Cytoprotective effects of hydrogen sulfide-releasing N-methyl-d-aspartate receptor antagonists mediated by intracellular sulfane sulfur

Article abstract of DOI:10.1039/c4md00180j

Hydrogen sulfide (H₂S) exerts a host of biological effects ranging from cytotoxicity to cytoprotection. Cytotoxicity of H₂S in neurodegenerative diseases may be mediated by N-methyl-d-aspartate receptor (NMDAR) activation. To exploit

Full text of DOI:10.1039/c4md00180j

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Cytoprotective effects of hydrogen sulfide-
releasing N-methyl-D-aspartate receptor
antagonists mediated by intracellular sulfane
sulfur†
Cite this: Med. Chem. Commun., 2014,
, 1577
5
a
a
b
c
d
Eizo Marutani,* Masahiro Sakaguchi, Wei Chen, Kiyoshi Sasakura, Jifeng Liu,
b
c
c
a
Ming Xian, Kenjiro Hanaoka, Tetsuo Nagano and Fumito Ichinose
Hydrogen sulfide (H
Cytotoxicity of H S in neurodegenerative diseases may be mediated by N-methyl-D-aspartate receptor
NMDAR) activation. To exploit cytoprotective effects of H S while minimizing its toxicity, we synthesized
2
S) exerts a host of biological effects ranging from cytotoxicity to cytoprotection.
2
(
2
a
series of
H
2
S-releasing NMDAR antagonists and examined their effects against 1-methyl-4-
+
phenylpyridinium (MPP )-induced cell death, a cellular model of Parkinson's disease. We observed that
Received 21st April 2014
Accepted 29th July 2014
2
cytoprotective effects of H S-releasing NMDAR antagonists correlated with their ability to increase
intracellular sulfane sulfur, but not H
2
S, levels. These studies suggest that H S-donor compounds that
2
DOI: 10.1039/c4md00180j
increase intracellular sulfane sulfur levels are potentially useful neuroprotective agents against
neurodegenerative diseases.
www.rsc.org/medchemcomm
13
substantia nigra. We found that inhaled H
2
S protected mice
1
. Introduction
from neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-
Hydrogen sulde (H S) is an endogenously produced gaseous tetrahydropyridine (MPTP), a toxin which causes mitochondrial
2
1
signaling molecule. In mammalian tissues, H S is generated by dysfunction in dopaminergic neurons of mammalian midbrain,
2
14
cystathionine b-synthase (CBS), cystathionine gamma-lyase (CSE) lead to Parkinson's disease-like symptoms. Recently, we have
2,3
and 3-mercaptopyruvate sulfurtransferase (3MST). On the other reported that a novel H
S-releasing hybrid N-methyl-D-aspartate
), thio- receptor (NMDAR) antagonist derivative, S-memantine, is more
) in reactions catalyzed by effective in protecting neurons against ischemic brain injury
S or its metabolites can also be converted to than the H S donor compound alone (i.e. the H S donor without
polysuldes that contain sulfane sulfur, a sulfur atom with six NMDAR antagonists) or memantine. S-mematine is a hybrid
2
2ꢀ
hand, H
sulfate (S
several enzymes. H
2
S is serially oxidized to persulde, sulte (SO
3
2ꢀ
2ꢀ
4
O
2 3
), and sulfate (SO
4
2
2
2
15
0
5,6
valence electrons but no charge (represented as S ). H S and derivative of a NMDAR antagonist memantine chemically
2
2
sulfane sulfur coexist and recent work suggests that sulfane combined with a slow releasing H S donor. However, mecha-
sulfur containing polysuldes, derived from H S, may be the nisms responsible for the cytoprotective effects of S-memantine
2
5–8
actual signaling molecules. Nonetheless, measurement of H
2
S
are incompletely dened.
or sulfur metabolites in biological samples has been technically
In the present study, we synthesized a series of H S-releasing
2
challenging and the role of sulfane sulfur in the cytoprotective NMDAR antagonists (Fig. 1) to compare their protective effects
6,9–11
2
effects of H S is incompletely understood.
