Synthesis of Amino-ADT Provides Access to Hydrolytically Stable Amide-Coupled Hydrogen Sulfide Releasing Drug Targets

Article abstract of DOI:10.1055/s-0035-1560603

As additional physiological functions of hydrogen sulfide (H₂S) are discovered, developing practical methods for exogenous H₂S delivery is important. In particular, nonsteroidal anti-inflammatory drugs (NSAIDs) functionalized with H₂S-releasing anethole dithiolethione (ADT-OH) through ester bonds are being investigated for their combined anti-inflammatory and antioxidant potential. The chemical robustness of the connection between drug and H₂S-delivery components, however, is a key and controllable linkage in these compounds. Because esters are susceptible to hydrolysis, particularly under acidic conditions such as stomach acid in oral drug delivery applications, we report here a simple synthesis of amino-ADT (ADT-NH₂) and provide conditions for successful ADT-NH₂ derivatization with the drugs naproxen and valproic acid. Using UV-vis spectroscopy and HPLC analysis, we demonstrate that amide-functionalized ADT derivatives are significantly more resistant to hydrolysis than ester-functionalized ADT derivatives.

Full text of DOI:10.1055/s-0035-1560603

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© Georg Thieme Verlag Stuttgart · New York
2016, 27, A–E
letter
A
M. D. Hammers et al.
Letter
Synlett
Synthesis of Amino-ADT Provides Access to Hydrolytically Stable
Amide-Coupled Hydrogen Sulfide Releasing Drug Targets
Matthew D. Hammers
Loveprit Singh
Cl
S
hydrolytically
stable
S
1. Pd(OAc)2, RuPhos
Cs2CO3, 4:1 THF–H2O
80 °C, 14 h
Boc
N
Leticia A. Montoya
Alan D. Moghaddam
Michael D. Pluth*
NSAID-CO2H
coupling agent
S
S
H
S
+
S
O
2. S8, DMA
BF3K
180 °C,18 h
Drug
N
H2N
H
ADT-NH2
H2S donor
Department of Chemistry and Biochemistry,
Institute of Molecular Biology, Materials Science
Institute, University of Oregon, Eugene, OR
97403-1253, USA
pluth@uoregon.edu
Received: 05.01.2016
Accepted: 20.01.2016
Published online: 04.02.2016
DOI: 10.1055/s-0035-1560603; Art ID: st-2016-r0003-l
rats, both anti-inflammatory effects.8 In models of septic
shock, however, inflammatory responses are observed in
conjunction with elevated levels of H2S,9 and inhibition of
enzymatic H2S production during endotoxemia mitigates
both inflammation and organ injury.10 Furthermore, en-
hanced levels of KATP activation have been linked with LPS-
induced models of hypotension and hyporeactivity.11 These
observations suggest that the interactions of H2S with KATP
channels during inflammation may be circumstantially ei-
ther beneficial or deleterious. In addition to inflammation,
signaling actions of H2S have significant effects on angio-
genesis. H2S promotes endothelial cell growth through up-
regulation of vascular endothelial growth factor (VEGF) ex-
pression,12 and topical H2S administration to burn wounds
has been shown to stimulate wound healing on rats.13 More
recently, H2S treatment was shown to enhance angiogene-
sis after ischemic stroke though cooperation of astrocytes
and endothelial cells.14 Taken together, these broad-reach-
ing roles of H2S highlight the pharmacological potential of
exogenously administered H2S.
Abstract As additional physiological functions of hydrogen sulfide
(H2S) are discovered, developing practical methods for exogenous H2S
delivery is important. In particular, nonsteroidal anti-inflammatory
drugs (NSAIDs) functionalized with H2S-releasing anethole dithiolethi-
one (ADT-OH) through ester bonds are being investigated for their
combined anti-inflammatory and antioxidant potential. The chemical
robustness of the connection between drug and H2S-delivery compo-
nents, however, is a key and controllable linkage in these compounds.
Because esters are susceptible to hydrolysis, particularly under acidic
conditions such as stomach acid in oral drug delivery applications, we
report here a simple synthesis of amino-ADT (ADT-NH2) and provide
conditions for successful ADT-NH2 derivatization with the drugs naprox-
en and valproic acid. Using UV-vis spectroscopy and HPLC analysis, we
demonstrate that amide-functionalized ADT derivatives are significant-
ly more resistant to hydrolysis than ester-functionalized ADT deriva-
tives.
