Development of Novel N-hydroxypyridone Derivatives as Potential Anti-Ischemic Stroke Agents

Article abstract of DOI:10.1021/acs.jmedchem.9b01338

Our previous study had identified ciclopirox (CPX) as a promising lead compound for treatment of ischemic stroke. To find better neuroprotective agents, a series of N-hydroxypyridone derivatives based on CPX were designed, synthesized, and evaluated in this study. Among these derivatives, compound 11 exhibits significant neuroprotection against oxygen glucose deprivation and oxidative stress-induced injuries in neuronal cells. Moreover, compound 11 possesses good blood-brain barrier permeability and superior antioxidant capability. In addition, a complex of compound 11 with olamine-11·Ola possesses good water solubility, negligible hERG inhibition, and superior metabolic stability. The in vivo experiment demonstrates that 11·Ola significantly reduces brain infarction and alleviates neurological deficits in middle cerebral artery occlusion rats. Hence, compound 11·Ola is identified in our research as a prospective prototype in the innovation of stroke treatment.

Full text of DOI:10.1021/acs.jmedchem.9b01338

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Article
Development of Novel N-hydroxypyridone
Derivatives as Potential Anti-Ischemic Stroke Agents
Linghao Hu, Hong-Xuan Feng, Hongguang Zhang, Songda Yu, Qinyuan Zhao, Wei
Wang, Fengxia Bao, Xun Ding, Jiajing Hu, Manjiong Wang, Yixiang Xu, Zengrui
Wu, Xiaokang Li, Yun Tang, Fei Mao, Xiaoyan Chen, Haiyan Zhang, and Jian Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01338 • Publication Date (Web): 07 Jan 2020
Downloaded from pubs.acs.org on January 7, 2020
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Development of Novel N-hydroxypyridone Derivatives as
Potential Anti-Ischemic Stroke Agents
Linghao Hu1,2,#, Hongxuan Feng3,4,#, Hongguang Zhang , Songda Yu , Qinyuan Zhao ,
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Wei Wang , Fengxia Bao , Xun Ding , Jiajing Hu , Manjiong Wang , Yixiang Xu ,
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1,2,
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Zengrui Wu , Xiaokang Li , Yun Tang , Fei Mao *, Xiaoyan Chen , Haiyan
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,4,
1,2,
Zhang *, Jian Li *
1State Key Laboratory of Bioreactor Engineering, East China University of Science and
Technology, 130 Mei Long Road, Shanghai 200237, China
2Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China
University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China
3CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
4University of Chinese Academy of Sciences, Beijing 100049, China
5Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong
Zhi Road, Shanghai 201203, China
#These authors contributed equally to this work.
*To whom correspondence should be addressed jianli@ecust.edu.cn,
hzhang@simm.ac.cn, maofei@ecust.edu.cn.
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ABSTRACT
Our previous study had identified ciclopirox (CPX) as a promising lead compound
for treatment of ischemic stroke. To find better neuroprotective agents, a series of N-
hydroxypyridone derivatives based on CPX were designed, synthesized and evaluated
in this study. Among these derivatives, compound 11 exhibits significant
neuroprotection against oxygen glucose deprivation (OGD) and oxidative stress
induced injuries in neuronal cells. Moreover, compound 11 possesses good blood-brain
barrier (BBB) permeability and superior antioxidant capability. In addition, complex of
compound 11 with olamine—11·Ola possesses good water solubility, negligible hERG
inhibition and superior metabolic stability. In vivo experiment demonstrates that
11·Ola significantly reduces brain infarction and alleviate neurological deficits in
middle cerebral artery occlusion (MCAO) rats. Hence, compound 11·Ola is identified
in our research as a prospective prototype in the innovation of stroke treatment.
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INTRODUCTION
Ischemic stroke, as the most common form of stroke, accounted for 2.57-2.82 million
deaths per year, and it is a major cause of severe disability among adults worldwide.1-3
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Currently, US Food and Drug Administration (FDA) only approved recombinant tissue
plasminogen activator (rtPA) for thrombolysis in the treatment of ischemic stroke.
However, rtPA must be administered within 4.5 h after stroke, and because of its short
therapeutic time window, only a limited number of ischemic stroke patients can receive
thrombolysis.4, 5 Furthermore, the subsequent ischemia-reperfusion (I/R) injury, which
happens when returning blood flow to ischemic brain tissue after thrombolysis, can
mitigate the benefits of thrombolysis. The clinical use of neuroprotective agents, such
as edaravone and NBP (racemic 3-n-butylphthalide, Figure 1), exerted beneficial
effects on outcome in stroke patients.6,7 Moreover, neuroprotective agents can be
administrated in a relatively wide therapeutic time window. This suggests that the
neuroprotection strategy, namely protecting the penumbra from I/R injury after
8
thrombolysis, is an effective therapy for stroke. Hence, in order to protect ischemic
neurons and minimize I/R injury, it is urgent to develop novel neuroprotective agents
with wide therapeutic time window to provide better prognosis for ischemic stroke
patients. The mechanisms and targets of reported neuroprotective agents include free
radical scavenging, alleviating oxidative stress, ionotropic glutamate receptors, anti-
inflammation and antiplatelet aggregation.9-14 Among these neuroprotective agents, the
well-known NXY-059, a spin-trapping free radical scavenging agent which was firstly
reported in 1990s significantly improved the overall distribution of scores on the
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modified Rankin scale (indicating its potential on reducing disability) in SAINT-I (a
clinical trial involving 1722 patients).15,16 Although NXY-059 had not been approved
17
at last, this work which demonstrated the ability of free radical scavenging agents to
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reduce oxidative stress-induced damage in I/R injury is still enlightening.
