Novel compounds useful for treatment of cardiovascular diseases

Abstract

The present invention provides novel compounds and pharmaceutical compositions thereof and methods of using the same for the treatment of cardiovascular diseases.

Description

[0001] This invention is a divisional application of the invention patent with application number 2019800657029, application date October 27, 2019, and invention name “New compounds suitable for treating cardiovascular diseases”. Technical Field

[0002] The present invention provides novel compounds and pharmaceutical compositions thereof for treating cardiovascular diseases and methods of using the same.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Non-Provisional Patent Application No. 16 / 600,476, filed on October 12, 2019, which is incorporated herein by reference in its entirety.

[0005] This application claims the benefit of U.S. Non-Provisional Patent Application No. 16 / 429,015, filed on June 2, 2019, which is incorporated herein by reference in its entirety.

[0006] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 755,516, filed on November 4, 2018, which is incorporated herein by reference in its entirety. Background Art

[0007] According to the 2019 American Heart Association Heart Disease and Stroke Statistics (Benjamin et al., 2019), cardiovascular diseases are responsible for most deaths in the United States. These diseases are found in 121,000,000 U.S. citizens over the age of 20 or 48% of the adult population. All of these cardiovascular diseases are associated with cellular hypoxia. Cellular hypoxia is defined as a relative lack of oxygen availability or utilization in living cells compared to normal physiological conditions.

[0008] By 2035, the annual cost of cardiovascular disease in the United States will reach $1.1 trillion. Despite tremendous progress in the management of cardiovascular disease, these same diseases remain the most common cause of death in the United States and must be considered a worldwide problem as well. In 2016, approximately 17.6 million people died worldwide from cardiovascular disease, an increase of 14.5% from 2006.

[0009] Health professionals are somewhat proud of the dramatic decline in mortality from cardiovascular disease in the United States in recent years. However, patients who once died acutely from these diseases are now chronic patients requiring long-term management. Our challenge as a society is to devise low-cost management strategies that will address this growing epidemic.

[0010] When oxygen supply is short, the metabolism of the cell, and therefore life itself, is compromised. Living cells respond to changes in the amount of available ambient oxygen with extremely sensitive mechanisms that reprogram pathways of gene expression. The nature of the response is specific to the situation and environment of each cell. Hypoxia-inducible factors (HIFs) are found in organisms from primitive worms to humans, everywhere oxygen delivery is an important variable in biological life, and there are currently many pharmaceutical development programs dedicated to altering HIF physiology. (Semenza, 2019) HIFs provide us with important descriptions of key components of hypoxia-mediated gene expression, but the scope of human cell responses to oxygen is not limited to the regulation of HIF expression.

[0011] Hypoxia is associated with inadequate perfusion and is noted in cardiovascular disease and other conditions associated with reduced blood flow to perfused organs. Reperfusion is associated with the return of blood flow. Typically, reperfusion is desired, but prolonged ischemia and subsequent return of blood flow can lead to local inflammation and cell damage due to toxic byproducts of oxidative metabolism. There is a way to mitigate this process. Repeated brief periods of inadequate perfusion and reperfusion can increase tolerance to future ischemic events. This phenomenon is known as preconditioning. Effective treatments for the adverse consequences of non-lethal oxygen delivery and severe reductions in perfusion have been strongly sought, and current treatment options are limited.

[0012] Most compounds currently developed clinically for managing hypoxia-related gene expression inhibit prolyl hydroxylases. Prolyl hydroxylases cause hypoxia-inducible factors to be targeted for degradation, so prolyl hydroxylase inhibitors prevent HIF degradation and actually enhance HIF-mediated events. Prolyl hydroxylase inhibitors are compounds that enhance the adaptive response of cells to hypoxia on a systemic basis via HIF. HIF enhanced by prolyl hydroxylase inhibitors can cause the kidneys to synthesize additional erythropoietin, which is an endogenous molecule that stimulates the development of red blood cells in the case of anemia to achieve a therapeutic effect. HIF enhancement also leads to angiogenesis, resulting in more blood vessels growing into hypoxic areas, which may be beneficial when, for example, coronary artery disease causes myocardial ischemia and related cardiac dysfunction. Disadvantageously, the same overall approach can simultaneously increase the arterial supply of dormant malignancies, and / or alter cell metabolism to conserve oxygen in the presence of oxygen. It is easy to underestimate the complexity of the pathophysiology of disease states associated with hypoxia. In order to develop safe and effective medicines, very selective methods will be needed to regulate hypoxia-mediated events. This will involve delivery within a limited organ-specific space or triggering activity under appropriate pathological circumstances.

[0013] Hypoxia transcriptionally regulates cyclooxygenase-2 (COX-2) expression in cultured human umbilical vein endothelial cells via the transactivators NF-κB p65, HMG I (Y), and Sp1, leading to increased production of PGE2. (Schmedtje, Jr., Ji, Liu, DuBois, & Runge, 1997; Xu, Ji, & Schmedtje, Jr., 2000; Ji, Xu, & Schmedtje, Jr., 1998) These findings reflect the fact that HIF is not sufficient to drive adaptation to hypoxia in humans, and that NF-κB is another important mediator of hypoxia-driven gene transactivation in the vascular endothelium.

[0014] Ischemic cardiovascular disease is associated with insufficient blood flow and is also associated with hypoxia. Ischemic preconditioning is part of the therapeutic effect attributed to the compounds of the present invention. Hypoxia increases endothelial expression of COX-2. COX-2 is essential for ischemic preconditioning as a partner in nitric oxide (NO) synthesis. (Li et al., 2007) Both are required to achieve the protection conferred to the heart in the late stages of ischemic preconditioning. (Guo et al., 2012)

[0015] The SLC14 (solute carrier 14) family of urea transporter genes regulates the transport of urea across cell membranes. UT-B (urea transporter, B, product of gene SLC14A1) facilitates the transport of urea, water, and urea analogs across cell membranes. (Shayakul, Clemencon, & Hediger, 2013) UT-B is widely expressed, including in the heart, vascular endothelium, and erythrocytes. UT-B-deficient mice have cardiac conduction abnormalities, increased brain urea concentrations, and no reduction in production. (Li, Chen, & Yang, 2012) Urea can freely permeate and passively enter cells, but equilibrium is slow, and UT-B promotes rapid excretion of urea from erythrocytes. (Sands, 1999) Urea is generally considered a waste product, and it carries nitrogen from amino acid decomposition to an opportunity for recycling. Renal failure is associated with decreased nitric oxide synthase (NOS) activity. However, when BUN is elevated to uremic levels, NOS activity in rats with normal renal function does not decrease. (Xiao, Erdely, Wagner, & Baylis, 2001) Urea may have cardioprotective properties in certain circumstances. (Wang et al., 1999)

[0016] Membrane UT-B is abundant on human vascular endothelium in cultures from different locations and appears to be involved in the regulation of nitric oxide (NO) synthesis. (Wagner, Klein, Sands, & Baylis, 2002) UT-B in vascular endothelium is usually used to expel urea from the cell membrane. Inhibition of UT-B in vascular endothelium leads to accumulation of urea in cells. It is believed that this leads to feedback inhibition of arginase (an enzyme that converts l-arginine into urea), which promotes the activity and expression of the l-arginine alternative pathway, in this case endothelial NOS (eNOS) that enables the production of NO to be increased. (Sun et al., 2016) Interestingly, it illustrates how the presence of urea enhances the production of NO.

