7-methoxy-1-tetralone (0.300 g, 1.0 mmol) and
hydrazine hydrate (0.3 mL) in acetic acid (3 mL) was refluxed at
120 °C for 15 h. The reaction mixture was concentrated and then
dissolved in ethyl acetate. The organic layer was washed sequen-
tially with water, saturated aqueous NaHCO3, water, and brine, dried
over anhydrous Na2SO4, filtered, and concentrated. The residue
obtained was purified by silica gel column chromatography using
ethyl acetate-hexane (3:7) to give 10 (0.051 g, 15%) and 11 (0.109
g, 32%), in addition to recovered starting material (95 mg).
Figure 3. A model of the necroptosis cell death pathway.
death pathways and potentially provide lead compounds for
therapeutic development.
Experimental Section
Chemistry Materials and Methods. Unless otherwise noted,
all reagents and solvents were purchased from commercial sources
and used without further purification. The NMR spectra were
obtained using a Bruker 400 MHz or Varian 400 or 500 MHz
1
Data for 10: mp 165-67 °C; H NMR (400 MHz, CDCl3) δ
1.94-1.99 (m, 1H), 2.29-2.31 (m, 1H), 2.32 (s, 3H), 2.40-2.42
(m, 2H), 3.16-3.22 (m, 1H), 3.89 (s, 3H), 4.95 (d, J ) 9.6 Hz,
1H), 6.93-6.96 (dd, J ) 2.8 and 8.4 Hz, 1H), 7.04-7.08 (t, J )
8.8 Hz, 2H), 7.12 (d, J ) 8.4 Hz, 1H), 7.28-7.32 (m, 2H), 7.46
(d, J ) 2.4 Hz, 1H). Anal. (C20H19FN2O2) C, H, N.
1
spectrometer. All H NMR spectra are reported in δ units (ppm)
and are referenced to the peak for tetramethylsilane (TMS) if
conducted in CDCl3, to the central line of the quintet at 3.30 ppm
for samples in CD3OD, or to the central line of the quintet at 2.49
ppm for samples in d6-DMSO. All 13C NMR spectra are reported
in δ units (ppm) and are referenced to the central line of the triplet
at 77.23 ppm if conducted in CDCl3, to the central line of the septet
at 49.0 ppm for samples in CD3OD, or to the central line of the
septet at 39.5 ppm for samples in d6-DMSO. Coupling constants
(J) are reported in hertz. Column chromatography was performed
on silica gel (Merck, grade 60, 230-400 mesh) or utilizing a
CombiFlash Sg 100c separation system (ISCO) with RediSep
disposable silica gel columns (ISCO). High-resolution mass spectra
were obtained by using an SX-102A mass spectrometer (JEOL
USA, Inc., Peabody, MA) or an LCT mass spectrometer (Micromass
Inc., Beverly, MA). All melting points were taken in glass capillary
tubes on a Mel-Temp apparatus and are uncorrected. The elemental
compositions of the compounds agreed to within (0.4% of the
calculated values.
1
Data for 11: mp 182-85 °C; H NMR (400 MHz, CDCl3) δ
1.02-1.06 (m, 1H), 1.75-1.78 (m, 1H), 2.48 (s, 3H), 2.81-2.87
(m, 2H), 3.54-3.57 (m, 1H), 3.90 (s, 3H), 5.72 (d, J ) 12.0 Hz,
1H), 6.93-7.11 (m, 6H), 7.56 (d, J ) 4.0 Hz, 1H); 13C NMR (400
MHz, CDCl3) δ 22.13, 24.50, 28.30, 48.77, 55.73, 62.61, 107.83,
112.70, 115.62, 118.72, 127.99, 128.21, 130.26, 132.25, 132.92,
155.79, 158.35, 161.13, 163.58, 168.72. Anal. (C20H19FN2O2) C,
H, N.
Data for (3R,3aS)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-8-methoxy-2H-benz[g]indazole (7): yield 25%; mp
1
146-48 °C; H NMR (400 MHz, CDCl3) δ 1.89-1.95 (m, 1H),
2.25-2.29 (m, 1H), 2.39 (s, 3H), 2.85-2.87 (m, 2H), 3.16-3.23
(m, 1H), 3.79 (s, 3H), 3.86 (s, 3H), 4.91 (d, J ) 9.2 Hz, 1H), 6.87-
6.93 (m, 3H), 7.09 (d, J ) 8.4 Hz, 1H), 7.22-7.25 (m, 2H), 7.44
(d, J ) 2.4 Hz, 1H). Anal. (C21H22N2O3) C, H, N.
General Procedure for the Synthesis of 2-Arylidene-1-
tetralones, 2-(Arylidene)chroman-4-ones, and 2-(Arylidene)-
thiochroman-4-ones under Basic Conditions. Exemplified for
2-(4-Fluorobenzylidene)-7-methoxy-1-tetralone. To a solution of
7-methoxy-1-tetralone (1.760 g, 0.01 mol) and 4-fluorobenzalde-
hyde (1.240 g, 0.01 mol) in ethanol (20 mL) was slowly added an
aqueous solution of NaOH (0.012 mol, 8 N) at room temperature.
The mixture was stirred for 2 h. The solid precipitate was collected
by filtration and then washed sequentially with ethanol, with water,
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-8-methoxy-2H-benz[g]indazole (8): yield 30%; mp
151-53 °C; H NMR (400 MHz, CDCl3) δ 1.03-1.11 (m, 1H),
1.72-1.76 (m, 1H), 2.44 (s, 3H), 2.77-2.91 (m, 2H), 3.47-3.55
(m, 1H), 3.75 (s, 3H), 3.87 (s, 3H), 5.66 (d, J ) 11.2 Hz, 1H),
6.80 (d, J ) 8.8 Hz, 2H), 6.89-6.92 (dd, J ) 3.2 and 8.8 Hz, 1H),
6.99 (d, J ) 8.4 Hz, 2H), 7.06 (d, J ) 8.4 Hz, 1H), 7.54 (d, J )
2.8 Hz, 1H); 13C NMR (400 MHz, CDCl3) δ 22.15, 24.42, 28.93,
48.84, 55.42, 55.70, 63.03, 107.76, 112.66, 114.15, 118.56, 127.44,
1

SAR Study of Tricyclic Necroptosis Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1891
127.88, 128.39, 129.26, 130.24, 132.32, 155.60, 158.28, 159.14,
168.56. Anal. (C21H22N2O3) C, H, N.
6.80 (d, J ) 8.5 Hz, 2H), 6.96 (d, J ) 3.0 Hz, 1H), 6.99 (d, J )
8.5 Hz, 2H), 7.07 (d, J ) 8.0 Hz, 2H), 7.33-7.47 (m, 4H), 7.64
(d, J ) 3.0 Hz, 1H). Anal. (C27H26N2O3) C, H, N.
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-fluo-
rophenyl)-2H-benz[g]indazole (9): yield 23%; mp 166-68 °C;
1H NMR (400 MHz, CDCl3) δ 1.06-1.11 (m, 1H), 1.77-1.81 (m,
1H), 2.47 (s, 3H), 2.87-2.95 (m, 2H), 3.56-3.63 (m, 1H), 5.72
(d, J ) 12.0 Hz, 1H), 7.00 (t, J ) 8.0 Hz, 2H), 7.06-7.10 (m,
2H), 7.19 (d, J ) 8.0 Hz, 1H), 7.28-7.36 (m, 2H), 8.09 (d, J )
8.0 Hz, 1H). Anal. (C19H17FN2O) C, H, N.
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-8-(4-morpholino)ethoxy-2H-benz[g]indazole (20):
yield 33%; mp 122-26 °C; 1H NMR (500 MHz, CDCl3) δ 1.02-
1.11 (m, 1H), 1.72 (m, 1H), 2.44 (s, 3H), 2.60 (t, J ) 4.5 Hz, 4H),
2.76-2.84 (m, 4H), 3.48-3.54 (m, 1H), 3.74-3.79 (m, 7H), 4.16-
4.19 (m, 2H), 5.66 (d, J ) 10.5 Hz, 1H), 6.80 (d, J ) 8.5 Hz, 2H),
6.90-6.93 (dd, J ) 3.0 and 9.0 Hz, 1H), 6.99 (d, H ) 8.5 Hz,
2H), 7.06 (d, J ) 9.0 Hz, 1H), 7.55 (d, J ) 3.0 Hz, 1H); HFABMS
m/z 450.2391 (calcd for C26H31N3O4, MH+, 450.2393).
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(3-fluo-
rophenyl)-8-methoxy-2H-benz[g]indazole (12): yield 39%; mp
1
176-77 °C; H NMR (400 MHz, CDCl3) δ 1.00-1.08 (m, 1H),
1.75-1.80 (m, 1H), 2.46 (s, 3H), 2.75-2.83 (m, 2H), 3.52-3.60
(m, 1H), 3.87 (s, 3H), 5.69 (d, J ) 11.2 Hz, 1H), 7.76-6.79 (dd,
J ) 2.4 and 9.6 Hz, 1H), 6.86-6.95 (m, 3H), 7.06 (d, J ) 8.8 Hz,
1H), 7.23-7.28 (m, 1H), 7.53 (d, J ) 3.2 Hz, 1H); 13C NMR (400
MHz, CDCl3) δ 22.18, 24.54, 28.97, 46.79, 55.75, 62.97, 107.18,
113.16, 114.65, 118.71, 121.92, 128.05, 130.27, 132.13, 139.57,
155.62, 156.22, 161.60, 164.24, 168.50. Anal. (C20H19FN2O2) C,
H, N.
