Discovery of a novel series of N-hydroxypyridone derivatives protecting astrocytes against hydrogen peroxide-induced toxicity via improved mitochondrial functionality

Article abstract of DOI:10.1016/j.bmc.2016.12.052

Astrocytes play a key role in brain homeostasis, protecting neurons against neurotoxic stimuli such as oxidative stress. Therefore, the neuroprotective therapeutics that enhance astrocytic functionality has been regarded as a promising strategy to reduce brain damage. We previously reported that ciclopirox, a well-known antifungal N-hydroxypyridone compound, protects astrocytes from oxidative stress by enhancing mitochondrial function. Using the N-hydroxypyridone scaffold, we have synthesized a series of cytoprotective derivatives. Mitochondrial activity assay showed that N-hydroxypyridone derivatives with biphenyl group have comparable to better protective effects than ciclopirox in astrocytes exposed to H₂O₂. N-hydroxypyridone derivatives, especially 11g, inhibited H₂O₂-induced deterioration of mitochondrial membrane potential and oxygen consumption rate, and significantly improved cell viability of astrocytes. The results indicate that the N-hydroxypyridone motif can provide a novel cytoprotective scaffold for astrocytes via enhancing mitochondrial functionality.

Full text of DOI:10.1016/j.bmc.2016.12.052

Accepted Manuscript
Discovery of a novel series of N-hydroxypyridone derivatives protecting astro-
cytes against hydrogen peroxide-induced toxicity via improved mitochondrial
functionality
Sarbjit Singh, Ja-Il Goo, Hyojin Noh, Sung Jae Lee, Myoung Woo Kim, Hyejun
Park, Hitesh B. Jalani, Kyeong Lee, Chunsook Kim, Won-Ki Kim, Chung Ju,
Yongseok Choi
PII:
S0968-0896(16)31045-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.052
BMC 13479
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 October 2016
28 December 2016
31 December 2016
Please cite this article as: Singh, S., Goo, J-I., Noh, H., Jae Lee, S., Woo Kim, M., Park, H., Jalani, H.B., Lee, K.,
Kim, C., Kim, W-K., Ju, C., Choi, Y., Discovery of a novel series of N-hydroxypyridone derivatives protecting
astrocytes against hydrogen peroxide-induced toxicity via improved mitochondrial functionality, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2016.12.052
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Graphical Abstract
Discovery of a novel series of N-
hydroxypyridone derivatives protecting
astrocytes against hydrogen peroxide-
induced toxicity via improved mitochondrial
functionality
Leave this area blank for abstract info.
Sarbjit Singh, Ja-Il Goo, Hyojin Noh, Sung Jae Lee, Myoung Woo Kim, Hyejun Park, Hitesh B. Jalani, Kyeong
Lee, Chunsook Kim, Won-Ki Kim, Chung Ju,* and Yongseok Choi,*

Bioorganic & Medicinal Chemistry
journal h omepage: www.els evier. com
Discovery of a novel series of N-hydroxypyridone derivatives protecting astrocytes
against hydrogen peroxide-induced toxicity via improved mitochondrial functionality
Sarbjit Singh,a,‡ Ja-Il Goo,b,‡ Hyojin Noh,c,‡ Sung Jae Lee,b Myoung Woo Kim,b Hyejun Park,b Hitesh B.
Jalani,b Kyeong Lee,a Chunsook Kim,d Won-Ki Kim,c Chung Juc,* and Yongseok Choib,*
a College of Pharmacy, Dongguk University-Seoul, Goyang 10326, Korea.
bSchool of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea.
cDepartment of Neuroscience, College of Medicine, Korea University, Seoul 02841, Korea.
dDepartment of Nursing, Kyungdong University, Wonju 24695, Kangwon-do, Korea.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Astrocytes play a key role in brain homeostasis, protecting neurons against neurotoxic stimuli
such as oxidative stress. Therefore, the neuroprotective therapeutics that enhance astrocytic
functionality has been regarded as a promising strategy to reduce brain damage. We previously
reported that ciclopirox, a well-known antifungal N-hydroxypyridone compound, protects
astrocytes from oxidative stress by enhancing mitochondrial function. Using the N-
Available online
hydroxypyridone scaffold, we have synthesized
a series of cytoprotective derivatives.
Mitochondrial activity assay showed that N-hydroxypyridone derivatives with biphenyl group
have comparable to better protective effects than ciclopirox in astrocytes exposed to H2O2. N-
hydroxypyridone derivatives, especially 11g, inhibited H2O2-induced detrioration of
mitochondrial membrane potential and oxygen consumption rate, and significantly improved
cell viability of astrocytes. The results indicate that the N-hydoxypyridone motif can provide a
novel cytoprotective scaffold for astrocytes via enhancing mitochondrial functionality.
Keywords:
Ciclopirox
N-hydroxypyridone
Astrocytes
Oxidative stress
Neuroprotection
2009 Elsevier Ltd. All rights reserved.
1. Introduction
range for fungi as secondary metabolites and exhibit an
extensive diversity of biological functions including anti-
Astrocytes play a key role in the maintenance of
homeostasis and function of the central nervous system. One
of major homeostatic function of astrocytes provides
defence mechanism against oxidative stress to neurons. In
various neurological disorders associated with oxidative
stress, astrocyte dysfunction has been implicated in the onset
and progression of neurodegenerative process.1 Furthermore,
recent evidence suggests that enhancing or recovering
astrocytic functions could represent a valuable strategy for
neuroprotection.2 For example, selective upregulation of
astrocyte-derived factors such as Nrf2, lactate, or heme
oxygenase-1 in astrocytes rescues activity or viability of
nearby neurons under neurotoxic stimuli.3–6 Therefore,
astrocytes has been emerging as an attractive target for novel
drug discovery, especially in neurodegenerative diseases
such as Alzheimer’s disease or Parkinson’s diseases, as well
as ischemic or traumatic brain injury.7
oxidant,8 anti-bacterial,9,10 anti-fungal,11 and anti-malarial
activities.12
Among synthetic N-hydroxypyridone derivatives, the
anti-fungal agent, ciclopirox [6-cyclohexyl-1-hydroxy-4-
methyl-2(1H)-pyridone] is one of the most well-known
compounds. Although it has been used as a topical treatment
for cutaneous fungal infections, recent phase I clinical study
in patients with refractory hematologic malignancies
demonstrated the oral systemic regimen was well tolerated
in humans.13 Furthermore, recent studies supported an
appealing possibility to repurpose ciclopirox as an antibiotic
against drug-resistant infections,14 anti-tumor agent,15 anti-
human immunodeficiency virus drug,16 or an agent to
enhance angiogenesis and wound healing in diabetic
environment.17
Another attractive possibility of repurposing ciclopirox is
as a cytoprotective drug against oxidative stress, especially
through protection of mitochondrial functionality.
Mitochondria are the source of free radicals and in turn, the
major target of oxidative stress. While impaired
mitochondrial functionality in astrocytes contributes to
neuronal injury,18–20 protection of mitochondria in astrocytes
enhances neuronal survival.21 For example, the
N-hydroxypyridone motif is included in the structure of a
variety of bioactive compounds. Naturally occurring N-
hydroxypyridone derivatives are often synthesized in a wide
‡These authors contributed equally to this work.
*Co-corresponding authors:
Email addresses: ychoi@korea.ac.kr (YS), cj2013@korea.ac.kr (CJ)

