Furoxans (Oxadiazole-4N-oxides) with Attenuated Reactivity are

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Cite This: J. Med. Chem. 2018, 61, 4593−4607

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*,† †

Department of Medicinal and Biological Chemistry and ‡Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, Ohio 43614, United States S Supporting Information *

ABSTRACT: 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, cGMPdependent 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.

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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 NO-sensitive soluble guanylyl cyclase (NO-sGC), resulting in increased production of cyclic guanosine monophosphate (cGMP) and kinase signal transduction, primarily via protein kinase G (PKG), leading to phosphorylation of cAMP response element binding protein (CREB) at Ser-133.1−3 CREB phosphorylation is required for synaptic plasticity and memory consolidation. Prolonged 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 the capacity for morphological flexibility in synaptic networks.4 Regulation of CREB was first identified as being controlled by cAMP/PKA kinase signaling but actually involves numerous coregulators. Importantly, inhibition of any component of the NO/cGMP/PKG system suppresses synaptic signal. This pathway is directly inhibited by amyloid beta (Aβ) in Alzheimer’s disease (AD).5−8 Reversal of Aβinduced deficits via agents that activate NO/cGMP signaling results in improved cognitive function, as demonstrated for nitrates, sGC activators, and phosphodiesterase inhibitors (PDEi).9−13 © 2018 American Chemical Society

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+, NO2−, or HONOO−). Here, we describe a class of synthetic, thioldependent NO mimetics known as furoxans. Furoxans have been investigated as potential therapeutics for a plethora of disease states.14−19 The majority of furoxan development has focused on relatively reactive furoxans that 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 that (1) reactivity and propensity for NO mimetic activity can be modulated via a predictable structure−activity relationship (SAR), (2) furoxans protect against oxygen glucose deprivation (OGD) in primary cortical Received: March 9, 2018 Published: April 23, 2018 4593

DOI: 10.1021/acs.jmedchem.8b00389 J. Med. Chem. 2018, 61, 4593−4607

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Figure 1. Overview of furoxan development for the CNS. The furoxans described here have more favorable physiochemical properties as potential CNS drugs than previously described furoxans. Furoxans have not been studied in the brain previously. *Abbreviations: MW = molecular weight; tPSA = total polar surface area; cLogP = calculated partition coefficient; Aβ = oligomeric amyloid beta; LTP = long-term potentiation; STPA = stepthrough passive avoidance.

Figure 2. (A) Design justification for furoxan library. (B) Review of furoxan designation nomenclature. Furoxans tautomerize when heated sufficiently (at least 100 °C for a sustained period of time depending upon substitution pattern) but do not interconvert at physiologically relevant temperatures.

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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 inter-related properties including (1) membrane permeability and solubility, (2) low propensity for efflux by P-glycoproteins (Pgp), and (3) adequate brain partitioning to provide a sufficient level of unbound drug at the site of action.22−24 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]). On the basis of these considerations, all of our compounds obey the following design criteria: (1) MW < 400 g/mol, (2) clogP = 2.5−4.5, and (3) tPSA = 45−85. A para-substituted aryl system was used to modulate furoxan reactivity while blocking potential metabolic para-hydroxylation, the main route of metabolism of phenyl-containing drugs.25 4-Methylpiperdine was incorporated as a cap group because it is small, relatively hydrophobic, and contains a tertiary amine (related 4alkylpiperdine reported pKa ∼ 9.0),26 which allows preparation of pharmaceutically acceptable salts that possess excellent water solubility. Importantly, despite predominantly existing as a charged species at physiological pH, similar 4-alkylpiperidines are present in several orally bioavailable CNS drugs, including

cell culture via a NO/cGMP-dependent mechanism, and (3) furoxans rescue oligomeric Aβ-induced deficits in long-term potentiation (LTP) (i.e., sustained strengthening of synaptic signal).20 Additionally, Chegaeve et al. reported restored LTP and disruption of Tau and Aβ aggregation using thiacarbocyanine-furoxan hybrids (Figure 1B).21 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 -withdrawing groups probed electronic sensitivity of the thiophilic furoxan ring with the goal of fine-tuning reactivity toward 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, and 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. 4594

