Gem-Dinitroalkyl Benzenes: A Novel Class of IOP-Lowering Agents for the Treatment of Ocular Hypertension

Article abstract of DOI:10.1021/acsmedchemlett.7b00264

Primary open angle glaucoma is the second most common cause of blindness worldwide. Nitric oxide has recently received particular attention as a potential antiglaucoma agent. In this work, gem-dinitroalkyl benzenes are evaluated for their capability to ac

Full text of DOI:10.1021/acsmedchemlett.7b00264

Letter
pubs.acs.org/acsmedchemlett
gem-Dinitroalkyl Benzenes: A Novel Class of IOP-Lowering Agents for
the Treatment of Ocular Hypertension
Marco Blangetti,† Barbara Rolando,† Elisabetta Marini,† Konstantin Chegaev,† Stefano Guglielmo,†
Loretta Lazzarato,*,† Laura Lucarini,§ Emanuela Masini,§ and Roberta Fruttero†
†Dipartimento di Scienza e Tecnologia del Farmaco, Universita
̀
degli Studi di Torino, Via P. Giuria 9, 10125 Turin, Italy
§Dipartimento di Neuroscienze, Area del Farmaco e Salute del Bambino (NEUROFARBA), Universita
50139 Florence (FI), Italy
̀
di Firenze, Viale Pieraccini 6,
S
* Supporting Information
ABSTRACT: Primary open angle glaucoma is the second most common cause of blindness
worldwide. Nitric oxide has recently received particular attention as a potential antiglaucoma
agent. In this work, gem-dinitroalkyl benzenes are evaluated for their capability to act as a new
class of IOP lowering agents. These derivatives have been endowed with a variety of NO-release
capacities and found to relax contracted rat aorta strips in a concentration-dependent manner.
They have been studied for their IOP-lowering activity in a transient ocular hypertensive rabbit
model at 1% dose. The most effective IOP-lowering products were compounds 9−11 and 13,
whose activity was similar to that of Molsidomine 120 min after administration. Compounds 9
and 13 were selected for evaluation using carbomer-induced glaucoma as the chronic model of
IOP. They cause a significant reduction in IOP in the first 24 h, and their activity is maintained over 5 days, displaying a
Molsidomine-like profile.
KEYWORDS: gem-Dinitroalkyl benzenes, nitric oxide, glaucoma, ocular hypertension
rimary open angle glaucoma (POAG), the most common
form of glaucoma, is the second most common cause of
A number of hybrid products that derive from the coupling of
antiglaucoma drugs with nitrooxy (−ONO2) NO-donor
moieties have therefore been developed.10 Two prostaglandin
F2alpha (PGF2α) hybrids are in clinical trials,11−13 and one has
been approved by FDA.14
P
blindness worldwide and shows higher incidence in developing
than in developed countries. Its prevention and treatment have
been major objectives for the World Health Organization
(WHO) VISION 2020 program.1 Current pharmacological
treatments for the disease aim to lower intraocular pressure
(IOP), the principal risk factor for the disease, which is caused
by an imbalance in the rate of aqueous humor (AH) production
by the ciliary process and/or by reduced AH drainage through
the trabecular meshwork, Schlemm’s canal, or the uveo-scleral
route.2 Several classes of drugs are used to achieve this aim,
including beta-blockers, alpha-agonists, carbonic anhydrase
inhibitors, miotics, and prostaglandin analogues.3 The use of
nitric oxide as a potential antiglaucoma agent has recently
become the focus of particular attention.4 NO is an endogenous
messenger that is almost ubiquitous in the human body where
it exerts a variety of effects. It is produced under the action of
NO synthase (NOS), an enzyme which exists in three isoforms;
the constitutively expressed endothelial NOS (e-NOS, NOS-
II), neuronal NOS (n-NOS, NOS-I) isoforms, and the
inducible NOS isoform (i-NOS, NOS-II).5,6 All three NOS
isoforms are present in the eye, where NO mediates a
multitude of ocular effects including IOP maintenance and the
control of basal ocular vascular tone through the activation of
the soluble guanylate cyclase (sGC) signaling pathway.4,7,8
Moreover, a number of products are able to produce NO in
physiological conditions (NO-donors) and can exert a variety
of effects in the eye, including ocular blood flow modulation. In
fact, some have been studied as potential antiglaucoma drugs.4,9
A new class of IOP lowering agents, 1,1-dinitroethyl/
dinitromethylbenzenes, are herein described as a continuation
of our studies into potential antiglaucoma agents.9,15 The
preparation of these products, their physical−chemical
characterization, and an investigation into their ability to
spontaneously release nitrite at physiological pH and via
reaction with cysteine are reported. The ability of this class of
compound to reduce IOP in transient ocular hypertensive
rabbit model (tOHT) and to relax rat aorta strips, which had
been precontracted with L-phenylephrine, is discussed herein.
