Furoxans (1,2,5-Oxadiazole- N -oxides) as novel no mimetic neuroprotective and procognitive agents

Article abstract of DOI:10.1021/jm201504s

Furoxans (1,2,5-oxadiazole-N-oxides) are thiol-bioactivated NO-mimetics that have not hitherto been studied in the CNS. Incorporation of varied substituents adjacent to the furoxan ring system led to modulation of reactivity toward bioactivation, studied

Full text of DOI:10.1021/jm201504s

Article
pubs.acs.org/jmc
Furoxans (1,2,5-Oxadiazole-N-Oxides) as Novel NO Mimetic
Neuroprotective and Procognitive Agents
Isaac T. Schiefer,† Lawren VandeVrede,† Mauro Fa’,‡ Ottavio Arancio,‡ and Gregory R. J. Thatcher*,†
†Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, (MC 781), 833
South Wood Street, Chicago, Illinois 60612-7231, United States
‡Department of Pathology, The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University, P&S
12-420D, 630 West 168th Street, New York, New York 10032, United States
S
* Supporting Information
ABSTRACT: Furoxans (1,2,5-oxadiazole-N-oxides) are thiol-
bioactivated NO-mimetics that have not hitherto been studied
in the CNS. Incorporation of varied substituents adjacent to
the furoxan ring system led to modulation of reactivity toward
bioactivation, studied by HPLC-MS/MS analysis of reaction
products. Attenuated reactivity unmasked the cytoprotective
actions of NO in contrast to the cytotoxic actions of higher
NO fluxes reported previously for furoxans. Neuroprotection was observed in primary neuronal cell cultures following oxygen
glucose deprivation (OGD). Neuroprotective activity was observed to correlate with thiol-dependent bioactivation to produce
−
NO2 , but not with depletion of free thiol itself. Neuroprotection was abrogated upon cotreatment with a sGC inhibitor, ODQ,
thus supporting activation of the NO/sGC/CREB signaling cascade by furoxans. Long-term potentiation (LTP), essential for
learning and memory, has been shown to be potentiated by NO signaling, therefore, a peptidomimetic furoxan was tested in
hippocampal slices treated with oligomeric amyloid-β peptide (Aβ) and was shown to restore synaptic function. The novel
observation of furoxan activity of potential therapeutic use in the CNS warrants further studies.
NO signaling via the second messenger molecule, cGMP, is
essential for normal brain function.21 NO stimulates cGMP
production through the activation of NO-sensitive soluble
guanylyl cyclase (sGC or NO-GC), which in turn leads to
activation of the memory-related transcription factor, cyclic-
AMP response element binding protein (CREB).22−25 More-
over, evidence indicates that NO may act directly in presynaptic
neurons.26 At physiological concentrations, NO possesses a
variety of neuroprotective and procognitive effects via a
combination of cGMP-dependent and independent pathways,
the latter proposed to include S-nitrosation of the active site
cysteine of caspase-3 inhibiting execution of apoptotic cell
death.27
Enhancement of NO/sGC signaling may provide a novel
approach to the treatment of Alzheimer’s disease (AD) and
other neurodegenerative disorders through neuroprotective and
procognitive actions.20,28−32 Brain derived neurotrophic growth
factor (BDNF), a downstream target of CREB, nourishes
neurons, resulting in increased neurogenesis and enhanced
synaptic plasticity.33−35 BDNF impairment has been demon-
strated in the AD brain, and Aβ, the major component of
amyloid plaques, has been shown to inhibit NO/sGC/CREB
signaling, leading to synaptic impairment.36 The importance of
NO signaling in modulating synaptic plasticity and its
correlation to enhanced learning and memory, as well as the
INTRODUCTION
■
The wide range of biological activity of compounds containing
a furoxan (1,2,5-oxadiazole-N-oxide) or benzofuroxan hetero-
cycle has been known for decades.1−3 Although thermally stable
and stable toward a range of acid−base conditions, furoxans are
thiophilic electrophiles.4 This reactivity may underlie observed
biological activity; for example, nitrobenzofuroxans were
described as “thiol-neutralizing agents” in early work on the
antileukemic properties of benzofuroxans and benzofurazans.5,6
However, the most commonly accepted mechanism underlying
furoxan activity involves thiol-dependent NO release.7 In recent
years, the furoxan moiety has been the subject of increased
attention, pioneered by Gasco and others, owing to a plethora
of interesting biological activities observed against a diverse
range of targets.8−19 The majority of these efforts have focused
on highly reactive furoxans that produce large fluxes of NO,
thereby eliciting the cytotoxic effects associated with NO at
high concentrations. Indeed, while NO possesses cytotoxic
effects at high concentrations, low levels of NO are potentially
protective, particularly in the CNS.20 Despite ample attempts to
develop furoxans as NO mimetics, efforts have not been
directed at the potential for neuroprotection in the CNS. While
the furoxan ring possesses inherent reactivity toward thiols, the
identity and pattern of furoxan substitution can potentially
dictate reactivity and the propensity for NO release, and
therefore may be manipulated to produce furoxans with
attenuated reactivity, which may hold potential as neuro-
protective agents.
Received: November 7, 2011
Published: March 19, 2012
© 2012 American Chemical Society
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Scheme 1
a
Scheme 2
a
Reagents and conditions: (i) NaNO2, acetic acid, <20 °C; (ii) TsCl, TEA, CH2Cl2, 0 °C; (iii) NaN3, DMF, 60 °C; (iv) CuI, DIPEA, toluene, with
phenylacetylene for 3, 2,8-dimethyl-5-(prop-2-ynyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole for 5, (S)-2-(4-fluorophenylsulfonamido)-3-methyl-
N-(prop-2-ynyl)butanamide for 6a−c, or CuSO4, sodium ascorbate, 3-ethynylaniline, t-BuOH/EtOH/H2O for 4; (v) toluene, 120 °C, 3 d.
neuroprotective effects of NO, support the development of NO
mimetics for the treatment of neurodegeneration and AD.28,34
While physiological concentrations of NO are essential for
neuronal function, high concentrations of NO may lead to cell
death. Therefore, an ideal neuroprotective and procognitive
NO mimetic would possess the ability to produce a lower flux
of NO over a sustained period of time. In this context, a
furoxan with attenuated reactivity, as a thiol-dependent,
nonspontaneous NO donor, is of interest (Scheme 1). To
date, limited attempts have been made to modulate furoxan
reactivity, and no reports have appeared on therapeutic
applications in the CNS.
RESULTS AND DISCUSSION
■
Design and Synthesis. The initial synthetic strategy, to
produce a variety of furoxans using divergent synthesis, started
from the aryl furoxan alcohols (1a−c) (Scheme 2). Furoxans
incorporating classical electron donating (p-OCH3, 1b) and
electron withdrawing (p-NO2, 1c) groups were designed to
examine the effect of modifying furoxan ring reactivity via
manipulations of electronic properties of the conjugated aryl
system. The 4-arylfuroxan-3-methanol compounds were
prepared by cyclization of the corresponding arylcinnamyl
alcohols by the action of sodium nitrite in acetic acid to yield
the corresponding 4-arylfuroxan-2N-oxide alcohols. The
alcohols were then activated using tosyl chloride to allow
conversion to the 4-arylfuroxan-2N-oxide azides (2a−c) using
NaN3 in DMF at 60 °C.
Furoxan reactivity is presumed to be most sensitive to
changes in direct substitution of the furoxan ring and its
conjugated systems. Contrary to modifications made in
substitution of the conjugated aryl system, a methylene linker
was maintained opposite to the aryl system in order to
minimize potential effects on reactivity by derivatization at this
end of the molecule. Recent evidence has shown the utility of
nonselective cysteine protease inhibitors for neurodegenerative
diseases,38,39 and it was speculated that a peptidomimetic
thiophilic furoxan may hold the potential to inhibit cysteine
proteases by direct reaction with active site cysteines. This
would allow inhibition of cysteine proteases associated with
neurodegeneration, concomitant with NO release. However,
before study of such a hypothesis, it was necessary to
In this paper, the design, synthesis, and evaluation of
peptidomimetic furoxans as attenuated NO-mimetic neuro-
protective agents was explored for the first time. Neuro-
protection was indeed observed for furoxans, whereas an
analogous thiophilic furazan analogue that cannot release NO
was neurotoxic. Upon coincubation of one furoxan with a sGC
inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ), the neuroprotective activity was abolished, implicating
activation of the NO/sGC cascade as a key mechanistic
pathway. Study of cysteine-mediated bioactivation of furoxans
further supported a NO-mediated mechanism, as cysteine
−
consumption did not correlate with NO2 production. One
neuroprotective NO-donating furoxan restored normal LTP in
hippocampal slices treated with oligomeric Aβ, replicating
previous data on restoration of synaptic plasticity and reversal
of cognitive deficits caused by oligomeric Aβ through a NO/
sGC/CREB pathway.28,37
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a
Scheme 3
a
Reagents and conditions: (i) PPh3, 35% NH4OH, DMF, rt; (ii) EDCI, DMAP, (S)-4-methyl-2-(phenylsulfonamido)pentanoic acid, CH2Cl2, 0 °C;
(iii) 9a, Zn, ammonium formate, MeOH, 60 °C.
investigate the effects of attenuated furoxans on neurons and
synapses, which is the focus of the current work.