2
and abilities to increase H S, or sulfane sulfur levels in culture
Parkinson's disease (PD) is one of the most common cells, or medium. We hypothesized that the protective effects of
12
neurodegenerative diseases. It is characterized by slow and
2
H S-releasing NMDAR antagonists may be related to the ability
progressive degeneration of dopaminergic neurons in the to increase intracellular levels of sulfane sulfur while inhibiting
NMDAR. To address this hypothesis, we examined the cyto-
protective effect and the ability to increase intracellular levels of
a
Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General
sulfane sulfur of H S-releasing NMDAR antagonists and their
2
Hospital and Harvard Medical School, Boston, Massachusetts, USA. E-mail:
parent H S donor. Unexpectedly, we observed that the protec-
2
emarutani@mgh.harvard.edu
b
tive effects of H
1-methyl-4-phenylpyridinium (MPP , a metabolite of MPTP)-
induced cell death were similar to those of the parent H S donor
compounds (H S-releasing compounds without NMDAR
2
S-releasing NMDAR antagonists against
Department of Chemistry, Washington State University, Pullman, Washington, USA
+
c
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-
ku, Tokyo 113-0033, Japan
2
d
Aberjona Laboratories, Inc., Beverly, MA, USA
2
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4md00180j
antagonists) alone. However, our study revealed that the
1
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2 2
Fig. 1 Structures of NMDAR antagonists, H S donors, and H S-releasing NMDAR antagonists.
2
cytoprotective effects of H S donor compounds correlated with 20 mM using crystal violet (CV) or 3-(4,5-dimethylthiazol-2-yl)-2,5-
their ability to increase intracellular levels of sulfane sulfur, but diphenyltetrazolium bromide (MTT) assay as described previ-
15
not H S.
ously. The result showed that CTBA (only in the MTT method
2
The present results indicate that sulfane sulfur, but not H S, but not in the CV method), ACS48-amantadine, and CTBA-
2
+
is the intracellular sulde metabolite that prevents MPP - memantine, but not ACS48, ACS81, S-memantine, ACS81-mem-
induced cytotoxicity and demonstrate the feasibility of drug antine, Na
2
S, or NMDAR antagonist alone, improved cell viability.
S- or However, there was no signicant difference in cell viability
between SH-SY5Y cells treated with H S-releasing NMDAR
antagonists and cells treated with their parent H S donor
compounds alone (Fig. 2). These observations indicate that,
development against neurodegenerative diseases using H
sulfane sulfur-based compounds.
2
2
2
2. Results and discussion
15
unlike in ischemic neuronal injury, a combination of a NMDAR
2.1. Chemistry
antagonist with a H S donor may not enhance the cytoprotective
2
+
4
-(3-Thioxo-3H-1,2-dithiol-4-yl)benzoic acid (ACS48) and N- effect of the H
2
S donor against MPP -induced toxicity.
(
(1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-
dithiol-4-yl)-benzamide (S-memantine) were prepared as
2
.3. H S donors increase intracellular levels of sulde and
2
15
described previously.
3-(Prop-2-en-1-yldisulfanyl)propanoic
sulfane sulfur in SH-SY5Y cells
acid (ACS81), 4-carbamothioylbenzoic acid (CTBA), 4-(3-thioxo-
2
To compare the ability of various H S donors (ACS48, ACS81,
3
,7
3
H-1,2-dithiol-4-yl)-N-[(1S,3R,5R,7S)-tricyclo[3.3.1.1 ]dec-1-yl]-
CTBA, S-memantine, ACS48-amantadine, ACS81-memantine,
CTBA-memantine, and Na S) and to increase H S or sulfane
benzamide (ACS48-amantadine), 3-(prop-2-en-1-yldisulfanyl)-N-
2
2
(
(
(1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)propanamide
ACS81-memantine), and 4-carbamothioyl-N-[(1R,3S,5S,7R)-3,5-
sulfur levels in the cell or medium in the absence of cells, we
measured the uorescence intensity of a novel H S-specic
probe HSip-1 or a sulfane sulfur-specic probe SSP4 loaded in
SH-SY5Y cells or medium aer the addition of H S donors at
0 mM to the medium (DMEM/F12, 10% FBS). Since the uo-
3
,7
2
dimethyltricyclo[3.3.1.1 ]-dec-1-yl]benzamide
(CTBA-mem-
antine) were prepared as shown in Schemes 1–3.
