Structure-activity relationship study of novel necroptosis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.07.077

Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling necrosis. It can be induced in a FADD-deficient variant of human Jurkat T cells treated with TNF-α. 5-(1H-Indol-3-ylmethyl)-2-thiohydantoins and 5-(1H-indol-3-ylmethyl)hydantoins were found to be potent necroptosis inhibitors (called necrostatins). A SAR study revealed that several positions of the indole were intolerant of substitution, while small substituents at the 7-position resulted in increased inhibitory activity. The hydantoin ring was also quite sensitive to structural modifications. A representative member of this compound class demonstrated moderate pharmacokinetic characteristics and readily entered the central nervous system upon intravenous administration.

Full text of DOI:10.1016/j.bmcl.2005.07.077

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5039–5044
Structure–activity relationship study of novel necroptosis inhibitors
Xin Teng,a Alexei Degterev,b Prakash Jagtap,a Xuechao Xing,a Sungwoon Choi,a
a
Regine Denu, Junying Yuan and Gregory D. Cuny
b
a,
*
´
aLaboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair,
Brigham & Womenꢀs Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA
bDepartment of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
Received 12 July 2005; accepted 29 July 2005
Available online 8 September 2005
Abstract—Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling
necrosis. It can be induced in a FADD-deficient variant of human Jurkat T cells treated with TNF-a. 5-(1H-Indol-3-ylmethyl)-2-
thiohydantoins and 5-(1H-indol-3-ylmethyl)hydantoins were found to be potent necroptosis inhibitors (called necrostatins). A
SAR study revealed that several positions of the indole were intolerant of substitution, while small substituents at the 7-position
resulted in increased inhibitory activity. The hydantoin ring was also quite sensitive to structural modifications. A representative
member of this compound class demonstrated moderate pharmacokinetic characteristics and readily entered the central nervous
system upon intravenous administration.
Ó 2005 Elsevier Ltd. All rights reserved.
Cells die in several morphologically distinct ways.1 One
of these processes, called apoptosis, is characterized by a
number of conserved and highly regulated steps includ-
ing concomitant nucleus and cytoplasm condensation,
DNA degradation, membrane blebbing, and caspase-
mediated cleavage of various cellular proteins. Apopto-
sis culminates in the formation of apoptotic bodies that
are phagocytosed by adjacent cells including macro-
phages in the periphery and microglial cells in the cen-
tral nervous system (CNS). This efficient process
prevents the accumulation of extracellular debris and
consequently does not lead to an inflammatory
response. Apoptosis is a genetically regulated process
that is necessary both during development and for main-
taining an organismꢀs homeostasis.2 However, under
certain pathological conditions this process, which
would normally be suppressed, is activated, leading to
cell death and dysfunction. Many key cellular targets
in this biochemical cascade have been identified and
some of them serve as potential targets for therapeutic
intervention, including caspases.3
A second morphologically distinct way that cells die,
called necrosis, is characterized by cell membrane and
organelle disruption, cell swelling, mitochondria impair-
ment, followed by cell lyses, which is accompanied by a
host inflammatory response.4 Unlike apoptosis, the
underlying biochemical events in necrosis are not well
understood. However, necrosis is known to play a
prominent role in many pathological conditions,5a
including ischemia (i.e., stroke5b and myocardial infarc-
tion5c), trauma, and possibly some forms of neurodegen-
eration.5d Recent studies also suggest that apoptosis
inhibition in many cases does not completely block cell
death, but rather results in a change from an apoptotic
to regulated caspase-independent cell death mechanisms
with morphological features resembling necrosis.6 One
of these mechanisms, called necroptosis, has recently
been described.7 Identifying and preparing low molecu-
lar weight molecules capable of inhibiting necroptosis
can assist in elucidating its role in the patho-physiology
of diseases and can provide lead compounds (i.e.,
necrostatins) for therapeutic development.
