Investigations into the carbonic anhydrase inhibition of COS-releasing donor core motifs

Article abstract of DOI:10.1016/j.bcp.2017.11.004

Carbonyl sulfide (COS) releasing scaffolds are gaining popularity as hydrogen sulfide (H₂S) donors through exploitation of the carbonic anhydrase (CA)-mediated hydrolysis of COS to H₂S. The majority of compounds in this emerging class of donors undergo triggerable decomposition (often referred to as self-immolation) to release COS, and a handful of different COS-releasing structures have been reported. One benefit of this donation strategy is that numerous caged COS-containing core motifs are possible and are poised for development into self-immolative COS/H₂S donors. Because the intermediate release of COS en route to H₂S donation requires CA, it is important that the COS donor motifs do not inhibit CA directly. In this work, we investigate the cytotoxicity and CA inhibition properties of different caged COS donor cores, as well as caged CO₂ and CS₂ motifs and non-self-immolative control compounds. None of the compounds investigated exhibited significant cytotoxicity or enhanced cell proliferation at concentrations up to 100 μM in A549 cells, but we identified four core structures that function as CA inhibitors, thus providing a roadmap for the future development of self-immolative COS/H₂S donor motifs.

Full text of DOI:10.1016/j.bcp.2017.11.004

Accepted Manuscript
Investigations into the Carbonic Anhydrase Inhibition of COS-Releasing Donor
Core Motifs
Andrea K. Steiger, Yu Zhao, Won Jin Choi, Alder Crammond, McKinna R.
Tillotson, Michael D. Pluth
PII:
S0006-2952(17)30668-8
https://doi.org/10.1016/j.bcp.2017.11.004
BCP 12949
DOI:
Reference:
Biochemical Pharmacology
To appear in:
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Accepted Date:
29 September 2017
9 November 2017
Please cite this article as: A.K. Steiger, Y. Zhao, W.J. Choi, A. Crammond, M.R. Tillotson, M.D. Pluth,
Investigations into the Carbonic Anhydrase Inhibition of COS-Releasing Donor Core Motifs, Biochemical
Pharmacology (2017), doi: https://doi.org/10.1016/j.bcp.2017.11.004
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Investigations into the Carbonic Anhydrase Inhibition of COS-Releasing Donor Core
Motifs
Andrea K. Steiger, Yu Zhao, Won Jin Choi, Alder Crammond, McKinna R. Tillotson, Michael
D. Pluth*
Department of Chemistry and Biochemistry, Institute of Molecular Biology.
1253 University of Oregon, Eugene, OR 97403, USA.
Email: pluth@uoregon.edu
Phone: (541) 346-7477
1

Abstract
Carbonyl sulfide (COS) releasing scaffolds are gaining popularity as hydrogen sulfide (H2S)
donors through exploitation of the carbonic anhydrase (CA)-mediated hydrolysis of COS to H2S.
The majority of compounds in this emerging class of donors undergo triggerable decomposition
(often referred to as self-immolation) to release COS, and a handful of different COS-releasing
structures have been reported. One benefit of this donation strategy is that numerous caged COS-
containing core motifs are possible and are poised for development into self-immolative
COS/H2S donors. Because the intermediate release of COS en route to H2S donation requires
CA, it is important that the COS donor motifs do not inhibit CA directly. In this work, we
investigate the cytotoxicity and CA inhibition properties of different caged COS donor cores, as
well as caged CO2 and CS2 motifs and non-self-immolative control compounds. None of the
compounds investigated exhibited significant cytotoxicity or enhanced cell proliferation at
concentrations up to 100 µM in A549 cells, but we identified four core structures that function as
CA inhibitors, thus providing a roadmap for the future development of self-immolative COS/H2S
donor motifs.
Keywords:
Hydrogen sulfide, carbonyl sulfide, carbonic anhydrase
2