2
10
2
2
.2. H S donors prevent cell death induced by 1-methyl-4-
2
rescence intensity of HSip-1 or SSP4 was almost saturated 3 h
+
phenylpyridinium (MPP ) in SH-SY5Y cells
aer the addition of the H S donor (Fig. S1 and S2†), we
2
To examine the protective effects of H S donors, we measured cell considered uorescence intensity 3 h aer the addition of the
2
+
viabilities of SH-SY5Y 24 h aer the addition of MPP (5 mM) with
H
2
S donor as an index of the total amount of H
S donors.
2
S or sulfane
H
2
S donor compounds with or without NMDAR antagonists at sulfur increased by H
2
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Scheme 1
Scheme 2
Scheme 3
The result showed that S-memantine and ACS48-amantadine
increased intracellular H
2 2
S levels, while all H S donors
increased H S levels in the medium (Fig. 3A and B). ACS48,
2
CTBA, ACS48-amantadine, and CTBA-memantine increased
intracellular sulfane sulfur levels, while ACS48, ACS81, S-
memantine, ACS48-amantadine, and ACS81-memantine
increased sulfane sulfur levels in the medium (Fig. 3C and D). A
simple sulde salt Na S did not increase sulfane sulfur levels in
2
cells or medium.
2
.4. Levels of intracellular sulfane sulfur, but not H S,
2
correlated with the ability of H
2
S donors to prevent cell death
induced by MPP in SH-SY5Y cells
To dene the role of H S or sulfane sulfur in the protective effect
of H S donors against MPP -induced cytotoxicity, we examined
+
+
Fig. 2 Cell viabilities of SH-SY5Y 24 h after the addition of MPP with
or without the H S donor. Cell viabilities were measured by the CV or
2
the MTT method (panel A or B). N ¼ 4 or 5 each; P < 0.001 control vs.
all the other groups, and *, or ** P < 0.05, or 0.01 vs. vehicle.
2
+
2
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measured by MTT assay was used for the calculation of corre-
+
lation since MPP caused mitochondrial dysfunction and the
mechanism of MTT assay is based on the ability of mitochon-
16
dria to metabolize MTT into formazan. We observed no
signicant relationship between the intracellular or medium

uorescence intensity of HSip-1 and cell viability (Fig. 4A
and B). However, we observed the positive relationship between
the intracellular, but not medium, uorescence intensity of
SSP4 and cell viability (Fig. 4C and D). These observations
indicate that the cytoprotective effects of H S donors against
2
+
MPP -induced toxicity correlate with their ability to produce
intracellular sulfane sulfur, but not H S.
2
3
. Conclusions
In the present study, we synthesized and characterized the
cytoprotective effects of S-memantine and novel H S-releasing
2
NMDAR antagonists (ACS48-amantadine, ACS81-memantine,
and CTBA-memantine). ACS48-amantadine and CTBA-mem-
antine, but not NMDAR antagonists, H
2
S donors alone, or other
H
2
S-releasing NMDAR antagonists, markedly improved the cell
+
viability of SH-SY5Y cells 24 h aer the addition of MPP .
However, we could not observe any H S-releasing NMDAR
2
antagonists that were signicantly more effective than parent
H S donor compounds alone by direct comparisons. Although
2
we have previously reported that S-memantine inhibits NMDAR
activation by glutamate, whether or not inhibition of NMDAR by
2
Fig. 3 Intracellular or medium levels of H S or sulfane sulfur. The
intracellular or medium fluorescence intensity (FI) was measured 3 h
after the addition of the H S donor to the medium loaded by a H
probe HSip-1 (panels A and B) or a sulfane sulfur probe SSP4 (panels C
2
2
S
##
###
and D). N ¼ 4 or 5; **, or *** P < 0.01, or 0.001 vs. baseline. , or
P
2
H S-releasing NMDAR antagonists promotes the benecial
†††
<
0.01, or 0.001 vs. parent H
2
S donor,
P < 0.001.
