Structure-activity relationship study of [1,2,3]thiadiazole necroptosis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.10.024

Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling non-regulated necrosis. This form of cell death can be induced in an array of cell types in apoptotic deficient conditions with death receptor family ligands. A series of [1,2,3]thiadiazole benzylamides was found to be potent necroptosis inhibitors (called necrostatins). A structure-activity relationship study revealed that small cyclic alkyl groups (i.e. cyclopropyl) and 2,6-dihalobenzylamides at the 4- and 5-positions of the [1,2,3]thiadiazole, respectively, were optimal. In addition, when a small alkyl group (i.e. methyl) was present on the benzylic position all the necroptosis inhibitory activity resided with the (S)-enantiomer. Finally, replacement of the [1,2,3]thiadiazole with a variety of thiophene derivatives was tolerated, although some erosion of potency was observed.

Full text of DOI:10.1016/j.bmcl.2007.10.024

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6836–6840
Structure–activity relationship study of [1,2,3]thiadiazole
necroptosis inhibitors
Xin Teng,a Heather Keys,b Arumugasamy Jeevanandam,c John A. Porco, Jr.,c
Alexei Degterev,b Junying Yuand and Gregory D. Cunya,*
aLaboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair, Brigham &
Women’s Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA
bDepartment of Biochemistry, Tufts University Medical School, 136 Harrison Avenue, Stearns 703, Boston, MA 02111, USA
cDepartment of Chemistry, Boston University and Center for Chemical Methodology and Library Development (CMLD-BU),
Boston, MA 02215, USA
dDepartment of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
Received 14 September 2007; revised 1 October 2007; accepted 5 October 2007
Available online 17 October 2007
Abstract—Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling
non-regulated necrosis. This form of cell death can be induced in an array of cell types in apoptotic deficient conditions with death
receptor family ligands. A series of [1,2,3]thiadiazole benzylamides was found to be potent necroptosis inhibitors (called necrosta-
tins). A structure–activity relationship study revealed that small cyclic alkyl groups (i.e. cyclopropyl) and 2,6-dihalobenzylamides at
the 4- and 5-positions of the [1,2,3]thiadiazole, respectively, were optimal. In addition, when a small alkyl group (i.e. methyl) was
present on the benzylic position all the necroptosis inhibitory activity resided with the (S)-enantiomer. Finally, replacement of the
[1,2,3]thiadiazole with a variety of thiophene derivatives was tolerated, although some erosion of potency was observed.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Cell death has traditionally been categorized as either
apoptotic or necrotic based on morphological character-
istics.1 These two modes of cell death were also initially
thought to fundamentally differ in underlying cellular
regulation, with the former representing a regulated cas-
pase-dependent mechanism,2 while the latter resulted
from non-regulated processes. However, more recent
studies demonstrate that the underlying basis of cellular
necrosis is more complex, as it can result in some in-
stances from regulated caspase-independent cellular
signaling.3
epithelial cells, and neurons). Necroptosis may represent
a significant contributor to and in some cases predomi-
nant mode of cellular demise under pathological condi-
tions involving excessive cell stress, rapid energy loss,
and massive oxidative species generation, where the
highly energy-dependent apoptosis process is not opera-
tive. The discovery of necroptosis, therefore, raises the
possibility of novel therapeutic intervention strategies
for the treatment of maladies where necrosis is known
to play a prominent role,5 including organ ischemia
(i.e. stroke6 and myocardial infarction7), trauma, and
possibly some forms of neurodegeneration.8
A regulated caspase-independent cell death pathway
with morphological features resembling necrosis, called
necroptosis, has recently been described.4 This manner
of cell death can be initiated with various stimuli (e.g.
TNF-a and Fas ligand) and in an array of cell types
(e.g. monocytes, fibroblasts, lymphocytes, macrophages,
The identification and optimization of low molecular
weight molecules capable of inhibiting necroptosis will
assist in elucidating its role in disease patho-physiology
and could provide lead compounds (i.e. necrostatins) for
therapeutic development. A series of hydantoin contain-
ing indole derivatives, exemplified by 1, were the first po-
tent in vitro and in vivo necroptosis inhibitors to be
described (Fig. 1).4,9 Since then, a series of tricyclic
derivatives, exemplified by 2,10 and substituted 3H-thie-
no[2,3-d]pyrimidin-4-ones, exemplified by 3,11 have also
been reported. In the course of continued screening for
Keywords: Necroptosis; Cell death; Caspase-independent; Necrosis;
[1,2,3]Thiadiazole.
