Chloro-oxime derivatives as novel small molecule chaperone amplifiers

Article abstract of DOI:10.1016/j.bmcl.2009.03.011

Chloro-oxime derivatives were investigated as novel small molecule chaperone amplifiers. Lead optimization led to the discovery of compounds that displayed potent HSF1 activation activity, significant cytoprotection in MG-132 stress, ER stress and PolyQ stress cell models (EC₅₀ < 10 μM).

Full text of DOI:10.1016/j.bmcl.2009.03.011

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3128–3135
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chloro-oxime derivatives as novel small molecule chaperone amplifiers
*
Yuefen Zhou , Khang Vu, Yongsheng Chen, John Pham, Thomas Brady, Gang Liu, Jinhua Chen,
Joonwoo Nam, P. S. Murali Mohan Reddy, Qingyan Au, Il Sang Yoon, Marie-Helene Tremblay, Gary Yip,
Charmian Cher, Bin Zhang, Jack R. Barber, Shi Chung Ng
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:
Chloro-oxime derivatives were investigated as novel small molecule chaperone amplifiers. Lead optimi-
zation led to the discovery of compounds that displayed potent HSF1 activation activity, significant cyto-
protection in MG-132 stress, ER stress and PolyQ stress cell models (EC50 < 10 lM).
Received 10 February 2009
Revised 2 March 2009
Accepted 4 March 2009
Available online 9 March 2009
Ó 2009 Published by Elsevier Ltd.
Keywords:
Small molecule chaperone amplifiers
Protein misfolding
HSF1, heat shock proteins (HSPs)
Stress granule
MG-132 stress
PolyQ stress
ER stress
Neurodegenerative diseases
Cytoprotection
Chloro-oxime derivatives
Neurodegenerative disease is a condition in which cells of the
brain and spinal cord become dysfunctional and ultimately die,
leading to disabilities in memory, movement, sensory processing
and decision making. At present there are few effective pharma-
ceuticals for the wide range of neurodegenerative diseases and
none of them can provide a cure. Therefore there are significant
unmet medical needs to find better therapies and approaches to
treat neurodegenerative diseases.
In recent years, substantial experimental evidence has accumu-
lated indicating that protein misfolding may be a universal under-
lying mechanism in the pathogenesis of several neurodegenerative
diseases including sporadic and familial amyotrophic lateral scle-
rosis (ALS and FALS), Alzheimer’s disease (AD), Parkinson’s disease
(PD), Huntington’s disease (HD) and related polyglutamine (polyQ)
expansion diseases as well as transmissible spongiform encepha-
lopathy (TSE) diseases.1–5 Heat shock proteins (HSPs) are molecu-
lar chaperones whose expressions are induced as part of the
cellular stress response. HSPs recognize and either repair or de-
stroy misfolded proteins.
Up-regulation of heat shock proteins (HSPs) via small molecules
has shown great therapeutic promise in a number of disease areas
where the probable common cause is the accumulation of mis-
folded proteins.10–12 For example, Arimoclomol is now in phase
II/III clinical trial in ALS patients,11 and Celastrol is in clinical trial
for treating rheumatoid arthritis.12 Several therapeutically active
small molecules that increase cellular HSP levels have been re-
ported (e.g., celastrol, radicicol, galdanamycin, 17-AAG). These
compounds seem to mediate their effect on HSPs by indirect acti-
vation of HSF1.13 Other compounds, exemplified by bimoclomol
arimoclomol, and iroxanadine do not activate HSF1 or induce
HSP expression in normal unstressed cells, but only amplify the al-
ready activated chaperone response in stressed or diseased cells
that accumulate misfolded proteins.10 Such a unique molecular
mechanism that specifically targets stressed cells potentially offers
greater safety than compounds that induce the chaperone response
in all cells indiscriminately.10 Our interest is to discover more
potent small molecule chaperone amplifiers to effectively treat
neurodegenerative diseases and other diseases of protein
misfolding.
Increased expression of many HSPs is regulated at least in part
by the activation of heat shock transcription factor-1 (HSF1).6–9
Previous investigation revealed that the transcriptional activity
of HSF1 in cultured cells correlated well with the formation of
intranuclear HSF1 granules.14,15 Hence we developed a quantita-
tive high-throughput assay for HSF1 transcriptional activity using
* Corresponding author. Tel. +1 858 273 2501x221; fax: +1 858 273 2697.
