Controllable hydrogen sulfide donors and their activity against myocardial ischemia-reperfusion injury

Article abstract of DOI:10.1021/cb400090d

Hydrogen sulfide (H₂S), known as an important cellular signaling molecule, plays critical roles in many physiological and/or pathological processes. Modulation of H₂S levels could have tremendous therapeutic value. However, the study on H₂S has been hindered due to the lack of controllable H₂S releasing agents that could mimic the slow and moderate H₂S release in vivo. In this work we report the design, synthesis, and biological evaluation of a new class of controllable H ₂S donors. Twenty-five donors were prepared and tested. Their structures were based on a perthiol template, which was suggested to be involved in H₂S biosynthesis. H₂S release mechanism from these donors was studied and proved to be thiol-dependent. We also developed a series of cell-based assays to access their H₂S-related activities. H9c2 cardiac myocytes were used in these experiments. We tested lead donors' cytotoxicity and confirmed their H₂S production in cells. Finally we demonstrated that selected donors showed potent protective effects in an in vivo murine model of myocardial ischemia-reperfusion injury, through a H ₂S-related mechanism.

Full text of DOI:10.1021/cb400090d

Articles
pubs.acs.org/acschemicalbiology
Controllable Hydrogen Sulfide Donors and Their Activity against
Myocardial Ischemia-Reperfusion Injury
Yu Zhao,† Shashi Bhushan,‡ Chuntao Yang,§ Hiroyuki Otsuka,‡ Jason D. Stein,† Armando Pacheco,†
Bo Peng,† Nelmi O. Devarie-Baez,† Hector C. Aguilar,∥ David J. Lefer,‡ and Ming Xian*,†
†Department of Chemistry and ∥Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington
99164, United States
‡Department of Surgery, Division of Cardiothoracic Surgery, Emory University School of Medicine, Carlyle Fraser Heart Center,
Atlanta, Georgia 30308, United States
§Department of Physiology, Guangzhou Medical University, Guangzhou 510182, China
S
* Supporting Information
ABSTRACT: Hydrogen sulfide (H2S), known as an im-
portant cellular signaling molecule, plays critical roles in many
physiological and/or pathological processes. Modulation of
H2S levels could have tremendous therapeutic value. However,
the study on H2S has been hindered due to the lack of
controllable H2S releasing agents that could mimic the slow
and moderate H2S release in vivo. In this work we report the
design, synthesis, and biological evaluation of a new class of controllable H2S donors. Twenty-five donors were prepared and
tested. Their structures were based on a perthiol template, which was suggested to be involved in H2S biosynthesis. H2S release
mechanism from these donors was studied and proved to be thiol-dependent. We also developed a series of cell-based assays to
access their H2S-related activities. H9c2 cardiac myocytes were used in these experiments. We tested lead donors’ cytotoxicity
and confirmed their H2S production in cells. Finally we demonstrated that selected donors showed potent protective effects in an
in vivo murine model of myocardial ischemia-reperfusion injury, through a H2S-related mechanism.
ydrogen sulfide (H2S) has been recognized as an
important cellular signaling molecule, much like nitric
simines, S-nitrosothiols, hydroxylamines, N-hydroxyguanidines,
etc. Moreover, many strategies, such as light, pH, enzymes, etc.,
can be used to trigger NO generation from these donors. In
contrast, currently available H2S donors are still very limited.
These donors include the following: (1) sulfide salts, such as
Na2S, NaHS, and CaS, have been widely used in the field.
These inorganic donors have the advantage of rapidly
enhancing H2S concentration. The maximum concentration
of H2S released from these salts can be reached within seconds.
However such a fast generation may cause acute changes in
blood pressure. In addition, since H2S is highly volatile in
solutions, the effective residence time of these donors in tissues
may be very short.15,16 (2) Naturally occurring polysulfide
compounds such as diallyl trisulfide (DATS) are also employed
as H2S donors in some studies. DATS can vasodilate rat
aortas17 and protect rat ischemic myocardium18 via a H2S-
related manner, but the simplicity of the structure limits its
application as H2S donors. (3) Synthetic H2S donors have
recently emerged as useful tools. GYY4137,19 which is a
Lawesson’s reagent derivative, releases H2S via hydrolysis both
in vitro and in vivo and exhibits some interesting biological
activities20,21 such as anti-inflammation.22−24 H2S release from
H
oxide (NO).1−6 The endogenous formation of H2S is attributed
to enzymes including cystathionine β-synthase (CBS),
cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfur-
transferase (MPST).7−10 These enzymes convert cysteine or
cysteine derivatives to H2S in different tissues and organs.