*
Corresponding author. Tel.: +1 617 768 8640; fax: +1 617 768
8606; e-mail: gcuny@rics.bwh.harvard.edu
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.024

X. Teng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6836–6840
6837
O
Me
O
N
a
b
H
N
N
N
N
OMe
N
N
MeO
O
N
H
OH
CO2Me
CHO
13
S
S
S
N
N
11
O
12
N
H
Me
2
Cl
Cl
1
H
N
N
c
N
Cl
O
N
Me
F
S
H
N
N
O
14
N
N
F
S
S
S
CN
Cl
O
d
e
H
N
N
N
N
N
N
N
3
4
Cl
CO2H
S
F
S
O
17
S
O
Figure 1. Necrostatins.
O
15
16
Scheme 2. Reagents and conditions: (a) NaBH4, MeOH, rt, 12 h; (b)
Dess–Martin reagent, CH2Cl2, rt, 1 h (65% over two steps); (c) 2-Cl-6-
F–PhCH2NH2, anhydrous MgSO4, Et3N, THF, rt, 2 h then
Na(OAc)3BH, ClCH2CH2Cl, rt, 6 h (41%); (d) oxalyl chloride, cat.
DMF, CH2Cl2, 0 ꢁC to rt, 1 h; (e) NaH, 2-Cl-6-F–PhC(@O)NH2,
THF, rt, 1 h (34% over two steps).
additional classes of necroptosis inhibitors, we discov-
ered that the [1,2,3]thiadiazole derivative 4 was a moder-
ately potent inhibitor (EC50 = 1.0 lM).12 Herein, we
report an initial structure–activity relationship (SAR)
study for this class of necroptosis inhibitors.
Many of the [1,2,3]thiadiazole derivatives evaluated
herein were prepared according to the procedure out-
lined in Scheme 1. Meldrum’s acid, 5, was treated with
acyl chlorides in the presence of pyridine to give b-keto-
esters 6.13 The esters were allowed to react with mono-
Boc-hydrazine in the presence of a catalytic amount of
p-toluenesulfonic acid (p-TsOH) to give imines 7.14
Cyclization in the presence of thionyl chloride yielded
the [1,2,3]thiadiazole esters 8. Acid hydrolysis of the es-
ters provided acids 9. These materials were coupled with
various amines utilizing HBTU (Method A), the corre-
sponding acyl chlorides (Method B) or through the use
of EDCI (Method C) to give amides 10.
orobenzylamine in the presence of anhydrous
magnesium sulfate to give an imine, which was subse-
quently used as crude material. The imine was then re-
duced with sodium triacetoxyborohydride to give the
secondary amine 14. The imide derivative 17 was also
prepared starting with acid 15, which was first converted
to the corresponding acid chloride 16. This material was
then allowed to react with the anion of 2-chloro-6-fluo-
robenzamide generated with sodium hydride to give
imide 17 in 34% yield.
The a-substituted ( )-2-chloro-6-fluorobenzylamines
were prepared according to Scheme 3. 2-Chloro-6-flu-
orobenzophenone, 18a, was reduced with borane–THF
complex to give the corresponding secondary alcohol.
