Plasminogen activator inhibitor-1 (PAI-1) inhibitor and method of use

Abstract

To provide plasminogen activator-1 (PAI-1) inhibitor compounds and uses thereof in the treatment of any disease or disorder associated with elevated PAI-1.SOLUTION: The disclosure includes, but is not limited to, the use of such compounds to prevent or reduce thrombosis and fibrosis, to promote thrombolysis, and to modulate lipid metabolism and treat diseases or disorders associated with elevated PAI-1, cholesterol, or lipid levels.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Government Support Statement This invention was made with government support under HL089407 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Provided herein are compounds and methods for modulating plasminogen activator inhibitor-1 (PAI-1) activity. More specifically, the disclosure relates to inhibitors of PAI-1 and the use of such inhibitors in modulating PAI-1 activity. The use of these inhibitors for the treatment of a number of diseases or disorders associated with PAI-1 activity is also provided. Such diseases or disorders include, but are not limited to, dysregulated lipid metabolism, obesity, diabetes, polycystic ovary syndrome, estrogen deficiency-induced bone loss, fibrosis and fibrotic diseases, inflammation, cell migration and migration-driven proliferation, angiogenesis, and thrombosis. Such inhibitors are also believed to be useful in modulating endogenous fibrinolysis and in conjunction with pharmacological thrombolysis. [Background technology]

[0003] Plasminogen activator inhibitor-1 (PAI-1) is a 50 kDa single-chain glycoprotein that is the major inhibitor of both urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA). PAI-1 exhibits a second-order rate constant of approximately 10, which is 10-1000 times faster than the rate of PA inhibition by other PAIs. 7 M -1 s -1It inhibits tPA and uPA. Furthermore, approximately 70% of total tPA in carefully collected normal human plasma is detected in complex with PAI-1, suggesting that inhibition of tPA by PAI-1 is a normal, ongoing process. PAI-1 can also directly inhibit plasmin. Thus, PAI-1 appears to be a major regulator of plasmin generation in vivo and, as such, plays an important role in both fibrotic and thrombotic diseases. PAI-1 has three potential N-linked glycosylation sites and contains 15–20% carbohydrate.

[0004] PAI-1 belongs to the serine protease inhibitor superfamily (serpins), a gene family that includes many protease inhibitors found in blood and other proteins with unrelated or unknown functions. Serpins are consumed during the inactivation process of proteases and function as "suicide inhibitors." Association of serpins with their target proteases occurs at amino acid residues called "bait" residues, located in a surface loop of the serpin called the active center loop (RCL). The "bait" residue, also known as the P1 residue, is thought to mimic the enzyme's normal substrate. When the P1 residue interacts with the S1 site of the target protease, cleavage of the RCL occurs. This is coupled to a large conformational change in the serpin, involving rapid insertion of the RCL into β-sheet A, a key structural feature of serpins. This allows the protease to tightly dock on the serpin surface, resulting in distortion of the enzyme's structure, including its active site. The insertion of the RCL also significantly increases the structural stability of the serpin, rigidifying the complex and trapping the protease in a covalent acyl-enzyme complex with the serpin.

[0005] Native PAI-1 exists in at least two distinct conformations: an active form that is produced and secreted by cells, and an inactive or latent form that accumulates in the cell culture medium over time. In blood and tissues, most PAI-1 is active; however, both active and latent forms of PAI-1 are found in platelets. In active PAI-1, the RCL is exposed on the surface of the molecule; upon reaction with proteases, the cleaved RCL is integrated into the center of β-sheet A. In the latent form, the RCL is intact, but instead of being exposed, the entire amino-terminal end of the RCL is inserted into β-sheet A as a central strand. This explains the increased stability of latent PAI-1 and its lack of inhibitory activity.

[0006] Active PAI-1 spontaneously converts to a latent form with a half-life of 1–2 hours at 37°C, and treatment with denaturants can revert latent PAI-1 to its active form. Negatively charged phospholipids can also convert latent PAI-1 to its active form, suggesting that the cell surface can regulate PAI-1 activity. The observation that latent PAI-1 injected into rabbits is clearly converted to its active form is consistent with this hypothesis. The spontaneous, reversible interconversion between the active and latent conformations is unique to PAI-1 and distinguishes it from other serpins, but the biological significance of the latent conformation remains unknown.

[0007] Other non-inhibitory forms of PAI-1 have also been identified. The first form results from the oxidation of one or more critical methionine residues within active PAI-1. This form differs from latent PAI-1 in that it can be partially reactivated by enzymes that specifically reduce the oxidized methionine residues. Oxidative inactivation of PAI-1 may be an additional mechanism of PAI-1 regulation; oxygen radicals generated locally by neutrophils or other cells may inactivate PAI-1, thus promoting plasmin generation at sites of infection or tissue remodeling. PAI-1 also exists in two distinct cleaved forms. As noted above, PAI-1 complexed with proteases is cleaved at its RCL. Uncomplexed PAI-1 can also be found in a state where its RCL is cleaved, resulting from dissociation of the PAI-1-PA complex or cleavage of the RCL by non-target proteases at sites other than P1. Neither of these forms of PAI-1 can inhibit protease activity; however, they can interact with other ligands.

[0008] Interactions between PAI-1 and non-protease ligands play an essential role in PAI-1 function. PAI-1 binds with high affinity to heparin, the cell adhesion protein vitronectin, and members of the endocytic low-density lipoprotein receptor (LDL-R) family, including lipoprotein receptor-related protein (LRP) and the very-low-density lipoprotein receptor (VLDL-R). These non-protease interactions are important for both PAI-1 localization and function, and they are primarily conformationally controlled through structural changes associated with RCL insertion. In the blood, most active PAI-1 circulates complexed with the glycoprotein vitronectin. The PAI-1-binding site on vitronectin is located in the region at the end of beta-sheet A of the PAI-1 structure. Binding sites for LDL-R family members have been identified, although less well characterized, in PAI-1 regions associated with alpha helix D adjacent to the vitronectin-binding domain. The heparin-binding domain of PAI-1 has also been mapped. This site is also located in alpha helix D of a region homologous to the heparin-binding domain of antithrombin III and may overlap with the binding site of LDL-R family members.

[0009] Vitronectin circulates in plasma and is present in the extracellular matrix primarily at sites of injury or remodeling. PAI-1 and vitronectin appear to have an important functional interdependence. Vitronectin stabilizes PAI-1 in its active conformation, thereby prolonging its biological half-life.