Peptidomimetic scaffolds were designed incorporating a
putative recognition motif, while having negligible impact on
the reactivity of the furoxan ring itself and allowing a divergent
synthetic approach. Terminal alkynes were synthesized using
standard peptide coupling procedures (see Supporting
Information for full experimental details) and then reacted
with 2a−c through a 2,3-dipolar cycloaddition to yield the
corresponding furoxan-triazole containing derivatives 3−5, as
well as a subset of analogues containing p-substituted arenes
(6a−c). The peptidomimetic oxadiazole-2N-oxides (6a−c)
were then tautomerized in boiling toluene to afford 7a−c.
Additionally, 2a and 2b were converted to their corresponding
amines (8a−8b) via a Staudinger reduction followed by DMAP
catalyzed coupling to a peptidomimetic synthon to give 9a−9b
(Scheme 3). The furazan analogue 10 was produced by
treatment of 9a with a combination of activated zinc and
ammonium formate at reflux.40
Crystallography. Although the oxadiazole-N-oxide ring
configuration can typically be assigned by comparing shifts of
C3 and C4 of the furoxan ring by 13C NMR, further analysis is
useful to confirm the position of the N-oxide moiety.
Crystallographic examination of 6c revealed two independent
molecules in the asymmetric unit (Figure 1), confirming the
presence of the oxadiazole-2N-oxide structure as well as the
stereochemistry of the L-valine residue incorporated into the
molecule (see Supporting Information for full crystallographic
details).
Reactivity. Attenuating the reactivity of the furoxan ring
Figure 1. Crystal structure of 6c confirming the oxadiazoles-2N-oxide
ring configuration.
through structural modification is of paramount importance in
NO-mimetic design. The majority of benzofuroxans are not
expected to release NO on the basis of a study by Medana and
41
−
and temperature in the presence of excess cysteine. After 2 h
reaction, products were identified by HPLC-MS/MS (Figure
2); the parent and daughter ions of the major product were
compatible with the bis-oxime hydrate D. The product
corresponding to the keto-oxime, E (or its enol tautomer),
and loss of NOx constituted 11% of the reaction mixture. A
compound corresponding to a cysteine-containing product (A)
was observed but constituted <1% of products from integration
of the UV absorbance chromatogram.
The comparable reaction of cysteine (2.5 mM) at pH 7.4
(PBS, 50 mM) and 37 °C was studied with 6a−c. After LC-
MS/MS identification of products, integration of the UV
absorbance chromatogram was used to quantify unreacted
furoxan starting material. The rate of reaction with cysteine was
observed to be lower for 6a−c than that of 9a based upon the
% of starting material remaining: 9a, 11%; 6a, 68%; 6b, 75%;
6c, 14% (see Supporting Information for additional chromato-
co-workers separately measuring NO2 , N2O, and NO. In
contrast, the formation of both NO and nitrosothiols from
furoxans has been postulated from attack of thiolate anion; the
furoxan impramidil was reported to yield nitrite, nitrate, and a
bis-oxime containing product.4,42,43 The exact mechanism of
furoxan degradation leading to NO bioactivity has not been
defined and is beyond the scope of the present work. A putative
mechanism is shown in Figure 2 for reaction of the furoxan 9a
with cysteine. In this mechanism: (1) formation of a keto-
oxime E necessarily requires loss of a nitrogen oxide (NOx),
(2) thiol may be regenerated after initial ring-opening, resulting
in C, and (3) initial ring-opening may result in thiol oxidation
to form a disulfide, producing B. Therefore, in the reaction of
furoxan with thiol, the consumption of thiol is not intrinsically
correlated with release of NOx.
Support for the mechanism shown in Figure 2 was gained
from study of the reaction of the furoxan 9a at physiological pH
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Figure 2. Putative mechanism describing reaction products observed using UV absorbance chromatogram at 250 nm for 9a (75 μM) in the presence
of excess cysteine (5 mM) in PBS (50 mM, pH 7.4) at 37 °C for 2 h. Approximate % of products determined by integration of UV absorbance peaks.
Structures proposed based on LC-MS/MS analysis of parent and daughter ions.
a
Scheme 4
a
Trend in reactivity based on % remaining parent furoxan (75 μM) after 2 h incubation with excess cysteine (2.5 mM) in PBS (50 mM, pH 7.4) at
37 °C. Analyzed by HPLC-UV(λ=250 nm)
.
−
grams and analysis). The higher reactivity of 9a is compatible
with intramolecular stabilization of the developing negative
charge in intermediate II, which has previously been reported
to accelerate furoxan breakdown (Scheme 4).44,45 More
importantly, the predicted modulation of reactivity by
substitution of the phenyl ring, with electron-donating (6b)
or electron-withdrawing (6c) substituents, was validated by the
data. Thus, analysis of the reaction products from 6a−c
demonstrated that structural modification modulates overall
furoxan reactivity.
out (see Supporting Information), and the rate of NO2
−
production corresponds with quantitative NO2 production,
which plateaus after 24 h. In agreement with the reported
stability of furoxans in aqueous media, control incubations
performed under identical conditions in the absence of cysteine
gave no measurable NOx production (data not shown).
The furoxans studied yielded between 2 and 80% mol
−
equivalent of NO2 over the reaction time studied (Table 1).
The oxadiazole-2N-oxides 6a−c yielded considerably more
−
NO2 than the tautomeric 7a−c. The p-OCH3 substituted
−
−
NO2 Production and Cysteine Depletion. The analysis
furoxans (4, 5, and 6b) also gave much larger amounts of NO2
of the reaction of 9a with cysteine identified reaction products
expected for thiol regeneration, thiol oxidation, and loss of
NOx. It has been reported previously that formation of
inorganic nitrite, as a measure of NOx release, in the presence
of excess cysteine (5 mM), showed reasonable correlation with
vascular tissue relaxation for a series of furoxans.46 In a similar
compared to both the tautomeric, 7a−c, and the methylen-
amine-containing 9a−b. For comparison, the bioactivation of a
well studied α-cyano-furoxan, 3-cyano-4-phenyl-1,2,5-oxadia-
zole 2-oxide (11), was analyzed under similar conditions. A
recent extensive study of furoxans as antiparasitic agents,
converged on 11 and other α-cyano-furoxans as drug
candidates.47 Previous studies of α-cyano-furoxans as anticancer
agents also included observations of cytotoxicity.48 As might be
expected if higher concentrations of NOx lead to toxicity, 11
−
fashion, the Griess assay for NO2 was used to analyze
reactions of furoxans (250 μM) with cysteine (2.5 mM) in a 1:1
solution of CH3CN:PBS (50 mM, pH 7.4) at 37 °C after 24 h.
Additional kinetic examination of NO2− production was carried
−
generated large amounts of NO2 on reaction with cysteine.
