Structure-activity relationship analysis of a novel necroptosis inhibitor, Necrostatin-5

Article abstract of DOI:10.1016/j.bmcl.2006.11.056

Necrostatin-5 (Nec-5) is a novel potent small-molecule inhibitor of necroptosis structurally distinct from previously described Necrostatin-1 (Nec-1), and therefore, represents a new direction for the inhibition of this cellular caspase-independent necrot

Full text of DOI:10.1016/j.bmcl.2006.11.056

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1455–1465
Structure–activity relationship analysis of a novel
necroptosis inhibitor, Necrostatin-5
Ke Wang,a Jinfeng Li,a Alexei Degterev,b Emily Hsu,b Junying Yuanb,
and Chengye Yuana,*,
aState Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 200032, China
bDepartment of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
Received 15 August 2006; revised 12 October 2006; accepted 7 November 2006
Available online 21 November 2006
Abstract—Necrostatin-5 (Nec-5) is a novel potent small-molecule inhibitor of necroptosis structurally distinct from previously
described Necrostatin-1 (Nec-1), and therefore, represents a new direction for the inhibition of this cellular caspase-independent
necrotic cell death mechanism. Here, we describe a series of structural modifications of Nec-5 and the structure–activity relationship
(SAR) of Nec-5 series in inhibiting necroptosis.
Ó 2006 Elsevier Ltd. All rights reserved.
Cell death is traditionally classified either as apoptosis
or necrosis. Apoptosis is regulated by an evolutionarily
conserved cellular mechanism that proceeds through
specific signal transduction pathways common to
different cell types. Necrosis, on the other hand, is
thought to be an unregulated cellular response to
overwhelming stress. Despite the prevalence of necrosis
under pathologic conditions, therapeutic strategies to
prevent cell death in pathological conditions have
targeted apoptosis rather than necrosis, because of
the perception that necrosis is an unregulated and
non-specific process, and therefore, difficult to be
targeted for therapeutic purposes.
death pathway, which we termed ‘necroptosis.’2–5 Nec-
roptosis is a regulated cell death pathway, activated
upon stimulation of FasL/TNFa family of death recep-
tor ligands under the conditions when apoptosis is
inhibited. Necroptosis is characterized by morphological
features normally attributed to unregulated necrosis.
The existence of a regulated cellular necrotic cell death
mechanism raised the possibility to specifically target
necrotic component of human diseases. As a first exam-
ple, we have used Nec-1 to investigate the pathological
importance of necroptosis in ischemic brain injury
which is known to involve both apoptosis and necro-
sis.6,7 We have found that treatment with Nec-1 reduced
the volume of the infarct and ameliorated the neurolog-
ical deficits in mouse 2 h middle cerebral artery occlu-
sion model. This study points toward the important
contribution of necroptosis to ischemic tissue injury
Apoptosis has been extensively characterized over the
past decade.1 However, there is an increasing awareness
that apoptosis is not the only regulated cell death mech-
anism. For example, although stimulation of the Fas/
TNFR death receptor (DR) family triggers a canonical
‘extrinsic’ apoptosis pathway, it was demonstrated that
in the absence of intracellular apoptotic signaling, Fas/
TNFR is capable of activating a common non-apoptotic
O
OMe
O
N
N
S
N
H
N
H
Keywords: Necroptosis; SAR; Inhibitor; Nec-5; Caspase-independent
cell death; Fused pyrimidinon-thiophene derivatives; Ischemic brain
injury.
S
N
SCH2CN
Nec-1
EC50=0.49 M
Nec-5
EC50 = 0.24 M
*
Corresponding
yuancy@mail.sioc.ac.cn
These two authors contributed equally to this work.
author.
Fax:
+86
21
54925379; e-mail:
Figure 1. Structure and activity of necrostatins: Nec-1 and Nec-5.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.056

1456
K. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1455–1465
and suggests that small molecule necrostatins may repre-
sent a novel class of therapeutically relevant molecules.
not been reported. Our synthetic protocol is as follows
(Scheme 1): on reacting 2-amino-3-carbethoxythiophene
(1)11,12 with p-methoxyphenyl isothiocyanate, a thiourea
analog was generated. Cyclization of the latter in ethano-
lic HCl provided 2-mercapto-3-p-methoxyphenyl-5,6-
tetramethylenothieno[2,3-d]pyrimidin-4-one (2),13 which
gave Nec-5 in 92% yield on reaction with BrCH2CN in
the presence of potassium hydroxide.14
To further define necroptosis pathway, we have screened
a chemical library of ꢀ100,000 compounds for chemical
inhibitors of the necrotic death of human monocytic
U937 cells induced by TNFa and zVAD-fmk,6 which
was used as an operational definition of necroptosis.
This screen resulted in the selection of several necropto-
sis inhibitors, including Necrostatin-1 (Nec-1), which
efficiently blocked necroptotic death.6 Here we describe
another novel necrostatin, Nec-5 (Fig. 1). Although
Nec-5 was selected in a screen in the presence of
zVAD-fmk, similarly to that of Nec-1,6 its action is
not dependent upon pharmacological inhibition of casp-
ases. Consistent with the direct activation of necroptosis
when induction of apoptosis is abolished by genetic
inactivation of apoptotic machinery,7–9 Nec-5 prevents
the death of TNFa treated FADD-deficient Jurkat cells,
which are unable to activate caspases in response to DR
signaling,10 even in the absence of zVAD-fmk. Because
the induction of necroptosis in FADD-deficient Jukart
cells does not rely on the presence of other chemicals,
for example, zVAD-fmk, we used this system to deter-
mine that the effective concentration for half-maximum
response (EC50) for Nec-5 was 0.24 lM, which exceeds
the activity of Nec-1 molecule.
