Pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione derivatives as novel small molecule chaperone amplifiers

Article abstract of DOI:10.1016/j.bmcl.2009.05.073

Pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione derivatives were investigated as novel small molecule amplifiers of heat shock factor 1 transcriptional activity. Lead optimization led to the discovery of compound 4A-13, which displayed potent HSF1 activit

Full text of DOI:10.1016/j.bmcl.2009.05.073

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4303–4307
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione derivatives as novel small
molecule chaperone amplifiers
a
a
a
a
a
Yuefen Zhou a, , Linyi Wei , Thomas P. Brady , P. S. Murali Mohan Redddy , Tram Nguyen , Jinhua Chen ,
*
Qingyan Au b, Il Sang Yoon b, Gary Yip b, Bin Zhang b, Jack R. Barber a,b, Shi Chung Ng a,b
a Department of Chemistry, CytRx Corporation, 3030 Bunker Hill Street, Suite 101, San Diego, CA 92109, USA
b Department of Biology, CytRx Corporation, 3030 Bunker Hill Street, Suite 101, San Diego, CA 92109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione derivatives were investigated as novel small molecule
Received 27 April 2009
Revised 15 May 2009
Accepted 20 May 2009
Available online 27 May 2009
amplifiers of heat shock factor 1 transcriptional activity. Lead optimization led to the discovery of com-
pound 4A-13, which displayed potent HSF1 activity under mild heat stress (EC50 = 2.5 lM) and significant
cytoprotection in both rotenone (EC50 = 0.23
lM) and oxygen-glucose deprivation cell toxicity models
(80% protection at 2.5 M).
l
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Small molecule chaperone amplifiers
Protein misfolding
HSF1
Heat shock proteins (HSPs)
Stress granule
Rotenone stress
OGD
Neurodegenerative diseases
Cytoprotection
Pyrimidotriazinedione derivatives
Protein misfolding has recently been implicated as one of the
fundamental mechanisms that link the pathogenesis of disparate
illnesses such as neurodegenerative diseases and prion-related
infectious diseases. Indeed, there is increasing experimental evi-
dence that links protein misfolding to sporadic and familial amyo-
trophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease
(PD), Huntington’s disease and related polyglutamine expansion
diseases as well as Transmissible Spongiform Encephalopathy dis-
eases.1–5 Molecular chaperones, many of which are also commonly
known as heat shock proteins (HSPs), play a major role in protein
folding and maintenance of proteome homeostasis against stress.
This cytoprotective mechanism involves stress-induced expression
of heat shock proteins such as HSP70, HSP90, HSP27 among others.
We are interested in identifying compounds similar to the cur-
rent clinical candidate arimoclomol,9 that do not induce chaperone
proteins de novo in non-stressed cells, but amplify the endogenous
induced chaperone expression in stressed or diseased cells. We
hypothesized that such a unique molecular mechanism with high
selectivity for diseased (stressed) cells may offer therapeutic
advantage with reduced side effects compared to general HSP
inducers that indiscriminately activate chaperone expression even
in non-stressed cells.
The pivotal transcription factor that regulates the expression of
these heat shock proteins is the heat shock transcription factor 1
(HSF1). It has been reported that the kinetics of HSF1 granule for-
mation correlates well with its transcriptional activity and is gen-
erally believed to be a visual indicator of the active transcriptional
complex.6,7 Based on these observations, we have developed a
quantitative high content image-based assay to measure the
HSF1 granule formation.8 We applied this assay as the primary
screen to identify and rank novel small molecule chaperone ampli-
fiers from chemical libraries. In this communication, we report the
discovery, synthesis, and initial structure–activity relationship
(SAR) of a series of pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione
derivatives as novel small molecule chaperone amplifiers that are
structurally distinct from arimoclomol. The biological profile of
this series of compounds suggests its potential therapeutic applica-
tion in a number of indications, including stroke and neurodegen-
erative diseases such as PD.