Recent studies have suggested that the production of
endogenous H2S and the exogenous administration of H2S
can exert protective effects in many pathologies.1,2 For example,
H2S has been shown to relax vascular smooth muscle, induce
vasodilation of isolated blood vessels, and reduce blood
pressure. H2S can also inhibit leukocyte adherence in
mesenteric microcirculation during vascular inflammation in
rats, suggesting H2S is a potent anti-inflammatory molecule. In
addition, H2S may intereact with S-nitrosothiols to form
thionitrous acid (HSNO), the smallest S-nitrosothiol, whose
metabolites, such as NO+, NO, and NO−, have distinct but
important physiological consequences.11 These results strongly
suggest that modulation of H2S levels could have potential
therapeutic values.
To explore the biological functions of H2S, researchers
started to use H2S releasing compounds (also known as H2S
donors) to mimic endogenous H2S generation.12−14 The idea is
similar to the well-studied nitric oxide (NO) donors. Currently
there are many options for NO donors, including organic
nitrates, nitrites, diazeniumdiolates, N-nitrosoamines, N-nitro-
Received: February 4, 2013
Accepted: April 2, 2013
© XXXX American Chemical Society
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GYY4137 is relatively low (<10% of H2S was released from this
molecule after 7 days).25 Dithiolthione is another structure that
releases H2S in aqueous solution.13 However the detailed
mechanism is still unclear. A major limitation of current donors
is that H2S release is largely uncontrollable. Modifications that
are made between the time that a solution is prepared and the
time that the biological effect is measured can dramatically
affect results. In our opinion, ideal H2S donors should be stable
by themselves in aqueous solutions. The release of H2S (both
the time and the rate) should be controllable (upon activation
by certain factors). Such donors will not only be useful research
tools for H2S researchers but also have unique therapeutic
benefits themselves.
The research in our laboratory focuses on the development
of controllable H2S donors. In 2011 we discovered a series of
N-(benzoylthio)benzamide derivatives as thiol-activated H2S
donors.26 These compounds are stable in aqueous solutions
and in the presence of some cellular nucleophiles. Upon
activation by cysteine or reduced glutathione (GSH), the
compounds could produce H2S (Scheme 1a).26 In this process,
group could allow us to develop different strategies to retrieve
perthiol, therefore achieving the regulation of H2S release. With
this idea in mind, we decided to test cysteine-based perthiol
derivatives 2 (Scheme 2). Acyl groups were used as the
protecting group on perthiol moieties.
The synthesis of four cysteine-based donors (2a−d) is
described in Supplementary Scheme S1. N-Benzoyl cysteine
methyl ester was first treated with 2-mercapto pyridine disulfide
to provide a cysteine-pyridine disulfide intermediate, which was
then treated with corresponding thioacids to give the desired
donor compounds. Using the procedure reported previously,26
we analyzed H2S release capabilities of these donors in the
presence of cysteine and GSH. As shown in Scheme 2, these
donors indeed could generate H2S in the presence of thiols.
Compared to N-(benzoylthio)-benzamide type donors, how-
ever, these donors showed much decreased ability of H2S
generation (read from their peaking concentrations). The initial
concentration of the donors was 150 μM, while the maximum
H2S concentration formed was less than 15% (by cysteine) or
8% (by GSH).
From the reaction mechanism point of view, we expected the
free SH of cysteine or GSH would undergo a thioester
exchange with the acyl group to produce perthiol 4 (pathway A,
Scheme 2b), which in turn should lead to H2S formation.
However, it was also possible that SH reacted with the acyl-
disulfide linkage to form a new disulfide 5 and thioacid 6
(pathway B). We found that thioacids could not release H2S
even in the presence of cysteine or GSH under the conditions
used in our experiments (see Supplementary Figure S1).31
Therefore, H2S release from donors 2 was diminished due to
the involvement of pathway B.