Influence of substituents on Nec-5 activity. In order to
investigate the structure–activity relationship, our strat-
egy for the structure modification of Nec-5 series was
primarily directed at three parts of the molecule (Fig. 2).
Three types of Nec-5 analogs, representing type 1, type
2, and type 3, were generated by changing R, Xn or
R1, R2, respectively. Since substitution of CH2CN moi-
ety by methyl group on sulfur atom of Nec-5 provided
potent compound, type 2 and type 3 molecules contain-
ing both mercaptoethylcyanide and methylthioether
moieties were generated.
Influence of substituent on the sulfur atom of Nec-5: syn-
thesis of 2-mercapto-3-p-methoxyphenyl-5,6-tetramethy-
len-othieno[2,3-d]pyrimidin-4-ones (3). For the study
of the influence of substituents of sulfur atom of Nec-5
on their bioactivities, a series of compounds 3a–x were
prepared by reaction of 2 with RX in the presence of
potassium hydroxide.
In this communication, we describe the structure–activ-
ity analysis of Nec-5 series.
As shown in Table 1, of all the compounds tested, only
few types of changes retained activity in the necroptosis
assay based on the treatment of FADD-deficient Jurkat
cells with TNFa, which is described above.6 It should be
Chemically, Nec-5 is known as 3-p-methoxyphenyl-5,6-
tetramethylenothieno[2,3-d]pyrimidin-4-one-2-merca-
ptoethylcyanide, however, its method of synthesis has
O
O
O
OEt
OEt
b
a
S
N
C
S
N
OMe
S
NH2
H
H
1
OMe
OMe
O
O
N
d
c
N
N
S
N
S
SH
SCH2CN
Nec-5
2
Scheme 1. Reagents and conditions: (a) cyanoacetate, S8, Et2NH, EtOH, reflux 12 h, yield 72%; (b) p-methoxyphenyl isothiocyanate, EtOH, reflux
5–6 h, yield 85%; (c) ethanolic HCl, reflux 12–24 h yield 78%; (d) KOH in 70% EtOH then BrCH2CN, 1–2 h, yield 92%.
various aryl units
OMe
Type 2
O
N
N
various substituents
Type 3
S
S CH2CN
various substituents
Type 1
Figure 2.

K. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1455–1465
Table 1. Structure and activity of compounds 3
1457
OMe
O
N
S
N
SR
3a-x
Yielda (%)
EC50 (lM)
Max protc (%)
b
Entry
Compound
R
1
2
Nec-5
3a
3b
3c
3d
3e
3f
CH2CN
Me
92
87
78
54
87
77
84
88
80
92
88
92
85
80
79
67
91
87
92
65
45
36
76
68
65
—
87
0.24
0.24
100
71
—
—
—
—
—
—
80.7
—
—
—
—
—
—
—
—
—
—
70
85
—
—
—
—
—
—
3
Et
n-Pr
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
6.08
4
5
n-Bu
n-Pent
n-Hex
6
7
8
3g
3h
3i
CH2CH@CH2
CH2C„CH
CH2C6H5
CH2(C6H4Me-4)
CH2(C6H4OMe-4)
CH2 (C6H4NO2-4)
CH2COMe
CH2COOMe
CH2CONH2
COMe
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
5.28
3j
3k
3l
3m
3n
3o
3p
3q
3r
COC3H7-n
COC6H5
3s
CH2CH2CN
CH2Cl
CH2NO2
3t
2.22
3u
3v
3w
3x
3y
3z
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
CH2C(O)NH(C6H4CF3-2)
CH2CH(OH)CH3
CH2COOH
Me (sulfoxide)
Me (sulfone)
a Yield% denotes percentage yield in the final reaction of synthesis.
b EC50 is the effective concentration for half-maximum response.
c Max protection represents max viability obtained in the presence of a compound.
noted that while some of the compounds afforded com-
plete 100% protection from necroptosis restoring viabil-
ity to control, a number of modifications resulted in not
only change in EC50 values, but also in decrease in the
degree of protection as determined by non-linear regres-
sion analysis of the viability data using GraphPad Prizm
scientific statistical software package.
ide (3y) or sulfone (3z)15 leads to a complete loss of
activity.
Influence of N-substituents of pyrimidinone part of Nec-5.
For the study of the influence of the aryl substituents,
3-aryl-5,6-tetramethylen-othieno[2,3-d]pyrimidin-4-one-
2-mercaptoethylcyanide (4) series in which Xn was
introduced into benzene ring were prepared. Since intro-
duction of methylmercapto moiety resulted in substan-
tial activity, the synthesis of 2-methylthio-3-aryl-5,6-
tetramethylenothieno[2,3-d]pyrimidin-4-one derivatives
(5) (Fig. 3) was also pursued.
Experimental data in Table 1 showed that substitution
of ethylcyanide moiety by methyl group in Nec-5 (3a,
entry 2 in Table 1) reserved its activity to a significant
extent. On the other hand, further extension of carbon
chain on sulfur 3b–3f resulted in the loss of activity.
Compound 3s in which CH2CN was replaced by
CH2CH2CN retained some activity. Compounds 3h
and 3t are still somewhat, albeit less, active, while intro-
duction of electron withdrawing group (EWG), for
example, 3i, 3n, and 3u, destroyed the activity of the
molecule completely. Overall, these data suggest that
this position of Nec-5 affords some limited flexibility,
that is, ethylcyanide side chain or S-methyl moiety,
and the presence of thioether bond is greatly preferred.
