Discovery of Highly Potent 2-Sulfonyl-Pyrimidinyl Derivatives for Apoptosis Inhibition and Ischemia Treatment

Article abstract of DOI:10.1021/acsmedchemlett.6b00489

A series of 2-sulfonyl-pyrimidinyl derivatives was developed as apoptosis inhibitors. These represent the first class of apoptosis inhibitors that function through stabilizing mitochondrial respiratory complex II. Starting from a phenotypic screen hit with micromolar activity, we optimized the cellular apoptosis inhibition activity of 2-sulfonyl-pyrimidinyl derivatives to picomolar level (compound 42, also named as TC9-305). The therapeutic potential of these new apoptosis inhibitors was further demonstrated by their neuroprotective effect on an ischemic animal model.

Full text of DOI:10.1021/acsmedchemlett.6b00489

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Letter
Discovery of Highly Potent 2-Sulfonyl-Pyrimidinyl
Derivatives for Apoptosis Inhibition and Ischemia Treatment
Li Li, Xian Jiang, Shaoqiang Huang, Zhengxin Ying, Zhao-Lan
Zhang, Chenjie Pan, Sisi Li, Xiaodong Wang, and Zhiyuan Zhang
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00489 • Publication Date (Web): 01 Mar 2017
Downloaded from http://pubs.acs.org on March 2, 2017
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Discovery of Highly Potent 2-Sulfonyl-Pyrimidinyl Derivatives for
Apoptosis Inhibition and Ischemia Treatment
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Li Li§,†, Xian Jiang†, Shaoqiang Huang†, Zhengxin Ying†, Zhaolan Zhang†, Chenjie Pan†, Sisi Li†,
Xiaodong Wang*,† and Zhiyuan Zhang*,†,#
§School of Life Sciences, Peking University, Beijing, 100871, China
†National Institute of Biological Sciences (NIBS), Beijing, 102206, China
#Collaborative Innovation Center for Cancer Medicine,Beijing, 102206, China
KEYWORDS. Apoptosis inhibitors; phenotypic screen; structure-activity optimization; ischemia treatment; mitochon-
dria protection.
ABSTRACT: A series of 2-sulfonyl-pyrimidinyl derivatives was developed as apoptosis inhibitors. These represent the first
class of apoptosis inhibitors that function through stabilizing mitochondrial respiratory complex II. Starting from a phe-
notypic screen hit with micromolar activity, we optimized the cellular apoptosis inhibition activity of 2-sulfonyl-
pyrimidinyl derivatives to picomolar levels. The therapeutic potential of these new apoptosis inhibitors was further
demonstrated by their neuroprotective effect on an ischemic animal model.
Excessive apoptosis is closely associated with immunode-
ficiency diseases, liver diseases, ischemia-associated injury,
and neurodegenerative diseases, including Alzheimer's
syndrome, Parkinson's syndrome, Huntington's syndrome,
and amyotrophic lateral sclerosis.1-7 Upon recognition of
stimulus signals that initiate mitochondria-mediated
apoptosis, the decision of whether or not to initiate apop-
tosis is determined via the complicated regulation of pro-
and anti-apoptotic BCL-2 (B-cell lymphoma-2) protein
family members.1 BH-3 only proteins (tBid, Bim, etc) trig-
ger the conformational activation of Bax and/or Bak,
which in turn oligomerize and translocate to the mito-
chondrial outer membrane and lead to mitochondrial
permeability changes.1,8 These changes enable the release
of pro-apoptotic proteins such as cytochrome c and Smac
to the cytoplasm.1,8,9 These pro-apoptotic factors then
mediate the activation of caspases and ultimately lead to
cell death.
are known to have complementary functions.1,8,9 There-
fore, there is an urgent need to develop more efficient
apoptosis inhibitors.
In this work, we report the medicinal chemistry effort in
the development of a series of 2-sulfonyl-pyrimidinyl de-
rivatives as highly potent apoptosis inhibitors. Starting
from a phenotypic screen hit compound with micromolar
activity, we optimized the cellular apoptosis inhibition
activity in a series of derivative compounds to picomolar
level. We also revealed the neuroprotective effect of this
class of compounds in a transient focal cerebral ischemia
model, further demonstrating their therapeutic potential
in treating diseases related to excessive apoptosis. To our
knowledge, this class of apoptosis inhibitors is by far the
most potent apoptosis inhibitors reported.
