Synthesis of nitric oxide donors derived from piloty’s acid and study of their effects on dopamine secretion from PC12 cells

Article abstract of DOI:10.3390/ph10030074

This study investigated the mechanisms and kinetics of nitric oxide (NO) generation by derivatives of Piloty’s acid (NO-donors) under physiological conditions. In order to qualitatively and quantitatively measure NO release, electron paramagnetic resonance (EPR) was carried out with NO spin trapping. In addition, voltammetric techniques, including cyclic voltammetry and constant potential amperometry, were used to confirm NO release from Piloty’s acid and its derivatives. The resulting data showed that Piloty’s acid derivatives are able to release NO under physiological conditions. In particular, electron-withdrawing substituents favoured NO generation, while electron-donor groups reduced NO generation. In vitro microdialysis, performed on PC12 cell cultures, was used to evaluate the dynamical secretion of dopamine induced by the Piloty’s acid derivatives. Although all the studied molecules were able to induce DA secretion from PC12, only those with a slow release of NO have not determined an autoxidation of DA itself. These results confirm that the time-course of NO-donors decomposition and the amount of NO released play a key role in dopamine secretion and auto-oxidation. This information could drive the synthesis or the selection of compounds to use as potential drugs for the therapy of Parkinson’s disease (PD).

Full text of DOI:10.3390/ph10030074

pharmaceuticals
Article
Synthesis of Nitric Oxide Donors Derived from
Piloty’s Acid and Study of Their Effects on Dopamine
Secretion from PC12 Cells
1
2
ID
1
3
2,
Daniele Sanna , Gaia Rocchitta
, Maria Serra , Marcello Abbondio , Pier Andrea Serra *,
2
4
4,
ID
5,
ID
Rossana Migheli , Lidia De Luca , Eugenio Garribba *
and Andrea Porcheddu *
1
Istituto CNR di Chimica Biomolecolare, Trav. La Crucca 3, 07040 Sassari, Italy;
Daniele.Sanna@icb.cnr.it (D.S.); Maria.Serra@icb.cnr.it (M.S.)
Department of Clinical and Experimental Medicine, Medical School, University of Sassari,
viale San Pietro 43/b, 07100 Sassari, Italy; grocchitta@uniss.it (G.R.); rmigheli@uniss.it (R.M.)
Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy; mabbondio@uniss.it
Department of Chemistry and Pharmacy, University of Sassari, via Vienna 2, 07100 Sassari, Italy;
ldeluca@uniss.it
2
3
4
5
Department of Chemical and Geological Sciences, University of Cagliari, S.S. 554, bivio per Sestu,
0
9042 Monserrato, Italy
*
Correspondence: paserra@uniss.it (P.A.S.); garribba@uniss.it (E.G.); porcheddu@unica.it (A.P.);
Tel.: +39-079-228-558 (P.A.S.); +39-079-229-487 (E.G.); +39-070-675-4410 (A.P.)
Received: 11 June 2017; Accepted: 30 August 2017; Published: 5 September 2017
Abstract: This study investigated the mechanisms and kinetics of nitric oxide (NO) generation by
derivatives of Piloty’s acid (NO-donors) under physiological conditions. In order to qualitatively
and quantitatively measure NO release, electron paramagnetic resonance (EPR) was carried out
with NO spin trapping. In addition, voltammetric techniques, including cyclic voltammetry
and constant potential amperometry, were used to confirm NO release from Piloty’s acid and its
derivatives. The resulting data showed that Piloty’s acid derivatives are able to release NO under
physiological conditions. In particular, electron-withdrawing substituents favoured NO generation,
while electron-donor groups reduced NO generation. In vitro microdialysis, performed on PC12
cell cultures, was used to evaluate the dynamical secretion of dopamine induced by the Piloty’s
acid derivatives. Although all the studied molecules were able to induce DA secretion from PC12,
only those with a slow release of NO have not determined an autoxidation of DA itself. These results
confirm that the time-course of NO-donors decomposition and the amount of NO released play a
key role in dopamine secretion and auto-oxidation. This information could drive the synthesis or the
selection of compounds to use as potential drugs for the therapy of Parkinson’s disease (PD).
Keywords: Piloty’s acid; nitric oxide; Parkinson’s disease; in vitro microdialysis
1
. Introduction
Almost 20 years ago, the first reports concerning the endogenous generation of nitric oxide (NO)
in mammalian systems appeared [
1–3]. Since these initial reports, the physiology, biochemistry and
pharmacology of NO and related compounds have received growing attention [
4
]. NO is a valuable
lead compound of a family of well-established signalling molecules. It is bio-synthesized via oxidation
of the amino group of L-arginine, yielding NO and citrulline, the reaction is catalysed by nitric oxide
synthases. Three NO synthases (NOS) are known: endothelial (eNOS), neuronal (nNOS), and inducible
(
iNOS) NOS [5]. Both nNOS and eNOS are constitutive isoforms, which generate low concentrations of
NO. In contrast, iNOS can be induced by various cytokines or lipopolysaccharides and produces large
amounts of NO over long time periods, often resulting in harmful effects. In the higher animals, NO
Pharmaceuticals 2017, 10, 74; doi:10.3390/ph10030074
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induces vasodilatation, neurotransmission and vessel homeostasis and is involved in the regulation
of respiration, digestion, sexual function, memory, sleep and cardiovascular function. Furthermore,
NO has also been correlated with several diseases, such as stroke, diabetes, cancer and cardiovascular
disease [
6
]. In recent years, it has been demonstrated that NO can be a fundamental actor in the central
] and tissue damage/protection [8,9]
nervous system (CNS), particularly in intracellular signalling [
7
.
Regulation of striatal dopamine (DA) neurotransmission is one of the most important roles for
endogenous NO in the CNS [10]. Several animal model studies have demonstrated that NO-donors
can increase extracellular levels of DA [11–13]. It has been previously shown that NO-mediated DA
release could depend on the source of NO, whether neuronal, glial, extracellular, exogenous [
8,9], and
the timing of NO generation [14].
From a strictly chemical point of view, NO is a simple hydrophobic gaseous molecule that is
highly diffusible and reactive. The following forms are important for biological action. NO free radical
is a highly versatile species with a low ionization potential that allows it to be easily oxidized to
+
−
nitrosonium cation (NO ), and quickly reduced to nitroxyl anion (NO ) [15]. Among them, NO is
by far the most stable and the one which has aroused tremendous biological interest and extensive
studies. It is known from the literature [8] that the nitrosonium ion can give rise to two main types
+
−
of compound. The first is ionic salts such as NO BF4 , while good example of the second type of
compound found are the nitroxyl halides. It deserves to be emphasized that both species are quickly
+
hydrolyzed forming nitrous acid (HNO ) and NO can be detected in aqueous solution only at very
2
low pH. Numerous reagents may also reduce NO radical and the degree of reduction may be more or
2
−
less effective with a wide range of products (N O, N O
2
−
, NO , etc.). Simple one-electron reduction
of NO affords the nitroxyl anion, NO . The average reducing potential of this redox process is about
0.25 V. The endogenous environment in which NO is generated may also regulate its biological
actions [16 19]. Despite a true understanding of the physiological role played by NO in vivo is an
2
2
−
+
–
important milestone, this task is far from being trivial [16,17]. Nowadays, while the chemistry of NO
is well-established, the exact biochemistry of NO is somewhat blurred [18]. To make matter worse,
real-time quantitative measurements of NO from NOS in biological media are analytically challenging
and time consuming as result of the labile nature of NO [20]. Even the choice of the most proper
analytical method for determining NO in biological matrices is not entirely off-set [21]. In this respect,
the application and comparison of different analytical techniques are needed to gain better insights
into the physiology and pathology of NO [22–26].
Direct administration of NO to mimic nNOS-mediated NO production for in vitro studies is
not possible due to its instability in aqueous media and/or when oxygen is present. Therefore,
chemical reagents that can continuously release NO under physiological conditions (NO-donors)
are increasingly important. Generally, NO-donors have different structures, which release NO at
different rates and through three different kinds of mechanisms. The first route is the one that releases
NO spontaneously via a thermal or photochemical self-decomposition process. In the second route
NO is released by a chemical reaction with acids, alkalis, metals and thiols. The third route is an
enzymatic oxidation process. Some NO donors can generate NO by following different pathways. The
most widely used NO-donors include sodium nitroprusside (SNP) [27], 3-morpholinosydnonimine
(
SIN-1) [28], S-nitroso-N-acetylpenicillamine (SNAP) [27] and sodium trioxodinitrate (Angeli’s salt) [29]
and glycerin trinitrate (GTN) [27]. First reported in 1896 by the German chemist, Oskar Piloty,
benzenesulfohydroxamic acid, commonly referred to as Piloty’s acid (PI), has been found to release
NO under certain condition [30]. From a structural point of view, Piloty’s acid is an N-substituted
hydroxylamine derivative (PhSO NHOH) bearing a good leaving group (a benzenesulfinate unit)
2
on nitrogen moiety (HONH-X, Figure 1). Depending on the nature of the X group and experimental
conditions (pH value, temperature, presence of oxidizing or reducing agents) Piloty’s acid can release
HNO, NO or both [31
–33]. Generally, nitroxyl (HNO) is generated from Piloty’s acid by spontaneous
decomposition in strongly basic (pH 13) solution and under anaerobic conditions [34].