[0017] Hypoxia increases the expression of UT-B in hypoxic vascular endothelium. The inventors found that in hypoxic (1% oxygen) cell cultures, the messenger RNA of the UT-B gene (SLC14A1) was significantly upregulated in human vascular endothelial cells. In this case, the upregulation of UT-B expression draws urea out of the endothelial cells and removes the arginase feedback inhibition that causes increased eNOS activity, reducing the net vasodilatory effect of nitrate administration. The source of urea or urea analogs should maintain effective intracellular urea substrate levels, overcome the upregulation of UT-B, and allow l-arginine to participate in the production of NO by eNOS, thereby increasing vasodilation in response to hypoxia and dilating adjacent vascular smooth muscle.

[0018] It would be advantageous to discover compounds that enhance the vasodilatory release of NO in the presence of hypoxia while enabling the process of ischemic preconditioning in a targeted (eg, localized) manner, thereby leading to the treatment and prevention of major adverse cardiac events in cardiovascular disease. Summary of the invention

[0019] Thus, in one aspect, the present invention provides novel compounds or pharmaceutically acceptable salts thereof that are responsive to areas of cellular hypoxia.

[0020] In another aspect, the present invention provides novel pharmaceutical compositions comprising: a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one compound of the present invention or a pharmaceutically acceptable salt thereof.

[0021] In another aspect, the present invention provides a novel method for treating a disease mediated by cellular hypoxia, comprising: administering to a mammal in need thereof a therapeutically effective amount of at least one compound of the present invention or a pharmaceutically acceptable salt thereof.

[0022] In another aspect, the present invention provides a process for preparing at least one compound of the present invention.

[0023] In another aspect, the present invention provides novel compounds or pharmaceutically acceptable salts for use in therapy.

[0024] In another aspect, the present invention provides the use of novel compounds for the manufacture of medicaments for treating diseases mediated by cellular hypoxia.

[0025] These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors' discovery that the presently claimed compounds, or pharmaceutically acceptable salts thereof, are expected to provide a therapeutic response localized in areas of cellular hypoxia. DETAILED DESCRIPTION

[0026] All references cited herein are incorporated by reference in their entirety.

[0027] In addition to urea or urea analogs (such as amides or hydroxyacetamides), the compounds of the present invention are also designed to release nitrates on the endothelial surface. They are small molecules with a central isosorbide portion attached to a urea portion (Formula I and IV), an amide portion (Formula II and V) or a hydroxyacetamide portion (Formula III and VI)) and a relative nitrate group (-NO3). Ethanolamide has the benefits of relatively high permeability and low toxicity through UT-B. (Zhao, Sonawane, Levin, & Yang, 2007) It is expected that the compounds of the present invention utilize urea and urea analogs to enter the passive movement of vascular endothelial cells across the cell membrane. In the case of vascular endothelial hypoxia, the expression of UT-B genes increases. The transport (removal) of urea and urea analogs mediated by UT-B occurs in hypoxia, which seems to limit the activity of eNOS and the synthesis of NO. Therefore, the replacement of intracellular urea will enhance the effect of nitrates provided to the vascular endothelium. It is expected that the combination of urea and / or urea analogs and nitrates will promote a series of events including vasodilation and ischemic preconditioning under hypoxic conditions.

[0028] The compounds of the present invention are characterized by a nitrate group that can be metabolized to NO via aldehyde dehydrogenase-2 or can be converted to nitrite and subsequently reduced by a series of enzymes including xanthine oxidase to form NO. Alternatively, intracellular L-arginine is oxidized by nitric oxide synthase to form NO. Historically, NO has been referred to as an endothelium-derived relaxing factor that induces the formation of cyclic guanosine monophosphate (cGMP) via activation of the NO receptor soluble guanylate cyclase. cGMP binds to and enhances the activity of protein kinase G, promoting the mobilization of calcium from smooth muscle cells, thereby reducing vascular tone because reduced intracellular calcium impairs the contractility of smooth muscle.

[0029] NO is also essential for ischemic preconditioning, although the exact intracellular mechanism of this effect is not as good as the vasodilation mechanism. COX-2 expression is an essential part of ischemic preconditioning, and COX-2 expression is induced by hypoxia via a mechanism that depends on the NF-κB transactivation system. In addition, as previously announced, when hypoxia causes COX-2 to rise, endothelial nitric oxide synthase appears to be slightly downregulated. (Schmedtje, Jr. et al., 1997) Therefore, the mechanism of increasing NO production by inducing COX-2 under hypoxic conditions has therapeutic value in the vascular endothelium.

[0030] Thus, in one aspect, the compounds of the invention will enhance the formation and release of vasodilatory NO while enabling ischemic preconditioning in a targeted manner.

[0031] In another aspect, the compounds of the invention provide additional substrate to overcome the consequences of hypoxia-induced UT-B expression and associated transport of urea and / or analogs out of the cell and across the cell membrane.

[0032] On the other hand, since the compounds of the present invention are not global regulators of HIF-(hypoxia inducible factor) and downstream effects, their therapeutic response will be concentrated in the area of ​​cellular hypoxia. By locally alleviating the consequences of cellular hypoxia, the compounds of the present invention can treat cardiovascular diseases with few or no side effects, which are not centrally administered therapeutic agents that non-selectively mimic the downstream effects of hypoxia.

[0033] In another aspect, the present invention provides novel compounds of Formula I, II, III, IV, V and / or VI:

[0034]

[0035] in:

[0036] R 1 does not exist;

[0037] Alternatively, R 1 Selected from (R 1 The right side of the isosorbide moiety is attached to the isosorbide moiety): (CH2)2O, (CH2)2NH, (CH2)3O, (CH2)3NH, CH2C(=O)O, and CH2C(=O)NH; and,

[0038] R 2 Selected from (R 2The right side of the isosorbide moiety is attached to the isosorbide moiety): (CH2)2O, (CH2)2NH, (CH2)3O, (CH2)3NH, CH2C(=O)O, CH2C(=O)NH, CH2OC(=O)O, CH2OC(=O)NH, CH2NHC(=O)O, and CH2NHC(=O)NH;

[0039] or a pharmaceutically acceptable salt thereof.

[0040] In another aspect, the compound is of Formula I or IV, or a pharmaceutically acceptable salt thereof.

[0041] In another aspect, the compound is of Formula I or IV, and R 1 Not present, or a pharmaceutically acceptable salt thereof.

[0042] In another aspect, the compound is of Formula I or IV, and R 1 Selected from: (CH2)2O, (CH2)2NH, (CH2)3O and (CH2)3NH, or a pharmaceutically acceptable salt thereof.

[0043] In another aspect, the compound is of Formula II or V, or a pharmaceutically acceptable salt thereof.

[0044] In another aspect, the compound is of Formula II or V, and R 2 Selected from: (CH2)2O, (CH2)2NH, (CH2)3O, (CH2)3NH, CH2OC(=O)O, CH2OC(=O)NH, CH2NHC(=O)O and CH2NHC(=O)NH, or a pharmaceutically acceptable salt thereof.

[0045] In another aspect, the compound is of Formula III or VI, or a pharmaceutically acceptable salt thereof.

[0046] In another aspect, the compound is of Formula III or VI, and R 1 Not present, or a pharmaceutically acceptable salt thereof.

[0047] In another aspect, the compound is of Formula III or VI, and R 1 Selected from: (CH2)2O, (CH2)2NH, (CH2)3O and (CH2)3NH, or a pharmaceutically acceptable salt thereof.

