Furoxans (Oxadiazole-4 N-oxides) with Attenuated Reactivity are Neuroprotective, Cross the Blood Brain Barrier, and Improve Passive Avoidance Memory

Article abstract of DOI:10.1021/acs.jmedchem.8b00389

Nitric oxide (NO) mimetics and other agents capable of enhancing NO/cGMP signaling have demonstrated efficacy as potential therapies for Alzheimer's disease. A group of thiol-dependent NO mimetics known as furoxans may be designed to exhibit attenuated reactivity to provide slow onset NO effects. The present study describes the design, synthesis, and evaluation of a furoxan library resulting in the identification of a prototype furoxan, 5a, which was profiled for use in the central nervous system. Furoxan 5a demonstrated negligible reactivity toward generic cellular thiols under physiological conditions. Nonetheless, cGMP-dependent neuroprotection was observed, and 5a (20 mg/kg) reversed cholinergic memory deficits in a mouse model of passive avoidance fear memory. Importantly, 5a can be prepared as a pharmaceutically acceptable salt and is observed in the brain 12 h after oral administration, suggesting potential for daily dosing and excellent metabolic stability. Continued investigation into furoxans as attenuated NO mimetics for the CNS is warranted.

Full text of DOI:10.1021/acs.jmedchem.8b00389

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Furoxans (oxadiazole-4N-oxides) with Attenuated
Reactivity are Neuroprotective, Cross the Blood Brain
Barrier, and Improve Passive Avoidance Memory
Austin Horton, Kevin Nash, Ethel Tackie-Yarboi, Alexander Kostrevski, Adam Novak, Aparna
Raghavan, Jatin Tulsulkar, Qasim Alhadidi, Nathan Wamer, Bryn Langenderfer, Kalee Royster,
Maxwell Ducharme, Katelyn Hagood, Megan Post, Zahoor A. Shah, and Isaac T. Schiefer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00389 • Publication Date (Web): 23 Apr 2018
Downloaded from http://pubs.acs.org on April 23, 2018
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Furoxans (oxadiazoleꢀ4Nꢀoxides) with Attenuated
Reactivity are Neuroprotective, Cross the Blood
Brain Barrier, and Improve Passive Avoidance
Memory
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†
‡
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Austin Horton , Kevin Nash , Ethel TackieꢀYarboi , Alexander Kostrevski , Adam Novak ,
†
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†
Aparna Raghavan , Jatin Tulsulkar , Qasim Alhadidi , Nathan Wamer , Bryn Langenderfer ,
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Kalee Royster , Maxwell Ducharme , Katelyn Hagood , Megan Post , Zahoor A. Shah , Isaac T.
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*
Schiefer
†
Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical
Sciences, University of Toledo, Toledo, OH, 43614
‡
Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and
Pharmaceutical Sciences, University of Toledo, Toledo, OH 43614
KEYWORDS. Furoxan, Nitric Oxide, NO mimetic, oxygen glucose deprivation, step through
passive avoidance, LCꢀMS/MS
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ABSTRACT
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Nitric oxide (NO) mimetics and other agents capable of enhancing NO/cGMP signaling have
demonstrated efficacy as potential therapies for Alzheimer’s disease. A group of thiolꢀdependent
NO mimetics known as furoxans may be designed to exhibit attenuated reactivity to provide
slow onset NO effects. The present study describes the design, synthesis, and evaluation of a
furoxan library resulting in the identification of a prototype furoxan, 5a, which was profiled for
use in the CNS. 5a demonstrated negligible reactivity toward generic cellular thiols under
physiological conditions. Nonetheless, cGMP dependent neuroprotection was observed. 5a (20
mg/kg) reversed cholinergic memory deficits in a mouse model of passive avoidance fear
memory. Importantly, 5a can be prepared as a pharmaceutically acceptable salt and is observed
in the brain 12 hr after oral administration, suggesting potential for daily dosing and excellent
metabolic stability. Continued investigation into furoxans as attenuated NO mimetics for the
CNS is warranted.
INTRODUCTION
Nitric oxide (NO) is a fundamental regulator of synaptic function. The ability of NO to
enhance synaptic plasticity is chiefly attributed to activation of NOꢀsensitive soluble guanylyl
cyclase (NOꢀsGC), resulting in increased production of cyclic guanosine monophosphate
(cGMP) and kinase signal transduction, primarily via protein kinase G (PKG), leading to
1
ꢀ3
phosphorylation of cAMP response element binding protein (CREB) at Serꢀ133. CREB
phosphorylation is required for synaptic plasticity and memory consolidation. Prolonged
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transcriptional activation of CREB results in the production of neurogenic gene products, such as
brain derived neurotrophic factor (BDNF), and the formation of new dendritic spines, ensuring
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the capacity for morphological flexibility in synaptic networks. Regulation of CREB was first
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identified as being controlled by cAMP/PKA kinase signaling but actually involves numerous
coꢀregulators. Importantly, inhibition of any component of the NO/cGMP/PKG system
suppresses synaptic signal. This pathway is directly inhibited by amyloid beta (Aβ) in
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Alzheimer’s disease (AD).
Reversal of Aβ induced deficits via agents which activate
NO/cGMP signaling results in improved cognitive function; as demonstrated for nitrates, sGC
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activators, and phosphodiesterase inhibitors (PDEi).
FIGURE 1, FIGURE 2
NO related biological chemistry is complex, and the term “NO donor” is often used to
•
describe multiple chemical classes, some of which do not directly yield molecular NO . Multiple
“NO donor” classes exhibit NO mimetic activity through one of several NO species (such as
•
ꢀ
+
ꢀ
ꢀ
NO , HNO, NO , NO , NO or HONOO ). Here, we describe a class of synthetic, thiolꢀ
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dependent NO mimetics, known as furoxans. Furoxans have been investigated as potential
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therapeutics for a plethora of disease states.
The majority of furoxan development has
focused on relatively reactive furoxans which harness the potent vasodilatory or cytotoxic effects
associated with large transient fluxes of NO. Indeed, of approximately 158 “furoxan” themed
original research articles in medicinal chemistry journals in the past 20 years, only two instances
focused on the use of furoxans for the central nervous system (CNS) (see Supplemental Table 1).
The pioneering effort by Schiefer et. al. used the furoxan moiety with a putative peptidomimetic
motif (Figure 1A) and demonstrated: 1) reactivity and propensity for NO mimetic activity can be
modulated via a predictable structure activity relationship (SAR), 2) furoxans protect against
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oxygen glucose deprivation (OGD) in primary cortical cell culture via a NO/cGMP dependent
mechanism, and 3) furoxans rescue oligomeric Aβ induced deficits in long term potentiation
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(LTP) (i.e., sustained strengthening of synaptic signal). Additionally, Chegaeve et. al. reported
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restored LTP and disruption of Tau and Aβ aggregation using thiacarbocyanineꢀfuroxan hybrids
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(
Figure 1B). Each of these studies incorporated the furoxan moiety within hybrid scaffolds and
confirmed efficacy with in vitro cell cultures and ex vivo hippocampal slices. Furoxans have not
hitherto been investigated in vivo for CNS applications.
In this paper a focused furoxan library was designed with rigorous consideration of
physiochemical properties for CNS penetration. Incorporating varying electron donating or
electron withdrawing groups probed electronic sensitivity of the thiophilic furoxan ring with the
goal of fineꢀtuning reactivity towards endogenous thiols, and thus the rate of NO release. The
furoxan, 5a, demonstrated negligible reactivity toward generic cellular thiols under physiological
conditions. Nonetheless, NO/cGMP dependent neuroprotection was observed with a hyperbolic
concentration response curve. 5a was shown to penetrate the blood brain barrier via multiple
administration routes and was capable of reversing cholinergic memory blockade in a mouse
model of passive avoidance memory.
