Design of barbiturate-nitrate hybrids that inhibit MMP-9 activity and secretion

Article abstract of DOI:10.1021/jm201352k

We describe a new type of barbiturate-based matrix metalloproteinase (MMP) inhibitor incorporating a nitric oxide (NO) donor/mimetic group (series 1). The compounds were designed to inhibit MMP at enzyme level and to attenuate MMP-9 secretion arising from

Full text of DOI:10.1021/jm201352k

Article
pubs.acs.org/jmc
Design of Barbiturate−Nitrate Hybrids that Inhibit MMP-9 Activity
and Secretion
Jun Wang, Shane O’Sullivan, Shona Harmon, Ray Keaveny, Marek W. Radomski, Carlos Medina,
and John F. Gilmer*
School of Pharmacy and Pharmaceutical Sciences, Trinity College, Dublin 2, Ireland
S
* Supporting Information
ABSTRACT: We describe a new type of barbiturate-based
matrix metalloproteinase (MMP) inhibitor incorporating a
nitric oxide (NO) donor/mimetic group (series 1). The
compounds were designed to inhibit MMP at enzyme level
and to attenuate MMP-9 secretion arising from inflammatory
signaling. To detect effects related to the nitrate, we prepared
and studied an analogous series of barbiturate C5-alkyl
alcohols that were unable to release NO (series 2). Both
series inhibited recombinant human MMP-2/9 activity with
nanomolar potency. Series 1 consistently inhibited the
secretion of MMP-9 from TNFα/IL1β stimulated Caco-2
cells at 10 μM, which could be attributed to NO related effects because the non-nitrate panel did not affect enzyme levels. Several
compounds from series 1 (10 μM) inhibited tumor cell invasion but none from the non-nitrate panel did. The work shows that
MMP-inhibitory barbiturates are suitable scaffolds for hybrid design, targeting additional facets of MMP pathophysiology, with
potential to improve risk-benefit ratios.
increased IL-1β mediated AP1 activation.17,18 At another
INTRODUCTION
The matrix metalloproteinase (MMP) enzymes and their tissue
■
level, NO is reported to destabilize MMP-9 mRNA, leading
to reduced enzyme levels.19 There are, on the other hand, some
inhibitors contribute to the regulation of the extracellular matrix
reports of NO activation of MMP activity.20 High levels of NO
and its byproduct can catalyze oxidation of the inhibitory
cysteine on its prodomain causing activation of proMMP-9.16,21
The interplay between NO and MMP-9 is evidently complex.
An MMP-inhibitory NO donor hybrid might be useful for
investigating this relationship, and it could have clinical
potential in pathological situations where inducible MMP-9
plays a significant role. NO-donating or NO-mimetic hybrids
have been designed for diverse drug types22−24 (particularly in
the anti-inflammatory field), leading in several instances to
clinical candidates with improved risk−benefit profiles.25
Usually, the NO-donating group abrogates activity and the
hybrid acts as a prodrug of the two moieties.23 However, the
topological features of the MMP-9 catalytic site suggested that
it might be possible to incorporate an NO-donor group into an
established MMP-inhibitor type without sacrificing enzyme
affinity.
and processing of myriad endogenous substances.1,2 Members
of the human MMP gene family (24) are classified according to
function and substrate specificity.3 Gelatinases A (MMP-2, EC
3.4.24.24) and B (MMP-9, EC 3.4.24.35) contain fibronectin-
like inserts, enabling them to bind and process gelatin. The
gelatinases degrade collagen type IV assisting cellular migration
processes4 that are relevant to the pathogenesis of cancer, heart
disease, and inflamation.5−8 MMP-9 is also linked with cancer
because this stromal-derived enzyme can promote tumor
angiogenesis. However, even in disease states, the gelatinases
continue to perform physiological functions, some of which
oppose the pathophysiology.9 Progress in the gelatinase
inhibitor field may therefore require approaches that can target
aberrantly regulated enzyme.
The promoter region of the MMP-9 gene contains cis-acting
regulatory elements with binding sites for NFkB, AP-1, and
PEA-3.10−13 MMP-9 expression is therefore responsive to
various cytokines including IL-1β, IL-2, IL-8, TNF-α, growth
factors, lipopolysaccharide, and phorbol esters.14,15 Endogenous
and pharmacologically derived nitric oxide (NO) is reported to
interact with these signaling pathways, leading in several
reported cases to inhibition of MMP-9 transcription/secretion.
For example, S-nitrosylation of its p50 subunit (NO+) has been
shown to reduce NFkB-binding in multiple cell lines.16,17
Incubation with the NO donor DETA-NO leads to reduced
AP1 binding, while inhibition of inducible NO synthase
C-5 disubstituted pyrimidine-2,4,6-triones (barbiturates) are
a relatively recently discovered MMP inhibitor drug-type with
attractive PK/PD properties (Figure 1A).26−29 Several high
resolution structures reveal a mode of binding for compounds
in this class that is similar to the more common hydroxamate
inhibitor type. The enolized barbiturate ring binds the Zn atom
Received: October 9, 2011
Published: January 16, 2012
© 2012 American Chemical Society
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selected in order to produce three amino-ethyl nitrates (1a−c),
as well as nitrates with greater separation between amino-
nitrogen and nitrate (1d−f). A parallel series of aminoalkyl
alcohol barbiturates (2) was independently produced by
reacting 3 with the corresponding aminoalkyl alcohols in
order to provide test compounds to isolate the contribution of
the nitrate group to inhibition of gelatinase secretion. The
identity and purity of the nitrates (series 1) and alcohols (series
2) was confirmed by IR, NMR, HPLC, and HRMS.
The inhibitory effects of series 1 and 2 on MMP-2 and
MMP-9 was measured using a fluorogenic substrate assay with
human recombinant MMP-2 and MMP-9. The compounds
were tested at 0.01−10000 nM following incubation for 45 min.
In general, the non-nitrate series 2 were more potent
inhibitors of MMP-2 and -9 than series 1 (Table 1). This
disparity was marked in the case of 2a, which was 86-fold more
potent for MMP-2 and 3.5-fold over MMP-9 than its nitrate
analogue 1a. The high potency of 2a against MMP-2 was a
somewhat surprising. However, the relatively high potency of
compounds in series 1 and 2 featuring piperazine or piperidines
is consistent with the literature on MMP inhibitory
barbiturates.26−30 Among the nitrates, 1b (IC50: MMP-2 =
53.05 nM, MMP-9 = 47.58 nM) was the most potent inhibitor
of both MMP-2 and MMP-9; indeed it departed from the
general pattern in being more potent against MMP-9 than its
non-nitrate analogue (2b). Another interesting nitrate, 1a
exhibited 3-fold selectivity for MMP-9 over MMP-2. The short
incubation time and test medium allow us to exclude any
contribution of nitrate activation to these results.
Figure 1. (A) C-5 piperazinyl barbiturates. (B) NO mimetic design
series 1 and parallel alcohol series 2. P1′, P2′ denote the Schechter and
Berger nomenclature for the pseudosubstrate positions occupied by
these groups on binding MMP-9
with one C-5 hydrophobic substituent occupying the deep S1′
pocket. The geminal C5′ group is directed toward the S2′
pocket, the outer rim of which is solvent exposed.30 These
observations suggested that an NO mimetic group such as an
organic nitrate could be placed at the terminus of either of the
C-5 substituents without significantly affecting binding.