Key words sulfur, dithiolethione, ADT-OH, hydrogen sulfide, H2S do-
nors
Although a variety of H2S-delivery methods exist, differ-
ent results are often observed depending upon which H2S
donor is used,6 and developing new types of synthetic H2S-
donating compounds is an active area of investigation.15 By
contrast to the more commonly utilized inorganic sulfide
sources such as Na2S and NaHS, which deliver an immediate
burst of H2S into an experimental system, small-molecule
donors are designed to more closely resemble continuous,
endogenous H2S release. One of the most extensively stud-
ied H2S donors is 5-(4-hydroxyphenyl)-3H-1,2-dithiol-3-
thione (ADT-OH), which contains the H2S-releasing dithio-
lethione moiety. This phenolic compound is easily func-
tionalized via ester linkages (Scheme 1), and a number of
ester-bound ADT derivatives of nonsteroidal anti-inflam-
matory (NSAIDs) and other drugs have been prepared and
are being investigated for their synergistic anti-inflamma-
Hydrogen sulfide (H2S) has joined carbon monoxide
(CO) and nitric oxide (NO) as the third endogenously pro-
duced gaseous signaling molecule, or gasotransmitter.1 Bio-
synthesized through enzymatic and nonenzymatic path-
ways, cellular H2S regulates critical cardiovascular, immune,
nervous, respiratory, and gastrointestinal system functions
and is implicated in a number of diseases.2 In addition to
central nervous system diseases, such as Down syndrome,3
Alzheimer’s,4 and Parkinson’s disease,5 the role of H2S in in-
flammation and angiogenesis has been of particular inter-
est.6,7 Different studies have shown H2S to exhibit either
anti-inflammatory or pro-inflammatory effects depending
on which model is being examined. For example, H2S-medi-
ated activation of KATP channels limits leukocyte adherence
to vascular endothelium and reduces edema formation in
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–E

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M. D. Hammers et al.
Letter
Synlett
tory and antioxidant potential. For example, an ADT-func-
tionalized diclofenac derivative, S-diclofenac, not only ex-
hibited greater anti-inflammatory effects than the parent
diclofenac in LPS-injected rats, but also displayed reduced
gastrointestinal toxicity.16 Other demonstrations of ester-
functionalized ADT drugs include derivatives of naproxen,17
valproic acid,18 aspirin,19 sildenafil,20 and mesalamine.21
One practical challenge associated with implementing
these ADT derivatives in drug delivery, however, is the po-
tential for hydrolysis of the ester bond linking the two drug
components. A recent report demonstrated that hydrolysis
of a polymeric ester-bound ADT in phosphate-buffered sa-
line occurred with a half-life of approximately 30 min-
utes.22 Therefore, stomach acid would also likely catalyze
hydrolysis and component fragmentation, which would be
particularly problematic in oral drug delivery systems. One
logical solution to this unwanted hydrolysis is to link ADT
to the drugs through amide, rather than ester, bonds.23
To access ADT-NH2, direct conversion of the phenolic
group in ADT-OH to an amine would be preferred. Our at-
tempts using a reported one-pot SNAr–Smiles rearrange-
ment strategy (Scheme 2,A)24 or palladium-catalyzed
Buchwald–Hartwig amination of triflated ADT (Scheme
2,B) were unsuccessful. We reasoned that the presence of
highly metalophilic sulfur atoms in the dithiolethione moi-
ety, combined with the sulfophilicity of palladium, makes
palladium-catalyzed cross-couplings with ADT problemat-
ic. Therefore, we reasoned that ADT-NH2 would need to be
constructed from an aniline precursor and that any metal-
mediated steps must be incorporated prior to installation of
the dithiolethione moiety (Scheme 2,C).
