Optimization of tricyclic Nec-3 necroptosis inhibitors for in vitro liver microsomal stability

Article abstract of DOI:10.1016/j.bmcl.2012.06.098

Necroptosis is a regulated caspase-independent cell death pathway with morphological features resembling passive non-regulated necrosis. Several diverse structure classes of necroptosis inhibitors have been reported to date, including a series of 3,3a,4,5-tetrahydro-2H-benz[g]indazoles (referred to as the Nec-3 series) displaying potent activity in cellular assays. However, evaluation of the tricyclic necroptosis inhibitor's stability in mouse liver microsomes indicated that they were rapidly degraded. A structure-activity relationship (SAR) study of this compound series revealed that increased liver microsomal stability could be accomplished by modification of the pendent phenyl ring and by introduction of a hydrophilic substituent (i.e., α-hydroxyl) to the acetamide at the 2-position of the tricyclic ring without significantly compromising necroptosis inhibitory activity. Further increases in microsomal stability could be achieved by utilizing the 5,5-dioxo-3-phenyl-2,3,3a,4- tetrahydro-[1]benzothiopyrano[4,3-c]pyrazoles. However, in this case necroptosis inhibitory activity was not maintained. Overall, these results provide a strategy for generating potent and metabolically stable tricyclic necrostatin analogs (e.g., 33, LDN-193191) potentially suitable for in vivo studies.

Full text of DOI:10.1016/j.bmcl.2012.06.098

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5685–5688
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of tricyclic Nec-3 necroptosis inhibitors for in vitro liver
microsomal stability
Sungwoon Choi a,e, Heather Keys b, Richard J. Staples c, Junying Yuan d, Alexei Degterev b,
Gregory D. Cuny a,f,
⇑
a Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham & Women’s Hospital and Harvard Medical School,
65 Landsdowne Street, Cambridge, MA 02139, USA
b Department of Biochemistry, Tufts University Medical School, 136 Harrison Avenue, Stearns 703, Boston, MA 02111, USA
c Department of Chemistry, Michigan State University, 578 S. Shaw Lane, East Lansing, MI 48824, USA
d Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
e Department of Scientific Criminal Investigation, Chungnam National University, Daejeon 305-764, Republic of Korea
f Department of Pharmacological and Pharmaceutical Sciences, University of Houston, College of Pharmacy, 549A Science and Research Bldg 2, Houston, TX 77204, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Necroptosis is a regulated caspase-independent cell death pathway with morphological features resem-
bling passive non-regulated necrosis. Several diverse structure classes of necroptosis inhibitors have been
reported to date, including a series of 3,3a,4,5-tetrahydro-2H-benz[g]indazoles (referred to as the Nec-3
series) displaying potent activity in cellular assays. However, evaluation of the tricyclic necroptosis inhib-
itor’s stability in mouse liver microsomes indicated that they were rapidly degraded. A structure–activity
relationship (SAR) study of this compound series revealed that increased liver microsomal stability could
be accomplished by modification of the pendent phenyl ring and by introduction of a hydrophilic substi-
Received 7 May 2012
Revised 25 June 2012
Accepted 27 June 2012
Available online 6 July 2012
Keywords:
Nec-3
Necroptosis
tuent (i.e., a-hydroxyl) to the acetamide at the 2-position of the tricyclic ring without significantly com-
promising necroptosis inhibitory activity. Further increases in microsomal stability could be achieved by
utilizing the 5,5-dioxo-3-phenyl-2,3,3a,4-tetrahydro-[1]benzothiopyrano[4,3-c]pyrazoles. However, in
this case necroptosis inhibitory activity was not maintained. Overall, these results provide a strategy
for generating potent and metabolically stable tricyclic necrostatin analogs (e.g., 33, LDN-193191) poten-
tially suitable for in vivo studies.
Inhibitors
Structure–activity relationship
Microsomal stability
Ó 2012 Elsevier Ltd. All rights reserved.