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Figure 1. Piloty’s acid (PI) molecular structure.
Briefly, the initial deprotonation at the nitrogen atom is followed by the heterolytic S-N bond
cleavage to afford HNO and benzenesulfinate anion (Scheme 1). Since pK
a
of HNO is approximately
−
11.4, it is reasonable to believe that nitroxyl anion (NO ) will be the predominant species capables of
existing under these conditions [35].
OH
O
S
O
O
S
O
NH
- H+
NH
kmax(25 °C): 4.22 x 10-4 s-1
S
HNO
+
O
O
pKa = 9.3
O
anaerobic conditions
benzenesulfinate
anion
Piloty’s acid
X-NHOH)
(
Scheme 1. Mechanism proposed for the spontaneous decomposition of Piloty’s acid (PI) in strongly
basic (pH 13) solution and under anaerobic conditions.
−
3
−1 ◦
at 37 C [36],
At pH 13, Piloty’s acid decomposes with the first-order rate constant of 1.8
×
10
s
1
/2
whereas the rate of HNO release in aqueous solutions at neutral pH significantly decreases (t
about
◦
8
0 h, at 25 C) making its use most effective above pH 8.0 [31
,
37] The results revealed that the half-life
of Piloty’s acid dramatically decreases with increasing pH values: 561 min at pH 8, 90 min at pH 9,
and 33 min at pH 10 [38 39]. The exclusion of oxygen is important since its presence causes oxidative
,
decomposition of Piloty’s acid (non-HNO pathway), which ultimately release nitric oxide (NO) rather
than HNO and becomes an NO donor [40]. In 1994, Grzesiok et al. observed that under physiological
conditions, Piloty’s acid is oxidized to a radical that spontaneously decomposes to give NO along with
the sulfinate leaving group (Scheme 2) [41]. This may explain the vasodilator and antiplatelet activity
of PI [42], as well as its potential anti-inflammatory activity. Under physiological conditions, however
Piloty’s acid derivatives can decompose though more than one route and it is very difficult entirely
rule out the presence/absence of NO or HNO [43].
O
S
O
S
Spontaneous
decomposition
O
Physiological
conditions
NH
NH
S
+ N
OH
O
OH
O
O
O
Scheme 2.
Mechanism proposed for the decomposition of Piloty’s acid (PI) under
physiological conditions.
This can rise to critical confusion in some experiments since HNO and NO shares similar effects,
although they follow different cellular signalling pathways. Recently, in a very interesting paper,
Miyata et al., prepared a series of Piloty’s acids derivatives and tested their HNO-releasing activity
under physiological conditions by GC-MS detection of nitrous oxide (N O), the dimerization product
2
of HNO (derived from the spontaneously dimerization of HNO) [44]. The results of this study

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confirmed the importance of initial Toscano’s findings [35
,37] and established that the presence of
electron-withdrawing groups at the ortho-position together with appropriate bulky alkyl residues at the
o-position promote HNO release at physiological pH. Among all Piloty’s acid derivatives evaluated,
N-Hydroxy-2-nitro-benzenesulfonamide and 2-bromo-N-hydroxybenzenesulfonamide have proven
the most efficient HNO donors at pH = 7 releasing HNO in aqueous solution without co-production of
NO [33]. A recent similar study conducted by Doctorovich et al., (2013) highlighted that, contrary to PI,
N-hydroxybenzenesulfonamides with electron-attracting or withdrawing substituents in para-position
on the aromatic ring behave preferentially as HNO donors operating at physiological pH and under
strictly anaerobic condition [45]. On the contrary, a study published in the same year by Miyata et al.,
pointed out that Piloty’s acid derivatives having electron-withdrawing groups on the aromatic ring at
the para-position released only a negligible amount of HNO by decomposition under physiological
oxidative conditions [44]. Therefore, some of these molecules might be ideal candidates for therapeutic
treatments responsive to NO and more in-depth investigations were necessary to further explore
those knowledge gaps [46–49]. In this study, we were intrigued by the possibility of using some
derivatives of the commercial available Piloty’s acid (PI) as potential source of solely NO. In this regard,
N-hydroxy-4-nitrobenzenesulfonamide (4-NO -PI) and N-hydroxy-4-methoxybenzene sulfonamide
2
(4-OMe-PI) have been synthesized and investigated in depth to exploit them as NO-donors under
oxidative stress (Figure 2).
O
O
S
O
S
H
H
H
S
N
O N
N
CH O
N
2
3
OH
OH
OH
O
O
O
PI
4-NO -PI
4-OMe-PI
2
Figure 2. Structure of N-hydroxy-benzenesulfonamide (PI), N-hydroxy-4-nitrobenzenesulfonamide
4-NO -PI) and N-hydroxy-4-methoxybenzenesulfonamide (4-OMe-PI).
(
2
Thus, to contribute to the elucidation of the decomposition mechanism of some select Piloty’s
molecular structures [50], the generation of NO species from these derivatives was investigated using
electron paramagnetic resonance (EPR) spectroscopy in association with cyclic voltammetry (CV)
and constant-potential amperometry (CPA). As NO is a paramagnetic free radical, EPR spectroscopy
is the most appropriate tool for its direct detection. The main advantage of EPR with respect to
other techniques is that it only detects paramagnetic molecules, and so NO can be measured without
the interference of other diamagnetic species; the disadvantage is that the release of HNO or other
derivatives cannot be revealed [51]. Because of the high reactivity of NO, spin-trapping techniques
have been developed to form relatively stable free radicals [52]. Spin trapping using Fe2+ complexes
with dithiocarbamates has been demonstrated an excellent procedure to reveal selectively NO [53].
Since the HNO eventually produced can be oxidized to NO and this process can be favoured by the
coordination with the spin trapper, EPR alone may not be able to clearly discriminate NO formed
directly by the decomposition of PI from that derived by the oxidation of HNO. Fortunately, a number
1
of studies reported in the literature on the reactivity of HNO with molecular oxygen seems to exclude
the possibility that the generation of NO species may takes place by this route since this reaction
3
−1 −1
is rather slow (k = 10 M
s ) [52
,53], mainly due to their different spin states. The combined
application of EPR and other techniques can help the interpretation of the experimental data. The
electrochemical sensors used in this study are able to detect NO with a high grade of sensitivity and
selectivity. In fact, while no scientific papers are present in literature about the HNO interference in NO
electrochemical detection assessed by poly-OPD/nafion amperometric sensors, Suárez and coworkers
developed specific sensors for the detection of HNO [54]. The mentioned authors showed that only in
the presence of a cobalt porphyrin it is possible to perform the amperometric detection of HNO.