[0048] In another aspect, the present invention provides a novel pharmaceutical composition comprising: a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0049] In another aspect, the present invention provides a novel method for treating diseases mediated by cellular hypoxia, comprising: administering a therapeutically effective amount of at least one compound of the present invention or a pharmaceutically acceptable salt thereof to a patient in need thereof.

[0050] In another aspect, the present invention provides a novel method for treating cardiovascular diseases, comprising: administering a therapeutically effective amount of at least one compound of the present invention or a pharmaceutically acceptable salt thereof to a patient in need thereof.

[0051] In another aspect, the cardiovascular disease is selected from the group consisting of coronary artery disease, myocardial infarction, heart failure, arrhythmia, cardiac electrophysiological disorders, congenital cardiovascular anomalies, developmental cardiovascular anomalies, inflammatory cardiomyopathy, Kawasaki disease, infectious cardiomyopathy, sudden death / cardiac arrest, atherosclerosis, atherosclerotic cardiovascular disease, valvular heart disease, venous insufficiency, cardiac thrombosis, vascular thrombosis, thromboembolism, peripheral arterial disease, aortic aneurysm, aortic dissection, vascular aneurysm, vascular dissection, stroke, systemic hypertension and pulmonary hypertension.

[0052] In another aspect, the present invention provides a compound of the present invention for use in therapy.

[0053] In another aspect, the present invention provides use of the present invention for the manufacture of a medicament for treating a disease mediated by cellular hypoxia.

[0054] In another aspect, the present invention provides use of the present invention for the manufacture of a medicament for treating cardiovascular disease.

[0055] Without departing from the spirit or essential attributes of the present invention, the present invention can be implemented in other specific forms. The present invention encompasses all combinations of aspects of the present invention mentioned herein. It should be understood that any and all embodiments of the present invention can be combined with any other embodiment to describe additional embodiments. It should also be understood that each individual element of the embodiment is intended to be considered as its own independent embodiment individually. In addition, any element of the embodiment is intended to be combined with any and all other elements from any embodiment to describe additional embodiments.

[0056] definition

[0057] Unless otherwise stated, the examples provided in the definitions present in this application are non-inclusive. They include, but are not limited to, the recited examples.

[0058] Cellular hypoxia is a lack of oxygen at the level of individual cells and is not necessarily associated with a lack of oxygen at the level of the intact organism or the environment.

[0059] The compounds described herein may have asymmetric centers, geometric centers (e.g., double bonds), or both. Unless a specific stereochemistry or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, and all geometric isomeric forms of the structure are intended. Compounds of the present invention containing asymmetrically substituted atoms may be separated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by the disappearance of racemic forms, by synthesis from optically active starting materials, or by the use of chiral auxiliaries. Geometric isomers of olefins, C=N double bonds, or other types of double bonds may be present in the compounds described herein, and all such stable isomers are included in the present invention. Specifically, cis and trans geometric isomers of the compounds of the present invention may also exist, and may be separated in mixtures of isomers or in separated isomeric forms. All methods for preparing the compounds of the present invention and intermediates therein are considered to be part of the present invention. All tautomers of the compounds shown or described are also considered to be part of the present invention.

[0060] "Mammal" and "patient" encompass warm-blooded mammals (e.g., humans and domestic animals) typically under medical care. Examples include cats, dogs, horses, cows, and humans, as well as humans alone.

[0061] "Treating" or "treatment" encompasses treating a disease state in a mammal, and includes: (a) preventing a mammal from developing a disease state, particularly when such mammal is susceptible to the disease state but has not yet been diagnosed with the disease; (b) inhibiting the disease state, such as curbing its development; and / or (c) alleviating the disease state, such as causing regression of the disease state, until a desired endpoint is reached. Treatment also includes ameliorating symptoms of a disease (e.g., alleviating pain or discomfort), where such improvement may or may not directly affect the disease (e.g., cause, spread, expression, etc.).

[0062] "Pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds in which the parent compound is modified by preparing an acid or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic and organic acids selected from the group consisting of: 1,2-ethanedisulfonic acid, 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonic acid, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, glycolyl-p-aminophenylarsonic acid, hexylresorcinolic acid, Hypogamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, naphthalenesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, and toluenesulfonic acid.

[0063] The pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent or in a mixture of the two; in general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are useful. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th edition, page 1445 (Gennaro & Remington, 1990), the disclosure of which is hereby incorporated by reference.

[0064] "Therapeutically effective amount" includes an amount of the compound of the present invention that is effective when administered alone or in combination to treat the indications listed herein. "Therapeutically effective amount" also includes the amount of the combination of the claimed compounds that is effective in treating the desired indication. The combination of compounds may be a synergistic combination. The described synergistic effect (Chou & Talalay, 1984) occurs when the effect of the compound is greater than the additive effect of the compound when a single dose is administered alone when administered in combination. In general, synergy is most clearly demonstrated at suboptimal concentrations of the compound. Compared to the individual components, the synergistic effect may be lower cytotoxicity, increased effect, or some other beneficial effect of the combination.

[0065] The compounds of the present invention are expected to have the activities as described herein.

[0066] Formulation and dosage

[0067] In the present invention, the compound(s) of the present invention may be administered in any suitable manner (e.g., enteral or parenteral). Examples of methods of administration include oral and transdermal. It is known to those skilled in the art that the routes of administration of the compounds of the present invention may vary significantly. In addition to other oral administrations, sustained release compositions may be advantageous. Other acceptable routes may include injections (e.g., intravenous, intramuscular, subcutaneous, and intraperitoneal); subcutaneous implants; and oral, sublingual, topical, rectal, vaginal, and intranasal administration. Examples of oral formulations include tablets, coated tablets, hard and soft gelatin capsules, solutions, emulsions, and suspensions. Bioerodible, non-bioerodible, biodegradable, and non-biodegradable administration systems may also be used, including drug eluting structures placed by catheters, such as stents, which can deliver the compounds of the present invention directly to the vessel wall.

[0068] If a solid composition in the form of a tablet is prepared, the main active ingredient can be mixed with a pharmaceutical vehicle, examples of which include silicon dioxide, starch, lactose, magnesium stearate and talc. Tablets can be coated with sucrose or another suitable substance, or can be treated to have a continuous or delayed activity, and thus continuously release a predetermined amount of active ingredient. Gelatin capsules can be obtained by mixing the active ingredient with a diluent and incorporating the resulting mixture into a soft or hard gelatin capsule. Syrups or elixirs can contain active ingredients and sweeteners (usually calorie-free), preservatives (such as methylparaben and / or propylparaben), flavorings and suitable pigments. Water-dispersible powders or granules can contain active ingredients mixed with dispersants or wetting agents or with suspending agents such as polyvinylpyrrolidone and with sweeteners or flavoring agents. Suppositories can be used for rectal administration, and the suppositories are prepared with a binder (such as cocoa butter and / or polyethylene glycol) that melts at rectal temperature. Parenteral administration can be carried out using aqueous suspensions, isotonic saline solutions or sterile solutions for injection containing pharmaceutically compatible dispersants and / or wetting agents (e.g., propylene glycol and / or polyethylene glycol). The active ingredient can also be formulated into microcapsules or microspheres, optionally with one or more carriers or additives. The active ingredient can also be present in the form of a complex with cyclodextrins such as α-, β- or γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin and / or methyl-β-cyclodextrin.

