Structure-activity relationship study of a novel necroptosis inhibitor, necrostatin-7

Article abstract of DOI:10.1016/j.bmcl.2008.08.058

Necroptosis is a regulated caspase-independent cell death mechanism characterized by morphological features resembling non-regulated necrosis. Necrotatin-7 (Nec-7), a novel potent small-molecule inhibitor of necroptosis, is structurally distinct from previously described necrostatins (Nec-1, Nec-3, Nec-4 and Nec-5). Here, we describe a series of structural modifications and the structure-activity relationship (SAR) of the Nec-7 series for inhibiting necroptosis.

Full text of DOI:10.1016/j.bmcl.2008.08.058

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4932–4935
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study of a novel necroptosis inhibitor,
necrostatin-7
Weihong Zheng a, Alexei Degterev b,c, Emily Hsu b, Junying Yuan 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
c Department of Biochemistry, Tufts University, 136 Harrision Avenue, Boston, MA 02111, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 May 2008
Revised 25 July 2008
Accepted 16 August 2008
Available online 22 August 2008
Necroptosis is a regulated caspase-independent cell death mechanism characterized by morphological
features resembling non-regulated necrosis. Necrotatin-7 (Nec-7), a novel potent small-molecule inhib-
itor of necroptosis, is structurally distinct from previously described necrostatins (Nec-1, Nec-3, Nec-4
and Nec-5). Here, we describe a series of structural modifications and the structure–activity relationship
(SAR) of the Nec-7 series for inhibiting necroptosis.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Necroptosis
Caspase-independent cell death
Necrostatin
Inhibitors
SAR
3-Aryl-pyrazole
Nec-7
Thiazolidin-4-one derivatives
Cell death has traditionally been subdivided into regulated and
unregulated forms. Apoptosis, a form of caspase-dependent regu-
lated cell death, reflects a cell’s decision to die in response to cues
and is executed by an intrinsic cellular machinery. Unregulated cell
death (often called necrosis) is believed to be caused by over-
whelming stress incompatible with cell survival. However, recent
studies revealed an ability of cells to activate regulated cell death
with morphological features closely resembling those of unregu-
lated necrosis. In particular, a regulated caspase-independent ne-
crotic cell death pathway called necroptosis, has recently been
described.1 Necroptosis is induced by tumor necrosis factor super-
family of death-domain receptors and characterized by necrotic
cell death morphology. Because, cell death with necrotic features
is commonly observed in a wide range of human pathologies,2
including stroke,3 myocardial infarction,4 brain trauma, and possi-
bly some forms of neurodegeneration,5 inhibition of necroptotic
cell death by specific small-molecule inhibitors, necrostatins,
may represent an exciting new direction for therapy.
multiple cell types. The identification and optimization of low
molecular weight molecules capable of inhibiting necroptosis is
instrumental in elucidating the role of this process in disease
patho-physiology and could provide lead compounds (i.e., necrost-
atins) for further therapeutic development. Up to this point, several
structurally distinct necrostatins (Nec-1, Nec-3,7 Nec-4,8 and Nec-
5,9) and corresponding modifications have been reported (Fig 1). In
this communication, we describe the initial structure–activity rela-
tionship analysis of a novel necrostatin (Nec-7, EC50 = 10.6 lm)
series. Biological activity of Nec-7 is different from that of Nec-1,
O
H
N
MeO
OMe
HN
N
S
N
O
N
H
Me
Nec-3
Nec-1
Necrostatin-1 (Nec-16) as well as other necrostatins have been
identified as specific and potent small-molecule inhibitor of
necroptotic cell death caused by death-domain receptors (DRs) in
O
O
Me
O
Cl
N
H
N
N
N
S
S
N
S
CN
O
F
* Corresponding author. Fax: +86 21 5492 5379.
E-mail address: yuancy@mail.sioc.ac.cn (C. Yuan).
These authors contributed equally to this work.