The synthesis of 1,1-dinitroethyl/dinitromethylbenzene
derivatives 8−14, bearing different electron-withdrawing and
electron-donating substituents on the aromatic ring, was easily
accomplished by means of a well-known procedure first
reported by Ponzio,16 and further reinvestigated by Suzuki in
1988.17 The treatment of stereoisomeric (E)/(Z) mixtures18 of
acetophenone oxime derivatives 1−5 with a solution of
dinitrogen tetroxide in dry diethyl ether (Et2O) at low
temperature afforded the corresponding 1,1-dinitroethylben-
zene derivatives 8−12 in moderate yields. The reaction also
Received: June 29, 2017
Accepted: September 13, 2017
Published: September 13, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.7b00264
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
proceeded with benzaldoxime derivatives 6 and 7 to afford the
corresponding 1,1-dinitromethylbenzenes 13 and 14 in slightly
lower yields (Scheme 1). Partition coefficient (logP) values for
−1.14) and 14 (logD7.4 = −1.21). The presence of the acidic
benzylic carbon atoms, which bear the two nitro groups, means
that derivatives 13 and 14 can exist in neutral or ionized forms,
depending on their ionization constants (pKas) and the pH of
the medium. Since this feature has an important influence on
the lipophilic−hydrophilic balance of the products, their pKas
were measured via potentiometric titration, giving values of
3.90 and 3.77, respectively. This means that the products exist
prevalently in the ionized form at physiological pH (Table 1).
The capacity of the gem-dinitro derivatives to release nitrite
was evaluated over 24 h of incubation in phosphate buffer
(PBS, pH 7.4) both in the absence and in the presence of an
excess of L-cysteine (1:50), by Griess reaction. The results
reported in Table 1 showed the extent of nitrite release by the
compounds at 1 and 6 h incubation, expressed as percent mol/
Scheme 1. Synthesis of gem-Dinitro Derivatives 8−14
−
mol of NO2 . No nitrite formation occurs for 1,1-dinitroethyl
derivatives 8−10 in the absence of the thiol cofactor in these
experiments, while the amount of nitrite released over 24 h of
incubation increases significantly, and in a linear time-
dependent manner, in the presence of a large excess of L-
cysteine (see Figure 1A for kinetic plots). The efficiency of
nitrite release occurs according to the series 10 > 8 ≥ 9. The
exact mechanism of the reaction will require additional
dedicated investigation, although the behavior could be
explained by the conventional nucleophilic displacement of a
nitro group under the action of the thiol cofactor, or perhaps
more probably, an electron transfer nucleophilic displacement
(electron transfer chain process).20 Derivative 11, which bears
the relatively efficient electron donating methoxy group (σp =
−0.27) in the para-position, is able to release nitrite
spontaneously over 24 h incubation in PBS (Figure 1B). The
thiol-independent mechanism is proposed to explain this
finding (Scheme 2) since p-OCH3 acetophenone was detected
as the predominant degradation product by HPLC. Similar
behavior is shown by compound 12, which bears a polar
glycolic alkoxy substituent in the para-position of the aromatic
ring (Figure 1B).
a
b
Isolated yields. OTEG = 2-(2-(2-methoxyethoxy)ethoxy)ethoxy.
the gem-dinitro derivatives 8−12, distribution coefficient values
at physiological pH (logD7.4) for compounds 13−14, obtained
using the shake-flask technique at room temperature, and
ionization constants determined by potentiometric titration for
derivatives 13 and 14 are reported in Table 1.