E-mail address: yuefen.zhou@gmail.com (Y. Zhou).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.03.011

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3128–3135
3129
(Methods A and B, Schemes 1 and 2)18 available to prepare the
compounds shown in Tables 2–4.
Table 1
The percentage of HSF1 granule formation (average) with or without heat shock stress
Oximes 2 were synthesized by the reaction of commercially
available aldehydes with hydroxylamine under heating conditions
in >90% yield. Alternatively oximes 2 could be provided by a one
step synthesis directly from ortho-hydroxyanilines 3 by treatment
with 2,2,2-trichloro-1-ethoxyethanol in good yields.19 Oximes 2
were then treated with thionyl chloride to form nitriles 4 which
were then converted to hydroxyimidamides 5 by treating with
hydroxylamine in good yield. Compounds 5 were then converted
to the key intermediates 8 in moderate yields by treating with qua-
ternary azetidinium salts 7 in the presence of a base under heating.
Azetidinium salts 7 were synthesized by reacting epichlorohydrin
with secondary amines 6 under heating in moderate yields.20 The
key intermediates 8 were then treated with NaNO2 and HCl to pro-
vide the desired chloro-oxime derivatives 9 in low to good yields.
Alternatively in Method B (Scheme 2), hydroxyimidamides 5 were
directly treated with epichlorohydrin to form compounds 10 in
moderate to good yields, which were then treated with either
amines 6 or methyl sulfonamide to form key intermediates 8 or
11 in P80% yields. Intermediates 8 and 11 were then converted
to the desired products 9 and 12 respectively in the same way as
that described in Method A. Methods A and B diverge at com-
pounds 5. For most substrates, both methods worked equally well.
However, in some cases reactions of amino-oximes 5 with 7 in
Method A gave very low yields while Method B resulted in fairly
good yields. On the other hand, Method A is more convergent.
Our lead optimization focused on the modification of the left
side (R1), right side (R2 and R3) of the molecules 9 and the replace-
ment of the Cl moiety in the chloro-oxime core to study the struc-
ture and activity relationship (SAR). We used the high content
image-based HSF1 granule formation in HeLa cells to measure
the HSF1 activation activity.21 Additionally, we developed the
MG-132 cell stress model in SK-N-SH neuroblastoma cells to
systematically measure the cytoprotection of the analogs in this
series.22 MG-132 is a potent and selective 26S proteasome inhibi-
Cl
N
N
O
N
O
OH
1
% of granule formation (standard deviation)
DMSO
Compound 1
No heat shock stress (37 °C)
Heat shock stress (41 °C)
5.6 ( 0.7)
7.3 ( 1.2)
6.7 ( 0.91)
58.0 ( 4.0)
high content image-based HSF1 granule formation in heat-shocked
HeLa cells in order to screen for potential amplifiers of HSF1.16 We
began our screening efforts with an in-house directed library con-
taining compounds structurally similar to arimoclomol, bimoclo-
mol, and iroxanadine. We discovered that chloro-oxime
1
showed potent HSF1 amplification activity (Table 1). Treatment
with 1 resulted in a significant increase in the percentage of cells
with active HSF1 granules following mild heat shock compared
to cells treated with 1 without heat shock (58% vs 7.3%, respec-
tively). However, there is no noticeable change in granule forma-
tion when cells were treated with 1 at non-stress conditions
(6.7% vs 5.6%), suggesting that 1 by itself is not a stressor. Thus,
1 is a bona fide amplifier of HSF1 activity, not a de novo activator
of HSF1. In addition, 1 also increased HSP70 expression under iden-
tical heat shock condition (41 °C with 2 h recovery time) with max-
imum percentage activation of 51% (EC50 = 32.9 lM) indicating
that compound 1 is a HSF1/HSP70 co-inducer.17 We focused on
lead optimization of this chemical series aiming to improve activ-
ity for chaperone amplification that would provide cytoprotection
in neurodegenerative disease cell models for potential use as tar-
geted chaperone therapies.