O
Me
N
S
N
H
Keywords: Necrosis; Necroptosis; Caspase-independent cell death;
Stroke; SAR; Ischemic brain injury; Indoles; Hydantoin; Central
nervous system.
N
H
*
Corresponding author. Tel.: +1 617 768 8640; fax: + 1 617 768
8606; e-mail: gcuny@rics.bwh.harvard.edu
1
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.077

5040
X. Teng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5039–5044
Recently, the 5-(1H-indol-3-ylmethyl)-2-thiohydantoin
1, termed necrostatin-1 (or Nec-1), was reported to
inhibit necroptosis induced by tumor necrosis factor
alpha (TNF-a).7 Furthermore, derivatives of this com-
pound demonstrated in vivo efficacy following intracere-
bralventricular (icv) administration in the transient
middle cerebral artery occlusion (MCAO) model of ische-
mic brain injury. Herein, we report a structure–activity
relationship (SAR) study of 5-(1H-indol-3-yl-methyl)-2-
thiohydantoin and 5-(1H-indol-3-ylmethyl)hydantoin
necrostatins. In addition, we report physical–chemical
data, and in vivo pharmacokinetic and brain penetration
evaluations of a represented member of this compound
class.
EtO2C
NMe2
CO2Et
b
a
NHCHO
N
H
N
H
N
R
Cl
Cl
Cl
4
5
6 R=H
7 R=Me
c
CO2R1
NH2
O
Me
N
f
d
S
N
H
N
R
N
R
Cl
Cl
8 R=R1=H; 9 R=Me, R1=H
10 R=H, R1=Me; 11 R=R1=Me
12 R=H
13 R=Me
e
The 5-(1H-indol-3-ylmethyl)-2-thiohydantoins were pre-
pared from tryptophans or tryptophan methyl esters,
according to Scheme 1. For example, tryptophan, 2,
was treated with methyl isothiocyanate in the presence
of aqueous pyridine and sodium hydroxide to give the
desired product 1 in low yield.8 However, the yield
was greatly enhanced by treating tryptophan methyl
ester, 3, with methyl isothiocyanate in the presence of
triethylamine in dichloromethane (DCM).
Scheme 2. Reagents and conditions: (a) 37% aq CH2O, Me2NH,
AcOH, rt, 16 h (86%); (b) (EtO2C)2CHNHCHO, NaOH, toluene, D,
3 d (65%); (c) MeI, KOH, DMSO, 0 °C, 4 h (77%); (d) aq NaOH,
THF, rt, 24 h, then AcOH, D, 24 h, then 3 N HCl, D, 24 h, then pH
adjusted to 6.0 with 2 N KOH (63%); (e) SOCl2, MeOH, D, 2 h (100%);
(f) MeNCS, Et3N, DCM, rt, 3 h (98%).
CHO
a
R1
R1
b
R1
Several tryptophan methyl esters were prepared from in-
doles, according to the method described by Rapoport
and co-workers.9 For example, 7-chloroindole, 4, was
treated with aqueous formaldehyde and dimethylamine
to give 7-chlorogramine, 5, (Scheme 2). The amine deriv-
ative was converted to 6 and then allowed to react with
diethyl 2-formylaminomalonate in the presence of sodi-
um hydroxide. Subsequently, the indole nitrogen was
alkylated upon treatment with methyl iodide to give 7.
Next, the 7-chlorotryptophans 8 and 9 were generated
from 6 and 7, respectively, by a sequence of reactions
involving base hydrolysis, acid-mediated de-carboxyla-
tion, followed by acid-mediated N-de-formylation. The
resulting amino acids were converted to methyl esters
10 and 11, with thionyl chloride in methanol. These
materials were cyclized to 1210 and 13, respectively, in
the presence of methyl isothiocyanate and triethylamine.
