Investigating the generation of hydrogen sulfide from the phosphonamidodithioate slow-release donor GYY4137

Article abstract of DOI:10.1039/c5md00170f

A combination of NMR spectroscopy, mass spectrometry and chemical synthesis was used to elucidate the two-step hydrolytic decomposition pathway of the slow-release hydrogen sulfide (H₂S) donor GYY4137 and the key decomposition product was also prepared by an independent synthetic route. The (dichloromethane-free) sodium salt of the phosphonamidodithioate GYY4137 was also produced as a pharmaceutically more acceptable salt. In contrast with GYY4137 and its sodium salt, the decomposition product did not generate H₂S or exert cytoprotective or anti-inflammatory effects in oxidatively stressed human Jurkat T-cells and LPS-treated murine RAW264.7 macrophages. The decomposition product represents a useful control compound for determining the biological and pharmacological effects of H₂S generated from GYY4137.

Full text of DOI:10.1039/c5md00170f

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CONCISE ARTICLE
Investigating the generation of hydrogen sulfide from
the phosphonamidodithioate slow-release donor
GYY4137
Cite this: DOI: 10.1039/x0xx00000x
a
b
c
a
Benjamin E. Alexander, Simon J. Coles, Bridget C. Fox, Tahmina F. Khan,
*c
a
a
b
Received 00th January 2012,
Accepted 00th January 2012
Joseph Maliszewski, Alexis Perry, Mateusz B. Pitak, Matthew Whiteman, and
*a
Mark E. Wood
DOI: 10.1039/x0xx00000x
A combination of NMR spectroscopy, mass spectrometry and chemical synthesis was used to
elucidate the twoꢀstep hydrolytic decomposition pathway of the slowꢀrelease hydrogen sulfide
www.rsc.org/
(
H S) donor GYY4137 and the key decomposition product was also prepared by an
2
independent synthetic route. The (dichloromethaneꢀfree) sodium salt of the
phosphonamidodithioate GYY4137 was also produced as a pharmaceutically more
acceptable salt. In contrast with GYY4137 and its sodium salt, the decomposition product
did not generate H S or exert cytoprotective or antiꢀinflammatory effects in oxidatively
2
stressed human Jurkat Tꢀcells and LPSꢀtreated murine RAW264.7 macrophages. The
decomposition product represents a useful control compound for determining the biological
and pharmacological effects of H S generated from GYY4137.
2
1
Introduction
Plasma concentrations as low as 0.4ꢀ0.9
been suggested from the results of fluorimetryꢀbased methods.
ꢀM have however,
1
3
Following on from the discovery of the biological significance The slow and sustained, enzymeꢀmediated release of H S leads
2
of nitric oxide (NO) and carbon monoxide (CO), hydrogen to these low concentrations of the gasotransmitter and it is
sulfide (H S) is the most recent addition to the family of becoming increasingly clear that for any proposed H S donor
2
2
1
ꢀ3
endogenous gasotransmitters. Its clinical importance has been molecules to give physiologically relevant results, it is very
highlighted in animal and human studies, where its involvement important that they are able to mimic this endogenous process.
in diverse processes including blood pressure regulation,
inflammation, diabetes and neuroprotection has been
demonstrated. The amino acids cysteine, homocysteine and
2
4
,5
6
7
8
cystathionine provide the principal biosynthetic origin of H S in
2
mammalian tissues, from pathways catalysed by cystathionineꢀ
9
β
ꢀsynthase (CBS) and cystathionineꢀ
γ
ꢀlyase (CSE) and in brain
1
0
11
homogenates and the macrovascular endothelium of CBS
knockꢀout mice a third, ꢀketoglutarateꢀand cysteine dependent
α
enzyme, 3ꢀmercaptopyruvate sulfurtransferase (MPST), has
also been shown to generate the gasotransmitter. Little is
currently known however, as to the possible involvement of the
latter pathway in human tissue.