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In this work, we have studied, in detail, the time profile of NO generation and the mechanism by
◦
which these two molecules generate NO under physiological conditions (pH 7.4, 37 C). The results
for 4-NO -PI and 4-OMe-PI were consistent with those of PI and another benchmark NO-donor
2
(S-nitroso-N-acetylpenicillamine, SNAP) [27]. Finally, an in vitro study of the neurochemical effects of
the new PI derivatives was also carried out. In particular, the capability of PI derivatives to induce the
dynamical secretion of dopamine on PC12 cell cultures was evaluated.
2
. Results
2
.1. Chemicals
Exploiting the propensity of sulfohydroxamic acids to release NO [55–59], we have designed an
environmentally friendly, multigram scale synthesis of Piloty’s acid analogues through the reaction of
commercially available sulfonyl chlorides with hydroxylamine hydrochloride in a basic medium.
A careful survey of the literature identified several factors that limit the scope of classical
sulfonylation reactions with NH OH [31
,
58]. In fact, the sulfonylation of weakly nucleophilic amines,
such as hydroxylamine, is difficult and sulfohydroxamic acids are often obtained in low yields after
several elaborate and tedious purification steps [60 61]. On the other hand, the use of activated
2
,
sulfohydroxamic acids with better leaving groups than chlorine does not improve the yield due to the
high reactivity of sulfonyl chlorides. Additionally, direct sulfonylation of hydroxylamine also gives
rise to a mixture of N- and O-sulfonylated side products (Scheme 3) [62–67].
O
O
O
S
O
S
O
S
O
S
OH
H
O
S
S
R
O
S
Base
O
H
S
R
.
R
Cl + HCl NH2OH
R
N
+
O
R
+
+
R
N
O
R
R
N
O
H N
2
O
O
O
O
O
O
O
R = Alkyl, Aryl
Side products
Scheme 3. Direct sulfonylation of hydroxylamine under basic condition.
In an attempt to address these problems, we have exploited the utility of MgO as a base for the
coupling of less reactive hydroxylamines with a set of commercially available sulfonyl chlorides. In the
presence of MgO, the sulfonyl chloride group selectively reacts with the more nucleophilic amino
group (the N in NH OH is a better electron donor than O) preventing the formation of side products.
2
We have observed that the sulfonylation reaction proceeds best with two equivalents of hydroxylamine
hydrochloride and three equivalents of magnesium oxide in MeOH/H O/THF (3:2:30). The reaction,
2
as monitored by TLC, was complete in only two hours at room temperature. This procedure does not
require any aqueous workup and it is potentially scalable, economical and environmentally friendly.
N-sulfohydroxamic acids are isolated at high yield and purity after a simple filtration [68,69].
2
.2. EPR Characterization of NO-Donors
NO is a diatomic free radical species, hence of paramagnetic nature; it follows that, electron
paramagnetic resonance spectroscopy (EPR) is believed the best technique for the direct monitoring of
NO in biological matrices [50 52]. The main advantage of EPR in comparison with other NO detection
,
techniques is that it only detects paramagnetic molecules irrespective of the optical appearance of
the sample [70] EPR results show that all the compounds studied release NO. Formation of NO is
indicated by the three-lines spectrum, due to the coupling between the unpaired electron and 14
N
nucleus (Figure S1 of Supplementary Materials). This indicates the trapping of NO by Fe(MGD) to
2
give the species Fe(MGD)2(NO) where MGD is N-methyl-D-glucamine dithiocarbamate. With elapsing
time, the intensity of the signal due to Fe(MGD) (NO) significantly increases, which is correlated with
2

Pharmaceuticals 2017, 10, 74
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the concentration of NO released by the specific NO-donor (Figure 3). Therefore, EPR spectroscopy
can be used to measure the amount and the rate of release of NO by the four compounds studied.
Figure 3. Increase of normalized intensity of EPR signal (measured in arbitrary units) due to
◦
Fe(MGD) (NO) as a function of the time in the experiments with PI (0.6 mM in PBS, pH 7.4, 37 C). All
2
spectra were recorded with the same instrumental parameters.
The decomposition of Piloty’s acid and its derivatives is strongly dependent on temperature.
◦
In particular, as expected, decomposition under physiological conditions (37 C) is much faster than at
◦
2
5 C (see Figure S2 of Supplementary Materials).
In Figure 4 the intensity of NO released by the four NO-donors and trapped by Fe(MGD)2
is reported. It is evident that the 4-nitro-derivative (4-NO -PI) releases NO faster and at higher
2
concentrations than Piloty’s acid (PI), while the 4-methoxy substituent (4-MeO-PI) induces a slower
release of NO and at lower concentrations. This different chemical behaviour can be attributed to
the characteristics of the aromatic ring substituents. The -NO group is electron withdrawing and
2
increases the rate and the amount of released NO by stabilizing the radical species involved in the
decomposition of PI (see Scheme 2) [42]. In contrast, the methoxy substituent is an electron releasing
group and disfavours NO release in comparison with the Piloty’s acid parent compound. This trend is
confirmed by N-hydroxy-4-fluorobenzenesulfonamide which, containing an electron withdrawing
substituent, behaves similarly to the nitro derivative [45].
As pointed out in the Introduction, with EPR spectroscopy the HNO eventually formed in the
decomposition of PI derivatives cannot be determined. In addition, it is possible to quantify only
the total amount of NO formed, but not its origin; in other words, we cannot demonstrate by EPR
measurements if all NO revealed comes from the decomposition of PI derivatives or from the oxidation
of HNO induced by its coordination to the Fe-based spin trapper.