RESULTS AND DISCUSSION
Library Design
The design motif for novel furoxans is summarized in Figure 2A. Groups at
GlaxoSmithKline, Eli Lilly, and others have described favorable CNS penetration as resulting
from several interꢀrelated properties, including: 1) membrane permeability and solubility, 2) low
propensity for efflux by Pꢀglycoproteins (Pgp), and 3) adequate brain partitioning to provide a
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sufficient level of unbound drug at the site of action.
Behavior with regard to these properties
is highly dependent upon the physiochemical properties of the molecule being studied (i.e.,
molecular weight [MW], partition coefficient [logP], and total polar surface area [tPSA]). Based
on these considerations, all of our compounds obey the following design criteria: 1) MW< 400
g/mol; 2) clogP 2.5ꢀ4.5; 3) tPSA 45ꢀ85. A paraꢀsubstituted aryl system was used to modulate
furoxan reactivity whilst blocking potential metabolic paraꢀhydroxylationꢀ the main route of
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metabolism of phenylꢀcontaining drugs. 4ꢀmethylpiperdine was incorporated as a cap group
because it is small, relatively hydrophobic, and contains a tertiary amine (related 4ꢀ
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alkylpiperdine reported pKa ~ 9.0) which allows preparation of pharmaceutically acceptable
salts that possess excellent water solubility. Importantly, despite predominantly existing as a
charged species at physiological pH, similar 4ꢀalkylpiperidines are present in several orally
bioavailable CNS drugs, including; haloperidol, risperidone, and the AD standard of careꢀ
donepezil.
SCHEME 1, SCHEME 2, FIGURE 3
Synthesis
Novel furoxans were synthesized from readily available cinnamic acids (Scheme 1)
beginning with anhydride formation and reduction with sodium borohydride to produce the
corresponding cinnamyl alcohols (1a-h). The cinnamyl alcohols were cyclized using an
optimized cyclization reaction with excess sodium nitrite performed in a 1:1 mixture of acetic
acid:dimethylformamide with gradual heating to yield 2a-h. A traditional Appel reaction
afforded the alkyl bromides 3a-h, and subsequent substitution with 4ꢀmethylpiperidine yielded
the oxadiazoleꢀ2Nꢀoxides (ODZꢀ2N), 4a-h. The final step of the synthesis involves
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tautomerization of the furoxan in refluxing toluene to yield oxadiazoleꢀ4Nꢀoxides (ODZꢀ4N).
Separation of the ODZꢀ2N/ODZꢀ4N tautomers was realized via reverse phase flash
chromatography to yield the ODZꢀ4N compounds 5a-h. Initial in vitro testing nominated 5a for
in vivo evaluation, vide infra. Accordingly, substitution of 3a with isotope labeled 4ꢀ
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methylpiperidineꢀ2,2,6,6ꢀd followed by tautomerization provided 7a as an appropriate internal
standard for LCꢀMS/MS studies (Scheme 2). Additionally, treatment of 5a with HCl in diethyl
ether provided the water soluble HCl salt for in vivo investigations.
Crystallographic examination of 5a (Figure 3A) confirmed the presence of the
oxadiazoleꢀ4Nꢀoxide (ODZꢀ4N) structure (see Supplemental Crystallographic Report). HPLC
purity analysis using a photodiode array detector (PDA) showed a noticeable shift in the λmax of
absorption for ODZꢀ2N analogs (~246ꢀ261 nm) versus ODZꢀ4N analogs (279ꢀ293 nm),
representative example shown for 4a and 5a in Figure 3B (see Supplemental Characterization for
PDA data for all compounds). This phenomenon may be used to easily distinguish furoxan
tautomeric form. In all instances, retention time (R ) comparison indicated that, despite
t
possessing greater aromaticity based on λmax, the ODZꢀ4N analogs are more polar than
corresponding ODZꢀ2N analogs. 5a was also profiled by 2DꢀNMR (see Supplemental Materials
for Homonuclear correlation spectroscopy [COSY] and Heteronuclear Multiple Quantum
Coherence [HMQC]).
Neuroprotection
Oxygen glucose deprivation (OGD) provides a cellular model of oxidative stress
resulting in apoptotic cell death, providing a model of neuronal death across neurodegenerative
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diseases, including AD. Expert perspectives suggest that experimental paradigms in drug
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discovery should model wellꢀunderstood target pathways rather than specific disease state.
NO mimetics have been benchmarked as being protective against oxidative stress induced by
OGD via multiple proposed mechanisms, including: 1) antioxidant effects, 2) mediating
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phosphorylation of prosurvival kinase signaling; and 3) Sꢀnitrosylation of proꢀapoptotic
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caspases.
Neuroprotection was evaluated by 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)2,5ꢀdiphenylꢀ
tetrazolium bromide (MTT) assay of cell viability in nonꢀdifferentiated PCꢀ12 cell cultures
subjected to OGD for 90 min followed by reoxygenation and replacement with nutrient rich
media containing furoxansꢀ screened at three concentrations (1, 10, and 25 ꢀM) for 24 hr.
Neuroprotection is reported as a percentage change relative to vehicle (DMSO) treated controls.
TABLE 1, FIGURE 4
The ODZꢀ2N compounds (except 4d) showed negligible neuroprotection (Table 1). A
few ODZꢀ4N analogs provided noticeable neuroprotective activity, compounds 5a (4ꢀF), 5c (4ꢀ
Br), and 5d (4ꢀOCHF ). The 4ꢀOCHF tautomers (4d and 5d) showed robust potency with
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significant efficacy at 1 ꢀM. 4d and 5d are considered to exhibit ‘outlierꢀlike’ behavior
compared to the rest of the library based upon: 1) 4d is the only active ODZꢀ2N from our series;
and 2) the potency of these compounds is an order of magnitude lower than any other compound.
Antioxidant effects, such as those traditionally seen for NO mimetics, result in potent in vitro
neuroprotection against oxidative stress associated with OGD. In NO related drug discovery the
most potent compounds in primary assays may not survive hitꢀtoꢀlead optimization due to
deleterious effects in animals. 4d and 5d warrant additional investigation, however, they were
not considered for proofꢀofꢀconcept in vivo investigation of attenuated furoxans because the
impetus for this work was to produce NO mimetics which activate NO/cGMP signaling without
producing significant fluxes of NOꢀ thereby reducing the potential for side effects. 5a and 5c
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demonstrate statistically equivalent efficacy and potency. 5a, rather than 5c, was chosen for in
vivo evaluation for multiple reasons, including the smaller molecular weight (a critical factor in
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brain penetration and ligand efficiency) and future potential for incorporation of F for
radioimaging. 7a was synthesized for use as a LCꢀMS/MS internal standard and was also tested
in the primary assay. Similar activity was expected compared to the nonꢀdeuterated 5a, however,
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a possessed no neuroprotective effects. UPLC separation suggests 7a is slightly more polar than
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a, based on R (Supplemental Figure S3). LCꢀMS/MS was used to measure membrane
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permeability in the primary assay (i.e., extracellular concentration versus intracellular
concentration) of 5a and 7a in the media (supernatant) and cytosol of control (no OGD) and
OGDꢀtreated PC12 cells following 24 hr incubation via subcellular fractionation. Each
compound was found in greatest amounts in the cytosol and submitting to OGD insult did not
significantly effect distribution (Supplemental Figure S4). These data demonstrates; 1) furoxans
readily cross cell membranes in the primary assay; and 2) significant amounts of furoxan after 24
hour incubation with PC12 cells; providing further evidence for the relatively unreactive nature
of this furoxan library.
An expanded doseꢀresponse assay of OGDꢀexposed PC12 cells treated with 5a revealed a
bellꢀshaped (i.e., hyperbolic) efficacy curve from which an EC = 1.9 ꢀM was calculated (Figure
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A). For concentration response studies a resorufin based dye was used as the viability readꢀout
due to a significantly shorter incubation time before colorimetric measurement which allows
more direct observation and increased throughput compared to MTT. The hyperbolic response
curve is in agreement with previous studies which identified a correlation between production of
ꢀ
NO by furoxans in the presence of cysteine (i.e., reactivity) with protection against OGD.