observed no cyclization or very poor yield if any. To address
this issue we designed another retrosynthetic plan as shown
in figure 1B. In this synthetic plan, the substituents on the
acetylene derivatives determines the side chain group on
final N-hydroxypyridone derivatives, which would be
employed to palladium catalyzed coupling with but-2-ynoate
to afford enyne esters. The enyne esters would be subjected
to electrophilic cyclization to allow the desired pyridones.
For this purpose, we first synthesized a variety of
acetylene derivatives as shown in scheme 1. Acetylenes
overexpression of antioxidant molecules, such as superoxide
dismutase 2 (SOD2), glutathione, or chaperones, such as
HSP70 or GRP 78 has proven to effectively reduce neuronal
death in experimental models of brain diseases.21 In line
with this view, we previously found that ciclopirox protects
astrocytes from peroxynitrite-induced cytotoxicity by
attenuating mitochondrial
inhibited peroxynitrite-induced
dysfunction.22
depolarization
Ciclopirox
of
mitochondrial transmembrane potential (Δψm) and resulting
ATP depletion. Ciclopirox also blocks H2O2-induced
mitochondrial injury in SK-HEP-1 cells by maintaining
Δψm and preventing mitochondrial permeability transition
pore (MPTP) opening.23 Ciclopirox acted directly on
isolated mitochondria and efficiently blocked H2O2-induced
depolarization of Δψm.
In this context, we synthesized a series of various N-
hydroxypyridone derivatives and explore the efficacy of
ciclopriox and its derivatives against oxidative stress-evoked
cytotoxicity in astrocytes. Here we demonstrate that
ciclopirox and selected derivatives effectively inhibited
H2O2-induced mitochondrial dysfunction and thus, resulting
cytotoxicity along with structure-activity relationship (SAR)
study of the derivatives.
4a
h
—
d
were commercially available whereas acetylenes 4e
were synthesized by Sonogashira coupling of
) with different bromobenzene
employing catalytic amount of
—
trimethylsilyl acetylene (
derivatives 1e
2
(
—h)
Pd(Ph3)2Cl2 and CuI in Et3N at 60°C, followed by
deprotection of trimethylsilyl group of corresponding
compounds 3e
1). Acetylene derivatives 4i
Sonogashira coupling of 1-bromo-4-iodobenzene (
ethynyltrimethylsilane ( ) using Pd(PPh3)2Cl2/CuI in Et3N
to afford ((4-bromophenyl)ethynyl)trimethylsilane ),
followed by coupling of later with different boronic acids
7i ) via Suzuki reaction to afford products 3i . The
phenylacetylene derivatives 4i were then obtained in
quantitative yields by deprotection of corresponding
compounds 3i by K2CO3 in MeOH:THF (1:1).
—
h
by K2CO3 in THF:MeOH (1:1) (scheme
were synthesized by
) with
—m
5
2
(
6
(
—
m
—m
2. Results and discussion
—
m
2.1. Chemistry
—
m
For the synthesis of N-hydroxypyridone derivatives, we
focused on the modification of cyclohexane moiety of
ciclopirox with diverse aromatic rings since multi-aromatic
ring containing compounds such as flavonoids are well
known for their anti-oxidant properties. For the purpose,
retrosynthetic analysis was carried out as shown in figure 1.
Figure 1A represents the classical approach of synthesis of
ciclopirox where the source of cyclohexyl group was a
substituent on the corresponding acid chloride.24-25
Scheme 1. Reagents and conditions: (a) Pd(PPh3)2Cl2, CuI, Et3N, 60°C (79-
90%); (b) K2CO3, THF:MeOH (1:1), r.t (90-98%); (c) Pd(PPh3)2Cl2, CuI,
Et3N, r.t (98%); (d) Pd(PPh3)2Cl2, aq. Na2CO3, DME, 120°C (69-91%); (e)
K2CO3, THF:MeOH (1:1), r.t (69-98%).
Synthesis of compounds 11a
First, different acetylene derivatives (4a
with ethylbut-2-ynoate ( ) using catalytic amount of
Pd(OAc)2 and TDMPP in benzene to afford corresponding
products 8a in moderate to high yields. Then the
electrophilic cyclization of products 8a was performed
—
m
is shown in scheme 2.
Fig. 1. Retrosynthetic plan for the synthesis of N-hydroxypyridone
derivatives; A) Classical approach; B) Our approach.
—
m) were coupled
7
—
m
When we attempted to synthesize different aromatic N-
hydroxypyridone derivatives with this approach; we
—
m

with ICl in DCM to afford iodo-substituted cyclized
products 9a in quantitative yields, followed by their de-
iodination with Pd(OAc)2/PPh3 in Et3N/HCOOH to give
products 10a Finally, compounds 11a were
synthesized by refluxing the corresponding compounds
10a with NH2OH·HCl in pyridine at 100°C (Scheme 2).
Table 1. The effects of N-hydroxypyridone derivatives on mitochondrial
metabolic activity in astrocytes exposed to H2O2.
—
m
PrestoBlue®
PrestoBlue®
assay
—
m.
—m
compds
Structure/R
assay
Max inhibitionb
(%)
IC50a (µM)
—
m
Ciclo-
pirox
43.72
(32.38–78.81)
57.92
0.33 ± 0.05
4.07 ± 1.46
2.74 ± 0.35
11a
11b
11c
(48.06–94.14)
26.83
(21.47–53.57)
0.75
-
(0.61–0.79)
72.58
11d
11e
1.99 ± 0.29
(38.35–85.07)
41.10
4.74 ± 1.16
0.45 ± 0.08
(37.54–70.68)
58.67
Scheme 2. Reagents and conditions: (a) Pd(OAc)2, TDMPP, benzene (44-
87%); (b) ICl, DCM (dry), r.t; (c) Pd(OAc)2, PPh3, Et3N, HCOOH, DMF,
62oC (21-60%); (d) Pd(PPh3)2Cl2, CuI, Et3N, 50oC (36-93%); (e)
NH2OH.HCl, pyridine, 100oC, 14 hr (50-71%).
11f
(35.02–74.27)
57.43
11g
11h
11i
0.21 ± 0.06
7.10 ± 2.71
1.86 ± 1.61
(42.26–71.14)
2.2. Biology
16.16
(5.24–54.21)
Mitochondrial metabolic activity assay The protective
effects of ciclopirox and all the synthesized N-
hydroxypyridone derivatives (11a—m) on oxidative stress
was initially evaluated in primary astrocyte cultures exposed
to H2O2. H2O2-induced mitochondrial dysfunction was
measured by resazurin-based PrestoBlue® assay, where the
non-fluorescent, blue resazurin reduces to the fluorescent
resorufin depending on mitochondrial activity.
All the N-hydroxypyridone derivatives were tested in a
series of dose escalation more than 8 points ranging from 1
nM to 10 µM. From the non-linear regression analysis of
dose-response curves, the concentration of drug at which
H2O2-induced deterioration of mitochondrial activity is half
inhibited (IC50, a measure of potency) and the maximum
inhibition (% as a measure of efficacy) that a drug can
achieve were calculated. As shown in Table 1, ciclopirox
protected mitochondrial metabolic activity against H2O2 in
astrocytes, having an IC50 value of 330 nM with a 43.72%
inhibition at maximum. To investigate the influence of the
cyclohexyl moiety at the N-hydroxypyridone nucleus,
systematic variations were carried out in this region. The
replacement of the cyclohexyl to the phenyl ring markedly
reduced the potency by 5 to 10 folds (compounds 11a—e).
Particularly, the addition of fluorine atom at position 3 of the
phenyl ring resulted in complete loss of activity.
48.70
(42.39–67.34)
44.63
11j
11k
11l
0.61 ± 0.14
0.34 ± 0.01
0.43 ± 0.17
0.30 ± 0.03
(31.98–58.52)
58.60
(34.09–66.38)
37.43
(34.35–66.45)
33.61
11m
(24.56–79.29)
aMitochondrial metabolic activity was determined by using Resazurin-based
presto blue® assay following manufacturer’s instructions. Cells were loaded
with presto blue® reagent at 37°C for 15 minutes and the corresponding
fluorescence change was measured (Ex = 540 nm, Em = 590 nm). Analysis
of dose-response curve was performed using the non-linear regression fit
analysis (log of dose vs. inhibitory response) to calculate the IC50 value of the
drug that inhibited 50% of H2O2-induced mitochondrial metabolic
dysfunction. IC50: the dose of derivative required to inhibit H2O2-induced
dysfunction of mitochondrial metabolic activity by 50%. Data are expressed
as the mean ± SEM from the dose-response curve of 3 to 7 independent
experiments; bMax inhibition (%): the maximal inhibitory response of the
derivatives estimated from the non-linear regression curve fit. Data are
expressed as a median (interquartile range) of inhibition percentage, N = 3 –
7.
Further structure-activity relationship around the N-
hydroxy-2-pyridone nucleus revealed that the addition of a
biphenyl ring was optimal for potency and efficacy
(compounds 11f, 11g, 11j—m). The most active example in
this series was the 4-biphenyl derivative 11g, which showed
an IC50 of 210 nM with a maximum inhibition value of