An analysis of the data shows that the partition coefficients
for compounds 8−12 are well-distributed over one logarithmic
unit interval (1.63 to 2.92). Compound 8, without any
substitution on the aromatic ring, shows a logP value of 2.34,
which falls in the optimal lipophilicity for corneal permeation.19
The introduction of a substituent in the para-position of the
aromatic ring modulates the lipophilicity, according to the
series 10 > 9 > 8 > 11 > 12. These data are in good accordance
with those calculated using the Hansch π constants of the
related substituents (πCF3 = 0.88, πCH3 = 0.56, πOCH3 = −0.02).
Compound 12 is the least hydrophobic of the series due to the
presence of the polar oxa-alkyl substituent, whose Hansch π
constant value is unknown. Our experimental results indicate
that it should be close to −0.70 (log P(12) − log P(8)).
As mentioned above, 1,1-dinitromethyl derivatives 13 and 14
are acidic compounds. Compound 13 is able to release nitrite
only under the action of cysteine (Table 1), while the para-
methoxy substituted compound 14 only releases nitrite
spontaneously at acidic pH. HPLC analyses of an acidic
solution of compound 14 after 24 h of incubation highlight the
presence of anisic acid as a final product, which can be
Lipophilicity differs markedly for products 13 (logD7.4
=
Table 1. Physico-chemical Properties (Lipophilicity Descriptors logP and logD7.4, Ionization Constant) and Extent of Nitrite
Release in the Presence and Absence of L-Cysteine at Physiological pH
c
−
% NO2
SE
physico-chemical properties
−L-cys
+L-cys
a
a
b
compound
logP SE
logD7.4 SE
pKa
1 h
6 h
1 h
<0.5
6 h
8
2.34 0.11
2.69 0.11
2.92 0.05
2.20 0.12
1.63 0.09
2.0 0.3
1.3 0.3
4.6 0.2
25.4 1.2
13.8 0.5
30.0 0.8
9
<0.5
10
11
12
13
14
0.8 0.1
3.5 0.1
2.7 0.3
3.0 0.1
2.6 0.2
24.9 1.2
12.8 1.2
−1.14 0.04
−1.21 0.04
3.77 0.01
3.90 0.01
3.0 0.07
d
d
1.3 0.1
4.0 0.2
a
b
Measured using the shake-flask method at room temperature. Determined by potentiometric titration with the GlpKa apparatus (n ≥ 3).
c
Percentage NO2− (mol/mol) SE, released with respect to the quantity of compound incubated at 37 °C in phosphate buffer (pH = 7.4) in the
d
absence and in the presence of an excess of L-cysteine (1:50), n ≥ 3. Determined after incubation at 37 °C in 30 mM HCl (pH 1.5) in the absence
of L-cysteine.
B
DOI: 10.1021/acsmedchemlett.7b00264
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
NO in the vasodilation (Table 2). This behavior could be due
to the ability of the compounds to release nitrite at
physiological pH under the action of the thiol cofactor
(compounds 8−10 and 13) or spontaneously (compounds
11, 12 and 14).
Nitrite, the principal metabolite of the aerobic oxidation of
NO, is a circulating store in blood of NO. Its transformation
from NO can either occur nonenzymatically via chemical
disproportionation or via reduction under the acidic and highly
reducing conditions, which occur in some disease states, as well
as via a redox reaction with a number of metal-containing
proteins.21,22 Nitrite can also react with thiols to give
nitrosothiols, which are involved in vascular smooth muscle
relaxation.23,24 It is able to relax aortic vessels under normoxic
conditions at high concentrations (100 to 1000 μM), via a NO-
dependent pathway,23,25 but its vasodilator potency is higher in
hypoxic vessels (10 μM), via both NO-dependent and
independent pathways.25−27 The vasodilator potency of tested
compounds follows the series 10 ≥ 14 > 11 ≥ 9 > 8 ≥ 12 > 13.
They are less potent than molsidomine but much more potent
than sodium nitrite. This behavior can be reasonably attributed
to the extremely slow passive diffusion rate of the nitrite anion
(HNO2 pKa = 3.14) across the highly lipophilic membrane of
smooth muscle cells. This should not be the case for the tested
compounds, which can more easily cross the membrane and
release nitrite inside the cell. The situation is different for 1,1-
dinitromethyl derivatives 13 and 14. Both products should
arrive inside the cell in low amounts since they are quite strong
acids (pKa= 3.77 and 3.90, respectively), which exist in large
prevalence as hydrophilic anions at physiological pH (7.4), and
are thus endowed with reduced membrane crossing capacity.
