Selective inhibitors of 12(S)-lipoxygenase (12-LOX) and methods of use thereof

Abstract

The present disclosure relates to small molecule inhibitors of 12(S)-lipoxygenase (12-LOX), and methods of using such small molecules to inhibit 12-LOX activation and treat diseases such as platelet hemostasis and thrombosis. Specifically, the present disclosure relates to compounds of formula (I) and pharmaceutically acceptable salts thereof: wherein the substituents are as described.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 658,936, filed on April 17, 2018, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to small molecule inhibitors of 12(S)-lipoxygenase (“12-LOX”), and methods of using such small molecules to inhibit 12-LOX activation and treat diseases such as platelet hemostasis and thrombosis. Background Art

[0004] Platelet adhesion and aggregation at the site of vascular injury are essential for maintaining normal hemostasis and preventing blood loss. However, when atherosclerotic plaque rupture compromises the integrity of the vessel wall, the same processes can also lead to the formation of arterial thrombosis and vascular occlusion. See Ruggeri ZM, Nat Med., 8:1227-1234 (2002). Excessive platelet activation and aggregation can lead to occlusive thrombosis and result in serious consequences such as myocardial infarction, ischemic stroke, and pulmonary embolism, which are leading causes of morbidity and mortality worldwide. See Go et al., Circulation, 129:399-410 (2014). Antiplatelet therapy is considered the gold standard due to its effectiveness in preventing abnormal platelet activation and plays a pivotal role in treating cardiovascular atherosclerotic events to reduce morbidity and mortality. See Steg et al., J Thromb Haemost, 9(suppl1):325-332 (2011). Currently approved antiplatelet therapies inhibit platelet function by targeting platelet enzymes, receptors, and glycoproteins. See Ikei et al., J. Lipid Res., 53:2546-2559 (2012). Although these treatments limit platelet function, they often result in an increased risk of concomitant bleeding. Thus, there is a need to identify novel antiplatelet therapeutic targets that limit bleeding.

[0005] The oxygenase platelet 12(S)-lipoxygenase ("12-LOX"), which is mainly expressed in human platelets, utilizes arachidonic acid released from phospholipids as a substrate to form bioactive metabolites (12-(S)-hydroperoxyeicosatetraenoic acid and 12-(S)-hydroxyeicosatetraenoic acid (12(S)-HETrE)), both of which have been shown to regulate biological processes such as integrin activation, vascular hypertension, and the 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 U S A., 95:13182-13187 (1998). The bioactive lipid products generated by 12-LOX oxidation have been shown to play a role in platelet activation, granule secretion, and in vitro clot retraction, indicating an important role of 12-LOX in regulating platelet function. See Ikei et al. Recently, a key role of 12-LOX activity in regulating protease-activated receptor-4-mediated ("PAR4-mediated") and glycoprotein VI-mediated ("GPVI-mediated") signaling pathways in platelets has been identified. 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).

[0006] Recently, a 12-LOX inhibitor, ML355, shown below, has been identified and has been shown to prevent thrombosis in vitro, with a selectivity >50-fold compared to the paralog 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type 1, and epithelial 15-human lipoxygenase type 2, and a selectivity >100-fold compared to bovine cyclooxygenase ("COX")-1 and human COX-2.

[0007]

[0008] In addition, it has recently been reported that 12-LOX is essential for FcγRIIa-mediated platelet activation, suggesting that 12-LOX may also be a potential therapeutic target for limiting immune-mediated thrombosis. See Luci et al. ML355 has also been shown to attenuate thrombus formation and vascular occlusion in vivo with minimal effects on hemostasis. See Adili et al., Arterioscler Thromb Vasc Biol. 37(10):1828-1839 (2017). Thus, ML355 treatment may be an ideal therapy for the pathogenesis of heparin-induced thrombocytopenia and thrombosis ("HITT") to prevent the occurrence and limit the progression of thrombotic complications of HITT because (1) inhibition of 12-LOX downregulates platelet reactivity and releases PF4 from platelet granules, (2) inhibition of 12-LOX prevents FcγRIIa-dependent immune-mediated platelet activation and platelet clearance, and (3) ML355 inhibits 12-LOX to effectively inhibit thrombosis without bleeding complications, translating into a safer alternative to current clinical treatment with thrombin inhibitors. ML355 has also been shown to have reasonable pharmacokinetics, relatively good tolerance in preclinical studies, and can be administered orally. However, the limited solubility of ML355 significantly reduces its clinical use.

[0009] Accordingly, there is a need for ML355 analogs that are capable of treating and / or preventing platelet hemostasis and thrombosis and that exhibit improved in vivo potency and solubility over ML355 without causing bleeding. SUMMARY OF THE INVENTION

[0010] The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0011] Wherein: R 2 is halo, C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl or OC 3-8 fluorocycloalkyl; Each of R 1 , R 3 , R 4 and R 5 is independently H, halo, C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl or OC 3-8 fluorocycloalkyl; Each of R a and R b is independently OH, OC 1-3 alkyl, C 1-3fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl, OC 3-8 fluorocycloalkyl or halo; R c 、R d 、R e each independently is H, OH, OC 1-3 alkyl, C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl, OC 3-8 fluorocycloalkyl or halo.

[0012] In some embodiments, R 2 is halo (e.g., Br, Cl or F). In various embodiments, R 2 is Cl or F. In some cases, R 2 is Cl. In various cases, R 2 is C 1-3 fluoroalkyl (e.g., CH2F, CHF2 or CF3), C 3-8 fluorocycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl substituted with F, CH2F, CHF2 or CF3), OC 1-3 fluoroalkyl or OC 3-8 fluorocycloalkyl. In some embodiments, R 2 is CF3 or In some cases, R 1 、R 3 、R 4 and R 5 each is H. In various cases, one or more of R 1 、R 3 、R 4 and R 5 is halo (e.g., Br, Cl or F), C 1-3 fluoroalkyl (e.g., CH2F, CHF2 or CF3), C 3-8 fluorocycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl substituted with F, CH2F, CHF2 or CF3), OC 1-3 fluoroalkyl or OC 3-8 fluorocycloalkyl. In some embodiments, one or more of R 1 、R 3 、R 4 and R 5 are F, Cl, CF3 or In various embodiments, R 1 is H or F; R 2 is Cl, CF3 or R 3is H or F; R 4 is H; and R 5 is H.

[0013] In some cases, R a and R b each independently is OH or OC 1-3 alkyl. In various cases, R a and R b one of them is OH or OC 1-3 alkyl; and R a and R b the other of them is C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl, OC 3-8 fluorocycloalkyl or halo group. In some embodiments, OC 1-3 alkyl is OCH3. In various embodiments, R a and R b each independently is C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl, OC 3-8 fluorocycloalkyl or halo group. In some cases, R a and R b at least one of them is CF3, F or Cl. In various cases, R c 、R d and R e are each H. In some embodiments, R c 、R d and R e one or more of them are halo group, C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl or OC 3-8 fluorocycloalkyl. In some embodiments, R c 、R d and R e one or more of them are F, Cl, CF3 or

[0014] Particularly contemplated compounds of the present disclosure include the compounds listed in Table A or pharmaceutically acceptable salts thereof. In some embodiments, compounds selected from the group consisting of A1, A2, A3, A4, A5 and A6 or salts thereof are provided herein. In some cases, the present disclosure provides a compound having the following structure or a salt thereof:

[0015] The present disclosure also provides a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier.