Besides, by chemical genetic methods using one of these
compounds (compound 33, also named compound A), we
recently reported the identification of the succinate de-
hydrogenase subunit B (SDHB) of mitochondrial respira-
tory complex II as the target protein, which is also a new
target for apoptosis inhibition.17 Compound 33 inhibits
apoptosis by stabilizing mitochondrial respiratory com-
plex II and protecting the integrity of the mitochondrial
electron transport chain.17 Together, we revealed a class of
new and potent apoptosis inhibitors that act on a novel
target in the apoptosis signaling pathway, and demon-
strated their therapeutic potential in excessive apoptosis-
related disease models.
The great majority of research into apoptosis inhibitors
has focused on targeting caspases or proteins of the pro-
apoptotic BCL-2 family.10 Blocking caspase activity can
stop the final execution step of apoptosis, but, by this
stage, cells have already suffered considerable damage
that typically induces other types of cell death such as
necrosis.11,12 Bax and Bid inhibitors have been developed to
target apoptosis.13-16 However, these apoptosis inhibitors
have only moderate potency, with EC50 values above or
around the micromolar level. Additionally, blocking a
single pro-apoptotic BCL-2 protein may have only limited
effects because other pro-apoptotic BCL-2 family proteins
A chemical library containing 200,000 small molecules
was screened for compounds that block apoptosis in a cell
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line (U2OS_Bim) where overexpression of a pro-apoptotic the necessity for the strong electron withdrawing proper-
BCL-2 member Bim is induced by Doxycycline (DOX).18
Several active hits were identified and further confirmed
as having EC50 values below 20 μM for maintaining cell
survival following Bim-induced apoptosis. All screening
hits were resynthesized, and their bioactivities were re-
confirmed. Of these hit compounds, N-(2-hydroxy-5-
methylphenyl)-4-((4-(thiophen-2-yl)-6-(trifluoromethyl)
pyrimidin-2-yl)sulfonyl)butanamide (Fig. 1a, EC50=4.0 μM)
could prevent mitochondrial cytochrome c release (Fig.
1b), which could not be achieved by caspase inhibitor
zVAD. This indicated the 2-sulfonyl-pyrimidinyl com-
pound functions upstream of caspase activation, and be-
tween BCL-2 family member regulation and cytochrome c
release. Therefore, this compound was selected for further
study.
ty of the original –CF3 group.
1
2
3
4
5
6
7
8
Table 1. SAR study of compounds 1-20
EC50
(nM)a
749
9
No.
1
R1
R2
R3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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
51
52
53
54
55
56
57
58
59
60
±11
1428
±291
2
1135
±242
3
1591
±206
4
∗
4207
±651
5
6
>20000
>20000
>20000
7
8
9
>20000
2201
±238
10
11
6642
±1261
Figure 1. The hit compound identified from a phenotypic
screen of apoptosis inhibition can block mitochondrial re-
lease of cytochrome c after Dox induced apoptosis. (a) Struc-
ture and apoptosis inhibiton activity of the hit compound,
and the the side groups (R1, R2, and R3) targeted in SAR de-
velopment. (b) Immunofluorescence imaging of cytochrome
c after treatment with the indicated compounds. The arrow
on the middle column indicated an example of cytochrome c
release. Scale bar: 20 μm.
3487
±418
12
13
6355
±469
7033
±421
14
3516
±446
15
16
To obtain more potent compounds to be used as both
target identification tools and potential drug candidates,
we undertook rational structure-activity relationship
(SAR) optimization. We started by optimization of frag-
ment R1 (Table 1). In each case, replacement of R1 with
aliphatic groups of varying sizes increased the apoptosis
inhibition activity by 3-5 fold (compounds 1-3). Aromatic
substitutes were also tested (compounds 4,5), and their
activities were similar to that of the hit compound. These
results indicate that fragment R1 was able to tolerate a
relatively large change in its structure and maintain the
activity. Because compound 1 had the strongest apoptosis
inhibition activity, we replaced the original structure of R1
with a methyl group for further SAR optimization of other
parts of the molecule (R2 and R3, Figure 1).
>20000
>20000
17
8756
±1213
18
19
>20000
1157
±66
20
(a), EC50: cellular apoptosis inhibition on U2OS_Bim cell line.