A
B
C
Table 2. The effects of N-hydroxypyridone derivatives on H2O2-induced
astrocytic cell death.
LDH assay
LDH assay
compounds
Structure/R
Max inhibitionb
(%)
IC50a (nM)
D
Ciclopirox
249.9 ± 65.7
104.5 ± 1.5
50.3 ± 9.1
32.6 ± 4.3
11f
E
11g
11j
71.6 ± 30.7
167.0 ± 13.7
156.1 ± 14.5
53.5 ± 10.9
29.6 ± 1.4
58.8 ± 15.3
11k
Fig. 2. Dose-response curves regarding protective effects of N-
hydroxypyridone derivatives against H2O2-induced astrocytic cell death
measured by the LDH assay. (A—E) Astrocytes were exposed to 500 µM
H2O2 in the presence or absence of ciclopirox or selected biphenyl N-
hydroxypyridone derivatives (11g, 11k—m) ranging 1 nM to 10 µM. Data
are expressed as the mean ± SEM (N=3) and the representative curves from 3
independent experiments were shown.
11l
106.5 ± 5.6
55.1 ± 9.4
11m
117.7 ± 63.7
59.1 ± 13.6
aCell death was assessed by measuring the amount of lactate dehydrogenase
(LDH) released into the culture supernatant using a diagnostic kit. The data
were expressed as the percentage of total LDH release measured in repeatedly
frozen and thawed parallel cell cultures. IC50:the dose of derivative required
to inhibit H2O2-induced astrocytic cell death by 50%. Data are expressed as
the mean ± SEM from the dose-response curve of 3 independent experiments;
bMax inhibition (%): the maximal inhibitory response of the derivatives
estimated from the non-linear regression curve fit. Data are expressed as the
mean ± SEM from the dose-response curve of 3 independent experiments.
A
C
D
B
57.43 % against H2O2-evoked mitochondrial metabolic
dysfunction. The replacement of 4-biphenyl to 3-biphenyl
ring as a substituent (11f) afforded two fold decrease in
potency with comparable efficacy. The modification of the
biphenyl ring by adding chlorine atom, or methoxy, methyl, or
tert-butyl group (compounds 11j, 11k, 11l, 11m, respectively)
was well-tolerated and slightly modulated the potency. While the
addition of the electron-withdrawing chlorine atom (11j) at 4-
position resulted in reduction of activity, while the effects of the
electron-donating methoxy, methyl or tert-butyl groups
E
(compounds 11k—m) were not dramatic, showing comparable
potencies. In contrast, replacement of one of phenyl ring of
compound 11g with the naphtyl ring (compound 11h) caused
noticeable deterioration of the activity. Substitution of the phenyl
to 2-naphthyl ring (compound 11i) also led to marked decrease of
the activity.
Fig. 3. Protective effects of N-hydroxypyridone derivatives in astrocytes
against H2O2-evoked Δψm depolarization. (A E) After loading with
500 nM TMRM at 37°C for 10 min, astrocytes were exposed to 500 µM
—

H2O2 in the presence or absence of ciclopirox or a selected biphenyl N-
hydroxypyridone derivative (11g, 11k—m) at the concentration of 1
µM. Fluorescence intensities (F. I.) of TMRM were measured every 5
min for 2 h. Data are expressed as the mean ± SEM (N=4). The
endpoint Δψm at 2 hours after treatment were determined based on F. I
values of TMRM. (Inlet) Data are expressed as the median (horizontal
bar), interquartile ranges (Q1 to Q3, vertical box), and min/max
(whiskers). Data were analysed by Kruskal-Wallis test followed by
Mann-Whitney test for multiple comparison (N=8). **P < 0.01 or ***P <
0.001 vs H2O2 control.
response to each modulator and the relative contribution of each
bioenergetic parameters. (B) Astrocytic OCR was measured in the
presence of ciclopirox or selected biphenyl N-hydroxypyridone
derivatives (11g, 11k—m) at the concentration 1 µM. Data are
expressed as the mean ± SEM (N=8). (C, D) ATP turnover-linked OCR
and maximum respiration capacity were calculated based on OCR
curve. Data are expressed as the median (horizontal bar), interquartile
ranges (Q1 to Q3, vertical box), and min/max (whiskers). Data were
analysed by Kruskal-Wallis test followed by Mann-Whitney test for
multiple comparison (N=8). **P < 0.01 vs control. (E) Astrocytes were
exposed to 500 µM H2O2 in the presence or absence of ciclopirox or
selected biphenyl N-hydroxypyridone derivatives (11g, 11k—m). The
effects on the mitochondrial bioenergetic profile were determined by
plotting OCR vs. ECAR for each treatment. Data shown are the mean ±
SEM (N = 8).
After the initial round of screening, the biphenyl N-
hydroxypyridone derivatives with high potency and efficacy
(compounds 11f, 11g, 11j—m) were selected for further
biological characterization. The protective effect of
derivatives against H2O2-induced cell death was assessed by
measuring the cell-injury-evoked release of lactate
dehydrogenase (LDH) from astrocytes (Table 2).
with both high potency and efficacy were shown in Fig. 2.
The curves of all derivatives exhibited a steep slope,26–28
with IC50 value ranging tens to several hundreds of nM and
50 to 60 % of maximum cell death inhibition. No apparent
cytotoxicity to astrocytes was observed at the tested
concentrations of all derivatives by themselves (up to 10
µM, data not shown).
Since the protective effects of ciclopirox on
peroxynitrite-induced astrocytic cell death was closely
related with its inhibitory effects on mitochondrial Δψm
depolarization,22 we next investigated whether the selected
When cells were treated with 500 µM H2O2 in the
presence or absence of biphenyl N-hydroxypyridone
derivatives, all tested compounds (11f, 11g, 11j—m) showed
substantial protective effects on cell viability in astrocytes.
As shown in Table 1, the 4-biphenyl derivative 11g showed
the highest potency in these series. Interestingly, the
replacement to 3-biphenyl ring (compound 11f) or the
addition of chlorine atom at the para position of the phenyl
ring (compound 11j) led to marked reduction in efficacy
biphenyl N-hydroxypyridone derivatives (11g, 11k—m)
affect H2O2-evoked Δψm depolarization in astrocytes (Fig.
3).
(39.1% or 44.6% reduction compared with compound 11g
,
respectively). The representative dose-response curves of
ciclopirox and other four biphenyl N-hydroxypyridone
derivatives (11g, 11k—m)
The change of Δψm was monitored by the fluorescence
change of the indicator, tetramethylrhodamine methyl ester
(TMRM). As shown in Fig. 3, H2O2 induced the dissipation
of Δψm in astrocytes prior to release of LDH. Ciclopirox
and all four derivatives significantly inhibited H2O2-induced
depolarization of Δψm at 1 µM concentration. Derivatives
did not induce Δψm depolarization alone (Data not shown).
We next determined the effects of N-hydroxypyridone
derivatives on mitochondrial respiration and glycolysis
using extracellular flux analysis. This non-invasive
technique allows the real-time measurements of O2
consumption rate (OCR, an indicator of oxidative
phosphorylation) and extracellular acidification rate (ECAR,
an indicator of glycolysis) simultaneously in intact cells. As
shown in a schematic diagram (Fig. 4A), the various
parameters of mitochondrial function were determined by
measuring OCR after sequential addition of pharmacological
inhibitors, oligomycin (a ATP synthase inhibitor to block
ATP synthesis), carbonyl cyanide p-[trifluoromethoxy]-
phenyl-hydrazone (FCCP, a proton ionophore to uncouple
ATP synthesis from the electron transport chain and allow
for maximum electron flux), and rotenone (a complex I
inhibitor to block the electron transport chain).
A
C
D
B
E
As shown in Fig. 4B, ciclopirox and biphenyl N-
hydroxypyridone derivatives
(11g, 11k—m) did not
significantly change ATP turnover-linked OCR (Fig. 4C)
and basal OCR (data not shown). Compound 11g and 11m
suppressed maximal respiratory capacity (Fig. 4D) and the
reserve capacity (data not shown) in astrocytes, without
affecting cell survival. As a bioenergetic profile was
Fig. 4. Modulation of mitochondrial bioenergetics by N-
hydroxypyridone derivatives in astrocytes. (A) Real-time mitochondrial
respiration was monitored using the Seahorse XF24 analyzer after each
addition of oligomycin, FCCP, and rotenone. The schematic curve
demonstrates resulting changes in oxygen consumption rate (OCR) in

