2.1. Assays

15-Lipoxygenase-1 qHTS Assay (AID 887). All screening operations were performed on a fully integrated robotic system (Kalypsys Inc., San Diego, CA). Three micoliter of enzyme (40 nM 15-hLO-1, final concentration) was dispensed into a 1536-well Greiner black clear-bottom assay plate. Then compounds and controls (23 nL) were transferred via Kalypsys pintool equipped with 1536-pin array. The plate was incubated for 15 min at room temperature, and then a 1 μL aliquot of substrate solution (final concentration of 50 μM arachidonic acid) was added to start the reaction. The reaction was stopped after 6.5 min by the addition of 4 μL of FeXO solution (final concentrations of 200 μM Xylenol Orange (XO) and 300 μM ferrous ammonium sulfate in 50 mM sulfuric acid). The assay plate was incubated at room temperature for 30 min. The absorbances at 405 and 573 nm were recorded using ViewLux high throughput CCD imager (Perkin-Elmer, Waltham, MA) using standard absorbance protocol settings. Plates containing DMSO only were included approximately every 50 plates throughout the qHTS to monitor any systematic trend in the assay signal associated with reagent dispenser variation or decrease in enzyme specific activity. Data was normalized to known positive control and DMSO treated wells for negative control.

Lipoxygenase UV-Vis Assay. The inhibitors were screened initially using one concentration point on a Perkin-Elmer Lambda 40 UV/Vis spectrophotometer. The percent inhibition was determined by comparing the enzyme rates of the control (DMSO solvent) and the inhibitor sample by following the formation of the conjugated diene product at 234 nm (ε = 25,000 M^-1cm^-1). The reactions were initiated by adding either of ∼ 40 nM 12-LOX, 40 nM 15-LOX-1, 0.5 μM 15-LOX-2 or 5-10 μL of 5-LOX crude extract to a cuvette with a 2 mL reaction buffer constantly stirred using a magnetic stir bar at room temperature (22 °C). Reaction buffers used for various lipoxygenase were as follows- 25 mM HEPES (pH 7.3), 0.3 mM CaCl₂, 0.1 mM EDTA, 0.2 mM ATP, 0.01% Triton X-100, 10 μM AA for the crude, ammonium sulfate precipitated 5-LOX; 25 mM Hepes (pH 8), 0.01% Triton X-100, 10 μM AA for 12-LOX and 25 mM Hepes buffer (pH 7.5), 0.01% Triton X-100, 10 µM AA for 15-LOX-1 and 15-LOX-2. The substrate concentration was quantitatively determined by allowing the enzymatic reaction to go to completion in the presence of 15-LOX-2. For the inhibitors that showed more than 50% inhibition at the one point screens, IC₅₀ values were obtained by determining the enzymatic rate at various inhibitor concentrations and plotted against inhibitor concentration, followed by a hyperbolic saturation curve fit (assuming total enzyme concentration [E] << K_i^app, so IC₅₀ ∼ K_i^app ).

HT22 Cell Culture. Glutathione depletion was induced in HT22 cells by glutamate treatment, and LDH release into the medium was measured to detect cell death as described. HT22 cells were cultured in DMEM containing 10% fetal bovine serum and penicillin/streptomycin. For viability experiments, cells were seeded at 1 × 10⁴ cells/well in 96-well plates and treated 18 hr later, when the cells were approximately 50-70% confluent. Treatment consisted of exchanging the medium to 100 μL fresh culturing medium and adding 5 mM glutamate in the presence or absence of DMSO (maximum 0.1% final concentration) as control or the indicated concentrations of ML351. Lactate dehydrogenase (LDH) content was determined separately for the cell extracts and corresponding media using a Cytotoxicity Detection Kit, and the percentage of LDH released to the medium calculated after subtracting the corresponding background value. To determine levels of the 12/15-LOX metabolite 12-hydroxy-eicosatetraenoic acid (12-HETE), we cultured HT22 cells in 75 cm² flasks in DMEM medium without phenol red, supplemented with 5% FBS, and treated the cells the next day when cells were 50-70% confluent. 24 hours later, the eicosanoid-containing fraction was isolated via Sep-Pak C-18 column, and 12-HETE was detected with a 12-HETE ELISA kit, used according to the manufacturer's instruction. Three independent experiments were evaluated.