Figure 1. Structures of NBP and Edaravone.
In order to find hit compound for ischemic stroke, an FDA-approved drugs library
(778 drugs) was screened through the oxygen glucose deprivation (OGD)-stimulated
ischemic cellular model in our previous study. Among them, the antifungal drug
ciclopirox (CPX) conferred significant neuroprotective effects against the ischemic
18
injury. CPX, which was first reported as an antifungal agent in 1970, is used for the
treatment of tinea pedis and tinea corporis caused by trichophyton rubrum. Later on, it
was reported that CPX could promote the survival of neuronally differentiated PC12
1
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cells in serum-free medium in 1998. In our previous studies, in vivo & in vitro results
had confirmed the effectiveness of CPX for ischemic brain injury. CPX treatment not
only reduced the brain infarction and neurological deficits in middle cerebral artery
occlusion (MCAO) rats significantly, but also ameliorated blood-brain barrier (BBB).20
Moreover, previous study proved that the neuroprotective effects of CPX was
associated with the enhancement of AKT and GSK3β phosphorylation.20
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In this study, CPX was selected as a lead compound for anti-stroke drug discovery.
As the course of NBP and edaravone treatment is typically 14 – 20 days, long-lasting
anti-ischemic effect is vital to ischemic stroke treatment. Thus, besides neuroprotective
activity, metabolic stability which contributes to long-lasting efficacy is also important
for anti-stroke agents. CPX consists of the N-hydroxyl pyridone ring, methyl at position
4 and cyclohexyl at position 6 (Figure 2). Therefore, in order to elucidate the structure
and activity relationship and improve neuroprotective activity and metabolic stability,
N-hydroxyl pyridone derivatives were designed and synthesized by replacing the
hydrogens or substituents at position 1, 3, 4, 5 and 6 with different substituents , and
their neuroprotective activities were evaluated through in vitro and in vivo experiments.
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Figure 2. Structures of CPX and Pyridone Derivatives.
Among them, compound 11 showed better neuroprotective activity, good BBB
permeability and superior antioxidant capacity. Moreover, 11·Ola (complex of 11 and
olamine) showed better metabolic stability, good water solubility, safety and better
efficacy in vivo, indicating that it possesses development potential as a prospective anti-
ischemic stroke agent.
RESULTS and DISCUSSION
Design and Synthesis
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To identify the pharmacophore of CPX, compounds 1-4 were designed and
synthesized, firstly. The hydroxyl at position 1 was replaced by ethoxyl, 2-methoxy-2-
oxoethoxy and carboxymethoxy to produce compounds 1–3 (ineffective, Table 1).
Compound 4 was given by replacing the pyridone of CPX with isoquinoline. After N-
hydroxypyridone was confirmed as the pharmacophore, the methyl at position 4 was
replaced by hydrogen and ethyl to produce compound 5 and compound 6. Because of
the inferior activity of compounds 5 and 6 (Table 1), methyl was fixed as the substituent
at position 4 for compounds 7-31. Next, to explore the ideal substituent at position 6,
compounds 7-19 were produced by replacing the cyclohexyl with alkyls, cycloalkyls,
norbornane, phenyl and benzyl. Then, in order to elucidate the effects of substituents at
position 3 and 5, halogen substituted derivatives (compounds 20-23) and formyl or
aminomethyl substituted derivatives (compounds 24-31) were designed and
synthesized.
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The synthetic route for the preparation of compounds 1-19 is shown in Scheme 1.
Reaction of CPX with iodoethane and methyl 2-bromoacetate produced compound 1
and 2, respectively (reactions a-b, Scheme1-I). Compound 3 was obtained by
hydrolyzing the methyl of compound 2 using lithium hydroxide (reaction c, Scheme1-
I). Reaction of commercially available 2-iodobenzoic acid and methanol in the presence
of sulfurous dichloride generated methyl 2-iodobenzoate (4a). Then Intermediate 4a
was used in the subsequent Sonogashira cross coupling with ethynylcyclohexane to
generate 4b, which was then refluxed in methanol with hydroxylamine hydrochloride
and hydroxyl potassium to afford compound 4 (Scheme1-II). Intermediate 5a was
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obtained through Kumada cross coupling of 2-bromo-6-methoxypyridine and
cyclohexylmagnesium bromide, which was oxidized by mCPBA to generate
intermediate 5b. Intermediate 5b was reflux in acetyl chloride and then in methanol to
afford compound 5. (Scheme 1-III). Using ethyl (Z)-3-methylpent-2-enoate (A6) and
cyclohexyl chloride as starting material, compound 6 was generated through reactions
j, k, m and n, this synthesis method is similar to that of compounds 7-19 as following.
Reaction of methyl 3-methylbut-2-enoate (A0, commercially available) with
corresponding acyl chloride in presence of aluminum trichloride provided different
substituted acyl α, β- unsaturated esters, which were then heated with a mixture of
sulfuric acid and acetic acid to give intermediates B7-B18 (Scheme 1-IV, reactions j,
k). The synthesis of B19 is an exception. The starting materials of 19 are bromoacetyl
chloride and methyl 3-methylbut-2-enoate, and the corresponding product 6-
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(bromomethyl)-4-methyl-2H-pyran-2-one was then used in the Suzuki cross coupling
with phenylboronic acid in the presence of Pd(dppf)Cl to generate intermediate B19
2
(Scheme 1-IV, reaction l). Intermediates B7-B17, B19 spontaneously formed C7-C17,
C19 when heated with tetraphosphorus decasulfide in toluene (Scheme 1-IV, reaction
m). Compounds C7-C17, C19 and hydroxylamine hydrochloride were refluxed in
pyridine to afford target compounds 7-17, 19 (Scheme 1-IV, reaction n). Intermediate
B18 directly formed compound 18 through an adjusted reaction n, see experiment
section.
