Secondary amines containing one aromatic nitro group: Preparation, nitrosation, sustained nitric oxide release, and the synergistic effects of released nitric oxide and an arginase inhibitor on vascular smooth muscle cell proliferation

Article abstract of DOI:10.1016/j.bmc.2012.12.043

Atherosclerosis, a leading cause of death worldwide, is associated with the excessive proliferation of vascular smooth muscle cells. Nitrogen monoxide, more commonly known as nitric oxide, inhibits this uncontrolled proliferation. Herein we report the preparation of two families of nitric oxide donors; beginning with the syntheses of secondary amine precursors, obtained through the reaction between 2 equiv of various monoamines with 2,4 or 2,6- difluoronitrobenzene. The purified secondary amines were nitrosated then subjected to a Griess reagent test to examine the slow and sustained nitric oxide release rate for each compound in both the absence and presence of reduced glutathione. The release rate profiles of these two isomeric families of NO-donors were strongly dependent on the number of side chain methylene units and the relative orientations of the nitro groups with respect to the N-nitroso moieties. The nitrosated compounds were then added to human aortic smooth muscle cell cultures, individually and in tandem with S-2-amino-6-boronic acid (ABH), a potent arginase inhibitor. Cell viability studies indicated a lack of toxicity of the amine precursors, in addition to anti-proliferative effects exhibited by the nitrosated compounds, which were enhanced in the presence of ABH.

Full text of DOI:10.1016/j.bmc.2012.12.043

Bioorganic & Medicinal Chemistry 21 (2013) 1123–1135
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Secondary amines containing one aromatic nitro group: Preparation,
nitrosation, sustained nitric oxide release, and the synergistic effects
of released nitric oxide and an arginase inhibitor on vascular smooth
muscle cell proliferation
Brandon Curtis a, Thomas J. Payne b, , David E. Ash a, Dillip K. Mohanty a,
⇑
a Department of Chemistry, Central Michigan University, Mt. Pleasant, MI 48858, USA
b Department of Biochemistry, University of TN, Knoxville, TN 37901, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Atherosclerosis, a leading cause of death worldwide, is associated with the excessive proliferation of vas-
cular smooth muscle cells. Nitrogen monoxide, more commonly known as nitric oxide, inhibits this
uncontrolled proliferation. Herein we report the preparation of two families of nitric oxide donors; begin-
ning with the syntheses of secondary amine precursors, obtained through the reaction between 2 equiv of
various monoamines with 2,4 or 2,6-difluoronitrobenzene. The purified secondary amines were nitrosat-
ed then subjected to a Griess reagent test to examine the slow and sustained nitric oxide release rate for
each compound in both the absence and presence of reduced glutathione. The release rate profiles of
these two isomeric families of NO-donors were strongly dependent on the number of side chain methy-
lene units and the relative orientations of the nitro groups with respect to the N-nitroso moieties. The
nitrosated compounds were then added to human aortic smooth muscle cell cultures, individually and
in tandem with S-2-amino-6-boronic acid (ABH), a potent arginase inhibitor. Cell viability studies indi-
cated a lack of toxicity of the amine precursors, in addition to anti-proliferative effects exhibited by
the nitrosated compounds, which were enhanced in the presence of ABH.
Received 17 October 2012
Revised 16 December 2012
Accepted 28 December 2012
Available online 9 January 2013
Keywords:
Sustained nitric oxide release
Cell culture study
Smooth muscle cell proliferation inhibition
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
role of insufficient endogenous NO concentrations in the establish-
ment and progression of atherosclerosis, the leading cause of
The small gaseous molecule nitrogen monoxide, commonly
known as nitric oxide (NO), is produced in vivo from -arginine,
and serves as a multifunctional signaling molecule. The production
of NO, a radical species, (and -citrulline) is catalyzed by nitric
cardiovascular disease worldwide.8 The onset of atherosclerosis is
commonly accompanied by increased levels of circulating low-
density lipoproteins (LDL), which culminates in the adhesion of
cholesterol to the inner blood vessel, composed of NO producing
vascular endothelial cells. Essentially smothered and isolated from
blood nutrients, these cells eventually undergo necrosis, and cease
NO production. Lower physiological NO concentrations have been
attributed to excessive SMC proliferation.9
The administration of NO-donors has been used to provide an
exogenous source of NO, and lessen the deleterious effects associ-
ated with reduced endogenous NO production. Several NO donors
including C, N, and S-nitroso, metal/NO complexes, and dia-
zeniumdiolates have been reported. However, these compounds
release NO rapidly in a single burst.10,11 Supplementation with such
exogenous NO donors and/or overproduction of endogenous NO are
known to confer harmful biological consequences. At elevated
concentrations, interactions between reactive oxygen species
(e.g., superoxide anion) and excess NO lead to the formation of
peroxynitrates—compounds capable of exerting a wide variety of
toxic effects.12 Furthermore, the prolonged use of glyceryl trinitrate,
L
L
oxide synthases (NOS) (three isoforms of which are known), in
the presence of additional co-factors. This same arginine pool also
serves as the substrate for the in vivo production of ornithine
catalyzed by arginase. Ornithine serves as the precursor to the
subsequent syntheses of various polyamines (Fig. 1).1–3 Though
structurally simple, the complex role of NO in maintaining multi-
ple homeostatic mechanisms allows it to exert several unrelated,
beneficial effects including vaso-relaxation,4 neurotransmission,5
and immune functions.6 Therefore it is not surprising that a variety
of disease states including inflammation, erectile dysfunction, and
multiple central nervous system disorders are associated with
decreased in vivo NO production.7 Of particular interest is the
⇑
Corresponding author. Tel.: +1 989 774 6445; fax: +1 989 774 3883.
E-mail address: Mohan1dk@cmich.edu (D.K. Mohanty).
Present address.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.043