In this study, we have prepared a panel of C-5 aminoalkyl
nitrates and the corresponding alcohols for comparison (Figure
1B). These were designed so that the C-5 phenyloxyphenyl
group would occupy the S1′ pocket with the alkyl nitrate group
directed into the S2′ pocket toward the solvent interface. We
provide evidence that the nitrates possess intrinsic MMP-9
inhibitory activity, interfere with cancer cell invasion, release
nitrite, and suppress tumor cell secretion of MMP-9.
SYNTHESIS AND EVALUATION OF GELATINASE
INHIBITORS
The key intermediate to generate series 1 was the barbiturate-5-
■
It was interesting to investigate the disposition of the nitrate
groups of series 1 on binding MMP-9, with the barbiturate and
5-phenyloxyphenyl group in their predicted orientations. Figure
2a shows representative conformations following docking
experiments (AutoDock 4) with 1a using the X-ray crystal
structure of a mutant MMP-9 bound to a 5-piperazinyl
barbiturate (PDB code 2OVX).30 Figure 2b shows a low
energy docking pose for 1c overlain with the piperazinyl ligand
in PDB 2OVX. The aminoalkyl nitrate groups of 1a and 1c are
directed through the S2′ pocket toward the enzyme surface.
The nitrate groups would be solvent exposed in this pose. The
greater potency of the aminoalkyl alcohols may be due to their
ability to form H-bonds with water and solvent surface. In
contrast, binding of series 1 in the high affinity mode places the
relatively lipophilic nitrate ester in a thermodynamically
unfavorable solvent exposed position. There is clearly scope
for enhancing potency in the nitrate-substituted class, possibly
by increasing polarity in the side chain or by moving the nitrate
further from the catalytic site. We did not try to improve
potency in this study because it was felt that the characteristics
of 1a−f represented an interesting balance of potential to
donate or mimic NO as well as gelatinase inhibitory potency.
The inhibitory data and docking analysis reinforce the idea that
barbiturate-based compounds could not only act as gelatinase
inhibitors by binding to MMP-2 and MMP-9, but scaffolds for
hybrid compounds possessing other pharmacological actions or
indeed NO-donating moieties such as furox(az)ans and
diazeniumdiolates.32
bromide 3 (Scheme 1). This is available in four steps from
Scheme 1. Preparation of 5-Phenoxyphenyl Barbiturate-5-
aminoalkyl Nitrates (1a−f) and Aminoalkylalcohols (2a−f)
a
a
(i) aminoalkyl nitrates or corresponding aminoalcohols, Et3N,
MeOH, RT, 24 h.
methyl 4-hydroxyphenylacetate as previously described.27 The
preparation of series 1 was achieved by allowing 3 to react with
various aminoalkyl nitrates in the presence of triethylamine and
MeOH at RT for 24 h (Scheme 1). The aminoalkyl nitrates
were themselves obtained by reacting the corresponding
commercially available aminoalkyl alcohols with fuming nitric
acid at −10 °C, over 20 min followed by addition of acetic
anhydride.31 The basic forms of the amines were isolated by
extraction of basic solutions of nitrate salts with ethyl acetate
and evaporation of the solvent. The aminoalkyl moieties were
MMPs can promote cancer cell invasion by degradation of
various extracellular matrix components resulting in release of
growth factor and cytokines. Inhibition of MMP activity,
especially MMP-9, has the potential to inhibit tumor growth
and metastasis.6 In addition, NO donors have been report to
inhibit cell proliferation and invasion, including pancreatic,
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a
Table 1. IC50 Values (nM) for Inhibition of Recombinant Human Gelatinases (95% CI)
a
The %I values represent the % reduction in the number of Caco-2 cells migrating across a matrigel membrane in response to stimulation with HGF
(100 ng/mL) in the presence of test compounds at 100 nM ( sd, n = 3 × 2).
prostate, and bladder cancer cells.33 Furthermore, NO can
inhibit the release of gelatinases from cancer cells. Therefore we
compared the effect of series 1 and 2 (100 nM) on cell
migration using an in vitro model of cancer cell invasiveness. In
this model, Caco-2 cells migrate through a matrigel membrane
following stimulation with hepatocyte growth factor (HGF, 100
ng/mL) (%I in Table 1). Relative amounts of cells on each side
of the membrane were counted by microscope following
staining. Both sets of compounds inhibited cell migration but
not in a manner that could be related to potency, gelatinase
selectivity, or ability to donate NO.
Because we had conducted a number of assays with the
compounds assessing biochemical effects that might be
attributable to NO related-effects, it was timely to make
some evaluation of their ability to undergo nitrate metabolism
in a cellular environment.
and the emergence of other NO donor types. Nitrate esters are
activated by mitochondrial aldehyde reductase-2 and by smooth
muscle cell P450 enzymes in a manner that is substrate
structure dependent.34 Indeed, the consequences of nitrate
metabolism and downstream effects are substrate and
concentration dependent.35,36 Nitrite and nitrate are prominent
metabolic byproducts and intermediates of these processes.37,38
Indeed, the NO donor/mimetic effects of organic nitrates may
be mediated via nitrite.34 We used a modified Griess assay to
measure the ability of the 1a−f to release nitrite/nitrate
(cumulatively NOx).39 In initial studies, we found that the
extent of NOx release was influenced by cell density. The
nitrate compounds 1a−f were incubated in the presence of
Caco-2 cells for 24 h at 200 and 10 μM (Table 2). A high level
of NOx was detected in the supernatants of cells treated with
the aminoethyl nitrate compounds 1a−c at 200 μM. This was
present as nitrite with 1a−b and nitrate in the case of 1c. At 10
μM, there were no apparent differences between the
compounds in capacity to generate NOx species, suggesting a
saturable metabolism pathway for the piperidine nitrates 1e−f.
Nitrate esters were selected for the present study because of
their ease of preparation, good stability, and relative lip-
ophilicity. Moreover, alkyl nitrates remain in widespread clinical
use despite concerns about tolerance or endothelial dysfunction
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phoretically. Then the gel was washed and incubated for 48 h to
allow gelatinolytic digestion to occur. The MMP-2 and MMP-9
activities of the resulting bands, as reflected in densitometry
values, provides an index of enzyme activity. We separately
showed with test inhibitors, and with the broad spectrum MMP
inhibitor phenanthroline, that under these conditions, the
density of the enzyme spot was unaffected by the inhibitor
which dissociates from the enzyme during separation and
becomes diluted to below its IC50. MMP-2 and MMP-9 were
highly expressed in PMA (10 μM) treated HT1080 cells. At
100 μM, compounds 1a, 1c, and 1e−f inhibited MMP-9
secretion by >80%. The compounds also inhibited MMP-2
expression, albeit to a lesser extent. Compounds 1b and 1d
caused modest inhibition of secretion of both enzymes. Because
MMP-2 is a housekeeping enzyme under indirect transcrip-
tional control (mainly through MTP-1/MMP1440), we
suspected that the apparently impressive effects on MMP
production could be partially due to effects on cell viability in
addition to dampening of pro-inflammatory signaling. There-
fore we evaluated the effect of series 1 and 2 on cell viability
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay (MTT) (Table 3). Several compounds from
both series exhibited low micromolar IC50 values for reducing
cell viability. Various chemically unrelated MMP inhibitors
(e.g., hydroxamates) have been reported to possess high
cytocidal activity, although it is not clear if this is related to
MMP inhibition; either way it may not be disadvantageous in a
cancer therapeutic.41 Notably, series 1 was overall of similar
cytocidal activity to the non-nitrates, although there were
potentially important differences in individual pairs. For
example, the piperazine nitrate 1c was cytotoxic but its non-
nitrate analogue 2c was not. Also the dinitrate 1a, which
exerted profound effects on both MMPs, was not cytotoxic but
its non-nitrate counterpart was (CC50: 8.2/6.8 μM in Caco-2/
HT1080).