assessed by plotting OCR vs ECAR for each treatment, the
control cells under basal conditions utilize both oxidative
phosphorylation and glycolysis (Fig. 4E, a grey square).
Ciclopirox and biphenyl N-hydroxypyridone derivatives did
not significantly change this metabolic profile under basal
conditions (Fig. 4E, white circles). In contrast, H2O2 caused
a marked decrease in OCR, suggesting mitochondrial
dysfunction consistent with loss of membrane potential in
astrocytes (Fig. 4E, a dark grey square). The treatment of
ciclopirox and biphenyl N-hydroxypyridone derivatives
(electrospray ionization MS) obtained on a G2 QTOF mass
spectrometer. TLC was performed on Merck silica gel 60
F254 plates. Column chromatography was conducted using
silica gel 60 N (Kanto, 100e210 mm) or silica gel 60 (Kanto,
40e50 mm). HPLC spectra were noted on Water-2695 by
using acetonitrile:water/60:40 as eluting system at flow rate
of 1 mL/min at 310 nM wavelength.
Synthesis of trimethyl((3-nitrophenyl)ethynyl)silane (3e)
In a two neck rbf containing TEA (90 ml) was added 1-
bromo-3-nitrobenzene 1e (9.00 g, 44.55 mmol) at rt under
nitrogen. The mixture was stirred for 5 minutes followed by
(11g, 11k—m) decreased the response of OCR to H2O2, and
stimulated ECAR (Fig. 4E, dark circles), suggesting the
restoration of mitochondrial bioenergetic function and an
acute stimulation of glycolytic metabolism against H2O2.
Glycolysis was known to supply glucose-6-phosphate,
which can shunt into the pentose phosphate pathway (PPP).
PPP drives regeneration of NADPH and Glutathione (GSH)
in response to oxidative stress, thus potentially contributing
to antioxidant redox status and survival of astrocytes, as
previously shown in neurons.29
Taken together, these data strongly suggest the protective
effects of ciclopirox and biphenyl N-hydroxypyridone
derivatives (11g, 11k—m) derivatives against H2O2-induced
cytotoxicity in astrocytes via inhibition of mitochondrial
dysfunction, by especially preserving mitochondrial Δψm
and bioenergetic profiles.
addition of ethynyltrimethylsilane
2 (6.93 ml, 49.01 mmol),
Pd(PPh3)2Cl2 (1.57 g, 2.23 mmol) and CuI (849 mg, 4.46
mmol). The resulting mixture was stirred for 17 hours at
60oC and then cooled down to rt. The reaction mixture was
evaporated in vacuum to remove excess of TEA followed by
addition of 1N HCl until the pH reached to 1. The mixture
was then extracted with EtOAc/water. The organic layer was
washed with brine and dried over anhydrous MgSO4. The
solvent was evaporated in vacuum and the residue was
purified by column chromatography (Hexane : EtOAc = 20 :
1) to give the desired compound 3e (7.68 g) as a light brown
1
solid; Yield = 79%; H NMR (CDCl3, 500 MHz): δ 8.02-
7.19 (4H, m, Ar-H), 0.00 (9H, s, -Si(CH3)3); 13C NMR
(CDCl3, 125 MHz): δ 148.0, 137.4, 129.2, 126.7, 124.9,
123.0, 102.1, 97.6, -0.3.
3. Conclusion
Synthesis of acetylene derivatives 4e-h
Acetylene derivatives 4a-4d were purchased from
Sigma-Aldrich and were used as such without any further
purification.
It was newly found that a novel class of N-hydroxy-2-
pyridone-based derivatives efficiently protected astrocytes
from reactive oxygen species like H2O2. On the basis SAR
studies, we discovered that the addition of biphenyl ring to
N-hydroxy-2-pyridone resulted in better potency and
efficacy. The protective effects of ciclopirox and the
biphenyl derivatives might be mainly due to their inhibitory
actions against mitochondrial dysfunction. Among all
compounds screened, compound 11g was found to be the
most potent showing IC50 values in the range of a few
hundred nM, which equal or higher than that of ciclopriox.
Considering the importance of astrocytes in neuronal
bioenergetics and survival in various neurological disorders,
the present study suggests that this new class of highly
potent and efficient N-hydroxy-2-pyridone derivatives open
an innovative therapeutic strategy for the treatment of
neurodegenerative diseases.
Synthesis of 1-ethynyl-3-nitrobenzene (4e)
In a two neck rbf containing THF (100 ml) and MeOH
(100 ml) was added compound 3e (7.55 g, 34.43 mmol) at rt.
The mixture was stirred for 5 minutes followed by addition
of K2CO3 (28.55 g, 206.58 mmol). The resulting mixture
was stirred for 1 hour at the same temperature. The reaction
mixture was filtered to remove K2CO3 and then extracted
with EtOAc/water. The organic layer was washed with brine
and dried over anhydrous MgSO4. The solvent was
evaporated and dried in vacuum to give desired compound
1
4e as a brown solid; Yield = 98 %; H NMR (CDCl3, 500
MHz): δ 8.34-7.52 (4H, m, Ar-H), 3.24 (1H, s, -CH); 13C
NMR (CDCl3, 125 MHz): δ 148.1, 137.7, 129.4, 126.9,
123.9, 123.5, 81.1, 79.9.
Experimental
Chemistry
3-Ethynylbiphenyl (4f)
According to the procedure described for the synthesis of
All melting points were determined on a Yamato melting
point apparatus (model MP-J3). The NMR spectra were
recorded on a VARIAN-500 MHz spectrometer. The
chemical shifts were reported in parts per million (d) relative
to TMS (0.0 ppm) as the internal standard, and the signals
are expressed as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), or br (broad). The values of the
coupling constant J are given in hertz. The mass spectra
were recorded using high-resolution mass spectrometry
(HRMS) (electron ionization MS) obtained on a JMS-700
mass spectrometer (Jeol, Japan) or using HRMS
1
4e, Yield = 90%; Yellow oil; H NMR (CDCl3, 500 MHz): δ
7.73-7.23 (9H, m, Ar-H), 3.09 (1H, s, -CH); 13C NMR
(CDCl3, 125 MHz): δ 140.6, 139.3, 130.0, 128.0, 126.8,
126.3, 121.7, 82.8.
4-ethynylbiphenyl (4g)
According to the procedure described for the synthesis of 4e
,
Yield = 90%; 1H NMR (CDCl3, 500 MHz): δ 7.58-7.34 (9H,
m, Ar-H), 3.11 (1H, s, -CH); 13C NMR (CDCl3, 125 MHz):
δ 141.6, 140.3, 132.6, 128.9, 127.7, 127.1, 121.0, 83.6, 77.7.