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B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
of SMC arginase I can effectively enhance NO biosynthesis by
increasing available concentrations of
-arginine to NOS.20 Coupling
Arginase
NOS
Urea
L-Ornithine
+
L-Arginine
L-Citrulline
NO
+
L
this endogenous NO synthesis with the exogenous supply from the
reported NO-donors is likely to further decrease HASMC prolifera-
tion, possibly in a synergistic manner. The anti-proliferative poten-
tial of each donor family, both independently and in combination
with ABH were determined. In addition to cellular proliferation
and viability assays, intracellular nitric oxide concentrations were
monitored using a third cellular assay—which utilized the cell
membrane permeable 4-amino-5-methylamino-20,70-difluorescein
diacetate (DAF-FM DA). The diacetate ester linkage is cleaved by
intracellular esterase to form 4-amino-5-methylamino-20,70-
difluorescein (DAF-FM), which detects NO following two major
pathways. The first involves its reactions with dinitrogen trioxide
(produced from the intra-cell membrane reactions of NO with
molecular oxygen) to form the triazole derivative (fluorescent sig-
nal). In the second pathway, the reaction of DAF-FM with reactive
oxygen species (ROS) forms a DAF-FM radical which then reacts
with NO to form the fluorescent triazole-containing compound.21
The chosen NO-donor, at its most efficient concentration, was
combined with effective concentrations of ABH to achieve an opti-
mal inhibition of HASMC proliferation. We report findings from
these studies below.
Figure 1. Conversion of
L
-arginine to nitric oxide and L-ornithine.
a clinically approved NO donor, is known to induce nitrate toler-
ance.13 Polymeric NO donors, capable of preventing rapid NO re-
lease have been reported as coating materials for implantable
devices.14–16
To circumvent these deleterious effects associated with rapid
NO release, compounds exhibiting a predictably slow and sustained
release of NO are necessary. We have recently reported the prepa-
ration and characterization of both a low molecular weight (LMW)
and a polymeric class of N-nitrosated secondary amines (NO do-
nors),7 each expressing the desirable attributes alluded to above.17
Most importantly, both newly developed classes exhibited NO re-
lease in three distinct phases: a brief, fast releasing preliminary
phase, an extended, more moderate second phase, and a slow and
sustained final phase. We need to point out the possible concerns
regarding the use of N-nitroso compounds as NO-donors. The amine
nitrogen attached methyl group is a suspected alkylating agent for
DNA modifications. For example, N-nitroso-N-methyl-N-alkyl
amines have been reported to induce bladder tumors in rats and
hamsters.18a The fact that N-nitroso-N-ethylaniline is not carcino-
genic lends further support to these hypotheses.18b Extensive
discussions regarding the structural effects of nitrosamines and
related compounds on their carcinogenicity can be found else-
where.18c The concentrations of the non-methylated N-nitroso
compounds reported in this study were effective at extremely low
levels, at or below the micro-molar range.
The polymeric NO donors were prepared from the correspond-
ing secondary amines—initially synthesized by the aromatic nucle-
ophilic substitution of aliphatic diamines with 1,5-difluoro-2,6-
dinitrobenzene (DFDNB). These polymers, along with select LMW
compounds, exhibited excellent solvent resistance due both to in-
ter- and intra-chain hydrogen bonding between the secondary
amine moiety and the aromatic nitro groups. Polymers were insol-
uble in common organic solvents, dissolving only in aqueous
concentrated sulfuric acid.19 While highly desirable for some appli-
cations, nitrosation of these polymers could only be carried out at
low temperatures in highly corrosive, aqueous concentrated sulfu-
ric acid. A reduction in inter- and intramolecular hydrogen bonding
would allow for the facile nitrosation of both the polymeric and
LMW secondary amines soluble in common organic solvents
(including tetrahydrofuran) at room temperature.
Reported herein are the detailed preparation, isolation, and
characterization of two isomeric families of LMW secondary
amines, and their subsequent nitrosation to yield the correspond-
ing NO-donors. The 20-day NO release rate profile and extended
half-life of each new donor were determined in a phosphate buf-
fered aqueous environment. In addition, because reduced glutathi-
one (GSH) is a known scavenger of intracellular NO, the altered
release rate profiles of representative donors from each family in
the presence of GSH are also reported.
Cultures of human aortic smooth muscle cells (HASMCs) were
employed to perform multiple cellular assays, to determine and
reinforce the effects of each donor and its metabolites on vascular
SMC proliferation and viability. After NO is released, the N-nitrosat-
ed compounds revert back to the corresponding secondary amine.
Therefore, to accurately gauge the inhibitory effects of each NO-
donor, it was crucial to determine any alteration of HASMC prolifer-
ation and/or viability attributable to these amine byproducts.
As discussed above, both NOS and arginase compete for the
2. Experimental
2.1. Chemicals and equipment
The difluoride 2,4-difluoronitrobenzene (2,4-DFNB) (Aldrich)
was distilled at reduced pressure, while 2,6-difluronitrobenzene
(2,6-DFNB) (Aldrich) was used as received. N,N-Dimethylacet-
amide (DMAC) and N-methylpyrrolidinone (NMP) (Aldrich) were
dried over calcium hydride and distilled at reduced pressure. Tolu-
ene was distilled over calcium hydride. The diamines (Aldrich),
with six to nine methylene units, were either purified by recrystal-
lization using low boiling ligroin (bp 35–50 °C) for solids or by dis-
tillation at reduced pressure for liquids. All other reagents for NO-
donor synthesis were used as received. Griess Reagent was pur-
chased from Oxford Biomed, Alexis, or Enzo Life Sciences. GSH
was purchased from Aldrich. MTT (3-(4,5-dimethylthiazole-2-yl)-
2,5-diphenyltetrazolium bromide) assay kit, and cellular dimethyl
sulfoxide (cDMSO) were purchased from ATCC. LIVE/DEAD™ via-
bility/cytotoxicity kit, DAF-FM and DAF-FM DA were purchased
from Invitogen Corporation. 2-(4-Carboxyphenyl)-4,4,5,5-tetrame-
thylimidazoline-1-oxyl-3-oxide, potassium salt (C-PTIO) was pur-
chased from Aldrich. ABH, ultra pure lipopolysaccharide (LPS),
interferon gamma (IFN-c) were supplied by Enzo Life Sciences,
Fisher, and PeproTech, respectively.
IR spectra were recorded on a Nicolet DxB FT-IR spectropho-
tometer using a thin film of the material on sodium chloride plate.
Mass spectra of the secondary amines were obtained on a Hewlett–
Packard Model 5995A gas chromatograph/mass spectrometer with
an ionization potential of 70 electron volts. Both 1H and 13C NMR
spectra of the compounds dissolved in deuterated trichlorome-
thane (CDCl3) were recorded using a Varian Mercury 300 MHz or
a Varian Inova 500 MHz instrument. Tetramethylsilane (TMS)
was used as the internal standard for these measurements.
2.2. General procedure for secondary amine preparation
(A6–A10 and B6–B10)
The preparation of compound A7 is provided as a representative
synthesis procedure of these compounds.
same substrate pool of L-arginine. In an effort to maximize intracel-
Heptylamine (1.3186 g, 0.0114 mol) and 2,4-DFNB, (0.9262 g,
0.0058 mol) were weighed into separate vials. Potassium
lular NO concentrations without causing cell death, the potent argi-
nase I inhibitor ABH was also tested in HASMC cultures. Inhibition