Necroptosis is a regulated caspase-independent cell death path-
way with morphological features resembling passive non-regu-
lated necrosis.1,2 This type of cell death can be initiated with
To date several diverse structure classes of necroptosis inhibi-
tors have been reported, including hydantoin containing indole
derivatives (i.e., 1),6 rel-(3R,3aR)-3-phenyl-3,3a,4,5-tetrahydro-
2H-benz[g]indazoles (i.e., 2),7 substituted 3H-thieno[2,3-d]pyrimi-
din-4-ones (i.e., 3),8 [1,2,3]thiadiazole benzylamides (i.e., 4)9 and
pyrrole benzylamides (i.e., 5)10 (Fig. 1). Many of these compounds
block necroptosis through inhibition of receptor interacting pro-
tein 1 (RIP1) kinase.11 In addition, ( )-1 has demonstrated in vivo
activity in the temporary and permanent middle cerebral artery
occlusion (MCAO) model of cerebral ischemia1, in a mouse model
of ischemia/reperfusion heart injury,12 in the controlled cortical
impact (CCI) model of traumatic brain injury (TBI),13 a retinal
ischemia-reperfusion injury model,14 a systemic inflammatory re-
sponse syndrome (SIRS) model,15 and a Huntington’s disease
model.16
various stimuli (e.g., TNF-a and Fas ligand) and in a variety of cell
types (e.g., monocytes, fibroblasts, lymphocytes, macrophages, epi-
thelial cells and neurons). Furthermore, necroptosis may represent
a significant contributor to and in some cases predominant mode
of cellular demise under pathological conditions involving exces-
sive cell stress, rapid energy loss and massive oxidative species
generation, not conducive for highly energy-dependent processes,
such as apoptosis. Regulated necrotic cell death mechanisms, such
as necroptosis, raises the possibility of novel therapeutic interven-
tion strategies for the treatment of conditions where necrosis is
known to play
a prominent role, such as organ ischemia
(i.e., stroke3 and myocardial infarction4), trauma and possibly some
forms of neurodegeneration.5
In order to evaluate the in vivo pharmacology of other necrop-
tosis inhibitors via preferred administration routes (i.e., oral, intra-
venous, intraperitoneal or subcutaneous) they must possess
adequate metabolic stability, in addition to in vitro potency. One
efficient and cost effective method of assessing a compound’s
⇑
Corresponding author. Tel.: +1 713 743 1274; fax: +1 713 743 1884.
E-mail address: gdcuny@central.uh.edu (G.D. Cuny).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.098

5686
S. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5685–5688
O
5
OMe
Me
O
a
R
O
c
N
H
3a
OMe
MeO
MeO
MeO
8
N
H
3
O
O
1 N
N
16
O
17 R = H
18 R = Me
N
H
b
Me
Cl
1
2
Me
d
OMe
O
Cl
MeO
O
N
H
N
N
N
N
N
O
N
F
S
19
Me
S
S
CN
O
3
4
Me
Me
OMe
+
OMe
MeO
MeO
Cl
N
NC
Me
N
N
N
H
N
O
O
N
Me
20a
Me
20b
F
Me
O
5
Scheme 2. Reagents and conditions: (a) NaN(TMS)2, THF, 0 °C, then 4-MeO-
PhC(O)Cl, 0 °C to rt (80%); (b) NaN(TMS)2, THF, 0 °C, then MeI, 0 °C to rt (91%); (c)
NH2NH2ꢁxH2O, DCM, 4 Å molecular sieves (52%); (d) MeC(O)Cl, DCM, ꢀ78 °C, then
NaBH(OAc)3, ꢀ78 °C to rt (95%).