We next conducted SAR optimization of fragment R3 (Ta-
ble 1). Although the replacement of the original thiophene
group with phenyl rings (compound 10) resulted in de-
creased activity, it offered more options for testing the
We next substituted the –CF3 group at the R2 position
with different groups (compounds 6-9); all such substitu-
tions led to the total loss of activity (Table 1). This may be
related to either a constrained binding environment or
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Table 2. SAR study of compounds 21-33
influence of substituents on different positions of the ring.
Compounds with –CH3 and –COOCH3 substitutions at
1
2
the 2-, 3-, and 4-positions of the phenyl ring had diver-
gent activities (compounds 11-16). Substitutions at both
the 2- and 4-positions (compounds 11, 13, 14, 16) led to
decreased activity compared with compound 10, whereas
substitutions at the 3-position (compounds 12, 15) re-
sulted in EC50 values similar to 10. This suggested more
open space around the 3-position of the phenyl group.
Compound 17 with a -CON(CH3)2 group had a complete
loss of activity, probably because of its polarity or unfa-
vourable conformation. Though compound 18, which has
a 3-oxybenzene group, had a 3-fold decrease in activity
compared with compound 10, the result agreed with our
assumption on more open space at this direction and en-
couraged us to do further SAR morphing at this position.
Considering the convenience of modification and synthe-
sis, we chose to replace the original thiophene group at
the R3 position with the pyridone ring (compound 19) as
an intermediate for further SAR exploration of the 3-
position. Compound 19 had no activity, possibly because
of the polarity of the pyridone ring. However, by adding a
benzyl group to the N atom of the pyridone we got com-
pound 20, of which the activity increased by 2-folds com-
pared with 10. This result further confirmed that a hydro-
phobic pocket has been reached by the addition of benzyl
group.
Encouraged by the result, SAR exploration on the benzyl
ring was then performed in more detail (Table 2). The
effects of chloro-substitution at different positions of the
benzyl ring (compounds 21-23) were evaluated. The 3- and
4-chloro substitutions of the benzyl ring (compounds 22,
23) led to increased apoptosis inhibition activity. We next
focused on substitutions at these two positions. Com-
pound 24, which has a 3-OH substitution, had the same
activity as compound 20 and was less potent than the 3-
chloro compound 22, which suggested a hydrophobic
environment around the benzyl ring. The 3-OMe substi-
tution of the benzyl ring (compound 25) increased activi-
ty by about 20-fold compared with compound 20. The –
OEt and –OPr substituted compounds 26 and 27 dis-
played similar activities to that of compound 25. We sub-
sequently examined the influence of 4-OMe substitution
(compound 28) and found that it increased activity by 12-
fold compared with compound 20, similar to the increase
observed for compound 25. In addition to oxyalkyl substi-
tution, we also tested the activities of ethynyl and cyano
groups at the 3- and 4- positions of the benzyl ring (com-
pounds 29-32). Only compound 30, which has an ethynyl
substitution at the 4-position of the benzyl ring, had an
increase in activity compared with compound 20, and this
increase was mild. Ethynyl substitution at the 3-position
and cyano substitution at both positions led to dimin-
ished activity. Based on these results, we next combined
the 3- and 4- position –OMe substitutions in compound
33 (compound A), which showed yet a further increase in
apoptosis inhibition activity, with an EC50 as low as 25 nM.
Thus compound 33 is 150-fold more potent than the initial
hit compound.
3
4
5
6
7
8
9
EC50 (nM)a
No.
R4
2-Cl
3-Cl
21
22
23
24
25
26
27
28
29
30
31
1143±235
257±37
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4-Cl
66±4.2
3-OH
1821±371
57±1.7
3-OMe
3-OEt
110±25
3-OPr
69±9.7
4-OMe
3-ethynyl
4-ethynyl
3-cyano
4-cyano
3,4-dimethoxy
94±9.6
4477±1040
817±189
4060±477
3489±184
25±0.57
32
33b
(a), EC50: cellular apoptosis inhibition in the
U2OS_Bim cell line.
(b), compound A.
To better understand the structural requirements for ac-
tivity and to further optimize potency, we undertook a
second round of SAR optimization at the original R1 posi-
tion (Table 3). Because the previous SAR analysis sug-
gested a large tolerance for various structures, we focused
on testing if extension of the R1 fragment could improve
activity.