Nec-5
Nec-4
Figure 1. Structures of necrostatins.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.058

W. Zheng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4932–4935
4933
Table 1
Nec-1 Nec-7 (6i)
Structure and activity of compounds 3
-
30 100 30 100
O
Y
N
R
X
32P-GST-RIP1
N
S
S
N
HN
N
H
Figure 2. Compound 6i (Nec-7) does not inhibit recombinant RIP1 kinase. RIP1
kinase autophosphorylation reactions were performed in the presence of the
indicated concentrations of Nec-1 and Nec-7 for 30 min at 30 °C. Reactions were
performed as described .10 Briefly, recombinant human GST-RIP1 (1–375 a.a.) was
expressed using Baculogold system in Sf9 cells and purified using glutathione-
sepharose beads. Protein was eluted in 50 mM Tris–HCl, pH 8.0. Two micrograms of
protein was used in each reaction. Reactions were performed in the presence of
3a-f
Compound
X
Y
R
EC50 (lM)
3a
3b
3c
3d
3e
3f
C
C
C
C
C
N
N
N
N
CMe
CH
H
Inactive
Inactive
Inactive
Inactive
5.25
4-F
3-F
2-F
4-F
4-F
20 mM ATP and 3 mCi c-32P-ATP. Following the reaction, products were separated
on 8% SDS–PAGE and visualized by autoradiography
CMe
23.07
Nec-3, Nec-4 and Nec-5 as Nec-7 does not inhibit RIP1 kinase
(Fig. 2), suggesting the possibility that Nec-7 may target an addi-
tional regulatory molecule in the pathway.
Table 2
Structure and activity of compounds 4
The synthesis of Nec-7 and most of its derivatives is outlined in
Scheme 1. 2-Aminothiazole was acylated with 2-chloroacetyl chlo-
ride followed by treatment with potassium thiocyanate to form the
thiazolidone 1.11 The semicarbazone of 4-fluoroacetophenone was
treated with Vilsmeier–Haack reagent to afford pyrrazole 212 Nec-
7 was derived from a Knoevenagel condensation of 1 and 2 with
the aid of sodium acetate anhydrous in glacial acetic acid.13
Influence of substituents on Nec-7 activity: To the strategy for
investigate the structure–activity relationship of the Nec-7, is
shown in Figure 2. Three portions of the molecule were examined:
(i) the thiazole; (ii) substituents on the phenyl group; and (iii) the
pyrazole.
Modification of the thiazole of Nec-7: The thiazole of Nec-7 was
replaced with a 1,3,4-thiadiazole and with the addition of methyl
substituents to various position on the thiazole. As shown in
Table 1, all of the thiadiazole derivatives were inactive. 3a–d in
Table 1. Adding a methyl on the thiazole 4-position increased the
activity slightly, but adding this group to the 5-position had a
negative effect (3e vs 3f in Table 1).
Influence of substituents on the phenyl ring of Nec-7: For the study
of the influence of substituents on the phenyl ring, a series of deriv-
atives of compound 3 were prepared according to Scheme 1.
As shown in Table 2, moving the fluoro group from the para-
position to the meta- or ortho-positions (4a and 4b) resulted in a
complete loss of activity. However, the fluoro group at the para-
position could be replaced with a variety of electron-donating or
electron-withdrawing substituents with retention of necroptosis
inhibitory activity. It is worth noting that replacing the fluoro
group by morpholine (4h) or phenyl (4l) increased activity. When
the fluoro group in the para-position was retained, adding and
additional fluoro group to the 3-position of phenyl ring enhanced
the activity slightly (4m vs Nec-7 and 4n vs 4h). Since derivative
O
N
R
N
S
S
N
HN
N
H
4a-o
Compound
R
EC50
10.6
Inactive
inactive
40.6
14.9
7.77
15.63
34.51
(lM)
Nec-7
4a
4b
4c
4d
4e
4-F
3-F
2-F
H
4-CH3O
4-CH3
4-Cl
4f
4g
4-Br
4h
2.93
4
N
O
O
4i
4j
4k
4l
4m
4-NO2
4-SCH3
4-OH
4-Ph
3,4-F2
22.96
—
32.67
1.29
6.32
4n
4o
2.90
3
N
4
F
3,4-O2(CH2)
34.83
4a with a fluoro group in the phenyl 3-position was inactive, the
para-substituent is crucial for maintaining the activity (Fig. 3).