DOI: 10.1021/acs.jmedchem.8b00389 J. Med. Chem. 2018, 61, 4593−4607

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Scheme 1. Library Synthesisa

(i) Ethyl chloroformate, triethylamine, sodium borohydride, 0 °C to r.t.; (ii) acetic acid:dimethylformamide (1:1), sodium nitrite, 0 to 60 °C; (iii) triphenylphosphine, carbon tetrabromide, 0 °C to r.t.; (iv) 4-methylpiperidine, triethylamine, r.t. to 60 °C; (v) toluene, reflux, 7 d.

a

Scheme 2. Synthesis of the LC-MS/MS Internal Standarda

a

(i) 4-Methylpiperidine-2,2,6,6-d4, triethylamine, r.t. to 60 °C; (ii) toluene, reflux, 7 d.

Figure 3. (A) Crystal structure of 5a confirming the oxadiazole-4N-oxide ring configuration. (B) HPLC-PDA analysis demonstrating differences in relative polarity and UV absorption properties for ODZ-2N and ODZ-4N furoxans 4a and 5a, respectively. Chromatograms represent overlay of UV signal at 254 nm with PDA analysis of the representative portion of the chromatogram shown above each peak.

isotope-labeled 4-methylpiperidine-2,2,6,6-d4 followed by tautomerization provided 7a as an appropriate internal standard for LC-MS/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 ODZ-4N structure (see Supplemental Crystallographic Report). HPLC purity analysis using a photodiode array detector (PDA) showed a noticeable shift in the λmax of absorption for ODZ-2N analogues (∼246−261 nm) versus that of ODZ-4N analogues (279−293 nm) with a 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 forms. In all instances, retention time (Rt) comparisons indicated that, despite possessing greater aromaticity based on λmax, the ODZ-4N analogues are more polar than the corresponding ODZ-2N analogues. Furoxan 5a

haloperidol, risperidone, and the AD standard of care donepezil. 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 4-methylpiperidine yielded the oxadiazole-2N-oxides (ODZ-2N) 4a−h. The final step of the synthesis involved tautomerization of the furoxan in refluxing toluene to yield oxadiazole-4N-oxides (ODZ-4N). Separation of the ODZ-2N/ODZ-4N tautomers was realized via reverse-phase flash chromatography to yield the ODZ-4N compounds 5a−h. Initial in vitro testing nominated 5a for in vivo evaluation (vide infra). Accordingly, substitution of 3a with 4595

DOI: 10.1021/acs.jmedchem.8b00389 J. Med. Chem. 2018, 61, 4593−4607

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Table 1. Protection against Oxygen Glucose Deprivation (OGD)a % change in viability relative to vehicle mean structure R1 = F R1 = CF3 R1 = Br R1 = OCHF2 R1 = OCH3 R1 = CH3 R1 = Cl R1 = H R1 = F d4 analogue

tautomer 4a 5a 4b 5b 4c 5c 4d 5d 4e 5e 4f 5f 4g 5g 4h 5h 6a 7a

(2N) (4N) (2N) (4N) (2N) (4N) (2N) (4N) (2N) (4N) (2N) (4N) (2N) (4N) (2N) (4N) (2N) (4N)

1 μM

10 μM

11% ± 2 −3% ± 4 −9% ± 7 2% ± 23% −7% ± 2 25% ± 6 54%** ± 11 67%*** ± 7 14% ± 6 11% ± 6 7% ± 12 22% ± 20 35% ± 16 −10% ± 12 9% ± 11 −21% ± 7 11% ± 8 8% ± 11

11% ± 3 46%*** ± 10% ± 7 −16% ± 7 −2% ± 1 77%*** ± 86%*** ± 61%*** ± 10% ± 6 12% ± 14 12% ± 15 13% ± 18 7% ± 8 19% ± 19 19% ± 6 6% ± 23 6% ± 7 20% ± 13

25 μM 7

5 15 7

5% ± 3 41%*** ± 5 0% ± 14 −22% ± 10 19% ± 5 96%*** ± 19 108%*** ± 24 53%** ± 2 −1% ± 9 5% ± 17 10% ± 11 8% ± 18 25% ± 16 4% ± 18 16% ± 15 −11% ± 6 −1% ± 12 7% ± 9