N
H
NO2
N
H
14
15
16
R2
O
R2
O
O
N
N
c
d
O
N
H
N
H
R1
R1
N
H
N
H
17
18
Scheme 3. Reagents and conditions: (a) CH2@CHMgBr, THF,
À40 °C, 20 min (40–65%); (b) POCl3, DMF, 0 °C to rt, 2 h (40–
98%); (c) piperidine, 110 °C, 4 h; (d) CoCl2 (2 equiv), NaBH4
(20 equiv), MeOH/THF (1:1), rt, 24 h (25–50% for two steps).
the presence of vinyl magnesium bromide.11 7-Ben-
zyloxyindole was prepared by a sequence of reactions
described by Harada et al.12 This material was converted
to 7-n-butoxyindole via removal of the benzyl group by
hydrogenation (10% Pd/C, EtOH, rt, 6 h) and subse-
quent alkylation (n-BuI, K2CO3, methyl ethyl ketone,
55 °C, 12 h). 1-Methyl-7-chloroindole was prepared by
alkylating 7-chloroindole (NaH, DMF, 0 °C to rt, then
MeI, 24 h). The 3-alkylhydantoins (Me, Et, n-Bu, and
i-Pr) were prepared utilizing published methods.13 3,5-
Dimethylhydantoin was prepared from 3-methylhydan-
toin by alkylation (MeI, MeOH, 10 N NaOH, reflux,
4 h). The alkenes 17 were then reduced with excess sodi-
um borohydride in the presence of cobalt dichloride to
give the desired products 18.
Many of the 5-(1H-indol-3-ylmethyl)hydantoins were
prepared according to Scheme 3. Indole-3-carboxalde-
hydes, 16, were condensed with 3-alkylhydantoins in
piperidine at 110 °C to give 17. Noncommercial
indole-3-carboxaldehydes were prepared from indoles
utilizing the Vilsmeier–Haack reaction. Noncommercial
indoles were prepared from nitrobenzene derivatives in
O
Me
CO2R
N
a, if R = H
or
NH2
S
N
H
b, if R = Me
N
H
N
H
The enantiomers 25 and 26 were prepared, according to
the procedure outlined in Scheme 4. Initially, 7-chloro-
indole, 4, was converted to the N-acetyltryptophan
derivative 19 upon treatment with serine in AcOH and
Ac2O. Kinetic resolution of this compound with D-ami-
noacylase in the presence of cobalt dichloride gave the
2 R=H
3 R=Me
1
Scheme 1. Reagents and conditions: (a) MeNCS, 50% aqueous
pyridine, 0.5 N NaOH, rt, 1 h, then concd HCl (pH 1), rt, 2 d (8%);
(b) MeNCS, Et3N, DCM, rt, 1 h (89%).

X. Teng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5039–5044
5041
CO2H
NHR
of sodium hydride to give 29. Next, the nitrile was re-
duced to an aldehyde with diisobutylaluminum hydride
(DIBAL), followed by acidification. The resulting alde-
hyde was converted to hydantoin 30 with ammonium
carbonate and potassium cyanide. Finally, regioselective
alkylation of 30 with dimethylacetamide–dimethylacetal
(DMA–DMA) gave 31.14 Derivative 36, which extends
the linker between the indole and hydantoin ring, was
prepared, according to Scheme 6. The a-keto ester
3217 was reduced with lithium aluminum hydride
(LAH). The resulting alcohol was converted to its
corresponding iodide and then displaced with cyanide
to give 33. The nitrile was reduced to aldehyde 34. This
aldehyde was cyclized to hydantoin 35 and then
alkylated regioselectively to give 36.
