BACKGROUND

Technical Field

The present disclosure relates to small molecule inhibitors of 12(S)-Lipoxygenase (“12-LOX”), and methods of using the small molecules to inhibit 12-LOX activation and to treat diseases, such as platelet hemostasis and thrombosis.

Description of Related Technology

Platelet adhesion and aggregation at the site of vascular injury are essential for maintaining normal hemostasis and preventing blood loss. However, the same processes can also lead to the development of arterial thrombosis and vessel occlusion when the integrity of the vessel wall is compromised by rupture of an atherosclerotic plaque. See Ruggeri Z M, Nat Med., 8:1227-1234 (2002). Excessive platelet activation and aggregation may lead to the formation of occlusive thrombi and result in severe consequences, such as myocardial infarction, ischemic stroke, and pulmonary embolism, which are the predominant causes of morbidity and mortality worldwide. See Go et al., Circulation, 129:399-410 (2014). Antiplatelet therapy is considered a gold standard for its effectiveness in preventing aberrant platelet activation and pivotal in the treatment of cardiovascular atherothrombotic events to reduce morbidity and mortality. See Steg et al., J Thromb Haemost, 9(suppl 1):325-332 (2011). Currently approved antiplatelet therapies inhibit platelet function by targeting platelet enzymes, receptors, and glycoproteins. See lkei et al., J. Lipid Res. 53:2546-2559 (2012). Although these therapeutic approaches limit platelet function, they often result in a concomitant increased risk of bleeding. Therefore, a need exists for the identification of novel antiplatelet therapeutic targets that limit bleeding.

Platelet 12(S)-lipoxygenase (“12-LOX”), an oxygenase predominantly expressed in human platelets, uses arachidonic acid released from the phospholipids as a substrate to form bioactive metabolites (12-(S)-hydroperoxyeicosatetraenoic acid and 12-(S)-hydroxyeicosatetraenoic acid (12(S)-HETrE)) that have been shown to regulate biological processes, such as integrin activation, vascular hypertension, and progression of certain types of cancer. See Steele et al., Cancer Epidemiol Biomarkers Prev, 8:467-483 (1999) and Ghosh et al., Proc Natl Acad Sci USA. 95:13182-13187 (1998). The bioactive lipid products resulting from 12-LOX oxidation have been shown to play a role in platelet activation, granule secretion, and clot retraction in vitro, suggesting an important role of 12-LOX in regulation of platelet function. See lkei et al. Recently, a key role for 12-LOX activity has been identified in regulation of protease-activated receptor-4-mediated (“PAR4-mediated”) and glycoprotein VI-mediated (“GPVI-mediated”) signaling pathways in the platelet. See Kenyon et al., J Med Chem. 54:5485-5497 (2011); Luci et al., J Med Chem. 57:495-506 (2014); Yeung et al., Thromb Haemost. 110:569-581 (2013).

A 12-LOX inhibitor, ML355, shown below, was recently identified and has been shown to prevent thrombosis in vitro, with a >50-fold selectivity versus the paralogs, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity versus bovine cyclooxygenase (“COX”)-1 and human COX-2.


Further, 12-LOX was recently reported to be essential for FcγRIIa-mediated platelet activation, suggesting 12-LOX may also be a potential therapeutic target to limit immune-mediated thrombosis. See Luci et al. ML355 also has been shown to impair thrombus formulation and vessel occlusion in vivo with minimal affects on hemostasis. See Adili et al., Arterioscler Thromb Vasc Biol. 37(10):1828-1839 (2017). Therefore, ML355 treatment can be an ideal therapy targeting the pathogenesis of heparin-induced thrombocytopenia and thrombosis (“HITT”) to prevent the onset, as well as limit the progression, of thrombotic complications of HITT because (1) inhibition of 12-LOX down regulates platelet reactivity and release of PF4 from platelet granules (2) inhibition of 12-LOX prevents FcγRIIa-dependent immune-mediated platelet activation and platelet clearance, and (3) ML355 inhibition of 12-LOX potently inhibits thrombosis without bleeding complications, which translates to a safer alternative to the currently clinical treatment with thrombin inhibitors. ML355 also has been shown to have reasonable pharmacokinetics, relatively good tolerance in preclinical studies, and can be given orally. However, ML355 suffers from limited solubility, which drastically diminishes its clinical use.

Therefore, there is a need for ML355 analogs capable of treating and/or preventing platelet hemostasis and thrombosis, which exhibit improved in vivo potency and solubility over ML355, without causing bleeding.