[0016] The present disclosure further provides a method of inhibiting the activation of (S)-lipoxygenase (“12-LOX”) in a cell, which comprises contacting the cell with an amount of a compound or composition described herein effective to inhibit 12-LOX activation.

[0017] In yet another aspect of the present disclosure, there is provided a method of inhibiting the release of platelet factor 4 (“PF4”) in a cell, which comprises contacting the cell with an amount of a compound or composition described herein effective to inhibit PF4 release.

[0018] The present disclosure also provides a method of inhibiting the formation of PF4-heparin complex in a cell, which comprises contacting the cell with an amount of a compound or composition described herein effective to inhibit the formation of PF4-heparin complex.

[0019] The present disclosure further provides a method of inhibiting platelet activation in a cell, which comprises contacting the cell with an amount of a compound or composition described herein effective to inhibit platelet activation. In some embodiments, the platelet activation is FcγRIIa-mediated platelet activation.

[0020] The present disclosure further provides a method of inhibiting thrombin, protease-activated receptor-4 (“PAR4”) and / or glycoprotein VI (“GPVI”) signaling in a cell, which comprises contacting the cell with an amount of a compound or composition described herein effective to inhibit PAR4 and / or GPVI signaling.

[0021] The present disclosure also provides a method of treating a thrombotic disorder in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a compound or composition described herein. Examples of thrombotic disorders 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”).

[0022] The present disclosure also provides a method of preventing thrombosis in a subject, which comprises administering to the subject a therapeutically effective amount of a compound or composition described herein.

[0023] The present disclosure further provides a method of treating thrombocytopenia in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a compound or composition described herein.

[0024] Those of ordinary skill in the art will recognize additional aspects and advantages upon review of the following detailed description in conjunction with the figures. The following description includes specific embodiments, and it is to be understood that the disclosure is illustrative and not intended to limit the invention to the specific embodiments described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Depicts the concentrations (30 mg / kg) of compounds A1, A2, and A4 administered orally or IV in the plasma of mice (n = 3) monitored at 3 time points (2 hours, 4 hours, and 7 hours), as further described in the Examples section.

[0026] Figure 2 Depicts the percentage of collagen-induced platelet aggregation of compounds A1 - A7 at 1 μM, 5 μM, 10 μM, and 20 μM, as further described in the Examples section.

[0027] Figure 3 Depicts the percentage of thrombin-induced platelet aggregation of compounds A1 - A7 at 1 μM, 5 μM, 10 μM, and 20 μM, as further described in the Examples section.

[0028] Figure 4 Depicts representative images of platelet accumulation (green) and fibrin formation (red) in the thrombi forming in the cremaster arterioles of wild-type ("WT") control animals treated with polyethylene glycol ("PEG"; control, top) and WT treated with compound A1 (3 mg / kg, twice daily for 2 days; bottom), as further described in the Examples section, indicating that inhibition of platelet 12(S)-lipoxygenase ("12-LOX") impairs thrombus formation in the laser-induced cremaster arteriole thrombosis model.

[0029] Figure 5 Depicts representative images of platelet accumulation (green) in the lungs of mice from control (PEG vehicle) and A1-treated (15 mg / kg) mice four hours after heparin-induced thrombocytopenia and thrombosis ("HITT"), as further described in the Examples section, indicating that the compounds disclosed herein can inhibit thrombocytopenia and thrombosis in the lungs of mice. DETAILED DESCRIPTION

[0030] Compounds having the structure of formula (I) are disclosed herein:

[0031]

[0032] or a pharmaceutically acceptable salt thereof, which can inhibit 12(S)-lipoxygenase (“12-LOX”) and is used for treating and preventing diseases related to platelet hemostasis and thrombosis. The compounds disclosed herein exhibit improved in vivo solubility and potency over ML355 (e.g., at least 10-fold higher potency than ML355) without causing bleeding.

[0033] The compounds disclosed herein can dose-dependently inhibit human platelet aggregation and the production of 12-LOX oxylipins. By pharmacokinetic evaluation, oral administration of the compounds disclosed herein in mice shows reasonable plasma drug levels. Similarly, the compounds disclosed herein can attenuate thrombus growth and vascular occlusion in FeCl3-induced mesenteric, laser-induced cremaster arteriolar thrombosis, and immune-mediated anti-GPIX in vivo models in mice. Importantly, in response to laser excision of the saphenous vein in mice or in a laser-induced rupture model of cremaster muscle microvessels, hemostatic embolism formation and bleeding are minimal after treatment with the compounds described herein.

[0034] Definitions

[0035] As used herein, the term “alkyl” refers to straight-chain and branched-chain saturated hydrocarbon groups containing from one to thirty carbon atoms (e.g., from one to twenty carbon atoms or from one to ten carbon atoms). The term C n means that the alkyl has “n” carbon atoms. For example, C4 alkyl refers to an alkyl having 4 carbon atoms. C 1-7 alkyl refers to an alkyl having a carbon atom number covering the entire range (e.g., 1 to 7 carbon atoms) and all subgroups (e.g., 1-6, 2-7, 1-5, 3-6, 1, 2, 3, 4, 5, 6, and 7 carbon atoms). Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), tert-butyl (1,1-dimethylethyl), 3,3-dimethylpentyl, and 2-ethylhexyl. Unless otherwise specified, the alkyl can be an unsubstituted alkyl or a substituted alkyl. For example, the alkyl can be substituted with one or more fluorine atoms to form a fluoroalkyl (e.g., methyl can be substituted with 1 to 3 fluorine atoms to provide CH2F, CHF2, or CF3).

[0036] As used herein, the term “cycloalkyl” refers to an aliphatic cyclic hydrocarbon group containing from three to eight carbon atoms (e.g., 3, 4, 5, 6, 7, or 8 carbon atoms). The term C n means that the cycloalkyl has “n” carbon atoms. For example, C5 cycloalkyl refers to a cycloalkyl having 5 carbon atoms in the ring. C 5-8A cycloalkyl group refers to a cycloalkyl group having a carbon atom number covering the entire range (e.g., 5 to 8 carbon atoms) and all subgroups (e.g., 5-6, 6-8, 7-8, 5-7, 5, 6, 7, and 8 carbon atoms). Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The cycloalkyl groups described herein can be separate or fused with another cycloalkyl group, heterocycloalkyl group, aryl group, and / or heteroaryl group. When a cycloalkyl group is fused with another cycloalkyl group, each cycloalkyl group can contain from three to eight carbon atoms. Unless otherwise specified, the cycloalkyl group can be substituted or unsubstituted, for example, optionally substituted with, for example, one to three groups independently selected from alkyl, alkylene-OH, C(O)NH2, NH2, oxo (=O), aryl, haloalkyl, halogen, and OH. In some cases, the cycloalkyl group can be substituted with one or more (e.g., 1 to 3) fluorine groups.

[0037] As used herein, the term "halogen" refers to a fluorine group, chlorine group, bromine group, or iodine group. The term "haloalkyl" refers to an alkyl group substituted with at least one (e.g., 1, 2, 3, 4, 5, or 1-5 or 1-3) halogen. "Fluoroalkyl" refers to a haloalkyl in which the halogen is a fluorine group.