During our in-house library screening using high content
image-based HSF1 granule assay, we discovered that 1-ethyl-6-
methyl-3-(thiophen-2-yl)pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-
* Corresponding author. Tel.: +1 858 273 2501x221; fax: +1 858 273 2697.
E-mail addresses: yuefen.zhou@gmail.com, yzhou@cytrx.com, yzhou01@yahoo.
com (Y. Zhou).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.073

4304
Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4303–4307
the presence of acetic acid to form a mixture of 3-(2-yl)pyrimi-
do[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione (4A) and 5,7-dioxo-3-(2-
yl)-1,5,6,7-tetrahydropyrimido[5,4-e][1,2,4]triazine 4-oxide (4B).12
Desired products of 4A and 4B were separated by HPLC purification.
Alternatively compounds 4B were reduced to form 4A by the treat-
ment of dithioerythritol (DTE) or Na2S2O4.
N
N
O
O
N
N
N
S
4A-1
Our lead optimization initially focused on the modification of R2
and R3 substituents (where R1 = Me) to study the structure and
activity relationship (SAR). The high content image-based HSF1
granule assay was used to measure the compound-induced HSF1
amplification activity.13 Rotenone is a broad-spectrum insecticide
and pesticide that is known to cause PD-like symptoms in rats.14
Therefore we developed a rotenone toxicity model in SK-N-SH neu-
roblastoma cells to measure the cytoprotection from rotenone in-
duced cellular damage.15 The results are summarized in Table 1.
As shown in Table 1, the N-oxide analogs of 4B were generally
slightly more potent than the corresponding 4A analogs in the
HSF1 granule assay (e.g., 4A-2 vs 4B-2, 4A-3 vs 4B-3, 4A-5 vs 4B-
5, 4A-7 vs 4B-7) but with the drawback of increased cell toxicity.
Therefore we subsequently focused our optimization on 4A
analogs.
Figure 1. HSF1 amplifier.
dione (4A-1 in Fig. 1) exhibited potent HSF1 amplifying activity
(Table 1). Therefore we focused on the lead optimization of this
chemical series aiming to discover more potent compounds that
amplify the HSF1-dependent stress response for cytoprotection in
neurodegenerative disease cell models for potential use as targeted
chaperone therapies.
The synthesis of the pyrimidotriazinedione derivatives was fairly
straightforward. It involved a 3-step synthesis. As shown in Scheme
1, first 6-chloro-pyrimidine-2,4(1H,3H)-dione (1), either commer-
cially available or synthesized according to literature methods,10
was converted to 6-hydrazinyl-pyrimidine-2,4(1H,3H)-dione (2)
by the treatment of hydrazine under heating.11 Compound 2 was
then reacted with an aldehyde to form an imine intermediate 3 un-
der heating which was further cyclized by the treatment of NaNO2 in
The R2 moiety had noticeable impact on HSF1 activity. Analogs
with n-butyl R2 group were generally more active than the analogs
Table 1
SAR in HSF1 granule and Rotenone assays
R2
R2
N
N
O
O
N
N
O
O
N
N
N
N
R3
N
O
CH3
R3
N
CH3
4A
4B
R2
R3
Producta
HSF1 EC50
(l
M)
Rotenone EC50
(l
M)
Increase in Rotenone
viabilityc (%)
HeLa CC50
(l
M)
SK CC50 (lM)
b
c
d
d
Et
Et
Et
n-Bu
n-Bu
n-Bu
n-Bu
Et
Et
Et
Et
Et
n-Bu
Et
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
isoamyle
i-Bu
n-Pr
Bn
2-Thiophene
5-Me-2-thiophene
5-Me-2-thiophene