15,17
2
effect of H S donors against PD remains to be elucidated.
We measured the levels of H S or sulfane sulfur which was
2
produced from H S donor compounds added to the medium
2
the correlation between the intracellular uorescence intensity
of HSip-1 or SSP4 and the cell viability of SH-SY5Y by calculating
Spearman's correlation coefficient. Cell viability which was
and examined the relationship between H
levels and cell viability. These results indicate that intracellular
levels of sulfane sulfur, but not H S, are associated with the
protective effects of H S against MPP -induced cytotoxicity.
2
S or sulfane sulfur
2
+
2
Since the hydrophobicity of CTBA (octanol/water partition
coefficient; c log P ¼ 1.53, calculated by ChemBioDraw Ultra
1
2
3.0, Perkin Elmer, Inc.) is lower than that of ACS48 (c log P ¼
.89) or ACS81 (c log P ¼ 2.03), the highest level of intracellular
sulfane sulfur by the addition of CTBA cannot be explained by
hydrophobicity or cell permeability alone. Caliendo and
colleagues have reported hypothetic hydrolysis mechanisms of
18
2
CTBA and ACS48 to release H S. CTBA may undergo hydrolysis
more markedly than does ACS48 since the aromatic ring in
CTBA makes the formed intermediates stable in the cell.
ACS48-amantadine increased the H S level in the cell or
2
medium as well as S-memantine, while ACS48-amantadine
increased sulfane sulfur levels in the cells and medium more
markedly than did S-memantine. The structural difference
between ACS48-amantadine and S-memantine is only the
NMDAR antagonist (amantadine or memantine). The c log P of
ACS48-amantadine or S-memantine is 4.52 or 5.56, respectively.
Therefore, hydrophobicity or cell permeability cannot explain
the reason for the higher level of intracellular sulfane sulfur by
the addition of ACS48-amantadine. The reason why just two
additional methyl moieties in the NMDAR antagonist dramati-
cally enhanced the ability of ACS48-amantadine to increase the
2
Fig. 4 Correlation between cell viability and H S, or sulfane sulfur
levels. Spearman's correlation coefficient was calculated using cell
viability (MTT method) and the fluorescence intensity of HSip-1 (panels
A and B) or SSP4 (panels C and D).
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intracellular sulfane sulfur levels remains to be elucidated in 124.3, 124.7, 125.2, 125.5, 129.3, 131.4, 132.5, 133.7, 140.1, 151.8,
+
+
the future study.
152.2, 153.2, 164.9, 169.4; MS (ESI ) m/z 627.6 (M + Na) .
4.1.2. Synthesis of ACS48-amantadine. P-Isopropyl benzoic
The present study indicates that a combination of a NMDAR
antagonist with a H S donor may not enhance the cytoprotective acid (2.0 g, 12 mmol) was dissolved in N,N-dimethylformamide
2
+
effect of H
2
S donors against MPP -induced toxicity. These (DMF, 41 ml) followed by addition of amantadine (2.21 g, 15
observations appear to contradict our recent nding that mmol) and N,N-diisopropylethylamine (DIPEA, 8.48 ml, 49
S-memantine exerted more potent neuroprotective effects than mmol) at room temperature (rt) under nitrogen (N ). The reac-
its parent compounds alone (ACS48 or memantine) against tion mixture was then cooled in an ice bath for 10 min. 2-(7-Aza-
2
15
ischemic neuronal injury. The reason for this apparent 1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexa-
contradiction may be the differing role of NMDAR in ischemic uorophosphate (HATU) was added in an ice bath and the
+
neuronal injury and MPP -induced cytotoxicity. NMDAR reaction mixture was stirred for 10 min at this temperature. The
antagonists have been shown to protect neurons from ischemic reaction mixture was warmed up to rt and stirred overnight. The
15
insults. In fact, in the previous study, we observed that reaction mixture was quenched with a solution of NaHCO
3
(200
memantine per se exerted cytoprotective effects against OGD- ml) and then extracted with EtOAc (100 ml ꢁ 3). The combined
15
induced cell death. In contrast, it has been reported that organic layers were washed with water 100 ml ꢁ 1 and brine 100
+
NMDAR antagonism may not be protective against MPP - ml ꢁ 1, and then dried over Na
2
SO
4
. The ltrate was concen-
+
induced cytotoxicity since the mechanism of MPP toxicity does trated and then the residue was puried by using the Biotage
19,20
not involve NMDAR activation.