The synthesis of the chloro-oxime derivatives was fairly
straightforward and there are a number of synthetic methods
Method A:
NH2
R'
3
OH
Cl
Cl
Cl
OH
O
HCl, H2O, NH2OH.HCl
NaOAc
O
SOCl2
OH
HONH2.HCl
R1
N
R1
N
R1
H
iPrOH / H2O
70 oC, 2h
reflux 1h
4
2
NaOH, EtOH / H2O
80oC, 3h
HONH2.HCl
NaHCO3
NH2
N
OH R2
N
NH2
O
OH
R1
R3
R1
N
EtOH / H2O
105 oC, 3h
OH
8
5
N+ Cl-
R2
HCl, NaNO2,
H2O, -5-0 oC, 1h
R3
7
O
Cl
OH R2
Cl
O
N
MeOH, reflux o.n
R1
N
R3
9
R2
HN
R3
6
Scheme 1. Synthetic method A for chloro-oxime derivative preparation.

3130
Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3128–3135
Method B:
R2
O
HN
NH2
N
OH R2
N
NH2
N
O
NH2
Cl
R3
6
O
O
R1
R3
R1
OH
R1
N
NaOH
H2O / DMSO
0 oC - r.t. o.n.
DMF, 60-100 oC, 3h
8
10
5
HCl, NaNO2,
H2O, -5-0 oC, 1h
O
O
S
H2N
Cs2CO3
DMF, 100 oC, 4h
Cl
OH R2
O
N
R1
N
R3
9
HCl, NaNO2,
H2O, -5-0 oC, 1h
NH2
N
OH
Cl
OH
H
H
N
O
O
N
R1
S
R1
N
S
O
O
O
O
11
12
Scheme 2. Synthetic method B for chloro-oxime derivative preparation.
tor that can cause protein misfolding and apoptosis because it
inhibits the degradation of ubiquitin-conjugated proteins,23 In
addition, MTS assays in both HeLa and SK-N-SH cells were run to
determine the CC50 (cytotoxic concentration at 50% of inhibition)
complete loss of HSF1 activity and weaker cytoprotection (34–
37). Overall the right side of the molecule (R4) had an impact on
the HSF1 activity as well as cytoprotection from MG-132 stress.
Next, the R1 SAR on the left side of the molecule was also stud-
ied and the results are summarized in Table 3.
As shown in Table 3, all the substituents on the benzoxazole
ring at various positions (4–7), electron withdrawing or donating,
resulted in similar or slightly improved HSF1 activity (38–47) than
the non-substituted benzoxazole (1). However, the majority of
them had improved cytoprotection effect from MG-132 stress with
values to evaluate the in vitro therapeutic index (TI = CC50
/
EC50).24 The results of the R4 (=–NR2R3) SAR on the right side of
the molecule are summarized in Table 2.
The initial hit (1) of the HSF1 amplifier also showed significant
cytoprotection in the MG-132 stress assay (Fig. 1).
As shown in Figure 1, when the DMSO control was treated with
MG-132, about 30% of cells survived compared with that without
MG-132 treatment. However, cell survival (rescue) increased three
folds in cells treated with compound 1 compared with MG-132
alone, indicating that the cells were almost fully rescued from
MG-132 toxicity.
EC50 of <10 lM. More importantly, they all showed better thera-
peutic index comparing with the initial hit (1). In addition, (4,5)-
naphathyl benzoxazole led to a more potent HSF1 activator (48)
with significant improvement in cytoprotection from MG-132
stress than the initial hit (1). However, its cell toxicity increased.
Interestingly, removing the N-atom in the benzoxazole or benzo-
thiazole ring (49–50) totally diminished the HSF1 activity and
cytoprotection effect suggesting that the benzoxazole ring of R1
is important to the HSF1 activity and cytoprotection from MG-
132 stress.
On the other hand, there was almost no separation between
EC50 value measured by HSF1 or MG-132 assays and cell toxicity
in HeLa or SK-N-SH cells measured by MTS assays for this com-
pound (1) (Table 2). Replacing the benzoxazole ring with a benzo-
thiazole moiety (13, X = S) retained both the HSF1 activity and
cytoprotection from MG-132. Reducing the ring size of R4 com-
pletely diminished the cytoprotection from MG-132 stress
although it had weak HSF1 activity (14). Interestingly, replacement
of piperidine with morpholine moiety of R4 resulted in a complete
loss of HSF1 activity and significant reduction of cytoprotection in
MG-132 assay (15). Addition of F-atoms on the piperidine ring also
reduced the HSF1 activity but restored much of the cytoprotection
with reduced cell toxicity in both HeLa and SK-N-SH cells (16–18).