Scheme 1. (a) Hydrogen Sulfide (H2S) Release from N-
(Benzoylthio)benzamide Derivatives; (b) H2S Biosynthesis
Catalyzed by Cystathionine γ-lyase (CSE); (c) Concept of
Perthiol-Based H2S Donors
Tertiary Perthiol-Based H2S Donors. We envisioned that
the steric hindrance on the α-carbon of the disulfide bridge
should prevent the reactions through pathway B and therefore
enhance H2S formation. As such we decided to prepare a series
of penicillamine-based perthiol derivatives 8 (Scheme 3a). The
general synthesis of this series of donors is described in
Supplementary Scheme S2. Briefly, C- and N-protected
penicillamine was first treated with 2,2′-dibenzothioazolyl
disulfide to provide a penicillamine-benzothioazolyl disulfide
intermediate and then treated with corresponding thioacids to
furnish the desired penicillamine-based donors. Both steps gave
good yields for all of the substrates. These donor compounds
proved to be stable in aqueous solutions. In the presence of
cysteine or GSH, we observed a time-dependent H2S
generation. The representative H2S release curves of 8a are
shown in Scheme 3. With an initial concentration at 100 μM,
the maximum H2S concentration formed was ∼80 μM (with
cysteine), which demonstrated the efficiency of this compound
as H2S donor. After reaching the maximum value, H2S
concentration started to drop due to volatilization.15
We expected that the change of acyl substitutions could affect
the rate of thioester exchange and regulate H2S generation.
Therefore a series of acyl substitution modified donors (19
compounds in total) were prepared and tested. The H2S
generation data of 9 representative compounds are summerized
in Scheme 3b. Data of the rest of the compounds are shown in
Supplementary Table S1. Generally, electron-withdrawing
groups on the phenyl group led to faster H2S generation,
while electron-donating groups led to slower H2S release. We
also observed significant steric effects on H2S formation. More
sterically hindered substrates (compounds 8o, 8r, 8s) resulted
in slower H2S release (with decreased H2S amounts) or even
cysteine perthiol (also known as thiocysteine) is believed to be
a key intermediate. It should be noticed that cysteine perthiol is
also involved in H2S biosynthesis catalyzed by CSE (Scheme
1b).13,27 These findings suggest that the perthiol (Scheme 1c)
can be a useful template for the design of controllable H2S
donors. Herein we report the development of perthiol-based
donors and their activities in myocardial ischemia-reperfusion
(MI/R) injury.
RESULTS AND DISCUSSION
■
Primary Perthiol-Based H2S Donors. Perthiols are known
to be unstable species.28−30 We expected a protecting group on
-SH could enhance the stability. In addition, the protecting
B
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Scheme 2. (a) H2S Generation Data of Cysteine-Based Donors 2; (b) Proposed Reactions of 2
no release at all. In addition, we found that cysteine always
caused higher/faster H2S release compared to GSH. This is
likely due to the fact that cysteine, compared with GSH, is a
smaller molecule and can react more quickly with the thioester
group. These results demonstrated that H2S release from these
perthiol-based donors could be regulated via structural
modifications.
H2S Release Mechanism Study. To understand the
mechanism of H2S release from these donors, we studied the
reaction between 8a and a cysteine derivative 9 (3 equiv). As
shown in Scheme 4, we confirmed the formation of a thioester
10, an asymmetric disulfide 12, a free thiol 13, as well as
cysteine disulfide 15. On the basis of these reaction products,
we proposed the mechanism as follows: the reaction is initiated
by a thioester exchange between 8a and 9 to form a new
thioester 10 and perthiol 11. Both S atoms of 11 can be
attacked by cysteine.32 Therefore two possible pathways exist:
(a) the cysteine attacks the internal S to yield disulfide 12 and
liberate H2S, or (b) the external S is attacked to form thiol 13
and cysteine perthiol 14. Then another molecule of cysteine 9
reacts with 14 to form disulfide 15 and release H2S. In this
process it is also possible that 13 reacts with 14 to form
disulfide 12 and release H2S.
can protect cardiovascular system against myocardial ischemia-
reperfusion (MI/R) injury.8,17,33−35 Several groups have shown
that H2S, when applied both at the time of reperfusion and as a
preconditioning reagent, exhibits the cardioprotection by
different mechanisms, such as preserving mitochondrial
function,36 reducing oxidative stress,34 decreasing myocardial
inflammation,37 and improving angiogenesis.38 We envisioned
that our perthiol-based donors might exhibit similar myocardial
protective effects in an in vivo model of murine MI/R injury.