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−
Table 1. NO2 Production from Furoxans after 24 h with
Table 2. Cysteine Remaining after 2 h Incubation with
Excess Cysteine
Furoxans under Hypoxic Conditions
a
b
a
b
−
−
compd
NO2 production (μM)
NO2 production as % (mol/mol)
compd
Cys as free thiol (mM)
Cys depletion as % (mol/mol)
2a
2b
2c
3
9.2 ( 0.1)
5.1 ( 0.1)
14.8 ( 0.8)
40.1 ( 0.8)
6.3 ( 0.1)
9.7 ( 0.1)
6.8 ( 0.1)
3.9 ( 0.7)
21.8 ( 0.5)
1.0 ( 0.4)
1.8 ( 0.1)
1.3 ( 0.1)
1.2 ( 0.2)
0.5 ( 0.1)
0.0 ( 0.1)
29.1 ( 0.3)
18.4
10.4
29.6
80.2
12.6
19.4
13.6
7.8
−
4
2267.2 ( 153.7)
1788.8 ( 18.7)
1410.1 ( 23.8)
1790.8 ( 38.6)
1830.4 ( 16.6)
1995.2 ( 26.1)
1724.4 ( 26.1)
1507.2 ( 29.5)
1620.4 ( 26.6)
1812.1 ( 33.4)
1724.4 ( 59.0)
1742.8 ( 23.3)
21.1
37.8
21.0
19.2
12.0
23.9
33.5
28.5
20.1
23.9
23.1
5
6a
6b
6c
7a
7b
7c
9a
9b
10
4
5
6a
6b
6c
7a
7b
7c
9a
9b
10
11
43.6
2.0
3.5
2.5
a
2.4
Free cysteine remaining in incubations containing furoxans (250 μM)
and cysteine (2.5 mM) after 2 h under hypoxic conditions at 37 °C in
PBS (50 mM, pH 7.4)/ CH3CN (1:1), with 1 mM EDTA. Cysteine
depletion relative to the control, represented as percent cysteine
depletion mol/mol.
1.0
b
0
58.2
a
Quantitative amount of NO2− produced from 250 μM of compound
after 24 h in the presence of excess cysteine (2.5 mM) in PBS (50 mM,
pH 7.4) at 37 °C; quantified using calibration curve generated with
b
−
−
NaNO2 using the Griess assay. NO2 production represented as %
mol/mol.
Again as anticipated, the furazan 10 provided a negative control
−
for NO2 release.
NO-mediated S-nitrosation of active site cysteines is an
important step in the regulation of several proteins involved in
cytoskeletal remodeling and cellular apoptosis.49−51 Moreover,
S-nitrosation may potentially result in NO facilitated cysteine
oxidation, leading to mixed disulfide formation. As discussed
earlier, thiol-induced breakdown of furoxans may lead to thiol
oxidation, thiol regeneration, or thiol alkylation. DTNB (5,5′-
dithiobis-2-nitrobenzoic acid or Ellmans Reagent) was used to
quantify free, reduced cysteine after incubation of selected
furoxans (250 μM) with cysteine (2.5 mM). In normoxic
solution, air oxidation of cysteine in the absence of furoxan
leads to complete loss of reduced thiol within 12 h, and a high
degree of background cysteine oxidation was observed upon
incubation of cysteine with selected furoxans over 4 h (see
Supporting Information).
−
Figure 3. Correlation between NO2 production (% mol/mol) and
−
cysteine depletion under hypoxic conditions (% mol/mol). NO2
measured using Griess assay and cysteine depletion determined using
DTNB.
control. After 24 h incubation, considerable neuroprotective
activity was seen for several compounds (Figure 4A), including
the 6a−c and 7a−c subsets, which demonstrated relative
neuroprotection in a manner that reflected the relative
reactivity of these analogues described in Scheme 4. E-64, a
well characterized neuroprotective cysteine protease inhib-
itor,54−56 was used as a positive control and gave significant
neuroprotection. The methylenamine containing 9a and 9b
were also shown to increase neuronal survival by 32% and 22%,
respectively. The neuroprotection elicited by 9a and 9b
suggests that even low amounts of NOx generation are capable
of eliciting a biological response. Compound 3 was not tested
for neuroprotection due to limitations in aqueous solubility.
Interestingly, while the majority of compounds showed
significant neuroprotective activity, a few furoxans showed
toxicity, including the azide family (2a−c) that elicited a ∼50−
75% loss of viability compared to the DMSO vehicle control,
and the carbazole derivative, 5, which resulted in total cell death
and reduced the MTT response to baseline levels (data not
shown). Furthermore, the furazan analogue, 10, was also
observed to be toxic. Because furazans are thiophilic but cannot
release NOx, this would again support a mechanism of
To obtain a more quantitative measure of furoxan-mediated
cysteine consumption, oxygen was depleted by bubbling with
O2-free N2 in sealed reaction vials, followed by assay of free
cysteine after reaction for 2 h (Table 2). A plot of cysteine
−
consumption versus NO2 production did not show a
correlation (Figure 3). For example, of the substituted-phenyl
furoxans, 6c gave the highest amounts of NOx release but the
lowest cysteine consumption, confirming the disconnection
between thiol consumption and NOx release, which is predicted
by the mechanism shown in Figure 2.
Neuroprotection. Neuroprotection was evaluated by 3-
(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide
(MTT) assay of cell viability in primary rat neuronal cell
cultures subjected to oxygen glucose deprivation (OGD).52
OGD provides a cellular model of ischemia and reperfusion
injury leading to apoptotic cell death, providing a model for
excitotoxic loss of neurons in neurodegenerative diseases such
as ischemic stroke and AD.53 Neurons were subjected to OGD
for 2 h, followed by reoxygenation and replacement with
nutrient rich media containing furoxans (50 μM) or vehicle
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protective mechanism driven by NOx production from an
attenuated furoxan core and are consistent with data presented
below from ex vivo LTP experiments.
LTP Restoration. LTP in the Schaffer collateral pathway of
the hippocampus is a cellular model of learning and memory
(for review see ref 57), which is significantly impaired after
perfusion with oligomers of Aβ.58 Aβ induced inhibition of LTP
is reversed by diazeniumdiolate NO-donors via a mechanism
shown to be mediated by the NO/sGC signaling pathway.28
CREB is essential for memory consolidation and NO has been
shown to activate CREB and to induce phosphorylation by
cGMP-dependent kinase activation.59 The peptidomimetic
furoxan 9a was selected for study of LTP restoration in
hippocampal slices on the basis of: (1) an excellent neuro-
protection profile (Figure 4A) (2) the confirmed involvement
of the NO/sGC pathway in the mechanism of 9a (Figure 4C),
and (3) desirable physiochemical properties for CNS
bioavailability compared to the other furoxan analogues studied
(MW < 450, clogP = 2.9). It was hypothesized that 9a should
possess the ability to restore synaptic function in hippocampal
slices coperfused with oligomeric Aβ. In accordance with
previous data and the hypothesis that NOx release from
furoxans is able to restore brain function, 9a (1 μM) was found
to completely abrogate Aβ induced synaptic deficits in LTP;
synaptic response to LTP induction was restored to levels
matching those of untreated wild-type controls (Figure 5). This
novel bioactivity of furoxans implicates the NO/sGC/CREB
pathway as being activated in hippocampal slices, in simile with
Figure 4. (A) Effect of selected furoxans on cell survival in primary rat
cortical neurons. Cells treated with compound (50 μM) for 24 h
following 2 h of OGD. Values represent survival as % of DMSO
treated control from MTT assay. (B) Tentative correlation between
−
NO2 production (% mol/mol) and cell survival (% of control). (C)
Effect on cell survival in primary rat cortical neurons by DMSO treated
control, the furoxan 9a (50 μM), 9a (50 μM) + ODQ (10 μM), and
ODQ (10 μM) treated control. Values represent survival as % of
DMSO treated control from MTT assay.
neuroprotection via NOx production, rather than other
mechanisms dependent on oxidation or alkylation of active
site protein thiols, such as those of cysteine proteases (i.e.,
calpain, cathepsin B, or apoptotic caspases). A trend between
−
NO2 release and neuroprotection was tentatively identified
(Figure 4B). This correlation is in agreement with the concept
that low levels of NO are essential for normal neuronal
physiological activity, whereas high concentrations of NO can
lead to cellular apoptosis and cell death. Notable outliers from
this tentative correlation (5, 6c) require special considerations:
(1) compounds may possess cytotoxic or neuroprotective
activity through pathways other than NO signaling, and this
seems to be the case for the highly toxic furoxan 5, (2) although
Figure 5. Restoration of long-term potentiation by 9a (1 μM) in
hippocampal slices perfused with oligomeric Aβ (200 nM) (for each
experiment n = 7).
−
NO2 production by furoxans has been reported to correlate
with NO-derived bioactivity, the formation of NO2− is possible
via routes that do not involve NO itself as an intermediate.
Furoxan 6c would be predicted to be toxic based upon high
other NO-donors and further supports development of
furoxans as therapeutics in dementia and neurodegeneration.
−
levels of NO2 formation, however, the product profile from
CONCLUSION
reaction with cysteine is very different from other furoxans
studied, including unique N,O-containing intermediates that
may be responsible for the copious NO2− production observed
(see Supporting Information for detailed discussion).