The alcohol was converted to the corresponding phthal-
imide via a Mitsunobu reaction followed by treatment
Compound 14 was prepared according to the procedure
outlined in Scheme 2. Ester 11 was reduced with sodium
borohydride to give 12. The alcohol was converted to
the corresponding aldehyde 13 utilizing Dess–Martin re-
agent. The aldehyde was condensed with 2-chloro-6-flu-
F
F
NH2
R2
R1
Cl
O
BocHN
R1
O
O
e
O
O
a
b
Me
Me
N
O
Cl
R1
OMe
OMe
a-c
d
18a R1 = C(O)Me
18b R1 = CN
19a R2 = Me
19b R2 = n-Bu
19c R2 = Ph
O
5
6
7
18c R1 = CN
R1
R1
R2
N
R3
N
N
c
F
F
F
Me
Me
N
Me
CN
Me
N
( )n
CO2R
S
f-h
S
e
NH2
CN
O
8 R = Me
9 R = OH
d
Cl
Cl
10
Cl
20a
20b
21
Scheme 1. Reagents and conditions: (a) RC(O)Cl, py, CH2Cl2, rt, 2 h,
then MeOH, 2 h (75%); (b) H2NNHBoc, cat. TsOH, toluene, 60 ꢁC,
4 h; (c) SOCl2, 60 ꢁC, 1 h (47% over two steps); (d) 6 N HCl, AcOH,
150 ꢁC, 4 h; (e) Method A: H2N(CH2)nR3, HBTU, i-Pr2NEt, CH2Cl2,
rt, 12 h (30–90%); Method B: oxalyl chloride, cat. DMF, CH2Cl2, 0 ꢁC
to rt, 1 h then H2N(CH2)nR3, EtOAc, saturated aqueous NaHCO3, rt,
2 h (20–75%); Method C: H2N(CH2)nR3, EDCI, HOBt, DMF, rt, 12 h
(60–90%).
Scheme 3. Reagents and conditions: (a) 1 M BH3ÆTHF, THF, rt, 2 h; (b)
diethyl azodicarboxylate, PPh3, phthalimide, THF, rt, 18 h (65% over
two steps); (c) H2NNH2ÆH2O, THF/EtOH (6:1), D, 11 h (50%); (d) 1 M
BH3ÆTHF, THF, 0 ꢁC to rt, 1.5 h then n-BuLi or PhLi, À78 ꢁC, 2 h (15%
when R2 = n-Bu, 20% when R2 = Ph); (e) NaO-t-Bu, MeI, NMP, THF,
rt, 48 h (85%); (f) 6 N HCl, 120 ꢁC, 12 h; (g) Boc2O, NaN3, n-Bu4NBr,
80 ꢁC, 24 h; (h) TFA, DCM, rt (58% over three steps).

6838
X. Teng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6836–6840
with hydrazine monohydrate to give 19a.15 Nitriles 18b
and 18c were treated with borane–THF complex fol-
lowed by addition of n-BuLi or PhLi to give amines
19b and 19c, respectively.16 The benzylnitrile 20a was
first dialkylated with methyl iodide to give 20b. This
material was hydrolyzed to the corresponding carbox-
ylic acid and then subjected to a one-pot Curtius rear-
rangement (via an in situ generated acyl azide) to give
a Boc-protected amine that upon deprotection yielded
amine 21.17
activity. Replacing the 2-chloro-6-fluorophenyl with a
1-naphthyl (45), 2-pyridyl (46) or substituted 2-pyridyl
(47) was detrimental to activity. However, addition of
a halogen to the 3-position of the 2,6-difluorophenyl
(49) gave an increase in necroptosis inhibition activity
with an EC50 value of 0.18 lM (Table 1).
Additional changes to the linker between the [1,2,3]thia-
diazole and the 2,6-dihalophenyl were also examined
(Table 2). The corresponding secondary amine (14)
and imide (17) derivatives of 32 were inactive. Also,
the benzylamide was necessary, with the homologous
phenethyl amide (51) and the truncated anilide (52)20
being significantly less active. Introduction of a methyl
group (53) onto the benzylic position gave a slight in-
crease in activity. Quite surprisingly, when the two enan-
tiomers of 53 were examined all of the necroptosis
activity resided in the (S)-enantiomer (55). However,
increasing the steric bulk of the benzylic substituent to
n-Bu (56), phenyl (57) or gem-dimethyl (58) resulted in
loss of activity.
(S)-1-(2-Chloro-6-fluorophenyl)ethylamine was pre-
pared by allowing 22 to react with methyl magnesium
chloride followed by treatment with acetic anhydride
to give a-enamide 23 (Scheme 4). Asymmetric hydroge-
nation in the presence of the chiral catalyst (S,S)-Me–
BPE–Rh gave amide 24.18 Acid hydrolysis of the amide
yielded the optically pure amine 25, isolated as the
hydrochloride salt. Similarly, (R)-25 was made utilizing
(R,R)-Me–BPE–Rh.