[0069] The dosage of the compound of the present invention used every day will vary from person to person, and can be determined according to the severity of the disease treated to a certain extent. The dosage of the compound of the present invention will also change according to the compound used. The example of the dosage of the compound of the present invention includes about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 76, 80, 85, 90, 95 to 100mg / kg mammal body weight. The compound can be used with a single dose or many smaller dosages over a period of time. The length of time for which the compound is administered varies from person to person and may continue until the desired result (i.e., reduction of body fat or prevention of body fat gain) is achieved. Thus, therapy may continue from 1 day to several weeks, months, or even years, depending on the subject being treated, the desired result, and the speed at which the subject responds to treatment according to the invention.

[0070] Possible examples of the tablet of the present invention are as follows.

[0071]

[0072]

[0073] Possible examples of the capsules of the present invention are as follows.

[0074]

[0075] In the above capsule, the active ingredient has a suitable particle size. Crystalline lactose and microcrystalline cellulose are uniformly mixed with each other, sieved, and then magnesium stearate is blended. The final mixture is filled into hard gelatin capsules of suitable size.

[0076] Possible examples of the injection solutions of the present invention are as follows.

[0077]

[0078] Other features of the present invention will become apparent in the course of the following description of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting of the invention.

[0079] Synthesis Example

[0080] The compounds of the present invention can be synthesized using the methods described below together with known synthetic methods in the field of synthetic organic chemistry methods, or variants thereof as understood by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reaction is carried out in a solvent suitable for the reagents and materials used and suitable for affecting the transformation. It should be understood by those skilled in the art of organic synthesis that the functional groups present on the molecule should be consistent with the proposed transformation. Sometimes a judgment needs to be made to modify the order of the synthetic steps or to select a specific process scheme rather than another in order to obtain the desired compound of the present invention. It will also be recognized that another major consideration in the planning of any synthetic route in the art is the wise choice of protecting groups for protecting the reactive functional groups present in the compounds described in the present invention. Authoritative discussions describing many alternatives for trained practitioners can be found in Protective Groups In Organic Synthesis. (Greene & Wuts, 1991) All references cited herein are incorporated herein by reference in their entirety.

[0081] Synthesis Examples 1-34 represent procedures that can be used to prepare compounds of the present invention. Synthesis Examples 1-17 utilize known compounds isosorbide-2-mononitrate (5-hydroxy-1,4:3,6-dianhydro-D-glucitol 2-nitrate) and 5-amino-isosorbide-2-mononitrate (5-amino-1,4:3,6-dianhydro-D-glucitol 2-nitrate), and Synthesis Examples 18-34 utilize isosorbide-5-mononitrate (2-hydroxy-1,4:3,6-dianhydro-D-glucitol 5-nitrate) and 2-amino-isosorbide-5-mononitrate (2-amino-1,4:3,6-dianhydro-D-glucitol 5-nitrate) as starting materials. In a specific embodiment of the present invention, Synthesis Examples 1-17 are used for Formulas I, II and III, and Synthesis Examples 18-34 are used for Formulas IV, V and VI.

[0082] Synthesis Example 1

[0083]

[0084] 5-Amino-isosorbide-2-mononitrate (1) IN HCl solution can be treated with potassium isocyanate with stirring at room temperature for 10-12 hours to give the 2-ureido derivative (2) after conventional workup via extraction.

[0085] Synthesis Example 2

[0086]

[0087] 5-Amino-isosorbide-2-mononitrate (1) can be treated with Nt-BOC-glycine (t-BOC = tert-butyl-oxycarbonyl) in the presence of N,N'-dicyclohexylcarbodiimide (DCC) and dimethyl-aminopyridine (DMAP) in dichloromethane. After stirring overnight at ambient temperature, the t-BOC amide (3) can be isolated in a conventional manner. Subsequent treatment of (3) with trifluoroacetic acid can provide the deprotected amino acid adduct (4), which upon treatment with potassium isocyanate and dilute HCl solution as previously described can afford the ureidoglycine adduct (5).

[0088] Synthesis Example 3

[0089]

[0090] Treatment of isosorbide-2-mononitrate (6) with 1-cyclohexyl-3-(2-morpholinyl)carbodiimide (CMC) in dichloromethane (DCM) in the presence of (N-pyrrolidinyl)pyrimidine can provide the protected amino acid adduct (7). Removal of the protecting group with TFA (trifluoroacetic acid ester) can provide the amino compound (8). Subsequent treatment with potassium isocyanate and dilute HCl solution at 0°C to ambient temperature can produce the urea compound (9).

[0091] Synthesis Example 4

[0092]

[0093] Treatment of 5-amino-isosorbide-2-mononitrate (1) with t-BOC-aminoethanol or t-BOC-3-aminopropanol in the presence of [Cp*Ir(Pro)Cl] (Pro = proline radical) (Cp = cyclopentadienyl) in toluene or water affords the protected diamino adduct (10). Removal of the protecting group with TFA affords the primary amine (11), which can be converted to the urea (12a, 12b) using potassium cyanate in dilute hydrochloric acid solution as described previously.

[0094] Synthesis Example 5

[0095]

[0096] Isosorbide-2-mononitrate (6) can be deprotonated with sodium hydride or lithium diisopropylamide in tetrahydrofuran (THF) and then treated with t-BOC-aminobromoethane or t-BOC-3-aminopropyl bromide to give the ether (12). Deprotection of the t-BOC group using TFA will give the primary amine (13), and subsequent treatment with potassium isocyanate as previously described can give the urea (14).

[0097] Synthesis Example 6

[0098]

[0099] Treatment of 5-amino-isosorbide-2-mononitrate (1) with ethyl cyanoacetate in THF or DCM can yield urea (15). Further reaction with anhydrous ammonia in methanol can yield ureidocarboxamide (16).

[0100] Synthesis Example 7

[0101]

[0102] Treatment of isosorbide-2-mononitrate (6) with tert-butyl cyanoacetate in acetonitrile in the presence of N-methylimidazole can produce the carbamate (17). Removal of the tert-butyl group with HCl in ether or with TFA can give the carboxylic acid (18). Amidation of the acid with ammonia in the presence of DCC can provide the carboxamide (19).

[0103] Synthesis Example 8

[0104]

[0105] Treatment of 5-amino-isosorbide-2-mononitrate (1) or isosorbide-2-mononitrate (6) with malonamide in the presence of DCC / DMAP or CMC / (N-pyrrolidinyl)pyrimidine in DCM can generate malonamide (20) or malonate-ester-amide (21), respectively.

[0106] Synthesis Example 9

[0107]

[0108] Treatment of 5-amino-isosorbide-2-mononitrate (1) with phosgene and pyridine at about 0°C can produce carbamoyl chloride (22a, X=NH). Reaction of this carbamoyl chloride with glycinamide in DCM in the presence of DMAP at low temperature can give the ureidocarboxamide (23). Alternatively, carbamoyl chloride (22) (X=NH) can be treated with hydroxyacetamide in DCM in the presence of DMAP and give the carbonate (24) with a terminal carboxamide group. If isosorbide-2-mononitrate (6) is used as the starting material for this sequence, the phosgene reaction can produce the carbonyl chloride (22, X=O) at low temperature, and if this intermediate is treated with glycinamide or hydroxyacetamide under the conditions described previously, the carbamate (25) or carbonate (26) can be prepared, respectively.

[0109] Synthesis Example 10

[0110]

[0111] Treatment of 5-amino-isosorbide-2-mononitrate (1) with 3-hydroxypropionamide or 4-hydroxybutyramide in toluene at elevated temperature in the presence of a cobalt catalyst and molecular sieves can yield formamido-amines (27) and (28), respectively.