[0038] As used herein, the term "substituted", when used to modify a chemical functional group, means that at least one hydrogen radical on the functional group is replaced by a substituent. Substituents can include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycloalkyl, thioether, polysulfide, aryl, heteroaryl, hydroxy, oxy, alkoxy, heteroalkoxy, aryloxy, heteroaryloxy, ester, thioester, carboxyl, cyano, nitro, amino, amide, acetamide, and halogen (e.g., fluorine, chlorine, bromine, or iodine). When a chemical functional group includes more than one substituent, these substituents can be bonded to the same carbon atom or two or more different carbon atoms. A substituted chemical functional group itself can include one or more substituents.

[0039] As used herein, the term "therapeutically effective amount" means an amount of a compound or combination of therapeutically active compounds that improves, alleviates, or eliminates one or more symptoms of a particular disease or condition or prevents or delays the onset of one of the multiple symptoms of a particular disease or condition.

[0040] As used herein, the terms "patient" and "subject" can be used interchangeably and mean animals (such as dogs, cats, cows, horses, and sheep (e.g., non-human animals)) and humans. A specific patient or subject is a mammal (e.g., human). The terms patient and subject include both males and females.

[0041] As used herein, the term "treating" (e.g., "treat" or "treatment") includes prophylactic (e.g., preventive) treatment and palliative treatment. In some cases, treating refers to treating the symptoms of a disorder or disease disclosed herein.

[0042] Compound

[0043] The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0044]

[0045] Wherein:

[0046] R 2 is a halogen group, C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl or OC 3-8 fluorocycloalkyl;

[0047] R 1 、R 3 、R 4 and R 5 each independently is H, a halogen group, C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl or OC 3-8 fluorocycloalkyl;

[0048] R a and R b each independently is OH, OC 1-3 alkyl, C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl, OC 3-8 fluorocycloalkyl or a halogen group; and

[0049] R c 、R d 、R e each independently is H, OH, OC 1-3 alkyl, C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl, OC 3-8 fluorocycloalkyl or a halogen group.

[0050] In some embodiments, R 2 is a halogen group (e.g., Br, Cl or F). In various embodiments, R 2 is Cl or F. In some cases, R 2is Cl. In various cases, R 2 is C 1-3 fluoroalkyl (e.g., CH2F, CHF2 or CF3), C 3-8 fluorocycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl substituted by F, CH2F, CHF2 or CF3), OC 1-3 fluoroalkyl or OC 3-8 fluorocycloalkyl. In some embodiments, R 2 is CF3 or In some cases, R 1 , R 3 , R 4 and R 5 each is H. In various cases, one or more of R 1 , R 3 , R 4 and R 5 are halo group (e.g., Br, Cl or F), C 1-3 fluoroalkyl (e.g., CH2F, CHF2 or CF3), C 3-8 fluorocycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl substituted by F, CH2F, CHF2 or CF3), OC 1-3 fluoroalkyl or OC 3-8 fluorocycloalkyl. In some embodiments, one or more of R 1 , R 3 , R 4 and R 5 are F, Cl, CF3 or In various embodiments, R 1 is H or F; R 2 is Cl, CF3 or R 3 is H or F; R 4 is H; and R 5 is H.

[0051] In some cases, each of R a and R b is independently OH or OC 1-3 alkyl. In various cases, one of R a and R b is OH or OC 1-3 alkyl; and the other of R a and R b is C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl, OC 3-8 fluorocycloalkyl or halo group. In some embodiments, OC 1-3The alkyl group is OCH3. In various embodiments, R a and R b each independently is C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl, OC 3-8 fluorocycloalkyl or a halogen group. In some cases, at least one of R a and R b is CF3, F or Cl. In various cases, R c , R d and R e are each H. In some embodiments, one or more of R c , R d and R e are a halogen group, C 1-3 fluoroalkyl, C 3-8 fluorocycloalkyl, OC 1-3 fluoroalkyl or OC 3-8 fluorocycloalkyl. In some embodiments, one or more of R c , R d and R e are F, Cl, CF3 or

[0052] Particularly contemplated compounds of the present disclosure include the compounds listed in Table A or pharmaceutically acceptable salts thereof:

[0053] Table A

[0054]

[0055]

[0056] In some embodiments, provided herein are compounds selected from the group consisting of A1, A2, A3, A4, A5 and A6 or salts thereof. In some cases, the present disclosure provides a compound having the following structure or a salt thereof:

[0057] Compound synthesis

[0058] The compounds provided herein can be synthesized using conventional techniques known to those skilled in the art and readily available starting materials. Generally, the compounds provided herein can be conveniently obtained by standard organic chemical synthesis methods.

[0059] For example, a compound having a m-chloroaniline group can be synthesized by reacting an appropriate 3-chloroaniline with 4-nitrobenzenesulfonyl chloride to form the desired N-(3-chlorophenyl)-4-nitrobenzenesulfonamide compound, reducing the nitro group to an amino group, and then reacting the amino group with an appropriate benzaldehyde group to form the desired compound, as shown in Scheme 1 in the Examples section.

[0060] Compounds having other substitution patterns can be synthesized by reacting 4-aminobenzenesulfonamide with an appropriate benzaldehyde group to form a 4-(benzyl)aminobenzenesulfonamide compound and coupling this compound with an appropriate aryl bromide group to obtain the desired compound, as shown in Scheme 2 in the Examples section.

[0061] Other synthetic methods for preparing the inhibitors disclosed herein can be found in the Examples section.

[0062] Method of use

[0063] Maintenance of hemostasis requires sufficient platelet reactivity. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi. Platelet 12(S)-lipoxygenase ("12-LOX"), an oxygenase highly expressed in platelets, has been shown to regulate platelet function and ex vivo thrombus formation, supporting a key role for 12-LOX in the regulation of in vivo thrombus formation. The compounds described herein (e.g., compounds of formula (I), compounds listed in Table A, and the aforementioned pharmaceutically acceptable salts) have been found to target 12-LOX in vivo, which has implications for thrombus formation and hemostasis. Accordingly, provided herein is a method of inhibiting 12-LOX in a cell, which comprises contacting the cell with an amount of a compound disclosed herein (e.g., a compound of formula (I), a compound listed in Table A, or the aforementioned pharmaceutically acceptable salt) effective to inhibit 12-LOX activation. In some embodiments, the contacting is in vivo. In various embodiments, the contacting is in vitro.

[0064] 12-LOX has been shown to attenuate the thrombin PAR4-mediated signaling pathway and the GPVI-mediated signaling pathway in human platelet activation. Accordingly, the present disclosure also provides a method of inhibiting thrombin PAR4 and / or GPVI signaling in a cell, which comprises contacting the cell with an amount of a compound disclosed herein (e.g., a compound of formula (I), a compound listed in Table A, or a pharmaceutically acceptable salt thereof) effective to inhibit PAR4 and / or GPVI signaling. In addition, the pharmacological inhibition of 12-LOX by the compounds described herein can attenuate platelet activation, such as FcγRIIa-mediated platelet activation. Accordingly, the present disclosure also provides a method of inhibiting platelet activation in a cell, such as FcγRIIa-mediated platelet activation, which comprises contacting the cell with an amount of a compound disclosed herein (e.g., a compound of formula (I), a compound listed in Table A, or a pharmaceutically acceptable salt thereof) effective to inhibit platelet activation. In some embodiments, the contacting is in vivo. In various embodiments, the contacting is in vitro.

