Human lipoxygenases (LOXs) are enzymes involved in catalyzing the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites as the end product [1-3]. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation and cell proliferation [4-6]. As a result, modulation of these responses through the inhibition of the lipoxygenase enzymes is of great interest. Our group has particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis and cancer [7-11]. However, despite the potential of 12-LOX as a therapeutic target, few potent and selective inhibitors have been reported. The lack of high quality 12-LOX inhibitors prompted us to initiate a high-throughput screening campaign as part of the MLPCN program which ultimately led to the discovery of ML127. While potent and selective, ML127 demonstrated limited tolerance for structural modifications, which hampered continued medicinal chemistry efforts thus a continued discovery efforts to develop additional novel inhibitors of 12-LOX is needed. Herein, we report the identification and medicinal chemistry optimization of an unrelated, second chemotype, ML355, which displays nM potency against 12-LOX and excellent selectivity over related lipoxygenases and cyclooxygenases. ML355 has favorable absorption, distribution, metabolism, and excretion (ADME) properties, inhibits PAR-4 induced aggregation and calcium mobilization in human platelets, and reduces 12-HETE in mouse/human beta cells suggesting its potential utility in animal models for antiplatelet therapy and diabetes.

Probe Structure & Characteristics

1. Recommendations for Scientific Use of the Probe

12-LOX has been implicated in the pathophysiology of a variety of diseases including arterial thrombosis and diabetes (T1D and T2D). Thus, targeted inhibition of 12-LOX has been proposed as a therapeutic strategy to mitigate the effects of these diseases by ultimately reducing the production of the bioactive metabolite 12-HETE. The ML355 probe displays potent and selective inhibition of 12-LOX in vitro and demonstrated good activity in cell-based assays. ML355 has been shown to decrease calcium mobilization and PAR-4 induced platelet aggregation in patient derived human platelets and to significantly inhibit AA/IONO-induced 12-HETE in mouse BTC3 cells and human islets. Hence the ML355 probe can be used by researchers to interrogate the role of 12-LOX in both diabetes and anti-platelet in vivo models via pharmacological inhibition. Moreover, ML355 has shown favorable ADME properties enabling the scientific community to explore its potential therapeutic applications for other diseases where the 12-LOX role is crucial.

2. Materials and Methods

General Methods for Chemistry: All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, N,N-dimethylformamide (DMF), acetonitrile, methanol and triethylamine were purchased from Sigma-Aldrich. Preparative purification was performed on a Waters semi-preparative HPLC system. The column used was a Phenomenex Luna C18 (5 micron, 30 × 75 mm) at a flow rate of 45 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection (220 nm). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50 °C. Method 2: A 3 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A Phenomenex Gemini Phenyl column (3 micron, 3 × 100 mm) was used at a temperature of 50 °C. Purity determination was performed using an Agilent Diode Array Detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. ¹H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical shifts are reported in ppm with undeuterated solvent (DMSO-d₆ at 2.49 ppm) as internal standard for DMSO-d₆ solutions. All of the analogs tested in the biological assays have purity greater than 95%, based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).

Biological Reagents: All commercial fatty acids (Sigma-Aldrich Chemical Company) were re-purified using a Higgins HAIsil Semi-Preparative (5 µm, 250 × 10mm) C-18 column. Solution A was 99.9% MeOH and 0.1% acetic acid; solution B was 99.9% H₂O and 0.1% acetic acid. An isocratic elution of 85% A:15% B was used to purify all fatty acids, which were stored at −80 ºC for a maximum of 6 months.

Human Platelets: Human platelets were obtained from healthy volunteers within the Thomas Jefferson University community and the Philadelphia area. These studies were approved by the Thomas Jefferson University Institutional Review Board, and informed consent was obtained from all donors before blood draw. Blood was centrifuged at 200 g for 13 min at room temperature. Platelet-rich plasma was transferred into a conical tube containing a 10% acid citrate dextrose solution (39 mM citric acid, 75 mM sodium citrate, and 135 mM glucose, pH 7.4) and centrifuged at 2000 g for 15 min at room temperature. Platelets were resuspended in Tyrode's buffer (12 mM NaHCO₃, 127 mM NaCl, 5 mM KCl, 0.5 mM NaH₂PO₄, 1 mM MgCl₂, 5 mM glucose, and 10 mM HEPES), and the final platelet concentration was adjusted to 3 × 10⁸ platelets/mL after counting with a ZI Coulter particle counter (Beckman Coulter, Fullerton, CA). Reported results are the data obtained using platelets from at least three different subjects. Agonists and inhibitors were used at concentrations indicated in the figures and figure legends.

