Novel amidrazone derivatives: Design, synthesis and activity evaluation

Article abstract of DOI:10.1016/j.bmc.2018.04.042

A series of new 6-styryl-naphthalene-2-amidrazone derivatives were synthesized and evaluated as potential ASIC1a inhibitors. Among them, compound 5e showed the most activity to inhibit [Ca²⁺]_i. elevation in acid-induced articular chondrocytes. Together with the important role of ASIC1a in the pathogenesis of tissue acidification diseases including rheumatoid arthritis, these results might provide a meaningful hint or inspiration in developing drugs targeting at tissue acidification diseases.

Full text of DOI:10.1016/j.bmc.2018.04.042

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel amidrazone derivatives: Design, synthesis and activity evaluation
⇑
⇑
Hua Zhou, Zhi Sen Wang, Xin Hua Liu , Fei Hu Chen
School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 6-styryl-naphthalene-2-amidrazone derivatives were synthesized and evaluated as
potential ASIC1a inhibitors. Among them, compound 5e showed the most activity to inhibit [Ca2+]i. ele-
vation in acid-induced articular chondrocytes. Together with the important role of ASIC1a in the patho-
genesis of tissue acidification diseases including rheumatoid arthritis, these results might provide a
meaningful hint or inspiration in developing drugs targeting at tissue acidification diseases.
Ó 2018 Elsevier Ltd. All rights reserved.
Received 14 January 2018
Revised 18 April 2018
Accepted 19 April 2018
Available online xxxx
Keywords:
ASIC activity
Amidrazone
Articular chondrocytes
[Ca2+
]
i
1. Introduction
physiological functions and excessive [Ca2+]i can cause a series of
pathological reaction. Our and other labs have recently reported
Rheumatoid arthritis is a systemic autoimmune disease, which
is characterized by inflammation synovitis and progressive joint
damage mainly in the small diarthrodial joints of the hands and
feet.1,2 Oxygen consumption and the metabolic rate are increased
in inflamed areas, thus promoting lactic acid secretion leading to
a drop in pH.3 Chronic inflammatory conditions such as RA exhibit
a degree of acidosis in the articular synovial fluid (SF), and the pH
of the SF may drop to < 6.0 in patients with active RA.4,5 Impor-
tantly, a low SF pH was shown to correlate with radiological joint
destruction in RA patients.6 And cartilage homeostasis is affected
by the local pH.7,8
that extracellular acidification could induce articular chondrocyte
injury through acid-sensing ion channel 1a (ASIC1a)-mediated
intracellular calcium ([Ca2+]i) overload, which suggested that
ASIC1a in chondrocytes may be a potential therapeutic target for
articular cartilage destruction in RA.12,13 In support of this, phar-
macological blockade of ASIC1a or deletion of the ASIC1 gene has
a protective effect for tissue acidification disease.
As we all know, amiloride 1 is a weak, non-selective ASIC1 inhi-
bitor with the IC50 30.2 l
M.14,15 Based on this scaffold, focused on
ASIC1, many analogues have been designed and synthesized.
Among them, amidine A-317567 2 was a potent blocker of ASIC1a
Local tissue acidosis subsequently activates some ion channels
such as acid sensing ion channels (ASICs), further leads to the
occurrence of diseases. ASICs, belonging to the degenerin/epithelial
sodium channel (DEG/ENaC) superfamily, can be activated by pro-
ton under acid condition. To date, at least seven different ASIC sub-
unit proteins (ASIC1a, ASIC1b, ASIC1b2, ASIC2a, ASIC2b, ASIC3,
ASIC4) have been cloned and identified in mammalian.9 They share
the same topology structures of their subunits (two transmem-
brane domains, a large extracellular loop, and short intracellular
N and C-termini), and the majority of ASIC subunit can form
amiloride-sensitive cation channels.10 Among them, ASIC1a is spe-
cial under acid condition on account of elevation intracellular
[Ca2+].11 Intracellular Ca2+ ([Ca2+]i) is a ubiquitous second messen-
ger in signal transduction pathways that modulates diverse
than amiloride with IC50 2.0 lM which was determined by the
patch clamp using dissociated adult rat dorsal root ganglion neu-
rons.16 Compounds 3 and 4 also showed the potent activity against
ASIC1a17(Fig. 1). Besides, there are already several venom peptides
with much greater selectivity than small molecules, such as
PcTx1,18 which was isolated from the venom of the South Ameri-
can tarantula Psalmopoeus cambridgei, of psalmotoxin 1. And there
is also a small molecule ASIC inhibitor NS383 that is claimed to be
selective for ASIC1a and ASIC3.19 These compounds are not selec-
tive for the ASIC1a except PcTx1.