Further support for the involvement of the NO/sGC
pathway was obtained using ODQ, a potent and selective
sGC inhibitor: after OGD, primary neurons were incubated
with 9a (50 μM) with or without ODQ (10 μM). Blockade of
cGMP formation by ODQ completely abolished the neuro-
protective activity observed for 9a alone (Figure 4C), while
treatment with ODQ in control groups showed no significant
impact on cell survival or toxicity. These findings strongly
support the involvement of a cGMP-dependent neuro-
■
The NO-mimetic furoxan ring was incorporated into a series of
peptidomimetic scaffolds; one novel approach used click
chemistry to demonstrate the potential for synthetic elabo-
ration of this novel peptidomimetic drug class. It was postulated
that substitution of the furoxan ring would lead to modulation
of reactivity toward thiol-mediated bioactivation, and further,
that attenuation of reactivity would unmask the cytoprotective
properties of NO. This was deemed important because
cytotoxicity has been associated with the higher fluxes of NO
deriving from known furoxan anti-infective agents. Attenuation
of reactivity and propensity for NO release was accomplished
by appropriate substitution of the furoxan ring. Neuro-
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Neuroprotection Assay. Use of animals was approved by the
Institutional Animal Care and Use Committee at the University of
Illinois at Chicago. Cell cultures were prepared from the cortex and
hippocampus of rat embryos at 16−18 d of gestation and grown in
neurobasal media supplemented with B27 and glutamine, a technique
shown to result in a population of greater than 99% pure neurons.59
After 9−11 DIV, cultures were transferred to hypoxic chamber (Coy
Lab, Grass Lake, MI; dimensions 41″ L × 23″ D × 23″ H) with an
atmosphere of 5% CO2/ 95% N2; oxygen tension being monitored by
electrode and kept to less than 1%. Culture media was replaced with a
salt-balanced solution containing the following (in mM): NaCl 116,
CaCl2 1.8, MgSO4 0.8, KCl 5.4, NaH2PO4 1, NaHCO3 14.7, HEPES
10. After 2 h, cells were removed from hypoxic chamber and
resupplied with neurobasal media and compounds at 50 μM or vehicle
control with or without ODQ (10 μM). After 24 h of incubation, cell
survival assay was performed by addition of MTT at 250 ng/mL,
followed by a 3 h incubation, followed by media aspiration and
dissolution of insoluble dye crystals in acidified organic propanol, with
final reading of absorbance performed on an ELISA plate reader at 590
nm.
protection by furoxans in primary rat neurons subjected to
ischemia-reperfusion injury correlated with NO2 production
−
but did not correlate with loss of free thiol. Neuroprotection
was abolished by coincubation with the sGC inhibitor, ODQ,
implicating the involvement of the NO/sGC cascade. One
furoxan, 9a, was observed to restore LTP following an Aβ-
induced synaptic deficit in mouse hippocampal slices,
compatible with the known capacity of NO/sGC/CREB
activation to restore LTP. As a whole, these findings strongly
suggest activation of the NO/sGC/CREB cascade as the
primary mechanism of furoxan elicited neuroprotection and
synaptic function restoration. Future efforts to develop
attenuated furoxans for the CNS should focus on developing
substitution patterns that decrease the reactivity of the furoxan
ring itself. The work represents the first description of furoxans
as neuroprotective and procognitive agents with potential
application in stroke and AD.
LTP Assay. Mice were decapitated and their hippocampi were
rapidly dissected out. then 400 μm slices were cut with a tissue
chopper and maintained in an interface chamber at 29 °C for 90 min
prior to recording. Slices were perfused with artificial CSF (in mM):
NaCl 124, KCl 4.4, Na2HPO4 1.0, NaHCO3 25, CaCl2 2.0, MgSO4 2.0,
and glucose 10, which was continuously bubbled with 95% O2 and 5%
CO2. Slices were permitted to recover from cutting for at least 90 min
before recordings. The fEPSPs were recorded from the CA1 region of
the hippocampus by placement of both the stimulating and the
recording electrodes in the CA1 stratum radiatum. Following
recording of a 15 min baseline in which individual fEPSP were
recorded every min at an intensity that evokes a response ∼35% of the
maximum evoked response, compounds and β-amyloid were perfused
for 20 min prior to LTP induction. LTP was elicited through a θ-burst
stimulation: 4 pulses at 100 Hz, with the bursts repeated at 5 Hz and
each tetanus including 3 ten-burst trains separated by 15 s.
General Methods. Unless stated otherwise, all reactions were
carried out under an atmosphere of dry argon in oven-dried glassware.
Indicated reaction temperatures refer to those of the reaction bath,
while room temperature (rt) is noted as 25 °C. Dichloromethane
(CH2Cl2) was distilled over CaH2 and THF distilled over Na(s). All
other solvents were of anhydrous quality purchased from Aldrich
Chemical Co. and used as received. Commercially available starting
materials and reagents were purchased from Aldrich, TCI, and Fisher
Scientific and were used as received unless specified otherwise. trans-
Epoxysucciny-L-leucyl-amido(4-guanidino) butane (E-64), 1H-[1,2,4]-
oxadiazole[4,3-a]quinoxalin-1-one (ODQ), and 4-phenyl-3-fuxoxan-
carbonitrile (11) were purchased from Sigma and used as received.
Analytical thin layer chromatography (TLC) was performed with (5
cm × 20 cm, 60 Å, 250 μm). Visualization was accomplished using a
254 nm UV lamp. 1H and 13C NMR spectra were recorded on either a
Bruker Avance 400 MHz spectrometer or Bruker DPX 400 MHz
spectrophotometer. Chemical shifts are reported in ppm with the
solvent resonance as internal standard ([CDCl3 7.27 ppm, 77.23
ppm], [DMSO-d6 2.5 ppm, 39.51 ppm], and [MeODd4 4.78, 49.0] for
1H, 13C, respectively). Data are reported as follows: chemical shift,
multiplicity (s = singlet, d = doublet, dd = doublet of doublet, t =
triplet, q = quartet, br = broad, m = multiplet, abq = ab quartet),
number of protons, and coupling constants. Low-resolution mass
spectra (LRMS) were acquired on an Agilent 6310 Ion-Trap LC/MS.
High resolution mass spectral (HRMS) data was collected in-house
using a Shimadzu QTOF 6500. All compounds submitted for
biological testing were found to be >95% pure by analytical HPLC.
Purity and/or low resolution mass spectral (ESI or APCI) analysis was
measured using an Advantage ARMOR C18; 5 μM; 150 mm × 4.6
mm analytical column on either a Shimadzu UFLC (Instrument 1) or
Agilent 1100 Series HPLC in tandem with an Agilent 6310 Ion Trap
LC/MS (Instrument 2). HPLC method: solvents, 0.05% FA in
CH3CN (solvent A); 0.05% FA in 9:1 mixture of H2O and MeOH
(solvent B); flow rate 1.0 mL/min. Gradient: t = 0 min, 10% B; t = 5
EXPERIMENTAL SECTION
■
All chemicals and reagents were purchased from Sigma-Aldrich (St.
Louis, MO), or Fisher Scientific unless stated otherwise.
Crystallography. The furoxan (6c) was chosen for crystallo-
graphic examination. Molecular formula, C23H23FN8O7S; formula
weight, 574.1418; crystal system, orthorhombic, P212121 (#19) with
unit cell dimensions a = 11.334 (1) Å, b = 21.427 (2) Å, c = 21.643 (1)
Å; volume = 5256.1 (6) Å3; Z, calculated density = 8, 1.452 mg/m3;
absorption coefficient = 0.190 mm−1. A colorless needle, with
dimensions 2 × 2 × 40 μm3, was selected for data collection. The
crystal was cooled to 100 K and aligned in the X-ray beam at sector 22
(SER-CAT) on the ID beamline at the Advanced Photon Source at
Argonne National Lab. Data collection was carried out at a wavelength
of 0.80 Å using 5° rotations and exposure time of 1 s and collected
with a MAR 300 mm CCD detector. Then 72 images were indexed
and integrated using XDS and averaged over mmm symmetry. The
structure was solved using SHELX-97 and refined using WinGX and
SHELXL; ORTEP was used to generate the molecular figures.
Hydrogen atoms were added to the model in idealized positions. The
final model gave agreement factors (R indices) of R1 = 0.0407, wR2 =
0.1030. We observed two independent molecules in the asymmetric
unit, confirming the presence of the oxadiazole-2N-oxide structure
within the structures.