4-Chloro-4'-ethynylbiphenyl (4j)
2-Ethynylnaphthalene (4h)
According to the procedure described for the synthesis of 4e
Yield = 91 %; Light brown solid; H NMR (CDCl3, 500
MHz) δ 8.00-7.44 (7H, m, Ar-H), 3.12 (1H, s, -CH); 13C
NMR (CDCl3, 125 MHz) δ 132.8, 132.6, 132.0, 128.3,
127.8, 127.5, 126.6, 126.3, 119.2, 83.8, 77.2.
According to the procedure described for the synthesis of
1
,
4i; Yield = 72%; Yellow solid; H NMR (CDCl3, 500 MHz):
δ 7.56-7.39 (8H, m, Ar-H), 3.13 (1H, s, -CH); 13C NMR
(CDCl3, 125 MHz): δ 140.2, 138.6, 133.8, 132.6, 129.0,
128.2, 126.8, 121.3, 83.3, 78.0.
1
4'-Ethynyl-2-methoxybiphenyl (4k)
((4-Bromophenyl)ethynyl)trimethylsilane (6)
According to the procedure described for the synthesis of
4i; Yield = 84%; Yellow solid; H NMR (CDCl3, 500 MHz):
1
In a two neck rbf containing TEA (200 ml) was added 1-
δ 7.69-7.10 (8H, m, Ar-H), 3.92 (3H, s, -OCH3), 3.23 (1H, s,
-CH); 13C NMR (CDCl3, 125 MHz): δ 155.3, 138.1, 130.7,
129.8, 129.6, 128.6, 128.1, 127.6, 127.0, 120.0, 119.5,
110.2, 82.9, 54.3
Bromo-4-iodobenzene
nitrogen. The mixture was stirred for 5 minutes followed by
addition of ethynyltrimethylsilane (13.19 ml, 93.31 mmol),
5 (24.00 g, 84.83 mmol) at rt under
2
Pd(PPh3)2Cl2 (2.98 g, 4.24 mmol) and CuI (1.62 g, 8.48
mmol). The reaction mixture was stirred for 30 minutes at
the same temperature. The reaction mixture was evaporated
in vaccum to remove excess of TEA followed by addition of
1N HCl until the pH reached to 1. The mixture was then
extracted with EtOAc/water. The organic layer was washed
with brine and dried over anhydrous MgSO4. The solvent
was evaporated and the residue was purified by column
4'-Ethynyl-2-methylbiphenyl (4l)
According to the procedure described for the synthesis of
1
4i; Yield = 91%; Yellow oil; H NMR (CDCl3, 500 MHz): δ
7.53-7.18 (8H, m, Ar-H), 3.08 (1H, s, -CH), 2.25 (3H, s, -
CH3); 13C NMR (CDCl3, 125 MHz): δ 142.3, 140.9, 134.9,
131.6, 130.2, 129.6, 129.4, 129.1, 127.4, 125.6, 120.4, 83.4,
77.1, 20.2.
chromatography (Hexane) to give the desired compound
6
1
(21.00 g) as a white solid; Yield = 98%; H NMR (CDCl3,
500 MHz): δ 7.19-7.06 (4H, m, Ar-H), 0.00 (9H, s, -
Si(CH3)3); 13C NMR (CDCl3, 125 MHz): δ 133.5, 131.6,
122.8, 122.3, 104.0, 95.7, 0.00.
4-tert-Butyl-4'-ethynylbiphenyl (4m)
According to the procedure described for the synthesis of
1
4i; Yield = 69%; Light yellow solid; H NMR (CDCl3, 500
MHz): δ 7.54-7.45 (8H, m, Ar-H), 3.10 (1H, s, -CH), 1.35
(9H, s, -C(CH3)3); 13C NMR (CDCl3, 125 MHz): δ 150.8,
141.3, 137.2, 132.4, 126.7, 126.59, 125.7, 120.6, 83.6, 77.5,
34.5, 31.2.
Synthesis
of
trimethyl((4-(naphthalen-1-
yl)phenyl)ethynyl)silane (3i)
In a sealed tube was taken compound
6
(7.36 g, 29.08
(E)-ethyl 3-methyl-5-phenylpent-2-en-4-ynoate (8a)
mmol), 1-naphthaleneboronic acid 7i (6.00 g, 34.89 mmol),
Pd(PPh3)2Cl2 (1.02 g, 1.45 mmol), Na2CO3 (15.41 g, 145.40
mmol), water (18 ml) and DME (60 ml). The reaction
mixture was stirred for 3 hours at 120oC and then cooled
down to rt. The mixture was then acidified with 1N HCl to
bring pH ~1. The mixture was then extracted with
EtOAc/water. The organic layer was washed with brine and
dried over anhydrous MgSO4. The solvent was evaporated in
vacuum and the residue was purified by column
chromatography (Hexane : EtOAc = 100 : 1) to give the
desired compound 3i (7.64 g) as a colourless oil; Yield =
87%; 1H NMR (CDCl3, 500 MHz): δ 7.58-7.06 (11H, m, Ar-
H), 0.00 (9H, s, -Si(CH3)3); 13C NMR (CDCl3, 125 MHz): δ
140.9, 139.3, 133.7, 132.7, 131.7, 129.8, 128.2, 128.0,
127.6, 126.7, 126.0, 125.8, 125.2, 122.0, 105.0, 94.6, 60.1, -
0.07.
In a two neck rbf was stirred a mixture of Pd(OAc)2 (275
mg, 1.22 mmol) and TDMPP (540 mg, 1.22 mmol) in
benzene (30 ml) for 15 minutes at rt under nitrogen. To this
mixture was added ethyl-2-butynoate
7 (2.84 ml, 24.48
mmol) and the resulting mixture was further stirred for 10
minutes at rt. Next, ethynylbenzene 4a (2.50 g, 24.48 mmol)
was added to this solution and the mixture was stirred for 5
to 10 minutes until reaction was completed. The workup of
reaction was performed by evaporating the solvent in
vacuum followed by column chromatography (Hexane :
EtOAc = 100 : 1) to give the desired compound 8a (2.62 g);
Yield = 50%; 1H NMR (CDCl3, 500 MHz): δ 7.47-7.45 (2H,
m, Ar-H), 7.36-7.33 (3H, m, Ar-H), 6.16 (1H, s, -CH), 4.21-
4.17 (2H, q, J = 7.5 Hz, -CH2CH3), 2.39 (3H, s, -CH3), 1.31-
1.28 (3H, t, J = 7.5 Hz, -CH2CH3); 13C NMR (CDCl3, 125
MHz): δ 164.9, 136.6, 130.7, 128.9, 127.8, 127.2, 122.9,
121.2, 92.4, 90.0, 58.9, 18.7, 13.1.
Synthesis of acetylene derivatives 4i-4m
5-Iodo-4-methyl-6-phenyl-2H-pyran-2-one (9a)
Synthesis of 1-(4-ethynylphenyl)naphthalene (4i)
According to the procedure described for the synthesis of
In a two neck rbf containing a solution of compound 8a
(2.62 g, 12.23 mmol) in CH2Cl2 (20 mL) was added 1 M
solution of ICl (18.35 ml, 18.35 mmol) dropwise under
nitrogen. The mixture was stirred at rt for 30 minutes
followed by its quenching with sodium thiosulfate solution
(20 ml) at same temperature. The reaction mixture was
extracted with CH2Cl2/water. The organic layer was washed
with brine and dried over anhydrous MgSO4. The solvent
was evaporated in vacuum and the residue was purified by
1
4e; Yield 98%, Yellow oil; H NMR (CDCl3, 500 MHz): δ
7.89-7.37 (11H, m, Ar-H), 3.13 (1H, s, -CH); 13C NMR
(CDCl3, 125 MHz): δ 141.1, 139.1, 133.6, 131.8, 131.1,
129.8, 128.1, 127.9, 127.8, 127.6, 126.62, 126.0, 125.5,
125.1, 120.8, 83.3, 77.3.