1. Introduction
Hydrogen sulfide (H2S), now well established as an important gaseous signaling
molecule, has been implicated in a wide variety of physiological processes since its initial
recognition in 1996.[1-3] H2S is produced endogenously and maintained at low (mid-nanomolar)
concentrations, and the administration of exogenous sulfide has been shown to provide a
therapeutic benefit in various applications, including reduction of myocardial infarct size,
vasodilation, and decrease in inflammation.[4, 5] For example, GYY4137 and AP39, which
undergo slow hydrolysis to release H2S, have been shown to exhibit anti-inflammatory
activity.[6, 7] Additionally, appending H2S donor scaffolds to non-steroidal anti-inflammatory
drugs (NSAIDs) has been used to generate a number of H2S-NSAID hybrids including ATB-346
and NBS-1120, which are based on naproxen and aspirin derivatives, respectively (Figure 1).[8,
9] Aligned with these positive impacts, new H2S donation strategies are emerging rapidly, and a
number of H2S-releasing compounds are currently in clinical trials.[10, 11] Recent goals in
improving upon H2S-based therapeutics have included triggerable donation in response to
specific (and variable) stimuli, controllable rates of H2S release, and the ability to append the
H2S-releasing moiety to a variety of scaffolds to potentially access H2S-drug hybrids.
Building from these needs, our lab recently introduced a new strategy for H2S donation
3

by using the triggered decomposition (often referred to as self-immolation) of carbonyl sulfide
(COS)-releasing motifs (Figure 2a), which leverages the rapid hydrolysis of COS to H2S by
carbonic anhydrase (CA), a ubiquitous mammalian zinc metalloenzyme that normally catalyzes
the hydrolysis of CO2 under near diffusion-limited rates to regulate blood pH levels and tissue
CO2 transport.[12, 13] Since our initial report,[14] a number of related COS-donating scaffolds
have been reported for H2S release.[15-21] Complementing slow-release COS donors,[16] a
common strategy has been to engineer self-immolative COS release, which can be activated by
different stimuli, including ROS,[21] esterase,[15, 18] light,[17, 20] and bio-orthogonal
triggers[19] (selected triggered examples are shown in Figure 2b). Moreover, we recently
reported on the kinetics of COS release from several different caged carbonyl sulfide isomers
and found that COS/H2S release profiles can be tuned by structural and isomeric
modifications.[22] This work expanded our growing knowledge of potential COS donor
scaffolds and provides a resource for researchers wishing to develop COS donor scaffolds to fit a
specific need. In addition to providing functional H2S donor platforms, the availability of such
COS donors may allow for the investigation of COS chemical biology, which has been largely
under-studied.[23] Critical to using these donor scaffolds for COS investigations or for
therapeutic H2S delivery is the general biocompatibility of the core motifs, including potential
direct interactions with CA. The efficient conversion of COS to H2S in all of the above scaffolds
requires CA, so it is imperative to not only understand the mechanism of CA activity in COS
hydrolysis, but also to determine whether any of the COS-donating motifs are functional CA
inhibitors. Significant prior research has focused on identifying CA inhibitors for therapeutic
uses, such as treatment of glaucoma, epilepsy, and mountain sickness, and is the topic of
numerous recent reviews.[13, 24, 25] Because the structural scope of active CA inhibitors is
4

broad and includes many sulfur-containing molecules, including dithiocarbamates and
trithiocarbonates,[26, 27] we wanted to determine whether the sulfur-containing cores of
possible COS-releasing scaffolds (Figure 2c) exhibit CA inhibition. If a COS-releasing motif
functions as a viable CA inhibitor, donor scaffolds built from such motifs are unlikely to find
widespread utility as functional H2S donors. Such investigations are important in understanding
the scope of potential COS-releasing structural motifs due to the necessity of CA activity for
efficient H2S release from COS donors
5