However, addition of electron donating groups on the piperidine
ring led to slight improvement in HSF1 activity and cytoprotection
from MG-132 stress with improved therapeutic index (19–20).
When R4 were acyclic amines, many of them (21–22, 24–26,
28–30) resulted in some improvement in both HSF1 activity and
cytoprotection from MG-132 toxicity compared with the piperi-
In addition to SAR of the left (R1) and right sides (R4) of the mol-
ecule, the effect of the middle part of the molecule (Y) on the HSF1
activity and cytoprotection from MG-132 stress was also evaluated
(Table 4).
As shown in Table 4, replacement of the Cl atom in the Cl-oxime
core with H, CH3, OCH3, CF3 or F moieties (51–55) completely
diminished HSF1 activity and MG-132 cytoprotection although re-
duced cell toxicity was observed. The amide analog also had no
HSF1 activity or MG-132 cytoprotection (56). In addition, removing
the hydroxyl group of initial hit 1 caused complete loss of HSF1
activity and significant reduction of the MG-132 cytoprotection
(57). These results suggest that both the Cl moiety in the Cl-oxime
core and the hydroxyl group are essential to the HSF1 activity and
the MG-132 cytoprotection effect.
dine (1). A few of them had single digit
l
M EC50 in the MG-132 as-
It is worth noting that all the compounds tested in this series
showed no activity in the HSF1 granule assay in the absence of
the heat shock stress (data not shown). Therefore, this class of
small molecule chaperone inducers acts like ‘smart drugs’ by a
selective interaction with only those cells in stress, and may pro-
vide therapeutic advantages.
In order to evaluate whether this class of compounds has more
general and broad cytoprotection effect, some of the compounds in
this series were also tested in the tunicamycin-induced endoplas-
mic reticulum (ER) stress assay.25 Tunicamycin blocks the matura-
tion of proteins that require glycosylation, resulting in protein
say. However, branching adjacent to the N-atom (27) of the tertiary
amine moiety (R4) or longer alkyl groups (23) led to decreased
HSF1 activity or cytoprotection from MG-132 stress. Furthermore,
replacing the terminal methyl group of 28 with a hydroxyl group
was well tolerated (31). The addition of an F-atom at the terminal
tertiary amine group of 28 or replacement of the terminal n-propyl
group of 28 with cyclopropyl methylene moiety retained HSF1
activity and cytoprotection (32–33). Other replacements of the
terminal tertiary amines including secondary amine, methyl sul-
fonamide, amide and reversed amide resulted in much weaker or

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3128–3135
3131
Table 2
SAR of R4 in HSF1 granule and MG-132 assays
Cl
N
R4
N
X
O
OH
Compounda
X
O
R4
EC50 (HSF1,
41.7
l
M)b
EC50 (MG-132,
15.7
l
M)c
CC50 (Hela,
41.7
l
M)d
CC50 (SK,
26
l
M)d
1
N
N
13
14
15
16
17
18
19
20
21
22
S
52.6
13.5
No activity
61.7
18.8
26.4
20
49.1
26.5
31.1
>80
52.6
>80
49.8
34
S
62.8
60.1
N
O
O
O
O
O
O
O
O
No activity
63% @ 80
49% @ 80
52.6
>80
N
O
F
35% @ 80
>80
N
F
N
N
F
52.7
F
17.4
9.3
55.3
N
32.4
11.1
10.2
10.2
78.9
36.2
NTe
46.4
N
27.1
NTe
N
23.9
66% @ 80
N
23
24
O
O
20.9
23
22.3
12.4
>80
41
N
N
40.7
42.1
25
26
O
O
25.5
16.6
14.7
9.9
45
30.8
N
N
45.7
40.3
(continued on next page)

3132
Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3128–3135
Table 2 (continued)
Compounda
X
O
R4
EC50 (HSF1,
62% @ 80
l
M)b
EC50 (MG-132,
20.9
l
M)c
CC50 (Hela,
38% @ 80
l
M)d
CC50 (SK,
54.2
l
M)d
27
N
N
N
N
28
O
O
O
O
O
O
O
O
24.1
8.9
57.6
67.8
59.9
50.2
33.8
24.4
55.4
48.5
50.4
25
29 (R)f
30 (S)f
31
28.6
7.1
31.9
7.8
31.1
30.6
46
OH
F
28.7
8.2
N
32
22.8
10.1
6.3
N
N
33
12.4
32.7
24.7
>80
34
25% @ 80
No activity
No activity
39.3
NH
O
35
O
HN
S
O
36
O
O
No activity
28% @ 80
41.9
43.6
>80
>80
N
H
O
37
46.9
59.6
N
a
The desired mass was found for each compound in LC–MS analysis. 1H NMR spectra are consistent with their structures. All the compounds showed no activity in HSF1
granule assay without heat shock stress.
b
See Ref. 21 (for HSF1 granule assay condition).