Before conducting animal experiments, we tested cytotoxicity
of two representative donors (8a/8l) in H9c2 cardiac
myocytes. The cell viability was detected using cell counter
kit (CCK)-8 assay (Figure 1). After 24-h exposure of H9c2 cells
to 8a and 8l at varied concentrations (0 to 100 μM), cell
viability did not decrease. Interestingly the exposure of cells to
8a and to 8l at concentrations of 12, 25, 50, and 100 μM,
increased cell viability percentage (at the level compared to 400
μM NaHS). The results of these studies indicate that these
perthiol-based donors do not promote cytotoxicity in cardiac
cells at the doses we have tested. We also determined cell
viability through the evaluation of reactive oxygen species
(ROS) concentration. The results are shown in Supplementary
Figure S3. Donors 8a and 8l did not lead to ROS increase,
which confirms the safety of the donors.
Biological Evaluation of Perthiol-Based H2S Donors.
With these donors in hand, we next explored their therapeutic
benefits. Recent studies with animal models suggested that H2S
We wondered if our donors indeed could release H2S when
interacting with myocytes and then conducted experiments to
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Scheme 3. (a) H2S Release Curve of Donor 8a; (b) H2S Generation Data of Selected Penicillamine-Based Donors
Scheme 4. Proposed Mechanism of H2S Generation
Figure 1. Effects of 8a and 8l on cell viability. H9c2 cells were treated
with different concentrations of 8a or 8l (12−100 μM) for 24 h. The
cell counter kit (CCK)-8 assay was performed to measure cell viability.
Data were shown as the mean SD (n = 8). **P < 0.01 versus control
group.
address this question. As shown in Figure 2, H9c2 cells were
incubated with the donors (8a and 8l) for 30 min, respectively.
After that a selective H2S fluorescent probe, WSP-1,39 was
applied into the cells to monitor the production of H2S. As
expected, donor-treated cells (Figure 2b and c) showed much
enhanced fluorescent signals compared to vehicle-treated cells
(Figure 2a). The image of positive control (with H2S) is shown
Figure 2. H2S production from 8a and 8l in H9c2 cells. Cells were
incubated with vehicle (A), 100 μM 8a (B), and 100 μM of 8l (C) for
30 min. After removal of excess donors, 250 μM concentration of a
H2S fluorescent probe (WSP-1) was added. Images were taken after 30
min.
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Figure 3. Cardioprotective effects of compounds 8a and 8l in myocardial ischemia-reperfusion injury. Compound 8a, 8l, or vehicle were injected in
vivo after 22.5 min of ischemia. (A) Structures of donors. (B) Circulating cardiac troponin I levels at following 45 min of MI and 2 h of reperfusion.
Troponin-I was significantly (p < 0.01) reduced with either 8a or 8l. (C) Myocardial area-at-risk (AAR) per left ventricle (AAR/LV) and infarct size
per area-at-risk (INF/AAR) were assessed in vehicle- (n = 14) and donor-treated animals (n = 14) at 24 h following MI/R. AAR/LV was similar
among all groups. INF/AAR was significantly (p < 0.05) smaller in animals treated with either 8a or 8l as compared to vehicle. (D) Representative
photomicrographs of a midventricular slice after MI/R stained with Evan’s blue and 2,3,5-triphenyltetrazolium chloride for both vehicle- and donor-
treated hearts.
in Supplementary Figure S2. In addition, we did not observe
shape changes of the cells after the treatment. These results
demonstrated that perthiol-based donors can release H2S when
interacting with H9c2 cells and H2S generation can be
evaluated by fluorescent image.
Finally we tested myocardial protective effects of donors 8a
and 8l against myocardial ischemia/reperfusion (MI/R) injury
in a murine model system. In these experiments, mice were
subjected to 45 min of left ventricular ischemia followed by 24
h reperfusion. Compounds 8a, 8l, or vehicle were administered
into the left ventricular lumen at the 22.5 min of myocardial
ischemia. All animal groups displayed similar area-at-risk per
left ventricle (AAR/LV), which means surgery caused similar
risk. However, compared to vehicle-treated mice, mice receiving
8a or 8l displayed a significant reduction in circulating levels of
cardiac troponin I and myocardial infract size per area-at-risk
(Figure 3B and C). For example, a 500 μg/kg bolus of 8l
maximally reduced INF/AAR by ∼50%, which was a significant
protection. These results demonstrated that perthiol-based
compounds can exhibit H2S-mediated cardiac protection in
MI/R injury and these compounds may have potential
therapeutic benefits.