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-2H-benz[g]indazole (13): yield 30%; mp 257-60 °C;
1H NMR (400 MHz, CDCl3) δ 1.01-1.11 (m, 1H), 1.70-1.75 (m,
1H), 2.40 (s, 3H), 2.76-2.89 (m, 2H), 3.47-3.54 (m, 1H), 3.71
(s, 3H), 5.63 (d, J ) 11.2 Hz, 1H), 6.76 (d, J ) 8.4 Hz, 2H), 6.96
(d, J ) 8.8 Hz, 2H), 7.11 (d, J ) 6.8 Hz, 1H), 7.23-7.29 (m, 2H),
8.02-8.04 (dd, J ) 1.6 and 7.6 Hz, 1H). Anal. (C20H20N2O2) C,
H, N.
Data for (3R,3aS)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-8-fluoro-2H-benz[g]indazole (21): required a second
silica gel column chromatography purification using 2% MeOH in
CH2Cl2; yield 26%; 1H NMR (500 MHz, CDCl3) δ 1.89-1.99 (m,
1H), 2.27-2.32 (m, 1H), 2.38 (s, 3H), 2.85-2.95 (m, 2H), 3.18-
3.24 (m, 1H), 3.70 (s, 3H), 4.92 (d, J ) 9.0 Hz, 1H), 6.88-6.91
(m, 2H), 7.01-7.05 (m, 1H), 7.14-7.16 (m, 1H), 7.23-7.25 (m,
2H), 7.63 (dd, J ) 9.5 and 2.5 Hz, 1H); HFABMS m/z 339.1523
(calcd for C20H20FN2O2, MH+, 339.1509).
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-8-fluoro-2H-benz[g]indazole (22): yield 29%; 1H
NMR (500 MHz, CDCl3) δ 1.03-1.12 (m, 1H), 1.74-1.79 (m,
1H), 2.44 (s, 3H), 2.78-2.89 (m, 2H), 3.49-3.55 (m, 1H), 3.76
(s, 3H), 5.67 (d, J ) 11.0 Hz, 1H), 6.81 (d, J ) 9.0 Hz, 2H), 6.97-
7.04 (m, 3H), 7.11-7.13 (m, 1H), 7.72 (dd, J ) 9.5 and 3.0 Hz,
1H); HFABMS m/z 339.1493 (calcd for C20H20FN2O2, MH+,
339.1509).
Data for (3R,3aS)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-8-nitro-2H-benz[g]indazole (23): yield 25%; 1H NMR
(500 MHz, CDCl3) δ 1.94-2.05 (m, 1H), 2.34-2.38 (m, 1H), 2.42
(s, 3H), 2.97-3.09 (m, 2H), 3.23-3.29 (m, 1H), 3.71 (s, 3H), 4.98
(d, J ) 9.5 Hz, 1H), 6.90 (d, J ) 9.0 Hz, 2H), 7.24 (d, J ) 7.0 Hz,
2H), 7.36 (d, J ) 8.5 Hz, 1H), 8.14 (dd, J ) 8.5 and 2.5 Hz, 1H),
8.79 (d, J ) 2.0 Hz, 1H); HFABMS m/z 366.1467 (calcd for
C20H20N3O4, MH+, 366.1454).
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-8-nitro-2H-benz[g]indazole (24): yield 45%; 1H NMR
(500 MHz, CDCl3) δ 1.07-1.16 (m, 1H), 1.81-1.86 (m, 1H), 2.47
(s, 3H), 2.95-2.97 (m, 2H), 3.56-3.62 (m, 1H), 3.76 (s, 3H), 5.72
(d, J ) 11.5 Hz, 1H), 6.82 (d, J ) 9.0 Hz, 2H), 6.97 (d, J ) 8.5
Hz, 2H), 7.33 (d, J ) 8.5 Hz, 1H), 8.13 (dd, J ) 8.5 and 2.5 Hz,
1H), 8.89 (d, J ) 2.5 Hz, 1H); HFABMS m/z 366.1467 (calcd for
C20H20N3O4, MH+, 366.1454).
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-6-methoxy-2H-benz[g]indazole (14): yield 43%; mp
1
203-207 °C; H NMR (500 MHz, CDCl3) δ 0.98-1.07 (m, 1H),
1.74-1.79 (m, 1H), 2.43 (s, 3H), 2.45-2.52 (m, 1H), 3.04-3.09
(m, 1H), 3.48-3.54 (m, 1H), 3.76 (s, 3H), 3.82 (s, 3H), 5.68 (d, J
) 11.0 Hz, 1H), 6.80 (d, J ) 8.5 Hz, 2H), 6.86 (d, J ) 8.0 Hz,
1H), 7.00 (d, J ) 8.5 Hz, 2H), 7.24 (t, J ) 8.0 Hz, 1H), 7.68 (d,
J ) 8.0 Hz, 1H). Anal. (C21H22N2O3) C, H, N.
Data for (3R,3aS)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-fluo-
rophenyl)-7-methoxy-2H-benz[g]indazole (15): yield 18%; mp
1
228-30 °C; H NMR (400 MHz, CDCl3) δ 1.89-2.00 (m, 1H),
2.24-2.30 (m, 1H), 2.38 (s, 3H), 2.88-2.94 (m, 2H), 3.12-3.19
(m, 1H), 3.83 (s, 3H), 4.89 (d, J ) 9.6 Hz, 1H), 6.68 (d, J ) 2.4
Hz, 1H), 6.83-6.85 (dd, J ) 2.4 and 8.4 Hz, 1H), 7.00-7.06 (m,
2H), 7.26-7.29 (m, 2H), 7.90 (d, J ) 8.8 Hz, 1H). Anal. (C20H19-
FN2O2) C, H, N.
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-fluo-
rophenyl)-7-methoxy-2H-benz[g]indazole (16): yield 20%; 1H
NMR (400 MHz, CDCl3) δ 1.00-1.07 (m, 1H), 1.74-1.76 (m,
1H), 2.42 (s, 3H), 2.76-2.89 (m, 2H), 3.49-3.56 (m, 1H), 3.81
(s, 3H), 5.67 (d, J ) 10.8 Hz, 1H), 6.65 (s, 1H), 6.83-6.85 (dd, J
) 6.0 and 2.4 Hz, 1H), 6.94-7.06 (m, 4H), 7.99 (d, J ) 9.2 Hz,
1H). Anal. (C20H19FN2O2) C, H, N.
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-ni-
troxyphenyl)-8-methoxy-2H-benz[g]indazole (25): yield 29%; mp
1
219-21 °C; H NMR (400 MHz, CDCl3) δ 0.91-1.01 (m, 1H),
1.71-1.75 (m, 1H), 2.43 (s, 3H), 2.75-2.82 (m, 2H), 3.57-3.64
(m, 1H), 3.84 (s, 3H), 5.74 (d, J ) 11.6 Hz, 1H), 6.88-6.91 (dd,
J ) 3.2 and 8.4 Hz, 1H), 7.22 (d, J ) 9.6 Hz, 2H), 7.48 (d, J )
3.2 Hz, 1H), 8.13 (d, J ) 9.6 Hz, 2H). Anal. (C20H19N3O4) C, H,
N.
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-fluo-
rophenyl)-7,8-dimethoxy-2H-benz[g]indazole (17): yield 33%;
1
mp 189-93 °C; H NMR (400 MHz, CDCl3) δ 1.01-1.08 (m,
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-ben-
zyloxyphenyl)-8-methoxy-2H-benz[g]indazole (26): yield 41%;
1H), 1.72-1.77 (m, 1H), 1.44 (s, 3H), 2.72-2.93 (m, 2H), 3.48-
3.55 (m, 1H), 3.88 (s, 3H), 3.95 (s, 3H), 5.67 (d, J ) 10.8 Hz,
1H), 6.60 (s, 1H), 6.94-6.99 (dd, J ) 9.2 and 8.8 Hz, 2H), 7.04-
7.07 (m, 2H), 7.47 (s, 1H). Anal. (C21H21FN2O3) C, H, N.
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-9-methoxy-2H-benz[g]indazole (18): yield 27%; mp
1
mp 156-61 °C; H NMR (400 MHz, CDCl3) δ 1.01-1.14 (m,
1H), 1.72-1.79 (m, 1H), 2.45 (s, 3H), 2.75-2.83 (m, 2H), 3.56-
3.64 (m, 1H), 3.87 (s, 3H), 4.99 (s, 2H), 5.67 (d, J ) 10.8 Hz,
1H), 6.87-6.92 (m, 2H), 6.99 (d, J ) 8.4 Hz, 1H), 7.06 (d, J )
8.8 Hz, 1H), 7.30-7.40 (m, 7H), 7.54 (d, J ) 3.2 Hz, 1H). Anal.
(C27H26N2O3) C, H, N.