However, these active compounds above give us preliminary
structure-activity relationships. We found that there was a com-
mon amidine group with a large polarity, which was easier to form
hydrogen bond with the ion channel protein. Therefore, based on
this, target compounds were designed by adding an amino group
to form another larger polarity group amidrazone. Moiety of pyra-
zine-2,6-diamine or 1,2,3,4-tetrahydroisoquinoline (compounds 1,
⇑
Corresponding authors.
E-mail addresses: xhliuhx@163.com (X.H. Liu), fhchen@sohu.com (F.H. Chen).
https://doi.org/10.1016/j.bmc.2018.04.042
0968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
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H. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 1. Preliminary structure-activity relationships as for target compounds.
2, 3, Fig. 1) containing nitrogen may be detrimental to selectivity,
more nitrogen of the six membered ring, the more weak to the
activity (compounds 1, 2), therefore a simple benzene ring was
used in this study. The authors reported that the isomeric Z-olefin
compound was ꢀ10-fold less potent than the E-olefin compound,
and the reduction of the double bond to alkane compound also
led to a marked decrease in ASIC-3 inhibition. From this point of
view, we designed and synthesized E-olefin compound for ASIC1a
for the reason that ASIC1a and ASIC-3 belong to the same super
family.
Based on all above, to find efficient lead compound with ASIC1a
inhibition activity, we designed and synthesized a series of 6-
styryl-naphthalene-2-amidrazone derivatives. In order to evaluate
whether the compounds had ASIC1a inhibition activity, we used
the laser scanning confocal microscope to observe the effect of tar-
get compounds on [Ca2+]i in rat articular chondrocytes. We hope
that the designed lead compounds may shed light on ASIC1a inhi-
bitors for medicinal chemistry in the field.
2.2. Activity evaluation of blocking ASIC1a
Whether direct Ca2+ entry through ASIC1a channels makes a
substantial and direct contribution to [Ca2+]i homeostasis is still
debatable in the some cells now.20,21 However, under appropriate
acid condition, activation of ASIC1a receptors can potentially cause
elevation intracellular Ca2+ in rat articular chondrocytes. Therefore,
if ASIC1a is blocked, the level of Ca2+ is lower than non-blocking
group. The potential effects of the synthesized compounds on
blocking ASIC1a activity were investigated by observing [Ca2+]i in
acid-induced rat articular chondrocytes, which were measured by
laser scanning confocal microscopy. For this purpose, we initially
observed the effects of compound 5 on chondrocytes viability to
determine concentration for the subsequent experiments. MTT
assays showed that apart from compound 5h and compound 5e
with the concentration of 50
significant influence on the viability of chondrocytes with the
concentration of 1.563–25 M (compounds 5a, 5b, 5d, 5f, 5g)
and 3.125–25 (compounds 5c, 5e) by 24 h treatment
lM, all other compound 5 had no
l
lM
a
(Fig. 2). Therefore, we selected those concentrations to observe
their effects on ASIC1a function in rat articular chondrocytes.
2. Results and discussion
2.1. Chemistry
2.2.1. Compound 5 decrease acid-induced protein expression of ASIC1a
In order to test whether compound 5 could inhibit acid-induced
protein expression of ASIC1a, we observed the effect of different
pH and time on ASIC1a protein expression. Western blotting
results showed that the ASIC1a protein expression in articular
chondrocytes was remarkably increased as the medium pH
decreased, with no significant difference between pH 6.0 and pH
5.5. The expression of ASIC1a protein increased within 3 h, but
gradually decreased after 3 h (Fig. 3). Exposured to acidic medium
(pH 6.0) for 3 h, the acidosis condition was selected as the follow-
ing western blotting experiments. Then, cultured chondrocytes
were pretreated with compound 5 for 24 h followed by stimulation
with acidic medium (pH 6.0) for 3 h. Western blotting results
6-Styryl-naphthalene-2-amidrazones (target compound 5)
were synthesized according to the protocol outlined in Scheme 1.