Reactivity Analysis. The appropriate furoxan (75 μM) was
incubated with L-cysteine (2.5 mM) in PBS (50 mM, pH 7.4) at 37 °C
for 2 h. The products were then separated and analyzed by HPLC-
MS/MS. Peak integration from UV absorbance spectra gives the
estimated relative abundance of each reaction product and is
correlated with the TIC chromatogram to identify peaks by MS/MS
analysis.
Griess Assay. The parent furoxan (250 μM) was dissolved in PBS
(50 mM, pH 7.4)/CH3CN (1:1); L-cysteine (2.5 mM) was added and
the mixture incubated at 37 °C for 24 h. Aliquots (taken at 1, 2, 4, 8,
12, 18, and 24 h) were incubated for 10 min with Griess reagent A
(1.0% sulfanilamide, 5.0% H3PO4 in dH2O)/ griess reagent B (0.1%
(N-1-naphthyl)ethylenediamine dihydrochloride in dH2O). UV
absorbance at 530 nm was measured using a Dynex MRX II
microplate spectrophotometer and calibrated using a standard curve
constructed with NaNO2 to yield nitrite concentration.
Cysteine Reactivity. The parent furoxan (250 μM) was dissolved
in a solution of PBS (50 mM, pH 7.4, over Chelex 100)/CH3CN (1:1)
containing EDTA (1%) and the mixture purged with N2 for 10 min; L-
cysteine (2.5 mM) was added and the mixture purged with N2 for an
additional 5 min followed by incubation at 37 °C for 2 h. Aliquots
were mixed with DTNB (750 μM, in PBS 50 mM, pH 7.0) for 5 min.
UV absorbance of DTNB mixture was measured at 412 nm using a
Dynex MRX II microplate spectrophotometer and calibrated using a
standard curve constructed using L-cysteine in the presence of DTNB
to yield total reduced cysteine concentration.
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min, 30% B; t = 15 min, 70% B; t = 30 min, 90% B; t = 35 min, 50% B,
t = 38, 10% B. Stop time 45 min.
Crude mixture of tosylated and chlorinated 1c (612 mg, 1.63 mmol)
and NaN3 (160 mg, 2.4 mmol) afforded 2c as a white solid (345 mg,
1
General Procedure for Arylfuroxan-3-methanol Cyclization. The
appropriate cinnamyl alcohol (100 mmol) was dissolved in acetic acid
(25 mL) under argon at 0 °C. NaNO2 (300 mmol) was added
portionwise over 30 min while maintaining reaction temperature <20
°C. The reaction was then allowed to warm to rt and monitored by
TLC. After 4 h, the reaction was diluted with ice water and extracted
with ethyl acetate (3 × 175 mL). The combined organic extracts were
washed with brine (2 × 75 mL), dried over Na2SO4, and concentrated
to afford orange oil. The residual oil was then purified by column
chromatography (hexane/ethyl acetate [10:1→ 5:1]) to yield the
desired products in each circumstance as described below.
85.8%). H NMR (CDCl3, 400 MHz): δ 8.45−8.42 (d, 2H), 8.01−
7.99 (d, 2H), 4.52 (s, 2H). 13C NMR (CDCl3, 100 MHz): 154.68,
149.55, 131.71, 128.81, 124.65, 111.22, 42.85. Analytical HPLC
(instrument 1): purity = 97.4%, tR = 23.4 min.
General Procedure for Click Reaction A. The azide (25 mg, 0.12
mmol) was dissolved in toluene (5 mL) followed by under argon, by
the addition of phenylacetylene (15 mg, 0.15 mmol) and copper(I)
iodide (10 mg, 0.032 mmol). N,N-Diisopropylethylamine (0.02 mL,
0.115 mmol) was then added to the solution. The reaction was
allowed to stir at room temperature until TLC analysis (hexane/ethyl
acetate [1:1]) indicated completion. The solution was filtered through
Celite and concentrated to give a crude brown solid, which was
purified by column chromatography to give the title compound.
General Procedure for Click Reaction B. The furoxan azide (1.0
equiv), 3-ethynylaniline (1.0 equiv), sodium ascorbate (0.4 equiv), and
CuSO4 (0.2 equiv) were stirred in t-BuOH/EtOH/H2O (3:1:1) for 12
h. Reaction was filtered through Celite and concentrated. Residual oil
was purified by column chromatography to give the title compound.
4-Phenyl-3-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)-1,2,5-oxadia-
zole 2-Oxide (3). Synthesized using the general click procedure with
the following values. Click reaction A: furoxan-azide (25 mg, 0.12
mmol), phenylacetylene (15 mg, 0.15 mmol), copper(I) iodide (10
mg, 0.032 mmol) and N,N-diisopropylethylamine (0.02 mL, 0.115
mmol) afforded 3 as a white solid (30 mg, 81.6%). 1H NMR (DMSO-
d6, 400 MHz): δ 8.69 (s, 1H), 7.83−7.81 (t, 4H), 7.61−7.59 (m, 3H),
7.44−7.31 (m, 3H), 5.81 (s, 2H). 13C NMR (DMSO-d6, 100 MHz):
157.46, 146.90, 131.94, 130.63, 129.82, 129.38, 128.56, 128.23, 125.84,
125.65, 122.93, 112.54, 42.62.
3-((4-(3-Aminophenyl)-1H-1,2,3-triazol-1-yl)methyl)-4-(4-me-
thoxyphenyl)-1,2,5-oxadiazole 2-Oxide (4). Click reaction B: p-
methoxyphenylfuroxan azide (65 mg, 0.26 mmol), 3-ethynylaniline
(30.8 mg, 0.26 mmol), sodium ascorbate (20 mg, 0.1 mmol), and
CuSO4 (8 mg, 0.05 mmol) afforded 4 as a white solid (72 mg, 75.0%).
1H NMR (CDCl3, 400 MHz): δ 8.02 (s, 1H), 7.87−7.84 (d, 2H, J =
6.8 Hz), 7.22−7.21 (t, 1H), 7.20−7.18 (d, 1H, J = 7.63 Hz), 7.17−7.15
(dt, 1H), 7.09−7.06 (d, 2H, J = 6.8 Hz), 6.68−6.55 (dq, 1H), 5.59 (s,
2H), 3.87 (s, 3H). 13C NMR (CDCl3, 100 MHz): 162.23, 156.27,
148.81, 146.96, 130.71, 129.85, 129.65, 120.74, 117.41, 116.05, 115.28,
115.06, 112.18, 111.61, 55.47, 42.09. ESI-HRMS (m/z): [M + H]+
calcd for C18H16N6O3, 365.1356; obsd, 365.1360. Analytical HPLC
(instrument 1): purity = 97.3%, tR = 12.7 min.
3-(Hydroxymethyl)-4-phenyl-1,2,5-oxadiazole 2-Oxide (1a). Yel-
1
low solid (4.8 g, 24.9%). H NMR (CDCl3, 400 MHz): δ 7.85−7.82
(m, 2H), 7.61−7.54 (m, 3H), 4.77−4.76 (d, 2H, J = 4.89 Hz), 2.82
(bs, 1H). 13C NMR (CDCl3, 100 MHz): 157.05, 131.58, 130.38,
129.62, 129.36, 127.99, 126.36, 115.07, 56.93.
3-(Hydroxymethyl)-4-(4-methoxyphenyl)-1,2,5-oxadiazole 2-
Oxide (1b). White solid (5.8 g, 26.1%). 1H NMR (CDCl3, 400
MHz): δ 7.79−7.77 (d, 2H, J = 9.75 Hz), 7.08−7.02 (d, 2H, J = 9.75
Hz), 4.77−4.75 (d, 2H), 3.90−3.86 (s, 3H), 2.64−2.60 (t, 1H). 13C
NMR (CDCl3, 100 MHz): 161.97, 156.48, 129.28, 118.42, 114.82,
114.74, 55.45, 53.42.
3-(Hydroxymethyl)-4-(4-nitrophenyl)-1,2,5-oxadiazole 2-Oxide
1
(1c). White solid (6.9 g, 29.1%). H NMR (CDCl3, 400 MHz): δ
8.44−8.42 (d, 2H, J = 8.8 Hz), 8.13−8.10 (d, 2H, J = 8.8 Hz), 4.79 (s,
2H), 2.45−2.18 (bs, 1H). 13C NMR (CDCl3, 100 MHz): 154.75,
149.15, 131.76, 128.55, 124.15, 113.21, 52.94.