Over expression and Purification of 12-Human Lipoxygenase, 5-Human Lipoxygenase, 12/15-Mouse Lipoxygenase and the 15-Human Lipoxygenases: Human platelet 12-lipoxygenase (12-LOX), human reticulocyte 15-lipoxygenase-1 (15-LOX-1), and human epithelial 15-lipoxygenase-2 (15-LOX-2), were expressed as N-terminally, His₆-tagged proteins and purified to greater than 90% purity, as evaluated by SDS-PAGE analysis. Human 5-lipoxygenase was expressed as a non-tagged protein and used as a crude ammonium sulfate protein fraction, as published previously. 12/15-mouse lipoxygenase (12/15-mLOX) was expressed as a non-tagged protein and purified on Bio-Rad Uno Q1 with NaCl as the eluent. Iron content of 12-LOX was determined with a Finnigan inductively coupled plasma mass spectrometer (ICP-MS), using cobalt-EDTA as an internal standard. Iron concentrations were compared to standardized iron solutions and used to normalize enzyme concentrations.

High-throughput Screen Materials: Dimethyl sulfoxide (DMSO) ACS grade was from Fisher, while ferrous ammonium sulfate, Xylenol Orange (XO), sulfuric acid, and Triton X-100 were obtained from Sigma-Aldrich.

2.2. Probe Chemical Characterization

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

N-(benzo[d]thiazol-2-yl)-4-((2-hydroxy-3-methoxybenzyl)amino) benzenesulfonamide (CID-70701426); ( LC-MS room temperature = 2.26 (Method 2) M+1 = 442.0; ¹H NMR (400 MHz, DMSO-d₆) δ 12.86 (s, 1H), 8.73 (d, J = 0.5 Hz, 1H), 7.75 (ddd, J = 7.9, 1.2, 0.6 Hz, 1H), 7.54 – 7.46 (m, 2H), 7.40 – 7.31 (m, 1H), 7.28 – 7.16 (m, 2H), 6.93 – 6.79 (m, 2H), 6.78 – 6.55 (m, 4H), 4.23 (d, J = 5.8 Hz, 2H) and 3.78 (s, 3H); ¹³C NMR (DMSO-d₆) δ ppm 152.4, 147.7, 144.3, 128.2, 125.7, 122.9, 120.4, 119.0, 111.4, 110.9, 56.2 and 40.6; HRMS (ESI) m/z (M+H)⁺ calcd. For C₂₁H₁₉N₃O₄S₂, 441.0817; found 441.0819.

Figure 1Stability of ML355 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at r.t. over the indicated time period in (A) pH 2 buffer , (B) PBS pH 7.4 buffer, (C) 1M HEPES pH 7.3 Lipoxygenase UV-Vis assay buffer, (D) and in the presence of 5 mM glutathione (reduced form)

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

Table 1List of probe ML355 and related analogs that have been submitted to the MLSMR

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Internal ID	MLS ID	SID	CID	ML #	Type	Source
NCGC00263773	MLS004813829	160844040	70701426	ML355	Probe	NCGC
NCGC00319027	MLS004813830	160844073	70701393		Analog	NCGC
NCGC00264087	MLS004813831	160844041	70701387		Analog	NCGC
NCGC00319012	MLS004813832	160844065	70701378		Analog	NCGC
NCGC00343716	MLS004813833	160844087	70701397		Analog	NCGC
NCGC00319030	MLS004813834	160844076	70701412		Analog	NCGC

2.3. Probe Preparation

Preparation of N-(benzo[d]thiazol-2-yl)-4-((2-hydroxy-3-methoxybenzyl)amino) benzenesulfonamide (ML355) is a two-step process described below and illustrated in Scheme 1.

Scheme 1

Synthetic route to ML355.

1. 4-aminobenzenesulfonamide, 2-hydroxy-3-methoxybenzaldehyde in ethanol (EtOH) was heated to reflux for 4 hr. During this time an orange precipitate form and the reaction mixture were cooled to room temperature before sodium borohydride (0.330 g, 8.71 mmol) was added. After addition of the sodium borohydride the reaction mixture turned to a clear solution and was allowed to stir at room temperature for 30 min. Slowly 1 M hydrochloric acid (HCl) in diethyl ether (Et₂O) was added yielding a colorless precipitate which was filtered and washed with ethanol followed by a wash with ether to give a colorless solid product 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide with a yield of 92%.
2. 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide, 2-bromobenzo[d]thiazole, potassium carbonate (K₂CO₃), N,N'-dimethylethylenediamine and copper (I) iodide were added to a 15 mL round bottom flask in DMF and the reaction mixture was heated for 18 hr at 90 ºC. The reaction mixture was cooled to room temperature, filtered through a pad of celite, and rinsed the ethyl acetate (EtOAc). The filtrate was transferred to a separation funnel, washed with water (3X), and saturated ammonium chloride solution (2X). The organic layer was concentrated to 2 mL of solvent and passed through a thiol cartridge, dried over sodium sulfate (Na₂SO₄), filtered and concentrated to a tan solid. The crude solid was purified on the Isco reverse phase 30 g gold^® column to provide a 30% yield of N-(benzo[d]thiazol-2-yl)-4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide (ML355) as an off-white solid.