Among them, compound 7 (6-bromo-naphthalene-2-carboxylic
acid amide) was prepared starting from a condensation of 6-
bromo-2-naphthoic acid with the ammonia solution. Then com-
pound 9 was synthesized from a Heck reaction of compound 8 with
styrene or substituted styrene. Subsequently, compound 9 was
treated with LiN(TMS)2 to produce compound 10 and then com-
pound 10 was reacted with hydrazine hydrate to gain the target
compound 5. Details of the synthesis process and structural char-
acterization were shown in support information.
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H. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
Scheme 1. Synthesis of compound 5. R = H (5a); R = 4-F (5b); R = 4-Cl (5c); R = 2-F (5d); R = 4-OCH3 (5e); R = 3-F (5f); R = 3-Cl (5g); R = 4-CH3 (5h). Reagent and Conditions:
(A) (COCl)2, CH2Cl2, rt; NH3ꢁH2O, ice bath; (B) POCl3, 80 °C; (C) Pd(OAc)2, 1,10-Bis(diphenylphosphino) ferrocene, Cs2CO3, DMF, 90 °C; (D) LiN(TMS)2, THF, rt, overnight; 2 M
EtOH/HCl, rt, 12 h; (E) N2H4ꢁH2O, MeOH, rt.
Fig. 2. Effect of target compounds 5 on articular chondrocytes viability. (a) DMSO group (5‰). (b) Amiloride (100, 50, 25, 12.5 and 6.25
(25, 12.5, 6.25, 3.125 and 1.563 M). (d) Compounds 5f and 5g (25, 12.5, 6.25, 3.125 and 1.563 M). (e) Compounds 5c, 5e and 5h (50, 25, 12.5, 6.25 and 3.125
viability was determined by MTT assay. Data were expressed as means SE obtained in three independent experiments.*p < 0.05, **p < 0.01 versus normal group.
l
M). (c) Compounds 5a, 5b and 5d
l
l
l
M). Cell
showed that with addition compound 5b, most of the compounds
could observably decreased the acid-induced protein expression of
ASIC1a and the highest concentration group had similar effect on
inhibition of ASIC1a protein expression compared with positive
drug amiloride group (Fig. 4). Para-F, the strong electron-with-
drawing group in compound 5b may be the reason that the com-
pound had no effect on ASIC1a expression compared with the
other compound 5. The strong electron-withdrawing group signif-
icantly decrease cloud density of benzene ring. This should be
affect the pi-pi interaction between the title compound and the
target.
2.2.2. Compound 5 inhibit [Ca2+]i elevation in acid-induced articular
chondrocytes
In order to further investigate the roles of compound 5 in influ-
encing ASIC1a function, we tested the effect of compound 5 and
amiloride, the ASIC1a non-specific inhibitor, on the acid-induced
articular chondrocytes. In our all experiments, 5 lM nimodipine
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H. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 3. Effect of different pH and time point on the protein expression of ASIC1a. (a) Different pH group. (b) Different time point under pH 6.0. Cultured chondrocytes were
treated with different pH medium or different time point under pH 6.0. The level of ASIC1a protein was assessed by Western blotting. b-Actin was chosen as an internal
control. Data were presented as mean SD obtained in three independent experiments. *p < 0.05, **p < 0.01 versus normal group (pH 7.4).
was added to all solutions as a blocker of voltage-gated Ca2+-chan-
nels to inhibit possible secondary activation of channels and
release of the internal Ca2+ stores. From the laser scanning confocal
micrographs and the representative traces, the results showed that
pH 6.0 solution significantly increased [Ca2+]i in articular chondro-
cytes. Furthermore, pretreated with compound 5 or blockade of
ASIC1a with amiloride could significantly reduce the fluorescence
intensity of intracellular calcium by acid-induced (Fig. 5). Among
the tested compounds and amiloride, inhibition rate of Ca2+ fluo-
4.1.1. General procedure for the synthesis of target compounds 5a–h
To a solution of the compound 10 (0.3 mmol) in methanol (10
mL) was added to 80% hydrazine hydrate (0.9 mmol). The reaction
mixture was stirred at room temperature overnight monitored by
TLC (methylene chloride, methanol, V/V = 10:1). As the reaction
progressed, solid was continuously produced. The product was col-
lected by filtration to give compound 5a–h as colorless solids.