Figure 1. Necrostatins. The numbering convention of 2 is also shown.
metabolic stability is to measure its resistance to metabolism over
time in the presence of liver microsomes.17 Utilizing this technique
with mouse liver microsomes, compound 2, which inhibits necrop-
give a mixture of two diastereomers, the (3R,3aR)-rel-isomers
8a–11a and the (3R,3aS)-rel-isomers 8b–11b. The diastereomers
were readily separated by column chromatography on silica gel
and the stereochemical assignments were made using 1H NMR.
Removal of the benzyl group in 9a was accomplished by hydroge-
nation in the presence of 10% Pd/C to give alcohol 12. Oxidation of
10a and 11a with m-chloroperoxybenzoic acid (m-CPBA) gave
sulfones 13 and 14, respectively. Sulfone 14 was subsequently con-
verted to alcohol 15 by hydrogenation.
tosis induced with TNF-
kat T cells with an EC50 value of 0.29
metabolic stability with a half-life (t1/2) of 8.2 min and intrinsic
clearance (CLint of 169 2.0 L/min/mg protein. Herein, we
a
in FADD-deficient variant of human Jur-
l
M, demonstrated poor
)
l
describe the results of a SAR study to optimize the in vitro liver
microsomal stability of the tricyclic (Nec-3) class of necroptosis
inhibitors.
Many of the tricyclic derivatives evaluated herein were pre-
pared according to the procedure outlined in Scheme 1.7 1-Tetral-
ones, 4-chromanones and 4-thiochromanones, 6, were treated
with benzaldehydes or phenylacetaldehyde under basic or acid
conditions to give 7. The chalcones were allowed to react with
hydrazine hydrate utilizing various acids (R3CO2H) as solvent to
Derivatives containing a methyl group at the 3a-position were
prepared according to the procedure outlined in Scheme 2. Initial
attempts to prepare these derivatives from an
a-methyl-1-tetra-
lone derivative utilizing the same synthetic strategy employed
for the preparation of the 3a-H derivatives were unsuccessful. In-
stead, 7-methoxy-1-tetralone, 16, was deprotonated with
NaN(TMS)2 and then allowed to react with 4-methyoxybenzoyl
chloride to give the 1,3-diketone 17. This compound was again
subjected to the same process, except that the anion was quenched
with iodomethane to give 1,3-diketone 18. Condensation of 18
with hydrazine hydrate in the presence of 4 Å molecular sieves
gave 19.18 Treatment of 19 at ꢀ78 °C with acetyl chloride followed
by reduction of the in situ generated acyl iminium gave a mixture
of diastereomers 20a and 20b in a ratio of 1:1.7, favoring isomer
X
X
R2
R1
a
R1
b
(CH2)n
O
O
6
7
X
X
R2
R2
R1
R1
H
H
+
(CH2)n
O
(CH2)n
O
N
N
N
R
N
R
8b R = Me or CF3, X = CH2
or O, n = 0 or 1
8a R = Me or CF3, X = CH2
or O, n = 0 or 1
9b R = CH2OBn, X = CH2, n = 0
10b R = Me, X = S, n = 0
11b R = CH2OBn, X = S, n = 0
9a R = CH2OBn, X = CH2, n = 0
10a R = Me, X = S, n = 0
c
d
11a R = CH2OBn, X = S, n = 0
12 R = CH2OH, X = CH2, n = 0
13 R = Me, X = SO2, n = 0
d
14 R = CH2OBn, X = SO2, n = 0
15 R = CH2OH, X = SO2, n = 0
c
Scheme 1. Reagents and conditions: (a) Ar(CH2)nCHO, 8 N NaOH, EtOH, rt, 2 h or
Ar(CH2)nCHO, concd HCl, MeOH,
120 °C, 15 h (70–80% when n = 0; 20% when n = 1); (c) H2 (1 atm), 10% Pd/C, EtOH
(47–75%); (d) m-CPBA, DCM, rt, 16 h (90–96%).
D
,
4 h (60–75%); (b) R3CO2H, NH2NH2ꢁxH2O,
Figure 2. Thermal ellipsoidal drawing of 20b as determined by single crystal X-ray
analysis.

S. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5685–5688
5687
Table 1
Table 2
Compounds prepared for microsomal stability studies and EC50 determinations for
EC50 determinations for necroptosis inhibition in FADD-deficient Jurkat T cells treated
with TNF- and mouse microsomal stability values
necroptosis inhibition in FADD-deficient Jurkat T cells treated with TNF-
a
a
Compound
EC50
0.29
> 100
15
0.39
0.46
12
0.28
0.75
> 100
0.16
0.090
0.28
0.33
0.64
27
(
l
M)a
t1/2 (min)
CLint (lL/min/mg protein)
X
2
8.2
2.9
7.2
15
10
11
12
40
3.0
33
8.6
27
169 2.0
476 53.9
194 5.9
90.7 13.4
135 9.3
120 39.3
115 18.5
34.6 2.6
463 78.1
42.4 5.5
162 5.7
50.5 3.6
68.7 18.0
25.5 3.8
12.1 1.4
9.38 1.4
R2
20a
20b
21
22
23
24
25
26
27
28
29
30
31
32
33
(CH2)n
O
R1
N
R3
N
R4
Compound
R1
R2
R3
R4
X
n
2
OMe
OMe
OMe
OMe
OMe
F
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
b-Ha
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
H
4-OMe
3-F, 4-OMe
3-F, 4-OMe
4-OCF3
4-OCF3
4-OCF3
3-F, 4-OCF3
Me
Me
Me
CF3
Me
Me
Me
Me
Me
CH2OH
Me
CH2OH
Me
CH2OH
CH2OH
CH2OH
CH2
CH2
CH2
CH2
O
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
20a
20b
21
22
23
24
25
26
27
28
29
30
31
32
33
b-Me
-Me
a
b-H
b-H
b-H
b-H
b-H
b-H
b-H
b-H
b-H
b-H
b-H
b-H
b-H
20
54
115
148
O
S
0.61
SO2
CH2
CH2
CH2
CH2
CH2
CH2
SO2
CH2
a
Standard deviation <10%.
8-position of the tricyclic ring was further replaced with a fluorine
(23) metabolic stability remained poor. Substitution of the benzylic
methylene with sulfur (24) similarly did not improve stability.
However, replacement with a sulfone (25) did result in a signifi-
cant increase in metabolic stability (t1/2 = 40 min and CLint = 34.6
a
a
-H refers to the (3R,3aS)-rel-diastereomer; b-H refers to the (3R,3aR)-rel-
diastereomer.
2.6 lL/min/mg protein) with a slight decrease in necroptosis inhib-
itory activity. Introduction of a methylene group between the 3-
position of the tricyclic ring and the pendent phenyl ring (26) sim-
ilarly did not increase stability, but did eliminate necroptosis
20b where the hydride anion approaches the acyl iminium inter-
mediate distal to the 3a-Me. The 3a-Me of 20b, which is syn to
the pendent phenyl, was shielded and appeared at d 0.69 in the
1H NMR spectra.19a The 3a-Me of 20a appeared further downfield
at d 1.59.19b The structure of 20b was confirmed by single crystal
X-ray analysis and reaffirmed the regioselectivity of the acylation
reaction and the stereochemical assignments of the diastereomers
(Fig. 2).20
In vitro microsomal stability was determined in pooled mouse
liver microsomes. Test compounds (Table 1) were incubated in
the presence and absence of NADPH for 0–60 min and the amount
of remaining compound was quantified.21 Necroptosis inhibitor
( )-1 demonstrated good metabolic stability in this assay with a
inhibitory activity. Addition of a hydroxyl group on the a-position
of the amide (27 vs 2 and 28 vs 29) or introduction of a trifluoro-
methoxy in place of a methoxy at the 4-position of the pendent
phenyl ring (30 vs 2) resulted in increased metabolic stability. A
combination of these changes (31) yielded a further increase in sta-
bility (t1/2 = 54 min and CLint = 25.5 3.8 lL/min/mg protein). A
similar result was also found with a sulfone derivative (25 vs
32), albeit necroptosis inhibitory activity was compromised.