2
Notably, although protective effect of 5a are lost at higher concentrations, no toxicity was
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observed at the highest concentration tested (50 ꢀM). Coꢀincubation with ODQ (1Hꢀ
[
1,2,4]oxadiazoloꢀ[4, 3ꢀa]quinoxalinꢀ1ꢀone), a selective sGC inhibitor, completely blocked the
neuroprotective effects of 5a (Figure 4B), providing evidence for enhanced NO/cGMP signaling
as being responsible for neuroprotective efficacy of 5a. This result is in agreement with previous
reports of ODQ blocking the ability of furoxans or nitrates to protect against oxidative stress in
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cell culture. The presence of a basic nitrogen in the structure of the furoxan may lead to the
interaction with a number of different targets in CNS. Hence, 5a (active) and 7a (inactive) were
sent to the Psychoactive Drug Screening Program for binding studies to assess offꢀtarget effects
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as described previously.
As shown in Supplementary Table 2, 5a does not interfere with
binding of any radioligands for all receptors screened, including GABA , serotonin receptors,
A
dopamine receptors, and muscarinic and nicotinic acetylcholine receptors. No hERG binding
was observed. For the most part 7a demonstrated as similar profile, but did significantly inhibit
binding at the delta opioid receptor and benzodiazepine (BZP) rat brain site.
Reactivity
5
a was incubated with Nꢀacetyl cysteine (5 mM) in PBS (50 mM, pH 7.4) to examine the
stability of the furoxan ring in the presence of excess free thiols (see Supplemental Figures S10ꢀ
S11). We hypothesized reactivity with excess thiol would correlate with neuroprotection in the
primary assay. However, no reaction was observed by HPLCꢀPDA analysis of all compounds,
while control incubations using the electrophile Nꢀethylmaleimide (NEM) showed complete
reaction of NEM after 2 and 12 hr. In agreement with the observed attenuated reactivity
ꢀ
negligible NO production was observed from 5a, in the form of NO , in the primary assay
2
(Supplemental Figure S11).
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FIGURE 5
Improved Memory in Step-through Passive Avoidance (STPA)
Pilot behavioral studies evaluated the ability of 5a to reverse scopolamine induced
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deficits in stepꢀthrough passive avoidance (STPA). STPA assesses the ability of drugs to reverse
36ꢀ38
hippocampalꢀdependent aversive memory deficits.
Scopolamine is a wellꢀcharacterized panꢀ
muscarinic antagonist which produces significant amnesic effects when administered 30 min
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prior to training in STPA.
Reversal of scopolamineꢀinduced amnesia is not a sophisticated
model, but it is well understood and remains useful as a resource efficient screen for procognitive
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agents. Importantly, reversal of scopolamine induced cholinergic deficits in STPA via
NO/cGMP enhancement is well characterized, and has been benchmarked for nitrates, sGC
42ꢀ46
activators, PKG activators, and several phosphodiesterase inhibitors (PDEi).
The STPA
apparatus consists of an acrylic box with an illuminated compartment connected to a darkened
compartment by a small guillotine door. Mice are placed in the illuminated side and are trained
against their natural tendency to translocate to the dark side using an aversive stimulus during
double trial acquisition. Memory consolidation is measured as the latency to enter the dark
compartment 24 hr after training (Figure 5). Testing latency 24 hr after treatment avoids
potential convolution from lethargy caused by drug exposure and lethargy is not reported for
47
agents acting via cGMP signaling, as shown for sildenafil in the open field test.
Furoxans have never been studied in the CNS. Hence, choices for dosing and
administration timing were not evident at the onset. A NOꢀchimeric nitrate reversed scopolamine
induced deficits in STPA when administered 20 min prior to training but had no effect when
given 60 min prior to training, demonstrating the short biological halfꢀlife of nitrates resulting in
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rapid onset of neuromodulatory efficacy at a low dose (1 mg/kg). Based on the knowledge that
attenuated furoxans likely have greater metabolic stability, we anticipated that a longer preꢀ
training treatment duration would be required to allow sufficient time for slowꢀonset NO
mimetic effects. With these considerations in mind, we examined varying doses at two preꢀ
training time points (1 or 2 hr). Scopolamine treated mice showed significantly less latency to
enter the dark compartment compared to wildꢀtype mice (Scopolamine treated: 63.5 s ± 30 s
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[
s.e.m] versus vehicle treated: 270 s ± 29 s [s.e.m], p<0.001). 5a was capable of reversing
scopolamine induced amnesia, with a statistically significant effect observed when administered
hr prior to training at 20 mg/kg (latency = 237 s ± 22 s [s.e.m], p<0.01). These data
2
demonstrates, for the first time, the ability of a furoxan to improve memory in mice by reversing
cholinergic memory blockade.
FIGURE 6
In Vivo Drug Stability, Brain Penetration, and Effect on Protein Phosphorylation
To understand the STPA behavioral results we attempted to correlate amount of 5a in the
forebrain with modulation of a hypothesized marker of efficacy, CREB phosphorylation. The
forebrain contains the primary neurocircuitry responsible for learning and memory, particularly
the hippocampus. Pilot studies analyzed the entire forebrain to ensure enough tissue was
available for both ELISA and LCꢀMS/MS analysis from the same animal (Figure 6A). 5a was
measured by LCꢀMS/MS analysis of plasma and forebrain samples after intraperitoneal (ip) and
oral (po) administration at varying time points (1, 2, or 12 hr [n = 4]). CREB phosphorylation
was also analyzed in the forebrain samples, as pCREB:tCREB ratio. Selected sacrifice times
were intended to provide a preliminary assessment of the in vivo t1/2 whilst providing a
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pharmacodynamic measurement in a system mimicking behavioral studies in which 5a (20
mg/kg) reversed scopolamine induced memory deficits when administered 2 hr prior to
behavioral training.
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Assessment of brain:plasma ratio was carried out via MRM using a LCꢀMS/MS. The
forebrain and plasma samples were processed using solid phase extraction (SPE). 5a was
observed in all drug treated animals, including detectable levels after 12 h (Figure 6). Deuterated
internal standards are desirable for absolute quantitation via LCꢀMS/MS because they have
similar physiochemical properties to their nonꢀdeuterated analog, but may be distinguished based
on molecular weight. Inclusion of 7a allowed for absolute quantitation of 5a in the forebrain of
treated mice. Extraction methodology and tandem LCꢀMS/MS parameters (triple quadrupole
utilizing optimized multiple reaction monitoring [MRM] transitions) were developed and
validated via extraction of untreated brain tissue spiked with varying concentrations of 5a and a
fixed concentration of 7a to develop a calibration curve. Systemic t1/2 for brain and plasma
measurements was between 50 min ꢀ 110 min, depending on administration route. There were no
significant differences in levels of 5a in the brain (B) versus plasma (P). Furthermore, there were
no significant differences in levels in the brain for po versus ip administration, suggesting a
consistent and favorable biodistribution profile with either administration route. Differences in
B:P based on administration route trended toward but did not reach significance (p = 0.16 using
unpaired tꢀtest) with an average oral administration B:P ratio of 1.49 (± 0.52, s.e.m) and
intraperitoneal administration average B:P ratio of 0.68 (± 0.03, s.e.m). We anticipate 5a
accesses the brain via passive diffusion based on a low molecular weight (291.1 g/mol) and a
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clogP ~ 3.1, both of which are predicted to allow passive membrane permeability.
These
findings are novel for several reasons: including, 1) this is the first report demonstrating a
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furoxan crossing the blood brain barrier; 2) to our knowledge, this is the first NO mimetic
observed in the brain 12 hr after administration (i.p. or p.o.); and 3) this is the first report
establishing a B:P ratio for a furoxan.