Issues connected with the lack of selectivity for different
sulfurꢀcontaining species, associated with the common methods
Fig. 1
2
Slow-release H S donors.
for determination of the level of H S in blood and its In the vast majority of the earlier reported studies, simple
2
production in tissue, has led to much, as yet unresolved, debate sulfide sources such as sodium hydrosulfide (NaSH), sodium
2
,5
in this area. A commonly used spectrometric/HPLC assay sulfide (Na S) and saturated aqueous solutions of H S were
2
2
based ultimately on the generation of methylene blue from H S, used. Although these facilitate the preparation of solutions of
2
1
2
hydrosulfide and aqueous sulfide suggests healthy adult accurately known sulfide concentration, they effectively give an
5
plasma serum levels of H S to be in the order of 20ꢀ60
ꢀ
M.
2
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Jou5Mrnal Name
instantaneous delivery of H S, which is not representative of containing moist air, develop an unmistakeable smell of H2S
2
1
4,15
the conditions within tissues and living cells.
after a few days and the headspace gas in the vials causes a
A number of "slowꢀrelease" H S donors have therefore, rapid blackening of moistened lead(II) acetateꢀimpregnated
2
been introduced and developed, which give a slow and paper. Aqueous solutions of GYY4137
3 which have been
protracted delivery of sulfide, suitable for in vivo allowed to decompose, have been used as a control in
2
,16
14,28
experiments.
hydroxythiobenzamide
dithioleꢀ3ꢀthione (ADTꢀOH)
in this regard, either on their own or in the production of neither the actual hydrolysis pathway nor the products formed
Putative H Sꢀreleasing moities such as 4ꢀ biological assays
and Park et al. have also observed the
2
3
1
1
and 5ꢀ(4ꢀhydroxyphenyl)ꢀ3
Hꢀ1,2ꢀ decomposition using P NMR in acetonitrile containing
(Fig. 1) have proved successful aqueous HEPES buffer but to the best of our knowledge,
2
1
2
1
7,18
modified versions of existing pharmaceutical products.
We have been reported previously. Clearly however, these
have also recently reported the preparation and evaluation of a degradation products have the potential to influence the
1
9,20
mitochondriaꢀtargeted derivative of ADTꢀOH
2
.
The interpretation of any biological results obtained with this H2S
(Fig. donor and hence, we carried out the experiments described
phosphonamidodithioate morpholinium salt GYY4137
3
1
) has however, become one of the most widely used slowꢀ herein, primarily in order to resolve this issue and to produce
1
5
release H S donor for biological studies. Other structurally useful control compounds in a pure state.
related derivatives have also been investigated
2
2
1ꢀ23
but despite
the widespread use of these compounds, surprisingly little has
been reported concerning their mode and rate of H S release.
2
Also, the nature of the degradation products arising from these
compounds after H S release have, thus far, only been the
2
16
subject of speculation and prediction,
although their
identification is crucial in unravelling the complex
physiological and pharmacological effects of these donors.
2
Results and Discussion
2
.1 Synthesis of, and hydrolysis pathway for, GYY4137
GYY4137
treatment of
Lawesson's reagent
3
a
is precipitated in crystalline form, by the
dichloromethane solution/suspension of
4
with 4 equiv. of morpholine (Scheme
The product is obtained as a dichloromethane complex,
generally showing a ca 2ꢀ3 : 1 stoichiometry of GYY4137 to
1
5,23
1
).
3
dichloromethane and commercial samples (e.g. SigmaAldrich)
also show this composition. An Xꢀray crystal structure of a
Fig. 2 ORTEP representation of the X-ray crystal structure of GYY4137 3.
Atomic displacement ellipsoids - 50% probability level.
dichloromethaneꢀfree
sample
recrystallised
from
chloroform/petroleum ether confirmed the morpholinium salt
2
1
31
In line with the results of Park et al.
spectrum of GYY4137
,
the P NMR
†
structure of
3 (Fig. 2).