The MTT assay results with HT1080 cells suggested that
some of the reduction in supernatant MMP-2 and MMP-9
observed previously was due to effects on cell viability.
However, if it was merely a cytotoxic effect, then the reduction
in the two enzyme levels should have been similar; instead, we
observed that MMP-9 activity in the supernatant was more
strongly affected than MMP-2 activity (Figure 3). Moreover, 1a
and 1e, which were not especially cytotoxic (CC50 72.0, 78.7
μM), nevertheless exerted significant inhibitory effects on
MMP-9 expression (38, 92%). We next sought to separate the
cytotoxic effects from those on gelatinase secretion on the
assumption that these were independent properties. The effect
of series 1 on cell viability was therefore further characterized at
10 μM using a flow cytometry approach to detect apoptotic
(Annexin V) and necrotic cells (propidium iodide). Com-
pounds 1a−f did not increase apoptotic or necrotic populations
Figure 2. Connolly surface on MMP-9 generated by the program
PyMOL based on the crystal structures of MMP-9 (PDB code 2OVX).
The ligand orientations of 1a (A) and 1c (B) had the lowest binding
energy of the conformations fitted into the designated pocket of the
MMP-9, calculated using Autodock4 based on the 100 runs in the
crystal structure of the MMP-9 (2OVX). The orientation of the
overlaid ligand 5-(4-phenoxyphenyl)-5-(4-pyrimidine-2-ylpiperazin-1-
yl-)pyrimidine-2,4,6-trione was directly taken from the mutant MMP-9
crystal structure file (2OVX).30 The surfaces were generated by the
programme PyMOL based on the crystal structures of MMP-9 (PDB
code 2VOX). Ligand orientations were used which had the lowest
binding energy of the conformations of 50 runs with 25000000 energy
evaluations in Autodock4 and displayed by PyMOL. The active site Zn
atoms appear as red spheres.
The impact of the hybrid compounds (100 μM) on
gelatinase secretion, a key proof-of-concept assay, was
performed using HT1080 fibrocarcinoma cells stimulated
with phorbolmyristate acetate (PMA, 10 μM). The flasks
were incubated at 5% CO2, 37 °C for 24 h, after which time the
supernatants were collected. Supernatants from the treated cells
were loaded onto a zymography gel at constant protein
concentration and MMP-2 and MMP-9 separated electro-
Table 2. Griess Assay Values for NO2− and NO3− Following Incubation of Series 1 at 200 and (10) μM over Caco-2 Monolayers
a
for 24 h (n = 3)
1a
1b
1c
1d
1e
1f
NOx
232.0 14.1,
(6.5 1.3)
128.7 8.8,
(5.7 0.7)
124.4 4.2,
(7.0 0.9)
44.5 10.8,
(6.9 1.6)
47.6 10.1,
(4.3 0.9)
43.3 12.0,
(7.3 0.9)
−
−
NO2
NO3
164.3 9.6
67.8 12.9
109.7 19.9
46.5 10.1
77.9 3.4
40.2 15.1
4.3 4.1
30.7 14.4
16.8 11.6
34.7 11.8
8.6 3.6
18.9 9.2
a
−
−
NOx values represent the sum of NO2 and NO3 .
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Table 3. Effect of Compounds on Cell Viability (MTT) of Caco-2 and HT1080 Cells Expressed As CC50 (Concentration
Causing 50% Reduction in Cell Viability (95% Confidence Interval)) or % Inhibition of Viability at a Given Concentration
a
Where There Was No Convergence with the Viability Data over a Concentration Range
CC50 (μM)
CC50 (μM)
Caco-2
HT1080
Caco-2
HT1080
1a
1b
1c
1d
1e
1f
>100
72.0
2a
2b
2c
2d
2e
2f
8.2
6.8
(6%, 100 μM)
(16.4−316.0)
(6.4−10.4)
(5.5−8.4)
>100
(28%, 100 μM)
48.5
(40.3−58.2)
12.8
(7.0−23.4)
78.7
(48.9−126.5)
11.8
(7.3−19.0)
76.5
(44.6−131.2)
>100
(25%, 100 μM)
78.5
(61.3−100.4)
>100
(33%, 100 μM)
26.0
(15.4−43.8)
>200
(100%, 200 μM)
25.2
(19.8−32.0)
47.8
(37.8−60.6)
33.1
(27.4−40.0)
30.1
(26.1−34.7)
66.57
(55.85−79.3)
46.4
(29.6−72.5)
>12.5
(12%, 2.5 μM)
>12.5
(37%, 12.5 μM)
8.1
(5.6−11.5)
a
CC50 values were estimated from sigmoidal curves fitted to data across eight concentration levels, 0.1−200 μM (n = 3)
Figure 3. Zymographic analysis of MMP-2/MMP-9 activity from PMA
(10 μM) stimulated HT1080 cell supernatants following treatment
with 1a−f at 24 h at 100 μM showing large reductions in the activity of
both enzymes.
Figure 4. Normalized densitometry of MMP-9 gelatinolytic activity
from supernatants of TNFα/IL1β (positive control (PC)) stimulated
Caco-2 cell following treatment with 1a−f or 2a−f at 24 h at 10 μM.
*p < 0.05.
following treatment of Caco-2 or HT1080 cells at 10 μM for 24
h (Supporting Information).
Thus, having validated a test concentration of 10 μM, the
effect of series 1 and 2 on gelatinase secretion was assessed over
24 h using Caco-2 cells. We used this cell line because it had
been less susceptible to loss of viability than the HT1080 cell
line in the MTT assay. A mixture of IL1β/TNFα was used to
stimulate cells in these experiments because this generates a
more robust inflammatory response than PMA in Caco-2 cells.
Series 1 and 2 were directly compared in an effort to exclude
effects arising from their differences in potency/selectivity
against the gelatinases or other MMPs (Figure 4). At 10 μM, all
members of series 1 reduced MMP-9 secretion relative to
control (p < 0.05). No effects were observed on MMP-2
levels.The nitrate compounds were consistently more effica-
cious inhibitors of MMP-9 secretion than series 2. These
differences were significant in pairs 1b/2b, 1c/2c, and 1f/2f.