column chromatography (Hexane : EtOAc = 25 : 1) to give
the desired compound 9a (1.53 g); Yield = 40%; 1H NMR
(CDCl3, 500 MHz): δ 7.65-7.63 (2H, m, Ar-H), 7.48-7.44
(3H, m, Ar-H), 6.26 (1H, s, Ar-H), 2.38 (3H, s, -CH3); 13C
NMR (CDCl3, 125 MHz): δ 161.2, 160.8, 158.4, 134.5,
130.6, 129.5, 128.1, 112.1, 29.1.
According to the procedure described for the synthesis of
10a; Yield = 36%; Yellow solid; H NMR (CDCl3, 500
1
MHz) δ 8.63-7.65 (4H, m, Ar-H), 6.66 (1H, s, Ar-H), 6.18
(1H, s, Ar-H), 2.29 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 161.2, 156.5, 155.5, 148.6, 133.0, 131.0, 130.0,
124.9, 120.3, 113.1, 105.4, 21.5; MS (TOF ES+): m/z calcd
for C12H10NO4 [M + 1]+, 232.0604; found, 232.0612.
Typical procedure for the synthesis of compounds 10a-
10m
6-(Biphenyl-3-yl)-4-methyl-2H-pyran-2-one (10f)
According to the procedure described for the synthesis of
1
4-Methyl-6-phenyl-2H-pyran-2-one (10a)
10a; Yield = 81%; Brown solid; H NMR (CDCl3, 500
In a two neck rbf was stirred a mixture of compound 9a
(1.53 g, 4.90 mmol), Pd(OAc)2 (22 mg, 0.098 mmol) and
Ph3P (51 mg, 0.196 mmol) in DMF (40 ml) for 5 minutes at
rt under nitrogen. TEA (2.05 ml, 14.70 mmol) was added to
this solution and the mixture was further stirred for 5
minutes followed by addition of 98% formic acid (0.41 ml,
10.78 mmol). The reaction mixture was stirred for 5 hours at
62oC. After completion of reaction, the mixture was cooled
down to rt and then extracted with EtOAc/water. The
organic layer was washed with brine and dried over
anhydrous MgSO4. The solvent was evaporated in vacuum
and the residue was purified by column chromatography
(Hexane : EtOAc = 20 : 1) to give the desired compound
MHz): δ 8.03-7.37 (9H, m, Ar-H), 6.57 (1H, s, Ar-H), 6.10
(1H, s, Ar-H), 2.24 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 162.3, 159.2, 156.3, 141.9, 140.0, 131.7, 129.3,
128.9, 127.8, 127.1, 124.3, 124.2, 111.7, 104.4, 21.6; MS
(TOF ES+): m/z calcd for C18H15O2 [M + 1]+, 263.1066;
found, 263.1068.
6-(Biphenyl-4-yl)-4-methyl-2H-pyran-2-one (10g)
According to the procedure described for the synthesis of
10a; Yield = 86%; Light-brown solid; 1H NMR (CDCl3, 500
MHz): δ 7.89-7.37 (9H, m, Ar-H), 6.55 (1H, s, Ar-H), 6.08
(1H, s, Ar-H), 2.23 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 162.3, 159.2, 155.9, 143.4, 139.8, 130.1, 128.9,
127.9, 127.4, 127.0, 126.0, 111.6, 103.9, 21.6; MS (TOF
ES+): m/z calcd for C18H15O2 [M + 1]+, 263.1066; found,
263.1073.
1
10a (730 mg); Yield = 80%; H NMR (CDCl3, 500 MHz): δ
7.83-7.81 (2H, m, Ar-H), 7.46-7.44 (3H, m, Ar-H), 6.52
(1H, s, Ar-H), 6.08 (1H, s, Ar-H), 2.23 (3H, s, -CH3); 13C
NMR (CDCl3, 125 MHz): δ 162.0, 159.2, 155.7, 131.1,
130.4, 128.6, 125.3, 111.4, 103.8, 21.3; MS (TOF ES+): m/z
calcd for C12H11O2 [M + 1]+, 187.0753; found, 187.0753.
4-Methyl-6-(naphthalen-2-yl)-2H-pyran-2-one (10h)
According to the procedure described for the synthesis of
1
10a; Yield = 87%; Brown solid; H NMR (CDCl3, 500
6-(4-Fluorophenyl)-4-methyl-2H-pyran-2-one (10b)
MHz): δ 8.37-7.51 (7H, m, Ar-H), 6.60 (1H, s, Ar-H), 6.08
(1H, s, Ar-H), 2.22 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 162.3, 159.3, 156.0, 134.1, 133.0, 128.8, 128.6,
128.3, 127.6, 127.5, 126.8, 126.0, 121.9, 111.6, 104.4,
21.56; MS (TOF ES+): m/z calcd for C16H13O2 [M + 1]+,
237.0909; found, 237.0908.
According to the procedure described for the synthesis of
1
10a; Yield 81%; H NMR (CDCl3, 500 MHz): δ 7.81-7.78
(2H, m, Ar-H), 7.14-7.10 (2H, m, Ar-H), 6.46 (1H, s, Ar-H),
6.05 (1H, s, Ar-H), 2.22 (3H, s, -CH3); 13C NMR (CDCl3,
125 MHz): δ 164.7, 162.7, 161.0, 159.7, 158.4, 131.8, 130.6,
115.4, 115.3, 112.2, 29.0; MS (TOF ES+): m/z calcd for
C12H10FO2 [M + 1]+, 205.0659; found, 205.0683.
4-Methyl-6-(4-(naphthalen-1-yl)phenyl)-2H-pyran-2-one
(10i)
6-(3-Fluorophenyl)-4-methyl-2H-pyran-2-one (10c)
According to the procedure described for the synthesis of
10a, Yield = 88%; Brown solid; H NMR (CDCl3, 500
1
According to the procedure described for the synthesis of
1
10a; Yield = 81%; Light brown solid; H NMR (CDCl3, 500
MHz): δ 7.95-7.42 (11H, m, Ar-H), 6.60 (1H, s, Ar-H), 6.11
(1H, s, Ar-H), 2.25 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 162.2, 159.2, 155.9, 143.3, 138.9, 133.7, 131.2,
130.5, 130.1, 128.3, 128.1, 126.8, 126.2, 125.8, 125.4,
125.2, 111.6, 104.0, 21.5; MS (TOF ES+): m/z calcd for
C22H17O2 [M + 1]+, 313.1222; found, 313.1226.
MHz): δ 7.59-7.11 (4H, m, Ar-H), 6.52 (1H, s, Ar-H), 6.09
(1H, s, Ar-H), 2.23 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 163.7, 161.8, 157.7, 155.6, 133.3, 130.3, 121.0,
117.4, 112.5, 112.1, 104.5, 21.3; MS (TOF ES+): m/z calcd
for C12H10FO2 [M + 1]+, 205.0659; found, 205.0672.
6-(4-Methoxyphenyl)-4-methyl-2H-pyran-2-one (10d)
6-(4'-Chlorobiphenyl-4-yl)-4-methyl-2H-pyran-2-one
(10j)
According to the procedure described for the synthesis of
1
10a; Yield = 72%; Yellow solid; H NMR (CDCl3, 500
According to the procedure described for the synthesis of
1
MHz): δ 7.77-7.75 (2H, d, J = 9.0 Hz, Ar-H), 6.95-6.93 (2H,
d, J = 9.0 Hz, Ar-H), 6.41 (1H, s, Ar-H), 6.00 (1H, s, Ar-H),
3.85 (3H, s, -OCH3), 2.21 (3H, s, -CH3); 13C NMR (CDCl3,
125 MHz): δ 162.6, 161.6, 159.5, 156.5, 127.2, 123.7, 114.2,
110.3, 102.7, 55.4, 21.6; MS (TOF ES+): m/z calcd for
C13H13O3 [M + 1]+, 217.0858; found, 217.0864.
10a; Yield = 88%; Brown solid; H NMR (CDCl3, 500
MHz): δ 7.86-7.40 (8H, m, Ar-H), 6.54 (1H, s, Ar-H), 6.07
(1H, s, Ar-H), 2.22 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 162.3, 159.0, 156.1, 142.1, 138.3, 134.2, 130.5,
129.2, 128.3, 127.3, 126.2, 111.8, 104.2, 21.7; MS (TOF
ES+): m/z calcd for C18H14ClO2 [M + 1]+, 297.0676; found,
297.0688.
4-Methyl-6-(3-nitrophenyl)-2H-pyran-2-one (10e)