The feeble vasodilating capacity of 13 could be justified by its
nonoptimal hydrophilic lipophilic balance (logD7.4 = −1.14).
By contrast, the good vasodilating capacity of 14 may be caused
by the spontaneous release of nitrite, which is a sGC-dependent
vasodilator.
−
Figure 1. (A) Nitrite (NO2 ) release kinetics for compounds 8, 9, and
10 in a phosphate buffer at pH 7.4 in the presence of L-cysteine (L-Cys,
50×) over time (24 h); nitrite release is not observed in the absence of
L-Cys. (B) Nitrite (NO2 ) release kinetics for compounds 11 and 12
−
□
○
in phosphate buffer at pH 7.4 in the absence ( 11, 12) and in the
■
●
presence of L-Cys (50×; 11, 12) over time (24 h). Results are
expressed as percentage (% mol/mol) of nitrite released. The symbols
represent data from three or more replicates, and error bars represent
standard deviation from the mean.
rationalized as being a Nef-like reaction mechanism, as
proposed in Scheme 3. However, the spontaneous nitrite
donor 11, which bears the same electron-donating substituent,
behaves as a non-pH-dependent spontaneous nitrite donor
(Figure 2).
The vasodilating activity of all the final products was assessed
on denuded rat aorta strips that had been precontracted with L-
phenylephrine under normoxic conditions. All 1,1-dinitroethyl
derivatives 8−12 were able to relax the contracted strips in a
concentration-dependent manner. Their EC50 values are
reported in Table 2. When the experiments were repeated in
the presence of 1 μM ODQ (1H-[1,2,4]oxadiazolo-[4,3-
a]quinoxalin-1-one), a well-known inhibitor of sGC, their
potencies were partly reduced according to the involvement of
Compounds 8−14 were studied in comparison with
molsidomine, a well-known NO-donor, for their IOP-lowering
effects in a transient ocular hypertensive rabbit (tOHT) model
following a 1% topical dose. The results are shown in Table 3.
IOP response 120 min after administration follows the series 9
≥ 10 ≥ 13 ≥ 11 ≫ 8 ≈ 14 > 12. The effect practically vanished
after 240 min. The most active compounds, 9−11 and 13,
showed potencies and profiles that were similar to molsido-
mine, which was selected as the reference long-lasting NO-
releaser.9,12
Scheme 2. Proposed Mechanism for Spontaneous Nitrite Release from Compounds 11 and 12
C
DOI: 10.1021/acsmedchemlett.7b00264
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Scheme 3. Proposed Nef-like Reaction Mechanism for the Spontaneous Nitrite Release from 14
−
Figure 2. pH-dependence of nitrite (NO2 ) release for compounds 11 (a) and 14 (b). The results are expressed as a percentage (% mol/mol) of
nitrite released with respect to the quantity of compound incubated. The bars represent data from three or more replicates, and error bars represent
standard deviation from the mean.
As mentioned above, these compounds can be considered
pro-drugs of nitrite, which can be released inside the cells. It is
known from the literature that nitrite displays an IOP-lowering
ability after topical administration in a normotensive rabbit
model.11 In a high IOP context, a significant reduction in ocular
blood flow in the retinic artery28,29 and shear stress at the
endothelial level both occur. These events are accompanied by
cellular hypoxia, which increases the vasodilator potency of NO
releasing compounds and aqueous humor drainage.28,30 This
behavior may partly explain the discrepancy between the
vasodilator potency of compound 13 and its ability to reduce
IOP in vivo. Moreover, this compound’s penetration into the
eye is quite different to cellular penetration into rat aorta strips
in vitro, which may be a plausible explanation for the observed
differences. The most active compounds in each series, 9
(nonionizable) and 13 (ionizable), were selected to be
evaluated using a carbomer-induced chronic model (see
experimental details in the Supporting Information) as
previously described by Supuran et al.31 The effects of 9 and
13 versus molsidomine on IOP in glaucomatous albino rabbits
were determined after repeated administrations over the course
of 5 days. The results are reported in Figure 3 as IOP change
(mmHg) versus time. As previously observed in the tOHT
model, both compounds cause a significant reduction in IOP in
the first 24 h, while their activity is retained over 5 days and
exhibits a molsidomine-like profile.