[0065] The compounds disclosed herein (e.g., a compound of formula (I), a compound listed in Table A, or a pharmaceutically acceptable salt thereof) can also prevent the release of platelet factor 4 (“PF4”) and the formation of PF4-heparin complexes in a cell. Accordingly, the present disclosure also provides a method of inhibiting PF4 release and / or PF4-heparin complex formation in a cell, which comprises contacting the cell with an amount of a compound disclosed herein (e.g., a compound of formula (I), a compound listed in Table A, or a pharmaceutically acceptable salt thereof) effective to inhibit PF4 release and / or PF4-heparin complex formation. In some embodiments, the contacting is in vivo. In various embodiments, the contacting is in vitro.

[0066] The present invention also provides administering to a subject in need thereof a therapeutically effective amount of a compound disclosed herein (e.g., a compound of formula (I), a compound listed in Table A, or a pharmaceutically acceptable salt thereof). The ability of the compounds disclosed herein to inhibit platelet activation, thrombocytopenia, and / or thrombosis in a subject in need thereof provides therapeutic efficacy in treating a variety of thrombotic disorders. Thrombotic disorders that can be particularly considered for treatment or prevention by administering the compounds disclosed herein 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"). The present invention further provides a method for preventing thrombosis and / or treating thrombocytopenia in a subject, which comprises administering to the subject a therapeutically effective amount of a compound disclosed herein (e.g., a compound of formula (I), a compound listed in Table A, or a pharmaceutically acceptable salt thereof), in an amount effective to prevent thrombosis and / or treat thrombocytopenia in the subject.

[0067] Further guidance on using the compounds disclosed herein to inhibit 12-LOX, such as a compound of formula (I), a compound listed in Table A, or a pharmaceutically acceptable salt thereof, can be found in the Examples section below.

[0068] Pharmaceutical formulations, dosages, and routes of administration

[0069] The methods provided herein include making and / or using a pharmaceutical composition, which comprises one or more of the compounds provided herein. The pharmaceutical composition itself is also included. The pharmaceutical composition generally comprises a pharmaceutically acceptable carrier. Accordingly, the present invention provides a pharmaceutical formulation, which comprises a compound described herein as previously described herein (e.g., a compound of formula (I), a compound listed in Table A, or a pharmaceutically acceptable salt thereof) and one or more pharmaceutically acceptable carriers.

[0070] The phrase "pharmaceutically acceptable" as used herein refers to those ligands, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0071] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. As used herein, the term "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with the administration of the drug. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) diols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions provided herein are pyrogen-free, i.e., do not induce a significant temperature increase when administered to a patient.

[0072] The term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic acid addition salts of the compounds provided herein. These salts can be prepared in situ during the final isolation and purification of the compounds provided herein, or by separately reacting the free base form of the compound with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurylsulfate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, dodecylsulfate, and amino acid salts, among others. See, e.g., Berge et al., (1977) "Pharmaceutical Salts", Journal of Pharmaceutical Sciences 66:1-19.

[0073] In some embodiments, the compounds provided herein may contain one or more acidic functional groups and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these instances, the term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic base addition salts of the compounds provided herein. These salts may equally be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth metal salts include salts of lithium, sodium, potassium, calcium, magnesium and aluminum, among others. Representative organic amines suitable for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc. (see, e.g., Berge et al., supra).

[0074] Wetting agents, emulsifying agents and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition.

[0075] As is well known in the art, the compositions prepared as described herein may be administered in various forms depending on the disorder to be treated and the age, condition and weight of the patient. For example, in the case of oral administration of the compositions, they may be formulated as tablets, capsules, granules, powders or syrups; or for parenteral administration, they may be formulated as injections (intravenous, intramuscular or subcutaneous), infusion preparations or suppositories. For administration by the ocular mucosal route, they may be formulated as eye drops or eye ointments. These formulations may be prepared by conventional means in combination with the methods described herein, and if desired, the active ingredient may be mixed with any conventional additives or excipients, such as binders, disintegrants, lubricants, correctives, solubilizers, suspending agents, emulsifying agents or coating agents.

[0076] The actual dosage level of the active ingredient in the pharmaceutical compositions provided herein may be varied so as to obtain a "therapeutically effective amount", which is the amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition and mode of administration and is non-toxic to the patient.

[0077] The concentration of the compounds provided herein in a pharmaceutically acceptable mixture will vary depending on several factors, including the dose of the compound to be administered, the pharmacokinetic properties of the compound(s) employed, and the route of administration. In some embodiments, the compositions provided herein may be provided as an aqueous solution containing from about 0.1 - 10% w / v of the compounds disclosed herein and other substances, for parenteral administration. Typical dosage ranges may include from about 0.01 to about 50 mg / kg body weight per day, given in 1 - 4 divided doses. Each divided dose may contain the same or different compounds. The dose will be a therapeutically effective amount depending on several factors, including the overall health of the patient and the formulation and route of administration of the compound(s) selected.

[0078] Dosage forms or compositions containing the compounds as described herein in the range of 0.005% to 100% may be prepared, with the remainder consisting of a non-toxic carrier. The methods for preparing these compositions are known to those skilled in the art. The expected compositions may contain from 0.001% - 100% active ingredient, in one embodiment, 0.1% - 95%, and in another embodiment, 75% - 85%. Although the dose will vary depending on the patient's symptoms, age, and weight, the nature and severity of the condition to be treated or prevented, the route of administration, and the form of the drug, generally, a daily dose of 0.01 to 2000 mg of the compound is recommended for adult patients, and this may be administered as a single dose or in divided doses. The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will generally be the amount of the compound that produces a therapeutic effect.

[0079] In jurisdictions where methods of treating humans are not patentable, the meaning of "administering" a composition to a human subject shall be limited to prescribing a controlled substance that a human subject can self-administer by any technique (e.g., orally, by inhalation, topically, by injection, by insertion, etc.). The broadest reasonable interpretation consistent with the laws or regulations defining patentable subject matter. In jurisdictions where methods of treating humans are not prohibited from being patented, "administration" of a composition includes both methods of treating humans and the foregoing activities.

[0080] Other embodiments

[0081] It should be understood that although the present disclosure has been described in connection with its specific description, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0082] Examples

[0083] The following examples are provided for illustration and are not intended to limit the scope of the invention.

[0084] Scheme 1. General scheme for m - chloro compounds

[0085]

[0086] Scheme 2. General protocol for other alternative modes

[0087]

[0088] Synthesis of N-(3 - chlorophenyl)-4-((2 - hydroxy - 3 - methoxybenzyl)amino)benzenesulfonamide (A1)

[0089]

[0090] A solution of 4-amino-N-(3-chlorophenyl)benzenesulfonamide (2A) (Zheng et al., Biorganic and Medicinal Chemistry 2007, 15, 1014 - 1021) (0.20 g, 0.71 mmol) in ethanol (5 mL) was treated with o-vanillin (0.14 g, 0.92 mmol). The resulting suspension was refluxed for 8 h. The mixture was cooled to room temperature, carefully treated with sodium borohydride (67 mg, 1.8 mmol), and stirred overnight at room temperature. The mixture was treated with methanol and water and stirred for 1.5 h. The solid was filtered, washed thoroughly with ethanol and dichloromethane, and the filtrate was concentrated to give an oil. The residue was purified by silica gel chromatography, eluting with a 5% methanol / dichloromethane solution to give 0.071 g of the title compound. 1 H NMR (400 MHz, methanol-d4) δ 7.47 - 7.38 (m, 2H), 7.18 - 7.04 (m, 2H), 6.97 (dddd, J = 7.8, 5.7, 2.1, 1.0 Hz, 2H), 6.78 (ddd, J = 14.1, 7.8, 1.7 Hz, 2H), 6.69 (t, J = 7.8 Hz, 1H), 6.60 - 6.49 (m, 2H), 4.30 (s, 2H), 3.83 (s, 3H). HRMS: Measured, m / z = 419.0825 (predicted m / z = 419.0827). HPLC: 93.6% (retention time, 6.89 min at 250 nm wavelength. Mobile phase: 90:10 water:acetonitrile → 90:10 acetonitrile:water over 13 min, adding 0.1% TFA to water and acetonitrile).

