Design, Synthesis, and Cardioprotective Effects of N-Mercapto-Based Hydrogen Sulfide Donors

Article abstract of DOI:10.1021/acs.jmedchem.5b01033

Hydrogen sulfide (H₂S) is a signaling molecule which plays regulatory roles in many physiological and/or pathological processes. Therefore, regulation of H₂S levels could have great potential therapeutic value. In this work, we report the design, synthesis, and evaluation of a class of N-mercapto (N-SH)-based H₂S donors. Thirty-three donors were synthesized and tested. Our results indicated that controllable H₂S release from these donors could be achieved upon structural modifications. Selected donors (NSHD-1, NSHD-2, and NSHD-6) were tested in cellular models of oxidative damage and showed significant cytoprotective effects. Moreover, NSHD-1 and NSHD-2 were also found to exhibit potent protective effects in a murine model of myocardial ischemia reperfusion (MI/R) injury.

Full text of DOI:10.1021/acs.jmedchem.5b01033

Subscriber access provided by UNIV OF CAMBRIDGE
Article
Design, Synthesis, and Cardioprotective Effects
of N-Mercapto-Based Hydrogen Sulfide Donors
Yu Zhao, Chuntao Yang, Chelsea Organ, Zhen Li, Shashi Bhushan, Hiro Otsuka,
Armando Pacheco, Jianming Kang, Hector Aguilar, David Lefer, and Ming Xian
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01033 • Publication Date (Web): 28 Aug 2015
Downloaded from http://pubs.acs.org on September 1, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 34
Journal of Medicinal Chemistry
1
2
3
4
Design, Synthesis, and Cardioprotective Effects of N-Mercapto-Based Hydrogen
5
6
Sulfide Donors
7
8
9
Yu Zhao,†, || Chuntao Yang,‡, || Chelsea Organ,§ Zhen Li,§ Shashi Bhushan,§ Hiro Otsuka,§
Armando Pacheco,† Jianming Kang,† Hector C. Aguilar,¶ David J. Lefer,§ and Ming
Xian*,†
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
† Department of Chemistry, Washington State University, Pullman, Washington, 99164,
United States
‡ Department of Physiology, Guangzhou Medical University, Guangzhou 511436, China
§ Cardiovascular Center of Excellence, Louisiana State University Health Science Center,
New Orleans, LA, 70112, United States
¶ Paul G. Allen School for Global Animal Health, Washington State University, Pullman,
WA 99164, United States
|| These authors contributed equally to this work.
ABSTRACT
Hydrogen sulfide (H2S) is a signaling molecule which plays regulatory roles in many
physiological and/or pathological processes. Therefore, regulation of H2S levels could
have great potential therapeutic value. In this work we report the design, synthesis, and
evaluation of a class of N-mercapto (N-SH)-based H2S donors. Thirty three donors were
synthesized and tested. Our results indicated that controllable H2S release from these
donors could be achieved upon structural modifications. Selected donors (NSHD-1,
NSHD-2, and NSHD-6) were tested in cellular models of oxidative damage and showed
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 34
1
2
3
4
5
6
7
8
9
significant cytoprotective effects. Moreover, NSHD-1 and NSHD-2 were also found to
exhibit potent protective effects in a murine model of myocardial ischemia reperfusion
(MI/R) injury.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
INTRODUCTION
Hydrogen sulfide (H2S), which is known for its characteristic odor of rotten eggs,
has been considered as a highly toxic air pollutant for centuries. However, this gaseous
molecule has been recently recognized as a member of a gasotransmitter family along
with nitric oxide (NO) and carbon monoxide (CO).1-7 Endogenous H2S formation has
been attributed to at least three enzymes: cystathionine -synthase (CBS), cystathionine
-lyase (CSE), and 3-mercaptopyruvate sulfur-transferase (MPST).8-12 These enzymes
convert cysteine or cysteine derivatives to H2S in different tissues and organs. A number
of studies have revealed a variety of biological effects of H2S, which include
vasodilation,13-15 anti-inflammation,16, 17 anti-cancer,18, 19 and cardioprotection.20-24 While
the exact mechanisms of these actions are still under investigation, some interesting
biochemical/catabolic reactions of H2S have been disclosed. For example, H2S reacts
readily with methemoglobin to yield sulfhemoglobin.25 H2S inhibits cytochrome c
oxidase, which accounts for its toxic action.26 H2S is an effective reducing agent and can
react with reactive oxygen species such as hydrogen peroxide, superoxide, and
peroxynitrite.27-29 It has been reported that H2S can cause protein S-sulfhydration (i.e to
form -S-SH)30-32 and this process is significant as it provides a possible route by which
H2S can alter functions of a wide range of cellular proteins and enzymes.33-35 H2S can
also interact with S-nitrosothiols to yield thionitrous acid (HSNO), the smallest S-
nitrosothiol, which can freely diffuse through membranes to facilitate transnitrosation of
2
ACS Paragon Plus Environment