1
157-60 °C; H NMR (500 MHz, CDCl3) δ 1.09-1.18 (m, 1H),
1.67-1.72 (m, 1H), 2.49 (s, 3H), 2.83-2.95 (m, 2H), 3.53-3.59
(m, 1H), 3.78 (s, 3H), 3.98 (s, 3H), 5.57 (d, J ) 11.0 Hz, 1H),
6.77 (d, J ) 7.0 Hz, 1H), 6.80 (d, J ) 9.0 Hz, 2H), 6.84 (d, J )
9.0 Hz, 1H), 7.04 (d, J ) 9.0 Hz, 2H), 7.25 (t, J ) 9.0 Hz, 1H).
Anal. (C21H22N2O3) C, H, N.
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-2-(2-meth-
oxyphenyl)-8-methoxy-2H-benz[g]indazole (27): yield 24%; 1H
NMR (500 MHz, CDCl3): δ 1.01-1.10 (m, 1H), 1.84-1.90 (m,
1H), 2.40 (s, 3H), 2.68-2.89 (m, 2H), 3.54-3.60 (m, 1H), 3.85
(s, 3H), 6.15 (d, J ) 11.4 Hz, 1H), 6.75-6.90 (m, 4H), 7.04 (d, J
) 8.5 Hz, 1H), 7.18-7.21 (m, 1H), 7.52 (d, J ) 2.5 Hz, 1H).
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-thi-
omethoxyphenyl)-8-methoxy-2H-benz[g]indazole (29): yield 22%;
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-8-benzyloxy-2H-benz[g]indazole (19): yield 36%; mp
1
155-57 °C; H NMR (500 MHz, CDCl3) δ 1.03-1.12 (m, 1H),
1
1.72-1.77 (m, 1H), 2.44 (s, 3H), 2.74-2.86 (m, 2H), 3.48-3.54
(m, 1H), 3.75 (s, 3H), 5.12 (s, 2H), 5.66 (d, J ) 10.5 Hz, 1H),
mp 144-47 °C; H NMR (500 MHz, CDCl3) δ 1.02-1.09 (m,
1H), 1.73-1.76 (m, 1H), 2.43 (s, 3H), 2.45 (s, 3H), 2.74-2.86

1892 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Jagtap et al.
(m, 2H), 3.50-3.56 (m, 1H), 3.87 (s, 3H), 5.66 (d, J ) 10.5 Hz,
1H), 6.90-6.92 (dd, J ) 3.0 and 8.5 Hz, 1H), 6.99 (d, J ) 8.5 Hz,
2H), 7.06 (d, J ) 8.5 Hz, 1H), 7.16 (d, J ) 8.5 Hz, 2H), 7.53 (d,
J ) 3.0 Hz, 1H). Anal. (C21H22N2O2S) C, H, N.
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(3,4-me-
thylenedioxyphenyl)-8-methoxy-2H-benz[g]indazole (31): yield
41%; mp 206-209 °C; 1H NMR (500 MHz, CDCl3) δ 1.08-1.17
(m, 1H), 1.75-1.80 (m, 1H), 2.42 (s, 3H), 2.76-2.84 (m, 2H),
3.47-3.53 (m, 1H), 3.87 (s, 3H), 5.62 (d, J ) 11.4 Hz, 1H), 5.90
(s, 2H), 6.52 (d, J ) 1.5 Hz, 1H), 6.57-6.59 (dd, J ) 1.5 and 8.0
Hz, 1H), 6.72 (d, J ) 7.5 Hz, 1H), 6.90-6.92 (dd, J ) 3.0 and 8.5
Hz, 1H), 7.07 (d, J ) 8.2 Hz, 1H), 7.52 (d, J ) 2.5 Hz, 1H). Anal.
(C21H20N2O4) C, H, N.
Data for (3R,3aR)-rel-2-acetyl-2,3,3a,4-tetrahydro-3-(4-meth-
oxyphenyl)-8-fluoro[1]benzopyrano[4,3-c]pyrazole (39): yield
53%; 1H NMR (500 MHz, CDCl3) δ 2.44 (s, 3H), 3.31 (dd, 1H, J
) 11.0, 13.0 Hz), 3.76 (s, 3H), 3.78-3.84 (m, 1H), 4.16 (dd, 1H,
J ) 5.8, 11.3 Hz), 5.73 (d, 1H, J ) 11.0 Hz), 6.82-6.86 (m, 3H),
6.98-7.05 (m, 3H), 7.57 (dd, 1H, J ) 3.0, 8.5 Hz); HFABMS m/z
341.1310 (calcd for C19H18FN2O3, MH+, 341.1301).
Data for (3R,3aS)-rel-2-acetyl-2,3,3a,4-tetrahydro-3-(4-meth-
oxyphenyl)-8-methoxy[1]benzothiopyrano[4,3-c]pyrazole (40):
1
yield 36%; H NMR (500 MHz, CDCl3) δ 2.39 (s, 3H), 3.07 (dd,
1H, J ) 4.5, 12.3 Hz), 3.33 (dd, 1H, J ) 12.3 Hz), 3.57-3.62 (m,
1H), 3.79 (s, 3H), 3.86 (s, 3H), 5.02 (d, 1H, J ) 8.5 Hz), 6.87-
6.90 (m, 2H), 7.09 (d, 1H, J ) 9.0 Hz), 7.20-7.23 (m, 2H), 7.52
(d, 1H, J ) 3.0 Hz); HFABMS m/z 369.1273 (calcd for C20H21N2O3S,
MH+, 369.1273).
Data for (3R,3aR)-rel-2-acetyl-3,3a,4,5-tetrahydro-3-(3,4-eth-
ylenedioxyphenyl)-8-methoxy-2H-benz[g]indazole (32): yield
1
Data for (3R,3aR)-rel-2-acetyl-2,3,3a,4-tetrahydro-3-(4-meth-
oxyphenyl)-8-methoxy[1]benzothiopyrano[4,3-c]pyrazole (41):
17%; mp 189-93 °C; H NMR (500 MHz, CDCl3) δ 1.06-1.15
(m, 1H), 1.78-1.80 (m, 1H), 2.41 (s, 3H), 2.75-2.86 (m, 2H),
3.46-3.52 (m, 1H), 3.87 (s, 3H), 4.22 (s, 4H), 5.60 (d, J ) 11.5
Hz, 1H), 6.55-6.58 (m, 2H), 6.76 (d, J ) 8.5 Hz, 1H), 6.89-6.91
(dd, J ) 3.0 and 9.0 Hz, 1H), 7.06 (d, J ) 8.0 Hz, 1H), 7.52 (d, J
) 3.0 Hz, 1H). Anal. (C22H22N2O4) C, H, N.
1
yield 52%; H NMR (500 MHz, CDCl3) δ 2.41-2.51 (m, 2H),
2.45 (s, 3H), 3.77 (s, 3H), 3.86 (s, 3H), 3.89-3.95 (m, 1H), 5.67
(d, 1H, J ) 11.0 Hz), 6.83-6.85 (m, 2H), 6.88 (dd, 1H, J ) 3.0,
8.8 Hz), 7.00-7.03 (m, 2H), 7.09 (d, 1H, J ) 9.0 Hz), 7.65 (d,
1H, J ) 3.0 Hz); HFABMS m/z 369.1267 (calcd for C20H21N2O3S,
MH+, 369.1273).
Data for (3R,3aS)-rel-2-acetyl-2,3,3a,4-tetrahydro-3-(4-fluo-
rophenyl)-8-methyl[1]benzopyrano[4,3-c]pyrazole (33): yield
15%; mp 218-20 °C; 1H NMR (400 MHz, CDCl3) δ 2.36 (s, 3H),
2.42 (s, 3H), 3.53-3.57 (m, 1H), 4.13-4.19 (m, 1H), 4.60-4.64
(m, 1H), 4.99 (d, J ) 8 Hz, 1H), 6.85 (d, J ) 8 Hz, 1H), 7.05-
7.09 (m, 2H), 7.16-7.31 (m, 3H), 7.63 (s, 1H); HFABMS m/z
325.1354 (calcd for C19H17FN2O2, MH+, 325.1352).
Data for (3R,3aR)-rel-2-acetyl-2,3,3a,4-tetrahydro-3-(4-fluo-
rophenyl)-8-methyl[1]benzopyrano[4,3-c]pyrazole (34): required
a second silica gel column chromatography purification using
hexane-EtOAc-MeCN (65:25:10); yield 42%; mp 243-46 °C;
1H NMR (400 MHz, CDCl3) δ 2.36 (s, 3H), 2.48 (s, 3H), 3.29 (t,
J ) 12 Hz, 1H), 3.72-3.83 (m, 1H), 4.14-4.18 (m, 1H), 5.77 (d,
J ) 12 Hz, 1H), 6.82 (d, J ) 8 Hz, 1H), 7.0-7.15 (m, 5H), 7.78
(s, 1H); HFABMS m/z 325.1359 (calcd for C19H17FN2O2, MH+,
325.1352).