5-Me-2-thiophene
5-Me-2-thiophene
5-Me-2-Furan
5-Me-2-Furan
5-Me-2-Furan
5-Me-2-Furan
2-Furan
2-Pyrrol
2-Pyrrol
2-Pyrrol
2-Imidazole
2-Imidazole
2-Thiazole
2-Benzimidazole
2-Quinoline
4A-1
4A-2
4B-2
4A-3
4B-3
4A-4
4B-4
4A-5
4B-5
4A-6
4A-7
4B-7
4B-8
4A-8
4A-9
4A-10
4A-11
4A-12
4A-13
4A-14
4A-15
4A-16
4A-17
4A-18
4A-19
4A-20
Celastrol
5.1
0.41
1.33
1.10
44%@1.25
0.19
0.33
49%@1.25
1.84
56%@1.25
64%@1.25
0.68
0.17
0.64
2.71
0.30
55%@0.63
1.9
0.29
0.23
0.24
53
50
57
44
27
23
49
50
56
64
44
35
49
62
56
55
59
71
53
44
67
55
27
NA
58
NA
24
10.5
22.2
15
15.3
6.6
7.8
9
22.3
9.8
5.6
10.7
5.2
6.3
11.9
3.6
2.5
76%@80
9.5
8.7
16.9
7.9
15.6
7.3
9.9
63.5
27.2
18.2
7.2
5.3
6.6
8.2
3.3
2.8
4.3
1.9
1.5
3.6
1.3
1.4
13.1
4.0
8.4
10.7
3.7
5.4
5.6
21.3
9.8
>80
1.6
17.3
11.6
16.8
8.6
9.9
10
46.3
20.6
10.1
35
24.7
14.9
39.8
7.7
2.1
40.8
2.5
2.5
2.5
1.3
25%@80
4.1
14.0
14.8
2-Benzothiazole
2-Benzothiazole
2-Benzothiazole
2-Benzothiazole
2-Benzothiazole
2-Benzothiazole
Phenyl
0.27
0.55
2.34
No activity
0.49
p-F-Bn
n-Bu
H
23.4
18.5
>80
3.0
2-Thiophene
NA
No activity
No activity
24%@0.16
NA
1.1f
a
The desired mass was found for each compound in LC–MS analysis and structure was confirmed by 1H NMR. All the compounds of 4A and 4B had no activity in HSF1
granule assay without heat shock stress (no de novo activation activity, data not shown). The data are the average of multiple testing.
b
See Ref. 13 for the HSF1 granule assay condition. EC50 is the concentration at which the percentage of cells that has positive HSF1 granules is half of the maximum.
See Ref. 15 for rotenone stress assay condition and the calculation of the maximum cytoprotection. EC50 is the concentration at which cytoprotection from rotenone is half
c
of maximal protection.
d
Viability was determined by the MTS assay described in Ref. 16. CC50 is the concentration at which cytotoxicity is half of maximum.
Isoamyl = 3,3-dimethylpropyl.
Celastrol has EC50 of 2.6 lM in HSF1 granule assay without heat shock stress (de novo activation).
e
f

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4303–4307
4305
R2
N
R2
N
H
N
H
N
H
N
O
O
Cl
O
N
H2N
b
a
N
N
R1
N
R3
R1
R1
O
O
O
3
2
1
c
R2
N
R2
N
N
O
N
O
N
N
+
N
N
R3
R1
R1
N
O
R3
N
O
O
4A
4B
d
i
Scheme 1. Reagents and conditions: (a) R2NHNH2Á2HCl, PrNEt2, EtOH, 100 °C, overnight; (b) R3CHO, EtOH, 80 °C, 1 h–overnight; (c) aq NaNO2, CH3CO2H, rt, overnight; (d)
DTE, MeOH, rt, 1 h–3 days.
with ethyl R2 group (e.g., 4A-4 vs 4A-5, 4B-4 vs 4B-5, 4B-7 vs 4B-8,
4A-8 vs 4A-9) without any effect on cell toxicity. n-Propyl R2
groups led to less active compounds than n-butyl R2 moiety (com-
pare 4A-16 with 4A-13). In addition, the R2 group with same size in
chain length as n-Bu normally led to compounds with similar HSF1
activity. For example, isoamyl R2 groups resulted in similar HSF1
activity to that of n-butyl moiety (4A-14 vs 4A-13). The compound
with iso-butyl R2 group (4A-15) was equipotent as celastrol, a ref-
erence compound that entered into clinical trial for treating rheu-
matoid arthritis.17 Interestingly, when R2 = H, HSF1 activity
diminished completely (4A-20) (as compared with 4A-1) suggest-
ing that the N-1 position needs to be substituted in order to gain
HSF1 amplifying activity.