Page 3 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
proteins.36 These reactions may be responsible for the biological functions of H2S. It is
also possible that additional important biological reactions of H2S will be discovered. All
of these findings suggest that regulation of H2S levels may have great therapeutic value.
In the H2S field, H2S releasing agents (also known as H2S donors) are important
research tools.37,38 Moreover H2S donors could potentially be unique therapeutic agents,
given the well-documented protective effects of H2S in many disease states.37,38 Among
these donors, sulfide salts, including sodium sulfide (Na2S) and sodium hydrogen sulfide
(NaHS), are most frequently used. These salts have the advantage of boosting H2S
concentrations rapidly. However, they release H2S spontaneously in aqueous solution,
making it hard to control the precise H2S concentrations. In addition, H2S concentrations
in aqueous solution can rapidly decrease due to volatilization, thus significantly limiting
the use of these two H2S precursors.39
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Considering these drawbacks, synthetic H2S donors have received considerable
attention and a number of donors have been reported.40, 41 For example, a widely used
donor is GYY4137, a derivative of Lawesson’s reagent.42 GYY4137 has been
demonstrated to release H2S both in vitro and in vivo. GYY4137 can induce H2S-related
biological effects such as vasodilation and anti-inflammation.42-45 However, the
mechanism of H2S release from GYY4137 is still unclear. The extremely slow release of
H2S from GYY4137 could also be problematic for its applications.46 Dithiolthiones
(DTTs) are another class of synthetic H2S donors.38 DTTs have been widely used to
prepare nonsteroidal anti-inflammatory drugs (NSAIDs)-H2S hybrids. The resultant HS-
NSAIDs, compared to the parent NSAIDs, showed similar anti-inflammatory effects, but
significantly decreased gastric damage.47-49 Although DTTs have shown promising H2S-
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 34
1
2
3
4
5
6
related biological activities, their H2S release is also uncontrollable and their H2S release
profile has not been explored yet.
7
8
9
The problem with the majority of currently available donors is that the H2S
generation from these compounds is uncontrollable. Commonly used donors such as
sulfide salts and GYY4137 release H2S spontaneously upon hydrolysis, making it very
difficult to obtain the precise physiological or pharmacological H2S levels needed.
Modifications made between the time that a solution is prepared and the time that the
biological effect is measured can dramatically affect research outcomes and sometimes
can even get disparate results. From a therapeutic perspective, and for applications in
H2S-related biological research, ideal H2S donors should release H2S slowly in moderate
amounts and/or in a highly controllable manner. To this end, our group introduced thiol-
activated H2S donors in 2009.50 The idea is described in Scheme 1. It is known that S-N
bonds are weak and can break under certain conditions.51-53 This property makes N-
mercapto (N-SH) species, such as 1, useful templates for the design of H2S donors. As N-
SH species are unstable and highly reactive, we expected that the protecting groups on –
SH should enhance their stability. Moreover, the removal of protecting groups can be
achieved by different strategies, making controllable H2S release possible. To prove this
hypothesis, twelve N-(benzoylthio)benzamides were prepared as the first thiol-activated
H2S donors. Some preliminary results regarding their H2S release profiles were
reported.50 Recently we expanded the scope of this type of donors and studied 33
substrates. Herein, we report the comprehensive results about these N-SH-based donors
(NSHDs), including their synthesis, H2S release evaluation/mechanism, and structure
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
ACS Paragon Plus Environment

Page 5 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
activity relationship (SAR). In addition, protective effects of selected donors on cellular
oxidative damages and myocardial ischemia reperfusion (MI/R) injury are also reported.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Design of NSHDs.
RESULTS AND DISCUSSION
Synthesis of N-(Acylthio)amides as NSHDs
In the design of thiol-activated NSHDs, acyl groups were selected as the
protecting groups on –SH. Both alkyl- and aryl-based acyl groups were used to prepare
the donors. The synthesis is illustrated in Scheme 2. Readily available thiocarboxylic
acids 2 were the starting materials. The treatment of 2 with hydroxylamine-O-sulfonic
acid gave S-acylthiohydroxylamines 3 as the reactive intermediates, which were further
reacted (without separation) with acid anhydrides to provide NSHDs 4. In total, 26
NSHDs were prepared using this route and overall yields were 61-81%.
Scheme 2. Synthesis of NSHDs.
H2S Release of N-(Acylthio)amides
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 34
1
2
3
4
5
6
7
8
9
With these compounds in hand, we first tested their H2S release ability in aqueous
buffers. N-(Acylthio)amides were found to be stable in buffers. Unlike hydrolysis-based
donors, N-(acylthio)amides did not release H2S spontaneously. In addition, no H2S
generation was observed in the presence of some potential cellular nucleophiles, such as
serine and lysine. However, a time-dependent H2S release was detected when cysteine
was added into the solutions, indicating that thiols are essential to trigger H2S release
from these molecules.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In order to systematically compare the H2S generation capabilities of these donors,
we studied the effects of donor concentrations, cysteine concentrations, solvent systems,
as well as reaction time/temperatures. Eventually the optimized conditions were found to
use 100 µM donors and 1000 µM cysteine in phosphate buffered saline (PBS) (pH 7.4)
containing 10% tetrahydrofuran (THF). H2S release was monitored at room temperature
for 2 hours. The well-known methylene blue (MB) method was used to measure H2S
generation. Using NSHD-1 as the example (Figure 1), a maximum of 56 µM of H2S
(peak H2S concentration) at 50 min (peak time) was detected. As shown in Figure 1,
higher concentrations of cysteine (i.e. 2 mM) did not significantly improve H2S release,
while lower concentrations of cysteine (i.e 0.5 mM) dramatically decreased H2S
production. In addition to cysteine, GSH’s ability to promote H2S release from NSHDs
was also evaluated. Results showed that GSH (1 mM) can also successfully trigger H2S
release at comparable levels as 1 mM cysteine.
6
ACS Paragon Plus Environment