[0010] Vitronectin enhances the efficacy of thrombin in inhibiting PAI-1 by approximately 300-fold. Similarly, binding of PAI-1 to vitronectin changes its conformation from its native plasma form, which does not support cell adhesion, to an "activated" form capable of binding integrins. However, integrin binding is blocked by the presence of PAI-1. As noted above, the association of PAI-1 with vitronectin is conformationally controlled, and upon protease inhibition, the conformational change in PAI-1 associated with RCL insertion results in a loss of its high affinity for vitronectin and an increase in its affinity for LDL-R family members. This occurs because the insertion of the RCL into PAI-1 disrupts the vitronectin-binding site and simultaneously exposes a cryptic receptor-binding site that is only revealed when PAI-1 is complexed with a protease, resulting in an approximately 100,000-fold shift in the relative affinity of PAI-1 from vitronectin to LDL-R family members and a subsequent shift in PAI-1 localization from vitronectin to the cellular receptor. Thus, the association of PAI-1 with vitronectin and the LDL-R is conformationally regulated.

[0011] High PAI-1 levels are associated with various diseases and disorders. For example, high PAI-1 levels are associated with acute diseases such as sepsis and myocardial infarction, as well as chronic diseases such as cancer, atherosclerosis, and type 2 diabetes. Furthermore, high PAI-1 levels are associated with cardiovascular disease; PAI-1 expression is significantly increased in severe atherosclerotic vessels, and PAI-1 protein levels consistently increase during disease progression from normal vessels to fatty streaks in atherosclerotic plaques. Increased PAI-1 levels are also associated with obesity and insulin resistance.

[0012] Furthermore, elevated plasma concentrations of PAI-1 have been associated with thrombotic events, and antibody neutralization of PAI-1 activity resulted in endogenous thrombolysis and enhanced reperfusion.12 Elevated levels of PAI-1 have also been implicated in polycystic ovary syndrome and estrogen deficiency-induced bone loss.

[0013] PAI-1 is synthesized in both mouse and human adipocytes. There is also a strong correlation between visceral fat mass and plasma levels of PAI-1 in humans and mice. This dramatic upregulation of PAI-1 in obesity suggests that adipose tissue itself can directly contribute to elevated systemic PAI-1 levels, increasing the probability of vascular disease through increased thrombosis and accelerated atherosclerosis. Notably, very recent data suggest that PAI-1 may also play a direct role in obesity.

[0014] In one study, genetically obese and diabetic ob / ob mice crossed onto a PAI-1-deficient background exhibited significantly reduced body weight and improved metabolic profiles compared with PAI-1-deficient ob / ob mice. Similarly, nutritionally induced obesity and insulin resistance were dramatically attenuated in mice genetically deficient in PAI-1 and in mice treated with an orally active PAI-1 inhibitor. The improved adiposity and insulin resistance in PAI-1-deficient mice may be related to the observation that PAI-1-deficient mice on a high-fat diet had increased metabolic rate and total energy expenditure compared with wild-type mice, while peroxisome proliferator-activated receptor γ (PPARγ) and adiponectin expression were preserved. However, because overexpression of PAI-1 in mice also impairs adipose tissue formation, the exact mechanisms involved remain unclear and may be complex. Collectively, these observations suggest that PAI-1 plays a previously unrecognized direct role in obesity and insulin resistance, including interactions beyond its identified activities in regulating fibrinolysis and tissue remodeling.

[0015] Indeed, if PAI-1 positively regulates adipose tissue development, the association between increased PAI-1 expression and the development of obesity may constitute a positive feedback loop that promotes adipose tissue expansion and dysregulation of normal cholesterol homeostasis. Thus, there is a need in the art for a deeper understanding of how PAI-1 is involved in metabolism, obesity, and insulin resistance. Summary of the Invention

[0016] In this specification, [ka] or a pharmaceutically acceptable salt thereof, wherein X is Cl or F. Optionally, the compound is [ka] or a pharmaceutically acceptable salt thereof. Optionally, the compound is [ka] or a pharmaceutically acceptable salt thereof. [ka] or a pharmaceutically acceptable salt thereof. [ka] Provided is a PAI-1 inhibitor having the structure: or a pharmaceutically acceptable salt thereof. Optionally, the compound is in the form of a pharmaceutically acceptable salt. Also provided is a pharmaceutical composition of one or more of the compounds or salts disclosed herein and a pharmaceutically acceptable excipient. Optionally, the composition is [ka] or a pharmaceutically acceptable salt thereof. In some cases, the composition comprises a compound having the structure [ka] or a pharmaceutically acceptable salt thereof.

[0017] Further provided are methods of inhibiting PAI-1 by contacting PAI-1 with a compound disclosed herein. Also provided are methods of treating a disorder associated with abnormal PAI-1 activity, comprising administering a compound disclosed herein to a subject in need thereof in an amount effective to treat the disorder. In some cases, the disorder is cancer, sepsis, obesity, insulin resistance, a disease or disorder associated with dysregulated lipid metabolism, a disease or disorder associated with elevated levels of VLDL or LDL, high cholesterol, a proliferative disease or disorder, fibrosis and fibrotic diseases, inflammatory bowel disease, coagulation homeostasis, cerebrovascular disease, microvascular disease, hypertension, dementia, atherosclerosis, osteoporosis, osteopenia, arthritis, asthma, heart failure, arrhythmia, angina pectoris, hormone deficiency, and the like. insufficiency, Alzheimer's disease, hypertension, inflammation, sepsis, fibrinolytic disorders, stroke, dementia, coronary heart disease, myocardial infarction, stable and unstable angina, vascular disease, peripheral arterial disease, acute vascular syndromes, thrombosis, prothrombosis, deep vein thrombosis, pulmonary embolism, cerebrovascular disease, microvascular disease, hypertension, diabetes, hyperglycemia, hyperinsulinemia, malignant lesions, premalignant lesions, gastrointestinal malignancies, liposarcoma, epithelial tumors, and psoriasis, disorders of extracellular matrix accumulation, angiogenesis (ne In some cases, the disease or disorder involving thrombosis or prothrombosis is atherosclerotic plaque formation, venous thrombosis, arterial thrombosis, myocardial ischemia, atrial fibrillation, deep vein thrombosis, coagulation syndrome, pulmonary thrombosis, cerebral thrombosis, thromboembolic complications of surgery, and peripheral arterial occlusion. In some cases, the disorder is fibrosis, more specifically, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, hepatic fibrosis, or scleroderma. In some cases, the disorder is inflammatory bowel disease, more specifically, Crohn's disease or ulcerative colitis.In some cases, the extracellular matrix accumulation disorder is renal fibrosis, chronic obstructive pulmonary disease, polycystic ovary syndrome, restenosis, renovascular disease, diabetic nephropathy, or organ transplant rejection.