[0112] Synthesis Example 11

[0113]

[0114] Treatment of isosorbide-2-mononitrate (6) with sodium hydride in THF or with lithium diisopropylamide in THF and subsequent alkylation of the alkoxide with 3-bromopropionamide or 4-bromobutanamide can give the carboxamido-ethers (29) and (30), respectively.

[0115] Synthesis Example 12

[0116]

[0117] Treatment of 2-amino-isosorbide-5-mononitrate (1) with acetoxyacetyl chloride in the presence of triethylamine in THF can yield the amide adduct (31). Hydrolysis of the acetate with sodium hydroxide solution can yield the hydroxyamide (32).

[0118] Synthesis Example 13

[0119]

[0120] Treatment of isosorbide-2-mononitrate (6) with acetoxyacetyl chloride in the presence of triethylamine in THF can produce the ester adduct (33). Hydrolysis of the acetate with dibutyltin oxide will produce the glycolic acid adduct (34).

[0121] Synthesis Example 14

[0122]

[0123] N-acetoxyacetylglycine can be prepared by treating benzylglycine with acetoxyacetyl chloride in the presence of triethylamine followed by hydrogenolysis of the benzyl ester in the presence of a palladium catalyst. Treatment of 5-amino-isosorbide-2-mononitrate (1) with acetoxyacetylglycine in the presence of DCC in DCM can produce the acetylated amide adduct (35). Removal of the acetate can be accomplished by reaction with KOH or with trimethyltin hydroxide in methanolic solution (36).

[0124] Synthesis Example 15

[0125]

[0126] Treatment of isosorbide-2-mononitrate (6) with acetoxyacetylglycine in the presence of DCC / DMP in DCM can produce the acetylated ester adduct (37). Removal of the acetate can be accomplished by reaction with trimethyltin hydroxide (38).

[0127] Synthesis Example 16

[0128]

[0129] Treatment of 5-amino-isosorbide-2-mononitrate (1) with Nt-BOC-aminoethanol or Nt-BOC-3-aminopropanol in toluene at elevated temperature in the presence of a cobalt catalyst and molecular sieves can provide the protected amino compound (39). Removal of the protecting group can be accomplished using TFA, which can afford the unprotected primary amine (40). Reaction of the amine (40) with acetoxyacetyl chloride in the presence of trimethylamine can afford the amide (41), and subsequent treatment with KOH in methanol can afford the hydroxyacetamides (42 and 43).

[0130] Synthesis Example 17

[0131]

[0132] Deprotonation of isosorbide-2-mononitrate (6) with sodium hydride or LDA in THF followed by addition of Nt-BOC-aminoethyl bromide or Nt-BOC-3-aminopropyl bromide can afford the protected ether (44). Deprotection of the amino group using TFA can afford the primary amine (45), which can afford the amide (46) upon treatment with ethoxyacetyl chloride. Hydrolysis of the acetate using KOH in MeOH can afford the glycolamides glycolamides (47 and 48).

[0133] Synthesis Example 18

[0134]

[0135] 2-Amino-isosorbide-5-mononitrate (49) in 1 N HCl solution can be treated with potassium isocyanate with stirring at room temperature for 10-12 hours to give the 2-ureido derivative (50) after conventional workup via extraction.

[0136] Synthesis Example 19

[0137]

[0138] 2-Amino-isosorbide-5-mononitrate (49) can be treated with Nt-BOC-glycine (t-BOC = tert-butyl-oxycarbonyl) in the presence of N,N'-dicyclohexylcarbodiimide (DCC) and dimethyl-aminopyridine (DMAP) in dichloromethane. After stirring overnight at ambient temperature, the t-BOC amide (51) can be isolated in a conventional manner. Subsequent treatment of (51) with trifluoroacetic acid can provide the deprotected amino acid adduct (52), which upon treatment with potassium isocyanate and dilute HCl solution as previously described can afford the ureidoglycine adduct (53).

[0139] Synthesis Example 20

[0140]

[0141] Treatment of isosorbide-5-mononitrate (54) with 1-cyclohexyl-3-(2-morpholinyl)carbodiimide (CMC) in the presence of (N-pyrrolidinyl)pyrimidine in dichloromethane (DCM) can afford the protected amino acid adduct (55). Removal of the protecting group with TFA (trifluoroacetic acid ester) can provide the amino compound (56). Subsequent treatment with potassium isocyanate and dilute HCl solution at 0°C to ambient temperature can afford the urea compound (57).

[0142] Synthesis Example 21

[0143]

[0144] Treatment of 2-amino-isosorbide-5-mononitrate (49) with t-BOC-aminoethanol or t-BOC-3-aminopropanol in the presence of [Cp*Ir(Pro)Cl] (Pro = proline group) (Cp = cyclopentadienyl) in toluene or water can give the protected diamino adduct (58). Removal of the protecting group with TFA can give the primary amine (59), which can be converted to the urea (60a, 60b) using potassium cyanate in dilute hydrochloric acid solution as described previously.

[0145] Synthesis Example 22

[0146]

[0147] Isosorbide-5-mononitrate (54) can be deprotonated with sodium hydride or lithium diisopropylamide in tetrahydrofuran (THF) and then treated with t-BOC-aminobromoethane or t-BOC-3-aminopropyl bromide to give the ether (60). Deprotection of the t-BOC group using TFA will give the primary amine (61), and subsequent treatment with potassium isocyanate as previously described can give the urea (62).

[0148] Synthesis Example 23

[0149]

[0150] Treatment of 2-amino-isosorbide-5-mononitrate (49) with ethyl cyanoacetate in THF or DCM can produce the urea (63). Further reaction with anhydrous ammonia in methanol can produce the ureidocarboxamide (64).

[0151] Synthesis Example 24

[0152]

[0153] Treatment of isosorbide-5-mononitrate (54) with tert-butyl cyanoacetate in acetonitrile in the presence of N-methylimidazole can produce the carbamate (65). Removal of the tert-butyl group with HCl or with TFA in ether can give the carboxylic acid (66). Amidation of the acid with ammonia in the presence of DCC can provide the carboxamide (67).

[0154] Synthesis Example 25

[0155]

[0156] Treatment of 2-amino-isosorbide-5-mononitrate (49) or isosorbide-5-mononitrate (54) with malonamide in the presence of DCC / DMAP CMC / (N-pyrrolidinyl)pyrimidine in DCM can generate malonamide (68) or malonate-ester-amide (69), respectively.

[0157] Synthesis Example 26

[0158]

[0159] Treatment of 2-amino-isosorbide-5-mononitrate (49) with phosgene and pyridine at about 0°C can produce carbamoyl chloride (70a, X=NH). Reaction of this carbamoyl chloride with glycinamide in DCM in the presence of DMAP at low temperature can give the ureidocarboxamide (71). Alternatively, carbamoyl chloride (70a, X=NH) can be treated with hydroxyacetamide in DCM in the presence of DMAP and give the carbonate (72) with a terminal carboxamide group. If isosorbide-5-mononitrate (54) is used as the starting material for this sequence, the phosgene reaction can produce the carbonyl chloride (70b, X=O) at low temperature, and if this intermediate is treated with glycinamide or hydroxyacetamide under the conditions described previously, the carbamate (73) or carbonate (74) can be prepared, respectively.

[0160] Synthesis Example 27

[0161]

[0162] Treatment of 2-amino-isosorbide-5-mononitrate (49) with 3-hydroxypropionamide or 4-hydroxybutyramide in toluene at elevated temperature in the presence of a cobalt catalyst and molecular sieves can yield the carboxamido-amines (75) and (76), respectively.