2. Materials and Methods
2.1 Synthetic Materials
Reagents were purchased from Sigma-Aldrich (St. Louis, MO), Alfa-Aesar (Haverhill, MA), or
Tokyo Chemical Industry (TCI, Portland, OR) and used as received. Compounds 1-15 were
synthesized as previously reported. Spectroscopic grade, inhibitor-free THF and DCM were
deoxygenated by sparging with argon followed by passage through a Pure Process Technologies
solvent purification system to remove water. Deuterated solvents were purchased from
Cambridge Isotope Laboratories (Tewksbury, MA) and used as received. Silica gel (SiliaFlash
1
F60, Silicycle, 230-400 mesh) was used for column chromatography. H, and 13C{1H} NMR
spectra were recorded on a Bruker 500 MHz instrument. Chemical shifts are reported in ppm
relative to residual protic solvent resonances.
2.2 Spectroscopic Materials
Tris-HCl buffer was prepared from Tris hydrochloride (Sigma Aldrich, 50 mM) and deionized
water obtained from a Synergy UV Millipore Water System and adjusted to pH 8.5 with a
SevenMulti pH Probe (Mettler Toledo). UV-visible spectra were acquired on a Cary 60 UV-vis
spectrometer equipped with a Quantum Northwest TC-1 temperature controller.
2.3 Cell Culture Materials
A549 cells obtained from ATCC (Manassas, VA) were grown in a 5% CO2 incubator at 37 °C in
Dulbecco’s Modified Eagle Medium (Gibco (Dublin, Ireland), high glucose, phenol red) with
6

10% FBS (VWR, Radnor, PA) and 1% penicillin streptomycin (Gibco). Cells were passaged (up
to p 25) every 1-3 days upon reaching 85-90% confluency. Absorbance measurements were
acquired using a Tecan Safire2 microplate reader.
2.4 Synthesis
2.4.1 General synthetic procedure A (Figure 3a)
The alcohol, thiol, or amine starting material (1.0 equiv.) was combined with phenyl
isothiocyanate or phenyl isocyanate (1.0 equiv.) in anhydrous THF (15 mL) at 0 °C, followed by
the addition of base (1.2 equiv.). The resultant mixture was stirred at 0 °C for 20 min, after
which the ice bath was removed, and the reaction mixture was stirred at r.t. until the completion
of the reaction as indicated by TLC. The reaction was quenched by adding brine (30 mL), and
the aqueous solution was extracted with ethyl acetate (3 × 15 mL). The organic layers were
combined, dried over MgSO4, and evaporated under vacuum. The crude products from each
reaction were purified by column chromatography.
Compound 1 was prepared from benzyl alcohol and phenyl isothiocyanate in the presence
of NaH (yield: 75%). Compound 2 was prepared from benzyl mercaptan and phenyl isocyanate
in the presence of NaH (yield: 38%). Compound 6 was prepared from benzyl alcohol and phenyl
isocyanate in the presence of TEA (yield: 20%). Compound 9 was prepared from benzyl
mercaptan and phenyl isothiocyanate in the presence of NaH (yield: 75%). Compound 14 was
prepared from benzyl amine and phenyl isocyanate in the presence of TEA (yield: 88%).
Compound 15 was prepared from benzyl amine and phenyl isothiocyanate in the presence of
TEA. (yield: 64%). Characterization data for previously-reported compounds 1, 2, 6, 9, 14, and
15 match the reported data. [21, 28-32]
7