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Given the knowledge that 5a reverses scopolamine induced deficits in STPA, we
expected 5a to elicit a change in CREB phosphorylation (pCREB). However, no effect on
pCREB was observed for any cohorts treated with 5a alone based on analysis by ELISA (Figure
6
C). A more rigorous evaluation was performed in followꢀup investigations studying
hippocampal protein phosphorylation by western blot. Mice were treated in an identical manner
to STPA studies except animals were sacrificed at the timeꢀpoint previously used for training in
STPA (i.e., at the time of memory acquisition). These studies included 4 cohorts (N = 8 per
cohort [with experimenters blinded to treatment]) with each receiving one i.p. injection 2 hr prior
to sacrifice (either 5a [20 mg/kg] or vehicle) and one i.p. injection 30 min prior to sacrifice
(either scopolamine [1 mg/kg] or vehicle). Blots for either phosphorylated proteins [pProtein] or
total protein [tProtein] were normalized to GAPDH loading controls prior to calculating
phosphorylation ratio as pProtein:tProtein, analyzed by oneꢀway ANOVA. Despite these efforts,
statistically significant changes in hippocampal protein phosphorylation was not observed in any
cohorts for pCREB:tCREB, or pErk:tErk (Supplementary Figure S16). Importantly, we did not
observe significant changes in protein phosphorylation for mice administered only scopolamineꢀ
the negative control group. Most articles utilizing acute scopolamine induced memory deficits do
not report measurements of protein phosphorylation. Those which do agree with our findings.
Indeed, acute scopolamine induces impaired learning and memory but has no effect on protein
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phosphorylation in normal mice (C57Bl6).
Furthermore, neither acute nor longꢀterm
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administration of PDEi’s effects hippocampal pCREB in normal mice.
Hence, our findings
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for furoxans are inꢀline with literature precedent for agents acting via NO/cGMP signaling.
Longꢀterm administration (> 3 weeks) of nitrates or PDEi’s to AD transgenic (ADꢀTg) mice
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results in increases in hippocampal pCREB relative to vehicle treated controls.
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Hippocampal pCREB is most appropriate as a marker of efficacy when studying longꢀterm
administration of these agents in AD transgenic mice (such as APP/PS1, 5xFAD, 3xTg).
We provide evidence for furoxan effects associated with activation of NO/cGMP
signaling; including, 1) protection against oxidative stress which is abolished upon blocking
cGMP production with a selective sGC inhibitor; and 2) reversal of scopolamine induced
memory impairmentꢀ reliably shown for NO mimetics, PDEI’s, and sGC activators. Furoxans
have been extensively described as thiolꢀdependent NO mimetics. Our pursuit of furoxans with
attenuated reactivity has resulted in the identification of efficacious furoxans which lack of
reactivity with generic thiols. Characterizing the reaction of an attenuated furoxan with thiol is
difficult due to lack of reactivity. The specific thiol(s) responsible for and mechanism of
bioactivation is not known at present. Based on our data, we present two proposed scenarios:
Scenario A) a subset of yet to be identified sufficiently reactive cellular thiols attack the furoxan
ring to activate NO release; or Scenario B) efficacious furoxans described here are metabolized,
in a CYP independent manner (based on activity in PC12 cells), to a yet to be identified active
metabolite. In scenario B, the active metabolite would contain the intact furoxan but would have
greater thiophilic reactivity. In medicinal chemistry it is wellꢀknown that instances in which a
deuterated derivative lacks efficacy of an equivalent nonꢀdeuterated molecule may be explained
by a requirement for metabolic activation for efficacy (i.e., an active metabolite is responsible for
4
efficacy). Hence, the lack of activity of the deuterated (d ) analog, 7a, provides support for the
notion of an active metabolite. Additional supportive data are provided by the STPA behavioral
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model in which activity was observed when 5a was administered 2 hr prior to training but not 1
hr. The foundational efforts described here provide a reasonable starting point for exploration of
these scenarios going forward.
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CONCLUSION
The neuroprotective properties of novel attenuated furoxans have been explored. A
furoxan, 5a, elicits a hyperbolic concentrationꢀresponse curve for protection against oxidative
stress which is completely blocked by coꢀincubation with ODQ, a selective sGC inhibitor, thus
implicating neuroprotection resulting from a cGMP dependent mechanism. The incorporation of
a tertiary amine into the cap group provides access to pharmaceutically acceptable salts.
Fortunately, the presence of the tertiary amine [calculated pKa ~9], which is likely charged at
physiologic pH, does not hinder ability to cross biological membranes based on levels of 5a in
the cytosolic fractions of PC12 cells and brain penetration following administration by oral
gavage. Memory improvement was observed for 5a when administered 2 hr prior to training in
STPA, showing statistically significant recovery of cognitive function after an induced deficit by
scopolamine. Cumulatively, our data indicates that 5a is a relatively stable attenuated (i.e,
weaklyꢀreactive) NO mimetic based on: 1) negligible reactivity with generic thiols at
physiological pH and temperature, 2) the presence of 5a in PC12 cells 24 hr after treatment, and
3
) the presence of 5a in the brain and plasma 12 hr after po and ip administration. Furthermore,
we hypothesize 5a works via a NO/cGMP/CREB dependent mechanism based upon: 1)
eliminated neuroprotection upon sGC blockage; 2) a hyperbolic concentration response curve;
and 3) reversal of cholinergic memory deficits in STPA. Our data suggests that free thiols alone
are not capable of activating release of NO from attenuated furoxans. Identification of the protein
target(s) or microenvironment(s) which facilitate bioactivation would allow for development of a
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quantitative structure activity relationship (QSAR) for analog refinement leading to candidate
nomination for IND enabling studies. Further investigation is warranted to elucidate the exact
mechanism of furoxan bioactivation leading to NO release.
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EXPERIMENTAL SECTION
All chemicals and reagents were purchased from SigmaꢀAldrich (St. Louis, MO), or Fisher
Scientific unless stated otherwise. All protocols and procedures involving the use of animals
were approved by the Institutional Animal Care and Use Committee at the University of Toledo.
General Methods. Reagents and solvents were purchased from common commercial suppliers
(Fisher Scientific or Sigma Aldrich) and used as received. All reactions were carried out under
atmospheric conditions at room temperature unless otherwise indicated. Reactions were
monitored by thinꢀlayer chromatography (TLC, LuxPlate silica gel 60 F254 plates) and revealed
by UV light (254 nm). Column chromatography was performed using Teledyne Combiflash Rf
with RediSepR Gold columns and RediSep Rf C18 columns for tautomerization purification.
f
HPLC analysis was performed using a Shimadzu Prominence HPLC (LCDꢀ20AD) with
temperature controlled autosampler (SILꢀ20AC), refractive index (RIDꢀ20A) and PDA (SPDꢀ
M20A) detectors. LCꢀMS/MS analysis was carried out with a Nexera XR UPLC coupled with a
Shimadzu 8050 triple quadrupole mass spectrometer (ESI, positive mode). Separations utilized a
®
Phenomenex Kinetix core column (2.6 µm, C18, 100 Å, 100 x 4.6 mm column). HPLC
conditions: Mobile phase A = water (0.1 % formic acid [FA]) and mobile phase B = acetonitrile
(0.1 % FA); 1.0 mL/min at 30 % B for 1 min followed by gradient increase to 95 % B over 5
minutes followed by 1 min at 95 % B and reꢀequilibration at 30 % B for 4 min resulting in a total
run time = 10 min. UPLCꢀMS/MS utilized same conditions substituting aqueous ammonium
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formate (25 mM, 0.1 % FA) as mobile phase A. Purity of tested compounds was found to be >
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5% pure at two wavelengths, 254 nm and 282 nm unless otherwise indicated. NMR ( H, C)
was taken using a Bruker Avance 600 MHz spectrometer (cryoprobe). High resolution mass
spectra (HRMS) was recorded using Waters Synapt high definition mass spectrometer (HDMS)
equipped with nanoꢀESI source positive mode.