Page 7 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Cysteine and GSH-mediated H2S release from NSHD-1
All NSHD substrates were then tested under the optimized conditions. Briefly, a
solution of each donor (0.1 mM) and cysteine (or GSH, 1 mM) was prepared in a mixed
solvent system PBS/THF (9:1, v:v). At different reaction times, 1.0 mL of reaction
aliquots were taken and mixed with MB cocktails (0.5 mL). The MB reactions were
carried out for 15 min and absorbance at 670 nm was then measured. H2S concentrations
were calculated based on a Na2S standard curve (please see experimental section for
detailed procedures). Each donor was tested three times, and their average results were
summarized in Table 1. The results showed that up to 68% of H2S can be detected within
a 2-hour experimental period, indicating that N-(acylthio)amides are potent H2S donors.
As for SAR, electronic effects on H2S release were observed. Compared to the parent
donor NSHD-1, donors with electron withdrawing groups (NSHD-2 – NSHD-5) showed
faster H2S release; donors with electron donating groups (NSHD-6 – NSHD-12)
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 34
1
2
3
4
5
6
7
8
9
exhibited slower H2S release. Steric effects were also observed as more sterically
hindered substrates showed slower H2S release (NSHD-13 – NSHD-15). In addition to
the thioester moiety, SAR studies were also conducted on amide terminals (NSHD-20 –
NSHD-26). Results showed that all of these amide-modified donors exhibited similar
H2S releasing abilities, suggesting the amide end had little effects on the reactivity. In all
GSH-treated groups, peak times were constantly 5–20-min slower than those in cysteine-
treated groups. This is presumably due to increased steric hindrance of GSH, therefore
causing slower reaction to release H2S. These findings demonstrated that structural
modifications can regulate H2S release from NSHDs, therefore, achieving controllable
H2S generation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. H2S release profiles of N-(acylthio)amides.
8
ACS Paragon Plus Environment

Page 9 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
O
H
CysSH/GSH (10 eq)
N
R2
H2S
R1
S
PBS (pH 7.4, 50 mM) / THF
(9:1), r.t. 2 h
O
M
100
7
8
9
Tpeak
(min)
Tpeak
(min)
[H2S]peak
( M)
[H2S]peak
( M)
R1
R2
R1
R2
Donors
Donors
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
55
65
55.8
51.2
65
70
40.7
58.4
H
F
NSHD-1
NSHD-2
NSHD-14
NSHD-15
80
90
37.5
48.1
50
55
39.5
40.6
45
60
48.3
28.1
65
80
48.1
68.2
NSHD-3 CF3
NSHD-16
Cl
50
60
49.4
31.3
60
65
40.9
49.9
NSHD-4
NSHD-17
NSHD-18
O
50
60
33.4
47.5
38.8
36.0
50
55
NSHD-5
O
60
65
41.8
46.8
60
70
48.2
55.6
NSHD-19
NSHD-20
O
NSHD-6
NSHD-7
60
80
29.0
34.0
55
65
51.4
57.6
CH3
O
55
80
47.0
39.5
NSHD-8
60
60
35.1
44.5
NSHD-21
NSHD-22
O
OAc
60
60
45.4
39.8
Cl
F
NSHD-9
60
70
25.2
29.9
60
70
29.4
19.8
NSHD-10
55
65
38.5
43.3
NSHD-23
NSHD-24
NSHD-25
55
65
49.3
39.3
45
50
46.4
58.3
NSHD-11
H3C
55
60
44.1
54.1
65
80
19.5
7.6
N
NSHD-12
NSHD-13
60
60
45.9
58.3
60
70
48.4
63.1
NSHD-26
H3C
a. Data were obtained with cysteine
b. Data were obtained with GSH
Synthesis and H2S Release of 1-(Benzoylthio)-3-phenylureas
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 34
1
2
3
4
5
6
7
8
9
To expand the scope of the donors, we next replaced the amide moiety with urea
templates. A series of 1-mercapto-3-phenylurea-based H2S donors (6) were synthesized
(Scheme 3). Briefly, readily available thiocarboxylic acids were treated with
hydroxylamine-O-sulfonic acid to form S-acylthiohydroxylamines 3, which were then
conjugated with phenyl isocyanate derivatives (5) to provide 1-(benzoylthio)-3-
phenylureas (6) as the desired products.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Chemical synthesis of 1-mercapto-3-phenylurea-based donors
H2S release from these donors was tested using the same protocol described above,
and the results are shown in Table 2. Interestingly, these 1-(benzoylthio)-3-phenylurea
based donors showed less and slower H2S release as compared to N-(acylthio)amides.
Table 2 H2S release profiles of 1-(benzoylthio)-3-phenylurea based donors
10
ACS Paragon Plus Environment

Page 11 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H2S Release Mechanism
To understand the mechanism of H2S release from these N-mercapto based
donors, a model reaction using NSHD-1 and cysteine (10 eq.) was studied. Three
products: N-benzoyl cysteine 8, benzamide 10, and cystine 12, were obtained. Based on
these products, the H2S release mechanism was proposed as follows (Scheme 4): a
reversible thiol exchange between NSHD-1 and cysteine initiates the reaction. The
resultant S-benzoyl cysteine 7 should then undergo a native chemical ligation (NCL)-type
acyl transfer to form a more stable N-benzoyl cysteine 8. Meanwhile, the free N-SH
species, N-mercaptobenzamide 9, should react with cysteine to yield benzamide 10 and
cysteine perthiol 11. The latter product can further react with cysteine to liberate H2S and
form cystine 12.
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 34
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4. Proposed mechanism of H2S release from NSHDs
Cytotoxicity of NSHDs
With these NSHDs in hand, we next explored their H2S-induced biological
activities. Recent studies have suggested that H2S can protect the cardiovascular system
against myocardial ischemia reperfusion (MI/R) injury. It has been demonstrated that H2S
(using Na2S or NaHS), when applied at the time of reperfusion or as a preconditioning
reagent, exhibits significant cardioprotection by different mechanisms, such as reducing
oxidative stress, preserving mitochondrial function, decreasing myocardial inflammation,
and improving angiogenesis.20-24 Among these, H2S’s effects as an antioxidant may play
a prominent role. We envisioned that NSHDs should exhibit similar cardioprotective
activities due to H2S release.
Before conducting experiments to investigate NSHD’s cardioprotective actions,
we tested cytotoxicity of three representative donors (NSHD-1, NSHD-2, and NSHD-6)
in H9c2 cardiomyoblasts. These three donors were selected because of their varied H2S
release abilities. The cells, originating from rat heart ventricular tissue, have been widely
used as an in vitro model for cardiac muscle in virtue of their morphological features and
12
ACS Paragon Plus Environment