Assay workflow: expose PC12 cells to OGD for 90 min → reoxygenate and add furoxan (1, 10, or 25 μM) → incubate for 24 h → MTT readout. Data represented as mean ± SEM (n = 3) of triplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle control using one-way ANOVA analysis with Dunnett’s posthoc test. The viability of the OGD vehicle group is 51.4 ± 4.1% compared to vehicle group. This number is based on N = 20 OGD/vehicle trials, reported as mean ± SEM. a

optimization due to deleterious effects in animals. Furoxans 4d and 5d warrant additional investigation; however, they were not considered for proof-of-concept in vivo investigation of attenuated furoxans because the impetus for this work was to produce NO mimetics that activate NO/cGMP signaling without producing significant fluxes of NO, thereby reducing the potential for side effects. Furoxans 5a and 5c demonstrate statistically equivalent efficacy and potency. Furoxan 5a, rather than 5c, was chosen for in vivo evaluation for multiple reasons, including the smaller molecular weight (a critical factor in brain penetration and ligand efficiency)32 and future potential for incorporation of 18F for radioimaging. Furoxan 7a was synthesized for use as an LC-MS/MS internal standard and was also tested in the primary assay. Similar activity was expected compared to nondeuterated 5a; however, 7a possessed no neuroprotective effects. UPLC separation suggests 7a is slightly more polar than 5a based on Rt (Supplemental Figure S3). LC-MS/MS was used to measure membrane 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 OGD-treated PC12 cells following 24 h 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 demonstrate (1) furoxans readily cross cell membranes in the primary assay and (2) significant amounts of furoxan after 24 h incubation with PC12 cells, providing further evidence for the relatively unreactive nature of this furoxan library. An expanded dose−response assay of OGD-exposed PC12 cells treated with 5a revealed a bell-shaped (i.e., hyperbolic) efficacy curve from which an EC50 = 1.9 μM was calculated (Figure 4A). For concentration response studies a resorufin based dye was used as the viability read-out due to a significantly shorter incubation time before colorimetric

was also profiled by 2D-NMR (see Supporting Information 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, which provides a model of neuronal death across neurodegenerative diseases, including AD.27 Expert perspectives suggest that experimental paradigms in drug discovery should model well-understood target pathways rather than specific disease state.28,29 NO mimetics have been benchmarked as being protective against oxidative stress induced by OGD via multiple proposed mechanisms including (1) antioxidant effects, (2) mediating phosphorylation of prosurvival kinase signaling, and (3) S-nitrosylation of proapoptotic caspases.30,31 Neuroprotection was evaluated by 3(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assays of cell viability in nondifferentiated PC-12 cell cultures subjected to OGD for 90 min followed by reoxygenation and replacement with nutrient-rich media containing furoxans screened at three concentrations (1, 10, and 25 μM) for 24 h. Neuroprotection is reported as percent change relative to vehicle (DMSO)-treated controls. The ODZ-2N compounds (except 4d) showed negligible neuroprotection (Table 1). A few ODZ-4N analogues provided noticeable neuroprotective activity, including compounds 5a (4-F), 5c (4-Br), and 5d (4-OCHF2). The 4-OCHF2 tautomers (4d and 5d) showed robust potency with significant efficacy at 1 μM. Furoxans 4d and 5d are considered to exhibit “outlierlike” behavior compared to the rest of the library based upon (1) 4d being the only active ODZ-2N from our series and (2) the potency of these compounds being 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 hit-to-lead 4596

DOI: 10.1021/acs.jmedchem.8b00389 J. Med. Chem. 2018, 61, 4593−4607

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NO production was observed from 5a in the form of NO2− in the primary assay (Supplemental Figure S11). Improved Memory in Step-Through Passive Avoidance (STPA). Pilot behavioral studies evaluated the ability of 5a to reverse scopolamine-induced deficits in STPA, which assesses the ability of drugs to reverse hippocampal-dependent aversive memory deficits.36−38 Scopolamine is a well-characterized pan-muscarinic antagonist that produces significant amnesic effects when administered 30 min prior to training in STPA.36,39,40 Reversal of scopolamine-induced amnesia is not a sophisticated model, but it is well understood and remains useful as a resource-efficient screen for procognitive agents.41 Importantly, reversal of scopolamine-induced cholinergic deficits in STPA via NO/cGMP enhancement is well characterized and has been benchmarked for nitrates, sGC activators, PKG activators, and several phosphodiesterase inhibitors (PDEi).42−46 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 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 h after training (Figure 5). Testing latency 24 h after treatment