Scheme 1. Synthesis of Compounds 1-19.
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O
O
O
OH
I.
O
N
OH
O
N
O
N
a
b
O
c
O
O
O
N
1
I
CPX
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O
OH
O
I
O
II.
O
O
N
d
e
f
OH
O
4a
4b
4
III.
O
N
OH
N
O
N
Br
O
N
O
O
g
h
i
5
5a
5b
O
O
*
OH
N
IV.
R2 Cl
j, k, l
R2
R2
R2
O
O
S
O
O
m
n
R0
R1
O
R1
R1
C6-17 & C19
R1
A0 or A6
B6-19
6-19
1
2
A0 = methyl 3-methylbut-2-enoate
B13, C13, 13, R =methyl, R =propyl
1
0
1
2
A6, R =ethyl, R =ethyl
B14, C14, 14, R =methyl, R =isobutyl
1
2
1
2
B6, C6, 6, R =ethyl, R =cyclohexyl
B15, C15, 15, R =methyl, R =pentyl
1
2
1
2
B7, C7, 7, R =methyl, R =cyclopropyl
B16, C16, 16, R =methyl, R =heptyl
1
2
1
2
B8, C8, 8, R =methyl, R =cyclobutyl
B17, C17, 17, R =methyl, R =nonyl
1
2
1
2
B9, C9, 9, R =methyl, R =cyclopentyl
B18, 18, R =methyl, R =phenyl
1
2
1
2
B10, C10, 10, R =methyl, R =cycloheptyl
B19, C19, 19, R =methyl, R =benzyl
1
2
B11, C11, 11, R =methyl, R =endo-bicyclo[2.2.1]heptan-2-yl
1
2
B12, C12, 12, R =methyl, R =methyl
The reactants of B19 are bromoacetyl chloride and A0.
*
Reagents and conditions: (a) iodoethane, K CO , DMF, 60 °C; (b) methyl 2-
2
3
bromoacetate, K CO , acetonitrile, reflux; (c) lithium hydroxide, water/THF, 40 °C; (d)
2
3
SOCl , then methanol and Et N; (e) ethynylcyclohexane, Et N, CuI, PdCl (PPh ) ,
2
3
3
2
3 2
toluene, 4.5 h, 40 °C under Ar; (f) NH OH, KOH, CH OH, reflux; (g)
2
3
cyclohexylmagnesium bromide, Pd(dppf)Cl ; (h) mCPBA, DCM, reflux; (i) acetyl
2
chloride, reflux, then MeOH; (j) AlCl , DCM, reflux; (k) H SO , acetic acid; (l) only
3
2
4
for B19: phenylboronic acid, Pd(dppf)Cl , Cs CO , 1,4-dioxane, reflux; (m) P S ,
2
2
3
4 10
toluene, reflux; (n) NH OH·HCl pyridine, reflux.
2
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The synthetic route for the preparation of compounds 20-31 is shown in Scheme
. CPX and N-halogen butanimide were refluxed in carbon tetrachloride in the presence
2
of initiator (AIBN) to afford halogen substituted (at position 3 or 5) pyridone
derivatives 20–23 (Scheme 2-II, reaction a). The sodium salt of compound 20 (prepared
by adding sodium hydride into the THF solution of compound 20) and tert-butyl lithium
were used in the halogen-lithium exchange reaction, then reacted with DMF to afford
the aldehyde, namely compound 24 (Scheme 2-II, reaction b). Compound 25-31 were
synthesized through reductive amination using compound 24 as reactant (Scheme 2-II,
reaction c).
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7
8
9
0
Scheme 2. Synthesis of Compounds 20-31.
I.
1
2
OH
N
OH
N
20, R =Br, R =H
a
O
1
2
O
21, R =H, R =Br
1
2
2
2, R =I, R =H
R1
R2
1
2
23, R =Cl, R =H
CPX
20-23
II.
OH
N
OH
N
R3 NH2
c
OH
N
b
O
Br
O
O
O
H
H
N
R3
20
24
25-31
3
3
2
2
2
2
5, R =methyl
29, R =cyclopentyl
3
3
6, R =propyl
30, R =phenyl
3
3
7, R =isopropyl
31, R =heptyl
3
8, R =propargyl
Reagents and conditions: (a) NBS/NIS/NCS, AIBN, CCl , reflux; (b) NaH, THF, 0 °C,
4
then tert-BuLi, DMF, THF, -78 °C; (c) Na(CH COO) BH, acetic acid, Cl(CH ) Cl, r.
3
3
2 2
t.
Pyridone Analogues Protecting SH-SY5Y Cells against Oxygen-Glucose
Deprivation (OGD) Damage
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Firstly, the neuroprotective effect of the compounds 1-31 was evaluated by
Oxygen-Glucose Deprivation (OGD) model, which is an in vitro model for ischemic
stroke, and it was widely used in the study of anti-stroke agents21-23. The results are
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
summarized in Tables 1-3 and Figure 3.
As shown in Table 1, compounds 1-3 with the substituents of ethoxyl, 2-methoxy-
1
2-oxoethoxy and carboxymethoxy at position 1 (R ) showed no neuroprotective activity.