6-(2'-Methoxybiphenyl-4-yl)-4-methyl-2H-pyran-2-one
(10k)
6.52 (1H, s, Ar-H), 6.17 (1H, s, Ar-H), 2.27 (3H, s, -CH3);
13C NMR (CDCl3, 125 MHz): δ 164.5, 162.6, 157.8, 148.8,
141.9, 131.1, 127.4, 115.6, 115.4, 114.1, 109.4, 21.5; HRMS
(ESI) [M-H]- Calcd for C12H9 FNO2: 218.0623, Found:
218.063.
According to the procedure described for the synthesis of
1
10a, Yield = 90%; Yellow solid; H NMR (CDCl3, 500
MHz): δ 7.84-6.98 (8H, m, Ar-H), 6.53 (1H, s, Ar-H), 6.05
(1H, s, Ar-H), 3.80 (3H, s, -OCH3), 2.20 (3H, s, -CH3); 13C
NMR (CDCl3, 125 MHz): δ 162.2, 159.3, 156.3, 155.9,
141.0, 130.4, 129.8, 129.5, 129.2, 129.1, 125.1, 120.8,
111.3, 103.7, 55.4, 21.4; MS (TOF ES+): m/z calcd for
C19H17O3 [M + 1]+, 293.1171; found, 293.1173.
6-(3-Fluorophenyl)-1-hydroxy-4-methylpyridin-2(1H)-
one (11c)
According to the procedure described for the synthesis of
11a; Yield = 57%; Yellow solid; m.pt. 149-150oC; HPLC
1
purity: 99.6%, tR = 3.3 min; H NMR (CDCl3, 500 MHz): δ
4-Methyl-6-(2'-methylbiphenyl-4-yl)-2H-pyran-2-one
(10l)
7.44-7.16 (4H, m, Ar-H), 6.54 (1H, s, Ar-H), 6.20 (1H, s,
Ar-H), 2.28 (3H, s, -CH3); 13C NMR (CDCl3, 125 MHz): δ
163.3, 161.3, 157.6, 148.7, 141.3, 133.2, 130.0, 124.7,
117.0, 114.4, 109.4, 21.4; HRMS (ESI) [M-H]- Calcd for
C12H9 FNO2: 218.0623, Found: 218.0616.
According to the procedure described for the synthesis of
1
10a, Yield = 87%; Light brown solid; H NMR (CDCl3, 500
MHz): δ 7.88-7.22 (8H, m, Ar-H), 6.56 (1H, s, Ar-H), 6.09
(1H, s, Ar-H), 2.28 (3H, s, -CH3), 1.24 (3H, s, -CH3); 13C
NMR (CDCl3, 125 MHz): δ 162.2, 159.3, 155.8, 144.4,
140.6, 135.0, 130.3, 129.7, 129.4, 127.6, 125.8, 125.3,
111.5, 103.8, 21.5, 20.2; MS (TOF ES+): m/z calcd for
C19H17O2 [M + 1]+, 277.1222; found, 277.1221.
1-Hydroxy-6-(4-methoxyphenyl)-4-methylpyridin-2(1H)-
one (11d)
According to the procedure described for the synthesis of
11a; Yield = 50%; Light yellow solid; m. pt. 175-176oC;
1
HPLC purity: 98.7%, tR = 3.3 min; H NMR (CDCl3, 500
6-(4'-tert-Butylbiphenyl-4-yl)-4-methyl-2H-pyran-2-one
(10m)
MHz): δ 7.61-7.59 (2H, d, J = 8.5 Hz, Ar-H), 6.97-6.95 (2H,
d, J = 9.0 Hz, Ar-H), 6.46 (1H, s, Ar-H), 6.15 (1H, s, Ar-H),
3.84 (3H, s, -OCH3), 2.23 (3H, s, -CH3); 13C NMR (CDCl3,
125 MHz): δ 160.8, 157.9, 148.6, 142.7, 130.4, 123.7, 113.8,
113.2, 109.0, 55.3, 21.4; HRMS (ESI) [M-H]- Calcd for
C13H12NO3: 230.0817, Found: 230.0823.
According to the procedure described for the synthesis of
1
10a, Yield = 93%; Brown solid; H NMR (CDCl3, 500
MHz) δ 7.88-7.47 (8H, m, Ar-H), 6.54 (1H, s, Ar-H), 6.07
(1H, s, Ar-H), 2.23 (3H, s, -CH3), 1.36 (9H, s, -C(CH3)3);
13C NMR (CDCl3, 125 MHz): δ 162.3, 159.3, 155.9, 151.2,
143.3, 136.8, 129.8, 127.2, 126.7, 126.0, 125.8, 111.5,
103.8, 34.6, 31.3, 21.6; MS (TOF ES+): m/z calcd for
C12H23O2 [M + 1]+, 319.1692; found, 319.1697.
1-Hydroxy-4-methyl-6-(3-nitrophenyl)pyridin-2(1H)-one
(11e)
According to the procedure described for the synthesis of
11a; Yield = 67%; Light yellow solid; m. pt. 172-173oC;
1
Typical procedure for the synthesis of compounds 11a-
11m
HPLC purity: 97.8%, tR = 3.0 min; H NMR (CDCl3, 500
MHz): δ 8.52-7.66 (4H, m, Ar-H), 6.60 (1H, s, Ar-H), 6.27
(1H, s, Ar-H), 2.31 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 157.4, 148.7, 148.0, 140.0, 134.7, 132.6, 129.4,
124.3, 123.8, 115.1, 109.5, 21.2; HRMS (ESI) [M-H]- Calcd
for C12H9N2O4: 245.0563, Found: 245.0570.
1-Hydroxy-4-methyl-6-phenylpyridin-2(1H)-one (11a)
In a two neck rbf was stirred a mixture of compound 10a
(730 mg, 3.92 mmol) and NH2OH.HCl (9.53 g, 137.20
mmol) in pyridine (20 mL) for 17 hours at 100oC under
nitrogen. After completion of reaction, the mixture was
cooled down to rt and extracted with CH2Cl2/water. The
organic layer was washed with brine and dried over
anhydrous MgSO4. The solvent was evaporated in vacuum
and the residue was recrystallized with ethanol to give the
desired compound 11a (425 mg) as a light yellow solid;
Yield = 54%; m.pt. 130-131oC; HPLC purity: 99.4%, tR =
6-(Biphenyl-3-yl)-1-hydroxy-4-methylpyridin-2(1H)-one
(11f)
According to the procedure described for the synthesis of
11a; Yield = 60%; Yellow semisolid; HPLC purity: 98.9%,
1
tR = 6.4 min; H NMR (MeOD, 500 MHz): δ 7.89-7.85 (1H,
m, Ar-H), 7.71-7.33 (8H, m, Ar-H), 6.50 (1H, s, Ar-H), 6.39
(1H, s, Ar-H), 2.28 (3H, s, -CH3); 13C NMR (MeOD, 125
MHz): δ 163.3, 159.0, 158.3, 141.9, 140.0, 131.7, 129.2,
129.1, 128.6, 127.5, 126.6, 124.0, 123.6, 110.7, 104.7, 20.2;
HRMS (ESI) [M-H]- Calcd for C18H14NO2: 276.1025,
Found: 276.1041
1
3.1 min; H NMR (CDCl3, 500 MHz): δ 7.66-7.64 (2H, m,
Ar-H), 7.48-7.46 (3H, m, Ar-H), 6.53 (1H, s, Ar-H), 6.19
(1H, s, Ar-H), 2.28 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 158.3, 148.8, 143.6, 131.6, 129.7, 129.1, 128.3,
114.5, 109.9, 21.4; HRMS (ESI) [M-H]- Calcd for
C12H10NO2: 200.0717, Found: 200.0724.
6-(Biphenyl-4-yl)-1-hydroxy-4-methylpyridin-2(1H)-one
(11g)
6-(4-Fluorophenyl)-1-hydroxy-4-methylpyridin-2(1H)-
one (11b)
According to the procedure described for the synthesis of
11a; Yield = 62%; Yellow solid; m. pt. 210-211oC; HPLC
1
According to the procedure described for the synthesis of
purity: 95.3%, tR = 8.2 min; H NMR (CDCl3, 500 MHz): δ
11a; Yield = 52%; Pale yellow solid; m.pt. 146-147oC;
7.75-7.25 (9H, m, Ar-H), 6.54 (1H, s, Ar-H), 6.24 (1H, s,
Ar-H), 2.30 (3H, s, -CH3); 13C NMR (CDCl3, 125 MHz): δ
157.4, 148.5, 142.8, 141.8, 140.0, 130.0, 129.3, 128.8,
1
HPLC purity: 98.8%, tR = 3.3 min; H NMR (CDCl3, 500
MHz): δ 7.66-7.64 (2H, m, Ar-H), 7.17-7.14 (2H, m, Ar-H),