We plan to examine cyto- system. The desired fractions were concentrated and then dried
protective effects of the series of H S-releasing NMDAR antag- using an oil pump to give 2.43 g pale yellow oil of amide (68%
2
+
onists against ischemic neuronal injury in future studies.
It has been suggested that sulfane sulfur containing poly-
yield, MS (ESI ) m/z 298 (M + 1)).
The amide (1.31 g, 4.4 mmol) was mixed with sulfur (3.94 g,
ꢂ
suldes, but not H
2
S itself, are the signaling molecules that 123 mmol) under N
2
. The mixture was then heated to 190 C in
mediate biological effects of “hydrogen sulde”. For example, an oil bath overnight. The reaction mixture was then cooled
Greiner and colleagues recently reported that polysuldes, but down. 50 ml toluene was added and stirred for 10 min. The
2
not H S, induce oxidation of lipid phosphatase and tensin mixture was then loaded onto a silica cartridge directly and
21
homolog (PTEN) modifying its activity. Similarly, Kimura and puried by using the Biotage system with gradient from pure
colleagues showed that polysuldes activate transient receptor hexanes to 50% EtOAc/hexanes. The desired fractions were
potential (TRP) A1 channels at an EC50 of 91 nM while H2S collected and then concentrated to give 0.389 g red solid (23%).
2
2
1
activates TRPA1 at an EC50 of 116 mM. Our current results
H NMR: d (CDCl
3
, 400 MHz), 8.47 (s, J ¼ 8.6 Hz, 1H); 7.79 (d, J ¼
support the important role of sulfane sulfur-containing poly- 8.6 Hz, 2H); 7.61 (d, J ¼ 8.2 Hz, 2H); 5.82 (s, 1H); 2.14 (s, 9H);
+
suldes by showing the correlation of the intracellular levels of 1.74 (bs, 6H). MS (ESI ) m/z 388 (M + 1).
sulfane sulfur and the cytoprotective effects of H
2
S-donor
Synthesis of ACS81. To a solution of disulallyl disulde (5.0
compounds in the cellular model of PD. These observations ml, 36.6 mmol) in a mixture of diethyl ether (21 ml) and
may form a foundation that enables future drug development methanol (44 ml) a solution of 3-mercaptopropanoic acid (1.08
against PD exploiting the ability of novel compounds that g, 10.2 mmol) was added in diethyl ether (11 ml), followed by a
augment intracellular levels of sulfane sulfur.
solution of 10 M NaOH (1.27 ml, 12.7 mmol). The reaction
mixture was stirred at rt overnight. The reaction mixture was
then concentrated and the residue was diluted with water (20
ml) and EtOAc (50 ml). A solution of 1 N HCl was added to
adjust pH ¼ 4 and then two layers were separated. Extract
4
. Experimental section
4.1. Chemistry
All moisture-sensitive reactions were performed under an argon aqueous with EtOAc (2 ꢁ 50 ml) and the combined organic
atmosphere using oven-dried glassware and anhydrous layers were washed with brine (50 ml) followed by drying over
solvents. Unless otherwise noted, commercially available Na
materials were used without purication. Products were puri- trated in a rota-evaporator. The residue is puried by using the
ed using a Biotage – Isolera System. Nuclear magnetic reso- Biotage system with EtOAc/hexanes. The desired fractions were
SO . The solid was ltered out and the ltrate was concen-
2
4

nance (NMR) splitting patterns are described as singlet (s), combined and concentrated to give 0.52 g pale yellow oil as the
+
doublet (d), triplet (t), and quartet (q); the values of chemical product (28% yield, MS (ESI ) m/z 179 (M + 1)).