This results indicate that hydroxyl at position 1 is necessary for the neuroprotective
activity of pyridone analogues. Compound 4 which was obtained by replacing pyridone
ring of CPX with isoquinoline showed good neuroprotective activity (78.49%) at 1 µM,
but no activity at 10 µM. Considered the activity of compounds 1-4, the N-
hydroxypyridone was fixed as the core of compounds 5-31. Compound 5 with hydrogen
at position 4 showed good neuroprotective activity (76.61%) at 10 µM, but no activity
at 1 µM. Compound 6 with ethyl substituent at position 4 showed moderate
neuroprotective activity at both 1 µM and 10 µM. The activities of compounds 5 and 6
is inferior to CPX. Therefore, methyl was fixed as the substituent at position 4 for
compounds 7-31.
Table 1. Effect of Compounds 1-6 on SH-SY5Y Cells Exposed to OGDa.
R1
N
OH
N
O
O
R4
1, 2, 3, 5, 6
4
Substituents
Cell Viability (% of Control)
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compd.
R1
R4
1 µM
10 µM
70.13 ± 0.91***
46.33 ± 9.22
49.94 ± 5.38
69.75 ± 4.17**
43.86 ± 6.76
CPX
-OH
-CH3
-CH3
-CH3
-CH3
1b
2
-OC H
2
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
56.44 ± 0.61
55.89 ± 3.71
61.62 ± 9.04
76.61 ± 1.64***
67.34 ± 5.25*
O
O
OH
3
56.05 ± 6.44
78.49 ± 5.81**
4
----
5
-OH
-OH
-H
51.75 ± 5.71
69.81 ± 4.08**
6
-C H
2
5
OGD
54.92 ± 2.12%###
aCell viability was measured by MTT assay at 24 h after OGD exposure. The compounds 1-6 at
concentrations of 1 µM or 10 µM were added to the cells immediately before OGD stimulus. The
data are presented as the percentage of surviving cells relative to control cells (100%) as the mean
±
SD, n = 3. ###p < 0.001 vs control, *p < 0.05, **p < 0.01, ***p < 0.001 vs OGD group, one-way
b
ANOVA, Dunnett's multiple comparison test. The data of compound 1 is gathered from another
assay (n = 3) in which the cell viability of OGD group was 49.91% of control.
The cell viability of groups treated with compounds 7-11 demonstrated that the
neuroprotective activity at 1 μM of compounds with large cycloalkyl substituents (11:
endo-norbornane, 10: cycloheptyl) at position 6 was better than compounds with
smaller substituents (7: cyclopropyl, 8: cyclobutyl). Compound 9 with cyclopentyl
substituent at position 6 showed good neuroprotective activity (70.66% at 10 µM), but
no activity at 1 μM. Compound 10 with cycloheptyl substituent at position 6) showed
good neuroprotective activity (71.01% at 1 µM). It is noteworthy that compound 11
1
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(endo-norbornane at position 6) showed good neuroprotective activity at both 1 µM
(70.62%) and 10 μM (64.46%). Compounds 12, 13, 15-17 showed no neuroprotective
activity (Table 2), revealing unbranched alkyl substitution at position 6 was adverse to
neuroprotective activity. Whereas compound 14 with branched alkyl substitution
isobutyl (Table 2) showed slightly better activity at 10 µM (73.30%) compared with
CPX, but no neuroprotective activity at 1 µM. So alkyl substitution at position 6 is not
favored compared to cycloalkyl or bicycloalkyl substitution. Compound 18 bearing a
phenyl substituent at position 6 showed no neuroprotective activity. Compound 19
bearing a benzyl substituent at position 6 showed comparable activity to that of CPX at
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0 µM, but no neuroprotective activity at 1 µM. Generally speaking, large cycloalkyl
or bicycloalkyl substitution at position 6 is better than small cycloalkyl, alkyl, phenyl
and benzyl substitution.
Table 2. Effect of Compounds 7-19 on SH-SY5Y cells Subjected to OGDa.
OH
O
N
R6
Cell Viability (% of Control)
Cell Viability (% of Control)
Compd.
CPX
R6
Compd.
R6
1
μM
10 μM
1 μM
10 μM
70.13 ± 0.91***
69.75 ± 4.17**
( )2
64.04 ± 5.58*
13
47.04 ± 2.90
7
8
9
58.77 ± 4.31
46.02 ± 7.0
52.36 ± 3.22
62.75 ± 15.02
71.15 ± 8.81*
70.66 ± 6.15**
14
15
16
47.67 ± 5.72
63.55 ± 2.28
71.68 ± 7.74
73.30 ± 3.55**
61.96 ± 13.60
58.52 ± 12.01
( )4
( )6
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
( )8
1
1
0
1
71.01 ± 2.40*
70.62 ± 3.83**
55.81 ± 5.82
38.42 ± 7.86
64.46 ± 4.26*
18.33 ± 4.84
17
18
19
52.98 ± 8.66
56.96 ± 2.52
46.46 ± 4.59
42.52 ± 8.19
20.13 ± 1.76
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CH3
12
76.77 ± 5.72**
OGD = 54.92 ± 2.12%###
aThe data are presented as the percentage of surviving cells relative to control cells and as the mean
SD, n=3. ###p < 0.001 vs control, *p < 0.05, **p < 0.01, ***p < 0.001 vs OGD group, one-way
±
ANOVA, Dunnett's multiple comparison test.