Page 13 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
biochemical properties.54 In our experiment, H9c2 cells were incubated with each donor
at different concentrations (20-160 µM) for 1 h. Cells were then washed with PBS twice
to remove extracellular donors and cell viability was measured by the standard CCK-8
assay. As shown in Figure 2, these donors did not exhibit significant cytotoxicity at low
concentrations (<40 µM). However, cytotoxicity was observed when higher
concentrations (>80 µM) were applied. Considering that cysteine and GSH are rich in
living systems, which are expected to react with NSHDs and trigger H2S release, we
envisioned NSHD-induced cytotoxicity could be diminished or avoided when cysteine or
GSH is presented. To prove this hypothesis, H9c2 cells were then incubated with each
donor (160 µM) in the presence of cysteine (480 µM). As expected NSHD’s cytotoxicity
was completed suppressed, suggesting that NSHDs should be safe in cysteine-rich
environments.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Cytotoxicity of NSHDs in the absence or presence of cysteine. H9c2 cells were
incubated with NSHD-1, NSHD-2, and NSHD-6 at varied concentrations (20-160 µM)
for 1 h in the absence or presence of cysteine (480 µM), respectively. Excess NSHDs
were then washed away with PBS. CCK-8 assay was applied to measure cell viability.
*P<0.05, **P<0.01 vs. Control group.
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 34
1
2
3
4
Establishment of an in vitro Model of MI/R Injury
5
6
7
8
Under the condition of MI/R injury, oxidative stress (OS) plays a central role.55
OS-induced cell injury can be triggered by reactive oxygen species (ROS), including
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
•−
superoxide (O2 ), hydroxyl radical (•OH), singlet oxygen (•O) and hydrogen peroxide
(H2O2).56, 57 Among these H2O2 is the dominant form due to its stability.58 Therefore,
H2O2-treated H9c2 cells are usually used as an in vitro model of MI/R injury.59, 60 To
establish the cellular model of MI/R injury, H9c2 cells were treated with increasing
concentrations of H2O2 (100-800 µM) for 5 h. As shown in Figure 3, exposure of H9c2
cells to H2O2 led to a dose-dependent inhibition in cell viability (P<0.01), indicating that
H2O2 was able to induce the oxidative injury in cardiomyocytes. This can partly imitate
in vivo I/R-triggered effects. Since the median lethal concentration of H2O2 in H9c2 cells
was approximately 400 µM, this concentration was used for the following studies.
Figure 3. Effects of H2O2 on cell viability. H9c2 cells were treated with varied
concentrations of H2O2 for 5 h. The CCK-8 assay was performed to detect cell viability.
Data were expressed as the mean ± SEM. **P<0.01 vs. Control group.
Effects of NSHDs on H2O2-induced Damage in H9c2 Cells
We next tested the protective capabilities of NSHDs against H2O2-induced
cellular damage. In these experiments, H9c2 cells were pretreated with NSHD-1, NSHD-
14
ACS Paragon Plus Environment

Page 15 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2, or NSHD-6 at varied concentrations (40, 80, and 160 µM) in the presence of cysteine
(480 µM) for 1 hour. Cells were then washed with PBS to remove extracellular NSHDs
and cysteine. The donor-treated cells were challenged by H2O2 (400 µM) for another 5 h,
after which cell viability was analyzed. Results showed that NSHDs exhibited significant
attenuation of H2O2-induced damage (Figure 4A, 4B and 4C). In order to compare these
new donors with known donors, we also tested the effects of GYY4137 and NaHS, two
well-studied H2S donors, under the same conditions. Similar protective effects were
observed with GYY4137 and NaHS (40, 80, and 160 µM) (Figure 4D and 4E). To further
prove the protective effects of NSHDs were due to H2S release we studied the effects of
cysteine only (480 µM) and by-products of NSHDs (i.e. N-benzoyl cysteine (NBC) and
benzamide (BZM), at 160 µM). These species did not exhibit protection against H2O2-
induced damage (Figure 4F). Taking together these results suggested that NSHDs can
protect cardiomyocytes from oxidative injury and their protective activities are likely due
to H2S release.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 34
1
2
3
4
5
6
7
8
9
Figure 4. Effects of H2S donors (A-E) and NSHD byproducts (F) on H2O2-induced
cellular damage. Prior to the 5-h H2O2 (400 μM) treatment, H9c2 cells were
preconditioned with NSHD-1 (A), NSHD-2 (B), and NSHD-6 (C) in the presence of
cysteine (480 µM) at indicated concentrations for 1 h, respectively. GYY4137 (D) and
NaHS (E) were used as positive controls under similar conditions. Effects of H2S
releasing byproducts, such as NBC and BZM (160 µM), and cysteine alone (480 µM)
were also investigated (F). CCK-8 assay was performed to detect cell viability. Data were
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
**
†
‡
expressed as the mean ± SEM. P<0.01 vs. Control group. P<0.05, P<0.01 vs. H2O2
treatment group.
Measurement of LDH and MMP Levels
In addition to the cell viability assay, several other methods were also used to
validate NSHD’s protective effects in cells. Lactate dehydrogenase (LDH) is a stable
cytoplasmic enzyme located in cytoplasm under normal conditions. When the heart
suffers from MI/R injury, the cell membrane of cardiomyocytes is damaged, leading to
the increase of cell membrane permeability and LDH leakage from the cells into the
blood.61 In the present study, by comparing the contents of LDH in cytoplasm and
medium, the LDH release rate can be used to indicate the extent of cellular injury. As
shown in Figure 5, preconditioning of H9c2 cells with cysteine (480 µM, 1 h) followed
by the exposure to H2O2 (400 µM, 5 h) remarkably enhanced LDH release, indicating
H2O2 induced severe damage to cells and cysteine (480 µM) did not provide protection.
However, LDH release was significantly reduced when cells were pretreated with 160
μM of NSHD donors in the presence of cysteine (480 µM), demonstrating that NSHDs
can protect cells from oxidative damage.
16
ACS Paragon Plus Environment