B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
1125
Table 1
carbonate (3.03 g, 0.022 mol, 50% excess) was transferred to a
100 mL three-necked round-bottomed flask fitted with a magnetic
stir bar, nitrogen inlet, thermometer, and a Dean–Stark trap fitted
with a condenser. Each glass vial used for weighing starting mate-
rials was rinsed with DMAC (5 mL) and the washings were trans-
ferred to the reaction vessel to ensure full transfer of any
residual reagents. Next, an additional 4 mL of DMAC was added
to the reaction vessel, followed by 15 mL of toluene. An initial exo-
therm of 5 °C was observed upon the dissolution of the starting
materials, rendering the reaction mixture bright yellow in color.
The reaction vessel was heated by an external temperature-con-
trolled oil bath (with mild stirring) for 17 h at 125 °C. At the com-
pletion of the reaction, the vessel was cooled, the reaction mixture
was diluted with dichloromethane, and then filtered through celite
to remove all salts. The filtrate was evaporated at reduced pressure
to yield a crude product that was subsequently dissolved in dichlo-
romethane and washed with water. The organic layer was dried
over anhydrous magnesium sulfate and filtered. The filtrate was
evaporated at reduced pressure, and the resulting solid was further
purified via recrystallization in absolute ethanol to yield yellow
needles. Spectroscopic data for the secondary amines are supplied
as Supplementary data.
Percent nitrosation, mole percentage of released NO, and half-lives of NO donors
prepared from model compounds
Model
compounds
# Carbons
% Nitrosated
NO released
(mol %)
Apparent
half-life (h)
A60
A70
A80
A90
A100
B60
6
7
8
9
10
6
7
8
9
10
6
100
100
100
100
100
100
100
100
100
100
100
100
100
100
2.34
2.48
2.63
2.83
3.00
1.85
2.02
2.13
2.32
2.60
0.84
1.29
1.32
1.90
105
87
83
69
60
140
126
93
80
62
B70
B80
B90
B100
A60
A90
B80
126
109
123
108
9
8
10
B100
Profiles obtained in PBS solution (top) and in the presence of GSH (bottom).
(ꢁ1 mg) previously washed with 0.1 M HCl and 0.1 M NH4OH solu-
tions. At t = 0, a volume of 1ꢂ PBS was added to create a 0.0125 M
solution (0.0250 M NO). Aliquots were withdrawn over the course
of the reaction to monitor NO release, from t = 0 h to t = 12 h every
1 h, from t = 12 h to t = 24 h, every 4 h, and from t = 24 h to 48 h,
every 8 h. After 48 h, aliquots were removed in 24 h increments.
2.3. Nitrosation of secondary amines and determination of the
extent of nitrosation
Compound B7 (0.0862 g, 0.00024 mol) was weighed in a 25 mL
round-bottomed flask, fitted with a stopper. Tetrahydrofuran (THF)
and glacial acetic acid (1:1 v/v) were added to dissolve the com-
pound. The reaction vessel was then submerged in a cold-water
bath prior to adding sodium nitrite in excess of greater than or
equal to eight moles (0.1361 g, 0.0019 mol). When gaseous com-
pounds were no longer visibly escaping the vessel, the water bath
was removed and the temperature of the reaction mixture was al-
lowed to rise to room temperature, and the vessel was stoppered.
The reaction was allowed to continue at this temperature for 5–8 h
for the A series of compounds and 24–48 h for B series. The pro-
gress of the reaction was monitored by IR spectroscopy. In both
cases (isomeric families A and B), the time required to achieve
100% nitrosation increased with length of the carbon chain substit-
uents. Upon completion, the reaction mixture was transferred to a
100 mL Erlenmeyer flask containing an ice-cold saturated aqueous
sodium chloride solution. The mixture was neutralized next with
saturated aqueous sodium bicarbonate solution. Water was dec-
anted and the solid product was dissolved in dichloromethane.
The solution was transferred to a separatory funnel and washed
twice with ice-cold saturated aqueous sodium chloride solution
and finally with ice water. The organic layer was then transferred
to a beaker submerged in ice, and anhydrous magnesium sulfate
was added. The solution was then filtered by gravity filtration into
a round-bottomed flask and the solvent was removed at reduced
pressure using a rotary evaporator. The nitrosated product, B70
was transferred and stored in a sealed container at 0 °C.
Aliquots (200
(Fisher), and centrifuged at 2000 rpm for 2 min. Next, three 50
aliquots of the supernatant were transferred to separate wells of
a 96-well plate and combined with 50 lL
of Griess Reagent. After a 5-min incubation period at room temper-
ature, the absorbance of each well was determined at 550 nm
using a micro plate reader (Molecular Devices) and SoftMax Pro
software. The NO release rate was determined by plotting the
absorbance versus time.
The same procedure was followed to determine the NO-release
profiles of A60, A90, B80, and B100 in 1ꢂ PBS solution containing re-
duced glutathione (GSH, 5 mM). Since GSH does not react with
Greiss Reagent, a new calibration curve (with GSH) was not neces-
sary for these measurements.
lL) were transferred to a 1.5 mL plastic microtube
l
L
lL of 1ꢂ PBS and 100
2.4.2. DAF-FM
Approximately 100 mg of each of the nitrosated material, A70,
B70, A100 (with GSH), and A100 (without GSH) was added to a
25 mL round bottomed flask, and diluted with 1ꢂ PBS solution.
The flask was fitted with a magnetic stir bar and a stopper,
wrapped in aluminum foil, and placed over a stir plate at a moder-
ate speed for the duration of the reaction. At the same designated
time intervals (Section 2.4.1), 200
1.5 mL Eppendorf micro tube and centrifuged for 2 min at 200 rpm.
Three 50 L aliquots of the resulting supernatant were added to
designated wells of a fluorescence grade 96-well plate (Corning)
and further diluted with an additional 50
L of fresh 1ꢂ PBS, for
a total volume of 100 L/well. Finally, 1.0 L of 1.0 mM DAF-FM/
cDMSO working solution was added, yielding a final DAF-FM con-
centration of 10 M per well. The plate was covered and incubated
lL aliquots were transferred to a
l
l
The extent of nitrosation was determined using IR spectroscopy.
The decrease in the absorbance due to the secondary amino groups
(3340 cmꢀ1) relative to the absorbance due to the aromatic double
bond (1655 cmꢀ1) was measured before and after nitrosation to
obtain the extent of nitrosation for each sample (Table 1).
l
l
l
at 37 °C for 30 min. Fluorescence excitation and emission maxima
were recorded at 495 and 515 nm, respectively using and Molecu-
lar Devices micro plate reader and SoftMax Pro software.
2.4. NO release rate determination
2.5. Verification of release of NO from A60 using C-PTIO
2.4.1. Griess reagent test
The detailed procedure has been provided elsewhere.17 Briefly,
nitrosated compounds (A60–100) and (B60–100), were weighed into
separate 25 mL round bottom flasks wrapped in aluminum foil,
and equipped with a magnetic stir bar and cadmium pellet
2.5.1. Analysis in 1ꢂ PBS solution
Three 50 mL, one necked, round-bottomed flasks (labeled 1, 2,
and 3) were equipped with a magnetic stir bar and previously
washed cadmium pellets as described above (Section 2.4.1). Next,