[0091] Synthesis of 4-((2 - hydroxy - 3 - methoxybenzyl)amino)-N-(3-(trifluoromethyl)phenyl)benzenesulfonamide (A2)

[0092]

[0093] A mixture of 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide (5A) (Luci et al., Journal of Medicinal Chemistry 2014, 57(2), 495 - 506) (0.22 g, 0.73 mmol), 1-bromo-3-(trifluoromethyl)benzene (0.20 g, 0.88 mmol), N,N-dimethylethylenediamine (0.032 g, 0.37 mmol), potassium carbonate (0.25 g, 1.8 mmol), copper(I) iodide (7.0 mg, 0.037 mmol) and acetonitrile (7 mL) was degassed with nitrogen in a sealed tube (for 15 minutes), and then heated to 70 °C for 24 hours. The mixture was cooled to room temperature and treated with saturated aqueous ammonium chloride (40 mL). The dark blue solution was extracted with ethyl acetate until the organic layer was clear (3 × 30 mL). The combined organic extracts were washed with water (25 mL) and brine (25 mL), dried over magnesium sulfate, and concentrated to give an oil. The crude product was purified by silica gel chromatography, eluting with 4% methanol / dichloromethane solution to give 0.117 g of the title compound. 1 1H NMR (400 MHz, methanol-d4) δ 7.50 - 7.40 (m, 2H), 7.40 - 7.21 (m, 4H), 6.82 - 6.73 (m, 2H), 6.67 (t, J = 7.8 Hz, 1H), 6.61 - 6.49 (m, 2H), 4.29 (s, 2H), 3.81 (s, 3H). HPLC: 100% (retention time, 6.97 minutes at 250 nm wavelength. Mobile phase: 90:10 water:acetonitrile → 90:10 acetonitrile:water over 13 minutes, adding 0.1% TFA to water and ACN). HRMS: measured m / z = 453.1089 [M + H]+ (predicted m / z = 453.1090).

[0094] Synthesis of N-(3 - chloro - 2 - fluorophenyl)-4-((2 - hydroxy - 3 - methoxybenzyl)amino)benzenesulfonamide (A3)

[0095]

[0096] Treat a solution of 3-chloro-2-fluoroaniline (0.50 g, 3.4 mmol) in pyridine (2 mL) with 4-nitrobenzenesulfonyl chloride (0.84 g, 3.8 mmol) in 3 equal portions. Heat the resulting mixture at 100 °C for 3 h and then cool to room temperature. Acidify the reaction mixture with 2N HCl and extract with ethyl acetate (3 × 25 mL). Wash the combined extracts with water and brine, dry over magnesium sulfate, and concentrate to give an oil. Dissolve the residue in hexanes and concentrate again to give an off-white powder, which is suspended in 1:10 ethyl acetate:hexanes and triturated. Filter off the solid, wash well with hexanes, and air dry to give 0.99 g of N-(3-chloro-2-fluorophenyl)-4-nitrobenzenesulfonamide (1B). 1 H NMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H), 8.44 - 8.34 (m, 2H), 8.03 - 7.94 (m, 2H), 7.42 (td, J = 7.4, 6.9, 2.3 Hz, 1H), 7.26 - 7.08 (m, 2H).

[0097] Treat a solution of N-(3-chloro-2-fluorophenyl)-4-nitrobenzenesulfonamide ( 1B )(0.95 g, 2.9 mmol) in THF (10 mL) with tin (1.2 g, 10 mmol) and concentrated HCl (4.3 mL) in this order; add the HCl gradually over 4 min. Reflux the resulting suspension for 2.5 h and then cool to room temperature. Add 2N NaOH dropwise to make the mixture basic. Filter the resulting suspension through celite; wash the celite cake well with water and ethyl acetate. Transfer the filtrate to a separatory funnel, separate the aqueous layer therein, and extract again with ethyl acetate (50 mL). Combine the organic extracts, wash with water (50 mL) and brine (50 mL), and dry over magnesium sulfate. Concentrate to give a semi-solid, which is triturated with ether to give 0.39 g of 4-amino-N-(3-chloro-2-fluorophenyl)benzenesulfonamide ( 2B ) as a solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H), 7.34 (d, J = 8.6 Hz, 2H), 7.28 (td, J = 7.4, 6.6, 1.6 Hz, 1H), 7.19 (td, J = 7.7, 7.1, 1.7 Hz, 1H), 7.10 (td, J = 8.1, 1.4 Hz, 1H), 6.57 - 6.48 (m, 2H), 6.02 (s, 2H).

[0098] Treat 4-amino-N-(3-chloro-2-fluorophenyl)benzenesulfonamide ( 2B(0.19 g, 0.63 mmol) in ethanol (5 mL). The resulting suspension was refluxed for 24 h and then cooled to room temperature. The reaction mixture was carefully treated with sodium borohydride (0.071 g, 1.9 mmol), stirred overnight at room temperature, treated with methanol and water, and stirred for 30 min. The solid was filtered through celite and the celite cake was washed thoroughly with ethanol and dichloromethane. The filtrate was concentrated to give an oil. The residue was purified by silica gel chromatography, eluting with 5% methanol / dichloromethane solution to give 0.101 g of the title compound. 1 H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H), 8.72 (s, 1H), 7.37 (d, J = 8.8 Hz, 2H), 7.26 (t, J = 7.3 Hz, 1H), 7.18 (t, J = 7.3 Hz, 1H), 7.08 (dd, J = 8.8, 7.5 Hz, 1H), 6.95 (t, J = 5.8 Hz, 1H), 6.87 - 6.79 (m, 1H), 6.76 - 6.65 (m, 2H), 6.56 (d, J = 8.9 Hz, 2H), 4.20 (d, J = 5.7 Hz, 2H), 3.77 (s, 3H). HRMS: Measured, m / z for [M + H]+ = 437.0736 (predicted m / z = 437.0733). HPLC: 94.8% (retention time, 6.86 min at 250 nm wavelength. Mobile phase: 90:10 water:acetonitrile → 90:10 acetonitrile:water over 13 min, adding 0.1% TFA to water and acetonitrile.)