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Figure 4. Normalized EPR intensity of the species Fe(MGD) (NO) (measured in arbitrary
2
units) as a function of time for: S-nitroso-N-acetylpenicillamine (SNAP), N-hydroxy-4-methoxy
benzenesulfonamide (4-MeO-PI), Piloty’s acid (PI) and N-hydroxy-4-nitrobenzenesulfonamide
(
4-NO -PI). The concentration of all NO-donors was 0.6 mM. The intensity, obtained after double
2
◦
integration of the spectra, was measured in PBS at 37 C and pH 7.4. All spectra were recorded with
the same instrumental parameters.
2
.3. Electrochemical Characterization of NO-Donors
Figure 5 shows the cyclic voltammograms (CVs) of PBS (dashed line) and NO-donors (continuous
lines) at bare epoxy carbon electrodes. All NO-donor molecules resulted in a clear increase of the
anodic current around +865 mV. This increase is due to the oxidation of released NO as previously
described [71
,72]. The absence of a definite peak for NO oxidation could be due to the composition of
the transducer material.

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Figure 5. Cyclic voltammograms of SNAP (
were carried out in fresh PBS/DMEM (10%), in the dark, at 37 C. All voltammograms were recorded
A
), PI (
B
), 4-OMe-PI (
C
) and 4-NO -PI (
D
). All experiments
2
◦
−
1
in the potential range
compare the current from PBS (dashed line) with that of PBS with 1 mM of the respective NO-donor
continuous line).
±
1.0 V vs. Ag/AgCl, using a scan rate of 250 mV s . Voltammograms shown
(
2
.4. In Vitro Calibration of NO Microsensor
As shown in Figure 6, constant potential amperometry (CPA) carried out in PBS/DMEM (10%)
−
1
2
with SNAP showed a sensitivity of 0.180
±
0.005 nA
µ
M
and R = 0.997. PI, 4-OMe-PI and
0.004, 0.079 0.003 and
, respectively, with R values of 0.987, 0.992 and 0.933, respectively, as shown
4
0
-NO -PI demonstrated similar results, yielding sensitivities of 0.086
±
±
2
−
µM
1
2
.096 ± 0.005 nA
in Table 1.
The LOD and LOQ values were determined as defined in Section 3.9. Sensors showed a LOD
of 38 7 nM and a LOQ of 125 9 nM. Amperometric experiments carried out on Piloty’s acid and
±
±
its derivatives, under the same conditions as the microdialysis experiments, confirmed that NO is
generated as a result of decomposition. Moreover, these molecules are able to liberate NO in the
short-term period based on an analysis of the slope values, that reflect the amount of NO released.
This finding is in agreement with the EPR experiments. Interestingly, 4-NO -PI was found to release
2
more NO than the other substances tested here.
Surprisingly, the slope of the SNAP CPA was greater than the other substances tested here. This is
likely due to the environment where SNAP decomposes. It has been widely demonstrated in the
literature that reductive conditions favour the degradation of S-nitrosothiols, such as SNAP [73,74].
In fact, PBS/DMEM (10%) provided a sufficiently reducing environment for the decomposition of
SNAP [71].

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Figure 6. CPAs of SNAP (
in fresh PBS, in the dark, at 37 C. Currents were recorded at a fixed potential of +865 mV vs. Ag/AgCl.
A
), PI (
B
), 4-OMe-PI (
C
) and 4-NO -PI (
D
). All experiments were carried out
2
◦
The concentration range was from 0 to 100 µM for all NO-donors.
2
Table 1. In Vitro NO calibration parameters (slope
±
SEM and R ) obtained from NO-donor molecules
on OPD/Nafion® (The Chemours Company FC, LLC) modified epoxy carbon electrodes .
a
NO-Donor
Slope (nA µM−1) ± SEM
R2
−1
NO Released (µM mM
donor
) b
SNAP
PI
0.180 ± 0.005
0.086 ± 0.004
0.079 ± 0.003
0.096 ± 0.005
0.997
0.987
0.992
0.993
14.0
7.7
6.9
4
4
-MeO-PI
-NO -PI
8.4
2
a
Data were obtained by applying a constant potential of +865 mV, in the dark at 37 C. b The NO released
◦
was calculated on the basis of decomposition of SNAP in CuCl, given a 90% NO yield (see ref. [41]) for 1 mM
of NO-donor.
2
.5. PC12 Cell Viability
A previous study [71] demonstrated that the effects of NO-donor drugs on DA secretion from
PC12 cells in vitro were dependent on the timing of NO generation and the extracellular environment
in which NO was generated. The study was performed using in vitro microdialysis of PC12 cell
suspensions. This is a suitable in vitro experimental model for studying neurodegenerative diseases
which involve a deficiency of dopamine, since PC12 cells are able to synthesize, secrete and metabolize
DA. In addition, endogenous NO production in PC12 cells is mediated by neuronal NO synthase
(nNOS) which positively modulates DA secretion [27].
PC12 cells were exposed to varying concentrations of SNAP, PI, 4-MeO-PI and 4-NO -PI at
2
concentrations that did not affect cell viability. As shown in Figure 7, all NO-donors induced a
concentration-dependent decrease in cell viability starting from 1.5 mM concentration (p < 0.05),

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as compared to vehicle, with the exception of 4-MeO-PI. From 2.5 mM, further significant differences
were observed, where 4-MeO-PI and 4-NO -PI were less toxic than SNAP and PI.
2
Figure 7. Effects of SNAP, PI, 4-MeO-PI and 4-NO -PI on PC12 cell viability. PC12 cells were incubated
2
3
2
in drug-free medium. At the start of each experiment, 100
later (time 0) with different concentrations of SNAP, PI, 4-MeO-PI and 4-NO -PI (0.1, 0.5, 1, 1.5, 2 mM).
×
10 cells/cm were plated and treated 24 h
2
After 24 h, the number of viable cells was determined in triplicate wells. Cell viability was established
by the MTT assay. Results are given as percent of control PC12 cells and represent the mean
of three experiments. * p < 0.05 compared with controls; # p < 0.05 compared with corresponding
±
SEM
ethanol concentrations.
The studies of PC12 viability demonstrated that Piloty’s acid and its derivatives are toxic to cells
only at concentrations above 1.0 mM. Furthermore, at higher concentrations, only Piloty’s acid was
particularly toxic, showing that nitro- and methoxy-derivatives of PI could be suitable for use in
biological models.
In view of these results, Piloty’s acid and its derivatives were used for microdialysis experiments,
in order to evaluate their effect on dopamine release from PC12 cell cultures. The cell culture results
were comparable with the results from EPR experiments.
2
.6. Effect of NO-Donor Molecules on Dialysate Levels of DA
All NO-donors were infused at 1.0 mM for 60 min (n = 4 cell suspensions). Data were obtained
from the outlet not perfused with the NO-donor. As shown in Figure 8, a 60 min SNAP infusion
induced a progressive and significant increase in DA concentration (1.6-fold increase). Under the same
conditions, 60 min infusions of PI and 4-MeO-PI induced a statistically significant increase in DA levels
(
6.2-fold and 3.3-fold, respectively). Conversely, 60 min infusion of 4-NO -PI resulted in a significant
2
decrease in DA concentration (70% decrease as compared to basal levels).