shis (d) are given in ppm relative to the residual solvent and
coupling constants (J) are given in hertz (Hz). The mass spectra mmol) was dissolved in DMF (4 ml) followed by addition of
were recorded on an Agilient/Micromass LC/MS.
memantane HCl (0.266 g, 1.23 mmol) and DIPEA (0.78 ml, 4.49
.1.1. Synthesis of SSP4. A uorescent probe for sulfane mmol) at rt under N . The reaction mixture was then cooled in
sulfur SSP4 (shown in Fig. S3†) was prepared using the same an ice bath for 10 min. HATU (0.640 g, 1.68 mmol) was added in
4.1.3. Synthesis of ACS81-memantine. ACS81 (0.2 g, 1.12
4
2
11
1
method as SSP2 reported previously.
H NMR (300 MHz, an ice bath and the reaction mixture was stirred for 10 min at
DMSO-d ) d 5.49 (s, 2H), 6.97 (d, J ¼ 9.0 Hz, 2H), 7.12 (d, J ¼ 9.0 this temperature. The reaction mixture was warmed up to rt and
6
Hz, 2H), 7.32 (t, J ¼ 6.0 Hz, 2H), 7.44–7.55 (m, 5H), 7.66 (d, J ¼ 9.0 stirred overnight. The reaction mixture was quenched with a
Hz, 2H), 7.76–7.88 (m, 2H), 8.09 (d, J ¼ 9.0 Hz, 1H), 8.19 (d, J ¼ solution of NaHCO
3
(50 ml) and then extracted with EtOAc
Cl) d 81.9, 110.9, 116.9, 118.2, (50 ml ꢁ 3). The combined organic layers were washed with
1
3
6
.0 Hz, 2H). C NMR (75 MHz, CD
3
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water 50 ml ꢁ 1 and brine 50 ml ꢁ 1, and then dried over Mediatech, Inc.) supplemented with 10% fetal bovine serum
Na SO . The ltrate was concentrated and then the residue was (FBS) and penicillin (100 units per ml)/streptomycin (100 mg
2
4
ꢀ
1
4
puried by using the Biotage system. The desired fractions were ml ). Briey, cells were seeded into 96 well plates (2 ꢁ 10 cells
concentrated and then dried using an oil pump to give 0.3 g pale per well). Cell culture medium was replaced every 2 days, and
1
ꢂ
yellow solid (79% yield). H NMR: d (CDCl
3
, 400 MHz), 5.70–5.85 the cultures were maintained at 37 C in 95% air/5% CO
2
in a
(m, 1H); 5.30 (bs, 1H), 5.154 (s, 1H), 5.01–5.0151 (m, 1H), 3.15 humidied incubator. Cells were used aer reaching 80%
(
1
1
m, 2H), 2.73 (t, J ¼ 7.3 Hz, 2H), 2.34 (t J ¼ 7.2 Hz, 2H), 2.14 (m, conuence.
H), 1.84 (m, 2H), 1.65 (m, 4H), 1.38 (m, 2H), 1.29 (m, 2H), 1.10–
4.2.2. Cell viability. CV and MTT assays were performed to
+
.20 (m, 2H), 0.85 (s, 6H). MS (ESI ) m/z 340 (M + 1).
examine cell viabilities of SH-SY5Y cells 24 h aer the addition
+
4
.1.4. Synthesis of CTBA. Under N , 4-cyano-benzoic acid of MPP (5 mM) with a vehicle (DMSO, nal concentration: 1%)
2
(
(
0.735 g, 5.0 mmol) was dissolved in MeOH (60 ml), a solution of or a H S donor (20 mM) as reported previously.