1126
B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
precisely 50 mg ( 0.1 mg) of A60 were added to flasks 1 and 2. A
1.3 mg sample of the solid probe was dissolved in 1ꢂ PBS to render
complete SMC medium to yield a 1% cDMSO/NO-donor solution (v/
v) (0.1, 1, 5, 10 and 20 M). After the removal of the serum free
medium, each solution was added to the predetermined wells in
a manner identical to that outlined in Section 2.6.2. Following
exposure, MTT, LIVE/DEAD, and DAF-FM DA analyses were
performed.
l
a 100
l
M C-PTIO solution. A 20 mL aliquot of the freshly prepared
solution was then delivered to flasks 1 and 3 using a volumetric
pipette. Flask 2 received an equal volume of pure 1ꢂ PBS. All three
flasks were capped and gently stirred at room temperature for
5 days. At 24 h increments, 500
individual micro-centrifuge tubes, and spun at 13,000 rpm for
2 min. Aliquots (100 L) of each supernatant were transferred to
lL aliquots were transferred to
2.6.4. HASMC exposure to ABH
l
Prior to the addition of ABH, LPS (50 lg/mL) and INF-c (50 ng/
separate wells of a 96-well plate and gently mixed with an equal
volume of Griess Reagent. The absorbance of each well was then
measured at 550 nm with mircoplate reader. Triplicate wells were
prepared for each sample, with the average absorbance being plot-
ted as a function of time.
mL) were added, and well plates were incubated at 37 °C for 6 h.
LPS and the cytokine were removed, and replaced with ABH/SMC
medium (100
lL) solution at varying concentrations (1, 10,
100 M, 1.0 and 5.0 mM). Similar protocols (outlined in Section
l
2.6.2) were followed for the incremental additions of ABH solution
to the cells.
2.5.2. Analysis in supplemented smooth muscle cell medium
Three round-bottomed flasks were fitted and solutions analyzed
using the same methods outline in Section 2.5.1 above with the fol-
lowing modifications: complete SMC medium (2% Fetal Bovine Ser-
um (FBS), 1% Growth Serum, 1% Penicillin) was used in place of 1ꢂ
PBS. The NO-donor (A60) was added as a 2 mM solution in cDMSO
2.6.5. Synergistic addition of ABH and NO-donors
To induce iNOS, cells were first incubated with LPS (50
and INF- (50 ng/mL) in SMC medium for 6 h at 37 °C. Cytokines
were then removed, and ABH (100 L, 5 mM) and varying concen-
lg/mL)
c
l
trations of each NO-donor (Table 2) in SMC medium were added to
(200
l
L, final concentration 20
lM). Centrifugation prior to addi-
each well, and cells were again incubated at 37 °C for 48 h. A sec-
tion to 96-well plate was omitted. Except for absorbance measure-
ond aliquot of 100 lL SMC medium solution (100 lL) containing
ments,24a all other procedures were performed in
environment.
a
sterile
the specified ABH/NO-donor combination was added, followed by
a final 48 h incubation period at 37 °C before undergoing subse-
quent viability and NO determination assays.
2.6. Cellular studies
2.6.6. MTT proliferation assay
2.6.1. Human aortic smooth muscle cell culture
At the end of the incubation period, 10
added to each well. Plates were incubated at 37 °C for 4–6 h. Final-
ly, MTT detergent (100 L) (ATCC) was added to each well. Plates
were incubated at room temperature, in the absence of light for
a minimum of 2 h (24 h maximum) yielding a homogeneous purple
solution in each well. The absorbance of each well was measured
using a plate reader at 570 nm. Comparisons between treatment
groups were made by t-test using the Minitab software program.
A value of p >0.05 was considered statistically equal.
lL MTT reagent was
Cultures of human aortic smooth muscle cells (HASMCs) were
developed and maintained in accordance with the procedural
guidelines recommended by both ScienCell and ATCC. To ensure
adequate attachment, the 75 cm2 culture flasks (Corning) required
a specially treated CellBINDÒ surface. Cells were grown in SMC
specialty medium (5% FBS, 1% Growth Serum, 1% Penicillin, (Scien-
Cell)) to P95% confluency before passage with trypsin. Following
passage six, cells were quantified (cells/mL) using a hematocytom-
eter, further diluted to a concentration of 30,000–35,000 cells/mL
l
using SMC medium, then transferred as 100
l
L aliquots to clear,
2.6.7. LIVE/DEAD assay
colorless, 96-well plates (Corning, with CellBIND surface) to pro-
vide 3000–3500 cells per well (cpw). The seeded plates were incu-
bated at 37 °C for 16–24 h to ensure stable cellular attachment. The
cellular population was synchronized to G0 by removing the com-
plete medium from each well and replacing it with serum-free
Optimum reagent concentrations for HASMCs, found in accor-
dance with the invitrogen protocol, were determined to be 0.50
and 0.25 lM for ethidium homodimer-1 (Eth-1) and calcein AM
(CAM), respectively.
Corning black, polystyrene 96-well microplates, with clear bot-
tom and CellBIND surface were prepared in the same manner as
the clear microplates mentioned above, and underwent identical
treatments with compound solutions for toxicity determination.
Because dead HASMCs become unattached, the medium containing
the dead cells was removed from each well, and transferred to indi-
vidual 0.5 mL micro-centrifuge tubes. Adherent cells were washed
SMC medium (100 lL). The plates were again incubated at 37 °C
for 24–48 h prior to subsequent testing.
2.6.2. HASMC exposure to secondary amines
Solutions of each secondary amine (A6–B10) were prepared in
cellular DMSO (cDMSO) at five concentrations (10, 100, 500 lM,
1.0 and 2.0 mM). An aliquot of each amine solution was combined
twice with PBS (100 lL) solution to remove residual medium.
with complete SMC medium to render a 1% cDMSO/medium solu-
The tubes containing the dead cells were centrifuged at
tion (v/v) (0.1, 1, 5, 10 and 20 lM), to be added to SMC cultures, in
2500 rpm for 3 min. The medium was removed, and the pellet of
addition to positive (medium only) and negative (medium and 1%
dead cells was washed in PBS solution (250 lL) and centrifuged.
DMSO) controls. The serum-free medium was replaced with
100
um, 1% Penicillin), and plates were incubated for 48 h, followed
by the addition of 100 L of fresh medium to each well for a final
volume (200 L). Plates were again incubated for 48 h, before MTT
lL of each sample medium solution (2% FBS, 1% Growth Ser-
Table 2
Extent of HASMC proliferation inhibition exhibited by NO-donors with and without
ABH (5 mM)
l
l
Concentration
A60
B60
ABH
or LIVE/DEAD analyses. The final testing concentration of cells after
20
lM
20
l
M
5 mM
96 h was 48,000–56,000 cpw.
%Viability reduction
28
—
—
—
—
Without ABH (RWO
Sum reduction (RWO + RABH
With ABH (RW
RWꢀ(RWO + RABH
)
23
51
64
13
18
46
48
2
2.6.3. HASMC exposure to NO-donors
Solutions of each nitrosated compounds (A60–A100 and B60–
B100) were prepared in cDMSO at the same five concentrations as
used with the secondary amines. Each sample was combined with
)
)
)