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Crystallography. Furoxan 5a was chosen for crystallographic examination. A clear colorless
irregular prismꢀlike specimen of C H FN O , approximate dimensions 0.058 mm x 0.156 mm x
1
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18
3
2
0
.270 mm, was used for the Xꢀray crystallographic analysis. The Xꢀray intensity data were
measured. The integration of the data using a monoclinic unit cell yielded a total of 26380
reflections to a maximum θ angle of 33.11° (0.65 Å resolution), of which 5531 were independent
(
average redundancy 4.769, completeness = 95.1 %, R = 4.72%, R = 4.02%) and 4041 (73.06
int sig
%) were greater than 2σ(F2). The final cell constants of a = 11.3374(14) Å, b = 7.2234(9) Å, c =
3
18.825(2) Å, β = 97.601(2)°, volume = 1528.1(3) Å , are based upon the refinement of the XYZꢀ
centroids of reflections above 20 σ(I). The final anisotropic fullꢀmatrix leastꢀsquares refinement
on F2 with 255 variables converged at R1 = 5.17 %, for the observed data and wR2 = 14.18 %
for all data. The methyl group (C15) is disordered. The occupancy of the C15 atoms was refined
to be 50 % and then fixed. Data were refined with isotropic atomic displacement parameters; the
bonded hydrogen atoms were calculated on idealized hydrogens and refined as riding atoms. The
goodnessꢀofꢀfit was 1.012. The largest peak in the final difference electron density synthesis was
3
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3
0.487 eꢀ/Å and the largest hole was ꢀ0.540 eꢀ/Å with an RMS deviation of 0.058 eꢀ/Å . On the
eꢀ
basis of the final model, the calculated density was 1.266 g/cm3 and F(000), 616 . Authors will
release the atomic coordinates and experimental data upon article publication.
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Oxygen Glucose Deprivation (OGD) Neuroprotection Assay. Viability is reported as %
change relative to vehicle treated control over at least three separate trials. Undifferentiated PC12
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cells (passage 12ꢀ14) were exposed to OGD as described previously. Briefly, 1x10 PC12 cells
were plated overnight in growth medium (DMEM/F12 1:1 containing 5 % FBS, 5 % HS,
penicillin/streptomycin). The medium was replaced with glucoseꢀfree buffered salt solution
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(HBSS phenol red medium), and the plate was placed in an anaerobic chamber under inert
atmosphere (argon) for 1.5 hr in a 37 °C incubator. After OGD, the glucoseꢀfree medium was
removed, and fresh growth medium was added along with drug or vehicle (DMSO) treatment for
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4 hr at 37 °C. Cell viability was assessed by MTT dye or PrestoBlue cell viability reagent
(ThermoFisher) according to manufacturer instructions. MTT (0.5 mg/mL) was added to each
well for 4 hr, the medium was aspirated, then the dye was reꢀsuspended in DMSO and read by
microplate reader (absorbance, 540 nm). Alternatively, PrestoBlue (10 %) was added to each
well for 30 min then read by microplate reader (fluorescence, excitation/emission 560/590 nm).
Reactivity Analysis. The appropriate furoxan (250 ꢁM) or Nꢀethylmaleimide (250 ꢁM) was
incubated ± Nꢀacetylcysteine (5 mM) in PBS (50 mM, pH 7.4) at 37 °C and aliquots taken at 2
and 12 h. The mixtures were analyzed by HPLCꢀPDA analysis according to general methods.
Peak integration determined the area under the curve (AUC) from UV absorbance spectra at the
λꢀmax of each analyte provided relative abundance of each starting material. Result can be found
in Supplemental Figures S10ꢀ11.
4
LC-MS/MS study of cell permeability. Due to different in vitro activity between 5a and its d ꢀ
counterpart, 7a, the cellular permeability of each compound was investigated by LCꢀMS/MS.
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Briefly, 1x10 PC12 cells were exposed to OGD as described in neuroprotection assay section
and treated with each analyte (25µM) or vehicle control for 24 hr. A control plate which was not
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submitted to OGD was also studied. The supernatant (1 mL) fractions were collected and stored
at ꢀ80 °C until further analysis. Cells were lysed using fractionation buffers for cytosolic. To
each sample, 150 µL Buffer A (HEPES [10 mM], MgCl [1.5 mM], KCl [10 mM], 1 % NPꢀ40,
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Dithiothreitol [0.5 mM], phenylmethylsulfonyl fluoride [0.2 mM], sodium orthovanadate [1
mM], NaF [50 mM], sodium pyrophosphate [10 mM], protease inhibitor cocktail [Thermo]) was
added, vortexed, and maintained at 0 °C for 15 min. The resulting solution was centrifuged, and
the supernatant collected as cytosolic fraction. Fractions were stored at ꢀ80 °C until further
analysis. Supernatant (extracellular) and cytosolic (intracellular) samples (100 µL) were diluted
with 200 ꢀL 2 % formic acid (FA) in dH O, and spiked with a corresponding internal standard
2
(7a for vehicle and 5aꢀtreated samples; 5a for 7aꢀtreated samples). Analysis was carried out as
described in the UPLCꢀMS/MS method described in the general methods section. The AUC for
the MRM transitions of the analytes were integrated, and normalized to their respective internal
standards. Result can be found in Supplemental Figure S4.
Step through passive avoidance. In the early morning (within 1 hr of light ‘on’ cycle begin),
male C57BL/6 mice were habituated in STPA apparatus to confirm photophobic phenotype.
Translocation in < 30 seconds considered normal. All mice exhibited normal photophobic
behavior. Mice were weighed and administered either vehicle (0.9 % NaCl with 1 % DMSO) or
the HCl salt of 5a (0.1, 1, 10, or 20 mg/kg) via ip injection at 60 min or 120 min prior to training
in STPA. Scopolamine (1 mg/kg) or vehicle (0.9 % NaCl), was administered 30 min prior to
training via ip injection. Mice were placed in the illuminated side of the Passive Avoidance
apparatus (Ugo Basile) and entry into the dark chamber (monitored by sensors in the STPA
apparatus) resulted in automatic closing of a vertical door and administration of a mild aversive
electrical shock (0.5 mA, 3 sec in duration) via electrical floor grid. The mouse was kept in the
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dark chamber for 30 sec and then returned to the holding cage for 120 sec before the second
training session. In the second training, in instances when the mice did not translocate in 300 sec
they were gently pushed into the dark compartment. Latency to enter the dark compartment was
measured 24 hr (± 30 min) after initial training to determine memory retention. No shock is
administered during the retention trial. Animals which did not translocate during the retention
trial were assigned a retention latency of 300 sec. Mice were euthenized by carbon dioxide
poisoning and cervical dislocation within 24 hr of retention testing completion. Notes: 1) Double
trial acquisition increases the statistical power of data from STPA and decreases requisite sample
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sizes from n = 12ꢀ14 to n = 7ꢀ9.
2) In four treatment cohorts (60 min [10 mg/kg], 60 min [20
mg/kg], [120 min 1 mg/kg], and [120 min 10 mg/kg]), upon opening the dark compartment
nd
following the 2 training a mouse jumped from the apparatus and landed on the floorꢀ these
mice were excluded from further analysis, hence the n = 8 for these groups. An oversight in
experimental setup resulted in the use of vehicle containing 1% DMSO, however, it should be
noted that the HCl salt of 5a readily dissolves in saline at the concentration used of these studies.