As shown in Table 3, compounds 20-31 are series of N-hydroxypyridone
derivatives with halogen, N-substituted aminomethyl or formyl substituents at position
3 or 5, and the substituents at position 4 and position 6 of compounds 20-31 are methyl
and cyclohexyl respectively. The introduction of bromine at position 5 (21) or iodine at
position 3 (22) produced a considerable activity improvement at 1 µM. Compounds 20
(
bromine at position 3), 23 (chlorine at position 3) and 24 (3-formyl derivative) showed
no activity at both 1 µM and 10 µM. Compounds 25-31 are a series of compounds
bearing N-substituted aminomethyl substitution at position 3 (Table 3). Compound 26
(
N-propyl aminomethyl), 27 (N-isopropyl aminomethyl) and 30 (N-phenyl
aminomethyl) exhibited neuroprotective activity at 10 µM, and 29 (N-cyclopentyl
aminomethyl) showed moderate activity at 1 µM. Compound 31 showed no activity at
1
µM, but good activity at 10 µM (87.18%) (Table 3). Compound 25 (N-methyl
aminomethyl) showed moderate activity at 10 μM, but no activity at 1 μM. However,
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compound 28 (N-propargyl aminomethyl) had no effects in OGD-exposed SH-SY5Y
cell. In general, the neuroprotective activity of compounds with aminomethyl
substitution at position 3 did not improve compared to CPX.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Among these 31 compounds, some compounds showed lower neuroprotective
effects at 10 μM than that at 1 μM. To evaluate whether these compounds have toxic
effect on normal cells at higher concentration, we tested the influence of the compounds
(1, 3, 4, 5, 6, 10, 11, 12, 15, 16, 17, 18, 20, 21, 22, 23, 28, 29 and CPX) on the cell
viability of SH-SY5Y cells without OGD insult. The results indicated CPX, 10, 11 and
29 reduce the viability at the concentration of 10 μM (Table S7), which might be related
with their lower neuroprotective activity at concentration of 10 μM.
Table 3. Effect of Compounds 20-31 on SH-SY5Y Cells Exposed to OGDa.
OH
O
N
R3
R5
20-31
Substituents
Cell Viability (% of Control)
Compd.
R3
-H
-Br
-H
-I
R5
1 µM
10 µM
69.75 ± 4.17**
59.87 ± 7.08
CPX
-H
-H
-Br
-H
-H
70.13 ± 0.91***
62.88 ± 5.41
2
2
0
1
81.04 ± 6.97*
76.35 ± 5.61**
63.40 ± 4.55
63.20 ± 11.04
55.12 ± 8.29
57.08 ± 4.07
22
2
3
-Cl
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
24
-CHO
-H
-H
54.77 ± 6.49
56.52 ± 4.99
67.47 ± 7.40*
H
N
2
5
60.90 ± 4.03
61.01 ± 4.51
60.95 ± 2.87
63.62 ± 5.22
67.04 ± 5.96*
56.81 ± 3.69
60.00 ± 12.37
H C
3
H
68.01 ± 2.47**
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
26
N
-H
-H
-H
-H
-H
-H
H
N
65.21 ± 4.25*
2
2
7
8
H
N
61.62 ± 8.41
H
29
61.87 ± 5.02
N
H
N
75.47 ± 7.12**
3
0
H
1b
N
87.18 ± 2.07**c
3
6
OGD = 54.92 ± 2.12%###
aThe data are presented as the percentage of surviving cells relative to control cells and as the mean
±
SD, n=3. ###p < 0.001 vs control, *p < 0.05, **p < 0.01, ***p < 0.001 vs OGD group, one-way
b
ANOVA, Dunnett's multiple comparison test. The data of compound 31 is gathered from another
c
assay of which the cell viability of OGD group was 54.23±4.48% of control. An exception, the
concentration was 4 µM.
In the first round OGD experiment, the concentrations of the tested compounds
are 1 µM and 10 µM. Among them, seven of the tested compounds (4, 6, 10, 11, 21,
2
2, 29) exerted neuroprotective effect at 1 µM, and they were selected for further
studies. Although the cell viability of SH-SY5Y cells treated with 1 µM of compound16
was beyond 70%, there was no statistical significance. An additional experiment
confirmed that it is ineffective at 1 µM (Figure S6). Some of the derivatives showed
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inferior neuroprotective effect at 10 µM compared to the effect at 1 µM, thus, in order
to further evaluate the activity of these seven compounds, it was necessary to set more
doses. Thus, we carried out another OGD experiment in which the doses of the
compounds were ranged from 0.25 µM to 4 µM (0.25, 0.5, 1, 2, 4 µM, Figure 3). There
were three compounds showed excellent neuroprotective activity (cell viability > 80%):
10 (at 2 µM), 11 (at 1, 2, 4 µM), 22 (at 2 µM). Among them, compound 11 provided
excellent neuroprotective effect at 1, 2, 4 µM (cell viability of these 3 doses are all
above 80%, p < 0.001).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Neuroprotective Effect of Compounds 4, 6, 10, 11, 21, 22, 29 at
Concentrations in the Range from 0.25 μM to 4 μM on SH-SY5Y Cells Exposed to
OGD Injury. The data are presented as the percentage of surviving cells relative to
control cells (100%) and as the mean ± SD, n=3, ###p < 0.001 vs control, *p < 0.05,
*
*p < 0.01, ***p < 0.001 vs OGD group, one-way ANOVA, Dunnett's multiple
comparison test.
Structure and Activity Relationships
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Analysis of the data revealed the structures and activities relationship (SAR) of
these derivatives: (I). The N-hydroxyl is necessary for the neuroprotective activity.