2.4.2 General synthetic procedure B (Figure 3b)
The alcohol, thiol, or amine starting material (1.0 equiv.) was combined with the phenyl
chloroformate reagent (1.0 equiv.) in anhydrous DCM (15 mL) at 0 °C, followed by the addition
of base (1.0 equiv.). The resultant solution was stirred at 0 °C for 20 min, after which the ice bath
was removed, and the reaction mixture was stirred at r.t. until the completion of the reaction as
indicated by TLC. The reaction was quenched by adding brine (30 mL), and the aqueous solution
was extracted with ethyl acetate (3 × 15 mL). The organic layers were combined, dried over
MgSO4, and evaporated under vacuum. The crude products from each reaction were purified by
column chromatography.
Compound 3 was prepared from benzyl alcohol and phenyl chlorothionoformate in the
presence of pyridine (yield: 57%). 1H NMR (500 MHz, CDCl3) δ (ppm): 7.55 (m, 7H), 7.33 (t, J
= 10.0 Hz, 1H), 7.16 (d, J = 10.0 Hz, 2H), 5.60 (s, 2H). C{1H} NMR (125 MHz, CDCl3) δ
13
(ppm): 195.0, 153.5, 134.2, 129.6, 128.8, 128.7, 128.6, 126.6, 122.0, 75.6. Compound 4 was
prepared from benzyl mercaptan and phenyl chloroformate in the presence of TEA (yield: 73%).
The characterization data match the reported data.[32] Compound 5 was prepared from benzyl
1
mercaptan and phenyl thiochloroformate in the presence of TEA (yield: 71%). H NMR (500
MHz, DMSO-d6) δ (ppm): 7.53 (m, 5H), 7.32 (m, 4H), 7.27 (m, 1H), 4.25 (s, 2H). 13C{1H}
NMR (125 MHz, DMSO-d6) δ (ppm): 188.6, 137.3, 135.8, 131.1, 130.2, 129.4, 129.1, 127.9,
126.5, 34.9. Compound 7 was prepared from benzyl alcohol and phenyl chloroformate in the
presence of TEA (yield: 62%). The characterization data match the reported data.[28] Compound
8 was prepared from benzyl alcohol and phenyl thiochloroformate in the presence of NaH (yield:
32%). 1H NMR (500 MHz, CDCl3) δ (ppm): 7.57 (m, 2H), 7.44 (m, 3H), 7.39 (m, 5H), 5.29 (s,
8

2H). 13C{1H} NMR (125 MHz, CDCl3) δ (ppm): 167.9,135.0, 134.9, 129.6, 129.2, 128.6, 128.5,
127.7, 69.4. Compound 10 was prepared from benzyl mercaptan and phenyl chlorothionoformate
in the presence of pyridine (yield: 43%). The characterization data match the reported data.[33]
Compound 11 was prepared from benzyl amine and phenyl chloroformate in the presence of
TEA (yield: 82%). The characterization data match the reported data.[34] Compound 12 was
prepared from benzyl amine and phenyl thiochloroformate in the presence of pyridine (yield:
57%). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.85 (s, 1H), 7.46 (m, 2H), 7.41 (s, 3H), 7.35 (t,
J = 5.0 Hz, 2H), 7.26 (d, J = 10.0 Hz, 3H), 4.32 (s, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ
(ppm): 164.6, 139.3, 135.4, 129.4, 129.3, 129.1, 128.8, 127.8, 127.5, 44.7. Compound 13 was
prepared from benzyl amine and phenyl chlorodithioformate in the presence of TEA (yield:
49%). 1H NMR (500 MHz, CDCl3) δ (ppm): 7.61 (m, 2H), 7.50 (m, 3H), 7.32 (m, 3H), 7.17 (d, J
= 10.0 Hz, 2H), 6.86 (br, 1H), 4.86 (d, J = 5.0 Hz, 2H). C{1H} NMR (125 MHz, CDCl3) δ
13
(ppm): 195.3, 135.8, 135.6, 131.2, 130.4, 128.9, 128.6, 128.0, 127.5, 50.2.
2.5 Cytotoxicity Assays
Stock solutions of the compounds (20 mM, 10 mM, 5 mM, 2 mM) were prepared in DMSO via
serial dilution and used to make 100 µM, 50 µM, 25 µM, and 10 µM solutions of each
compound in DMEM (no phenol red, no FBS). 96-Well plates were seeded with ~20,000
cells/well overnight prior to cytotoxicity experiments. Cells were washed with PBS (Gibco) and
incubated with the desired compound or with vehicle (0.5% DMSO) for one hour prior to being
washed with PBS and incubated with CCK-8 solution (10% in DMEM). After 1 hour, the
absorbance at 450 nm was measured on a plate reader, and the cell survival was calculated as a
percent of the control and normalized to the vehicle group. The results are expressed as mean
9