Because we had shown that the series 1 at 10 μM were
capable of inhibiting cytokine-induced MMP-9 secretion, we
repeated the tumor cell invasion assay at this higher
concentration. We also increased the stimulus concentration
(HGF (500 ng/mL) and PMA (10 μM)) in order to produce a
stronger MMP-9 release and a more aggressively invasive
cellular response. Under HGF stimulation, 1a significantly
inhibited tumor cell invasion (51%) whereas its non-nitrate
analogue 1b had no effect. The effects of the compounds on
PMA induced invasion are presented in Figure 5. Nitrate
analogues 1a, 1d, and 1e significantly inhibited cellular
migration under PMA stimulation (43.3%, 44.5%, and 64.9%,
Figure 5. Caco-2 cell invasion through a matrigel membrane following
stimulation with 10 μM PMA (positive control) and following
incubation in the presence of nitrate/non nitrate pairs with 1a−e or
2a−e for 48 h. Values are normalized to positive control. p < 0.05
(Anova with Bonferroni Multiple Comparisons (sd, n = 3)).
respectively). The pair 1c/2c inhibited cell migration, but the
effect was not significant. None of the non-nitrates significant
affected cell invasion under these conditions.
CONCLUSION
■
Several novel nitrate-substituted barbiturate-based gelatinase
inhibitors were prepared maintaining a classical MMP P1′
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5-(4-Phenoxyphenyl)-5-(4-(2-nitrooxy-ethyl)piperazin-1-yl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (1c). Product was obtained as off-
white solids (197 mg, 42.0%); mp 206−208 °C. HRMS: C22H24N5O7
phenoxyphenyl group at C5 but with a geminal P2′ nitroxyalkyl
amino group. The compounds retained intrinsic ability to
inhibit gelatinase activity of recombinant human gelatinases but
also liberated nitrite in a structure and concentration dependent
manner. Compounds from series 1 inhibited MMP-9 secretion
from Caco-2 cells stimulated with inflammatory cytokines,
whereas their non-nitrate analogues did not. Nitrate com-
pounds 1a, 1d, and 1e (10 μM) also inhibited tumor cell
invasion in response to PMA, whereas their non-nitrate
analogues did not. These interesting compounds merit further
exploration of the interplay of NO and MMP-inhibitory factors.
There is further scope within the general design for modulating
inhibitory potency, selectivity, and NO donor/mimetic proper-
ties. The balance between inhibitory potency and NO-mediated
anti-inflammatory effects will require study in appropriate
disease models.
1
requires 470.1676; found 470.1679. H NMR δ (DMSO-d6) ppm:
2.47−2.51 (m, 6H), 2.62−2.68 (m, 4H), 4.05−4.08 (m, 2H), 7.02−
7.07 (m, 4H), 7.17−7.21 (t, J = 7.53, 1H), 7.40−7.43 (m, 4H). 13C
NMR δ (DMSO-d6) ppm: 47.8, 52.8, 57.3, 71.0, 74.0, 118.2, 118.4,
119.2, 121.4, 124.2, 129.6, 130.2, 150.2, 155.9, 159.3. 170.1. IR (KBr)
ν (cm−1): 3066, 1710, 1684, 1640, 1281, 849.
5-(4-Phenoxyphenyl)-5-(4-(1-nitrooxy-ethyl)piperidin-1-yl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (1d). Product was obtained as off-
white solids (158 mg, 34.8%); mp 190−192 °C. HRMS: C22H23N4O7
1
requires 455.1561; found 455.1567. H NMR δ (CDCl3) ppm: 1.16−
1.27 (m, 2H) 1.47−1.58 (m, 3H), 2.32−2.37 (m, 2H), 2.77−2.83 (m,
2H), 4.84−4.90 (m, 2H), 6.92−6.95 (d, J = 8.53, 1H), 6.97−6.99 (d, J
= 8.53, 2H), 7.01−7.03 (d, J = 8.53, 1H), 7.15−7.18 (t, J = 7.53),
7.37−7.40 (t, J = 7.53, 1H), 7.43−7.48 (m, 3H). 13C NMR δ (CDCl3)
ppm: 27.4, 34.2, 49.1, 75.7, 76.1, 118.4, 119.8, 121.3, 124.3, 129.6,
130.2, 150.7, 155.9, 159.4, 170.6. IR (KBr) ν (cm−1): 3068, 1732,
1694, 1642, 1286, 849.
EXPERIMENTAL SECTION
■
5-(4-Phenoxyphenyl)-5-(4-(2-nitrooxy-ethyl)piperidin-1-yl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (1e). Product was obtained as off-
white solids (145 mg, 30.9%); mp 201−203 °C. HRMS: C23H25N4O7
requires 469.1723; found (M + H)+ = 469.1723. 1H NMR δ (CDCl3)
ppm: 1.33−1.39 (m, 2H,) 1.42−1.46 (m, 2H), 1.48−1.51 (m,
1H),1.73−1.78 (m, 2H), 2.57−2.64 (m, 2H), 2.76−2.79 (m, 2H),
4.78−4.82 (m, 2H), 6.92−6.94 (d, J = 8.53, 1H), 6.96−6.98 (d, J =
8.53, 2H), 7.03−7.05 (d, J = 8.53, 1H), 7.16−7.19 (t, J = 7.53, 1H),
7.36−7.40 (t, J = 7.53, 1H), 7.45−7.49 (m, 3H), 8.99 (s, 2H). 13C
NMR δ (CDCl3) ppm: 21.4, 32.2, 32.6, 48.1, 70.5, 76.0, 118.2, 119.7,
121.2, 124.2, 129.5, 129.9, 148.7, 155.8, 158.1, 169.8. IR (KBr) ν
(cm−1): 3068, 1732, 1704, 1633, 1285, 849.
All chemicals were purchased from Sigma-Aldrich (Ireland), except
where stated. All reactions were monitored using TLC. Uncorrected
melting points were measured on a Stuart Apparatus. Infrared (IR)
spectra were acquired on a Perkin-Elmer FT-IR Paragon 1000
spectrometer. 1H and 13C nuclear magnetic resonance (NMR) spectra
were recorded at 27 °C on a Bruker DPX 400 spectrometer (400.13
MHz, 1H; 100.61 MHz, 13C). Coupling constants are reported in
hertz. Electrospray ionization mass spectrometry (ESI-MS) was
performed in the positive ion mode on a liquid chromatography
time-of-flight mass spectrometer (Micromass LCT, Waters Ltd.,
Manchester, UK). The samples were introduced into the ion source
by an LC system (Waters Alliance 2795, Waters Corporation, USA) in
acetonitrile:water (60:40% v/v) at 200 μL/min. The capillary voltage
of the mass spectrometer was at 3 kV. The sample cone (declustering)
voltage was set at 40 V. For exact mass determination, the instrument
was externally calibrated for the mass range m/z 100 to m/z 1000. A
lock (reference) mass (m/z 556.2771) was used. Mass measurement
accuracies of < 5 ppm were obtained. RPHPLC was used to establish
compound purity. The stationary phase was a Waters Xbridge C18, 5
μm column (4.6 mm × 150 mm); the mobile phase consisted of a
mixture of methanol and water (60:40) at a flow rate of 1 mL/min.
HPLC was performed using a system consisting of a Waters 600 pump
and controller, Waters 717 autosampler and a Waters 996 photodiode
array detector controlled by Waters Empower Software. Chromato-
grams were extracted at 230 nm. All test compounds were >95% pure.