[0163] Synthesis Example 28

[0164]

[0165] Treatment of isosorbide-5-mononitrate (6) with sodium hydride in THF or with lithium diisopropylamide in THF and subsequent alkylation of the alkoxide with 3-bromopropionamide or 4-bromobutanamide can give the carboxamido-ethers (77) and (78), respectively.

[0166] Synthesis Example 29

[0167]

[0168] Treatment of 2-amino-isosorbide-5-mononitrate (49) with acetoxyacetyl chloride in the presence of triethylamine in THF can give the amide adduct (79). Hydrolysis of the acetate with sodium hydroxide solution can give the hydroxyamide (80).

[0169] Synthesis Example 30

[0170]

[0171] Treatment of isosorbide-5-mononitrate (54) with acetoxyacetyl chloride in the presence of triethylamine in THF can produce the ester adduct (81). Hydrolysis of the acetate with dibutyltin oxide will produce the glycolic acid adduct (82).

[0172] Synthesis Example 31

[0173]

[0174] N-acetoxyacetylglycine can be prepared by treating benzylglycine with acetoxyacetyl chloride in the presence of triethylamine followed by hydrogenolysis of the benzyl ester in the presence of a palladium catalyst. Treatment of 2-amino-isosorbide-5-mononitrate (49) with acetoxyacetylglycine in the presence of DCC in DCM can produce the acetylated amide adduct (83). Removal of the acetate (84) can be accomplished by reaction with KOH or with trimethyltin hydroxide in methanolic solution.

[0175] Synthesis Example 32

[0176]

[0177] Treatment of isosorbide-5-mononitrate (6) with acetoxyacetylglycine in the presence of DCC / DMP in DCM can produce the acetylated ester adduct (85). Removal of the acetate can be accomplished by reaction with trimethyltin hydroxide (86).

[0178] Synthesis Example 33

[0179]

[0180] Treatment of 2-amino-isosorbide-5-mononitrate (49) with Nt-BOC-aminoethanol or Nt-BOC-3-aminopropanol in toluene at elevated temperature in the presence of a cobalt catalyst and molecular sieves can provide the protected amino compound (87). Removal of the protecting group can be accomplished using TFA, which can afford the unprotected primary amine (88). Reaction of the amine (88) with acetoxyacetyl chloride in the presence of trimethylamine can provide the amide (89), and subsequent treatment with KOH in methanol can afford the hydroxyacetamides (90 and 91).

[0181] Synthesis Example 34

[0182]

[0183] Deprotonation of isosorbide-5-mononitrate (54) with sodium hydride or LDA in THF followed by addition of Nt-BOC-aminoethyl bromide or Nt-BOC-3-aminopropyl bromide can afford the protected ether (92). Deprotection of the amino group using TFA can afford the primary amine (93), which can afford the amide (94) upon treatment with ethoxyacetyl chloride. Hydrolysis of the acetate using KOH in MeOH can afford the hydroxyacetamides (95 and 96).

[0184] biology

[0185] The compounds of the invention have two main indications. The first indication is as an antianginal agent. The compounds are expected to improve exercise capacity in cases of coronary artery disease by acting as coronary vasodilators. The second indication is for secondary prevention of cardiovascular disease, and vasodilation and ischemic preconditioning are sought in this application.

[0186] The experiments described herein are used to test whether the compounds of the present invention are suitable for at least two major indications. The experiments are to determine the effects of the compounds of the present invention and hypoxia on the production of ROS (reactive oxygen species) and markers of inflammatory and ischemic preconditioning and vascular smooth muscle relaxation mediators using human aortic endothelial cells (HAEC) and peripheral blood mononuclear cells (PBMC) in vitro. The primary HAEC isolated from normal healthy adults can be purchased from Thermo Fisher Scientific (Grand Island, New York) and cultured in culture medium 200. Peripheral blood mononuclear cells can be used as a part of the in vitro determination of the effects of the compounds of the present invention, as described below.

[0187] Cell culture can be performed in a Biospherix (Lacona, NY) humidified cell culture incubator that can dynamically control oxygen and carbon dioxide in a closed environment to cover all aspects of cell manipulation. The culture medium can be prepared with a pH of 7.3. Incubator conditions can be normoxic (21% O2, 5% CO2) or hypoxic (1% O2, 5% CO2, balance N2) humidified incubator with an internal temperature of 37°C (using a controlled incubator with CO2 / O2 monitoring and CO2 / N2 gas source). The culture medium is equilibrated to ambient gas conditions overnight before exposure to the cells. Early on and in some cases in primary cultures, cells will be incubated with the experimental gas mixture for varying periods of time. A closed environmental chamber with a glove-type cell access device can be used to prevent reoxygenation. Regular analysis of the cell culture medium with pH, ​​pCO2, and pO2 electrodes ensures a controlled environment.

[0188] HAEC can be exposed to normoxic conditions (20% oxygen) or hypoxic conditions (1% oxygen) for 1 hour (for ROS measurement), 2 hours (for determining transcription factor activity), 4 hours (for determining inflammatory mediator genes) or 16 hours (for inflammatory mediator proteins) in the presence or absence of the compounds of the present invention. Dihydroethidium (DHE) and 2',7'-dichlorofluorescein (DCF) fluorescence assays can be performed to measure intracellular levels of ROS, such as superoxide anions and hydrogen peroxide. The DNA binding activity of proinflammatory transcription factors (NF-κB and AP-1) in HAEC can be analyzed by electrophoretic mobility shift assay (EMSA). As previously shown, the mRNA and protein expression levels of the inflammatory mediator IL-1β can be measured in HAEC cultured in normoxia versus hypoxia under lipopolysaccharide (LPS) stimulation. (Folco, Sukhova, Quillard, & Libby, 2014) Other markers (TNF-α, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-γ, UT-B, eNOS, COX-2, and MCP-1) can be examined in stimulated HAEC. The expression levels of mRNA and protein can be measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). After stimulating HAEC in primary culture, ELISA can be used to analyze selected mediators of relevant endothelial responses, such as PGE2. Metabolism can be comprehensively assessed using a combination of high-resolution respirometry and Seahorse extracellular flux analysis to indicate the contribution of mitochondria to glycolysis in patient cells using established protocols.

[0189] The activation level of the relevant transcription factor can be determined by measuring the DNA binding activity of the nuclear extract isolated from HAEC in EMSA. HAEC can be treated as indicated above, and the cells are then incubated with lysis buffer (10mM Tris-HCl, pH 8.0, 60mM KCl, 1mM ethylenediaminetetraacetic acid (EDTA), 1mM dithiothreitol, 100μM phenylmethylsulfonyl fluoride, 0.1% NP-40), lysed on ice for 5 minutes, and then centrifuged at 600×g for 4 minutes at 4°C to collect the nucleus. Then, the nuclear particles can be washed with lysis buffer without NP-40, in nuclear extract buffer (20mM Tris-HCl, pH 8.0, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 25% glycerol) on ice for 10 minutes, and centrifuged at 4°C for 15 minutes at 18,300×g. The supernatant containing the nuclear extract can be frozen immediately on dry ice and transferred to -80°C until analysis. 4 μg of nuclear protein extract, 10 mM Tris-Cl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol and 2 μg poly [dI-dC] can be used for binding reaction. After adding the reagent, the mixture can be incubated at room temperature for 25 minutes. Then, biotin-labeled specific oligonucleotide probes and / or unlabeled controls can be added, and the binding mixture is then incubated at room temperature for 25 minutes. Competition studies can be performed by adding molar excess of unlabeled oligonucleotides to the binding reaction. Protein-DNA complexes obtained by electrophoresis on a non-denaturing 5% polyacrylamide gel for 3 hours at 150 V can be used. This is a variation of the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, Rockford, IL).

