A novel necroptosis inhibitor - Necrostatin-21 and its SAR study

Article abstract of DOI:10.1016/j.bmcl.2013.06.073

An initial structure-activity relationship study of the novel necroptosis inhibitor Nec-21 was described. Any changes of the tetracyclic scaffold were detrimental for the activity. Introduction of a substituent to 7 or 8 position (e.g., cyano or methoxy group, respectively), would increase the activity. The 7 and 8-position disubstituted compound 17b was 35-fold as potent as the lead, while EC₅₀ reached 14 nM.

Full text of DOI:10.1016/j.bmcl.2013.06.073

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel necroptosis inhibitor—necrostatin-21 and its SAR study
Zhijie Wu a, Ying Li b, Yu Cai a, Junying Yuan a,b, Chengye Yuan a,
⇑
a State Key Laboratory of Bio-organic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
b Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An initial structure–activity relationship study of the novel necroptosis inhibitor Nec-21 was described.
Any changes of the tetracyclic scaffold were detrimental for the activity. Introduction of a substituent to 7
or 8 position (e.g., cyano or methoxy group, respectively), would increase the activity. The 7 and
8-position disubstituted compound 17b was 35-fold as potent as the lead, while EC50 reached 14 nM.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 14 May 2013
Revised 21 June 2013
Accepted 26 June 2013
Available online xxxx
Keywords:
Necroptosis
Nec-21
Inhibitor
SAR study
RIP1
JNK3
For a long time, apoptosis was taken as the sole form of pro-
grammed cell death during development, homeostasis and disease,
whereas necrosis was considered as an unregulated and uncontrol-
lable process. Evidence now reveals that necrosis can also occur in a
regulated manner, which was termed ‘necroptosis’ by Yuan1 in
2005. Different from that of apoptosis, necroptosis is mediated by
a RIP1 kinase-dependent, caspase-independent pathway.2,3 Nec-
roptosis has been implicated in mediating various diseases, includ-
ing ischemic injury, neurodegeneration and viral infection, and may
be a new promising target for the treatment of these diseases.
In 2005, Nec-1, the first small-molecule inhibitor of necroptosis,
was reported by Yuan and her co-workers at Harvard Medical
School.4 Further study demonstrated that Nec-1 is a highly specific
inhibitor of RIP1 kinase.5 Several structurally distinct necrostatins
(Nec-36, Nec-47, Nec-58 and Nec-79) and NecroX series10 (e.g., Nec-
roX-18) were developed (Fig. 1). In this Letter, we describe the ini-
tial structure–activity relationship study of a novel tetracyclic
the desired phenylhydrazone 2 in good yield, followed by treat-
ment with 1.8 M H2SO4 under refluxing condition to afford tricyclic
compound 3.11 Enol 4, prepared through another condensation of 3
with ethyl formate, was converted to Nec-21 by closing the pyra-
zole ring with hydrazine hydrate.12
The general route was also applicable for derivatives with
electron-donating group or halogen in ring A from corresponding
anilines. But derivatives with strong electron-withdrawing group
(such as nitro or cyano group) in ring A had to be synthesized under
more strenuous conditions. For example, phenylhydrazone 5, pre-
pared according to Scheme 1, must be heated to reflux in TFA to af-
ford compound 6, followed by condensation with DMF DEA to give
enamine 7. Subsequently, 7-cyano-Nec-21 16k was obtained by
refluxing 7 and hydrazine hydrate in ethanol13 (Scheme 2).
The derivatives with replaced active hydrogen (N2-H and/or
N10-H) were synthesized according to Scheme 3. Compound 15a
and 11, obtained by introduction of methyl and tosyl on N2 atom,
respectively, could be easily converted into compound 15c and 12
by methylation on N10 atom. Subsequently, detosylation of 12 with
Na-naphthalene solution gave 15b.
Ring D could be changed into isoxazole (15d) if 4 was treated
with hydroxylamine hydrochloride in refluxing alcohol. Compound
15e, with a pyrrole ring, was prepared by detosylation of 13, which
was obtained through the Fischer indole synthesis reaction of 1214
and phenylhydrazine hydrochloride (Scheme 4).
Ester group at 3-position could be introduced by condensation
of 3 with diethyl oxalate, followed by cyclization with hydrazine
hydrate in refluxing acetic acid.15 Compound 16g was obtained
by LiAlH4 reduction of 16f (Scheme 5).
necrostatin (Nec-21, EC50 = 0.50 lM) series. The preliminary chem-
ical biology study suggested that Nec-21 was a dual inhibitor of
RIP1 and JNK3 kinases, whose mechanism of action may differ
from that of previously reported inhibitors.
Nec-21, namely 2,4,5,10-tetrahydropyrazolo[3,4-a]carbazole
(Fig. 2), and most derivatives were prepared according to Scheme 1
(general route). Enol 1, synthesized from the condensation of
cyclohexanone and ethyl formate in the presence of NaH, was
treated with diazonium salt, generated in situ from aniline, to give
⇑
Corresponding author. Tel.: +86 21 54925120; fax: +86 21 54925379.
E-mail address: yuancy@mail.sioc.ac.cn (C. Yuan).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.073
Please cite this article in press as: Wu, Z.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.06.073