2.2. Probe Chemical Characterization

*Purity >98% as determined by LC/MS and ¹H NMR analyses.

5-(methylamino)-2-(naphthalen-1-yl)oxazole-4-carbonitrile (CID 664510): LC-MS Retention Time: t₁ (Method 1) = 6.011 min and t₂ (Method 2) = 2.42 min; ¹H NMR (400 MHz, DMSO-d₆) δ 9.15 (dq, J = 8.7 and 0.9 Hz, 1H), 8.44 (brs, 1H), 8.10 – 7.99 (m, 3H), 7.74 – 7.57 (m, 3H), 3.07 – 3.01 (m, 3H).; ¹³C NMR (400 MHz, DMSO-d₆) δ 161.9, 161.9, 149.5, 134.1, 134.0, 131.2, 129.2, 128.3, 127.2, 127.2, 127.0, 126.9, 125.8, 125.8, 125.8, 125.7, 122.1, 116.5, 116.5, 84.1, 84.1, 84.1, 29.7, 29.7, 29.6; HRMS (ESI) m/z (M+H)⁺ calcd. for C₁₅H₁₂N₃O, 250.0975; found 250.0975.

Figure 1Stability of ML351 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at room temperature over 48 hr in (A) DPBS pH 7.4 buffer, (b) Lipoxygenase UV-Vis assay buffer of 1M HEPES pH 7.3, (C) pH 2 buffer and (D) pH 10 buffer

Results showed good stability of ML351 in different types of buffers.

Figure 2Structures of the five ML351 analogs that have been submitted to the MLSMR with their corresponding Compound IDs and MLS IDs listed in Table 1

2.3. Probe Preparation

Preparation of 5-(methylamino)-2-(naphthalen-1-yl)oxazole-4-carbonitrile (ML351) is a two-step process described below and illustrated in Scheme 1.

Scheme 1

Synthetic route to ML351.

1. A mixture of 1-naphthoic acid and 2-aminomalononitrile.TsOH in ethyl acetate was added to triethanolamine (TEA) followed by 50 % solution of propylphosphonic anhydride (T₃P^®) in ethyl acetate. The reaction was allowed to stir at room temperature for 12 hr and was then diluted with ethyl acetate. The organic layer was successively washed with water, saturated bicarbonate solution, brine and dried with magnesium sulfate (MgSO₄), and concentrated in vacuo. The crude product was purified on a biotage flash^® system eluting with 50% ethyl acetate in hexanes containing 0.1 % triethylamine to provide the yellow solid 5-amino-2-(naphthalen-1-yl)oxazole-4-carbonitrile intermediate with 81% yield.
2. A mixture of 5-amino-2-(naphthalen-1-yl)oxazole-4-carbonitrile, paraformaldehyde and sodium methoxide in methanol was stirred at 65 °C in for 3 hr until a clear mixture was obtained. The reaction mixture was cooled and sodium borohydride was added slowly and stirred further at room temperature for 1 hr. The crude product was extracted with ethyl acetate and successively washed with water and brine. The ethyl acetate layer was dried with MgSO₄, and concentrated in vacuo. The crude product was purified on a biotage flash^® system eluting with 30% ethyl acetate in hexanes containing 0.1 % triethylamine to provide 5-(methylamino)-2-(naphthalen-1-yl)oxazole-4-carbonitrile (ML351), a colorless solid end product with 27 % yeild.

3.1. Dose Response Curves for Probe

Figure 3Dose response curves for probe ML351 showing inhibition of 15-LOX-1 in the UV-Vis assay

3.2. Cellular Activity

Figure 4 (A) Inhibition of 12-HETE production in HT-22 cells by ML351 following treatment with glutamate (5 mM). (B) Protection of Glutamate (5 mM) induced HT-22 death by increasing amounts of ML351 (** p < 0.1 *** P < 0.001). Result showed 12% death rate with no glutamate added (normalized to 100%).