See Ref. 22 (for MG-132 stress assay condition).
See Ref. 24 (for MTS assay condition).
NT=not tested.
c
d
e
f
>95% ee based on the chiral HPLC analysis.
retention and stress induction in the endoplasmic reticulum. In
this assay, the differentiated PC12 cells were treated with tunica-
mycin in the presence of selected compounds at 37 °C for 43 h. Cell
viability was measured with ATPlite and percentage of increased
ATP was calculated by comparing with the DMSO control with
tunicamycin treatment alone. For the compounds tested (1, 25
and 28), excellent cytoprotection from ER stress was observed
(>200%, >150% and ꢀ300%, respectively) as shown in Figure 2.
Next, in order to further evaluate the potential applications of
this series of chloro-oxime derivatives, the cytoprotection effect
of selected compounds in biologically relevant cell models was also
investigated. The polyQ-Htt model that expresses the misfolded
Huntingtin protein is a cell-based model system for Huntington’s
disease. In this assay,26 stably transfected and doxycycline (DOX)
inducible polyQ 145-Htt cells were used. When protein expression
was induced by DOX, Q145 expressing cells showed about 40% cell
viability compared with non-induced cells measured by MTS. In
the presence of either compounds 1 or 25, significant cell protec-
tion was observed resulting in 77% and 58% increase in cell num-
ber, respectively, as compared with the DMSO control with DOX
treatment indicating that the cells were fully rescued from polyQ
toxicity (Fig. 3). This result encourages us to evaluate more com-
pounds of this class in the polyQ cell model in the future.
In order to evaluate the possibility that the increase in cell via-
bility observed above may be caused by stimulation of cell prolif-
eration rather than inhibition of cell death by the compounds
tested, additional experiments were run using Click-iT cell prolifer-
ation assay (data not shown) with selected compounds. We found

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3128–3135
3133
Table 3
SAR of R1 in HSF1 granule and MG-132 assays
Cl
N
N
R1
O
OH
Compound
R1
EC50 (HSF1,
41.7
l
M)
EC50 (MG-132,
15.7
l
M)
CC50 (Hela,
41.7
lM)
CC50 (SK,
26
lM)
N
O
1
F
N
38
39
40
51.3
29.4
20.2
12
11
6.9
>80
>80
40.2
62.4
37.3
20.3
O
N
O
F
F
N
O
N
41
18.5
3.8
19.3
16.9
O
F
N
42
43
44
45
41.6
31
9.5
6.1
6.3
9
65% @ 80
18.8
34.6
28
O
N
O
Cl
Cl
N
20.9
22.9
31.4
23.2
20
O
N
44.2
O
O
O
S
N
O
46
42.9
5.9
41.4
44.2
N
47
48
49
50
41.6
7.9
45.1
18.9
>80
>80
42.4
17.4
>80
N
O
S
O
O
N
O
12.1
5.2
No activity
No activity
No activity
No activity
O
S
67.7

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Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3128–3135
Table 4
SAR of Y in HSF1 granule and MG-132 assays
Y
N
N
N
O
O
OH
Compound
Product or Y
Cl
EC50 (HSF1,
41.7
lM)
EC50 (MG-132,
15.7
l
M)
CC50 (Hela,
41.7
l
M)
CC50 (SK,
26
lM)
1
51
52
53
54
55
H
No activity
No activity
No activity
No activity
No activity
No activity
No activity
No activity
No activity
No activity
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
CH3
OCH3
CF3
F
N
N
N
O
N
O
O
OH
OH
O
N
O
56
57
No activity
No activity
No activity
>80
>80
>80
>80
HN
Cl
N
N
N
O
58.7
O
120%
100%
80%
60%
40%
20%
0%
Compound 1
100%
90.6%
450
400
350
300
250
200
150
100
50
Compound 25
Compound 28
30%
DMSO
DMSO + MG-132
1 + MG-132
0
Figure 1. The percentage of cell numbers of compound 1 with MG-132 treatment
compared with the DMSO control.