5a: 6-Styryl-naphthalene-2-amidrazone, light yellow solid,
yield, 28%, mp 221–222 °C; 1H NMR (400 MHz, DMSO-d6) d: 8.15
(s, 1H), 8.01–7.77 (m, 5H), 7.66 (d, J = 7.5 Hz, 2H), 7.49–7.37 (m,
4H), 7.29 (dd, J = 14.7, 7.3 Hz, 1H), 5.76 + 5.23 (2brs, 4H,
AC(@NH)NHNH2). ESI-MS: 288.1 (C19H17N3, [M+H]+).
rescence intensity was 20.83% (5c, 50
l
M), 33.37% (5a, 25
M), 63.23% (5h, 50
M), and 23.49% (amiloride, 100 M). Results
l
M),
52.07% (5d, 25
68.92% (5e, 25
l
l
M), 59.51% (5g, 25
l
l
M)
l
showed that most compounds were superior to amiloride and
compound 5e was the most potent.
5b: 6-[2-(4-Fluoro-phenyl)-vinyl]-naphthalene-2-amidrazone,
light yellow solid, yield, 31%; mp 235–236 °C; 1H NMR (400 MHz,
DMSO-d6) d: 8.15 (s, 1H), 7.98–7.77 (m, 5H), 7.71 (dd, J = 8.0, 5.9
Hz, 2H), 7.42 (d, J = 16.6 Hz, 1H, ACH@CHA), 7.36 (d, J = 16.6 Hz,
1H, ACH@CHA), 7.24 (t, J = 8.7 Hz, 2H), 5.75 + 5.25 (2brs, 4H,
AC(@NH)NHNH2). ESI-MS: 306.1 (C19H16FN3, [M+H]+).
5c: 6-[2-(4-Chloro-phenyl)-vinyl]-naphthalene-2-amidrazone,
light yellow solid, yield, 25%, mp 249–250 °C; 1H NMR (600 MHz,
DMSO-d6) d: 8.14 (s, 1H), 7.99–7.77 (m, 5H), 7.67 (t, J = 8.6 Hz, 2H),
7.48–7.35 (m, 4H), 5.95 + 5.74 (2brs, 4H, AC(@NH)NHNH2). HR-ESI-
MS: m/z [M+H]+ calcd for C19H16ClN3: 322.1106; found: 322.1105.
5d: 6-[2-(2-Fluoro-phenyl)-vinyl]-naphthalene-2-amidrazone,
light yellow solid, yield, 33%; mp 239–240 °C; 1H NMR (600 MHz,
DMSO-d6) d: 8.14 (s, 1H), 8.01 (s, 1H), 7.91 (d, J = 8.7 Hz, 1H),
7.85 (dt, J = 28.0, 8.7 Hz, 4H), 7.49 (d, J = 16.5 Hz, 1H, ACH@CHA),
7.42 (d, J = 16.5 Hz, 1H, ACH@CHA), 7.33 (dd, J = 13.6, 7.2 Hz,
1H), 7.24 (t, J = 8.3 Hz, 2H), 5.80 + 5.25 (2brs, 4H, AC(@NH)
NHNH2). HR-ESI-MS: m/z [M+H]+ calcd for C19H16FN3: 306.1401;
found: 306.1403.
3. Conclusion
In summary, we designed and synthesized some 6-styryl-naph-
thalene-2- amidrazone derivatives as potential ASIC1a inhibitors,
followed by chemical synthesis and biological evaluated for them.
Among them, compound 5e exhibited strong inhibition effect on
ASIC1a, whose inhibition rate was 68.92% at 25 lM. These results
preliminarily showed that some title compounds had potential
inhibition effect on function, which suggested that 6-styryl- naph-
thalene-2-amidrazone derivatives may have a potential blocking
effect of ASIC1a, but this still need further experiments to confirm
through patch clamp and their selectivity on ASICs also require fur-
ther study.