3 showed a single peak at 90.4 ppm in
d6ꢀacetone and 89.0 ppm in CDCl3 and such values are
consistent with the chemical shifts observed for structurally
2
9
similar arylphosphonamidodithioates. In order to follow the
course of the hydrolysis of GYY4137 and ultimately, to
determine the phosphorusꢀcontaining species produced, a ca
3
1
3
1
M solution was prepared in CDCl and examined by P NMR
3
on a daily basis, relying on adventitious water in the solvent to
§
effect reaction. As observed by Park et al. when using buffered
2
1
d3ꢀacetonitrile as solvent, after 24 h, a clear additional singlet
was observed at 64.9 ppm, whose intensity increased steadily
1
with time. The aromatic proton region of the H NMR spectrum
also exhibited signals consistent with the formation of a new
, 0 °C compound containing a 1,4ꢀdisubstituted benzene ring and
integration of distinct doubleꢀdoublets corresponding to
2 2
Scheme 1 Synthesis of GYY4137. Conditions: Morpholine (4 equiv.), CH Cl
to RT (45-70%). (x = 0.3 to 0.5)
GYY4137
3 and this putative hydrolysis product, suggested a
Although samples of GYY4137
room temperature in air, the fact that it releases H S on contact
with water is in no doubt. Samples stored in sealed vials,
3 can be stored normally at
conversion of 10% after 24 h and 14% after 48 h. 50%
conversion was achieved in approximately 13 days, with
essentially complete loss of the P signal corresponding to
2
31
2
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A
00
R
1
T
7
I
0
C
F
LE
GYY4137
3 after 71 days. (Note: The solution volume in the D O) and a molecular ion of m/z = 272.0524, corresponding to
2
NMR sample tube was kept constant by regular addition of the expected molecular formula of the anionic component of
additional CDCl , which also ensured the provision of the C H NO PS.
3
11 15
3
required small quantities to water to maintain the steady
hydrolysis.)
A solution of
GYY4137 , was carefully layered on top of a similar volume
in
as solvent, again relying on hydrolysis by adventitious water. A D O, with care being taken to avoid initial mixing of the two
5 in d ꢀacetone, produced by hydrolysis of
6
3
Essentially identical results were obtained using d ꢀacetone of an approximately equimolar solution of potassium salt
6
6
2
3
1
reaction sample from this solvent showing ca 50% conversion solutions. A P NMR spectrum of this sample of layered
1
by H NMR was analysed by mass spectrometry under negative solutions revealed two closelyꢀspaced singlets at 62.6 and 63.2
ion electrospray conditions and was found to contain two clear ppm, which coalesced into a single resonance at 65.4 ppm after
molecular ion peaks. The first at m/z = 288.0281 corresponded thorough mixing of the two solutions, confirming the presence
to the anionic component of GYY4137
3 with the expected of the same phosphorusꢀcontaining species in both of the
molecular formula of C H NO PS and an identical mass original solutions, as suggested from mass spectrometry.
1
1
15
2
2
spectrum was obtained for a pure sample of
3. The second
molecular ion was observed at m/z = 272.0514, consistent with
the replacement of one of the sulfur atoms in the starting
material
3 with oxygen, producing a new species with
molecular formula C H NO PS. This suggested the formation
1
1
15
3
of compound
GYY4137
consistent with the formation of H S by straightforward sulfurꢀ
5
(Scheme 2) as the initial hydrolysis product of
3
and importantly, its formation would be fully
2
3
1
oxygen exchange. The P chemical shift of 64.9 ppm observed
for this compound was also consistent with the proposed
2
9
arylphosphonamidothioate structure
5
.
A sample analysed
after 71 days of hydrolysis in moist CDCl showed only the
3
molecular ion corresponding to this new product
same mass spectrometry conditions.
5 under the
Scheme 3 Preparation of authentic 4-methoxyphenylphosphonamidothioate
Reagents and conditions: (i) SO Cl (3.3 equiv.), CCl , 0 °C, 1 h then RT, 1 h (72%
crude), (ii) morpholine (0.9 equiv.), Et N (0.9 equiv.), CH Cl , RT, 1 h (83% based
on morpholine), (iii) KOSiMe (2.9 equiv.), Et O, RT, 7 days (94%).
6.
2
2
4
3
2
2
3
2
1
3
These results, in conjunction with C NMR spectra,
therefore confirmed unambiguously, the structure of the
proposed, initial hydrolysis product
reaction of GYY4137 with water.
5, resulting from the
3
In both of the chloroform and acetoneꢀbased hydrolysis
solutions described above, a further, minor peak appeared in the
Scheme 2 Two-step hydrolytic degradation of GYY4137
each structure is morpholinium.)