We also tested in vivo H2S production from donors 8a and 8l.
As such, donors (1 mg/kg for 8a and 500 μg/kg for 8l) were
injected intravenously via tail vein injection. Blood and hearts
Figure 4. In vivo H2S levels (μM) in blood (A) and hearts (B)
obtained from mice treated with 8a and 8l.
were obtained at 15 min following injection. H2S levels were
determined using previously described gas chromatography and
chemiluminesence methods.40 As shown in Figure 4, blood and
myocardial levels of H2S were significantly (p < 0.01) increased
following injection of the donors as compared to controls.
Conclusion. In summary, we have developed a series of new
H2S donors based on the perthiol template. Their H2S
generation is regulated by thiols such as cysteine or GSH.
We also demonstrated that H2S release capability from these
donors can be manipulated by structural modifications.
Moreover, these donors are nontoxic to cardiac cells and
their H2S production upon interacting with myocytes can be
detected. Some donors exhibited potent myocardial protective
effects in MI/R injury, presumably due to H2S generation. It
should be noted that H2S generation from these donors is not
dependent on specific enzymes such as CBS and CSE. Recently
our group tested the effects of some H2S donors on CBS/CSE
activity and we did not notice any changes even after weeks.41
Taken together these donors may be potential therapeutic
agents. In our in vivo experiments, 8l exhibited better
cardioprotective effects compared to those of 8a (Figure 3).
Interestingly, 8l also exhibited better activity in cell viability test
and H2S generation test (Figures 1 and 2). These data suggest
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Product Analysis. A 100 mg portion of 8a (0.26 mmol) was
dissolved in 10.0 mL of THF/phosphate buffer (pH 7.4) (1:1, v/v).
Then cysteine derivative 9 (187 mg, 0.78 mmol) was added into the
solution. The mixture was stirred at RT for 1 h. The reaction mixture
was extracted with DCM 3 times. The organic layers were combined,
dried with MgSO4, and concentrated. Products 10, 12, 13, and 15
were isolated by flash column chromatography (1% v/v MeOH in
DCM).
that in vitro evaluation of donors may allow us to predicate
donors’ in vivo behaviors. Further development of this type of
donors and evaluation of their other H2S related biological
activities are currently ongoing in our laboratory.
METHODS
■
Synthesis of Compounds 2a−2d. 2-Mercapto pyridine disulfide
(2.2 g, 10 mmol) was dissolved in 50 mL of CHCl3. To this solution
was added N-benzoyl cysteine methyl ester (1.2 g, 5 mmol). The
reaction was stirred at RT for 1 h and then concentrated under
vacuum, and 1.48 g of compound a was obtained as a white solid by
flash chromatography (hexane/ethyl acetate = 2:1). Please see
Supporting Information for characterization data of a. Synthetic
intermediate a, 83 mg, 0.24 mmol, was dissolved in 5 mL of CHCl3.
To this solution was added thiobenzoic acid (42 mg, 0.3 mmol). The
mixture was stirred at RT for 1 h. The excess thiobenzoic acid was
removed by washing with aqueous NaHCO3 solution. The organic
layer was separated, dried, and concentrated under vacuum. The final
product 2a was purified as white solid by flash chromatography
10 and 15 are known compounds. Their data are given in
Supporting Information.
1
12 (1:1 mixture of diastereoisomers): mp 73−75 °C; H NMR
(300 MHz, CDCl3) δ 7.84 (d, J = 6.6 Hz, 2H), 7.82 (d, J = 6.9 Hz,
2H), 7.46 (m, 6H), 7.29 (m, 2H), 6.96 (br, 1H), 6.78 (br, 1H), 6.69
(d, J = 9.3 Hz, 2H), 5.09 (m, 2H), 4.68 (d, J = 9..9 Hz, 1H), 4.65 (d, J
= 9.9 Hz, 1H), 3.78 (s, 6H), 3.29 (m, 6H), 3.07 (m, 2H), 1.97 (s, 6H),
1.35 (m, 20H), 0.86 (t, J = 7.2 Hz, 6H); 13C NMR (75 MHz, CDCl3)
δ 171.4, 171.2, 170.4 (2C), 169.3, 169.2, 167.4, 167.3, 133.8, 133.7,
132.2, 132.1, 128.8 (2C), 127.5, 127.4, 58.6, 58.4, 53.3, 53.1, 53.0, 52.8,
42.3, 42.2, 39.6, 34.9, 31.8 (2C), 31.5 (2C), 29.3, 25.5, 25.4, 25.3, 24.2,
23.5, 22.9, 20.3, 14.4, 14.0; IR (thin film) cm−1 3300, 3072, 2962,
2934, 2871, 1739, 1645, 1535, 1366, 1228; mass spectrum (ESI/MS)
m/z 506.1 [M + Na]+; HRMS m/z 506.1752 [M + Na]+; calcd for
C22H33N3NaO5S2 506.1759; yield 20%.