Page 17 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Effects of NSHDs on H2O2-induced LDH release. H9c2 cells were
preconditioned with NSHD-1, NSHD-2, and NSHD-6 (160 μM) in the presence of
cysteine (480 µM) for 1 h followed by treatment with H2O2 (400 µM) for 5 h. LDH in
cells and medium was measured with a commercial kit. Data were expressed as the mean
± SEM. **P<0.01 vs. Control group. ‡P<0.01 vs. H2O2 treatment group.
Mitochondria are the energy factory of cells and sensitive to noxious stimuli.
Mitochondrial membrane potential (MMP) usually reflects whether mitochondria are
healthy or not, which further indicates whether cells are suffering from noxious damages.
Rh-123 staining is a useful tool to observe MMP. Figure 6A shows that under normal
condition H9c2 cells had bright green fluorescence. When cells were preconditioned with
cysteine (480 µM) for 1 h, and then treated with H2O2 (400 µM) for 5 h, a dramatic MMP
loss was observed, evidenced by weak green fluorescence, suggesting that H2O2 induced
severe damage to cells and cysteine (480 µM) did not provide significant protection
(Figure 6B). However, preconditioning with 160 µM of NSHDs greatly impeded this
MMP loss (Figures 6C-6F) by preserving mitochondrial functions. These results further
confirmed that NSHDs exhibited potent cellular protection against oxidative injury.
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 34
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Effects of H2S donors on H2O2-induced MMP loss in H9c2 cells. (A-E) Rh123
staining followed photofluorography to observe MMP in H9c2 cells. (A) Control group.
(B) Cells were pretreated with cysteine (480 µM) for 1 h, then exposed to H2O2 (400 μM)
for 5 h. Cells were preconditioned with 160 μM of NSHD-1 (C), NSHD-2 (D), NSHD-6
(E) in the presence of cysteine (480 µM) for 1 h prior to H2O2 treatment. (F) Quantitative
analysis for Rh123 in panels A-E. Mean fluorescence intensities (MFI) measured using
‡
image software were expressed as the mean ± SEM. **P<0.01 vs. Control group. P<0.01
vs. H2O2 treated group.
Cardioprotective Effects of NSHD-1 and NSHD-2 in MI/R Injury
Finally, we tested the protective effects of two representative donors, i.e. NSHD-1
and NSHD-2, 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. NSHD-1, NSHD-2, or vehicle was administered by direct
intracardiac (i.c.) injection at the time of reperfusion at different doses. All animal groups
displayed similar area-at-risk per left ventricle (AAR/LV), which means surgery caused
18
ACS Paragon Plus Environment

Page 19 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
similar risk. However, compared to vehicle treated mice, mice receiving NSHD-1 and
NSHD-2 displayed a dose-dependent reduction in infarct size per area-at-risk (INF/AAR),
assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining (Figure 7). A 100 g/kg
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
bolus of NSHD-1 maximally reduced INF/AAR by ~35%. Similarly, 50 g/kg and 100
g/kg of NSHD-2 reduced INF/AAR by ~ 50% (p < 0.01, p < 0.001, respectively).
Moreover, circulating cardiac troponin I (TnI) level, the marker for acute myocardial
infarction, were significantly lowered with NSHD-2 treated animals (p < 0.05 for 50
ug/kg and p < 0.01 for 100 ug/kg) (Figure 8). These results strongly suggest that N-
mercapto compounds are potent H2S donors and may have some pharmacological
benefits in biological systems. In the present study we injected these H2S donors directly
into the left ventricular cavity at the time of reperfusion. In the clinical setting we believe
that these agents could be administered via a number of routes to patients suffering from
an acute myocardial infarction. These compounds could be administered via intravenous
bolus injection or infusion or via intracoronary injection during coronary catheterization
to open the occluded coronary artery. In previous studies we have administered H2S
donors into the systemic circulation with excellent results.62
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 34
1
2
3
4
5
6
7
8
9
Figure 7. Cardioprotective effects of NSHD-1 and NSHD-2 in myocardial ischemia-
reperfusion injury. Doses of NSHD-1, NSHD-2 or vehicle were injected at the time of
reperfusion. (Left) Myocardial area-at-risk (AAR) per left ventricle (AAR/LV) and
infarct per area-at-risk (INF/AAR) were assessed in vehicle (n = 12), NSHD-1 (50 g/kg)
treated animal (n = 6), and NSHD-1 (100 g/kg) treated animal (n = 12). AAR/LV was
similar among all groups. INF/AAR was significantly smaller in the 100 g/kg dose
treated animal. (Right) Myocardial area-at-risk (AAR) per left ventricle (AAR/LV) and
infarct per area-at-risk (INF/AAR) were assessed in the vehicle (n = 12), NSHD-2 (50
g/kg) treated (n = 12), and NSHD-2 (100 g/kg) treated animals (n = 17). AAR/LV was
similar among all groups. INF/AAR was significantly smaller in animals treated with
either dose.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. Blood was collected at 4 h of reperfusion, and circulating cardiac troponin I
levels were measured. Troponin-I level was significantly reduced with either 50 g/kg or
100 g/kg of NSHD-2 treatment.
CONCLUSIONS
In summary, we have developed a series of H2S donors based on a N-mercapto
template. Their H2S release is triggered by cellular thiols such as cysteine or GSH. H2S
release from these donors can be regulated by structural modifications. Moreover, we
demonstrated that these donors are safe in cysteine-rich environments. Their protective
20
ACS Paragon Plus Environment