[0190] Real-time quantitative polymerase chain reaction (QRT-PCR) can be used for determination. Fluorescent 5'-nuclease assay technology with probes and primers (Applied Biosystems, Foster City, CA) can be used for gene expression analysis. HAEC can be treated as indicated above, and total RNA can be isolated from HAEC using RNeasy mini kit (Qiagen, Valencia, CA). 1 μg of total RNA can be reverse transcribed in 20 μL 5mM MgCl2, 10mM Tris-HCl, pH 9.0, 50mM KCl, 0.1% Triton X-100, 1mM dNTP, 1 unit / μL recombinant RNasin, 15 units / μg of avian myeloblastosis virus (AMV) reverse transcriptase and 0.5 μg random hexamer at 25°C for 15 minutes, at 42°C for 45 minutes, and at 99°C for 5 minutes. Individual genes can be amplified on an Applied Biosystems 7300 Real-Time PCR System using a universal PCR master mix and a standard thermal cycler protocol (50°C for 2 minutes, 95°C for 15 seconds and 60°C for 1 minute, repeated 45 times before the first cycle). Gene expression assay reagents for human TNF-α, IL-1, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-α, TB, COX-2, eNOS, MCP-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be used for specific probes and primers for PCR amplification. The threshold cycle (CT) can be determined from each well using the Applied Biosystems Sequence Detection Software, which represents the fractional cycle number at which the amount of amplified target gene reaches a fixed threshold.

[0191] The intracellular level of ROS can be measured by DHE and DCF fluorescence staining using a fluorescence microscope. HAEC can be treated as indicated above, and DHE or carboxyl-H2DCF-DA (Invitrogen Corp., Carlsbad, CA) is loaded into cells at a concentration of 5 μM in PBS at 37°C in a 5% CO2 / 95% air, humidified cell culture incubator for 30 minutes. HAEC can be washed with PBS and examined on a fluorescence microscope. The DHE or DCF fluorescence intensity of the acquired digital image can be quantified accordingly.

[0192] The effect of the compounds of the invention on hypoxia-mediated endothelial NO release can be measured by isolating cells with membrane-permeable 4,5-diaminofluorescein diacetate (DAF-2 diacetate), which results in the accumulation of membrane-impermeable intracellular DAF-2. DAF-2 reacts with NO to form highly fluorescent triazolofluorescein (DAF-2T), and this can be detected by fluorescence spectroscopy of the supernatant. (Leikert, Müller, Vollmar, & Dirsch, 2001)

[0193] Parallel studies can be used to test the effects of compounds of the invention on cellular ROS homeostasis and metabolism in cell metabolism assays. Plated cells from different experimental groups can be studied on a Seahorse extracellular flux analyzer. Oxygen consumption rates (mitochondrial function) and extracellular acidification rates (glycolysis) can be measured under basal conditions, under maximal respiration conditions, and under conditions of inhibited mitochondrial bioenergetics using established methods. (Dai et al., 2016) Parallel subsets of cells can be placed in Oroboros high-resolution respirometer chambers to simultaneously measure cellular ROS and metabolism using established protocols. (Alleman et al., 2016)

[0194] Peripheral blood mononuclear cells and serum can be obtained from human volunteers. In clinical trials designed to test the compounds of the invention in patients with ischemic cardiomyopathy (including a history of myocardial infarction) and secondary prevention indications, patients can be used as their own controls. This study was limited to 5 × 8 mL tubes per blood draw, from which 2 mL of plasma-derived serum and 8 × 10 per tube were expected to be drawn using a BD Vacutainer CPT and sodium heparin (Franklin Lakes, NJ). 6 PBMC. Contains sodium heparin anticoagulant and FICOLL Hypaque density liquid, with a polyester gel barrier separating the two liquids. The tube can be centrifuged at 1500g for 15 minutes at room temperature. Plasma can be obtained as the supernatant above the mononuclear cell and platelet layer on top of the density solution. Serum can be derived from the plasma supernatant by adding CaCl2 to a final concentration of 20mM and allowing it to clot for 4 hours at room temperature, followed by freezing the clotted plasma at -20°C overnight, thawing, and centrifuging the remaining serum at 5000g for at least 5 minutes. The serum can be filtered, aliquoted and frozen at -20°C. PBMC can be separated by centrifugation at 400g for 10-15 minutes at 18°C-20°C, washed twice in phosphate-buffered saline, and then centrifuged at 8×10 6 The cells / 2 mL were resuspended in AIM V medium (Life Technologies) and then split into 1×10 6Aliquots of cells. Cultures can be performed in Biospherix (Lacona, NY) humidified cell culture incubators that dynamically control oxygen and carbon dioxide in a closed environment, covering all aspects of cell manipulation.

[0195] DHE / DCF fluorescence assays, real-time RT-PCR, ELISA, EMSA, and reporter gene assays can be performed to determine ROS generation, mRNA, and protein expression of mediators of ischemic preconditioning, including downstream products of endothelial nitric oxide synthase and cyclooxygenase-2 expression. Compared to controls, products of eNOS and COX-2 can demonstrate the therapeutic value of the compounds of the invention. Toxicity can be assessed by the metabolic effects of the compounds of the invention using Seahorse technology.

[0196] Tables 1-6 show the structures of compounds of the invention that can be synthesized as described above.

[0197] Table 1

[0198]

[0199] Examples# <![CDATA[R 1 ]]> Does not exist 2. <![CDATA[(CH2)2O]]> 3. <![CDATA[(CH2)2NH]]> 4. <![CDATA[(CH2)3O]]> 5. <![CDATA[(CH2)3NH]]> 6. <![CDATA[CH2C(=O)O]]> 7. <![CDATA[CH2C(=O)NH]]>

[0200] Table 2

[0201]

[0202] Examples# <![CDATA[R 2 ]]> 1. <![CDATA[(CH2)2O]]> 2. <![CDATA[(CH2)2NH]]> 3. <![CDATA[(CH2)3O]]> 4. <![CDATA[(CH2)3NH]]> 5. <![CDATA[CH2C(=O)O]]> 6. <![CDATA[CH2C(=O)NH]]> 7. <![CDATA[CH2OC(=O)O]]> 8. <![CDATA[CH2OC(=O)NH]]> 9. <![CDATA[CH2NHC(=O)O]]> 10. <![CDATA[CH2NHC(=O)NH]]>

[0203] Table 3

[0204]

[0205]

[0206]

[0207] Table 4

[0208]

[0209] Examples# <![CDATA[R 1 ]]> 1. Does not exist 2. <![CDATA[(CH2)2O]]> 3. <![CDATA[(CH2)2NH]]> 4. <![CDATA[(CH2)3O]]> 5. <![CDATA[(CH2)3NH]]> 6. <![CDATA[CH2C(=O)O]]> 7. <![CDATA[CH2C(=O)NH]]>

[0210] Table 5

[0211]

[0212]

[0213]

[0214] Table 6

[0215]

[0216] Examples# <![CDATA[R 1 ]]> 1. Does not exist 2. <![CDATA[(CH2)2O]]> 3. <![CDATA[(CH2)2NH]]> 4. <![CDATA[(CH2)3O]]> 5. <![CDATA[(CH2)3NH]]> 6. <![CDATA[CH2C(=O)O]]> 7. <![CDATA[CH2C(=O)NH]]>

[0217] Many modifications and variations of the present invention are possible in light of the above teachings.It is therefore to be understood that within the scope of the appended claims, the present invention may be practiced otherwise than as specifically described herein.