SD (n = 6).
2.6 Determination of CA Inhibition
Stock solutions of each compound (20 mM in DMSO), acetazolamide (20 mM in DMSO), p-
nitrophenylacetate (pNPA, 15 mM in ethanol), and CA (7.5 mg/mL in Tris buffer) were prepared
and stored at 2-4 °C until immediately prior to use. pNPA (20 µL) was injected into the Tris HCl
buffer (3 mL) immediately followed by the desired compound of interest (7.5 µL) with stirring at
37.00
0.05 °C. Data collection began immediately prior to injection of CA (20 µL) and
continued until the production of pNP was complete, as determined by a plateau in absorbance at
405 nm. Absorption data was fit to a first-order exponential equation in Origin 8 to obtain
pseudo first-order rate data, which are reported in units of s-1. All trials were completed in
triplicate and rates are reported as mean SD (n = 3).
3. Results
3.1 Synthesis of Model Compounds
The 15 model compounds used in our investigations were prepared using two general
procedures. In synthetic procedure A (Figure 3a), one equivalent of an alcohol, thiol, or amine
was combined with one equivalent of phenyl isothiocyanate or phenyl isocyanate and base and
was stirred until completion of the reaction. Following an extraction and purification by column
chromatography, compounds 1, 2, 6, 9, and 14-15 were isolated in 20-88% yield. In synthetic
procedure B (Figure 3b), one equivalent of the alcohol, thiol, or amine starting material was
combined with one equivalent of phenyl chloroformate and base. When the reaction was
complete, the product was washed, extracted, and purified by column chromatography.
Compounds 3-5, 7-8, and 10-13 were isolated in 32-82% yield.
10

Our initial report of caged COS release utilized an O-alkyl thiocarbamate scaffold, but S-
alkyl thiocarbamates and thiocarbonates have also been reported.[15, 17, 22] Depending on the
placement of the COS moiety in the caged core, there are different constitutional isomers that
can release COS, which motivated our selection of model compounds 1-5 (Figure 3c). Similarly,
the availability of caged CO2 compounds, which generate CO2/H2O rather than COS/H2S,
prompted our investigation of control compounds 6-8 (Figure 3d). Additionally, the recent
interest in CS2 donor development prompted our inclusion of dithiocarbamates 9-10 (Figure
3e).[35] Finally, we included isomers 11-15 as control compounds, which have similar
functional motifs but lack the caged COS core (Figure 3f). For each of these model compounds,
our goal was to determine whether the core structures exerted cytotoxicity or cell proliferative
effects and also to measure the CA inhibition profiles for each compound to better guide future
COS donor development.
11

3.2 Cytotoxicity Evaluation
Prior to measuring CA inhibition profiles, we first measured the cytotoxicity of each
compound in A549 human lung adenocarcinoma cells using the CCK-8 cell proliferation assay.
We chose concentrations ranging from 10 – 100 µM for each compound, which corresponds to
the general concentration ranges typically used in biological experiments that use H2S-donating
compounds. Incubation of A549 with 10, 25, 50, or 100 µM of each compound did not result in
significant cytotoxicity or proliferation, suggesting that the inherent core structures from each
model compound do not significantly impact cell viability (Figure 4).
3.3 Determination of CA Inhibitors
To investigate the potential CA inhibition properties of each of the selected scaffolds, we
used CA-mediated p-nitrophenyl acetate (pNPA) hydrolysis as a model system for measuring
CA activity / inhibition. The CA-mediated hydrolysis of pNPA is slower than the near diffusion-
12