Replacing the N-hydroxyl with ethoxyl, 2-methoxy-2-oxoethoxy and carboxymethoxy
(compounds 1-3) leads to the loss of activity (Table 1). (II). Methyl is the optimal
substituent at positon 4. Replacing methyl with hydrogen and ethyl reduced the
neuroprotective activity of compounds 5-6 (Table 1). (III). Large cycloalkyl and
bicycloalkyl substituents at position 6 are better than small cycloalkyl, alkyl, phenyl
and benzyl substituents (compounds 10-11 vs 7-9, 12-19, Table 2). (IV). Halogen
substituents at position 3 or 5 has no obvious influence to the neuroprotective activity
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(compounds 20-23, Table 3). (V). The N-substituted aminomethyl at position 3 could
not improve the neuroprotective activity.
Antioxidant Ability of Pyridone Analogues.
In the pathological process of ischemia-reperfusion, there are excessive free
radicals including ROS generated within the penumbra, causing oxidative damage.21
Therefore the antioxidant property of compound is considered as an important indicator
for its neuroprotective activity.22, 23 The antioxidant activities of compounds with good
neuroprotective activities in OGD model (exhibited significant improvement in cell
viability compared with OGD group at 1 µM) were evaluated by the oxygen radical
absorbance capacity assay (ORAC).24 In this experiment, the fluorescent probe
fluorescein can react with the peroxide radical induced by AAPH, which is widely used
as the free radical reactions initiator, to generate a non-fluorescent substance, and the
quenching of fluorescence can be slowed by antioxidants. This process closely mimics
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the production and scavenging of in vivo peroxide radicals. Compared with other assays
like DPPH assay, the ORAC method combines both inhibition time and degree of
inhibition into a single quantity though measuring the kinetics of the decrease in
fluorescence for each sample compared to a blank and building an antioxidant curves
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5
(
fluorescence versus time, normalized to the curve of the blank). In general, ORAC
values were expressed as trolox equivalents which were calculated through plugging
the area under the curve (AUC) into the regression equations between the net AUC and
Trolox concentrations, the higher values indicated the better antioxidant activity. The
results in Table 4 manifested that compound 11, which exhibited the best
neuroprotective effect in OGD model, also demonstrated superior antioxidant activity
(1.13 ± 0.19) compared to CPX (0.52 ± 0.32).
BBB Permeability of Pyridone Analogues
Good BBB penetration ability is required for central nervous system drugs. To
evaluate the BBB penetration ability of pyridone analogues, the permeability (Pe)
values of compounds with good neuroprotective activities in OGD model (4, 6, 10, 11,
21, 22, 29) were determined through the parallel artificial membrane permeation assay
(PAMPA, using lipid extract of porcine brain covered membrane). 13 marketed drugs
-6
were tested to build the standard that compounds with P values greater than 3.08 × 10
e
cm/s could cross the BBB by passive permeation (marked CNS+). Among them,
−
6
−6
−6
compounds 11 (7.50 × 10 cm/s), 22 (7.65 × 10 cm/s) and CPX (8.17 × 10 cm/s)
show good BBB permeation. In short, compound 11 demonstrated the best
neuroprotective activity in OGD model, and it also has good BBB permeability ability
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1
1
1
1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
−
6
(7.50 × 10 cm/s) and better antioxidants capability (1.13 ± 0.19) compared to lead
compound CPX (0.52 ± 0.32). Thus, compound 11 was selected for further studies.
Table 4. Oxygen Radical Absorbance Capacity (ORAC, Trolox Equivalents) and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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8
9
0
1
2
3
4
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6
7
8
9
0
−
6
−1
Permeability Results (P ×10 cm s ) from the PAMPA-BBB Assay for Compounds
e
4
, 6, 10, 11, 21, 22, 29.
−6
−1 b
Compd.
ORACa
P (10 cm s )
Prediction
e
CPX
0.52 ± 0.32
NA
8.17 ± 1.43
4.43 ± 0.72
4.53 ± 0.34
4.42 ± 0.57
7.50 ± 0.33
3.97 ± 0.25
7.65 ± 0.52
3.89 ± 2.01
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
4
6
NA
1
0
NA
11
1.13 ± 0.19
NA
2
2
1
2
0.20 ± 0.17
NA
29
aValues are expressed as the mean ± SD of three independent experiments, NA means no activity.
b
Data are expressed as μmol of Trolox equivalent/μmol of tested compounds. Values are expressed
as the mean ± SD of three independent experiments.
Water-Solubility of Compounds 11 and 11·Ola
Since marketed anti-stroke drugs include butylphthalide and edaravone are
administered through injection, the water-solubility is important for anti-stroke agent.
Therefore the solubility of 11 and its complex with olamine in normal saline (0.9 % for
intravenous injection) was calculated by comparing the HPLC peak area to the
calibration curves which were plotted as peak areas versus concentrations. However,
compound 11 shows poor water-solubility (1.22 mg/mL). Ciclopirox olamine is the
complex consist of ciclopirox and 2-aminoethan-1-ol (olamine is USAN-approved
contraction for ethanolamine), it is water-soluble and widely used in external
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medicament. Olamine is not only used in external medicament but also used in oral
medicaments like eltrombopag olamine, and it is considered a low toxic compound.
Thus, the complex of compound 11 and olamine (named 11·Ola for short) was
synthesized and it processed good water-solubility (10.90 mg/mL), as shown in Table
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
. Compound 11·Ola was an ideal form for intravenously administration in the in vivo
experiment.
Table 5. Water-solubility of Compounds 11 and 11·Ola (n = 3, mean ± SD).
OH
N
OH
N
O
O
.
H N
2
OH
Compd.