Table 2. Vasodilating Activity of Compounds 8-14
compound
EC50 (μM) SE
EC50 (μM) SE + ODQ 1 μM
Molsidomine
0.10 0.02
18
a
3
8
50
24
2
4
9
99
55
64
b
5
3
3
10
11
12
13
14
4.0 0.7
20
54
c
2
6
4.4 0.7
64
4
a
b
c
% relax at 100 μM: 19 2. % relax at 100 μM: 40 5. % relax at
100 μM: 24 1.
Table 3. IOP-Lowering Effects of Tested Compounds in a
Transient Ocular Hypertensive Rabbit (tOHT) Model
a
IOP lowering efficacy (mmHg)
compound (1%)
60 min
120 min
240 min
molsidomine
−6.9 0.7
−4.3 0.2
−5.3 0.7
−6.5 0.3
−5.7 0.2
−3.7 0.2
−5.6 0.1
−4.9 0.2
−10.4 1.8
−7.3 0.1
−12.7 0.6
−11.7 1.4
−10.7 1.8
−4.6 0.6
−10.5 0.2
−6.7 0.7
−0.34 0.02
−0.11 0.01
+0.11 0.02
−2.3 0.1
−1.1 0.1
+0.12 0.01
8
9
10
11
12
13
14
+0.3 0.1
−0.04 0.01
In summary, we have developed a new class of IOP lowering
agents that are endowed with varying NO-release capacity. The
synthetic procedure allows a series of products to be easily
obtained. These compounds were able to relax the contracted
rat aorta strips in a concentration-dependent manner and show
a
Ocular hypotensive efficacy is expressed in mmHg, as the average
difference in IOP (intraocular pressure) between compound-treated
eyes or vehicle-treated eyes and their respective pretreatment value, as
shown in the following formula: efficacy = (IOPdrug − IOPpredose drug) −
(IOPveh − IOPpredose veh).
D
DOI: 10.1021/acsmedchemlett.7b00264
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
ACKNOWLEDGMENTS
■
We thank the University of Turin (ricerca locale ex-60%) for
financial support. The authors wish to thank Prof. Alberto
Gasco (Universita
̀
degli Studi di Torino).
REFERENCES
■
(1) Moore, D. B.; Tomkins, O.; Ben-Zion, I. A review of primary
congenital glaucoma in the developing world. Surv. Ophthalmol. 2013,
58, 278−285.
(2) Henderer, J. D.; Rapuano, C. J. Ocular Pharmacology. In
Goodman’s, The Pharmacological Basis of Therapeutic, 11th ed.;
Brunton, L., Lazo, J. S., Parker, K. L., Eds.; McGraw-Hill: New
York, 2006; p 1707.
(3) Sambhara, D.; Aref, A. A. Glaucoma management: relative value
and place in therapy of available drug treatments. Ther. Adv. Chronic
Dis. 2014, 5, 30−43.
(4) Cavet, M. E.; Vittitow, J. L.; Impagnatiello, F.; Ongini, E.; Bastia,
E. Nitric oxide (NO): an emerging target for the treatment of
glaucoma. Invest. Ophthalmol. Visual Sci. 2014, 55, 5005−5015 and
references therein.
(5) Kerwin, J. F., Jr.; Heller, M. The arginine-nitric oxide pathway a
target for new drugs. Med. Res. Rev. 1994, 14, 23−74.
(6) Wink, D. A.; Mitchell, J. R. Chemical biology of nitric oxide:
insights into regulatory, cytotoxic and cytoprotective mechanism of
nitric oxide. Free Radical Biol. Med. 1998, 25, 434−456.
(7) Chiou, G. C. Effects of nitric oxide on eye diseases and their
treatment. J. Ocul. Pharmacol. Ther. 2001, 17, 189−198.
(8) Haefliger, I. O.; Meyer, P.; Flammer, J.; Luscher, T. F. The
vascular endothelium as a regulator of the ocular circulation: a new
concept in ophthalmology? Surv. Ophthalmol. 1994, 39, 123−132.