Evaluation of necroptosis inhibitory activity was per-
formed using a FADD-deficient variant of human Jur-
Finally, modifications to the [1,2,3]thiadiazole heterocy-
cle were examined (Table 3). Replacement with a variety
of thiazoles (59–61) or an oxazole (62) was detrimental
to activity. Likewise, the pyridazine (63), which at-
tempted to replace the sulfur of the [1,2,3]thiadiazole
with a CH@CH, was also inactive. However, moderate
kat
T cells treated with TNF-a as previously
described.4,10 Utilizing these conditions the cells effi-
ciently underwent necroptosis, which was completely
and selectively inhibited by 1 (EC50 = 0.050 lM). For
EC50 value determinations, cells were treated with
10 ng/mL of human TNF-a in the presence of increasing
concentration of test compounds for 24 h followed by
ATP-based viability assessment.19
Table 1. EC50 determinations of necroptosis inhibition in FADD-
deficient Jurkat T cells treated with TNF-a
R1
R2
The initial SAR revealed that the amide NH was crucial
for activity. For example, simple methylation (26 vs 4
and 50 vs 32) resulted in significant loss of activity.
Introduction of branching into the alkyl group at the
4-position of the [1,2,3]thiadiazole increased activity,
with i-Pr (31), c-Pr (32), and c-Bu (33) being optimal.
However, introduction of a t-Bu (36) or phenyl (37) at
this position resulted in decreased activity. The 2-
chloro-6-fluoro substitution of the phenyl ring also ap-
peared to be necessary for potent activity. For example,
compounds with a 2-methylphenyl (28) or 2-methoxy-
phenyl (29) were less active. In addition, the 2,6-dichloro
(38) or 2,6-difluoro (39) substituted derivatives were also
less active in some cases compared to 2-chloro-6-fluoro
substitution (32). Consistent with these findings, remov-
ing one of the halogens (40) or replacing one of the hal-
ogens with small (41) or large (42) electron-donating
groups also resulted in decreased activity. Replacing
one of the halogens with other electron-withdrawing
groups, such as cyano (43) or CF3 (44), did not restore
R3
N
N
N
S
O
a
Compound
R1
R2
R3
EC50 (lM)
4
Me
Me
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
2-Cl-6-F–Ph
2-Cl-6-F–Ph
2,6-di-F–Ph
2-Me–Ph
1.0
11
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Me
Me
3.5
27
Me
2-OMe–Ph
>100
4.1
n-Pr
i-Pr
2-Cl-6-F–Ph
2-Cl-6-F–Ph
2-Cl-6-F–Ph
2-Cl-6-F–Ph
2-Cl-6-F–Ph
2-Cl-6-F–Ph
2-Cl-6-F–Ph
2-Cl-6-F–Ph
2,6-di-Cl–Ph
2,6-di-F–Ph
2-F–Ph
0.58
0.50
0.60
1.9
c-Pr
c-Bu
c-Pentyl
c-Hex
t-Bu
Ph
6.0
18
>100
6.0
1.5
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
1.5
10
2-Cl-6-Me–Ph
2-Cl-6-(OPh)–Ph
2-Cl-6-CN–Ph
2-F-6-CF3–Ph
1-Naphthyl
2-Py
F
>100
>100
>100
>100
40
F
Me
F
CN
Cl
b
a
NHAc
R
Cl
23
Cl
24 R = NHAc
22
3-F-2-Py
9.6
c
25 R = NH2•HCl
2-Cl-3,6-di-F–Ph
3-Cl-2,6-di-F–Ph
2-Cl-6-F–Ph
0.52
0.18
16
Scheme 4. Reagents and conditions: (a) MeMgCl, THF, rt, 24 h then
Ac2O, 120 ꢁC, 20 min (43%); (b) (S,S)-Me–BPE–Rh (1 mol%), H2
(60 psi), rt, 12 h (90%); (c) 4 N HCl, 120 ꢁC, 6 h (100%).
a Standard deviation <10%.