[0099] 4-((2 - hydroxy - 3 - methoxybenzyl)amino)-N-(3-(1-(trifluoromethyl)cyclopropyl)phenyl)benzenesulfonamide (A4) synthesis

[0100]

[0101] 4 - ((2 - Hydroxy - 3 - methoxybenzyl)amino)benzenesulfonamide 5A(Luci et al., Journal of Medicinal Chemistry 2014, 57(2), 495 - 506) (0.40 g, 1.3 mmol), 1 - bromo - 3-(1-(trifluoromethyl)cyclopropyl)benzene (0.41 g, 1.6 mmol), N,N - dimethylethylenediamine (0.057 g, 0.65 mmol), potassium carbonate (0.45 g, 3.2 mmol), copper(I) iodide (12 mg, 0.065 mmol), and dioxane (5 mL) were degassed with nitrogen in a sealed tube (for 10 minutes), and then heated at 80 °C for 2 days. The mixture was cooled to room temperature and treated with saturated aqueous ammonium chloride (50 mL). The dark solution was extracted with dichloromethane (4 × 40 mL). The combined organic extracts were washed with water (50 mL) and brine (50 mL), dried over magnesium sulfate, and concentrated to give an oil. The crude product was purified by silica gel chromatography, eluting with 4% methanol / dichloromethane solution to give 0.091 g of the title compound. 1 1H NMR (400 MHz, methanol - d4) δ 7.44 - 7.34 (m, 2H), 7.22 - 7.10 (m, 3H), 7.02 (ddd, J = 7.8, 2.2, 1.3 Hz, 1H), 6.79 (ddd, J = 18.8, 7.9, 1.6 Hz, 2H), 6.70 (t, J = 7.8 Hz, 1H), 6.59 - 6.51 (m, 2H), 4.31 (s, 2H), 3.84 (s, 3H), 1.31 - 1.23 (m, 2H), 0.94 - 0.83 (m, 2H). Trace amounts of diethyl ether. HPLC: 98.9% (retention time, 7.39 minutes at 250 nm. Mobile phase: 90:10 water:acetonitrile → 90:10 acetonitrile:water over 13 minutes, with 0.1% TFA added to water and acetonitrile.) HRMS: Measured, m / z for [M + H]+ = 493.1402 (predicted m / z = 493.1403).

[0102] Synthesis of N-(3 - chloro - 4 - fluorophenyl)-4-((2 - hydroxy - 3 - methoxybenzyl)amino)benzenesulfonamide (A5)

[0103]

[0104] A solution of 3-chloro-4-fluoroaniline (0.50 g, 3.4 mmol) in pyridine (1.7 mL) was treated with 4-nitrobenzenesulfonyl chloride (0.84 g, 3.8 mmol) in three equal portions. The resulting mixture was heated at 100 °C for 3.5 h. The solution was then cooled to room temperature, acidified with 2 N HCl and extracted with ethyl acetate (2 × 25 mL). The combined extracts were washed with water and brine, dried over magnesium sulfate and concentrated to give a solid. The material was triturated with ethyl acetate:hexane 1:10, filtered and air-dried to give 1.0 g of N-(3-chloro-4-fluorophenyl)-4-nitrobenzenesulfonamide (3). 1 H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.43 - 8.31 (m, 2H), 8.04 - 7.89 (m, 2H), 7.31 (t, J = 9.0 Hz, 1H), 7.23 (dd, J = 6.5, 2.6 Hz, 1H), 7.05 (ddd, J = 8.8, 4.1, 2.6 Hz, 1H).

[0105] A solution of N-(3-chloro-4-fluorophenyl)-4-nitrobenzenesulfonamide (3) (1.0 g, 3.0 mmol) in THF (10 mL) was treated with tin (1.3 g, 11 mmol) and concentrated HCl (4.5 mL) in that order; the HCl was added gradually over 4.5 min. The resulting suspension was refluxed for 3.5 h and then stirred overnight at room temperature. 2 N NaOH was added dropwise to render the mixture basic and the thick suspension formed was filtered through diatomaceous earth. The diatomaceous earth cake was washed thoroughly with ethyl acetate and the filtrate was transferred to a separatory funnel; the aqueous layer was separated and the mixture was extracted again with ethyl acetate (3 × 30 mL). The combined organic layers were washed with water (2 × 50 mL) and brine (50 mL), dried over magnesium sulfate. Concentration gave an oil which solidified on standing. The crude product was purified by silica gel chromatography, eluting with hexane:ethyl acetate 1:1 to give 0.45 g of 4-amino-N-(3-chloro-4-fluorophenyl)benzenesulfonamide (4). 1 H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 7.40 - 7.33 (m, 2H), 7.27 (t, J = 9.1 Hz, 1H), 7.14 (dd, J = 6.6, 2.7 Hz, 1H), 7.01 (ddd, J = 8.9, 4.2, 2.7 Hz, 1H), 6.56 - 6.49 (m, 2H), 6.02 (s, 2H).

[0106] 4-Amino-N-(3-chloro-4-fluorophenyl)benzenesulfonamide was treated with o-vanillin (0.17 g, 1.1 mmol). 4(0.23 g, 0.75 mmol) in ethanol (5 mL); the resulting suspension was refluxed for 24 h. The suspension was cooled to room temperature, treated carefully with sodium borohydride (0.085 g, 2.3 mmol), and stirred overnight at room temperature. The mixture was treated with methanol and water and stirred for 30 min. The solid was filtered through celite, and the celite cake was washed thoroughly with ethanol and dichloromethane. The filtrate was concentrated to give an oil. The product was purified by silica gel chromatography, eluting with 2% methanol / dichloromethane solution to give 0.93 g of the title compound. 1 1H NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.76 (s, 1H), 7.39 (s, 2H), 7.26 (t, J = 9.1 Hz, 1H), 7.14 (dd, J = 6.6, 2.6 Hz, 1H), 7.08 - 6.91 (m, 2H), 6.83 (dd, J = 7.6, 1.9 Hz, 1H), 6.76 - 6.60 (m, 2H), 6.56 (d, J = 8.7 Hz, 2H), 4.19 (d, J = 5.8 Hz, 2H), 3.77 (s, 3H). HPLC: 98.3% (retention time, 7.13 min at 250 nm wavelength. Mobile phase: 90:10 water:acetonitrile → 90:10 acetonitrile:water over 13 min while adding 0.1% TFA to both water and ACN). HRMS: measured m / z for [M + H]+ = 437.0728 (predicted m / z = 437.0733).

[0107] 4-((2 - hydroxy - 3-(trifluoromethyl)benzyl)amino)-N-(3-(trifluoromethyl)phenyl)benzenesulfonamide (A6) Synthesis

[0108]

[0109] A solution of 4-aminobenzenesulfonamide (0.40 g, 2.3 mmol), 2-hydroxy-3-(trifluoromethyl)benzaldehyde (Filippova et al., Tetrahedron 2016, 72(41), 6572 - 6577) (0.51 g, 2.7 mmol) and ethanol (10 mL) was refluxed overnight and then cooled to room temperature. The resulting suspension was treated with sodium borohydride (0.13 g, 3.5 mmol) and stirred overnight at room temperature. The solution was concentrated and the residue was partitioned between water and dichloromethane. The aqueous layer was separated and extracted with ethyl acetate (3 × 25 mL). The combined organic extracts were dried over magnesium sulfate and concentrated. The material was purified by silica gel chromatography, eluting with 5% methanol / dichloromethane solution to give 0.25 g of 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide ( 5B ). 11H NMR (400 MHz, methanol-d4) δ 7.66 - 7.58 (m, 2H), 7.43 (dd, J = 7.3, 5.5 Hz, 2H), 6.93 (t, J = 7.7 Hz, 1H), 6.70 - 6.65 (m, 2H), 4.43 (s, 2H).