0
20
40
60
80
100
Concentration of compounds (µM)
Figure 2. Effect of compounds on the viability of differentiated PC12 cells treated
with tunicamycin for 43 h as compared with the DMSO control treated with
tunicamycin (normalized to 100%).
that neither compound 1 nor 28 caused any cell proliferation up to
20
pounds significantly decreased cell proliferation in the compound
concentration range tested (0–20 M). These results ruled out
lM tested in HeLa cells while in SK-N-SH cells both above com-
l
from MG-132 stress. In addition, both left (R1) and right sides
(R4) of the molecules had effects on activity and cell toxicity. Many
compounds under study showed excellent cytoprotection in the
MG-132 stress cell model with single digit micro molar potency.
Among them, a few compounds further showed great cytoprotec-
tion in ER stress and polyQ-Htt cell models, suggesting that they
may have broad utility in neurodegenerative diseases. As the pol-
the possibility of compound-induced proliferation effect in these
assays, and the cytoprotection observed is likely via the molecular
chaperone amplification mechanism.
In summary, we synthesized a number of chloro-oxime deriva-
tives as novel small molecule chaperone amplifiers. Our SAR stud-
ies revealed that the Cl moiety at the chloro-oxime core played the
essential role in the HSF1 activation activity and cytoprotection

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3128–3135
3135
to eliminate compounds that induce heat shock response in the non-stress
cells. The overall dilution of compound was 200-fold, with a concentration
ranging from 0.3 lM to 80 lM. Immunocytochemical staining for HSF1 in HeLa
120%
100%
80%
60%
40%
20%
0%
106. 2%
100%
94.8%
cells was performed following the previous publication with some
modifications (Ref.: Zhang, B.; Gu, X.; Uppalapati, U; Ashwell, M. A.; Leggett,
D. S.; Li, C. J. J. Biomol. Screen 2008, 13, 538). Image acquisition was 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, granule
area and nuclear intensity coefficient of variation (CV) 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. The maximum percentage of cells that have
positive granule formation >20% is considered active. The compounds were
60%
DMSO
DMSO + DOX
1 + DOX
25 + DOX
Figure 3. The percentage of polyQ 145-Htt cell numbers of compound 1 and 25
induced by DOX compared with the DMSO control.
tested in the concentration range of 0.3–80 lM with twofold dilution. EC50
value is the concentration at which 50% of cells have positive granule
formation. The average standard deviation for the data in Tables 2 and 3
generated from this assay was 34% for multiple testing of same compounds in
different runs.
yQ-Htt cell stress model is directly relevant to the Huntington’s
disease (HD), this class of compounds potentially could be used
as a new therapeutic approach in treating polyglutamine expan-
sion neurodegenerative diseases such as Huntington’s disease.
22. MG-132 stress assay condition: SK-N-SH cells at 12,500 cells/well were
pretreated with compounds one hour before addition of MG-132 at a final
concentration of 5 lM for 24 h. Compound 1, an in house compound serves as a
positive control and DMSO as a negative control. Cell viability was measured
with ATPlite-1step (Perkin Elmer) according to manufacturer’s protocol.
Percentage of increased ATP was calculated using the following formula
(RLU, relative luminescent unit): Increased ATP% = (RLUcompound À RLUDMSO)/
RLUDMSO Â 100%. EC50 values and curve fitting were calculated using PRISM 4.0
References and notes
(GRAPHPAD Software, San Diego, CA) with nonlinear regression analysis. EC50
1. Leigh, P. N.; Whitwell, H.; Garofalo, O.; Buller, J.; Swash, M.; Martin, J. E.; Gallo,
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value is the concentration at which the cell numbers increase 50% by
comparing with MG-132 treatment alone without compound. The
compounds were tested in the concentration range of 0.3–80 lM with
twofold dilution and the maximum percentage of the cytoprotection >60% is
considered active. The average standard deviation for the data in Tables 2 and 3
generated from this assay was 26% for multiple testing of same compounds in
different runs.