3
. (Note: Counterion for
31
P NMR spectrum after 7 days at 15.8 (CDCl ) and 13.1 ppm
3
(
d ꢀacetone). This showed a slight but steady increase in
6
3
1
relative intensity in comparison with the
P
signal
In order to confirm this structural assignment, we prepared
an authentic sample of the potassium salt of the anionic
component of by a modification of a previously reported
method for the synthesis of this class of compound (Scheme
corresponding to
5 but even after 71 days, this additional
6
product was clearly still a very minor component of the reaction
mixture. A negative ion electrospray mass spectrum of the
5
crude product of hydrolysis of GYY4137
acetone) revealed, in addition to the peaks corresponding to
and a trace of , a third molecular ion at m/z = 187.0166,
consistent with the molecular formula C H O P. This strongly
3
after 90 days (in d6ꢀ
3
0
3
). Treatment of Lawesson's reagent
4
with sulfuryl chloride
5
3
1
gave phosphonothioic dichloride
7
(72% crude yield), which
3
provided the corresponding morpholinophosphinothioic
chloride on reaction with morpholine, in the presence of
triethylamine (83% yield).
Reaction of with 2.9 equiv. of potassium
trimethylsilanolate in diethyl ether gave a precipitate of pure
potassium salt in 94% yield after 7 days. Salt showed a
7
8
4
8
suggests the occurrence of a much slower, extensive hydrolysis
of the arylphosphonamidothioate to the phosphonate (with
concurrent loss of morpholine and a second equiv. of H S)
5
9
8
3
2
2
3
1
(
Scheme 2), the P chemical shift observed for this product
6
6
being in reasonable agreement with that reported for 4ꢀ
3
1
single peak at 64.5 ppm in its P NMR spectrum (recorded in
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Jou5Mrnal Name
methoxyphenylphosphonic acid. (
δ
= 15.06 ppm in d6ꢀ
H S generation from 3, 6 and 11 was determined using 5,5'ꢀ
2
3
3
15
DMSO).
dithiobisꢀ(2ꢀnitrobenzoic acid) (Fig. 3[A]) and resulted in the
and 11
. Intracellular H2S
unlikely to make any significant contribution to biological was also determined using the sulfideꢀspecific probe 3'ꢀ
The very slow rate of formation of any appreciable formation of 2.1 ± 0.6 µM and 2.6 ± 0.3 µM H S from
3
2
quantities of
9 during the hydrolysis process suggests that it is respectively. H S was not detected from 6
2
experiments that use GYY4137
The first hydrolysis product
capability to act as a very slowꢀrelease H S donor in its own human Jurkat Tꢀcells. In both assays,
3 as a slowꢀrelease H S donor. methoxyꢀ3ꢀoxoꢀ3Hꢀspiro[isobenzofuranꢀ1,9'ꢀxanthen]ꢀ6'ꢀyl 2ꢀ
2
1
9,34
5
however, clearly has the (pyridinꢀ2ꢀyldisulfanyl)benzoate (WSPꢀ1)
(Fig. 3[B]) in
failed to generate
6 represent potentially detectable levels of H S when used at the same concentration as
6
2
right and hence it and potassium salt
important and useful control compounds for such studies.
2
3
, whereas H S production was detected from GYY4137
3
and
outside (Fig. 3[A]) and inside (Fig. 3[B]) of cells. These data
may be useful as a control compound for and
The hydrolysis experiments revealed the fact that the 11 and for determining whether the biological and
phosphorusꢀbound morpholine is released extremely slowly pharmacological effects of these compounds are due to H2S
from GYY4137 and is therefore, unlikely to pose any (and/or intermediates that may be formed from them under
problems in the use of this compound. The morpholinium physiological conditions) from those of the hydrolysis products,
2
1
1
2
.2 Preparation of the sodium salt equivalent of GYY4137
suggest that
6
3
3
counterion however, represents a potential complication to such as
6
.
results obtained using this H S donor and therefore, in order to
2
eliminate this issue, we also investigated the preparation of a
more pharmaceutically acceptable salt. Removal of the
dichloromethane from the salt was also a priority and
maintaining samples of GYY4137
3 under high vacuum for
long periods of time unfortunately has little, if any, effect on its
composition.
Treatment of a cold, aqueous solution of GYY4137
2ꢀfold excess of glacial acetic acid produced a white, powdery
precipitate of the phosphinodithioic acid 10 in high (84%) yield
Scheme 4). As expected, 10 proved to be only sparingly
3 with a
1
(
soluble in water.