Figure 4. (A) Neuroprotection concentration response curve for 5a. PC12 cells were submitted to OGD for 90 min followed by addition of 5a (0.1, 1, 5, 10, 25, or 50 μM) and incubated under normoxic conditions for 24 h. Data represent percent change in viability relative to vehicle-treated control mean = 1, Prestoblue readout. Vehicle control viability range (assay baseline [blue]) and viability range control plates not submitted to OGD are shown in yellow. (B) Neuroprotection of 5a is dependent on sGC signaling. ODQ (10 μM) abolished neuroprotective activity of 5a in a PC12-OGD assay using MTT as a viability readout. Data represented as mean ± SEM (n = 3) of duplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle control using one-way ANOVA analysis with Dunnett’s posthoc test. Average viability of the OGD vehicle cells was 51.4% (±4.1) that of vehicle cells based on 20 OGD/vehicle trials, represented as mean ± SEM.

measurement, which allows more direct observation and increased throughput compared to MTT. The hyperbolic response curve is in agreement with previous studies that identified a correlation between production of NO2− by furoxans in the presence of cysteine (i.e., reactivity) with protection against OGD. Notably, although protective effects of 5a are lost at higher concentrations, no toxicity was observed at the highest concentration tested (50 μM). Co-incubation with ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one), 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 cell culture.33 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 off-target effects as described previously.34,35 As shown in Supplementary Table 2, 5a does not interfere with binding of any radioligands for all receptors screened, including GABAA, serotonin receptors, dopamine receptors, and muscarinic and nicotinic acetylcholine receptors. No hERG binding was observed. For the most part 7a demonstrated a similar profile but significantly inhibited binding at the delta opioid receptor and benzodiazepine (BZP) rat brain site. Reactivity. Furoxan 5a was incubated with N-acetyl 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 S10 and S11). We hypothesized reactivity with excess thiol would correlate with neuroprotection in the primary assay. However, no reaction was observed by HPLC-PDA analysis of all compounds, whereas control incubations using the electrophile N-ethylmaleimide (NEM) showed complete reaction of NEM after 2 and 12 h. In agreement with the observed attenuated reactivity, negligible

Figure 5. Reversal of cholinergic memory deficits in STPA by 5a. Male C57BL/6 mice administered either vehicle or 5a (0.1, 1, 10, or 20 mg/ kg) via ip injection at 1 or 2 h prior to training. Scopolamine (1 mg/ kg) was administered 30 min prior to training. Memory was measured as latency to enter a dark chamber 24 h after training. Data represented as mean and SEM (n = 8−9) for n = 8, see exclusion criteria in Experimental Section. **p < 0.01 and ***p < 0.001 compared to negative scopolamine-treated control using one-way ANOVA analysis with Dunnett’s posthoc test.

avoids potential convolution from lethargy caused by drug exposure, and lethargy is not reported for agents acting via cGMP signaling as shown for sildenafil in the open field test.47 Furoxans have never been studied in the CNS. Hence, choices for dosing and administration timing were not evident at the onset. A NO-chimeric nitrate reversed scopolamineinduced 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 half-life of nitrates resulting 4597