2
Z. Wu et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
O
Me
S
Me
Cl
N
H
N
OMe
H
N
MeO
N
N
H
S
N
N
O
O
F
N
H
Me
Nec-3
Nec-4
Nec-1
F
N
N
O
O
S
N
O
O
O
H
N
O
N
NH
N
S
O
S
N
S
HN
N
H
S
N
CN
O
NecroX-18
Nec-7
Nec-5
Figure 1. Structures of necrostatins and NecroX-18.
5
4
6
a
b
3
7
8
C
N
Ts
A
B
D
N
H
13
N
Ts
NH
N
H
N
H
N
N
O
H
9
12
15e
10
Nec-21
Scheme 4. Reagents and conditions: (a) PhNHNH2, AcOH, reflux, 34%; (b) MeONa,
MeOH, reflux, 46%.
EC50 = 0.50µM
Figure 2. Structure of Nec-21 (each substituent position and each ring are
numbered).
We set the overall research strategy to first determine the
optimal molecular scaffold and then study the modification of
substituent at the optimal scaffold. Inspired by the SAR study of
Nec-14, we investigated three units in Nec-21, namely the active
hydrogens (N2-H and N10-H), the ring D and the linker ring C to
draw the optimal scaffold (Table 1).
The NH groups in lead compounds are usually considered of
great importance, for their activities due to the hydrogen-bond
interaction with the target proteins. As we forecasted, methylation
either on N2 or N10 resulted in complete loss of the necrosis
inhibitory activity (15a, 15b and 15c).
a
NH2
N2Cl
OH
c
d
N
O
N
H
O
O
b
2
1
For the modification of ring D, the pyrazole ring was replaced
with its bioisosteres, such as isoxazole (15d) and pyrrole ring
(15e). Unfortunately, both derivatives lost their activity com-
pletely, meanwhile, 15f, resulted from methylation of 15d, was
also inactive.
e
f
NH
N
N
H
OH
N
H
N
H
O
O
4
3
Nec-21
Ring C, as a linker, can adjust the relative position of the indole
ring and the pyrazole ring, consequently modulating the activity of
the lead compound. The constraction, expansion, opening or
Scheme 1. General route. Reagents and conditions: (a) NaNO2, concd HCl, À10 °C to
À0 °C; (b) HCOOEt, NaH, Et2O, rt, 92%; (c) NaOAc, MeOH, 0 °C, 77%; (d) 1.8 M H2SO4,
MeCN, reflux, 58%; (e) HCOOEt, NaH, THF, 99%; (f) N2H4ÁH2O, EtOH, rt, 87%.
NC
NC
NC
NC
a
b
c
N
N
O
N
H
N
H
N
H
6
N
H
NH
N
O
O
5
7
16k
Scheme 2. Reagents and conditions: (a) TFA, reflux, 90%; (b) (EtO)2CHNMe2 (DMF DEA), reflux, 68%; (c) N2H4ÁH2O, EtOH, reflux, 80%.
a for R = Me
b for R = Ts
c, d
NH
N
R
N
R
15a
11 R = Ts
N
N
H
O
O
8
R = Me
9 R = Ts
3
R = Me
e
f for R = Ts
N
N
N
R
N
Me
N
H
N
Me
15c
R = Me
15b
12 R = Ts
Scheme 3. Reagents and conditions: (a) MeI, NaH, acetone, rt, 100%; (b) TsCl, KOH, DMF, rt, 50%; (c) HCOOEt, NaH, THF, 75%; (d) N2H4ÁH2O, EtOH, rt, 47%; (e) MeI, LiHMDS,
THF, À78 °C to rt, 80% for R = Me, 76% for R = Ts; (f) Na, naphthalene, rt, 33%.
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Z. Wu et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
COOEt
COOEt
CH2OH
NH
a
b
c
NH
N
H
OH
N
H
N
N
H
N
H
N
O
O
16g
16f
3
14
Scheme 5. Reagents and conditions: (a) (COOEt)2, NaH, THF, 66%; (b) N2H4ÁH2O, AcOH, reflux, 97%; (c) LiAlH4, THF, 0 °C, 64%.
Table 2
Table 1
SAR of derivatives with single substituent at each position
SAR of the active hydrogens, ring D and ring C
6
Z
Z
7
3
R
Y
Y
NH
8
N
H
N
N
R
X
N
R
X
9
16a - 16o
15a - 15c, 15g - 15k
15d - 15f
Compound
R
EC50 (lM)
Compound
R
X
Y
Z
EC50
0.50
(lM)
Nec-21
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
16o
H
9-F
0.50
0.86
0.76
6.32
0.67
1.45
4.86
2.11
Nec-21
15a
15b
15c
15d
15e
15f
15g
15h
15i
H
Me
H
Me
H
H
Me
H
H
H
H
N
N
N
N
O
NH
NH
N-Me
N-Me
N
–(CH2)2–
–(CH2)2–
–(CH2)2–
–(CH2)2–
–(CH2)2–
–(CH2)2–
–(CH2)2–
–CH2–
Inactivea
Inactive
Inactive
Inactive
Inactive
Inactive
5.79
9-Me
9-NO2
6-Cl
6-OMe
3-COOEt
3-CH2OH
7-F
NH
O
CH
N
N
N
N
N
N
NH
NH
NH
NH
NH
0.14a
0.78