B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
1127
The supernatant was removed, and the cells were again suspended
in fresh PBS solution (100 L), and then returned to the original
well (with adherent cells). A PBS solution (100 L) of Eth-1
(0.5 M) and CAM (0.25 M) were added to each well. Plates were
incubated at room temperature for 45 min. The percentages of
both live and dead cells were determined using a fluorescent plate
reader (Molecular Devices) at ex/em 495/530 nm (CAM) and 528/
645 nm (Eth-1), respectively.
ate) was observed. These findings suggest that attempts to form
high molecular weight polymers using diamines at elevated tem-
peratures (necessary for the reactions to proceed to completion
in an increasingly more viscous medium and with decreasing con-
centrations of reactive ends) would be unsuccessful. Details of
these side reactions will be reported separately.
l
l
l
l
3.2. Nitrosation of secondary amines A6–A10, B6–B10 and
extent of nitrosation determination
2.6.8. Intracellular NO determination using DAF-FM DA
Corning black, polystyrene 96-well microplates, with CellBIND
surface were prepared as described in Section 2.5.1 and then trea-
ted with the predetermined compound solutions (NO-donors, ABH,
or a combination of ABH and NO-donor). After exposure, a DAF-FM
Nitrosation of compounds A6–A10 and B6–B10 performed in
glacial acetic acid/THF (1:1 v/v) in the presence of excess sodium
nitrite (Scheme 1) could be achieved quantitatively (Table 1). The
rate of nitrosation for the B (2,6-) family of isomeric diamines
was substantially slower than the corresponding A (2,4-) isomers
due to the closer proximity of the nitro group to both amine groups
compared to that of the corresponding 2,4-substituted compounds.
This phenomenon is typical when NO acts as the nitrosating agent
of weakly basic, N-alkyl aryl secondary amines.22 In addition, both
families exhibit a decrease in the rate of nitrosation due to steric
interferences with increasing aliphatic chain lengths. For example,
the conversion time of B6 to B60 was approximately 24 h, in con-
trast to 48 h required for complete nitrosation of B10.
DA (10 lM) solution in PBS (100 lM) was added to each well.
Plates were incubated at room temperature in total darkness for
60 min. The DAF-FM DA solution was then removed, and cells were
washed (3ꢂ) with PBS to remove excess probe. Fresh medium (or
PBS) was added, and plates were again incubated under the same
conditions to allow for complete de-esterification of DAF-FM DA.
Fluorescence was then recorded at ex/em 495 and 515 nm,
respectively.
3. Results and discussion
3.3. NO release rate determination
3.1. Preparation of secondary amines A6–A10 and B6–B10 from
aliphatic homologous monoamines and 2,4-/2,6-DFNB
The amount of NO released from each nitrosated compound
(A60–A100 and B60–B100) was measured using Griess reagent17 over
a period of 20 days (Fig. 2a and b). From an examination of these
figures, two trends are clearly discernible. First, the rate of NO re-
lease increases as the number of methylene units increases. Sec-
ond, the release of NO occurs in three distinct stages; an initial
fast phase (ꢁ0–10 h), a moderately fast phase (ꢁ10–200 h) and a
very slow and sustained phase (ꢁ200–480 h), respectively. These
observations are consistent with previously reported data based
on nitrosated model compounds prepared from DFDNB.17 The cal-
culated apparent half-lives ranged from 60 h to 105 h for A60–A100
and from 62–140 h for B60–B100 (Table 1), which are significantly
longer than many currently available commercial NO donors.10,23
The NO release profiles of compounds A70 and B70 were also deter-
mined using DAF-FM, a NO and /or N2O3 probe (Fig. 2c). An analy-
sis of Figure 2c indicates that irrespective of the NO-probe used
(Griess reagent (Fig. 2a and b) or DAF-FM), both the release profiles
and the rates (A70 and B70) remain unchanged. A control experi-
ment with a solution of DAF-FM in PBS did not exhibit an enhanced
fluorescent signal.
Secondary amines were prepared from homologous aliphatic
primary amines, containing six to ten carbon atoms, and 2,4 or
2,6-dinitroflurobenzenes (2,4-DFNB or 2,6-DFNB) (Scheme 1). All
reactions were carried out at 120 °C for ꢁ24 h. Under these reac-
tion conditions, the desired compounds could be prepared in high
yields (Supplementary data). Thin layer chromatography (TLC)
coupled with GC/MS analyses of the reaction mixtures indicated
the absence of side products. This scenario changed rapidly when
any one of these reactions was conducted above 130 °C. At these
temperatures, in addition to the desired product, the formation
of numerous other compounds (derived from one major intermedi-
A comparison of half-lives between any two isomeric NO do-
nors (e.g., A80 and B80) indicates that the 2,6-isomer exhibits a
higher half-life compared to the corresponding 2,4-isomer. This
phenomenon possibly due to the loss of the radical species NO,
results in the formation of electron deficient nitrogen radical in
closer proximity to the electron withdrawing nitro group (energet-
ically less favorable) than that formed from the corresponding
2,4-isomers. In addition, the differing steric environments of the
N–NO moieties in these two families may further contribute to
the differing NO release rates.
In order to ascertain the nature of the NO species being released
from the N-aryl-N-alkyl-N-nitrosamines, A60–A100 and B60–B100,
C-PTIO and Griess reagent were employed (Section 2.4.1).24 C-PTIO
is also known to interact with peroxynitrite anion in vivo where
superoxide radical anions are present.25 In the present instance
our experiments were carried out in solvent systems of PBS solu-
tion or using the SMC culture medium. Under these conditions,
superoxide radical anions are not formed, which excludes the for-
mation of peroxynitrite anion and the resulting interference during
the C-PTIO tests. Data from these studies in PBS solution and in a
Scheme 1. Syntheses and nitrosation of secondary amines A6–A10 and B6–B10.

1128
B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
Figure 3. (a) Analyses of nitrite production from NO-donor, A60 in the presence and
the absence of C-PTIO in 1ꢂ PBS. (b) Analyses of nitrite production from NO-donor,
A60 in the presence and the absence of C-PTIO in supplemented SMC culture
medium. Aliquots were removed every 24 h, treated with Greiss reagent and
absorbances measured at 550 nm (data displayed in 3a). Data for 3b were obtained
from absorbances measured at 550 nm with a background subtraction at 630 nm.
C-PTIO rapidly captures released NO radical species before a fraction can escape and
converts it to nitrite (detected by Griess reagent). Consequently, the presence of C-
PTIO results in significantly higher measured nitrite concentrations (higher
absorbance values) than in its absence at any point in time during the span of
the experiment (5 days).
unexpected in view of the earlier findings.26 It has been shown that
N-aryl-N-alkyl-N-nitrosamines with linear alkyl groups and elec-
tron withdrawing nitro substituent in the ortho or para position
to the nitrogen atoms of the amine moieties, structural features
similar to the NO-donors under consideration, undergo homolytic
cleavage to form NO. The ease of homolytic cleavage was attrib-
uted to the delocalization of electron density on the nitrogen atoms
of the amine groups and consequent weakening of the N–NO bond
due to the loss of double bond characteristics.26 Additional theoret-
ical calculations and experimental studies have been reported in
support of the favorable formation of NO radical species over that
of nitrosonium ion (from heterolytic cleavage), with increasing
delocalization of the lone pair on the nitrogen atom of the amine
nitrogen atom.27
Figure 2. The release profiles were determined in PBS solution at room temper-
ature using Griess reagent to measure the nitrite concentration produced from the
released NO. Absorbance values at 550 nm were determined using a Molecular
Devices plate reader. (a) NO release profiles of NO-donors, A60–A100, (b) NO release
profiles of NO-donors, B60–B100. A fluorescent probe DAF-FM was used as an
alternate probe to detect NO and/or N2O3. Fluorescent signals were detected using
the same micro-plate reader at 495/515 (ex/em) (c) NO release profiles of A70 and
B70 determined using DAF-FM.
solution of supplemented culture medium are displayed in Figure
3a and b, respectively. From an examination of these figures one
observes the significant increase in nitrite concentration in the
presence of C-PTIO than in its absence, which is indicative of the
rapid capture of the NO radical species (and its subsequent conver-
sion to nitrite anion)25a released from the NO-donor, A60. The for-
mation of NO from the NO-donors under consideration is not
The subsequent reactions of nitric oxide and oxygen in an
aqueous environment produce mono and dinitrogen species
including nitrite, nitrate, dinitrogen trioxide and dinitrogen tetrox-
ide. Dinitrogen oxides may undergo further decompositions to
produce nitrites and nitrates.28 Nitrites and nitrates undergo intra-
cellular reduction to nitric oxide by mitochondrial aldehyde
dehydrogenase.29