Water-solubility (mg/mL)
1
1
1.22 ± 0.01
10.90 ± 1.53
1.43 ± 0.03
6.82 ± 0.45
11·Ola
CPX
CPX Olamine
The Anti-apoptotic Effect of Compound 11·Ola on OGD-exposed SH-SY5Y Cells
Considering that inflammation and oxidative damage caused by the I/R injury can
lead to neuronal apoptosis which results in neuron death in the penumbra, reduction of
apoptotic rate is another important indicator of neuroprotective capability.21, 26 Thus,
Annexin-V-FITC and PI staining was performed to examine whether the
neuroprotective effects of compound 11·Ola on SH-SY5Y cells correlated with
reduced apoptosis. The distribution of SH-SY5Y cells to the lower left quadrant
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indicated that these cells were viable and without measurable apoptosis. OGD insults
induced obvious increases in cell numbers in the lower and upper right quadrants, which
indicated that these SH-SY5Y cells underwent early and late apoptosis. (Figure 4A)
The percentage of SH-SY5Y cells at early and late stages of apoptosis increased from
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5.88% to 40.98% in the OGD group compared to the control group (Figure 4B).
Treatment with compound 11·Ola at 4 μM significantly reduced the apoptotic ratio to
5.91% (Figure 4B). Moreover, the OGD treatment induced obvious morphological
2
changes of SH-SY5Y cells, including neurite disappearance, bright and shrinking cell
body, and reduced cell number. 11·Ola treatment remarkably attenuated the cell
morphological changes (Figure 4C). The results above indicated that treatment with
11·Ola not only reduce the cell apoptosis but also protect the cell morphology against
OGD injury.
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Figure 4. Compound 11·Ola Decreases OGD-induced Apoptosis in SH-SY5Y Cells.
(A) Representative Plot of Annexin V-FITC/PI Staining; (B) Statistical Results of
Apoptotic rate (n = 4, error bars represent standard error of the mean (SEM), ###p <
1
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0.001 vs control, ***p < 0.001 vs OGD group, t-test); (C) Photomicrographs of cell
morphology (scale bar = 100 μm).
Neuroprotective Effect of Compound 11·Ola on Glutamate-exposed HT22 Cells
and H O -exposed SH-SY5Y Cells.
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2
In the process of I/R injury, the release of glutamate activates ionotropic glutamate
receptors and causes excitotoxicity which can result in neuronal death. In addition, the
consequent reactive oxygen species (ROS) are also account for neuronal death in the
ischemic penumbra. Therefore, in vitro models using glutamate and H O stimulus are
2
2
widely used to mimic ischemic stroke injury.27-30 In present study, we further evaluated
the neuroprotection of optimal compound 11·Ola on glutamate and H O stimulated
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2
cellular models. As shown in Figure 5A, excessive glutamate exposure significantly
reduced the cell viability of HT22 cells to 28.53% of the control, but incubation with
11·Ola at concentrations of 0.5 μM, 1 μM, 2 μM and 4 μM significantly protected the
cells against the glutamate insult, and the protective effect is similar to that of ciclopirox
olamine. Moreover, as shown in Figure 5B, 100 μM H O reduced cell viability to 45.73%
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of the control. Compound 11·Ola at 1 μM, 2 μM and 4 μM promoted cell viability up
to 80%, which is also similar to that of ciclopirox olamine. These results suggested that
compound 11·Ola could attenuate oxidative damage in SH-SY5Y cells and glutamate-
induced toxicity in HT22 cells.
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Figure 5. Neuroprotective Effect of Compound 11·Ola on HT22 Cells Subjected to
Glutamate Stimulus (A) and SH-SY5Y Cells Exposed to H O Injury (B). The data are
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presented as the percentage of surviving cells relative to control cells (100%) and as the
mean ± SD, n = 3 for (A), n = 4 for (B). ###p < 0.001 vs control, **p < 0.01, ***p <
0
.001 vs glutamate group or H O group, one-way ANOVA, Dunnett's multiple
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comparison test.
Iron Chelation and bSDTNBI Prediction Results
The hypoxia-inducible factor (HIF)-1α plays an important role in the regulation of
gene expression in response to hypoxia and ischemia. It induces transcription of genes
involved in energy metabolism, angiogenesis and erythropoiesis which promotes the
neuronal survival under ischemic conditions.31 HIF-1α is regulated by HIF-prolyl
hydroxylases (PHDs), in other words, hydroxylation of the proline residue leads to the
degradation of HIF-1α. Iron ion which is utilized as a cofactor in the hydroxylation
process is necessary for the activity of PHDs. Moreover, it is reported that stabilizing
HIF-1α through the inhibition of PHDs significantly reduced the infarct volumes and
3
2
improved behavior after MCAO. Previous study had confirmed that ciclopirox could
induce HIF-1α stability through iron chelating.33 In this study, in silico bSDTNBI
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prediction also suggested the strong correlation between HIF1A gene (encoded HIF-
1
α) and the neuroprotective activity of CPX and compound 11 (CPX: HIF1A ranked
34
4
th, 11: HIF1A ranked 5th, Figure 6A). The bSDTNBI (balanced substructure–drug–
1
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target network-based inference) is a network-based method which can identify new
drug–target interactions (DTIs) for further understanding the mechanism of action
(MoA). Derived from recommendation algorithms for networks, it uses resource
diffusion processes to predict potential targets for both known drugs and new chemical
entities and does not rely on either three-dimensional structures of target proteins or
negative samples. Furthermore, it can predict potential ligands for targets for the
development of potential targeted therapies (Figure S2 Schematic diagram of
bSDTNBI). Here, iron chelating experiments was performed to clarify the relationship
between the iron chelation and neuroprotective activity of 11, CPX and 1, and the ion
chelating ability was determined by UV−visual spectroscopy. As shown in Table 1 &
2, both CPX olamine and 11·Ola exerted good neuroprotective activity, and compound
1
showed no activity. As shown in Figure 6B & 6C, the absorption between 350 nm-
500 nm increased after the addition of ammonium iron(III) citrate and FeSO to both
4
1
1 solutions and the CPX solutions. The change of the absorption curves of 11 and CPX
demonstrated the production of the iron chelating complex. In addition, the color charge
of these solutions also corresponded with the curve change (Figure S3. photographs).