General Procedure for Alcohol Activation. Triethylamine (0.62
mL, 4.43 mmol) was added to furoxan alcohol (284 mg, 1.48 mmol) in
freshly distilled CH2Cl2 (10 mL) at 0 °C. TsCl (380 mg, 2.2 mmol)
was separately dissolved in CH2Cl2 (5 mL) and added dropwise over
10 min. The mixture was allowed to warm to rt and stirred for 3 h. The
reaction was quenched w/saturated aqueous NH4OH (10 mL) and
extracted with ethyl acetate (3 × 75 mL). Combined organic layers
washed with brine (2 × 15 mL), dried over Na2SO4, and concentrated
to give yellow oil. Reaction products were isolated by column
chromatography (hexane/ethyl acetate [8:1 → 4:1]). Once products
were confirmed by NMR, subsequent crude mixtures of the tosyl and
chloro intermediates were submitted to next reaction without further
purification.
General Procedure for Preparation of the Azide. NaN3 (1.5 equiv)
was added to the a mixture of the tosyl and chloro derivatives (1.0
equiv) in DMF (10 mL) and the reaction brought to 50 °C and
reaction progress monitored by TLC. After completion of reaction
(typically 4−12 h), the reaction was quenched with ice water (150
mL) and extracted with ethyl acetate (3 × 50 mL). The combined
organic extracts were washed with brine (2 × 50 mL), water (3 × 250
mL), dried over Na2SO4, and concentrated to give orange oil. Crude
oil was purified by column chromatography (hexane/ethyl acetate
[5:1]) to give a mixture of the desired azides described below.
3-(Azidomethyl)-4-phenyl-1,2,5-oxadiazole 2-Oxide (2a). Synthe-
sized using the general procedure with the following values. Crude
mixture of tosylated and chlorinated 1a (366 mg, 1.06 mmol) and
NaN3 (100 mg, 1.6 mmol) afforded 2a as a white solid (175 mg,
3-((4-((2,8-Dimethyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)-
methyl)-1H-1,2,3-triazol-1-yl)methyl)-4-(4-methoxyphenyl)-1,2,5-
oxadiazole 2-Oxide (5). Click reaction A: carbazole alkyne (26 mg,
0.105 mmol [see Supporting Information for preparation]), furoxan
azide (34 mg, 0.143 mmol), DIPEA (0.03 mL, 0.18 mmol), and CuI
1
(20 mg, 0.06 mmol) yielded 5 as a white solid (52 mg, 75.1%). H
NMR (CDCl3, 400 MHz): δ 7.79−7.76 (d, 2H, 8.83 Hz), 7.50 (s,
1H), 7.22−7.19 (m, 2H), 7.05−7.02 (d, 2H, J = 8.83 Hz), 7.01−6.99
(d, 1H, J = 8.28 Hz), 5.40 (s, 2H), 5.31 (s, 2H), 3.88 (s, 3H), 3.80 (s,
2H), 3.00 (s, 4H), 2.89 (s, 3H), 2.63 (s, 3H), 2.43 (s, 3H). 13C NMR
(CDCl3, 100 MHz): 161.81, 155.80, 145.21, 134.28, 131.96, 129.20,
128.57, 125.62, 122.65, 122.61, 117.30, 117.04, 114.65, 110.84, 108.31,
106.64, 55.09, 51.60, 51.00, 44.38, 41.67, 38.17, 21.69, 21.00. ESI-
HRMS (m/z): [M + H]+ calcd for C26H27N7O3, 486.2248; obsd,
486.2258. Analytical HPLC (instrument 1): purity = 99.0%, tR = 8.3
min.
( R ) - 3 - ( ( 4 - ( ( 2 - ( 4 - F l u o r o p h e n y l s u l f o n a m i d o ) - 3 -
methylbutanamido)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4-phenyl-
1,2,5-oxadiazole 2-Oxide (6a). Click reaction A: peptidomimetic
alkyne (140 mg, 0.44 mmol [see Supporting Information for
preparation]), 2a (100 mg, 0.40 mmol), DIPEA (0.07 mL, 0.44
mmol), and CuI (10 mg, 0.04 mmol) yielded 6a (Rf = 0.37, hexane/
ethyl acetate [1:2]) as a white solid (174 mg, 76.8%). 1H NMR
(DMSO-d6, 400 MHz): δ 8.80−8.60 (t, 1H), 7.94 (s, 1H), 7.89−7.81
(bs, 1H), 7.89−7.76 (m,4H), 7.65−7.58 (m, 3H), 7.34−7.29 (t, 2H),
5.72 (s, 2H), 4.19−3.93 (abq, 2H), 3.44 (bs, 1H), 1.78−1.76 (q, 1H),
0.77−0.70 (dd, 6H). 13C NMR (DMSO-d6, 100 MHz): 170.27,
1
76.3%). H NMR (CDCl3, 400 MHz): δ 7.76−7.74 (d, 2H), 7.63−
7.56 (m, 3H), 4.48 (s, 2H). 13C NMR (CDCl3, 100 MHz): 156.51,
131.54, 129.51, 127.67, 125.80, 111.75, 42.83. Analytical HPLC
(instrument 1): purity = 97.9%, tR = 22.8 min.
3-(Azidomethyl)-4-(4-methoxyphenyl)-1,2,5-oxadiazole 2-Oxide
(2b). Synthesized using general the procedure with the following
values. Crude mixture of tosylated and chlorinated 1b (560 mg, 1.5
mmol) and NaN3 (150 mg, 2.2 mmol) afforded 2b as a white solid
(310 mg, 84.3%). 1H NMR (CDCl3, 400 MHz): δ 7.70−7.67 (d, 2H),
7.09−7.05 (d, 2H), 4.47 (s, 2H), 3.90 (s, 3H). 13C NMR (CDCl3, 100
MHz): 162.10, 156.20, 129.15, 118.01, 114.94, 111.71, 55.47, 42.91.
Analytical HPLC (instrument 1): purity = 99.2%, tR = 23.5 min.
3-(Azidomethyl)-4-(4-nitrophenyl)-1,2,5-oxadiazole 2-oxide (2c).
Synthesized using the general procedure with the following values.
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157.41, 145.02, 131.97, 130.54, 129.95, 129.86, 128.20, 125.84, 124.34,
116.27, 116.05, 112.64, 62.25, 42.18, 34.21, 31.12, 19.36, 18.78. ESI-
HRMS (m/z): [M + H]+ calcd for C23H24FN7O5S, 530.1567; obsd,
530.1636; [M − H]+ calcd for C23H24FN7O5S, 528.1519; obsd,
528.1541. Analytical HPLC (instrument 1): purity = 97.1%, tR = 24.4
min.
1H), 7.63−7.61 (d, 2H),7.52−7.48 (t, 1H), 7.12−7.04 (m, 5H), 6.82
(bs, 1H), 5.76−5.74 (q, 2H), 5.59−5.50 (m, 2H), 3.60 (s, 3H), 3.54−
3.51 (m, 2H), 2.10−2.02 (m, 3H), 0.82−0.79 (q, 6H). 13C NMR
(CDCl3, 100 MHz): 170.50, 161.57, 151.4, 145.07, 135.48, 133.57,
130.13, 130.01, 129.23, 128.47, 122.60, 116.27, 116.04, 115.06, 62.20,
55.47, 45.29, 34.73, 31.38, 29.67, 19.09, 17.27. ESI-HRMS (m/z): [M
+ H]+ calcd for C24H26FN7O6S, 560.1673; obsd, 560.1723; [M − H]+
calcd for C24H26FN7O6S, 558.1625; obsd, 558.1664. Analytical HPLC
(instrument 2): purity = 95.9%, tR = 23.9 min.
( R ) - 4 - ( ( 4 - ( ( 2 - ( 4 - F l u o r o p h e n y l s u l f o n a m i d o ) - 3 -
methylbutanamido)methyl)-1H-1,2,3-triazol-1-yl)methyl)-3-(4-ni-
trophenyl)-1,2,5-oxadiazole 2-Oxide (7c). White solid (52%
conversion); Rf = 0.28 (hexane/ethyl acetate [1:2]). 1H NMR
(CDCl3, 400 MHz): δ 8.41−8.39 (d, 2H, J = 8.96), 7.96−7.94 (d, 2H,
J = 8.96), 7.86−7.82 (q, 2H), 7.77 (s, 1H), 7.17−7.13 (t, 2H), 6.76
(bs, 1H), 5.84−5.76 (q, 2H), 5.41−5.39 (d, 1H, J = 8.00), 4.46−4.41
(q, 2H), 3.50−3.46 (q, 1H), 2.06−2.01 (m, 1H), 0.80−0.78 (q, 6H).