X. Teng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6836–6840
6839
Table 2. EC50 determinations of necroptosis inhibition in FADD-
deficient Jurkat T cells treated with TNF-a
by TNF-a in mouse fibrosarcoma L929 cells, but was
ineffective against zVAD.fmk-induced necroptosis in
the same cell line.10 Therefore, a similar analysis with
the [1,2,3]thiadiazole series was performed. Compound
55 showed the same activity profile as 2, providing effec-
tive protection of Jurkat or L929 cells from TNF-a-in-
duced necroptosis, while lacking activity in zVAD.fmk
treated L929 cells (Fig. 2). However unlike 2, [1,2,3]thia-
diazole 55 was fully active in SV40-transformed mouse
adult lung fibroblasts stimulated to undergo necroptosis
with a combination of TNF-a and zVAD.fmk, in a sim-
ilar manner to 1. Collectively, these results demonstrate
that the [1,2,3]thiadiazole series possess a distinct mode
of necroptosis inhibition compared to the previously de-
scribed necrostatins. These data further illustrate that
cell-based screening for necrostatins allows for identifi-
cation of both ‘universal’ (i.e. 1) and diverse cell type/
stimulus specific necroptosis inhibitors (i.e. 2 and 55).
It remains to be determined whether cell type specificity
observed in vitro translates into in vivo models of path-
ologic injury. If it does, then cell type/stimulus specific
inhibitors of necroptosis, such as the tricyclic (i.e. 2)
and the [1,2,3]thiadiazole series (i.e. 55), may offer
advantages under conditions where molecule specificity
may be beneficial, such as treating chronic conditions
like neurodegenerative diseases.
F
H
N
N
Y
N
X
R
S
a
Compound
X
Y
R
(R)/(S)
EC50 (lM)
14
17
51
52
53
54
55
56
57
58
CH2
CH2
Cl
Cl
Cl
F
—
—
>100
>100
27
C@O C@O
C@O CH2CH2
C@O
C@O CH(Me)
C@O CH(Me)
C@O CH(Me)
C@O CH(n-Bu)
C@O CH(Ph)
C@O C(Me)2
—
—
—
>100
0.40
Cl
Cl
Cl
Cl
Cl
Cl
(R)/(S)
(R)
(S)
>100
0.28
(R)/(S)
(R)/(S)
—
>100
>100
>100
a Standard deviation <10%.
Table 3. EC50 determinations of necroptosis inhibition in FADD-
deficient Jurkat T cells treated with TNF-a
R1
F
H
N
Z
Y
Cl
R2
X
In conclusion, a series of [1,2,3]thiadiazole benzylamides
was found to inhibit TNF-a-induced necroptosis in
FADD-deficient variant of human Jurkat T cells. A
SAR study revealed that: (i) secondary 2,6-dihalo substi-
tuted benzylamides were required; (ii) when a small alkyl
group (i.e. methyl) was present in the benzylic position
all the necroptosis inhibitory activity resided with the
(S)-enantiomer; (iii) small branched or cyclic alkyl
groups (i.e. i-Pr, c-Pr or c-Bu) were optimal in the 4-po-
sition of the [1,2,3]thiadiazole; (iv) replacement of the
[1,2,3]thiadiazole with a variety of thiophene derivatives
was tolerated, although with some erosion of potency.
In addition, the [1,2,3]thiadiazole series showed a unique
cell type/stimulus necroptosis inhibition profile com-
pared with two previously described classes of inhibi-
tors. Studies are currently underway to evaluate the
O
a
Compound
X
Y
Z
R1
R2 EC50
(lM)
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
S
S
S
O
CH
CMe
N
N
N
N
N
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
20
>100
>100
>100
>100
7.0
C(4-ClPh)
CH
CH@CH
N
CH
S
S
S
S
S
S
S
S
S
S
S
S
S
O
CH
CH
CBr
CH
CH
Me Me 3.9
Me
H
H
H
H
H
H
H
H
H
H
H
H
1.3
1.2
CH
CH
CCN Me
CH c-Pr
CMe Cl
5.1
3.9
CH
CH
CH
CH
CH
CMe
Cl
H
H
H
9.6
3.9
CMe
CH
CH
9.4
3.7
120
100
CH
CH
C(CH2)4
0.48
>100
>100
13
CH
CR3
CH
OEt
Jurkat/TNF
L929/TNF
L929/zVAD
CH
CH
Me
Me
80
60
40
20
0
R3 = SO2-4-Cl–Ph.
a Standard deviation <10%.