Together with the important role of ASIC1a in the pathogenesis
of tissue acidification diseases including rheumatoid arthritis, we
hope that these results might provide a meaningful hint or inspira-
tion in developing drugs targeting at tissue acidification diseases.
5e:
6-[2-(4-Methoxy-phenyl)-vinyl]-naphthalene-2-amidra-
zone, light yellow solid, yield, 71%; mp 228–229 °C; 1H NMR
(600 MHz, DMSO-d6) d: 8.12 (s, 1H), 7.93–7.86 (m, 2H), 7.81 (dt,
J = 20.6, 8.6 Hz, 3H), 7.58 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 16.4 Hz,
1H, ACH@CHA), 7.24 (d, J = 16.4 Hz, 1H, ACH@CHA), 6.96 (t, J =
6.9 Hz, 2H), 5.84 + 5.26 (2brs, 4H, AC(@NH)NHNH2), 3.77 (s, 3H,
AOCH3). HR-ESI-MS: m/z [M+H]+ calcd for C20H19N3O: 318.1601;
found: 318.1601.
4. Experimental section
4.1. Chemistry
The degree of reactions was monitored by thin layer chro-
matography (TLC) on Merck pre-coated silica GF254 plates. Melt-
5f: 6-[2-(3-Fluoro-phenyl)-vinyl]-naphthalene-2-amidrazone,
light yellow solid, yield, 65%; mp 261–262 °C; 1H NMR (600 MHz,
DMSO-d6) d: 8.14 (s, 1H), 7.97 (d, J = 7.4 Hz, 1H), 7.93–7.89 (m,
1H), 7.83 (ddd, J = 13.4, 12.6, 6.9 Hz, 3H), 7.51 (d, J = 7.9 Hz, 1H),
7.48–7.45 (m, 2H), 7.44–7.36 (m, 2H), 7.10 (t, J = 8.4 Hz, 1H),
5.80 + 5.25 (2brs, 4H, AC(@NH)NHNH2). HR-ESI-MS: m/z [M+H]+
calcd for C19H16FN3: 306.1401; found: 306.1401.
ing points (approximately) were confirmed by
a XT4MP
apparatus (Taike Corp., Beijing, China). 1H NMR and 13C NMR spec-
tra were collected on PX400 spectrometer at room temperature
with TMS and solvent signals allotted as internal standards. Chem-
ical shifts were reported in ppm (d). Mass spectra were performed
on an Agilent 1260–6221 TOF mass spectrometer.
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H. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
Fig. 4. Target compound 5 and amiloride (100 lM) inhibited acid medium (pH 6.0) modulation of ASIC1a protein expression. Cultured chondrocytes were pretreated with or
without target compound for 24 h followed by stimulation with acid medium (pH 6.0) for 3 h (Fig. 4a–h). The level of ASIC1a protein was assessed by western blotting. b-
Actin was chosen as an internal control. Data were presented as mean SD obtained in three independent experiments. **p < 0.01 versus control group; &&p < 0.01 versus pH
6.0 + DMSO group.
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H. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5g: 6-[2-(3-Chloro-phenyl)-vinyl]-naphthalene-2-amidrazone,
(s, 2H), 2.30 (s, 3H, ACH3). HR-ESI-MS: m/z [M+H]+ calcd for
light yellow solid, yield, 37%; mp 213–214 °C; 1H NMR (600 MHz,
DMSO-d6) d: 8.16 (s, 1H), 8.00 (s, 1H), 7.97–7.81 (m, 4H), 7.75
(s, 1H), 7.62 (t, J = 8.2 Hz, 1H), 7.53 (d, J = 16.5 Hz, 1H), 7.42 (dd,
J = 19.6, 12.0 Hz, 2H), 7.34 (d, J = 7.9 Hz, 1H), 5.94 + 5.39 (2brs,
4H, AC(@NH)NHNH2). HR-ESI-MS: m/z [M+H]+ calcd for
C
20H19N3: 302.1652; found:302.1650.