limited hydrolysis of CO2, and the p-nitrophenol (pNP) product has a characteristic absorbance at
405 nM, thus providing a simple method to measure CA activities by UV-vis spectroscopy
(Figure 5a)[36]. For each compound, we measured the rate of pNPA hydrolysis to pNP and
compared these observed rates to those of CA alone, as well as the rate in the presence of the
common CA inhibitor acetazolamide (AAA, Figure 5b). In each case, the rate of production of
pNP was then fit directly to a first-order exponential equation. Of the 15 compounds tested, 11
failed to produce significant changes in the rate of pNPA hydrolysis, suggesting that these
compounds do not exhibit significant CA inhibition at concentrations typically used in H2S
donor experiments. Four compounds, however, were found to inhibit CA activity: 2, 9, 12, and
13. Of these compounds, 2 and 9 were moderate inhibitors and decreased the rate of pNPA
hydrolysis by about 50%. By comparison, compounds 12 and 13 were much more effective CA
inhibitors and decreased the rate of hydrolysis to approximately that seen in the presence of
AAA, which is recognized as a strong CA inhibitor (Figure 5c-f).
-
13

3.4 Inhibition studies with potential decomposition products
In addition to the 15 compounds examined above, we also investigated the CA inhibition
properties of thiophenol and benzyl thiol because these thiols could potentially be generated as
hydrolysis and/or decomposition products of 2, 9, 12, and 13. (vide infra). We found both
thiophenol and benzyl thiol to inhibit CA (Figure 5g). The rate of hydrolysis in the presence of
14

thiophenol alone was found to be similar to that of 12 and 13, both of which, if deprotonated,
would decompose to thiophenol. Similarly, the rate of pNPA hydrolysis observed in the presence
of benzyl thiol is similar to that of 2 and 9, which would decompose to benzyl thiol.
4. Discussion
The recent introduction and adoption of triggered COS-releasing compounds for H2S
donation highlights the potential of this strategy to access a broad class of H2S donors with
potential therapeutic properties. In addition to providing functional COS/H2S donor platforms,
the use of analogous CO2/H2O-releasing compounds provide key control compounds that
undergo the same self-immolative chemistry to generate the same organic byproducts as the
canonical donors, but which do not release COS/H2S. For all such scaffolds, a key component of
the COS/H2S donor strategy is the requirement of CA-mediated COS hydrolysis to H2S. Because
of this requirement, the donor motifs should not interfere with CA activity. With these
considerations in mind, we wanted to measure cytotoxicity profiles and CA inhibition data for
these potential COS-releasing moieties to help determine the most promising scaffolds for caged
COS release.
We identified a library of 15 compounds that include known and potential caged COS
scaffolds, caged CO2 and CS2 motifs, and related control compounds as described in the Results
section. Although each of these compounds represents a scaffold that could be used in a donor or
control compound, they do not contain triggering moieties to allow for decomposition, meaning
that they should not release COS spontaneously. By investigating the cytotoxicity and CA
inhibition profile of each core motif, we hoped to identify the core structures that do not affect
CA activity for future COS/H2S donor design.
15

As outlined in the Results section, none of the prepared model compounds were found to
be cytotoxic in A549 cells at concentrations below 100 µM, suggesting that the core motifs are
not inherently cytotoxic at concentration ranges typically used in H2S donation studies. Of the 15
compounds investigated, however, we did identify four compounds that inhibit CA activity: 2, 9,
12, and 13 (Figure 6a). At first, the only similarity between these compounds appeared to be the
presence of a sulfur atom adjacent to the thiocarbonyl moiety. We also noticed, however, that
deprotonation of the amide NH in these compounds could lead to decomposition to form a thiol-
containing byproduct (benzyl thiol for 2 and 9 or thiophenol for 12 and 13, Figure 6b). We
hypothesized that these thiol byproducts may be responsible for the observed inhibitory
properties, rather than the thiocarbamate/dithiocarbamate scaffolds directly. Further supporting
this hypothesis, we measured the inhibitory effects of benzyl thiol and thiophenol. Benzyl thiol
exhibited similar CA inhibitory profiles to 2 and 9, and thiophenol inhibited CA similarly to 12
and 13. Aside from the shown CA inhibition properties, this additionally suggests the instability
of these scaffolds at even moderately basic conditions, making them less reliable as COS donor
constructs or as suitable controls.
In combination with recent work investigating stability and rate of COS/H2S release from
16