control with rotenone treatment) with 76–103% cell survival com-
pared with that of the DMSO control without rotenone treatment
(e.g., 4A-12 has cell viability of 103%, Fig. 2). Both the EC50 value
and the maximum percent cell viability increase (compared with
the DMSO control with rotenone treatment, for example, 4A-12
has a maximum cytoprotection of 71%) are listed in Table 1.
The majority of the compounds tested were very potent in the
rotenone stress assay with EC50 < 1
lM. However, addition of a
methyl group at the ortho-position of a thiophene ring as R3 group
resulted in less potent compounds with EC50 > 1 lM (compared
4A-2 or 4A-3 with 4A-1). The R3 moieties such as 2-benzimidazole,
2-imidazole (R2 = Et) and 5-methyl-2-furan (R2 = Et) also led to less
potent compounds with EC50 > 1
thermore, benzyl and para-F-benzyl R2 moieties led to either a less
potent compound with EC50 > 1 M (4A-17) or complete loss of
lM (4A-11, 4A-8 and 4A-5). Fur-
R3 groups also had significant impact on HSF1 activity. The
addition of a methyl group at the ortho-position of the thiophene
ring resulted in significant activity loss (compare 4A-2 or 4A-3
with 4A-1). A similar SAR trend was observed when 2-furan was
the R3 group (4A-5 vs 4A-6). Replacement of 2-thiophene with
either 2-pyrrol or 2-imidazole ring led to much less potent com-
pounds (compare 4A-7 or 4A-8 with 4A-1). In addition, both 2-thi-
azole and 2-benzothiazole R3 groups led to more potent
compounds (4A-10 and 4A-13) with the later being less toxic in
both HeLa and SK-N-SH cells. However, replacement of the 5-
membered thiophene ring of 4A-1 with a 6-membered phenyl ring
as R3 (4A-19) resulted in a significant loss of HSF1 activity.
It is important to note that none of the compounds tested in this
series of pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione deriva-
tives showed activity in HSF1 granule assay in the absence of heat
shock stress while celastrol displayed potent HSF1 activity
l
cytoprotection from rotenone toxicity (4A-18). Without substitu-
tion (R2 = H), the cytoprotection diminished completely suggesting
that the R2 group is critical to the cytoprotection effect which was
consistent with the diminished HSF1 amplifying activity of this
compound (4A-20). Overall both R2 and R3 groups have effects
on the level of cytoprotection from rotenone-induced toxicity.
In general, the therapeutic window (CC50/EC50) of the com-
pounds in SK cells in this series is significant, up to 48-fold (4A-
2) suggesting significant therapeutic potential of this series of com-
pounds for PD. The HSF1 EC50/CC50 ratio in HeLa cells is generally
smaller than the corresponding ratio in the rotenone protection as-
say in SK cells (sevenfold or less), mostly because the EC50 for cyto-
protection is substantially lower than the EC50 for HSF1 activation.
This suggests that relatively small increases in HSF1 activation are
sufficient to protect against rotenone cytotoxicity. Since the
(EC50 = 2.6 lM) without heat shock stress. Therefore, this class of
novel small molecules, with a selective chaperone amplifying ef-
fect only to those cells under stress, may thus provide therapeutic
advantages with potentially fewer side effects compared to chaper-
one inducers like celastrol.
120%
103%
100%
100%
Next, in order to further evaluate the potential applications of
this series of pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione
derivatives, the cytoprotective effects of these compounds in bio-
logically relevant cell models were also investigated. The rotenone
model of PD is a cell-based system for study of protein misfolding
induced cytotoxicity.18
In the control experiment, when 200 nM of rotenone was ap-
plied with DMSO for 48 h, about 40% of SK-N-SH cells were killed.