LC-MS/MS Method Development. Synthesized standards of 5a and 7a were used to study LCꢀ
MS/MS fragmentation to provide parent to daughter ion transitions for MRM analysis as
follows: 5a (292.2 ꢀ112.1, 292.2ꢀ99.1 292.2ꢀ154.1) and 7a (296.2ꢀ158.1, 296.2 ꢀ116.2,
2
96.2 ꢀ102.2). MRM transitions are shown in Supplemental Figure 10ꢀ11. During method
development, blank brain homogenate (100 ꢀL each) was added to 200 ꢀL of 2 % aqueous FA
spiked with a fixed concentration of 7a (0.5 ꢀMꢀ final concentration based on reconstitution in
1
00 ꢀL LCꢀMS load solvent) and vary amounts of 5a (0.0001, 0.001, 0.01, 0.1, 1, or 10 ꢀMꢀ
final concentration based on reconstitution in 100 ꢀL LCꢀMS load solvent). Each mixture was
vortexed briefly and applied to a solid phase extraction resin (Evolute Express ABN cartridge;
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size = 30 mg/1 mL [Biotage]) using a Vacmaster manifold (Biotage). Cartridges were washed
with dH O (1 mL), analytes eluted with MeOH (1 mL), and evaporated to dryness via high flow
2
air purge. Samples were reconstituted in 100 ꢁL of ACN:dH O (1:1) for LCꢀMS/MS analysis
2
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(injection volume of 20 ꢁL). All samples were run in one batch, and then the entire batch was
repeated. The internal standard AUC deviation was < 10 % for all samples. For each sample, the
AUC for 5a was divided by the intraꢀrun TIC 7a to provide 5a:7a ratios which were averaged
between the two runs and plotted against corresponding concentrations and demonstrated
2
appropriate linearity, R = 0.99. The lower limit of quantitation (LLOQ) for 5a was < 0.1 nM
(signalꢀtoꢀnoise [S/N] to be > 10:1). Determination of the exact limit of detection (LOD) and
LLOQ was not required based on the presence of 5a in high concentrations.
Bioavailability Study. C57Bl6 mice were weighed and administered the HCl salt of 5a (20
mg/kg) via oral gavage (vehicle = sesame oil) or intraperitoneal injection (vehicle = 0.9 % NaCl
with 1 % DMSO). Mice were returned to holding cages and monitored prior to euthenisia by
carbon dioxide poisoning and cervical dislocation after 1, 2, or 12 h. No signs of lethargy or
distress were noted during monitoring. Blood was collected into K EDTA coated tubes (BD
2
®
Microtainer ) by cardiac puncture, flash frozen in liquid N , and stored at ꢀ80 °C for further
2
analysis. Each brain was removed and immediately placed on a metal dissection surface that lay
atop of bed of dry ice in order to minimize protein degradation and two portions dissected out: 1)
the forebrain (containing cerebral cortex and hippocampus), and 2) the cerebellum. Dissected out
tissue was placed in a preꢀweighed 5 ml eppendorf tube and flash frozen with liquid N and
2
stored at ꢀ80 °C for further analysis.
Each forebrain was placed in 1.5 ml RINO tube (Next Advance Inc.) followed by addition of
tissue extraction reagent (1 mL [Invitrogen]ꢀ containing a protease/phosphatase inhibitor
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cocktail). The mixture was homogenized with The Bullet Blender Storm (Next Advance Inc.)
for 1 min at 4 °C (speed 8) followed by centrifugation at 13k rpm for 2 min at 4 °C. A
homogenate aliquot of 100 ꢀL was extracted and the rest of the homogenate stored at ꢀ80 °C for
further analysis. Homogenate (100 ꢀL) or plasma (50 ꢀL) was added to 2 % FA (200 µL,
containing 7a [0.5 ꢀMꢀ final concentration upon reconstitution in 100 ꢀL] and applied to a solid
phase extraction resin (Evolute Express ABN cartridge; size = 30 mg/1 mL [Biotage]) using a
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Vacmaster manifold (Biotage). Cartridges were washed with dH O (1 mL), analytes eluted with
2
MeOH (1 mL), and evaporated to dryness via high flow air purge. Samples were reconstituted in
100 ꢁL of load solvent for LCꢀMS/MS analysis (injection volume of 10 ꢁL). All samples were
run in one batch and then the entire LC batch was repeated. For each sample, the AUC for 5a
was divided by the intraꢀrun AUC of 7a to provide 5a:7a ratios which were averaged between
the two runs. Forebrain homogenate extraction was repeated a second time and comparison of
2
analyte concentration from both extractions provided a strong correlation (R = 0.99), suggesting
remarkable consistency for the solid phase extraction method. Plasma extraction was only
performed once due to limited amount of material and the reliability shown for forebrain
extractions. n = 4 for all PK data except 1 h ip group. Note: for 1 h ip group we measured 5a at
levels 50ꢀ100x greater than any other mouse (in plasma and brain). Experimenters hypothesized
potential mistaken bladder injection. We have not investigated this outlier further. It is possible
that the alkaline pH of the bladder deprotonated the piperidine ring allowing increased
membrane permeability and greater distribution.
Measuring pCREB:tCREB ratio by ELISA
pCREB and tCREB were measured using ELISA kits purchased from Invitrogen according to
provided protocol. Briefly, protein concentration for forebrain homogenates from the
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bioavailability study were analyzed by Bradford assay. Each homogenate (4 ꢀL) was added to
standard diluent buffer (96 ꢀL) and incubated for 2 hr alongside calibration curve standard
solutions. Wells were washed with wash buffer (4 x 300 ꢀL) followed by 1 hr incubation with
appropriate primary antibodies (CREB [pS133] Detection Antibody or CREB (Total) detection
antibody). Wells were washed with wash buffer (4 x 300 ꢀL) followed by 30 min incubation
with secondary antibody (antiꢀRabbit IgG HRP). Wells were washed with wash buffer (4 x 300
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ꢀ
L) followed by 30 min incubation with the chromogen (100 ꢀL) and subsequent addition of the
stop solution (100 ꢀL). Absorbance was measured at 450 nm using a Biotek Synergy H2 plate
reader. Levels of pCREB and tCREB were normalized to total protein concentration to provide
reported pCREB:tCREB ratio’s. Data represents SEM, analysis by oneꢀway ANOVA with
Tukey’s test; N = 4.
In vivo study of protein phosphorylation by Western blot
5
3
Protein phosphorylation was measured according to previously described procedures. C57Bl6
mice were weighed and administered the HCl salt of 5a (20 mg/kg) via ip injection (vehicle = 0.9
%
NaCl [1 % DMSO]) 2 hr prior to sacrifice. Scopolamine (1 mg/kg) or vehicle (0.9 % NaCl),
was administered 30 min prior to sacrifice via ip injection. Mice were returned to holding cages
and monitored prior to euthenisia by carbon dioxide poisoning and cervical dislocation. No signs
of lethargy or distress were noted during monitoring. Hippocampal tissue was dissected out and
flash frozen in liquid nitrogen until transfer to ꢀ80 ⁰C for storage. Hippocampi were
homogenized with cell extraction reagent (Thermofisher) containing protease/phosphatase
inhibitor cocktail, centrifuged at 13k rpm for 5 min, and supernatant collected and frozen at ꢀ80
⁰C. Protein concentration was determined by Bradford assay and samples analyzed by loading
equivalent amounts of protein onto 10ꢀ20 % TrisꢀGlycine Mini gels (Novex WedgeWell,
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ThermoFisher). Proteins were transferred from to PVDF membranes and blocked with 5 % BSA
for 1 h at room temperature followed by overnight incubation at 4 °C with the following
antibodies: rabbit antiꢀpCREB (Ser133) (1:1000, Cell Signaling), rabbit antiꢀCREB (1:1000, Cell
Signaling), rabbit antiꢀpꢀp44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:2000, Cell Signaling),
rabbit antiꢀp44/42 MAPK (ERK1/2) (1:1000, Cell Signaling), and rabbit antiꢀGAPDH (1:2000,
Millipore). After washing three times with TBST (5 min/wash), membranes were incubated with
secondary antibody, goat antiꢀrabbit (1:10000 in 5 % milk, Jackson ImmunoResearch). Blots
were imaged using BioꢀRad ChemiDoc XRS. The images were analyzed using NIH ImageJ and
the values were normalized with respect to GAPDH and expressed as ratio of phosphorylated
protein to total protein. See supplementary information for detailed experimental procedure.