[0018] Further provided are methods for modulating cholesterol, lipid clearance, and / or lipid uptake in a subject with elevated levels of PAI-1, comprising administering to the subject an effective amount of a compound disclosed herein in an amount effective to reduce the subject's elevated PAI levels and thereby modulate cholesterol, lipid clearance, and / or lipid uptake. In some cases, the compound increases circulating high-density lipoprotein (HDL) and / or decreases circulating very-low-density lipoprotein (VLDL) in the subject. In various cases, the compound inhibits the binding of apolipoprotein E (ApoE) or apolipoprotein A (ApoA) to VLDL-R. In various cases, the compound reduces the binding of HDL or apolipoprotein E (ApoE) or apolipoprotein A (ApoA) to the ApoA receptor. In various cases, the compound reduces the binding of PAI-1 to apolipoprotein E (ApoE). In various cases, the compound reduces the binding of PAI-1 to apolipoprotein A (ApoA). In various cases, the compound reduces binding of PAI-1 to VLDL. In various cases, the compound binds to PAI-1 in the presence of vitronectin. In various cases, the compound binds to PAI-1 in the presence of urokinase-type plasminogen activator (uPA).

[0019] In any of the methods disclosed herein, the subject can be a human.

[0020] The foregoing summary is not intended to define all aspects of the invention, and additional aspects are described in other sections, such as the Detailed Description. The entire document is intended to be linked as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if those combinations of features are not found together in the same sentence, paragraph, or section of this document. [Brief explanation of the drawings]

[0021] [Figure 1] Serpin activity in the presence of various concentrations of CDE-517 is shown. [Figure 2] Serpin activity in the presence of various concentrations of CDE-252 is shown. [Figure 3] Serpin activity in the presence of various concentrations of CDE-519 is shown. [Figure 4] Serpin activity in the presence of various concentrations of CDE-520 is shown. [Figure 5] Serpin activity in the presence of various concentrations of CDE-264 is shown. [Figure 6] Serpin activity in the presence of various concentrations of CDE-295 is shown. [Figure 7] Serpin activity in the presence of various concentrations of CDE-234 is shown. [Figure 8] Serpin activity in the presence of various concentrations of CDE-241 is shown. [Figure 9] Serpin activity in the presence of various concentrations of CDE-246 is shown. [Figure 10] Serpin activity in the presence of various concentrations of CDE-413 is shown. [Figure 11] Serpin activity in the presence of various concentrations of CDE-415 is shown. [Figure 12] Serpin activity in the presence of various concentrations of CDE-412 is shown. [Figure 13] Serpin activity in the presence of various concentrations of CDE-248 is shown. [Figure 14] Serpin activity in the presence of various concentrations of CDE-266 is shown. [Figure 15] Serpin activity in the presence of various concentrations of CDE-301 is shown. [Figure 16] Serpin activity in the presence of various concentrations of CDE-307 is shown. [Figure 17] Serpin activity in the presence of various concentrations of CDE-340 is shown. [Figure 18] Serpin activity in the presence of various concentrations of CDE-422 is shown. [Figure 19] Serpin activity in the presence of various concentrations of CDE-423 is shown. [Figure 20] Serpin activity in the presence of various concentrations of CDE-424 is shown. [Figure 21] Serpin activity in the presence of various concentrations of CDE-446 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0022] As used herein, the structure: [ka] or a pharmaceutically acceptable salt thereof, wherein X is Cl or F. Optionally, the compound has the structure [ka] or a pharmaceutically acceptable salt thereof. In some cases, the compound has the structure [ka] or a pharmaceutically acceptable salt thereof. [ka] Further provided is a PAI-1 inhibitor having the structure: [ka] or a pharmaceutically acceptable salt thereof. Pharmaceutical compositions comprising one or more of these compounds or salts thereof are also provided.

[0023] As used herein, the term "pharmaceutically acceptable salts" refers to those salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanoate, and the like. Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base.Such salts include alkali metal, alkaline earth metal, aluminum salts, ammonium, N. + (C 1-4 Examples of suitable salts include, but are not limited to, salts of organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and salts of basic amino acids such as lysine and arginine. The present disclosure also contemplates the quaternization of any basic nitrogen-containing group of the compounds disclosed herein. Water- or oil-soluble or dispersible products may be obtained by such quaternization. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0024] How to use PAI-1 inhibitors As described hereinabove, the methods disclosed herein may include treating a disease or disorder associated with elevated PAI-1 levels, comprising administering a PAI-1 inhibitor. In one embodiment, the subject is a mammal. In some cases, the mammalian subject is a human.

[0025] In some embodiments, the present disclosure provides PAI-1 inhibitor compounds and methods for using the compounds in the treatment of a number of diseases or disorders associated with PAI-1 activity. Such conditions, e.g., diseases or disorders, include, but are not limited to, dysregulated lipid metabolism, obesity, diabetes, polycystic ovary syndrome, bone loss induced by estrogen deficiency, fibrosis and fibrotic diseases, inflammation, cell migration and migration-driven proliferation, and angiogenesis or thrombosis. In some aspects, such inhibitors are also believed to be useful in regulating endogenous fibrinolysis and in conjunction with pharmacological thrombolysis. In various aspects, the present disclosure provides PAI-1 inhibitor compounds and methods for using the compounds in the treatment of acute diseases associated with elevated PAI-1 levels, such as, but not limited to, sepsis, myocardial infarction, and thrombosis, compared to the PAI-1 levels of normal subjects known not to have sepsis, myocardial infarction, or thrombosis. In some embodiments, the PAI-1 inhibitor compounds disclosed herein are used in methods for treating diseases and disorders associated with elevated PAI-1 levels, including, but not limited to, cancer, atherosclerosis, insulin resistance, type 2 diabetes, and fibrotic diseases, as compared to the PAI-1 levels of normal subjects known not to be afflicted with these diseases or disorders. In various embodiments, provided herein are PAI-1 inhibitor compounds for modulating lipid metabolism, including increasing circulating HDL and / or decreasing circulating VLDL in a subject.

[0026] In various embodiments, PAI-1 inhibitors are useful in treating any condition, including a disease or disorder, in which a reduction in PAI-1 levels would provide benefit. PAI-1 inhibitors are useful alone or in combination with other compounds that can act to promote a reduction in PAI-1 levels.

[0027] The PAI-1 inhibitor may be formulated into a suitable formulation and administered to one or more sites in a subject in a therapeutically effective amount. In some embodiments, the PAI-1 inhibitor-based therapy is administered via continuous or intermittent intravenous administration. In various embodiments, the PAI-1 inhibitor-based therapy is administered via continuous or intermittent intramuscular or subcutaneous administration. In other aspects, the PAI-1 inhibitor-based therapy is administered via oral or buccal administration. An "effective amount" refers to an amount of the PAI-1 inhibitor compound sufficient to support an observable change in the level of one or more biological activities of PAI-1, plasminogen activator, HDL, LDL, or VLDL and / or an observable change in the indication for which the treatment method is intended. The change may be a decrease in the level of PAI-1 activity. In some embodiments, the change is an increase in plasminogen activator and / or HDL, and / or a decrease in LDL and VLDL.