However, treatment with compounds 4A or 4B in the presence of
rotenone stress resulted in significant cytoprotection (27–71%
maximum percent cell viability increase compared with the DMSO
80%
60%
60%
40%
20%
0%
DMSO
DMSO + Rotenone
4A-12 + Rotenone
Figure 2. Percent cell survival with rotenone treatment in the presence or absence
of the compound (0.625
rotenone treatment.
lM) as compared with the DMSO control with or without

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Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4303–4307
Table 2
the product was extracted with dichloromethane (3 Â 50 mL). The organic
phase was dried (Na2SO4) and concentrated in vacuo. The residue was purified
by automated flash chromatography (ISCO, CombiFlash Companion purchased
from Teledyne ISCO) on silica gel (0–10% of MeOH in DCM) to give the desired
products (2). The yields ranged from 44% to 95%. The synthesis was referred to
the procedure as described in WO 2004/007499 A1 and WO04/065387.
Cytoprotective effect in OGD cell model in SH-SY-5Y cells
Compound
Cytoprotection (%)
0.5
lM
1.0
l
M
2.5 lM
12. General procedure for synthesis of 4A and 4B where
(0.75 mmol) was added to solution of 6-(1-hydrazinyl)-3-methyluracil
R
1 = Me: an aldehyde
Celastrol
4A-1
4A-10
4A-13
8
18
7
18
48
23
38
À22
14
39
a
(0.75 mmol) in ethanol (6 mL) and the mixture was stirred at 80 °C for 1 h–
overnight. The solid precipitate was collected by filtration, washed with
hexanes, and dried in vacuo. The imine intermediate thus obtained was of
sufficient purity to be carried to the next step without further purification. This
imine intermediate was then treated with glacial acetic acid (2 mL). An
14
80
aqueous solution of sodium nitrite (37 mg in 93 lL of H2O) was added and the
rotenone assay is a functional assay and a disease relevant cell
model system, we consider the therapeutic window related to
cytoprotection in SK cells to be more important and relevant than
the corresponding pharmacodynamic effects in the HeLa cells used
for screening.
In addition, the correlation between HSF1 activity and rotenone
cytoprotection is not linear. This lack of direct correlation may sug-
gest that HSF1 is not the direct molecular target for this series of
compounds but is likely downstream from the direct target in this
pathway. We believe that the initial compound modulates at least
two separate mechanistic pathways, one that amplifies the HSF1
response and one that is toxic. When structural changes are made
in the molecule, some presumably allow for improved binding to
one of the targets versus the other.
Oxygen and glucose deprivation (OGD) is an in vitro cell-based
model system of ischemia and stroke. Cytotoxicity induced by OGD
is at least partly due to protein misfolding and aggregation.19 A few
selected compounds along with the reference compound celastrol
were tested in an OGD cell model in SH-SY-5Y cells and the results
are summarized in Table 2.20 Compounds 4A-1, 4A-10 and 4A-13
started to show some cytoprotective effect at concentration as
resulting mixture was stirred at room temperature overnight, neutralized by
aqueous Na2CO3 and extracted by 5% MeOH in DCM. The residue was purified
by preparative thin layer chromatography on silica gel (40% toluene in ethyl
acetate) or by reverse-phase HPLC (Gilson 215 liquid handler) (5–95% ACN in
water with 0.05% TFA) to afford pure desired products 4A and 4B. Alternatively,
the mixture after work-up was first dissolved in methanol (10 mL), then
dithioerythritol (2.25 mmol) was added and the mixture was stirred at room
temperature for 72 h. The crude mixture was purified by reversed-phase HPLC
(5–95% ACN in water with 0.05% TFA) to afford the desired product 4A as a
solid after evaporation. The isolated yields ranged from 6–75%. The desired
mass was found by LC–MS analysis for each product listed in Table 1. Example
(4A-1): 1-ethyl-6-methyl-3-thiophen-2-yl-1H-pyrimido[5,4-e][1,2,4]triazine-
5,7-dione: yield: 75%; LC–MS (ES+, m/z) = 290 [M+1]+; 1H NMR (DMSO-d6,
400 MHz): d 1.43 ppm (t, 3H, J = 5.72 Hz), 3.28 (s, 3H), 4.43 (q, 2H, J = 5.79),
7.26 (t, 1H, J = 3.06 Hz), 7.87 (d, 1H, J = 4.25 Hz), 7.88 (d, 1H, J = 2.47 Hz). The
synthesis was referred to the procedure described in Lacrampe, J. F. A., et al.