1
(hexane/ethyl acetate = 10: 4). Mp 94−96 °C; H NMR (300 MHz,
CDCl3) δ 7.97 (d, J = 6.9 Hz, 2H), 7.90 (d, J = 7.8 Hz, 2H), 7.80 (d, J
= 7.5 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.47 (m, 5H), 5.06 (m, 1H),
3.70 (s, 3H), 3.57 (dd, J = 14.4, 4.8 Hz, 1H), 3.30 (dd, J = 14.4, 4.8
Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 191.3, 170.8, 167.3, 135.3,
134.7, 133.7, 132.1, 129.2, 128.8, 128.0, 127.6, 53.0, 51.8, 40.9; IR
(thin film) cm−1 3326, 3056, 2955, 2927, 1748, 1683, 1638, 1520,
1489, 1319, 1203, 883; mass spectrum (ESI/MS) m/z 398.1 [M +
Na]+; HRMS m/z 398.0500 [M + Na]+; calcd for C18H17NNaO4S2
398.0497; yield 81%.
13: mp 176−177 °C; 1H NMR (300 MHz, CDCl3) δ 6.92 (br, 1H),
6.77 (d, J = 9.3 Hz, 1H), 4.51 (d, J = 9.3 Hz, 1H), 3.21 (m, 2H), 2.65
(s, 1H), 2.04 (s, 3H), 1.48 (m, 5H), 1.32 (m, 5H), 0.90 (t, J = 7.2 Hz,
3H); 13C NMR (75 MHz, CDCl3) δ 170.4, 169.9, 60.4, 46.3, 39.4,
31.5, 31.2, 28.7, 23.6, 20.3, 14.0; IR (thin film) cm−1 3267, 3084, 2967,
2935, 2874, 2558, 1667, 1638, 1537, 1456, 1371, 1241, 1136; mass
spectrum (ESI/MS) m/z 269.1 [M + Na]+; HRMS m/z 247.1473 [M
+ H]+; calcd for C11H23N2O2S 247.1480; yield 55%.
Cell Viability Assay. H9c2 (2-1) cardiomyocytes (H9c2 cells)
were cultured in DMEM high glucose medium supplemented with
10% fetal bovine serum (FBS) at 37 °C under an atmosphere of 5%
CO2 and 95% air. H9c2 cells at a concentration of 1 × 105/mL were
inoculated in 96-well plates and cultured overnight. H2S donor (8a or
8l) in FBS-free medium was administered and cultured for 24 h. The
cell viability was measured by cell counter kit (CCK)-8. The
absorbance at 450 nm was measured with a microplate reader
(Molecular Devices, Sunnyvale, CA, USA). Optical density (OD) of
the 8 wells in the indicated groups was used to calculate percentage of
cell viability according to the formula below:
Compounds 2b−2d were prepared from the corresponding
thiolacids using the same procedure as 2a. Their data are reported
in the Supporting Information.
Synthesis of Compounds 8a−8s. 2,2′-Dibenzothiazolyl disulfide
(4.32g, 13 mmol) was dissolved into 500 mL of CHCl3. To this
solution was added D,L-penicillamine derivative 13 (2.36 g, 9.6 mmol).
The reaction mixture was stirred at RT for 48 h. Solvent was then
removed, and the crude mixture was then purified by flash column
chromatography (3% v/v MeOH in DCM) to provide the
intermediate d as white solid. To a 15 mL CHCl3 solution containing
d (822 mg, 2 mmol) was added thiobenzoic acid (1.10 g, 8 mmol).