CO2H
NHAc
a
b
4
N
H
N
H
Cl
Cl
20 (R); R = H
21 (S); R = Ac
22 (S); R = H
19
c
O
CO2Me
NH2HCl
NHMe
NH2
f
e
d
20
N
H
N
H
Cl
Cl
24
23
O
O
Me
O
Several other compounds were prepared that altered the
hydantoin portion of the molecule. 7-Chloroindole, 4,
was converted to the a-hydroxy acid 38 in low yield
(Scheme 7).18 This material was transformed to its cor-
responding N-methylamide utilizing HBTU19 coupling,
followed by cyclization in the presence of CDI to give
39. Tryptophan derivative 8 was converted to 40 by first
protecting the amine with a Boc group and then convert-
ing the carboxylate to a Weinreb amide. The amide 40
was reduced with LAH to aldehyde 41. Following
reductive amination with methylamine, removal of the
carbamate-protecting group with HCl in methanol and
then cyclization in the presence of CDI 43 was obtained.
Me
N
N
d, e, f
O
N
H
N
H
22
N
H
N
H
Cl
Cl
26
25
Scheme 4. Reagents and conditions: (a) Ac2O, AcOH, 75 °C, 2 h
(40%); (b) D-aminoacylase, phosphate buffer (pH 7.8), CoCl2, 37 °C,
48 h (100%); (c) 3 N HCl, D, 6 h (100%); (d) SOCl2, MeOH, 0 °C to rt
(100%); (e) MeNH2, MeOH, 24 h (96%); (f) triphosgene, Et3N, DCM,
0 °C, 2 h, (34%).
N-Acetyltryptophan derivative 19 was also converted
to 45, as shown in Scheme 8. Initially, the acid was
reduced to alcohol 44 with BH3ÆTHF. Then, the acetyl
group was removed by hydrolysis. Finally, cyclization
with CDI provided 45.
(R)-tryptophan derivative 20 and (S)-N-acetyl trypto-
phan derivative 21, which were readily separated.14
The amino acid 20 was then converted to its correspond-
ing methyl ester with thionyl chloride in methanol. The
ester 23 upon treatment with methyl amine in methanol
provided amide 24. Finally, cyclization of the amide
with triphosgene in the presence Et3N in DCM gave
25.15 The (S)-N-acetyl tryptophan derivative 21
was hydrolyzed in 3 N HCl to give 22. This material
was converted to 26 in a similar manner as the R-
enantiomer.15
Evaluation of necroptosis inhibitory activities was
performed using a FADD-deficient variant of human
Jurkat T cells treated with TNF-a, as previously
described.7 Under these conditions, the cells efficiently
underwent necroptosis, which was completely and
selectively inhibited by 1. For EC50 value determina-
tions, cells were treated with 10 ng/mL of human TNFa
in the presence of increasing concentrations of test
compounds for 24 h followed by an ATP-based viability
assessment.20
Derivative 31, which altered the linker between the
indole and hydantoin rings, was prepared according to
Scheme 5.16 Nitrile 27 was first converted to N-Boc
derivative 28. The cyclobutane ring was installed by
treating 28 with 1,3-dibromopropane in the presence
R
O
N
O
R
O
CO2Et
NH
O
R
e
N
a - c
CN
CN
O
b
c,d
N
H
N
H
N
H
Cl
N
H
N
H
N
R
Cl
N
H
32
Cl
30 R = H
31 R = Me
35 R = H
36 R = Me
33 R = CN
34 R = CHO
27 R = H
28 R = Boc
29
f
a
e
d
Scheme 5. Reagents and conditions: (a) Boc2O, DMAP, CH2Cl2, rt
(76%); (b) NaH, Br(CH2)3Br, DMSO, 0 °C to rt, 16 h (75%); (c)
DIBAL, toluene, À50 °C then Et2O–NH4Cl (1:1) then 2 N H2SO4
(68%); (d) (NH4)2CO3, KCN, EtOH, H2O, 80 °C (63%); (e) DMA–
DMA, toluene/DMF (5:1), 110 °C, 2 h (98%).