127.8, 127.1, 113.4, 109.0, 21.4; HRMS (ESI) [M-H]- Calcd
for C18H14NO2: 276.1025, Found: 276.1035.
109.2, 21.4, 20.3; HRMS (ESI) [M-H]- Calcd for
C19H16NO2: 290.1186, Found: 290.119.
1-Hydroxy-4-methyl-6-(naphthalen-2-yl)pyridin-2(1H)-
one (11h)
6-(4'-tert-Butylbiphenyl-4-yl)-1-hydroxy-4-
methylpyridin-2(1H)-one (11m)
According to the procedure described for the synthesis of
According to the procedure described for the synthesis of
11a; Yield = 56%; Yellow solid; m. pt. 205-206oC; HPLC
11a; Yield = 67%; Light yellow solid; m. pt. 170-171oC;
1
1
purity: 99.4%, tR = 4.7 min; H NMR (DMSO-d6, 500
HPLC purity: 99.6%, tR = 3.3 min; H NMR (CDCl3, 500
MHz): δ 8.12 (1H, s, Ar-H), 7.98-7.95 (3H, m, Ar-H), 7.68-
7.67 (1H, d, J = 8 Hz, Ar-H), 7.59-7.57 (2H, m, Ar-H), 6.41
(1H, s, Ar-H), 6.25 (1H, s, Ar-H), 2.19 (3H, s, -CH3); 13C
NMR (DMSO-d6, 125 MHz): δ 158.6, 148.6, 145,5, 133.3,
132.8, 130.5, 128.8, 128.7, 128.0, 127,7, 127.6, 127.1,
126.9, 116.3, 108.9, 21.1; HRMS (ESI) [M-H]- Calcd for
C16H12NO2: 250.0873, Found: 250.0884.
MHz): δ 7.73-7.48 (8H, m, Ar-H), 6.53 (1H, s, Ar-H), 6.24
(1H, s, Ar-H), 2.28 (3H, s, -CH3), 1.37 (9H, s, -C(CH3)3);
13C NMR (CDCl3, 125 MHz): δ 157.5, 150.9, 148.5, 142.6,
142.1, 137.1, 129.7, 129.2, 126.8, 126.7, 125.8, 113.4,
109.1, 34.5, 31.2, 21.4; HRMS (ESI) [M-H]- Calcd for
C22H22NO2: 332.1651, Found: 332.1664.
Biological methods
1-Hydroxy-4-methyl-6-(4-(naphthalen-1-
yl)phenyl)pyridin-2(1H)-one (11i)
Cell culture
Mixed glial cells were prepared from the prefrontal cortices
of 1 to 2-day-old Sprague-Dawley rat pups as previously
described30. In brief, meninges-free cerebral hemispheres were
dissociated by titurating through a Pasteur pipettes. Cells were
then plated on poly-D-lysine (1 µg/ml)-coated 175-cm2 flask
and maintained in MEM supplemented with 10% FBS
(Hyclone, Logan, UT) for a week. To prepare pure astrocytic
cultures, the flasks were shaken at 260 rpm overnight at 37°C
on day 8 of the mixed glial cultures. After removal of
supernatant containing detached microglial cells and
oligodendrocytes, the remaining astrocytes were trypsinized,
washed, and replated at a density 3.0 × 102 cells/mm2 in the
growth medium on poly-D-lysine (10 µg/ml)-coated plates. All
experimental procedures involving animals were performed in
accordance with the NIH Guide for the Care and Use of
Laboratory Animals and were approved by Korea University
Institutional Animal Care & Use Committee (KUIACUC-2014-
233). Cells were used for experiments 4 or 5 day after plating.
According to the procedure described for the synthesis of
11a; Yield = 61 %; Light yellow solid; m. pt. 242-243oC;
1
HPLC purity: 98.0%, tR = 11.4 min; H NMR (CDCl3, 500
MHz): δ 7.93-7.44 (11H, m, Ar-H), 6.56 (1H, s, Ar-H), 6.30
(1H, s, Ar-H), 2.31 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 157.7, 148.6, 142.6, 142.2, 139.2, 133.8, 131.4,
130.2, 128.9, 128.4, 128.1, 127.0, 126.3, 125.9, 125.7,
125.4, 113.7, 109.3, 21.5; HRMS (ESI) [M-H]- Calcd for
C22H16NO2: 326.1186, Found: 326.1185.
6-(4'-Chlorobiphenyl-4-yl)-1-hydroxy-4-methylpyridin-
2(1H)-one (11j)
According to the procedure described for the synthesis of
11a; Yield = 56.6%; Light yellow solid; m. pt. 198-199oC;
1
HPLC purity: 91.8%, tR = 9.6 min; H NMR (CDCl3, 500
MHz): δ 7.74-7.43 (8H, m, Ar-H), 6.54 (1H, s, Ar-H), 6.23
(1H, s, Ar-H), 2.29 (3H, s, -CH3); 13C NMR (CDCl3, 125
MHz): δ 157.3, 148.3, 141.6, 141.3, 138.3, 133.9, 130.2,
129.2, 128.9, 128.1, 126.6, 113.4, 108.9, 21.2; HRMS (ESI)
[M-H]- Calcd for C18H13ClNO2: 310.0640, Found: 310.0634.
Presto Blue assay
Mitochondrial metabolic activity was determined by
Resazurin-based presto blue assay (Invitrogen, Grand Island,
NY), following manufacturer’s instructions. In brief, cells were
loaded for 15 minutes with presto blue reagent at 37°C. The
fluorescence change was determined by using a microplate
reader (Ex = 540 nm, Em = 590 nm; SpectraMax GeminiEM,
Molecular Devices, Sunnyvale, CA). Analysis of dose-response
curve was performed using the non-linear regression fit (log of
dose vs. inhibitory response) function of GraphPad Prism® v.
6.07 to calculate the IC50 value of the drug that inhibited 50% of
H2O2-induced mitochondrial metabolic dysfunction.
1-Hydroxy-6-(2'-methoxybiphenyl-4-yl)-4-
methylpyridin-2(1H)-one (11k)
According to the procedure described for the synthesis of
11a; Yield = 71%; Yellow solid; m. pt. 242-243oC; HPLC
1
purity: 97.8%, tR = 8.3 min; H NMR (CDCl3, 500 MHz): δ
7.71-7.00 (8H, m, Ar-H), 6.53 (1H, s, Ar-H), 6.24 (1H, s,
Ar-H), 3.84 (3H, s, -OCH3), 2.29 (3H, s, -CH3); 13C NMR
(CDCl3, 125 MHz): δ 157.5, 156.4, 148.4, 142.1, 140.3,
130.7, 129.5, 129.4, 129.1, 128.4, 120.9, 113.2, 111.3,
109.1, 55.5, 21.4; HRMS (ESI) [M-H]- Calcd for
C19H16NO3: 306.1135, Found: 306.1139.
LDH assay
Cell death were assessed by measuring the amount of lactate
dehydrogenase (LDH) released into the culture supernatant
using a diagnostic kit (Sigma-Aldrich, St. Louis, MO), as
described previously31. The data were expressed as the
percentage of total LDH release, as measured with repeatedly
frozen and thawed parallel cell cultures.
1-Hydroxy-4-methyl-6-(2'-methylbiphenyl-4-yl)pyridin-
2(1H)-one (11l)
According to the procedure described for the synthesis of
11a; Yield = 66%; Yellow solid; m. pt. 184-185oC; HPLC
1
purity: 97.8%, tR = 8.2 min; H NMR (CDCl3, 500 MHz): δ
7.72-7.25 (8H, m, Ar-H), 6.54 (1H, s, Ar-H), 6.26 (1H, s,
Ar-H), 2.31 (3H, s, -CH3), 2.29 (3H, s, -CH3); 13C NMR
(CDCl3, 125 MHz): δ 157.5, 148.5, 143.6, 142.1, 140.8,
135.1, 130.3, 129.6, 129.1, 128.6, 127.6, 125.8, 113.4,
Measurement of mitochondrial membrane potential
Mitochondrial membrane potential was assessed by the
mitochondrial uptake of Tetramethylrhodamine methyl ester

(TMRM, Molecular probes) as previously described32. In brief,
cells were loaded with 500 nM TMRM at 37°C for 10 min and
the changes in the mitochondrial accumulation of TMRM was
analysed by using a microplate reader (SpectraMax GeminiEM,
Molecular Devices).
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Measurement of oxygen consumption rate
Mitochondrial oxygen consumption rate (OCR), an indicator
for mitochondrial respiration functionality, was measured using
the XF24 extracellular flux analyser (Seahorse Bioscience,
Billerical, MA), as previously described31. After sequential
addition of oligomycin (an ATP synthase inhibitor; Sigma-
Aldrich), FCCP (a protonophore; Sigma-Aldrich), and rotenone
(a complex I inhibitor; Sigma-Aldrich) in the presence or
absence of each derivative and H2O2 to astrocytes, OCR was
assessed according to the manufacturer’s protocol. The
extracellular acidification rate (ECAR), an indicator for cellular
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oxygen-independent
glycolysis
was
also
measured
14.
15.
16.
17.
simultaneously. Bioenergetic parameters were normalized to
baseline and calculated as ATP turnover (the difference
between the starting basal OCR and oligomycin-repressed
OCR) as well as maximal respiration capacity (the difference
between FCCP and rotenone-induced OCR changes).
Statistical analysis
Data were expressed as mean ± standard error of the mean
(SEM) and analysed for statistical significance by using either
non-parametric Kruskal-Wallis test followed by Mann-Whitney
U test or an analysis of variance (ANOVA) followed by the
post-hoc Bonferroni test for multiple comparisons, depending
on the Levene test results (SPSS; release20.0.0.1, IBM Corp.).
18.
19.
20.
21.
22.
23.
Acknowledgments
This study was supported by grants from the Bio & Medical
Technology Development Program (2012M3A9C1053532 and
2015M3A6A4065734 to K. Lee) and Basic Science Research
Program (2015R1A2A2A01004202 to W.-K. Kim and
2014R1A1A1037088 to C. Ju) through the National Research
Foundation of Korea (NRF) funded by the Ministry of Science,
ICT & Future Planning, Republic of Korea.
24.
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