3. Muchowski, P. J.; Wacker, J. L. Nat. Rev. Neurosci. 2005, 6, 11–22.
4. Walsh, D. M.; Selkoe, D. J. Protein Pept. Lett. 2004, 11, 213.
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23. Lee, D. H.; Goldberg, A. L. Trends Cell Biol. 1998, 8, 397.
24. MTS assay condition: HeLa (4000 cells/well) and SK-N-SH (14,000 cells/well)
were seeded in DMEM containing 10% FBS for 18 h before experiment.
Compounds or DMSO control were added to the culture at 1–200 dilutions
with a final concentration ranging from 0.3 lM and 80 lM. MTS assay was
performed according to a published protocol (Ref.: Zhang, B.; Gu, X. Uppalapati,
U.; Ashwell, M. A.: Leggett, D. S.; Li, C. J., J Biomol. Screen 2008, 13, 538).
Percentage of inhibition was determined using the following
formula:(MTSDMSO À MTScompound)/MTSDMSO Â 100%. CC50 values and curve
fitting were calculated using PRISM 4.0 (GRAPHPAD Software, San Diego, CA) with
nonlinear regression analysis. The average standard deviation for the data in
Tables 2 and 3 generated from MTS_Hela and MTS_SK assays were 34% and
14%, respectively, for multiple testing of same compounds in different runs.
25. Tunicamycin-induced endoplasmic reticulum (ER) stress assay condition: PC12
cells were plated at a density of 5000 cells/well in 96-well plates. NGF was
added to a final concentration of 50 ng/ml to induce differentiation for 5–
7 days with neurite outgrowth observed. The differentiated PC12 cells were
treated with compound (1:20 dilution) and Tunicamycin (final concentration
at 750 ng/mL) for 43 h at 37 °C. Cell viability was measured with ATPlite
(PerkinElmer) according to the manufacture’s instruction. Percentage of
increased ATP was calculated using the following formula (RLU, relative
11. Boggs, J. BioWorld Today 2009, 20, 1.
12. Corson, T. W.; Crews, C. M. Cell 2007, 130, 769.
13. Westerheide, S. D.; Morimoto, R. I. J. Biol. Chem. 2005, 280, 33097.
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15. Nonaka, T.; Akimoto, T.; Mitsuhashi, N.; Tamaki, Y.; Nakano, T. Cancer Lett.
2003, 202, 89.
16. Au, Q.; Kanchanastit, P.; Barber, J. R.; Ng, S. C.; Zhang, B. J. Biomol. Screen 2008,
13, 953.
17. HSP70 granule assay condition: The assay was performed according to the
previous publication (Ref. 16) which was very similar to the HSF1 granule
assay as described in Ref. 21. Briefly, immunocytochemical staining for HSP70
was performed 2 h after heat shock at 41 °C. Automatic image acquisition was
performed using an INcell 1000 analyzer and analysis was carried out using
Multi Target Analysis (MTA) module from Workstation 3.6 for quantification of
the HSP70 granule count and total granule area.
˘
18. Nagy, P. L.; Balvázs, B.; Boross, M.; Szilbereky, J.; Zsila, G.; Abrahám L.; Blaskó,
luminescent unit): Increased ATP% = (RLUcompound À RLUDMSO)/RLUDMSO
Â
G.; Gachályi, B.; Almási, A.; Német, G. U.S. Patent 5,147,879, 1992.
19. Huang, F.-C. U.S. Patent 4,368,201, 1983.
100%. EC50 values and curve fitting were calculated using PRISM 4.0 (GRAPHPAD
Software, San Diego, CA) with nonlinear regression analysis.
20. a Dressman, B. A.; Godfrey, A. G. WO 00/38685, 2000.; (b) Gaertner, V. R. J. Org.
Chem. 1968, 33, 523; (c) Gaertner, V. R. Tetrahedron Lett. 1967, 343.
21. HSF1 granule assay condition: The assay was performed according to the
previous publication (Ref. 16). Briefly, HeLa cells were pretreated with
compounds 1 h before 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
26. PolyQ-Htt induction model condition: The stable polyQ 145-Htt cells (5000 cells
per well) were treated with doxycycline (10
lg/mL) for 6 days in the presence or
absence of test compounds (0.3–60 M). Cell viability was then measured with
l
MTS assay. The cytoprotection of compounds in polyQ-Htt induction model is
defined as following: (MTScompound À MTSpolyQ alone)/MTSpolyQ alone  100 (%).