The reaction was stirred at RT for 10 min. Excess thiobenzoic acid was
removed by washing with NaHCO3. The organic layer was separated,
dried, and concentrated. The final product 8a was purified by flash
column chromatography (1% v/v MeOH in DCM) as white solid. Mp
132−134 °C; 1H NMR (300 MHz, CDCl3) δ 8.16 (m, 1H), 8.03 (d, J
= 7.5 Hz, 2H), 7.64 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 7.5 Hz, 2H), 7.04
(d, J = 8.1 Hz, 1H), 4.46 (d, J = 8.4 Hz, 1H), 3.36 (m, 2H), 2.01 (s,
3H), 1.62 (m, 2H), 1.44 (m, 5H), 1.25 (s, 3H), 0.95 (t, J = 7.2 Hz,
3H); 13C NMR (75 MHz, CDCl3) δ 194.0, 170.4, 168.8, 135.5, 134.8,
129.2, 128.3, 58.7, 53.8, 39.8, 31.6, 27.0, 24.0, 23.5, 20.5, 14.0; IR (thin
film) cm−1 3285, 3085, 2962, 2929, 2868, 1684, 1636, 1561, 1527,
1445, 1379, 1202, 1174, 1118, 890, 676; mass spectrum (ESI/MS) m/
z 405.1 [M + Na]+; HRMS m/z 383.1411 [M + H]+; calcd for
C18H27N2O3S2 383.1463; yield 94%.
OD treatment group
OD control group
% cell viability =
× 100
H2S Release in H9c2 Cells. H9c2 cells were inoculated in 6-well
plates and cultured overnight. The cells were co-incubated with 100
μM H2S donor, 8a, or 8l dissolved in phosphate buffered solution
(PBS) at 37 °C for 30 min, and then the solution in the wells was
removed. The cells were then co-incubated with a H2S probe (WSP-1)
solution (250 μM in PBS) and surfactant CTAB (500 μM) in PBS at
37 °C for 30 min. After the PBS was removed, the fluorescence signal
was observed by AMG fluorescent microscope (Advanced Microscopy
Group, USA).
Cardioprotective Effects in MI/R. Animals. Male C57BL/6J
mice, 10−12 weeks of age (Jackson Laboratories, Bar Harbor, ME),
were used in the present study. All animals were housed in a
temperature-controlled animal facility with a 12-h light/dark cycle,
with water and rodent chow provided ad libitum. All animals received
humane care in compliance with the Principles of Laboratory Animal
Care formulated by the National Society of Medical Research and the
Guide for the Care and Use of Laboratory Animals published by the
National Institutes of Health (Publication 85-23, Revised 1996). All
animal procedures were approved by the Emory University Institu-
tional Animal Care and Use Committee.
Compounds 8b−8s were prepared from corresponding thiolacids
using the same procedure as 8a.
H2S Measurement. The reaction was initiated by adding 75 μL of
stock solution of the donor (40 mM, in THF) into pH 7.4 phosphate
buffer (30 mL) containing cysteine (1.0 mM). Then 1.0 mL of
reaction aliquots were periodically taken and transferred to 4.0-mL
vials containing zinc acetate (1% w/v, 100 μL) and N,N-dimethyl-1,4-
phenylenediamine sulfate (20 mM, 200 μL) in 7.2 M HCl and ferric
chloride (30 mM, 200 μL) in 1.2 M HCl. The absorbance (670 nm) of
the resulted solution (1.5 mL) was determined 15 min thereafter using
a UV−vis spectrometer (Thermo Evolution 300). The H2S
concentration of each sample was calculated against a calibration
curve of Na2S. The H2S releasing curve was obtained by plotting H2S
concentration versus time.
Drug Preparation. On the day of experimentation, test compounds
(8a or 8l) were diluted in 0.5 mL of 100% THF solution. For in vivo
experiments, the test compounds were further diluted in sterile saline
to obtain the correct dosage to be delivered in a volume of 50 μL. The
F
dx.doi.org/10.1021/cb400090d | ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Articles
resulting concentration of THF in this dosage was 0.5% v/v. Vehicle
consisted of a solution of 0.5% v/v THF in sterile saline.
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ASSOCIATED CONTENT
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: mxian@wsu.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is supported by an American Chemical Society-Teva
USA Scholar Grant to M.X. and National Institutes of Health
(R01GM088226 to M.X. and R01HL092141, R01HL093579,
R01HL079040, P20HL113452, and 5U24HL094373 to D.J.L.).
D.J.L. is also supported by the Carlyle Fraser Heart Center of
Emory University Hospital Midtown. The authors would like to
acknowledge the expert assistance of A. L. King during the
course of these experimental studies.
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