Synthetic Chemistry
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General reduction procedure: The appropriate cinnamic acid (1 eq) was dissolved in
anhydrous tetrahydrofuran (THF, 20 mL) and brought to 0 °C under argon. Triethylamine (TEA,
1
eq) was added dropwise over 2 min. After an additional 5 min, ethyl chloroformate (1 eq) was
added dropwise over 5 min to yield a white precipitate. The solution was filtered through a glass
frit and NaBH (3.8 eq) was added portion wise to the filtrate at 0 °C with stirring (in some
4
instances additional equivalents of NaBH were addedꢀ as indicated for each analog below). 30
4
min after the final NaBH addition the reaction was quenched with MeOH (20 mL) added over
4
3
0 min using a syringe pump and the reaction stirred overnight. HCl (1 N) was added dropwise
over 30 min until solution became acidic (~30 mL). The reaction was extracted with CH Cl (3 x
2
2
5
0 mL) and the combined organic extracts washed with brine (1 x 100 mL). The combined
organic layers were concentrated in vacuo and purified by flash chromatography (SiO2,
hexanes/ethyl acetate) to give the corresponding cinnamyl alcohols as described below.
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(
E)-3-(4-Fluorophenyl)prop-2-en-1-ol (1a). Synthesized using the general procedure with the
following quantities: (E)ꢀ3ꢀ(4ꢀFluorophenyl)acrylic acid (2.06 g, 12.4 mmol); TEA (1.26 g, 12.4
mmol); ethyl chloroformate (1.35 g, 12.4 mmol); NaBH (4.70 g, 124.2 mmol); afforded 1a as
4
1
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white solid (762 mg, 40.3 %). H NMR (CDCl , 400 MHz):
δ 7.36ꢀ7.33 (2H, qd, J = 12.0 Hz);
3
7
.03ꢀ6.99 (2H, t); 6.60ꢀ6.56 (1H, d, J = 16.0 Hz); 6.32ꢀ6.25 (1H, m); 4.33ꢀ4.31 (2H, d, J = 8.0
Hz).
(E)-3-(4-(Trifluoromethyl)phenyl)prop-2-en-1-ol (1b). Synthesized using the general
procedure with the following quantities: (E)ꢀ3ꢀ(4ꢀ(Trifluoromethyl)phenyl)acrylic acid (2.15 g,
9
.25 mmol); TEA (936 mg, 9.25 mmol); ethyl chloroformate (1.00 g, 9.25 mmol); NaBH (1.29
4
1
g, 35.2 mmol); afforded 1b as white solid (1.84 g, 91.2 %). H NMR (CDCl , 400 MHz):
δ 7.57ꢀ
3
7
.55 (2H, d, J = 8.0 Hz); 7.47ꢀ7.45 (2H, d, J = 8.0 Hz); 6.68ꢀ6.64 (1H, d, J = 16.0 Hz); 6.48ꢀ
.42 (1H, m); 4.37ꢀ4.35 (2H, d, J = 8.0 Hz).
6
(E)-3-(4-Bromophenyl)prop-2-en-1-ol (1c). Synthesized using the general procedure with the
following quantities: (E)ꢀ3ꢀ(4ꢀBromophenyl)acrylic acid (3.00 g, 13.2 mmol); TEA (1.34 g, 13.2
mmol); ethyl chloroformate (1.43 g, 13.2 mmol); NaBH (1.85 g, 50.2 mmol); afforded 1c as
4
1
white solid (2.27 g, 81.6 %). H NMR (CDCl , 400 MHz):
δ 7.44ꢀ7.42 (2H, d, J = 8.0 Hz);
3
7
.25ꢀ7.23 (2H, d, J = 8.0 Hz); 6.57ꢀ6.53 (1H, d, J = 16.0 Hz); 6.38ꢀ6.31 (1H, m); 4.32ꢀ4.30 (2H,
d, J = 8 Hz).
(E)-3-(4-(Difluoromethoxy)phenyl)prop-2-en-1-ol (1d). Synthesized using the general
procedure with the following quantities: (E)ꢀ3ꢀ(4ꢀ(Difluoromethoxy)phenyl)acrylic acid (1.14 g,
5
.32 mmol); TEA (540 mg, 5.32 mmol); ethyl chloroformate (579 mg, 5.32 mmol); NaBH (2.24
4
1
g, 60.7 mmol); afforded 1d as white solid (923 mg, 86.3 %). H NMR (CDCl , 400 MHz):
δ
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.34ꢀ7.32 (2H, d, J = 8.0 Hz); 7.05ꢀ7.03 (2H, d, J = 8.0 Hz); 6.68ꢀ6.49 (2H, m); 6.32ꢀ6.25 (1H,
m); 4.31ꢀ4.30 (2H, d, J = 4.0 Hz).
(
E)-3-(4-Methoxyphenyl)prop-2-en-1-ol (1e). Synthesized using the general procedure with the
following quantities: (E)ꢀ3ꢀ(4ꢀMethoxyphenyl)acrylic acid (3.00 g, 16.8 mmol); TEA (1.70 g,
6.8 mmol); ethyl chloroformate (1.83 g, 16.8 mmol); NaBH (4.46 g, 118 mmol); afforded 1e as
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white solid (1.95 g, 70.7 %). H NMR (DMSOꢀd , 400 MHz):
δ 7.37ꢀ7.35 (2H, d, J = 8.0 Hz);
6.90ꢀ6.88 (2H, d, J = 8.0 Hz); 6.52ꢀ6.48 (1H, d, J = 16.0 Hz); 6.26ꢀ6.19 (1H, m); 4.11ꢀ4.10 (2H,
d, J = 4.0 Hz); 3.75 (3H, s).
(E)-3-(p-Tolyl)prop-2-en-1-ol (1f). Synthesized using the general procedure with the following
quantities: (E)ꢀ3ꢀ(pꢀTolyl)acrylic acid (3.04 g, 18.7 mmol); TEA (1.90 g, 18.7 mmol); ethyl
chloroformate (2.03 g, 18.7 mmol); NaBH (2.69 g, 71.2 mmol); afforded 1f as white solid (1.57
4
1
g, 56.5 %). H NMR (CDCl , 400 MHz):
δ 7.28ꢀ7.26 (2H, d, J = 8.0 Hz); 7.13ꢀ7.11 (2H, d, J =
3
8.0 Hz); 6.59ꢀ6.55 (1H, d, J = 16.0 Hz); 6.34ꢀ6.27 (1H, m); 4.30ꢀ4.28 (2H, d, J = 8.0 Hz); 2.33
1H, s).
(
(
E)-3-(4-Chlorophenyl)prop-2-en-1-ol (1g). Synthesized using the general procedure with the
following quantities: (E)ꢀ3ꢀ(4ꢀChlorophenyl)acrylic acid (2.00 g, 11.0 mmol); TEA (1.11 g, 11.0
mmol); ethyl chloroformate (1.19 g, 11.0 mmol); NaBH (1.57 g, 41.6 mmol); afforded 1g as
4
1
white solid (1.30 g, 70.6 %). H NMR (CDCl , 400 MHz):
δ 7.31ꢀ7.26 (4H, d, J = 8.0 Hz); 6.58ꢀ
3
6
.54 (1H, d, J = 16.0 Hz); 6.36ꢀ6.30 (1H, m); 4.32ꢀ4.31 (2H, d, J = 4.0 Hz).
(E)-3-Phenylprop-2-en-1-ol (1h). Synthesized using the general procedure with the following
quantities: (E)ꢀ3ꢀPhenylpropꢀ2ꢀenoic acid (2.00 g, 13.5 mmol); TEA (1.50 g, 14.9 mmol); ethyl
1
chloroformate (1.61 g, 14.9 mmol); afforded 1h as white solid (1.46 g, 80.5 %). H NMR
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(
CDCl , 400MHz):
δ
7.42ꢀ7.41 (2H, d, J = 4.0 Hz); 7.36ꢀ7.34 (2H, t); 7.29ꢀ7.28 (1H, t); 6.66ꢀ
3
6
.63 (1H, d, J = 12.0 Hz); 6.42ꢀ6.38 (1H, m); 4.36ꢀ4.35 (2H, d, J = 4.0 Hz).