Fortunately, organotrifluoroborate salts have emerged
as excellent substrates for Suzuki–Miyaura reactions due to
their improved air-stability and reduced cost.25 For our pur-
poses, they offer an attractive alternative to expensive vinyl
boronic acid and boronate substrates.26 Utilizing this strate-
gy, we found that ADT-NH2 is accessible in two steps from
commercially available starting materials with 56% overall
yield (Scheme 3,A). Palladium-catalyzed coupling of 4-
chloro-(N-Boc)aniline with potassium trans-1-propenyltri-
fluoroborate installs a propenyl dithiolethione precursor
onto the protected aniline to form compound 1.27 Subse-
quent reaction with elemental sulfur at 180 °C both forms
the dithiolethione and deprotects the aniline to give ADT-
NH2.28,29 Methylated ADT-NMe2 (Scheme 3,D) was synthe-
sized with the same methodology using 4-bromo-N,N-di-
methylaniline as starting material. ADT-OH and ADT-OMe
were prepared using previously reported methods,16 and
we report an updated purification method in the Support-
ing Information. Importantly, dithiolethione formation
from reaction of elemental sulfur with an aryl propenyl
group appears to be general, as similar reaction conditions
may be used to form ADT-NH2, ADT-NMe2, and ADT-OMe
from their respective propenyl starting materials.
S
S
S
S
Drug-COOH
S
S
O
(coupling agent)
ADT-OH
HO
Drug
O
Scheme 1 Esterification of ADT-OH to form H2S-releasing drugs
To better enable access to more robust ADT-functional-
ized NSAIDs, we report here a straightforward synthesis
and purification of 5-(4-aminophenyl)-3H-1,2-dithiol-3-
thione (amino-ADT, ADT-NH2) and compare the hydrolytic
stability between amide- and ester-coupled ADT. We also
provide conditions for coupling of ADT-NH2 with common
drugs, including naproxen and valproic acid and demon-
strate similar cytotoxicities for the ester- and amide-linked
versions of these drug conjugates.
A)
O
S
S
S
S
S
Br
NH2
S
NH2
O
S
S
S
NaOH
HO
O
H2N
S
S
S
B)
[Pd]
[Pd]
S
S
S
TfO
H2N
S
C)
S
S8
X
S
H2N
H2N
H2N
X = halide
Scheme 2 Potential synthetic routes to ADT-NH2
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–E

C
M. D. Hammers et al.
Letter
Synlett
Cl
S
S
A)
Boc
S8
N
Pd(OAc)2, RuPhos
H
S
+
Cs2CO3, 4:1 THF–H2O
80 °C, 14 h
DMA, 180 °C
18 h
Boc
BF3K
N
H
H2N
1
ADT-NH2
S
S
S
S
B)
AcCl, Et3N
S
S
O
Y = O: (ADT-OAc)
NH: (ADT-NAc)
THF, r.t., 22 h
X
Y
X = OH (ADT-OH)
NH2 (ADT-NH2)
S
S
S
O
Y
C)
S
Y = O: (ADT-OVal)
NH: (ADT-NVal)
valproic acid or naproxen
EDC·HCl, DMAP
S
S
THF, r.t., 24 h (X = OH)
Me
Y
CHCl3, 120 °C, 24 h (X = NH2)
X
X = OH (ADT-OH)
NH2 (ADT-NH2)
O
MeO
S
Y = O: (ADT-ORox)
NH: (ADT-NRox)
S
S
S
D)
S
S
S
S
S
MeO
Me2N
ADT-NMe2
ADT-OMe
Scheme 3 Synthesis of ADT-containing compounds including (A) ADT-NH2, (B) ADT-OAc and ADT-NAC, (C) ADT drug conjugates, and (D) ADT-NMe2
and ADT-OMe
Having developed a facile route to access ADT-NH2, our
next objective was to demonstrate effective functionaliza-
tion of the amine. Acetamide (ADT-NAc) and acetate (ADT-
OAc) ADT derivatives were synthesized as model com-
pounds for hydrolysis study, both through reactions with
acetyl chloride (Scheme 3,B). Ester- and amide-coupled
ADT derivatives of naproxen and valproic acid (ADT-ORox,
ADT-OVal, ADT-NRox, and ADT-NVal) were coupled using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC·H-
Cl) and catalytic 4-dimethylaminopyridine (DMAP) as cou-
pling agents (Scheme 3,C). Both ADT-NH2 and ADT-OH are
weak nucleophiles, making substitution reactions challeng-
ing. The dithiolethione group is inductively electron-with-
drawing, as reflected in the increased acidity of ADT-OH
compared with phenol (pKa of 7.86 vs. 10.00), slightly more
so than a nitrile group (pKa of 4-cyanophenol is 7.95).30 Res-
onance contributions from lone-pair electron delocaliza-
tion produce partial zwitterionic character in these com-
pounds, which further decreases their nucleophilicities
(Scheme S1, Supporting Information). We found that ADT-
NH2 is less reactive than ADT-OH, which suggests a higher
degree of electron delocalization. For example, esterifica-
tion of ADT to form esters ADT-ORox and ADT-OVal pro-
ceed at room temperature, whereas 120 °C temperatures
are required for the corresponding amide couplings of ADT-
NRox and ADT-NVal. Nonetheless, ADT-NH2 is readily
linked through amide bonds using these standard method-
ologies, which can likely be extended toward coupling with
other carboxylic acid containing NSAIDs.