B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
1129
Additionally, we have investigated if and how the NO-release
profiles may be affected under aerobic conditions in the presence
of intracellular glutathione (GSH).30 The concentration of GSH
was maintained at 5 mM31 in PBS solution and the NO-release pro-
files of four NO donors, A60, A90, and B80, B100, determined over a
period of 20 days, are displayed in Figure 4a and b, respectively.
When compared with Figure 2a and b, the NO release rate patterns
in the presence of GSH are similar; NO is still released in three dis-
tinct stages and the NO release rate still increases with the number
of methylene units. However, important differences exhibited by
all four compounds tested are apparent. Of considerable impor-
tance is the overall decrease in the amount of NO released to
approximately one third of that observed in the absence of GSH.
Furthermore, the initial ‘fast’ phase and second moderately fast
phase are considerably shorter in the presence of GSH.
S-Nitrosothiols (RSNO), including nitrosated glutathione
(GSNO), act as NO storage and transport vehicles in vivo. GSH is
known to react with NO, either produced endogenously or released
from an exogenous source(s). It has been previously reported that
the addition of a catalytic amount of copper(I) induces NO release
from GSNO.32 We have confirmed these earlier findings with the
addition of a catalytic amount (<0.5 mg) of solid cuprous chloride
to the reaction mixture containing B100 with GSH. One hour after
the addition of copper, it was observed that the total amount of
NO released into solution was equal to that determined for B100
in the absence of GSH (Fig. 4b). These observations clearly suggest
that the presence of GSH does not affect the NO-donor0s ability to
release NO. Similar results were obtained when a different probe,
DAF-FM was used to monitor the release rate profile for A100 with
and without GSH (Fig. 4c).33 A lack of increased fluorescent signal
from a mixture of GSH and DAF-MF in PBS solution confirmed that
the trends observed in the fluorescent intensities (Fig. 4c) are due
to NO or related species.
The secondary amines were nitrosated using sodium nitrite
(Section 2.2), in an acidic medium. Griess reagent detects NO indi-
rectly, by determining the total nitrite (formed from NO) concen-
tration.17 To ensure the NO concentrations determined using
Griess reagent were solely due to NO released from the NO-donors,
rather than from any residual nitrite from the reaction mixture, a
control experiment was undertaken. A PBS solution of sodium ni-
trite (0.0125 M) was treated with Griess reagent. The nitrite con-
centration measured over 20 days remained constant (Fig. 2a),
unlike the three-stage release of NO from the NO-donors. These
observations clearly confirm that the measured concentrations of
NO derived nitrite are solely due to the NO-donors.
3.4. Cell culture studies
3.4.1. Secondary amine toxicity
After the release of NO, the N-nitroso compounds revert to the
corresponding secondary amine precursors, which remain exposed
to cells in the culture medium. Therefore, it was imperative to
examine the potential influences A6–A10 and B6–B10 may exert
towards HASMCs. An MTT assay was performed to identify
whether the secondary amines exert any anti-proliferative or toxic
effects on HASMCs. Because the amines were sparingly soluble in
aqueous medium, a combination of cDMSO and SMC Cellular Med-
ium (DMSO/medium, 1% v/v) was used as the vehicle. Data ob-
tained from MTT studies are shown in Figure 5a (A6–A10) and
Figure 4. The release profiles were determined in PBS solution at room temper-
ature using Griess reagent to measure the nitrite concentration produced from the
released NO in the presence and absence of GSH. Absorbance values at 550 nm were
determined using a Molecular Devices Plate Reader. (a) NO release profiles of A60
and A90 in the presence (A60G, A90G) and the absence of GSH (5 mM), determined by
Griess reagent. (b) NO release profiles of B80 and B100 in the presence (B80G, B100G)
and the absence of GSH (5 mM). Addition of catalytic amount of Cu+ to B100G at
t = 430 h resulted in the release of NO from GSNO. A fluorescent probe DAF-FM was
used as an alternate probe to detect NO and/or N2O3. Fluorescent signals were
detected using the same micro-plate reader at 495/515 (ex/em) (c) NO release
profiles of A100 in the presence (A100G) and the absence of GSH (5 mM) determined
using DAF-FM.
5b (B6–B10), respectively. For clarity, data from 0.1 to 1 lM amine
concentrations are not shown in the figures, however the complete
data set is displayed in Table S.B1, Appendix B (Supplementary
data). An examination of these figures first illustrates that 1%
DMSO ((ꢀ) control)) results in a decrease in the number of viable
cells when compared to SMC medium alone ((+) control).34 Sec-
ondly, all of the amines tested, with the exception of B9 and B10,
exhibited equal or less toxicity than the vehicle at four or more
concentrations, suggesting any decrease in cell viability is a conse-
quence of the vehicle0s presence. For example, compound A8