General cyclization procedure: The appropriate cinnamyl alcohol (1 eq) was dissolved in
AcOH:DMF (1:1) and brought to 0 °C under argon. Sodium nitrite (10 eq) was then added. The
reaction was stirred for 1 hr at 0 °C. The reaction mixture was heated, at 40 °C sodium nitrite
was added (5 eq), and reaction heated to 80 °C over 1.5 hr. The reaction was diluted with ice
water (100 mL) and extracted with EA (3 x 50 mL). Combined organic layers were washed with
brine (1 x 100 mL) and the organic layer concentrated by rotary evaporation. The combined
organic layers were concentrated in vacuo and purified by flash chromatography (SiO2,
hexanes/ethyl acetate) to give the corresponding furoxan as indicated below.
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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0
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0
1
2
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9
0
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0
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0
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8
9
0
4
-(4-Fluorophenyl)-3-(hydroxymethyl)-1,2,5-oxadiazole 2-oxide (2a). Synthesized using the
general procedure with the following quantities: 1a (762 mg, 5.01 mmol); NaNO (5.19 g, 75.2
2
1
mmol); afforded 2a as yellow solid (278 mg, 26.4 %). H NMR (CDCl , 400 MHz):
δ 7.92ꢀ7.88
3
(2H, qd, J = 16.0 Hz); 7.31ꢀ7.29 (2H, t); 4.78 (2H, s).
3
-(Hydroxymethyl)-4-(4-(trifluoromethyl)phenyl)-1,2,5-oxadiazole 2-oxide (2b). Synthesized
using the general procedure with the following quantities: 1b (1.51 g, 7.47 mmol); NaNO (5.15
2
1
g, 74.7 mmol); afforded 2b as yellow solid (699 mg, 36.0 %). H NMR (CDCl , 400 MHz):
δ
3
8
.24 ꢀ8.22 (2H, d, J = 8.0 Hz); 8.02ꢀ8.00 (2H, d, J = 8.0 Hz); 4.76ꢀ4.72 (2H, d, J = 16.0 Hz);
4
.01 (1H, s).
4
-(4-Bromophenyl)-3-(hydroxymethyl)-1,2,5-oxadiazole 2-oxide (2c). Synthesized using the
general procedure with the following quantities: 1c (1.11 g, 5.21 mmol); NaNO (5.39 g, 78.1
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1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
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3
3
3
3
3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
mmol); afforded 2c as reddish brown solid (993 mg, 70.4 %). H NMR (CDCl , 400 MHz):
δ
3
7
.96ꢀ7.95 (2H, d, J = 4.0 Hz); 7.85ꢀ7.80 (2H, d, J = 20.0 Hz); 4.65 (2H, s).
4-(4-(Difluoromethoxy)phenyl)-3-(hydroxymethyl)-1,2,5-oxadiazole
2-oxide
(2d).
0
1
2
3
4
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0
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0
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2
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0
1
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9
0
Synthesized using the general procedure with the following quantities: 1d (923 mg, 4.61 mmol);
1
NaNO (2.47 g, 35.8 mmol); afforded 2d as yellow solid (306 mg, 25.7 %). H NMR (CDCl ,
2
3
400 MHz):
δ
7.88ꢀ7.86 (2H, d, J = 8.0 Hz); 7.30ꢀ7.28 (2H, d, J = 8.0 Hz); 6.80ꢀ6.43 (1H, m);
4
.73 (2H, s).
3
-(Hydroxymethyl)-4-(4-methoxyphenyl)-1,2,5-oxadiazole 2-oxide (2e). Synthesized using
the general procedure with the following quantities: 1e (1.88 g, 11.5 mmol); NaNO (7.89 g, 114
2
1
mmol); afforded 2e as yellow solid (523 mg, 20.6 %). H NMR (CDCl , 400 MHz):
δ 7.92ꢀ7.90
3
(
2H, d, J = 8.0 Hz); 7.21ꢀ7.19 (2H, d, J = 8.0 Hz); 6.03 (1H, s); 4.57 (2H, s); 3.89 (1H, s).
3
-(Hydroxymethyl)-4-(p-tolyl)-1,2,5-oxadiazole 2-oxide (2f). Synthesized using the general
procedure with the following quantities: 1f (2.28 g, 15.2 mmol); NaNO (10.6 g, 154 mmol);
2
1
afforded 2f as yellow solid (687 mg, 21.7 %). H NMR (CDCl , 400 MHz):
δ 7.69ꢀ7.68 (2H, d, J
3
=
4.0 Hz); 7.33ꢀ7.31 (2H, d, J = 8.0 Hz); 4.7 (2H, s); 2.47ꢀ2.36 (4H, qd).
4
-(4-Chlorophenyl)-3-(hydroxymethyl)-1,2,5-oxadiazole 2-oxide (2g). Synthesized using the
general procedure with the following quantities: 1g (1.67 g, 9.90 mmol); NaNO (13.7 g, 198
2
1
mmol); afforded 2g as yellow solid (495 mg, 22.1 %). H NMR (CDCl3, 400 MHz):
δ 7.81ꢀ7.79
(2H, d, J = 8.0 Hz); 7.53ꢀ7.51 (2H, d, J = 8.0 Hz); 4.72 (2H, s).
3-(Hydroxymethyl)-4-phenyl-1,2,5-oxadiazole 2-oxide (2h). Synthesized using the general
procedure with the following quantities: 1h (1.00 g, 7.45 mmol); NaNO (5.14 g, 74.5 mmol);
2
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afforded 2h as yellow solid (727 mg, 50.9 %). H NMR (CDCl , 400 MHz):
δ
7.81ꢀ7.80 (2H, m);
3
7
.59ꢀ7.56 (3H, m); 4.75 (2H, s).
General bromination procedure: The appropriate furoxan (1 eq) was dissolved in anhydrous
1
1
1
1
1
1
1
1
1
1
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2
2
2
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2
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5
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0
1
2
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0
1
2
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8
9
0
1
2
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8
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CH Cl at 0 °C under argon. PPh (1.2 eq) was added in one portion. Reaction was stirred for 15
2
2
3
min followed by addition of CBr (1.2 eq). The ice bath was removed and reaction maintained at
4
room temp for 2 hr. The combined organic layers were concentrated in vacuo and purified by
flash chromatography (SiO , hexanes/ethyl acetate) to give the corresponding furoxans.
2
3
-(Bromomethyl)-4-(4-fluorophenyl)-1,2,5-oxadiazole 2-oxide (3a). Synthesized using the
general procedure with the following quantities: 2a (408 mg, 2.12 mmol); PPh (668 mg, 2.55
3
1
mmol); CBr (844 mg, 2.55 mmol); afforded 3a as a yellow solid (306 mg, 57.8 %). H NMR
4
(
CDCl , 400 MHz): δ 7.83ꢀ7.79 (2H, qd); 7.31ꢀ7.29 (2H, t); 4.40 (2H, s).
3
3
-(Bromomethyl)-4-(4-(trifluoromethyl)phenyl)-1,2,5-oxadiazole 2-oxide (3b). Synthesized
using the general procedure with the following quantities: 2b (877 mg, 3.37 mmol); PPh (1.06
3
1
g, 4.05 mmol); CBr (1.34 g, 4.05 mmol); afforded 3b as yellow solid (366 mg, 33.6 %). H
4
NMR (CDCl , 400 MHz):
δ 7.98ꢀ7.96 (2H, d, J = 8.0 Hz); 7.88ꢀ7.86 (2H, d, J = 8.0 Hz); 4.45
3
(2H, s).
3-(Bromomethyl)-4-(4-bromophenyl)-1,2,5-oxadiazole 2-oxide (3c). Synthesized using the
general procedure with the following values: 2c (772 mg, 2.85 mmol); PPh (897 mg, 3.42
3
1
mmol); CBr (1.13 g, 3.42 mmol); afforded 3c as yellow solid (554 mg, 58.3 %). H NMR
4
(CDCl , 400 MHz):
δ
7.79ꢀ7.76 (2H, m); 7.60ꢀ7.57 (2H, m); 4.39 (2H, s).
3
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