Preparation of (3R,3aR)-rel-2-Acetyl-3,3a,4,5-tetrahydro-3-
(4-hydroxyphenyl)-8-methoxy-2H-benz[g]indazole (28). A mix-
ture of 26 (0.370 mg, 0.86 mmol) and 5% Pd-C (10 mg) in ethyl
acetate (20 mL) was stirred under a hydrogen atmosphere for 20 h
and then filtered though a pad of Celite. The Celite was then washed
sequentially with ethyl acetate and acetic acid. The combined
filtrates were concentrated. The solid obtained was recrystallized
from ethyl acetate to give 28 (0.185 g, 63%): mp 240-41 °C; 1H
NMR (400 MHz, d6-DMSO-CDCl3) δ 0.99-1.11 (m, 1H), 1.72-
1.76 (m, 1H), 2.43 (s, 3H), 2.67-2.83 (m, 2H), 3.46-3.53 (m,
1H), 3.87 (s, 3H), 5.61 (d, J ) 10.8 Hz, 1H), 6.74 (d, J ) 8.4 Hz,
2H), 6.87 (d, J ) 8.8 Hz, 2H), 6.61 (m, 1H), 7.07 (d, J ) 8.4 Hz,
1H), 7.43-7.46 (m,1H), 7.52 (d, J ) 3.2 Hz, 1H). Anal.
(C20H20N2O3) C, H, N.
Preparation of (3R,3aR)-rel-2-Acetyl-3,3a,4,5-tetrahydro-3-
(4-hydroxyphenyl)-8-hydroxy-2H-benz[g]indazole (30). To a
solution of 28 (0.100 g, 0.28 mmol) under an argon atmosphere in
dichloromethane (5 mL) was added BBr3 (1.0 mL, 1.0 M solution
in dichloromethane) at -78 °C. The reaction mixture was stirred
at room temperature for 1 h and then poured into an ice cold solution
of HCl (1.0 N). The mixture was extracted in ethyl acetate. The
organic layer was washed with water, dried over anhydrous Na2-
SO4, filtered, and concentrated. The solid obtained was recrystallized
Data for (3R,3aR)-rel-2-acetyl-2,3,3a,4-tetrahydro-3-(4-fluo-
rophenyl)-8-methoxy[1]benzopyrano[4,3-c]pyrazole (35): yield
1
32%; mp 245 °C; H NMR (500 MHz, CDCl3) δ 2.45 (s, 3H),
3.21-3.27 (m, 1H), 3.84 (s, 3H), 3.82-3.87 (m, 1H), 4.10-4.14
(dd, J ) 10.5 and 12.5 Hz, 1H), 4.55-4.59 (dd, J ) 6.0 and 10.5
Hz, 1H), 5.75 (d, J ) 10.5 Hz, 1H), 6.82 (d, J ) 9.0 Hz, 1H),
6.91-6.94 (dd, J ) 3.5 and 9.5 Hz, 1H), 6.98-7.01 (dd, J ) 8.5
and 8.5 Hz, 2H), 7.06-7.08 (m, 2H), 7.33 (d, J ) 3.5 Hz, 1H);
HFABMS m/z 341.1304 (calcd for C19H17FN2O3, MH+, 341.1301).
1
from ethyl acetate to give 30 (62 mg, 67%): mp 164-67 °C; H
Data for (3R,3aS)-rel-2-acetyl-2,3,3a,4-tetrahydro-3-(4-meth-
oxyphenyl)-8-methoxy[1]benzopyrano[4,3-c]pyrazole (36): yield
NMR (400 MHz, DMSO-CDCl3) δ 0.70-0.90 (m, 1H), 1.41-
1.46 (m, 1H), 2.41-2.57 (m, 5H), 3.15-3.22 (m, 1H), 5.29 (d, J
) 11.2 Hz, 1H), 6.43 (d, J ) 6.8 Hz, 2H), 6.53-6.57 (m, 3H),
6.68 (d, J ) 8.4 Hz, 1H), 7.19 (s, 1H); HFABMS m/z 323.1399
(calcd for C19H18N2O3, MH+, 323.1396).
1
39%; H NMR (500 MHz, CDCl3) δ 2.39 (s, 3H), 3.53-3.59 (m,
1H), 3.80 (s, 3H), 3.84 (s, 3H), 4.11 (dd, 1H, J ) 10.3, 12.8 Hz),
4.57 (dd, 1H, J ) 6.0, 10.5 Hz), 4.95 (d, 1H, J ) 10.0 Hz), 6.86
(d, 1H, J ) 9.0 Hz), 6.88-6.91 (m, 2H), 6.93 (dd, 1H, J ) 2.8,
9.3 Hz), 7.21-7.24 (m, 2H), 7.26-7.27 (m, 1H); HFABMS m/z
353.1485 (calcd for C20H21N2O4, MH+, 353.1501).
Preparation of (3R,3aS)-rel-2-Acetyl-2,3,3a,4-tetrahydro-3-
(4-methoxyphenyl)-5,5-dioxo-8-methoxy[1]benzothiopyrano[4,3-
c]pyrazole (42) and (3R,3aR)-rel-2-Acetyl-2,3,3a,4-tetrahydro-
3-(4-methoxyphenyl)-5,5-dioxo-8-methoxy[1]benzothiopyrano[4,3-
c]pyrazole (43). A solution of 40 (21 mg, 0.057 mmol) in
dichloromethane (5 mL) at 0 °C was treated with MCPBA (60 mg).
The reaction mixture was stirred at room temperature for 16 h,
then diluted with ethyl acetate (50 mL), washed sequentially with
saturated NaHCO3 and brine, dried over anhydrous sodium sulfate,
filtered, and concentrated. The residue was purified by column
chromatography on silica gel using ethyl acetate-hexane (2:3) as
the eluent to give 42 (96%) as a white solid. 43 (92%) was prepared
in a similar manner from 41.
Data for (3R,3aR)-rel-2-acetyl-2,3,3a,4-tetrahydro-3-(4-meth-
oxyphenyl)-8-methoxy[1]benzopyrano[4,3-c]pyrazole (37): yield
50%; 1H NMR (500 MHz, CDCl3) δ 2.45 (s, 3H), 3.30 (dd, 1H, J
) 11.0, 13.0 Hz), 3.76 (s, 3H), 3.78-3.83 (m, 1H), 3.85 (s, 3H),
4.13 (dd, 1H, J ) 5.8, 10.8 Hz), 5.72 (d, 1H, J ) 11.0 Hz), 6.81-
6.84 (m, 3H), 6.91 (dd, 1H, J ) 3.0, 9.5 Hz), 7.00-7.02 (m, 2H),
7.34 (d, 1H, J ) 3.5 Hz); HFABMS m/z 353.1492 (calcd for
C20H21N2O4, MH+, 353.1501).
Data for (3R,3aS)-rel-2-acetyl-2,3,3a,4-tetrahydro-3-(4-meth-
oxyphenyl)-8-fluoro[1]benzopyrano[4,3-c]pyrazole (38): yield
1
33%; H NMR (500 MHz, CDCl3) δ 2.38 (s, 3H), 3.53-3.59 (m,
Data for 42: 1H NMR (500 MHz, CDCl3) δ 2.41 (s, 3H), 3.54-
3.62 (m, 2H), 3.81 (s, 3H), 3.95 (s, 3H), 4.09-4.15 (m, 1H), 5.03
(d, 1H, J ) 10.5 Hz), 6.89-6.92 (m, 2H), 7.14 (dd, 1H, J ) 2.3,
8.8), 7.21-7.24 (m, 2H), 7.56 (d, 1H, J ) 2.5 Hz), 7.88 (d, 1H, J
) 9.0 Hz); HFABMS m/z 401.1159 (calcd for C20H21N2O5S, MH+,
401.1171).
1H), 3.80 (s, 3H), 4.13 (dd, 1H, J ) 10.3, 12.8 Hz), 4.60 (dd, 1H,
J ) 5.8, 10.3 Hz), 4.97 (d, 1H, J ) 9.5 Hz), 6.87-6.91 (m, 3H),
7.02-7.06 (m, 1H), 7.21-7.24 (m, 2H), 7.49 (dd, 1H, J ) 3.0, 8.0
Hz); HFABMS m/z 341.1312 (calcd for C19H18FN2O3, MH+,
341.1301).

SAR Study of Tricyclic Necroptosis Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1893
1
Data for 43: H NMR (500 MHz, CDCl3) δ 2.48 (s, 3H), 2.68
separated, washed sequentially with water and brine, dried over
anhydrous Na2SO4, filtered, and concentrated. The residue was
purified by column chromatography on silica gel column using ethyl
acetate-hexane (2:3) as the eluent to give (3R,3aS)-rel-54 (11%)
and 54 (68%).
(dd, 1H, J ) 13.8 Hz), 3.11 (dd, 1H, J ) 4.3, 13.8 Hz), 3.77 (s,
3H), 3.95 (s, 3H), 4.40-4.46 (m, 1H), 5.80 (d, 1H, J ) 11.5 Hz),
6.84-6.86 (m, 2H), 6.96-6.98 (m, 2H), 7.14 (dd, 1H, J ) 3.0, 9.0
Hz), 7.64 (d, 1H, J ) 2.5 Hz), 7.86 (d, 1H, J ) 9.5 Hz); HFABMS
m/z 401.1160 (calcd for C20H21N2O5S, MH+, 401.1171).