[0028] In various embodiments, administration of the composition is systemic or local, and in still other embodiments, comprises a single site injection of a therapeutically effective amount of a PAI-1 inhibitor composition. Any route known to those of skill in the art for administration of the therapeutic compositions disclosed herein is contemplated, including, for example, intravenous, intramuscular, subcutaneous, oral, or catheter for chronic administration.

[0029] In some cases, the therapeutic composition may be delivered to the patient at multiple sites. Multiple doses may be administered simultaneously or over several hours. Similarly, the therapeutic composition may be taken orally on a regular basis. In certain cases, it is beneficial to provide a continuous flow of the therapeutic composition. Additional therapy may be administered on a periodic basis, such as daily, weekly, or monthly.

[0030] In addition to therapies based solely on delivery of PAI-1 inhibitor compositions, combination therapies are specifically contemplated, and PAI-1 inhibitor composition therapy may be used in combination with other agents commonly used to treat elevated PAI-1, LDL, and VLDL levels.

[0031] To achieve appropriate therapeutic results using the methods and compositions disclosed herein, it is further contemplated that a composition comprising a PAI-1 inhibitor and at least one other therapeutic agent (second therapeutic agent) is administered to a subject in need thereof. Such therapeutic agents include, but are not limited to, agents used in the management of cardiovascular disease, such as statins, anti-inflammatory agents, and cholesterol-lowering agents, such as ACE inhibitors. Such agents also include agents targeting neurological disorders, including, but not limited to, agents targeting stroke, seizures, and Alzheimer's disease. In another embodiment, the additional agent includes, but is not limited to, agents targeting diabetes. These are all disorders associated with elevated PAI-1 levels, and therefore, it is contemplated that combination therapy can be used with a PAI-1 inhibitor and other known therapies.

[0032] The combination therapy composition is provided in a combined amount effective to produce the desired therapeutic result in treating increased levels of PAI-1, VLDL, or LDL and / or to produce a detectable change in the indications described herein. In this process, the PAI-1 inhibitor and a second agent or factor are administered simultaneously. Thus, the method can involve administering a single composition or pharmacological formulation containing both agents, or simultaneously administering two different compositions or formulations, one containing the PAI-1 inhibitor therapeutic composition and the other containing the second therapeutic agent.

[0033] Alternatively, the PAI-1 inhibitor treatment may precede or follow the second therapeutic treatment by an interval ranging from minutes to weeks. In embodiments in which the second therapeutic and the PAI-1 inhibitor are administered separately, it is generally desirable to ensure that no significant period of time lapses between the time of delivery, allowing the second therapeutic and the PAI-1 inhibitor to exert their beneficially combined effects. In such cases, it is contemplated to administer both modalities within about 12-24 hours of each other, or within about 6-12 hours of each other, or alternating, with only about a 12-hour delay. In some situations, however, where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between each administration, it may be desirable to significantly extend the treatment period.

[0034] Systemic delivery of PAI-1 inhibitors to patients is a highly efficient method for delivering a therapeutically effective amount of compound to counteract the immediate clinical symptoms of a disease or disorder. Alternatively, in certain situations, local delivery of PAI-1 inhibitors and / or second therapeutic agents is appropriate. In certain embodiments, it is contemplated that PAI-1 inhibitors will be delivered to patients over a long period of time. Furthermore, it is contemplated that PAI-1 inhibitors will be taken throughout the patient's lifetime to reduce PAI-1, VLDL, and / or LDL levels.

[0035] Pharmaceutical Compositions As described above, methods are provided herein for using pharmaceutical compositions comprising an effective amount of a PAI-1 inhibitor and pharmaceutically acceptable excipients, such as diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers useful in PAI-1 inhibitor therapy. Such compositions may include additives such as diluents of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, detergents and solubilizers (e.g., Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimersol, benzyl alcohol), and bulking agents (e.g., lactose, mannitol), as well as the incorporation of materials into particulate formulations of polymeric compounds, such as polylactic acid, polyglycolic acid, or associated with liposomes or micelles. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the PAI-1 inhibitor. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990) Mack Publishing Co., Easton, PA, pages 1435-1712, incorporated herein by reference.

[0036] Sterile liquid compositions include solutions, suspensions, emulsions, syrups, and elixirs. The compounds disclosed herein can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile organic solvents, or a mixture of both. In one embodiment, the liquid carrier is suitable for parenteral injection. If the compound is sufficiently soluble, it can be dissolved directly in normal saline, with or without a suitable organic solvent, such as propylene glycol or polyethylene glycol. If necessary, a dispersion of the finely divided compound can be made into an aqueous starch solution, a sodium carboxymethylcellulose solution, or a suitable oil, such as peanut oil. Liquid pharmaceutical compositions that are sterile solutions or suspensions can be administered intramuscularly, intraperitoneally, or subcutaneously. In many cases, liquid composition forms can be used instead of the preferred solid oral administration methods.

[0037] For standard dosing regimens, it is preferable to prepare unit dosage forms of the compound. In this way, the composition can be easily subdivided into smaller doses at the physician's discretion. For example, unit dosages may be in the form of packeted powders, vials, or ampoules, or, in one embodiment, capsules or tablets. The active compound present in these unit dosage forms of the composition may be present in amounts ranging from about 1 gram to about 15 grams or more for one or more daily administrations, depending on the patient's specific needs. The daily dose of the active compound will vary depending on the route of administration, the patient's size, age, sex, and severity of the condition, and the patient's response to treatment as monitored by blood analysis and the patient's recovery rate.

[0038] The exact dosage employed will depend on several factors, including the host, whether veterinary or human, the nature and severity of the condition, e.g., the disease or disorder being treated, the mode of administration, and the specific active substance used. The compounds can be administered by any conventional route, particularly enterally, and in one embodiment, orally in tablet or capsule form. The administered compounds can be in free form or in pharmaceutically acceptable salt form, as appropriate, for use as pharmaceuticals, particularly for the preventive or therapeutic treatment of atherosclerosis and its sequelae (angina pectoris, myocardial infarction, arrhythmia, heart failure, renal failure, stroke, peripheral arterial occlusion, and related disease states). These measures slow the progression of disease states and help the body reverse the process in a natural way.

[0039] The PAI-1 inhibitor or derivative thereof can be formulated for injection, or for oral, nasal, pulmonary, topical, or other types of administration, as will be appreciated by those skilled in the art. The formulations may be liquid or solid, such as lyophilized, for reconstitution.