Pharmaceuticals 2017, 10, 74
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Figure 8. Effect of SNAP (A), PI (B), 4-MeO-PI (C) and 4-NO -PI (D) perfusion on DA concentrations
2
in dialysates from PC12 cells suspension. All NO-donors (1.0 mM, n = 3 cell suspensions) were infused
for 60 min (open bar, 80–120 min). Dialysates were collected, at 20-min intervals before, during and
after NO-donor infusion. The collected dialysates were analysed by HPLC. Values are the mean
S.E.M. * p < 0.05 compared with baseline values.
±
Cell viability was assessed by trypan blue exclusion before filling the capillary apparatus and at
the end of each experiment. Non-viable cells were 15.3 1.7%, 13.9 1.6% 3.4 0.7% and 4.4 0.8%
in the SNAP, PI, 4-NO -PI and 4-MeO-PI experiments respectively.
±
±
±
±
2
As previously demonstrated [71], DA concentrations in the dialysates depended largely on both
the timing of NO generation and the extracellular environment in which NO is generated. NO is an
unstable, but highly diffusible, simple molecule. In aqueous solution, NO undergoes autoxidation by a
third-order rate equation [75]. As previously shown, both the amount and timing of NO generation
allowed NO to enter the cells and reach a concentration suitable for activation of the sGC/cyclic GMP
pathway [76].
The presence of an electron withdrawing substituent, as in 4-NO -PI, resulted in a rapid and
2
consistent release of NO. Infusion of 4-NO -PI resulted in a significant decrease in DA, of more
2
than 70% compared to basal levels. Most likely, this decrease reflects that a greater amount of NO
diffuses across the cell membrane than either undergoes extracellular autoxidation [74] and/or induces
extracellular DA nitration [77,78], with a consequent decrease in dialysate DA concentration.
Conversely, in agreement with EPR results, the presence of an electron-donor group, as in
4-MeO-PI, resulted in a slow release of NO. Infusion of 4-MeO-PI resulted in a significant and
long-lasting increase in dialysate DA levels, confirming that the timing, and in some ways the amount,
of NO generation influences DA release from cells.

Pharmaceuticals 2017, 10, 74
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Piloty’s acid had a similar effect to 4-MeO-PI, but resulted in a greater increase in dialysate DA
levels. This greater increase could reflect the balance between DA release, due to NO passage through
the cell membrane, and that arising from cell death due to the toxicity of PI.
From the microdialysis experiments, it is possible infer the toxicity of the molecules tested as
the DA dialysate content results from both release due to NO stimulation, as well as lysis due to cell
death. DA release measured with perfusion of SNAP and 4-NO -PI increased and did not return to
2
basal levels. This could be due to the toxicity of these compounds, as demonstrated by the trypan blue
exclusion results. MTT data at 24 h showed that SNAP is toxic, while 4-NO -PI seems to be less toxic
2
over the same time frame.
In relation to DA release, Piloty’s acid and 4-MeO-PI were less toxic in the short term as DA in the
dialysates approached baseline values. The higher amount of DA release with Piloty’s acid perfusion
could be due to the relatively higher toxicity observed, even in long-time viability experiments.
These results can be applied to the development of new NO-donor drugs for neurodegenerative
pathologies, involving a deficiency of dopamine and where an inflammatory component is overt,
such as Parkinson’s disease. Therapies for such diseases should be able to slowly release NO at
concentrations that allow entry of NO into the cells resulting in DA release, but that reduce DA
auto-oxidation or cell death.
3
. Materials and Methods
3
.1. Chemicals
Starting materials, reagents, and dry solvent were purchased from Sigma-Aldrich (Milano, Italy)
and used without further purification. Insulated copper wires were purchased from Advent Research
Materials, Suffolk, U.K. Thin layer chromatography (TLC) was performed on Merck Kieselgel 60 TLC
plates. 1H NMR and C NMR spectra were recorded in the solvents indicated at 300 MHz and
13
75 MHz, respectively, unless otherwise noted. Chemical shifts are reported in parts per million
(
ppm,
δ), were measured from Tetramethylsilane (0.0 ppm) and are referenced to the solvent CDCl
3
1
13
(7.26 ppm), DMSO-d (2.49 ppm), for H NMR, and CDCl (77.0 ppm), DMSO-d (39.5 ppm) for
C
6
3
6
NMR. Data are reported as follows: chemical shifts, multiplicity (s = singlet, d= doublet, t = triplet,
q = quartet, br = broad, brs = broad singlet, m = multiplet, p = pseudo), coupling constants J (Hz),
relative integration value. High resolution mass spectra (HRMS) were obtained using electron-impact
(
EI) or electrospray ionization (ESI). All newly synthesized compounds gave satisfactory elemental
analysis. HPLC analysis was performed on a Waters Alliance HT 2795 Separation Module with a
Waters 2996 Photodiode Array Detector using an Xterra MS C-18 column (5 M, 4.6 150 mm)
µ
×
eluting with aqueous acetonitrile (v/v 1/1) containing 0.05% CF3COOH at a flow rate of 1 mL/min.
Melting points were recorded in open capillary tubes using a Buchi apparatus and the values reported
are uncorrected.
3
.2. Organic Synthesis
3
.2.1. Synthesis of N-Methyl-D-glucamine Dithiocarbamate (MGD)
MGD was synthesized according to an established procedure [79,80]. A solution of sodium
hydroxide 2.5 N was prepared by dissolving NaOH pellets (20 g, 0.5 mol) in distilled water (200 mL)
◦
at 0 C. The reaction vessel containing sodium hydroxide solution was cooled with an ice/salt bath,
and then N-methyl-D-glucamine (NMG) (97.6 g, 0.5 mol) was added dropwise to the alkaline solution
◦
◦
and dissolved with stirring. The solution was warmed from
−
5 C to 5 C. A separate solution of
◦
carbon disulfide (CS 150 mL, 0.83 mol) in absolute ethanol (50 mL) was prepared and cooled to 0 C.
2,
The cold solution of CS was slowly added into the previously prepared cold solution of NMG in
2
NaOH with stirring over a period of about 15 min. The temperature of the reaction mixture was kept
◦
◦
between 0 C and 5 C during the carbon disulfide addition to minimize the formation of coloured side