Oxidation of methylthio group to corresponding sulfox-
Synthesis of compounds 4 and 5. To prepare 4, compound
1 was reacted with aryl isothiocyanate. Resulting thiourea
analog was cyclized smoothly in ethanolic HCl to form
2-mercapto-3-aryl-5,6-tetramethylenothieno[2,3-d]pyrimi-
din-4-one.13 The later was reacted with BrCH2CN in the
presence of potassium hydroxide to give 4.
As shown in Table 2, 4a with unsubstituted phenyl ring
was inactive, while introduction of 4-methyl group to
the benzene ring (4g) or replacement of methoxyl with

1458
K. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1455–1465
tive. These data confirm that ethylcyanide moiety is
greatly preferred over methylthio group in fused pyrim-
idone ring.
O
O
N
Xn
Xn
N
N
S
N
5
S CH3
S
S
CH2CN
Influence of substituents on thiophene ring of Nec-5. For
the study of the influence of substituents on thiophene
ring of Nec-5, 3-p-methoxyphenyl-5,6-disubstituted
thieno[2,3-d]pyrimidin-4-one-2-mercaptoethylcyanide (6)
analogs in which cyclohexyl ring fused to thiophene
molecule was replaced by R1 and R2 were preapred.
Synthesis of 2-methylthio-3-p-methoxyphenyl-5,6-disub-
stituted thieno [2,3-d]pyrimidin-4-one (7) derivatives was
also pursued.
4
Figure 3.
ethoxy moiety (4d) retained some activity. Further in-
crease in Xn size, that is, X = 4-OBn in 4e, eliminated
the activity, indicating important role of X = 4-OMe in
Nec-5 binding to the target and showing that increasing
the carbon number of R in OR is not favorable to activ-
ity, likely due to steric hindrance. Interestingly, X = 4-
OCF3, inactivated the molecule (4t), while X = 4-F,
4h, retained significant activity. Meanwhile, the activity
of 4j was significantly decreased when 4-F was replaced
by 4-Cl and 4-Br derivative (4k) was completely inactive.
Compound 4q (X = 3, 4-O2(CH2)) showed good activi-
ty, indicating highly restricted nature of the target’s
binding pocket interacting with this part of Nec-5 mol-
ecule with high preference toward methoxy group.
Synthesis of compounds 6 and 7. Variation of R1 and R2
led to derivatives with different aliphatic ring. Com-
pounds of 6 series were synthesized starting from corre-
sponding 2-amino-3-carbethoxythiophenes,11,12 which
were reacted with p-methoxy-phenylisothiocyanate, to
obtain thiourea analog and then cyclized smoothly in
ethanolic solution saturated with dried hydrogen chlo-
ride to form 2-mercapto[2,3-d]pyrimidin-4-one.13 The
later gave target molecule 6 on reacting with BrCH2CN
in the presence of potassium hydroxide (Scheme 2).
Synthesis of compound 5. A series of 2-methylthio-3-aryl-
5,6-tetramethylenothieno [2,3-d]pyrimidin-4-one (5)
analogs was also prepared by reacting 4 with MeI in
the presence of potassium hydroxide.
As shown in Table 4, 6a, 6b, 6c, and 6d which contain
hydrogen in the R1 and/or R2 position of 5,6-thiophene
ring were completely inactive. When R1, R2 are both
methyl groups (6e), high degree of activity is retained.
Limited extension of R1 preserved activity to a
significant extent (6j), while extending R2 position was
significantly more detrimental: 6f (R2 = Et) displayed
As shown in Table 3, while 5b and 5g showed some
activity, albeit significantly lower than corresponding 4
analogs (4h and 4p), all of other derivatives were inac-
Table 2. Structure and activity of compounds 4
O
N
Xn
N
SCH2CN
S
4
Entry
Compound
Xn
Yield (%)
EC50 (lM)
Max prot (%)
1
2
3
4a
4b
4c
Nec-5
4d
4e
4f
H
87
58
87
92
85
83
87
80
81
85
90
87
91
76
74
47
52
79
55
74
81
Inactive
17.8
1.46
—
51.2
87.7
100
83.8
—
2-OMe
3-OMe
4-OMe
4-OEt
0.24
0.55
4
5
4-OBn
2-Me
4-Me
Inactive
Inactive
1.80
6
—
7
4g
4h
4i
48.6
85.1
66.9
55.0
—
8
4-F
3-Cl
0.24
5.10
9
10
11
12
13
14
15
16
17
18
19
20
4j
4-Cl
4-Br
8.50
4k
4l
Inactive
16.7
5.70
3,4-Me2
3,4-Cl2
3,4-F2
45.0
39.0
—
4m
4n
4o
4p
4q
4r
Inactive
Inactive
5.70
2,4-(OMe)2
3,4-(OMe)2
3,4-O2(CH2)
4-SMe
2-Me, 4-Cl
4-OCF3
—
57.0
100
70.0
—
0.89
2.33
4s
Inactive
Inactive
4t
—

K. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1455–1465
Table 3. Structure and activity of compounds 5
1459
O
Xn
N
S
N
SCH3
5
Entry
Compound
Xn
Yield (%)
EC50 (lM)
Max prot (%)
1
2
3a
5a
5b
5c
5d
5e
5f
4-OCH3
4-OBn
4-F
87
75
64
77
82
71
58
69
75
67
71
84
0.24
Inactive
1.57
71
—
3
61.2
—
4
4-Br
3,4-Me2
3,4-Cl2
Inactive
16.7
5
38.0
—
6
Inactive
Inactive
16.7
7
2,4-(OMe)2
3,4-(OMe)2
3,4-O2(CH2)
3-SMe
—
8
5g
5h
5i
54.5
—
9
Inactive
Inactive
Inactive
Inactive
10
11
12
—
—
5j
5k
2-Me, 4-Cl
4-OCF3
—
O
O
R1
R2
R1
R2
OEt
a
OEt
b
S
N
C
S
N
OMe
S
NH2
O
H
H
OMe
OMe
O
N
R1
R2
R1
R2
N
c
N
S
N
SH
S
SCH2CN
6
Scheme 2. Reagents and conditions: (a) p-methoxyphenyl isothiocyanate, EtOH, reflux; (b) ethanolic HCl, reflux; (c) KOH in 70% EtOH then
BrCH2CN rt, 1–2 h.