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B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
Figure 6. Determination of cytotoxic effects by secondary amines on HASMC
cultures using a LIVE/DEAD assay. Two distinct fluorescent measurements at em
495/ex 515 and em 495/ex 635 provided data points for calculating reported
Figure 5. Determination of anti-proliferative effects exerted on cultures of HASMCs
by secondary amines. Changes in HASMC proliferation were evaluated using an
MTT viability assay, after a 96 h incubation period with a given secondary amine (in
cDMSO). Absorbance measurements of each well (570 nm) provided data points,
which were expressed as a percentage of the positive control (100%), in which cells
were grown in the absence of any test agents. In addition, cDMSO acted as the
vehicle to deliver amines; therefore a negative control of 1% cDMSO was also tested
for HASMC proliferation inhibition. (⁄) Indicates a statistically significant difference
(p <0.05) between the given compound/concentration combination and the vehicle
alone. The graphical data represents the mean SE of triplicate determinations on
the extent of HASMC proliferation inhibition in the presence of (a) A6–A10 and (b)
B6–B10.
percentages of LIVE and DEAD cells, respectively. (⁄) Indicates
a statistically
significant difference (p <0.05) between the given compound/concentration com-
bination and the vehicle alone. The concurrent decrease in LIVE cells and increase in
DEAD cells exhibited by the negative control suggests 1% DMSO induces cell death.
Therefore the use of DMSO as the vehicle accounts for the percentage of DEAD cells
present in all tested samples. The graphical data represents the mean SE of
triplicate determinations, and reveal the percentages of LIVE and DEAD cells
resulting from exposure to (a) A6–A10 and (b) B6–B10.
centage of live and dead cells as the vehicle. This suggests that
the vehicle, rather than the secondary amine (barring the above
exceptions) is responsible for the decrease in cell viability. Com-
pounds B9 and B10, on the other hand, caused a statistically signif-
icant decrease in the number of live cells, while maintaining
statistically equal numbers of dead cells as the vehicle. These
observations indicate that both B9 and B10 exert anti-proliferative
effects rather than causing cell death. Collectively the amines
tested do not cause cell death irrespective of the concentrations
used in these experiments. Details of the performed statistical
analyses (p values and t-stat values) are supplied as supplementary
data in Appendix C.
exhibited higher toxicity at a lower concentration (0.1
lM); how-
ever at higher concentrations (1, 5 and 20.0 M), it was found to
l
be equally or less toxic than the vehicle. These variations can be
attributed to experimental artifacts. Conversely, the presence of
B9 and B10 at the three highest, and all tested concentrations
respectively, induces a statistically significant decrease in cell via-
bility compared to the vehicle alone.
In order to further confirm the trends observed in the MTT as-
say, a LIVE/DEAD assay was performed to ascertain whether the
secondary amines were decreasing cell proliferation rates or induc-
ing cell death. Results from these studies are displayed in Figure 6
(A6–A10) and 6b (B6–B10). Consistent with the MTT assay, treat-
ment with cDMSO alone (the vehicle) results in a statistically sig-
nificant decrease (ꢁ12%) in the number of live cells. Additionally,
the concurrent increase in the number of dead cells (Fig. 6a and
b) indicates that the presence of the vehicle results in cell death
in contrast to inhibition of proliferation. Furthermore, each second-
ary amine, except B9, and B10, showed a statistically equal per-
3.4.2. Effects of exogenous NO-donors (A60–B100) on HASMC
proliferation
The addition of our NO-donors (A60–A100 and B60–B80) at vari-
ous concentrations (Section 2.6.3) to cultures of HASMCs was per-
formed to determine the most effective concentrations required to
obtain maximum proliferation inhibition, without initiating cell

B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
1131
death (e.g. necrosis or apoptosis). The results obtained from the
MTT, LIVE/DEAD, and DAF-FM DA assays are displayed in Fig. 7–
9, respectively. In Figure 7, for the purpose of clarity, data from
0.1 to 1 lM N-nitrosated amine concentrations are not shown,
however the complete data set is shown in Appendix B, Table
S.B2 (Supplementary data). An examination of both the MTT assay
and LIVE portion of the LIVE/DEAD test results, Figures 7 and 8a
and c, respectively, indicates that all of the NO-donors confer
anti-proliferative effects on HASMCs at concentrations less than
or equal to 20 lM. Furthermore, for all of the compounds tested
in both isomeric families (excluding B70), the extent of prolifera-
tion inhibition is enhanced with increasing concentrations of NO-
donors. In contrast, B70 hindered proliferation more effectively
with increasing concentrations from 0.1 to 5 lM, however, further
increases in the concentrations had less salutary effects.
Of equal importance are the DEAD test results (Fig. 8b and d). As
mentioned in Section 3.4.1, the presence of cDMSO initiates death
in a small, yet significant percentage of cells. However, with the
addition of a given NO donor, the amount of dead cells remains sta-
tistically constant, attributable to the presence of the vehicle.
These observations indicate the presence of the NO-donors is
responsible for a statistically significant decrease in viable (LIVE)
Figure 8. Determination of cytotoxic effects on HASMCs due to presence of NO-
donors at varying concentrations, using
a a
LIVE/DEAD assay. (⁄) Indicates
statistically significant difference (p <0.05) between the given compound/concen-
tration combination and the vehicle alone. A statistically significant decrease in the
percentage of LIVE cells, accompanied by no change in the percentage of DEAD cells
Figure 7. Determination of anti-proliferative effects exerted on cultures of HASMCs
by NO-donors, using an MTT viability assay. (⁄) Indicates a statistically significant
difference (p <0.05) between the given compound/concentration combination and
the vehicle alone. The graphical data represents the mean SE of triplicate
determinations on the extent of HASMC proliferation inhibition in the presence of
(a) A60–A100 and (b) B60–B100.
in the presence of the same compound/concentration combination, verifies a
reduction of viable HASMCs due to the inhibition of proliferation. The graphical data
represents the mean SE of triplicate determinations, and illustrate (a) the
percentage of LIVE cells after exposure to A60–A100, (b) the percentage of DEAD
cells after exposure to A60–A100, (c) the percentage of LIVE cells after exposure to
B60–B100 and (d) the percentage of DEAD cells after exposure to B60–B100.

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B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
are illustrated in Figure 9a (2,4-isomers) and 9b (2,6-isomer). An
examination of these figures demonstrates an increase in intracel-
lular NO (increasing fluorescence signal) with increasing NO-donor
concentrations with the exception of B70. Irrespective of the vari-
ous transformation reactions the released NO may have under-
gone, these findings reiterate the fact that NO is being released,
Figure 9. Monitoring of intracellular NO and/or N2O3 concentrations of HASMCs
exposed to NO-donors, using the membrane-permeable probe DAF-FM DA.
Fluorescence measurements (ex 495/em 515) provided data points, which were
reported using the given arbitrary units. (⁄) Indicates a statistically significant
difference (p <0.05) between the given compound/concentration combination and
the vehicle alone. The graphical data represents the mean SE of triplicate
determinations in the presence of (a) A60–A100 and (b) B60–B100.
cells by influencing a decrease in cell proliferation rather than ini-
tiating cell death. It should be pointed out that under mild NO in-
duced oxidative stress, SMCs undergo apoptosis rather than a
termination in cell proliferation.35,36 LIVE/DEAD assays conducted
by us indicate a lack of cell death when NO-donors are adminis-
tered, which has led us to attribute the decrease in viable cell pop-
ulation to the inhibition of proliferation. Furthermore, the
maximum concentration of NO delivered from the highest dose
of a NO-donor (20
lM) to 48,000–56,000 SMCs did not exceed
40 M (0.7–0.8 nM/cell). These calculations are based on the
l
hypothesis that a NO-donor loses both the NO moieties over a
96 h period and all NO or NO derived species enter the cells. The
calculated intracellular concentration of NO is significantly less
than 130 10 nM, which a single smooth muscle cell can with-
stand without induction of oxidative stress.37 These observations
coupled with the data from LIVE/DEAD assays establish the de-
crease in viable cells to the inhibition of proliferation only.
Finally, the addition of NO-donors to HASMC culture should in-
crease the overall presence of NO within the cells being treated.
DAF-FM DA was used to access the inner cellular environment,
and assess the intracellular NO and/or N2O3 concentration.21 The
results of the DAF-FM DA assays for both families of compounds
Figure 10. Determination of anti-proliferative and or cytotoxic effects exerted on
cultures of HASMCs by ABH at varying concentrations utilizing (a) MTT Viability
Assay and (b) LIVE/DEAD assay. (c) Additionally, changes in HASMC intracellular NO
and/or N2O3 concentrations were determined in the presence of varying ABH
concentrations using DAF-FM DA. (⁄) Indicates a statistically significant difference
(p <0.05) between the given compound/concentration combination and the positive
control. The graphical data represents the mean SE of duplicate determinations.