Scheme 6. Reagents and conditions: (a) LiAlH4, THF, D, 18 h (75%);
(b) PPh3, imidazole, I2, DCM, rt, 1.5 h (85%); (c) KCN, DMF, 110 °C
4 h (95%); (d) DIBAL, toluene, À70 °C to rt, 5 h (51%); (e)
(NH4)2CO3, KCN, EtOH, H2O, 60 °C, 20 h (78%); (f) DMA–DMA,
toluene, D, 2 h (65%).

5042
X. Teng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5039–5044
O
was observed at the highest compound concentrations
employed. In general, the SAR of the hydantoins was
similar to that of the thiohydantoins. Introduction of
small electron-withdrawing (e.g., 66) or electron-donat-
ing (e.g., 59) substituents to the indole 7-position
increased necroptosis inhibitory activity. Furthermore,
substitution at the 2, 4, 5, or 6-positions of the indole
was detrimental. Interestingly, the 5,7-dichloro deriva-
tive 65 was also inactive. Removing the substituent on
the imide nitrogen of the hydantoin (54)21 was also
detrimental to activity.
Me
O
CO2H
OH
N
O
a
b, c
O
CO2H
N
H
N
H
Cl
Cl
37
38
39
O
NH(Me)OMe
CHO
NHBoc
NHBoc
d, e
f
8
N
N
Next, the SAR of the hydantoin ring was examined in
more detail (Table 2). In this SAR study, the 7-chloroin-
dole was held constant. First, the (R)-enantiomer (25)
was found to be ꢀ4-fold more active than the (S)-enan-
tiomer (26). In addition, small alkyl substituents (e.g., 66
and 68) were best on the imide nitrogen compared to
larger groups (e.g., 69 and 70). Substitution of the amide
nitrogen (71) also resulted in loss of activity. However,
replacement of the hydantoin ring with an N-methyl
oxazolidine-2,4-dione (39) only resulted in a slight
decrease in activity. Removal of a carbonyl group to
give either a cyclic urea (43) or a cyclic carbamate (45)
resulted in complete loss of activity.
H
H
Cl
Cl
40
41
Me
N
NHMe
NHBoc
N
H
g
O
h, i
N
H
N
H
Cl
Cl
42
43
Scheme 7. Reagents and conditions: (a) 4, SnCl4, CCl4, 0 °C, 1.5 h
(7%); (b) HBTU, MeNH2 in THF, CH2Cl2, i-Pr2EtN, rt, 12 h (25%);
(c) CDI, THF, rt, 36 h (38%); (d) Boc2O, satd aq NaHCO3, EtOAc/
THF (10:3), rt, 12 h (80%); (e) HBTU, NH(Me)OMeHCl, CH2Cl2,
i-Pr2EtN, rt, 1 h (78%); (f) LiAlH4, THF, À50 °C, 1 h; (g) MeNH2 in
THF, MgSO4, rt, 3 h then NaBH4 (10 equiv), 0 °C, 2 h (40% for two
steps); (h) satd HCl in MeOH, rt, 2 h; (i) CDI, Et3N, 0 °C to rt, 24 h
(45% for two steps).
The bridge between the indole and the hydantoin was
briefly evaluated. Increasing the steric bulk of the meth-
Table 1. EC50 determinations for inhibition of necroptosis in FADD-
deficient Jurkat T cells treated with TNF-a
OH
NHAc
O
R2
O
O
N
H
N
a
b, c
19
4
7
X
N
H
N
H
N
H
R1
2
Cl
Cl
Y
44
45
Scheme 8. Reagents and conditions: (a) BH3ÆTHF, À5 °C to rt, 4 h
(74%); (b) 3 N HCl, D, 4 h; (c) CDI, Et3N, À5 °C to rt, 24 h (42% over
two steps).