Table 4. Structure and activity of compounds 6
Entry
Compound
R1
R2
Yield (%)
EC50 (lM)
Max prot (%)
1
2
6a
6b
6c
H
H
80
65
70
88
91
89
72
75
71
77
81
92
65
72
44
37
54
Inactive
Inactive
Inactive
Inactive
0.45
—
—
H
Me
Et
3
H
—
—
4
6d
6e
Me
Me
Me
Me
Me
Me
Et
H
5
Me
Et
100
86.8
—
6
6f
5.26
7
6g
6h
6i
n-Pr
i-Pr
Inactive
Inactive
Inactive
1.08
8
—
—
9
C14H29
Me
10
11
6j
100
100
100
83
6k
Nec-5
6l
–(CH2)3–
–(CH2)4–
–(CH2)5–
0.45
0.24
12
13
14
15
16
0.96
Inactive
0.18
6m
6n
6o
6p
CH2CH2CHCH3CH2
–CH@CHCH@CH–
CH2CH2NEtCH2
—
83.3
—
Inactive
Inactive
CH2CH2N(i-Pr)CH2
—
EC50 of 5.26 lM and 86.8% protection and compounds
6g and 6h were inactive. Thus, experimental data
demonstrate that while R1 and R2 contribute to
compound activity, extension of hydrocarbon chain
beyond methyl at the position 6 and, to a lesser
extent, at position 5 is detrimental for activity. Changing

1460
K. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1455–1465
the size of aliphatic ring of compound Nec-5 was
also investigated. 6k with five-membered ring retained
most of the activity, while 6l (seven-membered ring) was
less active and compounds 6m and 6o were essentially
inactive. These data indicated that increased size of the
aliphatic ring was inactivating, consistent with the
side-chain extension data, from these series. Interestingly,
substitution of phenyl ring for the cyclohexane ring (6n)
retained most of the compound activity.
As shown in Table 5, overall activities of these series
paralleled those of 6 series, yet were generally lower,
consistent with previously generated data for other types
of modifications.
Influence of substituents on sulfur and thiophene
ring of Nec-5: synthesis of compound 8. Since 6k
showed activity, synthesis of compound 8 analogs
was carried out to determine if varying thiophene
ring will translate into different SAR for the sul-
fur moiety. Reacting 2-mercapto-3-p-methoxyphenyl-
5,6-trimethylenothieno[2,3-d]pyrimidin-4-one with RX
in the presence of potassium hydroxide led to the
formation of the corresponding compounds of 8
series.
Synthesis of compound 7. A series of 2-methylthio-3-p-
methoxyphenyl-5,6-disubstituted thieno[2,3-d] pyrimi-
din-4-ones were also prepared by the procedure shown
in Scheme 2 using MeI instead of BrCH2CN as S-alkyl-
ation reagent.
Table 5. Structure and activity of compounds 7
OMe
O
R1
N
R2
S
N
SCH3
7
Entry
Compound
R1
R2
Yield (%)
EC50 (lM)
Max prot (%)
1
2
7a
7b
7c
7d
7e
7f
H
H
H
86
88
92
77
90
92
88
81
92
78
74
69
71
66
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
0.24
—
—
—
—
—
—
—
—
97.0
—
—
77
—
—
Me
Me
Et
4
Me
Me
Et
5
6
Me
Me
Me
Me
7
n-Pr
i-Pr
8
7g
7h
7i
9
C14H29
10
11
12
13
14
15
–(CH2)3–
–(CH2)5–
7j
Inactive
Inactive
0.24
7k
7l
CH2CH2CHCH3CH2
–CH@CHCH@CH–
CH2CH2NEtCH2
7m
7n
Inactive
Inactive
CH2CH2N(i-Pr)CH2
Table 6. Structure and activity of compounds 8
OMe
O
N
N
S
SR
8
Entry
Compound
R
Yield (%)
EC50 (lM)
Max prot (%)
1
2
8a
8b
8c
8d
8e
8f
Et
n-Pr
88
87
91
78
76
85
84
76
65
71
77
Inactive
Inactive
Inactive
Inactive
Inactive
2.49
—
—
3
n-But
n-Pent
—
—
4
5
CH2CH@CH2
CH2C„CH
CH2C6H5
CH2(C6H4NO2-4)
CH2COMe
CH2NO2
CH2CH2OH
—
6
63.4
—
7
8g
8h
8i
Inactive
Inactive
Inactive
Inactive
7.14
8
—
—
9
10
11
8j
8k
—
100

K. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1455–1465
Table 7. Structure and activity of compounds 9
1461
OMe
O
R1
R2
N
S
N
SR
9
Entry
Compound
R
R1
R2
Yield (%)
EC50 (lM)
Max prot (%)
1
2
9a
9b
9c
9d
9e
9f
CH2C„CH
CH2C„CH
CH2C„CH
CH2C„CH
CH2C„CH
CH2C„CH
Et
H
H
85
77
78
79
81
76
93
90
90
49
55
80
64
88
51
Inactive
Inactive
4.86
—
—
90.3
—
—
—
—
—
—
—
78
—
—
—
—
Me
Me
Me
Me
H
3
Me
Et
4
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
7.26
5
n-Pr
6
–(CH2)5–
7
9g
9h
9i
Me
Me
Me
Et
8
Et
Et
9
–(CH2)5–
10
11
12
13
14
15
9j
CH2CH2CN
CH2CH2OH
CH2CH2OH
CH2CH2OH
Et
Me
Me
Me
Me
Me
Me
Me
Me
Et
9k
9l
Inactive
Inactive
Inactive
Inactive
9m
9n
9o
n-Pr
n-Pr
Me
CH2C(O)OH
As shown in Table 6, introduction of the five-membered
ring generally maintained previously described SAR.