Fig. 3
2 2
Generation of H S from 3, 6 and 11. [A] H S generation in the
absence of cells using 5,5'-dithiobis-(2-nitrobenzoic acid) and [B] intracellular
generation of H S in human Jurkat cells using WSP-1. Data are mean ± S. D. of six
or more determinations. *p < 0.05 c.f. oxidant treatment.
2
‡
2
.4 Effects of compounds 3, 6 and 11 on oxidative stress-induced
cytotoxicity
We have shown previously that GYY4137 protected human
cells in culture from oxidative stressꢀinduced toxicity induced
by 4ꢀhydroxynonenal and SINꢀ1 but the contribution of the
Scheme 4 Synthesis
of
sodium
4-
methoxyphenyl(morpholino)phosphinodithioate 11. Reagents and Conditions: (i)
CH CO H (12 equiv.), H O, 0-5 °C (84%). (ii) NaH (1 equiv.), Et O, 0 °C to RT (98%).
,
3
2
2
2
Addition of sodium hydride to a suspension/solution of 10
35
hydrolysis product
order to determine whether
compound, we exposed human Jurkat Tꢀcells to 4ꢀ
6
had not been evaluated. Therefore in
in diethyl ether resulted in substantial degradation of the
material but portionwise addition of the acid 10 to 1 equiv. of
sodium hydride, ensuring that the base was always in excess,
gave an excellent (98%) yield of the corresponding (highly
waterꢀsoluble) sodium salt 11 (Scheme 4). H NMR spectra
revealed the acid 10 (and therefore, the salt 11) to be
6
would be suitable as a control
34
35
hydroxynonenal (4ꢀHNE) (20 µM) and SINꢀ1 (100 µM)
the presence of and 10 and determined cellular viability
after 24 h. Fig. 4 shows that 200 µM and 11 both significantly
inhibited oxidative stress induced cell death. In contrast, no
significant inhibition of cell death was observed with
suggesting that the cytoprotective effects of and 11 were due
to H S. Control experiments showed that , or 11 alone did
,
in
3, 6
1
3
-
dichloromethaneꢀfree.
6
,
3
3, 6
2
.3 H S release from GYY4137 and related compounds
2
2
4
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not induce significant cytotoxicity or induce significant cellular potassium salt 6. This salt has the potential to be used as a control
proliferation (Trypan blue assay; data not shown).
compound in future evaluation of H2S donors. The more
pharmaceutically acceptable sodium salt equivalent 11 of GYY4137
3
was prepared and characterised and the rates of H S generation
2
Fig. 4 Inhibition of oxidative-stress induced cytotoxicity by
human Jurkat T-cells. Cells were incubated with
3, 6 and 11 in
or 11 (200 µM) for 1 h and
3, 6
either SIN-1 (100 µM) or 4-HNE (20 µM). Cell viability was assessed by Trypan
Blue assay after 24 h. Data are mean ± S. D. of six or more determinations. *p <
‡
0.05 c.f. oxidant treatment.
Fig. 5
Inhibition of lipolysaccharide (LPS)-induced nitric oxide and
prostaglandin E
incubated with
for 24 h. After this time, cell culture media was collected and assayed for [A]
2
synthesis by
3, 6 and 11 in murine RAW264.7 cells. Cells were
, 6 or 11 (200 µM) for 1 h prior to the addition of LPS (1 µg/ml)
2
.5 Effects of compounds 3, 6 and 11 on inflammation in vitro:
3
Nitric oxide (NO) and prostaglandin E (PGE ) synthesis
2
2
-
2 2
NO (as an index of nitric oxide generation) and [B] PGE by commercial ELISA.
Data are mean ± S. D. of six or more determinations. *p < 0.05 c.f. oxidant
Bacterial lipopolysaccharide (LPS) is
a wellꢀknown
‡
inducer of proꢀinflammatory enzyme and inducible nitric oxide treatment.