WO 2004/007498 A2, 2004.
13. HSF1 granule assay: The assay was performed according to the previous
publications (Ref. 8 and Zhou, Y. et al. Bioorg. Med. Chem. Lett. 2009, 19, 3128).
Briefly, HeLa cells were pretreated with compounds 1 h before mild heat shock
at 41 °C for 2 h with no recovery time. As a control, HeLa cells were pretreated
with compounds at 37 °C for 3 h in order to eliminate compounds that induce
heat shock response in non-stressed cells. The overall dilution of compound
was 200-fold, with
Immunocytochemical staining for HSF1 in HeLa cells was performed as
described in the Ref. with some modifications. Image acquisition was
a concentration ranging from 0.3 lM to 80 lM.
8
performed using an INcell 1000 analyzer (GE Healthcare, Piscataway, NJ) with a
10Â object. Image analysis was carried out using Multi Target Analysis module
from Workstation 3.6. Algorithms for the HSF1 total granule counts were
established according to assay conditions and manufacture instructions. EC50
values and curve fitting were calculated using Prism 4.0 (GraphPad Software,
San Diego, CA) with nonlinear regression analysis. A maximum percentage
<20% HSF1 granule positive cells (compared to 7% in the DMSO negative
control) was considered inactive.
low as 0.5
Among them compound 4A-13 displayed the maximum cytopro-
tection of 80% at 2.5 M compared with the OGD DMSO control
and it provided much higher cytoprotection than celastrol at
M (38% vs 18%) and at 2.5
M (80% vs À22%).
In summary, we synthesized number of pyrimido[5,4-
lM with the maximum cytoprotection at 1 or 2.5 lM.
l
1
l
l
14. Caboni, P.; Sherer, T.; Zhang, N.; Taylor, G.; Na, H.; Greenamyre, J.; Casida, J.
Chem. Res. Toxicol. 2004, 17, 1540.
a
e][1,2,4]triazine-5,7(1H,6H)-dione derivatives as novel small mole-
cule chaperone amplifiers. Our SAR studies revealed that both R2
and R3 substituents had effects on HSF1 amplifying activity. Most
compounds under study showed potent cytoprotection in the rote-
none stress cell model. One of the compounds (4A-13) exhibited
potent HSF1 amplifying activity and excellent cytoprotection effect
in both rotenone and OGD cell models. As the rotenone and OGD
cell models are directly relevant to PD and stroke respectively,
these compounds could potentially be used in treating these
indications.
15. Rotenone stress assay condition: SK-N-SH neuroblastoma cells (within passage
6, purchased from ATCC, Manassas, VA) were seeded in a 96 well plate with
8000 cells/well in DMEM containing 0.1% FBS for 18 h before Rotenone
treatment. Using Sciclone liquid handling workstation (Caliper Life Science,
Hopkinton, MA), fresh media containing rotenone with or without testing
compounds were added to the assay plates with a final concentration of
rotenone at 200 nM and compound concentration ranging from 0.019
10 M. Cells were incubated for 48 h followed by MTS viability assay. Percent
cytoprotection was determined using the following formula:
lM to
l
(MTScompound À MTSrotenone alone)/MTSrotenone alone  100%. EC50 values and
curve fitting were calculated using Prism 4.0 (GraphPad Software, San Diego,
CA) with nonlinear regression analysis. Maximum percent cytoprotection <20%
was considered inactive.