[0110] 4-((2-Hydroxy-3-methoxybenzyl)amino)benzenesulfonamide ( 5B )(0.12 g, 0.36 mmol), 1-bromo-3-(trifluoromethyl)benzene (0.097 g, 0.43 mmol), N,N-dimethylethylenediamine (16 mg, 0.18 mmol), potassium carbonate (0.13 g, 0.90 mmol), copper(I) iodide (3 mg, 0.018 mmol) and acetonitrile (5 mL) were degassed with nitrogen in a sealed tube (for 10 minutes), then heated to 70 °C for 24 hours. The mixture was cooled to room temperature and treated with saturated aqueous ammonium chloride (30 mL). The dark blue solution was extracted with ethyl acetate until the organic layer was clear (3 × 30 mL). The combined organic extracts were washed with water (25 mL) and brine (25 mL), dried over magnesium sulfate and concentrated to give an oil. The crude product was purified by preparative thin layer chromatography (prep TLC), eluting with 4% methanol / dichloromethane solution to give 0.060 g of the title compound. 1 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.59 (s, 1H), 7.44 (dd, J = 12.3, 8.3 Hz, 4H), 7.31 (d, J = 13.6 Hz, 4H), 7.01 (s, 1H), 6.92 (t, J = 7.8 Hz, 1H), 6.58 (d, J = 8.7 Hz, 2H), 4.31 (d, J = 5.4 Hz, 2H). HPLC: 94.2% (retention time, 7.54 minutes at 250 nm. Mobile phase: 90:10 water:acetonitrile → 90:10 acetonitrile:water over 13 minutes, adding 0.1% TFA to water and ACN). HRMS: measured m / z = 491.0859 [M+H]+ (predicted m / z = 491.0859).

[0111] 4-((2 - hydroxy - 3 - methoxybenzyl)amino)-N-(1 - methyl - 1H - indol - 2 - yl)benzenesulfonamide (A7) synthesis Synthesis

[0112]

[0113] 4-((2-Hydroxy-3-methoxybenzyl)amino)benzenesulfonamide ( 5A ) 2(Luci et al., Journal of Medicinal Chemistry 2014, 57(2), 495 - 506) (0.40 g, 1.3 mmol) (0.40 g, 1.3 mmol), a mixture of 2 - bromo - 1 - methylbenzimidazole (0.33 g, 1.6 mmol), N,N - dimethylethylenediamine (0.057 g, 0.65 mmol), potassium carbonate (0.45 g, 3.2 mmol), copper(I) iodide (12 mg, 0.065 mmol) and dioxane (5 mL) was degassed with nitrogen in a sealed tube (for 10 minutes), and then heated to 80 °C for 3 days. The mixture was cooled to room temperature and treated with saturated aqueous ammonium chloride solution (50 mL). The solution was extracted with dichloromethane (5 × 40 mL). The combined organic extracts were washed with water (50 mL) and brine (50 mL), dried over magnesium sulfate and concentrated. The residue was triturated with ethyl acetate and the formed solid was filtered and washed thoroughly with ethyl acetate. The filtrate was concentrated and the residue was purified by silica gel chromatography, eluting with 5% methanol / dichloromethane solution. Yield: 0.11 g of the title compound. 1 1H NMR (400 MHz, DMSO - d6) δ 11.61 (s, 1H), 8.73 (s, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.42 - 7.29 (m, 2H), 7.14 (ddd, J = 7.3, 5.2, 1.5 Hz, 2H), 6.86 - 6.77 (m, 1H), 6.74 (dd, J = 9.0, 7.0 Hz, 2H), 6.66 (t, J = 7.8 Hz, 1H), 6.54 (d, J = 8.6 Hz, 2H), 4.20 (d, J = 5.8 Hz, 2H), 3.76 (s, 3H), 3.38 (s, 3H). HRMS: Measured m / z for [M + H]+ = 439.1437 (predicted m / z = 439.1435).

[0114] Pharmacokinetic study of the inhibitor in mouse plasma

[0115] After oral (\"PO\") and intravenous (\"IV\") administration of the compound, the plasma concentrations of compounds A1, A2 and A4 in mice were determined.

[0116] Specificity. Chromatograms of blank plasma and blank plasma / spiked with internal standard (CE302) indicated that blank plasma did not significantly interfere with the determination of compounds A1, A2 and A4 and IS.

[0117] Calibration curves. The concentration ranges were evaluated as follows, 2.5 - 5000 ng / mL for compound A2, 1 - 5000 ng / mL for compound A4, and 5 - 5000 ng / mL for compound A1. Curves were constructed using weighted (1 / X2) linear regression. Linear regression analysis was performed by plotting the peak area ratio (y) against the concentration (x) in ng / mL. The linearity of the relationship between the peak area ratio and the concentration was demonstrated by the correlation coefficient (R) obtained from the linear regression.

[0118] Instrument conditions. The LC - MS and mass spectrometry analysis conditions for the tested compounds are shown below.

[0119]

[0120] Results. The individual and mean compound A1, A2, and A4 concentration - time data for the IV and PO dosing groups are listed in Table 1 and presented graphically in Figure 1 .

[0121] Table 1. Concentrations of compounds A1, A2, and A4 in mouse plasma at 2, 4, and 7 hours after PO and IV administration.

[0122]

[0123]

[0124] *Outlier

[0125]

[0126] *Outlier

[0127] Preparation of washed human platelets

[0128] Citrated whole blood was centrifuged (200 g for 10 minutes) to separate platelet - rich plasma. Platelet - rich plasma was treated with acid - citrate - dextrose (2.5% sodium citrate, 1.5% citric acid, 2.0% D - glucose) and adenosine triphosphate diphosphatase (0.02 U / mL) and then centrifuged (2000 g for 10 minutes) to pellet the platelets. Unless otherwise stated, platelets were resuspended at 3.0×10 8 platelets / mL in Tyrode’s buffer (10 mM HEPES, 12 mM NaHCO3, 127 mM NaCl, 5 mM KCl, 0.5 mM NaH2PO4, 1 mM MgCl2, and 5 mM glucose).

[0129] Platelet aggregation

[0130] The washed human platelets were prepared at 3×10 8 platelets / ml and aggregation was measured in a 4-channel Lumi aggregometer (Chonolog Inc, model 700D) under stirring conditions at 1100 RPM at 37°C. The platelets were incubated with the compounds described herein at increasing concentrations (1 μM to 20 μM) for 10 minutes, and platelet aggregation was induced by EC 80 concentrations of thrombin or collagen. Each condition was repeated with platelets from 5 independent volunteers (N = 5). Aggregation inhibition was considered statistically significant if aggregation was significantly reduced compared to the ML355-treated condition. *, P <.05; **, P <.01; ***, P <.001; ****, P <.0001. See Figure 2 and Figure 3 .

[0131] In vivo pharmacokinetics after oral administration in mice

[0132] Compound A1 (30 mg / kg) was administered orally to mice, and the plasma drug concentration in the mice (n = 3) was monitored at 8 time points (0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours) and the plasma drug concentration in the mice was evaluated by PK analysis as described above.