General Synthetic Method. A solution of 5-bromo-5-(4-
phenoxyphenyl)pyrimidine-2,4,6(1H,3H,5H)-trione (3) in MeOH
was treated with the appropriate alkyl amine (1.5 equiv) and Et3N
(3 equiv) at room temperature for 24 h. The solvents were evaporated
and the products separated directly using flash chromatography.
5-(4-Phenoxyphenyl)-5-(bis-(2-nitrooxy-ethyl)-amino)pyrimidine-
2,4,6(1H,3H,5H)-trione (1a). Product obtained as off-white solids (194
mg, 39.6%); mp 184−186 °C. HRMS: C20H19N5O10 [M + Na+]
requires 512.1182; found 512.1168. 1H NMR δ (MeOH-d4) ppm:
2.84−2.87 (t, J = 5.02, 4H), 4.64−4.67 (t, J = 5.02, 4H), 6.91−7.01
(m, 4H), 7.12−7.15 (t, J = 7.53, 1H), 7.34−7.38 (t, J = 7.53, 3H),
7.46−7.51 (m, 3H). 13C NMR δ (MeOH-d4) ppm: 46.7, 72.0, 100.8,
119.3, 120.6, 122.2, 125.2, 128.7, 131.1, 150.6, 157.6, 160.2, 170.5. IR
(KBr) ν (cm−1): 3068, 1728, 1706, 1648, 1281, 849.
5-(4-Phenoxyphenyl)-5-(3-(1-nitrooxy-methyl)piperidin-1-yl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (1f). Product was obtained as off-
white solids (181 mg, 39.8%); mp 190−192 °C. HRMS: C22H23N4O7
1
requires 455.1568; found 455.1567. H NMR δ (CDCl3) ppm: 1.28−
1.30 (m, 1H), 1.3−1.40 (m, 1H), 1.56−1.59 (m, 1H), 1.64−1.68 (m,
1H), 1.71−1.76 (m, 1H, CH), 2.08−2.13 (m, 1H), 2.51−2.57 (m,
1H), 2.61−2.68 (m, 1H), 2.71−2.75 (m, 1H), 4.37−4.41 (m, 1H),
4.52−4.57 (m, 1H), 6.92−6.94 (d, J = 8.53, 1H), 6.96−6.99 (d, J =
8.53, 1H), 7.04−7.06 (d, J = 8.53, 1H), 7.16−7.20 (t, J = 7.53, 1H),
7.36−7.40 (t, J = 7.53, 2H), 7.45−7.49 (m, 2H), 9.15 (s, 1H), 9.16 (s,
1H). 13C NMR δ (CDCl3) ppm: 26.6, 34.2, 48.8, 50.7, 53.7, 73.7, 74.6.
118.5, 119.7, 121.3, 124.2, 129.4, 129.9, 148.9, 155.7, 158.9, 170.0. IR
(KBr) ν (cm−1): 3066, 1723, 1688, 1640, 1281, 853.
Biological Methods. Cell Culture and Treatment. Human
fibrosarcoma HT-1080 cells were cultured in Eagle’s Minimal
Essential Medium (MEM) containing 10% fetal bovine serum
(FBS), 100 IU/mL penicillin, and 50 μg/mL streptomycin. The
cultures were incubated at 37 °C in a humidified 5% CO2
atmosphere until confluence. Caco-2 cells were cultivated in
Eagle’s minimal essential medium (MEM) containing 20% fetal
bovine serum (FBS), 100 IU/mL penicillin, and 50 μg/mL
streptomycin.
Zymography. HT1080 cells were grown in 25 mL tissue culture
flasks until approximately 75% confluent. The media was then changed
to serum-free media, and the cells were treated with test compounds
(100 μM) and incubated for 3 h. PMA (10 μM) was then added, and
the flasks were incubated for 24 h at 37 °C in a humidified 5% CO2
atmosphere. The compounds were introduced in DMSO such that the
final concentration did not exceed 0.1% DMSO. After 24 h, the
supernatant was removed and centrifuged at 13000 rpm for 5 min to
remove dead cells or floating debris. Supernatants were stored at −80
°C until analysis. Caco-2 cells were grown in 25 mL tissue culture
flasks in 20% FBS media until approximately 75% confluent. The
media was then changed to serum-free, and the cells were then treated
with test compounds at 100 nM or 10 μM and incubated for 30 min.
Cytokines TNF-α and Il-1β were then added at 10 ng/mL, and the
flasks were incubated for 24 h at 37 °C in a humidified 5% CO2
atmosphere. The compounds were dissolved in DMSO to give a final
5-(4-Phenoxyphenyl)-5-(methyl-(2-nitrooxy-ethyl)-amino)-
pyrimidine-2,4,6(1H,3H,5H)-trione (1b). Product was obtained as off-
white solids (134 mg, 32.3%); mp 143−145 °C. C19H18N4O7 [M +
1
Na+] requires 437.1073; found 437.1078. H NMR δ (CDCl3) ppm:
2.41 (s, 3H), 2.91−2.96 (m, 2H), 4.37−4.42 (m, 2H), 6.90−6.92 (d, J
= 8.03, 2H), 7.00−7.02 (d, J = 8.03, 1H), 7.15−7.18 (t, J = 7.53, 1H),
7.34−7.38 (t, J = 7.53, 1H), 7.40−7.46 (m, 3H). 13C NMR δ (CDCl3)
ppm: 38.2, 49.8, 70.8, 100.8, 118.3, 119.8, 121.4, 124.6, 129.5, 130.0,
150.8, 155.6, 159.1, 171.2. IR (KBr) ν (cm-1): 3066, 1720, 1698, 1644,
1280, 842.
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DMSO concentration of <1%. At 24 h, the conditioned media were
collected and centrifuged at 13000 rpm for 5 min at RT to remove any
dead cells or floating debris. The resultant supernatants were stored at
−80 °C until analysis. Samples were normalized with respect to
protein content using a Bradford assay. The enzymatic activities of
MMP-2 and MMP-9 were assayed by gelatin zymography in serum-
free media. The samples were electrophoresed on an SDS−PAGE
containing 2% gelatin. The gels were washed with 2.5% Triton X three
times for 20 min cycles. The gels were then washed twice and finally
incubated with zymography buffer (0.15 M NaCl, 5 mM CaCl2, 0.05%
NaN3, and 50 mM Tris-HCl buffer, pH 7.5) at 37 °C for 48 h. After
incubation, the gels were stained with 0.025% Coomassie Brilliant Blue
G250 in 25% MeOH, 10% acetic acid, and H2O and destained with
acetic acid 8%, methanol 4%, and H2O. The gelatinolytic activity was
detected as a band of gelatin digestion and was quantified by
densitometry using gel documentation system r (Bio-RAD, Universal
hood II and Quantity One 4.6 software) and expressed as a percentage
of the positive control (HT-1080 cells incubated with serum free
media and cytokines in the absence of inhibitors).
removed from T25 flasks and washed with 10× binding buffer (0.1 M
Hepes (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) before being
resuspended in 100 mL of 1× binding buffer. A 20 mL aliquot of
these cells was stained using 5 mL of Annexin V-FITC and 5 mL of
propidium iodide and incubated at room temperature for 15 min in
the dark. Following incubation, samples were diluted in 1× binding
buffer and analyzed within 5 min using a BD FACSArray (BD
Biosciences, Oxford, UK). Flow cytometry was performed on double-
stained cell samples (Caco-2 and HT1080 cells). The instrument was
set up to measure the size (forward scatter), granularity (side scatter),
and cell fluorescence. Antibody binding was measured by analyzing
individual cells for fluorescence. Data were analyzed using BD FACS
Array software and expressed as a percentage of control fluorescence in
arbitrary units.