4.2. Isolation of articular chondrocytes and treatment
Rat articular chondrocytes were isolated using the method
described previously.22 Cartilage tissue from knee joint of three
or four rats was merged for each isolation. Cartilage from the knee
joint was cutted into small pieces (about 1 ꢂ 1 mm) and firstly
digested with Trypsin-EDTA Solution (Beyotime, Hangzhou, China)
and then digested with 0.2% type II collagenase (Sigma Chemical
C19H16ClN3: 322.1106; found: 322.1106.
5h:
6-(2-p-Tolyl-vinyl)-naphthalene-2-amidrazone,
light
yellow solid, yield, 25%; mp 243–244 °C; 1H NMR (600 MHz,
DMSO-d6) d: 8.13 (s, 1H), 7.95–7.77 (m, 5H), 7.54 (t, J = 8.5 Hz,
2H), 7.40–7.33 (m, 2H), 7.20 (d, J = 7.7 Hz, 2H), 5.92 (s, 2H), 5.35
Fig. 5. Blockade of ASIC1a with compounds 5 and amiloride reduced acid-induced elevation of [Ca2+]i level in articular chondrocytes. Cellular confocal micrographs of the
same scale showed the changes in the [Ca2+]i intensity, as visualized by Fluo-3-AM, in articular chondrocytes. (a) Acid-induced elevation of [Ca2+]i in Ca2+-free extracellular
solution; (b) Acid-induced elevation of [Ca2+]i in extracellular Ca2+ solution; (c) Acid-induced elevation of [Ca2+]i in chondrocytes treated with compound 5a (25
l
M); (d)
M); (e) Acid-induced elevation of [Ca2+]i in chondrocytes treated with compound 5d (25
M); (f) Acid-induced elevation of [Ca2+]i in chondrocytes treated with compound 5e (25 M); (g) Acid-induced elevation of [Ca2+]i in chondrocytes treated with compound
5g (25 in chondrocytes treated with compound 5h (50 in chondrocytes treated with
M); (h) Acid-induced elevation of [Ca2+ M); (i) Acid-induced elevation of [Ca2+
amiloride (100
Acid-induced elevation of [Ca2+]i in chondrocytes treated with compound 5c (50
l
l
l
l
]
l
]
i
i
l
M). The amplitude of [Ca2+]i intensity in chondrocytes induced by acid treatment was quantified as the maximal rise of [Ca2+]i above basal levels. i: Before
exposure to acid solution. ii: Increased Ca2+ intensity when pH was decreased to 6.0. iii: A few minutes after exposure to the acid solution (n = 3–4).
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H. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
Fig. 5 (continued)
Co., St. Louis, MO, USA) in PBS. After digestion, isolated cells were
the cells were incubated in complete DMEM solution at 37 °C for 4
h. At the end of incubation, MTT solutions were removed, 150 lL
centrifuged and washed three times with PBS. The freshly isolated
chondrocytes were plated at a density of 2 ꢂ 104 cells/well, which
had been immersed previously in plastic dishes filled with Dul-
becco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO,
USA) supplemented with 10% fetal bovine serum (FBS, Sigma, St.
dimethyl sulfoxide (DMSO) was added to each well and stirred to
dissolve the water-insoluble formazan salt. Then, the optical den-
sity (OD) of each well was measured at 492 nm using an
enzyme-linked immunosorbent assay (ELISA) microplate reader
(Biotek, Vermont, USA), and the well without cells served as blank
control. All experiments were performed in triplicate and should
be repeated at least three times.
Louis, MO, USA), 100 IU/ml penicillin, and 100 lg/mL strepto-
mycin. These cultures were maintained under sterile conditions
at 37 °C in a humidified 5% CO2 incubator. Primary rat articular
chondrocytes were subjected to different treatments with
increased concentrations of compound 5a–h (1.563–50 lM) in
4.4. Laser scanning confocal microscopy
DMEM supplemented with 10% FBS for 24 h. After exposure to a
normal extracellular solution (pH 7.4) with compound 5a–h, chon-
drocytes were cultured in a low pH solution (pH 6.0) for 3 h. Chon-
drocytes cultured in the pH 7.4 solution were used as control
group. After treatment, the cells were washed three times with
PBS and cell lysates were subjected to Western blot.