The addition of the other three iron salts (ammonium iron(III) sulfate, ammonium
iron(II) sulfate, FeCl ) led to similar changes in the absorption curve of the solutions,
3
which indicated that these changes result from iron ion, not anion. (Figure S4). On the
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contrary, the curve of compound 1 was almost unchanged when FeSO and ammonium
4
iron(III) citrate were added into the solutions (Figure 6D), this indicated that compound
1
(no neuroprotection) did not chelate iron ion. These findings suggested that the
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neuroprotective activity of 11 and CPX might correlate with iron chelation and
stabilization of HIF1-α.
Figure 6. The bSDTNBI Prediction (A), Absorption Spectra of CPX, 11, and 1 (60μM)
Alone and in the Presence of FeSO , Ammonium Iron(III) Citrate (120 μM) (B, C, D).
4
Compound 11·Ola Attenuated Brain Injury of MCAO Rats.
In view of compound 11·Ola possesses superior neuroprotective, good antioxidant
ability, BBB permeability and water-solubility in vitro, its in vivo neuroprotective
effect was further evaluated through a widely-used MCAO model for ischemic stroke
study.28, 35, 36 In our previous studies, the dose for CPX olamine in rat which afford the
best efficacy was 3 mg/kg (i.v.), hence, this dose was used in our experiment for
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comparison. Due to the minimum effective dose of compound 11 is much lower than
CPX (Figure 3), two experiment groups treated with 0.3 mg/kg (i.v.) and 1 mg/kg (i.v.)
of compound 11·Ola were set, respectively. As shown in Figure 7A, B, an obvious
infarction (39%) was induced by MCAO insult, which displayed as the white region of
brain sections. Treatment with 1 mg/kg of compound 11·Ola significantly reduced the
infarct volume to 23% (Figure 7A, B, p < 0.01 vs MCAO group) and decreased the
neurological severity scores to 5 (p < 0.01 vs MCAO group, Figure 7C), and 0.3 mg/kg
dose of compound 11·Ola reduced the infarct volume to 25% (p < 0.05 vs MCAO group,
Figure 7B). As a comparison, treatment with 3 mg/kg of CPX reduced the infarcted
volume by 26% (p < 0.01 vs MCAO group) and decreased the neurological severity
scores to 6 (p < 0.05 vs MCAO group). Above experiments indicated that compound
1
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0
1
1·Ola can significantly protected the brain and attenuated neurological deficits after
I/R injury in vivo and its initial dose of efficacy is lower than that of CPX olamine. The
protective effects of 11·Ola at the dose of 1 mg/kg was comparable to that of CPX
olamine at 3 mg/kg, and taken together the similar cytotoxicity of 11·Ola and CPX
olamine (see below Table 6), suggesting that 11·Ola possesses superior safety window
compared to CPX olamine.
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NNN
Figure 7. Effect of Compound 11·Ola on Infarct Area and Neurological Function after
2 h of MCAO and 24 h of Reperfusion. (A) Representative Picture of TTC staining, (B)
Quantification of Infarct Volumes, (C) Neurological Severity Scores. (n=15-18, data
are presented as mean  standard error of the mean (SEM), ###p < 0.001 vs sham group,
*
p < 0.05, **p < 0.01, vs vehicle-treated MCAO group, one-way ANOVA, Dunnett's
multiple comparison test.).
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The hERG Potassium Channels Inhibition, Cytotoxicity and Metabolic Stability
of Compound 11·Ola and CPX Olamine
The safety is important for drug candidates, so we further evaluated the optimal
compound 11·Ola through hERG potassium channel inhibition assay and cytotoxicity
assay. The hERG potassium channels inhibition can lead to the prolongation of Q-T
interval, therefore, it is an important indicator of cardiotoxicity. The hERG IC value
1
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of compound 11·Ola and CPX olamine is assessed through automated patch-clamp
37
assay, and the results in Table 6 shows that the hERG inhibition of compound 11·Ola
and CPX olamine is negligible (IC values of them are above 40 μM, Table 6). This
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suggests that these two compounds are unlikely to cause cardiovascular safety risk
arising from hERG inhibition.
MRC-5 is a human cell line composed of fibroblasts which were derived from the
embryonal lung tissue, and it is widely used in the evaluation of the toxicity in
medicinal chemistry research.38, 39 The IC values in SH-SY5Y cell line were assessed
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as the indicator of neurotoxicity. Therefore, we assessed the cytotoxicity of 11·Ola and
CPX olamine against MRC-5 and SH-SY5Y as indicators of the safety of these two
compounds. As shown in Table 6, the IC of both 11·Ola and CPX olamine are all
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above 100 μM. So these two compounds are safe for neurons at their therapeutic doses.
Considering that neuroprotective agents are administered continuously during the
middle and late stages, we prolonged the incubating time to 72 h for MRC-5, and the
IC values of 11·Ola and CPX olamine in MRC-5 cell line at 72 h are 23.60 ± 1.15
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μM and 23.47 ± 4.19 μM respectively (Table 6). To compare the activity of 11·Ola and
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