[0040] PAI-1 inhibitors or derivatives thereof are useful for treating either acute or chronic diseases or disorders associated with increased levels of PAI-1, LDL, or VLDL. In some embodiments, the condition (e.g., disease or disorder) alleviated or regulated by administration of a PAI-1 inhibitor is characterized by increased levels of VLDL and LDL. Such conditions may be induced as a therapeutic process for other purposes, such as chemotherapy or radiation therapy. Such conditions may be due to genetic inheritance or may be a side effect of another condition or drug.

[0041] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not cause adverse reactions, allergic reactions, or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vector or cell, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0042] The active compositions used in the methods disclosed herein include classical pharmaceutical preparations.These compositions can be administered via any common route, as long as the target tissue is accessible via that route.The pharmaceutical composition can be introduced into the subject by any conventional method, for example, intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary (e.g., sustained release), oral, sublingual, nasal, anal, vaginal, or transdermal delivery, or by surgical placement at a specific site.Treatment can consist of a single administration or multiple administrations over a period of time.

[0043] The active compound can be prepared for administration as a solution of free base or pharmacologically acceptable salt in water, suitably mixed with surfactant such as hydroxypropylcellulose.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, and their mixture, and in oil.Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0044] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is desirable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0045] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with some of the other ingredients listed above as needed, and then sterilize by filtration.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the other ingredients listed above as needed.For the preparation of sterile powder for sterile injectable solution, the preferred method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient plus any additional desired ingredients from its solution that has been previously sterilized by filtration.

[0046] "Pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the composition.

[0047] For oral administration of the composition, the PAI-1 inhibitor may be mixed with an excipient and used in the form of a non-ingestible mouthwash or dentifrice. Mouthwashes can be prepared by incorporating the required amount of active ingredient into a suitable solvent, such as sodium borate solution (Dobell's solution). Alternatively, the active ingredient may be incorporated into an antiseptic rinse containing sodium borate, glycerin, and potassium bicarbonate. The active ingredient can also be dispersed in dentifrices, including gels, pastes, powders, slurries, and the like. The active ingredient can be added in a therapeutically effective amount to a paste dentifrice, which may contain water, a binder, an abrasive, a flavoring agent, a foaming agent, and a humectant.

[0048] The composition used in the present method can be formulated in neutral or salt form.Pharmaceutically acceptable salts include, for example, acid addition salts (formed with free amino groups of protein) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0049] The composition used in this method can be formulated in micelles or liposomes.Such formulations include sterically stabilized micelles or liposomes and sterically stabilized mixed micelles or liposomes.The lipid bilayer of liposomes and micelles is known to fuse with the plasma membrane of cells, and deliver the enclosed contents to intracellular compartments, so such formulations can promote intracellular delivery.

[0050] Once formulated, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.The formulations are easily administered in a variety of dosage forms, such as injection solutions, drug-release capsules, etc.For example, for parenteral administration in aqueous solution, the solution should be appropriately buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose.These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0051] Generally, the effective amount of a PAI-1 inhibitor or its derivative is determined by the age, weight, and condition or severity of the recipient's disease or disorder. See Remington's Pharmaceutical Sciences, supra, pages 697-773, incorporated herein by reference. Typically, a dosage of about 0.001 μg / kg body weight / day to about 1000 μg / kg body weight / day can be used, although those skilled in the art will recognize that more or less frequent dosages may be used. Administration may be once or more times daily or less frequently, and may be combined with other compositions described herein. Note that the present disclosure is not limited to the dosages listed herein.

[0052] By initiating a treatment regimen with a minimum daily dose of approximately 1 gram, PAI-1 blood levels and symptom relief analysis of the patient can be used to determine whether a higher dose is necessary. Those skilled in the art will understand that appropriate dosage levels for treatment will vary, in part, depending on the molecule being delivered, the indication for which the PAI-1 inhibitor compound is being used, the route of administration, and the patient's size (patient's weight, body surface, or organ size) and condition (age and general health). Thus, clinicians may titrate dosages and modify the route of administration to achieve optimal therapeutic efficacy. Typical dosages may range from about 0.1 μg / kg to up to about 100 mg / kg or more, depending on the factors discussed above. In other embodiments, dosages may range from 0.1 μg / kg to up to about 100 mg / kg, or from 1 μg / kg to up to about 100 mg / kg, or from 5 μg / kg to up to about 100 mg / kg.

[0053] A "unit dose" is defined as a discrete amount of a therapeutic composition dispersed in a suitable carrier. Parenteral administration can be performed by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of the drug. Those skilled in the art will readily optimize effective dosages and administration schedules as determined by good medical practice and the clinical condition of each individual patient.

[0054] The frequency of administration depends on the pharmacokinetic parameters and route of administration of the drug. The optimal pharmaceutical formulation will be determined by those skilled in the art depending on the route of administration and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra, pages 1435-1712, incorporated herein by reference. Such formulations may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered drug. Depending on the route of administration, appropriate doses are calculated based on body weight, body surface area, or organ size. Further refinement of the calculations required to determine an appropriate therapeutic dose can be routinely performed by those skilled in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein and the pharmacokinetic data observed in animal or human clinical trials.

[0055] Appropriate dosages can be confirmed by using established assays for determining the level of myocardial infarction in conjunction with relevant dose-response data. The final dosing regimen will be determined by the attending physician, taking into account factors that modify the drug's action, such as the drug's specific activity, the severity of the injury, the patient's responsiveness, the patient's age, condition, weight, sex, and diet, the severity of the infection, the time of administration, and other clinical factors. As studies are conducted, more information regarding appropriate dose levels and duration of treatment will become available.

[0056] It will be appreciated that the pharmaceutical compositions and methods of treatment disclosed herein are useful in human and veterinary medicine. Thus, the subject to be treated is, in one embodiment, a mammal. In a representative embodiment, the mammal is a human.

[0057] Additionally, further contemplated are kits containing components comprising a composition comprising a PAI-1 inhibitor and, optionally, at least one additional agent useful in the treatment of acute and chronic diseases and disorders discussed herein.

[0058] Use of the Compounds in Treating Diseases or Disorders Provided herein is the use of the disclosed compounds for the manufacture of a medicament for the treatment or prevention of any of the diseases or disorders discussed herein.

[0059] The compounds are inhibitors of the serine protease inhibitor PAI-1 and are therefore useful for treating or preventing processes involving the production and / or action of PAI-1. Thus, in various embodiments, the compounds are useful for preventing or reducing thrombosis, promoting thrombolysis, and reducing fibrosis by regulating lipid metabolism, as described herein. In some embodiments, the compounds are useful for treating diseases or disorders associated with high cholesterol and elevated PAI-1 levels. In various embodiments, the compounds are useful for treating high levels of VLDL or LDL. In some embodiments, the compounds are useful for increasing HDL.