The intracellular calcium level of isolated rat articular chondro-
cytes was monitored using fluorescence imaging.23 1 ꢂ 105 cells
were planted into small plate that was only used for laser scanning
confocal microscopy and after 24 h, cells were washed three times
with D-Hanks’ solution and incubated with 10 lM Fluo-3-AM and
0.02% Pluronic F-127 (Biotium, Hayward, California, USA) for 30
min at 37 °C. After incubation, the cells were washed three times
with Hank’s solution to remove the extracellular Fluo3-AM. To
eliminate the effects of voltage-gated Ca2+ channels from intracel-
4.3. Cell viability assay
Cells were planted into 96-well plates (5 ꢂ 103 cells/well) and
incubated for 24 h. After treatment, 20
l
L of MTT (Sigma, St. Louis,
lular stores, nimodipine (5
lM) was added to the extracellular
MO, USA) reagent solution (5 mg/mL) was added to each well, and
fluid. Then cells were perfused initially with D-Hank’s solution
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(pH 6.0), or pretreated with Amiloride (100
l
M) or compounds 5a–
M; compounds 5c, 5h 50
M) and then perfused with Hank’s solution (pH 6.0). The fluores-
h (compounds 5a, 5b, 5d, 5e, 5f, 5g 25
l
l
cence of intracellular Fluo-3 was quantitated by confocal laser
scanning fluorescence microscopy (Zeiss 710, Germany) with exci-
tation at 488 nm and emission at 525 nm. Gray scale images with
0–50 steps were collected at different time points before and up to
5 min after fluorescence microscopy and archived as TIFF image
files for later analysis. Digitized images were acquired, stored
and analyzed on a computer controlled by Leica-sp5 LAS AF soft-
ware. Images (340/380 ratio) were analyzed by averaging pixel
ratio values in circumscribed regions of cells in the field of view.
The values were exported from Axon Imaging Workbench software
to SigmaPlot for additional analysis and plotting.
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Author contributions
Xin-Hua Liu and Fei-Hu Chen designed the research; Zhi Sen
Wang and Hua Zhou conducted the studies and prepared the
manuscript; all authors read and approved the manuscript.
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physiological
and
therapeutical
perspectives.
Neuropharmacology.
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11. Sherwood TW, Frey EN, Askwith CC. Structure and activity of the acid-sensing
ion channels. AJP Cell Physiol. 2012;303:C699–C710.
12. Yuan FL, Chen FH, Lu WG, et al. Acid-sensing ion channel 1a mediates acid-
induced increases in intracellular calcium in rat articular chondrocytes. Mol Cell
Biochem. 2010;340:153–159.
Additional informations
13. Li X, Wu FR, Xu RS, et al. Acid-sensing ion channel 1a-mediated calcium influx
regulates apoptosis of endplate chondrocytes in intervertebral discs. Expert
Opin Ther Targets. 2014;18:1–14.
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articular chondrocytes by amiloride attenuates articular cartilage destruction
in rats with adjuvant arthritis. Inflammation Res. 2010;59:939–947.
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characterization of A-317567, a novel blocker of acid sensing ion channels.
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Supplemental Informations include synthesis of all intermedi-
ates, 1H NMR and HR-ESI-MS of title compounds. Supplementary
information
accompanies
this
paper
at
http://
www.nature.com/scientificreports.
Competing financial interests
16. Kuduk SD, Di Marco CN, Bodmer-Narkevitch V, et al. Synthesis, structure-
activity relationship, and pharmacological profile of analogs of the ASIC-3
inhibitor A-317567. ACS Chem Neurosci. 2010;1:19–24.
The authors declare no competing financial interests.
17. Buta A, Maximyuk O, Kovalskyy D, et al. Novel potent orthosteric antagonist of
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The authors wish to thank the National Natural Science Founda-
tion of China (No. 21572003, 21172048).
A. Supplementary data
21. Tian Y, Bresenitz P, Reska A, EI Moussaoui L, Beier CP, Grunder S. Glioblastoma
cancer stem cell lines express functional acid sensing ion channels ASI-C1a and
ASIC3. Sci Rep. 2017;7 13674.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2018.04.042.
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Please cite this article in press as: Zhou H., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.042