3.3. Profiling Assays

Selective profiling of ML351 and selected analogs showed inactivity of ML351 against 15-LOX-2, 12-LOX and 5-LOX, but good inhibition against 15-LOX-1 with 0.02 μM (Table 2). Moreover, ML355 represents a vast PK and ADME property improvement over the majority of compounds reported previously (vide infra). Despite the low molecular weight (249 Da), and favorable log D (pH 7.4) of 2.6, most analogs exhibited poor solubility. The aqueous kinetic solubility in PBS buffer was determined to be 1.2 μM, which is about 7 times the in vitro IC₅₀. Empirically, a vast improvement in the solubility in the 15-LOX assay buffer was observed which was encouraging and suggests that solubility was not a detrimental factor in the biochemical studies. Importantly, the compound demonstrated favorable PAMPA permeability (passive) and acceptable Caco-2 permeability of >1 (1.5 cm/s^-6) with no evidence of efflux (efflux ratio: 0.7) suggesting the compound is not susceptible to the action of P-glycoprotein 1 (Pgp), a well-characterized ABC-transporter. Moreover, ML351 was stable in various aqueous solutions (pH 2, pH 7.4, pH 9) and mouse plasma. In addition, ML351 exhibited minimal CYP inhibition of the 2D6 and 3A4 isoforms at 10.3% and 3.5% inhibition respectively. Microsomal stability appears to be species dependent with ML351 possessing moderate stability to rat liver microsomes (T_1/2 = 18 minutes) while being less stable to mouse liver microsomes (5.5 min). The compounds were completely stable in the absence of NADPH, suggesting a CYP-mediated degradation. Given the interest in testing compound ML351 in proof of concept mouse models of stroke, in vivo PK data on ML351 was obtained and found a suitable formulation. ML351 has a relatively fast half-life in both plasma and brain (T_1/2 = ∼ 1 hr) with a C_max of 13.8 μM in plasma (69 times in vitro IC₅₀) and 28.8 μM in brain (144 times in vitro IC₅₀). Encouragingly, ML351 has a brain/plasma ratio of 2.8 which demonstrates favorable BBB permeability and suggested that this compound was suitable for in vivo proof of concept (POC) models of ischemic stroke (vide infra).

Table 2

Selectivity profiling of ML351 and other top compounds.

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Figure 5Prior Art Inhibitors of 15-Lipoxygenase-1 (15-LOX-1)

Previously reported natural product based inhibitors of 15-LOX-1, such as boswellic acid, baicalein [10], and nor-dihydroguairetic acid (NDGA) [11] all possess several liabilities. These compounds are less potent and less selective towards 15-LOX-1, and are not easily amendable to further optimization as a result of their polyphenolic or terpene-based structures. In comparison, ML351 is potent (200 nM), selective (no activity against related isozymes or COX-1/2), chemically tractable and drug-like. Our previous chemical probe for 15-LOX-1, ML094, was found to be extremely potent (14 nM) and selective but lacked activity in cell-based assays, possibly due to poor cell permeability, intracellular hydrolysis of the terminal ester moiety, or inactivity against m12-LOX [12]. Tryptamine (37l) and imidazole-based (21n) inhibitors are potent and seemingly selective against 5-LO and 12-LO but a more comprehensive selectivity study is not reported. These compounds were reported to have unfavorable solubility and log P values, and while 21n seemed to possess improved physicochemical properties, with a ClogP of >5, there was no in vitro ADME or in vivo PK properties reported. Finally, the pyrazole derivative (15i) has improved solubility and cLogP, as compared to analogs 37n and 21n, suggesting inconclusive in vivo PK properties so despite the improved properties these compounds remain problematic. Additionally, the compounds reported by the BMS researchers, and in particular 21n and 15i, are quite complex structurally and possess at least one chiral center which limits their utility as probe compounds unless they are willing to provide academic researcher with the sample. In contrast, ML351 has much better ligand efficacy and can be synthesized in 2-3 steps from commercially available starting material. While ML351 does possess some aforementioned ADME liabilities (solubility, microsomal stability), this report represents the first account of a selective 15-LOX-1 inhibitor with demonstrated BBB permeability and activity in in vivo efficacy models for ischemic stroke.