[0060] In some embodiments, these inhibitors are provided for the treatment of diseases or disorders related to PAI-1 activity.Such diseases or disorders include, but are not limited to, inflammation, cell migration and cell migration-driven proliferation, and angiogenesis or thrombosis.Such inhibitors are also believed to be useful in regulating endogenous fibrinolysis and in conjunction with pharmacological thrombolysis.

[0061] The compounds are useful for the treatment or prevention of insulin resistance, obesity, non-insulin-dependent diabetes mellitus, cardiovascular disease, coronary artery-related thrombosis and cerebrovascular disease.The compounds are also useful for inhibiting disease processes involving thrombotic and prothrombotic conditions, including, but not limited to, the formation of atherosclerotic plaques, venous and arterial thrombosis, myocardial ischemia, atrial fibrillation, deep vein thrombosis, coagulation syndrome, pulmonary thrombosis, cerebral thrombosis, thromboembolic complications of surgery (such as joint replacement surgery), and peripheral arterial occlusion.These compounds are also useful for the treatment of stroke associated with or resulting from atrial fibrillation.

[0062] The compounds are also used in the treatment or prevention of high cholesterol and diseases or disorders associated with such conditions.

[0063] The compounds can also be used to treat diseases or disorders associated with extracellular matrix accumulation, including, but not limited to, renal fibrosis, chronic obstructive pulmonary disease, polycystic ovary syndrome, restenosis, renal vascular disease, and organ transplant rejection.

[0064] The compounds may also be used to treat fibrosis, including, but not limited to, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, liver fibrosis, and scleroderma.

[0065] The compounds may also be used to treat inflammatory bowel diseases, including but not limited to Crohn's disease and ulcerative colitis.

[0066] The compounds can also be used to treat malignancies and diseases or disorders associated with angiogenesis, such as diabetic retinopathy.

[0067] The compounds may also be used in conjunction with and after processes or procedures involving the maintenance of vascular patency, including vascular surgery, vascular graft and stent patency, organ, tissue and cell implantation and transplantation.

[0068] The compound can also be used to treat Alzheimer's disease. This method can also be characterized as inhibiting plasminogen activation by PAI-1 in a mammal, particularly a human, suffering from or affected by Alzheimer's disease. This method can also be characterized as increasing or normalizing the level of plasmin concentration in a mammal, particularly a mammal suffering from or affected by Alzheimer's disease.

[0069] The compounds can be used to treat myelofibrosis associated with myeloid metaplasia by modulating stromal cell hyperplasia and the increase in extracellular matrix proteins.

[0070] The compounds may also be used in combination with highly active antiretroviral therapy (HAART) including protease inhibitors to treat diseases or disorders resulting from fibrinolytic dysfunction and hypercoagulation in HIV-1 infected patients receiving such therapy.

[0071] The compounds may be used in the treatment of diabetic nephropathy and renal dialysis associated with nephropathy.

[0072] The compounds are intended to be used in the treatment of cancer, sepsis, proliferative diseases such as psoriasis, for the improvement of coagulation homeostasis, for the treatment of cerebrovascular disease, microvascular disease, hypertension, dementia, atherosclerosis, osteoporosis, arthritis, asthma, heart failure, arrhythmias, angina pectoris, etc., as hormone replacement agents to treat, prevent, or reverse the progression of atherosclerosis, Alzheimer's disease, osteoporosis, osteopenia, for the reduction of inflammatory markers, for impaired fibrinolysis, for the reduction of C-reactive protein, or for the prevention or treatment of low-grade vascular inflammation, stroke, dementia, coronary heart disease, for the primary and secondary prevention of myocardial infarction, and for the treatment of chronic vascular diseases such as cerebrovascular disease, microvascular disease, hypertension, dementia, atherosclerosis, osteoporosis, arthritis, asthma, heart failure, arrhythmias, angina pectoris, etc. and unstable angina pectoris, for the primary prevention of coronary events, for the secondary prevention of cardiovascular events, for peripheral vascular disease, peripheral arterial disease, acute vascular syndromes, deep vein thrombosis, pulmonary embolism, for reducing the risk of undergoing myocardial revascularization, for microvascular diseases such as nephropathy, neuropathy, retinopathy, nephrotic syndrome, for hypertension, for type 1 and type 2 diabetes and related diseases, for obesity, insulin resistance, hyperglycemia, hyperinsulinemia, proliferative diseases such as malignancies, premalignant lesions, gastrointestinal malignancies, liposarcoma and epithelial tumors, psoriasis, for improving coagulation homeostasis and / or for improving endothelial function, and for all forms of cerebrovascular disease.

[0073] The compounds disclosed herein can be used for topical application in wound healing to prevent scarring.

[0074] The compounds disclosed herein can be used to treat vascular damage associated with inflammatory diseases, septic shock, and infections, and to treat blood and blood products used in dialysis, liquid-phase blood storage, and especially ex vivo platelet aggregation. The compounds can also be used in combination with thrombolytic, fibrinolytic, and anticoagulant agents. The compounds of the present invention can also be added to human plasma during blood chemistry analysis in a hospital setting to determine their fibrinolytic ability.

[0075] Further provided herein are methods for treating, preventing, ameliorating, or inhibiting each of the diseases mentioned herein in a mammal, and in one embodiment, a human, each comprising administering to a mammal in need of such treatment, prevention, amelioration, or inhibition a pharmaceutically or therapeutically effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof.

[0076] The compounds disclosed herein can also be used as imaging agents to treat cancer, including but not limited to breast and ovarian cancer, and for the identification of metastatic cancers.

[0077] A pharmaceutically or therapeutically effective amount of a compound herein will be understood to refer to that amount of the compound in question that sufficiently inhibits the serine protease inhibitor PAI-1 in a mammal in need thereof to a degree sufficient to provide the desired improvement in the condition in question, or to provide sufficient inhibition of the serine protease inhibitor PAI-1 to prevent, inhibit or limit the onset of the physiological basis of the disease or condition in question. [Example]

[0078] Synthesis of compound CDE517: [ka]

[0079] Ethyl 2-((4-chloro-3-(trifluoromethoxy)benzyl)amino)-2-oxoacetate: A stirred solution of 4-chloro-3-(trifluoromethoxy)benzylamine (771.9 mg, 3.42 mmol) and pyridine (830 μL, 10.26 mmol) in dichloromethane (10 mL) was cooled in an ice bath. Ethyl oxalyl chloride (385 μL, 3.42 mmol) was added dropwise, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate and washed with 0.2 N HCl (2×), saturated aqueous NaHCO3 (2×), and brine (1×). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo to give 1.0726 g (96% yield) of the product as a clear oil. 1 H-NMR(DMSO-d6,400MHz)δ9.5(t,J=6Hz,1H),7.62(d,J=8.2Hz,1H),7.44(bs,1H),7.3(d d,J=8.7,1.8Hz,1H),4.33(d,J=6.4Hz,2H),4.21(q,J=6.9Hz,2H),1.23(t,J=6.9Hz,3H). [ka]