Pharmaceuticals 2017, 10, 74
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products. After addition of the CS solution, cooling and stirring of the reaction mixture was continued
2
for an additional 20 min. Also, after addition of the CS solution, MeOH (500 mL) was added and
2
the vessel covered to prevent solvent evaporation. The mixture was allowed to stand overnight to
facilitate the formation of larger crystals. The MGD precipitate was collected by vacuum filtration. The
filter cake was washed with solutions of ethanol with progressively higher ethanol contents, as follows:
first, with 70% ethanol (500 mL), then with of 95% ethanol (1000 mL), and then with absolute ethanol
(500 mL). To dry the collected precipitate, the vacuum was maintained for 2 h, followed by air drying
to assure desiccation. The filtrate and washings were refrigerated to obtain a second crop of crystals.
The first crop of precipitate air dried to a white powder with a yield of 107.4 g (69%). The second
crop of precipitate was slightly yellow in colour with a yield of 17.1 g (11%). Analysis of this powder
◦
indicated that it was a monohydrate form. The melting point was 231–232 C, which is consistent with
◦
the melting point of commercially available MGD (232–233 C). The experimental elemental analysis
results (C, 30.74; H, 5.90; N, 4.54) were consistent with the expected values (C, 30.86; H, 5.82; N, 4.49)
+
for C H NO S Na
8
18
6 2
3
.2.2. Synthesis of N-Hydroxy-4-methoxybenzenesulfonamide
Hydroxylamine hydrochloride (0.72 g, 10 mmol) in MeOH/H O (3:2, 5 mL) was treated with
2
MgO (0.34 g, 8.6 mmol), followed by a solution of p-Methoxybenzenesulfonyl chloride (0.89 g,
.3 mmol) in THF (30 mL), and then MgO (0.17 g, 4.3 mmol). The reaction was vigorously stirred
at room temperature until the sulfonyl chloride had completely disappeared, as determined by TLC
EtOAc–hexane, 1:1; 2 h). Then, the mixture was filtered through a pad of Celite first, and then on a
4
(
short plug of silica gel. The clear filtrate was dried over MgSO and evaporated to dryness to give the
4
resulting N-hydroxy-4-methoxybenzenesulfonamide (0.81 g, 93%) as a crystalline white solid (99%
◦
purity). The melting point of the isolated solid was 123–127 C, which is consistent with the literature
◦
1
value (126–128 C) [81]. H NMR results are shown in Figure S3 of the Supplementary Materials
DMSO-d6, = 9.52 (d, J = 3.0 Hz, 1H), 9.40 (d, J = 3.0 Hz, 1H), 7.78 (d, J = 9.0 Hz, 2H), 7.13 (d, J = 9.0 Hz
H), 3.82 (s, 3H)). 13C NMR results are shown in Figure S4 of the Supplementary Materials (CDCl3,
=163.5, 131.0, 129.6, 114.8, 56.4). IR (thin film) analysis gave the following results: 3440, 2107,
(
δ
,
2
δ
ν
−
1
+
+
1
640, 1491, 1452, 1321, 1250, 1094, 1015 cm . HRMS (ES ) experimental results (MNa : 226.0144)
+
were consistent with the calculated expected results for C H NO SNa (226.0144). The experimental
7
9
4
elemental analysis results (C, 41.40; H, 4.52; N, 6.95) were consistent with the expected results for
C H NO S (C, 41.37; H, 4.46; N, 6.89).
7
9
4
3
.2.3. Synthesis of N-Hydroxy-4-nitrobenzenesulfonamide
N-hydroxy-4-nitrobenzenesulfonamide was prepared as previously reported [68]. A crystalline
◦
yellow solid was obtained at 97% yield (98% purity), with a melting point of 158–160 C, which
◦
1
is consistent with that reported in the literature (154–155 C) [82]. H NMR results are shown in
Figure S5 of the Supplementary Materials (CDCl , = 9.69 (s, 1H), 9.52 (s, 1H), 8.37 (d, J = 8.95, 2H),
.16 (d, J = 8.93 Hz, 2H)). 13C NMR results are shown in Figure S6 of the Supplementary Materials
CDCl3, = 149.4, 142.56 129.2, 122.9). The experimental elemental analysis results (C, 33.10; H, 2.86; N,
2.75) were consistent with the expected results for C H N O S (C, 33.03; H, 2.77; N, 12.84). HRMS
δ
3
8
(
δ
1
(
6
6
2
5
+
+
ES ) experimental results (MNa : 240.9887) were consistent with the calculated expected results for
+
C H N O SNa (240.9890).
6
6
2
5
3
.3. Electron Paramagnetic Spectroscopy
3
.3.1. Theory Background and Spin Trapping
EPR (Electron Paramagnetic Resonance) is a resonance spectroscopy, which can be applied to
paramagnetic species (i.e., chemical species with unpaired electrons), such as NO [83]. In the absence
of any hyperfine coupling with other nuclei, only a transition (recorded as the first derivative of the

Pharmaceuticals 2017, 10, 74
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absorption curve) is expected. Instead, when an unpaired electron couples with a nucleus with angular
momentum of nuclear spin I, (2I + 1) signals with identical intensity are expected. Although NO is
a paramagnetic species, it cannot be detected directly using EPR spectroscopy because of its high
reactivity and very short lifetime in aqueous solution. Therefore, spin trapping must be used. The spin
trap is a chemical species that reacts with the short-lived NO radical to form a relatively stable free
radical. One of the first spin traps for NO was deoxyhemoglobin [84]. Subsequently, Mordvintcev et
al., proposed the use of Fe2 complexes with diethyldithiocarbamate [85], but this approach is limited
+
2
+
by their poor solubility in water. Komarov et al., improved the technique by using a species of Fe
with N-methyl-D-glucamine dithiocarbamate (Fe(MGD) ), which is soluble in water [86]. In this work,
2
we used Fe(MGD) as the spin trapping agent. NO released by the decomposition of the NO-donors is
2
trapped by Fe(MGD) , forming iron-nitroxyl species (Fe(MGD) (NO)) (Figure 9). This reaction results
2
2
in a stable form of NO that is easily detectable as a three-line EPR signal, arising from the hyperfine
coupling between the unpaired electron and 14N nucleus (I = 1). The intensity ratio between the three
14
resonances is 1:1:1 and the value of the hyperfine splitting constant, a( N) is 12.5 G (Figure S1 of
Supplementary Materials).
O
N
R
S
S
S
S
R'
R
Fe
R'
Figure 9. Structure of the complex Fe(MGD) (NO).
2
The intensity of the EPR signal due to the trapped NO increases as a function of time as the
concentration of the species Fe(MGD) (NO) increases (see Figure 4). Therefore, EPR spectroscopy can
2
be used to measure the amount and the rate of NO release from a series of NO-donors. It must be
observed that under the experimental conditions used in this study PI derivatives can release also
HNO as discussed in the Introduction [44,45].
3
.3.2. Experimental
EPR spectra were recorded in aqueous solutions with an X-band (9.40 GHz) Bruker EMX
spectrometer. A solution of Fe(MGD) was prepared in PBS, by dissolving MGD (0.045 M) and
2
FeSO ion (FeSO , 0.015 M). Solutions containing Piloty’s acid were prepared in DMSO or H O using a
4
4
2
ratio 1:1 with Fe(MGD) . Different concentrations of PI and Fe(MGD) were used (0.12, 0.20, 0.50, 0.60,
2
2
0
.90 and 1.20 mM). Approximately 1 mL of solution was kept with a Pasteur pipette, and subsequently
inserted in the cavity for EPR analysis. The acquisition time between spectra was 4 min and the
total duration of the experiments was between 1 and 10 h. Experiments were carried out at room
◦
◦
temperature (25 C) or physiological temperature (37 C). An identical procedure was followed for the
PI derivatives, 4-NO -PI and 4-OMe-PI. The results were compared with those for SNAP in H O. The
2
2
instrumental EPR parameters were as follows: modulation frequency 100 kHz, modulation amplitude
3
4
Gauss, sweep time 168 s, microwave power 20 mW; this microwave power, using the Bruker ER
119HS resonator, is below the saturation level. The spectrum in Figure S1 was simulated with the
computer program WinEPR SimFonia 1.25 (Bruker Analytische Messtechnik GmbH) with the following
14
parameters a( N) 12.5 G, g 2.0428, linewidth 4 G, Lorentzian/Gaussian 0.10. The intensity of EPR
signals (Figures 2 and S2) was estimated after double integration of the spectra.