13C NMR (CDCl3, 100 MHz): 170.69, 150.75, 148.82, 145.70, 130.09,
( R ) - 3 - ( ( 4 - ( ( 2 - ( 4 - F l u o r o p h e n y l s u l f o n a m i d o ) - 3 -
methylbutanamido)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4-(4-me-
thoxyphenyl)-1,2,5-oxadiazole 2-Oxide (6b). Click reaction A:
peptidomimetic alkyne (140 mg, 0.44 mmol), 2b (100 mg, 0.40
mmol), DIPEA (0.07 mL, 0.44 mmol), and CuI (10 mg, 0.04 mmol)
yielded 6b (Rf = 0.36, hexane/ethyl acetate [1:2]) as a white solid
1
(yield = 58%). H NMR (CD3CN-d3, 400 MHz): δ 7.85−7.82 (q,
2H), 7.79−7.77 (d, 2H, J = 8.83), 7.69 (s, 1H), 7.24−7.20 (t, 2H),
7.15−7.13 (d, 2H, J = 8.78), 6.91 (bs, 1H), 5.93−5.91 (d, 1H, J =
8.80), 5.57 (s, 2H), 4.24−4.07 (qd, 2H), 3.89 (s, 3H), 3.55−3.47 (t,
1H), 1.95−1.78 (m, 1H), 0.82−0.79 (q, 6H). 13C NMR (CD3CN-d3,
100 MHz): 170.29, 163.11, 162.08, 157.02, 145.07, 137.88, 130.05,
129.95, 129.78, 124.26, 117.97, 116.27, 116.05, 115.33, 112.53, 62.26,
55.92, 49.03, 42.24, 34.23, 31.11, 19.34, 18.77. ESI-HRMS (m/z): [M
+ H]+ calcd for C24H26FN7O6S, 560.1673; obsd, 560.1739; [M − H]+
calcd for C24H26FN7O6S, 558.1625; obsd, 558.1649. Analytical HPLC
(instrument 1): purity = 98.5%, tR = 24.9 min.
130.00, 128.77, 124.54, 122.81, 116.40, 116.17, 112.81, 62.36, 60.38,
45.12, 34.90, 31.07, 19.07, 17.26, 14.17. ESI-HRMS (m/z): [M + H]+
calcd for C23H23FN8O7S: 575.1418; obsd, 575.1456; [M − H]+ calcd
for C23H23FN8O7S, 573.1370; obsd, 573.1380. Analytical HPLC
(instrument 2): ourity = 95.1%, tR = 24.2 min.
( R ) - 3 - ( ( 4 - ( ( 2 - ( 4 - F l u o r o p h e n y l s u l f o n a m i d o ) - 3 -
methylbutanamido)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4-(4-ni-
trophenyl)-1,2,5-oxadiazole 2-Oxide (6c). 2c (200 mg, 1 mmol)
dissolved in toluene (5 mL) was added the terminal alkyne (340 mg,
1.1 mmol) in 4 mL of toluene/methanol (3:1) at room temperature.
To this suspension, CuI (30 mg, 0.1 mmol) was added followed by
DIPEA (0.18 mL, 1 mmol) and the reaction stirred for 8 h. The
reaction was then filtered through Celite and concentrated. The
corresponding oil was dissolved in CH2Cl2 and successively washed
with 1 N HCl (2 × 15 mL), brine (2 × 30 mL), dried over Na2SO4,
and concentrated to give a yellow solid. The solid was then washed
with CHCl3, giving the desired product as a white solid (352 mg,
General Procedure for Staudinger Reduction, Yielding 8a and
8b. The appropriate azide (1 equiv) and P(Ph)3 (15.8 equiv) were
dissolved in DMF and stirred at rt for 1 h. Then 35% NH4OH (4
equiv) was added dropwise and reaction continued for an additional
hour. The mixture was then diluted with ice water and extracted with
ethyl acetate (3 × 100 mL). Combined organic layers were then
extracted with 1 N HCl (3 × 100 mL). Combined acidic extracts were
washed with CHCl3 (3 × 75 mL), brought to pH ∼ 10 using 1 N
NaOH, extracted with ethyl acetate (3 × 150 mL), concentrated in
vacuo, and purified by column chromatography to give the desired
products as white solids.
3-(Aminomethyl)-4-phenyl-1,2,5-oxadiazole 2-Oxide (8a). Syn-
thesized following the general procedure with the following quantities:
2a (70 mg, 0.32 mmol), P(Ph)3 (1.33 g, 5.06 mmol), 35% NH4OH
(1.26 mL), and DMF (8.4 mL, 0.038 M reaction concentration)
yielded 8a (78 mg, 33.3%). 1H NMR (CDCl3, 400 MHz): δ 7.66−7.61
(m, 3H), 7.57−7.52 (m, 2H), 3.94 (s, 2H), 1.60 (bs, 2H). 13C NMR
(CDCl3, 100 MHz): 156.54, 131.54, 129.51, 127.64, 125.79, 111.78,
42.82.
1
63%). Rf = 0.38 (hexane/ethyl acetate [1:2]). H NMR (DMSO-d6,
400 MHz): δ 8.39−8.37 (d, 2H, J = 8.0 Hz), 8.34−8.31 (t, 1H), 8.26
(s, 1H), 8.07−8.05 (d, 2H, J = 8.0 Hz), 7.92 (s, 1H), 7.85−7.83 (d,
1H, J = 9.2 Hz), 7.31−7.26 (t, 2H), 5.78 (s, 2H), 4.08−3.89 (qd, 2H),
3.42−3.38 (t, 1H), 1.76−1.71 (m, 1H), 0.73−0.66 (dd, 6H). 13C
NMR (DMSO-d6, 100 MHz): 170.29, 156.09, 149.57, 145.04, 131.77,
130.05, 129.95, 129.91, 124.81, 124.42, 116.27, 116.24, 112.66, 62.26,
34.21, 31.09, 19.34. ESI-HRMS (m/z): [M + H]+ calcd for
C23H23FN8O7S, 575.1418; obsd, 575.1477; [M − H]+ calcd for
C23H23FN8O7S, 573.1370; obsd, 573.1384. Analytical HPLC (instru-
ment 1): purity = 95.7%, tR = 24.7 min.
General Procedure for the Isomerization to the Tautomeric
Oxadiazole-N-oxides. The appropriate oxadiazole-2N-oxide (1 mmol)
was suspended in anhydrous toluene (10 mL) and heated to 120 °C
for 3 d. The reaction was allowed to cool to rt, toluene removed under
vacuo, and the mixture of isomers was purified by column
chromatography (hexane/ethyl acetate [2:1 → 1:2]) to give the
corresponding tautomeric furoxans.
3-(Aminomethyl)-4-(4-methoxyphenyl)-1,2,5-oxadiazole 2-Oxide
(8b). Synthesized following the general procedure with the following
quantities: 2b (284 mg, 1.48 mmol), P(Ph)3 (6.13 g, 23.4 mmol), 35%
NH4OH(aq) (6.0 mL), and DMF (32 mL) yielded 8b (98 mg, 38.5%).
1H NMR (CDCl3, 400 MHz): δ 7.71−7.68 (d, 2H), 7.09−7.05 (d,
2H), 4.10 (s, 2H). 13C NMR (CDCl3, 100 MHz): 161.10, 155.15,
129.19, 118.11, 114.54, 112.71, 48.52.
General Coupling Procedure. The appropriate carboxylic acid (1
equiv) was dissolved in anhydrous CH2Cl2 and brought to 0 °C under
argon. DMAP (0.5 equiv) and EDCI (1.1 equiv) were added and the
suspension stirred for 15 min at 0 °C. The furoxan amine (1.0 equiv)
dissolved in anhydrous CH2Cl2 (5 mL) was added slowly at 0 °C. The
reaction was allowed to warm to rt and stirred overnight. The mixture
was quenched w/1 N HCl, diluted with ice water, and extracted with
CH2Cl2 (3 × 50 mL). Combined organic layers were washed with
brine (2 × 25 mL), dried over Na2SO4, and concentrated to give crude
yellow oil. The residual oil purified by column chromatography to give
the corresponding furoxan peptidomimetics.