Compound
R1
R2
X
Y
EC50 (lM)
1
46
47
48
12
13
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
H
Me
Me
Me
H
S
NH
NMe
S
0.49
>10
4.8
H
S
H
S
Nec-1 (1) had an EC50 value of 0.49 lM (Table 1). Sub-
stitution of the indole nitrogen (46 and 13), introduction
of a chlorine substituent to the indole 6-position (49),
replacement of the indole with a benzothiophene (47),
or removal of the imide methyl on the thiohydantoin
(48)21 was detrimental to necroptosis inhibitory activity.
However, introduction of an electron-withdrawing (e.g.,
51, 12, 50) or an electron-donating substituent (e.g., 52)
to the indole 7-position increased the activity.
H
S
NH
NH
NMe
NH
NH
NH
NH
NH
NH
S
>10
0.18
>10
>10
0.16
0.26
0.63
1.3
7-Cl
7-Cl
6-Cl
Me
Me
Me
Me
Me
Me
Me
H
S
S
S
7-Br
7-F
S
S
7-OMe
H
S
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
>10
5.6
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
2-Me
4-Cl
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NMe
>10
9.2
Although
5-(1H-indol-3-ylmethyl)-2-thiohydantoins
were capable of protecting cells from necroptosis, cyto-
toxicity was observed at higher concentrations
(ꢀ100 lM). We attribute this toxicity to the thiohydan-
toin portion of the molecules and hypothesize that
cytotoxicity is mechanistically distinct from the anti-nec-
roptosis activity.22 An approach to reduce the cytotoxic-
ity of the compounds without compromising on the
desired activity would be to replace the thiohydantoin
with a hydantoin. Although such a change resulted in
a 3-fold decrease in activity (1 vs 53), no cytotoxicity
5-OMe
7-OMe
7-OBn
7-O-n-Bu
7-Me
6-Cl
>10
0.71
>10
0.27
0.33
5.7
7-F
0.59
>10
0.21
3.8
5,7-di-Cl
7-Cl
7-Cl

X. Teng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5039–5044
5043
Table 2. EC50 determinations for inhibition of necroptosis in FADD-
deficient Jurkat T cells treated with TNF-a
In conclusion, 5-(1H-indol-3-ylmethyl)-2-thiohydanto-
ins and 5-(1H-indol-3-ylmethyl)hydantoins were found
to be potent necrostatins. A SAR study revealed that
several positions of the indole were intolerant of substi-
tution, while smaller, substituents (i.e., OMe or Cl) at
the 7-position resulted in increased activity. The hydan-
toin ring was also quite sensitive to structural modifica-
tions. A representative member (66) of this compound
class demonstrated moderate pharmacokinetic charac-
teristics and readily entered the CNS upon intravenous
administration. Studies are currently underway to eval-
uate further these compounds in other in vivo animal
models of disease where necroptosis is likely to play a
substantial role. Furthermore, these compounds can
serve as molecular tools to interrogate further the
mechanism of necroptotic cell death.
Z
Y
O
X
N
H
Cl
Compound (R or S)
X
Y
Z
EC50 (lM)
26
25
68
69
70
71
39
43
45
S
NH
NH
NH
NH
NH
NMe
NMe
NEt
C@O 0.23
C@O 0.05
C@O 0.18
R
R
R/S
R/S
R/S
R/S
R/S
R/S
N-n-Bu C@O 0.85
N-i-Pr
C@O >10
C@O >10
C@O 0.38
NMe NMe
O
NMe
NMe
O
NH
NH
CH2
CH2
>10
>10
Acknowledgments
X.T., X.X., S.C., P.J., R.D., and G.D.C. thank the
Harvard Center for Neurodegeneration 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. We
thank Dr. Michael Furlong (Absorption Systems) for
insightful discussions about the pharmacokinetic
studies. We thank Amano Enzyme Inc. for a supply
of D-aminoacylase.
ylene bridge (31) and extending the linker between the
indole and the hydantoin (36) were both found to be
detrimental for necroptosis inhibitory activity
(EC50 > 10 lM).