Pharmaceuticals 2017, 10, 74
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3
.4. NO Microsensors
3
.4.1. Preparation of NO Microsensors
NO microsensors (Figure 10) were made using insulated silver wires (50 mm in length) using a
modified approach of previously described procedures [87 88]. Approximately 3.0 mm of silver wire
was exposed at both ends of the wire and one end was inserted into a silica capillary tube (20 mm in
length; i.d. 180 m, Polymicro Technologies, Phoenix, AZ, USA). A 180 m diameter carbon-composite
disk electrode (area: 2.5 10 cm ) was then fabricated by mixing 840 mg of graphite with 500 mg
of Araldite-M and 200 mg of hardener and filling the silica capillary tubing with the mixture. After
,
µ
µ
−
4
2
×
◦
®
2
4 h at 40 C, the sensor surface was polished using a high speed drill (Dremel 300, Dremel Europe,
Breda, The Nederlands) equipped with an aluminum oxide grinding wheel [60]. The surface of the
fabricated sensors was then polished with alumina. These bare electrodes were used for a preliminary
electrochemical characterization of NO-donors by cyclic voltammetry.
Figure 10. Scheme of NO microsensor. The active surface is a 180
The disk surface is then modified with p-OPD and Nafion to ensure selectivity of the sensors for NO.
µ
m diameter epoxy-carbon disk.
®
The sensors were then modified with O-phenylenediamine (OPD) and Nafion® using a
combination of previously described protocols [26,89–93] in order to make them selective for NO and
to avoid electrochemical responses from the most common physiological, electroactive molecules.
After fabrication, the electrodes were characterized and only electrodes with NO detection limits
<50 nM and selectivity against ascorbic acid (>1000:1), dopamine (>250:1), and nitrite (>800:1) were
used [59].
3
.4.2. Microsensor Characterization of NO Donors
Cyclic voltammetry and constant potential amperometry, were used in addition to EPR
experiments, in order to confirm the generation of NO by the compounds under study. As previously
described [88], a three-electrode electrochemical cell was used for both electropolymerisation of OPD
and electrochemical measurements of NO release. All amperometric experiments were carried out
by connecting epoxy-carbon microsensors, as working electrodes (WE), Ag/AgCl (NaCl, 3 M) as a
reference electrode (RE) and a high surface area stainless steel electrode as an auxiliary electrode (AE),
to a four-channel equipment (eDAQ QuadStat, e-Corder 410, eDAQ, 6 Doig Ave, Denistone East NSW
2
112, Australia).
Cyclic voltammetry (CV) was carried out in order to investigate the redox properties of the newly
synthetized NO-donors in a buffered aqueous solution (PBS) at physiological conditions (pH 7.4,

Pharmaceuticals 2017, 10, 74
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◦
3
7 C) and for confirming the oxidation of released nitric oxide. Voltammograms were recorded in
−
1
the potential range
±
1.0 V using a scan rate of 250 mV s . Bare epoxy carbon electrodes were used
during acquisition of CVs to obtain a complete electrochemical profile of the NO-donors. SNAP was
used as reference molecule for comparison with the other NO-donor molecules studied here. SNAP
CVs were obtained in the dark at 37 C in fresh PBS over a concentration range between 0.5 and
◦
2
.5 mM (Figure 3). NO-donor solutions were freshly prepared immediately before the experiments by
dissolving the compounds in DMSO to obtain 100 mM solutions. These stock solutions were injected
into the experimental cells to obtain final concentrations between 0.5 and 2.5 mM. For amperometric
calibrations, OPD/Nafion modified electrodes were used.
SNAP was also used as a reference compound for constant potential amperometry (CPA) as the
decomposition kinetics and yield of NO are well characterized. SNAP was used as a standard for
microsensor calibration by measuring the relative NO release in a CuCl saturated solution in the dark
by applying a constant potential of +865 mV vs. Ag/AgCl and adding known aliquots of SNAP stock
solution for final concentrations from 0 to 2.5 µM [91].
CPAs for all NO-donor molecules were carried out in fresh PBS/DMEM 10% (see Section 3.7)
◦
and under physiological conditions (pH = 7.4 and 37 C) in order to assess the yield of released NO
compared to SNAP. A constant potential of +865 mV vs. Ag/AgCl was applied and, after a stable
baseline was reached, known amounts of NO-donor stock solutions were added to the PBS in order to
obtain final concentrations between 0 and 100 µM.
3
.5. Cell Culture and Capillary Tube Construction for In Vitro Microdialysis
◦
PC12 cells were maintained at 37 C in 60-mm plastic culture plates in Dulbecco’s modified
Eagle’s medium (DMEM)/F12 supplemented with 10% horse serum and 5% fetal calf serum, in a
humidified atmosphere of 5% CO /95% air [94]. After short-term culture (15–20 passages), the cells
2
were washed twice in 2 mL of phosphate buffered saline PBS/DMEM 10% containing glucose (10 mM),
NaCl (137 mM), CaCl (1.17 mM) and MgCl (1.0 mM), harvested by trituration and centrifuged (94 g
2
2
for 5 min). Cells were re-suspended in PBS/DMEM and the number of cells/50 µL was assessed using
trypan blue in a Burker’s chamber. The initial volume of the cell suspension was adjusted to reach a
3
final concentration of 900 × 10 cells/50 µL.
The capillary tube for cell culture suspension microdialysis has been described in detail in a
previous study [27 95]. Briefly, the probe was made using two sections of plastic-coated silica tubing
,94
,
(
o.d. 150 m, i.d. 75 µm, Scientific Glass Engineering, Milton Keynes, UK), each placed in the center of
µ
a semi-permeable, polyacrylonitrile dialysis fiber (AN-69; Hospal Industrie, Meyzieu CEDEX, France,
5 mm length). Each semi-permeable membrane had an active length of 40 mm. Thereafter, each
section of plastic-coated silica tubing was positioned in the center of a section of polythene tubing (i.d.
.58 mm; Portex, Hythe, Kent, UK, 35 mm length). The two sections of silica tubing served as inlets for
4
0
connections with the dialysis pump. Dialysates from each polyacrylonitrile dialysis fiber were collected
from the sections of polythene tubing, which served as outlets. A third section of plastic-coated silica
tubing (40 mm length) was sealed outside the polythene tubings to permit the aspiration of PC12
cell suspension into the microdialysis probe. All probe components were coated with quick drying
epoxy glue. Thereafter, the microdialysis apparatus (semi-permeable polyacrylonitrile dialysis fibers
positioned side-by-side plus sealed plastic coated silica tubing) was placed in an un-heparinized
micro-hematocrit capillary tube (7.5 mm length, 1.1 mm i.d.; Chase Scientific Glass Inc., Rockwood, TN,
USA), having a volume of 70 µL. The side-by-side positioning of the dialysis fibers permits separate
co-infusion of drugs. In addition, the two distinct outlets permit separate dialysate sample collection
from the same PC12 cell suspension. Separate sample collection is useful when, for example, one or
more drugs which may have either pro-oxidant or antioxidant properties are infused at the same time.