(
(
NO) synthase and cyclooxygenaseꢀ2 in a number of cell types
such as macrophages), leading to the synthesis of NO and from
3
,
6
and 11 were monitored using colourimetric and
and 11
reported to inhibit LPSꢀinduced NO and PGE synthesis, it is proved to be equally effective in this regard, whereas
PGE respectively. Although GYY4137 has previously been fluorescence assays. Phosphonamidodithioate salts
3
2
2
not clear whether these effects were due to H S (from generation of the gasotransmitter from hydrolysis product
6
is
2
GYY4137) or from products of GYY4137 hydrolysis such as too low to be significant when compared with the original
1
4
6
.
To investigate this, we incubated murine RAW264.7 donors (Fig. 3). Furthermore,
macrophages with LPS (1 µg/ml) in the presence of and 11 induced cell death (Fig. 4) and LPSꢀinduced NO and PGE2
and analysed cell culture media for nitrite by Griess assay (as generation (Fig. 5) suggesting
may be useful as a control
an index of NO synthesis) and PGE by commercial ELISA. In compound in studying the biological effects of
and 11 in vitro
6 failed to prevent oxidantꢀ
3, 6
6
2
3
these studies, significant inhibition of NO (Fig. 5[A]) and PGE2 and in vivo
Fig. 5[B]) synthesis were observed with both and 11 but not
, suggesting H S from
and 11 was responsible for the Acknowledgements
inhibition of NO and PGE synthesis, rather than hydrolysis We wish to thank the EPSRC UK National Mass Spectrometry
.
(
3
6
3
2
2
products such as
6
.
Facility at Swansea University for mass spectrometric studies
and Dr Stephen J. Simpson (Exeter, UK) for assistance with the
recording of NMR spectra.
3
Conclusions
In summary, we have elucidated the hydrolysis pathway by
which the commonly used slowꢀrelease phosphonamidodithioate Notes and references
a
donor GYY4137 3 produces H S. This is twoꢀstep process, the first
Biosciences, College of Life and Environmental Sciences, University of
2
of which involves straightforward sulfurꢀoxygen exchange with Exeter, Geoffrey Pope Building, Stocker Road, Exeter, EX4 4QD, UK. Eꢀ
water to give an arylphosphonamidothioate 5 and the second, slower mail: m.e.wood@exeter.ac.uk; Tel: +44 (0)1392 723450.
b
step results in complete hydrolysis to an arylphosphonate 9. On the
EPSRC UK National Crystallography Service, Chemistry, University of
normal timescale of biological experiments which employ GYY4137 Southampton, Highfield, Southampton, SO17 1BJ, UK.
c
3
. as a source of H S, the first hydrolysis product 5 is clearly the
University of Exeter Medical School, St. Luke's Campus, Magdelen
2
most significant byꢀproduct and its structure was confirmed by an Road, Exeter, EX1 2LU, UK. Eꢀmail: m.whiteman@exeter.ac.uk; Tel:
independent synthesis of the arylphosphonamidothioate moiety as its +44 (0)1392 722942.
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Jou al Name
5Mrn
†
XꢀRay crystallographic data of 3 were collected on a Bruker APEXII 13 E. A. Wintner, T. L. Deckwerth, W. Langston, A. Bengtson, D.
CCD diffractometer mounted at the window of a Bruker FR591 rotating
anode (MoKα, λ = 0.71073 Å) and equipped with an Oxford Cryosystems
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Tombs and C. Szabo, Br. J. Pharmacol., 2010, 160, 941.
2
4
Cryostream device. Data were processed using the COLLECT package
14 M. Whiteman, L. Li, P. Rose, C. H. Tan, D. Parkinson and P. K.
Moore, Antioxid. Redox Signalling, 2010, 12, 1147.
and unit cell parameters were refined against all data. An empirical
2
5
absorption correction was carried out using SADABS. crystal structure 15 L. Li, M. Whiteman, Y. Y. Guan, K. L. Neo, Y. Cheng, S. W. Lee, Y.
2
was solved by direct methods and refined on F
o
by fullꢀmatrix leastꢀ
Zhao, R. Baskar, C. H. Tan and P. K. Moore, Circulation, 2008, 117,
2
6
squares refinements using SHELXꢀ97 program suite. Graphics were
2531.
2
7
generated using OLEX2. The corresponding CIF has been deposited 16 G. Caliendo, G. Cirino, V. Santagada and J. L. Wallace, J. Med.
with the Cambridge has been deposited with the Cambridge
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