Preparation of (3R,3aS)-rel-2-Formyl-3,3a,4,5-tetrahydro-3-
(4-methoxyphenyl)-8-methoxy-2H-benz[g]indazole ((3R,3aS)-rel-
52) and (3R,3aR)-rel-2-Formyl-3,3a,4,5-tetrahydro-3-(4-meth-
oxyphenyl)-8-methoxy-2H-benz[g]indazole (52). A solution of
2-(4-methoxybenzylidene)-7-methoxy-1-tetralone (2.0 g, 0.0068
mol) and hydrazine hydrate (2.0 mL) in formic acid (20 mL) was
heated at 120 °C for 2 days. The reaction mixture was concentrated
and then extracted with ethyl acetate. The organic layer was washed
sequentially with water, saturated aqueous NaHCO3, water, and
brine, dried over anhydrous Na2SO4, filtered, and then concentrated.
The residue was purified by column chromatography on silica gel
using ethyl acetate-hexane (3:7) as the eluent to give (3R,3aS)-
rel-52 (0.740 g, 32%) and 52 (0.905 g, 40%).
Data for (3R,3aS)-rel-54: mp 159-61 °C; 1H NMR (400 MHz,
CDCl3) δ 1.87-1.98 (m, 1H), 2.13 (s, 3H), 2.26-2.31 (m, 1H),
2.86-2.89 (m, 2H), 3.17-3.24 (m, 1H), 3.78 (s, 3H), 3.86 (s, 3H),
4.90 (d, J ) 9.2 Hz, 1H), 5.09 (d, J ) 15.6 Hz, 1H), 5.22 (d, J )
15.6 Hz, 1H), 6.87-6.95 (m, 3H), 7.10 (d, J ) 8.8 Hz, 1H), 7.24
(d, J ) 8.8 Hz, 2H), 7.41 (d, J ) 3.2 Hz, 1H). Anal. (C23H24N2O5)
C, H, N.
1
Data for 54: mp 201-204 °C; H NMR (400 MHz, CDCl3) δ
1.05-1.16 (m, 1H), 1.73-1.77 (m, 1H), 2.16 (s, 3H), 2.72-2.89
(m, 2H), 3.48-3.55 (m, 1H), 3.75 (s, 3H), 3.87 (s, 3H), 5.16-
5.25 (m, 2H), 5.64 (d, J ) 11.2 Hz, 1H), 6.80 (d, J ) 8.8 Hz, 2H),
6.91-6.94 (dd, J ) 2.0 and 8.4 Hz, 1H), 6.99 (d, J ) 8.8 Hz, 2H),
7.07 (d, J ) 8.4 Hz, 1H), 7.49 (d, J ) 3.2 Hz, 1H). Anal.
(C23H24N2O5) C, H, N.
Data for (3R,3aS)-rel-52: mp 166-68 °C; 1H NMR (400 MHz,
CDCl3) δ 1.90-1.99 (m, 1H), 2.27-2.31 (m, 1H), 2.86-2.90 (m,
2H), 3.21-3.28 (m, 1H), 3.80 (s, 3H), 3.86 (s, 3H), 4.89 (d, J )
9.6 Hz, 1H), 6.90-6.98 (m, 3H), 7.10 (d, J ) 8.8 Hz, 1H), 7.25-
7.27 (m, 2H), 7.43 (d, J ) 2.4 Hz, 1H), 8.97 (s, 1H). Anal.
(C20H20N2O3) C, H, N.
Data for (3R,3aR)-rel-2-(methoxyacetyl)-3,3a,4,5-tetrahydro-
3-(4-methoxyphenyl)-8-methoxy-2H-benz[g]indazole (56): yield
16%; mp 157-59 °C; H NMR (500 MHz, CDCl3) δ 1.06-1.12
(m, 1H), 1.75-1.77 (m, 1H), 2.76-2.88 (m, 2H), 3.46-3.50 (m,
1H), 3.51 (s, 3H), 3.75 (s, 3H), 3.87 (s, 3H), 4.54 (d, J ) 16.0 Hz,
1H), 4.66 (d, J ) 16.0 Hz, 1H), 5.68 (d, J ) 11.0 Hz, 1H), 6.80
(d, J ) 9.5 Hz, 2H), 6.91-6.93 (dd, J ) 3.0 and 9.0 Hz, 1H), 6.98
(d, J ) 9.5 Hz, 2H), 7.07 (d, J ) 8.0 Hz, 1H), 7.50 (d, J ) 3.0 Hz,
1H); HFABMS m/z 381.1810 (calcd for C22H24N2O4, MH+,
381.1814).
1
1
Data for 52: mp 155-59 °C; H NMR (400 MHz, CDCl3) δ
1.01-1.12 (m, 1H), 1.69-1.74 (m, 1H), 2.68-2.85 (m, 2H), 3.49-
3.76 (m, 1H), 3.76 (s, 3H), 3.81 (s, 3H), 5.58 (d, J ) 11.2 Hz,
1H), 6.78-7.04 (m, 6H), 7.48 (d, J ) 3.2 Hz, 1H), 8.94 (s, 1H).
Anal. (C20H20N2O3) C, H, N.
Data for (3R,3aR)-rel-2-(trifluoroacetyl)-3,3a,4,5-tetrahydro-
3-(4-methoxyphenyl)-8-methoxy-2H-benz[g]indazole (57): yield
Preparation of (3R,3aS)-rel-2-Propionyl-3,3a,4,5-tetrahydro-
3-(4-methoxyphenyl)-8-methoxy-2H-benz[g]indazole ((3R,3aS)-
rel-53) and (3R,3aR)-rel-2-Propionyl-3,3a,4,5-tetrahydro-3-(4-
methoxyphenyl)-8-methoxy-2H-benz[g]indazole (53). A solution
of 2-(4-methoxybenzylidene)-7-methoxy-1-tetralone (0.500 g, 1.7
mmol) and hydrazine hydrate (0.5 mL) in propionic acid (5 mL)
was refluxed at 140 °C for 2 days. The reaction mixture was
concentrated and the residue dissolved in ethyl acetate (25 mL).
The mixture was washed sequentially with water, saturated aqueous
NaHCO3, and water, dried over anhydrous Na2SO4, filtered, and
concentrated. The residue was purified by column chromatography
on silica gel using ethyl acetate-hexane (3:7) as the eluent to give
(3R,3aS)-rel-53 (0.217 g, 35%) and 53 (0.252 g, 41%).
1
22%; mp 220-22 °C; H NMR (500 MHz, CDCl3) δ 1.09-1.18
(m, 1H), 1.75-1.80 (m, 1H), 2.77-2.89 (m, 2H), 3.53-3.59 (m,
1H), 3.76 (s, 3H), 3.87 (s, 3H), 5.68 (d, J ) 10 Hz, 1H), 6.84 (d,
J ) 8.5 Hz, 2H), 6.94-6.97 (dd, J ) 3.0 and 8.5 Hz, 1H), 7.01 (d,
J ) 8.5 Hz, 2H), 7.07 (d, J ) 8.5 Hz, 1H), 7.56 (d, J ) 3.0 Hz,
1H); HFABMS m/z 405.1432 (calcd for C21H19F3N2O3, MH+,
405.1426).
Preparation of (3R,3aR)-rel-2-(Hydroxyacetyl)-3,3a,4,5-tet-
rahydro-3-(4-methoxyphenyl)-8-methoxy-2H-benz[g]indazole (55).
To a solution of 54 (0.150 g, 0.36 mmol) in methanol (10 mL)
was added NaOH (50%, 2 mL) at room temperature. The reaction
mixture was stirred at room temperature for 24 h before being
diluted with ethyl acetate. The organic layer was washed with water,
dried over anhydrous Na2SO4, filtered, and concentrated. The
residue obtained was purified by column chromatography on a silica
gel column using ethyl acetate-hexane (2:3) as the eluent to furnish
55 (0.105 g, 78%): mp 163-64 °C; 1H NMR (400 MHz, CDCl3)
δ 1.06-1.17 (m, 1H), 1.72-1.78 (m, 1H), 2.76-2.89 (m, 2H), 3.76
(s, 3H), 3.87 (s, 3H), 4.55-7.70 (m, 2H), 5.66 (d, J ) 10.0 Hz,
1H), 6.82 (d, J ) 9.2 Hz, 2H), 6.92-6.95 (dd, J ) 2.4 and 8.8 Hz,
1H), 6.99 (d, J ) 9.2 Hz, 2H), 7.07 (d, J ) 8.4 Hz, 1H), 7.50 (d,
J ) 2.4 Hz, 1H); 13C NMR (400 MHz, CDCl3) δ 24.38, 28.90,
48.57, 55.46, 55.76, 61.40, 69.61, 108.02, 114.34, 119.12, 127.53,
127.84, 128.47, 130.32, 132.61, 157.67, 158.36, 159.42, 168.66.
Anal. (C21H22N2O4) C, H, N.
Data for (3R,3aS)-rel-53: mp 133-36 °C; 1H NMR (400 MHz,
CDCl3) δ 1.15 (t, J ) 7.6 Hz, 3H), 1.85-1.96 (m, 1H), 2.23-2.29
(m, 1H), 2.74-2.87 (m, 4H), 3.15-3.22 (m, 1H), 3.78 (s, 3H),
3.86 (s, 3H), 4.89 (d, J ) 9.6 Hz, 1H), 6.87-6.92 (m, 3H), 7.08
(d, J ) 8.4 Hz, 1H), 7.23 (d, J ) 8.8 Hz, 2H), 7.44 (d, J ) 2.4 Hz,
1H). Anal. (C22H24N2O3) C, H, N.