[0133] Compound pretreatment of experimental mice for in vivo studies

[0134] C57BL / 6 wild-type (WT) control mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and housed in the research facility of the University of Michigan. Compound A1 was synthesized and formulated specifically in polyethylene glycol 300 (PEG 300) for oral gavage in mice for in vivo thrombosis and hemostasis studies. For the laser-induced cremaster arteriole thrombosis model, mice were treated with compound A1 (3 mg / kg) or with PEG 300 by oral administration twice a day for 2 days, and then intravital microscopy studies were performed on the third day.

[0135] Laser - induced cremaster arteriole thrombosis model

[0136] Adult mice (10 - 12 weeks old) were anesthetized as described above, surgically prepared as detailed, and an endotracheal tube was inserted to facilitate respiration. Throughout the experiment, the cremaster muscle was prepared and perfused with pre-warmed bicarbonate-buffered saline. DyLight 488-conjugated rat anti-mouse platelet GP1bβ antibody (0.1 μg / g; EMFRET Analytics) and AlexaFluor 647-conjugated anti-fibrin (0.3 μg / g) or Alexa Flour 647 rat anti-mouse CD62P (3 μg / mouse) were administered via jugular vein cannulation prior to vascular injury. Multiple independent thrombi were induced in the arterioles (30 - 50 μm in diameter) of each mouse by a laser ablation system (Ablate! Photoablation System; Intelligent Imaging Innovations, Denver, CO, USA). Images of thrombus formation at the site of the injured arteriole were acquired in real time using a Zeiss AxioExaminer Z1 fluorescence microscope equipped with a solid-state laser emission system (LaserStack; Intelligent Imaging Innovations) and a high-speed sCMOS camera under a 63x water immersion objective. Using the Slidebook program, changes in fluorescence intensity during thrombus formation were analyzed in all captured images after subtracting the fluorescence background defined on the uninjured part of the blood vessel.

[0137] Representative images of platelet accumulation (green) and fibrin formation (red) in thrombi forming in cremaster arterioles of wild-type ("WT") control animals treated with polyethylene glycol ("PEG"; control, top) and WT treated with compound A1 (3 mg / kg, twice daily for 2 days; bottom) are shown in Figure 4 Figure.

[0138] Anti - GPIX - induced HITT pulmonary thrombosis model

[0139] Adult transgenic mice (10 - 12 weeks old) expressing the human FcγRIIa receptor on their platelets were anesthetized as described above, and 15 mg / kg of A1 or control vehicle was administered by oral gavage 30 minutes prior to an IV bolus of 0.75 mg / kg anti-GPIX to induce activation of the FcγRIIa receptor on platelets, thus mimicking heparin-induced thrombocytopenia and thrombosis ("HITT") in mice. The anti-GPIX antibody was conjugated with DyLight 488 to observe its binding to platelets and platelet accumulation in the lung. Four hours after anti-GPIX induction, the animals were sacrificed and the lungs were observed using a Li-COR Odyssey imager.

[0140] Representative images of platelet accumulation (green) in the lungs of mice from control (PEG vehicle) and A1-treated (15 mg / kg) mice are shown in Figure 5 in

[0141] The foregoing description is given only for a clear understanding, and should not be construed as an unnecessary limitation therefrom, as modifications within the scope of the invention may be apparent to those of ordinary skill in the art.

[0142] Throughout this specification and the appended claims, unless the context otherwise requires, the word "comprise" and variations thereof (such as "comprises" or "comprising") shall be understood to imply the inclusion of the stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0143] Throughout the specification, unless otherwise described, when a composition is described as including components or materials, it is contemplated that the composition may also consist essentially of or consist of any combination of the stated components or materials. Similarly, unless otherwise described, when a method is described as including specific steps, it is contemplated that the method may also consist essentially of or consist of any combination of the stated steps. In the absence of any elements or steps not specifically disclosed herein, the invention illustratively disclosed herein may be practiced appropriately.

[0144] The practice of the methods and their individual steps disclosed herein can be performed manually and / or with the aid of an electronic device or automation provided by an electronic device. Although various methods have been described with reference to specific embodiments, those of ordinary skill in the art will readily appreciate that other modes of implementation associated with the methods can be used. For example, unless otherwise described, the order of the various steps can be changed without departing from the scope or spirit of the method. Additionally, some of the individual steps can be combined, omitted, or further subdivided into additional steps.

[0145] All patents, publications, and references cited herein are hereby incorporated by reference in their entirety. In the event of a conflict between this disclosure and the incorporated patents, publications, and references, this disclosure shall govern.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: Wherein: R 2 is a halogen group, C 1-3 fluoroalkyl or C 3-8 fluorocycloalkyl; R 1 is H or a halogen group; R 3 is H or a halogen group; R 4 is H; R 5 is H; R a and R b are independently OH or OC 1-3 alkyl; and R c 、R d 、R e Each of them is H.

2. The compound or salt according to claim 1, wherein R 2 is a halogen group.

3. The compound or salt according to claim 2, wherein R 2 is Cl or F.

4. The compound or salt according to claim 3, wherein R 2 is Cl.

5. The compound or salt according to claim 1, wherein R 2 is C 1-3 fluoroalkyl or C 3-8 fluorocycloalkyl.

6. The compound or salt according to claim 5, wherein R 2 is CF3 or 7. The compound or salt according to claim 1, wherein each of R 1 , R 3 , R 4 and R 5 is H.

8. The compound or salt according to claim 1, wherein one or both of R 1 and R 3 are halogen groups.

9. The compound or salt according to claim 8, wherein one or both of R 1 and R 3 is or are F or Cl.

10. The compound or salt according to claim 1, wherein R 1 is H or F; R 2 is Cl, CF3 or R 3 is H or F; R 4 is H; and R 5 is H.

11. The compound or salt according to claim 1, wherein OC 1-3 alkyl is OCH3.

12. A compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound or salt according to any one of claims 1 to 12.

14. Use of a compound or salt according to any one of claims 1 to 12 in the manufacture of a medicament for inhibiting the activation of (S)-lipoxygenase in cells.

15. Use of a compound or salt according to any one of claims 1 to 12 in the manufacture of a medicament for inhibiting the release of platelet factor 4 in cells.

16. Use of a compound or salt according to any one of claims 1 to 12 in the manufacture of a medicament for inhibiting the formation of PF4-heparin complexes in cells.

17. Use of a compound or salt according to any one of claims 1 to 12 in the manufacture of a medicament for inhibiting platelet activation in cells.

18. Use according to claim 17, wherein the platelet activation is FcγRIIa-mediated platelet activation.

19. Use of a compound or salt according to any one of claims 1 to 12 in the manufacture of a medicament for inhibiting thrombin, protease-activated receptor-4 and / or glycoprotein VI signaling in cells.

20. Use of a compound or salt according to any one of claims 1 to 12 in the manufacture of a medicament for treating a thrombotic disorder in a subject in need thereof.

21. Use according to claim 20, wherein the thrombotic disorder is selected from arterial thrombosis, deep vein thrombosis, pulmonary embolism, ischemic stroke, immune thrombocytopenia, heparin-induced thrombocytopenia, and heparin-induced thrombocytopenia and thrombosis.

22. Use of a compound or salt according to any one of claims 1 to 12 in the manufacture of a medicament for preventing thrombosis in a subject.

23. Use of a compound or salt according to any one of claims 1 to 12 in the manufacture of a medicament for treating thrombocytopenia in a subject in need thereof.

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