Although introduction of fluorine at the 3-position of the pendent
phenyl ring did not increase stability in one case (2 vs 28), in an-
other instance this change in combination with a hydroxyl group
on the
sition of the pendent phenyl (2 vs 33) resulted in a significant (18-
times) stability increase (t1/2 = 148 min and CLint = 9.38 1.4 L/
a-position of the amide and a trifluoromethoxy at the 4-po-
t1/2 = 59.1 min and CLint = 23.5 2.1
lL/min/mg protein. Evaluation
of necroptosis inhibitory activity was performed using a FADD-
l
deficient variant of human Jurkat T cells treated with TNF-
a
as pre-
min/mg protein) with only a modest (twofold) decrease in necrop-
tosis inhibitory activity.
In conclusion, increased liver microsomal stability as well as
improvement in activity were accomplished for the tricyclic
(Nec-3) series of necroptosis inhibitors by modification of the
pendent phenyl and by introduction of a hydrophilic substituent
viously described.1,7 Utilizing these conditions the cells efficiently
underwent necroptosis, which was completely and selectively
inhibited by ( )-1 (EC50 = 0.21
cells were treated with 10 ng/mL of human TNF-
of increasing concentration of test compounds (11 doses between
30 nM and 100 M) for 24 h followed by ATP-based viability
lM). For EC50 value determinations,
a
in the presence
l
(i.e., a-hydroxyl) to the acetamide at the 2-position, resulting in
assessment (Table 2).
inhibitor 33 (LDN-193191).19c The benzylic position of the tricyclic
ring also appeared to influence metabolic stability. Although
replacement of the methylene group with a sulfone increased met-
abolic stability, it significantly decreased necroptosis inhibitory
activity. Additional optimization of tricyclic necroptosis inhibitors
utilizing the information from this study may result in further in-
creases in metabolic stability and provide a unique set of necropto-
sis inhibitors suitable for in vivo analysis of the pathologic role of
necroptosis following acute and potentially chronic injury.
Several regions of the tricyclic necroptosis inhibitor 2 were con-
sidered potential liability sites responsible for the compound’s
poor metabolic stability in mouse liver microsomes. These sites in-
cluded the methoxy groups at both the 8-position of the tricyclic
ring and the 4-position of the pendent phenyl, the dihydropyrazole
ring, the benzylic carbon at the 5-positions and the amide at the
2-position.
Introduction of a methyl group at the 3a-position of the tricyclic
ring (20a and 20b), which was envisioned to block potential oxida-
tion of the dihydropyrazole, did not result in improved stability.
Interestingly, necroptosis inhibitory actively was dramatically
decreased for both diastereomers. Replacement of the benzylic
methylene at the 5-position with oxygen (22) did not result in an
increase in metabolic stability. When the methoxy group at the
Acknowledgments
S.C. and G.D.C. thank the Harvard NeuroDiscovery Center
(HNDC) for financial support. A.D. and J.Y. thank the National Insti-
tute on Aging, National Institute of General Medical Sciences and

5688
S. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5685–5688
14. Rosenbaum, D. M.; Degterev, A.; David, J.; Rosenbaum, P. S.; Roth, S.; Grotta, J.
C.; Cuny, G. D.; Yuan, J.; Savitz, S. I. J. Neurosci. Res. 2010, 88, 1569.
15. Duprez, L.; Takahashi, N.; Van Hauwermeiren, F.; Vandendriessche, B.;
Goossens, V.; Vanden Berghe, T.; Declercq, W.; Libert, C.; Cauwels, A.;
Vandenabeele, P. Immunity 2011, 35, 908.