We first replaced the methyl group in compound 33 with
n-pentyl and n-heptyl groups (compounds 34 and 35), but
lengthening the alkyl chain largely reduced cellular activi-
ty. Ether structures (compounds 36 and 37) were then
tried; however, these did not improve the cellular activity
much, either. An amide structure was then tested. Com-
pound 38, which has N, N-dimethylbutyramide at the R1
position, had an EC50 of 164 nM; this is a mild increase
compared with the activities of compounds 36 and 37.
Replacement of the dimethylamine with pyrrolidine (39)
further increased the activity by ~5-fold compared with
compound 38. We then tried the piperidine at the amide
bond (compound 40), and activity increased dramatically
to an EC50 of 1.4 nM, which is much higher than observed
for compounds 33 and 39. These observations indicate
that large aliphatic moieties at the amide position are
favorable for activity, and we speculate that the extended
R1 moiety possibly interacts hydrophobically with the tar-
get protein. Consistent with our assumption, the intro-
duction of an unsubstituted amide, a butyramide struc-
ture, at R1 (compound 41), was detrimental to activity.
Therefore, to increase the hydrophobicity of the R1 frag-
ment, we synthesized compound 42, which has a highly
hydrophobic adamantane moiety. The activity of this
compound increased to an EC50 of 0.42 nM, which is 50-
fold higher than the parent compound 33.
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assay (Table S2).22 Before apoptosis induction and cell
Table 3. SAR study of compounds 34-42
1
2
3
4
5
6
7
8
viability determination, two substets of cells were incu-
bated with tested compounds, and then one subset of
cells was washed several times to eliminate the unbound
test compounds while the other subset of cells was not
washed. Tested compounds all had similar EC50 values
under these two conditions, indicating irreversible bind-
ing modes for these compounds.
CF3
N
O
R1
N
S
O
O
N
O
O
No.
R1
EC50
(nM)a
In addition to blocking mitochondrial-mediated apoptosis,
this class of 2-sulfonyl-pyrimidinyl derivative apoptosis
inhibitors could also block the dysfunction of mitochon-
dria that occurs following the induction of apoptosis. To
evaluate mitochondrial function, we measured changes in
mitochondrial membrane potential under different condi-
tions using tetramethylrhodamine methyl ester (TMRM),
a membrane potential sensitive red fluorescent dye. Dox
induction of Bim overexpression is known to result in the
loss of mitochondrial membrane potential and thus di-
minish TMRM enrichment during apoptosis.23 All of the
tested compounds maintained TMRM enrichment and
mitochondria membrane potential, an outcome that can-
not be achieved with the caspase inhibitor zVAD (Figure
2a). The mitochondrial protective effect of the 2-sulfonyl-
pyrimidinyl derivative apoptosis inhibitors was further
supported by measurements of reactive oxygen species
(ROS) levels taken following the induction of apoptosis.
ROS levels are known to increase the following interrup-
tion of the electron transport chain and the resulting
changes in membrane permeability that occur during
apoptosis. Such ROS increases are understood to be stim-
ulative factors for apoptosis induction and to cause fur-
ther damage to cells.24 Treatment with the novel apopto-
sis inhibitor compounds maintained normal ROS levels,
even upon apoptosis induction, in a dose-dependent
manner (Figure 2b).
9
34
35
36
364±13.4
253±17.5
216±20.9
282±27.9
10
11
12
13
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21
22
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24
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42
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37
38
N
164±12.5
27±2.43
∗
O
39
40
N
∗
O
N
1.4±0.19
>20000
∗
O
41
42
0.42±0.05
NH
∗
O
(a), cellular apoptosis inhibiton activity.
In addition to testing the inhibition activity of the 2-
sulfonyl-pyrimidinyl derivative compounds on Bim over-
expression induced apoptosis, we also evaluated if they
could block apoptosis induced by the overexpression of
tBid, another pro-apoptotic protein of the Bcl-2 family.
Most compounds displayed similar apoptosis inhibition
activities, and compound 42 had an EC50 of 0.23 nM (Ta-
ble S1).