( R ) - 4 - ( ( 4 - ( ( 2 - ( 4 - F l u o r o p h e n y l s u l f o n a m i d o ) - 3 -
methylbutanamido)methyl)-1H-1,2,3-triazol-1-yl)methyl)-3-phenyl-
1,2,5-oxadiazole 2-Oxide (7a). White solid (33% conversion); Rf =
1
0.27 (hexane/ethyl acetate [1:2]). H NMR (CDCl3, 400 MHz): δ
7.85−7.81 (q, 2H), 7.71 (s, 1H), 7.66−7.65 (m, 2H), 7.61−7.54 (m,
3H), 7.13−7.08 (t, 2H), 6.84 (bs, 1H), 5.80−5.72 (q, 2H), 5.60−5.58
(d, 1H, J = 8.54), 4.45−4.36 (m, 2H), 3.55−3.51 (q, 1H), 2.06−2.01
(m, 1H), 0.83−0.79 (q, 6H). 13C NMR (CDCl3, 100 MHz): 170.55,
151.25, 145.15, 131.20, 130.08, 129.87, 129.54, 127.69, 122.78, 121.33,
116.27, 116.05, 62.24, 45.17, 34.76, 31.33, 19.09, 17.31. ESI-HRMS
(m/z): [M + H]+ calcd for C23H24FN7O5S, 530.1567; obsd, 530.1624;
[M − H]+ calcd for C23H24FN7O5S, 528.1519; obsd, 528.1557.
Analytical HPLC (instrument 2): purity = 97.0%, tR = 23.68 min.
( S ) - 4 - ( ( 4 - ( ( 2 - ( 4 - F l u o r o p h e n y l s u l f o n a m i d o ) - 3 -
methylbutanamido)methyl)-1H-1,2,3-triazol-1-yl)methyl)-3-(4-me-
thoxyphenyl)-1,2,5-oxadiazole 2-Oxide (7b). White solid (25%
conversion); Rf = 0.26 (hexane/ethyl acetate [1:2]). 1H NMR
(CDCl3, 400 MHz): δ 8.14−8.12 (d, 1H), 7.84−7.81 (t, 3H), 7.69 (s,
(R)-3-((4-Methyl-2-(phenylsulfonamido)pentanamido)methyl)-4-
phenyl-1,2,5-oxadiazole 2-Oxide (9a). Synthesized using the general
procedure with the following values: 8a (111 mg, 0.58 mmol), EDCI
(130 mg, 0.69 mmol), DMAP (40 mg, 0.3 mmol), and peptidomimetic
acid (190 mg, 0.69 mmol) afforded 9a as a white solid (244 mg,
1
95.4%). H NMR (CDCl3, 400 MHz): δ 7.85−7.79 (m, 4H), 7.59−
7.42 (m, 6H), 6.85−6.83 (t, 1H), 5.154−5.135 (d, 1H), 4.53−4.30 (m,
2H), 3.75−3.71 (m, 1H), 1.59−1.52 (m, 1H), 1.45−1.41 (m, 2H),
0.851−0.834 (d, 3H), 0.703−0.687 (3H). 13C NMR (CDCl3, 100
MHz): 171.91, 156.24, 139.00, 133.17, 131.30, 129.37, 129.05, 127.93,
3084
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Journal of Medicinal Chemistry
Article
127.24, 125.80, 112.94, 55.39, 42.25, 32.28, 24.27, 22.88, 20.98. ESI-
HRMS (m/z): [M + H]+ calcd for C21H24N4O5S, 445.1491; obsd,
445.1556; [M − H]+ calcd for C21H24N4O5S, 443.1443; obsd,
REFERENCES
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(R)-4-(4-Methoxyphenyl)-3-((4-methyl-2-(phenylsulfonamido)-
pentanamido)methyl)-1,2,5-oxadiazole 2-Oxide (9b). Synthesized
using the general procedure with the following values: 8b (40 mg, 0.21
mmol), EDCI (48 mg, 0.25 mmol), DMAP (20 mg, 0.25 mmol), and
peptidomimetic acid (68 mg, 0.25 mmol) afforded 9b as a white solid
1
(75 mg, 80.7%). H NMR (MeOD-d4, 400 MHz): δ 7.81−7.80 (m,
2H), 7.70−7.68 (d, 2H, J = 9.2 Hz), 7.57−7.46 (m, 3H), 7.09−7.07
(d, 2H, J = 9.2 Hz), 5.5 (s, 1H), 4.28−4.05 (q, 2H), 3.68−3.65 (q,
1H), 1.49−1.41 (m, 1H), 1.29−1.21 (m, 1H), 1.08−1.03 (m, 1H),
0.78−0.76 (d, 3H, J = 6.8 Hz), 0.66−0.65 (d, 3H, J = 6.8 Hz). 13C
NMR (MeOD-d4, 100 MHz): 171.91, 161.97, 155.83, 150.45, 145.07,
138.95, 133.20, 130.85, 129.42, 129.04, 128.78, 127.23, 117.96, 114.84,
112.85, 55.39, 42.25, 32.28, 24.27, 22.88, 20.98. ESI-HRMS (m/z): [M
+ H]+ calcd for C22H26N4O6S, 475.1597; obsd, 475.1650; [M − H]+
calcd for C22H26N4O6S, 473.1549; obsd, 473.1530. Analytical HPLC
(instrument 2): purity = 99.5%, tR = 25.7 min.
(R)-4-Methyl-N-((4-phenyl-1,2,5-oxadiazol-3-yl)methyl)-2-
(phenylsulfonamido)pentanamide (10). Activated zinc dust (40 mg,
0.5 mmol) was added to a solution of 9a (120 mg, 0.27 mmol) in
anhydrous MeOH (5 mL) and the reaction stirred for 15 min.
Ammonium formate (50 mg, 0.8 mmol) was then added in one
portion and the reaction was refluxed at 60 °C for 8 h. The reaction
mixture was then cooled to room temperature, filtered through Celite,
concentrated, and purified by column chromatography to give the
1
desired product as a white solid (15 mg, 13.0%). H NMR (CDCl3,
400 MHz): δ 7.85−7.85 (d, 2H, J = 1.6 Hz), 7.69−7.66 (m, 2H),
7.57−7.44 (m, 6H), 6.76−6.75 (t, 1H), 4.73−4.60 (m, 2H), 3.76−3.72
(m, 1H), 1.51−1.46 (m, 2H), 1.39−1.36 (m, 1H), 0.84−0.81 (d, 3H, J
= 6.8 Hz), 0.68−0.63 (d, 3H, J = 6.8 Hz). 13C NMR (CDCl3, 100
MHz): 171.55, 153.05, 150.29, 139.05, 133.15, 130.85, 129.33, 129.19,
128.14, 127.27, 125.03, 55.43, 41.98, 34.11, 24.26, 22.86, 21.01. ESI-
HRMS (m/z): [M + H]+ calcd for C21H24N4O4S, 429.1542; obsd,
429.1573. Analytical HPLC (instrument 1): purity = 95.2%, tR = 19.9
min.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional experimental methods and adjunct results/discus-
sion. This material is available free of charge via the Internet at
http://pubs.acs.org.
(14) Sorba, G.; Galli, U.; Cena, C.; Fruttero, R.; Gasco, A.; Morini,
G.; Adami, M.; Coruzzi, G.; Brenciaglia, M. I.; Dubini, F. A new
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AUTHOR INFORMATION
Corresponding Author
*Phone: 312-355-5282. Fax: 312 996 7107. E-mail: thatcher@
uic.edu.
■
Notes
The authors declare no competing financial interest.
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and biological evaluation of 3,4-diphenyl-1,2,5-oxadiazole-2-oxides and
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ACKNOWLEDGMENTS
The work was supported by NIH grant U01 AG028713.
■
ABBREVIATIONS USED
■
sGC, soluble guanylyl cyclase; cGKs, cGMP-dependent protein
kinases; CREB, cyclic-AMP response element binding protein;
LTP, long-term potentiation; Aβ, amyloid β; AD, Alzheimer’s
disease; BDNF, brain derived neurotropic growth factor; OGD,
oxygen glucose deprivation; MTT, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; GSNO, S-nitrosoglutathione;
DTNB, 5,5′-dithiobis-2-nitrobenzoic acid; ODQ, 1H-[1,2,4]-
oxadiazolo[4,3-a]quinoxalin-1-one; NOx, a nitrogen oxide
−
−
(including NO, NO2 , and NO3 )
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