3. Results

3.1. Dose Response Curves for Probe

Figure 3Dose response curves for probe ML355 showing inhibition in the UV-Vis assay

3.2. Cellular Activity

Figure 4Dose dependent inhibition of stimulated 12-HETE by ML355

Mouse beta cells (BTC3) were treated with arachidonic acid and calcium ionophore (AA/IONO) alone or in the presence of ML355. Graphed are the levels of 12-HETE expressed as a percentage of that detected in cells stimulated with AA/IONO alone. 12-HETE was measured by ELISA. Result showed inhibition of 12-HETE expression by the ML355 probe.

Figure 5Histogram of 12-HETE expression (measured by ELISA) in human primary donor islets stimulated with Arachidonic acid and calcium ionophore (AA/IONO) alone or in the presence of 10 µM of ML355

Result showed inhibition of the of the 12-HETE expression on stimulated human islets upon treatment of ML355.

3.3. Profiling Assays

Selective profiling of ML355 and selected analogs showed inactivity of ML355 against 15-LOX-1, 15-LOX-2 and 5-LOX but good 12-LOX with 0.29 µM (Table 2). Moreover, ML355 demonstrated excellent microsomal stability with both rat (T_1/2 >30 minutes) and mouse (T_1/2 >300 minutes) and was found to be stable to mouse plasma over a 2 hour period (100% remaining). ML355 showed no degradation over various aqueous buffers (pH 2-9) and was stable to 5 mM glutathione suggesting excellent stability. One remaining liability is the aqueous solubility which is <5 µM, however improved solubility is observed in the assay buffer (qualitative analysis). ML355 showed moderate permeability in the Caco-2 assay (1.5 × 10⁻⁶ cm/s) and does not appear to be a substrate for Pgp given the efflux ratio of <2. In vivo PK studies where ML355 was administered as a solution via IV (3mpk) and PO (30mpk) demonstrated that ML355 is orally bioavailable (%F = 20) with good half-life (T_1/2 = 2.9 hours). At 30 mpk dosing, ML355 achieves a C_max of over 135 times the in vitro IC₅₀ and remains over IC₅₀ value for over 12 hours. The compound has low clearance (3.4 mL/min/kg) and good overall exposure (AUC_inf) of 38 µM. Although, the volume of distribution (V_D) observed was low (0.55 L/kg), the rest of the PK profiling results suggested a reasonable distribution between tissue and blood. These profiling results provided informative data necessary to develop an appropriate dosing regimen for future in vivo studies.

Table 2

Selectivity profiling of ML355 and other top compounds. NT = not tested while NI = No inhibition.

4. Discussion

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

Previously reported inhibitors of 12-LOX (Figure 6) such as baicalein [12] and nor-dihydroguairetic acid (NDGA) [13], “bromo-phenols” or “pyrazole derivatives” all possess several liabilities. These compounds are not only less potent, selective, but are also not easily amendable to further optimization. Our previously described 12-LOX inhibitor (ML127) demonstrates potent inhibition (<500 nM) and excellent selectivity but is not very tolerant of structural modifications [14]. Few regions of the molecule are amendable for optimization limiting available avenues for medicinal chemistry efforts. In addition, while the 8-HQ scaffold of ML127 has not yet emerged as a liability for the series, the known promiscuous metal chelation for related compounds of that type suggested one should proceed with caution. In comparison, the new ML355 probe series demonstrated potent (290 nM) activity towards 12-LOX and excellent selectivity against related enzymes 15-LOX-1, 5-LOX, 15-LOX-2, and COX ½ (Table 3). Additionally, this chemotype is structurally distinct from all previously reported inhibitors (including ML127) and it possesses a very drug-like scaffold. This series is readily amendable to structural modifications and displays clear and tractable SAR. Most importantly, ML355 exhibits a favorable in vitro ADME and in vivo PK profile with activity in disease relevant cell-based systems like diabetes through12-HETE reduction in β-cells.

Figure 6

Prior Art Inhibitors of 12-Lipxygenase.

Table 3

Comparison of ML355 to previously identified 12-LOX inhibitors. ND = Not Determined

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