Griess Assay. A series of nitrite and nitrate standard solutions were
prepared with sodium nitrite and sodium nitrate from 3 to 200 μM. A
200 μL aliquot of each nitrite standard solution and 200 μL of water
was added to a 96-well plate.42 For nitrate standard solutions, 200 μL
of 4 °C saturated solution of VCl3 was added instead of water. The
blank wells contained only 400 μL of water. All the wells were treated
at 4 °C with 100 μL of sulfanilamide (2% w/v) and 100 μL of N-1-
napthylethylenediamine dihydrochloride (0.1 w/v). The plate was
shaken for 45 min, and the absorbance was measured at 540 nm. The
supernatants of the barbiturate-based nitrate treated Caco-2 cells
following PMA stimulation (10 μM) were tested in similar manner.
The resulting absorbance values of the supernatants were applied to
the calibration line formula. The blank cellular data was generated by
treating the cells with PMA in the absence of test article.
Molecular Modeling. The structure of MMP-9 (PDB code 2VOX)
used was an MMP-9 active site mutant with barbiturate inhibitor. The
barbiturate inhibitor and water molecules were removed. Docking
calculations were carried out using AutoDock version 4.0 with the
Lamarckian genetic algorithm (LGA). The molecular models of each
inhibitor were built using the builder function of MOE and minimized
with MOPAC 7 (AM1 method) interfaced to MOE. The Zn
parameters were changed to zinc radius, 0.87 Å; well depth, 0.35
kcal/mol; and Zn atomic charge, +0.95. The 3D affinity grid box was
designed to include the full active site and possible residues. The
setting of the center of grid boxes was based on the value of active Zn.
Docking calculations were set to 20 runs. Then 25000000 energy
evaluations were allowed as a maximum in each run. At the end of the
calculation, AutoDock performed cluster analysis. Docking solutions
with ligand all-atom root-mean-square deviation (rmsd) within 2.0 Å
of each other were clustered together and ranked by the lowest energy
representative.
Statistical Analysis. All data are presented as group of means with
standard error of the mean of n ≥ 3. Statistical analysis of the mean
difference between multiple groups was determined by one-way
ANOVA followed by Tukey−Kramer multiple comparison post test. A
P value <0.05 was considered to be statistically significant. All
statistical analyses were performed using Prism version 4 for Windows,
GraphPad Software, La Jolla, California, USA.
MMP-2 and MMP-9 Fluorogenic Assay. Recombinant proMMP-2
and proMMP-9 (R&D Systems, Ireland) were activated by treatment
with p-aminophenylmercuric acetate at 37 °C for 1 and 24 h,
respectively. The synthetic broad-spectrum fluorogenic substrate (7-
methoxycoumarin-4-yl)-acetyl-pro-Leu-Gly-Leu-(3-(2,4-dinitrophen-
yl)-L-2,3-diaminopropionyl)-Ala-Arg-NH2 (R&D Systems, UK) was
used to assay MMP-2 and MMP-9 activity, as described.26
Invasion Assay. The BD BioCoat Matrigel Invasion Chamber assay
system was used to study the pharmacological effects of the nitrates
and alcohols on tumor cell migration. Briefly, the assay utilizes an
invasion chamber consisting of a tissue culture plate with cell culture
inserts and an 8 μm pore size polycarbonate membrane. The upper
surface of the insert membrane is coated with a thin layer of Matrigel
Basement Membrane Matrix. Matrigel is an extract of basement
membrane derived from a murine tumor, with identical components,
both chemically and immunologically, to authentic basement
membrane. The layer occludes the pores of the membrane, blocking
noninvasive cells from migrating through the pores. Invasive cells, on
the other hand, are able to degrade the matrix proteins that occlude
the pores and this ability allows them to pass through the pores. The
invasion assay was performed according to the manufacturer’s
instructions. Briefly, aliquots of 0.5 mL of cell suspensions consisting
of 2 × 105 Caco-2 cells/mL were added to the inserts and allowed to
invade for 48 h in a humidified tissue culture incubator, at 37 °C, 5%
CO2 atmosphere. The tumor cells were stimulated with either PMA
(10 μM) or HGF (100 ng/mL or 500 ng/mL). The noninvading cells
were afterward removed by scrubbing with a cotton tipped swab and
the cells on the lower surface of the membrane stained with Diff-Quik
stain. The membrane was then examined by light microscopy using an
Olympus CKX41 microscope (Olympus America Inc., Melville, NY,
USA). Photomicrographs were captured using a digital camera and
MicroFire (Olympus America Inc.) software. All the experiments were
carried out in duplicate.
MTT Assay. Cell viability was determined by MTT assay. Cells were
seeded (Caco-2 at 8 × 103 cells/well and HT1080 at 12 × 104 cells/
well) into 96-well plates and grown until 70% confluent. Cells were
serum starved for 1 h and then treated with test compounds at 0.1−
200 μM under serum-free conditions for 24 h. At the end of this
incubation, MTT (0.5 mg/mL) was added and incubated for 3 h at 37
°C. After 3 h, supernatants were removed, 100 μL of DMSO was
added, and the plates were incubated with shaking for 10 min. The
formazan crystals formed inside the viable cells were solubilized in
DMSO, and the optical density was read on a plate reader at 540 nm.
The % cell viability was determined as follows: (optical density of
treated cells/optical density of nontreated cells) × 100. The
experiments were performed a minimum of three times with three
replicates per concentration.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis and characterization data for compounds 2a−f; flow
cytometry analysis of apoptosis in Caco-2 and HT1080 cells.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Annexin V Flow Cytometry Assay. Cells were grown to 80%
confluence in T25 flasks and treated with test compounds for 24 h in
serum-free medium; control cells were treated only with vehicle in
serum-free media for 24 h. Following 24 h treatment, cells were
Corresponding Author
*Phone: +353-1-896 2795. Fax: +353-1-896 2793. E-mail:
gilmerjf@tcd.ie.
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Article
(15) Li, W.; Li, H.; Bocking, A. D.; Challis, J. R. Tumor necrosis
factor stimulates matrix metalloproteinase 9 secretion from cultured
human chorionic trophoblast cells through TNF receptor 1 signaling
to IKBKB-NFKB and MAPK1/3 pathway. Biol. Reprod. 2010, 83,
481−487.
ACKNOWLEDGMENTS
■
This work was supported in part by a Science Foundation of
Ireland (SFI) Research Frontiers grant (RFP/BMT2781)
awarded to C.M. C.M. is SFI Stokes lecturer.
(16) Marshall, H. E.; Stamler, J. S. Inhibition of NF-kappa B by S-
nitrosylation. Biochemistry 2001, 40, 1688−1693.