Compounds 8a, 8b, 8c, 8d (R = Et, Pr, Bu, Pent) with
the extension of carbon chain were inactive. Introduc-
tion of EWG, that is, R = Bz, CH2COOMe, completely
eliminated activity. It is interesting to note that coordi-
nated changes in R groups on thiophene ring resulted
in surprising preservation of activity, that is, five-mem-
bered ring and methyl group combined together (7i) dis-
played higher activity than each change separately 3a
and 6k, which may indicate somewhat different topology
of the different Nec-5 analogs in the active center
depending on the combination of substituents in differ-
ent parts of the molecule.
din-4-ones was carried out according to Scheme 2,
except RX in the presence of potassium hydroxide were
used as alkylating agents.
As shown in Table 7, compounds 9c and 9k possess
some activity, but are significantly less potent than com-
pound Nec-5. Notably, compound 9g was inactive, sug-
gesting that reduced size of the thiophene ring did not
translate into higher degree of flexibility in the tolerated
size of the sulfur substituent.
Influence of substituents on thiophene ring and N-pyrimid-
inone part. Influence of the substituents of Nec-5 was
studied by changing thiophene ring and pyrimidinone
part together. Since compounds 4h, 4q, 6n, and 6e
showed substantial activity, synthesis of their derivatives
was pursued.
Synthesis of compound 9. Preparation of 2-mercapto-3-p-
methoxyphenyl-5,6-disubstituted thieno[2,3-d]pyrimi-
Table 8. Structure and activity of compounds 10 and 11
F
O
N
R1
N
R2
S
SCH2CN / CH3
10 / 11
Entry
Compound
R1
R2
Yield (%)
EC50 (lM)
Max prot (%)
1
2
3
4
5
6
7
8
10a
10b
10c
10d
11a
11b
11c
11d
Me
Me
Me
Et
86
84
89
88
91
90
81
74
4.43
Inactive
0.89
87.6
—
–(CH2)3–
–(CH2)5–
88.7
—
Inactive
2.60
Me
Me
Me
Et
69.2
—
Inactive
3.00
Inactive
–(CH2)3–
–(CH2)5–
95
—

1462
K. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1455–1465
Table 9. Structure and activity of compounds 12, 13, and 14
O
O
O
O
O
O
R1
R1
R2
N
N
R2
S
N
SCH2CN / CH3
S
N
SCH2CN
12 / 13
14
Entry
Compound
R1
R2
Yield (%)
EC50 (lM)
Max prot (%)
1
2
12a
12b
12c
13a
13b
13c
14a
14b
14c
14d
Me
Et
Me
Me
91
89
86
92
93
90
91
90
85
93
1.65
1.90
100
100
100
67.0
—
3
–(CH2)3–
1.18
1.11
4
Me
Et
Me
Me
5
Inactive
Inactive
0.25
6
–(CH2)3–
–(CH2)3–
–(CH2)4–
—
7
100
100
100
100
8
0.22
0.15
9
10
–CH@CH–CH@CH–
Me
Me
0.25
Synthesis of compounds 10 and 11. The 3-p-fluorophenyl-
5,6-disubstituted thieno[2,3-d]pyrimidin-4-one-2-merca-
ptoethylcyanide 10 and corresponding methylthioether
11 were generated through reacting mercapto derivatives
with BrCH2CN or MeI, respectively, in the presence of
potassium hydroxide.
was carried out. Synthesis of 3-(30,40)-methylene-dioxy-
phenyl-5,6-disubstituted thieno[2,3-d]pyrimidin-4-one-
2-mercaptoethylcyanide (12) and corresponding methyl-
thio ether (13) was performed through S-alkylation with
BrCH2CN or MeI in the presence of potassium hydrox-
ide, respectively. And synthesis of 3-(30,40)-ethylene-
dioxyphenyl-5,6-disubstituted thieno[2,3-d]pyrimidin-4-
one-20-mereaptoethylcyanide (14) was performed by
the usual procedure of S-alkylation of corresponding
mercapto compounds.
As shown in Table 8, the introduction of fluorine atom
at the phenyl ring, where seven-membered ring-contain-
ing molecules completely lacked activity (10d and 11d),
in contrast to methoxy analog (6l). The latter result is
reminiscent of the lack of activity displayed by com-
pound 7j. These results point that all three major moie-
ties, targeted by our analysis (Fig. 2), make important
contributions to binding, and multiple unfavorable
changes result in synergistic loss of activity indicative
of the inability of the resulting molecules to properly
occupy the binding pocket.