[0080] N-(4-chloro-3-(trifluoromethoxy)benzyl)-2-hydrazinyl-2-oxoacetamide (CDE-517): To a solution of ethyl 2-((4-chloro-3-(trifluoromethoxy)benzyl)amino)-2-oxoacetate (1.0726 g, 3.29 mmol) in absolute ethanol (30 mL), 50% hydrazine hydrate (425 μL, 6.59 mmol) was added dropwise and stirred for 2 h. The solid was filtered, dried in vacuo, and then triturated with boiling deionized water to give 0.7191 g (70.2% yield) of the product as a white solid. 1 H-NMR(DMSO-d6,400MHz)δ10.1(bs,1H),9.37(t,J=6.4Hz,1H),7.61(d,J=8.2Hz,1H), 7.42(bs,1H),7.28(dd,J=8.3,1.8Hz,1H),4.5(d,J=3.6Hz,2H),4.31(d,J=6.4Hz,2H); 13C-NMR (DMSO-d6, 100MHz) δ160.6,158.3,144.3,141.1,131.4,128.5,124.8,122.4,120.6(q,J=256.5Hz),41.8.

[0081] Synthesis of compound CDE-415: N-(3-chloro-4-fluorobenzyl)-2-hydrazinyl-2-oxoacetamide (CDE-415): To a solution of 3-chloro-4-fluorobenzylamine (230 μL, 1.83 mmol) and pyridine (296 μL, 3.66 mmol) in dichloromethane (5 mL) was added ethyl 2-chloro-2-oxoacetate (215 μL, 1.92 mmol) dropwise over an ice bath. After 5 minutes, the solution was removed from the ice bath and allowed to warm to room temperature. The reaction mixture was diluted with ethyl acetate, washed with 0.2 N HCl (2×) and saturated NaHCO₃ (1×), dried over MgSO₄, filtered, and concentrated in vacuo to give 0.336 g of ethyl 2-(3-chloro-4-fluorobenzylamino)-2-oxoacetate as a white solid (71% yield). 1 H NMR (CDCl3, 400 MHz) δ 7.42 (s, 1H), 7.34 (dd, J = 2.3, 6.9 Hz, 1H), 7.17 (M, 1H), 7.10 (t, J = 8.7 Hz, 1H), 4.46 (d, J = 6.0 Hz, 2H), 4.35 (q, J = 7.3 Hz, 2H), 1.39 (t, J = 6.9 Hz, 3H). To a solution of ethyl 2-(3-chloro-4-fluorobenzylamino)-2-oxoacetate (211.9 mg, 0.816 mmol) in ethanol (6 mL) was added dropwise 50% hydrazine hydrate (102 μL). The mixture was stirred at room temperature overnight. The product was filtered from the mixture and dried under vacuum to give 0.188 g of N-(3-chloro-4-fluorobenzyl)-2-hydrazinyl-2-oxoacetamide as a white solid (94% yield). 1 HNMR(DMSO-d6,400MHz)δ10.01(s,1H),9.28(t,J=6.4Hz,1H),7.42(dd,J=1.8,5. 5Hz,1H),7.32(t,J=8.7Hz,1H),7.23(m,1H),4.48(s,2H),4.25(d,J=6.4Hz,2H); 13CNMR(DMSO-d6,100MHz)δ160.48,158.43,156.75(d,J=244Hz),137.32,129.97,128.63,119.62(J=18Hz),117.25(J=20Hz),41.66.

[0082] Synthesis of compound CDE-412: N-(4-chloro-3-fluorobenzyl)-2-hydrazinyl-2-oxoacetamide (CDE-412): To a solution of 4-chloro-3-fluorobenzylamine (225 μL, 1.83 mmol) and pyridine (296 μL, 3.66 mmol) in dichloromethane (5 mL) was added ethyl 2-chloro-2-oxoacetate (215 μL, 1.92 mmol) dropwise over an ice bath. After 10 minutes, the solution was removed from the ice bath and allowed to warm to room temperature. The reaction mixture was diluted with ethyl acetate, washed with 0.2 N HCl (2×) and saturated NaHCO₃ (1×), dried over MgSO₄, filtered, and concentrated in vacuo to give 0.3878 g (82% yield) of ethyl 2-(4-chloro-3-fluorobenzylamino)-2-oxoacetate as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 7.45 (s, 1H), 7.36 (t, J = 7.96 Hz, 1H), 7.09 (d, J = 9.6 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 4.48 (d, J = 6.4 Hz, 2H), 4.35 (q, J = 6.9 Hz, 2H), 1.38 (t, J = 6.9 Hz, 3H). To a solution of ethyl 2-(4-chloro-3-fluorobenzylamino)-2-oxoacetate (119.7 mg, 0.461 mmol) in ethanol (6 mL) was added dropwise 50% hydrazine hydrate (57 μL). The mixture was stirred at room temperature overnight. The product was filtered from the mixture and dried under vacuum to give 59.0 mg of N-(4-chloro-3-fluorobenzyl)-2-hydrazinyl-2-oxoacetamide as a white solid (52% yield). 1HNMR(DMSO-d6,400MHz)δ10.02(s,1H),9.30(t,J=6.4Hz,1H),7.49(t,J=8.24Hz,1H),7.24 (dd,J=1.8,10.5Hz,1H),7.08(dd,J=1.36,8.24Hz,1H),4.51(s,2H),4.27(d,J=6.4Hz,2H); 13 CNMR(DMSO-d6,100MHz)δ160.54,158.74,158.40,156.29,141.29,141.23,130.99,125.08,125.05,118.36,118.19,116.31,116.10,41.86.

[0083] Fluorescent IC50 plate assay of PAI-1 inhibitors: To assay PAI-1 inhibitor activity in plasma, recombinant active human PAI-1 (Molecular Innovations) was added at concentrations up to 20 nM to PAI-1-depleted human plasma (Molecular Innovations) containing 10 μg / mL aprotinin (Roche). Next, 10 μL of this human plasma (with or without PAI-1) was added to wells containing 80 μL of buffer containing increasing concentrations of PAI-1 inhibitors and incubated for 15 min at 23°C (buffer: 40 mM HEPES, 100 mM NaCl, 0.005% Tween-20, pH 7.4, and 10% DMSO). Next, 10 μL of 25 nM UPA (rheotrombopag) (final concentration: 2.5 nM) was added to each reaction well and incubated for an additional 30 min at 24 °C, resulting in a final PAI-1 concentration of 2 nM and a final uPA concentration of 2.5 nM. After this incubation, 100 μL of buffer containing 100 mM uPA fluorescent substrate Z-Gly-Gly-Arg-AMC (Calbiochem) was added to a final concentration of 50 μM. Residual uPA activity in each reaction mixture was determined from the rate of AMC release by uPA measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm for 10 min at 23 °C. Data are expressed as residual PAI-1 activity as a percentage of control PAI-1 activity.