Page 21 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
effects on cellular oxidative injury and myocardial ischemia reperfusion injury were also
observed. Taking together, our studies demonstrate that N-mercapto species are potent
H2S donors and they possess potential therapeutic benefits.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
METHODS
Chemical Synthesis
All solvents were reagent grade. Tetrahydrofuran (THF) was freshly distilled from
sodium/benzophenone under argon. Reactions were magnetically stirred and monitored
by thin layer chromatography (TLC) with 0.25 mm pre-coated silica gel plates. Flash
chromatography was performed with silica gel 60 (particle size 0.040-0.062 mm). Yields
refer to chromatographically and spectroscopically pure compounds, unless otherwise
stated. Proton and carbon-13 NMR spectra were recorded on a 300 MHz spectrometer.
1
Chemical shifts are reported relative to chloroform (δ7.26) for H NMR and chloroform
13
(δ77.0) for C NMR. The purity of all the compounds was at least 95%, determined by
LC/MS. Compounds were also characterized by TLC, melting points, and NMR
spectroscopy.
Synthesis of (N-acylthio)amides (NSHD-1 – NSHD-26)
General procedure: thiocarboxylic acids (1 mmol) were mixed with KOH (5
mmol) in water (10 mL). To this mixture was added hydroxylamine-O-sulfonic acids (3
mmol). The reaction mixture was stirred at room temperature for 5 min and then
extracted with 15 mL of CH2Cl2 three times. Organic layers were then combined, dried
(by MgSO4), and concentrated to give the crude product S-acylthiohydroxylamines 3.
Without further purification 3 was dissolved in CH2Cl2 (5 mL) containing acid anhydride
(2 mmol). The reaction solution was stirred at room temperature for 12 h. Crude products
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 34
1
2
3
4
5
6
7
8
9
were precipitated out and collected. Purification by column chromatography or re-
crystallization gave the final products. The characterization data of each donor is
provided in the supporting information.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of 1-(benzoylthio)-3-phenylureas (NSHD-27 – NSHD-33)
Each freshly prepared S-acylthiohydroxylamine 3 (1 mmol, see procedure
described above) was mixed with the corresponding phenyl isocyanate derivative 5 (2
mmol) in CH2Cl2 (2 mL). The mixture was stirred at room temperature for 12 h. The
crude products were precipitated out and collected. Recrystallization in CH2Cl2/hexanes
gave the final products. The characterization data of each donor is provided in the
supporting information.
H2S Measurement
H2S generation was initiated by adding 100 μL of a donor’s stock solution (30
mM in THF) into a 30 mL PBS (pH 7.4, 50 mM)/THF (9:1) solution containing cysteine
or GSH (1.0 mM). Then 1.0 mL of reaction aliquots were periodically taken and
transferred to UV cuvettes containing MB cocktail (100 μL of zinc acetate (1% w/v), 200
μL of N, N-dimethyl-1,4-phenylenediamine sulfate (20 mM in 7.2 M HCl) and 200 μL of
ferric chloride (30 mM in 1.2 M HCl)). The MB reaction was carried out for 15 min and
the absorbance (670 nm) of the resultant solution was determined using an UV−Vis
spectrometer (Thermo Evolution 300). The H2S concentration of each sample was
calculated against a calibration curve, which was obtained with a series of Na2S solutions.
Product Analysis
NSHD-1 (129 mg 0.5 mmol) in 2 mL of MeOH was added to a stirred solution of
L-cysteine (605 mg, 5 mmol) in 50 mL of PBS buffer (pH 7.4, 20 mM). After 1 hour, 0.1
mL of solution was taken via syringe equipped with a 45 μM syringe filter, diluted with
22
ACS Paragon Plus Environment

Page 23 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
0.4 mL of MeOH and analyzed by HPLC (Thermo Scientific Surveyor, Hypersil gold
column, 4.6 × 100 mm, water/CH3CN 80/20 2 min; 60/40 4 min; 20/80 2 min; 80/20
2min, flow rate 1 mL/min). Compounds 8, 10 and 12 were identified and quantified as
compared to standard compounds. These compounds were also isolated from the reaction
mixture and characterized. The yields were 80%, 96%, and 95%, respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cell Culture
H9c2 cells were purchased from the American Type Culture Collection
(Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) high glucose medium supplemented with 10% fetal bovine serum (FBS) at 37℃
under 5% CO2.
Cell Viability Assay
The cell counting kit (CCK)-8 (Dojindo Laboratory, Kumamoto, Japan) was
applied to measure the cell viability of H9c2 cells cultured in 96-well plates. After the
indicated cell treatment, 100 μL of CCK-8 solution at 1:10 dilution was added to each
well and cells were incubated for a further 3 h at 37℃. Absorbance was measured at 450
nm with a microplate reader (Tecan Infinite F200, Switzerland). The mean optical density
(OD) of 4 wells in each group was used to calculate cell viability as follows:
(experiments were performed in triplicates)
% Cell viability ꢀ ꢁꢂ ꢃꢄꢅꢆꢃꢇꢅꢈꢃ ꢉꢄꢊꢋꢌ ×100
ꢁꢂ ꢍꢊꢈꢃꢄꢊꢎ ꢉꢄꢊꢋꢌ
Protective Activities of NSHDs on H2O2-Induced Cellular Damage
H9c2 cells were treated with NSHD-1, NSHD-2, and NSHD-6 at varied
concentrations (40-160 µM) for 1 h in the presence of cysteine (480 µM), respectively.
Excess donors and cysteine were washed away with PBS (pH 7.4). Cells were then
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 34
1
2
3
4
5
6
7
8
9
incubated with H2O2 (400 µM) for an additional 5 h. The control group was set up by
incubating H9c2 cells with H2O2 (400 µM) only. Cell viability was measured using the
CCK-8 assay. GYY4137 and NaHS were used as positive controls and experiments were
set up in a similar manner in the absence of cysteine. Cysteine (480 µM), NBC (160 µM)
and BZM (160 µM) were incubated with cells as well to rule out their effects on cell
viability.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
LDH Measurement
The rate of lactate dehydrogenase (LDH) release was determined with a
commercial LDH kit supplied by Thermo Fisher Scientific Inc. (Pittsburgh PA, US).
Briefly, H9c2 cells were inoculated 96-well plate and grew to about 70% confluence.
After the indicated treatments, 50 μL of supernatant per well was carefully removed and
transferred into corresponding wells for determination of extracellular LDH levels. Then,
100 μl of DMEM with 2% Triton X-100 was added to the adherent cells to lyse the cells.
Fifty microliters of cell lysate was transferred to a 96-well plate to determine intracellular
LDH levels. The same volume of prepared reaction mixture was added to supernatant or
homogenate, separately, and reacted for 10 min at room temperature by gentle shaking.
Stopping solution was added to stop the reaction. Absorbance was measured at 490 nm
and 600 nm with a microplate reader (Tecan Infinite F200, Switzerland). LDH release
rate was calculated as follows (experiments were performed 4 times):
% LDH release = extracellular LDH / (extracellular LDH + intracellular LDH) × 100
MMP Measurement
Mitochondrial membrane potential (MMP) was observed using a fluorescent dye
rhodamine 123 (Rh123), a cell-permeable cationic dye that preferentially enters the
24
ACS Paragon Plus Environment