It is now firmly established that excessive apoptotic cell
death is prominent in neurological disorders, such as Alz-
heimer's disease and Parkinson’s disease, and it is also a
main cause for cerebral ischemic stroke.25 Following cere-
bral ischemia, BH3-only proteins are upregulated and
activate the intrinsic apoptosis pathway.26 Compound 33
has already been shown to promote cell survival in a Par-
kinson’s disease model.17 Here, we tested the potential
efficiency of this series of apoptosis inhibitor compounds
in a rat transient focal ischemia model.27 A nylon suture
was introduced to occlude the origin of the middle cere-
bral artery (MCA). A 5μL solution of different concentra-
tions of compound 33 or 42 was injected into the left stria-
tum after suture insertion. The suture was removed 1hr
after insertion to allow reperfusion. After 24hr, animals
were sacrificed. Evaluation of the brain infarct volume in
different groups showed that both compounds 33 and 42
had obvious protective effects on brain tissues and re-
duced the brain infarct volume due to induction of focal
cerebral ischemia (Fig. 3). Consistent with the SAR study,
at injection concentrations of 50μM and 200μM, com-
pound 42 had a better protective effect than compound 33.
By chemical genetic methods using a biotinylated deriva-
tive of compound 33 as bioaffinitive probe, we previously
reported that compound 33 covalently binds the 243 cys-
teine of SDHB through an SN2 substitution at the sulfone
position. 17 Sulfone can serve as a good leaving group in
SN2 substitution reactions when linked to electron-
withdrawing aromatic rings.19-21 In the SAR study, we ob-
served that the replacement of -CF3 at the R2 position
with less powerful electron-withdrawing groups totally
abolished activity (Table 1, compounds 6-9). In model
reactions with different nucleophilic reagents (cysteine,
lysine, and glutathione) (Figure S1, S2), we observed that
compounds 1, 33 and 42 reacted with cysteine and gluta-
thione but not lysine, whereas compound 6, of which the
electron-withdrawing group -CF3 was replaced by an elec-
tron-donating CH3, didn’t react with any of them. This
indicated the reactivity of the sulfone group in the tested
compounds was consistent with the cellular activity of the
corresponding compounds. We also confirmed that active
2-sulfonyl-pyrimidinyl derivative compounds shared the
covalent binding property using a cellular wash/no-wash
In conclusion, using medicinal chemistry we systematical-
ly developed a novel and highly potent series of apoptosis
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Figure 2. Mitochondrial protective effects of a series of apoptosis inhibitors. (a) TMRM staining of mitochondria in US2OS-Bim
cells with (1),no apoptosis induction and treatment with DMSO, and with apoptosis inducted by the addition of DOX in the
presence of the following treatments: (2), DMSO; (3), zVAD (10μM); (4), compound 20 (10μM); (5), compound 22 (1μM); (6),
compound 23 (1μM); (7), compound 33 (100nM); (8), compound 42 (3 nM). Scale: 100 μm. (b) Mean cellular ROS levels in U2OS-
Bim cells with (red bars) and without (blue bars) Dox induced apoptosis under the indicated compound treatments. Top: ROS
levels under treatment with various apoptosis inhibitor compounds, DMSO, and zVAD. Bottom: ROS levels under treatment
with different concentrations of compound 42, DMSO, and zVAD (20µM). The error bars represent the standard error of results
from two separate experiments. P values are based on comparisons of each sample to the DMSO-treated apoptosis induction
group. *: P<0.05; **: P<0.01; ***: P<0.001.
Figure 3. Compound 33(a) and compound 42 (b) have protective effects against ischemia-reperfusion injury. left: Representative
2, 3, 5-triphenyltetrazolium chloride (TTC) stained sections from middle cerebral artery occlusion (MCAO) brains injected with
5µl of the indicated concentrations of compounds 33 and 42. The white color represents the infarct and the red color represents
the normal tissue. Right: Quantification of relative infarct volume. The numbers 1-7 on the x-axis correspond to the treatments
in the top panel. Bars represent means ± standard deviation, n=3-6. P values are based on comparisons of each sample to the
DMSO-treated ischemia-reperfusion(+) group.*: P,0.05; **: P<0.01; ***: P<0.001.
inhibitors. Based on an initial hit compound from pheno-
typic screening, we improved the cellular apoptosis inhi-
bition activity of 2-sulfonyl-pyrimidinyl derivatives from
low micromolar to picomolar levels through SAR optimi-
zation, and verified their covalent binding mode. This
series of compounds showed mitochondrial protective
effect as well as apoptosis inhibition. Following our previ-
ous work on the identification of SDHB subunit of mito-
chondrial respiratory complex II as the interacting target
of compound 33 and the demonstration of protective ef-
fect on Parkinson model, we further showed that com-
pounds 33 and 42 are also highly efficacious on transient
focal cerebral ischemia model, indicating the great thera-
peutic potential in the development of novel therapies to
treat apoptosis-related diseases.