B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
1133
and the concentration available for cellular use is determined by
the initial concentration of the NO-donor used in these experi-
ments. In the case of B70, the backbone of each side chain consists
of an even number of sp3 hybridized atoms (a nitrogen and seven
carbon atoms). In contrast, B60 and B80, are each flanked by two
chains composed of an odd number of atoms. We speculate that
these attributes dictate the solubilities of these three compounds
in cDMSO. It is not unlikely that B70 (side chains with an even num-
ber of atoms) can achieve a greater degree of packing resulting in
decreased solubility compared to B60 and B80. At concentrations
65
tion. However, when the concentrations of B70 were increased to
10 and 20 M, the average absorbances decreased, due possibly
l
M, B70 exhibited the expected pattern of proliferation inhibi-
l
to the precipitation of B70 (Fig. 9b). On the other hand, B60 and
B80 being devoid of this solubility issue release increasing amounts
of NO with increasing concentrations. These effects due to the sol-
ubility differences are also observed with MTT (Fig. 7b) and LIVE/
DEAD (Fig. 8c) assays.
3.4.3. Effects of ABH induced, endogenous NO increase on
HASMC proliferation
The introduction of ABH, the most potent inhibitor of argi-
nase,20 should increase the bioavailability of
L-arginine to NOS
thereby enhancing endogenous NO production. In order to verify
this established hypothesis, PBS solutions of ABH were added in
various concentrations to HASMC culture (Section 2.6.4). Solutions
of LPS and the cytokine IFN-c were added to the culture medium to
activate inducible NOS (iNOS) 6 h prior to ABH administration.38
The cells were subsequently analyzed by both MTT and LIVE/DEAD
assays. Results from these studies are displayed in Figure 10a and
b, respectively. Analyses of these figures indicate that the inhibi-
tion of HASMC proliferation increases with increasing concentra-
tions of ABH. The highest concentration of ABH was maintained
at 5 mM (48% inhibition),39 which was more effective in inhibiting
cell proliferation than the 1 mM ABH concentration (35%). ABH
(5 mM) was used in combination with the selected NO-donor (at
optimum concentrations) to identify any synergistic effects. Find-
ings from these studies are delineated in Section 3.4.4 below.
DAF-FM DA was used again to determine the intracellular con-
centrations of NO and/or N2O3.21 The results from these experi-
ments are displayed in Figure 10c; the amount of intracellular
NO rises with increasing concentrations of ABH. Control experi-
ments using solutions of ABH, DAF-FM DA and ABH, DAF-FM in
the supplemented culture medium failed to produce enhanced
fluorescent signals. These findings clearly indicate that sufficient
inhibition of arginase, and the consequent build-up of the L-argi-
nine pool, allows for increased production of intracellular NO.
3.4.4. Synergistic effects of NO-donors and ABH on HASMC
proliferations
Of the eight NO-donors tested, compounds A60 and B60 (20
lM)
were chosen for in tandem addition with ABH (5 mM) to investi-
gate their combined effects on HASMC proliferation. To be consid-
ered synergistic, the reduction in proliferation rendered must be
greater than the sum of reduction caused by each component indi-
vidually. Results from both the MTT viability assay (Fig. 11a) and
the LIVE/DEAD test (Fig. 11b) reveal that both NO-donors in the
presence of ABH more effectively inhibit HASMC proliferation than
any of the three compounds alone. Furthermore, Figure 11b illus-
trates that the combined supplementation does not initiate addi-
tional cell death compared to the vehicle alone. To further
support these observations, the amount of intracellular NO and/
or N2O3 present, as detected by DAF-FM DA (Fig. 11c),21 is greater
in cells treated with the NO-donor or ABH than the untreated cells.
Tandem supplementation with these two reagents results in a lar-
ger increase of intercellular NO (compared to the negative control)
Figure 11. Determination of anti-proliferative and or cytotoxic effects exerted on
cultures of HASMCs by A60 (20 M) and/or B60 (20
l lM) in combination with ABH
(5 mM) utilizing (a) MTT Viability Assay and (b) LIVE/DEAD assay. (c) Additionally,
changes in HASMC intracellular NO and/or N2O3 concentrations were determined in
the presence of A60 (20 M) and/or B60 (20 M) + ABH (5 mM) using DAF-FM DA. (⁄)
l l
Indicates a statistically significant difference (p <0.05) between the given com-
pound/ABH combination and the negative control. The graphical data represents
the mean SE of duplicate determinations.
than the sum of singular NO-donor or ABH additions. Table 2 sum-
marizes the comparison between the singular and in tandem addi-
tions. The data contained allows for the determination of
synergistic, additive, or simply a more effective inhibition of
HASMC proliferation. For example, A60 with ABH exhibited an addi-

1134
B. Curtis et al. / Bioorg. Med. Chem. 21 (2013) 1123–1135
tional 13% decrease (64%) in cell proliferation when compared to
the sum of percent decreases (51%) from each of the components
alone. On the other hand, the combination of B60 and ABH brought
about an additional proliferation reduction of only 2% (48%, com-
pared to 46%); less than the combined effects of A60 and ABH,
which may be attributable to the higher half-life of B60. The anti-
proliferative effects of two arginase inhibitors, S-(2-boronoethyl)-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.043.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
L
-cysteine (BEC) and L L-arginine (L-NOHA) have been
-NG-hydroxy-
References and notes
reported.40 The anti-proliferative effects of ABH are exerted in a
similar manner by simultaneously increasing endogenous NO pro-
duction and inhibiting polyamine formation. The latter is accom-
plished as ABH inhibits arginase I, down regulating the
production of ornithine-the precursor to polyamines. Since poly-
amines are known to increase NO-independent HASMC prolifera-
tion,40,41 a decrease in polyamine concentrations would result in
a decrease in HASMC proliferation as well. Therefore, the co-
administration of a NO-donor along with a potent arginase inhibi-
tor is likely to be synergistic rather than additive.
1. Ignarro, L. J. In Nitric Oxide Biology and Pathobiology; Ignarro, L. J., Ed.; Academic
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4. Conclusions
Collectively, the above results verify the successful synthesis of
two different isomeric families of secondary amines via nuceleo-
philic aromatic substitution reactions, from two activated aromatic
difluorides. The resulting secondary amines were subsequently
nitrosated, with 100% efficiency to prepare the desired NO-donors.
Due to the strategic placement of the strong electron withdrawing
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Acknowledgments
The work was supported in part by Bridge to Commercialization
Fund (CMU) and National Institute of Health (NIH) Award Number
R15HL 106600 from the National Heart, Lung and Blood Institute
(NHLB). The content is solely the responsibility of the authors
and does not represent the official views of NHLB or NIH.

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