16. MTS viability assay: HeLa (8000 cells/well) and SK-N-SH (14,000 cells/well)
cells were seeded in DMEM containing 10% FBS in 96-well plates (Catalog #
Costar 3598, Cornings, MA) for 18 h before experiment. Compounds or DMSO
control were added to the culture at 1–200 dilutions with a final concentration
References and notes
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J.-M.; Weller, R. O.; Anderton, B. H. Brain 1991, 114, 775.
ranging from 0.3 lM and 80 lM (final DMSO concentration is 0.5% v/v). Cells
were incubated with compounds for 72 h followed by MTS/PMS addition into
the plates and incubated for four additional hours. SDS was added to a final
concentration of 1.4% (w/v%). Plates were then measured for absorbance at
492 nm using envision excite (Perkin Elmer, Wellesley, MA). The absorbance at
492 nm is directly proportional to the living cells in the culture. Percent
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inhibition
was
determined
using
the
following
formula:
(MTSDMSO À MTScompound)/MTSDMSO Â 100%. IC50 values and curve fitting were
calculated using Prism 4.0 (GraphPad Software, San Diego, CA) with nonlinear
regression analysis.
17. Corson, T. W.; Crews, C. M. Cell 2007, 130, 769.
9. (a) Sõti, C.; Nagy, E.; Giricz, Z.; Vígh, L.; Csermely, P.; Ferdinandy, P. British J.
Pharmacol. 2005, 146, 769; (b) Boggs, J. Bioworld Today 2009, 20, 1.
10. (a) Shamim, M. T.; Ukena, D.; Padgett, W. L.; Daly, J. W. J. Med. Chem. 1989, 32,
1231; (b) Petry, S.; Barimghaus, K.-H.; Tennagels, N.; Mueller, G. U.S. Patent
2004/0242583 Al, 2004.
18. (a) Greenamyre, J. T.; Betarbet, R.; Sherer, T. B. Parkin. Relat. Disord. 2003, S59;
(b) Lim, K.-L. et al Human Mol. Gen. 2005, 14, 3885; (c) Wang, X.; Qin, Z.-H.;
Leng, Y.; Wang, Y.; Jin, X.; Chase, T. N.; Bennett, M. C. J. Neurochem. 2002, 83,
1094.
19. Giffard, R. G.; Xu, L.; Zhao, H.; Carrico, W.; Ouyang, Y.; Qiao, Y.; Sapolsky, R.;
Steinberg, G.; Hu, B.; Yenari, M. A. J. Exp. Biol. 2004, 207, 3213.
20. Oxygen glucose deprivation (OGD) assay: The assay was performed according to
a previous publication (Ref. 8). Briefly, SHSY5Y cells (purchased from ATCC,
11. General procedure for synthesis of
2 where R
1 = Me: diisopropylethylamine
(5.2 mL, 30 mmol) and dry ethanol (30 mL) were mixed with 6-chloro-3-
methyluracil (10 mmol) and a hydrazine salt (15 mmol) in a sealed tube. The
mixture was stirred at 100 °C overnight. After cooling to the room temperature,

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4303–4307
4307
Manassas, VA) were plated at a density of 25,000 cells/well in 96-well plates
pre-coated with collagen I (BD Biosciences, San Diego, CA) and grown for 16–
24 h in complete medium. For the induction of oxygen glucose deprivation
(OGD), cells were washed twice in pre-deoxygenated medium with no glucose
or serum. Selected compounds were added to the cells after medium change
within 1 h before OGD stress, and the plates were placed in modular incubator
chambers (Billups-Rothenberg, Del Mar, CA). The chambers were flushed with
a gas mixture of 95% N2/5% CO2 at a flow rate of 10 L/min for 30 min at room
temperature. The residual O2 concentration was monitored by a PO2 meter
which was connected to the deoxygen chamber with a final concentration less
than 1%. After flushing, the chambers were sealed and maintained in a 37 °C
incubator for 28 h. MTS viability assay was performed. Percent cytoprotection
(the percent of the cell viability increase) compared with the OGD DMSO
control was determined using the following formula: (MTScompound
À
MTSDMSO)/MTSDMSO Â 100%.