Pharmaceuticals 2017, 10, 74
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3
.6. Microdialysis Procedure
The microdialysis procedure has been described in detail previously [94
,
95]. Briefly, the cellular
microdialysis probe was perfused with PBS/DMEM 10% by means of a peristaltic microinfusion
double channel pump (P720 peristaltic pump; Instech, Plymouth Meeting, PA, USA) which pumped
◦
PBS/DMEM from a 60 mm plastic culture plate in an IR incubator at 37 C in a humidified atmosphere
of 5% CO /95% air at a flow rate of 3.0
µL/min. The pump channels were connected to the probe
2
inlets by a length of polythene tubing. The perfusion apparatus was then filled with 50 L of the PC12
µ
cell suspension by manual aspiration, using a 1.0 mL syringe connected to the plastic coated silica
◦
tubing sealed outside the polythene tubings. Thereafter, the perfusion apparatus was kept at 37 C.
Samples were collected manually in 250
from both outlets. Then, a 30 L aliquot of the dialysate, recovered from the probe outlet not perfused
with the NO-donor was analysed by HPLC. The first sample was collected after 60 min of stabilization
time 0). After this, dialysates were collected at 20-min intervals, for 40 min prior to the infusion of
µL micro-centrifuge tubes (Alpha Laboratories, Eastleigh, UK)
µ
(
NO-donors. The concentration of dopamine detected after the first 20 min of perfusion was taken as
the concentration at time 0. No significant decrease in cell viability was observed at the end of the
experiments, as previously observed [27,94,95]. Cell viability was assessed before filling the capillary
apparatus and at the end of each experiment by trypan blue exclusion. For each experiment, the cell
viability was given as the difference between final and initial percentages of non-viable cells.
3
.7. Chromatographic Analysis of Dialysates from PC12 Cell Suspension
DA was quantified by high performance liquid chromatography coupled with electrochemical
detection (HPLC-EC), as previously described [27,94], using an Alltech 426 HPLC pump equipped with
a Rheodyne injector (model 7725), a C18 column (150 mm × 4.6 mm i.d., Thoso Haas ODS80TM C18,
Im Leuschnerpark 4D-64347 Griesheim, Germany ), an electrochemical detector (BAS modeldeleted
LC4B, 2701 Kent Avenue West Lafayette, IN, USA) and a PC-based analog-to-digital converter system
(
(
Varian Star Chromatographic Workstation, Valnut Creek, CA, USA). The mobile phase was citric acid
0.1 M), sodium acetate (0.1 M), EDTA (1.0 mM), MeOH (9%) and sodium octylsulphate (50 mg/L) at
pH = 2.9. The flow rate was 1.3 mL/min.
3
.8. Assessment of Cell Viability
Cell viability was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay after 24 h of incubation. In the MTT assay, viable cells convert the soluble
dye MTT to insoluble blue formazan crystals. At the start of each experiment and at selected intervals
thereafter, the cell number was determined in triplicate wells. In brief, 1 mg of MTT was added per
◦
mL of medium, and the cultures were incubated at 37 C for 4 h. The cells were rinsed with PBS
and centrifuged at 4000 rpm for 20 min to remove the supernatant containing MTT. Thereafter, the
supernatant was discarded and the pellet was dissolved in 2 mL of isopropanol and centrifuged at
4000 rpm for 5 min. The absorbance of the supernatant was then determined at 600 nm using a Bauty
Diagnostic microplate reader. A standard curve was constructed utilizing known concentrations of
cells at the start of every experiment.
3
.9. Statistical Analysis
Concentrations of DA in dialysates from PC12 cell suspension are reported in nM and given as
the mean
±
SEM. Electrochemical currents are reported in nA while concentrations of NO generated
M. NO-donor effects on DA were statistically evaluated in
from NO-donors in PBS are reported in
µ
terms of changes in absolute dialysate concentrations. Statistical significance was assessed using an
ANOVA with Newman-Keuls post-hoc analysis. The null hypothesis was rejected when p < 0.05.

Pharmaceuticals 2017, 10, 74
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Limits of detection and quantification (LOD and LOQ, Equations (1) and (2)) have been calculated
by means of a statistical method based on the standard deviation (
σ
) of the response and the linear
region slope (LRS) of the calibration curve [96] as follows:
LOD = 3.3σ/LRS
LOQ = 10σ/LRS
(1)
(2)
4
. Conclusions
In this study the NO release from Piloty’s acid and its derivatives (4-NO -PI and 4-OMe-PI) with
2
different electronic properties have been investigated, and the results have been compared with a
benchmark NO-donor (SNAP). An efficient multigram synthesis procedure was developed that allows
preparation of a sulfohydroxamic acid library without any tedious purification steps associated with
previously published strategies. The synthetic methods presented here can be, in principle, applied
to the preparation of any Piloty’s acid derivative. NO release was investigated using spectroscopic
and electrochemical techniques. EPR spectroscopy used in spin trapping experiments with Fe(MGD)2
certainly detects NO, but it is difficult to establish with certainly if the HNO eventually released by the
PI derivatives can be coordinated to Fe(II) favouring its oxidation to NO and therefore resulting in the
contemporaneous detection of both these species. The results of EPR spectroscopy should be combined
with those obtained with other techniques, such as the electrochemical studies [97]. Experimental data
showed that Piloty’s acid derivatives with electron-withdrawing substituents favoured NO generation
in physiological conditions, while electron-donor groups disfavoured this process. However, in the
light of the results of the tests on the dopamine release from PC12 cells induced by PI derivatives and
of the potential application in the therapy of Parkinson’s disease, we would like to highlight here
that we are interested not in the detailed mechanism of decomposition of these compounds, but in
the effect of the total NO generated (i.e., formed directly by the decomposition of PI derivatives or
indirectly by the oxidation of HNO) in biological systems.
In this regard, 4-MeO-PI and, probably, all derivatives with electron-donor groups on the aromatic
ring could represent an interesting class of NO donors, because PI derivatives are not toxic over
the concentration range used here and decomposes to yield NO at a rate that allows the passage
of NO through the membranes and the release of dopamine, but that does not induce considerable
cell damage or death. Based on these preliminary data, we intend to design and synthesize new
Piloty’s acid derivatives with electron-donating groups on the phenyl ring and evaluate the dopamine
release induced by these molecules. Although the potential anti-inflammatory properties of the
newly-synthesized molecules were not investigated in this study, these will be characterized in
future research as Cyclo-oxygenase Inhibitor Nitric Oxide Donors (CINODs) have recently been
shown to be effective in the treatment of neurodegenerative diseases [98,99], such as Alzheimer’s and
Parkinson’s disease.
Supplementary Materials: The following are available online at www.mdpi.com/1424-8247/10/3/74/s1,
Figure S1: Experimental (in blue) and simulated (in pink) spectra of the species, Figure S2: EPR intensity of the
1
13
species Fe(MGD) (NO), Figure S3: H NMR of N-hydroxy-4-methoxybenzenesulfonamide, Figure S4: C NMR
2
1
of N-hydroxy-4-methoxybenzenesulfonamide, Figure S5: H NMR of N-hydroxy-4-nitrobenzenesulfonamide,
Figure S6: C NMR of N-hydroxy-4-nitrobenzenesulfonamide.
1
3
Acknowledgments: This work was financially supported by Autonomous Region of Sardinia, Project CRP1_409
“
2
Effetti sul SNC di una libreria di donatori di nitrossido derivati dall’acido di Piloty”, Legge Regionale 7 Agosto
007, N. 7: “Promozione della Ricerca Scientifica e dell’Innovazione Tecnologica in Sardegna”.
Author Contributions: Pier Andrea Serra, Eugenio Garribba and Andrea Porcheddu conceived and designed
the experiments; Lidia De Luca, Gaia Rocchitta, Marcello Abbondio and Maria Serra performed the experiments;
Daniele Sanna and Rossana Migheli analyzed the data.
Conflicts of Interest: The authors declare no conflict of interest.

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