1
Data for 53: mp 150-52 °C; H NMR (400 MHz, CDCl3) δ
1.05-1.10 (m, 1H), 1.21 (t, J ) 8.0 Hz, 3H), 1.70-1.78 (m, 1H),
2.74-2.88 (m, 4H), 3.47-3.52 (m, 1H), 3.75 (s, 3H), 3.87 (s, 3H),
5.65 (d, J ) 11.2 Hz, 1H), 6.80 (d, J ) 8.4 Hz, 2H), 6.89-6.92
(dd, J ) 3.2 and 8.4 Hz, 1H), 6.98 (d, J ) 8.8 Hz, 2H), 7.06 (d, J
) 8.4 Hz, 1H), 7.54 (d, J ) 3.2 Hz, 1H). Anal. (C22H24N2O3) C,
H, N.
General Procedure for the Preparation of 2-Substituted (3R,-
3aS)-rel- and (3R,3aR)-rel-3,3a,4,5-Tetrahydro-3-(4-methox-
yphenyl)-8-methoxy-2H-benz[g]indazoles. Exemplified for (3R,-
3aS)-rel-2-(Acetoxyacetyl)-3,3a,4,5-tetrahydro-3-(4-
methoxyphenyl)-8-methoxy-2H-benz[g]indazole ((3R,3aS)-rel-
54) and (3R,3aR)-rel-2-(Acetoxyacetyl)-3,3a,4,5-tetrahydro-3-(4-
methoxyphenyl)-8-methoxy-2H-benz[g]indazole (54). A solution
of 2-(4-methoxybenzylidene)-1-tetralone (1.0 g, 3.4 mmol), trim-
ethylacetic acid (5.0 g), and hydrazine hydrate (1.0 mL) in ethanol
(5 mL) was refluxed for 6 h and then concentrated. The residue
obtained was dissolved in ethyl acetate (10 mL) and then washed
with water. The organic layer was slowly poured into a saturated
NaHCO3 solution (10 mL). The resulting solution was slowly treated
with acetoxyacetyl chloride (1.83 mL, 17 mmol). The mixture was
stirred at room temperature for 30 min. The ethyl acetate layer was
General Procedure for the Preparation of 2-Carbothioamides
and 2-Carboxamides of (3R,3aR)-rel-3,3a,4,5-Tetrahydro-3-(4-
methoxyphenyl)-8-methoxy-2H-benz[g]indazoles. Exemplified
for (3R,3aR)-rel-2-Carbothioamide-3,3a,4,5-tetrahydro-3-(4-
methoxyphenyl)-8-methoxy-2H-benz[g]indazole (59). A suspen-
sion of 2-(4-methoxybenzylidene)-7-methoxy-1-tetralone (0.850 g,
0.0028 mol) and thiosemicarbazide (0.790 g, 0.0084 mol) in ethanol
(30 mL) and concentrated HCl (2 mL) was refluxed for 6 h. The
resulting solution was cooled to room temperature. The solid
precipitate was isolated by filtration, washed with ethanol, and then
recrystallized from ethanol to give 59 (0.985 g, 96%): mp 236-
1
39 °C; H NMR (500 MHz, CDCl3) δ 1.04-1.13 (m, 1H), 1.76-
1.81 (m, 1H), 2.75-2.87 (m, 2H), 3.60-3.66 (m, 1H), 3.76 (s,
3H), 3.85 (s, 3H), 6.07 (d, J ) 10.5 Hz, 1H), 6.83 (d, J ) 9.0 Hz,
2H), 6.93-6.95 (dd, J ) 3.0 and 8.5 Hz, 1H), 6.99 (d, J ) 9.0 Hz,

1894 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Jagtap et al.
2H), 7.08 (d, J ) 8.5 Hz, 1H), 7.49 (d, J ) 3.0 Hz, 1H); HFABMS
m/z 368.1439 (calcd for C20H21N3O2S, MH+, 368.1433).
(5 mL). The solvent was evaporated, and the residue was washed
with ether (20 mL) three times and dried to afford the oxalate salt
of 46: 1H NMR (500 MHz, DMSO-d6) δ 1.06-1.12 (m, 1H),
1.95-1.98 (m, 1H), 2.36 (s, 3H), 3.71 (s, 3H), 3.86 (s, 3H), 3.95-
4.01 (m, 1H), 4.52-4.53 (m, 1H), 5.73 (d, J ) 11.0 Hz, 1H), 6.86-
6.89 (m, 2H), 6.93-6.95 (m, 2H), 7.14 (dd, J ) 3.0, 8.5 Hz, 1H),
7.46 (d, J ) 8.5 Hz, 1H), 7.49 (d, J ) 3.0 Hz, 1H); HFABMS m/z
366.1818 (calcd for C21H24N3O3, MH+, 366.1818).
Preparation of (3R,3aR,5S)-rel-2-Acetyl-5-(N,N-dimethylamino)-
3,3a,4,5-tetrahydro-3-(4-methoxyphenyl)-8-methoxy-2H-benz-
[g]indazole (47). To a solution of 46 (210 mg, 0.57 mmol) in THF
(10 mL) was added formic acid (0.150 mL) at 0 °C, followed by
slow addition of aqueous formaldehyde (37 wt %). The reaction
mixture was refluxed for 24 h. The solution was allowed to cool,
made basic with aqueous NaOH (25 wt %), and extracted with
ethyl acetate. The organic layer was washed with water, dried over
anhydrous Na2SO4, filtered, and concentrated. The residue was
purified by column chromatography on silica gel using ethyl acetate
as the eluent to give 47 (105 mg, 47%): mp 123-28 °C; 1H NMR
(500 MHz, CDCl3) δ 0.9-1.02 (m, 1H), 2.21 (s, 6H), 2.43 (s, 3H),
3.23 (m, 1H), 3.73 (s, 3H), 3.89 (s, 3H), 4.03-4.09 (m, 1H), 5.66
(d, J ) 11.0 Hz, 1H), 6.78 (d, J ) 8.5 Hz, 2H), 6.90-6.92 (dd, J
) 2.5 and 8.5 Hz, 1H), 6.96 (d, J ) 8.5 Hz, 2H), 7.12 (d, J ) 8.5
Hz, 1H), 7.58 (d, J ) 2.5 Hz, 1H). Anal. (C23H27N3O3) C, H, N.
Preparation of (3R,3aR)-rel-2-Ethyl-3,3a,4,5-tetrahydro-3-(4-
methoxyphenyl)-8-methoxy-2H-benz[g]indazole (62). To a solu-
tion of 8 (180 mg, 0.51 mmol) in THF (5 mL) under an argon
atmosphere was added LiAlH4 (2.056 mL, 2.056 mmol, 1 M in
THF) at room temperature. The reaction mixture was stirred at room
temperature for 1 h and then quenched with methanol followed by
ice. The reaction mixture was extracted with ethyl acetate. The
organic layer was washed with water, dried over anhydrous sodium
sulfate, filtered, and concentrated. The residue was purified by
column chromatography on silica gel using 25% ethyl acetate-
hexane as the eluent to give 62 (30 mg, 17%): thick oil; 1H NMR
(500 MHz, CDCl3) δ 1.40 (t, J ) 7.0 Hz, 3H), 1.72-2.04 (m, 2H),
2.62-2.85 (m, 2H), 3.87 (s, 6H), 4.15 (q, J ) 7.0 Hz, 2H), 4.75
(d, J ) 10.5 Hz, 1H), 6.75-6.77 (dd, J ) 8.0 and 3.0 Hz, 1H),
7.01 (d, J ) 8.0 Hz, 2H), 7.12 (d, J ) 8.5 Hz, 1H), 7.29 (d, J )
8.0 Hz, 2H), 7.44 (d, J ) 3.0 Hz, 1H); LRMS m/z 336.3 (calcd for
C21H24N2O2, M+, 336.2).
Evaluation of Necroptosis Inhibitory Activity in an FADD-
Deficient Variant of Human Jurkat T Cells Treated with TNF-
r. Necroptosis activity was performed using an FADD-deficient
variant of human Jurkat T cells treated with TNF-R as previously
described.17 For EC50 value determinations, cells (500000 cells/
mL, 100 µL/well in a 96-well plate) were treated with 10 ng/mL
human TNF-R in the presence of increasing concentrations of test
compounds for 24 h at 37 °C in a humidified incubator with 5%
CO2 followed by ATP-based viability assessment. Stock solutions
(30 mM) in DMSO were initially prepared and then diluted with
DMSO to give testing solutions, which were added to each test
well. The final DMSO concentration was 0.5%. Eleven compound
test concentrations (0.029, 0.058, 0.12, 0.23, 0.46, 0.93, 1.9, 3.7,
11.1, 33, and 100 µM) were used. Each concentration was done in
duplicate. Cell viability assessments were performed using a
commercial luminescent ATP-based assay kit (CellTiter-Glo,
Promega, Madison, WI) according to the manufacturer’s instruc-
tions. Briefly, 40 µL of the cell lysis/ATP detection reagent was
added to each well. The plates were incubated on a rocking platform
for 10 min at room temperature, and luminescence was measured
using a Wallac Victor 3 plate reader (Perkin-Elmer, Wellesley, MA).