Experimental data in Table 9 showed that with
exception of inactive 13b and 13c, all of the compounds
investigated either with methylene or with ethylene
dioxy moiety attached to phenyl ring on pyrimidone
nitrogen atom inhibited enhanced activity, particularly
for compounds of 14 series. In the latter case, consistent
with our previous conclusion the Xn moiety afforded
significant flexibility to the structure of the thiophene
ring.
Synthesis of compounds 12, 13, and 14. Since 3-(30,40)-
methylene-dioxyphenyl-5,6-tetramethylenothieno [2,3-d]
pyrimidin-4-one-2-mercaptoethylcyanide (4q), in which
dioxalane ring is attached to the phenyl moiety, showed
significant activity, synthesis of its derivatives (13 and 14)
Synthesis of compounds 15 and 16. Synthesis of 15 and 16
analogs was performed in order to study the influence of
substituents of benzene ring on activity (Table 10).
Table 10. Structure and activity of compounds 15 and 16
O
N
R1
N
R2
S
SCH2CN / CH3
15 / 16
R2
Me
Entry
Compound
R1
Yield (%)
EC50 (lM)
Max prot (%)
1
2
3
4
5
6
15a
15b
15c
16a
16b
16c
Me
87
81
87
78
85
88
2.79
16.7
90.7
38.0
—
CH2CH2CH(CH3)CH2
–(CH2)3–
Inactive
Inactive
Inactive
Inactive
Me
Me
—
—
CH2CH2CH(CH3)CH2
–(CH2)3–
—

K. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1455–1465
Table 11. Structure and activity of compounds 17 and 18
1463
O
Xn
SCH2CN / CH3
Yield (%)
N
S
N
17 / 18
Entry
Compound
Xn
EC50 (lM)
Max prot (%)
1
2
17a
17b
17c
17d
17e
17f
18a
18b
18c
18d
18e
H
4-F
79
77
82
82
83
67
82
78
81
88
76
NDa
3.06
NDa
—
3
4-OEt
3,4-O2(CH2)
4-OCF3
4-NMe2
H
Inactive
0.27
—
4
100
—
5
Inactive
Inactive
NDa
6
—
7
NDa
—
8
4-F
3.06
9
4-OEt
3,4-O2(CH2)
4-OCF3
Inactive
Inactive
Inactive
—
—
10
11
—
a Not determined.
Interestingly, compound 15a showed higher activity
than 4l, proving a first example of coordinated changes
in the left and right part of the molecules displaying
compensatory, rather than synergistic effect. It is possi-
ble that 3,4-Me-substituted molecule may assume alter-
native binding position in the presence of the smaller R1/
R2 substituents, resulting in retention of the activity.
However, this effect is limited to a particular combina-
tion of R1/R2 as 15b and 15c are essentially inactive (Ta-
ble 11).
Compound 17d is a potent inhibitor along with 6n and
7l consistent with previously defined SAR for other
types of thiophene ring substituents. Therefore, substitu-
tion of phenyl for the cyclohexane ring does not appear
to significantly change Nec-5 activity.
Synthesis of compounds 19 and 20. Since methyl groups
in R1 and R2 positions showed significant activity (com-
pound 6e), analogs with additional phenyl ring substitu-
tions (Xn, compounds 19 and 20) were prepared by
reacting corresponding mercapto derivative with
BrCH2CN or MeI, respectively, in the presence of potas-
sium hydroxide.
Synthesis of compounds 17 and 18. Since compound 6n
showed good activity, synthesis of its analogs with var-
ious phenyl ring substituents (17 and 18) was carried
out, by reacting 2-amino-benzo[b]thiophene-3-carboxyl-
ic acid ethyl ester and arylisothiocyanate with NaOH in
DMF to generate corresponding mercapto compound
and, subsequently, S-alkylation with BrCH2CN or
MeI, respectively, in the presence of potassium hydrox-
ide to generate target molecules.
Analysis of analogs of 19 and 20 as well as previously
described derivatives with R1 = Me, R2 = Me (Table
12) suggests that such modifications are not favorable
for activity, with all the analogs studied being generally
less active than the corresponding cyclohexane moiety
(R1, R2 = –(CH2)4–) analogs of Nec-5. Furthermore,
Table 12. Structure and activity of compounds 19 and 20
O
Xn
N
S
N
SCH2CN / CH3
19 / 20
Entry
Compound
Xn
Yield (%)
EC50 (lM)
Max prot (%)
1
2
3
4
5
6
7
8
19a
19b
19c
19d
20a
20b
20c
20d
4-OEt
87
79
81
83
90
84
87
84
7.77
Inactive
3.70
97
—
63
—
—
—
—
—
4-OBn
4-OCF3
4-NMe2
4-OEt
Inactive
Inactive
Inactive
Inactive
Inactive
4-OBn
4-OCF3
4-NMe2

1464
K. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1455–1465
Table 13. Structure and activity of compounds 21
O
Xn
R1
R2
N
S
N
SCH2CN
21
Entry
Compound
R1
H
R2
Xn
Yield (%)
EC50 (lM)
Max prot (%)
1
2
3
4
5
6
7
8
21a
21b
21c
21d
21e
21f
21g
21h
H
H
H
85
90
67
87
78
88
85
71
Inactive
Inactive
Inactive
Inactive
Inactive
3.70
—
—
—
—
—
70
—
—
–(CH2)3–
–(CH2)3–
–(CH2)5–
4-OEt
H
CH2CH2CH(CH3)CH2
–(CH2)3–
–(CH2)5–
3,4-(OMe)2
4-OCF3
4-OCF3
Inactive
Inactive
–(CH2)3–
3,4-(OMe)2
unlike results obtained with 3-substituents (series 14/15),
again, synergistic loss of activity was observed for unfa-
vorable changes in thiophene ring and in 4-position, for
example, compounds 4d and 6e showed significantly
higher activity compared to 19a.