[0084] For assays in buffer or buffer containing 1.5% bovine serum albumin (BSA), the assays were performed as described above, except that neither plasma nor aprotinin was added, and activity was measured in either the same buffer containing 100 mM NaCl, 40 mM HEPES, 0.005% Tween-20, 10% DMSO, pH 7.4, or 1.5% BSA. The results are shown in Figure 1. [ka] This is the case for the compound (CDE-517). Figure 2 shows the structure [ka] This is the case for the compound (CDE-252). Figure 3 shows the structure [ka] This is the case for the compound (CDE-519). Figure 4 shows the structure [ka] This is the case for the compound (CDE-520). Figure 5 shows the structure [ka] This is the case for the compound (CDE-264). Figure 6 shows the structure [ka] This is the case for the compound (CDE-295). Figure 7 shows the structure [ka] This is the case for the compound (CDE-234). Figure 8 shows the structure [ka] This is the case for the compound (CDE-241). [ka] This is the case for the compound (CDE-246). [ka] This is the case for the compound (CDE-413).

[0085] Figure 11 shows the structure [ka] This is the case for the compound (CDE-415). [ka] This is the case for compound (CDE-412). These data are compared with those for the monohalophenyl compounds CDE-248 and CDE-266 (Figures 13 and 14, respectively), as well as a variety of other dihalophenyl compounds: [1] (CDE-301, Figure 15), [2] (CDE-307, Figure 16), [3] (CDE-340, Figure 17), [4] (CDE-422, Figure 18), [5] (CDE-423, Figure 19), [6] (CDE-424, Figure 20), and [7] (CDE-446, Figure 21).

Claims

1. A compound having the structure: or a pharmaceutically acceptable salt thereof. 【Chemical 1】

2. 10. A composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof for use in a method of inhibiting plasminogen activator inhibitor-1 (PAI-1), the method comprising contacting PAI-1 with the compound or a pharmaceutically acceptable salt thereof in an amount effective to inhibit the PAI-1.

3. 10. A composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof for use in a method for treating a disorder associated with abnormal PAI-1 activity, said method comprising administering to a subject in need thereof said compound or a pharmaceutically acceptable salt thereof in an amount effective to treat said disorder.

4. The disorder may be cancer, sepsis, obesity, insulin resistance, a disease or disorder associated with dysregulated lipid metabolism, a disease or disorder associated with elevated VLDL or LDL levels, high cholesterol, a proliferative disease or disorder, fibrosis and fibrotic diseases, inflammatory bowel disease, coagulation homeostasis, cerebrovascular disease, microvascular disease, hypertension, dementia, atherosclerosis, osteoporosis, osteopenia, arthritis, asthma, heart failure, arrhythmias, angina, hormone deficiency, 4. The composition of claim 3, wherein the therapeutic agent is selected from the group consisting of: insufficiency, Alzheimer's disease, hypertension, inflammation, sepsis, fibrinolytic disorders, stroke, dementia, coronary heart disease, myocardial infarction, stable and unstable angina, vascular disease, peripheral arterial disease, acute vascular syndrome, thrombosis, prothrombosis, deep vein thrombosis, pulmonary embolism, cerebrovascular disease, microvascular disease, hypertension, diabetes, hyperglycemia, hyperinsulinemia, malignant lesions, premalignant lesions, gastrointestinal malignancies, liposarcoma, epithelial tumors, and psoriasis, disorders of extracellular matrix accumulation, neoangiogenesis, myelofibrosis, fibrinolytic dysfunction, polycystic ovary syndrome, estrogen deficiency-induced bone loss, angiogenesis, angiogenesis, myelofibrosis, or fibrinolytic dysfunction.

5. 5. The composition of claim 4, wherein the disease or disorder involving thrombosis or prothrombosis is atherosclerotic plaque formation, venous thrombosis, arterial thrombosis, myocardial ischemia, atrial fibrillation, deep vein thrombosis, coagulation syndrome, pulmonary thrombosis, cerebral thrombosis, thromboembolic complications of surgery, and peripheral arterial occlusion.

6. The composition of claim 4, wherein the disorder is fibrosis.

7. The composition of claim 6, wherein the fibrosis is pulmonary fibrosis, renal fibrosis, cardiac fibrosis, hepatic fibrosis, or scleroderma.

8. The composition of claim 4, wherein the disorder is inflammatory bowel disease.

9. 9. The composition of claim 8, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

10. 5. The composition of claim 4, wherein the extracellular matrix accumulation disorder is renal fibrosis, chronic obstructive pulmonary disease, polycystic ovary syndrome, restenosis, renovascular disease, diabetic nephropathy, or organ transplant rejection.

11. 10. A composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof for use in a method for modulating cholesterol, lipid clearance, and / or lipid uptake in a subject having elevated levels of PAI-1, the method comprising administering to the subject an effective amount of the compound or a pharmaceutically acceptable salt thereof in an amount effective to lower the elevated PAI-1 level in the subject and thereby modulate cholesterol, lipid clearance, and / or lipid uptake.

12. 12. The composition of claim 11, wherein the compound or salt increases circulating high density lipoprotein (HDL) and / or decreases circulating very low density lipoprotein (VLDL) in the subject.

13. 12. The composition of claim 11, wherein the compound or salt inhibits the binding of apolipoprotein E (ApoE) or apolipoprotein A (ApoA) to VLDL-R.

14. 12. The composition of claim 11, wherein the compound or salt reduces binding of HDL or apolipoprotein E (ApoE) or apolipoprotein A (ApoA) to the ApoA receptor.

15. 12. The composition of claim 11, wherein the compound or salt reduces binding of PAI-1 to apolipoprotein E (ApoE).

16. 12. The composition of claim 11, wherein the compound or salt reduces binding of PAI-1 to apolipoprotein A (ApoA).

17. 12. The composition of claim 11, wherein the compound or salt reduces binding of PAI-1 to VLDL.

18. 12. The composition of claim 11, wherein the compound or salt binds to PAI-1 in the presence of vitronectin.

19. 12. The composition of claim 11, wherein the compound or salt binds to PAI-1 in the presence of urokinase-type plasminogen activator (uPA).

20. The composition of any one of claims 2 to 19, wherein the subject is a human.

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