A representative member of the 5-(1H-indol-3-ylmethyl)
hydantoins (66)23 was characterized further. This
compound exhibited 78.7% protein binding in human
plasma, had a LogD7.4 of 1.90, and solubility in phos-
phate buffer (pH 7.4) of 130 lg/mL. The pharmacoki-
netics of 66, following intravenous administration to
24
mice, is shown in Table 3. Gender-specific differences
References and notes
were noted with more rapid clearance from females.
This difference may be due to gender-specific differences
in compound metabolism.25 The brain concentration
and brain- to -plasma ratio of 66 were also determined
in male mice following intravenous administration.
These results are shown in Table 4.24 Compound 66 par-
titions into the brain resulting in a brain- to -plasma ra-
tio of 2.4 to 1. However, the compound was cleared
from both the brain and plasma within 180 min post-
injection.
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Table 3. Pharmacokinetic data for 66 following intravenous admin-
istration to mice
Gender AUC8h
Cmax
T1/2
Vss
CLs
(min ng/mL) (ng/L) (min) (mL/kg) (mL/min/kg)
Male
Female
16461
9921
873
573
67
<60
2495
3345
61
100
Table 4. Brain concentration and brain to plasma ratio of 66 following
intravenous administration to male mice
Post-injection
time
(min)
Brain
concentration
(lM)
Plasma
concentration
(lM)
Brain to
plasma
ratio
7. 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.
30
180
0.74
0
0.31
0.003
2.4
—

5044
X. Teng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5039–5044
8. Ramachandran, L. K.; McConnell, W. B. J. Am. Chem.
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Sadowski, S.; Ball, R. G.; Hoogsteen, K. J. Med. Chem.
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9. Hurt, C. R.; Lin, R.; Rapoport, H. J. Org. Chem. 1999, 64,
225.
19. Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D.
Tetrahedron Lett. 1989, 30, 1927.
20. Compound concentrations were 0.03–100 lM, except
for compounds 25 and 26 where the lowest concentra-
tion was 2 nM. Cell viability assessments were per-
10. Compound 12: mp 249–253 °C; 1H NMR (500 MHz,
DMSO-d6–CDCl3) d 3.02 (s, 3H), 3.18–3.22 (dd, 1H,
J1 = 14.5 Hz,
J2 = 5.5 Hz);
3.30–3.34
(dd,
1H,
J1 = 14.5 Hz, J2 = 5.5 Hz); 4.35 (dd, 1H, J1 = 5.5 Hz,
J2 = 4.5 Hz); 7.00 (t, 1H, J = 7.5 Hz); 7.13 (d, 1H,
J = 7.5 Hz); 7.19 (d, 1H, J = 2.0 Hz); 7.52 (d, 1H,
J = 7.5 Hz); 9.92 (s, 1H), 10.45 (s, 1H); 13C NMR,
(100 MHz, DMSO-d6) d 25.92, 26.61, 59.45, 108.84,
115.64, 117.56, 119.45, 120.46, 125.44, 129.19, 132.59,
174.43, 182.83; Elemental analysis calculated for
C13H12ClN3OS: C, 53.15; H, 4.12; N, 14.30. Found: C,
53.16; H, 4.21; N, 14.01.
formed using a commercial luminescent ATP-based
assay kit (CellTiter-Glo, Promega, Madison, WI). Cell
viability values were adjusted to account for nonspecific
toxicity, which in most cases were <10%. EC50 values
were calculated using, nonlinear regression analysis of
sigmoid dose–response (variable slope) curves from
plots of logarithm of inhibitor concentrations versus
viability values.
21. Compound 48 was prepared by treating 3 first with Me3Si-
NCS (10 equiv) and i-Pr2EtN (10 equiv) in CH2Cl2 at
room temperature for 24 h and then heating at 110 °C in
acetic acid for 5 h. Compound 54 was prepared in an
analogous manner using Me3Si-NCO.