As shown in Table 13, combining changes to thiophene
and phenyl rings was detrimental to activity, as only
compound 21f showed some, yet greatly reduced,
activity.
Influence of substituents of sulfur and N-pyrimidinone of
Nec-5. For the study of the influence of substituents
on sulfur and N-pyrimidinone of Nec-5, 2-mercapto-3-
aryl-5,6-tetramethylenothieno[2,3-d]pyrimidin-4-ones (22)
which combined unsubstituted phenyl ring and various
substituents on sulfur were prepared.
Synthesis of compound 21. For the study of the influence
of the combination of the substituents on thiophene ring
and N-pyrimidinone of Nec-5, derivatives of 3-aryl-5,
6-disubstituted thieno[2,3-d]pyrimidin-4-one-2- merca-
ptoethylcyanide (21) were prepared.
As shown in Table 14, all the derivatives are inactive, con-
sistent with the requirement for Xn = 40-OMe and prefer-
ence for 2-mercaptoethylcyanide moiety as R group.
Table 14. Structure and activity of compounds 22
O
Xn
N
Influence of substituents of sulfur, N-pyrimidinone as well as
thiophene ring. For the study of the influence of simulta-
neous substitution on sulfur in thiophene ring and N-pyri-
midinone of Nec-5, 2-mercapto-3-aryl-5,6-disubstituted
thieno[2,3-d] pyrimidin-4-ones (23) were synthesized.
S
N
SR
22
Entry
Compound
R
Xn
Yield (%)
1
2
3
22a
22b
22c
CH2CH@CH2
CH2Ph
CH2C6H4NO2
H
H
H
58
87
83
As shown in Table 15, consistent with the preference for
R1, R2 = –(CH2)4–, R = CH2CN and Xn = 40-OMe, all
the analogs of 23 were completely inactive.
Table 15. Structure and activity of compounds 23
O
N
Xn
R1
N
R2
S
SR
23
Entry
Compound
R
R1
H
R2
H
Xn
Yield (%)
1
2
23a
23b
23c
23d
23e
23f
23g
23h
23i
Me
Me
H
90
88
89
69
79
84
85
79
88
81
CH2CH2CH(CH3)CH2
–(CH2)5–
3,4-(OMe)2
H
3
Et
4
CH2C„CH
CH2C„CH
Me
Me
Et
Me
Me
3,4-O2(CH2)
3,4-O2(CH2)
4-OEt
5
6
–(CH2)3–
–(CH2)3–
7
Me
3,4-O2(CH2)
3,4-O2(CH2)
H
8
CH2CH2OH
Me
Me
Et
Me
9
10
CH2CH2NEtCH2
–(CH2)3–
23j
4-OCF3

K. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1455–1465
1465
6. (a) Degterev, A.; Huang, Z.; Boyce, M.; Li, Y.; Jagtap, P.;
Mizushima, N.; Cuny, G. D.; Moskowitz, M.; Yuan, J.
Nat. Chem. Biol. 2005, 2, 112; (b) Teng, X.; Degterev, A.;
Jagtap, P.; Xing, X.; Choi, S.; Denu, R.; Yuan, J.; Cuny,
G. D. Bioorg. Med. Chem. Lett. 2005, 15, 5039.
7. (a) Lo, E. H.; Dalkara, T.; Moskowitz, M. A. Nat. Rev.
Neurosci. 2003, 224, 29; (b) Gwag, B. J.; Lobner, D.; Koh,
J. Y.; Wie, M. B.; Chio, D. W. Neuroscience 1995, 68, 615;
(c) Rosenbaum, D. M.; Gupta, G.; D’Amore, J.; Singh,
M.; Weidenheim, K.; Zhang, H.; Kessler, J. A.
J. Neurosci. Res. 2000, 61, 686; (d) Martin-Villalba, A.;
Herr, I.; Jeremias, J.; Hahne, M.; Brandt, R.; Vogel, J.;
Schenkel, J.; Herdegen, T.; Debatin, K. M. J. Neurosci.
1999, 19, 3809; (e) Martin-Villalba, A.; Hahne, M.;
Kleber, S.; Vogel, J.; Falk, W.; Schenkel, J.; Krammer,
P. H. Cell Death Differ. 2001, 8, 679.
Our preliminary SAR study demonstrated that the EC50
value for inhibition of necroptosis in FADD-deficient
Jurkat T cells treated with TNFa of Nec-5 is closely
related to the chemical structure of the molecule. First
of all, the presence of thioethylcyanide moiety on the
a-position of fused pyrimidone-4 part is essential, substi-
tution of this moiety results in complete loss of activity.