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in rapid onset of neuromodulatory efficacy at a low dose (1 mg/kg).9 On the basis of the knowledge that attenuated furoxans likely have greater metabolic stability, we anticipated that a longer pretraining treatment duration would be required to allow sufficient time for slow-onset NO mimetic effects. With these considerations in mind, we examined varying doses at two pretraining time points (1 or 2 h). Scopolamine-treated mice showed significantly less latency to enter the dark compartment compared to that of wild-type mice (scopolamine-treated: 63.5 ± 30 s [SEM] versus vehicle-treated: 270 ± 29 s [SEM], p < 0.001). Furoxan 5a was capable of reversing scopolamine-induced amnesia with a statistically significant effect observed when administered 2 h prior to training at 20 mg/kg (latency = 237 ± 22 s [SEM], p < 0.01). These data demonstrate, for the first time, the ability of a furoxan to improve memory in mice by reversing cholinergic memory blockade. In Vivo Drug Stability, Brain Penetration, and Effect on Protein Phosphorylation. To understand the STPA behavioral results, we attempted to correlate the 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 LC-MS/MS analysis from the same animal (Figure 6A). Furoxan 5a was measured by LC-MS/MS analysis of plasma and forebrain samples after intraperitoneal (ip) and oral (po) administration at varying time points (1, 2, or 12 h [n = 4]). CREB phosphorylation was also analyzed in the forebrain samples as the pCREB:tCREB ratio. Selected sacrifice times were intended to provide a preliminary assessment of the in vivo t1/2 while providing a pharmacodynamic measurement in a system mimicking behavioral studies in which 5a (20 mg/kg) reversed scopolamine-induced memory deficits when administered 2 h prior to behavioral training. Assessment of brain:plasma ratio was carried out via MRM (multiple reaction monitoring) using LC-MS/MS. The forebrain and plasma samples were processed using solid-phase extraction (SPE). Furoxan 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 LC-MS/MS because they have similar physiochemical properties to their nondeuterated analogue 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 LC-MS/MS parameters (triple quadrupole utilizing optimized 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 and 110 min depending on the 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 t test) with an average oral administration B:P ratio of 1.49 (±0.52, SEM) and intraperitoneal administration average B:P ratio of 0.68 (±0.03, SEM). We anticipate 5a accesses the brain via passive diffusion

Figure 6. (A) Male C57BL/6 mice (N = 4 per cohort) were treated with vehicle or 5a (20 mg/kg), and at the specified time, plasma and forebrain were collected and processed as indicated. (B) At the specified time (1, 2, or 12 h), plasma (triangles) and forebrain (circles) were collected, spiked with 7a, and extracted. Oral gavage (po) is indicated by solid lines and yellow symbols. Intraperitoneal injection (ip) is indicated by dotted lines and red symbols. Quantitation based on the ratio of the AUC of the MRM transitions for 5a versus 7a relative to a calibration curve. Concentrations (μM) were calculated after normalizing for tissue weight (assuming tissue/plasma density = 1). Data shown as mean ± SEM (n = 4) for all cohorts except 1 h ip (n = 3), see exclusion criteria in Experimental Section. (C) Forebrains were homogenized, normalized by bradford, and analyzed for pCREB and tCREB by ELISA (V = veh, D = drug [5a]). The ratio of absorbance values was normalized to Veh mean = 1 for each treatment group. Data represent mean ± SEM (n = 4), analysis by one-way ANOVA with Tukey’s test.

based on a low molecular weight (291.1 g/mol) and a clogP of ∼3.1, both of which are predicted to allow passive membrane permeability.48 These findings are novel for several reasons, including being the first report (1) demonstrating a furoxan crossing the blood brain barrier, (2) to our knowledge, demonstrating the first NO mimetic observed in the brain 12 h after administration (ip or po), and (3) establishing a B:P ratio for a furoxan. 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 6C). A more rigorous evaluation was 4598

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foundational efforts described here provide a reasonable starting point for exploration of these scenarios going forward.