ABBREVIATIONS USED
■
(17) Pfeilschifter, J.; Eberhardt, W. Molecular mechanisms of nitric
oxide-dependent inhibition of TPA-induced matrix metalloproteinase-
9 (MMP-9) in MCF-7 cells. J. Cell Physiol. 2009, 219, 276−287.
(18) Shin, C. Y.; Lee, W. J.; Choi, J. W.; Choi, M. S.; Ryu, J. R.; Oh,
S. J.; Cheong, J. H.; Choi, E. Y.; Ko, K. H. Down-regulation of matrix
metalloproteinase-9 expression by nitric oxide in lipopolysaccharide-
stimulated rat primary astrocytes. Nitric Oxide 2007, 16, 425−432.
(19) Akool, el-S.; Kleinert, H.; Hamada, F. M.; Abdelwahab, M. H.;
AP-1, activator protein 1; annexin V FTC, annexin V
fluorescein conjugate; DETA-NO, diethylenetriamine NON-
Oate; FBS, fetal bovine serum; HRMS, high resolution mass
spectrometry; HGF, hepatic growth factor; HPLC, high
performance liquid chromatography; IL-1β, interleukin-1β;
IL-2, interleukin-2; IL-8, interleukin-8; MTP-1, membrane
type matrix metalloproteinase-1; MTT, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide assay; NfKB, nuclear
factor kB; PK/PD, pharmacokinetic/pharmacodynamic; PMA,
phorbolmyristate acetate; TNF-α, tumor necrosis factor-α
Forstermann, U.; Pfeilschifter, J.; Eberhardt, W. Nitric oxide increases
̈
the decay of matrix metalloproteinase 9 mRNA by inhibiting the
expression of mRNA-stabilizing factor HuR. Mol. Cell. Biol. 2003, 23,
4901−4916.
(20) Ridnour, L. A.; Windhausen, A. N.; Isenberg, J. S.; Yeung, N.;
Thomas, D. D.; Vitek, M. P.; Roberts, D. D.; Wink, D. A. Nitric oxide
regulates matrix metalloproteinase-9 activity by guanylyl-cyclase-
dependent and -independent pathways. Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 16898−16903.
(21) McCarthy, S. M.; Bove, P. F.; Matthews, D. E.; Akaike, T.; van
der Vliet, A. Nitric oxide regulation of MMP-9 activation and its
relationship to modifications of the cysteine switch. Biochemistry 2008,
47, 5832−5840.
(22) Martelli, A.; Rapposelli, S.; Calderone, V. NO-releasing hybrids
of cardiovascular drugs. Curr. Med. Chem. 2006, 13, 609−625.
(23) Jones, M.; Inkielewicz, I.; Medina, C.; Santos-Martinez, M. J.;
Radomski, A.; Radomski, M. W.; Lally, M. N.; Moriarty, L. M.;
Gaynor., J.; Carolan, C. G.; Khan, D.; O’Byrne, P.; Harmon, S.;
Holland, V.; Clancy, J. M.; Gilmer, J. F. Isosorbide-based aspirin
prodrugs: integration of nitric oxide releasing groups. J. Med. Chem.
2009, 52, 6588−6598.
REFERENCES
■
(1) Clark, I. M.; Swingler, T. E.; Sampieri, C. L.; Edwards, D. R. The
regulation of matrix metalloproteinases and their inhibitors. Int. J.
Biochem. Cell Biol. 2008, 40, 1362−1378.
(2) Malemud, C. J. Matrix metalloproteinases (MMPs) in health and
disease: an overview. Front. Biosci. 2006, 11, 1696−1701.
(3) Brew, K.; Nagase, H. The tissue inhibitors of metalloproteinases
(TIMPs): an ancient family with structural and functional diversity.
Biochim. Biophys. Acta 1803, 55−71.
(4) Olson, M. W.; Toth, M.; Gervasi, D. C.; Sado, Y.; Ninomiya., Y.;
Fridman, R. High affinity binding of latent matrix metalloproteinase-9
to the alpha2(IV) chain of collagen IV. J. Biol. Chem. 1998, 273,
10672−10681.
(5) Back, M.; Ketelhuth, D. F.; Agewall, S. Matrix metalloproteinases
in atherothrombosis. Prog. Cardiovasc. Dis. 2010, 52, 410−428.
(6) (a) Gialeli, C.; Theocharis, A. D.; Karamanos, N. K. Roles of
matrix metalloproteinases in cancer progression and their pharmaco-
logical targeting. FEBS J. 2011, 278, 16−27. (b) Rudek, M. A.; Venitz,
J.; Figg, W. D. Matrix metalloproteinase inhibitors: do they have a
place in anticancer therapy? Pharmacotherapy 2002, 22, 705−720.
(7) Medina, C.; Radomski, M. W. Role of matrix metalloproteinases
in intestinal inflammation. J. Pharmacol. Exp. Ther. 2006, 318, 933−
938.
(8) Naito, Y.; Yoshikawa, T. Role of matrix metalloproteinases in
inflammatory bowel disease. Mol. Aspects Med. 2005, 26, 379−390.
(9) Martin, M. D.; Matrisian, L. M. The other side of MMPs:
protective roles in tumor progression. Cancer Metastasis Rev. 2007, 26,
717−724.
(10) Chakraborti, S.; Mandal, M.; Das, S.; Mandal, A.; Chakraborti,
T. Regulation of matrix metalloproteinases: an overview. Mol. Cell.
Biochem. 2003, 253, 269−285.
(11) Gum, R.; Lengyel, E.; Juarez, J.; Chen, J. H.; Sato, H.; Seiki, M.;
Boyd, D. Stimulation of 92-kDa gelatinase B promoter activity by ras is
mitogen-activated protein kinase kinase 1-independent and requires
multiple transcription factor binding sites including closely spaced
PEA3/ets and AP-1 sequences. J. Biol. Chem. 1996, 271, 10672−
10680.
(12) Simon, C; Simon, M; Vucelic, G; Hicks, M. J.; Plinkert, P. K.;
Koitschev, A; Zenner, H. P. The p38 SAPK pathway regulates the
expression of the MMP-9 collagenase via AP-1-dependent promoter
activation. Exp. Cell Res. 2001, 271, 344−355.
(24) (a) Thatcher, G. R.; Bennett, B. M.; Reynolds, J. N. NO
chimeras as therapeutic agents in Alzheimer’s disease. Curr. Alzheimer
Res. 2006, 3, 237−245. (b) Boschi, D.; Tron, G. C.; Lazzarato, L.;
Chegaev, K.; Cena, C.; Di Stilo, A.; Giorgis, M.; Bertinaria, M.;
Fruttero, R.; Gasco, A. NO-donor phenols: a new class of products
endowed with antioxidant and vasodilator properties. J. Med. Chem.
2006, 49, 2886−2897. (c) Abdellatif, K. R.; Chowdhury, M. A.;
́
Velazquez, C. A.; Huang, Z.; Dong, Y.; Das, D.; Yu, G.; Suresh, M. R.;
Knaus, E. E. Celecoxib prodrugs possessing a diazen-1-ium-1,2-diolate
nitric oxide donor moiety: synthesis, biological evaluation and nitric
oxide release studies. Bioorg. Med. Chem. Lett. 2010, 20, 4544−4549.
(d)
(25) Schnitzer, T. J.; Kivitz, A.; Frayssinet, H.; Duquesroix, B.
