2
S. Acharya et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
N
O
HN
HN
N
N
O
NO2
O
N
O
O
OH
N
O
N
N
O2
N
O2
OH
N
N
N
N
N
N
O
N
H
N
N
O
OH
> pH 5.0
N
OH
O
.
O
O
O
O2NO
N
N
SIN-1A
H
SIN-1
4-Hydroxy
OH
4-Nitro
SIN-1B
Nipradilol
TEMPOL (C3)
SA-2
TEMPOL-H
NH
N
NO
N
N
Figure 1. Hybrid NO donor compounds.
O
Literature report by Anggard and Haj-Yehia16 supported our
O
SIN-1C
observation that the presence of nitroxide may be responsible for
enhanced NO bioavailability via scavenging the surrounding ROS
and/or up regulating the SOD enzyme. The 4-nitro TEMPOL com-
pound and reported analogs have shown better NO mediated bio-
logical actions and act as superior antioxidants. However, organic
nitrates are poor nitric oxide donors in ocular tissues and are not
efficacious in lowering IOP in a therapeutic manner. In contrast,
3-(4-morpholino)-sydnonimine hydrochloride (linsidomine, SIN-
1, Fig. 2), a mesoionic class of heterocyclic (1,2,3-oxadiazole amine)
NO donor is reported to effectively lower intra ocular pressure
(IOP) in rabbit eyes.4 However the short duration of action (3 h
post dose) of SIN-I suggested that there is need to design robust
compounds with improved in vivo efficacy.
To continue our innovation in discovering small molecules as
anti-glaucoma targets,17–19 we disclose here for the first time the
design and synthesis of a novel dual acting nitric oxide donor-
antioxidant hybrid compound, SA-2. The goal is to provide the
additive activity to protect RGC and photoreceptor cells against
oxidative stress and enhance the duration of IOP lowering via
improved NO availability.
In a simple aerobic aqueous solution, 1,2,3-oxadiazole amine
containing NO donor SIN-1 undergoes base-catalyzed hydrolysis
spontaneously to release one mole of NO and one mole of superox-
ide radical (Fig. 3). Therefore, to improve the drug shelf life, SIN-1
is used as a carbamate20 (molsidomine) or amide21 (ciclosidomine)
prodrug (Fig. 2) for treatment of angina pectoris. Both the com-
pounds are hydrolyzed in liver to release SIN-1.22 It is apparent
that the stoichiometric production of superoxide (OÅ2À) generated
from SIN-1A may combine with NO to form the undesired toxic
peroxynitrite (ONOOÅÀ) resulting in reduced NO availability.
Our goal was to incorporate a radical scavenger functionality in
sydnonimine core structure to enhance the NO availability via
scavenging both the superoxide by-product produced during NO
release as well as the pathological ROS during IOP mediated oxida-
tive stress. For this purpose, we choose piperidine nitroxide to be
the appropriate radical scavenger. Piperidine nitroxide stable rad-
icals as in TEMPOL (compound C3, Fig. 1) demonstrated effective
antioxidant activity in various biological systems ranging from
molecular, cellular and laboratory animal level, and act as catalytic
SOD mimics. Besides the catalytic SOD mimetic activity of
Figure 3. NO release from SIN-1 (1,2,3-oxadiazole amine) at biological pH.
nitroxides, they can oxidize reduced transition metals that poten-
tiate damage by producing reactive oxygen-derived species
(ROS), detoxify hypervalent metals such as the ferryl-heme species,
facilitate heme-mediated catalytic removal of H2O2, trap carbon-
centered radicals, and terminate radical chain reactions. Through
competing with NO for OÅ2À the nitroxides might also indirectly
lower the production of peroxynitrite and elevate NO.23,24 Com-
pounds such as OT-440, OT-551 containing such nitroxide and N-
hydroxyl amine functionality have been shown to protect RPE cells
as well as RGC against oxidative stress in animal models.25 The
nitroxide radical and N-hydroxyl amine are interchangeable inside
the cell to render same antioxidant activity.26,27
The 4-amino TEMPOL-H sydnonimine SA-2 was prepared from
4-amino TEMPO (1, Scheme 1) following the literature proce-
dure.28 4-Amino TEMPO was treated with formaldehyde bisulfite
in H2O followed by heating with KCN at 50 °C to produce the nitrile
intermediate 2 which was nitrosylated at 0 °C using NaNO2/concd
HCl. The product formation was confirmed from LC/MS. The nitro-
sylated intermediate 3 is unstable and immediately converted to
the 1,2,3-oxadiazole amine hydrochloride salt SA-2 on treatment
with excess methanolic HCl.
Compounds SIN-1, SA-2 and 4-nitro TEMPOL-H (compound C3,
Fig. 1) were analyzed for total nitrate production in reference to
the reported Greiss assay protocol.29 Sodium nitrite solution
(1 mM) in water was used as reference standard. Both SIN-1 and
hybrid compound SA-2 released nitric oxide similarly as shown
in Figure 4.
We believe that the target tissue/cell for NO mediated IOP low-
ering effect is at the trabecular meshwork cells, the site that
appears to be affected during open angle glaucoma where increase
in IOP and other pathological stress contribute to glaucoma, and
neural cell death. Cell viability study has been done to demonstrate
the lack of overt toxicity of this class of molecules in NTM-5 (tra-
becular meshwork cell line) and 661 W photoreceptor cells. Here
we have demonstrated that (Figs. 5 and 6), treatment with SA-2
did not compromise the viability of neither the NTM5 cells nor
ClHHN
O
N
O
HN
O
N
NH2
N
NC
NH
N
N
O
NC
O
N
O
N
N
N
O
a
O
b
c
N
N
N
N
N
N
N
N
O
1
N
O
N
O
OH
SA-2
O
O
2
3
O
Ciclosidomine
Molsidomine
Linsidomine (SIN-1)
Scheme 1. Reagents and conditions: (a) HCHO, KCN, H2O, 50 °C, 6 h, 93%, (b)
NaNO2, HCl, H2O, 0 °C, 1 h, 88%, (c) 1 M HCl in MeOH, 12 h, 23 °C, 80%.
Figure 2. Sydnonimine NO donor.