Page 25 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mitochondria based on the highly negative MMP. Depolarization of MMP usually leads
to a decreased intake of Rh123 and an intracellular weak fluorescence. After the indicated
treatments, 10 mg/L Rh123 was added to cell cultures for 15 min at 37 ℃ and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
fluorescence was measured over the entire field of vision using a fluorescent microscope
(Advanced Microscopy Group, Seattle, US). The mean fluorescent intensity (MFI) of
Rh123 from four random fields was analyzed using IMAGEJ 1.41o software.
Cardioprotective Effects in MI/R Injury
Animals: Male C57BL/6J mice, 10–12 weeks of age (Jackson Labs, Bar Harbor, ME)
were used in the present study. All animals were housed in a temperature-controlled
animal facility with a 12-hour 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. All animal procedures were approved by the Louisiana State University Health
Science Center Institutional Animal Care and Use Committee.
Myocardial Ischemia/Reperfusion (MI/R) Protocol: Surgical occlusion of the left
coronary artery (LCA), infarct size determination and troponin I (TnI) measurements
were performed as previously described.63 C57BL6/J male mice (10-12 weeks of age)
were anesthetized using pentobarbital (50 mg/kg) and ketamine (60 mg/kg) and
maintained under anesthesia throughout the procedure with additional administration of
pentobarbital (50 mg/kg). Mice were intubated and mechanically ventilated using a Hugo
Sachs type 845 minivent. A hair removal agent was used to remove the hair on the chest
of mice. The area was then cleaned using betadine and alcohol wipes alternating 3 times.
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 34
1
2
3
4
5
6
7
8
9
An incision was made proximally left of the midline of the chest, exposing the second
and third ribs. The chest was opened by cauterizing through the second and third rib. The
LCA was visualized and using a 7-0 silk suture a 3-5 mm piece of PE-10 tubing was
secured over the LCA, occluding the artery. Mice were subjected to 45 minutes of
myocardial injury and at the time of reperfusion an injection of vehicle (0.5% THF),
NSHD-1 (50 µg/kg and 100 µg/kg), or NSHD-2 (50 µg/kg) was given i.c. The chest was
closed in layers, closing the muscle layer first, then the skin. Mice were removed from
ventilation once pedal reflex returned and it was determine respiration was spontaneous.
Ice were placed in a recovery cage and supplied with 100% O2. Reperfusion was allowed
for 24 hours, blood samples for TnI measurements were taken at 4 hours after time of
reperfusion. At 24 hours of reperfusion the LV area at risk (AAR) and infarct size were
determined by evans blue and 2,3,5-tetrazolium chloride stainin, as previously
described.63
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Drug preparation: On the day of experimentation, test compounds (NSHD-1 or NSHD-
2) were diluted in 0.2 mL of 100% THF solution. For in vivo experiments, the test
compounds were further diluted in sterile saline to final concentrations of 0.05 mg/ml or
0.1 mg/ml. Then, various volumes were administered based on animals’ weights to obtain
the correct dosage. The resulting concentration of THF in each dosage was 0.5% v/v.
Vehicle consisted of a solution of 0.5% v/v THF in sterile saline.
Cardiac Troponin-I Assay: Blood samples were collected via a tail vein at 4 h of
reperfusion. Cardiac troponin-I level was measured in serum using the Life Diagnostic
highsensitivity mouse cardiac troponin-I ELISA kit (Mouse Cardiac Tn-I ELISA Kit;
Life Diagnostics, West Chester, PA) following the manufacturer instructions.
26
ACS Paragon Plus Environment