2
NH ) S (3.3 ml, 20% aqueous solution, 5.5 mmol) was added and
4.2.3. Measurement of H S or sulfane sulfur levels. H S
2 2
4
2
the reaction mixture was stirred overnight. The reaction mixture levels in SH-SY5Y cells or medium (DMEM/F12, 10% FBS)
was then concentrated to dryness. The residue was participated without cells were measured using H S-specic uorescent probe
in H O (100 ml) and EtOAc (150 ml). Two layers are separated and HSip-1 DA for intracellular H S or HSip-1 for extracellular H S,
2
2
2
2
the aqueous was extracted with EtOAc. The combined organic respectively, as described previously. Briey, the uorescence
layers were washed with water, brine, and then dried over intensity of HSip-1-loaded SH-SY5Y cells or medium without cells
Na SO . The ltrate was concentrated and then the residue was was measured by using a SpectraMax M5 Microplate reader
2
4
puried by using the Biotage system. The desired fractions were (Molecular Devices, Inc.) before or 1.5 h, 3 h, or 6 h aer the
concentrated and then dried using an oil pump to give 0.41 g addition of the vehicle (DMSO) or the H S donor at 20 mM to the
2
+
yellow solid (45% yield). MS (ESI ) m/z 182 (M + 1).
medium. The nal concentration of DMSO was adjusted to 1%.
4
.1.5. Synthesis of CTBA-memantane. 4-Cyanobenzoic acid Sulfane sulfur levels in SH-SY5Y cells or medium without cells
1.0 g, 6.8 mmol) was dissolved in DMF (23 ml) followed by were measured using sulfane sulfur specic uorescent probe
addition of memantine HCl (1.76 g, 8.16 mmol) and DIPEA (4.73 SSP4 instead of HSip-1 in the protocol of H S measurement.
ml, 27.19 mmol) at rt under N . The reaction mixture was then
4.2.4. Statistical analysis. All data are presented as means ꢃ
(
2
2
cooled in an ice bath for 10 min. HATU (3.88 g, 10.2 mmol) was SE. Data were analyzed by ANOVA using Sigmastat 3.01a (Systat
added in an ice bath and the reaction mixture was stirred for 10 Soware Inc., Chicago, IL) and Prism 5 soware package
min at this temperature. The reaction mixture was warmed up to (GraphPad Soware, La Jolla, CA). A Newman–Keuls multiple
rt and stirred overnight. The reaction mixture was quenched comparison post hoc test was performed aer One-way ANOVA
with a solution of NaHCO
EtOAc (100 ml ꢁ 3). The combined organic layers were washed viability and the H
with water 100 ml ꢁ 1 and brine 100 ml ꢁ 1, and then dried over correlation coefficient was calculated. The P values smaller than
Na SO . The ltrate was concentrated and then the residue was 0.05 were considered signicant.
3
(100 ml) and then extracted with as required. To determine the correlation between the cell
2
S level, or the sulfane sulfur level, Spearman's
2
4
puried by using the Biotage system. The desired fractions were
concentrated and then dried using an oil pump to give 1.7 g pale
yellow solid of amide (81% yield, MS (ESI ) m/z 309 (M + 1)).
Acknowledgements
+
Under a nitrogen atmosphere, the amide (0.616 g, 2.0 mmol) This work was supported by the National Institutes of Health
was dissolved in MeOH (20 ml), a solution of (NH S (1.31 ml, (NIH) grant HL101930 to F. Ichinose and ACS-Teva USA Scholar
0% aqueous solution, 2.2 mmol) was added and the reaction Award, and NIH grant HL116571 to M. Xian.
mixture was stirred overnight. The reaction mixture was then
concentrated to dryness. The residue was participated in H O (50
4 2
)
2
2
Notes and references
ml) and EtOAc (50 ml). Two layers are separated and the aqueous
was extracted with EtOAc. The combined organic layers were
washed with water 100 ml ꢁ 1 and brine 100 ml ꢁ 1, and then
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