Influence of N-contained groups at the phenyl para-position of
Nec-7: Since 4h (EC50 = 2.93 lM) with a morpholine group at the
4-position of the phenyl ring displayed increased inhibitory activ-
ity compared to that of original Nec-7, several derivatives with
substituting groups containing a N atom at the phenyl para-posi-
tion were examined. These derivatives were also synthesized
O
N
N
N
S
a, b
F
NH2
S
S
HN
O
N
1
e
N
S
F
S
N
HN
N
H
c, d
F
COCH3
OHC
Nec-7
N
N
H
2
Scheme 1. Reagents and conditions: (a) 2-chloroacetyl chloride, Net3, CH2Cl2, 0 °C–rt, 53%; (b) KSCN, EtOH, reflux, 85%; (c) H2NHNCONH2ÁHCl, NaOAc, CH3OH/H2O, 98%; (d)
i—POCl3–DMF; ii—10% NaOH; iii—10 N HCl, 86%; (e) NaOAc, HOAc, reflux, 91%.

4934
W. Zheng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4932–4935
various subsituents
F
O
N
Type 2
various substituents
Types 1
N
S
S
N
HN
N
H
various heterocycles
Type 3
Nec-7
Figure 3. Substituents of Nec-7 derivatives.
Table 3
in place of the phenyl ring was also active. Various other
Structure and activity of compounds 5a–g
R1
X
replacements were also examined. As shown in Table 4, most
replacements were well tolerated, except p-chloro- (6d) and o-flu-
orobiphenyls (6f), suggesting that this type of conjugated system
may be preferable. However, the disruption of the biphenyl struc-
ture had little influence on the activity (6i). Thus, the conjugated
structure is not important for optimal activity of the biphenyl.
Notably, another conjugated bi-cyclic derivative with naphthyl
R2
N
O
N
N
S
S
N
HN
N
H
5a-g
R2
group displayed greatly decreased activity (EC50 = 56.8 lM).
Compound
R1
X
EC50 (lM)
Influence of pyrazole ring of Nec-7: For the study of the influence
of pyrazole ring of Nec-7, derivatives with other types of aromatic
heterocycles in place of pyrazole were synthesized, such as triazole
and isoxazole. Phenylpropiolaldehyde15 were prepared from corre-
sponding para-substituted benzaldehydes through a Corey–Fuchs
reaction, and then cyclized with sodium azide in DMSO at room
temperature to form 5-aryl-4-carbaldehyde-1,2,3-trizazoles,16
7a–d (Scheme 2).
5a
5b
5c
5d
5e
5f
–(CH2)5–
–(CH2)4–
—
—
—
—
—
CAO
CAO
6.36
9.53
3.30 0.047
—
–(C2H4)2S
Me
Me
Et
Et
—
–(C2H4)2O
–(CH2)5–
7.19 0.280
2.09 0.060
5g
The N-hydroxybenzimidoyl chloride derived from chlorination
of benzaldehyde oxime by N-chlorosuccinimide (NCS)17 was trea-
ted with 3-dimethylaminoacrolein in the presence of triethylamine
to generate the isoxazole carboxaldehydes 8 via a 1,3-dipolar
cycloaddition18 (Scheme 3).
The N atom of pyrazole can easily react with electrophilic re-
agents in the present of potassium carbonate at room temperature,
such as iodomethane, bromoacetonitrile and benzyl bromide. The
main products were N-substituting 3-aryl-4-carboxaldehydepy-
razoles 9 (Scheme 4).
according to Scheme 1. The acetophenones for compounds 5a–c
were prepared by a SNAr reaction of N-contained heterocycles with
4-fluroacetophenone in the present of potassium carbonate.14
while the staring material for 5d and 5c was derived from alkyl-
ation of 4-aminoacetophenone by iodomethane or iodoethane.