0.17
0.080
0.25
0.25
0.10
0.11a
–(CH2)3–
0.64
Inactive
3.62
7-Et
N.A.b
7-OMe
7-CN
8-Cl
8-CF3
8-OMe
8-CN
15j
15k
–CH@CH–
–CH2CH(CH3)–
H
0.81
a
Cell viability <30% at 20
The linkage is disconnected.
l
M.
b
a
Cytotoxicity at high concentration.
aromatization of ring C (15h–15j) or introduction of methyl at
4-position (15k)16, all of these means led to decrease of the activity
to varying degrees. So, modification of the tetracyclic scaffold, such
as the active hydrogens, ring D and ring C, could not improve the
inhibitory activity.
cyclic disubstitution at 7 and 8 position did not perform as well
as 17a. As 17d was only three fold as potent as the lead, while
17e was five fold less active (Table 3).
Then we focused on the substituent modification after determi-
nation of the optimal scaffold (Table 2). Introduction of halogen
(e.g., 16a), alkyl group (e.g., 16b) or strong electron-withdrawing
group (e.g., 16c) to 9-position were all detrimental to necroptosis
inhibitory activity. Introduction of chlorine (16d) or methoxy
group (16e) at 6-position also resulted in a slight decrease in activ-
ity. As the result of introduction of ester (16f) or hydroxyl group
(16g) at 3-postion, the activity of corresponding derivatives was
dropped 4–10 times.
In conclusion, a SAR study of the novel necroptosis inhibitor
Nec-21 revealed that changes of the tetracyclic scaffold were det-
rimental, introduction of a substituent to the 7- or 8-position could
increase the activity, especially, the cyano and methoxy group. Fur-
ther improvement in activity could be achieved if two substituents
were introduced to the 7- and 8-position, respectively. The most
potent compound 17b was 35-fold as potent as the lead, while
EC50 reached 14 nM. These compounds can serve as molecular
tools to investigate further the mechanism of necroptotic cell
death.
Compared to the 6, 9 and 3-postion, the 7 and 8-postion were
better derivative sites. Compound 16h with 7-F substituent was
three fold more potent than the lead, but cytotoxicity was ob-
served at high concentration (>10 lM). Introduction of alkyl
groups to 7-position (e.g., 16i) was detrimental to the activity.
However, introduction of a methoxy group (16j) or a cyano group
(16k) to 7-position would bring increase of the activity by three or
six folds, respectively. For 8-substituented derivatives, introduc-
tion of an electron-withdrawing (e.g., 16l, 16m and16o) or an elec-
tron-donating (e.g., 16n) group could both increase the potency,
Table 3
SAR of disubstituted derivatives
R1
7
NH
R2
N
H
N
9
although 16o was cytotoxic at high concentration (>1.0 lM).
17a - 17e
Upon examination of the SAR of the single substituent at each
position, we combined these well performed substituents (i.e.,
methoxy and cyano group at 7-position and methoxy group at
8-positon) and synthesized some disubstituted derivatives. The
7,8-dimethoxy derivative 17a was 11-fold as potent as the lead;
The 7-cyano-8-methoxy derivative 17b could be more than 35-fold
in activity, however severe cytotoxicity was observed at high
Compound
R1
H
7-OMe
7-CN
6-OMe
7, 8-OCH2O
7, 8-OCH2CH2O
R2
EC50
0.50
(lM)
Nec-21
17a
17b
17c
17d
H
8-OMe
8-OMe
8-OMe
0.047
0.014a
0.11
0.15
2.64
17e
concentration (>0.1
comparable activity to 16n with 8-methoxy group. However the
lM); The 6,8-disubstituted derivate 17c had
a
Cytotoxicity at high concentration (>0.1 lM).
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Z. Wu et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.06.
073.
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