To compare the hydrolytic stability of ester- and amide-
bound ADT derivatives in an acidic environment akin to the
stomach, the hydrolysis of model compounds ADT-OAc and
ADT-NAc in 0.10 M HCl was investigated using UV-vis spec-
troscopy and HPLC. After addition of ADT-OAc (20 μM) to a
cuvette containing 0.10 M HCl, a decrease in absorption at
320 nm was observed with concomitant growth of a new
peak at 358 nm, which is consistent with formation of ADT-
OH after ester hydrolysis (Figure 1,A). By contrast, treat-
ment of ADT-NAc (20 μM) resulted in only minimal hydro-
lysis to form ADT-NH2 (λmax = 311 nm, Figure 1,B). Over the
course of the 18 h experiment, 69% of the ester-bound ADT
was hydrolyzed, whereas only 9% of the amide was hydro-
lyzed under identical conditions (Figure 1,C). ADT-OH and
ADT-NH2 fragments were also identified as reaction by-
products using HPLC analysis. (Figure S1, Supporting Infor-
mation). As expected, these results clearly demonstrate the
enhanced chemical stability of amide linkages toward hy-
drolysis in ADT derivatives by contrast to ester linkages. As
a preliminary demonstration of the biological activity of
these amide-functionalized ADT-based donors, we also
tested the cytotoxicity of the amide- and ester-linked ADT
conjugates in HeLa cells using the MTT assay.31 As a whole,
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M. D. Hammers et al.
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Synlett
Figure 1 Hydrolysis of acyl ADT derivatives (20 μM) in 0.10 M HCl at 37 °C. A) UV-vis timecourse of ADT-OAc (blue = initial, black = final) over 18 h
overlaid with the absorbance spectrum of ADT-OH (red); B) time course of ADT-NAc (blue = initial, black = final) over 18 h overlaid with the absorbance
spectrum of ADT-NH2 (red); C) calculated percent hydrolysis of ADT-OAc and ADT-NAc.
the amide and ester ADT derivatives generally displayed
similar toxicity profiles (Figure S2, Supporting Informa-
tion), suggesting that these compounds may find similar bi-
ological efficacies in future investigations.
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In conclusion, amide-coupled ADT derivatives help pro-
vide a solution to challenges associated with undesired
acid-catalyzed hydrolysis of ester-bound anethole dithiole-
thione NSAIDs in oral drug delivery applications. By utiliz-
ing advances in Suzuki–Miyaura cross-coupling using tri-
fluoroborate salts, we successfully accessed the key inter-
mediate 1 toward our target H2S donor, ADT-NH2. We hope
that the proof of concept syntheses of ADT-NVal and ADT-
NRox will assist future investigations toward unlocking the
therapeutic potential of H2S-releasing drugs.
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Acknowledgment
Research reported in this publication was supported by the NIH
(R01GM113030) and the Sloan Foundation. The NMR facilities at the
University of Oregon are supported by NSF/ARRA CHE-0923589. The
Biomolecular Mass Spectrometry Core of the Environmental Health
Sciences Core Center at Oregon State University is supported, in part,
by the NIEHS (P30ES000210) and the NIH.
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P. K. Free Radical Biol. Med. 2007, 42, 706.
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macol. 2010, 159, 1236.
Supporting Information
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Soldato, P.; Sowers, A. L.; Yeh, G. C.; Moody, T. W.; Wink, D. A.;
Ramchandran, R.; Roberts, D. D. Br. J. Pharmacol. 2007, 151, 142.