As shown in Table 3, heterocyclic groups without an oxygen
atom (5a and 5b) also increased activity, but less so compared to
the morpholine ring. A derivative with a six-membered ring dis-
played better activity compared to a five-membered ring (5a vs
5b). Thiomorpholine substitution improved activity similar to that
of morpholine (5c vs 4h). Finally, addition of the N-containing
heterocycles to the phenyl ring through an acyl moiety, for exam-
ple, piperidine ring, resulted in further improvement in activity (5f
vs 5g).
Finally, derivatives 10 were prepared through condensation
of these various carboxaldehyde derivatives 7,
8 or 9 with
a
b
R
c
R
CHO
Br
Influence of bipheny, biaryl ethers, biaryl thioethers and biarylsulf-
Br
N
ones of Nec-7: Compound 4l (EC50 = 1.29
l
M) with a biphenyl
R
OHC
R
CHO
Table 4
N
Structure and activity of compounds 6a–i
N
H
X
R
N
O
R= -H, -Cl, -OCH3, -F
7a-d
S
N
Scheme 2. Reagents and conditions: (a) CBr4, PPh3, CH2Cl2, 0 °C–rt, 81–88%; (b) i—
n-BuLi, À78 °C, ii—DMF, iii—10% KH2PO4, 64–71%; (c) i—NaN3, DMSO, rt; ii—15%
KH2PO4, 41–61%.
S
HN
N
N
H
6a-i
b
a
Compound
X
R
EC50 (lM)
R
R
CHO
Cl
N
OH
6a
6b
6c
6d
6e
6f
6g
6h
6i
—
—
—
—
—
—
–O–
–S–
–SO2–
o-OMe
p-OMe
p-Me
p-Cl
p-F
m-F
–H
–H
–H
2.41 0.235
2.59 0.476
4.38 1.296
>100
3.87 0.752
47.97 0.255
—
R
c
OHC
R
N
OH
N
O
8a-d
—
Scheme 3. Reagents and conditions: (a) HO–NH2ÁHCl, K2CO3, EtOH, rf, 80–94%; (b)
1.78 0.078
NCS, CH2Cl2, rt, 50–54%; (c) 3-dimethylaminoacrolein, Et3N, THF, rt, 42–93%.

W. Zheng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4932–4935
4935
may interact with the target protein through hydrogen bonding.
These new derivatives will be used to further interrogate the
mechanism(s) of necroptotic cell death.
F
F
O
O
F
O
a
+
N
N
N
R
N
N
H
R
N
Acknowledgments
9a-c
2
The authors thank Dr. Greg Cuny for editing this manuscript.
This project was supported in part by the Chinese Academy of Sci-
ences, National Institute of General Medical Sciences (USA), and
National Institute of Neurological Disorders and Stroke (USA) (to
J.Y.) and National Natural Science Foundation of China (Nos.
20272075, 20372076 to C.Y.).
Scheme 4. Reagents and condition: (a) RX, K2CO3, CH3CN, rt, 65–91%.
Table 5
structure and activity of compounds 10a–l
N
O
R
S
N
References and notes
S
HN
N
X
Y
1. Degterev, A.; Huang, Z.; Boyce, M.; Li, Y.; Jagtap, P.; Mizushima, N.; Cuny, G. D.;
Mitchison, T.; Moskowitz, M.; Yuan, J. Nat. Chem. Biol. 2005, 1, 112.
2. Nieminen, A. L. Int. Rev. Cytol. 2003, 224, 29.
10a-l
Compound
X
Y
R
EC50 (lM)
3. Lo, E. H.; Dalkara, T.; Moskowitz, M. A. Nat. Rev. Neurosci. 2003, 4, 399.
4. McCully, J. D.; Wakiyama, H.; Hsieh, Y. J.; Jones, M.; Levitsky, S. Am. J. Physiol.
Heart Circ. Physiol. 2004, 286, H1923.
5. Yuan, J.; Lipinski, M.; Degterev, A. Neuron 2003, 40, 401.
6. Teng, X.; Degterev, A.; Jagtap, P.; Xing, X.; Choi, S.; Denu, R.; Yuan, J.; Cuny, G. D.
Bioorg. Med. Chem. Lett. 2007, 17, 1455.