[0218] References

[0219] Alleman, RJ, Tsang, AM, Ryan, TE, Patteson, DJ, McClung, JM, Spangenburg, EE, et al. (2016). Exercise-induced protection against reperfusion arrhythmia involves stabilization of mitochondrial energetics. American Journal of Physiology-Heart and Circulatory Physiology, 310, H1360-H1370.

[0220] Benjamin, EJ, Muntner, P., Alonso, A., Bittencourt, MS, Callaway, CW, Carson, AP, et al. (2019). Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. Circulation, 139, e56-e528.

[0221] Chou, TC, Talalay, P. (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Advances in Enzyme Regulation, 22, 27-55

[0222] Dai, W., Cheung, E., Alleman, RJ, Perry, JB, Allen, ME, Brown, DA, et al. (2016). Cardioprotective effects of mitochondria-targeted peptide SBT-20 in two different models of rat ischemia / reperfusion. Cardiovascular Drugs and Therapy, 30, 559-566.

[0223] Folco, EJ, Sukhova, GK, Quillard, T., & Libby, P. (2014). Moderate hypoxia potentiates interleukin-1beta production in activated human macrophages. Circulation Research, 115, 875-883.

[0224] Gennaro, AR & Remington, JP (1990). Remington's Pharmaceutical Sciences. In (18th ed., p. 1445). Easton, PA: Mack.

[0225] Greene, TW & Wuts, PGM (1991). Protective groups in organic synthesis. (2nd ed.) New York: Wiley.

[0226] Guo, Y., Tukaye, DN, Wu, WJ, Zhu, X., Book, M., Tan, W., et al. (2012). The COX-2 / PGI2 receptor axis plays an obligatory role in mediating the cardioprotection conferred by the late phase of ischemic preconditioning. PLoS One, 7, e41178.

[0227] Ji, Y.-S., Xu, Q., & Schmedtje, JF, Jr. (1998). Hypoxia induces high-mobility-group protein I(Y) and transcription of the cyclooxygenase-2 gene in human vascular endothelium. Circulation Research, 83, 295-304.

[0228] Leikert, JF, TR, Müller, C., Vollmar, AM, & Dirsch, VM (2001). Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein. FEBS Letters, 506, 131-134.

[0229] Li, Q., Guo, Y., Tan, W., Ou, Q., Wu, W. J., Sturza, D. et al. (2007). Cardioprotection Afforded by Inducible Nitric Oxide Synthase Gene Therapy Is Mediated by Cyclooxygenase-2 via a Nuclear Factor-κB Dependent Pathway. Circulation, 116, 1577-1584.

[0230] Li, X., Chen, G, Yang, B. (2012) Urea transporter physiology studied in knockout mice. Frontiers in Physiology, 3: 217.

[0231] Sands, JM (1999). Regulation of Renal Urea Transporters. Journal of the American Society of Nephrology, 10, 635-646.

[0232] Schmedtje, JF, Jr., Ji, Y.-S., Liu, W.-L., DuBois, RN, & Runge, MS (1997). Hypoxia induces cyclooxygenase-2 via the NF-κB p65 transcription factor in human vascular endothelial cells. Journal of Biological Chemistry, 272, 601-608.

[0233] Semenza, GL (2019). Pharmacologic Targeting of Hypoxia-Inducible Factors. Annual Review of Pharmacology and Toxicology, 59, 379-403.

[0234] Shayakul, C., Clemencon, B., & Hediger, M.A. (2013). The urea transporter family (SLC14): physiological, pathological and structural aspects. Molecular Aspects of Medicine, 34, 313-322.

[0235] Sun, Y., Lau, C. W., Jia, Y., Li, Y., Wang, W., Ran, J., Li, F., Huang, Y., Zhou, H., Yang, B. (2016) Functional inhibition of urea transporter UT-B enhances endothelial-dependent vasodilatation and lowers blood pressure via L-arginine-endothelial nitric oxide synthase-nitric oxide pathway. Scientific Reports 6, 18697.

[0236] Wagner, L., Klein, JD, Sands, JM, & Baylis, C. (2002). Urea transporters are distributed in endothelial cells and mediate inhibition of L-arginine transport. American Journal of Physiology: Renal Physiology, 283, F578-F582.

[0237] Wang, X., Wu, L., Aouffen, M., Mateescu, M., Nadeau, R., & Wang, R. (1999). Novel cardiac protective effects of urea: from shark to rat. British Journal of Pharmacology, 128, 1477-1484.

[0238] Xiao, S.; Erdely, A.; Wagner, L.; Baylis, C. (2001) Uremic levels of BUN do not cause nitric oxide deficiency in rats with normal renal function. Am J Physiol Renal Physiol 280, F996-F1000.

[0239] Xu, Q., Ji, Y. S., & Schmedtje, J. F., Jr. (2000). Sp1 increases expression of cyclooxygenase-2 in hypoxic vascular endothelium-Implications for the mechanisms of aortic aneurysm and heart failure. Journal of Biological Chemistry, 275, 24583-24589.

[0240] Zhao, D., Sonawane, ND, Levin, MH, & Yang, B. (2007). Comparative transport efficiencies of urea analogues through urea transporter UT-B. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1768, 1815-1821.

Claims

1. A compound of formula II or V: in: R 1 does not exist; Alternatively, R 1 is selected from the group consisting of: (CH2)2O, (CH2)2NH, (CH2)3O, (CH2)3NH, CH2C(=O)O, and CH2C(=O)NH; and, R 2 Selected from: (CH2)2O, (CH2)2NH, (CH2)3O, (CH2)3NH, CH2C(=O)O, CH2C(=O)NH, CH2OC(=O)O, CH2OC(=O)NH, CH2NHC(=O)O and CH2NHC(=O)NH; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein the compound is of formula II or V or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 2, wherein: R 2 Selected from: (CH2)2O, (CH2)2NH, (CH2)3O, (CH2)3NH, CH2OC(=O)O, CH2OC(=O)NH, CH2NHC(=O)O and CH2NHC(=O)NH; or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 2, wherein: R 2 is CH2C(=O)O, or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 2, wherein: R 2 is CH2C(=O)NH, or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition comprising: a therapeutically effective amount of the compound according to claim 1 and a pharmaceutically acceptable carrier.

7. A method for treating a disease, comprising: administering a therapeutically effective amount of the compound according to claim 1 to a mammal in need thereof, wherein the disease is cardiovascular disease.

8. The method for treating a disease according to claim 7, wherein the cardiovascular disease is selected from the group consisting of coronary artery disease, myocardial infarction, heart failure, arrhythmia, cardiac electrophysiological disorders, congenital cardiovascular anomalies, developmental cardiovascular anomalies, inflammatory cardiomyopathy, Kawasaki disease, infectious cardiomyopathy, sudden death / cardiac arrest, atherosclerosis, atherosclerotic cardiovascular disease, valvular heart disease, venous insufficiency, cardiac thrombosis, vascular thrombosis, thromboembolism, peripheral arterial disease, aortic aneurysm, aortic dissection, vascular aneurysm, vascular dissection, stroke, systemic hypertension and pulmonary hypertension.

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