Cell viability was expressed as a ratio of the signal in the well
treated with TNF-R and compound to the signal in the well treated
with compound alone. This was done to account for nonspecific
toxicity, which in most cases was <10%. EC50 values were
calculated using nonlinear regression analysis of sigmoid dose-
response (variable slope) curves from plots of log(I) verses viability
values. The reported EC50 values ( SD were determined from at
least two independent experiments.
Data for (3R,3aR)-rel-2-carboxamide-3,3a,4,5-tetrahydro-3-
(4-methoxyphenyl)-8-methoxy-2H-benz[g]indazole (58): yield
1
75%; mp 217-20 °C; H NMR (500 MHz, CDCl3) δ 1.02-1.11
(m, 1H), 1.74-1.79 (m, 1H), 2.73-2.86 (m, 2H), 3.54-3.60 (m,
1H), 3.75 (s, 3H), 3.86 (s, 3H), 5.58 (d, J ) 10.5 Hz, 1H), 6.81 (d,
J ) 8.5 Hz, 2H), 6.88-6.90 (dd, J ) 3.0 and 8.5 Hz, 1H), 7.01 (d,
J ) 8.5 Hz, 2H), 7.05 (d, J ) 8.5 Hz, 1H), 7.47 (d, J ) 3.0 Hz,
1H); HFABMS m/z 352.1663 (calcd for C20H21N3O3, MH+,
352.1663).
Data for (3R,3aR)-rel-2-carbothioamide-3,3a,4,5-tetrahydro-
3-(4-methoxyphenyl)-8-methoxy-N-methyl-2H-benz[g]inda-
zole (60): yield 28%; mp 199-200 °C; 1H NMR (500 MHz,
CDCl3) δ 1.02-1.11 (m, 1H), 1.76-1.81 (m, 1H), 2.75-2.86 (m,
2H), 3.21 (d, J ) 5.0 Hz, 3H), 3.56-3.62 (m, 1H), 3.75 (s, 3H),
3.87 (s, 3H), 6.10 (d, J ) 11.0 Hz, 1H), 6.80 (d, J ) 8.5 Hz, 2H),
6.91-6.94 (dd, J ) 3.0 and 8.5 Hz, 1H), 6.96 (d, J ) 8.5 Hz, 2H),
7.07 (d, J ) 8.5 Hz, 1H), 7.46 (m, 1H), 7.48 (d, J ) 3.0 Hz, 1H);
HFABMS m/z 382.1579 (calcd for C21H23N3O2S, MH+, 382.1589).
Preparation of (3R,3aR)-rel-2-Carboxamidine-3,3a,4,5-tet-
rahydro-3-(4-methoxyphenyl)-8-methoxy-2H-benz[g]indazole (61).
A solution of 59 (0.5 g, 1.3 mmol) and methyl iodide (1.0 mL) in
ethanol (10 mL) was refluxed for 2 h. When the reaction mixture
was concentrated to one-fourth of its volume, a solid precipitate
formed. The mixture was filtered, and the solid was washed with
cold ethanol to give 65 (565 mg, 85%), which was used without
further purification. A solution of 65 (175 mg, 0.34 mmol) and
ammonia (2.0 N solution in ethanol, 15 mL) in ethanol (15 mL)
was stirred at 60 °C for 16 h. The reaction mixture was concentrated.
The solid was recrystallized from methanol-diethyl ether to give
61 (105 mg, 88%): mp 170-75 °C; 1H NMR (500 MHz, CDCl3)
δ 1.07-1.15 (m, 1H), 1.74-1.78 (m, 1H), 2.76-2.89 (m, 2H), 3.77
(s, 3H), 3.79-3.84 (m, 1H), 3.88 (s, 3H), 6.19 (d, J ) 10.5 Hz,
1H), 6.86 (d, J ) 8.5 Hz, 2H), 6.97-6.99 (dd, J ) 3.0 and 8.5 Hz,
1H), 7.10 (d, J ) 8.5 Hz, 2H), 7.50 (d, J ) 3.0 Hz, 1H); HFABMS
m/z 351.1825 (calcd for C20H22N4O2, MH+, 351.1821).
Preparation of (3R,3aR,5S)-rel-2-Acetyl-5-azido-3,3a,4,5-tet-
rahydro-3-(4-methoxyphenyl)-8-methoxy-2H-benz[g]indazole (44)
and 2-Acetyl-3-(4-methoxyphenyl)-8-methoxy-2H-benz[g]inda-
zole (45). To a solution of 8 (800 mg, 2.2 mmol) in acetonitrile
(10 mL) under an argon atmosphere was added trimethylsilyl azide
(1.517 mL, 5.0 equiv) followed by slow addition of [bis(trifluoro-
acetoxy)iodo]benzene (2.948 g, 3.0 equiv). The reaction mixture
was stirred at room temperature for 24 h. The reaction mixture
was concentrated to give a dark brown residue that was purified
by column chromatography on silica gel using ethyl acetate-hexane
(1:3) as the eluent to give 44 (252 mg, 29%), 45 (239 mg, 31%),
and recovered starting material 8 (45 mg).
1
Data for 44: thick oil; H NMR (500 MHz, CDCl3) δ 1.24-
1.28 (m, 1H), 1.82-1.88 (m, 1H), 2.46 (s, 3H), 3.74 (s, 3H), 3.86-
3.89 (m, 1H), 3.91 (s, 3H), 4.66 (m, 1H), 5.70 (d, J ) 11.5 Hz,
1H), 6.80 (d, J ) 8.5 Hz, 2H), 6.96 (d, J ) 8.5 Hz, 2H), 7.0-7.02
(dd, J ) 3.0 and 9.0 Hz, 1H), 7.19 (d, J ) 9.0 Hz, 1H), 7.60 (d, J
) 3.0 Hz, 1H); HFABMS m/z 392.1722 (calcd for C21H22N5O3,
MH+, 392.1723).
1
Data for 45: mp 169-73 °C; H NMR (500 MHz, CDCl3) δ
3.02 (s, 3H), 3.91 (s, 3H), 4.04 (s, 3H), 7.10 (d, J ) 8.5 Hz, 2H),
7.27-7.29 (dd, J ) 3.0 and 8.5 Hz, 1H), 7.74 (d, J ) 2.5 Hz, 2H),
7.87 (d, J ) 8.5 Hz, 1H), 7.92 (d, J ) 8.5 Hz, 2H), 8.96 (d, J )
2.5 Hz, 1H); HFABMS m/z 347.1365 (calcd for C21H19N2O3, MH+,
347.1396).
Preparation of (3R,3aR,5S)-rel-2-Acetyl-5-amino-3,3a,4,5-
tetrahydro-3-(4-methoxyphenyl)-8-methoxy-2H-benz[g]inda-
zole Oxalate Salt (46). A solution of 44 (0.240 g, 0.61 mmol) and
10% Pd-C (50 mg) in ethanol (10 mL) was stirred under hydrogen
(1 atm) for 24 h. The solvent was evaporated, and the residue was
purified by silica gel column chromatography utilizing 5% methanol
in dichloromethane to afford the free base of 46 (85 mg, 38%).
The free base of 46 (44 mg) in ethyl acetate (20 mL) was treated
with a solution of oxalic acid (12 mg, 1.1 equiv) in ethyl acetate

SAR Study of Tricyclic Necroptosis Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1895
(13) Chipuk, J. E.; Green, D. R. Do inducers of apoptosis trigger caspase-
independent cell death? Nat. ReV. Mol. Cell Biol. 2005, 6, 268-
275.
(14) Bro¨ker, L. E.; Kruyt, F. A. E.; Giaccone, G. Cell death independent
of caspases: a review. Clin. Cancer Res. 2005, 11, 3155-3162.
(15) Fink, S. L.; Cookson, B. T. Apoptosis, pyroptosis, and necrosis:
mechanistic description of dead and dying eukaryotic cells. Infect.
Immun. 2005, 73, 1907-1916.
Evaluation of Necroptosis Inhibitory Activity in L929 Cells
Treated with TNF-r or zVAD.fmk. L929 cells (100000 cells/
mL, 100 µL/well in a 96-well plate) were treated with 10 ng/mL
human TNF-R or 100 µM zVAD.fmk in the presence of DMSO
(control), 30 µM (()-1, or 8 for 24 h at 37 °C in a humidified
incubator with 5% CO2 followed by ATP-based viability assessment
as described in the previous experiment. Stock solutions (30 mM)
in DMSO were initially prepared and then diluted with DMSO to
give testing solutions. Each sample was done in duplicate. The final
DMSO concentration was 0.5%. Cell viability values were adjusted
to account for nonspecific toxicity, which in most cases was <10%.
The reported cell viability values (%) ( SD were determined from
two independent experiments.
(16) Kroemer, G.; Martin, S. J. Caspase-independent cell death. Nat. Med.
2005, 11, 725-730.
(17) Degterev, A.; Huang, Z.; Boyce, M.; Li, Y.; Jagtap, P.; Mizushima,
N.; Cuny, G. D.; Mitchison, T.; Moskowitz, M.; Yuan, J. Chemical
inhibitor of nonapoptotic cell death with therapeutic potential for
ischemic brain injury. Nat. Chem. Biol. 2005, 1, 112-119.
(18) Teng, X.; Degterev, A.; Jagtap, P.; Xing, X.; Choi, S.; Denu, R.;
Yuan, J.; Cuny, G. D. Structure-activity relationship study of novel
necroptosis inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 5039-
5044.