Lung
fibroblasts/TNF+zVAD
activity could be obtained with a variety of thiophene
derivatives (64–74), except for the ethoxy derivative 75
and the sulfone derivative 76. In one case (74) the nec-
roptosis activity approached that seen for the most po-
tent [1,2,3]thiadiazoles. However, replacement of the
[1,2,3]thiadiazole with a furan (77) was less effective.
DMSO
1
2
55
Figure 2. Cell type/stimulus specific activities of necrostatins. FADD-
deficient Jurkat, L929 and mouse adult lung fibroblast cells were
treated for 24 h with 10 ng/mL human TNF-a and/or 100 lM
zVAD.fmk as indicated in the presence of 30 lM of necrostatin 1, 2
or 55. Cell viability was determined using an ATP-based assessment
method. Values were normalized to cells treated with necrostatins in
the absence of necroptotic stimulus, which were set as 100% viability.
Error bars reflect standard deviation values (N = 2).
In our previous analyses, we discovered that although 1
showed activity in a broad range of necroptosis cellular
systems, 2 was restricted to specific cell types/stimuli.9,10
For example, 2 efficiently inhibited necroptosis initiated

6840
X. Teng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6836–6840
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pharmacology of these compounds in animal models of
disease where necroptosis is likely to play a substantial
role (i.e. cerebral ischemia, traumatic brain injury, and
liver injury). Additionally, these compounds are being
used to further interrogate the mechanism(s) of necrop-
totic cell death.
Acknowledgments
10. Jagtap, P. G.; Degterev, A.; Choi, S.; Keys, H.; Yuan, J.;
Cuny, G. D. J. Med. Chem. 2007, 50, 1886.
11. Wang, K.; Li, J.; Degterev, A.; Hsu, E.; Yuan, J.; Yuan,
C. Bioorg. Med. Chem. Lett. 2007, 17, 1455.
X.T. and G.D.C. thank the Harvard Center for Neuro-
degeneration and Repair (HCNR) for financial support.
A.D. and J.Y. thank the National Institute on Aging,
National Institute of General Medical Sciences and
American Health Assistance Foundation for financial
support. X.T., G.D.C., and J.Y. thank the National
Institute of Neurological Disorders and Stroke
(NINDS) for financial support. J.A.P. Jr. thanks the
National Institutes of Health and Bristol-Myers Squibb
for financial support. A.D. is a recipient of NIH Men-
tored Scientist Development Award from the National
Institute on Aging (NIA). The SV40-transformed adult
mouse lung fibroblasts were a generous gift of Dr. Philip
Tsichlis (Tufts University).
12. Several other biological activities of 4-alkyl-[1,2,3]thiadi-
azole-5-benzylamides have been reported, including inhi-
bition of I-kappa B kinase complex (IKK), see: Pitts, W.
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19. For EC50 value determinations, FADD-deficient variant
of human Jurkat T cells (5 · 105 cells/mL, 100 lL per well
in a 96-well plate) was treated with 10 ng/mL of human
TNF-a in the presence of increasing concentration 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 prepared and
then diluted with DMSO to give testing solutions, which
were added to each test well. The final DMSO concentra-
tion was 0.5%. Eleven compound test concentrations
(0.030–100 lM) were used. Each concentration was done
in duplicate. Cell viability assessments were performed
using a commercial luminescent ATP-based assay kit
(CellTiter-Glo) according to the manufacturer’s instruc-
tions. Cell lysis/ATP detection reagent (40 lL) was added
to each well. 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). Cell viability was expressed as a ratio of the signal
in the well treated with TNF-a 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.
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20. Compound 52 was prepared in low yield (10%) by
allowing 16 to react with 2,6-difluoroaniline in THF and
pyridine at room temperature. The reaction with 2-chloro-
6-fluoroaniline was unsuccessful presumably due to
increased steric hindrance.