Page 27 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
ACKNOWLEDGEMENTS
7
8
9
We would like to thank Orleiquis Guerra Mulgado for technical support. This
work is supported by an American Chemical Society-Teva USA Scholar Grant to M.X.,
National Institutes of Health (R01HL116571 to M.X. and D.J.L.), and Natural Science
Foundation of China (81200606) to C.-t. Y.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Supporting Information
Detailed synthetic procedures, characteristic data, and experimental procedures
are provided. This material is available free of charge via the Internet at
http://pubs.acs.org.
Corresponding Author
Ming Xian: 509-335-6073, mxian@wsu.edu
Abbreviations Used
H2S, hydrogen sulfide; NO, nitric oxide; CO, carbon monoxide; CBS, cystathionine -
synthase; CSE, cystathionine -lyase; MPST, 3-mercaptopyruvate sulfur-transferase; S-
SH, S-sulfhydration; HSNO, thionitrous acid; Na2S, sodium sulfide; NaHS, sodium
hydrogen sulfide; DTTs, dithiolthiones; NSAIDs, nonsteroidal anti-inflammatory drugs;
N-SH, N-mercapto; NSHDs, N-SH-based donors; SAR, structure activity relationship;
MI/R, myocardial ischemia reperfusion; MB, methylene blue; GSH, glutathione; PBS,
phosphate buffered saline; NCL, native chemical ligation; CCK-8, Cell Counting Kit-8;
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 34
1
2
3
4
•−
OS, oxidative stress; ROS, reactive oxygen species; O2 , superoxide; •OH, hydroxyl
5
6
7
8
radical; •O, singlet oxygen; H2O2, hydrogen peroxide; NBC, N-benzoyl cysteine; BZM,
benzamide; LDH, lactate dehydrogenase; MMP, mitochondrial membrane potential; i.c.,
intracardiac; AAR/LV, area-at-risk per left ventricle; INF/AAR, infarct size per area-at-
risk; TTC, 2,3,5-triphenyltetrazolium chloride; TnI, troponin I; AAR, area-at-risk; THF,
tetrahydrofuran; TLC, thin layer chromatography; DMEM, Dulbecco’s modified Eagle’s
medium; FBS, fetal bovine serum; OD, optical density; MFI, mean fluorescent intensity;
LCA, left coronary artery
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
REFERENCES:
1. Li, L.; Moore, P. K. H2S and cell signaling. Annu. Rev. Pharmacol. Toxicol. 2011, 51,
169−187.
2. Fukuto, J. M.; Carrington, S. J.; Tantillo, D. J.; Harrison, J. G.; Ignarro, L. J.;
Freeman, B. A.; Chen, A.; Wink, D. A. Small molecule signaling agents: the
integrated chemistry and biochemistry of nitrogen oxides, oxides of carbon, dioxygen,
hydrogen sulfide, and their derived species. Chem. Res. Toxicol. 2012, 25, 769−793.
3. Vandiver, M. S.; Snyder, S. H. Hydrogen sulfide: a gasotransmitter of clinical
relevance. J. Mol. Med. 2012, 90, 255−263.
4. Wang, R. Physiological implications of hydrogen sulfide: a whiff exploration that
blossomed. Physiol. Rev. 2012, 92, 791−896.
5. Olson, K. R.; Donald, J. A.; Dombkowski, R. A.; Perry, S. F. Evolutionary and
comparative aspects of nitric oxide, carbon monoxide and hydrogen sulfide. Respir.
Physiol. Neurobiol. 2012, 184, 117−129.
6. Kolluru, G. K.; Shen, X.; Bir, S. C.; Kevil, C. G. Hydrogen sulfide chemical biology:
Pathophysiological roles and detection. Nitric Oxide 2013, 35, 5-20.
7. Ono, K.; Akaike, T.; Sawa, T.; Kumagai, Y.; Wink, D.; Tantillo, D. J.; Hobbs, A. J.;
Nagy, P.; Xian, M.; Lin, J.; Fukuto, J. M. The redox chemistry and chemical biology
of H2S, hydropersulfides, and derived species: implications to their possible
biological activity and utility. Free Radical Biol. Med. 2014, 77, 82-94.
28
ACS Paragon Plus Environment

Page 29 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
8. Kabil, O.; Banerjee, R. Enzymology of H2S biogenesis, decay and signaling. Antioxid.
Redox. Signal. 2014, 20, 770-782.
6
7
8
9
9. King, B. S. Potential biological chemistry of hydrogen sulfide (H2S) with the nitrogen
oxides. Free Radic. Biol. Med. 2013, 55, 1-7.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10. Szabo, C. Roles of hydrogen sulfide in the pathogenesis of diabetes mellitus and its
complications. Antioxid. Redox. Signal. 2012, 17, 68-80.
11. Kimura, H. Production and physiological effects of hydrogen sulfide. Antioxid. Redox.
Signal. 2014, 20, 783-793.
12. Kolluru, G. K.; Shen, X.; Kevil, C. G. A tale of two gases: NO and H2S, foes or
friends for life? Redox. Biol. 2013, 1, 313-318.
13. Köhn, C.; Dubrovska, G.; Huang, Y.; Gollasch, M. Hydrogen sulfide: potent
regulator of vascular tone and stimulator of angiogenesis. Int. J. Biomed. Sci. 2012, 8,
81-86.
14. Jackson-Weaver, O.; Osmond, J. M.; Riddle, M. A.; Naik, J. S.; Gonzalez Bosc, L.
V.; Walker, B. R.; Kanagy, N. L. Hydrogen sulfide dilates rat mesenteric arteries by
activating endothelial large-conductance Ca2+-activated K+ channels and smooth
muscle Ca2+ sparks. Am. J. Physiol. Heart Circ. Physiol. 2013, 304, H1446-H1454.
15. Ariyaratnam, P.; Loubani, M.; Morice, A. H. Hydrogen sulphide vasodilates human
pulmonary arteries: a possible role in pulmonary hypertension. Microvasc. Res. 2013,
90, 135-137.
16. Wallace, L.; Vong, L.; Mcknight, W.; Dicay, M.; Martin, G. R. Endogenous and
exogenous hydrogen sulfide promotes resolution of colitis in rats. Gastroenterology
2009, 137, 569-578.
17. Wallace, J. L. Hydrogen sulfide-releasing anti-inflammatory drugs. Trends
Pharmacol. Sci. 2007, 28, 501-505.
18. Kashfi, K. Anti-cancer activity of new designer hydrogen sulfide-donating hybrids.
Antioxid. Redox. Signal. 2014, 20, 831-846.
19. Kaium, M. A.; Liu, Y.; Zhu, Q.; Liu, C.; Duan, J.; Tan, B.; Zhu, Y. Z. H2S donor, S-
propargyl-cysteine, increases CSE in SGC-7901 and cancer-induced mice: evidence
for a novel anti-cancer effect of endogenous H2S? PLoS One 2011, 6, e20525.
20. Lefer, D. J. A new gaseous signaling molecule emerges: cardioprotective role of
hydrogen sulfide. Proc. Natl. Acad. Sci. U S A. 2007, 104, 17907-17908.
29
ACS Paragon Plus Environment