(9) Blangetti, M.; Rolando, B.; Chegaev, K.; Guglielmo, S.; Lazzarato,
L.; Durante, M.; Masini, E.; Almirante, N.; Bastia, E.; Impagnatiello, F.;
Fruttero, R.; Gasco, A. New furoxan derivatives for the treatment of
ocular hypertension. Bioorg. Med. Chem. Lett. 2017, 27, 479−483.
(10) Steele, R. M.; Benedini, F.; Biondi, S.; Borghi, V.; Carzaniga, L.;
Impagnatiello, F.; Miglietta, D.; Chong, W. K. M.; Rajapakse, R.;
Cecchi, A.; Temperini, C.; Supuran, C. T. Nitric oxide-donating
carbonic anhydrase inhibitors for the treatment of open-angle
glaucoma. Bioorg. Med. Chem. Lett. 2009, 19, 6565−6570.
(11) Borghi, V.; Bastia, E.; Guzzetta, M.; Chiroli, V.; Toris, C. B.;
Batugo, M. R.; Carreiro, S. T.; Chong, W. K. M.; Gale, D. C.; Kucera,
D. J.; Jia, L.; Prasanna, G.; Ongini, E.; Krauss, A. H. P.; Impagnatiello,
F. A novel nitric oxide releasing prostaglandin analog, NCX 125,
reduces intraocular pressure in rabbit, dog, and primate models of
glaucoma. J. Ocul. Pharmacol. Ther. 2010, 26, 125−131.
(12) Impagnatiello, F.; Borghi, V.; Gale, D. C.; Batugo, M.; Guzzetta,
M.; Brambilla, S.; Carreiro, S. T.; Chong, W. K. M.; Prasanna, G.;
Chiroli, V.; Ongini, E.; Krauss, A. H. P. A dual acting compound with
latanoprost amide and nitric oxide releasing properties, shows ocular
hypotensive effects in rabbits and dogs. Exp. Eye Res. 2011, 93, 243−
249.
Figure 3. Effect on IOP of topical treatment with one drop (50 μL) of
1% 9 (top) or 1% 13 (bottom) versus 1% molsidomine in carbomer-
induced glaucoma in New Zealand albino rabbits. Data are presented
as means SD (n = 4). ***p < 0.001, **p < 0.01, *p < 0.05 versus
molsidomine (t-test).
activity similar to molsidomine in both transient ocular
hypertensive rabbits and in a chronic model of IOP.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.7b00264.
Synthetic procedures and experimental details (PDF)
(13) Impagnatiello, F.; Toris, C. B.; Batugo, M.; Prasanna, G.; Borghi,
V.; Bastia, E.; Ongini, E.; Krauss, A. H. P. Intraocular pressure-
lowering activity of NCX 470, a novel nitric oxide-donating
bimatoprost in preclinical models. Invest. Ophthalmol. Visual Sci.
2015, 56, 6558−6564.
(14) http://www.nicox.com/rd/ (last accessed August 2017).
(15) Chegaev, K.; Lazzarato, L.; Tamboli, Y.; Boschi, D.; Blangetti,
M.; Scozzafava, A.; Carta, F.; Masini, E.; Fruttero, R.; Supuran, C. T.;
Gasco, A. Furazan and furoxan sulfonamides are strong α-carbonic
anhydrase inhibitors and potential antiglaucoma agents. Bioorg. Med.
Chem. 2014, 22, 3913−3921.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: loretta.lazzarato@unito.it.
ORCID
Konstantin Chegaev: 0000-0001-9962-1218
Stefano Guglielmo: 0000-0002-3313-4369
Loretta Lazzarato: 0000-0001-7100-8593
Author Contributions
(16) Ponzio, G. Behavior of the Benzaldoxime against so- called
″nitrous acid″ and against nitrogen tetroxide. Gazz. Chim. Ital. 1906,
36, 287−298.
(17) Suzuki, H.; Takaoka, H.; Yamamoto, H.; Ogawa, T. Synthesis
and properties of dinitromethylated arenes. Reinvestigation of the
Ponzio reaction. Bull. Chem. Soc. Jpn. 1988, 61, 2927−2931.