SUMMARY

Provided herein are compounds of Formula (I), or pharmaceutically acceptable salts thereof:


wherein: R2is halo, C1-3fluoroalkyl, C3-8fluorocyloalkyl, OC1-3fluoroalkyl, or OC3-8fluorocyloalkyl; each of R1, R3, R4, and R5independently is H, halo, C1-3fluoroalkyl, C3-8fluorocyloalkyl, OC1-3fluoroalkyl, or OC3-8fluorocyloalkyl; each of Raand Rbindependently is OH, OC1-3alkyl, C1-3fluoroalkyl, C3-8fluorocyloalkyl, OC1-3fluoroalkyl, OC3-8fluorocyloalkyl, or halo; each of Rc, Rd, and Reindependently is H, OH, OC1-3alkyl, C1-3fluoroalkyl, C3-8fluorocyloalkyl, OC1-3fluoroalkyl, OC3-8fluorocyloalkyl, or halo.

In some embodiments, R2is halo (e.g., Br, Cl, or F). In various embodiments, R2is Cl or F. In some cases, R2is Cl. In various cases, R2is C1-3fluoroalkyl (e.g., CH2F, CHF2, or CF3), C3-8fluorocyloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl substituted with F, CH2F, CHF2, or CF3), OC1-3fluoroalkyl, or OC3-8fluorocyloalkyl. In some embodiments, R2is CF3or


In some cases, each of R1, R3, R4, and R5is H. In various cases, one or more of R1, R3, R4, and R5is halo (e.g., Br, Cl, or F), C1-3fluoroalkyl (e.g., CH2F, CHF2, or CF3), C3-8fluorocyloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl substituted with F, CH2F, CHF2, or CF3), OC1-3fluoroalkyl, or OC3-8fluorocyloalkyl.

In some embodiments, one or more of R1, R3, R4, and R5is F, Cl, CF3or


In various embodiments, R1is H or F; R2is Cl, CF3, or


R3is H or F; R4is H; and R5is H.

In some cases, each of Raand Rbindependently is OH or OC1-3alkyl. In various cases, one of Raand Rbis OH or OC1-3alkyl; and the other of Raand Rbis C1-3fluoroalkyl, C3-8fluorocyloalkyl, OC1-3fluoroalkyl, OC3-8fluorocyloalkyl, or halo. In some embodiments, OC1-3alkyl is OCH3. In various embodiments, each of Raand Rbindependently is C1-3fluoroalkyl, C3-8fluorocyloalkyl, OC1-3fluoroalkyl, OC3-8fluorocyloalkyl, or halo. In some cases, at least one of Raand Rbis CF3,


F, or Cl. In various cases, Rc, Rd, and Reare each H. In some embodiments, one or more of Rc, Rd, and Reis halo, C1-3fluoroalkyl, C3-8fluorocyloalkyl, OC1-3fluoroalkyl, or OC3-8fluorocyloalkyl. In some embodiments, one or more of Rc, Rd, and Reis F, Cl, CF3or



Specifically contemplated compounds of the disclosure include a compound listed in Table A, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is a compound or salt thereof selected from the group consisting of A1, A2, A3, A4, A5, and A6. In some cases, the disclosure provides a compound or salt thereof having a structure:



The disclosure also provides a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier.

The disclosure further provides a method of inhibiting (S)-Lipoxygenase (“12-LOX”) activation in a cell comprising contacting the cell with a compound or composition described herein in an amount effective to inhibit 12-LOX activation.

The disclosure still further another aspect of the disclosure provides a method of inhibiting platelet factor 4 (“PF4”) release in cell comprising contacting the cell with a compound or composition described herein in an amount effective to inhibit PF4 release.

The disclosure also provides a method of inhibiting PF4-heparin complex formation in cell comprising contacting the cell with a compound or composition described herein in an amount effective to inhibit PF4-heparin complex formation.

The disclosure further provides a method of inhibiting platelet activation in a cell comprising contacting the cell with a compound or composition described herein in an amount effective to inhibit platelet activation. In some embodiments, the platelet activation is FcγRIIa-mediated platelet activation.

The disclosure still further provides a method of inhibiting thrombin, protease-activated receptor-4 (“PAR4”), and/or glycoprotein VI (“GPVI”) signaling in a cell comprising contacting the cell with a compound or composition described herein in an amount effective to inhibit PAR4 and/or GPVI signaling.

This disclosure also provides a method of treating a thrombotic disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound or composition described herein. Examples of the thrombotic disorder include arterial thrombosis, deep vein thrombosis (“DVT”), pulmonary embolism (“PE”), ischemic stroke, immune thrombocytopenia (“ITP”), Heparin-induced thrombocytopenia (“HIT”), and Heparin-induced thrombocytopenia and thrombosis (“HITT”).

Also provided herein is a method of preventing thrombosis in a subject comprising administering to the subject a therapeutically effective amount of a compound or composition described herein.

Further provided herein is a method of treating thrombocytopenia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound or composition described herein.

Further aspects and advantages will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. The description hereafter includes specific embodiments with the understanding that the disclosure is illustrative, and is not intended to limit the invention to the specific embodiments described herein.