(19) 7-Fluoro-1-tetralone was prepared from 7-nitro-1-tetralone by reduc-
tion (SnCl2‚2H2O, THF, 60 °C, 1 h) followed by a Sandmeyer
reaction (HCl, NaNO2, H2O, 5 °C, then NaBF4, 0 °C, 1 h, then switch
solvent to toluene, 110 °C until 1 h after evolution of nitrogen gas
ceases) according to the literature procedure; see: Yoshihama, M.;
Nakakoshi, M.; Nakamura, J.; Nakayama, S. WO 9832724, 1998.
(20) Many literature references utilize the cis/trans-isomer nomenclature
for the 3,3a,4,5-tetrahydro-2H-benzo[g]indazole ring system. In these
references the relative orientation of the C-3 and C-3a protons was
used for the assignment of the cis/trans-isomer nomenclature. For
example, 5 was referred to as the trans-isomer and 6 as the cis-
isomer. However, herein the new naming convention of Chemical
Abstracts is used. Isomer 5 is the (3R,3aS)-rel-isomer, and 6 is the
(3R,3aR)-rel-isomer.
Acknowledgment. P.G.J., S.C., and G.D.C. thank the
Harvard Center for Neurodegeneration and Repair (HCNR) for
financial support. A.D. and J.Y. thank the National Institute on
Aging, National Institute of General Medical Sciences, and the
American Health Assistance Foundation for financial support.
We thank Dr. Jin Hong for her assistance in obtaining and
interpreting the NOE and HH-COSY experiments.
Supporting Information Available: Detailed experimental
procedures for the preparation of tetralones, characterization of
2-arylidene-1-tetralones,2-(arylidene)chroman-4-ones,and2-(arylidene)-
thiochroman-4-ones, combustion analyses, HPLC methods, purity
assessments, and characterization of the (3R,3aS)-rel-isomers of 9,
12-14, 17-20, 25, 26, 29, 31, 32, 35, and 56. This material is
available free of charge via the Internet at http://pubs.acs.org.
(21) Lo´ra´nd, T.; Aradi, F.; Szo¨llo¨sy, AÄ .; To´th, G.; Ko´nya, T. Isomerization
of substituted tricyclic 4,5-dihydropyrazoles. Monatsh. Chem. 1996,
127, 971-977.
(22) Szo¨llo¨sy, AÄ ; To´th, G.; Lo´ra´nd, T.; Ko´nya, T.; Aradi, F.; Le´vai, A.
Fused heterocycles. Part 4. Synthesis and stereochemistry of
hexahydrobenzo[6,6]cyclohepta[1,2-c]pyrazoles. J. Chem. Soc., Per-
kin Trans. 2 1991, 489-493.
(23) Sinha, A. K.; Rastogi, S. N. Synthesis of 3,3a-trans/cis-2-phenyl/
acetyl-3-aryl-tetrahydroindeno/naphtha[1,2-c]- and hexahydrobenzo-
[6,7]cyclohepta[1,2-c]pyrazoles as antiimplantation agents. Indian J.
Chem. 1991, 30B, 684-692.
References
(1) Kanduc, D.; Mittelman, A.; Serpico, R.; Sinigaglia, E.; Sinha, A.
A.; Natale, C.; Santacroce, R.; Di Corcia, M. G.; Lucchese, A.; Dini,
L., Pani, P.; Santacroce, S.; Simone, S.; Bucci, R., Farber, E. Cell
death: apoptosis verses necrosis (review). Int. J. Oncol. 2002, 21
(1), 165-170.
(2) (a) Yuan, J.; Yankner, B. A. Apoptosis in the nervous system. Nature
2000, 407, 802-809. (b) Cryns, V.; Yuan, J. Proteases to die for.
Genes DeV. 1998, 12, 1551-1570.
(3) (a) Talanian, R. V.; Brady, K. D.; Cryns, V. L. Caspases as targets
for anti-inflammatory and anti-apoptotic drug discovery. J. Med.
Chem. 2000, 43, 3351-3371. (b) Moore, J. D.; Rothwell, N. J.;
Gibson, R. M. Involvement of caspases and calpains in cerebrocortical
neuronal cell death is stimulus-dependent. Br. J. Pharmacol. 2002,
135, 1069-1077 and references therein. (c) Boyce, M.; Degterev,
A.; Yuan, J. Caspases: an ancient cellular sword of Damocle. Cell
Death Differ. 2004, 11, 29-37.
(4) (a) Ding, H. F.; Fisher, D. E. Induction of apoptosis in cancer: new
therapeutic opportunities. Ann. Med. 2002, 34, 451-469. (b) Row-
insky, E. K. Targeted induction of apoptosis in cancer management:
the emerging role of tumor necrosis factor-related apoptosis-inducing
ligand receptor activating agents. J. Clin. Oncol. 2005, 23, 9394-
9407.
(5) Martin, L. J.; Al-Abdulla, N. A.; Brambrink, A. M.; Kirsch, J. R.;
Sieber, F. E.; Portera-Cailliau, C. Neurodegeneration in excitotoxicity,
global cerebral ischemia, and target deprivation: A perspective on
the contributions of apoptosis and necrosis. Brain Res. Bull. 1998,
46, 281-309.
(6) Lo, E. H.; Dalkara, T.; Moskowitz, M. A. Mechanisms, challenges
and opportunities in stroke. Nat. ReV. Neurosci. 2003, 4, 399-415.
(7) McCully, J. D.; Wakiyama, H.; Hsieh, Y. J.; Jones, M.; Levitsky, S.
Differential contribution of necrosis and apoptosis in myocardial
ischemia-reperfusion injury. Am. J. Physiol. Heart Circ. Physiol.
2004, 286, H1923-H1935.
(24) To´th, G.; Szo¨llo¨sy, AÄ .; Lo´ra´nd, T.; Ko´nya, T.; Szabo´, D.; Fo¨ldesi,
A.; Le´vai, A. Fused heterocycles. Part 3. Synthesis and stereochem-
istry of benzopyrano- and benzothiapyrano-[4,3-c]pyrazoles. J. Chem.
Soc., Perkin Trans. 2 1989, 319-323.
(25) Sangwan, N. Use of characteristic 1H N.M.R. chemical shifts to
differentiate diastereoisomeric [1]benzopyrano-[4,3-c]pyrazoles, pyra-
zolo[4,3-c]quinolines and related compounds. J. Chem. Res., Synop.
1987, 22-23.
(26) El-Rayyes, N. R.; Al-Jawhary, A. Heterocycles. Part VIII. Synthesis
of new substituted benz[g]indazoles. J. Heterocycl. Chem. 1986, 23,
135-140.
(27) Sangwan, N.; Rastogi, S. N. Studies in antifertility agents: Part
XXXII - synthesis & stereochemistry of 3,3a-trans & cis-2(H)-
acetyl-3-aryl-3,3a-dihydropyrazolo[4,3-c][2H]chromenes, pyrazolo-
[3,4-a]benzocycloalk-1-enes & 3,3a-trans- & cis-2-(2H)-acetyl-3-aryl-
8-methoxy-5-tosyl-3,3a,4,5-tetrahydropyrazolo[4, 3-c]quinolines. Indian
J. Chem. 1981, 20B, 135-139.
(28) Kita, Y.; Tohma, H.; Takada, T.; Mitoh, S.; Fujita, S.; Gyoten, M.
A novel and direct alkyl azidation of p-alkylanisoles using phenyl
iodine(III) bis(trifluoroacetate) (PIFA) and trimethylsilyl azide. Synlett
1994, 427-428.
(29) Yu, L.; Alva, A.; Su, H.; Dutt, P.; Freundt, E.; Welsh, S.; Baehrecke,
E. H.; Lenardo, M. J. Regulation of the ATG7-beclin 1 program of
autophagic cell death by caspse-8. Science 2004, 304, 1500-1502.
(30) For examples of zVAD.fmk-induced caspase-independent cell death,
see: (a) Vandenabeele, P.; Vanden Berghe, T.; Festjens, N. Caspase
inhibitors promote alternative cell death pathways. Sci. STKE 2006,
358, pe44. (b) Martinet, W.; Schrijvers, D. M.; Herman, A. G.; De
Meyer, G. R. z-VAD-fmk-induced non-apoptotic cell death of
macrophages: possibilities and limitations for atherosclerotic plaque
stabilization. Autophagy 2006, 2, 312-314.
(8) Yuan, J.; Lipinski, M.; Degterev, A. Diversity in the mechanisms of
neuronal cell death. Neuron 2003, 40, 401-413.
(9) Kitanaka, C.; Kuchino, Y. Caspase-independent programmed cell
death with necrotic morphology. Cell Death Differ. 1999, 6, 508-
515.
(10) Fiers, W.; Beyaert, R.; Declercq, W.; Vandenabeele, P. More than
one way to die: apoptosis, necrosis and reactive oxygen damage.
Oncogene 1999, 18, 7719-7730.
(11) Borner, C.; Monney, L. Apoptosis without caspases: an inefficient
molecular guillotine? Cell Death Differ. 1999, 6, 497-507.
(12) Edinger, A. L.; Thompson, C. B. Death by design: apoptosis, necrosis
and autophagy. Curr. Opin. Cell Biol. 2004, 16, 663-669.
JM061016O