different COS-releasing scaffolds,[22] the present work provides an important resource for
selecting the best scaffolds for COS donation in the design of H2S therapeutics. Four scaffolds
(1, 3, 4, and 5) were identified that have either been reported to release COS or are likely to
release COS through self-immolation that do not inhibit CA. These motifs are likely ideal
starting points in the design of new COS/H2S donor motifs. Although this work provides the first
insights into the potential CA inhibition properties of COS/H2S donor scaffolds, any structural
modifications could impact the extent of inhibition, which will likely require future constructs to
be tested for CA inhibition directly. Additionally, the identification of thiocarbamate 2 as a CA
inhibitor should not completely eliminate this S-alkyl thiocarbamate motif from consideration as
a COS/H2S donor core, because further functionalization of the core structure may impact or
reduce the CA inhibition profiles. Indeed, similar scaffolds have been shown to be successful
COS donors in recent reports.[15, 17] Future work is required to determine the precise
consequences of using a COS donor that inhibits CA as an H2S donor, but the efficiency, rate of
decomposition, and rate of H2S production may all be affected. Through the rational selection of
COS donor cores that provide efficacious COS/H2S release rates, suitable stability in the
presence of biological milieu, and minimal CA inhibition, we expect the field of COS/H2S
donors will continue to expand rapidly and provide access to new motifs with
pharmacologically-relevant activities.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
17

Research reported in this publication was supported in part by the Sloan Foundation, Dreyfus
Foundation, the NIH (MDP; R01GM113030), and the NSF/GRFP (AKS; DGE-1309047). NMR
infrastructure at the UO is supported by the NSF (CHE-1427987).
18

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Figure 1. Selected examples of H2S-releasing therapeutics currently in various stages of clinical
development.
Figure 2. (a) General strategy for H2S generation from COS-releasing molecules. A caged
thiocarbamate is shown as an example system. (b) Selected examples of triggered COS donors
with different core structural motifs. (c) Potential thiocarbonate, thiocarbamate, and
dithiocarbonate motifs that could function as COS/H2S donors.
Figure 3: (a), (b) General synthetic procedures to prepare model compounds. Structures of the
model compounds prepared and investigated in this study include (c) COS donor scaffolds (1-5),
(d) CO2 donor scaffolds (6-8), (e) CS2 donor scaffolds (9-10), and (f) control scaffolds (11-15).
Figure 4. CCK-8 cytotoxicity assay of compounds 1-15. The cytotoxicity of each compound was
investigated in A549 cells at 10, 25, 50 and 100 µM using the CCK-8 cell counting assay and
compared to the DMSO vehicle. (a) caged COS scaffolds (1-5), (b) caged CO2 (6-8) and CS2 (9-
10) scaffolds, and (c) control scaffolds (11-15).
Figure 5. (a) CA-mediated hydrolysis of pNPA to form pNP. (b) Strucutre of known CA
inhibitor AAA. pNPA assay of (c) caged COS scaffolds, (d) caged CO2 scaffolds, (e) caged CS2
scaffolds, and (f) control scaffolds. (g) Absorbance data was fit to a first order exponential to
determine rate of hydrolysis in the presence of each compound and compared to AAA, PhSH,
and BnSH.
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Figure 6. (a) Structures of compounds identified as CA inhibitors. (b) Mechanism for production
of thiophenol or benzyl thiol following deprotonation of 2, 9, 12, or 13.
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