American Health Assistance Foundation for financial support. S.C.,
G.D.C. and J.Y. thank the National Institute of Neurological Disor-
ders and Stroke (NINDS) for financial support. A.D. is a recipient
of NIH Mentored Scientist Development Award from the National
Institute on Aging (NIA).
16. Zhu, S.; Zhang, Y.; Bai, G.; Li, H. Cell Death Dis. 2011, 2, e115.
´
17. Baranczewski, P.; Stanczak, A.; Sundberg, K.; Svensson, R.; Wallin, A.; Jannson,
J.; Garberg, P.; Postlind, H. Pharmacol. Rep. 2006, 58, 453.
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References and notes
19. (a) 1H NMR of 20a (500 MHz, CDCl3): d 1.59 (s, 3H), 1.78–1.82 (m, 2H), 2.35 (s,
3H), 2.35–2.45 (m, 2H), 3.78 (s, 3H), 3.85 (s, 3H), 5.39 (s, 1H), 6.74 (dd,
J1 = 8.5 Hz, J2 = 3.0 Hz, 1H), 6.92–6.95 (m, 3H), 7.51 (d, J = 3.0 Hz, 1H), 7.70 (d,
J = 8.5 Hz, 2H). (b) 1H NMR of 20b (500 MHz, CDCl3): d 0.69 (s, 3H), 2.05–2.14
(m, 2H), 2.46 (s, 3H), 2.85–3.01 (m, 2H), 3.80 (s, 3H), 3.86 (s, 3H), 4.93 (s, 1H),
6.88 (d, J = 9.0 Hz, 1H), 6.92 (dd, J1 = 9.0 Hz, J2 = 2.5 Hz, 1H), 7.10–7.12 (m, 3H),
7.41 (d, J = 2.5 Hz, 1H). (c) 1H NMR of 33 (500 MHz, CDCl3): d 1.01–1.10 (m, 1H),
1.79–1.84 (m, 1H), 2.81–2.88 (m, 2H), 3.58–3.64 (m, 1H), 3.88 (s, 3H), 4.59–
4.74 (m, 2H), 5.69 (d, J = 11.0 Hz, 1H), 6.89–6.97 (m, 3H), 7.09 (d, J = 8.5 Hz, 1H),
7.24–7.28 (m, 1H), 7.48 (d, J = 2.5 Hz, 1H).
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Dev. 2010, 24, 2592; Vandenabeele, P.; Galluzzi, L.; Vanden Berghe, T.; Kroemer,
G. Nat. Rev. Mol. Cell Biol. 2010, 11, 700; (c) Christofferson, D. E.; Yuan, J. Curr.
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20. CCDC 875792 contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
21. Microsomal stability was determined in pooled mouse liver microsomes. Test
compound (3 lM final concentration) along with 0.5 mg/mL microsome
protein and 1 mM NADPH was incubated for 0, 5, 15, 30 and 60 min.
Incubation of test compound and microsomes in the absence of NADPH
served as a negative control. The samples were quenched with methanol and
centrifuged for 20 min at 2500 rpm to precipitate proteins. Sample
supernatants were analyzed (N = 3) by LC/MS. The ln peak area ratio
(compound peak area/internal standard peak area) was plotted against time
and the slope of the line determined to give the elimination rate constant
[k = (ꢀ1)(slope)]. The half life (t1/2 in minutes), and the in vitro intrinsic
clearance (CLint in
lL/min/mg protein) were calculated according to the
following equations, where V = incubation volume in
lL/mg protein:
0:693
k
Vð0:693Þ
:
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A.; Yuan, J. Nat. Chem. Biol. 2008, 4, 313.
t1=2
¼
; CLint
¼
t1=2
12. Smith, C. C. T.; Davidson, S. M.; Lim, S. Y.; Simpkin, J. C.; Hothersall, J. S.; Yellon,
D. M. Cardiovasc. Drugs Ther. 2007, 21, 227.
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