ASSOCIATED CONTENT
Supporting Information
Detailed experimental procedure, supporting figures and
tables, synthesis and characterization of all compounds and
intermediates, are listed in Supporting Information. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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(16) Becattini, B.; Culmsee, C.; Leone, M.; Zhai, D.; Zhang, X.;
AUTHOR INFORMATION
Crowell, K. J.; Rega, M. F.; Landshamer, S.; Reed, J. C.; Plesnila, N.,
Structure–activity relationships by interligand NOE-based design
and synthesis of antiapoptotic compounds targeting Bid. Proceedings
of the National Academy of Sciences 2006, 103 (33), 12602-12606.
(17) Jiang, X.; Li, L.; Ying, Z.; Pan, C.; Huang, S.; Li, L.; Dai, M.; Yan,
B.; Li, M.; Jiang, H., A Small Molecule That Protects the Integrity of
the Electron Transfer Chain Blocks the Mitochondrial Apoptotic
Pathway. Molecular Cell 2016, 63 (2), 229-239.
1
2
3
4
5
6
Corresponding Author
*Email: zhangzhiyuan@nibs.ac.cn
Author Contributions
All authors have given approval to the final version of the
manuscript.
7
8
9
(18) Jiang, X.; Jiang, H.; Shen, Z.; Wang, X., Activation of
mitochondrial protease OMA1 by Bax and Bak promotes cytochrome
c release during apoptosis. Proceedings of the National Academy of
Sciences 2014, 111 (41), 14782-14787.
Funding Sources
This work is supported by National High Technology Project
973 (2011CB504300).
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Prof. Zhenhua Zhang (China Agricultural Universi-
ty) for providing several azide reagents (synthesized in the
National Key Technologies R&D Program of China,
2015BAK45B01, CAU) and Mr. Qinsi Ji for his synthesis work.
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Figure 1. The hit compound identified from a phenotypic screen of apoptosis inhibition can block
mitochondrial re-lease of cytochrome c after Dox induced apoptosis. (a) Structure and apoptosis inhibiton
activity of the hit compound, and the the side groups (R1, R2, and R3) targeted in SAR development. (b)
Immunofluorescence imaging of cytochrome c after treatment with the indicated compounds. The arrow on
the middle column indicated an example of cytochrome c release. Scale bar: 20 µm.
Of these hit compounds, N-(2-h
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Figure 2. Mitochondrial protective effects of a series of apoptosis inhibitors. (a) TMRM staining of
mitochondria in US2OSꢀBim cells with (1),no apoptosis induction and treatment with DMSO, and with
apoptosis inducted by the addition of DOX in the presence of the following treatments: (2), DMSO; (3),
zVAD (10ꢁM); (4), compound 20 (10ꢁM); (5), compound 22 (1ꢁM); (6), compound 23 (1ꢁM); (7),
compound 33 (100nM); (8), compound 42 (3 nM). Scale: 100 ꢁm. (b) Mean cellular ROS levels in U2OSꢀBim
cells with (red bars) and without (blue bars) Dox induced apoptosis under the indicated compound
treatments. Top: ROS levels under treatment with various apoptosis inhibitor compounds, DMSO, and zVAD.
Bottom: ROS levels under treatment with different concentrations of compound 42, DMSO, and zVAD
(20µM). The error bars represent the standard error of results from two separate experiments. P values are
based on comparisons of each sample to the DMSOꢀtreated apoptosis induction group. *: P<0.05; **:
P<0.01; ***: P<0.001.
All of the tested compounds ma
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Figure 3. Compound 33(a) and compound 42 (b) have protective effects against ischemiaꢀreperfusion injury.
left: Representative 2, 3, 5ꢀtriphenyltetrazolium chloride (TTC) stained sections from middle cerebral artery
occlusion (MCAO) brains injected with 5µl of the indicated concentrations of compounds 33 and 42. The
white color represents the infarct and the red color represents the normal tissue. Right: Quantification of
relative infarct volume. The numbers 1ꢀ7 on the xꢀaxis correspond to the treatments in the top panel. Bars
represent means ± standard deviation, n=3ꢀ6. P values are based on comparisons of each sample to the
DMSOꢀtreated ischemiaꢀreperfusion(+) group.*: P,0.05; **: P<0.01; ***: P<0.001.
Evaluation of the brain infarc
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