(19) Giustarini, D.; Del Soldato, P.; Sparatore, A.; Rossi, R. Free Radical
Biol. Med. 2010, 48, 1263.
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1560603.
S
u
p
p
ortioInfgrmoaitn
S
u
p
p
ortiInfogrmoaitn
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G. D.; Shukla, N. Br. J. Pharmacol. 2008, 155, 984.
References and Notes
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(b) Doucet, H. Eur. J. Org. Chem. 2008, 2013.
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(28) Synthesis of ADT-NH2
Compound 1 (100 mg, 0.429 mmol) and sulfur (96 mg, 3.0
mmol) were added to an oven-dried pressure vessel and dis-
solved in DMA (6 mL) under N2, and the reaction was stirred at
180 °C for 18 h. After stirring, the solvent was removed under
vacuum while heated at 40 °C. The crude residue was diluted
with H2O, extracted into EtOAc. The combined organic fractions
were washed with brine and dried with Na2SO4. The compound
was purified using column chromatography (3% MeOH–CH2Cl2)
to afford the pure product as a dark brown solid (66 mg, 68%
yield). 1H NMR (500 MHz, CD2Cl2): δ = 7.51 (d, J = 8.7 Hz, 2 H),
7.36 (s, 1 H), 6.71 (d, J = 8.7 Hz, 2 H), 4.27 (br, 2 H) ppm. 13C{1H}
NMR (125 MHz, CD2Cl2): δ = 215.05, 174.86, 151.60, 133.66,
129.25, 121.63, 115.29 ppm. HRMS: m/z [M + H]+ calcd for
[C9H8NS3]+: 225.9819; found: 225.9823.
(27) Synthesis of 1
In a glovebox, 4-chloro(N-Boc)aniline (350 mg, 1.54 mmol),
potassium trans-1-propenyltrifluoroborate (273 mg, 1.85
mmol), Pd(OAc)2 (35 mg, 0.16 mmol), RuPhos (144 mg, 0.308
mmol), and Cs2CO3 (1.5 g, 4.6 mmol) were added to an oven-
dried three-neck round-bottom flask fitted with a reflux con-
denser. The sealed apparatus was removed from the glovebox,
and a degassed solvent mixture of 4:1 THF–water (10 mL) was
added via syringe; minimal solvent aids with full conversion
and overall yield. The reaction mixture was stirred at 80 °C
under N2 for 14 h, after which the solvent was removed by
rotary evaporation. The residue was dissolved in EtOAc and fil-
tered through Celite. The filtrate was washed with H2O, brine,
and dried with Na2SO4. The crude product was purified using
column chromatography (20–50% hexane–EtOAc gradient) and
further purified by recrystallization from hexanes to afford the
pure product as a light brown solid (318 mg, 88% yield). 1H NMR
(500 MHz, DMSO): δ = 9.31 (s, 1 H), 7.38 (d, J = 8.5 Hz, 2 H), 7.24
(d, J = 8.6 Hz, 2 H), 6.31 (dd, J = 15.8, 1.43 Hz, 1 H), 6.11–6.18 (m,
1 H), 1.81 (dd, J = 6.56, 1.5 Hz, 3 H), 1.47 (s, 9 H) ppm. 13C{1H}
NMR (125 MHz, DMSO): δ = 152.67, 138.26, 131.34, 130.41,
125.94, 123.34, 118.08, 78.95, 28.11, 18.19 ppm. HRMS: m/z [M
+ Na]+ calcd for [NaC14H19NO2]+: 256.1313; found: 256.1309.
(29) (a) We note that although the synthesis of ADT-NH2 is sug-
gested in a patent,29b the precursor to compound 1 in the patent
application is not a known compound, and no preparation is
presented. (b) Wallace, J.; Cirino, G.; Caliendo, G.; Sparatore, A.;
Santagada, V.; Fiorucci, S. US Patent 2007/0197479 A1, 2007.
(30) Chollet, M.; Legouin, B.; Burgot, J.-L. J. Chem. Soc., Perkin Trans. 2
1998, 2227.
(31) Mosmann, T. J. Immunol. Methods 1983, 65, 55.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–E