The exceptions are corresponding methylthio ethers as
3a and 7i exhibit same EC50 value as Nec-5, although
it provides significantly lower, max protection value of
71% and 97%. Meanwhile, oxidation of the sulfur atom,
either to sulfoxide 3y or to sulfone 3z completely elimi-
nates the activity. Second, presence of –OMe group in
the para-position of the benzene ring located on pyrim-
idone nitrogen is also important. Compound with para-
bromophenyl group exhibited (4k) loss of the activity
since variation of the electronic effect of the aryl substit-
uents including modification of –OMe group position
gave only less even inactive compound. Compound with
para-fluorophenyl group (4h) displayed significant EC50
value and a slightly decreased max protection of 85.1%,
while larger halides were not tolerated. Furthermore,
ethylene dioxy group is preferable to methoxy with 14c
showing almost twofold increase in activity. Finally,
cyclopentyl (6k), cycloheptyl (6l), and even benzene ring
(6n) exhibit certain degree of activity. It is worthy to
point out that introduction of two methyl groups to
the a and b position of thiophene ring will bring com-
pound with significant activities. Our results suggest that
while Nec-5 displays a stringent SAR, there are also
positions in the molecule especially phenyl ring attached
to the N-pyrimidone, which can be potentially further
optimized to generate more active Nec-5 analogs.
8. Li, M.; Beg, A. A. J. Virol. 2000, 74, 7470.
9. (a) Lin, Y.; Choksi, S.; Shen, H. M.; Yang, Q. F.; Hur, G.
M.; Kim, Y. S.; Tran, J. H.; Nedospasov, S. A.; Liu, Z. G.
J. Biol. Chem. 2004, 279, 10822; (b) Wilson, C. A.;
Browning, J. L. Cell Death Differ. 2002, 9, 1321.
10. Chan, F. K. J. Biol. Chem. 2003, 278, 51613.
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99, 94; (b) Tranberg, C.; Zickgraf, A.; Giunta, B. N.;
Luetjens, H.; Figler, H.; Murphree, L. J.; Falke, R.;
Fleischer, H.; Linden, J.; Scammells, P. J.; Olsson, R. A.
J. Med. Chem. 2002, 45, 382; (c) Gutschow, M.; Kuerschner,
L.; Neumann, U.; Pietsch, M.; Loser, R.; Koglin, N.;
Eger, K. J. Med. Chem. 1999, 42, 5437; (d) Gewald, K.;
Neumann, G. Chem. Ber. 1968, 101, 1933.
12. (a) Sabnis, R. W. Sulfur Rep. 1994, 16, 1; (a) Sabnis, R.
W.; Rangnekar, D. W. J. Heterocycl. Chem. 1999, 36, 333.
13. (a) Vishnu, J. R.; Hrishi, K. P.; Arnold, J. V.
J. Heterocycl. Chem. 1981, 18, 1277; (b) Devani, M. B.;
Shishoo, C. J.; Pathak, U. S.; Parikh, S. H.; Saha, G. F.;
Padhya, A. C. J. Pharm. Sci. 1976, 65, 660; (c) Leistner, S.;
Gutschow, M.; Wagner, G. Synthesis 1987, 466; (d)
Modica, M.; Santagati, M.; Santagati, A.; Russo, F.;
Cagnotto, A.; Goegan, M.; Mennini, T. Bioorg. Med.
Chem. Lett. 2000, 10, 1089; (e) Duval, E.; Case, A.; Stein,
R. L.; Cuny, G. D. Bioorg. Med. Chem. Lett. 2005, 15,
1885.
Acknowledgments
This Project was supported in part by the Chinese Acad-
emy of Sciences, National Institute of General Medical
Sciences (USA), and National Institute of Neurological
Disorders and Stroke (USA) (to J.Y.) and the National
Natural Science Foundation of China (Nos. 20272075,
20372076 to C.Y.)
14. Nec-5: mp 212–214 °C. 1H NMR(CDCl3) (d): 1.82–1.90
(m, 4H, CH2), 2.77–2.97 (m, 4H, CH2), 3.89 (s, 5H,
OCH3 + CH2CN), 7.18 (d, J = 8.7 Hz, 2H, OCH3–PhH),
7.36 (d, J = 8.7 Hz, 2H, OCH3–PhH). IR (KBr, cmꢁ1):
2939, 2848, 2249(C„N), 1630(s, C@O), 1444, 1321, 1262,
872. MS m/z (rel intensity): 383 (M+) (31.21), 311 (27.60),
146(base). Anal. Calcd for C19H17N3O2S2: C, 59.51; H,
4.47; N, 10.96. Found: C, 59.13; H, 4.48; N, 10.80.
15. Oxidation of compound 3 was carried out as follows: a
mixture of 3a (1 mmol) and m-chloroperoxybenzoic acid
(m-CPBA) (2.2 mmol) in CH2Cl2 (20 ml) was stirred for
48 h. The reaction was completed as monitored by TLC.
The product was separated by silica gel column chroma-
tography using chloroform as eluent. The product 3z was
crystallized from chloroform–ethanol as colorless crystal,
yield 87%, mp 230 °C. 1H NMR(d): 1.86–1.92 (m, 4H,
CH2), 2.47 (s, S(O)2CH3), 2.73–2.78 (m, 2H, CH2), 2.91–
2.97 (m, 2H, CH2), 3.87 (s, 3H, OCH3), 7.02 (d,
J = 9.0 Hz, 2H, ArH), 7.19 (d, J = 9.0 Hz, 2H, ArH).
IR(KBr)cmax: 3199, 2938, 2837, 1712, 1655 (s, C@O),
1510, 1246, 829. MS m/z (rel intensity): 390 (M+), 359, 311
(base), 199, 159. Anal. Calcd for C18H18N2O4S2: C, 55.37;
H, 4.65; N, 7.17. Found: C, 55.98; H, 4.79; N, 6.84.
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