Efficacy and safety of naproxcinod in the treatment of patients with
osteoarthritis of the knee: a 13-week prospective, randomized,
multicenter study. Osteoarthritis Cartilage 2010, 18, 629−639.
̀
(26) Grams, F.; Brandstetter, H.; D’Alo, S.; Geppert, D.; Krell, H. W.;
Leinert, H.; Livi, V.; Menta, E.; Oliva, A.; Zimmermann, G.
Pyrimidine-2,4,6-triones: a new effective and selective class of matrix
metalloproteinase inhibitors. Biol. Chem. 2001, 382, 1277−1285.
(27) Wang, J.; Medina, C.; Radomski, M. W.; Gilmer, J. F. N-
Substituted homopiperazine barbiturates as gelatinase inhibitors.
Bioorg. Med. Chem. 2011, 19, 4985−4999.
(28) Breyholz, H. J.; Schafers, M.; Wagner, S.; Holtke, C.; Faust, A.;
̈
̈
Rabeneck, H.; Levkau, B.; Schober, O.; Kopka, K. C-5-disubstituted
barbiturates as potential molecular probes for noninvasive matrix
metalloproteinase imaging. J. Med. Chem. 2005, 48, 3400−3409.
(13) Zhao, X.; Benveniste, E. N. Transcriptional activation of human
matrix metalloproteinase-9 gene expression by multiple co-activators. J.
Mol. Biol. 2008, 383, 945−956.
(14) Balasubramanian, S.; Fan, M.; Messmer-Blust, A. F.; Yang, C.
H.; Trendel, J. A.; Jeyaratnam, J. A.; Pfeffer, L. M.; Vestal, D. J. The
interferon-gamma-induced GTPase, mGBP-2, inhibits tumor necrosis
factor alpha (TNF-alpha) induction of matrix metalloproteinase-9
(MMP-9) by inhibiting NF-kappaB and Rac protein. J. Biol. Chem.
2011, 286, 20054−20064.
(29) Breyholz, H. J.; Wagner, S.; Faust, A.; Riemann, B.; Holtke, C.;
̈
Hermann, S.; Schober, O.; Schafers, M.; Kopka, K. Radiofluorinated
̈
pyrimidine-2,4,6-triones as molecular probes for noninvasive MMP-
targeted imaging. ChemMedChem 2010, 5, 777−789.
(30) Tochowicz, A.; Maskos, K.; Huber, R.; Oltenfreiter, R.; Dive, V.;
Yiotakis, A.; Zanda, M.; Pourmotabbed, T.; Bode, W.; Goettig, P.
2161
dx.doi.org/10.1021/jm201352k | J. Med. Chem. 2012, 55, 2154−2162

Journal of Medicinal Chemistry
Article
Crystal structures of MMP-9 complexes with five inhibitors:
contribution of the flexible Arg424 side-chain to selectivity. J. Mol.
Biol. 2007, 371, 989−1006.
(31) Gilmer, J. F.; Moriarty, L. M.; Clancy, J. M. Evaluation of
nitrate-substituted pseudocholine esters of aspirin as potential nitro-
aspirins. Bioorg. Med. Chem. Lett. 2007, 17, 3217−3220.
(32) (a) Keefer, L. K. Fifty Years of Diazeniumdiolate Research.
From Laboratory Curiosity to Broad-Spectrum Biomedical Advances.
ACS Chem. Biol. 2011, 6, 1147−1155. (b) Buonsanti, M. F.; Bertinaria,
M.; Stilo, A. D.; Cena, C.; Fruttero, R.; Gasco, A. Nitric oxide donor
beta2-agonists: furoxan derivatives containing the fenoterol moiety and
related furazans. J. Med. Chem. 2007, 50, 5003−5011.
(33) Sugita, H.; Kaneki, M.; Furuhashi, S.; Hirota., M.; Takamori, H.;
Baba, H. Nitric oxide inhibits the proliferation and invasion of
pancreatic cancer cells through degradation of insulin receptor
substrate-1 protein. Mol. Cancer Res. 2010, 8, 1152−1163.
(34) Daiber, A.; Wenzel, P.; Oelze, M.; Munzel, T. New insights into
bioactivation of organic nitrates, nitrate tolerance and cross-tolerance.
Clin. Res. Cardiol. 2008, 97, 12−20.
(35) (a) Schuhmacher, S.; Schulz, E.; Oelze, M.; Konig, A.; Roegler,
̈
C.; Lange, K.; Sydow, L.; Kawamoto, T.; Wenzel, P.; Munzel, T.;
̈
Lehmann, J.; Daiber, A. A new class of organic nitrates: investigations
on bioactivation, tolerance and cross-tolerance phenomena. Br. J.
Pharmacol. 2009, 158, 510−520. (b) Roegler, C.; Konig, A.; Glusa, E.;
̈
Lehmann, J. A novel class of nitrovasodilators: potency and in vitro
tolerance of organic aminoalkylnitrates. J. Cardiovasc. Pharmacol. 2010,
56, 484−490.
(36) Oelze, M.; Knorr, M.; Schell., R.; Kamuf, J.; Pautz., A.; Art, J.;
Wenzel, P.; Munzel, T.; Kleinert, H.; Daiber, A. Regulation of human
̈
mitochondrial aldehyde dehydrogenase (ALDH-2) activity by electro-
philes in vitro. J. Biol. Chem. 2011, 286, 8893−8900.
(37) Schiefer, I. T.; Abdul-Hay, S.; Wang, H.; Vanni, M.; Qin, Z.;
Thatcher., G. R. Inhibition of amyloidogenesis by nonsteroidal anti-
inflammatory drugs and their hybrid nitrates. J. Med. Chem. 2011, 54,
2293−2306.
(38) Dos Santos, J. L.; Lanaro, C.; Lima, L. M.; Gambero, S.; Franco-
Penteado, C. F.; Alexandre-Moreira, M. S.; Wade, M.; Yerigenahally,
S.; Kutlar, A.; Meiler, S. E.; Costa, F. F.; Chung, M. Design, synthesis,
and pharmacological evaluation of novel hybrid compounds to treat
sickle cell disease symptoms. J. Med. Chem. 2011, 54, 5811−5819.
(39) Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.;
Tannenbaum, S. R. Analysis of nitrate, nitrite, and [15N]nitrate in
biological fluids. Anal. Biochem. 1982, 126, 131−138.
(40) Sato, H.; Takino, T. Coordinate action of membrane-type
matrix metalloproteinase-1 (MT1-MMP) and MMP-2 enhances
pericellular proteolysis and invasion. Cancer Sci. 2010, 101, 843−847.
(41) Rossello, A.; Nuti, E.; Catalani, M. P.; Carelli, P.; Orlandini, E.;
Rapposelli, S.; Tuccinardi, T.; Atkinson, S. J.; Murphy, G.; Balsamo, A.
A new development of matrix metalloproteinase inhibitors: twin
hydroxamic acids as potent inhibitors of MMPs. Bioorg. Med. Chem.
Lett. 2005, 15, 2311−2314.
(42) Miranda, K. M.; Espey, M. G.; Wink, D. A. A rapid, simple
spectrophotometric method for simulataneous detection of nitrate and
nitrite. Nitric Oxide 2001, 5, 62−71.
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