11. Bartoli, G.; Palmieri, G.; Bosco, M.; Dalpozzo, R.
Tetrahedron Lett. 1989, 30, 2129.
12. Harada, H.; Fujii, A.; Kato, S. Synth. Commun. 2003, 33,
507.
13. For the methyl derivative, see: (a) Janin, Y. L.; Huel, C.;
Flad, G.; Thirot, S. Eur. J. Org. Chem. 2002, 1, 1763; For
the ethyl and n-butyl derivatives see: (b) Harries, C.;
Weiss, M. Liebigs Ann. Chem. 1903, 327, 355; For the
i-propyl derivative see: (c) Park, K.-H.; Kurth, M. J. J.
Org. Chem. 2000, 65, 3520.
14. Konda-Yamada, Y.; Okada, C.; Yoshida, K.; Umeda, Y.;
Arima, S.; Sato, N.; Kai, T.; Takayanagi, H.; Harigaya, Y.
Tetrahedron 2002, 58, 7851.
15. The enantiomeric purity of 25 and 26 was determined to
be >98% by 1H NMR using a 0.02 M solution in CDCl3 in
the presence of 0.02 M europium tris-[3-(trifluoromethyl-
hydroxymethylene)-(+)-camphorate].
16. Horwell, D. C.; McKiernan, M. J.; Osborne, S. Tetrahe-
dron Lett. 1998, 39, 8729.
22. (a) Suzen, S.; Buyukbingol, E. Farmaco 1998, 53, 525; (b)
Durant, G. J.; Emmett, J. C.; Ganellin, C. R.; Miles, P. D.;
Parsons, M. E.; Prain, H. D.; White, G. R. J. Med. Chem.
1977, 20, 901.
23. Compound 66: mp 173–175 °C; 1H NMR (500 MHz,
CDCl3) d 8.43 (br s, 1H), 7.50 (d, 1H, J = 8.0 Hz), 7.22 (d,
1H, J = 7.5 Hz), 7.13 (d, 1H, J = 2.0 Hz), 7.06 (t, 1H,
J = 7.8 Hz), 5.69 (br s, 1H), 4.27 (ddd, 1H, J1 = 1.0 Hz,
J2 = 3.5 Hz, J3 = 8.8 Hz), 3.43 (dd, 1H, J1 = 3.5 Hz,
J2 = 14.5 Hz), 3.01 (dd, 1H, J1 = 9.3 Hz, J2 = 14.8 Hz),
2.95 (s, 3H); 13C NMR, (100 MHz, CDCl3) d 173.4,
157.2, 133.6, 128.4, 123.6, 122.0, 120.8, 117.2, 116.9,
111.0, 58.0, 28.1, 24.5; Elemental analysis calculated for
C13H21ClN3O2: C, 56.22; H, 4.36; N, 15.13. Found: C,
56.19; H, 4.41; N, 15.10.
17. Faul, M. M.; Engler, T. A.; Sullivan, K. A.; Grutsch, J. L.;
Clayton, M. T.; Martinelli, M. J.; Pawlak, J. M.;
LeTourneau, M.; Coffey, D. S.; Pedersen, S. W.; Kolis,
S. P.; Furness, K.; Malhotra, S.; Al-awar, R. S.; Ray, J. E.
J. Org. Chem. 2004, 69, 2967.
24. N = 3 per gender for pharmacokinetic study; N = 3 per
time point for brain concentration and brain- to -plasma
ratio study; mice: CD-1; dose: 1 mg/kg; dosing vehicle:
10% propylene glycol in normal saline; dosing concentra-
tion: 0.16 mg/mL.
18. Lewis, R. T.; Macleod, A. M.; Merchant, K. J.; Kelleher,
F.; Sanderson, I.; Herbert, R. H.; Cascieri, M. A.;
25. Mugford, C. A.; Kedderis, G. L. Drug Metab. Rev. 1998,
30, 441.