performed in follow-up investigations studying hippocampal protein phosphorylation by Western blotting. Mice were treated in an identical manner to STPA studies except the animals were sacrificed at the time-point previously used for training in STPA (i.e., at the time of memory acquisition). These studies included four cohorts (N = 8 per cohort [with experimenters blinded to treatment]) with each receiving one ip injection 2 h prior to sacrifice (either 5a [20 mg/kg] or vehicle) and one ip injection 30 min prior to sacrifice (either scopolamine [1 mg/kg] or vehicle). Blots for either phosphorylated protein [pProtein] or total protein [tProtein] were normalized to GAPDH loading controls prior to calculating the phosphorylation ratio as pProtein:tProtein analyzed by one-way ANOVA. Despite these efforts, statistically significant changes in hippocampal protein phosphorylation were 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 scopolamine, the negative control group. Most articles utilizing acute scopolamine-induced memory deficits do not report measurements of protein phosphorylation, and those that do, agree with our findings. Indeed, acute scopolamine induces impaired learning and memory but has no effect on protein phosphorylation in normal mice (C57BL/6).49,50 Furthermore, neither acute nor long-term administration of PDEi’s affects hippocampal pCREB in normal mice.39,51 Hence, our findings for furoxans are in line with literature precedent for agents acting via NO/cGMP signaling. Long-term administration (>3 weeks) of nitrates or PDEi’s to AD transgenic (AD-Tg) mice results in increases in hippocampal pCREB relative to vehicle-treated controls.10,12,13 Hippocampal pCREB is most appropriate as a marker of efficacy when studying long-term 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 impairment reliably shown for NO mimetics, PDEI’s, and sGC activators. Furoxans have been extensively described as thiol-dependent NO mimetics. Our pursuit of furoxans with attenuated reactivity has resulted in the identification of efficacious furoxans that lack reactivity with generic thiols. Characterizing the reaction of an attenuated furoxan with thiol is difficult due to the lack of reactivity. The specific thiol(s) responsible for and mechanism of bioactivation is not known at present. On the basis of our data, we present two proposed scenarios: (A) a subset of yet to be identified sufficiently reactive cellular thiols attack the furoxan ring to activate NO release, or (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 well-known that instances in which a deuterated derivative lacks efficacy of an equivalent nondeuterated molecule may be explained by a requirement for metabolic activation for efficacy (i.e., an active metabolite is responsible for efficacy). Hence, the lack of activity of the deuterated (d4) analogue, 7a, provides support for the notion of an active metabolite. Additional supportive data are provided by the STPA behavioral model in which activity was observed when 5a was administered 2 h prior to training but not 1 h. The

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CONCLUSIONS The neuroprotective properties of novel attenuated furoxans have been explored. The furoxan 5a elicits a hyperbolic concentration response curve for protection against oxidative stress that is completely blocked by coincubation 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 the 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 h 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, weakly reactive) NO mimetic based on (1) negligible reactivity with generic thiols at physiological pH and temperature, (2) the presence of 5a in PC12 cells 24 h after treatment, and (3) the presence of 5a in the brain and plasma 12 h 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 suggest that free thiols alone are not capable of activating release of NO from attenuated furoxans. Identification of the protein target(s) or microenvironment(s) that facilitate bioactivation would allow for the development of a quantitative structure−activity relationship (QSAR) for analogue 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 Sigma-Aldrich (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. Isotope labeled starting materials were purchased from Cambridge Isotope Laboratories. All reactions were carried out under atmospheric conditions at room temperature unless otherwise indicated. Reactions were monitored by thin-layer chromatography (TLC, LuxPlate silica gel 60 F254 plates) and revealed by UV light (254 nm). Column chromatography was performed using Teledyne Combiflash Rf with RediSepRf Gold columns and RediSep Rf C18 columns for tautomerization purification. HPLC analysis was performed using a Shimadzu Prominence HPLC (LCD-20AD) with temperature-controlled autosampler (SIL-20AC), refractive index (RID-20A), and PDA (SPD-M20A) detectors. LC-MS/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 × 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 min followed by 1 min at 95% B and re4599

DOI: 10.1021/acs.jmedchem.8b00389 J. Med. Chem. 2018, 61, 4593−4607

Journal of Medicinal Chemistry

Article

methylsulfonyl fluoride [0.2 mM], sodium orthovanadate [1 mM], NaF [50 mM], sodium pyrophosphate [10 mM], protease inhibitor cocktail [Thermo]), which was then vortexed and maintained at 0 °C for 15 min. The resulting solution was centrifuged, and the supernatant was 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 μL of 2% formic acid (FA) in dH2O and spiked with a corresponding internal standard (7a for vehicle and 5a-treated samples; 5a for 7atreated samples). Analysis was carried out as described in the UPLCMS/MS method described in the General Methods section. The AUCs for the MRM transitions of the analytes were integrated and normalized to their respective internal standards. The result can be found in Supplemental Figure S4. Step-Through Passive Avoidance. In the early morning (within 1 h of the light “on” cycle beginning), male C57BL/6 mice were habituated in the STPA apparatus to confirm the photophobic phenotype. Translocation in