7. Jagtap, P. G.; Degterev, A.; Choi, S.; Keys, H.; Yuan, J.; Cuny, G. D. J. Med. Chem.
2007, 50, 1886.
8. Teng, X.; Keys, H.; Jeevanandam, A.; Porco, J. A.; Degterev, A.; Yuan, J.; Cuny, G.
D. Bioorg. Med. Chem. Lett. 2007, 17, 6836.
9. Wang, K.; Li, J.; Degterev, A.; Hsu, E.; Yuan, J.; Yuan, C. Bioorg. Med. Chem. Lett.
2007, 17, 1455.
10. Degterev, A.; Hitomi, J.; Germscheid, M.; Ch’en, I. L.; Korkina, O.; Teng, X.;
Abbott, D.; Cuny, D.; Yuan, C.; Wagner, G.; Hedrick, S. M.; Gerber, S. A.;
Lugovskoy, A.; Yuan, J. Nat. Chem. Biol. 2008, 4, 313.
10a
10b
10c
10d
10e
10f
10g
10h
10i
N
N
N
N
C
C
C
C
C
C
C
C
NH
NH
NH
NH
O
O
O
O
C-Me
H
Cl
OCH3
F
Cl
F
CH3
NO2
F
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
10j
10k
10l
C-CH2CONH2
C-Bn
C-Ph
F
F
F
11. Byoung, M. Kwon; Jeen, Woo Park J. Heterocycl. Chem. 1983, 20, 1725.
12. (a) Kira, M. A.; Aboul-Enein, M. N.; Korkor, M. I. J. Heterocycl. Chem. 1970, 2, 25;
(b) Baraldi, P. G.; Cacciari, B.; Spalluto, G., et al Synthesis 1997, 10, 1140.
13. Compound Nec-7: mp >300 °C; 1H NMR(300 MHz, DMSO) d 7.42–7.50 (m, 4H),
7.60–7.65 (m, 2H), 7.73 (d, 1H, J = 3.6 Hz), 7.91 + 7.19 (br s, 1H), 12.54 (br s, 1H,
@NH), 13.68 + 13.86 (br s, 1H, –NH–); EIMS m/z (rel intensity): 371(M+) (4.87),
370 (17.68), 234 (24.34), 209 (21.97), 207 (21.96), 165 (40.10), 91 (100, base),
44 (37.30), 41 (20.31); IR (KBr, cmÀ1) 3200 (N–H), 1713, 1593 (C@O), 1498,
1438, 1304, 1192, 1132, 846; Anal. Calcd for C16H10FN5OS2: C, 51.74; H, 2.71;
N, 18.86. Found: C, 51.68; H, 2.87; N, 19.06.
14. Meciarova, M.; Toma, S.; Podlesna, J., et al Monatsh. Chem. 2003, 134, 37.
15. Journet, M.; Cai, D.; DiMichele, L. M.; Larsen, R. D. Tetrahedron Lett. 1998, 39,
6427.
16. Journet, M.; Cai, D.; Kowal, J. J.; Larsen, R. D. Tetrahedron Lett. 2001, 42, 9117.
17. Shang, Y.; Wang, Y. Synthesis 2002, 12, 1663.
thiazolidone 1. As shown in Table 5, all of these modifications
resulted in complete loss of necroptosis inhibitory activity. Consid-
ering that the bioisosteric replacements of pyrazole ring were
detrimental to activity, this ring represents an important
determinant of Nec-7 activity.
In conclusion, a number of derivatives of Nec-7 were found to
inhibit TNF-a-induced necroptosis in FADD-deficient variant of hu-
man Jurkat T cells. A SAR study revealed that (i) substituent groups
at phenyl 4-position are essential; (ii) the para-position of the phe-
nyl ring was tolerant of substitution, and larger groups (i.e., mor-
pholine and additional phenyl rings) were better; (iii) the
pyrazole ring was quite sensitive to structural modification and
18. Balsamo, A.; Coletta, I.; Guglielmotti, A., et al Eur. J. Med. Chem. Chim. Ther.
2003, 38, 157.