The manuscript was written with contributions from all
authors, who all gave their approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
E
DOI: 10.1021/acsmedchemlett.7b00264
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(18) The isomeric ratios were determined by 1H NMR analysis
comparing the downfield-shifted CH3 singlet signal of the (E) isomer
with the upfield-shifted methyl singlet of the (Z) isomer.
(19) Shirasaki, Y. Molecular design for enhancement of ocular
penetration. J. Pharm. Sci. 2008, 97, 2462−2496.
(20) Miyake, H.; Yamamura, K. Replacement of the activated nitro
group by phenylthio group. Bull. Chem. Soc. Jpn. 1986, 59, 89−91.
(21) Zweier, J. L.; Samouilov, A.; Kuppusamy, P. Non-enzymatic
nitric oxide synthesis in biological systems. Biochim. Biophys. Acta,
Bioenerg. 1999, 1411, 250−262.
(22) Lundberg, J. O.; Weitzberg, E. NO-synthase independent NO
generation in mammal. Biochem. Biophys. Res. Commun. 2010, 396,
39−45.
(23) Ignarro, L. J.; Lippton, H.; Edwards, J. C.; Baricos, W. H.;
Hyman, A. L.; Kadowitz, P. J.; Gruetter, C. A. Mechanism of vascular
smooth muscle relaxation by organic nitrates, nitrite, nitroprusside and
nitric oxide: evidence for the involvement of S-nitrosothiols as active
intermediates. J. Pharmacol. Exp. Ther. 1981, 218, 739−749.
(24) Grossi, L.; Montevecchi, C. S-nitroso cysteine and cystine from
reaction of cysteine with nitrous acid. A kinetic investigation. J. Org.
Chem. 2002, 67, 8625−8630.
(25) Maher, A. R.; Milsom, A. B.; Gunaruwan, P.; Abozguia, K.;
Ahmed, I.; Weaver, R. A.; Thomas, P.; Ashrafian, H.; Born, G. V. R.;
James, P. E.; Frenneaux, M. P. Hypoxic modulation of exogenous
nitrite-induced vasodilation in humans. Circulation 2008, 117, 670−
677 and references therein.
(26) Pinder, A. G.; Pittaway, E.; Morris, K.; James, P. E. Nitrite
directly vasodilates hypoxic vasculature via nitric oxide-dependent and
-independent pathways. Br. J. Pharmacol. 2009, 157, 1523−1530.
(27) Allen, J. D.; Gow, A. J. Nitrite, NO and hypoxic vasodilation. Br.
J. Pharmacol. 2009, 158, 1653−1654.
(28) Galassi, F.; Renieri, G.; Sodi, A.; Ucci, F.; Vannozzi, L.; Masini,
E. Nitric oxide proxies and ocular perfusion pressure in primary open
angle glaucoma. Br. J. Ophthalmol. 2004, 88, 757−760.
(29) Impagniatello, F.; Giambene, B.; Lanzi, C.; Pini, A.; Sanna, T.;
Bastia, E.; Ongini, E.; Galassi, F.; Masini, E. The nitric oxide donating
triancinolone acetonide NCX 434 does not increase intraocular
pressure and reduced endothelin-1 induced biochemical and functional
changes in the rabbit eye. Br. J. Ophthalmol. 2012, 96, 757−761.
(30) Vullo, D.; Durante, M.; Di Leva, F. S.; Cosconati, S.; Masini, E.;
Scozzafava, A.; Novellino, E.; Supuran, C. T.; Carta, F. Mono-
thiocarbamates strongly inhibit carbonic anhydrase in vitro and
possess intraocular pressure lowering activity in an animal model of
glaucoma. J. Med. Chem. 2016, 59, 5857−5867.
(31) Fabrizi, F.; Mincione, F.; Somma, T.; Scozzafava, G.; Galassi, F.;
Masini, E.; Impagnatiello, F.; Supuran, C. T. A new approach to
antiglaucoma drugs: carbonic anhydrase inhibitors with or without NO
donating moieties. Mechanism of action and preliminary pharmacol-
ogy. J. Enzyme Inhib. Med. Chem. 2012, 27, 138−147.
F
DOI: 10.1021/acsmedchemlett.7b00264
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX